TWI812677B - Novel compounds for the treatment, alleviation or prevention of disorders associated with tau aggregates - Google Patents

Abstract

The present invention relates to novel compounds that can be employed in the treatment, alleviation or prevention of a group of disorders and abnormalities associated with Tau (Tubulin associated unit) protein aggregates including, but not limited to, Neurofibrillary Tangles (NFTs), such as Alzheimer’s disease (AD).

Description

Novel compounds for treating, ameliorating or preventing conditions associated with TAU aggregates

The present invention relates to novel compounds useful in the treatment, amelioration or prevention of a group of conditions and abnormalities associated with Tau (tubulin-associated unit) protein aggregates, including but not limited to neurofibrillary tangles (NFTs), such as Alzheimer's disease Alzheimer's disease (AD).

Many diseases of aging are based on or related to extracellular or intracellular deposition of amyloid or amyloid-like proteins that contribute to pathogenesis and disease progression. The best characterized amyloid protein that forms extracellular aggregates is amyloid beta (Aβ). Other examples of amyloid proteins that form extracellular aggregates are prion, ATTR (transthyretin) or ADan (ADanPP). Amyloid proteins that mainly form intracellular aggregates include (but are not limited to) Tau, α-synuclein, TAR DNA-binding protein 43 (TDP-43), and huntingtin (htt). Diseases involving tau aggregates are often classified as tauopathies (eg, AD).

Amyloid or amyloid-like deposits result from the misfolding and subsequent aggregation of proteins to produce beta-pleated plate assemblies in which multiple peptides or proteins are held together by intermolecular hydrogen bonds. Although amyloid or amyloid-like proteins have different primary amino acid sequences, their deposits usually contain many shared molecular components, especially the presence of β-pleated plate quaternary structures. The link between amyloid deposits and disease remains largely unclear. A wide variety of protein aggregates, both those related to disease pathology and those unrelated to disease pathology, have been found to be toxic, suggesting that common molecular features of amyloid species are associated with or contribute to disease pathogenesis (Bucciantini et al., Nature, 2002, 416, 507-11). Various polymers of β-pleated plate aggregates of peptides or proteins are also associated with the toxicity of different peptides or proteins ranging from dimers to soluble low molecular weight oligomers, fibrils or insoluble fibrillar deposits.

Alzheimer's disease (AD) is a neurological disorder thought to be caused by amyloid plaques, the extracellular accumulation of abnormal deposits of (amyloid-β) Aβ aggregates in the brain. Other major neuropathological hallmarks of AD are intracellular neurofibrillary tangles (NFTs), which originate from the aggregation of hyperphosphorylated Tau protein, misfolded Tau, or pathological Tau and its conformational isomers. AD shares its pathogenesis with many neurodegenerative tauopathies, particularly with certain types of frontotemporal dementia (FTD). Tau protein is a readily soluble "naturally unfolded" protein that tightly binds to microtubules (MTs) to promote their assembly and stability. MTs are essential for the integrity of the cellular scaffold of neurons and thus for the proper formation and function of neuronal circuits and therefore for learning and memory. The association of Tau with MT is controlled by dynamic phosphorylation and dephosphorylation, as shown primarily in vitro and in non-neuronal cells. In the AD brain, tau pathology (tau pathology) occurs later than amyloid pathology, but there is still discussion within the industry as to whether Aβ protein is the causative agent of AD (this constitutes the essence of the so-called amyloid cascade hypothesis). Quite controversial (Hardy et al., Science 1992, 256, 184-185; Musiek et al., Nature Neurosciences 2015, 18(6), 800-806). The exact mechanisms linking amyloid and tau pathology remain largely unknown, but are proposed to involve activation of neuronal signaling pathways acting on GSK3 and cdk5 as major "Tau-kinases" or By which it acts (Muyllaert et al., Rev. Neurol. (Paris), 2006, 162, 903-7; Muyllaert et al., Genes Brain and Behav. 2008, Suppl. 1, 57-66). Even though tauopathy occurs later than amyloid, it is not just a harmless side effect, but the main pathological executor of AD. In experimental mouse models, cognitive deficits caused by amyloid pathology are almost completely ameliorated by the absence of Tau protein (Roberson et al., Science, 2007, 316(5825), 750-4), and cognitive dysfunction and Dementia severity correlates with tauopathy but not amyloid pathology.

Diseases involving tau aggregates are generally classified as tauopathies and include, but are not limited to, Alzheimer's disease (AD), familial AD, PART (primary age-related tauopathy), library Creutzfeldt-Jacob disease, boxer dementia, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease (GSS), inclusion bodies Myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Parkinsonism-dementia complex of Guam , non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic granulopathy, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, associated with chromosome 17 Frontotemporal dementia with Parkinson's disease (FTDP-17), Hallervorden-Spatz disease (Hallervorden-Spatz disease), multiple system atrophy (MSA), Niemann-Pick disease type C (Niemann- Pick disease type C), pallidum-pontine-substantia nigra degeneration, Pick's disease (PiD), progressive subcortical gliomatosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis inflammation, tangle-dominant dementia, postencephalitic Parkinson's disease, myotonic dystrophy, subacute sclerosing panencephalopathy, LRRK2 mutations, chronic traumatic encephalopathy (CTE), familial British dementia familial British dementia, familial Danish dementia, other frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with iron accumulation in the brain, SLC9A6 correlation Mental retardation, white matter tauopathy with glial inclusions, epilepsy, Lewy body dementia (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease ), HIV-related dementia, adult-onset diabetes, senile cardiac amyloidosis, glaucoma, ischemic stroke, psychosis in AD, and Huntington's disease. (Williams et al., Intern. Med. J., 2006, 36, 652-60; Kovacs et al., J Neuropathol Exp Neurol. 2008; 67(10): 963-975; Higuchi et al., Neuropsychopharmacology - 5th Generation of Progress , 2002, Part 9, Chapter 94:1339-1354; Hilton et al., Acta Neuropathol. 1995;90(1):101-6; Iqbal et al., Biochimica et Biophysica Acta 1739 (2005) 198-210; McQuaid et al. Humans, Neuropathol Appl Neurobiol. 1994 Apr;20(2):103-10; Vossel et al., Lancet Neurol 2017; 16: 311-22; Stephan et al., Molecular Psychiatry (2012) 17, 1056-1076; Anderson et al., Brain (2008), 131, 1736-1748; Savica et al., JAMA Neurol. 2013;70(7):859-866; Brown et al., Molecular Neurodegeneration 2014, 9:40; El Khoury et al., Front . Cell. Neurosci., 2014, Vol. 8, Article No. 22: 1-18; Tanskanen et al., Ann. Med. 2008;40(3):232-9; Gupta et al., CAN J OPHTHALMOL, Vol. 43 , Issue 1, 2008: 53-60; Dickson et al., Int J Clin Exp Pathol 2010;3(1):1-23; Fernández-Nogales et al., Nature Medicine, 20, 881-885 (2014); Bi et al., Nature Communications Volume 8, Article Number: 473 (2017); Murray et al., Biol Psychiatry. 2014 April 1; 75(7): 542-552).

Of all agents in clinical trials for the treatment of Alzheimer's disease in 2017, agents targeting tau were extremely scarce and accounted for only 8% of phase II clinical trials (Cummings et al., Alzheimer's & Dementia: Translational Research & Clinical Interventions 3 (2017) 367-384). Current treatments targeting Tau protein mainly include antibody-based methods, which are mainly limited to targeting only extracellular Tau. Using a small molecule approach, several Tau kinase inhibitors have been developed, although they are extremely challenging in terms of toxicity and specificity. However, only one kinase inhibitor, Nilotinib, is currently being tested in clinical trials. Finally, among Tau aggregation inhibitors, only one, LMTX, is currently in clinical trials (Cummings et al., 2017). Although Tau-based therapies have become the focus of increasing attention in recent years, there is still a great need in the industry for other therapeutic agents targeting pathological Tau conformational isomers known or presumed to cause tauopathies.

WO2011/128455 relates to specific compounds suitable for the treatment of amyloid or amyloid-related conditions.

WO2010/080253 relates to dipyridyl-pyrrole derivative compounds which can be used to treat diseases suitable for inhibition, regulation and/or modulation of protein kinase signal transduction.

It is an object of the present invention to provide compounds that can be used to treat, ameliorate or prevent a group of disorders and abnormalities associated with Tau protein aggregates, including (but not limited to) NFT, such as Alzheimer's disease (AD). ). Additionally, there is a need in the industry for compounds that can be used as therapeutic agents for: (a) reducing Tau aggregates/NFTs by recognizing aggregated Tau and depolymerizing Tau (e.g., by changing the molecular conformation of the Tau aggregates), and/or (b) prevent the formation of Tau aggregates, and/or (c) interfere with Tau aggregates intracellularly, and/or (d) reduce Tau misfolding and hyperphosphorylation in vivo and/or (f) reduce neuronal Inflammation markers. The inventors have surprisingly found that these objectives can be achieved by compounds of formula (I) as set out below.

The compounds of formula (I) (a) exhibit a high ability to reduce Tau aggregates by recognizing aggregated Tau and deaggregating Tau (e.g., by changing the molecular configuration of the Tau aggregates), and/or (b) preventing Formation of Tau aggregates, and/or (c) intracellular interference with Tau aggregates, and/or (d) reduction of Tau misfolding and hyperphosphorylation in vivo and/or (f) reduction of neuroinflammatory markers. Although not wishing to be bound by theory, it is postulated that compounds of formula (I) inhibit Tau aggregation or deaggregate preformed Tau aggregates, including when present within cells. Due to their unique design features, these compounds exhibit properties such as appropriate lipophilicity and molecular weight, brain uptake and pharmacokinetics, cell permeability, solubility and metabolic stability, making them useful candidates for the treatment, amelioration or prevention of tauopathies. The potion of success.

The accumulation of Tau NFT lesions has been shown to be well correlated with cognitive deficits in AD, both through histopathological analysis and through in vivo Tau PET imaging. Compounds of the present invention may prevent the formation of Tau aggregates or deaggregate pre-existing Tau aggregates, and thus may be expected to prevent or reduce associated cognitive deficits in AD.

Ultrastructural analysis has shown that the Tau inclusion system consists of paired helical filaments (PHF) or straight filaments (SF). High-resolution structural analysis has shown that these silk systems are composed of a core region containing amino acids 306-378 of Tau, which adopts a crossed β/β-helical structure. Compounds of the present invention can recognize aggregated Tau and deaggregate Tau (eg, by changing the molecular conformation of Tau aggregates), and thus are expected to promote Tau clearance.

Additionally, it has been shown that Tau can spread from cell to cell and that certain forms of Tau (which act as seeds) can induce structural changes in the native Tau protein within healthy cells to undergo misfolding and aggregation. Aggregated Tau is thought to be responsible for seeding and thus causing the spread of Tau pathology. Compounds of the present invention may interfere with aggregated Tau intracellularly and thus may be expected to reduce the spread of Tau pathology and ultimately prevent or reduce associated cognitive deficits in AD.

The Tau aggregation cascade begins with Tau misfolding and hyperphosphorylation. These events are believed to precede the formation of intracellular Tau neuronal inclusions and, therefore, the establishment and spread of Tau pathology. The compounds of the present invention can reduce Tau misfolding and hyperphosphorylation in vivo, and thus are expected to be useful in the treatment, amelioration or prevention of diseases associated with Tau pathology.

Finally, the link between Tau pathology and neuroinflammation is now well established. Neuroinflammation is a critical event already in the early stages of AD and is believed to be one of the triggers for Tau accumulation in PHF. Furthermore, once Tau pathology is well established in the brain, several mouse models of tauopathy display significant neuroinflammation, indicating that Tau pathology can also induce neuroinflammatory processes. These two findings indicate that tau pathology and neuroinflammation are linked in a positive feedback loop. In Tau pathology, compounds of the invention reduce markers of neuroinflammation.

The present invention discloses novel compounds of formula (I) that have the ability to: (a) reduce Tau aggregates, recognize aggregated Tau, and deaggregate Tau (e.g., by changing the molecular configuration of Tau aggregates), and/or (b) Prevent the formation of Tau aggregates, and/or (c) interfere with Tau aggregates within cells, and/or (d) reduce Tau misfolding and hyperphosphorylation in vivo and/or (f) reduce neuroinflammatory markers. The present invention provides methods of using compounds of formula (I) or pharmaceutical compositions thereof to treat conditions and abnormalities associated with Tau protein aggregates, including (but not limited to) NFTs. The present invention further provides pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier or excipient.

The invention is summarized under the following entries: 1. A compound of formula (I), and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates and polymorphs thereof; wherein A is selected from the group consisting of: and ,in and Can be connected to Q at any available location, where and Substituted with one or more substituents ^Rj , and wherein and Optionally substituted by one or more substituents R ; B is selected from the group consisting of O and NR ^a ; E and V are independently selected from the group consisting of: N, NR ⁵ , O and S; G is selected from the following The group consisting of: benzene ring, pyrimidine ring and pyridine ring; J is selected from the group consisting of O and NR ¹ ; Q is selected from the group consisting of N and CR ¹ ; Y is selected from the group consisting of CZ and N, the condition is When Y is N and Y ¹ , Y ² and Y ³ are CZ, B is N-alkyl or O; Y ¹ is selected from the group consisting of CZ and N; Y ² is selected from the group consisting of CZ and N; Y ³ is selected from the group consisting of CZ and N; Z is independently selected from the group consisting of: H, halogen, O-alkyl, alkyl and CN; R is independently selected from the group consisting of and -NR ³ R ⁴ ; R ^a is selected from the group consisting of H and alkyl groups; R ^b , R ^c , R ^d , R ^e , R ^f and R ^g are independently selected from the group consisting of H and alkyl groups group, or any two of R ^b , R ^c , R ^d , ^Re , R ^f and R ^g may be linked to form a 3- to 8-membered ring; R ^j is independently selected from the group consisting of: - halogen, -O-alkyl, -CF ₃ , -CN, -NR ³ R ⁴ , and , where a bridge containing C _1-2 carbon atoms can exist between a carbon atom and c or d carbon atom or where a bridge containing C _1-2 carbon atoms can exist between b carbon atom and c or d carbon atom ; R ¹ is selected from the group consisting of H and alkyl; R ² is independently selected from the group consisting of alkyl, F and =O, wherein the alkyl is optionally substituted by halogen, -OH or -O-alkyl, And if two R ² are twin-positioned, they can be connected to form a 3- to 6-membered ring; R ³ and R ⁴ are independently selected from the group consisting of H and an alkyl group, wherein the alkyl group may be optionally treated with halogen , -OH or -O-alkyl substitution; R ⁵ is selected from the group consisting of H and alkyl; n is 0, 1, 2, 3 or 4. r and s are independently 0, 1, 2, or 3; and t and u are independently 1, 2, or 3. 2. For example, the compound of item 1 is a compound of formula (Ia): Among them, A , B , ^Rb , ^Rc , ^Rd , ^Re , ^Rf , ^Rg , Y and Z are as defined in item 1. 3. For example, the compound of item 1 is a compound of formula (Ib): Where A , B , ^Rb , ^Rc , ^Rd , ^Re , ^Rf , ^Rg and Z are as defined in item 1. 4. A compound as in any one of items 1 to 3, wherein A is and ,in and Can be connected to Q or N at any available location, where Substituted with one or more substituents ^Rj , and wherein Optionally substituted with one or more substituents R. 5. A compound as in any one of items 1 to 3, wherein A is ,in Can be connected to Q or N at any available location, and where Optionally substituted with one or more substituents R. 6. A compound of any one of items 1 to 5, which is a compound of formula (Ic): Where E , R , V and Z are as defined in item 1. 7. A compound of any one of items 1 to 4, which is a compound of formula (Id): where R ^a , R ^j and Z are as defined in item 1 and p is 1 or 2. 8. The compound of item 1, wherein the compound is selected from the group consisting of: . 9. A pharmaceutical composition comprising a compound as in any one of items 1 to 8 and optionally a pharmaceutically acceptable carrier or excipient. 10. A compound according to any one of items 1 to 8 for use as a medicament. 11. A compound of formula (I), and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates and polymorphs thereof; for the treatment, improvement or prevention of aggregation with Tau protein A disease or abnormality related to the body, wherein series A is selected from the group consisting of: and ,in and Can be connected to Q at any available location, where and Substituted with one or more substituents ^Rj , and wherein and Optionally substituted by one or more substituents R ; B is selected from the group consisting of O and NR ^a ; E and V are independently selected from the group consisting of: N, NR ⁵ , O and S; G is selected from the following The group consisting of: benzene ring, pyrimidine ring and pyridine ring; J is selected from the group consisting of O and NR ¹ ; Q is selected from the group consisting of N and CR ¹ ; Y is selected from the group consisting of CZ and N; Y ¹ Y ² is selected from the group consisting of CZ and N; Y ³ is selected from the group consisting of CZ and N; Z is independently selected from the group consisting of: H, halogen, O- Alkyl, alkyl and CN; R is independently selected from and -NR ³ R ⁴ ; R ^a is selected from the group consisting of H and alkyl groups; R ^b , R ^c , R ^d , R ^e , R ^f and R ^g are independently selected from the group consisting of H and alkyl groups group, or any two of R ^b , R ^c , R ^d , ^Re , R ^f and R ^g may be linked to form a 3- to 8-membered ring; R ^j is independently selected from the group consisting of: - halogen, -O-alkyl, -CF ₃ , -CN, -NR ³ R ⁴ , and , where a bridge containing C _1-2 carbon atoms can exist between a carbon atom and c or d carbon atom or where a bridge containing C _1-2 carbon atoms can exist between b carbon atom and c or d carbon atom ; R ¹ is selected from the group consisting of H and alkyl; R ² is independently selected from the group consisting of alkyl, F and =O, wherein the alkyl is optionally substituted by halogen, -OH or -O-alkyl, And if two R ² are twin-positioned, they can be connected to form a 3- to 6-membered ring; R ³ and R ⁴ are independently selected from the group consisting of H and an alkyl group, wherein the alkyl group may be optionally treated with halogen , -OH or -O-alkyl substitution; R ⁵ is selected from the group consisting of H and alkyl; n is 0, 1, 2, 3 or 4; r and s are independently 0, 1, 2 or 3; And t and u are independently 1, 2 or 3. 12. A compound as used in entry 11, wherein the compound of formula (I) is as defined in any of entries 1 to 8. 13. A compound according to any one of items 1 to 8 or 11 for reducing Tau aggregation. 14. A compound according to any one of items 1 to 8 or 11 for preventing the formation of Tau aggregates and/or for inhibiting Tau aggregation. 15. A compound according to any one of items 1 to 8 or 11 for intracellular interference with Tau aggregates. 16. A compound according to any one of items 1 to 8 or 11, which is used to reduce Tau misfolding and hyperphosphorylation in vivo. 17. A compound according to any one of items 1 to 8 or 11 for reducing markers of neuroinflammation. 18. A method of treating, preventing or ameliorating a condition or abnormality associated with Tau protein aggregates, the method comprising administering an effective amount of a compound of any one of items 1 to 8 or 11 to an individual in need thereof. 19. A method of reducing Tau aggregation, the method comprising administering to an individual in need thereof an effective amount of a compound of any one of items 1 to 8 or 11. 20. A method of preventing the formation of Tau aggregates and/or inhibiting Tau aggregation, the method comprising administering an effective amount of a compound of any one of items 1 to 8 or 11 to an individual in need thereof. 21. A method of intracellularly interfering with Tau aggregates, the method comprising administering to an individual in need thereof an effective amount of a compound of any one of items 1 to 8 or 11. 22. A method of reducing Tau misfolding and hyperphosphorylation in vivo, the method comprising administering an effective amount of a compound of any one of items 1 to 8 or 11 to an individual in need thereof. 23. A method of reducing markers of neuroinflammation, the method comprising administering to an individual in need thereof an effective amount of a compound of any one of items 1 to 8 or 11. 24. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for the treatment, prevention or amelioration of a condition or abnormality associated with Tau protein aggregates. 25. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for reducing Tau aggregation. 26. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for preventing the formation of Tau aggregates and/or for inhibiting Tau aggregation. 27. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for intracellular interference with Tau aggregates. 28. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for reducing Tau misfolding and hyperphosphorylation in vivo. 29. Use of a compound according to any one of items 1 to 8 or 11 for the manufacture of a medicament for reducing markers of neuroinflammation. 29. A mixture comprising a compound as in any one of items 1 to 8 and at least one other biologically active compound selected from a compound different from any one of items 1 to 8 Therapeutic agent, wherein the mixture further comprises at least one of a pharmaceutically acceptable carrier, diluent and excipient. 30. A mixture as in item 29, wherein the other biologically active compound is a compound used to treat amyloidosis. 31. A mixture as in item 29 or 30, wherein the compound and/or the other biologically active compound is present in a therapeutically effective amount. 32. A mixture as in any one of items 29 to 31, wherein the other biologically active compound is selected from the group consisting of: compounds that resist oxidative stress; anti-apoptotic compounds; metal chelators; DNA repair inhibitors, for example Pirenzepine and metabolites; 3-amino-1-propanesulfonic acid (3APS); 1,3-propanedisulfonate (1,3PDS); α-secretase activator; β-secretase And gamma-secretase inhibitors; glycogen synthase kinase 3 inhibitors; O-linked N-acetyl glucosamine hydrolase (O-glcnacase, OGA) inhibitors; neurotransmitters; β-pleat plate disruptors; Amyloid β scavenging/eliminating cellular component attractant; inhibitor of N-terminally truncated amyloid β-like (including pyroglutamylated amyloid β-like 3-42), anti-inflammatory molecule, or cholinesterase inhibitor ( ChEI), such as tacrine, rivastigmine, donepezil and/or galantamine; M1 agonists; other drugs, including any amyloid or Tau-modified drugs and nutritional supplements; antibodies, including any functionally equivalent antibodies or functional portions thereof; or vaccines. 33. A mixture as in item 32, wherein the other biologically active compound is a cholinesterase inhibitor (ChEI). 34. For example, mixture 32 of item 32, wherein the other biologically active compound is selected from the group consisting of: tacnin, rivastigmine, donepezil, galantamine, nicotinic acid and memantine ). 35. A mixture as in item 32, wherein the other biologically active compound is an antibody, in particular a monoclonal antibody, including any functionally equivalent antibody or functional part thereof. 36. A mixture as in any one of items 29 to 35, wherein the compound and/or the other biologically active compound is present in a therapeutically effective amount. 37. A compound as used in item 11, a method as in item 18 or a use as in item 24, wherein the disorder is selected from the group consisting of Alzheimer's disease (AD), familial AD, primary age-related tauopathy (PART), Creutzfeldt-Jakob disease, boxer's dementia, Down syndrome, Gertzmann-Steusler-Steinke disease (GSS), inclusion body myositis, prion protein cerebral amyloid angiopathy, Traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Guam Parkinson's disease-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic granulopathy, corticobasal CBD, diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and parkinsonism (FTDP-17), Hallerwarden-Spatz disease, multiple system atrophy (MSA), Niemann-Pick disease type C, pallidum-pontine-nigra degeneration, Pick's disease (PiD), progressive subcortical gliomatosis, progressive supranuclear palsy (PSP), subacute sclerosing Panencephalitis, tangle-dominant dementia, postencephalitic parkinsonism, myotonic dystrophy, subacute sclerosing panencephalopathy, LRRK2 mutations, chronic traumatic encephalopathy (CTE), familial British type Dementia, familial Danish dementia, other frontotemporal degenerations, Guadeloupe Parkinson's disease, neurodegeneration with iron accumulation in the brain, SLC9A6-related mental retardation, white matter tauopathy with glomerulocytes Inclusion bodies, epilepsy, Lewy body dementia (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes, senile cardiac amyloidosis , glaucoma, ischemic stroke, psychosis in AD and Huntington's disease, preferably Alzheimer's disease (AD), corticobasal degeneration (CBD), Pick's disease (PiD) and progressive Supranuclear palsy (PSP). 38. Use of a compound according to any one of items 1 to 8 or 11 as an analytical reference or as an in vitro screening tool.

Definitions Within the meaning of this application, the following definitions apply: "Alkyl" means a saturated linear or branched organic moiety consisting of carbon and hydrogen atoms. Examples of suitable alkyl groups have 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and include methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl and isobutyl . Alkyl groups are optionally substituted with halogen (preferably F), -OH or -O alkyl (preferably -OMe). "Hal" or "halogen" refers to F, Cl, Br and I. "3- to 8-membered ring" means a 3-, 4-, 5-, 6-, 7- or 8-membered ring in which there are no carbon atoms in the ring and one or more carbon atoms have 1 or 2 (for 3-member ring), 1, 2 or 3 (for 4-member ring), 1, 2, 3 or 4 (for 5-member ring) or 1, 2, 3, 4 or 5 (for 6-member ring), 1, 2, 3, 4, 5 or 6 (for a 7-membered ring) or 1, 2, 3, 4, 5, 6 or 7 (for an 8-membered ring) the same or different heteroatoms are substituted, where these The heteroatoms are selected from O, N and S.

Compounds of the present invention having one or more optically active carbons may exist as: racemates and racemic mixtures (including mixtures in all ratios), stereoisomers (including diastereomeric mixtures and individual non-enantiomeric mixtures). Enantiomers, enantiomer mixtures and single enantiomers, mixtures of configuration isomers and single configuration isomers), tautomers, atropisomers and rotamers. All isomeric forms are included in the present invention. Compounds containing olefin double bonds described in this invention include E and Z geometric isomers. Also included in the present invention are all pharmaceutically acceptable salts, prodrugs, polymorphs, hydrates and solvates.

The term "polymorph" refers to the various crystalline structures of the compounds of the invention. This may include, but is not limited to, crystalline forms (and amorphous materials) and all lattice forms. The salts of the present invention may be crystalline and may exist as more than one polymorph.

The invention also encompasses solvates, hydrates and anhydrous forms of the salts. The solvent included in the solvate is not specifically limited and can be any pharmaceutically acceptable solvent. Examples include water and C _1-4 alcohols (such as methanol or ethanol).

"Pharmaceutically acceptable salts" are defined as derivatives of the disclosed compounds wherein the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, basic or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the customary non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. By way of example, such customary nontoxic salts include those derived from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and from organic acids. Salts prepared from acids such as (but not limited to) acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, parmoic acid, maleic acid, Hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfamic acid, 2-acetyloxybenzoic acid, fumaric acid, toluenesulfonic acid, methane sulfonic acid, ethane disulfonic acid, oxalic acid, Isethionic acid and the like. The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing basic or acidic moieties. Generally, such salts may be prepared by reacting the free acid or base form of the compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two. Organic solvents include, but are not limited to, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, PA, 1990, page 1445, the disclosure of which is incorporated herein by reference.

The compounds of the present invention may also be provided in the form of prodrugs, ie, compounds that are metabolized in vivo to active metabolites. As used below in describing and patenting the invention, the term "prodrug" means any covalently bonded compound that releases the active parent drug as a result of biotransformation in vivo. References to Goodman and Gilman describing prodrugs (The Pharmacological Basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", pp. 13-15) are generally incorporated by reference. in this article.

"Pharmaceutically acceptable" is defined as suitable for use in contact with human and animal tissue within the scope of reasonable medical judgment without undue toxicity, irritation, allergic reactions or other problems or complications and proportional to a reasonable benefit/risk ratio Those compounds, materials, compositions and/or dosage forms.

The patient or individual of the present invention is generally an animal, specifically a mammal, and more specifically a human being.

"Tau" as used herein refers to the highly soluble microtubule-binding protein found primarily in neurons and includes the main 6 isoforms, cleaved or truncated forms and other modified forms, such as those derived from phosphorylation, sugar Sylation, glycation, prolyl isomerization, nitration, acetylation, polyamination, ubiquitination, small ubiquitination (sumoylation) and oxidation.

"Aggregated Tau" refers to aggregated monomers of Tau peptide or protein that fold into oligomeric or polymeric structures.

"Neurofibrillary tangles" (NFTs) as used herein refer to insoluble aggregates of highly phosphorylated Tau protein containing paired helical filaments (PHF) and straight filaments. Its presence is a hallmark of AD and other diseases called tauopathies.

The term "antibody or antibodies" as used herein is an industry-recognized term and should be understood to refer to a molecule or active fragment of a molecule that binds to a known antigen, or specifically to immunoglobulin molecules and immunoglobulins. The antigen-binding portion of a protein molecule. In particular, mixtures of the invention include a compound of the invention and an anti-Tau or anti-Abeta antibody.

The term "functionally equivalent antibody or functional portion thereof" as used herein shall be understood to refer to an equivalent molecule or active fragment of a molecule that binds to a known antigen, or in particular to immunoglobulin molecules and immunoglobulins The antigen-binding portion of a molecule that has substantially the same (biological) activity as the antibody from which it is derived.

The "vaccines" reported in the mixtures of the invention are in particular Tau or Abeta vaccines.

Unless otherwise stated, the definitions and preferred definitions given in the "Definitions" section apply to all embodiments set forth below.

The compounds of the present invention are described below. It is understood that all possible combinations of the following definitions are also contemplated.

In one embodiment, the invention relates to compounds of formula (I):

and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates and polymorphs thereof.

The preferred embodiment of the compound of formula (I) is

Another preferred embodiment of the compound of formula (I) is

Another preferred embodiment of the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

Even more preferably, the compound of formula (I) is or

Even better,

The following definition of A applies to the compounds of formula (I) and their preferred embodiments.

Series A is selected from the group consisting of: and , where G is selected from benzene ring, pyrimidine ring and pyridine ring. therefore Covers the following preferred embodiments: and . and therefore, The following examples are covered: and . In a preferred embodiment, Department selected from and . The following rings can be imagined: and . Preferred embodiments include and . In these structural formulas, ring G is not shown completely, but only consists of part of the bond instruct.

In a preferred embodiment, Series A is selected from and .

In another preferred embodiment, A series and . In a preferred embodiment, A series .

In a more preferred embodiment, Series A is selected from and . In a more preferred embodiment, Series A is selected from and .

In another preferred embodiment, A is selected from and .

In an even more preferred embodiment, Series A .

In the above definition of A and its preferred embodiments, and Connect to Q at any available location.

In the above definition of A and its preferred embodiments, and Substituted with one or more substituents ^Rj .

In the above definition of A and its preferred embodiments, and Optionally substituted with one or more substituents R.

Where appropriate, the following definitions apply to formula (I) and its preferred embodiments.

System B is selected from the group consisting of O and NR ^a . More preferably, B is NR ^a , most preferably NH.

E and V are independently selected from the group consisting of: N, NR ⁵ , O and S. Since the ring system containing E and V is unsaturated, at least one of E and V is N.

J is selected from the group consisting of O and NR ¹ , preferably O.

Q is selected from the group consisting of N and CR ¹ . Preferably, Q is selected from the group consisting of N and CH, and more preferably, Q is N.

Y is selected from the group consisting of CZ and N. More preferably, Y is selected from the group consisting of CH and N. Even better, Y is CH. In one embodiment, if Y is N and Y ¹ , Y ² and Y ³ are CZ, then B is N-alkyl or O, preferably B is N-alkyl.

Y ¹ is selected from the group consisting of CZ and N. Preferably, Y ¹ is CZ.

Y ² is selected from the group consisting of CZ and N. Preferably, Y ² is CZ.

Y ³ is selected from the group consisting of CZ and N. Preferably, Y ³ is CZ.

Z is independently selected from the group consisting of: H, halogen (preferably F), O-alkyl, alkyl and CN, preferably selected from the group consisting of H, halogen (preferably F) and O-alkyl. In a preferred embodiment, one Z is independently halogen (preferably F) or O-alkyl, and the other Z is H. In a more preferred embodiment, one Z is halogen (preferably F) and the other Z is H.

R is independently chosen from and -NR ³ R ⁴ or less, preferably the R series is free. And - a group composed of NR ³ R ⁴ , better R series ,For example . In these embodiments, Better series .

R ^a is selected from the group consisting of H and alkyl groups, more preferably H and Me, even more preferably H.

R ^b , R ^c , R ^d , ^Re , R ^f , and R ^g are independently selected from the group consisting of H and alkyl, or any of R ^b , R ^c , R ^d , ^Re , R ^f , and R ^g The two (eg, which are attached to the same or adjacent ring atoms) can be linked to form a 3- to 8-membered ring. More preferably, R ^b , R ^c , R ^d , ^Re , R ^f , R ^g are independently H or alkyl, even more preferably H.

R ^j is independently selected from the group consisting of: -halogen, -O-alkyl, -CF ₃ , -CN, -NR ³ R ⁴ , and , where a bridge containing C _1-2 carbon atoms can exist between a carbon atom and c or d carbon atom or where a bridge containing C _1-2 carbon atoms can exist between b carbon atom and c or d carbon atom . More preferably, R ^j is selected from the group consisting of: -halogen, -O-alkyl, -NR ³ R ⁴ and , or even better R ^j system .

R ¹ is selected from the group consisting of H and alkyl groups, preferably an alkyl group, more preferably CH ₃ .

R ² is independently selected from the group consisting of alkyl, F, and =O, wherein the alkyl group is optionally substituted by halogen, -OH or -O-alkyl, and where two R ² are geminal, then Can be linked to form a 3 to 6 member ring. In one embodiment, R ² is optionally substituted alkyl, in another embodiment, R ² is F, in another embodiment, R ² is =O.

R ³ and R ⁴ are independently selected from the group consisting of H and alkyl, wherein the alkyl is optionally substituted with halogen, -OH or -O-alkyl. In one embodiment, R ³ or R ⁴ is optionally substituted alkyl and the other is H. In another embodiment, R ³ is alkyl and R ⁴ is optionally substituted alkyl. In another embodiment, R ³ and R ⁴ are H.

R ⁵ is selected from the group consisting of H and alkyl. In one embodiment, R ⁵ is H. In another embodiment, R ⁵ is alkyl.

n is 0, 1, 2, 3 or 4, preferably n is 0 or 1, more preferably n is 0.

p represents 1 or 2, preferably 1.

r and s are independently 0, 1, 2 or 3.

t and u are independently 1, 2 or 3.

Preferred compounds of formula (I) are

Preferred compounds are also illustrated in the Examples.

Any combination of the embodiments, preferred embodiments and further preferred embodiments disclosed herein is also contemplated by the present invention.

Pharmaceutical Compositions Although the compounds of the present invention may be administered alone, they are preferably formulated into pharmaceutical compositions in accordance with standard pharmaceutical practice. Accordingly, the present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula (I), optionally in admixture with a pharmaceutically acceptable carrier, diluent, adjuvant or excipient.

Pharmaceutically acceptable excipients are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 15th ed., Mack Publishing Co., New Jersey (1975). Pharmaceutical excipients can be selected based on the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense that it is not harmful to the recipient.

Pharmaceutically acceptable excipients that can be used to formulate the pharmaceutical compositions of the present invention may include, for example, carriers; vehicles; diluents; solvents, such as monohydric alcohols (such as ethanol, isopropyl alcohol) and polyhydric alcohols (such as dihydric alcohols). alcohol) and edible oils (such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil), oily esters (such as ethyl oleate, isopropyl myristate), binders; adjuvants; solubilizers; Thickener; stabilizer; disintegrant; glidant; lubricant; buffer; emulsifier; wetting agent; suspending agent; sweetener; colorant; flavoring agent; coating agent; preservative; antioxidant ; Processing agents; drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, right Spinose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point wax and ion exchange resin.

Routes of administration (delivery) of the compounds of the invention include, but are not limited to, one or more of the following: oral (e.g., as a lozenge, capsule, or as an ingestible solution), topical, transmucosal (e.g., as an inhalation) nasal spray or aerosol), nasal, parenteral (e.g., by injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, tracheal Internal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, transocular (including intravitreal or intraanterior chamber), transcutaneous, rectal, transbuccal, epidural and sublingual.

For example, the compounds may be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions containing flavoring or coloring agents for immediate, delayed, modified, continuous, pulsed or Controlled release applications.

Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably corn, potato or tapioca starch), hydroxyl Sodium starch acetate, croscarmellose sodium and certain complex silicates; and granulated binders, such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be incorporated. Solid compositions of a similar type may also be used as fillers in gelatin capsules. In this regard, preferred excipients include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agents may be combined with various flavorings or correctives, coloring substances or dyes, emulsifying and/or suspending agents and diluents such as water, ethanol, propylene glycol and glycerol. and combinations thereof.

If a compound of the invention is administered parenterally, examples of such administration include one or more of the following: intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, cranial The compounds may be administered intramuscularly, intramuscularly or subcutaneously; and/or by using infusion techniques. For parenteral administration, the compounds are preferably used in the form of sterile aqueous solutions, which may contain other substances, such as sufficient salt or glucose to render the solution isotonic with blood. Aqueous solutions should be appropriately buffered if necessary (preferably to a pH of 3 to 9). Suitable parenteral formulations are readily prepared under sterile conditions by standard pharmaceutical techniques well known to those skilled in the art.

The compounds of the present invention may be administered intranasally or by inhalation, as indicated, and are administered using solvents such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkane (e.g., 1, In the case of suitable propellants such as 1,1,2-tetrafluoroethane (HFA134AT) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA)), carbon dioxide or other suitable gases, self-adding Pressurized containers, pumps, sprays or nebulizers are conveniently delivered in the form of dry powder inhalers or aerosol spray delivery. In the case of pressurized aerosols, the dosage unit may be determined by providing a valve to deliver the dose. Pressurized containers, pumps, sprays or atomizers may contain a solution or suspension of the active compound, for example using a mixture of ethanol and propellant as solvent, which may additionally contain a lubricant, such as sorbitan trioleate. Capsules and cartridges (eg, made from gelatin) for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base (eg, lactose or starch).

Alternatively, the compounds of the present invention may be administered in the form of a suppository or pessary, or they may be administered topically in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting. The compounds of the present invention may also be administered transdermally or transdermally, for example, through the use of transdermal patches.

It may also be administered via the pulmonary or rectal route. It can also be administered via the eye route. For ocular use, the compounds may be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline, with a preservative as appropriate. (e.g. benzalkonium chloride) combination. Alternatively, it can be formulated in an ointment such as paraffin grease.

For topical application to the skin, the compounds of the invention may be formulated as a suitable ointment containing the active compound suspended or dissolved in a mixture such as one or more of: mineral oil, liquid paraffin, white petrolatum, propylene glycol, emulsifying wax And water. Alternatively, it may be formulated as a suitable lotion or cream, suspended or dissolved in a mixture of, for example, one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, Polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Generally, the physician will determine the actual dosage that is most appropriate for the individual individual. The specific dosage and dosage frequency for any particular individual may vary and will depend on a variety of factors, including the activity of the particular compound employed, the metabolic stability and length of action of the compound, age, weight, general health, sex, diet , mode and timing of administration, rate of excretion, drug combinations, severity of the specific condition and individuals undergoing treatment.

Recommended dosages of the compounds of the invention for administration to humans (approximately 70 kg body weight) are 0.1 mg to 3 g, 0.1 mg to 2 g, 0.1 mg to 1 g, preferably 1 mg to 500 mg of active ingredient per unit dose. Unit doses may be administered, for example, from 1 to 4 times per day. Dosage will depend on the route of administration. It should be understood that routine changes in dosage may be necessary depending on the age and weight of the patient and the severity of the condition to be treated. The precise dosage and route of administration will ultimately be determined by the attending physician or veterinarian.

The compounds of the invention may also be used in combination with other therapeutic agents. When a compound of the present invention is used in combination with a second therapeutic agent active against the same disease, the dosage of each compound may be different than the dosage of that compound alone.

The combinations mentioned above can be conveniently presented and used in the form of pharmaceutical preparations. The individual components of these combinations may be administered sequentially or simultaneously in separate or combined pharmaceutical formulations by any convenient route. When administered sequentially, the compound of the invention or the second therapeutic agent can be administered first. When administered simultaneously, the combination may be administered as the same or different pharmaceutical compositions. When combined in the same formulation, it is understood that the two compounds must be stable and compatible with each other and with the other components of the formulation. When formulated separately, they may be provided in any convenient formulation, conveniently, in a manner such as is known in the art for such compounds.

Pharmaceutical compositions of the present invention may be produced in a manner known per se to those skilled in the art, as set forth, for example, in Remington's Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., New Jersey (1975).

Diseases or conditions that may be treated, ameliorated, or prevented using the compounds of the invention are those associated with Tau protein aggregates, such as neurodegenerative disorders. Examples of diseases and conditions that can be treated, ameliorated or prevented are caused by or related to the formation of nerve fiber lesions. This is the main brain pathology of tauopathies. Such diseases and conditions include a heterogeneous population of neurodegenerative diseases or conditions, including those showing the coexistence of Tau and amyloid pathology.

Examples of diseases and conditions that can be treated, ameliorated or prevented include (but are not limited to) Alzheimer's disease (AD), familial AD, PART (primary age-related tauopathy), Creutzfeldt-Jakob disease, Boxer dementia, Down syndrome, Gertzmann-Steusler-Steinke disease (GSS), inclusion body myositis, prion cerebral amyloid vasculopathy, traumatic brain injury (TBI), Amyotrophic lateral sclerosis (ALS), Guam Parkinson's disease-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic granulopathy, corticobasal degeneration (CBD), diffuse Neurofibrillary tangles with calcification, frontotemporal dementia with Parkinson's disease associated with chromosome 17 (FTDP-17), Hallerwarden-Spatz disease, multiple system atrophy (MSA), Niemann type C Pick's disease, pallidal-pontine-nigra degeneration, Pick's disease (PiD), progressive subcortical gliomatosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-predominant form Dementia, postencephalitic parkinsonism, myotonic dystrophy, subacute sclerosing panencephalopathy, LRRK2 mutations, chronic traumatic encephalopathy (CTE), familial British dementia, familial Danish form Dementia, other frontotemporal degenerations, Guadeloupe Parkinson's disease, neurodegeneration with iron accumulation in the brain, SLC9A6-related mental retardation, white matter tauopathy with glial inclusions, epilepsy, Lewy bodies Dementia (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes, senile cardiac amyloidosis, glaucoma, ischemic stroke, Psychosis and Huntington's disease in AD. Preferably, diseases and conditions that can be treated, improved or prevented include Alzheimer's disease (AD), and other neurodegenerative tauopathies, such as Creutzfeldt-Jakob disease, Boxer's dementia, muscular dystrophy Lateral sclerosis (ALS), argyrophilic granulopathy, corticobasal degeneration (CBD), frontotemporal dementia with Parkinson's disease associated with chromosome 17 (FTDP-17), Pick's disease (PiD) ), progressive supranuclear palsy (PSP), tangle-dominant dementia, Guam Parkinson's disease dementia complex, Hallwarden-Spatz disease, chronic traumatic encephalopathy (CTE), trauma brain injury (TBI) and other frontotemporal lobar degeneration. More preferably, Alzheimer's disease (AD), corticobasal degeneration (CBD), Pick's disease (PiD) and progressive supranuclear palsy (PSP).

The compounds of the invention may also be used to reduce protein aggregation, in particular Tau aggregation. The ability of a compound to reduce Tau aggregation can be determined, for example, using a ThT assay (Hudson et al., FEBS J., 2009, 5960-72).

The compounds of the present invention can be used to treat a variety of disorders in which neuroinflammatory processes are associated with misfolding and/or pathological aggregation of Tau protein.

The compounds of the invention can be used as analytical references or in vitro screening tools for characterizing tissues with Tau pathology and for testing compounds targeting Tau pathology on such tissues.

The compounds of the present invention can be used as photoprobes for cross-linking compounds to targets, and in in vitro screening assays to identify and characterize the mode of action of compounds, including localizing binding sites. Some examples of photoprobes for the compounds of the present invention are reported below:

The compounds of the invention may also be provided in the form of mixtures with at least one other biologically active compound and/or pharmaceutically acceptable carriers and/or diluents and/or excipients. These compounds and/or other biologically active compounds are preferably present in therapeutically effective amounts.

The nature of the other biologically active compounds will depend on the intended use of the mixture. Other biologically active substances or compounds may exert their biological effects through the same or similar mechanism as the compounds of the present invention, or through an unrelated mechanism of action, or through multiple related and/or unrelated mechanisms of action.

In general, other bioactive compounds may include neurotransmission enhancers, psychotherapeutic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine sedatives, acetylcholine synthesis, Storage or release enhancer, acetylcholine postsynaptic receptor agonist, monoamine oxidase-A or monoamine oxidase-B inhibitor, N-methyl-D-aspartate glutamate receptor antagonist, non- Steroid anti-inflammatory drugs, antioxidants and serotonergic receptor antagonists. In particular, other bioactive compounds may be selected from the group consisting of: compounds useful in treating amyloidosis; compounds that resist oxidative stress; anti-apoptotic compounds; metal chelators; DNA repair inhibitors, such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS); α-secretase activator; β-secretase and γ-secretase inhibitors Agent; glycogen synthase kinase 3 inhibitor; O-linked N-acetylglucosamine hydrolase (OGA) inhibitor; neurotransmitter; beta pleated plate disruptor; amyloid beta scavenging/eliminating cellular component attractant Agents; inhibitors of N-terminally truncated starch-like β (including pyroglutamic acid-like starch β 3-42); anti-inflammatory molecules, or cholinesterase inhibitors (ChEI), such as tacnin, rivan Stimamine, donepezil and/or galantamine; M1 agonists; other drugs, including any starch-like or Tau-modified drugs and nutritional supplements; antibodies, including any functionally equivalent antibodies or functional parts thereof; or vaccine.

In another embodiment, the mixture of the invention may comprise niacin or memantine together with a compound of the invention and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment of the invention, there is provided a mixture comprising, for example, for the treatment of positive and negative psychotic symptoms, including hallucinations, delusions, thought disorders manifested by marked incoherence, derailment, and speech disturbances. tangentiality) and bizarre or deconstructive behavior, as well as amusia, flattened affect, apathy, and social withdrawal with clozapine, ziprasidone, risperidone, and Aripiprazole or olanzapine as another biologically active compound "atypical antipsychotic", as well as the compound of the present invention and optionally pharmaceutically acceptable carriers and/or diluents and/or Excipients.

Other compounds which may suitably be combined with the compounds of the invention for use in mixtures are described, for example, in WO 2004/058258 (see in particular pages 16 and 17), including therapeutic drug targets (pages 36 to 39), Alkanesulfonic acids and alkanol sulfates (pages 39 to 51), cholinesterase inhibitors (pages 51 to 56), NMDA receptor antagonists (pages 56 to 58), estrogens (pages 58 to 59 ), nonsteroidal anti-inflammatory drugs (pages 60 and 61), antioxidants (pages 61 and 62), peroxisome proliferator-activated receptor (PPAR) agonists (pages 63 to 67), cholesterol-lowering agents (pages 63 to 67) Pages 68 to 75), amyloid inhibitors (pages 75 to 77), inhibitors of amyloid formation (pages 77 to 78), metal chelators (pages 78 and 79), antipsychotics and antidepressants ( pages 80 to 82), nutritional supplements (pages 83 to 89) and compounds that increase the availability of bioactive substances in the brain (see pages 89 to 93) and prodrugs (pages 93 and 94), the document is Incorporated herein by reference.

The invention also includes all suitable isotopic variations of the compounds of the invention. Isotopic variations of the compounds of the present invention are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from that usually or found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, respectively. ¹⁸ O, ³⁵ S, ¹⁸ F and ³⁶ Cl. Certain isotopic variations of the present invention, such as those in which radioactive isotopes (eg, ³ H or ¹⁴ C) are incorporated, may be used in drug and/or substrate tissue distribution studies. Isotopes containing tritium (i.e. ³ H) and carbon-14 (i.e. ¹⁴ C) are particularly preferred because of their ease of preparation and detectability. ¹⁸ F-labeled compounds are particularly suitable for imaging applications such as PET. In addition, substitution with isotopes such as deuterium (ie, ² H) may provide certain therapeutic advantages, such as increased half-life in vivo or reduced dosage requirements, due to greater metabolic stability, and may therefore be preferred in some circumstances. Isotopic variations of the compounds of the present invention may generally be prepared by conventional procedures, for example by the illustrative methods or by preparation as set forth in the Examples and Preparations below, using appropriate isotopic variations of suitable reagents.

Compounds of the invention may be synthesized by one of the general methods shown in the following schemes. These methods are given for illustrative purposes only and should not be construed as limiting.

General synthetic scheme for preparing the building blocks of the invention:

plan 1

Heating commercially available phenylhydrazine derivatives ( Z = H, F, Cl, Me or OMe; R ^a = H, CH ₃ ) and commercially available 4-side oxyhexahydropyridine-1 in a suitable solvent under acidic conditions -Tertiary butyl formate (Fischer-Indole synthesis), which after purification gives tricyclic 2,3,4,5-tetrahydro- 1H -pyrido[4,3- b ] Indole building block. In the case of using 2- or 3-substituted phenylhydrazine derivatives, the regioisomers are separated by supercritical fluid chromatography (SFC). The tricyclic building block bearing the NH-moiety on the indole ring was further treated with _Boc2O to selectively protect the aliphatic secondary amine moiety and was obtained after purification.

Scenario 2 To avoid the formation of regioisomers resulting from the use of 2- or 3-substituted phenylhydrazine derivatives, the corresponding phenylhydrazine derivatives with additional halogen atoms (Br, Cl) adjacent to the hydrazine moiety are used. Therefore, Fisher's indole synthesis using commercially available tert-butyl 4-pendantoxyhexahydropyridine-1-carboxylate in a suitable solvent under acidic conditions yields only a single product with an additional halogen atom after purification. The aliphatic secondary amine was Boc protected and the product was obtained after purification. The additional halogen atoms are then removed by hydrogenation with a suitable base in an appropriate solvent using a palladium catalyst to obtain the desired tricyclic 2,3,4,5-tetrahydro-1 H -pyrido[4,3 - b ] indole building blocks.

Option 3 The NH-portion of the indole moiety is then treated with methyl iodide or tosyl chloride using a suitable base in an appropriate solvent to obtain an N-methyl or N-toluenesulfonyl derivative after purification. The Boc-protecting group is removed by acid treatment in an appropriate solvent to obtain the desired tricyclic 2,3,4,5-tetrahydro- 1H -pyrido[4,3- b ]indole building block after purification . In the absence of alkali treatment, the corresponding salt is obtained.

Option 4 Commercially available fluoropyridine derivatives having an F atom at position 3, 4 or 5 and an additional halogen atom (Br, Cl) at position 2 are treated with methylhydrazine in an appropriate solvent to obtain the corresponding N- Methylhydrazine derivatives. Fisher indole synthesis using commercially available tert-butyl 4-pentanoxyhexahydropyridine-1-carboxylate in a suitable solvent under acidic conditions gave the desired tricyclic 9-methyl-6,7 after purification. ,8,9-tetrahydro- 5H -pyrrolo[2,3- b :4,5- c ']dipyridine derivatives.

Option 5 The commercially available phenylhydrazine derivative ( Z = F) and the commercially available 3-pentanoxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl are heated in a suitable solvent under acidic conditions. ester (Fisher indole synthesis), and after purification, tricyclic 5,6,7,8,9,10-hexahydro-7,10-cycloiminocycloheptyl [ b ] indole derivatives were obtained. In the case of 2- or 3-substituted phenylhydrazine derivatives, the regioisomers must be separated by supercritical fluid chromatography (SFC). The aliphatic secondary amine moiety is then Boc protected and the product is obtained after purification. The NH-portion of the indole moiety is then treated with toluenesulfonyl chloride using a suitable base in an appropriate solvent to obtain the N-toluenesulfonyl derivative after purification. The Boc-protecting group is removed by acid treatment in an appropriate solvent to obtain the desired tricyclic 5,6,7,8,9,10-hexahydro-7,10-cycloiminocycloheptane after purification [ b ]Indole building block. In the absence of alkali treatment, the corresponding salt is obtained.

Option 6 Use N-hydroxyphthalimide, copper(I) chloride and pyridine to treat commercially available phenyl groups in appropriate solvents The acid ester derivative (Z = F) is purified to obtain the corresponding hydroxylamine derivative containing a phthalimine protecting group. The phthalimine protecting group is cleaved using hydrazine hydrate and the corresponding hydroxylamine derivative is obtained in salt form. The hydroxylamine derivative and commercially available 1,4-dioxa-8-azaspiro[4.5]decane (Fisher Indole Synthesis) were heated in a suitable solvent under acidic conditions to obtain tricyclic 1, 2,3,4-Tetrahydrobenzofuro[3,2- c ]pyridine derivatives.

Option 7 Commercially available benzo[ d ]thiazole (G = Ph) or benzo[ d ]oxazole (G = Ph) derivatives. The leaving group In the case of less reactive amines, the desired benzo[ d ]thiazole or benzo[ d ]oxazole derivatives are obtained by performing a nucleophilic substitution reaction under microwave conditions. The corresponding thiazolo[5,4- b ]pyridines (G = Py) and thiazolo[4,5- b ]pyridines containing two halogen (Br, Cl) atoms were treated with morpholine in an appropriate solvent and with an additional base. (G = Py) derivatives to obtain the corresponding morpholino-thiazolo[5,4- b ]pyridine (G = Py) and morpholino-thiazolo[4,5- b ]pyridine (G = Py) derivatives. The corresponding thiazolo[5,4- d ]pyrimidine (G = pyrimidine) derivative containing two halogen (Br, Cl) atoms is treated with morpholine in an appropriate solvent and with an additional base to obtain the corresponding morpholine after purification Gyl-thiazolo[5,4- d ]pyrimidine (G = pyrimidine) derivatives.

Option 8 Commercially available [1,2,4]triazolo[1,5- a ]pyridine derivatives containing two halogen (Br, Cl) atoms were treated with morpholine under microwave conditions to obtain nucleophilic substitution after purification product.

Option 8a Utilizing morpholine, 3-oxa-8-azabicyclo[3.2.1]octane or 8-oxa-3-azabicyclo[3.2.1]octane under microwave conditions containing two bromines Atomized commercially available 3,6-dibromopyridazine and 2,5-dibromopyrazine derivatives to obtain nucleophilic substitution products after purification.

Option 8b Using morpholine to treat commercially available 2,7-dichloroquinoline, 2-bromo-6-chloro-1,7-naphthyridine, and 2-bromo-6-containing two halogen (Br, Cl) atoms under microwave conditions Chloro-1,8-naphthyridine and 2,6-dichloroquinoline derivatives to obtain nucleophilic substitution products after purification.

General synthetic scheme for preparing compounds of the invention:

Option 9 The tricyclic building blocks with B= _NCH3 or B=O are reacted with amino-substituted benzo[ d ]thiazole or benzo[ d ]oxazole derivatives or substituted pyridine derivatives via palladium chemistry using appropriate palladium contacts. (Chloro-(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II) )-Methyl-tert-butyl ether adduct; Pd(RuPhos) G1), ligand (2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl; RuPhos) and base (Lithium bis(trimethylsilyl)amide; LiHMDS) is coupled in a suitable solvent (tetrahydrofuran; THF) to obtain the desired compound of formula (I) after purification.

Option 10 The tricyclic building block containing N-toluenesulfonyl group at the indole/azaindole moiety and the amino-substituted benzo[ d ]thiazole, benzo[ d ]oxazole, thiazolo[5,4- b ]pyridine (G = Py), thiazolo[4,5- b ]pyridine (G = Py) derivatives, thiazolo[5,4- d ]pyrimidine (G = pyrimidine) or amino-substituted [1, 2,4]Triazolo[1,5- a ]pyridine derivatives or amino-substituted pyridine (L = Py), pyrazine (L = pyrazine), pyridazine (L = pyridazine), pyrimidine (L =pyrimidine) derivatives or amino-substituted isoquinoline, naphthyridine, quinazoline derivatives via palladium chemistry using a suitable palladium catalyst (see (dibenzylideneacetone) dipalladium (0); Pd ₂ (dba) ₃ ), ligand (2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl; RuPhos) and base (sodium tert-butoxide; NaOtBu) in a suitable solvent (1,4- Dioxane) coupling to obtain the desired compound of formula (I) after purification. Alternatively, benzo[ d ]thiazole, benzo[ d ]oxazole, thiazolo[5, 4- b ]pyridine (G = Py), thiazolo[4,5- b ]pyridine (G = Py) derivatives, thiazolo[5,4- d ]pyrimidine (G = pyrimidine) or amino-substituted [ 1,2,4]Triazolo[1,5- a ]pyridine derivatives or amino-substituted pyridine (L = Py), pyrazine (L =pyrazine), pyridazine (L =pyridazine), pyrimidine (L = pyrimidine) derivatives or amino-substituted isoquinoline, naphthyridine, quinazoline derivatives via palladium chemistry using a suitable palladium catalyst (see (dibenzylideneacetone) dipalladium (0); Pd ₂ ( dba) ₃ ), ligand (2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl; RuPhos) and weak base (cesium carbonate; Cs ₂ CO ₃ ) in a suitable solvent (1, 4-dioxane) to obtain the compound protected by N-toluenesulfonyl group after purification. The tosyl protecting group is then removed using a suitable base (cesium carbonate; Cs ₂ CO ₃ ) in a suitable solvent (2-methylTHF, methanol) at elevated temperature (reflux) to obtain the desired product after purification Compounds of formula (I).

Plan 11 Using a suitable base (potassium carbonate; K ₂ CO ₃ ) in a suitable solvent (DMF), a tricyclic building block containing an N-toluenesulfonyl group at the indole moiety is combined with di-halogenated benzo[ d ]thiazole and di- Halogenated benzo[ d ]oxazoles are coupled to give nucleophilic displacement products. The product is then subjected to palladium chemistry with a suitable palladium catalyst (see (dibenzylideneacetone) dipalladium (0); Pd ₂ (dba) ₃ ), ligand (2-dicyclohexylphosphino-2′,6′ - Diisopropoxybiphenyl; RuPhos) and a base (sodium tert-butoxide; NaOtBu) are coupled in a suitable solvent (1,4-dioxane) to obtain the desired compound of formula (I) after purification .

General synthetic scheme for preparing tritium-labeled compounds of the invention:

Plan 12 Catalyst is added to the tritium reaction vessel, followed by a solution of the compound of the invention in dichloromethane. Attach the container to the tritium line and pressurize the tritium gas at -200°C. The solution was stirred at room temperature for 8 hours, cooled to -200°C and excess gas removed. The reaction flask was rinsed with methanol and the combined organic phases were evaporated under vacuum. The crude material was purified by HPLC, the mobile phase was evaporated under vacuum and the product was redissolved in absolute ethanol. Specific activity was determined by mass spectrometry. Depolymerization of Tau K18 and full-length (fl) Tau can be measured using any suitable assay known in the art. Describes standard in vitro assays for measuring disaggregation capacity.

Examples All reagents and solvents were obtained from commercial sources and used without further purification. ¹ H NMR spectra were recorded on Bruker AV 300 and 400 MHz spectrometers in deuterated solvents. Chemical shifts (δ) are reported in parts per million and coupling constants (J values) are reported in Hertz. Spin multiplicity is indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet). Mass spectra were acquired on an Agilent 1290 Infinity II spectrometer with a 6130 Chemstation and an Agilent 1200 Infinity II spectrometer with a 6130 Chemstation. GC-MS data were collected using an Agilent 7890B gas chromatograph and 5977B mass spectrometer. Infrared spectra were obtained on a PerkinElmer spectrometer. Chromatography was performed using silica gel (Fluka: Silica gel 60, 0.063-0.2 mm) and appropriate solvents as indicated in the specific examples. Flash purification was performed using a Biotage Isolera (Biotage) with an HP-Sil or KP-NH SNAP column and a solvent gradient as indicated in the specific examples. Thin layer chromatography (TLC) was performed on silica plates using UV detection.

Preparation Example 1 Step A 4-Fluorophenylhydrazine (1 g, 7.9 mmol) and 4-pentyloxyhexahydropyridine-1-carboxylic acid tert-butyl ester (1.2 g, 8.3 mmol) were added to 1,4-di To a solution in oxane (10 mL) was added concentrated H ₂ SO ₄ (1 mL). The reaction mixture was then heated at 110 °C for 3 h. The reaction mixture was cooled to room temperature and the precipitate was filtered off. The solid was dissolved in water, basified with NaOH solution and extracted with DCM (dichloromethane). _The organic phase was separated and dried over _Na2SO4 and the solvent was removed to give the title compound as a pale yellow solid (950 mg, 59%). MS: 191 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.91 (s, 1H), 7.23-7.24 (m, 1H), 7.09-7.09 (m, 1H), 6.80-6.81 (m, 1H), 3.91 (s, 2H), 3.11 (t, J = 5.56 Hz, 2H), 2.75 (d, J = 4.96 Hz, 2H). Step B To a solution of the title compound from Step A above (0.95 g, 4.77 mmol) in THF (tetrahydrofuran) was added di-tert-butyl dicarbonate (Boc ₂ O) (1.5 g) and the mixture was stirred Stay overnight. After the reaction was complete, as confirmed by TLC, the solvent was removed and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (10/80 => 80/20), The title compound (1.1 g, 78%) was obtained as a pale yellow viscous liquid. MS: 291 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.00 (s, 1H), 7.26 (q, J = 4.52 Hz, 1H), 7.18 (t, J = 8.12 Hz, 1H), 6.83-6.83 ( m, 1H), 4.49 (s, 2H), 3.69 (t, J = 5.64 Hz, 2H), 2.76 (s, 2H), 1.43 (s, 9H). Step C To a solution of the title compound from Step B above (0.41 g, 1.41 mmol) in THF (5 mL) was added sodium hydride (0.15 g, 6.25 mmol), followed by p-toluenesulfonyl chloride (TsCl) (0.29 g, 1.45 mmol). The reaction mixture was stirred for 10 min. The mixture was dissolved in EtOAc ₍ 20 ml) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (20/80 => 80/20) to obtain the title compound (0.45 g, 72%). MS: 445 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.03-8.04 (m, 1H), 7.77 (d, J = 8.20 Hz, 2H), 7.36-7.38 (m, 3H), 7.15-7.16 (m , 1H), 4.43 (s, 2H), 3.69 (t, J = 5.64 Hz, 2H), 3.08 (s, 2H), 2.32 (s, 3H), 1.43 (s, 9H). Step D To a solution of the title compound from Step C above (0.42 g, 0.915 mmol) in dichloromethane was added 2N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (0.2 g, 58%). MS: 345 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.61 (s, 1H), 8.01-8.02 (m, 1H), 7.82 (d, J = 8.40 Hz, 2H), 7.45-7.45 (m, 1H ), 7.39 (d, J = 8.40 Hz, 2H), 7.20-7.21 (m, 1H), 4.25 (s, 2H), 3.49 (s, 2H), 3.35 (d, J = 3.60 Hz, 2H), 2.34 (s, 3H). Step E To a solution of the title compound from Step D above (5.0 g, 13 mmol) in dichloromethane (50 mL) was added triethylamine (5 mL) and stirred for 10 min. The reaction mixture was diluted with dichloromethane (20 mL), washed with water (2 × 30 mL) and saturated NaCl solution (30 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to afford the title compound as the free base (quantitative yield).

Preparation Example 2 Step A To a solution of the title compound from Preparative Example 1, Step B (0.41 g, 1.41 mmol) in THF (5 mL) was added sodium hydride (0.067 g, 2.82 mmol) and the suspension was stirred at room temperature for 20 min. The reaction mixture was cooled again to 0°C and methyl iodide (0.24 g, 1.45 mmol) was added. The reaction mixture was stirred for 3 h. The reaction mixture was dissolved in EtOAc (15 mL) and washed with water and brine and _dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (20/80 => 80/20) to obtain the title compound (0.3 g, 70%). MS: 304 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.38-7.39 (m, 1H), 7.22 (dd, J = 2.40, 10.00 Hz, 1H), 6.90-6.91 (m, 1H), 4.49 (s , 2H), 3.71 (t, J = 6.00 Hz, 2H), 3.62 (s, 3H), 2.79 (t, J = 5.20 Hz, 2H), 1.40 (s, 9H) . Step B To a solution of the title compound from Step A above (0.3 mg, 0.986 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred overnight. The reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (0.12 g, 60%). MS: 205.08 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.45 (br, 1H), 7.45-7.46 (m, 1H), 7.30-7.31 (m, 1H), 6.98-7.00 (m, 1H), 4.26 (s, 2H), 3.66 (s, 3H), 3.49 (d, J = 4.80 Hz, 2H), 3.06 (t, J = 6.00 Hz, 2H), 2.50 (s, 9H).

Preparation Example 3 Step A : 1-(4-fluorophenyl)-1-methylhydrazine (2 g, 14.0 mmol) and 4-side oxyhexahydropyridine-1-carboxylic acid tert-butyl ester (2.84 g, 14.0 mmol) in 1,4-dioxane (10 mL) was added concentrated H ₂ SO ₄ (1 mL). The reaction mixture was then heated at 110°C overnight. The reaction mixture was cooled to room temperature and the precipitate was filtered off. Discard the filtrate. The solid was dissolved in water (10 mL), the pH was adjusted to 14 using NaOH solution, and the mixture was extracted with dichloromethane (150 mL). The organic phase was washed with water and brine and _dried over _Na2SO4 . The solvent was removed to give the title compound as the free base (1.3 g, 46%). MS: 205.08 (M+H) ⁺ .

Preparation Example 4 Step A : Add (4-methoxyphenyl)hydrazine (1 g, 5.6 mmol) and 4-side oxyhexahydropyridine-1-carboxylic acid tert-butyl ester (1.13 g, 5.6 mmol) at ice bath temperature. To a solution in 1,4-dioxane (10 mL) was added _{concentrated H2SO4} (1 mL). The reaction mixture was then heated at 110 °C for 3 h. The reaction mixture was allowed to cool to room temperature and the precipitate was filtered off. The solid was dissolved in water, basified with NaOH solution and extracted with dichloromethane. The organic phase was separated and dried over _Na2SO4 , filtered and the solvent removed to give the title compound (0.60 g, 53%) as _a pale yellow viscous liquid. The crude product was used as such in the next step. MS: 203 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (0.60 mg, 2.9 mmol) in THF was added di-tert-butyl dicarbonate (0.65 g, 2.9 mmol) and the mixture was stirred overnight. The solvent was removed and the crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (80/20) to give the title compound as a pale yellow solid (0.55 g, 62%). MS: 303 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.71 (bs, 1H), 7.17 (d, J = 8.40 Hz, 1H), 6.89 (s, 1H), 6.65-6.66 (m, 1H), 4.49 (s, 2H), 3.75 (s, 3H), 3.69 (t, J = 5.60 Hz, 2H), 2.74 (t, J = 5.60 Hz, 2H), 1.44 (s, 9H). Step C To a solution of the title compound from Step B above (0.55 g, 1.8 mmol) in THF (5 mL) was added sodium hydride (0.087 g, 3.6 mmol) followed by p-toluenesulfonyl chloride (0.342 g, 1.8 mmol). The reaction mixture was stirred for 45 minutes. After the reaction was complete, the reaction mixture was dissolved in EtOAc ₍ 200 mL) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system and an EtOAc/hexane gradient (20/80) to obtain the title compound (0.45 g, 45%) as an off-white solid. MS: 457 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.92 (d, J = 9.20 Hz, 1H), 7.71 (d, J = 8.40 Hz, 2H), 7.35 (d, J = 8.00 Hz, 2H) , 7.01 (s, 1H), 6.91-6.92 (m, 1H), 4.42 (s, 2H), 3.77 (s, 3H), 3.68 (t, J = 5.60 Hz, 2H), 3.05 (bs, 2H), 2.31 (s, 3H), 1.43 (s, 9H). Step D To a solution of the title compound from Step C above (0.450 g, 0.986 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to obtain the compound as an off-white solid (0.23 g, 65%). MS: 357 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.67 (bs, 1H), 7.89 (d, J = 9.20 Hz, 1H), 7.77 (d, J = 8.40 Hz, 2H), 7.36 (d, J = 8.00 Hz, 2H), 7.11 (d, J = 2.40 Hz, 1H), 6.93-6.94 (m, 1H), 4.24 (s, 2H), 3.77 (s, 3H), 3.48-3.49 (m, 2H ), 3.34-3.35 (m, 2H), 2.33 (s, 3H).

Preparation Example 5 Step A To a solution of the title compound from Preparative Example 4, Step B (0.55 g, 1.8 mmol) in THF (5 mL) was added sodium hydride (0.087 g, 3.6 mmol) at 0°C. The suspension was stirred at room temperature for 20 minutes. The reaction mixture was cooled again to 0°C and iodomethane (0.255 g, 1.8 mmol) was added. The reaction mixture was stirred for 45 minutes. After the reaction was complete, the reaction mixture was dissolved in EtOAc ₍ 20 ml) and washed with water and brine and dried over _Na2SO4 , filtered and concentrated. The crude mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (80/20) to obtain the title compound (0.40 g, 45%) as a light brown solid. MS: 317 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.29 (d, J = 8.80 Hz, 2H), 6.93 (s, 1H), 6.72-6.73 (m, 1H), 4.50 (s, 2H), 3.76 (s, 3H), 3.70-3.72 (m, 2H), 3.59 (s, 3H), 2.78-2.79 (m, 2H), 1.44 (s, 9H). Step B To a solution of the title compound from Step A above (0.4 g, 1.26 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as a white solid (0.2 g, 73%). MS: 217 (M+H) ⁺ .

Preparation Example 6 Step A : Irradiate a mixture of 2-chloro-5-fluoropyridine (10 g, 76.34 mmol) and N- methylhydrazine (6 mL) in a microwave oven at 180°C for 1 h. After the reaction was completed, the reaction mixture was allowed to cool and poured into ice-cold water and extracted with dichloromethane (2 × 50 mL). The organic phase was separated and dried over _Na2SO4 and the solvent was removed to give the title compound (10 g, crude) _as a light brown solid. The crude product was used as such in the next step. MS: 143 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (10 g, 70.84 mmol) in methanol (100 mL) was added tert-butyl 4-pentanoxyhexahydropyridine-1-carboxylate (16.9 g, 85 mmol) and stir overnight. The solvent was removed, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using a methanol/DCM gradient (1/99) to obtain the title compound (5 g, 22%) as a brown viscous oil. . MS: 323 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.82-7.84 (m, 1H), 7.13-7.14 (m, 1H), 6.72-6.73 (m, 1H), 4.51 (bs, 2H), 3.77 (s, 3H), 3.71-3.73 (m, 2H), 2.76-2.78 (m, 2H), 1.43 (s, 9H). Step C A mixture of the title compound from Step B above (5 g, 15.50 mmol) and diethylene glycol (5 mL) was irradiated in a microwave oven at 180°C for 1 h. After the reaction was completed, the reaction mixture was allowed to cool and poured into ice-cold water and extracted with dichloromethane (2 × 50 mL). The organic phase was separated and dried over Na ₂ SO ₄ and the solvent was removed by preparative HPLC (column: Phenomenex Gemini C18 (150*4.6) mm, 3.0 μm. Mobile phase A: 10 mM acetic acid in Milli-Q water Ammonium. Mobile phase B: acetonitrile. Flow rate: 1.0 ml\min). The crude mixture was purified to obtain the title compound (0.9 g, 29%) as a brown solid. MS: 206 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.87-7.89 (m, 1H), 7.46-7.47 (m, 1H), 4.53 (bs, 2H), 3.77 (s, 3H), 3.71-3.73 (m, 2H), 2.76-2.78 (m, 2H).

Preparation Example 7 Step A To 3-(fluorophenyl)hydrazine (1 g, 6.1 mmol) and 4-pentyloxyhexahydropyridine-1-carboxylic acid tert-butyl ester (1.2 g, 6.1 mmol) in 1,4- To a solution in dioxane (10 mL) was added concentrated H ₂ SO ₄ (1 mL). The reaction mixture was then warmed to 25 °C and heated at 110 °C for 3 h. The reaction mixture was cooled to room temperature and the precipitate was filtered off. The solid was dissolved in water, basified with NaOH solution and extracted with dichloromethane. The organic phase was separated and dried over _Na2SO4 and the solvent was removed to give a mixture of regioisomers as _a pale yellow solid (0.65 g, 56%). MS: 191.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.87 (bs, 1H), 7.26-7.30 (m, 1H), 7.02-7.05 (m, 1H), 6.74-6.79 (m, 1H), 3.83 (bs, 2H), 2.99-3.02 (m, 2H), 2.65-2.66 (m, 2H). Step B To a solution of the regioisomer mixture (0.65 g, 3.15 mmol) in THF was added di-tert-butyl dicarbonate (0.757 g, 3.47 mmol), and the mixture was stirred for 12 h. After the reaction was complete (monitored by TLC), the solvent was concentrated under reduced pressure to yield crude product. The crude product was purified by silica gel (60-120 mesh) column chromatography using hexane: EtOAc (70:30) to obtain the regioisomer 7-fluoro-1,3,4,5-tetrakis as a yellow solid. Hydro- 2H -pyrido[4,3- b ]indole-2-carboxylic acid tert-butyl ester and 9-fluoro-1,3,4,5-tetrahydro- 2H -pyrido[4,3 - b ] A mixture of tert-butyl indole-2-carboxylate (0.750 g, 61%) in a ratio of approximately 70:30 respectively. MS: 291.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.01 (bs, 1H), 7.36-7.39 (m, 1H), 7.06-7.09 (m, 1H), 6.79-6.84 (m, 1H), 4.51 (bs, 2H), 3.68-3.71 (m, 2H), 2.74-2.76 (m, 2H), 1.38 (s, 9H). Step C is carried out by SFC chiral column (Chiracel OJ-H; column: X-bridge C8 (50 × 4.6) mm, 3.5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA Acetonitrile) separated the regioisomer mixture (0.750 mg, 70:30) to obtain the second eluted title compound 7-fluoro-1,3,4,5-tetrahydro as a pale yellow solid with 100% chiral purity. -2H -pyrido[4,3- b ]indole-2-carboxylic acid tert-butyl ester (0.4 mg, 53%). First elution of the title compound 9-fluoro-1,3,4,5-tetrahydro- 2H -pyrido[4,3- b ]indole-2-carboxylic acid tert-butyl ester was isolated as having 100% chiral Light yellow solid with high purity (0.25 g, 33%). The title compound was eluted second: MS: 291.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.01 (bs, 1H), 7.36-7.39 (m, 1H), 7.06-7.09 (m, 1H), 6.79-6.84 (m, 1H), 4.51 (bs, 2H), 3.68-3.71 (m, 2H), 2.74-2.77 (m, 2H), 1.44 (s, 9H). RT=2.08 min. First eluate the title compound: MS: 291.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.22 (s, 1H), 7.13 (d, J = 8.08 Hz, 1H), 6.96-6.97 (m, 1H), 6.69-6.71 (m, 1H ), 4.63 (s, 2H), 3.69-3.70 (m, 2H), 2.68-2.76 (m, 2H), 1.44 (s, 9H). RT=1.74 min. Step D To a second solution of the title compound from Step C above (0.4 g, 1.37 mmol) in THF (5 mL) was added sodium hydride (0.099 mg, 4.137 mmol) followed by p-toluenesulfonyl chloride ( 0.288 g, 1.51 mmol). The reaction mixture was stirred for 30 minutes. The mixture was dissolved in EtOAc ₍ 20 ml) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (20/80 => 80/20) to obtain the title compound (0.3 g, 49%). MS: 445 (M+H) ⁺ . ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.92-7.94 (m, 1H), 7.68-7.70 (m, 1H), 7.25-7.29 (m, 4H), 7.00-7.04 (m, 1H), 4.50 (bs, 2H), 3.76 (bs, 2H), 3.12 (bs, 2H), 2.38 (s, 3H), 1.51 (s, 9H). Step E To a solution of the title compound from Step D above (0.3 g, 0.676 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred for 12 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (0.2 g 78%). MS: 345 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.50 (bs, 2H), 7.80-7.87 (m, 2H), 7.78 (d, J = 2.00 Hz, 1H), 7.57-7.61 (m, 1H ), 7.40 (d, J = 8.16 Hz, 2H), 7.18-7.23 (m, 1H), 4.27 (bs, 2H), 3.56 (bs, 2H), 3.47 (bs, 2H), 2.34 (s, 3H) .

Preparation Example 8 Step A : (2-Chloro-3-fluorophenyl)hydrazine (10 g, 62.5 mmol) and 4-pentanoxyhexahydropyridine-1-carboxylic acid tert-butyl ester (12 g, 62.5 mmol) at 0°C ) in 1,4-dioxane (100 mL) was added concentrated H ₂ SO ₄ (10 mL). The reaction mixture was then warmed to 25 °C and heated at 110 °C for 3 h. The reaction mixture was allowed to cool to room temperature and the precipitate was filtered off. The solid was dissolved in water, basified with NaOH solution and extracted with dichloromethane. The organic phase was separated and dried over _Na2SO4 and _the solvent was removed to give the title compound as a pale yellow solid (10 g, 72%). MS: 225 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.23 (bs, 1H), 7.27-7.28 (m, 1H), 6.94-6.96 (m, 1H), 3.82 (s, 2H), 2.98-3.00 (m, 2H), 2.68 (d, J = 4.72 Hz, 2H). Step B To a solution of the title compound from Step A above (10 g, 44.5 mmol) in THF (100 mL) was added di-tert-butyl dicarbonate (10.5 g, 46.5 mmol) and the mixture was stirred for 12 h. After the reaction was complete (monitored by TLC), the solvent was concentrated under reduced pressure to yield crude product. The crude product was purified by silica gel (60-120 mesh) column chromatography to obtain the title compound (12 g, 85%) as a yellow solid. MS: 325.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.43 (s, 1H), 7.36-7.38 (m, 1H), 6.97-7.00 (m, 1H), 4.51 (s, 2H), 3.68-3.69 (m, 2H), 2.76-2.78 (m, 2H), 1.43 (s, 9H). Step C To a solution of the title compound from Step B above (5 g, 15.3 mmol) in anhydrous methanol (50 mL) was added triethylamine (6.74 mL, 46.18 mmol) and 10% Pd/C (0.2 mg, 20 %wt). The hydrogenation was carried out at a pressure of 10 bar for 16 hours. The reaction mixture was filtered through a pad of celite and concentrated in vacuo to give the title compound (4 g, 90%). MS: 291.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.01 (bs, 1H), 7.36-7.39 (m, 1H), 7.06-7.09 (m, 1H), 6.79-6.84 (m, 1H), 4.51 (bs, 2H), 3.68-3.71 (m, 2H), 2.74-2.77 (m, 2H), 1.44 (s, 9H). Step D To a solution of the title compound from Step C above (4 g, 13.7 mmol) in THF (40 mL) was added sodium hydride (9.9 g, 41.23 mmol) followed by p-toluenesulfonyl chloride (2.88 g, 15.1 mmol). The reaction mixture was stirred for 30 min. The mixture was dissolved in EtOAc ₍ 200 ml) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (20/80 => 80/20) to obtain the title compound (5 g, 82%). MS: 445 (M+H) ⁺ . ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.92-7.94 (m, 1H), 7.68-7.70 (m, 1H), 7.25-7.29 (m, 4H), 7.00-7.04 (m, 1H), 4.50 (bs, 2H), 3.76 (bs, 2H), 3.12 (bs, 2H), 2.38 (s, 3H), 1.51 (s, 9H). Step E To a solution of the title compound from Step D above (3 g, 6.76 mmol) in dichloromethane (30 mL) was added 2 N HCl in 1,4-dioxane (15 mL). The reaction mixture was stirred for 12 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (2 g, 78%). MS: 345 (M+H) ⁺ . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ = 9.50 (bs, 2H), 7.80-7.87 (m, 2H), 7.78 (d, J = 2.00 Hz, 1H), 7.57-7.61 (m, 1H) , 7.40 (d, J = 8.16 Hz, 2H), 7.18-7.23 (m, 1H), 4.27 (bs, 2H), 3.56 (bs, 2H), 3.47 (bs, 2H), 2.34 (s, 3H).

Preparation Example 9 Step A To a solution of the title compound from Preparative Example 8, Step C (0.4 mg, 1.37 mmol) in THF (5 mL) was added sodium hydride (0.099 g, 4.137 mmol) followed by methyl iodide (0.102 ml, 1.64 mmol). . The reaction mixture was stirred for 2 h. The mixture was dissolved in EtOAc ₍ 20 ml) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (20/80 => 80/20) to obtain the title compound (0.3 mg, 73%). MS: 305.37 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (0.3 mg, 0.986 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (0.18 g 75%). MS: 205 (M+H) ⁺ .

Preparation Example 10 Step A To the first solution of the title compound (0.2 mg, 0.67 mmol) in THF (5 mL) from Preparative Example 7, Step C, was added sodium hydride (0.048 g, 2.137 mmol) followed by p-toluenesulfonyl chloride. (0.144 g, 0.76 mmol). The reaction mixture was stirred for 30 minutes. The mixture was dissolved in EtOAc ₍ 20 ml) and washed with water and brine and dried over _Na2SO4 . The crude product was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (20/80 => 80/20) to obtain the title compound (0.155 g, 50%). MS: 445 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.80-7.82 (m, 3H), 7.31-7.32 (m, 3H), 7.07-7.09 (m, 1H), 4.56 (s, 2H), 3.68 -3.69 (m, 2H), 3.09 (bs, 2H), 2.33 (s, 3H), 1.43 (s, 9H). Step B To a solution of the title compound from Step A above (0.15 g, 0.337 mmol) in dichloromethane was added 2 N HCl in 1,4-dioxane (5 mL). The reaction mixture was stirred for 12 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound as an off-white solid (0.1 g, 71%). MS: 345 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.49 (bs, 2H), 7.85-7.87 (m, 3H), 7.35-7.36 (m, 3H), 7.12-7.14 (m, 1H), 4.39 (s, 2H), 3.48 (bs, 2H), 3.17 (bs, 2H), 2.35 (s, 3H).

Preparation Example 11 Step A : 2-(4-Fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5g, 22.51 mmol) in dichloroethane (250 mL), add N-hydroxyphthalimide (7.34 g, 45.03 mmol), copper (I) chloride (2.22 g, 22.51 mmol), pyridine (2.75 mL, 33.75 mmol), and molecular sieve (5 g ), and the reaction mixture was heated to 70 °C for 12 h. After the reaction was complete, the reaction mixture was filtered through a bed of celite and washed with ethyl acetate. The ethyl acetate layer was concentrated and the crude product was purified on silica gel using a Biotage Isolera One purification system using a hexane/EtOAc gradient (90/10 => 80/20) to give the title compound as a white solid (1.4 g, 24 %). MS: 258.22 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.92-7.93 (m, 4H), 7.33-7.34 (m, 2H), 7.20-7.22 (m, 2H). Step B To a solution of the title compound from Step A above (1.4 g, 5.44 mmol) in chloroform:methanol (9:1, 100 mL) was added hydrazine hydrate (0.81 g, 16.32 mmol), and the reaction mixture was heated at 25 Stir for 12 h at ℃. After the reaction was complete, the reaction mixture was filtered through a bed of celite and washed with dichloromethane. The dichloromethane layer was concentrated and the crude product was added to diethyl ether (5 mL) and 2 N HCl in diethyl ether (2 mL) at 0 °C, and the reaction mixture was stirred at 25 °C for 30 min. After the reaction was completed, the reaction mixture was filtered and dried under vacuum to obtain an off-white solid (0.44 g, 50%). MS: 128.12 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.03-8.04 (m, 2H), 7.76-7.76 (m, 2H). Step C To the title compound from Step B above (0.44 g, 2.7 mmol) and 1,4-dioxa-8-azaspiro[4.5]decane (0.44 g, 3.07 mmol) were added to 1 , to a solution of 4-dioxane (5 mL) was added concentrated H ₂ SO ₄ (0.5 mL). The reaction mixture was warmed to 25°C and then further heated at 150°C for 1 h under microwave conditions. The reaction mixture was cooled to 25°C and the precipitate was filtered off. The solid was dissolved in water, basified with NaOH solution and extracted with dichloromethane. The organic phase was separated and dried over _Na2SO4 , and _the solvent was concentrated and the crude product was used unchanged in the next step (0.270 g, 52%). MS: 192.21 (M+H) ⁺ .

Preparation Example 12 Step A : Add 4-(fluorophenyl)hydrazine hydrochloride (10 g, 0.061 mol) and 3-side oxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid at 0°C. To a solution of tributyl ester (13.9 g, 0.61 mol) in 1,4-dioxane (100 mL) was added concentrated _H2SO4 (10 mL), and the mixture was then heated to 100 °C for 12 _h . After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed by high vacuum to obtain crude material. The crude material was basified using 30% sodium hydroxide solution and a solid precipitated. The precipitated solid was filtered, washed with water (100 mL) and diethyl ether (100 mL), and the solid was dried under line vacuum for 16 h to obtain the title compound (11 g, 83%) as a light brown solid. MS: 216.10 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (11 g, 0.051 mol) in THF (50 mL) was added triethylamine (11 mL, 0.076 mol) and di-tert-butyl dicarbonate at 0°C. ester (11.13 g, 0.051 mol) and then stirred at room temperature for 12 h. After the reaction was complete, as confirmed by TLC, the solvent was removed and the crude reaction mixture was purified via a silica column using 40% to 50% ethyl acetate in petroleum ether to give the title compound (7.4) as a pale yellow solid. g, 46%). MS: 316.15 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.94 (bs, 1H), 7.23-7.24 (m, 2H), 6.80-6.81 (m, 1H), 5.09-5.11 (m, 1H), 4.46 (bs, 1H), 3.23-3.27 (m, 1H), 2.51-2.57 (m, 1H), 2.22-2.26 (m, 1H), 2.00-2.07 (m, 1H), 1.59-1.60 (m, 1H) , 1.41-1.44 (m, 2H), 1.20-1.27 (m, 9H). Step C To a solution of the title compound from Step B above (3 g, 0.009 mol) in THF (30 mL) was added sodium hydride (60% in mineral oil; 0.57 g, 0.014 mol) portionwise at 0°C. . After the addition was complete, the reaction mixture was stirred at room temperature for 30 minutes, and then the reaction mixture was cooled to 0°C again. To this mixture was added dropwise p-toluenesulfonyl chloride (2.16 g, 0.11 mol) dissolved in THF (20 mL) at 0°C. After the addition was complete, the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete, as confirmed by TLC, the reaction mixture was cooled to 0°C and quenched with ice water, followed by extraction with ethyl acetate (100 mL). The ethyl acetate layer was washed with water (30 mL) and brine solution (30 mL). The organic layer was dried over _Na2SO4 , filtered and evaporated to give the crude product, which was purified by a silica column using 15% to 25% ethyl acetate in petroleum ether to give the title compound (2.9 ₎ as an off-white solid. g, 65%). MS: 470.16 (M+H) ⁺ . Step D To a solution of the title compound from Step C above (2.9 g, 0.006 mol) in dichloromethane (10 mL) was added 4 N HCl in 1,4-dioxane (20 mL) at 0°C. ). The reaction mixture was stirred at ambient temperature for 12 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether (10 mL) to afford the title compound as an off-white solid (2.4 g, 98%). MS: 370.15 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.07-8.08 (m, 1H), 7.78-7.80 (m, 1H), 7.30-7.31 (m, 3H), 7.10-7.11 (m, 1H) , 5.13 (d, J = 4.80 Hz, 1H), 4.52 (d, J = 4.80 Hz, 1H), 3.64-3.65 (m, 4H), 3.30-3.30 (m, 1H), 2.22-2.24 (m, 7H ), 1.92 (bs, 1H).

Preparation Example 13 Step A To a solution of 2,6-dichlorobenzo[d]thiazole (5 g, 24.5 mmol) in dry dichloromethane (50 mL) was added morpholine (3.19 g, 36.6 mmol) and the mixture was cooled to 0℃. To the cold reaction mixture, triethylamine (3.71 g, 36.7 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid, which was triturated with diethyl ether to give the title compound (5 g, 96%). MS: 213.4 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.80 (d, J = 8.00 Hz, 1H), 7.53(d, J = 2.00 Hz, 1H), 7.13-7.14 (m, 1H), 3.74- 3.75 (m, 4H), 3.56-3.57 (m, 4H).

Preparation Example 14 Step A To a solution of 2,5-dichlorobenzo[d]thiazole (5 g, 24.5 mmol) in dry dichloromethane (50 mL) was added morpholine (3.19 g, 36.6 mmol) and the mixture was cooled to 0℃. To the cold reaction mixture, triethylamine (3.71 g, 36.7 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (4.5 g, 86%). MS: 255.4 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.82 (d, J = 8.00 Hz, 1H), 7.50 (d, J = 2.00 Hz, 1H), 7.11-7.11 (m, 1H), 3.72- 3.73 (m, 4H), 3.55-3.56 (m, 4H).

Preparation Example 15 Step A To a solution of 2,6-dichlorobenzo[d]oxazole (5 g, 26.8 mmol) in dry dichloromethane (50 mL) was added morpholine (3.50 g, 40.3 mmol) and the mixture was cooled to 0℃. To the cold reaction mixture, triethylamine (4.0 g, 39.6 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (5 g, 78%). MS: 239.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.59 (d, J = 2.80 Hz, 1H), 7.30 (d, J = 11.20 Hz, 1H), 7.21 (dd, J = 2.80, 11.20 Hz, 1H), 3.71-3.74 (m, 4H), 3.57-3.60 (m, 4H).

Preparation Example 16 Step A To a solution of 2,5-dichlorobenzo[d]oxazole (5 g, 26.8 mmol) in anhydrous dichloromethane (50 mL) was added morpholine (3.50 g, 40.3 mmol) and the mixture was cooled to 0℃. To the cold reaction mixture, triethylamine (4.0 g, 39.6 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (5.2 g, 81%). MS: 239.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.44 (d, J = 8.40 Hz, 1H), 7.36 (d, J = 2.40 Hz, 1H), 7.06 (dd, J = 2.00, 8.40 Hz, 1H), 3.71-3.73 (m, 4H), 3.59-3.61 (m, 4H).

Preparation Example 17 Step A To a solution of 2,6-dichlorobenzo[d]thiazole (5 g, 24.5 mmol) in dry dichloromethane (50 mL) was added 2 M dimethylamine in THF (18.37 mL, 36.65 mol ), and the mixture was cooled to 0°C. To the cold reaction mixture, triethylamine (6.8 mL, 49 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (4.8 g, 94%). MS: 213.4 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.88 (d, J = 2.10 Hz, 1H), 7.41 (d, J = 8.70 Hz, 1H), 7.25-7.26 (m, 1H), 3.14 ( s, 6H).

Preparation Example 18 Step A To a solution of 2,5-dichlorobenzo[d]thiazole (5 g, 24.5 mmol) in dry dichloromethane (50 mL) was added 2 M dimethylamine in THF (18.37 mL, 36.65 mol ), and the mixture was cooled to 0°C. To the cold reaction mixture, triethylamine (6.8 mL, 49 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated to give a white solid which was triturated with diethyl ether to give the title compound (4.5 g, ₈₈ %). MS: 213.4 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.77 (d, J = 11.20 Hz, 1H), 7.46 (d, J = 2.40 Hz, 1H), 7.05-7.05 (m, 1H), 3.14 ( s, 6H).

Preparation Example 19 Step A. To a solution of 6-bromo-2-chlorobenzo[d]thiazole (0.45 g, 1.81 mmol) in ethanol (12 mL) was added 2 M dimethylamine solution (3 mL) and the mixture was microwaved using a Biotage microwave. Heat at 100°C for 60 minutes. The reaction mixture was cooled to room temperature. The solvent was removed, and the crude product was purified on a silica column using an ethyl acetate and heptane gradient (40/60 => 60/40) to obtain the title compound as a solid (0.441 g, 95%). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.70 (d, J = 1.9 Hz, 1H), 7.43 - 7.35 (m, 2H), 3.20 (s, 6H).

Preparation Example 20 Step A To a solution of 2,6-dichlorobenzo[d]oxazole (5 g, 26.6 mmol) in dry dichloromethane (50 mL) was added 2 M dimethylamine in THF (26.6 mL, 53.2 mmol) and the mixture was cooled to 0 °C. To the cold reaction mixture, triethylamine (5.6 mL, 39.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (5 g, 96%). MS: 197.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.56 (s, 1H), 7.23-7.24 (m, 1H), 7.16-7.16 (m, 1H), 3.13 (s, 6H).

Preparation Example 21 Step A To a solution of 2,5-dichlorobenzo[d]oxazole (5 g, 26.6 mmol) in dry dichloromethane (50 mL) was added 2 M dimethylamine in THF (26.6 mL, 53.2 mmol) and the mixture was cooled to 0 °C. To the cold reaction mixture, triethylamine (5.6 mL, 39.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was treated with H ₂ O (2 × 20 mL) and extracted with dichloromethane. The organic layer was separated, dried over _Na2SO4 , filtered and evaporated _to give a white solid which was triturated with diethyl ether to give the title compound (4.9 g, 94%). MS: 197.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.40 (d, J = 8.40 Hz, 1H), 7.30 (d, J = 2.00 Hz, 1H), 6.99-6.99 (m, 1H), 3.13 ( s, 6H).

Preparation Example 22 Step A. To a solution of 6-bromo-2-chlorobenzo[d]thiazole (0.8 g, 3.2 mmol) in ethanol (12 mL) was added isopropylamine solution (1 mL), and the mixture was heated at 100°C using a Biotage microwave oven. Heat for 45 minutes. The reaction mixture was cooled to room temperature. The solvent was removed and the crude product was crystallized from a mixture of EtOAc and n-heptane to give the title compound as a solid (0.663 g, 75.8%). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.69 (t, J = 1.3 Hz, 1H), 7.38 (d, J = 1.3 Hz, 2H), 7.27 (s, 1H), 5.51 - 5.26 (m , 1H), 3.92 (h, J = 6.5 Hz, 1H), 1.33 (d, J = 6.4 Hz, 6H).

Preparation Example 23 Step A. To a solution of 6-bromo-2-chlorobenzo[d]thiazole (1 g, 4.0 mmol) in ethanol (6 mL) was added isopropylamine solution (1.5 mL), and the mixture was heated at 100°C using a Biotage microwave oven. Heat for 90 minutes. The reaction mixture was cooled to room temperature. The solvent was removed and the crude product was dissolved in dichloromethane (150 mL) and washed with 1 M NaOH solution, water and brine and _dried over _Na2SO4 . The solvent was removed under reduced pressure to obtain the crude product, which was purified on a silica column using an EtOAc and heptane gradient (20/80 => 50/50) to obtain the title compound as a solid (0.35 g, 36 %). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.72 (s, 1H), 7.41 (s, 1H), 7.29 (d, J = 3.3 Hz, 1H), 5.40 (s, 1H), 3.13 (s , 3H).

Preparation Example 24 Step A To a solution of 5-bromo-2-chlorobenzo[d]thiazole (0.9 g, 3.62 mmol) in ethanol (12 mL) was added 4 M methylamine solution (1 mL) and the mixture was incubated using a Biotage microwave. Heat at 100°C for 90 minutes. The reaction mixture was cooled to room temperature. The solvent was removed and the crude product was purified on a silica column using an EtOAc and heptane gradient (20/80 => 50/50) to obtain the title compound as a solid (0.57 g, 65%). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.68 (t, J = 2.0 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.22 (dd, J = 8.4, 1.9 Hz, 1H ), 5.90 (s, 1H), 3.13 (s, 3H).

Preparation Example 25 Step A. To a solution of 5-bromo-2-chlorobenzo[d]thiazole (0.87 g, 3.5 mmol) in ethanol (12 mL) was added isopropylamine solution (1.5 mL), and the mixture was heated at 100°C using a Biotage microwave oven. Heat for 60 minutes. The reaction mixture was cooled to room temperature. The solvent was removed and the crude product was dissolved in dichloromethane (150 mL) and washed with 1 M NaOH solution, water and brine and _dried over _Na2SO4 . The solvent was removed under reduced pressure to obtain the crude product, which was purified on a silica column using an EtOAc and heptane gradient (20/80 => 50/50) to obtain the title compound as a solid (0.75 g, 79 %). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.67 (d, J = 1.9 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.19 (dd, J = 8.3, 1.9 Hz, 1H ), 5.47 (s, 1H), 4.04 - 3.79 (m, 1H), 1.34 (d, J = 6.5 Hz, 6H).

Preparation Example 26 Step A : Prepare a solution of 5-bromo-3-iodopyridin-2-amine (5 g, 16.72 mmol) and benzyl isothiocyanate (3.29, 20.07 mmol) in acetone (10 mL) at 60°C. Stir for 12 hours at low temperature and monitor the reaction by TLC. The solvent was evaporated and the solid was filtered, washed with n-hexane (200 mL) and dried to give the title compound as an off-white solid (4 g, 52%). MS: 461.5 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 12.35 (s, 1H), 11.86 (s, 1H), 8.64-8.65 (m, 2H), 7.99-7.99 (m, 2H), 7.67 (s , 1H), 7.56 (d, J = 9.40 Hz, 2H). Step B To a solution of the title compound from Step A above (4 g, 12.1 mmol) in 1,4-dioxane (60 mL) was added potassium carbonate (2.5 g, 18.15 mmol), L-proline ( 0.28 g, 2.43 mmol) and copper(I) iodide (0.462 g, 2.43 mmol). The reaction mixture was then stirred at 80°C for 16 hours and the reaction was monitored by TLC. The reaction mixture was poured into 1.0 L water and 1.0 L saturated aqueous NH ₄ Cl solution. The suspension was stirred at room temperature for 1 hour. The solid was filtered, washed with saturated aqueous NH ₄ Cl (2 × 300 mL) and water (2 × 300 mL) and dried to give the title compound (2.5 g, crude) as an off-white solid. MS: 334.51 (M+H) ⁺ . Step C A suspension of the title compound from Step B above (2 g, 5.98 mmol) in ₇₀ % _H2SO4 (6, 3.0 vol) was heated at 120°C for 2 hours. The reaction mixture was cooled to room temperature and slowly poured into 100 mL of cold water (0°C). The reaction mixture was then adjusted to alkaline pH by adding 50% aqueous NaOH solution. Then, the compound was extracted with EtOAc (6 × 150 mL). The combined organic layers were dried over _Na2SO4 and filtered, and the solution was concentrated to give the title compound as a pale yellow solid (0.3 g _, 23%). MS: 230.4 (M) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.27-8.31 (m, 2H), 8.11 (s, 2H). Step D To a suspension of the title compound from Step C above (0.3 mg, 1.3 mmol) in acetonitrile (5 mL) was added tert-butyl nitrite ( 0.2 ml) using a syringe over a period of 10 min at 0°C. , 1.95 mmol). Then, copper(II) chloride (0.2 g, 1.56 mmol) was added portionwise. After 30 minutes at 0°C, the reaction mixture was warmed to room temperature for 1 hour and heated to 65°C, then stirred for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated and the product was diluted with water (20 mL) and 5% MeOH/DCM (3 × 20 mL). The combined organics were washed with brine (10 mL), dried over _Na2SO4 , filtered and concentrated under reduced pressure _. The crude compound was purified by silica gel (60-120) column chromatography and elution with 1% methanol/dichloromethane to obtain the title compound (0.15 g, 46%) as an off-white solid. MS: 250.9 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.91 (d, J = 2.40 Hz, 1H), 8.82 (d, J = 1.60 Hz, 1H). Step E To a solution of the title compound from Step D above (0.18 g, 0.72 mmol) in dry dichloromethane (5 mL) was added triethylamine (0.3 mL, 2.16 mmol) and morpholine (0.074 g, 0.86 mmol) ), and the mixture was stirred at room temperature for 6 hours. The reaction mixture was concentrated in vacuo. The crude compound was purified by silica gel (60-120) column chromatography and elution with petroleum ether/ethyl acetate to obtain the title compound (0.18 g, 83%) as a grayish-yellow solid. MS: 300.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.49 (d, J = 2.00 Hz, 1H), 8.38 (d, J = 1.60 Hz, 1H), 3.72-3.74 (m, 4H), 3.61- 3.62 (m, 4H).

Preparation Example 27 Step A : A solution of 2-bromo-6-chloropyridin-3-amine (5 g, 24.1 mmol) and potassium thiocyanate (7 g, 72.3 mmol) in ethanol (50 mL) and concentrated hydrochloric acid (37%, 100 mL) and stir at 100°C for 40-45 h. The reaction was confirmed to be complete by TLC (PE/EA = 7.5/2.5). The reaction mixture was cooled to room temperature and concentrated to provide a brown solid, which was partitioned between dichloromethane (150 mL) and 1 N aqueous NaOH (50 mL). The solid was filtered and dried to give the title compound as a pale yellow solid (3.5 g, 79% yield). MS: 186.1 (M+H) ⁺ . Step B To a suspension of the title compound from Step A above (1.5 g, 8.08 mmol) in acetonitrile (25 mL) was added tert-butyl nitrite ( 1.4 ml) using a syringe over a period of 10 min at 0°C. , 12.12 mmol). Then, copper (II) bromide (2.16 g, 9.69 mmol) was added portionwise. After 30 minutes at 0°C, the reaction mixture was allowed to warm to room temperature for 2 minutes. The progress of the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated and the mixture was diluted with water (20 mL) and 5% MeOH/DCM (3 × 20 mL). The combined organics were washed with brine (10 mL), dried over _Na2SO4 , filtered and concentrated under reduced pressure _. The crude compound was purified by silica gel (60-120) column chromatography and elution with 1% methanol/dichloromethane to obtain the title compound (0.65 g, 32%) as a light yellow solid. MS: 248.5 (M+H) ⁺ . Step C To a solution of the title compound from Step B above (0.65 g, 2.61 mmol) in dry dichloromethane (5 mL) was added triethylamine (1.1 mL, 7.83 mmol) and morpholine (0.34 g, 3.91 mmol) ), and the mixture was stirred at room temperature for 6 hours. The reaction mixture was concentrated in vacuo. The crude compound was purified by silica gel (60-120) column chromatography and elution with petroleum ether/ethyl acetate to obtain the title compound (0.6 g, 90%) as a grayish-yellow solid. MS: 256.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.83 (d, J = 8.40 Hz, 1H), 7.41 (d, J = 8.44 Hz, 1H), 3.72-3.74 (m, 2H), 3.59- 3.60 (m, 2H).

Preparation Example 28 Step A : Reflux a mixture of 2,7-dibromo-[1,2,4]triazolo[1,5-a]pyridine (0.4 g, 1.44 mmol) and morpholine (4 mL) under _N2 atmosphere 4 hours. The reaction mixture was concentrated to dryness. The crude compound was purified by silica gel (60-120) column chromatography, using petroleum ether/ethyl acetate 10%-100% as the eluent, to obtain the title compound as a white solid (0.27 g, 66% ). MS: 285.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.64 (d, J = 7.08 Hz, 1H), 7.83 (s, 1H), 7.13-7.14 (m, 1H), 3.69-3.71 (m, 2H ), 3.45-3.46 (m, 2H).

Preparation Example 29 Dissolve 3,6-dibromopyridazine (0.2 g, 0.841 mmol) in acetonitrile (2.5 mL). Morpholine (0.110 mL, 1.261 mmol) and triethylamine (0.176 mL, 1.261 mmol) were then added, and the suspension was irradiated in the microwave at 160 °C for 1 h and 20 min. Dilute the reaction mixture with dichloromethane and water. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/0 -> 0/100) to obtain the title compound (0.168 g, 82%) as a white solid. . MS: 245.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.35 (d, J = 9.5 Hz, 1H), 6.79 (d, J = 9.5 Hz, 1H), 3.88 - 3.79 (m, 4H), 3.66 - 3.55 (m, 4H).

Preparation Example 30 To a suspension of sodium hydride (0.0404 g, 1.682 mmol) in dry THF (3 mL) was added morpholine (0.146 mL, 1.682 mmol) under nitrogen at 0°C. The reaction mixture was stirred at 0 °C for 15 min, then 2,5-dibromopyrazine (0.400 g, 1.682 mmol) dissolved in anhydrous THF (3 mL) was added. The reaction mixture was stirred under reflux overnight. It was quenched with water and the product was extracted three times with EtOAc. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/0 -> 0/100) to obtain the title compound (0.245 g, 60%) as a white solid. . ¹ H-NMR (400 MHz, chloroform- d ) δ = 8.15 (d, J = 1.5 Hz, 1H), 7.86 (d, J = 1.5 Hz, 1H), 3.89 - 3.76 (m, 4H), 3.60 - 3.45 (m, 4H).

Preparation Example 31 Step A To a hot solution of 2-chloro-5-nitro-4-cyanothiopyrimidine (10 g, 46.2 mmol) in acetic acid (264 mL, 4617 mmol) at 120 °C was added iron (15.47 g, 277 mmol). The mixture was stirred at the same temperature for 1 hour. The mixture was allowed to cool to room temperature and insoluble material was removed by filtration and washed with acetic acid. The filtrate was concentrated under reduced pressure and dissolved in THF (200 mL) and EtOAc (400 mL), and washed with saturated aqueous NH ₄ Cl solution (100 mL), saturated NaHCO ₃ solution (100 mL), and washed with Na ₂ SO ₄ Drying and removal of solvent under reduced pressure gave the title compound (6.82 g, 79%). ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.42 (s, 1H). Step B To a suspension of the title compound from step A above (8.2 g, 43 mmol) in acetonitrile (20 mL) was added tert-butyl nitrite (6.8 g, 65.9 mmol) at 0°C over 30 min. Then, copper (II) bromide (11.78 g, 52.8 mmol) was added in several portions. After 30 minutes at 0°C, the reaction mixture was allowed to warm to room temperature and stirred for 12 hours. Water (50 mL) and EtOAc (100 mL) were added and the phases separated. The aqueous layer was extracted twice with EtOAc and the organic layers were _combined , dried over _Na2SO4 and the solvent was evaporated under reduced pressure. The residue was purified on a HP-Sil SNAP column using EtOAc/n-heptane (20/80) using a Biotage Isolera One purification system to give the title compound (4.94 g, 45%). ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.54 (s, 1H), 8.30 (s, 2H). Step C The title compound from step B above (0.157 g, 0.627 mmol) was dissolved in morpholine (3 mL, 0.627 mmol) and stirred at room temperature for 2 h. Dilute the reaction mixture with dichloromethane and water. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/0 -> 0/100) to obtain the title compound (0.048 g, 30%) as a white solid. . MS: 258.6 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.71 (s, 1H), 3.74 (dd, J = 6.0, 3.6 Hz, 4H), 3.70 - 3.58 (m, 4H).

Preparation Example 32 To a suspension of sodium hydride (0.077 g, 3.21 mmol) in dry THF (5 mL) was added (1 R ,5 S )-3-oxa-8-azabicyclo [3.2 .1] Octane hydrochloride (0.457 g, 3.06 mmol). The reaction mixture was stirred for 15 minutes, then 2,5-dibromopyrazine (0.800 g, 3.36 mmol) dissolved in anhydrous THF (5 mL) was added. The reaction mixture was stirred under reflux overnight. The reaction mixture was quenched with water and the product was extracted three times with ethyl acetate. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/0 -> 0/100) to obtain the title compound (0.038 mg, 5%) as a white solid. . MS: 271.8 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.24 (d, J = 1.4 Hz, 1H), 8.11 (d, J = 1.4 Hz, 1H), 4.49 (s, 2H), 3.62 (d, J = 10.9 Hz, 2H), 3.52 (d, J = 11.0 Hz, 2H), 2.02 - 1.95 (m, 2H), 1.91 (m, 2H).

Preparation Example 33 Step A: Place palladium(II) acetate (0.00651 g, 0.029 mmol) and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos) (0.0415 g, 0.087 mmol). To the reaction vial, add degassed 1,4-dioxane (2 mL). The resulting solution was briefly degassed. Heat the suspension at 100°C (on a preheated heating block) for less than 1 minute until the color of the solution changes from orange to dark pink. The vial was then removed from the heating block and the title compound from Preparative Example 1 (0.110 g, 0.290 mmol) and the title compound from Preparative Example 29 (0.078 g, 0.319 mmol) and cesium carbonate (0.331 g, 1.015 mmol) were added ). The reaction vial was filled with argon before being closed. The reaction mixture was heated at 100 °C for 12 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (90/10 -> 0/100) to obtain the title compound (0.072 g, 49%) as a yellow solid. . MS: 508.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.03 (dd, J = 9.1, 4.5 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 9.9 Hz, 1H), 7.38 (dd, J = 8.9, 2.6 Hz, 1H), 7.32 (dd, J = 9.0, 6.1 Hz, 2H), 7.17 (td, J = 9.2, 2.6 Hz, 1H), 6.93 (s, 1H ), 4.55 (s, 2H), 3.88 (t, J = 5.6 Hz, 2H), 3.78 - 3.65 (m, 4H), 3.41 - 3.34 (m, 4H), 3.21 - 3.14 (m, 2H), 2.30 ( s, 3H).

Preparation Examples 34 to 41f Following the palladium coupling procedure as set forth in Preparative Example 33, except using the tricyclic amine derivatives and the bromo/chlorine derivatives indicated in the table below, the following compounds were prepared: Table 1

Preparation Example 42 Step A Palladium(II) acetate (0.0045 g, 0.020 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethyl 𠮿 (XANTPHOS) (0.035 g, 0.061 mmol) was placed in the reaction vial and degassed 1,4-dioxane (4 mL) was added. The resulting solution was briefly degassed. Heat the suspension at 100°C (on a preheated heating block) for less than 1 minute until the color of the solution changes from orange to dark pink. The vial was then removed from the heating block and the title compound from Preparative Example 1, Step E (70 mg, 0.203 mmol) and the title compound from Preparative Example 26 (0.067 g, 0.224 mmol) and cesium carbonate (0.232 g, 0.771 mmol). The reaction vial was filled with argon before being closed. The reaction mixture was heated at 100 °C for 18 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude product was purified on a silica column using a Biotage Isolera One purification system and methylene chloride/methanol (100/00 -> 95/05) to obtain the title compound (0.052 g, 45%) as a yellow solid. ¹ H NMR (400 MHz, chloroform- d ) δ = 8.24 (d, J = 2.7 Hz, 1H), 8.16 - 8.03 (m, 1H), 7.64 - 7.59 (m, 2H), 7.55 (d, J = 2.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.02 (dd, J = 8.6, 1.9 Hz, 2H), 4.23 (d, J = 2.0 Hz, 2H), 3.84 - 3.78 (m, 4H ), 3.69 - 3.63 (m, 4H), 3.59 (t, J = 5.6 Hz, 2H), 3.48 (s, 1H), 3.25 (dq, J = 5.7, 3.5, 2.8 Hz, 2H), 2.33 (s, 3H).

Preparation Example 43 Step A Palladium(II) acetate (0.0045 g, 0.020 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethyl 𠮿 (XANTPHOS) (0.035 g, 0.061 mmol) was placed in the reaction vial and degassed 1,4-dioxane (4 mL) was added. The resulting solution was briefly degassed. Heat the suspension at 100°C (on a preheated heating block) for less than 1 minute until the color of the solution changes from orange to dark pink. The vial was then removed from the heating block and the title compound from Preparative Example 1, Step E (70 mg, 0.203 mmol) and the title compound from Preparative Example 27 (0.067 g, 0.224 mmol) and cesium carbonate (0.232 g, 0.771 mmol). The reaction vial was filled with argon before being closed. The reaction mixture was heated at 100 °C for 18 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude product was purified on a silica column using a Biotage Isolera One purification system using methylene chloride/methanol (100/00 -> 95/05) to obtain the title compound (0.048 g, 42%) as a yellow solid. ¹ H NMR (400 MHz, chloroform- d ) δ = 8.10 (dd, J = 9.0, 4.4 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.63 - 7.57 (m, 2H), 7.15 ( d, J = 8.2 Hz, 2H), 7.09 - 6.97 (m, 2H), 6.75 (d, J = 8.9 Hz, 1H), 4.51 (d, J = 2.0 Hz, 2H), 3.94 (t, J = 5.6 Hz, 2H), 3.82 (q, J = 5.2 Hz, 4H), 3.57 (dd, J = 5.8, 4.0 Hz, 4H), 3.25 (td, J = 5.5, 2.7 Hz, 2H), 2.31 (s, 3H ).

Preparation Example 44 Step A Dissolve 3,6-dibromopyridazine (0.2 g, 0.841 mmol) in acetonitrile (2.5 mL). Then 8-oxa-3-azabicyclo[3.2.1]octane (0.105 g, 0.925 mmol) and triethylamine (0.176 mL, 1.261 mmol) were added, and the suspension was irradiated in the microwave to 160 ℃ for 1 h and 20 minutes. Dilute the reaction mixture with dichloromethane and water. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/0 -> 0/100) to obtain the title compound (0.152 g, 67%) as a beige solid. . MS: 271.5 (M+H) ⁺ . ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.32 (d, J = 9.5 Hz, 1H), 6.70 (d, J = 9.5 Hz, 1H), 4.60 - 4.43 (m, 2H), 3.83 (d , J = 12.8 Hz, 2H), 3.23 (dd, J = 12.4, 2.7 Hz, 2H), 2.08 - 1.94 (m, 2H), 1.90 - 1.76 (m, 2H).

Preparation Example 45 Step A To a solution of 2,6-dichlorobenzo[ d ]oxazole (1.2 g, 6.3 mmol) was added a 33 wt.% methylamine solution in absolute ethanol (8 mL), and the mixture was heated in a Biotage microwave oven. Heat at 100°C for 30 minutes. The reaction mixture was cooled to room temperature and dissolved in dichloromethane (150 mL). _The organic phase was washed with water, 1 N NaOH solution and brine, and then dried over _Na2SO4 . The solvent was removed and the title compound was obtained (1.08 g, 94%). ¹ H NMR (400 MHz, chloroform- d ) δ 7.30 - 7.24 (m, 2H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H), 5.35 (s, 1H), 3.14 (s, 3H). Step B To a stirred suspension of the title compound from Step A above (39 mg, 1.643 mmol) in anhydrous THF (1.5 mL) was slowly added 6-chloro-N-methylbenzo[d]oxazole at 0°C. A solution of azole-2-amine (100 mg, 0.548 mmol) in anhydrous THF (2 mL) was stirred at the same temperature for 30 min. Then a solution of 4-toluene-1-sulfonyl chloride (107 mg, 0.561 mmol) in anhydrous THF (1.5 mL) was added dropwise at 0 °C, and the reaction mixture was stirred at 0 °C for 2 h. The reaction mixture was quenched with ice water (4 mL) at 0 °C. The product was then extracted three times with ethyl acetate. The combined organic extracts were washed with water, brine, dried over _Na2SO4 , filtered and evaporated under reduced pressure to give the title compound as _a beige solid (154 mg, 83%). MS: 337.14 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.93 - 7.89 (m, 2H), 7.88 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.49 - 7.44 (m, 2H), 7.36 (dd, J = 8.5, 2.0 Hz, 1H), 3.49 (s, 3H), 2.39 (s, 3H).

Preparation Example 46 Step A To a suspension of NaH (0.47 g, 19.55 mmol) in THF (20 mL) was added dropwise commercially available 1,3,4,5-tetrahydro-2H-pyrido[4,3- b] Indole-2-carboxylic acid tert-butyl ester (2.0 g, 7.35 mmol) (dissolved in THF). The mixture was then stirred at room temperature for 1 h. Afterwards, tosyl chloride (1.5 g, 7.82 mmol) (dissolved in THF) was added at 0 °C, and the mixture was then stirred at room temperature for 2 h. After the reaction was complete as checked by TLC, the reaction mixture was quenched with ice water, followed by extraction with ethyl acetate. The organic layer was concentrated to give the title compound (1.8 g, 65%). The product was used as received in the next step. MS: 427.1 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (1.8 g, 4.22 mmol) in DCM (20 mL) was added 4 M HCl in dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and the residue was washed with diethyl ether to give the title compound as an off-white solid (1.0 g, 66%). MS: 361.3 (M+H) ⁺ .

Preparation Example 47 Step A : Commercially available (2-fluorophenyl)hydrazine hydrochloride (2 g, 12.34 mmol) and 4-side oxyhexahydropyridine-1-carboxylic acid tert-butyl ester (2.45 g, 12.34 mmol) were prepared at 0°C. mmol) in dioxane (20 mL) was added concentrated H ₂ SO ₄ (2 mL). The reaction mixture was then heated at 100 °C for 4 h. After the reaction was complete as confirmed by TLC, the reaction mixture was cooled to 25°C and then concentrated. The crude mixture was basified by 10% NaOH solution and the precipitate was filtered off. The solid was washed with water and dried under vacuum to give the title compound (1.7 g, 75%). MS: 191.0 (M+H) ⁺ . Step B To a stirred solution of the title compound from Step A above (1.7 g, crude) in THF (20 mL) was added TEA (3.76 mL, 26.82 mmol) and di-tert-butyl dicarbonate at room temperature. (2.34 mL, 10.73 mmol). The mixture was stirred for 12 h. After the reaction was complete, as confirmed by TLC, the solvent was removed and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/heptane gradient (10/80 => 80/20), The title compound was obtained as a pale yellow solid (700 mg, 27%). MS: 291.1 (M+H) ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ = 11.38 (bs, 1H), 7.22 (d, J = 7.60Hz, 1H), 6.96-6.85 (m, 2H), 4.53 (s, 2H), 3.70 -3.71(m,2H),2.78(bs,2H),1.44(s,9H).

Step C

To a suspension of NaH (144 mg, 3.61 mmol) in THF (15 mL) was added the title compound from step B above (700 mg, 2.41 mmol) (dissolved in THF) dropwise at 0°C. The mixture was then stirred at room temperature for 1 h. Afterwards, tosyl chloride (549 mg, 2.89 mmol) (dissolved in THF) was added at 0 °C, and the mixture was then stirred at room temperature for 2 h. After the reaction was complete, as confirmed by TLC, the reaction mixture was quenched with ice water and then extracted with ethyl acetate. The organic layer was concentrated, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (10/80=>80/20) to give the title compound (900 mg, 84%).

MS: 345.1(M-Boc).

¹ H-NMR(400MHz, DMSO- d ₆ )δ=7.79(d, J =8.04Hz,2H),7.41(d, J =8.24Hz,2H),7.34(d, J =7.68Hz,2H), 7.22-7.23(m,1H),7.07-7.09(m,1H),4.50(s,2H),3.70-3.72(m,2H),3.17(bs,2H),2.36(s,3H),1.40( s,9H).

Step D

To a solution of the title compound from Step C above (900 mg, 2.02 mmol) in DCM (10 mL) was added 4N HCl in dioxane (5 mL). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and the residue was washed with diethyl ether to give the title compound as a light brown solid (450 mg, 65%).

MS: 345.1(M+H) ⁺ .

Preparation Example 48 Step A To a suspension of NaH (300 mg, 12.39 mmol) in THF (15 mL) was added the title compound from Preparative Example 47, Step B (1.2 g, 4.13 mmol) (dissolved in THF) dropwise at 0°C. . The reaction mixture was then stirred at room temperature for 1 h. Afterwards, methyl iodide (0.5 mL, 8.26 mmol) was added at 0 °C, and the mixture was then stirred at room temperature for 2 h. After completion, the reaction mixture was quenched with ice water and extracted with ethyl acetate. The organic layer was concentrated, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (10/80 => 80/20) to give the title compound (900 mg, 72%) . MS: 305.3 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.23 (d, J = 9.60 Hz, 1H), 6.88-6.91 (m, 2H), 4.51 (s, 2H), 3.71-3.73 (m, 5H ), 2.73-2.77 (m, 2H), 1.50 (s, 9H). Step B To a solution of the title compound from Step A above (900 mg, 2.96 mmol) in DCM (10 mL) was added 4 M HCl in dioxane (5 mL). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and the residue was washed with diethyl ether to give the title compound as a light brown solid (500 mg, 83%). MS: 205.1 (M+H) ⁺ .

Preparation Example 49 Step A : To a solution of commercially available 2-bromo-6-chloro-1,8-naphthyridine (0.2 g, 0.821 mmol) in anhydrous acetonitrile (5 mL), potassium carbonate (0.335 mg, 2.46 mmol) and morpholine ( 0.11 g, 1.23 mmol). The reaction mixture was heated to 100°C for 3 hours. The reaction mixture was concentrated under vacuum, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (10/80 => 80/20) to obtain the title product as a pale yellow solid. compound (170 mg, 83%). MS: 250.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.71 (s, 1H), 8.07-8.08 (m, 1H), 7.82-7.95 (m, 1H), 7.55-7.56 (m, 1H), 3.72 (s, 8H).

Preparation Example 50 Step A : To a solution of commercially available 2-bromo-6-chloro-1,7-naphthyridine (0.5 g, 2.05 mmol) in anhydrous acetonitrile (5 mL), potassium carbonate (0.837 mg, 6.16 mmol) and morpholine ( 0.27 g, 3.08 mmol). The reaction mixture was heated to 100°C for 3 hours. The reaction mixture was concentrated under vacuum, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (10/80 => 80/20) to obtain the title product as a pale yellow solid. compound (270 mg, 53%). MS: 250.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 9.01 (s, 1H), 7.91-7.93 (m, 2H), 6.92-6.93 (m, 1H), 3.71 (s, 8H).

Preparation Example 51 Step A. To a solution of commercially available 2,7-dichloroquinoline (0.5 g, 2.52 mmol) in anhydrous DMF (5 mL), potassium carbonate (1 g, 7.52 mmol) and morpholine (0.32 g, 3.78 mmol) were added. The reaction mixture was heated to 100°C for 3 hours. The reaction mixture was concentrated in vacuo to give the title compound as a pale yellow solid (500 mg, 80%). MS: 249.1 (M+H) ⁺ .

Preparation Example 52 Step A. To a solution of commercially available 2,6-dichloroquinoline (0.5 g, 2.52 mmol) in anhydrous DMF (5 mL) was added potassium carbonate (1 g, 7.52 mmol) and morpholine (0.32 g, 3.78 mmol). The reaction mixture was heated to 100°C for 3 hours. The reaction mixture was concentrated under vacuum, and the crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using an EtOAc/hexane gradient (10/80 => 80/20) to obtain the title product as a pale yellow solid. compound (500 mg, 80%). MS: 249.1 (M+H) ^{+ 1} H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.06 (d, J = 12.40 Hz, 1H), 7.84 (d, J = 2.40 Hz, 1H), 7.51- 7.52 (m, 2H), 7.31 (d, J = 12.40 Hz, 1H), 3.66-3.67 (m, 8H).

Preparation Example 53 Step A : Add a solution of commercially available 4,6-dichloropyridin-3-amine (8.0 g, 49.07 mmol) and benzyl isothiocyanate (7.3 mL, 53.98 mmol) in acetone (120 mL). Stir at 60°C for 3 hours. The reaction was monitored by TLC. The solvent was evaporated and the solid was filtered, washed with n-hexane (100 mL) and dried to give the desired product as an off-white solid (14.0 g, 87%). MS: 328.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ =12.39 (s, 1H), 12.02 (s, 1H), 8.74 (s, 1H), 7.98-7.99 (m, 3H), 7.67-7.68 (m , 1H), 7.56 (t, J = 7.60 Hz, 2H). Step B To a solution of the title compound from Step A above (14.0 g, 42.94 mmol) in N-methyl-2-pyrrolidone (NMP) (70 mL) was added sodium methoxide (NaOMe) (4.6 g, 85.88 mmol). The mixture was then heated to 120°C and stirring continued for 4 hours. The reaction was monitored by TLC. The reaction mixture was poured into cold water (300 mL) and a white precipitate was obtained. The solid was filtered and washed with water (300 mL) and n-hexane (200 mL). The compound was dried under vacuum for 6 h to obtain the desired product (14.0 g, crude) as a white solid. The product was used as received in the next step. MS: 290.0 (M+H) ⁺ . Step C A suspension of the title compound from Step B _above (14.0 g, 48.4 mmol) in 70% _H2SO4 (50.0 mL) was heated at 110°C for 4 hours. The reaction mixture was cooled to room temperature, and the reaction mixture was slowly poured into 200 mL of cold water (0°C). The reaction mixture was then adjusted to alkaline pH by adding 50% aqueous NaOH solution. Then, the compound was extracted with EtOAc (6 × 100 mL). The combined organic layers were dried over _Na2SO4 and filtered, and the solvent was concentrated to afford the desired product as _a pale yellow solid (6 g, 67%). MS: 186.1 (M+H) ^{+ 1} H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.30 (s, 1H), 7.86 (s, 1H). Step D To a suspension of the title compound from Step C above (5.0 g, 27.02 mmol) in acetonitrile (120 mL) was added tert-butyl nitrite ( 4.8 mL, 4.8 mL, via syringe over 10 min at 0°C). 40.54 mmol). Then, copper (II) bromide (9.0 g, 40.54 mmol) was added portionwise. After 30 minutes at 0°C, the reaction mixture was warmed to room temperature for 2.5 hours and the progress of the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated and diluted with water (200 mL) and 5% MeOH/DCM (3 × 200 mL). The combined organics were washed with brine (50 mL), dried over _Na2SO4 , filtered and concentrated under reduced pressure to give the title compound as a white solid ( _6.5 g). The product was used as received in the next step. MS: 250.9 (M+H) ⁺ Step E To a solution of the title compound from Step D above (6.5 g, 26.09 mmol) in anhydrous DCM (100 mL) was added triethylamine (11.2 mL, 81.5 mmol) and methyl Phenoline (2.8 mL, 28.13 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo. The crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system and an EtOAc/hexane gradient (10/80 => 80/20) to obtain the title compound (4.7 g, 71%) as a pale yellow solid. MS: 256.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.48 (s, 1H), 8.05 (s, 1H), 3.73-3.74 (m, 4H), 3.60-3.61 (m, 4H).

Preparation Example 54 To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.25 g, 0.0013 mol) in acetonitrile (15 mL) was added K ₂ CO ₃ (0.538 g, 0.0039 mol) and 5-oxa -8-Azaspiro[3.5]nonane (0.178 g, 0.0014 mol). Afterwards, the reaction mixture was heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a white solid (0.30 g, 80.21%). MS: 279.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ =7.44 (d, J = 8.40 Hz, 1H), 7.36-7.37 (m, 1H), 7.06 (dd, J = 2.00, 8.40 Hz, 1H), 3.64-3.65 (m, 2H), 3.60 (s, 2H), 3.54-3.55 (m, 2H), 1.96-1.97 (m, 4H), 1.71-1.74 (m, 2H).

Preparation Example 55 To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.60 g, 0.0032 mol) in acetonitrile (15 mL) was added K ₂ CO ₃ (1.32 g, 0.0096 mol) and 2-methoxy Ethane-1-amine (0.266 g, 0.0035 mol). Afterwards, the reaction mixture was heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a brown solid (0.41 g, 56.24%). MS: 226.9 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.26 (s, 1H), 7.35 (d, J = 11.20 Hz, 1H), 7.28 (d, J = 2.80 Hz, 1H), 6.99 (dd, J = 2.80, 11.20 Hz, 1H), 3.48 (d, J = 7.20 Hz, 4H), 3.27 (s, 3H).

Preparation Example 56 To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.719 g, 0.0038 mol) in DCM (15 mL) was added TEA (2.67 ml, 0.0191 mol) and (2R)-2-methyl morpholine (0.5 g, 0.00494 mol) and stirred at 25°C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a white solid (0.6 g, 45.4%). MS: 253.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.43 (d, J = 11.20 Hz, 1H), 7.35 (d, J = 2.40 Hz, 1H), 7.05 (dd, J = 2.80, 11.20 Hz, 1H), 3.89-3.90 (m, 3H), 3.56-3.59 (m, 2H), 3.17-3.18 (m, 1H), 2.86-2.89 (m, 1H), 1.15 (d, J = 8.40 Hz, 3H) .

Preparation Example 57 To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.360 g, 0.0019 mol) in DCM (15 mL) was added TEA (1.33 ml, 0.0095 mol) and (2S)-2-methyl morpholine (0.25 g, 0.00247 mol) and stirred at 25°C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated. The crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using 20%-25% ethyl acetate in petroleum ether to obtain the title compound as a white solid (0.3 g, 45.5%). MS: 253.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.43 (d, J = 8.48 Hz, 1H), 7.34-7.35 (m, 1H), 7.05 (dd, J = 2.08, 8.46 Hz, 1H), 3.90-3.90 (m, 3H), 3.61-3.62 (m, 2H), 3.24-3.25 (m, 1H), 2.87-2.89 (m, 1H), 1.15 (d, J = 6.20 Hz, 3H).

Preparation Example 58 To a solution of 2,5-dichloro-1,3-benzoxazole (1.20 g, 6.38 mmol) in anhydrous DCM (50 mL) at 0 °C was added (3R)-3-methylmorpholine (0.775 g, 7.65 mmol) and Et ₃ N (1.94 g, 19.10 mmol), and stirred at 25°C for 4 h. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with water (20 mL) and extracted with DCM (20 mL × 2). The combined organic extracts were dried over _Na2SO4 , filtered and evaporated under reduced _pressure . The crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using 10%-20% ethyl acetate in petroleum ether to give the title compound (1.0 g, 59.9%). MS: 252.9 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.43 (d, J = 11.20 Hz, 1H), 7.35 (d, J = 2.80 Hz, 1H), 7.04 (dd, J = 2.80, 11.40 Hz, 1H), 4.17-4.19 (m, 1H), 3.69-3.76 (m, 3H), 3.42-3.43 (m, 5H), 1.30 (d, J = 9.20 Hz, 3H).

Preparation Example 59 To a solution of 2,5-dichloro-1,3-benzoxazole (1.20 g, 6.38 mmol) in anhydrous DCM (50 mL) at 0 °C was added (3S)-3-methylmorpholine (0.775 g, 7.65 mmol) and Et ₃ N (1.94 g, 19.10 mmol), and stirred at 25°C for 4 h. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with _H2O (20 mL) and extracted with DCM (20 mL × 2). The combined organic extracts were dried over _Na2SO4 , filtered and evaporated under reduced _pressure . The crude reaction mixture was purified on a silica column using a Biotage Isolera One purification system using 10%-20% ethyl acetate in petroleum ether to give the title compound (0.8 g, 49.6%). MS: 252.9 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.43 (d, J = 11.20 Hz, 1H), 7.35 (d, J = 2.80 Hz, 1H), 7.04 (dd, J = 2.80, 11.20 Hz, 1H), 4.17-4.19 (m, 1H), 3.73-3.75 (m, 4H), 3.46-3.47 (m, 2H), 1.30 (d, J = 9.20 Hz, 3H), .

Preparation Example 60 To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.5 g, 0.00265 mol) in acetonitrile (10 mL) was added K ₂ CO ₃ (1.1 g, 0.00797 mol) and 2-methoxy 1-N-Methylethane-1-amine (0.284 g, 0.00319 mol). Afterwards, the reaction mixture was heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a brown liquid (0.61 g, 95%). MS: 241.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.40 (d, J = 11.20 Hz, 1H), 7.30 (d, J = 2.80 Hz, 1H), 7.00 (dd, J = 2.80, 11.20 Hz, 1H), 3.68 (t, J = 6.40 Hz, 2H), 3.58 (t, J = 7.20 Hz, 2H), 3.27 (s, 3H), 3.16 (s, 3H).

Preparation Example 61 Step A. To a stirred solution of 2-amino-3-chlorophenol (4 g, 0.0280 mol) in ethanol (80 mL) was added O-ethyldithiocarbonate potassium salt (4.49 g, 0.0280 mol), then Heat to 85 °C under _N for 12 h. After the reaction was completed according to LCMS, the reaction mixture was concentrated, the crude product was acidified by using acetic acid (pH = 5) and the solid was filtered, washed with water, and dried under vacuum for 6 h to obtain 4-chlorobenzene as a light brown solid. and[d]oxazole-2-thiol (4.6 g, 89%). MS: 186.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 14.44 (bs, 1H), 7.46 (d, J = 10.80 Hz, 1H), 7.35 (d, J = 11.20 Hz, 1H), 7.23 (t, J = 10.80 Hz, 1H). Step B To a solution of the title compound from Step A above (4.6 g, 0.0244 mol) in DCM (90 mL) was added oxalate chloride (3.20 mL, 0.0374 mol) at 0°C, followed by DMF (1 mL) , and then stirred at 25°C for 1 h. Then TEA (10 mL, 0.0734 mol) and morpholine (2.5 mL, 0.0293 mol) were added at 0 °C and stirred at 25 °C for 2 h under nitrogen. After the reaction was followed to completion by TLC, water (40 mL) was added and extracted with DCM (2×50 mL). The organic layer was concentrated and the crude material was purified on a silica column using a Biotage Isolera One purification system using 10%-15% ethyl acetate in petroleum ether to obtain the title compound as a pale yellow solid (3.4 g, 58% ). MS: 239.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.40 (d, J = 10.80 Hz, 1H), 7.23 (d, J = 10.40 Hz, 1H), 7.03 (t, J = 10.80 Hz, 1H) , 3.71-3.73 (m, 4H), 3.60-3.62 (m, 4H).

Preparation Example 62 Step A To a suspension of NaH (0.47 g, 19.8 mmol) in THF (10 mL) was added dropwise 1,3,4,5-tetrahydro-2H-pyrido[4,3-b] at 0°C. Indole-2-carboxylic acid tert-butyl ester (1.8 g, 6.61 mmol) (dissolved in THF) and then stirred at room temperature for 1 h. Afterwards, methyl iodide (0.7 mL, 11.76 mmol) was added at 0 °C, and then stirred at room temperature for 2 h. After the reaction was completed (monitored by TLC), the reaction mixture was quenched with ice water and extracted with ethyl acetate. The organic layer was concentrated to give a crude reaction mixture 5-methyl-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2-carboxylic acid tert-butyl ester (1.5 g , crude). The product was used as received in the next step. MS: 287.2 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (1.5 g, 5.24 mmol) in DCM (20 mL) was added 4.0 M HCl in dioxane (5 mL). The reaction mixture was stirred overnight. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and washed with diethyl ether to give the title compound (800 mg, crude) as a brown solid. MS: 187.1 (M+H) ⁺ .

Preparation Example 63 Step A: Add (3-methoxyphenyl)hydrazine hydrochloride (10 g, 57.4 mmol) and 4-pentyloxyhexahydropyridine-1-carboxylic acid tert-butyl ester (11.3 g, 57.4 mmol) in ethanol (100 mL) was added tartaric acid. Dimethylurea (30:70, 10 g) was added and heated at 70 °C for 16 h. The reaction mixture was cooled to 25°C and concentrated in vacuo. The residue was dissolved in water and basified with NaOH solution (30%) to pH = 14 and extracted with DCM. The organic phase was separated and dried over _Na2SO4 , filtered and the solvent was evaporated under reduced pressure to obtain 7-methoxy-2,3,4,5-tetrahydro _- 1H-pyridine as a pale yellow gum. and [4,3-b]indole (2.5 g, 21%). The crude product was used as such in the next step. MS: 203.0 (M+H) ⁺ . Step B To a solution of the title compound from Step A above (600 mg, 2.9 mmol) in THF (6 mL) was added triethylamine (0.8 mL, 5.8 mmol) and Boc anhydride (650 mg, 3 mmol) and stirred for 12 h. After the reaction was completed (monitored by TLC), the reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column chromatography using Hexane:EtOAc (80:20) to obtain 7-methane as a pale yellow solid. Oxy-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2-carboxylic acid tert-butyl ester (610 mg, 68%). MS: 303.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.69 (bs, 1H), 7.25 (d, J = 11.60 Hz, 1H), 6.80 (d, J = 2.80 Hz, 1H), 6.61 (dd, J = 2.80, 11.60 Hz, 1H), 4.48 (s, 2H), 3.81 (s, 3H), 3.67-3.68 (m, 2H), 2.73 (bs, 2H), 1.44 (s, 9H). MS: 187.1 (M+H) ⁺ . Step C To a solution of the title compound from Step B above (550 mg, 1.8 mmol) in THF (5 mL) was added NaH (60% mineral oil, 0.14 g, 3.6 mmol) at 0 °C and stirred for 30 min. . Then p-toluenesulfonyl chloride (342 mg, 1.8 mmol) was added and stirred for 45 min. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water and extracted with EtOAc (20 mL × 3). _The combined organic extracts were washed with water, brine, and dried over _Na2SO4 . Filtration and evaporation of the solvent under reduced pressure yielded 7-methoxy-5-toluenesulfonyl-1,3,4,5-tetrahydro-2H-pyrido[4,3-b] as an off-white solid. Indole-2-carboxylic acid tert-butyl ester (550 mg, 66%). The product was used as received in the next step. MS: 357.0 (M+H) ⁺ . Step D To a solution of the title compound from Step C above (550 mg, 1.204 mmol) in anhydrous DCM (5 mL) was slowly added HCl (g) in dioxane (2 M, 5 mL) at 0°C. ) and stir at 25°C for 12 h. After the reaction was complete (monitored by TLC), the reaction mixture was evaporated under reduced pressure to yield crude product. The crude product was washed with diethyl ether to obtain 7-methoxy-5-toluenesulfonyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole ( 350 mg, 81%). MS: 357.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.73 (d, J = 8.40 Hz, 1H), 7.56 (s, 1H), 7.37 (d, J = 8.00 Hz, 2H), 7.27 (d, J = 8.40 Hz, 1H), 6.85-6.86 (m, 1H), 3.82 (s, 3H), 3.72 (s, 2H), 2.90-2.97 (m, 4H), 2.32 (s, 3H).

Preparation Example 64 Step A 8-Methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (1 g, 5.37 mmol) in DCM (20 mL) at 0 °C TEA (2.25 mL, 16.1 mmol) and BOC anhydride (1.85 mL, 8.05 mol) were added to the stirred solution, and then stirred at 25°C for 12 h. After the reaction was completed according to TLC, water was added to the reaction mixture, followed by extraction with DCM. The organic layer was separated and washed with brine solution, concentrated and washed with hexane to give the title compound as an off-white solid (1.1 g, 71%). MS: 287.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.74 (s, 1H), 7.16 (d, J = 8.44 Hz, 1H), 6.85 (d, J = 8.28 Hz, 1H), 4.49 (s, 2H), 3.69 (t, J = 5.64 Hz, 2H), 2.74 (t, J = 5.28 Hz, 2H), 2.35 (s, 3H), 1.47 (s, 9H). Step B To a suspension of sodium hydride (0.125 g, 3.13 mmol) in THF (10 mL) was added the title compound from Step A above (0.6 g, 2.08 mmol) (dissolved in 20 mL THF) dropwise at 0°C. medium), then stir at room temperature for 30 min. Afterwards, tosyl chloride (1.19 g, 6.25 mmol) (dissolved in 20 mL THF) was added dropwise at 0 °C, and then stirred at room temperature for 3 h. After the reaction was complete according to TLC, the reaction mixture was quenched with ice water and the solid formed was filtered and washed with water and dried to give the title compound as a white solid (0.550 g, 57%). MS: 341.1 (M+H) ⁺ -Boc. ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.91 (d, J = 8.52 Hz, 1H), 7.73 (d, J = 8.20 Hz, 2H), 7.34 (d, J = 8.40 Hz, 2H) , 7.26 (s, 1H), 7.15 (d, J = 8.56 Hz, 1H), 4.41 (s, 2H), 3.68 (t, J = 5.60 Hz, 2H), 3.06 (s, 2H), 2.29-2.30 ( m, 6H), 1.43 (s, 9H), Step C To a solution of the title compound from Step B above (0.55 g, 1.20 mmol) in DCM was added 4 M HCl in dioxane ( 4 mL). The reaction mixture was stirred for 2 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and filtered with diethyl ether to give the title compound as a white solid (0.40 g, 85.9%).

Preparation Example 65 Step A. 8-Chloro-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (1 g, 4.84 mmol) in DCM (20 mL) at 0 °C. TEA (2.02 mL, 14.5 mmol) and BOC anhydride (1.67 mL, 7.26 mmol) were added to the stirred solution, and then stirred at 25°C for 12 h. After the reaction was completed according to TLC, water was added to the reaction mixture, followed by extraction with DCM. The organic layer was separated, washed with brine solution, and concentrated to obtain 8-chloro-1,3,4,5-tetrahydropyrido[4,3-b]indole-2-carboxylic acid tert-butyl ester ( 1 g, 66.5%). MS: 305.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.12 (bs, 1H), 7.46 (d, J = 1.68 Hz, 1H), 7.30 (d, J = 8.56 Hz, 1H), 7.03 (dd, J = 1.96, 8.52 Hz, 1H), 4.51 (s, 2H), 3.70 (t, J = 5.64 Hz, 2H), 2.77 (t, J = 5.32 Hz, 2H), 1.47 (s, 9H). Step B To a suspension of sodium hydride (0.096 g, 2.42 mmol) in THF (10 mL) cooled to 0°C was added dropwise the title compound from Step A above (0.5 g, 1.61 mol) at 0°C ( Dissolve in 20 mL THF) and stir at room temperature for 30 min. Afterwards, tosyl chloride (0.921 g, 48.3 mol) (dissolved in 20 mL THF) was added dropwise at 0 °C, and then stirred at room temperature for 3 h. After the reaction was completed according to TLC, the reaction mixture was quenched with ice water and then extracted with ethyl acetate (100 mL). The organic layer was separated, dried over sodium sulfate, filtered and then concentrated. The product was purified by silica gel column chromatography using petroleum ether:ethyl acetate (75:25) to obtain 8-chloro-5-(p-tolylsulfonyl)-3,4-dihydro-1H as a white solid. -Pyrido[4,3-b]indole-2-carboxylic acid tert-butyl ester (0.450 g, 60%). MS: 361.1 (M+H) ⁺ -Boc. ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.05 (d, J = 8.88 Hz, 1H), 7.78 (d, J = 8.16 Hz, 2H), 7.64 (d, J = 1.96 Hz, 1H) , 7.35-7.36 (m, 3H), 4.44 (s, 2H), 3.68 (t, J = 5.60 Hz, 2H), 3.08 (bs, 2H), 2.32 (s, 3H), 1.43 (s, 9H). Step C To a solution of the title compound from Step B above (0.45 g, 0.97 mol) in DCM was added 4 M HCl in dioxane (3 mL) at 0°C. The reaction mixture was stirred for 2 h. After the reaction was complete, the reaction mixture was evaporated to remove the solvent and concentrated with diethyl ether to afford the title compound as a white solid (0.32 g, 91%). The product was used as received in the next step. MS: 361.1 (M+H) ⁺ .

Preparation Example 66 Step A To a solution of 5-bromo-2-chlorobenzo[d]oxazole (1 g, 4.30 mmol) in anhydrous DCM (10 mL) at 0°C was added morpholine (0.56 g, 6.42 mmol) and Et ₃ N (1.7 mL, 12.9 mmol) and stirred at 25°C for 4 h. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with _H2O (10 mL) and extracted with DCM (10 mL × 2). The combined organic extracts were dried over _Na2SO4 , filtered and evaporated under reduced pressure to give _crude product. The crude product was triturated with diethyl ether (100 mL), filtered, washed with diethyl ether (5 mL) and dried to give the title compound as an off-white solid (0.85 g, 71%). ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.48 (d, J = 2.40 Hz, 1H), 7.34-7.38 (m, 1H), 7.16-7.17 (m, 1H), 3.70-3.72 (m , 4H), 3.58-3.59 (m, 4H).

Preparation Example 67 Step A : To a stirred solution of 4,6-dichloropyridin-3-amine (2.5 g, 15.3 mmol) in THF (50 mL) was added triphosgene (4.55 g, 15.3 mol) in THF dropwise, followed by TEA (4.28 mL, 30.7 mol) and heated to reflux for 2 h. The reaction mixture was concentrated in vacuo. The residue was dissolved in acetonitrile (50 mL) and toluene (50 mL), and morpholine (1.34 g, 15.3 mmol) was added and heated to 110 °C for 12 h. Afterwards, TLC was checked, the crude material was concentrated and purified by silica column chromatography using petroleum ether:ethyl acetate (20:80) to obtain N-(4,6-dichloro-3-pyridyl) as a white solid ) Morpholine-4-methamide (3.0 g, 70.1%). MS: 276.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.55 (s, 1H), 8.38-8.40 (m, 1H), 7.78-7.79 (m, 1H), 3.57-3.58 (m, 4H), 3.40 -3.42 (m, 4H). Step B To a solution of the title compound from Step A above (3.0 g, 10.8 mmol) in 1,4-dioxane (5 mL) was added Cs ₂ CO ₃ (10.5 g, 32.4 mol), 1,10- phenanthroline (0.972 g, 5.40 mol) and copper iodide (1.03 g, 5.40 mol), and then heated to 120°C for 12 h. The reaction mixture was filtered through celite and washed with DCM/MeOH, concentrated and the crude material was purified on a silica column using a Biotage Isolera One purification system with an EtOAc/hexane gradient (40/60) to give the title compound as an off-white solid (0.150 g, crude). The crude product was used as such in the next step. MS: 240.1 (M+H) ⁺ .

Preparation Example 68 Step A 8-Ethoxy-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (1 g, 4.62 mmol) in DCM (20 mL) at 0 °C TEA (1.97 mL, 13.87 mmol) and BOC anhydride (1.5 mL, 6.93 mol) were added to the stirring solution, and then stirred at 25°C for 12 h. After the reaction was completed according to TLC, water was added to the reaction mixture, followed by extraction with DCM. The organic layer was separated and washed with brine solution, concentrated and washed with hexane to obtain 8-ethoxy-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole as a brown solid -Tert-Butyl 2-carboxylate (0.8 g, 54%). MS: 317.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.69 (bs, 1H), 7.16 (d, J = 8.68 Hz, 1H), 6.87 (s, 1H), 6.66 (t, J = 6.76 Hz, 1H), 4.48 (s, 1H), 3.97-3.99 (m, 2H), 3.69 (t, J = 5.48 Hz, 2H), 2.74 (bs, 2H), 1.44 (s, 9H), 1.23 (t, J = 6.84 Hz, 3H). Step B To a suspension of sodium hydride (0.136 g, 2.84 mmol) in THF (5 mL) was added the title compound from Step A above (0.3 g, 0.95 mmol) (dissolved in 10 mL THF) dropwise at 0°C. medium), then stir at room temperature for 30 min. Afterwards, tosyl chloride (0.27 g, 1.42 mmol) (dissolved in 10 mL THF) was added dropwise at 0 °C, and then stirred at room temperature for 3 h. After the reaction is completed according to TLC, the reaction mixture is quenched with ice water, the solid formed is filtered, washed with water and dried to obtain 8-ethoxy-5-toluenesulfonyl-1,3,4 as a white solid , tert-butyl 5-tetrahydro-2H-pyrido[4,3-b]indole-2-carboxylate (0.32 g, 72%). The product was used as received in the next step. MS: 371.2 (M+H) ⁺ -Boc. Step C To a solution of the title compound from Step B above (0.32 g, 0.68 mmol) in DCM was added 4 M HCl in dioxane (4 mL) at 0°C. The reaction mixture was stirred for 2 h. After the reaction was completed, the reaction mixture was evaporated to remove the solvent and filtered with diethyl ether to give the title compound as a brown solid (0.13 g, 56%). The product was used as received in the next step. MS: 371.2 (M+H) ⁺ .

Preparation Example 69 Step A : To a stirred solution of tert-butyl 4-pentanoxyhexahydropyridine-1-carboxylate (10.00 g, 0.0502 mol) in ethanol (100 mL) was added hydroxylamine hydrochloride (6.98 g, 0.100 mol) and CH ₃ COONa (8.23 g, 0.100 mol) and then heated to 90 °C under nitrogen atmosphere for 12 h. After the reaction was completed according to LCMS, the reaction mixture was concentrated and water (100 mL) was added to the crude material, followed by extraction with dichloromethane (250 mL). The organic layer was concentrated and the crude product was purified through a silica gel column (Biotage) using 18%-30% ethyl acetate in petroleum ether to obtain 4-(hydroxyimino)hexahydropyridine-1- as a white solid. tert-Butyl formate (5 g, 46.4%). MS: 159.1 (M+H) ⁺ -tert-butyl ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.45 (s, 1H), 3.33-3.36 (m, 4H), 2.42-2.44 (m , 2H), 2.20-2.22 (m, 2H), 1.41 (s, 9H). Step B To a suspension of sodium hydride (0.268 g, 7.00 mmol) in DMF (3 mL) was added the title compound from Step A above (0.500 g, 2.33 mmol) (dissolved in 5 mL DMF) dropwise at 0°C. medium), then stir at room temperature for 60 min. Afterwards, 2-fluoropyridine (0.340 g, 3.50 mmol) (dissolved in 2 mL DMF) was added dropwise at 0 °C, and then stirred at room temperature for 3 h. After the reaction was completed according to TLC, the reaction mixture was quenched with ice water, and then extracted with ethyl acetate (30 mL). The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to obtain 4-Pendantoxy-3-(2-Pendantoxy-1,2-dihydropyridin-3-yl)hexahydrogen as a light brown solid tert-Butylpyridine-1-carboxylate (300 mg, crude). The crude product was used as such in the next step. MS: 293.2 (M+H) ⁺ . Step C A suspension _of the title compound from Step B above (0.5 g, 1.71 Mmol) in concentrated _H2SO4 (2.0 mL) was stirred at room temperature under a nitrogen atmosphere for 16 h. The LCMS was checked afterwards, which indicated only starting material, and the reaction mixture was heated to 60 °C under nitrogen atmosphere for 16 h. The LCMS was then checked and indicated 80% product quality. The reaction mixture was cooled to room temperature, 10% acetonitrile in water (20.0 mL) was added, and the reaction mixture was _basified by using solid _K2CO3 , and the solid was filtered. The filtrate was concentrated to give the title compound (250 mg, crude) as a brown viscous oil. The product was used as received in the next step. MS: 175.1 (M+H) ⁺ . Step D To a stirred solution of the title compound from Step C above (0.6 g, 3.15 mmol) in tetrahydrofuran (10.0 mL) was added TEA (1.32 mL, 9.46 mol) and BOC anhydride (0.869 mL, 3.78 mmol) at 0°C. ) and then stirred at 25°C for 12 h under nitrogen atmosphere. After the reaction was completed according to TLC, the reaction mixture was concentrated and the crude product was purified through a silica gel column (Biotage) using 15%-20% ethyl acetate in petroleum ether to obtain -7,8-dihydrogen as an off-white solid. Furo[2,3-b:4,5-c']bipyridine-6(5H)-carboxylic acid tert-butyl ester (500 mg, 53%). MS: 275.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.21-8.22 (m, 1H), 8.05-8.06 (m, 1H), 7.30-7.31 (m, 1H), 4.51 (s, 2H), 3.76 (t, J = 5.60 Hz, 2H), 2.86 (t, J = 5.60 Hz, 2H), 1.44 (s, 9H). Step E To a stirred solution of the title compound from Step D above (0.5 g, 1.67 mmol) in dichloromethane (5 mL) was added 4.0 M HCl in dioxane (2 mL) at 0°C and then Stir at 0°C-20°C for 2 h. After the reaction was completed according to TLC and LCMS, the reaction mixture was concentrated to obtain the title compound (350 mg, quantitative) as an off-white solid. MS: 175.1 (M+H) ⁺ .

Preparation Example 70 Step A To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.25 g, 0.0013 mol) in acetonitrile (15 mL) was added K ₂ CO ₃ (0.55 g, 0.0040 mol) and (2S ,6R)-2,6-dimethylmorpholine (0.17 g, 0.0014 mol). The reaction mixture was then heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to the title compound as a white solid (0.2 g, 56%). MS: 267.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.43 (d, J = 8.44 Hz, 1H), 7.34 (d, J = 1.96 Hz, 1H), 7.04-7.04 (m, 1H), 3.99 ( d, J = 13.16 Hz, 2H), 3.66-3.67 (m, 2H), 2.82 (t, J = 10.84 Hz, 2H), 1.00 (d, J = 6.28 Hz, 6H).

Preparation Example 71 Step A. To a stirred solution of 2,5-dichloro-1,3-benzoxazole (0.6 g, 0.0032 mol) in acetonitrile (15 mL) was added K ₂ CO ₃ (1.32 g, 0.0096 mol) and a third Butylamine (0.255 g, 0.0035 mol). The reaction mixture was then heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a brown solid (0.4 g, 55%). MS: 225.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 7.97 (s, 1H), 7.31-7.32 (m, 2H), 6.96-6.97 (m, 1H), 1.40 (s, 9H).

Preparation Example 72 Step A. To a stirred solution of 6-bromo-2-chloroquinazoline (0.3 g, 1.234 mmol) in acetonitrile (10 mL) was added K ₂ CO ₃ (0.34 g, 2.467 mmol) and morpholine (0.17 g, 1.85 mmol). ). The reaction mixture was then heated to 70 °C for 12 h. After the reaction was complete according to TLC, DCM and water (50 mL) were added to the reaction mixture. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give the title compound as a pale yellow solid (0.25 g, 69%). The product was used as received in the next step. MS: 294.0 (M+H) ⁺ .

Preparation Example 73 Step A. To a stirred solution of 2,6-dichloro-1,5-naphthyridine (100 mg, 0.502 mmol) in dioxane (3 mL) was added triethylamine (0.210 ml, 1.507 mmol) and morpholine ( 0.052 ml, 0.603 mmol). The reaction mixture was then stirred at 110°C. After 4 h, the reaction was not complete, so triethylamine (0.210 ml, 1.507 mmol) was added and the reaction mixture was further stirred at 110 °C overnight. The reaction mixture was concentrated to dryness and then dissolved in dichloromethane and washed with saturated aqueous _NH4Cl solution. The aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried over _Na2SO4 , filtered and evaporated under reduced pressure to give the title product _as a beige solid (93 mg, 74%). MS: 250.02 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.05 (d, J = 9.4 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H) , 7.53 (d, J = 9.5 Hz, 1H), 3.71 (hept, J = 3.3, 2.6 Hz, 8H).

Preparation Example 74 Step A Dissolve 2-bromo-5-chloropyridine (200 mg, 1.039 mmol) in acetonitrile (2.5 mL), and add (1S,4S)-2-oxa-5-azabicyclo [2.2. 1] Heptane hydrochloride (211 mg, 1.559 mmol) and triethylamine (0.362 mL, 2.60 mmol), and the suspension was irradiated in microwave to 160°C for 1h20. The sample was then extracted between water (20 mL) and dichloromethane (20 mL). The aqueous layer was washed twice with dichloromethane. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using a methylene chloride/methanol gradient (100/0 -> 90/10) to obtain the product as a beige solid (57 mg, 26%). MS: 211.03 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.06 (d, J = 2.6 Hz, 1H), 7.56 (dd, J = 9.0, 2.7 Hz, 1H), 6.57 (d, J = 9.0 Hz, 1H), 4.80 (d, J = 2.3 Hz, 1H), 4.65 (d, J = 2.4 Hz, 1H), 3.76 (dd, J = 7.3, 1.5 Hz, 1H), 3.61 (d, J = 7.3 Hz, 1H), 3.43 (dd, J = 10.1, 1.5 Hz, 1H), 3.20 (d, J = 10.3 Hz, 1H), 1.90 (dd, J = 9.7, 2.3 Hz, 1H), 1.87 - 1.81 (m, 1H ).

Preparation Example 75 Step A Dissolve 2-bromo-5-chloropyridine (200 mg, 1.039 mmol) in acetonitrile (2.5 mL), and add (1R,4R)-2-oxa-5-azabicyclo [2.2. 1] Heptane hydrochloride (211 mg, 1.559 mmol) and triethylamine (0.362 mL, 2.60 mmol), and the suspension was irradiated in microwave to 160°C for 1h20. The sample was then extracted between water (20 mL) and dichloromethane (20 mL). The aqueous layer was washed twice with dichloromethane. The combined organic layers were dried _{over Na2SO4} , filtered and concentrated under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using a methylene chloride/methanol gradient (100/0 -> 90/10) to obtain the product as a beige solid (51 mg, 23%). MS: 211.04 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 8.06 (dd, J = 2.7, 0.7 Hz, 1H), 7.56 (dd, J = 9.0, 2.7 Hz, 1H), 6.63 - 6.50 (m, 1H ), 4.80 (s, 1H), 4.64 (s, 1H), 3.75 (dd, J = 7.3, 1.5 Hz, 1H), 3.61 (d, J = 7.3, 0.9 Hz, 1H), 3.43 (dd, J = 10.1, 1.6 Hz, 1H), 3.20 (d, J = 10.1, 1.1 Hz, 1H), 1.94 - 1.78 (m, 2H).

Example 1 Step A To degassed tetrahydrofuran (5 mL) was added chloro-(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2- Aminoethyl)phenyl]palladium(II)-methyl-tert-butyl ether adduct (PdRuPhos G1) (0.017 g, 0.024 mmol), 2-dicyclohexylphosphino-2′,6′- Diisopropoxybiphenyl (RuPho) (0.011 g, 0.024 mmol), the title compound from Preparative Example 2 (0.05 g, 0.024 mmol) and commercially available 4-(6-bromobenzo[d]thiazole-2- base) morpholine (0.073 g, 0.029 mmol). Then, a 1 M solution of lithium bis(trimethylsilyl)amide (LiHMDS) in tetrahydrofuran (1 mL, 1 mmol) was added. The resulting reaction mixture was heated at reflux for 2 hours. The reaction mixture was cooled to room temperature and dissolved in dichloromethane (100 mL). The organic phase was washed with water and brine and _dried over _Na2SO4 . The solvent was removed under reduced pressure. The crude product was purified on a silica column using a Biotage Isolera One purification system using an ethyl acetate/n-heptane gradient (80/20 => 100/0) to obtain the title compound (0.070 g, 69%). ¹ H-NMR (400 MHz, chloroform- d ) δ = 7.51 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.27 (s, 1H), 7.20 (dd, J = 8.8, 4.2 Hz, 1H), 7.14 (td, J = 8.6, 2.4 Hz, 2H), 6.94 (td, J = 9.1, 2.5 Hz, 1H), 4.40 (s, 2H), 3.88 - 3.81 (m, 4H), 3.70 (t, J = 5.7 Hz, 2H), 3.64 (s, 3H), 3.59 (t, J = 4.9 Hz, 4H), 2.92 (t, J = 5.7 Hz, 2H).

Example 2 Step A To a stirred solution of the title compound of Preparative Example 1 (0.150 g, 1 eq) in anhydrous 1,4-dioxane (5 mL) was added commercially available 4-(6-bromobenzo[d]thiazole-2 -ethyl)morpholine (1 eq), sodium tert-butoxide (3 eq), and the mixture was degassed under _N2 atmosphere for 10 min. To this reaction mixture were added Pd ₂ (dba) ₃ (0.05 eq) and Ru-Phos (0.1 eq), and the mixture was heated to 100°C until the reaction was complete. After the reaction was complete, the reaction mixture was filtered through a bed of celite and washed with EtOAc. The filtrate was concentrated and the crude product was purified by column chromatography or preparative HPLC to afford the title compound 6 as indicated in Table 2.

Examples 3 to 96e The following compounds were prepared following the palladium coupling procedure as set forth in Examples 1 and 2 , except using the tricyclic amine derivatives and bromo/chlorine derivatives indicated in the table below. Examples 71 and 72 were prepared following the procedures set forth in the preparation of Examples 42 and 43 , respectively, followed by the deprotection procedure set forth in Example 97 . Table 2

Example 97 The title compound from Preparative Example 33 (0.072 g, 0.142 mmol) and cesium carbonate (0.107 g, 0.328 mmol) were placed in a microwave tube, followed by MeOH (1.5 mL) and THF (3 mL). The reaction mixture was irradiated in the microwave to 110°C for 30 minutes and then allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (80/20->0/100) to obtain the title product as a yellow solid. compound (0.027 g, 53%). MS: 354.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 10.96 (s, 1H), 7.39 (d, J = 9.9 Hz, 1H), 7.28 (s, 1H), 7.26 (t, J = 4.4 Hz, 1H), 7.20 (dd, J = 10.0, 2.6 Hz, 1H), 6.85 (td, J = 9.2, 2.6 Hz, 1H), 4.62 (s, 2H), 3.91 (t, J = 5.7 Hz, 2H), 3.79 - 3.67 (m, 4H), 3.37 - 3.32 (m, 4H), 2.88 (t, J = 5.6 Hz, 2H).

Examples 98 to 105f Following the tosylate cleavage procedure as set forth in Example 97, except using the tosylate protected precursor indicated in the table below, the following compounds were prepared: Table 3

Example 106 Step A To a solution of the title compound from Preparative Example 7 (500 mg, 1.31 mmol) in DMF (5 mL) was added 2,5-dichlorobenzoxazole (327 mg, 1.74 mmol) and potassium carbonate (602 mg , 4.36 mmol) and heated to 100°C for 8 hours. After the reaction was completed, the reaction mixture was poured into ice-cooled water; the precipitated solid was filtered and dried to obtain the title compound. The product was used as received in the next step without further purification (0.500 g, 77%). MS: 496.1 (M+H) ⁺ . Step B : Add anhydrous 1,4-dioxane (5 mL) to a 50 mL double-neck round-bottom flask and degas for 20 min. To this were added palladium acetate (67 mg, 0.1008 mmol) and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (144 mg, 0.302 mmol). The resulting solution was orange in color and degassed for an additional 10 min. Heat the suspension at 100°C for a few seconds (20-30 seconds) on a preheated oil bath. The color changes to dark green. Remove the oil bath and degas for 5-10 minutes. To this were added the title compound from step A above (500 mg, 1.008 mmol), morpholine (88 mg, 1.008 mmol) and cesium carbonate (0.98 g, 3.024 mmol) and the reaction mixture was heated to 100°C for 3 hours . After 3 h, LC-MS showed that the product was the main peak. The reaction mixture was filtered through a pad of celite and concentrated in vacuo to give the title compound. The product was used as received in the next step without further purification (0.450 g, 82%). MS: 547.3 (M+H) ⁺ . Step C To a solution of the title compound from Step B above (450 mg, 0.82 mmol) in 1,4-dioxane:methanol (1:1; 5 mL) was added sodium tert-butoxide (230 mg, 2.4 mmol) and heated to 70°C for 16 hours. The reaction mixture was concentrated in vacuo. Purification was performed on a silica column using a Biotage Isolera One purification system using a petroleum ether/EtOAc gradient (0 - 100%) to give the title compound as a solid (0.270 g, 84%). MS: 393.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.10 (s, 1H), 7.47-7.48 (m, 1H), 7.28 (d, J = 8.72 Hz, 1H), 7.08-7.09 (m, 1H ), 6.92 (d, J = 2.20 Hz, 1H), 6.83-6.84 (m, 1H), 6.62-6.63 (m, 1H), 4.81 (s, 2H), 3.98-4.00 (m, 2H), 3.73- 3.74 (m, 4H), 3.04-3.05 (m, 4H), 2.94-2.95 (m, 2H).

Example 107 To a solution of the title compound from Preparative Example 7 (500 mg, 1.318 mmol) in DMF (5 mL) was added 2,5-dichlorobenzothiazole (268 mg, 1.32 mmol) and potassium carbonate (545 mg, 3.95 mmol) ), and heated to 100°C for 8 hours. After the reaction was completed, the reaction mixture was poured into ice-cooled water, and the precipitated solid was filtered and dried to obtain the title compound (500 mg, 74%). The product was used as received in the next step without further purification. MS: 513.1 (M+H) ⁺ . Step B To a stirred solution of the title compound from Step A above (200 mg, 0.39 mmol) in anhydrous dioxane (5 mL) was added morpholine (51 mg, 0.58 mmol), sodium tert-butoxide (112 mg , 1.17 mmol) and degassed under _N atmosphere for 10 min. To this reaction mixture were added ginseng(dibenzylideneacetone)dipalladium(0) (17.9 mg, 0.019 mmol) and 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (18.2 mg , 0.039 mmol) and heated to 100°C until the reaction was completed. After the reaction was complete (monitored by LCMS), the reaction mixture was filtered through celite and washed with EtOAc. The filtrate was concentrated under reduced pressure to give crude product. Purification was performed on a silica column using a Biotage Isolera One purification system using a petroleum ether/EtOAc gradient (0 - 100%) to give the title compound as a solid (50 mg, 33%). MS: 409.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.11 (s, 1H), 7.60 (d, J = 8.68 Hz, 1H), 7.48-7.49 (m, 1H), 7.07-7.08 (m, 2H ), 6.86-6.88 (m, 2H), 4.77 (s, 2H), 3.97-3.98 (m, 2H), 3.74-3.76 (m, 4H), 3.11-3.12 (m, 4H), 2.95-2.96 (m , 2H).

Example 108 Step A To a solution of the title compound from Preparative Example 7 (1 g, 2.63 mmol) in DMF (10 mL) was added 2,6-dichlorobenzothiazole (0.537 g, 2.63 mmol) and potassium carbonate (1.09 g, 7.89 mmol) and heated to 100°C for 8 hours. After the reaction was completed, the reaction mixture was poured into ice-cooled water, and the precipitated solid was filtered and dried to obtain the title compound (1.0 g, 74%). The product was used as received in the next step without further purification. MS: 513.2 (M+H) ⁺ . Step B To a stirred solution of the title compound from Step A above (150 mg, 0.293 mmol) in anhydrous dioxane (5 mL) was added morpholine (39 mg, 0.44 mmol), sodium tert-butoxide (85 mg , 0.87 mmol) and degassed under _N2 atmosphere for 10 min. To this reaction mixture were added ginseng(dibenzylideneacetone)dipalladium(0) (13.4 mg, 0.0015 mmol) and 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (13.65 mg , 0.029 mmol) and heated to 100°C until the reaction was completed. After the reaction was complete (monitored by LCMS), the reaction mixture was filtered through celite and washed with EtOAc. The filtrate was concentrated under reduced pressure to give crude product. Purification was performed on a silica column using a Biotage Isolera One purification system using a petroleum ether/EtOAc gradient (0 - 100%) to give the title compound as a solid (60 mg, 50%). MS: 409.1 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.10 (s, 1H), 7.48-7.50 (m, 1H), 7.36-7.38 (m, 2H), 7.08-7.09 (m, 1H), 6.96 -6.97 (m, 1H), 6.85-6.86 (m, 1H), 4.74 (s, 2H), 3.94-3.95 (m, 2H), 3.74-3.75 (m, 4H), 3.06-3.07 (m, 4H) , 2.94-2.95 (m, 2H).

Example 109 Step A To a solution of the title compound from Preparative Example 7 (250 mg, 0.725 mmol) in DMF (5 mL) was added 2,6-dichlorobenzoxazole (166 mg, 0.88 mmol) and potassium carbonate (326 mg , 2.36 mmol) and heated to 100°C for 8 hours. After the reaction was completed, the reaction mixture was poured into ice-cooled water, and the precipitated solid was filtered and dried to obtain the title compound (250 mg, 70%). The product was used as received in the next step without further purification. MS: 496.1 (M+H) ⁺ . Step B To a stirred solution of the title compound from Step A above (250 mg, 0.505 mmol) in anhydrous dioxane (5 mL) was added morpholine (53 mg, 0.60 mmol), sodium tert-butoxide (145 mg , 1.52 mmol) and degassed under _N atmosphere for 10 min. To this reaction mixture were added ginseng(dibenzylideneacetone)dipalladium(0) (23.1 mg, 0.0252 mmol) and 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (23.53 mg , 0.051 mmol) and heated to 100°C until the reaction was completed. After the reaction was complete (monitored by LCMS), the reaction mixture was filtered through celite and washed with EtOAc. The filtrate was concentrated under reduced pressure to give crude product. Purification was performed on a silica column using a Biotage Isolera One purification system using a petroleum ether/EtOAc gradient (0 - 100%) to give the title compound as a solid (50 mg, 25%). MS: 393.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ = 11.09 (s, 1H), 7.45-7.46 (m, 1H), 7.17 (d, J = 8.56 Hz, 1H), 7.08-7.09 (m, 2H ), 6.87-6.79 (m, 2H), 4.77 (s, 2H), 3.95-3.96 (m, 2H), 3.72-3.74 (m, 4H), 3.03-3.04 (m, 4H), 2.91-2.93 (m , 2H).

Example 110 Step A To a stirred solution of the title compound from Preparative Example 1 (0.15 g, 0.43 mmol) in anhydrous 1,4-dioxane (5 mL) was added the title compound from Preparative Example 50 (0.17 g, 0.43 mmol) and Cs ₂ CO ₃ (0.420 g, 1.29 mmol). The reaction mixture was degassed under _N2 atmosphere for 10 min. Then Pd(OAc) ₂ (0.009 g, 0.043 mmol) and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl‎ (0.062 g; 0.129 mmol) were added, and the reaction was The mixture was heated to 100°C until the reaction was complete. After the reaction was complete (monitored by LCMS), the reaction mixture was filtered through celite and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give crude product. The crude material is purified by flash column chromatography or preparative HPLC to obtain the tosyl-protected compound. To a solution of tosyl compound (1.0 eq) in dioxane:MeOH (1:1, 10 vol) was added NaOtBu (3 eq) and heated to 70°C for 6 h. The reaction mixture was concentrated in vacuo and the crude product was column purified to give the desired product. The crude material was purified by flash column chromatography or preparative HPLC to afford the title compound (0.042 g, 24%). ¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 11.00 (bs, 1H), 8.70 (s, 1H), 7.96 (d, J = 9.60 Hz, 1H), 7.45-7.46 (m, 1H), 7.39 (d, J = 9.20 Hz, 1H), 7.07-7.08 (m, 2H), 6.82-6.83 (m, 1H), 4.64 (s, 2H), 4.02-4.04 (m, 2H), 3.73 (d, J = 4.80 Hz, 4H), 3.59 (d, J = 4.40 Hz, 4H), 2.90 (bs, 2H). MS: 404.2 (M+H) ⁺ .

Examples 111 and 112 Following deprotection of the toluenesulfonyl group, the following compounds were prepared following the palladium coupling procedure as set forth in Example 110 . Table 4 Example 113 Step A : Palladium(II) acetate (0.013 g, 0.058 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylmethacrylate (XANTPHOS) (0.101 g, 0.174 mmol) was placed in the reaction vial and degassed. Add 1,4-dioxane (6 mL). The resulting solution was briefly degassed. Heat the suspension at 100°C (on a preheated heating block) for less than 1 minute until the color of the solution changes from orange to dark pink. The vial was then removed from the heating block and the title compound from Preparative Example 52 (148 mg, 0.581 mmol), the title compound from Preparative Example 1 (0.200 g, 0.581 mmol) and cesium carbonate (0.662 g, 2.033 mmol) were added ). The reaction vial was filled with argon before being closed. The reaction mixture was heated at 100 °C for 18 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate (100/00 --> 50/50) gradient. The obtained crude reaction mixture (0.261 g, 0.469 mmol) and cesium carbonate (0.214 g, 0.658 mmol) were placed into a microwave tube, followed by MeOH (4 mL) and THF (8 mL). The reaction mixture was irradiated in the microwave to 110°C for 30 minutes and then allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was purified on a silica column using a Biotage Isolera One purification system using a methylene chloride/methanol gradient (100/00 --> 95/05) to obtain the title compound as a yellow solid. (0.020 g, 9%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.97 (s, 1H), 7.95 (d, 1H), 7.54 (d, 2H), 7.33 - 7.10 (m, 4H), 6.86 (td, 1H), 4.41 (s, 2H), 3.72 (dt, 6H), 3.55 (t, 4H), 2.95 (d, 2H). MS: 403.19 (M+H) ⁺ .

Example 114 Step A : Palladium(II) acetate (0.013 g, 0.058 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylmethacrylate (XANTPHOS) (0.101 g, 0.174 mmol) was placed in the reaction vial and degassed. Add 1,4-dioxane (6 mL). The resulting solution was briefly degassed. Heat the suspension at 100°C (on a preheated heating block) for less than 1 minute until the color of the solution changes from orange to dark pink. The vial was then removed from the heating block and the title compound from Preparative Example 51 (144 mg, 0.581 mmol), the title compound from Preparative Example 1 (0.200 g, 0.581 mmol) and cesium carbonate (0.662 g, 2.033 mmol) were added ). The reaction vial was filled with argon before being closed. The reaction mixture was heated at 100 °C for 18 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude product was purified on a silica column using a Biotage Isolera One purification system using n-heptane/ethyl acetate gradient (100/00 --> 50/50). The obtained crude reaction mixture (0.122 g, 0.219 mmol) and cesium carbonate (0.214 g, 0.658 mmol) were placed into a microwave tube, followed by MeOH (4 mL) and THF (8 mL). The reaction mixture was irradiated in the microwave to 110°C for 30 minutes and then allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was purified on a silica column using a Biotage Isolera One purification system using a methylene chloride/methanol gradient (100/00 --> 95/05) to obtain the title compound as a yellow solid. (0.081 g, 35%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.97 (s, 1H), 7.88 (d, 1H), 7.56 (d, 1H), 7.40 - 7.13 (m, 3H), 7.02 (d, 1H), 6.97 - 6.79 (m, 2H), 4.49 (s, 2H), 3.80 (t, 2H), 3.72 (t, 4H), 3.60 (t, 4H), 2.93 (d, 2H). MS: 403.20 (M+H) ⁺ .

Example 115 Step A Add Pd(OAc) ₂ (16 mg, 0.072 mmol) and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl‎ (XPhos; 103 mg, 0.2177 mmol) to the reaction vial and add degassed dioxane (5 ml). The vial was filled with argon and sealed. The suspension was heated at 100 °C for 1 min and 8-fluoro-5-toluenesulfonyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (Preparation Example 1 ) (250 mg, 0.725 mmol), 4-(4-bromophenyl)morpholine (210 mg, 0.87 mmol) and Cs ₂ CO ₃ (707 mg, 2.177 mmol), and the solution was heated at 100°C 18 hours. Dilute the reaction mixture with ethyl acetate (30 mL) and water (30 mL). The organic phase was separated and the aqueous phase was extracted twice more with ethyl acetate. The combined organic phases were dried over _Na2SO4 , filtered and the solvent evaporated under reduced _pressure . The crude material was purified on a HP-Sil column (Biotage) using a DCM/MeOH gradient (100/0 -> 95/05) to obtain 4-(4-(8-fluoro-5-toluene) as a yellow solid Sulfonyl-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)phenyl)morpholine (235 mg, 64%). To 4-(4-(8-fluoro-5-toluenesulfonyl-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)phenyl) A solution of morpholine (0.235 mg, 0.465 mmol) in dioxane:MeOH (1:1, 5 vol) was added NaOtBu (133 mg, 1.39 mmol) and heated to 70°C for 6 h. The reaction mixture was concentrated in vacuo and the crude product was column purified on a HP-Sil column (Biotage) using a DCM/MeOH gradient (100/0 -> 95/05) to give the title compound as an off-white solid (57 mg, 35%) MS: 352.0 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 10.93 (bs, 1H), 7.21-7.24 (m, 2H), 6.99-7.01 (m, 2H), 6.85-6.86 (m, 3H), 4.24 ( s, 2H), 3.73 (bs, 4H), 3.52-3.53 (m, 2H), 2.98 (bs, 4H), 2.86 (bs, 2H).

Example 116 To 7-fluoro-5-toluenesulfonyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (Preparation Example 7 ) (250 mg, 0.7267 mmol) was dissolved in anhydrous To a stirred solution in dioxane (5 mL) was added 5-chloro-N-(2-methoxyethyl)-N-methylbenzo[d]oxazole-2-amine (Preparation Example 60 ) (190 mg, 0.7994 mmol), sodium tert-butoxide (202 mg, 2.180 mmol), and degassed under _N2 atmosphere for 10 min. To this reaction mixture was added Ruphos G4 Pd (60 mg, 0.0726 mmol) and heated to 100°C until the reaction was complete. After the reaction was complete, the reaction mixture was filtered through a bed of celite, washing with EtOAc. The filtrate was concentrated and the crude material was purified by column chromatography on a HP-Sil column (Biotage) using a DCM/MeOH gradient (100/0 -> 95/05) to give the title compound (37 mg, 13 %) MS: 395.0 (M+H) ⁺ . 1H-NMR (400 MHz, DMSO- d ₆ ) δ 10.94 (bs, 1H), 7.43-7.44 (m, 1H), 7.23 (d, J = 8.80 Hz, 1H), 7.06-7.06 (m, 1H), 6.99 (d, J = 2.40 Hz, 1H), 6.80-6.81 (m, 1H), 6.71-6.72 (m, 1H), 4.32 (s, 2H), 3.64-3.65 (m, 2H), 3.56-3.58 ( m, 4H), 3.27 (s, 3H), 3.13 (s, 3H), 2.86 (t, J = 5.20 Hz, 2H).

Examples 117 to 145 Following the procedures reported in Example 115 and Example 116 , the following compounds were prepared. Table 5

Example 146 Step A To a suspension of sodium hydride (0.0293 g, 0.764 mmol) in DMF (3 mL) was added dropwise 5-(7-fluoro-1,3,4,5-tetrahydropyrido[4 ,3-b]indol-2-yl)-2-morpholinyl-1,3-benzoxazole (Example 45 ) (0.100 g, 0.255 mmol) (dissolved in 3 mL DMF), then incubated in the chamber Stir at room temperature for 60 minutes. Afterwards, ethyl iodide (0.0596 g, 0.382 mmol) (dissolved in 2 mL DMF) was added dropwise at 0 °C, and then stirred at room temperature for 3 h. After the reaction was completed according to TLC, the reaction mixture was quenched with ice water, and then extracted with ethyl acetate (20 mL). The organic layer was separated, dried over sodium sulfate, filtered and then concentrated to give crude material, which was then purified by silica column (Biotage) using 40%-50% ethyl acetate in petroleum ether to 5 as an off-white solid. -(5-ethyl-7-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-2-yl)-2-morpholinyl-1,3-benzox Azole. (15 mg, 14%) MS: 421.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 7.47-7.48 (m, 1H), 7.26-7.28 (m, 2H), 7.06 (d, J = 2.40 Hz, 1H), 6.84-6.85 (m, 1H), 6.78-6.79 (m, 1H), 4.33 (s, 2H), 4.10 (q, J = 7.20 Hz, 2H), 3.55-3.57 (m, 10H), 2.90 (t, J = 4.80 Hz, 2H ), 1.22 (t, J = 7.20 Hz, 3H).

Examples 147 to 153 The following compounds were prepared following the palladium coupling procedure as set forth in Examples 1 , 2 , and 115 , except using the tricyclic amine derivatives and bromo/chlorine derivatives indicated in the table below. Table 5a

Example of HCl salt of the compound of the present invention 89 HCl Step A : 4-(6-(7-fluoro-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)thiazolo[ A solution of 4,5-c]pyridin-2-yl)morpholine (Example 89 ) (0.1 g, 0.244 mmol) in anhydrous DCM (5 mL) was added 4 M HCl in dioxane (0.2 mL) and stirred for 1 hours. The reaction mixture was concentrated in vacuo and triturated with diethyl ether to give the title compound as a brown solid (0.09 g, 83%). MS: 410.2 (M+H) ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 11.14 (bs, 1H), 8.28 (s, 1H), 7.98 (s, 1H), 7.38-7.40 (m, 1H), 7.11-7.12 (m, 1H), 6.85-6.86 (m, 1H), 4.75 (s, 2H), 4.02 (t, J = 5.64 Hz, 2H), 3.73-3.75 (m, 8H), 2.97 (s, 2H).

EXAMPLE Following the hydrochloride salt procedure as set forth in Example 89 HCl , the following compounds were prepared. Table 6

Example ³ H-12 for the synthesis of radioactive ligands of the compounds of the present invention The catalyst was added to the tritium reaction vessel, followed by a solution of Example 12 starting material (1.0 mg, 0.003 mmol) in dichloromethane. Attach the container to the tritium line and pressurize the tritium gas at -200°C. The solution was stirred for 8 hours, cooled to -200°C and excess gas removed. Rinse the reaction flask with 4 × 1 mL of methanol and transfer each to a 100 mL recovery flask. The combined organic phases were evaporated in vacuo (crude yield: 124 mCi). The crude material was purified by HPLC, the mobile phase was evaporated under vacuum and the product was redissolved in absolute ethanol (yield: 17 mCi, purity >97%). The specific activity was determined by mass spectrometry to be 80.6 Ci/mmol.

Example ³ H-45 The catalyst was added to the tritium reaction vessel, followed by a solution of Example 45 starting material (1.0 mg, 0.003 mmol) in dichloromethane. Attach the container to the tritium line and pressurize the tritium gas at -200°C. The solution was stirred for 8 hours, cooled to -200°C and excess gas removed. Rinse the reaction flask with 4 × 1 mL of methanol and transfer each to a 100 mL recovery flask. The combined organic phases were evaporated in vacuo (crude yield: 124 mCi). The crude material was purified by HPLC, the mobile phase was evaporated under vacuum and the product was redissolved in absolute ethanol (yield: 17 mCi, purity >99%). The specific activity was determined by mass spectrometry to be 96.8 Ci/mmol.

Bioassay Description Full-length Tau (flTau) depolymerization assay by thioflavin T (ThT) The longest isoform of human Tau (2N4R; 441 amino acids) was expressed in bacteria and purified. For analysis of Tau depolymerization by ThT, 35 µM recombinant full-length (fl) Tau in PBS was incubated at 37°C in the presence of 50 µM heparin (Sigma-Aldrich) and 10 mM DTT (Sigma-Aldrich) at 750 RPM. Aggregate under shaking for 24 hours. Compounds were dissolved in anhydrous dimethylsulfoxide (DMSO, Sigma-Aldrich) to a concentration of 10 mM. Serial dilutions of flTau aggregates and compounds were mixed together in PBS (50 µL volume) to a final concentration of 2 µM flTau aggregates and 160 µM to 0.04 µM compound. The mixture was incubated at room temperature (RT) for 30 minutes, then 40 µL of this mixture was transferred to a black 384-well assay plate (Perkin-Elmer) and mixed with 10 µL of 100 µM ThT (250 mM Glycerol in PBS). Amino acids (both from Sigma-Aldrich) were mixed. Fluorescence (relative fluorescence units; RFU) was measured in duplicate or in duplicate on a Tecan reader (excitation: 440 nm; emission: 485 nm). The percent flTau depolymerization was then calculated and the half-maximum effective concentration ( _EC50 ) was determined assuming a binding site fitting model using GraphPad Prism version 5 (GraphPad Software). Tau K18 Depolymerization Analysis by ThT Tau K18 fragments covering amino acids 244 to 372 of the longest isoform of human Tau441 (2N4R) were expressed in bacteria and purified, or purchased from SignalChem . For K18 depolymerization assay by ThT, 35 µM recombinant K18 in PBS was aggregated at 37°C in the presence of 50 µM heparin (Sigma-Aldrich) and 10 mM DTT (Sigma-Aldrich) with shaking at 750 RPM 24 hours. Compounds were dissolved in anhydrous dimethylsulfoxide (DMSO, Sigma-Aldrich) to a concentration of 10 mM. Serial dilutions of K18 aggregates and compounds were mixed together in PBS (volume 50 µL) to a final concentration of 2 μM K18 aggregates and 160 µM to 0.04 µM compound. The mixture was incubated at room temperature (RT) for 30 minutes, then 40 µL of this mixture was transferred to a black 384-well assay plate (Perkin-Elmer) and mixed with 10 µL of 100 µM ThT (250 mM Glycerol in PBS). Amino acids (both from Sigma-Aldrich) were mixed. Fluorescence (relative fluorescence units; RFU) was measured in duplicate or in duplicate on a Tecan reader (excitation: 440 nm; emission: 485 nm). The percent K18 depolymerization was then calculated and the half-maximum effective concentration ( _EC50 ) was determined assuming a binding site fitting model using GraphPad Prism version 5 (GraphPad Software).

The following example compounds were measured: Table 7 Legend: +++ EC ₅₀ ＜ 10 uM; ++ EC ₅₀ 10＜x＜25 uM; + EC ₅₀ 25＜x＜50 uM.

Intracellular Tau aggregation is reduced compared to complete medium [DMEM-F12 supplemented with 2.5 µg/ml G418 (Sigma-Aldrich) selection antibiotic 4.5 g/L Glutamax (Invitrogen), 15% FBS (Biochrom), 1% Peni/Strep ( A human neuroblastoma cell line expressing the full-length form of human Tau carrying the P301L mutation was cultured in Invitrogen. The day before the experiment, 5 × 10 ⁵ cells/well were plated in 3 mL of complete culture medium in a 6-well plate. The next day, cells were incubated with DMSO or 5 µM of a compound of the invention for an additional 24 h at 37°C. After incubation, cells were trypsinized and resuspended in 100 µl of homogenization buffer [25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM containing phosphatase inhibitors (30 mM NaF, 0.2 mM _Na VO ₄ , 1 nM Okadaic acid, 1 mM PMSF, 5 mM Na ₄ P ₂ O ₇ ) in EGTA and protease inhibitor mixture (Complete ^TM , Roche)], and then rapidly frozen and thawed three times Cycle for physical dissolution. The samples are then tested directly in the AlphaLISA assay.

Phosphorylation, aggregation and total Tau were quantified by AlphaLisa using the following antibody pairs: · HT7-Receiver Beads + Biotin (BT)-Tau13-Donor Beads: Total Tau · HT7-recipient beads + biotin (BT)-HT7-donor beads: Aggregation of human Tau

Tau13 (Abcam) was biotinylated using the EZ-Link® NHS-PEO Solid Phase Biotinylation Kit (Thermo Scientific), while the HT7-biotin system was from a commercial source (Thermo Scientific).

For each antibody pair, the concentrations of acceptor beads and biotinylated antibodies were optimized. All samples were first tested in a dilution series in PBS to identify the linear range and optimal dilution for each sample and analysis. For the final protocol, the following reagents were added to a 384-well white OptiPlate (PerkinElmer): · 5 µL test dilution sample · Mixed biotin-mAb receptor beads at a final concentration below 20 µL: · Combination of 1.25 nM HT7-BT and 10 µg/ml HT7-Acc beads · Combination of 5 nM Tau13-BT and 2.5 µg/ml HT7-Acc beads

After the mixture was incubated for 1 h at room temperature, 25 µL of 25 µg/mL Streptavidin donor beads (Perkin Elmer) was added in the dark. After 30 min incubation, plates were analyzed using the EnSpire Alpha instrument and EnSpire Workstation version 3.00. Data for aggregated Tau were normalized to total Tau and then expressed as percentage of DMSO-treated cells.

The following example compounds were measured: Table 8 Legend: +++%＞50; ++% 50＜x＜25; +% 25＜x＜10.

In vivo efficacy test of the compounds of the present invention . In vivo study design of Example 12 and Example 45. Double transgenic rTg4510 mice express full-length human Tau carrying the P301L (Tau4RON-P301L) mutation under the control of the tet-inducible CaMKII promoter (Ramsden et al., J. Neurosci., 2005 • 25(46):10637-10647). A single transgenic mouse expressing only the tetracycline-controlled transactivator (tTA) was used as a genotype control. The study contained 4 treatment groups (n=15 females for tTa group and n=11 female mice/group for treatment) with the following group allocation (see Table 9). Beginning at 5 months of age, compound or vehicle control was administered via gastric tube feeding once daily or twice daily for 1 month. All mice received 200 ppm deoxytetracycline in the feed for 3 weeks, plus a loading dose of 1.5 mg/mL in 4% sucrose in the drinking water for the first two days. Dosing of deoxytetracycline began at week 2 and continued throughout the dosing period.

Cerebrospinal fluid (CSF) analysis was performed on all animals, and histology of misfolded Tau (MC1), total microglia (Iba1), and phagocytic microglia (CD68) was performed on 10 animals/group. Table 9 : In vivo study design for Test Example 12 and Example 45 (a) Vehicle: 0.5% w/v Hydroxypropyl Methylcellulose 4000 cps; 0.5% w/v Tween 80

Cerebrospinal fluid (CSF) collection and analysis Mice were deeply anesthetized by standard injectable anesthesia and perfused transcardially with cold PBS. Use surgical scissors to remove the fur and skin from the neck (the mice are not decapitated). The tissue surrounding the skull base and brainstem is carefully removed via blunt dissection with minimal bleeding. Once the meninges are exposed in the foramen magnum region of the skull base, a 27ga sphenoidal needle is held perpendicular to the skull and inserted laterally into the cerebrospinal fluid (CSF) space. Samples were quickly immersed in liquid nitrogen and stored at -80°C until biochemical analysis by AlphaLISA. Total human Tau in CSF Tau was quantified using the following antibody pair: HT7-recipient beads + biotin (BT)-Tau13-donor beads. Tau13 (Abcam) antibody was biotinylated using the EZ-Link® NHS-PEO Solid Phase Biotinylation Kit (Thermo Scientific). For the final protocol, add the following reagents to a 384-well white OptiPlate (PerkinElmer): 5 µL of test dilution sample 20 µL of mixed biotin-mAb receptor beads, final concentration: 0.6 nM Tau13-BT with 2.5 µg/ ml HT7-Acc Beads Combination

After the mixture was incubated for 1 h at room temperature, 25 µL of 25 µg/mL streptavidin donor beads (Perkin Elmer) was added in the dark. After a 30 minute incubation, the plates were analyzed using the EnSpire Alpha instrument and EnSpire Workstation version 3.00. Data were analyzed by one-way ANOVA followed by post hoc comparisons, and statistics shown refer to differences compared to vehicle-treated rTg4510 mice.

As shown in Figure 1, both Example 12 and Example 45 reduced Tau content in CSF.

Histological evaluation of rTg4510 mice treated with Example 12 and Example 45 After perfusion, the brains were then removed and hemisectioned hemisagittally. The right hemisphere was fixed in 4% paraformaldehyde in PBS for 3 hours at room temperature, refrigerated by immersion in 15% sucrose at 4°C for 3 days, and prepared for cryosectioning. The fixed hemispheres were then frozen in dry ice on OCT media in a cryo-mold and sagittal cryosections (10 mm thickness) were performed using a Leica CM3050 cryomicrotome. Sections from 10 mice/group were collected from approximately 12 medial and lateral planes. Systematic randomized sets of sections from seven sagittal levels/animal were used to quantify Iba1 and CD68 immunoreactivity, while 1 section/animal from medial and lateral levels 4 was used to evaluate Tau misfolding (MC1 monoclonal antibody). Frozen sections were removed from -20°C and air-dried at room temperature for 25 minutes. Brain tissue was circled using Pap Pen Liquid Blocker and washed once for 5 minutes and once for 10 minutes in PBS at room temperature. Block and permeabilize using 10% normal goat serum (NGS) and 0.25% Triton X-100 in PBS for 2 hours at room temperature. The sections were then blotted dry with paper and incubated overnight in a humidity chamber at 4°C with mouse monoclonal MC1 antibody diluted 1:1000 in PBS containing 5% NGS and 0.25% Triton X-100. Sections were washed three times for 10 min in PBS at room temperature and incubated with Cy3-labeled goat anti-mouse IgG (H+L) secondary antibody (Jackson) diluted 1:1000 in PBS at room temperature. Incubate in light for 30 minutes. After washing three times in PBS for 10 minutes, the sections were incubated with a solution of 0.1% Sudan Black (Sigma) in 70% ethanol for 30 seconds at room temperature to reduce tissue autofluorescence. The sections were washed three times in PBS for 10 minutes, mounted using ProLong Gold anti-fade reagent (Molecular Probes) containing DAPI, and covered with coverslips. Sections were imaged using a digital slide scanner (Panoramic 250 Flash, 3D Histech Ltd.) and quantified using the image visualization software Visiopharm.

MC1 staining was analyzed in the frontal cortex among the upper cortical layers. A region of interest (ROI) corresponding to a field of view at 20× magnification was drawn around the frontal cortex. This ROI was analyzed in Visiopharm using predetermined thresholds for MC1 staining, where the pixel intensity threshold was above 30 (8-bit image) and all detected objects ^smaller than 20 μm were excluded. Data were analyzed by one-way ANOVA followed by post hoc tests.

As shown in Figure 2, both Example 12 and Example 45 reduced MC1 positive area. These data indicate that treatment with both Example 12 and Example 45 in rTg4510 reduced the amount of misfolded Tau in this aggressive Tau transgenic model.

In order to evaluate whether the above-mentioned effects of the tested compounds on Tau also have effects on neuroinflammation markers, sections were labeled with rat anti-mouse CD68 pure line FA-11 (BD Biosciences) and goat multi-strain anti-Iba1 antibodies (Abcam). And counterstained with DAPI. Antibody binding was visualized using highly cross-absorbing fluorescently labeled secondary antibodies (Thermo Fisher). Antibodies were diluted in Antibody Diluent (Dako) and non-specific endogenous IgG binding was blocked using M.O.M. serum (Vector) before initial incubation. Mounted sections were imaged in their entirety at 20× magnification (plan apochromatic objective) on an Axio Scan Z1 slide scanner driven by ZEN software using LED (Colibri2) illumination and a sensitive Orca Flash 4.0 monochrome camera. Data were analyzed by one-way ANOVA followed by post hoc comparisons, and statistics shown refer to differences compared to vehicle-treated rTg4510 mice.

As shown in Figure 3A, both Example 12 and Example 45 reduced microgliosis as measured by Iba1 immunoreactive area. Furthermore, both Examples 12 and 45 reduced highly activated phagocytic microglia that were positive for CD68 (Figure 3B). Taken together, these data indicate that treatment with both Example 12 and Example 45 in rTg4510 reduced the extent of neuroinflammation in this aggressive Tau transgenic model.

Reduction of Tau misfolding in neurofibrillary tangles (NFTs) from human AD and PSP brain sections Fresh frozen tissue sections from one AD case and two PSP cases were obtained from a commercial distributor (Tissue Solutions, UK). Incubate tissue sections with 100 µM of Example 45 or DMSO in a humidity chamber at room temperature for approximately 60 hours. After the cultured sections were washed three times in PBS, they were fixed with paraformaldehyde (PFA; Sigma) for 10 min at 4°C and then washed three more times in PBS. Sections were then permeabilized in blocking buffer (PBS, 10% pure goat serum (NGS), 0.25% Triton X) for 1 hour at room temperature. After blocking, sections were incubated with Tau conformational antibody (MC1 monoclonal antibody) in blocking buffer (5% NGS, 0.25% Triton X) overnight at 4°C. The next day, sections were washed three times for 5 min in PBS and incubated with Cy3-conjugated AffiniPure goat anti-mouse antibody (Jackson laboratories) for 1 h at room temperature. Wash three times in PBS for 5 minutes to wash out excess antibody. To reduce autofluorescence, sections were incubated with 0.1% Sudan black (Sigma) dissolved in 70% ethanol for 8 min at room temperature. Finally, the sections were washed 5 times in PBS for 5 min and mounted under coverslips using ProLong Gold anti-fade reagent (Invitrogen) containing DAPI. The sections were then dried at room temperature for 24 hours before being imaged using a digital slide scanner (Pannoramic P250 Flash III, 3D Histech Ltd.) and using Visiopharm software to quantify Tau misfolding in NFTs. As shown in Figure 4, Example 45 reduced Tau misfolding in NFTs by approximately 30%, with similar efficacy on AD and PSP human brain samples.

Ex vivo high-resolution autoradiography in human AD brain sections using ³ H- Example 45. Fresh frozen tissue sections from an AD case were obtained from a commercial distributor (Tissue Solutions, UK). For autoradiography, brain sections were fixed with 4% PFA (Sigma) for 15 min at 4°C. Sections were incubated with 20 nM [ ^3H ]-Example 45 alone or together with 5 μM non-radioactive Example 45 for 1 hour at room temperature. The sections were then washed as follows: first in ice-cold buffer for 1 minute, then twice in ice-cold 70% ethanol for 1 minute, in ice-cold buffer for 1 minute and finally with a brief rinse in ice-cold distilled water. Sections were then allowed to dry under airflow for 1 hour and then exposed to Ilford core latex type K5 (Agar Scientific) for 5 days at 4°C in a light-tight slide storage box. Exposure to latex was carried out in a dark room illuminated by a safety light according to the manufacturer's instructions, and latex fragments were melted in an equal volume of 40°C preheated water. After 5 days, sections were developed according to the manufacturer's instructions. Sections were mounted using ProLong Gold anti-fade reagent (Invitrogen) and imaged using a digital slide scanner (Pannoramic P250 Flash III, 3D Histech Ltd.).

As shown in Figure 5, Example 45 shows specific target engagement on Tau NFTs in human AD brain samples.

Figure 1 : Tau in CSF quantified in rTg4510 mice treated with Example 12 and Example 45. Figure 2 : Quantification of Tau misfolding in rTg4510 mice treated with Example 12 and Example 45. Figure 3 : Quantification of Ibal (A) and CD68 (B) in rTg4510 mice treated with Example 12 and Example 45. Figure 4 Example 45 reduces Tau misfolding in NFTs in human AD and PSP brain slices. Figure 5 High-resolution autoradiography of human AD brain sections using ^3H -Example 45 (A) and ^3H -Example 45 and Cold Example 45 (B).

Claims (29)

A compound of formula (I) and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof,

Among them, series A is selected from the group consisting of:

,

,

,

,

,

,

,

and

,in

,

,

,

,

,

,

,

and

Can be connected to Q at any available location, where

,

,

and

Substituted with one or more substituents R ^j , where when A is

When , R ^j is not -CN, halogen or - CF ₃ ; where A is

or

When , R ^j is not halogen, and where

,

,

and

Optionally substituted by one or more substituents R ; B is selected from the group consisting of O and NR ^a ; E and V are independently selected from the group consisting of: N, NR ⁵ , O and S; G is selected from the following The group consisting of: benzene ring and pyridine ring; J is selected from the group consisting of O and NR ¹ ; Q is selected from the group consisting of N and CR ¹ ; Y is selected from the group consisting of CZ and N, provided that Y is When N and Y ¹ , Y ² and Y ³ are CZ, B is N-alkyl or O; Y ¹ is selected from the group consisting of CZ and N; Y ² is selected from the group consisting of CZ and N; Y ³ is selected from the group consisting of CZ and N Selected from the group consisting of CZ and N; Z is independently selected from the group consisting of: H, halogen, O-alkyl, alkyl, and CN; R is independently selected from

and -NR ³ R ⁴ ; R ^a is selected from the group consisting of H and alkyl groups; R ^b , R ^c , R ^d , R ^e , R ^f and R ^g are independently selected from the group consisting of H and alkyl groups group, or any two of R ^b , R ^c , R ^d , Re , R ^f and R ^g can be connected to form a 3- to 8-membered ring; R ^j is independently selected from the group consisting of ^: - halogen, - O-alkyl, -CF ₃ , -CN, - NR ³ R ⁴ ,

and

, where a bridge containing C _1-2 carbon atoms can exist between a carbon atom and c or d carbon atom or where a bridge containing C _1-2 carbon atoms can exist between b carbon atom and c or d carbon atom ; R ¹ is selected from the group consisting of H and alkyl; R ² is independently selected from the group consisting of alkyl, F and =O, wherein the alkyl is optionally substituted by halogen, -OH or -O-alkyl, And if two R ² are twin-positioned, they can be connected to form a 3- to 6-membered ring; R ³ and R ⁴ are independently selected from the group consisting of H and an alkyl group, wherein the alkyl group may be optionally treated with halogen, -OH or -O-alkyl substitution; R ⁵ is selected from the group consisting of H and alkyl; n is 0, 1, 2, 3 or 4, r and s are independently 0, 1, 2 or 3; and t and u are independently 1, 2 or 3. For example, the compound of claim 1 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof, wherein the compound is a compound of formula (Ia):

Among them ^, A , B , Rb ^, Rc , Rd ^, Re , Rf , ^Rg , Y ^and Z are as defined in claim ¹ . For example, the compound of claim 1 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are compounds of formula (Ib):

Among them, A , B , Rb , Rc, Rd, Re, Rf , ^Rg and ^Z are ^as defined ^{in claim 1} . For example, the compound of any one of claims 1 to 3 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof, wherein A is

,in

Can be connected to Q or N at any available location, and where

Optionally substituted with one or more substituents R. For example, the compound of any one of claims 1 to 3 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are compounds of formula (Ic) :

Where E , R , V and Z are as defined in claim 1. For example, the compound of claim 1 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof, wherein the compound is selected from the group consisting of:

A compound and all its stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs, selected from the group consisting of:

A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof, and Pharmaceutically acceptable carriers or excipients, as appropriate. For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used as Potion. The use of a compound of formula (I) and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof,

It is used for manufacturing, for treating, ameliorating or preventing diseases or abnormalities related to Tau protein aggregates, for manufacturing, for reducing Tau aggregation, for preventing the formation of Tau aggregates and/or for inhibiting Tau aggregation, for Agents that interfere with Tau aggregates in cells, are used to reduce Tau misfolding and hyperphosphorylation in vivo, or are used to reduce neuroinflammatory markers, wherein A is selected from the group consisting of:

,

,

,

and

,in

,

,

,

,

,

,

,

and

Can be connected to Q at any available location, where

,

,

and

Substituted with one or more substituents R ^j , where when A is

, R ^j is not -CN, halogen or -CF ₃ , and where

,

,

and

Optionally substituted by one or more substituents R ; B is selected from the group consisting of O and NR ^a ; E and V are independently selected from the group consisting of: N, NR ⁵ , O and S; G is selected from the following The group consisting of: benzene ring, pyrimidine ring and pyridine ring; J is selected from the group consisting of O and NR ¹ ; Q is selected from the group consisting of N and CR ¹ ; Y is selected from the group consisting of CZ and N; Y ¹ Y ² is selected from the group consisting of CZ and N; Y ³ is selected from the group consisting of CZ and N; Z is independently selected from the group consisting of: H, halogen, O- Alkyl, alkyl and CN; R is independently selected from

and -NR ³ R ⁴ ; R ^a is selected from the group consisting of H and alkyl groups; R ^b , R ^c , R ^d , R ^e , R ^f and R ^g are independently selected from the group consisting of H and alkyl groups group, or any two of R ^b , R ^c , R ^d , Re , R ^f and R ^g can be connected to form a 3- to 8-membered ring; R ^j is ^{independently} selected from the group consisting of: - halogen, - O-alkyl, -CF ₃ , -CN, -NR ³ R ⁴ ,

and

, where a bridge containing C _1-2 carbon atoms can exist between a carbon atom and c or d carbon atom or where a bridge containing C _1-2 carbon atoms can exist between b carbon atom and c or d carbon atom ; R ¹ is selected from the group consisting of H and alkyl; R ² is independently selected from the group consisting of alkyl, F and =O, wherein the alkyl is optionally substituted by halogen, -OH or -O-alkyl, And if two R ² are twin-positioned, they can be connected to form a 3- to 6-membered ring; R ³ and R ⁴ are independently selected from the group consisting of H and an alkyl group, wherein the alkyl group may be optionally treated with halogen, -OH or -O-alkyl substitution; R ⁵ is selected from the group consisting of H and alkyl; n is 0, 1, 2, 3 or 4; r and s are independently 0, 1, 2 or 3; and t and u are independently 1, 2 or 3. The use of claim 10, wherein the compound of formula (I) is as defined in any one of claims 1 to 7. The use of claim 10 or 11, wherein the disease is selected from Alzheimer's disease (AD), familial AD, primary age-related tauopathy (PART), Creutzfeldt- Jacob disease), boxer dementia, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease (GSS), inclusion body myositis, Prion Protein (prion protein) cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic granulopathy, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, chromosome 17-related Frontotemporal dementia with Parkinson's disease (FTDP-17), Hallervorden-Spatz disease, multiple system atrophy (MSA), Niemann-Pick disease type C disease type C), pallidum-pontine-substantia nigra degeneration, Pick's disease (PiD), progressive subcortical gliomatosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis , tangle-dominant dementia, post-encephalitic Parkinson's disease, Myotonic dystrophy, subacute sclerosing panencephalopathy, LRRK2 mutation, chronic traumatic encephalopathy (CTE), familial British dementia, familial Danish dementia ), other frontotemporal degeneration, Guadeloupe Parkinsonism, neurodegeneration with iron accumulation in the brain, SLC9A6-related mental retardation, white matter tauopathy with glomerular inclusions, epilepsy, Lewy Lewy body dementia (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes, senile heart disease Amylase, glaucoma, ischemic stroke, AD psychosis and Huntington's disease. The use of claim 10 or 11, wherein the disease is selected from Alzheimer's disease (AD), corticobasal degeneration (CBD), Pick's disease (PiD) and progressive supranuclear palsy (PSP) . For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used for Reduce Tau aggregation. For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used for To prevent the formation of Tau aggregates and/or to inhibit Tau aggregation. For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used for thin Intracellular interference with Tau aggregates. For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used for Reduce Tau misfolding and hyperphosphorylation in vivo. For example, the compounds of any one of claims 1 to 3, 6 and 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof are used for Reduce markers of neuroinflammation. A compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof or a medicament as claimed in claim 8 Use of the composition for the manufacture of a medicament for reducing Tau aggregation. A compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof or a medicament as claimed in claim 8 Use of the composition for manufacturing agents for preventing the formation of Tau aggregates and/or for inhibiting Tau aggregation. A compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof or a medicament as claimed in claim 8 Use of compositions for the manufacture of agents for interfering with Tau aggregates within cells. A compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof or a medicament as claimed in claim 8 Use of the composition for manufacturing agents for reducing Tau misfolding and hyperphosphorylation in vivo. A compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof or a medicament as claimed in claim 8 Use of a composition for the manufacture of a medicament for reducing markers of neuroinflammation. A mixture comprising a compound as claimed in any one of claims 1 to 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof and at least one Other biologically active compounds selected from therapeutic agents different from the compounds of any one of claims 1 to 7, which are selected from the group consisting of: compounds that resist oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitor, pirenzepine or its metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activation Agents, β- and γ-secretase inhibitors, glycogen synthase kinase 3 inhibitors, O-linked N-acetylglucosamine hydrolase (O-glcnacase, OGA) inhibitors, neurotransmitters, β-pleated plates β-sheet breakers, amyloid β-like scavenging/depleting cellular component attractants, N-terminally truncated starch-like β-like (including pyroglutamate-like β-amyloid 3-42) inhibitors , anti-inflammatory molecules, cholinesterase inhibitors (ChEI), tacrine, rivastigmine, donepezil, galantamine, M1 agonist , starch-like or Tau-modified drugs and nutritional supplements, antibodies, functionally equivalent antibodies or functional parts thereof, or vaccines, The mixture further includes at least one of a pharmaceutically acceptable carrier, diluent and excipient. The mixture of claim 24, wherein the compound and/or the other biologically active compound is present in a therapeutically effective amount. The mixture of claim 24 or 25, wherein the other biologically active compound is a cholinesterase inhibitor (ChEI). Such as the mixture of claim 24 or 25, wherein the other biologically active compound is selected from the group consisting of: tacnin, rivastigmine, donepezil, galantamine, nicotinic acid and memantine ( memantine). Such as the mixture of claim 24 or 25, wherein the other biologically active compound is a monoclonal antibody or a functionally equivalent antibody or a functional part thereof. The use of a compound as claimed in any one of claims 1 to 6 or 7 and all stereoisomers, racemic mixtures, tautomers, pharmaceutically acceptable salts, hydrates and polymorphs thereof, Analytical reference or in vitro screening tools for characterizing tissues with Tau pathology and for testing compounds targeting Tau pathology on such tissues.