WO2025199092A1 - Ship1 modulators and methods of treatment and uses thereof - Google Patents

Ship1 modulators and methods of treatment and uses thereof

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Publication number
WO2025199092A1
WO2025199092A1 PCT/US2025/020356 US2025020356W WO2025199092A1 WO 2025199092 A1 WO2025199092 A1 WO 2025199092A1 US 2025020356 W US2025020356 W US 2025020356W WO 2025199092 A1 WO2025199092 A1 WO 2025199092A1
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compound
group
alkyl
stereoisomer
pharmaceutically acceptable
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Timothy Richardson
Daniel Beck
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Purdue Research Foundation
Indiana University
Indiana University Bloomington
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Purdue Research Foundation
Indiana University
Indiana University Bloomington
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07D417/02—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 two hetero rings
    • C07D417/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the general field of the present disclosure is novel approaches to the treatment of Alzheimer's and other neurodegenerative disorders using novel therapeutics comprising SHIP1 phosphatase modulators.
  • AD Alzheimer’s disease
  • a ⁇ extracellular 0-amyloid
  • NFTs intra-neuronal neurofibrillary tangles
  • amyloid cascade hypothesis has come under increased scrutiny due to inadequate efficacy of drugs targeting A ⁇ peptide processing and various forms of A ⁇ . See Panza et al., “A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease,” (2019) Nat Rev Neurol 15: pp. 73-88.
  • GWAS genome-wide association studies
  • APOE ABCA7, PLCG2, and INPP5D
  • Microglia are the non-neuronal, macrophage-like cells that serve as resident immune cells in the brain. See Vaughan et al., “Neuroglial cells in the cerebral cortex of rats from young adulthood to old age: an electron microscope study,” (1974) J Neurocytol 3: pp. 405-429.
  • microglia originate from stem cells in the yolk sac and differentiate into CD45 , CX3CR1 immune cells that migrate to the central nervous system (CNS).
  • CNS central nervous system
  • Kierdorf et al. “Microglia emerge from erythromyeloid precursors via Pu. 1 - and lrf8- dependent pathways,” (2013) Nat Neurosci 16: pp. 273-280. Once resident, these cells renew slowly in humans at a rate of approximately 28 percent per year, thus providing a mechanism to renew microglia. See Reu et al., “The Lifespan and Turnover of Microglia in the Human Brain.” (2017) Cell Rep 20: pp. 779-784.
  • AD Disease associated microglia
  • (2017) Cell 169: pp. 1276-1290 Although their relevance to human microglia in AD remains a current area of intense study, they have gene signatures associated with lipid metabolism and phagocytosis hypothesized to reflect the neuroprotective role of microglia in the clearance of extracellular toxins. See Olah et al., “Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease,” (2020) Nat Commun 11 : pp. 6129.
  • a two-state model of DAM induction has been proposed, in which homeostatic microglia that are associated with and support the health of neurons become activated with increased expression of DAP 12.
  • TREM2 ligands such as apolipoproteins (including, e.g., APOE) and A ⁇ induce microglial differentiation into stage-2 DAMs with increased expression of LPI. CST7. and AXL. Deczkowska et al.. “Disease-Associated Microglia: A Universal Immune Sensor of Neurodegeneration,” (2016) Cell 173: pp. 1073-1081; Keren-Shaul et al. 2017.
  • TREM2 is a receptor expressed on the surface of microglia. Genetic evidence suggests that lower TREM2 expression and inactivating variants increase risk of AD. See Jonsson et al., “Variant of TREM2 associated with the risk of Alzheimer's disease,” (2013) N Engl J Med 368: pp. 107-116. TREM2 binds A ⁇ and APOE, which activates microgliosis and the clearance of extracellular debris. See Yeh et al., “TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia.” (2016) Neuron 91 : pp. 328-340.
  • the TREM2 R4/H variant reduces the affinity of TREM2 ligands and cellular activation downstream, which requires DAP 12, an adapter protein on the intracellular side of the plasma membrane that associates with numerous signal transduction mediators.
  • DAP 12 an adapter protein on the intracellular side of the plasma membrane that associates with numerous signal transduction mediators.
  • the INPP5D gene encodes the Src homology 2 (SH2) domain-containing phosphatase- 1 (SH1P1). which is a phosphatidylinositol phosphatase that plays a key role regulating pathways downstream from TREM2.
  • SH2 Src homology 2
  • SH1P1 Src homology 2 domain-containing phosphatase- 1
  • SHIP1 is a complex, multidomain protein with a phosphatase (Ptase) domain flanked by a pleckstrin-homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ] and a C2 domain that binds phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2 ].
  • Ptase phosphatase
  • PH pleckstrin-homology
  • the C2 domain is essential for cellular function and interactions between the Ptase and C2 domains modulate enzymatic activity. See Le Coq et al., “Structural basis for interdomain communication in SHIP2 providing high phosphatase activity,” (2017) eLife 6: p. 26640.
  • SHIP1 converts PI(3,4,5)P 3 to PI(3,4)P 2 .
  • SHIP1 also contains an N-terminal SH2 domain that binds immunoreceptor tyrosine-based activation motifs (IT AMs) and a C-terminal proline rich domain that binds many other proteins including PLC ⁇ 2 and the Tec and Syk family kinases.
  • PI(3,4,5)P 3 binds and activates other PH-containing proteins such as PLC ⁇ 2, PDK1, and AKT.
  • PH PH-containing proteins
  • AKT AKT
  • SHIP1 binds ITAMs, competes with kinases, and converts PI(3,4,5)P 3 to PI(3,4)P 2 , it limits downstream signaling in multiple ways, and is therefore understood as a brake on microglia activation.
  • the present disclosure provides novel compounds that are SHIP1 inhibitors that address the need for a potent and effective treatment for Alzheimer’s disease and Alzheimer’s disease-related dementias.
  • the present disclosure also provides a pharmaceutical composition for the prevention of Alzheimer’s disease and Alzheimer's disease-related dementias.
  • the present disclosure provides one or more compounds of Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond; U and X are C, V is CR V . and Y and Z are N; or X and Z are C, V is CR V .
  • R X is selected from the group consisting of wherein o is an integer 0 or 1 ;
  • R V selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, and C 3 -C 6 cycloalkyl: wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 -C 6 alkoxy;
  • R 1 is selected from the group consisting of hydrogen, -C(O)
  • alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, -NR a R b , C 1 -C 6 alkyl, and C 1 -C 6 alkoxy;nR a and R b are each independently selected from the group consisting of hydrogen C 1 -C 6 alkyl.
  • the compound may be represented by:
  • R 2 and R 3 may each be hydrogen. In any embodiment, R 2 and R 3 may be taken together to form oxo.
  • R X may be selected from the group consisting of:
  • n may be an integer 0, 1, or 2.
  • R 4 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • R 5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • R V may be selected from the group consisting of hydrogen, - CH 3 , and -CH(CH 3 ) 2 .
  • R 4 and R 5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • the present disclosure provides a compound selected from the group consisting of:
  • the present disclosure provides a compound selected from the group consisting of:
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a pharmaceutical composition, which may comprise any compound of any aspect described herein, or a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; further comprising a pharmaceutically acceptable excipient.
  • the composition may further comprise one or more additional neurodegenerative disorder therapeutic agents.
  • composition may further comprise one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
  • the present disclosure provides a method of preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any aspect described herein, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or comprising the pharmaceutical composition of any aspect described herein.
  • the neurodegenerative disorder may be selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
  • the Alzheimer’s disease-related dementia may be selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the subject may be a mammal. In any embodiment, the subject may be a human patient. In any embodiment, the human patient may be an adult.
  • the present disclosure provides the use of a compound of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • a neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment.
  • the Alzheimer's disease-related dementia is selected from the group consisting of Lewy body dementia (LBD). frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
  • the present disclosure provides the use of a pharmaceutical composition of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • a neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment.
  • the Alzheimer's disease-related dementia is selected from the group consisting of Lewy body dementia (LBD). frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
  • the disclosure provides for a composition comprising a SHIP1 inhibitor for use in combination with an antibody or antigen-binding fragment for treating Alzheimer’s disease and Alzheimer’s disease-related dementias.
  • FIG. 1 is a schematic depicting the SHIP1 complex and associated pathways.
  • FIG. 2 is a schematic depicting the malachite green enzy me assay.
  • items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).
  • items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).
  • SH1P1 is a complex, multi-domain protein with a phosphatase (Ptase) domain flanked by a pleckstrin- homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ] and a C2 domain that binds phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2 ].
  • Ptase phosphatase
  • PH pleckstrin- homology
  • the C2 domain (SEQ ID NO: 1) is essential for cellular function and interactions between the Ptase and C2 domains modulate enzymatic activity. See Le Coq et al., 2017. SHIP1 converts PI(3,4,5)P 3 to PI(3,4)P 2 . SHIP1 also contains an N-terminal SH2 domain that binds immunoreceptor tyrosine-based activation motifs (ITAMs) and a C-terminal proline rich domain that binds many other proteins including PLC ⁇ 2 and the Tec and Syk family kinases. PI(3,4,5)P 3 binds and activates other PH-containing proteins such as PLC ⁇ 2, PDK1, and AKT. See Scheffzek et al. 2012.
  • SHIP1 binds receptor ITAMs, competes with kinases, and converts PI(3,4,5)P 3 to PI(3,4)P 2 , it limits downstream signaling in multiple ways, and is therefore understood as a brake on microglia activation. Therefore, our therapeutic hypothesis is that inhibition of SHIP1 early in disease would increase microglial protective functions and reduce the rate of disease progression and cognitive decline in Alzheimer’s patients.
  • the investigators of the present disclosure developed a novel class of SHIP1 modulating compounds, which are described in International Patent Application No. PCT/US2023/078035, the contents of which are incorporated by reference in their entirety herein.
  • the present disclosure provides new compounds, compositions comprising such compounds, methods of treating, preventing, or inhibiting the progression of neurodegenerative disease in a subject, and uses of such compounds and compositions.
  • the term “treating the progression of mild cognitive impairment to Alzheimer's disease” includes restraining, slowing, stopping, or reversing the progression of mild cognitive impairment to Alzheimer's disease in a patient.
  • the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.
  • One or more compounds of the present disclosure can react to form pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts and common methodology for preparing them are well know n in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties. Selection and Use (Manual of Pharmaceutical Salts: Properties. Selection and Use). 2nd revised edition (Wiley-VCH. 2011); SM Berge, et al.. "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.
  • the present disclosure provides one or more compounds of Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond; U and X are C, V is CR V .
  • R X is selected from the group consisting of wherein o is an integer 0 or 1; R V selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 2 - C 6 alkenyl, C 2 -C 6 alkynyl, and C 3 -C 6 cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 -C 6
  • alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 -C 6 alkoxy;
  • R 2 and R 3 are each hydrogen, or R 2 and R 3 are taken together to form oxo;
  • R 4 and R 5 are each independently selected for each occurrence from the group consisting of halogen, hydroxyl.
  • each alky l, alkenyl, alky nyl, alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, -NR a R b , C 1 -C 6 alkyl, and C 1 -C 6 alkoxy;nR a and R b are each independently selected from the group consisting of hydrogen C 1 -C 6 alkyl, and - CH 2 -phenyl; wherein C 1 -C 6 alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 - C 6 alkoxy; or R a and R b , together with the nitrogen to which they are attached, may be joined together to form a 4-7 membered heterocyclyl optionally substituted by one or more
  • the compound may be represented by:
  • R 2 and R 3 may each be hydrogen. In any embodiment, R 2 and R 3 may be taken together to form oxo.
  • R X may be selected from the group consisting of: .
  • m may be an integer 0, 1 , or 2.
  • n may be an integer 0, 1, or 2.
  • R 4 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • R 5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • R V may be selected from the group consisting of hydrogen, - CH 3 , and -CH(CH 3 ) 2 .
  • R 4 and R 5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a compound selected from the group consisting of:
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a compound selected from the group consisting of:
  • the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • the present disclosure provides a pharmaceutical composition, which may comprise any compound of any aspect described herein, or a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; further comprising a pharmaceutically acceptable excipient.
  • the composition may further comprise one or more additional neurodegenerative disorder therapeutic agents.
  • the composition may further comprise one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
  • the present disclosure provides a method of preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any aspect described herein, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or comprising the pharmaceutical composition of any aspect described herein.
  • the neurodegenerative disorder may be selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
  • the Alzheimer’s disease-related dementia may be selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the subject may be a mammal. In any embodiment, the subject may be a human patient. In any embodiment, the human patient may be an adult.
  • the present disclosure provides the use of a compound of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • a neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment.
  • the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
  • the present disclosure provides the use of a pharmaceutical composition of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • a neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment.
  • the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
  • excipients or earners include sodium citrate or dicalcium phosphate and/or a) one or more fillers or extenders (a filler or extender may be, but is not limited to, one or more selected from starches, lactose, sucrose, glucose, mannitol, and silicic acid), b) one or more binders (binders may be selected from, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), c) one or more humectants (a humectant may be, but is not limited to, glycerol), d) one or more disintegrating agents (disintegrating agents may be selected from, but are not limited to, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates, and sodium carbonate), e) one or more solution retarding agents (for example, but not
  • Effective or therapeutic amounts of the compositions of this disclosure include any amount sufficient to inhibit (e.g., slow or stop) the progression of a neurodegenerative disorder. In some embodiments, effective amounts of the compositions include any amount sufficient to inhibit (e.g., slow or stop) the deterioration of the cognitive function of a patient.
  • the amount of the active ingredient that may be combined with the optional carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration.
  • the specific dose level for any particular patient may depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disorder or disease undergoing therapy.
  • a therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
  • V is CR V .
  • Y and Z are N;
  • X and Z are C
  • V is CR V .
  • V and U are N; or U, X and Z are C,
  • V is N
  • Y is S or O
  • V is NR V .
  • Y is N;
  • X and Z are C
  • U, V and Y are N; or
  • R X is selected from the group consisting of R V selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, and C 3 -C 6 cycloalkyk wherein alky l, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 -C 6 alkoxy;
  • R 2 and R 3 are each hydrogen, or R 2 and R 3 are taken together to form oxo;
  • each alkyl, alkenyl, alkynyl, alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently- selected from the group consisting of halogen, hydroxyl, -NR a R b , C 1 -C 6 alkyl, and C 1 -C 6 alkoxy;
  • R a and R b are each independently selected from the group consisting of hydrogen C 1 - C 6 alkyl, and -CH 2 -phenyl; wherein C 1 -C 6 alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C 1 -C 6 alkoxy; or
  • R a and R b together with the nitrogen to which they are attached, may be joined together to form a 4-7 membered heterocyclyl optionally substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy; m is an integer 0. 1, 2, 3, 4, or 5; and n is an integer 0, 1, 2, 3, 4, or 5.
  • Clause 8 The compound of any one of clauses 1-7. wherein R V is selected from the group consisting of hydrogen and C 1 -C 6 alkyl.
  • Clause 10 The compound of any one of clauses 1-9, wherein m is an integer 0, 1, or 2.
  • Clause 11 The compound of any one of clauses 1-10, wherein R 4 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • Clause 13 The compound of any one of clauses 1-12, wherein R 5 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • R 2 and R 3 are each independently selected from halogen and C 1 -C 6 alkyl; m is an integer 0, 1, or 2; and n is an integer 0, 1 , or 2.
  • Clause 17 The compound of any one of clauses 14-16, wherein R 4 and R 5 are independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH 3 .
  • Clause 22 A compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
  • Clause 23 A pharmaceutical composition comprising a compound of any one of clauses 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound, and a pharmaceutically acceptable excipient.
  • Clause 24 The composition of clause 23, further comprising one or more additional neurodegenerative disorder therapeutic agents.
  • Clause 25 The composition of either of clauses 23 or 24, further comprising one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
  • Clause 26 A method of treating, preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject an effective amount of the compound of any one of clauses 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or the pharmaceutical composition of any one of clauses 23- 25.
  • Clause 27 The method of clause 26, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
  • Clause 28 The method of clause 27, wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • LBD Lewy body dementia
  • FDD frontotemporal degeneration
  • VCID vascular cognitive impairment and dementia
  • Clause 29 The method of any one of clauses 26-28, wherein the subject is a mammal.
  • Clause 30 The method of any one of clauses 26-28, wherein the subject is human patient.
  • Clause 31 The method of any one of clause 30, wherein the human patient is an adult.
  • Clause 32 Use of a compound of any of clauses 1-22 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • Clause 33 The use of clause 32, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
  • Clause 35 The use of any one of clauses 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal.
  • Clause 36 The use of any one of clauses 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
  • Clause 37 The use of clause 36, wherein the human patient is an adult.
  • Clause 38 Use of a pharmaceutical composition of any of clauses 23-25 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
  • Clause 39 The use of clause 38, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
  • Clause 40 The use of clause 39. wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
  • LBD Lewy body dementia
  • FDD frontotemporal degeneration
  • VCID vascular cognitive impairment and dementia
  • Clause 41 The use of any one of clauses 38-40, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal.
  • Clause 42 The use of any one of clauses 38-40, w herein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
  • Clause 43 The use of clause 42, wherein the human patient is an adult.
  • Flash normal phase (NP) or reversed-phase (RP) chromatography were performed on a Teledyne ISCO NextGen 300 instrument using prepacked silica gel or Cl 8-functionalized silica gel columns available from Teledyne ISCO, or on a Teledyne ISCO CombiFlash® using prepacked silica gel columns available from Agela or Welch.
  • RP HPLC reverse-phase
  • a WatersTM AutoPurification HPLC system equipped with PDA and ELSD detectors and a WatersTM XBridge Cl 8 Prep column (10 ⁇ m, 250 mm x 19 mm).
  • High-resolution mass spectra were obtained on an Agilent 6550 Q-TOF instrument. All compounds had >95% purity as determined by LC-MS.
  • One of the following specified LC-MS methods was used to determine test compound purity:
  • Method 1 gradient table A (below); column, WatersTM ACQUITY® HSS-T3 (1.8 ⁇ m, 100 mm x 2.1 mm); mobile phase A, 0.1% trifluoroacetic acid (TFA) in H 2 O; mobile phase B, acetonitrile (ACN); flow rate. 0.3 mL/min; detection wavelength, 214 nm; column temperature, 35 °C.
  • TFA trifluoroacetic acid
  • ACN acetonitrile
  • Method 2 gradient table A; column, WatersTM ACQUITY® BEH C-18 (1.7 ⁇ m, 100 mm x 2. 1 mm) [or C-8 (1.7 ⁇ m, 100 mm x 2. 1 mm) for 11]; mobile phase A, 5 rnM NH4OAC in H 2 O; mobile phase B, ACN; flow rate, 0.3 mL/min; detection wavelength, 214 nm; column temperature. 35 °C.
  • Method 3 gradient table B; column.
  • WatersTM ACQUITY® BEH C-18 (1.7 gm, 100 mm x 2.1 mm); mobile phase A, 5 mM NH 4 OAc in H 2 O; mobile phase B, ACN; flow rate, 0.3 mL/min; detection wavelength, 214 nm; column temperature, 35 °C.
  • Method 4 gradient table C; column, WatersTM ACQUITY® BEH C-18 (1.7 ⁇ m, 50 mm x 2.1 mm); mobile phase A, 0.1% formic acid in H 2 O; mobile phase B, 0.1% formic acid in ACN; flow rate, 0.6 mL/min; detection wavelength, 254 nm; column temperature, 40 °C.
  • N-bromosuccinimide (NBS) (912 mg. 5.12 mmol, 1.2 eq). The reaction mixture was stirred at 60 °C for 16 hours. The reaction mixture was diluted with H 2 O, extracted with DCM, washed with brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo.
  • Reaction mixture was monitored by TLC and LCMS. Reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Crude was purified by flash chromatography using 20% EtOAc in hexane as an eluent, desired fractions were concentrated to afford l-[4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-l-yl]ethan-l-one (120 mg, 256 ⁇ mol) as a brown semi solid. MS ES+ 361.17.
  • the crude material was purified by column chromatography eluted with 70% ethyl acetate in heptane.
  • the crude was purified by Prep-HPLC using TFA buffer solution to provide l-(4-methyl- 1.5-di phenyl- 1 H-pyrazol-3-yl)piperazine trifluoroacetate salt (20 mg, 62.8 ⁇ mol) as an off white solid.
  • reaction mixture was purged with N 2 for 15 minutes, palladium — triphenylphosphine (1/4) (73mg, 0.1 eq., 63.9 ⁇ mol) was added and purging continued for 5 minutes, then the reaction mixture was heated at 100 °C and stirred for 16 hours. After completion of the reaction (TLC monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated to obtain crude.
  • reaction mixture was purged with N 2 for 15 minutes, RuPhos Pd G3 (66.8 mg, 0.05 eq., 79.8 ⁇ mol) was then added and purging continued for another 5 minutes.
  • the reaction mixture was heated at 130 °C and stirred for 16 hours. After completion of the reaction (TLC and LCMS monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated to obtain crude.
  • reaction mixture was quenched with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated to obtain crude which was purified by PREP HPLC using ABC buffer to afford 3,3-dimethyl-l-(4-(4-methyl-l,5- diphenyl-1H-pyrazol-3-yl)piperidin-l-yl)butan-l-one (83 mg, 0.2 mmol) as a white solid.
  • reaction mixture was purged with N 2 for 15 minutes, then was added Catacxium A Pd G3 (42.9 mg, 0.05 eq.. 58.9 prnol) and purging continued for 20 minutes, then the reaction mixture was heated at 120 °C and stirred for 16 hours. After completion of the reaction (TLC & LCMS monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • reaction mixture was purged with N 2 for 15 minutes, after that palladium — triphenylphosphine (1/4) (64.6 mg, 0.05 eq., 55.9 ⁇ mol) was added and purging continued for 20 minutes, then the reaction mixture was heated at 100 °C and stirred for 16 hours. After completion of the reaction (TLC monitoring), reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • palladium — triphenylphosphine (1/4) 64.6 mg, 0.05 eq., 55.9 ⁇ mol
  • reaction mixture was diluted with cold water and extracted with EtOAc. Organic layer was combined and washed with water followed by brine. It was then dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The resulting mixture was purified by flash column chromatography on SiO 2 gel column using 0-5% EtOAc in heptanes and fractions containing the desired product were concentrated to afford 3,5- dibromo-1 -phenyl- 1H-1, 2, 4-triazole (1.9 g, 6.27 mmol) as a white solid.
  • the resulting mixture was purged with N 2 for 15 minutes, then Pd(PPh3) 4 (99.8 mg, 86.4 ⁇ mol) was added and purging continued for additional 20 minutes.
  • the reaction mixture was then heated at 80 °C under vigorous stirring for 16 hours. After completion of the reaction (TLC & LCMS monitoring) it was diluted with chilled water, extracted with EtOAc, washed with water followed by brine. The organic extract was dried over anhydrous Na 2 SO 4 . filtered and concentrated under reduced pressure.
  • the resulting mixture was purified by Si-gel flash column chromatography on 12 g Welch SiO 2 gel LS column using 30% EtOAc/ heptanes as eluant.
  • Enzymatic inhibitory potencies (IC 50 ) (see table below) were determined using PI(3,4,5)P 3 -diC 8 as a substrate at 25 °C in 50 mM HEPES buffer (pH7.4, 150 mM NaCl, 2 mM MgCl 2 ). See FIG. 2. Compounds diluted in DMSO were added to 384-well plates. Human SHIP1 1-899 multidomain enzyme solution was added. After a 20-minute incubation period, the reaction was initiated by addition of PI(3,4,5)P 3 -diC 8 . Final compound concentrations ranged from 50 nM to 950 ⁇ M.
  • CETSA Cellular Thermal Shift Assay
  • a split Nano Luciferase assay (SplitLuc CETSA) was used to demonstrate target engagement of SHIP1 inhibitors in a physiologically relevant cellular context by quantifying changes in the thermal stability of a HiBit-labeled full length SHIP1 protein in intact cells. See Martinez et al. (2016) Sci Rep 8: p. 9472; Oh-Hashi et al. (2017) Biochem Biophys Rep 12: pp. 40- 45. This assay was run in the following two formats with HMC3/HiBit-INPP5D stably transfected cells.
  • Compound dose response Run at target T m (44.2 °C for SHIP1) with compound dosing from 80 ⁇ M or 100 ⁇ M with 1 :3 serial dilutions to generate an 8-point curve. Cells were treated for 60 minutes before being heated at target T m for 3 minutes before luminescence detection. The concentration that induced a half-maximum response (AC 50 ) was calculated using a four-parameter logistic curve regression model with change at highest concentration noted when difference from control >3SD. Activities are shown in Tables 2. below.
  • pHrodo-Myelin Phagocytosis/Cell Health Assay with Microglial Cells This 384-well plate high content imaging assay was developed to quantify phagocytosis and cell health simultaneously using either BV2 or HMC3 immortalized microglial cell lines or primary microglia isolated from mouse brain. See Mason, et al., "‘Microglial Phagocytosis/Cell Health High-Content Assay,” (2023) Curr Protoc 3: e724. Briefly, cells were cultured in DMEM GlutaMax media (ThermoFisher) containing 10% FBS and Pen-Strep in 37 °C 5% CO 2 incubator.
  • DMEM GlutaMax media ThermoFisher
  • Day 1 Cells were plated (Coming Falcon 384 well Optilux Black and clear bottom plates for imaging) with BV2 at 400 cells/45pl/welk HMC3 at 600 cells/45pl/well, or primary at 2000 cell/45pl/well.
  • Day 2 Cells were treated with 10x serially diluted compounds in a dose range of 60 ⁇ M to 3 nM for 48 hrs at 37 °C.
  • Dav 3 Cells were seeded with pHrodo- myelin (for total 20 hrs) 24 hrs after starting compound treatment. The pHrodo-myelin stocks were at 1 mg/ml (protein equivalent) stored in -20 °C or -80 °C freezer.
  • Cortical tissue from C57BL/6J neonatal mice was homogenized in Dulbecco's Modified Eagle Medium (DMEM), filtered through 250 and 100 ⁇ m mesh, and cultured in Advanced DMEM/F12 supplemented with 10% fetal bovine serum, lx GlutaMAX and lx Penicillin/Streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • the cultures were subjected to mild trypsinization using 0.083% Trypsin-EDTA in DMEM for 30 mins to detach an intact layer of astrocytes.
  • the microglia attached to the bottom were used as described in above to measure myelin phagocytosis and cell health. Activities are shown in table 3 below. [00187] Table 3

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Abstract

Compounds and pharmaceutical compositions for the treatment of neurodegenerative diseases and related conditions, in particular through modulation of the activity of SHIP1 phosphatase, and methods of treating, preventing, and inhibiting the progression of neurodegenerative disease and related conditions using the same.

Description

SHIP1 MODULATORS AND METHODS OF TREATMENT AND USES THEREOF
REFERENCE TO GOVERNMENT GRANTS
[0001] This invention was made with government support under AG065181 awarded by National Institutes of Health. The Government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION
[0002] This application claims priority to U.S. Application Serial No. 63/567,560, filed March 20, 2024, which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0003] This application incorporates by reference the material in the Extensible Markup Language (XML) file titled IUIC-164_Sequence-Listing.xml, created March 18, 2025, which is 2,252 bytes in size.
FIELD
[0004] The general field of the present disclosure is novel approaches to the treatment of Alzheimer's and other neurodegenerative disorders using novel therapeutics comprising SHIP1 phosphatase modulators.
BACKGROUND
[0005] Alzheimer’s disease (AD) is a fatal, neurodegenerative disorder, characterized by histopathological accumulation of extracellular 0-amyloid (Aβ) plaques and intra-neuronal neurofibrillary tangles (NFTs), which have been hypothesized to result in neurotoxicity and progressive cognitive decline. See Kumar et al., “A review on Alzheimer's disease pathophysiology and its management: an update, (2015) Pharmacol Rep 67: pp. 195-203; Hardy et al., “Alzheimer's disease: the amyloid cascade hypothesis: an update and reappraisal,” (2006) J Alzheimers Dis 9: pp. 151-153; Shen et al, “Complement activation by neurofibrillary tangles in Alzheimer's disease,” (2001) Neurosci Lett 305: pp. 165-168; Scheltens et al., “Alzheimer's disease, 2021) Lancet 397 : pp. 1577-1590. The amyloid cascade hypothesis postulates that various forms of Aβ oligomers and plaques are instrumental in a neuropathological process that triggers subsequent NFT pathology, neuroinflammation, and neuronal loss; however, the mechanisms by which Aβ influences neurotoxic signaling including NFT formation remain an area of intense study. Recently, the amyloid cascade hypothesis has come under increased scrutiny due to inadequate efficacy of drugs targeting Aβ peptide processing and various forms of Aβ. See Panza et al., “A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease,” (2019) Nat Rev Neurol 15: pp. 73-88.
[0006] More recent evidence including genome-wide association studies (GWAS), whole genome sequencing, differential gene expression, and gene-expression network analyses comparing normal to affected Alzheimer’s brain tissue have identified risk and protective variants in genes such as TREM2, CD33. APOE, ABCA7, PLCG2, and INPP5D, which are essential to microglia function. See Malik et al., "Genetics ignite focus on microglial inflammation in Alzheimer's disease.” (2015) Mol Neurodegener 10: p. 52. Microglia are the non-neuronal, macrophage-like cells that serve as resident immune cells in the brain. See Vaughan et al., “Neuroglial cells in the cerebral cortex of rats from young adulthood to old age: an electron microscope study,” (1974) J Neurocytol 3: pp. 405-429.
[0007] During development microglia originate from stem cells in the yolk sac and differentiate into CD45 , CX3CR1 immune cells that migrate to the central nervous system (CNS). See Kierdorf et al., “Microglia emerge from erythromyeloid precursors via Pu. 1 - and lrf8- dependent pathways,” (2013) Nat Neurosci 16: pp. 273-280. Once resident, these cells renew slowly in humans at a rate of approximately 28 percent per year, thus providing a mechanism to renew microglia. See Reu et al., “The Lifespan and Turnover of Microglia in the Human Brain.” (2017) Cell Rep 20: pp. 779-784. Disease associated microglia (DAM) have been characterized at sites of Aβ plaques and neurodegeneration in animal models. See Keren-Shaul et al., “A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease,” (2017) Cell 169: pp. 1276-1290. Although their relevance to human microglia in AD remains a current area of intense study, they have gene signatures associated with lipid metabolism and phagocytosis hypothesized to reflect the neuroprotective role of microglia in the clearance of extracellular toxins. See Olah et al., “Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease,” (2020) Nat Commun 11 : pp. 6129. A two-state model of DAM induction has been proposed, in which homeostatic microglia that are associated with and support the health of neurons become activated with increased expression of DAP 12. APOE. and Triggering receptor expressed on myeloid cells-2 (TREM2). TREM2 ligands such as apolipoproteins (including, e.g., APOE) and Aβ induce microglial differentiation into stage-2 DAMs with increased expression of LPI. CST7. and AXL. Deczkowska et al.. “Disease-Associated Microglia: A Universal Immune Sensor of Neurodegeneration,” (2018) Cell 173: pp. 1073-1081; Keren-Shaul et al. 2017.
[0008] TREM2 is a receptor expressed on the surface of microglia. Genetic evidence suggests that lower TREM2 expression and inactivating variants increase risk of AD. See Jonsson et al., “Variant of TREM2 associated with the risk of Alzheimer's disease,” (2013) N Engl J Med 368: pp. 107-116. TREM2 binds Aβ and APOE, which activates microgliosis and the clearance of extracellular debris. See Yeh et al., “TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia.” (2016) Neuron 91 : pp. 328-340. The TREM2R4/H variant reduces the affinity of TREM2 ligands and cellular activation downstream, which requires DAP 12, an adapter protein on the intracellular side of the plasma membrane that associates with numerous signal transduction mediators. See Sudom et al., “Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant ofthe immune receptor TREM2,” (2018) J Biol Chem 293: pp. 12634-12646. For example, recruitment of SYK to phosphorylated tyrosine residues at the C-terminus of DAP 12 mediates signaling through phosphorylation of PLCγ2 and subsequent formation of intracellular IP3 and Ca2+ release. See Konishi et al., “Microglial TREM2/DAP12 Signaling: A Double-Edged Sword in Neural Diseases,” (2018) Front Cell Neurosci 12: p. 206. Importantly, an activating variant of PLCG2, PLCG2P522R is protective in AD. See Magno et al., “Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph,” (2019) Alzheimer ’s Res Ther 11 : p. 16. This human genetic evidence suggests that dampened microglia activity increases risk of neurodegeneration while activated microglia are protective, clearing Aβ oligomers and plaques, and mitigating an inflammatory microenvironment that is toxic to neurons. See Deczkowska et al., 2018.
[0009] The recent regulatory approval of the anti-amyloid antibody aducanumab provides evidence for the role of activated microglia in the treatment of AD. See Dunn et al., “Approval of Aducanumab for Alzheimer Disease-The FDA's Perspective.” (2021) JAMA Intern Med 181: pp. 1276-1278. Cell surface Fey receptors (FcγRs) on microglia recognize the Fc portion of IgG antibodies triggering downstream effector functions. See Boumazos et al., “The role of IgG Fc receptors in antibody-dependent enhancement,” (2020) Nat Rev Immunol 20: pp. 633-643. The clearance of Aβ deposits by aducanumab has been demonstrated to occur by targeting Aβ via the variable region of the antibody coupled to FcγR-mediated enhancement of microglia recruitment and phagocytosis. See Sevigny et al., “The antibody aducanumab reduces Abeta plaques in Alzheimer's disease,” (2016) Nature 537: pp. 50-56.
[0010] The INPP5D gene encodes the Src homology 2 (SH2) domain-containing phosphatase- 1 (SH1P1). which is a phosphatidylinositol phosphatase that plays a key role regulating pathways downstream from TREM2. See Peng et al., “TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1,” (2010) Sei Signal 3: p. 38; Pauls et al., “Regulation of immune cell signaling by SHIP1 : A phosphatase, scaffold protein, and potential therapeutic target,” (2017) Eur J Immunol 47: pp. 932-945. SHIP1 is a complex, multidomain protein with a phosphatase (Ptase) domain flanked by a pleckstrin-homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and a C2 domain that binds phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. See Damen et al., “The 145-kDa protein induced to associate with She by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5 -phosphatase,” (1996) Proc Natl Acad Sci USA 93: pp. 1689-1693; Blunt et al., “Pharmacological targeting of phosphoinositide lipid kinases and phosphatases in the immune system: success, disappointment, and new opportunities,” (2012) Front Immunol 3: p. 226. The PH and C2 domains locate and orient the catalytic site towards its PI(3,4,5)P3 substrate at the intracellular side of the membrane. The C2 domain is essential for cellular function and interactions between the Ptase and C2 domains modulate enzymatic activity. See Le Coq et al., “Structural basis for interdomain communication in SHIP2 providing high phosphatase activity,” (2017) eLife 6: p. 26640. SHIP1 converts PI(3,4,5)P3 to PI(3,4)P2. SHIP1 also contains an N-terminal SH2 domain that binds immunoreceptor tyrosine-based activation motifs (IT AMs) and a C-terminal proline rich domain that binds many other proteins including PLCγ2 and the Tec and Syk family kinases. PI(3,4,5)P3 binds and activates other PH-containing proteins such as PLCγ2, PDK1, and AKT. See Scheffzek et al., “Pleckstrin homology (PH) like domains — versatile modules in protein-protein interaction platforms,” (2012) FEBS Lett 586: pp. 2662-2673. Because SHIP1 binds ITAMs, competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it limits downstream signaling in multiple ways, and is therefore understood as a brake on microglia activation. See Pauls et al. 2017.
[0011] Taken together, this understanding of AD risk and protective variants in genes critical to the activity of microglia, the role of microglia in the clearance of Aβ, and SHIP1 as a limiting node downstream from TREM2 and FCγRIIB, suggests that inhibition of SHIP1 would activate microglia, and would therefore be an effective therapeutic strategy in disease. This therapeutic intervention could be synergistically combined with anti-amyloid and/or TREM2 agonist antibodies. Therefore, inhibitors of SHIP1 would increase the protective functions of microglia and could therefore be used to prevent or treat disease, reduce the rate of disease progression and cognitive decline in patients, and reverse neurodegeneration.
[0012] The present disclosure provides novel compounds that are SHIP1 inhibitors that address the need for a potent and effective treatment for Alzheimer’s disease and Alzheimer’s disease-related dementias. The present disclosure also provides a pharmaceutical composition for the prevention of Alzheimer’s disease and Alzheimer's disease-related dementias.
SUMMARY
[0013] Recent evidence including genome-wide association studies (GWAS), whole genome sequencing, differential gene expression, and gene-expression network analyses comparing normal to affected Alzheimer’s brain tissue have identified risk and protective variants in genes such as TREM2, PLCG2 and INPP5D that are essential to microglia function. INPP5D encodes SHIP1, a multi-domain protein with a phosphatase that converts PI(3,4,5)P3 to PI(3,4)P2, a SH2 domain that interacts with receptor ITAMs and competes with SYK, and a proline rich region that binds many other proteins. SHIP1 therefore limits microglia activation in multiple ways. Inhibition of SH1P1 early in disease would increase microglial protective functions and reduce the rate of disease progression and cognitive decline in Alzheimer’s patients.
[0014] In an aspect, the present disclosure provides one or more compounds of Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond; U and X are C, V is CRV. and Y and Z are N; or X and Z are C, V is CRV. and Y and U are N; or U, X and Z are C, V is N, and Y is S or O; or U, X, and Z are C, V is NRV, and Y is N; or X and Z are C, and U, V and Y are N; or U and Z are C, and V, X and Y are N; RX is selected from the group consisting of wherein o is an integer 0 or 1 ; RV selected from the group consisting of hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, and C3-C6cycloalkyl: wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; R1 is selected from the group consisting of hydrogen, -C(O)Ra, - (C=O)ORa, -C(=O)NRaRb, -S(O)2NRaRb, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, and C3- C6cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; R2 and R3 are each hydrogen, or R2 and R3 are taken together to form oxo; R4 and R5 are each independently selected for each occurrence from the group consisting of halogen, hydroxyl, -CN, -NRaRb, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C3- C6cycloalkyl, -C(=O)NRaRb, -NRa(C=O)Rb, -O(C=O)NRaRb, -NRa(C~O)ORb. -NRa(C=O)NRaRb, -(C=O)C1-C6alkyl, -(C=O)OC1-C6alkyl, -O(C=O)C1-C6alkyl, -SH, -SC1-C6alkyl, -S(O)2NRaRb, and -NRaS(O)2C1-C6alkyl; wherein each alkyl, alkenyl, alkynyl. alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, -NRaRb, C1-C6alkyl, and C1-C6alkoxy;nRa and Rb are each independently selected from the group consisting of hydrogen C1-C6alkyl. and -CH2-phenyl; wherein C1-C6alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; or Ra and Rb, together with the nitrogen to which they are attached, may be joined together to form a 4- 7 membered heterocyclyl optionally substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl. C1-CCalkyl. and C1-C6alkoxy; m is an integer 0. 1, 2, 3. 4, or 5; and n is an integer 0. 1, 2, 3. 4, or 5.
[0015] In any embodiment, the compound may be represented by:
[0016] In any embodiment, R2 and R3 may each be hydrogen. In any embodiment, R2 and R3 may be taken together to form oxo.
[0017] In any embodiment, RX may be selected from the group consisting of:
[0018] In any embodiment, R1 may be selected from the group consisting of hydrogen. - Me, -C(O)Ra, and -(C=O)ORa. In any embodiment, R1 may be selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3. In any embodiment, RV may be selected from the group consisting of hydrogen and C1-C6alkyl. In any embodiment, RV may be selected from the group consisting of hydrogen, -CH3. and -CH(CH3)2.
[0019] In any embodiment, m may be an integer 0, 1 , or 2. In any embodiment, n may be an integer 0, 1, or 2.
[0020] In any embodiment, R4 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3. In any embodiment, R5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0021] In another aspect, the present disclosure provides a compound represented by: or a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: RX is selected from RV is selected from hydrogen and C1-C6alkyl; R1 is selected from hydrogen, -C(O)C1-C6alkyl, and -(C=O)OC1-C6alkyl; R2 and R3 are each independently selected from halogen and C1-C6alkyl; m is an integer 0, 1, or 2; and n is an integer 0, 1, or 2.
[0022] In any embodiment, RV may be selected from the group consisting of hydrogen, - CH3, and -CH(CH3)2.
[0023] In any embodiment, R1 may be selected from the group consisting of hydrogen, - C(O)CH3, and -(C=O)OC(CH3)3.
[0024] In any embodiment, R4 and R5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0025] In a further aspect, the present disclosure provides a compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound. [0026] In still another aspect, the present disclosure provides a compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0027] In another aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0028] In an additional aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0029] In another aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0030] In yet another aspect, the present disclosure provides a pharmaceutical composition, which may comprise any compound of any aspect described herein, or a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; further comprising a pharmaceutically acceptable excipient. In any embodiment, the composition may further comprise one or more additional neurodegenerative disorder therapeutic agents.
[0031] In any embodiment, the composition may further comprise one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
[0032] In another aspect, the present disclosure provides a method of preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any aspect described herein, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or comprising the pharmaceutical composition of any aspect described herein.
[0033] In any embodiment, the neurodegenerative disorder may be selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer’s disease-related dementia may be selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
[0034] In any embodiment, the subject may be a mammal. In any embodiment, the subject may be a human patient. In any embodiment, the human patient may be an adult.
[0035] In another aspect, the present disclosure provides the use of a compound of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition. In any embodiment, such neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer's disease-related dementia is selected from the group consisting of Lewy body dementia (LBD). frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
[0036] In another aspect, the present disclosure provides the use of a pharmaceutical composition of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition. In any embodiment, such neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer's disease-related dementia is selected from the group consisting of Lewy body dementia (LBD). frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
[0037] In some embodiments, the disclosure provides for a composition comprising a SHIP1 inhibitor for use in combination with an antibody or antigen-binding fragment for treating Alzheimer’s disease and Alzheimer’s disease-related dementias.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a schematic depicting the SHIP1 complex and associated pathways.
[0039] FIG. 2 is a schematic depicting the malachite green enzy me assay.
DETAILED DESCRIPTION
[0040] Various quantities, such as amounts, sizes, dimensions, proportions, and the like, are presented in a range format throughout this disclosure. It should be understood that the description of a quantity in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example. 1.1, 2, 2.3, 4.62, 5. and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.
[0041] The terminology used herein is to describe particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).
[0042] Unless expressly stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers i s understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
[0043] Recent evidence including genome-wide association studies (GWAS) and differential gene expression comparing normal to affected Alzheimer’s brain tissue have identified risk and protective variants in genes such as TREM2. PLCG2. and INPP5D that are essential to microglia function. The INPP5D gene encodes the Src homology 2 (SH2) domain-containing phosphatase- 1 (SHIP1), which is a phosphatidylinositol phosphatase that plays a key role regulating pathways downstream from TREM2. See Peng et al. 2010; Pauls et al., 2017. SH1P1 is a complex, multi-domain protein with a phosphatase (Ptase) domain flanked by a pleckstrin- homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and a C2 domain that binds phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. See FIG. 1 ; Damen et al. 1996; Blunt et al., 2012. The PH and C2 domains locate and orient the catalytic site towards its PI(3,4,5)P3 substrate at the intracellular side of the membrane. The C2 domain (SEQ ID NO: 1) is essential for cellular function and interactions between the Ptase and C2 domains modulate enzymatic activity. See Le Coq et al., 2017. SHIP1 converts PI(3,4,5)P3 to PI(3,4)P2. SHIP1 also contains an N-terminal SH2 domain that binds immunoreceptor tyrosine-based activation motifs (ITAMs) and a C-terminal proline rich domain that binds many other proteins including PLCγ2 and the Tec and Syk family kinases. PI(3,4,5)P3 binds and activates other PH-containing proteins such as PLCγ2, PDK1, and AKT. See Scheffzek et al. 2012. Because SHIP1 binds receptor ITAMs, competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it limits downstream signaling in multiple ways, and is therefore understood as a brake on microglia activation. Therefore, our therapeutic hypothesis is that inhibition of SHIP1 early in disease would increase microglial protective functions and reduce the rate of disease progression and cognitive decline in Alzheimer’s patients.
[0044] The investigators of the present disclosure developed a novel class of SHIP1 modulating compounds, which are described in International Patent Application No. PCT/US2023/078035, the contents of which are incorporated by reference in their entirety herein. [0045] The present disclosure provides new compounds, compositions comprising such compounds, methods of treating, preventing, or inhibiting the progression of neurodegenerative disease in a subject, and uses of such compounds and compositions.
[0046] In any of the embodiments disclosed herein, the term “treating the progression of mild cognitive impairment to Alzheimer's disease” includes restraining, slowing, stopping, or reversing the progression of mild cognitive impairment to Alzheimer's disease in a patient.
[0047] In any of the embodiments disclosed herein, the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.
[0048] One or more compounds of the present disclosure can react to form pharmaceutically acceptable salts. Pharmaceutically acceptable salts and common methodology for preparing them are well know n in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties. Selection and Use (Manual of Pharmaceutical Salts: Properties. Selection and Use). 2nd revised edition (Wiley-VCH. 2011); SM Berge, et al.. "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977. [0049] In an aspect, the present disclosure provides one or more compounds of Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond; U and X are C, V is CRV. and Y and Z are N; or X and Z are C, V is CRV, and Y and U are N; or U, X and Z are C, V is N, and Y is S or O; or U, X, and Z are C, V is NRV, and Y is N; or X and Z are C, and U, V and Y are N; or U and Z are C, and V, X and Y are N; RX is selected from the group consisting of wherein o is an integer 0 or 1; RV selected from the group consisting of hydrogen, C1-C6alkyl, C2- C6alkenyl, C2-C6alkynyl, and C3-C6cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; R1 is selected from the group consisting of hydrogen. -C(O)Ra. -(C=O)ORa, -C(=O)NRaRb. -S(O)2NRaRb, C1-C6alkyl, C2-C6alkenyl, C2- C6alkynyl, and C3-C6cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; R2 and R3 are each hydrogen, or R2 and R3 are taken together to form oxo; R4 and R5 are each independently selected for each occurrence from the group consisting of halogen, hydroxyl. -CN, -NRaRb. C1-C6alkyl, C2-C6alkenyl, C2-C6alkymyl, C1- C6alkoxy, C3-C6cycloalkyl, -C(=O)NRaRb, -NRa(C=O)Rb, -O(C=O)NRaRb, -NRa(C=O)ORb, - NRa(C=O)NRaRb, -(C=O)C1-C6alkyl, -(C=O)OC1-C6alkyl, -O(C=O)C1-C6alkyl, -SH, -SC1-C6alkyl, -S(O)2NRaRb. and -NRaS(O)2C1-C6alkyl; wherein each alky l, alkenyl, alky nyl, alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, -NRaRb, C1-C6alkyl, and C1-C6alkoxy;nRa and Rb are each independently selected from the group consisting of hydrogen C1-C6alkyl, and - CH2-phenyl; wherein C1-C6alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1- C6alkoxy; or Ra and Rb, together with the nitrogen to which they are attached, may be joined together to form a 4-7 membered heterocyclyl optionally substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl. C1-C6alkyl, and C1- C6alkoxy; m is an integer 0, 1, 2, 3, 4, or 5; and n is an integer 0, 1, 2, 3, 4, or 5.
[0050] In any embodiment, the compound may be represented by:
[0051] In any embodiment, R2 and R3 may each be hydrogen. In any embodiment, R2 and R3 may be taken together to form oxo.
[0052] In any embodiment, RX may be selected from the group consisting of: .
[0053] In any embodiment, R1 may be selected from the group consisting of hydrogen. - Me, -C(O)Ra, and -(C=O)ORa. In any embodiment, R1 may be selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3. In any embodiment, RV may be selected from the group consisting of hydrogen and C1-C6alkyl. In any embodiment, RV may be selected from the group consisting of hydrogen, -CH3. and -CH(CH3)2.
[0054] In any embodiment, m may be an integer 0, 1 , or 2. In any embodiment, n may be an integer 0, 1, or 2. [0055] In any embodiment, R4 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3. In any embodiment, R 5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0056] In another aspect, the present disclosure provides a compound represented by: or a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: RX is selected from RV is selected from hydrogen and C1-C6alkyl; R1 is selected from hydrogen, -C(O)C1-C6alkyl, and -(C=O)OC1-C6alkyl; R2 and R3 are each independently selected from halogen and C1-C6alkyl; m is an integer 0, 1, or 2; and n is an integer 0, 1. or 2.
[0057] In any embodiment, RV may be selected from the group consisting of hydrogen, - CH3, and -CH(CH3)2.
[0058] In any embodiment, R1 may be selected from the group consisting of hydrogen, - C(O)CH3, and -(C=O)OC(CH3)3.
[0059] In any embodiment, R4 and R5 may be independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0060] In a further aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0061] In still another aspect, the present disclosure provides a compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0062] In another aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0063] In an additional aspect, the present disclosure provides a compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0064] In another aspect, the present disclosure provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0065] In yet another aspect, the present disclosure provides a pharmaceutical composition, which may comprise any compound of any aspect described herein, or a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; further comprising a pharmaceutically acceptable excipient. In any embodiment, the composition may further comprise one or more additional neurodegenerative disorder therapeutic agents. [0066] In any embodiment, the composition may further comprise one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
[0067] In another aspect, the present disclosure provides a method of preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any aspect described herein, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or comprising the pharmaceutical composition of any aspect described herein.
[0068] In any embodiment, the neurodegenerative disorder may be selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer’s disease-related dementia may be selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD). vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
[0069] In any embodiment, the subject may be a mammal. In any embodiment, the subject may be a human patient. In any embodiment, the human patient may be an adult.
[0070] In another aspect, the present disclosure provides the use of a compound of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition. In any embodiment, such neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
[0071] In another aspect, the present disclosure provides the use of a pharmaceutical composition of any aspect described herein for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition. In any embodiment, such neurodegenerative disorder or related condition may be selected from the group consisting of Alzheimer’s disease, Alzheimer's disease-related dementia, and mild cognitive impairment. In any embodiment, the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. In any embodiment, the medicament may be formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient. In any embodiment, the human patient may be an adult.
[0072] Excipients
[0073] Illustrative, non-limiting examples of excipients or earners include sodium citrate or dicalcium phosphate and/or a) one or more fillers or extenders (a filler or extender may be, but is not limited to, one or more selected from starches, lactose, sucrose, glucose, mannitol, and silicic acid), b) one or more binders (binders may be selected from, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), c) one or more humectants (a humectant may be, but is not limited to, glycerol), d) one or more disintegrating agents (disintegrating agents may be selected from, but are not limited to, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates, and sodium carbonate), e) one or more solution retarding agents (for example, but not limited to, paraffin), f) one or more absorption accelerators (selected from, but not limited to, quaternary ammonium compounds), g) one or more wetting agents (for example, but not limited to, acetyl alcohol and glycerol monostearate), h) one or more absorbents (selected from, but not limited to, kaolin and bentonite clay), and i) one or more lubricants (selected from, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate). In the case of capsules, tablets and pills, for example, the dosage form may also comprise buffering agents.
[0074] "Effective or Therapeutic Amount’'
[0075] Effective or therapeutic amounts of the compositions of this disclosure include any amount sufficient to inhibit (e.g., slow or stop) the progression of a neurodegenerative disorder. In some embodiments, effective amounts of the compositions include any amount sufficient to inhibit (e.g., slow or stop) the deterioration of the cognitive function of a patient.
[0076] The amount of the active ingredient that may be combined with the optional carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. The specific dose level for any particular patient may depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disorder or disease undergoing therapy. A therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
[0079] Embodiments of the compositions and methods of the presents disclosure may be appreciated, without limitation, by reference to the following clauses:
[0080] Clause 1. A compound represented by Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond;
U and X are C,
V is CRV, and
Y and Z are N; or
X and Z are C,
V is CRV, and
V and U are N; or U, X and Z are C,
V is N, and
Y is S or O; or
U, X, and Z are C,
V is NRV, and
Y is N; or
X and Z are C, and
U, V and Y are N; or
U and Z are C, and
V, X and Y are N; RX is selected from the group consisting of RV selected from the group consisting of hydrogen, C1-C6alkyl, C2-C6alkenyl, C2- C6alkynyl, and C3-C6cycloalkyk wherein alky l, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy;
R1 is selected from the group consisting of hydrogen, -C(O)Ra, -(C=O)ORa, -C(=O)NRaRb, -S(O)2NRaRb, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, and C3-C6cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1- C6alkoxy;
R2 and R3 are each hydrogen, or R2 and R3 are taken together to form oxo;
R4 and R5 are each independently selected for each occurrence from the group consisting of halogen, hydroxyl, -CN, -NRaRb, C1-C6alkyl. C2-C6alkenyl, C2-C6alkynyl. C1-C6alkoxy. C3- C6cycloalkyl, -C(=O)NRaRb, -NRa(C=O)Rb, -O(C=O)NRaRb, -NRa(C=O)ORb, -
NRa(C=O)NRaRb, -(C=O)C1-C6alkyl, -(C=O)OC1-C6alkyl, -O(C=O)C1-C6alkyl, -SH, -SC1-C6alkyl. -S(O)2NRaRb, and -NRaS(O)2C1-C6alkyk wherein each alkyl, alkenyl, alkynyl, alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently- selected from the group consisting of halogen, hydroxyl, -NRaRb, C1-C6alkyl, and C1-C6alkoxy;
Ra and Rb are each independently selected from the group consisting of hydrogen C1- C6alkyl, and -CH2-phenyl; wherein C1-C6alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; or
Ra and Rb, together with the nitrogen to which they are attached, may be joined together to form a 4-7 membered heterocyclyl optionally substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, C1-C6alkyl, and C1-C6alkoxy; m is an integer 0. 1, 2, 3, 4, or 5; and n is an integer 0, 1, 2, 3, 4, or 5.
[0081] Clause 2. The compound of clause 1, wherein the compound is represented by:
[0082] Clause 3. The compound of clause 1 or 2, wherein R2 and R3 are each hydrogen.
[0083] Clause 4. The compound of clause 1 or 2, wherein R2 and R3 are taken together to form oxo.
[0084] Clause 5. The compound of any one of clauses 1-4, wherein RX is selected from the group consisting of:
[0085] Clause 6. The compound of any one of clauses 1-5, wherein R1 is selected from the group consisting of hydrogen, -Me, -C(O)Ra, and -(C=O)ORa. [0086] Clause 7. The compound of any one of clauses 1-6, wherein R1 is selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3.
[0087] Clause 8. The compound of any one of clauses 1-7. wherein RV is selected from the group consisting of hydrogen and C1-C6alkyl.
[0088] Clause 9. The compound of any one of clauses 1-8, wherein RV is selected from the group consisting of hydrogen, -CH3, and -CH(CH3)2.
[0089] Clause 10. The compound of any one of clauses 1-9, wherein m is an integer 0, 1, or 2.
[0090] Clause 11. The compound of any one of clauses 1-10, wherein R4 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0091] Clause 12. The compound of any one of clauses 1-11, wherein n is an integer 0, 1, or 2.
[0092] Clause 13. The compound of any one of clauses 1-12, wherein R5 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0093] Clause 14. A compound represented by: or a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: RX is selected from RV is selected from hydrogen and C1-C6alkyl;
R1 is selected from hydrogen, -C(O)C1-C6alkyl, and -(C=O)OC1-C6alkyl;
R2 and R3 are each independently selected from halogen and C1-C6alkyl; m is an integer 0, 1, or 2; and n is an integer 0, 1 , or 2.
[0094] Clause 15. The compound of clause 14, wherein RV is selected from the group consisting of hydrogen, -CH3, and -CH(CH3)2.
[0095] Clause 16. The compound of clause 14 or 15, wherein R1 is selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3.
[0096] Clause 17. The compound of any one of clauses 14-16, wherein R4 and R5 are independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
[0097] Clause 18. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0098] Clause 19. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[0099] Clause 20. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[00100] Clause 21. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[00101] Clause 22. A compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
[00102] Clause 23. A pharmaceutical composition comprising a compound of any one of clauses 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound, and a pharmaceutically acceptable excipient.
[00103] Clause 24. The composition of clause 23, further comprising one or more additional neurodegenerative disorder therapeutic agents.
[00104] Clause 25. The composition of either of clauses 23 or 24, further comprising one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers. [00105] Clause 26. A method of treating, preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject an effective amount of the compound of any one of clauses 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or the pharmaceutical composition of any one of clauses 23- 25.
[00106] Clause 27. The method of clause 26, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
[00107] Clause 28. The method of clause 27, wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
[00108] Clause 29. The method of any one of clauses 26-28, wherein the subject is a mammal.
[00109] Clause 30. The method of any one of clauses 26-28, wherein the subject is human patient.
[00110] Clause 31. The method of any one of clause 30, wherein the human patient is an adult.
[00111] Clause 32. Use of a compound of any of clauses 1-22 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
[00112] Clause 33. The use of clause 32, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
[00113] Clause 34. The use of clause 33. wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
[00114] Clause 35. The use of any one of clauses 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. [00115] Clause 36. The use of any one of clauses 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
[00116] Clause 37. The use of clause 36, wherein the human patient is an adult.
[00117] Clause 38. Use of a pharmaceutical composition of any of clauses 23-25 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
[00118] Clause 39. The use of clause 38, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
[00119] Clause 40. The use of clause 39. wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
[00120] Clause 41. The use of any one of clauses 38-40, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal. [00121] Clause 42. The use of any one of clauses 38-40, w herein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
[00122] Clause 43. The use of clause 42, wherein the human patient is an adult.
[00123] Further reference is made to the following experimental examples.
[00124] EXAMPLES
[00125] The following examples are provided for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.
[00126] General Methods and Procedures [00127] Chemical Synthesis and Validation. For each of the examples provided herein, reagents and solvents were purchased from commercial sources and used without further purification. All reactions involving air- or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE™ NEO 400 MHz or Bruker DRX500-1 500 MHz instruments.
[00128] For 1H NMR, chemical shifts in ppm relative to the residual solvent peak, multiplicities, coupling constants in Hertz, and numbers of protons are indicated. Reactions were routinely monitored by analytical thin layer chromatography (TLC) or liquid chromatographymass spectroscopy (LC-MS). Analytical TLC was performed on silica gel 60 F254 silica gel plates, and 254 nm ultraviolet (UV) light and/or I2 staining were used for visualization. Reaction monitoring LC-MS and high-performance liquid chromatography (HPLC) purity data were obtained on a Waters™ ACQUITY® ultra-performance liquid chromatography (UPLC) system equipped with a Waters™ ACQUITY® BEH C18 column (1.7 μm. 50 mm x 2.1 mm) or other specified column (see below), and either TUV and SQD2 detectors or PDA and QDa detectors. Flash normal phase (NP) or reversed-phase (RP) chromatography were performed on a Teledyne ISCO NextGen 300 instrument using prepacked silica gel or Cl 8-functionalized silica gel columns available from Teledyne ISCO, or on a Teledyne ISCO CombiFlash® using prepacked silica gel columns available from Agela or Welch. Preparative reverse-phase (RP) HPLC was performed on a Waters™ AutoPurification HPLC system equipped with PDA and ELSD detectors and a Waters™ XBridge Cl 8 Prep column (10 μm, 250 mm x 19 mm). High-resolution mass spectra were obtained on an Agilent 6550 Q-TOF instrument. All compounds had >95% purity as determined by LC-MS. One of the following specified LC-MS methods was used to determine test compound purity:
[00129] Method 1: gradient table A (below); column, Waters™ ACQUITY® HSS-T3 (1.8 μm, 100 mm x 2.1 mm); mobile phase A, 0.1% trifluoroacetic acid (TFA) in H2O; mobile phase B, acetonitrile (ACN); flow rate. 0.3 mL/min; detection wavelength, 214 nm; column temperature, 35 °C.
[00130] Method 2: gradient table A; column, Waters™ ACQUITY® BEH C-18 (1.7 μm, 100 mm x 2. 1 mm) [or C-8 (1.7 μm, 100 mm x 2. 1 mm) for 11]; mobile phase A, 5 rnM NH4OAC in H2O; mobile phase B, ACN; flow rate, 0.3 mL/min; detection wavelength, 214 nm; column temperature. 35 °C. [00131] Method 3: gradient table B; column. Waters™ ACQUITY® BEH C-18 (1.7 gm, 100 mm x 2.1 mm); mobile phase A, 5 mM NH4OAc in H2O; mobile phase B, ACN; flow rate, 0.3 mL/min; detection wavelength, 214 nm; column temperature, 35 °C.
[00132] Method 4: gradient table C; column, Waters™ ACQUITY® BEH C-18 (1.7 μm, 50 mm x 2.1 mm); mobile phase A, 0.1% formic acid in H2O; mobile phase B, 0.1% formic acid in ACN; flow rate, 0.6 mL/min; detection wavelength, 254 nm; column temperature, 40 °C.
[00133] The following intermediate synthesis steps and species may be applied to the Examples described herein:
Intermediate 1
(E)-2-methyl-3-phenylacrylaldehyde
[00134] To a stirring, 0 °C solution of potassium hydroxide (KOH) (529 mg, 9.42 mmol, 0.5 eq) in MeOH (10 mL) and H2O (10 mL) was added benzaldehyde (2 g, 18.8 mmol, 1 eq), followed 5 minutes later by propanal (also sometimes called “propionaldehyde”) (2.19 g, 37.7 mmol, 2 eq). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated and the residue was taken up with dichloromethane (DCM). The organic solution was washed with H2O and then with brine. The organic solution was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash LC on silica gel, eluting with 17% ethyl acetate in heptane, to provide (E)-2-methyl-3-phenylacrylaldehyde (1 g, 36%) as a semisolid. 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 7.68-7.58 (m, 2H), 7.55-7.40 (m. 4H), 1.99 (s, 3H).
Intermediate 2
4-methyl-l,5-diphenyl-1H-pyrazole
[00135] To a stirring solution of (E)-2-methyl-3-phenylacrylaldehyde (1 g. 6.84 mmol, 1 eq) and phenylhydrazine (labeled in the Synthesis Scheme 1 diagram as (ii)) (1.11 g, 10.3 mmol, 1.5 eq) in EtOH (10 mL) was added I2 (1.3 g, 10.3 mmol, 1.5 eq). The reaction mixture was heated to 80 °C for 16 hours. The reaction mixture was concentrated and the residue was taken up with DCM. The organic solution was washed with H2O and then with saturated aqueous NaHSO4 solution. The organic solution was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash LC on silica gel, eluting with 40% ethyl acetate (EtOAc) in heptane, to provide 4-methyl-l,5-diphenyl-lH-pyrazole (0.8 g, 50%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 7.64 (s, 1H), 7.42-7.24 (m, 6H), 7.21-7.13 (m, 4H), 2.05 (s, 3H); LRMS m/z (ES~) 235.2 [M+H]+.
Intermediate 3
3-Bromo-4-methyl-l,5-diphenyl-1H-pyrazole
[00136] To a stirring solution of 4-methyl-l,5-diphenyl-1H-pyrazole (1.00 g, 4.27 mmol, 1 eq) in ACN (7 mL) at 0 °C was added N-bromosuccinimide (NBS) (912 mg. 5.12 mmol, 1.2 eq). The reaction mixture was stirred at 60 °C for 16 hours. The reaction mixture was diluted with H2O, extracted with DCM, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash LC on silica gel eluting with 17% EtOAc in heptane to afford 3- bromo-4-methyl-1.5-diphenyl-lH-pyrazole (0.5 g. 37%) as a brown semisolid. 1H NMR (400 MHz, DMSO-d6) δ 7.50 - 7.30 (m, 6H). 7.30 - 7.15 (m, 4H). 1.99 (s. 3H).
Intermediate 4
Tert-butyl 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)-3,6-dihydropyridine-l(2H)- carboxylate
[00137] To a stirring solution of 3-bromo-4-methyl-l,5-diphenyl-1H-pyrazole (1.00 g, 3. 19 mmol, 1 eq) and tert-butyl 4-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolan-2-yl)-l,2,3,6- tetrahydropyridine-1 -carboxylate (1.18 g, 3.83 mmol, 1.2 eq) in EtOH (15 mL) was added K2CO3 (1.32 g, 9.58 mmol, 3 eq) and reaction mixture was purged with N2 for 20 min. Then palladium- tetrakis(triphenyl phosphine) (Pd(PPh3)4) (369 mg, 0.319 mmol, 0.1 eq) was added and reaction mixture was stirred and heated to reflux for 16h. The reaction mixture was diluted with H2O, extracted with EtOAc, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash LC on silica gel eluting with 20% EtOAc in hexane to afford tert-butyl 4-(4-methyl-1.5-diphenyl-lH-pyrazol-3-yl)-3,6-dihydropyridine-l(2H)-carboxylate (470 mg, 35%) as a brown semisolid. 1H NMR (400 MHz, DMSO-d6) δ 7.45 - 7.10 (m, 10H), 6.20 - 6.10 (m, 1H), 4.10 - 4.00 (m, 2H), 3.60 - 3.45 (m, 2H), 2.65 - 2.55 (m, 2H), 2.08 (s, 3H), 1.44 (s, 9H); LRMS m/z (ES+) 416.21 [M+H]+.
Example 1 tert- Butyl 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidine-l -carboxylate
[00138] To a stirring solution of tert-butyl 4-(4-methyl-l,5-diphenyl-lH-pyrazol-3-yl)-3,6- dihydropyridine-l(2H)-carboxylate (0.30 g, 0.722 mmol, 1 eq) in MeOH (7 mL) was added 10% w/w Pd/C (307 mg, 0.289 mmol. 0.4 eq) under N2 atmosphere at room temperature. Then the reaction mixture was stirred at room temperature for 16 hours under H2. The reaction mixture was filtered through celite, and the celite was washed with MeOH. The filtrate was concentrated in vacuo. The residue w as purified by flash LC on silica gel eluting with 70% EtOAc in heptane to afford tert-butyl 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidine-l-carboxylate (168 mg, 56%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.42 - 7.09 (m, 10H), 4.10 - 4.00 (m, 2H), 3.00 - 2.80 (m, 3H), 2.00 (s, 3H), 1.90 - 1.80 (m, 2H), 1.70 - 1.55 (m, 2H), 1.41 (s, 9H); LRMS m/z (ES+) 418.4 [M+H]+; HPLC purity 96.30% (Method 3).
[00139] The following compounds were prepared in a similar manner to Example 1.
Example 4
4-(4-inethyl-l .5-diphenyl- 1H-pyrazol-3-yl)piperi(line trifluoroactetate salt
[00140] To a stirring solution of tert-butyl 4-(4-methyl-l,5-diphenyl-lH-pyrazol-3- yl)piperidine-l -carboxylate (350 mg, 0.838 mmol, 1 eq) in DCM (5 mL) at 0 °C was added dropwise TFA (1.28 mL, 16.8 mmol, 20 eq). The reaction mixture was stirred with warming to room temperature for 16 hours. The reaction mixture was concentrated in vacuo and the residue was washed three times with Et2O to afford 4-(4-methyl- 1.5-diphenyl-1H-pyrazol-3-yl)piperidine (106 mg, 40%) as an off-white TFA salt. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.31 (s, 1H), 7.45 - 7.09 (m, 10H), 3.45 - 3.37 (m, 2H), 3.15 - 3.00 (m, 3H), 2.10 - 1.85 (m. 7H); LRMS m/z (ES+) 318.25 [M+H]+; HPLC purity 97.46% (Method 1).
[00141] The following compounds were prepared in a similar manner to Example 4.
Example 19 4-(l-(2-chlorophenyl)-4-methyl-5-phenyl-1H-pyrazol-3-yl)-l,2,3,6-tetrahydropyridine
[00142] To a stirred solution of tert-butyl 4-(l-(2-chlorophenyl)-4-methyl-5-phenyl-1H- pyrazol-3-yl)-3.6-dihydropyridine- 1 (2H)-carboxylale (prepared in a similar manner to Intermediate 4) (0.2 g, 444 μmol) in dichloromethane (3.35 mL, 52.3 mmol) was added trifluoroacetic acid (170 μL, 5 eq., 2.22 mmol) at 0 °C. The reaction mixture was stirred for 2 hours at room temperature. After completion of the reaction (TLC monitoring), reaction mixture was concentrated under reduced pressure. The crude was purified by PREP HPLC in TFA buffer, desired fractions were concentrated to afford 4-(l-(2-chlorophenyl)-4-methyl-5-phenyl-1H- pyrazol-3-yl)-l,2,3,6-tetrahydropyridine as a white TFA salt (64 mg, 138 μmol). LRMS m/z (ES+) 350.3 [M+H]+; HPLC purity 99.68% (Method 2).
[00143] The following compounds were prepared in a similar manner to Example 19.
Example 35 l-methyl-4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidine
[00144] To a stirred solution of tert-butyl 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3- yl)piperidine-l -carboxylate (250 mg, 599 μmol) in tetrahydrofuran (5 mL, 61.4 mmol) was added LiAIH4 (34. 1 mg, 1.5 eq., 898 μmol) at 0 °C and reaction mixture was stirred at 50 °C for 16 hours. After completion (TLC & LCMS monitoring), reaction mixture was diluted with ice cold water, extracted with DCM. washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude which was purified by preparative HPLC using TFA buffer to provide l-methyl-4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidine trifluoroacetate salt (75 mg, 215 μmol) as a colorless semi-solid. LRMS m/z (ES+) 332.0 [M+H]+; HPLC purity 95.8% (Method 1). l-[4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-l-yl]ethan- 1-one
[00145] To a stirred solution of 3-bromo-4-methyl-l,5-diphenyl-1H-pyrazole (0.5 g, 1.6 mmol) and 1 -(piperazin- l-yl)ethan-l -one (307 mg, 1.5 eq., 2.39 mmol) in toluene (10 mL. 94.1 mmol) was added cesium carbonate (1.56 g, 3 eq., 4.79 mmol) and reaction mixture was purged with nitrogen for 20 minutes. Then XPhos Pd G2 (126 mg, 0.1 eq., 160 μmol) was added and reaction mixture was heated at 110 °C and stirred for 16 hours. Reaction mixture was monitored by TLC and LCMS. Reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Crude was purified by flash chromatography using 20% EtOAc in hexane as an eluent, desired fractions were concentrated to afford l-[4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-l-yl]ethan-l-one (120 mg, 256 μmol) as a brown semi solid. MS ES+ 361.17.
[00146] The following compounds were prepared in a similar manner to Intermediate 5.
Example 37 l-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazine
[00147] To a stirred solution of l-[4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-l- yl]ethan-l-one (140 mg, 388 μmol) in water (3 mL, 167 mmol), the reaction mixture was cooled at 0 °C, and hydrogen chloride (HCl) (3 mL) was added. Reaction mixture was stirred at 100 °C for 4 hours, reaction was monitored by TLC. After completion, reaction mixture concentrated then quenched by saturated solution of NaHCO3 and extracted with ethyl acetate, washed with water with organic layer dry over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The crude material was purified by column chromatography eluted with 70% ethyl acetate in heptane. The crude was purified by Prep-HPLC using TFA buffer solution to provide l-(4-methyl- 1.5-di phenyl- 1 H-pyrazol-3-yl)piperazine trifluoroacetate salt (20 mg, 62.8 μmol) as an off white solid. LRMS m/z (ES+) 319.3 [M+H]+; HPLC purity 96.56% (Method 2).
Example 38 4-(4-methyl-l,5-diphenyl-lH-pyrazol-3-yl)pyridine
[00148] To a stirred solution of 3-bromo-4-methyl-l-phenyl-5-phenylpyrazole (0.2 g, 639 μmol) and pyridin-4-ylboronic acid (118mg, 1.5 eq.. 958 μmol) in 1,4-dioxane (5 mL, 58.6 mmol) and water (0.5 mL, 27.8 mmol) was added dipotassium carbonate (177mg, 2 eq., 1.28 mmol). Then the reaction mixture was purged with N2 for 15 minutes, palladium — triphenylphosphine (1/4) (73mg, 0.1 eq., 63.9 μmol) was added and purging continued for 5 minutes, then the reaction mixture was heated at 100 °C and stirred for 16 hours. After completion of the reaction (TLC monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated to obtain crude. Crude was purified by prep HPLC in ABC buffer to provide 4-(4- methyl-l,5-diphenyl-lH-pyrazol-3-yl)pyridine (26 mg, 82.7 μmol, 13%) as an off-white solid. LRMS m/z (ES+) 312.3 [M+H]+; HPLC purity 99.95% (Method 2).
Intermediate 6 tert-butyl 3-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)-8-azabicyclo[3.2.1 ]oct-2-ene-8- carboxylate
[00149] To a pressure vessel with stir bar was added 3-bromo-4-methyl-l-phenyl-5- phenylpyrazole (230 mg. 734 μmol). tert-butyl 3-(4.4.5.5-tetramethyl-l,3,2-dioxaborolan-2-yl)-8- azabicyclo[3.2.1]oct-2-ene-8-carboxylate (295 mg, 1.2 eq., 881 μmol), (lE,4E)-l,5-diphenyl-l,4- pentadien-3-one — l,5-diphenyl-l,4-pentadien-3-one — palladium (1/2/2) (34 mg, 0.051 eq., 37.1 μmol), 2,2'-bis(diphenylphosphino)-l, 1'-binaphthyl (69 mg, 0.15 eq., I l l μmol), dipotassium carbonate (203 mg, 2 eq., 1.47 mmol), 1,4-dioxane (21 mL), and H2O (2 mL). The vial was sealed and the atmosphere exchanged for N2, then the screw-cap was put in place and the reaction mixture was stirred at 100 °C for 16 hours. The reaction mixture was cooled to room temperature and filtered through a bed of Celite, washing with EtOAc. The filtrate was concentrated in vacuo and the residue was purified by flash LC eluting with 90: 10 hexanes:EtOAc to provide tert-butyl 3-(4- methyl-l,5-diphenyl-1H-pyrazol-3-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate as ayellow oil (357 mg, 808 umol, 110%). ES+ 442.62
Example 39
3-(4-Methyl-l,5-diphenyl-1H-pyrazoI-3-yl)-8-azabicyclo[3.2.1]oct- 2-ene dihydrochloride
[00150] To a round bottom flask containing tert-buty l 3-(4-methyl-l-phenyl-5-phenyl-3- pyrazolyl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (243 mg, 550 μmol) was added a solution of 4N HC1 in 1,4-dioxane (3 mL). The reaction mixture was stirred at room temperature overnight. TLC and LC-MS showed completion. The reaction mixture was concentrated under a stream of N2. Then the residue was dried under high vacuum to provide the dihydrochloride salt of 3-(4- methyl-l,5-diphenyl-1H-pyrazol-3-yl)-8-azabicyclo[3.2.1]oct-2-ene (167 mg, 403 μmol, 73%) as ayellow solid. ES+ 342.51
Intermediate 7 l-[(p-methoxyphenyl)methyl]-4-(4-methyl-l-phenyl-5-phenyl-3-pyrazolyl)-2-piperazinone [00151] To a stirred solution of 3- bromo-4-methyl-l-phenyl-5-phenylpyrazole (0.5 g, 1.6 mmol) and l-[(p-methoxyphenyl)methyl]-2-piperazinone — hydrogen chloride (1/1) (492 mg, 1.2 eq.. 1.92 mmol) in toluene (5 mL, 47 mmol) was added cesium carbonate (1.04 g, 2 eq.. 3.19 mmol). The reaction mixture was purged with N2 for 15 minutes, RuPhos Pd G3 (66.8 mg, 0.05 eq., 79.8 μmol) was then added and purging continued for another 5 minutes. The reaction mixture was heated at 130 °C and stirred for 16 hours. After completion of the reaction (TLC and LCMS monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated to obtain crude. Crude was purified by flash column chromatography using 10% MeOH in DCM as an eluent, desired fractions were concentrated to afford l-[(p- methoxyphenyl)methyl]-4-(4-methyl-l-phenyl-5-phenyl-3-pyrazolyl)-2-piperazinone (380 mg, 588 μmol, 37%) as white solid. DUIS+ 453. 15
Example 40 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-2-one
[00152] To the compound l-[(p-methoxyphenyl)methyl]-4-(4-methyl-l-phenyl-5-phenyl- 3-pyrazolyl)-2-piperazinone (350 mg, 773 μmol) in trifluoromethanesulfonic acid (205 μL, 3 eq., 2.32 mmol) was added trifluoroacetic acid (59.2 μL, 773 μmol) dropwise at 0 °C. The reaction mixture was heated at 70 °C for 3 hours. The solvent was evaporated by using reduced pressure and resultant residue was washed 2-3 times with Et2O and n-pentane to obtain crude. Crude was purified by prep HPLC using ABC buffer to afford 4-(4-methyl- l .5-diphenyl- lH-pyrazol-3- yl)piperazin-2-one (15 mg, 43.3 μmol, 7%) as a white solid. LRMS m/z (ES+) 333.3 [M+H]+; HPLC purity 96. 17% (Method 2).
Intermediate 8
Tert-butyl 4-(4-methyl-l,5-diphenyl-lH-pyrazol-3-yl)-3-oxopiperazine-l-carboxylate
[00153] In each of 8 parallel reactions combine 3-bromo-4-methyl-l .5-diphenyl- l rt- pyrazole (10 mg, 31.9 μmol, 1 eq), tert-butyl 3 -oxopiperazine- 1 -carboxylate (6 mg, 30 μmol, 1 eq), Cui (6 mg, 31.5 μmol. 1 eq), trans-N,N'-dimethylcyclohexane-l,2-diamine (5 mg, 35.2 μmol, 1 eq), K3PO4 (14 mg, 66 μmol, 2 eq), 1-2 activated 4A molecular sieves, and 1,4-dioxane (1 mL, anhydrous). The vial was sealed, the atmosphere was exchanged for N2 using high vacuum followed by N2 backfill, then stirred at 150 °C overnight. The reaction mixture was cooled to room temperature, diluted with EtOAc, and filtered through a Celite pad, washing the Celite pad with EtOAc. The filtrate was washed with saturated aq NH4CI solution, then with H2O, and then with brine. The organic solution was dried over MgSCL and filtered and concentrated in vacuo. The residue was purified by flash LC on silica gel, eluting with a 20 to 30 to 40% EtOAc in hexanes gradient, providing tert-butyl 4-(4-methyl-l,5-diphenyl-lH-pyrazol-3-yl)-3-oxopiperazine-l- carboxylate (51 mg, 255 μmol, 46%) as a beige solid. ES+ 433.69
Example 41 l-(4-Methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-2-one HC1
[00154] To tert-butyl 4-(4-methyl-l-phenyl-5-phenyl-3-pyrazolyl)-3-oxo-l- piperazinecarboxylate (60 mg, 139 μmol) in a vial with stir bar was added a solution of 4N HCl in 1,4-dioxane (2 mL). The reaction mixture was stirred at room temperature for 2 hrs. The reaction mixture was concentrated under a stream of N2. The obtained brown oil was next triturated by adding Et2O (approx. 1 mL) and then scraping the residue with a spatula then sonicating. The obtained suspension was filtered with vacuum suction and the collected solid was dried under high vacuum to provide l-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperazin-2-one HC1 salt (34 mg, 92.2 μmol. 66%) as a beige solid. ES+ 333.58.
Example 42 3,3-dimethyl-l-(4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidin-l-yl)butan- 1-one
[00155] To a stirred solution of 4-(4-methyl-l,5-diphenyl-1H-pyrazol-3-yl)piperidine — trifluoroacetic acid (1/1) (150 mg. 348 μmol) in dimethylformamide (5 mL. 64.6 mmol) at 0 °C under N2 atmosphere, was added l,l,3,3-tetramethyl-2-(3H-l,2,3,4-tetraazainden-3-yl)-3- isoureaium hexafluoridophosphate(l-) (198 mg, 1.5 eq., 521 μmol) and N- ethylbis(isopropyl)amine (135 mg, 3 eq., 1.04 mmol). The reaction mixture was stirred 30 minutes, then was added 3,3-dimethylbutyric acid (40.4 mg, 348 μmol) and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (TLC and LCMS monitoring), reaction mixture was quenched with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated to obtain crude which was purified by PREP HPLC using ABC buffer to afford 3,3-dimethyl-l-(4-(4-methyl-l,5- diphenyl-1H-pyrazol-3-yl)piperidin-l-yl)butan-l-one (83 mg, 0.2 mmol) as a white solid. LRMS m/z (ES+) 416.5 [M+H]+; HPLC purity 99.9% (Method 3).
Intermediate 9 3-Chl oro- 1 ,5-d iphenyl- 1 H-py razole
[00156] To a stirred l,5-diphenylpyrazolidin-3-one (3.3 g, 13.8 mmol) was added phosphoryl trichloride (10.8 mL. 8.3 eq., 115 mmol). The resulting reaction mixture was stirred at 100 °C for 16 hours. The progress of reaction was monitored by TLC & LCMS. The reaction mixture was quenched with NaHCO3, and ice, and extracted with EtOAc, washed with brine, dried overNa2SO4, filtered and concentrated under reduced pressure. The resulting mixture was purified by column chromatography in 1 % EtOAc/ heptane, the desired fraction was concentrated under reduced pressure to get 3-Chloro-l,5-diphenyl-1H-pyrazole (675 mg, 1.99 mmol) as semi solid which was used crude in the next reaction. DUIS+ MS 254.95
Intermediate 10 tert- butyl 4-(l-phenyl-5-phenyl-3-pyrazolyl)-l,2,3,6-tetrahydro-l-pyridinecarboxylate [00157] To a stirred solution of 3-chloro-l-phenyl-5-phenylpyrazole (0.3 g, 1.18 mmol) and tert-butyl-4-(4.4.5.5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-l ,2,3,6-tetrahydro-l - pyridinecarboxylate (401 mg, 1.1 eq., 1.3 mmol) in 1,4 dioxane (3 mL) and water (0.5 mL, 27.8 mmol) was added dipotassium carbonate (488 mg, 3 eq., 3.53 mmol). Then the reaction mixture was purged with N2 for 15 minutes, then was added Catacxium A Pd G3 (42.9 mg, 0.05 eq.. 58.9 prnol) and purging continued for 20 minutes, then the reaction mixture was heated at 120 °C and stirred for 16 hours. After completion of the reaction (TLC & LCMS monitoring), reaction mixture was concentrated and residue was diluted with chilled water, extracted with EtOAc, washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting mixture was purified by column chromatography in 3-5% EtOAc/ Heptane, desired fraction was concentrated under reduced pressure to get tert-butyl 4-(l-phenyl-5-phenyl-3- pyrazolyl)-l,2,3,6-tetrahydro-l-pyridinecarboxylate (350 mg, 872 prnol) as yellow solid. ES+ 402.4
Example 43 4-(l,5-diphenyl-1H-pyrazol-3-yl)-l,2,3,6-tetrahydropyridine
[00158] To a stirred solution tert-butyl 4-(l-phenyl-5-phenyl-3-pyrazolyl)-l, 2,3,6- tetrahydro-1 -pyridinecarboxylate (35 mg, 87.2 μmol) in trifluoroacetic acid (33.4 μL, 5 eq., 436 μmol) was added trifluoroacetic acid (33.4 μL, 5 eq., 436 μmol) at 0 °C. Then the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction (TLC & LCMS monitoring), reaction mixture was washed with pentane and concentrated under reduced pressure to afford l-phenyl-5-phenyl-3-(l,2,3,6-tetrahydro-4- pyridyl)pyrazole — trifluoroacetic acid (1/1) (41 mg. 98.7 μmol) as white solid. LRMS m/z (ES+) 302.3 [M+H]+; HPLC purity 99.46% (Method 2).
Intermediate 11
2-bromo-l-rnethyl-4,5-diphenyl-1H-imidazole
[00159] To a stirred solution 2-bromo-4,5-diphenylimidazole (550 mg, 1.84 mmol) was added sodium hydride (88.2 mg, 2 eq., 3.68 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 20 minutes, then was added iodomethane (137 μL, 1.2 eq., 2.21 mmol) at 0 °C and the reaction mixture was stirred for 2 hours at room temperature. After completion of the reaction (TLC&LCMS monitoring), reaction mixture quenched by water and extracted with DCM, washed with water. The combined organic layer dry over Na2SO4, filtered and evaporated under reduced pressure to obtained the crude compound. The crude was purified by column chromatography eluted with 45% EtOAc in heptane, desired fractions were concentrated to obtained 2-bromo-l-methyl-4,5-diphenylimidazole (350 mg, 1.07 mmol, 58.3%) as light brown solid. ES+ 313.19
Intermediate 12
2-bromo- 1 -methy 1-4, 5-diphenyl-1H-imidazole
[00160] To a stirred solution of l-phenyl-5-phenyl-1H-l,2,4-triazol-3-ylamine (0.9 g, 2.57 mmol) in acetonitrile (10 mL, 191 mmol) was added 3-methyl-l-nitrosooxybutane (377 μL, 1.1 eq.. 2.83 mmol) at 0 °C, followed by addition of copper dibromide (631 mg, 1.1 eq., 2.83 mmol). The reaction mixture was heated at 60 °C for 1 hour. The progress of reaction was monitored by TLC & LCMS, after completion reaction mixture was filtered through celite bed and concentrated. Crude was purified by flash column chromatography using 30% EtOAc in heptane as an eluent, desired fractions were concentrated reduced pressure to get 3-bromo-l-phenyl-5-phenyl-lH-l,2,4- triazole (0.1 g, 316 μmol, 12.3%) as brown solid. ES+ 302.16
Example 44 tert-butyl 4-(l-methyl-4,5-diphenyl-1H-imidazol-2-yl)-3,6-dihydropyridine-l(2H)- carboxylate
[00161] To a stirred solution of 2-bromo-l-methyl-4,5-diphenylimidazole (350 mg, 1.12 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,2,3,6-tetrahydro-l- pyridinecarboxylate (346 mg, 1.12 mmol) in 1,4 dioxane (8 mL) and water (2 mL, 111 mmol) was added disodium carbonate (355 mg, 3 eq., 3.35 mmol). Then the reaction mixture was purged with N2 for 15 minutes, after that palladium — triphenylphosphine (1/4) (64.6 mg, 0.05 eq., 55.9 μmol) was added and purging continued for 20 minutes, then the reaction mixture was heated at 100 °C and stirred for 16 hours. After completion of the reaction (TLC monitoring), reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting mixture was purified by flash chromatography using 25% EtOAc in n-heptane as an eluent, desired fractions were concentrated under reduced pressure to afford tert-butyl 4-(l-methyl-4,5-diphenyl-2-imidazolyl)-l,2,3,6- tetrahydro-1 -pyridinecarboxylate (450 mg, 1.04 mmol) as semi solid. LRMS m/z (ES+) 416.4 [M+H]+; HPLC purity 95.22% (Method 2).
[00162] The following compounds were prepared in a similar manner to Example 44.
Example 49 4-(l-methyl-4,5-diphenyl-1H-imidazol-2-yl)-l,2,3,6-tetrahydropyridine
[00163] To a stirred solution of tert-butyl 4-(l-methyl-4,5-diphenyl-2-imidazolyl)-l,2,3,6- tetrahydro-1 -pyridinecarboxylate (50 mg, 120 μmol) in dichloromethane (4 mL, 62.5 mmol) was added trifluoroacetic acid (46 μL, 5 eq., 602 μmol) at 0 °C. Reaction mixture was stirred at room temperature for 2 hours. Reaction was monitored by TLC. After completion, reaction mixture evaporated under reduced pressure to obtained the crude compound. The crude residue was purified by prep HPLC using TFA buffer solution to obtained l-methyl-4,5-diphenyl-2-(l,2,3,6- tetrahydro-4-pyridyl)imidazole — trifluoroacetic acid (1/1) (35 mg, 79.9 μmol. 66.4%) as yellow semi solid. LRMS m/z (ES+) 316.3 [M+H]+; HPLC purity 95.22% (Method 2).
[00164] The following compounds were prepared in a similar manner to Example 49.
Example 55 tert-butyl 4-( l-methyl-4,5-diphenyl-1H-iniidazol-2-yl)piperidine-l -carboxylate
[00165] To a stirred solution of tert-butyl 4-(l-methyl-4,5-diphenyl-2-imidazolyl)-l,2,3,6- tetrahydro-1 -pyridinecarboxylate (375 mg, 902 μmol) in ethyl acetate (20 mL, 203 mmol), was added platinum dioxide (205 mg, 902 μmol) into the reaction mixture under nitrogen atmosphere at room temperature Then reaction mixture was stirred at room temperature for 2 hours under H2. The reaction mixture was monitored by TLC and LCMS data. After completion of the reaction, the reaction mass was filter through celite bed and washed with EtOAc, concentrated to obtained crude. The crude was purified by column chromatography eluted with 15% EtOAc in heptane, desired fractions were concentrated to obtained tert-butyl 4-(l-methyl-4,5-diphenyl-2- imidazolyl)-l -piperidinecarboxylate (0.3 g, 704 μmol, 78%) as off white solid. LRMS m/z (ES ) 418.4 [M+H]+: HPLC purity 98.96% (Method 2).
[00166] The following compounds were prepared in a similar manner to Example 55.
Example 59
4-( l-methyl-4,5-dipheny 1- 1 H-imidazol-2-y l)piperidine
[00167] To a stirred solution of tert-butyl 4-(l-methyl-4,5-diphenyl-2-imidazolyl)-l- piperidinecarboxylate (150 mg, 359 μmol) in dichloromethane (7 mL, 109 mmol) was added trifluoroacetic acid (137 μL, 5 eq., 1.8 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours, reaction was monitored by TLC, after completion, reaction mixture evaporated under reduced pressure to obtained the crude compound. The crude residue was purified by prep HPLC using TFA buffer solution to obtained l-methyl-4,5-diphenyl-2-(4- piperidyl)imidazole — trifluoroacetic acid (1/1) (75 mg, 170 μmol, 50.6%) as white solid. LRMS m/z (ES+) 318.2 [M+H]+; HPLC purity 99.81% (Method 1).
[00168] The following compounds were prepared in a similar manner to Example 59.
Tert-butyl 4-(4,5-dibromo-2H-l, 2, 3-triazol-2-yl)piperidine-l -carboxylate
[00169] To a solution of 4,5-dibromo-2H-l,2,3-triazole (3.00 g, 13.2 mmol) and tert-butyl 4-bromo-l -piperidinecarboxylate (3.84 g, 1.1 eq., 14.5 mmol) inN, N-dimethylformamide (DMF) (125 mL) was added potassium iodide (220 mg, 0.1 eq., 1.32 mmol) and dicesium carbonate (12.9 g, 3 eq., 39.7 mmol). The reaction mixture was stirred and heated at 105 °C for 16 hours, then cooled to room temperature. The reaction mixture was diluted with H2O and extracted using EtOAc. The combined organic layers were washed with H2O then brine, then dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash LC on silica gel eluting with 90: 10 to 50:50 hexanes:EtOAc to provide tert-butyl 4-(4,5-dibromo-2H-l,2,3-triazol-2- yl)piperidine-l -carboxylate (2.7 g, 6.58 mmol, 50%) as a white solid. ES+ 311.30 (M-Boc+H+), 352.35 (M-tBu+H ).
4-(4.5-diphenyl-2H- 1.2.3-triazol-2-yl)piperidine dihydrochloride
[00170] In microwave vial with stir bar were combined tert- butyl 4-(4.5-dibromo-2H-l,2,3- triazol-2-yl)-l -piperidinecarboxylate (250 mg, 610 μmol), phenylboronic acid (186 mg, 2.5 eq., 1.52 mmol), dipotassium carbonate (168 mg, 2 eq., 1.22 mmol), (lE,4E)-l,5-diphenyl-l,4- pentadien-3-one — l,5-diphenyl-l,4-pentadien-3-one — palladium (1/2/2) (28 mg, 0.05 eq., 30.6 μmol), 4,5-bis(diphenylphosphino)-9,9-dimethyl-9H-xanthene (38 mg. 0.11 eq.. 65.7 μmol) and 1,4-dioxane (9 mL) and H2O (1 mL). Three cycles of hi-vac evacuation and N2 backfill were performed. The mixture was stirred and heated to 110 °C for 3 days. The reaction mixture was cooled to room temperature and then concentrated in vacuo. The residue was directly purified by flash LC on silica gel eluting with 5 to 10 to 15% EtOAc in hexanes, from which 221 mg Boc- protected intermediate was obtained as a white solid. To the Boc-protected intermediate was added 4N HC1 in 1,4-dioxane (2 mL). The reaction mixture was stirred at room temperature for 3 days. Then the reaction mixture was dried down under N2, suspended in Et2O, and filtered with vacuum suction to provide the dihydrochloride salt of 4-(4,5-diphenyl-2H-l,2,3-triazol-2-yl)piperidine (182 mg, 484 μmol, 79%) as a white solid. ES+ 305.53
Intermediate 14
3,5-dibromo-l-phenyl-1H-l,2,4-triazole
[00171] To a stirred solution of 3,5-dibromo-lH-l,2,4-triazole (3 g. 1.0 eq, 13.2 mmol) in toluene (20.0 mL) and pyridine (1.28 mL, 15.9 mmol) was added phenyl boronic acid (1.77 g, 14.5 mmol) and sodium carbonate (1.68 g, 15.9 mmol). The reaction mixture was purged with O2 for 15 minutes and copper bis(acetate) (2.4 g, 13.2 mmol) was added. Oxygen purging was continued for 20 minutes and the reaction mixture was heated at 70 °C under stirring. After 16 hours, reaction was complete based on TLC & LCMS analysis. The reaction mixture was diluted with cold water and extracted with EtOAc. Organic layer was combined and washed with water followed by brine. It was then dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting mixture was purified by flash column chromatography on SiO2 gel column using 0-5% EtOAc in heptanes and fractions containing the desired product were concentrated to afford 3,5- dibromo-1 -phenyl- 1H-1, 2, 4-triazole (1.9 g, 6.27 mmol) as a white solid.
Intermediate 15 tert-butyl 4-(3-bromo-l-phenyl-1H-l,2,4-triazol-5-yl)-3,6-dihydropyridine-l(2H)- carboxylate
[00172] To a stirred solution of 3,5-dibromo-l-phenyl-lH-l,2,4-triazole (1 g, 3.13 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,2,3,6-tetrahydro-l- pyridinecarboxylate (1.16 g. 3.76 mmol) in 1,4 dioxane (12. 1 mL) and water (1.21 mL) was added sodium carbonate (997 mg, 9.4 mmol) and the reaction mixture was purged with N2 for 15 minutes. To this suspension, Pd(PPh3)4 (181 mg, 157 μmol) was added and nitrogen purging was continued for additional 5 minutes. The reaction mixture was heated under vigorous stirring at 90 °C. After 16 hours, reaction was complete (TLC monitoring). The reaction mixture was concentrated and residue diluted with chilled water and extracted with EtOAc. The combined organics were washed with water followed by brine and then dried over anhydrous Na2SO4. The organics were filtered and concentrated. Crude was purified by Si-gel flash column chromatography (12 g Welch SiO2 gel LS column) using 0-10% EtOAc/heptanes as an eluant. The desired fractions were concentrated to afford tert-butyl 4-(3-bromo-l -phenyl- 1H-1, 2.4-triazol-5-yl)-3.6-dihydropyridine- l(2H)-carboxylate (800 mg, 1.97 mmol) as light brown solid.
Intermediate 16 tert-butyl 4-( 1.3-diphenyl- 1H-1.2.4-ti iazol-5-yl)-3.6-dihydi opyridine-l (2H)-carboxylate [00173] To a stirred solution of tert-butyl 4-(3-bromo-l-phenyl-lH-l,2,4-triazol-5-yl)- 1,2,3,6-tetrahydro-l-pyridinecarboxylate (700 mg, 1.0 eq, 1.73 mmol) in toluene (3.66 mL) and water (1.57 mL) was added phenyl boronic acid (232 mg, 1.9 mmol) and sodium carbonate (549 mg, 5.18 mmol). The resulting mixture was purged with N2 for 15 minutes, then Pd(PPh3)4 (99.8 mg, 86.4 μmol) was added and purging continued for additional 20 minutes. The reaction mixture was then heated at 80 °C under vigorous stirring for 16 hours. After completion of the reaction (TLC & LCMS monitoring) it was diluted with chilled water, extracted with EtOAc, washed with water followed by brine. The organic extract was dried over anhydrous Na2SO4. filtered and concentrated under reduced pressure. The resulting mixture was purified by Si-gel flash column chromatography on 12 g Welch SiO2 gel LS column using 30% EtOAc/ heptanes as eluant. The desired fractions were concentrated to afford tert-butyl 4-(l,3-diphenyl-lH-l,2,4-triazol-5-yl)-3,6- dihy dropyri dine- l(2H)-carboxy late as white solid (650 mg, 1.61 mmol). 1H-NMR (400 MHz, DMSO-d6): δ 8.06-8.04 (m. 2H), 7.59-7.55 (m, 5H). 7.51-7.43 (m, 3H). 6.05 (bs. 1H), 3.89 (bs, 2H), 3.44 (t, J= 5.12 Hz, 2H), 2.33-2.32 (bs, 2H), 1.40 (s, 9H) and LCMS: [M+H] + 403.40.
Example 65 4-(l,3-diphenyl-lH-l,2,4-triazol-5-yl)-l,2,3,6-tetrahydropyridine
[00174] To a stirred solution of tert-butyl 4-(l,3-diphenyl-1H-l,2,4-triazol-5-yl)-3,6- dihydropyridine-l(2H)-carboxylate (50 mg. 124 μmol) in dichloromethane (1.0 mL) was added trifluoroacetic acid (47.5 μL, 621 μmol) at 0 °C and the reaction was allowed to stir at room temperature for 2 hours. After completion (TLC & LCMS monitoring), reaction mixture was directly concentrated under reduced pressure. The crude residue was washed with pentane followed by diethyl ether and dried to afford l-phenyl-5-phenyl-3-(l,2,3,6-tetrahydro-4-pyridyl)-1H-l,2,4- triazole as TFA salt (30 mg, 72 μmol) as brown solid. 1H-NMR (400 MHz. DMSO-d6): δ 8.87 (s, 1H), 8.05-8.04 (d, J = 6.72 Hz, 2H), 7.60-7.57 (m, 5H), 7.52-7.47 (m, 3H), 6.01 (bs, 1H), 3.68- 3.58 (m, 2H), 3.27 (m, 2H), 2.60 (m, 2H) and LCMS: [M+H] + 303.40.
Intermediate 16 tert-butyl 4-( 1.3-diphenyl-1H-l .2.4-triazol-5-yl)-3.6-dihydropyridine-l (2H)-carboxylate
[00175] To a stirred solution of tert-butyl 4-(l-phenyl-3-phenyl-lH-l,2,4-triazol-5-yl)- 1 .2.3.6-tetrahydro- 1 -pyridinecarboxylate (200 mg, 497 μmol) in ethyl acetate (22.0 mL) was added 85% platinum dioxide (113 mg, 497 μmol). The reaction was flushed with nitrogen and then with hydrogen. Reaction was continued under hydrogen atmosphere (balloon pressure) for 1 hour. TLC and LCMS monitoring indicated complete consumption of starting material. The reaction mixture was fdtered by Celite and residue washed with ethyl acetate. The fdtrate was concentrated under reduced pressure and crude residue was purified by RP prep HPLC using ammonium bicarbonate buffer to afford tert-butyl 4-(l-phenyl-3-phenyl-lH-l,2,4-triazol-5-yl)-l-piperidinecarboxylate (100 mg. 247 μmol) as white solid. 1H-NMR (400 MHz. DMSO-d6): δ 8.04-8.02 (m. 2H), 7.63- 7.57 (m, 5H), 7.50-7.41 (m, 3H), 3.99-3.95 (d, J= 12.64 Hz, 2H), 3.07-3.01 (m, 1H), 2.78 (m, 2H), 1.85-1.82 (m, 2H), 1.72-1.64 (m, 2H), 1.41 (s, 9H) and LCMS: [M+H] + 405.40.
Example 66
4-(l,3-diphenyl-1H-l,2,4-triazol-5-yl)piperidine
[00176] To a stirred solution of tert-butyl 4-(l-phenyl-3-phenyl-lH-l,2,4-triazol-5-yl)-l- piperidinecarboxylate (80 mg, 198 μmol) in dichloromethane (1.5 mL) was added trifluoroacetic acid (75.7 μL, 989 μmol) at 0 °C and the reaction mixture was allowed to stir at room temperature for 2 hours. After completion (TLC & LCMS monitoring), the reaction mixture was concentrated under reduced pressure. Resulting gummy product was triturated and washed with pentane, then dried under vacuum to afford l-phenyl-3-phenyl-5-(4-piperidyl)-lH-l,2,4-triazole as TFA salt (45 mg, 108 μmol) as off white solid. 1H-NMR (400 MHz, DMSO-d6): δ 8.67 (bs, 1H), 8.39 (bs, 1H), 8.04-8.03 (m, 2H). 7.63 (m. 5H), 7.49-7.47 (m, 3H). 3.37-3.22 (m, 3H), 2.99 (m. 2H), 2.03 (m, 4H) and LCMS: [M+H] + 305.40.
Example 67
[00177] Biological Activity.
[00178] Malachite Green Enzyme Assay
Enzymatic inhibitory potencies (IC50) (see table below) were determined using PI(3,4,5)P3-diC8 as a substrate at 25 °C in 50 mM HEPES buffer (pH7.4, 150 mM NaCl, 2 mM MgCl2). See FIG. 2. Compounds diluted in DMSO were added to 384-well plates. Human SHIP11-899 multidomain enzyme solution was added. After a 20-minute incubation period, the reaction was initiated by addition of PI(3,4,5)P3-diC8. Final compound concentrations ranged from 50 nM to 950 μM. Final reaction concentrations for the PI(3,4,5)P3-diC8 substrate and the enzyme were 52 μM and 10 nM respectively. The reaction was quenched after 10 minutes by adding Malachite BioMol Green (Enzo Lifesciences, PA, USA). Plates were then incubated for 30 minutes at room temperature. Absorbance (620nm) was measured using a SpectraMax® Me5 Microplate Reader (Molecular Devices, LLC, USA). IC50 values were calculated by fitting absorbance versus inhibitor concentration and are reported as the geometric mean and standard error of mean with the number of repeats (n) indicated. Activities are shown in Table 1, below.
[00179] Table 1
[00180] Cellular Thermal Shift Assay (CETSA)
[00181] A split Nano Luciferase assay (SplitLuc CETSA) was used to demonstrate target engagement of SHIP1 inhibitors in a physiologically relevant cellular context by quantifying changes in the thermal stability of a HiBit-labeled full length SHIP1 protein in intact cells. See Martinez et al. (2018) Sci Rep 8: p. 9472; Oh-Hashi et al. (2017) Biochem Biophys Rep 12: pp. 40- 45. This assay was run in the following two formats with HMC3/HiBit-INPP5D stably transfected cells.
Thermal shift: Cells were treated with 40 μM compound for 60 minutes, then heated with a temperature gradient covering 38-52 °C for 3 minutes before luminescence detection. Mean and standard deviation of control Tm were determined to generate a "Mean+3SD" ΔTm, which was used as threshold to determine a significant ΔTm of compound from control. When the difference of ΔTm of compound treated cells from control Tm > 3SD, the compound was considered positive for target engagement, otherwise negative. SD = average standard deviation. Activities are shown in Tables 2, below'.
Compound dose response: Run at target Tm (44.2 °C for SHIP1) with compound dosing from 80 μM or 100 μM with 1 :3 serial dilutions to generate an 8-point curve. Cells were treated for 60 minutes before being heated at target Tm for 3 minutes before luminescence detection. The concentration that induced a half-maximum response (AC50) was calculated using a four-parameter logistic curve regression model with change at highest concentration noted when difference from control >3SD. Activities are shown in Tables 2. below.
[00182] Table 2
[00183] pHrodo-Myelin Phagocytosis/Cell Health Assay with Microglial Cells [00184] This 384-well plate high content imaging assay was developed to quantify phagocytosis and cell health simultaneously using either BV2 or HMC3 immortalized microglial cell lines or primary microglia isolated from mouse brain. See Mason, et al., "‘Microglial Phagocytosis/Cell Health High-Content Assay,” (2023) Curr Protoc 3: e724. Briefly, cells were cultured in DMEM GlutaMax media (ThermoFisher) containing 10% FBS and Pen-Strep in 37 °C 5% CO2 incubator. Day 1: Cells were plated (Coming Falcon 384 well Optilux Black and clear bottom plates for imaging) with BV2 at 400 cells/45pl/welk HMC3 at 600 cells/45pl/well, or primary at 2000 cell/45pl/well. Day 2: Cells were treated with 10x serially diluted compounds in a dose range of 60 μM to 3 nM for 48 hrs at 37 °C. Dav 3: Cells were seeded with pHrodo- myelin (for total 20 hrs) 24 hrs after starting compound treatment. The pHrodo-myelin stocks were at 1 mg/ml (protein equivalent) stored in -20 °C or -80 °C freezer. Stocks were thawed and diluted with culture media into 10x seeding solution (50pg/ml) and added 5pl/well to 384 well cell plate. Day 4: Cell staining and imaging. Nuclear staining solution was prepared by adding 1 pl of 10 mg/mL Hoechst-33342 to every 1 ml culture media that will be added to cell plate at 20 pl/well. The final concentration of Hoechst-33342 to cells was about 2.5 pg/ml. Cell plates were incubated for >30 min at 37 °C before imaging. Cell plates were scanned with an ArrayScan automatic high content imaging system using a 10x objective lens, 4 fields/well collected. Three measurements were obtained 1) mean total phagocytosis spot intensity per cell, 2) total cell counts per well, and 3) mean average nuclear intensify per cell for cell health. Apoptotic cells showed nuclear intensity increase (early apoptosis) or decrease (later apoptosis). Activities are shown in table 3 below.
[00185] Primary Mouse Microglia
[00186] Cortical tissue from C57BL/6J neonatal mice (P0-P3) was homogenized in Dulbecco's Modified Eagle Medium (DMEM), filtered through 250 and 100 μm mesh, and cultured in Advanced DMEM/F12 supplemented with 10% fetal bovine serum, lx GlutaMAX and lx Penicillin/Streptomycin. At 21 days in vitro (DIV), the cultures were subjected to mild trypsinization using 0.083% Trypsin-EDTA in DMEM for 30 mins to detach an intact layer of astrocytes. The microglia attached to the bottom were used as described in above to measure myelin phagocytosis and cell health. Activities are shown in table 3 below. [00187] Table 3
[00188] INCORPORATION BY REFERENCE
[00189] The contents of all publications cited herein are incorporated by reference in their entireties.

Claims

IN THE CLAIMS
1. A compound represented by Formula (I): a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: denotes a single bond or a double bond;
U and X are C,
V is CRV, and
Y and Z are N; or
X and Z are C,
V is CRV, and
Y and U are N; or
U, X and Z are C,
V is N, and
Y is S or O; or
U, X, and Z are C,
V is NRV, and
Y is N; or
X and Z are C, and
U, V and Y are N; or
U and Z are C, and
V, X and Y are N; RX is selected from the group consisting of wherein o is an integer 0 or 1 ; RV is selected from the group consisting of hydrogen, C1-C6alkyl, C2-C6alkenyl, C2- C6alkynyl, and C3-C6cycloalkyl; wherein alky l, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy;
R1 is selected from the group consisting of hydrogen, -C(O)Ra, -(C=O)ORa, -C(=O)NRaRb, -S(O)2NRaRb, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, and C3- C6cycloalkyl; wherein alkyl, alkenyl, alkynyl, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy;
R2 and R3 are each hydrogen, or R2 and R3 are taken together to form oxo;
R4 and R5 are each independently selected for each occurrence from the group consisting of halogen, hydroxyl, -CN. -NRaRb, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1- C6alkoxy, C3-C6cycloalkyl, -C(=O)NRaRb, -NRa(C=O)Rb. -O(C=O)NRaRb. - NRa(C=O)ORb, -NRa(C=O)NRaRb, -(C=O)C1-C6alkyl, -(C=O)OC1-C6alkyl, -O(C=O)C1- C6alkyl, -SH, -SC1-C6alkyl, -S(O)2NRaRb, and -NRaS(O)2C1-C6alkyl; wherein each alkyl, alkenyl, alkynyl, alkoxy, and cycloalkyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl. -NRaRb, C1-C6alkyl, and C1-C6alkoxy;
Ra and Rb are each independently selected from the group consisting of hydrogen C1- C6alkyl. and -CH2-phenyl; wherein C1-C6alkyl and phenyl may optionally be substituted with one or more substituents each independently selected from the group consisting of halogen, hydroxyl, and C1-C6alkoxy; or
Ra and Rb, together with the nitrogen to which they are attached, may be joined together to form a 4-7 membered heterocyclyl optionally substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, C1-C6alkyl, and C1-C6alkoxy; m is an integer 0, 1, 2, 3, 4, or 5; and n is an integer 0, 1. 2, 3, 4, or 5.
2. The compound of claim 1, wherein the compound is represented by:
3. The compound of claim 1 or 2, wherein R2 and R3 are each hydrogen.
4. The compound of claim 1 or 2, wherein R2 and R3 are taken together to form oxo.
5. The compound of any one of claims 1-4, wherein RX is selected from the group consisting of:
6. The compound of any one of claims 1-5, wherein R1 is selected from the group consisting of hydrogen, -Me, -C(O)Ra, and -(C=O)ORa.
7. The compound of any one of claims 1-6. wherein R1 is selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3.
8. The compound of any one of claims 1-7, wherein RV is selected from the group consisting of hydrogen and C1-C6alkyl.
9. The compound of any one of claims 1-8, wherein RV is selected from the group consisting of hydrogen, -CH3, and -CH(CH3)2.
10. The compound of any one of claims 1-9, wherein m is an integer 0, 1, or 2.
11. The compound of any one of claims 1-10, wherein R4 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
12. The compound of any one of claims 1-11, wherein n is an integer 0, 1, or 2.
13. The compound of any one of claims 1-12, wherein R5 is independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
14. A compound represented by: or a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound, or a salt of a stereoisomer of the compound, wherein: RX is selected from RV is selected from hydrogen and C1-C6alkyl;
R1 is selected from hydrogen, -C(O)C1-C6alkyl, and -(C=O)OC1-C6alkyl; R2 and R3 are each independently selected from halogen and C1-C6alkyl; m is an integer 0, 1, or 2; and n is an integer 0, 1, or 2.
15. The compound of claim 14, wherein RV is selected from the group consisting of hydrogen, - CH3. and -CH(CH3)2.
16. The compound of claim 14 or 15, wherein R1 is selected from the group consisting of hydrogen, -C(O)CH3, and -(C=O)OC(CH3)3.
17. The compound of any one of claims 14-16, wherein R4 and R5 are independently selected for each occurrence from the group consisting of fluoro, chloro, and -CH3.
18. A compound selected from the group consisting of
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
19. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
20. A compound selected from the group consisting of:
or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
21. A compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
22. A compound selected from the group consisting of: or a pharmaceutically acceptable salt of such compound, or a stereoisomer of such compound, or a pharmaceutically acceptable salt of a stereoisomer of such compound.
23. A pharmaceutical composition comprising a compound of any one of claims 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound, and a pharmaceutically acceptable excipient.
24. The composition of claim 23, further comprising one or more additional neurodegenerative disorder therapeutic agents.
25. The composition of either of claims 23 or 24, further comprising one or more pharmaceutically acceptable adjuvants, binders, carriers, diluents, or fillers.
26. A method of treating, preventing, or inhibiting the progression of a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject an effective amount of the compound of any one of claims 1-22, a pharmaceutically acceptable salt of the compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt of a stereoisomer of the compound; or the pharmaceutical composition of any one of claims 23-25.
27. The method of claim 26, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
28. The method of claim 27, wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
29. The method of any one of claims 26-28, wherein the subject is a mammal.
30. The method of any one of claims 26-28, wherein the subject is human patient.
31. The method of any one of claim 30. w herein the human patient is an adult.
32. Use of a compound of any of claims 1-22 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
33. The use of claim 32, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer's disease, Alzheimer's disease-related dementia, and mild cognitive impairment.
34. The use of claim 33, wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
35. The use of any one of claims 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal.
36. The use of any one of claims 32-34, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
37. The use of claim 36, wherein the human patient is an adult.
38. Use of a pharmaceutical composition of any of claims 23-25 for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition.
39. The use of claim 38. wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer’s disease, Alzheimer’s disease-related dementia, and mild cognitive impairment.
40. The use of claim 39. wherein the Alzheimer’s disease-related dementia is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and multiple etiology dementias.
41. The use of any one of claims 38-40, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a mammal.
42. The use of any one of claims 38-40, wherein the medicament is formulated for the treatment or prevention of a neurodegenerative disorder or related condition, or to inhibit progression of such neurodegenerative disorder or related condition in a human patient.
43. The use of claim 42, wherein the human patient is an adult.
PCT/US2025/020356 2024-03-20 2025-03-18 Ship1 modulators and methods of treatment and uses thereof Pending WO2025199092A1 (en)

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Citations (6)

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US20080318070A1 (en) * 2005-05-24 2008-12-25 Shikoku Chemicals Corporation Water-Soluble Preflux and Usage of the Same
US20090149463A1 (en) * 2004-02-20 2009-06-11 Leifeng Cheng Therapeutic agents
US20130345268A1 (en) * 2009-02-13 2013-12-26 The Trustees Of Dartmouth College Methods and Compositions for the Treatment of RAS Associated Disorders
WO2024015759A1 (en) * 2022-07-14 2024-01-18 The Trustees Of Indiana University Crizotinib analogues as ship1 inhibitors useful to treat alzheiner's diseases
WO2024092205A1 (en) * 2022-10-27 2024-05-02 The Trustees Of Indiana University Inhibition of ship1 as a therapeutic strategy for the treatment of alzheimer's disease

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US20090149463A1 (en) * 2004-02-20 2009-06-11 Leifeng Cheng Therapeutic agents
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WO2007106721A2 (en) * 2006-03-10 2007-09-20 Jenrin Discovery Cannabinoid receptor antagonists/inverse agonists useful for treating obesity
US20130345268A1 (en) * 2009-02-13 2013-12-26 The Trustees Of Dartmouth College Methods and Compositions for the Treatment of RAS Associated Disorders
WO2024015759A1 (en) * 2022-07-14 2024-01-18 The Trustees Of Indiana University Crizotinib analogues as ship1 inhibitors useful to treat alzheiner's diseases
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