WO2021123291A1 - Oga inhibitor compounds - Google Patents

Oga inhibitor compounds Download PDF

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WO2021123291A1
WO2021123291A1 PCT/EP2020/087197 EP2020087197W WO2021123291A1 WO 2021123291 A1 WO2021123291 A1 WO 2021123291A1 EP 2020087197 W EP2020087197 W EP 2020087197W WO 2021123291 A1 WO2021123291 A1 WO 2021123291A1
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mmol
group
disease
mixture
compound
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PCT/EP2020/087197
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French (fr)
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José Manuel Bartolomé-Nebreda
Ana Isabel De Lucas Olivares
Andrés Avelino TRABANCO-SUÁREZ
Juan Antonio Vega Ramiro
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Janssen Pharmaceutica Nv
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/048—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being five-membered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention relates to O-GlcNAc hydrolase (OGA) inhibitors, having the structure shown in Formula (I) wherein the radicals are as defined in the specification.
  • OGA O-GlcNAc hydrolase
  • the invention is also directed to pharmaceutical compositions comprising such compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which inhibition of OGA is beneficial, such as tauopathies, in particular Alzheimer’s disease or progressive supranuclear palsy; and neurodegenerative diseases accompanied by a tau pathology, in particular amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations; or alpha synucleinopathies, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease.
  • tauopathies in particular Alzheimer’s disease or progressive
  • O-GlcNAcylation is a reversible modification of proteins where N-acetyl-D- glucosamine residues are transferred to the hydroxyl groups of serine- and threonine residues yield O-GlcNAcylated proteins. More than 1000 of such target proteins have been identified both in the cytosol and nucleus of eukaryotes. The modification is thought to regulate a huge spectrum of cellular processes including transcription, cytoskeletal processes, cell cycle, proteasomal degradation, and receptor signalling.
  • O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA) are the only two proteins described that add (OGT) or remove (OGA) O-GlcNAc from target proteins.
  • OGA was initially purified in 1994 from spleen preparation and 1998 identified as antigen expressed by meningiomas and termed MGEA5, consists of 916 amino (102915 Dalton) as a monomer in the cytosolic compartment of cells. It is to be distinguished from ER- and Golgi-related glycosylation processes that are important for trafficking and secretion of proteins and different to OGA have an acidic pH optimum, whereas OGA display highest activity at neutral pH.
  • the OGA catalytic domain with its double aspartate catalytic center resides in the N- terminal part of the enzyme which is flanked by two flexible domains.
  • the C-terminal part consists of a putative HAT (histone acetyl transferase domain) preceded by a stalk domain. It has yet still to be proven that the HAT-domain is catalytically active.
  • O-GlcNAcylated proteins as well as OGT and OGA themselves are particularly abundant in the brain and neurons suggesting this modification plays an important role in the central nervous system. Indeed, studies confirmed that O-GlcNAcylation represents a key regulatory mechanism contributing to neuronal communication, memory formation and neurodegenerative disease. Moreover, it has been shown that OGT is essential for embryogenesis in several animal models and ogt null mice are embryonic lethal. OGA is also indispensible for mammalian development. Two independent studies have shown that OGA homozygous null mice do not survive beyond 24-48 hours after birth. Oga deletion has led to defects in glycogen mobilization in pups and it caused genomic instability linked cell cycle arrest in MEFs derived from homozygous knockout embryos. The heterozygous animals survived to adulthood however they exhibited alterations in both transcription and metabolism.
  • Oga heterozygosity suppressed intestinal tumorigenesis in an Ape- mouse cancer model and the Oga gene ( MGEA5 ) is a documented human diabetes susceptibility locus.
  • O-GlcNAc-modifications have been identified on several proteins that are involved in the development and progression of neurodegenerative diseases and a correlation between variations of O-GlcNAc levels on the formation of neurofibrillary tangle (NFT) protein by Tau in Alzheimer’s disease has been suggested.
  • O-GlcNAcylation of alpha-synuclein in Parkinson’s disease has been described (Levine, PM, et al. PNAS January 29, 2019, Vol. 116, No. 5, pp 1511-1519; Lewis, YE et al. ACS Chem Biol. 2017 Apr 21, Vol. 2, No. 4, pp 1020-1027; Marotta, NP et al. Nat Chem. 2015 Nov, Vol. No. 11, pp. 913-20).
  • tau is encoded on chromosome 17 and consists in its longest splice variant expressed in the central nervous system of 441 amino acids. These isoforms differ by two N-terminal inserts (exon 2 and 3) and exon 10 which lie within the microtubule binding domain. Exon 10 is of considerable interest in tauopathies as it harbours multiple mutations that render tau prone to aggregation as described below.
  • Tau protein binds to and stabilizes the neuronal microtubule cytoskeleton which is important for regulation of the intracellular transport of organelles along the axonal compartments. Thus, tau plays an important role in the formation of axons and maintenance of their integrity. In addition, a role in the physiology of dendritic spines has been suggested as well.
  • Tau aggregation is either one of the underlying causes for a variety of so called tauopathies like PSP (progressive supranuclear palsy), Down’s syndrome (DS), FTLD (frontotemporal lobe dementia), FTDP-17 (frontotemporal dementia with Parkinsonism- 17), Pick’s disease (PD), CBD (corticobasal degeneration), agryophilic grain disease (AGD), and AD (Alzheimer’s disease).
  • tau pathology accompanies additional neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) or FTLD cause by C90RF72 mutations.
  • tau is post- translationally modified by excessive phosphorylation which is thought to detach tau from microtubules and makes it prone to aggregation.
  • O-GlcNAcylation of tau regulates the extent of phosphorylation as serine or threonine residues carrying O- GlcNAc-residues are not amenable to phosphorylation. This effectively renders tau less prone to detaching from microtubules and reduces aggregation into neurotoxic tangles which ultimately lead to neurotoxicity and neuronal cell death.
  • This mechanism may also reduce the cell-to-cell spreading of tau-aggregates released by neurons via along interconnected circuits in the brain which has recently been discussed to accelerate pathology in tau-related dementias. Indeed, hyperphosphorylated tau isolated from brains of AD-patients showed significantly reduced O-GlcNAcylation levels.
  • OGA inhibitor administered to TNPL3 tau transgenic mice successfully reduced NFT formation and neuronal loss without apparent adverse effects. This observation has been confirmed in another rodent model of tauopathy where the expression of mutant tau found in FTD can be induced (tg4510). Dosing of a small molecule inhibitor of OGA was efficacious in reducing the formation of tau-aggregation and attenuated the cortical atrophy and ventricle enlargement.
  • amyloid precursor protein APP
  • O-GlcNAcylation of the amyloid precursor protein (APP) favours processing via the non-amyl oidogenic route to produce soluble APP fragment and avoid cleavage that results in the AD associated amyloid-beta (Ab) formation.
  • Maintaining O-GlcNAcylation of tau by inhibition of OGA represents a potential approach to decrease tau-phosphorylation and tau-aggregation in neurodegenerative diseases mentioned above thereby attenuating or stopping the progression of neurodegenerative tauopathy-diseases.
  • WO2012/117219 (Summit Corp. pic., published 7 September 2012) describes N-[[5- (hydroxymethyl)pyrrolidin-2-yl]methyl]alkylamide and N-alkyl-2-[5- (hydroxymethyl)pyrrolidin-2-yl]acetamide derivatives as OGA inhibitors.
  • WO2014/159234 (Merck Patent GMBH, published 2 October 2014) discloses mainly 4-phenyl or benzyl-piperidine and piperazine compounds substituted at the 1 -position with an acetamido-thiazolylmethyl or acetamidoxazolylmethyl substituent and the compound N-[5-[(3-phenyl-l-piperidyl)methyl]thiazol-2-yl]acetamide;
  • WO20 16/0300443 (Asceneuron S.A., published 3 March 2016), WO2017/144633 and W02017/0114639 (Asceneuron S.A., published 31 August 2017) disclose 1,4- disubstituted piperidines or piperazines as OGA inhibitors;
  • WO2017/144637 discloses more particular 4-substituted l-[l-(l,3-benzodioxol-5-yl)ethyl]-piperazine; l-[l-(2,3- dihydrobenzofuran-5-yl)ethyl]-; l-[l-(2,3-dihydrobenzofuran-6-yl)ethyl]-; and 1-[1- (2,3-dihydro-l,4-benzodioxin-6-yl)ethyl]-piperazine derivatives as OGA inhibitors;
  • WO2017/106254 Merck Sharp & Dohme Corp. describes substituted N-[5-[(4- methylene-l-piperidyl)methyl]thiazol-2-yl]acetamide
  • WO2018/217558 (Eli Lilly and Company) describes 5-methyl-l,3,4-oxadiazol-2-yl and WO2019/178191 (Biogen Ma Inc) discloses [(hetero)aryl-3-ylmethyl]pyrrolidin-l-ylmethyl- and [(hetero)aryl-3- ylmethyl]piperidin-l-ylmethyl- derivative compounds as OGA inhibitors
  • WO2018/140299 (Eli Lilly and Company) discloses N-[fhioro-5-[[(2S,4S)-2-methyl-4- [(5-methyl-l,2,4-oxadiazol-3-yl)methoxy[-l-piperidyl]methyl]thiazol-2-yl]acetamide as OGA inhibitor.
  • R A is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thienyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- aj
  • R a is selected from hydrogen and Ci- 4alkyl
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and methyl;
  • R 2 represents H or C3 ⁇ 4
  • R B is a radical selected from the group consisting of (b-1), (b-2), and (b-3): wherein R bl represents hydrogen or fluoro; and the pharmaceutically acceptable salts and the solvates thereof.
  • Illustrative of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the compounds described above.
  • An illustration of the invention is a pharmaceutical composition made by mixing any of the compounds described above and a pharmaceutically acceptable carrier.
  • Illustrating the invention is a process for making a pharmaceutical composition comprising mixing any of the compounds described above and a pharmaceutically acceptable carrier.
  • Exemplifying the invention are methods of preventing or treating a disorder mediated by the inhibition of O-GlcNAc hydrolase (OGA), comprising administering to a subject in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
  • O-GlcNAc hydrolase O-GlcNAc hydrolase
  • An example of the invention is a method of preventing or treating a disorder selected from a tauopathy, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or a neurodegenerative disease accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations, or preventing or treating a disorder selected from an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of
  • tauopathy in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or a neurodegenerative disease accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations or for use in preventing or treating a disorder selected from an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease,, in a subject in need thereof.
  • a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal
  • the present invention is directed to compounds of Formula (I), as defined herein before, and pharmaceutically acceptable addition salts and solvates thereof.
  • the compounds of Formula (I) are inhibitors of O-GlcNAc hydrolase (OGA) and may be useful in the prevention or treatment of tauopathies, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or maybe useful in the prevention or treatment of neurodegenerative diseases accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations; or may be useful in the prevention or treatment of alpha synucleinopathies, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia
  • the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein
  • R A is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- ajpyridinyl, imidazo[l,5-a]pyridinyl, imidazo[l,5-b]pyridazinyl,
  • R a is selected from hydrogen and Ci- 4alkyl
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and methyl;
  • R 2 represents H or C3 ⁇ 4
  • R B is a radical selected from the group consisting of (b-1), (b-2), and (b-3): wherein R bl represents hydrogen or fluoro.
  • the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein R A is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, imidazo[l,2-a]pyridinyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and methyl;
  • R 2 represents H or C3 ⁇ 4
  • R B is a radical selected from the group consisting of (b-1), (b-2), and (b-3): wherein R bl represents hydrogen or fluoro.
  • the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein
  • R A is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from quinolinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents;
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la and R lb are each independently selected from the group consisting of hydrogen and Ci-4alkyl, in particular hydrogen and methyl; and R lc and R ld are each hydrogen;
  • R 2 represents H or C3 ⁇ 4
  • R B is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
  • the invention is directed to compounds of Formula (I) as described herein, wherein xx
  • R A is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; and Ci- 4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are all hydrogen;
  • R 2 represents CFF; and R B is a radical selected from the group consisting of (b-1) and (b-2):
  • the invention is directed to compounds of Formula (I) as described herein, wherein
  • R A is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl; and Ci-4alkyloxy; R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are all hydrogen;
  • R 2 represents CFF
  • R B is a radical selected from the group consisting of (b-1) and (b-2):
  • the invention is directed to compounds of Formula (I) as described herein, wherein
  • R A is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with a substituent selected from the group consisting of cyano and Ci-4alkyl;
  • R la , R lb , R lc , and R ld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, R la , R lb , R lc , and R ld are all hydrogen; R 2 represents C3 ⁇ 4; and
  • R B is a radical selected from the group consisting of (b-1) and (b-2):
  • Halo shall denote fluoro, chloro and bromo
  • Ci ⁇ alkyl shall denote a straight or branched saturated alkyl group having 1, 2, 3 or 4 carbon atoms, respectively e.g. methyl, ethyl, 1 -propyl, 2-propyl, butyl, 1 -methyl-propyl, 2-methyl- 1 -propyl, 1,1-dimethylethyl, and the like
  • Ci-4alkyloxy shall denote an ether radical wherein Ci-4alkyl is as defined before.
  • substituted in general, whenever the term “substituted” is used in the present invention, it is meant, unless otherwise indicated or is clear from the context, to indicate that one or more hydrogens, in particular 1 to 3 hydrogens, preferably 1 or 2 hydrogens, more preferably 1 hydrogen, on the atom or radical indicated in the expression using “substituted” are replaced with a selection of substituents from the indicated group, provided that the normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.
  • subject refers to an animal, preferably a mammal, most preferably a human, who is or has been the object of treatment, observation or experiment. As used herein, the term “subject” therefore encompasses patients, as well as asymptomatic or presymptomatic individuals at risk of developing a disease or condition as defined herein.
  • terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • prophylactically effective amount means that amount of active compound or pharmaceutical agent that substantially reduces the potential for onset of the disease or disorder being prevented.
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
  • compound of Formula (I) is meant to include the addition salts, the solvates and the stereoisomers thereof.
  • the invention includes all stereoisomers of the compound of Formula (I) either as a pure stereoisomer or as a mixture of two or more stereoisomers.
  • Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If a compound contains a double bond, the substituents may be in the E or the Z configuration. If a compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration. Therefore, the invention includes enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof.
  • the absolute configuration is specified according to the Cahn-Ingold-Prelog system.
  • the configuration at an asymmetric atom is specified by either R or S.
  • Resolved compounds whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate plane polarized light.
  • stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1%, of the other isomers.
  • a compound of formula (I) is for instance specified as (R)
  • a compound of formula (I) is for instance specified as E
  • E this means that the compound is substantially free of the Z isomer
  • a compound of formula (I) is for instance specified as cis, this means that the compound is substantially free of the trans isomer.
  • addition salts of the compounds of this invention refer to non toxic "pharmaceutically acceptable addition salts".
  • Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable addition salts.
  • Suitable pharmaceutically acceptable addition salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • suitable pharmaceutically acceptable addition salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
  • acids which may be used in the preparation of pharmaceutically acceptable addition salts include, but are not limited to, the following: acetic acid, 2,2-dichloroactic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucoronic acid, L-glutamic acid, beta- oxo-glutaric acid, glycolic acid, hippuric acid
  • Representative bases which may be used in the preparation of pharmaceutically acceptable addition salts include, but are not limited to, the following: ammonia, L-arginine, benethamine, benzathine, calcium hydroxide, choline, dimethylethanol- amine, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylene-diamine, l-methyl-glucamine, hydrabamine, 1 //-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, l-(2-hydroxyethyl)-pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.
  • the compounds according to the invention can generally be prepared by a succession of steps, each of which is known to the skilled person.
  • the compounds can be prepared according to the following synthesis methods.
  • the compounds of Formula (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following art-known resolution procedures.
  • the racemic compounds of Formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid.
  • Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali.
  • An alternative manner of separating the enantiomeric forms of the compounds of Formula (I) involves liquid chromatography using a chiral stationary phase.
  • Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.
  • the final compounds of Formula (I) can be prepared by reacting an intermediate compound of Formula (II) with a compound of Formula (III) according to reaction scheme (1).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, dichloromethane, a metal hydride, such as, for example sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride and may require the presence of a suitable base, such as, for example, triethylamine, and/or a Lewis acid, such as, for example titanium tetraisopropoxide or titanium tetrachloride, under thermal conditions, such as, 0 °C or room temperature, or 140 °C, for example for 1 hour or 24 hours.
  • a suitable reaction-inert solvent such as, for example, dichloromethane
  • a metal hydride such as, for example sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride
  • a suitable base such as, for
  • final compounds of Formula (I) can be prepared by reacting an intermediate compound of Formula (II) with a compound of Formula (IV), wherein halo is chloro, bromo or iodo, according to reaction scheme (2).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, acetonitrile or DMF, a suitable base, such as, for example, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine, under thermal conditions, such as, 75 °C or 80 °C, for example for 1 hour or 24 hours.
  • a suitable reaction-inert solvent such as, for example, acetonitrile or DMF
  • a suitable base such as, for example, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine, under thermal conditions, such as, 75 °C or 80 °C, for example for 1 hour or 24 hours.
  • reaction scheme (2) all variables are defined as in Formula
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, anhydrous dichloromethane, a Lewis acid, such as, for example titanium tetraisopropoxide or titanium tetrachloride, under thermal conditions, such as, 0 °C or room temperature, for example for a period of 1 hour to 24 hours.
  • a suitable reaction-inert solvent such as, for example, anhydrous dichloromethane, a Lewis acid, such as, for example titanium tetraisopropoxide or titanium tetrachloride, under thermal conditions, such as, 0 °C or room temperature, for example for a period of 1 hour to 24 hours.
  • halo is
  • final compounds of Formula (I) can be prepared by a copper coupling reaction of an intermediate compound of Formula (VII) with a compound of Formula (VIII) according to reaction scheme (4).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, isopropanol, a copper catalyst, such as, copper (I) iodide and a suitable base, such as, potassium phospate, under thermal conditions, such as, for example, 100 °C, for example for 48 hours.
  • halo is preferably bromo or iodo.
  • final compounds of Formula (I) can be prepared by “Buchwald- Hartwig cross coupling reaction” of an intermediate compound of Formula (VII) with a compound of Formula (VIII) according to reaction scheme (4).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, toluene or tetrahydrofuran, a palladium catalyst, such as, BrettPhos Pd G3 methasulfonate (CAS: 1470372-59-8), a ligand, such as, BrettPhos (CAS: 1070663-78-3) and a suitable base, such as, sodium tert-butoxide, under thermal conditions, such as, for example, 70 °C, for example for 48 hours.
  • halo is preferably chloro, bromo or iodo.
  • PG is a suitable protecting group of the nitrogen function such as, for example, p- toluenesulfonyl (Tos).
  • Suitable methods for removing such protecting group comprise but are not limited to: treatment with a base, such as, for example, sodium hydroxide, in a reaction inert solvent, such as, for example, ethanol, under thermal conditions, such as, for example, 60 °C, for a period of, for example for 30 minutes.
  • a base such as, for example, sodium hydroxide
  • a reaction inert solvent such as, for example, ethanol
  • thermal conditions such as, for example, 60 °C, for a period of, for example for 30 minutes.
  • Intermediate compounds of Formula (II) can be prepared cleaving a protecting group (PG) in an intermediate compound of Formula (X) according to reaction scheme (6).
  • PG is a suitable protecting group of the nitrogen function such as, for example, tert- butoxycarbonyl (Boc), ethoxycarbonyl, benzyl, benzyloxycarbonyl (Cbz).
  • Suitable methods for removing such protecting groups are widely known to the person skilled in the art and comprise but are not limited to: Boc deprotection: treatment with a protic acid, such as, for example, trifluoroacetic acid or hydrochloric acid, in a reaction inert solvent, such as, for example, di chi orom ethane or 1,4-dioxane; ethoxycarbonyl deprotection: treatment with a strong base, such as, for example, sodium hydroxide, in a reaction inert solvent such as for example wet tetrahydrofuran; benzyl deprotection: catalytic hydrogenation in the presence of a suitable catalyst, such as, for example, palladium on carbon, in a reaction inert solvent, such as, for example, methanol or ethanol; benzyloxycarbonyl deprotection: catalytic hydrogenation in the presence of a suitable catalyst, such as, for example, palladium on carbon, in a reaction inert solvent
  • Intermediate compounds of Formula (X) can be prepared by reaction of a compound of Formula (XI) and a halo derivative of Formula (VIII) according to reaction scheme (7).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, dimethylformamide, dimethylsulfoxide, 1,4-dioxane or 1 -butanol, and a suitable base, such as, potassium carbonate, DIPEA or Et3N, under thermal conditions, such as, for example, 60 °C or 120 °C, for example for 4 to 48 hours.
  • halo is preferably chloro, bromo or iodo.
  • PG is defined as in Formula (X).
  • intermediate compounds of Formula (X) can be prepared by “Buchwald-Hartwig cross coupling reaction” of an intermediate compound of Formula (XI) with an intermediate compound of Formula (VIII) according to reaction scheme (7).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, toluene or 1,4-dioxane, a palladium catalyst, such as, Pd 2 (dba) 3 (CAS: 51364-51-3), a ligand, such as, XPhos (CAS: 564483-18-7) orDavePhos (CAS: 213697-53-1) and a suitable base, such as, cesium carbonate or sodium tert-butoxide, under thermal conditions, such as, for example, 90 °C or 120°C, for example for 12 to 48 hours.
  • halo is preferably chloro, bromo or iodo.
  • PG is defined as in Formula (X).
  • the reaction is performed in a suitable reaction-inert solvent, such as, for example, dimethylformamide, a copper catalyst, such as, CuCL (CAS: 7447-39-4), a source of sulfur, such as, carbon disulfide and a suitable base, such as, potassium carbonate, under thermal conditions, such as, for example, 120 °C, for example for 16 hours.
  • a suitable reaction-inert solvent such as, for example, dimethylformamide
  • a copper catalyst such as, CuCL (CAS: 7447-39-4)
  • a source of sulfur such as, carbon disulfide
  • a suitable base such as, potassium carbonate
  • Intermediate compounds of Formula (VII) can be prepared by reacting an intermediate compound of Formula (Il-a), wherein R A is a suitable protecting group (PG) defined as in Formula (X), with an intermediate compound of Formula (IV) according to reaction scheme (2) and followed by cleavage of the protected group (PG) according to reaction scheme (6).
  • R A is a suitable protecting group defined as in Formula (X)
  • Intermediates of Formula (Il-a), (Il-b), (III), (IV), (V), (VI), (VIII), (XI) and (XII) are commercially available or can be prepared by known procedures to those skilled in the art.
  • O-GlcNAc hydrolase O-GlcNAc hydrolase
  • Such diseases include, but are not limited to Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler- Scheinker disease, Parkinson’s disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical glio
  • the compounds of the present invention and the pharmaceutically acceptable compositions thereof inhibit O-GlcNAc hydrolase (OGA) and therefore may be also useful in the treatment or prevention of diseases involving an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease.
  • OAA O-GlcNAc hydrolase
  • treatment is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting or stopping of the progression of a disease or an alleviation of symptoms, but does not necessarily indicate a total elimination of all symptoms.
  • prevention is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting or stopping of the onset of a disease.
  • the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in the treatment or prevention of diseases or conditions selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non-Guamanian motor neuron disease with neurofibrill
  • the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in the treatment, prevention, amelioration, control or reduction of the risk of diseases or conditions selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler- Scheinker disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non- Guamanian
  • the diseases or conditions may in particular be selected from a tauopathy, more in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or the diseases or conditions may in particular be neurodegenerative diseases accompanied by a tau pathology, more in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations.
  • a tauopathy more in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease
  • the diseases or conditions may in particular be neurodegenerative diseases accompanied by a
  • the diseases or conditions may in particular be selected from an alpha synuclinopathy, more in particular a tauopathy selected from the group consisting of Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease.
  • an alpha synuclinopathy more in particular a tauopathy selected from the group consisting of Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease.
  • Amyloid-positive (Ab+) clinically normal individuals consistently demonstrate evidence of an “AD-like endophenotype” on other biomarkers, including disrupted functional network activity in both functional magnetic resonance imaging (MRI) and resting state connectivity, fluorodeoxyglucose 18 F (FDG) hypometabolism, cortical thinning, and accelerated rates of atrophy.
  • MRI magnetic resonance imaging
  • FDG fluorodeoxyglucose 18 F
  • MCI mild cognitive impairment
  • AD dementia Alzheimer’s scientific community is of the consensus that these Ab+ clinically normal individuals represent an early stage in the continuum of AD pathology.
  • Alzheimer’s disease at a preclinical stage before the occurrence of the first symptoms.
  • All the different issues relating to preclinical Alzheimer’s disease such as, definitions and lexicon, the limits, the natural history, the markers of progression and the ethical consequences of detecting the disease at the asymptomatic stage, are reviewed in Alzheimer’s & Dementia 12 (2016) 292-323.
  • Two categories of individuals may be recognized in preclinical Alzheimer’s disease or tauopathies.
  • Cognitively normal individuals with amyloid beta or tau aggregation evident on PET scans, or changes in CSF Abeta, tau and phospho-tau are defined as being in an “asymptomatic at-risk state for Alzheimer’s disease (AR-AD)” or in a “asymptomatic state of tauopathy”.
  • AR-AD Alzheimer’s disease
  • Individuals with a fully penetrant dominant autosomal mutation for familial Alzheimer’s disease are said to have “presymptomatic Alzheimer’s disease”.
  • Dominant autosomal mutations within the tau-protein have been described for multiple forms of tauopathies as well.
  • the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in control or reduction of the risk of preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, or tau-r elated neurodegeneration as observed in different forms of tauopathies.
  • the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in control or reduction of the risk of prodromal Parkinson’s disease.
  • treatment does not necessarily indicate a total elimination of all symptoms, but may also refer to symptomatic treatment in any of the disorders mentioned above.
  • a method of treating subjects such as warm-blooded animals, including humans, suffering from or a method of preventing subjects such as warm blooded animals, including humans, suffering from any one of the diseases mentioned hereinbefore.
  • Said methods comprise the administration, i.e. the systemic or topical administration, preferably oral administration, of a prophylactically or a therapeutically effective amount of a compound of Formula (I), a stereoisomeric form thereof, a pharmaceutically acceptable addition salt or solvate thereof, to a subject such as a warm-blooded animal, including a human.
  • the invention also relates to a method for the prevention and/or treatment of any of the diseases mentioned hereinbefore comprising administering a prophylactically or a therapeutically effective amount of a compound according to the invention to a subject in need thereof.
  • the invention also relates to a method for modulating O-GlcNAc hydrolase (OGA) activity, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of a compound according to the invention and as defined in the claims or a pharmaceutical composition according to the invention and as defined in the claims.
  • OAA O-GlcNAc hydrolase
  • a method of treatment may also include administering the active ingredient on a regimen of between one and four intakes per day.
  • the compounds according to the invention are preferably formulated prior to administration.
  • suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients.
  • Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound of Formula (I) and one or more additional therapeutic agents, as well as administration of the compound of Formula (I) and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.
  • a compound of Formula (I) and a therapeutic agent may be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.
  • NBDs neurocognitive disorders
  • the present invention also provides compositions for preventing or treating diseases in which inhibition of O-GlcNAc hydrolase (OGA) is beneficial, such as Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, agryophilic grain disease, amyotrophic lateral sclerosis, frontotemporal lobe dementia caused by C90RF72 mutations, Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease, said compositions comprising a therapeutically effective amount of a compound according to formula (I) and a pharmaceutically acceptable carrier or diluent.
  • OAA O-GlcNAc hydrolase
  • the present invention further provides a pharmaceutical composition comprising a compound according to the present invention, together with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
  • compositions of this invention may be prepared by any methods well known in the art of pharmacy.
  • a therapeutically effective amount of the particular compound, in base form or addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form of preparation desired for administration.
  • a pharmaceutically acceptable carrier which may take a wide variety of forms depending on the form of preparation desired for administration.
  • These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous, or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
  • the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
  • Injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
  • These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.
  • Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
  • the exact dosage and frequency of administration depends on the particular compound of Formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
  • the pharmaceutical composition will comprise from 0.05 to 99% by weight, preferably from 0.1 to 70% by weight, more preferably from 0.1 to 50% by weight of the active ingredient, and, from 1 to 99.95% by weight, preferably from 30 to 99.9% by weight, more preferably from 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
  • the present compounds can be used for systemic administration such as oral, percutaneous or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like.
  • the compounds are preferably orally administered.
  • the exact dosage and frequency of administration depends on the particular compound according to Formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art.
  • said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
  • suitable unit doses for the compounds of the present invention can, for example, preferably contain between 0.1 mg to about 1000 mg of the active compound.
  • a preferred unit dose is between 1 mg to about 500 mg.
  • a more preferred unit dose is between 1 mg to about 300 mg.
  • Even more preferred unit dose is between 1 mg to about 100 mg.
  • Such unit doses can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration.
  • a preferred dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years.
  • the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
  • a typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient.
  • the time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • the invention also provides a kit comprising a compound according to the invention, prescribing information also known as “leaflet”, a blister package or bottle, and a container. Furthermore, the invention provides a kit comprising a pharmaceutical composition according to the invention, prescribing information also known as “leaflet”, a blister package or bottle, and a container.
  • the prescribing information preferably includes advice or instructions to a patient regarding the administration of the compound or the pharmaceutical composition according to the invention.
  • the prescribing information includes advice or instruction to a patient regarding the administration of said compound or pharmaceutical composition according to the invention, on how the compound or the pharmaceutical composition according to the invention is to be used, for the prevention and/or treatment of a tauopathy in a subject in need thereof.
  • the invention provides a kit of parts comprising a compound of Formula (I) or a stereoisomeric for thereof, or a pharmaceutically acceptable salt or a solvate thereof, or a pharmaceutical composition comprising said compound, and instructions for preventing or treating a tauopathy.
  • the kit referred to herein can be, in particular, a pharmaceutical package suitable for commercial sale.
  • compositions, methods and kits provided above, one of skill in the art will understand that preferred compounds for use in each are those compounds that are noted as preferred above. Still further preferred compounds for the compositions, methods and kits are those compounds provided in the non-limiting Examples below.
  • m.p.” means melting point
  • min means minutes
  • ACN means acetonitrile
  • aq.” means aqueous
  • Boc means /cvV-butyloxy carbonyl
  • DCM means dichloromethane
  • DMF means dimethylformamide
  • DMSO means dimethylsulfoxide
  • dppf means l,l'-ferrocenediyl-bis(diphenylphosphine)
  • CuCh means copper (II) chloride
  • Pd(PPh3)4 means tetrakis(triphenylphosphine)palladium(0)
  • Pd 2 (dba) 3 means tris(dibenzylideneacetone)dipalladium(0)
  • X-Phos means 2-dicyclohexylphosphino- 2',4',6'-tri-isopropyl-l,r-biphenyl
  • RS Whenever the notation “RS” is indicated herein, it denotes that the compound is a racemic mixture at the indicated centre, unless otherwise indicated.
  • the stereochemical configuration for centres in some compounds has been designated “A” or “A’ when the mixture(s) was separated; for some compounds, the stereochemical configuration at indicated centres has been designated as “A*” or “ V*” when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure.
  • the enantiomeric excess of compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separated enantiomer(s).
  • Microwave assisted reactions were performed in a single-mode reactor: Initiator TM Sixty EXP microwave reactor (Biotage AB).
  • TLC Thin layer chromatography
  • Tosyl Chloride (CAS: 98-59-9; 298 mg, T56 mmol) was added to a solution of 5- bromo-2-methyl-li -benzo[d]imidazole (300 mg, 1.42 mmol) and Et3N (0.22 mL, T56 mmol) in DCM (10 mL). The mixture was stirred at room temperature for 16h. Then a saturated solution of NaHC0 3 was added and the mixture was extracted with DCM.
  • reaction mixture was stirred at 75 °C for 12 h. Additional amount of intermediate 10 (99 mg, 0.45 mmol) was added and the mixture was stirred at 75 °C for 12 h. Then H 2 O was added and the mixture was extracted with EtOAc. The organic layer was separated, dried (MgS0 4 ), filtered and the solvent removed in vacuo. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from
  • Intermediate 1-3 was prepared following an analogous procedure to the one described for the synthesis of intermediate 1-2 using intermediate 28 (316 mg, 1.37 mmol) and intermediate B2.23 (707 mg, 1.51 mmol) as starting materials and stirring the mixture at 80 °C for 48 h.
  • Acetic anhydride (CAS: 108-24-7; 19.4 g, 190.05 mmol) was added to a solution of 2- amino-3 -bromo-5-fluoropyri dine (CAS: 869557-43-7; 33 g, 172.77 mmol) in toluene
  • Phosphorus pentasulfide (1.70 g, 7.67 mmol) was added to a suspension of intermediate 20 (1.38 g, 5.90 mmol) in THF (32.2 mL). The mixture was stirred at rt for 16 h and an additional amount of phosphorus pentasulfide (0.39 g, 1.77 mmol) was added. The mixture was stirred at rt for another 16 h and then CS2CO3 (3.08 g, 9.44 mmol) was added. The mixture was stirred at 70 °C for 16 h and then additional quantity of CS2CO3 (3.08 g, 9.44 mmol) was added.
  • 6-methylpyridine (CAS: 1083169-00-9; 3.58 g, 17.72 mmol), X-Phos (0.61 g, 1.29 mmol) and CS2CO3 (13.12 g, 40.27 mmol) in toluene (98 mL) under nitrogen atmosphere.
  • the mixture was stirred at 90 °C for 16 h.
  • the mixture was filtered through a pad of Celite® and washed with EtOAc.
  • the filtrate was washed with a saturated NaHC0 3 solution and the organic layer was separated, dried (Na 2 S0 4 ), filtered and the solvents evaporated in vacuo.
  • Intermediate B1.3 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 1.08 g, 5.81 mmol), 2-bromo-4-methylpyridine (CAS: 4926-28-7; 0.5 g,
  • Intermediate B1.4 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using /V-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 455 mg, 2.44 mmol), 2-bromo-5-methylpyridine (CAS: 3510-66-5; 400 mg, 2.33 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 64 mg, 0.07 mmol), DavePhos (CAS: 213697-53-1; 60 mg, 0.15 mmol) andNaO*Bu (447 mg, 4.65 mmol) in 1,4-dioxane (20 mL) as starting materials and stirring the mixture at 100 °C for 16.
  • /V-tert-butoxycarbonylpiperazine CAS: 57260-71-6; 455 mg, 2.44 mmol
  • 2-bromo-5-methylpyridine CAS: 3510-66-5; 400 mg, 2.33 mmol
  • Pd 2 (dba) 3
  • Intermediate B1.6 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.68 g, 3.66 mmol), 6-chloro-4-methoxynicotinonitrile (CAS: 1187190- 69-7; 0.41 g, 2.44 mmol) and Et3N (1.66 mL, 12.19 mmol) as starting materials.
  • A-tert-butoxycarbonylpiperazine CAS: 57260-71-6; 0.68 g, 3.66 mmol
  • 6-chloro-4-methoxynicotinonitrile CAS: 1187190- 69-7; 0.41 g, 2.44 mmol
  • Et3N (1.66 mL, 12.19 mmol
  • Intermediate B1.7 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.32 g, 1.71 mmol), 5 -bromo-2-i sopropylpyrimi di ne (CAS : 1060813-04-8; 0.33 g, 1.63 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 0.77 g, 0.08 mmol), X-Phos (0.80 g g, 0.16 mmol) and CS2CO3 (1.61 g, 4.88 mmol) as starting materials.
  • A-tert-butoxycarbonylpiperazine CAS: 57260-71-6; 0.32 g, 1.71 mmol
  • 5 -bromo-2-i sopropylpyrimi di ne CAS : 1060813-04-8; 0.33 g, 1.63 mmol
  • Intermediate B1.8 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.54 g, 2.86 mmol), 4-chloro-2,6-dimethylpyrimidine (CAS: 4472-45-1; 0.28 g, 1.90 mmol) and DIPEA (0.67 mL, 3.81 mmol) in 1,4-dioxane (6 mL) as starting materials and stirring the mixture at 50 °C for 16b. Additional amount of A-tert- butoxycarbonylpiperazine (CAS: 57260-71-6; 0.5 eq) was added and the mixture was stirred at 80 °C for 20 h.
  • A-tert-butoxycarbonylpiperazine CAS: 57260-71-6; 0.54 g, 2.86 mmol
  • 4-chloro-2,6-dimethylpyrimidine CAS: 4472-45-1; 0.28 g, 1.90
  • Intermediate B1.10 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.37 g, 1.93 mmol), 3 -chloro-6-(tri fluoromethyl)pyri dazine (CAS: 258506-68-2; 0.3 g, 1.61 mmol) and DIPEA (0.56 mb, 3.22 mmol) in 1-butanol (4 mL) as starting materials and stirring the mixture at 90 °C for 16h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.37 g, 1.93 mmol
  • 3 -chloro-6-(tri fluoromethyl)pyri dazine CAS: 258506-68-2; 0.3 g, 1.61 mmol
  • DIPEA 0.56 mb, 3.22 mmol
  • Intermediate B 1.11 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.61 g, 3.27 mmol), 2-chloro-6-methylpyrazine (CAS: 38557-71-0; 0.4 g, 3.11 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 85 mg, 0.09 mmol), DavePhos (80 mg, 0.20 mmol) and NaCfBu (0.6 g, 6.22 mmol) as starting materials.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.61 g, 3.27 mmol
  • 2-chloro-6-methylpyrazine CAS: 38557-71-0
  • Pd 2 (dba) 3 CAS: 51364-51-3; 85 mg, 0.09 mmol
  • DavePhos 80 mg, 0.20 mmol
  • NaCfBu 0.6
  • Intermediate B1.12 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.4 g, 2.15 mmol), 2-chloro-5-(trifluoromethyl)pyrazine (CAS: 799557-87-2; 0.39 g, 2.15 mmol) and K2CO3 (0.5 g, 3.65 mmol) as starting materials and stirring the mixture at 120 °C for 3h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.4 g, 2.15 mmol
  • 2-chloro-5-(trifluoromethyl)pyrazine CAS: 799557-87-2; 0.39 g, 2.15 mmol
  • K2CO3 0.5 g, 3.65 mmol
  • Intermediate B1.13 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.56 g, 3.02 mmol), 2-chloro-3 , 5 -di methyl pyrazine (CAS: 32779- 36-5; 0.4 g, 2.88 mmol), Pd2(dba)3 (CAS: 51364-51-3; 79 mg, 0.09 mmol), DavePhos (74 mg, 0.19 mmol) and NaO*Bu (0.55 g, 5.75 mmol) as starting materials.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.56 g, 3.02 mmol
  • 2-chloro-3 , 5 -di methyl pyrazine CAS: 32779- 36-5; 0.4 g, 2.88 mmol
  • Pd2(dba)3 CAS: 51364-51-3
  • Intermediate B1.15 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.32 g, 1.67 mmol), 6-chloropyridazine-3-carbonitrile (CAS: 35857-89-7; 0.2 g, 1.39 mmol) and DIPEA (0.48 mL, 2.78 mmol) in 1-butanol (4 mL) as starting materials and stirring the mixture at 90 °C for 16h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.32 g, 1.67 mmol
  • 6-chloropyridazine-3-carbonitrile CAS: 35857-89-7; 0.2 g, 1.39 mmol
  • DIPEA 0.48 mL, 2.78 mmol
  • Intermediate B1.16 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.59 g, 3.17 mmol), 2,5-bibromopyridine (CAS: 624-28-2; 0.5 g,
  • Intermediate B1.19 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.17 using intermediate B1.18 (1 g, 2.81 mmol), dppf (CAS: 12150-46-8; 140 mg, 0.25 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3;
  • Intermediate B1.20 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 11.98 g, 64.31 mmol), ethyl 2-chloropyrimidine-5-carboxylate (CAS: 89793-12-4; lOg, 53.59 mmol) and K 2 CO 3 (7.41 g, 53.59 mmol) as starting materials and stirring the mixture at 90 °C for 24 h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 11.98 g, 64.31 mmol
  • ethyl 2-chloropyrimidine-5-carboxylate CAS: 89793-12-4; lOg, 53.59 mmol
  • K 2 CO 3 7.41 g, 53.59 mmol
  • Intermediate B1.23 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 419 mg, 2.25 mmol), 4-chloroquinaldine (CAS: 4295-06-1; 0.46 mL, 2.25 mmol), Pd2(dba)3 (CAS: 51364-51-3; 103 mg, 0.11 mmol), XPhos (CAS: 564483-18-7; 107 mg, 0.23 mmol) and CS2CO3 (2.2 g, 6.76 mmol) as starting materials and stirring the mixture at 95 °C for 24 h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 419 mg, 2.25 mmol
  • 4-chloroquinaldine CAS: 4295-06-1; 0.46 mL, 2.25 mmol
  • Pd2(dba)3 CAS: 51364-51-3;
  • Intermediate B1.24 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 2.17 g, 11.63 mmol), 3 -chloro-6-( 1 H-pyrazol - 1 -yl )pyridazine
  • Intermediate B1.25 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 1.32 g, 7.11 mmol), 7-bromo-2-methylimidazo[l,2-a]pyridine (CAS: 1194375-40-0; 0.5 g, 2.37 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 46 mg, 0.05 mmol), XPhos (CAS: 564483-18-7; 86 mg, 0.15 mmol) and NaO*Bu (0.35 g, 3.55 mmol) as starting materials and stirring the mixture at 100 °C for 12 h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 1.32 g, 7.11 mmol
  • 7-bromo-2-methylimidazo[l,2-a]pyridine CAS: 1194375-40-0
  • Intermediate B1.26 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 177 mg, 0.95 mmol), intermediate 1 (347 mg, 0.95 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 44 mg, 0.05 mmol), XPhos (CAS: 564483-18-7; 55 mg, 0.095 mmol) and Cs 2 C0 3 (929 mg, 2.85 mmol) as starting materials and stirring the mixture at 105 °C for 18 h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 177 mg, 0.95 mmol
  • intermediate 1 347 mg, 0.95 mmol
  • Pd 2 (dba) 3 CAS: 51364-51-3; 44 mg, 0.05 mmol
  • XPhos CAS: 564483-18-7; 55 mg, 0.095 mmol
  • Intermediate B1.27 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.5 g, 2.68 mmol), intermediate 10 (0.58 g, 2.68 mmol) and DIPEA (1.85 mL, 10.74 mmol) in acetonitrile (10 mL) as starting materials and stirring the mixture at 80 °C for 48 h.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 0.5 g, 2.68 mmol
  • intermediate 10 (0.58 g, 2.68 mmol
  • DIPEA 1.85 mL, 10.74 mmol
  • Intermediate B1.28 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (S)- l -A'-tert-butoxy carbonyl -2- methylpiperazine (CAS: 149488-78-8; 3 g, 14.98 mmol), 2, 5 -di chloropyrazine (CAS: 19745-07-4; 1.34 g, 13.02 mmol) and DIPEA (6.46 mL, 39.07 mmol) in DMF (10 mL) as starting materials and stirring the mixture at 110 °C for 16h.
  • (S)- l -A'-tert-butoxy carbonyl -2- methylpiperazine CAS: 149488-78-8; 3 g, 14.98 mmol
  • 2, 5 -di chloropyrazine CAS: 19745-07-4; 1.34 g, 13.02 mmol
  • DIPEA 6.46 mL, 39.07 mmol
  • Intermediate B 1.29 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.17 using intermediate B 1.28 (4.13 g, 12.14 mmol), dppf (CAS: 12150-46-8; 0.61 g, 1.09 mmol), Pd 2 (dba) 3 (CAS: 51364-51- 3; 445 mg, 0.49 mmol), Zn (CAS: 7440-66-6; 159 mg, 2.43 mmol) and Zn(CN) 2 (CAS: 557-21-1; 0.8 g, 6.8 mmol) as starting materials and stirring the mixture at 100 °C for 16 h.
  • Intermediate B1.31 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-b utoxy carbonyl piperazine (CAS: 57260-71-6; 12.42 g, 66.66 mmol), 5-bromo-l, 3, 4-thiadiazol -2-amine (CAS: 37566-39-5; 10 g, 55.55 mmol) and K2CO3 (7.68 g, 55.55 mmol) in DMF (100 mL) as starting materials and stirring the mixture at 90 °C for 16h.
  • a -tert-b utoxy carbonyl piperazine CAS: 57260-71-6; 12.42 g, 66.66 mmol
  • 5-bromo-l, 3, 4-thiadiazol -2-amine CAS: 37566-39-5; 10 g, 55.55 mmol
  • K2CO3 7.68 g, 55.55 mmol
  • Acetic anhydride (CAS: 108-24-7; 8.05 g, 78.85 mmol) was added to a mixture of intermediate B1.31 (15 g, 52.56 mmol), pyridine (6.24 g, 78.85 mmol) and 4- dimethylaminopyridine (0.3 g, 2.42 mmol) in DCM (200 mL) at 0 °C. The mixture was stirred at room temperature for 16 h. Then, H2O (200 mL) was added and the mixture was extracted with DCM (6 x 300 mL). The organic layer was separated, dried (Na SCri), filtered and the solvent removed in vacuo to yield intermediate B 1.32 (15 g, 86%) as a reddish brown solid.
  • Intermediate B1.33 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 1 g, 5.37 mmol), 4-bromo-2,6-dimethylpyri dine (CAS: 5093-70-9; 1.20 g, 6.44 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 393 mg, 0.43 mmol), XPhos (CAS:
  • Intermediate B1.34 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 2.5 g, 13.42 mmol), 2-chloro-4-iodo-6-(trifluoromethyl)pyridine (CAS: 205444-22-0; 4.54 g, 14.77 mmol), Pd(OAc) 2 (CAS: 3375-31-3; 151 mg, 0.67 mmol), Xantphos (CAS: 161265-03-8; 388 mg, 0.67 mmol) and Cs 2 C0 3 (8.75 g, 26.85 mmol) in DMF (5 mL) and 1,4-dioxane (60 mL) as starting materials and stirring the mixture at 140 °C for 20 minutes under microwave irradiation.
  • a -tert-butoxy carbonyl piperazine CAS: 57260-71-6; 2.5 g, 13.42 mmol
  • Intermediate B1.39 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 169447-70-5; 1.5 g, 8.05 mmol), 5-chloropyrazine-2-carbonitrile (CAS: 36070- 75-4; 1.26 g, 8.86 mmol) and DIPEA (4.16 mL, 24.16 mmol) in DMF (20 mL) as starting materials and stirring the mixture at 60 °C for 16 h.
  • a -tert-butoxy carbonyl piperazine CAS: 169447-70-5; 1.5 g, 8.05 mmol
  • 5-chloropyrazine-2-carbonitrile CAS: 36070- 75-4; 1.26 g, 8.86 mmol
  • DIPEA 4.16 mL, 24.16 mmol
  • Intermediate B1.40 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (R)-l-/V-tert-butoxy carbonyl-2- methylpiperazine (CAS: 169447-70-5; 2 g, 9.99 mmol), 5-chloropyrazine-2- carbonitrile (CAS: 36070-75-4; 1.21 g, 8.68 mmol) and DIPEA (4.3 mL, 26.05 mmol) in DMF (7 mL) as starting materials and stirring the mixture at 70 °C for 16 h.
  • (R)-l-/V-tert-butoxy carbonyl-2- methylpiperazine CAS: 169447-70-5; 2 g, 9.99 mmol
  • 5-chloropyrazine-2- carbonitrile CAS: 36070-75-4; 1.21 g, 8.68 mmol
  • DIPEA 4.3 mL, 26.05 mmol
  • Intermediate B 1.42 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using fcrt-butyl 2,2-dimethylpiperazine-l- carboxylate (CAS: 674792-07-5; 400 mg, 1.87 mmol), 5-bromo-2-methylpyrimidine (CAS: 7752-78-5; 388 mg, 2.24 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 86 mg, 0.09 mmol), X-Phos (108 mg, 0.19 mmol) and Cs 2 C0 3 (1.82 g, 5.60 mmol) in toluene (8 mL) as starting materials and stirring the mixture at 110 °C for 16 h.
  • fcrt-butyl 2,2-dimethylpiperazine-l- carboxylate CAS: 674792-07-5; 400 mg, 1.87 mmol
  • 5-bromo-2-methylpyrimidine CAS: 7752-78-5;
  • Intermediate B 1.44 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (R)-l -A -tert-butoxy carbonyl -2- methylpiperazine (CAS: 169447-70-5; 0.5 g, 2.5 mmol), 2-chl oro-5 -fluoropyri mi dine (CAS: 62802-42-0; 0.4 g, 3 mmol) and DIPEA (0.86 mL, 5 mmol) in 2 -propanol (7 mL) as starting materials and stirring the mixture at 120 °C for 30 minutes and at 130 °C for 45 minutes under microwave irradiation.
  • Intermediate B 1.45 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using (R)-l -A -tert-butoxy carbonyl -2- methylpiperazine (CAS: 169447-70-5; 2.32 g, 11.56 mmol), 5-bromo-2- methylpyrimidine (CAS: 7752-78-5; 2 g, 11.56 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.53 g, 0.58 mmol), X-Phos (0.67 g, 1.16 mmol) and CS2CO3 (11.3 g, 34.68 mmol) in toluene (44 mL) as starting materials and stirring the mixture at 110 °C for 48 h.
  • Intermediate B 1.46 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using (S)-l -L -tert-butoxy carbonyl -2- methylpiperazine (CAS: 149488-78-8; 2.3 g, 11.48 mmol), 2-chloropyrimidine (CAS: 1722-12-9; 1.32 g, 11.48 mmol), Pd 2 (dba) 3 (CAS: 51364-51-3; 0.52 g, 0.57 mmol), X-
  • Intermediate B2.3 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B1.3 as starting material.
  • Intermediate B2.4 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.4 as starting material.
  • Intermediate B2.6 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.6 as starting material.
  • Intermediate B2.7 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.7 as starting material.
  • Intermediate B2.8 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B1.8 as starting material.
  • Intermediate B2.9 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.9 as starting material.
  • Intermediate B2.10 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.10 as starting material.
  • Intermediate B2.11 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.11 as starting material.
  • Intermediate B2.14 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.14 as starting material.
  • Intermediate B2.15 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.15 as starting material.
  • Intermediate B2.16 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.17 as starting material.
  • Intermediate B2.17 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.19 as starting material.
  • Intermediate B2.19 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.22 as starting material.
  • Intermediate B2.20 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.23 as starting material.
  • Intermediate B2.21 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.24 as starting material.
  • Intermediate B2.22 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.25 as starting material.
  • Intermediate B2.24 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using 1 -(5-methyl- 1 ,2,4-oxadiazol-3 - yl)piperazine (prepared as described in Bioorganic & Medicinal Chemistry, 16(4), 1613-1631; 2008) as starting material.
  • Intermediate B2.25 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.1 using intermediate B 1.27 as starting material.
  • Intermediate B2.32a was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.29a using 4-[5-(acetylamino)-2- pyrimidinyl]-l-piperazinecarboxylic acid , 1,1-dimethylethyl ester (prepared as described in PCT Int. Appl., 2016030443; 5 g, 15.56 mmol) as starting material.
  • intermediate B2.32a (x HC1 salt, 400 mg, 1.55 mmol) in MeOH (6 mL) and EhO (2 mL) was passed through an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of N3 ⁇ 4 in MeOH. The desired fractions were collected and concentrated in vacuo to afford intermediate B2.32b (280 mg, 82%) as a brown solid.
  • Intermediate B2.36 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.32 using intermediate B 1.41 as starting material.
  • Intermediate B2.38 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.43 as starting material.
  • Intermediate B2.39 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.44 as starting material.
  • Compound 5 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.5 (150 mg, 0.65 mmol) as starting materials and heating the mixture at 75 °C for 18 h.
  • the mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo.
  • Product 9 (118 mg) was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the residue was triturated with Et 2 0 to afford product 9a (HC1 salt, 119 mg) as a white solid.
  • Compound 11 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.11 (151 mg, 0.55 mmol) as starting materials and heating the mixture at 80 °C for 24 h.
  • the mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96).
  • Compound 14 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (133 mg, 0.61 mmol) and intermediate B2.14 (165 mg, 0.73 mmol) as starting materials and heating the mixture at 80 °C for 24 h.
  • the mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96).
  • Compound 16 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (276 mg, 1.27 mmol) and intermediate B2.15 (120 mg, 0.63 mmol) as starting materials and heating the mixture at 75 °C for 24 h.
  • the mixture was purified by flash column chromatography (silica, dry load in silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo to yield product 16 (45 mg, 18%) as a light yellow oil.
  • the residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 70% [25mM NH4HCO3] - 30% [ACNMeOH (1:1)] to 27% [25mM NH4HCO3] - 73% [ACNMeOH (1 : 1)]).
  • the desired fractions were evaporated in vacuo and the residue was triturated in
  • Compound 20 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (119 mg, 0.55 mmol) and intermediate B2.19 (100 mg, 0.45 mmol) as starting materials and heating the mixture at 75 °C for 40 h.
  • the mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 2.3/97.7). The desired fractions were evaporated in vacuo.
  • the residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 95% [0.1% HCOOH] - 5% [MeCN:MeOH (1:1)] to 70% [0.1% HCOOH] - 30% [MeCN:MeOH (1:1)]).
  • the desired fractions were neutralized with a saturated NaHC0 3 solution, partially evaporated in vacuo and extracted with DCM.
  • the organic layer was separated, dried (MgS0 4 ), filtered and the solvent removed in vacuo to yield product 20 (62 mg, 34%) as a white foamy solid.
  • Compound 23 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (115 mg, 0.53 mmol) and intermediate B2.22 (88 mg, 0.41 mmol) as starting materials and heating the mixture at 75 °C for 40 h.
  • the mixture was purified by flash column chromatography (silica, NH 3 (7M in MeOH)/MeOH/DCM, gradient from 0/0/100 to 20/1/1). The desired fractions were evaporated in vacuo.
  • the residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm), from 95% [0.1% TFA] - 5% [MeCNMeOH (1:1)] to 63% [0.1% TFA] - 37% [MeCNMeOH (1:1)]).
  • the desired fractions were evaporated in vacuo.
  • the residue was purified by reverse phase chromatography from 72% [25mM NH 4 HCO 3 ] - 28% [MeCNMeOH (1:1)] to 36% [25mM NH 4 HCO 3 ] - 64% [MeCNMeOH (1:1)]).
  • the desired fractions were evaporated in vacuo to yield product 23 (26 mg, 16%) as a white foamy solid.
  • Ethylene glycol (CAS: 107-21-1; 0.0125 mL, 0.22 mmol), copper(I) iodide (CAS: 7681-65-4; 9 mg, 0.05 mmol) and potassium phosphate tribasic (191 mg, 0.90 mmol) were added to a solution of l-methyl-4-iodo-lH-pyrazole (CAS: 39806-90-1; 56 mg, 0.27 mmol) and intermediate B2.25 (60 mg, 0.23 mmol) in ‘PrOH (0.8 mL). The mixture was stirred at 100 °C for 48 h. The solvents were evaporated in vacuo. Then the reaction was treated with LEO and extracted with DCM.
  • Product 27 (38 mg) was taken into EEO and treated with HC1 (2N in EEO, 4 eq). The mixture was stirred at room temperature for 1 h. The solvents were evaporated in vacuo and the residue was triturated with EEO to afford product 27 (HC1 salt, 32 mg, 76%) as a white solid.
  • Product 28 (80 mg) and product 29 (86 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et 2 0, 4 eq). The mixtures were stirred at room temperature for 30 minutes. The solvents were evaporated in vacuo and the products were triturated with Et 2 0 to yield product 28 (HC1 salt, 82 mg, 93%) and product 29 (HC1 salt, 85 mg, 90%) both as white solids.
  • the mixture was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: from 70% NH 4 HCO 3 0.25% solution in water, 30% C3 ⁇ 4CN to 35% NH 4 HCO 3 0.25% solution in Water, 65% CH3CN) to yield products 30 (30 mg, 9%), 31 (30 mg, 9%), 32 (25 mg, 7%) and 33 (22 mg, 6%) as solids.
  • the crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo.
  • the residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C3 ⁇ 4CN to 60% NH4HCO3 0.25% solution in Water, 40% CFECN).
  • the aqueous phase was extracted with EtOAc, separated, dried (Na 2 S04), filtered and the solvent evaporated in vacuo to yield product 36 (29 mg, 8%) as a white solid.
  • the mixture was treated with a saturated solution of NFECl and DCM and filtered through a pad of Celite®.
  • the organic layer was separated, dried (MgSCE), filtered and the solvents evaporated in vacuo.
  • the crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo.
  • Ti(Oi-Pr)4 (CAS: 546-68-9; 1.5 eq, 0.18 mL) was added to a stirred solution of intermediate B2.31 (100 mg, 0.41 mmol) and 2,3-dihydro-[l,4]-dioxino[2,3-b]pyridine- 6-carbaldehyde (CAS: 615568-24-6; 1.2 eq, 81 mg) in DCM (1.8 mL) under nitrogen atmosphere. The mixture was stirred at room temperature for 16 h. Then, the mixture was cooled at 0 °C and methylmagnesium bromide (1.4M in THF and toluene, 5 eq, 1.46 mL) was added dropwise.
  • intermediate B2.31 100 mg, 0.41 mmol
  • 2,3-dihydro-[l,4]-dioxino[2,3-b]pyridine- 6-carbaldehyde (CAS: 615568-24-6; 1.2 eq, 81 mg
  • the mixture was stirred at 0 °C for 15 minutes and at room temperature for 2 h. Then the mixture was treated with a saturated solution of NH4CI and extracted with DCM. The mixture was filtered through a pad of Celite®. The organic layer was separated, dried (NaiSCE), filtered and the solvents evaporated in vacuo. The residue was treated with 2,3-dihydro-[l,4]-dioxino[2,3-b]pyridine-6- carbaldehyde (0.72 eq, 48 mg), Ti(Oi-Pr)4 (0.9 eq, 0.11 mL) and DCM (1.1 mL) under nitrogen atmosphere. The mixture was stirred at rt for 16 h.
  • the residue was purified by flash column chromatography (silica, EtOAc in DCM, gradient from 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo.
  • the residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 67% NH4HCO3 0.25% solution in Water, 33% C3 ⁇ 4CN to 50% NH4HCO3 0.25% solution in Water,
  • Product 44 was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 60% NH4HCO3 0.25% solution in Water, 40% MeOH to 37% NH4HCO3 0.25% solution in Water, 63% MeOH). The desired fractions were collected and concentrated in vacuo to yield product 44 (46 mg, 14%) as a light yellow oil.
  • Product 45 was purified by RP HPLC (stationary phase: XBridge C18 50 x 100 mm, 5 pm, mobile phase: gradient from 67% NH4HCO3 0.25% solution in Water, 33% C3 ⁇ 4CN to 50% NH4HCO3 0.25% solution in Water, 50% C3 ⁇ 4CN). The desired fractions were collected and concentrated in vacuo to yield product 45 (98 mg, 30%) as a yellow oil.
  • Product 48 was purified via chiral SFC (stationary phase: Chiralpak IC 5pm 250*21.2mm, mobile phase: 50% CO2, 50% iPOH(0.3% iPrNFb)) to give product 49 (45 mg, 18%) and product 50 (46 mg, 18%).
  • Product 49 was triturated with Et 2 0 to yield product 49 (35 mg, 14%) as a white solid.
  • Product 50 was triturated with Et 2 0 to yield product 50 (37 mg, 15%) as a cream solid.
  • product 55 (x HC1 salt, 25 mg) as a white solid.
  • Product 55 (130 mg) was purified via chiral SFC (stationary phase: CHIRACEL OJ-H 5pm 250*20mm, mobile phase: 70% CO2, 30% EtOH (0.3% iPrNFL)) to yield product 56 (46 mg, 18%) and product 57 (45 mg, 18%).
  • Product 56 (46 mg) was taken up in diethyl ether (2 mL) and treated with HC1 (1M in Et 2 0, 0.14 mL) and the mixture was stirred at room temperature for 30 minutes. The solid was filtered to yield product 56 (x 2HC1 salt, 52 mg) as a white solid.
  • Product 61 was passed through an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of NH 3 in MeOH. The desired fractions obtained with 7M solution of NH 3 in MeOH were collected and concentrated in vacuo to afford product 61 (15 mg, 13%) as a white solid.
  • Compound 64 was prepared following an analogous procedure to the one described for the synthesis of compound 36 using intermediate B2.32b (80 mg, 0.36 mmol), intermediate 26 (80 mg, 0.38 mmol), Ti(Oi-Pr)4 (CAS: 546-68-9; 0.16 mL, 0.54 mmol) and sodium cyanoborohydride (CAS: 25895-60-7; 27 mg, 0.43 mmol) in THF (3 mL) as starting materials.
  • the crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo.
  • Product 65 was purified via chiral SFC (Stationary phase: Chiralcel OD-H 5pm 250x21.2mm, Mobile phase: 68% CO2, 32% iPrOH) to yield two fractions which were purified via preparative LC (Stationary phase: irregular bare silica 24g, Mobile phase: 95% DCM, 5% MeOH) to yield product 66 (68 mg, 19%) and 67 (60 mg).
  • Product 67 was purified by RP HPLC (Stationary phase: Cl 8 XB ridge 30 x 100 mm 5 pm, Mobile phase: Gradient from 90% NH 4 HCO 3 0.25% solution in Water, 10% CH 3 CN to 60% NH 4 HCO 3 0.25% solution in Water, 40% CH 3 CN) to yield product 67 (30 mg, 8%).
  • Product 71 was purified via chiral SFC (Stationary phase: CHIRACEL OJ-H 5pm 250*30mm, Mobile phase: 70% CO2, 30% EtOH) to yield product 72 (30 mg, 14%) and product 73 (27 mg, 13%).
  • PREPARATION OF PRODUCT 82 Compound 82 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.15 (163 mg, 0.43 mmol) and intermediate 28 (109 mg, 0.47 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 mih, mobile phase: from 81% [25mM NH4HCO3] - 19% [100% de ACN] to 45% [25mM NH4HCO3] - 55% [100% de ACN]). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 82 (70 mg, 42%) E65. PREPARATION OF PRODUCT 83
  • Product 84 (75 mg) was purified via chiral SFC (stationary phase: CHIRALPAK AD-H 5pm 250*21.2mm, mobile phase: 60% CO2, 40% MeOH) to yield product 86 (42 mg) and product 87 (39 mg) both as light yellow solids.
  • chiral SFC stationary phase: CHIRALPAK AD-H 5pm 250*21.2mm, mobile phase: 60% CO2, 40% MeOH
  • Compound 102 was prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (464 mg, 2.13 mmol), intermediate B2.36 (400 mg, 1.94 mmol) and DIPEA (1.34 mL, 7.76 mmol) as starting materials and heating the mixture at 70 °C for 72 h.
  • the residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo.
  • the residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C3 ⁇ 4CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo. The residue was treated with methyl tert- butyl ether to yield product 102 (20 mg, 3%) as a pale yellow solid.
  • Compound 103 was prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (230 mg, 1.06 mmol), intermediate B2.37 (168 mg, 0.81 mmol) and DIPEA (0.56 mL, 3.26 mmol) as starting materials and heating the mixture at 70 °C for 72 h.
  • the residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo.
  • Product 104 (45 mg) and product 105 (44 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et 2 0, 4 eq). The mixture was stirred at room temperature for 30 minutes. The mixture was filtered off to yield product 104 (HC1 salt, 37 mg, 75%) and product 105 (HC1 salt, 43 mg, 89%) both as white solids.
  • Product 106 60 mg and product 107 (49 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et 2 0, 4 eq). The mixture was stirred at room temperature for 30 minutes. The mixture was filtered off to yield product 106 (HC1 salt, 52 mg, 79%) and product 107 (HC1 salt, 52 mg, 96%) both as white solids.
  • Values are peak values and are obtained with experimental uncertainties that are commonly associated with this analytical method.
  • Mettler Toledo MP50 (B) For a number of compounds, melting points were determined in open capillary tubes on a Mettler FP 81HT / FP90 apparatus. Melting points were measured with a temperature gradient of 1, 3, 5 or 10 °C/minute. Maximum temperature was 300 °C. The melting point was read from a digital display (B).
  • HPLC High Performance Liquid Chromatography
  • MS Mass Spectrometer
  • the SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO2) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS). It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time%) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software. TABLE 5.
  • SFC Analytical Supercritical fluid chromatography
  • Analytical SFC-MS Methods (Flow expressed in mL/min; column temperature (T) in °C; run time in minutes; backpressure (BPR) in bars. TABLE 6.
  • Analytical SFC data - R t means retention time (in minutes), [M+H] + means the protonated mass of the compound, method refers to the method used for (SFC)MS analysis of enantiomerically pure compounds.
  • the assay is based on the inhibition of the hydrolysis of fluorescein mono-B-D-N- Acetyl-Glucosamine (FM-GlcNAc) (Mariappa et al. 2015, Biochem J 470:255) by the recombinant human Meningioma Expressed Antigen 5 (MGEA5), also referred to as O-GlcNAcase (OGA).
  • MGEA5 Meningioma Expressed Antigen 5
  • O-GlcNAcase O-GlcNAcase
  • the hydrolysis FM-GlcNAc Marker Gene technologies, cat # M1485) results in the formation of B-D-N-glucosamineacetate and fluorescein.
  • the fluorescence of the latter can be measured at excitation wavelength 485 nm and emission wavelength 538nm. An increase in enzyme activity results in an increase in fluorescence signal.
  • Full length OGA enzyme was purchased at OriGene (cat # TP322411). The enzyme was stored in 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol at -20 °C. Thiamet G and GlcNAcStatin were tested as reference compounds (Yuzwa et al. 2008 Nature Chemical Biology 4:483; Yuzwa et al. 2012 Nature Chemical Biology 8:393). The assay was performed in 200mM Citrate/phosphate buffer supplemented with 0.005% Tween-20. 35.6 g Na 2 HP0 4 2 3 ⁇ 40 (Sigma, # C0759) were dissolved in 1 L water to obtain a 200 mM solution.
  • citric acid (Merck, # 1.06580) was dissolved in 1 L water to obtain a 100 mM solution. pH of the sodiumphosphate solution was adjusted with the citric acid solution to 7.2.
  • the buffer to stop the reaction consists of a 500 mM Carbonate buffer, pH 11.0. 734 mg FM-GlcNAc were dissolved in 5.48 mL DMSO to obtain a 250 mM solution and was stored at -20 °C. OGA was used at a 2nM concentration and FM-GlcNAc at a lOOuM final concentration. Dilutions were prepared in assay buffer.
  • HEK293 cells inducible for P301L mutant human Tau were established at Janssen.
  • Thiamet-G was used for both plate validation (high control) and as reference compound (reference EC50 assay validation).
  • OGA inhibition is evaluated through the immunocytochemical (ICC) detection of O-GlcNAcylated proteins by the use of a monoclonal antibody (CTD110.6; Cell Signaling, #9875) detecting O- GlcNAcylated residues as previously described (Dorfmueller et al. 2010 Chemistry & biology, 17:1250). Inhibition of OGA will result in an increase of O- GlcNAcylated protein levels resulting in an increased signal in the experiment.
  • ICC pictures are imaged with a Perkin Elmer Opera Phenix plate microscope and quantified with the provided software Perkin Elmer Harmony 4.1.
  • Cells were propagated in DMEM high Glucose (Sigma, #D5796) following standard procedures. 2 days before the cell assay cells are split, counted and seeded in Poly-D- Lysine (PDL) coated 96-wells (Greiner, #655946) plate at a cell density of 12,000 cells per cm 2 (4,000 cells per well) in IOOmI of Assay Medium (Low Glucose medium is used to reduce basal levels of GlcNAcylation) (Park et al. 2014 The Journal of biological chemistry 289: 13519). At the day of compound test medium from assay plates was removed and replenished with 90pl of fresh Assay Medium.
  • PDL Poly-D- Lysine
  • Imaging is performed using Perkin Elmer Phenix Opera using a water 20x objective and recording 9 fields per well.
  • Intensity readout at 488nm is used as a measure of O-GlcNAcylation level of total proteins in wells.
  • IC o-values are calculated using parametric non-linear regression model fitting. As a maximum inhibition Thiamet G at a 200uM concentration is present on each plate. In addition, a concentration response of Thiamet G is calculated on each plate.

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Abstract

The present invention relates to O-GlcNAc hydrolase (OGA) inhibitors. The invention is also directed to pharmaceutical compositions comprising such compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which inhibition of OGA is beneficial, such as tauopathies, in particular Alzheimer's disease or progressive supranuclear palsy; and neurodegenerative diseases accompanied by a tau pathology, in particular amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C9ORF72 mutations; or alpha synucleinopathies, in particular Parkinson's disease, dementia due to Parkinson's (or neurocognitive disorder due to Parkinson's disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher's disease.

Description

OGA INHIBITOR COMPOUNDS
FIELD OF THE INVENTION The present invention relates to O-GlcNAc hydrolase (OGA) inhibitors, having the structure shown in Formula (I)
Figure imgf000002_0001
wherein the radicals are as defined in the specification. The invention is also directed to pharmaceutical compositions comprising such compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which inhibition of OGA is beneficial, such as tauopathies, in particular Alzheimer’s disease or progressive supranuclear palsy; and neurodegenerative diseases accompanied by a tau pathology, in particular amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations; or alpha synucleinopathies, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease. BACKGROUND OF THE INVENTION
O-GlcNAcylation is a reversible modification of proteins where N-acetyl-D- glucosamine residues are transferred to the hydroxyl groups of serine- and threonine residues yield O-GlcNAcylated proteins. More than 1000 of such target proteins have been identified both in the cytosol and nucleus of eukaryotes. The modification is thought to regulate a huge spectrum of cellular processes including transcription, cytoskeletal processes, cell cycle, proteasomal degradation, and receptor signalling.
O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA) are the only two proteins described that add (OGT) or remove (OGA) O-GlcNAc from target proteins. OGA was initially purified in 1994 from spleen preparation and 1998 identified as antigen expressed by meningiomas and termed MGEA5, consists of 916 amino (102915 Dalton) as a monomer in the cytosolic compartment of cells. It is to be distinguished from ER- and Golgi-related glycosylation processes that are important for trafficking and secretion of proteins and different to OGA have an acidic pH optimum, whereas OGA display highest activity at neutral pH.
The OGA catalytic domain with its double aspartate catalytic center resides in the N- terminal part of the enzyme which is flanked by two flexible domains. The C-terminal part consists of a putative HAT (histone acetyl transferase domain) preceded by a stalk domain. It has yet still to be proven that the HAT-domain is catalytically active.
O-GlcNAcylated proteins as well as OGT and OGA themselves are particularly abundant in the brain and neurons suggesting this modification plays an important role in the central nervous system. Indeed, studies confirmed that O-GlcNAcylation represents a key regulatory mechanism contributing to neuronal communication, memory formation and neurodegenerative disease. Moreover, it has been shown that OGT is essential for embryogenesis in several animal models and ogt null mice are embryonic lethal. OGA is also indispensible for mammalian development. Two independent studies have shown that OGA homozygous null mice do not survive beyond 24-48 hours after birth. Oga deletion has led to defects in glycogen mobilization in pups and it caused genomic instability linked cell cycle arrest in MEFs derived from homozygous knockout embryos. The heterozygous animals survived to adulthood however they exhibited alterations in both transcription and metabolism.
It is known that perturbations in O-GlcNAc cycling impact chronic metabolic diseases such as diabetes, as well as cancer. Oga heterozygosity suppressed intestinal tumorigenesis in an Ape- mouse cancer model and the Oga gene ( MGEA5 ) is a documented human diabetes susceptibility locus.
In addition, O-GlcNAc-modifications have been identified on several proteins that are involved in the development and progression of neurodegenerative diseases and a correlation between variations of O-GlcNAc levels on the formation of neurofibrillary tangle (NFT) protein by Tau in Alzheimer’s disease has been suggested. In addition, O-GlcNAcylation of alpha-synuclein in Parkinson’s disease has been described (Levine, PM, et al. PNAS January 29, 2019, Vol. 116, No. 5, pp 1511-1519; Lewis, YE et al. ACS Chem Biol. 2017 Apr 21, Vol. 2, No. 4, pp 1020-1027; Marotta, NP et al. Nat Chem. 2015 Nov, Vol. No. 11, pp. 913-20).
In the central nervous system six splice variants of tau have been described. Tau is encoded on chromosome 17 and consists in its longest splice variant expressed in the central nervous system of 441 amino acids. These isoforms differ by two N-terminal inserts (exon 2 and 3) and exon 10 which lie within the microtubule binding domain. Exon 10 is of considerable interest in tauopathies as it harbours multiple mutations that render tau prone to aggregation as described below. Tau protein binds to and stabilizes the neuronal microtubule cytoskeleton which is important for regulation of the intracellular transport of organelles along the axonal compartments. Thus, tau plays an important role in the formation of axons and maintenance of their integrity. In addition, a role in the physiology of dendritic spines has been suggested as well.
Tau aggregation is either one of the underlying causes for a variety of so called tauopathies like PSP (progressive supranuclear palsy), Down’s syndrome (DS), FTLD (frontotemporal lobe dementia), FTDP-17 (frontotemporal dementia with Parkinsonism- 17), Pick’s disease (PD), CBD (corticobasal degeneration), agryophilic grain disease (AGD), and AD (Alzheimer’s disease). In addition, tau pathology accompanies additional neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) or FTLD cause by C90RF72 mutations. In these diseases, tau is post- translationally modified by excessive phosphorylation which is thought to detach tau from microtubules and makes it prone to aggregation. O-GlcNAcylation of tau regulates the extent of phosphorylation as serine or threonine residues carrying O- GlcNAc-residues are not amenable to phosphorylation. This effectively renders tau less prone to detaching from microtubules and reduces aggregation into neurotoxic tangles which ultimately lead to neurotoxicity and neuronal cell death. This mechanism may also reduce the cell-to-cell spreading of tau-aggregates released by neurons via along interconnected circuits in the brain which has recently been discussed to accelerate pathology in tau-related dementias. Indeed, hyperphosphorylated tau isolated from brains of AD-patients showed significantly reduced O-GlcNAcylation levels.
An OGA inhibitor administered to TNPL3 tau transgenic mice successfully reduced NFT formation and neuronal loss without apparent adverse effects. This observation has been confirmed in another rodent model of tauopathy where the expression of mutant tau found in FTD can be induced (tg4510). Dosing of a small molecule inhibitor of OGA was efficacious in reducing the formation of tau-aggregation and attenuated the cortical atrophy and ventricle enlargement.
Moreover, the O-GlcNAcylation of the amyloid precursor protein (APP) favours processing via the non-amyl oidogenic route to produce soluble APP fragment and avoid cleavage that results in the AD associated amyloid-beta (Ab) formation.
Maintaining O-GlcNAcylation of tau by inhibition of OGA represents a potential approach to decrease tau-phosphorylation and tau-aggregation in neurodegenerative diseases mentioned above thereby attenuating or stopping the progression of neurodegenerative tauopathy-diseases.
WO2012/117219 (Summit Corp. pic., published 7 September 2012) describes N-[[5- (hydroxymethyl)pyrrolidin-2-yl]methyl]alkylamide and N-alkyl-2-[5- (hydroxymethyl)pyrrolidin-2-yl]acetamide derivatives as OGA inhibitors.
WO2014/159234 (Merck Patent GMBH, published 2 October 2014) discloses mainly 4-phenyl or benzyl-piperidine and piperazine compounds substituted at the 1 -position with an acetamido-thiazolylmethyl or acetamidoxazolylmethyl substituent and the compound N-[5-[(3-phenyl-l-piperidyl)methyl]thiazol-2-yl]acetamide;
WO20 16/0300443 (Asceneuron S.A., published 3 March 2016), WO2017/144633 and W02017/0114639 (Asceneuron S.A., published 31 August 2017) disclose 1,4- disubstituted piperidines or piperazines as OGA inhibitors;
WO2017/144637 (Asceneuron S.A, published 31 August 2017) discloses more particular 4-substituted l-[l-(l,3-benzodioxol-5-yl)ethyl]-piperazine; l-[l-(2,3- dihydrobenzofuran-5-yl)ethyl]-; l-[l-(2,3-dihydrobenzofuran-6-yl)ethyl]-; and 1-[1- (2,3-dihydro-l,4-benzodioxin-6-yl)ethyl]-piperazine derivatives as OGA inhibitors;
WO2017/106254 (Merck Sharp & Dohme Corp.) describes substituted N-[5-[(4- methylene-l-piperidyl)methyl]thiazol-2-yl]acetamide; WO2018/217558 (Eli Lilly and Company) describes 5-methyl-l,3,4-oxadiazol-2-yl and WO2019/178191 (Biogen Ma Inc) discloses [(hetero)aryl-3-ylmethyl]pyrrolidin-l-ylmethyl- and [(hetero)aryl-3- ylmethyl]piperidin-l-ylmethyl- derivative compounds as OGA inhibitors; and WO2018/140299 (Eli Lilly and Company) discloses N-[fhioro-5-[[(2S,4S)-2-methyl-4- [(5-methyl-l,2,4-oxadiazol-3-yl)methoxy[-l-piperidyl]methyl]thiazol-2-yl]acetamide as OGA inhibitor.
There is still a need for OGA inhibitor compounds with an advantageous balance of properties, for example with improved potency, good bioavailability, pharmacokinetics, and brain penetration, and/or better toxicity profile. It is accordingly an object of the present invention to provide compounds that overcome at least some of these problems. SUMMARY OF THE INVENTION
The present invention is directed to compounds of Formula (I)
Figure imgf000006_0001
and the tautomers and the stereoisomeric forms thereof, wherein RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thienyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- ajpyridinyl, imidazo[l,5-a]pyridinyl, imidazo[l,5-b]pyridazinyl, indolizinyl, 1H- indolyl, lH-indazolyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; cyano; hydroxy; phenyl; pyrazolyl; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
-NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci- 4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or C¾; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000007_0001
wherein Rbl represents hydrogen or fluoro; and the pharmaceutically acceptable salts and the solvates thereof.
Illustrative of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the compounds described above. An illustration of the invention is a pharmaceutical composition made by mixing any of the compounds described above and a pharmaceutically acceptable carrier. Illustrating the invention is a process for making a pharmaceutical composition comprising mixing any of the compounds described above and a pharmaceutically acceptable carrier. Exemplifying the invention are methods of preventing or treating a disorder mediated by the inhibition of O-GlcNAc hydrolase (OGA), comprising administering to a subject in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
Further exemplifying the invention are methods of inhibiting OGA, comprising administering to a subject in need thereof a prophylactically or a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
An example of the invention is a method of preventing or treating a disorder selected from a tauopathy, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or a neurodegenerative disease accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations, or preventing or treating a disorder selected from an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
Another example of the invention is any of the compounds described above for use in preventing or treating a tauopathy, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or a neurodegenerative disease accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations or for use in preventing or treating a disorder selected from an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease,, in a subject in need thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compounds of Formula (I), as defined herein before, and pharmaceutically acceptable addition salts and solvates thereof. The compounds of Formula (I) are inhibitors of O-GlcNAc hydrolase (OGA) and may be useful in the prevention or treatment of tauopathies, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or maybe useful in the prevention or treatment of neurodegenerative diseases accompanied by a tau pathology, in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations; or may be useful in the prevention or treatment of alpha synucleinopathies, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease.
In a particular embodiment, the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein
RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- ajpyridinyl, imidazo[l,5-a]pyridinyl, imidazo[l,5-b]pyridazinyl, indolizinyl, 1H- indolyl, lH-indazolyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; cyano; pyrazolyl; Ci- 4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
-NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci- 4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or C¾; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000009_0001
wherein Rbl represents hydrogen or fluoro.
In a further embodiment, the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, imidazo[l,2-a]pyridinyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; pyrazolyl; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents; -NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci-4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or C¾; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000010_0001
wherein Rbl represents hydrogen or fluoro.
In a further embodiment, the invention is directed to compounds of Formula (I) as referred to herein, and the tautomers and the stereoisomeric forms thereof, wherein
RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from quinolinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents;
Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents; -NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is and Ci-4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla and Rlb are each independently selected from the group consisting of hydrogen and Ci-4alkyl, in particular hydrogen and methyl; and Rlc and Rld are each hydrogen;
R2 represents H or C¾; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000011_0001
In a further embodiment, the invention is directed to compounds of Formula (I) as described herein, wherein xx
RA is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; and Ci- 4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are all hydrogen;
R2 represents CFF; and RB is a radical selected from the group consisting of (b-1) and (b-2):
Figure imgf000012_0001
(b-1) (b-2) wherein Rbl represents fluoro.
In a further embodiment, the invention is directed to compounds of Formula (I) as described herein, wherein
RA is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl; and Ci-4alkyloxy; Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are all hydrogen;
R2 represents CFF; and
RB is a radical selected from the group consisting of (b-1) and (b-2):
Figure imgf000012_0002
(b-1) (b-2) wherein Rbl represents fluoro.
In a further embodiment, the invention is directed to compounds of Formula (I) as described herein, wherein
RA is a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; each optionally substituted with a substituent selected from the group consisting of cyano and Ci-4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are all hydrogen; R2 represents C¾; and
RB is a radical selected from the group consisting of (b-1) and (b-2):
Figure imgf000013_0001
DEFINITIONS
“Halo” shall denote fluoro, chloro and bromo; “Ci^alkyl” shall denote a straight or branched saturated alkyl group having 1, 2, 3 or 4 carbon atoms, respectively e.g. methyl, ethyl, 1 -propyl, 2-propyl, butyl, 1 -methyl-propyl, 2-methyl- 1 -propyl, 1,1-dimethylethyl, and the like; “Ci-4alkyloxy” shall denote an ether radical wherein Ci-4alkyl is as defined before.
In general, whenever the term “substituted” is used in the present invention, it is meant, unless otherwise indicated or is clear from the context, to indicate that one or more hydrogens, in particular 1 to 3 hydrogens, preferably 1 or 2 hydrogens, more preferably 1 hydrogen, on the atom or radical indicated in the expression using “substituted” are replaced with a selection of substituents from the indicated group, provided that the normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.
The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, who is or has been the object of treatment, observation or experiment. As used herein, the term “subject” therefore encompasses patients, as well as asymptomatic or presymptomatic individuals at risk of developing a disease or condition as defined herein.
The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. The term "prophylactically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that substantially reduces the potential for onset of the disease or disorder being prevented.
As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
Hereinbefore and hereinafter, the term “compound of Formula (I)” is meant to include the addition salts, the solvates and the stereoisomers thereof.
The terms “stereoisomers” or “stereochemically isomeric forms” hereinbefore or hereinafter are used interchangeably.
The invention includes all stereoisomers of the compound of Formula (I) either as a pure stereoisomer or as a mixture of two or more stereoisomers.
Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If a compound contains a double bond, the substituents may be in the E or the Z configuration. If a compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration. Therefore, the invention includes enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof.
The absolute configuration is specified according to the Cahn-Ingold-Prelog system. The configuration at an asymmetric atom is specified by either R or S. Resolved compounds whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate plane polarized light.
When a specific stereoisomer is identified, this means that said stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1%, of the other isomers. Thus, when a compound of formula (I) is for instance specified as (R), this means that the compound is substantially free of the (S) isomer; when a compound of formula (I) is for instance specified as E, this means that the compound is substantially free of the Z isomer; when a compound of formula (I) is for instance specified as cis, this means that the compound is substantially free of the trans isomer. For use in medicine, the addition salts of the compounds of this invention refer to non toxic "pharmaceutically acceptable addition salts". Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable addition salts. Suitable pharmaceutically acceptable addition salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable addition salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
Representative acids which may be used in the preparation of pharmaceutically acceptable addition salts include, but are not limited to, the following: acetic acid, 2,2-dichloroactic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucoronic acid, L-glutamic acid, beta- oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene- 1,5- disulfonic acid, 1 -hydroxy -2 -naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L- pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoromethylsulfonic acid, and undecylenic acid. Representative bases which may be used in the preparation of pharmaceutically acceptable addition salts include, but are not limited to, the following: ammonia, L-arginine, benethamine, benzathine, calcium hydroxide, choline, dimethylethanol- amine, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylene-diamine, l-methyl-glucamine, hydrabamine, 1 //-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, l-(2-hydroxyethyl)-pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.
The names of compounds were generated according to the nomenclature rules agreed upon by the Chemical Abstracts Service (CAS) or according to the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC).
PREPARATION OF THE FINAL COMPOUNDS
The compounds according to the invention can generally be prepared by a succession of steps, each of which is known to the skilled person. In particular, the compounds can be prepared according to the following synthesis methods.
The compounds of Formula (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following art-known resolution procedures. The racemic compounds of Formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid.
Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali. An alternative manner of separating the enantiomeric forms of the compounds of Formula (I) involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.
EXPERIMENTAL PROCEDURE 1
The final compounds of Formula (I) can be prepared by reacting an intermediate compound of Formula (II) with a compound of Formula (III) according to reaction scheme (1). The reaction is performed in a suitable reaction-inert solvent, such as, for example, dichloromethane, a metal hydride, such as, for example sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride and may require the presence of a suitable base, such as, for example, triethylamine, and/or a Lewis acid, such as, for example titanium tetraisopropoxide or titanium tetrachloride, under thermal conditions, such as, 0 °C or room temperature, or 140 °C, for example for 1 hour or 24 hours. In reaction scheme (1) all variables are defined as in Formula (I).
Figure imgf000017_0001
Reaction scheme 1
EXPERIMENTAL PROCEDURE 2
Additionally, final compounds of Formula (I) can be prepared by reacting an intermediate compound of Formula (II) with a compound of Formula (IV), wherein halo is chloro, bromo or iodo, according to reaction scheme (2). The reaction is performed in a suitable reaction-inert solvent, such as, for example, acetonitrile or DMF, a suitable base, such as, for example, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine, under thermal conditions, such as, 75 °C or 80 °C, for example for 1 hour or 24 hours. In reaction scheme (2) all variables are defined as in Formula (I).
Figure imgf000017_0002
Reaction scheme 2
EXPERIMENTAL PROCEDURE 3 Additionally, final compounds of Formula (I), wherein R2 = CEP, herein referred to as (I-a), can be prepared by reacting an intermediate compound of Formula (II) with a compound of Formula (V) followed by reaction of the formed imine derivative with and intermediate compound of Formula (VI) according to reaction scheme (3). The reaction is performed in a suitable reaction-inert solvent, such as, for example, anhydrous dichloromethane, a Lewis acid, such as, for example titanium tetraisopropoxide or titanium tetrachloride, under thermal conditions, such as, 0 °C or room temperature, for example for a period of 1 hour to 24 hours. In reaction scheme (3) all variables are defined as in Formula (I), and wherein halo is chloro, bromo or iodo. Reaction scheme 3
EXPERIMENTAL PROCEDURE 4
Additionally, final compounds of Formula (I) can be prepared by a copper coupling reaction of an intermediate compound of Formula (VII) with a compound of Formula (VIII) according to reaction scheme (4). The reaction is performed in a suitable reaction-inert solvent, such as, for example, isopropanol, a copper catalyst, such as, copper (I) iodide and a suitable base, such as, potassium phospate, under thermal conditions, such as, for example, 100 °C, for example for 48 hours. In reaction scheme (4) all variables are defined as in Formula (I), and halo is preferably bromo or iodo.
Additionally, final compounds of Formula (I) can be prepared by “Buchwald- Hartwig cross coupling reaction” of an intermediate compound of Formula (VII) with a compound of Formula (VIII) according to reaction scheme (4). The reaction is performed in a suitable reaction-inert solvent, such as, for example, toluene or tetrahydrofuran, a palladium catalyst, such as, BrettPhos Pd G3 methasulfonate (CAS: 1470372-59-8), a ligand, such as, BrettPhos (CAS: 1070663-78-3) and a suitable base, such as, sodium tert-butoxide, under thermal conditions, such as, for example, 70 °C, for example for 48 hours. In reaction scheme (4) all variables are defined as in Formula (I), and halo is preferably chloro, bromo or iodo.
Figure imgf000018_0001
Reaction scheme 4
EXPERIMENTAL PROCEDURE 5
Additionally, final compounds of Formula (I-b), wherein RA = 2 -methyl- \H- benzo[d]imidazole, herein referred to as (I-b), can be prepared cleaving a protecting group (PG) in an intermediate compound of Formula (IX) according to reaction scheme (5). In reaction scheme (5) all variables are defined as in Formula (I), and PG is a suitable protecting group of the nitrogen function such as, for example, p- toluenesulfonyl (Tos). Suitable methods for removing such protecting group are widely known to the person skilled in the art and comprise but are not limited to: treatment with a base, such as, for example, sodium hydroxide, in a reaction inert solvent, such as, for example, ethanol, under thermal conditions, such as, for example, 60 °C, for a period of, for example for 30 minutes.
Figure imgf000019_0001
EXPERIMENTAL PROCEDURE 6
Intermediate compounds of Formula (II) can be prepared cleaving a protecting group (PG) in an intermediate compound of Formula (X) according to reaction scheme (6). In reaction scheme (6) all variables are defined as in Formula (I), and PG is a suitable protecting group of the nitrogen function such as, for example, tert- butoxycarbonyl (Boc), ethoxycarbonyl, benzyl, benzyloxycarbonyl (Cbz). Suitable methods for removing such protecting groups are widely known to the person skilled in the art and comprise but are not limited to: Boc deprotection: treatment with a protic acid, such as, for example, trifluoroacetic acid or hydrochloric acid, in a reaction inert solvent, such as, for example, di chi orom ethane or 1,4-dioxane; ethoxycarbonyl deprotection: treatment with a strong base, such as, for example, sodium hydroxide, in a reaction inert solvent such as for example wet tetrahydrofuran; benzyl deprotection: catalytic hydrogenation in the presence of a suitable catalyst, such as, for example, palladium on carbon, in a reaction inert solvent, such as, for example, methanol or ethanol; benzyloxycarbonyl deprotection: catalytic hydrogenation in the presence of a suitable catalyst, such as, for example, palladium on carbon, in a reaction inert solvent, such as, for example, methanol or ethanol. (X) (II)
Reaction scheme 6 EXPERIMENTAL PROCEDURE 7
Intermediate compounds of Formula (X) can be prepared by reaction of a compound of Formula (XI) and a halo derivative of Formula (VIII) according to reaction scheme (7). The reaction is performed in a suitable reaction-inert solvent, such as, for example, dimethylformamide, dimethylsulfoxide, 1,4-dioxane or 1 -butanol, and a suitable base, such as, potassium carbonate, DIPEA or Et3N, under thermal conditions, such as, for example, 60 °C or 120 °C, for example for 4 to 48 hours. In reaction scheme (7) all variables are defined as in Formula (I), and halo is preferably chloro, bromo or iodo. PG is defined as in Formula (X).
Additionally, intermediate compounds of Formula (X) can be prepared by “Buchwald-Hartwig cross coupling reaction” of an intermediate compound of Formula (XI) with an intermediate compound of Formula (VIII) according to reaction scheme (7). The reaction is performed in a suitable reaction-inert solvent, such as, for example, toluene or 1,4-dioxane, a palladium catalyst, such as, Pd2(dba)3 (CAS: 51364-51-3), a ligand, such as, XPhos (CAS: 564483-18-7) orDavePhos (CAS: 213697-53-1) and a suitable base, such as, cesium carbonate or sodium tert-butoxide, under thermal conditions, such as, for example, 90 °C or 120°C, for example for 12 to 48 hours. In reaction scheme (7) all variables are defined as in Formula (I), and halo is preferably chloro, bromo or iodo. PG is defined as in Formula (X).
Figure imgf000020_0001
(XI) (X)
Reaction scheme 7
EXPERIMENTAL PROCEDURE 8
Additionally, intermediate compounds of Formula (X), wherein RA = thiazolo[4,5-b]pyridine, herein referred to as (X-a), can be prepared by reaction of an intermediate compound of Formula (XI) with a compound of Formula (XII) according to reaction scheme (8). The reaction is performed in a suitable reaction-inert solvent, such as, for example, dimethylformamide, a copper catalyst, such as, CuCL (CAS: 7447-39-4), a source of sulfur, such as, carbon disulfide and a suitable base, such as, potassium carbonate, under thermal conditions, such as, for example, 120 °C, for example for 16 hours. In reaction scheme (8) all variables are defined as in Formula (I), and halo is preferably bromo. PG is defined as in Formula (X).
Figure imgf000021_0001
Reaction scheme 8
Intermediate compounds of Formula (VII) can be prepared by reacting an intermediate compound of Formula (Il-a), wherein RA is a suitable protecting group (PG) defined as in Formula (X), with an intermediate compound of Formula (IV) according to reaction scheme (2) and followed by cleavage of the protected group (PG) according to reaction scheme (6).
Intermediate compounds of Formula (IX) can be prepared by following an analogous procedure to the one described for the synthesis of final compounds (I) starting from compound (Il-b), wherein RA = 2-methyl- l//-benzo[d]imidazole, according to reaction scheme (2). Intermediates of Formula (Il-a), (Il-b), (III), (IV), (V), (VI), (VIII), (XI) and (XII) are commercially available or can be prepared by known procedures to those skilled in the art.
PHARMACOLOGY The compounds of the present invention and the pharmaceutically acceptable compositions thereof inhibit O-GlcNAc hydrolase (OGA) and therefore may be useful in the treatment or prevention of diseases involving tau pathology, also known as tauopathies, and diseases with tau inclusions. Such diseases include, but are not limited to Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler- Scheinker disease, Parkinson’s disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, SLC9A6-related mental retardation, subacute sclerosing panencephalitis, tangle-only dementia, and white matter tauopathy with globular glial inclusions.
The compounds of the present invention and the pharmaceutically acceptable compositions thereof inhibit O-GlcNAc hydrolase (OGA) and therefore may be also useful in the treatment or prevention of diseases involving an alpha synucleinopathy, in particular Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease.
As used herein, the term “treatment” is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting or stopping of the progression of a disease or an alleviation of symptoms, but does not necessarily indicate a total elimination of all symptoms. As used herein, the term “prevention” is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting or stopping of the onset of a disease.
The invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in the treatment or prevention of diseases or conditions selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, SLC9A6-related mental retardation, subacute sclerosing panencephalitis, tangle- only dementia, white matter tauopathy with globular glial inclusions, Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease.
The invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in the treatment, prevention, amelioration, control or reduction of the risk of diseases or conditions selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler- Scheinker disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non- Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, SLC9A6-related mental retardation, subacute sclerosing panencephalitis, tangle-only dementia, white matter tauopathy with globular glial inclusions, Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease. In particular, the diseases or conditions may in particular be selected from a tauopathy, more in particular a tauopathy selected from the group consisting of Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, and agryophilic grain disease; or the diseases or conditions may in particular be neurodegenerative diseases accompanied by a tau pathology, more in particular a neurodegenerative disease selected from amyotrophic lateral sclerosis or frontotemporal lobe dementia caused by C90RF72 mutations. In particular, the diseases or conditions may in particular be selected from an alpha synuclinopathy, more in particular a tauopathy selected from the group consisting of Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease.
Preclinical states in Alzheimer’s and tauopathy diseases:
In recent years the United States (US) National Institute for Aging and the International Working Group have proposed guidelines to better define the preclinical (asymptomatic) stages of AD (Dubois B, et al. Lancet Neurol. 2014;13:614-629; Sperling, RA, et al. Alzheimers Dement. 2011;7:280-292). Hypothetical models postulate that Ab accumulation and tau-aggregation begins many years before the onset of overt clinical impairment. The key risk factors for elevated amyloid accumulation, tau-aggregation and development of AD are age (i.e, 65 years or older), APOE genotype, and family history. Approximately one third of clinically normal older individuals over 75 years of age demonstrate evidence of Ab or tau accumulation on PET amyloid and tau imaging studies, the latter being less advanced currently. In addition, reduced Abeta-levels in CSF measurements are observed, whereas levels of non-modified as well as phosphorylated tau are elevated in CSF. Similar findings are seen in large autopsy studies and it has been shown that tau aggregates are detected in the brain as early as 20 years of age and younger. Amyloid-positive (Ab+) clinically normal individuals consistently demonstrate evidence of an “AD-like endophenotype” on other biomarkers, including disrupted functional network activity in both functional magnetic resonance imaging (MRI) and resting state connectivity, fluorodeoxyglucose 18F (FDG) hypometabolism, cortical thinning, and accelerated rates of atrophy. Accumulating longitudinal data also strongly suggests that Ab+ clinically normal individuals are at increased risk for cognitive decline and progression to mild cognitive impairment (MCI) and AD dementia. The Alzheimer’s scientific community is of the consensus that these Ab+ clinically normal individuals represent an early stage in the continuum of AD pathology. Thus, it has been argued that intervention with a therapeutic agent that decreases Ab production or the aggregation of tau is likely to be more effective if started at a disease stage before widespread neurodegeneration has occurred. A number of pharmaceutical companies are currently testing BACE inhibition in prodromal AD.
Thanks to evolving biomarker research, it is now possible to identify Alzheimer’s disease at a preclinical stage before the occurrence of the first symptoms. All the different issues relating to preclinical Alzheimer’s disease such as, definitions and lexicon, the limits, the natural history, the markers of progression and the ethical consequences of detecting the disease at the asymptomatic stage, are reviewed in Alzheimer’s & Dementia 12 (2016) 292-323.
Two categories of individuals may be recognized in preclinical Alzheimer’s disease or tauopathies. Cognitively normal individuals with amyloid beta or tau aggregation evident on PET scans, or changes in CSF Abeta, tau and phospho-tau are defined as being in an “asymptomatic at-risk state for Alzheimer’s disease (AR-AD)” or in a “asymptomatic state of tauopathy”. Individuals with a fully penetrant dominant autosomal mutation for familial Alzheimer’s disease are said to have “presymptomatic Alzheimer’s disease”. Dominant autosomal mutations within the tau-protein have been described for multiple forms of tauopathies as well.
Thus, in an embodiment, the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in control or reduction of the risk of preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, or tau-r elated neurodegeneration as observed in different forms of tauopathies.
Prodromal states of Parkinson’s disease have also been studied. Thus, in an embodiment, the invention also relates to a compound according to the general Formula (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for use in control or reduction of the risk of prodromal Parkinson’s disease.
As already mentioned hereinabove, the term “treatment” does not necessarily indicate a total elimination of all symptoms, but may also refer to symptomatic treatment in any of the disorders mentioned above. In view of the utility of the compound of Formula (I), there is provided a method of treating subjects such as warm-blooded animals, including humans, suffering from or a method of preventing subjects such as warm blooded animals, including humans, suffering from any one of the diseases mentioned hereinbefore.
Said methods comprise the administration, i.e. the systemic or topical administration, preferably oral administration, of a prophylactically or a therapeutically effective amount of a compound of Formula (I), a stereoisomeric form thereof, a pharmaceutically acceptable addition salt or solvate thereof, to a subject such as a warm-blooded animal, including a human.
Therefore, the invention also relates to a method for the prevention and/or treatment of any of the diseases mentioned hereinbefore comprising administering a prophylactically or a therapeutically effective amount of a compound according to the invention to a subject in need thereof.
The invention also relates to a method for modulating O-GlcNAc hydrolase (OGA) activity, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of a compound according to the invention and as defined in the claims or a pharmaceutical composition according to the invention and as defined in the claims.
A method of treatment may also include administering the active ingredient on a regimen of between one and four intakes per day. In these methods of treatment the compounds according to the invention are preferably formulated prior to administration. As described herein below, suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients.
The compounds of the present invention, that can be suitable to treat or prevent any of the disorders mentioned above or the symptoms thereof, may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound of Formula (I) and one or more additional therapeutic agents, as well as administration of the compound of Formula (I) and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, a compound of Formula (I) and a therapeutic agent may be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.
A skilled person will be familiar with alternative nomenclatures, nosologies, and classification systems for the diseases or conditions referred to herein. For example, the fifth edition of the Diagnostic & Statistical Manual of Mental Disorders (DSM-5™) of the American Psychiatric Association utilizes terms such as neurocognitive disorders (NCDs) (both major and mild), in particular, neurocognitive disorders due to Alzheimer’s disease. Such terms may be used as an alternative nomenclature for some of the diseases or conditions referred to herein by the skilled person.
PHARMACEUTICAL COMPOSITIONS
The present invention also provides compositions for preventing or treating diseases in which inhibition of O-GlcNAc hydrolase (OGA) is beneficial, such as Alzheimer’s disease, progressive supranuclear palsy, Down’s syndrome, frontotemporal lobe dementia, frontotemporal dementia with Parkinsonism- 17, Pick’s disease, corticobasal degeneration, agryophilic grain disease, amyotrophic lateral sclerosis, frontotemporal lobe dementia caused by C90RF72 mutations, Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, or alpha synucleinopathy caused by Gaucher’s disease, said compositions comprising a therapeutically effective amount of a compound according to formula (I) and a pharmaceutically acceptable carrier or diluent.
While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition comprising a compound according to the present invention, together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
The pharmaceutical compositions of this invention may be prepared by any methods well known in the art of pharmacy. A therapeutically effective amount of the particular compound, in base form or addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous, or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
The exact dosage and frequency of administration depends on the particular compound of Formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99% by weight, preferably from 0.1 to 70% by weight, more preferably from 0.1 to 50% by weight of the active ingredient, and, from 1 to 99.95% by weight, preferably from 30 to 99.9% by weight, more preferably from 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
The present compounds can be used for systemic administration such as oral, percutaneous or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like. The compounds are preferably orally administered. The exact dosage and frequency of administration depends on the particular compound according to Formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
The amount of a compound of Formula (I) that can be combined with a carrier material to produce a single dosage form will vary depending upon the disease treated, the mammalian species, and the particular mode of administration. However, as a general guide, suitable unit doses for the compounds of the present invention can, for example, preferably contain between 0.1 mg to about 1000 mg of the active compound. A preferred unit dose is between 1 mg to about 500 mg. A more preferred unit dose is between 1 mg to about 300 mg. Even more preferred unit dose is between 1 mg to about 100 mg. Such unit doses can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration. A preferred dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
A typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to start, interrupt, adjust, or terminate therapy in conjunction with individual patient response.
The invention also provides a kit comprising a compound according to the invention, prescribing information also known as “leaflet”, a blister package or bottle, and a container. Furthermore, the invention provides a kit comprising a pharmaceutical composition according to the invention, prescribing information also known as “leaflet”, a blister package or bottle, and a container. The prescribing information preferably includes advice or instructions to a patient regarding the administration of the compound or the pharmaceutical composition according to the invention. In particular, the prescribing information includes advice or instruction to a patient regarding the administration of said compound or pharmaceutical composition according to the invention, on how the compound or the pharmaceutical composition according to the invention is to be used, for the prevention and/or treatment of a tauopathy in a subject in need thereof. Thus, in an embodiment, the invention provides a kit of parts comprising a compound of Formula (I) or a stereoisomeric for thereof, or a pharmaceutically acceptable salt or a solvate thereof, or a pharmaceutical composition comprising said compound, and instructions for preventing or treating a tauopathy. The kit referred to herein can be, in particular, a pharmaceutical package suitable for commercial sale.
For the compositions, methods and kits provided above, one of skill in the art will understand that preferred compounds for use in each are those compounds that are noted as preferred above. Still further preferred compounds for the compositions, methods and kits are those compounds provided in the non-limiting Examples below.
EXPERIMENTAL PART
Hereinafter, the term “m.p.” means melting point, “min” means minutes, “ACN” means acetonitrile, “aq.” means aqueous, “Boc” means /cvV-butyloxy carbonyl, “DCM” means dichloromethane, “DMF” means dimethylformamide, “DMSO” means dimethylsulfoxide, “dppf’ means l,l'-ferrocenediyl-bis(diphenylphosphine), “CuCh” means copper (II) chloride, “Pd(PPh3)4” means tetrakis(triphenylphosphine)palladium(0), “Pd2(dba)3” means tris(dibenzylideneacetone)dipalladium(0), “X-Phos” means 2-dicyclohexylphosphino- 2',4',6'-tri-isopropyl-l,r-biphenyl, “BrettPhos” means dicyclohexyl[3,6-dimethoxy-2', 4',6'-tris(l-methylethyl)[l,T-biphenyl]-2-yl]phosphine, “DavePhos” means 2'-
(dicyclohexylphosphino)-/V,/V-dimethyl[l,l'-biphenyl]-2-amine, “rt” or “RT” means room temperature, “rac” or “RS” means racemic, “LC-MS” means liquid chromatography/mass spectrometry, “HPLC” means high-performance liquid chromatography, “RP” means reversed phase, “Rt” means retention time (in minutes), “[M+H]+” means the protonated mass of the free base of the compound, “wt” means weight, “EtOAc” means ethyl acetate, “MeOH” means methanol, “sat” means saturated, “soltn” or “sol.” means solution, “TFA” means trifluoroacetic acid, “TMDA” means N,N,N',N'-tetramethylethylenediamine, “SFC” means supercritical fluid chromatography, “SFC-MS” means supercritical fluid chromatography/mass spectrometry.
Whenever the notation “RS” is indicated herein, it denotes that the compound is a racemic mixture at the indicated centre, unless otherwise indicated. The stereochemical configuration for centres in some compounds has been designated “A” or “A’ when the mixture(s) was separated; for some compounds, the stereochemical configuration at indicated centres has been designated as “A*” or “ V*” when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure. The enantiomeric excess of compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separated enantiomer(s).
Microwave assisted reactions were performed in a single-mode reactor: Initiator TM Sixty EXP microwave reactor (Biotage AB).
Thin layer chromatography (TLC) was carried out on silica gel 60 F254 plates (Merck) using reagent grade solvents. Open column chromatography was performed on silica gel, particle size 60 A, mesh = 230-400 (Merck) using standard techniques.
Automated flash column chromatography was performed using ready-to-connect cartridges, on irregular silica gel, particle size 15-40 pm (normal phase disposable flash columns) on different flash systems: either a SPOT or LAFLASH systems from Armen Instrument, or PuriFlash® 430evo systems from Interchim, or 971-FP systems from Agilent, or Isolera 1 SV systems from Biotage. PREPARATION OF THE INTERMEDIATES
PREPARATION OF INTERMEDIATE 1
Figure imgf000032_0001
Tosyl Chloride (CAS: 98-59-9; 298 mg, T56 mmol) was added to a solution of 5- bromo-2-methyl-li -benzo[d]imidazole (300 mg, 1.42 mmol) and Et3N (0.22 mL, T56 mmol) in DCM (10 mL). The mixture was stirred at room temperature for 16h. Then a saturated solution of NaHC03 was added and the mixture was extracted with DCM.
The organic layer was separated, dried (MgS04), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 90/10). The desired fractions were collected and concentrated in vacuo to yield intermediate 1 (347 mg, 67%) as a yellowish oil. PREPARATION OF INTERMEDIATE 2
Figure imgf000032_0002
1-2
Intermediate 10 (118 mg, 0.54 mmol) was added to a stirred solution of intermediate B2.23 (168 mg, 0.45 mmol) and K2CO3 (188 mg, 1.36 mmol) in acetonitrile (4 mL).
The reaction mixture was stirred at 75 °C for 12 h. Additional amount of intermediate 10 (99 mg, 0.45 mmol) was added and the mixture was stirred at 75 °C for 12 h. Then H2O was added and the mixture was extracted with EtOAc. The organic layer was separated, dried (MgS04), filtered and the solvent removed in vacuo. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from
0/100 to 50/50). The desired fractions were evaporated in vacuo to yield intermediate 2 (187 mg, 75%) as a pale white solid. PREPARATION OF INTERMEDIATE 3
Figure imgf000033_0001
1-3
Intermediate 1-3 was prepared following an analogous procedure to the one described for the synthesis of intermediate 1-2 using intermediate 28 (316 mg, 1.37 mmol) and intermediate B2.23 (707 mg, 1.51 mmol) as starting materials and stirring the mixture at 80 °C for 48 h.
PREPARATION OF INTERMEDIATE 4
Figure imgf000033_0002
1-4
Sodium borohydride (3.54 g, 94 mmol) was added to a solution of 1 -(2, 3 -dihydro- [l,4]dioxino[2,3-b]pyridin-6-yl)ethenone (CAS: 1254044-25-1; 4.5 g, 23.4 mmol) in EtOH (109 mL) at 0 °C. The mixture was stirred at rt for 10 min. Water was added and the mixture was extracted with DCM (80 mL x 3). The organic layers were combined, dried (Na2S04), filtered and concentrated in vacuo to yield intermediate 4 (4.04 g, 95%) as a pale-yellow oil. PREPARATION OF INTERMEDIATE 5
Figure imgf000033_0003
1-5
Thionyl chloride (6.51 mL, 89 mmol) was added to a solution of intermediate 4 (4.04 g, 22.3 mmol) in DCM (150 mL) at 0 °C. The mixture was stirred at rt for 12 h. Water (80 mL) was added and the mixture was extracted with DCM (80 mL x 3). The combined organic layers were dried (Na2S04), filtered and evaporated in vacuo to yield crude intermediate 5 (3.53 g, 79%) as a brown oil that solidified upon standing. PREPARATION OF INTERMEDIATE 6
Figure imgf000034_0001
1-6
(2-Bromoethoxy)-/er/-butyldimethylsilane (CAS: 86864-60-0; T51 mL, 7.06 mmol) was added to a stirred suspension of 2,6-dichloro-5-fluoropyridin-3-ol (CAS: 2228660- 663-5; 1.13 g, 6.21 mmol) and K2C03 (1.22 g, 8.82 mmol) in DMF (5.95 mL). The reaction mixture was stirred at 90 °C for 16 h, diluted with water and extracted with EtOAc (twice). The combined organic layers were dried (Na SOA, filtered and the solvents were evaporated in vacuo. The crude product was purified by flash column chromatography (Si 02, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were collected and the solvents were evaporated in vacuo to afford intermediate 6 (1.095 g, 78%) as white solid.
PREPARATION OF INTERMEDIATE 7
Figure imgf000034_0002
1-7
To a mixture of intermediate 6 (1.30 g, 5.77 mmol) in /-BuOH (32.6 mL) was added t- BuOK (777 mg, 6.92 mmol). The reaction mixture was heated at 90 °C for 1 h, cooled down and the solvent was removed in vacuo. The residue was diluted with water and EtOAc. The mixture was filtered through a pad of Celite® and washed with EtOAc. The organic layer was washed with brine, dried (Na SOA, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (S1O2, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to yield intermediate 7 (470 mg, 43%) as a white solid. PREPARATION OF INTERMEDIATE 8
Figure imgf000034_0003
To a mixture of intermediate 7 (900 mg, 4.75 mmol) in toluene (16.7 mL) were added Pd(PPh3)2Cl2 (366 mg, 0.52 mmol) and tributyl(l -ethoxy vinyl)tin (2.25 mL, 6.65 mmol). The reaction mixture was stirred at 92 °C for 16 h. Then HC1 (2N, 1 mL) was added and the mixture was stirred at rt for 3 h. The mixture was neutralized with NaHCC (sat.) and extracted with EtOAc. The organic layer was dried (MgSCE), filtered and concentrated in vacuo. The crude mixture was purified by flash column chromatography (SiCh, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were collected and concentrated in vacuo to afford intermediate 8 (563 mg, 60%) as a brown solid.
PREPARATION OF INTERMEDIATE 9
Figure imgf000035_0001
Intermediate 9 was prepared following an analogous procedure to the one described for the synthesis of intermediate 4 using intermediate 8 as starting material. Intermediate 9 was obtained in 81% yield as a yellowish oil.
PREPARATION OF INTERMEDIATE 10
Figure imgf000035_0002
Intermediate 10 was prepared following an analogous procedure to the one described for the synthesis of intermediate 5 using intermediate 9 as starting material. Intermediate 10 was obtained in 80% yield as a brown oil.
PREPARATION OF INTERMEDIATE 11
Figure imgf000035_0003
A mixture of sodium hydride (25.7 g, 641.4 mmol) in THF (900 mL) was stirred at 0 °C under N2. Then, 3-butyn-l-ol (CAS: 927-74-2; 39.7 g, 566 mmol) was added dropwise and the mixture was stirred at 0 °C for 0.5 h. After that time, a solution of 2- chloro-5-fluoropyrimidine (CAS: 62802-42-0; 50 g, 377.3 mmol) in THF (100 mL) was added dropwise and the mixture was stirred at 0 °C. The reaction mixture was stirred at rt for 5 h. Then, water (1 L) was added and the mixture was extracted with DCM (1000 mL x 3). The organic layer was separated, dried over Na2S04 filtered and concentrated in vacuo to give the crude product as yellow oil. The crude product was purified by column chromatography (silica; petroleum ether/EtOAc 100/0 to 1/1). The pure fractions were collected and the solvent was evaporated under vacuum to give the desired product intermediate 11 (37.5 g, 56.7%) as a white solid.
PREPARATION OF INTERMEDIATE 12
Figure imgf000036_0001
A mixture of intermediate 11 (60 g, 361.1 mmol) in nitrobenzene (50 mL) was heated at 230 °C for 2 h. The reaction mixture was connected via cannula to a bubbler containing a sat. aq. soltn of NaOH (to trap the HCN released in the reaction). After the reaction was completed the crude was cooled and treated with an aq. solution of HC1 2N. The mixture was stirred for 1 h and the aq. layer was separated and then was treated with NaHC03 to basic pH, the crude was extracted with EtOAc (200ml x 3).
The organic layer was separated, dried over Na2S04, filtered and concentrated in vacuo. The crude product was purified by column chromatography (silica; petroleum ether/EtOAc 100/0 to 1/2). The pure fractions were collected, and the solvent was evaporated under vacuum to give intermediate 12 (46.6 g, 92.8%) as a white solid. PREPARATION OF INTERMEDIATE 13
Figure imgf000036_0002
A solution of BuLi (2.5 M in hexane; 2.87 mL, 7.19 mmol) was added a stirred solution of TMDA (CAS: 110-18-9, 816.5 pL, 5.39 mmol) in dry Et20 (40 mL) at -20 °C, and the mixture was stirred at -20 °C for 1 h. The mixture was cooled down to -78 °C, and then intermediate 12 (500 mg, 3.59 mmol) diluted in dry Et20 (5 mL) was added dropwise, and the mixture was stirred at -78 °C for 1 h. Then, L -for yl pi peri dine (806.2 pL, 7.19 mmol) diluted in dry Et20 (5 mL) was added dropwise to the previous mixture and the reaction mixture was stirred at -78 °C for 10 min. The reaction mixture was diluted with water at 0 °C and extracted with EtOAc. The organic layer was separated, washed with water, dried (MgSCri), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 40/60). The desired fractions were collected and concentrated in vacuo to yield intermediate 13 (213 mg, 35.5%) as a yellow solid. PREPARATION OF INTERMEDIATE 14
Figure imgf000037_0001
To a mixture of 5-fluoro-2,3-dihydrofuro[2,3-b]pyridine (CAS: 1356542-41-0; 500 mg, 3.6 mmol) in DCM (15 mL), meta-chloroperbenzoic acid (806 mg, 4.7 mmol) was added at rt. The mixture was stirred at 25 °C for 36 h. The solvent was removed in vacuo, and the residue thus obtained was purified by silica gel column chromatography (silica; EtOAc in heptane 0/100 to 30/70 then DCM in MeOH 0/100 to 6/94). The desired fractions were collected and concentrated in vacuo to afford intermediate 14 (400 mg, 72%) as white solid.
PREPARATION OF INTERMEDIATE 15
Figure imgf000037_0002
To a mixture of intermediate 14 (400 mg, 2.57 mmol) in acetonitrile (7 mL), trimethylsilyl cyanide (CAS:7677-24-9; 1.29 mL, 10.3 mmol) and triethylamine (0.9 mL, 6.47 mmol) were added. The mixture was stirred at 90 °C for 24 h. The mixture was cooled and treated with water and extracted with EtOAc (2 x 10 mL). The combined organic extracts were dried over MgS04 and the solvent was evaporated in vacuo to give a residue that was purified by flash column chromatography (S1O2, EtOAc in heptane 0/100 to 30/60). The desired fractions were collected and concentrated in vacuo to intermediate 15 (320 mg, 76%) as an oil. PREPARATION OF INTERMEDIATE 16
Figure imgf000038_0001
To a mixture of intermediate 15 (340 mg, 2.071 mmol) in dry THF (20 mL), methyl magnesium bromide (2.071 mL, 2.9 mmol, 1.4 M in THF) was added at 0°C. After completion of the addition, the reaction was stirred for 16 h at rt. The mixture was quenched with 1M aq HC1 and stirred for 30 min, then the crude was basified with ME OH until pH 8. The solution was extracted with EtOAc (2x5 mL) The combined organic extracts were dried (Na2S04), filtered and evaporated to dryness to give a residue that was purified by flash column chromatography (S1O2, EtOAc in heptane 0/100 to 20/80). The desired fractions were collected and concentrated to yield intermediate 16 (150 mg, 40%) as a colorless oil.
PREPARATION OF INTERMEDIATE 17
Figure imgf000038_0002
NaBH4 (55.5 mg, 1.47 mmol) was added to a solution of intermediate 16 (133 mg, 0.73 mmol) in EtOH (3 mL) at 0 °C. The reaction mixture was stirred at room temperature for 30 min and the reaction was quenched with NH4CI (sat., aq.). The mixture was extracted with DCM. The combined organic layers were dried (MgS04), filtered and concentrated in vacuo to afford intermediate 17 (130 mg, 97%) as an oil. PREPARATION OF INTERMEDIATE 18
Figure imgf000038_0003
Thionyl chloride (0.8 mL, 11.0 mmol) was added to a solution of intermediate 17 (500 mg, 2.73 mmol) in DCM (12 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 h, diluted with water and extracted with DCM. The combined organic layers were dried (MgS04), filtered and evaporated in vacuo to yield intermediate 18 (520 mg) which was used without any purification in the next step.
PREPARATION OF INTERMEDIATE 19
Figure imgf000039_0001
Methanesulfonyl chloride (CAS: 124-63-0; 0.055 mL, 0.71 mmol) was added to a solution of intermediate 17 (100 mg, 0.55 mmol), Et3N (0.19 mL, 1.37 mmol) in DCM (5 mL) at 0° C. The mixture was stirred at 0 °C for 2 h. The reaction crude was treated with a saturated NaHC03 solution and extracted with DCM. The organic layer was separated, dried (Na2S04), filtered and the solvent removed in vacuo to afford intermediate 19 (130 mg, 91%) as a transparent oil.
PREPARATION OF INTERMEDIATE 20
Figure imgf000039_0002
Acetic anhydride (CAS: 108-24-7; 19.4 g, 190.05 mmol) was added to a solution of 2- amino-3 -bromo-5-fluoropyri dine (CAS: 869557-43-7; 33 g, 172.77 mmol) in toluene
(500 mL) and the mixture was stirred at 100 °C for 48 h under nitrogen atmosphere. After cooling, the solvents were evaporated in vacuo. The residue was purified by flash column chromatography (silica; EtOAc in petroleum ether, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo to yield intermediate 20 (28 g, 70%) as a yellow solid. PREPARATION OF INTERMEDIATE 21
Figure imgf000040_0001
1-21
Phosphorus pentasulfide (1.70 g, 7.67 mmol) was added to a suspension of intermediate 20 (1.38 g, 5.90 mmol) in THF (32.2 mL). The mixture was stirred at rt for 16 h and an additional amount of phosphorus pentasulfide (0.39 g, 1.77 mmol) was added. The mixture was stirred at rt for another 16 h and then CS2CO3 (3.08 g, 9.44 mmol) was added. The mixture was stirred at 70 °C for 16 h and then additional quantity of CS2CO3 (3.08 g, 9.44 mmol) was added. The mixture was stirred at 70 °C for a further 16 h and then water was added. The mixture was extracted with EtOAc and the organic layer was separated, dried (Na2SC>4), filtered and the solvents were evaporated in vacuo. The residue was purified by flash column chromatography (S1O2; EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were collected and concentrated in vacuo to yield intermediate 21 as a pale-orange solid (0.78 g, 78%).
PREPARATION OF INTERMEDIATE 22
Figure imgf000040_0002
1-22 w-Chloroperbenzoic acid (1.13 g, 6.42 mmol) was added to a mixture of intermediate 21 (0.72 g, 4.28 mmol) in DCM (24 mL). The mixture was stirred at rt for 16 h and then more w-chloroperbenzoic acid (1.13 g, 6.42 mmol) was added. The mixture was stirred at rt for a further 3 days and then water was added, and the mixture extracted with DCM. The organic layer was separated, dried (MgS04), filtered and the solvents were evaporated in vacuo. The residue was taken up into DCM and the solid formed was filtered off and discarded. The filtrate was evaporated in vacuo and the residue purified by flash column chromatography (S1O2; MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield intermediate 22 as a white solid (0.51 g, 65%). PREPARATION OF INTERMEDIATE 23
Figure imgf000041_0001
1-23
A mixture of intermediate 22 (1.93 g, 8.92 mmol), tetrabutylammonium bromide (4.31 g, 13.38 mmol) and molecular sieves 4 A in DCM (80 mL) was stirred at rt for 10 min. Then, / oluenesulfonic anhydride (4.37 g, 13.38 mmol) was added and the mixture was stirred at rt for 20 h under N2. The reaction mixture was filtered off and the filtrate was evaporated in vacuo. The crude was purified by flash column chromatography (S1O2; DCM). The desired fractions were collected and concentrated to give intermediate 23 as a white solid (907 mg, 41.2%).
PREPARATION OF INTERMEDIATE 24
Figure imgf000041_0002
1-24
A 10% aq. solution of K2CO3 (18 mL) followed by vinylboronic acid pinacol ester (CAS: 75927-49-0; 0.43 mL, 2.51 mmol) and Pd(PPh3)4 (217 mg, 0.19 mmol) were added to a stirred solution of intermediate 23 (0.9 g, 3.64 mmol) in 1,4-dioxane (11 mL) in a sealed tube under N2. The mixture was stirred at 130 °C for 30 minutes under microwave irradiation. The mixture was treated with water and EtOAc and filtered through celite®. The filtrate was extracted with EtOAc. The organic layer was separated, dried (Na2S04), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (S1O2, EtOAc in heptane 0/100 to 60/40). The desired fractions were collected and concentrated in vacuo to yield intermediate 24 (571 mg, 81%) as an orange solid.
PREPARATION OF INTERMEDIATE 25
Figure imgf000041_0003
Intermediate 24 (521 mg, 2.68 mmol) was added to a mixture of CH3CN (36 mL) and water (1.9 mL) containing three drops of indicator Sudan III (CAS: 85-86-9). The solution was cooled to 0 °C and a mixture of O3/O2 was passed through the flask until the red color dissipated. The reaction was purged with N2 for 10 min. Then, the reaction was diluted with water and DCM. The organic layer was separated, dried (MgSCri), filtered and the solvents removed in vacuo to yield intermediate 25 (498 mg, 95%) as a yellow solid.
PREPARATION OF INTERMEDIATE 26
Figure imgf000042_0001
To a mixture of intermediate 23 (1000 mg, 4.05 mmol) in toluene (20 mL) were added Pd(PPh3)2Cl2 (284 mg, 0.40 mmol) and tributyl(l -ethoxy vinyl)tin (1.64 mL, 4.86 mmol). The reaction mixture was stirred at 80 °C for 48 h. Then HC1 (2N, 2 mL) was added and the mixture was stirred at 70 °C for 7 h. The mixture was neutralized with NaHC03 (sat.) and extracted with EtOAc. The organic layer was dried (Na2S04), filtered and concentrated in vacuo. The crude mixture was purified by flash column chromatography (S1O2, EtOAc in heptane, gradient from 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to afford intermediate 26 (620 mg, 73%) as a light orange solid.
PREPARATION OF INTERMEDIATE 27
Figure imgf000042_0002
NaBFE (357 mg, 9.43 mmol) was added to a solution of intermediate 26 (991 mg, 4.72 mmol) in EtOH (24 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 90 min. Water was added and the mixture was extracted with DCM. The combined organic layers were dried (Na2S04), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (S1O2, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were collected and concentrated in vacuo to afford intermediate 27 (834 mg, 83%) as a white solid. PREPARATION OF INTERMEDIATE 28
Figure imgf000043_0001
CCU (3.8 mL) was added to a solution of intermediate 27 (834 mg, 3.93 mmol) and PPI13 (CAS: 603-35-0; 2.06 g, 7.86 mmol) in CHCh (3.3 mL) at 0 °C. The mixture was stirred at room temperature for 22 h. Then, the solvents were evaporated in vacuo. The residue was purified by flash column chromatography (S1O2, EtOAc in heptane, gradient from 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to afford intermediate 28 (783 mg, 86%) as a white solid.
PREPARATION OF INTERMEDIATES I-Bl.l, B1.2, B1.3, B1.4, B1.5, B1.6, B1.7, B1.8, B1.9, B1.10, Bi l l, B1.12, B1.13, B1.14, B1.15, B1.16, B1.17, B1.18, B1.19, B1.20, B1.21, B1.22, B1.23, B1.24, B1.25, B1.26, B1.27, B1.28, B1.29, B1.30, B1.31, B1.32, B1.33, B1.34, B1.35, B1.36, B1.37, B1.38, B1.39, B1.40, B1.41, B1.42, B1.43, B1.44, B1.45 and B 1.46
Figure imgf000043_0002
Pd2(dba)3 (CAS: 51364-51-3; 1.18 g, 1.29 mmol) was added to a mixture of /V-tert- butoxycarbonylpiperazine (CAS: 57260-71-6; 3 g, 16.11 mmol), 4-bromo-2-m ethoxy -
6-methylpyridine (CAS: 1083169-00-9; 3.58 g, 17.72 mmol), X-Phos (0.61 g, 1.29 mmol) and CS2CO3 (13.12 g, 40.27 mmol) in toluene (98 mL) under nitrogen atmosphere. The mixture was stirred at 90 °C for 16 h. Then the mixture was filtered through a pad of Celite® and washed with EtOAc. The filtrate was washed with a saturated NaHC03 solution and the organic layer was separated, dried (Na2S04), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 15/85). The desired fractions were collected and concentrated in vacuo to yield intermediate Bl.l as a yellow oil (3.52 g, 71% yield).
Figure imgf000044_0001
/V-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 2.63 g, 14.11 mmol) was added to a solution of 4-chloro-2-methylpyridine (CAS: 3678-63-5; 1.2 g, 9.41 mmol) and Et3N (1.57 mL, 11.29 mmol) in DMSO (31 mL). The mixture was stirred at 120 °C for 24 h. Then, the mixture was washed with H2O and extracted with EtOAc. The organic layer was separated, dried (NaiSCE), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to yield intermediate B1.2 (1.83 g, 70%) as a yellow oil.
Figure imgf000044_0002
Intermediate B1.3 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 1.08 g, 5.81 mmol), 2-bromo-4-methylpyridine (CAS: 4926-28-7; 0.5 g,
2.91 mmol), and Et3N (0.61 mL, 4.36 mmol) as starting materials.
I-B1.4
Intermediate B1.4 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using /V-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 455 mg, 2.44 mmol), 2-bromo-5-methylpyridine (CAS: 3510-66-5; 400 mg, 2.33 mmol), Pd2(dba)3 (CAS: 51364-51-3; 64 mg, 0.07 mmol), DavePhos (CAS: 213697-53-1; 60 mg, 0.15 mmol) andNaO*Bu (447 mg, 4.65 mmol) in 1,4-dioxane (20 mL) as starting materials and stirring the mixture at 100 °C for 16.
Figure imgf000045_0001
I-B1.5 Intermediate B1.5 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.78 g, 4.13 mmol), 2-chloro-5-(trifluoromethyl)pyridine (CAS: 4926-28- 7; 0.5 g, 2.75 mmol) and DIPEA (0.71 mL, 5.51 mmol) as starting materials.
Figure imgf000045_0002
I-B1.6
Intermediate B1.6 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.68 g, 3.66 mmol), 6-chloro-4-methoxynicotinonitrile (CAS: 1187190- 69-7; 0.41 g, 2.44 mmol) and Et3N (1.66 mL, 12.19 mmol) as starting materials.
Figure imgf000046_0001
Intermediate B1.7 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.32 g, 1.71 mmol), 5 -bromo-2-i sopropylpyrimi di ne (CAS : 1060813-04-8; 0.33 g, 1.63 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.77 g, 0.08 mmol), X-Phos (0.80 g g, 0.16 mmol) and CS2CO3 (1.61 g, 4.88 mmol) as starting materials.
Figure imgf000046_0002
Intermediate B1.8 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using A-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.54 g, 2.86 mmol), 4-chloro-2,6-dimethylpyrimidine (CAS: 4472-45-1; 0.28 g, 1.90 mmol) and DIPEA (0.67 mL, 3.81 mmol) in 1,4-dioxane (6 mL) as starting materials and stirring the mixture at 50 °C for 16b. Additional amount of A-tert- butoxycarbonylpiperazine (CAS: 57260-71-6; 0.5 eq) was added and the mixture was stirred at 80 °C for 20 h.
Figure imgf000046_0003
I-B1.9 Intermediate B1.9 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using /V-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 2.22 g, 11.91 mmol), 5-bromo-2-methylpyrimidine (CAS: 7752-78-5; 2 g, 11.56 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.53 g, 0.58 mmol), X-Phos (0.55 g, 1.16 mmol) and CS2CO3 (11.3 g, 34.68 mmol) as starting materials.
Figure imgf000047_0001
I-B1.10
Intermediate B1.10 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.37 g, 1.93 mmol), 3 -chloro-6-(tri fluoromethyl)pyri dazine (CAS: 258506-68-2; 0.3 g, 1.61 mmol) and DIPEA (0.56 mb, 3.22 mmol) in 1-butanol (4 mL) as starting materials and stirring the mixture at 90 °C for 16h.
Figure imgf000047_0002
I-B1.11
Intermediate B 1.11 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.61 g, 3.27 mmol), 2-chloro-6-methylpyrazine (CAS: 38557-71-0; 0.4 g, 3.11 mmol), Pd2(dba)3 (CAS: 51364-51-3; 85 mg, 0.09 mmol), DavePhos (80 mg, 0.20 mmol) and NaCfBu (0.6 g, 6.22 mmol) as starting materials.
Figure imgf000048_0001
Intermediate B1.12 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.4 g, 2.15 mmol), 2-chloro-5-(trifluoromethyl)pyrazine (CAS: 799557-87-2; 0.39 g, 2.15 mmol) and K2CO3 (0.5 g, 3.65 mmol) as starting materials and stirring the mixture at 120 °C for 3h.
Figure imgf000048_0002
I-B1.13
Intermediate B1.13 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.56 g, 3.02 mmol), 2-chloro-3 , 5 -di methyl pyrazine (CAS: 32779- 36-5; 0.4 g, 2.88 mmol), Pd2(dba)3 (CAS: 51364-51-3; 79 mg, 0.09 mmol), DavePhos (74 mg, 0.19 mmol) and NaO*Bu (0.55 g, 5.75 mmol) as starting materials.
Figure imgf000048_0003
I-B1.14
Intermediate B1.14 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine
(CAS: 57260-71-6; 0.76 g, 4.08 mmol), 2-chl oro-3 -m ethyl pyrazine (CAS: 95-58-9; 0.4 niL, 3.89 mmol), Pd2(dba)3 (CAS: 51364-51-3; 107 mg, 0.12 mmol), DavePhos (100 mg, 0.25 mmol) and NaCfBu (0.75 g, 7.78 mmol) as starting materials. I-B1.15
Intermediate B1.15 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.32 g, 1.67 mmol), 6-chloropyridazine-3-carbonitrile (CAS: 35857-89-7; 0.2 g, 1.39 mmol) and DIPEA (0.48 mL, 2.78 mmol) in 1-butanol (4 mL) as starting materials and stirring the mixture at 90 °C for 16h.
Figure imgf000049_0001
I-B1.16
Intermediate B1.16 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.59 g, 3.17 mmol), 2,5-bibromopyridine (CAS: 624-28-2; 0.5 g,
2.11 mmol) and DIPEA (0.92 mL, 5.28 mmol) as starting materials and stirring the mixture at 100 °C for 16h.
Figure imgf000049_0002
I-B1.17
Dppf (CAS: 12150-46-8; 66 mg, 0.12 mmol) and Pd2(dba)3 (CAS: 51364-51-3; 47 mg, 0.05 mmol) were added to a stirred solution of intermediate B 1.16 (440 mg, 1.29 mmol) in dry DMA (15 mL) in a sealed tube and under nitrogen atmosphere at 45 °C. The mixture was stirred at 45 °C for 5 minutes. Then, Zn (CAS: 7440-66-6; 17 mg, 0.26 mmol) and Zn(CN)2 (CAS: 557-21-1; 85 mg, 0.72 mmol) were added under nitrogen at 45 °C. The mixture was stirred at 100 °C for 2h. Additional dppf (0.09 eq), Pd2(dba)3 (0.04 eq), Zn (0.2 eq) and Zn(CN)2 (0.6 eq) were added under nitrogen at 45 °C and the mixture was stirred at 100 °C for 24 h. The mixture was diluted with a saturated NaHCC solution and extracted with EtOAc. The organic layer was separated, dried (MgSCE), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to yield intermediate Bl.17 (267 mg, 72%) as a yellow solid.
Figure imgf000050_0001
I-B1.18 Intermediate B 1.18 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 2.71 g, 14.53 mmol), 5 -brom o-2-chl oro-4 -m ethy lpy ri di ne (CAS:
778611-64-6; 2 g, 9.69 mmol) and Et3N (4.05 niL, 29.06 mmol) as starting materials and stirring the mixture at 120 °C for 24 h.
Figure imgf000050_0002
Intermediate B1.19 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.17 using intermediate B1.18 (1 g, 2.81 mmol), dppf (CAS: 12150-46-8; 140 mg, 0.25 mmol), Pd2(dba)3 (CAS: 51364-51-3;
103 mg, 0.11 mmol), Zn (CAS: 7440-66-6; 37 mg, 0.56 mmol) and Zn(CN)2 (CAS: 557-21-1; 330 mg, 2.81 mmol) as starting materials and stirring the mixture at 90 °C for
16 h. Intermediate B1.20 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 11.98 g, 64.31 mmol), ethyl 2-chloropyrimidine-5-carboxylate (CAS: 89793-12-4; lOg, 53.59 mmol) and K2CO3 (7.41 g, 53.59 mmol) as starting materials and stirring the mixture at 90 °C for 24 h.
Figure imgf000051_0001
A mixture of intermediate B1.20 (15 g, 44.59 mmol) and methylamine (33% solution in EtOH, CAS: 74-89-5; 18.47 g, 71.82 mmol) was stirred at room temperature for 16 h. The mixture was filtered and the solvent was evaporated in vacuo to give the desired product as a white solid. The crude product was purified by flash column chromatography (silica, EtOAc in petroleum ether, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo to yield intermediate B1.21 (5 g, 35%) as a white solid.
Figure imgf000051_0002
/V-tert-butoxycarbonylpiperazine (CAS: 57260-71-6; 0.51 g, 2.69 mmol) was added to a stirred solution of 2-amino-3-bromopyridine (CAS: 13534-99-1; 0.32 g, 1.79 mmol), CS2 (CAS: 75-15-0; 0.13 mL, 2.15 mmol), CuCl2 (CAS: 7447-39-4; 0.24 g, 1.79 mmol) and K2CO3 (0.75 g, 5.38 mmol) in DMF (3.6 mL). The mixture was stirred at 120 °C for 16 h. Then the mixture was diluted with a saturated solution of NaHCCb solution and extracted with EtOAc. The organic layer was separated, dried (MgSCri), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 45/55). The desired fractions were collected and concentrated in vacuo to give intermediate B 1.22 (347 mg, 59%) as a dark brown solid.
Figure imgf000052_0001
Intermediate B1.23 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 419 mg, 2.25 mmol), 4-chloroquinaldine (CAS: 4295-06-1; 0.46 mL, 2.25 mmol), Pd2(dba)3 (CAS: 51364-51-3; 103 mg, 0.11 mmol), XPhos (CAS: 564483-18-7; 107 mg, 0.23 mmol) and CS2CO3 (2.2 g, 6.76 mmol) as starting materials and stirring the mixture at 95 °C for 24 h.
Figure imgf000052_0002
Intermediate B1.24 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 2.17 g, 11.63 mmol), 3 -chloro-6-( 1 H-pyrazol - 1 -yl )pyridazine
(CAS: 29334-66-5; 0.35 g, 1.94 mmol) and Et3N (0.32 mL, 2.33 mmol) in acetonitrile (10 mL) as starting materials and stirring the mixture at 82 °C for 16h.
Figure imgf000053_0001
I-B1.25
Intermediate B1.25 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 1.32 g, 7.11 mmol), 7-bromo-2-methylimidazo[l,2-a]pyridine (CAS: 1194375-40-0; 0.5 g, 2.37 mmol), Pd2(dba)3 (CAS: 51364-51-3; 46 mg, 0.05 mmol), XPhos (CAS: 564483-18-7; 86 mg, 0.15 mmol) and NaO*Bu (0.35 g, 3.55 mmol) as starting materials and stirring the mixture at 100 °C for 12 h.
Figure imgf000053_0002
Intermediate B1.26 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 177 mg, 0.95 mmol), intermediate 1 (347 mg, 0.95 mmol), Pd2(dba)3 (CAS: 51364-51-3; 44 mg, 0.05 mmol), XPhos (CAS: 564483-18-7; 55 mg, 0.095 mmol) and Cs2C03 (929 mg, 2.85 mmol) as starting materials and stirring the mixture at 105 °C for 18 h.
Figure imgf000053_0003
Intermediate B1.27 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 0.5 g, 2.68 mmol), intermediate 10 (0.58 g, 2.68 mmol) and DIPEA (1.85 mL, 10.74 mmol) in acetonitrile (10 mL) as starting materials and stirring the mixture at 80 °C for 48 h.
Figure imgf000054_0001
I-B1.28
Intermediate B1.28 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (S)- l -A'-tert-butoxy carbonyl -2- methylpiperazine (CAS: 149488-78-8; 3 g, 14.98 mmol), 2, 5 -di chloropyrazine (CAS: 19745-07-4; 1.34 g, 13.02 mmol) and DIPEA (6.46 mL, 39.07 mmol) in DMF (10 mL) as starting materials and stirring the mixture at 110 °C for 16h.
Figure imgf000054_0002
I-B1.29 Intermediate B 1.29 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.17 using intermediate B 1.28 (4.13 g, 12.14 mmol), dppf (CAS: 12150-46-8; 0.61 g, 1.09 mmol), Pd2(dba)3 (CAS: 51364-51- 3; 445 mg, 0.49 mmol), Zn (CAS: 7440-66-6; 159 mg, 2.43 mmol) and Zn(CN)2 (CAS: 557-21-1; 0.8 g, 6.8 mmol) as starting materials and stirring the mixture at 100 °C for 16 h. Additional Zn(CN)2 (0.56 eq) and Zn (0.2 eq) were added and the mixture was stirred at 100 °C for 20 h. Additional dppf (0.09 eq), Pd2(dba)3 (0.04 eq), Zn(CN)2 (0.56 eq) and Zn (0.2 eq) were added and the mixture was stirred at 120 °C for 24 h.
Figure imgf000054_0003
I-B1.30 Intermediate B1.30 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using (S)-l -A'-tert-b utoxy carbonyl -2- methylpiperazine (CAS: 149488-78-8; 2.32 g, 11.56 mmol), 5-bromo-2- methylpyrimidine (CAS: 7752-78-5; 2 g, 11.56 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.53 g, 0.58 mmol), XPhos (CAS: 564483-18-7; 0.67 g, 1.16 mmol) and Cs2C03 (11.3 g, 34.68 mmol) as starting materials and stirring the mixture at 110 °C for 48 h.
Figure imgf000055_0001
Intermediate B1.31 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-b utoxy carbonyl piperazine (CAS: 57260-71-6; 12.42 g, 66.66 mmol), 5-bromo-l, 3, 4-thiadiazol -2-amine (CAS: 37566-39-5; 10 g, 55.55 mmol) and K2CO3 (7.68 g, 55.55 mmol) in DMF (100 mL) as starting materials and stirring the mixture at 90 °C for 16h.
Figure imgf000055_0002
Acetic anhydride (CAS: 108-24-7; 8.05 g, 78.85 mmol) was added to a mixture of intermediate B1.31 (15 g, 52.56 mmol), pyridine (6.24 g, 78.85 mmol) and 4- dimethylaminopyridine (0.3 g, 2.42 mmol) in DCM (200 mL) at 0 °C. The mixture was stirred at room temperature for 16 h. Then, H2O (200 mL) was added and the mixture was extracted with DCM (6 x 300 mL). The organic layer was separated, dried (Na SCri), filtered and the solvent removed in vacuo to yield intermediate B 1.32 (15 g, 86%) as a reddish brown solid.
Figure imgf000056_0001
Intermediate B1.33 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 1 g, 5.37 mmol), 4-bromo-2,6-dimethylpyri dine (CAS: 5093-70-9; 1.20 g, 6.44 mmol), Pd2(dba)3 (CAS: 51364-51-3; 393 mg, 0.43 mmol), XPhos (CAS:
564483-18-7; 205 mg, 0.43 mmol) and CS2CO3 (4.37 g, 13.42 mmol) as starting materials and stirring the mixture at 90 °C for 16 h.
Figure imgf000056_0002
Intermediate B1.34 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.1 using A -tert-butoxy carbonyl piperazine (CAS: 57260-71-6; 2.5 g, 13.42 mmol), 2-chloro-4-iodo-6-(trifluoromethyl)pyridine (CAS: 205444-22-0; 4.54 g, 14.77 mmol), Pd(OAc)2 (CAS: 3375-31-3; 151 mg, 0.67 mmol), Xantphos (CAS: 161265-03-8; 388 mg, 0.67 mmol) and Cs2C03 (8.75 g, 26.85 mmol) in DMF (5 mL) and 1,4-dioxane (60 mL) as starting materials and stirring the mixture at 140 °C for 20 minutes under microwave irradiation.
Figure imgf000056_0003
Pd(OAc)2 (CAS: 3375-31-3; 18 mg, 0.08 mmol) and tricyclohexylphosphine tetrafluorob orate (CAS: 58656-04-5; 59 mg, 0.16 mmol) were added to a stirred solution of intermediate B1.34 (1.6 g, 4.37 mmol), trimethylboroxine (CAS: 823-96-1; 0.40 mL, 2.89 mmol) and K2CO3 (443 mg, 3.21 mmol) in 1,4-dioxane (12 mL). The mixture was deoxygenated with a nitrogen flow for 5 minutes. Then the reaction was stirred at 100 °C for 3.5 h under N2, at room temperature for 24 h and then at 100 °C for 2.5 h. Additional trimethylboroxine (0.22 eq, 0.13 mL), Pd(OAc)2 (0.0061 eq, 6 mg), tricyclohexylphosphine tetrafluorob orate (0.012 eq, 20 mg) and K2CO3 (0.24 eq, 148 mg) were added and the mixture was stirred at 100 °C for 2 h and then at 120 °C for 20 minutes under microwave irradiation and at 140 °C for 20 minutes under microwave irradiation. After cooling the mixture was washed with water and extracted with DCM. The organic layer was separated, dried (MgSCL), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo to give intermediate B 1.35 (338 mg, 21%) as a white solid.
Figure imgf000057_0001
I-B1.36 Intermediate B 1.36 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using 1-tert-butoxy carbonyl-2 - methylpiperazine (CAS: 120737-78-2; 12.56 g, 62.69 mmol), 2-chloro-5- nitropyrimidine (CAS: 10320-42-0; 10 g, 62.68 mmol) and EtsN (18.74 g, 185.23 mmol) in DMF (100 mL) as starting materials and stirring the mixture at 90 °C for 12 h.
Figure imgf000057_0002
I-B1.37
A mixture of intermediate B1.36 (12 g, 37.11 mmol), iron (CAS: 7439-89-6; 10.36 g, 185.56 mmol) and NH4C1 (9.93 g, 185.56 mmol) in H20 (60 mL) and THF (60 mL) was stirred at room temperature for 36 h. The mixture was filtered and quenched with H2O (60 mL). The mixture was extracted with DCM (3 x 180 mL). The organic layer was separated, dried (NaiSCri), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in petroleum ether, gradient from 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to yield intermediate B1.37 (9 g, 83%) as a yellow oil.
Figure imgf000058_0001
I-B1.38
Intermediate B1.38 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.32 using intermediate B 1.37 as starting material.
Figure imgf000058_0002
I-B1.39
Intermediate B1.39 was prepared following an analogous procedure to the one described for the synthesis of intermediate B 1.2 using A -tert-butoxy carbonyl piperazine (CAS: 169447-70-5; 1.5 g, 8.05 mmol), 5-chloropyrazine-2-carbonitrile (CAS: 36070- 75-4; 1.26 g, 8.86 mmol) and DIPEA (4.16 mL, 24.16 mmol) in DMF (20 mL) as starting materials and stirring the mixture at 60 °C for 16 h.
Figure imgf000058_0003
I-B1.40
Intermediate B1.40 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (R)-l-/V-tert-butoxy carbonyl-2- methylpiperazine (CAS: 169447-70-5; 2 g, 9.99 mmol), 5-chloropyrazine-2- carbonitrile (CAS: 36070-75-4; 1.21 g, 8.68 mmol) and DIPEA (4.3 mL, 26.05 mmol) in DMF (7 mL) as starting materials and stirring the mixture at 70 °C for 16 h.
Figure imgf000059_0001
I-B1.41 Intermediate B 1.41 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using fe/ -butyl ci s-2, 6-dimethylpi perazi ne- 1 - carboxyl ate (CAS: 180975-66-0; 1 g, 4.67 mmol), 5-bromo-2-methylpyrimidine (CAS: 7752-78-5; 0.97 g, 5.60 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.21 g, 0.23 mmol), X- Phos (0.27 g, 0.47 mmol) and CS2CO3 (4.56 g, 14 mmol) in toluene (15 mL) as starting materials and stirring the mixture at 110 °C for 16 h.
Figure imgf000059_0002
I-B1.42
Intermediate B 1.42 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using fcrt-butyl 2,2-dimethylpiperazine-l- carboxylate (CAS: 674792-07-5; 400 mg, 1.87 mmol), 5-bromo-2-methylpyrimidine (CAS: 7752-78-5; 388 mg, 2.24 mmol), Pd2(dba)3 (CAS: 51364-51-3; 86 mg, 0.09 mmol), X-Phos (108 mg, 0.19 mmol) and Cs2C03 (1.82 g, 5.60 mmol) in toluene (8 mL) as starting materials and stirring the mixture at 110 °C for 16 h.
Figure imgf000059_0003
I-B1.43 Intermediate B 1.43 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (R)-l -A -tert-butoxy carbonyl -2- methylpiperazine (CAS: 169447-70-5; 0.4 g, 2 mmol), 2-chloro-5-methylpyrimidine
(CAS: 22536-61-4; 0.31 g, 2.4 mmol) and DIPEA (0.69 mL, 4 mmol) in 2-propanol (7 niL) as starting materials and stirring the mixture at 180 °C for 30 minutes under microwave irradiation.
Figure imgf000060_0001
I-B1.44
Intermediate B 1.44 was prepared following an analogous procedure to the one described for the synthesis of intermediate B1.2 using (R)-l -A -tert-butoxy carbonyl -2- methylpiperazine (CAS: 169447-70-5; 0.5 g, 2.5 mmol), 2-chl oro-5 -fluoropyri mi dine (CAS: 62802-42-0; 0.4 g, 3 mmol) and DIPEA (0.86 mL, 5 mmol) in 2 -propanol (7 mL) as starting materials and stirring the mixture at 120 °C for 30 minutes and at 130 °C for 45 minutes under microwave irradiation.
Figure imgf000060_0002
Intermediate B 1.45 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using (R)-l -A -tert-butoxy carbonyl -2- methylpiperazine (CAS: 169447-70-5; 2.32 g, 11.56 mmol), 5-bromo-2- methylpyrimidine (CAS: 7752-78-5; 2 g, 11.56 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.53 g, 0.58 mmol), X-Phos (0.67 g, 1.16 mmol) and CS2CO3 (11.3 g, 34.68 mmol) in toluene (44 mL) as starting materials and stirring the mixture at 110 °C for 48 h.
Figure imgf000061_0001
I-B1.46
Intermediate B 1.46 was prepared following an analogous procedure to the one described for the synthesis of intermediate Bl.l using (S)-l -L -tert-butoxy carbonyl -2- methylpiperazine (CAS: 149488-78-8; 2.3 g, 11.48 mmol), 2-chloropyrimidine (CAS: 1722-12-9; 1.32 g, 11.48 mmol), Pd2(dba)3 (CAS: 51364-51-3; 0.52 g, 0.57 mmol), X-
Phos (0.66 g, 1.15 mmol) and CS2CO3 (11.2 g, 34.45 mmol) in toluene (44 mL) as starting materials and stirring the mixture at 110 °C for 24 h.
PREPARATION OF INTERMEDIATES I-B-2.1, B2.2, B2.3, B2.4, B2.5, B2.6, B2.7,
Figure imgf000061_0002
A solution of intermediate Bl.l (3.52 g, 11.45 mmol) in methanol (36 mL) was added to a solid phase reactor containing amberlyst ® 15 hydrogen form (CAS: 39389-20-3, 12.18 g, 57.24 mmol) form in it. The mixture was shaken in at room temperature for 16 h. The mixture was filtered, and the resin washed with MeOH. The combined filtrates were discarded. Then a 7N solution of NH3 in MeOH was added to the resin. The mixture was shaken in the solid phase reactor for 2 h. The resin was filtered off and washed twice with more 7N solution of NH3 in MeOH (3 x 33.6 ml; 30 min shaken each time). The filtrates were combined and concentrated in vacuo to yield intermediate B2.1 as a brown oil used in the following step without further purification (2.04 g,
86%).
Figure imgf000062_0001
TFA (2.68 mL, 36.05 mmol) was added to a solution of intermediate B1.2 (500 mg,
I.80 mmol) in DCM (5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 1.5 h. The reaction was concentrated to dryness in vacuo. The residue was dissolved in MeOH (20 mL) and amberlyst ® 26 hydroxy form (CAS: 39339-85-0,
I I .1 g, 35.52 mmol) was added. The mixture was stirred at room temperature until pH was basic. The mixture was filtered, and the resin washed with MeOH. The combined filtrates were evaporated in vacuo to yield intermediate B2.2 (312 mg, 99%).
Figure imgf000062_0002
Intermediate B2.3 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B1.3 as starting material.
Figure imgf000062_0003
Intermediate B2.4 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.4 as starting material.
Figure imgf000062_0004
I-B2.5 Intermediate B2.5 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B1.5 as starting material.
Figure imgf000063_0001
Intermediate B2.6 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.6 as starting material.
Figure imgf000063_0002
Intermediate B2.7 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.7 as starting material.
Figure imgf000063_0003
Intermediate B2.8 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B1.8 as starting material.
Figure imgf000063_0004
Intermediate B2.9 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.9 as starting material.
Figure imgf000064_0001
Intermediate B2.10 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.10 as starting material.
Figure imgf000064_0002
I-B2.11
Intermediate B2.11 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.11 as starting material.
Figure imgf000064_0003
I-B2.12 Intermediate B2.12 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.12 as starting material.
Figure imgf000064_0004
I-B2.13
Intermediate B2.13 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.13 as starting material.
Figure imgf000065_0001
I-B2.14
Intermediate B2.14 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.14 as starting material.
Figure imgf000065_0002
Intermediate B2.15 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.15 as starting material.
Figure imgf000065_0003
Intermediate B2.16 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.17 as starting material.
Figure imgf000065_0004
Intermediate B2.17 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.19 as starting material.
Figure imgf000066_0001
A mixture of intermediate B 1.21 (5 g, 15.56 mmol) and HC1 (4N in 1,4-dioxane, 100 mL, 400 mmol) was stirred at room temperature for 1 h. The mixture was filtered off and the solvent was evaporated in vacuo. The residue was washed with methyl tert- butyl ether (250 mL) to yield intermediate B2.18a (x 2 HC1 salt) (4.26 g, 93%) as a white solid.
Intermediate B2.18a (x 2 HC1 salt) (0.5 g, 1.70 mmol) in methanol (14 mL) was added to a solid phase reactor containing amberlyst ® 15 hydrogen form (CAS: 39389-20-3, 1.81 g) form in it. The mixture was shaken in at room temperature for 16 h. The mixture was filtered, and the resin washed with MeOH. The combined filtrates were discarded. Then a 7N solution of NIL in MeOH was added to the resin. The mixture was shaken in the solid phase reactor. The resin was filtered off and washed twice with more 7N solution of ML in MeOH. The filtrates were combined and concentrated in vacuo to yield intermediate B2.18b (free base) as a brown foam used in the following step without further purification (0.38 g, 91%).
Figure imgf000066_0002
Intermediate B2.19 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.22 as starting material.
Figure imgf000066_0003
Intermediate B2.20 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.23 as starting material.
Figure imgf000067_0001
Intermediate B2.21 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.24 as starting material.
Figure imgf000067_0002
Intermediate B2.22 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.25 as starting material.
Figure imgf000067_0003
Intermediate B2.23 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using intermediate B 1.26 as starting material. I-B2.24
Intermediate B2.24 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.2 using 1 -(5-methyl- 1 ,2,4-oxadiazol-3 - yl)piperazine (prepared as described in Bioorganic & Medicinal Chemistry, 16(4), 1613-1631; 2008) as starting material.
Figure imgf000068_0001
Intermediate B2.25 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.1 using intermediate B 1.27 as starting material.
Figure imgf000068_0002
I-B2.26
2-Methylpiperazine (CAS: 109-07-9; 5.25 g, 52.39 mmol) and Et3N (6.63 g, 65.48 mmol) were added to a mixture of 2-chloropyrimidine (CAS: 1722-12-9; 5 g, 43.66 mmol) in CH3CI (50 mL). The mixture was stirred at room temperature for 4 h. Then H2O (100 mL) was added and the mixture was extracted with DCM (3 x 100 mL). The organic layer was separated, dried (NaiSCL), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 100/0). The desired fractions were collected and concentrated in vacuo to yield intermediate B2.26 (6.56 g, 82%) as a yellow solid.
Figure imgf000069_0001
60/40
I-B2.27
A mixture of intermediate B1.29 (1.38 g, 4.55 mmol) and TFA (6.96 mL, 90.98 mmol) was stirred at room temperature for 7 h. The solvent was evaporated in vacuo. The residue was dissolved in methanol (14 mL) and was added to a solid phase reactor containing amberlyst ® 15 hydrogen form (CAS: 39389-20-3, 4.84 g) form in it. The mixture was shaken in at room temperature for 16 h. The mixture was filtered, and the resin washed with MeOH. The combined filtrates were discarded. Then a 7N solution of NIL in MeOH was added to the resin. The mixture was shaken in the solid phase reactor for 2 h. The resin was filtered off and washed twice with more 7N solution of N¾ in MeOH. The filtrates were combined and concentrated in vacuo to yield intermediate B2.27 (925 mg, quant., 60/40 mixture) as a yellow solid.
Figure imgf000069_0002
I-B2.28
TFA (5.24 mL, 68.40 mmol) was added to a stirred solution of intermediate B 1.30 (lg, 3.42 mmol) in DCM (18 mL) at 0 °C. The mixture was stirred at room temperature for 90 minutes. Then, the solvent was evaporated in vacuo. The residue was treated with toluene and concentrated in vacuo to yield intermediate B2.28a (trifluoroacetate salt, 1.1 g, quant.) as a brown solid.
Intermediate B2.28a (trifluoroacetate salt) was converted into the free base following an analogous procedure to the one described for the synthesis of intermediate B2.18b yielding intermediate B2.28b (free base) as an oil.
Figure imgf000070_0001
A mixture of intermediate B 1.32 (15 g, 45.82 mmol) and HC1 (4N in 1,4-dioxane, 100 mL) was stirred at room temperature for 1 h. The mixture was filtered and the solvent was evaporated in vacuo. The residue was washed with methyl tertbutyl ether (250 mL) to yield intermediate B2.29a (x 2 HC1, 8.15 g, 57%) as a reddish solid.
A solution of intermediate B2.29a (x 2 HC1, 1.5 g, 5 mmol) in MeOH (33 mL), DMF (3 mL) and LLO (12 mL) was added to a solid phase reactor containing amberlyst ® 15 hydrogen form (CAS: 39389-20-3; 5.32 g) form in it. The mixture was shaken in at room temperature for 16 h. The mixture was filtered, and the resin washed with MeOH. The combined filtrates were discarded. Then a 7N solution of ML in MeOH was added to the resin. The mixture was shaken in the solid phase reactor. The resin was filtered off and washed twice with more 7N solution of ML in MeOH. The filtrates were combined and concentrated in vacuo to yield intermediate B2.29b (1.09 g, 96%) as a pale pink solid.
Figure imgf000070_0002
Compound B2.30 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.29b starting from B 1.33 as starting material.
Figure imgf000070_0003
Intermediate B2.31 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.1 using intermediate B 1.35 as starting material. I-B2.32
Intermediate B2.32a was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.29a using 4-[5-(acetylamino)-2- pyrimidinyl]-l-piperazinecarboxylic acid , 1,1-dimethylethyl ester (prepared as described in PCT Int. Appl., 2016030443; 5 g, 15.56 mmol) as starting material.
A solution of intermediate B2.32a (x HC1 salt, 400 mg, 1.55 mmol) in MeOH (6 mL) and EhO (2 mL) was passed through an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of N¾ in MeOH. The desired fractions were collected and concentrated in vacuo to afford intermediate B2.32b (280 mg, 82%) as a brown solid.
Figure imgf000071_0001
I-B2.33
Intermediate B2.33a and B2.33b were prepared following an analogous procedure to the one described for the synthesis of intermediate B2.32a and B2.32b using intermediate B 1.38 as starting material.
Figure imgf000071_0002
85/15
I-B2.34
Intermediate B2.34 (85/15 mixture) was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.39 as starting material.
Figure imgf000072_0001
94/6
I-B2.35
Intermediate B2.35 (94/6 mixture) was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.40 as starting material.
Figure imgf000072_0002
I-B2.36
Intermediate B2.36 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.32 using intermediate B 1.41 as starting material.
Figure imgf000072_0003
I-B2.37 Intermediate B2.37 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.32 using intermediate B1.42 as starting material.
Figure imgf000073_0001
I-B2.38
Intermediate B2.38 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.43 as starting material.
Figure imgf000073_0002
I-B2.39
Intermediate B2.39 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.44 as starting material.
Figure imgf000073_0003
I-B2.40 Intermediate B2.40 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.32 using intermediate B 1.45 as starting material.
Figure imgf000073_0004
I-B2.41 Intermediate B2.41 was prepared following an analogous procedure to the one described for the synthesis of intermediate B2.27 using intermediate B 1.46 as starting material.
PREPARATION OF FINAL COMPOUNDS
El. PREPARATION OF PRODUCT 1
Figure imgf000074_0001
Intermediate 10 (95 mg, 0.44 mmol) was added to a solution of intermediate B2.1 (100 mg, 0.48 mmol) and K2CO3 (181 mg, 1.31 mmol) in CH3CN (3.5 mL). The reaction mixture was stirred at 80 °C for 24 h. Then the reaction was diluted with DCM and washed with H2O. The organic layer was separated, dried (Na2SC>4), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were collected and concentrated in vacuo to give product 1 (59 mg, 35%) as a cream solid.
E2. PREPARATION OF PRODUCT 2
Figure imgf000074_0002
Compound 2 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.2 (115 mg, 0.65 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were collected and a saturated solution of NaHCCb solution was added until pH was basic. The organic phase was separated, dried (MgS04), filtered and evaporated in vacuo to yield product 2 (47 mg, 28%) as a white solid.
E3. PREPARATION OF PRODUCT 3
Figure imgf000075_0001
Compound 3 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.3 (120 mg, 0.68 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo to yield a colorless oil (100 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 3 (HC1 salt, 70 mg, 38%) as a white solid.
E4. PREPARATION OF PRODUCT 4
Figure imgf000075_0002
Compound 4 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.4 (68 mg, 0.38 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 72% [25mM NH4HCO3] - 28% [ACN:MeOH (1:1)] to 36% [25mM NH4HCO3] - 64% [ACN:MeOH (1:1)]). The desired fractions were evaporated in vacuo to yield a white foamy solid (47 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 4 (37 mg, 27%) as a white solid. E5. PREPARATION OF PRODUCT 5
Figure imgf000076_0001
Compound 5 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.5 (150 mg, 0.65 mmol) as starting materials and heating the mixture at 75 °C for 18 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 59% [25mM NH4HCO3] - 41% [ACNMeOH (1:1)] to 17% [25mM NH4HCO3] - 83% [ACNMeOH (1 : 1)]). The desired fractions were evaporated in vacuo to yield a white foamy solid (103 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 5 (HC1 salt, 114 mg, 54%) as a white solid.
E6. PREPARATION OF PRODUCT 6
Figure imgf000076_0002
Compound 6 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.6 (120 mg, 0.55 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo. The residue was triturated in DIPE to afford product 6 (79 mg, 42%) as a white solid. E7. PREPARATION OF PRODUCT 7
Figure imgf000077_0001
Compound 7 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (109 mg, 0.50 mmol) and intermediate B2.7 (129 mg, 0.63 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo to afford product 7 (152 mg, 77%) as a white solid.
E8. PREPARATION OF PRODUCT 8
Figure imgf000077_0002
Compound 8 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (136 mg, 0.62 mmol) and intermediate B2.8 (100 mg, 0.52 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 3.5/96.5). The desired fractions were evaporated in vacuo to afford product 8 (130 mg, 66%) as a beige foamy solid.
E9. PREPARATION OF PRODUCT 9 and 9a
Figure imgf000077_0003
Compound 9 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (983 mg, 4.52 mmol) and intermediate B2.9 (966 mg, 5.42 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo and the residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 6.5/93.5). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE and the solid was filtered and dry in vacuo to yield product 9 (free base) (1.15 g, 70%) as a beige solid.
Product 9 (118 mg) was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the residue was triturated with Et20 to afford product 9a (HC1 salt, 119 mg) as a white solid.
E10. PREPARATION OF PRODUCT 10
Figure imgf000078_0001
Compound 10 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (102 mg, 0.47 mmol) and intermediate B2.10 (107 mg, 0.39 mmol) as starting materials and heating the mixture at 75 °C for 24 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 20/80). The desired fractions were evaporated in vacuo. The residue was triturated with Et20 to yield product 10 (43 mg, 26%) as a white solid.
Ell. PREPARATION OF PRODUCT 11
Figure imgf000078_0002
Compound 11 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.11 (151 mg, 0.55 mmol) as starting materials and heating the mixture at 80 °C for 24 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo and the residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 70% [25mM NH4HCO3] - 30% [ACN:MeOH (1:1)] to 27% [25mM NH4HCO3] - 73% [ACN:MeOH (1:1)]). The desired fractions were evaporated in vacuo to yield a yellow sticky solid (66 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 11 (HC1 salt, 36 mg, 20%) as a white solid.
E12. PREPARATION OF PRODUCT 12
Figure imgf000079_0001
Compound 12 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (183 mg, 0.84 mmol) and intermediate B2.12 (150 mg, 0.65 mmol) as starting materials and heating the mixture at 75 °C for 12 h. Additional amount of intermediate 10 (42 mg, 0.19 mmol) was added and the mixture was stirred at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo to yield product 12 (160 mg, 59%) as a pale white solid.
El 3. PREPARATION OF PRODUCT 13
Figure imgf000079_0002
Compound 13 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (101 mg, 0.46 mmol) and intermediate B2.13 (125 mg, 0.65 mmol) as starting materials and heating the mixture at 75 °C for 18 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo to yield a white foamy solid (74 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 13 (HC1 salt, 50 mg, 26%) as a white solid. E14. PREPARATION OF PRODUCT 14
Figure imgf000080_0001
Compound 14 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (133 mg, 0.61 mmol) and intermediate B2.14 (165 mg, 0.73 mmol) as starting materials and heating the mixture at 80 °C for 24 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo and the residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 72% [25mM NH4HCO3] - 28% [ACN:MeOH (1 : 1)] to 36% [25mM NH4HCO3] - 64% [ACN:MeOH (1 : 1)]). The desired fractions were evaporated in vacuo to yield a yellow sticky solid (147 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1 eq). The solvents were evaporated in vacuo and the products were triturated in Et20 to afford product 14 (HC1 salt, 119 mg, 49%) as a white solid. E 15. PREPARATION OF PRODUCT 15
Figure imgf000080_0002
Compound 15 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (109 mg, 0.50 mmol) and intermediate B2.15 (79 mg, 0.42 mmol) as starting materials and heating the mixture at 75 °C for 24 h. The mixture was purified by flash column chromatography (silica,
MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo to yield product 15 (33 mg, 21%). E16. PREPARATION OF PRODUCT 16
Figure imgf000081_0001
Compound 16 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (276 mg, 1.27 mmol) and intermediate B2.15 (120 mg, 0.63 mmol) as starting materials and heating the mixture at 75 °C for 24 h. The mixture was purified by flash column chromatography (silica, dry load in silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo to yield product 16 (45 mg, 18%) as a light yellow oil.
E17. PREPARATION OF PRODUCT 17
Figure imgf000081_0002
Compound 17 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (89 mg, 0.41 mmol), intermediate B2.16 (64 mg, 0.34 mmol) and K2CO3 (94 mg, 0.68 mmol) as starting materials and heating the mixture at 75 °C for 12 h. Additional amount of K2CO3 (1 eq) was added and the mixture was stirred at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 80/20). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 70% [25mM NH4HCO3] - 30% [ACNMeOH (1:1)] to 27% [25mM NH4HCO3] - 73% [ACNMeOH (1 : 1)]). The desired fractions were evaporated in vacuo and the residue was triturated in
DIPE to afford product 17 (92 mg, 73%) as a white solid.
El 8. PREPARATION OF PRODUCT 18
Figure imgf000081_0003
Compound 18 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.17 (74 mg, 0.37 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo. The residue was triturated with Et20 and filtered to yield product 18 (40 mg, 28%) as a white solid.
E19. PREPARATION OF PRODUCT 19
Figure imgf000082_0001
Compound 10 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (159 mg, 0.73 mmol) and intermediate B2.18b (200 mg, 0.81 mmol) as starting materials and heating the mixture at 70 °C for 20 h. The mixture was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 1.5/98.5). The desired fractions were evaporated in vacuo to yield product 19 (104 mg, 32%) as a white solid.
E20. PREPARATION OF PRODUCT 20
Figure imgf000082_0002
Compound 20 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (119 mg, 0.55 mmol) and intermediate B2.19 (100 mg, 0.45 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 2.3/97.7). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 95% [0.1% HCOOH] - 5% [MeCN:MeOH (1:1)] to 70% [0.1% HCOOH] - 30% [MeCN:MeOH (1:1)]). The desired fractions were neutralized with a saturated NaHC03 solution, partially evaporated in vacuo and extracted with DCM. The organic layer was separated, dried (MgS04), filtered and the solvent removed in vacuo to yield product 20 (62 mg, 34%) as a white foamy solid.
E21. PREPARATION OF PRODUCT 21
Figure imgf000083_0001
Compound 21 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (120 mg, 0.55 mmol) and intermediate B2.20 (96 mg, 0.42 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo to yield product 21 (58 mg, 34%) as a pale white solid.
E22. PREPARATION OF PRODUCT 22
Figure imgf000083_0002
Compound 22 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.21 (127 mg, 0.55 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 95% [0.1% HCOOH] - 5% [MeCNMeOH (1:1)] to 63% [0.1% HCOOH] - 37% [MeCNMeOH (1 : 1)]). The desired fractions were evaporated in vacuo and the residue was purified by
RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: (from 72% [65mM NH4OAc + ACN (90: 10)] - 28% [ACN: MeOH 1 : 1] to 36% [65mM NH4OAC + ACN (90: 10)] - 64% [ACN: MeOH 1:1]). The desired fractions were neutralized with a saturated NaHC03 solution and extracted with DCM. The organic layer was separated, dried (MgS04), filtered and the solvent removed in vacuo to yield product 22 (35 mg, 18%) as a white solid. E23. PREPARATION OF PRODUCT 23
Figure imgf000084_0001
Compound 23 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (115 mg, 0.53 mmol) and intermediate B2.22 (88 mg, 0.41 mmol) as starting materials and heating the mixture at 75 °C for 40 h. The mixture was purified by flash column chromatography (silica, NH3 (7M in MeOH)/MeOH/DCM, gradient from 0/0/100 to 20/1/1). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm), from 95% [0.1% TFA] - 5% [MeCNMeOH (1:1)] to 63% [0.1% TFA] - 37% [MeCNMeOH (1:1)]). The desired fractions were evaporated in vacuo. The residue was purified by reverse phase chromatography from 72% [25mM NH4HCO3] - 28% [MeCNMeOH (1:1)] to 36% [25mM NH4HCO3] - 64% [MeCNMeOH (1:1)]). The desired fractions were evaporated in vacuo to yield product 23 (26 mg, 16%) as a white foamy solid.
E24. PREPARATION OF PRODUCT 24
Figure imgf000084_0002
NaOH (15 mg, 0.37 mmol) was added to a solution of intermediate 2 (187 mg, 0.34 mmol) in EtOH (20 mL) and the mixture was stirred at 60 °C for 30 minutes. The solvents were evaporated in vacuo. Then H2O was added and the mixture was extracted with EtOAc. The organic layer was separated, dried (MgS04), filtered and the solvent removed in vacuo. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo to yield product 24 (124 mg, 91%) as a white crystalline powder. E25. PREPARATION OF PRODUCT 25
Figure imgf000085_0001
Compound 25 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (100 mg, 0.46 mmol) and intermediate B2.24 (60 mg, 0.35 mmol) as starting materials and heating the mixture at 75 °C for 12 h. The mixture was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 95% [0.1% HCOOH] - 5% [MeCNMeOH (1:1)] to 63% [0.1% HCOOH] - 37% [MeCNMeOH (1:1)]). The desired fractions were evaporated in vacuo. The residue was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1 eq). The solvents were evaporated in vacuo to afford product 25 (HC1 salt, 33 mg, 24%) as a white solid.
E26. PREPARATION OF PRODUCT 26
Figure imgf000085_0002
THF (0.5 mL) followed by 5-bromo-2-methylpyridine (CAS: 3430-13-5; 39 mg, 0.22 mmol) were added to a mixture of intermediate B2.25 (50 mg, 0.19 mmol), Brettphos Pd G3 methanesulfonate (CAS: 1470372-59-8; 17 mg, 0.02 mmol), Brettphos (CAS: 1070663-78-3; 10 mg, 0.02 mmol) and NaO*Bu (54 mg, 0.56 mmol) in a sealed tube and under nitrogen atmosphere. The mixture was stirred at 70 °C for 48 h. Then the reaction was treated with H2O and extracted with EtOAc. The organic layer was separated, dried (MgSCri), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 6/94). The desired fractions were collected and concentrated in vacuo to yield product 26 (30 mg, 45%) as an oil.
Product 26 (25 mg) was taken into Et20 and treated with HC1 (2N in Et20, 4 eq). The mixture was stirred at room temperature for 1 h. The solvents were evaporated in vacuo and the residue was triturated with Et20 to afford product 26 (HC1 salt, 25 mg, 91%) as a white solid. E27. PREPARATION OF PRODUCT 27
Figure imgf000086_0001
Ethylene glycol (CAS: 107-21-1; 0.0125 mL, 0.22 mmol), copper(I) iodide (CAS: 7681-65-4; 9 mg, 0.05 mmol) and potassium phosphate tribasic (191 mg, 0.90 mmol) were added to a solution of l-methyl-4-iodo-lH-pyrazole (CAS: 39806-90-1; 56 mg, 0.27 mmol) and intermediate B2.25 (60 mg, 0.23 mmol) in ‘PrOH (0.8 mL). The mixture was stirred at 100 °C for 48 h. The solvents were evaporated in vacuo. Then the reaction was treated with LEO and extracted with DCM. The organic layer was separated, dried (NaiSCE), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 6/94). The desired fractions were collected and concentrated in vacuo to yield product 27 (50 mg, 64%) as an oil.
Product 27 (38 mg) was taken into EEO and treated with HC1 (2N in EEO, 4 eq). The mixture was stirred at room temperature for 1 h. The solvents were evaporated in vacuo and the residue was triturated with EEO to afford product 27 (HC1 salt, 32 mg, 76%) as a white solid.
E28. PREPARATION OF PRODUCTS 28 and 29
Figure imgf000086_0002
28 29
Compounds 28 and 29 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 10 (287 mg, 1.32 mmol), intermediate B2.26 (220 mg, 1.20 mmol) and DIPEA (0.83 mL, 4.79 mmol) as starting materials and heating the mixture at 70 °C for 5 days. The solvents were evaporated in vacuo. The residue was taken into DCM (3 mL) and LEO (1 mL) was added. The mixture was passed through an ISOLUTE ® HM-N disposable liquid-liquid extraction column (5 mL). The fraction eluted with DCM was evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: from 70% NH4HCO3 0.25% solution in water, 30% C¾CN to 35% NH4HCO30.25% solution in Water, 65% CH3CN) to yield products 28 (87 mg, 20%) and 29 (92 mg, 21%) both as oils.
Product 28 (80 mg) and product 29 (86 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et20, 4 eq). The mixtures were stirred at room temperature for 30 minutes. The solvents were evaporated in vacuo and the products were triturated with Et20 to yield product 28 (HC1 salt, 82 mg, 93%) and product 29 (HC1 salt, 85 mg, 90%) both as white solids.
Figure imgf000088_0001
33
Compounds 30, 31, 32 and 33 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (200 mg, 0.92 mmol), intermediate B2.27 (170 mg, 0.84 mmol) and DIPEA (0.58 mL, 3.35 mmol) as starting materials and heating the mixture at 70 °C for 20 h. The mixture was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: from 70% NH4HCO3 0.25% solution in water, 30% C¾CN to 35% NH4HCO30.25% solution in Water, 65% CH3CN) to yield products 30 (30 mg, 9%), 31 (30 mg, 9%), 32 (25 mg, 7%) and 33 (22 mg, 6%) as solids.
Figure imgf000089_0001
Compounds 34 and 35 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (235 mg, 1.08 mmol), intermediate B2.28a (300 mg, 0.98 mmol) and DIPEA (1 mL, 5.80 mmol) as starting materials and heating the mixture at 70 °C for 48 h. Additional B2.28a (150 mg, 0.49 mmol) and DIPEA (0.17 mL, 0.99 mmol) were added and the mixture was stirred at 70 °C for 3 days. The mixture was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 6/94). The desired fractions were collected and concentrated in vacuo to yield fraction A (42 mg) and fraction B (27 mg).
Fraction A was further purified by reverse phase (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 70% NH4HCO3 0.25% solution in Water, 30% CH3CN to 35% NH4HCO3 0.25% solution in Water, 65% CH3CN), to give product
34 (19 mg, 5%) as a white solid.
Fraction B was further purified by reverse phase (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 85% NH4HCO3 0.25% solution in Water, 15% CH3CN to 55% NH4HCO3 0.25% solution in Water, 45% CH3CN), to yield product 35 (18 mg, 5%) as a white solid. E31. PREPARATION OF PRODUCT 36
Figure imgf000090_0001
A solution of intermediate B2.29b (200 mg, 0.88 mmol), intermediate 8 (178 mg, 0.90 mmol) and Ti(Oi-Pr)4 (CAS: 546-68-9; 0.52 mL, 1.76 mmol) in DCE (4 mL) was stirred at 80 °C for 20 h under nitrogen atmosphere. Then, sodium cyanoborohydride (CAS: 25895-60-7; 66 mg, 1.06 mmol) was added and the mixture was stirred at 80 °C for 3 h. The mixture was treated with LEO and EtOAc and filtered through a pad of Celite®. The organic layer was separated, dried (MgSCL), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C¾CN to 60% NH4HCO3 0.25% solution in Water, 40% CFECN). The aqueous phase was extracted with EtOAc, separated, dried (Na2S04), filtered and the solvent evaporated in vacuo to yield product 36 (29 mg, 8%) as a white solid.
E32. PREPARATION OF PRODUCT 37
Figure imgf000090_0002
2,3-Dihydro-[l,4]-dioxino[2,3-b]pyridine-6-carbaldehyde (CAS: 615568-24-6; 126 mg, 0.76 mmol) and Ti(Oi-Pr)4 (CAS: 546-68-9; 0.32 mL, 1.09 mmol) were added to a solution of intermediate B2.1 (150 mg, 0.72 mmol) in DCM (2 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction was cooled at 0 °C and methylmagnesium bromide (1.4M in THF and toluene, 2.58 mL, 3.62 mmol) was added dropwise and the mixture was stirred at room temperature for 2 h. The mixture was treated with a saturated solution of NFECl and DCM and filtered through a pad of Celite®. The organic layer was separated, dried (MgSCE), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 80% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 20% C¾CN to 60% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 40% CH3CN) to yield product 37 (79 mg, 29%) as a colorless sticky solid.
E33. PREPARATION OF PRODUCTS 38 and 39
Figure imgf000091_0001
38 39 Compounds 38 and 39 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 5 (201 mg, 1.01 mmol) and intermediate B2.1 (230 mg, 1.11 mmol) as starting materials and heating the mixture at 70 °C for 16 h. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo. The residue was purified by chiral SFC (stationary phase: CHIRALPAK AD-H 5pm 250*30mm, mobile phase: 50% CO2, 50% EtOH (0.3% iPrME)) to yield product 38 (34 mg, 9%) and product 39 (36 mg, 10%).
Product 38 (24 mg) and product 39 (26 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et20, 3 eq). The mixture was stirred at room temperature for 5 minutes. The mixture was filtered off to yield product 38 (x 2 HC1 salt, 20 mg, 70%) and product 39 (x 2 HC1 salt, 23 mg, 75%) both as white solids. E34. PREPARATION OF PRODUCT 40
Figure imgf000092_0001
Compound 40 was prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.4 (150 mg, 0.85 mmol) as starting material. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 80% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 20% CH3CN to 60% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 40% CH3CN) to yield product 40 (254 mg, 88%).
Product 40 (30 mg) was taken up in diethyl ether (0.2 mL) and treated with HC1 (6N in PrOH, 0.5 mL). The solvents were evaporated in vacuo to yield product 40 (x 2 HC1 salt, 6 mg) as a white solid. E35. PREPARATION OF PRODUCT 41
Figure imgf000092_0002
Compound 41 was prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.30 (136 mg, 0.82 mmol) as starting material. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 80% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 20% CEPCN to 60% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 40% CH3CN). The residue was dissolved in DCM and washed with a saturated solution of NaHCCb. The organic layer was separated, dried (NaiSCE), filtered and the solvents evaporated in vacuo to yield product 41 (62 mg, 22%). E36. PREPARATION OF PRODUCT 42
Figure imgf000093_0001
Ti(Oi-Pr)4 (CAS: 546-68-9; 1.5 eq, 0.18 mL) was added to a stirred solution of intermediate B2.31 (100 mg, 0.41 mmol) and 2,3-dihydro-[l,4]-dioxino[2,3-b]pyridine- 6-carbaldehyde (CAS: 615568-24-6; 1.2 eq, 81 mg) in DCM (1.8 mL) under nitrogen atmosphere. The mixture was stirred at room temperature for 16 h. Then, the mixture was cooled at 0 °C and methylmagnesium bromide (1.4M in THF and toluene, 5 eq, 1.46 mL) was added dropwise. The mixture was stirred at 0 °C for 15 minutes and at room temperature for 2 h. Then the mixture was treated with a saturated solution of NH4CI and extracted with DCM. The mixture was filtered through a pad of Celite®. The organic layer was separated, dried (NaiSCE), filtered and the solvents evaporated in vacuo. The residue was treated with 2,3-dihydro-[l,4]-dioxino[2,3-b]pyridine-6- carbaldehyde (0.72 eq, 48 mg), Ti(Oi-Pr)4 (0.9 eq, 0.11 mL) and DCM (1.1 mL) under nitrogen atmosphere. The mixture was stirred at rt for 16 h. Then, the mixture was cooled at 0 °C and methylmagnesium bromide (1.4M in THF and toluene, 3 eq, 0.87 mL) was added dropwise. The mixture was stirred at 0 °C for 15 minutes and at room temperature for 2 h. Then the mixture was treated with a saturated solution of NH4CI and extracted with DCM. The mixture was filtered through a pad of Celite®. The organic layer was separated, dried (NaiSCL), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 1/99). The desired fractions were collected and concentrated in vacuo. The residue was purified by flash column chromatography (silica, EtOAc in DCM, gradient from 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 67% NH4HCO3 0.25% solution in Water, 33% C¾CN to 50% NH4HCO3 0.25% solution in Water,
50% CH3CN). The desired fractions were collected and the solvents partially concentrated in vacuo. The aqueous phase was extracted with DCM. The organic layer was separated, dried (NaiSCL), filtered and the solvents evaporated in vacuo to yield product 42 (47 mg, 28%) as a pale yellow oil. E37. PREPARATION OF PRODUCT 43
Figure imgf000094_0001
Compound 43 was prepared following an analogous procedure to the one described for the synthesis of compound 37 using 2-(l-piperazinyl)-pyrimidine (CAS: 20980-22-7; 100 mg, 0.61 mmol) as starting material. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo to yield product 43 (93 mg, 46%) as a yellow oil.
E38. PREPARATION OF PRODUCTS 44 and 45
Figure imgf000094_0002
44 45
Compounds 44 and 45 were prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.26 (170 mg, 0.95 mmol) as starting material. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 50/50). The desired fractions were evaporated in vacuo to yield product 44 and product 45 both as yellow oils.
Product 44 was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 60% NH4HCO3 0.25% solution in Water, 40% MeOH to 37% NH4HCO3 0.25% solution in Water, 63% MeOH). The desired fractions were collected and concentrated in vacuo to yield product 44 (46 mg, 14%) as a light yellow oil.
Product 45 was purified by RP HPLC (stationary phase: XBridge C18 50 x 100 mm, 5 pm, mobile phase: gradient from 67% NH4HCO3 0.25% solution in Water, 33% C¾CN to 50% NH4HCO3 0.25% solution in Water, 50% C¾CN). The desired fractions were collected and concentrated in vacuo to yield product 45 (98 mg, 30%) as a yellow oil.
E39. PREPARATION OF PRODUCT 46
Figure imgf000095_0001
Compound 46 was prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.18 (150 mg, 0.68 mmol) as starting material. The crude product was purified by flash column chromatography (silica, 7M solution of ammonia in MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC
(stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 80% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 20% CH3CN to 60% lOmM NH4HCO3/NH4OH pH=9 solution in Water, 40% CH3CN). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 46 (48 mg, 18%) as a white solid.
E40. PREPARATION OF PRODUCT 47
Figure imgf000095_0002
Compound 47 was prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.29b (175 mg, 0.77 mmol) as starting material. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 2/98). The desired fractions were evaporated in vacuo. The residue was precipitated with CEPCN to yield product 47 (176 mg, 59%) as a white solid. E41. PREPARATION OF PRODUCTS 48, 49 and 50
Figure imgf000096_0001
48 49 50
Compounds 48, 49 and 50 were prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.32b (144 mg, 0.65 mmol) as starting material. The crude product was precipitated with MeOH to yield product 48 (109 mg, 43%) as a pale yellow solid.
Product 48 was purified via chiral SFC (stationary phase: Chiralpak IC 5pm 250*21.2mm, mobile phase: 50% CO2, 50% iPOH(0.3% iPrNFb)) to give product 49 (45 mg, 18%) and product 50 (46 mg, 18%). Product 49 was triturated with Et20 to yield product 49 (35 mg, 14%) as a white solid. Product 50 was triturated with Et20 to yield product 50 (37 mg, 15%) as a cream solid.
E42. PR
Figure imgf000097_0001
51 52
Compounds 51 and 52 were prepared following an analogous procedure to the one described for the synthesis of compound 37 using intermediate B2.33b (125 mg, 0.53 mmol) as starting material. The crude product was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm, Mobile phase: Gradient from 85% NH4HCO3 0.25% solution in Water, 15% CEPCN to 60% NH4HCO3 0.25% solution in Water, 40% CH3CN). The desired fractions were collected and concentrated in vacuo to yield product 51 (47 mg, 22%) and 52 (81 mg, 38%) both as white solids. E43. PREPARATION OF PRODUCT 53
Figure imgf000097_0002
Et3N (0.27 mL, 1.95 mmol) was added to a stirred suspension of intermediate B2.33a (x 2HC1 salt; 150 mg, 0.49 mmol) in DCM (3 mL). The mixture was stirred for 2 minutes. Then, 2,3-Dihydro-[l,4]-dioxino[2,3-b]pyridine-6-carbaldehyde (CAS: 615568-24-6; 88 mg, 0.54 mmol) and sodium triacetoxyborohydride (CAS: 56553-60-
7; 310 mg, 1.46 mmol) were added and the mixture was stirred at room temperature for 24 h. The mixture was diluted with a saturated solution of NaHC03 and extracted with DCM. The organic layer was separated, dried (MgS04), filtered and the solvents evaporated in vacuo. The crude product was purified by RP HPLC (Stationary phase: XBridge Cl 8 50 x 100 mm, 5 pm, Mobile phase: Gradient from 90% NH4HCO3 0.25% solution in Water, 10% CH3CN to 66% NH4HCO3 0.25% solution in Water, 34% CH3CN). The desired fractions were collected and concentrated in vacuo. The residue was triturated with Et20 to yield product 53 (123 mg, 66%) as a white solid.
E44. PREPARATION OF PRODUCT 54
Figure imgf000098_0001
Compound 54 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.29a (x 2 HC1 salt; 125 mg, 0.42 mmol), intermediate 19 (130 mg, 0.50 mmol) and Et3N (0.26 mL, 1.87 mmol) as starting materials and heating the mixture at 70 °C for 24 h. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to afford product 54 (20 mg, 12%) as a cream solid.
E45. PREPARATION OF PRODUCTS 55, 56 and 57
Figure imgf000098_0002
Compounds 55, 56 and 57 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate 18 (150 mg, 0.74 mmol) as starting material. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo to yield product 55 (167 mg, 66%) as an oil. Product 55 (30 mg) was taken up in diethyl ether (3 mL) and treated with HC1 (1M in Et20, 0.05 mL) and the mixture was stirred at room temperature for 30 minutes. The solid was filtered to yield product 55 (x HC1 salt, 25 mg) as a white solid. Product 55 (130 mg) was purified via chiral SFC (stationary phase: CHIRACEL OJ-H 5pm 250*20mm, mobile phase: 70% CO2, 30% EtOH (0.3% iPrNFL)) to yield product 56 (46 mg, 18%) and product 57 (45 mg, 18%).
Product 56 (46 mg) was taken up in diethyl ether (2 mL) and treated with HC1 (1M in Et20, 0.14 mL) and the mixture was stirred at room temperature for 30 minutes. The solid was filtered to yield product 56 (x 2HC1 salt, 52 mg) as a white solid.
Product 57 (45 mg) was taken up in diethyl ether (2 mL) and treated with HC1 (1M in Et20, 0.12 mL) and the mixture was stirred at room temperature for 30 minutes. The solid was filtered to yield product 56 (x 2HC1 salt, 51 mg) as a white solid. E46. PREPARATION OF PRODUCT 58
Figure imgf000099_0001
Ti(Oi-Pr)4 (CAS: 546-68-9; 0.27 mL, 0.94 mmol) was added to a solution of intermediate B2.28b (120 mg, 0.62 mmol) and intermediate 13 (104 mg, 0.62 mmol) in DCM (4 mL). The reaction mixture was stirred at room temperature for 16 h. Then, sodium triacetoxyborohydride (CAS: 56553-60-7; 265 mg, 1.25 mmol) was added and the reaction mixture was stirred at room temperature for 3 h. Then a saturated solution of NaHCC was added and the mixture was extracted with DCM. The organic layer was separated, dried (MgSCL), filtered and the solvents evaporated in vacuo. The crude product was purified by RP HPLC (stationary phase: Cl 8 XB ridge 30 x 100 mm 5 pm, Mobile phase: gradient from 85% NH4HCO3 0.25% solution in Water, 15% CFPCN to 55% NH4HCO3 0.25% solution in Water, 45% CH3CN) to yield product 58 (10 mg, 4%) as a solid. E47. PREPARATION OF PRODUCTS 59 and 60
Figure imgf000100_0001
59 60
Compounds 59 and 60 were prepared following an analogous procedure to the one described for the synthesis of compound 58 using intermediate B2.27 (120 mg, 0.59 mmol) as starting material. The crude product was purified by RP HPLC (stationary phase: Cl 8 XBridge 50 x 100 mm 5 pm, mobile phase: gradient from 70% NH4HCO3 0.25% solution in Water, 30% C¾CN to 35% NH4HCO30.25% solution in Water, 65% CH3CN) to yield products 59 (75 mg, 36%) and 60 (58 mg, 23%) both as light yellow solids.
Figure imgf000101_0001
61 62
Compounds 61 and 62 were prepared following an analogous procedure to the one described for the synthesis of compound 58 using intermediate B2.27 (60 mg, 0.30 mmol) and intermediate 25 (58 mg, 0.30 mmol) as starting materials. The product was purified by RP HPLC (stationary phase: C18 XBridge 50 x 100 mm 5 pm, mobile phase: Gradient from 70% NH4HCO3 0.25% solution in Water, 30% CFPCN to 35% NH4HCO3 0.25% solution in Water, 65% CFPCN) to yield product 61 (20 mg) and product 62 (15 mg, 11%) both as light yellow solids. Product 61 was passed through an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of NH3 in MeOH. The desired fractions obtained with 7M solution of NH3 in MeOH were collected and concentrated in vacuo to afford product 61 (15 mg, 13%) as a white solid.
E49. PREPARATION OF PRODUCTS 63
Figure imgf000101_0002
Compound 63 was prepared following an analogous procedure to the one described for the synthesis of compound 36 using intermediate B2.29b (80 mg, 0.35 mmol), intermediate 26 (78 mg, 0.37 mmol), Ti(Oi-Pr)4 (CAS: 546-68-9; 0.15 mL, 0.53 mmol) and sodium cyanoborohydride (CAS: 25895-60-7; 27 mg, 0.42 mmol) in THF (3 mL) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo. The residue was purified by using an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of ME in MeOH. The desired fractions obtained with 7M solution of ME in MeOH were collected and concentrated in vacuo to afford product 63 (5 mg, 3%) as a colorless oil.
E50. PREPARATION OF PRODUCT 64
Figure imgf000102_0001
Compound 64 was prepared following an analogous procedure to the one described for the synthesis of compound 36 using intermediate B2.32b (80 mg, 0.36 mmol), intermediate 26 (80 mg, 0.38 mmol), Ti(Oi-Pr)4 (CAS: 546-68-9; 0.16 mL, 0.54 mmol) and sodium cyanoborohydride (CAS: 25895-60-7; 27 mg, 0.43 mmol) in THF (3 mL) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo. The residue was purified by purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 nm 5 pm, mobile Phase: Gradient from 74% 0.1% NH4CO3H/NH4OH pH = 9 solution in water, 26% OECN to 58 % 0.1% NH4CO3H/NH4OH pH = 9 solution in water, 42% CH3CN) yielding product 64 (35 mg, 23%) as a white solid.
E51. PREPARATION OF PRODUCTS 65, 66 and 67
Figure imgf000103_0001
Compounds 65, 66 and 67 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.18 (230 mg, 1.04 mmol) and intermediate 28 (200 mg, 0.87 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 3/97). The desired fractions were evaporated in vacuo to yield product 65 (238 mg, 66%) as a cream solid.
Product 65 was purified via chiral SFC (Stationary phase: Chiralcel OD-H 5pm 250x21.2mm, Mobile phase: 68% CO2, 32% iPrOH) to yield two fractions which were purified via preparative LC (Stationary phase: irregular bare silica 24g, Mobile phase: 95% DCM, 5% MeOH) to yield product 66 (68 mg, 19%) and 67 (60 mg).
Product 67 was purified by RP HPLC (Stationary phase: Cl 8 XB ridge 30 x 100 mm 5 pm, Mobile phase: Gradient from 90% NH4HCO3 0.25% solution in Water, 10% CH3CN to 60% NH4HCO3 0.25% solution in Water, 40% CH3CN) to yield product 67 (30 mg, 8%).
E52. PREPARATION OF PRODUCT 68
Figure imgf000103_0002
Compound 68 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.9 (151 mg, 0.85 mmol) and intermediate 28 (130 mg, 0.56 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0 and then MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were evaporated in vacuo. The residue was triturated with heptane and filtered to yield product 68 (93 mg, 44%) as a white solid.
E53. PREPARATION OF PRODUCT 69
Figure imgf000104_0001
Compound 69 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.8 (95 mg, 0.49 mmol) and intermediate 28 (126 mg, 0.54 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo to yield product 69 (115 mg, 60%) as a beige foamy solid.
E54. PREPARATION OF PRODUCT 70
Figure imgf000104_0002
Compound 70 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.7 (125 mg, 0.61 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 70 (131 mg, 75%) as a white solid. E55. PREPARATION OF PRODUCTS 71, 72 and 73
Figure imgf000105_0001
Compounds 71, 72 and 73 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.1 (121 mg, 0.58 mmol) and intermediate 28 (122 mg, 0.53 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo to yield product 71 (81 mg, 38 %) as a white solid.
Product 71 was purified via chiral SFC (Stationary phase: CHIRACEL OJ-H 5pm 250*30mm, Mobile phase: 70% CO2, 30% EtOH) to yield product 72 (30 mg, 14%) and product 73 (27 mg, 13%).
E56. PREPARATION OF PRODUCT 74
Figure imgf000105_0002
Compound 74 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.5 (120 mg, 0.52 mmol) and intermediate 28 (132 mg, 0.57 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 25/75). The desired fractions were evaporated in vacuo to yield a colorless sticky solid (172 mg) which was taken into DCM and treated with HC1 (4N in 1,4- dioxane, 1.05 eq). The solvents were evaporated in vacuo and the product was triturated with Et20 to afford product 74 (HC1 salt, 156 mg, 64%) as a white solid. E57. PREPARATION OF PRODUCT 75
Figure imgf000106_0001
Compound 75 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.2 (83 mg, 0.47 mmol) and intermediate 28 (90 mg, 0.39 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo to yield a colorless sticky solid (172 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo to afford product 75 (88 mg, 60%) as a white solid.
E58. PREPARATION OF PRODUCT 76
Figure imgf000106_0002
Compound 76 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.4 (90 mg, 0.51 mmol) and intermediate 28 (90 mg, 0.39 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Phenomenex Gemini Cl 8 100x30mm 5pm Column; from 72% [25mM NH4HCO3] - 28% MeCN to 36% [25mM NH4HCO3] - 64% MeCN). The desired fractions were evaporated in vacuo to yield a white foam (55 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the residue was triturated with Et20 and filtered to afford product 76 (HC1 salt, 47 mg, 29%) as a white solid.
E59. PREPARATION OF PRODUCT 77
Figure imgf000106_0003
Compound 77 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.3 (77 mg, 0.43 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo to yield product 77 (58 mg, 36%) as a white solid.
E60. PREPARATION OF PRODUCT 78
Figure imgf000107_0001
Compound 78 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.16 (95 mg, 0.42 mmol), intermediate 28 (117 mg, 0.51 mmol) and K2CO3 (117 mg, 0.85 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 20/80 to 100/0). The desired fractions were evaporated in vacuo. The residue was triturated with Et20 to yield product 78 (119 mg, 72%) as a white solid.
E61. PREPARATION OF PRODUCT 79
Figure imgf000107_0002
Compound 79 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.17 (91 mg, 0.45 mmol), intermediate 28 (104 mg, 0.45 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 60/40). The desired fractions were evaporated in vacuo to yield a white solid (82 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the residue was triturated with Et20 and filtered to afford product 79 (HC1 salt, 74 mg, 37%) as a white solid. E62. PREPARATION OF PRODUCT 80
Figure imgf000108_0001
Compound 80 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.6 (123 mg, 0.56 mmol), intermediate 28 (100 mg, 0.43 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 80 (72 mg, 40%) as a white solid.
E63. PREPARATION OF PRODUCT 81
Figure imgf000108_0002
Compound 81 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.10 (101 mg, 0.43 mmol), intermediate 28 (110 mg, 0.48 mmol) and K2CO3 (120 mg, 0.87 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 95/5). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 81 (59 mg, 32%) as a white solid.
E64. PREPARATION OF PRODUCT 82
Figure imgf000108_0003
Compound 82 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.15 (163 mg, 0.43 mmol) and intermediate 28 (109 mg, 0.47 mmol) as starting materials. The crude product was purified by flash column chromatography (silica, EtOAc). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 mih, mobile phase: from 81% [25mM NH4HCO3] - 19% [100% de ACN] to 45% [25mM NH4HCO3] - 55% [100% de ACN]). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 82 (70 mg, 42%) E65. PREPARATION OF PRODUCT 83
Figure imgf000109_0001
Compound 83 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.12 (77 mg, 0.33 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini 08 30 x 100 mm 5 pm, mobile phase: from 59% [25mM NH4HCO3] - 41% [ACN: MeOH 1:1] to 17% [25mM NH4HCO3] - 83% [ACN: MeOH 1:1]). The desired fractions were evaporated in vacuo to yield product 83 (50 mg, 35%) as a white solid.
E66. PREPARATION OF PRODUCTS 84, 85, 86 and 87
Figure imgf000110_0001
87
Compound 84, 85, 86 and 87 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.34 (90 mg, 0.48 mmol) and intermediate 28 (121 mg, 0.52 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, mobile phase: gradient from 70% NH4HCO3 0.25% solution in Water, 30% CEPCN to 35% NH4HCO3 0.25% solution in Water, 65% CH3CN) to yield product 84 (129 mg) and product 85 (28 mg, 13%). Product 84 was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo to yield product 84 (104 mg, 57%).
Product 84 (75 mg) was purified via chiral SFC (stationary phase: CHIRALPAK AD-H 5pm 250*21.2mm, mobile phase: 60% CO2, 40% MeOH) to yield product 86 (42 mg) and product 87 (39 mg) both as light yellow solids.
E67. PREPARATION OF PRODUCT 88
Figure imgf000111_0001
Compound 88 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.13 (66 mg, 0.34 mmol) and intermediate 28 (87 mg, 0.38 mmol) as starting materials. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo to yield a beige foamy solid (77 mg) which was taken into DCM and treated with HC1 (4N in 1,4-dioxane, 1.05 eq). The solvents were evaporated in vacuo and the residue was triturated with Et20 and filtered to afford product 88 (HC1 salt, 55 mg, 37%) as a white solid.
E68. PREPARATION OF PRODUCT 89
Figure imgf000111_0002
Compound 89 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.11 (146 mg, 0.53 mmol) and intermediate 28 (102 mg, 0.44 mmol) as starting materials. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from (70% [25mM NH4HCO3] - 30% [ACN: MeOH 1 : 1]) to (27% [25mM NH4HCO3] -
73% [ACN: MeOH 1:1]). The desired fractions were evaporated in vacuo to yield a white sticky solid (65 mg) which was taken into DCM and treated with HC1 (4N in 1,4- dioxane, 1 eq). The solvents were evaporated in vacuo and the residue was triturated with Et20 and filtered to afford product 89 (HC1 salt, 59 mg, 32%) as a white solid.
E69. PREPARATION OF PRODUCT 90
Figure imgf000112_0001
Compound 90 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.14 (117 mg, 0.52 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from (70% [25mM NH4HCO3] - 30% [ACN: MeOH 1:1]) to (27% [25mM NH4HCO3] - 73% [ACN: MeOH 1:1]). The desired fractions were evaporated in vacuo. The residue was triturated with DIPE to yield product 90 (79 mg, 48%) as a white solid.
Figure imgf000112_0002
7N solution of NH3 in MeOH (5 mL) was added to intermediate 3 (653 mg, 1.16 mmol) and the reaction mixture was stirred at 33 °C for 16 h and at 37 °C for 24 h. The solvents were evaporated in vacuo. The mixture was diluted with a saturated NaHC03 solution and extracted with EtOAc. The organic layer was separated, dried (MgS04), filtered and the solvent removed in vacuo. The crude product was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield product 91 (249 mg, 51%) as a white solid.
Product 91 (238 mg) was purified by SFC (LUX Amylose- 1, 150 x 21.2 mm, 5 pm, Isocratic 40% Methanol (60% C02) + 0.1% DEA at l=220hih). The desired fractions were collected and concentrated in vacuo to yield product 92 (68 mg) and product 93 (59 mg) both as white solids.
E71. PREPARATION OF PRODUCT 94
Figure imgf000113_0001
Compound 94 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.20 (83 mg, 0.37 mmol) and intermediate 28 (110 mg, 0.48 mmol) as starting materials. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 5/95). The desired fractions were evaporated in vacuo to yield product 94 (91 mg, 57%) as a white solid.
E72. PREPARATION OF PRODUCT 95
Figure imgf000113_0002
Compound 95 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.21 (120 mg, 0.52 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 70% [25mM NH4HCO3] - 30% [ACN: MeOH 1:1] to 27% [25mM NH4HCO3] - 73% [ACN: MeOH 1:1]). The desired fractions were collected and concentrated in vacuo to yield product 95 (50 mg, 27%) as a white solid. E73. PREPARATION OF PRODUCT 96
Figure imgf000114_0001
Compound 96 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.19 (100 mg, 0.45 mmol) and intermediate 28 (106 mg, 0.46 mmol) as starting materials. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 2/98). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from (70% [25mM NH4HCO3] - 30% [ACN: MeOH 1:1]) to (27% [25mM NH4HCO3] - 73% [ACN: MeOH 1:1]). The desired fractions were evaporated in vacuo with CH3CN to yield product 96 (65 mg, 34%) as a white solid.
E74. PREPARATION OF PRODUCT 97
Figure imgf000114_0002
Compound 97 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.22 (65 mg, 0.30 mmol) and intermediate 28 (83 mg, 0.36 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 49% [0.1% HCOOH] - 51% [MeCN:MeOH (1:1)] to 6% [0.1% HCOOH] - 94% [MeCN:MeOH (1:1)]). The desired fractions were collected, neutralized with sat. NaHCCb, partially evaporated in vacuo and extracted with DCM. The organic layer was separated, dried (MgSCE), filtered and the solvent evaporated in vacuo. The crude product was purified by flash column chromatography (silica, NH3 (7M in MeOH)/MeOH/DCM, gradient from 0/0/100 to 20/1/1). The desired fractions were collected and concentrated in vacuo to yield product 97 (4 mg, 3%) as a brown sticky solid. E75. PREPARATION OF PRODUCT 98
Figure imgf000115_0001
Compound 98 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.24 (56 mg, 0.33 mmol) and intermediate 28 (100 mg, 0.43 mmol) as starting materials. The residue was purified by flash column chromatography (silica, EtOAc in heptane, gradient from 0/100 to 100/0). The desired fractions were evaporated in vacuo to yield product 98 (71 mg, 57%) as a white solid.
E76. PREPARATION OF PRODUCT 99
Figure imgf000115_0002
Compound 99 was prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.34 (100 mg, 0.53 mmol) and intermediate 10 (138 mg, 0.63 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: Phenomenex Gemini C18 30 x 100 mm 5 pm, mobile phase: from 70% 25mM NH4HCO3 - 30% MeCNMeOH (1 : 1) to 27% 25mM
NH4HCO3 - 73% MeCNMeOH (1:1)). The desired fractions were evaporated in vacuo to yield a colorless sticky solid which was triturated with Et20 and filtered to afford product 99 (54 mg, 27%) as a white solid.
E77. PREPARATION OF PRODUCTS 100 and 101
Figure imgf000116_0002
100 101
Compounds 100 and 101 were prepared following an analogous procedure to the one described for the synthesis of compound 1 using intermediate B2.35 (170 mg, 0.84 mmol) and intermediate 10 (237 mg, 1.09 mmol) as starting materials. The residue was purified by RP HPLC (stationary phase: C18 XBridge 30 x 100 mm 5 pm, Mobile phase: Gradient from 70% NH4HCO3 0.25% solution in Water, 30% CFPCN to 35% NH4HCO3 0.25% solution in Water, 65% CH3CN). The desired fractions were collected and concentrated in vacuo to yield product 100 (48 mg, 15%) as an oil and product 101 (50 mg, 16%) as an off white solid.
E78. PREPARATION OF PRODUCT 102
Figure imgf000116_0001
Compound 102 was prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (464 mg, 2.13 mmol), intermediate B2.36 (400 mg, 1.94 mmol) and DIPEA (1.34 mL, 7.76 mmol) as starting materials and heating the mixture at 70 °C for 72 h. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C¾CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo. The residue was treated with methyl tert- butyl ether to yield product 102 (20 mg, 3%) as a pale yellow solid.
E79. PREPARATION OF PRODUCT 103
Figure imgf000117_0001
Compound 103 was prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (230 mg, 1.06 mmol), intermediate B2.37 (168 mg, 0.81 mmol) and DIPEA (0.56 mL, 3.26 mmol) as starting materials and heating the mixture at 70 °C for 72 h. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% CH3CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo. The residue was treated with methyl te/7-butyl ether to yield product 103 (40 mg, 13%) as a pale yellow solid. E80. PREPARATION OF PRODUCTS 104 and 105
Figure imgf000118_0001
Compounds 104 and 105 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (294 mg, 1.35 mmol), intermediate B2.38 (200 mg, 1.04 mmol) and DIPEA (0.72 mL, 4.16 mmol) as starting materials and heating the mixture at 70 °C for 72 h. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C¾CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo to afford products 104 (76 mg, 20%) and 105 (44 mg, 11%) as yellow sticky solids. Product 104 (76 mg) was passed through an Isolute® SCX-2 cartridge eluting first with MeOH and then with 7M solution of NH3 in MeOH. The desired fractions obtained with 7M solution of NH3 in MeOH were collected and concentrated in vacuo to afford product 104 (45 mg, 12%) and as a yellow sticky solid
Product 104 (45 mg) and product 105 (44 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et20, 4 eq). The mixture was stirred at room temperature for 30 minutes. The mixture was filtered off to yield product 104 (HC1 salt, 37 mg, 75%) and product 105 (HC1 salt, 43 mg, 89%) both as white solids.
E81. PREPARATION OF PRODUCTS 106 and 107
Figure imgf000119_0001
Compounds 106 and 107 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (288 mg,
1.33 mmol), intermediate B2.39 (200 mg, 1.02 mmol) and DIPEA (0.70 mL, 4.08 mmol) as starting materials and heating the mixture at 70 °C for 72 h. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase:
Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C¾CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo to yield products 106 (60 mg, 16%) and 107 (49 mg, 12%) as yellow sticky solids.
Product 106 (60 mg) and product 107 (49 mg) were independently taken up in diethyl ether and treated with HC1 (2N in Et20, 4 eq). The mixture was stirred at room temperature for 30 minutes. The mixture was filtered off to yield product 106 (HC1 salt, 52 mg, 79%) and product 107 (HC1 salt, 52 mg, 96%) both as white solids.
E82. PREPARATION OF PRODUCTS 108 and 109
Figure imgf000120_0001
Compounds 108 and 109 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (237 mg, 1.09 mmol), intermediate B2.40 (161 mg, 0.84 mmol) and DIPEA (0.58 mL, 3.35 mmol) as starting materials and heating the mixture at 70 °C for 72 h. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 80% NH4HCO3 0.25% solution in Water, 20% C¾CN to 0% NH4HCO3 0.25% solution in Water, 100% CH3CN). The desired fractions were collected and concentrated in vacuo to afford products 108 (40 mg, 13%) and 109 (30 mg, 10%) both as white solids.
E83. PREPARATION OF PRODUCTS 110 and 111
Figure imgf000120_0002
110 111
Compounds 110 and 111 were prepared following an analogous procedure to the one described for the synthesis of compounds 28 and 29 using intermediate 10 (317 mg, 1.46 mmol), intermediate B2.41 (200 mg, 1.12 mmol) and DIPEA (0.77 mL, 4.49 mmol) as starting materials and heating the mixture at 70 °C for 5 days. The residue was purified by flash column chromatography (silica, MeOH in DCM, gradient from 0/100 to 4/96). The desired fractions were evaporated in vacuo. The residue was purified by RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 pm), Mobile phase: Gradient from 70% NH4HCO3 0.25% solution in Water, 30% C¾CN to 35% NH4HCO3 0.25% solution in Water, 65% CH3CN), yielding. The desired fractions were collected and concentrated in vacuo to afford products 110 (70 mg, 17%) and 111 (85 mg, 21%) as white solids. The following compounds were prepared following the methods exemplified in the Experimental Part. In case no salt form is indicated, the compound was obtained as a free base. Έc. No.’ refers to the Example number according to which protocol the compound was synthesized. ‘Co. No.’ means compound number. The values of salt stoichiometry or acid content in the compounds as provided herein, are those obtained experimentally. The content of acid reported herein was determined by 'H NMR integration and/or elemental analysis.
TABLE 1
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
ANALYTICAL PART MELTING POINTS
Values are peak values and are obtained with experimental uncertainties that are commonly associated with this analytical method.
DSC823e (A): For a number of compounds, melting points were determined with a DSC823e (Mettler-Toledo) apparatus. Melting points were measured with a temperature gradient of 10 °C/minute. Maximum temperature was 300 °C. Values are peak values (A).
Mettler Toledo MP50 (B) For a number of compounds, melting points were determined in open capillary tubes on a Mettler FP 81HT / FP90 apparatus. Melting points were measured with a temperature gradient of 1, 3, 5 or 10 °C/minute. Maximum temperature was 300 °C. The melting point was read from a digital display (B).
LCMS
GENERAL PROCEDURE The High Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).
Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time...) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW) and/or exact mass monoisotopic molecular weight. Data acquisition was performed with appropriate software. Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H]+ (protonated molecule) and/or [M-H] (deprotonated molecule). All results were obtained with experimental uncertainties that are commonly associated with the method used. Hereinafter, “SQD” Single Quadrupole Detector, “MSD” Mass Selective Detector, “QTOF” Quadrupole-Time of Flight, “rt” room temperature, “BEH” bridged ethylsiloxane/silica hybrid, HSS” High Strength Silica, “CSH” charged surface hybrid, “UPLC” Ultra Performance Liquid Chromatography, “DAD” Diode Array Detector. TABLE 2. LC-MS Methods (Flow expressed in mL/min; column temperature (T) in °C;
Run time in min).
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
TABLE 3. Analytical data - melting point (M.p.) and LCMS. “n.m ” means that was not measured, “n.d.” means that could not be determined. [M+H]+ means the protonated mass of the free base of the compound, [M-H] means the deprotonated mass of the free base of the compound or the type of adduct specified [M+CH3COO] )· Rt means retention time (in min). For some compounds, exact mass was determined.
Figure imgf000158_0002
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
OPTICAL ROTATIONS
Optical rotations were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and reported as follows: [a]° (l, c g/lOOml, solvent, T °C). [a]l T = (100a) / (lx c): where / is the path length in dm and c is the concentration in g/100 ml for a sample at a temperature T (°C) and a wavelength l (in nm). If the wavelength of light used is 589 nm (the sodium D line), then the symbol D might be used instead. The sign of the rotation (+ or -) should always be given. When using this equation, the concentration and solvent are always provided in parentheses after the rotation. The rotation is reported using degrees and no units of concentration are given (it is assumed to be g/100 mL).
TABLE 4 Optical Rotation data.
Figure imgf000162_0001
SFCMS-METHODS GENERAL PROCEDURE FOR SFC-MS METHODS
The SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO2) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS). It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time...) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software. TABLE 5. Analytical SFC-MS Methods (Flow expressed in mL/min; column temperature (T) in °C; run time in minutes; backpressure (BPR) in bars.
Figure imgf000163_0001
TABLE 6. Analytical SFC data - Rt means retention time (in minutes), [M+H]+ means the protonated mass of the compound, method refers to the method used for (SFC)MS analysis of enantiomerically pure compounds.
Figure imgf000164_0001
NMR For a number of compounds, 'H NMR spectra were recorded on a Bruker AV III HD spectrometer operating at 400 MHz, on a Bruker Avance NEO operating at 500 MHz, or on a Bruker Avance NEO spectrometer operating at 400 MHz, using CHLOROFORM-r/ (deuterated chloroform, CDCh) or DMSO-r/r, (deuterated DMSO, dimethyl-d6 sulfoxide) as solvent. Chemical shifts (d) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as internal standard.
TABLE 7. ¾ NMR results
Figure imgf000165_0001
Figure imgf000166_0001
PHARMACOLOGICAL EXAMPLES
1) OGA - BIOCHEMICAL ASSAY
The assay is based on the inhibition of the hydrolysis of fluorescein mono-B-D-N- Acetyl-Glucosamine (FM-GlcNAc) (Mariappa et al. 2015, Biochem J 470:255) by the recombinant human Meningioma Expressed Antigen 5 (MGEA5), also referred to as O-GlcNAcase (OGA). The hydrolysis FM-GlcNAc (Marker Gene technologies, cat # M1485) results in the formation of B-D-N-glucosamineacetate and fluorescein. The fluorescence of the latter can be measured at excitation wavelength 485 nm and emission wavelength 538nm. An increase in enzyme activity results in an increase in fluorescence signal. Full length OGA enzyme was purchased at OriGene (cat # TP322411). The enzyme was stored in 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol at -20 °C. Thiamet G and GlcNAcStatin were tested as reference compounds (Yuzwa et al. 2008 Nature Chemical Biology 4:483; Yuzwa et al. 2012 Nature Chemical Biology 8:393). The assay was performed in 200mM Citrate/phosphate buffer supplemented with 0.005% Tween-20. 35.6 g Na2HP042 ¾0 (Sigma, # C0759) were dissolved in 1 L water to obtain a 200 mM solution. 19.2 g citric acid (Merck, # 1.06580) was dissolved in 1 L water to obtain a 100 mM solution. pH of the sodiumphosphate solution was adjusted with the citric acid solution to 7.2. The buffer to stop the reaction consists of a 500 mM Carbonate buffer, pH 11.0. 734 mg FM-GlcNAc were dissolved in 5.48 mL DMSO to obtain a 250 mM solution and was stored at -20 °C. OGA was used at a 2nM concentration and FM-GlcNAc at a lOOuM final concentration. Dilutions were prepared in assay buffer.
50 nl of a compound dissolved in DMSO was dispensed on Black Proxiplate TM 384 Plus Assay plates (Perkin Elmer, #6008269) and 3 pi fl-OGA enzyme mix added subsequently. Plates were pre-incubated for 60 min at room temperature and then 2 mΐ FM-GlcNAc substrate mix added. Final DMSO concentrations did not exceed 1%. Plates were briefly centrifuged for 1 min at 1000 rpm and incubate at room temperature for 6 h. To stop the reaction 5 mΐ STOP buffer were added and plates centrifuge again 1 min at lOOOrpm. Fluorescence was quantified in the Thermo Scientific Fluoroskan Ascent or the PerkinElmer EnVision with excitation wavelength 485 nm and emission wavelength 538 nm.
For analysis a best-fit curve is fitted by a minimum sum of squares method. From this an IC50 value and Hill coefficient was obtained. High control (no inhibitor) and low control (saturating concentrations of standard inhibitor) were used to define the minimum and maximum values.
2) OGA - CELLULAR ASSAY
HEK293 cells inducible for P301L mutant human Tau (isoform 2N4R) were established at Janssen. Thiamet-G was used for both plate validation (high control) and as reference compound (reference EC50 assay validation). OGA inhibition is evaluated through the immunocytochemical (ICC) detection of O-GlcNAcylated proteins by the use of a monoclonal antibody (CTD110.6; Cell Signaling, #9875) detecting O- GlcNAcylated residues as previously described (Dorfmueller et al. 2010 Chemistry & biology, 17:1250). Inhibition of OGA will result in an increase of O- GlcNAcylated protein levels resulting in an increased signal in the experiment. Cell nuclei are stained with Hoechst to give a cell culture quality control and a rough estimate of immediate compounds toxicity, if any. ICC pictures are imaged with a Perkin Elmer Opera Phenix plate microscope and quantified with the provided software Perkin Elmer Harmony 4.1.
Cells were propagated in DMEM high Glucose (Sigma, #D5796) following standard procedures. 2 days before the cell assay cells are split, counted and seeded in Poly-D- Lysine (PDL) coated 96-wells (Greiner, #655946) plate at a cell density of 12,000 cells per cm2 (4,000 cells per well) in IOOmI of Assay Medium (Low Glucose medium is used to reduce basal levels of GlcNAcylation) (Park et al. 2014 The Journal of biological chemistry 289: 13519). At the day of compound test medium from assay plates was removed and replenished with 90pl of fresh Assay Medium. 10m1 of compounds at a lOfold final concentration were added to the wells. Plates were centrifuged shortly before incubation in the cell incubator for 6 hours. DMSO concentration was set to 0.2%. Medium is discarded by applying vacuum. For staining of cells medium was removed and cells washed once with 100 mΐ D-PBS (Sigma, #D8537). From next step onwards unless other stated assay volume was always 50m1 and incubation was performed without agitation and at room temperature. Cells were fixed in 50m1 of a 4% paraformaldehyde (PFA, Alpha aesar, # 043368) PBS solution for 15 minutes at room temperature. The PFA PBS solution was then discarded and cells washed once in lOmM Tris Buffer (LifeTechnologies, # 15567-027), 150mMNaCl (LifeTechnologies, #24740-0110, 0.1% Triton X (Alpha aesar, # A16046), pH 7.5 (ICC buffer) before being permeabilized in same buffer for 10 minutes. Samples are subsequently blocked in ICC containing 5% goat serum (Sigma, #G9023) for 45-60 minutes at room temperature. Samples were then incubated with primary antibody (1/1000 from commercial provider, see above) at 4°C overnight and subsequently washed 3 times for 5 minutes in ICC buffer. Samples were incubated with secondary fluorescent antibody (1/500 dilution, Lifetechnologies, # A-21042) and nuclei stained with Hoechst 33342 at a final concentration of lpg/ml in ICC (Lifetechnologies, # H3570) for 1 hour. Before analysis samples were washed 2 times manually for 5 minutes in ICC base buffer.
Imaging is performed using Perkin Elmer Phenix Opera using a water 20x objective and recording 9 fields per well. Intensity readout at 488nm is used as a measure of O-GlcNAcylation level of total proteins in wells. To assess potential toxicity of compounds nuclei were counted using the Hoechst staining. IC o-values are calculated using parametric non-linear regression model fitting. As a maximum inhibition Thiamet G at a 200uM concentration is present on each plate. In addition, a concentration response of Thiamet G is calculated on each plate.
TABLE 8. Results in the biochemical and cellular assays. Representative compounds of the present invention were tested according to the procedure as described above, with results as listed in the table below (n.d. means not determined). The values reported in the table below are subject to error margins associated with the assay used and the equipment.
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Representative compounds of the present invention were tested according to the procedure as described above, with results as listed in the table below (n.d. means not determined). The values reported in the table below are subject to typical error margins, and are averaged values over several runs of a particular compound, that were obtained after recalibration of the equipment.
Figure imgf000172_0002

Claims

1. A compound of Formula (I)
Figure imgf000173_0001
or a tautomer or a stereoisomeric form thereof, wherein RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thienyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- ajpyridinyl, imidazo[l,5-a]pyridinyl, imidazo[l,5-b]pyridazinyl, indolizinyl, 1H- indolyl, lH-indazolyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; cyano; hydroxy; phenyl; pyrazolyl; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents;
Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
-NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci- 4alkyl; Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or CFF; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000174_0001
(b-1) (b-2) (b-3) wherein Rbl represents hydrogen or fluoro; or a pharmaceutically acceptable addition salt or a solvate thereof.
2. The compound according to claim 1, wherein
RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, imidazolyl, thiazolyl, oxazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, 1,8-naphthyridinyl, pyrazolo[l,5-a]pyridinyl, imidazo[l,2- ajpyridinyl, imidazo[l,5-a]pyridinyl, imidazo[l,5-b]pyridazinyl, indolizinyl, 1H- indolyl, lH-indazolyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; cyano; pyrazolyl; Ci- 4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents;
-NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci- 4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or CfR; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000175_0001
(b-1) (b-2) (b-3) wherein Rbl represents hydrogen or fluoro.
3. The compound according to claim 1 or 2, wherein
RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of pyrazolyl, thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from the group consisting of lH-benzo[d]imidazolyl, imidazo[l,2-a]pyridinyl, quinolinyl, isoquinolinyl, and thiazolo[4,5-b]pyridinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; pyrazolyl; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents; Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents; -NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is selected from hydrogen and Ci-4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and methyl;
R2 represents H or CfR; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000175_0002
(b-1) (b-2) (b-3) wherein Rbl represents hydrogen or fluoro.
4. The compound according to any one of claims 1 to 3, wherein
RA is selected from the group consisting of a) a six-membered monocyclic heteroaryl radical selected from the group consisting of pyridyl, pyrimidinyl, and pyrazinyl; b) a five-membered monocyclic heteroaryl radical selected from the group consisting of thiadiazolyl, and oxadiazolyl; and c) a 9- to 10-membered bicyclic heteroaryl radical selected from quinolinyl; wherein each of a), b) or c) may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of cyano; Ci-4alkyl optionally substituted with 1, 2, or 3 independently selected halo substituents;
Ci-4alkyloxy optionally substituted with 1, 2, or 3 independently selected halo substituents; -NH-C(=0)-Ra; and -C(=0)-NH-Ra; wherein Ra is and Ci-4alkyl;
Rla, Rlb, Rlc, and Rld are each independently selected from the group consisting of hydrogen and Ci-4alkyl; in particular, Rla and Rlb are each independently selected from the group consisting of hydrogen and Ci-4alkyl, in particular hydrogen and methyl; and Rlc and Rld are each hydrogen;
R2 represents H or C¾; and
RB is a radical selected from the group consisting of (b-1), (b-2), and (b-3):
Figure imgf000176_0001
wherein Rbl represents fluoro.
5. A pharmaceutical composition comprising a prophylactically or a therapeutically effective amount of a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
6. A process for preparing the pharmaceutical composition according to claim 5, comprising mixing a pharmaceutically acceptable carrier with a prophylactically or a therapeutically effective amount of a compound according to any one of claims 1 to 4.
7. A compound as defined in any one of claims 1 to 4, or the pharmaceutical composition as defined in claim 5, for use as a medicament.
8. A compound as defined in any one of claims 1 to 4, or the pharmaceutical composition as defined in claim 5, for use in the treatment or prevention of a tauopathy, or an alpha synucleinopathy.
9. The compound or the pharmaceutical composition for use according to claim 8, wherein the tauopathy is selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration
(some cases caused by C90RF72 mutations), Gerstmann-Straussler-Scheinker disease, Parkinson’s disease, Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non- Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, SLC9A6-related mental retardation, subacute sclerosing panencephalitis, tangle-only dementia, and white matter tauopathy with globular glial inclusions; and the alpha synucleinopathy is selected from the group consisting of Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease.
10 A compound as defined in any one of claims 1 to 4, or the pharmaceutical composition as defined in claim 5, for use in the control or reduction of the risk of preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, or tau-related neurodegeneration as observed in different forms of tauopathies.
11. A compound as defined in any one of claims 1 to 4, or the pharmaceutical composition as defined in claim 5, for use in the control or reduction of the risk of prodromal Parkinson’s disease.
12. A method of preventing or treating a disorder selected from the group consisting of tauopathy or an alpha synucleinopathy, in particular a tauopathy selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, Down’s syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia and parkinsonism linked to chromosome 17 (caused by MAPT mutations), Frontotemporal lobar degeneration (some cases caused by C90RF72 mutations), Gerstmann-Straussler-Scheinker disease, Parkinson’s disease,
Guadeloupean parkinsonism, myotonic dystrophy, neurodegeneration with brain iron accumulation, Niemann-Pick disease, type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, SLC9A6-related mental retardation, subacute sclerosing panencephalitis, tangle-only dementia, and white matter tauopathy with globular glial inclusions, or in particular an alpha synucleinopathy selected from the group consisting of Parkinson’s disease, dementia due to Parkinson’s (or neurocognitive disorder due to Parkinson’s disease), dementia with Lewy bodies, multiple system atrophy, and alpha synucleinopathy caused by Gaucher’s disease, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of a compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.
13. A method for inhibiting O-GlcNAc hydrolase, comprising administering to a subject in need thereof, a prophylactically or a therapeutically effective amount of a compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5.
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