Alpha-synuclein binding agents and methods of use

Abstract

The present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, which can be suitable for imaging alpha-synuclein pathology-and thus for binding and imaging alpha-synuclein aggregates in patients suffering from Parkinson's disease. More particularly, the invention relates to a method for studying alpha-synuclein in the brain in vivo in positron emission tomography (PET) imaging using the compounds of the invention as tracers to allow the diagnosis of Parkinson's disease and other neurodegenerative diseases characterized by alpha-synuclein pathology. The invention also relates to a method of measuring the clinical efficacy of a therapeutic agent for Parkinson's disease and other neurodegenerative diseases characterized by alpha-synuclein pathology.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of or priority to U.S. Provisional Application No. 63 / 485,159, filed February 15, 2023, the entire contents of which are incorporated herein by reference. BACKGROUND

[0002] Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, amyotrophic lateral sclerosis, and prion diseases, are debilitating diseases that affect cognition and / or muscle control. These diseases are a subset of protein misfolding diseases. Protein folding is an essential process for protein function in all organisms, and conditions that disrupt protein folding pose a threat to cell viability. In some cases, disease onset is because a particular protein no longer functions when it adopts a misfolded state. In other diseases, the pathogenic state arises because misfolding occurs simultaneously with aggregation, and the underlying aggregation is deleterious. Although neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are caused by different proteins, both involve the accumulation of insoluble fibrillar protein deposits called amyloids. For example, Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), collectively known as “synucleinopathies,” are associated with the accumulation of aggregated forms of alpha-synuclein in neurons of the brain (see Nat. Rev. Neuro. 2013, 9 , 13-24 and J. Parkinson’s Disease 2013 , 3 , 565-567). As the cardinal neuropathological change in PD, the degeneration of dopaminergic neurons occurs in the substantia nigra, as well as Lewy bodies (LBs) and Lewy neurites (LN). To date, the pathogenic mechanisms of PD have not been fully discovered.

[0003] Alpha-synuclein is a presynaptic terminal protein consisting of 140 amino acids that plays important roles in the central nervous system, including synaptic vesicle recycling and synthesis, vesicle storage, and neurotransmitter release. It is specifically upregulated in discrete populations of presynaptic terminals in the brain during acquisition of relevant synaptic rearrangements. Alpha-synuclein naturally exists in a highly soluble, unfolded state. Evidence suggests that filamentous aggregates of alpha-synuclein accumulate at presynaptic membranes and trigger synaptic dysfunction and neuronal cell death in synucleinopathies, and can be the cause of Parkinson’s disease and DLB. Alpha-synuclein aggregation into the main component of Lewy bodies, microscopic protein deposits in deteriorating nerve cells, has been identified by antibody immunohistochemical studies. Accumulation of misfolded fibrillar alpha-synuclein in Lewy bodies (LBs) and Lewy neurites (LN) is considered a hallmark of PD.

[0004] Diagnosis of PD is mainly based on clinical symptoms such as resting tremor, bradykinesia, and rigidity, although these methods have their limitations (see J. Neurology 2019, 266 , 1927-1936). The current desired treatment for PD is to slow disease progression and minimize the disease symptoms in patients. Thus, a method to diagnose PD at a very early stage can greatly help physicians to design a treatment paradigm accordingly and slow disease progression. There is still a need for improved diagnostic methods for identifying aggregation of misfolded proteins, including alpha-synuclein, for early detection and continued monitoring of PD in a subject (see J. Parkinson’s Disease 2013, 3 , 565-567).

[0005] Alpha-synuclein positron emission tomography (PET) tracers would be a valuable non-invasive diagnostic biomarker for spatial and temporal quantification of aggregated pathological alpha-synuclein in the human brain as a biomarker for Parkinson’s disease. In addition, alpha-synuclein PET tracers can be used for patient selection for PD clinical trials. In this modality, alpha-synuclein tracers can be developed as companion diagnostic agents for co-registration of therapeutic agents. In addition, alpha-synuclein PET tracers can be a disease relevant tool for quantification of stabilization or reduction of alpha-synuclein formation for key disease improvement PD therapeutics.

[0006] Thus, there is a need for a neuroimaging radiotracer that allows for in vivo imaging of alpha-synuclein pathology, providing insight into alpha-synuclein aggregate deposition in the human brain. A successful neuroimaging radiotracer must cross the blood-brain barrier, clear rapidly from tissue and plasma, and have high affinity and specificity for alpha-synuclein aggregates with high selectivity over binding to beta-amyloid and tau aggregation proteins that 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 alpha synuclein binding ligands have been described with reduced selectivity over aggregated beta-amyloid (WO 2019 / 121661), there is a need for compounds with high levels of selectivity over co-expressed aggregation proteins in PD in order to quantify alpha synuclein specific signal in in vivo imaging studies in PD patients.

[0007] The present invention facilitates these interests by providing compounds of Formula I as binding ligands for aggregated alpha-synuclein with high selectivity over binding to aggregated beta-amyloid pathology. The present invention also relates to a method of using compounds of Formula I as tracers in PET imaging to study alpha-synuclein deposits in the brain in vivo, thereby allowing diagnosis of neurodegenerative diseases characterized by alpha-synuclein pathology. The present invention also relates to a method of measuring the clinical efficacy of a therapeutic agent targeting alpha-synuclein pathology.

[0008] SUMMARY The present invention relates to compounds of Formula I, pharmaceutically acceptable salts thereof, pharmaceutical compositions containing them, diagnostic and therapeutic uses, and methods of preparing these compounds. One embodiment of the present invention provides compounds of Formula I: I or pharmaceutically acceptable salts thereof, wherein: R is independently selected from H, -Ci-6alkyl, OR c or halo, wherein the alkyl is optionally substituted with one to three groups from -Ci-6alkyl, OR c or halo; Ra is independently selected from unsubstituted or substituted -Ci-6alkyl, the alkyl being optionally substituted with 1 to 3 R groups; Rb is independently selected from -Ci-6alkyl, halo, -(CH2)nOR c , -CN, -NRc2, -(CH2)nhalo, or -O(CH2)nhalo; Rc is independently selected from H or -Ci-6alkyl, wherein the alkyl is optionally substituted with one to three groups from Ci-6alkyl, OR d or halo; Rd is independently selected from H or -Ci-6alkyl; R1 is independently selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c , halo, NR2, unsubstituted or substituted Ci-6alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl, wherein the alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted with one to three Rb groups; R2 is selected from hydrogen, OR c , NO2, halo, or -Ci-6alkyl; Ring A 1selected from pyridinyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; Ring A 2 selected from pyrimidinyl, phenyl, pyridinyl, pyrazinyl, or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, pyrazinyl, or pyridazinyl is optionally substituted with 1 to 3 R groups; Ring A 3 selected from pyridinyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl, or pyrimidinyl; 2H - pyrido[3,2, b [1,4]oxazine or phenyl; Ring B is selected from a) , b) or c) ; 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; The present application also relates to isotopically-labeled compounds of Formula I. In addition, the present application provides pharmaceutical compositions comprising a compound of Formula I and at least one pharmaceutically acceptable carrier.

[0009] The present application relates to compounds of Formula I, which are useful for binding to alpha-synuclein aggregated proteins and / or tau aggregated proteins, and thus for binding and imaging of alpha-synuclein aggregated protein pathology in PD and non-PD synucleinopathy patients, as well as aggregated Tau protein pathology in Alzheimer’s Disease (AD) and non-AD tauopathy patients via PET imaging techniques generally known in the art (see J. Nucl. Med. 2019, 60 , 93-99 and 107-114). The present application also relates to methods of using compounds of Formula I to identify patients with abnormal levels of aggregated alpha-synuclein pathology in the brain. The present application also relates to methods of using compounds of Formula I as biomarkers to measure the progression of alpha-synuclein pathology over time for the clinical evaluation of potential therapeutic agents that can alter the progression of Parkinson’s Disease.

[0010] The compounds of the present application can also be used for imaging and detection of other neurodegenerative diseases characterized by deposition of alpha-synuclein aggregates, such as multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). BRIEF DESCRIPTION OF DRAWINGS Figure 1 : against 3Saturation binding experiments of [H]-105 in aggregated β-amyloid- rich AD tissue homogenates.

[0012] Figure 2 Saturation binding experiments of [H]-105 in aggregated β-amyloid- rich AD tissue homogenates. 3 Saturation binding experiments of [H]-1 in tritium nuclear insoluble fractions from PD tissue homogenates rich in aggregated α-synuclein.

[0013] Figure 3 Saturation binding experiments of [H]-1 in tritium nuclear insoluble fractions from PD tissue homogenates rich in aggregated α-synuclein. 3 [H]-1 radioligand saturation binding data.

[0014] Figure 4 [H]-1 radioligand saturation binding data. 3 [H]-1 radioligand saturation binding data.

[0015] Figure 5 [H]-24 radioligand saturation binding data. 3 [H]-24 radioligand saturation binding data.

[0016] Figure 6 [H]-24 radioligand saturation binding data. 3 [H]-24 radioligand saturation binding data. DETAILED DESCRIPTION The present invention provides novel substituted heterocyclic piperazine amide compounds, synthetic methods for making the compounds, pharmaceutical compositions containing them, isotopically labeled compounds, and methods of using the compounds as imaging agents.

[0018] In one embodiment, the present invention relates to a compound of Formula I: I or a pharmaceutically acceptable salt thereof, wherein: R is independently selected from H, -C1-6alkyl, OR c or halo, wherein the alkyl is optionally substituted with one to three groups selected from -C1-6alkyl, OR c or halo; Ra is independently selected from unsubstituted or substituted -C1-6alkyl, the alkyl being optionally substituted with 1 to 3 R groups; Rb is independently selected from -C1-6alkyl, halo, -(CH2)nOR c , -CN, -NRc2, -(CH2)nhalo, or -O(CH2)nhalo; Rc is independently selected from H or -Ci-6alkyl, wherein said alkyl is optionally substituted with one to three groups independently selected from -Ci-6alkyl, OR d or halo; Rd is independently selected from H or -Ci-6alkyl; R1 is independently selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c , halo, NR2, unsubstituted or substituted Ci-6alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclyl, wherein said alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted with one to three Rb groups; R2 is selected from hydrogen, OR c , NO2, halo or -Ci-6alkyl; 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, wherein said pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl is optionally substituted with 1 to 3 R groups; 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 selected from a) , b) or c) ; 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; In a further embodiment, the present application relates to a compound of formula IA, IA or pharmaceutically acceptable salts thereof, wherein: R is independently selected from H, -Ci-6alkyl or halo, wherein said alkyl is optionally substituted with one to three groups independently selected from -Ci-6alkyl, ORc or halo-substituted groups; Ra is independently selected from unsubstituted or substituted -Ci-6alkyl or -(CH2)i-3O-(CH2)o-3R, said alkyl being optionally substituted with one to three R groups; Rb is independently selected from -Ci-6alkyl, halo, -(CH2)nOR c -CN, -NRc2, -(CH2)nhalo or -O(CH2)nhalo; Rc is independently selected from H or -Ci-6alkyl, wherein said alkyl is optionally substituted with one to three groups from -Ci-6alkyl, OR d or halo-substituted groups; Rd is independently selected from H or -Ci-6alkyl; R1 is independently selected from -(CH2)nOR c -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c halo, -NR2, unsubstituted or substituted Ci-6alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl or unsubstituted or substituted heterocyclyl, wherein said alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted with one to three Rb groups; R2 is selected from hydrogen, OR c halo or -Ci-6alkyl; Ring A 1 is selected from pyridinyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridinyl, pyrazinyl or pyridazinyl, wherein said pyrimidinyl, phenyl, pyridinyl, pyrazinyl or pyridazinyl is optionally substituted with one to three R groups; Ring A 3 is selected from pyridinyl, 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.

[0019] In a further embodiment, the present application relates to a compound of Formula IA, or a pharmaceutically acceptable salt thereof, wherein: R is independently selected from H, -Ci-6alkyl, OR cor halo, wherein the alkyl is optionally substituted with one to three groups from -C1-6alkyl, OR c or halo; Ra is independently selected from unsubstituted or substituted -C1-6alkyl or -(CH2)i-3O-(CH2)o-3R, the alkyl is optionally substituted with 1 to 3 R groups; Rb is independently selected from -C1-6alkyl, halo, -(CH2)nOR c , -CN, -NRc2, -(CH2)nhalo or -O(CH2)nhalo; Rc is independently selected from H or -C1-6alkyl, wherein the alkyl is optionally substituted with one to three groups from -C1-6alkyl, OR d or halo; Rd is independently selected from H or -C1-6alkyl; R1 is independently selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c , halo, -NR2, unsubstituted or substituted C1-6alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl or unsubstituted or substituted heterocyclyl, wherein the alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted with one to three Rb groups; R2 is selected from hydrogen, OR c , -NO2, halo or -C1-6alkyl; 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, wherein the pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl is optionally substituted with 1 to 3 R groups; 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; 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; In a further embodiment, the present invention relates to a compound of formula IA or a pharmaceutically acceptable salt thereof, wherein R is independently selected from H, -C1-6 alkyl, OR c Or halogenated, wherein the alkyl group is optionally converted by one to three derivatives from -C1-6 alkyl, OR c Or substituted with halogenated groups; Ra is independently selected from unsubstituted or substituted -C1-6 alkyl groups, wherein the alkyl group is optionally substituted by 1 to 3 R groups; Ring A 1 Selected from pyridyl, pyrazinyl, or pyrimidinyl; Ring A 2 Selected from pyrimidinyl, phenyl, or pyridinyl, wherein the pyrimidinyl, phenyl, or pyridinyl group is optionally substituted with 1 to 3 R groups; Ring A 3 Selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl; m is selected from 1 or 2; p is selected from 0, 1, or 2; Furthermore, all other substituents and variables are as defined above in Equation I.

[0020] In another embodiment, the present invention relates to compounds of formula IB: IB Or its medicinal salts, wherein: R is independently selected from H, -C1-6 alkyl, or halogenated, wherein the alkyl group is optionally divided by one to three derivatives from -C1-6 alkyl, OR c Or substituted with halogenated groups; Ra is an unsubstituted or substituted -C1-6 alkyl group, wherein the alkyl group is optionally substituted by 1 to 3 R groups; Rb is independently selected from -C1-6 alkyl, halogenated, and -(CH2)nOR. c -CN, -(CH2)n halogens or -O(CH2)n halogens; Rc is independently selected from H or -C1-6 alkyl; R1 is selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c Halogenated, NR2, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 Cycloalkyl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted heterocyclic group, wherein the alkyl, phenyl, cycloalkyl, heteroaryl, or heterocyclic group may be substituted with one to three Rb groups; R2is selected from hydrogen, OR c , halo or -Ci-6alkyl; Ring A 1 is selected from pyridyl, pyrazinyl, pyrazolyl, oxazolyl, thiazolyl or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridyl, pyrazinyl or pyridazinyl is optionally substituted with 1 to 3 R groups; Ring A 3 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 application relates to a compound of Formula IC: IC or pharmaceutically acceptable salts thereof, wherein: R is independently selected from H, -Ci-6alkyl or halo, wherein the alkyl is optionally substituted with one to three groups from -Ci-6alkyl, OR c or halo; Ra is independently selected from unsubstituted or substituted -Ci-6alkyl, the alkyl being optionally substituted with 1 to 3 R groups; Rb is independently selected from -Ci-6alkyl, halo, -(CH2)nOR c , -CN, -(CH2)nhalo or -0(CH2)nhalo; Rc is independently selected from H or -Ci-6alkyl; R1is independently selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR c , halo, NR2, unsubstituted or substituted Ci-6alkyl, unsubstituted or substituted phenyl, unsubstituted or substituted C3-C 10 cycloalkyl, unsubstituted or substituted heteroaryl or unsubstituted or substituted heterocyclyl, wherein the alkyl, phenyl, cycloalkyl, heteroaryl or heterocyclyl can be substituted with one to three Rbgroups; R2is selected from hydrogen, OR c , halo or -Ci-6alkyl; Ring A 1 is selected from pyridyl, pyrazinyl, thiazolyl, pyrazolyl, oxazolyl or pyrimidinyl; Ring A 2 It is selected from pyrimidinyl, phenyl, pyridinyl, pyrazinyl or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, pyrazinyl or pyridazinyl is optionally substituted by 1 to 3 R groups; Ring A 3 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 of formula IC or a pharmaceutically acceptable salt thereof, wherein: R is independently selected from H, -C1-6 alkyl, or halogenated, wherein the alkyl group is optionally divided by one to three derivatives from -C1-6 alkyl, OR c Or substituted with halogenated groups; Ra is independently selected from unsubstituted or substituted -C1-6 alkyl groups, wherein the alkyl group is optionally substituted by 1 to 3 R groups; Rb is independently selected from -C1-6 alkyl, halogenated, and -(CH2)nOR. c -CN, -(CH2)n halogens or -O(CH2)n halogens; Rc is independently selected from H or -C1-6 alkyl; R1 is selected from -(CH2)nOR c -(CH2)nO(CH2)nR, -NR2, pyridinyl, pyrazolyl, aziridine, pyrrolidinyl or furanyl, wherein the pyridinyl, pyrazolyl, aziridine, pyrrolidinyl or furanyl may optionally be substituted with one to three Rb groups; R2 is selected from hydrogen, OR c , halogenated or -C1-6 alkyl; Ring A 1 Selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl; Ring A 2 Selected from pyrimidinyl, pyridinyl, or pyrazinyl, wherein the pyrimidinyl, pyridinyl, or pyrazinyl is optionally substituted with 1 to 3 R groups; Ring A 3 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 application provides a compound of Formula I, IA, IB, or IC, wherein ring A is 1 selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl. In another embodiment, ring A is 1 selected from pyridyl, pyrazinyl, pyrazolyl, or pyrimidinyl. In another embodiment, ring A is 1 selected from pyridyl or pyrazinyl.

[0024] In one embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein ring A is 2 selected from pyrimidinyl, pyridyl, or pyrazinyl, wherein said pyrimidinyl, pyridyl, or pyrazinyl is optionally substituted with one to three R groups. In one embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein ring A is 2 selected from pyrimidinyl or pyrazinyl, wherein said pyrimidinyl or pyrazinyl is optionally substituted with one to three R groups. In a further embodiment, ring A is 2 pyrimidinyl optionally substituted with one to three R groups. In a further embodiment, ring A is 2 pyrazinyl optionally substituted with one to three R groups.

[0025] In one embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein ring A is 3 selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl. In another embodiment, ring A is 3 selected from pyridyl, pyrazinyl, or phenyl.

[0026] In one embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein Rcis independently selected from H or -Ci-6alkyl, wherein said alkyl is optionally substituted with one to three groups from -Ci-6alkyl, OR d or halo. In another embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein Rcis independently selected from H or -Ci-6alkyl.

[0027] In one embodiment, the application provides a compound of Formula I, IA, IB, or IC, wherein R1is selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -(CH2)nO(CH2)nOR chalo, -NR2, -C1-6alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicyclo-heptanyl, furanyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolopyrazolyl, morpholinyl, tetrazolyl, or piperazinyl, wherein said alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicyclo-heptanyl, furanyl, thiazolyl, pyrimidinyl, oxa-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolopyrazolyl, morpholinyl, tetrazolyl, piperazinyl can be optionally substituted with one to three R b groups.

[0028] In one embodiment, the present application provides a compound of Formula I, IA, IB, or IC, wherein R1is selected from -(CH2)nOR c , -(CH2)nO(CH2)nR, -NR2, pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, furanyl, wherein said pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furanyl can be optionally substituted with one to three R b groups.

[0029] Representative compounds of the present application include a compound selected from the group consisting of:

[0030] or a pharmaceutically acceptable salt thereof.

[0031] The present application relates to compounds of Formula I for use as imaging agents.

[0032] One embodiment of the present application includes a compound selected from the group consisting of Example Nos. 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 a pharmaceutically acceptable salt thereof. A further embodiment of the present application includes a compound selected from the group consisting of Example Nos. 39, 47, 51, 78, 79, 96, 112, 116, and 141, or a pharmaceutically acceptable salt thereof. Another embodiment of the present application includes a compound selected from the group consisting of Example Nos. 96, 112, 113, 115, 116, 118, 134, 138, and 141, or a pharmaceutically acceptable salt thereof. A further embodiment of the present application includes a compound selected from the group consisting of Example Nos. 96, 112, 116, and 141, or a pharmaceutically acceptable salt thereof.

[0033] Another aspect of the application relates to a compound of Formula I, or a pharmaceutically acceptable salt thereof, labeled with an isotope selected from2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36CL,82Br,76Br,77Br,123I,124I, or131I. In a further aspect of the application, the compound of Formula I is isotopically labeled with3H,11C, or18F. Examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to3H-1,3H-24,18F-39,18F-47,18F-51,18F-78,18F-79,11C-94,18F-96,11C-97,18F-116,11C-117,11C-118,18F-141, and11C-143, and the like. Further examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to3H-1,3H-24,18F-39,18F-47,18F-51,18F-78,18F-79,18F-96,18F-116, and18F-141, and the like. Further examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to11C-118, and11C-143, and the like. Further examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to18F-96,18F-116, and18F-141, and the like. Further examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to18F-78,18F-79,18F-96,18F-116, and18F-141, and the like. Further examples of isotopically labeled compounds of Formula I, or a pharmaceutically acceptable salt thereof, include, but are not limited to18F-96,18F-116, and18F-141, and the like.

[0034] Another aspect of the application relates to a compound of Formula I, or a pharmaceutically acceptable salt thereof, labeled with an isotope selected from2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36CL,82Br,76Br,77Br,123I,124I, or131I, for use as an imaging agent.

[0035] In one embodiment, the application provides a pharmaceutical composition comprising a compound of the application, e.g., a compound of Formula I, and at least one pharmaceutical excipient.

[0036] Compounds of Formula I are inhibitors and / or binders of aggregated alpha-synuclein or tau protein. Compounds of Formula I and isotopically-labeled variants thereof are useful in the diagnosis and / or treatment of Parkinson's disease and / or Alzheimer's disease. Methods of detecting labels are well known to those skilled in the art. For example, isotopic labels can be detected using imaging techniques, photographic film, or scintillation counters. 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).

[0037] Compounds of Formula (I) can also form components of bifunctional compounds that are targeted protein degrader compounds that bind aggregated alpha-synuclein protein. Such targeted alpha-synuclein protein degrader compounds contain a target protein binding moiety formed from a compound of Formula (I) and an E3 ubiquitin ligase binding moiety. Targeted alpha-synuclein protein degrader compounds typically contain a linker group that links the alpha-synuclein protein binding moiety and the E3 ubiquitin ligase binding moiety. The E3 ubiquitin ligase-binding moiety in alpha-synuclein targeted protein degrader compounds can be, but is not limited to, a binder of the E3 ligase von Hippel-Lindau protein, a binder of the E3 ligase cereblon protein, or a binder of the MDM2 protein. Such compounds can be administered in pharmaceutical compositions to treat disease conditions, including but not limited to the conditions disclosed herein.

[0038] In the subsequent description, conventional structural representations are employed, and include conventional stereochemical notation for certain asymmetric carbon centers. Thus, structural representations of compounds of the present application include conventional stereochemical notation for some of the asymmetric carbon centers shown in the example compounds. Thus, in this context, a solid black "wedge" bond represents a bond projecting out of the plane of the representation medium, a "hashed wedge" bond represents a bond descending into the plane of the representation medium, and a "wavy" line attached to a carbon with a double bond represents inclusion of both the cis and trans orientations. As is conventional, a plain unbroken line represents all spatial configurations for the bond depicted. Thus, where no particular stereochemical notation is provided, the representation contemplates all stereochemical and spatial orientations of the structural features.

[0039] As shown in and described above in the Examples of the application, the use of conventional "solid wedges" and "open wedges" bond representations structurally indicate specific asymmetric carbon centers. To a large extent, the absolute configuration of the Example compounds has not been determined, but has been assigned by analogy to specific Example compounds of known stereochemical configuration (determined by X-ray crystallography) that were prepared using the same or similar reaction conditions and starting reagents and isolated under the same chromatographic conditions. Thus, the specific assignment of configuration structurally represented herein is intended to identify the particular compound prepared with an excess of one particular stereoisomer, and does not necessarily constitute a statement that the stereochemical structure of the compound is absolutely determined, unless otherwise indicated in the data presented.

[0040] It is understood that when mixtures of isomers are obtained, the mixtures can be separated on a practical basis into individual isomers using conventional methods such as by chromatography or crystallization, or by preparing the described synthesis using stereochemically uniform starting materials, or by stereoselective synthesis, if desired, to produce a single stereoisomer in substantially pure form. Optionally, derivatization can be performed before the isolation of stereoisomers. The separation of stereoisomeric mixtures can be performed as an intermediate step in a

[0041] When indicated herein, the absolute stereochemistry is determined by X-ray crystallography of crystalline products or crystalline intermediates, if appropriate, derivatized with a reagent containing a chiral center of known configuration. Unless otherwise indicated, the application is meant to include all such isomers, as well as mixtures thereof, their racemates, salts, solvates (including hydrates) and solvated salts, of the racemates, enantiomers, and diastereomers.

[0042] When a wavy line terminates a conventional bond (as opposed to connecting two atoms within a structure), it indicates the point of attachment to the rest of the structure, e.g.: indicates that the sec-butyl moiety is bonded via the methylene group via the bond terminated by the wavy line. When letter designations are used to depict substituent moieties, a dash is used to indicate the point of bonding to the indicated substrate, e.g. -CH2-C(O)-CH2Cl indicates that the acetyl chloride moiety is bonded via the methylene portion of the moiety.

[0043] When the compounds of Formula I are capable of tautomerism, all individual tautomers and mixtures thereof are included within the scope of the application.

[0044] When any variable (e.g., R, R 1, n, heteroaryl, alkyl, etc.) at any occurrence in any component or in Formula I are independent of the definition of each occurrence of that variable or moiety unless otherwise indicated at that definition. One of ordinary skill in the art will recognize that the selection of combinations of various substituents (i.e., R 1 , R 2 , etc.) defined in structural representations is selected in compliance with well- known principles of chemical structure connectivity and stability, and that combinations of substituents and / or variables are only permissible if such combinations result in stable compounds.

[0045] A "stable" compound is one that can be prepared and isolated, and that retains its structure and properties for a period of time sufficient to allow use of the compound for the purposes described herein (e.g., therapeutic administration to a subject). The compounds of the present application are limited to stable compounds encompassed by Formula I.

[0046] When any variable or moiety is indicated to occur more than one time in any component or in Formula I, the definition of that variable or moiety at each occurrence is independent of the definition of that variable or moiety at every other occurrence unless otherwise indicated at that definition. 1-4 , including the extremes of the specified ranges (i.e., 1 and 4 in the example) and all integer values therebetween (i.e., 2 and 3 in the example).

[0047] It is understood that reference to "Formula I" also includes compounds of Formula IA, Formula IB, and Formula IC, unless otherwise indicated.

[0048] "Alkyl" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

[0049] "Halo" or "halogen" as used herein means fluoro, chloro, bromo, and iodo.

[0050] "Cycloalkyl" as used herein is intended to include cyclic saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Preferably, cycloalkyl is C3-C10 cycloalkyl. Examples of such cycloalkyl elements include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0051] "Aryl" as used herein is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl, or acenaphthyl. In one embodiment of the application, aryl is phenyl or naphthyl. In another embodiment, aryl is phenyl.

[0052] The term heterocyclyl, heterocyclic, or heterocyclic as used herein represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and one to four heteroatoms selected from the group consisting of N, O, and S, and includes any bicyclic groups in which any of the above-defined heterocyclic rings are fused with benzene rings. The heterocyclic ring can be attached at any heteroatom or carbon atom which results in the creation of a stable structure. When two rings are fused together, the term heterocyclyl, heterocyclic, or heterocyclic can include a heteroaryl moiety. Examples of heterocyclic elements include, but are not limited to, azabicyclo[2.2.1]heptanyl, azacycloheptyl, azacyclobutyl, benzodioxolyl, chromanyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydro-pyrrolo[l,2-b]pyrazolyl, dioxolanyl, imidazolidinyl, indolinyl, isochromanyl, isoindolinyl, morpholinyl, oxa-5-azabicyclo[2.2.1]heptanyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, and thiamorpholinyl.

[0053] In one embodiment, the heterocyclyl is selected from azabicyclo[2.2.1]heptanyl, azacycloheptyl, azacyclobutyl, dihydro-pyrrolo[l,2-b]pyrazolyl, morpholinyl, oxa-5-azabicyclo[2.2.1]heptanyl, piperidinyl, piperazinyl, pyrazolidinyl, pyrrolidinyl, pyrrolyl, and tetrahydrofuranyl. In another embodiment, the heterocyclyl is selected from azabicyclo[2.2.1]heptanyl, azacycloheptyl, azacyclobutyl, dihydro-pyrrolo[l,2-b]pyrazolyl, oxa-5-azabicyclo[2.2.1]heptanyl, piperazinyl, and pyrrolidinyl.

[0054] "Heteroaryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring wherein at least one ring is aromatic and wherein one to four carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S. Examples of such heterocyclic elements include, but are not limited to, azepinyl, furanyl, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthridinyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, 5H-pyrrolo[2,3-b]pyrazinyl, pyrrolyl, quinazolinyl, quinolinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiazolyl, thienofuranyl, thienothiophenyl, thienyl, triazolyl, and the like. In one embodiment, the heteroaryl is selected from furanyl, imidazolyl, indolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, 5H-pyrrolo[2,3-b]pyrazinyl, tetrazolyl, thiazolyl, thienyl, triazolyl, and the like.

[0055] For use in medicine, the salts of the compounds of formula I will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of compounds according to this application or of their pharmaceutically acceptable salts. When the compounds of this application are basic, "pharmaceutically-acceptable salts" means salts of pharmaceutically-acceptable inorganic or organic acids. Salts of such inorganic acids include hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric salts. Preferred are the salts of hydrochloric acid. Salts of such organic acids as ascorbic, tartaric, maleic, acetic, succinic, fumaric, gluche- dric, formic, glycolic, stearic, lactic, malic, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, ethanesulfonic, p-toluenesulfonic, salicylic, sulfanilic, cy- clohexanesulfamic, nicotinic, isothionic, benzenesulfonic acids, and the like, are also preferred. Salts of an amino acid, such as arginate, asparginate, glutamate, and the like, are also preferred. 1 - benzathines, betaines, caffeine, choline, N,N'-dibenzylethylenediamine, di- ethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethy- lene-diamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0056] When the compound of the application is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0057] The preparation of such pharmaceutically acceptable salts and other typical salts of pharmaceutical compounds is described more fully in Berg et al., "Pharmaceutical Salts," et al. , J. Pharm. Sci. 1977:66:1-19. J. Pharm. Sci.,

[0058] If the compound of Formula I contains both a basic moiety, such as a diethylamino or piperidinyl group, and an acidic moiety, such as a carboxylic acid, both the salt and zwitterion can exist. All such possible salts and zwitterions are within the scope of this application.

[0059] The present application also includes isotopically-labelled compounds of the present application which are identical to those recited herein but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The present application also relates to the use of isotopically-labelled compounds as radiotracers for in vivo imaging of the brain for alpha-synuclein aggregates in the diagnosis, monitoring and / or treatment of Parkinson's Disease (PD). Another aspect of the present application is the use of isotopically-labelled compounds in PET, an in vivo analytical technique in the diagnosis, monitoring and / or treatment of PD. 3 H、 11 C or 18 F-labelled compounds can be used in in vitro and in vivo methods for determining binding, receptor occupancy, and metabolism studies, including covalent labelling.

[0060] Another aspect of the present application relates to the use of isotopically-labelled compounds for the screening of new chemical entities. In particular, various isotopically-labelled compounds are used in magnetic resonance imaging, autoradiography and other similar analytical tools. The present application is meant to include all suitable isotopic variations of a compound of Formula I. Examples of isotopes that can be preferentially incorporated in the compounds of the application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, iodine, fluorine, and chlorine, such as, but not limited to 2H, 3H, 13C, 11C, 14C, 15N, 18F, 32P, 123I, 124I, and 125I respectively. Certain isotopically-labelled compounds of the present application, for example those incorporating 14C or 18F, are useful in drug and / or substrate tissue distribution assays. Further, certain isotopically-labelled compounds of the present application, for example those incorporating 3H, 13C, and / or 18F, are useful in metabolic assays. 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 I-labeled substituted heterocyclic derivative compounds. It will be appreciated that other isotopes can also be incorporated by known means. In particular, the present application relates to compounds of formula I 11 C、 13 C、 14 C、 18 F、 15 O、 13 N、 35 S、 2 H and 3 H isotopes, compositions and methods of preparation, and use as radiotracers or PET tracers in the diagnosis and measurement of the role of compounds in PD therapy. In a further embodiment, the present application relates to compounds of formula I labeled with 3 H、 11 C or 18 F isotopes, together with compositions and methods of preparation, and use as PET tracers in the diagnosis and measurement of the role of compounds in PD therapy. The present application also relates to non-toxic alpha-synuclein protein binding compounds that are capable of rapidly crossing the blood-brain barrier, have low non-specific binding properties and are rapidly cleared from the system. This and other aspects of the present application will be realized upon reading the entire content of the specification.

[0061] Isotopically enriched compounds within formula I can be prepared by conventional techniques well known to those skilled in the art or by methods analogous to those described in the Schemes and Examples herein, using appropriate isotopically enriched reagents and / or intermediates without undue experimentation.

[0062] As described herein, the present application includes isotopically labeled compounds of the present application. An "isotopically labeled," "radiolabeled," "tracer," "radiotracer," "labeled tracer," or "radioligand" compound is a compound in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (i.e., naturally occurring). Suitable radionuclides (i.e., "detectable isotopes") that can be incorporated into the compounds of the present application include, but are not limited to, 2H, 3H, 11C, 13C, 14C, 13N, 15N, 150, 170, 180, 18F, 35S, 36CI, 82Br, 76Br, 77Br, 123I, 124I, and 131I. An isotopically labeled compound of the present application need only be enriched in a detectable isotope to or above the level that permits detection with techniques appropriate for the particular application. The radionuclide incorporated into the radiolabeled compound will depend upon the particular application of that radiolabeled compound. In another embodiment of the present application, the radionuclide is selected from the group consisting of 11 C, 13 C, 14 C, 18 F, 15 O, 13 N, 35 S, 2 H and 3 H, preferably 11 C, 3 H and 18 F.

[0063] Isotopically labeled compounds of the present application are prepared by incorporating a selected isotope into the substrate molecules. This is accomplished by utilizing reagents which must contain one or more atoms which become radioactive by placing them in a source of radiation (e.g., nuclear reactor, cyclotron, etc.). Additionally, many isotopically labeled reagents, such as 2 H2O, 3 H3CI, 14 C6H5Br, ClCH2 14 COCl, etc., are commercially available. The isotopically labeled reagents are then used in standard organic chemical synthesis techniques to incorporate one or more isotopic atoms into the compounds of Formula I as described below. The following schemes illustrate how to prepare compounds of Formula I.

[0064] The present application also relates to pharmaceutical compositions comprising an effective amount of at least one compound of Formula I and a pharmaceutically acceptable carrier. The compositions can include, but are not limited to, one or more buffers, wetting agents, emulsifiers, suspending agents, lubricants, adsorbents, surfactants, preservatives, and the like. The compositions can be formulated as solids, liquids, gels, or suspensions for oral administration (e.g., drenches, pills, tablets, powders, capsules, oral sprays, emulsions); parenteral administration (e.g., subcutaneous, intramuscular, intravenous, epidural injection); topical administration (e.g., creams, ointments, controlled release patches, sprays); intravaginally, rectally, transdermally, ocularly, or nasally. In a further embodiment, the pharmaceutical compositions of the present application can be formulated for parenteral administration, e.g., intravenous formulations.

[0065] The present application provides radiolabeled compounds of Formula I as alpha-synuclein imaging agents and synthetic precursor compounds from which they are derived. The compounds of Formula I bind aggregated alpha-synuclein to potentially track the progression of age-related diseases such as PD as well as other synucleinopathies and neurodegenerative diseases such as multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and the like. The compounds of the present application can also be used in combination with a wide range of cognitive deficit enhancers. Thus, in another embodiment of the present application, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or formulation comprising a compound of Formula (I) is administered concurrently, simultaneously, sequentially or separately with another or more pharmaceutically active compounds useful in AD / PD therapy including, for example, donepezil, memantine, tacrine, carvidopa, levodopa, MOA-B inhibitors, catechol O-methyl transferase (COMT) inhibitors, and the like, and equivalents and pharmaceutically active isomers and metabolites thereof.

[0066] It is an object of the present application to provide a radiopharmaceutical, e.g., an isotopically labeled compound of Formula I, which is useful for alpha-synuclein imaging and has high specific radioactivity and high target tissue selectivity due to its high affinity properties for alpha-synuclein aggregates.

[0067] According to the present application, a method of imaging alpha-synuclein deposits in a patient, wherein an isotopically labeled compound of Formula I is used as an imaging agent, comprises the steps of: a) positioning a human patient in a PET camera in a supine position; b) intravenously administering to the patient about 0.1 to about 10 mCi of an isotopically labeled compound of Formula I; and c) performing an emission scan of the brain region of the patient's head to identify aggregation of alpha-synuclein in the patient's brain tissue. Techniques for performing emission scans of the head are well known to those skilled in the art. PET techniques are described in Freeman et al., PET Imaging and Metabolism, 3rd Edition, Springer-Verlag: New York, 1993.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).

[0068] The term "labeled tracer" refers to any molecule that can be used to track or detect a defined activity in vivo, for example, preferred tracers are tracers that accumulate in areas where alpha-synuclein aggregates can be found. Preferably, the labeled tracer is one that can be observed in a live laboratory animal, healthy human or patient (referred to as a subject), for example, by positron emission tomography (PET) scanning. Suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins, including enzymes.

[0069] The present application also provides methods for determining the in vivo activity of enzymes or other molecules. In one embodiment, an isotopically labeled compound of Formula I is used as a tracer to track the binding activity of aggregated alpha-synuclein protein in the brain and central nervous system.

[0070] Biomarkers of Parkinson's disease state, prognosis and progression can be used for both general diagnostic applications of Parkinson's disease therapeutics as well as clinical development programs. Compounds of Formula I can be used to provide biomarker information for patients in clinical trials for new symptomatic and disease-modifying Parkinson's disease therapies and to help patient selection and allocation into cohorts. The present application will be used as one of the biomarkers of disease state to allow the correct patients to enter the appropriate PhIIb trial cohort. In addition, the present application can act as a marker of disease prognosis as an inclusion criterion to increase the probability that the disease will progress in the placebo treatment arm, a problem that continues to plague Parkinson's disease clinical trials. Finally, the present application can act as a biomarker of disease progression to monitor the clinical course of patients in treatment and can provide an independent biomarker measure of treatment response of the therapeutic drug. The tracer can be selected depending on the detection method chosen. A diagnostic effective amount of a labeled or unlabeled compound of the present application is administered to a living subject, including humans, prior to performing the methods of the present application.

[0071] The present application also provides a method of measuring the clinical efficacy of a therapeutic agent for the treatment of Parkinson's Disease (PD), comprising the steps of a) administering an isotopically labeled compound of formula I to a patient diagnosed with PD prior to treatment with the therapeutic agent, b) measuring the amount of alpha-synuclein aggregate formation in the brain tissue of the patient, c) administering an isotopically labeled compound of formula I to the patient after treatment with the therapeutic agent, d) measuring the amount of alpha-synuclein aggregate formation in the brain tissue of the patient after treatment, and e) analyzing whether the therapeutic agent terminates or reduces the progression of alpha-synuclein aggregate formation in the brain tissue of the patient.

[0072] The diagnostically effective amount of a labeled or unlabeled compound of the present application administered prior to performing the in vivo methods of the present application is in the range of 0.1 ng to 100 mg / kg body weight, preferably in the range of 1 ng to 10 mg / kg body weight.

[0073] The compounds of the present application can be used for the diagnosis, monitoring and measurement of Parkinson's Disease and other non-PD synucleinopathies, such as multiple system atrophy (MSA), dementia with Lewy bodies (DLB).

[0074] In preferred embodiments, the compounds of the present application can be used for the diagnosis, monitoring or measurement of Parkinson's Disease, non-PD synucleinopathies, neurodegenerative diseases, cognitive disorders, schizophrenia, pain disorders and sleep disorders.

[0075] The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts, in close confinement, such as the combination, complexation or aggregation of two or more ingredients, or the dissociation of one or more ingredients, or other types of reactions or interactions of one or more ingredients. In pharmaceutical compositions, the active compound, which is a compound of formula I, is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. Accordingly, the pharmaceutical compositions of the present application encompass any composition made by admixing a compound of the present application and a pharmaceutically acceptable carrier.

[0076] Generally, pharmaceutical compositions are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. In pharmaceutical compositions, the active compound, which is a compound of formula I, is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. Accordingly, the pharmaceutical compositions of the present application encompass any composition made by admixing a compound of the present application and a pharmaceutically acceptable carrier.

[0077] The term "patient" (or "subject") as used herein refers to an animal, preferably a mammal, particularly a human, in need of assessment via an imaging study. The term "administering" and variants thereof (e.g., "administering" a compound), as used herein with respect to a compound of Formula I, means providing the compound, or a pharmaceutically acceptable salt thereof, to a subject in need of treatment.

[0078] The present application also provides methods of synthesizing compounds that are useful as intermediates in the preparation of the compounds of the present application.

[0079] The compounds described herein can be prepared according to the methods of the following Schemes and Examples, using appropriate materials, and are further illustrated by the specific examples below. Deuterated forms of the compounds of the present application can be prepared by substituting isotopically labeled reagents for non-isotopically labeled reagents. The compounds set forth in the examples, however, are not to be construed as forming the only genus that is considered as the application. The examples further set forth details regarding specific compounds of the present application. Those skilled in the art will readily understand that known variations of the conditions and processes described can be used to prepare these compounds. Unless otherwise indicated, the reagents and starting materials used to prepare the intermediates and example compounds are commercially available. Unless otherwise indicated, all temperatures are in degrees Celsius. Mass spectra (MS) were measured by electrospray ionization mass spectrometry (ESI). 1 H NMR spectra were recorded at 300-500 MHz.

[0080] ABREVIATIONS AD = Alzheimer's disease (ipcADI) NiBr2= N,N'-bis(lR,2R,3R,5S)-(-)-isopinocampheyl-2,3- butanediamine nickel (II) bromide Anal. = analytical calc. = calculated BSA = bovine serum albumin Cellulose SC = stationary polysaccharide stationary phase [cellulose tris(3,5- dichlorophenylcarbamate) selector] CPME = cyclopentyl methyl ether Crabtree's catalyst = (1,5-cyclooctadiene)(pyridine)(tricyclohexylphosphine)iridium(i) hexafluorophosphate Cs2CO3 = cesium carbonate CsF = cesium fluoride DAST = diethylaminosulfur trifluoride DCM = dichloromethane DEA = diethylamine DIPEA = N,N - diisopropylethylamine DMF = dimethylformamide DMSO = dimethylsulfoxide DMA = dimethylacetamide DPBS = Dulbecco’s Phosphate Buffered Saline EDTA = ethylenediaminetetraacetic acid EtOAc = ethyl acetate EtOH = ethanol h = hour HATU = (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HPLC = high pressure liquid chromatography IHC = immunohistochemistry IPA = isopropyl alcohol IPAc = isopropyl acetate K2CO3 = potassium carbonate K3PO4 = potassium phosphate tribasic LCMS = liquid chromatography mass spectrometry mCi = millicurie MeCN = acetonitrile MeOH = methanol MgSO4 = magnesium sulfate MS = mass spectrometry NaHBEt3 = sodium triethylborohydride NaHCO3 = sodium bicarbonate Na2SO4 = sodium sulfate NH4Cl = ammonium chloride NH4OH = ammonium hydroxide NMP = N-methylpyrrolidone NMR = nuclear magnetic resonance spectroscopy PD = Parkinson’s disease Pd / C = 10 wt% carbon supported palladium PdCl2(dppf) = 1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II) PE or Pet Ether = petroleum ether PEI = polyethylenimine RuPhos Pd G2 = 2nd generation RuPhos precatalyst, chloro(2- dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'- biphenyl)]palladium(II) rt = room temperature SNAr = nucleophilic aromatic substitution reaction TFA = trifluoroacetic acid TLC = thin layer chromatography tR = retention time THF = tetrahydrofuran wt% = weight percent XPhos Palladacycle-G2 = 2nd generation XPhos precatalyst, chloro(2- dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'- biphenyl)]palladium(II) Unless otherwise noted in the experimental procedures, the compounds described herein are synthesized as racemic mixtures. In some cases, the final product can be further modified, for example, by manipulation of substituents. These manipulations can include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions that are commonly known by those skilled in the art. In some cases, the order of carrying out the foregoing reaction schemes can be varied to facilitate the reaction or to avoid unwanted reaction products. The following schemes and examples are provided to more fully enable those skilled in the art to practice the application. These examples are merely illustrative and are not to be construed as limiting the application in any way.

[0081] General Scheme A Substituted piperazines (A-1) can be converted to aryl piperazines A-2 via SNAr or Pd-mediated C-N coupling reactions. Deprotection followed by SNAr or Pd-mediated C-N coupling with aryl halides gives intermediates A-3. If Y is nitro in A-3, reduction (e.g., hydrogenation) can give anilines intermediates A-4 and subsequent amide coupling gives target molecules A-5.

[0082] Alternatively, if Y is bromo (or Cl), intermediates A-6 can be generated from Pd-mediated C-N coupling with Boc-amines and subsequent amide coupling gives target molecules A-7.

[0083] General Scheme B Substituted piperazines (B-1) can be converted to aryl piperazines B-2 via SNAr or Pd-mediated C-N coupling reaction followed by deprotection. B-2 can undergo SNAr or Pd-mediated C-N coupling with aryl halides to give intermediates B-3. B-3 can participate in a reduction reaction to give aniline intermediates B-4 and subsequent amide coupling to give target molecules B-5.

[0084] General Scheme C Intermediates C-1 can undergo SNAr reaction with amines or NH-containing heterocycles to give target compounds C-2.

[0085] General Scheme D Intermediates D-1 can participate in Pd-mediated C-C coupling reaction to give target compounds D-2.

[0086] General Scheme E Substituted piperazines (E-1) can be converted to aryl piperazines E-2 via SNAr or Pd-mediated C-N coupling reaction. Deprotection followed by SNAr or Pd-mediated C-N coupling with aryl halides to give intermediates E-3. Halides E-3 can undergo Pd-mediated C-N coupling with pre-formed primary amides to give target molecules E-4.

[0087] PREPARATION OF INTERMEDIATES Synthesis of Intermediate A: (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine 1-2: Synthesis of (S)-tert-butyl 3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-1-carboxylate To a solution of (S)-tert-butyl 3-methylpiperazine-l-carboxylate (1-1, 13.6 g, 67.9 mmol) in DMF (150 mL) was added K2CO3(14.08 g, 102 mmol) and 2-chloro-5-nitropyrimidine (12.46 g, 78 mmol). The mixture was stirred at 25 °C under a N2balloon for 12 h. TLC showed the starting material was completely consumed. Water (450 mL) was added and the mixture was stirred at 25 °C (room temperature) for 30 min. The precipitated solid was collected by filtration, washed with water (100 mL x 3) and dried to give 1-2 as a solid.

[0088] 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)。

[0089] 1-3: Synthesis of (S)-2-(2-methylpiperazin-1-yl)-5-nitropyrimidine To a solution of 1-2 (21 g, 64.9 mmol) in DCM (160 mL) was added TFA (40 mL) at 0 °C. The mixture was stirred at 25 °C for 2 h. TLC showed most of the starting material was consumed completely. The mixture was concentrated under reduced pressure to give the crude product (S)-2-(2-methylpiperazin-l-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). The organic layer was dried over Na2SO4, filtered and concentrated to give 1-3 as a solid.

[0090] 1 H 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] + .

[0091] 1-4: Synthesis of (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)-5-nitropyrimidine ​To a solution of 1-3 (2.5 g, 11.20 mmol) in dioxane (50 mL) was added 2-bromopyridine (3.72 g, 23.52 mmol), Cs2C03(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). The mixture was stirred at 110 °C for 12 h under a N2balloon. TLC showed most of the starting material was completely consumed. The mixture was filtered and concentrated. The residue was extracted with EtOAc (100 mL) and H20 (60 mL). The combined organic extracts were washed with brine (100 mL), dried over anhydrous Na2S04and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using silica gel column eluting with 15-30% EtOAc / PE to give 1-4 as a solid. .

[0092] 1 H 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] + .

[0093] Synthesis of (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine (Int A) To a solution of 1-4 (2.5 g, 8.32 mmol) in MeOH (40 mL) was added Pd / C (0.2 g, 1.879 mmol). The mixture was stirred at 25 °C for 2 h under a H2balloon. TLC showed most of the starting material was completely consumed. The mixture was filtered and washed with MeOH ( The filter cake was washed. The combined organic extracts were concentrated under reduced pressure to give Int A as an oil.

[0094] 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] + .

[0095] Synthesis of Intermediate B: (R)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine (R)-2-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrimidin-5-amine (Int B) was isolated as an oil by a similar sequence to the synthesis of Int A.

[0096] 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, 5.2 Hz, 1H),4.54~4.72 (m, 3H), 4.12~4.25 (m, 3H), 3.05~3.13 (m, 1H), 2.79~2.91 (m, 1H),1.02 (d, J = 6.8 Hz, 3H)。MS (ESI) m / z: 271.0 [M+H]+ .

[0097] Synthesis of Intermediate C: (R)-2-(3-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine 3-2: Synthesis of (R)-tert-butyl 3-methyl-4-(pyridin-2-yl)piperazine-1-carboxylate To a solution of (R)-tert-butyl-3-methylpiperazine-l-carboxylate (3-1, 2 g, 9.99 mmol) in dioxane (40 mL) was added 2-bromopyridine (3.31 g, 20.97 mmol), Cs2C03(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). The mixture was stirred at 110 °C for 12 h under a N2balloon. TLC showed 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 eluting with 15% EtOAc / PE to give 3-2 as an oil.

[0098] 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] + .

[0099] 3-3: Synthesis of (R)-2-methyl-1-(pyridin-2-yl)piperazine To a solution of 3-2 (2 g, 7.21 mmol) in EtOAc (17 mL) was added EtOAc / HCl (34 mL). The mixture was stirred at 25 °C for 5 h. TLC showed the starting material was completely consumed. The resulting solid was collected by filtration. The filter cake was washed with EtOAc (5 mL) and dried to give 3-3 as an oil.

[0100] 1 H 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] 3-4: Synthesis of (R)-2-(3-methyl-4-(pyridin-2-yl)piperazin-1-yl)-5-nitropyrimidine To a solution of 3-3 (1.7 g, 9.59 mmol) in DMF (20 mL) was added 2-chloro-5-nitropyrimidine (1.836 g, 11.51 mmol) and K2CO3(5.30 g, 38.4 mmol). The mixture was stirred at 80 °C for 2 h under a N2balloon. TLC showed most of the starting material was consumed completely. Water (150 mL) was added and the mixture was stirred at 25 °C (r.t.) for 30 min. The precipitated solid was collected by filtration, washed with water (100 mL x 3) and dried to give 3-4 as an oil.

[0102] 1 H NMR (400 MHz, DMSO-d6): δ = 9.15 (d, J = 4.4 Hz, 2H), 8.14 (d, J = 3.6Hz, 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] + .

[0103] Synthesis of (R)-2-(3-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine (Int C) To a solution of 3-4 (1.8 g, 5.99 mmol) in MeOH (30 mL) was added Pd / C (0.2 g, 1.879 mmol). The mixture was stirred under a H2balloon at 25 °C for 2 h. TLC showed most of the starting material was completely consumed. The mixture was filtered and the filter cake was washed with MeOH (5 mL). The combined organic extracts were concentrated under reduced pressure to give Int C as an oil.

[0104] 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] + .

[0105] Synthesis of Intermediate D: (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine (S)-2-(2-Methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrimidin-5-amine was isolated as an oil in a similar synthetic sequence as described above for Int C.

[0106] 1 H 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] + .

[0107] Synthesis of Intermediate E: (S)-2-(4-(6-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-amine (S)-2-(4-(6-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)pyrimidin-5-amine was isolated as an oil in a similar synthetic sequence as described for Int A.

[0108] Synthesis of Intermediate F: (S)-6-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyridin-3-amine 6-2: Synthesis of (S)-tert-butyl 2-methyl-4-(pyridin-2-yl)piperazine-1-carboxylate A mixture of (S)-l-N-BOC-2-methylpiperazine (15 g, 74.9 mmol), 2-fluoropyridine (36.4 g, 374 mmol) was heated at 135 °C for 48 h. LCMS showed starting material remaining and new product formed. C 15 H 23 N3O2[M+H] + LCMS (ESI) of 278.1 found 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 / EtOAc = 4: 1 to give 6-2 as a colorless liquid.

[0109] 6-3: Synthesis of (S)-3-methyl-1-(pyridin-2-yl)piperazine dihydrochloride A solution of hydrogen chloride in EtOAc (37.9 mL, 151 mmol) was added to 6-2 (7 g, 25.2 mmol) at room temperature, which was stirred at 20 °C for 18 h. LCMS (ESI) showed starting material consumed and new product formed. C 10 H 15 N3·2ClH [M+H] + LCMS (ESI) of 178.1 found 178.1, tR= 0.170 min. The mixture was evaporated under reduced pressure. Then, EtOAc (5 mL) was added to the residue. Evaporate it under reduced pressure to obtain 6-3 as a solid.

[0110] 6-4: Synthesis of (S)-2-methyl-1-(5-nitropyridin-2-yl)-4-(pyridin-2-yl)piperazine 2-Chloro-5-nitropyridine (0.697 g, 4.40 mmol) was added to a stirred mixture of 6-3 (1 g, 4.00 mmol) and K2CO3 (2.210 g, 15.99 mmol) in DMF (12 mL), and the mixture was stirred at 80 °C for 15 h. LCMS (ESI) showed that the starting material was consumed and a new product was formed. 15 H 17 N5O2[M+H] + The theoretical LCMS (ESI) value was 300.1, the measured value was 300.1, and tR = 0.351 min. Then, the mixture was poured into 30 mL of ice water, filtered, and the filter cake was rinsed with water (…). The product was washed, dried under vacuum, and then dried to obtain 6-4, which is a brown solid.

[0111] Synthesis of (S)-6-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyridin-3-amine (Int F) At room temperature, iron (0.933 g, 16.70 mmol) was added to a stirred mixture of 6-4 (1.0 g, 3.34 mmol), ammonium chloride (1.787 g, 33.4 mmol), EtOH (20 mL), and water (10 mL), and the mixture was stirred at room temperature (Temp: 40 °C) under a N2 atmosphere. LCMS (ESI) showed that the starting material was consumed and a new product was formed. 15 H 19 N5[M+H] + The theoretical LCMS (ESI) value was 270.1, the measured value was 270.1, and the tR value was 0.128 min. 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 then fertigated with EtOAc (…). Extraction. The combined organic fractions were dried (Na2SO4), filtered, and the solvent was evaporated under reduced pressure. The solvent was dissolved in EtOAc (10 mL), and 0.5 mL of HCl (4M EtOAc solution) was added dropwise. The solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC (reversed-phase C-18 column) by elution with acetonitrile / water + 0.1% TFA to give Int F as a brown solid.

[0112] 1 H NMR (400 MHz, Methanol- d 4) 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); C15H19N5 [M+H] + LCMS (ESI) for C15H19N5 [M+H] expected: 270.1, found: 270.1.

[0113] Synthesis of Intermediate G: (S)-5-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyridin-2-amine 7-1 : Synthesis of (S)-2-methyl-l-(6-nitropyridin-3-yl)-4-(pyridin-2-yl)piperazine To 5-fluoro-2-nitropyridine (1.2 g, 8.45 mmol) and 6-3 (2.42 g, 13.65 mmol) in DMA (20 mL) was added K2CO3(4.67 g, 33.8 mmol). The mixture was stirred at 100 °C for 18 h. TLC (petroleum ether: ethyl acetate = 1 : 1) showed very little starting material remained. C 15 H 17 N5O2 [M+H] + LCMS (ESI) for C15H19N5 [M+H] expected: 270.1, found: 270.1. R = 0.57 min. The mixture was cooled, diluted with ethyl acetate (20 mL) and water (20 mL), the organic layer washed with aqueous Na2SO4(saturated, 10 ml), dried (Na2SO4), filtered and the solvent evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with petroleum ether / ethyl acetate = 1 : 1 to give 7-1 as a solid.

[0114] 1H 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); C 15 H 17 N5O2[M+H] + LCMS (ESI) of the theoretical value: 300.1, the measured value: 300.5.

[0115] (S)-5-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyridin-2-amine (Int G) Synthesis of 7-1 (1.1 g, 3.67 mmol) and Pd-C (300 g, 2819 mmol) in MeOH (20 mL) was treated with H2(15 Psi) and the mixture was stirred at 20 °C for 18 hours. TLC (DCM:MeOH = 10:1) showed no starting material remained. The desired mass was detected. C 15 H 19 N5[M+H] + LCMS (ESI) of the theoretical value: 270.2, the measured value: 269.8, tR = 0.596 min. The solvent was filtered, the filtrate was concentrated, and the residue was purified by silica gel column flash chromatography eluting with ethyl acetate:EtOH = 10:1 to 20:1 to give Int G as a solid.

[0116] 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, 2.8Hz, 1H), 6.68 (d, J6.58 - 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); C 15 H 19 N5[M+H] + LCMS (ESI) for C14H16BrN5 [M+H] expected: 336.0, found: 335.9.

[0117] Intermediate H: Synthesis of (S)-5-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrazin-2- amine 8-1 : Synthesis of (S)-2-bromo-5-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrazine In a clean dry sealed tube, to 2,5-dibromopyrazine (1.046 g, 4.40 mmol) and 6-3 (1 g, 4.00 mmol) in DMSO (10 mL) was added CsF (3.04 g, 19.99 mmol), then the resulting mixture was stirred at 90 °C for 18 h. LCMS (ESI) showed the starting material was consumed and a new product was formed. The mixture was cooled to room temperature, filtered and the filter cake was washed with DCM (10 mL). The solvent was evaporated under reduced pressure. The residue was purified by silica gel column flash chromatography eluted with petroleum ether / EtOAc = 4:1 to give (S)-2-bromo-5-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrazine as a gum. LCMS (ESI) for C14H16BrN5 [M+H] expected: 336.0, found: 335.9. + LCMS (ESI) for C14H16BrN5 [M+H] expected: 336.0, found: 335.9.

[0118] 8-2: Synthesis of (S)-tert-butyl (5-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrazin-2- yl)carbamate Synthesis of (S)-5-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrazin-2-amine (Int H) ​(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (0.519 g, 0.898 mmol) and diacetoxy palladium (0.101 g, 0.449 mmol) were added 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) at room temperature under N2 atmosphere and the mixture was heated (Temp: 70 °C) for 18 h while stirring. LCMS (ESI) showed the starting material was consumed and a new product was formed. The mixture was filtered and concentrated, the residue was purified by silica gel flash chromatography (ISCOR F150; Sepa Flash Column) eluting with petroleum ether / EtOAc = 3:1 to give 8-2 as a solid.

[0119] 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);C 18 H 24 N6O2[M+H] + LCMS (ESI) for C26H26N6O2 [M+H] theoretical value: 457.2, found: 457.2.

[0120] Preparation of Compound Intermediate I: (S)-2-(4-(4-fluoropyridin-2-yl)-2-methylpiperazin-l- yl)pyrimidin-5-amine HCl / EtOAc (0.675 ml, 2.70 mmol) was added to a stirred mixture of 8-2 (1.0 g, 2.70 mmol) in ethyl acetate (5 mL) at room temperature and the mixture was stirred at room temperature (Temp: 20 °C) for 48 h. LCMS (ESI) showed the starting material was consumed and a new product was formed. Then concentrated, the residue was adjusted to pH 9 with saturated Na2CO3, extracted with EtOAc (20 mL, 3 times), then washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel flash chromatography (ISCORP 150; Sepa Flash Column) eluted with petroleum ether / EtOAc = 1:1 to give Int H as a solid.

[0121] 1H NMR (500 MHz, Chloroform-d) δ 8.18-8.22 (m, 1H), 7.72 (d, J = 1.53Hz, 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);C 14 H 18 N6[M+H] + LCMS (ESI) for C17H21N6O4 [M+H]+theor: 397.2, found: 397.1.

[0122] I-2: Synthesis of tert-butyl (S)-4-(4-fluoropyridin-2-yl)-2-methylpiperazine-l-carboxylate I-3: Synthesis of (S)-l-(4-fluoropyridin-2-yl)-3-methylpiperazine I-4: Synthesis of (S)-2-(4-(4-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)-5-nitropyrimidine To a stirred solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (1-1, 5 g, 24.96 mmol) in toluene (200 mL) purged with argon for 10 min was added 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(ll) (19.39 g, 24.96 mmol) at room temperature. The reaction mixture was again purged with argon for 10 min and stirred at 120 °C for 12 h. The reaction mixture was diluted with water (80 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over Na2S04and concentrated under reduced pressure to get the crude product. The crude product was purified by silica gel (300 g column) using 20% EtOAc / pet. ether as a gradient. The pure fractions were concentrated under reduced pressure to get compound 1-2 as a yellow liquid. M / Z (ESI): 296.37 [M+H] + .

[0123] Synthesis of (S)-2-(4-(4-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)pyrimidin-5-amine (Int I) To a stirred solution of compound 1-2 (5 g, 16.93 mmol) in DCM (80 mL) was added TFA (6.52 mL, 85 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to get compound 1-3 as a light yellow liquid. M / Z (ESI): 196.23 [M+H] + .

[0124] Preparation of Compound Intermediate J: 6-(3,3-difluoroazetidin-l-yl)nicotinic acid To a stirred solution of compound 1-3 (3.5 g, 17.93 mmol) in DMF (80 mL) was added 2-chloro-5-nitropyrimidine (3.43 g, 21.51 mmol) and K2C03(9.91 g, 71.7 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 2 h. The reaction mixture was diluted with ice-cold water (20 mL), the resulting solid was filtered and washed with water (2 x 50 mL) and dried under reduced pressure to get compound 1-4 as a light yellow solid. M / Z (ESI): 319.20 [M+H] + .

[0125] J-2: Synthesis of methyl 6-(3,3-difluoroazetidin-l-yl)nicotinate To a stirred solution of compound I-4 (3.5 g, 11 mmol) in EtOH (50 mL) was added 10% Pd-C (1.170 g, 5.50 mmol) at room temperature. The reaction mixture was degassed with nitrogen gas for 3 times and stirred under balloon pressure of H2 gas at room temperature for 16 h. The reaction mixture was filtered through a bed of celite on a Buckner funnel, washed with EtOAc (50 mL) and concentrated under reduced pressure to get compound Int I as a yellow liquid. M / Z (ESI): 289.17 [M+H] + .

[0126] Synthesis of 6-(3,3-difluoroazetidin-l-yl)nicotinic acid (Int J) Preparation of Compound Intermediate K: (S)-2-(4-(4-fluoropyridin-2-yl)-2-methylpiperazin-l- yl)pyrimidin-5-amine To a stirred solution of 3,3-difluoroazetidine hydrochloride (8.35 g, 64.5 mmol) in DMF (250 mL) was added K2CO3 (26.7 g, 193 mmol) followed by methyl 6-fluoro nicotinate (J-1, 10 g, 64.5 mmol) at room temperature. The reaction mixture was stirred at 90 °C for 12 h. The resulting reaction mixture was quenched with ice cold water (200 mL), the separated solid was filtered on a Buchner funnel, the solid was washed with water (100 mL x 2), dried under reduced pressure to get compound J-2 as a white solid. M / Z (ESI): 229.04 [M+H] + .

[0127] K-1 : Synthesis of tert-butyl (S)-3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-l-carboxylate To a stirred solution of compound J-2 (10 g, 43.5 mmol) in THF (70 mL), water (140 mL) was added LiOH (3.13 g, 131 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, acidified with saturated KHSO4 solution (PH = 5) and extracted with DCM (3 x 200 mL), dried over Na2SO4, filtered and concentrated, washed with pentane, dried under reduced pressure to get compound Int J as an off-white solid. M / Z (ESI): 215.04 [M+H] + .

[0128] K-2: Synthesis of tert-butyl (S)-4-(5-amino pyrimidin-2-yl)-3-methylpiperazine-l-carboxylate K-3: Synthesis of tert-butyl (S)-4-(5-(6-fluoronicotinamido)pyrimidin-2-yl)-3-methylpiperazine- 1-carboxylate K-4: Synthesis of tert-butyl (S)-4-(5-(6-(3,3-difluoroazetidin-l-yl)nicotinamido)pyrimidin-2- yl)-3-methylpiperazine-l-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (5 g, 24.96 mmol) in DMF (50 mL) was added potassium carbonate (6.90 g, 49.9 mmol) and 2-chloro-5-nitropyrimidine (4.78 g, 30.0 mmol) and stirred at 60 °C under N2atmosphere for 3 h. The reaction mixture was quenched with ice cold water (200 mL) and stirred at room temperature for 30 min. The solid precipitate obtained was filtered, washed with water (100 mL x 3) and dried under vacuum to get K-l as a light yellow solid.

[0129] 1 H NMR (400 mHz, 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=11.8Hz, 1H), 2.90-2.99 (m, 1H), 1.50 (s, 9H), 1.27 (d, J=6.8 Hz, 3H)。

[0130] Synthesis of (S)-6-(3,3-difluoroazetidin-l-yl)-N-(2-(2-methylpiperazin-l-yl)pyrimidin-5- yl)nicotinamide (Int K) To a stirred solution of K-l (3 g, 9.28 mmol) in THF / MeOH / ethyl acetate (1:1:1) (50 mL) was added palladium on carbon (1.975 g, 18.56 mmol) and stirred at 25 °C under hydrogen balloon atmosphere for 15 h. The reaction mixture was filtered, washed with MeOH / THF (3 x 50 mL), the combined organic layer was concentrated under reduced pressure to get K-2 as a yellow solid. M / Z (ESI): 294.15 [M+H] + .

[0131] Preparation of Compound Intermediate L: (S)-2-(4-(2-fluoropyridin-4-yl)-2-methylpiperazin-l- yl)pyrimidin-5-amine To a stirred solution of K-2 (1.20 g, 4.09 mmol) and 6-fluoro nicotinic acid (1.154 g, 8.18 mmol) in DMF (20 mL) was added 50% 1-propanephosphonic anhydride solution in EtOAc (5.21 g, 8.18 mmol) and TEA (1.710 mL, 12.27 mmol) at room temperature and stirred at room temperature for 15 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with brine (100 ml) and saturated NaHC03solution (100 ml), the organic phase was dried over Na2S04, filtered and concentrated under reduced pressure to get K-3 as a solid. M / Z (ESI): 417.42 [M+H] + .

[0132] L-1 : Synthesis of tert-butyl (S)-4-(2-fluoropyridin-4-yl)-2-methylpiperazine-l-carboxylate L-2: Synthesis of (S)-l-(2-fluoropyridin-4-yl)-3-methylpiperazine To a stirred solution of 3,3-difluoroazetidine hydrochloride (641 mg, 4.95 mmol) in DMF (10 mL) was added K2C03(912 mg, 6.60 mmol), K-3 (700 mg, 1.649 mmol) at room temperature and stirred at 100 °C overnight. The reaction mixture was quenched with ice cold water (30 mL), extracted with EtOAc (3 x 25 mL), the organic layer was washed with brine solution (2 x 25 mL), dried over Na2S04, filtered and concentrated to get K-4 as a brown solid. M / Z (ESI): 490.45 [M+H] + .

[0133] ​ ​ To a stirred solution of K-4 (700 mg, 1.158 mmol) in DCM (10 mL) was added TFA (0.268 mL, 3.47 mmol) at 0 °C and stirred at 0 °C for 5 h. The reaction mixture was concentrated and co-distilled with toluene twice to get Int K as a yellow semi-solid. M / Z (ESI): 390.18 [M+H] + .

[0134] ​ ​ ​ At room temperature, chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-diphenyl)]palladium(II) (1.939 g, 2.496 mmol), tert-butyl(S)-2-methylpiperazine-1-carboxylic acid ester (1 g, 4.99 mmol), and sodium tert-butoxide (1.200 g, 12.48 mmol) were added to a stirred solution of 4-bromo-2-fluoropyridine (1.142 g, 6.49 mmol) in toluene (20 mL), and the mixture was stirred at 110 °C for 18 h. The reaction mixture was quenched with ice-cold water (10 mL), extracted with ethyl acetate (2 × 200 mL), and the combined organic layers were carefully washed with a brine solution (2 × 30 mL), dried over sodium sulfate, filtered, concentrated under reduced pressure, and the crude compound was purified by 100-200 silica gel Biotage rapid column chromatography using 50% EtOAc / petroleum ether as eluent. The pure fraction was concentrated under reduced pressure to give compound L-1 as a yellow viscous solid. M / Z (ESI): 296.31 [M+H] + .

[0135] ​ At 0 °C, TFA (0.777 mL, 10.16 mmol) was added to a stirred solution of compound L-1 (1.0 g, 3.39 mmol) in DCM (12 mL), and the mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with NaHCO3 solution (10 mL), extracted with DCM (2 × 50 mL), washed with water (2 × 30 mL), and the combined organic layers were washed with brine solution (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound L-2 as a yellow solid. M / Z (ESI): 196.07 [M+H] + .

[0136] Synthesis of (S)-2-(4-(2-fluoropyridin-4-yl)-2-methylpiperazin-l-yl)-5-nitropyrimidine At room temperature, potassium carbonate (1487 mg, 10.76 mmol) and 2-chloro-5-nitropyrimidine (858 mg, 5.38 mmol) were added to a stirred solution of compound L-2 (700 mg, 3.59 mmol) in DMF (12 mL), and the mixture was stirred at 80 °C for 3 h. The reaction mixture was quenched with ice-cold water (20 mL), the precipitated solid was filtered, and dried under vacuum to give compound L-3 as a grayish-white solid. M / Z (ESI): 319.24 [M+H] + .

[0137] Synthesis of (S)-2-(4-(2-fluoropyridin-4-yl)-2-methylpiperazin-l-yl)-5-nitropyrimidine To a stirred solution of compound L-3 (600 mg, 1.885 mmol) in EtOH (15 mL) was added Pd-C (241 mg, 2.262 mmol) at room temperature and stirred under hydrogen balloon for 12 h at room temperature. The reaction mixture was filtered on Buckner funnel through celite bed, washed with EtOAc (2 x 50 mL), concentrated under reduced pressure to get compound Int L as a gummy solid. M / Z (ESI): 289.25 [M+H] + .

[0138] Examples Example 1 : Synthesis of (S)-6-methoxy-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-l- yl)pyrimidin-5-yl)nicotinamide Example 1 : Synthesis of (S)-6-methoxy-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-l- yl)pyrimidin-5-yl)nicotinamide To a solution of Int A (185 mg, 0.684 mmol) in DMF (3422 μL) was added 6-methoxy nicotinic acid (131 mg, 0.855 mmol), DIEA (359 μL, 2.053 mmol) and HATU (325 mg, 0.855 mmol). The mixture was stirred at 25 °C for 16 h. Then, the mixture was purified by preparative HPLC (reverse phase C-18 column) eluting with acetonitrile / water + 0.1% TFA to afford 1 as a solid.

[0139] 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, 3.6Hz, 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, 3.8 Hz,1H), 3.34 (dd, J = 12.8, 3.9 Hz, 1H), 3.11 (td, J = 12.2, 3.8 Hz, 1H), 1.30(d, J = 6.6 Hz, 3H). MS (ESI) m / z: 406.4 [M+H] + .

[0140] By similar methods, the compounds contained in Table 1 were synthesized by the synthetic sequence used to prepare the intermediates, utilizing the amide coupling reaction shown in Example 1. Where necessary, commercially available reagents were substituted, resulting in the following examples.

[0141] Table 1

[0142] Example 37: Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]-6-pyrazol-l-yl-pyridine-3-carboxamide Example 37: Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]-6-pyrazol-l-yl-pyridine-3-carboxamide Example 37: Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]-6-pyrazol-l-yl-pyridine-3-carboxamide Example 37: Synthesis of N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]-6-pyrazol-l-yl-pyridine-3-carboxamide 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 Hunig's base (517 μL, 2.96 mmol) in DMF (2466 μL) was prepared at 50 °C. After 18 h, the reaction was determined to be complete by LCMS. The reaction was diluted with EtOAc and washed with saturated NaHCO3, water, and brine; the organic phase was dried over MgSO4, filtered, and concentrated. The material was purified by normal phase column chromatography (0 to 100% EtOAc in hexanes, ISCO 24 g column, 25 min gradient) to give 9-1 as a solid.

[0143] 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). 20 H 20 FN7O [M+H] + LCMS (ESI) for C21H21FN4O2: theoretical 394.2, found 394.3.

[0144] Table 2 Example 46: Synthesis of 6-(3,3-difluoroazetidin-l-yl)-N-[2-[(2S)-2-methyl-4-(2- pyridyl)piperazin-l-yl]pyrimidin-5-yl]pyridine-3-carboxamide To a solution of 9-1 (25 mg, 0.064 mmol) and 1H-pyrazole (8.65 mg, 0.127 mmol) in DMF (635 μL) was added LiHMDS (63.5 μL, 0.095 mmol; 1.5M in THF) at ambient temperature and the reaction was heated to 100 °C. After 4 h, the reaction was complete. The reaction was diluted with EtOAc and quenched with water and brine; the organic phase was dried over MgS04, filtered and concentrated. The material was purified by normal phase column chromatography (0 to 100% EtOAc in hexanes, ISCO 12g column; 20 minute gradient) to give 37 as a solid.

[0145] 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). C 23 H 23 N9O [M+H] + LCMS (ESI) for C25H23FN6O [M+H] expected: 441.2, found: 441.3 The compounds contained in Table 2 were synthesized by a similar method from the synthetic sequence in Example 37. Where necessary, commercially available reagents were replaced to yield the following examples.

[0146] Table 3

[0147] Example 63: Synthesis of 6-(3-fluorophenyl)-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin- l-yl]pyrimidin-5-yl]pyridine-3-carboxamide Synthesis of 6-bromo-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]pyridine-3-carboxamide (20) A solution of 9-1 (30 mg, 0.076 mmol) and Hunig's base (80 μl, 0.458 mmol) in DMF (763 μl) was added to 3,3-difluorozacyclobutane hydrochloride (29.6 mg, 0.229 mmol) and heated to 100 °C. The reaction was completed by LCMS after 18 h. The reaction was cooled and diluted with EtOAc. The organic phase was washed with water / saturated NH4Cl, dried over MgSO4, and concentrated. The residue was purified by normal-phase column chromatography (20 to 100% EtOAc in hexane, ISCO 12 g column; 20 min gradient) to give 46 as a solid.

[0148] 1H NMR (500 MHz, DMSO- d 6) 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); C 23 H 24 F2N8O [M+H] + The theoretical value of LCMS (ESI) is 467.2, and the measured value is 467.3.

[0149] The compounds included in Table 3 were synthesized using a similar method, following the synthesis sequence in Example 46. If necessary, commercially available reagents were substituted to produce the following examples.

[0150] Synthesis of 6-bromo-N-[2-[(2S)-2-methyl-4-(2-pyridyl)piperazin-l-yl]pyrimidin-5- yl]pyridine-3-carboxamide (20)

[0151] Table 4 Example 87: (S)-6-(3,3-difluoroazetidin-l-yl)-N-(2-(4-(4-fluoropyridin-2-yl)-2- methylpiperazin-l-yl)pyrimidin-5-yl)nicotinamide Example 88: (S)-6-(3,3-difluoroazetidin-l-yl)-N-(2-(2-methyl-4-(4-nitropyridin-2- yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide Example 88: (S)-6-(3,3-difluoroazetidin-l-yl)-N-(2-(2-methyl-4-(4-nitropyridin-2- yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide 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 Hunig's base (517 μL, 2.96 mmol) in DMF (2466 μL) was prepared and heated to 50 °C. After 18 h, the reaction was complete by LCMS. The reaction was diluted with EtOAc and washed with saturated NaHCO3, water, and brine; the organic phase was dried over MgSO4, filtered, and concentrated. The material was purified by normal phase column chromatography (20 to 100% EtOAc in hexanes, ISCO 24 g column; 25 min gradient) to give 20 as a solid.

[0152] 1H NMR (500 MHz, DMSO- d 6) 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 (dd, J =23.9, 12.8 Hz, 2H), 3.32 – 3.25 (s, 1H), 3.18 (dd, J = 13.0, 4.1 Hz, 1H), 3.07– 2.86 (m, 1H), 1.15 (d, J = 6.6 Hz, 3H). C 20 H 20 BrN7O [M+H] + LCMS (ESI) for C21H19BrN7O [M+H] theoretical value: 454.1, found: 454.2.

[0153] Example 89: (S)-N-(2-(4-(2-fluoropyridin-4-yl)-2-methylpiperazin-l-yl)pyrimidin-5- yl)-6-(lH-pyrazol-l-yl)nicotinamideExample 90: (S)-N-(2-(2-methyl-4-(2-nitropyridin-4-yl)piperazin-l-yl)pyrimidin-5- yl)-6-(lH-pyrazol-l-yl)nicotinamide A solution of 20 (8.2 mg, 0.018 mmol), 3-fluorophenyl pinacol borate (15.9 mg, 0.072 mmol), SPhos-Pd-G2 (2.6 mg, 0.033 mmol), and K3PO4 (23 μL of a 1.5 M solution in water, 0.036 mmol) in DMF (2466 μL) was prepared and heated to 80 °C. After 18 h, the reaction was complete by LCMS. The reaction 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 modifier, Phenomenex C18 Luna column, 100 x 21.2 mm, 5 microns) to give 63 as a solid after concentration.

[0154] 1H NMR (500 MHz, DMSO- d 6) 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)。C 26 H 24 FN7O [M+H] +LCMS (ESI) found: 470.2, calc. 470.2.

[0155] The compounds contained in Table 4 were synthesized by analogous methods from the synthetic sequence in Example 63. Where necessary, commercially available reagents were replaced to yield the following examples.

[0156] ​

[0157] ​ ​ To a stirred solution of compound intermediate I (500 mg, 1.734 mmol) in DMF (5 mL) was added compound intermediate J (557 mg, 2.60 mmol), DIPEA (0.909 mL, 5.20 mmol) and 50% 1-propanephosphonic anhydride solution in EtOAc (1104 mg, 3.47 mmol) at room temperature. The reaction mixture was stirred at 50 °C for 12 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4and concentrated under reduced pressure to get the crude compound. The crude compound was purified by preparative HPLC (Method: Mobile Phase - 10 mM Ammonium bicarbonate in H2O: MeCN, Column - LUNA pack C18 (21.2 x 250) mm, 5 um Flow - 18 ml / min, Gradient: 0 / 45, 12 / 70, 12.05 / 98, 14 / 98, 14.05 / 45, 17 / 45) to get 87 as a light brown solid. M / Z (ESI): 485.30 [M+H] + .

[0158] 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)。

[0159] ​ ​ ​ ​ To a stirred solution of Int K (250 mg, 0.465 mmol) in dioxane (3 mL) was added Cs2CO3(455 mg, 1.396 mmol), 2-bromo-4-nitropyridine (123 mg, 0.605 mmol), degassed for 10 min under nitrogen, after which 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 stirred at 100 °C for 16 h. The reaction mixture was filtered over a bed of celite, washed with ethyl acetate. The filtrate was dried over sodium sulfate and evaporated under reduced pressure, the crude compound was purified by preparative HPLC (Method: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - LUNA Omega C18 (21.2 x 250) mm 5um flow - 18 ml / min, gradient method - 0 / 50, 10.2 / 84, 10.25 / 100, 12 / 100, 12.05 / 50, 16 / 50) and lyophilized to give 88 as a yellow solid. M / Z (ESI): 512.35 [M+H] + .

[0160] 1H NMR (400 mHz, 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).

[0161] ​ ​ To a stirred solution of compound Int L (150 mg, 0.520 mmol) in THF (3 mL) was added TEA (0.290 ml, 2.081 mmol), 50% 1-propane phosphonic anhydride solution in EtOAc (0.312 ml, 1.040 mmol), 6-(1H-pyrazol-1-yl)nicotinic acid (128 mg, 0.676 mmol) at 0°C and stirred at room temperature for 12 h. The reaction mixture was quenched with NaHC03solution (10 ml), extracted with EtOAc (2 x 50 mL), washed with water (2 x 10 mL), the combined organic layer was washed with brine (20 mL), dried over Na2S04, filtered, concentrated under reduced pressure, subjected to preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN column - X-Select Pheny Hexyl (19 x 250) mm 5u flow - 18 ml / min gradient method - 0 / 35, 9.3 / 70, 9.4 / 99, 11 / 99, 11.05 / 35, 15 / 35). The pure fractions were concentrated and lyophilized to get 89 as off-white solid. M / Z (ESI): 460.15 [M+H] + .

[0162] 1H NMR (400 mHz, 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).

[0163] ​ ​ K-6: Synthesis of tert-butyl (S)-4-(5-(6-(lH-pyrazol-l-yl)nicotinamido)pyrimidin-2-yl)-3- methylpiperazine- 1 -carboxylate K-7: Synthesis of (S)-N-(2-(2-methylpiperazin-l-yl)pyrimidin-5-yl)-6-(lH-pyrazol-l- yl)nicotinamide To a stirred solution of 1H-pyrazole (82 mg, 1.201 mmol), cesium carbonate (391 mg, 1.201 mmol) in DMF (5 mL) was added K-3 (250 mg, 0.600 mmol) under nitrogen atmosphere at room temperature and stirred at 90 °C for 3 h. The progress of the reaction was monitored by LCMS and TLC. TLC showed completion of the reaction. Then, the reaction mixture was diluted with ethyl acetate (100 mL) and water (60 mL). The organic layer was separated, the aqueous layer was extracted with ethyl acetate (2 x 40 mL) and the combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get compound K-6 as a light brown gummy solid. M / Z (ESI): 465.36 [M+H] + .

[0164] 90: Synthesis of (S)-N-(2-(2-methyl-4-(2-nitropyridin-4-yl)piperazin-l-yl)pyrimidin-5-yl)-6- (lH-pyrazol-l-yl)nicotinamide To a stirred solution of compound K-6 (190 mg, 0.409 mmol) in DCM (5.7 mL) was added TFA (1.565 mL, 20.45 mmol) at room temperature and stirred for 2 h at room temperature under nitrogen atmosphere. The progress of the reaction was monitored by TLC. TLC showed the completion of the reaction. Then the reaction mixture was concentrated under reduced pressure and the crude residue was diluted with DCM (100 mL) and H2O (60 mL). Then Na2C03 was added to adjust the pH to 7-8 and extracted with DCM (100 mL x 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to get compound K-7 as a light brown solid. M / Z (ESI): 365.15 [M+H] + .

[0165] Example 91 : (S)-6-(lH-imidazol-l-yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-l-yl)pyrimidin- 5-yl)nicotinamide Table 5 To a stirred solution of compound K-7 (105 mg, 0.288 mmol), potassium carbonate (119 mg, 0.864 mmol) in DMF (5 mL) was added 4-chloro-2-nitropyridine (91 mg, 0.576 mmol) at room temperature under nitrogen atmosphere and stirred for 6 h at 50 °C. The progress of the reaction was monitored by LCMS and TLC. TLC showed the completion of the reaction. Then the reaction mixture was 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 x 30 mL) and the combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure, the crude material was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, column - X-BRIDGE C18 (19 x 250) mm 5u flow - 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 method (conditions: column: Chiralpak IG (250 x 4.6) mm 5μ mobile phase - A: MeOH / DCM / DEA (50 / 50 / 0.2), Isocratic A: 100% flow rate: 1.0 mL / min, diluent: EtOH). The pure fractions were concentrated and lyophilized to get 90 as a yellow solid. M / Z (ESI): 487.12 [M+H] . + .

[0166] 1H NMR (500 mHz, 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).

[0167] Example 112: (S)-6-(3,3-difluoroazetidin-l-yl)-N-(5-(4-(6-fluoropyrimidin-4-yl)-2- methylpiperazin- 1 -yl)pyrazin-2-yl)nicotinamide M-3: Synthesis of tert-butyl (S)-4-(6-fluoropyrimidin-4-yl)-2-methylpiperazine- 1 -carboxylate To a stirred solution of intermediate A (150 mg, 0.555 mmol), 6-(1H- imidazol-1-yl)nicotinic acid (157 mg, 0.832 mmol) in THF (30 ml) was added TEA (0.193 mL, 1.387 mmol), 1-propanephosphonic anhydride (0.495 mL, 0.832 mmol) at 25 °C and stirred for 16 h at 25 °C. The reaction mixture was quenched with ice cold water (10 mL), extracted with ethyl acetate (2 x 100 mL), the combined organic layer was washed with brine solution (2 x 10 mL), dried over 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 x 250) mm 5u, flow - 18 ml / min, gradient - 0 / 45, 6.9 / 76, 6.95 / 100, 9 / 100, 9.05 / 45, 12 / 45). The pure fractions were concentrated and lyophilized to get 91 as a light yellow solid. M / Z (ESI): 442.14 [M+H] + .

[0168] 1H NMR (400 mHz, 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).

[0169] The compounds contained in Table 5 were synthesized by similar methods from the above synthetic sequence. Where necessary, commercially available reagents were replaced to produce the following examples.

[0170] M-4: Synthesis of (S)-4-fluoro-6-(3-methylpiperazin-l-yl)pyrimidine

[0171] M-6: Synthesis of (S)-4-(4-(5-bromopyrazin-2-yl)-3-methylpiperazin-l-yl)-6-fluoropyrimidine M-7: Synthesis of tert-butyl (S)-(5-(4-(6-fluoropyrimidin-4-yl)-2-methylpiperazin-l-yl)pyrazin- 2-yl)carbamate M-8: Synthesis of (S)-5-(4-(6-fluoropyrimidin-4-yl)-2-methylpiperazin-l-yl)pyrazin-2-amine To a stirred solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (M-l) (2.0 g, 9.99 mmol) in DMA (20 mL) was added N,N-diisopropylethylamine (5.2 mL, 30.0 mmol) followed by 4,6-difluoropyrimidine (M-2) (1.28 g, 11.0 mmol) at 0 °C and the mixture was stirred at 110 °C for 18 h. The reaction mixture was poured into crushed ice, extracted with EtOAc, the organic layer was washed with brine solution and dried over Na2S04, evaporated under reduced pressure to get compound M-3. M / Z (ESI): 297.12 [M+H+].

[0172] 112: Synthesis of (S)-6-(3,3-difluoroazetidin-l-yl)-N-(5-(4-(6-fluoropyrimidin-4-yl)-2- methylpiperazin- 1 -yl)pyrazin-2-yl)nicotinamide To a stirred solution of M-3 (2.2 g, 7.42 mmol) in DCM (10 mL) was added 4M HC1 in 1,4-dioxane (9.25 g, 74.2 mmol) at 0 °C and stirred for 18 h. The reaction mixture was concentrated under reduced pressure and washed with saturated NaHC03solution, extracted with EtOAc. The organic phase was washed with brine solution, dried over Na2S04and concentrated under reduced pressure to get compound M-4. M / Z (ESI): 197.06 [M+H+].

[0173] Example 113: (S)-6-(azetidin-l-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-l- yl)pyrimidin-5-yl)nicotinamide To a stirred solution of M-4 (1.9 g, 9.68 mmol) in DMSO (20 mL) was added cesium fluoride (2.94 g, 19.4 mmol) and 2,5-dibromopyrazine (M-5) (1.843 g, 7.75 mmol) at 0 °C and stirred at 80 °C for 18 h. The reaction mixture was poured into ice-cold water, extracted with EtOAc and the organic layer was washed with brine solution, dried over Na2S04and evaporated under vacuum to get compound M-6. M / Z (ESI): 353.04 [M+H+].

[0174] Example 114: (R)-6-(4-(fluoromethyl)-lH-pyrazol-l-yl)-N-(2-(2-(methoxymethyl)-4-(pyridin- 2-yl)piperazin- 1 -yl)pyrimidin-5-yl)nicotinamide O-3: Synthesis of (R)-2-(2-(methoxymethyl)-4-(pyridin-2-yl)piperazin-l-yl)-5-nitropyrimidine To a stirred solution of M-6 (800 mg, 2.265 mmol) in toluene (10 mL) was added 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) at 0 °C and stirred at 70 °C for 18 h. The reaction mixture was poured into ice-cold water (5 mL), extracted with EtOAc (5 mL x 3) and the organic layer washed with brine solution, dried over anhydrous Na2SO4, evaporated under reduced pressure to get compound M-7. M / Z (ESI): 390.17 [M+H+].

[0175] O-4: Synthesis of (R)-2-(2-(methoxymethyl)-4-(pyridin-2-yl)piperazin-l-yl)pyrimidin-5-amine To a stirred solution of M-7 (600 mg, 1.541 mmol) in DCM (8 mL) was added TFA (0.590 mL, 7.70 mmol) at 0 °C and stirred at room temperature for 18 h. The reaction mixture was evaporated, quenched with saturated NaHCO3solution (10 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine solution, dried over Na2SO4and evaporated under reduced pressure to get M-8. M / Z (ESI): 290.21 [M+H+].

[0176] Example 114: (R)-6-(4-(fluoromethyl)-lH-pyrazol-l-yl)-N-(2-(2-(methoxymethyl)-4-(pyridin- 2-yl)piperazin- 1 -yl)pyrimidin-5-yl)nicotinamide Example 115: (S)-N-(5-(4-(6-fluoropyrimidin-4-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6- (l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of int J (89 mg, 0.415 mmol) in DCM (5 mL) was added 1- methylimidazole (0.083 mL, 1.037 mmol) followed by methanesulfonyl chloride (0.035 mL, 0.456 mmol) at 0 °C and stirred for 15 min, then M-8 (60 mg, 0.207 mmol) was added and stirred at 45 °C for 4 h. The reaction mixture was poured into ice-cold water (2 mL) and extracted with DCM (3 mL x 3). The organic layer was washed with brine solution, dried over 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 x 250) mm 5u, flow - 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 get 112. M / Z (ESI): 484.18 [M+H+]. 1 H NMR (400 mHz, DMSO-d6) δ: 10.57 (s, 1H), 8.82 (br dd, J=17.7, 1.6Hz, 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).

[0177] ​ ​ To a stirred solution of N-1 (prepared in a similar manner to M-8) (0.060 g, 0.208 mmol) and N-2 (prepared in a similar manner to J) (0.074 g, 0.416 mmol) in DMF (2 ml) was added HATU (0.158 g, 0.416 mmol) and DIPEA (0.109 ml, 0.624 mmol) at 0 °C and stirred at 100 °C for 1 h. The reaction mixture was quenched with ice cold water (10 mL), extracted with ethyl acetate (2 x 50 mL) and the combined organic layer washed with brine solution (2 x 20 mL), dried over sodium sulphate, filtered and concentrated under reduced pressure and the crude compound purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN, column - X-Bridge C18 (19 x 250) mm 5u, flow - 8 ml / min, gradient - 0 / 40, 8 / 73, 8.05 / 99, 10 / 99, 10.05 / 40, 13 / 40). The pure fractions were concentrated and lyophilized to get 113. M / Z (ESI): 449.46 [M+H] + .

[0178] 1H NMR (400 mHz, 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).

[0179] ​ ​ ​ To a stirred solution of O-1 (prepared in a similar manner as int3-3) (300 mg, 1.231 mmol) in DMF (4 ml) was added K2CO3(851 mg, 6.15 mmol) and 2-chloro-5-nitropyrimidine (O-2) (236 mg, 1.477 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 2 h under nitrogen atmosphere. The reaction mixture was quenched with H2O (25 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (2 x 25 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to get O-3. M / Z (ESI): 331.17 [M+H] + .

[0180] ​ To a stirred solution of O-3 (50 mg, 0.151 mmol) in MeOH (5 mL) was added 10% Pd-C (16.11 mg, 0.015 mmol) at room temperature. The reaction mixture was stirred under hydrogen atmosphere at room temperature for 4 h. The reaction mixture was diluted with EtOAc (15 mL), filtered through a pad of celite and washed with EtOAc (2 x 15 mL). The filtrate was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to get O-4. M / Z (ESI): 301.24 [M+H] + .

[0181] ​ ​ To a stirred solution of O-4 (40 mg, 0.133 mmol) in DMF (1 mL) was added HATU (50.6 mg, 0.133 mmol), O-5 (32.4 mg, 0.146 mmol) and DIPEA (0.070 mL, 0.4 mmol) at room temperature. The reaction mixture was stirred under nitrogen atmosphere at room temperature for 2 h. The reaction mixture was quenched with water (15 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (2 x 10 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure. The crude compound was purified by preparative-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O: MeCN, column - X-Select C18 (19 x 250) mm 5u, flow - 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 get 114. M / Z (ESI): 504.32 [M+H] + .

[0182] 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, 3.49 (t, J = 9.0 Hz, 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)。

[0183] ​ ​ P-3: Synthesis of tert-butyl (S)-4-(6-fluoropyrimidin-4-yl)-3-methylpiperazine-1- carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (P-l) (5 g, 24.96 mmol) in DMF (50 mL) was added DIPEA (13.08 mL, 74.9 mmol) and 4,6-difluoropyrimidine (P-2) (3.48 g, 30.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was quenched with ice-cold water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 80 g silica (230-400 silica) cartridge and the compound was eluted with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under vacuum to get P-3. M / Z (ESI): 297.16 [M+H] + .

[0184] P-4: Synthesis of (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) was added 4M HC1 in 1,4-dioxane (9.70 mL, 38.8 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated and dried under reduced pressure to get P-4. M / Z (ESI): 197.09 [M+H] + .

[0185] P-6: Synthesis of (S)-4-(4-(5-bromopyrazin-2-yl)-2-methylpiperazin-1-yl)-6- fluoropyrimidine To a stirred solution of P-4 (500 mg, 2.149 mmol) in DMSO (8 mL) was added cesium fluoride (1958 mg, 12.89 mmol) and 2,5-dibromopyrazine (P-5) (613 mg, 2.58 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 6 h under argon atmosphere. The reaction mixture was quenched with ice cold water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using silica gel column and eluting the compound with 25% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get P-6. M / Z (ESI): 353.06 [M+H] + .

[0186] P-7: Synthesis of 6-(1-methyl-1H-pyrazol-4-yl)nicotinamide To a solution of 6-bromonicotinamide (3 g, 14.92 mmol) in 1,4-dioxane (40 mL) and H2O (10 mL) was added 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (3.73 g, 17.91 mmol), Na2CO3 (4.75 g, 44.8 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 5 min. To this reaction mixture was then added PdCl2(dppf) (1.092 g, 1.492 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 12 h. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 80 g silica (230-400 mesh) column and eluting the compound with 10-15% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to get P-7. M / Z (ESI): 203.02 [M+H] + .

[0187] Example 115: (S)-N-(5-(4-(6-fluoropyrimidin-4-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide Example 115: (S)-N-(5-(4-(6-fluoropyrimidin-4-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide To a stirred solution of P-6 (100 mg, 0.283 mmol) in 1,4-dioxane (2 mL) was added P-7 (69 mg, 0.341 mmol), Cs2C03(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) at room temperature. The reaction mixture was degassed and purged with argon for 10 min. The reaction mixture was stirred in microwave at 130 °C for 30 min. The reaction mixture was quenched with water (30 mL) and extracted with DCM (3 x 20 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was triturated with diethyl ether (2 x 10 mL) and dried under reduced pressure. The resulting compound was purified again by preparative HPLC (conditions: mobile phase - 0.1% FA in H20: MeCN, column - X-Bridge C18 (19 x 250) mm, 5 μ, 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 purified compounds were combined, concentrated under reduced pressure and lyophilized to get 115. M / Z (ESI): 475.18 [M+H] + .

[0188] 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).

[0189] Example 116: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin- 1-yl)pyrazin-2-yl)nicotinamide Q-3: Synthesis of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-3-methylpiperazine-1-carboxylate Q-4: Synthesis of (S)-1-(5-fluoropyridin-2-yl)-2-methylpiperazine To a stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (Q-1) (1 g, 4.99 mmol) in toluene (20 mL) was added 2-chloro-5-fluoropyridine (Q-2) (0.995 mL, 9.99 mmol) and sodium tert-butoxide (1.440 g, 14.98 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 25 min. To this reaction mixture was then added RuPhos Pd G2 (0.388 g, 0.499 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h under nitrogen atmosphere in a sealed tube. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 85 mL). The combined organic layers were washed with brine (2 x 40 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 80 g silica (230-400 mesh) column and eluted with 15% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford Q-3. M / Z (ESI): 296.11 [M+H] + .

[0190] Q-6: Synthesis of (S)-2-bromo-5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrazine To a stirred solution of Q-3 (100 mg, 0.339 mmol) in DCM (2 mL) was added 4M HC1 in 1,4-dioxane (0.423 mL, 1.693 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h under nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure. The crude compound was triturated with 20% EtOAc in ethyl ether and dried under reduced pressure to afford Q-4. M / Z (ESI): 196.12 [M+H] + .

[0191] Example 116: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin- 1-yl)pyrazin-2-yl)nicotinamide To a stirred solution of Q-4 (80 mg, 0.345 mmol) in DMSO (2 mL) was added CsF (157 mg, 1.036 mmol) and 2,5-dibromopyrazine (Q-5) (164 mg, 0.691 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h in a sealed tube under nitrogen atmosphere. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2 x 35 mL). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 80 g silica (230-400 mesh) column and eluting the compound with 25% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get Q-6. M / Z (ESI): 352.97 [M+H] + .

[0192] Example 117: (R)-N-(5-(4-(5-fluoropyridin-2-yl)-3-(methoxymethyl)piperazin-1-yl)pyrazin-2- yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide R-3: Synthesis of benzyl (R)-4-(5-fluoropyridin-2-yl)-3-(methoxymethyl)piperazine-1- carboxylate To a stirred solution of Q-6 (60 mg, 0.170 mmol) in 1,4-dioxane (1.5 mL) was added Cs2C03(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 a similar manner as X-5) (61.4 mg, 0.170 mmol) at room temperature. The reaction mixture was stirred in a microwave at 150 °C for 2 h under nitrogen atmosphere. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 75 mL). The combined organic layers were washed with brine (2 x 40 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 40 g silica (230-400 mesh) column and eluting the compound with 3% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure. The resulting compound was further purified by chiral prep purification [Cellulose SC (250 x 30 x 5 μ), MeCN:MeOH (90:10)]. The pure fractions were combined, concentrated under reduced pressure and lyophilized to get 116. M / Z (ESI): 467.28 [M+H] + .

[0193] 1 H 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 = 9.4Hz, 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)。

[0194] R-4: Synthesis of (R)-1-(5-fluoropyridin-2-yl)-2-(methoxymethyl)piperazine R-6: Synthesis of (R)-2-bromo-5-(4-(5-fluoropyridin-2-yl)-3-(methoxymethyl)piperazin-1- yl)pyrazine Example 117: (R)-N-(5-(4-(5-fluoropyridin-2-yl)-3-(methoxymethyl)piperazin-1-yl)pyrazin-2- yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide To a stirred solution of R-1 (2 g, 7.57 mmol) in 1,4-dioxane (30 mL) was added sodium tert-butoxide (2.182 g, 22.70 mmol), 2-chloro-5-fluoropyridine (R-2) (1.194 g, 9.08 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 5 min. To this reaction mixture was then added (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'- biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) mesylate (0.633 g, 0.757 mmol) at room temperature. The reaction mixture was stirred in a sealed tube at 110 °C for 16 h. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column and eluted with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get R-3. M / Z (ESI): 360.07 [M+H] + .

[0195] Example 118: (S)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6- (1-methyl-1H-pyrazol-4-yl)nicotinamide To a stirred solution of R-3 (800 mg, 2.226 mmol) in MeOH (10 mL) was added 10% Pd / C (237 mg, 0.223 mmol). The reaction mixture was stirred at room temperature for 16 h under hydrogen atmosphere. The reaction mixture was filtered through a pad of celite, the filtrate was concentrated and dried under reduced pressure to get R-4. M / Z (ESI): 226.00 [M+H] + . S-2: Synthesis of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of R-4 (600 mg, 2.66 mmol) in DMSO (10 mL) was added CsF (1214 mg, 7.99 mmol) and 2,5-dibromopyrazine (R-5) (1267 mg, 5.33 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h in a sealed tube under nitrogen atmosphere. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column and eluted with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get R-6. M / Z (ESI): 382.17 [M+H] + S-3: Synthesis of (S)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine S-4: Synthesis of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-iodopyrimidine 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) was added 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) at room temperature. The reaction mixture was degassed and purged with argon for 10 min. The reaction mixture was stirred in the microwave at 150 °C for 2 h. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (5 mL), dried over 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 (25 x 250) mm, 5 μ flow - 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 lyophilized to yield 117. M / Z (ESI): 504.15 [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), 8.34 (dd, J = 8.4 Hz, 2.4Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 8.08-8.15 (m, 2H), 7.78 (dd, J = 8.4 Hz, 0.8Hz, 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)。

[0197] Example 118: (S)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6- ​ ​ To a stirred solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (S-1) (2 g, 9.99 mmol) and 2-bromo-5-fluoropyridine (2.109 g, 11.98 mmol) in toluene (50 mL) was added sodium tert-butoxide (2.88 g, 30.0 mmol) at room temperature and degassed with argon for 5 min. Then, RuPhos Pd G2 (0.776 g, 0.999 mmol) was added to the reaction mixture at room temperature and degassed with argon for 1 min again. The reaction mixture was stirred at 110 °C for 16 h. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 24 g silica gel column and eluting the compound with 50% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford S-2. M / Z (ESI): 296.11 [M+H] + .

[0198] ​ To a stirred solution of S-2 (1.5 g, 5.08 mmol) in DCM (20 mL) was added 4M HC1 in 1,4-dioxane (5.08 mL, 20.31 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to afford S-3. M / Z (ESI): 196.01 [M+H] + .

[0199] ​ To a stirred solution of S-3 (1.2 g, 5.18 mmol) in DMF (20 mL) under argon was added 2-chloro-5-iodopyrimidine (1.494 g, 6.21 mmol) and DIPEA (2.71 mL, 15.54 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by silica column and eluted with 10% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford S-4. M / Z (ESI): 400.19 [M+H] + .

[0200] ​ (1 -methyl- 1 H-pyrazol-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) was added Cs2C03(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) at room temperature and degassed with argon for 10 min. The reaction mixture was stirred at 150 °C for 2 h under microwave irradiation. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (5 mL), dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN, column - X-Bridge, C18 (19 x 250) mm, 5 μ flow - 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 afford 118. M / Z (ESI): 474.21 [M+H] + .

[0201] 1 H 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)。

[0202] Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide To a solution of T-1 (prepared in a similar manner to R-6) (100 mg, 0.225 mmol) in 1,4-dioxane (2 mL) was added P-7 (54.6 mg, 0.270 mmol), Cs2C03(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) at room temperature. The reaction mixture was degassed and purged with argon for 10 min. Then, the reaction mixture was stirred in a microwave at 150 °C for 2 h.

[0203] The reaction mixture was quenched with water (20 mL) and extracted with DCM (3 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was triturated with diethyl ether (2 x 5 mL) and concentrated under reduced pressure. The resulting compound was purified by preparative HPLC (conditions: Instrument ID ANL-MCL5-PREP-020 Column Name Betasil Phenyl Hexyl (21.2 x 250) MM, 5μ Column Number 1, 20 mL / min, 40% to 80% ACN in Water over 10 min, 254 nm). 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) Purification. The pure fractions were combined and concentrated under reduced pressure to afford 119. M / Z (ESI): 519.31 [M+H] + .

[0204] Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide To a solution of P-7 (100 mg, 0.232 mmol) in 1,4-dioxane (2 mL) was added U-1 (prepared in a similar manner as Q-6) (56.4 mg, 0.279 mmol), Cs2C03(227 mg, 0.697 mmol) and trans-N,N'-dimethylcyclohexane-1,2-diamine (3.31 mg, 0.023 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 10 min. The reaction mixture was stirred in the microwave at 150 °C for 2 h. The reaction mixture was quenched with water (30 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was triturated with diethyl ether (2 x 10 mL) and concentrated under reduced pressure. The resulting compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20:MeCN column - X-Bridge, C18 (19 x 250) mm, 5 μ flow - 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 lyophilized to afford 120. M / Z (ESI): 505.25 [M+H] + .

[0205] 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)。

[0206] Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide Synthesis of (R)-2-(2-(methoxymethyl)piperazin-l-yl)pyrimidine Synthesis of (R)-4-chloro-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-l-yl)-5- (trimethylsilyl)pyrimidine To a stirred solution of V-1 (prepared in a similar manner as 3-2) (2 g, 6.49 mmol) in DCM (40 mL) was added HC1 in 1,4-dioxane (3.08 mL, 25.9 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated and dried under reduced pressure to afford V-2. M / Z (ESI): 209.18 [M+H] + .

[0207] Synthesis of (R)-4-chloro-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-l-yl)-5- (trimethylsilyl)pyrimidine Synthesis of (R)-4-chloro-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-l-yl)-5- (trimethylsilyl)pyrimidine To a stirred solution of V-2 (1.5 g, 6.13 mmol) in DMF (30 mL) was added 2,4-dichloro-5-(trimethylsilyl)pyrimidine (1.627 g, 7.36 mmol) and DIPEA (3.21 mL, 18.39 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 3 h. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by 100 g silica gel (60-120 mesh) column and eluted with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford V-3. M / Z (ESI): 393.21 [M+H] + .

[0208] Example 119: (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin- 1 -yl)pyrimidin-5-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide To a stirred solution of V-3 (1.2 g, 3.05 mmol) in MeCN (10 mL) and DCM (5 mL) was added ICI (0.230 mL, 4.58 mmol) at -10 °C. The reaction mixture was stirred at -10 °C for 3 h. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by 100 g silica gel (100-200 mesh) column and eluted the compound with 25% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford V-4. M / Z (ESI): 446.92 [M+H] + .

[0209] Example 122: (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2- yl)-6-(pyrrolidin- 1 -yl)nicotinamide To a stirred solution of V-4 (500 mg, 1.119 mmol) in DMSO (10 mL) was added potassium fluoride (325 mg, 5.60 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 12 h. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by 100 g silica gel (100-200 mesh) column and eluted the compound with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford V-5. M / Z (ESI): 431.00 [M+H] + .

[0210] Synthesis of (S)-5-fluoro-2-(2-methylpiperazin-l-yl)pyrimidine hydrochloride Synthesis of (S)-5-fluoro-2-(2-methylpiperazin-l-yl)pyrimidine hydrochloride 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) was added 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) at room temperature. The reaction mixture was stirred at room temperature for 48 h. The reaction mixture was quenched with water (30 mL) and extracted with EtOAc (3 x 40 mL). The combined organic layers were dried over anhydrous 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 x 250 mm), 5 μ flow - 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) purification. The pure fractions were combined, concentrated under reduced pressure and lyophilized to get 121. M / Z (ESI): 505.32 [M+H] + .

[0211] 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)。

[0212] Synthesis of (S)-5-fluoro-2-(2-methylpiperazin-l-yl)pyrimidine hydrochloride Synthesis of 6-(pyrrolidin-l-yl)nicotinamide Example 122: (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2- yl)-6-(pyrrolidin- 1 -yl)nicotinamide A stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (X-l) (1 g, 4.99 mmol) in toluene (20 mL) was purged with argon for 10 min. 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 and purged with argon again for another 10 min. The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 25 g silica gel cartridge and eluting the compound with 30% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get X-2. M / Z (ESI): 297.27 [M+H] + .

[0213] Example 123: (S)-6-(l-(2-fluoroethyl)-lH-pyrazol-4-yl)-N-(2-(4-(5-fluoropyridin-2-yl)- 2-methylpiperazin- 1 -yl)pyrimidin-5-yl)nicotinamide To a stirred solution of X-2 (1.1 g, 3.71 mmol) in DCM (20 mL) was added 4M HC1 in 1,4-dioxane (3.71 mL, 14.85 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to get X-3. M / Z (ESI): 197.02 [M+H] + .

[0214] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide To a stirred solution of X-3 (800 mg, 3.44 mmol) in DMSO (20 mL) was added CsF (1567 mg, 10.31 mmol) and 2,5-dibromopyrazine (981 mg, 4.13 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was quenched with water (120 mL) and extracted with EtOAc (3 x 120 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 25 g silica gel column and eluting the compound with 30% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get X-4. M / Z (ESI): 353.09 [M+H] + .

[0215] Synthesis of (R)-(4-(5-bromopyrazin-2-yl)-l-(5-fluoropyridin-2-yl)piperazin-2-yl)methanol To a stirred solution of 6-chloronicotinamide (1 g, 6.39 mmol) in DMF (30 mL) was added pyrrolidine (0.681 g, 9.58 mmol) and K2C03(2.65 g, 19.16 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was quenched with water (80 mL) and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was washed with diethyl ether (2 x 30 mL) and concentrated under reduced pressure to get X-5. M / Z (ESI): 192.00 [M+H] + .

[0216] ​ ​ 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) was added 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) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was quenched with water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over 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 x 250 mm), 5 μ flow - 7 mL / min, gradient - 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 lyophilized to afford 122. M / Z (ESI): 464.28 [M+H] + .

[0217] 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).

[0218] ​ ​ To a stirred solution of S-4 (50 mg, 0.125 mmol) and Y-1 (prepared in a similar manner as P-7) (29.3 mg, 0.125 mmol) in 1,4-dioxane (1 mL) was added Cs2C03(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) at room temperature and degassed with argon for 10 min. The reaction mixture was stirred at 150 °C for 2 h under microwave irradiation. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (5 mL), dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 2.5 mM ammonium bicarbonate in H20: MeCN, column - X-Bridge, C18 (19 x 250) mm, 5 μ flow - 15.0 mL / min gradient method - 0 / 45, 2 / 45, 10.5 / 62, 10.55 / 100, 13 / 100, 13.05 / 45, 17 / 45 ANL-PREP-023). The pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 123. M / Z (ESI): 506.21 [M+H] + .

[0219] 1 H 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, 3.4Hz, 1H), 1.16 (d , J = 6.8 Hz, 3H)。

[0220] ​ ​ ​ To a stirred solution of R-6 (200 mg, 0.523 mmol) in DCM (2 mL) was added BBr3(1.570 mL, 1.570 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated, quenched with ice-cold water (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with saturated aqueous NaHC03solution (20 mL) and brine (2 x 20 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 40 g silica (100-200 mesh) column and eluting the compound with 10% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford Z-1. M / Z (ESI): 369.12 [M+H] + .

[0221] Synthesis of Z-2: (R)-2-bromo-5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2- yl)piperazin-l-yl)pyrazine Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of Z-1 (120 mg, 326 μmol) in DMF (3 mL) was added NaH (39.1 mg, 1.63 mmol) at 0 °C for 10 min. Then, to this reaction mixture was added 1-fluoro-2-iodoethane (567 mg, 3.26 mmol) at 0 °C. The reaction mixture was stirred at 60 °C for 16 h. The reaction mixture was quenched with ice-cold water (25 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were washed with brine (25 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 40 g silica (100-200 mesh) column and eluting the compound with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford Z-2. M / Z (ESI): 415.15 [M+H] + .

[0222] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-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) was added cesium carbonate (189 mg, 579 μmol) at room temperature. The reaction mixture was degassed and purged with nitrogen for 10 min. Then, to this reaction mixture was added copper (I) iodide (3.68 mg, 19.3 μmol) and trans-(1 r,2r)-N,N'-dimethyl-1,2-cyclohexanediamine (305 mL, 0.966 μmol). The reaction mixture was stirred at 150 °C in the microwave for 2 h. The reaction mixture was filtered through a pad of celite and concentrated under reduced pressure. The residue was quenched with water (2 mL) and extracted with 10% MeOH in DCM (2 x 2 mL). The combined organic layers were washed with brine (2 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20:MeCN, column - X-Select, C18 (10 x 150) mm, 5 μ flow - 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 afford 124. M / Z (ESI): 536.31 [M+H] + .

[0223] 1H 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)。

[0224] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of M-6 (200 mg, 566 μmol) in 1,4-dioxane (3 mL) was added Cs2CO3(553 mg, 1.70 mmol), Cul (10.8 mg, 56.6 μmol), (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (4.03 mg, 28.3 μmol) and AA-1 (prepared in a similar manner as X-5) (182 mg, 849 μmol) at room temperature. The reaction mixture was stirred in a microwave at 150 °C for 2 h under nitrogen atmosphere. The reaction mixture was quenched with (20 mL) and extracted with 10% MeOH in DCM (2 x 45 mL). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous 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 x 250) mm, 5 μ flow - 15.0 mL / min, gradient - 0 / 40, 3 / 40, 10.5 / 65, 10.55 / 100, 13 / 100, 13.05 / 40, 17 / 40). The pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 125. M / Z (ESI): 487.30 [M+H] + .

[0225] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.77 (br s, 1H), 9.00 (s, 2H),8.87 (s, 1H), 8.33 (d, J = 2.4 Hz, 1H), 8.15 (s, 1H), 6.90 (br s, 1H), 6.64 (s,1H), 4.58 (t, J = 12.4 Hz, 4H), 4.04-4.52 (m, 3H), 3.44 (dd, J = 13.4 Hz, 3.4 Hz,1H), 3.20-3.28 (m, 2H), 1.07 (d, J = 6.8 Hz, 3H)。

[0226] Synthesis of BB-3: tert-butyl (S)-4-(6-fluoropyridin-3-yl)-2-methylpiperazine-l-carboxylate Synthesis of BB-4: (S)-l-(6-fluoropyridin-3-yl)-3-methylpiperazine Synthesis of BB-5: (S)-2-bromo-5-(4-(6-fluoropyridin-3-yl)-2-methylpiperazin-l-yl)pyrazine To a stirred solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (5 g, 25 mmol) in 1,4-dioxane (100 mL) was added sodium 2-methylpropan-2-olate (8.03 mL, 74.9 mmol) and 5-bromo-2-fluoropyridine (4.39 g, 25 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 15 min. To this reaction mixture was then added tris(dibenzylideneacetone)dipalladium (1.14 g, 1.25 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.44 g, 2.5 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 12 h in a sealed tube under nitrogen atmosphere. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 75 mL). The combined organic layers were washed with brine (2 x 30 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 80 g silica (230-400 mesh) column and eluted with 20% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford BB-3. M / Z (ESI): 296.28 [M+H] + .

[0227] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of BB-3 (2.5 g, 8.46 mmol) in DCM (30 mL) was added 4M HC1 in 1,4-dioxane (309 mg, 8.46 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated and dried under reduced pressure to afford BB-4. M / Z (ESI): 196.24 [M+H] + .

[0228] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of BB-4 (500 mg, 2.16 mmol) in DMSO (10 mL) was added CsF (983 mg, 6.47 mmol) and 2,5-dibromopyrazine (513 mg, 2.16 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h in a sealed tube. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using a 12 g silica (230-400 mesh) column and eluting the compound with 30% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford BB-5. M / Z (ESI): 352.23 [M+H] + .

[0229] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide To a stirred solution of BB-5 (150 mg, 426 μmol) and BB-6 (prepared in a similar manner as X-5) (90.8 mg, 426 μmol) in 1,4-dioxane (2 mL) was added Cul (8.11 mg, 42.6 μmol), trans-N,N'-dimethyl-1,2-cyclohexanediamine (6.72 μL, 21.3 μmol) and Cs2C03(416 mg, 1.28 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 40 h in a sealed tube. The reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: Column: XBridge C18 (4.6 x 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 Diluent: MeCN: Water (90: 10) V / V). The pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 126. M / Z (ESI): 485.15 [M+H] + .

[0230] 1 H 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)。

[0231] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide Synthesis of FF-2: (S)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)pyrimidin-5- yl)-6-(3-(hydroxymethyl)azetidin-l-yl)nicotinamide To a stirred solution of CC-1 (prepared in a similar manner as BB-5) (150 mg, 376 µmol) and P-7 (76 mg, 376 µmol) in 1,4-dioxane (2 mL) was added cesium carbonate (367.0 mg, 1128 µmol), CuI (7.16 mg, 37.6 µmol) and trans-N,N’-dimethyl-1,2-cyclohexanediamine (5.93 µL, 18.8 µmol) at room temperature. The reaction mixture was stirred in a sealed tube at 110 °C for 40 h. The reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: Column: X Bridge Amide (4.6 x 150) mm, 3.5 µm Mobile Phase-A: 20 mM Ammonium Hydroxide in water Mobile Phase-B: 50:50 MeCN:MEOH Gradient (T / %B): 0 / 5, 1 / 5, 12 / 60, 15 / 70, 20 / 100, 22.1 / 5, 24 / 5, Flow Rate: 1.0 mL / min, Column Oven Temperature: Ambient Diluent: MeCN:H2O). The pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 127. M / Z (ESI): 474.19 [M+H] + .

[0232] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.32 (s, 1H), 9.05 (d, J = 1.6 Hz,1H), 8.71 (s, 1H), 8.41 (s, 1H), 8.28 (dd, J = 8.2 Hz, 2.2 Hz, 1H), 8.10 (s,1H), 7.85-7.91 (m, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.62-7.69 (m, 1H), 7.06 (dd, J = 9.2 Hz, 3.4 Hz, 1H), 4.85-4.95 (m, 1H), 4.48 (d, J = 10.4 Hz, 1H), 3.91 (s,3H), 3.71 (d, J = 12.0 Hz, 1H), 3.61 (d, J = 12.0 Hz, 1H), 3.33-3.39 (m, 1H),2.97 (dd,J = 12.0 Hz, 4.0 Hz, 2H), 2.77 (td, J = 11.6 Hz, 3.6 Hz, 1H), 1.26 (d, J = 6.8 Hz, 3H)。

[0233] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide Table 6 To a stirred solution of DD-1 (prepared in a similar manner as BB-5) (100 mg, 284 μmol) in 1,4-dioxane (1.5 mL) was added Cs2CO3(278 mg, 852 μmol), Cul (5.41 mg, 28.4 μmol), P-7 (68.9 mg, 341 μmol) and trans-N,N'-dimethyl-1,2-cyclohexanediamine (4.48 μL, 14.2 μmol). The reaction mixture was stirred at 110 °C in a sealed tube under nitrogen atmosphere for 16 h. The reaction mixture was quenched with water (45 mL) and extracted with EtOAc (2 x 85 mL). The combined organic layers were washed with brine (2 x 45 mL), dried over anhydrous 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 - Princeton Spher ULTIMA C18 (21.2 x 250), 55 μ flow - 15.0 ml / min, gradient method: - 0 / 48, 3 / 48, 14 / 59, 14.05 / 98, 17 / 98, 17.05 / 48, 21.0 / 48). The pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 128. M / Z (ESI): 474.36 [M+H] + .

[0234] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.83 (s, 1H), 9.09 (dd, J = 2.2 Hz,0.6 Hz, 1H), 8.90 (d, J = 1.2 Hz, 1H), 8.41 (s, 1H), 8.34 (dd, J = 8.4 Hz, 2.4Hz, 1H), 8.19 (d, J= 8.4 Hz, 0.8 Hz, 1H), 7.62-7.71 (m, 1H), 7.06 (dd, J = 8.8 Hz, 3.4 Hz, 1H),4.58-4.75 (m, 1H), 4.14 (d, J = 10.0 Hz, 1H), 3.91 (s, 3H), 3.74 (d, J = 12.0 Hz,1H), 3.63 (d, J = 12.0 Hz, 1H), 3.27 (dd, J = 12.8 Hz, 3.6 Hz, 1H), 3.03 (dd, J =12.0 Hz, 3.6 Hz, 1H), 2.84 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.24 (d, J = 6.4 Hz,3H)。 J

[0235] Example 124: (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-l- yl)pyrazin-2-yl)-6-(l-methyl-lH-pyrazol-4-yl)nicotinamide ​ ​To a stirred solution of EE-1 (prepared in a similar manner as S-4) (100 mg, 250 pmol) and P-7 (60.8 mg, 301 pmol) in 1,4-dioxane (2 mL) was added trans-N,N'-dimethyl-1,2-cyclohexanediamine (3.95 pL, 12.5 pmol), Cs2CO3 (245 mg, 751 pmol) and Cul (4.77 mg, 25.0 pmol) at room temperature. The reaction mixture was stirred at 120 °C for 16 h under nitrogen atmosphere. The reaction mixture was quenched with saturated aqueous Na2CO3 (20 mL) and extracted with 10% MeOH in DCM (2 x 35 mL). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous 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 - YMC-Actus (20 x 100) mm, 3 pm flow - 12 mL / min, gradient - 0 / 43, 3 / 43, 9.0 / 62, 9.05 / 100, 12 / 100, 12.05 / 43, 15 / 43). The pure fractions were combined, concentrated under reduced pressure and lyophilized to get 129. M / Z (ESI): 474.26 [M+H] + .

[0236] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.31 (s, 1H), 9.05 (d, J = 2.0 Hz,1H), 8.70 (s, 2H), 8.41 (s, 1H), 8.28 (dd, J = 8.2 Hz, 2.2 Hz, 1H), 8.08-8.15(m, 2H), 7.81 (dd, J = 8.4 Hz, 0.4 Hz, 1H), 7.49-7.59 (m, 1H), 6.92 (dd, J = 9.4Hz, 3.4 Hz, 1H), 4.80-4.93 (m, 1H), 4.44 (dt, J = 13.2 Hz, 3.2 Hz, 1H), 4.16(dd, J = 21.4 Hz, 12.6 Hz, 2H), 3.91 (s, 3H), 3.27 (d, J = 3.6 Hz, 1H), 3.15 (dd,J = 13.0 Hz, 3.8 Hz, 1H), 2.92 (td, J = 12.2 Hz, 3.6 Hz, 1H), 1.16 (d, J = 6.8 Hz,3H)。

[0237] ​ ​ ​ ​ To a stirred solution of S-4 (215 mg, 0.501 mmol) and FF-1 (prepared in a similar manner as X-5) (104 mg, 0.501 mmol) in 1,4-dioxane (3 mL) was added Cs2CO3(490 mg, 1.503 mmol), copper (I) iodide (9.54 mg, 0.050 mmol), trans-N,N'-dimethylcyclohexane-1,2-diamine (3.56 mg, 0.025 mmol) and degassed with argon for 10 min at room temperature. The reaction mixture was stirred at 150 °C for 2 h under microwave irradiation. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by silica column and eluted the compound with 10% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to get FF-2. M / Z (ESI): 477.07 [M-H] - .

[0238] ​ ​ To a stirred solution of FF-2 (70 mg, 0.146 mmol) in DCM (1 mL) was added DAST (0.039 mL, 0.293 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min under argon atmosphere. The reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (5 mL), dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN, Column - X-Bridge, C18 (19 x 250) mm, 5 μ flow - 2.0 mL / min, Gradient - 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 lyophilized to afford 130. M / Z (ESI): 481.14 [M+H] + .

[0239] 1 H 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, 3.6Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H)。

[0240] The compounds included in Table 6 were synthesized by similar methods from the above synthetic sequence. Where necessary, commercially available reagents were replaced to produce the following examples.

[0241] ​

[0242] ​ ​ GG-2: Synthesis of tert-butyl(S)-2-methyl-4-(pyrimidin-2-yl)piperazine-1-carboxylic acid ester To a solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (GG-1, 1.2 g, 1 Eq, 5.99 mmol) in DMF (24 mL) was added potassium carbonate (2.484 g, 3 Eq, 17.97 mmol) followed by 2-chloropyrimidine (1.029 g, 1.5 Eq, 8.99 mmol). The mixture was stirred at 22 °C for 3 h. Water (100 mL) was added to the mixture, stirred at 22 °C for 30 min, and extracted with EtOAc (100 mL x 3). The organic layer was dried over MgS04, filtered through a sintered filter, and concentrated under reduced pressure. The resulting residue was dissolved in DCM (3 mL) and purified using normal phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexanes, EtOAc, 26 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to give GG-2. MS (ESI) m / z: 279.3 [M+H] + .

[0243] Synthesis of GG-3: (S)-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride To a stirred solution of tert-butyl (S)-2-methyl-4-(pyrimidin-2-yl)piperazine-l- carboxylate (GG-2, 1.20 g, 1 Eq, 4.31 mmol) in 1,4-dioxane (12.0 mL) was added 4M hydrogen chloride in 1,4-dioxane (2.16 mL, 4.00 molar, 2 Eq, 8.62 mmol). The mixture was stirred at 22 °C for 18 h. The mixture was concentrated under reduced pressure to give GG-3. MS (ESI) m / z: 179.4 [M+H] + .

[0244] GG-4: Synthesis of (S)-2-(2-methyl-4-(pyrimidin-2-yl)piperazin-1-yl)-5-nitropyrimidine To a solution of (S)-2-(3-methylpiperazin-l-yl)pyrimidine hydrochloride (GG-3, 1.00 g, 1 Eq, 3.98 mmol) in DMF (18.0 mL) was added potassium carbonate (2.20 g, 4 Eq, 15.9 mmol) and 2-chloro-5-nitropyrimidine (762 mg, 1.2 Eq, 4.78 mmol). The mixture was stirred at 60 °C for 18 h. To the mixture was added water and stirred at 22 °C for 30 min. The resulting precipitated solid was collected by filtration through a sintered filter, washed with water (100 mL x 3), and dried to give GG-4. MS (ESI) m / z: 302.5 [M+H] + .

[0245] GG-5: Synthesis of (S)-2-(2-methyl-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-amine A solution of (S)-2-(2-methyl-4-(pyrimidin-2-yl)piperazin-l-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 and purged with N2(x3), then 10% carbon supported palladium (177 mg, 0.5 Eq, 1.66 mmol) was added and degassed and purged with H2(x3). The mixture was stirred at 22 °C under a H2-filled balloon for 18 h. The mixture was filtered through a sintered filter, washed with MeOH (5 mL x 3), and concentrated under vacuum to give GG-5. MS (ESI) m / z: 272.4 [M+H] + .

[0246] GG-6: (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinyl Amine Synthesis To a solution of (S)-2-(2-methyl-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-amine (GG-5, 250.0 mg, 1 Eq, 921.4 μmol) in DCM (6.0 mL) was added 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). The mixture was stirred at 22 °C for 30 min. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm - 30x250 mm column, 10-100% 5 mM NH4HCO3 in water: acetonitrile, 30 min gradient). Fractions containing product were combined and extracted between water (100 mL) and DCM (100 mL x 3). The collected organic layer was dried over MgSO4, then concentrated under vacuum to give GG-6. MS (ESI) m / z: 473.5 [M+H] + .

[0247] Example 134: 6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-2-fluoro-N-(2-(4-(pyrimidine- Synthesis of 2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide To a solution of (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidin-2-yl)piperazin- 1-yl)pyrimidin-5-yl)nicotinamide (GG-6, 60.0 mg, 1 Eq, 127 pmol) in 1,4-dioxane (0.50 mL) was added 1M potassium phosphate tribasic (31.5 pL, 3 Eq, 380 pmol) and 3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazole (44.5 mg, 1.5 Eq, 190 pmol). The mixture was purged with N2for 5 min before adding XPhos Palladacycle-G2 (9.97 mg, 0.1 Eq, 12.7 pmol). The mixture was stirred at 80 °C for 3 h. The mixture was concentrated and purified using normal phase chromatography (ISCO 12g RediSep Gold High Performance Silica, 10-100% hexanes, EtOAc, 18 min gradient). Fractions containing the desired product were combined and concentrated under reduced pressure. LCMS showed a major side product so it was re-dissolved in DCM (1 mL) and placed in a TLC chromatography chamber (1:4 hexanes, EtOAc). The desired product was collected from the TLC plate, stirred in EtOAc, and filtered through a fritted filter to give 134. MS (ESI) m / z: 501.6 [M+H] + .

[0248] 1H 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 = 7.9Hz, 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 = 7.3Hz, 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).

[0249] Example 135: 6-(4-aminophenyl)-2-fluoro-N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl) Niacinamide To a solution of (S)-6-bromo-2-fluoro-N-(2-(2-methyl-4-(pyrimidin-2-yl)piperazin-1- yl)pyrimidin-5-yl)nicotinamide (GG-6, 60.0 mg, 1 Eq, 127 μmol) in 1,4-dioxane (0.50 mL) was added 1M potassium phosphate tribasic (0.064 mL, 1.00 molar, 2 Eq, 0.064 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (10.5 mg, 1.15 Eq, 0.048 mmol). The mixture was purged with N2for 1 min, then XPhos Palladacycle-G2 (2.52 mg, 0.1 Eq, 0.003 mmol) was added. The mixture was stirred at 80 °C for 3 h. The mixture was filtered through a frit, concentrated, and purified using reverse phase HPLC (XBridge Prep OBD C18 column, 40-100% water with 0.1% NH4OH, acetonitrile). Fractions containing the desired product were combined and concentrated under reduced pressure to give 135. MS (ESI) m / z: 487.5 [M+H] + .

[0250] 1H NMR (500 MHz, DMSO): δ 8.73 (d, J = 2.3 Hz, 1H), 8.67 (s, 2H),8.38 (d, J = 4.7 Hz, 2H), 8.24 (dd, J = 9.8, 8.0 Hz, 1H), 8.10 (dd, J = 8.8,2.5 Hz, 1H), 7.91 (dd, J = 8.0, 1.9 Hz, 1H), 6.65 (t, J = 4.7 Hz, 1H), 6.56(d, J = 10.6 Hz, 3H), 4.87 (d, J = 2.6 Hz, 1H), 4.60 (d, J = 13.1 Hz, 1H),4.56 (d, J = 13.0 Hz, 1H), 4.47 – 4.40 (m, 1H), 3.24 (ddd, J = 17.1, 11.3,3.8 Hz, 2H), 3.07 (td, J = 12.5, 3.7 Hz, 1H), 1.09 (d, J = 6.6 Hz, 3H)。

[0251] Intermediate HH-1 : 6-(5,6-dihydro-4 H - pyrrolo [1,2- b ] pyrazol-3-yl)-2-fluoronicotinic acid HH-1 : 6-(5,6-dihydro-4 H - pyrrolo [1,2- b ] pyrazol-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 min. 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 EtOAc (3 x 30 mL) and DCM (3 x 20 mL). The aqueous layer was acidified with 1 M HCl and the resulting precipitated solid was collected by filtration, washed with water (10 mL x 2) and dried to give HH-1. MS (ESI) m / z: 248.2 [M+H] + .

[0252] Example 136: S )-6-(5,6-dihydro-4 H - pyrrolo [1,2- b ]pyrazol-3-yl)-2-fluoro- N -(2-(4-(5- Synthesis of Fluoropyrazin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide HH-2: tert-butyl (S)-4-(5-aminopyrimidin-2-yl)-3-methylpiperazin-1-ylcarboxylate S Synthesis of tert-butyl (S)-4-(5-aminopyrimidin-2-yl)-3-methylpiperazin-1-ylcarboxylate To a solution of 1-2 (8.88 g, 27.5 mmol) in methanol (137 mL) was added 10% Pd / C (2.00 g, 1.88 mmol). The mixture was stirred at 25 °C for 16 h under a hydrogen balloon. Ice water (300 mL) was added, and the mixture was stirred for 30 min. The precipitated solid was collected by filtration, washed with water (20 mL x 2) and dried to give HH-2. MS (ESI) m / z: 294.3 [M+H] + .

[0253] HH-3: tert-butyl( S )-4-(5-(6-(5,6-dihydro-4 H - pyrrolo [1,2- b ] pyrazol-3-yl)-2- fluoronicotinamide Synthesis of (amino)pyrimidine-2-yl)-3-methylpiperazine-1-carboxylic acid ester 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 h. The reaction was concentrated and purified by normal phase column chromatography [0 to 100% EtOAc / EtOH (3:1 mixture) in hexanes] to give HH-3. MS (ESI) m / z: 523.5 [M+H] + .

[0254] HH-4: (S)-3-amino-6-fluoro-5-(2,2,2-trifluoro-ethyl)-4,5-dihydro-isoxazole-4-carboxylic acid tert-butyl ester S )-6-(5,6-dihydro-4H-pyrrolo[1,2- b ]pyrazol-3-yl)-2-fluoro- N -(2-(2-methylpiperidin-1-yl)ethyl)-2-oxo-5-oxa-6- azaspiro[3. Synthesis of (azin-1-yl)pyrimidin-5-yl)nicotinamide To a stirred solution of compound HH-3 (822 mg, 1.57 mmol) in DCM (3.2 mL) was added 4M HC1 in dioxane (3.93 mL, 15.7 mmol) at 25 °C. The mixture was stirred at room temperature for 2 h, then poured into saturated aqueous sodium bicarbonate solution (20 mL) and extracted with DCM (3 x 10 mL). The combined organic layers were dried over Na2S04, filtered and concentrated under reduced pressure to give HH-4. M / Z (ESI): 423.4 [M+H] + .

[0255] 136: S )-6-(5,6-dihydro-4 H - pyrrolo [1,2- b ]pyrazol-3-yl)-2-fluoro- N -(2-(4-(5-fluoropyr Synthesis of (-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide To a solution of HH-4 (10.0 mg, 0.024 mmol) in DMF (0.4 mL) was added 2,5-difluoropyrazine (4.1 mg, 0.036 mmol) and DIPEA (1.0 μL, 0.12 mmol). The mixture was stirred at 80 °C for 16 h. The reaction mixture was purified by reverse-phase HPLC (on an XBridge Prep OBD C18 column, 40% to 100% MeCN / H2O w / 0.1% NH4OH gradient). The desired fractions were concentrated to give 136. MS (ESI) m / z: 519.2 [M+H] + .

[0256] Example 137: S )-6-(5,6-dihydro-4 H - pyrrolo [1,2- b ] pyrazol-3-yl)-2-fluoro- N - (2-(4-(6- Fluoropyrimidine-4-yl)-2-methylpiperazine-1-yl)pyrimidine-5-yl)nicotinamide 137:( S )-6-(5,6-dihydro-4 H - pyrrolo [1,2- b ] pyrazol-3-yl) -2- fluoro- N - (2- (4- (6- fluoro pyrimidin-4-yl) phenyl) -4- methylpiperidin- Synthesis of pyridin-4-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide To a solution of HH-4 (20.0 mg, 0.0473 mmol) in DMF (0.473 mL) was added 4,6-difluoropyrimidine (8.24 g, 0.0710 mmol) and DIPEA (0.0412 mL, 0.237 mmol). The mixture was stirred at 22 °C for 2 h. The reaction mixture was purified by reverse-phase HPLC (on a Gemini-NX column, 40-100% MeCN / H2O w / 0.1% NH4OH gradient). The desired fractions were concentrated to give 137. MS (ESI) m / z: 519.4 [M+H] + .

[0257] 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, 3.6Hz, 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)。

[0258] Example 138: (R)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-2-fluoro-N-(2-(3-(methyl) 4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide II-2: Synthesis of tert-butyl(R)-3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazine-1-carboxylic acid ester To a solution of tert-butyl (R)-3-(methoxymethyl)piperazine-l-carboxylate (II- 1, 1.0 g, 1 Eq, 4.34 mmol) in DMF (15 mL) was added diisopropylethylamine (1.68 g, 2.24 mL, 3 Eq, 13.0 mmol) followed by 2-chloropyrimidine (746 mg, 1.5 Eq, 6.51 mmol). The mixture was stirred at 130 °C for 18 h. To the mixture was added water (50 mL), which was stirred at 22 °C for 30 min, and then extracted with EtOAc (60 mL x 3). The organic layer was dried over MgS04, filtered through a sintered filter, and concentrated under reduced pressure. The resulting residue was dissolved in DCM (3 mL) and purified using normal phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexanes, EtOAc, 28 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to give II-2. MS (ESI) m / z: 309.5 [M+H] + .

[0259] II-3: Synthesis of (R)-2-(2-(methoxymethyl)piperazin-1-yl)pyrimidine hydrochloride To a solution of tert-butyl (R)-3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazine-l- carboxylate (II-2, 870 mg, 1 Eq, 2.82 mmol) in 1,4-dioxane (12.0 mL) was added 4M hydrogen chloride in 1,4-dioxane (1.41 mL, 2 Eq, 5.64 mmol). The mixture was stirred at 22 °C for 2 h. Additional 4M hydrogen chloride in 1,4-dioxane (4 mL) was added to the mixture and stirred for 1 h. The mixture was concentrated under reduced pressure to give II-3. MS (ESI) m / z: 209.4 [M+H] + .

[0260] II-4: Synthesis of (R)-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)-5-nitropyrimidine To a solution of (R)-2-(2-(methoxymethyl)piperazin-l-yl)pyrimidine hydrochloride (II-3, 790 mg, 1 Eq, 2.81 mmol) in DMF (15.0 mL) was added potassium carbonate (1.55 g, 4 Eq, 11.2 mmol) and 2-chloro-5-nitropyrimidine (538 mg, 1.2 Eq, 3.37 mmol). The mixture was stirred at 60 °C for 18 h. Water was added to the mixture and stirred at 22 °C for 30 min. The resulting precipitated solid was collected by filtration through a sintered filter, washed with water (100 mL x 3), and dried to give II-4. MS (ESI) m / z: 332.4 [M+H] + .

[0261] II-5: Synthesis of (R)-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-amine A solution of (R)-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-l-yl)-5- nitropyrimidine (II-4, 820 mg, 1 Eq, 2.47 mmol) in THF (8.00 mL) and MeOH (8.00 mL) was degassed and purged with N2(x3), then 10% carbon supported palladium (132 mg, 0.5 Eq, 1.24 mmol) was added, degassed, and purged with H2(x3). The mixture was stirred at 22 °C for 18 h under a H2-filled balloon. The mixture was filtered through a sintered filter, washed with MeOH (5 mL x 3), and concentrated under vacuum to give II-5. MS (ESI) m / z: 302.5 [M+H] + .

[0262] II-6:(R)-6-bromo-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin- Synthesis of 5-yl)nicotinamide To a solution of (R)-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-amine (II-5, 100.0 mg, 1 Eq, 331.8 pmol) in DCM (2.0 mL) was added diisopropylethylamine (286 pL, 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 pmol), and 6-bromo-2-fluoronicotinic acid (87.61 mg, 1.2 Eq, 398.2 pmol). The mixture was stirred at 22 °C for 1 h. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 5mm - 30 x 250 mm column, 5 mM NH4HCO3 in water: acetonitrile 10-100%, 26 min gradient). Fractions containing product were combined and extracted between water (80 mL) and DCM (80 mL x 3). The collected organic layer was dried over MgSO4, then concentrated under vacuum to give II-6. MS (ESI) m / z: 503.5 [M+H] + .

[0263] Example 138: (R)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-2-fluoro-N-(2-(3-(methyl) 4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide To a solution of (R)-6-bromo-2-fluoro-N-(2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide (II-6, 20.0 mg, 1 Eq, 39.7 pmol) in 1,4-dioxane (0.50 mL) was added 1 M potassium phosphate tribasic (9.87 pL, 3 Eq, 119 pmol) 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 pmol). The mixture was purged with N2for 5 min, then XPhos Palladacycle-G2 (3.13 mg, 0.1 Eq, 3.97 pmol) was added. The mixture was stirred at 80 °C for 2 h. The mixture was filtered through a frit, concentrated, and purified using reverse-phase HPLC (XBridge Prep OBD C18 column, 40%-100% water with 0.1% NH4OH, acetonitrile). Fractions containing the desired product were combined and concentrated under reduced pressure to give 138. MS (ESI) m / z: 531.6 [M+H] + .

[0264] 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)。

[0265] Example 139: (S)-6-(3-fluorozacricyclobutane-1-yl)-N-(6-(4-(6-fluoropyrimidin-4-yl)-2-methyl) Piperazin-1-yl)pyridin-3-yl)nicotinamide JJ-2: Synthesis of (S)-6-bromo-N-(6-(4-(6-fluoropyrimidin-4-yl)-2- methylpiperazin-1-yl)pyridin-3-yl)nicotinamide Example 139: (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(6-fluoropyrimidin-4-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide To a stirred solution of JJ-1 (500 mg, 1.734 mmol) in DMF (20 mL) was added 6-bromonicotinic acid (525 mg, 2.60 mmol), HATU (1319 mg, 3.47 mmol) and DIPEA (0.909 mL, 5.20 mmol) at room temperature and stirred at 60 °C for 16 h. Then, the reaction mixture was cooled, diluted with ethyl acetate (20 mL), washed with cold brine, dried over Na2S04, filtered, concentrated under reduced pressure and purified using Prep. HPLC (Prep. HPLC conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN, column - X-Select C18 (19 x 250) mm 5u, flow - 18 ml / min, gradient - 0 / 20, 9 / 78, 9.05 / 99, 11 / 99, 11.02 / 20, 14 / 20 against the crude material. The obtained compound was further purified by SFC method (conditions: column: Chiralpak IG (250 x 4.6 mm) 5u, mobile phase: 0.1% DEA in CO2: MeOH, flow rate: 4.0 ml / min, temperature: 40 °C, wavelength: 220 nm). ), 5 μ flow phase-A: MeOH / DCM / DEA (50 / 50 / 0.2), isocratic A: 100% flow rate: 1.0 mL / min diluent: EtOH. The pure fractions were concentrated and lyophilized to get JJ-2. M / Z (ESI): 472.14 [M+H] + .

[0266] 1 H NMR (400 mHz, 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).

[0267] Piperazin-1-yl)pyridin-3-yl)nicotinamide Example 139: (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(6-fluoropyrimidin-4-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide To a stirred solution of JJ-2 (90 mg, 0.210 mmol) in toluene (3 mL) was added 3- fluorazetidine hydrochloride (25.8 mg, 0.231 mmol), Cs2C03(93 mg, 0.284 mmol), 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (12.17 mg, 0.021 mmol) at room temperature, purged with argon for 10 min, after which Pd2(dba)3(9.63 mg, 10.52 μmol) was added, purged with argon again for another 10 min and stirred at 110 °C for 16 h. Then, the reaction mixture was filtered over a bed of celite and washed with EtOAc. The filtrate was washed with cold brine, dried over Na2S04, filtered, concentrated under reduced pressure and the crude purified by Prep-HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H20: MeCN, column - X-Select C18 (19 x 250) mm 5u, flow - 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 method (conditions: column: , 5 μ flow phase - A: MeOH / DCM / DEA (50 / 50 / 0.2) Isocratic A: 100% flow rate: 0.70 mL / m, in diluent: EtOH. The pure fractions were concentrated under reduced pressure and lyophilized to get 139. M / Z (ESI): 467.18 [M+H] + .

[0268] 1 H NMR (400 mHz, 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, 2.2Hz, 1H), 7.87-7.94 (m, 1H), 6.88 (br d, J=8.1 Hz, 1H), 6.61 (s, 1H), 6.53 (br

[0269] Example 141: (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6- (1-methyl-1H-pyrazol-4-yl)nicotinamide KK-2: Synthesis of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate To a stirred solution of NN-4 (70 mg, 164 μmol) in DMF (1 mL) was added DIPEA (0.18 mL, 1 mmol) and 2, 6-difluoropyridine (23 mg, 200 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h under 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 preparative HPLC (conditions: mobile phase - 10 mM ammonium bicarbonate in H2O:MeCN, column - X-Bridge C18 (19 x 250) mm, 5 μ flow - 15.0 mL / min, gradient method 0 / 55, 2 / 55, 10 / 67, 10.05 / 100, 12 / 100, 12.05 / 55, 15 / 55) purification. The pure fractions were combined and concentrated under reduced pressure to afford 140. M / Z (ESI): 485.29 [M+H] + .

[0270] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.58 (s, 1H), 8.86 (d, J = 1.2Hz, 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)。

[0271] KK-3: Synthesis of (S)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine hydrochloride KK-4: Synthesis of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine KK-5: Synthesis of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine To a solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (KK-1, 1.00 g, 1 Eq, 4.99 mmol) in toluene (30.0 mL) was added sodium 2-methylpropan-2-olate (1.44 g, 1.61 mL, 3 Eq, 15.0 mmol) and tert-butyl (S)-2-methylpiperazine-l-carboxylate (1.00 g, 1 Eq, 4.99 mmol). The mixture was purged with N2for 5 min, then chloro(2-dicyclohexylphosphino-2',6'-di-isopropoxy-l,l'- biphenyl)(2'-amino-l,l'-biphenyl-2-yl)palladium(II) (194 mg, 0.05 Eq, 250 μmol) was added. The mixture was stirred at 110 °C for 48 h. To the mixture was added water (50 mL), and it was extracted with EtOAc (100 mL x 3). The organic layer was dried over MgSO4, filtered through a sintered filter, and concentrated under reduced pressure. The resulting residue was dissolved in DCM (3 mL) and purified using normal phase chromatography (ISCO 80 g RediSepGold High Performance Silica, 0-100% hexanes, EtOAc, 26 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to give KK-2. MS (ESI) m / z: 296.4 [M+H] + .

[0272] KK-6: Synthesis of (S)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin- 2-yl)nicotinamide To a solution of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-l- carboxylate (KK-2, 1.00 g, 1 Eq, 3.39 mmol) in 1,4-dioxane (14.0 mL) was added 4M hydrogen chloride in 1,4-dioxane (3.39 mL, 4 Eq, 13.5 mmol). The mixture was stirred at 22 °C for 18 h. The mixture was concentrated under reduced pressure to give KK-3. MS (ESI) m / z: 196.3 [M+H] + .

[0273] Example 141: (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6- (1-methyl-1H-pyrazol-4-yl)nicotinamide To a solution of (S)-l-(5-fluoropyridin-2-yl)-3-methylpiperazine hydrochloride (KK-3, 900 mg, 1 Eq, 3.36 mmol) in DMF (15.0 mL) was added potassium carbonate (1.86 g, 4 Eq, 13.4 mmol) and 2-chloro-5-nitropyrazine (642 mg, 1.2 Eq, 4.03 mmol). The mixture was stirred at 60 °C for 18 h. To the mixture was added water and stirred at 22 °C for 30 min. The resulting precipitated solid was collected by filtration through a sintered filter, washed further with water (100 mL x 3), and dried to give KK-4. MS (ESI) m / z: 319.3 [M+H] + .

[0274] Example 142: (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide A solution of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)-5-nitropyrazine (KK-4, 579 mg, 1 Eq, 1.82 mmol) in THF (5.00 mL) and MeOH (5.00 mL) was degassed and purged with N2(x3), then 10% carbon supported palladium (96.8 mg, 0.5 Eq, 909 μmol) was added, degassed, and purged with H2(x3). The mixture was stirred at 22 °C under a H2-filled balloon for 18 h. The mixture was filtered through a sintered filter, washed with MeOH (5 mL x 3), and concentrated under vacuum to give KK-5. MS (ESI) m / z: 289.3 [M+H] + .

[0275] Example 143: (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6- (1-methyl-1H-pyrazol-4-yl)nicotinamide LL-2: Synthesis of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1-carboxylate To a solution of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-l-yl)-5- nitropyrazine (KK-5, 520.00 mg, 1 Eq, 1.8035 mmol) in DMF (8.0 mL) was added diisopropylethylamine (1.165 g, 1.57 mL, 5 Eq, 9.0174 mmol), 2,4,6-tripropyl- 1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (1.1477 g, 1.22 mL, 50% Wt, 1 Eq, 1.8035 mmol), and 6-chloronicotinic acid (340.97 mg, 1.2 Eq, 2.1642 mmol). The mixture was stirred at 22 °C for 1.5 h. The crude mixture was purified using basic reverse phase chromatography (Waters XBridge Prep C18 5mm-50x250 mm column, 5 mM NH4HCO3 in water:acetonitrile, 10-100%, 33 min gradient). Fractions containing product were combined and extracted between water (100 mL) and DCM (100 mL x 3). The collected organic layer was dried over MgSO4and then concentrated under vacuum to give KK-6. MS (ESI) m / z: 428.4 [M+H] + .

[0276] LL-3: Synthesis of (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride LL-4: Synthesis of (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazin-2-yl)piperazin-1-yl)pyrimidine To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide (KK-6, 20.00 mg, 1 Eq, 46.74 pmol) in 1,4-dioxane (0.30 mL) was added 1M potassium phosphate tribasic (140.2 pL, 1.00 mole, 3 Eq, 140.2 pmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazole (11.67 mg, 1.2 Eq, 56.74 pmol). The mixture was purged with N2for 5 min, then XPhos Palladacycle-G2 (3.678 mg, 0.1 Eq, 4.674 pmol) was added. The mixture was stirred at 80 °C for 2 h. After the reaction time, LCMS showed high conversion of desired product. Next, the mixture was filtered through a frit, concentrated, and purified using reverse phase HPLC (XBridge Prep OBD C18 column, 40-100% water with 0.1% NH4OH, acetonitrile). Fractions containing the desired product were combined and concentrated under reduced pressure to give 141. MS (ESI) m / z: 474.5 [M+H] + .

[0277] 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 = 3.0Hz, 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).

[0278] LL-5: Synthesis of (S)-5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-amine LL-6: Synthesis of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin- 2-yl)nicotinamide To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-l-yl)pyrazin-2-yl)nicotinamide (KK-6, 20.00 mg, 1 Eq, 46.74 μmol) in DMF (0.50 mL) was added 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). The mixture was concentrated under vacuum, redissolved in DCM (1 mL) and purified using normal phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% hexanes, EtOAc, 26 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to give 142. MS (ESI) m / z: 485.4 [M+H] + .

[0279] 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 = 11.8Hz, 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).

[0280] ​ ​ ​ To a solution of tert-butyl (S)-2-methylpiperazine-l-carboxylate (LL-1, 5.43 g, 1.00 Eq, 27.1 mmol) in DMF (67.8 mL) was added 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). The mixture was stirred at 80 °C for 16 h. To the mixture was added water (300 mL) and extracted with diethyl ether (100 mL x 3). The organic layer was dried over Na2S04, filtered through a sintered filter, and concentrated under reduced pressure. The resulting residue was purified using normal phase chromatography (ISCO 330 g RediSep Gold High Performance Silica, 0-25% hexanes, EtOAc). Fractions containing the desired product were combined and concentrated under vacuum to give LL-2. MS (ESI) m / z: 297.1 [M+H] + .

[0281] ​ To a solution of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-l- carboxylate (LL-2, 7.35 g, 1 Eq, 24.8 mmol) in DCM (124 mL) was added 4M hydrogen chloride in DCM (62.0 mL, 4.00 moles, 10 Eq, 248 mmol). The mixture was stirred at 22 °C for 2 h. The mixture was concentrated under reduced pressure to give LL-3. MS (ESI) m / z: 320.2 [M+H] + .

[0282] ​ To a solution of (S)-5-fluoro-2-(3-methylpiperazin-l-yl)pyrimidine hydrochloride (LL-3, 1.00 g, 1 Eq, 5.10 mmol) in DMF (25.5 mL) was added potassium carbonate (2.82 g, 4.00 Eq, 20.4 mmol) and 2-chloro-5-nitropyrazine (976 mg, 1.20 Eq, 6.12 mmol). The mixture was stirred at 60 °C for 16 h. To the mixture was added ice water and the resulting precipitated solid was collected through a sintered filter, washed with water (5 mL x 3), and dried to give LL-4. MS (ESI) m / z: 290.2 [M+H] + .

[0283] ​ A solution of (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazin-2-yl)piperazin-l- yl)pyrimidine (LL-4, 1.20 g, 1 Eq, 3.76 mmol) in DCM (9.40 mL) and MeOH (9.40 mL) was degassed and purged with N2(x3) before 10% carbon supported palladium (200.0 mg, 0.500 Eq, 1.879 mmol) was added, degassed and purged with H2(x3). The mixture was stirred under a H2-filled balloon at 22 °C for 6 h. The mixture was filtered through a frit and concentrated under vacuum to give LL-5. MS (ESI) m / z: 290.2 [M+H] + ​ ​ To a solution of (S)-5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-l-yl)pyrazin-2- amine (LL-5, 520.00 mg, 1 Eq, 1.8035 mmol) in DMF (8.0 mL) was added diisopropylethylamine (670.11 mg, 903 μL, 5 Eq, 5.1846 mmol), 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphospha cyclononane 2,4,6-trioxide (659.85 mg, 699 μL, 50% Wt, 1 Eq, 1.0369 mmol), and 6-chloronicotinic acid (196.04 mg, 1.2 Eq, 1.2443 mmol). The mixture was stirred at 22 °C for 3 h. The crude mixture was purified using basic reverse phase chromatography (Waters XBridge Prep C18 5 mm-50 x 250 mm column, 5 mM NH4HCO3 in water:acetonitrile 10-100%, 33 min gradient). Fractions containing product were combined and extracted between water (100 mL) and DCM (150 mL x 3). The collected organic layer was dried over MgSO4and then concentrated under vacuum to give LL-6. MS (ESI) m / z: 429.3 [M+H] + .

[0284] Example 143: (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-l-yl)pyrazin-2-yl)-6- ( 1 -methyl- 1 H-pyrazol-4-yl)nicotinamide To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2- methylpiperazin-l-yl)pyrazin-2-yl)nicotinamide (LL-6, 20.00 mg, 1 Eq, 46.64 pmol) in 1,4-dioxane (0.50 mL) was added 1M potassium phosphate tribasic (139.9 pL, 1.00 molar, 3 Eq, 139.9 pmol) and l-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH- pyrazole (11.64 mg, 1.2 Eq, 55.96 pmol). The mixture was purged with N2for 5 min before adding XPhos Palladacycle-G2 (3.669 mg, 0.1 Eq, 4.664 pmol). The mixture was stirred at 80 °C for 2 h. The mixture was concentrated and purified using normal phase chromatography (ISCO 12 g RediSep Gold High Performance Silica, 10-100% hexanes, EtOAc, 18 min gradient). Fractions containing the desired product were combined and concentrated under reduced pressure. LCMS showed major side products and so it was re-dissolved in DMSO (1 mL) and re-purified using basic reverse phase chromatography (Gilson Waters XBridge Prep OBD C18 5 pm 10-100% 0.1% NH4OH in water: acetonitrile, 10 min gradient). Fractions containing the product were combined and concentrated to give 143. MS (ESI) m / z: 475.4 [M+H] + .

[0285] 1 H 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).

[0286] Example 144: (S)-6-( 1 -(2-fluoroethyl)- 1 H-pyrazol-4-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)- 2-methylpiperazin-l-yl)pyrazin-2-yl)nicotinamide Example 144: (S)-6-( 1 -(2-fluoroethyl)- 1 H-pyrazol-4-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)- 2-methylpiperazin-l-yl)pyrazin-2-yl)nicotinamide To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide (LL-6, 20.00 mg, 1 Eq, 46.64 pmol) in 1,4-dioxane (0.50 mL) was added 1M potassium phosphate tribasic (139.9 pL, 1.00 molar, 3 Eq, 139.9 pmol) 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 pmol). The mixture was purged with N2for 5 min, then XPhos Palladacycle-G2 (3.669 mg, 0.1 Eq, 4.664 pmol) was added. The mixture was stirred at 80 °C for 2 h. The mixture was concentrated under a concentrator, dissolved in DCM (1 mL), and purified using normal phase chromatography (ISCO 12 g RediSepGold High Performance Silica, 10-100% hexanes, EtOAc, 18 min gradient). Fractions containing the desired product were combined and concentrated under reduced pressure to give 144. MS (ESI) m / z: 507.4 [M+H] + .

[0287] 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 = 13.0Hz, 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).

[0288] Synthesis of radiolabeled intermediates: Intermediate NN-5: (S)-N-(5-(4-(6-chloropyrimidin-4-yl)-2-methylpiperazin-l-yl)pyrazin-2-yl)-6- (3,3-difluoroazetidin-l-yl)nicotinamide Synthesis of (S)-6-(3,3-difluoroazetidin-l-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2- methylpiperazin-l-yl)pyrazin-2-yl)nicotinamide NN-1: Synthesis of tert-butyl (S)-4-(5-bromopyrazin-2-yl)-3-methylpiperazine-l-carboxylate To a stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (5 g, 24.96 mmol) in DMSO (100 mL) was added CsF (11.38 g, 74.9 mmol) and 2,5-dibromopyrazine (7.13 g, 30.0 mmol) at room temperature. The reaction mixture was stirred at 80 °C under argon atmosphere for 6 h. The reaction mixture was quenched with cold water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using silica (230-400 mesh) column and eluted the compound with 15% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford NN-1. M / Z (ESI): 357.15 [M+H] + .

[0289] NN-3: Synthesis of tert-butyl (S)-4-(5-(6-(3,3-difluoroazetidin-l-yl)nicotinamido)pyrazin-2-yl)- 3-methylpiperazine-l-carboxylate NN-4: Synthesis of (S)-6-(3,3-difluoroazetidin-l-yl)-N-(5-(2-methylpiperazin-l-yl)pyrazin-2- yl)nicotinamide To a stirred solution of NN-1 (1.50 g, 4.20 mmol) in 1,4-dioxane (30 mL) was added NN-2 (985 mg, 4.62 mmol), Cs2C03(4.10 g, 12.6 mmol), Cul (80.0 mg, 420 μmol) and trans-(lr,2r)-N,N'-dimethyl-l,2-cyclohexanediamine (66.2 μL, 210 μmol) at room temperature and degassed with argon for 10 min. The reaction mixture was stirred at 150 °C under microwave irradiation for 2 h. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using silica (230-400 mesh) column and eluted the compound with 5% MeOH in DCM. The pure fractions were combined and concentrated under reduced pressure to afford NN-3. M / Z (ESI): 490.77 [M+H] + .

[0290] NN-5: Synthesis of (S)-N-(5-(4-(6-chloropyrimidin-4-yl)-2-methylpiperazin-l-yl)pyrazin-2-yl)-6- (3,3-difluoroazetidin-l-yl)nicotinamide OO-7A: Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid To a stirred solution of NN-3 (900 mg, 1.84 mmol) in DCM (10 mL) was added 4M HC1 in 1,4-dioxane (2.30 mL, 9.19 mmol) at 0 °C. The reaction mixture was stirred at 25 °C under argon atmosphere for 4 h. The reaction mixture was concentrated under reduced pressure to afford NN-4. M / Z (ESI): 390.23 [M+H] + .

[0291] Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid Synthesis of tert-butyl (S)-3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-l-carboxylate To a stirred solution of NN-4 (150 mg, 352 μmol) in DMF (2 mL) was added DIPEA (0.38 mL, 2.2 mmol) and 4,6-dichloropyrimidine (63.0 mg, 423 μmol) at room temperature. The reaction mixture was stirred at 25 °C under argon atmosphere for 6 h. The reaction mixture was quenched with cold water (10 mL). The precipitate was stirred for 10 min, filtered and dried under reduced pressure. The crude compound was triturated with diethyl ether (10 mL) to afford NN-5. M / Z (ESI): 502.21 [M+H] + .

[0292] 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 = 13.2Hz, 3.6 Hz, 1H), 3.20-3.29 (m, 2H), 1.07 (d, J = 6.4 Hz, 3H).

[0293] Synthesis of tert-butyl (S)-3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-l-carboxylate Synthesis of tert-butyl (S)-3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-l-carboxylate Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methylpiperazin-l-yl)pyrimidin-5-yl)nicotinamide Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid To a stirred solution of tert-butyl (S)-3-methylpiperazine-l-carboxylate (10 g, 49.9 mmol) in DMF (100 mL) was added 2-chloro-5-nitropyrimidine (9.56 g, 59.9 mmol) and cesium carbonate (16.3 g, 49.9 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 120 g of silica gel column and eluting the compound with 70% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get OO-l. M / Z (ESI): 324.23 [M+H] + .

[0294] Synthesis of (S)-6-(azetidin-l-yl)-N-(2-(2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid To a stirred solution of OO-l (15 g, 46.4 mmol) in EtOH (100 mL) and THF (100 mL) was added 10% Pd / C (4.94 g, 46.4 mmol) at room temperature. The reaction mixture was degassed and purged with nitrogen. The reaction mixture was stirred at room temperature for 16 h under hydrogen atmosphere. The reaction mixture was filtered through a pad of celite, the filtrate was concentrated and dried under reduced pressure. The crude compound was triturated with n-pentane and dried under reduced pressure to get OO-2. M / Z (ESI): 294.22 [M+H] + .

[0295] ​ ​ 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) was added DIPEA (1.78 mL, 10.2 mmol) and HATU (1.94 g, 5.11 mmol) at room temperature. The reaction mixture was stirred at room temperature for 8 h. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (20 mL). The combined organic layers were dried over Na2S04, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 12 g of silica gel column and eluting the compound with 40% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to get OO-3. M / Z (ESI): 417.29 [M+H] + .

[0296] ​​ To a stirred solution of OO-3 (800 mg, 1.92 mmol) in DMSO (5 mL) was added azetidine hydrochloride (270 mg, 2.88 mmol) and K2CO3 (796 mg, 5.76 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at 120 °C for 24 h. The reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by Biotage using 12 g silica gel column and eluting the compound with 80% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford OO-4. M / Z (ESI): 454.33 [M+H] + .

[0297] ​ ​ To a stirred solution of OO-4 (3 g, 6.61 mmol) in DCM (50 mL) was added 4M HC1 in 1,4-dioxane (6.61 mL, 26.5 mmol) at 0 °C under argon atmosphere. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated and dried under reduced pressure to afford OO-5. M / Z (ESI): 354.33 [M+H] + .

[0298] ​ ​ 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) was added potassium carbonate (2.13 g, 15.4 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was quenched with water (30 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column and eluting the compound with 80% EtOAc in petroleum ether. The pure fractions were combined and concentrated under reduced pressure to afford OO-6. M / Z (ESI): 509.23 [M+H] + .

[0299] 1H 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).

[0300] ​ 2-dioxolanyl-pyridin-2-yl)piperazin-l-yl)pyrimidin-5-yl)nicotinamide and OO-7B (S)-(6-(4- Synthesis of (5-(6-(azetidin-l-yl)nicotinamido)pyrimidin-2-yl)-3-methylpiperazin-l-yl)pyridin-3-yl)boronic acid Synthesis of PP-3: (S)-l-(5-((5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)aminomethyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate To a stirred solution of OO-6 (300 mg, 588.9 μmol) in 1,4-dioxane (5 mL) was added potassium acetate (173.4 mg, 1.767 mmol) and bis(pinacolato)diboron (179.5 mg, 706.7 μmol) at room temperature under an atmosphere of argon. The reaction mixture was degassed and purged with argon for 2 min. To this reaction mixture was then added PdCl2(dppf)-CH2Cl2 adduct (48.09 mg, 58.89 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was filtered through a pad of celite and the filtrate was 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 x 250) mm, 5 μ flow - 15.0 mL / min, gradient - 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, concentrated under reduced pressure and lyophilized separately to afford OO-7A and OO-7B.

[0301] OO-7A: M / Z (ESI): 557.44 [M+H] + .

[0302] 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)。

[0303] OO-7B: M / Z (ESI): 473.32 [M-H] - 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 = 12.0Hz, 3.6 Hz, 1H), 2.34-2.41 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H)。

[0304] Synthesis of PP-1: 6-(3-hydroxyazetidin-l-yl)nicotinamide Synthesis of PP-2: (S)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6-(3-hydroxyazetidin-l-yl)nicotinamide Synthesis of PP-3: (S)-l-(5-((5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)aminomethyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate At 0 °C, K₂CO₃ (2.65 g, 19.16 mmol) and 3-hydroxyazacyclobutane hydrochloride (0.840 g, 7.66 mmol) were added to a stirred solution of 6-chloronicotinamide (1 g, 6.39 mmol) in DMF (40 mL). The reaction mixture was stirred at 100 °C under a nitrogen atmosphere for 16 h. The reaction mixture was diluted with EtOAc (50 mL), filtered through a diatomaceous earth mat, and washed with EtOAc (2 × 50 mL). The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using a 40 g silica (230-400 mesh) column, eluting with 12% MeOH in DCM. The purified fractions were combined and concentrated under reduced pressure to give PP-1. M / Z (ESI): 194.06 [M+H] + .

[0305] Radiosynthesis method Synthesis of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6-(lH-pyrazol-l-yl)nicotinamide At room temperature, Cs₂CO₃ (833 mg, 2.56 mmol), cuprous iodide (I) (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 to a stirred solution of Q-6 (300 mg, 0.852 mmol) in 1,4-dioxane (3 mL). The reaction mixture was stirred at 150 °C under a nitrogen atmosphere for 2 h under microwave irradiation. The reaction mixture was quenched with water (80 mL) and extracted with EtOAc (2 × 150 mL). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using an 80 g silica (230-400 mesh) column, and eluted with 3% MeOH in DCM. The purified fractions were combined and concentrated under reduced pressure to give PP-2. M / Z (ESI): 465.34 [M+H] + .

[0306] HPLC Prep separation conditions Synthesis of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6-(lH-pyrazol-l-yl)nicotinamide At 0 °C, TEA (0.180 mL, 1.292 mmol), 4-dimethylaminopyridine (26.3 mg, 0.215 mmol), and p-toluenesulfonyl-Cl (246 mg, 1.292 mmol) were added to a stirred solution of PP-2 (200 mg, 0.431 mmol) in DCM (4 mL). The reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 3 h. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 × 85 mL). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using an 80 g silica (230-400 mesh) column and eluted with 70% EtOAc in petroleum ether. The purified fractions were combined and concentrated under reduced pressure. The obtained compound was ground in 10% diethyl ether in pentane and dried under reduced pressure to give PP-3. M / Z (ESI): 619.33 [M+H] + .

[0307] 1 H 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 = 12.8Hz, 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).

[0308] HPLC Prep separation conditions [ 3 H]-1 : [3H]-(S)-6-methoxy-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5- Synthesis of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6-(lH-pyrazol-l-yl)nicotinamide Crabtree's catalyst (0.15 mg) was added to a tritium reaction vessel, followed by a solution of compound 1 (0.5 mg) in CH2Cl2 (0.2 mL). The vessel was connected to a 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 flasks were rinsed with 4 × 1 mL CH3OH and transferred to separate 100 mL recovery flasks. The combined CH3OH was removed under vacuum. Crude yield: 90 mCi. The substance was purified by HPLC. The mobile phase was removed under vacuum, and the product was redissolved in anhydrous ethanol. Yield: 20.6 mCi, purity >99%. The specific activity was determined by mass spectrometry to be 81.04 Ci / mmol; C 21 H 20 T3N7O2[M+H] + MW: 411.5, measured value: 412.4.

[0309] HPLC Prep separation conditions Method: 0-70% within 20 minutes Column: Phenomenex Luna 5flm C18 100 x 21.2mm Flow rate: 4 mL / min Injection volume: 0.5 mL Detection: UV @ 254 nm Mobile phase A: 0.05% TFA in H2O Mobile phase B: CH3CN Product elution time: 16.7 minutes [ 3 H]-24: [3H]-2-methoxy-N-[2-[(2S)-2-methyl-4-(2-pyridinyl)piperazin-l-yl]pyrimidin-5- Synthesis of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-l-yl)pyrazin-2-yl)-6-(lH-pyrazol-l-yl)nicotinamide Crabtree catalyst (0.3 mg) was added to a tritium reaction vessel, followed by a solution of compound 24 (0.4 mg) in CH2Cl2 (0.2 mL). The vessel was connected to a 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 flasks were rinsed with 4 × 1 mL CH3OH and transferred to separate 100 mL recovery flasks. The combined CH3OH was removed under vacuum. Crude yield: 125 mCi. The substance was purified by HPLC. The mobile phase was removed under vacuum, and the product was redissolved in anhydrous ethanol. Yield: 26 mCi, purity >99%. Specific activity was determined by mass spectrometry to be 150.01 Ci / mmol; C 21 H20 T5N7O2[M+H] + MW: 415.5, measured value: 416.3.

[0310] HPLC Prep separation conditions Method: 10% for 5 minutes; 10-90% for 20 minutes. Column: Phenomenex Luna Sum C18 100 x 21.2mm Flow rate: 4 mL / min Injection volume: 0.5 mL Detection: UV @ 254 nm Mobile phase A: 0.05% TFA in H2O Mobile phase B: CH3CN Product elution time: 18.1 minutes [ 3 H]-89: [3H]-( S )-N-(2-(4-(2-fluoropyridin-4-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)- Assay protocol Compound 89 in CH2Cl2 was washed with 0.5 M Na2CO3 aqueous solution, the organic layer was then dried 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, the nickel pre-catalyst ( ipc NiBr2 (6.65 mg) was dissolved in CPME (670 μL) and treated with NaHBEt3 (1 M, 23 μL) in toluene, followed by stirring for 5 min. The substrate solution (100 μL) was added to the active catalyst solution (100 μL) in the tritium ionization vessel and fixed using a portable Swagelok® valve. The valve was connected to the Trisorber, and two freeze-pump-thaw cycles were performed before introducing tritium gas at 155 mmHg. The reaction was thawed and then placed in an oil bath at 45 °C with stirring overnight. After capturing the used tritium on a waste bed, the reaction was transferred to a vial containing 10 mL of saturated sodium bicarbonate aqueous solution. The mixture was extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 247.2 mCi; RCP: 90%. The substance was purified by HPLC. The collected fraction was diluted with an equal volume of water, concentrated on a pair of C18 columns, and eluted with EtOH. Yield: approximately 20 mL of ethanol solution @ 4.22 mCi / mL. Specific activity was determined by mass spectrometry to be 55.52 Ci / mmol; C 23 H 19T4FN9O [M+H] + MW: 468.2, measured value: 468.3.

[0311] Obtaining post-mortem human tissue samples for in vitro binding assays Method: 10-95% B within 15 minutes, rebalanced within 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1 mL / min Injection volume: 1 uL Detection: UV @ 298 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 7.08 minutes Preparation of detergent-insoluble fractions of human brain tissue for in vitro binding studies Column: Gemini NX C18, 10 x 250 mm @ 40℃ Flow rate: 5 mL / min Injection volume: 0.4 mL Detection: UV @ 298 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN [ 3 H]-87: 3 H]- S )-6-(3,3-difluoroazetidin-1-yl)-N-(2-(4-(4-fluoropyridin-2-yl)- Procedure for alpha-synuclein tissue homogenate binding assay (Assay 1) Compound 87 (0.50 mg, 1.03 μol) was weighed into a tritium-concentrating vessel. Crabtree catalyst (1.12 mg, 0.51 μol) was prepared as a solution in CH₂Cl₂ (1.4 mL). The catalyst solution (500 μL) was added to the tritium-concentrating vessel connected to the ultrator port of the Trisorber and subjected to two freeze-pump-thaw cycles before introducing tritium gas at 84 mmHg. The reaction was thawed to room temperature and then stirred for 4 hours. After capturing the used tritium in a waste bed, the reaction was transferred to a vial containing 10 mL of saturated sodium bicarbonate aqueous solution. The mixture was extracted three times with dichloromethane. The combined organic layers were dried over 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 fraction was diluted with an equal volume of water, concentrated on a pair of C18 columns, and eluted with EtOH. A portion of the purified batch was allocated and diluted to 20.0 mL. Yield: 20.0 mL ethanol solution @ 2.45 mCi / mL. Specific activity was determined by mass spectrometry to be 131.4 Ci / mmol; C 23 H 19 T5F3N8O [M+H] + MW: 495.2, measured value: 495.3.

[0312] Figure 2 Method: 10-95% B within 15 minutes, rebalanced within 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1 mL / min Injection volume: 1.5-2.0 uL Detection: UV @ 304 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 7.63 minutes Figure 2 Method: Isoconcentration (A:B = 55:45) Column: Gemini NX C18, 10 x 250 mm @ 40℃ Flow rate: 5 mL / min Injection volume: 0.5 mL Detection: UV @ 304 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN [ 3 H]-91 : [3H]-(S)-6-(1 H-imidazol-1 -yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-1 -yl)ethyl)pyridazine-3-carboxamide Figure 2 In a glove box, compound 91 (1.33 mg, 3.0 μol) was dissolved in CPME (75 μL) and NMP (25 μL). A nickel precatalyst ( ipc ADI)NiBr2 (6.73 mg) was dissolved in CPME (670 μL) and treated with NaHBEt3 in toluene (1 M, 25 μL), followed by stirring for 5 min. In a tritium-containing vessel, the substrate solution (100 μL) was combined with the active catalyst solution (200 μL, 3.5 μmol) and fixed using a portable Swagelok® valve. The valve was connected to a Trisorber, and two freeze-pump-thaw cycles were performed before introducing 102 mm tritium gas. The reaction was thawed and then placed in an oil bath at 45 °C with stirring overnight. After capturing the used tritium in a waste bed, the reaction was transferred to a vial containing 10 mL of saturated sodium bicarbonate aqueous solution. The mixture was extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 120 mCi; RCP: 67%. The substance was purified by HPLC. The collected fraction was diluted with an equal volume of water, concentrated on a pair of C18 columns, and eluted with EtOH. Yield: approximately 20 mL of ethanol solution @ 3.18 mCi / mL. Specific activity was determined by mass spectrometry to be 44.9 Ci / mmol; C 23 H 17 T7N9O [M+H] + MW: 456.3, measured value: 456.0.

[0313] Competitive radioligand binding to pathologically aggregated beta amyloid in AD tissue (Assay 2): Method: 10-95% B is incubated for 12 minutes, maintained for 3 minutes, and then rebalanced for 6 minutes. Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40℃ Flow rate: 1 mL / min Injection volume: 1.0 uL Detection: UV @ 294 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN Product elution time: 6.32 minutes Figure 1 Method: Isoconcentration (A:B = 65:35) Column: Gemini NX C18, 10 x 250 mm @ 40℃ Flow rate: 5 mL / min Injection volume: 0.5 mL Detection: UV @ 295 nm Mobile phase A: 0.05 M pH 10 TEAA in H2O Mobile phase B: CH3CN Figure 1 Radioligand binding data in tritium-extracted alpha-synuclein from PD tissue (Assay 1) and A beta-rich AD tissue (Assay 2) - Table 1 Frozen human brain tissue from patients with Parkinson's disease (PD) was purchased from Analytical Biological Services Inc. Samples were postmortem tissue from donors clinically diagnosed with advanced PD. Alpha-synuclein, tau, and amyloid loads were determined by a combination of immunohistochemistry on frozen thin coronal sections and α-LISA-based quantification of protein levels in detergent-insoluble protein fractions. Tissue samples from the temporal lobe cortex of one donor were identified as having moderate to high α-synuclein loads, low amyloid levels, and minimal to no tau pathology. Determination-insoluble fractions from the temporal lobe cortex of this patient were used to support homogenate binding studies.

[0314] (ND = not determined) Gray matter was dissected from the temporal lobe cortex using a scalpel blade and minced with fine anatomical scissors. To prepare insoluble fractions, the minced tissue was homogenized using a glass Dounce tissue homogenizer 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). The homogenate was centrifuged at 100,000 × g for 45 min. At 4 °C, the pellet was resuspended in TBS-TX buffer using a Polytron at the highest setting for 30 sec. The homogenate was centrifuged at 100,000 × g for 45 min, and the pellet was resuspended in TBS-TX buffer. The final homogenate was analyzed using the BCA protein assay to determine protein concentration. The homogenate was aliquoted into 0.5 mL tubes and stored at -70 °C until use.

[0315] Figure 3 - 6 For the heat-saturated binding assay, radioligands of various concentrations are prepared in the assay buffer (DPBS with 0.1% BSA) to achieve a final concentration range of 0.84 to 50 nM. 3 H]-1. Add 25 μL of the radioligand to an insoluble fraction of 200 μL of PD brain homogenate, and dilute to 150 μg / ml in assay buffer (the incubation, filtration, and determination of the amount of radioligand used in the assay are described below). Non-specific binding was determined using self-blocking of the unlabeled compound. Saturation data were analyzed using GraphPad / Prism software.

[0316] Figure 3 Describes the use of [ 3 Saturation binding assays of tritium nuclei-insoluble fractions in temporal cortical PD tissue rich in aggregated α-synuclein [H]-1. These data support the use of this ligand in radioligand binding assays to optimize the efficacy of tactic binding to pathological α-synuclein. Figure 4 It shows [ 3 An example of thermally saturated binding of H-1, in which the radioactive ligand showed a high affinity for α-synuclein in PD brain homogenate, with a measured dissociation constant of 25 nM.

[0317] For the substitutional α-synuclein binding assay, the unlabeled test compound was dissolved in DMSO at 10 mM. Dilutes of the test compound were prepared in 100% DMSO at 1000× the final assay concentration to achieve various concentrations, and 0.225 μL aliquots were allocated to the assay plate. Insoluble fractions of PD brain homogenate were diluted in assay buffer from an initial 10 mg / mL to 50 μg / mL, and 200 μL was added to the assay plate, resulting in a final concentration of 10 μg / well. [The final assay concentration was prepared in assay buffer at 10× the final assay concentration.] 3H]-1, and added 25 μl to the assay plate to bring the final assay concentration to 2.0 nM. 200 μL of homogenate and 25 μL of radioligand were added to the assay plate containing the titrant. The plate was incubated at RT for 90 min. Unbound and bound ligands were separated by filtering the GF / C filter plate using a Perkin Elmer FilterMate Harvester Unifilter-96 (pretreated with 0.2% PEI at 4°C for 60 min), and unbound ligands were washed three times with 1 ml of ice-cold DPBS. The filter plate was dried (in an oven at 47°C under vacuum for 1 hour or at room temperature overnight). 50 μL / well of Microscint-20 (Perkin Elmer) was added to the plate, and the plate was counted per well for 1 minute using a Perkin Elmer TopCount. Data were analyzed using IDBS Activity Base to determine the K values ​​shown in Table 1. i Value (K) d The value was 25 nM, and the ligand concentration was 2.0 nM. Figure 5 ).

[0318] Figure 6 Frozen human brain samples for Alzheimer's disease (AD) were purchased from Analytical Biological Services Inc. The samples were post-mortem tissue from donors clinically diagnosed with AD, with most of the white matter dissected from the frontal cortex for preparation of gray-rich tissue homogenates. Gray-rich frontal cortex brain homogenates were prepared by homogenizing the tissue in ice-cold phosphate-buffered saline (PBS), pH 7.4, at 80 mg wet tissue per mL, on a Polytron setting 16 at 4°C for 45 seconds. The homogenates were further diluted with ice-cold PBS to 30 mg wet tissue per mL and homogenized again for 1 minute as described above. The homogenates were aliquoted in 5 mL tubes and stored at -70°C until use.

[0319] This assay uses radioligands prepared as described in ACS Med. Chem. Lett., Vol. 2, pp. 498-502. 3 H]-105.

[0320] For the heat-saturated binding assay, radioligands of various concentrations are prepared in assay buffer (PBS with 0.1% BSA) with 20% DMSO at concentrations ranging from 3.9 to 500 nM. 3[H]-105. Add 25 μL of the radioligand to 200 μL of crude brain homogenate (diluted to 0.5 mg / ml in assay buffer) to achieve a final radioligand concentration ranging from 0.39 to 50 nM and a final crude brain homogenate volume of 100 μg wet weight / well (the incubation, filtration, and determination of the amount of radioligand used in the assay are described below). Nonspecific binding was determined using self-blocking of the unlabeled compound. Saturation data were analyzed using Graphpad / Prism software.

[0321] ​ The high-affinity saturation binding of [3H]-105 to AD cortical tissue homogenates rich in aggregated β-amyloid pathology was described. ​ It shows [ 3 An example of thermally saturated binding of H-105 was demonstrated, in which the radioligand exhibited a high affinity for aggregated β-amyloid (Aβ) in AD brain homogenate, with a measured dissociation constant of 11 nM. This data supports the use of this ligand in radioligand binding assays to screen for binding to aggregated β-amyloid.

[0322] For assay 2, the unlabeled test compound was dissolved in DMSO at 10 mM. Dilutes of the test compound were prepared to various concentrations in 100% DMSO at 1000x the final assay concentration, and 0.225 μL aliquots were allocated to the assay plate. Brain homogenate was diluted in assay buffer from an initial 30 mg / mL to 0.5 mg / mL, and 200 μL was added to the assay plate to bring the final concentration to 100 μg wet weight / well. The final concentration was prepared in assay buffer with 20% DMSO at 10x the final concentration. 3 H]-105, and 25 μL was added to the assay plate to make the final assay concentration 3.0 nM. The plate was incubated at 37 °C for 90 min. Unbound and bound ligands were separated by filtering the bound ligands onto a GF / B filter plate (pretreated with 0.1% PEI for 30 min) using Packard Filtermate and washing away unbound ligands with 2.5 mL of ice-cold 5 mM Tris at pH 7.4. The filter plate was dried at 57 °C for 1 hr, and 50 μL of Microscint was added to each well of the plate. The plate was counted using PerkinElmer TopCount at 3 H cpm for 1 min per well. The data were analyzed using IDBS Activity Base to determine K i The values ​​are displayed in the following data table 1 (K) d (Value 11.0 nM, ligand concentration 3.5 nM).

[0323] ​​ ​

[0324] Methods for obtaining radioligand saturation binding data in human cortical PD tissue homogenates rich in aggregated α-synuclein and human cortical AD tissue homogenates rich in aggregated Aβ pathology: Banner PD brain homogenate was obtained through the Michael J. Fox Foundation (MJFF) consortium. Characterization and tissue preparation of this type of brain tissue were performed as described in US 2019 / 0256492, paragraphs 0242-0244 for conjugation studies. Banner PD brain homogenate, derived from post-mortem brain tissue of donors diagnosed with PD, was prepared using cingulate cortex collected from multiple PD brains. It was rich in α-synuclein pathology but lacked amyloid and tau pathologies as verified by neuropathology. The final concentration of Banner PD brain homogenate was 333 mg wet tissue per 1 mL buffer. The homogenate was aliquoted 1 mL / tube and stored at -70°C before use.

[0325] Frozen human brain samples for Alzheimer's disease (AD) were purchased from Analytical Biological Services Inc. These were post-mortem tissue samples from donors clinically diagnosed with AD and pathologically validated for amyloid plaques by IHC. Frontal cortex brain homogenates were prepared by homogenizing the frontal cortex in ice-cold phosphate-buffered saline (PBS), pH 7.4, at 4°C on a Polytron set 6 for 30 seconds. The final concentration of the brain homogenate was 10 mg of wet tissue per 1 mL of buffer. The homogenate was aliquoted into 5 mL tubes and stored at -70°C before use.

[0326] Synthesize as described in the methods listed above [ 3 H]-1. In a volume of 3.9 mCi / mL, [ 3 The specific activity of H]-1 is 196.6 Ci / mmol.

[0327] A heat-saturated binding assay was performed at nine concentrations ranging from 60.0 nM to 0.6 nM to evaluate the binding of radioligands to Banner PD and ABS AD brain homogenates. Brain homogenates were diluted with PBS buffer from an initial volume of 30 mg / mL to 2.0 mg / mL (ABS AD) or 2.8 mg / mL (Banner PD). Total binding was defined in the absence of competing compounds, and non-displacement 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. The test tubes were preheated to room temperature for 30 minutes, and then 10 μl of radioligand dilution (10X) was added to each tube to a final volume of 100 μl. Incubate at 37°C for 120 minutes, then filter the assay sample onto a GF / C filter using a Skatron 12-well collector and wash with ice-cold buffer (30 nM Tris pH 7.5) set to 5-5-5 (~3 × 2 ml). Before use, pre-soak the GF / C filter paper used for the Skatron collector in 0.1% BSA at room temperature for 1 hour. Flush the filter into a scintillation vial and count for 1 minute in 2 mL of Ultima Gold on a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in triplicate and in a laboratory designated for studies using human tissue.

[0328] With [ 3 H]-1 is processed in a similar way to [ 3 Determination of H]-24.

[0329] Will use [ 3 H]-1 and [ 3 The data graphs of these saturation binding assays for H]-24 are shown in ​ In particular, ​ Describes the use of [ 3 Saturation binding assays of H-1 and PD cingulate cortical tissue homogenates rich in aggregated α-synuclein. These data confirm strong binding to pathological β-synuclein in tissue homogenates. ​ Describes the use of [ 3 Saturation binding assays of H]-1 and pathological AD tissue homogenates rich in aggregated β-amyloid. (ND = not determined due to incomplete saturation of binding signal.) These data confirm weak binding to pathological β-amyloid in tissue homogenates. ​ Describes the use of [ 3Saturation binding assays of H]-24 and PD cortical tissue homogenates rich in aggregated α-synuclein. These data confirm strong binding to pathological α-synuclein in tissue homogenates. ​ A saturation binding assay was performed using [3H]-24 and pathological AD tissue homogenates rich in aggregated β-amyloid. (ND = Not determined due to incomplete saturation of binding signal.) The data confirm weak binding to pathological β-amyloid in the tissue homogenate.

[0330] In vitro binding of α-synuclein tracers in human PD brain tissue homogenates Frozen human brain samples for Parkinson's disease (AD) were provided by the Banner Sun Health Institute (USA) in collaboration with the Michael J Fox Foundation (MJFF). These were post-mortem tissue samples from donors clinically diagnosed with PD and neuropathologically confirmed as PD pathology. Brain homogenates of the cerebral cortex were prepared by homogenizing the cortex in ice-cold phosphate-buffered saline (PBS), pH 7.4, at 4°C on a Polytron Set 6 for 30 seconds. The final concentration of the brain homogenate was 30 mg of wet tissue per 1 mL of buffer. The homogenate was aliquoted into 1 mL tubes and stored at -70°C before use.

[0331] synthesis[ 3 H]-87 and [ 3The specific activity of H]-87 was 131.5 Ci / mmol. For the heat-saturated binding assay, nine concentrations of radioligand were used, ranging from 50 nM to 0.5 nM and 10 nM to 0.1 nM. Brain homogenate was diluted from an initial volume of 30 mg / mL to a final concentration of 2.8 mg / mL using assay buffer (Tris, pH 7.5, 0.1% BSA), and 250 μL per assay tube was used in the assay. The unlabeled test compound was dissolved in DMSO at 1 mM. Dilutes of the test compound were prepared to various concentrations using assay buffer containing 2% DMSO. Total binding was defined in the absence of competing compounds, and non-displacement binding was determined in the presence of 1 μM of unlabeled self-blocking. Add the compound diluent (10X) to each test tube containing 200 μL of brain homogenate diluent (25 μL per tube), and incubate at room temperature for 30 minutes. Then add the radioligand diluent (10X) to each test tube (25 μL per tube) to a final volume of 250 μL per tube. Incubate at 37°C for 120 minutes, then filter the test sample through a GF / C filter using a Skatron 12-well collector and wash with ice-cold buffer (Tris, pH 7.5) set to 5-5-5 (~3 × 2 ml). Before use, pre-soak the GF / C filter paper for the Skatron collector in 0.1% BSA at room temperature for 1 hour. Flush the filter into scintillation vials. Add liquid scintillation solution (2 mL Ultima Gold) to each vial, immerse in the filter for 4 hours, and count for 1 minute on a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in duplicate or triplicate (depending on assay settings) in laboratories designated for human tissue studies.

[0332] Data Table 2 - [ 3 In vitro binding data of H-87 in human Parkinson's disease tissue homogenate

[0333] synthesis[ 3 H]-89 and [ 3The specific activity of H]-89 was 55.5 Ci / mmol. For the heat-saturated binding assay, nine concentrations of radioligands ranging from 50 nM to 0.5 nM were used. Brain homogenate was diluted from an initial volume of 30 mg / mL to a final concentration of 2.8 mg / mL using assay buffer (Tris, pH 7.5, 0.1% BSA), and 250 μL per assay tube was used in the assay. The unlabeled test compound was dissolved in DMSO at 1 mM. Dilutes of the test compound were prepared to various concentrations using assay buffer containing 2% DMSO. Total binding was defined in the absence of competing compounds, and non-displacement binding was determined in the presence of 1 μM of unlabeled self-blocking. Add the compound diluent (10X) to each test tube containing 200 μL of brain homogenate diluent (25 μL per tube), and incubate at room temperature for 30 minutes. Then add the radioligand diluent (10X) to each test tube (25 μL per tube) to a final volume of 250 μL per tube. Incubate at 37°C for 120 minutes, then filter the test sample through a GF / C filter using a Skatron 12-well collector and wash with ice-cold buffer (Tris, pH 7.5) set to 5-5-5 (~3 × 2 ml). Before use, pre-soak the GF / C filter paper for the Skatron collector in 0.1% BSA at room temperature for 1 hour. Flush the filter into scintillation vials. Add liquid scintillation solution (2 mL Ultima Gold) to each vial, immerse in the filter for 4 hours, and count for 1 minute on a Perkin Elmer Tri-Carb 2900TR. Data analysis was performed using Prism software. All assays were performed in duplicate or triplicate (depending on assay settings) in laboratories designated for human tissue studies.

[0334] Data Table 3 - [ 3 In vitro binding data of H-89 in human Parkinson's disease tissue homogenate

[0335] [ 18 Radiochemical synthesis of F] ligands General methods Will[ 18 [F] Fluoride is concentrated on an anion exchange resin and eluted before use. Unless otherwise specified, it will contain [ 18The anion exchange resin containing fluoride was eluted with Kryptofix 222 (7 mg, 19 μmol) and K2CO3 (2.1 mg, 15 μmol) in acetonitrile / water (80 / 20, 0.7 mL) and transferred to a 1-mL V-shaped vial vented in a microwave cavity. The fluoride was dried under an argon flow and microwave heating (35 W / 90 °C). Additional aliquots of acetonitrile (3 × 0.5 mL) were added for azeotropic drying at 35 W / 90 °C.

[0336] 18 Synthesis of F]-87​ A solution of 88 (0.5 mg, 1.0 μmol) in DMSO (0.3 mL) was added to a container containing dried [ 18 In a microwave-safe vial containing the fluoride flotation unit, the vent tube was removed, and the reaction mixture was heated at 170 °C (60 W) for 3 min. After cooling to <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 Gemini, C6-phenyl, 110A, 150 × 10 mm column at a flow rate of 5 mL / min. The mobile phase was acetonitrile / 0.1% trifluoroacetic acid, from 50% to 95%. The radioactive fraction eluted between 12.5 and 13.5 min was collected, diluted with 20 mL of water for injection, and loaded into a Waters Sep-Pak Classic C18 column (Waters, Milford, MA, USA). Sep-Pak was washed with 10 mL of water, then eluted with ethanol (0.5 mL) into a 10 mL sterile vial and diluted to the desired formulation. The final products were analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic, 5μm, C18, 50×3mm (Phenomenex) at a flow rate of 1 mL / min. The mobile phase consisted of a mixture of acetonitrile / 0.1% trifluoroacetic acid in water, increasing from 10% to 90% over 10 min. The purity was determined using a UV detector (254 nm). 18 The concentration of F]-87 was determined. The product was identified by co-injecting a sample with compound 87, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-87 was 6.3 min.

[0337] 18 Synthesis of F]-89​ A solution of 90 (0.5 mg, 1.0 μmol) in DMSO (0.3 mL) was added to a container containing dried [ 18 In a microwave-safe vial containing the fluoride, the vent tube was removed, and the reaction mixture was heated at 170 °C (60 W) for 3 min. After cooling to <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 Gemini, C6-phenyl, 110A, 150 × 10 mm column at a flow rate of 5 mL / min. The mobile phase was acetonitrile / 0.1% trifluoroacetic acid: 65 / 35. The radioactive fraction eluted between 7.5 and 8.5 min was collected, diluted with 20 mL of water for injection, and loaded into a Waters Sep-Pak Classic C18 column (Waters, Milford, MA, USA). Sep-Pak was washed with 10 mL of water, then eluted with ethanol (0.5 mL) into a 10 mL sterile vial and diluted to the desired formulation. The final products were analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic, 5μm, C18, 50×3 mm (Phenomenex) at a flow rate of 1 mL / min. The mobile phase consisted of a mixture of acetonitrile in water and 0.1% trifluoroacetic acid, increasing from 10% to 90% within 10 min. The final product was determined using a UV detector (254 nm). 18 The concentration of F]-89 was determined. The product was identified by co-injecting a sample of compound 89, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-89 was 5.1 min.

[0338] 18 Synthesis of F]-112​ A solution of NN-5 (1.4 mg, 2.40 μmol) in DMF (0.3 mL) was added to a container containing dried [ 18In a microwave-safe vial containing fluoride, the vent tube was removed, and the reaction mixture was heated at 120 °C (60 W) for 3 min. After cooling to <50 °C, the reaction 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), eluting from 30% to 70% over 15 min. The radioactive fraction eluted between 14 and 15 min was collected in a flask containing 30% β-cyclodextrin solution (1 mL), evaporated under negative pressure, diluted with brine, and transferred to a sterile container. The final products were analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic, 5μm, C18, 50×3 mm (Phenomenex) at a flow rate of 1.5 mL / min. The mobile phase consisted of a mixture of acetonitrile in water and 0.1% formic acid, increasing from 5% to 50% over 7 min. The final product was determined using a UV detector (254 nm). 18 The concentration of F]-112 was determined. The product was identified by co-injecting a sample with compound 112, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-112 was 6.0 min.

[0339] 18 Synthesis of F]-113​ Will[ 18 F] Fluoride is concentrated on the anion exchange resin, which is pretreated before use by rinsing with EtOH (10 mL), followed by rinsing with 0.5 M KOTf (10 mL) and H2O (10 mL).

[0340] will contain [ 18The F-fluoride anion exchange resin was eluted with tetrabutylammonium trifluoromethanesulfonate (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) into a vented 2.5 mL V-vial and dried at 100 °C under conventional heating with an argon flow. An additional CH3CN aliquot (2 × 0.5 mL) was added for azeotropic drying. The V-vial was rinsed with air from a syringe (10 mL) and heated to 120 °C, then a solution of OO-7B (2.0 mg, 4.2 mmol), copper(II) tetra(pyridine)trifluoromethanesulfonate (11.3 mg, 17 mmol), and pyridine (32 mL, 40 mmol) in 1,3-dimethyl-2-imidazolinone (DMI, 0.5 mL) was added. The reaction mixture was heated at 120 °C for 20 min, then diluted at room temperature to a vial containing 10% CH3CN / 10 mM Na2HPO4 pH 7.4 in H2O (1.0 mL), mixed, and injected onto 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 and a mobile phase of CH3CN / 10 mM Na2HPO4 pH 7.4 in a gradient of 30–50%. The radioactive fraction eluted between 16.3 and 16.4 min 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, 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 8.1 at a gradient of 35%–45%. The purity was determined using a UV detector (254 nm). 18 The concentration of F]-113 was determined. The product was identified by co-injecting a sample with compound 113, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-113 was 6.8 min.

[0341] By isotopic exchange 18 F]-115 synthesis will contain [ 18The anion exchange resin containing 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) into a ventilated 2.5 mL V-shaped vial and dried at 100 °C under conventional heating with an argon flow. An aliquot of CH3CN (2 × 0.5 mL) was added and azeotropically dried. The dried […] 18 The vial of F Et4NF was heated to 130°C, and then 115 (0.3 mg, 0.6 mmol) of the solution in DMSO (0.5 mL) was added. The reaction mixture was heated at 130°C for 10 min, and then transferred to a vial containing H2O (0.8 mL) for dilution at room temperature. The mixture was then injected onto 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 at pH 7.4. The radioactive fraction eluted between 14.3 and 14.7 min 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 analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent), employing 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 at pH 8.0, with a gradient of 30–40%. The purity was determined using a UV detector (254 nm). 18 The concentration of F]-115 was determined. The product was identified by co-injecting a sample with compound 115, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-115 is 4.5 min.

[0342] 18 Synthesis of F]-116​ Will[ 18 F] Fluoride is concentrated on the anion exchange resin, which is pretreated before use by rinsing with EtOH (10 mL), followed by rinsing with 0.5 M K3PO4 (10 mL) and H2O (10 mL).

[0343] will contain [ 18The anion exchange resin containing fluoride was eluted with tetrabutylammonium methanesulfonate (6.8 mg, 20 mmol) in CH3CN / H2O 1:1 (1.0 mL), followed by elution with CH3CN (0.5 mL) into a ventilated 2.5 mL V-shaped vial and dried at 100 °C under conventional heating with an argon flow. An aliquot of the sample was then added with additional CH3CN (2 × 0.5 mL) and azeotropically dried. The dried […] 18 The vial of F]Bu4NF was heated to 120°C, and then 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 min, then transferred to a vial containing H2O (1.0 mL) for dilution at room temperature, mixed, and injected onto 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 and a mobile phase of 30% CH3CN / 10 mM Na2HPO4 in H2O, pH 7.4. The radioactive fraction eluted between 21.5 and 22.1 min 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, 4 mm EC-C18100 × 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, in a gradient of 5–95%. The purity was determined using an ultraviolet detector (254 nm). 18 The concentration of F]-116 was determined. The product was identified by co-injecting a sample with compound 116, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-116 was 6.6 min.

[0344] By isotopic exchange 18 F]-134 synthesis A solution of 134 (1.2 mg, 2.40 μmol) in DMF (0.3 mL) was added to a solution containing dried [ 18In a microwave-safe vial containing fluoride, the vent tube was removed, and the reaction mixture was heated at 140 °C (25 W) for 3 min. After cooling to <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), eluting from 40% to 70% over 15 min. The radioactive fraction eluted between 10 and 10.8 min was collected in a flask containing 30% β-cyclodextrin solution (1 mL), evaporated under negative pressure, diluted with saline, and transferred to a sterile container. The final products were analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent) with an ONYX Monolithic, 5 μm, C18, 50 × 3 mm (Phenomenex) at a flow rate of 1.5 mL / min. The mobile phase consisted of a mixture of acetonitrile / 0.1% formic acid in water, increasing from 5% to 90% over 7 min. The final product was determined using a UV detector (254 nm). 18 The concentration of F]-134 was determined. The product was identified by co-injecting a sample with compound 134, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-134 was 4.4 min.

[0345] By isotopic exchange 18 Synthesis of [F]-138 will contain [ 18 The 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) into a ventilated 2.5 mL V-shaped vial and dried at 100 °C under conventional heating with an argon flow. An aliquot of CH3CN (2 × 0.5 mL) was added and azeotropically dried. The dried […] 18The vial of F]Et4NF was heated to 130°C, and then 138 (0.2 mg, 0.4 mmol) of a solution in DMSO (0.5 mL) was added. The reaction mixture was heated at 130°C for 10 min, and then diluted at room temperature to a vial containing 10% CH3CN / 10 mM Na2HPO4 pH 7.4 in H2O (0.8 mL), mixed, and injected onto 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 and a mobile phase of 35% CH3CN / 10 mM Na2HPO4 pH 7.4 in H2O. The radioactive fraction eluted between 16.7 and 17.2 min was collected in a round-bottom flask containing 10% captisol (0.5 mL) in H2O, evaporated under negative pressure to remove CH3CN, diluted with saline, and transferred to 10 mL sterile vials. The final product was analyzed for chemical and radiochemical purity using an analytical HPLC system (Agilent), employing 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 gradient of 35–45%. The final product was determined using an ultraviolet detector (254 nm). 18 The concentration of F]-138 was determined. The product was identified by co-injecting a sample with compound 138, and its radiochemical purity was determined using a sodium iodide detector (Bioscan). Compound [ 18 The retention time of F]-138 was 5.6 min.

[0346] Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adjustments, changes, modifications, substitutions, deletions or additions can be made to the methods and solutions without departing from the spirit and scope of the invention. Therefore, the present invention is intended to be defined by the scope of the appended claims, and it is reasonable that these claims be interpreted broadly.

Claims

1. Compounds of Formula I: Or its medicinal salts, wherein: R is independently selected from H, -C 1-6 alkyl, OR c or halo, wherein the alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR c or halo; R a independently selected from unsubstituted or substituted -C 1-6 alkyl, said alkyl being optionally substituted with 1 to 3 R groups; R b independently selected from -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -NR c 2, -(CH2) n halogen or -O(CH2) n halo; R c independently selected from H or -C 1-6 alkyl, wherein said alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR d or halogenated groups; R d independently selected from H or -C 1-6 alkyl; R 1 independently selected from -(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, wherein the alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted by one to three R b groups; R 2 selected from hydrogen, OR c , NO2, 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, pyridinyl, pyrazinyl, or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, pyrazinyl, or pyridazinyl is optionally substituted with 1 to 3 R groups; 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][l,4]oxazine, or phenyl; Ring B is selected from a) b) c) 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.

2. The compound of claim 1, having the structure of formula IA: Or its medicinal salts, wherein: R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR c or halo; R a is independently selected from the group consisting of unsubstituted or substituted -C 1-6 alkyl, said alkyl being optionally substituted with 1 to 3 R groups; R b independently selected from -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -NR c 2, -(CH2) n halogen or -O(CH2) n halo; R c independently selected from H or -C 1-6 alkyl, wherein said alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR d or halogenated groups; R d independently selected from H or -C 1-6 alkyl; R 1 independently selected from -(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, wherein the alkyl, cycloalkyl, phenyl, heteroaryl or heterocyclyl can be substituted by one to three R b groups; R 2 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, pyridinyl, pyrazinyl, or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, pyrazinyl, or pyridazinyl is optionally substituted with 1 to 3 R groups; 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][l,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.

3. The compound of claim 1, wherein Ring A 1 is selected from pyridyl, pyrazinyl or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl or pyridinyl, wherein the pyrimidinyl, phenyl or pyridinyl is optionally substituted with 1 to 3 R groups; Ring A 3 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; Or its medicinal salt.

4. The compound of claim 1, having the structure of formula IB: Or its medicinal salts, wherein: R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR c or halo; R a is unsubstituted or substituted -C 1-6 alkyl, which alkyl is optionally substituted with 1 to 3 R groups; R b independently selected from -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -NR c 2, -(CH2) n halogen or -O(CH2) n halo; R c independently selected from H or -C 1-6 alkyl; R 1 -(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, wherein the alkyl, phenyl, cycloalkyl, heteroaryl or heterocyclyl can be substituted by one to three R b groups; R 2 selected from hydrogen, OR c , halo, or -C 1-6 alkyl; Ring A 1 is selected from pyridyl, pyrazinyl, pyrazolyl, oxazolyl, thiazolyl, or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, pyridinyl, or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, or pyridazinyl is optionally substituted with 1 to 3 R groups; Ring A 3 is selected from pyridyl, pyrazinyl, pyrimidinyl, indolyl, pyrrolopyrazinyl, oxadiazolyl, 3,4-dihydro-2H-pyrido[3,2,b][l,4]oxazinyl, or phenyl; n is independently selected from 0, 1, 2, 3, or 4; and p is selected from 1 or 2.

5. The compound of claim 1, having the structure of formula IC: Or its medicinal salts, wherein: R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl is optionally substituted with one to three groups independently selected from -C 1-6 alkyl, OR c or halo; R a independently selected from unsubstituted or substituted -C 1-6 alkyl, said alkyl being optionally substituted with 1 to 3 R groups; R b independently selected from -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -(CH2) n halogen or -O(CH2) n halo; R c independently selected from H or -C 1-6 alkyl; R 1 independently selected from -(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, wherein the alkyl, phenyl, cycloalkyl, heteroaryl or heterocyclyl can be substituted by one to three R b groups; R 2 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, pyridinyl, pyrazinyl, or pyridazinyl, wherein the pyrimidinyl, phenyl, pyridinyl, pyrazinyl, or pyridazinyl is optionally substituted with 1 to 3 R groups; 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][l,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.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is 2 is selected from pyrimidinyl, pyridinyl, or pyrazinyl, wherein the pyrimidinyl, pyridinyl, or pyrazinyl is optionally substituted with 1 to 3 R groups.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is ###00007### wherein the pyrimidinyl is optionally substituted with 1 to 3 R groups. 2 is a pyrimidinyl group, wherein the pyrimidinyl group is optionally substituted with 1 to 3 R groups.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is 3 is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl, or phenyl.

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is ###00017### 3 is selected from pyridyl, pyrazinyl, or phenyl.

10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is ###00009### 1 is selected from pyridyl, pyrazinyl, pyrazolyl, or pyrimidinyl.

11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is 1 is selected from pyridyl or pyrazinyl.

12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from -(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, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicyclo-heptanyl, furanyl, thiazolyl, pyrimidinyl, oxo-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolopyrazolyl, morpholinyl, tetrazolyl, or piperazinyl, wherein the alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicyclo-heptanyl, furanyl, thiazolyl, pyrimidinyl, oxo-azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, dihydropyrrolopyrazolyl, morpholinyl, tetrazolyl, or piperazinyl can be optionally substituted with one to three R b groups.

13. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from -(CH2) n OR c , -(CH2) n O(CH2) n R, -NR2, pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furanyl, wherein the pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furanyl can be substituted with one to three R b groups.

14. The compound of claim 4, wherein R is independently selected from H, -C 1-6 alkyl or halo, wherein the alkyl is optionally substituted with one to three groups from -C 1-6 alkyl, OR c or halo. R a independently selected from unsubstituted or substituted -C 1-6 alkyl, said alkyl being optionally substituted with 1 to 3 R groups; R b independently selected from -C 1-6 alkyl, halo, -(CH2) n OR c , -CN, -(CH2) n halogen or -O(CH2) n halo; R c independently selected from H or -C 1-6 alkyl; R 1 -(CH2) n OR c -(CH2) n O(CH2) n R, -NR2, pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furanyl, wherein the pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furanyl can be substituted with one to three R b groups; R 2 selected from hydrogen, OR c , halo, or -C 1-6 alkyl; Ring A 1 is selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, or pyrazolyl; Ring A 2 is selected from pyridyl, pyrazinyl, or phenyl, wherein the pyridyl, pyrazinyl, or phenyl is optionally substituted with 1 to 3 R groups; Ring A 3 is selected from pyrimidinyl, pyridinyl or pyrazinyl; n is independently selected from 0, 1, 2, 3, or 4; and p is selected from 0, 1, 2, or 3. Or its medicinal salt.

15. Compounds, selected from Or its medicinal salt.

16. The compound of claim 15, 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 a pharmaceutically acceptable salt thereof.

17. The compound of claim 15, selected from examples numbered 39, 47, 51, 78, 79, 96, 112, 116 and 141, or pharmaceutically acceptable salts thereof.

18. The compound of claim 1 or 15, or a pharmaceutically acceptable salt thereof, which is labeled with an isotope selected from the group consisting of 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 170, 180, 18F, 35S, 36CI, 82Br r , 76Br r , 77Br r , 123I, 124I, or 131I.

19. The compound of claim 15, or a pharmaceutically acceptable salt thereof, labeled with an isotope of 3H, 11C or 18F.

20. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

21. A method for imaging α-synuclein deposits in a human patient using the compound of claim 18 or a pharmaceutically acceptable salt thereof as an imaging agent, comprising the following steps: a) Positioning the human patient supine in a PET camera; b) Administering to a patient about 0.1 to about 10 mCi of the compound of claim 17; and c) Perform emission scanning on brain regions of the patient's head to identify the aggregation of α-synuclein in the patient's brain tissue.

22. A method for measuring the clinical efficacy of a therapeutic agent for Parkinson's disease, comprising the following steps: a) Prior to treatment with the therapeutic agent, administer the isotopically labeled compound of formula I according to claim 18 to a patient diagnosed with PD. b) Measure the amount of α-synuclein aggregates formed in the brain tissue of the patients. c) Following treatment with the therapeutic agent, administer to the patient the isotopically labeled compound of formula I according to claim 18. d) Measure the amount of α-synuclein aggregates formed in the patient's brain tissue after treatment, and e) Analyze whether the therapeutic agent terminates or reduces the progression of α-synuclein aggregate formation in the patient's brain tissue.

23. The compound of claim 1, or a pharmaceutically acceptable salt thereof, as an imaging agent.

24. A compound of claim 18, or a pharmaceutically acceptable salt thereof, for use as an imaging agent.

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