α-Synuclein binder and method of use

Abstract

The present invention relates to compounds of formula (I) or pharmaceutically acceptable salts thereof, which may be suitable for imaging α-synuclein pathology. Accordingly, these compounds or pharmaceutically acceptable salts thereof are useful for binding and imaging α-synuclein aggregates in patients suffering from Parkinson's disease. More specifically, the present invention relates to methods of using the compounds of the present invention as tracers in positron emission tomography (PET) imaging to study α-synuclein in the brain in vivo, enabling the diagnosis of Parkinson's disease and other neurodegenerative diseases characterized by α-synuclein pathology. The present invention also relates to methods for measuring the clinical efficacy of therapeutic agents for Parkinson's disease and other neurodegenerative diseases characterized by α-synuclein pathology. [Formula 1] TIFF2025538915000179.tif31126

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims the benefit or priority of U.S. Provisional Application No. 63 / 485,159, filed on 15 February 2023, the disclosure thereof being incorporated herein in its entirety. [Background technology]

[0002] Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis (ALS), and prion diseases are debilitating diseases that affect cognitive and / or muscle control. These diseases are a subset of protein misfolding diseases. Protein folding is an essential process for protein function in all living organisms, and conditions that disrupt protein folding threaten the viability of cells. In some cases, the above diseases arise because a particular protein becomes non-functional when it is misfolded. In other diseases, misfolding occurs simultaneously with aggregation, and pathological symptoms arise because the underlying aggregates are harmful. Even though neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are caused by different proteins, both involve the accumulation of insoluble fibrous protein deposits called amyloid. For example, Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA), collectively known as "synucleinopathy," are associated with the accumulation of aggregated forms of α-synuclein protein in neurons in the brain (see "Nat. Rev. Neuro. 2013, 9, 13-24" and "J. Parkinson's Disease 2013, 3, 565-567"). Major neuropathological changes in PD include degeneration of dopaminergic neurons in the substantia nigra, as well as the formation of Lewy bodies (LBs) and Lewy neurites (LNs). To date, the pathogenesis of PD is not fully understood.

[0003] Alpha-synuclein is a presynaptic terminal protein consisting of 140 amino acids, playing crucial roles in the central nervous system, including synaptic vesicle recycling and synthesis, vesicle storage, and neurotransmitter release. It is specifically upregulated in individual populations of presynaptic terminals in the brain during synaptic rearrangement associated with acquisition. Alpha-synuclein is naturally highly soluble and exists in an unfolded state. Evidence suggests that in synucleinopathy, filamentous aggregates of alpha-synuclein accumulate on the presynaptic membrane, causing synaptic dysfunction and neuronal cell death, and that these filamentous aggregates may contribute to Parkinson's disease and DLB. Antibody immunohistochemical studies have identified alpha-synuclein aggregates as a major component of Lewy bodies, which are microscopic protein deposits in degraded nerve cells. The accumulation of misfolded fibrous α-synuclein in Lewy bodies (LBs) and Lewy neurites (LNs) is considered a characteristic feature of Parkinson's disease (PD).

[0004] The diagnosis of Parkinson's disease (PD) is primarily based on clinical symptoms such as resting tremor, bradykinesia, and rigidity, but these methods have limitations (see "J. Neurology 2019, 266, 1927-1936"). Current treatment goals for PD are to slow disease progression and minimize patient symptoms. Therefore, methods for diagnosing PD at a very early stage can greatly assist physicians in designing treatment paradigms accordingly and slowing disease progression. Improved diagnostic methods for identifying the aggregation of misfolded proteins containing α-synuclein are still needed for early detection and continuous monitoring of PD in the population (see "J. Parkinson's Disease 2013, 3, 565-567").

[0005] Alpha-synuclein positron emission tomography (PET) tracers may be valuable non-invasive diagnostic biomarkers for spatial and temporal quantification of aggregated pathological alpha-synuclein in the human brain, serving as a Parkinson's disease biomarker. Furthermore, alpha-synuclein PET tracers may be useful for patient selection in PD clinical trials. In this manner, alpha-synuclein tracers can be developed as companion diagnostics for the co-registration of therapeutic agents. Moreover, alpha-synuclein PET tracers may be important disease-related tools for quantifying the stabilization or reduction of alpha-synuclein formation in relation to disease-modifying PD therapeutic agents.

[0006] Therefore, there is a need for neuroimaging radiotracers that enable in vivo imaging of α-synuclein pathology and thereby provide insights into the deposition of α-synuclein aggregates in the human brain. Successful neuroimaging radiotracers must cross the blood-brain barrier, be rapidly cleared from tissues and plasma, and possess high affinity and specificity for α-synuclein aggregates with higher selectivity than binding to β-amyloid and tau aggregate proteins, which are co-expressed in many PD patient populations (see "Biol Psychiatry 2015, 78, 672-683" and "J Neuropath Exper Neurol 2003, 62, 389-397"). While α-synuclein-binding ligands with reduced selectivity for aggregated β-amyloid have been described (WO2019 / 121661), compounds with high selectivity for aggregated proteins co-expressed in PD are needed to quantify α-synuclein-specific signals in in vivo imaging studies of PD patients. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2019 / 121661 [Non-patent literature]

[0008] [Non-Patent Document 1] Nat. Rev. Neuro. 2013, 9, 13-24 [Non-Patent Document 2] J. Parkinson's Disease 2013, 3, 565-567 [Non-Patent Document 3] J. Neurology 2019, 266, 1927-1936 [Non-Patent Document 4] Biol Psychiatry 2015, 78, 672-683 [Non-Patent Document 5] J Neuropath Exper Neurol 2003, 62, 389-397 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention advances these concerns by providing a compound represented by formula I as an aggregated α-synuclein binding ligand having higher selectivity than the binding of aggregated β-amyloid. The present invention further relates to a method of using the compound represented by formula I as a tracer in PET imaging to study α-synuclein deposition in the brain in vivo, in order to enable the diagnosis of neurodegenerative diseases characterized by α-synuclein pathology. The present invention further relates to a method of measuring the clinical efficacy of therapeutic agents targeting α-synuclein pathology. [Means for solving the problem]

[0010] The present invention relates to compounds represented by formula I, pharmaceutically salts thereof, pharmaceutical compositions containing them, diagnostic and therapeutic uses, and processes for producing such compounds. One embodiment of the present invention relates to a compound represented by formula I: [ka] Provided is a compound represented by or a pharmaceutically acceptable salt thereof, wherein, R is independently selected from H, -C , , d , 1-6 , 1-6 , c , n , n , n , c , c , 1 , , n , 10 , 1-6 , c , d , n , 1-6 , n , n , n , c alkyl, OR c or halo, wherein the alkyl may be substituted with one to three groups selected from -C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted -C 1-6 alkyl, wherein the alkyl may be substituted with one to three groups of R; R b is -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -NR c 2, -(CH2) n halogen or -O(CH2) n halo; R c is independently selected from H or -C 1-6 alkyl, wherein the alkyl may be substituted with one to three groups selected from -C 1-6 alkyl, OR d or halo; R d is independently selected from H or -C 1-6 alkyl; R 1 is -(CH2) n OR c , -(CH2) n O(CH2) n R, -(CH2) n O(CH2)​​​​​​​A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocycline is independently selected from cycloalkyl, cycloalkyl, phenyl, heteroaryl, or heterocycline, where R b It can be replaced by 1 to 3 of the following: R 2 is hydrogen, OR c NO2, Halo or C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, imidazopyridinyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; Ring B is, [ka] Selected from; m is selected from 1, 2, or 3; n is independently selected from 0, 1, 2, 3, or 4; p is selected from 0, 1, 2, or 3; and, q is selected from 1, 2, or 3.

[0011] The present invention also covers isotope-labeled compounds represented by formula I. Furthermore, the present invention provides pharmaceutical compositions comprising a compound represented by formula I and at least one pharmaceutically acceptable carrier.

[0012] The present invention relates to a compound represented by Formula I that can be useful for binding α-synuclein aggregated protein and / or tau aggregated protein, and is therefore useful for binding and imaging α-synuclein aggregated protein pathogenesis in patients with PD and non-PD synucleopathies, and aggregated tau protein pathogenesis in patients with Alzheimer's disease (AD) and non-AD tauopathy, by PET imaging techniques commonly known in the art (see "Nucl. Med. 2019, 60, 93-99 and 107-114"). The present invention further relates to a method of using the compound represented by Formula I to identify patients with abnormal levels of aggregated α-synuclein pathogenesis in the brain. The present invention further relates to a method of using the compound represented by Formula I to measure the progression of α-synuclein pathogenesis over time as a biomarker in the clinical evaluation of potential therapeutic agents that can modify the progression of Parkinson's disease.

[0013] The compounds of the present invention may also be useful for imaging and detection of other neurodegenerative diseases characterized by the deposition of α-synuclein aggregates, such as multiple system atrophy (MSA) and Lewy body dementia (DLB). [Brief explanation of the drawing]

[0014] [Figure 1] Saturation binding experiments for [3H]-105 in aggregated β-amyloid-rich AD tissue homogenates. [Figure 2] Saturation binding experiments on [3H]-1 in the triton-insoluble fraction of aggregated α-synuclein-rich PD tissue homogenate. [Figure 3] [3H]-1 radioactive ligand saturation binding data in human PD cortical tissue homogenates rich in aggregated α-synuclein pathology. [Figure 4] [3H]-1 radioactive ligand saturation binding data in aggregated Aβ-rich human AD cortical tissue homogenate. [Figure 5][3H]-24 radioligand saturation binding data in aggregated α-synuclein-rich human PD cortical tissue homogenates. [Figure 6] [3H]-24 radioligand saturation binding data in aggregated Aβ-rich human cortical AD tissue homogenate. [Modes for carrying out the invention]

[0015] The present invention provides novel substituted heterocyclic piperazine amide compounds, methods for synthesizing these compounds, pharmaceutical compositions containing them, isotope-labeled compounds, and methods for using these compounds as imaging agents.

[0016] In one embodiment, the present invention is based on formula I: [ka] The subject is the compound represented by or its pharmaceutically acceptable salt, where, In the above formula, R is H, -C 1-6 Alkyl, OR c Alternatively, it is independently selected from the halo, where the alkyl is -C 1-6 Alkyl, OR c Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R a -C is either not substituted or has been substituted. 1-6 A alkyl group is independently selected, where the alkyl group may be substituted with 1-3 groups of R; R b is -C 1-6 Alkyl, halo, -(CH2) n Ure c -CN, -NR c 2, -(CH2) n Halogen or -O(CH2) n Selected independently from Halo; R c is H or -C 1-6 Selected independently of alkyl, where the alkyl is -C 1-6Alkyl, OR d Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R d is H or -C 1-6 Selected independently of alkyl; R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2) n R, -(CH2) n O(CH2) n Ure c , Halo, NR2, C that is not substituted or is substituted 1-6 Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocycline is independently selected from cycloalkyl, cycloalkyl, phenyl, heteroaryl, or heterocycline, where R b It can be replaced by 1 to 3 of the following: R 2 is hydrogen, OR c NO2, Halo or C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, imidazopyridinyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; Ring B is, [Chemical formula] selected from TIFF0007881829000005.tif30143; m is selected from 1, 2 or 3; n is independently selected from 0, 1, 2, 3 or 4; p is selected from 0, 1, 2 or 3; and, q is selected from 1, 2 or 3.

[0017] In a further embodiment, the present invention relates to a compound of formula IA: [Chemical formula] or a pharmaceutically acceptable salt thereof, wherein, in the above formula, R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl is optionally substituted with 1 to 3 groups selected from -C 1-6 alkyl, OR c or halo; R a is unsubstituted or substituted -C 1-6 alkyl or -(CH2) 1-3 O-(CH2) 0-3 R, wherein the alkyl is optionally substituted with 1 to 3 groups of R; R b is -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -NR c 2, -(CH2) n halogen or -O(CH2) n halo, independently selected; R c is independently selected from H or -C <000,0088>alkyl, wherein the alkyl is optionally substituted with 1 to 3 groups selected from -C 1-6 alkyl, OR d or halo; R d is independently selected from H or -C 1-6 alkyl; R 1 is -(CH2) n OR c , -(CH2) n O(CH2) n R, -(CH2) n O(CH2) n OR c , halo, -NR2, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl or unsubstituted or substituted heterocyclyl, where the alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted with 1 to 3 groups of R b ; R 2 is selected from hydrogen, OR c , halo or C 1-6 alkyl; Ring A 1 is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl may be substituted with 1 to 3 groups of R; Ring A 3 is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; m is selected from 1, 2 or 3; n is independently selected from 0, 1, 2, 3 or 4; and, p is selected from

[0018] In further embodiments, the present invention relates to a compound represented by formula IA or a pharmaceutically acceptable salt thereof, wherein, R is H, -C 1-6 Alkyl, OR c Alternatively, it is independently selected from the halo, where the alkyl is -C 1-6 Alkyl, OR c Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R a -C is either not substituted or has been substituted. 1-6 Alkyl or -(CH2) 1-3 O-(CH2) 0-3 Selected independently of R, where the alkyl group may be substituted with 1-3 groups of R; R b is -C 1-6 Alkyl, halo, -(CH2) n Ure c -CN, -NR c 2, -(CH2) n Halogen or -O(CH2) n Selected independently from Halo; R c is H or -C 1-6 Selected independently of alkyl, where the alkyl is -C 1-6 Alkyl, OR d Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R d is H or -C 1-6 Selected independently of alkyl; R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2) n R, -(CH2) n O(CH2) n Ure c , Halo, -NR2, C that is not substituted or is substituted 1-6Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocycline is independently selected from cycloalkyl, cycloalkyl, phenyl, heteroaryl, or heterocycline, where R b It can be replaced by 1 to 3 of the following: R 2 is hydrogen, OR c -NO2, Halo or -C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, imidazopyridinyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; m is selected from 1, 2, or 3; n is independently selected from 0, 1, 2, 3, or 4; p is selected from 0, 1, 2, or 3; and, q is selected from 1, 2, or 3.

[0019] In further embodiments, the present invention relates to a compound represented by formula IA or a pharmaceutically acceptable salt thereof, wherein, R is H, -C 1-6 Alkyl, OR c Alternatively, it is independently selected from the halo, where the alkyl is -C 1-6 Alkyl, OR cAlternatively, it may be substituted with 1 to 3 elements selected from the halo; R a -C is either not substituted or has been substituted. 1-6 A alkyl group is independently selected, where the alkyl group may be substituted with 1-3 groups of R; Ring A 1 It is selected from pyridyl, pyrazinyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, or pyridyl, where the pyrimidinyl, phenyl, or pyridyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl; m is selected from 1 or 2; p is selected from 0, 1, or 2; and, All other substituents and variable parts are as defined above in Formula I.

[0020] In another embodiment, the present invention relates to formula IB: [ka] The subject is the compound represented by or its pharmaceutically acceptable salt, where, In the above formula, R is H, -C 1-6 A molecule independently selected from alkyl or halo, where the alkyl is -C 1-6 Alkyl, OR c Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R a -C is either not substituted or has been substituted. 1-6 It is an alkyl group, where the alkyl group may be substituted with R1-3 groups; R b is -C 1-6 Alkyl, halo, -(CH2) n Ure c -CN, -(CH2) n Halogen or -O(CH2)n Selected independently from Halo; R c is H or -C 1-6 Selected independently of alkyl; R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2) n R, -(CH2) n O(CH2) n Ure c , Halo, NR2, C that is not substituted or is substituted 1-6 Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 Selected from cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl, where the alkyl, phenyl, cycloalkyl, heteroaryl, or heterocyclyl is R b It can be replaced by 1 to 3 of the following: R 2 is hydrogen, OR c , Halo or C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, pyrazolyl, oxazolyl, thiazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, pyrrolopyrazinyl, oxadiazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine, or phenyl; n is independently selected from 0, 1, 2, 3, or 4; and, p is selected from 1 or 2.

[0021] In another embodiment, the present invention is directed to a compound represented by Formula IC: [Chemical formula] or a pharmaceutically acceptable salt thereof, wherein in the above formula R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl may be substituted with 1 to 3 groups selected from -C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted -C 1-6 alkyl, wherein the alkyl may be substituted with 1 to 3 groups of R; R b is -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -(CH2) n halogen or -O(CH2) n halo, independently selected; R c is independently selected from H or -C 1-6 alkyl; R 1 is -(CH2) n OR c , -(CH2) n O(CH2) n R, -(CH2) n O(CH2) n OR c , halo, NR2, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl or unsubstituted or substituted heterocyclyl, independently selected, wherein the alkyl, phenyl, cycloalkyl, heteroaryl or heterocyclyl may be substituted with 1 to 3 groups of R b ; R 2 is hydrogen, OR c , Halo or C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl, where the pyrimidinyl, phenyl, pyridyl, pyrazinyl, or pyridazinyl may be substituted with groups R1 to R3; Ring A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; n is independently selected from 0, 1, 2, 3, or 4; and, p is selected from 0, 1, 2, or 3.

[0022] In another embodiment, the present invention relates to a compound represented by formula IC or a pharmaceutically acceptable salt thereof, wherein, R is H, -C 1-6 A molecule independently selected from alkyl or halo, where the alkyl is -C 1-6 Alkyl, OR c Alternatively, it may be substituted with 1 to 3 elements selected from the halo; R a -C is either not substituted or has been substituted. 1-6 A alkyl group is independently selected, where the alkyl group may be substituted with 1-3 groups of R; R b is -C 1-6 Alkyl, halo, -(CH2) n Ure c -CN, -(CH2) n Halogen or -O(CH2) n Selected independently from Halo; R cis H or -C 1-6 Selected independently of alkyl; R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2) n Selected from R, -NR2, pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl, where pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl may be R b It can be replaced by 1 to 3 of the following: R 2 is hydrogen, OR c , Halo or C 1-6 Selected from alkyl groups; Ring A 1 It is selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl; Ring A 2 is selected from pyrimidinyl, pyridyl, or pyrazinyl, where the pyrimidinyl, pyridyl, or pyrazinyl may be substituted with groups R1 to R3; Ring A 3 It is selected from pyridyl, pyrazinyl, or phenyl; n is independently selected from 0, 1, 2, 3, or 4; and, p is selected from 0, 1, 2, or 3.

[0023] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, wherein ring A 1 ring A is selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl. In another embodiment, ring A 1 This is selected from pyridyl, pyrazinyl, pyrazolyl, or pyrimidinyl. In another embodiment, ring A 1 It is selected from pyridyl or pyrazinyl.

[0024] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, wherein ring A 2is selected from pyrimidinyl, pyridyl, or pyrazinyl, where the pyrimidinyl, pyridyl, or pyrazinyl may be substituted with groups 1 to 3 of R. In one embodiment, the present invention provides compounds represented by formula I, formula IA, formula IB, or formula IC, where the ring A 2 a is selected from pyrimidinyl or pyrazinyl, where the pyrimidinyl or pyrazinyl may be substituted with 1 to 3 groups of R. In further embodiments, ring A 2 is a pyrimidinyl, which may be substituted with groups 1-3 of R. In further embodiments, ring A 2 is pyrazinyl, which may be substituted with R1-3 groups.

[0025] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, where A 3 This is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl. In another embodiment, ring A 3 This is selected from pyridyl, pyrazinyl, or phenyl.

[0026] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, wherein R c is H or -C 1-6 Selected independently of alkyl, where the alkyl is -C 1-6 Alkyl, OR d Alternatively, it may be substituted with 1 to 3 groups selected from halos. In another embodiment, the present invention provides compounds represented by formula I, formula IA, formula IB, or formula IC, where R c is H or -C 1-6 It is selected independently of alkyl.

[0027] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, wherein R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2)n R, -(CH2) n O(CH2) n Ure c , Halo, -NR2, C 1-6 Selected from alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicycloheptanyl, furyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridadinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolylpyrazolyl, morpholinyl, tetrazolyl, or piperazinyl, here And, the alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicycloheptanyl, furyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolylpyrazolyl, morpholinyl, tetrazolyl, and piperazinyl may be R b It can be substituted with 1 to 3 of the following groups.

[0028] In one embodiment, the present invention provides a compound represented by formula I, formula IA, formula IB, or formula IC, wherein R 1 is, -(CH2) n Ure c ,-(CH2) n O(CH2) n Selected from R, -NR2, pyridyl, pyrazolyl, azetidinil, pyrrolidinil, and furyl, where pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl may be R b It can be substituted with 1 to 3 of the following groups.

[0029] Representative compounds of the present invention include compounds selected from the following or pharmaceutically acceptable salts thereof. [Table 1] TIFF0007881829000010.tif220165 TIFF0007881829000011.tif222166 TIFF0007881829000012.tif228166 TIFF0007881829000013.tif235166 TIFF0007881829000014.tif238166 TIFF0007881829000015.tif237166 TIFF0007881829000016.tif234166 TIFF0007881829000017.tif228166 TIFF0007881829000018.tif214166 TIFF0007881829000019.tif225166 TIFF0007881829000020.tif215166 TIFF0007881829000021.tif218165 TIFF0007881829000022.tif198166 TIFF0007881829000023.tif208166 TIFF0007881829000024.tif207166 TIFF0007881829000025.tif193166 TIFF0007881829000026.tif187166 TIFF0007881829000027.tif204166 TIFF0007881829000028.tif229166 TIFF0007881829000029.tif236166 TIFF0007881829000030.tif237166 TIFF0007881829000031.tif236166 TIFF0007881829000032.tif227166 TIFF0007881829000033.tif211166

[0030] This invention relates to a compound represented by formula I for use as an imaging agent.

[0031] One embodiment of the present invention includes compounds selected from Examples No. 1, 6, 9, 11, 12, 37, 39, 47, 51, 57, 58, 64, 72, 75, 78, 79, 80, 91, 96, 112, 113, 115, 116, 118, 134, 138, and 141 or pharmaceutically acceptable salts thereof. Further embodiments of the present invention include compounds selected from Examples No. 39, 47, 51, 78, 79, 96, 112, 116, and 141 or pharmaceutically acceptable salts thereof. Another embodiment of the present invention includes compounds selected from Examples No. 96, 112, 113, 115, 116, 118, 134, 138, and 141 or pharmaceutically acceptable salts thereof. Further embodiments of the present invention include compounds selected from Examples No. 96, 112, 116, and 141 or pharmaceutically acceptable salts thereof.

[0032] Another aspect of the present invention is, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 CL, 82 Br, 76 Br, 77 Br, 123 I, 124 I or 131 The present invention relates to compounds represented by formula I or pharmaceutically acceptable salts labeled with an isotope selected from I. In a further aspect of the present invention, the compounds represented by formula I are 3 H, 11 C or 18It is isotope-labeled with F. Examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 3 H-1, 3 H-24, 18 F-39, 18 F-47, 18 F-51, 18 F-78, 18 F-79, 11 C-94, 18 F-96, 11 C-97, 18 F-116, 11 C-117, 11 C-118, 18 F-141 and 11 Examples include C-143. Further examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 3 H-1, 3 H-24, 18 F-39, 18 F-47, 18 F-51, 18 F-78, 18 F-79, 18 F-96, 18 F-116 and 18 Examples include F-141. Further examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 11 C-118 and 11 Examples include C-143. Further examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 18 F-96, 18 F-116 and 18 Examples include F-141. Further examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 18 F-78, 18 F-79, 18 F-96, 18 F-116 and 18Examples include F-141. Further examples of isotope-labeled compounds represented by formula I or their pharmaceutically acceptable salts include, but are not limited to, 18 F-96, 18 F-116 and 18 Examples include the F-141.

[0033] Another aspect of the present invention is for use as an imaging agent, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 CL, 82 Br, 76 Br, 77 Br, 123 I, 124 I or 131 The target compounds represented by formula I, labeled with an isotope selected from I, or pharmaceutically acceptable salts thereof.

[0034] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention (for example, a compound represented by formula I) and at least one pharmaceutically active ingredient.

[0035] The compound represented by formula I is an inhibitor and / or binder of aggregated α-synuclein or tau protein. The compound represented by formula I and its isotopically labeled variants may be useful in the diagnosis and / or treatment of Parkinson's disease and / or Alzheimer's disease. Means for detecting the label are well known to those skilled in the art. For example, the isotopic label can be detected using imaging techniques, photographic film or a scintillation counter. In a preferred embodiment, the label is detected in vivo in the brain of a subject by imaging techniques, such as positron emission tomography (PET).

[0036] The compound represented by formula (I) may also form a component of a bifunctional compound that is a targeted proteolytic compound that binds to aggregated α-synuclein protein. Such a targeted α-synuclein proteolytic compound comprises a target protein-binding moiety formed from the compound represented by formula (I) and an E3 ubiquitin ligase-binding moiety. The targeted α-synuclein proteolytic compound typically comprises a linker group that connects the α-synuclein protein-binding moiety and the E3 ubiquitin ligase-binding moiety. The E3 ubiquitin ligase-binding moiety in the α-synuclein targeted proteolytic compound may be, but are not limited to, a conjugate to the E3 ligase von Hipperrindau protein, a conjugate to the E3 ligase cereblon protein, or a conjugate to the MDM2 protein. Such compounds may be administered in pharmaceutical compositions to treat pathological conditions (which include, but are not limited to, the conditions disclosed herein).

[0037] In the following descriptions, conventional structural representations are employed, including conventional stereochemical notations for specific chiral carbon centers. Therefore, the structural representations of the compounds of the present invention include conventional stereochemical notations for some chiral carbon centers shown in the exemplary compounds. Thus, in such examples, a blacked-out “wedge” bond represents a bond protruding from the plane of the regeneration medium, a “finely engraved wedge” bond represents a bond descending into the plane of the regeneration medium, and a “wavy” line attached to a carbon with a double bond indicates the inclusion of both possible cis and trans orientations. As in the conventional practice, a plain solid line represents all spatial arrangements of the depicted bond. Therefore, where no specific stereochemical notation is given, the representation is intended to include all stereochemical and spatial orientations of the structural feature in question.

[0038] As shown in the examples of the present invention and as mentioned above, certain chiral carbon centers are structurally represented using the conventional “solid wedge” and “finely etched wedge” bonding representations. In most cases, the absolute configuration of the exemplary compounds has not been confirmed, but is assigned by analogy with specific exemplary compounds whose stereochemical configuration is known (confirmed by X-ray crystallography) and which were prepared using the same or similar reaction conditions and starting reagents and isolated under the same chromatographic conditions. Accordingly, the specific assignment of configurations structurally represented herein is intended to identify that a particular compound prepared has an excess of one particular stereoisomer, and is not necessarily stated herein as a description of the absolute determination of the stereochemical structure of the compound unless otherwise indicated in the presented data.

[0039] If an isomer mixture is obtained, it will be understood that the preparation of individual stereoisomers in significant enantiomer excess can be carried out, if desired, by separating the mixture using conventional methods (e.g., by chromatography or crystallization), by using stereochemically homogeneous starting materials for the described synthesis, or by stereoselective synthesis. In some cases, derivatization can be performed before separation of stereoisomers. Separation of a mixture of stereoisomers can be carried out at an intermediate stage during the synthesis of the compound represented by formula I, or it can be carried out on the final racemic product.

[0040] Where otherwise specified, absolute stereochemistry is determined by X-ray crystallography of the crystalline product or intermediate, where the crystalline product or intermediate is derivatized, if necessary, with a reagent containing a stereoisomer center of known configuration. Unless otherwise specified, the present invention encompasses all such isomers, salts, solvates (including hydrates), or solvated salts of such racemic compounds, enantiomers, or diastereomers.

[0041] When a wavy line terminates a conventional bond (rather than connecting two atoms within a structure), it indicates a bond point to the structure. For example, [ka] The tilde indicates that the secondary butyl moiety is bonded via a methylene group through a bond terminated by a wavy line. When alphabetical notation is used to represent a substitution, a dash is used to indicate the bond point to the indicated substrate. For example, -CH2-C(O)-CH2Cl indicates that the acetyl chloride moiety is bonded via the methylene moiety of that moiety.

[0042] If the compound represented by formula I is tautomerizable, all individual tautomers and mixtures thereof are included within the scope of the present invention.

[0043] Any variable part (e.g., R, R) 1 If any substituent (i.e., R, n, heteroaryl, alkyl, etc.) appears more than once in any component or in Formula I, the definition of that substituent in each appearance is independent of the definition of that substituent in all other appearances, unless otherwise specified at the time of the definition. Those skilled in the art will know that various substituents (i.e., R) as defined in the structural representation are independent of the definition of that substituent in all other appearances. 1 , R 2It will be recognized that the selection of combinations (such as) should be made in accordance with well-known principles of bonding and stability of chemical structures, and that combinations of substituents and / or variable parts are only permissible if such combinations result in a stable compound.

[0044] A “stable” compound is one that can be prepared and isolated, and whose structure and properties remain essentially unchanged or can be kept essentially unchanged for a period of time sufficient to enable the compound to be used for the purposes described herein (e.g., therapeutic administration to a subject). The compounds of the present invention are limited to stable compounds encompassed by Formula I.

[0045] Any variable part or substructure is in the form of a range (e.g., (-CH) 2- ) 1-4 When expressed as ), it includes not only the two extremes of the specified range (i.e., 1 and 4 in the example above), but also all integer values ​​in between (i.e., 2 and 3 in the example above).

[0046] Unless otherwise indicated, it is understood that references to "Formula I" also include compounds represented by Formulas IA, IB, and IC.

[0047] Where used herein, “alkyl” is intended to encompass both branched and linear saturated aliphatic hydrocarbon groups having a specified number of carbon atoms.

[0048] As used herein, "halogen" or "halo" means fluoro, chloro, bromo, and iodine.

[0049] Where used herein, “cycloalkyl” is intended to encompass a cyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms. Preferably, the cycloalkyl is C3-C 10It is a cycloalkyl compound. Examples of such cycloalkyl compounds include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0050] As used herein, “aryl” is intended to mean any stable monocyclic or bicyclic carbocyclic ring with up to seven members in each ring, wherein at least one ring is aromatic. Examples of such aryl components include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl, or acenaphthyl. In one embodiment of the present invention, the aryl is phenyl or naphthyl. In a further embodiment, the aryl is phenyl.

[0051] The terms “heterocyclyl,” “heterocycle,” or “heterocyclic formula,” as used herein, refer to a stable 5-7 membered monocyclic heterocyclic ring or a stable 8-11 membered bicyclic heterocyclic ring, which is either saturated or unsaturated and consists of a carbon atom and 1-4 heteroatoms selected from the group consisting of N, O, and S, and encompass any bicyclic group formed by the condensation of any of the above-defined heterocyclic rings to a benzene ring. The heterocyclic ring may be bonded with any heteroatoms or carbon atoms resulting in a stable structure. The terms “heterocyclyl,” “heterocycle,” or “heterocyclic formula” may include a heteroaryl moiety when two rings are condensed with each other. Examples of heterocyclic components include, but are not limited to, azabicyclo[2.2.1]heptanil, azepanil, azetidinil, benzodioxolyl, chromanil, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranil, dihydrobenzothiopyranylsulfone, dihydro-pyrrolo[1,2-b]pyrazolyl, 1,3-dioxolanil, imidazolidinil, indolinil, isochromanil, isoindolinil, morpholinil, oxa-5-azabicyclo[2.2.1]heptanil, 2-oxopiperazinil, 2-oxopiperidinil, 2-oxopyrrolidinil, piperidyl, piperazinil, pyrazolidinil, pyrrolidinil, tetrahydrofuryl, tetrahydroisoquinolinil, tetrahydroquinolinil, and thiamorpholinil.

[0052] In one embodiment, the heterocyclil is selected from azabicyclo[2.2.1]heptanil, azepanil, azetidinil, dihydro-pyrrolo[1,2-b]pyrazolyl, morpholinil, oxa-5-azabicyclo[2.2.1]heptanil, piperidyl, piperazinil, pyrazolidinil, pyrrolidinil, pyrrolyl, and tetrahydrofuryl. In another embodiment, the heterocyclil is selected from azabicyclo[2.2.1]heptanil, azepanil, azetidinil, dihydro-pyrrolo[1,2-b]pyrazolyl, oxa-5-azabicyclo[2.2.1]heptanil, piperazinil, and pyrrolidinil.

[0053] The term "heteroaryl" is intended to mean any stable monocyclic or bicyclic carbocyclic ring with up to seven members in each ring, where at least one ring is aromatic and 1 to 4 carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S. Examples of such heterocyclic components include, but are not limited to, azepinyl, furanyl, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthylidinyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridadinyl, pyrimidinyl, 5H-pyrrolo[2,3-b]pyrazinyl, pyrrolyl, quinazolinyl, quinolinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiazolyl, thienofuryl, thienothienyl, thienyl, and triazolyl. In one embodiment, the heteroaryl is selected from furyl, imidazolyl, indolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridadinyl, pyrimidinyl, 5H-pyrrolo[2,3-b]pyradinyl, tetrazolyl, thiazolyl, thienyl, triazolyl, and the like.

[0054] When used in pharmaceuticals, salts of the compound represented by formula I are pharmaceutically acceptable salts. However, other salts may also be useful in the preparation of the compound or its pharmaceutically acceptable salt according to the present invention. When the compound of the present invention is acidic, a suitable "pharmaceutically acceptable salt" refers to a salt prepared from pharmaceutically acceptable, non-toxic bases, including inorganic and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese salts, manganese, potassium, sodium, and zinc. Particularly preferred are ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts. Salts derived from pharmaceutically acceptable, non-toxic organic bases include salts of primary, secondary, and tertiary amines, salts of substituted amines (including naturally occurring substituted amines), salts of cyclic amines, and basic ion exchange resins (e.g., arginine, betaine, caffeine, choline, N,N). 1 Examples include salts of dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydravamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0055] When the compound of the present invention is basic, the salt can be prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucoic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Particularly preferred are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid.

[0056] The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is described in more detail by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977:66:1-19.

[0057] When a compound represented by formula I contains both an acidic group and a basic group within its molecule, the present invention also includes zwitterions in addition to the salt forms described above.

[0058] The present invention further encompasses isotope-labeled compounds of the present invention that are structurally identical to those described herein, except that a statistically significant proportion of one or more atoms in the compound in its form are replaced by atoms having a different atomic mass or mass number than the most abundant isotope commonly found in nature, and therefore the naturally occurring amount of that isotope present in the compound of the present invention is altered. Another aspect of the present invention relates to the use of isotope-labeled compounds as neuroimaging radiotracers for in vivo imaging of the brain with respect to α-synuclein aggregates in the diagnosis, monitoring, and / or treatment of Parkinson's disease (PD). Another aspect of the present invention relates to the use of isotope-labeled compounds in PET, an in vivo analytical technique in the diagnosis, monitoring, and / or treatment of PD. 3 H, 11 C or 18 Compounds labeled with F can be used in in vitro and in vivo methods for measuring binding, receptor occupancy, and metabolic studies, including covalent labeling.

[0059] Another aspect of the present invention relates to the use of isotope-labeled compounds for screening new chemical substances. In particular, various isotope-labeled compounds find usefulness in magnetic resonance imaging, autoradiography, and other similar analytical tools. The present invention is intended to encompass all suitable isotope variants of the compound represented by formula I. Examples of isotopes that can be preferentially incorporated into the compounds of the present invention include, but are not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine, fluorine, and chlorine. 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I or 131 Examples include substituted heterocyclic derivatives represented by formula I, which are isotope-labeled with I. It should be understood that other isotopes can also be incorporated by known means. In particular, the present invention relates to compounds represented by formula I. 11 C, 13 C, 14 C, 18 F, 15 O, 13 N, 35 S, 2 H and 3 The present invention relates to 1H isotopes, compositions, methods for preparing them, and their use as radiotracers or PET tracers in the diagnosis and measurement of the effects of compounds in the treatment of PD. In further embodiments, the present invention relates to 3 H, 11 C or 18The present invention relates to compounds represented by formula I, isotope-labeled with 1F, as well as compositions and methods for preparing them, and their use as PET tracers in the diagnosis and measurement of the effects of compounds in the treatment of PD. The present invention also relates to non-toxic α-synuclein protein-binding compounds that can rapidly cross the blood-brain barrier, have low nonspecific binding characteristics, and are rapidly removed from the system. This and other aspects of the present invention will be understood by examining the entire specification.

[0060] The isotope-enriched compounds contained in Formula I can be prepared without excessive experimentation by conventional techniques well known to those skilled in the art, or by processes similar to those described in the schemes and examples herein, using suitable isotope-enriched reagents and / or intermediates.

[0061] As described herein, the present invention encompasses the isotope-labeled compounds of the present invention. An “isotope-labeled” compound, a “radio-labeled” compound, a “tracer” compound, a “radio-tracer” compound, a “labeled tracer” compound, or a “radio-ligand” compound is a compound in which one or more atoms are replaced or substituted by atoms having atomic masses or mass numbers different from those commonly found in nature (i.e., naturally occurring). Suitable radionuclides (i.e., “detectable isotopes”) that can be incorporated into the compounds of the present invention include, but are not limited to, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 76 Br, 77 Br, 123 I, 124 I and 131Examples include I. The isotope-labeled compounds of the present invention are only required to be enriched with detectable isotopes to a degree or beyond that allows detection by techniques suitable for a particular application. The radionuclides incorporated into the radiolabeled compounds of the present invention depend on the specific application of the radiolabeled compound. In another embodiment of the present invention, the radionuclides are, 11 C, 13 C, 14 C, 18 F, 15 O, 13 N, 35 S, 2 H and 3 Represented by H, preferably, 11 C, 3 H and 18 It is represented by F.

[0062] The isotope-labeled compounds of the present invention are prepared by incorporating a selected isotope into a substrate molecule. This is achieved by using a reagent in which one or more atoms contained in the reagent have been radioactive, by placing the reagent in a radioactive source such as a nuclear reactor or cyclotron. Furthermore, 2 H2O, 3 H3CI, 14 C6H5Br, ClCH2 14 Many isotope-labeling reagents, such as COCl, are commercially available. Then, using standard organic chemical synthesis techniques, the isotope atoms are incorporated into the compound represented by formula I. The following scheme illustrates a method for producing the compound represented by formula I.

[0063] The present invention further relates to a pharmaceutical composition comprising an effective amount of at least one compound represented by formula I and a pharmaceutically acceptable carrier. The composition may, but is not limited to, contain one or more buffers, wetting agents, emulsifiers, suspending agents, lubricants, adsorbents, surfactants, preservatives, and the like. The composition may be formulated as a solid, liquid, gel, or suspension for oral administration (e.g., drench agents, bolus agents, tablets, powders, capsules, mouth sprays, emulsions), parenteral administration (e.g., subcutaneous injection, intramuscular injection, intravenous injection, epidural injection), topical application (e.g., creams, ointments, controlled-release patches, sprays), vaginal administration, rectal administration, transdermal administration, intraocular administration, or intranasal administration. In further embodiments, the pharmaceutical composition of the present invention may be formulated for parenteral administration (e.g., intravenous formulations).

[0064] The present invention provides radiolabeled compounds represented by formula I as α-synuclein imaging agents and synthetic precursor compounds for preparing them. The compounds represented by formula I bind to aggregated α-synuclein and potentially track the progression of age-related diseases such as PD, as well as other synucleinopathy and neurodegenerative diseases such as multiple system atrophy (MSA) and Lewy body dementia (DLB). The compounds of the present invention can also be used in combination with a wide range of cognitive deficit enhancement agents. Accordingly, in another embodiment of the present invention, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or formulation comprising a compound represented by formula (I), is administered concurrently, simultaneously, sequentially, or separately with another pharmaceutically active compound used in the treatment of AD / PD (which includes, for example, donepezil, memantine, tacrine, carbidopa, levodopa, MOA-B inhibitors, catechol O-methyltransferase (COMT) inhibitors, etc.) and their equivalents, pharmaceutically active isomers, and metabolites.

[0065] The object of the present invention is to provide a radiopharmaceutical agent (for example, an isotope-labeled compound represented by formula I) that is useful in α-synuclein imaging and has high specific radioactivity and high target tissue selectivity due to its high affinity for α-synuclein aggregates.

[0066] According to the present invention, a method for imaging α-synuclein deposits in a patient (in which an isotope-labeled compound represented by formula I is used as an imaging agent) comprises the following steps: (a) positioning a human patient in a supine position inside a PET camera; (b) intravenously administering approximately 0.1 to approximately 10 mCi of the isotope-labeled compound represented by formula I to the patient; and (c) performing a vascular scan of the cerebral region of the patient's head to confirm the aggregation of α-synuclein in the patient's brain tissue. Techniques for performing vascular scans of the head are well known to those skilled in the art. PET techniques are described in: Freeman et al., Freeman and Johnson's Clinical Radionuclide Imaging, 3rd. Ed. Vol. 1 (1984); Grune & Stratton, New York; Ennis et Q. Vascular Radionuclide Imaging: A Clinical Atlas, John Wiley & Sons, New York (1983).

[0067] The term “labeled tracer” refers to any molecule that can be used to track or detect defined activity in vivo. For example, a preferred tracer is one that accumulates in a region where α-synuclein aggregates may be found. Preferably, the labeled tracer is one that can be seen, for example, by positron emission tomography (PET) scans in living experimental animals, healthy humans, or patients (also referred to as subjects). Suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, pigments, and proteins (including enzymes).

[0068] The present invention also provides a method for confirming the in vivo activity of an enzyme or other molecule. In one embodiment, an isotope-labeled compound represented by formula I is used as a tracer to track the binding activity of aggregated α-synuclein protein in the brain and central nervous system.

[0069] Biomarkers of Parkinson's disease symptoms, prognosis, and progression are all useful not only for general diagnostics but also for the clinical development planning of Parkinson's disease treatments. The compound represented by Formula I can be used to provide biomarker information to patients in clinical trials of novel symptomatic and disease-modifying Parkinson's disease therapies, and to assist in patient selection and cohort allocation. The present invention serves as one of the disease symptom biomarkers for enrolling eligible patients into appropriate PhIIb trial cohorts. In addition, the present invention can serve as one of the disease prognosis markers as an entry inclusion criterion to increase the probability of disease progression in the placebo group, a problem that continues to plague Parkinson's disease clinical trials. Finally, the present invention can serve as one of the disease progression biomarkers for monitoring the clinical course of patients during treatment and can provide an independent biomarker index of therapeutic response to therapeutic drugs. The tracer can be selected according to a chosen detection method. Before carrying out the method of the present invention, a diagnostically effective amount of the labeled or unlabeled compound of the present invention is administered to a living organism (this includes humans).

[0070] The present invention also provides a method for measuring the clinical efficacy of a therapeutic agent useful for treating Parkinson's disease (PD), comprising the following steps: (a) administering an isotope-labeled compound represented by formula I to a patient diagnosed with PD before treatment with the therapeutic agent; (b) measuring the amount of α-synuclein aggregates formed in the patient's brain tissue; (c) administering an isotope-labeled compound represented by formula I to the patient after treatment with the therapeutic agent; (d) measuring the amount of α-synuclein aggregates formed in the patient's brain tissue after treatment; and (e) analyzing whether the therapeutic agent stopped or reduced the progression of α-synuclein aggregate formation in the patient's brain tissue.

[0071] The diagnostically effective amount of the labeled or unlabeled compound of the present invention to be administered before performing the in vivo method of the present invention is in the range of 0.1 ng to 100 mg per kg of body weight, preferably in the range of 1 ng to 10 mg per kg of body weight.

[0072] The compounds of the present invention are useful in the diagnosis, monitoring, and measurement of Parkinson's disease and other non-PD synuclein disorders (e.g., multiple system atrophy (MSA), Lewy body dementia (DLB), etc.).

[0073] In preferred embodiments, the compounds of the present invention are useful in the diagnosis, monitoring, or measurement of Parkinson's disease, non-PD synucleinopathy, neurodegenerative diseases, cognitive impairment, schizophrenia, pain disorders, and sleep disorders.

[0074] As used herein, the term “composition” is intended to encompass products containing specific components in predetermined amounts or proportions, and any products obtained directly or indirectly from specific combinations of specific components. In relation to pharmaceutical compositions, the term is intended to encompass products containing one or more active components and, in the case of a support, inert components, and any products obtained directly or indirectly from any combination of two or more components, from complex formation or aggregation, from the dissociation of one or more components, or from another type of reaction or interaction of one or more components.

[0075] Generally, pharmaceutical compositions are prepared by uniformly and tightly combining the active ingredient with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, molding the product into a desired formulation. In such pharmaceutical compositions, the active compound (which is the compound represented by formula I) is present in an amount sufficient to produce the desired effect on the disease process or symptoms. Accordingly, the pharmaceutical compositions of the present invention encompass any composition produced by mixing the compound of the present invention with a pharmaceutically acceptable carrier.

[0076] Where used herein, “patient” (or alternatively, “subject”) refers to an animal, preferably a mammal, in particular a human, that requires evaluation via imaging studies. Where used herein, the terms “administer” and its variation (e.g., “administer” the compound) relating to the compound represented by Formula I mean providing the compound or a pharmaceutically acceptable salt thereof to a subject in need of treatment.

[0077] The present invention further provides a method for synthesizing compounds useful as intermediates in the preparation of the compounds of the present invention.

[0078] The compounds described herein can be prepared using appropriate materials and following the procedures of the following schemes and examples, and are further illustrated by the following specific examples. Deuterated versions of the compounds of the present invention can be prepared by replacing the non-isotope-labeled reagent with a suitable isotope-labeled reagent. However, the compounds illustrated in the examples should not be construed as forming the only genus considered as part of the present invention. The examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparation procedures can be used to prepare these compounds. Reagents and starting materials for preparing intermediates and exemplary compounds are commercially available unless otherwise indicated. All temperatures are in degrees Celsius unless otherwise indicated. Mass spectra (MS) were measured by electrospray ion mass spectrometry (ESI). 1 1H NMR spectra were recorded at 300-500 MHz.

[0079] [Table 2] TIFF0007881829000036.tif246165TIFF0007881829000037.tif50165

[0080] The compounds described herein were synthesized as racemic mixtures unless otherwise indicated in the experimental procedure. The final product may be further modified, for example, by manipulating substituents. These manipulatives may include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions, which are generally known to those skilled in the art. The order in which the above reaction schemes are carried out may be changed to accelerate the reaction or avoid undesirable reaction products. The following schemes and examples are provided to allow for a better understanding of the present invention. These examples are merely illustrative and should not be construed as limiting the present invention.

[0081] General Scheme A [ka]

[0082] Substitutive piperazine (A-1) can be converted to arylpiperazine A-2 by a CN coupling reaction via SNAr or Pd. Deprotection is followed by a CN coupling reaction with an aryl halide via SNAr or Pd to obtain intermediate A-3. If Y in A-3 is nitro, reduction (e.g., hydrogenation) can be performed to obtain aniline intermediate A-4, and subsequent amide coupling yields the target molecule A-5.

[0083] [ka]

[0084] Alternatively, if Y is a bromide (or Cl), intermediate A-6 can be obtained by CN coupling with a Boc-amine via Pd, and the target molecule A-7 can be obtained by subsequent amide coupling.

[0085] General Scheme B [ka]

[0086] Substituted piperazine (B-1) can be converted to arylpiperazine B-2 by a CN coupling reaction via SNAr or Pd, followed by deprotection. Intermediate B-3 can be obtained by CN coupling of B-2 with an aryl halide via SNAr or Pd. By reducing B-3, an aniline intermediate B-4 can be obtained, and the target molecule B-5 can be obtained by subsequent amide coupling.

[0087] General Scheme C [ka]

[0088] Intermediate C-1 can be subjected to a SNAr reaction with an amine or an NH-containing heterocycle to obtain the target compound C-2.

[0089] General Scheme D [ka] Intermediate D-1 can be subjected to a Pd-mediated CC coupling reaction to obtain the target compound D-2.

[0090] General Scheme E [ka]

[0091] Substituted piperazine (E-1) can be converted to arylpiperazine E-2 by a CN coupling reaction via SNAr or Pd. Deprotection is followed by a CN coupling reaction with an aryl halide via SNAr or Pd to obtain intermediate E-3. Halide E-3 is then subjected to a Pd-mediated CN coupling reaction with a pre-formed primary amide to obtain the target molecule E-4.

[0092] Preparation of intermediates Synthesis of intermediate A: (S)-2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-amine [ka]

[0093] Synthesis of 1-2: (S)-tert-butyl 3-methyl-4-(5-nitropyrimidine-2-yl)piperazine-1-carboxylate To a solution of (S)-tert-butyl 3-methylpiperazine-1-carboxylate (1-1, 13.6 g, 67.9 mmol) in DMF (150 mL), K2CO3 (14.08 g, 102 mmol) and 2-chloro-5-nitropyrimidine (12.46 g, 78 mmol) were added. The mixture was stirred under an N2 balloon at 25°C for 12 hours. TLC showed that the starting material was completely consumed. Water (450 mL) was added, and the mixture was stirred at 25°C (rt) for 30 minutes. The precipitated solid was collected by filtration, washed with water (100 mL x 3), and dried to obtain 1-2 as a solid. 1 ¹H NMR (500 MHz, chloroform-d): δ = 9.07 (s, 2H), 5.06 (br s, 1H), 4.67 (br s, 1H), 3.91~4.29 (m, 2H), 3.28~3.36 (m, 1H), 3.13 (br s, 1H), 2.83~3.01 (m, 1H), 1.45~1.52 (m, 9H), 1.26 (d, J = 6.5 Hz, 3H)

[0094] Synthesis of 1-3: (S)-2-(2-methylpiperazin-1-yl)-5-nitropyrimidine To a solution of 1-2 (21 g, 64.9 mmol) in DCM (160 mL), TFA (40 mL) was added at 0°C. The mixture was stirred at 25°C for 2 hours. TLC showed that most of the starting material had been completely consumed. The mixture was concentrated under reduced pressure to obtain the crude product (S)-2-(2-methylpiperazin-1-yl)-5-nitropyrimidine (25 g, 78 mmol) as an oil. The product was diluted with DCM (200 mL) and H2O (160 mL). Then, Na2CO3 was added to the solution to adjust the pH to 7-8. The solution was extracted with DCM (200 mL x 2). The organic layer was dehydrated with Na2SO4, filtered, and concentrated to obtain 1-3 as a solid. 1H NMR (400 MHz, DMSO-d6): δ = 9.44 (s, 1H), 8.92~9.10 (m, 1H), 5.13~5.24 (m, 1H), 4.82 (d, J = 14.4 Hz, 1H), 3.31~3.49 (m, 3H), 3.26 (d, J = 7.2 Hz, 1H), 3.06 (d, J = 8.8 Hz, 1H), 1.52 (s, 1H), 1.33 (d, J = 7.2 Hz, 3H). MS (ESI) m / z: 224.0 [M+H] + .

[0095] Synthesis of 1-4: (S)-2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)-5-nitropyrimidine To a solution of 1-3 (2.5 g, 11.20 mmol) in dioxane (50 mL), 2-bromopyridine (3.72 g, 23.52 mmol), Cs2CO3 (14.96 g, 45.9 mmol), and chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (0.968 g, 1.344 mmol) were added. The mixture was stirred under N2 balloon at 110°C for 12 hours. TLC showed that most of the starting material was completely consumed. The mixture was filtered and concentrated. The residue was extracted with ELISA (3 × 50 mL) and H2O (60 mL). The combined organic extracts were washed with brine (100 mL), dehydrated with anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a silica gel column (eluted with 15-30% ethyl acetate / PE) to obtain solids 1-4. 1H NMR (400 MHz, chloroform-d): δ = 9.10 (s, 2H), 8.17~8.24 (m, 1H), 7.47~7.59 (m, 1H), 6.62~6.71 (m, 2H), 5.11 (dt, J = 6.4, 3.2 Hz, 1H), 4.74 (dt, J = 13.6, 3.6 Hz, 1H), 4.24 (d, J = 12.8 Hz, 1H), 4.13 (d, J = 13.2 Hz, 1H), 3.53~3.63 (m, 1H), 3.37 (dd, J = 13.2, 4.0 Hz, 1H), 3.12 (td, J = 12.0, 3.6 Hz, 1H), 1.34 (d, J = 6.8 Hz, 3H). MS (ESI) m / z: 301.0 [M+H] + .

[0096] Synthesis of (S)-2-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyrimidine-5-amine (Int A) To a solution of 1-4 (2.5 g, 8.32 mmol) in MeOH (40 mL), Pd / C (0.2 g, 1.879 mmol) was added. The mixture was stirred under an H2 balloon at 25°C for 2 hours. TLC showed that most of the starting material had been completely consumed. The mixture was filtered, and the filtered cake was washed with MeOH (3 × 200 mL). The combined organic extract was concentrated under reduced pressure to obtain Int A as an oily substance. 1 H NMR (400 MHz, chloroform-d): δ = 8.19 (dd, J = 4.8, 1.2 Hz, 1H), 8.01 (s, 2H), 7.45~7.50 (m, 1H), 6.66 (d, J = 8.8 Hz, 1H), 6.60 (dd, J = 6.8, 5.2 Hz, 1H), 4.76~4.85 (m, 1H), 4.32~4.40 (m, 1H), 4.19~4.26 (m, 1H), 4.10 (dt, J = 12.8, 2.0 Hz, 1H), 3.23~3.41 (m, 2H), 3.15 (s, 2H), 3.01~3.08 (m, 1H), 1.22 (d, J = 6.8 Hz, 3H). MS (ESI) m / z: 271.1 [M+H]+ .

[0097] Synthesis of intermediate B: (R)-2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-amine [ka] (R)-2-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyrimidine-5-amine (Int B) was isolated as an oily substance using a synthetic procedure similar to that used for the synthesis of Int A. 1 H NMR (400 MHz, DMSO-d6): δ = 8.07 (dd, J = 4.8, 1.3 Hz, 1H), 7.90 (s, 2H), 7.48~7.54 (m, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.59 (dd, J = 6.8, MS (ESI) m / z: 271.0 [M+H] + .

[0098] Synthesis of intermediate C: (R)-2-(3-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-amine [ka]

[0099] Synthesis of 3-2: (R)-tert-butyl 3-methyl-4-(pyridine-2-yl)piperazine-1-carboxylate To a solution of (R)-tert-butyl 3-methylpiperazine-1-carboxylate (3-1, 2 g, 9.99 mmol) in dioxane (40 mL), 2-bromopyridine (3.31 g, 20.97 mmol), Cs2CO3 (13.34 g, 40.9 mmol), and chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (0.864 g, 1.198 mmol) were added. The mixture was stirred under N2 balloon at 110°C for 12 hours. TLC showed that most of the starting material was completely consumed. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified using a silica gel column (eluted with 15% siRNA / PE) to obtain 3-2 as an oily substance. 1 H NMR (400 MHz, chloroform-d): δ = 8.18 (dd, J = 4.8, 1.3 Hz, 1H), 7.42~7.52 (m, 1H), 6.54~6.64 (m, 2H), 4.46 (br s, 1H), 3.78~4.25 (m, 3H), 2.93~3.23 (m, 3H), 1.48 (s, 9H), 1.12 (d, J = 6.8 Hz, 3H). MS (ESI) m / z: 278.1 [M+H] + .

[0100] Synthesis of 3-3: (R)-2-methyl-1-(pyridine-2-yl)piperazine To a solution of 3-2 (2 g, 7.21 mmol) in HCl (17 mL), HCl / HCl (34 mL) was added. The mixture was stirred at 25°C for 5 hours. TLC showed that the starting material was completely consumed. The resulting solid was collected by filtration. It was washed with the cake HCl (5 mL) and dried to obtain 3-3 as an oily substance. 1H NMR (400 MHz, DMSO-d6): δ = 10.07 (br d, J = 7.2 Hz, 1H), 9.72 (br s, 1H), 8.02~ 8.12 (m, 2H), 7.40 (br d, J = 9.2 Hz, 1H), 7.03 (t, J = 6.4 Hz, 1H), 4.79 (br s, 1H), 4.38 (br d, J = 14.4 Hz, 1H), 3.58 (br t, J = 12.0 Hz, 1H), 3.22~3.36 (m, 3H), 3.09 (br d, J = 10.4 Hz, 1H), 1.41 (d, J = 6.8 Hz, 3H).

[0101] Synthesis of 3-4: (R)-2-(3-methyl-4-(pyridine-2-yl)piperazine-1-yl)-5-nitropyrimidine To a solution of 3-3 (1.7 g, 9.59 mmol) in DMF (20 mL), 2-chloro-5-nitropyrimidine (1.836 g, 11.51 mmol) and K2CO3 (5.30 g, 38.4 mmol) were added. The mixture was stirred at 80°C for 2 hours under an N2 balloon. TLC showed that most of the starting material had been completely consumed. Water (150 mL) was added, and the mixture was stirred at 25°C (rt) for 30 minutes. The precipitated solid was collected by filtration, washed with water (100 mL x 3), and dried to obtain 3-4 as an oily substance. 1 H NMR (400 MHz, DMSO-d6): δ = 9.15 (d, J = 4.4 Hz, 2H), 8.14 (d, J = 3.6 Hz, 1H), 7.51~7.60 (m, 1H), 6.82 (s, 1H), 6.65 (dd, J = 6.8, 5.2 Hz, 1H), 4.62~4.76 (m, 3H), 4.13~4.24 (m, 1H), 3.53 (d, J = 4.0 Hz, 1H), 3.33~3.40 (m, 1H), 3.11~3.23 (m, 1H), 1.02 (d, J = 6.4 Hz, 3H). MS (ESI) m / z: 301.1 [M+H] + .

[0102] Synthesis of (R)-2-(3-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyrimidine-5-amine (Int C) Pd / C (0.2g, 1.879 mmol) was added to a solution of 3-4 (1.8g, 5.99 mmol) in MeOH (30 mL). The mixture was stirred under an H2 balloon at 25°C for 2 hours. TLC showed that most of the starting material had been completely consumed. The mixture was filtered, and the filtered cake was washed with MeOH (3 × 200 mL). The combined organic extract was concentrated under reduced pressure to obtain Int C as an oily substance. 1 H NMR (400 MHz, DMSO-d6): δ = 8.12 (dd, J = 4.8, 1.2 Hz, 1H), 7.91 (s, 2H), 7.50~7.55 (m, 1H), 6.78 (d, J = 8.8 Hz, 1H), 6.60 (dd, J = 6.8, 5.2 Hz, 1H), 4.54~4.64 (m, 3H), 4.27~4.45 (m, 2H), 4.08 (s, 1H), 2.99~3.10 (m, 2H), 2.83~290 (m, 1H), 1.03 (d, J = 6.6 Hz, 3H). MS (ESI) m / z: 271.0 [M+H] + .

[0103] Synthesis of intermediate D: (S)-2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-amine [ka]

[0104] (S)-2-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyrimidine-5-amine was isolated as an oily substance using the same synthetic procedure as for the synthesis of Int C described above. 1H NMR (400 MHz, DMSO-d6): δ = 8.09 (dd, J = 4.8, 1.2 Hz, 1H), 7.89 (s, 2H), 7.48~7.52 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.58 (dd, J = 6.8, 5.2 Hz, 1H), 4.51~4.60 (m, 3H), 4.28~4.41 (m, 2H), 3.98~4.11 (m, 1H), 2.99~3.08 (m, 2H), 2.84 (td, J = 12.0, 3.6 Hz, 1H), 1.01 (d, J = 6.8 Hz, 3H). MS (ESI) m / z: 271.1 [M+H] + .

[0105] Synthesis of intermediate E: (S)-2-(4-(6-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine [ka] (S)-2-(4-(6-fluoropyridine-2-yl)-2-methylpiperazin-1-yl)pyrimidine-5-amine was isolated as an oily substance using a synthetic procedure similar to that described for Int A.

[0106] Synthesis of intermediate F: (S)-6-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyridine-3-amine [ka]

[0107] Synthesis of 6-2: (S)-tert-butyl 2-methyl-4-(pyridine-2-yl)piperazine-1-carboxylate A mixture of (S)-1-N-BOC-2-methylpiperazine (15 g, 74.9 mmol) and 2-fluoropyridine (36.4 g, 374 mmol) was heated at 135°C for 48 hours. LC-MS showed that the starting material remained and a new product was formed. LCMS (ESI) C 15 H 23 Calculated values ​​for N3O2 [M+H] + : 278.1, Measured value: 278.2, tR = 0.684 min The mixture was evaporated under reduced pressure. The residue was purified by silica gel column flash chromatography (eluting with petroleum ether / alkyl group = 4:1) to obtain 6-2 as a colorless liquid.

[0108] Synthesis of 6-3: (S)-3-methyl-1-(pyridine-2-yl)piperazine dihydrochloride Hydrogen chloride (37.9 mL, 151 mmol) in SiO was added to 6-2 (7 g, 25.2 mmol) at room temperature, and the mixture was stirred at 20°C for 18 hours. LC-MS (ESI) showed that the starting material was consumed and a new product was formed. LCMS (ESI) C 10 H 15 Calculated value for N3·2ClH [M+H] + : 178.1, Measured value: 178.1, tR = 0.170 min The mixture was evaporated under reduced pressure. Then, toluene (25 mL x 3) was added to the residue. This was evaporated under reduced pressure to obtain 6-3 as a solid.

[0109] Synthesis of 6-4: (S)-2-methyl-1-(5-nitropyridine-2-yl)-4-(pyridine-2-yl)piperazine To a stirred mixture of 6-3 (1 g, 4.00 mmol) and K2CO3 (2.210 g, 15.99 mmol) in DMF (12 mL), 2-chloro-5-nitropyridine (0.697 g, 4.40 mmol) was added, and the mixture was stirred at 80°C for 15 hours. LC-MS (ESI) showed that the starting material was consumed and a new product was formed. LCMS (ESI) C 15 H 17 Calculated values ​​for N5O2 [M+H] + : 300.1, Measured value: 300.1, tR = 0.351 min Next, the mixture was poured into 30 mL of ice water, filtered, and the filtered cake was washed with water (15 mL x 3). It was dried under reduced pressure, and 6-4 was obtained as a brown solid.

[0110] Synthesis of (S)-6-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyridine-3-amine (Int F) To a stirred mixture of 6-4 (1.0 g, 3.34 mmol) and ammonium chloride (1.787 g, 33.4 mmol) in EtOH (20 mL) and water (10 mL), iron (0.933 g, 16.70 mmol) was added at room temperature, and the mixture was stirred at room temperature (temperature: 40 °C) under an N2 atmosphere. LC-MS (ESI) showed that the starting material was consumed and a new product was formed. LCMS (ESI) C 15 H 19 Calculated value for N5 [M+H] + : 270.1, Measured value: 270.1, tR = 0.128 min

[0111] The mixture was cooled to room temperature, filtered, and the solvent was evaporated under reduced pressure. Water (25 mL) was added, and the mixture was extracted with dimethyl phosphate (30 mL x 3). The combined organic fraction was dehydrated (Na2SO4), filtered, the solvent was evaporated under reduced pressure, dissolved in dimethyl phosphate (10 mL), 0.5 mL of HCl (4 M dimethyl phosphate solution) was added dropwise, and the solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC (reverse-phase C-18 column) (eluted with acetonitrile / water + 0.1% TFA) to obtain Int F as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ ppm 1.31 (d, J=6.26 Hz, 3 H) 3.61 - 3.76 (m, 2 H) 3.86 (dd, J=13.69, 3.91 Hz, 1 H) 4.01 - 4.16 (m, 3 H) 4.52 (br d, J=6.65 Hz, 1 H) 7.03 (t, J=6.65 Hz, 1 H) 7.10 (d, J=9.39 Hz, 1 H) 7.40 (d, J=9.39 Hz, 1 H) 7.64 (dd, J=9.39, 3.13 Hz, 1 H) 7.81 (s, 1 H) 7.97 - 8.11 (m, 2 H); Calculated value for LCMS (ESI) C15H19N5 [M+H] + : 270.1, Measured value: 270.1

[0112] Synthesis of intermediate G: (S)-5-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyridine-2-amine [ka]

[0113] Synthesis of 7-1: (S)-2-methyl-1-(6-nitropyridine-3-yl)-4-(pyridine-2-yl)piperazine 5-fluoro-2-nitropyridine (1.2 g, 8.45 mmol) and 6-3 (2.42 g, 13.65 mmol) were added to DMA (20 mL), to which K2CO3 (4.67 g, 33.8 mmol) was added. The mixture was stirred at 100°C for 18 hours. TLC (petroleum ether:ethyl acetate = 1:1) showed that almost no starting materials remained. LCMS (ESI) C 15 H 17 Calculated values ​​for N5O2 [M+H] + : 300.1, Measured value: 300.5, t R = 0.57 minutes

[0114] The mixture was cooled, diluted with ethyl acetate (20 mL) and water (20 mL), the organic layer was washed with aqueous Na2CO3 (saturated, 10 mL), dehydrated (Na2SO4), filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column flash chromatography (eluting with petroleum ether / ethyl acetate = 1:1) to obtain 7-1 as a solid. 1 H NMR (400 MHz, chloroform-d) δ 8.36 - 8.58 (m, 1H) 8.17 - 8.28 (m, 2H) 8.12 (d, J = 3.1 Hz, 1H) 7.46 - 7.72 (m, 1H) 7.18 (dd, J=9.2, 2.9 Hz, 1H) 6.50 - 6.82 (m, 2H) 4.17 - 4.38 (m, 2H) 4.04 - 4.43 (m, 1H) 3.69 - 3.95 (m, 1H) 3.44 - 3.62 (m, 2H) 3.27 - 3.38 (m, 1H) 3.01 (s, 7H) 2.94 (s, 7H) 2.08 (s, 7H) 1.30 (d, J=6.7 Hz, 4H); LCMS (ESI) C 15 H17 Calculated values ​​for N5O2 [M+H] + : 300.1, Measured value: 300.5.

[0115] Synthesis of (S)-5-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyridine-2-amine (Int G) 7-1 (1.1 g, 3.67 mmol) and Pd-C (300 g, 2819 mmol) in MeOH (20 mL) were treated with H2 (15 Psi), and the mixture was stirred at 20°C for 18 hours. TLC (DCM:MeOH = 10:1) showed that no starting material remained. The desired mass was detected. LCMS (ESI) C 15 H 19 Calculated value for N5 [M+H] + : 270.2, Measured value: 269.8, tR = 0.596 min

[0116] The solvent was filtered, the filtrate was concentrated, and the residue was purified by silica gel column flash chromatography (ethyl acetate:EtOH = 10:1 → 20:1 elution) to obtain Int G as a solid. 1 H NMR (500 MHz, chloroform-d) δ 8.20 (dd, J = 4.9, 1.1 Hz, 1H) 7.86 (d, J = 2.4 Hz, 1H) 7.48 (ddd, J = 8.6, 7.1, 2.0 Hz, 1H) 7.26 (dt, J = 5.8, 2.8 Hz, 1H), 6.68 (d, J = 8.5 Hz, 1H) 6.58 - 6.63 (m, 1H) 6.50 (d, J=8.7 Hz, 1H) 4.30 (br s, 2H) 3.92 (m, 1H) 3.73 - 3.81 (m, 1H) 3.53 (m, 1H) 3.32 (m, 1H) 3.21 (dd, J=12.4, 7.4 Hz, 1H) 3.08 - 3.14 (m, 1H) 3.00 - 3.07 (m, 1H) 0.97 (d, J=6.3 Hz, 3H); LCMS (ESI) C 15 H 19 Calculated value for N5 [M+H] +: 270.2, Measured value: 269.8.

[0117] Synthesis of intermediate H: (S)-5-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrazine-2-amine [ka] Synthesis of 8-1: (S)-2-bromo-5-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrazine In a clean, dry, sealed tube, 2,5-dibromopyrazine (1.046 g, 4.40 mmol) and 6-3 (1 g, 4.00 mmol) in DMSO (10 mL) were mixed with CsF (3.04 g, 19.99 mmol), and the resulting mixture was stirred at 90°C for 18 hours. LC-MS (ESI) showed that the starting materials had been consumed and a new product had been formed. The mixture was cooled to room temperature, filtered, and the filtered cake was washed with DCM (10 mL x 3). The solvent was evaporated under reduced pressure. The residue was purified by silica gel column flash chromatography (eluting with petroleum ether / SiO=4:1) to obtain (S)-2-bromo-5-(2-methyl-4-(pyridine-2-yl)piperazin-1-yl)pyrazine as a gum-like substance. Calculated values ​​for LCMS (ESI) C14H16BrN5 [M+H] + : 336.0, Measured value: 335.9.

[0118] Synthesis of 8-2: (S)-tert-butyl (5-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrazine-2-yl)carbamate To a stirred mixture of 8-1 (1.5 g, 4.49 mmol), tert-butyl carbamate (1.052 g, 8.98 mmol), and cesium carbonate (4.39 g, 13.46 mmol) in dioxane (25 mL), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (0.519 g, 0.898 mmol) and diacetoxypalladium (0.101 g, 0.449 mmol) were added at room temperature under an N2 atmosphere, and the mixture was heated with stirring (temperature: 70 °C) for 18 hours. LC-MS (ESI) showed that the starting material was consumed and a new product was formed. The mixture was filtered, concentrated, and the residue was purified by silica gel flash chromatography (ISCORF150; Sepa flash column) (eluted with petroleum ether / SiO1 = 3:1) to obtain 8-2 as a solid. 1 H NMR (400 MHz, chloroform-d) δ 8.72 (brs, 1H), 8.10-8.28 (m, 1H), 7.74 (s, 1H), 7.41-7.55 (m, 1H), 6.99 (brs, 1H), 6.51-6.74 (m, 2H), 4.47 (brdd, J = 3.06, 5.99 Hz, 1H), 4.22 (brd, J = 12.23 Hz, 1H), 4.10 (brdd, J = 1.47, 12.72 Hz, 1H), 3.97 (brdd, J = 2.69, 12.72 Hz, 1H), 3.26-3.42 (m, 2H), 3.05-3.20 (m, 1H), 1.51-1.58 (m, 9H), 1.19 (dd, J = 2.32, 6.48 Hz, 3H); LCMS (ESI) C 18 H 24 Calculated values ​​for N6O2 [M+H] + : 357.2, Measured value: 357.2.

[0119] Synthesis of (S)-5-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrazine-2-amine (Int H) To a stirred mixture of 8-2 (1.0 g, 2.70 mmol) in ethyl acetate (5 mL), HCl / siRNA (0.675 mL, 2.70 mmol) was added at room temperature, and the mixture was stirred at room temperature (temperature: 20°C) for 48 hours. LC-MS (ESI) showed that the starting material was consumed and a new product was formed. The mixture was then concentrated, the residue was adjusted to pH 9 with saturated Na2CO3, extracted with siRNA (20 mL, 3 times), washed with brine (20 mL), dehydrated with Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (ISCORF150; Sepa flash column) (eluting with petroleum ether / siRNA = 1:1) to obtain Int H as a solid. 1H NMR (500 MHz, chloroform-d) δ 8.18-8.22 (m, 1H), 7.72 (d, J = 1.53 Hz, 1H), 7.67 (d, J = 1.22 Hz, 1H), 7.46-7.53 (m, 1H), 6.68 (d, J = 8.54 Hz, 1H), 6.63 (dd, J = 5.19, 7.02 Hz, 1H), 5.47-5.50 (m, 1H), 4.34 (td, J = 3.32, 6.48 Hz, 1H), 4.20 (brdd, J = 1.68, 12.05 Hz, 1H), 3.96-4.07 (m, 3H), 3.76 (td, J = 3.55, 12.13 Hz, 1H), 3.38 (dd, J = 3.81, 12.66 Hz, 1H), 3.27 (dt, J = 3.51, 11.52 Hz, 1H), 3.15-3.21 (m, 1H), 1.15 (d, J = 6.71 Hz, 3H); LCMS (ESI) C 14 H 18 Calculated value for N6 [M+H] + : 271.2, Measured value: 271.1.

[0120] Preparation of compound intermediate I: (S)-2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine [ka] Synthesis of I-2: tert-butyl (S)-4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (I-1, 5 g, 24.96 mmol) in toluene (200 mL) (purged with argon gas for 10 minutes), 2-bromo-4-fluoropyridine (4.39 g, 24.96 mmol), sodium tert-butoxide (2.399 g, 24.96 mmol), and chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (19.39 g, 24.96 mmol) were added at room temperature. The reaction mixture was again purged with argon for 10 minutes and stirred at 120°C for 12 hours. The reaction mixture was diluted with water (80 mL) and extracted with ELISA (3 × 100 mL). The combined organic layers were dehydrated with Na2SO4 and concentrated under reduced pressure to obtain the crude product. This crude product was purified using 100-200 mesh silica gel (300 g cartridge) (eluted with 20% siRNA / petroleum ether as a gradient). The pure fraction was concentrated under reduced pressure to obtain compound I-2 as a yellow liquid. M / Z (ESI): 296.37 [M+H] + .

[0121] Synthesis of I-3: (S)-1-(4-fluoropyridine-2-yl)-3-methylpiperazine To a stirred solution of compound I-2 (5 g, 16.93 mmol) in DCM (80 mL), TFA (6.52 mL, 85 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain compound I-3 as a pale yellow liquid. M / Z (ESI): 196.23 [M+H] + .

[0122] Synthesis of I-4: (S)-2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)-5-nitropyrimidine Compound I-3 (3.5 g, 17.93 mmol) was stirred in DMF (80 mL), to which 2-chloro-5-nitropyrimidine (3.43 g, 21.51 mmol) and K2CO3 (9.91 g, 71.7 mmol) were added at room temperature. The reaction mixture was stirred at 80 °C for 2 hours. The reaction mixture was diluted with ice-cold water (20 mL), the resulting solid was filtered, washed with water (2 × 50 mL), and dried under reduced pressure to obtain compound I-4 as a pale yellow solid. M / Z (ESI): 319.20 [M+H] + .

[0123] Synthesis of intermediate I: (S)-2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine To a solution of compound I-4 (3.5 g, 11 mmol) in EtOH (50 mL), 10% Pd-C (1.170 g, 5.50 mmol) was added at room temperature. The reaction mixture was degassed three times with nitrogen and stirred under H2 gas balloon pressure at room temperature for 16 hours. The reaction mixture was filtered through a Celite bed using a Buckner funnel, washed with ELISA (50 mL), and concentrated under reduced pressure to obtain compound Int I as a yellow liquid. M / Z (ESI): 289.17 [M+H] + .

[0124] Preparation of compound intermediate J: 6-(3,3-difluoroazetidine-1-yl)nicotinic acid [ka] Synthesis of J-2: Methyl 6-(3,3-difluoroazetidine-1-yl)nicotinate To a stirred solution of 3,3-difluoroazetidine hydrochloride (8.35 g, 64.5 mmol) in DMF (250 mL), K2CO3 (26.7 g, 193 mmol) was added at room temperature, followed by the addition of methyl 6-fluoronicotinate (J-1, 10 g, 64.5 mmol) at room temperature. The reaction mixture was stirred at 90°C for 12 hours. The resulting reaction mixture was quenched with ice-water (200 mL), the separated solid was filtered through a Buchner funnel, the solid was washed with water (100 mL x 2), and dried under reduced pressure to obtain compound J-2 as a white solid. M / Z (ESI): 229.04 [M+H] + .

[0125] Synthesis of intermediate J: 6-(3,3-difluoroazetidine-1-yl)nicotinic acid To a stirred solution of compound J-2 (10 g, 43.5 mmol) in THF (70 mL) and water (140 mL), LiOH (3.13 g, 131 mmol) was added at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated, acidified with a saturated solution of KHSO4 (pH=5), extracted with DCM (3 × 200 mL), dehydrated with Na2SO4, filtered, concentrated, washed with pentane, and dried under reduced pressure to obtain compound Int J as an off-white solid. M / Z (ESI): 215.04 [M+H] + .

[0126] Preparation of compound intermediate K: (S)-2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine [ka] Synthesis of K-1: tert-butyl (S)-3-methyl-4-(5-nitropyrimidine-2-yl)piperazine-1-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (5 g, 24.96 mmol) in DMF (50 mL), potassium carbonate (6.90 g, 49.9 mmol) and 2-chloro-5-nitropyrimidine (4.78 g, 30.0 mmol) were added, and the mixture was stirred at 60°C for 3 hours under an N2 atmosphere. The reaction mixture was quenched with ice water (200 mL) and stirred at room temperature for 30 minutes. The resulting solid precipitate was filtered, washed with water (100 mL x 3), and dried under vacuum to obtain K-1 as a pale yellow solid. 1 ¹H NMR (400MHz, chloroform-d) δ: 9.08 (s, 2H), 4.97-5.14 (m, 1H), 4.61-4.76 (m, 1H), 3.87-4.32 (m, 2H), 3.26-3.43 (m, 1H), 3.14 (br d, J=11.8 Hz, 1H), 2.90-2.99 (m, 1H), 1.50 (s, 9H), 1.27 (d, J=6.8 Hz, 3H).

[0127] Synthesis of K-2: tert-butyl (S)-4-(5-aminopyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a solution of K-1 (3 g, 9.28 mmol) in THF / MeOH / ethyl acetate (1:1:1) (50 mL), carbon-supported palladium (1.975 g, 18.56 mmol) was added and the mixture was stirred at 25°C for 15 hours under a hydrogen balloon atmosphere. The reaction mixture was filtered, washed with MeOH / THF (3 × 50 mL), and the combined organic layers were concentrated under reduced pressure to obtain K-2 as a yellow solid. M / Z (ESI): 294.15 [M+H] + .

[0128] Synthesis of K-3: tert-butyl (S)-4-(5-(6-fluoronicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate A stirred solution of K-2 (1.20 g, 4.09 mmol) and 6-fluoronicotinic acid (1.154 g, 8.18 mmol) in DMF (20 mL) was mixed with toluene (5.21 g, 8.18 mmol) and a 50% 1-propanephosphonic acid anhydride solution in TEA (1.710 mL, 12.27 mmol) at room temperature, and the mixture was stirred at room temperature for 15 hours. The reaction mixture was diluted with toluene (100 mL), washed with brine (100 mL) and saturated NaHCO3 solution (100 mL), the organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain K-3 as a solid. M / Z (ESI): 417.42 [M+H] + .

[0129] Synthesis of K-4: tert-butyl (S)-4-(5-(6-(3,3-difluoroazetidine-1-yl)nicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of 3,3-difluoroazetidine hydrochloride (641 mg, 4.95 mmol) in DMF (10 mL), K2CO3 (912 mg, 6.60 mmol) and K-3 (700 mg, 1.649 mmol) were added at room temperature, and the mixture was stirred overnight at 100°C. The reaction mixture was quenched with ice-cold water (30 mL), extracted with siRNA (3 × 25 mL), the organic layer was washed with brine solution (2 × 25 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain K-4 as a brown solid. M / Z (ESI): 490.45 [M+H] + .

[0130] Synthesis of intermediate K: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(2-(2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide To a stirred solution of K-4 (700 mg, 1.158 mmol) in DCM (10 mL), TFA (0.268 mL, 3.47 mmol) was added at 0°C and the mixture was stirred at 0°C for 5 hours. The reaction mixture was concentrated and distilled twice with toluene to obtain Int K as a yellow semi-solid. M / Z (ESI): 390.18 [M+H] + .

[0131] Preparation of compound intermediate L: (S)-2-(4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine [ka] Synthesis of L-1: tert-butyl (S)-4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of 4-bromo-2-fluoropyridine (1.142 g, 6.49 mmol) in toluene (20 mL), chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (1.939 g, 2.496 mmol), tert-butyl (S)-2-methylpiperazine-1-carboxylate (1 g, 4.99 mmol), and sodium tert-butoxide (1.200 g, 12.48 mmol) were added at room temperature, and the mixture was stirred at 110 °C for 18 hours. The reaction mixture was quenched with ice-cold water (10 mL), extracted with ethyl acetate (2 × 200 mL), washed with brine solution (2 × 30 mL), dehydrated with sodium sulfate, filtered, concentrated under reduced pressure, and the crude compound was purified by 100-200 silica gel Biotage flash column chromatography (using 50% dimethyl ether as the eluent). The pure fraction was concentrated under reduced pressure to obtain compound L-1 as a yellow, viscous solid. M / Z (ESI): 296.31 [M+H] + .

[0132] Synthesis of L-2: (S)-1-(2-fluoropyridine-4-yl)-3-methylpiperazine To a stirred solution of compound L-1 (1.0 g, 3.39 mmol) in DCM (12 mL), TFA (0.777 mL, 10.16 mmol) was added at 0°C and the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with NaHCO3 solution (10 mL), extracted with DCM (2 × 50 mL), washed with water (2 × 30 mL), washed the combined organic layer with brine solution (20 mL), dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure to obtain compound L-2 as a yellow solid. M / Z (ESI): 196.07 [M+H] + .

[0133] Synthesis of L-3: (S)-2-(4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-yl)-5-nitropyrimidine To a stirred solution of compound L-2 (700 mg, 3.59 mmol) in DMF (12 mL), potassium carbonate (1487 mg, 10.76 mmol) and 2-chloro-5-nitropyrimidine (858 mg, 5.38 mmol) were added at room temperature, and the mixture was stirred at 80°C for 3 hours. The reaction mixture was quenched with ice-cold water (20 mL), the precipitated solid was filtered, and dried under reduced pressure to obtain compound L-3 as an off-white solid. M / Z (ESI): 319.24 [M+H] + .

[0134] Synthesis of intermediate L: (S)-2-(4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-amine To a stirred solution of compound L-3 (600 mg, 1.885 mmol) in EtOH (15 mL), Pd-C (241 mg, 2.262 mmol) was added at room temperature, and the mixture was stirred at room temperature under a hydrogen balloon for 12 hours. The reaction mixture was filtered through a Celite bed using a Buckner funnel, washed with SiO2 (2 × 50 mL), and concentrated under reduced pressure to obtain compound Int L as a viscous solid. M / Z (ESI): 289.25 [M+H] + . [Examples]

[0135] Examples Example 1: Synthesis of (S)-6-methoxy-N-(2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Int A (185 mg, 0.684 mmol) was dissolved in DMF (3422 μL) and 6-methoxynicotinic acid (131 mg, 0.855 mmol), DIEA (359 μL, 2.053 mmol), and HATU (325 mg, 0.855 mmol) were added. The mixture was stirred at 25°C for 16 hours. The mixture was then purified by preparative HPLC (reverse-phase C-18 column) (eluted with acetonitrile / water + 0.1% TFA) to obtain compound 1 as a solid. 1H NMR (500 MHz, chloroform-d) δ 8.72 (d, J = 2.2 Hz, 1H), 8.57 (s, 2H), 8.22 (dd, J = 4.9, 1.2 Hz, 1H), 8.11 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (ddd, J = 8.9, 7.2, 2.0 Hz, 1H), 7.43 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.69 (d, J = 8.6 Hz, 1H), 6.65 (dd, J = 6.8, 5.2 Hz, 1H), 4.96 (dt, J = 6.6, 3.6 Hz, 1H), 4.55 (dt, J = 13.5, 3.4 Hz, 1H), 4.24 (d, J = 12.5 Hz, 1H), 4.14 (dd, J = 12.7, 2.0 Hz, 1H), 4.03 (s, 3H), 3.48 (ddd, J = 13.5, 11.3, MS (ESI) m / z: 406.4 [M+H] + .

[0136] The compounds listed in Table 1 were synthesized using a method similar to the synthetic procedure used to prepare the intermediates in Example 1, utilizing the amide coupling reaction described therein. Commercially available reagents were used as needed to prepare the compounds in the following examples. [Table 3] TIFF0007881829000058.tif197166 TIFF0007881829000059.tif233166 TIFF0007881829000060.tif222166 TIFF0007881829000061.tif217165 TIFF0007881829000062.tif204165 TIFF0007881829000063.tif211166 TIFF0007881829000064.tif212166

[0137] Example 37: Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-1-yl]pyrimidine-5-yl]-6-pyrazole-1-ylpyridine-3-carboxamide [ka] Synthesis of 9-1: (S)-6-fluoro-N-(2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide A solution of Int A (200 mg, 0.740 mmol), 6-fluoronicotinic acid (157 mg, 1.110 mmol), HATU (422 mg, 1.110 mmol), and Hünig base (517 μL, 2.96 mmol) was prepared in DMF (2466 μL) at 50°C. After 18 hours, the reaction was confirmed to be complete by LC-MS. The reaction product was diluted with SiO2, washed with saturated NaHCO3, water, and brine; the organic layer was dehydrated with MgSO4, filtered, and concentrated. The substance was purified by normal-phase column chromatography (0 → 100% SiO2 in hexane, ISCO 24 g column; 25-minute gradient) to obtain 9-1 as a solid. 1H NMR (500 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.83 (d, J = 2.0 Hz, 1H), 8.69 (s, 2H), 8.51 (td, J = 8.3, 2.4 Hz, 1H), 8.12 (d, J = 3.5 Hz, 1H), 7.60-7.47 (m, 1H), 7.39 (dd, J = 8.6, 2.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.64 (dd, J = 6.8, 5.1 Hz, 1H), 4.95-4.74 (m, 1H), 4.44 (d, J = 13.4 Hz, 1H), 4.24 (dd, J = 24.0, 12.8 Hz, 2H), 3.33-3.25 (m, 1H), 3.18 (dd, J = 12.9, 3.8 Hz, 1H), 2.95 (td, J = 12.1, 3.7 Hz, 1H), 1.15 (d, J = 6.6 Hz, 3H). LCMS (ESI) C 20 H 20 Calculated value for FN7O [M+H] + : 394.2, Measured value: 394.3.

[0138] Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazine-1-yl]pyrimidine-5-yl]-6-pyrazole-1-ylpyridine-3-carboxamide (37) To a solution of 9-1 (25 mg, 0.064 mmol) and 1H-pyrazole (8.65 mg, 0.127 mmol) in DMF (635 μL), LiHMDS (63.5 μL, 0.095 mmol; 1.5 M in THF) was added at ambient temperature, and the reaction mixture was heated to 100°C. After 4 hours, the reaction was complete. The reaction mixture was diluted with ethyl acetate, quenched with water and brine; the organic layer was dehydrated with MgSO4, filtered, and concentrated. The substance was purified by normal-phase column chromatography (0 → 100% ethyl acetate in hexane, ISCO 12 g column; 20-minute gradient) to obtain 37 as a solid. 1H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.03 (d, J = 1.8 Hz, 1H), 8.72 (d, J = 4.4 Hz, 2H), 8.52 (dd, J = 8.6, 2.2 Hz, 1H), 8.18 - 8.02 (m, 2H), 7.97 - 7.82 (m, 1H), 7.61 - 7.46 (m, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.69 - 6.56 (m, 2H), 4.94 - 4.77 (m, 1H), 4.52 - 4.36 (m, 1H), 4.24 (dd, J = 24.2, 12.8 Hz, 2H), 3.29 (d, J = 3.7 Hz, 1H), 3.19 (dd, J = 13.0, 3.8 Hz, 1H), 2.96 (td, J = 12.2, 3.8 Hz, 1H), 1.16 (d, J = 6.6 Hz, 3H). LCMS (ESI) C 23 H 23 Calculated values ​​for N9O [M+H] + : 442.2, Measured value: 442.3.

[0139] The compounds listed in Table 2 were synthesized using a method similar to that of Example 37, following the synthesis sequence. Commercially available reagents were used as needed to prepare the compounds in the following examples. [Table 4] TIFF0007881829000067.tif219166

[0140] Example 46: Synthesis of 6-(3,3-difluoroazetidine-1-yl)-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazine-1-yl]pyrimidine-5-yl]pyridine-3-carboxamide [ka]

[0141] A solution of 9-1 (30 mg, 0.076 mmol) and Hünig base (80 μL, 0.458 mmol) in DMF (763 μL) was added to 3,3-difluoroazetidine hydrochloride (29.6 mg, 0.229 mmol) and heated to 100°C. After 18 hours, the reaction was confirmed to be complete by LC-MS. The reaction product was cooled and diluted with SiO2. The organic layer was washed with water / saturated NH4Cl, dehydrated with MgSO4, and concentrated. The residue was purified by normal-phase column chromatography (20 → 100% SiO2 in hexane, ISCO 12 g column; 20 min gradient) to obtain 46 as a solid. 1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.76 (d, J = 1.9 Hz, 1H), 8.67 (s, 2H), 8.25 - 8.03 (m,2H), 7.60 - 7.47 (m, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.72 - 6.56 (m, 2H), 4.90 - 4.79 (m, 1H), 4.57 - 4.35(m, 6H), 4.24 (dd, J = 24.8, 12.7 Hz, 2H), 3.31 - 3.24 (m, 1H), 3.22 - 3.11 (m, 1H), 2.95 (td, J = 12.1, 3.7Hz, 1H), 1.14 (d, J = 6.6 Hz, 3H); LCMS (ESI) C 23 H 24 Calculated values ​​for F2N8O [M+H] + : 467.2, Measured value: 467.3.

[0142] The compounds included in Table 3 were synthesized using a method similar to the synthesis sequence in Example 46. Commercially available reagents were used as needed to prepare the compounds in the following examples. [Table 5] TIFF0007881829000070.tif202166 TIFF0007881829000071.tif235166 TIFF0007881829000072.tif148166

[0143] Example 63: Synthesis of 6-(3-fluorophenyl)-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-1-yl]pyrimidine-5-yl]pyridine-3-carboxamide [ka] Synthesis of 6-bromo-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-1-yl]pyrimidine-5-yl]pyridine-3-carboxamide (20) A solution of Int A (200 mg, 0.740 mmol), 6-bromonicotinic acid (224 mg, 1.110 mmol), HATU (422 mg, 1.110 mmol), and Hünig base (517 μL, 2.96 mmol) was prepared in DMF (2466 μL) and heated to 50°C. After 18 hours, the reaction was confirmed to be complete by LC-MS. The reaction product was diluted with SiO and washed with saturated NaHCO3, water, and brine; the organic layer was dehydrated with MgSO4, filtered, and concentrated. The substance was purified by normal-phase column chromatography (20 in hexane → 100% SiO, ISCO 24 g column; 25 min gradient) to obtain 20 as a solid. 1H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.93 (d, J = 2.1 Hz, 1H), 8.69 (s, 2H), 8.24 (dd, J = 8.3, 2.5 Hz, 1H), 8.16 - 8.03 (m, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.60 - 7.45 (m, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.63 (dd, J = 6.8, 5.1 Hz, 1H), 4.84 (dd, J = 6.3, 3.2 Hz, 1H), 4.52 - 4.35 (m, 1H), 4.23 LCMS (ESI)C 20 H 20 Calculated values ​​for BrN7O [M+H] + : 454.1, Measured value: 454.2.

[0144] Synthesis of 6-(3-fluorophenyl)-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-1-yl]pyrimidine-5-yl]pyridine-3-carboxamide (63) A solution of 20 (8.2 mg, 0.018 mmol), 3-fluorophenyl pinacol boronate (15.9 mg, 0.072 mmol), SPhos-Pd-G2 (2.6 mg, 0.033 mmol), and K3PO4 (1.5 M aqueous solution, 23 μL, 0.036 mmol) was prepared in DMF (2466 μL) and heated to 80°C. After 18 hours, the reaction was confirmed to be complete by LC-MS. The reaction product was diluted with DMF (1.0 mL), and the mixture was purified by RP HPLC (reverse-phase column chromatography; MeCN in water, 0.1% NH4OH regulator, Phenomenex C18 Luna column, 100 × 21.2 mm, 5 micron). After concentration, 63 was obtained as a solid. 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.22 (s, 1H), 8.73 (s, 2H), 8.43 (dd, J = 8.4, 1.9 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 4.4 Hz, 1H), 8.04 (dd, J = 25.7, 9.0 Hz, 2H), 7.68 - 7.49 (m, 2H), 7.36 (t, J = 7.4 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.73 - 6.57 (m, 1H), 4.96 - 4.75 (m, 1H), 4.44 (d, J = 13.4 Hz, 1H), 4.25 (dd, J = 24.5, 12.8 Hz, 2H), 3.32 - 3.25 (m, 1H) 3.19 (dd, J = 12.8, 3.6 Hz, 1H), 2.96 (td, J = 12.0, 3.5 Hz, 1H), 1.16 (d, J = 6.6 Hz, 4H).; LCMS (ESI) C 26 H 24 Calculated value for FN7O [M+H] + : 470.2, Measured value: 470.2.

[0145] The compounds listed in Table 4 were synthesized using a method similar to that of Example 63, following the synthesis sequence. Commercially available reagents were used as needed to prepare the compounds in the following examples. [Table 6] TIFF0007881829000075.tif211166 TIFF0007881829000076.tif235166 TIFF0007881829000077.tif217166 TIFF0007881829000078.tif225165 TIFF0007881829000079.tif177166

[0146] Example 87: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Compound intermediate I (500 mg, 1.734 mmol) was stirred in THF (10 mL) to which compound intermediate J (557 mg, 2.60 mmol), DIPEA (0.909 mL, 5.20 mmol), and a 50% 1-propanephosphonic anhydride solution in SiO (1104 mg, 3.47 mmol) were added at room temperature. The reaction mixture was stirred at 50 °C for 12 hours. The reaction mixture was diluted with water (10 mL) and extracted with SiO (3 × 30 mL). The combined organic layer was dehydrated with Na₂SO₄ and concentrated under reduced pressure to obtain the crude compound. The crude compound was subjected to preparative HPLC purification (Method: Mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, Column - LUNA Pack C18 (21.2 × 250) mm 5 μm, Flow rate - 18 mL / min, Gradient method: 0 / 45, 12 / 70, 12.05 / 98, 14 / 98, 14.05 / 45, 17 / 45) to obtain 87 as a light brown solid. M / Z (ESI): 485.30 [M+H] + . 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 8.73 (t, J = 2 Hz, 1H), 8.65 (s, 2H), 8.09-8.15 (m, 2H), 6.65-6.71 (m, 2H), 6.51-6.55 (m, 1H), 4.81 (br s, 1H), 4.32-4.53 (m, 5H), 4.22 (t, J = 13.2 Hz, 2H), 3.21-3.32 (m, 2H), 2.95-3.2 (m, 1H), 1.12 (d, J = 2 Hz, 3H).

[0147] Example 88: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(2-(2-methyl-4-(4-nitropyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of 88: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(2-(2-methyl-4-(4-nitropyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide To a stirred solution of Int K (250 mg, 0.465 mmol) in dioxane (3 mL), Cs2CO3 (455 mg, 1.396 mmol) and 2-bromo-4-nitropyridine (123 mg, 0.605 mmol) were added at room temperature, and the mixture was degassed under nitrogen for 10 minutes. Subsequently, chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (18.08 mg, 0.023 mmol) was added, and the mixture was stirred at 100°C for 16 hours. The reaction mixture was filtered through a Celite bed and washed with ethyl acetate. The filtrate was dehydrated with sodium sulfate and evaporated under reduced pressure. The crude compound was purified by Prep-HPLC (Method: Mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, Column - LUNA Omega C18 (21.2 × 250) mm 5 μm, Flow rate - 18 mL / min, Gradient method - 0 / 50, 10.2 / 84, 10.25 / 100, 12 / 100, 12.05 / 50, 16 / 50), and lyophilized to obtain 88 as a yellow solid. M / Z (ESI): 512.35 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 10.07 (s, 1H), 8.76 (d, J=2.0 Hz, 1H), 8.68 (s, 2H), 8.41 (d, J=5.4 Hz, 1H), 8.15 (dd, J=8.8, 2.4 Hz, 1H), 7.49 (d, J=1.5 Hz, 1H), 7.26 (dd, J=5.5, 1.8 Hz, 1H), 6.66 (d, J=8.8 Hz, 1H), 4.85 (dt, J=6.3, 3.3 Hz, 1H), 4.29-4.59 (m, 7H), 3.33-3.41 (m, 2H), 3.11-3.20 (m, 1H), 1.14 (d, J=6.6 Hz, 3H).

[0148] Example 89: (S)-N-(2-(4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1H-pyrazole-1-yl)nicotinamide [ka] To a stirred solution of compound Int L (150 mg, 0.520 mmol) in THF (3 mL), TEA (0.290 mL, 2.081 mmol), a 50% 1-propanephosphonic acid anhydride solution in  (0.312 mL, 1.040 mmol), and 6-(1H-pyrazole-1-yl)nicotinic acid (128 mg, 0.676 mmol) were added at 0°C, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was quenched with NaHCO3 solution (10 mL), extracted with Depositphotos (2 × 50 mL), washed with water (2 × 10 mL), the combined organic layer was washed with brine solution (20 mL), dehydrated with Na2SO4, filtered, concentrated under reduced pressure, and subjected to preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN column - X-Select phenylhexyl (19 × 250) mm 5u flow rate - 18 mL / min gradient method - 0 / 35, 9.3 / 70, 9.4 / 99, 11 / 99, 11.05 / 35, 15 / 35). The pure fraction was concentrated and lyophilized to obtain 89 as an off-white solid. M / Z (ESI): 460.15 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 10.44 (s, 1H), 9.03 (d, J=2.2 Hz, 1H), 8.72 (s, 3H), 8.51 (dd, J=8.6, 2.2 Hz, 1H), 8.08 (d, J=8.6 Hz, 1H), 7.87-7.96 (m, 1H), 7.81 (d, J=6.1 Hz, 1H), 6.81 (br d, J=6.1 Hz, 1H), 6.61-6.68 (m, 1H), 6.49 (s, 1H), 4.78 (dt, J=6.7, 3.1 Hz, 1H), 4.33-4.41 (m, 1H), 3.86-3.98 (m, 2H), 3.34-3.48 (m, 2H), 3.09-3.18 (m, 1H), 1.16 (d, J=6.6 Hz, 3H).

[0149] Example 90: (S)-N-(2-(2-methyl-4-(2-nitropyridine-4-yl)piperazine-1-yl)pyrimidine-5-yl)-6-(1H-pyrazole-1-yl)nicotinamide [ka] Synthesis of K-6: tert-butyl (S)-4-(5-(6-(1H-pyrazole-1-yl)nicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of 1H-pyrazole (82 mg, 1.201 mmol) and cesium carbonate (391 mg, 1.201 mmol) in DMF (5 mL), K-3 (250 mg, 0.600 mmol) was added at room temperature under a nitrogen atmosphere, and the mixture was stirred at 90°C for 3 hours. The progress of the reaction was monitored by LC-MS and TLC. TLC indicated that the reaction was complete. The reaction mixture was then diluted with ethyl acetate (100 mL) and water (60 mL). The organic layer was separated, the aqueous layer was re-extracted with ethyl acetate (2 × 40 mL), and the combined organic layer was dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound K-6 as a light brown, viscous solid. M / Z (ESI): 465.36 [M+H] + .

[0150] Synthesis of K-7: (S)-N-(2-(2-methylpiperazin-1-yl)pyrimidine-5-yl)-6-(1H-pyrazole-1-yl)nicotinamide To a stirred solution of compound K-6 (190 mg, 0.409 mmol) in DCM (5.7 mL), TFA (1.565 mL, 20.45 mmol) was added at room temperature, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The progress of the reaction was monitored by TLC. TLC indicated that the reaction was complete. The reaction mixture was then concentrated under reduced pressure, and the crude residue was diluted with DCM (100 mL) and H2O (60 mL). Na2CO3 was then added to adjust the pH to 7-8, and the mixture was extracted with DCM (100 mL x 3). The combined organic layer was dehydrated with sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound K-7 as a light brown solid. M / Z (ESI): 365.15 [M+H] + .

[0151] Synthesis of 90: (S)-N-(2-(2-methyl-4-(2-nitropyridine-4-yl)piperazine-1-yl)pyrimidine-5-yl)-6-(1H-pyrazole-1-yl)nicotinamide To a stirred solution of compound K-7 (105 mg, 0.288 mmol) and potassium carbonate (119 mg, 0.864 mmol) in DMF (5 mL), 4-chloro-2-nitropyridine (91 mg, 0.576 mmol) was added at room temperature under a nitrogen atmosphere, and the mixture was stirred at 50 °C for 6 hours. The progress of the reaction was monitored by LC-MS and TLC. TLC indicated that the reaction was complete. The reaction mixture was then diluted with ethyl acetate (60 mL) and water (50 mL). The organic layer was separated, the aqueous layer was re-extracted with ethyl acetate (2 × 30 mL), the combined organic layers were dehydrated with sodium sulfate, filtered, concentrated under reduced pressure, and the crude material was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-BRIDGE C18 (19 × 250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 30, 9 / 75, 9.05 / 100, 11 / 100, 11.05 / 30, 13.5 / 30). The obtained compound was further purified by SFC (conditions: column - Chiralpak IG (4.6 × 250 mm), 5 μ, mobile phase - A: MeOH / DCM / DEA (50 / 50 / 0.2), fixed composition A: 100%, flow rate: 1.0 mL / min, diluent: EtOH). The pure fraction was concentrated and freeze-dried to obtain 90 as a yellow solid. M / Z (ESI): 487.12 [M+H]+ . 1 H NMR (500MHz, DMSO-d6) δ: 10.45 (s, 1H), 9.00-9.05 (m, 1H), 8.69-8.76 (m, 3H), 8.52 (dd, J=8.5, 2.4 Hz, 1H), 8.18 (d, J=5.8 Hz, 1H), 8.05-8.10 (m, 1H), 7.89-7.94 (m, 1H), 7.63 (d, J=2.4 Hz, 1H), 7.23 (dd, J=6.1, 2.4 Hz, 1H), 6.65 (dd, J=2.6, 1.7 Hz, 1H), 4.77-4.85 (m, 1H), 4.35-4.42 (m, 1H), 3.98-4.09 (m, 2H), 3.45-3.55 (m, 2H), 3.22-3.29 (m, 1H), 1.17 (d, J=6.4 Hz, 3H).

[0152] Example 91: (S)-6-(1H-imidazole-1-yl)-N-(2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Intermediate A (150 mg, 0.555 mmol) and 6-(1H-imidazole-1-yl)nicotinic acid (157 mg, 0.832 mmol) were mixed in THF (30 mL) and TEA (0.193 mL, 1.387 mmol) and 1-propanephosphonic anhydride (0.495 mL, 0.832 mmol) were added at 25°C and the mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with ice-cold water (10 mL), extracted with ethyl acetate (2 × 100 mL), washed with brine solution (2 × 10 mL), dehydrated with sodium sulfate, filtered, concentrated under reduced pressure, and the crude compound was purified by preparative HPLC (mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Select C18 (19 × 250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 45, 6.9 / 76, 6.95 / 100, 9 / 100, 9.05 / 45, 12 / 45). The pure fraction was concentrated and lyophilized to obtain 91 as a pale yellow solid. M / Z (ESI): 442.14 [M+H]+ . 1 H NMR (400MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.04 (d, J=2.0 Hz, 1H), 8.71 (s, 2H), 8.66 (s, 1H), 8.52 (dd, J=8.7, 2.3 Hz, 1H), 8.12 (dd, J=4.9, 1.2 Hz, 1H), 8.06 (t, J=1.2 Hz, 1H), 8.01 (d, J=8.6 Hz, 1H), 7.55 (ddd, J=8.6, 7.0, 2.2 Hz, 1H), 7.18 (s, 1H), 6.86 (d, J=8.8 Hz, 1H), 6.60-6.67 (m, 1H), 4.85 (dt, J=6.4, 3.2 Hz, 1H), 4.41-4.49 (m, 1H), 4.17-4.31 (m, 2H), 3.28 (br d, J=3.7 Hz, 1H), 3.19 (br dd, J=13.1, 3.8 Hz, 1H), 2.90-3.01 (m, 1H), 1.16 (d, J=6.6 Hz, 3H).

[0153] The compounds included in Table 5 were synthesized using a method similar to the synthesis sequence described above. Commercially available reagents were used as needed to prepare the following examples. [Table 7] TIFF0007881829000086.tif216166 TIFF0007881829000087.tif211166 TIFF0007881829000088.tif192166 TIFF0007881829000089.tif205165 TIFF0007881829000090.tif205166 TIFF0007881829000091.tif140166

[0154] Example 112: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] Synthesis of M-3: tert-butyl (S)-4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (M-1) (2.0 g, 9.99 mmol) in DMA (20 mL), N,N-diisopropylethylamine (5.2 mL, 30.0 mmol) was added at 0°C, followed by the addition of 4,6-difluoropyrimidine (M-2) (1.28 g, 11.0 mmol). The mixture was stirred at 110°C for 18 hours. The reaction mixture was poured onto crushed ice, extracted with ELISA, washed with brine solution, dehydrated with Na2SO4, and evaporated under reduced pressure to obtain compound M-3. M / Z (ESI): 297.12 [M+H+].

[0155] Synthesis of M-4: (S)-4-fluoro-6-(3-methylpiperazin-1-yl)pyrimidine To a stirred solution of M-3 (2.2 g, 7.42 mmol) in DCM (10 mL), 4 M HCl (9.25 g, 74.2 mmol) in 1,4-dioxane was added at 0°C and the mixture was stirred for 18 hours. The reaction mixture was concentrated under reduced pressure, washed with saturated NaHCO3 solution, and extracted with siRNA. The organic layer was washed with brine solution, dehydrated with Na2SO4, and concentrated under reduced pressure to obtain compound M-4. M / Z (ESI): 197.06 [M+H+].

[0156] Synthesis of M-6: (S)-4-(4-(5-bromopyrazine-2-yl)-3-methylpiperazine-1-yl)-6-fluoropyrimidine To a stirred solution of M-4 (1.9 g, 9.68 mmol) in DMSO (20 mL), cesium fluoride (2.94 g, 19.4 mmol) and 2,5-dibromopyrazine (M-5) (1.843 g, 7.75 mmol) were added at 0°C, and the mixture was stirred at 80°C for 18 hours. The reaction mixture was poured into ice-cold water, extracted with phenylethylamine, washed with brine solution, dehydrated with Na2SO4, and evaporated under reduced pressure to obtain compound M-6. M / Z (ESI): 353.04 [M+H+].

[0157] Synthesis of M-7: tert-butyl (S)-(5-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)carbamate To a stirred solution of M-6 (800 mg, 2.265 mmol) in toluene (10 mL), cesium carbonate (996 mg, 3.06 mmol), tris(dibenzylideneacetone)dipalladium(0) (104 mg, 0.113 mmol), xantphos (131 mg, 0.227 mmol), and tert-butyl carbamate (292 mg, 2.492 mmol) were added at 0°C and stirred at 70°C for 18 hours. The reaction mixture was poured into ice water (5 mL), extracted with siRNA (5 mL x 3), the organic layer was washed with brine, dehydrated with anhydrous Na2SO4, and evaporated under reduced pressure to obtain compound M-7. M-7. M / Z (ESI): 390.17 [M+H+].

[0158] Synthesis of M-8: (S)-5-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-amine To a stirred solution of M-7 (600 mg, 1.541 mmol) in DCM (8 mL), TFA (0.590 mL, 7.70 mmol) was added at 0°C and the mixture was stirred at room temperature for 18 hours. The reaction mixture was evaporated, quenched with saturated NaHCO3 solution (10 mL), and extracted with toluene (10 mL x 3). The organic layer was washed with brine, dehydrated with Na2SO4, and evaporated under reduced pressure to obtain M-8. M / Z (ESI): 290.21 [M+H+].

[0159] Synthesis of 112: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide: To a stirred solution of Int J (89 mg, 0.415 mmol) in DCM (5 mL), 1-methylimidazole (0.083 mL, 1.037 mmol) was added at 0°C, followed by methanesulfonyl chloride (0.035 mL, 0.456 mmol) at 0°C. The mixture was stirred for 15 minutes, then M-8 (60 mg, 0.207 mmol) was added, and the mixture was stirred at 45°C for 4 hours. The reaction mixture was poured into ice-cold water (2 mL) and extracted using DCM (3 mL x 3). The organic layer was washed with brine solution, dehydrated with anhydrous Na2SO4, evaporated under reduced pressure, and the crude compound was purified by preparative HPLC (mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, column - X-Bridge C18 (19×250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 30, 9.2 / 78, 9.25 / 99, 11.2 / 99, 11.25 / 30, 15.2 / 30), and lyophilized to obtain compound 112. M / Z (ESI): 484.18 [M+H+]. 1 H NMR (400MHz, DMSO-d6) δ: 10.57 (s, 1H), 8.82 (br dd, J=17.7, 1.6 Hz, 2H), 8.32 (br d, J=2.7 Hz, 1H), 8.20 (br dd, J=8.8, 2.4 Hz, 1H), 8.12 (br d, J=1.2 Hz, 1H), 6.58-6.65 (m, 2H), 4.60 (br dd, J=6.4, 2.7 Hz, 1H), 4.42-4.54 (m, 4H), 4.31-4.40 (m, 2H), 4.06-4.11 (m, 1H), 3.75-3.81 (m, 1H), 3.43 (br dd, J=13.7, 3.7 Hz, 1H), 3.24 (br dd, J=8.4, 4.5 Hz, 2H), 1.06 (d, J=6.4 Hz, 3H).

[0160] Example 113: (S)-6-(azetidine-1-yl)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] To a stirred solution in DMF (2 mL) containing N-1 (prepared in the same manner as M-8) (0.060 g, 0.208 mmol) and N-2 (prepared in the same manner as J) (0.074 g, 0.416 mmol), HATU (0.158 g, 0.416 mmol) and DIPEA (0.109 mL, 0.624 mmol) were added at 0°C, and the mixture was stirred at 100°C for 1 hour. The reaction mixture was quenched with ice-cold water (10 mL), extracted with ethyl acetate (2 × 50 mL), washed with brine solution (2 × 20 mL), dehydrated with sodium sulfate, filtered, concentrated under reduced pressure, and the crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Bridge C18 (19 × 250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 40, 8 / 73, 8.05 / 99, 10 / 99, 10.05 / 40, 13 / 40). The pure fraction was concentrated and lyophilized to obtain compound 113. M / Z (ESI): 449.46 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 9.94 (s, 1H), 8.61-8.75 (m, 3H), 8.10 (d, J=3.2 Hz, 1H), 8.03 (dd, J=8.9, 2.3 Hz, 1H), 7.53 (td, J=8.8, 3.2 Hz, 1H), 6.92 (dd, J=9.4, 3.3 Hz, 1H), 6.40 (d, J=8.8 Hz, 1H), 4.84 (br s, 1H), 4.42 (br d, J=13.4 Hz, 1H), 4.10-4.24 (m, 2H), 4.04 (t, J=7.5 Hz, 4H), 3.23-3.30 (m, 1H), 3.13 (dd, J=13.0, 3.9 Hz, 1H), 2.91 (br d, J=3.7 Hz, 1H), 2.30-2.42 (m, 2H), 1.15 (d, J=6.6 Hz, 3H).

[0161] Example 114: (R)-6-(4-(fluoromethyl)-1H-pyrazole-1-yl)-N-(2-(2-(methoxymethyl)-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of O-3: (R)-2-(2-(methoxymethyl)-4-(pyridine-2-yl)piperazine-1-yl)-5-nitropyrimidine To a stirred solution of O-1 (prepared in the same manner as Int 3-3) (300 mg, 1.231 mmol) in DMF (4 mL), K2CO3 (851 mg, 6.15 mmol) and 2-chloro-5-nitropyrimidine (O-2) (236 mg, 1.477 mmol) were added at room temperature. The reaction mixture was stirred at 80°C for 2 hours under a nitrogen atmosphere. The reaction mixture was quenched with H2O (25 mL) and extracted with RINKAN (2 × 50 mL). The combined organic layers were washed with brine (2 × 25 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain O-3. M / Z (ESI): 331.17 [M+H] + .

[0162] Synthesis of O-4: (R)-2-(2-(methoxymethyl)-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-amine To a stirred solution of O-3 (50 mg, 0.151 mmol) in MeOH (5 mL), 10% Pd-C (16.11 mg, 0.015 mmol) was added at room temperature. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 4 hours. The reaction mixture was diluted with HCl (15 mL), filtered through a Celite pad, and washed with HCl (2 × 15 mL). The filtrate was dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain O-4. M / Z (ESI): 301.24 [M+H] + .

[0163] Example 114: (R)-6-(4-(fluoromethyl)-1H-pyrazole-1-yl)-N-(2-(2-(methoxymethyl)-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide To a stirred solution of O-4 (40 mg, 0.133 mmol) in DMF (1 mL), HATU (50.6 mg, 0.133 mmol), O-5 (32.4 mg, 0.146 mmol), and DIPEA (0.070 mL, 0.4 mmol) were added at room temperature. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction mixture was quenched with water (15 mL) and extracted with pharmaceutically acceptable phosphate (2 × 20 mL). The combined organic layers were washed with brine (2 × 10 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, column - X-Select C18 (19×250) mm 5u, flow rate - 18 mL / min gradient, method - 0 / 45, 8 / 81, 8.05 / 99, 10 / 99, 10.05 / 45, 13 / 45). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain compound 114. M / Z (ESI): 504.32 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.46 (s, 1H), 9.05 (s, 1H), 8.90 (d, J = 3.2 Hz, 1H), 8.71 (s, 2H), 8.53 (d, J = 8.4 Hz, 1H), 7.98-8.17 (m, 3H), 7.56 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.65 (t, J = 6.4 Hz, 1H), 5.32-5.55 (m, 2H), 4.88 (br s, 1H), 4.48 (d, J = 13.6 Hz, 1H), 4.37 (d, J = 13.2 Hz, 1H), 4.26 (d, J = 12.4 Hz, 1H), 3.49 (t, J = 9.0 Hz, 1H), 3.38-3.45 (m, 1H), 3.23 (s, 4H), 3.14 (dd, J = 13.2 Hz, 3.6 Hz, 1H), 2.89-3.04 (m, 1H).

[0164] Example 115: (S)-N-(5-(4-(6-fluoropyrimidine-4-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] Synthesis of P-3: tert-butyl (S)-4-(6-fluoropyrimidine-4-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (P-1) (5 g, 24.96 mmol) in DMF (50 mL), DIPEA (13.08 mL, 74.9 mmol) and 4,6-difluoropyrimidine (P-2) (3.48 g, 30.0 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was quenched with ice-cold water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was washed with brine (100 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using an 80 g silica (230-400 silica) cartridge, and the compound was eluted with 20% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain P-3. M / Z (ESI): 297.16 [M+H] + .

[0165] Synthesis of P-4: (S)-4-fluoro-6-(2-methylpiperazin-1-yl)pyrimidine To a stirred solution of P-3 (2.3 g, 7.76 mmol) in DCM (30 mL), 4 M HCl (9.70 mL, 38.8 mmol) in 1,4-dioxane was added at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated and dried under reduced pressure to obtain P-4. M / Z (ESI): 197.09 [M+H] + .

[0166] Synthesis of P-6: (S)-4-(4-(5-bromopyrazine-2-yl)-2-methylpiperazine-1-yl)-6-fluoropyrimidine To a stirred solution of P-4 (500 mg, 2.149 mmol) in DMSO (8 mL), cesium fluoride (1958 mg, 12.89 mmol) and 2,5-dibromopyrazine (P-5) (613 mg, 2.58 mmol) were added at room temperature. The reaction mixture was stirred at 80°C for 6 hours under an argon atmosphere. The reaction mixture was quenched with ice-cold water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a silica gel cartridge, and the compound was eluted with 25% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain P-6. M / Z (ESI): 353.06 [M+H] + .

[0167] Synthesis of P-7: 6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a solution of 6-bromonicotinamide (3 g, 14.92 mmol) in 1,4-dioxane (40 mL) and H2O (10 mL), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (3.73 g, 17.91 mmol) and Na2CO3 (4.75 g, 44.8 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon for 5 minutes. Then, PdCl2 (dppf) (1.092 g, 1.492 mmol) was added to the reaction mixture at room temperature. The reaction mixture was stirred at 100 °C for 12 hours. The reaction mixture was quenched with water (100 mL) and extracted with ELISA (3 × 50 mL). The combined organic layers were dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified using Biotage with an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 10-15% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to obtain P-7. M / Z (ESI): 203.02 [M+H] + .

[0168] Example 115: (S)-N-(5-(4-(6-fluoropyrimidine-4-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a stirred solution of P-6 (100 mg, 0.283 mmol) in 1,4-dioxane (2 mL), P-7 (69 mg, 0.341 mmol), Cs2CO3 (277 mg, 0.849 mmol), copper(I) iodide (5 mg, 0.026 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (4 mg, 0.028 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon gas for 10 minutes. The reaction mixture was stirred in a microwave at 130°C for 30 minutes. The reaction mixture was quenched with water (30 mL) and extracted with DCM (3 × 20 mL). The combined organic layer was washed with brine (20 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was ground with diethyl ether (2 × 10 mL) and dried under reduced pressure. The obtained compound was purified again by prep-HPLC (conditions: mobile phase - 0.1% FA:MeCN in H2O, column - X-Bridge C18 (19×250) mm, 5 μm, flow rate - 15.0 mL / min, gradient method - 0 / 35, 2 / 35, 6 / 45, 10.5 / 48.7, 10.55 / 100, 12.5 / 100, 12.55 / 35, 16 / 35). The pure compounds were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 115. M / Z (ESI): 475.18 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.84 (s, 1H), 9.09 (dd, J = 2.4 Hz, 0.8 Hz, 1H), 8.88 (d, J = 1.6 Hz, 1H), 8.41 (s, 1H), 8.32-8.39 (m, 2H), 8.23 ​​(d, J = 1.6 Hz, 1H), 8.10 (d, J = 0.4 Hz, 1H), 7.78 (dd, J = 8.4 Hz, 0.8 Hz, 1H), 6.58 (d, J = 1.2 Hz, 1H), 4.69 (br s, 1H), 4.15-4.39 (m, 3H), 3.91 (s, 3H), 3.35-3.48 (m, 2H), 3.08-3.20 (m, 1H), 1.19 (d, J = 6.4 Hz, 3H).

[0169] Example 116: (S)-6-(3-fluoroazetidine-1-yl)-N-(5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] Synthesis of Q-3: tert-butyl (S)-4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (Q-1) (1 g, 4.99 mmol) in toluene (20 mL), 2-chloro-5-fluoropyridine (Q-2) (0.995 mL, 9.99 mmol) and sodium tert-butoxide (1.440 g, 14.98 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon gas for 25 minutes. RuPhos Pd G2 (0.388 g, 0.499 mmol) was then added to the reaction mixture at room temperature. The reaction mixture was stirred at 110°C for 16 hours under a nitrogen atmosphere in a sealed tube. The reaction mixture was quenched with water (50 mL) and extracted with ELISA (2 × 85 mL). The combined organic layers were washed with brine (2 × 40 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified using Biotage with an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 15% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain Q-3. M / Z (ESI): 296.11 [M+H] + .

[0170] Synthesis of Q-4: (S)-1-(5-fluoropyridine-2-yl)-2-methylpiperazine To a stirred solution of Q-3 (100 mg, 0.339 mmol) in DCM (2 mL), 4 M HCl (0.423 mL, 1.693 mmol) in 1,4-dioxane was added at 0°C. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was ground with 20% ethyl ether in diethyl ether and dried under reduced pressure to obtain Q-4. M / Z (ESI): 196.12 [M+H] + .

[0171] Synthesis of Q-6: (S)-2-bromo-5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine To a stirred solution of Q-4 (80 mg, 0.345 mmol) in DMSO (2 mL), CsF (157 mg, 1.036 mmol) and 2,5-dibromopyrazine (Q-5) (164 mg, 0.691 mmol) were added at room temperature. The reaction mixture was stirred at 100°C for 16 hours under a nitrogen atmosphere in a sealed tube. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 35 mL). The combined organic layers were washed with brine (2 × 20 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 25% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain Q-6. M / Z (ESI): 352.97 [M+H] + .

[0172] Example 116: (S)-6-(3-fluoroazetidine-1-yl)-N-(5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide To a stirred solution of Q-6 (60 mg, 0.170 mmol) in 1,4-dioxane (1.5 mL), Cs2CO3 (167 mg, 0.511 mmol), copper(I) iodide (3.24 mg, 0.017 mmol), trans-N,N'-dimethylcyclohexane-1,2-diamine (1.212 mg, 8.52 μmol), and (Q-7) (prepared in the same manner as X-5) (61.4 mg, 0.170 mmol) were added at room temperature. The reaction mixture was stirred under a nitrogen atmosphere in a microwave at 150 °C for 2 hours. The reaction mixture was quenched with water (50 mL) and extracted with SiO2 (2 × 75 mL). The combined organic layers were washed with brine (2 × 40 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using a 40 g silica (230-400 mesh) cartridge, and the compound was eluted with 3% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure. The resulting compound was further purified by achiral prep-purification [Cellulose SC (250 × 30 × 5 μm), MeCN:MeOH (90:10)]. The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 116. M / Z (ESI): 467.28 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.83 (d, J = 1.6 Hz, 1H), 8.76 - 8.82 (m, 1H), 8.10 - 8.26 (m, 3H), 7.49 - 7.59 (m, 1H), 6.87 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 5.40 - 5.72 (m, 1H), 4.50 - 4.63 (m, 1H), 4.32 - 4.49 (m, 2H), 3.98 - 4.32 (m, 5H), 3.16 - 3.30 (m, 2H), 3.00 - 3.12 (m, 1H), 1.09 (d, J = 6.4 Hz, 3H).

[0173] Example 117: (R)-N-(5-(4-(5-fluoropyridine-2-yl)-3-(methoxymethyl)piperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0174] Synthesis of R-4: (R)-1-(5-fluoropyridine-2-yl)-2-(methoxymethyl)piperazine 10% Pd / C (237 mg, 0.223 mmol) was added to a stirred solution of R-3 (800 mg, 2.226 mmol) in MeOH (10 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 16 hours. The reaction mixture was filtered through a Celite pad, the filtrate was concentrated, and dried under reduced pressure to obtain R-4. M / Z (ESI): 226.00 [M+H] +.

[0175] Synthesis of R-6: (R)-2-bromo-5-(4-(5-fluoropyridine-2-yl)-3-(methoxymethyl)piperazine-1-yl)pyrazine To a stirred solution of R-4 (600 mg, 2.66 mmol) in DMSO (10 mL), CsF (1214 mg, 7.99 mmol) and 2,5-dibromopyrazine (R-5) (1267 mg, 5.33 mmol) were added at room temperature. The reaction mixture was stirred at 100°C for 16 hours under a nitrogen atmosphere in a sealed tube. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 30 mL). The combined organic layer was washed with brine (10 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography, and the compound was eluted with 20% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain R-6. M / Z (ESI): 382.17 [M+H] +

[0176] Example 117: (R)-N-(5-(4-(5-fluoropyridine-2-yl)-3-(methoxymethyl)piperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a stirred solution of R-6 (70 mg, 0.183 mmol) and P-7 (37.0 mg, 0.183 mmol) in 1,4-dioxane (1 mL), Cs2CO3 (179 mg, 0.549 mmol), copper(I) iodide (3.49 mg, 0.018 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (1.302 mg, 9.16 μmol) were added at room temperature. The reaction mixture was degassed and purged with argon gas for 10 minutes. The reaction mixture was stirred in a microwave at 150°C for 2 hours. The reaction mixture was quenched with water (10 mL) and extracted with ethyl acetate (2 × 20 mL). The combined organic layer was washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN; column - X-Bridge, C18 (25×250) mm, 5 μm; flow rate - 15.0 mL / min; gradient method 0 / 35, 1 / 35, 10.3 / 70, 10.35 / 98, 13.5 / 98, 13.55 / 35, 16 / 35). The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 117. M / Z (ESI): 504.15 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.84 (s, 1H), 9.09 (dd, J = 2.4 Hz, 0.8 Hz, 1H), 8.88 (d, J = 1.6 Hz, 1H), 8.41 (s, 1H), 8.34 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 8.08 - 8.15 (m, 2H), 7.78 (dd, J = 8.4 Hz, 0.8 Hz, 1H), 7.49 - 7.59 (m, 1H), 6.88 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 4.54 - 4.63 (m, 1H), 4.38 (d, J = 13.2 Hz, 1H), 4.23 (d, J = 12.8 Hz, 1H), 3.99 - 4.13 (m, 1H), 3.91 (s, 3H), 3.46 (t, J = 9.2 Hz, 1H), 3.33 - 3.39 (m, 1H), 3.20 - 3.29 (m, 5H), 3.11 - 3.20 (m, 1H).

[0177] Example 118: (S)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0178] Synthesis of S-3: (S)-1-(5-fluoropyridine-2-yl)-3-methylpiperazine To a stirred solution of S-2 (1.5 g, 5.08 mmol) in DCM (20 mL), 4 M HCl (5.08 mL, 20.31 mmol) in 1,4-dioxane was added at 0°C. The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain S-3. M / Z (ESI): 196.01 [M+H] + .

[0179] Synthesis of S-4: (S)-2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)-5-iodopyrimidine To a stirred solution of S-3 (1.2 g, 5.18 mmol) in DMF (20 mL), 2-chloro-5-iodopyrimidine (1.494 g, 6.21 mmol) and DIPEA (2.71 mL, 15.54 mmol) were added under argon at room temperature. The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed with brine (10 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by silica column chromatography, and the compound was eluted with 10% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain S-4. M / Z (ESI): 400.19 [M+H] + .

[0180] Example 118: (S)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a stirred solution of S-4 (50 mg, 0.125 mmol) and P-7 (25.3 mg, 0.125 mmol) in 1,4-dioxane (1 mL), Cs2CO3 (122 mg, 0.376 mmol), copper(I) iodide (2.385 mg, 0.013 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (0.891 mg, 6.26 μmol) were added at room temperature, and the mixture was degassed with argon for 10 minutes. The reaction mixture was stirred at 150°C for 2 hours under microwave irradiation. The reaction mixture was quenched with water (10 mL) and extracted with ethyl acetate (2 × 20 mL). The combined organic layers were washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN; column - X-Bridge, C18 (19×250) mm, 5 μm; flow rate - 12.0 mL / min; gradient method: 0 / 50, 2 / 50, 8.66 / 60, 8.7 / 100, 12 / 100, 12.05 / 50, 16 / 50). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain compound 118. M / Z (ESI): 474.21 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ = 10.31 (s, 1H), 9.05 (d, J = 2.0 Hz, 1H), 8.70 (s, 2H), 8.41 (s, 1H), 8.28 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 8.07-8.15 (m, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.49-7.58 (m, 1H), 6.92 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 4.80-4.90 (m, 1H), 4.10-4.50 (m, 3H), 3.91 (s, 3H), 3.25-3.30 (m, 1H), 3.15 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 2.92 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.16 (d, J = 6.8 Hz, 3H).

[0181] Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] To a solution of T-1 (prepared in the same manner as R-6) (100 mg, 0.225 mmol) in 1,4-dioxane (2 mL), P-7 (54.6 mg, 0.270 mmol), Cs2CO3 (220 mg, 0.675 mmol), copper(I) iodide (4.29 mg, 0.023 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (3.20 mg, 0.023 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon for 10 minutes. The reaction mixture was then stirred in a microwave at 150°C for 2 hours.

[0182] The reaction mixture was quenched with water (20 mL) and extracted with DCM (3 × 20 mL). The combined organic layer was washed with brine (20 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was ground with diethyl ether (2 × 5 mL) and concentrated under reduced pressure. The resulting compound was purified by prep-HPLC (conditions: instrument ID ANL-MCL5-PREP-020, column name Betasil phenylhexyl (21.2 × 250) MM, 5 μm, column No. #250 × 19, mobile phase A 10 mM ammonium bicarbonate in water, mobile phase B acetonitrile, gradient program (T / %B) 0 / 35, 2 / 35, 10 / 55, 11.63 / 55, 11.65 / 100, 15 / 100, 15.01 / 35, 18 / 35). The pure fractions were combined and concentrated under reduced pressure to obtain 119. M / Z (ESI): 519.31 [M+H] + .

[0183] Example 120: (R)-N-(2-(4-(6-fluoropyrimidine-4-yl)-3-(methoxymethyl)piperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] To a solution of P-7 (100 mg, 0.232 mmol) in 1,4-dioxane (2 mL), U-1 (prepared in the same manner as Q-6) (56.4 mg, 0.279 mmol), Cs2CO3 (227 mg, 0.697 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (3.31 mg, 0.023 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon for 10 minutes. The reaction mixture was stirred in a microwave at 150 °C for 2 hours. The reaction mixture was quenched with water (30 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (20 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was ground with diethyl ether (2 × 10 mL) and concentrated under reduced pressure. The obtained compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN column - X-Bridge, C18 (19×250) mm, 5 μm flow rate - 14.0 mL / min gradient method: -0 / 35, 2 / 35, 8.60 / 45, 8.65 / 100, 11.65 / 100, 11.70 / 35, 15.0 / 35). The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 120. M / Z (ESI): 505.25 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.33 (s, 1H), 9.05 (d, J = 2.0 Hz, 1H), 8.71 (s, 2H), 8.41 (s, 1H), 8.35 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 8.2 Hz, 2.2 Hz, 1H), 8.10 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 6.58 (s, 1H), 4.57 - 4.96 (m, 2H), 4.11 - 4.56 (m, 2H), 3.91 (s, 3H), 3.43 - 3.50 (m, 2H), 3.11 - 3.29 (m, 6H).

[0184] Example 121: (R)-N-(4-fluoro-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] Synthesis of V-2: (R)-2-(2-(methoxymethyl)piperazin-1-yl)pyrimidine To a stirred solution of V-1 (prepared in the same manner as 3-2) (2 g, 6.49 mmol) in DCM (40 mL), HCl in 1,4-dioxane (3.08 mL, 25.9 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated and dried under reduced pressure to obtain V-2. M / Z (ESI): 209.18 [M+H] + .

[0185] Synthesis of V-3: (R)-4-chloro-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)-5-(trimethylsilyl)pyrimidine To a stirred solution of V-2 (1.5 g, 6.13 mmol) in DMF (30 mL), 2,4-dichloro-5-(trimethylsilyl)pyrimidine (1.627 g, 7.36 mmol) and DIPEA (3.21 mL, 18.39 mmol) were added at room temperature. The reaction mixture was stirred at 80°C for 3 hours. The reaction mixture was quenched with water (100 mL) and extracted with siRNA (3 × 100 mL). The combined organic layer was dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified using a 100 g silica gel (60-120 mesh) cartridge, and the compound was eluted with 20% siRNA in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain V-3. M / Z (ESI): 393.21 [M+H] + .

[0186] Synthesis of V-4: (R)-4-chloro-5-iodo-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine To a stirred solution of V-3 (1.2 g, 3.05 mmol) in MeCN (10 mL) and DCM (5 mL), ICl (0.230 mL, 4.58 mmol) was added at -10°C. The reaction mixture was stirred at -10°C for 3 hours. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified using a 100 g silica gel (100-200 mesh) cartridge, and the compound was eluted with 25% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain V-4. M / Z (ESI): 446.92 [M+H] + .

[0187] Synthesis of V-5: (R)-4-fluoro-5-iodo-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine Potassium fluoride (325 mg, 5.60 mmol) was added at room temperature to a stirred solution of V-4 (500 mg, 1.119 mmol) in DMSO (10 mL). The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was dehydrated with anhydrous sodium 2 SO4, filtered, and concentrated under reduced pressure. The crude compound was purified using a 100 g silica gel (100-200 mesh) column, and the compound was eluted with 20% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain V-5. M / Z (ESI): 431.00 [M+H] + .

[0188] Example 121: (R)-N-(4-fluoro-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a stirred solution of V-5 (50 mg, 0.116 mmol) and P-7 (28.2 mg, 0.139 mmol) in 1,4-dioxane (1 mL), Cs2CO3 (114 mg, 0.349 mmol), copper(I) iodide (2.213 mg, 0.012 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (0.827 mg, 5.81 μmol) were added at room temperature. The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 × 40 mL). The combined organic layer was dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, column - X-Bridge C18 (10 × 250 mm), 5 μF flow rate - 7 mL / min, gradient method 0 / 52, 2 / 52, 7.5 / 55.5, 10 / 55.5, 10.05 / 100, 12 / 100, 12.05 / 52, 16 / 52). The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 121. M / Z (ESI): 505.32 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.15 (s, 1H), 9.05 (d, J = 1.6 Hz, 1H), 8.54 (d, J = 13.2 Hz, 1H), 8.38-8.45 (m, 3H), 8.28 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 8.10 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 6.68 (t, J = 4.6 Hz, 1H), 4.87-4.49 (m, 1H), 4.66 (d, J = 13.2 Hz, 1H), 4.49-4.58 (m, 1H), 4.43 (d, J = 11.2 Hz, 1H), 3.91 (s, 3H), 3.39-3.48 (m, 3H), 3.27 (d, J = 2.4 Hz, 1H), 3.24 (s, 3H), 3.15-3.21 (m, 1H).

[0189] Example 122: (S)-N-(5-(4-(5-fluoropyrimidine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)-6-(pyrrolidine-1-yl)nicotinamide [ka]

[0190] Synthesis of X-2: tert-butyl (S)-4-(5-fluoropyrimidine-2-yl)-3-methylpiperazine-1-carboxylate A stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (X-1) (1 g, 4.99 mmol) in toluene (20 mL) was purged with argon gas for 10 minutes. Then, 2-chloro-5-fluoropyrimidine (0.993 g, 7.49 mmol)), sodium tert-butoxide (1.440 g, 14.98 mmol) and chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (0.388 g, 0.499 mmol) were added to the reaction mixture at room temperature, and the mixture was again purged with argon for another 10 minutes. The reaction mixture was stirred at 100 °C for 16 hours. The reaction mixture was quenched with water (100 mL) and extracted with dimethyl (3 × 100 mL). The combined organic layer was dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 25 g silica gel cartridge, and the compound was eluted with 30% dimethyl in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain X-2. M / Z (ESI): 297.27 [M+H] + .

[0191] Synthesis of X-3: (S)-5-fluoro-2-(2-methylpiperazin-1-yl)pyrimidine hydrochloride To a stirred solution of X-2 (1.1 g, 3.71 mmol) in DCM (20 mL), 4 M HCl (3.71 mL, 14.85 mmol) in 1,4-dioxane was added at 0°C. The reaction mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to obtain X-3. M / Z (ESI): 197.02 [M+H] + .

[0192] Synthesis of X-4: (S)-2-(4-(5-bromopyrazine-2-yl)-2-methylpiperazine-1-yl)-5-fluoropyrimidine To a stirred solution of X-3 (800 mg, 3.44 mmol) in DMSO (20 mL), CsF (1567 mg, 10.31 mmol) and 2,5-dibromopyrazine (981 mg, 4.13 mmol) were added at room temperature. The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was quenched with water (120 mL) and extracted with pharmaceutically acceptable phosphate (3 × 200 mL). The combined organic layer was dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 25 g silica gel cartridge, and the compound was eluted with 30% pharmaceutically acceptable phosphate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain X-4. M / Z (ESI): 353.09 [M+H] + .

[0193] Synthesis of X-5: 6-(pyrrolidine-1-yl)nicotinamide To a stirred solution of 6-chloronicotinamide (1 g, 6.39 mmol) in DMF (30 mL), pyrrolidine (0.681 g, 9.58 mmol) and K2CO3 (2.65 g, 19.16 mmol) were added at room temperature. The reaction mixture was stirred at 80°C for 16 hours. The reaction mixture was quenched with water (80 mL) and extracted with toluene (3 × 150 mL). The combined organic layers were dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was washed with diethyl ether (2 × 30 mL) and concentrated under reduced pressure to obtain X-5. M / Z (ESI): 192.00 [M+H] + .

[0194] Example 122: (S)-N-(5-(4-(5-fluoropyrimidine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)-6-(pyrrolidine-1-yl)nicotinamide To a stirred solution of X-4 (60 mg, 0.170 mmol) and X-5 (40 mg, 0.204 mmol) in 1,4-dioxane (1 mL), Cs2CO3 (166 mg, 0.510 mmol), copper(I) iodide (3.24 mg, 0.017 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (2.416 mg, 0.017 mmol) were added at room temperature. The reaction mixture was stirred at 100 °C for 16 hours. The reaction mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN; column - X-Bridge C18 (10 × 250 mm), 5 μF; flow rate - 7 mL / min; gradient method - 0 / 52, 2 / 52, 7.5 / 55.5, 10 / 55.5, 10.05 / 100, 12 / 100, 12.05 / 52, 16 / 52). The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain compound 122. M / Z (ESI): 464.28 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ = 10.38 (s, 1H), 8.83 (d, J = 1.2 Hz, 1H), 8.77 (d, J = 2.0 Hz, 1H), 8.50 (d, J = 0.8 Hz, 2H), 8.18 (d, J = 1.6 Hz, 1H), 8.11 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 4.78-4.87 (m, 1H), 4.38-4.48 (m, 1H), 4.17-4.30 (m, 2H), 3.46 (s, 4H), 3.36-3.40 (m, 1H), 3.22 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 3.02 (td, J = 11.8 Hz, 4.0 Hz, 1H), 1.96 (t, J = 6.6 Hz, 4H), 1.16 (d, J = 6.8 Hz, 3H).

[0195] Example 123: (S)-6-(1-(2-fluoroethyl)-1H-pyrazole-4-yl)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] To a stirred solution of S-4 (50 mg, 0.125 mmol) and Y-1 (prepared in the same manner as P-7) (29.3 mg, 0.125 mmol) in 1,4-dioxane (1 mL), Cs2CO3 (122 mg, 0.376 mmol), copper(I) iodide (2.385 mg, 0.013 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (0.891 mg, 6.26 μmol) were added at room temperature, and the mixture was degassed with argon for 10 minutes. The reaction mixture was stirred at 150°C for 2 hours under microwave irradiation. The reaction mixture was quenched with water (10 mL) and extracted with dimethyl ammonium compound (2 × 20 mL). The combined organic layers were washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 2.5 mM ammonium bicarbonate in H2O:MeCN; column - X-Bridge, C18 (19×250) mm, 5 μm; flow rate - 15.0 mL / min; gradient method - 0 / 45, 2 / 45, 10.5 / 62, 10.55 / 100, 13 / 100, 13.05 / 45, 17 / 45 ANL-MCL-PREP-023). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain compound 123. M / Z (ESI): 506.21 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ = 10.33 (s, 1H), 9.07 (d, J = 1.6 Hz, 1H), 8.70 (s, 2H), 8.49 (s, 1H), 8.30 (dd, J = 8.2 Hz, 2.2 Hz, 1H), 8.18 (s, 1H), 8.10 (d, J = 2.8 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.53 (td, J = 8.6 Hz, 2.9 Hz, 1H), 6.92 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 4.40-4.91 (m, 6H), 4.10-4.25 (m, 2H), 3.24-3.30 (m, 1H), 3.15 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 2.93 (td, J = 11.8 Hz, 3.4 Hz, 1H), 1.16 (d, J = 6.8 Hz, 3H).

[0196] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridine-2-yl)piperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] Synthesis of Z-1: (R)-(4-(5-bromopyrazine-2-yl)-1-(5-fluoropyridine-2-yl)piperazine-2-yl)methanol To a stirred solution of R-6 (200 mg, 0.523 mmol) in DCM (2 mL), BBr3 (1.570 mL, 1.570 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, quenched with ice-cold water (10 mL), and extracted with ethyl acetate (2 × 10 mL). The combined organic layers were washed with aqueous saturated NaHCO3 (20 mL) and brine (2 × 20 mL). The combined organic layers were dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 40 g silica (100-200 mesh) cartridge, and the compound was eluted with 10% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain Z-1. M / Z (ESI): 369.12 [M+H] + .

[0197] Synthesis of Z-2: (R)-2-bromo-5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridine-2-yl)piperazine-1-yl)pyrazine To a stirred solution of Z-1 (120 mg, 326 μmol) in DMF (3 mL), NaH (39.1 mg, 1.63 mmol) was added at 0°C for 10 minutes. Then, 1-fluoro-2-iodoethane (567 mg, 3.26 mmol) was added to this reaction mixture at 0°C. The reaction mixture was stirred at 60°C for 16 hours. The reaction mixture was quenched with ice-cold water (25 mL) and extracted with ethyl acetate (2 × 25 mL). The combined organic layer was washed with brine (25 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 40 g silica (100-200 mesh) cartridge, and the compound was eluted with 20% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain Z-2. M / Z (ESI): 415.15 [M+H] + .

[0198] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridine-2-yl)piperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide To a stirred solution of Z-2 (80.0 mg, 193 μmol) and P-7 (43.0 mg, 212 μmol) in 1,4-dioxane (2 mL), cesium carbonate (189 mg, 579 μmol) was added at room temperature. The reaction mixture was degassed and purged with nitrogen gas for 10 minutes. Then, copper(I) iodide (3.68 mg, 19.3 μmol) and trans-(1r,2r)-N,N'-bismethyl-1,2-cyclohexanediamine (305 mL, 0.966 μmol) were added to the reaction mixture. The reaction mixture was stirred in a microwave at 150 °C for 2 hours. The reaction mixture was filtered through a Celite pad and concentrated under reduced pressure. The residue was quenched with water (2 mL) and extracted with 10% MeOH (2 × 2 mL) in DCM. The combined organic layers were washed with brine (2 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H₂O:MeCN column - X-Select, C18 (10 × 150) mm, 5 μm flow rate - 6.0 mL / min gradient method: -0 / 40, 14 / 40, 14.1 / 100, 17.9 / 100, 18 / 40, 22 / 40). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain compound 124. M / Z (ESI): 536.31 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.84 (s, 1H), 9.09 (d, J = 1.6 Hz, 1H), 8.88 (d, J = 1.6 Hz, 1H), 8.41 (s, 1H), 8.34 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 8.07 - 8.21 (m, 3H), 7.78 (d, J = 8.4 Hz, 1H), 7.54 (td, J = 8.8 Hz, 3.2 Hz, 1H), 6.89 (dd, J = 9.2 Hz, 3.2 Hz, 1H), 4.55 - 4.65 (m, 1H), 4.38 - 4.55 (m, 3H), 4.25 (d, J = 12.4 Hz, 1H), 3.99 - 4.10 (m, 1H), 3.91 (s, 3H), 3.45 - 3.71 (m, 5H), 3.24 - 3.28 (m, 1H), 3.12 - 3.20 (m, 1H).

[0199] Example 125: (S)-2-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)pyrimidine-5-carboxamide

Chem.

[0200] Example 126: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] Synthesis of BB-3: tert-butyl (S)-4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (5 g, 25 mmol) in 1,4-dioxane (100 mL), sodium 2-methylpropane-2-oleate (8.03 mL, 74.9 mmol) and 5-bromo-2-fluoropyridine (4.39 g, 25 mmol) were added at room temperature. The reaction mixture was degassed and purged with argon gas for 15 minutes. Then, tris(dibenzylideneacetone)dipalladium (1.14 g, 1.25 mmol) and 4,5-bis(diphenylphosphin)-9,9-dimethylxanthene (1.44 g, 2.5 mmol) were added to the reaction mixture at room temperature. The reaction mixture was stirred at 110 °C for 12 hours under a nitrogen atmosphere in a sealed tube. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2 × 75 mL). The combined organic layers were washed with brine (2 × 30 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 20% siRNA in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain BB-3. M / Z (ESI): 296.28 [M+H] + .

[0201] Synthesis of BB-4: (S)-1-(6-fluoropyridine-3-yl)-3-methylpiperazine To a stirred solution of BB-3 (2.5 g, 8.46 mmol) in DCM (30 mL), 4 M HCl (309 mg, 8.46 mmol) in 1,4-dioxane was added at 0°C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated and dried under reduced pressure to obtain BB-4. M / Z (ESI): 196.24 [M+H] + .

[0202] Synthesis of BB-5: (S)-2-bromo-5-(4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-yl)pyrazine To a stirred solution of BB-4 (500 mg, 2.16 mmol) in DMSO (10 mL), CsF (983 mg, 6.47 mmol) and 2,5-dibromopyrazine (513 mg, 2.16 mmol) were added at room temperature. The reaction mixture was stirred in a sealed tube at 80°C for 16 hours. The reaction mixture was quenched with water (10 mL) and extracted with pharmaceutically acceptable phosphate (2 × 25 mL). The combined organic layer was washed with brine (2 × 20 mL), dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 12 g silica (230-400 mesh) cartridge, and the compound was eluted with 30% pharmaceutically acceptable phosphate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain BB-5. M / Z (ESI): 352.23 [M+H] + .

[0203] Example 126: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide To a stirred solution of BB-5 (150 mg, 426 μmol) and BB-6 (prepared in the same manner as X-5) (90.8 mg, 426 μmol) in 1,4-dioxane (2 mL), CuI (8.11 mg, 42.6 μmol), trans-N,N'-bismethyl-1,2-cyclohexanediamine (6.72 μL, 21.3 μmol), and Cs2CO3 (416 mg, 1.28 mmol) were added at room temperature. The reaction mixture was stirred in a sealed tube at 110 °C for 40 hours. The reaction mixture was quenched with water (5 mL) and extracted with SiO2 (2 × 25 mL). The combined organic layer was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: column: XBridge C18 (4.6 × 150) mm, 3.5 μm; mobile phase-A: 10 mM ammonium bicarbonate in water; mobile phase-B: 100% acetonitrile; gradient (T / %B): 0 / 10, 12 / 98, 16 / 98, 16.1 / 10, 20 / 10; flow rate: 1.0 mL / min; column; oven temperature: ambient temperature; diluent: MeCN:water (90:10) V / V). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain compound 126. M / Z (ESI): 485.15 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.58 (s, 1H), 8.86 (d, J = 1.2 Hz, 1H), 8.80 - 8.85 (m, 1H), 8.22 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.16 (d, J = 1.6 Hz, 1H), 7.87 - 7.94 (m, 1H), 7.63 - 7.72 (m, 1H), 7.06 (dd, J = 8.8 Hz, 3.4 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 4.60 - 4.69 (m, 1H), 4.50 (t, J = 12.4 Hz, 4H), 4.12 (d, J = 10.0 Hz, 1H), 3.73 (d, J = 12.0 Hz, 1H), 3.63 (d, J = 12.0 Hz, 1H), 3.24 - 3.30 (m, 1H), 3.02 (dd, J = 12.4 Hz, 3.6 Hz, 1H), 2.84 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.23 (d, J = 6.4 Hz, 3H).

[0204] Example 127: (S)-N-(2-(4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0205] Example 128: (R)-N-(5-(4-(6-fluoropyridine-3-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0206] Example 129: (R)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0207] Example 130: (S)-6-(3-(fluoromethyl)azetidine-1-yl)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide

Chem.

[0208] Example 130: (S)-6-(3-(fluoromethyl)azetidine-1-yl)-N-(2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide DAST (0.039 mL, 0.293 mmol) was added at 0°C to a stirred solution of FF-2 (70 mg, 0.146 mmol) in DCM (1 mL). The reaction mixture was stirred at 0°C for 30 minutes under an argon atmosphere. The reaction mixture was quenched with water (5 mL) and extracted with Depositphotos (2 × 20 mL). The combined organic layer was washed with brine (5 mL), dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H₂O:MeCN column - X-Bridge, C18 (19 × 250) mm, 5 μm flow rate - 12.0 mL / min gradient method - 0 / 50, 2 / 50, 10.5 / 66.5, 10.6 / 100, 13 / 100, 13.1 / 50, 16 / 50). The pure fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain 130. M / Z (ESI): 481.14 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ = 9.99 (s, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.66 (s, 2H), 8.10 (d, J = 2.8 Hz, 1H), 8.05 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.48-7.57 (m, 1H), 6.92 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.45 (d, J = 8.8 Hz, 1H), 4.80-4.89 (m, 1H), 4.54-4.75 (m, 2H), 4.38-4.48 (m, 1H), 4.09-4.25 (m, 4H), 3.80-3.90 (m, 2H), 3.23-3.30 (m, 1H), 3.04-3.20 (m, 2H), 2.91 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H).

[0209] The compounds included in Table 6 were synthesized using a method similar to the synthesis sequence described above. Commercially available reagents were used as needed to prepare the following examples. [Table 8]

[0210] Example 134: 6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of GG-2: tert-butyl (S)-2-methyl-4-(pyrimidine-2-yl)piperazine-1-carboxylate To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (GG-1, 1.2 g, 1 Eq, 5.99 mmol) in DMF (24 mL), potassium carbonate (2.484 g, 3 Eq, 17.97 mmol) was added, followed by the addition of 2-chloropyrimidine (1.029 g, 1.5 Eq, 8.99 mmol). The mixture was stirred at 22°C for 3 hours. Water (100 mL) was added to the mixture, and it was stirred at 22°C for 30 minutes. The mixture was extracted with siRNA (100 mL x 3). The organic layer was dehydrated with MgSO4, filtered through a frit filter, and concentrated under reduced pressure. The resulting residue was dissolved in DCM (3 mL) and purified by normal-phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexane, siRNA, 26-minute gradient). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain GG-2. MS (ESI) m / z: 279.3 [M+H] +

[0211] Synthesis of GG-3: (S)-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride A solution of tert-butyl (S)-2-methyl-4-(pyrimidine-2-yl)piperazine-1-carboxylate (GG-2, 1.20 g, 1 Eq, 4.31 mmol) in 1,4-dioxane (12.0 mL) was prepared by adding 4 M hydrogen chloride (2.16 mL, 4.00 mol, 2 Eq, 8.62 mmol) in 1,4-dioxane. The mixture was stirred at 22°C for 18 hours. The mixture was concentrated under reduced pressure to obtain GG-3. MS (ESI) m / z: 179.4 [M+H] +

[0212] Synthesis of GG-4: (S)-2-(2-methyl-4-(pyrimidine-2-yl)piperazine-1-yl)-5-nitropyrimidine (S)-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride (GG-3, 1.00 g, 1 Eq, 3.98 mmol) was dissolved in DMF (18.0 mL) and potassium carbonate (2.20 g, 4 Eq, 15.9 mmol) and 2-chloro-5-nitropyrimidine (762 mg, 1.2 Eq, 4.78 mmol) were added. The mixture was stirred at 60°C for 18 hours. Water was added to the mixture and stirred at 22°C for 30 minutes. The resulting precipitated solid was collected by filtration through a frit filter, washed with water (100 mL x 3), and dried to obtain GG-4. MS (ESI) m / z: 302.5 [M+H] +

[0213] Synthesis of GG-5: (S)-2-(2-methyl-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-amine A solution of (S)-2-(2-methyl-4-(pyrimidine-2-yl)piperazin-1-yl)-5-nitropyrimidine (GG-4, 1.00 g, 1 Eq, 3.32 mmol) in THF (8.00 mL) and MeOH (8.00 mL) was degassed, purged with N2 (×3), then carbon-supported 10% palladium (177 mg, 0.5 Eq, 1.66 mmol) was added, degassed, and purged with H2 (×3). The mixture was stirred under an H2-filled balloon at 22°C for 18 hours. The mixture was filtered through a frit filter, washed with MeOH (5 mL × 3), and concentrated under reduced pressure to obtain GG-5. MS (ESI) m / z: 272.4 [M+H] +

[0214] Synthesis of GG-6: (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide To a solution of (S)-2-(2-methyl-4-(pyrimidine-2-yl)piperazin-1-yl)pyrimidine-5-amine (GG-5, 250.0 mg, 1 Eq, 921.4 μmol) in DCM (6.0 mL), diisopropylethylamine (595.4 mg, 793 μL, 5 Eq, 4.607 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (700.7 mg, 2 Eq, 1.843 mmol), and 6-bromo-2-fluoronicotinic acid (243.2 mg, 1.2 Eq, 1.106 mmol) were added. The mixture was stirred at 22°C for 30 minutes. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm - 30×250mm column, 10-100% 5mM NH4HCO3 aqueous solution:acetonitrile, 30 min gradient). The fractions containing the product were combined and extracted between water (100 mL) and DCM (100 mL × 3). The collected organic layer was dehydrated with MgSO4 and then concentrated under reduced pressure to obtain GG-6. MS (ESI) m / z: 473.5 [M+H] +

[0215] Synthesis of Example 134: 6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidine-2-yl)piperazin-1-yl)pyrimidine-5-yl)nicotinamide (GG-6, 60.0 mg, 1 Eq, 127 μmol) was added to a solution of (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidine-2-yl)piperazin-1-yl)pyrimidine-5-yl)nicotinamide (GG-6, 60.0 mg, 1 Eq, 127 μmol) in 1,4-dioxane (0.50 mL) to which 1 M tribasic potassium phosphate (31.5 μL, 3 Eq, 380 μmol) and 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (44.5 mg, 1.5 Eq, 190 μmol) were added. The mixture was purged with N2 for 5 minutes, and then XPhos Palladacycle-G2 (9.97 mg, 0.1 Eq, 12.7 μmol) was added. The mixture was stirred at 80°C for 3 hours. The mixture was concentrated and purified using normal-phase chromatography (ISCO 12 g RediSep Gold High Performance Silica, 10-100% hexane, RINKAN, 18 min gradient). The fractions containing the desired product were combined and concentrated under reduced pressure. LC-MS showed the main by-products, so it was redissolved in DCM (1 mL) and placed in a TLC chromatography chamber (1:4 hexane, RINKAN). The desired product was collected from the TLC plate, stirred in RINKAN, and filtered through a frit filter to obtain 134. MS (ESI) m / z: 501.6 [M+H] + 11H NMR (500 MHz, CDCl3): δ 8.60 (s, 2H), 8.58 - 8.52 (m, 1H), 8.33 (d, J = 4.7 Hz, 2H), 8.22 (d, J = 15.6 Hz, 1H), 8.07 (s, 1H), 7.42 (d, J = 7.9 Hz, 1H), 6.51 (t, J = 4.7 Hz, 1H), 4.98 (s, 1H), 4.69 (d, J = 12.7 Hz, 1H), 4.63 (d, J = 13.2 Hz, 1H), 4.55 (d, J = 13.3 Hz, 1H), 4.22 (t, J = 7.3 Hz, 2H), 3.42 - 3.28 (m, 2H), 3.24 (t, J = 7.3 Hz, 2H), 3.16 (td, J = 12.2, 3.5 Hz, 1H), 2.74 (p, J = 7.3 Hz, 2H), 1.23 (d, J = 6.8 Hz, 3H).

[0216] Example 135: 6-(4-aminophenyl)-2-fluoro-N-(2-(4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide

Chem.

[0217] Synthesis of intermediate HH-1: 6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoronicotinic acid [ka] Synthesis of HH-1: 6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoronicotinic acid A mixture of 6-bromo-2-fluoronicotinic acid (1.90 g, 8.64 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (3.03 g, 12.9 mmol), and 1 M aqueous K3PO4 (25.9 mL, 25.9 mmol) in dioxane (57 mL) was purged with nitrogen for 10 minutes. XPhos-Pd-G2 (680 mg, 864 μmol) was added, and the mixture was stirred at 100°C for 2.5 hours. The mixture was cooled, poured into water (100 mL), and extracted with ELISA (3 × 30 mL) and DCM (3 × 20 mL). The aqueous layer was acidified with 1M HCl, the resulting precipitated solid was collected by filtration, washed with water (10 mL x 2), and dried to obtain HH-1. MS (ESI) m / z: 248.2 [M+H] +

[0218] Synthesis of Example 136: (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(5-fluoropyrazine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of HH-2: tert-butyl (S)-4-(5-aminopyrimidine-2-yl)-3-methylpiperazine-1-carboxylate A solution of 1-2 (8.88 g, 27.5 mmol) in methanol (137 mL) was mixed with 10% Pd / C (2.00 g, 1.88 mmol). The mixture was stirred under a hydrogen balloon at 25°C for 16 hours. Ice water (300 mL) was added, and the mixture was stirred for 30 minutes. The precipitated solid was collected by filtration, washed with water (20 mL x 2), and dried to obtain HH-2. MS (ESI) m / z: 294.3 [M+H] + .

[0219] Synthesis of HH-3: tert-butyl (S)-4-(5-(6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoronicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate A solution of HH-2 (2.14 g, 7.30 mmol), HH-1 (1.80 g, 7.30 mmol), HATU (3.33 g, 8.75 mmol), and DIPEA (6.35 mL, 36.5 mmol) in DCM (73 mL) was stirred at 25°C for 2 hours. The reaction product was concentrated and purified by normal-phase column chromatography [0 → 100% Â / EtOH (3:1 mixture) in hexane] to obtain HH-3. MS (ESI) m / z: 523.5 [M+H] + .

[0220] Synthesis of HH-4: (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide To a stirred solution of compound HH-3 (822 mg, 1.57 mmol) in DCM (3.2 mL), 4 M HCl (3.93 mL, 15.7 mmol) in dioxane was added at 25°C. The mixture was stirred at room temperature for 2 hours, then poured into saturated sodium bicarbonate aqueous solution (20 mL) and extracted with DCM (3 × 10 mL). The combined organic layer was dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure to obtain HH-4. M / Z (ESI): 423.4 [M+H] +

[0221] Synthesis of 136: (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(5-fluoropyrazine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide To a solution of HH-4 (10.0 mg, 0.024 mmol) in DMF (0.4 mL), 2,5-difluoropyrazine (4.1 mg, 0.036 mmol) and DIPEA (1.0 μL, 0.12 mmol) were added. The mixture was stirred at 80°C for 16 hours. The reaction mixture was purified by reverse-phase HPLC (40 → 100% MeCN / H2O w / 0.1% NH4OH gradient on an XBridge Prep OBD C18 column). The desired fraction was concentrated to obtain compound 136. MS (ESI) m / z: 519.2 [M+H] + .

[0222] Example 137: (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of 137: (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide To a solution of HH-4 (20.0 mg, 0.0473 mmol) in DMF (0.473 mL), 4,6-difluoropyrimidine (8.24 g, 0.0710 mmol) and DIPEA (0.0412 mL, 0.237 mmol) were added. The mixture was stirred at 22°C for 2 hours. The reaction mixture was purified by reverse-phase HPLC (on a Gemini-NX column with a 40 → 100% MeCN / H2O w / 0.1% NH4OH gradient). The desired fraction was concentrated to obtain compound 137. MS (ESI) m / z: 519.4 [M+H] + . 1 H NMR (500 MHz, CDCl3) δ 8.62 (s, 2H), 8.55 (dd, J = 10.3, 8.0 Hz, 1H), 8.35 (d, J = 2.5 Hz, 1H), 8.23 ​​(d, J = 15.7 Hz, 1H), 8.07 (s, 1H), 7.42 (dd, J = 8.0, 2.7 Hz, 1H), 6.02 (s, 1H), 4.99 - 4.94 (m, 1H), 4.56 (dt, J = 13.7, 3.6 Hz, 1H), 4.33 - 4.06 (m, 4H), 3.55 - 3.42 (m, 2H), 3.30 - 3.21 (m, 3H), 2.74 (p, J = 7.4 Hz, 2H), 1.23 (d, J = 6.6 Hz, 3H).

[0223] Example 138: (R)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Synthesis of II-2: tert-butyl (R)-3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-carboxylate To a solution of tert-butyl (R)-3-(methoxymethyl)piperazine-1-carboxylate (II-1, 1.0 g, 1 Eq, 4.34 mmol) in DMF (15 mL), diisopropylethylamine (1.68 g, 2.24 mL, 3 Eq, 13.0 mmol) was added, followed by the addition of 2-chloropyrimidine (746 mg, 1.5 Eq, 6.51 mmol). The mixture was stirred at 130°C for 18 hours. Water (50 mL) was added to the mixture, and it was stirred at 22°C for 30 minutes, after which it was extracted with siRNA (60 mL x 3). The organic layer was dehydrated with MgSO4, filtered through a frit filter, and concentrated under reduced pressure. The resulting residue was dissolved in DCM (3 mL) and purified by normal-phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexane, siRNA, 28-minute gradient). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain II-2. MS (ESI) m / z: 309.5 [M+H] +

[0224] Synthesis of II-3: (R)-2-(2-(methoxymethyl)piperazin-1-yl)pyrimidine hydrochloride A solution of tert-butyl (R)-3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-carboxylate (II-2, 870 mg, 1 Eq, 2.82 mmol) in 1,4-dioxane (12.0 mL) was mixed with 4 M hydrogen chloride (1.41 mL, 2 Eq, 5.64 mmol) in 1,4-dioxane. The mixture was stirred at 22°C for 2 hours, and an additional 4 M hydrogen chloride (4 mL) in 1,4-dioxane was added to the mixture and stirred for 1 hour. The mixture was concentrated under reduced pressure to obtain II-3. MS (ESI) m / z: 209.4 [M+H] +

[0225] Synthesis of II-4: (R)-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)-5-nitropyrimidine (R)-2-(2-(methoxymethyl)piperazin-1-yl)pyrimidine hydrochloride (II-3, 790 mg, 1 Eq, 2.81 mmol) was dissolved in DMF (15.0 mL) and potassium carbonate (1.55 g, 4 Eq, 11.2 mmol) and 2-chloro-5-nitropyrimidine (538 mg, 1.2 Eq, 3.37 mmol) were added. The mixture was stirred at 60°C for 18 hours. Water was added to the mixture and stirred at 22°C for 30 minutes. The resulting precipitated solid was collected by filtration through a frit filter, washed with water (100 mL x 3), and dried to obtain II-4. MS (ESI) m / z: 332.4 [M+H] +

[0226] Synthesis of II-5: (R)-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-amine A solution of (R)-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazin-1-yl)-5-nitropyrimidine (II-4, 820 mg, 1 Eq, 2.47 mmol) in THF (8.00 mL) and MeOH (8.00 mL) was degassed, purged with N2 (×3), then carbon-supported 10% palladium (132 mg, 0.5 Eq, 1.24 mmol) was added, degassed, and purged with H2 (×3). The mixture was stirred under an H2-filled balloon at 22°C for 18 hours. The mixture was filtered through a frit filter, washed with MeOH (5 mL × 3), and concentrated under reduced pressure to obtain II-5. MS (ESI) m / z: 302.5 [M+H] +

[0227] Synthesis of II-6: (R)-6-bromo-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide To a solution of (R)-2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazin-1-yl)pyrimidine-5-amine (II-5, 100.0 mg, 1 Eq, 331.8 μmol) in DCM (2.0 mL), diisopropylethylamine (286 μL, 5 Eq, 1.659 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (252.4 mg, 2 Eq, 663.7 μmol), and 6-bromo-2-fluoronicotinic acid (87.61 mg, 1.2 Eq, 398.2 μmol) were added. The mixture was stirred at 22°C for 1 hour. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm-30×250mm column, 10-100% 5mM NH4HCO3 aqueous solution:acetonitrile, 26 min gradient). The fractions containing the product were combined and extracted between water (80 mL) and DCM (80 mL × 3). The collected organic layer was dehydrated with MgSO4 and then concentrated under reduced pressure to obtain II-6. MS (ESI) m / z: 503.5 [M+H] +

[0228] Example 138: (R)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide (R)-6-bromo-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidine-2-yl)piperazin-1-yl)pyrimidine-5-yl)nicotinamide (II-6, 20.0 mg, 1 Eq, 39.7 μmol) was dissolved in 1,4-dioxane (0.50 mL) to which 1 M tribasic potassium phosphate (9.87 μL, 3 Eq, 119 μmol) and 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (14.0 mg, 1.5 Eq, 59.6 μmol) were added. The mixture was purged with N2 for 5 minutes, and then XPhos Palladacycle-G2 (3.13 mg, 0.1 Eq, 3.97 μmol) was added. The mixture was stirred at 80°C for 2 hours. The mixture was filtered through a frit filter, concentrated, and purified using reverse-phase HPLC (XBridge Prep OBD C18 column, 40-100% water (containing 0.1% NH4OH), acetonitrile). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain 138. MS (ESI) m / z: 531.6 [M+H] + 1H NMR (500 MHz, CDCl3): δ 8.61 (s, 2H), 8.58 - 8.52 (m, 1H), 8.35 (d, J = 4.7 Hz, 2H), 8.21 (d, J = 15.5 Hz, 1H), 8.07 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H), 6.53 (t, J = 4.7 Hz, 1H), 5.06 (s, 1H), 4.88 (d, J = 13.4 Hz, 1H), 4.62 (t, J = 13.1 Hz, 2H), 4.22 (t, J = 7.2 Hz, 2H), 3.51 (dd, J = 15.6, 6.5 Hz, 3H), 3.33 (s, 3H), 3.29 (dd, J = 13.6, 3.9 Hz, 1H), 3.26 - 3.16 (m, 3H), 2.78 - 2.69 (m, 2H).

[0229] Example 139: (S)-6-(3-fluoroazetidine-1-yl)-N-(6-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyridine-3-yl)nicotinamide [ka] Synthesis of JJ-2: (S)-6-bromo-N-(6-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyridine-3-yl)nicotinamide To a stirred solution of JJ-1 (500 mg, 1.734 mmol) in DMF (20 mL), 6-bromonicotinic acid (525 mg, 2.60 mmol), HATU (1319 mg, 3.47 mmol), and DIPEA (0.909 mL, 5.20 mmol) were added at room temperature, and the mixture was stirred at 60°C for 16 hours. Next, the reaction mixture was cooled, diluted with ethyl acetate (20 mL), washed with cold brine, dehydrated with Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified using prep-HPLC (Prep. HPLC conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Select C18 (19 × 250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 20, 9 / 78, 9.05 / 99, 11 / 99, 11.02 / 20, 14 / 20). The obtained compound was further purified by SFC (Conditions: column - Chiralpak IG (4.6 × 250 mm), 5 μ, mobile phase A - MeOH / DCM / DEA (50 / 50 / 0.2), fixed composition A - 100%, flow rate - 1.0 mL / min, diluent - EtOH). The pure fraction was concentrated and freeze-dried to obtain JJ-2. M / Z (ESI): 472.14 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 10.37 (s, 1H), 8.91 (d, J=2.0 Hz, 1H), 8.46 (d, J=2.7 Hz, 1H), 8.32 (d, J=2.4 Hz, 1H), 8.23 (dd, J=8.3, 2.4 Hz, 1H), 7.91 (dd, J=9.3, 2.7 Hz, 1H), 7.85 (d, J=8.3 Hz, 1H), 6.87 (d, J=9.0 Hz, 1H), 6.61 (s, 1H), 4.54-4.60 (m, 1H), 4.26-4.43 (m, 2H), 4.06 (br dd, J=9.5, 3.2 Hz, 1H), 3.41 (br dd, J=13.4, 3.4 Hz, 1H), 3.15-3.25 (m, 2H), 1.03 (d, J=6.4 Hz, 3H).

[0230] Example 139: (S)-6-(3-fluoroazetidine-1-yl)-N-(6-(4-(6-fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyridine-3-yl)nicotinamide To a stirred solution of JJ-2 (90 mg, 0.210 mmol) in toluene (3 mL), 3-fluoroazetidine hydrochloride (25.8 mg, 0.231 mmol), Cs2CO3 (93 mg, 0.284 mmol), and 4,5-bis(diphenylphosphin)-9,9-dimethylxanthene (12.17 mg, 0.021 mmol) were added at room temperature, the mixture was purged with argon for 10 minutes, followed by the addition of Pd2(dba)3 (9.63 mg, 10.52 μmol) at room temperature, the mixture was purged again with argon for another 10 minutes, and the mixture was stirred at 110°C for 16 hours. The reaction mixture was then filtered through a Celite bed and washed with phenylethylamine. The filtrate was washed with cold brine, dehydrated with Na2SO4, filtered, concentrated under reduced pressure, and the crude material was purified by Prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Select C18 (19×250) mm 5u, flow rate - 18 mL / min, gradient method - 0 / 60, 8 / 80, 8.02 / 100, 10 / 100, 10.08 / 60, 14 / 60). The obtained compound was further purified by SFC (conditions: column - Chiralpak IE (4.6×250 mm), 5 μ, mobile phase A - MeOH / DCM / DEA (50 / 50 / 0.2), fixed composition A - 100%, flow rate - 0.70 mL / min, diluent - EtOH). The pure fraction was concentrated under reduced pressure and lyophilized to obtain compound 139. M / Z (ESI): 467.18 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 9.95 (s, 1H), 8.70 (br d, J=2.0 Hz, 1H), 8.45 (br d, J=2.4 Hz, 1H), 8.32 (d, J=2.7 Hz, 1H), 8.09 (br dd, J=8.8, 2.2 Hz, 1H), 7.87-7.94 (m, 1H), 6.88 (br d, J=8.1 Hz, 1H), 6.61 (s, 1H), 6.53 (br d, J=8.6 Hz, 1H), 5.42-5.63 (m, 1H), 4.50-4.56 (m, 1H), 4.43 (br d, J=4.4 Hz, 8H), 3.16-3.24 (m, 2H), 2.54 (s, 1H), 1.03 (br d, J=6.4 Hz, 3H).

[0231] Example 140: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(6-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] To a stirred solution of NN-4 (70 mg, 164 μmol) in DMF (1 mL), DIPEA (0.18 mL, 1 mmol) and 2,6-difluoropyridine (23 mg, 200 μmol) were added at room temperature. The reaction mixture was stirred at 100 °C for 16 hours under an argon atmosphere. The reaction mixture was quenched with ice-cold water (5 mL), the precipitated solid was filtered, and dried under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Bridge C18 (19 × 250) mm, 5 μm, flow rate - 15.0 mL / min, gradient method 0 / 55, 2 / 55, 10 / 67, 10.05 / 100, 12 / 100, 12.05 / 55, 15 / 55). The pure fractions were combined and concentrated under reduced pressure to obtain 140. M / Z (ESI): 485.29 [M+H] + 11H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.58 (s, 1H), 8.86 (d, J = 1.2 Hz, 1H), 8.82 (d, J = 2.0 Hz, 1H), 8.22 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.13 (d, J = 1.2 Hz, 1H), 7.69 (q, J = 8.0 Hz, 1H), 6.75 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.27 (dd, J = 7.8 Hz, 2.6 Hz, 1H), 4.57 - 4.65 (m, 1H), 4.50 (t, J = 12.4 Hz, 4H), 4.02 - 4.18 (m, 3H), 3.26 (dd, J = 14.4 Hz, 3.2 Hz, 2H), 3.09 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.11 (d, J = 6.4 Hz, 3H).

[0232] Example 141: (S)-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide

Chem.

[0233] Synthesis of KK-3: (S)-1-(5-fluoropyridine-2-yl)-3-methylpiperazine hydrochloride To a solution of tert-butyl (S)-4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-carboxylate (KK-2, 1.00 g, 1 Eq, 3.39 mmol) in 1,4-dioxane (14.0 mL), 4 M hydrogen chloride (3.39 mL, 4 Eq, 13.5 mmol) in 1,4-dioxane was added. The mixture was stirred at 22°C for 18 hours. The mixture was concentrated under reduced pressure to obtain KK-3. MS (ESI) m / z: 196.3 [M+H] +

[0234] Synthesis of KK-4: (S)-2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)-5-nitropyrazine To a solution of (S)-1-(5-fluoropyridine-2-yl)-3-methylpiperazine hydrochloride (KK-3, 900 mg, 1 Eq, 3.36 mmol) in DMF (15.0 mL), potassium carbonate (1.86 g, 4 Eq, 13.4 mmol) and 2-chloro-5-nitropyrazine (642 mg, 1.2 Eq, 4.03 mmol) were added. The mixture was stirred at 60°C for 18 hours. Water was added to the mixture and stirred at 22°C for 30 minutes. The resulting precipitated solid was collected by filtration through a frit filter, washed with water (100 mL x 3), and dried to obtain KK-4. MS (ESI) m / z: 319.3 [M+H] +

[0235] Synthesis of KK-5: (S)-2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)-5-nitropyrazine A solution of (S)-2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine (KK-4, 579 mg, 1 Eq, 1.82 mmol) in THF (5.00 mL) and MeOH (5.00 mL) was degassed, purged with N2 (×3), then carbon-supported 10% palladium (96.8 mg, 0.5 Eq, 909 μmol) was added, degassed, and purged with H2 (×3). The mixture was stirred under an H2-filled balloon at 22°C for 18 hours. The mixture was filtered through a frit filter, washed with MeOH (5 mL × 3), and concentrated under reduced pressure to obtain KK-5. MS (ESI) m / z: 289.3 [M+H] +

[0236] Synthesis of KK-6: (S)-6-chloro-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide To a solution of (S)-2-(4-(5-fluoropyridine-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine (KK-5, 520.00 mg, 1 Eq, 1.8035 mmol) in DMF (8.0 mL), diisopropylethylamine (1.165 g, 1.57 mL, 5 Eq, 9.0174 mmol), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosfinan 2,4,6-trioxide (1.1477 g, 1.22 mL, 50% by weight, 1 Eq, 1.8035 mmol) and 6-chloronicotinic acid (340.97 mg, 1.2 Eq, 2.1642 mmol) were added. The mixture was stirred at 22°C for 1.5 hours. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm-50×250mm column, 10-100% 5mM NH4HCO3 aqueous solution:acetonitrile, 33 min gradient). The fractions containing the product were combined and extracted between water (100 mL) and DCM (100 mL × 3). The collected organic layer was dehydrated with MgSO4 and then concentrated under reduced pressure to obtain KK-6. MS (ESI) m / z: 428.4 [M+H] +

[0237] Example 141: (S)-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide (S)-6-chloro-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide (KK-6, 20.00 mg, 1 Eq, 46.74 μmol) was dissolved in 1,4-dioxane (0.30 mL) to which 1 M tribasic potassium phosphate (140.2 μL, 1.00 mol, 3 Eq, 140.2 μmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (11.67 mg, 1.2 Eq, 56.09 μmol) were added. The mixture was purged with N2 for 5 minutes, and then XPhos Palladacycle-G2 (3.678 mg, 0.1 Eq, 4.674 μmol) was added. The mixture was stirred at 80°C for 2 hours. After the reaction time, LC-MS showed a high conversion rate of the desired product. Next, the mixture was filtered through a frit filter, concentrated, and purified using reverse-phase HPLC (XBridge Prep OBD C18 column, 40-100% water (containing 0.1% NH4OH), acetonitrile). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain 141. MS (ESI) m / z: 474.5 [M+H] + 1 H NMR (500 MHz, DMSO): δ 10.84 (s, 1H), 9.09 (s, 1H), 8.89 (s, 1H), 8.41 (s, 1H), 8.34 (dd, J = 8.3, 2.1 Hz, 1H), 8.16 (s, 1H), 8.12 (d, J = 3.0 Hz, 1H), 8.10 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.54 (td, J = 8.7, 3.1 Hz, 1H), 6.95 (dd, J = 9.3, 3.3 Hz, 1H), 4.62 (s, 1H), 4.25 - 4.09 (m, 3H), 3.91 (s, 3H), 3.30 - 3.23 (m, 1H), 3.20 (dd, J = 12.8, 3.5 Hz, 1H), 2.99 (td, J = 12.0, 3.7 Hz, 1H), 1.14 (d, J = 6.5 Hz, 3H).

[0238] Example 142: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] (S)-6-chloro-N-(5-(4-(5-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide (KK-6, 20.00 mg, 1 Eq, 46.74 μmol) was dissolved in DMF (0.50 mL) and potassium carbonate (19.38 mg, 3 Eq, 140.2 μmol), potassium fluoride (8.147 mg, 3.284 μL, 3 Eq, 140.2 μmol), and 3,3-difluoroazetidine (6.526 mg, 1.5 Eq, 70.11 μmol) were added. The mixture was concentrated under reduced pressure, redissolved in DCM (1 mL), and then purified by normal-phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexane, HCl, 26-minute gradient). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain 142. MS (ESI) m / z: 485.4 [M+H] + 1 H NMR (500 MHz, CDCl3): δ 9.17 (s, 1H), 8.73 (d, J = 2.1 Hz, 1H), 8.07 (d, J = 5.7 Hz, 3H), 7.82 (s, 1H), 7.32 - 7.28 (m, 1H), 6.65 (dd, J = 9.2, 3.2 Hz, 1H), 6.42 (d, J = 8.7 Hz, 1H), 4.55 (s, 1H), 4.46 (t, J = 11.8 Hz, 4H), 4.15 (d, J = 12.1 Hz, 1H), 4.06 (dd, J = 17.3, 13.6 Hz, 2H), 3.40 (td, J = 12.1, 3.6 Hz, 1H), 3.34 (dd, J = 12.7, 3.7 Hz, 1H), 3.13 (qd, J = 12.2, 4.4 Hz, 1H), 1.26 (d, J = 6.5 Hz, 3H).

[0239] Example 143: (S)-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide [ka] Synthesis of LL-2: tert-butyl (S)-4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-carboxylate To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (LL-1, 5.43 g, 1.00 Eq, 27.1 mmol) in DMF (67.8 mL), diisopropylethylamine (17.5 g, 23.6 mL, 5.00 Eq, 136 mmol) and 2-chloro-5-fluoropyrimidine (5.39 g, 3.75 mL, 1.50 Eq, 40.7 mmol) were added. The mixture was stirred at 80°C for 16 hours. Water (300 mL) was added to the mixture, and it was extracted with ether (100 mL x 3). The organic layer was dehydrated with Na2SO4, filtered through a frit filter, and concentrated under reduced pressure. The resulting residue was purified by normal-phase chromatography (ISCO 330 g RediSep Gold High Performance Silica, 0-25% hexane, siRNA). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain LL-2. MS (ESI) m / z: 297.1 [M+H] +

[0240] Synthesis of LL-3: (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride A solution of tert-butyl (S)-4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-carboxylate (LL-2, 7.35 g, 1 Eq, 24.8 mmol) in DCM (124 mL) was mixed with 4 M hydrogen chloride (62.0 mL, 4.00 mol, 10 Eq, 248 mmol) in DCM. The mixture was stirred at 22°C for 2 hours. The mixture was concentrated under reduced pressure to obtain LL-3. MS (ESI) m / z: 320.2 [M+H] +

[0241] Synthesis of LL-4: (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazine-2-yl)piperazine-1-yl)pyrimidine (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride (LL-3, 1.00 g, 1 Eq, 5.10 mmol) was dissolved in DMF (25.5 mL) to which potassium carbonate (2.82 g, 4.00 Eq, 20.4 mmol) and 2-chloro-5-nitropyrazine (976 mg, 1.20 Eq, 6.12 mmol) were added. The mixture was stirred at 60°C for 16 hours. Ice water was added to the mixture, and the resulting precipitated solid was collected through a frit filter, washed with water (5 mL x 3), and dried to obtain LL-4. MS (ESI) m / z: 290.2 [M+H] +

[0242] Synthesis of LL-5: (S)-5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-amine A solution of (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazine-2-yl)piperazin-1-yl)pyrimidine (LL-4, 1.20 g, 1 Eq, 3.76 mmol) in DCM (9.40 mL) and MeOH (9.40 mL) was degassed, purged with N2 (×3), then carbon-supported 10% palladium (200.0 mg, 0.500 Eq, 1.879 mmol) was added, degassed, and purged with H2 (×3). The mixture was stirred under an H2-filled balloon at 22°C for 6 hours. The mixture was filtered through a frit filter and concentrated under reduced pressure to obtain LL-5. MS (ESI) m / z: 290.2 [M+H] +

[0243] Synthesis of LL-6: (S)-6-chloro-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide To a solution of (S)-5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazin-1-yl)pyrazine-2-amine (LL-5, 520.00 mg, 1 Eq, 1.8035 mmol) in DMF (8.0 mL), diisopropylethylamine (670.11 mg, 903 μL, 5 Eq, 5.1846 mmol), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosfinan 2,4,6-trioxide (659.85 mg, 699 μL, 50% by weight, 1 Eq, 1.0369 mmol), and 6-chloronicotinic acid (196.04 mg, 1.2 Eq, 1.2443 mmol) were added. The mixture was stirred at 22°C for 3 hours. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm-50×250mm column, 10-100% 5mM NH4HCO3 aqueous solution:acetonitrile, 33 min gradient). The fractions containing the product were combined and extracted between water (100 mL) and DCM (150 mL × 3). The collected organic layer was dehydrated with MgSO4 and then concentrated under reduced pressure to obtain LL-6. MS (ESI) m / z: 429.3 [M+H] +

[0244] Example 143: (S)-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(1-methyl-1H-pyrazole-4-yl)nicotinamide (S)-6-chloro-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide (LL-6, 20.00 mg, 1 Eq, 46.64 μmol) was dissolved in 1,4-dioxane (0.50 mL) to which 1 M tribasic potassium phosphate (139.9 μL, 1.00 mol, 3 Eq, 139.9 μmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (11.64 mg, 1.2 Eq, 55.96 μmol) were added. The mixture was purged with N2 for 5 minutes, and then XPhos Palladacycle-G2 (3.669 mg, 0.1 Eq, 4.664 μmol) was added. The mixture was stirred at 80°C for 2 hours. The mixture was concentrated and purified using normal-phase chromatography (ISCO 12 g RediSep Gold High Performance Silica, 10-100% hexane, siRNA, 18 min gradient). The fractions containing the desired product were combined and concentrated under reduced pressure. LC-MS showed the main by-products, so it was dissolved again in DMSO (1 mL) and purified again using basic reverse-phase chromatography (Gilson Waters XBridge Prep OBD C18 5 μm 10-100% 0.1% NH4OH water:acetonitrile, 10 min gradient). The fractions containing the product were combined and concentrated to obtain 143. MS (ESI) m / z: 475.4 [M+H] + 1H NMR (500 MHz, CDCl3): δ 9.20 (s, 1H), 9.06 (d, J = 2.1 Hz, 1H), 8.29 - 8.16 (m, 4H), 8.02 (s, 2H), 7.83 (s, 1H), 7.56 (d, J = 8.3 Hz, 1H), 4.63 (d, J = 13.2 Hz, 1H), 4.56 (d, J = 13.0 Hz, 2H), 4.06 (d, J = 12.7 Hz, 1H), 3.99 (s, 3H), 3.42 - 3.29 (m, 2H), 3.23 (td, J = 12.3, 3.6 Hz, 1H), 1.20 (d, J = 6.5 Hz, 3H).

[0245] Example 144: (S)-6-(1-(2-fluoroethyl)-1H-pyrazole-4-yl)-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide [ka] (S)-6-chloro-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide (LL-6, 20.00 mg, 1 Eq, 46.64 μmol) was added to a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidine-2-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide (LL-6, 20.00 mg, 1 Eq, 46.64 μmol) in 1,4-dioxane (0.50 mL) and 1 M tribasic potassium phosphate (139.9 μL, 1.00 mol, 3 Eq, 139.9 μmol) and 1-(2-fluoroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (13.44 mg, 1.2 Eq, 55.96 μmol). The mixture was purged with N2 for 5 minutes, and then XPhos Palladacycle-G2 (3.669 mg, 0.1 Eq, 4.664 μmol) was added. The mixture was stirred at 80°C for 2 hours. The mixture was concentrated in a concentrator, dissolved in DCM (1 mL), and purified by normal-phase chromatography (ISCO 12 g RediSep Gold High Performance Silica, 10-100% hexane, siRNA, 18 min gradient). The fractions containing the desired product were combined and concentrated under reduced pressure to obtain 144. MS (ESI) m / z: 507.4 [M+H] + 1 H NMR (500 MHz, CDCl3): δ 9.20 (s, 1H), 9.07 (d, J = 1.9 Hz, 1H), 8.25 (s, 1H), 8.23 ​​(s, 2H), 8.20 (dd, J = 8.3, 2.0 Hz, 1H), 8.13 (s, 1H), 8.09 (s, 1H), 7.82 (s, 1H), 7.58 (d, J = 8.3 Hz, 1H), 4.87 (t, J = 4.7 Hz, 1H), 4.78 (t, J = 4.7 Hz, 1H), 4.63 (d, J = 12.9 Hz, 1H), 4.56 (d, J = 13.0 Hz, 2H), 4.49 (dt, J = 26.7, 4.8 Hz, 3H), 4.06 (d, J = 12.7 Hz, 1H), 3.41 - 3.30 (m, 2H), 3.23 (td, J = 12.3, 3.6 Hz, 1H), 1.20 (d, J = 6.5 Hz, 3H)

[0246] Synthesis of radioactively labeled intermediates: Synthesis of intermediate NN-5: (S)-N-(5-(4-(6-chloropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(3,3-difluoroazetidine-1-yl)nicotinamide [ka] Synthesis of NN-1: tert-butyl (S)-4-(5-bromopyrazine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (5 g, 24.96 mmol) in DMSO (100 mL), CsF (11.38 g, 74.9 mmol) and 2,5-dibromopyrazine (7.13 g, 30.0 mmol) were added at room temperature. The reaction mixture was stirred at 80°C for 6 hours under an argon atmosphere. The reaction mixture was quenched with cold water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was washed with brine (100 mL), dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a silica column (230-400 mesh), and the compound was eluted with 15% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain NN-1. M / Z (ESI): 357.15 [M+H] +

[0247] Synthesis of NN-3: tert-butyl (S)-4-(5-(6-(3,3-difluoroazetidine-1-yl)nicotinamide)pyrazine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of NN-1 (1.50 g, 4.20 mmol) in 1,4-dioxane (30 mL), NN-2 (985 mg, 4.62 mmol), Cs2CO3 (4.10 g, 12.6 mmol), CuI (80.0 mg, 420 μmol), and trans-(1r,2r)-N,N'-bismethyl-1,2-cyclohexanediamine (66.2 μL, 210 μmol) were added at room temperature, and the mixture was degassed with argon for 10 minutes. The reaction mixture was stirred at 150 °C for 2 hours under microwave irradiation. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a silica (230-400 mesh) column, and the compound was eluted with 5% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to obtain NN-3. M / Z (ESI): 490.77 [M+H] + .

[0248] Synthesis of NN-4: (S)-6-(3,3-difluoroazetidine-1-yl)-N-(5-(2-methylpiperazine-1-yl)pyrazine-2-yl)nicotinamide To a stirred solution of NN-3 (900 mg, 1.84 mmol) in DCM (10 mL), 4 M HCl (2.30 mL, 9.19 mmol) in 1,4-dioxane was added at 0°C. The reaction mixture was stirred under an argon atmosphere at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to obtain NN-4. M / Z (ESI): 390.23 [M+H] +

[0249] Synthesis of NN-5: (S)-N-(5-(4-(6-chloropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrazine-2-yl)-6-(3,3-difluoroazetidine-1-yl)nicotinamide To a stirred solution of NN-4 (150 mg, 352 μmol) in DMF (2 mL), DIPEA (0.38 mL, 2.2 mmol) and 4,6-dichloropyrimidine (63.0 mg, 423 μmol) were added at room temperature. The reaction mixture was stirred under an argon atmosphere at 25°C for 6 hours. The reaction mixture was quenched with cold water (10 mL). The precipitated solution was stirred for 10 minutes, filtered, and dried under reduced pressure. The crude compound was ground with diethyl ether (10 mL) to obtain NN-5. M / Z (ESI): 502.21 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ = 10.59 (s, 1H), 8.76-8.94 (m, 2H), 8.37 (s, 1H), 8.22 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.13 (s, 1H), 7.06 (s, 1H), 6.62 (d, J = 8.8 Hz, 1H), 4.21-4.70 (m, 7H), 4.01-4.17 (m, 1H), 3.42 (dd, J = 13.2 Hz, 3.6 Hz, 1H), 3.20-3.29 (m, 2H), 1.07 (d, J = 6.4 Hz, 3H).

[0250] Synthesis of OO-7A: (S)-6-(azetidine-1-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide, and synthesis of OO-7B: (S)-(6-(4-(5-(6-(azetidine-1-yl)nicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-yl)pyrimidine-3-yl)boronic acid [ka]

[0251] Synthesis of OO-1: tert-butyl (S)-3-methyl-4-(5-nitropyrimidine-2-yl)piperazine-1-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (10 g, 49.9 mmol) in DMF (100 mL), 2-chloro-5-nitropyrimidine (9.56 g, 59.9 mmol) and cesium carbonate (16.3 g, 49.9 mmol) were added at room temperature under an argon atmosphere. The reaction mixture was stirred at 80°C for 8 hours. The reaction mixture was quenched with crushed ice and extracted with ELISA (2 × 100 mL). The combined organic layers were washed with brine, dehydrated with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 120 g silica gel cartridge, and the compound was eluted with 70% ELISA in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain OO-1. M / Z (ESI): 324.23 [M+H] + .

[0252] Synthesis of OO-2: tert-butyl (S)-4-(5-aminopyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of OO-1 (15 g, 46.4 mmol) in EtOH (100 mL) and THF (100 mL), 10% Pd / C (4.94 g, 46.4 mmol) was added at room temperature. The reaction mixture was degassed and purged with nitrogen gas. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 16 hours. The reaction mixture was filtered through a Celite pad, the filtrate was concentrated, and dried under reduced pressure. The crude compound was ground with n-pentane and dried under reduced pressure to obtain OO-2. M / Z (ESI): 294.22 [M+H] + .

[0253] Synthesis of OO-3: tert-butyl (S)-4-(5-(6-fluoronicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of OO-2 (1 g, 3.41 mmol) and 6-fluoronicotinic acid (577 mg, 4.09 mmol) in THF (20 mL), DIPEA (1.78 mL, 10.2 mmol) and HATU (1.94 g, 5.11 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was quenched with water (20 mL) and extracted with SiO2 (20 mL). The combined organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 12 g silica gel cartridge, and the compound was eluted with 40% SiO2 in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain OO-3. M / Z (ESI): 417.29 [M+H] + .

[0254] Synthesis of OO-4: tert-butyl (S)-4-(5-(6-(azetidine-1-yl)nicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylate To a stirred solution of OO-3 (800 mg, 1.92 mmol) in DMSO (5 mL), azetidine hydrochloride (270 mg, 2.88 mmol) and K2CO3 (796 mg, 5.76 mmol) were added under an argon atmosphere at room temperature. The reaction mixture was stirred at 120 °C for 24 hours. The reaction mixture was quenched with crushed ice and extracted with siRNA (2 × 20 mL). The combined organic layer was washed with brine, dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 12 g silica gel cartridge, and the compound was eluted with 80% siRNA in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain OO-4. M / Z (ESI): 454.33 [M+H] +

[0255] Synthesis of OO-5: (S)-6-(azetidine-1-yl)-N-(2-(2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide To a stirred solution of OO-4 (3 g, 6.61 mmol) in DCM (50 mL), 4 M HCl (6.61 mL, 26.5 mmol) in 1,4-dioxane was added under an argon atmosphere at 0°C. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated and dried under reduced pressure to obtain OO-5. M / Z (ESI): 354.33 [M+H] + .

[0256] Synthesis of OO-6: (S)-6-(azetidine-1-yl)-N-(2-(4-(5-bromopyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide Potassium carbonate (2.13 g, 15.4 mmol) was added at room temperature to a stirred solution of OO-5 (2 g, 5.13 mmol) and 5-bromo-2-fluoropyridine (1.08 g, 6.16 mmol) in DMSO (10 mL). The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was quenched with water (30 mL) and extracted with toluene (2 × 50 mL). The combined organic layer was washed with brine (20 mL), dehydrated with anhydrous sodium 2 SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography, and the compound was eluted with 80% toluene in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to obtain OO-6. M / Z (ESI): 509.23 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.94 (s, 1H), 8.64-8.72 (m, 3H), 8.17 (d, J = 2.4 Hz, 1H), 8.03 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.69 (dd, J = 9.0 Hz, 2.6 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.39 (d, J = 8.4 Hz, 1H), 4.77-4.90 (m, 1H), 4.36-4.48 (m, 1H), 4.13-4.29 (m, 2H), 4.04 (t, J = 7.4 Hz, 4H), 3.21-3.30 (m, 2H), 2.96-3.06 (m, 1H), 2.32-2.43 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H).

[0257] Synthesis of OO-7A: (S)-6-(azetidine-1-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide, and synthesis of OO-7B: (S)-(6-(4-(5-(6-(azetidine-1-yl)nicotinamide)pyrimidine-2-yl)-3-methylpiperazine-1-yl)pyrimidine-3-yl)boronic acid To a stirred solution of OO-6 (300 mg, 588.9 μmol) in 1,4-dioxane (5 mL), potassium acetate (173.4 mg, 1.767 mmol) and bis(pinacolate)diborone (179.5 mg, 706.7 μmol) were added at room temperature under an argon atmosphere. The reaction mixture was degassed and purged with argon gas for 2 minutes. Then, PdCl2(dppf)-CH2Cl2 adduct (48.09 mg, 58.89 μmol) was added to the reaction mixture at room temperature. The reaction mixture was stirred at 100 °C for 16 hours. The reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude compound was purified by prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN; column - X-Bridge C18 (19×250) mm, 5 μm; flow rate - 15.0 mL / min; gradient method: 0 / 40, 2 / 40, 15 / 75, 13 / 75, 13.05 / 100, 15 / 100, 15.05 / 40, 18 / 40 prep-020). The pure fractions were combined separately, concentrated under reduced pressure, and lyophilized to obtain OO-7A and OO-7B.

[0258] OO-7A: M / Z (ESI): 557.44 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ = 9.94 (s, 1H), 8.60-8.73 (m, 3H), 8.34 (d, J = 1.2 Hz, 1H), 8.03 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.71 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 4.75-4.85 (m, 1H), 4.25-4.46 (m, 3H), 4.04 (t, J = 7.6 Hz, 4H), 3.27 (d, J = 3.2 Hz, 2H), 3.05 (td, J = 11.8 Hz, 3.6 Hz, 1H), 2.34-2.40 (m, 2H), 1.27 (s, 12H), 1.10 (d, J = 6.8 Hz, 3H). OO-7B: M / Z (ESI): 473.32 [MH] - 1 H NMR (400 MHz, DMSO-d6) δ = 9.94 (s, 1H), 8.63-8.72 (m, 3H), 8.47 (d, J = 1.2 Hz, 1H), 8.03 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.82-7.91 (m, 3H), 6.80 (d, J = 8.8 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 4.76-4.89 (m, 1H), 4.22-4.47 (m, 3H), 4.04 (t, J = 7.4 Hz, 4H), 3.20-3.30 (m, 2H), 2.99 (td, J = 12.0 Hz, 3.6 Hz, 1H), 2.34-2.41 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H).

[0259] Synthesis of PP-3: (S)-1-(5-((5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)carbamoyl)pyridine-2-yl)azetidine-3-yl 4-methylbenzenesulfonate [ka] Synthesis of PP-1: 6-(3-hydroxyazetidine-1-yl)nicotinamide To a stirred solution of 6-chloronicotinamide (1 g, 6.39 mmol) in DMF (40 mL), K2CO3 (2.65 g, 19.16 mmol) and 3-hydroxyazetidine hydrochloride (0.840 g, 7.66 mmol) were added at 0°C. The reaction mixture was stirred at 100°C for 16 hours under a nitrogen atmosphere. The reaction mixture was diluted with toluene (50 mL), filtered through a Celite pad, and washed with toluene (2 × 50 mL). The filtrate was dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using a 40 g silica (230-400 mesh) cartridge, and the compound was eluted with 12% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to obtain PP-1. M / Z (ESI): 194.06 [M+H] +

[0260] Synthesis of PP-2: (S)-N-(5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)-6-(3-hydroxyazetidine-1-yl)nicotinamide To a stirred solution of Q-6 (300 mg, 0.852 mmol) in 1,4-dioxane (3 mL), Cs2CO3 (833 mg, 2.56 mmol), copper(I) iodide (16.22 mg, 0.085 mmol), trans-N,N'-dimethylcyclohexane-1,2-diamine (6.66 μL, 0.043 mmol), and PP-1 (181 mg, 0.937 mmol) were added at room temperature. The reaction mixture was stirred at 150 °C for 2 hours under a nitrogen atmosphere and microwave irradiation. The reaction mixture was quenched with water (80 mL) and extracted with RINKAN (2 × 150 mL). The combined organic layers were washed with brine (2 × 80 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified using Biotage with an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 3% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to obtain PP-2. M / Z (ESI): 465.34 [M+H] +

[0261] Synthesis of PP-3: (S)-1-(5-((5-(4-(5-fluoropyridine-2-yl)-3-methylpiperazine-1-yl)pyrazine-2-yl)carbamoyl)pyridine-2-yl)azetidine-3-yl 4-methylbenzenesulfonate To a stirred solution of PP-2 (200 mg, 0.431 mmol) in DCM (4 mL), TEA (0.180 mL, 1.292 mmol), 4-dimethylaminopyridine (26.3 mg, 0.215 mmol), and tosyl-Cl (246 mg, 1.292 mmol) were added at 0°C. The reaction mixture was stirred at 25°C for 3 hours under a nitrogen atmosphere. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2 × 85 mL). The combined organic layer was washed with brine (2 × 40 mL), dehydrated with anhydrous sodium 2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by biotage using an 80 g silica (230-400 mesh) cartridge, and the compound was eluted with 70% ethyl acetate in petroleum ether. The pure fractions were combined and concentrated under reduced pressure. The obtained compound was ground with 10% diethyl ether in pentane and dried under reduced pressure to obtain PP-3. M / Z (ESI): 619.33 [M+H] + 1H NMR (400 MHz, DMSO-d6) δ = 10.49 (s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.74 (d, J = 2.4 Hz, 1H), 8.06-8.25 (m, 3H), 7.86 (d, J = 8.4 Hz, 2H), 7.47-7.60 (m, 3H), 6.87 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.45 (d, J = 8.8 Hz, 1H), 5.27-5.36 (m, 1H), 4.50-4.60 (m, 1H), 4.16-4.40 (m, 4H), 4.03 (d, J = 12.8 Hz, 1H), 3.95 (dd, J = 10.0 Hz, 2.8 Hz, 2H), 3.16-3.29 (m, 2H), 3.04 (td, J = 11.8 Hz, 3.4 Hz, 1H), 2.46 (s, 3H), 1.08 (d, J = 6.4 Hz, 3H).

[0262] Radiosynthesis procedure [ 3 Synthesis of [3H]-1: [3H]-(S)-6-methoxy-N-(2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Crabtree catalyst (0.15 mg) was added to the tritium reaction vessel, followed by the addition of a solution of compound 1 (0.5 mg) in CH2Cl2 (0.2 mL). The vessel was attached to the tritium line and pressurized to 0.5 atm with tritium gas at -200°C. The solution was stirred for 17 hours, cooled to -200°C, and excess gas was removed. The reaction flask was rinsed with 4 × 1 mL of CH3OH, and each was transferred to a 100 mL recovery flask. The combined CH3OH was removed under reduced pressure. Crude yield: 90 mCi. This substance was purified by HPLC. The mobile phase was removed under reduced pressure, and the product was redissolved in anhydrous ethanol. Yield: 20.6 mCi, purity >99%. The specific activity was determined to be 81.04 Ci / mmol by mass spectrometry. C 21 H 20 MW [M+H] for T3N7O2+ : 411.5, Measured value: 412.4.

[0263] HPLC preparatory separation conditions Method: 0-70% in 20 minutes Column: Phenomenex Luna 5flm C18 100×21.2mm Flow rate: 4mL / min Injection volume: 0.5mL Detection: UV @ 254nm Mobile phase A: 0.05% TFA in H2O Mobile phase B: CH3CN Product elution time: 16.7 minutes.

[0264] [ 3 Synthesis of H]-24: [3H]-2-methoxy-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazine-1-yl]pyrimidine-5-yl]pyridine-4-carboxamide [ka] Crabtree catalyst (0.3 mg) was added to the tritium reaction vessel, followed by the addition of a solution of compound 24 (0.4 mg) in CH2Cl2 (0.2 mL). The vessel was attached to the tritium line and pressurized to 0.5 atm with tritium gas at -200°C. The solution was stirred for 17 hours, cooled to -200°C, and excess gas was removed. The reaction flask was rinsed with 4 × 1 mL of CH3OH, and each was transferred to a 100 mL recovery flask. The combined CH3OH was removed under reduced pressure. Crude yield: 125 mCi. This substance was purified by HPLC. The mobile phase was removed under reduced pressure, and the product was redissolved in anhydrous ethanol. Yield: 26 mCi, purity >99%. The specific activity was determined to be 150.01 Ci / mmol by mass spectrometry.

[0265] C 21 H 20 MW [M+H] for T5N7O2 + : 415.5, Measured value: 416.3.

[0266] HPLC preparative Method: 10% for 5 minutes; 10-90% for 20 minutes Column: Phenomenex Luna Sum C18 100×21.2mm Flow rate: 4mL / min Injection volume: 0.5mL Detection: UV @ 254nm Mobile phase A: 0.05% TFA in H2O Mobile phase B: CH3CN Product elution time: 18.1 minutes.

[0267] [ 3 Synthesis of H]-89: [3H]-(S)-N-(2-(4-(2-fluoropyridine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)-6-(1H-pyrazole-1-yl)nicotinamide [ka] Compound 89 in CH2Cl2 was washed with a 0.5 M Na2CO3 aqueous solution, and the organic layer was then dehydrated with Na2SO4, and the filtrate was evaporated to dryness. The resulting residue (4 mg) was dissolved in CPME (75 μL) and NMP (50 μL). In a glove box, nickel pre-catalyst ( ipc ADI)NiBr2 (6.65 mg) was dissolved in CPME (670 μL), treated with NaHBEt3 (1 M, 23 μL) in toluene, and then stirred for 5 minutes. The substrate solution (100 μL) was added to the activated catalyst solution (100 μL) in the tritiation vessel and fixed with a portable Swagelok® valve. The valve was attached to a Trisorber, and two freeze-pump-thaw cycles were performed before introducing 155 mmHg of tritium gas. The reaction mixture was thawed and then placed in an oil bath at 45°C and stirred overnight. After capturing spent tritium in the wastewater bed, the reaction mixture was transferred to a vial containing 10 mL of saturated aqueous sodium bicarbonate. The mixture was extracted three times with dichloromethane. The combined organic layer was dehydrated with sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 247.2 mCi; RCP: 90%. This substance was purified by HPLC. The collected fraction was diluted with an equal volume of water, concentrated in a pair of C18 cartridges, and eluted with EtOH. Yield: approximately 20 mL ethanol solution @ 4.22 mCi / mL. The specific activity was determined to be 55.52 Ci / mmol by mass spectrometry. C 23 H 19 MW [M+H] for T4FN9O + : 468.2, Measured value: 468.3.

[0268] HPLC analysis conditions Method: 10-95% B in 15 minutes, re-equilibrium for 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1mL / min Injection volume: 1uL Detection: UV @ 298nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 7.08 minutes.

[0269] HPLC preliminary separation conditions Column: Gemini NX C18, 10×250mm @ 40℃ Flow rate: 5mL / min Injection volume: 0.4mL Detection: UV @ 298nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN.

[0270] [ 3 Synthesis of H]-87: [3H]-(S)-6-(3,3-difluoroazetidine-1-yl)-N-(2-(4-(4-fluoropyridine-2-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Compound 87 (0.50 mg, 1.03 umol) was weighed and placed in a tritiation vessel. Crabtree catalyst (1.12 mg, 0.51 umol) was prepared as a solution in CH2Cl2 (1.4 mL). 500 μL of this catalyst solution was added to the tritiation vessel attached to the ultra-tall port of the Trisorber, and two freeze-pump-thaw cycles were performed before introducing 84 mmHg of tritium gas. The reaction mixture was thawed to room temperature and then stirred for 4 hours. After capturing spent tritium in the wastewater bed, the reaction mixture was transferred to a vial containing 10 mL of saturated aqueous sodium bicarbonate. The mixture was extracted three times with dichloromethane. The combined organic layer was dehydrated with sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 204.4 mCi; RCP: 89%. The substance was purified by HPLC. The collected fractions were diluted with an equal volume of water, concentrated in a pair of C18 cartridges, and eluted with EtOH. A portion of the purified batch was aliquoted and diluted to 20.0 mL. Yield: 20.0 mL ethanol solution @ 2.45 mCi / mL. The specific activity was determined to be 131.4 Ci / mmol by mass spectrometry. C 23 H 19 MW [M+H] for T5F3N8O + : 495.2, Measured value: 495.3.

[0271] HPLC analysis conditions Method: 10-95% B in 15 minutes, re-equilibrium for 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1mL / min Injection volume: 1.5-2.0uL Detection: UV @ 304nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 7.63 minutes.

[0272] HPLC preliminary separation conditions Method: Isometric (A:B=55:45) Column: Gemini NX C18, 10×250mm @ 40℃ Flow rate: 5mL / min Injection volume: 0.5mL Detection: UV @ 304nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN.

[0273] [ 3 Synthesis of H]-91: [3H]-(S)-6-(1H-imidazole-1-yl)-N-(2-(2-methyl-4-(pyridine-2-yl)piperazine-1-yl)pyrimidine-5-yl)nicotinamide [ka] Compound 91 (1.33 mg, 3.0 umol) was dissolved in CPME (75 μL) and NMP (25 μL) in a glove box. Nickel pre-catalyst ( ipc ADI)NiBr2 (6.73 mg) was dissolved in CPME (670 μL), treated with NaHBEt3 (1 M, 25 μL) in toluene, and then stirred for 5 minutes. The substrate solution (100 μL) was combined with the activated catalyst solution (200 μL, 3.5 μL) in a tritiation vessel and fixed with a portable Swagelok® valve. The valve was attached to a Trisorber, and two freeze-pump-thaw cycles were performed before introducing 102 ml of tritium gas. The reaction mixture was thawed and then placed in an oil bath at 45°C and stirred overnight. After capturing spent tritium in the wastewater bed, the reaction mixture was transferred to a vial containing 10 mL of saturated aqueous sodium bicarbonate. The mixture was extracted three times with dichloromethane. The combined organic layer was dehydrated with sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 120 mCi; RCP: 67%. This substance was purified by HPLC. The collected fraction was diluted with an equal volume of water, concentrated in a pair of C18 cartridges, and eluted with EtOH. Yield: approximately 20 mL ethanol solution @ 3.18 mCi / mL. The specific activity was determined to be 44.9 Ci / mmol by mass spectrometry. C 23 H 17MW [M+H] for T7N9O + : 456.3, Measured value: 456.0.

[0274] HPLC analysis conditions Method: 10-95% B over 12 minutes, hold for 3 minutes, re-equilibrium for 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1mL / min Injection volume: 1.0uL Detection: UV @ 294nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 6.32 minutes.

[0275] HPLC preliminary separation conditions Method: Isometric (A:B=65:35) Column: Gemini NX C18, 10×250mm @ 40℃ Flow rate: 5mL / min Injection volume: 0.5mL Detection: UV @ 295nm Mobile phase A: 0.05M pH10 TEAA in H2O Mobile phase B: CH3CN.

[0276] Assay protocol Acquisition of human postmortem tissue samples for in vitro binding assays Frozen human brain tissue from Parkinson's disease (PD) patients was purchased from Analytic Biological Services Inc. The samples were postmortem tissue from donors clinically diagnosed with late-stage PD. Alpha-synuclein, tau, and amyloid loads were determined by a combination of immunohistochemistry of frozen thin coronal sections and quantification based on α-Lisa protein levels in surfactant-insoluble protein fractions. A temporal cortical tissue sample from one donor showed moderate to high alpha-synuclein load, low amyloid, and minimal tau pathology. The surfactant-insoluble fraction of the temporal cortex from this patient was used to support homogenate-binding studies.

[0277] Preparation of surfactant-insoluble fractions of human brain tissue for in vitro binding studies. Gray matter was extracted from the temporal cortical tissue using a dissecting knife and finely chopped with thin dissecting scissors. To prepare the insoluble fraction, the minced tissue was homogenized in ice-cold TBS-TX buffer (50 mM Tris + 150 mM NaCl + 1% Triton X100 + 1 mM EDTA + 1 tablet / 10 mL of complete protease inhibitor + 1 tablet / 10 mL of PHOSSTOP phosphatase inhibitor tablet) using a glass Dounce tissue grinder. The homogenate was centrifuged at 100,000 × g for 45 minutes. The pellet was resuspended in TBS-TX buffer using a Polytron at its highest setting for 30 seconds at 4°C. The homogenate was centrifuged at 100,000 × g for 45 minutes, and the pellet was resuspended in TBS-TX buffer. The final homogenate was subjected to a BCA protein assay to measure the protein concentration. The homogenate was divided into equal portions of 0.5 mL / tube and stored at -70°C until use.

[0278] Procedure for the α-synuclein tissue homogenate binding assay (Assay 1) In the hot saturated binding assay, the final concentration of the radioactive ligand is in the range of 0.84 to 50 nM. 3Radioactive ligands of various concentrations were prepared in assay buffer (DPBS + 0.1% BSA) to achieve [H]-1. 25 μL of radioactive ligand was added to 200 μL of the insoluble fraction of PD brain homogenate diluted to 150 μg / mL in assay buffer (incubation, filtration, and measurement of the amount of radioactive ligand used in the assay are described below). Nonspecific binding was measured using self-blocking with an unlabeled compound. Saturation data were analyzed using GraphPad / Prism software.

[0279] Figure 2 is [ 3 This figure shows a saturation binding experiment using Triton-insoluble fractions of temporal cortical PD tissue rich in aggregated α-synuclein with H-1. This data supports the use of this ligand in radioligand binding assays to optimize its efficacy in binding to pathological α-synuclein. Figure 2 shows [ 3 This shows an example of hot saturated binding of [H]-1, where the radioactive ligand exhibits high affinity for α-synuclein in PD brain homogenate, with a measured dissociation constant of 25 nM.

[0280] For the substitution α-synuclein binding assay, the unlabeled test compound was dissolved in DMSO at 10 mM. Dilution of the test compound to various concentrations was performed in 100% DMSO at a concentration 1000 times the final assay concentration, and 0.225 μL aliquots were dispensed into assay plates. The insoluble fraction of PD brain homogenate was diluted in assay buffer from the original 10 mg / mL to 50 μg / mL, and 200 μL was added to the assay plate to achieve a final concentration of 10 μg per well. 3[H]-1 was prepared in assay buffer to 10 times its final concentration, and 25 μL was added to the assay plate to achieve a final assay concentration of 2.0 nM. 200 μL of homogenate and 25 μL of radioactive ligand were added to the assay plate containing the titrated compound. The plate was incubated at room temperature for 90 minutes. Unbound and bound ligands were separated by filtering a GF / C filter plate (pre-treated with 0.2% PEI at 4°C for 60 minutes) through a Perkin Elmer FilterMate Harvester Unifilter-96, and washing the unbound ligands three times with 1 mL of ice-cold DPBS. The filter plate was dried (in an oven under reduced pressure at 47°C for 1 hour, or overnight at room temperature). 50 μL / well of Microscint-20 (Perkin Elmer) was added to the plate, and the plate was counted on a Perkin Elmer TopCount for 1 minute per well. The data was analyzed using IDBS Activity Base to determine the Ki values ​​shown in Data Table 1 (K d Value 25 nM, ligand concentration 2.0 nM; Figure 2).

[0281] Competitive binding of radioactive ligands to pathologically aggregated β-amyloid in AD tissue (Assay 2): Frozen human brain samples from Alzheimer's disease (AD) were purchased from Analytic Biological Services Inc. The samples were postmortem tissue from a clinically diagnosed AD donor, and a large portion of the white matter was excised from the frontal cortex to concentrate the gray matter tissue preparation. A gray matter-rich frontal cortex brain homogenate was prepared by homogenizing the tissue in ice-cold phosphate-buffered saline (PBS) (pH 7.4) with 80 mg of wet tissue per mL at 4°C for 45 seconds using a Polytron set to 16. The homogenate was further diluted with ice-cold PBS to 30 mg of wet tissue per mL and homogenized for another minute as described above. The homogenate was divided into 5 mL / tube portions and stored at -70°C until use.

[0282] Radiating ligand [ 3H]-105 was prepared in this assay as described in "ACS Med. Chem. Lett., Vol. 2, pages 498-502". [ka]

[0283] In the hot saturation binding assay, various concentrations of radioactive ligands [ 3 [H]-105 was prepared in assay buffer (PBS + 0.1% BSA) with 20% DMSO to a concentration ranging from 3.9 to 500 nM. 25 μL of radioactive ligand was added to 200 μL of crude brain homogenate (diluted to 0.5 mg / mL with assay buffer) to achieve a final radioactive ligand concentration ranging from 0.39 to 50 nM, resulting in the final crude brain homogenate at 100 μg wet weight / assay well (incubation, filtration, and measurement of the amount of radioactive ligand used in the assay are described below). Nonspecific binding was measured using self-blocking with an unlabeled compound. Saturation data were analyzed using GraphPad / Prism software.

[0284] Figure 1 shows the relationship between [AD cortical tissue homogenate rich in aggregated β-amyloid pathology] 3 This represents the high affinity saturated bond of H]-105. Figure 1 shows [ 3 This shows an example of hot saturated binding of [H]-105, where the radioactive ligand exhibits high affinity for aggregated β-amyloid (abeta) in AD brain homogenate, with a measured dissociation constant of 11 nM. This data supports the use of this ligand in radioactive ligand binding assays for screening binding to aggregated β-amyloid.

[0285] In Assay 2, the unlabeled test compound was dissolved in DMSO at 10 mM. Dilution of the test compound to various concentrations was performed in 100% DMSO at a concentration 1000 times the final assay concentration, and 0.225 μL aliquots were dispensed into assay plates. The brain homogenate was diluted from the original 30 mg / mL volume in assay buffer to 0.5 mg / mL, and 200 μL was added to the assay plate to obtain a final concentration of 100 μg wet weight / assay well. 3 H]-105 was prepared to a 10-fold final concentration in assay buffer + 20% DMSO, and 25 μL was added to the assay plate to a final assay concentration of 3.0 nM. The plate was incubated at 37°C for 90 minutes. Unbound and bound ligands were separated by filtering the bound ligands on a GF / B filter plate (pre-treated with 0.1% PEI for 30 minutes) using a Packard Filtermate, and washing the unbound ligands with 2.5 mL of ice-cold 5 mM Tris (pH 7.4). The filter plate was dried at 57°C for 1 hour, and 50 μL of Microscint was added to each well of the plate. The plate was counted for 3H cpm for 1 minute per well using a PerkinElmer TopCount. The data were analyzed using the IDBS Activity Base, and the K data is shown in Table 1 below. i Value determined (K d (Value 11.0 nM, ligand concentration 3.5 nM).

[0286] [Table 9] TIFF0007881829000134.tif241166 TIFF0007881829000135.tif241166 TIFF0007881829000136.tif241166 TIFF0007881829000137.tif220165

[0287] Procedure for processing radioactive ligand saturation binding data in human cortical PD tissue homogenates rich in aggregated α-synuclein and human cortical AD tissue homogenates rich in aggregated Aβ pathogenesis.

[0288] Banner Parkinson's disease (PD; Braak stage 5 / 6) brain homogenates were obtained through the Michael J. Fox Foundation (MJFF) tissue consortium. Tissue preparation for characterization and integration studies of such brain tissues was performed using the method described in U.S. Patent No. 2019 / 0256492, paragraphs 0242-0244. Banner PD brain homogenates, derived from postmortem brain tissue of PD-diagnosed donors, were prepared using cingulate cortex samples taken from multiple PD brains that were rich in α-synuclein pathogenesis but lacked amyloidopathogenesis and tauopathy upon neuropathological verification. The final concentration of Banner PD brain homogenates was 333 mg wet weight per 1 mL of buffer. The homogenates were divided into 1 mL / tube portions and stored at -70°C before use.

[0289] Frozen human brain samples from Alzheimer's disease (AD) were purchased from Analytic Biological Services Inc. These were postmortem tissues from donors clinically diagnosed with AD and were validated for amyloid pathology by intravascular heating (IH). Frontal cortical brain homogenates were prepared by homogenizing the frontal cortex in ice-cold phosphate-buffered saline (PBS) (pH 7.4) at 4°C for 30 seconds using a Polytron set to 6. The final concentration of the brain homogenate was 10 mg of moist tissue per 1 mL of buffer. The homogenate was divided into 5 mL / tube portions and stored at -70°C before use.

[0290] [ 3 H]-1 was synthesized as described in the procedure briefly outlined above. 3 The specific activity of H-1 is 196.6 Ci / mmol in a volume of 3.9 mCi / mL.

[0291] Hot saturated binding assays were performed individually at nine concentrations ranging from 60.0 nM to 0.6 nM to evaluate the binding of radioligands to Banner PD brain homogenate and ABS AD brain homogenate. Brain homogenates were diluted from their original 30 mg / mL volume to 2.0 mg / mL (ABS AD) or 2.8 mg / mL (Banner PD) with PBS buffer. Total binding was defined in the absence of competing compounds, and non-substitutable binding was measured in the presence of 1 μM of unlabeled self-blocking. The assay buffer was 30 nM Tris (pH 7.5) containing 0.1% BSA. Assay tubes were pre-incubated at room temperature for 30 minutes, and then diluted radioligands (10-fold) were added to the assay tubes (10 μL each, separately) to a final volume of 100 μL per tube. Incubation was performed at 37°C for 120 minutes, after which the assay samples were filtered onto a GF / C filter using a Skatron 12-well harvester and washed with ice-cold buffer (30 nM Tris pH 7.5) at a setting of 5-5-5 (approximately 3 × 2 ml). The GF / C filter paper for the Skatron harvester was pre-soaked in 0.1% BSA at room temperature for 1 hour before use. The filters were punched into scintillation vials and counted for 1 minute on a 2 mL Ultima Gold on a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in 3 replicates in a laboratory designated for human tissue studies.

[0292] [ 3 The assay for H]-24 is, 3 The procedure was carried out using the same method as in H-1.

[0293] [ 3 H]-1 and [ 3 The data from these saturated binding assays using H]-24 are shown in Figures 3 to 6. Specifically, Figure 3 shows the PD zonal cortical tissue homogenate and [ 3This shows a saturated binding experiment using H]-1. This data shows strong binding to pathological α-synuclein in tissue homogenates. Figure 4 shows AD tissue homogenates and [ rich in aggregated β-amyloid pathology]. 3 This shows a saturated binding experiment using H]-1. (ND = Unconfirmed due to incomplete saturation of the binding signal) This data shows weak binding to pathological β-amyloid in tissue homogenates. Figure 5 shows PD cortical tissue homogenates and [ rich in aggregated α-synuclein. 3 This shows a saturated binding experiment using H]-24. This data shows strong binding to pathological α-synuclein in tissue homogenates. Figure 6 shows AD tissue homogenates and [ rich in aggregated β-amyloid pathology]. 3 This represents a saturated binding experiment using [H]-24. (ND = Unconfirmed due to incomplete saturation of the binding signal) This data indicates weak binding to pathological β-amyloid in tissue homogenates.

[0294] In vitro binding of α-synuclein tracers to human PD brain tissue homogenates. Frozen human brain samples from Parkinson's disease (PD) patients were provided by the Banner Sun Health Institute (USA) through a collaborative research project with the Michael J Fox Foundation (MJFF). These were postmortem tissues from donors clinically diagnosed with PD and whose PD pathology was confirmed by neuropathological verification. Cortical brain homogenates were prepared by homogenizing the cortex in ice-cold phosphate-buffered saline (PBS) (pH 7.4) at 4°C for 30 seconds using a Polytron set to 6. The final concentration of the brain homogenate was 30 mg of moist tissue per 1 mL of buffer. The homogenates were divided into 1 mL / tube portions and stored at -70°C before use.

[0295] [ 3 H]-87 is synthesized, 3The specific activity of H]-87 was 131.5 Ci / mmol. For the hot saturated binding assay, nine concentrations of radioligands ranging from 50 nM to 0.5 nM and 10 nM to 0.1 nM were used. Brain homogenates were diluted from their original 30 mg / mL volume with assay buffer (Tris, pH 7.5, 0.1% BSA) to a final concentration of 2.8 mg / mL, with 250 μL per assay tube used in the assay. Unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of the test compounds to various concentrations were performed using assay buffer containing 2% DMSO. Total binding was defined in the absence of competing compounds, and unsubstitutable binding was measured in the presence of 1 μM of unlabeled self-blocking. A 10-fold dilution of the compound was added to an assay tube containing 200 μL of diluted brain homogenate (25 μL each, separately) and the tube was pre-incubated at room temperature for 30 minutes. Then, a 10-fold dilution of the radioactive ligand was added to the assay tube (25 μL each, separately) to a final volume of 250 μL per tube. Incubation was performed at 37°C for 120 minutes. The assay samples were then filtered onto a GF / C filter using a Skatron 12-well harvester and washed with ice-cold buffer (Tris, pH 7.5) at a setting of 5-5-5 (approximately 3 × 2 ml). The GF / C filter paper for the Skatron harvester was pre-soaked in 0.1% BSA at room temperature for 1 hour before use. The filters were punched into scintillation vials. Liquid scintillation solution (2 mL of Ultima Gold) was added to each vial, filtered for 4 hours, and counted for 1 minute using a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in 2 or 3 replicates, depending on the assay setup, in laboratories designated for human tissue studies.

[0296] [Table 10]

[0297] [ 3 H]-89 is synthesized, 3The specific activity of H]-89 was 55.5 Ci / mmol. Nine concentrations of radioligands ranging from 50 nM to 0.5 nM were used in the hot saturated binding assay. Brain homogenates were diluted from their original 30 mg / mL volume with assay buffer (Tris, pH 7.5, 0.1% BSA) to a final concentration of 2.8 mg / mL, with 250 μL per assay tube used in the assay. Unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of the test compounds to various concentrations were performed using assay buffer containing 2% DMSO. Total binding was defined in the absence of competing compounds, and unsubstitutable binding was measured in the presence of 1 μM of unlabeled self-blocking. A 10-fold dilution of the compound was added to an assay tube containing 200 μL of diluted brain homogenate (25 μL each, separately) and the tube was pre-incubated at room temperature for 30 minutes. Then, a 10-fold dilution of the radioactive ligand was added to the assay tube (25 μL each, separately) to a final volume of 250 μL per tube. Incubation was performed at 37°C for 120 minutes. The assay samples were then filtered onto a GF / C filter using a Skatron 12-well harvester and washed with ice-cold buffer (Tris, pH 7.5) at a setting of 5-5-5 (approximately 3 × 2 ml). The GF / C filter paper for the Skatron harvester was pre-soaked in 0.1% BSA at room temperature for 1 hour before use. The filters were punched into scintillation vials. Liquid scintillation solution (2 mL of Ultima Gold) was added to each vial, filtered for 4 hours, and counted for 1 minute using a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in 2 or 3 replicates, depending on the assay setup, in laboratories designated for human tissue studies.

[0298] [Table 11]

[0299] [ 18 Radiochemical synthesis of [F]-ligand General method [ 18 [F]Fluoride was concentrated using an anion exchange resin and eluted before use. Unless otherwise specified, [ 18 An anion exchange resin containing [F] fluoride was eluted with Cryptofix 222 (7 mg, 19 μmol) and K2CO3 (2.1 mg, 15 μmol) in acetonitrile / water (80 / 20, 0.7 mL) and transferred to a vented 1 mL V-type vial in a microwave cavity. The fluoride was dried under argon flow by microwave heating (35 W / 90 °C). Additional aliquots of acetonitrile (3 × 0.5 mL) were added for azeotropic drying at 35 W / 90 °C.

[0300] [ 18 Synthesis of F]-87 [ka] A solution of 88 (0.5 mg, 1.0 μmol) in DMSO (0.3 mL) is dried [ 18The reaction mixture was added to a microwave vial containing [F] fluoride, the vent line was removed, and the reaction mixture was heated at 170°C (60W) for 3 minutes. After cooling to below 50°C, the reaction mixture was diluted with H2O (0.8 mL), mixed, and injected into a semi-preparative HPLC column. The product was purified at a flow rate of 5 mL / min using a Gemini (C6-Phenyl, 110A, 150 × 10 mm) column. The mobile phase was 50 to 95% acetonitrile / 0.1% trifluoroacetic acid. The radioactive fraction eluted between 12.5 and 13.5 minutes was collected, diluted with 20 mL of sterile water for injection, and loaded into a Waters Sep-Pak Classic C18 cartridge (Waters, Milford, MA, USA). The Sep-Pak was rinsed with 10 mL of water, then eluted with ethanol (0.5 mL), and placed in a 10 mL sterile vial to dilute to the desired formulation. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic (5μ, C18, 50×3mm) (Phenomenex) at a flow rate of 1 mL / min. The mobile phase was a mixture of acetonitrile / 0.1% trifluoroacetic acid in water (from 10% to 90% in 10 minutes). 18 The concentration of F]-87 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 87, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-87 was 6.3 minutes.

[0301] [ 18 Synthesis of F]-89 [ka]

[0302] The solution of 90 (0.5 mg, 1.0 μmol) in DMSO (0.3 mL) is dried [ 18The reaction mixture was added to a microwave vial containing [F] fluoride, the vent line was removed, and the reaction mixture was heated at 170°C (60W) for 3 minutes. After cooling to below 50°C, the reaction mixture was diluted with H2O (0.8 mL), mixed, and injected into a semi-preparative HPLC column. The product was purified at a flow rate of 5 mL / min using a Gemini (C6-Phenyl, 110A, 150 × 10 mm) column. The mobile phase was acetonitrile / 0.1% trifluoroacetic acid (65 / 35). The radioactive fraction eluted between 7.5 and 8.5 minutes was collected, diluted with 20 mL of sterile water for injection, and loaded into a Waters Sep-Pak Classic C18 cartridge (Waters, Milford, MA, USA). The Sep-Pak was rinsed with 10 mL of water, then eluted with ethanol (0.5 mL), and placed in a 10 mL sterile vial to dilute to the desired formulation. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic (5μ, C18, 50×3mm) (Phenomenex) at a flow rate of 1 mL / min. The mobile phase was a mixture of acetonitrile / 0.1% trifluoroacetic acid in water (from 10% to 90% in 10 minutes). 18 The concentration of [F]-89 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 89, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-89 was 5.1 minutes.

[0303] [ 18 Synthesis of F-112 [ka] A solution of NN-5 (1.4 mg, 2.40 μmol) in DMF (0.3 mL) is dried [ 18The reaction mixture was added to a microwave vial containing [F] fluoride, the vent line was removed, and the reaction mixture was heated at 120°C (110W) for 3 minutes. After cooling to below 50°C, the reaction mixture was diluted with H2O (0.8 mL), mixed, and injected into a semi-preparative HPLC column. The product was purified using a Zorbax Eclipse XDB-C18 (Agilent) (5 μm, 9.4 × 250 mm) HPLC column at a flow rate of 5 mL / min. The mobile phase was acetonitrile / Na2HPO4 (10 mM) (from 30% to 70% in 15 minutes). The radioactive fraction eluted between 14 and 15 minutes was collected in a flask containing 30% β-cyclodextrin solution (1 mL), evaporated under negative pressure, diluted with physiological saline, and transferred to a sterile container. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic (5μ, C18, 50×3mm) (Phenomenex) at a flow rate of 1.5 mL / min. The mobile phase was a mixture of acetonitrile / 0.1% formic acid in water (from 5% to 50% in 7 minutes). 18 The concentration of [F]-112 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 112, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-112 was 6.0 minutes.

[0304] [ 18 Synthesis of F-113 [ka] [ 18 [F] fluoride was concentrated on an anion exchange resin. This anion exchange resin was pre-treated before use by flushing with EtOH (10 mL), followed by flushing with 0.5 M KOTf in H2O (10 mL) and H2O (10 mL).

[0305] [ 18An anion exchange resin containing [F] fluoride was eluted with tetrabutylammonium triflate (7.5 mg, 19 mmol) and cesium carbonate (0.1 mg, 0.3 mmol) in H2O (0.5 mL), followed by elution with CH3CN (1.0 mL), and placed in a vented 2.5 mL V-shaped vial. The vial was dried under argon flow by heating at normal temperatures of 100 °C. Additional aliquots of CH3CN (2 × 0.5 mL) were added for azeotropic drying. The V-vial was flushed with air from a syringe (10 mL), heated to 120 °C, and then a solution of OO-7B (2.0 mg, 4.2 mmol), tetrakis(pyridine)copper(II) triflate (11.3 mg, 17 mmol), and pyridine (32 mL, 40 mmol) in 1,3-dimethyl-2-imidazolidinone (DMI; 0.5 mL) was added. The reaction mixture was heated at 120°C for 20 minutes, then transferred to a vial containing 10 mM Na2HPO4 (pH 7.4) (1.0 mL) in 10% CH3CN / H2O at room temperature, diluted, mixed, and injected into a semi-preparative HPLC column. The product was purified using a Gemini (C18, 5 mm, 110A, 150 × 10 mm) HPLC column (Phenomonex) at a flow rate of 5 mL / min, mobile phase CH3CN / 10 mM Na2HPO4 (pH 7.4), and gradient 30-50%. The radioactive fraction eluted between 16.3 and 16.4 minutes was collected in a round-bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure, and transferred to a 10 mL sterile vial. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with a Poroshell 120 (4mm EC-C18 100×4.6mm) HPLC column (Agilent) at a flow rate of 1.5 mL / min and a mobile phase of CH3CN / 10mM NH4OAc (pH 8.1) with a 35-45% gradient. 18 The concentration of [F]-113 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 113, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-113 was 6.8 minutes.

[0306] [By isotope exchange] 18 Synthesis of F-115 [ka] [ 18 An anion exchange resin containing [F] fluoride was eluted with tetraethylammonium bicarbonate (4.2 mg, 22 mmol) in CH3CN / H2O (1:1) (1.0 mL), followed by elution with CH3CN (0.5 mL). The mixture was placed in a vented 2.5 mL V-shaped vial and dried under argon flow at normal heating temperature of 100 °C. Additional aliquots of CH3CN (2 × 0.5 mL) were added for azeotropic drying. 18 A vial containing [F]Et4NF was heated to 130°C, and then a solution of 115 (0.3 mg, 0.6 mmol) in DMSO (0.5 mL) was added. The reaction mixture was heated at 130°C for 10 minutes, then transferred to a vial containing H2O (0.8 mL) at room temperature, diluted, mixed, and injected into a semi-preparative HPLC column. The product was purified using a Zorbax XDB-C18 (5 mm, 150 × 9.4 mm) HPLC column (Agilent) at a flow rate of 5 mL / min with a mobile phase of 30% CH3CN / 10 mM Na2HPO4 (pH 7.4). The radioactive fraction eluted between 14.3 and 14.7 minutes was collected in a round-bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure to remove CH3CN, and transferred to a 10 mL sterile vial. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with a Poroshell 120 (4mm EC-C18 100×4.6mm) HPLC column (Agilent) at a flow rate of 1.5 mL / min and a mobile phase of CH3CN / 10mM NH4OAc (pH 8.0) with a 30-40% gradient. 18 The concentration of [F]-115 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 115, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-115 was 4.5 minutes.

[0307] [ 18 Synthesis of F-116 [ka]

[0308] [ 18 [F] fluoride was concentrated on an anion exchange resin. Before use, this anion exchange resin was pre-treated by flushing with EtOH (10 mL), followed by flushing with 0.5 M K3PO4 in H2O (10 mL) and H2O (10 mL).

[0309] [ 18 An anion exchange resin containing [F] fluoride was eluted with tetrabutylammonium mesylate (6.8 mg, 20 mmol) in CH3CN / H2O (1:1) (1.0 mL), followed by elution with CH3CN (0.5 mL). The mixture was placed in a vented 2.5 mL V-shaped vial and dried under argon flow at normal heating temperature of 100 °C. Additional aliquots of CH3CN (2 × 0.5 mL) were added for azeotropic drying. 18A vial containing [F]Bu4NF was heated to 120°C, and then a solution of PP-3 (0.9 mg, 1.5 mmol) in DMSO / isoamyl alcohol (1:1) (0.5 mL) was added. The reaction mixture was heated at 120°C for 10 minutes, then transferred to a vial containing H2O (1.0 mL) at room temperature, diluted, mixed, and injected into a semi-preparative HPLC column. The product was purified using a Zorbax XDB-C18 (5 mm, 150 × 9.4 mm) HPLC column (Agilent) at a flow rate of 5 mL / min with 10 mM Na2HPO4 (pH 7.4) in a mobile phase of 30% CH3CN / H2O. The radioactive fraction eluted between 21.5 and 22.1 minutes was collected in a round-bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure to remove CH3CN, and transferred to a 10 mL sterile vial. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with a Poroshell 120 (4mm EC-C18 100×4.6mm) HPLC column (Agilent) at a flow rate of 1.5 mL / min and a mobile phase of CH3CN / 10mM NH4OAc (pH 8.0) with a gradient of 5-95%. 18 The concentration of [F]-116 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 116, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-116 was 6.6 minutes.

[0310] [By isotope exchange] 18 Synthesis of F-134 [ka] The solution of 134 (1.2 mg, 2.40 μmol) in DMF (0.3 mL) was dried [ 18The reaction mixture was added to a microwave vial containing [F] fluoride, the vent line was removed, and the reaction mixture was heated at 140°C (25W) for 3 minutes. After cooling to below 50°C, the reaction mixture was diluted with H2O (0.8 mL), mixed, and injected into a semi-preparative HPLC column. The product was purified using a Zorbax Eclipse XDB-C18 (Agilent) (5 μm, 9.4 × 250 mm) HPLC column at a flow rate of 5 mL / min. The mobile phase was acetonitrile / Na2HPO4 (10 mM) from 40 to 70% after 15 minutes. The radioactive fraction eluted between 10 and 10.8 minutes was collected in a flask containing 30% β-cyclodextrin solution (1 mL), evaporated under negative pressure, diluted with physiological saline, and transferred to a sterile container. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic (5μ, C18, 50×3mm) (Phenomenex) at a flow rate of 1.5 mL / min. The mobile phase was a mixture of acetonitrile / 0.1% formic acid in water (from 5% to 90% in 7 minutes). 18 The concentration of [F]-134 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 134, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F]-134 was 4.4 minutes.

[0311] [By isotope exchange] 18 Synthesis of F-138 [ka] [ 18 An anion exchange resin containing [F] fluoride was eluted with tetraethylammonium bicarbonate (3.9 mg, 19 mmol) in CH3CN / H2O (1:1) (1.0 mL), followed by elution with CH3CN (0.5 mL). The mixture was placed in a vented 2.5 mL V-shaped vial and dried under argon flow at normal heating temperature of 100 °C. Additional aliquots of CH3CN (2 × 0.5 mL) were added for azeotropic drying.18 A vial containing [F]Et4NF was heated to 130°C, and then a solution in 138 (0.2 mg, 0.4 mmol) DMSO (0.5 mL) was added. The reaction mixture was heated at 130°C for 10 minutes, then transferred to a vial containing 10 mM Na2HPO4 (pH 7.4) (0.8 mL) in 10% CH3CN / H2O at room temperature, diluted, mixed, and injected into a semi-preparative HPLC column. The product was purified using a Zorbax XDB-C18 (5 mm, 150 × 9.4 mm) HPLC column (Agilent) at a flow rate of 5 mL / min with 10 mM Na2HPO4 (pH 7.4) in 35% CH3CN / H2O mobile phase. The radioactive fraction eluted between 16.7 and 17.2 minutes was collected in a round-bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure to remove CH3CN, diluted with physiological saline, and transferred to a 10 mL sterile vial. The final product was tested for chemical and radiochemical purity using an analytical HPLC system (Agilent) with a Poroshell 120 (4 mm EC-C18 100 × 4.6 mm) HPLC column (Agilent) at a flow rate of 1.5 mL / min and a mobile phase of CH3CN / 10 mM NH4OAc (pH 8.0) with a 35-45% gradient. 18 The concentration of [F]-138 was measured using an ultraviolet detector (254 nm). The characteristics of the product were confirmed by simultaneous injection of a sample of compound 138, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time for F-138 was 5.6 minutes.

[0312] While the present invention has been described and illustrated with reference to specific embodiments thereof, those skilled in the art will understand that various adaptations, changes, modifications, substitutions, deletions, or additions to the procedures and protocols can be made without departing from the spirit and scope of the invention. Accordingly, the present invention is defined by the claims that follow below, and such claims are intended to be interpreted broadly, insofar as they are reasonable.

Claims

1. Formula I: 【Chemistry 1】 [During the ceremony, R is H, -C １－６ Alkyl, OR ｃ Alternatively, it is independently selected from the halo, where the alkyl is -C １－６ Alkyl, OR ｃ Alternatively, it may be substituted with 1 to 3 groups selected from the halo; R ａ -C is either not substituted or is substituted. １－６ A alkyl group is independently selected, where the alkyl group may be substituted with one to three groups of R; R ｂ is selected independently from -C １－６ alkyl, halo, -(CH ２ ), ｎ OR ｃ , -CN, -NR ｃ ２ , -(CH ２ ), ｎ halogen or -O(CH ２ ), ｎ halo; R ｃ is H or -C １－６ Selected independently of alkyl, where the alkyl is -C １－６ Alkyl, OR ｄ Alternatively, it may be substituted with 1 to 3 groups selected from the halo; R ｄ is H or -C １－６ Selected independently of alkyl; R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, - (CH ２ ) ｎ O(CH ２ ) ｎ OR ｃ Hello, NR ２ , C that is not substituted or has been substituted １－６ Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C ３ -C １０ A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl is independently selected from cycloalkyl, cycloalkyl, phenyl, heteroaryl, or heterocyclyl, where R ｂ It can be substituted with 1 to 3 of the following groups; R ２ is hydrogen, OR ｃ NO ２ , Halo or C １－６ Selected from alkyl groups; Ring A １ This is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A ２ is selected from pyrimidinyl, pyridyl, or pyrazinyl, where the pyrimidinyl, pyridyl, or pyrazinyl may be substituted with groups 1 to 3 of R; Ring A ３ is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, imidazopyridinyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; Ring B is, 【Chemistry 2】 Selected from; m is selected from 1, 2, or 3; n is independently selected from 0, 1, 2, 3, or 4; p is selected from 0, 1, 2, or 3; and, [q is selected from 1, 2, or 3] A compound represented by or a pharmaceutically acceptable salt thereof.

2. Formula IA: 【Transformation 3】 [During the ceremony, R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, - (CH ２ ) ｎ O(CH ２ ) ｎ OR ｃ Hello, -NR ２ , C that is not substituted or has been substituted １－６ Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C ３ -C １０ A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl is independently selected from cycloalkyl, cycloalkyl, phenyl, heteroaryl, or heterocyclyl, where R ｂ It can be substituted with 1 to 3 of the following groups; R ２ is hydrogen, OR ｃ , Halo or C １－６ Selected from alkyl groups; Ring A １ This is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A ３ This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; m is selected from 1, 2, or 3; n is independently selected from 0, 1, 2, 3, or 4; and, p is selected from 0, 1, 2, or 3; and R, R ａ , R ｂ , R ｃ , R ｄ and ring A ２ This is the same as in claim 1. A compound according to claim 1 or a pharmaceutically acceptable salt thereof having the structure represented by .

3. Ring A １ It is selected from pyridyl, pyrazinyl, or pyrimidinyl; Ring A ３ This is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl; m is selected from 1 or 2; and, p is selected from 0, 1, or 2; and, Ring A ２ This is the same as claim 1. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

4. Formula IB: 【Chemistry 4】 [During the ceremony, R is H, -C １－６ A molecule independently selected from alkyl or halo, where the alkyl is -C １－６ Alkyl, OR ｃ Alternatively, it may be substituted with 1 to 3 groups selected from the halo; R ａ -C is either not substituted or is substituted. １－６ It is an alkyl group, where the alkyl group may be substituted with R1 to R3 groups; R ｂ is selected independently from -C １－６ alkyl, halo, -(CH ２ ) ｎ OR ｃ , -CN, -NR ｃ ２ , -(CH ２ ) ｎ halogen or -O(CH ２ ) ｎ halo; R ｃ is independently selected from H or -C １－６ alkyl; R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, - (CH ２ ) ｎ O(CH ２ ) ｎ OR ｃ Hello, NR ２ , C that is not substituted or has been substituted １－６ Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C ３ -C １０ Selected from cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl, where the alkyl, phenyl, cycloalkyl, heteroaryl, or heterocyclyl is R ｂ It can be substituted with 1 to 3 of the following groups; R ２ is hydrogen, OR ｃ , Halo or C １－６ Selected from alkyl groups; Ring A １ This is selected from pyridyl, pyrazinyl, pyrazolyl, oxazolyl, thiazolyl, or pyrimidinyl; Ring A ３ This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, pyrrolopyrazinyl, oxadiazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine, or phenyl; n is independently selected from 0, 1, 2, 3, or 4; p is selected from 1 or 2; and Ring A ２ This is the same as in claim 1. A compound according to claim 1 or a pharmaceutically acceptable salt thereof having the structure represented by .

5. Formula IC: 【Transformation 5】 [During the ceremony, R is H, -C １－６ A molecule independently selected from alkyl or halo, where the alkyl is -C １－６ Alkyl, OR ｃ Alternatively, it may be substituted with 1 to 3 groups selected from the halo; R ａ -C is either not substituted or is substituted. １－６ A alkyl group is independently selected, where the alkyl group may be substituted with one to three groups of R; R ｂ is, -C １－６ Alkyl, halo, -(CH ２ ) ｎ OR ｃ ,-CN,-(CH ２ ) ｎ Halogen or -O(CH) ２ ) ｎ Selected independently from Halo; R ｃ is H or -C １－６ Selected independently of alkyl; R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, - (CH ２ ) ｎ O(CH ２ ) ｎ OR ｃ Hello, NR ２ , C that is not substituted or has been substituted １－６ Alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C ３ -C １０ A cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocycline is independently selected from cycloalkyl, phenyl, cycloalkyl, heteroaryl, or heterocycline, where R ｂ It can be substituted with 1 to 3 of the following groups; R ２ is hydrogen, OR ｃ , Halo or C １－６ Selected from alkyl groups; Ring A １ This is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A ３ This is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, imidazolyl, pyrrolopyrazinyl, oxadiazolyl, triazolyl, thiazolyl, isoxazolyl, oxazolyl, 3,4-dihydro-2H-pyrido[3,2,b][1,4]oxazine or phenyl; n is independently selected from 0, 1, 2, 3, or 4; p is selected from 0, 1, 2, or 3; and Ring A ２ This is the same as in claim 1. A compound according to claim 1 or a pharmaceutically acceptable salt thereof having the structure represented by .

6. Ring A ２ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein is a pyrimidinyl, wherein the pyrimidinyl may be substituted with groups R1 to R3.

7. Ring A ３ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl.

8. Ring A ３ is a compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from pyridyl, pyrazinyl, or phenyl.

9. Ring A １ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from pyridyl, pyrazinyl, pyrazolyl, or pyrimidinyl.

10. Ring A １ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from pyridyl or pyrazinyl.

11. R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, - (CH ２ ) ｎ O(CH ２ ) ｎ OR ｃ Hello, -NR ２ , C that is not substituted or has been substituted １－６ Selected from alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicycloheptanyl, furyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridadinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolylpyrazolyl, morpholinyl, tetrazolyl, or piperazinyl. Here, the alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicycloheptanyl, furyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolylpyrazolyl, morpholinyl, tetrazolyl, or piperazinyl is R ｂ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which can be substituted with 1 to 3 of the groups.

12. R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, -NR ２ Selected from pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl, where pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl is R ｂ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which can be substituted with 1 to 3 of the groups.

13. R is H, -C １－６ A molecule independently selected from alkyl or halo, where the alkyl is -C １－６ Alkyl, OR ｃ Alternatively, it may be substituted with 1 to 3 groups selected from the halo; R ａ -C is either not substituted or is substituted. １－６ A alkyl group is independently selected, where the alkyl group may be substituted with one to three groups of R; R ｂ is, -C １－６ Alkyl, halo, -(CH ２ ) ｎ OR ｃ ,-CN,-(CH ２ ) ｎ Halogen or -O(CH) ２ ) ｎ Selected independently from Halo; R ｃ is H or -C １－６ Selected independently of alkyl; R １ is, -(CH ２ ) ｎ OR ｃ ,-(CH ２ ) ｎ O(CH ２ ) ｎ R, -NR ２ Selected from pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl, where pyridyl, pyrazolyl, azetidinil, pyrrolidinil, or furyl is R ｂ It can be substituted with 1 to 3 of the following groups; R ２ is hydrogen, OR ｃ , Halo or C １－６ Selected from alkyl groups; Ring A １ This is selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl; Ring A ２ is pyrazinyl, where the pyrazinyl may be substituted with groups R1 to R3; Ring A ３ It is selected from pyrimidinyl, pyridyl, or pyrazinyl; n is independently selected from 0, 1, 2, 3, or 4; and, p is selected from 0, 1, 2, or 3; The compound according to claim 4 or a pharmaceutically acceptable salt thereof.

14. the below described: Table 1 A compound selected from or a pharmaceutically acceptable salt thereof.

15. A compound according to claim 14 or a pharmaceutically acceptable salt thereof, selected from Example numbers 1, 6, 9, 11, 12, 37, 39, 47, 51, 57, 58, 64, 72, 75, 78, 79, 80, 91, 96, 112, 113, 115, 116, 118, 134, 138 and 141; Table 2

16. A compound according to claim 14 or a pharmaceutically acceptable salt thereof, selected from Examples No. 39, 47, 51, 78, 79, 96, 112, 116 and 141; Table 3

17. A pharmaceutical composition comprising the compound described in claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

18. A composition comprising the compound described in claim 1 or a pharmaceutically acceptable salt thereof, for use as an imaging agent.

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