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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
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  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/46—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino or carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
  • C07C229/50—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino or carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups and carboxyl groups bound to carbon atoms being part of the same condensed ring system
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  • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
  • C07C49/753—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups
  • C07C49/755—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups a keto group being part of a condensed ring system with two or three rings, at least one ring being a six-membered aromatic ring
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  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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Definitions

• the present invention relates to spirocyclic acylguanidines and their use as inhibitors of the ⁇ -secretase enzyme (BACE1) activity, pharmaceutical compositions containing the same, and methods of using the same as therapeutic agents in the treatment of neurodegenerative disorders, disorders characterized by cognitive decline, cognitive impairment, dementia and diseases characterized by production of ⁇ -amyloid deposits or neurofibrillary tangles.
• BACE1 ⁇ -secretase enzyme
• AD Alzheimer's disease
• APP amyloid precursor protein
• APP amyloid precursor protein
• BACE activity is correlated directly to the generation of ⁇ peptide from APP, and studies increasingly indicate that the inhibition of BACE inhibits the production of ⁇ peptide.
• Amyloidogenic plaques and vascular amyloid angiopathy also characterize the brains of patients with Trisomy 21 (Down Syndrome), Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-type (HCHWA-D), and other neurodegenerative disorders. Neurofibrillary tangles also occur in other neurodegenerative disorders including dementia-inducing disorders.
• WO 2010/021680, WO2011/106414 and WO 2010/105179 disclose spirocyclic acylguanidines with a spirocyclic scaffold as inhibitors of beta-secretase.
• the present invention provides compounds that are BACEl inhibitors and are useful as therapeutic agents in the treatment of a disease or disorder characterized by elevated ⁇ -amyloid deposits or ⁇ -amyloid levels in a patient.
• the disclosed BACEl inhibitors are not only highly potent inhibitors of the BACEl enzyme (assay 1) but also show:
• the present invention provides compounds which show a combination of high potency as BACEl inhibitors, high selectivity against the cardiac hERG channel, and low phospholipidosis activity.
• Another embodiment of the invention is a compound of the present invention or a pharmaceutically acceptable salt thereof for use as a medicament.
• Another embodiment of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
• Another embodiment of the invention is a compound of the present invention or a pharmaceutically acceptable salt thereof for use in the treatment of a BACEl mediated disorder or disease in a subject.
• Another embodiment of the invention is the use of a compound of the present invention or a
• Another embodiment of the invention is a pharmaceutical composition for treatment of a BACEl mediated disorder or disease in a subject comprising a compound of the present invention.
• Another embodiment of the invention is a method of treating a subject with a BACEl mediated disease or disorder, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.
• the compounds of the present invention exhibit potent activity against the BACEl enzyme and Abeta formation together with the selectivity against the hERG channel and the low propensity to cause phospholipidosis.
• the compounds of the present invention show a BACEl inhibition with an IC 50 ⁇ 15 nM, a cellular Abeta production inhibition with an IC 50 of ⁇ 2 nM, a hERG inhibition of ⁇ 50 at 10 ⁇ , and phospholipidosis with a First Effect Concentration (FEC) of > 150 ⁇ .
• FEC First Effect Concentration
• hERG human Ether-a-go-go-Related Gene
• xenobiotics and subsequent delayed cardiac repolarization are associated with an increased risk for a specific polymorphic ventricular tachyarrhythmia, torsade de pointes, as established by Sanguinetti et al. (1995, Cell, Apr. 21 , 81(2):299- 307) and a large body of subsequent evidence.
• Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Drug- induced phospholipidosis is an undesirable drug reaction. Therefore, in order to avoid detrimental side effects, compounds with low phospholipidosis potential are preferred for human therapeutic use.
• WO 2010/105179 do not have this combination of desirable properties.
• Table 2 provides data showing that certain compounds of the present invention have significantly lower IC 50 inhibitory values in a BACE1 enzymatic assay and also in a cellular Abeta assay relative to certain comparator compounds described in WO 2010/105179.
• the compound and its pharmaceutically acceptable salts shall encompass all stereo, optical and geometrical isomers (e.g., enantiomers, diastereomers, E/Z isomers, etc.) and racemates thereof, as well as mixtures in different proportions of the separate enantiomers, mixtures of diastereomers, or mixtures of any of the foregoing forms.
• stereo, optical or geometric isomer When a stereo, optical or geometric isomer is depicted by name or structure, it is to be understood that the stereo, optical and/or geometric isomeric purity of the named or depicted stereo, optical or geometric isomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight. Stereo, optical and geometric isomeric purity is determined by dividing the weight of the named or depicted stereo, optical and geometric isomer in a mixture by the total weight of all stereo, optical and geometric isomers in the mixture.
• solvates refer to crystalline forms wherein solvent molecules are incorporated into the crystal lattice during crystallization.
• Solvate may include water or nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and EtOAc.
• Solvates wherein water is the solvent molecule incorporated into the crystal lattice, are typically referred to as "hydrates.” Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. "Anhydrous form” refers to compounds with no solvent or water or substantially no solvent or water incorporated into the crystal structure (e.g., less than 1 : 10, 1 :20; 1 : 100; or 1 :200 molar ratio of solvent or water to compound).
• phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio.
• pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of
• salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
• such salts include salts from ammonia, L-arginine, betaine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine (2,2' -iminobis(ethanol)), diethylamine, 2- (diethylamino)-ethanol, 2-aminoethanol, ethylenediamine, N-ethyl-glucamine, hydrabamine, 1H- imidazole, lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, l-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide, triethanolamine (2,2' ,2"- nitrilotris(ethanol)), tromethamine,
• the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.
• the inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE1 activity using commercially available substrate HiLyte FluorTM488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys- (QXLTM 520)-OH (SEQ ID NO: l) AnaSpec, San Jose, CA) and truncated human beta-secretase, BACE1 (amino acids 1-454) fused to a myc-his tag and secreted from ⁇ 293/ ⁇ ⁇ . cells into OptiMEMTM (Invitrogen). The substrate was dissolved at 1 mg/ml in DMSO.
• the assay was performed in the presence of OptiMEM (supernatant collected over 24 h and cleared from cellular debris by centrifugation) containing the ectodomain of BACE1, 25 ⁇ water containing the desired 2-fold concentration of test compound and 2% DMSO, 1 ⁇ substrate peptide, 20 mM NaOAc, pH 4.4, and 0.04% Triton-X100 in a total assay volume of 50 ⁇ in a 384 well plate.
• 25 ⁇ of compound dilution were given to the plate followed by the addition of 10 ⁇ of BACE1 containing
• OptiMEMTM diluted 1: 10 in water with 0.2% Triton X-100 The reaction was started with the addition of 15 ⁇ substrate in NaOAc buffer. The reaction was incubated at rt (dark) in an Envision ® multilabel reader (Perkin Elmer) and the cleavage of the substrate was recorded as kinetic for 60 min at ex: 485 nm, em: 538 nm. Blank wells containing no enzyme were included on each plate.
• Cellular potency of the compounds of the invention was assessed in an assay monitoring production of Abetal-x peptides in the H4 neuroglioma cell line (ATCC, Cat. #HTB-148) stably expressing human APP, using an immunoassay such as AlphaLISA (PerkinElmer, Cat.# AL288).
• Tested compounds were dissolved in DMSO and pre -diluted in the culture medium (DMEM containing 10% FBS and 1% penicillin/streptomycin) to achieve twice the final concentration of the compounds in the assay. Equal volumes of the 2x solutions of the tested compounds and cell suspension were added to a 96-well culture plate, so that each well contained -10,000 cells in a final volume of 200 ⁇ .
• HEK (human embryonic kidney) 293 cells were stably transfected with hERG cDNA.
• Membrane currents were recorded using an EPC-10 patch clamp amplifier and PatchMaster software. hERG-mediated membrane currents were recorded at 35 °C, using the whole-cell configuration of the patch-clamp technique. Transfected HEK293 cells were clamped at a holding potential of -60 mV and hERG-mediated inactivating tail currents were elicited using a pulse pattern with fixed amplitudes
• Compound preparation and application The different concentrations of the test compound were applied sequentially on each of the different cells investigated. A steady state level of baseline current was measured for at least 6 sweeps prior to the application of the first test compound concentration.
• test compound was dissolved in DMSO to yield a master stock solution which was diluted further in DMSO to stock solutions needed for the lower concentrations.
• Final dilutions in extracellular buffer were prepared freshly from these stocks by a 1 : 1000 dilution step each before starting the experiments.
• Peak current amplitudes were measured 3 ms after the ramp to +40 mV. For baseline and each concentration the peak currents of the three last sweeps before application of the next concentration were averaged. Residual currents (I I 0 ) were calculated for each cell as the fraction of actual average peak current and average baseline peak current. Results are presented as percent ( ) inhibition (l-I/Io)*100 at 10 ⁇ .
• test compounds The phospholipidogenic potential of test compounds was assayed using the human hematopoetic U937 cell line.
• the test principle was to analyze the phospholipid content by staining the cells with the fluorescent dye Nile red.
• U937 cells were seeded into cell culture plates at 0.5 x 10 6 cells/mL in RPMI medium containing 10 % FBS, 1 % DMSO, and 0.005 % gentamicin. The cells were then cultivated with or without different concentrations of test compounds for 48 h under standard culture conditions.
• the cells were centrifuged at 130x g for 4 min and washed once with PBS. Then 2x 0.5 mL cell suspension was prepared for non-fixed cell measurement (0.5 mL for propidium iodide (PI) viability measurement and 0.5 mL for Nile red measurement).
• PI propidium iodide
• Nile red staining of the 0.5 mL non-fixed cell samples 50 of a ready to use Nile red solution (10 ⁇ g/mL) were added per sample. Samples were kept on ice for 5 min. Thereafter, they were washed once with 4 mL PBS (4°C, 250x g for 8 min) and finally resuspended in 400 ⁇ PBS and kept for flow cytometry measurement.
• each Nile red sample was measured by flow cytometry at channel 1 (504-541 nm) and channel 4 (660-680 nm).
• the cerebrum was homogenized (5 volumes per weight) in 20 mM Tris-HCL, pH 8.5, 0.2% Triton-XlOO supplemented with protease inhibitors (cOmplete, Roche Applied Science) at 4 ° C using a glass Dounce homogenizer. The homogenate was centrifuged at 120,000xg for 60 min at 4 ° C, and the supernatant was collected and analyzed for Abl-x using immunoassay with chemiluminescent detection (Meso-Scale Discovery, Rockville, MD (MSD)).
• the wells were washed 4 times with PBS, and 25 ⁇ of the detection antibody (Sulfo- TAG labeled 3 ⁇ - ⁇ 40 antibody supplied by MSD) was added and incubated for 1 hr at rt. After 4 washes with PBS, 150 ⁇ of the chemiluminescence detection reagent (Read Buffer T, MSD) was added, and the plate was read on an MSD Sector Imager 6000 instrument. The calibration curve was fit into a non-linear four-parameter regression model, and the ⁇ - ⁇ concentrations were calculated for each well containing the cleared brain lysates. The percent of ⁇ lowering was calculated based on the difference with the average ⁇ concentration obtained for the brains from the animals treated with vehicle alone.
• the detection antibody Sulfo- TAG labeled 3 ⁇ - ⁇ 40 antibody supplied by MSD
• Table 1 shows the following properties of the compounds of the present invention: BACEl inhibitory potency as measured in assay 1, cellular inhibitory potency as measured in assay 2, hERG inhibition as measured in assay 3, and first effect concentration (FEC) of phospholipidosis as measured in assay 4.
• BACEl inhibitory potency as measured in assay 1
• cellular inhibitory potency as measured in assay 2
• hERG inhibition as measured in assay 3
• FEC first effect concentration
• Table 2 provides data showing that certain compounds of the present invention have significantly lower IC 50 inhibitory values in the BACEl enzymatic assay (Assay 1) and in the cellular Abeta assay (Assay 2) relative to certain comparator compounds described in WO 2010/105179.
• Example 5 Example 14 Example 15 example 281 in
• Example 8 Example 11 example 121 in
• Example 12 Example 18 example 320 in
• the present invention is directed to compounds which are useful in the treatment of disorders or diseases characterized by elevated ⁇ -amyloid deposits or ⁇ -amyloid levels in a subject wherein the inhibition of the activity of the ⁇ -secretase enzyme (BACE1) is of therapeutic benefit, including but not limited to the treatment, amelioration or prevention of neurodegenerative disorders, disorders characterized by cognitive decline, cognitive impairment, dementia, and diseases characterized by production of ⁇ -amyloid deposits or neurofibrillary tangles.
• BACE1 ⁇ -secretase enzyme
• Compounds of the present invention are useful for treatment of Alzheimer's disease, Trisomy 21 (Down Syndrome), Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-type (HCHWA-D), senile dementia, cerebral amyloid angiopathy, degenerative dementia, dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy, dementia associated with cortical basal degeneration, diffuse Lewy body type of Alzheimer's disease, dry age related macular degeneration (AMD), and glaucoma.
• HHWA-D Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-type
• senile dementia cerebral amyloid angiopathy
• degenerative dementia dementias of mixed vascular and degenerative origin
• dementia associated with Parkinson's disease dementia associated with progressive supranuclear palsy
• dementia associated with cortical basal degeneration dementia associated with cortical basal degeneration
• the "dry” form of AMD also known as "central geographic atrophy” results from atrophy to the retinal pigment epithelial layer below the neurosensory retina, which causes vision loss through loss of photoreceptors (rods and cones) in the central part of the eye.
• No medical or surgical treatment is currently available for this condition.
• Treatments available so far include the use of vitamin supplements with high doses of antioxidants, lutein and zeaxanthin, which may slow the progression of dry macular degeneration.
• Glaucoma is a disease where fluid pressure inside the eye increases, causing irreversible damage to the optic nerve and loss of vision. Abeta colocalizes with apoptotic retinal ganglion cells in experimental glaucoma and induces significant retinal ganglion cell apoptosis in a dose- and time-dependent manner.
• the present invention relates to a compound or a pharmaceutically acceptable salt thereof for use as a medicament.
• the present invention relates to the use of a compound in the treatment of a disease and/or condition wherein the inhibition of the activity of the ⁇ -secretase enzyme (BACE1) is of therapeutic benefit.
• BACE1 ⁇ -secretase enzyme
• the present invention relates to the use of a compound in the treatment of neurodegenerative disorders, disorders characterized by cognitive decline, cognitive impairment, dementia, and diseases characterized by production of ⁇ -amyloid deposits or neurofibrillary tangles.
• the present invention relates to the use of a compound of the present invention in the treatment of Alzheimer's disease, Trisomy 21 (Down Syndrome), Hereditary Cerebral Hemorrhage with
• HCVWA-D Dutch-type
• senile dementia cerebral amyloid angiopathy
• degenerative dementia dementias of mixed vascular and degenerative origin
• dementia associated with Parkinson's disease dementia associated with progressive supranuclear palsy
• dementia associated with cortical basal degeneration dementia associated with cortical basal degeneration
• diffuse Lewy body type of Alzheimer's disease dry AMD, and glaucoma.
• the present invention also provides a method for the treatment of a disorder related to or associated with excessive BACE1 activity in a patient in need thereof which comprises administering to said patient an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.
• the present invention also provides methods for inhibiting the activity of BACE1 in a subject in need thereof, comprising administering to a subject and/or contacting a receptor thereof with an effective amount of at least one disclosed compound or a pharmaceutically acceptable salt thereof.
• the present invention also provides methods of ameliorating ⁇ -amyloid deposits in a subject in need thereof, comprising
• the invention includes a therapeutic method for treating or ameliorating a BACE1 mediated disorder in a subject in need thereof comprising administering to a subject in need thereof an effective amount of a compound of the invention described herein, or pharmaceutically acceptable salts thereof or composition thereof.
• the term "subject” and “patient” may be used interchangeably, and means a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
• companion animals e.g., dogs, cats, and the like
• farm animals e.g., cows, pigs, horses, sheep, goats and the like
• laboratory animals e.g., rats, mice, guinea pigs and the like.
• the subject is a human in need of treatment.
• the term "treating" or 'treatment” refers to obtaining desired pharmacological and/or physiological effect.
• the effect can be prophylactic (i.e., reducing the likelihood of developing the disorder or disease) or therapeutic, which includes achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, disorder or syndrome;
• the dose range of the compounds according to the present invention applicable per day is usually from 0.1 to 3000 mg, preferably from 1 to 2000 mg , more preferably from 10 to 1000 mg, most preferably, 50 or 500 mg.
• Each dosage unit may conveniently contain from 0.1 to 1000 mg, preferably 25 to 250 mg.
• the actual pharmaceutically effective amount or therapeutic dosage will of course depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease. In any case, the combination will be administered at dosages and in a manner which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.
• Suitable preparations for administering the compounds of the present invention will be apparent to those with ordinary skill in the art and include for example tablets, pills, capsules, suppositories, lozenges, troches, solutions, syrups, elixirs, sachets, injectables, inhalatives and powders, etc.
• the content of the pharmaceutically active compound(s) should be in the range from 0.1 to 95 wt.- , preferably 5 to 90 wt.- % of the composition as a whole.
• Suitable tablets may be obtained, for example, by mixing one or more compounds of the invention with known excipients, for example inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and/or lubricants.
• excipients for example inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and/or lubricants.
• the tablets may also consist of several layers.
• the present invention includes combination therapy for treating or ameliorating a disease or a disorder described herein.
• the combination therapy comprises administering a combination of at least one compound of the present invention with one or more agent selected from the group of, for example, gamma-secretase inhibitors or modulators; amyloid aggregation inhibitors blocking the formation of Abeta oligomers or Abeta fibrils (e.g., ELND-005); directly or indirectly acting
• anti-oxidants e.g., vitamin E or ginkolide
• antiinflammatory substances e.g., Cox inhibitors, NSAIDs additionally or exclusively having Abeta lowering properties
• HMG-CoA reductase inhibitors statins
• acetylcholinesterase inhibitors e.g., donepezil, rivastigmine, tacrine, galantamine,; tacrine
• NMDA receptor antagonists e.g., memantine
• AMPA receptor agonists e.g., AMPA receptor positive modulators, AMPAkines, monoamine receptor reuptake inhibitors, substances modulating the concentration or release of neurotransmitters
• substances inducing the secretion of growth hormone e.g., ibutamoren mesylate and capromorelin
• antibiotics e.g., minocyclin or rifampicin
• PDE2 PDE4, PDE5, PDE
• histamine H3 antagonists 5 HT-4 agonists or partial agonists, 5HT-6 antagonists, alpha2-adrenoreceptor antagonists, calcium antagonists, muscarinic receptor Ml agonists or partial agonists or positive modulators, muscarinic receptor M2 antagonists, muscarinic receptor M4 antagonists, metabotropic glutamate -receptor 5 positive modulators, antidepressants, such as citalopram, fluoxetine, paroxetine, sertraline and trazodone; anxiolytics, such as lorazepam and oxazepam; antiphychotics, such as aripiprazole, clozapine, haloperidol, olanzapine, quetiapine, risperidone and ziprasidone, and other substances that modulate receptors or enzymes in a manner such that the efficacy and/or safety of the compounds according to the invention is increased and/or unwanted side effects are reduced.
• the compounds according to the invention may also be used in combination with immunotherapies (e.g., active immunisation with Abeta or parts thereof or passive immunisation with humanised anti-Abeta antibodies or nanobodies) for the treatment of the above-mentioned diseases and conditions.
• immunotherapies e.g., active immunisation with Abeta or parts thereof or passive immunisation with humanised anti-Abeta antibodies or nanobodies
• Combination therapy includes co-administration of the compound of the invention with one or more other agent, sequential administration of the compound and one or more other agent, administration of a composition containing a compound and one or more other agent, or simultaneous administration of separate compositions containing the compound and one or more other agent.
• Microwave reactions were carried out in CEM reactor using discovery SP system.
• Mobile phase A water with 0.05% ammonia solution
• Mobile phase B ACN
• Flow rate 25 mL/min
• Detection UV 220 nm / 254 nm
• Mobile phase A water with 0.05% ammonia solution
• Mobile phase B ACN
• Flow rate 25 mL/min
• Detection UV 220 nm / 254 nm
• Column Durashell CI 8 250*30mm*5um; Column temperature: 30°C
• Mass Spectrometer Waters SQD; Ionization: Positive Electrospray Ionization (ESI); Mode Scan (100-1400 m z in every 0.2 second); ES Capillary Voltage:3.5 kv; ES Cone Voltage: 25 v Source Temperature : 120 °C; Disolvation Temperature: 500 °C; Desolvation Gas Flow: Nitrogen Setting 650 (L/h); Cone Gas Flow: Nitrogen Setting 50 (L/h)
• ESI Positive Electrospray Ionization
• Mode Scan 100-1400 m z in every 0.2 second
• ES Capillary Voltage 3.5 kv
• ES Cone Voltage 25 v
• Source Temperature 120 °C
• Disolvation Temperature 500 °C
• Desolvation Gas Flow Nitrogen Setting 650 (L/h)
• Cone Gas Flow Nitrogen Setting 50 (L/h)
• Example 27 the following chromatographic conditions and instrumentation were used:
• Example 3 was synthesized from intermediate 11B from Example 1 following the same procedure as in Example 1 and utilizing intermediate 18 in step 9 of Example 1.
• Example 4 was synthesized from intermediate 11B following the same procedure as described for Example 1 utilizing intermediate 25 instead of 2-pyrimidylmethanamine in step 9.
• reaction mixture was quenched by addition of saturated aqueous NH 4 C1 (100 mL) and H 2 0 (200 mL) at -78 °C with stirring. The mixture was slowly allowed to warm to ambient temperature. The mixture was extracted with EtOAc (3 x 150 mL). The combined organic layers were washed with H 2 0 (2 x 200 mL), brine (2 x 200 mL), dried, filtered and concentrated under vacuum, the residue was purified by column chromatography on silica gel eluting with petroleum ether: EtOAc (20: 1 to 3: 1) to give intermediate 28 (7.5 g, 75%).
• Step 2 Synthesis of intermediate 30A & 30B
• Example 8 This was synthesized by a procedure similar as described in Example 6. (3-methyloxetan-3- yl)methanamine was used as described in Example 1 in step 9 followed by oxidation as described in step 10 to yield Example 8.
• Example 9 This was synthesized by a procedure similar as described in Example 6. 4-(aminomethyl)pyrimidine was used as described in Example 1 in step 9, followed by oxidation as described in step 10 in Example 6 to yield Example 9.
• reaction mixture was stirred at about -10 °C for 30 min, quenched with 6N aq HC1 solution (10 mL), stirred at about 0 °C for 30 min, and then allowed to warm to room temperature.
• the mixture was concentrated to remove THF, and toluene (60 mL) was added.
• the aqueous layer was removed, and the organic phase was washed with water (3 x 60 mL).
• the organic phase was concentrated to 1 ⁇ 2 volume, heated to 50 °C to obtain a solution, and then cooled to 0 °C over 1 h and held at 0 °C for 1 h.
• the solid was filtered and washed with cold (0 °C) toluene (12 mL), and dried under vacuum to give compound 33 (9.93 g, 83%).
• Example 12 was synthesized as per procedure described Example 10. In step 9, 2-aminooxetane was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 12
• Example 13 was synthesized as per procedure described Example 10. In step 9, oxetan-3-ylmethanamine was used instead of (5-fluorpyriminde)-2methylamine to yield Example 13.
• Example 14 was synthesized as per procedure described Example 10. In step 9, (5)-2-(aminomethyl)- tetrahydrofuran was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 14.
• LC-MS: t R 1.02 min, MS (ESI) m/z 437 [M+H] + .
• Example 15 was synthesized as per procedure described in Example 10. In step 9, (R)-2-(aminomethyl)- tetrahydrofuran was used instead of (5-fluoropyrimidine)-2-methylamine to yield example 15.
• LC-MS: t R 1.02 min, MS (ESI) m/z 437 [M+H] + .
• Example 16 was synthesized as per procedure described in Example 10. In step 9, 2-(aminomethyl)- tetrahydropyran was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 16
• Example 17 was synthesized as per procedure described in Example 10. In step 9, intermediate 18 was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 17.
• Example 19 was synthesized as per procedure described in Example 10. In step 9, intermediate 45 was used to yield Example 19.
• Example 20 (12.8 mg, 25%) as a white solid.
• Example 21 was synthesized as per procedure described in Example 10. In step 9, (R)-(l,4-dioxan-2- yl)methanamine was used to yield Example 21.
• Example 22 was synthesized as per procedure described in Example 10. In step 9, (S)-(l,4-dioxan-2- yl)methanamine was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 22.
• Example 23 was synthesized as per procedure described in Example 10. In step 9, intermediate 25 was used to yield Example 23.
• Example 24 was synthesized as per procedure described in Example 10. In step 9, 3-pyrimidyl- methanamine was used instead of (5-fluoropyrimidine)-2-methylamine to yield Example 24.
• Example 25 was synthesized in a method similar to Example 18. In step 9, (tetrahydrofuran-3- yl)methanamine was utilized and the two diastereomers were separated by SFC-Method B. Further elaboration of intermediate arising out of the second peak from the SFC-Method B yielded Example 26.
• LCMS: tR 0.845 min; m/z 430.3[M+H] + .
• Example 26 was synthesized in a method similar to Example 1. In the synthesis of Example 28, in step 1 of Example 1, methyl methacrylate was used instead of methyl acrylate. In step 3, the corresponding polar isomer 6B was isolated and further elaborated as described in Example 1. In step 9, intermediate 17 was utilized to yield Example 28.
• Example 27 was synthesized by a method as described in Example 6. (l,4-dioxan-2-yl)methanamine was used in step 9 followed by oxidation as described in step 10 to yield Example 27.
• Example 28 was synthesized from intermediate 11B from example 1 following the same procedure as in example 1 and utilizing-5-2-(aminomethyl) dioxane in step 9 of example 1.
• Example 29 was synthesized in a method similar to Example 20.
• LC-MS t R 1.02 min; MS (ESI) m/z 446 [M+H] + .

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