Crizotinib analogues as SHIP1 inhibitors useful for the treatment of Alzheimer's disease

Abstract

The general field of the present disclosure is novel treatments for Alzheimer's disease and other neurodegenerative disorders using novel therapeutic agents, including SHIP1 phosphatase inhibitors. Specifically, the present disclosure provides compounds, pharmaceutical compositions, and methods for treating and preventing such diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 389,039, filed July 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] References to government subsidies

[0004] This invention was made with government support awarded under the National Institutes of Health under AG065181. The government has certain rights in this invention.

[0005] Field

[0006] The general field of the present disclosure is novel treatments for Alzheimer's disease and other neurodegenerative disorders using novel therapeutic agents, including SHIP1 phosphatase inhibitors. [Background technology]

[0007] Background technology

[0008] Alzheimer's disease (AD) is a fatal neurodegenerative disorder characterized by the histopathological accumulation of extracellular β-amyloid (Aβ) plaques and intraneuronal neurofibrillary tangles (NFTs), which are hypothesized to lead to neurotoxicity and progressive cognitive decline. 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. Alzheimer's Dis 9: pp.151-153, Shen et al. 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 involved in neuropathological processes that lead to subsequent NFT lesions, neuroinflammation, and neuronal loss; however, the mechanisms by which Aβ influences neurotoxic signaling, including NFT formation, remain an area of intense investigation. In recent years, the amyloid cascade hypothesis has come under increasing scrutiny due to the failure of drugs targeting Aβ peptide processing and Aβ plaques. See Panza et al., “A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease,” (2019) Nat Rev Neurol 15:73-88.

[0009] More recent evidence includes genome-wide association studies (GWAS) and differential gene expression studies comparing normal brain tissue with Alzheimer's disease brain tissue, which have identified risk and protective variants in genes essential for microglial function, such as TREM2, CD33, APOE, ABCA7, PLCG2, and INPP5D. See Malik et al., "Genetics ignite focus on microglial inflammation in Alzheimer's disease," (2015) Mol Neurodegener 10:52. Microglia are 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 3:405-429.

[0010] During development, microglia arise from stem cells in the yolk sac and express CD45 + , CX3CR1 +They differentiate into immune cells, which migrate to the CNS. See Kierdorf et al., “Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways,” (2013) Nat Neurosci 16:273-280. Once settled, these cells slowly regenerate at a rate of approximately 28% per year in humans, thus providing a mechanism for microglia renewal. See Reu et al., “The Lifespan and Turnover of Microglia in the Human Brain,” (2017) Cell Rep 20:779-784. Disease-associated microglia (DAMs) 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 169:1276–1290. Their association with human microglia in AD remains an area of intense research; they possess genetic signatures related to lipid metabolism and phagocytosis, hypothesized to reflect microglia's neuroprotective role 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 11:6129. A two-state model of DAM induction has been proposed, in which homeostatic microglia, associated with and supportive of neuronal health, become activated through increased expression of DAP12, APOE, and triggering receptor expressed on myeloid cells-2 (TREM2). TREM2 ligands, such as apolipoprotein E (APOE) and Aβ, induce microglial differentiation into stage 2 DAMs with increased expression of LP1, 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.

[0011] TREM2 is a receptor expressed on the surface of microglia. Genetic evidence suggests that reduced TREM2 expression and inactivating variants increase the risk of AD. See Jonsson et al., “Variant of TREM2 associated with the risk of Alzheimer's disease,” (2013) N Engl J Med 368:107-116. TREM2 binds Aβ and APOE, activating microgliosis and 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 91:328-340. TREM2 R47H The mutant reduces TREM2 ligand affinity and downstream cellular activation, which requires DAP12, an adaptor protein on the intracellular side of the plasma membrane that associates with multiple signaling mediators. See Sudom et al., “Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant of the immune receptor TREM2,” (2018) J Biol Chem 293:12634-12646. For example, recruitment of SYK to phosphotyrosine residues in the C-terminus of DAP12 promotes phosphorylation of PLCγ2 and subsequent formation of intracellular IP3 and Ca2+. 2+It mediates signal transduction through release of TREM2 / DAP12. See Konishi et al., “Microglial TREM2 / DAP12 Signaling: A Double-Edged Sword in Neural Diseases,” (2018) Front Cell Neurosci 12:206. Importantly, an activating mutant of PLCG2, PLCG2 P522R 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 11:16. This human genetic evidence suggests that activated microglia are protective, clearing Aβ oligomers and plaques and mitigating the inflammatory microenvironment that is toxic to neurons, while attenuating microglial activity increases the risk of neurodegeneration. See Deczkowska et al., 2018.

[0012] The recent 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 181:1276-1278. Cell surface Fcγ receptors (FcγRs) on microglia recognize the Fc portion of IgG antibodies, triggering downstream effector functions. See Bournazos et al., “The role of IgG Fc receptors in antibody-dependent enhancement,” (2020) Nat Rev Immunol 20:633-643. Clearance of Aβ deposits by aducanumab has been demonstrated to occur by targeting Aβ through enhanced microglial recruitment and phagocytosis mediated by the variable region of the antibody linked to FcγR. See Sevigny et al., “The antibody aducanumab reduces Abeta plaques in Alzheimer's disease,” (2016) Nature 537; pp. 50-56.

[0013] The INPP5D gene encodes Src homology 2 (SH2) domain-containing phosphatase-1 (SHIP1), a phosphatidylinositol phosphatase that plays a key role in regulating pathways downstream of TREM2. See Peng et al., “TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1,” (2010) Sci Signal 3:38; Pauls et al., “Regulation of immune cell signaling by SHIP1: A phosphatase, scaffold protein, and potential therapeutic target,” (2017) Eur J Immunol 47:932-945. SHIP1 contains 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 phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2-[The text then shifts to a more detailed description of the PH and C2 domains, which are complex multidomain proteins. See Damen et al., "The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase," (1996) Proc Natl Acad Sci USA 93:1689-1693; Blunt et al., "Pharmacological targeting of phosphoinositide lipid kinases and phosphatases in the immune system: success, disappointment, and new opportunities," (2012) Front Immunol 3:226. The PH and C2 domains position and orient the catalytic site toward its PI(3,4,5)P3 substrate on the intracellular side of the membrane. The C2 domain is essential for cellular function and interactions between Ptase enzymes, and the C2 domain regulates enzymatic activity. See Le Coq et al., "Structural basis for interdomain communication in SHIP2 providing high phosphatase activity," (2017) Elife 6:26640. SHIP1 converts PI(3,4,5)P3 to PI(3,4)P-2. SHIP1 also contains an N-terminal SH2 domain that binds to immunoreceptor tyrosine-based activation motifs (ITAMs) and a C-terminal proline-rich domain that binds to many other proteins, including PLCγ2 and 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 586:2662-2673. SHIP1 binds to ITAMs, competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P. -2 This is understood to limit downstream signaling in various ways, thereby suppressing microglial activation, thereby converting them into ATP. See Pauls et al. 2017.

[0014] Taken together, this understanding of AD risk and protective variants in genes critical for microglial activity, their role in Aβ clearance, and SHIP1 as a downstream restrictive node of TREM2 and FCγRIIB suggests that inhibiting SHIP1 activates microglia and thus represents an effective therapeutic strategy in the disease. We also anticipate that this therapeutic intervention could be synergistically combined with anti-amyloid and / or TREM2 agonist antibodies. Therefore, SHIP1 inhibitors are likely to enhance the protective function of microglia and thus could be used to prevent or treat disease, reduce the rate of disease progression and cognitive decline, and reverse neurodegeneration in patients.

[0015] The present disclosure provides novel compounds that are SHIP1 inhibitors that address the need for potent and effective treatments for Alzheimer's disease and dementia associated with Alzheimer's disease. The present disclosure also provides pharmaceutical compositions for preventing Alzheimer's disease and dementia associated with Alzheimer's disease. Summary of the Invention

[0016] overview

[0017] Recent evidence, including GWAS and differential gene expression studies comparing normal brain tissue with brain tissue affected by Alzheimer's disease, has identified risk and protective variants in genes essential for microglial function, such as TREM2, PLCG2, and INPP5D. INPP5D encodes SHIP1, a multidomain protein containing a phosphatase that converts PI(3,4,5)P3 to PI(3,4)P2, an SH2 domain that interacts with receptor ITAMs and competes with SYK, and a proline-rich region that binds to many other proteins. Thus, SHIP1 limits microglial activity in multiple ways. Inhibition of SHIP1 early in the disease process enhances microglial protective function and reduces the rate of disease progression and cognitive decline in Alzheimer's disease patients.

[0018] We performed a 50,000-compound screen on SHIP1 phosphatase, analyzed publicly available fragment-based screens, and evaluated inhibitors reported in the literature. We used a malachite green assay with PtdIns(3,4,5)P3-diC8 and SHIP1 Ptase-C2 to measure inhibitory potency. A cellular thermal shift assay was used to confirm intracellular target engagement. A phospho-AKT assay was used to provide additional evidence of on-target activity. High-content imaging assays measuring phagocytosis, cell count, and nuclear intensity were performed using BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Mouse microglia were analyzed to demonstrate similar activity in primary cells. Inhibitors predicted to have drug-like properties were subjected to assays measuring solubility, cell permeability, and mouse microsomal stability. Physiologically based pharmacokinetic models were compared to in vivo measured exposure for selected compounds upon oral administration in mice.

[0019] Inhibition of SHIP1 is a novel therapeutic strategy for the treatment of Alzheimer's disease. Structurally distinct molecular scaffolds with varying degrees of enzyme inhibition, cellular activity, and exposure in mice were identified.

[0020] More specifically, the present disclosure provides a compound of formula I: [ka]

[0021] wherein each X is independently C or N;

[0022] n1+n2=4,

[0023] R1, R2 are independently H, F, Cl, Br or methyl;

[0024] R3 is H, amide, carbamate, or alkyl;

[0025] R4 is H or CH3

[0026] or a pharmaceutically acceptable salt thereof.

[0027] In other embodiments, the present disclosure includes a method of treating Alzheimer's disease in a patient, comprising administering to a patient in need thereof a compound of the present disclosure or a pharmaceutically acceptable salt thereof. Additionally, the present disclosure includes a method of treating dementia associated with Alzheimer's disease in a patient, comprising administering to a patient in need thereof a compound of the present disclosure or a pharmaceutically acceptable salt thereof. The present disclosure further provides a method of treating the progression of mild cognitive impairment to Alzheimer's disease or a related dementia in a patient, comprising administering to a patient in need thereof an effective amount of a compound of the present disclosure. The present disclosure further provides a method of preventing Alzheimer's disease or dementia associated with Alzheimer's disease, comprising administering to a patient in need thereof an effective amount of a compound of the present disclosure.

[0028] The present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. In another embodiment, the composition further comprises one or more additional therapeutic agents. In a further embodiment, the present disclosure provides a pharmaceutical composition for the treatment of Alzheimer's disease, comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. In yet another embodiment, the present disclosure provides a pharmaceutical composition for the treatment of dementia associated with Alzheimer's disease, comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more carriers, diluents, or pharmaceutically acceptable excipients. In yet another embodiment, the present disclosure provides a pharmaceutical composition for the prevention of Alzheimer's disease and dementia associated with Alzheimer's disease, comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more carriers, diluents, or pharmaceutically acceptable excipients.

[0029] Additionally, the present disclosure provides compounds of the present disclosure, or pharmaceutically acceptable salts thereof, for use in therapy, particularly for the treatment of Alzheimer's disease. Additionally, the present disclosure provides compounds of the present disclosure, or pharmaceutically acceptable salts thereof, for use in the treatment of Alzheimer's disease. In a further embodiment, the present disclosure provides the use of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of Alzheimer's disease.

[0030] In other embodiments, the present disclosure provides a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for use in therapy, particularly for the treatment of dementia associated with Alzheimer's disease. Additionally, the present disclosure provides a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for use in the treatment of dementia associated with Alzheimer's disease. In a further embodiment, the present disclosure provides the use of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of dementia associated with Alzheimer's disease.

[0031] The present disclosure further provides a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for use in preventing Alzheimer's disease and dementia associated with Alzheimer's disease. The present disclosure further provides a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for use in preventing Alzheimer's disease and dementia associated with Alzheimer's disease. In a further embodiment, the present disclosure provides the use of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in preventing Alzheimer's disease and dementia associated with Alzheimer's disease.

[0032] The present disclosure also includes intermediates and processes useful in the synthesis of the disclosed compounds.

[0033] Alzheimer's disease-related dementia (ADRD) includes dementia with Lewy bodies (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and dementia of multiple etiologies. Mild cognitive impairment is defined as a potential prodromal stage of Alzheimer's disease-related dementia based on clinical features and the progression of patients with mild cognitive impairment to Alzheimer's disease over time. See Morris et al., "Mild cognitive impairment represents early-stage Alzheimer's disease," (2001) Arch Neurol 58:397-405; Petersen et al., "Mild cognitive impairment: clinical characterization and outcome," (1999) Arch Neurol 56:303-308.

[0034] These and other embodiments and features of the present disclosure will become more apparent with reference to the following description, the accompanying drawings, and the claims. Furthermore, it will be understood that the features of the various embodiments described herein can exist in various combinations and permutations without being mutually exclusive.

[0035] In some embodiments, the present disclosure provides compositions comprising a SHIP1 inhibitor for use in combination with an antibody or antigen-binding fragment for treating Alzheimer's disease and dementia associated with Alzheimer's disease. [Brief explanation of the drawings]

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] [Figure 1] FIG. 1 is a schematic diagram showing the SHIP1 protein domain and other signaling proteins downstream of the TREM2 receptor.

[0038] [Figure 2] FIG. 1 is a schematic diagram showing the Malachite Green enzyme assay.

[0039] [Figure 3] 1 shows the activity of compounds 9 and 10 in primary mouse microglia.

[0040] [Figure 4] 1 is a graph showing the pharmacokinetics of compounds 9 and 10 in C57BL / 6J mice. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description

[0042] Various quantities, such as amounts, sizes, dimensions, proportions, etc., are presented in range format throughout this disclosure. It should be understood that the description of quantities 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 possible subranges as well as every individual numerical value within that range, unless the context clearly dictates otherwise. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values within that range, e.g., 1.1, 2, 2.3, 4.6, 2, 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 limits in the stated ranges. 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.

[0043] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. As used herein, the singular forms "a," "an," and "the" are intended to encompass the plural forms unless the context clearly dictates otherwise. It will be further understood that the terms "includes," "comprises," "including," and / or "comprising," as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude 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 will be understood that items in a list of the form "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, an item listed in the form "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).

[0044] Unless expressly specified or clear from the context, the term "about" as used herein in reference to a number or range of numbers is understood to mean the stated number and that number + / - 10%, or 10% below the stated lower limit or 10% above the stated upper limit of the values recited for a range.

[0045] Recent evidence includes GWAS and differential gene expression studies comparing normal brain tissue with brain tissue affected by Alzheimer's disease, which have identified risk and protective variants in genes essential for microglial function, such as TREM2, PLCG2, and INPP5D. The INPP5D gene encodes Src homology 2 (SH2) domain-containing phosphatase-1 (SHIP1), a phosphatidylinositol phosphatase that plays a key role in regulating pathways downstream of TREM2. See Peng et al., 2010; Pauls et al., 2017. SHIP1 contains pleckstrin homology (PH) domains on either side of the phosphatase (Ptase) domain that bind phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P]. 2- SHIP1 is a complex multidomain protein with a C2 domain that binds to PI(3,4,5)P3 and a PH domain that binds to PI(3,4,5)P3. See Figure 1, Damen et al. 1996; Blunt et al., 2012. The PH and C2 domains position and orient the catalytic site toward its PI(3,4,5)P3 substrate on the intracellular side of the membrane. The C2 domain is essential for cellular function and interactions between PTases, and the C2 domain regulates enzymatic activity. See Le Coq et al., 2017. SHIP1 converts PI(3,4,5)P3 to PI(3,4)P-2. SHIP1 also contains an N-terminal SH2 domain that binds to immunoreceptor tyrosine-based activation motifs (ITAMs) and a C-terminal proline-rich domain that binds to many other proteins, including PLCγ2 and Tec and Syk family kinases. PI(3,4,5)P3 binds and activates other PH-containing proteins, such as PLCγ2, PDK1, and AKT. See Sscheffzek et al. 2012. SHIP1 binds to receptor ITAMs and competes with kinases to convert PI(3,4,5)P3 to PI(3,4)P. -2Therefore, our therapeutic hypothesis is that inhibiting SHIP1 early in the disease process will enhance microglial protective function and reduce the rate of disease progression and cognitive decline in Alzheimer's disease patients.

[0046] In any of the embodiments disclosed herein, the term "treating the progression of mild cognitive impairment to Alzheimer's disease" includes inhibiting, slowing, halting, or reversing the progression of mild cognitive impairment to Alzheimer's disease in a patient.

[0047] In any of the embodiments disclosed herein, the term "treating" or "to treat" includes inhibiting, slowing, halting, or reversing the progression or severity of an existing condition or disease.

[0048] In any of the embodiments disclosed herein, the term "patient" refers to a human.

[0049] The compounds of the present disclosure can react to form pharmaceutically acceptable salts. Pharmaceutically acceptable salts and general methods for preparing them are well known in the art. For example, see 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.

[0050] excipients

[0051] Illustrative, non-limiting examples of excipients or carriers include sodium citrate or dicalcium phosphate and / or a) one or more fillers or extenders (fillers or extenders may be one or more selected from, but are not limited to, starch, lactose, sucrose, glucose, mannitol, and silicic acid), b) one or more binders (binders may be selected from, but are not limited to, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), c) one or more humectants (humectants may be, but are not limited to, glycerol), d) one or more disintegrants (disintegrants may be selected from, but are not limited to, agar, calcium carbonate, potato or may be selected from, but not limited to, tapioca starch, alginic acid, silicates, and sodium carbonate), e) one or more solution retarders (for example, but not limited to, paraffin), f) one or more absorption accelerators (for example, 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 (for example, but not limited to, kaolin and bentonite clay), and i) one or more lubricants (for example, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate). In the case of capsules, tablets, and pills, for example, the dosage form may also include buffering agents.

[0052] "Effective or therapeutic amount"

[0053] An effective or therapeutic amount of a composition of the present disclosure includes any amount sufficient to inhibit (e.g., slow or halt) the progression of a neurodegenerative disorder, hi some embodiments, an effective amount of a composition includes any amount sufficient to inhibit (e.g., slow or halt) the decline in cognitive function in a patient.

[0054] The amount of active ingredient that can be combined with optional carrier materials to produce a single dosage form can vary depending on the host treated and the specific mode of administration. The specific dosage level for any particular patient can depend on various factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease or illness being treated. The therapeutically effective amount for a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician.

[0055] General Methods and Assays

[0056] Malachite Green Enzyme Assay. Enzyme Inhibitory Potency (IC 50 The activity of PI(3,4,5)P3-diC8 was determined using PI(3,4,5)P3-diC8 as a substrate in 50 mM HEPES buffer (pH 7.4, 150 mM NaCl, 2 mM MgCl2) at 25 °C. Compounds diluted in DMSO were added to a 384-well plate. Enzyme solution was then added. After a 20-minute incubation period, the reaction was initiated by the addition of PI(3,4,5)P3-diC8. Final compound concentrations ranged from 50 nM to 950 μM. The final reaction concentrations of PI(3,4,5)P3-diC8 substrate and enzyme were 52 μM and 10 nM, respectively. After 10 minutes, the reaction was quenched by adding Malachite BioMol Green (Enzo Lifesciences, PA, USA). The plate was then incubated at room temperature for 30 minutes. The absorbance (620 nm) was measured using a SpectraMax Me5 Microplate Reader (Molecular Devices, LLC, USA). 50 Values were calculated by fitting absorbance versus inhibitor concentration.

[0057] Cellular thermal shift assay (CETSA). Split nanoluciferase assay (SplitLuc CETSA) was used to demonstrate target engagement of SHIP1 inhibitors in physiologically relevant cellular contexts by quantifying changes in the thermal stability of HiBit-tagged full-length SHIP1 protein in intact cells. This assay was performed in two formats using HMC3 / HiBit-INPP5D stably transformed cells:

[0058] 1. Thermal shift: Cells were treated with 40 μM compound for 60 minutes, then heated in a temperature gradient spanning 38-52°C for 3 minutes, followed by luminescence detection. The mean and standard deviation of the control Tm were determined to generate a "mean + 3SD" ΔTm, which was used as a threshold to determine a significant ΔTm of the compound from the control. If the difference in ΔTm of compound-treated cells from the control Tm was >3SD, the compound was considered positive for target engagement; otherwise, negative. SD = mean standard deviation.

[0059] 2. Compound dose response: Compounds were administered in a 1:3 serial dilution starting from 80 μM or 100 μM and run at the target Tm (44.2°C for SHIP1) to generate 8-point curves. Cells were treated for 60 min, then heated at the target Tm for 3 min, followed by luminescence detection. AC50 was calculated using a 4-parameter logistic curve regression model, where the change at the highest concentration was indicated if the difference from the control was >3 SD; otherwise, the AC50 was recorded as "nc" (no calculation).

[0060] PhosphoAKT assay. THP1 cells were treated with inhibitors for 90 min, and then the levels of phosphorylated and total AKT (pAKT / tAKT) were detected using the Perkin Elmer Alpha SureFire Ultra Multiplex PhosphoAKT (S473) kit according to the manufacturer's instructions. 50 Values were calculated by fitting the ratio of pATK / tAKT versus inhibitor concentration.

[0061] pHrodo-Myelin Phagocytosis / Cell Health Assay Using Microglial Cells. This 384-well plate high-content imaging assay was developed to simultaneously quantify phagocytosis and cell health using either BV2 or HMC3 immortalized microglial cell lines or primary microglia isolated from mouse brain. Cells were cultured in DMEM GlutaMax medium (ThermoFisher) containing 10% FBS and Pen-Strep at 37°C in a 5% CO2 incubator.

[0062] Assay timing

[0063] Day 1: Cells were plated at 400 cells / 45 μl / well with BV2, 600 cells / 45 μl / well with HMC3, or primary at 2000 cells / 45 μl / well (Corning Falcon 384-well Optilux black and clear bottom plates for imaging).

[0064] Day 2: Cells were treated with 10-fold serial dilutions of compounds at doses ranging from 60 μM to 3 nM for 48 hours at 37°C.

[0065] Day 3: After 24 hours of treatment with starting compound, cells were seeded with pHrodo-myelin (20 hours total). pHrodo-myelin stocks were stored at 1 mg / ml (protein equivalent) in a -20°C or -80°C freezer. Stocks were thawed and diluted to a 10x seeding solution (50 μg / ml) with culture medium, and 5 μl / well was added to 384-well cell plates.

[0066] Day 4: Cell staining and imaging. A nuclear staining solution was prepared by adding 1 μl of 10 mg / mL Hoechst-33342 to every 1 ml of culture medium, which was added to the cell plate at 20 μl / well. The final concentration of Hoechst-33342 on the cells was approximately 2.5 μg / ml. The cell plate was incubated at 37°C for >30 minutes before imaging. The cell plate was scanned with an ArrayScan automated high-content imaging system using a 10x objective, collecting four fields per well. Three measurements were obtained: 1) average total phagocytic spot intensity per cell, 2) total cell number per well, and 3) average nuclear intensity per cell for cell health. Apoptotic cells showed either increased (early apoptosis) or decreased (late apoptosis) nuclear intensity.

[0067] Activity of Compound 9 in Primary Mouse Microglia. Cortical tissue from C57BL / 6J neonatal mice (P0–P3) was homogenized in Dulbecco's Modified Eagle's Medium (DMEM), filtered through 250 μm and 100 μm meshes, and cultured in Advanced DMEM / F12 supplemented with 10% fetal bovine serum, 1x GlutaMAX, and 1x penicillin / streptomycin. At 21 days in vitro (DIV), cultures were subjected to mild trypsinization for 30 minutes using 0.083% Trypsin-EDTA in DMEM to detach an intact layer of astrocytes. Adherent microglia were used in the pHrodo-myelin phagocytosis assay as described to measure phagocytosis and cell health. Activity of Compound 10 in primary mouse microglia was obtained in a similar manner to that for Compound 9.

[0068] Pharmacokinetics of Compound 9. Compound 9 was formulated at 5 mg / ml in HPMC (1%) / Tween 80 (0.25%) / purified water. Male C57BL / 6J mice, 8-12 weeks old, from The Jackson Laboratory were dosed by oral gavage (100 mg / kg, 20 ml / kg). Plasma exposures were obtained at 0.5, 1, 2, 4, 6, 8, and 12 hours, and the 24-hour endpoint. Brain exposures were obtained at the 4-hour and 24-hour endpoints. Pharmacokinetics of Compound 10 were obtained in the same manner as Compound 9.

[0069] See further the experimental examples below.

[0070] example

[0071] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not meant to limit the disclosure in any way. The examples, along with the methods described herein, are currently representative of preferred embodiments and are offered by way of example only and are not intended to limit the scope of the invention. Modifications therein and other uses will occur to those skilled in the art that are encompassed within the spirit of the disclosure as defined by the claims.

[0072] compound

[0073] [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0074] Example 1

[0075] Intermediate 1

[0076] tert-Butyl 4-(4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0077] To a stirred solution of 5-bromopyridin-3-ol (1 g, 5.75 mmol) and tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (2.17 g, 5.75 mmol) in toluene (5 mL, 42.3 mmol), ethanol (5 mL, 85.6 mmol), and water (2 mL, 111 mmol), dipotassium carbonate (1.59 g, 2 equiv., 11.5 mmol) was added, and the reaction mixture was purged with nitrogen for 20 minutes. Then, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (234 mg, 0.05 equiv., 287 μmol) was added, and the reaction mixture was heated to 100 °C and stirred for 16 hours. The reaction mixture was monitored by TLC and LCMS. The reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash chromatography using 40% EtOAc in hexane as the eluent, and the desired fractions were concentrated to give tert-butyl 4-[4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (0.5 g, 1.39 mmol, 24.2%) as a white solid. MS 343.12.

[0078] Example 2

[0079] compound 1

[0080] tert-Butyl 4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0081] To a stirred solution of tert-butyl 4-[4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (0.4 g, 1.16 mmol) and 1-(bromomethyl)-2-chlorobenzene (263 mg, 1.1 equiv, 1.28 mmol) in dimethylformamide (7 mL, 90.4 mmol) was added dipotassium carbonate (321 mg, 2 equiv, 2.32 mmol) in portions and the reaction mixture was heated at 100° C. for 16 h. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash chromatography using 40% EtOAc in hexanes as eluent, and the desired fractions were concentrated to give tert-butyl 4-(4-(5-((2-chlorophenyl)methoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (180 mg, 365 μmol, 31.4%) as an off-white solid. MS 469.22.

[0082] Compounds 2-8 were all prepared in the same manner as compound 1. [Table 3]

[0083] Example 3

[0084] compound 9

[0085] 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine HCl [ka]

[0086] To a stirred solution of tert-butyl 4-(4-(5-[(2-chlorophenyl)methoxy]pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (180 mg, 384 μmol) in dichloromethane (5 mL, 78.1 mmol) was added 4 M HCl in dioxane (2 mL) in small portions at 0° C., and the reaction mixture was stirred at room temperature for 3 h. After completion (monitored by TLC and LCMS), the reaction mixture was concentrated and triturated with n-pentane and diethyl ether to afford 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine hydrochloride (91 mg, 222 μmol, 57.9%) as an off-white solid. MS 369.25.

[0087] Example 4

[0088] compound 10

[0089] 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine HCl [ka]

[0090] To a stirred solution of tert-butyl 4-(4-(5-[(2,4-dichlorophenyl)methoxy]pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (250 mg, 497 μmol) in dichloromethane (5 mL, 78.1 mmol) was added 4 M HCl in dioxane (2 mL) in small portions at 0° C., and the reaction mixture was stirred at room temperature for 3 h. After completion (monitored by TLC and LCMS), the reaction mixture was concentrated and triturated with n-pentane and diethyl ether to afford 3-[(2,4-dichlorophenyl)methoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridine hydrochloride (170 mg, 422 μmol, 85%) as an off-white solid. MS 403.10.

[0091] Compounds 11 and 12 were prepared in a similar manner to compound 10. [Table 4]

[0092] Example 5

[0093] compound 13

[0094] 3-((2,6-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine trifluoroacetate [ka]

[0095] To a stirred solution of tert-butyl 4-(4-(5-((2,6-dichlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (225 mg, 447 μmol) in dichloromethane (5 mL, 78.1 mmol), trifluoroacetic acid (1 mL) was added portionwise at 0° C., and the reaction mixture was stirred at room temperature for 3 hours. After completion (monitored by TLC and LCMS), the reaction mixture was concentrated and triturated with n-pentane and diethyl ether. The compound was purified by preparative HPLC using 1% TFA / acetonitrile in water to give 3-((2,6-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (18 mg, 42.8 μmol, 9.6%) as an off-white trifluoroacetic acid salt. MS 403.20.

[0096] Compound 14 was prepared in a similar manner to compound 10. [Table 5]

[0097] Example 6

[0098] compound 15

[0099] 3-((2-chlorobenzyl)oxy)-5-(1-(1-(3,3-dimethylbutyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0100] To a stirred solution of 3-((2-chlorophenyl)methoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (150 mg, 407 μmol) and 1-bromo-3,3-dimethylbutane (67.1 mg, 407 μmol) in dimethylformamide (3 mL, 38.7 mmol) was added dipotassium carbonate (169 mg, 3 equiv, 1.22 mmol) in portions and the reaction mixture was heated at 100° C. for 16 h. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash chromatography using 40% EtOAc in hexanes as eluent, and the desired fractions were concentrated to give 3-((2-chlorobenzyl)oxy)-5-(1-(1-(3,3-dimethylbutyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (15 mg, 33.1 μmol, 8.1%) as an off-white solid. MS 453.35.

[0101] Example 7

[0102] compound 16

[0103] 1-(4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-3,3-dimethylbutan-1-one [ka]

[0104] To a stirred solution of 3-((2-chlorophenyl)methoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (0.1 g, 271 μmol) in dichloromethane (2 mL, 31.2 mmol) was added triethylamine (114 μL, 3 equiv., 813 μmol) followed by 3,3-dimethylbutanoyl chloride (54.7 mg, 1.5 equiv., 407 μmol) at 0° C. The reaction mixture was stirred at room temperature for 16 hours. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water and extracted with DCM. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified using 10% MeOH in DCM as eluent and the desired fractions were concentrated to give 1-(4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-3,3-dimethylbutan-1-one (38 mg, 79.2 μmol, 29.2%) as a yellowish semi-solid. MS 467.37.

[0105] Example 8

[0106] compound 17

[0107] Methyl 4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0108] To a stirred solution of 3-[(2-chlorophenyl)methoxy)-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridine (150 mg, 407 μmol) in dichloromethane (3 mL, 46.9 mmol) was added triethylamine (170 μL, 3 equiv., 1.22 mmol) followed by methyl chloroformate (57.6 mg, 1.5 equiv., 610 μmol) at 0° C. The reaction mixture was stirred at room temperature for 16 hours. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water and extracted with DCM. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified using 10% MeOH in DCM as the eluent and the desired fractions were concentrated to give methyl 4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (53 mg, 118 μmol, 29.0%) as a yellowish semi-solid. MS 427.30.

[0109] Example 9

[0110] compound 18

[0111] (4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)(phenyl)methanone [ka]

[0112] To a stirred solution of 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine hydrochloride (75 mg, 170 μmol) in tetrahydrofuran (2 mL, 24.6 mmol) was added benzoyl chloride (24 μL, 1.2 equiv., 207 μmol) and N,N-diisopropylethylamine (125 μL, 4.2 equiv., 718 μmol) at room temperature. The reaction mixture was stirred for 2 hours, at which time LCMS indicated the reaction was complete. The reaction mixture was diluted with water (2 mL) and extracted with ethyl acetate (2 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude compound. The crude material was purified by silica gel flash chromatography using 0-20% MeOH in dichloromethane to give the desired product (4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)(phenyl)methanone (62 mg, 131 μmol, 77%) as a yellow oil. MS 473.91.

[0113] Example 10

[0114] compound 19

[0115] Benzyl 4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0116] To a stirred solution of 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine hydrochloride (75 mg, 170 μmol) in tetrahydrofuran (2 mL, 24.6 mmol) was added benzyl chloroformate (29 μL, 1.2 equiv., 204 μmol) and N,N-diisopropylethylamine (149 μL, 5 equiv., 856 μmol) at room temperature. The reaction mixture was stirred for 1.5 hours, at which time LCMS indicated the reaction was complete. The reaction mixture was diluted with water (2 mL) and extracted with ethyl acetate (3×2 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude compound. The crude material was purified by silica gel flash chromatography using 0-85% EtOAc in n-hexane to give the desired product benzyl 4-(4-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (32 mg, 63.6 μmol, 37%) as a yellow oil. MS 504.47.

[0117] Example 11

[0118] compound 20

[0119] 3-((3-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0120] Intermediate 1 (starting material for Intermediate 2, which is the starting material for Compound 20) (3-bromo-5-((3-chlorobenzyl)oxy)pyridine) [ka]

[0121] To a stirred solution of cesium carbonate (206 mg, 1.1 equiv., 632 μmol) and 5-bromopyridin-3-ol (100 mg, 575 μmol) in dimethylformamide (12 mL, 155 mmol) was added 1-(bromomethyl)2-chlorobenzene (118 mg, 1 equiv., 575 μmol). The reaction mixture was heated and stirred in an Anton Paar Monowave 450 microwave at 70° C. for 2 hours. Upon completion, the solution was diluted with ethyl acetate and washed five times with brine. The reaction mixture was concentrated. The crude product was purified by flash chromatography using 60% ethyl acetate in hexanes as the eluent. The desired fractions were concentrated to give 3-bromo-5-((3-chlorebenzyl)oxy)pyridine (163 mg, 544 μmol). MS 296.96.

[0122] Intermediate 2 (starting material for compound 20) (tert-butyl 4-(4-(5-((3-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0123] To a reaction vessel was added 3-bromo-5-((3-chlorobenzyl)oxy)pyridine (170 mg, 0.98 equiv., 569 μmol), tert-butyl 4-[4,5,6,6-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (262 mg, 1.1 equiv., 640 μmol), 1,4-dioxane (5.82 mL, 68.3 mmol), dipotassium carbonate (262 mg, 3.3 equiv., 1.89 mmol), and water (1.11 mL, 61.7 mmol). The reaction mixture was frozen, evacuated, purged with nitrogen, and thawed. This cycle was repeated three times. Pd(dppf)Cl2 (21.6 mg, 0.05 equiv., 29.1 μmol) was added, and the mixture was heated to 90 °C for 4 h. After the reaction was complete (monitored by LCMS), the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine. The organics were dried over Na2SO4, filtered, and concentrated to give the crude material. The crude product was purified by flash chromatography using a gradient of 0 to 100% EtOAc in hexanes as the eluent, and the desired fractions were concentrated to give tert-butyl 4-(4-(5-((3-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate as an off-white solid (126 mg, 269 μmol). MS (M+AcCN) 510.45.

[0124] 3-((3-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (Compound 20)

[0125] To a stirred solution of tert-butyl 4-(4-(5-((3-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (126 mg, 269 μmol) in dichloromethane (2 mL, 31.4 mmol) was added 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) at room temperature. After 2 hours, additional 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) was added, and the reaction mixture was stirred overnight. After completion (monitored by LCMS), the solution was concentrated in vacuo and washed with methyl tert-butyl ether (2×2 mL). The remaining solid was dried in vacuo to give the HCl salt of 3-((3-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine as a white solid. MS 369.35.

[0126] Example 12

[0127] compound 21

[0128] (S)-3-(1-(2-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0129] Intermediate 3 (starting material for Intermediate 4, which is the starting material for Compound 21) (tert-butyl 4-(4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0130] To a stirred solution of 5-bromopyridin-3-ol (2.01 g, 11.6 mmol) and tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (4.36 g, 11.6 mmol) in toluene (16 mL, 151 mmol) was added dipotassium carbonate (3.2 g, 2 equiv., 23.1 mmol). The reaction mixture was purged with argon for 20 minutes. Bis(diphenylphosphino)ferrocene palladium dichloride (472 mg, 0.05 equiv., 578 μmol) was then added, and the reaction mixture was heated to 100° C. and stirred for 16 hours. The reaction mixture was monitored by TLC and LCMS. The reaction mixture was concentrated. The crude product was purified by flash chromatography using 60% ethyl acetate in hexane as the eluent. The desired fractions were concentrated to give tert-butyl 4-[4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (1.2 g, 3.31 mmol) as an off-white solid. MS 345.25.

[0131] Intermediate 4 (starting material for compound 21) (tert-butyl (S)-4-(4-(5-(1-(2-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0132] To a stirred solution of tert-butyl 4-(4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.5 g, 1.45 mmol) and (1R)-1-(2-chlorophenyl)ethan-1-ol (238 mg, 1.52 mmol) in tetrahydrofuran (20 mL, 246 mmol), triphenylphosphane (799 mg, 2 equiv., 3.04 mmol) was added portionwise, and the reaction mixture was stirred at room temperature for 1 hour. Then, (E)-N-[(ethoxycarbonyl)imino]ethoxyformamide (530 mg, 2 equiv., 3.04 mmol) was added portionwise to a stock solution in tetrahydrofuran, and the reaction mixture was stirred at room temperature for 16 hours. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography using 70% EtOAc in hexanes as the eluent, and the desired fractions were concentrated to give tert-butyl (S)-4-(4-(5-(1-(2-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (180 mg, 373 μmol) as a sticky brown solid. MS 482.90.

[0133] (S)-3-(1-(2-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (Compound 21)

[0134] To a stirred solution of tert-butyl (S)-4-(4-(5-(1-(2-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (180 mg, 373 μmol) in dichloromethane (4 mL, 62.5 mmol), trifluoroacetic acid (1 mL) was added portionwise at 0° C., and the reaction mixture was stirred at room temperature for 3 hours. After completion (monitored by TLC and LCMS), the reaction mixture was concentrated and triturated with n-pentane and diethyl ether. Preparative HPLC using TFA buffer afforded the TFA salt of (S)-3-(1-(2-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (45 mg, 89.7 μmol) as an off-white solid. MS 383.30.

[0135] Example 13

[0136] compound 22

[0137] (R)-3-(1-(2-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0138] Prepared in essentially the same manner as (S)-3-(1-(2-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine. MS 383.30.

[0139] Example 14

[0140] compound 23

[0141] (R)-3-(1-(4-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0142] Intermediate 5 (starting material for compound 23) (tert-butyl (R)-4-(4-(5-(1-(4-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0143] To a stirred solution of tert-butyl 4-(4-(5-hydroxy-3-pyridyl)-1-pyrazolyl)-1-piperidinecarboxylate (440 mg, 1.28 mmol) in dimethylformamide (12.8 mL, 165 mmol) was added dipotassium carbonate (353 mg, 2 equiv., 2.56 mmol) and the reaction mixture was stirred for 5 minutes. (S)-1-(p-chlorophenyl)-1-(mesyloxy)ethane (0.3 g, 1.28 mmol) was then added and the reaction mixture was heated at 70° C. and stirred for 12 hours. The reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography using a gradient of 0-100% EtOAc in hexanes as eluent, and the desired fractions were concentrated to give tert-butyl (R)-4-(4-(5-(1-(4-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (129.24 mg, 1 μmol) as a yellow oil. MS (M+AcCN) 524.50.

[0144] (R)-3-(1-(4-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (Compound 23)

[0145] To a stirred solution of tert-butyl (R)-4-(4-(5-(1-(4-chlorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (130 mg, 269 μmol) in dichloromethane (2 mL, 31.4 mmol) was added 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) at room temperature. After 2 hours, additional 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) was added, and the reaction mixture was stirred overnight. Upon completion (monitored by LCMS), the solution was concentrated in vacuo and washed with methyl tert-butyl ether (2×2 mL). The remaining solid was dried in vacuo to give the HCl salt of (R)-3-(1-(4-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine. MS 383.24.

[0146] Example 15

[0147] compound 24

[0148] (S)-3-(1-(4-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0149] Prepared in essentially the same manner as (R)-3-(1-(4-chlorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine. MS 383.30.

[0150] Example 16

[0151] compound 25

[0152] 3-((2-methylphenyl)methoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine [ka]

[0153] Intermediate 6 (starting material for compound 25) (tert-butyl 4-(4-(5-((2-methylbenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0154] To a stirred solution of tert-butyl 4-(4-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.5 g, 1.45 mmol) in N,N-dimethylformamide (5 mL) was added dipotassium carbonate (602 mg, 3 equiv., 4.36 mmol), followed by 1-(bromomethyl)-2-methylbenzene (403 mg, 1.5 equiv., 2.18 mmol). The reaction mixture was heated and stirred at 100°C for 16 hours. The reaction was monitored by TLC and LCMS. Upon completion, the reaction mixture was quenched with water (15 mL) and extracted with ethyl acetate (50 mL). The organic layer was washed with ice water and brine solution to remove DMF, then dried over sodium sulfate and distilled to give the crude product. The crude product was purified by Combiflash chromatography using 100-200 silica. The pure compound was eluted in 40% ethyl acetate in hexane to give a pale yellow gum, which was further triturated with diethyl ether to give tert-butyl 4-(4-(5-((2-methylbenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (230 mg, 492 μmol) as a white solid. MS 449.38.

[0155] 3-((2-methylphenyl)methoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (Compound 25)

[0156] To a stirred solution of tert-butyl 4-(4-(5-((2-methylbenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (100 mg, 223 μmol) was added trifluoroacetic acid (0.5 mL) at 0° C. The reaction mixture was then stirred at room temperature for 3 hours. The reaction was monitored by TLC and LCMS. Upon completion, the reaction mixture was distilled to dryness to give the crude product. The crude product was purified by Combiflash chromatography using 100-200 silica. The pure compound was eluted with 15% MeOH in DCM to give 3-((2-methylphenyl)methoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine as a pale green gum. MS 349.37.

[0157] Example 17

[0158] compound 26

[0159] 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-3-yl)pyridine [ka]

[0160] Intermediate 7 (starting material for intermediate 8, which is the starting material for compound 26) (tert-butyl 4-(3-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0161] To a stirred solution of 5-bromopyridin-3-ol (1 g, 5.75 mmol) and tert-butyl 4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (2.17 g, 5.75 mmol) in toluene (3 mL, 25.4 mmol), ethanol (3 mL, 51.4 mmol), and water (1 mL, 55.5 mmol), dipotassium carbonate (278 mg, 2 equiv., 2.01 mmol) was added, and the reaction mixture was purged with nitrogen for 20 minutes. 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex (469 mg, 0.1 equiv., 575 μmol) was then added, and the reaction mixture was heated to 100°C and stirred for 16 hours. The reaction mixture was monitored by TLC and LCMS. The reaction mixture was concentrated. The crude product was purified by flash chromatography using 50% EtOAc in hexane as an eluent, and the desired fractions were concentrated to give tert-butyl 4-[3-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (0.3 g, 775 μmol, 13.5%) as an off-white solid. MS 345.25.

[0162] Intermediate 8 (starting material for compound 26) (tert-butyl 4-(3-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0163] To a stirred solution of tert-butyl 4-(3-(5-hydroxypyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (250 mg, 726 μmol) and 1-(bromomethyl)-2-chlorobenzene (149 mg, 726 μmol) in dimethylformamide (5 mL, 64.6 mmol) was added dipotassium carbonate (301 mg, 3 equiv, 2.18 mmol) in portions and the reaction mixture was heated at 100° C. for 16 h. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water, extracted with EtOAc, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography using 40% EtOAc in hexanes as eluent, and the desired fractions were concentrated to give tert-butyl 4-(3-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.1 g, 198 μmol) as an off-white solid. MS 469.25.

[0164] 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-3-yl)pyridine (Compound 26)

[0165] To a stirred solution of tert-butyl 4-(3-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.2 g, 426 μmol) in dichloromethane (5 mL, 78.1 mmol) was added trifluoroacetic acid (0.4 mL, 4 mmol) in portions at 0° C., and the reaction mixture was stirred at room temperature for 3 hours. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, and concentrated. The crude product was purified by preparative HPLC using TFA as a buffer to give the TFA salt of 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-3-yl)pyridine (20 mg, 40.6 μmol) as an off-white solid. MS 369.25.

[0166] Example 18

[0167] compound 27

[0168] 3-((2-chlorobenzyl)oxy)-5-(3-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine [ka]

[0169] Intermediate 9 (starting material for Intermediate 10, which is the starting material for Compound 27) (3-bromo-5-((2-chlorobenzyl)oxy)pyridine) [ka]

[0170] To a stirred solution of 5-bromopyridin-3-ol (5.28 g, 30.3 mmol) in N,N-dimethylformamide (100 mL, 1.29 mol) was added cesium carbonate (10.9 g, 33.4 mmol) and 2-chlorobenzyl bromide (3.94 mL, 30.3 mmol). The reaction mixture was stirred at room temperature for 1 h. After the reaction was complete (monitored by LCMS), the mixture was diluted with water (100 mL), and the resulting mixture was extracted with methyl tert-butyl ether (3 × 75 mL). The combined extracts were washed with water (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography using a gradient of 0% to 15% ethyl acetate in hexanes as the eluent. The desired fractions were concentrated to give 3-bromo-5-((2-chlorobenzyl)oxy)pyridine (5.3 g, 17.8 mmol) as a white solid. MS 298.15.

[0171] Intermediate 10 (starting material for compound 27) (tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-3-yl)piperidine-1-carboxylate) [ka]

[0172] To a reaction vessel was added tert-butyl 4-(1H-pyrazol-3-yl)piperidine-1-carboxylate (210 mg, 836 μmol), 3-bromo-5-((2-chlorobenzyl)oxy)pyridine (498 mg, 1.67 mmol), copper(I) iodide (27 mg, 142 μmol), potassium carbonate (294 mg, 2.13 mmol), tribasic potassium phosphate (289 mg, 1.36 mmol), acetonitrile (9 mL, 172 mmol), and (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (132 μmol, 836 μmol). The vessel was purged with nitrogen, sealed, and heated at 120° C. for 4 hours. After the reaction was complete (monitored by LCMS), the mixture was cooled to room temperature and diluted with saturated aqueous ammonium chloride (5 mL). The mixture was extracted with ethyl acetate (3 × 5 mL). The combined extracts were washed with saturated aqueous ammonium chloride (2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography using a gradient of 0% to 50% ethyl acetate in hexanes as the eluent. The desired fractions were concentrated to give tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-3-yl)piperidine-1-carboxylate (341 mg, 727 μmol) as a clear, colorless oil. MS 469.46.

[0173] 3-((2-chlorobenzyl)oxy)-5-(3-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine (Compound 27)

[0174] To a stirred solution of tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-3-yl)piperidine-1-carboxylate (280 mg, 597 μmol) in dichloromethane (3 mL, 47.1 mmol) was added 4N hydrochloric acid in dioxane (3 mL, 12 mmol) at room temperature. After 1 hour, the reaction was complete (monitored by LCMS) and a white solid had settled to the bottom of the vessel. The supernatant was removed and the remaining solid was washed with dichloromethane (3×5 mL). The remaining solid was dried in vacuo to give 3-((2-chlorobenzyl)oxy)-5-(3-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine (259 mg, 586 μmol) as a white solid. MS 369.38.

[0175] Example 19

[0176] compound 28

[0177] 3-((2-chlorobenzyl)oxy)-5-(4-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine [ka]

[0178] Intermediate 11 (starting material for compound 28) (tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-4-yl)piperidine-1-carboxylate) [ka]

[0179] To a reaction vessel was added tert-butyl 4-(1H-pyrazol-4-yl)piperidine-1-carboxylate (100 mg, 398 μmol), 3-bromo-5-((2-chlorobenzyl)oxy)pyridine (238 mg, 796 μmol), copper(I) iodide (12 mg, 63.0 μmol), potassium carbonate (137 mg, 995 μmol), tribasic potassium phosphate (135 mg, 637 μmol), acetonitrile (4 mL, 76.5 mmol), and (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (63 μmol, 400 μmol). The vessel was purged with nitrogen, sealed, and heated at 120° C. for 5 hours. After completion of the reaction (monitored by LCMS), the mixture was cooled to room temperature and diluted with saturated aqueous ammonium chloride (3 mL). The mixture was extracted with ethyl acetate (3×3 mL). The combined extracts were washed with water (3 × 1 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography using a gradient of 0% to 40% ethyl acetate in hexane as the eluent. The desired fractions were concentrated to give tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-4-yl)piperidine-1-carboxylate (169 mg, 360 μmol) as a yellow oil. MS 469.50.

[0180] 3-((2-chlorobenzyl)oxy)-5-(4-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine (Compound 28)

[0181] To a stirred solution of tert-butyl 4-(1-(5-((2-chlorobenzyl)oxy)pyridin-3-yl)-1H-pyrazol-4-yl)piperidine-1-carboxylate (150 mg, 320 μmol) in dichloromethane (2 mL, 31.4 mmol) was added 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) at room temperature. After 2 hours, additional 4N hydrochloric acid in dioxane (0.5 mL, 2 mmol) was added, and the reaction mixture was stirred overnight. After completion (monitored by LCMS), the solution was concentrated in vacuo and washed with methyl tert-butyl ether (2×2 mL). The remaining solid was dried in vacuo to give the HCl salt of 3-((2-chlorobenzyl)oxy)-5-(4-(piperidin-4-yl)-1H-pyrazol-1-yl)pyridine (118 mg, 267 μmol) as a white solid. MS 369.38.

[0182] Example 20

[0183] compound 29

[0184] 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine [ka]

[0185] Intermediate 12 (starting material for intermediate 13, which is the starting material for compound 29) (tert-butyl 4-(4-(3-hydroxyphenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0186] To a stirred solution of 3-bromophenol (1 g, 5.78 mmol) in toluene (80.5 mL, 68 mmol) and ethanol (8.05 mL, 138 mmol) was added dipotassium carbonate (1.6 g, 2 equiv., 11.6 mmol) and tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (2.62 g, 1.2 equiv., 6.94 mmol). The reaction mixture was then purged with N for 10 minutes, followed by the addition of tetrakis(triphenylphosphine)palladium(0) (334 mg, 0.05 equiv., 289 μmol), and the resulting reaction mass was heated at 100° C. for 16 hours. After completion (monitored by TLC and LCMS), it was diluted with water, extracted with ethyl acetate, washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography using 0-40% ethyl acetate in n-heptane as eluent and the desired fractions were concentrated to give tert-butyl 4-(4-(3-hydroxyphenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (700 mg, 2.04 mmol) as a white solid. MS 344.36.

[0187] Intermediate 13 (starting material for compound 29) (tert-butyl 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate) [ka]

[0188] To a stirred solution of tert-butyl 4-(4-(3-hydroxyphenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (500 mg, 1.4 equiv, 1.45 mmol) in dimethylformamide (5 mL, 64.6 mmol) was added dipotassium carbonate (420 mg, 3 equiv, 3.04 mmol) and 1-(bromomethyl)-2-chlorobenzene (312 mg, 1.5 equiv, 1.52 mmol), and the reaction was then heated at 100° C. for 16 h. After completion (monitored by TLC and LCMS), the mixture was diluted with cold water, extracted with ethyl acetate, washed with water, brine, dried over NaSO, filtered, and concentrated. The crude product was purified by flash chromatography using 0-20% ethyl acetate as eluent and the desired fractions were concentrated to give tert-butyl 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (250 mg, 534 μmol) as an off-white solid. MS 468.35.

[0189] 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine (Compound 29)

[0190] To a stirred solution of tert-butyl 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (130 mg, 277 μmol) in dichloromethane (4.85 mL, 75.8 mmol) was added trifluoroacetic acid (2 mL) in small portions at 0° C., and the reaction was then stirred at room temperature for 3 hours. After the reaction was complete (monitored by LCMS), the mixture was concentrated and the resulting solid was triturated with diethyl ether to give the TFA salt of 4-(4-(3-((2-chlorobenzyl)oxy)phenyl)-1H-pyrazol-1-yl)piperidine trifluoroacetate (102 mg, 245 μmol, 88%) as a white solid. MS 368.30.

[0191] Example 21

[0192] Malachite Green Enzyme Assay

[0193] Enzyme inhibitory potency (IC 50 ) (see table below) was determined using PI(3,4,5)P3-diC8 as a substrate in 50 mM HEPES buffer (pH 7.4, 150 mM NaCl, 2 mM MgCl2) at 25 °C. See Figure 2. Compounds diluted in DMSO were added to a 384-well plate. Enzyme solution was added. After a 20-minute incubation period, the reaction was initiated by the addition of PI(3,4,5)P3-diC8. Final compound concentrations ranged from 50 nM to 950 μM. Final reaction concentrations of PI(3,4,5)P3-diC8 substrate and enzyme were 52 μM and 10 nM, respectively. After 10 minutes, the reaction was quenched by adding Malachite BioMol Green (Enzo Lifesciences, PA, USA). The plate was then incubated at room temperature for 30 minutes. The absorbance (620 nm) was measured using a SpectraMax Me5 Microplate Reader (Molecular Devices, LLC, USA). 50 Values were calculated by fitting absorbance versus inhibitor concentration and are reported as geometric means and standard errors with the number of replicates (n) indicated. Compounds and activities are as shown in Tables 1 and 2 above.

[0194] Example 22

[0195] Cellular Thermal Shift Assay (CETSA)

[0196] We demonstrated target engagement of SHIP1 inhibitors in physiologically relevant cellular contexts by quantifying the change in thermostability of HiBit-tagged full-length SHIP1 protein in intact cells using a split nanoluciferase assay (SplitLuc CETSA). See Martinez et al. (2018) Sci Rep 8:9472 and Oh-Hashi et al. (2017) Biochem Biophys Rep 12:40-45. This assay was performed in two formats using HMC3 / HiBit-INPP5D stably transfected cells. The results are shown in the table below.

[0197] Thermal shift: Cells were treated with 40 μM compound for 60 minutes, then heated in a temperature gradient spanning 38-52°C for 3 minutes, followed by luminescence detection. The mean and standard deviation of the control Tm were determined to generate a "mean + 3SD" ΔTm, which was used as a threshold to determine a significant ΔTm for a compound from the control. If the difference in ΔTm of compound-treated cells from the control Tm was >3SD, the compound was considered positive for target engagement; otherwise, it was considered negative. SD = mean standard deviation.

[0198] Compound dose response: Compounds were administered in a 1:3 serial dilution from 80 μM or 100 μM and run at the target Tm (44.2°C for SHIP1) to generate 8-point curves. Cells were treated for 60 min, then heated at the target Tm for 3 min, followed by luminescence detection. Half-maximal responses (AC) were calculated using a 4-parameter logistic regression model where a difference from control of >3 SD indicated a change at the highest concentration. 50 The concentration of α-glucan that induced the α-glucan-1-phosphate dehydrogenase (AGD) was calculated. The activity is shown in Table 3 below.

[0199] Example 23

[0200] Phospho-AKT assay

[0201]

[0202] THP1 cells were treated with inhibitors for 90 min, and then the levels of phosphorylated and total AKT (pAKT / tAKT) were detected using the Perkin Elmer Alpha SureFire Ultra Multiplex PhosphoAKT (S473) kit according to the manufacturer's instructions. 50 Values were calculated by fitting the ratio of pATK / tAKT versus inhibitor concentration.

[0203] Example 24

[0204] pHrodo-Myelin Phagocytosis / Cell Health Assay Using Microglial Cells

[0205] This 384-well plate high-content imaging assay was developed to simultaneously quantify phagocytosis and cell health using either BV2 or HMC3 immortalized microglial cell lines or primary microglia isolated from mouse brain. Cells were cultured in DMEM GlutaMax medium (ThermoFisher) containing 10% FBS and Pen-Strep at 37°C in a 5% CO2 incubator. Day 1: Cells were plated at 400 cells / 45 μl / well with BV2, 600 cells / 45 μl / well with HMC3, or primary at 2000 cells / 45 μl / well in Corning Falcon 384-well Optilux black and clear-bottom imaging plates. Day 2: Cells were treated with 10-fold serial dilutions of compounds at doses ranging from 60 μM to 3 nM for 48 hours at 37°C. Day 3: After 24 hours of treatment with the starting compound, cells were seeded with pHrodo-myelin (20 hours total). pHrodo-myelin stocks were stored at 1 mg / ml (protein equivalent) in a -20°C or -80°C freezer. The stocks were thawed and diluted with culture medium to a 10x seeding solution (50 μg / ml), and 5 μl / well was added to a 384-well cell plate. Day 4: Cell staining and imaging. A nuclear staining solution was prepared by adding 1 μl of 10 mg / ml Hoechst-33342 for every 1 ml of culture medium added to the cell plate at 20 μl / well. The final concentration of Hoechst-33342 on the cells was approximately 2.5 μg / ml. The cell plate was incubated at 37°C for >30 minutes before imaging. Cell plates were scanned with an ArrayScan automated high-content imaging system using a 10x objective, collecting 4 fields per well. Three measurements were obtained: 1) mean total phagocytic spot intensity per cell, 2) total cell number per well, and 3) mean nuclear intensity per cell for cell health. Apoptotic cells showed either increased (early apoptosis) or decreased (late apoptosis) nuclear intensity. Activity is shown in Table 3 below.

[0206] Example 25

[0207] Activity of Compound 9 in primary mouse microglia

[0208] Cortical tissue from C57BL / 6J neonatal mice (P0–P3) was homogenized in Dulbecco's modified Eagle's medium (DMEM), filtered through 250 μm and 100 μm meshes, and cultured in Advanced DMEM / F12 supplemented with 10% fetal bovine serum, 1x GlutaMAX, and 1x penicillin / streptomycin. At 21 days in vitro (DIV), the cultures were subjected to mild trypsinization for 30 minutes using 0.083% Trypsin-EDTA in DMEM to detach an intact layer of astrocytes. Adherent microglia were used to measure myelin phagocytosis and cell health as described in Example 13. The results are shown in Figure 3.

[0209] Example 26

[0210] Activity of compound 10 in primary mouse microglia

[0211] Primary mouse microglia for compound 10 were obtained in the same manner as for compound 9. The results are shown in Figure 3.

[0212] Example 27

[0213] Pharmacokinetics of Compound 9 in C57BL / 6J mice

[0214] Compounds were formulated at 5 mg / ml in hydroxyethylcellulose (1%) / Tween 80 (0.25%) / antifoam (0.05%) / purified water. 100 mg / kg, 20 ml / kg, was administered by oral gavage. Plasma exposures were obtained at 0.25, 0.5, 1, 2, and 8 hours, and at the 24-hour endpoint. Brain exposures were obtained at the 4-hour and 24-hour endpoints. Results are shown in Figure 4.

[0215] Example 28

[0216] Pharmacokinetics of Compound 10 in C57BL / 6J Mice

[0217] The pharmacokinetics of compound 10 in C57BL / 6J mice was obtained in a similar manner to that for compound 9. The results are shown in Figure 4.

[0218] [Table 6]

[0219] Y = ΔTm difference of cells treated with 40 μM compound from control Tm is >3 SD; NT = not tested; NC = not calculated

[0220] overview

[0221] SHIP1 phosphatase is generally understood to suppress microglial activation downstream of TREM2 and Fc receptors. The SHIP1 inhibitors described herein increase phagocytosis in mouse (BV2) and human (HMC3) microglial cell lines by 1.2-2-fold over baseline at μM concentrations, with minimal impact on cell health. In primary mouse microglia, compound 9 increased phagocytosis by 1.8-fold, resulting in a 1.8-fold increase in EC 50 The EC was 1.3 μM. Compound 10 increased phagocytosis by 1.5-fold, 50 The plasma and brain concentrations of compound 9 in C57BL / 6J mice administered orally at 100 mg / kg were 0.54 μM. Compound 9 achieved plasma and brain concentrations of 5 μM and 0.5 μM, respectively, demonstrating sufficient target engagement for in vivo pharmacodynamic studies. Compound 10 achieved plasma and brain concentrations of 2 μM and 6 μM, respectively.

[0222] As will be understood from the description herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, but are not limited to, the aspects and embodiments listed below.

[0223] The present disclosure provides methods and compounds directed to inhibiting SHIP1 to activate microglia. The methods and compounds disclosed herein enable inhibition of SHIP1 early in neurodegenerative disease, resulting in increased microglial protective function and reducing the rate of disease progression and cognitive decline in, for example, Alzheimer's disease patients.

[0224] More specifically, the present disclosure:

[0225] Compounds of Formula I: [ka]

[0226] wherein each X is independently C or N;

[0227] n1+n2=4,

[0228] R1, R2 are independently H, F, Cl, Br or methyl;

[0229] R3 is H, amide, carbamate, or alkyl;

[0230] R4 is H or CH3

[0231] or a pharmaceutically acceptable salt thereof.

[0232] A method of treating a neurodegenerative disorder or related condition in a patient, alone or in combination with any other embodiment, comprising administering to a patient in need thereof an effective amount of a compound of formula I.

[0233] A method of treating the progression of a neurodegenerative disorder or related condition in a patient, alone or in combination with any other embodiment, comprising administering to a patient in need of such treatment an effective amount of a compound of formula I.

[0234] A method of preventing a neurodegenerative disorder or related condition in a patient, alone or in combination with any other embodiment, comprising administering to a patient in need of such treatment an effective amount of a compound of formula I.

[0235] The compound or a pharmaceutically acceptable salt thereof, alone or in combination with any other embodiment, is formulated into a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

[0236] Alone or in combination with any other embodiment, the method further comprises the administration of one or more additional therapeutic agents.

[0237] Pharmaceutical compositions comprising a compound of formula (I), alone or in combination with any other embodiment, further comprise one or more pharmaceutically acceptable carriers, diluents or excipients.

[0238] Alone or in combination with any other embodiment, the pharmaceutical composition is administered to a patient in need thereof to treat or prevent a neurological disorder or related condition, or to inhibit the progression of said neurological disorder or related condition.

[0239] Alone or in combination with any other embodiment, the pharmaceutical composition further comprises the administration of one or more additional therapeutic agents.

[0240] Alone or in combination with any other embodiment, a compound of formula II: [ka]

[0241] wherein each X is independently C or N;

[0242] R1, R2 are independently H, F, Cl, Br or methyl;

[0243] R3 is H, amide, carbamate, or alkyl;

[0244] R4 is H or CH3

[0245] or a pharmaceutically acceptable salt thereof, is used for manufacturing a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition.

[0246] Alone or in combination with any other embodiment, a compound of formula III: [ka]

[0247] wherein each X is independently C or N;

[0248] R1, R2 are independently H, F, Cl, Br or methyl;

[0249] R3 is H, amide, carbamate, or alkyl;

[0250] R4 is H or CH3

[0251] or a pharmaceutically acceptable salt thereof, is used for manufacturing a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition.

[0252] Alone or in combination with any other embodiment, a compound of formula IV: [ka]

[0253] wherein each X is independently C or N;

[0254] R1, R2 are independently H, F, Cl, Br or methyl;

[0255] R3 is H, amide, carbamate, or alkyl;

[0256] R4 is H or CH3

[0257] or a pharmaceutically acceptable salt thereof, is used for manufacturing a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition.

[0258] Alone or in combination with any other embodiment, [ka] [ka] [ka] and [ka] a compound selected from

[0259] or a pharmaceutically acceptable salt thereof, is used for manufacturing a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition.

[0260] Alone or in combination with any other embodiment, the compound [ka]

[0261] or a pharmaceutically acceptable salt thereof, is used for manufacturing a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition.

[0262] Alone or in combination with any other embodiment, the neurodegenerative disorder or associated condition is selected from the group consisting of Alzheimer's disease, dementia associated with Alzheimer's disease, or mild cognitive impairment.

[0263] Alone or in combination with any other embodiment, the dementia associated with Alzheimer's disease is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and dementia of multiple etiologies.

[0264] Alone or in combination with any other embodiment, a method for synthesizing a compound of formula I.

[0265] Alone or in combination with any other embodiment, the compound of formula I is: [ka] is.

[0266] Alone or in combination with any other embodiment, the method of treating Alzheimer's disease in a patient comprises administering to a patient in need thereof a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0267] Alone or in combination with any other embodiment, a method of treating dementia associated with Alzheimer's disease in a patient comprises administering to a patient in need thereof a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0268] A method of treating the progression of mild cognitive impairment to Alzheimer's disease or related dementia in a patient, alone or in combination with any other embodiment, comprises administering to a patient in need of such treatment an effective amount of a compound of the present disclosure.

[0269] Alone or in combination with any other embodiment, a method for preventing Alzheimer's disease or dementia associated with Alzheimer's disease comprises administering to a patient in need of such treatment an effective amount of a compound of the present disclosure.

[0270] Pharmaceutical compositions, alone or in combination with any other embodiment, include compounds of the present disclosure, or pharmaceutically acceptable salts thereof, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0271] Alone or in combination with any other embodiment, the pharmaceutical compositions include compounds of the present disclosure, or pharmaceutically acceptable salts thereof, in combination with one or more additional therapeutic agents.

[0272] The pharmaceutical compositions, alone or in combination with any other embodiment, include a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for the treatment of Alzheimer's disease, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0273] Pharmaceutical compositions for the treatment of dementia associated with Alzheimer's disease, alone or in combination with any other embodiment, comprise a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, in combination with one or more carriers, diluents, or pharmaceutically acceptable excipients.

[0274] Pharmaceutical compositions for the prevention of Alzheimer's disease and dementia associated with Alzheimer's disease, alone or in combination with any other embodiment, include a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more carriers, diluents, or pharmaceutically acceptable excipients.

[0275] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used in therapy, particularly for treating Alzheimer's disease.

[0276] Alone or in combination with any other embodiment, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, are used in the treatment of Alzheimer's disease.

[0277] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used to manufacture a medicament for the treatment of Alzheimer's disease.

[0278] Alone or in combination with any other embodiment, the compounds of the present disclosure or pharmaceutically acceptable salts thereof are used in therapy, particularly for the treatment of dementia associated with Alzheimer's disease.

[0279] Alone or in combination with any other embodiment, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, are used in the treatment of dementia associated with Alzheimer's disease.

[0280] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used to manufacture a medicament for the treatment of dementia associated with Alzheimer's disease.

[0281] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used to prevent Alzheimer's disease and dementia associated with Alzheimer's disease.

[0282] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used to prevent Alzheimer's disease and dementia associated with Alzheimer's disease.

[0283] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, alone or in combination with any other embodiment, are used to manufacture a medicament for use in the prevention of Alzheimer's disease and dementia associated with Alzheimer's disease.

[0284] The present disclosure also includes intermediates and processes useful in the synthesis of the disclosed compounds.

[0285] While embodiments of the present disclosure have been described herein, those skilled in the art will recognize that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. Compounds of Formula I: 【Chemical 1】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

2. A method of treating a neurodegenerative disorder or related condition in a patient, comprising administering to a patient in need thereof an effective amount of a compound of formula I: 【Chemistry 2】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

3. A method for treating the progression of a neurodegenerative disorder or related condition in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of formula I: 【Chemistry 3】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

4. A method of preventing a neurodegenerative disorder or related condition in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of formula I: 【Chemistry 4】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

5. 3. The method of claim 2, wherein the compound or a pharmaceutically acceptable salt thereof is formulated in a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

6. 4. The method of claim 3, wherein the compound or a pharmaceutically acceptable salt thereof is formulated in a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

7. 5. The method of claim 4, wherein the compound or a pharmaceutically acceptable salt thereof is formulated in a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

8. 6. The method of claim 5, further comprising administering one or more additional therapeutic agents.

9. 10. The method of claim 6, further comprising administering one or more additional therapeutic agents.

10. 8. The method of claim 7, further comprising administering one or more additional therapeutic agents.

11. Compounds of Formula I: 【Chemistry 5】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients.

12. 12. The pharmaceutical composition of claim 11, administered to a patient in need thereof for the treatment or prevention of a neurological disorder or related condition, or to inhibit the progression of said neurological disorder or related condition.

13. 12. The pharmaceutical composition of claim 11, further comprising one or more additional therapeutic agents.

14. 1. The method of claim 1, wherein the compound of formula II is a compound of formula II: 【Chemistry 6】 wherein each X is independently C or N; R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

15. 1. The use of a compound of formula III: 【Chemistry 7】 wherein each X is independently C or N; R1 is H, F, Cl, Br or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

16. 1. The method of claim 1, wherein the compound of formula IV is a compound of formula IV: 【Chemistry 8】 wherein each X is independently C or N; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a pharmaceutically acceptable salt thereof.

17. for the manufacture of a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition, 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 and 【Chemistry 10】 10. Use of a compound selected from:

18. 1. A compound for the manufacture of a medicament for treating or preventing a neurodegenerative disorder or related condition or for inhibiting the progression of said neurodegenerative disorder or related condition: 【Chemistry 11】 or a pharmaceutically acceptable salt thereof.

19. 11. The method of any one of claims 2 to 10, wherein the neurodegenerative disorder or associated condition is selected from the group consisting of Alzheimer's disease, dementia associated with Alzheimer's disease, or mild cognitive impairment.

20. 20. The method of claim 19, wherein the dementia associated with Alzheimer's disease is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and dementia of multiple etiologies.

21. 14. The pharmaceutical composition of any one of claims 11 to 13, wherein the neurodegenerative disorder or related condition is selected from the group consisting of Alzheimer's disease, dementia associated with Alzheimer's disease, or mild cognitive impairment.

22. 22. The pharmaceutical composition of claim 21, wherein the dementia associated with Alzheimer's disease is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and dementia of multiple etiologies.

23. 19. The use according to any one of claims 14 to 18, wherein the neurodegenerative disorder or associated condition is selected from the group consisting of Alzheimer's disease, dementia associated with Alzheimer's disease or mild cognitive impairment.

24. 24. The use of claim 23, wherein the dementia associated with Alzheimer's disease is selected from the group consisting of Lewy body dementia (LBD), frontotemporal degeneration (FTD), vascular cognitive impairment and dementia (VCID), and dementia of multiple etiologies.

25. Compounds of Formula I: 【Chemistry 12】 wherein each X is independently C or N; n1+n2=4, R1 and R2 are independently H, F, Cl, Br, or methyl; R3 is H, amide, carbamate, or alkyl; R4 is H or CH ３ is) or a method for synthesizing a pharmaceutically acceptable salt thereof.

26. The compound of formula I is: 【Chemistry 13】 2. The compound of claim 1, wherein:

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