Patent Grant
11034696
This application is a 371 of International Application No. PCT/CN2018/073833, filed Jan. 23, 2018, which claims the benefit of International Application No. PCT/CN2017/072589, filed Jan. 25, 2017, and International Application No. PCT/CN2017/072614, filed Jan. 25, 2017, which are incorporated herein in their entireties.
The present invention relates to novel compounds that inhibit LRRK2 kinase activity, processes for their preparation, compositions containing them and their use in the treatment of diseases associated with or characterized by LRRK2 kinase activity, for example, Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS).
Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective degeneration and cell death of dopaminergic neurons in the substantial nigra region of the brain. Parkinson's disease was generally considered to be sporadic and of unknown etiology, but, in the last 15 years, there has been an important development of the understanding of the genetic basis of this disease and associated pathogenic mechanisms. One area of the development is the understanding of leucine rich repeat kinase 2 (LRRK2) protein. A number of mis-sense mutations in the LRRK2 gene have been strongly linked with autosomal dominant Parkinson's disease in familial studies (See WO2006068492 and WO2006045392; Trinh and Farrer 2013, Nature Reviews in Neurology 9: 445-454; Paisan-Ruiz et al., 2013, J. Parkinson's Disease 3: 85-103). The G2019S mutation in LRRK2 is the most frequent mis-sense mutation and is associated with a clinical phenotype that closely resembles sporadic Parkinson's disease. The LRRK2 G2019S mutation is also present in approximately 1.5% of sporadic Parkinson's disease cases (See Gilks et al., 2005, Lancet, 365: 415-416). In addition to the known pathogenic coding mutations in LRRK2, additional amino acid coding variants of LRRK2 have been identified that are also associated with risk of developing Parkinson's disease (See Ross et al., 2011 Lancet Neurology 10: 898-908). Furthermore, genome-wide association studies (GWAS) have identified LRRK2 as a Parkinson's disease susceptibility locus, which indicates that LRRK2 may be also relevant to sporadic Parkinson's disease cases without mutations that cause amino acid substitutions in the LRRK2 protein. (See Satake et al., 2009 Nature Genetics 41:1303-1307; Simon-Sanchez et al 2009 Nature Genetics 41: 1308-1312)
LRRK2 is a member of the ROCO protein family and all members of this family share five conserved domains. The most common pathogenic mutation G2019S occurs in the highly conserved kinase domain of LRRK2. This mutation confers an increase in the LRRK2 kinase activity in in vitro enzyme assays of recombinant LRRK2 proteins (See Jaleel et al., 2007, Biochem J, 405: 307-317) and in LRRK2 proteins purified from G2019S PD patient-derived cells (See Dzamko et al., 2010 Biochem. J. 430: 405-413). A less frequent LRRK2 pathogenic mutation that confers amino acid substitution at a different residue, R1441, has also been shown to elevate LRRK2 kinase activity by decreasing the rate of GTP hydrolysis by the GTPase domain of LRRK2 (See Guo et al., 2007 Exp Cell Res. 313: 3658-3670; West et al., 2007 Hum. Mol Gen. 16: 223-232). Moreover, phosphorylation of Rab protein physiologic substrates of LRRK2 has been shown to be increased by a range of Parkinson's disease pathogenic mutations of LRRK2 (See Steger et al., 2016 eLife 5 e12813). Therefore, the evidence indicates that the kinase and GTPase activities of LRRK2 are important for pathogenesis, and that the LRRK2 kinase domain may regulate overall LRRK2 function (See Cookson, 2010 Nat. Rev. Neurosci. 11: 791-797).
There is evidence to show that the increased LRRK2 kinase activity is associated with neuronal toxicity in cell culture models (See Smith et al., 2006 Nature Neuroscience 9: 1231-1233) and kinase inhibitor compounds protect against LRRK2-mediated cell death (See Lee et al., 2010 Nat. Med. 16: 998-1000). LRRK2 has also been reported to act as a negative regulator of microglial-mediated clearance of alpha-synuclein (See Maekawa et al., 2016 BMC Neuroscience 17:77), suggesting a possible utility of LRRK2 inhibitors in promoting clearance of neurotoxic forms of alpha-synuclein in the treatment of Parkinson's disease.
Induced pluripotent stem cells (iPSCs) derived from LRRK2 G2019S Parkinson's disease patients have been found to exhibit defects in neurite outgrowth and increased susceptibility to rotenone, that may be ameliorated by either genetic correction of the G2019S mutation or treatment of cells with small molecule inhibitors of LRRK2 kinase activity (See Reinhardt et al., 2013 Cell Stem Cell 12: 354-367). Mitochondrial DNA damage has been reported as a molecular marker of vulnerable dopamine neurons in substantia nigra of postmortem Parkinson's disease specimens (See Sanders et al 2014 Neurobiol. Dis. 70: 214-223). Increased levels of such mitochondrial DNA damage associated with LRRK2 G2019S mutation in isPCs is blocked by genetic correction of the G2019S mutation (See Sanders et al., 2014 Neurobiol. Dis. 62: 381-386).
Additional evidence links LRRK2 function and dysfunction with autophagy-lysosomal pathways (See Manzoni and Lewis, 2013 Faseb J. 27:3234-3429). LRRK2 proteins confer defects in chaperone-mediated autophagy that negatively impact the ability of cells to degrade alpha-synuclein (Orenstein et al., 2013 Nature Neurosci. 16 394-406). In other cell models, selective LRRK2 inhibitors have been shown to stimulate macroautophagy (See Manzoni et al., 2013 BBA Mol. Cell Res. 1833: 2900-2910). These data suggest that small molecule inhibitors of LRRK2 kinase activity may have utility in the treatment of diseases characterized by defects in cellular proteostasis that result from aberrant autophagy/lysosomal degradation pathways including forms of Parkinson's disease associated with GBA mutations (See Swan and Saunders-Pullman 2013 Curr. Neurol. Neurosci Rep. 13: 368), other alpha-synucleinopathies, tauopathies, Alzheimer's disease (See Li et al., 2010 Neurodegen. Dis. 7: 265-271) and other neurodegenerative diseases (See Nixon 2013 Nat. Med. 19: 983-997) and Gaucher disease (See Westbroek et al., 2011 Trends. Mol. Med. 17: 485-493). As promoters of autophagy, small molecule inhibitors of LRRK2 kinase may also have utility in treatment of other diseases including diabetes, obesity, motor neuron disease, epilepsy and some cancers (See Rubinsztein et al., 2012 Nat. Rev. Drug Discovery 11: 709-730), pulmonary diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (See Araya et al., 2013 Intern. Med. 52: 2295-2303) and autoimmune diseases susch as systemic lupus erythematosus (See Martinez et al., 2016 Nature 533: 115-119). As promoters of autophagy and phagocytic processes, small molecule inhibitors of LRRK2 kinase may also have utility in augmenting host responses in treatment of a range of intracellular bacterial infections, parasitic infections and viral infections, including diseases such as tuberculosis (See Rubinsztein et al., 2012 Nat. Rev. Drug Discovery 11: 709-730; Araya et al., 2013 Intern. Med. 52: 2295-2303; Gutierrez, Biochemical Society Conference; Leucine rich repeat kinase 2: ten years along the road to therapeutic intervention, Henley Business School, UK 12 Jul. 2016), HIV, West Nile Virus and chikungunya virus (see Shoji-Kawata et al., 2013 Nature 494: 201-206). LRRK2 inhibitors may have utility in treatment of such diseases alone, or in combination with drugs that directly target the infectious agent. Further, significantly elevated levels of LRRK2 mRNA have also been observed in fibroblasts of Niemann-Pick Type C (NPC) disease patients compared with fibroblasts of normal subjects, which indicates that aberrant LRRK2 function may play a role in lysosomal disorders (See Reddy et al., 2006 PLOS One 1 (1):e19 doi: 10.1371/journal.pone.0000019—supporting information Dataset S1). This observation suggests that LRRK2 inhibitors may have utility for treatment of NPC.
The PD-associated G2019S mutant form of LRRK2 has also been reported to enhance phosphorylation of tubulin-associated Tau (See Kawakami et al., 2012 PLoS ONE 7: e30834, doi 10.1371), and disease models have been proposed in which LRRK2 acts upstream of the pathogenic effects of Tau and alpha-synuclein (See Taymans & Cookson, 2010, BioEssays 32: 227-235). In support of this, LRRK2 expression has been associated with increased aggregation of insoluble Tau, and increased Tau phosphorylation, in a transgenic mouse model (See Bailey et al., 2013 Acta Neuropath. 126:809-827). Over-expression of the PD pathogenic mutant protein LRRK2 R1441G is reported to cause symptoms of Parkinson's disease and hyperphosphorylation of Tau in transgenic mouse models (See Li, Y. et al. 2009, Nature Neuroscience 12: 826-828). Therefore, these data suggest that LRRK2 inhibitors of kinase catalytic activity may be useful for the treatment of tauopathy diseases characterized by hyperphosphorylation of Tau such as argyrophilic grain disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy and inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (See Goedert, M and Jakes, R (2005) Biochemica et Biophysica Acta 1739, 240-250). In addition, LRRK2 inhibitors may have utility in treatment of other diseases characterized by diminished dopamine levels such as withdrawal symptoms/relapse associated with drug addiction (See Rothman et al., 2008, Prog. Brain Res, 172: 385).
Other studies have also shown that overexpression of the G2019S mutant form of LRRK2 confers defects in subventricular zone (SVZ) neuroprogenitor cell proliferation and migration in transgenic mouse models (See Winner et al., 2011 Neurobiol. Dis. 41: 706-716) and reduces neurite length and branching cell culture models (See Dachsel et al., 2010 Parkinsonism & Related Disorders 16: 650-655). Moreover, it was reported that agents that promote SVZ neuroprogenitor cell proliferation and migration also improve neurological outcomes following ischemic injury in rodent models of stroke (See Zhang et al., 2010 J. Neurosci. Res. 88: 3275-3281). These findings suggest that compounds that inhibit aberrant activity of LRRK2 may have utility for the treatments designed to stimulate restoration of CNS functions following neuronal injury, such as ischemic stroke, traumatic brain injury, spinal cord injury.
Mutations in LRRK2 have also been identified that are clinically associated with the transition from mild cognitive impairment (MCI) to Alzheimer's disease (See WO2007149798). These data suggest that inhibitors of LRRK2 kinase activity may be useful for the treatment diseases such as Alzheimer's disease, other dementias and related neurodegenerative disorders.
Aberrant regulation of normal LRRK2 proteins is also observed in some disease tissues and models of disease. Normal mechanisms of translational control of LRRK2 by miR-205 are perturbed in some sporadic PD cases, where significant decreases in miR-205 levels in PD brain samples concur with elevated LRRK2 protein levels in those samples (See Cho et al., (2013) Hum. Mol. Gen. 22: 608-620). Therefore, LRRK2 inhibitors may be used in treatment of sporadic PD patients who have elevated levels of normal LRRK2 proteins.
In an experimental model of Parkinson's disease in marmosets, an elevation of LRRK2 mRNA is observed in a manner that correlates with the level of L-Dopa induced dyskinesia (See Hurley, M. J et al., 2007 Eur. J. Neurosci. 26: 171-177). This suggests that LRRK2 inhibitors may have a utility in amelioration of such dyskinesias.
Significantly elevated levels of LRRK2 mRNA have been reported in ALS patient muscle biopsy samples (See Shtilbans et al., 2011 Amyotrophic Lateral Sclerosis 12: 250-256) It is suggested that elevated levels of LRRK2 kinase activity may be a characteristic feature of ALS. Therefore, this observation indicated that LRRK2 inhibitor may have utility for treatment of ALS.
There is also evidence indicating that LRRK2 kinase activity may play a role in mediating microglial proinflammatory responses (See Moehle et al., 2012, J. Neuroscience 32: 1602-1611). This observation suggests a possible utility of LRRK2 inhibitors for treatment of aberrant neuroinflammatory mechanisms that contribute a range of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, multiple sclerosis, HIV-induced dementia, amyotrophic lateral sclerosis, ischemic stroke, traumatic brain injury and spinal cord injury. Some evidence also indicates that LRRK2 plays a role in regulating neuronal progenitor differentiation in vitro (See Milosevic, J. et al., 2009 Mol. Neurodegen. 4: 25). This evidence suggests that inhibitors of LRRK2 may have a utility in production of neuronal progenitor cells in vitro for consequent therapeutic application in cell based-treatment of CNS disorders.
It has been reported that Parkinson's disease patients bearing LRRK2 G2019S mutation display increased frequency of non-skin cancers, including renal, breast, lung, prostate cancers as well as acute myelogenous leukemia (AML). Since there is evidence to show that G2019S mutation in LRRK2 increases catalytic activity of the LRRK2 kinase domain, small molecule inhibitors of LRRK2 may have a utility in treatment of cancers, for example kidney cancer, breast cancer, lung cancer, prostate cancer (e.g. solid tumors) and blood cancer (See. AML; Saunders-Pullman et al., 2010, Movement Disorders, 25:2536-2541; Inzelberg et al., 2012 Neurology 78: 781-786). Amplification and over-expression of LRRK2 has also been reported in papillary renal and thyroid carcinomas, where co-operativity between LRRK2 and the MET oncogene may promote tumor cell growth and survival (See Looyenga et al., 2011 PNAS 108: 1439-1444.)
Some studies suggested that genetic association of common LRRK2 variants with susceptibility to ankylosing spondylitis (See Danoy P, et al., 2010. PLoS Genet.; 6(12):e1001195; and leprosy infection. (See Zhang F R, et al. 2009, N Engl J Med. 361:2609-18.) These findings suggest that inhibitors of LRRK2 may have a utility in the treatment of ankylosing spondylitis and leprosy infection.
Meta-analysis of three genome wide associated scans for Crohn's disease identified a number of loci associated with the disease, including the locus containing the LRRK2 gene (See Barrett et al., 2008, Nature Genetics, 40: 955-962). Evidence has also emerged that LRRK2 is an IFN-γ target gene that may be involved in signaling pathways relevant to Crohn's disease pathogenesis (See Gardet et al., 2010, J. Immunology, 185: 5577-5585). These findings suggest that inhibitors of LRRK2 may have utility in the treatment of Crohn's disease.
As an IFN-γ target gene, LRRK2 may also play a role in T cell mechanisms that underlie other diseases of the immune system such as multiple sclerosis and rheumatoid arthritis. Further potential utility of LRRK2 inhibitors comes from the reported finding that B lymphocytes constitute a major population of LRRK2 expressing cells (See Maekawa et al. 2010, BBRC 392: 431-435). This suggests that LRRK2 inhibitors may be effective in treatment of diseases of the immune system for which B cell depletion is, or may be, effective in diseases such as lymphomas, leukemias, multiple sclerosis (See Ray et al., 2011 J. Immunol. 230: 109), rheumatoid arthritis, systemic lupus erythematosus, autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis, bullous skin disorders, type 1 diabetes mellitus, Sjogren's syndrome, Devic's disease and inflammatory myopathies (See Engel et al., 2011 Pharmacol. Rev. 63: 127-156; Homam et al., 2010 J. Clin. Neuromuscular Disease 12: 91-102).
WO2016036586 and WO2017012576 disclose a series of compounds described as inhibitors of LRRK2 kinase and their use in the treatment of diseases, including, inter alia, Parkinson's disease. Unmet needs exist for new treatments that will halt or slow disease progression both in terms of motor (e.g. control of gait dysfunction, freezing, and postural imbalance) and non-motor symptoms (e.g. PD-associated dementia), reducing the need for escalating use of symptomatic medications and associated long-term adverse effects of currently available treatment (e.g. dyskinesia and on/off fluctuations) maintaining independence for longer.
The present invention provides, in a first aspect, compounds of Formula (I) and salts thereof:
wherein
In a further aspect of the invention, the invention provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
A further aspect of the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of Parkinson's disease or Alzheimer's disease.
The foregoing and other aspects of the present invention will now be described in more detail with respect to the description and methodologies provided herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the embodiments of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, biology described herein are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there is a plurality of definitions for a term used herein, those in this section prevail unless stated otherwise.
As used herein, “alkyl” refers to a monovalent, saturated hydrocarbon chain having a specified number of carbon atoms. For example, C1-3 alkyl refers to an alkyl group having from 1 to 3 carbon atoms. Alkyl groups may be straight or branched. In some embodiments, branched alkyl groups may have one, two, or three branches. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, and propyl (n-propyl and isopropyl).
As used herein, “alkoxy” refers to the group —O-alkyl. For example, C1-6 alkoxy groups contain from 1 to 6 carbon atoms. C1-3 alkoxy groups contain from 1 to 3 carbon atoms. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxyl, pentyloxy, and hexyloxy.
As used herein, “cycloalkyl” refers to a saturated monocyclic hydrocarbon ring having a specified number of carbon atoms. For example, C3-6 cycloalkyl contains 3 to 6 carbon atoms as member atoms in the ring. Examples of C3-6 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, “halogen” refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). “Halo” refers to the halogen radicals: fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).
As used herein, “haloalkyl” refers to an alkyl group, as defined above, having one or more halogen atoms selected from F, Cl, Br, or I, which are substituted on any or all of the carbon atoms of the alkyl group by replacing hydrogen atoms attached to the carbon atoms and which may be the same or different. For example, C1-3haloalkyl refers to a C1-3alkyl group substituted with one or more halogen atoms. In some embodiments, “haloalkyl” refers to an alkyl group substituted with one or more halogen atoms independently selected from F or Cl. Exemplary haloalkyl groups include, but are not limited to, chloromethyl, bromoethyl, trifluoromethyl, and dichloromethyl.
As used herein, “haloalkoxy” refers to the group —O-haloalkyl. For example, C1-3 haloalkoxy groups contain from 1 to 6 carbon atoms.
As used herein, “heterocyclyl” is a monovalent radical derived by removal of a hydrogen atom from a saturated monocyclic ring, which ring consists of ring carbon atoms and 1 or more ring heteroatoms independently selected from nitrogen, oxygen or sulphur. In one embodiment, the ring-heteroatoms are independently selected from nitrogen or oxygen. The number of ring atoms may be specified. For example, a “5-6 membered heterocyclyl” is a heterocyclyl as defined above consisting of 5-6 ring atoms. The term nitrogen containing heterocyclyl refers to heterocyclyl ring as defined above that contains at least one nitrogen ring atom. The term oxygen containing heterocyclyl should be construed in an analogous manner. The expression —O-4-6 membered heterocyclyl ring refers to heterocyclyl ring as defined above consisting of 4-6 ring atoms that is linked to the core through an oxygen atom. Other ring heteroatoms (nitrogen, oxygen or sulphur) may additionally be present. Examples of herterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl (including, for example, tetrahydrofuran-2-yl and tetrahydrofuran-3-yl), pyrrolidinyl (including, for example, pyrrolidin-1-yl and pyrrolidin-3-yl), tetrahydro-2H-pyrany or oxanyl (including, for example, tetrahydro-2H-pyran-3-yl or oxayn-3-yl and tetrahydro-2H-pyran-4-yl or oxan-4-yl), piperidinyl (including, for example, piperidin-3-yl and piperidin-4-y) and morpholinyl (including, for example, morpholin-2-yl and morpholin-4-yl).
The term “fused bicyclic heterocyclyl” refers to a monovalent radical derived by removal of a hydrogen atom from a saturated bicyclic ring (two rings sharing one bond), which consists of carbon atoms and 1, 2 or 3 heteroatoms independently selected from nitrogen, oxygen or sulphur with the proviso that only one heteroatom may be oxygen. The number of atoms in the bicyclic heterocyclyl may be specified. For example, the term 6-9 membered fused bicyclic heterocyclyl refers to a fused bicyclic heterocyclyl that has a total of 6 to 9 atoms in the two rings. The term N-linked 6-9 membered fused bicyclic heterocyclyl refers to a 6-9 membered fused bicyclic heterocyclyl as defined above that contains at least one nitrogen ring atom through which it is linked to the core. One or two other ring heteroatoms (nitrogen, oxygen or sulphur) may additionally be present with the proviso that only one ring heteroatom may be oxygen. In one embodiment, the term N-linked 6-9 membered fused bicyclic heterocyclyl refers to a 6-9 membered fused bicyclic heterocyclyl as defined above that contains at least one nitrogen ring atom through which it is linked to the core. One or two other ring heteroatoms (nitrogen or oxygen) may additionally be present with the proviso that only one ring heteroatom may be oxygen. Examples of fused bicyclic heterocyclyl rings and N-linked fused bicyclic heterocyclyl rings include, but are not limited to,
As used herein, the term “heterospirane ring” refers to a monovalent radical derived by removal of a hydrogen atom from two rings connected through just one atom. The heterospirane ring consists of carbon atoms and 1 or more heteroatoms independently selected from nitrogen and oxygen but the spiro atom must be carbon. The number of atoms in the heterospirane ring may be specified. For example, a 7-10 membered heterospirane ring refers to a heterospirane ring that has a total of 7 to 10 atoms in the two rings (including the spiro-atom). The term “N-linked 7-10 membered heterospirane ring” refers to a 7-10 membered heterospirane ring as defined above that contains one nitrogen ring atom through which it is linked to the core. Examples of heterospirane rings and N-linked heterospirane rings include, but are not limited to,
As used herein, “substituted” in reference to a group indicates that one or more hydrogen atom attached to a member atom (e.g., carbon atom) within the group is replaced with a substituent selected from the group of defined substituents. It should be understood that the term “substituted” includes the implicit provision that such substitution is in accordance with the permitted valence of the substituted atom and the substituent and that the substitution results in a stable compound (i.e. one that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination and that is sufficiently robust to survive isolation from a reaction mixture). When it is stated that a group may contain one or more substituent, one or more (as appropriate) member atom within the group may be substituted. In addition, a single member atom within the group may be substituted with more than one substituent as long as such substitution is in accordance with the permitted valence of the atom.
As used herein, “optionally substituted” indicates that a particular group may be unsubstituted, or may be substituted as further defined.
As used herein, the term “disease” refers to any alteration in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease can also include a distemper, ailing, ailment, malady, disorder, sickness, illness, complain, interdisposition and/or affectation.
As used herein, “treat”, “treating” or “treatment” in reference to a disease means: (1) to ameliorate the disease or one or more of the biological manifestations of the disease, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the disease or (b) one or more of the biological manifestations of the disease, (3) to alleviate one or more of the symptoms or effects associated with the disease, (4) to slow the progression of the disease or one or more of the biological manifestations of the disease, and/or (5) to diminish the likelihood of severity of a disease or biological manifestations of the disease. Symptomatic treatment refers to treatment as referred to in point (1), (3) and (5). Disease modifying treatment refers to treatment as defined in point (2) and (4).
As used herein, “prevent”, “preventing” or “prevention” means the prophylactic administration of a drug to diminish the likelihood of the onset of or to delay the onset of a disease or biological manifestation thereof.
As used herein, “subject” means a mammalian subject (e.g., dog, cat, horse, cow, sheep, goat, monkey, etc.), and human subjects including both male and female subjects, and including neonatal, infant, juvenile, adolescent, adult and geriatric subjects, and further including various races and ethnicities including, but not limited to, white, black, Asian, American Indian and Hispanic.
As used herein, “pharmaceutically acceptable salt(s)” refers to salt(s) that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.
As used herein, “therapeutically effective amount” in reference to a compound of the invention or other pharmaceutically-active agent means an amount of the compound sufficient to treat or prevent the patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A therapeutically effective amount of a compound will vary with the particular compound chosen (e.g. consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the disease being treated; the severity of the disease being treated; the age, size, weight, and physical disease of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.
This invention provides, in a first aspect, a compound of Formula (I) or a salt thereof:
wherein
In one embodiment, R1 is an N-linked 6-9 membered fused bicyclic heterocyclyl optionally substituted with one, two or three substituents independently selected from the group consisting of: oxo, halo, hydroxyl, C1-3alkyl and C1-3alkoxy.
In one embodiment, R1 is an N-linked 6-9 membered fused bicyclic heterocyclyl selected from the group consisting of: hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, hexahydropyrrolo[1,2-a]pyrazine-6(2H)-yl, 3-azabicyclo[3.1.0]hexanyl and hexahydro-2H-furo[2,3-c]pyrrolyl, which fused bicyclic heterocyclyl is optionally substituted with one, two or three substituents independently selected from the group consisting of: oxo, halo, hydroxyl, C1-3alkyl and C1-3alkoxy.
In one embodiment, R1 is an N-linked 6-9 membered fused bicyclic heterocyclyl selected from the group consisting of: hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-6(2H)-yl, 3-azabicyclo[3.1.0]hexanyl, hexahydro-2H-furo[2,3-c]pyrrolyl and octahydropyrazino[2,1-c][1,4]oxazinyl, which fused bicyclic heterocyclyl is optionally substituted with one, two or three substituents independently selected from the group consisting of: oxo and hydroxyl.
In one embodiment, R1 is an N-linked 6-9 membered fused bicyclic heterocyclyl selected from the group consisting of: hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-2(2H)-yl, 3-azabicyclo[3.1.0]hexane-3-yl, hexahydro-2H-furo[2,3-c]pyrrole-5-yl and octahydropyrazino[2,1-c][1,4]oxazine-8-yl, which fused bicyclic heterocyclyl is optionally substituted with one, two or three substituents independently selected from the group consisting of: oxo and hydroxyl.
In one embodiment, R1 is an N-linked 6-9 membered fused bicyclic heterocyclyl selected from the group consisting of: hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one, 3-azabicyclo[3.1.0]hexan-1-ol, hexahydro-2H-furo[2,3-c]pyrrolyl and octahydropyrazino[2,1-c][1,4]oxazinyl.
In one embodiment, R1 is an N-linked 7-10 membered heterospirane ring, which heterospirane ring is optionally substituted with one, two or three substituents independently selected from the group consisting of: oxo, halo, hydroxyl, C1-3 alkoxyl and C1-3alkyl, wherein said C1-3 alkoxyl and C1-3alkyl is optionally substituted with one or two substituents independently selected from the group consisting of halo and hydroxyl, and with the proviso that R1 is not 2-oxa-6-azaspiro[3.4]octan-6-yl;
R1 is an N-linked 7-10 membered heterospirane ring, which heterospirane ring is optionally substituted with one substituent selected from the group consisting of: oxo, halo, hydroxyl, C1-3 alkoxyl and C1-3alkyl, and with the proviso that R1 is not 2-oxa-6-azaspiro[3.4]octan-6-yl.
In one embodiment, R1 is an N-linked 7-10 membered heterospirane ring selected from the group consisting of: oxazasprio[2.5]octanyl, dioxazaspiro[2.6]nonanyl, dioxazaspiro[3.5]nonanyl, dioxazaspiro[4.4]nonanyl, diazaspiro[2.7]decanyl, diazasprio[3.6]decanyl, diazasprio[4.5]decanyl, oxaadiazaspiro[2.7]decanyl, oxadiazasprio[3.6]decanyl and oxadiazasprio[4.5]decanyl, which heterospirane ring is optionally substituted with one substituent selected from the group consisting of: oxo, halo, hydroxyl, C1-3 alkoxyl and C1-3alkyl.
In one embodiment, R1 is an N-linked 7-10 membered heterospirane ring selected from the group consisting of: oxazasprio[2.5]octanyl, dioxazaspiro[2.6]nonanyl, dioxazaspiro[3.5]nonanyl, dioxazaspiro[4.4]nonanyl, diazaspiro[2.7]decanyl, diazasprio[3.6]decanyl, diazasprio[4.5]decanyl, oxaadiazaspiro[2.7]decanyl, oxadiazasprio[3.6]decanyl and oxadiazasprio[4.5]decanyl, which heterospirane ring is optionally substituted with one oxo group.
In one embodiment, R1 is an N-linked 7-10 membered heterospirane ring selected from the group consisting of: 1-oxa-4,8-diazaspiro[4.5]decan-8-yl, 3-oxa-1,8-diazaspiro[4.5]decan-8-yl, 1,8-diazaspiro[4.5]decan-8-yl, 2,8-diazaspiro[4.5]decan-8-yl, 2,5-dioxa-8-azaspiro[3.5]nonan-8-yl and 4-oxa-7-azaspiro[2.5]octan-7-yl, which heterospirane ring is optionally substituted with one oxo group.
In one embodiment, R1 is 3-oxo-1-oxa-4,8-diazaspiro[4.5]decan-8-yl, 2-oxo-3-oxa-1,8-diazaspiro[4.5]decan-8-yl, 2-oxo-1,8-diazaspiro[4.5]decan-8-yl, 1-oxo-2,8-diazaspiro[4.5]decan-8-yl, 2,5-dioxa-8-azaspiro[3.5]nonan-8-yl and 4-oxa-7-azaspiro[2.5]octan-7-yl.
In one embodiment, R2 is selected from the group consisting of:
In one embodiment, R2 is selected from the group consisting of:
In one embodiment, R2 is a 4-7 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a 5-6 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a 5-6 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a 5-6 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a 5-6 membered heterocyclyl ring selected from the group consisting of: piperazinyl, piperidinyl, pyrrolidinyl, tetrahydropyridinyl, tetrahydropyranyl, tetrahydrofuranyl and morpholinyl, which heterocyclyl ring is optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is piperidinyl or pyrrolidinyl ring, which ring is optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a piperidinyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a piperidinyl or pyrrolidinyl ring, which ring is optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a piperidinyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In one embodiment, R2 is a pyrrolidinyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3alkyl and hydroxyl.
In one embodiment, R2 is 1-(oxetan-3-yl)piperidin-4-yl or 1-(oxetan-3-yl)-3-fluoro-piperidin-4-yl.
In one embodiment, R2 is 1-(oxetan-3-yl)piperidin-4-yl.
In one embodiment, R2 is tetrahydropyran-4-yl.
In one embodiment, R2 is C3-6 cycloalkyl optionally substituted with one or two substituents independently selected from the group consisting of cyano, halo, hydroxyl, C1-3alkyl, C1-3alkoxyl, CO2H and a 4-6 membered heterocyclyl ring; In one embodiment, R2 is C3-6 cycloalkyl optionally substituted with one or two substituents independently selected from the group consisting of cyano, halo, hydroxyl, C1-3alkyl, C1-3alkoxyl and CO2H.
In one embodiment, R2 is C3-6 cycloalkyl optionally substituted with one or two substituents independently selected from the group consisting of hydroxyl and C1-3alkyl.
In one embodiment, R2 is cyclohexyl optionally substituted with one or two substituents independently selected from the group consisting of hydroxyl and C1-3alkyl.
In one embodiment, R2 is O-4-6 membered heterocyclyl ring wherein the heterocyclyl ring is optionally substituted with one or two substituents independently selected from the group consisting of: cyano, hydroxyl, C1-3alkyl, C1-3alkoxyl, CH2OH and —CO2H.
In one embodiment, R2 is O-tetrahydrofuran wherein the tetrahydrofuran ring is optionally substituted with one or two substituents independently selected from the group consisting of: cyano, hydroxyl, C1-3alkyl, C1-3alkoxyl, CH2OH and —CO2H.
In one embodiment, R3 is selected from the group consisting of CN, C1-3 alkyl, C1-3 alkoxy, C1-3haloalkyl, and C3 cycloalkyl. In one embodiment, R3 is selected from the group consisting of C1-3 alkyl and C1-3 alkoxyl. In one embodiment, R3 is selected from the group consisting of methyl and methoxy.
In one embodiment, R4 is selected from the group consisting of H, halo, CN, C1-3alkyl and C1-3haloalkyl. In one embodiment, R4 is selected from the group consisting of H, halo and C1-3alkyl. In one embodiment, R4 is selected from the group consisting of H, halo and methyl. In one embodiment, R4 is selected from the group consisting of H, fluoro, chloro and methyl. In one embodiment, R4 is selected from the group consisting of H, chloro and methyl. In one embodiment, R4 is selected from the group consisting of chloro and methyl. In one particular embodiment, R4 is methyl.
In one embodiment, the invention provides a compound of Formula (I) or a salt thereof wherein R1, R3 and R4 are as defined above, and R2 is a 5-6 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In this embodiment, R1, R3 and R4 may be further defined as in any of the preceding embodiments. For example, R3 may be selected from the group consisting of methyl and methoxy and/or R4 may be selected from the group consisting of chloro and methyl.
In one embodiment, the invention provides a compound of Formula (I) or a salt thereof wherein R1, R3 and R4 are as defined above and R2 is a 5-6 membered heterocyclyl ring optionally substituted with one, two or three substituents independently selected from the group consisting of:
In this embodiment, R1, R3 and R4 may be further defined as in any of the preceding embodiments. For example, R3 may be selected from the group consisting of methyl and methoxy and/or R4 may be selected from the group consisting of chloro and methyl.
In one embodiment, the invention provides a compound of Formula (I) or a salt thereof wherein R1, R3 and R4 are as defined above and R2 is C3-6 cycloalkyl optionally substituted with one or two substituents independently selected from the group consisting of cyano, halo, hydroxyl, C1-3alkyl, C1-3alkoxyl and CO2H. In this embodiment, R1, R3 and R4 may be further defined as in any of the preceding embodiments. For example, R3 may be selected from the group consisting of methyl and methoxy and/or R4 may be selected from the group consisting of chloro and methyl.
In one embodiment, the invention provides a compound of Formula (I) or a salt thereof wherein R1, R3 and R4 are as defined above and R2 is O-4-6 membered heterocyclyl ring wherein the heterocyclyl ring is optionally substituted with one or two substituents independently selected from the group consisting of: cyano, hydroxyl, C1-3alkyl, C1-3alkoxyl, CH2OH and —CO2H. In this embodiment, R1, R3 and R4 may be further defined as in any of the preceding embodiments. For example, R3 may be selected from the group consisting of methyl and methoxy and/or R4 may be selected from the group consisting of chloro and methyl.
In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound of any one of Examples A-1 to A-13 or a pharmaceutically acceptable salt thereof. In one embodiment, the compound of formula (I) is a compound of any one of Examples A-1 to A-13.
In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound of any one of Examples B-1 to B-10 or a pharmaceutically acceptable salt thereof. In one embodiment, the compound of formula (I) is a compound of any one of Examples B-1 to B-10.
In addition to the free base form or free acid form of the compounds described herein, the salt form of the compounds is also within the scope of the present invention. The salts or pharmaceutically-acceptable salts of the compounds described herein may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid form or free base form with a suitable base or acid, respectively. For reviews on suitable pharmaceutical salts see Berge et al, J. Pharm, Sci., 66, 1-19, 1977; P L Gould, International Journal of Pharmaceutics, 33 (1986), 201-217; and Bighley et al, Encyclopedia of Pharmaceutical Technology, Marcel Dekker Inc, New York 1996, Volume 13, page 453-497.
Certain compounds of formula (I) contain a basic group and are therefore capable of forming pharmaceutically-acceptable acid addition salts by treatment with a suitable acid. Suitable acids include pharmaceutically-acceptable inorganic acids and pharmaceutically-acceptable organic acids. Exemplary pharmaceutically-acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicyclate, glycollate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), and napthalene-2-sulfonate. In some embodiments, the pharmaceutically acceptable salts include the L-tartrate, ethanedisulfonate (edisylate), sulfate, phosphate, p-toluenesulfonate (tosylate), hydrochloride salt, methanesulfonate, citrate, fumarate, benzenesulfonate, maleate, hydrobromate, L-lactate, malonate, and S-camphor-10-sulfonate. In certain embodiments, some of these salts form solvates. In certain embodiments, some of these salts are crystalline.
Certain compounds of formula (I) contain an acidic group and are there for capable of forming pharmaceutically-acceptable base addition salts by treatment with a suitable base. Exemplary pharmaceutically acceptable base addition salts include, but are not limited to, aluminium, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N′-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl-2-pyrrolildine-1′-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine, and zinc.
Certain compounds of Formula (I) or salts thereof may exist in stereoisomeric forms (e.g., they may contain one or more asymmetric carbon atoms). The individual stereoisomers (enantiomers and diastereomers) and mixtures of these are included within the scope of the present invention. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.
Certain compounds of Formula (I) are capable of existing in tautomeric forms. For example, certain compounds exhibit keto-enol tautomerism. In some cases, only one of a pair of tautomeric forms fall within Formula (I). Such alternative tautomers also form part of the invention.
The invention also includes isotopically-labelled compounds and salts, which are identical to compounds of Formula (I) or salts thereof, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of Formula (I) or salts thereof isotopes of hydrogen, carbon, nitrogen, fluorine, such as 3H, 11C, 14C and 18F. Such isotopically-labelled compound of Formula (I) or salts thereof are useful in drug and/or substrate tissue distribution assays. For example, 11C and 18F isotopes are useful in PET (positron emission tomography). PET is useful in brain imaging. Isotopically-labelled compounds of Formula (I) and salts thereof can generally be prepared by carrying out the procedures disclosed below, by substituting a readily available isotopically-labelled reagent for a non-isotopically labelled reagent. In one embodiment, compounds of Formula (I) or salts thereof are not isotopically labelled.
Certain compounds of Formula (I) or salts thereof may exist in solid or liquid form. In the solid state, compounds of Formula (I) or salts may exist in crystalline or noncrystalline form, or as a mixture thereof. For compounds of Formula (I) or salts that are in crystalline form, the skilled artisan will appreciate that pharmaceutically-acceptable solvates may be formed wherein solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent that is incorporated into the crystalline lattice are typically referred to as “hydrates.” Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water.
The skilled artisan will further appreciate that certain compounds of Formula (I), pharmaceutically acceptable salts or solvates thereof that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as “polymorphs.” Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.
The skilled artisan also appreciates that this invention may contain various deuterated forms of compounds of Formula (I), or pharmaceutically acceptable salts thereof. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. A person of ordinary skill in the art will know how to synthesize deuterated forms of compounds of Formula (I), or pharmaceutically acceptable salts thereof. Commercially available deuterated starting materials may be employed in the preparation of deuterated forms of compounds of Formula (I) or pharmaceutically acceptable salts thereof, or they may be synthesized using conventional techniques employing deuterated reagents (e.g. lithium aluminum deuteride).
Compounds of Formula (I) or pharmaceutically acceptable salts thereof are inhibitors of LRRK2 kinase activity and are thus believed to be of potential use in the treatment of or prevention of the following neurological diseases: Parkinson's disease, Alzheimer's disease, dementia (including Lewy body dementia and vascular dementia, HIV-induced dementia), amyotrophic lateral sclerosis (ALS), age related memory dysfunction, mild cognitive impairment, argyrophilic grain disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), withdrawal symptoms/relapse associated with drug addiction, L-Dopa induced dyskinesia, ischemic stroke, traumatic brain injury, spinal cord injury and multiple sclerosis. Other diseases potentially treatable by inhibition of LRRK2 include, but are not limited to, lysosomal disorders (for example, Niemann-Pick Type C disease, Gaucher disease), Crohn's disease, cancers (including thyroid, renal (including papillary renal), breast, lung and prostate cancers, leukemias (including acute myelogenous leukemia (AML)) and lymphomas), rheumatoid arthritis, systemic lupus erythematosus, autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis, bullous skin disorders, type 1 diabetes mellitus, obesity, epilepsy, pulmonary diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, Sjogren's syndrome, Devic's disease, inflammatory myopathies, ankylosing spondylitis, bacterial infections (including leprosy), viral infections (including tuberculosis, HIV, West Nile virus and chikungunya virus) and parasitic infections.
One aspect of the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in therapy. In one embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of or prevention of the above disorders (i.e. the neurological diseases and other diseases listed above). In one embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of or prevention of Parkinson's disease. In one embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of Parkinson's disease. In another embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of or prevention of Alzheimer's disease. In one embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of Alzheimer's disease. In another embodiment, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of tuberculosis.
A further aspect of the invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of or prevention of the above disorders (i.e. the neurological diseases and other diseases listed above). A further aspect of the invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of or prevention of Parkinson's disease. A further aspect of the invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of Parkinson's disease. In another embodiment, the invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of treatment of or prevention of Alzheimer's disease. In one embodiment, the invention provides the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of Alzheimer's disease. In another embodiment, the invention provides use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of tuberculosis.
A further aspect of the invention provides a method of treatment or prevention of a disorder listed above (i.e. selected from the neurological diseases and other diseases listed above), which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of treatment or prevention of Parkinson's disease, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of treatment of Parkinson's disease, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of treatment or prevention of Alzheimer's disease, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of treatment of Alzheimer's disease, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of treatment of tuberculosis, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In an embodiment, the subject is human.
In the context of the present invention, treatment of Parkinson's disease refers to the treatment of sporadic Parkinson's disease, and/or familial Parkinson's disease. In one embodiment, treatment of Parkinson's disease refers to treatment of familial Parkinson's disease. Familial Parkinson's disease patients are those expressing one or more of the following LRRK2 kinase mutations: G2019S mutation, N1437H mutation, R1441G mutation, R1441C mutation, R1441H mutation, Y1699C mutation, S1761R mutation, or I2020T mutation. In another embodiment, familial Parkinson's disease patients express other coding mutations (such as G2385R) or non-coding single nucleotide polymorphisms at the LRRK2 locus that are associated with Parkinson's disease In a more particular embodiment, familial Parkinson's disease includes patients expressing the G2019S mutation or the R1441G mutation in LRRK2 kinase. In one embodiment, treatment of Parkinson's disease refers to the treatment of familial Parkinson's disease includes patients expressing LRRK2 kinase bearing G2019S mutation. In another embodiment, familial Parkinson's disease patients express aberrantly high levels of normal LRRK2 kinase.
In the context of the present invention, treatment of Parkinson's disease refers to the treatment of sporadic Parkinson's disease, and/or familial Parkinson's disease. In one embodiment, treatment of Parkinson's disease refers to treatment of familial Parkinson's disease. Familial Parkinson's disease patients are those expressing one or more of the following LRRK2 kinase mutations: G2019S mutation, N1437H mutation, R1441G mutation, R1441C mutation, R1441H mutation, Y1699C mutation, S1761R mutation, or I2020T mutation. In another embodiment, familial Parkinson's disease patients express other coding mutations (such as G2385R) or non-coding single nucleotide polymorphisms at the LRRK2 locus that are associated with Parkinson's disease In a more particular embodiment, familial Parkinson's disease includes patients expressing the G2019S mutation or the R1441G mutation in LRRK2 kinase. In one embodiment, treatment of Parkinson's disease refers to the treatment of familial Parkinson's disease includes patients expressing LRRK2 kinase bearing G2019S mutation. In another embodiment, familial Parkinson's disease patients express aberrantly high levels of normal LRRK2 kinase.
In one embodiment, the invention provides a method of treatment of Parkinson's disease, which comprises administering to a human expressing the G2019S mutation in LRRK2 kinase in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
In one embodiment, the invention provides a method of treatment of Parkinson's disease, which comprises testing in a human for the G2019S mutation in LRRK2 kinase and administering to the human expressing the G2019S mutation in LRRK2 kinase in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
Treatment of Parkinson's disease may be symptomatic or may be disease modifying. In one embodiment, treatment of Parkinson's disease refers to symptomatic treatment. In one embodiment, treatment of Parkinson's disease refers to disease modifying treatment.
Compounds of the present invention may also be useful in treating patients identified as susceptible to progression to severe Parkinsonism by means of one or more subtle features associated with disease progression such as family history, olfaction deficits, constipation, cognitive defects, gait or biological indicators of disease progression gained from molecular, biochemical, immunological or imaging technologies. In this context, treatment may be symptomatic or disease modifying.
In the context of the present invention, treatment of Alzheimer's disease refers to the treatment of sporadic Alzheimer's disease and/or familial Alzheimer's disease. Treatment of Alzheimer's disease may be symptomatic or may be disease modifying. In one embodiment, treatment of Alzheimer's disease refers to symptomatic treatment.
In the context of the present invention, treatment of dementia (including Lewy body dementia and vascular dementia, HIV-induced dementia), amyotrophic lateral sclerosis (ALS), age related memory dysfunction, mild cognitive impairment, argyrophilic grain disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), multiple sclerosis, lysosomal disorders (for example, Niemann-Pick Type C disease, Gaucher disease), Crohn's disease, cancers (including thyroid, renal (including papillary renal), breast, lung and prostate cancers, leukemias (including acute myelogenous leukemia (AML)) and lymphomas), rheumatoid arthritis, systemic lupus erythematosus, autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis, bullous skin disorders, type 1 diabetes mellitus, obesity, epilepsy, pulmonary diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, Sjogren's syndrome, Devic's disease, inflammatory myopathies, ankylosing spondylitis, may be symptomatic or disease modifying. In certain embodiments, treatment of these disorders refers to symptomatic treatment.
The invention also provides the use of inhibitors of LRRK2 in the production of neuronal progenitor cells in vitro for consequent therapeutic application in cell based-treatment of CNS disorders.
When a compound of Formula (I) or a pharmaceutically acceptable salt thereof is intended for use in the treatment of Parkinson's disease, it may be used in combination with medicaments alleged to be useful as symptomatic treatments of Parkinson's disease. Suitable examples of such other therapeutic agents include L-dopa, and dopamine agonists (e.g. pramipexole, ropinirole).
When a compound of Formula (I) or a pharmaceutically acceptable salt thereof is intended for use in the treatment of Alzheimer's disease, it may be used in combination with medicaments claimed to be useful as either disease modifying or symptomatic treatments of Alzheimer's disease. Suitable examples of such other therapeutic agents may be symptomatic agents, for example those known to modify cholinergic transmission such as M1 muscarinic receptor agonists or allosteric modulators, M2 muscarinic antagonists, acetylcholinesterase inhibitors (such as tetrahydroaminoacridine, donepezil hydrochloride rivastigmine, and galantamine), nicotinic receptor agonists or allosteric modulators (such as α7 agonists or allosteric modulators or α4β2 agonists or allosteric modulators), PPAR agonists (such as PPARγ agonists), 5-HT4 receptor partial agonists, 5-HT6 receptor antagonists e.g. SB-742457 or 5HT1A receptor antagonists and NMDA receptor antagonists or modulators, or disease modifying agents such as β or γ-secretase inhibitors e.g semagacestat, mitochondrial stabilizers, microtubule stabilizers or modulators of Tau pathology such as Tau aggregation inhibitors (e.g. methylene blue and REMBER™), NSAIDS, e.g. tarenflurbil, tramiprosil; or antibodies for example bapineuzumab or solanezumab; proteoglycans for example tramiprosate.
When a compound of Formula (I) or a pharmaceutically acceptable salt thereof is intended for use in the treatment of bacterial infections, parasitic infections or viral infections, it may be used in combination with medicaments alleged to be useful as symptomatic treatments that directly target the infectious agent.
When a compound of Formula (I) or a pharmaceutically acceptable salt thereof is used in combination with other therapeutic agents, the compound may be administered either sequentially or simultaneously by any convenient route.
The invention also provides, in a further aspect, a combination comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof together with one or more further therapeutic agent or agents.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.
When a compound of Formula (I) or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.
Compounds of Formula (I) or pharmaceutically acceptable salts thereof may be formulated into pharmaceutical compositions prior to administration to a subject. According to one aspect, the invention provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. According to another aspect, the invention provides a process for the preparation of a pharmaceutical composition comprising admixing a compound of Formula (I) or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable excipient.
Pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain, for example, 0.1 mg, 0.5 mg, or 1 mg to 50 mg, 100 mg, 200 mg, 250 mg, 500 mg, 750 mg or 1 g of a compound of the present invention, depending on the disease being treated, the route of administration and the age, weight and condition of the subject, or pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. In other embodiments, the unit dosage compositions are those containing a daily dose or sub-dose as described herein, or an appropriate fraction thereof, of an active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well-known to one skilled in the art.
A therapeutically effective amount of a compound of Formula (I) will depend upon a number of factors including, for example, the age and weight of the intended recipient, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant prescribing the medication. However, a therapeutically effective amount of a compound of formula (I) for the treatment of diseases described in the present invention will generally be in the range of 0.1 to 100 mg/kg body weight of recipient per day and more usually in the range of 1 to 10 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day would usually be from 70 to 700 mg and this amount may be given in a single dose per day or in a number of sub-doses per day as such as two, three, four, five or six doses per day. Or the dosing can be done intermittently, such as once every other day, once a week or once a month. A therapeutically effective amount of a pharmaceutically acceptable salt or solvate, etc., may be determined as a proportion of the therapeutically effective amount of the compound of Formula (I) per se. It is envisaged that similar dosages would be appropriate for treatment of the other diseases referred to above.
The pharmaceutical compositions of the invention may contain one or more compounds of Formula (I). In some embodiments, the pharmaceutical compositions may contain more than one compound of the invention. For example, in some embodiments, the pharmaceutical compositions may contain two or more compounds of Formula (I). In addition, the pharmaceutical compositions may optionally further comprise one or more additional pharmaceutically active compounds.
As used herein, “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient may be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a subject and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided.
The compounds of the invention and the pharmaceutically-acceptable excipient or excipients may be formulated into a dosage form adapted for administration to the subject by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration (including buccal or sublingual) such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration (including subcutaneous, intramuscular, intravenous or intradermal) such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) nasal inhalation such as dry powders, aerosols, suspensions, and solutions; and (6) topical administration (including buccal, sublingual or transdermal) such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels. Such compositions may be prepared by any methods known in the art of pharmacy, for example by bringing into association a compound of Formula (I) with the carrier(s) or excipient(s). Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
Suitable pharmaceutically-acceptable excipients may vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically-acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate carrying or transporting the compound or compounds of the invention once administered to the subject from an organ, or a portion of the body, to another organ, or a portion of the body. Certain pharmaceutically-acceptable excipients may be chosen for their ability to enhance patient compliance.
Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically-acceptable excipients may serve more than one function and may serve alternative functions depending on how much the excipient is present in the formulation and what other ingredients are present in the formulation.
Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically-acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically-acceptable excipients and may be useful in selecting suitable pharmaceutically-acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).
The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).
In one aspect, the invention is directed to a solid oral dosage form such as a tablet or capsule comprising a therapeutically effective amount of a compound of the invention and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g. corn starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.
In certain embodiment, the present invention is directed to a pharmaceutical composition comprising 0.01 to 1000 mg of one or more of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and 0.01 to 5 g of one or more pharmaceutically acceptable excipients.
In another embodiment, the present invention is directed a pharmaceutical composition for the treatment of neurodegeneration disease comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In another embodiment, the present invention is directed a pharmaceutical composition for the treatment of Parkinson's disease comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
The process to be utilized in the preparation of compounds of formula (I) described herein depends upon the desired compounds. Such factors as the selection of the specific substituent and various possible locations of the specific substituent all play a role in the path to be followed in the preparation of the specific compounds of this invention. Those factors are readily recognized by one of ordinary skill in the art.
In general, the compounds of the present invention may be prepared by standard techniques known in the art and by known processes analogous thereto. General methods for preparing compounds of formula (I) are set forth below. All starting material and reagents described in the below general experimental schemes are commercially available or can be prepared by methods known to one skilled in the art.
The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. Suitable protecting groups and the methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley & Sons, NY (1999). In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.
General Scheme 1 provides exemplary processes of synthesis for preparing compounds of the present invention.
General Scheme 1 provides an exemplary synthesis for preparing compound 3 which represents compounds of Formula (I). In Scheme 1, R1, R2, R3 and R4 are as defined in Formula I.
Step (i) may be a substitution reaction by reacting compound 1 with compound 2 using appropriate base such as Cs2CO3 in an appropriate solvent such as N, N-dimethylformamide (DMF) under suitable temperature such as about 100° C. to provide compound 3.
Step (i) may alternatively be a coupling reaction using appropriate reagents such as CuI and N,N′-dimethyl-cyclohexane-1,2-diamine in the presence of suitable base such as K3PO4 in a suitable solvent such as toluene at suitable temperature such as reflux condition to provide compound 3.
Step (i) may alternatively be a coupling reaction using appropriate reagents such as Pd2dba3 and di-tert-butyl(2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphine in the presence of suitable base such as sodium tert-butoxide in a suitable solvent such as toluene at suitable temperature such as 100° C. to provide compound 3.
General Scheme 2 provides an exemplary synthesis for preparing intermediate 1 where R2 is connected to the indazole ring though a carbon atom of R2. R2a represents R2 where this is connected to the indazole ring though a carbon atom of R2. The protecting group, P1, can be any suitable protecting groups for example, tetrahydro-2H-pyran-2-yl (THP), (trimethylsilyl)ethoxy)methyl (SEM) or Acetyl (Ac).
Intermediate 5 can be obtained in step (i) by reacting starting material 4 with suitable reagents such as DHP in the presence of suitable acids such as TsOH in appropriate solvents such as DCM under suitable temperatures such as 20° C. to 40° C.
Step (ii) is a cross-coupling reaction between intermediate 5 and boronic acid or esters using appropriate palladium catalysts such as Pd(dppf)Cl2 in the presence of suitable bases such as Na2CO3 in appropriate solvents such as 1,4-dioxane at suitable temperatures such as 60° C. to 100° C.
Step (iii) involves reaction with suitable oxidation reagents such as H2O2 in a suitable solvent such as THF under suitable temperatures such as −60° C. to −10° C. to provide intermediate 7.
Step (iv) is a reaction with a suitable reducing reagent such as hydrogen in the presence of suitable catalysts such Pd/C in polar solvents such as MeOH at appropriate temperatures such as 25° C. to 80° C.
Step (v) may be an oxidation reaction with oxidants such as DMP in suitable solvents such as DCM under suitable temperatures such as 0° C. to 25° C. to give intermediate 8.
Steps (vi) and (viii) involve reaction with a fluridizer such as DAST in suitable solvents such as DCM under suitable temperatures such as −78° C. to 0° C.
Steps (viii) (x) and (xi) are de-protection reactions. Typically, the intermediate is reacted with suitable acids such HCl in suitable solvents such as 1,4-dioxane under suitable temperatures such as 25° C. to 40° C. to give intermediate 1.
General Scheme 3 provides an exemplary synthesis for preparing intermediate 1. when R2 connects to the indazole ring through an oxygen or nitrogen atom of R2. R2b represents R2 when R2 connects to the indazole ring through an oxygen atom of R2. R2c represents R2 when R2 connects to the indazole ring through the nitrogen atom of R2. The protecting group, P1, can be any suitable protecting group for example, tetrahydro-2H-pyran-2-yl (THP), (trimethylsilyl)ethoxy)methyl (SEM) or Acetyl (Ac).
Step (i) is a reaction with suitable reagents such as 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) using appropriate catalysts such as Pd(PPh3)4 in the presence of appropriate bases such as KOAc in appropriate solvents such as DMF at a suitable temperatures such as 80° C. to 120° C., followed by reaction with suitable reagents such H2O2 in the presence of appropriate bases such as NaOH in suitable solvents such as THF at appropriate temperatures such as 25° C. to 80° C.
Step (ii) is a reaction with a suitable alkylating reagent such as 2-iodopropane in the presence of suitable bases such as Cs2CO3 in appropriate solvents such as CH3CN at a suitable temperatures such as 25° C. to 100° C.
Step (iii) can be a Buchwald coupling reaction with different amines such as 1-methylpiperazine using appropriate palladium catalysts such as Pd2(dba)3 in the presence of appropriate bases such as Cs2CO3 and appropriate ligands such as BINAP in appropriate solvents such as PhMe under suitable temperatures such as 80° C. to 130° C., or an Ullman coupling reaction with different amides such as 4-hydroxy-4-methylpiperidin-2-one using appropriate copper catalysts such as Cu(OAc)2 in the presence of appropriate bases such as LiHMDS and appropriate ligands such as DMEDA (or in absence of ligand) in appropriate solvents such as DCM under suitable temperatures such as 80° C. to 130° C.
Steps (iv) and (v) are deprotection reactions with suitable acids such HCl in suitable solvents such as 1,4-dioxane under suitable temperatures such as 25° C. to 40.
General Scheme 4 provides an exemplary synthesis for preparing intermediates 2. Step (i) can be a reaction with different amines using appropriate bases such as TEA in appropriate solvents such as EtOH under suitable temperatures such as 25° C. to 100° C. to provide intermediate 2.
The following descriptions and examples illustrate the invention. These examples are not intended to limit the scope of the present invention, but rather to provide guidance to the skilled chemist to prepare and use the compounds, compositions and methods of the present invention. While particular embodiments of the present invention are described, the skilled chemist will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention.
The chemical names of compounds described in the present application follows the principle of IUPAC nomenclature.
Heating of reaction mixtures with microwave irradiations was carried out on a Smith Creator (purchased from Personal Chemistry, Forboro/MA, now owned by Biotage), an Emrys Optimizer (purchased from Personal Chemistry) or an Explorer (provided by CEM Discover, Matthews/NC) microwave.
Conventional techniques may be used herein for work up of reactions and purification of the products of the Examples.
References in the Examples below relating to the drying of organic layers or phases may refer to drying the solution over magnesium sulfate or sodium sulfate and filtering off the drying agent in accordance with conventional techniques. Products may generally be obtained by removing the solvent by evaporation under reduced pressure.
Purification of the compounds in the examples may be carried out by conventional methods such as chromatography and/or re-crystallization using suitable solvents. Chromatographic methods are known to the skilled person and include e.g. column chromatography, flash chromatography, HPLC (high performance liquid chromatography), and MDAP (mass directed auto-preparation, also referred to as mass directed LCMS purification). MDAP is described in e.g. W. Goetzinger et al, Int. J. Mass Spectrom., 2004, 238, 153-162.
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Both flash and gravity chromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel. Preparative HPLC were performed using a Gilson Preparative System using a Luna 5 u C18(2) 100A reverse phase column eluting with a 10-80 gradient (0.1% TFA in acetonitrile/0.1% aqueous TFA) or a 10-80 gradient (acetonitrile/water). The CombiFlash system used for purification in this application was purchased from Isco, Inc. CombiFlash purification was carried out using a pre-packed SiO2 column, a detector with UV wavelength at 254 nm and mixed solvents.
The terms “CombiFlash”, “Biotage®”, “Biotage 75” and “Biotage SP4®” when used herein refer to commercially available automated purification systems using pre-packed silica gel cartridges.
Final compounds were characterized with LCMS (conditions listed below) or NMR. 1H NMR or 19FNMR spectra were recorded using a Bruker Avance 400 MHz spectrometer. CDCl3 is deuteriochloroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and CD3OD is tetradeuteriomethanol. Chemical shifts are reported in parts per million (ppm) downfield from the internal standard tetramethylsilane (TMS) or the NMR solvent. Abbreviations for NMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad. J indicates the NMR coupling constant measured in Hertz.
All temperatures are reported in degrees Celsius. All other abbreviations are as described in the ACS Style Guide (American Chemical Society, Washington, D.C., 1986).
Absolute stereochemistry can be determined by methods known to one skilled in the art, for example X-ray or Vibrational circular dichroism (VCD).
When an enantiomer or a diasteroisomer is described and the absolute stereochemistry of a chiral center is not known, the use of “*” at the chiral centre denotes that the absolute stereochemistry of the chiral center is not known, i.e. the compound as drawn may be either a single R enantiomer or a single S enantiomer. Where the absolute stereochemistry at a chiral center of an enantiomer or a diasteroisomer is known, a bold wedge symbol () or a hashed wedge symbol (
) is used as appropriate, without the use of “*” at the chiral centre.
When a geometric or cis-trans isomer is described and the absolute configuration of the isomer is not known, the use of “*” at one of the atoms relevant to the geometric or cis-trans isomerism denotes that the absolute configuration at or around that atom is not known, i.e. the compound as drawn may be either a single cis isomer or a single trans enantiomer.
In the procedures that follow, after each starting material, reference to an intermediate is typically provided. This is provided merely for assistance to the skilled chemist. The starting material may not necessarily have been prepared from the batch referred to.
LCMS Conditions:
1) Acidic method:
a. Instruments: HPLC: Waters UPC2 and MS: Qda
Mobile phase: water containing 0.1% FA/0.1% MeCN
Column: ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm and 1.7 μm 2.1×100 mm
Detection: MS and photodiode array detector (PDA)
b. Instruments: HPLC: Shimadzu and MS: 2020
Mobile phase: water containing 0.1% FA/0.1% MeCN
Column: Sunfire C18 5 μm 50×4.6 mm and Sunfire C18 5 μm 150×4.6 mm
Detection: MS and photodiode array detector (PDA)
2) Basic conditions:
Instruments: HPLC: Agilent 1260 and MS: 6120
Mobile phase: 0.1% NH4OH in H2O/0.1% NH4OH in ACN
Column: Xbridge C18 5 μm 50×4.6 mm and Xbridge C18 5 μm 150×4.6 mm
Detection: MS and photodiode array detector (DAD)
Prep-HPLC Conditions
Instrument: Waters instrument
Column: Xbridge Prep C18 column OBD (10 μm, 19×250 mm), Xbrige prep C18 10 μm OBD™ 19×150 mm, Sunfire Prep C18 10×25 0 mm 5 μm, XBRIDGE Prep C18 10×150 mm 5 μm, etc
Acidic method:
Mobile phase: water containing 0.1% TFA/acetonitrile.
Basic method:
Mobile phase: water containing 0.1% NH4OH/acetonitrile.
Chiral Prep-HPLC:
Thar SFC Prep 80 (TharSFC ABPR1, TharSFC SFC Prep 80 CO2 Pump, TharSFC Co-Solvent Pump, TharSFC Cooling Heat Exchanger and Circulating Bath, TharSFC Mass Flow Meter, TharSFC Static Mixer, TharSFC Injection Module, Gilson UV Detector, TharSFC Fraction Collection Module
Chiral-HPLC Analysis:
Instrument: Thar SFC Prep 80 (TharSFC ABPR1, TharSFC SFC Prep 80 CO2Pump, TharSFC Co-Solvent Pump, TharSFC Cooling Heat Exchanger and Circulating Bath, TharSFC Mass Flow Meter, TharSFC Static Mixer, TharSFC Injection Module, Gilson UV Detector, TharSFC Fraction Collection Module
Column and mobile phase: are described in below examples.
To a solution of NaI (11.9 g, 79.7 mmol) in HI (55%, 50 mL) was added 4,6-dichloro-2-methylpyrimidine (10.0 g, 61.3 mmol) in portions. The resulting suspension was heated to 40° C. and stirred for 1 hour. The reaction mixture was cooled and filtered. The solid was washed with water and then triturated with methanol (50 mL). The mixture was filtered to give the title compound (9.0 g, yield 42%) as white solid.
1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 2.67 (s, 3H).
LCMS (mobile phase: 5-95% acetonitrile in 2.5 min): Rt=1.59 min, MS Calcd: 346; MS Found: 347 [M+H]+.
To a solution of NaI (5.5 g, 36.3 mmol) in HI (55% in water, 30 mL) was added 4,6-dichloro-2-methoxypyrimidine (5 g, 27.9 mmol). The mixture was heated to 40° C. and stirred for 14 h. The reaction mixture was cooled to room temperature and poured into ice water (50 mL). The filtered was washed with ice water three times to give product as a white solid (3.2 g, yield 32%).
LC-MS [mobile phase: from 80% water (0.1% TFA) and 20% CH3CN (0.1% TFA) to 20% water (0.1% TFA) and 80% CH3CN (0.1% TFA) in 10 min]: purity 100%, Rt=4.72 min; MS Calcd.: 362, MS Found: 363 [M+H]+.
To a solution of (R)-octahydropyrrolo[1,2-a]pyrazine (209 mg, 1.66 mmol) and DIPEA (643 mg, 4.97 mmol) in EtOH (20 mL) was added 4,6-diiodo-2-methoxypyrimidine (600 mg, 1.66 mmol). The reaction was stirred at room temperature for 36 h. Solvent was removed in vacuum and the residue was purified by silica gel chromatography (eluted with PE/EtOAc=1:1) to give product (377 mg, yield 63.1%) as a pale yellow solid.
1H NMR (400 MHz, CDCl3): δ 6.65 (s, 1H), 4.54˜4.17 (m, 2H), 3.90 (s, 3H), 3.14˜3.02 (m, 3H), 2.67 (t, J=11.2 Hz, 1H), 2.23˜2.13 (m, 2H), 1.98˜1.74 (m, 4H), 1.51˜1.41 (m, 1H).
To a solution of (S)-octahydropyrrolo[1,2-a]pyrazine (209 mg, 1.66 mmol) and DIPEA (643 mg, 4.97 mmol) in EtOH (20 mL) was added 4,6-diiodo-2-methoxypyrimidine (600 mg, 1.66 mmol). The reaction was stirred at room temperature for 36 h. Solvent was removed in vacuum and the residue was purified by silica gel chromatography (eluted with PE/EtOAc=1:1) to give product (385 mg, yield 65%) as a pale yellow solid.
1H NMR (400 MHz, CDCl3): δ 6.65 (s, 1H), 4.49˜4.17 (m, 2H), 3.90 (s, 3H), 3.14˜3.00 (m, 3H), 2.67 (t, J=10.0 Hz, 1H), 2.23˜2.13 (m, 2H), 1.98˜1.75 (m, 4H), 1.51˜1.41 (m, 1H).
To a solution of 4,6-diiodo-2-methylpyrimidine (300 mg, 0.87 mmol) and (R)-octahydropyrrolo[1,2-a]pyrazine (110 mg, 0.87 mmol) in THF/EtOH (5 mL/5 mL) was added DIEA (338 mg, 2.61 mmol). Then the reaction was stirred at room temperature for one day. Then the reaction was concentrated and purified by column (PE:EtOAc=7:1-4:1-1:1) to get a yellow solid (270 mg, yield: 90.5%).
LC-MS [mobile phase: from 60% water (0.1% TFA) and 40% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 2 min]: Rt=0.24 min; MS Calcd: 344, MS Found: 345 [M+H]+.
To a solution of 4,6-diiodo-2-methylpyrimidine (200 mg, 0.58 mmol) and (S)-octahydropyrrolo[1,2-a]pyrazine dihydrochloride (115 mg, 0.58 mmol) in THF/EtOH (5 mL/5 mL) was added DIPEA (225 mg, 1.74 mmol). Then the reaction was stirred at room temperature for two days. Then the reaction was concentrated and purified by column chromatography (PE:EtOAc=4:1-1:1) to get desired product as off-white solid (90 mg, yield: 45).
LC-MS [mobile phase: from 60% water (0.1% TFA) and 40% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 2 min]: Rt=0.26 min; MS Calcd: 344, MS Found: 345 [M+H]+.
Run 1: To a stirred solution of rel-(3aR,6aR)-hexahydro-2H-furo[3,2-c]pyrrole (98 mg, 0.866 mmol) in DMF (1.0 ml) were added 4,6-diiodo-2-methylpyrimidine (300 mg, 0.866 mmol) and DIPEA (0.70 ml, 4.24 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was combined with reaction mixture of next run for further workup procedures.
Run 2: To a stirred solution of rel-(3aR,6aR)-hexahydro-2H-furo[3,2-c]pyrrole (130 mg, 1.16 mmol) in DMF (1.5 ml) were added 4,6-diiodo-2-methylpyrimidine (400 mg, 1.16 mmol) and DIPEA (1.0 ml, 6.05 mmol). The reaction was stirred at room temperature overnight. The reaction solution was combined with reaction mixture of previous run, concentrated to dryness and the yellow residue oil was diluted with ice sat. aqueous Na2CO3 (20 ml). The resultant was extracted with EtOAc (3×25 ml) and the combined organic layers were washed by brine (20 ml), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography (wet-loaded with CH2Cl2) eluted with PE/EtOAc=3/1 afforded pure desired product as a yellow thick oil (514 mg, yield over 2 batches: 76%).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 2.6 min]: Rt=1.08 min; MS Calcd: 331, MS Found: 332 [M+H]+.
1H NMR (400 MHz, CDCl3) δ6.59 (s, 1H), 4.60 (s, 1H), 3.99 (q, J=7.6 Hz, 1H), 3.92-3.86 (m, 1H), 3.72 (br s, 2H), 3.52 (br s, 1H), 3.29 (br s, 1H), 3.00 (t, J=6.0 Hz, 1H), 2.46 (s, 3H), 2.23-2.14 (m, 1H), 1.92-1.88 (m, 1H).
A solution of 2-methylpyrazine (6.58 g, 70.0 mmol) in THF (20 mL) was added dropwise to a solution of LDA (2.0 M in THF, 45.5 mL, 91.0 mmol) in THF (60 mL) at −78° C. under nitrogen atmosphere. After the mixture was stirred at −78° C. for 30 min, tert-butyl 2-bromoacetate (13.7 g, 70.0 mmol) was added and the resulting mixture was stirred at −78° C. for 2 hrs. Then the mixture was poured into water (100 mL) and extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated in reduced pressure. The residue was purified by column chromatography (petroleum ether:EtOAc=6:1) to give the title compound (5.4 g, yield 37%) as a pale yellow oil.
1H NMR (300 MHz, CDCl3): δ 8.49-8.47 (m, 2H), 8.40 (s, 1H), 3.09 (t, J=7.2 Hz, 2H), 2.72 (t, J=6.9 Hz, 2H), 1.40 (s, 9H).
A suspension of tert-butyl 3-(pyrazin-2-yl)propanoate (5.49 g, 26.4 mmol) and Pd/C (10% wet, 1.5 g) in methanol (150 mL) was hydrogenated over 55 psi at 50° C. for 18 hrs. The reaction mixture was filtered and the filtrate was concentrated in reduced pressure to give the title compound (4.7 g, 83%) as pale yellow oil which was used for the next step without further purification.
1H NMR (300 MHz, CDCl3): δ 2.97-2.59 (m, 6H), 2.39-2.24 (m, 3H), 1.60-1.53 (m, 2H), 1.43 (s, 9H).
A mixture of tert-butyl 3-(piperazin-2-yl)propanoate (0.57 g, 2.66 mmol) and 4,6-diiodo-2-methoxypyrimidine (962 mg, 2.66 mmol) and triethylamine (0.40 g, 4.0 mmol) in methanol (10 mL) was stirred at 70° C. for 1 h. The reaction mixture was directly concentrated in reduced pressure. The residue was partitioned with water (80 mL) and EtOAc (60 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography (EtOAc) to give the title compound (0.84 g, yield 70%) as pale yellow oil.
1H NMR (300 MHz, CDCl3): δ 6.62 (s, 1H), 4.13-4.08 (m, 2H), 3.90 (s, 3H), 3.09-2.60 (m, 5H), 2.36-2.32 (m, 2H), 1.77-1.66 (m, 2H), 1.45 (s, 9H).
To a solution of tert-butyl 3-(4-(6-iodo-2-methoxypyrimidin-4-yl)piperazin-2-yl)propanoate (840 mg, 1.88 mmol) and triethylamine (380 mg, 3.75 mmol) in THF (10 mL) was added Boc2O (490 mg, 2.25 mmol). The reaction mixture was stirred at room temperature for 5 hrs. The mixture was poured into water (70 mL) and extracted with EtOAc (50 mL×2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4 and concentrated in reduced pressure. The residue was purified by column chromatography (petroleum ether:EtOAc=6:1) to give the title compound (0.92 g, yield 90%) as a pale yellow oil.
1H NMR (300 MHz, CDCl3): δ 6.61 (s, 1H), 4.25-3.98 (m, 4H), 3.90 (s, 3H), 3.19-2.99 (m, 3H), 2.29-2.16 (m, 2H), 1.90-1.87 (m, 1H), 1.73-1.68 (m, 1H), 1.48 (s, 9H), 1.43 (s, 9H).
To a solution of 5-bromo-2,4-dimethylaniline (15.0 g, 75.0 mmol) in chloroform (150 mL) was added Ac2O (15.0, 150 mmol) under ice bath. KOAc (8.00 g, 82.5 mmol), 18-crown-6 (10.0 g, 37.5 mmol) and isoamyl nitrite (26.3 g, 225 mmol) were added. The mixture was refluxed for 36 hrs. The reaction mixture was concentrated and the residue was dissolved in EtOAc (500 mL). The organic solution was washed with water (100 mL), dried over Na2SO4 and concentrated. The residue was dissolved in THF (100 mL) and NaOH (4 M, 40.0 mL, 160 mmol) was added. The mixture was stirred at rt for 1 h. The solvent was removed under vacuum and the residue was partitioned between EtOAc (400 mL) and water (200 mL). The organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc from 10:1 to 5:1) to give the title compound (5.1 g, yield 32%) as an orange solid.
1H NMR (300 MHz, CDCl3): δ 10.20 (br, 1H), 7.99 (s, 1H), 7.75 (s, 1H), 7.61 (s, 1H), 2.50 (s, 3H).
To a solution of 6-bromo-5-methyl-1H-indazole (5.10 g, 24.2 mmol) in dry DCM (120 mL) was added DHP (4.10 g, 48.4 mmol), TsOH (0.800 g, 4.80 mmol) and Mg2SO4 (5.0 g) at rt. The reaction mixture was heated to 35° C. and stirred for an hour. The reaction mixture was filtered and the filtrate was washed with Na2CO3 (10%, 100 mL), dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc from 50:1 to 20:1) to give the title compound (6.0 g, yield 84%) as an orange solid.
1H NMR (300 MHz, CDCl3): δ 7.90 (s, 1H), 7.84 (s, 1H), 7.55 (s, 1H), 5.63 (dd, J=9.6, 3.0 Hz, 1H), 4.05-4.00 (m, 1H), 3.78-3.70 (m, 1H), 2.58-2.44 (m, 4H), 2.20-2.02 (m, 2H), 1.78-1.65 (m, 3H).
LCMS (mobile phase: 5-95% CH3CN): Rt=2.19 min in 3 min; MS Calcd: 294; MS Found: 295 [M+H]+.
To a suspension of 6-bromo-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (5.50 g, 18.6 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (6.90 g, 22.3 mmol) and Na2CO3 (4.90 g, 46.5 mmol) in dioxane (150 mL) and water (130 mL) was added Pd(dppf)Cl2 (658 mg, 0.900 mmol). The mixture was degassed with N2 for 3 times and then stirred at 80° C. overnight. The solvent was removed under vacuum and the residue was partitioned between EtOAc (300 mL) and water (200 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc=10:1) to give the title compound (7.3 g, yield 99%) as a slight brown solid.
1H NMR (400 MHz, CDCl3): b 7.92 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 5.67 (dd, J=9.6, 2.8 Hz, 1H), 5.63 (br s, 1H), 4.07-4.01 (m, 3H), 3.78-3.70 (m, 1H), 3.67-3.64 (m, 2H), 2.62-2.53 (m, 1H), 2.45-2.39 (m, 2H), 2.34 (s, 3H), 2.18-2.12 (m, 1H), 2.07-2.02 (m, 1H), 1.81-1.73 (m, 2H), 1.69-1.61 (m, 1H), 1.52 (s, 9H).
To a solution of tert-butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-5,6-dihydropyridine-1(2H)-carboxylate (33.0 g, 83.0 mmol) in dry THF (300 mL) was added BH3-THF (1 M, 332 mL, 332 mmol) at 10° C. The mixture was gradually warmed to rt and stirred overnight. The reaction mixture was cooled to 0° C. and NaOH (aq, 2 M, 125 mL, 249 mmol) was added carefully. H2O2(30%, 87 mL, 830 mmol) was followed. The temperature was kept below 10° C. during the addition of NaOH and H2O2. The mixture was stirred for an hour at rt. Na2SO3 (10%, 100 mL) was added to the reaction mixture and stirred for 20 min. The organic layer was separated and the aqueous was extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated. The crude was purified by column chromatography (PE:EtOAc from 3:1 to 1:1) to give tert-butyl 3-hydroxy-4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate as major product (D A-15) (23 g, yield 67%) as a white solid and tert-butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (D A-16) as minor product (6.7 g, yield 20%) as a slight brown solid.
1H NMR (300 MHz, CDCl3): δ 7.90 (s, 1H), 7.48 (s, 1H), 7.34 (s, 1H), 5.68 (dd, J=9.6 Hz, 2.7 Hz, 1H), 4.33-4.28 (m, 2H), 4.06-4.02 (m, 1H), 3.80-3.72 (m, 1H), 3.00-2.82 (m, 3H), 2.65-2.51 (m, 1H), 2.44 (s, 3H), 2.22-2.11 (m, 1H), 2.08-2.00 (m, 1H), 1.88-1.80 (m, 2H), 1.77-1.63 (m, 5H), 1.51 (s, 9H).
tert-Butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (1.0 g, 2.5 mmol) was dissolved in HCl/MeOH (5 mol/L, 10 mL). Then the mixture was stirred for 6 hrs. The mixture was concentrated under reduced pressure to afford the title compound (820 mg, yield >100%) as a light yellow solid used for next step without purification.
LC-MS: 5-95% CH3CN, Rt=1.13 min, MS Calcd.: 215, MS Found: 216 [M+H]+.
To a solution of 5-methyl-6-(piperidin-4-yl)-1H-indazole hydrochloride (600 mg, 2.39 mmol) in CH3OH (10 mL) and H2O (2 mL) was added KOH (268 mg, 4.78 mmol) and (Boc)2O (781 mg, 3.58 mmol) under ice bath. The reaction mixture was stirred at rt for 2 hrs. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by column chromatograph (PE:EtOAc from 10:1 to 4:1) to give the title compound (353 mg, yield 47%) as a yellow oil.
1H NMR (300 MHz, CDCl3): δ 10.15 (br s, 1H), 7.95 (s, 1H), 7.53 (s, 1H), 7.29 (s, 1H), 4.34 (br s, 2H), 2.95-2.81 (m, 3H), 2.45 (s, 3H), 1.86-1.81 (m, 2H), 1.69-1.61 (m, 2H), 1.51 (s, 9H).
To a solution of (trans)-tert-Butyl 3-hydroxy-4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (24.5 g, 59.0 mmol) in dry DCM (200 mL) was added DAST (38.0 g, 236 mmol) under N2 at −65° C. The mixture was gradually warmed to rt and stirred for 2 hrs. The reaction mixture was carefully poured into Na2CO3 aqueous solution (10%, 300 mL) and stirred for 20 min. The organic layer was separated and the aqueous was extracted with DCM (250 mL×2). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated. The crude was purified by column chromatography (PE:EtOAc=10:1) to give the title compound (11.8 g, yield 48%) as a white solid.
1H NMR (400 MHz, CDCl3): δ7.92 (s, 1H), 7.52 (s, 1H), 7.41 (s, 1H), 5.74-5.67 (m, 1H), 4.80-4.59 (m, 2H), 4.21 (br, 1H), 4.07-3.99 (m, 1H), 3.80-3.71 (m, 1H), 3.25-3.19 (m, 1H), 2.89-2.79 (m, 2H), 2.65-2.51 (m, 1H), 2.45 (s, 3H), 2.19-2.15 (m, 1H), 2.15-2.04 (m, 1H), 1.93-1.88 (m, 1H), 1.80-1.74 (m, 5H), 1.52 (s, 9H).
LCMS: 5-95% CH3CN, Rt=2.25 min in 3 min; MS Calcd: 417; MS Found: 418 [M+H]+.
A mixture of (cis)-tert-butyl 3-fluoro-4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl) piperidine-1-carboxylate (2.50 g, 6.00 mmol) in HCl/dioxane (6 mol/L, 40 mL) was stirred at rt for 6 hrs. The reaction mixture was cooled to 0° C. and filtered. The solid was washed with cold 1,4-dioxane (5 mL) to get the title compound (1.4 g, yield 100%) as a white solid which was used for next step directly.
LC-MS: 5-95% CH3CN, Rt=1.73 min; MS Calcd.: 233, MS Found: 234 [M+H]+.
To a solution of (cis)-6-(3-fluoropiperidin-4-yl)-5-methyl-1H-indazole hydrochloride (1.40 g, 6.00 mmol) and oxetan-3-one (2.16 g, 30.0 mmol) in methanol (5 mL) and 1,2-dichloroethane (50 mL) was added NaBH3CN (1.13 g, 18.0 mmol). Then the mixture was stirred at rt for 3 hrs. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (100 mL×3). The combined organic layers were dried over MgSO4, filtered and concentrated. The crude was purified by column chromatography (DCM:MeOH=30:1) to give the title compound (1.0 g, yield 57.6%) as a white solid.
LC-MS: 5%-95% CH3CN, Rt=1.85 min; MS Calcd.: 289, MS Found: 290 [M+H]+.
To a mixture of 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (939 mg, 4.47 mmol), 6-bromo-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (1200 mg, 4.07 mmol), PdCl2(dppf)-CH2Cl2adduct (332 mg, 0.407 mmol) and tripotassium phosphate (2589 mg, 12.20 mmol) was added DMF (10 mL) and water (2.500 mL). The reaction mixture was heated to 100° C. for 3 h. The reaction mixture was diluted with ethyl acetate and filtered. the filtrate was concentrated and purified by silica column (30% EA in PE) to give 6-(3,6-dihydro-2H-pyran-4-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (610 mg, 2.044 mmol, 50.3% yield).
MS: 299.0 [M+H]+.
A mixture of 6-(3,6-dihydro-2H-pyran-4-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (610 mg, 2.044 mmol), Pd—C (435 mg, 0.409 mmol, 10%) and methanol (10 mL) was stirred under hydrogen balloon atmosphere at rt for 16 h, then filtered. The filtrate was concentrated to afford a crude 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-6-(tetrahydro-2H-pyran-4-yl)-1H-indazole (614 mg, 2.044 mmol, 100% yield).
MS: 301.1 [M+H]+.
A solution of 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-6-(tetrahydro-2H-pyran-4-yl)-1H-indazole (610 mg, 2.031 mmol), HCl (4.06 mL, 20.31 mmol) and methanol (5 mL) was stirred at rt for 16 h. The reaction solution was neutralized by aq. NaHCO3 to pH=7 and extracted with ethyl acetate. The combined organic phases were dried and concentrated to give 5-methyl-6-(tetrahydro-2H-pyran-4-yl)-1H-indazole (430 mg, 1.988 mmol, 98% yield).
MS: 217.1 [M+H]+.
A mixture of 5-methyl-6-(tetrahydro-2H-pyran-4-yl)-1H-indazole (0.22 g, 1.0 mmol) and tert-butyl 2-(3-(tert-butoxy)-3-oxopropyl)-4-(6-iodo-2-methoxypyrimidin-4-yl)piperazine-1-carboxylate (0.55 g, 1.0 mmol), CuI (0.15 g. 0.80 mmol), N,N′-dimethyl-cyclohexane-1,2-diamine (0.23 g, 1.6 mmol) and potassium phosphate (0.34 g, 1.6 mmol) in toluene (8 mL) was stirred at 110° C. nitrogen atmosphere for 18 hrs. After cooled down to rt the mixture was poured into diluted ammonia solution (10%, 50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography (petroleum ether:EtOAc=4:1) to give the mixture of the crude product, which was further purified by prep-HPLC (ACN/H2O, 20-95%) to give the title compound (0.22 g, yield 35%) as a white solid.
1H NMR (300 MHz, CDCl3): δ 8.76 (s, 1H), 8.06 (d, J=3.0 Hz, 1H), 7.52 (s, 1H), 6.84 (s, 1H), 4.38-3.99 (m, 9H), 3.65-3.57 (m, 2H), 3.27-3.06 (m, 4H), 2.48 (s, 3H), 2.30-2.19 (m, 2H), 1.99-1.79 (m, 6H), 1.49 (s, 9H), 1.41 (s, 9H).
tert-Butyl 2-(3-(tert-butoxy)-3-oxopropyl)-4-(2-methoxy-6-(5-methyl-6-(tetrahydro-2H-pyran-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)piperazine-1-carboxylate (0.22 g, 0.35 mmol) was purified by chiral prep. column (IC, Hex; EtOH=60:40, 12 mL/min, 254 nm) to give Peak 1 (D A-26, 85 mg, yield 39%) as white solid and Peak 2 (D A-27, 80 mg, yield 36%) as white solid.
Peak 1: 1H NMR (300 MHz, CDCl3): δ 8.76 (s, 1H), 8.06 (s, 1H), 7.52 (s, 1H), 6.84 (s, 1H), 4.38-4.11 (m, 8H), 4.02-4.00 (m, 1H), 3.65-3.57 (m, 2H), 3.27-3.04 (m, 4H), 2.48 (s, 3H), 2.30-2.19 (m, 2H), 1.97-1.75 (m, 6H), 1.49 (s, 9H), 1.41 (s, 9H). Chiral HPLC: Chiral pak IC 5 μm 4.6×250 mm, Hex:EtOH=60:40, Flow: 1.0 ml/min, 230 nm, T=30° C. Rt=10.189 min, 100% ee.
Peak 2: 1H NMR (300 MHz, CDCl3): δ 8.76 (s, 1H), 8.07 (s, 1H), 7.52 (s, 1H), 6.83 (s, 1H), 4.38-4.14 (m, 8H), 4.03-4.00 (m, 1H), 3.64-3.57 (m, 2H), 3.27-3.04 (m, 4H), 2.48 (s, 3H), 2.30-2.19 (m, 2H), 1.99-1.75 (m, 6H), 1.49 (s, 9H), 1.41 (s, 9H). Chiral condition: Chiral pak IC 5 μm 4.6×250 mm, Hex:EtOH=60:40, Flow: 1.0 ml/min, 230 nm, T=30° C. Rt=12.626 min, 100% ee.
To a solution of 5-methyl-6-(piperidin-4-yl)-1H-indazole hydrochloride (820 mg of crude, 2.50 mmol) in DCE (15 mL) was added oxetan-3-one (1.80 g, 25.0 mmol). The mixture was stirred at room temperature for 40 min. Then the mixture was cooled under ice bath and NaBH3CN (473 mg, 7.50 mmol) was added to the mixture. The mixture was warmed to room temperature and stirred for 2 hrs. Then the reaction mixture was poured into Na2CO3 aqueous solution (10%, 100 mL) and stirred for 15 min. Then the mixture was extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by column chromatography (DCM:MeOH=40:1) to afford the title compound (473 mg, yield 70%) as a white solid 1H NMR (300 MHz, CDCl3): δ 10.11 (br, 1H), 7.96 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 4.72-4.69 (m, 4H), 3.61-3.52 (m, 1H), 2.96-2.93 (m, 2H), 2.88-2.78 (m, 1H), 2.44 (s, 3H), 2.06-1.98 (m, 2H), 1.90-1.82 (m, 4H).
LC-MS: [mobile phase: 5-95% Acetonitrile in 2.5 min]: Rt=1.37 min; MS Calcd: 271; MS Found: 272 [M+H]+.
To a solution of ethyl 2-(allyl(benzyl)amino)acetate (2.00 g, 8.58 mmol) in THF (160 mL) was added TiCl(OiPr)3 (1.0 mL, 3.42 mmol) in one portion at rt. Then isopropylmagnesium chloride (2 M in THF, 21.6 mL, 43.0 mmol) was added dropwise via a syringe pump over 3 hrs. After the addition, the mixture was stirred at rt overnight. The reaction was quenched with water (20 mL). The mixture was diluted with ethyl acetate (300 mL) and water (100 mL). The organic solution was washed brine (100 mL×2), dried over Na2SO4 and concentrated. The residue was purified by reverse phase chromatography (ACN/water: 30-100%) to give the crude (710 mg with 70% purity by NMR). The crude was further purified by normal phase chromatography (CH2Cl2:methanol=80:1) to give the desired compound (320 mg, yield 20%) as a slight yellow oil.
1H NMR (300 MHz, CDCl3): δ 7.30-7.22 (m, 5H), 3.65 (s, 2H), 3.16-3.13 (m, 1H), 2.80-2.77 (m, 1H), 2.67-2.61 (m, 2H), 1.47-1.41 (m, 1H), 1.19-1.16 (m, 1H), 0.95-0.91 (m, 1H).
To a solution of 3-benzyl-3-azabicyclo[3.1.0]hexan-1-ol (320 mg, 1.69 mmol) in methanol (20 mL) was added Pd(OH)2 (50 mg) at rt. The mixture was stirred under H2 (balloon) for 3 hrs. The mixture was filtered and the filtrate was concentrated under vacuum to give the desired compound (150 mg, yield 90%) with 90% purity by NMR as a colorless oil.
1H NMR (300 MHz, CDCl3): δ 3.15-3.08 (m, 2H), 3.01-2.97 (m, 1H), 2.74-2.70 (m, 1H), 1.43-1.38 (m, 1H), 1.09-1.04 (m, 1H), 0.67-0.64 (m, 1H).
To a mixture of tert-butyl 4-(5-methyl-1H-indazol-6-yl)piperidine-1-carboxylate (780 mg, 2.48 mmol) in toluene (20 mL) was added 4,6-diiodo-2-methylpyrimidine (1.10 g, 3.22 mmol), K3PO4 (2.60 g, 12.40 mmol), CuI (476 mg, 2.50 mmol) and N,N′-dimethyl-cyclohexane-1,2-diamine (298 mg, 2.09 mmol). The reaction mixture was heated to 100° C. and stirred for 3 hrs. The reaction mixture was cooled to room temperature and poured into water (100 mL). The desired was extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4 and concentrated. The residue was purified by column (PE:EtOAc=12:1) to give the title compound (800 mg, yield 62%) as a white solid.
1H NMR (300 MHz, CDCl3): δ 8.65 (s, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 7.53 (s, 1H), 4.39-4.24 (m, 2H), 2.99-2.81 (m, 3H), 2.77 (s, 3H), 2.49 (s, 3H), 1.90-1.1.79 (m, 2H), 1.77-1.63 (m, 2H), 1.51 (s, 9H).
To a mixture of tert-butyl 4-(1-(6-iodo-2-methylpyrimidin-4-yl)-5-methyl-1H-indazol-6-yl)piperidine-1-carboxylate (400 mg, 0.750 mmol), 3-azabicyclo[3.1.0]hexan-1-ol (150 mg crude, 1.21 mmol), Pd2(dba)3 (69 mg, 0.075 mmol) and Cs2CO3 (975 mg, 3.00 mmol) in THF (20 mL) was added Xphos (38 mg, 0.079 mmol) under N2 atmosphere. The reaction mixture was refluxed for 8 hrs. The reaction mixture was concentrated. The residue was partitioned with EtOAc (100 mL) and water (50 mL). The organic layer was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by column (PE:EtOAc=5:1) to give crude product (75 mg) as a brown oil. The crude was further purified by prep. TLC (PE:EtOAc=2:1) to give the title compound (40 mg, yield 11%) as a slight yellow solid. 1H NMR (300 MHz, CDCl3): δ 8.74 (s, 1H), 8.04 (s, 1H), 7.49 (s, 1H), 6.66 (s, 1H), 4.37-4.25 (m, 2H), 3.68-3.63 (m, 2H), 2.97-2.84 (m, 3H), 2.60 (s, 3H), 2.46 (s, 3H), 1.90-1.76 (m, 6H), 1.51 (s, 9H), 1.30-1.26 (m, 2H), 0.71-0.67 (m, 1H).
A mixture of 4,6-diiodo-2-methoxypyrimidine (200 mg, 0.55 mmol), octahydropyrazino[2,1-c][1,4]oxazine (119 mg, 0.55 mmol) and DIEA (357 mg, 2.76 mmol) in THF (20 mL) and i-PrOH (20 mL) was stirred at 50° C. overnight. The reaction solution was concentrated and the residue was diluted with EtOAc (30 mL). The resulting mixture was washed with brine (30 mL), dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography column (CH2Cl2:MeOH=20:1) to give product as a yellow solid (160 mg, 77% yield)
LC-MS [mobile phase: from 60% water (0.1% FA) and 40% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 2.0 min]: Rt=0.49 min; MS Calcd.: 376, MS Found: 377 [M+H]+.
To a solution of NaI (11.9 g, 79.7 mmol) in HI (55%, 50 mL) was added 4,6-dichloro-2-methylpyrimidine (10.0 g, 61.3 mmol) in portions. The resulting suspension was heated to 40° C. and stirred for 1 hour. The reaction mixture was cooled and filtered. The solid was washed with water and then triturated with methanol (50 mL). The mixture was filtered to give the title compound (9.0 g, yield 42%) as white solid.
1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 2.67 (s, 3H).
LCMS (mobile phase: 5-95% acetonitrile in 2.5 min): Rt=1.59 min, MS Calcd: 346; MS Found: 347 [M+H]+.
To a solution of NaI (5.5 g, 36.3 mmol) in HI (55% in water, 30 mL) was added 4,6-dichloro-2-methoxypyrimidine (5 g, 27.9 mmol). The mixture was heated to 40° C. and stirred for 14 h. The reaction mixture was cooled to room temperature and poured into ice water (50 mL). The filtered was washed with ice water three times to give product as a white solid (3.2 g, yield 32%).
LC-MS [mobile phase: from 80% water (0.1% TFA) and 20% ACN (0.1% TFA) to 20% water (0.1% TFA) and 80% ACN (0.1% TFA) in 10 min]: Rt=4.72 min; MS Calcd.: 362, MS Found: 363 [M+H]+.
To a solution of 4,6-diiodo-2-methylpyrimidine (300 mg, 0.87 mmol) and 2,5-dioxa-8-azaspiro[3.5]nonane hemioxalate (303 mg, 0.87 mmol) in THF/EtOH (5 mL/5 mL) was added DIPEA (338 mg, 2.61 mmol). The reaction was stirred at room temperature for 48 hours. The reaction was heated to 40° C. and stirred another 4 hours. The reaction mixture was concentrated and purified by column chromatography (PE:EtOAc=7:1-4:1) to get desired product as yellow solid (230 mg, yield: 76%).
LC-MS [mobile phase: from 60% water (0.1% TFA) and 40% ACN (0.1% TFA) to 5% water (0.1% TFA) and 95% ACN (0.1% TFA) in 2 min]: Rt=0.33 min; MS Calcd: 347, MS Found: 348 [M+H]+.
DIPEA (496 mL, 3.0 mmol) was added to a solution of 4,6-diiodo-2-methylpyrimidine (488 mg, 1.4 mmol) and 3-oxa-1,8-diazaspiro[4.5]decan-2-one (200 mg, 1.4 mmol) in EtOH/THF (7 mL/7 mL). The reaction was stirred at rt. for 2 days. The solvent was removed and the residue was purified by column chromatography (PE:EtOAc=6:1) to gave desired product as a white solid (300 mg, yield: 57%).
LC-MS [mobile phase: from 60% water (0.1% FA) and 40% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.0 min]: Rt=0.25 min; MS Calcd: 374, MS Found: 375 [M+H]+.
A suspension of 4,6-diiodo-2-methylpyrimidine (0.5 g, 1.45 mmol) and 1,8-diazaspiro[4.5]decan-2-one hydrochloride (275 mg, 1.45 mmol) in EtOH/THF (8 mL/8 mL) was added DIPEA (561 mg, 4.35 mmol). The mixture was stirred at rt. for 2 days. The reaction mixture was concentrated. The residue was purified by column chromatography on silica gel (DCM:MeOH=50:1) to give title compound as a yellow solid (380 mg, yield: 70%).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2 min]: Rt=1.01 min; MS Calcd: 372 MS Found: 373 [M+H]+.
To a stirred solution of 4,6-diiodo-2-methylpyrimidine (300 mg, 0.867 mmol) and 4-oxa-7-azaspiro[2.5]octane hydrochloride (130 mg, 0.869 mmol) in THF/EtOH (5 ml/5 ml) was added DIPEA (450 mg, 3.48 mmol). The reaction was stirred at room temperature overnight. The mixture was concentrated and the residue was purified by column chromatography eluted with PE/EtOAc=7/1 to afford the desired product as pink solid (210 mg, yield: 73%). LC-MS [mobile phase: from 60% water (0.1% TFA) and 40% ACN (0.1% TFA) to 5% water (0.1% TFA) and 95% ACN (0.1% TFA) in 2.0 min]: Rt=0.61 min; MS Calcd: 331, MS Found: 332 [M+H]+.
A mixture of 1 4,6-diiodo-2-methylpyrimidine (346 mg, 1.0 mmol) and 2,8-diazaspiro[4.5]decan-1-one (272 mg, 1.2 mmol) and TEA (303 mg, 3.0 mmol) in DMSO (6 mL) was stirred at 60° C. for 4 hours. The mixture was diluted with H2O (30 mL) and extracted with EtOAc (30 mL×3). The extracts were combined and dried over Na2SO4. The organic phase was filtered and concentrated. The residue was purified by silica gel chromatography column (petroleum ether/EtOAc=10:1) to give the title compound (319 mg, 86%) as a yellow solid.
1HNMR (300 MHz, CDCl3): δ 6.80 (s, 1H), 6.09 (br, 1H), 4.16 (d, J=10.5 Hz, 2H), 3.40-3.29 (m, 4H), 2.44 (s, 3H), 2.12-2.07 (m, 2H), 1.96-1.88 (m, 4H).
A mixture of benzyl 4-oxopiperidine-1-carboxylate (2.33 g, 10 mmol), 2-hydroxyacetamide (825 mg, 11 mmol) and TsOH—H2O (95 mg, 0.5 mmol) in benzene (25 ml) was stirred at 120° C. under Ar for 72 hours with Dean-Stark apparatus. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC (MeCN/H2O=0:100-100:0) to give desired product as white solid (725 mg, yield: 25%).
LC-MS [mobile phase: from 80% water (0.1% FA) and 20% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.0 min]: Rt=1.11 min; MS Calcd: 290, MS Found: 291 [M+H]+.
A mixture of benzyl 3-oxo-1-oxa-4,8-diazaspiro[4.5]decane-8-carboxylate (725 mg, 2.5 mmol) and Pd(OH)2 (100 mg) in MeOH (50 mL) was stirred at room temperature under H2 for 18 hours. The reaction mixture was filtered and the filtrate was concentrated to give crude desired product as oil (351 mg, yield: 90%).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.0 min]: Rt=0.23 min; MS Calcd: 156, MS Found: 157 [M+H]+.
To a solution of 1-oxa-4,8-diazaspiro[4.5]decan-3-one (351 mg, 2.25 mmol) and 4,6-diiodo-2-methylpyrimidine (778 mg, 2.25 mmol) in THF/EtOH (40 ml/40 ml) was added DIPEA (887 mg, 6.88 mmol). The reaction was stirred at room temperature for 18 hours. The reaction mixture was concentrated and the residue was purified by column chromatography (eluent: PE/EtOAc=6/1 to 1/1, followed by CH2Cl2/MeOH=40/1) to get desired product as a solid (680 mg, yield: 81%).
LC-MS [mobile phase: from 70% water (0.1% FA) and 30% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.0 min]: Rt=0.36 min; MS Calcd: 374, MS Found: 375 [M+H]+.
To a solution of 5-bromo-2,4-dimethylaniline (15.0 g, 75.0 mmol) in chloroform (150 mL) was added Ac2O (15.0, 150 mmol) under ice bath. KOAc (8.00 g, 82.5 mmol), 18-crown-6 (10.0 g, 37.5 mmol) and isoamyl nitrite (26.3 g, 225 mmol) were added. The mixture was refluxed for 36 hrs. The reaction mixture was concentrated and the residue was dissolved in EtOAc (500 mL). The organic solution was washed with water (100 mL), dried over Na2SO4 and concentrated. The residue was dissolved in THF (100 mL) and NaOH (4 M, 40.0 mL, 160 mmol) was added. The mixture was stirred at rt for 1 h. The solvent was removed under vacuum and the residue was partitioned between EtOAc (400 mL) and water (200 mL). The organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc from 10:1 to 5:1) to give the title compound (5.1 g, yield 32%) as an orange solid.
1H NMR (300 MHz, CDCl3): δ 10.20 (br, 1H), 7.99 (s, 1H), 7.75 (s, 1H), 7.61 (s, 1H), 2.50 (s, 3H).
To a solution of 6-bromo-5-methyl-1H-indazole (5.10 g, 24.2 mmol) in dry DCM (120 mL) was added DHP (4.10 g, 48.4 mmol), TsOH (0.800 g, 4.80 mmol) and Mg2SO4(5.0 g) at rt. The reaction mixture was heated to 35° C. and stirred for an hour. The reaction mixture was filtered and the filtrate was washed with Na2CO3 (10%, 100 mL), dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc from 50:1 to 20:1) to give the title compound (6.0 g, yield 84%) as an orange solid.
1H NMR (300 MHz, CDCl3): δ 7.90 (s, 1H), 7.84 (s, 1H), 7.55 (s, 1H), 5.63 (dd, J=9.6, 3.0 Hz, 1H), 4.05-4.00 (m, 1H), 3.78-3.70 (m, 1H), 2.58-2.44 (m, 4H), 2.20-2.02 (m, 2H), 1.78-1.65 (m, 3H).
LCMS: (mobile phase: 5-95% ACN), Rt=2.19 min in 3 min; MS Calcd: 294; MS Found: 295 [M+H]+.
To a suspension of 6-bromo-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (5.50 g, 18.6 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1 (2H)-carboxylate (6.90 g, 22.3 mmol) and Na2CO3 (4.90 g, 46.5 mmol) in dioxane (150 mL) and water (130 mL) was added Pd(dppf)Cl2 (658 mg, 0.900 mmol). The mixture was degassed with N2 for 3 times and then stirred at 80° C. overnight. The solvent was removed under vacuum and the residue was partitioned between EtOAc (300 mL) and water (200 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude was purified by column chromatography (PE:EtOAc=10:1) to give the title compound (7.3 g, yield 99%) as a slight brown solid.
1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 5.67 (dd, J=9.6, 2.8 Hz, 1H), 5.63 (br, 1H), 4.07-4.01 (m, 3H), 3.78-3.70 (m, 1H), 3.67-3.64 (m, 2H), 2.62-2.53 (m, 1H), 2.45-2.39 (m, 2H), 2.34 (s, 3H), 2.18-2.12 (m, 1H), 2.07-2.02 (m, 1H), 1.81-1.73 (m, 2H), 1.69-1.61 (m, 1H), 1.52 (s, 9H).
To a solution of tert-butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-5,6-dihydropyridine-1(2H)-carboxylate (33.0 g, 83.0 mmol) in dry THF (300 mL) was added BH3-THF (1 M, 332 mL, 332 mmol) at 10° C. The mixture was gradually warmed to rt and stirred overnight. The reaction mixture was cooled to 0° C. and NaOH (aq, 2 M, 125 mL, 249 mmol) was added carefully. H2O2(30%, 87 mL, 830 mmol) was followed. The temperature was kept below 10° C. during the addition of NaOH and H2O2. The mixture was stirred for an hour at rt. Na2SO3 (10%, 100 mL) was added to the reaction mixture and stirred for 20 min. The organic layer was separated and the aqueous was extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated. The crude was purified by column chromatography (PE:EtOAc from 3:1 to 1:1) to give trans tert-butyl 3-hydroxy-4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate as major product (D B-14) (23 g, yield 67%) as a white solid and tert-butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (D B-15) as minor product (6.7 g, yield 20%) as a slight brown solid.
D B-14: 1H NMR (300 MHz, CDCl3): δ 7.90 (s, 1H), 7.48 (s, 1H), 7.34 (s, 1H), 5.68 (dd, J=9.6 Hz, 2.7 Hz, 1H), 4.33-4.28 (m, 2H), 4.06-4.02 (m, 1H), 3.80-3.72 (m, 1H), 3.00-2.82 (m, 3H), 2.65-2.51 (m, 1H), 2.44 (s, 3H), 2.22-2.11 (m, 1H), 2.08-2.00 (m, 1H), 1.88-1.80 (m, 2H), 1.77-1.63 (m, 5H), 1.51 (s, 9H).
tert-Butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (1.0 g, 2.5 mmol) was dissolved in HCl/MeOH (5 mol/L, 10 mL). Then the mixture was stirred for 6 hrs. The mixture was concentrated under reduced pressure to afford the title compound (820 mg, yield >100%) as a light yellow solid used for next step without purification.
LC-MS: 5-95% ACN, Rt=1.13 min, MS Calcd.: 215, MS Found: 216 [M+H]+.
HCl/MeOH (5M, 200 mL) was added to a solution of tert-butyl 4-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)piperidine-1-carboxylate (55.4 g, 138.8 mmol) in MeOH (150 mL). The reaction was stirred at rt overnight. The solution was concentrated and then Na2CO3 aq. and NaOH aq. were added until pH >12. The mixture was filtered and the solid was dried to give product as a white solid. (29.3 g, yield=98%)
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.0 min]: Rt=0.85 min; MS Calcd.: 215, MS Found: 216 [M+H]+.
To a solution of 5-methyl-6-(piperidin-4-yl)-1H-indazole hydrochloride (600 mg, 2.39 mmol) in CH3OH (10 mL) and H2O (2 mL) was added KOH (268 mg, 4.78 mmol) and (Boc)2O (781 mg, 3.58 mmol) under ice bath. The reaction mixture was stirred at rt for 2 hrs. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by column chromatograph (PE:EtOAc from 10:1 to 4:1) to give the title compound (353 mg, yield 47%) as a yellow oil.
1H NMR (300 MHz, CDCl3): b 10.15 (br, 1H), 7.95 (s, 1H), 7.53 (s, 1H), 7.29 (s, 1H), 4.34 (br, 2H), 2.95-2.81 (m, 3H), 2.45 (s, 3H), 1.86-1.81 (m, 2H), 1.69-1.61 (m, 2H), 1.51 (s, 9H).
To a solution of 5-methyl-6-(piperidin-4-yl)-1H-indazole hydrochloride (820 mg of crude, 2.50 mmol) in DCE (15 mL) was added oxetan-3-one (1.80 g, 25.0 mmol). The mixture was stirred at room temperature for 40 min. Then the mixture was cooled under ice bath and NaBH3CN (473 mg, 7.50 mmol) was added to the mixture. The mixture was warmed to room temperature and stirred for 2 hrs. Then the reaction mixture was poured into Na2CO3 aqueous solution (10%, 100 mL) and stirred for 15 min. Then the mixture was extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by column chromatography (DCM:MeOH=40:1) to afford the title compound (473 mg, yield 70%) as a white solid
1H NMR (300 MHz, CDCl3): δ 10.11 (br, 1H), 7.96 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 4.72-4.69 (m, 4H), 3.61-3.52 (m, 1H), 2.96-2.93 (m, 2H), 2.88-2.78 (m, 1H), 2.44 (s, 3H), 2.06-1.98 (m, 2H), 1.90-1.82 (m, 4H).
LC-MS [mobile phase: 5-95% Acetonitrile in 2.5 min]: Rt=1.37 min; MS Calcd: 271; MS Found: 272 [M+H]+.
To a solution of 1,4-dioxaspiro[4.5]decan-8-one (10 g, 64.1 mmol) and N,N-bis(trifluoromethylsulfonyl)aniline (25.2 g, 70.5 mmol) in THF (150 mL) was added LiHMDS (70.5 mL, 70.5 mmol) drop-wise under N2 at −78° C. The mixture was stirred at −78° C. for 30 min and warmed to room temperature. The reaction was quenched with aq.NH4Cl (150 mL) and the mixture was extracted with EtOAc (150 mL×3). The combined organic layers were washed with brine (150 mL×3), dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by silica gel chromatography (PE→PE:EtOAc=10:1) to give the product 1,4-dioxaspiro[4.5]dec-7-en-8-yltrifluoromethanesulfonate as a light yellow oil. (22.9 g, quantitative).
1H NMR (400 MHz, CDCl3): δ 5.66 (t, J=4.0 Hz, 1H), 3.99 (br, 4H), 2.53 (t, J=6.0 Hz, 2H), 2.41 (s, 2H), 1.91 (t, J=6.4 Hz, 2H).
To a mixture of 6-bromo-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (5.78 g, 19.6 mmol), bis(pinacolato)diboron (9.95 g, 39.2 mmol) and CH3COO− K+ (7.68 g, 78.3 mmol) in dioxane (50 mL) was added Pd(PPh3)4(6.60 g, 5.09 mmol). The mixture was heated to 90° C. and stirred under N2 overnight. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica column (PE:EtOAc from 100:1 to 60:1) to give the desired product (2.7 g, yield 41%) as a yellow solid.
1H NMR (400 MHz, CDCl3): δ 7.98 (s, 1H), 7.94 (s, 1H), 7.47 (s, 1H), 5.77 (dd, J=9.6, 2.8 Hz, 1H), 4.05-4.02 (m, 1H), 3.81-3.75 (m, 1H), 2.61 (s, 3H), 2.19-2.13 (m, 1H), 2.04-2.00 (m, 1H), 1.84-1.72 (m, 2H), 1.65-1.59 (m, 2H), 1.38 (s, 12H).
LC-MS: [mobile phase: from 90% water (0.02% NH4OAc) and 10% ACN to 5% water (0.02% NH4OAc) and 95% ACN in 4 min], Rt=2.914 min MS Calcd.: 342, MS Found: 343 [M+H]+.
To a solution of 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (3.0 g, 8.77 mmol), 1,4-dioxaspiro[4.5]dec-7-en-8-yl trifluoromethanesulfonate (3.0 g, 10.52 mmol) and K2CO3 (3.6 g, 26.30 mmol) in 1,4-dioxane/H2O (50 mL/10 mL) was added Pd(dppf)Cl2 (642 mg, 0.88 mmol). The mixture was stirred at 100° C. for 4 hrs under N2. The reaction mixture was poured into water (300 mL) and the mixture was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by silica gel chromatography eluted with EtOAc: Petroleum Ether=1:10 to 1:3 to give the product 5-methyl-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (2.3 g, 74.0% yield) as a white solid.
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.5 min]: Rt=1.30 min; MS Calcd.: 354; MS Found: 355 [M+H]+.
The mixture of 5-methyl-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (2.3 g, 6.49 mmol) and 10% Pd/C (230 mg) in MeOH (30 mL) was stirred at room temperature overnight under H2. The reaction mixture was filtered and the filter cake was washed with MeOH (30 mL×3). The filtrate was concentrated to dryness to give the product 5-methyl-6-(1,4-dioxaspiro[4.5]decan-8-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (1.8 g, 77.6% yield) as a white solid.
LC-MS [mobile phase: mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.5 min]: Rt=1.295 min; MS Calcd: 356, MS Found: 357 [M+H]+
To a solution of 5-methyl-6-(1,4-dioxaspiro[4.5]decan-8-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (120 mg, 0.34 mmol) in CH2Cl2(10 mL) was added TFA (4 mL). The reaction mixture was stirred at room temperature overnight. The reaction was concentrated to dryness, the residue was dissolved in CH2Cl2(100 mL). The solution was washed with sat. NaHCO3 (50 mL) and brine (50 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated to dryness. The residue was purified by silica gel chromatography (eluted with, EtOAc: Petroleum Ether=1:10 to 1:2) to give the product 4-(5-methyl-1H-indazol-6-yl)cyclohexanone (60 mg, 77.3% yield) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 7.97 (s, 1H), 7.56 (s, 1H), 7.32 (s, 1H), 3.36-3.28 (m, 1H), 2.59-2.55 (m, 4H), 2.51 (s, 3H), 2.26-2.21 (m, 2H), 1.96-1.89 (m, 2H).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 2.5 min]: Rt=1.23 min; MS Calcd.: 228; MS Found: 229 [M+H]+.
The solution of 4-(5-methyl-1H-indazol-6-yl)cyclohexanone (7.0 g, 30.7 mmol), 4,6-diiodo-2-methylpyrimidine (20.0 g, 57.8 mmol) in THF (500 mL) was degassed and then CuI (2.0 g, 10.5 mmol) and K3PO4 (21.2 g, 100.00 mmol) were added. The mixture was degassed and N1,N2-dimethylethane-1,2-diamine (2 g, 22.7 mmol) was added. The reaction was stirred at RT overnight and then the reaction was diluted with EtOAc (500 mL). The mixture was washed with aq. NH4Cl (200 mL×2) and brine (200 mL). The solution was dried and concentrated. The residue was purified by chromatography (silica EtOAc:DCM=0:100˜5:100) to give a white solid. The solid was re-crystallized with DMF/water to give the product as a white solid (4.5 g, 33% yield).
1H NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 8.27 (s, 1H), 8.13 (s, 1H), 7.57 (s, 1H), 3.37 (t, J=12.0 Hz, 1H), 2.76 (s, 3H), 2.62-2.59 (m, 4H), 2.55 (s, 3H), 2.30-2.26 (m, 2H), 2.09-1.98 (m, 2H).
MeMgBr (0.4 mL, 1.2 mmol) was added to the solution of 4-(1-(6-iodo-2-methylpyrimidin-4-yl)-5-methyl-1H-indazol-6-yl)cyclohexanone (223 mg, 0.5 mmol) in THF (5 mL) at RT and the reaction was stirred at rt for 10 min. The reaction was quenched with sat. NH4Cl (20 mL) and the mixture was extracted with EtOAc (2×20 mL). The solution was dried and concentrated. The residue was purified by chromatography (EtOAc:PE=1:3, 15 g of silica gel) to give two white solids.
Peak 1: 99 mg, 43% yield
LC-MS [mobile phase: from 70% water (0.1% FA) and 30% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 10 min]: Rt=2.153 min; MS Calcd: 462, MS Found: 463 [M+H]+.
Peak 2: 80 mg, 35% yield
LC-MS [mobile phase: from 70% water (0.1% FA) and 30% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 10 min]: Rt=2.027 min; MS Calcd: 462, MS Found: 463 [M+H]+.
To a solution of 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (2.70 g, 7.89 mmol) in THF (80 mL) and NaOH aqueous solution (1 N, 40 mL) was added H2O2 aqueous (37%, 4.48 g, 39.5 mmol) at 0-15° C. The mixture was diluted with sat. NaHSO3 (100 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica column (PE:EtOAc=6:1) and the crude was slurried with PE (3.5 mL) to give the desired product (1.70 g, yield 94%) as a yellow solid.
1H NMR (300 MHz, CDCl3): δ 7.85 (s, 1H), 7.42 (s, 1H), 6.93 (s, 1H), 5.58 (dd, J=9.6, 2.7 Hz, 1H), 5.44 (s, 1H), 4.04-3.99 (m, 1H), 3.75-3.66 (m, 1H), 2.60-2.47 (m, 1H), 2.32 (s, 3H), 2.17-2.01 (m, 2H), 1.81-1.63 (m, 3H).
LC-MS [mobile phase: from 90% water (0.02% NH4OAc) and 10% ACN to 5% water (0.02% NH4OAc) and 95% ACN in 4 min]: Rt=2.113 min, MS Calcd.: 232, MS Found: 233 [M+H]+.
To a solution of tetrahydrofuran-3-ol (500 mg, 5.68 mmol) and Et3N (860 mg, 8.52 mmol) in CH2Cl2 (10 mL) at 0° C. was added MsCl (842 mg, 7.39 mmol) dropwise. The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with CH2Cl2 (150 mL), washed with sat. NaHCO3 (50 mL), dried over Na2SO4 and concentrated to give the title compound (700 mg, 74%) as yellow oil.
1H NMR (300 MHz, CDCl3): δ 5.36-5.31 (m, 1H), 4.06-3.86 (m, 4H), 3.06 (s, 3H), 2.29-2.22 (m, 2H).
A mixture of 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-ol (150 mg, 0.65 mmol), tetrahydrofuran-3-yl methanesulfonate (129 mg, 0.78 mmol) and K2CO3 (180 mg, 1.3 mmol) in DMF (2.0 mL) was stirred at 80° C. overnight under N2 atmosphere. The reaction was diluter with water (50 mL), extracted with EtOAc (150 mL×2), washed with brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (Petroleum ether:EtOAc=3:1) to give the title compound (150 mg, 77%) as yellow oil.
1H NMR (300 MHz, CDCl3): δ 7.86 (s, 1H), 7.44 (s, 1H), 6.82 (s, 1H), 5.66 (dd, J=9.3, 2.1 Hz, 1H), 5.09-5.04 (m, 1H), 4.14-3.96 (m, 5H), 3.79-3.71 (m, 1H), 2.63-2.51 (m, 1H), 2.28 (s, 3H), 2.17-2.04 (m, 4H), 1.81-1.69 (m, 3H).
To a solution of 5-methyl-1-(tetrahydro-2H-pyran-2-yl)-6-((tetrahydrofuran-3-yl)oxy)-1H-indazole (150 mg, 0.5 mmol) in DCM (2 mL) was added TFA (2 mL). The resulting mixture was stirred at room temperature for 5 hours. The reaction was dilutre with DCM (100 mL), washed with brine (100 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (Petroleum ether:EtOAc=2:3) to give the title compound (95 mg, 86%) as a yellow solid.
1H NMR (300 MHz, CDCl3): δ 7.92 (s, 1H), 7.48 (s, 1H), 6.74 (s, 1H), 5.02-4.96 (m, 1H), 4.13-3.93 (m, 4H), 2.29 (s, 3H), 2.27-2.20 (m, 2H).
A mixture of 2,5-dioxa-8-azaspiro[3.5]nonane oxalate (500 mg, 1.44 mmol), 4,6-diiodo-2-methoxypyrimidine (453 mg, 1.37 mmol), TEA (415 mg, 4.11 mmol) in i-PrOH (10 mL) and DMSO (4 mL) was stirred at room temperature for 4 hours. The mixture was added H2O (50 mL), extracted EtOAc with (30*3 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated. The crude was purified by flash chromatography (petroleum ether/EtOAc=2:1) to give compound (408 mg, 82%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 6.70 (s, 1H), 4.63 (d, J=7.2 Hz, 2H), 4.42 (d, J=7.2 Hz, 2H), 3.93 (s, 3H), 3.86 (s, 2H), 3.71 (t, J=4.8 Hz, 2H), 3.54 (s, 2H).
A mixture of 8-(6-iodo-2-methoxypyrimidin-4-yl)-2,5-dioxa-8-azaspiro[3.5]nonane (127 mg, 0.35 mmol), tert-butyl-4-(5-methyl-1H-indazol-6-yl) piperidine-1-carboxylate (100 mg, 0.32 mmol), CuI (30 mg, 0.15 mmol), K3PO4 (133 mg, 0.63 mmol) and N,N′-dimethylcyclohexane-1,2-diamine (45 mg, 0.32 mmol) in toluene (3 mL) was stirred at 100° C. for 5 hours. The mixture was added EtOAc (50 mL), washed with NH3H2O (30 mL×3). The organic layer was dried over Na2SO4, filtered and concentrated. The crude was purified by flash chromatography (petroleum ether/EtOAc=1/1) to give compound (170 mg, 97%) as a yellow oil. 1HNMR (400 MHz, CDCl3): δ 8.71 (s, 1H), 8.09 (s, 1H), 7.53 (s, 1H), 6.91 (s, 1H), 4.64 (d, J=6.8 Hz, 2H), 4.48 (d, J=7.2 Hz, 2H), 4.31 (br, 2H), 4.12 (s, 3H), 3.96 (s, 2H), 3.76-3.74 (m, 6H), 3.69-3.67 (m, 6H), 3.63-3.61 (m, 4H), 3.58-3.56 (m, 4H), 2.48 (s, 3H).
To a suspension of 6-cis-3-fluoro-1-(oxetan-3-yl)piperidin-4-yl)-5-methyl-1H-indazole (70 mg, 0.24 mmol) (peak 1), (R)-2-(6-iodo-2-methoxypyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (104 mg, 0.29 mmol), CuI (46 mg, 0.24 mmol) and K3PO4 (108 mg, 0.51 mmol) in dry toluene (5 mL) was added N,N-dimethyl-1,2-ethanediamine (43 mg, 0.48 mmol). The suspension was degassed with N2 and stirred at 85° C. for 2 h. EtOAc (50 mL) was added and the resulting mixture was washed with brine (2×50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by pre-HPLC (Waters 2767 Sepax Amethyst 21.2×100 mm 5 μm, Phase: MeCN/H2O (0.1% TFA): 5%˜95%, Flow rate: 15 mL/min, 214 nm/254 nm) to give product (24 mg, yield 19.0%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.88 (s, 1H), 8.07 (s, 1H), 7.54 (s, 1H), 6.87 (s, 1H), 4.95-4.81 (m, 1H), 4.81-4.63 (m, 4H), 4.48-4.41 (br, 1H), 4.14 (s, 3H), 3.69-3.62 (m, 1H), 3.25-3.07 (m, 5H), 2.84-2.72 (m, 2H), 2.48 (s, 3H), 2.31-1.70 (m, 10H), 1.55-1.45 (m, 2H).
19F NMR (376 MHz, CDCl3): δ −184.0 (s, 1F).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 10 min]: Rt=5.17 min; MS Calcd.: 521, MS Found: 522 [M+H]+.
Chiral purity: Rt=8.915 min; ee %=99.5%.
Chiral method: Chiralpak AD-H 5 μm 4.6×250 mm, Phase: Hexane:Isopropanol (0.2% diethylamine)=65/35, F: 1.0 mL/min, W: 214 nm, T: 25° C.
To a suspension of (cis)-6-(3-Fluoro-1-(oxetan-3-yl)piperidin-4-yl)-5-methyl-1H-indazole (70 mg, 0.24 mmol) (peak 2), (S)-2-(6-iodo-2-methoxypyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (104 mg, 0.29 mmol), CuI (46 mg, 0.24 mmol) and K3PO4 (108 mg, 0.51 mmol) in dry toluene (5 mL) was added N,N-dimethyl-1,2-ethanediamine (43 mg, 0.48 mmol). The suspension was degassed with N2 and stirred at 85° C. for 2 h. EtOAc (50 mL) was added and the resulting mixture was washed with brine (2×50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by pre-HPLC (Waters 2767, Sepax Amethyst 21.2×100 mm 5 μm, Phase: MeCN/H2O (0.1% TFA): 5%-95%, Flow rate: 15 mL/min, 214 nm/254 nm) to give product (50 mg, yield 39.7%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.88 (s, 1H), 8.07 (s, 1H), 7.54 (s, 1H), 6.87 (s, 1H), 4.95-4.81 (m, 1H), 4.81-4.63 (m, 4H), 4.48-4.41 (br, 1H), 4.14 (s, 3H), 3.69-3.62 (m, 1H), 3.25-3.07 (m, 5H), 2.84-2.72 (m, 2H), 2.48 (s, 3H), 2.31-1.70 (m, 10H), 1.55-1.45 (m, 2H).
19F NMR (376 MHz, CDCl3): δ −184.0 (s, 1F).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 10 min]: Rt=5.35 min; MS Calcd.: 521, MS Found: 522 [M+H]+.
Chiral purity: Rt=12.450 min; ee %=99.1%.
Chiral method: Chiralpak AD-H 5 μm 4.6×250 mm, Phase: Hexane:Isopropanol (0.2% diethylamine)=65/35, F: 1.0 mL/min, W: 214 nm, T: 25° C.
To a suspension of (cis)-6-(3-Fluoro-1-(oxetan-3-yl)piperidin-4-yl)-5-methyl-1H-indazole (70 mg, 0.24 mmol) (peak 1), (S)-2-(6-iodo-2-methoxypyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (104 mg, 0.29 mmol), CuI (46 mg, 0.24 mmol) and K3PO4 (108 mg, 0.51 mmol) in dry toluene (5 mL) was added N,N-dimethyl-1,2-ethanediamine (43 mg, 0.48 mmol). The suspension was degassed with N2 and stirred at 85° C. for 2 h. EtOAc (50 mL) was added and the resulting mixture was washed with brine (2×50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by pre-HPLC (Waters 2767/Qda, Waters XBridge 30×150 mm 5 μm, Phase: MeCN/H2O (0.1% TFA): 5%˜95%, Flow rate: 20 mL/min, 214 nm/254 nm) to give product (70 mg, yield 55.5%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.88 (s, 1H), 8.07 (s, 1H), 7.54 (s, 1H), 6.87 (s, 1H), 4.95-4.81 (m, 1H), 4.81-4.63 (m, 4H), 4.48-4.41 (br, 1H), 4.14 (s, 3H), 3.69-3.62 (m, 1H), 3.25-3.07 (m, 5H), 2.84-2.72 (m, 2H), 2.48 (s, 3H), 2.31-1.70 (m, 10H), 1.55-1.45 (m, 2H).
19F NMR (376 MHz, CDCl3): δ −184.0 (s, 1F).
LC-MS (mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 10 min]: Rt=5.15 min; MS Calcd.: 521, MS Found: 522 [M+H]+.
Chiral purity: Rt=9.697 min; ee %=99.8%.
Chiral method: Chiralpak AD-H 5 μm 4.6×250 mm, Phase: Hexane:Isopropanol (0.2% diethylamine)=65/35, F: 1.0 mL/min, W: 214 nm, T: 25° C.
To a suspension of (cis)-6-(3-Fluoro-1-(oxetan-3-yl)piperidin-4-yl)-5-methyl-1H-indazole (70 mg, 0.24 mmol) (peak 2), (R)-2-(6-iodo-2-methoxypyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (104 mg, 0.29 mmol), CuI (46 mg, 0.24 mmol) and K3PO4 (108 mg, 0.51 mmol) in dry toluene (5 mL) was added N,N-dimethyl-1,2-ethanediamine (43 mg, 0.48 mmol). The suspension was degassed with N2 and stirred at 85° C. for 2 h. EtOAc (50 mL) was added and the resulting mixture was washed with brine (2×50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by pre-HPLC (Waters 2767 Sepax Amethyst 21.2×100 mm 5 μm, Phase: MeCN/H2O (0.1% TFA): 5%˜95%, Flow rate: 15 mL/min, 214 nm/254 nm) to give product (70 mg, yield 55.5%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.88 (s, 1H), 8.07 (s, 1H), 7.54 (s, 1H), 6.87 (s, 1H), 4.95˜4.81 (m, 1H), 4.81˜4.63 (m, 4H), 4.48˜4.41 (br, 1H), 4.14 (s, 3H), 3.69˜3.62 (m, 1H), 3.25˜3.07 (m, 5H), 2.84˜2.72 (m, 2H), 2.48 (s, 3H), 2.31˜1.70 (m, 10H), 1.55˜1.45 (m, 2H).
19F NMR (376 MHz, CDCl3): δ−184.0 (s, 1F).
LC-MS (mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 10 min]: Rt=5.16 min; MS Calcd.: 521, MS Found: 522 [M+H]+.
Chiral purity: Rt=11.898 min; ee %=99.4%.
Chiral method: Chiralpak AD-H 5 μm 4.6×250 mm, Phase: Hexane:Isopropanol (0.2% diethylamine)=65/35, F: 1.0 mL/min, W: 214 nm, T: 25° C.
A solution of tert-butyl 2-(3-(tert-butoxy)-3-oxopropyl)-4-(2-methoxy-6-(5-methyl-6-(tetrahydro-2H-pyran-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)piperazine-1-carboxylate (85 mg, 0.13 mmol, Peak 1) in MeOH (5 mL) was added HCl/MeOH (7 M, 5 mL). The mixture was stirred at rt for 1 h. The mixture was directly concentrated in reduced pressure to give the crude solid as a mixture of the desired acid and the corresponding methyl ester (62 mg). The crude solid was treated with lithium hydroxide monohydrate (42 mg, 1.0 mmol) in THF/H2O (2 mL/2 mL) and stirred for 1 h. The reaction mixture was purified by prep-HPLC (ACN/H2O, 05-95%) to give example 5 (Enantiomer 1, 7 mg, yield 11%) as a white solid.
1H NMR (300 MHz, DMSO-d6): δ 8.65 (s, 1H), 8.32 (s, 1H), 7.62 (s, 1H), 6.92 (s, 1H), 4.60-4.46 (m, 2H), 4.01-3.85 (m, 6H), 3.57-3.46 (m, 3H), 3.12-3.05 (m, 1H), 2.89-2.69 (m, 3H), 2.43 (s, 3H), 2.28-2.12 (m, 3H), 1.76-1.58 (m, 5H).
LC-MS [XB-C18, ¢4.6×50 mm 5 μm; mobile phase: from 95% water (0.02% NH4OAc) and 5% CH3CN to 5% water (0.02% NH4OAc) and 95% CH3CN in 6.5 min]: Rt=4.180 min; MS Calcd.: 462; MS Found: 463 [M+H]+.
A solution of tert-butyl 2-(3-(tert-butoxy)-3-oxopropyl)-4-(2-methoxy-6-(5-methyl-6-(tetrahydro-2H-pyran-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)piperazine-1-carboxylate (80 mg, 0.13 mmol) (Peak 2) in MeOH (5 mL) was added HCl/MeOH (7 M, 5 mL). The mixture was stirred at rt for 1 h. The reaction was directly concentrated in reduced pressure to give the crude yellow solid as the mixtures of the desired acid and the corresponding methyl ester. The crude solid was treated with lithium hydroxide monohydrate (42 mg, 1.0 mmol) in THF/H2O (2 mL/2 mL) and stirred for 1 h. The mixture was purified by prep-HPLC (ACN/H2O, 05-95%) to give example E A-6 (Enantiomer 2, 7 mg, yield 12%) as a white solid.
1H NMR (300 MHz, DMSO-d6): δ 8.67 (s, 1H), 8.32 (s, 1H), 7.62 (s, 1H), 6.92 (s, 1H), 4.61-4.45 (m, 2H), 4.01-3.84 (m, 6H), 3.57-3.45 (m, 3H), 3.12-3.06 (m, 1H), 2.89-2.69 (m, 3H), 2.43 (s, 3H), 2.27-2.12 (m, 3H), 1.72-1.57 (m, 5H).
LC-MS [XB-C18, ¢4.6×5 0 mm 5 μm; mobile phase: from 95% water (0.02% NH4OAc) and 5% CH3CN to 5% water (0.02% NH4OAc) and 95% CH3CN in 6.5 min]: Rt=4.176 min; MS Calcd.: 462; MS Found: 463 [M+H]+.
To a solution of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (79 mg, 0.29 mmol) and (R)-2-(6-iodo-2-methylpyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (100 mg, 0.29 mmol), K3PO4 (123 mg, 0.58 mmol), CuI (55 mg, 0.29 mmol) in toluene(4 ml) was added N1,N2-dimethylethane-1,2-diamine (51 mg, 0.58 mmol) under Ar. Then the reaction mixture was stirred at 95° C. for 3 hours. TLC showed the reaction was completed. Then it was filtered and washed with DCM. The filtrate was concentrated and purified by column(PE:EtOAc=1:1 followed by DCM:MeOH=50:1-40:1-30:1-20:1) to give desired product as a white solid (94 mg, yield: 66.3%).
1H NMR (400 MHz, CDCl3): δ 8.78 (s, 1H), 8.05 (s, 1H), 7.50 (s, 1H), 6.95 (s, 1H), 4.71 (d, J=6.8 Hz, 4H), 4.70-4.49 (m, 2H), 3.56 (t, J=6.4 Hz, 1H), 3.17-3.15 (m, 4H), 2.98-2.95 (m, 2H), 2.85-2.78 (m, 3H), 2.65 (s, 3H), 2.45 (s, 3H), 2.31-2.22 (m, 3H), 2.17 (s, 1H), 2.06-2.04 (m, 3H), 1.84-1.80 (m, 2H), 1.56-1.52 (m, 2H).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 10 min]: Rt=4.60 min; MS Calcd: 487.31, MS Found: 488.0 [M+H]+.
To a solution of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (71 mg, 0.26 mmol) and (S)-2-(6-iodo-2-methylpyrimidin-4-yl)octahydropyrrolo[1,2-a]pyrazine (90 mg, 0.26 mmol), K3PO4 (110 mg, 0.52 mmol), CuI (50 mg, 0.26 mmol) in toluene (2 ml) was added N1,N2-dimethylethane-1,2-diamine (46 mg, 0.52 mmol) under Ar. Then the reaction was stirred at 95° C. for 4 hours. TLC showed the reaction was completed. Then it was filtered and washed with DCM. The filtrate was concentrated and purified by column(DCM:MeOH=50:1-40:1-30:1-20:1) to get a yellow solid. (20 mg, Yield: 15.7%).
1H NMR (400 MHz, CDCl3): δ 8.78 (br, 1H), 8.07 (s, 1H), 7.50 (s, 1H), 6.95 (s, 1H), 4.71 (d, J=6.8 Hz, 4H), 4.70-4.49 (m, 2H), 3.56 (t, J=6.4 Hz, 1H), 3.17-3.15 (m, 2H), 2.98-2.95 (m, 2H), 2.85-2.78 (m, 2H), 2.65 (s, 3H), 2.45 (s, 3H), 2.31-2.22 (m, 2H), 2.07-1.94 (m, 8H), 1.70-1.65 (m, 1H), 1.70-1.50 (m, 3H).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 10 min]: Rt=4.58 min; MS Calcd: 487, MS Found: 488 [M+H]+.
To a solution of tert-butyl 4-(1-(6-(1-hydroxy-3-azabicyclo[3.1.0]hexan-3-yl)-2-methylpyrimidin-4-yl)-5-methyl-1H-indazol-6-yl)piperidine-1-carboxylate (70 mg, 0.13 mmol) in CH2Cl2 (2 mL) was added TFA (1 mL) at 0° C. After added the mixture was warmed to rt and stirred for 2 hrs. The mixture was concentrated to give the title compound (35 mg, yield >100%) which was used for next step directly.
LCMS (mobile phase: 5-95% Acetonitrile in 4 min): Rt=2.565 min; MS Calcd: 404; MS Found: 405 [M+H]+.
To a solution of 3-(2-methyl-6-(5-methyl-6-(piperidin-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)-3-azabicyclo[3.1.0]hexan-1-ol 2,2,2-trifluoroacetate (70 mg, 0.13 mmol) in ClCH2CH2Cl (4 mL) and methanol (0.5 mL) was added oxetan-3-one (13 mg, 0.18 mmol) at rt. The solution was stirred at rt for 30 min and then NaBH3CN (22 mg, 0.35 mmol) was added. The resulting mixture was stirred at rt overnight. The mixture was poured into sat. Na2CO3 solution (5 mL). The desired was extracted with ethyl acetate (5 mL×3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4 and concentrated. The residue was purified by prep. TLC (CH2Cl2:methanol=30:1) to give the title compound (4.7 mg) as a white solid.
1H NMR (300 MHz, CDCl3): δ 8.81 (s, 1H), 8.04 (s, 1H), 7.49 (s, 1H), 6.65 (s, 1H), 4.72-4.70 (m, 4H), 3.67-3.55 (m, 4H), 2.99-2.96 (m, 2H), 2.87-2.83 (m, 1H), 2.63 (s, 3H), 2.45 (s, 3H), 2.08-1.94 (m, 7H), 1.88-1.79 (m, 1H), 1.30-1.27 (m, 1H), 0.72-0.68 (m, 1H).
LC-MS [mobile phase: from 95% water (0.1% TFA) and 5% CH3CN to 5% water (0.1% TFA) and 95% CH3CN in 6.5 min]: Rt=2.992 min; MS Calcd.: 460; MS Found: 461 [M+H]+.
N,N′-dimethylethylenediamine (80 mg, 0.90 mmol) was added to a mixture of cis-5-(6-iodo-2-methylpyrimidin-4-yl)hexahydro-2H-furo[2,3-c]pyrrole (183 mg, 0.553 mmol), 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (150 mg, 0.553 mmol), CuI (86 mg, 0.453 mmol) and K3PO4 (289 mg, 1.360 mmol) in toluene (4 ml) under Ar. The reaction was stirred at 100° C. for 3 h. The cooled reaction mixture was filtered and the filtrate was concentrated. The residue was purified by column chromatography (eluent: CH2Cl2:MeOH=20:1) to afford the desired product as a white solid (200 mg, yield: 76%).
1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.05 (s, 1H), 7.49 (s, 1H), 6.71 (s, 1H), 4.71 (d, J=6.8 Hz, 4H), 4.65 (t, J=5.2 Hz, 1H), 4.02 (q, J=7.6 Hz, 1H), 3.93-3.83 (m, 3H), 3.66 (dd, J=12.4, 4.8 Hz, 1H), 3.56 (t, J=5.2 Hz, 1H), 3.46-3.42 (m, 1H), 3.06-3.03 (m, 1H), 2.97 (d, J=9.2 Hz, 2H), 2.85-2.82 (m, 1H), 2.65 (s, 3H), 2.45 (s, 3H), 2.20-2.17 (m, 1H), 2.03-1.94 (m, 7H).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 12.0 min]: Rt=5.01 min; MS Calcd: 474, MS Found: 475 [M+H]+.
Chiral separation of racemic cis-5-(2-methyl-6-(5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)hexahydro-2H-furo[2,3-c] (202 mg) by chiral prep-HPLC (Method: Column: AD-H; Column size: 0.46 cm I.D.×15 cm L; Mobile phase: CO2:ETOH (0.1% NH3.H2O)=60:40; Flow rate: 0.5 ml/min; Wave length: UV 254 nm; Temperature: 25° C.; Sample solution in EtOH) afforded peak 1 as a pale yellow solid (Rt=5.893 min, 79.9 mg, yield: 39%) and peak 2 as a pale yellow solid (Rt=7.095 min, 90.8 mg, yield: 44%)
Peak 1: 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.05 (s, 1H), 7.49 (s, 1H), 6.71 (s, 1H), 4.71 (d, J=6.4 Hz, 4H), 4.65 (t, J=5.2 Hz, 1H), 4.02 (q, J=7.6 Hz, 1H), 3.93-3.88 (m, 3H), 3.66 (dd, J=12.4, 4.4 Hz, 1H), 3.56 (t, J=6.6 Hz, 1H), 3.46-3.42 (m, 1H), 3.06-3.03 (m, 1H), 2.97 (d, J=11.8 Hz, 2H), 2.85-2.81 (m, 1H), 2.65 (s, 3H), 2.45 (s, 3H), 2.20-2.17 (m, 1H), 2.04-1.94 (m, 7H).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 12.0 min]: Rt=4.88 min; MS Calcd: 474, MS Found: 475 [M+H]+.
Peak 2 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.05 (s, 1H), 7.49 (s, 1H), 6.71 (s, 1H), 4.71 (d, J=6.8 Hz, 4H), 4.65 (t, J=5.6 Hz, 1H), 4.02 (q, J=7.6 Hz, 1H), 3.93-3.86 (m, 3H), 3.66 (dd, J=12.4, 4.4 Hz, 1H), 3.56 (t, J=6.6 Hz, 1H), 3.46-3.42 (m, 1H), 3.06-3.03 (m, 1H), 2.97 (d, J=11.2 Hz, 2H), 2.85-2.81 (m, 1H), 2.65 (s, 3H), 2.45 (s, 3H), 2.20-2.17 (m, 1H), 2.04-1.94 (m, 7H).
LC-MS [mobile phase: from 90% water (0.1% TFA) and 10% CH3CN (0.1% TFA) to 5% water (0.1% TFA) and 95% CH3CN (0.1% TFA) in 12.0 min]: Rt=4.96 min; MS Calcd: 474, MS Found: 475 [M+H]+.
To a suspension of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (115 mg, 1.43 mmol), 8-(6-iodo-2-methoxypyrimidin-4-yl)octahydropyrazino[2,1-c][1,4]oxazine (160 mg, 0.63 mmol), CuI (81 mg, 0.43 mmol) and K3PO4 (181 mg, 0.85 mmol) in toluene/THF (10 mL/2 mL) was added DMEDA (75 mg, 0.85 mmol). The resulting mixture was degassed with N2 three times. The reaction was stirred at 80° C. for 2 hour. EtOAc (40 mL) was added and the resulting mixture was washed with sat. NH4Cl (30 mL) and brine (30 mL). The organic solution was dried over anhydrous Na2SO4 and concentrated. The residue was purified by prep-HPLC (Gilson 281, YMC-Actus Triart Prep C18-S 250×20 mm 10 μm, Mobile Phase: MeCN/H2O (0.05% TFA): 15-95%, Flow rate: 20 ml/min, 254 nm) to give the product as a pale yellow solid (200 mg, yield: 91%).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 2.0 min]: Rt=1.13 min; MS Calcd.: 519, MS Found: 520 [M+H]+.
The compound 8-(2-methoxy-6-(5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazol-1-yl) pyrimidin-4-yl)octahydropyrazino[2,1-c][1,4]oxazine (200 mg, 0.38 mmol) was purified by chiral prep-HPLC (AD-H, 0.46 cm I.D.×15 cm L, Mobile phase: CO2:EtOH (0.1% NH3.H2O)=60:40, Flow rate: 0.5 mL/min, Wave length: UV 254 nm, Temperature: 25° C.) to give product as two pale yellow solids.
(1.4 mg, yield: 1%).
1H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H), 8.07 (s, 1H), 7.51 (s, 1H), 6.84 (s, 1H), 4.70 (d, J=6.4 Hz, 4H), 4.40-4.27 (m, 2H), 4.15 (s, 3H), 3.89˜3.81 (m, 2H), 3.76˜3.71 (m, 1H), 3.59˜3.52 (m, 1H), 3.32 (t, J=10.8 Hz, 1H), 3.18 (t, J=11.6 Hz, 1H), 2.93 (d, J=10.4 Hz, 2H), 2.87˜2.80 (m, 2H), 2.74 (d, J=11.2 Hz, 1H), 2.68˜2.61 (m, 1H), 2.45 (s, 3H), 2.44˜2.27 (m, 3H), 2.05˜1.99 (m, 2H), 1.92˜1.83 (m, 4H).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 9 min]: Rt=3.79 min; MS Calcd: 519, MS Found: 520 [M+H]+.
Chiral HPLC [AD 4.6×250 mm, 5 μm (Daicel) (CA-HPLC-023), Phase: Hexane/EtOH (0.2% DEA)=60/40, flow rate: 0.5 mL/min, temperature: 35° C.]: Rt: 2.471 min, ee: 100%
(109.3 mg, yield: 55%)
1H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H), 8.06 (s, 1H), 7.51 (s, 1H), 6.84 (s, 1H), 4.70 (d, J=6.4 Hz, 4H), 4.40-4.27 (m, 2H), 4.15 (s, 3H), 3.90-3.81 (m, 2H), 3.74 (t, J=10.0 Hz, 1H), 3.58˜3.51 (m, 1H), 3.32 (t, J=10.4 Hz, 1H), 3.18 (t, J=12.0 Hz, 1H), 2.94 (d, J=10.8 Hz, 2H), 2.87˜2.80 (m, 2H), 2.73 (d, J=11.6 Hz, 1H), 2.65 (t, J=11.2 Hz, 1H), 2.45 (s, 3H), 2.42˜2.27 (m, 3H), 2.04˜1.98 (m, 2H), 1.91˜1.85 (m, 4H).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% CH3CN (0.1% FA) to 5% water (0.1% FA) and 95% CH3CN (0.1% FA) in 9 min]: Rt=3.77 min; MS Calcd: 519, MS Found: 520 [M+H]+.
Chiral HPLC [AD 4.6×250 mm, 5 μm (Daicel) (CA-HPLC-023), Phase: Hexane/EtOH (0.2% DEA)=60/40, flow rate: 0.5 mL/min, temperature: 35° C.]: Rt: 2.873 min, ee: 99.71%
To a mixture of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (217 mg, 0.8 mmol), 8-(6-iodo-2-methylpyrimidin-4-yl)-1-oxa-4,8-diazaspiro[4.5]decan-3-one (300 mg, 0.8 mmol), K3PO4 (339 mg, 1.6 mmol) and CuI (152 mg, 0.8 mmol) in toluene (8 ml) under Ar was added N,N′-dimethylethylenediamine (141 mg, 1.6 mmol). The reaction was stirred at 100° C. for 3 hours. TLC showed reaction was complete. The reaction mixture was filtered and the filter cake was washed with CH2Cl2. The combined filtrate was concentrated and the residue was purified by column chromatography (eluent: PE/EtOAc=1/1 to 0/1, followed by CH2Cl2/MeOH=30/1 to 25/1) afforded pure desired product as yellow solid (30 mg, yield: 7%).
1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.05 (s, 1H), 7.50 (s, 1H), 7.00 (s, 1H), 6.66 (s, 1H), 4.71 (d, J=6.4 Hz, 4H), 4.32 (s, 2H), 4.32-4.25 (m, 2H), 3.58-3.48 (m, 3H), 2.97 (d, J=10.4 Hz, 2H), 2.84 (br, 1H), 2.64 (s, 3H), 2.45 (s, 3H), 2.05-1.93 (m, 8H), 1.86-1.79 (m, 2H).
LC-MS [mobile phase: from 60% water (0.1% NH4OH) and 40% ACN (0.1% NH4OH) to 5% water (0.1% NH4OH) and 95% ACN (0.1% NH4OH) in 10.0 min]: Rt=3.61 min; MS Calcd: 517, MS Found: 518 [M+H]+.
DMEDA (65 mg, 0.74 mmol) was added to a solution of 8-(6-iodo-2-methylpyrimidin-4-yl)-3-oxa-1,8-diazaspiro[4.5]decan-2-one (118 mg, 0.31 mmol), 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (100 mg, 0.37 mmol), CuI (70 mg, 0.37 mmol) and K3PO4 (156 mg, 0.74 mmol) in toluene (8 mL) under Ar. The reaction was stirred at 100° C. for 3.5 h. The mixture was filtered and purified by column chromatography (DCM:MeOH=50:1) to gave product as a white solid (50 mg, yield: 31%).
LC-MS [mobile phase: from 90% water (0.1% FA) and 10% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 10.0 min]: Rt=5.612 min; MS Calcd: 517, MS Found: 518 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.05 (s, 1H), 7.50 (s, 1H), 6.99 (s, 1H), 6.31 (s, 1H), 4.71 (d, J=6.5 Hz, 4H), 4.22 (s, 2H), 3.96-3.79 (m, 2H), 3.79-3.63 (m, 2H), 3.64-3.45 (m, 1H), 2.97 (d, J=10.3 Hz, 2H), 2.89-2.77 (m, 1H), 2.64 (s, 3H), 2.45 (s, 3H), 2.07-1.82 (m, 10H).
A mixture of cis-4-(1-(6-iodo-2-methylpyrimidin-4-yl)-5-methyl-1H-indazol-6-yl)-1-methylcyclohexanol (70 mg, 0.15 mmol), 2,6-diazaspiro[3.4]octan-7-one hydrochloride (122 mg, 0.75 mmol) and DIEA (194 mg, 1.50 mmol) in NMP (3 mL) was stirred at 30° C. overnight, then poured into water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic phase was washed with brine (3×50 mL), dried over Na2SO4 and concentrated to obtain the crude. The crude was purified by prep-HPLC to give product as a white solid (35 mg, yield: 50%)
1H NMR (400 MHz, CDCl3): δ 8.851 (s, 1H), 8.061 (s, 1H), 7.492 (s, 1H), 6.615 (s, 1H), 6.218 (s, 1H), 4.180-4.250 (m, 4H), 2.781 (s, 1H), 2.629 (s, 3H), 2.454 (s, 6H), 1.987-1.924 (t, 2H), 1.866-1.764 (m, 4H), 1.661-1.603 (t, 4H), 1.324 (s, 3H).
LC-MS [mobile phase: from 70% water (0.1% TFA) and 30% ACN (0.1% TFA) to 95% water (0.1% TFA) and 5% ACN (0.1% TFA) in 10 min]: Rt=6.47 min; MS Calcd.: 460, MS Found: 461 [M+H]+.
Prep-HPLC method:
Waters 2767/Qda, Waters XBridge 30×150 mm 5 μm 20 ml/min A: H2O, B: ACN
To a solution of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (179 mg, 0.66 mmol) and 8-(6-iodo-2-methylpyrimidin-4-yl)-2,5-dioxa-8-azaspiro[3.5]nonane (230 mg, 0.66 mmol), K3PO4 (280 mg, 1.32 mmol), CuI (126 mg, 0.66 mmol) in toluene (4 ml) was added N1,N2-dimethylethane-1,2-diamine (116 mg, 1.32 mmol) under Ar. The reaction was stirred at 95° C. for 3.5 hours. TLC showed the reaction was complete. The reaction mixture was filtered and washed with DCM. The filtrate was concentrated and purified by column chromatography (PE:EtOAc=1:1 followed by DCM:MeOH=50:1) to get desired product as white solid (240 mg, yield: 74%).
1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.07 (s, 1H), 7.51 (s, 1H), 7.02 (s, 1H), 4.71 (d, J=6.4 Hz, 4H), 4.65 (d, J=6.8 Hz, 2H), 4.49 (d, J=7.2 Hz, 2H), 3.98 (s, 2H), 3.76 (t, J=4.6 Hz, 2H), 3.67 (d, J=4.8 Hz, 2H), 3.58-3.55 (m, 1H), 2.98 (d, J=10.4 Hz, 2H), 2.90-2.85 (m, 1H), 2.67 (s, 3H), 2.46 (s, 3H), 2.05-1.84 (m, 6H).
LC-MS [mobile phase: from 80% water (0.1% TFA) and 20% ACN (0.1% TFA) to 5% water (0.1% TFA) and 95% ACN (0.1% TFA) in 10 min]: Rt=4.85 min; MS Calcd: 490, MS Found: 491 [M+H]+.
A mixture of 8-(6-iodo-2-methylpyrimidin-4-yl)-1,8-diazaspiro[4.5]decan-2-one (223 mg, 0.6 mmol) and 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (164 mg, 0.6 mmol) in toluene (10 mL) was added K3PO4 (254 mg, 1.2 mmol), CuI (114 mg, 0.6 mmol) and N1,N2-dimethylethane-1,2-diamine (53 mg, 0.6 mmol). The mixture was degassed and protected with Ar then heated at 100° C. for 5 h. the reaction mixture was filtered and the filtrate was concentrated. The residue was purified by prep-HPLC to give title compound as a yellow solid as a TFA salt (50 mg)
1H NMR (400 MHz, DMSO-d6): δ 10.60 (br, 1H), 8.73 (s, 1H), 8.35 (s, 1H), 8.09 (s, 1H), 7.68 (d J=8.8 Hz, 1H), 7.02 (s, 1H), 4.86-4.78 (m, 4H), 4.34-4.32 (m, 1H), 3.82-3.57 (m, 6H), 3.24-3.10 (m, 3H), 2.64 (s, 3H), 2.47 (s, 3H), 2.27-1.63 (m, 12H).
LC-MS [mobile phase: from 80% water (0.1% FA) and 20% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 10 min]: Rt=4.44 min; MS Calcd: 515, MS Found: 516 [M+H]+.
Instrument: Waters 2767/Qda
Column: Waters sunfire C18 20×250 mm 10 μm, Rate: 30 ml/min
Wavelength: 214 nm/254 nm, Trigger: 254 nm
Mobile Phase A: H2 (0.1% TFA), Mobile Phase B: ACN
Gradient Method:
To a solution of 8-(6-iodo-2-methylpyrimidin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (140 mg, 0.376 mmol) in toluene (15 mL) were added 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (103 mg, 0.376 mmol), CuI (108 mg, 0.57 mmol), N,N′-dimethylethylenediamine (67 mg, 0.75 mmol) and K3PO4 (160 mg, 0.75 mmol). The reaction mixture was stirred at 100° C. for 3 h with N2 protection. The reaction mixture was diluted with EtOAc (60 mL) and then filtered. The filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (EtOAc:MeOH=5:1˜2:1) to give product (120 mg). The product was further purified by prep-TLC (EtOAc:MeOH=3:1) to give product (57 mg) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.30 (s, 1H), 7.62 (s, 2H), 7.00 (s, 1H), 4.58-4.30 (m, 6H), 3.48-3.44 (m, 1H), 3.25-3.16 (m, 3H), 2.90-2.82 (m, 3H), 2.55 (s, 3H), 2.43 (s, 3H), 2.05 (t, J=6.8 Hz, 2H), 1.96-1.91 (m, 2H), 1.85-1.82 (m, 2H), 1.74-1.62 (m, 5H), 1.47-1.43 (m, 2H).
LC-MS [mobile phase: from 95% water (0.1% FA) and 5% ACN (0.1% FA) to 5% water (0.1% FA) and 95% ACN (0.1% FA) in 9 min]: Rt=4.54 min; MS Calcd: 515, MS Found: 516 [M+H]+.
To a mixture of 5-methyl-6-(1-(oxetan-3-yl)piperidin-4-yl)-1H-indazole (81 mg, 0.298 mmol), 7-(6-iodo-2-methylpyrimidin-4-yl)-4-oxa-7-azaspiro[2.5]octane (100 mg, 0.302 mmol), K3PO4 (127 mg, 0.60 mmol) and CuI (57 mg, 0.30 mmol) in toluene (2 ml) under Ar was added N,N′-dimethylethylenediamine (53 mg, 0.60 mmol). The reaction was stirred at 95° C. for 3 hours.
The reaction mixture was filtered and the filter cake was washed with CH2Cl2. The combined filtrate was concentrated and the residue was purified by column chromatography eluted with CH2Cl2/MeOH=15/1 afforded pure desired product as white solid (76 mg, yield: 53%).
LC-MS [mobile phase: from 80% water (0.1% TFA) and 20% ACN (0.1% TFA) to 5% water (0.1% TFA) and 95% ACN (0.1% TFA) in 10.0 min]: Rt=5.11 min; MS Calcd: 474, MS Found: 475 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.05 (s, 1H), 7.50 (s, 1H), 6.90 (s, 1H), 4.71 (d, J=6.8 Hz, 4H), 3.87 (t, J=4.4 Hz, 2H), 3.80 (d, J=4.4 Hz, 2H), 3.66 (s, 2H), 3.57 (t, J=6.4 Hz, 1H), 2.97 (d, J=10.4 Hz, 2H), 2.85-2.81 (m, 1H), 2.64 (s, 3H), 2.45 (s, 3H), 2.05-2.00 (m, 2H), 1.94 (br, 4H), 0.90-0.55 (m, 4H), 0.68 (m, 2H).
A mixture of 8-(6-iodo-2-methylpyrimidin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (119 mg, 0.32 mmol), 5-methyl-6-((tetrahydrofuran-3-yl)oxy)-1H-indazole (70 mg, 0.32 mmol), N,N′-dimethylcyclohexane-1,2-diamine (90 mg, 0.64 mmol), CuI (61 mg, 0.32 mmol) and K3PO4 (136 mg, 0.64 mmol) in toluene (3 mL) was stirred at 100° C. for 2 hours. The mixture was filtered and concentrated. The residue was purified by prep-HPLC (gilson-2 Kinete EVO C18, 5 μm 21.2×150 mm; 20-50% B, A: H2O (0.1% HCl), B: ACN, UV: 214 nm, flow rate 20 ml/min) to give the title compound (97 mg, 66%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 8.16 (s, 1H), 8.09 (s, 1H), 7.48 (s, 1H), 7.16 (s, 1H), 5.54 (br, 1H), 5.07 (br, 1H), 4.40-4.37 (m, 2H), 4.13-3.95 (m, 6H), 3.40 (t, J=6.4, 2H), 3.03 (s, 3H), 2.34-2.27 (m, 5H), 2.14-2.07 (m, 4H), 1.81-1.76 (m, 2H).
LC-MS [column: C18; column size: 4.6×50 mm; mobile phase: B (ACN): A (0.1% TFA); gradient (B %) in 6 min]: Rt=3.088 min, MS Calcd.: 462, MS Found: 463 [M+H]+.
To a mixture of 8-(2-methoxy-6-(5-methyl-6-(piperidin-4-yl)-1H-indazol-1-yl)pyrimidin-4-yl)-2,5-dioxa-8-azaspiro[3.5]nonane (139 mg, 0.31 mmol), oxetan-3-one (111 mg, 1.55 mmol), Na(CN)BH3 (39 mg, 0.62 mmol) in DCM (4 mL), was added AcOH (2 drops). The mixture was stirred at room temperature for 4 hours. The mixture was concentrated. The crude was purified by prep-HPLC (sunfire C18, 5 μm, 19×150 mm, 05-50% ACN-H2O (0.1% HCl), flow rate: 15 ml/min, GT 10 mins.) to give solution A, The solution A was washed with NaHCO3 (aq). The organic layer was concentrated to give the title compound (27 mg, 17%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.80 (s, 1H), 8.12 (s, 1H), 7.56 (s, 1H), 6.95 (s, 1H), 4.74-4.71 (M, 4H), 4.69 (d, J=7.2 Hz, 2H), 4.52 (d, J=6.8 Hz, 2H), 4.20 (s, 3H), 4.01 (s, 2H), 3.81-3.80 (m, 2H), 3.71 (t, J=5.2 Hz, 2H), 3.61-3.58 (m, 1H), 2.97 (d, J=11.2 Hz, 2H), 2.88 (t, J=3.6 Hz, 1H), 2.50 (s, 3H), 2.09-2.03 (m, 2H), 1.98-1.91 (m, 4H).
LC-MS [Phenomenex Kinetex 5 μm EVO C18, 50×4.6 mm; mobile phase: B (ACN): A (0.02% NH4OAc); gradient (B %) in 6 min]: Rt=3.795 min; MS Calcd.: 506, MS Found: 507 [M+H]+.
To a solution of tert-butyl 4-(1-(2-methoxy-6-(2,5-dioxa-8-azaspiro[3.5]nonan-8-yl)pyrimidin-4-yl)-5-methyl-1H-indazol-6-yl)piperidine-1-carboxylate (170 mg, 0.31 mmol) in TFA (1 mL) and DCM (4 mL) was stirred at room temperature for 5 hours. Sat. NaHCO3 was added to the reaction to adjust pH>7. The mixture was diluted with H2O (50 mL), extracted with EtOAc (30 mL×3). The combined organic layer was dried over Na2SO4, filtered and concentrated to give the compound (139 mg, 100%) as white solid.
As stated above, the compounds of the present invention are LRRK2 kinase inhibitors, and are useful in the treatment of diseases mediated by LRRK2. The biological activities of the compounds of the present invention can be determined using any suitable assay for determining the activity of a candidate compound as a LRRK2 kinase inhibitor, as well as tissue and in vivo models.
1) Full Length G2019 Human LRRK2 Inhibition Mass Spectrometry Assay
Insect Cell Cultures
Sf9 insect cells (Invitrogen Life Technologies, Carlsbad, Calif.) were maintained at 27° C. in SF 900 II SFM in 500-ml stationary flasks. The cells were maintained in exponential growth phase and subcultured twice per week. For larger volumes, cells were grown in 2-liter shaker flasks (Erlenmeyer, Corning) while being agitated with 120 rpm at 27° C. incubator shaker.
Human G2019 LRRK2 Plasmids Preparation
Primers used for PCR cloning:
The primers described above were used to amplify full length human LRRK2 (amino acids from 1-2527) with N terminal Flag tag from BioCat 116313 using high fidelity PCR polymerase KOD-Plus according to the manufacturer's instructions (KOD-211, TOYOBO). The PCR product was digested with BamHI and KpnI and ligated into BamHI and KpnI digested pHTBV1mcs3(BioCat 127555). The sequence of the target fragment was confirmed by DNA sequencing. The construct BioCat141351 was generated by Novoprotein.
The G2019 full length Flag-LRRK2 coding sequence is SEQ ID No: 8.
The translated protein sequence for human G2019 full length LRRK2 flag tagged protein is SEQ ID No: 9.
Generation of the BacMam Virus
To generate the recombinant BacMam virus, SF9 cells were seeded in 6-well tissue culture dishes at 9×105 cells/well and allowed to attach for 20 min. Transfection procedure was followed using AESOP AP5911v2. Briefly, DH10Bac competent cells (10361-012, Invitrogen) were transformed by the genotypically normal human LRRK2 BacMam plasmid to generate the recombinant baculovirus DNA. The Sf9 insect cells were co-transfected with the mixture of recombinant baculovirus DNA and cellfectin (10362-100, Invitrogen). After 4 h of incubation at 27° C., the transfection media was replaced with Sf-900 III SFM medium containing 5% HI FBS (Ser. No. 10/100,147, Invitrogen). The cells were further incubated for 4 days. The infected cell culture medium containing the baculovirus (PO virus stock) was collected and amplified by further infecting the Sf9 cells at multiplicity of infection (MOI) (pfu/cell) of 0.1.
Quantification of BacMam Viral Titre by BacPAKRapid Titer
The viral titre, measured as plaque forming unit (pfu)/ml was determined using BacPAK Papid Titer Kit (631406, Clontech) according to the manufacturer's protocol. The Sf9 cells seeded in 96-well plate with 3×105 cells per well were incubated with serial dilution of the viral stocks for 1 h at 27° C., 50 μl methyl cellulose overlay was added to each well followed by 43-47 h incubation. The cells were then fixed in 4% paraformaldehyde (PFA). After blocking the cells with diluted normal goat serum, Mouse anti-gp64 antibody was added to the cells. After 30 min incubation, the cells were washed with phosphate buffered saline containing 0.1% Triton-X100 (PBST) and incubated for another 30 min with goat anti-mouse antibody/HRP conjugate. This was followed by blue peroxidase substrate which detects the single infected cells and foci of infected cells by their dark blue color.
Protein Expression
Expression of Flag Tagged Full Length G2019 Human LRRK2
HEK293 6E cells (BioCat: 120363) were incubated in a 37° C. incubator with a humidified atmosphere of 5% CO2 on an orbital shaker rotating at 110 rpm. On the day of transduction, the cell viability was higher than 98% and the cell density was in the range of 1×106˜1.5×106 cells/ml.
HEK293 6E cells were centrifuged at 1,000 rpm for 10 min, and then the cells were resuspended in fresh Freestyle 293 expression medium(Invitrogen:12338) with 0.1% F-68(Invitrogen:24040-032) but without antibiotics(G418) at density of 1×106 cells/mi. BacMam virus with Flag-hu LRRK2 (genotypically normal) gene was centrifuged at 40,000 g for 2 hours, then resuspended in fresh Freestyle 293 expression medium. The resuspended virus was added into the cells in at MOI of 10. The cells were incubated in a 37° C. incubator with a humidified atmosphere of 5% CO2 in air on an orbital shaker rotating at 110 rpm. Cultures were harvested at approximately 48 hours post-transduction by centrifugation at 4,000 rpm for 20 min and pellets were frozen for purification.
Protein Purification
Purification of Flag Tagged Full Length G2019 Human LRRK2
The cell pellet was resuspended in 20 mL lysis buffer (50 mM TrisHCl pH7.5 at 4° C., 500 mM NaCl, 0.5 mM EDTA, 0.1% TritonX-100, 10% glycerol, freshly add 2 mM DTT), with protease inhibitors (Roche: 04693132001) and benzonase(Merck Millipore: 70746-3CN) at recommended concentration suggested by suppliers. The suspended cells were lysed by sonication on ice for 30 min (2 secs on/4 sec off, 20% amplitude), and centrifuged at 10,000 rpm for 30 minutes at 4° C. The supernatant was incubated with 1 mL per litre of cell culture of anti-Flag magnetic beads (Sigma-Aldrich: M8823) at 4° C. for 3 hours, then the beads were washed by 5 mL per litre of cell culture binding buffer (50 mM Tris pH7.5@ 4C, 500 mM NaCl, 0.5 mM EDTA, 0.1% TritonX-100, 10% glycerol, freshly add 2 mM DTT) for three times. The Flag tagged LRRK2 proteins were eluted by Elution buffer (50 mM Tris pH7.5@ 4C, 500 mM NaCl, 0.5 mM EDTA, 0.1% TritonX-100, 10% glycerol, freshly add 2 mM DTT, 250 ug/ml Flag peptide (Sigma-Aldrich:F3290)) at 4° C. for 2 hours. Flag peptide was removed by Zeba Spin Desalting Columns, 7K MWCO(Thermo-Fisher: 89893) and the buffer of eluted LRRK2 proteins was exchanged into Storage Buffer (50 mM Tris pH7.5@4C, 150 mM NaCl, 0.5 mM EDTA, 0.02% Triton X-100, 2 mM DTT and 50% Glycerol) using Amicon Ultra Centrifugal Filter Units (100 kD) (Merck: UFC910096). Fractions containing LRRK2 proteins were pooled, aliquoted and stored at −80° C. Protein concentration was determined by Bradford protein assay, and protein purity was analyzed by NuPAG Novex 4-12% Bis-Tris Protein Gels (Invitrogen: NP0322BOX).
Full length G2019 human LRRK2 Inhibition Mass Spectrometry Assay
This assay for Leucine Rich Repeat Kinase 2 (LRRK2) inhibition is based on the direct measurement of the peptide ‘LRRKtide’ (LRRKtide: RLGRDKYKT*LRQIRQ and “*” refers to the site of phosphorylation.) and phosphorylated ‘LRRKtide’ using a high throughput RapidFire mass spectrometry assay. Inhibitors are defined as compounds which reduce the conversion of LRRKtide to phospho-LRRKtide.
Assay Protocol
RapidFire Settings:
2) Recombinant Cellular LRRK2 AlphaScreen Assay
To determine the activity of compounds against LRRK2 kinase activity in cells, the observed LRRK2 kinase-dependent modulation of LRRK2 Ser 935 phosphorylation (Dzamko et al., 2010, Biochem. J. 430: 405-413) was utilized to develop a quantitative 384 well plate-based immunoassay of LRRK2 Ser935 phosphorylation in the human neuroblastoma cell line SH-SY5Y, engineered to over-express recombinant full length LRRK2 protein.
A BacMam virus expressing full length recombinant LRRK2 was purchased from Invitrogen and amplified by inoculation of SF-9 cells at MOI 0.3 for 4-5 days in Sf-900 III SFM medium supplemented with 3% fetal bovine serum. Infected cell cultures were then centrifuged at 2000 g for 20 minutes, viral supernatant titer determined by anti-gp64 plaque assay and stored at 4° C.
Affinity-purified anti-phospho LRRK2 Ser935 sheep polyclonal antibody (Dzamko et al., 2010, Biochem. J. 430: 405-413) was biotinylated by standard methods (PerkinElmer). Anti-LRRK2 rabbit polyclonal antibody was purchased from Novus Biologicals. AlphaScreen Protein A IgG Kit (including acceptor and donor beads) was purchased from Perkin Elmer.
SH-SY5Y cells were grown in DMEM/F12 medium with 10% dialysed fetal bovine serum and harvested by treatment with 0.5% trypsin-EDTA for 5 minutes at 37° C. followed by centrifugation at 1000 rpm for 4 minutes. The cell pellet was resuspended in Opti-MEM reduced serum media (Invitrogen) at 200,000 cells/ml and mixed with the BacMam LRRK2 virus at MOI=50. 50 μl cell solutions were then dispensed to each well of a 384-well plate and incubated at 37° C., 5% CO2 for 24 hours.
Serial dilutions of test compounds were prepared in Opti-MEM reduced serum media (Invitrogen) and 5.6 ul transferred from compound plate to cell assay plate to achieve a top final assay concentration of 10 uM. DMSO was used in certain wells as controls. Cells were incubated at 37° C., 5% CO2 for 60 minutes. The medium was then removed and cells lysed by addition of 20 ul cell lysis buffer (Cell Signaling Technology) and incubation at 4° C. for 20 minutes. 10 ul of antibody/acceptor bead mix [(1/1000 biotinylated-pS935 LRRK2 antibody, 1/1000 total-LRRK2 antibody, 1/100 Acceptor beads in AlphaScreen detection buffer (25 mM Hepes (pH 7.4), 0.5% Triton X-100, 1 mg/ml Dextran 500 and 0.1% BSA)] was then added to each well and plates incubated for 2 hours at ambient temperature in the dark. 10 μl of donor beads solution (1/33.3 donor beads in AlphaScreen detection buffer) was then added to each well. Following incubation for a further 2 hours at ambient temperature in the dark, plates were read on an EnVision™ plate reader at emission 520-620 nm with excitation 680 nm. Dose response curve data was based on sigmoidal dose-response model.
Pharmacological Data
Compounds of Examples A-1-A-13 were tested in the recombinant cellular LRRK2 AlphaScreen assay and exhibited a pIC50>6. Compounds of Examples A-1-A-4 and A-13 exhibited a pIC50≥7 in the recombinant cellular LRRK2 AlphaScreen assay. The compound of example A-13 exhibited a pIC50 of 8.1 in the recombinant cellular LRRK2 AlphaScreen assay.
Compounds of Examples A-1-A-7 were additionally tested in the full length G2019 human LRRK2 Inhibition Mass Spectrometry assay and exhibited a pIC50≥6.5.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/072589 | Jan 2017 | WO | international |
| PCT/CN2017/072614 | Jan 2017 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/073833 | 1/23/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/137618 | 8/2/2018 | WO | A |
| Number | Date | Country |
|---|---|---|
| 105980388 | Sep 2016 | CN |
| WO 2014134772 | Sep 2014 | WO |
| WO 2014134774 | Sep 2014 | WO |
| WO 2016036586 | Mar 2016 | WO |
| WO 2017012576 | Jan 2017 | WO |
| Entry |
|---|
| Layzer, Degenerative Diseases of the Nervous System, Cecil Textbook of Medicine, 20th Edition, vol. 2, pp. 2050-2057 (1996). |
| Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., 1983, New York, p. 4. |
| Dermeret al., Bio/Technology, 1994, 12:320. |
| Number | Date | Country | |
|---|---|---|---|
| 20190359618 A1 | Nov 2019 | US |
