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  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0459—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two nitrogen atoms as the only ring hetero atoms, e.g. piperazine
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  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
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  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/08—Bridged systems

Definitions

• ALPHA-SYNUCLEIN BINDERS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit or priority to U.S. Provisional Application No. 63/485,159, filed February 15, 2023, the disclosure of which is incorporated herein by 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.
• the disease arises because a specific protein is no longer functional when adopting a misfolded state.
• the pathological state originates because misfolding occurs concomitantly with aggregation, and the underlying aggregates are detrimental.
• 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.
• Parkinson's Disease PD
• Dementia with Lewy Bodies DLB
• MSA multiple system atrophy
• 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).
• LB Lewy bodies
• LN Lewy neurites
• Alpha-synuclein is a presynaptic terminal protein that consists of a140-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 are microscopic 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.
• LB Lewy bodies
• LN Lewy neurites
• 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.
• 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.
• 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.
• an alpha-synuclein PET tracer could be useful for patient selection for PD clinical trials.
• an alpha-synuclein tracer could be developed as a companion diagnostic for co- registration of a therapeutic agent.
• 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.
• 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.
• the invention 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, –C 1-6 alkyl, OR c or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; R b is independently selected from –C 1-6 alkyl, halo, -(CH 2 ) n OR c , -CN, -NRc 2, -(CH 2 ) n halogen, or -O(CH 2 ) n halo; R c is independently selected from H or –C 1-6 alkyl, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR d or halo; R d is independently selected from H or
• 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.
• 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).
• MSA multiple system atrophy
• DLB dementia with Lewy Bodies
• FIG.1 Saturation binding experiment in aggregated beta-amyloid rich AD tissue homogenate for [ 3 H]-105.
• FIG.2 Saturation binding experiment in triton-insoluble fraction from PD tissue homogenate enriched in aggregated alpha-synuclein for [ 3 H]-1.
• FIG.3 [ 3 H]-1 radioligand saturation binding data in human cortical PD tissue homogenate enriched in aggregated alpha-synuclein pathology.
• FIG.4 [ 3 H]-1 radioligand saturation binding data in human cortical AD tissue homogenate enriched in aggregated A ⁇ .
• FIG.5 [ 3 H]-24 radioligand saturation binding data in human cortical PD tissue homogenate enriched in aggregated alpha-synuclein pathology.
• FIG.6 [ 3 H]-24 radioligand saturation binding data in human cortical AD tissue homogenate enriched in aggregated A ⁇ .
• DETAILED DESCRIPTION OF THE INVENTION [0018] 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.
• the present invention is directed to a compound of Formula I: or a pharmaceutically acceptable salt thereof wherein; R is independently selected from H, –C 1-6 alkyl, OR c or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; R b is independently selected from –C 1-6 alkyl, halo, -(CH 2 ) n OR c , -CN, -NRc 2, -(CH 2 ) n halogen, or -O(CH 2 ) n halo; R c is independently selected from H or –C 1-6 alkyl, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR d or halo; 25002 R
• the present invention is directed to a compound of Formula IA, or a R is independently selected from H, –C 1-6 alkyl or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo;
• R a is independently selected from unsubstituted or substituted –C 1-6 alkyl or -(CH 2 ) 1-3 O-(CH 2 ) 0-3 R, said alkyl optionally substituted with 1 to 3 groups of R;
• R b is independently selected from –C alkyl, hal c c 1 -6 o, -(CH 2 ) n OR , -CN, -NR 2, -(CH 2 ) n halogen, or -O(CH 2 ) n halo;
• R c is independently selected from H or –C 1-6 alkyl, where said alkyl is optionally substituted with one to three groups from –C 1-6
• the present invention is directed to a compound of Formula IA, or a pharmaceutically acceptable salt thereof wherein;
• R is independently selected from H, –C 1-6 alkyl, OR c or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo;
• R a is independently selected from unsubstituted or substituted –C 1-6 alkyl or -(CH 2 ) 1-3 O-(CH 2 ) 0-3 R, said alkyl optionally substituted with 1 to 3 groups of R;
• R b is independently selected from –C alkyl c c 1 -6 , halo, -(CH 2 ) n OR , -CN, -NR 2 , -(CH 2 ) n halogen, or 25002 -O(CH 2 ) n halo;
• R c is independently selected from H or –C 1-6 alkyl, where said alkyl
• the present invention is directed to a compound of Formula IA, or a pharmaceutically acceptable salt thereof wherein; 25002 R is independently selected from H, –C 1-6 alkyl, OR c or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; Ring A 1 is selected from pyridyl, pyrazinyl or pyrimidinyl; Ring A 2 is selected from pyrimidinyl, phenyl, or pyridyl, where said pyrimidinyl, phenyl, or pyridyl is optionally substituted with 1 to 3 groups of R; Ring A 3 is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl,
• the present invention is directed to compounds of Formula IB or a pharmaceutically acceptable salt thereof wherein; R is independently selected from H, –C 1-6 alkyl or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; 25002 R b is independently selected from –C 1-6 alkyl, halo, -(CH 2 ) n OR c , -CN, -(CH 2 ) n halogen, or -O(CH 2 ) n halo; R c is independently selected from H or –C 1-6 alkyl; R 1 is selected from -(CH 2 ) n OR c , -(CH 2 ) n O(CH 2 ) n R, -(CH 2 ) n O
• the present invention is directed to compounds of Formula IC 25002 or a pharmaceutically acceptable salt thereof wherein: R is independently selected from H, –C 1-6 alkyl or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; R b is independently selected from –C 1-6 alkyl, halo, -(CH 2 ) n OR c , -CN, -(CH 2 ) n halogen, or -O(CH 2 ) n halo; R c is independently selected from H or –C 1-6 alkyl; R 1 is independently selected from -(CH 2 ) n OR c , -(CH 2 ) n O(CH 2 ) n R, -(CH 2 )
• the present invention is directed to compounds of Formula IC, or a pharmaceutically acceptable salt thereof wherein: R is independently selected from H, –C 1-6 alkyl or halo, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR c or halo; R a is independently selected from unsubstituted or substituted –C 1-6 alkyl, said alkyl optionally substituted with 1 to 3 groups of R; R b is independently selected from –C 1-6 alkyl, halo, -(CH 2 ) n OR c , -CN, -(CH 2 ) n halogen, or -O(CH 2 ) n halo; R c is independently selected from H or –C 1-6 alkyl; R 1 is selected from -(CH 2 ) n OR c , -(CH 2 ) n O(CH 2 ) n R, -NR 2 ,
• the invention provides a compound of Formula I, IA, IB or IC, wherein Ring A 1 is selected from pyridyl, pyrazinyl, pyrimidinyl, thiazolyl or pyrazolyl. In another embodiment, Ring A 1 is selected from pyridyl, pyrazinyl, pyrazolyl or pyrimidinyl. In another embodiment, Ring A 1 is selected from pyridyl or pyrazinyl.
• the invention provides a compound of Formula I, IA, IB or IC, wherein Ring A 2 is selected from pyrimidinyl, pyridyl or pyrazinyl, where said pyrimidinyl, pyridyl or pyrazinyl is optionally substituted with 1 to 3 groups of R.
• the invention provides a compound of Formula I, IA, IB or IC, wherein Ring A 2 is selected from pyrimidinyl or pyrazinyl, where said pyrimidinyl or pyrazinyl is optionally substituted with 1 to 3 groups of R.
• Ring A 2 is pyrimidinyl, which is optionally substituted with 1 to 3 groups of R.
• Ring A 2 is pyrazinyl, which is optionally substituted with 1 to 3 groups of R.
• the invention provides a compound of Formula I, IA, IB or IC, A 3 is selected from pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, triazolyl or phenyl.
• Ring A 3 is selected from pyridyl, pyrazinyl or phenyl.
• the invention provides a compound of Formula I, IA, IB or IC, wherein R c is independently selected from H or –C 1-6 alkyl, where said alkyl is optionally substituted with one to three groups from –C 1-6 alkyl, OR d or halo.
• the invention provides a compound of Formula I, IA, IB or IC, wherein R c is independently selected from H or –C 1-6 alkyl.
• the invention provides a compound of Formula I, IA, IB or IC, wherein R 1 is selected from -(CH 2 ) n OR c , -(CH 2 ) n O(CH 2 ) n R, -(CH 2 ) n O(CH 2 ) n OR c , halo, - NR2, -C 1-6 alkyl, cyclopropyl, imidazolyl, pyridyl, indolyl, pyrazolyl, triazolyl, azetidinyl, phenyl, azepanyl, pyrrolopyrazinyl, pyrrolidinyl, azabicyclo-heptanyl, furyl, thiazolyl, pyrimidinyl, oxa- azabicycloheptanyl, pyridazinyl, thienyl, isoxazolyl, oxazolyl, di
• the invention provides a compound of Formula I, IA, IB or IC, wherein R 1 is selected from -(CH 2 ) n OR c , -(CH 2 ) n O(CH 2 ) n R, -NR2, pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, furyl, where said pyridyl, pyrazolyl, azetidinyl, pyrrolidinyl, or furyl can be optionally substituted with one to three groups of R b .
• Representative compounds of the present invention include compounds selected from 25002 Ex. Structure Name No.
• the present invention is directed to compound of Formula I for use as an imaging agent.
• An embodiment of the invention comprises a compound selected from Ex.
• a further embodiment of the invention comprises a compound selected from Ex. No.39, 47, 51, 78, 79, 96, 112, 116 and 141 or a pharmaceutically acceptable salt thereof.
• Another embodiment of the invention comprises a compound selected from Ex. No.96, 112, 113, 115, 116, 118, 134, 138 and 141, or a pharmaceutically acceptable salt thereof.
• a further embodiment of the invention comprises a compound selected from Ex. No.96, 112, 113, 115, 116, 118, 134, 138 and 141, or a pharmaceutically acceptable salt thereof.
• a further embodiment of the invention comprises a compound selected from Ex.
• Another aspect of the invention is directed to compounds of Formula I, or a pharmaceutically acceptable salt thereof, that are labeled with an isotope selected from 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 CL, 82 Br, 76 Br, 77 Br, 123 I, 124 I or 131 I.
• the compounds of Formula I are isotopically labeled with 3 H, 11 C or 18 F.
• Examples of isotopically labeled a compound of Formula I, or 25002 pharmaceutically acceptable salts thereof include, but are not limited to, 3 H-1, 3 H-24, 18F- 39, 18F- 47, 18F- 51, 18F- 78, 18F- 79, 11C- 94, 18F- 96, 11C- 97, 18F- 116, 11C- 117, 11C- 118, 18F- 141, and 11C- 143, and the like.
• isotopically labeled a compound of Formula I, or pharmaceutically acceptable salts thereof include, but are not limited to 3 H-1, 3 H-24, 18F- 39, 18F- 47, 18F- 51, 18F- 78, 18F- 79, 18F- 96, 18F- 116, and 18F- 141, and the like.
• isotopically labeled a compound of Formula I, or pharmaceutically acceptable salts thereof include, but are not limited to 11C- 118, and 11C- 143, and the like.
• isotopically labeled a compound of Formula I, or pharmaceutically acceptable salts thereof include, but are not limited to 18F- 96, 18F- 116, and 18F- 141, and the like. Further examples of isotopically labeled a compound of Formula I, or pharmaceutically acceptable salts thereof, include, but are not limited to 18F- 78, 18F- 79, 18F- 96, 18F- 116, and 18F- 141, and the like. Further examples of isotopically labeled a compound of Formula I, or pharmaceutically acceptable salts thereof, include, but are not limited to 18F- 96, 18F- 116, and 18F- 141, and the like.
• Another aspect of the invention is directed to compounds of Formula I, or a pharmaceutically acceptable salt thereof, that are labeled with an isotope selected from 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 CL, 82 Br, 76 Br, 77 Br, 123 I, 124 I or 131 I, for use as an imaging agent.
• 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.
• 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.
• isotopic labels may be detected using imaging techniques, photographic film or scintillation counters.
• the label is detected in vivo in the brain of the subject by imaging techniques, for example positron emission tomography (PET).
• PET positron emission tomography
• 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 25002 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.
• conventional structural representation is employed and includes conventional stereochemical notation for certain asymmetric carbon centers.
• structural representation of compounds of the invention includes conventional stereochemical notation for some asymmetric carbon centers shown in the example compounds.
• 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
• a “wavey” line appended to a carbon bearing a double bond indicates both possible cis and trans orientations
• 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.
• 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 a statement of the absolute determination of the stereochemical structure of said compound unless otherwise noted in the data presented.
• 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.
• 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.
• 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.: secondary-butyl moiety is bonded via the methylene group via the bond terminated with the wavey line.
• a dash is employed to indicate the point of bonding to the indicated substrate, e.g.: -CH 2 - C(O)-CH2Cl indicates the acetyl chloride moiety is bonded via the methylene portion of the moiety.
• 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 25002 administration to a subject).
• the compounds of the present invention are limited to stable compounds embraced by Formula I.
• Halogen or "halo” as used herein means fluoro, chloro, bromo and iodo.
• cycloalkyl is intended to include cyclic saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Preferably, cycloalkyl is C 3 - C 10 cycloalkyl. Examples of such cycloalkyl elements include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic.
• aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
• aryl is phenyl or naphthyl.
• aryl is phenyl.
• heterocyclyl, heterocycle or heterocyclic represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
• the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
• heterocyclyl, heterocycle or heterocyclic can include heteroaryl moieties when two rings are fused together.
• heterocyclic elements include, but are not limited to, azabicyclo[2.2.1]heptanyl, azepanyl, azetidinyl, benzodioxolyl, chromanyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydro-pyrrolo[1,2-b]pyrazolyl, 1,3-dioxolanyl, imidazolidinyl, indolinyl, isochromanyl, isoindolinyl, morpholinyl, oxa-5-azabicyclo[2.2.1]heptanyl, 2-oxopiperazinyl, 2- oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuryl,
• heterocyclyl is selected from azabicyclo[2.2.1]heptanyl, azepanyl, azetidinyl, dihydro-pyrrolo[1,2-b]pyrazolyl, morpholinyl, oxa-5-azabicyclo[2.2.1]heptanyl, piperidyl, piperazinyl, pyrazolidinyl, pyrrolidinyl, pyrrolyl, and tetrahydrofuryl.
• heterocyclyl is selected from azabicyclo[2.2.1]heptanyl, azepanyl, azetidinyl, dihydro-pyrrolo[1,2-b]pyrazolyl, oxa-5-azabicyclo[2.2.1]heptanyl, piperazinyl, and pyrrolidinyl.
• Heteroaryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic and wherein from one to four carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S.
• heterocyclic elements include, but are not limited to, azepinyl, furanyl, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, 5H-pyrrolo[2,3-b]pyrazinyl, pyrrolyl, quinazolinyl, quinolinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiazolyl, thienofuryl, thienothienyl, thienyl, triazolyl and the like.
• heteroaryl is selected from furyl, imidazolyl, indolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, 5H-pyrrolo[2,3- b]pyrazinyl, tetrazolyl, thiazolyl, thienyl, triazolyl and the like.
• 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.
• 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,N 1 -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.
• basic ion exchange resins such as arginine, be
• salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
• 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.
• 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 of the 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).
• PD Parkinson’s Disease
• 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.
• the 3 H, 11 C or 18 F labeled compounds can be used in in vitro and in vivo methods for the determination of binding, receptor occupancy and metabolic studies including covalent labeling.
• Another aspect of the invention relates to the use of the isotopically labeled compounds to screen for new chemical matter.
• 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.
• 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: 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I or 131 I isotopically labeled substituted heterocyclic derivative 25002 compounds of Formula I. It will be appreciated that other isotopes may be incorporated by known means also.
• the present invention is directed to 11 C, 13 C, 14 C, 18 F, 15 O, 13 N, 35 S, 2 H, and 3 H 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.
• the present invention is directed to compounds of Formula I that are isotopically labeled with 3 H, 11 C or 18 F, 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.
• 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.
• 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 number typically found in nature (i.e., naturally occurring).
• Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 76 Br, 77 Br, 123 I, 124 I and 131 I.
• the isotopically labeled compounds of the invention need only to be enriched 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.
• the radionuclides are represented by 11 C, 13 C, 14 C, 18 F, 15 O, 13 N, 35 S, 2 H, and 3 H, preferably 11 C, 3 H, and 18 F.
• 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.
• 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.
• 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, oin
• the pharmaceutical composition of the present invention may be formulated for parenteral administration, such as an intravenous formulation.
• This invention provides radiolabeled compounds of Formula I as alpha-synulcein 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.
• MSA Multiple Systems Atrophy
• DLB Dementia with Lewy Bodies
• the compounds of this invention may also be used in combination with a broad range of cognition deficit enhancement agents.
• 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.
• 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.
• 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.
• a radiopharmaceutical agent such as an isotopically labeled compound of Formula I
• a method for imaging alpha-synuclein deposits in a patient comprises the steps of: a) placing a human patient in a supine position in a PET camera; b) 25002 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.
• 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.
• 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.
• the present invention also provides methods of determining in vivo activity of an enzyme or other molecule.
• 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.
• 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 for Parkinson’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.
• 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, an issue that continues to plague Parkinson’s disease clinical trials.
• 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.
• a diagnostically effective amount of a labeled or unlabeled compound of the invention is administered to a living body, including a human.
• 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.
• PD Parkinson’s Disease
• 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.
• 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).
• MSA Multiple Systems Atrophy
• DLB Dementia with Lewy Bodies
• 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.
• 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 specified amounts.
• 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.
• 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.
• the active compound which is a compound of Formula I
• the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
• the term "administration" and variants thereof 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.
• the present invention also provides a method for the synthesis of compounds useful as intermediates in the preparation of compounds of the invention.
• 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.
• Anal. analytical calc.
• the final product may be further modified, for example, by manipulation of substituents.
• substituents may include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions which are commonly known to those skilled in the art.
• 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 25002 fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way.
• Generic Scheme A R r-[F/Cl/Br] R a R a a A 1.
• Generic Scheme B can be to B-2 via SNAr or Pd-mediated C-N coupling reactions followed by deprotection.
• B-2 can undergo SNAr or Pd- mediated C-N couplings with aryl halides provide intermediates B-3.
• B-3 can be engaged in reduction reactions to provide aniline intermediates B-4 and subsequent amide couplings provide target molecules B-5.
• Generic Scheme C a O X X R R R a O X X R R N X X F H X X N R [0086]
• Intermediates C-1 can engage in SNAr reactions with amines or NH-containing heterocycles to afford target compounds C-2.
• reaction mixture was again purged with Argon for 10 min and stirred at 120°C for 12h.
• the reaction mixture was diluted with water (80 mL) and extracted with EtOAc (3 x 100 mL). Combined organic layer was dried over Na 2 SO 4 and concentrated under reduced pressure to get crude product.
• the crude product was purified by 100-200 mesh silica gel (300 g cartridge), eluted with 20% EtOAc/Pet ether as gradient. Pure fractions concentrated under reduced pressure to afford compound I-2 as a yellow liquid.
• reaction was complete by 25002 LCMS.
• the reaction was diluted with DMF (1.0 mL) and the mixture was purified by RP HPLC (reverse phase column chromatography; MeCN in water, 0.1% NH4OH modifier, Phenomenex C18 Luna column, 100 x 21.2 mm, 5 micron) to afford 63 as a solid after concentration.
• Reaction mixture was filtered on celite bed, washed with ethyl acetate. Filtrate was dried over sodium sulfate and evaporated under reduced pressure and crude compound was purified by Prep-HPLC (method: MOBILE PHASE - 10mM Ammonium Bicarbonate IN H2O: MeCN, COLUMN - LUNA Omega C18 (21.2X250) mm 5um Flow- 18ml/min, GRADIENT METHOD-0/50, 10.2/84, 10.25/100, 12/100, 12.05/50, 16/50) and lyophilized to afford 88 as a yellow solid.
• reaction mixture was diluted with ethyl acetate (60 mL) and water (50 mL). Organic layer was separated and aqueous layer was re extracted with ethyl acetate (2 x 30 mL) and combined organic layer dried over sodium sulphate, filtered and concentrated under reduced pressure and crude was purified by Prep HPLC (conditions: MOBILE PHASE - 10mM Ammonium Bicarbonate IN H2O: MeCN COLUMN - X-BRIDGE C18 (19X250) mm 5u Flow-18ml/min GRADIENT METHOD-0/30, 9/75, 9.05/100, 11/100, 11.05/30, 13.5/30.
• Example 91 (S)-6-(1H-imidazol-1-yl)-N-(2-(2-methyl-4-(pyridin-2-yl)piperazin-1-yl)pyrimidin- 5-yl)nicotinamide A (150 mg, 0.555 mmol), 6-(1H-imidazol-1- yl)nicotinic acid (157 mg, 0.832 mmol) in THF (30 ml) was added TEA (0.193 ml, 1.387 mmol), 1-propanephosphonic anhydride (0.495 ml, 0.832 mmol) at 25 °C and stirred for 16 h at 25 °C.
• Example 114 (R)-6-(4-(fluoromethyl)-1H-pyrazol-1-yl)-N-(2-(2-(methoxymethyl)-4-(pyridin-2- yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide 25002 - - - [0149] To a stirred solution of O-1 (made in an analogous method as int 3-3) (300 mg, 1.231 mmol) in DMF (4 mL) were added K2CO3 (851 mg, 6.15 mmol) and 2-chloro-5-nitropyrimidine (O-2) (236 mg, 1.477 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 2 h under nitrogen atmosphere.
• reaction mixture was stirred at room temperature for 4 h under hydrogen atmosphere. Reaction mixture was diluted with EtOAc (15 mL), filtered through celite pad and washed with EtOAc (2 x 15 mL). Filtrate was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to afford O-4.
• Example 114 (R)-6-(4-(fluoromethyl)-1H-pyrazol-1-yl)-N-(2-(2-(methoxymethyl)-4-(pyridin-2- yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide [0151] To a stirred solution of O-4 (40 mg, 0.133 mmol) in DMF (1 mL) were added HATU (50.6 mg, 0.133 mmol), O-5 (32.4 mg, 0.146 mmol) and DIPEA (0.070 mL, 0.4 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h under nitrogen atmosphere.
• Reaction mixture was degassed and purged with argon gas for 10 min. The reaction mixture was stirred in a microwave at 130 °C for 30 min. Reaction mixture was quenched with water (30 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 (2 x 10 mL) and dried under reduced pressure.
• Example 116 (S)-6-(3-fluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-3-methylpiperazin-1- yl)pyrazin-2-yl)nicotinamide
• Q-6 60 mg, 0.170 mmol
• 4-dioxane 1.5 mL
• Cs2CO3 167 mg, 0.511 mmol
• copper(I) iodide (3.24 mg, 0.017 mmol)
• trans-N, N'- dimethylcyclohexane-1, 2-diamine (1.212 mg, 8.52 ⁇ mol)
• Q-7) made in an analogous manner as X-5) (61.4 mg, 0.170 mmol) at room temperature.
• reaction mixture was stirred in a microwave at 150 °C for 2 h under nitrogen atmosphere. Reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 75 mL). Combined organic layer was washed with brine (2 x 40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 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 further re-purified by achiral prep-purification [Cellulose SC (250X30X5 ⁇ ), MeCN:MeOH (90:10)].
• reaction mixture (2-dicyclohexylphosphino-2', 6'-diisopropoxy-1, 1'- biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate (0.633 g, 0.757 mmol) at room temperature.
• the reaction mixture was stirred at 110 °C for 16 h in a sealed tube.
• 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 Na 2 SO 4 , filtered and concentrated under reduced pressure.
• Reaction mixture was degassed and purged with argon gas for 10 min. The reaction mixture was stirred in a microwave at 150 °C for 2 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 Na 2 SO 4 , filtered and concentrated under reduced pressure.
• Example 118 (S)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(1- methyl-1H-pyrazol-4-yl)nicotinamide [0168] To a stirred solution of S-4 (50 mg, 0.125 mmol) and P-7 (25.3 mg, 0.125 mmol) in 1,4-dioxane (1 mL) were added Cs2CO3 (122 mg, 0.376 mmol), copper(I) iodide (2.385 mg, 0.013 mmol) and trans-N,N'-dimethylcyclohexane-1,2-diamine (0.891 mg, 6.26 ⁇ mol) at room temperature and degassed with argon for 10 min.
• Cs2CO3 122 mg, 0.376 mmol
• copper(I) iodide 2.385 mg, 0.013 mmol
• Example 119 (R)-N-(2-(3-((2-fluoroethoxy)methyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin- 5-yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide [0169] To solution of T-1 (prepared in an analogous manner as R-6) (100 mg, 0.225 mmol) in 1, 4-dioxane (2 mL) were added P-7 (54.6 mg, 0.270 mmol), Cs 2 CO 3 (220 mg, 0.675 mmol), copper (I) iodide (4.29 mg, 0.023 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (3.20 mg, 0.023 mmol) at room temperature.
• reaction mixture was degassed and purged with argon for 10 min. Then this reaction mixture was stirred in microwave at 150 °C for 2 h. 25002 [0170] 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 (2 x 5 mL) and concentrated under reduced pressure.
• Obtained compound was purified by prep-HPLC purification (conditions: Instrument ID ANL-MCL5-PREP-020 Column Name Betasil Phenyl Hexyl (21.2X250)MM, 5 ⁇ Column No# 250*19 Mobile Phase-A 10mM Ammonium BiCarbonate in water Mobile Phase-B Acetonitrile Gradient program (T/%B) 0/35, 2/35, 10/55, 11.63/55, 11.65/100, 15/100, 15.01/35, 18/35). Pure fractions were combined and concentrated under reduced pressure to afford 119. M/Z (ESI): 519.31 [M+H] + .
• Example 120 (R)-N-(2-(4-(6-fluoropyrimidin-4-yl)-3-(methoxymethyl)piperazin-1-yl)pyrimidin- 5-yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide N F N [0171] To added U-1 (prepared in an analogous manner as Q-6) (56.4 mg, 0.279 mmol), Cs2CO3 (227 mg, 0.697 mmol), and trans-N,N'-dimethylcyclohexane-1,2-diamine (3.31 mg, 0.023 mmol) at room temperature. The reaction mixture was degassed and purged with argon for 10 min.
• reaction mixture was stirred in microwave at 150 °C for 2 h. Reaction mixture was quenched with water (30 mL) and extracted with DCM (3 x 30 mL). Combined organic layer was washed with brine 25002 (20 mL), dried over anhydrous Na 2 SO 4, filtered and concentrated under reduced pressure. Crude compound was triturated with diethyl ether (2 x 10 mL) and concentrated 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, 5 ⁇ Flow-14.0 ml/min Gradient Method :- 0/35,2/35,8.60/45,8.65/100,11.65/100,11.70/35,15.0/35). Pure fractions were combined, concentrated under reduced pressure and lyophilized to afford 120. M/Z (ESI): 505.25 [M+H] + .
• Example 121 (R)-N-(4-fluoro-2-(3-(methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1- yl)pyrimidin-5-yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide [0172] To a stirred solution of V-1 (made in an analogous manner as 3-2) (2 g, 6.49 mmol) in DCM (40 mL) was added HCl in 1, 4-dioxane (3.08 mL, 25.9 mmol) at 0 °C. The reaction 25002 mixture was stirred at room temperature for 12 h. Reaction mixture was concentrated and dried under reduced pressure to afford V-2.
• reaction mixture was stirred at 80 °C for 12 h. Reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 100 mL). Combined organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. Crude compound was purified by 100 g silica gel (100-200 mesh) column and compound eluted with 20% EtOAc in petroleum ether. Pure fractions were combined and concentrated under reduced pressure to afford V-5. M/Z (ESI): 431.00 [M+H] + .
• Example 122 (S)-N-(5-(4-(5-fluoropyrimidin-2-yl)-3-methylpiperazin-1-yl)pyrazin-2-yl)-6- (pyrrolidin-1-yl)nicotinamide [0181] To a stirred solution of X-4 (60 mg, 0.170 mmol) and X-5 (40 mg, 0.204 mmol) in 1,4- dioxane (1 mL) were added Cs2CO3 (166 mg, 0.510 mmol), copper(I) iodide (3.24 mg, 0.017 mmol) and trans-N,N'-dimethylcyclohexane-1,2-diamine (2.416 mg, 0.017 mmol) at room temperature.
• Cs2CO3 166 mg, 0.510 mmol
• copper(I) iodide 3.24 mg, 0.017 mmol
• Example 124 (R)-N-(5-(3-((2-fluoroethoxy)methyl)-4-(5-fluoropyridin-2-yl)piperazin-1- yl)pyrazin-2-yl)-6-(1-methyl-1H-pyrazol-4-yl)nicotinamide 25002 a mg, was BBr 3 (1.570 mL, 1.570 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. Reaction mixture was concentrated and quenched with ice cold water (10 mL) and extracted with EtOAc (2 x 10 mL).
• Reaction mixture was degassed and purged with argon gas for 15 min. Then to this reaction mixture were added Tris(dibezylideneacetone)dipalladium (1.14 g, 1.25 mmol) and 4,5-bis(diphenylphosphino)-9,9- dimethyl xanthene (1.44 g, 2.5 mmol) at room temperature. The reaction mixture was stirred at 110 °C for 12 h under nitrogen atmosphere in a sealed tube. Reaction mixture was quenched with water (50 mL) and extracted with EtOAc (2 x 75 mL). Combined organic layer was washed with brine (2 x 30 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced 25002 pressure.
• Example 126 (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(6-fluoropyridin-3-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide
• BB-5 150 mg, 426 ⁇ mol
• BB-6 made in an analogous manner as X-5)
• CuI 8.11 mg, 42.6 ⁇ mol
• trans-N,N'-bismethyl-1,2-cyclohexanediamine (6.72 ⁇ L, 21.3 ⁇ mol
• Cs 2 CO 3 (416 mg, 1.28 mmol) at room temperature.
• Example 127 (S)-N-(2-(4-(6-fluoropyridin-3-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(1- methyl-1H-pyrazol-4-yl)nicotinamide [0191] To a stirred solution of CC-1 (prepared in an as BB-5) (150 mg, 376 ⁇ mol) and P-7 (76 mg, 376 ⁇ mol) in 1,4-dioxane (2 mL) were added cesium carbonate (367.0 mg, 1128 ⁇ mol), CuI (7.16 mg, 37.6 ⁇ mol) and trans-N,N'-bismethyl-1,2-cyclohexanediamine (5.93 ⁇ L, 18.8 ⁇ mol) at room temperature.
• reaction mixture was stirred at 110 °C for 40 h in a sealed tube. Reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 25 mL). Combined organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
• Example 128 (R)-N-(5-(4-(6-fluoropyridin-3-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(1- methyl-1H-pyrazol-4-yl)nicotinamide [0192] To a stirred solution of DD-1 (prepared in an analogous manner as 5) (100 mg, 284 ⁇ mol) in 1, 4-dioxane (1.5 mL) were added Cs 2 CO 3 (278 mg, 852 ⁇ mol), CuI (5.41 mg, 28.4 ⁇ mol), P-7 (68.9 mg, 341 ⁇ mol) and trans-N,N'-bismethyl-1,2-cyclohexanediamine (4.48 ⁇ L, 14.2 ⁇ mol) at room temperature.
• reaction mixture was stirred at 110 °C for 16 h under nitrogen atmosphere in a sealed tube. Reaction mixture was quenched with water (45 mL) and extracted with EtOAc (2 x 85 mL). Combined organic layer was washed with brine (2 x 45 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
• Example 129 (R)-N-(2-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)-6-(1- methyl-1H-pyrazol-4-yl)nicotinamide
• EE-1 prepared in an analogous manner (100 mg, 250 ⁇ mol) and P-7 (60.8 mg, 301 ⁇ mol) in 1, 4-dioxane (2 mL) were added trans-N,N'-bismethyl- 1,2-cyclohexanediamine (3.95 ⁇ L, 12.5 ⁇ mol), Cs 2 CO 3 (245 mg, 751 ⁇ mol) and CuI (4.77 mg, 25.0 ⁇ mol) at room temperature.
• reaction mixture was stirred at 120 °C for 16 h under nitrogen atmosphere. Reaction mixture was quenched with aqueous saturated Na 2 CO 3 (20 mL) and extracted with 10% MeOH in DCM (2 x 35 mL). Combined organic layer was washed with brine (2 x 20 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
• Example 130 (S)-6-(3-(fluoromethyl)azetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide 5-yl)-6- (3-(hydroxymethyl)azetidin-1-yl)nicotinamide [0194] To a stirred solution of S-4 (215 mg, 0.501 mmol) and FF-1 (prepared in an analogous manner as X-5) (104 mg, 0.501 mmol) in 1,4-dioxane (3 mL) were added Cs 2 CO 3 (490 mg, 1.503 mmol), copper(I) iodide (9.54 mg, 0.050 mmol), trans-N,N'-dimethylcyclohexane-1,2- diamine (3.56 mg, 0.025 mmol) at room temperature and degassed with argon for 10 min.
• reaction mixture was stirred at 150 °C for 2 h under microwave irradiation.
• Reaction mixture was 25002 quenched with water (10 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 silica column and compound eluted with 10% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford FF-2.
• Example 130 (S)-6-(3-(fluoromethyl)azetidin-1-yl)-N-(2-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide [0195] To a stirred solution of FF-2 (70 mg, 0.146 mmol) in DCM (1 mL) was added DAST (0.039 mL, 0.293 mmol) at 0 °C. The reaction mixture was stirred under argon atmosphere at 0 °C for 30 min. Reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 20 mL).
• Example 135 6-(4-aminophenyl)-2-fluoro-N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5- yl)nicotinamide yl)pyrimidin-5-yl)nicotinamide (GG-6, 15.1 mg, 1 Eq, 0.032 mmol) in 1,4-dioxane (0.50 mL) was added 1M potassium phosphate, tribasic (0.064 mL, 1.00 molar, 2 Eq, 0.064 mmol) and 5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (10.5 mg, 1.15 Eq, 0.048 mmol).
• XPhos-Pd-G2 (680 mg, 864 ⁇ mol) was added and the mixture was stirred for 2.5 hours at 100 °C. The mixture was cooled and poured onto water (100 mL) and extracted with EtOAc (3 x 30 mL) and DCM (3 x 20 mL). The aqueous layer was acidified with 1M HCl and the resulting precipitated solid was collected by filtration, washed with water (10 mL x 2) and dried to give HH-1.
• reaction mixture was purified by reverse phase HPLC (40 to 100% MeCN/H2O w/ 0.1% NH4OH gradient on an XBridge Prep OBD C18 column). The desired fractions were concentrated to yield 136. MS (ESI) m/z: 519.2 [M+H] + .
• Example 137 (S)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-2-fluoro-N-(2-(4-(6- fluoropyrimidin-4-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide fluoropyrimidin-4-yl)-2-methylpiperazin-1-yl)pyrimidin-5-yl)nicotinamide [0209] To a solution of HH-4 (20.0 mg, 0.0473 mmol) in DMF (0.473 mL) was added 4,6- difluoropyrimidine (8.24 g, 0.0710 mmol) and DIPEA (0.0412 mL, 0.237 mmol).
• Example 138 (R)-6-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-2-fluoro-N-(2-(3- (methoxymethyl)-4-(pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-yl)nicotinamide 25002 [0210] To a solution of tert-butyl (R)-3-(methoxymethyl)piperazine-1-carboxylate (II-1, 1.0 g, 1 Eq, 4.34 mmol) in DMF (15 mL) was added diisopropylethylamine (1.68 g, 2.24 mL, 3 Eq, 13.0 mmol) followed by 2-chloropyrimidine (746 mg, 1.5 Eq, 6.51 mmol).
• the mixture was stirred for 1 h at 22°C.
• the crude mixture was purified using basic reverse-phase chromatography (Waters XBridge Prep C185mm - 30x250mm column, 10-100% 5 mM NH 4 HCO 3 water solution: acetonitrile, 26 min gradient). Fractions containing the product were combined and extracted between water (80 mL) 25002 and DCM (80 mL x 3). The collected organic layer was dried over MgSO 4 and then concentrated under vacuum to yield II-6.
• JJ-2 (S)-6-bromo-N-(6-(4-(6-fluoropyrimidin-4-yl)-2-methylpiperazin-1-yl)pyridin- 3-yl)nicotinamide
• 6- bromonicotinic acid 525 mg, 2.60 mmol
• HATU 1319 mg, 3.47 mmol
• DIPEA DIPEA
• Example 139 (S)-6-(3-fluoroazetidin-1-yl)-N-(6-(4-(6-fluoropyrimidin-4-yl)-2-methylpiperazin- 1-yl)pyridin-3-yl)nicotinamide [0217] To a stirred solution of JJ-2 (90 mg, 0.210 mmol) in toluene (3 ml) was added 3- fluoroazetidine hydrochloride (25.8 mg, 0.231 mmol), Cs2CO3 (93 mg, 0.284 mmol), 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (12.17 mg, 0.021 mmol) at room temperature and purged with argon for 10 min, followed by the addition of Pd2(dba)3 (9.63 mg, 10.52 ⁇ mol) at room temperature and again purged with argon for another 10 min and stirred for 16 h at 110 °C
• Example 140 (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(6-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide [0218] To a stirred mL) were added DIPEA (0.18 mL, 1 mmol) and 2, 6-difluoropyridine (23 mg, 200 ⁇ mol) 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 (5 mL), precipitated solid was filtered and dried under reduced pressure.
• Example 141 (S)-N-(5-(4-(5-fluoropyridin-2-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)-6-(1- methyl-1H-pyrazol-4-yl)nicotinamide
• KK-2 tert-butyl (S)-4-(5-fluoropyridin-2-yl)-2-methylpiperazine-1-carboxylate
• Example 142 (S)-6-(3,3-difluoroazetidin-1-yl)-N-(5-(4-(5-fluoropyridin-2-yl)-2- methylpiperazin-1-yl)pyrazin-2-yl)nicotinamide yl)pyrazin-2-yl)nicotinamide (KK-6, 20.00 mg, 1 Eq, 46.74 ⁇ mol) in DMF (0.50 mL) was added 25002 potassium carbonate (19.38 mg, 3 Eq, 140.2 ⁇ mol), potassium fluoride (8.147 mg, 3.284 ⁇ L, 3 Eq, 140.2 ⁇ mol)and 3,3-difluoroazetidine (6.526 mg, 1.5 Eq, 70.11 ⁇ mol).
• 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 LL-6.
• NN-3 tert-butyl (S)-4-(5-(6-(3,3-difluoroazetidin-1-yl)nicotinamido)pyrazin-2-yl)-3- methylpiperazine-1-carboxylate 25002 [0234] To a stirred solution of NN-1 (1.50 g, 4.20 mmol) in 1,4-dioxane (30 mL) were added NN-2 (985 mg, 4.62 mmol), Cs2CO3 (4.10 g, 12.6 mmol), CuI (80.0 mg, 420 ⁇ mol) and trans- (1r,2r)-N,N'-bismethyl-1,2-cyclohexanediamine (66.2 ⁇ L, 210 ⁇ mol) at room temperature and degassed with argon for 10 min.
• reaction mixture was stirred at 150 °C for 2 h under microwave irradiation. Reaction mixture was quenched with water (50 mL) and extracted with DCM (3 x 50 mL). Combined organic layer was washed with brine (50 mL), dried over Na 2 SO 4, filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using silica (230-400 mesh) column and compound eluted with 5% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford NN-3. M/Z (ESI): 490.77 [M+H] + .
• reaction mixture was stirred at 80 °C for 8 h. Reaction mixture was quenched with crushed ice and extracted with EtOAc (2 x 100 mL). Combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. Crude compound was purified by Biotage using 120 g silica gel cartridge and compound eluted with 70% EtOAc in pet ether. Pure fractions were combined and concentrated under reduced pressure to afford OO-1. M/Z (ESI): 324.23 [M+H] + .
• Crude compound was purified by prep-HPLC purification (conditions: MOBILE PHASE – 10mM Ammonium Bicarbonate in H 2 O: MeCN Column – X-Bridge C18 (19X250) mm, 5 ⁇ Flow-15.0 ml/min Gradient Method : 0/40, 2/40, 15/75, 13/75, 13.05/100, 15/100, 15.05/40, 18/40 prep-020). Pure fractions were combined, concentrated under reduced pressure and lyophilized separately to afford OO-7A and OO-7B.
• OO-7A M/Z (ESI): 557.44 [M+H] + .
• reaction mixture was stirred under nitrogen atmosphere at 150 °C for 2 h under microwave irradiation. 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 80 mL), 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 3% MeOH in DCM. Pure fractions were combined and concentrated under reduced pressure to afford PP-2. M/Z (ESI): 465.34 [M+H] + .
• the vessel was attached to the Tritium line and pressurized to 0.5 atm with Tritium gas at -200° C. The solution was stirred for 17 hours, 25002 cooled to -200° C and excess gas removed. The reaction flask was rinsed with 4 x 1 mL CH 3 OH transferring each to a 100 mL recovery flask. The combined CH3OH was removed under vacuum. Crude yield: 125 mCi. The material was purified by HPLC. Mobile phase was removed under vacuum and the product was re-dissolved in absolute Ethanol. Yield: 26 mCi, purity >99%.
• the Specific Activity was determined to be 150.01 Ci/mmol by mass spectrometry; MW for C 21 H 20 T 5 N 7 O 2 [M+H] + : 415.5, found: 416.3.
• HPLC Prep Method 10%8 for 5 minutes; 10-90%8 in 20 minutes
• the resulting residue (4 mg) was dissolved in CPME (75 ⁇ L) and NMP (50 ⁇ L).
• the nickel precatalyst ( ipc ADI)NiBr 2 (6.65 mg) was dissolved in CPME (670 ⁇ L) and treated with NaHBEt 3 in toluene (1 M, 23 ⁇ L) then stirred for 5 minutes.
• the substrate solution 100 ⁇ L was added to the active catalyst solution (100 ⁇ L) 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 155 mmHg 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 25002 sodium sulfate and evaporated. The residue was dissolved in EtOH for LSC and radio-HPLC analysis. Crude yield: 247.2 mCi; RCP: 90%. 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.
• the catalyst solution (500 ⁇ L) was added to the tritiation vessel which was attached to the ultra torr port on 25002 the Trisorber and subjected to two freeze-pump-thaw cycles before 84 mmHg tritium gas was introduced.
• the reaction was thawed to room temperature then stirred 4 hours. 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: 204.4 mCi; RCP: 89%. 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. A portion of the purified batch was dispensed and diluted to 20.0 mL. Yield: 20.0 mL ethanol soln @ 2.45 mCi/mL.
• the Specific Activity was determined to be 131.4 Ci/mmol by mass spectrometry; MW for C23H19T5F3N8O [M+H] + : 495.2, found: 495.3.
• 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 4 o C on setting 16 of Polytron.
• PBS Phosphate Buffered Saline
• the 25002 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 -70 o C until use.
• Radioligand [ 3 H]-105 prepared as described in ACS Med. Chem. Lett., Vol.2, pages 498-502, was used in this assay.
• Assay Buffer PBS plus 0.1% BSA
• 20% DMSO ranging from 3.9 to 500 nM.
• FIG.1 shows an example of hot saturation binding of [ 3 H]-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.
• 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 ul 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 ul were added to the assay plate for a final concentration of 100 ug wet weight/assay well.
• [ 3 H]-105 was prepared at 10x final concentration in Assay Buffer plus 20% DMSO and 25 ul was added to the assay plate for final assay concentration of 3.0 nM. The plates were incubated at 37 o C 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.
• the Banner PD brain homogenates which are postmortem brain tissue from donors who were diagnosed with PD, were prepared using cingulate cortex collected from multiple PD brains, which were rich of alpha-synuclein pathology, but free from amyloid pathology and tauopathy by neuropathological validation.
• the final concentration of Banner PD brain homogenates was 333 mg wet tissue per 1mL buffer. Homogenates were aliquoted in 1mL/tube and stored at -70°C prior to use.
• the 25002 assay tubes were pre-incubated at room temperature for 30 minutes, then radioligand dilutions (10X) were added into the assay tube (10 ⁇ L each / per tube, separately) to a final volume of 100 ⁇ L per tube. Incubation was carried out at 37oC for 120 minutes, and then the assay samples were filtered onto GF/C filters using Skatron 12 well harvester, washing on setting 5 – 5 – 5 ( ⁇ 3x2ml) ice cold buffer (30 nM Tris pH 7.5). The GF/C filter papers for Skatron harvester were pre- soaked in 0.1% BSA for 1 hour at room temperature before use.
• FIGS.3-6 Data for these saturation binding assays using [ 3 H]-1 and [ 3 H]-24 are illustrated in FIGS.3-6. Specifically, FIG.3 depicts a saturation binding experiment using [ 3 H]-1 and PD cingulate cortical tissue homogenate enriched in aggregated alpha-synuclein.
• FIG.5 depicts a saturation binding experiment using [ 3 H]-24 and PD cortical tissue homogenate enriched in aggregated alpha-synuclein. This data demonstrates potent binding to pathological alpha-synuclein in tissue homogenate.
• the final product was tested for chemical and radiochemical purity by means of an analytical HPLC system (Agilent) using a ONYX Monolithic, 5 ⁇ , C18, 50X3 mm (Phenomenex) at a flow rate of 1 mL/min.
• the mobile phase was a mixture consisting of acetonitrile / 0.1% trifluoroacetic acid in water from 10 to 90 % in 10 min.
• Concentration of [ 18 F]-89 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 89, and radiochemical purity was determined using a sodium iodide detector (Bioscan).
• the retention time for compound [ 18 F]-89 was 5.1 min.
• the radioactive fraction eluting between 14 and 15 minutes was collected in a flask 25002 containing a 30% ß-cyclodextrin solution (1mL), evaporated under negative pressure diluted with saline and transferred into a sterile container.
• the final product was tested for chemical and radiochemical purity by means of an analytical HPLC system (Agilent) using a ONYX Monolithic, 5 ⁇ , C18, 50X3 mm (Phenomenex) at a flow rate of 1.5 mL/min.
• the mobile phase was a mixture consisting of acetonitrile / 0.1% formic acid in water from 5 to 50 % in 7 min. Concentration of [18F]-112 was determined by means of an ultraviolet detector (254 nm).
• v-vial was flushed with air from a syringe (10 mL) and heated to 120 °C after which a solution of OO-7B (2.0 mg, 4.2 mmol), tetrakis(pyridine)copper(II) triflate (11.3 mg, 17 mmol) and pyridine (32 mL, 40 mmol) in 1,3-dimethyl-2-imidazolidinone (DMI ; 0.5 mL) was added.
• OO-7B 2.0 mg, 4.2 mmol
• tetrakis(pyridine)copper(II) triflate (11.3 mg, 17 mmol
• pyridine 32 mL, 40 mmol
• DMI 1,3-dimethyl-2-imidazolidinone
• the reaction mixture was heated at 120 °C for 20 min followed by transfer to a vial containing 10% CH3CN/10 mM Na2HPO4 in H2O pH 7.4 (1.0 mL) at room temperature for dilution, mixing and injection onto a semi-prep HPLC column.
• the product was purified using a Gemini C18, 5 mm, 110A ,150x10mm HPLC column (Phenomonex) with a flowrate of 5 ml/min and a mobile phase of CH 3 CN / 10 mM Na 2 HPO 4 pH 7.4 at a gradient of 30 – 50%.
• the radioactive fraction that eluted between 16.3 and 16.4 min was collected into a round bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure and 25002 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-C18 100x4.6mm HPLC column (Agilent) with a flowrate of 1.5 ml/min and a mobile phase of CH3CN / 10 mM NH4OAc pH 8.1 at a gradient of 35 – 45%. Concentration of [ 18 F]-113 was determined by means of an ultraviolet detector (254 nm).
• the vial containing dried [ 18 F]Et4NF was heated to 130 °C after which a solution of 115 (0.3 mg, 0.6 mmol) in DMSO (0.5 mL) was added.
• the reaction mixture was heated at 130 °C for 10 min followed by transfer to a vial containing H2O (0.8 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% CH 3 CN / 10 mM Na 2 HPO 4 pH 7.4.
• the radioactive fraction that eluted between 14.3 and 14.7 min was collected into a round bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure to remove CH 3 CN 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 30 – 40%. Concentration of [ 18 F]-115 was determined by means of an ultraviolet detector (254 nm).
• 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 Na2HPO4 in 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 [ 18 F]-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 [ 18 F]-116 was 6.6 min.
• the mobile phase was acetonitrile / Na2HPO4 (10 mM) from 40 to 70% in 15 min.
• the radioactive fraction eluting between 10 and 10.8 minutes was collected in a flask containing a 30% ß-cyclodextrin solution (1mL), evaporated under negative pressure diluted with saline and transferred into a sterile container.
• the final product was tested for chemical and radiochemical purity by means of an analytical HPLC system (Agilent) using a ONYX Monolithic, 5 ⁇ , C18, 50X3 mm (Phenomenex) at a flow rate of 1.5 mL/min.
• the mobile phase was a mixture consisting of acetonitrile / 0.1% formic acid in water from 5 to 90 % in 7 min.
• Concentration of [ 18 F]-134 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 134, and radiochemical purity was determined using a sodium iodide detector (Bioscan). The retention time for compound [ 18 F]-134 was 4.4 min.
• the reaction mixture was heated at 130 °C for 10 min followed by transfer to a vial containing 10% CH3CN/10 mM Na2HPO4 in H2O pH 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 Zorbax XDB-C18, 5 mm, 150x9.4mm HPLC column (Agilent) with a flowrate of 5 ml/min and a mobile phase of 35% CH3CN / 10 mM Na2HPO4 in H2O pH 7.4.
• the radioactive fraction that eluted between 16.7 and 17.2 min was collected into a round bottom flask containing 10% captisol in H2O (0.5 mL), evaporated under negative pressure to remove CH 3 CN, diluted with saline 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 35 – 45%.

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  • 2024-02-12 WO PCT/US2024/015346 patent/WO2024173219A1/en not_active Ceased
  • 2024-02-12 EP EP24757482.5A patent/EP4665331A1/de active Pending
  • 2024-02-12 KR KR1020257030256A patent/KR20250145090A/ko active Pending
• 2025
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