Targeted epigenetic therapies for disorders of progranulin deficiency

Abstract

The present disclosure relates to compounds, e.g., compounds of Formula (I) and Formula (II), and pharmaceutically acceptable salts thereof, and their use for modulating levels of progranulin.

Description

TARGETED EPIGENETIC THERAPIES FOR DISORDERS OF PROGRANULIN DEFICIENCY STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under R01NS108115 awarded by National Institute of Neurological Disorders & Stroke / National Institute of Health. The government has certain rights in the invention. PRIORITY CLAIM

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 524,837, filed on July 3, 2023 and U.S. Provisional Application No. 63 / 610,275, filed on December 14, 2023, each of which is incorporated by reference in its entirety, including any drawings and exhibits. FIELD

[0003] The present disclosure relates to compounds, e.g., compounds of Formula (I) and Formula (II), and pharmaceutically acceptable salts thereof, and their use for modulating levels of progranulin. BACKGROUND

[0004] Frontotemporal dementia (FTD) is characterized by the selective degeneration of the frontal and temporal lobes and is the second most common cause of presenile dementia, accounting for 5-15% of all dementia cases. Clinically, frontotemporal lobar degeneration (FTLD) patients commonly present with severe personality and behavioral changes, as well as fluent or non-fluent aphasias. Pathologically, frontotemporal lobar degeneration (FTLD) cases near ubiquitously show evidence of abnormal intracellular protein aggregation, and the composition of their aggregating proteins can distinguish FTLD subtypes.

[0005] One of the most significant genetic risk factors for FTLD is mutations in the GRN gene, which encodes the progranulin (PGRN) protein. FTLD caused by mutations in GRN characteristically presents with an aberrant accumulation of ubiquitin and TAR DNA-binding protein (TDP-43)-positive inclusions. GRN mutation frequency in FTLD is between 1-11%, with a large range due to significant differences in mutation frequency among various populations. By the age of sixty, over half of GRN mutation carriers are affected by FTLD; by 70, over 90% are affected. To date, at least seventy pathogenic mutations in GRN havebeen identified, with the majority introducing a premature stop codon. These mutations result in a reduction of GRN mRNA, and corresponding PGRN protein, resulting in PGRN haploinsufficiency.

[0006] PGRN is a 68.5 kDa protein that is processed via multiple N-linked glycosylation events to produce a mature 88 kDa protein. PGRN is then secreted and cleaved either extracellularly or after re-internalization into fragments (granulins) via the actions of several proteases such as elastase, proteinase 3, and cathepsins B, D, and L. PGRN modulates a variety of biological processes, such as mediating wound repair and inflammation, promoting epithelial cell growth, and inhibiting transcriptional elongation. PGRN deficiency causes broad lysosomal dysfunction characterized by inclusion-filled lysosomes and increased expression of lysosomal proteins such as cathepsin D and LAMP1, and homozygous loss of GRN causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Reduced lysosomal function is a common feature observed in neurodegenerative diseases, and PGRN deficiency is known to cause lysosomal dysfunction through alterations in lysosomal protein composition and a reduction in lipid homeostasis. An additional mechanism by which PGRN may protect against neurodegeneration is through moderating neuroinflammation. Recent research has pointed toward the importance of microglial PGRN in CNS homeostasis. SUMMARY

[0007] Some embodiments provide a compound of Formula (I)or a pharmaceutically acceptable salt thereof, wherein Ring A is 5-6 membered heteroaryl or phenyl; Ring B is 6-10 membered heteroaryl, 9-14 membered heterocyclyl, or phenyl; R1is C1-C6 alkyl; R2is: (i) phenyl optionally substituted with 1-2 independently selected R2A, (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected ,(iii) C1-C6 alkyl, (iv) C3-C6 cycloalkyl, (v) -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl), (vi) -(C=O)C3-C6 cycloalkyl, (vii) 4-10 membered heterocyclyl, and (viii) -NR2B(C3-C6 cycloalkyl); R2Ais halogen or hydroxyl; R2Bis hydrogen or C1-C6 alkyl; R3is halogen or -NR3A(C=O)C1-C6 alkyl; R3Ais hydrogen or C1-C6 alkyl; L is a bond, -O-, -S-, -S(=O)-, -SO2-, -N(RA)-, -C(RARB)-, or -C(=O)-; RAand R2Bare each independently hydrogen or C1-C6 alkyl; RBis hydrogen, hydroxyl, or C1-C6 alkoxyl; and n is 0 or 1.

[0008] Some embodiments provide a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0009] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (II), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0010] Some embodiments provide a method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0011] Some embodiments provide a method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein :(i) phenyl optionally substituted with 1-3 independently selected R2A, (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A, (iii) C1-C6 alkyl, (iv) C3-C6 cycloalkyl, (v) -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl), (vi) –(C=O)C3-C6 cycloalkyl, and (vii) 4-10 membered heterocyclyl, and (viii) -NR2B(C3-C6 cycloalkyl); R2Ais halogen, C1-C6 alkyl, or hydroxyl; R3is halogen and -NR3A(C=O)C1-C6 alkyl, -NR3BSO2R3C, or C1-C6 alkyl optionally substituted with hydroxyl; R3Aand R3Bare each independently hydrogen or C1-C6 alkyl; R3Cis C1-C6 alkyl; L is bond, -O-, -S-, -S(=O)-, -SO2-, -N(RA)-, -C(RARB)-, or -C(=O)-; RAis hydrogen or C1-C6 alkyl; RBis hydrogen, hydroxyl, and C1-C6 alkoxyl; RCis absent or -NRC1(C=O)C1-C6 alkyl; RC1is H or C1-C6 alkyl; and n is 0 or 1.

[0012] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0013] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising administering to the subject an effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0014] The details of one or more embodiments of the subject matter of this disclosure are set forth in the accompanying drawings and the description. Other features, aspects, and advantages of the subject matter will become apparent from the description, the drawings, and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0015] FIGs. 1A-F Shows bromodomain inhibitors enhance cellular PGRN in human neural progenitor cells. (A) Assay strategy for identifying novel small-molecule enhancers of PGRN. Assay 1: qPCR to identify GRN enhancers. Assay 2: Immunoblotting to validate enhancement at the protein level. (B) Bromodomain inhibitors (10 µM) enhance GRN mRNA in human NPCs after 24 hours. Data are expressed as mean ± S.E. of n = 2 treatment biological replicates and n = 6 DMSO biological replicates, normalized to GAPDH and relative to DMSO. Compound concentrations used: Bromosporine, 10 µM; I-BET151, 10 µM; PFI-1, 10 µM. (C) Structures of BET inhibitor probes used for further investigations. (D) BET inhibitors enhance GRN mRNA in NPCs and in 18-day neurons after 24 hours of treatment. (E) BET inhibitors enhance PGRN protein levels in NPCs and in neurons after 24 hours of treatment. Representative Western shown along with quantification in (F). * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

[0016] FIGs. 2A-H. Shows dependencies of BET inhibitors as enhancers of PGRN. (A) Schematic representation of human BRD4 with selected domains and interactors.32 (B) Mechanistic models for BET inhibition enchanting progranulin protein levels. (C) Human NPCs in various co-treatments with MLN-4924 (1 µM), Mivebresib (1 µM), and dBET6 (300 nM). Cells were treated with 1 µM MLN-4924 or DMSO only for 2 hours prior to addition of 300 nM dBET6 or DMSO. Cells were then allowed to then incubate for four hours prior to addition of 1 µM Mivebresib or DMSO for 24 hours. Data are expressed as mean ± S.E. of 3 biological replicates. (D) Immunoblots against BRD4 for human NPCs treated as described in (C). (E) Quantification of (D). Data normalized to β-actin. (F) Diagram of experimental setup for data presented in panels G and H. (G) Mivebresib (1 µM) is able to potently upregulate PGRN protein levels at the 8-hour and 24-hour timepoints in human NPCs. Data are expressed as mean ± S.E. of 2 biological replicates. (H) Treatment with Mivebresib for short timeframes, followed by compound washout and then lysis after 24 hours from initial compound addition, enhances PGRN protein levels in human NPCs. Data are expressed as mean ± S.E. of 3-6 biological replicates. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

[0017] FIGs. 3A-E Shows the novel CNS-optimized BET inhibitors as enhancers of PGRN. (A) Structures of select RAY03 series compounds. (B) RAY03-series compounds (blue) are score highly in CNS MPO scoring for use in CNS disorders in comparison to other BET inhibitors (orange). (C-D) RAY03-series compounds can potently engage BRD4 derived from both HEK-293T and NPC lysates. Data are expressed as mean ± S.E. of 2biological replicates. (E) RAY03-series compounds (1 µM) can enhance PGRN in human NPCs after 24 hours of treatment. Data are expressed as mean ± S.E. of 3 biological replicates. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

[0018] FIGs. 4A-E Shows RAY03-B series compounds potently engage BRD4 and can enhance cellular PGRN. (A) GlideXP lowest-energy conformer of Mivebresib and RAY03- 18B in BRD4 BD2. (B) Structures of select RAY03-B series analogs. (C) RAY03-B series analogs can engage BRD4 derived from HEK-293T and NPC lysates. (D) RAY03-B series analogs can engage BRD4 BD1 and BD2 with varying potency. (E) RAY03-B series analogs (1 µM) can enhance PGRN in human NPCs after 24 hours of treatment.

[0019] FIGs. 5A-D Shows BET inhibitors can enhance progranulin in GRN- haploinsufficient NPCs and neurons. (A) 2-week differentiated GRNR493X / + neurons express MAP2, neurofilaments, and TUJ1 as detected by ICC. (B-D) Mivebresib and Panobinostat are able to rescue progranulin haploinsufficiency in GRN-haploinsufficient NPCs and neurons, but not in iPSCs.

[0020] FIG. 6 shows the strategy for generation of a C-terminal PGRN-Luciferase reporter line.

[0021] FIG. 7 shows the engineering strategy for the PGRN-Luciferase reporter.

[0022] FIG. 8A shows primer validation strategy, and FIG. 8B shows the PCR validation of reporter line.

[0023] FIG. 9A shows the effect of internal HDAC inhibitors and FIG. 9B shows the effect of secreted progranulin-luciferase signal. Data plotted as mean ± SEM (n=3). DMSO is plotted as first data point for visualization.

[0024] FIG. 10 shows BET inhibitors enhance overall PGRN-Luc signal. Data plotted as mean ± SEM (n=4).

[0025] FIG. 11 shows the PGRN-Luc assay can distinguish enhancers from negative controls in a 384-well format (n=192 for DMSO, n=128 for Panobinostat).

[0026] FIG. 12 shows results of a targeted epigenetic screen in the HMC3 PGRN-Luc reporter line.

[0027] FIG. 13 shows validation of hits from the PGRN-Luc screen. Data plotted as mean ± SEM (n=3).

[0028] FIG. 14A and 14B show time-activity curves (TAC) in whole brain in C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq), with injected dose per gram (ID / g) and standard uptake values (SUV), respectively, shown to quickly increase immediately after injection and gradually decrease over 60 min.

[0029] FIG. 15A and 15B show TAC in blood of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq), with ID / g and SUV, respectively, shown to quickly decrease then stabilize over 60 min.

[0030] FIG. 16 shows image-derived brain / blood ratio of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq).

[0031] FIG. 17 shows [18F]Ray03-18B levels (ID / g) in brain regions of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0032] FIG. 18A and 18B show [18F]Ray03-18B level (ID / g) levels in tissues of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq), with levels in each tissue and levels in each tissue over timepoints 5 min – 60 min, respectively.

[0033] FIG. 19 shows whole body images in C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0034] FIG. 20 shows brain images (sagittal view) of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0035] FIG. 21 shows brain images (coronal view) of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0036] FIG. 22 shows brain images (coronal view) of C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0037] FIG. 23. Validation of Jess automated western blotting for the quantification of PGRN. PGRN can be visualized across a wide range of lysate and antibody concentrations.

[0038] FIG. 24. Quantification of data from figure 23 showing linearity of signal response.

[0039] FIG. 25. iPSCs bearing a heterozygous and homozygous GRN R493X mutation show decreased expression of PGRN.

[0040] FIG. 26. Quantification of figure 25.

[0041] FIG. 27. RAY03-18B potently binds to BRD4 in a commercial TR-FRET assay. [Note that this is the BPS Biosciences TR-FRET assay. Dose-response curve for this experiment was determined by using the reagents from the commercially available BRD4 (BD1+BD2) TR-FRET Assay Kit in a 384-well format (Proxiplate-384 Plus, PerkinElmer #6008280) following the manufacturer’s protocol (BPS Biosciences, Catalog #BPS-326120). Final well DMSO content: 0.2%].

[0042] FIG. 28. Pharmacokinetic profile of 18A (A) and 18B (B) injected intravenously (5 mg / kg) in CD-1 mice.

[0043] FIG. 29 shows calculated KD and KDapp values (with 95% confidence interval) for compounds against recombinant, GST-tagged BRD4-BD1 and BRD4-BD2.

[0044] FIG. 30 shows the pharmacokinetic properties of RAY03-18A and RAY03-18B injected intraveneously in CD-1 mice.

[0045] FIG. 31A and 31B show in vitro autoradiography results. FIG. 31A is baseline, self-blocking, and AD mouse brain tissue autograms (sagittal), Self-blocking studies (10 μM); FIG. 31B shows quantification (expressed as Gray value 0-255). All data are the mean ± SD, n = 4 for baseline, n = 4 for self-blocking, n = 4 for AD mice. DETAILED DESCRIPTION

[0046] In some aspects, provided herein is a compound Formula (I) and Formula (II), or a pharmaceutically acceptable salt or composition thereof, and to methods of use thereof for the detection and / or treatment of a PGRN-deficient associated disease. In some aspects, the compound of Formula (I) and Formula (II) targets the BET family of bromodomain- containing epigenetic readers that regulate progranulin (PGRN) protein levels. Definitions

[0047] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification and the attached appendices are incorporated herein by reference in their entireties.

[0048] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCHPublishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0049] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0050] Compounds described herein can comprise one or more asymmetric centers or double bonds, and thus can exist in various isomeric forms, e.g., enantiomers, diastereomers, racemates, geometric isomers, stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)-. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw–Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure includes compounds in racemic and optically pure forms. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0051] When a range of values is listed, it is intended to encompass each value and sub– range within the range. For example “C1-C6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0052] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present disclosure.

[0053] “Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (“C1-C50 alkyl”). In some embodiments, an alkyl group has, for example, 1 to 20 carbon atoms (“C1-C20 alkyl”), 1 to 10 carbon atoms (“C1- C10 alkyl”), or 1 to 6 carbom atoms (“C1-C6 alkyl”). Examples of C1-C6 alkyl groupsinclude methyl (C1), ethyl (C2), n–propyl (C3), isopropyl (C3), n–butyl (C4), tert–butyl (C4), sec–butyl (C4), iso–butyl (C4), n–pentyl (C5), 3–pentanyl (C5), amyl (C5), neopentyl (C5), 3–methyl–2–butanyl (C5), tertiary amyl (C5), and n–hexyl (C6). Additional examples of alkyl groups include n–heptyl (C7), n–octyl (C8) and the like. Common alkyl abbreviations include Me (–CH3), Et (–CH2CH3), iPr (–CH(CH3)2), nPr (–CH2CH2CH3), nBu (– CH2CH2CH2CH3), or i–Bu (–CH2CH(CH3)2).

[0054] “Halo” or “halogen,” independently or as part of another substituent, means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom. The term “halide” by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom. In certain embodiments, the halo group is fluorine.

[0055] “Haloalkyl” refers to an alkyl group as described herein (e.g., a C1-C6 alkyl group) in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono- haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chloro- difluoroalkyl, and 2-fluoroisobutyl.

[0056] “Alkoxy” or “alkoxyl” refers to an alkyl group as described herein (e.g., a C1-C6 alkyl group), which is attached to a molecule via oxygen atom. This includes moieties where the alkyl part may be linear or branched, such as methoxy, ethoxy, n-propoxy, iso-propoxy, n- butoxy, sec-butoxy, tert-butoxy, n-pentoxy and n-hexoxy.

[0057] “Haloalkoxy” refers to an alkoxy group as described herein (e.g., a C1-C6 alkoxy group), in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono- haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloro-fluoroalkoxy, chloro-difluoroalkoxy, and 2-fluoroisobutoxy.

[0058] “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1–naphthyl and 2–naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a C6-C10 aryl. Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl. As used herein, the term “arylene”, employed alone or in combinationwith other terms, refers to a divalent aryl linking group having 6 to 14 ring carbon atoms. Examples of arylene group include phenylene and naphthylene.

[0059] “Heteroaryl” refers to a radical of a 5–14 membered monocyclic, bicyclic or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“6–10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl groups can also contain one or more oxidized atoms such as carbon (oxo), sulfur (sulfone, sulfoxide), and nitrogen (N-oxide). Heteroaryl bicyclic ring systems can include one or more heteroatoms in one, two or three rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic or tricylic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2–indolyl) or the ring that does not contain a heteroatom (e.g., 5–indolyl). A heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. As used herein, the term “heteroarylene”, employed alone or in combination with other terms, refers to a divalent heteroaryl linking group having 5 to 14 ring atoms. Examples of heteroarylene group include indolylene, pyridinylene, and quinolinylene.

[0060] In some embodiments, a heteroaryl group is a 6–10 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“6–10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5–8 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5–6 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–6 membered heteroaryl”). In some embodiments, the 5–6 membered heteroaryl has 1–3 ring heteroatoms selected from nitrogen, oxygen, andsulfur. In some embodiments, the 5–6 membered heteroaryl has 1–2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, the heteroaryl group is unsubstituted 5–14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5–14 membered heteroaryl.

[0061] Exemplary 5–membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5–membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5–membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5–membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6–membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6–membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6–membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7–membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6–bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6– bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0062] “Cycloalkyl” refers to a radical of a saturated or partially unsaturated cyclic hydrocarbon group having from 3 to 12 ring carbon atoms (“C3-C6 cycloalkyl”) and zero heteroatoms in the non–aromatic ring system. In some embodiments, a cycloalkyl group has, for example, 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. As used herein, the term “cycloalkylene”, employed alone or in combination with other terms, refers to a divalentcycloalkyl linking group having 3 to 10 ring carbon carbons. Examples of cycloalkylene groups include cyclopropylene and cyclohexylene.

[0063] “Heterocyclyl” refers to a radical of a 4-12 membered saturated or partially unsaturated ring system having ring carbon atoms and 1 to 4 ring heteroatomic groups, wherein each heteroatomic group is independently selected from nitrogen, oxygen, sulfur and oxidized forms of sulfur (for example, S, S(O) and S(O)2), boron, phosphorus, and silicon (“3–12 membered heterocyclyl”). Heterocyclyl groups can also contain one or more oxidized carbon atoms (oxo groups). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon, nitrogen, phosphorus, or silicon atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged, or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”). Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 4-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, and sulfur and oxidized forms of sulfur (for example, S, S(O) and S(O)2), within the moiety. As used herein, the term “heterocyclylene”, employed alone or in combination with other terms, refers to a divalent heterocyclyl linking group having 4 to 12 ring atoms. Examples of heterocyclylene groups include piperazinylene, tetrahydrofuranylene, and pyrrolidinylene.

[0064] Exemplary 4–membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5–membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl–2,5–dione. Exemplary 5–membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin–2–one. Exemplary 5–membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6– membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6–memberedheterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6–membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7–membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8–membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5– membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6– bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6– membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6–bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0065] “Hydroxy” or “hydroxyl” refers to the radical -OH.

[0066] Whenever a group is described as being “optionally substituted”, that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being “substituted” the substituent(s) may be selected from one or more the indicated substituents. If no substituents are indicated, it is meant that the indicated “optionally substituted” or “substituted” group may be substituted with one or more individually and independently selected group(s) that are stable and chemically acceptable for the group being substituted. Non-limiting examples of optional substituents are halogen, cyano, hydroxyl, nitro, sulfhydryl, amino, acyl, alkyl, hydroxyalkyl, aminoalkyl, haloalkyl, alkenyl, alkynyl, alkoxy, alkenoxy, alkynoxy, haloalkoxy, haloalkenoxy, haloalkynoxy, cycloalkyl, halocycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aralkyl, cycloalkylalkyl, cycloalkylalkoxy, heteroaralkyl, alkoxyalkyl, heterocyclylalkyl, O-carbamyl, N-carbamyl, alkoxycarbonyl, C-amido, N- amido, alkyl phosphine oxide, SF5, S-sulfonamido, N-sulfonamido, C-carboxy, sulfoxide, and sulfone.

[0067] In some embodiments one or more of the nitrogen atoms of a disclosed compound if present are oxidized to the corresponding N-oxide.

[0068] The term "pharmaceutically acceptable salts" is meant to include salts that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. In some instances, pharmaceutically acceptable salts are obtained by reacting a compound having acidic group described herein with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassiumsalt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts with amino acids such as arginine, lysine, and the like, or by other methods previously determined. The pharmacologically acceptable salts not specifically limited as far as it can be used in medicaments. Examples of a salt that the compounds described herein form with a base include the following: salts thereof with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts thereof with organic bases such as methylamine, ethylamine and ethanolamine; salts thereof with basic amino acids such as lysine and ornithine; and ammonium salt.

[0069] The term "pharmaceutically acceptable excipients" refers to a carrier or an adjuvant that may be administered to a patient, together with a compound of the present disclosure, or a pharmaceutically acceptable salt, solvate, salt of the solvate or prodrug thereof, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0070] The term “pharmaceutical composition” refers to a mixture of a compound described herein with other chemical components (referred to collectively herein as “pharmaceutically acceptable carriers”), such as stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or other excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

[0071] The term “tautomer” as used herein refers to compounds whose structures differ markedly in arrangement of atoms, but which exist in easy and rapid equilibrium, and it is to be understood that compounds provided herein may be depicted as different tautomers, and when compounds have tautomeric forms, all tautomeric forms are intended to be within the scope of the disclosure, and the naming of the compounds does not exclude any tautomer. An example of a tautomeric forms includes the following example:

[0072] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure.

[0073] Compounds provided herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. That is, an atom, in particular when mentioned in relation to a compound according to Formula (I), comprises allisotopes and isotopic mixtures of that atom, either naturally occurring or synthetically produced, either with natural abundance or in an isotopically enriched form. For example, when hydrogen is mentioned, it is understood to refer to1H,2H,3H or mixtures thereof; when carbon is mentioned, it is understood to refer to11C,12C,13C,14C or mixtures thereof; when nitrogen is mentioned, it is understood to refer to13N,14N,15N or mixtures thereof; when oxygen is mentioned, it is understood to refer to14O,15O,16O,17O,18O or mixtures thereof; and when fluoro is mentioned, it is understood to refer to18F,19F or mixtures thereof; unless expressly noted otherwise. For example, in deuteroalkyl and deuteroalkoxy groups, where one or more hydrogen atoms are specifically replaced with deuterium (2H). As some of the aforementioned isotopes are radioactive, the compounds provided herein therefore also comprise compounds with one or more isotopes of one or more atoms, and mixtures thereof, including radioactive compounds, wherein one or more non-radioactive atoms has been replaced by one of its radioactive enriched isotopes. Radiolabeled compounds are useful as additional agents, e.g., therapeutic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds provided herein, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure. In some embodiments, the compounds described herein are labeled with18F.

[0074] “Treating” or “treatment” refers to reducing the symptoms or arresting or inhibiting further development of the disease (in whole or in part). “Treating” or “treatment” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the disease and the like. For example, certain methods herein treat a PGRN- associated disease by decreasing or reducing the progression, re-occurrence, or symptoms of the a PGRN-associated disease.

[0075] An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce protein activity, reduce or increase protein levels, detect the presence and / or amount of a protein, or reduce one or more symptoms of a disease). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”

[0076] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to reduction inthe progression of a disease and / or symptoms of disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In some embodiments, inhibition refers to a decrease in the activity of bromodomain resulting in enhancement of GRN protein levels.

[0077] A “subject,” as used herein, refers to a living organism suffering from or prone to a disease that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non-limiting examples include mammals such as humans. In some embodiments, a subject is human. In some embodiments, a subject is a newborn human. In some embodiments, a subject is an elderly human. In some embodiments, the subject is a pediatric subject (e.g., a subject 21 years of age or less).

[0078] “PGRN”, as used herein, means progranulin.

[0079] The term “PGRN-associated disease” as used herein refers to diseases associated with reduction of GRN mRNA, and corresponding PGRN protein levels. Non-limiting examples of PGRN-associated disease include neuroinflammatory and lysosomal dysfunction diseases generally. Specific non-limiting examples include frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, and Lewy body dementia (LBD).

[0080] One potential therapeutic approach for the detection and / or treatment of PGRN- deficient disease is to increase the expression of PGRN from the remaining wild-type allele. In support of this therapeutic hypothesis, enhancing PGRN protein levels has shown benefit in several animal models. However, an important consideration for PGRN-enhancing therapies is that PGRN overexpression may enhance the risk for various malignancies. Most recently, it was demonstrated that delivering PGRN to the brains of Grn- / - mice via an engineered protein transport vehicle (PTV) fusion protein can rescue several pathological phenotypes, including restoring lipid homeostasis, reducing gliosis and preventing neurodegeneration. However, an essential requirement for therapeutic approaches that upregulate PGRN may be long-term, chronic dosing, prior to the emergence of symptoms. Thus, strategies that attempt to enhance PGRN via genetic methods, or through intravenous administration of recombinant PGRN, may not be optimal from a therapeutic perspective.

[0081] Histone deacetylase (HDAC) inhibitors can enhance PGRN protein levels and HDAC inhibitors possessing varying selectivity and kinetic profiles have been shown to enhance PGRN protein levels both in mouse primary neurons and Neuro-2A cells, as well asin human NPCs and neurons. However, while HDAC inhibitors have seen common clinical use in neoplastic diseases, their tolerability and safety profiles are not ideal for chronic dosing in a pre-symptomatic population. Moreover, targeting epigenetic regulators to modulate PGRN expression with small-molecule epigenetic enhancers of cellular PGRN represents a particularly attractive strategy as many small-molecule drugs can be taken orally, which improves patient compliance.

[0082] Herein is disclosed a targeted epigenetic screen for compounds that increase GRN mRNA and protein levels in human neural progenitor cells (NPCs). Surprisingly, this screen revealed bromodomain and extra-terminal domain (BET) inhibitors (BETi) are enhancers of GRN mRNA and PGRN protein levels. The finding that a bromodomain inhibitor enhances the expression of a gene of interest is atypical. In various cell types, typically gene expression is downregulated, although a minority of genes may be upregulated. In some embodiments, the loss of BRD4 abrogates the ability of BET inhibitors to enhance progranulin. In some embodiments, the properties of the BET inhibitors are optimized for CNS exposure. In some embodiments, the BET inhibitors rescue PGRN protein levels in iPSC-derived neurons bearing the GRN493X / + haploinsufficiency mutation.

[0083] As a screening strategy, an expandable neural progenitor cell line (8330-8 RC1) was employed that was generated through an iPSC intermediate from human fibroblasts. This cell line was chosen over other commonly employed model cell lines in order to most accurately recapitulate the regulation of PGRN in a human, CNS-relevant cell type. The ability of HDAC inhibitors to enhance GRN mRNA in NPCs, including Panobinostat (1 µM), Abexinostat (1 µM), and Rocilinostat (10 µM) was assessed (see FIG. 1B).

[0084] It was discovered that selected bromodomain inhibitors were active enhancers of GRN mRNA: Bromosporine (10 µM), I-BET151 (10 µM), and PFI-1 (10 µM) (FIG. 1B). The finding that a bromodomain inhibitor enhances gene expression is atypical. In various cell types, typically gene expression is downregulated, although a minority of genes appear to be upregulated. The BET-selective probes I-BET151 and PFI-1 enhanced GRN mRNA levels prompted investigation of the BET family of bromodomain-containing proteins. For this secondary screening, Bromodomain and Extra-terminal domain (BET) family specific probes I-BET151, PFI-1, and JQ1 were employed (FIG. 1C). Moreover, in both NPCs and in 18-day differentiated neurons, PFI-1 (10 µM), I-BET151 (2.5 µM), and JQ1 (1 µM) increased GRN mRNA by a factor of ~2.5-fold (FIG. 1D). Next was evaluated whether this effect could be recapitulated at the protein level and it was found that 24-hour treatment of both NPCs and 18-day neurons with PFI-1 (10 µM), I-BET151 (2.5 µM), and JQ1 (1 µM) enhanced cellularprotein levels that varied by both compound and cell type (FIGS. 1E-F). Collectively, these results demonstrate the potential of targeting BET family members for the enhancement of PGRN in CNS-relevant cell types. BET inhibitors as enhancers of cellular PGRN

[0085] Bromodomains are modular protein domains that bind to acetylated lysine residues on histones and other proteins. BET family members include the related proteins BRD2, BRD3, BRD4, and BRDT, which each contain two bromodomains, BD1 and BD2.33 Through the Extra-Terminal (ET) domain, BET family members can interact with a variety of transcriptional regulators including CHD4, ATAD5, and JMJD6. BRD4 in particular interacts with the Mediator complex which has been found preferentially in ‘super-enhancer’ regions as well as associated with active transcriptional regions across a variety of cell types. BRD4 also directs CDK9 and Cyclin T1, which collectively constitute the Positive Transcription Elongation Factor B (P-TEFb), to primary response genes to promote productive transcriptional elongation by RNA polymerase II (FIG. 2A).

[0086] For the progranulin quantification described herein, the Protein Simple Jess Automated Western Blot System was utilized for automated capillary gel electrophoresis. First, the ability of a PGRN antibody to linearly quantify increases across a wide variation in lysate concentration in the microglia-like HMC3 cells (0.125 mg / mL – 1 mg / mL) was validated (see Supplemental FIGS. 1A, 1B). Then the specificity of this detection method was demonstrated by showing a ~50% reduction in signal in a human iPSC line bearing a heterozygous GRN R403X mutation, and a complete loss of signal in a human iPSC line bearing the homozygous GRN R403X mutation, the most prevalent nonsense mutation causing haploinsufficiency in humans (Supplemental FIGS. 1C, 1D).

[0087] Upon validation of this assay system, neural progenitor cells were used as a model cell system for investigating the dependencies of BET inhibitors as enhancers of progranulin. Three simplified model systems were implemented to elucidate the possible mechanisms by which a BET inhibitor could augment GRN expression (FIG. 2B). In the Repressive model, a BET family member such as BRD4 may direct a negative regulatory complex to the GRN promoter / enhancer region, thereby repressing GRN transcription. An analogous mechanism can be observed in the manner by which BRD4 guides G9a to impose a repressive methylation mark on autophagy-related genes. For the second proposed model, it was posited that BET family members could function as Indirect Repressors by facilitating a protein that inhibits GRN transcription. In the third model, it was hypothesized that BETfamily members may not bind to GRN under basal conditions; however, they could be recruited to GRN to activate transcription upon exposure to BET inhibitors.

[0088] To test whether BET proteins are operating as negative regulators of GRN, it was reasoned that BET degradation ought to recapitulate the effects of a BET inhibitor if BET proteins are operating as negative regulators of GRN. The previously-characterized BET degrader dBET6, was used to probe whether BET degradation can recapitulate the enhancement of PGRN as seen with Mivebresib. NPCs were first treated with dBET6 or DMSO for 4 hours, followed by 24 hours treatment with Mivebresib or DMSO, without removing dBET6 from the media if present. It was found that dBET6 alone was unable to enhance progranulin (FIG. 2C). Furthermore, it was found that degradation of BET family members through dBET6 attenuates the ability of Mivebresib to enhance PGRN (FIG. 2C). It was next determined whether this effect could be rescued upon pre-treatment with the NEDD8 neddylation inhibitor MLN-4924, which ought to inhibit CUL4-E3 ligase activity and thus prevent degradation of BET proteins. Indeed, it was found that MLN-4924 rescues the ability of Mivebresib to enhance progranulin with dBET6 treatment (FIG. 2C). Collectively these results reveal that BET proteins are necessary for the ability of Mivebresib to enhance progranulin.

[0089] Immunoblotting was used to validate the ability of the perturbagen combinations to modulate BRD4 protein levels. It was found that treatment with degrader dBET6 significantly reduced BRD4 protein levels, which was rescued by treatment with MLN-4924 (FIGS. 2D, 2E). It was also found that co-treatment with MLN-4924 and either degrader or Mivebresib enhanced BRD4 protein levels by ~2.5-fold and ~4-fold, respectively. It was found that BRD4 protein levels alone did not correlate with progranulin protein levels (Supplemental FIG. 2), suggesting that pharmacological manipulation of BRD4, as opposed to increasing BRD4 protein levels, is key to the enhancement of progranulin, or that BRD4 is not the most important BET family member in the regulation of PGRN protein levels.

[0090] A few experiments were designed and executed in order to better characterize the kinetics of PGRN enhancement in response to BET inhibition. In the first experiment, human NPCs were treated with either Mivebresib or DMSO for various timeframes, and the cells were lysed cells after the duration of each time-course (FIGS. 2F, G). It was found that Mivebresib treatment induced PGRN upregulation in a time-dependent manner, with ~1.4- fold and ~2.7-fold increases in PGRN observed after 8 and 24 hours, respectively (FIG. 2E). In the second experiment, NPCs were treated with Mivebresib or DMSO for 2 hours or 8hours, and then compounds were washed out, and the cells lysed after 24 hours had passed since initial compound treatment (FIGS. 2F, H). It was revealed that treatment of NPCs with Mivebresib for 2 hours, followed by 22 hours of incubation post-washout, was sufficient to significantly enhance PGRN protein levels >2-fold, with a more pronounced effect after 8 hours of Mivebresib exposure (FIG. 2G). Collectively, these results suggest that limited exposure to a BET inhibitor is sufficient to produce therapeutic increases in progranulin protein levels. CNS-optimized bromodomain inhibitors for the detection and / or treatment of GRN-deficient FTD

[0091] While well-characterized BET inhibitors such as JQ1 and I-BET151 are known to cross the blood-brain-barrier, there still exists a lack of diverse chemotypes of CNS permeable bromodomain inhibitors. Herein is described novel BET inhibitors demonstrated to both engage lysate-derived endogenous BRD4 as well as to enhance PGRN in human NPCs. Also disclosed herein is that BET inhibition can rescue PGRN protein levels in iPSC- derived neurons with the GRN R493X haploinsufficiency mutation.

[0092] Although several chemical probes for BET proteins have emerged over the past decade, none have been explicitly optimized for use in CNS disorders. Accordingly, it was sought to address this gap through the synthesis of BET inhibitors inspired by the pyrrolo- pyridinone core of Mivebresib. To guide these medicinal chemistry efforts, CNS Multiparameter Optimization (MPO) scoring was utilized, which has previously been used to guide the optimization of drug candidates for desirable ADME attributes, safety profiles, and CNS permeability.

[0093] Compounds were triaged for their ability to bind to BRD4 using a commercially available TR-FRET assay which utilizes a donor / acceptor-labeled BRD4 and acetylated peptide pair. These efforts led to the identification of RAY03-18A as a potent binder of BRD4, with an IC50of 23 nM in this assay (FIGS. 3A, 3B). Continued optimization from this scaffold led to the identification of RAY03-81A, 82A, 84A, 85A, and 86A (FIG. 3A). These compounds, along with other analogs, generally occupy the molecular weight space of <400 Da and have high CNS MPO scoring compared to other BET inhibitors (FIG. 3C).

[0094] To analyze the ability of these compounds to bind to BRD4, a TR-FRET assay was utilized, which allows for characterization of target engagement toward endogenous BRD4 derived from cellular lysates of cell types of interest. This assay overcomes a key limitation of commercial in vitro BRD4 TR-FRET assays, which generally utilize arecombinantly-expressed protein that may lack critical post-translational modifications or native binding partners of BRD4 in cells. It was found that the RAY03-A-series analogs were able to potently engage BRD4 derived from both HEK-293T and NPC lysates, with KD,app values ranging from 30 nM to 559 nM, validating the ability of these compounds to engage endogenous BRD4 from relevant cell lines (FIG. 3D, Table 1). Furthermore, it was found that the A-series compounds generally bound to recombinant BRD4 BD1 with similar affinities as to BD2, except for RAY03-85A, which was ~5.5-fold selective for BD1 over BD2 (FIG. 3E, Table 1). These assays demonstrate the ability of RAY-series compounds to engage endogenous BRD4, and provide an easy platform for further optimization of selectivity properties. Table 1. Calculated KD and KD,app values (with 95% confidence interval) for RAY03-A-series compounds against HEK-293T and NPC derived BRD4, as well as the individual bromodomains of BRD4. ENDOGENOUS BRD4 ENDOGENOUS BRD4 (NPC), (HEK293T), KD,APP (NM) KD,APP (NM) 18A 44 (40, 49) 46 (39, 53) 81A 30 (28, 32) 32 (29, 36) 82A 49 (45, 53) 45 (40, 50) 84A 370 (325, 422) 378 (316, 453) 85A 93 (84, 102) 100 (93, 108) 86A 559 (505, 620) 559 (482, 649) 18B121 (110, 134) 131 (119, 144) 81B538 (458, 632) 532 (439, 643) 82B519 (479, 563) 430 (357, 517) 84B196 (175, 220) 195 (170, 222) 85B3909 (3418, 4529) 3813 (2938, 5316) 86B7154 (5735, 10001) 5900 (3734, 16424)

[0095] Finally, it was determined whether these compounds could enhance PGRN protein levels in human NPCs. It was found that 24 hours of 1 µM compound treatment was sufficient to induce >2-fold increase in PGRN protein levels for RAY03-18A, 81A, 82A, and 85A, while 84A and 86A produced a less substantial response (FIG. 3F), likely reflecting the reduced potencies of these compounds. These results demonstrate that a new class of CNS- optimized BET inhibitors can engage BRD4 from cellular lysates and enhance PGRN protein levels.BET inhibitor chemotype can engage BRD4 and enhance PGRN protein levels in human NPCs

[0096] The docked structure of ABBV-744 in BRD4 BD2 was found to be nearly identical to the published crystal structure, PDB: 6ONY (FIG. 4, left panel), which served as a positive control for docking studies. ABBV-744 was validated as a potent binder using Glide Docking through the Schrodinger Modeling Suite, with an XP GScore of -12.4 kcal / mol. The binding pocket had space to accommodate modifications off the pyrrole moiety. Accordingly, modifications were pursued at this position, which led to the identification of RAY03-18B as a putative hit compound (XP Gscore: -8.7 kcal / mol) (FIG. 4A, right panel).

[0097] RAY03-18B was synthesized and validated for its ability to bind BRD4 in a commercial TR-FRET assay with an IC50 of 527 nM (Supplemental FIG. 3). Guided by docking studies as well as CNS MPO scoring, additional RAY03-B series analogs were prepared. A select set of the most promising RAY03-B series compounds, RAY03-18B, 81B, 82B, 84B, 85B, and 86B were advanced into in vitro target engagement / selectivity profiling assays (FIG. 4B). These compounds differ from their “A” series compounds only in terms of their connectivity on the core, allowing for direct comparisons between the A / B-series isomers.

[0098] It was found that RAY03-18B, 81B, 82B, and 84B can potently engage both BRD4 derived from HEK293T and NPC lysates, and BRD4 BD1 and BD2 in vitro (FIGS. 4C, 4D, Supplemental Table 1). The most potent compounds, RAY03-18B and RAY03-84B, were able to engage BRD4 derived from NPCs with KD,app values of 131 nM and 196 nM, respectively. These compounds were also able to engage BRD4 BD1 and BD2 with similar potency.

[0099] It was then determined whether these compounds could enhance PGRN protein levels in human NPCs. It was found that 24 hours of treatment with RAY03-18B, 81B, 82B, and 84B significantly enhanced PGRN protein levels (FIG. 4E). It was also found that RAY03-85B and RAY03-86B were unable to upregulate PGRN, which was consistent with their relatively lower potency in the in vitro target engagement assays.

[0100] The pharmacokinetic properties of RAY03-18A and RAY03-18B were assessed in CD-1 mice. It was found that in CD-1 mice, both RAY03-18A and RAY03-18B can readily cross the blood-brain barrier (Supplemental FIGS. 4A and 4B, Supplemental Table 2). However, both compounds were cleared rapidly.Bromodomain inhibitors can rescue loss of PGRN in iPSCs and iPSC-derived neurons bearing the GRN R493X mutation

[0101] To determine whether Mivebresib could rescue progranulin haploinsufficiency in the GRNR493X / + model, haploinsufficient iPSCs, NPCs, and neurons were treated with either Panobinostat or Mivebresib. It was found that both Mivebresib and Panobinostat were able to enhance progranulin protein levels in both the NPCs and neurons, but not in the iPSCs. These data demonstrate the ability of BET inhibitors to rescue progranulin haploinsufficiency. Methods of Use

[0102] Mutations in GRN are one of the strongest genetic risk factors for the development of FTD. Mutations in GRN have also been associated with the development of ALS, Parkinson’s, and Alzheimer’s Disease. That GRN mutations are associated with other forms of neurodegenerative disease suggests a common mechanism for PGRN in protecting against neurodegeneration. A putative mechanism by which PGRN may exert its neuroprotective functions is through maintaining proper lysosomal function. Reduced lysosomal function is a common feature observed in neurodegenerative diseases, and PGRN deficiency is known to cause lysosomal dysfunction through alterations in lysosomal protein composition and a reduction in lipid homeostasis. An additional mechanism by which PGRN may protect against neurodegeneration is through moderating neuroinflammation. Recent research has pointed toward the importance of microglial PGRN in CNS homeostasis. For individuals bearing heterozygous GRN mutations, the goal of PGRN enhancement would be to restore PGRN expression to wild-type levels. In the (Grn wild-type) mouse brain, PGRN expression is age-dependent, with aged mice expressing ~50% less PGRN in the hippocampus and hypothalamus when compared to same regions of young mice. While it is not yet known how GRN expression in humans is altered with age, in aged individuals, a therapeutic opportunity may exist to correct the age-dependent decreases in PGRN expression via a PGRN enhancing small-molecule. In some embodiments, the compounds disclosed herein, or a pharmaceutically acceptable salt or composition thereof can be used to treat age-dependent decreases in PGRN expression. Furthermore, GRN is highly expressed in myeloid cells, and is more highly expressed in microglia than in neurons, astrocytes, or endothelial cells in the mouse thalamus. Accordingly, enhancing PGRN expression may help to maintain lysosomal homeostasis and to tame inflammatory processes, which may have therapeutic applications in neurodegenerative disease. In some embodiments, the compoundsdisclosed herein, or a pharmaceutically acceptable salt or composition thereof can be used to treat an inflammatory disease. In some embodiments, the compounds disclosed herein, or a pharmaceutically acceptable salt or composition thereof can be used to treat a neurodegenerative disease.

[0103] The rate by which a BET inhibitor can upregulate PGRN suggests that GRN may be a gene that is ‘poised’ for translation by RNA Polymerase II, which BRD4 regulates the processivity of through recruitment and activation of the positive transcription elongation factor P-TEFb. A BET inhibitor may possibly redistribute BRD4 from super-enhancer regions of the genome, and increase the availability of free non-chromatin-associated BRD4 that may then complex with P-TEFb and allow for downstream transcriptional activation.

[0104] The mechanistic investigations disclosed herein suggest that BD2-selective inhibition is sufficient to enhance PGRN protein levels in human NPCs. As BD2-selective inhibitors appear to have a more tolerable safety profile than pan-BD inhibitors, these data indicate that there may be a therapeutic opportunity for a BET inhibitor as an enhancer of PGRN.

[0105] Some embodiments provide a method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0106] Some embodiments provide a method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0107] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0108] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising administering to the subject an effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0109] In some embodiments, the PGRN-associated disease is a neuroinflammatory disease.

[0110] In some embodiments, the PGRN-associated disease is a lysosomal dysfunction disease.

[0111] In some embodiments, the PGRN-associated disease is frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD).

[0112] In some embodiments, the PGRN-associated disease is frontotemporal dementia (FTD).

[0113] In some embodiments, the PGRN-associated disease is Alzheimer’s disease.

[0114] In some embodiments, the PGRN-associated disease is bipolar disorder.

[0115] In some embodiments, the PGRN-associated disease is schizophrenia.

[0116] In some embodiments, the PGRN-associated disease is Lewy body dementia (LBD).

[0117] In some embodiments, the subject is suspected of having a PGRN-associated disease.

[0118] In some embodiments, the subject has a family history of PGRN-associated disease.

[0119] In some embodiments, the subject has a clinical record indicative of the subject having a PGRN-associated disease.

[0120] Some embodiments provide a method of treating frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0121] Some embodiments provide a method of treating a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject suspected of having the disease, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0122] Some embodiments provide a method of treating a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject having a clinical record indicating a diagnosis of the disease, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0123] Some embodiments provide a method of treating a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject previously determined to have the disease, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0124] Some embodiments provide a method of treating FTD in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0125] Some embodiments provide a method of treating FTD in a subject suspected of having FTD, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0126] Some embodiments provide a method of treating FTD in a subject having a clinical record indicating a diagnosis of FTD, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0127] Some embodiments provide a method of treating FTD in a subject previously determined to have FTD, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0128] Some embodiments provide a method of treating FTD in a subject in need thereof, comprising administering to the subject a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0129] Some embodiments provide a method of treating FTD in a subject in need thereof, comprising administering to the subject a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the subject was previously determined to have FTD. For example, through one or more genetic tests, one or more clinical evaluations, or a combination thereof.

[0131] Some embodiments provide a method of detecting frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0132] Some embodiments provide a method of detecting a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject suspected of having the disease, comprising administering to the subject an effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0133] Some embodiments provide a method of detecting a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject having a clinical record indicating a diagnosis of thedisease, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0134] Some embodiments provide a method of detecting a disease selected from frontotemporal dementia (FTD), Alzheimer’s disease, bipolar disorder, schizophrenia, or Lewy body dementia (LBD) in a subject previously determined to have the disease, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0135] Some embodiments provide a method of detecting FTD in a subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0136] Some embodiments provide a method of detecting FTD in a subject suspected of having FTD, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0137] Some embodiments provide a method of detecting FTD in a subject having a clinical record indicating a diagnosis of FTD, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0138] Some embodiments provide a method of detecting FTD in a subject previously determined to have FTD, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0139] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising contacting a sample from the subject with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0140] Some embodiments provide a method of detecting a PGRN-associated disease in a subject, comprising contacting a sample from the subject with an effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the detecting comprises obtaining one or more images of the sample after the contacting step, for example, one or more PET images.

[0142] In some embodiments, the detecting comprises obtaining one or more images of the subject after the administering step, for example, one or more PET images.

[0143] In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample.

[0144] In some embodiments, the compound is radiolabeled. In some embodiments, the compound is18F-labeled.Compounds of Formula (I)

[0145] To the extent that Formula (I) and Formula (II) share substituent numbering (e.g., R1, R2, etc.), the identity of the substituents in the embodiments is intended to apply to both Formulae as appropriate. In the compounds described herein, the connection of Ring B (shown below) to the remainder of the compound indicates that Ring B can be connected to Ring A or to the R1-containing ring in Formula (I) and Formula (II).

[0146] Some embodiments provide a compound of Formula (I)or a pharmaceutically acceptable salt thereof, wherein Ring A is 5-6 membered heteroaryl or phenyl; Ring B is 6-10 membered heteroaryl, 9-14 membered heterocyclyl, or phenyl; R1is C1-C6 alkyl; R2is: (i) phenyl optionally substituted with 1-2 independently selected R2A, (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A, (iii) C1-C6 alkyl, (iv) C3-C6 cycloalkyl, (v) -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl), (vi) -(C=O)C3-C6 cycloalkyl, (vii) 4-10 membered heterocyclyl, and (viii) -NR2B(C3-C6 cycloalkyl); R2Ais halogen or hydroxyl; R2Bis hydrogen or C1-C6 alkyl; R3is halogen or -NR3A(C=O)C1-C6 alkyl; R3Ais hydrogen or C1-C6 alkyl; L is a bond, -O-, -S-, -S(=O)-, -SO2-, -N(RA)-, -C(RARB)-, or -C(=O)-; RAand R2Bare each independently hydrogen or C1-C6 alkyl; RBis hydrogen, hydroxyl, or C1-C6 alkoxyl; andn is 0 or 1.

[0147] In some embodiments, Ring A is 5 membered heteroaryl. In some embodiments, Ring A is 6 membered heteroaryl. In some embodiments, Ring A is furanyl. In some embodiments, Ring A is thiophenyl. In some embodiments, Ring A is pyridinyl.

[0148] In some embodiments, Ring A is phenyl.

[0149] In some embodiments, Ring B is 6-10 membered heteroaryl. In some embodiments, Ring B is 6 membered heteroaryl. In some embodiments, Ring B is 9-10 membered heteroaryl. In some embodiments, Ring B is pyridinyl or pyrimidinyl. In some embodiments, Ring B is pyridinyl. In some embodiments, Ring B is pyrimidinyl.

[0150] In some embodiments, Ring B is 9-14 membered heterocyclyl.

[0151] In some embodiments, Ring B is phenyl.

[0152] In some embodiments, R1is C3-C6 alkyl. In some embodiments, R1is C1-C2 alkyl. In some embodiments, R1is methyl.

[0153] In some embodiments, R2is phenyl optionally substituted with 1-2 independently selected R2A. In some embodiments, R2is phenyl substituted with 1-2 independently selected R2A.

[0154] In some embodiments, R2is 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A.

[0155] In some embodiments, R2is 5-6 membered heteroaryl substituted with 1-2 independently selected R2A. In some embodiments, R2is unsubstituted 5-6 membered heteroaryl.

[0156] In some embodiments, R2is C1-C6 alkyl. In some embodiments, R2is methyl.

[0157] In some embodiments, R2is C3-C6 cycloalkyl. In some embodiments, R2is cyclopropyl or cyclobutyl.

[0158] In some embodiments, R2is -(C=O)(C1-C6 alkyl optionally substituted with C1- C6 alkoxyl). In some embodiments, R2is -(C=O)(C1-C6 alkyl).

[0159] In some embodiments, R2is -(C=O)C3-C6 cycloalkyl.

[0160] In some embodiments, R2is 4-10 membered heterocyclyl.

[0161] In some embodiments, R2is -NR2B(C3-C6 cycloalkyl).

[0162] In some embodiments, R2Ais halogen. In some embodiments, R2Ais fluoro or chloro.

[0163] In some embodiments, R2Ais hydroxyl.

[0164] In some embodiments, R2Bis hydrogen.

[0165] In some embodiments, R2Bis C1-C6 alkyl. In some embodiments, R2Bis methyl.

[0166] In some embodiments, R3is halogen. In some embodiments, R3is fluoro or chloro.

[0167] In some embodiments, R3is -NR3A(C=O)C1-C6 alkyl.

[0168] In some embodiments, R3Ais hydrogen.

[0169] In some embodiments, R3Ais C1-C6 alkyl. In some embodiments, R3Ais methyl.

[0170] In some embodiments, L is a bond.

[0171] In some embodiments, L is -O-.

[0172] In some embodiments, L is -S-.

[0173] In some embodiments, L is -S(=O)-.

[0174] In some embodiments, L is -SO2-.

[0175] In some embodiments, L is -N(RA)-.

[0176] In some embodiments, L is -C(RARB)-.

[0177] In some embodiments, L is -C(=O)-.

[0178] In some embodiments, RAis hydrogen. In some embodiments, RAis C1-C6 alkyl.

[0179] In some embodiments, RBis hydrogen. In some embodiments, RBis hydroxyl. In some embodiments, RBis C1-C6 alkoxyl. In some embodiments, R2Bis hydrogen. In some embodiments, R2Bis C1-C6 alkyl.

[0180] In some embodiments, n is 0.

[0181] In some embodiments, n is 1.

[0182] In some embodiments, L is -O-, R1is methyl, and Ring B is phenyl, pyridinyl, or pyrimidinyl.

[0183] In some embodiments, L is -O-, R1is methyl, Ring B is phenyl, pyridinyl, or pyrimidinyl and R2is (i) phenyl optionally substituted with 1-2 independently selected R2Aor (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A.

[0184] In some embodiments, Ring B is connected directly to Ring A.

[0185] In some embodiments, Ring B is connected to the pyridone ring of Formula (I).

[0186] Some embodiments provide a compound of Formula (I-A), or a pharmaceutically acceptable salt thereof:

[0187] Some embodiments provide a compound of Formula (I-B), or a pharmaceutically acceptable salt thereof:

[0188] Some embodiments provide a compound of Formula (I-C), or a pharmaceutically acceptable salt thereof:

[0189] Some embodiments provide a compound of Formula (I-D), or a pharmaceutically acceptable salt thereof: (I-D).

[0190] Some embodiments provide a compound of Formula (I-E), or a pharmaceutically acceptable salt thereof:

[0191] Some embodiments provide a compound of Formula (I-F), or a pharmaceutically acceptable salt thereof:

[0192] Some embodiments provide a compound of Formula (I-G), or a pharmaceutically acceptable salt thereof:

[0193] Some embodiments provide a compound of Formula (I-H), or a pharmaceutically acceptable salt thereof:

[0194] Some embodiments provide a compound of Formula (I-J), or a pharmaceutically acceptable salt thereof:

[0195] Some embodiments provide a compound of Formula (I-K), or a pharmaceutically acceptable salt thereof:

[0196] Some embodiments provide a compound of Formula (I-L), or a pharmaceutically acceptable salt thereof:

[0197] Some embodiments provide a compound of Formula (I-M), or a pharmaceutically acceptable salt thereof:

[0198] In some embodiments, the compounds of Formula (I) are the compounds shown in Table 2, or a pharmaceutically acceptable salt thereof. Table 2. Exemplary compounds of Formula (I)*Indicates18F-labeled compound

[0199] In some embodiments, any of the fluorinated compounds described herein are18F- labeled, such as Compounds 1-16, 20-23, 25-32, 48-52, or 59 (e.g., Compound 60).

[0200] In some embodiments, the compound is [18F]Ray03-18B, or a pharmaceutically acceptable salt thereof, having the chemical name 3-(2-(4-(fluoro-18F)phenoxy)pyridin-3-yl)- 6-methyl-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, or a pharmaceutically acceptable salt thereof, and having the structure:, or a pharmaceutically acceptable salt thereof. Pharmaceutical Compositions

[0201] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0202] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (II), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. EXAMPLES Materials and Methods

[0203] The general methods for the preparation of the compounds of Formula (I) and Formula (II) have been described in an illustrative manner and is intended to be description, rather than of limitation. Thus, it will be appreciated that conditions such as choice of solvent, temperature of reaction, volumes, reaction time may vary while still producing the desired compounds. In addition, it will be appreciated that many of the reagents provided in the following examples may be substituted with other suitable reagents. See, e.g., Smith & March, Advanced Organic Chemistry, 7th Ed. (2013). Such changes and modifications, including without limitation, those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and / or methods of use provided herein, may be made without departing from the spirit and scope thereof.

[0204] All commercially available reagents were used without further purification unless otherwise stated.1H and13C NMR spectra were recorded on a Bruker AV-III-400 or 500 MHz NMR spectrometer. Chemical shifts are reported as δ values in ppm downfield from TMS as the internal standard.1H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, b= broad, m = multiplet), coupling constant (Hz), integration. Low resolution mass spectra were obtained on Waters Acquity Ultra Performance LC with electrospray ionization and SQ detector by injecting sample in a steady flow of 1 mM ammonium acetate in 20% water-acetonitrile at the rate of 0.2 mL min-1. The purity of compounds were determined by analytical HPLC, performed on a Shimadzu Prominence-HPLC with ELSD PDA multi, and a Gemini NX C-18 column (250 x 4.6 mm, 5μ) with mobile phase (A) 0.1% Formic Acid in water and mobile phase (B) 0.1% Formic Acid in Acetonitrile using following gradient of B / A (0 min, 10%), (5 min, 90%), (6 min, 95%), (10 min, 95%), (10 min, 10%) and (14 min, 10%) at 1.0 mL min-1flow rate. Analytical thin layer chromatography was performed on 250 μM silica gel F254plates. Preparative thin layer chromatography was performed on 1000 μM silica gel F254 plates. Flash column chromatography was performed employing 230-400 mesh silica gel.

[0205] All mouse studies were carried out at Massachusetts General Hospital (PHS Assurance of Compliance No. A3596-01). The Subcommittee on Research Animal Care (SRAC) serves as the Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital. SRAC reviewed and approved all procedures detailed in this paper. All mice were socially housed in cages appropriate for the physical and behavioral health of the individual animal and were given unlimited access to food and water, with additional nutritional supplements provided as prescribed by the attending veterinary staff.

[0206] The compounds described herein may be prepared according to the following schemes:Scheme 1. Synthetic route A Reagents and conditions: (a) LiOMe,1,1-dimethoxy-N,N-dimethylmethanamine, DMF, 100 °C; (b) Fe, NH4Cl, THF-EtOH-water; (c) NaH, pTsCl,THF, RT; (d) 4 M HCl, 1,4-dioxane, 65 °C; (e) Cs2CO3, MeI, 1,4-dioxane, RT; (f) bis(pinacolato)diboron, KOAc, Pd2dba3, X- phos, 1,4-dioxane, 90 °C; (g) Aryl bromides (6a-f), K2CO3, Pd2dba3, meCgPPh, 1,4-dioxane- water (4:1), 65 °C; (h) K2CO3, MeOH-water, 85 °C.Scheme 2. Synthetic route B Reagents and conditions: (a) NaH, DMF, MeI, 0 °C; (b) K3PO4, Pd-XPhos-G3, SPhos, 1,4- dioxane-water, 95oC.Scheme 3. Synthetic route C Reagents and conditions: (a) NBS, ACN, 60°C, 15 h; (b) MeI, Cs2CO3, DMF, 0 °C, 4 h; (c) B2Pin2, KOAc, XPhos, Pd2dba3; (d) K3PO4, Pd-XPhos-G3, SPhos, dioxane-water, 90oC, 16 h.Scheme 4. Synthetic route D Reagents and conditions: (a) (1,3-dioxolan-2-yl)-N-methylmethanamine, DMF, RT; (b) MsOH, 60 °C; (c) NaH, pTsCl, THF, 0 °C; (d) bis(pinacolato)diboron, KOAc, Pd2dba3, X- phos, 1,4-dioxane, 95 °C; (e) 6a-e, K2CO3, Pd2dba3, meCgPPh, 1,4-dioxane-water (4:1), 65 °C; (f) K2CO3, methanol-water, 85 °C.Scheme 5. Synthetic route E Reagents and conditions: (a) 2 / 3 / 4-bromobenzaldehyde, K2CO3, Pd2dba3, meCgPPh, 1,4- dioxane-water (4:1), 65 °C; (b) spiroamine salt, Et3N, AcOH, DCE, methanol, 60oC, then NaBH3CN; (c) K2CO3, methanol-water, 85 °C. Selected Intermediates 3-Bromo-4-(4-fluorophenoxy)pyridine (6)

[0207] To a solution of 4-fluorophenol (160 mg, 1.43 mmol) in dry DMF, 60% sodium hydride in mineral oil (60 mg, 1.5 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-4-chloropyridine (276 mg, 1.43 mmol) was added and heated at 150oC for 2 h. The reaction mixture was cooled, concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (CHCl3) to provide title compound 6 (300 mg, 77% yield). MS (ESI+) m / z 267.8 (M + H)+.1H NMR (500 MHz, CDCl3) 8.69 (s, 1H), 8.32 (d, J = 5.5 Hz, 1H), 7.15-7.08 (m, 4H), 6.59 (d, J =5.5 Hz, 1H). 3-Bromo-5-(4-fluorophenoxy)pyridine (7)

[0208] To a cooled (0-5oC) solution of 4-flurophenol (708mg, 6.31 mmol) in DMF (7mL) under nitrogen was added sodium hydride (270 mg, 11.25 mmol, 60% mineral oil). The mixture was stirred at room temperature for 2 h, treated with 3,5-dibromopyridine (750 mg, 3.16 mmol), and heated at 100 °C for 6 h. The reaction mixture was cooled to room temperature, poured into a mixture of cold water (20 mL) and 5 M sodium hydroxide (2 mL), and extracted with diethyl ether (3 X 30 mL). The combined ether extracts were dried using Na2SO4, filtered, and concentrated by rotary evaporation. This was purified by silica gel column chromatography with 20% ethyl acetate in hexanes as eluent to provide 7 (80 mg, 9% yield). MS (ESI+) m / z 267.8 (M + H)+.1H NMR (500 MHz, CDCl3) d 8.40 (d, J = 2.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.37 (t, J = 2.5 Hz, 1H), 7.14-7.01 (m, 4H). 5-(2-Bromophenoxy)-2-fluoropyridine (8)

[0209] To a mixture of 2-bromophenol (171 mg, 0.98 mmol), 2-fluoropyridinyl-5-boronic acid (280 mg, 1.98 mmol), anhydrous Cu(OAc)2(192 mg, 1.06 mmol) and hot 4A molecular sieves (500 mg) were added anhydrous dichloromethane (5 mL) and anhydrous pyridine (320 mg, 4.05 mmol) under nitrogen. The mixture was stirred at ambient temperature under oxygen atmosphere for 18 h. The reaction mixture was filtered through celite, washed with methanol (10 mL), concentrated and purified by silica gel column chromatography (20-100% chloroform in hexanes) to provide title compound 8 (90 mg, 34% yield). MS (ESI+) m / z 268.0 (M + H)+.1H NMR (500 MHz, CDCl3) d 7.91 (dd, J = 1.5, 2.5 Hz, 1H), 7.66 (dd, J =2.0, 8.0 Hz, 1H), 7.40-7.35 (m, 1H), 7.31 (dt, J = 1.5, 7.5 Hz, 1H), 7.08 (dt, J = 1.5, 8.0 Hz, 1H), 6.98 (dd, J =1.5, 8.5 Hz, 1H), 6.91 (dd, J =4.0, 9.0 Hz, 1H). 5-(3-Bromophenoxy)-2-fluoropyridine (9)

[0210] To a mixture of 3-bromophenol (80 mg, 0.46 mmol), 2-fluoropyridinyl-5-boronic acid (155 mg, 1.10 mmol), anhydrous Cu(OAc)2 (84 mg, 0.46 mmol) and hot 4A molecular sieves (500 mg) were added anhydrous dichloromethane (5 mL) and anhydrous pyridine (240 mg, 3.04 mmol) under nitrogen. The mixture was stirred at ambient temperature under oxygen atmosphere for 5 h. The reaction mixture was filtered through celite, washed withmethanol (10 mL), concentrated and purified by preparative TLC (chloroform) to provide title compound 9 (30 mg, 24% yield). MS (ESI+) m / z 267.8 (M + H)+.1H NMR (500 MHz, CDCl3) d 7.99 (dd, J = 1.5, 2.5 Hz, 1H), 7.49-7.44 (m, 1H), 7.31-7.28 (m, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.14 (t, J = 2.0 Hz, 1H), 6.97-6.92 (m, 2H). N-[5-Bromo-6-(4-fluorophenoxy)pyridin-3-yl]acetamide (10)

[0211] Step-a: To a solution of 4-fluorophenol (240 mg, 2.14 mmol) in dry DMF (10 mL), 60% sodium hydride in mineral oil (60 mg, 1.50 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 2,3-Dibromo-5-nitropyridine (400 mg, 1.42 mmol) was added and stirred at ambient temperature for 1 h. The reaction mixture was concentrated, diluted with ethyl acetate (30 mL), washed with saturated NaHCO3, water (10 mL), and brine (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-60% chloroform in hexanes) to provide3-bromo-2-(4-fluorophenoxy)-5- nitropyridine (380 mg, 84% yield). MS (ESI+) m / z 324.9 (M + H)+.1H NMR (500 MHz, CDCl3) d 8.43 (d, J = 2.5 Hz, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.38 (s, 1H), 7.12-7.06 (m, 4H), 2.18 (s, 3H).

[0212] Step-b and step-c: To a solution of 3-bromo-2-(4-fluorophenoxy)-5-nitropyridine (300 mg, 0.96 mmol) in a 3:3:1 mixture of THF-MeOH-H2O (14 mL), ammonium chloride (55 mg, 1.03 mmol) and iron-powder (230 mg) were added and refluxed for 1.5 h. The reaction mixture was cooled, filtered, concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4and concentrated to provide (300 mg) crude. The crude product was dissolved in dry dichloromethane (5 mL) and cooled. To the cooled solution acetyl chloride (150 mg, 1.91 mmol) and triethylamine (0.6 mL, 4.2 mmol) were added and stirred for 1 h while warming upto ambient temperature. The reaction mixture was concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-5 % MeOH in chloroform) to provide title compound 10 (250 mg, 95% over two steps). MS (ESI+) m / z 324.9 (M + H)+.1H NMR (500 MHz, CDCl3) d 8.43 (d, J = 2.5 Hz, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.38 (s, 1H), 7.12-7.06 (m, 4H), 2.18 (s, 3H).N-[5-Bromo-6-(4-fluorophenoxy)pyridin-3-yl]-N-methylacetamide (11)

[0213] To a solution of compound 10 (110 mg, 0.34 mmol) in anhydrous THF (2 mL), 60% sodium hydride in mineral oil (22 mg, 0.55 mmol) was added at ambient temperature. After 5 min, iodomethane (85 mg, 0.60 mmol) was added drop-wise to the reaction mixture and stirred at ambient temperature for 2 h. The reaction mixture was concentrated and purified by preparative TLC (1% MeOH in CHCl3) to provide title compound 11 (100 mg, 87% yield). MS (ESI+) m / z 339.0 (M + H)+.1H NMR (500 MHz, DMSO-d6) d 8.36 (s, 1H), 8.13 (s, 1H), 7.29-7.7.25 (m, 4H), 3.11 (s, 3H), 1.79 (s, 3H). N-[3-Bromo-4-(4-fluorophenoxy)phenyl]acetamide (12)

[0214] Step a: To a solution of 4-fluorophenol (224 mg, 2.0 mmol) in dry DMF (10 mL), 60% sodium hydride in mineral oil (80 mg, 2.0 mmol) was added portion-wise at ambient temperature. After 10 min, 3, 4-dibromonitrobenzene (420 mg, 1.5 mmol) was added and heated at 80 °C for 30 min. The reaction mixture was cooled, concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-20% MeOH in chloroform) to provide 1-(2-bromo- 4-nitrophenoxy)-4-fluorobenzene (400 mg, 85% yield).

[0215] Step-b and step-c: To a degassed solution of 1-(2-bromo-4-nitrophenoxy)-4- fluorobenzene (400 mg, 1.28 mmol) in ethyl acetate (10 mL), 10% Pd / charcoal (40 mg) was added. The mixture was stirred under H2-gas at ambient temperature for 2 h. The reaction mixture was filtered and concentrated. The crude product was dissolved in dry dichloromethane (5 mL) and cooled. To the cooled solution acetyl chloride (0.2 mL, 2.8 mmol) and triethylamine (0.4 mL, 2.8 mmol) were added. The reaction mixture was then allowed to warm to ambient temperature over 4 h, evaporated and purified by silica gel column chromatography (0-10% MeOH in chloroform) to provide title compound 12 (400 mg, 96% yield). MS (ESI+) m / z 324.0(M + H)+.1H NMR (500 MHz, CDCl3) d 7.84 (d, J = 3.0 Hz, 1H), 7.39 (dd, J = 2.5, 9.0 Hz, 1H), 7.25 (s, 1H), 7.04-6.98 (m, 2H), 6.93-6.87 (m, 3H), 2.18 (s, 3H).N-[3-Bromo-4-(4-fluorophenoxy)phenyl]-N-methylacetamide (13)

[0216] To a solution of compound 12 (130 mg, 0.4 mmol) in anhydrous THF (2 mL), 60% sodium hydride in mineral oil (26 mg, 0.65 mmol) was added at ambient temperature. After 5 min, iodomethane (98 mg, 0.7 mmol) was added drop-wise to the reaction mixture and stirred at ambient temperature for 2 h. The reaction mixture was then concentrated and purified by preparative TLC (2.5% MeOH-CHCl3) to provide title compound 13 (100 mg, 72% yield). MS (ESI+) m / z 338.0 (M + H)+.1H NMR (500 MHz, CDCl3) d 7.49 (d, J = 2.0 Hz, 1H), 7.10-7.04 (m, 3H), 7.02-6.97 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 3.24 (s, 3H), 1.91 (s, 3H). 3-Bromo-4-[(cyclopent-1-en-1-yl)oxy]pyridine (14)

[0217] To a mixture of 3-Bromo-4-hydroxypyridine (85 mg, 0.49 mmol), cyclopentenyl- 1-boronic acid (112 mg, 1.00 mmol), anhydrous Cu(OAc)2 (90 mg, 0.49 mmol) and hot 4A molecular sieves (500 mg) were added anhydrous DCM (5 mL) and anhydrous pyridine (165 mg, 2.02 mmol) were added to the reaction mixture under nitrogen and stirred at ambient temperature under nitrogen for 18 h. The reaction mixture was filtered through celite, washed with methanol (10 mL), concentrated and purified by preparative TLC (8% MeOH in chloroform) to provide the title compound 14 (60 mg, 50% yield). MS (ESI+) m / z 239.9 (M + H)+.1H NMR (500 MHz, CDCl3) d 7.99 (d, J = 2.5 Hz, 1H), 7.55 (dd, J = 2.0, 7.5 Hz, 1H), 6.49 (d, J = 7.5 Hz, 1H), 5.74-5.70 (m, 1H), 2.76-2.71 (m, 2H), 2.58-2.54 (m, 2H), 2.18-2.11 (m, 2H). N-(3-Bromo-4-fluorophenyl)acetamide (16)

[0218] To a cooled solution of 3-bromo-4-fluoroaniline (630 mg, 3.31 mmol) in dry dichloromethane (5 mL) acetyl chloride (0.3 mL, 4.20 mmol) and triethylamine (1.0 mL, 7.24mmol) were added and allowed to warm to ambient temperature over 4 h. The reaction mixture was concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-100% chloroform in EtOAc) to provide title compound 16 (400 mg, 52% yield). MS (ESI+) m / z 231.9 (M + H)+.1H NMR (500 MHz, CDCl3) d 7.80 (dd, J = 3.0, 6.5 Hz, 1H), 7.59 (bs, 1H), 6.36 (m, 1H), 7.05 (t, J = 8.0 Hz, 1H), 2.17 (s, 3H). 2-Bromo-6-fluoro-1-(pyridin-4-ylmethyl)-1H-benzo[d]imidazole (17)

[0219] Step-a: To a solution of 4-picolylamine (340 mg, 3.14 mmol) in toluene (5 mL), potassium carbonate (228 mg, 4.78 mmol) was added at ambient temperature and stirred for 10-15 min. The temperature of the reaction mixture was raised up to 50oC. 2, 4-difluoro nitrobenzene (500 mg, 3.14 mmol) was added to the reaction mixture slowly and stirred at 45-50oC for 1-2 h. The reaction mixture was cooled and diluted with water (5 mL). Organic layer was separated and dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-5% chloroform in MeOH) to provide 5-fluoro-2-nitro-N-(pyridin-4- ylmethyl)benzenamine (600 mg, 77% yield). MS (ESI+) m / z 248.1 (M + H)+.

[0220] Step-b: To a degassed solution of 5-fluoro-2-nitro-N-(pyridin-4- ylmethyl)benzenamine (500 mg, 2.02 mmol) in methanol (5 mL) was added 10% Pd / charcoal (50 mg). The mixture was stirred under H2-gas at ambient temperature for 4 h. The reaction mixture was filtered, concentrated and purified by silica gel column chromatography (0-5% chloroform in MeOH) to provide 5-fluoro-N1-(pyridin-4-ylmethyl)benzene-1,2-diamine (200 mg, 46% yield). MS (ESI+) m / z 218.1 (M + H)+.

[0221] Step-c: To a cooled solution of KOH (18 mg, 1.38 mmol) in ethanol (2 mL), 5- fluoro-N1-(pyridin-4-ylmethyl)benzene-1,2-diamine (200 mg, 0.92 mmol) and carbon disulfide (211 mg, 2.76 mmol) was added and stirred for 5-10 min. The mixture was stirred and refluxed for 4 h. The reaction mixture was then concentrated and the crude was purified by silica gel column chromatography (0-5% chloroform in MeOH) to provide 6-fluoro-1- (pyridin-4-ylmethyl)-1H-benzo[d]imidazole-2(3H)-thione (60 mg, 25% yield). MS (ESI+) m / z 260.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) d 13.08 (bs, 1H), 8.51 (d, J = 5.5 Hz,2H), 7.29 (dd, J = 2.0, 8.5 Hz, 1H), 7.25 (d, J = 5.5 Hz, 2H), 7.22 (dd, J = 4.0, 8.0 Hz, 1H), 7.05 (dt, J = 2.0, 9.0 Hz, 1H), 5.51 (s, 2H).

[0222] Step-d: To a cooled solution of 6-fluoro-1-(pyridin-4-ylmethyl)-1H- benzo[d]imidazole-2(3H)-thione(60 mg, 0.231mmol), 48% aqueous HBr (26 mg, 0.312mmol), and acetic acid (1 mL) was added bromine (132 mg, 0.83mmol) and stirred at ambient temperature for 4 h. The reaction mixture was cooled and diluted with ethyl acetate (10 mL). 5 mL water was added and organic layer was separated and dried over Na2SO4, concentrated, and purified by preparative TLC (0-5% chloroform in MeOH) to provide title compound 17 (30 mg, 42% yield). MS (ESI+) m / z 305.9 (M + H)+.1H NMR (500 MHz, DMSO-d6) d 8.53 (d, J = 6.0 Hz, 2H), 7.69 (dd, J = 5.0, 8.0 Hz, 1H), 7.60 (dd, J = 2.0, 9.0 Hz, 1H), 7.13 (dt, J = 2.0, 9.0 Hz, 1H), 7.08 (d, J = 5.0 Hz, 2H), 5.58 (s, 2H). 3-Bromo-2-(4-fluorophenoxy)pyridine

[0223] To a solution of 4-fluorophenol (160 mg, 1.43 mmol) in dry DMF, 60% sodium hydride in mineral oil (60 mg, 1.5 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloropyridine (276 mg, 1.43 mmol) was added to the reaction mixture and heated at 150oC for 2 h. The reaction mixture was cooled, concentrated, diluted with ethyl acetate (30 mL), washed with water (10 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide title compound (300 mg, 77%). MS (ESI+) m / z 267.9 (M + H)+.1H NMR (500 MHz, CDCl3) d 8.05 (dd, J = 1.5, 5.0 Hz, 1H), 7.93 (dd, J = 1.5, 7.5 Hz, 1H), 7.15-7.08 (m, 4H), 6.89 (dd, J = 5.0, 8.0 Hz, 1H). 2-Bromo-α-(4-fluorophenyl)benzenemethanol

[0224] To a suspension of Mg turnings (240 mg, 10 mmol) in anhydrous THF (10 mL) was added dibromoethane (75 mg, catalytic amount) and stirred at rt. After 10 min, 4- bromofluorobenzene (1.75 g, 10 mmol) was added and stirred for 1 h. To the reaction mixture was drop-wise added a solution of 2-bromobenzaldehyde (1.85 g, 10 mmol) dissolved inanhydrous THF (5 mL) and stirred for 2h at rt. The reaction was quenched by addition saturated NH4Cl solution (2 mL). The reaction mixture was concentrated, diluted with ethyl acetate (60 mL), and washed with water (10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-50% CHCl3in hexane) to provide title compound (1.28 g, 47%).1H NMR (500 MHz, CDCl3) d 7.59-7.52 (m, 2H), 7.39-7.33 (m, 3H), 7.19-7.14 (m, 1H), 7.05-6.90 (m, 2H), 7.17 (d, J = 3.5 Hz, 1H), 2.38 (d, J = 3.5 Hz, 1H). 1-Bromo-2-[(4-fluoro-phenyl)-methoxy-methyl]-benzene

[0225] To a cooled solution of 6b (280 mg, 1.0 mmol) in anhydrous THF (5 mL) was added 60% sodium hydride in mineral oil (40 mg, 1.0 mmol) and stirred. After 10 min, iodomethane (86 uL, 1.4 mmol) was added to the reaction and stirred for 2 h. The reaction was quenched by addition saturated NH4Cl solution (2 mL). The reaction mixture was concentrated, diluted with ethyl acetate (60 mL), and washed with water (10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-50% CHCl3in hexane) to provide title compound (250 mg, 84%).1H NMR (500 MHz, CDCl3) d 7.56-7.50 (m, 2H), 7.37-7.32 (m, 3H), 7.14 (dt, J = 1.5, 7.5 Hz, 1H), 7.03-6.96 (m, 2H), 5.63 (s, 1H), 3.38 (s, 3H). 2-Bromo-4'-fluorobenzophenone

[0226] To a solution of 6b (250 mg, 0.9 mmol) in anhydrous DMSO (5 mL) was added IBX-reagent (500 mg, 1.8 mmol) and stirred for 3h. The reaction mixture was diluted with ethyl acetate (60 mL), and washed with water (3 x 10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-50% CHCl3in hexane) to provide title compound (180 mg, 64%).1H NMR (500 MHz, CDCl3) d 7.86-7.81 (m, 2H), 7.66 (dd, J = 1.5, 9.0 Hz, 1H), 7.43 (dt, J = 1.5, 7.5 Hz, 1H), 7.39-7.32 (m, 2H), 7.17-7.11 (m, 2H).2-Bromo-4'-fluorodiphenylmethane

[0227] To a solution of 6b (250 mg, 0.9 mmol) in anhydrous DCM (2.5 mL) was added triethylsilane (313 mg, 2.7 mmol) followed by TFA (0.4 mL) and stirred for 3h. The reaction mixture was concentrated, diluted with ethyl acetate (60 mL), and washed with water (10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-10% CHCl3 in hexane) to provide title compound (240 mg, 90%).1H NMR (500 MHz, CDCl3) d 7.57 (dd, J = 1.5, 9.0 Hz, 1H), 7.26-7.22 (m, 1H), 7.17-7.06 (m, 4H), 7.00-6.95 (m, 2H), 4.08 (s, 2H). 3-Bromo-2-(4-fluorophenylthio)pyridine

[0228] To a solution of 3-bromo-2-chloropyridine (380 mg, 2.0 mmol) and 4- fluorothiophenol (260 mg, 2 mmol) in anhydrous DMF (10 mL) was added Cs2CO3 (1.3 g, 4.0 mmol) and heated at 100oC for 3 h. The reaction mixture was cooled, concentrated, diluted with ethyl acetate (30 mL), and washed with water (10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-50% CHCl3 in hexane) to provide title compound (300 mg, 50%).1H NMR (500 MHz, CDCl3) d 8.22 (dd, J = 1.5, 5.0 Hz, 1H), 7.73 (dd, J = 1.5, 8.0 Hz, 1H), 7.55-7.50 (m, 2H), 7.14-7.08 (m, 2H), 8.22 (dd, J = 4.5, 8.0 Hz, 1H). 3-Bromo-N-(4-fluorophenyl)-2-pyridinamine

[0229] A solution of 4-fluoroiodobenzene (222 mg, 1.0 mmol), 3-bromo-2-pyridinamine (175 mg, 1.0 mmol) and sodium tert-butoxide (182 mg, 2 mmol) in anhydrous dioxane (10 mL) was degassed by purging N2-gas for 30 min. To the solution was added Pd2dba3 (46 mg, 0.05 mmol) and xantphos (58 mg, 0.1 mmol) and heated at 110oC for 2 h. The reaction mixture was cooled, concentrated, diluted with ethyl acetate (30 mL), and washed with water(10 mL). The organic layer was dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0-10% ethyl acetate in hexane) to provide title compound (211 mg, 79%).1H NMR (500 MHz, CDCl3) d 8.17-8.11 (m, 1H), 7.75-7.72 (m, 1H), 7.63-7.51 (m, 2H), 7.34 (t, J = 8.0 Hz, 0.5H), 7.08-7.02 (m, 2H), 6.92 (bs, 0.5 H), 6.63 (dd, J = 5.0, 7.5 Hz, 1H). Obtained as 3:1 mixture of fluoro and defluoro product that cannot be separated. N-(7-bromo-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)cyclopropanecarboxamide

[0230] Step-a: To a cooled (0-5 °C) solution of 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (218 mg, 1.44 mmol) and catalytic amount of acetic acid (5 mg, 0.08 mmol) in anhydrous acetonitrile (2.5 mmol) was added NBS (275 mg, 1.55 mmol) portion-wise and allowed to warm up to ambient temperature over 2 h while stirring. The reaction mixture was diluted with water (20 mL) and extracted with chloroform (3 x 20 mL). The organic phase was dried over Na2SO4, concentrated and purified by silica gel column chromatography (0-30% ethyl acetate in hexane) followed by prep TLC (20% ethyl acetate in hexane) to provide 7-bromo- 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (100 mg) as yellow solid.

[0231] Step-b: To a cooled solution of above crude (100 mg, 0.43 mmol) and cyclopropane carboxylic acid (80 mg, 0.93 mmol) in anhydrous DCM (2.5 mL) was added triethylamine (220 mg, 2.2 mmol) followed by BOP-reagent (300 mg, 0.68 mmol) in portions and allowed to warm-up to ambient temperature over 16 h while stirring. The reaction mixture was diluted with ethyl acetate (40 mL), and then washed with water (5 mL), saturated NaHCO3solution (5 mL) and brine (5 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by prep TLC (20% ethyl acetate in hexane) to provide target compound (90 mg, 68%) as yellow solid.1H NMR (400 MHz, CDCl3) δ 7.89 (bs, 1H), 7.58 (bs, 1H), 7.04 (s, 1H), 4.26-4.18 (m, 4H), 1.59-1.51(m, 1H), 1.11-1.06 (m, 2H), 0.90- 0.82 (m, 2H). 8-bromo-N-cyclopropyl-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide

[0232] Step-a: To a cooled solution of 3-bromo-4-hydroxy-5-methoxybenzaldehyde (510 mg, 2.21 mmol) in anhydrous DCM (6.0 mL) was added 1M borontribromide in heptane (5.0 mL, 5.0 mmol) drop-wise and allowed to warm-up to ambient temperature over 1 h while stirring. The reaction mixture was cooled to (0-5 ^C) and quenched with dropwise addition of methanol (1 mL). The mixture was concentrated and co-evaporated with CHCl3to obtain 3- bromo-4,5-dihydroxybenzaldehyde (500 mg) as black solid. The crude solid was dissolved in anhydrous DMF (10 mL). To the solution was added cesium carbonate (2.30 g, 7.07 mmol) and stirred for 30 min at ambient temperature. 1,2-Dibromoethane (510 mg, 2.71 mmol) was added to the solution dropwise and heated to 90 ^C for 4.5 h. The mixture was concentrated, diluted with ethyl acetate (50 mL), washed with water (10 mL) and brine (10 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by prep silica gel column chromatography (0-5% methanol in CHCl3) to provide target compound (100 mg, 18%) as white solid.1H NMR (400 MHz, CDCl3) δ 9.78 (s, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 4.47-4.43 (m, 2H), 4.34-4.00 (m, 2H).

[0233] Step-b: To a stirring solution of 8-bromo-2,3-dihydrobenzo[b][1,4]dioxine-6- carbaldehyde (95 mg, 0.39 mmol) in acetone (2.5 mL) was added 0.5 M aqueous solution of KMnO4(2.0 mL, 1.0 mmol) drop-wise and heated at 80 °C for 1 h. The resultant suspension was filtered through a celite pad, washed with 50% acetone-water mixture (2 x 10 mL). The combined filtrate was concentrated, suspended in 1 M HCl solution (20 mL) and extracted with ethyl acetate (2 x 30 mL). The organic phase was dried over dry Na2SO4, concentrated to obtain 8-bromo-2, 3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (100 mg) as white solid, which was used without further purification. To a cooled solution of above crude (95 mg, 0.37 mmol) in anhydrous DCM (2.0 mL) was added anhydrous triethylamine (125 mg, 1.25 mmol) followed by TBTU (170 mg, 0.53 mmol) and stirred for 30 min. Then cyclopropylamine (82 mg, 1.43 mmol) was added to the reaction mixture and allowed to warm-up to ambient temperature over 2 h while stirring. The reaction mixture was diluted with ethyl acetate (40 mL), washed with saturated NaHCO3solution (10 mL) and brine (10 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by preparative TLC (4% methanol in CHCl3) to obtain target compound (70 mg, 63%) as white solid.1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 2.0 Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H), 6.15 (bs, 1H), 4.45-4.36 (m, 2H), 4.30-4.26 (m, 2H), 2.91-2.83 (m, 1H), 0.91-0.83 (m, 2H), 0.64-0.58 (m, 2H). 1-(6-bromo-3,4-dihydro-2(1H)-isoquinolinyl)-2-methoxy-1-propanone

[0234] Step-a: To a cooled solution of 6-Bromo-1,2,3,4-tetrahydroisoquinoline (290 mg, 1.37 mmol), DL-lactic acid (230 mg, 2.56 mmol) in anhydrous DCM (5 mL)) was added trimethylamine (330 mg, 3.3 mmol) followed by BOP-reagent (720 mg, 1.63 mmol) in portions and allowed to warm-up to ambient temperature over 5 h while stirring. The reaction mixture was diluted with CHCl3(40 mL), wash with water (10 mL), saturated NaHCO3 solution (10 mL) and brine (10 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by column chromatography (ethyl acetate in Hexanes) to provide 1- (6-bromo-3,4-dihydro-2(1H)-isoquinolinyl)-2-hydroxy-1-propanone (360 mg, 93%) as clear liquid.1H NMR (400 MHz, CDCl3) δ 7.37-7.29 (m, 2H), 7.06-6.96 (m, 1H), 4.71 (s, 1H), 4.58-4.48 (m, 2H), 4.00-3.58 (m, 3H), 2.93-2.86 (m, 2H), 1.40-1.32 (m, 3H).

[0235] Step-b: To a cooled solution of 1-(6-bromo-3,4-dihydro-2(1H)-isoquinolinyl)-2- hydroxy-1-propanone (350 mg, 1.23 mmol) in anhydrous THF (5.0 mL) was added 60% sodium hydride suspension in mineral oil (66 mg, 1.65 mmol) in portions. After 10 min iodomethane (310 mg, 2.18 mmol) was added dropwise and allowed to warm-up to ambient temperature over 4 h. The reaction mixture was diluted with CHCl3(40 mL), wash with water (10 mL), saturated NaHCO3 solution (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, concentrated and purified by column chromatography (0-5% methanol in chloroform) to obtain the title compound (250 mg, 68%) as clear liquid. N-(6-Bromo-3,4-dihydro-2H-chromen-4-yl)-N-methylacetamide

[0236] Step-a: To a solution of 6-Bromo-4-chromanone (340 mg, 1.37 mmol) in anhydrous methanol (7 mL) was added sodium borohydride (150 mg, 4.0 mmol) in portions over 5 min and allowed to stir for 2 h. The reaction mixture was quenched with 1M HCl (10 mL) and extracted with ethyl acetate (2 x 25 mL). The organic phase was dried over Na2SO4, concentrated. The residue was dissolved in anhydrous DCM (5 mL) and cooled. To a cooled solution was added anhydrous trimethylamine (335 mg, 3 mmol) and methanesulfonyl chloride (180 mg, 1.57 mmol) dropwise and allowed to stir for 2 h. The reaction mixture wasdiluted with DCM (30 mL), washed with water (10 mL) and brine (10 mL). The organic phase was dried over dry Na2SO4 and concentrated to provide corresponding mesyl intermediate (470 mg) as red liquid. The residue wad suspended in 33% methylamine in methanol and stirred at 70oC for 18 h. The reaction mixture was diluted with 50% brine (10 mL) and extracted with ethyl acetate (2 x 30 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by column chromatography (0-5% methanol in CHCl3) to provide the title compound (235 mg, 65% over 3 steps) as yellow liquid.1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 3.0 Hz, 1H), 7.23 (dd, J = 3.5, 9.0 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 4.31-4.15 (m, 2H), 3.64 (t, J = 4.5 Hz, 1H), 2.51 (s, 3H), 2.06-1.90 (m, 2H).

[0237] Step-b: To a cooled solution of N-(6-Bromo-3,4-dihydro-2H-chromen-4-yl)- methylamine (235 mg, 0.97 mmol) in anhydrous DCM (2.5 mL) was added anhydrous trimethylamine (250 mg, 2.5 mmol) and acetyl chloride (145 mg, 1.86 mmol) dropwise and allowed to stir for 2 h. The reaction mixture was diluted with DCM (30 mL), washed with water (10 mL) and brine (10 mL). The organic phase was dried over dry Na2SO4, concentrated and purified by silica gel column chromatography to provide title compound (160 mg, 57%) as clear liquid.1H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 9.0, 1H), 7.05 (s, 1H), 6.72 (d, J = 9.0 Hz, 1H), 6.05-6.00 (m, 1H), 4.35-4.30 (m, 1H), 4.22-4.14 (m, 1H), 2.73 (s, 3H), 2.21 (s, 3H), 2.11-2.01 (m, 2H). 3-Bromo-2-(3,4-difluorophenoxy)pyridine

[0238] To a solution of 3,4-difluorophenol (500 mg, 3.85 mmol) in dry DMF, 60% sodium hydride in mineral oil (138 mg, 5.78 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloropyridine (730 mg, 3.80 mmol) was added to the reaction mixture and heated at 150 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide the title compound as a colorless liquid at room temperature (1 g, 90.9% yield).1H NMR (400 MHz, CDCl3) δ 8.07 (q, J = 2.1 Hz, 1H), 7.94 (dd, J1=7.9 Hz, J2=1.7 Hz, 1H), 7.19 (q, J = 9.2 Hz, 1H), 7.04 (dq, J1=10.8 Hz, J2=3.2 Hz, 1H), 6.93 (m, 2H).3-bromo-2-(3,5-difluorophenoxy)pyridine

[0239] To a solution of 3,5-difluorophenol (500 mg, 3.85 mmol) in dry DMF, 60% sodium hydride in mineral oil (138 mg, 5.78 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloropyridine (730 mg, 3.80 mmol) was added to the reaction mixture and heated at 150 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide the title compound as an ivory color solid substance (800 mg, 72.7% yield).1H NMR (500 MHz, CDCl3) δ 8.12 (q, J = 2.1 Hz, 1H), 7.96 (dd, J1=7.6 Hz, J2=1.4 Hz, 1H), 6.98 (q, J = 4.1 Hz, 1H), 6.71 (m, 3H). 3-bromo-2-((4-fluorophenyl)thio)pyridine

[0240] To a solution of 4-fluorobenzenethiol (183 mg, 1.43 mmol) in dry DMF, Potassium carbonate (395 mg, 2.86 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloropyridine (276 mg, 1.43 mmol) was added to the reaction mixture and heated at 140 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide the title compound as yellow semi liquid crystalline substance at room temperature (265 mg, 65.2% yield).1H NMR (500 MHz, CDCl3) δ 8.24 (q, J = 2.1 Hz, 1H), 7.74 (dd, J1=8.3 Hz, J2=1.4 Hz, 1H), 7.54 (td, J1=6.2 Hz, J2=2.5 Hz, 2H), 7.13 (m, 2H), 6.89 (q, J = 4.1 Hz, 1H). 3-bromo-N-(4-fluorophenyl)pyridin-2-amine

[0241] To a solution of 4-fluoroaniline (159 mg, 1.43 mmol) in dry DMF, 60% sodium hydride in mineral oil (50 mg, 2.1 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloropyridine (276 mg, 1.43 mmol) was added to the reaction mixture and heated at 150 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide the title compound as yellowish liquid crystalline material at room temperature (307 mg, 80.3% yield).1H NMR (500 MHz, CDCl3) δ 8.12 (q, J = 2.1 Hz, 1H), 7.73 (dd, J1=7.9 Hz, J2=1.7 Hz, 1H), 7.54 (m, 2H), 7.04 (m, 2H), 6.92 (bs, 1H), 6.63 (q, J = 4.4 Hz, 1H). 3-bromo-5-fluoro-2-(4-fluorophenoxy)pyridine

[0242] To a solution of 4-fluorophenol (160 mg, 1.43 mmol) in dry DMF, 60% sodium hydride in mineral oil (50 mg, 2.1 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 3-bromo-2-chloro-5-fluoropyridine (300 mg, 1.43 mmol) was added to the reaction mixture and heated at 150 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gel column chromatography (0-5% methanol in CHCl3) to provide the title compound as an ivory solid substance at room temperature (368 mg, 90% yield).1H NMR (500 MHz, CDCl3) δ 8.10 (d, J= 2.8 Hz, 1H), 7.50 (d, J= 2.8 Hz, 1H), 7.11 (m, 2H), 7.04 (m, 2H). 5-bromo-4-(4-fluorophenoxy)pyrimidine

[0243] To a solution of 4-fluorophenol (100 mg, 0.89 mmol) in dry DMF, 60% sodium hydride in mineral oil (50 mg, 2.1 mmol) was added portion-wise at ambient temperature and stirred for 10 min. 5-bromo-4-chloropyrimidine (172 mg, 0.89 mmol) was added to the reaction mixture and heated at 150 °C for 2 h. The reaction mixture was cooled, concentrated and then diluted again with ethyl acetate (30 mL). Thereafter, the crude product was washed with water (10 mL), dried over Na2SO4, concentrated, and finally, purified by silica gelcolumn chromatography (0-5% methanol in CHCl3) to provide the title compound as an ivory solid substance at room temperature (180 mg, 75% yield).1H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H), 8.62 (s, 1H), 7.15 (s, 2H), 7.13 (m, 2H). 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (5A)

[0244] Step-a: To a solution of 5-Bromo-2-methoxy-4-methyl-3-nitropyridine (1A) (10.0 g, 40.5 mmol) in DMF (100 mL) was added 1 M lithium methanolate in methanol (2.5 mL, 2.5 mmol, 1 M) and heated to 100 °C. To this reaction mixture 1,1-dimethoxy-N,N- dimethylmethanamine (40 mL, 296 mmol) was added dropwise over 10 min. The reaction mixture was further stirred at 100 °C for 5 h and then allowed to cool to ambient temperature. Then 600 mL water was added gradually to the reaction mixture. The resulting precipitate was collected by vacuumfiltration, washed with water (50 mL), and dried to provide (E / Z)-2- (5-bromo-2-methoxy-3-nitropyridin-4-yl)-N,N-dimethylethenamine (1A-a) (10.5 g, 86% yield) as bright red solid. It was reacted further without purification. MS (ESI+) m / z 302.0 (M + H)+.

[0245] Step-b: (E / Z)-2-(5-Bromo-2-methoxy-3-nitropyridin-4-yl)-N,N- dimethylethenamine (5 g, 16.6 mmol), iron (7 g, 125.4 mmol) and ammonium chloride (15 g, 280.4 mmol) were dissolved in 5:3:2 mixture of THF: Ethanol: Water (50 mL). The reaction mixture was stirred at room temperature for 12 h. After TLC analysis (20% EtOAc in Hexanes) showed complete consumption of starting material, the reaction mixture was filteredfilter and extract product with ethyl acetate (30 mL×2 times). Finally, purify the concentrated residue by silica gel column chromatography (5-40% EtOAc in Hexane) to provide compound 2A. (5 g, 38% yield over two steps). MS (ESI+) m / z 226.9 (M + H)+.1H NMR (500 MHz, CDCl3) δ 8.70 (bs, 1H), 7.84 (s, 1H), 7.31 (t, J = 2.5 Hz, 1H), 6.57 (t, J = 2.5 Hz, 1H), 4.08 (s, 3H).

[0246] Step-c: To a cooled (0-5 °C) solution of 2A (3.63 g, 16.1 mmol) in dry THF (50 mL) under nitrogen was added sodium hydride (0.72 g of 60% dispersion in oil, 18.0 mmol) and stirred for 10 min. p-Toluenesulfonyl chloride (3.3 g, 17.4 mmol) was then addedportion-wise over 5 min, and the reaction mixture was allowed to warm to ambient temperature and stirred under nitrogen for 1 h. The reaction mixture was concentrated, diluted with ethyl acetate (50 mL) and washed with water (2 x 20 mL). The organic layer was concentrated and purified by silica gel column chromatography (2-20% EtOAc in hexanes) to provide 4-bromo-7-methoxy-1-tosyl-1H-pyrrolo[2,3-c]pyridine (2A-a) (4.9 g, 80% yield). MS (ESI+) m / z 381.1 (M + H)+.

[0247] Step-d: 4-bromo-7-methoxy-1-tosyl-1H-pyrrolo[2,3-c]pyridine (4.9 g, 12.8 mmol) in 1,4-dioxane (50 mL) and 4 M HCl in 1,4-dioxane (50 mL) were stirred at 65 °C for 5 h. The reaction mixture was then cooled to ambient temperature. The reaction mixture was filtered, rinsed with water (10 mL), and dried to provide compound 3A (4.35 g, 92% yield) as a beige color solid. MS (ESI+) m / z 367.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 11.50 (bs, 1H), 8.03 (d, J = 3.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 6.59 (d, J = 3.5 Hz, 1H), 2.37 (s, 3H).

[0248] Step-e: To a suspension of compound 3A (4.34 g, 11.8 mmol) and cesium carbonate (5.35 g, 16.4 mmol) in dioxane (75 mL), iodomethane (3.9 g, 16.5 mmol) was added dropwise and stirred at ambient temperature for 4 h. The reaction mixture was concentrated, diluted with ethyl acetate (75 mL) and washed with water (2 x 25 mL). The organic layer was concentrated and purified by silica gel column chromatography (0-5% MeOH in CHCl3) to provide compound 4A as white solid (3.6 g, 78% yield). MS (ESI+) m / z 380.8 (M + H)+.1H NMR (500 MHz, CDCl3) δ 8.00 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 3.5 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.17 (s, 1H), 6.51 (d, J = 3.5 Hz, 1H), 3.50 (s, 3H), 2.40 (s, 3H).

[0249] Step-f: A mixture of Compound 4A (1.04 g, 2.6 mmol), 4,4,4′,4′,5,5,5′,5′- octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.80 g, 3.2 mmol), potassium acetate (0.50 g, 5.1 mmol), tris(dibenzylideneacetone)dipalladium(0) (60 mg, 0.065 mmol), and 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-PHOS, 123 mg, 0.26 mmol) was degassed by vacuum and filled with nitrogen (repeated three times). Degassed dry 1,4- dioxane (20 mL) was added to the reaction mixture and heated under argon at 90 °C for 5 h. The consumption of starting material was monitored by TLC (1% MeOH in CHCl3). The reaction mixture was cooled to ambient temperature, concentrated, and partitioned between ethyl acetate (40 mL) and water (2 x 20 mL). The ethyl acetate layer was washed with brine (2 x 10 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (10−80% ethyl acetate in hexanes) to provide compound 5A as waxy solid (5.4 g, 73% yield). MS (ESI+) m / z 429.1 (M + H)+.1H NMR (500 MHz,DMSO-d6) δ 7.98 (d, J = 3.5 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.72 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 3.5 Hz, 1H), 3.43 (s, 3H), 2.37 (s, 3H), 1.29 (s, 12H).

[0250] The intermediates shown in Table A were prepared from commercially available starting materials using methods analogous to those used to prepare 6-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one. Table A. Additional Boronic ester intermediates6-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (5B)

[0251] Prepared according to route D

[0252] Step-a: To a solution of 1-(4-bromo-1H-pyrrol-2-yl)-2,2,2-trichloroethanone (1B) (0.94 g, 3.2 mmol) in dry DMF (2.5 mL), (1,3-dioxolan-2-yl)-N-methylmethanamine (0.45 g, 3.8 mmol) was added and stirred at ambient temperature for 18 h. Another batch of (1,3- dioxolan-2-yl)-N-methylmethanamine (0.45 g, 3.8 mmol) was added and heated to 70 °C for 5 h. The reaction mixture was evaporated, diluted with ethyl acetate (30 mL) and washed with water (2 x 15 mL). The organic phase was dried over Na2SO4, evaporated and purifiedby silica gel column chromatography (20-100% ethyl acetate in hexane) to afford N-((1,3- Dioxolan-2-yl)methyl)-4-bromo-N-methyl-1H-pyrrole-2-carboxamide (2B) (0.84 g, 90%) as beige solid. MS (ESI+) m / z 289.0 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 11.78 (bs, 1H), 7.03-7.01 (m, 1H), 6.65 (s, 1H), 5.00 (s, 1H), 3.95-3.82 (m, 4H), 3.61 (s, 2H), 3.22 (s, 3H).

[0253] Step-b: A solution of 2B (370 mg, 1.3 mmol) in methanesulfonic acid (2.5 mL) was stirred at 60 °C for 24 h. The reaction mixture was cooled and poured into 3M NaOH solution (15 mL) and stirred for 30 min. The resultant suspension was filtered and purified by preparative TLC (4% methanol in CHCl3) to provide 3-bromo-6-methyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (80 mg, 27%) as white solid. MS (ESI+) 226.9 m / z (M + H)+.1H NMR (500MHz, DMSO-d6) δ 12.39 (bs, 1H), 7.47 (s, 1H), 7.28 (d, J = 6.5 Hz, 1H), 6.33 (d, J = 7.5 Hz, 1H), 3.50 (s, 3H).

[0254] Step-c: To a cooled solution of 3B (150 mg, 0.66 mmol) in dry THF (5 mL), sodium hydride (32 mg of 60% dispersion in oil, 0.80 mmol) was added portion-wise. After 10 min, tosyl chloride (160 mg, 0.84 mmol) was added to the solution in portions and stirred for 1 h at RT. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL). The organic phase was dried over Na2SO4 and dried to provide 3-bromo-6- methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one (4B) (250 mg, 98%) as white solid. MS (ESI+) m / z 381.0 (M + H)+.1H NMR (500MHz, DMSO-d6) δ 8.18 (s, 1H), 8.00 (d, J= 8.5 Hz, 2H), 7.53 (d, J = 7.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 6.35 (d, J = 7.0 Hz, 1H), 3.41 (s, 3H), 2.38 (s, 3H).

[0255] Step-d: 4B (127 mg, 0.35 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2- dioxaborolane) (0.42 g, 1.65 mmol), dried potassium acetate (74 mg, 0.75 mmol), Xphos (17 mg, 0.04 mmol) and Pd2dba3 (8 mg, 0.02 mmol) were taken in an RBF and degassed by vacuum and filled with nitrogen (repeated three times). To the reaction mixture, dry 1, 4- dioxane (2.0 mL) was added and stirred at 95 °C for 3 h. The reaction was diluted with ethyl acetate (30 mL) and washed with water (2 x 15 mL) and brine (2 x 10 mL). The organic phase was evaporated, and purified by preparative TLC (1% methanol in chloroform) to provide the title compound (120 mg, 80%) and proto-dehalogenated by-product. The crude 6- methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1,6-dihydro-7H-pyrrolo[2,3- c]pyridin-7-one (5B) was used for the subsequent Suzuki coupling reactions (see Scheme 4) without further purification. A small quantity of material was further purified for analytical sample preparation. MS (ESI+) m / z 429.1 (M + H)+.1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 7.2 Hz, 1H), 6.77 (d, J = 7.2 Hz, 1H), 3.51 (s, 3H), 2.39 (s, 3H), 1.35 (s, 12H).4-(6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-3-yl)benzaldehyde

[0256] A mixture of 5B (500 mg, 1.17 mmol), 4-bromobenzaldehyde (230 mg, 1.24 mmol), K2CO3(500 mg, 3.62 mmol) was dissolved in dioxane (10.0 mL) and water (2.5 mL). The solution was degassed by purging with N2-gas for 30 min. Then, meCgPPh (14 mg, 0.048 mmol) and Pd2dba3 (21 mg, 0.022 mmol) were added and stirred at 65oC for 2 h. Reaction mixture was diluted with water (20 mL), extracted with CHCl3(2 x 50 mL). The organic layer was washed with brine and evaporated to dryness. The residue was suspended in minimum amount of Ethylacetate and filtered to provide title compound as off-white solid (270 mg, 56%).1H NMR (400 MHz, CDCl3) δ 10.06 (s, 1H), 8.12 (s, 1H), 8.08 (d, J = 6.8 Hz, 2H), 7.99 (d, J = 6.8 Hz, 2H), 7.71 (d, J = 6.8 Hz, 2H), 7.34 (d, J = 6.8 Hz, 2H), 7.10 (d, J = 7.2 Hz, 1H), 6.59 (d, J = 7.2 Hz, 1H), 3.55 (s, 3H), 2.42 (s, 3H). 3-(6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-3-yl)benzaldehyde

[0257] A mixture of 5B (500 mg, 1.17 mmol), 3-bromobenzaldehyde (246 mg, 1.33 mmol), K2CO3 (500 mg, 3.62 mmol) was dissolved in dioxane (10.0 mL) and water (2.5 mL). The solution was degassed by purging with N2-gas for 30 min. Then, meCgPPh (14 mg, 0.048 mmol) and Pd2dba3(21 mg, 0.022 mmol) were added and stirred at 60oC for 2 h. Reaction mixture was diluted with water (20 mL), extracted with CHCl3 (2 x 50 mL). The organic layer was washed with brine and evaporated to dryness. The residue was purified by silica gel column chromatography (0-10% methanol in CHCl3) to provide title compound (400 mg, 85%) as white solid.1H NMR (400 MHz, CDCl3) δ 10.09 (s, 1H), 8.09 (s, 1H), 8.01 (d, J = 8.0 Hz, 2H), 8.04 (t, J = 1.6 Hz, 1H), 7.89 (td, J = 1.6, 6.8 Hz, 1H), 7.81 (td, J = 1.6,8.0 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 7.2 Hz, 1H), 6.58 (d, J = 7.2 Hz, 1H), 3.55 (s, 3H), 2.42 (s, 3H). 2-(6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-3-yl)benzaldehyde

[0258] A mixture of 5B (330 mg, 0.77 mmol), 2-bromobenzaldehyde (166 mg, 0.89 mmol), K2CO3 (310 mg, 2.25 mmol) was dissolved in dioxane (10.0 mL) and water (2.5 mL). The solution was degassed by purging with N2-gas for 30 min. Then, meCgPPh (9 mg, 0.031 mmol) and Pd2dba3 (12 mg, 0.013 mmol) were added and stirred at 60oC for 2 h. Reaction mixture was diluted with water (20 mL), extracted with CHCl3 (2 x 50 mL). The organic layer was washed with brine and evaporated to dryness. The residue was purified by silica gel column chromatography (0-10% methanol in CHCl3) to provide title compound (250 mg, 71%) as white solid.1H NMR (500MHz, CDCl3) δ 10.00 (s, 1H), 8.11 (d, J = 8.5 Hz, 2H), 8.01 (d, J = 7.5 Hz, 1H), 7.96 (s, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 7.0 Hz, 1H), 6.18 (d, J = 7.5 Hz, 1H), 3.54 (s, 3H), 2.43 (s, 3H). Compounds Example 1. General Procedure for Suzuki coupling

[0259] Aryl bromide intermediate (0.10-0.20 mmol), anhydrous K2CO3 (3.0 eq.), meCgPPh (0.06 eq.) and Pd2dba3(0.03 eq.) were added to a three-neck flask and the reaction mixture was degassed by vacuum and back-filled with nitrogen (repeated three times). 1.1 M solution of boronic acid pinacol ester intermediate 5A or 5B (1.5 eq.) in degassed 4:1 dioxane-water mixture was then added to the reaction mixture and stirred at 65 °C for 2-4 h. Reaction was monitored using TLC analysis (80% EtOAc in hexanes or 5% MeOH in CHCl3) till the disappearance of boronic acid pinacol ester intermediate (2-4 h). The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over Na2SO4,evaporated to dryness and purified using preparative TLC (5% MeOH in CHCl3).

[0260] The following tosylate adducts were prepared from pinacol ester intermediate 5A and aryl bromides using route A, Step g and the general procedure Suzuki coupling:

[0261] 4-(4-(4-Fluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin- 7(6H)-one (6a) was obtained in 71% yield following general procedure step g. MS (ESI+) m / z 490.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.45 (d, J = 6.5 Hz, 1H), 8.01-7.96 (m, 2H), 7.95 (d, J = 3.5 Hz, 1H), 7.64 (s, 1H), 7.44 (d, J = 7.0 Hz, 2H), 7.32-7.25 (m, 4H), 6.71 (d, J = 6.0 Hz, 1H), 6.63 (d, J = 3.5 Hz, 1H), 3.47 (s, 3H), 2.38 (s, 3H).

[0262] 4-(5-(4-Fluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin- 7(6H)-one (7a) was obtained in 72% yield following general procedure step g. MS (ESI+) m / z 490.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.51 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 2.5 Hz, 1H), 8.04 (d, J = 4.0 Hz, 1H), 7.98-7.93 (m, 2H), 7.73 (s, 1H), 7.51 (t, J = 2.0 Hz, 1H), 7.44-7.39 (m, 2H), 7.31-7.20 (m, 4H), 6.67 (d, J = 3.5 Hz, 1H), 3.46 (s, 3H), 2.39 (s, 3H).

[0263] 4-(2-(6-Fluoropyridin-3-yloxy)phenyl)-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (8a) was obtained in 67 % yield following general procedure step g. MS (ESI+) m / z 490.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 7.96-7.92 (m, 3H), 7.87-7.85 (m, 1H), 7.61-7.56 (m, 1H), 7.51 (s, 1H), 7.46-7.40 (m, 4H), 7.28 (dt, J = 1.5, 7.5 Hz, 1H), 7.11 (dd, J = 3.5, 9.0 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H) 6.52 (d, J = 3.5 Hz, 1H), 3.41 (s, 3H), 2.38 (s, 3H).

[0264] 4-(3-(6-Fluoropyridin-3-yloxy)phenyl)-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (9a) was obtained in 40% yield following general procedure step g. MS (ESI+) m / z 490.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.10 (t, J = 1.0 Hz, 1H), 8.03 (d, J = 3.5 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.81-7.75 (m, 1H), 7.63 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.24 (dd, J = 3.5, 9.0 Hz, 1H), 7.18 (t, J = 2.0 Hz, 1H), 7.04 (dd, J = 2.5, 8.0 Hz, 1H), 6.68 (d, J = 3.0 Hz, 1H), 3.45 (s, 3H), 2.38 (s, 3H).

[0265] N-(6-(4-Fluorophenoxy)-5-(6-methyl-7-oxo-6,7-dihydro-1-tosyl-1H-pyrrolo[2,3- c]pyridin-4-yl)pyridin-3-yl)acetamide (10a) was obtained in 79% yield following general procedure step g. MS (ESI+) m / z 547.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 10.17 (bs, 1H), 8.22 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 2.5 Hz, 1H), 8.00-7.96 (m, 3H), 7.66 (s, 1H),7.42 (d, J = 8.5 Hz, 2H), 7.21-7.11 (m, 4H), 6.63 (d, J = 3.5 Hz, 1H), 3.47 (s, 3H), 2.38 (s, 3H), 2.06 (s, 3H).

[0266] N-(6-(4-Fluorophenoxy)-5-(6-methyl-7-oxo-6,7-dihydro-1-tosyl-1H- pyrrolo[2,3c] pyridine-4-yl)pyridin-3-yl)-N-methyl acetamide (11a) was obtained in 50%yield following general procedure step g. MS (ESI+) m / z 561.2 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.00-7.95 (m, 3H), 7.91 (s, 1H), 7.71 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 6.5 Hz, 4H), 6.70 (d, J = 3.5 Hz, 1H), 3.48 (s, 3H), 3.16 (s, 3H), 2.38 (s, 3H), 1.85 (s, 3H).

[0267] N-(4-(4-Fluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1-tosyl-1H-pyrrolo[2,3- c]pyridin-4-yl)phenyl)acetamide (12a) was obtained in 84% yield following general procedure step g. MS (ESI+) m / z 546.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 10.05 (bs, 1H), 7.96 (d, J = 4.5 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 2.5 Hz, 1H), 7.56 (dd, J = 2.5, 9.0 Hz, 1H), 7.48 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.08-7.02 (m, 2H), 6.97 (d, J = 9.0 Hz, 1H), 6.86-6.82 (m, 2H), 6.52 (d, J = 3.5 Hz, 1H), 3.39 (s, 3H), 2.38 (s, 3H), 2.04 (s, 3H).

[0268] N-(4-(4-Fluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1-tosyl-1H- pyrrolo[2,3c] pyridin-4-yl)phenyl)-N-methyl acetamide (13a) was obtained in 34% yield following general procedure step g. MS (ESI+) m / z 560.2 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 7.98-7.92 (m, 3H), 7.55 (s, 1H), 7.46-7.38 (m, 4H), 7.15 (t, J = 9.0 Hz, 2H), 7.01 (s, 2H), 6.94 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 3.5 Hz, 1H), 3.42 (s, 3H), 3.16 (s, 3H), 2.38 (s, 3H), 1.84 (s, 3H).

[0269] 4-(4-(Cyclopentenyloxy)pyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin- 7(6H)-one (14a) was obtained in 50 % yield following general procedure step g. MS (ESI+) m / z 462.2 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.01-7.98 (m, 2H), 7.95 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 3.5 Hz, 1H), 7.50 (s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 6.36 (d, J = 3.5 Hz, 1H), 6.27 (d, J = 8.0 Hz, 1H), 5.96 (s, 1H), 3.42 (s, 3H), 2.80-2.76 (m, 2H), 2.64-2.61 (m, 2H), 2.38 (s, 3H), 2.02-1.98 (m, 2H).

[0270] 4-[2-Fluoro-5-(methanesulfonyl)phenyl]-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (15a):

[0271] The title compound was obtained in 77% yield following general procedure step g. MS (ESI+) m / z 475.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) d 8.40-8.00 (m, 3H), 7.98 (d, J = 8.5 Hz, 2H), 7.73 (s, 1H), 7.65 (t, J = 5.0 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 6.48 (t, J = 3.5 Hz, 1H), 3.48 (s, 3H), 3.28 (s, 3H), 2.39 (s, 3H).

[0272] N-(4-Fluoro-3-(6-methyl-7-oxo-6,7-dihydro-1-tosyl-1H-pyrrolo[2,3-c]pyridin-4- yl)phenyl)acetamide (16a):

[0273] The title compound was obtained in 50% yield following general procedure step g. MS (ESI+) m / z 454.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.02 (d, J= 3.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.70 (dd, J = 2.0, 7.0 Hz, 1H), 7.60-7.54 (m, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.27 (t, J = 9.0 Hz, 1H), 6.47 (dd, J = 2.0, 4.0 Hz, 1H), 3.64 (s, 3H), 2.39 (s, 3H), 2.04 (s, 3H).

[0274] 4-(6-Fluoro-1-(pyridin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-6-methyl-1-tosyl- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (17a) was obtained in 50% yield following general procedure step g. MS (ESI+) m / z 528.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 8.39 (d, J = 5.5 Hz, 2H), 8.01 (d, J = 3.5 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.78-7.72 (m, 2H), 7.49- 7.42 (m, 3H), 7.16-7.11 (m, 1H), 6.88 (d, J = 5.5 Hz, 2H), 6.73 (d, J = 3.5 Hz, 1H), 5.59 (s, 2H), 3.40 (s, 3H), 2.38 (s, 3H).

[0275] 4-(2-(4-Fluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin- 7(6H)-one (18a) was obtained in 47% yield following general procedure step g. MS (ESI+) m / z 490.1 (M + H)+.1H NMR (500 MHz, DMSO-d6) δ 7.97 (dd, J = 3.5, 5.5 Hz, 1H), 7.99- 7.95 (m, 3H), 7.85 (dd, J = 2.5, 7.5 Hz, 1H), 7.64 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.24-7.16 (m, 5H), 6.63 (d, J = 3.5 Hz, 1H), 3.47 (s, 3H), 2.38 (s, 3H).

[0276] 4-(4-(4-Hydroxyphenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (26a) was obtained as white solid in 86% yield following the general procedure step g. MS (ESI+) m / z 488.1 (M + H)+.1H NMR (500 MHz, CDCl3) d 8.47 (s, 1H), 8.40 (d, J = 5.5 Hz, 1H), 8.04 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 3.5 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.16 (s, 1H), 6.92-6.86 (m, 4H), 6.70 (d, J = 5.5 Hz, 1H), 6.41 (d, J = 3.5 Hz, 1H), 3.58 (s, 3H), 2.39 (s, 3H). 4-(5-Acetamido-3-(6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4- yl)pyridin-2-yloxy)phenyl acetate (27a)

[0277] 4-(2-(4-Hydroxyphenoxy)-5-nitropyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one was obtained from 5 as brown solid in 58% yield following the general procedure step g as impure intermediate and used without further purification. MS (ESI-) m / z 531.0 (M - H)-.

[0278] 4-(2-(4-Hydroxyphenoxy)-5-nitropyridin-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (150 mg, 0.28 mmol), iron powder (78.7 mg, 1.4 mmol), and ammonium chloride (22.6 mg, 0.42 mmol) were combined in THF (4 mL), ethanol (4 mL), and water (1.3 mL). The mixture was heated at 100 °C with vigorous stirring for 1 h. The reaction mixture was cooled to ambient temperature and filtered through a plug of Celite to remove the solids. The plug was rinsed repeatedly with methanol. The filtrate was concentrated, andthe residue partitioned between ethyl acetate and water. The ethyl acetate layer was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to afford 4-(5-amino-2-(4-hydroxyphenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (100 mg, 70%). 4-(5-Amino-2-(4-hydroxyphenoxy) pyridine-3-yl)-6-methyl-1-tosyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one was dissolved in dry CH2Cl2 (3 mL) and cooled the solution to 0 °C. To the cooled solution acetyl chloride (57.8 mg, 0.73 mmol) and triethylamine (70.5 mg, 0.69 mmol) were added. The reaction mixture was then allowed to warm to ambient temperature over 3 h. The reaction mixture was concentrated, diluted with EtOAc (30 mL), washed with water (2 x 10 mL) and brine (10 mL), dried over Na2SO4, concentrated, and purified by prep TLC chromatography (7% MeOH in CHCl3) to provide title compound 27a (65 mg, 60% yield) as gummy solid. MS (ESI+) m / z 587.2 (M + H)+.1H NMR (500 MHz, DMSO-d6) d 10.18 (s, 1H), 8.23 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 8.00-7.95 (m, 3H), 7.65 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.16-7.08 (m, 4H), 6.63 (d, J = 3.0 Hz, 1H), 3.46 (s, 3H), 2.37 (s, 3H), 2.26 (s, 3H), 2.05 (s, 3H).

[0279] 4-(4-Acetamido-2-(6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3- c]pyridin-4-yl)phenoxy)phenyl acetate (28a):

[0280] The compound was obtained as white solid in 77% yield following the general procedure step g. MS (ESI+) m / z 586.1(M + H)+.1H NMR (500MHz, CDCl3) d 8.01 (d, J = 7.5 Hz, 2H), 7.82 (d, J = 3.0 Hz, 1H), 7.52 (s, 1H), 7.47-7.43 (m, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.06 (s, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 6.49 (d, J = 3.5 Hz, 1H), 3.45 (s, 3H), 2.39 (s, 3H), 2.26 (s, 3H), 2.21(s, 3H).

[0281] 4-(2-(3,4-difluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-81A):

[0282] The title compound was obtained in 70% yield following the general Suzuki coupling procedure as described in step g.1H NMR (500 MHz, CDCl3) δ 8.17 (q, J = 2.3 Hz, 1H), 8.06 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 3.4 Hz, 1H), 7.71 (dd, J1 = 7.6 Hz, J2 = 2.1 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.15 (m, 3H), 6.96 (m, 1H), 6.82 (m, 1H), 6.39 (d, J = 3.4 Hz, 1H), 3.58 (s, 3H), 2.41 (s, 3H).

[0283] 4-(2-(3,5-difluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-82A):

[0284] The title compound was obtained in 74% yield following the general Suzuki coupling procedure as described in step g.1H NMR (500 MHz, CDCl3) δ 8.22 (q, J = 2.3 Hz,1H), 8.05 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 3.4 Hz, 1H), 7.74 (dd, J1= 7.2 Hz, J2= 1.7 Hz, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.17 (dd, J1 = 7.2 Hz, J2 = 5.2 Hz, 1H), 7.15 (s, 1H), 6.64 (qd, J1 = 4.8 Hz, J2 = 2.8 Hz, 3H), 6.38 (d, J = 3.4 Hz, 1H), 3.58 (s, 3H), 2.41 (s, 3H).

[0285] 4-(2-((4-fluorophenyl)thio)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-84A):

[0286] The title compound was obtained in 69% yield following the general Suzuki coupling procedure as described in step g.1H NMR (500 MHz, CDCl3) δ 8.36 (q, J = 2.3 Hz, 1H), 8.08 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 3.4 Hz, 1H), 7.46 (td, J1= 6.7 Hz, J2= 4.4 Hz, 1H), 7.30 (dd, J1 = 10.7 Hz, J2 = 2.4 Hz, 2H), 7.10 (m, 3H), 7.02 (m, 3H), 6.24 (d, J = 3.4 Hz, 1H), 3.58 (s, 3H), 2.43 (s, 3H).

[0287] 4-(2-((4-fluorophenyl)amino)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-85A):

[0288] The title compound was obtained in 42% yield following the general Suzuki coupling procedure as described in step g.1H NMR (400 MHz, CDCl3) δ 8.25 (dd, J1= 5.0 Hz, J2= 1.9 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 3.6 Hz, 1H), 7.38 (m, 5H), 7.08 (s, 1H), 6.98 (t, J = 8.7 Hz, 2H), 6.81 (dd, J1 = 7.4 Hz, J2 = 5.0 Hz, 1H), 6.27 (d, J = 3.4 Hz,1H), 6.15 (s, 1H), 3.58 (s, 3H), 2.42 (s, 3H).

[0289] 4-(5-fluoro-2-(4-fluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-86A):

[0290] The title compound was obtained in 61% yield following the general Suzuki coupling procedure as described in step g.1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 2.9 Hz, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 3.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 3.0 Hz, 1H), 7.07 (m, 5H), 6.22 (d, J = 3.6 Hz, 1H), 3.56 (s, 3H), 2.42 (s, 3H).

[0291] 4-(2-(4-fluorophenoxy)pyridin-3-yl)-6-methylthieno[2,3-c]pyridin-7(6H)-one (Ray-03-92A): The title compound was obtained as a white solid with 66% yield following the general Suzuki coupling procedure as described in step g.1H NMR (400 MHz, CDCl3) δ 8.16 (dd, J1=4.9 Hz, J2= 1.9 Hz, 1H), 8.02 (d, J = 5.2 Hz, 1H), 7.90 (dd, J1=7.4 Hz, J2= 1.9 Hz, 1H), 7.77 (s, 1H), 7.29 (d, J = 5.2 Hz, 1H), 7.25 (m, 1H), 7.19 (m, 4H), 3.61 (s, 3H). HPLC: tR = 7.719 min, 94.581%.

[0292] 4-(2-(4-fluorophenoxy)pyridin-3-yl)-2-methylisoquinolin-1(2H)-one (Ray-03- 95A):

[0293] The title compound was obtained as a white solid with 74% yield following the general Suzuki coupling procedure as described in step g.1H NMR (400 MHz, CDCl3) δ 8.52 (dd, J1=8.1 Hz, J2= 1.0 Hz, 1H), 8.23 (dd, J1=4.9 Hz, J2= 1.9 Hz, 1H), 7.71 (d, J1=7.3 Hz,J2= 2.1 Hz, 1H), 7.63 (m, 1H), 7.53 (m, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.13 (m, 2H), 7.02 (m, 4H), 3.67 (s, 3H). HPLC: tR = 8.073 min, 94.581%. Example 2. Synthesis of 4-(2-(3,4-difluorophenoxy)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-81A)

[0294] Obtained using the following general procedure for removal of the tosylate (tosyl) protecting group (i.e., detosylation):

[0295] The tosylate adduct was added to 5 mL methanol-water (4:1), then 3 eq. of K2CO3was added, and the reaction mixture was stirred at 85 °C for 2-3 h. The reaction was monitored using TLC (4% MeOH in CHCl3) until the disappearance of starting material (tosyl adduct). The reaction mixture was then evaporated to dryness. The crude was suspended in 10 mL water and extracted with 10% MeOH-CHCl3. The organic layer was dried over Na2SO4, evaporated to dryness and purified using preparative TLC (4% MeOH in CHCl3)The title compound was obtained as white solid with 80% yield using the general procedure for detosylation. MS (ESI+) m / z 354.1 (M + H)+.1H NMR (500 MHz, CDCl3) δ 9.80 (bs, 1H), 8.17 (q, J = 2.3 Hz, 1H), 7.84 (dd, J1 = 7.2 Hz, J2 = 1.7 Hz, 1H), 7.27 (t, J = 2.8 Hz, 1H), 7.15 (m, 3H), 6.99 (m, 1H), 6.85 (m, 1H), 6.36 (t, J = 2.4 Hz, 1H), 3.71 (s, 3H). HPLC: tR= 7.454 min, 93.546%. Example 3. Synthesis of 4-(2-(3,5-difluorophenoxy)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (RAY-03-82A)

[0296] The title compound was obtained as white waxy solid with 82% yield following the general procedure for detosylation. MS (ESI+) m / z 353.1 (M+).1H NMR (500 MHz,CDCl3) δ 10.01 (bs, 1H), 8.22 (q, J = 2.1 Hz, 1H), 7.87 (dd, J1= 7.2 Hz, J2= 1.7 Hz, 1H), 7.29 (m, 1H), 7.19 (dd, J1 = 6.9 Hz, J2 = 4.8 Hz, 1H), 7.12 (s, 1H), 6.67 (td, J1 = 7.4 Hz, J2 = 2.5 Hz, 2H), 6.62 (m, 1H), 6.35 (d, J = 2.1 Hz, 1H), 3.70 (s, 3H). HPLC: tR = 7.641 min, 97.941%. Example 4. Synthesis of 4-(2-((4-fluorophenyl)thio)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-84A)

[0297] The title compound was obtained as white solid with 89% yield following the general procedure for detosylation. MS (ESI+) m / z 352.17 (M + H)+.1H NMR (500 MHz, CDCl3) δ 10.34 (bs, 1H), 8.37 (q, J = 2.1 Hz, 1H), 7.58 (dd, J1 = 7.6 Hz, J2 = 2.1 Hz, 1H), 7.43 (qd, J1 = 5.7 Hz, J2 = 3.1 Hz, 2H), 7.30 (t, J = 2.4 Hz, 1H), 7.12 (q, J = 4.1 Hz, 1H), 7.05 (m, 3H), 6.22 (t, J = 2.4 Hz, 1H), 3.73 (s, 3H). HPLC: tR= 7.469 min, 99.136%. Example 5. Synthesis of 4-(2-((4-fluorophenyl)amino)pyridin-3-yl)-6-methyl-1,6- dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-85A)

[0298] The title compound was obtained as light yellow waxy solid with 81% yield following the general procedure for detosylation.1H NMR (500 MHz, CDCl3) δ 10.41 (bs, 1H), 8.26 (dd, J1 = 5.0 Hz, J2 = 1.9 Hz, 1H), 7.49 (dd, J1 = 7.3 Hz, J2 = 1.9 Hz, 1H), 7.42 (m, 2H), 7.31 (t, J = 2.8 Hz, 1H), 7.03 (s, 1H), 6.96 (m, 2H), 6.83 (dd, J1= 7.3 Hz, J2= 4.9 Hz, 1H), 6.40 (s, 1H), 6.27 (t, J = 2.5 Hz, 1H), 3.73 (s, 3H). HPLC: tR = 5.426 min, 99.380%. Example 6. Synthesis of 4-(5-fluoro-2-(4-fluorophenoxy)pyridin-3-yl)-6-methyl-1,6- dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (RAY-03-86A)

[0299] The title compound was obtained as white waxy substance with 65% yield following the general procedure for detosylation. MS (ESI+) m / z 353.4 (M+).1H NMR (500 MHz, CDCl3) δ 10.24 (s, 1H), 8.17 (d, J = 2.8 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 7.27 (t, J = 2.8 Hz, 1H), 7.07 (m, 5H), 6.18 (t, J = 2.4 Hz, 1H), 3.70 (s, 3H). HPLC: tR = 7.802 min, 94.018%. Example 7. Synthesis of 5-(2-(4-fluorophenoxy)pyridin-3-yl)-7-methyl-1,7-naphthyridin- 8(7H)-one (Ray-03-93A)

[0300] Prepared according to Route B:

[0301] Step-a: To a suspension of compound 5-bromo-1,7-naphthyridin-8(7H)-one (250 mg, 1.11 mmol) and sodium hydride (37 mg, 1.55 mmol) in N,N-dimethyl formamide (5 mL), was added iodomethane (235 mg, 1.66 mmol) dropwise at 0 °C and the reaction mixture was stirred at ambient temperature for 6 h. The reaction mixture was concentrated, diluted with ethyl acetate (15 mL) and washed with water (2 x 15 mL). The organic layer was concentrated and purified by silica gel column chromatography (0-5% Methanol in Chloroform) to provide compound 5-bromo-7-methyl-1,7-naphthyridin-8(7H)-one as white solid (207 mg, 78% yield).1H NMR (400 MHz, CDCl3) δ 8.92 (dd, J1 = 4.4 Hz, J2 = 1.5 Hz, 1H), 8.18 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H), 7.66 (q, J = 4.3 Hz, 1H), 7.47 (s, 1H), 3.68 (s, 3H).

[0302] Step-b: A mixture of compound 5-bromo-7-methyl-1,7-naphthyridin-8(7H)-one (150 mg, 0.63 mmol), anhydrous tripotassium phosphate (401 mg, 1.89 mmol), 2- Dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (SPhos) (16 mg, 0.038 mmol), and Pd- XPhos-G3 (16 mg, 0.019 mmol) were added to a three-necked flask and the reaction mixturewas degassed by vacuum and back-filled with nitrogen (repeated three times). 1.1 M solution of 2-(4-fluorophenoxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (298 mg, 0.95 mmol) in degassed 4:1 (v / v) ratio of 1, 4-dioxane-water mixture was then added to the reaction mixture and stirred at 95 °C for 6 h. Reaction was monitored using TLC analysis (80% Ethyl acetate in hexane) till the disappearance of boronic acid pinacol ester intermediate. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over Na2SO4, evaporated to dryness and purified using preparative TLC (50% Ethyl acetate in Hexane) to provide the title compound as a white solid (36 mg, 16% yield). MS (ESI+) m / z 348.23 (M+1)+.1H NMR (400 MHz, CDCl3) δ 8.94 (d, J = 3.0 Hz, 1H), 8.25 (dd, J1 = 4.9 Hz, J2 = 1.9 Hz, 1H), 7.73 (m, 2H), 7.55 (q, J = 4.2 Hz, 1H), 7.24 (s, 1H), 7.15 (dd, J1= 7.3 Hz, J2= 4.9 Hz, 1H), 7.02 (m, 4H), 3.75 (s, 3H). HPLC (Method- B): tR = 7.475 min, 91.430%.

[0303] Example 8. Synthesis of 4-(2-(4-fluorophenoxy)pyridin-3-yl)-6- methylfuro[2,3-c]pyridin-7(6H)-one (Ray-03-96A)

[0304] Prepared according to Route C:

[0305] Step-a: To a suspension of furo[2,3-c]pyridin-7(6H)-one(250 mg, 1.85 mmol) in acetonitrile (5 mL), N-bromosuccinimide (495 mg, 2.78 mmol) was added dropwise and stirred at 60 °C for overnight. The reaction mixture was concentrated, diluted with dichloromethane (15 mL) and washed with aqueous sodium thiosulfate solution (2 x 15 mL). The organic layer was concentrated and purified by silica gel column chromatography (0-5% Methanol in Chloroform) to provide 4-bromofuro[2,3-c]pyridin-7(6H)-one as white solid (308 mg, 78% yield). MS (ESI+) m / z 214.1 (M+).1H NMR (500 MHz, CDCl3) δ 11.31 (bs, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.31 (s, 1H), 6.77 (d, J = 2.1 Hz, 1H).

[0306] Step-b: 4-bromo-6-methylfuro[2,3-c]pyridin-7(6H)-one (Ray-03-96A-3):

[0307] To a suspension of 4-bromofuro[2,3-c]pyridin-7(6H)-one (250 mg, 1.17 mmol) and Cesium carbonate (762 mg, 2.34 mmol) in N,N-dimethylformamide (5 mL), Iodomethane (249 mg, 1.75 mmol) was added dropwise at 0 °C and stirred at ambient temperature for 4 h. The reaction mixture was concentrated, diluted with Ethyl acetate (15mL) and washed with water (2 x 15 mL). The organic layer was concentrated and purified by silica gel column chromatography (0-5% Methanol in Chloroform) to provide 4-bromo-6- methylfuro[2,3-c]pyridin-7(6H)-one as white solid (236 mg, 88% yield). MS (ESI+) m / z 227.9 (M)+.1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 2.1 Hz, 1H), 7.30 (s, 1H), 6.70 (d, J = 2.1 Hz, 1H), 3.65 (s, 3H). Step-c: A mixture of 4-bromo-6-methylfuro[2,3-c]pyridin-7(6H)-one (228 mg, 1.0 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (762 mg, 3.0 mmol), Potassium acetate (294 mg, 3.0 mmol), tris(dibenzylideneacetone)dipalladium(0) (27 mg, 0.03 mmol), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-Phos, 29 mg, 0.06 mmol) was degassed by vacuum and filled with nitrogen (repeated three times). Degassed dry 1, 4- dioxane (5 mL) was added to the reaction mixture and heated under nitrogen at 95 °C for 16 h. The consumption of starting material was monitored by TLC (1% Methanol in Chloroform). The reaction mixture was cooled to ambient temperature, concentrated, and partitioned between Ethyl acetate (20 mL) and water (2 x 20 mL). The Ethyl acetate layer was washed with brine (2 x 10 mL), dried over anhydrous Na2SO4, and concentrated. The yellow semi solid (264 mg) residue was used for the next step as 6-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)furo[2,3-c]pyridin-7(6H)-one without further purification to avoid any degradation of the boronic acid pinacol ester intermediate.

[0308] Step-d: 3-bromo-2-(4-fluorophenoxy)pyridine (170 mg, 0.63 mmol), anhydrous Tripotassium phosphate (401 mg, 1.89 mmol), 2-Dicyclohexylphosphino-2′,6′- dimethoxybiphenyl (SPhos) (16 mg, 0.038 mmol), and Pd-XPhos-G3 (16 mg, 0.019 mmol) were added to a three-necked flask and the reaction mixture was degassed by vacuum and back-filled with nitrogen (repeated three times). 1.1 M solution of 6-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)furo[2,3-c]pyridin-7(6H)-one (264 mg) in degassed 4:1 (v / v) ratio of 1, 4-dioxane-water mixture was then added to the reaction mixture and stirred at 90 °C for overnight. Reaction was monitored using TLC analysis (80% Ethyl acetate in Hexane) till the disappearance of boronic acid pinacol ester intermediate (15-16 h). The reaction mixture was diluted with Ethyl acetate and washed with water. The organic layer was dried over Na2SO4, evaporated to dryness and purified using preparative TLC (5% Methanol in Chloroform). The title compound was obtained in (32 mg, 15% yield). MS (ESI+) m / z 337.12 (M+1)+.1H NMR (500 MHz, CDCl3) δ 8.17 (q, J = 2.3 Hz, 1H), 7.76 (m, 2H), 7.34 (s, 1H), 7.12 (q, J = 4.1 Hz, 1H), 7.08 (d, J = 6.2 Hz, 4H), 6.69 (d, J = 1.4 Hz, 1H), 3.74 (s, 3H). HPLC (Method-A): tR= 7.318 min, 98.131%.Example 9. General procedure for reductive amination (Route E, step b):

[0309] A mixture of aryl aldehyde intermediate (0.2 mmol, 1eq), spiroamine salt (0.25 mmol, 1.25 eq) and freshly activated 3A molecular sieve (600 mg) was dissolved in dry 1,2- dichloroethane (2.0 mL). To the solution was added dry methanol (1.0 ml), anhydrous Et3N (40 mg, 0.4 mmol) and glacial acetic acid (30 mg, 0.5 mmol). Resulting solution was heated at 60oC for 1 h and then cooled to 0-5oC using ice-bath. To the cooled solution was added NaBH3CN (18 mg, 0.28 mmol) and stirred for 2-3h. The reaction was quenched by addition of water (10 mL) and extracted with CHCl3(3 x 10 mL). The organic layer was dried over Na2SO4, evaporated to dryness and purified using preparative TLC.

[0310] The following tosylate adducts were prepared from aryl aldehyde intermediates and spiroamine salts via reductive amination according to route E, Step b as described above: 6-Methyl-3-(4-(1-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H-pyrrolo[2,3- c]pyridin-7(6H)-one (11a) was obtained as white solid in 79% yield.1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.45-7.37 (m, 4H), 6.67 (d, J = 7.2 Hz, 1H), 4.36 (t, J = 7.6 Hz, 2H), 3.47 (s, 2H), 3.42 (s, 3H), 2.45 (bs, 2H), 2.38 (s, 3H), 2.30 (t, J = 7.6 Hz, 2H), 2.22 (bs, 2H), 1.86-1.71 (m, 4H).

[0311] 6-Methyl-3-(3-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11b)

[0312] 6-Methyl-3-(4-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11c)

[0313] 6-Methyl-3-(3-(8-oxa-2-azaspiro[4.5]dec-2-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11d) was obtained as white solid in 50% yield.1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.60-7.51 (m, 2H), 7.50 (d, J = 7.2 Hz, 1H), 7.47-7.39 (m, 3H), 7.36-7.28 (m, 1H), 6.63 (d, J = 7.2 Hz, 1H), 3.62 (bs, 2H), 3.51 (t, J = 4.8 Hz, 4H), 3.42 (s, 3H), 2.57 (bs, 2H), 2.38 (s, 5H), 1.63 (bs, 2H), 1.58-1.42 (m, 4H)

[0314] 6-Methyl-3-(4-(8-oxa-2-azaspiro[4.5]dec-2-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11e) was obtained as white solid in 58% yield.1H NMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.02 (d, J = 9.0 Hz, 2H), 7.61 (bs, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 4H), 6.67 (d, J = 7.0 Hz, 1H), 3.58 (bs, 2H), 3.51 (t, J = 5.0 Hz, 4H), 3.50 (s, 3H), 2.55 (bs, 2H), 2.44-2.34 (m, 5H), 1.64 (bs, 2H), 1.57-1.43 (m, 4H).

[0315] 6-Methyl-3-(3-(7-oxa-2-azaspiro[3.5]non-2-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11f) was obtained as white solid in 64% yield.1H NMR(400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.55-7.47 (m, 3H), 7.46-7.39 (m, 3H), 7.30 (d, J = 7.6 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H), 3.67 (bs, 2H), 3.51-3.43 (m, 4H), 3.42 (s, 3H), 3.02 (bs, 4H), 2.38 (s, 3H), 1.69-1.63 (m, 4H).

[0316] 6-Methyl-3-(4-(7-oxa-2-azaspiro[3.5]non-2-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11g) was obtained as white solid in 67% yield.1H NMR (500 MHz, DMSO-d6) δ 8.15 (s, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 6.5 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 7.5 Hz, 2H), 6.65 (d, J = 7.0 Hz, 1H), 3.63 (bs, 2H), 3.51-3.45 (m, 4H), 3.42 (s, 3H), 3.00 (bs, 4H), 2.38 (s, 3H), 1.69-1.63 (m, 4H).

[0317] 6-Methyl-3-(3-(1-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11h) was obtained as white solid in 61% yield.1H NMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.56 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.46-7.41 (m, 3H), 7.32 (d, J = 7.5 Hz, 1H), 6.63 (d, J = 7.0 Hz, 1H), 3.68 (t, J = 7.0 Hz, 2H), 3.53 (bs, 2H), 3.42 (s, 3H), 2.51-2.40 (m, 2H), 2.38 (s, 3H), 2.38-2.33 (m, 2H), 1.83 (quint, J = 7.0 Hz, 2H), 1.66-1.61(m, 2H), 1.58-1.52 (m, 4H).

[0318] 6-Methyl-3-(4-(1-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11i) was obtained as white solid in 59% yield.1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 6.8 Hz, 1H), 7.41 (t, J = 8.8 Hz, 4H), 6.67 (d, J = 6.8 Hz, 1H), 3.68 (t, J = 6.4 Hz, 2H), 3.50 (s, 2H), 3.42 (s, 3H), 2.45 (bs, 2H), 2.38 (s, 3H), 2.34 (bs, 2H), 1.83 (quint, J = 7.2 Hz, 2H), 1.66-1.59 (m, 2H), 1.54 (t, J = 5.6 Hz, 4H).

[0319] 6-Methyl-3-(3-(2-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11j) was obtained as white solid in 56% yield.1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.46-7.41 (m, 3H), 7.32 (d, J = 7.5 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 3.71 (t, J = 7.0 Hz, 2H), 3.52 (bs, 2H), 3.42 (s, 3H), 3.42 (s, 2H), 2.41-2.34 (m, 7H), 1.66-1.61(t, J = 7.0 Hz, 2H), 1.56-1.51 (m, 4H).

[0320] 6-Methyl-3-(4-(2-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11k) was obtained as white solid in 57% yield.1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 6.8 Hz, 1H), 7.41 (t, J = 8.8 Hz, 4H), 6.66 (d, J = 7.2 Hz, 1H), 3.71 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 3.45-3.41 (m, 5H), 2.38 (s, 3H), 2.35 (bs, 4H), 1.66 (t, J = 7.2 Hz, 2H), 1.56-1.42(m, 4H).

[0321] 6-Methyl-3-(3-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11l) was obtained as white solid in 53% yield.1H NMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.58 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 7.5 Hz, 1H), 6.67 (d, J = 7.0 Hz, 1H), 4.39-4.30 (m, 2H), 3.58 (dd, J = 13.5, 20.5 Hz, 2H), 3.42 (s, 3H), 2.76-2.71 (m, 1H), 2.38 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H), 2.28-2.21 (m, 1H), 2.13-2.08 (m, 1H), 1.86-1.81 (m ,1H), 1.64-1.59 (m, 1H), 1.52-1.31 (m, 3H).

[0322] 6-Methyl-3-(4-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11m)

[0323] 6-Methyl-3-(2-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11n) was obtained as white solid in 41% yield.1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.44-7.30 (m, 6H), 7.01 (d, J = 7.2 Hz, 1H), 6.30 (d, J = 7.2 Hz, 1H), 4.41 (s, 4H), 3.53 (s, 3H), 3.29 (bs, 2H), 2.41 (s, 3H), 2.34 (bs, 4H), 1.95-1.85 (m, 4H).

[0324] 6-Methyl-3-(2-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)-1-tosyl-1H- pyrrolo[2,3-c]pyridin-7(6H)-one (11o) was obtained as white solid in 62% yield.1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.07 (d, J = 6.8 Hz, 2H), 7.53-7.47 (m, 1H), 7.41-7.30 (m, 5H), 7.01 (d, J = 6.8 Hz, 1H), 6.27 (d, J = 6.8 Hz, 1H), 4.52-4.38 (m, 2H), 3.53 (s, 3H), 3.46 (d, J = 12.8 Hz, 1H), 3.36 (d, J = 12.8 Hz, 1H), 2.87 (d, J = 10.4 Hz, 1H), 2.56 (d, J = 10.4 Hz, 1H), 2.41 (s, 3H), 2.33 (t, J = 8.0 Hz, 2H), 2.18-1.92 (m, 4H), 1.61-1.52 (m, 2H).

[0325] The following tosylate adducts were prepared from pinacol ester intermediate 5B and aryl bromides using synthetic route D and the general procedure for Suzuki coupling:

[0326] 3-(2-(3,4-difluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (RAY-03B-081B):1H NMR (400 MHz, CDCl3) δ 8.17 (dd, J1=4.8 Hz, J2=1.8 Hz, 1H), 8.05 (dd, J1=20.1 Hz, J2=8.4 Hz, 2H), 7.86 (m, 1H), 7.32 (m, 2H), 7.16 (m, 2H), 7.03 (m, 2H), 6.89 (m, 1H), 6.45 (m, 1H), 3.52 (s, 3H), 2.41 (s, 3H).

[0327] 3-(2-(3,5-difluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (RAY-03B-082B):1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 8.07 (m, 3H), 7.88 (d, J = 3.4 Hz, 1H), 7.31 (m, 3H), 7.03 (m, 2H), 6.77 (d, J = 6.9 Hz, 1H), 6.44 (m, 1H), 6.36 (d, J = 7.0 Hz, 1H), 3.52 (s, 3H), 2.41 (s, 3H).

[0328] 3-(2-((4-fluorophenyl)thio)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (RAY-03B-084B):1H NMR (400 MHz, CDCl3) δ 8.36 (dd, J1=4.8 Hz, J2=1.8 Hz, 1H), 8.10 (m, 3H), 7.52 (dd, J1=7.6 Hz, J2=1.8 Hz, 1H), 7.46 (m, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.09 (m, 4H), 6.26 (d, J = 7.1 Hz, 1H), 3.55 (s, 3H), 2.42 (s, 3H).

[0329] 3-(2-((4-fluorophenyl)amino)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (RAY-03B-085B):1H NMR (500 MHz, CDCl3) δ 8.45 (t, J=2.6 Hz, 1H), 8.08 (dd, J1=11.7 Hz, J2=8.3 Hz, 3H), 7.75 (dd, J1=7.6 Hz, J2=2.1 Hz, 1H), 7.35 (q, J = 4.1 Hz, 3H), 7.05 (d, J= 6.9 Hz, 1H), 6.26 (d, J = 7.6 Hz, 1H), 3.54 (s, 3H), 2.43 (s, 3H).

[0330] 3-(5-fluoro-2-(4-fluorophenoxy)pyridin-3-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-086B):1H NMR (500 MHz, CDCl3) δ 8.16 (d, J = 2.8 Hz, 1H), 8.07 (d, J=6.2 Hz, 3H), 7.34 (d, J = 8.3 Hz, 2H), 7.31 (s, 1H), 7.07 (m, 5H), 6.22 (d, J = 6.9 Hz, 1H), 3.53 (s, 3H), 2.42 (s, 3H).

[0331] 3-(4-(4-fluorophenoxy)pyrimidin-5-yl)-6-methyl-1-tosyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-087B):1H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 8.2 Hz, 2H), 8.26 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.16 (s, 2H), 7.14 (d, J = 1.5 Hz, 2H), 7.10 (d, J = 7.1 Hz, 1H), 6.49 (d, J = 7.1 Hz, 1H), 3.55 (s, 3H), 2.42 (s, 3H). Example 10. Synthesis of 6-Methyl-3-(4-(1-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-061, 12a)

[0332] Obtained as a white solid in 66% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 6.8 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 4.35 (t, J = 8.0 Hz, 2H), 3.53 (s, 3H), 3.43 (s, 2H), 2.48-2.38 (m, 2H), 2.30 (t, J = 8.0 Hz, 2H), 2.25-2.15 (m, 2H), 1.85-1.66 (m, 4H) . HPLC: tR = 4.89 min, 99.2%. Example 11. Synthesis of 6-Methyl-3-(3-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-062, 12b)

[0333] Obtained as white solid in 80% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.21 (bs, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.55-7.45 (m, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 7.2 Hz, 1H), 4.27 (s, 4H), 3.53 (s, 3H), 3.46 (bs, 2H), 2.29 (bs, 4H), 1.77 (bs, 4H). HPLC: tR = 4.93 min, 99.6%.Example 12. Synthesis of 6-Methyl-3-(4-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-063, 12c)

[0334] Obtained as white solid in 80% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.20 (bs, 1H), 7.61 (s, 1H), 7.56 (d, J = 6.4 Hz, 2H), 7.32 (d, J = 6.4 Hz, 2H), 7.26 (d, J = 7.2 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 4.27 (s, 4H), 3.53 (s, 3H), 3.42 (bs, 2H), 2.28 (bs, 4H), 1.77 (bs, 4H). HPLC: tR = 4.87 min, 98.2%. Example 13. Synthesis of 6-Methyl-3-(3-(8-oxa-2-azaspiro[4.5]dec-2-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-064, 12d)

[0335] Obtained as white solid in 74% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.22 (bs, 1H), 7.67-7.45 (m, 3H), 7.43-7.33 (m, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.21 (bs, 1H), 6.74 (d, J = 6.0 Hz, 1H), 3.56-3.46 (m, 9H), 1.63(bs, 2H), 1.48 (bs, 4H). HPLC: tR= 4.77 min, 97.9%. Example 14. Synthesis of 6-Methyl-3-(4-(8-oxa-2-azaspiro[4.5]dec-2-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-065, 12e)

[0336] Obtained as white solid in 43% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.19 (bs, 1H), 7.61 (bs, 1H), 7.55 (bs, 2H), 7.36 (bs, 2H), 7.25 (d, J = 7.2 Hz, 1H), 6.76 (d, J = 7.2 Hz, 1H), 3.58-3.48 (m, 9H), 3.30 (bs, 4H), 1.63(bs, 2H), 1.48 (bs, 4H). HPLC: tR = 4.96 min, 98.3%.Example 15. Synthesis of 6-Methyl-3-(3-(7-oxa-2-azaspiro[3.5]non-2-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-066, 12f)

[0337] Obtained as white solid in 72% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.21 (bs, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.57-7.45 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 3.77 (bs, 2H), 3.53 (s, 3H), 3.51-3.43 (m, 4H), 3.19 (bs, 4H), 1.72-1.64 (m, 4H). HPLC: tR= 4.96 min, 98.2%. Example 16. Synthesis of 6-Methyl-3-(4-(7-oxa-2-azaspiro[3.5]non-2-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-067, 12g)

[0338] Obtained as white solid in 42% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.19 (bs, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 1H), 6.74 (d, J = 7.2 Hz, 1H), 3.66 (bs, 2H), 3.52 (s, 3H), 3.51-3.43 (m, 4H), 3.10 (bs, 4H), 1.71-1.63 (m, 4H). HPLC: tR= 4.91 min, 99.4%. Example 17. Synthesis of 6-Methyl-3-(3-(1-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-068, 12h)

[0339] Obtained as white solid in 91% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.20 (bs, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.51 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H), 3.69 (t, J = 6.4 Hz, 2H), 3.56-3.48 (m, 5H), 2.52-2.32 (m, 4H), 1.82 (quint, J = 6.8 Hz, 2H), 1.66-1.60 (m, 2H), 1.59-1.51 (m, 4H). HPLC: tR= 4.82 min, 98.5%.Example 18. Synthesis of 6-Methyl-3-(4-(1-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-069, 12i)

[0340] Obtained as white solid in 81% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 7.6 (d, J = 3.0 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 7.5 Hz, 2H), 7.25 (d, J = 7.0 Hz, 1H), 6.75 (d, J = 7.5 Hz, 1H), 3.68 (t, J = 6.5 Hz, 2H), 3.53 (s, 3H), 3.46 (s, 2H), 2.48-2.28 (d, 4H), 1.83 (quint, J = 7.0, 2H), 1.62 (t, J = 7.5 Hz, 2H), 1.58-1.51 (m, 4H). HPLC: tR= 5.04 min, 98.8%. Example 19. Synthesis of 6-Methyl-3-(3-(2-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-070, 12j)Obtained as white solid in 80% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.19 (bs, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.51 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H), 3.70 (t, J = 6.8 Hz, 2H), 3.53 (s, 3H), 3.50 (bs, 2H), 3.41 (s, 2H), 2.36 (bs, 4H), 1.66 (t, J = 6.8 Hz, 2H), 1.57-1.48 (m, 4H). HPLC: tR= 4.87 min, 99.1%. Example 20. Synthesis of 6-Methyl-3-(4-(2-oxa-8-azaspiro[4.5]dec-8-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-071, 12k)

[0341] Obtained as white solid in 83% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.18 (bs, 1H), 7.59 (s, 1H), 7.55 (d, J = 7.5 Hz, 2H), 7.33 (d, J = 7.5 Hz, 2H), 7.25 (d, J = 7.5 Hz, 1H), 6.75 (d, J = 7.0 Hz, 1H), 3.71 (t, J = 7.0 Hz, 2H), 3.53 (s, 3H), 3.45(s, 2H), 3.41 (s, 2H), 2.42-2.26 (m, 4H), 1.67 (t, J = 7.0 Hz, 2H), 1.56-1.45(m, 4H). HPLC: tR= 4.91 min, 98.7%.Example 21. Synthesis of 6-Methyl-3-(3-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-072, 12l)

[0342] Obtained as white solid in 93% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.20 (bs, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.54 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 4.39-4.28 (m, 2H), 3.60-3.59 (m, 5H), 2.78-2.71 (m, 1H), 2.51-2.43 (m, 1H), 2.30 (t, J = 8.0 Hz, 2H), 2.26-2.02 (m, 2H), 1.87- 1.78 (m, 1H), 1.63-1.54 (m, 1H), 1.52-1.35 (m, 2H). HPLC: tR= 4.87 min, 99.6%. Example 22. Synthesis of 6-Methyl-3-(4-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (RAY-03B-073, 12m)

[0343] Obtained as white solid in 80% yield using the general procedure for removal of the tosyl protecting group.1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.57(d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.25 (d,J = 7.6 Hz, 1H), 6.77 (d, J = 7.2 Hz, 1H), 4.40-4.28 (m, 2H), 3.54 (d, J = 13.2, 1H), 3.53 (s, 3H), 3.46 (d, J = 13.2, 1H), 2.78-2.72 (m, 1H), 2.49-2.43 (m, 1H), 2.35-2.27 (m, 2H), 2.20-2.12 (m, 1H), 2.10-1.98 (m, 1H), 1.88-1.78 (m, 1H), 1.62-1.53 (m, 1H), 1.49-1.34 (m, 2H). HPLC: tR= 4.89 min, 99.2%. Example 23. Synthesis of 6-Methyl-3-(2-(2-oxa-7-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (Ray-03B-74, 12n)

[0344] Obtained as white solid in 80% yield using the general procedure for removal of the tosyl protecting group.1H NMR (500 MHz, DMSO-d6) δ 12.11 (bs, 1H), 7.55 (d, J = 3.0 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.34-7.25 (m, 3H), 7.18 (d, J = 7.0 Hz, 1H), 6.32 (d, J = 8.0Hz, 1H), 4.23 (s, 4H), 3.51 (s, 3H), 2.24-2.15 (m, 4H), 1.72 (bs, 4H). HPLC: tR= 4.92 min, 99.4%. Example 24. Synthesis of 6-Methyl-3-(2-(1-oxa-6-azaspiro[3.5]non-7-ylmethyl)phenyl)- 1H-pyrrolo[2,3-c]pyridin-7(6H)-one (Ray-03B-75, 12o)

[0345] Obtained as white solid in 77% yield using the general procedure for removal of the tosyl protecting group.1H NMR (500 MHz, DMSO-d6) δ 12.16 (bs, 1H), 7.63 (d, J = 3.5 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.37-7.28 (m, 3H), 7.19 (d, J = 7.0 Hz, 1H), 6.34 (d, J = 7.5 Hz, 1H), 4.39-4.31 (m, 2H), 3.52 (s, 3H), 3.37 (s, 2H), 2.68-2.62 (m, 1H), 2.39-2.32 (m, 1H), 2.27 (t, J = 8.0 Hz, 2H), 2.19-2.12 (m, 1H), 2.05-1.97 (m, 1H), 1.84-1.77 (m, 1H), 1.58-1.51 (m, 1H), 1.50-1.39 (m, 2H). HPLC: tR = 4.98 min, 98.9%. Example 25. Synthesis of 3-(2-(3,4-difluorophenoxy)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-081B)

[0346] Obtained in 80% yield following the general detosylation procedure. MS (ESI+) m / z 354.14 (M + H)+.1H NMR (500 MHz, CDCl3) δ 10.17 (bs, 1H), 8.08 (q, J = 2.3 Hz, 1H), 7.90 (dd, J1=7.6 Hz, J2=2.1 Hz, 1H), 7.63 (d, J = 2.8 Hz, 1H), 7.14 (m, 2H), 7.01 (m, 2H), 6.87 (m, 1H), 6.70 (d, J=7.6 Hz, 1H), 3.66 (s, 3H). HPLC: tR = 7.534 min, 97.354%. Example 26. Synthesis of 3-(2-(3,5-difluorophenoxy)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-082B)

[0347] Obtained as white solid in 76% yield following the the general detosylation procedure. MS (ESI+) m / z 354.14 (M + H)+.1H NMR (500 MHz, CDCl3) δ 10.60 (bs, 1H), 8.14 (q, J = 2.1 Hz, 1H), 7.93 (dd, J1=7.2 Hz, J2=1.7 Hz, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.18 (q, J = 4.1 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.69 (m, 3H), 6.62 (tt, J1=9.1 Hz, J2=2.4 Hz, 1H), 3.68 (s, 3H). HPLC: tR = 7.641 min, 97.941%. Example 27. Synthesis of 3-(2-((4-fluorophenyl)thio)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-084B)

[0348] Obtained as white solid in 96% yield following the general detosylation. MS (ESI+) m / z 352.30 (M + H)+.1H NMR (500 MHz, CDCl3) δ 10.49 (bs, 1H), 8.31 (q, J = 2.3 Hz, 1H), 7.56 (dd, J1=7.6 Hz, J2=2.1 Hz, 1H), 7.52 (d, J = 2.8 Hz, 1H), 7.47 (m, 2H), 7.07 (m, 3H), 7.01 (d, J = 6.9 Hz, 1H), 6.51 (d, J = 6.9 Hz, 1H), 3.70 (s, 3H). HPLC: tR= 7.528 min, 95.010%. Example 28. Synthesis of 3-(2-((4-fluorophenyl)amino)pyridin-3-yl)-6-methyl-1,6-dihydro- 7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-085B)

[0349] Obtained as white solid in 96% yield following the general detosylation procedure. MS (ESI+) m / z 335.3 (M + H)+.1H NMR (500 MHz, CDCl3) δ 10.79 (bs, 1H), 8.21 (q, J = 2.3 Hz, 1H), 7.48 (m, 5H), 6.98 (m, 3H), 6.83 (dd, J1=7.2 Hz, J2=5.2 Hz, 1H), 6.60 (m, 1H), 6.50 (d, J = 6.9 Hz, 1H), 3.69 (s, 3H). HPLC: tR= 5.384 min, 94.261%. Example 29. Synthesis of 3-(5-fluoro-2-(4-fluorophenoxy)pyridin-3-yl)-6-methyl-1,6- dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (RAY-03-86B)

[0350] Obtained as white solid in 96% yield following the general detosylation procedure. MS (ESI+) m / z 353.4 (M+).1H NMR (500 MHz, CDCl3) δ 10.70 (bs, 1H), 8.09 (d, J = 2.8 Hz, 1H), 7.55 (d, J = 2.8 Hz, 1H), 7.36 (d, J = 3.4 Hz, 1H), 7.08 (m, 4H), 6.99 (d, J = 6.9 Hz, 1H), 6.46 (d, J = 7.6 Hz, 1H), 3.67 (s, 3H). HPLC: tR = 7.833 min, 97.276%. Example 30. Synthesis of 3-(4-(4-fluorophenoxy)pyrimidin-5-yl)-6-methyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (Ray-03-87B)

[0351] The title compound was obtained as white solid in 85% yield following the general detosylation procedure at room temperature as mentioned in step-f. MS (ESI+) m / z337.34 (M+1)+.1H NMR (400 MHz, CDCl3) δ 10.25 (bs, 1H), 8.84 (s, 1H), 8.66 (s, 1H), 7.75 (d, J = 2.9 Hz, 1H), 7.13 (m, 4H), 7.06 (m, 2H), 6.85 (dd, J1=6.5 Hz, J2=2.1 Hz, 1H), 6.75 (d, J = 7.3 Hz, 1H), 3.69 (s, 3H). HPLC: tR = 6.725 min, 97.500%. Example 31. Synthesis of 3-(2-((4-fluorophenyl)sulfinyl)pyridin-3-yl)-6-methyl-1,6- dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-90B)

[0352] The title compound was obtained as white solid with 33% yield by treating the detosylated product, Ray-03-84B compound, in dichloromethane with 3-chloroperbenzoic acid at 0 °C for 3 h. MS (ESI+) m / z 368.30 (M+1)+.1H NMR (500 MHz, CDCl3) δ 11.19 (bs, 1H), 8.78 (d, J = 3.4 Hz, 1H), 7.68 (dd, J1=7.6 Hz, J2=1.4 Hz, 1H), 7.45 (q, J = 4.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.29 (q, J = 4.6 Hz, 2H), 6.99 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 8.3 Hz, 2H), 6.30 (d, J = 6.9 Hz, 1H), 3.72 (s, 3H). HPLC: tR= 5.900 min, 99.166%. Example 32. Synthesis of 3-(2-((4-fluorophenyl)sulfonyl)pyridin-3-yl)-6-methyl-1,6- dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (Ray-03-91B)

[0353] The title compound was obtained as white solid with 8% yield by treating the detosylated product, Ray-03-84B compound, in dichloromethane with 3-chloroperbenzoic acid at 0 °C for 3 h. MS (ESI+) m / z 384.26 (M+1)+.1H NMR (500 MHz, CDCl3) δ 10.80 (bs, 1H), 8.58 (t, J = 2.4 Hz, 1H), 7.81 (d, J = 6.9 Hz, 1H), 7.66 (m, 3H), 7.53 (q, J = 4.1Hz, 1H), 7.01 (t, J = 8.6 Hz, 2H), 6.90 (t, J = 6.5 Hz, 1H), 6.19 (d, J = 6.9 Hz, 1H), 3.67 (s, 3H). HPLC: tR= 6.487 min, 89.309%.Example 33. Radiolabeled synthesis of RAY03-18B

[0354] No-carrier-added18F-fluoride was produced from water 97% enriched in 180 (Sigma-Aldrich) by the nuclear reaction 18O(p, n)18F with a Siemens Eclipse HP cyclotron and a silver-bodied target at Athinoula A. Martinos Center for Biomedical Imaging. The produced18F-fluoride in water was transferred from the cyclotron target by helium push.

[0355] Precursor 3-(2-(4-hydroxyphenoxy)pyridin-3-yl)-6-methyl-1,6-dihydro-7H- pyrrolo[2,3-c]pyridin-7-one (5 mg; prepared from 5A and 4-((3-bromopyridin-2- yl)oxy)phenol via route A, MS (ESI+) m / z 334.17 (M+1)+) and Ru complex CpRu(COD)Cl (25 mg) were added to an ethanol (50 μL) in a 0.5 dram (1.8 mL) borosilicate glass vial. The vial was capped and then heated at 85oC while stirring for 30 minutes. The vial was cooled down at 23oC for 3 minutes and needed no further purification. After then, CIIm (29 mg) and 150 μL of MeCN were added to the vial, and the resulting mixture was drawn into a 1.0 mL polypropylene syringe for elution of18F-fluoride ion.

[0356] 18F-Fluoride solution from the cyclotron was directly loaded with a syringe onto a QMA anion exchange cartridge (Chromafix 30-Ps-HC03-), and the trapped activity was measured. The cartridge was first washed with MeCN (1.0 mL) and then inverted and fitted with a female x female Luer adapter. With the prepared mixture,18F-fluoride was eluted into a dram (3.7 ml) borosilicate vial. The cartridge was washed with DMSO (150 μL), followed by DMSO: MeCN (50 μL, 1:1 (v / v)). The reaction vial, which contained 400 μL of the reaction mixture, was sealed with a Teflon-lined cap and heated at 125oC for 30 minutes. After reaction finished, the vial was cooled down at 23oC for 3 minutes, The reaction mixture was subsequently with water containing 0.1% trifluoroacetic acid and then purified by reverse phase semipreparative HPLC (Agilent Eclipse XDB-C18, 5 um, 9,4x250 mm, flow rate = 5.0 mL / min, mobile phase= 0.1% TFA in water / 0.1% TFA in acetonitrile, 45 / 55, v / v), and the desired fraction was collected.

[0357] The final product was reformulated by loading onto a solid-phase exchange (SPE) C-18 cartridge, rinsing with H2O (5 ml), eluting with MeOH (1 mL), and diluting with salinesolution (0.9%, 9 mL). including formulation, [18F]Ray03-18B were prepared in 80 to 100 minutes after the end of bombardment (E0B), and the average radiochemical yield was 40%. Chemical and radiochemical purities were >95%. Biological Examples Example 34. PGRN Enhancement Assays

[0358] Human iPSC-Derived Neural Progenitor Cell Culture:

[0359] The derivation of healthy control NPCs from iPSCs reprogrammed according to Sheridan et. al (2011) from the clinically unaffected human fibroblast cell line GM08330 (8330-8, Coriell Institute for Medical Research, Camden, NJ) has been described (Cheng et al., 2017). Isogenic iPSC containing an allelic series of a pathogenic GRN mutation, (GRN+ / +, GRNR493X / +(clone G05), and GRNR493X / R493X(clone A02) were acquired from the Jackson Laboratory, which generated these iPSC as part of their project with the NIH iPSC Neurodegenerative Disease Institute. NPC were derived from the GRNR493Xallelic series as described by Cheng et al. (2017), with the exception that nascent NPC were purified by magnetic-activated cell sorting as described by Bowles et al. (2019). Culturing of NPCs occurred as previously described. Briefly, NPCs were cultured in T75 flasks (Corning #353110, #353133, or #353136) and 6-well plates (Corning #353046, CellTreat #229105, or CytoOne CC7682-7506), or 24-well plates (Corning #353047) which were coated first with 20 µg / mL poly-ornithine (Sigma Aldrich #P35307) in ddH2O for 2-4 hours at 37˚C and then with 5 μg / mL laminin (Sigma Aldrich #L2020) in PBS overnight at 37˚C (coating volumes: 15 mL for T-75 flasks, 2 mL for 6-well plates, 1 mL for 24-well plates). Coated plates were stored at 4˚C until use for up to 5 weeks.

[0360] Media for culturing NPCs (“NS media”) consists of 70% DMEM (Dulbecco's modified Eagle's Medium, Gibco #11995), 30% Ham’s F12 with L-glutamine (Modified Cellgro / Mediatech #10-080-CV), with 1X penicillin / streptomycin (100X, Gibco #15140- 122), and 1X B27 Supplement (50X, Gibco #17504-044). Media was supplemented with EGF (20 ng / mL, Epidermal Growth Factor, Sigma Aldrich #E9644, prepared as 1000X stock in DMEM), bFGF (20 ng / mL, basic Fibroblast Growth Factor, ReproCELL #03-0002, prepared as 1000X stock in PBS), and heparin (5 μg / mL, Sigma Aldrich #H3149, prepared as 1000X stock in Ham's F12) immediately before use. Growth factor stock solutions were stored at 4˚C for up to one month.

[0361] NPCs were maintained at 37°C with 5% CO2in a humidified atmosphere and passaged at a 1:2 or 1:3 ratio, or seeded with 3-4 x 106cells per T75 flask. For passaging,confluent cultures were washed with PBS and then treated with TrypLE Select (Life Technologies #12563029) until cell detachment. TrypLE treatment was stopped via addition of NPC media. Cells were gently triturated to obtain a single-cell suspension and were centrifuged at 300 rcf for 5 minutes and then resuspended in NPC media (with growth factors). Coated flasks / plates were first washed with PBS, and then the flasks / plates were allowed to equilibrate at 37˚C / 5% CO2 with NPC media with growth factors prior to the addition of cells.

[0362] Human iPSC-derived Neuron Culture. Human iPSC-derived neurons were derived from NPCs by growth factor withdrawal. NPCs were grown on plastic tissue culture ware in 6-well plates (Corning #353046, CellTreat #229105, or CytoOne CC7682-7506) that were coated with concurrently with 20 μg / mL polyornithine (Sigma Aldrich #P3655) and 5 μg / mL laminin (Sigma Aldrich #L2020) in dPBS (Thermo Fisher #NC9655718). Plates were stored at 4°C and washed with 1 mL DPBS prior to plating cells.

[0363] PGRN quantification using the Jess Automated Western Blot System:

[0364] Select immunoblotting experiments were performed using Jess (Bio-Techne) and data were processed and analyzed using Compass for Simple Western software (version 6.0). Runs were conducted using EZ Standard Pack 1 12-230 kDa (Bio-Techne PS-ST01EZ-8), 12-230 kDa pre-filled plates (PS-PP03), 12-230 kDa separation module (SMW004), anti- rabbit HRP detection (DM-001), RePlex (RP-001), and total protein detection modules (DM- TP01, biotin labeling reagent 042-973) according to the manufacturer’s instructions, and anti- PGRN primary (Abcam ab208777) diluted 1:50 in milk-free antibody dilution buffer (043- 524). The following run settings were used for each experiment: separation time: 25 min, separation voltage: 375 V, RePlex purge time: 30 min, biotin labeling time: 30 min, antibody diluent time: 5 min, primary antibody time: 30 min, secondary antibody time: 30 min, total protein HRP time: 30 min. All intensity quantifications are normalized to the total protein assay quantification unless otherwise indicated.

[0365] Compound treatment of NPCs followed by PGRN quantification:

[0366] NPCs were cultured as described above. NPCs were seeded on coated 6-well plates at 300,000 cells per well in 2 mL NPC media with growth factors, and were allowed to grow to confluency over 3 nights at 37˚C with 5% CO2. After cells reached confluency, compound stocks (10 mM) were diluted 1:10,000 into NPC media with growth factors. Wells were aspirated, and to each well is added 2 mL compound-media (or DMSO-containing media) at indicated concentrations.

[0367] After 24 hours of compound treatment, media was aspirated, cells were washed with 2 mL PBS, and then to each well is added 1 mL PBS. Cells were then scraped using a cell lifter (Costar 3008), and then transferred to 1.5-mL eppendorfs where they were then spun down at 400 rcf at room temperature for 5 minutes. The supernatant was then aspirated off, and pellets were either immediately lysed or stored in the -80˚C until further analysis.

[0368] Pellets were lysed for 10-15 minutes at 4˚C with 100 µL M-PER lysis buffer with 1 cOmplete Mini, EDTA-free protease inhibitor. Lysates were then clarified via spinning at 20k rcf at 4˚C for 10 minutes, and then the supernatant was then transferred to new 2-mL Eppendorf tubes. Concentrations were determined via a BCA assay, and samples were normalized to 500 µg / mL or 250 µg / mL prior to PGRN quantification via the Jess Automated Western Blot System. Example 35. BPS Biosciences BRD4 TR-FRET Assay

[0369] Dose-response curves for BRD4 inhibitors were determined by using the reagents from the commercially available BRD4 (BD1+BD2) TR-FRET Assay Kit in a 384-well format (Proxiplate-384 Plus, PerkinElmer #6008280) following the manufacturer’s protocol (BPS Biosciences, Catalog #BPS-326120). Final well DMSO content: 0.2%.

[0370] TR-FRET measurements:

[0371] Unless otherwise noted, experiments were performed in white, 384-well microtiter plates (Corning 3572 or PerkinElmer ProxiPlate-384 Plus) in 30 μL or 10 μL assay volume, respectively. TR-FRET measurements were acquired on a Tecan SPARK plate reader with SPARKCONTROL software version V2.1 (Tecan Group Ltd.), with the following settings: 340 / 50 nm excitation, 490 / 10 nm (Tb), 520 / 10 nm (FITC) emission, 100 μs delay, and 400 μs integration. The 490 / 10 and 520 / 10 nm emission channels were acquired with a 50% mirror and a dichroic 510 mirror, respectively, using independently optimized detector gain settings unless specified otherwise. The TR-FRET ratio was taken as the 520 / 490 nm intensity ratio on a per-well basis.

[0372] Determination of equilibrium dissociation constant (KD) of JQ1-FITC toward individual recombinant bromodomains and KD,app toward endogenous BRD4 in HEK293T lysate: Recombinant BRD4(BD1) and BRD4(BD2) were purchased from BPS Biosciences, Inc and Epicypher, Inc (GST-BRD4(BD1), 31040; GST-BRD4(BD2), 15-0013, respectively). Recombinant bromodomains were diluted to 0.5 nM in assay buffer (25 mM HEPES, 150 mM NaCl, 0.5 mg / mL BSA, 0.005% TWEEN-20, pH 7.5) with 2 nM CoraFluor-1-labeled GST VHH (ChromoTek ST-250), then JQ1-FITC was added in serialdilution (cmax= 100 nM) using an HP D300 digital dispenser and allowed to equilibrate for 2 h at room temperature before TR-FRET measurements were taken. Nonspecific signal was determined with 50 μM JQ1-Acid, and data were fitted to a One Site – Specific Binding model in Prism 9. The KDdetermined from the one-site model was then used in Equation 1 to adjust for a two-site model due to the dimeric GST protein:

[0373] For the profiling of endogenous BRD4, HEK293T cell lysate as prepared above was diluted to 0.8 mg / mL total protein in 1:3 lysis buffer:dilution buffer with 0.5 nM rabbit anti-BRD4 antibody (Cell Signaling Technology; E2A7X) and 1 nM CoraFluor-1-labeled anti-rabbit Nano-Secondary (ChromoTek CTK0101). JQ1-FITC was added in serial dilution (cmax= 200 nM) using an HP D300 digital dispenser and allowed to equilibrate for 2 h at room temperature before TR-FRET measurements were taken. Nonspecific signal was determined with 50 μM JQ1-Acid, and data were fitted to a One Site – Specific Binding model in Prism 9. The KD,appdetermined from the one-site model was then adjusted for a two- site model using Equation 1.

[0374] TR-FRET ligand displacement assays for recombinant bromodomains and endogenous BRD4 in HEK293T / NPC lysate:

[0375] The following assay parameters have been used: (i) 4 nM GST-BRD4(BD1), 4 nM CoraFluor-labeled GST VHH, 20 nM JQ1-FITC in assay buffer, (ii) 4 nM GST- BRD4(BD2), 4 nM CoraFluor-labeled GST VHH, 20 nM JQ1-FITC in assay buffer, (iii) HEK293T or NPC cell lysate at 0.8 mg / mL total protein, 1 nM rabbit anti-BRD4 antibody, 2 nM CoraFluor-labeled anti-rabbit Nano-Secondary, 20 nM JQ1-FITC. For all experiments, test compounds were added in serial dilution (cmax= 10 μM) using an HP D300 digital dispenser and allowed to equilibrate for 2 h at room temperature before TR-FRET measurements were taken. The assay floor (background) was defined with the 10 μM Mivebresib dose, and the assay ceiling (top) was defined via a no-inhibitor control. Data were fitted to a four-parameter dose-response model in Prism 9.

[0376] Calculation of inhibitor KD and KD,app values from measured TR-FRET IC50: For TR-FRET ligand displacement assays with recombinant bromodomains, the KD of the respective fluorescent tracer (JQ1-FITC) under each assay condition was determined. For endogenous BRD4 in whole cell extract, the KD,app of JQ1-FITC under each assay conditionwas determined. Inhibitor KDand KD,appvalues were calculated using Cheng-Prusoff principles, outlined in Equation 2 below:

[0377] Where IC50is the measured IC50value, [S] is the concentration of fluorescent tracer, and KX is the KD or KD,app of the fluorescent tracer for a given condition.

[0378] HMC-3 cell culture: HMC3 cells (ATCC CRL-3304) were cultured in 90% EMEM with EBSS and L-Glutamine (Lonza), 10% FBS (Sigma), 1% penicillin-streptomycin (Gibco), with 1 mM sodium pyruvate (Sigma) and allowed to proliferate at 37˚C with 5% CO2. Cells were typically sub-cultured between 1:3 and 1:8, and media was replaced upon acidification. Example 36. Luciferase Reporter assay

[0379] Loss of function mutations in GRN, which encodes progranulin (PGRN), are some of the most common genetic risk factors for frontotemporal dementia. In particular, emerging evidence points to the role of progranulin haploinsufficiency in microglia as a major contributor to neurodegenerative disease. For example, progranulin-haploinsufficient mouse microglia secrete neurotoxic factors that promote neuronal nuclear pore defects, causing TDP-43 mislocalization. Specifically, loss of progranulin in mice causes increased complement protein production, which results in the degradation of neuronal circuits through excessive synaptic pruning.

[0380] There is significant interest in identifying strategies to upregulate the expression of the remaining wild-type allele, aiming to normalize progranulin protein concentrations. However, the development of such approaches has been hindered by the lack of sensitive methods for progranulin quantification suitable for high-throughput screening. In order to identify small molecules that may rescue progranulin expression in microglia, it was decided to generate a progranulin-luciferase reporter line in HMC-3 cells (ATCC CRL-3304), an immortalized human microglia line.

[0381] The goal was to develop an endogenously-tagged fusion between the C-terminus of progranulin and a small, modified luciferase enzyme, TurboLuc16. It was posited that this strategy would offer clear advantages over synthetic reporter-gene constructs, such as a luciferase or a PGRN-luciferase fusion governed by a synthetic GRN promoter, as theseartificial systems might not precisely replicate the chromatin arrangement of the native GRN locus. Moreover, utilizing an endogenously-tagged fusion protein facilitates tracking of progranulin through the differentiation of secreted versus internal luminescent signal. Identifying modulators of progranulin localization would not be possible with an approach that solely reporters on GRN expression.

[0382] To generate the reporter line, it was envisioned generating a fusion protein between progranulin and a luciferase enzyme. This was accomplished with a gRNA to the 3’ end of GRN, adjacent to the sequence encoding for the stop codon, then introducing an exogenous DNA homology-directed repair template with 5’ and 3’ overhangs to GRN flanking the sequence encoding for a luciferase enzyme (FIG. 6).

[0383] TurboLuc16 was chosen as the luciferase enzyme, which is a 16-kDa engineered Metridia-family luciferase designed for reduced size and increased brightness (Thermo- Fisher). The reporter was engineered to contain a P2A self-cleaving peptide, followed by a puromycin resistance cassette flanked by loxP sites (FIG. 7). It was reasoned that including the antibiotic resistance cassette would allow for facile selection of edited cells, while the loxP sites would then allow for the possibility of removal of the puromycin resistance cassette via introduction of Cre recombinase.

[0384] HMC-3 cell culture:

[0385] HMC3 cells (ATCC CRL-3304) were cultured in 90% EMEM with EBSS and L- Glutamine (Lonza), 10% FBS (Sigma), 1% penicillin-streptomycin (Gibco), with 1 mM sodium pyruvate (Sigma) and allowed to proliferate at 37˚C with 5% CO2. Cells were typically sub-cultured between 1:3 and 1:8, and media was replaced upon acidification.

[0386] Generation of luciferase homology arm:

[0387] Two PCR reactions were set up using reagents provided by the TrueTag Donor DNA Kit (Invitrogen A42990). Each PCR reaction contained 25 µL of 2x Phusion Master Mix, 22 µL nuclease-free water, 1 µL of 10 µM forward and reverse primers (Invitrogen 10336022), and 1 µL of universal C-terminal luciferase template (TrueTag Donor DNA Kit) at 20 ng / µL.

[0388] Generation of endogenously tagged luciferase reporter line:

[0389] A 6-well plate of HMC-3 media was prepared and warmed in the incubator at 37˚C / 5% CO2. To three 500 µL tubes is added 20 µL of supplemented Nucleofector Solution (Amaxa Cell Line Nucleofector Kit V, cat VCA-1003). To two tubes is added 1250 ng Cas9 (0.25 µL, 5 mg / mL, Invitrogen A36498). To one Cas9-containing tube is added 7 µL ofluciferase homology arm, prepared as described above, and to the other Cas9-containing tube is added 7 µL DI water. To these two tubes is then added 7.5 pmol sgRNA (Invitrogen A35534, sequence: G*C*C*UUGAGACAGCUGCUGUG with modified scaffold) (1µL, 100 pmol, was diluted into 9 µL nucleofector solution, and then 0.75 µL was added to each tube). To the third tube is added 1 µL of supplied pMaxGFP vector at 0.5 µg / µL. Reagents were allowed to sit at room temperature for 15 minutes.

[0390] HMC-3 cells at passage 6 were trypsinized from a T-75 flask after washing with PBS. Prior to aspiration and trypsinization, the flask appeared to be ~90% confluent. Cells were counted via the manual hemocytometer and were at 350,000 cells / mL. Cells were centrifuged at 250 rcf for 5 minutes at RT. The cell pellet was then re-suspended in 400 µL of supplemented nucleofector solution. After light mixing to ensure homogeneity, 100 µL of this cell suspension was then added to the above tubes containing transfection reagents. Then, using the supplied pipettes, these solutions were transferred into the supplied cuvettes, ensuring that the bottom of the cuvette does not have air bubbles. The cuvette was then capped and labeled. The cuvettes were then taken over to the Amaxa Nucleofector II, and the procedure for THP-1 cells was run using "V-001" protocol for high expression. After each cuvette was nucleofected, 500 µL of HMC-3 media was added to each cuvette. The entire volume was then transferred using the supplied pipettes to one well of the 6-well plate that had been prepared above and warming in the incubator. Plate returned to the incubator.

[0391] Tagged HMC-3 enrichment

[0392] Lines were passaged according to normal HMC-3 cell culture protocol. Lines were scaled up to subculturing in T-75 flasks. After one month of passaging, a T-75 flask was washed with PBS, trypsinized, and then re-suspended in HMC-3 media to 170 cells per mL. 30 µL of this suspension was then seeded onto a white, transparent bottom 384-well plate (Thermo 164610) using the Multidrop Combi 384 (Thermo Scientific). After four days, 15 µL of media was removed from each well, replaced with fresh media, and transferred to a fresh 384-well plate. Active luciferase reagent (Thermo 88263) was prepared according to manufacturer’s protocol and 15 µL was dispensed to each well using the Multidrop Combi 384. Plate was allowed to nutate at RT for 15 minutes. Luminescence then read using a Tecan Spark. A number of wells were confirmed to have luciferase activity above media alone. After growing to near confluency, one positive well and one negative well were aspirated, washed with 30 µL PBS, and then trypsinized with 40 µL tryplE. The cell suspension was transferred using 40 µL of HMC-3 to a fresh 96-well plate containing 200 µL HMC-3 media. Cultures were grown into progressively larger plate formats over the upcoming weeks.Luciferase activity was validated via sampling the culture media of cells grown in T-75 flasks. Aliquots of cells were frozen down in HMC-3 media with 10% DMSO and are referred to as the “HMC3 GRN-Luc Enriched” line.

[0393] Validation of endogenous tagging

[0394] PCR Validation of endogenous knock-in: Cell pellets of the wild-type HMC-3 and enriched GRN-Luc lines were generated from two confluent wells of a 6-well plate and stored at -80˚C. DNA was isolated from each pellet using the Qiagen DNeasy Blood and Tissue Kit (Qiagen 69504) following the instructions for cell pellets. 100 ng of isolated genomic DNA for each cell line was then amplified using the following primer pairs, with each primer set containing one primer that amplifies from the insert and the other from the endogenous loci. Primers were purchased from Integrated DNA Technologies.

[0395] For validation via agarose gel electrophoresis, PCR was conducted via addition of 100 ng isolated genomic DNA (<1 µL each sample) to 25 µL Phusion Flash HF Master Mix, 2X (Invitrogen F548L) followed by addition of 1 µL of each primer pair as 25 µM stock solutions in TE buffer (to 500 nM final concentrations). Solutions were then filled with DI water to 50 µL and then run on the Thermocycler (Bio-Rad T100) with the following protocol: 98˚C for 1 minute, [98 ˚C for 15 seconds, 68˚C for 10 seconds, 72˚C for 30 seconds] with the bracketed component repeated 32x, followed by 72˚C for 10 minutes and a hold at 4˚C.

[0396] PCR products were visualized on a 2% agarose gel (UltraPure Agarose, Invitrogen 16500-500) made in 1x TAE buffer (Thermo Fisher Scientific 15-558-026) with 1 µL ethidium bromide (Primer design, FIG. 8A, results in FIG. 8B). PCR reactions were diluted into a 6x loading buffer containing Xylene Cyanol (Sigma-Aldrich X4126). 30 µL sample was loaded into each lane, and Quick-Load Purple 100 bp DNA Ladder (NEB N0551S). Gel was run for 2 hours in TAE buffer at 90V.

[0397] For sequencing, PCR was conducted via addition of 100 ng isolated genomic DNA (<1 µL each sample) to 25 µL Phusion Flash HF Master Mix, 2X (Invitrogen F548L) followed by addition of 1 µL primers (forward 1 and reverse 2) and separately (forward 2 and reverse 1) from 25 µM stock solutions in TE buffer (to 500 nM final concentrations). Solutions were then filled with DI water to 50 µL and then run on the Thermocycler (Bio- Rad T100) with the following protocol: 98˚C for 10 seconds, [98 ˚C for 1 second, 68˚C for 55 seconds, 72˚C for 15 seconds] with the bracketed component repeated 40x, followed by 72˚C for 1 minute and a hold at 4˚C.

[0398] PCR products were visualized on a 2% agarose gel (UltraPure Agarose, Invitrogen 16500-500) made in 1x TAE buffer (Thermo Fisher Scientific 15-558-026) with 1 µL ethidium bromide. PCR reactions were diluted into a 6x loading buffer containing Xylene Cyanol (Sigma-Aldrich X4126). 30 µL sample was loaded into each lane, and Quick-Load Purple 100 bp DNA Ladder (NEB N0551S). Gel was run for 2 hours in TAE buffer at 90V.

[0399] The F1-R2 and F2-R1 bands were excised, and DNA was isolated using the Qiaquick PCR Purification Kit (Qiagen 28104) following manufacturer’s instructions. A small aliquot of purified DNA was then validated on a 2% agarose gel, and the remainder of the aliquot was sequenced using Next-Generation Sequencing by the CCIB DNA Core Facility at Massachusetts General Hospital (Cambridge, MA). Example 37. PGRN-Luc compound screening assays

[0400] For the initial assays testing HDAC inhibitors, HMC3 PGRN-Luc cells were trypsinized from confluent T-75 flasks and re-suspended in HMC-3 media at 150,000 cells / mL. 30 µL of cell suspension was then seeded onto each well of black, 384-well clear- bottom plates (Corning 3712) using the Multidrop Combi (Thermo Fisher). Cells were allowed to adhere to plates in the incubator at 37˚C / 5% CO2for 8 hours. After 8 hours, compounds were added to each well using the D300e (Tecan) compound dispenser, and all wells were DMSO-normalized (0.3%). Cells were allowed to incubate with compounds for 20 hours.

[0401] After 20 hours, plates were removed from the incubator and allowed to cool to RT. 15 µL of media was removed from each well using a multichannel pipette and added to separate Corning 3712 plates for a read of “secreted” progranulin. To both the media- containing plates as well as the cell-containing plates is then added 15 µL of active luminescence reagent (Thermo 18636) which was generated according to manufacturer’s instructions. Plates were allowed to nutate at RT for 15 minutes prior to reading using the EnVision (PerkinElmer).

[0402] For future experiments testing BET inhibitors, the previous procedure was replicated except cells were re-suspended to 200,000 cells / mL, cells were seeded on white, opaque-bottom (Corning 3570) plates, cells were given 4 hours to attach to plates, and compounds were allowed to incubate for 24 hours. For experiments where secreted progranulin signal was not independently read, a volume of active luminescence reagent equivalent to that of culture media was added to each well to measure total PGRN-Luc signal.

[0403] PGRN-Luc assay optimization and calculation of Z’ score:

[0404] The previous procedure was replicated except cells were seeded at 10,000 cells / well in 30 µL media on a white, opaque-bottom (Corning 3570) plate. 30 µL of 300 µM Panobinostat was added to the inner wells of 8 columns of the 384-well plate, and DMSO only was added to the inner wells of 12 columns using a multichannel pipette. To the HMC-3 PGRN-Luc cells which were allowed to attach for 4 hours is then added compound (or DMSO) using a manual pin transfer tool (V&P Scientific, VP386) which was washed with MeOH x3 and allowed to dry prior to use. Compounds were allowed to incubate for 24 hours prior to reading total luminescence signal according to previous procedure. The Z’ score was calculated using the following equation: Z’ = 1 – (3 (σp + σn) / |μp – μn|) where σp is the standard deviation of the Panobinostat conditions, σn is the standard deviation of the DMSO conditions, and μpand μnare the means of the panobinostat and DMSO conditions, respectively.

[0405] PGRN-Luc screen proof-of-concept and dose-response validation:

[0406] The above procedure for assay optimization was replicated for this experiment, with the exception that the compounds used were from an internally-curated neuroepigenetic-targeted and bioactive compound library which was stored at -20˚C in DMSO. For dose-response validations, compounds were re-purchased from various vendors and tested according to the above dose-response compound testing procedures using white, solid-bottom plates (Corning 3570) with 4 hours of cell attachment and 24 hours of compound treatment. Example 38. Proof-of-concept studies

[0407] After generation of the enriched reporter line, the ability of known enhancers of progranulin to enhance luminescence signal was validated. The ability of the pan-histone deacetylase (HDAC) inhibitor Panobinostat was evaluated, which has previously been shown to enhance progranulin in neural progenitor cells, neurons, and in Neuro-2a cells, to enhance luminescence signal. However, whether HDAC inhibitors can enhance progranulin in microglia has yet to be shown. It was decided to profile the ability of structurally distinct HDAC inhibitors to enhance progranulin in HMC-3 cells. Of macrocyclic HDAC inhibitors, the slow-on / slow-off inhibitors rhomidepsin and trapoxin A were profiled, and the fast- on / fast-off inhibitor apicidin. The ortho-aminoanilides BML-210, entinostat, Cpd-60, and CI- 994, as well as the hydroxamic acids riclinostat, belinostat, abexinostat, trichostatin A, panobinostat, and tubacin were also tested. Internal and secreted progranulin signal was evaluated after 20 hours of compound treatment (FIGS. 9A, 9B).

[0408] It was found that the macrocyclic inhibitors apicidin and romidepsin were potent enhancers of both internal and secreted progranulin with low-nM EC50s, while trapoxin A was slightly less potent. None of the ortho-amino anilides tested were able to enhance progranulin protein expression. This result is consistent with findings in neural progenitor cells and neurons. The lack of activity of this structural class may be due to differences in the ability of these compounds to bind to specific HDAC-containing epigenetic protein complexes. Finally, it was found that the hydroxamic acids panobinostat, trichostatin A, belinostat, and abexinostat are able to enhance progranulin with sub-µM EC50s, while riclinostat and tubacin were less potent in their ability to do so.

[0409] BET inhibitors enhance progranulin in HMC3 cells

[0410] It was next sought to determine whether BET inhibitors can enhance progranulin in HMC3 cells. BET inhibitors have previously been shown to enhance progranulin in both NPCs and in neurons. However, whether BET inhibitors can enhance progranulin in a microglia-like cell type is yet unknown. In this regard, it was found that Mivebresib is able to enhance progranulin levels in HMC3 cells at low-nM concentrations, although not to the same extent as Panobinostat (FIG. 10).

[0411] BET proteins contain two structurally similar bromodomains, BD1 and BD2, that function as non-redundant readers of acetyllysine residues. Inhibitors of individual bromodomains produce distinct biological effects, which may produce differing clinical profiles. It was thus decided to test the BD-1 selective inhibitor iBET-BD1 along with the BD-2 selective inhibitor ABBV-744. It was found that the BD2-selective inhibitor ABBV- 744 was sufficient to enhance progranulin at concentrations selective for BD2 over BD1 (<300 nM), while the BD-1 selective inhibitor was unable to enhance progranulin at any concentrations tested.

[0412] It was next sought to demonstrate the utility of this reporter line to screen for novel enhancers of progranulin expression. The sensitivity of the assay in distinguishing PGRN-Luc enhancers from negative controls was evaluated. Accordingly, PGRN-Luc signal was evaluated after overnight treatment with either DMSO (n=192) or 1 µM Panobinostat (n=128). It was found that the Z’ score for this assay was 0.36 which was suitable for the identification of enhancers of PGRN-Luc signal (FIG. 11).

[0413] After validating the ability of this assay to distinguish progranulin enhancers from negative controls, a proof-of-concept compound screen was carried out. The reporter line was ceded in a 384-well format, and used a pin-transfer tool to treat cells with various doses of compounds from a targeted epigenetic library. After overnight incubation, the totalPGRN-Luc signal was evaluated and a subset of compounds were found which enhanced the PGRN-Luc signal (FIG. 12).

[0414] As a positive control for the assay, it found that the histone deacetylase inhibitors trichostatin A, M-344, Nullscript, SAHA, apicidin, SB939, among others at various doses were able to enhance PGRN-Luc signal >2-fold above the standard deviation from the DMSO average (Table 3). Additionally, the methyltransferase inhibitors BIX-01294, UNC0638, and (R)-PFI-2 at various doses were active as enhancers of progranulin.

[0415] Table 3. Screening results

[0416] Validation of dose dose-response format:

[0417] The ability of UNC0638 and BIX-02194 as well as GSK-1070916 to enhance progranulin at low-µM concentrations validated (Figure 8). Collectively, these data support the use of this line in a high-throughput format to screen for novel modulators of both internal and secreted progranulin.Example 39. In vitro autoradiography

[0418] Baseline and AD Mice brain tissue was cut and stored at -80 °C until they were used for experiment. The tissue sections were preincubated with Tris·HCl buffer (pH 7.4, 50 mM) solution for 30 min at ambient temperature, followed by incubation with [18F]Ray03- 18B (185 MBq / L, ~ 1 nM) for 20 min at ambient temperature (n = 4). For blocking studies (n = 4), unlabeled Ray03-18B (10 µM) was added to incubation solution in advance to determine the specificity of radioligand binding. After incubation, tissue sections were washed with Tris·HCl buffer (3 × 5 min). The tissue sections were dried with cold air, then placed on imaging plates for 120 min. Autoradiograms were obtained and ROIs were carefully drawn with the reference of naked-eye observation. Data were analyzed using Image J software and expressed as Gray value (0-255). Finally, the data was imported into software Graphpad Prism (10.1.2) for analysis and the processed images were exported. The results are shown in FIG. 31. Example 40. PET / CT acquisition and post processing

[0419] PET / CT imaging was performed in C57BL / 6 mice (25-30 g, male; n = 4 for baseline, and n = 3 for blocking with the pre-treatment of Ray03-18B). All mice were anesthetized with inhalational isoflurane at 3% in a carrier of 2 L / min medical oxygen, and maintained at 2% isoflurane for the duration of the scan. Highly trained animal technicians monitored animal safety throughout all procedures. The mice were fixed on the bed of a Triumph Trimodality PET / CT scanner (Gamma Medica, Northridge, CA) in the prone position, and injected with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) via a lateral tail vein catheterization at the start of PET acquisition. For blocking studies, Ray03-18B (3 mg / kg, iv) was injected at 10 min prior to [18F]Ray03-18B injection. Dynamic PET acquisition lasted for 60 min and was followed by CT for anatomic coregistration. PET data were reconstructed using a 3D-MLEM method resulting in a full width at half-maximum resolution of 1 mm. The correction for uniformity, scatter and attenuation was applied. Reconstructed images were exported from the scanner in DICOM format along with an anatomic CT. These files were imported to PMOD 3.2 software (PMOD Technology, Zurich, Switzerland).

[0420] PET / CT image analysis:

[0421] PET images were analyzed using the PMOD 3.2 software (PMOD Technology, Zurich, Switzerland). Volumes of interest (VOIs) were drawn manually as spheres guided by high-resolution CT structural images and summed PET data, with a radius no less than 1 mmto minimize partial volume effects. Time-activity curves (TACs) were exported in terms of decay-corrected activity at specified time points with gradually increasing intervals. The whole-body biodistribution data was obtained in a same way as the brain. Finally, the data was imported into software Graphpad Prism (10.1.2) for analysis and the processed images were exported.

[0422] FIGs. 14-18 show [18F]Ray03-18B levels (ID / g) levels in C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq). FIGs. 19-22 show images in C57BL / 6 mice after i.v (tail vein) injection with [18F]Ray03-18B (150-200 μL, ~5.6 MBq) over 60 min.

[0423] Data from figures 14-16, 18, and 19 demonstrate CNS and peripheral exposure to the radiolabeled analog. Figures 17, 20-22 demonstrate broad compound exposure throughout the brain. These results provide evidence of CNS tissue exposure, which is an essential requirement for compounds that aim to enhance CNS progranulin levels as a therapeutic for FTD-GRN and other neurodegenerative disorders.

[0424] Although this disclosure contains many specific embodiment details, these should not be construed as limitations on the scope of the subject matter or on the scope of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments. Certain features that are described in this disclosure in the context of separate embodiments can also be implemented, in combination, in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments, separately, or in any suitable sub-combination. Moreover, although previously described features may be described as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can, in some cases, be excised from the combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.

[0425] Particular embodiments of the subject matter have been described. Other embodiments, alterations, and permutations of the described embodiments are within the scope of the following claims as will be apparent to those skilled in the art. While operations are depicted in the drawings or claims in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed (some operations may be considered optional), to achieve desirable results.

[0426] Accordingly, the previously described example embodiments do not define or constrain this disclosure. Other changes, substitutions, and alterations are also possible without departing from the spirit and scope of this disclosure.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula (I)or a pharmaceutically acceptable salt thereof, wherein: Ring A is 5-6 membered heteroaryl or phenyl; Ring B is 6-10 membered heteroaryl, 9-14 membered heterocyclyl, or phenyl; R1is C1-C6 alkyl; R2is: (i) phenyl optionally substituted with 1-2 independently selected R2A, (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A, (iii) C1-C6 alkyl, (iv) C3-C6 cycloalkyl, (v) -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl), (vi) -(C=O)C3-C6 cycloalkyl, (vii) 4-10 membered heterocyclyl, and (viii) -NR2B(C3-C6 cycloalkyl); R2Ais halogen or hydroxyl; R2Bis hydrogen or C1-C6 alkyl; R3is halogen or -NR3A(C=O)C1-C6 alkyl; R3Ais hydrogen or C1-C6 alkyl; L is a bond, -O-, -S-, -S(=O)-, -SO2-, -N(RA)-, -C(RARB)-, or -C(=O)-; RAand R2Bare each independently hydrogen or C1-C6 alkyl; RBis hydrogen, hydroxyl, or C1-C6 alkoxyl; and n is 0 or 1.

2. The compound of claim 1, wherein Ring A is 5 membered heteroaryl.

3. The compound of claim 1, wherein Ring A is 6 membered heteroaryl.

4. The compound of claim 1, wherein Ring A is phenyl.

5. The compound of any one of claims 1-4, wherein Ring B is 6-10 membered heteroaryl.

6. The compound of any one of claims 1-5, wherein Ring B is 6 membered heteroaryl.

7. The compound of any one of claims 1-5, wherein Ring B is 9-10 membered heteroaryl.

8. The compound of any one of claims 1-4, wherein Ring B is 9-14 membered heterocyclyl.

9. The compound of any one of claims 1-4, wherein Ring B is phenyl.

10. The compound of any one of claims 1-9, wherein R1is C3-C6 alkyl.

11. The compound of any one of claims 1-9, wherein R1is C1-C2 alkyl.

12. The compound of any one of claims 1-9 and 11, wherein R1is methyl.

13. The compound of any one of claims 1-12, wherein R2is phenyl optionally substituted with 1-2 independently selected R2A.

14. The compound of any one of claims 1-13, wherein R2is phenyl substituted with 1-2 independently selected R2A.

15. The compound of any one of claims 1-12, wherein R2is 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A.

16. The compound of any one of claims 1-12 and 15, wherein R2is 5-6 membered heteroaryl substituted with 1-2 independently selected R2A.

17. The compound of any one of claims 1-12 and 15, wherein R2is unsubstituted 5-6 membered heteroaryl.

18. The compound of any one of claims 1-12, wherein R2is C1-C6 alkyl.

19. The compound of any one of claims 1-12, wherein R2is C3-C6 cycloalkyl.

20. The compound of any one of claims 1-12, wherein R2is -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl).

21. The compound of any one of claims 1-12 and 20, wherein R2is -(C=O)(C1-C6 alkyl).

22. The compound of any one of claims 1-12, wherein R2is -(C=O)C3-C6 cycloalkyl.

23. The compound of any one of claims 1-12, wherein R2is 4-10 membered heterocyclyl.

24. The compound of any one of claims 1-12, wherein R2is -NR2B(C3-C6 cycloalkyl).

25. The compound of any one of claims 1-24, wherein R2Ais halogen.

26. The compound of any one of claims 1-24, wherein R2Ais hydroxyl.

27. The compound of any one of claims 1-26, wherein R2Bis hydrogen.

28. The compound of any one of claims 1-26, wherein R2Bis C1-C6 alkyl.

29. The compound of any one of claims 1-28, wherein R3is halogen.

30. The compound of any one of claims 1-28, wherein R3is -NR3A(C=O)C1-C6 alkyl.

31. The compound of any one of claims 1-30, wherein R3Ais hydrogen.

32. The compound of any one of claims 1-30, wherein R3Ais C1-C6 alkyl.

33. The compound of any one of claims 1-32, wherein L is a bond.

34. The compound of any one of claims 1-32, wherein L is -O-.

35. The compound of any one of claims 1-32, wherein L is -S-.

36. The compound of any one of claims 1-32, wherein L is -S(=O)-.

37. The compound of any one of claims 1-32, wherein L is -SO2-.

38. The compound of any one of claims 1-32, wherein L is -N(RA)-.

39. The compound of any one of claims 1-32, wherein L is -C(RARB)-.

40. The compound of any one of claims 1-32, wherein L is -C(=O)-.

41. The compound of any one of claims 1-40, wherein RAis hydrogen.

42. The compound of any one of claims 1-40, wherein RAis C1-C6 alkyl.

43. The compound of any one of claims 1-42, wherein RBis hydrogen.

44. The compound of any one of claims 1-42, wherein RBis hydroxyl.

45. The compound of any one of claims 1-42, wherein RBis C1-C6 alkoxyl.

46. The compound of any one of claims 1-45, wherein R2Bis hydrogen.

47. The compound of any one of claims 1-45, wherein R2Bis C1-C6 alkyl.

48. The compound of any one of claims 1-47, wherein n is 0.

49. The compound of any one of claims 1-47, wherein n is 1.

50. A pharmaceutical composition comprising the compound of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

51. A pharmaceutical composition comprising the compound of Formula (II), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

52. A method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

53. A method of treating a PGRN-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein R2is: (i) phenyl optionally substituted with 1-3 independently selected R2A, (ii) 5-6 membered heteroaryl optionally substituted with 1-2 independently selected R2A, (iii) C1-C6 alkyl, (iv) C3-C6 cycloalkyl, (v) -(C=O)(C1-C6 alkyl optionally substituted with C1-C6 alkoxyl), (vi) –(C=O)C3-C6 cycloalkyl, and(vii) 4-10 membered heterocyclyl, and(viii) -NR2B(C3-C6 cycloalkyl);R2Ais halogen, C1-C6 alkyl, or hydroxyl;R3is halogen and -NR3A(C=O)C1-C6 alkyl, -NR3BSO2R3C, or C1-C6 alkyl optionally substituted with hydroxyl;R3Aand R3Bare each independently hydrogen or C1-C6 alkyl;R3Cis C1-C6 alkyl;L is bond, -O-, -S-, -S(=O)-, -SO2-, -N(RA)-, -C(RARB)-, or -C(=O)-;RAis hydrogen or C1-C6 alkyl;RBis hydrogen, hydroxyl, and C1-C6 alkoxyl;Rcis absent or -NRcl(C=O)Cl-C6 alkyl;RC1is H or C1-C6 alkyl; and n is 0 or 1.

54. The method of Claim 52 or 53, wherein the PGRN-associated disease is frontotemporal dementia.

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