Alpha-synuclein binders and methods of use

Abstract

The invention is directed to compounds of Formula: (I) or their pharmaceutically acceptable salts, which may be suitable for imaging alpha-synuclein pathology and hence are useful in binding and imaging alpha-synuclein aggregates in patients with Parkinson's Disease. More specifically, this invention relates to a method of using the compounds of this invention as tracers in positron emission tomography (PET) imaging to study alpha-synuclein in brain in vivo to allow diagnosis of Parkinson's Disease and other neurodegenerative diseases characterized by alpha-synuclein pathology. The invention further relates to a method of measuring clinical efficacy of therapeutic agents for Parkinson's Disease and other neurodegenerative diseases characterized by alpha-synuclein pathology.

Description

ALPHA-SYNUCLEIN BINDERS AND METHODS OF USE CROSS-REERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 683,354, filed August 15, 2024, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis and prion diseases are debilitating diseases which 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 present a threat to cell viability. In some cases, the disease arises because a specific protein is no longer functional when adopting a misfolded state. In other diseases, the pathological state originates because misfolding occurs concomitantly with aggregation, and the underlying aggregates are detrimental. Even though neurodegenerative diseases such as Alzheimer's and Parkinson's are caused by different proteins, both involve the accumulation of insoluble fibrous protein deposits, called amyloids. For example, Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA), which are collectively referred to as "synucleinopathies," have been linked to the accumulation of aggregated forms of the alpha-synuclein protein in neurons in the brain (see Nat. Rev. Neuro. 2013, 9, 13-24 and J. Parkinson’s Disease 2013, 3, 565-567). As the primary neuropathologic change of PD, the degeneration of dopaminergic neurons occurs in the substantia nigra, as well as Lewy bodies (LB) and Lewy neurites (LN). To date, the pathogenic mechanism of PD has not been fully discovered.

[0003] Alpha-synuclein is a presynaptic terminal protein that consists of a 140-amino acid protein that plays an important function in the central nervous system including synaptic vesicle recycling and synthesis, vesicular storage, and neurotransmitter release. It is specifically upregulated in a discrete population of presynaptic terminals of the brain during acquisition- related synaptic rearrangement. Alpha-synuclein naturally exists in a highly soluble, unfolded state. Evidence suggests that filamentous aggregates of alpha-synuclein accumulate at the pre- synaptic membrane and trigger synapse dysfunction and neuronal cell death in synucleinopathies and may be the cause of Parkinson's and DLB. Alpha-synuclein aggregation has been identified by antibody immunohistological studies as the major component of Lewy bodies, which aremicroscopic protein deposits in deteriorating nerve cells. Accumulation of misfolded, fibrillar alpha-synuclein in Lewy bodies (LB) and Lewy neurites (LN) is considered a hallmark of PD.

[0004] The diagnosis of PD is mainly based on the clinical symptoms such as rest 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 the disease progression and minimize the disease symptoms in the patients. Therefore, a method of diagnosing PD in the very early stage can greatly help the physicians to design the therapeutic paradigm accordingly, and to slow the disease progression. There remains a need for improved diagnostic methods for identifying aggregations of misfolded proteins, including alpha-synuclein for early detection and ongoing monitoring of PD in subjects (see J. Parkinson’s Disease 2013, 3, 565-567).

[0005] An alpha-synuclein positron emission tomography (PET) tracer would be a valuable non-invasive diagnostic biomarker for spatial and temporal quantification of aggregated pathological alpha-synuclein in human brain as a Parkinson’s Disease biomarker. Additionally, an alpha-synuclein PET tracer could be useful for patient selection for PD clinical trials. In this mode, an alpha-synuclein tracer could be developed as a companion diagnostic for co- registration of a therapeutic agent. Additionally, an alpha-synuclein PET tracer could be a critical disease-relevant tool for quantifying a stabilization or decrease of alpha-synuclein formation for disease-modifying PD therapeutics.

[0006] Therefore, a need exists for neuroimaging radiotracers that would allow in vivo imaging of alpha-synuclein pathology thereby providing insight into the deposition of alpha-synuclein aggregates in the human brain. The successful neuroimaging radiotracer must cross the blood- brain barrier, have rapid clearance from tissue and plasma, and possess high affinity and specificity for alpha-synuclein aggregates with high selectivity over binding to beta-amyloid and tau aggregated proteins due to co-expression 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 that have reduced selectivity over aggregated beta-amyloid (WO 2019 / 121661), there is a need for compounds with high levels of selectivity over co- expressed aggregated proteins in PD in order to quantify an alpha synuclein specific signal in an in vivo imaging study for PD patients.

[0007] The present invention advances these interests by providing compounds of Formula I as aggregated alpha-synuclein binding ligands with high selectivity over binding of aggregated beta-amyloid pathology. The instant invention also relates to a method of using the compounds of Formula I as tracers in PET imaging to study alpha-synuclein deposits in brain in vivo to allow diagnosis of neurodegenerative diseases characterized by alpha-synuclein pathology. Theinvention further relates to a method of measuring clinical efficacy of therapeutic agents targeting alpha-synuclein pathology. SUMMARY OF THE INVENTION

[0008] The invention is directed to compounds of Formula I, pharmaceutical salts thereof, pharmaceutical compositions comprising them, diagnostic and therapeutic uses and processes for making such compounds. An embodiment of the invention provides a compound of Formula I:or a pharmaceutically acceptable salt thereof wherein; R is independently selected from H, –C1-6alkyl, or halo, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORcor halo; Rais selected from unsubstituted or substituted –C1-6alkyl, said alkyl optionally substituted with 1 to 3 groups of R; Rbis selected from a) halo, b) OH, ,Rcis independently selected from H or –C1-6alkyl, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORdor halo; Rdis independently selected from H or –C1-6alkyl; X is CH or N; and Ring A is selected from pyrazinyl or pyrimidinyl.

[0009] The present invention is also directed to isotopically-labeled compounds of Formula I. Additionally, the present invention provides pharmaceutical compositions comprising a compound of Formula I and at least one pharmaceutically acceptable carrier.

[0010] The present invention is directed to compounds of Formula I which may be useful for binding alpha-synuclein aggregated proteins and / or tau aggregated proteins, and hence are useful in binding and imaging alpha-synuclein aggregated protein pathology in PD and non-PD synucleinopathy patients as well as aggregated Tau protein pathology Alzheimer’s Disease (AD) and non-AD tauopathy patients via PET imaging techniques known commonly in the field (see J. Nucl. Med.2019, 60, 93-99 and 107-114). This invention also relates to methods of using compounds of Formula I to identify patients with abnormal levels of aggregated alpha-synuclein pathology in the brain. This invention also relates to methods of using a compound of Formula I as a to measure progression of alpha-synuclein pathology over time as a biomarker in clinical assessment of potential therapeutic agents that can modify Parkinson’s Disease progression.

[0011] Compounds of this invention may also be useful for imaging and detecting for other neurodegenerative diseases characterized by the deposition of alpha-synuclein aggregates such as multiple system atrophy (MSA) and dementia with Lewy Bodies (DLB).BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1: Saturation binding experiment in aggregated beta-amyloid rich AD tissue homogenate for [3H]-105.

[0013] FIG.2: Sagittal slice of a PET image of [18F]-8 in rhesus monkey brain.

[0014] FIG.3: Sagittal slice of a PET image of [18F]-13 in rhesus monkey brain. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] An embodiment of the invention provides a compound of Formula I:or a pharmaceutically acceptable salt thereof wherein; R is independently selected from H, –C1-6alkyl, or halo, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORcor halo; Rais selected from unsubstituted or substituted –C1-6alkyl, said alkyl optionally substituted with 1 to 3 groups of R; Rbis selected from a) halo, b) OH,Rcis independently selected from H or –C1-6alkyl, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORdor halo; Rdis independently selected from H or –C1-6alkyl; X is CH or N; and Ring A is selected from pyrazinyl or pyrimidinyl.

[0017] A further embodiment of the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein; R is halo, Rais–C1-6alkyl, and X is CH.

[0018] A further embodiment of the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein; R is halo, Rais–C1-6alkyl, and X is N.

[0019] A further embodiment of the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein Rbis selected from halo, OH, or, A further embodiment of the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein Rbis selected from F orCompounds of the present invention include:or a pharmaceutically acceptable salt thereof.

[0020] The present invention is directed to compound of Formula I for use as an imaging agent.

[0021] An embodiment of the invention comprises a compound selected from Ex. No.2, 8, 13 and 14, or a pharmaceutically acceptable salt thereof. A further embodiment of the invention comprises a compound selected from Ex. No.8, or a pharmaceutically acceptable salt thereof. A further embodiment of the invention comprises a compound selected from Ex. No.13 or a pharmaceutically acceptable salt thereof.

[0022] Another aspect of the invention is directed to compounds of Formula I, or apharmaceutically acceptable salt thereof, that are 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 invention, the compounds of Formula I are isotopically labeledwith3H,11C or18F. Examples of isotopically labeled a compound of Formula I, orpharmaceutically acceptable salts thereof, include, but are not limited to,18F-2,18F-8,18F-13,18F-14, and the like. Further examples of isotopically labeled a compound of Formula I, orpharmaceutically acceptable salts thereof, include, but are not limited to18F-8 and18F-13, andthe like.

[0023] Another aspect of the invention is directed to compounds of Formula I, or apharmaceutically acceptable salt thereof, that are 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.

[0024] In one embodiment the present invention provides pharmaceutical compositions comprising a compound of the invention, for example, a compound of Formula I, and at least one pharmaceutical excipient.

[0025] 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, may be useful for the diagnosis and / or treatment of Parkinson's disease and / or Alzheimer's disease. Means of detecting labels are well known to those skilled in the art. For example, isotopic labels may bedetected using imaging techniques, photographic film or scintillation counters. In a preferred embodiment, the label is detected in vivo in the brain of the subject by imaging techniques, for example positron emission tomography (PET).

[0026] The compounds of Formula (I) may also form a component of bifunctional compounds that are targeted protein degrader compounds that bind aggregated alpha-synuclein proteins. Such targeted alpha-synuclein protein degrader compounds contain a target protein binding moiety which is formed from a compound of Formula (I) and an E3 ubiquitin ligase-binding moiety. The targeted alpha-synuclein protein degrader compounds typically contain a linker group joining the alpha-synuclein protein binding moiety and the E3 ubiquitin ligase-binding moiety. The E3 ubiquitin ligase-binding moieties in the alpha-synuclein targeted protein degrader compounds can be, but are not limited to, binders to the E3 ligase von Hippel-Lindau protein, binders to the E3 ligase cereblon protein, or binders to the MDM2 protein. Such compounds can be administered in pharmaceutical compositions to treat disease conditions, including but not limited to, the conditions disclosed herein.

[0027] In the description that follows conventional structural representation is employed and includes conventional stereochemical notation for certain asymmetric carbon centers. Thus, structural representation of compounds of the invention includes conventional stereochemical notation for some asymmetric carbon centers shown in the example compounds. Accordingly, in such instances, solid black “wedge” bonds represent bonds projecting from the plane of the reproduction medium, “hashed wedge” bonds representing descending bonds into the plane of the reproduction medium, and a “wavey” line appended to a carbon bearing a double bond indicates both possible cis and trans orientations are included. As is conventional, plain solid lines represent all spatial configurations for the depicted bonding. Accordingly, where no specific stereochemical notation is supplied the representation contemplates all stereochemical and spatial orientations of the structural features.

[0028] As is shown in the examples of the invention, and mentioned above, particular asymmetric carbon centers are structurally represented using conventional “Solid Wedge” and “Hash Wedge” bonding representation. For the most part, absolute configuration has not been determined for the example compounds, but has been assigned by analogy to specific example compounds of known stereochemical configurations (determined by X-ray crystallography) prepared using the same or analogous reaction conditions and starting reagents and isolated under the same chromatographic conditions. Accordingly, specific assignment of the configurations structurally represented herein is meant to identify the specific compounds prepared has having an excess of one particular stereoisomer and is not put forth herein necessarily as being astatement of the absolute determination of the stereochemical structure of said compound unless otherwise noted in the data presented.

[0029] It will be appreciated that where isomeric mixtures are obtained, the preparation of individual stereoisomers in significant percentages of enantiomeric excess can be carried out, if desired, by separation of the mixture using customary methods, for example by chromatography or crystallization, or by the use of stereochemically uniform starting materials for the synthesis described, or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of Formula I or it can be done on a final racemic product.

[0030] Where indicated herein, absolute stereochemistry is determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Unless a particular isomer, salt, solvate (including hydrates) or solvated salt of such racemate, enantiomer, or diastereomer is indicated, the present invention includes all such isomers, as well as salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and mixtures thereof.

[0031] Where a wavey line terminates a conventional bond (as opposed to connecting two atoms within a structure) it indicates a point of bonding to a structure, e.g.:indicates a the secondary-butyl moiety is bonded via the methylene group via the bond terminated with the wavey line. Where an alphabetical notation is used to depict a substituent moiety, a dash is employed to indicate the point of bonding to the indicated substrate, e.g.: -CH2- C(O)-CH2Cl indicates the acetyl chloride moiety is bonded via the methylene portion of the moiety.

[0032] Where compounds of Formula I are capable of tautomerization, all individual tautomers as well as mixtures thereof are included in the scope of this invention.

[0033] When any variable (e.g., R, alkyl, etc.) occurs more than one time in any constituent or in Formula I, its definition on each occurrence is independent of its definition at every other occurrence unless otherwise specified at the point of definition. One of ordinary skill in the art will recognize that choice of combinations of the various substituents defined in a structural representation are to be chosen in conformity with well-known principles of chemical structureconnectivity and stability, and combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0034] A "stable" compound is a compound which can be prepared and isolated and whose structure and properties remain or can be caused to remain essentially unchanged 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 invention are limited to stable compounds embraced by Formula I.

[0035] Where any variable or moiety is expressed in the form of a range, e.g., (-CH2-)1-4, both of the extrema of the specified range are included (i.e., 1 and 4 in the example) as well as all of the whole number values in between (i.e., 2 and 3 in the example).

[0036] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

[0037] "Halogen" or "halo" as used herein means fluoro, chloro, bromo and iodo (or fluoride, chloride, bromide and iodide).

[0038] In an embodiment of the instant invention, R is halo. In a further embodiment, R is chloride or fluoride. In another embodiment, R is fluoride.

[0039] In an embodiment of the instant invention, Rais–C1-6alkyl. In a further embodiment of the instant invention, Rais methyl.

[0040] In an embodiment of the instant invention, Rbis selected from halo, OH,embodiment of the instant invention, Rbis selected from halo,embodiment of the instant invention, Rbis halo. In an embodiment of the instant invention, Rbis F. In anembodiment of the instant invention, Rb is OH or.

[0041] In an embodiment of the instant invention, X is CH. In an embodiment of the instant invention, X is N.

[0042] In an embodiment of the instant invention, Ring A is pyrazinyl. In an embodiment of the instant invention, Ring A is pyrimidinyl.

[0043] 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 the compounds according to the invention or of their pharmaceutically acceptable salts. When the compound of the present invention is acidic, suitable “pharmaceutically acceptable salts” refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, N,N1-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, 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.

[0044] When the compound of the present invention is basic, salts may 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.

[0045] The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977:66:1-19.

[0046] If the compounds of Formula I simultaneously contain acidic and basic groups in the molecule the invention also includes zwitterions, in addition to the salt forms described above.

[0047] The present invention also embraces isotopically-labeled compounds of the present invention which are structurally identical to those recited herein, but for the fact that a statistically significant percentage of one or more atoms in that form of the compound are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number of the most abundant isotope usually found in nature, thus altering the naturally occurring abundance of that isotope present in a compound of the invention. Another aspect ofthe invention relates to use of the isotopically labeled compounds as neuroimaging 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 invention is use of the isotopically labeled compounds in PET, which is an in vivo analysis technique in the diagnosis,monitoring, and / or treatment of PD. The3H,11C or18F labeled compounds can be used in invitro and in vivo methods for the determination of binding, receptor occupancy and metabolic studies including covalent labeling.

[0048] Another aspect of the invention relates to the use of the isotopically labeled compounds to screen for new chemical matter. In particular, various isotopically labeled compounds find utility in magnetic resonance imaging, autoradiography and other similar analytical tools. The present invention is meant to include all suitable isotopic variations of the compounds of Formula I. Examples of isotopes that can be preferentially incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine, fluorine and chlorine, for example, but not limited to:2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36Cl,82Br,76Br,77Br,123I,124I,125I or131I isotopically labeled substituted heterocyclic derivative compounds of Formula I. It will be appreciated that other isotopes may be incorporated by known means also. In particular, the present invention is directed to11C,13C,14C,18F,15O,13N,35S,2H, and3H isotopes of compounds of Formula I, compositions and methods of their preparation and use as radiotracers or PET tracers in diagnosing and measuring the effects of a compound in the treatment of PD. In a further embodiment, the present invention is directed to compounds of Formula I that are isotopically labeled with3H,11C or18F, along with compositions and methods of their preparation and use as PET tracers in diagnosing and measuring the effects of a compound in the treatment of PD. The present invention also relates to non-toxic alpha-synuclein protein binding compounds that can rapidly cross the blood brain barrier, have low non-specific binding properties and are rapidly cleared from the system. This and other aspects of the invention will be realized upon review of the specification in its entirety.

[0049] Isotopically-enriched compounds within Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates.

[0050] As indicated herein the present invention includes isotopically labeled compounds of the invention. An "isotopically-labeled", "radio-labeled", “tracer”, “radiotracer”, “labeled tracer” or “radioligand” compound, is a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass numbertypically found in nature (i.e., naturally occurring). Suitable radionuclides (i.e., "detectable isotopes") that may be incorporated in compounds of the present invention include but are notlimited to2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36Cl,82Br,76Br,77Br,123I,124I and131I. The isotopically labeled compounds of the invention need only to beenriched with a detectable isotope to, or above, the degree which allows detection with a technique suitable for the particular application. The radionuclide that is incorporated in the instant radiolabeled compounds will depend on the specific application of that radiolabeled compound. In another embodiment of the invention the radionuclides are represented by11C,13C,14C,18F,15O,13N,35S,2H, and3H, preferably11C,3H, and18F.

[0051] The isotopically labeled compounds of this invention are prepared by incorporating a selected isotope into the substrate molecule. This is accomplished by utilizing reagents that have had one or more of the atoms contained therein made radioactive by placing them in a source of radioactivity such as a nuclear reactor, a cyclotron and the like. Additionally, many isotopically labeled reagents, such as2H2O,3H3CI,14C6H5Br, ClCH214COCl and the like, are commercially available. The isotopically labeled reagents are then used in standard organic chemistry synthetic techniques to incorporate the isotope atom, or atoms, into a compound of Formula I as described below. The following Schemes illustrate how to make the compounds of Formula I.

[0052] This invention further relates to a pharmaceutical composition comprising an effective amount of at least one compound of Formula I and a pharmaceutically acceptable carrier. The composition may comprise, but is not limited to, one or more buffering agents, wetting agents, emulsifiers, suspending agents, lubricants, adsorbents, surfactants, preservatives and the like. The composition may be formulated as a solid, liquid, gel or suspension for oral administration (e.g., drench, bolus, tablet, powder, capsule, mouth spray, emulsion); parenteral administration (e.g., subcutaneous, intramuscular, intravenous, epidural injection); topical application (e.g., cream, ointment, controlled-released patch, spray); intravaginal, intrarectal, transdermal, ocular, or nasal administration. In a further embodiment, the pharmaceutical composition of the present invention may be formulated for parenteral administration, such as an intravenous formulation.

[0053] This invention provides radiolabeled compounds of Formula I as alpha-synuclein imaging agents and synthetic precursor compounds from which they are prepared. 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 Systems Atrophy (MSA), Dementia with Lewy Bodies (DLB), etc. The compounds of this invention may also be used in combination with a broad range ofcognition deficit enhancement agents. Thus, in another embodiment of this invention 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 pharmaceutically active compound or compounds used in AD / PD therapies including for example donepezil, memantine, tacrine, carvidopa, levodopa, MOA-B inhibitors, catechol O-methyltransferase (COMT) inhibitors, etc. and equivalents and pharmaceutically active isomer(s) and metabolite(s) thereof.

[0054] An objective of the present invention is to provide a radiopharmaceutical agent, such as an isotopically labeled compound of Formula I, that is useful in alpha-synuclein imaging and has high specific radioactivity and high target tissue selectivity by virtue of its high affinity for alpha- synuclein aggregates.

[0055] In accordance with the present invention, a method for imaging alpha-synuclein deposits in a patient, wherein an isotopically-labeled compound of Formula I is employed as the imaging agent, comprises the steps of: a) placing a human patient in a supine position in a PET camera; b) administering, intravenously, about 0.1 to about 10 mCi of an isotopically-labeled compound of Formula I to the patient; and c) performing an emission scan of the cerebral region of the patient’s head to identify aggregations of alpha-synuclein in the brain tissue of the patient. The technique for performing an emission scan of the head is well known to those of skilled in the art. PET techniques are described in Freeman et al., Freeman and Johnson's Clinical Radionuclide Imaging, 3rd. Ed. Vol.1 (1984); Grune & Stratton, New York; Ennis et Q. Vascular Radionuclide Imaging: A Clinical Atlas, John Wiley & Sons, New York (1983).

[0056] The term "labeled tracer" refers to any molecule which can be used to follow or detect a defined activity in vivo, for example, a preferred tracer is one that accumulates in the regions where alpha-synuclein aggregates may be found. Preferably, the labeled tracer is one that can be viewed in a living experimental 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, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.

[0057] The present invention also provides methods of determining in vivo activity of an enzyme or other molecule. In an 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.

[0058] Biomarkers of Parkinson’s disease state, prognosis and progression will all be useful for general diagnostic utilities as well as for clinical development plans for therapeutic agents forParkinson’s disease. Compounds of Formula I may be used to provide biomarker information for patients in clinical trials for novel symptomatic and disease-modifying Parkinson’s disease treatments and to assist in patient selection and assignment to cohorts. The present invention will serve as one of the biomarkers of disease state in order to get the correct patients into the proper PhIIb trial cohort. In addition, the present invention can serve as one marker of disease prognosis as an entry inclusion criterion in order to enhance the probability that the disease will progress in the placebo treatment arm. Finally, the present invention can serve as one biomarker of disease progression to monitor the clinical course of patients on therapy and could provide an independent biomarker measure of treatment response by a therapeutic drug. The tracer can be selected in accordance with the detection method chosen. Before conducting the method of the present invention, a diagnostically effective amount of a labeled or unlabeled compound of the invention is administered to a living body, including a human.

[0059] The present invention also provides a method of measuring the clinical efficacy of therapeutic agents useful for treating Parkinson’s Disease (PD) comprising the steps of: a) administering an isotopically-labeled compound of Formula I to the patient diagnosed with PD before treatment with said therapeutic agent, b) measuring the amount of alpha-synuclein aggregate formation in the patient’s brain tissue, c) administering an isotopically-labeled compound of Formula I to the patient after treatment with said therapeutic agent, d) measuring the amount of alpha-synuclein aggregate formation in the patient’s brain tissue after treatment, and e) analyzing whether said therapeutic agent stopped or decreased the progression of alpha- synuclein aggregate formation in the patient’s brain tissue.

[0060] The diagnostically effective amount of the labeled or unlabeled compound of the invention to be administered before conducting the in-vivo method for the present invention is within a range of from 0.1 ng to 100 mg per kg body weight, preferably within a range of from 1 ng to 10 mg per kg body weight.

[0061] The compounds of the present invention have utility in diagnosing, monitoring, and measuring Parkinson’s disease and other non-PD synucleinopathies such as Multiple Systems Atrophy (MSA), Dementia with Lewy Bodies (DLB).

[0062] In preferred embodiments, the compounds of the invention are useful in diagnosing, monitoring or measuring Parkinson’s Disease, non-PD synucleinopathies, neurodegenerative disease, cognitive disorders, schizophrenia, pain disorders and sleep disorders.

[0063] The term "composition" as used herein is intended to encompass a product comprising specified ingredients in predetermined amounts or proportions, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specifiedamounts. This term in relation to pharmaceutical compositions is intended to encompass a product comprising one or more active ingredients, and an optional carrier comprising inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

[0064] In general, pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition 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 invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

[0065] As the term is used herein, “patients” (alternatively “subjects”) refers to an animal, preferably a mammal, and in particular a human, in need of assessment via an imaging study. As used herein, the term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of Formula I means providing the compound, or a pharmaceutically acceptable salt thereof, to a subject in need of treatment.

[0066] The present invention also provides a method for the synthesis of compounds useful as intermediates in the preparation of compounds of the invention.

[0067] The compounds described herein can be prepared according to the procedures of the following schemes and examples, using appropriate materials and are further exemplified by the following specific examples. Deuterated versions of the compounds of the invention can be prepared by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent. The compounds illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. Reagents and starting materials for preparing the intermediates and example compounds are commercially available, unless indicated otherwise. All temperatures are degrees Celsius unless otherwise noted. Mass spectra (MS) were measured by electrospray ion-mass spectroscopy (ESI).1H NMR spectra were recorded at 300-500 MHz.List of Abbreviations AD = Alzheimer's disease (ipcADI)NiBr2 = N,N′-bis(1R,2R,3R,5S)-(−)-isopinocampheyl-2,3-butanediimine nickel(II) bromide Anal. = analytical calc. = calculated BSA = Bovine Serum Albumin CPME = cyclopentyl methylether DCM = dichloromethane DEA = diethylamine DIPEA = N,N-diisopropylethylamine DMA = dimethylacetamide DMAP = dimethylaminopyridine DMF = dimethylformamide DMSO = dimethyl sulfoxide DPBS = Dulbecco's phosphate-buffered saline EDTA = Ethylenediaminetetraacetic acid Eq = equivalent EtOAc = ethyl acetate EtOH = ethanol h = hour(s) HPLC = high-pressure liquid chromatography LCMS = Liquid Chromatography coupled to Mass Spectrometry mCi = millicurie MeCN = acetonitrile MeOH = methyl alcohol MS = mass spectroscopy MW = Microwave NaHBEt3 = sodium triethylborohydride NaOtBu = sodium tert-butoxide NMP = N-methylpyrolidone NMR = nuclear magnetic resonance spectroscopy PD = Parkinson’s DiseasePd / C = 10% palladium on carbon by weight 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 T3P = propanephosphonic anhydride TEA = triethylamine TFA = trifluoroacetic acid TLC = thin-layer chromatography tR = retention time TRIS = tris(hydroxymethyl)aminomethane THF = tetrahydrofuran TsCl = p-toluene sulfonylchloride Ts = tosylate wt% = percentage by weight

[0068] Compounds described herein were synthesized as a racemic mixture unless otherwise stated in the experimental procedures. In some cases, the final product may be further modified, for example, by manipulation of substituents. These manipulations may include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions which are commonly known to those skilled in the art. In some cases, the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. The following schemes and examples are provided so that the invention might be more fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way.

[0069] While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. It is intended, therefore, thatthe invention be defined by the scope of the claims that follow and that such claims be interpreted as broadly as is reasonable. Synthesis of Intermediates: Synthesis of Intermediate A: 6-(3-fluoroazetidin-1-yl)nicotinamide

[0070] To a stirred solution of 6-chloronicotinamide (A-1, 6.0 g, 38.3 mmol) in DMSO (60 mL) were added K2CO3(26.5 g, 192 mmol) and 3-fluoroazetidine hydrochloride (8.55 g, 76.6 mmol) at rt. The reaction mixture was stirred under nitrogen atmosphere at 110 °C for 16 h. Reaction mixture was concentrated under reduced pressure. Crude compound was purified by Biotage using 80 g silica (230-400 mesh) cartridge and compound eluted with 90% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford A. M / Z (ESI): 196.06 [M+H]+. Synthesis of Intermediate B: 6-(3-hydroxyazetidin-1-yl)nicotinamide

[0071] To a stirred solution of 6-chloronicotinamide (1 g, 6.39 mmol) in DMF (40 mL) were added K2CO3 (2.65 g, 19.16 mmol) and 3-hydroxyazetidine hydrochloride (0.840 g, 7.66 mmol) at 0 °C. The reaction mixture was stirred under nitrogen atmosphere at 100 °C for 16 h. Reaction mixture was diluted with EtOAc (50 mL), filtered through celite pad and washed with EtOAc (2 x 50 mL). Filtrate was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 12% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford B. M / Z (ESI): 194.06 [M+H]+. EXAMPLES Example 1: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyridin-2-yl)-3-methylpiperazine-1-carboxylate (1-3):

[0072] To a stirred solution of tert-butyl (R)-3-methylpiperazine-1-carboxylate (1-1) (5 g, 25 mmol) in toluene (100 mL) were added sodium tert-butoxide (7.2 g, 74.9 mmol) and 2-chloro-5- fluoropyridine (1-2) (3.01 mL, 30 mmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture was added RuPhos Pd G2 (1.94 g, 2.5 mmol) at room temperature. The reaction mixture was stirred at 120 °C for 16 h under nitrogen atmosphere. Reaction mixture was quenched with water (200 mL) and extracted with EtOAc (2 x 500 mL). Combined organic layer was washed with brine (2 x 150 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 10%EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 1-3. M / Z (ESI): 296.22 [M+H]+. Synthesis of (R)-1-(5-fluoropyridin-2-yl)-2-methylpiperazine (1-4):

[0073] To a stirred solution of 1-3 (5 g, 16.9 mmol) in DCM (50 mL) was added 4 M HCl in 1, 4-dioxane (21.2 mL, 84.6 mmol) drop wise at 0 °C. The reaction mixture was stirred at room temperature for 6 h under nitrogen atmosphere. Reaction mixture was concentrated under reduced pressure. Crude compound was triturated with diethyl ether (2 x 100 mL) and dried under reduced pressure to afford 1-4. M / Z (ESI): 196.10 [M+H]+. Synthesis of (R)-2-bromo-5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrazine (1-6):

[0074] To a stirred solution of 1-4 (4 g, 17.3 mmol) in DMSO (60 mL) were added CsF (7.87 g, 51.8 mmol) and 2,5-dibromopyrazine (1-5) (4.93 g, 20.7 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 5 h under nitrogen atmosphere. Reaction mixture was quenched with water (150 mL) and extracted with EtOAc (2 x 500 mL). Combined organic layer was washed with brine (2 x 200 mL), dried over anhydrous Na2SO4,filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 20% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 1-6. M / Z (ESI): 352.05 [M+H]+. Example 1: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0075] To a stirred solution of 1-6 (200 mg, 568 μmol) in 1, 4-dioxane (5 mL) were added Cs2CO3(555 mg, 1.70 mmol), CuI (10.8 mg, 56.8 μmol) and Int A (133 mg, 681 μmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (4.48 μL, 28.4 μmol) at room temperature. The reaction mixture was stirred in microwave at 150 °C for 2 h. Reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 x 250 mL). Combined organic layer was washed with brine (2 x 80 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 20 g silica (230-400 mesh) cartridge and compound eluted with 3% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 1. M / Z (ESI): 467.13 [M+H]+.

[0076] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.83 (d, J = 1.2 Hz, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.20 (d, J = 1.2 Hz, 1H), 8.10-8.18 (m, 2H), 7.53 (td, J = 9.0 Hz, 2.8 Hz, 1H), 6.87 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 5.43-5.64 (m, 1H), 4.51-4.60 (m, 1H), 4.31-4.44 (m, 2H), 4.18-4.30 (m, 2H), 4.12-4.18 (m, 1H), 3.99-4.11 (m, 2H), 3.15-3.27 (m, 2H), 3.04 (td, J = 12.0 Hz, 4.0 Hz, 1H), 1.09 (d, J = 6.4 Hz, 3H). Example 2: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate (2-2):

[0077] To a solution of tert-butyl (R)-2-methylpiperazine-1-carboxylate (2-1, 350 mg, 1 Eq, 1.75 mmol) in toluene (11.7 mL) was added sodium 2-methylpropan-2-olate (420 mg, 2.5 Eq, 4.37 mmol) and 2-bromo-5-fluoropyridine (461 mg, 1.5 Eq, 2.62 mmol). The mixture was purged with N2 three times then chloro(2-dicyclohexylphosphino-2',6'-di-i-propoxy-1,1'- biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (67.9 mg, 0.05 Eq, 87.4 μmol) was added. The mixture was stirred for 16 h at 100°C. Next, the reaction mixture was filtered through a celite filter, extracted with EtOAc (5 mL x 3), and concentrated under reduced pressure. The resulting residue was purified using normal-phase chromatography (ISCO 80 g RediSep Gold High Performance Silica, 0-100% Hexane, EtOAc / EtOH (3:1mixture)). Fractions containing the desired product were combined and concentrated under vacuum to yield 2-2. MS (ESI) m / z: 296.2 [M+H]+. Synthesis of (R)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine dihydrochloride (2-3):

[0078] To a solution of tert-butyl (R)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1- carboxylate (2-2, 482 mg, 1 Eq, 1.63 mmol) in DCM (5.44 mL) was added 4M hydrogen chloride (4.08 mL, 10 Eq, 16.3 mmol). The mixture was stirred for 2 h at 100°C. The mixture was concentrated under reduced pressure to yield 2-3. MS (ESI) m / z: 196.1 [M+H]+. Synthesis of (R)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine (2-4):

[0079] To a solution of (R)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine dihydrochloride (2-3, 438 mg, 1 Eq, 1.63 mmol) in DMF (8.17 mL) was added potassium carbonate (903 mg, 4 Eq, 6.53 mmol) and 2-chloro-5-nitropyrazine (313 mg, 1.2 Eq, 1.96 mmol). The mixture was stirred for 16 h at 60°C. The reaction mixture was cooled to room temperature and water (20 mL) was added. The resulting precipitated solid was collected by filtration through a fritted filter, washed with water (3 mL x 3), and dried to give 2-4. MS (ESI) m / z: 319.1 [M+H]+. Synthesis of (R)-5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-amine (2-5):

[0080] A solution of (R)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine (2-4, 420 mg, 1 Eq, 1.32 mmol) in THF (4.00 mL) and MeOH (4.00 mL) was degassed and purged with N2three times then added with 10% palladium on carbon (70.2 mg, 0.5 Eq, 660 μmol) and degassed and purged with H2 an additional three times. The mixture was stirred for 16 h at 22°C under a H2filled balloon. The mixture was filtered through a fritted filter, washed with EtOAc (5 mL x 3), and concentrated under vacuum to yield 2-5. MS (ESI) m / z: 289.3 [M+H]+.Synthesis of (R)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)nicotinamide (2-6):

[0081] To a solution of (R)-5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- amine (2-5, 300 mg, 1 Eq, 1.04 mmol) in DMF (6.0 mL) was added diisopropylethylamine (672 mg, 906 µL, 5 Eq, 5.20 mmol), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (662 mg, 701 µL, 50% Wt, 1 Eq, 1.04 mmol), and 6-chloronicotinic acid (197 mg, 1.2 Eq, 1.25 mmol). The mixture was stirred for 16 h at 22°C. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C1830x250 mm column, 10-100% 5 mM NH4HCO3water solution: acetonitrile, 20 minute gradient). Fractions containing the product were combined and extracted between water (100 mL) and DCM (100 mL x 3). The collected organic layer was dried over MgSO4then concentrated under vacuum to yield 2-6. MS (ESI) m / z: 428.4 [M+H]+. Example 2: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0082] To a solution of (R)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide (2-6, 50.0 mg, 1 Eq, 117 μmol) in DMA (1.0 mL) was added potassium fluoride (20.4 mg, 3 Eq, 351 μmol), and 3-fluoroazetidine hydrochloride (13.0 mg, 1 Eq, 117 μmol). The mixture was stirred for 48 h at 80ºC. The mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C18 - 30x250 mm column, 10-100% water with 0.1% NH4OH: acetonitrile, 18 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 2. MS (ESI) m / z: 467.5 [M+H]+

[0083] 1H NMR (500 MHz, CDCl3): δ 9.17 (s, 1H), 8.71 (s, 1H), 8.07 (s, 2H), 8.01 (d, J = 8.7 Hz, 1H), 7.81 (s, 1H), 7.29 (d, J = 7.7 Hz, 1H), 6.64 (dd, J = 9.1, 2.6 Hz, 1H), 6.35 (d, J = 8.7 Hz, 1H), 5.48 (d, J = 56.7 Hz, 1H), 4.54 (s, 1H), 4.42 (dq, J = 19.2, 6.1 Hz, 2H), 4.25 (dd, J = 23.9, 8.7 Hz, 2H), 4.15 (d, J = 12.2 Hz, 1H), 4.05 (t, J = 14.0 Hz, 2H), 3.44 – 3.36 (m, 1H), 3.34 (dd, J = 12.5, 3.2 Hz, 1H), 3.12 (dt, J = 11.7, 5.7 Hz, 1H), 1.25 (d, J = 6.3 Hz, 3H). Example 3: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of 3-2: tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate

[0084] To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (3-1, 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-1-carboxylate (1.00 g, 1 Eq, 4.99 mmol). The mixture was purged with N2for 5 minutes then chloro(2-dicyclohexylphosphino-2',6'-di-i- propoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (194 mg, 0.05 Eq, 250 μmol) was added. The mixture was stirred for 48 h at 110°C. Water (50 mL) was added to the mixture and it was extracted with EtOAc (100 mL x 3). The organic layer was dried over MgSO4, filtered through a fritted 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% Hexane, EtOAc, 26 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 3-2. MS (ESI) m / z: 296.4 [M+H]+. Synthesis of 3-3: (S)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine hydrochloride

[0085] To a solution of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1- carboxylate (3-2, 1.00 g, 1 Eq, 3.39 mmol) in 1,4-dioxane (14.0 mL) was added 4M hydrogen chloride (3.39 mL, 4 Eq, 13.5 mmol) in 1,4-dioxane. The mixture was stirred for 18 h at 22°C. The mixture was concentrated under reduced pressure to yield 3-3. MS (ESI) m / z: 196.3 [M+H]+. Synthesis of (S)-2-bromo-5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazine (3-4):

[0086] To a stirred solution of 3-3 (1.5 g, 6.5 mmol) in DMSO (10 mL) were added CsF (5.9 g, 39 mmol) and 2, 5-dibromopyrazine (1.8 g, 7.8 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (75 mL) and extracted with EtOAc (2 x 100 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 24 g silica (100-200 mesh) cartridge and compound eluted with 40% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 3-4 (1 g, 2.8 mmol, 43% yield, LCMS: 98.95%) as a light brown solid. M / Z (ESI): 352.18 [M+H]+. Synthesis of 3: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0087] To a stirred solution of 3-4 (1 g, 2.8 mmol) and Int A (0.67 g, 3.4 mmol) in 1, 4-dioxane (10 mL) was added Cs2CO3(2.8 g, 8.5 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture were added CuI (54 mg, 0.28 mmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (22 μL, 0.14 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (120 mL) and extracted with EtOAc (2 x 130 mL). Combined organic layer was washed with brine (120 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate in H2O: MeCN Column – Hypersil C18 (30X250) mm, 5µ Flow-25.0 ml / min Gradient Method –0 / 60,2 / 60,11.3 / 67,11.35 / 100,12 / 100,12.05 / 60,16 / 60,prep-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 3. M / Z (ESI): 467.34 [M+H]+.

[0088] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.49 (s, 1H), 8.85 (d, J = 1.2 Hz, 1H), 8.77 (d, J = 2.0 Hz, 1H), 8.06-8.20 (m, 3H), 7.53 (td, J = 9.0 Hz, 3.2 Hz, 1H), 6.95 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 5.42-5.66 (m, 1H), 4.55-4.64 (m, 1H), 4.31-4.44 (m, 2H), 4.03-4.26 (m, 5H), 3.14-3.30 (m, 2H), 2.98 (td, J = 12.0 Hz, 3.2 Hz, 1H), 1.13 (d, J = 6.8 Hz, 3H). Example 4: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamideSynthesis of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-3-methylpiperazine-1-carboxylate (4-2):

[0089] To a stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (4-1, 5 g, 0.02 mol) and 2-bromo-5-fluoropyridine (5 g, 0.03 mol) in 1, 4-dioxane (15 mL) at room temperature. Reaction mixture was degassed and purged with argon gas for 10 min. To this reaction mixture were added sodium tert-butoxide (7 g, 0.07 mol) and chloro(2-dicyclohexylphosphino-2',6'-di-i- propoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (2 g, 2 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with EtOAc (50 mL). Combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound (8 g) was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 70% EtOAc in pet ether. Pure fractions were combined, concentrated under reduced pressure to afford 4-2. M / Z (ESI): 296.26 [M+H]+. Synthesis of (S)-1-(5-fluoropyridin-2-yl)-2-methylpiperazine (4-3):

[0090] To a stirred solution of 4-2 (2 g, 7 mmol) in DCM (5 mL) was added 4M HCl in 1, 4- dioxane (7 mL, 0.03 mol) drop wise at 0 °C under argon atmosphere. The reaction mixture was stirred at room temperature for 12 h. Reaction mixture was concentrated under reduced pressure. Crude compound was triturated with diethyl ether (15 mL) and dried under reduced pressure to afford 4-3. M / Z (ESI): 196.14 [M+H]+. Synthesis of (S)-2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)-5-iodopyrimidine (4-4):

[0091] To a stirred solution of 4-3 (1.4 g, 6.0 mmol) in DMF (20 mL) were added 2-chloro-5- iodopyrimidine (1.7 g, 7.3 mmol) and DIPEA (1.1 mL, 6.0 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 50%EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 4-4. M / Z (ESI): 400.21 [M+H]+. Example 4: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamide

[0092] To a stirred solution of 4-4 (250 mg, 626 μmol) in 1, 4-dioxane (2 mL) were added CuI (11.9 mg, 62.6 μmol), Cs2CO3 (612 mg, 1.88 mmol) and Int A (147 mg, 751 μmol) at roomtemperature. Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (4.94 μL, 31.3 μmol) drop wise at room temperature. The reaction mixture was stirred in microwave at 150 °C for 2 h under nitrogen atmosphere. Reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 80 mL). Combined organic layer was washed with brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 20 g silica (230-400 mesh) cartridge and compound eluted with 3% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 4. M / Z (ESI): 467.17 [M+H]+.

[0093] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.64 (s, 2H), 8.06-8.14 (m, 2H), 7.48-7.56 (m, 1H), 6.85 (dd, J = 9.6 Hz, 3.2 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.44-5.65 (m, 1H), 4.48-4.60 (m, 3H), 4.30-4.44 (m, 2H), 3.96-4.17 (m, 3H), 3.27 (d, J =13.2 Hz, 3.6 Hz, 1H), 3.05-3.20 (m, 2H), 1.02 (d, J = 6.4 Hz, 3H). Example 5: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyridin-2-yl)-3-methylpiperazine-1-carboxylate (5-3):

[0094] To a stirred solution of tert-butyl (R)-3-methylpiperazine-1-carboxylate (5-1, 5 g, 0.02 mol) and 2-chloro-5-fluoropyridine (5-2, 3 g, 0.02 mol) in 1, 4-dioxane (100 mL) were added sodium 2-methylpropan-2-olate (8 mL, 0.07 mol) and chloro(2-dicyclohexylphosphino- 2',6'-di-i-propoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (2 g, 2 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenchedwith ice cold water (100 mL) and extracted with EtOAc (2 x 100 mL). Combined organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (Pre-packed) cartridge and compound eluted with 40% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 5-3. M / Z (ESI): 296.30 [M+H]+. Synthesis of (R)-1-(5-fluoropyridin-2-yl)-2-methylpiperazine (5-4):

[0095] To a stirred solution of 5-3 (2 g, 7 mmol) in DCM (40 mL) was added 4M HCl in 1, 4- dioxane (2 mL, 7 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h. Reaction mixture was concentrated and dried under reduced pressure to afford 5-4. M / Z (ESI): 196.14 [M+H]+. Synthesis of (R)-2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)-5-iodopyrimidine (5-6):

[0096] To a stirred solution of 5-4 (1.8 g, 9.2 mmol) in DMF (30 mL) were added DIPEA (4.8 mL, 28 mmol) and 2-chloro-5-iodopyrimidine (5-5, 2.2 g, 9.2 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with ice cold water (200 mL) and extracted with EtOAc (2 x 200 mL). Combined organic layer was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (Pre-packed) cartridge and compound eluted with 40% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 5-6. M / Z (ESI): 400.21 [M+H]+. Example 5: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamide

[0097] To a stirred solution of 5-6 (200 mg, 501 μmol) and Int A (97.8 mg, 501 μmol) in 1, 4- dioxane (4 mL) were added Cs2CO3 (490 mg, 1.5 mmol), copper(I) iodide (9.54 mg, 50.1 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (3.95 μL, 25 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with ice cold water (50 mL) and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – YMC Actus Trait Ex-RS C18 (20X150) mm, 5µ Flow – 15.0 ml / min GRADIENT METHOD- 0 / 40, 2 / 40,12 / 70, 12.05 / 98, 15 / 98, 15.05 / 40, 18 / 40 PREP-023). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 5. M / Z (ESI): 467.30 [M+H]+.

[0098] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.65 (s, 2H), 8.05-8.15 (m, 2H), 7.53 (td, J = 8.8 Hz, 2.8 Hz, 1H), 6.85 (dd, J = 9.6 Hz, 3.2 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.40-5.65 (m, 1H), 4.46-4.65 (m, 3H), 4.30-4.46 (m, 2H), 3.95-4.20 (m, 3H), 3.23-3.29 (m, 1H), 3.05-3.18 (m, 2H), 1.02 (d, J = 6.4 Hz, 3H). Example 6: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate (6-2):

[0099] A stirred solution of tert-butyl (R)-2-methylpiperazine-1-carboxylate (6-1, 5 g, 0.025 mol) and 2-chloro-5-fluoropyridine (3.28 g, 0.025 mol) in toluene (60 mL) at room temperature was degassed and purged with argon gas for 10 min. To this reaction mixture were added sodium 2-methylpropan-2-olate (7.2 g, 0.075 mol) and (2-Dicyclohexylphosphino-2',6'-diisopropoxy- 1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate (2.1 g, 2.5 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with EtOAc (50 mL). Combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 70% EtOAc in pet ether. Pure fractions were combined, concentrated under reduced pressure to afford 6-2. M / Z (ESI): 296.26 [M+H]+. Synthesis of (R)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine (6-3):

[0100] To a stirred solution of 6-2 (3.8 g, 12.9 mmol) in DCM (40 mL) was added 4M HCl in 1, 4-dioxane (7 mL, 0.03 mol) drop wise at 0 °C under argon atmosphere. The reaction mixture was stirred at room temperature for 12 h. Reaction mixture was concentrated under reduced pressure. Crude compound was triturated with diethyl ether (15 mL) and dried under reduced pressure to afford 6-3. M / Z (ESI): 196.01 [M+H]+. Synthesis of (R)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-iodopyrimidine (6-4):

[0101] To a stirred solution of 6-3 (1.0 g, 5.12 mmol) in DMF (45 mL) were added 2-chloro-5- iodopyrimidine (1.23 g, 75.12 mmol) and DIPEA (1.1 mL, 6.0 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 80 g silica (230-400 mesh) cartridge and compound eluted with 50%EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 6-4. M / Z (ESI): 400.12 [M+H]+. Example 6: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamide

[0102] To a stirred solution of 6-4 (100 mg, 250 μmol) and Int A (48.9 mg, 250 μmol) in 1,4- dioxane (2 mL) was added Cs2CO3(245 mg, 751 μmol), CuI (4.77 mg, 25 μmol) and trans-N,N`- dimethylcyclohexane-1,2-diamine (1.98 μL, 12.5 μmol) at room temperature. The reactionmixture was stirred at 120 °C for 16 h. Reaction mixture was filtered through celite pad and washed with EtOAc (30 mL). Filtrate was quenched with water (30 mL) and extracted with EtOAc (2 x 30 mL). Combined organic layer was washed with brine (1 x 30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: X-Bridge, C18 (19X250) mm, 5µ: Mobile Phase – 10mM Ammonium Bicarbonate IN H2O: MeCN, Column – X-Bridge C18 (19X250) mm, 5µ Flow-15.0 ml / min, Gradient Method –0 / 40,2 / 40,12 / 65,13 / 65,13.1 / 100,15.5 / 100,15.6 / 40,19 / 40. PREP-023). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 6. M / Z (ESI): 467.30 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.66 (s, 2H), 8.05-8.13 (m, 2H), 7.53 (td, J = 9.0 Hz, 2.8 Hz, 1H), 6.92 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.43-5.65 (m, 1H), 4.78-4.89 (m, 1H), 4.30-4.47 (m, 3H), 4.04-4.23 (m, 4H), 3.23-3.30 (m, 1H), 3.13 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 2.91 (td, J = 12.0 Hz, 4.0 Hz, 1H), 1.15 (d, J = 6.8 Hz, 3H). Example 7: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7-2):

[0103] A stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (7-1) (1 g, 4.99 mmol) in toluene (20 mL) was purged with argon gas for 10 min. Then 2-chloro-5- fluoropyrimidine (0.993 g, 7.49 mmol)), sodium tert-butoxide (1.440 g, 14.98 mmol) andchloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'- biphenyl)]palladium(II) (0.388 g, 0.499 mmol) were added to the reaction mixture at room temperature and again purged with argon for another 10 min. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 25 g silica gel cartridge and compound eluted with 30% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 7-2. M / Z (ESI): 297.27 [M+H]+. Synthesis of (S)-5-fluoro-2-(2-methylpiperazin-1-yl)pyrimidine hydrochloride (7-3):

[0104] To a stirred solution of 7-2 (1.1 g, 3.71 mmol) in DCM (20 mL) was added 4M HCl in 1,4-dioxane (3.71 mL, 14.85 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 4 h. Reaction mixture was concentrated under reduced pressure to afford 7-3. M / Z (ESI): 197.02 [M+H]+. Synthesis of (S)-2-(4-(5-bromopyrazin-2-yl)-2-methylpiperazin-1-yl)-5-fluoropyrimidine (7-4):

[0105] To a stirred solution of 7-3 (800 mg, 3.44 mmol) in DMSO (20 mL) were 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. Reaction mixture was quenched with water (120 mL) and extracted with EtOAc (3 x 200 mL). Combined organic layer was dried over Na2SO4,filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 25 g silica gel cartridge and compound eluted with 30% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 7-4. M / Z (ESI): 353.09 [M+H]+. Example 7: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0106] To a stirred solution of 7-4 (150 mg, 425 μmol) in 1, 4-dioxane (3 mL) were added Int A (100 mg, 510 μmol), Cs2CO3(415 mg, 1.27 mmol) and copper(I) iodide (8 mg, 42 μmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 5 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (3.35 μL, 21.2 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h in a sealed tube. Reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 20 mL). Combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filteredand concentrated under reduced pressure. Crude compound was triturated with diethyl ether (10 mL) and dried under reduced pressure. Obtained compound was purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate in H2O: MeCN COLUMN – X-Bridge C18 (19X250) mm, 5µ Flow-15.0 ml / min Gradient Method – 0 / 54,9 / 54,9.05 / 100,12 / 100,12.05 / 54,15 / 54. PREP-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 7. M / Z (ESI): 468.22 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.83 (d, J = 1.6 Hz, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.50 (s, 2H), 8.12-8.22 (m, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.40-5.66 (m, 1H), 4.71-4.86 (m, 1H), 4.30-4.44 (m, 3H), 4.19-4.30 (m, 2H), 4.11-4.18 (m, 1H), 4.05-4.11 (m, 1H), 3.35-3.40 (m, 1H), 3.23 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 2.99-3.08 (m, 1H), 1.16 (d, J = 6.4 Hz, 3H). Example 8: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (8-3):

[0107] To a stirred solution of tert-butyl (R)-3-methylpiperazine-1-carboxylate (8-1, 5 g, 25 mmol) in toluene (100 mL) were added 2-chloro-5-fluoropyrimidine (8-2, 3.31 g, 25 mmol) and NaOtBu (7.2 g, 74.9 mmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture was added chloro(2- dicyclohexylphosphino-2',6'-di-i-propoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2- yl)palladium(II) (1.94 g, 2.50 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h in a sealed tube. Reaction mixture was filtered through celite pad, filtrate was concentrated and dried under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 10% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 8-3. M / Z (ESI): 241.10 [M+H-56]+. Synthesis of (R)-5-fluoro-2-(2-methylpiperazin-1-yl)pyrimidine (8-4):

[0108] To a stirred solution of 8-3 (4 g, 13.5 mmol) in DCM (70 mL) were added 4M HCl in 1, 4-dioxane (27 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h under nitrogen atmosphere. Reaction mixture was concentrated and dried under reduced pressure to afford 8-4. M / Z (ESI): 197.10 [M+H]+. Synthesis of (R)-2-(4-(5-bromopyrazin-2-yl)-2-methylpiperazin-1-yl)-5-fluoropyrimidine (8-6):

[0109] To a stirred solution of 8-4 (3.5 g, 17.84 mmol) in DMSO (60 mL) was added CsF (8.128 g, 53.51 mmol) and 2, 5-dibromopyrazine (8-5, 4.243 g, 17.84 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h under nitrogen atmosphere. Reaction mixture was quenched with water (250 mL), precipitated solid was filtered and dried under reduced pressure to afford 8-6. M / Z (ESI): 355.12 [M+H]+. Example 8: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0110] To a stirred solution of 8-6 (150 mg, 425 μmol) and Int A (99.5 mg, 510 μmol) in 1, 4- dioxane (4 mL) was added Cs2CO3(415 mg, 1.27 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture were added CuI (8.09 mg, 42.5 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (3.35 μL, 21.2 μmol) at room temperature. The reaction mixture was stirred in microwave at 120 °C for 16 h. Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filteredand concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate in H2O: MeCN COLUMN – YMC-Triart Actus (20X150) mm, 5µ Flow - 15 ml / min Gradient Method- 45,2 / 45,10 / 85,10.05 / 100,11.5 / 100,11.55 / 45,15 / 45, prep-023). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 8. M / Z (ESI): 468.21 [M+H]+.

[0111] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.83 (d, J = 1.6 Hz, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.50 (s, 2H), 8.14-8.21 (m, 2H), 6.49 (d, J= 8.8 Hz, 1H), 5.43-5.65 (m, 1H), 4.78-4.85 (m, 1H), 4.33-4.44 (m, 3H), 4.18-4.30 (m, 2H), 4.04-4.17 (m, 2H), 3.33-3.38 (m, 1H), 3.20-3.27 (m, 1H), 3.02 (td, J = 12.0 Hz, 3.6 Hz, 1H), 1.16 (d, J = 6.4 Hz, 3H). Example 9: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamideSynthesis of tert-butyl (R)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1-carboxylate (9-3):

[0112] To a stirred solution of tert-butyl (R)-2-methylpiperazine-1-carboxylate (9-1, 5 g, 0.02 mol) and 2-chloro-5-fluoropyrimidine (9-2, 3 g, 0.02 mol) in DMF (50 mL) was added diisopropylethylamine (0.01 L, 0.07 mol) at room temperature. The reaction mass stirred at 120 °C for 16 h under nitrogen atmosphere. Reaction mixture was filtered through celite pad, filtrate was concentrated under reduced pressure. Crude compound (10 g) was purified by silica gel column and compound eluted with 10% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 9-3 M / Z (ESI): 241.18 [M+H-56]+. Synthesis of (R)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine (9-4):

[0113] To a stirred solution of 9-3 (5 g, 0.02 mol) in DCM (40 mL) was added TFA (5 mL, 0.07 mol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h under nitrogen atmosphere. Reaction mixture was concentrated under reduced pressure. Crude compound (6 g) was triturated with diethyl ether and dried under reduced pressure to afford 9-4. M / Z (ESI): 197.01 [M+H]+. Synthesis of (R)-2-(4-(5-bromopyrazin-2-yl)-3-methylpiperazin-1-yl)-5-fluoropyrimidine (9-5):

[0114] To a stirred solution of 9-4 (5 g, 0.02 mol) in DMSO (30 mL) were added CsF (7 g, 0.05 mol) and 2,5-dibromopyrazine (4 g, 0.02 mol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h under nitrogen atmosphere. Reaction mixture was quenched with ice cold water (80 mL) and extracted with EtOAc (3 x 80 mL). Combined organic layer was washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (100-200 mesh) cartridge and compound eluted with 30% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 9-5. M / Z (ESI): 353.23 [M+H]+. Example 9: (R)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0115] To a stirred solution of 9-5 (300 mg, 849 μmol) and Int A (182 mg, 934 μmol) in 1, 4- dioxane (15 mL) was added Cs2CO3(830 mg, 2.55 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture were added CuI (16.2 mg, 84.9 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (6.70 μL, 42.5 μmol) at room temperature and again degassed with nitrogen gas for 2 min. The reaction mixturewas stirred at 80 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (3 x 50 mL). Combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Crude compound was again purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – YMC ExRS C1820x150mm 5µ Flow- 15.0 ml / min Gradient Method –0 / 53,4 / 53,10 / 59.5,10.05 / 100,13 / 100,13.05 / 53,15 / 53. PREP-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 9. M / Z (ESI): 468.41 [M+H]+.

[0116] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.85 (d, J = 1.2 Hz, 1H), 8.77 (d, J = 2.4 Hz, 1H), 8.48 (s, 2H), 8.10-8.18 (m, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.42-5.64 (m, 1H), 4.58-4.68 (m, 1H), 4.30-4.58 (m, 4H), 4.04-4.18 (m, 3H), 3.29-3.32 (m, 1H), 3.10-3.28 (m, 2H), 1.07 (d, J = 6.4 Hz, 3H). Example 10: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin- 1-yl)pyrazin-2-yl)nicotinamideSynthesis of 10-2: tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1-carboxylate

[0117] To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (10-1, 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, 136mmol) and 2-chloro-5-fluoropyrimidine (5.39 g, 3.75 mL, 1.50 Eq, 40.7 mmol). The mixture was stirred for 16 h at 80°C. Water (300 mL) was added to the mixture and extracted with ether (100 mL x 3). The organic layer was dried over Na2SO4, filtered through a fritted 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% Hexane, EtOAc). Fractions containing the desired product were combined and concentrated under vacuum to yield 10-2. MS (ESI) m / z: 297.1 [M+H]+. Synthesis of 10-3: (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride

[0118] To a solution of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1- carboxylate (10-2, 7.35 g, 1 Eq, 24.8 mmol) in DCM (124 mL) was added 4M hydrogen chloride (62.0 mL, 4.00 molar, 10 Eq, 248 mmol) in DCM. The mixture was stirred for 2 h at 22°C. The mixture was concentrated under reduced pressure to yield 10-3. MS (ESI) m / z: 197.2 [M+H]+. Synthesis of 10-4: (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazin-2-yl)piperazin-1-yl)pyrimidine

[0119] To a solution of (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine hydrochloride (10-3, 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 for 16 h at 60 °C. Ice water was added to the mixture and the resulting precipitated solid was collected through a fritted filter, washed with water (5 mL x 3), and dried to give 10-4. MS (ESI) m / z: 320.2 [M+H]+. Synthesis of 10-5: (S)-5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-amine

[0120] A solution of (S)-5-fluoro-2-(3-methyl-4-(5-nitropyrazin-2-yl)piperazin-1-yl)pyrimidine (10-4, 1.20 g, 1 Eq, 3.76 mmol) in DCM (9.40 mL) and MeOH (9.40 mL) was degassed and purged with N2 (x 3) then added 10% palladium on carbon (200.0 mg, 0.500 Eq, 1.879 mmol) and degassed and purged with H2(x 3). The mixture was stirred for 6 h at 22°C under a H2filled balloon. The mixture was filtered through a fritted filter and concentrated under vacuum to yield 10-5. MS (ESI) m / z: 290.2 [M+H]+. Synthesis of 10-6: (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin- 2-yl)nicotinamide

[0121] To a solution of (S)-5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- amine (10-5, 520 mg, 1 Eq, 1.80 mmol) in DMF (8.0 mL) was added diisopropylethylamine (670mg, 903 μL, 5 Eq, 5.18 mmol), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (659 mg, 699 μL, 50% Wt, 1 Eq, 1.03 mmol), and 6-chloronicotinic acid (196 mg, 1.2 Eq, 1.24 mmol). The mixture was stirred for 3 h at 22°C. The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C185mm - 50x250mm column, 10-100% 5 mM NH4HCO3 water solution: acetonitrile, 33 min gradient). Fractions containing the product were combined and extracted between water (100 mL) and DCM (150 mL x 3). The collected organic layer was dried over MgSO4 then concentrated under vacuum to yield 10-6. MS (ESI) m / z: 429.3 [M+H]+. Example 10: (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide

[0122] To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide (10-6, 40.0 mg, 1 Eq, 93.3 μmol) in DMA (1.0 mL) was added potassium fluoride (16.3 mg, 3 Eq, 280 μmol), and 3-fluoroazetidine hydrochloride (10.4 mg, 1 Eq, 93.3 μmol). The mixture was stirred for 16 h at 100ºC. Post reaction time, LCMS showed high conversion of desired product. The reaction mixture was concentrated, purified using normal phase-chromatography (ISCO 24 g RediSep Gold High Performance Silica, 0-100% hexane: EtOAC:EtOH (3:1 mixture), 18 minute gradient), then re-purified using basic reverse- phase chromatography (Waters Sunfire Prep C18 OBD 19x150 mm column, 30-100% water with 0.1% NH4OH: acetonitrile, 15 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to afford 10. MS (ESI) m / z: 468.5 [M+H]+.

[0123] 1H NMR (500 MHz, CDCl3): δ 9.18 (s, 1H), 8.74 (d, J = 1.8 Hz, 1H), 8.24 (s, 2H), 8.20 (s, 1H), 8.09 (d, J = 8.7 Hz, 1H), 7.83 (s, 1H), 6.40 (d, J = 8.7 Hz, 1H), 5.51 (d, J = 56.7 Hz, 1H), 4.65 (d, J = 12.8 Hz, 1H), 4.60 – 4.46 (m, 4H), 4.32 (dd, J = 23.6, 9.7 Hz, 2H), 4.06 (d, J = 12.6 Hz, 1H), 3.40 (dd, J = 13.5, 4.0 Hz, 1H), 3.34 (td, J = 12.1, 3.5 Hz, 1H), 3.24 (td, J = 12.3, 3.5 Hz, 1H), 1.21 (d, J = 6.5 Hz, 3H). Example 11: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamideSynthesis of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (11-2):

[0124] A stirred solution of tert-butyl (S)-3-methylpiperazine-1-carboxylate (11-1) (1 g, 4.99 mmol) in toluene (20 mL) was purged with argon gas for 10 min. Then 2-chloro-5- fluoropyrimidine (0.993 g, 7.49 mmol)), sodium tert-butoxide (1.440 g, 14.98 mmol) andchloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'- biphenyl)]palladium(II) (0.388 g, 0.499 mmol) were added to the reaction mixture at room temperature and again purged with argon for another 10 min. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 25 g silica gel cartridge and compound eluted with 30% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 11-2. M / Z (ESI): 297.27 [M+H]+. Synthesis of (S)-5-fluoro-2-(2-methylpiperazin-1-yl)pyrimidine hydrochloride (11-3):

[0125] To a stirred solution of 11-2 (1.1 g, 3.71 mmol) in DCM (20 mL) was added 4M HCl in 1,4-dioxane (3.71 mL, 14.85 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 4 h. Reaction mixture was concentrated under reduced pressure to afford 11-3. M / Z (ESI): 197.02 [M+H]+. Synthesis of (S)-5-fluoro-2-(4-(5-iodopyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidine (11-4):

[0126] To a stirred solution of 11-3 (2.0 g, 8.6 mmol) in DMF (20 mL) were added 2-chloro-5- iodopyrimidine (2.6 g, 10.3 mmol) and DIPEA (7.5 mL, 43.0 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound (2.2 g) was purified by Biotage using 40 g silica (230-400 mesh) cartridge and compound eluted with 50%EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 11-4. M / Z (ESI): 401.21 [M+H]+. Example 11: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1- yl)pyrimidin-5-yl)nicotinamide

[0127] To a stirred solution of 11-4 (150 mg, 374.8 μmol) and Int A (87.80 mg, 449.8 μmol) in 1, 4-dioxane (4 mL) was added Cs2CO3(366.4 mg, 1.124 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture was added CuI (7.138 mg, 37.48 μmol) and trans-N,N`-dimethylcyclohexane-1,2- diamine (2.96 μL, 18.74 μmol) at room temperature. The reaction mixture was stirred at 120 °C for 16 h. Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4,filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – YMC-Triart Actus (20X150) mm, 5µ Flow - 15 ml / min GRADIENT METHOD- 45,2 / 45,10 / 85,10.1 / 100,11.5 / 100,11.6 / 45,15 / 45, PREP-023). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 11. M / Z (ESI): 468.14 [M+H]+.

[0128] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.0 (s, 1H), 8.711 (d, J = 2.0 Hz, 1H), 8.649 (s, 2H), 8.49 (s, 2H), 8.083 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.44-5.64 (m, 1H), 4.77-4.87 (m, 1H), 4.48-4.60 (m, 2H), 4.32-4.44 (m, 3H), 4.05-4.18 (m, 2H), 3.20-3.30 (m, 2H), 3.065 (td, J = 12.0 Hz, 4.0 Hz, 1H), 1.09 (d, J = 6.8 Hz, 3H). Example 12: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamideSynthesis of 12-3: (R)-5-fluoro-2-(4-(5-iodopyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidine

[0129] To a stirred solution of 12-1 (1.2 g, 6.12 mmol) in DMF (10 mL) were added DIPEA (1.07 mL, 6.12 mmol) and 2-chloro-5-iodopyrimidine (12-2, 1.47 g, 6.12 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with water (300 mL) and extracted with EtOAc (2 x 500 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 40 g silica (100-200 mesh)cartridge and compound eluted with 40% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 12-3. M / Z (ESI): 401.17 [M+H]+. Example 12: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamide

[0130] To a stirred solution of 12-3 (200 mg, 500 μmol) and Int A (146 mg, 750 μmol) in 1, 4- dioxane (5 mL) were added Cs2CO3 (488 mg, 1.5 mmol), CuI (9.52 mg, 50 μmol) and trans- N,N`-dimethylcyclohexane-1,2-diamine (3.94 μL, 25 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with water (250 mL), and extracted with EtOAc (2 x 250 mL). Combined organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 80 g silica (100-200 mesh) cartridge and compound eluted with 68% EtOAc in hexane. Pure fractions were combined and concentrated under reduced pressure to afford 12. M / Z (ESI): 468.21 [M+H]+.

[0131] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.65 (s, 2H), 8.49 (s, 2H), 8.08 (dd, J = 8.6 Hz, 2.2 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.42-5.66 (m, 1H), 4.78-4.85 (m, 1H), 4.48-4.60 (m, 2H), 4.32-4.44 (m, 3H), 4.05-4.18 (m, 2H), 3.20-3.30 (m, 2H), 3.01-3.12 (m, 1H), 1.09 (d, J = 6.8 Hz, 3H). Example 13: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamideNSynthesis of tert-butyl (R)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1-carboxylate (13-3):

[0132] To a stirred solution of tert-butyl (R)-2-methylpiperazine-1-carboxylate (13-1, 1 g, 4.99 mmol) and 2-chloro-5-fluoropyrimidine (13-2, 794 mg, 5.99 mmol) in DMF (20 mL) was added DIPEA (2.61 mL, 15 mmol) at 0 °C. The reaction mixture was stirred at 120 °C for 16 h.Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 10% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 13-3. M / Z (ESI): 197.02 [M+H-Boc]+. Synthesis of (R)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine (13-4):

[0133] To a stirred solution of 13-3 (1.2 g, 4.05 mmol) in DCM (20 mL) was added 4M HCl in 1, 4-dioxane (4.05 mL, 16.2 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was concentrated and dried under reduced pressure to afford 13-4. M / Z (ESI): 197.06 [M+H]+. Synthesis of (R)-5-fluoro-2-(4-(5-iodopyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidine (13-6):

[0134] To a stirred solution of 13-4 (1 g, 4.30 mmol) and 2-chloro-5-iodopyrimidine (13-5, 1.03 g, 4.30 mmol) in DMF (20 mL) was added DIPEA (2.25 mL, 12.9 mmol) at 0 °C. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 5% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 13-6. M / Z (ESI): 401.13 [M+H]+. Example 13: (R)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamide

[0135] To a stirred solution of 13-6 (150 mg, 375 μmol) and Int A (88 mg, 0.45 mmol) in 1, 4- dioxane (4 mL) were added Cs2CO3 (366 mg, 1.12 mmol) and CuI (7 mg, 0.04 mmol) at 0oC. Reaction mixture was degassed and purged with argon gas for 5 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (3 μL, 0.02 mmol) at 0oC. The reaction mixture was stirred at 120 °C for 16 h. Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: X-Bridge C18 (19X150) mm, 5µ Mobile Phase – 10mM Ammonium Bicarbonate INH2O: MeCN COLUMN – X-Bridge C18 (19X150) mm, 5µ Flow-15.0 ml / min Gradient Method –0 / 45,2 / 45,9.50 / 68.4,9.60 / 100,11 / 100,11.1 / 45,14 / 45. PREP-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 13. M / Z (ESI): 468.35 [M+H]+.

[0136] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.67 (s, 2H), 8.47 (s, 2H), 8.08 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 5.43-5.64 (m, 1H), 4.81-4.42 (m, 1H), 4.30-4.60 (m, 5H), 4.02-4.19 (m, 2H), 3.19-3.30 (m, 2H), 3.07 (td, J = 12.2 Hz, 3.2 Hz, 1H), 1.09 (d, J = 6.4 Hz, 3H). Example 14: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamideSynthesis of tert-butyl (S)-4-(5-fluoropyrimidin-2-yl)-2-methylpiperazine-1-carboxylate (14-3):

[0137] To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (14-1, 1 g, 4.99 mmol) in DMF (20 mL) were added 2-chloro-5-fluoropyrimidine (14-2, 728 mg, 5.49 mmol), DIPEA (2.61 mL, 15 mmol) at room temperature. The reaction mixture was stirred at 120 °C for 16 h under argon atmosphere. Reaction mixture was quenched with ice cold water (50 mL) and extracted with EtOAc (3 x 50 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 24 g silica (230-400 mesh) cartridge and compound eluted with 25% EtOAc in pet ether. Pure fractions were combined, concentrated under reduced pressure to afford 14-3. M / Z (ESI): 241.10 [M+H-56]+. Synthesis of (S)-5-fluoro-2-(3-methylpiperazin-1-yl)pyrimidine (14-4):

[0138] To a stirred solution of 14-3 (1.5 g, 5.06 mmol) in DCM (20 mL) was added TFA (1.95 mL, 25.3 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h under argon atmosphere. Reaction mixture was concentrated and dried under reduced pressure to afford 14-4. M / Z (ESI): 197.05 [M+H]+. Synthesis of (S)-5-fluoro-2-(4-(5-iodopyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidine (14-6):

[0139] To a stirred solution of 14-4 (1.5 g, 4.83 mmol) in DMF (20 mL) were added DIPEA (4.21 mL, 24.2 mmol), 2-chloro-5-iodopyrimidine (14-5, 1.39 g, 5.8 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h under argon atmosphere. Reaction mixture was quenched with ice cold water (50 mL) and extracted with EtOAc (3x 50 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 24 g silica (230-400 mesh) cartridge and compound eluted with 25% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 14-6. M / Z (ESI): 401.13 [M+H]+. Example 14: (S)-6-(3-fluoroazetidin-1-yl)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin- 1-yl)pyrimidin-5-yl)nicotinamide

[0140] To a stirred solution of 14-6 (150 mg, 375 μmol) in 1,4-dioxane (3 mL) were added Int A (88 mg, 451 μmol), Cs2CO3 (366 mg, 1.12 mmol), copper(I) iodide (8 mg, 42 μmol), trans- N,N`-dimethylcyclohexane-1,2-diamine (3.33 μL, 21.1 μmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 10 min. The reaction mixture was stirred at110 °C for 24 h. Reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 20 mL). Combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was triturated with diethyl ether (10 mL) and dried under reduced pressure. Obtained compound was purified by prep-HPLC purification (conditions: X-Bridge, C18 (19X150) mm, 5µ. Mobile Phase – 10mM Ammonium Bicarbonate in H2O: MeCN column – X-Bridge, C18 (19X150) mm, 5µ Flow-15.0 ml / min Gradient Method – 0 / 45,2 / 45,10 / 85,12.50 / 85,12.51 / 99,13 / 99,13.10 / 45,16 / 45. PREP-014). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 14. M / Z (ESI): 468.35 [M+H]+.

[0141] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.67 (s, 2H), 8.47 (s, 2H), 8.08 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 5.43-5.64 (m, 1H), 4.83-4.91 (m, 1H), 4.33-4.55 (m, 5H), 4.06-4.17 (m, 2H), 3.16-3.29 (m, 2H), 3.07 (td, J = 12.2 Hz, 3.6 Hz, 1H), 1.09 (d, J = 6.4 Hz, 3H). Synthesis of Radiolabeling intermediates: Synthesis of (R)-1-(5-((5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (1-8):Synthesis of (R)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (1-7):

[0142] To a stirred solution of 1-6 (500 mg, 1.42 mmol) in 1, 4-dioxane (8 mL) were added Cs2CO3(1.39 g, 4.26 mmol), CuI (27 mg, 142 μmol) and Int B (329 mg, 1.70 mmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (11.2 μL, 71.0 μmol) at room temperature. The reaction mixture was stirred in microwave at 150 °C for 2 h. Reaction mixture was quenched with water (80 mL) and extracted with EtOAc (2 x 150 mL). Combined organic layer was washed with brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotageusing 40 g silica (230-400 mesh) cartridge and compound eluted with 3% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure. Obtained compound was again purified by prep-HPLC purification (conditions: Prep-HPLC Purification conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate IN H2O: MeCN

[0143] COLUMN – X-Bridge C18 (19X150) mm, 5µ, Flow-15.0 ml / min, GRADIENT METHOD –0 / 30, / 2 / 30,8.35 / 61.5,8.40 / 100,10.50 / 100,10.55 / 30,10.60 / 30,13 / 30). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 1-7. M / Z (ESI): 465.14 [M+H]+.

[0144] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.44 (s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.74 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.09-8.14 (m, 2H), 7.50-7.56 (m, 1H), 6.87 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.74 (br s, 1H), 4.51-4.61 (m, 2H), 4.17-4.31 (m, 4H), 3.98-4.09 (m, 1H), 3.77 (dd, J = 9.6 Hz, 4.4 Hz, 2H), 3.16-3.27 (m, 2H), 3.04 (td, J = 12.2 Hz, 3.2 Hz, 1H), 1.09 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (1-8):

[0145] To a stirred solution of 1-7 (250 mg, 538 μmol) in DCM (3 mL) were added TEA (225 μL, 1.61 mmol), DMAP (6.58 mg, 53.8 μmol) and Ts-Cl (205 mg, 1.08 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 5 h under nitrogen atmosphere. Reaction mixture was quenched with water (80 mL) and extracted with EtOAc (2 x 120 mL). Combined organic layer was washed with brine (2 x 80 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 20 g silica (230-400 mesh) cartridge and compound eluted with 2% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure. Obtained compound was triturated with 2% DCM in diethyl ether (2 x 50 mL) and dried under reduced pressure to afford 1-8. M / Z (ESI): 619.43 [M+H]+.

[0146] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.49 (s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.74 (d, J = 1.6 Hz, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.11-8.15 (m, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.50- 7.56 (m, 3H), 6.87 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.45 (d, J = 8.8 Hz, 1H), 5.27-5.33 (m, 1H), 4.51-4.58 (m, 1H), 4.16-4.34 (m, 4H), 3.99-4.06 (m, 1H), 3.95 (dd, J = 10.0 Hz, 2.8 Hz, 2H), 3.15-3.27 (m, 2H), 3.04 (td, J = 12.0 Hz, 3.2 Hz, 1H), 2.46 (s, 3H), 1.08 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (2-8):Synthesis of (R)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (2-7):

[0147] To a solution of (R)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide (2-6, 50.0 mg, 1 Eq, 117 μmol) in DMA (0.5 mL) was added potassium fluoride (20.4 mg, 3 Eq, 351 μmol), and azetidin-3-ol hydrochloride (12.8 mg, 1 Eq, 117 μmol). The mixture was stirred for 16 h at 80ºC. Post reaction time, LCMS showed high conversion of desired product. The reaction mixture was purified using basic reverse-phase chromatography (Waters Sunfire Prep C18 OBD 19x150 mm column, 30-100% water with 0.1% NH4OH: acetonitrile, 15 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 2-7. MS (ESI) m / z: 465.6 [M+H]+. Synthesis of (R)-1-(5-((5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (2-8):

[0148] To a solution of ((R)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)-6-(3-hydroxyazetidin-1-yl)nicotinamide (2-7, 50.0 mg, 1 Eq, 108 μmol) in DCM (0.5 mL) was added triethylamine (45.0 µL, 32.7 mg, 3 Eq, 323 μmol), 4-dimethylaminopyridine (1.32 mg, 0.1 Eq, 810.8 μmol), followed by p-toluenesulfonyl chloride (32.8 µL, 41.0 mg, 2 Eq, 215 µmol). The mixture was stirred for 16 h at 22ºC. The reaction mixture was purified using normal phase chromatography (ISCO 24 g RediSep Gold High Performance Silica, 0-100% hexane: EtOAc / EtOH (3:1 mixture), 15 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 2-8. MS (ESI) m / z: 619.6 [M+H]+. Synthesis of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (3-9):Synthesis of 3-2: tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate

[0149] To a solution of tert-butyl (S)-2-methylpiperazine-1-carboxylate (3-1, 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-1-carboxylate (1.00 g, 1 Eq, 4.99 mmol). The mixture was purged with N2 for 5 minutes then chloro(2-dicyclohexylphosphino-2',6'-di-i- propoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (194 mg, 0.05 Eq, 250 μmol) was added. The mixture was stirred for 48 h at 110°C. Water (50 mL) was added to the mixture and it was extracted with EtOAc (100 mL x 3). The organic layer was dried over MgSO4, filtered through a fritted 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% Hexane, EtOAc, 26 min gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 3-2. MS (ESI) m / z: 296.4 [M+H]+. Synthesis of 3-3: (S)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine hydrochloride

[0150] To a solution of tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1- carboxylate (3-2, 1.00 g, 1 Eq, 3.39 mmol) in 1,4-dioxane (14.0 mL) was added 4M hydrogen chloride (3.39 mL, 4 Eq, 13.5 mmol) in 1,4-dioxane. The mixture was stirred for 18 h at 22°C. The mixture was concentrated under reduced pressure to yield 3-3. MS (ESI) m / z: 196.3 [M+H]+.Synthesis of 3-5: (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine

[0151] To a solution of (S)-1-(5-fluoropyridin-2-yl)-3-methylpiperazine hydrochloride (3-4, 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 for 18 h at 60 °C. Water was added to the mixture and stirred for 30 min at 22°C. The resulting precipitated solid was collected by filtration through a fritted filter, washed with water (100 mL x 3), and dried to give 3-5. MS (ESI) m / z: 319.3 [M+H]+. Synthesis of 3-6: (S)-5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-amine

[0152] A solution of (S)-2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)-5-nitropyrazine (3-5, 579 mg, 1 Eq, 1.82 mmol) in THF (5.00 mL) and MeOH (5.00 mL) was degassed and purged with N2 (x 3) then added 10% palladium on carbon (96.8 mg, 0.5 Eq, 909 μmol) and degassed and purged with H2(x 3). The mixture was stirred for 18 h at 22°C under a H2filled balloon. The mixture was filtered through a fritted filter, washed with MeOH (5 mL x 3), and concentrated under vacuum to yield 3-6. MS (ESI) m / z: 289.3 [M+H]+. Synthesis of 3-7: (S)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)nicotinamide

[0153] To a solution of (S)-5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-amine (3-6, 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 for 1.5h at 22°C The crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C185mm - 50x250mm column, 10-100% 5 mM NH4HCO3 water solution: acetonitrile, 33 min gradient). Fractions containing the 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 yield 3-7. MS (ESI) m / z: 428.4 [M+H]+. Synthesis of 3-8: (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide

[0154] To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide (3-7, 50.0 mg, 1 Eq, 117 μmol) in DMA (1.0 mL) was addedpotassium fluoride (20.4 mg, 3 Eq, 350 μmol), and azetidin-3-ol hydrochloride (12.8 mg, 1 Eq, 117 μmol). The mixture was stirred for 16 h at 80ºC. Post reaction time, LCMS showed high conversion of desired product. The reaction mixture was purified using basic reverse-phase chromatography (Waters XBridge C1830x150 mm column, 10-100% 5 mM NH4HCO3 water solution: acetonitrile, 22 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 3-8. MS (ESI) m / z: 465.5 [M+H]+. Synthesis of 3-9 (S)-1-(5-((5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate

[0155] To a solution of (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)-6-(3-hydroxyazetidin-1-yl)nicotinamide (3-8, 40.0 mg, 1 Eq, 86.1 μmol) in DCM (1.0 mL) was added triethylamine (36.0 µL, 26.1 mg, 3 Eq, 258 μmol), 4-Dimethylaminopyridine (1.05 mg, 0.1 Eq, 8.61 μmol), followed by p-Toluenesulfonyl chloride (26.2 µL, 32.8 mg, 2 Eq, 172 µmol). The mixture was stirred for 16 h at 22ºC. The reaction mixture was purified using normal phase chromatography (ISCO 40 g RediSep Gold High Performance Silica, 0-100% hexane: EtOAc / EtOH (3:1 mixture), 18 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to 3-9. MS (ESI) m / z: 619.6 [M+H]+. Synthesis of (S)-1-(5-((2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (4-6)Synthesis of (S)-N-(2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (4-5):

[0156] To a stirred solution of 4-4 (800 mg, 2 mmol) and Int B (465 mg, 2.40 mmol) in 1, 4- dioxane (10 mL) at room temperature in a sealed tube. Reaction mixture was degassed and purged with nitrogen gas for 10 min. Then to this reaction mixture were added Cs2CO3(1.96 g,6.01 mmol), CuI (38.2 mg, 200 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (63.2 μL, 200 μmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (2 x 30 mL). Combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound (800 mg) was purified by Biotage using 12 g silica (230-400 mesh) cartridge and compound eluted with 5% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 4-5. M / Z (ESI): 465.41 [M+H]+. Synthesis of (S)-1-(5-((2-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (4-6):

[0157] To a stirred solution of 4-5 (250 mg, 538 μmol) in DCM (5 mL) were added TEA (300 μL, 2.15 mmol), p-TsCl (308 mg, 1.61 mmol) and DMAP (6.58 mg, 53.8 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h. Reaction mixture was quenched with water (20 mL) and extracted with DCM (2 x 30 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound (300 mg) was purified by Biotage using 12 g silica (230-400 mesh) cartridge and compound eluted with 3% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure. Obtained compound was further purified by prep-HPLC purification (conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – ymc-exrs C18 (19X150) mm, 5µ Flow-15.0 ml / min, GRADIENT METHOD – 0 / 50,2 / 50,10 / 65,15 / 65,15.1 / 99,18 / 99,18.1 / 50,21 / 50). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 4-6. M / Z (ESI): 619.44 [M+H]+.

[0158] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.99 (s, 1H), 8.60-8.70 (m, 3H), 8.11 (d, J = 2.8 Hz, 1H), 8.06 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.49-7.56 (m, 3H), 6.85 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 5.26-5.33 (m, 1H), 4.46-4.60 (m, 3H), 4.31 (dd, J = 6.8 Hz, 9.8 Hz, 2H), 3.90-4.05 (m, 3H), 3.25-3.30 (m, 1H), 3.09-3.09 (m, 2H), 2.46 (s, 3H), 1.02 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (6-3):Synthesis of (R)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (6-5):

[0159] To a stirred solution of 6-4 (300 mg, 751 μmol) and Int B (145 mg, 751 μmol)in 1, 4- dioxane (4 mL) were added Cs2CO3 (735 mg, 2.25 mmol), CuI (14.3 mg, 75.1 μmol) and trans- N,N`-dimethylcyclohexane-1,2-diamine (5.93 μL, 37.6 μmol) at room temperature. The reaction mixture was stirred at 120 °C for 16 h. Reaction mixture was filtered through celite pad, washed with EtOAc (50 mL), filtrate was quenched with water (50 mL) and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine (1 x 30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate in H2O: MeCN, COLUMN – X-Bridge C18 (19X250) mm, 5µ, Flow-15.0 ml / min, Gradient Method – 0 / 33,2 / 33,10 / 65,10.01 / 100,11.5 / 100,11.55 / 33,15 / 33 Prep-023). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 652. M / Z (ESI): 465.20 [M+H]+.

[0160] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.95 (s, 1H), 8.68 (d, J = 2.0 Hz, 1H), 8.66 (s, 2H), 8.10 (d, J = 2.8 Hz, 1H), 8.04 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.53 (td, J = 8.8 Hz, 2.8 Hz, 1H), 6.92 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 5.74 (d, J = 6.4 Hz, 1H), 4.80- 4.87 (m, 1H), 4.57-4.65 (m, 1H), 4.39-4.45 (m, 1H), 4.10-4.28 (m, 4H), 3.77 (dd, J = 9.6 Hz, 4.4 Hz, 2H), 3.27-3.29 (m, 1H), 3.10-3.18 (m, 1H), 2.85-2.98 (m, 1H), 1.15 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (6-6):

[0161] To a stirred solution of 6-5 (60 mg, 129 μmol) in ACN (0.3 mL) and THF (0.6 mL) were added TEA (54 μL, 388 μmol), DMAP (1.58 mg, 12.9 μmol) and Ts-Cl (39.3 μL, 258 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 48 h under nitrogen atmosphere. Reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2 x 30 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: X-Bridge, C18 (19X250) mm, 5µ Mobile Phase – 10mM Ammonium Bicarbonate IN H2O: CAN column – X-Bridge C18 (19X250) mm, 5µ Flow-15.0 ml / min, Gradient Method –0 / 55,2 / 55,7 / 62,12.5 / 62,14 / 99,18 / 55). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 6-6. M / Z (ESI): 619.39 [M+H]+.

[0162] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.99 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.65 (s, 2H), 8.03-8.12 (m, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.48-7.58 (m, 3H), 6.91 (dd, J = 9.4 Hz, 3.4 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 5.27-5.33 (m, 1H), 4.80-4.87 (m, 1H), 4.37-4.47 (m, 1H), 4.31 (dd, J = 9.3 Hz, 6.8 Hz, 2H), 4.08-4.22 (m, 2H), 3.96 (dd, J = 10.4 Hz, 2.8 Hz, 2H), 3.22-3.29 (m, 1H), 3.08-3.19 (m, 1H), 2.91 (td, J = 12.4 Hz, 3.2 Hz, 1H), 2.46 (s, 3H), 1.14 (d, J = 6.4 Hz, 3H). Synthesis of (S)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (7-6):Synthesis of (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (7-5):

[0163] To a stirred solution of 7-4 (0.4 g, 1.1 mmol) and Int B (0.26 g, 1.4 mmol) in 1, 4- dioxane (6 mL) was added Cs2CO3 (1.1 g, 3.4 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture was added CuI (22 mg, 0.11 mmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (8.9 μL, 57 μmol) drop wise at room temperature. The reaction mixture was stirred in microwave at 110 °C for 2 h. Reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 12 g silica (100-200 mesh) cartridge and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 7-5. M / Z (ESI): 466.25 [M+H]+. Synthesis of (S)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (7-6):

[0164] To a stirred solution of 7-5 (280 mg, 601.5 μmol) and TEA (419 μL, 3.008 mmol) in DCM (5 mL) was added DMAP (36.74 mg, 300.8 μmol) at 0 °C. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture was added Ts-Cl (344 mg, 1.805 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was quenched with water (20 mL), and extracted with EtOAc (2 x 30 mL). Combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using Biotage using 12 g silica (100-200 mesh) cartridge and compound eluted with 20% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 7-6. M / Z (ESI): 620.44 [M+H]+.

[0165] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.82 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.50 (s, 2H), 8.18 (d, J = 1.2 Hz, 1H), 8.13 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.8 Hz, 1H), 5.25-5.33 (m, 1H), 4.76- 4.85 (m, 1H), 4.20-4.43 (m, 5H), 3.92-3.98 (m, 2H), 3.29-3.31 (m, 1H), 3.22 (dd, J = 12.8 Hz, 4.0 Hz, 1H), 3.00-3.07 (m, 1H), 2.46 (s, 3H), 1.08 (d, J = 7.2 Hz, 3H).Synthesis of (R)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (8-8):Synthesis of (R)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (8-7):

[0166] To a stirred solution of 8-6 (366 mg, 1.04 mmol) in 1, 4-dioxane (5 mL) and Int B (200 mg, 1.04 mmol) were added Cs2CO3(1.01 g, 3.11 mmol), CuI (19.7 mg, 104 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (8.16 μL, 51.8 μmol) at room temperature. The reaction mixture was stirred at 110 °C for 16 h in a sealed tube. Reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 25 mL). Combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 80% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford 8-7. M / Z (ESI): 466.33 [M+H]+.

[0167] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.44 (s, 1H), 8.83 (d, J = 1.2 Hz, 1H), 8.74 (d, J = 2.0 Hz, 1H), 8.50 (s, 2H), 8.18 (d, J = 1.6 Hz, 1H), 8.11 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 6.4 Hz, 1H), 4.75-4.86 (m, 1H), 4.55-4.65 (m, 1H), 4.35-4.44 (m, 1H), 4.16-4.30 (m, 4H), 3.72-3.80 (m, 2H), 3.35-3.40 (m, 1H), 3.22 (dd, J = 13.0 Hz, 3.8 Hz, 1H), 3.02 (td, J = 12.2 Hz, 3.2 Hz, 1H), 1.16 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (8-8):

[0168] To a stirred solution of 8-7 (150 mg, 322 μmol) in DCM (3 mL) were added TEA (135 μL, 967 μmol), Ts-Cl (123 mg, 644 μmol) and DMAP (3.94 mg, 32.2 μmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h under nitrogen atmosphere. Reaction mixture was quenched with water (10 mL) and extracted with DCM (2 x 25 mL). Combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification. Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 8-8. M / Z (ESI): 620.40 [M+H]+. Prep-HPLC Purification Conditions: Column : XBridge C18 (4.6x150) mm, 3.5µm Mobile Phase-A : 10mM 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 ovenTemp : Ambient Diluent : ACN:WATER (90 / 10) V / V.

[0169] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.82 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.50 (s, 2H), 8.18 (d, J = 1.2 Hz, 1H), 8.13 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.8 Hz, 1H), 5.55-5.33 (m, 1H), 4.75- 4.86 (m, 1H), 4.15-4.44 (m, 5H), 3.90-4.00 (m, 2H), 3.35-3.40 (m, 1H), 3.22 (d, J = 12.8 Hz, 4.0 Hz, 1H), 3.02 (td, J = 12.2 Hz, 3.6 Hz, 1H), 2.46 (s, 3H), 1.16 (d, J = 6.8 Hz, 3H). Synthesis of (R)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (9-7):Synthesis of (R)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (9-6):

[0170] To a stirred solution of 9-5 (500 mg, 1.42 mmol) and Int B (410 mg, 2.12 mmol) in 1, 4-dioxane (10 mL) was added Cs2CO3 (1.38 g, 4.25 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture was added CuI (27 mg, 142 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (11.2 μL, 70.8 μmol) at room temperature and again degassed with nitrogen gas for 2 min. The reaction mixture was stirred at 80 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (3 x 50 mL). Combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 25 g silica (100-200 mesh) cartridge and compound eluted with 6% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 9-6. M / Z (ESI): 466.46 [M+H]+. Synthesis of (R)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (9-7):

[0171] To a stirred solution of 9-6 (400 mg, 859 μmol) in DCM (20 mL) were added TEA (599 μL, 4.30 mmol) and DMAP (31.5 mg, 258 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 5 min. Then to this reaction mixture was added Ts-Cl (328 mg, 1.72 mmol) slowly at 0oC. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (3 x 30 mL). Combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 12 g silica (100-200 mesh) cartridge and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure. Crude compound was again purified by prep-HPLC purification (conditions: Mobile Phase – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – X-Select, C18 PACKED (25X150) mm, 5µ Flow-20.0 ml / min Gradient Method – 0 / 60,2 / 60,8 / 75,9.75 / 75,9.8 / 100,13 / 100,13.05 / 60,16 / 60. PREP-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 9-7. M / Z (ESI): 620.44 [M+H]+.

[0172] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.50 (s, 1H), 8.84 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.48 (s, 2H), 8.09-8.15 (m, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.4 Hz, 1H), 5.25-5.33 (m, 1H), 4.57-4.66 (m, 1H), 4.43-4.56 (m, 2H), 4.26-4.34 (m, 2H), 4.06-4.12 (m, 1H), 3.90-4.00 (m, 2H), 3.30-3.31 (m, 1H), 3.10-3.26 (m, 2H), 2.46 (s, 3H), 1.07 (d, J = 6.8 Hz, 3H). Synthesis of (S)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (10-8):Synthesis of (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (10-7):

[0173] To a solution of (S)-6-chloro-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide (10-6, 30.0 mg, 1 Eq, 69.9 μmol) in DMA (1.0 mL) was added potassium fluoride (12.2 mg, 3 Eq, 210 μmol), and azetidin-3-ol hydrochloride (7.66 mg, 1 Eq, 69.9 μmol). The mixture was stirred for 16 h at 100ºC. The reaction mixture was purified using basic reverse-phase chromatography (Waters Sunfire Prep C18 OBD 19x150 mm column, 30- 100% water with 0.1% NH4OH: acetonitrile, 15 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 10-7. MS (ESI) m / z: 466.6 [M+H]+.Synthesis of (S)-1-(5-((5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (10-8):

[0174] To a solution of (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2- yl)-6-(3-hydroxyazetidin-1-yl)nicotinamide (10-7, 25.0 mg, 1 Eq, 53.7 μmol) in DCM (0.5 mL) was added triethylamine (22.5 µL, 16.3 mg, 3 Eq, 161 μmol), 4-Dimethylaminopyridine (1.00 mg, 0.2 Eq, 8.00 μmol), followed by p-Toluenesulfonyl chloride (16.4 µL, 20.5 mg, 2 Eq, 107 µmol). The mixture was stirred for 16 h at 22ºC. The reaction mixture was purified using normal phase chromatography (ISCO 24 g RediSep Gold High Performance Silica, 0-100% hexane: EtOAc / EtOH (3:1 mixture), 16 minute gradient). Fractions containing the desired product were combined and concentrated under vacuum to yield 10-8. MS (ESI) m / z: 620.6 [M+H]+. Synthesis of (S)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (11-6):Synthesis of (S)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (11-5):

[0175] To a stirred solution of 11-4 (500 mg, 1.25 mmol) and Int B (241 mg, 1.25 mmol) in 1, 4-dioxane (10 mL) was added Cs2CO3 (1.22 g, 3.75 mmol) at room temperature. Reaction mixture was degassed and purged with nitrogen gas for 5 min. Then to this reaction mixture were added CuI (23.8 mg, 125 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (9.85 μL,62.5 μmol) at room temperature and again degassed with nitrogen gas for 2 min. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (3 x 50 mL). Combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 25 g silica (100-200 mesh) cartridge and compound eluted with 6% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 11-5. M / Z (ESI): 466.42 [M+H]+. Synthesis of (S)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (11-6):

[0176] To a stirred solution of 11-5 (400 mg, 859 μmol) in DCM (20 mL) was added TEA (599 μL, 4.30 mmol) and DMAP (31.5 mg, 258 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 5 min. Then to this reaction mixture was added Ts-Cl (328 mg, 1.72 mmol) slowly at 0oC. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was quenched with water (30 mL) and extracted with 10% MeOH in DCM (3 x 30 mL). Combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 12 g silica (100-200 mesh) cartridge and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 11-6. M / Z (ESI): 620.33 [M+H]+.

[0177] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.99 (s, 1H), 8.60-8.70 (m, 3H), 8.49 (s, 2H), 8.06 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.24-5.33 (m, 1H), 4.75-4.86 (m, 1H), 4.46-4.60 (m, 2H), 4.26-4.60 (m, 3H), 3.92-4.00 (m, 2H), 3.19-3.28 (m, 2H), 3.07 (td, J = 12.6 Hz, 3.6 Hz, 1H), 2.46 (s, 3H), 1.09 (d, J = 6.4 Hz, 3H). Synthesis of (R)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (12-5):Synthesis of (R)-N-(2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (12-4): To a stirred solution of 12-3 (200 mg, 500 μmol) and Int B (96.6 mg, 500 μmol) in 1, 4-dioxane (4 mL) were added Cs2CO3(488 mg, 1.50 mmol), copper(I) iodide (9.52 mg, 50 μmol) and trans- N,N`-dimethylcyclohexane-1,2-diamine (3.94 μL, 25 μmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 h. Reaction mixture was quenched with ice cold water (50 mL) and extracted with EtOAc (2 x 50 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4,filtered and concentrated under reduced pressure. Crudecompound was purified by Biotage using 24 g silica (pre-packed) cartridge and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 12-4. M / Z (ESI): 466.42 [M+H]+. Synthesis of (R)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (12-5):

[0178] To a stirred solution of 12-4 (200 mg, 430 μmol) in DCM (0.2 mL) were added TEA (180 μL, 1.29 mmol), Ts-Cl (164 mg, 859 μmol) and DMAP (5.25 mg, 43.0 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was quenched with ice cold water (50 mL) and extracted with DCM (2 x 50 mL). Combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by prep-HPLC purification (conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN – YMC-Actus TRAIRT C18 (20X150) mm, 5µ Flow - 15 ml / min GRADIENT METHOD- 0 / 50,2 / 50,10 / 85,11.1 / 100,11.9 / 100,12 / 50,15 / 50. APMS-009). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 12-5. M / Z (ESI): 620.40 [M+H]+.

[0179] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.99 (s, 1H), 8.62-8.69 (m, 3H), 8.49 (s, 2H), 8.06 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.25-5.33 (m, 1H), 4.67-4.85 (m, 1H), 4.47-4.60 (m, 2H), 4.35-4.42 (m, 1H), 4.27- 4.33 (m, 2H), 3.90-4.00 (m, 2H), 3.20-3.30 (m, 2H), 3.07 (td, J = 12.6 Hz, 3.6 Hz, 1H), 2.46 (s, 3H), 1.09 (d, J = 6.8 Hz, 3H).Synthesis of (R)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (13-8):Synthesis of (R)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (13-7):

[0180] To a stirred solution of 13-6 (300 mg, 750 μmol) and Int B (145 mg, 750 μmol) in 1, 4- dioxane (5 mL) were added Cs2CO3 (733 mg, 2.25 mmol) and CuI (14.3 mg, 75 μmol) at 0oC.Reaction mixture was degassed and purged with argon gas for 5 min. Then to this reaction mixture was added trans-N,N`-dimethylcyclohexane-1,2-diamine (5.91 μL, 37.5 μmol) at 0oC. The mixture was stirred at 110 °C for 16 h. Reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 10 mL). Combined organic layer was washed with brine (5 mL), dried over anhydrous Na2SO4 and filtered and concentrated under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 13-7. M / Z (ESI): 466.15 [M+H]+.

[0181] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.96 (s, 1H), 8.61-8.70 (m, 3H), 8.47 (s, 2H), 8.04 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 5.74 (d, J = 6.4 Hz, 1H), 4.81-4.92 (m, 1H), 4.56-4.67 (m, 1H), 4.38-4.56 (m, 3H), 4.20-4.29 (m, 2H), 3.77 (dd, J = 9.6 Hz 4.4 Hz, 2H), 3.20-3.30 (m, 2H), 3.01-3.12 (m, 1H), 1.09 (d, J = 6.8 Hz, 3H). Synthesis of (R)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (13-8):

[0182] To a stirred solution of 13-7 (120 mg, 258 μmol) in DCM (5 mL) were added TEA (180 μL, 1.29 mmol), DMAP (3.15 mg, 25.8 μmol) and Ts-Cl (98.3 mg, 516 μmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Reaction mixture was quenched with water (10 mL) and extracted with DCM (2 x 20 mL). Combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by silica gel column and compound eluted with 100% EtOAc. Pure fractions were combined and concentrated under reduced pressure to afford 13-8. M / Z (ESI): 620.31 [M+H]+.

[0183] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.65 (s, 2H), 8.47 (s, 2H), 8.06 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.26-5.33 (m, 1H), 4.81-4.90 (m, 1H), 4.39-4.55 (m, 3H), 4.27-4.34 (m, 2H), 3.91-3.99 (m, 2H), 3.19-3.29 (m, 2H), 3.07 (td, J = 12.6 Hz, 3.2 Hz, 1H), 2.46 (s, 3H), 1.09 (d, J = 6.8 Hz, 3H). Synthesis of (S)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (14-8):Synthesis of (S)-N-(2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(3- hydroxyazetidin-1-yl)nicotinamide (14-7):

[0184] To a stirred solution of 14-6 (250 mg, 625 μmol) in 1, 4-dioxane (4 mL) were added Int B (145 mg, 750 μmol), Cs2CO3(611 mg, 1.87 mmol), copper(I) iodide (12.0 mg, 63 μmol) and trans-N,N`-dimethylcyclohexane-1,2-diamine (5.54 μL, 35.2 μmol) at room temperature. Reaction mixture was degassed and purged with argon gas for 10 min. The reaction mixture was stirred at 110 °C for 16 h. Reaction mixture was filtered through celite pad, washed with water (20 mL), DCM (10 mL) and extracted with DCM (3 x 20 mL). Combined organic layer was withbrine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 24 g silica (230-400 mesh) cartridge and compound eluted with 5% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford 14-7. M / Z (ESI): 466.33 [M+H]+.

[0185] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.95 (s, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.66 (s, 2H), 8.47 (s, 2H), 8.03 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 5.74 (d, J = 6.8 Hz, 1H), 4.84-4.90 (m, 1H), 4.55-4.65 (m, 1H), 4.41-4.55 (m, 3H), 4.21-4.28 (m, 2H), 3.77 (dd, J = 9.6 Hz, 4.4 Hz, 2H), 3.16-3.29 (m, 2H), 3.03-3.12 (m, 1H), 1.09 (d, J = 6.8 Hz, 3H). Synthesis of (S)-1-(5-((2-(4-(5-fluoropyrimidin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5- yl)carbamoyl)pyridin-2-yl)azetidin-3-yl 4-methylbenzenesulfonate (14-8): 14505

[0186] To a stirred solution of 14-7 (150 mg, 322 μmol) in DMF (1 mL), THF (2 mL) were added TEA (135 μL, 967 μmol), Ts-Cl (123 mg, 644 μmol), DMAP (3.94 mg, 32.2 μmol) at room temperature. The reaction mixture was stirred at room temperature for 40 h under argon atmosphere. Reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 20 mL). Combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 12 g silica gel column and compound eluted with 80% EtOAc in pet ether as eluent. Pure fractions were combined and concentrated under reduced pressure to afford 14-8. M / Z (ESI): 620.40 [M+H]+.

[0187] 1H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.00 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.65 (s, 2H), 8.47 (s, 2H), 8.06 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.27-5.33 (m, 1H), 4.83-4.90 (m, 1H), 4.40-4.54 (m, 3H), 4.27-4.35 (m, 2H), 3.94-3.99 (m, 2H), 3.16-3.29 (m, 2H), 3.07 (td, J = 12.2 Hz, 3.5 Hz, 1H), 2.46 (s, 3H), 1.09 (d, J = 6.4 Hz, 3H). Radiosynthesis Procedures Synthesis of Compound 91: (S)-6-(1H-imidazol-1-yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin- 1-yl)pyrimidin-5-yl)nicotinamideSynthesis of 91-2: (S)-tert-butyl 3-methyl-4-(5-nitropyrimidin-2-yl)piperazine-1-carboxylate

[0188] To a solution of (S)-tert-butyl 3-methylpiperazine-1-carboxylate (91-1, 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 for 12 h at 25 °C under N2balloon. TLC showed the starting material was consumed completely. Water (450 mL) was added and the mixture wasstirred at 25 °C (rt) for 30 min. The precipitated solid was collected by filtration, washed with water (100 mL x 3) and dried to give the 91-2.

[0189] 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). Synthesis of 91-3: (S)-2-(2-methylpiperazin-1-yl)-5-nitropyrimidine

[0190] To a solution of 91-2 (21 g, 64.9 mmol) in DCM (160 mL) was added TFA (40 mL) at 0 °C. The mixture was stirred for 2 h at 25 °C. 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-1-yl)-5-nitropyrimidine (25 g, 78 mmol) as an oil. The product was diluted with DCM (200 mL) and H2O (160 mL). Then the Na2CO3was added to solution to adjust pH to 7~8. The solution was extracted with DCM (200 mL*2). The organic layer was dried over Na2SO4, filtered and concentrated to give 91-3.1H NMR (400 MHz, DMSO-d6): δ = 9.44 (s, 1H), 8.92~9.10 (m, 1H), 5.13~5.24 (m, 1H), 4.82 (d, J = 14.4 Hz, 1H), 3.31~3.49 (m, 3H), 3.26 (d, J = 7.2 Hz, 1H), 3.06 (d, J = 8.8 Hz, 1H), 1.52 (s, 1H), 1.33 (d, J = 7.2 Hz, 3H). MS (ESI) m / z: 224.0 [M+H]+. Synthesis of 91-4: (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)-5-nitropyrimidine

[0191] To a solution of 91-3 (2.5 g, 11.20 mmol) in Dioxane (50 mL) was added 2- bromopyridine (3.72 g, 23.52 mmol), Cs2CO3 (14.96 g, 45.9 mmol) and chloro(2- dicyclohexylphosphino-2’, 6’-dimethoxy-1, 1’-biphenyl)[2-(2’-amino-1,1’- biphenyl)]palladium(II) (0.968 g, 1.344 mmol). The mixture was stirred for 12 h at 110 °C under N2 balloon. TLC showed most of the starting material was consumed completely. The mixture was filtered and concentrated. The residue was extracted with EtOAc (3*50 mL) and H2O (60 mL). The combined organic extracts were washed with brine (100 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a silica gel column eluting with 15~30% EtOAc / PE to give the 91-4.1H NMR (400 MHz, Chloroform -d): δ = 9.10 (s, 2H), 8.17~8.24 (m, 1H), 7.47~7.59 (m, 1H), 6.62~6.71 (m, 2H), 5.11 (dt, J = 6.4, 3.2 Hz, 1H), 4.74 (dt, J = 13.6, 3.6 Hz, 1H), 4.24 (d, J = 12.8 Hz, 1H), 4.13 (d, J = 13.2 Hz, 1H), 3.53~3.63 (m, 1H), 3.37 (dd, J = 13.2, 4.0 Hz, 1H), 3.12 (td, J = 12.0, 3.6 Hz, 1H), 1.34 (d, J = 6.8 Hz, 3H). MS (ESI) m / z: 301.0 [M+H]+. Synthesis of (S)-2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-5-amine (91-5)

[0192] To a solution of 91-4 (2.5 g, 8.32 mmol) in MeOH (40 mL) was added Pd / C (0.2 g, 1.879 mmol). The mixture was stirred for 2 h at 25 °C under H2 balloon. TLC showed most of the starting material was consumed completely. The mixture was filtered, and the filter cake was washed with MeOH (3*200 mL). The combined organic extracts were concentrated under reduced pressure to give the 91-5.

[0193] 1H 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]+. Synthesis of 91: (S)-6-(1H-imidazol-1-yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-1- yl)pyrimidin-5-yl)nicotinamide

[0194] To a stirred solution of 91-5 (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 quenched with ice cold water (10 mL), extracted with ethyl acetate (2 x 100 mL), combined organic layer washed with brine solution (2 x 10 mL), dried over sodium sulfate, filtered, concentrated under reduced pressure and crude compound purified by Prep HPLC (Mobile Phase - 10mM Ammonium Bicarbonate in H2O: MeCN COLUMN - X-Select C18 (19X250) mm 5u Flow-18ml / min Gradient Method-0 / 45, 6.9 / 76, 6.95 / 100, 9 / 100, 9.05 / 45, 12 / 45). Pure fractions concentrated and lyophilized to afford 91. M / Z (ESI): 442.14 [M+H]+.1H NMR (400MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.04 (d, J=2.0 Hz, 1H), 8.71 (s, 2H), 8.66 (s, 1H), 8.52 (dd, J=8.7, 2.3 Hz, 1H), 8.12 (dd, J=4.9, 1.2 Hz, 1H), 8.06 (t, J=1.2 Hz, 1H), 8.01 (d, J=8.6 Hz, 1H), 7.55 (ddd, J=8.6, 7.0, 2.2 Hz, 1H), 7.18 (s, 1H), 6.86 (d, J=8.8 Hz, 1H), 6.60-6.67 (m, 1H), 4.85 (dt, J=6.4, 3.2 Hz, 1H), 4.41-4.49 (m, 1H), 4.17-4.31 (m, 2H), 3.28 (br d, J=3.7 Hz, 1H), 3.19 (br dd, J=13.1, 3.8 Hz, 1H), 2.90-3.01 (m, 1H), 1.16 (d, J=6.6 Hz, 3H). Synthesis of [3H]-91: [3H]-(S)-6-(1H-imidazol-1-yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin- 1-yl)pyrimidin-5-yl)nicotinamide

[0195] In a glove box, Compound 91 (1.33 mg, 3.0 μmol) was dissolved in CPME (75 µL) and NMP (25 µL). The nickel pre-catalyst (ipcADI)NiBr2(6.73 mg) was dissolved in CPME (670 µL) and treated with NaHBEt3 in toluene (1 M, 25 µL) then stirred for 5 minutes. The substrate solution (100 µL) was combined with the active catalyst solution (200 µL, 3.5 μmol) in a tritiation vessel and secured with a portable Swagelok® valve. The valve was attached to the Trisorber and subjected to two freeze -pump-thaw cycles before 102 mm tritium gas was introduced. The reaction was thawed, then placed in an oil bath at 45 °C and stirred overnight. After capture of spent tritium on the waste bed, the reaction was transferred into a vial with 10 mL saturated aqueous sodium bicarbonate. The mixture was extracted three times with dichloromethane. The combined organic layers were dried with sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 120 mCi; RCP: 67%. The material was purified by HPLC. The collected fractions were diluted with an equal volume of water, concentrated on a pair of C18 cartridges and eluted with EtOH. Yield: approx.20 mL ethanol soln @ 3.18 mCi / mL. The Specific Activity was determined to be 44.9 Ci / mmol by mass spectrometry; MW for C23H17T7N9O [M+H]+: 456.3, found: 456.0. HPLC Analytic Conditions Method: 10-95%B in 12 minutes, hold 3 min, 6 min re-equilibration Column: Gemini NX C18, 4.6 x 50 mm, 3.5 mm @ 40 °C Flow Rate: 1 mL / min Injection Volume: 1.0 μL Detection: UV @ 294 nm Mobile Phase A: 0.05 M pH 10 TEAA in H2OMobile Phase B: CH3CN Product Elution Time: 6.32 minutes HPLC Preparatory Separation Conditions Method: Isocratic (A:B = 65:35) Column: Gemini NX C18, 10 x 250 mm @ 40 °C 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 Assay Protocols Acquisition of Human Post-Morten Tissue Samples for In Vitro Binding Assays

[0196] Frozen human brain tissues from Parkinson’s Disease (PD) patients were purchased from Analytic Biological Services Inc. The samples were postmortem tissues from donors with clinical diagnosis of late stage of PD. Alpha-synuclein, tau and amyloid burden was determined through a combination of immunohistochemistry on frozen thin coronal sections, as well as alpha Lisa-based quantification of protein levels in a detergent insoluble protein fraction. A tissue sample of temporal cortex was identified from one donor as having moderate to high alpha- synuclein burden, low amyloid and minimal to no tau pathology. The detergent insoluble fraction of temporal cortex from this patient was used to support homogenate binding studies. Preparation Detergent Insoluble Fraction of Human Brain Tissue for In Vitro Binding Studies

[0197] Grey matter was dissected out of the temporal cortex tissue with a dissecting blade and minced with fine dissecting scissors. To prepare insoluble fractions, minced tissue was homogenized in ice cold TBS-TX buffer (50mM Tris + 150 mM NaCl + 1% Triton X100 + 1mM EDTA + 1 tablet / 10mL of Complete Protease inhibitor + 1 tablet / 10mL PHOSSTOP phosphatase inhibitor tablet) with glass Dounce tissue grinder. Homogenates were centrifuged at 100,000 x g for 45 minutes. The pellet was resuspended in TBS-TX buffer, using a Polytron at highest setting for 30 seconds at 4 °C. Homogenates were centrifuged at 100,000 x g for 45 minutes and the pellet was resuspended in TBS-TX buffer. A BCA protein assay was performed on the final homogenate to determine the protein concentration. Homogenates were aliquoted in 0.5 ml / tube and stored at -70 °C until use.Procedures for Alpha-Synuclein Tissue Homogenate Binding Assays (Assay 1)

[0198] For displacement α-synuclein binding assay, compounds and control were solvated in dimethyl sulfoxide (DMSO) and transferred using focused acoustic energy by an Echo 655 liquid handling instrument (Beckman Coulter, Indianapolis, IN) into designated wells of uniquely bar coded 96-well v-bottom low binding polypropylene microplates (Thermo Scientific, 249946). Compound dose response curves were prepared in a 10-point, 3-fold fashion within columns 2 - 11 of the microplate from high to low compound concentrations. The final assay concentration of dose response curves when starting at 1 mM ranged from 1.2 µM to 0.061 nM (0.12 % DMSO final assay concentration, 270 nL / well). Controls included no inhibitor (DMSO only) dispensed into wells A1 – D1, A12 – D12 for minimum efficacy signal and Compound 91 at a final assay concentration of 12 µM into wells E1 – H1, E12 – H12 for maximum efficacy signal. Liquid- handling steps for dispensing insoluble fractions of PD brain homogenates and radioligand were performed using a Bravo automated liquid handling platform equipped with a 96LT disposable tip head (Agilent Technologies, Santa Clara, CA). Insoluble fractions of PD brain homogenates were diluted to 50 µg / mL in the Assay Buffer, and 200 µL was dispensed to the assay plate for a final concentration of 10 µg / well.25 µL of (9X) [3H]-91 was dispensed to the assay plate for final assay concentration of 3.0 nM. Sealed assay plates were incubated at room temperature for 90 minutes with gentle agitation. The incubation was terminated by rapid filtration through UniFilter-96 GF / C microplates (pre-treated for 30 minutes with 0.2% Polyethylenimine at 4oC) by using a FilterMate Harvester (PerkinElmer). The microplates were subsequently washed four times with a total volume of 3.75 mL using ice-cold Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco 14190136 ) before drying 90 minutes at 47oC with a vacuum oven (Fisher Scientific Isotemp 285A) or overnight at room temperature. The bottom of each UniFilter-96 GF / C microplate was adhesively sealed (PerkinElmer 6005199) prior to the addition of 50 μL MicroScint-20 liquid scintillation cocktail (PerkinElmer 6013621) to each well. A clear adhesive seal (TopSeal-A PLUS, PerkinElmer 6050185) was then applied to the top of each microplate and counted 1 minute / well on MicroBeta2system (PerkinElmer, Model: 2450-0120). Data was analyzed using IDBS ActivityBase XE Runner (version 9.6.0.148) to determine Kivalues shown in Data Table 1 (Kd value 0.90 nM, ligand concentration 3.0 nM). Competitive radioligand binding to pathological aggregated beta amyloid in AD tissue (Assay 2):

[0199] Frozen human brain samples of Alzheimer’s disease (AD) were purchased from Analytic Biological Services Inc. The samples were postmortem tissue from donors with clinicaldiagnosis of AD and much of the white matter was dissected out of the frontal cortex in order to enrich the tissue preparations for gray matter. Brain homogenates of gray matter enriched frontal cortex were prepared by homogenizing the tissue in ice cold Phosphate Buffered Saline (PBS), pH 7.4 at 80 mg wet weight tissue per 1 ml for 45 seconds at 4oC on setting 16 of Polytron. The homogenate was further diluted with ice cold PBS to 30 mg wet weight tissue per 1 ml and homogenized for an additional minute as described above. Homogenates were aliquoted in 5 ml / tube and stored at -70oC until use.

[0200] Radioligand [3H]-105, prepared as described in ACS Med. Chem. Lett., Vol.2, pages 498-502, was used in this assay.

[0201] For hot saturation binding assay, various concentrations of radioligand, [3H]-105 were prepared in Assay Buffer (PBS plus 0.1% BSA) plus 20% DMSO ranging from 3.9 to 500 nM. 25 μl of radioligand was added to 200 μl of crude brain homogenates (diluted to 0.5 mg / ml in Assay Buffer) for final concentration of radioligand ranging from 0.39 to 50 nM and final crude brain homogenates of 100 μg wet weight / assay well (incubation, filtration, and determination of amount of radioligand used in assay are described below). Self-block with unlabeled compound was used to determine non-specific binding. Saturation data was analyzed using Graphpad / Prism software.

[0202] FIG.1 depicts high affinity saturation binding of [3H]-105 to AD cortical tissue homogenate enriched in aggregated beta-amyloid pathology. FIG.1 shows an example of hot saturation binding of [3H]-105, where the radioligand shows high affinity for aggregated beta amyloid (abeta) in AD brain homogenates with measured dissociation constant of 11 nM. This data supports the use of this ligand in radioligand binding assays to screen for binding to aggregated beta-amyloid.

[0203] For Assay 2 unlabeled test compounds were dissolved in DMSO at 10 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000x final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg / ml from original 30 mg / ml volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight / assay well. [3H]-105 was prepared at 10x final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 3.0 nM. The plates were incubated at 37oC for 90 minutes.Unbound and bound ligand were separated by filtration of bound onto GF / B filter plates (pre- treated for 30 min with 0.1% PEI) using a Packard Filtermate and washing away unbound with 2.5 ml ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hr at 57°C and 50 μl Microscint was added to each well of the plate. The plates were counted for 3H cpm for 1 min per well using PerkinElmer TopCount. Data was analyzed using IDBS Activity Base to determine Kivalues shown below in Data Table 1 (Kdvalue 11.0 nM, ligand concentration 3.5 nM). Radioligand Binding Data in triton-extracted alpha-synuclein from PD Tissue (Assay 1) and Aβ- rich AD Tissue (Assay 2) – Data Table 1 (ND = Not Determined)In vitro binding of [18F]-alpha synuclein tracers in human PD brain tissue homogenates

[0204] The frozen human brain samples of Parkinson's disease (PD) were provided by Banner Sun Health Institute (USA) through collaboration with Michael J Fox Foundation (MJFF). They are postmortem tissue from donors with clinical diagnosis of PD and neuropathological validation of PD pathology. Brain homogenates of cerebral cortex were prepared by homogenizing the cortex in ice cold Phosphate Buffered Saline (PBS), pH 7.4, for 30 seconds at 4°C on setting 6 of Polytron. The final concentration of brain homogenates was 30mg wet tissue per 1mL buffer. Homogenates were aliquoted in 1mL / tube and stored at -70°C prior to use. Brain homogenates of brainstem were prepared by homogenizing the brainstem in ice cold 30mM TRIS, pH 7.5, for 30 seconds at 4°C on setting 6 of Polytron. The final concentration ofbrain homogenates was 30mg wet tissue per 1mL buffer. Homogenates were aliquoted in 1mL / tube and stored at -70°C prior to use.

[0205] [18F]-8, and [18F]-13 were synthesized as described below. [18F]-116 was synthesized as described below. For hot saturation binding assay, 12 concentrations of radioligand were used, ranging from 30nM to 0.3 nM. Brain homogenates (samples identified below) were diluted from original 30 mg / mL volume to final concentration of 2.5 mg / mL with assay buffer (Tris, pH 7.5, 0.1% BSA), and 250 μl per assay tube was used in assay. Unlabeled test compounds were dissolved in DMSO at 10mM. Total binding was defined in the absence of competing compound, and non-displaceable binding was determined in the presence of 1µM unlabeled self block. DMSO or unlabeled compound were added into the assay tube (25µL each / per tube, separately) containing 200µL brain homogenate dilution, and pre-incubate the tubes at room temperature for 30 minutes, then radioligand dilutions (10X) were added into the assay tube (25µL each / per tube, separately) to a final volume of 250µL per tube. Incubation was carried out at 37ºC for 60 minutes, and then the assay samples were filtered onto GF / C filters using Skatron 12 well harvester, washing with8 ml ice cold buffer (Tris, pH 7.5). GF / C filter papers for Skatron harvester were pre-soaked in 0.1% BSA for 30 min at 4C before use. Filters were punched into scintillation vials. Liquid scintillation fluid (2mL Ultima Gold) was added into each vial, allowing to soak into filters for 4 hours and counted on Perkin Elmer Tri-Carb 2900TR for 1 minute. The data analysis was done with Prism software. All assays were done in duplicate in the laboratory designated for studies using human tissues. Data Table 2- In Vitro Binding data for [18F] ligands in human tissue homogenatesRadiochemical Synthesis of [18F]-LigandsHPLC Preparatory Separation Conditions Flow Rate: 5 ml / min Column 1: Gemini C6-Phenylhexyl 5μ 250x10mm (Phenomonex) Mobile Phase A: CH3CNMobile Phase B: Na2HPO410 mM HPLC Analytical Separation Conditions Flow Rate: 1.5 ml / min Column 2: Onyx Monolithic C18  100x3.0 mm (Phenomenex) Column 3: Poroshell 120 EC-C18100x4.6mm 5µ (Agilent) Mobile Phase A: CH3CN Mobile Phase C: 0.1% HCOOH Mobile Phase D: 10 mM NH4OAc pH 8.0 Synthesis of [18F]-8

[0206] The [18F]fluoride containing anion exchange resin was eluted with tetraethylammonium bicarbonate (5.5 mg, 29 mmol) in CH3CN / H2O 1:1 (1.0 mL), followed by CH3CN (0.5 mL) into a vented 2.5 mL v-shaped vial and dried under argon flow using conventional heating at 100°C. Additional aliquots of CH3CN (2 x 0.5 mL) were added for azeotropic drying.8-8 (1.6 mg, 2.6 mmol) in DMF (0.5 mL) was added the dried [18F]Et4NF. The reaction mixture was heated at 120°C for 15 min followed by transfer to a vial containing 20% CH3CN / 10 mM Na2HPO4pH 7.4 (0.8 mL) at room temperature for dilution, mixing and injection onto a semi-prep HPLC column. The product was purified using a C6-PhenylHexyl, 5 mm, 250x10mm HPLC column (Phenomonex) with a flowrate of 5 ml / min and a mobile phase of 42% CH3CN / 10 mM Na2HPO4pH 7.4. The radioactive fraction that eluted between 21.1 and 22.4 min was collected into a round bottom flask containing sodium ascorbate (10 mg), evaporated under negative pressure followed by transfer to a 10 mL sterile vial. The final product was tested for chemical and radiochemical purity by means of an analytical HPLC system (Agilent) using a Poroshell 120, 4 mm EC-C18100x4.6mm HPLC column (Agilent) with a flowrate of 1.5 ml / min and a mobile phase of CH3CN / 10 mM NH4OAc pH 8.0 at a gradient of 40- 45%. Concentration of [18F]-8 was determined by means of an ultraviolet detector (254 nm). Confirmation of the identity of the product was determined by coinjection of a sample of compound [18F]-8, andradiochemical purity was determined using a sodium iodide detector (Bioscan). The retention time for compound [18F]-8 was 5.20 min.  Data Table 3 – Purification and Analysis conditions for synthesis of18F ligands.Synthesis of [18F]-116

[0207] [18F]Fluoride was transported to the radiochemistry lab on an anion exchange resin which was pretreated by flushing with EtOH (10 mL) followed by 0.5M K3PO4 in H2O (10 mL) and H2O (10 mL) before use.

[0208] The [18F]fluoride containing anion exchange resin was eluted with tetrabutylammonium mesylate (6.8 mg, 20 mmol) in CH3CN / H2O 1:1 (1.0 mL), followed by CH3CN (0.5 mL) into a vented 2.5 mL v-shaped vial and dried under argon flow using conventional heating at 100°C. Additional aliquots of CH3CN (2 x 0.5 mL) were added for azeotropic drying. The vial containing dried [18F]Bu4NF was heated to 120°C after which a solution of PP-3 (0.9 mg, 1.5 mmol) in DMSO / iso-amyl alcohol 1:1 (0.5 mL) was added. The reaction mixture was heated at 120°C for 10 min followed by transfer to a vial containing H2O (1.0 mL) at room temperature for dilution, mixing and injection onto a semi-prep HPLC column. The product was purified using a Zorbax XDB-C18, 5 mm, 150x9.4mm HPLC column (Agilent) with a flowrate of 5 ml / min and a mobile phase of 30% CH3CN / 10 mM Na2HPO4in H2O pH 7.4. The radioactive fraction that eluted between 21.5 and 22.1 min was collected into 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 by means of an analytical HPLC system (Agilent) using a Poroshell 120, 4 mm EC-C18100x4.6mm HPLC column (Agilent) with a flowrate of 1.5 ml / min and a mobile phase of CH3CN / 10 mM NH4OAc pH 8.0 at a gradient of 5 – 95%. Concentration of [18F]-116 was determined by means of an ultraviolet detector (254 nm). Confirmation of the identity of the product was determined by coinjection of a sample of compound 116, and radiochemical purity was determined using a sodium iodide detector (Bioscan). The retention time for compound [18F]-116 was 6.6 min. [18F]-8 Imaging in rhesus monkey

[0209] A fasted rhesus monkey (7-11 kg) was anesthetized with ketamine I.M. (15 mpk) and the monkey was placed in the PET camera bed. An I.V. catheter was inserted into the rightsaphenous vein. For arterial sampling, the right femoral area was aseptically prepared and an arterial catheter was placed and fixed with sutures.

[0210] Subsequent anesthesia was maintained with Propofol. Induction dose was 5 mg / kg I.V., followed by an infusion at 0.4-0.6mg / kg / min for the duration of the scanning procedure. The animal was intubated and positioned inside the camera gantry supine, head first. Animal was maintained on ventilated medical grade air:oxygen gas mixture at approximately 23 respirations per minute for the duration of the study. Ventilation I:E ratio, volume and rate of respiration was adjusted to maintain CO2levels about 40 mmHg and SpO2levels 95 to 100%. A temperature probe, pulse oximeter, non-invasive blood pressure cuff, and end tidal CO2 monitor were connected. Body temperature was maintained by placing K-module heating pads on dorsal and ventral sides of animal. General fluid therapy was maintained with 10ml / kg / hr Lactated Ringer’s, IV throughout scanning procedure. Another line was placed in lower saphenous artery for sampling and connected to an Instech automated blood sampling system. An aliquot of [18F]- 8 was injected IV over 2 min via a syringe pump, with positron emission imaging beginning at the time of injection and continuing for 90 minutes.

[0211] Whole blood samples were collected via arterial catheter into Heparin tubes for determination of radioactivity in whole blood and plasma. Samples were centrifuged and 20 μl whole blood and plasma were counted 20, 40, 80, 100, 120, and 150 seconds post PET ligand injection. Samples of blood (0.8 ml) were taken for metabolite correction and determination of radioactivity in plasma and whole blood at 3, 5, 15, 30, 60, and 90 minutes.

[0212] FIG.2 shows a sagittal slice of a PET image of [18F]-8 in rhesus monkey brain. The image is averaged over 30-90 minutes post injection and overlaid on a brain MRI template. This demonstrates suitable distribution of [18F]-8 in monkey brain.

[0213] FIG.3 shows a sagittal slice of a PET image of [18F]-13 in rhesus monkey brain. The image is averaged over 30-90 minutes post injection and overlaid on a brain MRI template. This demonstrates suitable distribution of [18F]-13 in monkey brain.

Claims

1. WHAT IS CLAIMED IS:

1. A compound of Formula I:or a pharmaceutically acceptable salt thereof wherein; R is independently selected from H, –C1-6alkyl, or halo, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORcor halo; Rais selected from unsubstituted or substituted –C1-6alkyl, said alkyl optionally substituted with 1 to 3 groups of R; Rbis selected from a) halo, b) OH,Rcis independently selected from H or –C1-6alkyl, where said alkyl is optionally substituted with one to three groups from –C1-6alkyl, ORdor halo; Rdis independently selected from H or –C1-6alkyl; X is CH or N; and Ring A is selected from pyrazinyl or pyrimidinyl.

2. The compound of Claim 1, or a pharmaceutically acceptable salt thereof, wherein; R is halo, Rais–C1-6alkyl, and X is CH.

3. The compound of Claim 3, or a pharmaceutically acceptable salt thereof, wherein; R is halo, Rais–C1-6alkyl, and X is N.

4. The compound of any one of Claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein Rbis halo.

5. The compound of any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Rbis F.

6. A compound selected fromor a pharmaceutically acceptable salt thereof.

7. The compound of Claim 6 selected fromor a pharmaceutically acceptable salt thereof.

8. The compound of any one of Claims 1-7, or a pharmaceutically acceptable saltthereof, which is labeled with an isotope selected from2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36Cl,82Br,76Br,77Br,123I,124I or131I.

9. The compound of any one of Claims 1-7, or a pharmaceutically acceptable saltthereof, which is labeled with an isotope selected from3H,11C or18<sub>F.

10. A pharmaceutical composition comprising a compound of any one of Claims 1-9, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

11. A method of imaging alpha-synuclein deposits in a human patient, using a compound of any one of Claims 1-9, or a pharmaceutically acceptable salt thereof, as the imaging agent, comprising the following steps: a) placing a human patient in a supine position in the PET camera;b) administering about 0.1 to about 10 mCi of a compound of Claim 1 to the patient; and c) performing an emission scan of the cerebral region of the patient’s head to identify aggregations of alpha-synuclein in the brain tissue of the patient.

12. A method of measuring the clinical efficacy of therapeutic agents for Parkinson’s disease comprising the steps of a) administering an isotopically-labeled compound of any one of Claims 1-9, to the patient diagnosed with PD before treatment with said therapeutic agent, b) measuring the amount of alpha-synuclein aggregate formation in the patient’s brain tissue, c) administering an isotopically-labeled compound of any one of Claims 1-9, to the patient after treatment with said therapeutic agent, d) measuring the amount of alpha-synuclein aggregate formation in the patient’s brain tissue after treatment, and e) analyzing whether said therapeutic agent stopped or decreased the progression of alpha-synuclein aggregate formation in the patient’s brain tissue.

13. A compound of any one of Claims 1-9 for use as an imaging agent.

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