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    • 专利摘要:
    the present invention provides a compound of formula (i), or a pharmaceutically acceptable salt thereof, and the use of compounds of formula (i) for the treatment of neurodegenerative diseases and disorders, such as alzheimer's disease. formula i
    公开号:BR112019013535A2
    申请号:R112019013535-9
    申请日:2018-01-19
    公开日:2020-01-07
    发明作者:Jacques Francois Dreyfus Nicolas；James LINDSAY-SCOTT Peter
    申请人:Eli Lilly And Company；
    IPC主号:
    专利说明:

    Invention Patent Specification Report for N-4FLUOR-5 - [[((2S, 4S) -2-METHYL-4 - [(5-METHYL-1,2,4-OXADIAZOL-3YL) METHYXY] -1 -PIPERIDIL] METHIL] TI AZOL-2-IL] ACETAMIDE AS
    OGA INHIBITOR.
    [001] The present invention relates to new 5-methyl-1,2,4-oxadiazol-3-yl compounds, to pharmaceutical compositions comprising the compounds, to methods of using the compounds to treat physiological disorders and to useful intermediates and processes in the synthesis of compounds.
    [002] The present invention is in the field of the treatment of Alzheimer's disease, progressive supranuclear palsy (PSP) and other diseases and disorders involving tau-mediated neurodegeneration, collectively known as tauopathies.
    [003] Alzheimer's disease is a devastating neurodegenerative disorder that affects millions of patients worldwide. In view of the agents currently approved on the market that provide only transient and symptomatic benefits to the patient, there is a significant unmet need in the treatment of Alzheimer's disease.
    [004] The oligomerization of the tau protein associated with microtubules in filamentary structures, such as the paired helical filaments (PHFs) and the straight or twisted filaments, which give rise to neurofibrillary tangles (NFTs) and the filaments of neuropiles (NTs), is one of the defining pathological features of Alzheimer's disease and other tauopathies. The number of NFTs in the brains of individuals with Alzheimer's disease has been found to correlate closely with the severity of the disease, suggesting that tau plays a key role in neuronal dysfunction and neurodegeneration (Nelson et al., J Neuropathol Exp Neurol., 71 (5), 362-381 (2012)). The pathology of tau has been shown to correlate with
    Petition 870190060631, of 06/28/2019, p. 16/55
    2/32 disease duration in PSP; cases with a more aggressive course of disease have a higher tau load than cases with a slower progression. (Williams et al., Brain, 130, 1566-76 (2007)).
    [005] Recent studies (Yuzwa et al., Nat Chem Biol, 4 (8), 483490 (2008)) support the therapeutic potential of OGlcNAcase inhibitors (OGA) to limit hyperphosphorylation and the aggregation of tau in pathological tau treatment of Alzheimer's disease and related tau-mediated neurodegeneration disorders. Specifically, the OGA inhibitor Thiamet-G has been linked to delayed loss of motor neurons in the JNPL3 mouse tau model (Yuzwa et al., NatChem Biol, 8, 393-399 (2012)) and to a reduction in pathology of tau and dystrophic neurites in the Tg4510 tau mouse model (Graham et al., Neuropharmacology, 79, 307-313 (2014)). Consequently, OGA inhibitors are recognized as a valid therapeutic approach to reduce the accumulation of hyperphosphorylated pathological forms of tau, such as NFTs and NTs.
    [006] US Patent No. 9,120,781 discloses hexahydrobenzo-oxazole and hexahydrobenzothiazole derivatives that have OGA inhibitory activity and are further disclosed as useful in the treatment of diseases and disorders related to OGA deficiency or overexpression , and / or the accumulation or deficiency of 2acetamido-2-deoxy-58-D-glycopyranoside (O-GIcNAc). In addition, US 2016/0031871 discloses certain glycosidase inhibitors for the treatment of Alzheimer's disease.
    [007] OGA inhibitors that are penetrating into the brain are desired to provide treatments for tau-mediated neurodegeneration disorders, such as Alzheimer's disease and PSP. The present invention provides certain new compounds that are OGA inhibitors.
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    3/32 [008] Accordingly, the present invention provides a compound of Formula I:
    Formula I or a pharmaceutically acceptable salt thereof.
    [009] Furthermore, the present invention provides a compound of Formula Ia:
    Formula Ia
    H or a pharmaceutically acceptable salt thereof.
    [0010] The present invention also provides a method of treating Alzheimer's disease in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof.
    [0011] The present invention further provides a method of treating the progression from moderate cognitive impairment to Alzheimer's disease in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of Formulas I or Ia, or a pharmaceutically salt. acceptable value. .
    [0012] The present invention also provides a method of treating progressive supranuclear palsy in a patient in need of such treatment, comprising administering to the patient
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    4/32 an effective amount of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof. The present invention also provides a method of treating tau-mediated neurodegenerative disorders in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof.
    [0013] Furthermore, this invention provides a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for use in the treatment of Alzheimer's disease or for use in preventing progression from moderate cognitive impairment to Alzheimer's disease. In addition, this invention provides a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for use in the treatment of progressive supranuclear palsy. The invention also provides a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for use in the treatment of tau-mediated neurodegenerative disorders.
    [0014] Furthermore, this invention provides the use of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of Alzheimer's disease or for the prevention of the progression of cognitive impairment moderate to Alzheimer's disease. In addition, this invention provides the use of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of progressive supranuclear palsy. The invention also provides for the use of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of tau-mediated neurodegenerative disorders.
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    The invention further provides a pharmaceutical composition, comprising a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable vehicles, diluents or excipients. The invention further provides a process for preparing a pharmaceutical composition, comprising mixing a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable vehicles, diluents or excipients. This invention also encompasses new intermediates and processes for the synthesis of the compounds of Formulas I and Ia.
    [0016] Moderate cognitive impairment has been defined as a potential prodromal stage of dementia associated with Alzheimer's disease, based on the clinical presentation and progression of patients exhibiting moderate cognitive impairment to Alzheimer's dementia over time. The term "preventing the progression from moderate cognitive impairment to Alzheimer's disease" includes restricting, delaying, stopping or reversing the progression from moderate cognitive impairment to Alzheimer's disease in a patient.
    [0017] As used in this document, the terms "treatment" or "treat" include containing, delaying, stopping or reversing the progression or severity of an existing symptom or disorder.
    [0018] As used in this document, the term patient refers to a human being.
    [0019] As used herein, the term "effective amount" refers to the amount or dose of the compound of the invention, or a pharmaceutically acceptable salt thereof, which, after administration of a single or multiple dose to the patient, provides the effect desired in the patient under diagnosis or treatment.
    [0020] An effective amount can be readily determined by a person skilled in the art through the use of known techniques and
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    6/32 by observing the results obtained under similar circumstances. In determining the effective amount for a patient, several factors are considered, including, but not limited to: the species of patient; their size, age and general health; the specific disease or disorder involved; the degree, or involvement, or severity, of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the selected dose regimen; the use of concomitant medication; and other relevant circumstances.
    [0021] The compounds of the present invention are generally effective over a wide range of dosages. For example, dosages per day usually range from about 0.1 to about 15 mg / kg of body weight. In some cases, dosage levels below the lower limit of the above range may be more than adequate, while in other cases even higher doses with acceptable side effects may be employed and therefore the above dosage range is not intended to limit the scope of the invention in any way.
    [0022] The compounds of the present invention are preferably formulated as pharmaceutical compositions administered by any route that makes the compound bioavailable, including the oral and transdermal routes. Most preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing them are well known in the art (See, e.g., Remington: The Science and Practice of Pharmacy, L.V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012).
    [0023] The compounds of Formulas I and la, or the pharmaceutically acceptable salts thereof, are particularly useful in the treatment methods of the invention, however certain configurations
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    7/32 are preferred. The following paragraphs describe these preferred settings. It will be understood that these preferences are applicable to both treatment methods and the compounds of the invention.
    [0024] The compounds of the present invention include:
    N ° N
    O
    Formula
    Ia

    Formula Id and the pharmaceutically acceptable salts thereof.
    [0025] The compound of Formula I in which the methyl and oxygen substituents on the piperidine ring are in the cis or trans configuration, or the pharmaceutically acceptable salt thereof, is included in the scope of the invention, with the cis configuration being preferred. For example, a person skilled in the art will appreciate that the methyl at position 2 is in the cis configuration relative to the oxygen at position 4, as shown in Scheme A below:
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    8/32
    Scheme A
    Formula la
    Formula lc [0026] Additionally, a person skilled in the art will appreciate that the methyl in position 2 is in the trans configuration in relation to oxygen in position 4, as shown in Scheme B below:
    Scheme B
    Formula Ib
    Formula Id [0027] Compounds where the chiral center at position 2 of the piperidine ring is in the S configuration are additionally preferred. Although the present invention contemplates all individual enantiomers and diastereoisomers, as well as mixtures of the enantiomers
    Petition 870190060631, of 06/28/2019, p. 23/55
    9/32 of said compounds, including racemates, compounds with the absolute configuration as shown below are particularly preferred:
    [0028] A N- [4-fluoro-5 - [[(2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4-oxadiazol3-yl) methoxy] -1-piperidyl] methyl] thiazol-2-yl] acetamide and the pharmaceutically acceptable salts thereof; and N- [4-fluoro-5 - [[((2S, 4S) 2-methyl-4 - [(5-methyl-1,2,4-oxadiazol-3-yl) methoxy] -1-piperidyl] methyl ] thiazol-2yl] acetamide are particularly preferred.
    [0029] The crystalline form of N- [4-fluorine-5 - [[((2S, 4S) -2-methyl-4 - [(5methyl-1,2,4-oxadiazol-3-yl) methoxy] -1 -piperidyl] methyl] thiazol-2-yl] acetamide is especially preferred. The crystalline form of N- [4-fluor-5 - [[(2S, 4S) -2methyl-4 - [(5-methyl-1,2,4-oxadiazol-3-yl) methoxy] -1-piperidyl] methyl] thiazol-2yl] acetamide, which is characterized by a peak in the powder X-ray diffraction spectrum at a 12.1 ° 2-theta diffraction angle in combination with one or more peaks selected from the group consisting of 15, 3 °, the 21.6, 22.2 °, 22.7 °, 23.5 °, 24.3 ° and 26.8 °, with a tolerance for the diffraction angles of 0.2 degrees is further preferred .
    [0030] The individual isomers, enantiomers and diastereoisomers can be separated or resolved by a person skilled in the art at any convenient point in the synthesis of the compounds of the invention, by methods such as selective crystallization techniques or chiral chromatography (See, for example, J. Jacques, etal., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, Inc., 1981, and EL Eliel and SH Wilen ”, Stereochemistry of Organic Compounds, WileyInterscience, 1994).
    [0031] A pharmaceutically acceptable salt of the compounds of the invention can be formed, for example, by reacting an appropriate free base of a compound of the invention and an appropriate pharmaceutically acceptable acid, in a suitable solvent, under standard conditions well known in the art. The formation of such salts is well known and
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    10/32 appreciated in the technique. See, for example, Gould, P.L., "Salt selection for basic drugs", International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4: 427-435 (2000); and Berge, S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).
    [0032] The compounds of the present invention, or their salts, can be prepared by a variety of procedures known to a person skilled in the art, some of which are illustrated in the schemes, preparations and examples below. One skilled in the art recognizes that the synthetic steps specific to each of the described routes can be combined in different ways, or in combination with the steps of different schemes, to prepare the compounds of the invention, or their salts. The products of each step in the schemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding and crystallization. In the diagrams below, all substituents, unless otherwise indicated, are as previously defined. Reagents and starting materials are readily available to a person skilled in the art. Without limiting the scope of the invention, the following schemes, preparations and examples are provided to further illustrate the invention. In addition, one skilled in the art appreciates that compounds of Formulas la, lb, Ic and Id can be prepared using the starting material with the corresponding stereochemical configuration that can be prepared by a person skilled in the art. For example, the Schemes below use the starting materials with the configuration ultimately corresponding to Formula Ia.
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    11/32 [0033] In general, a Formula Ia compound can be prepared from a Formula II compound (Scheme 1). More specifically, a compound of Formula 11 is alkylated reductively with N- (4-fluoro-5-formylthiazol-2-yl) acetamide in the presence of a suitable reducing agent, such as sodium triacetoxyborohydride, in a suitable solvent, to provide a compound of Formula Ia in a suitable solvent, such as ethyl acetate. N- (4-fluoro5-formylthiazol-2-yl) acetamide can be prepared by methods known in the chemical techniques, as well as by the methods provided in the Preparations and the Examples that follow.
    [0034] A compound of Formula IIa can be prepared from a compound of Formula lla, where Pg is a suitable amine protecting group. More specifically, a compound of Formula IIa, where Pg is tert-butyl carboxylate (t-BOC), is reacted with an acid, such as hydrochloric acid or trifluoroacetic acid, in a suitable solvent, such as dioxane or dichloromethane, to provide a compound of Formula IIa. Suitable amine protecting groups are known in the chemical arts and include t-BOC and Cbz, as well as those discussed in T. W. Green, P. G. M. Wuts, Protective Groups in Organic Synthesis Wiley-Interterscience, New York, 1999.
    Layout 1
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    12/32 [0035] A compound of Formula Illa, where Pg is a suitable amine protecting group, can be prepared from a compound of Formula IVa (Scheme 2). More specifically, a compound of Formula IVa, where Pg is tert-butyl carboxylate, is reacted with 3 (chloromethyl) -5-methyl-1,2,4-oxadiazole in the presence of a base, such as tert- sodium butoxide, to provide a compound of Formula Illa. The reaction is conveniently carried out in a solvent, such as acetonitrile or dimethylformamide. A compound of Formula IVa, where Pg is tert-butyl carboxylate, can be prepared essentially as described in WO 2004/094380 A1. More specifically, a Formula Va compound is reacted with a reducing agent, such as lithium tri (sec-butyl) borohydride, in a solvent, such as tetrahydrofuran, to provide a Formula IVa compound, where Pg is tert-butyl carboxylate. A compound of Formula Va, where Pg is a suitable amine protecting group, can be prepared by processes known in the chemical techniques, including those described in WO 2004/094380 A1.
    Layout 2
    Va iva Hla
    Preparation 1
    Synthesis of tert-butyl N- (4-fluor-5-formyl-thiazol-2-yl) carbamate.
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    13/32
    Η
    [0036] c cesium fluoride (227 g, 1480 mmols) is added to a solution of tert-butyl N- (4-chloro-5-formyl-thiazol-2-yl) carbamate (38.8 g, 148 mmols for the preparation of tert-butyl N- (4-chloro-5-formyl-thiazol-2yl) carbamate, see, for example, N. Masuda, et al., Bioorg MedChem, 12, 6171-6182 (2004)) , in DMSO (776 mL), at room temperature. The reaction mixture is stirred in a 145 Ό heating block with an internal temperature of 133 Ό for 48 hours, then the mixture is cooled in an ice water bath. To the mixture is added the saturated aqueous solution of sodium bicarbonate (500 ml), brine (500 ml) and ethyl acetate (500 ml). The mixture is stirred at room temperature for 10 minutes, then it is filtered through diatomaceous earth, washing with ethyl acetate (500 mL). The filtrate is transferred to a separating funnel and the layers are separated, then the aqueous layer is extracted with ethyl acetate (1 L). The combined organics are washed with brine (1 L), then the brine layer is extracted with ethyl acetate (300 ml). The combined organics are dried over sodium sulfate, filtered and concentrated to give a residue. The residue is passed through a filling of silica gel (330 g) eluting with 5% ethyl acetate in dichloromethane (1.5 L) and the filtrate is concentrated to give a residue (24.2 g).
    [0037] The residue (32.7 g of combined batches, 133 mmols) is dissolved in isopropanol (303 mL), filtered and then purified by SFC (Supercritical Fluid Chromatography) using an IC column (derived from cellulose polysaccharide: tris (3,5-dichlorophenylcarbamate, 30 x 250 mm, 5 u) with 10% IPA (without additive), at 180 mL / minute, with injections
    Petition 870190060631, of 06/28/2019, p. 28/55
    14/32 of 3 ml. Fractions containing the product are concentrated to give the title compound (16.1 g MS m / z 247.0 (M + H).
    Preparation 2
    Synthesis of N- (4-fluor-5-formyl-thiazol-2-yl) acetamide (Method A)
    H [0038] In a jacketed vessel, zinc bromide (91.9 g, 408 mmols) is added in one portion to a mixture of tert- N- (4-fluor-5formyl-thiazol-2-yl) carbamate butyl (33.5 g, 136 mmols) and dichloromethane (503 ml), at room temperature. The reaction mixture is stirred overnight at an internal temperature of 37 Ό, then the jacket temperature is adjusted to -10 O and tetrahydrofuran (111 mL) is added dropwise over 15 minutes, maintaining an internal temperature below 6 Ό. The jacket temperature is then adjusted to -30 O and pyridine (110 mL, 1360 mmols) is added dropwise over 5 minutes, maintaining an internal temperature below 5 O. The jacket temperature is adjusted to 0 Ό and the acetic anhydride (116 ml, 1220 mmols) is added dropwise over 5 minutes. The reaction mixture is stirred overnight at an internal temperature of 37 Ό, then cooled to room temperature and passed through a small pad of diatomaceous earth, eluting with tetrahydrofuran (500 mL). The filtrate is transferred to a flask and the mixture is concentrated to give a residue, which is concentrated from toluene (50 ml). To the residue is added a solution of citric acid monohydrate (57.2 g, 272 mmols) in water (400 ml) and 2-methyltetrahydrofuran (400 ml) and the mixture is stirred at 40 Ό for 5 minutes, then passed through a short filling of diatomaceous earth, eluting with 2-methyltetrahydrofuran (100 mL). The filtrate is transferred to a separating funnel and the
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    15/32 layers are separated. The aqueous layer is extracted with 2methyltetrahydrofuran (2 x 250 ml) and the combined organics are diluted with water (500 ml). To the mixture is added solid sodium bicarbonate, in portions, for 5 minutes, with stirring until the evolution of gas ceases. The mixture is transferred to a separating funnel and the layers are separated, then the aqueous layer is extracted with 2methyltetrahydrofuran (200 ml and 100 ml). The combined organics are dried over sodium sulfate, filtered and concentrated to give a residue, which is diluted with 2-methyltetrahydrofuran (100 ml) and the mixture is passed through a short silica gel filling (250 g), eluting with 2 -methyltetrahydrofuran (2.5 L). The filtrate is concentrated to give a residue which is suspended in a 1: 1 mixture of dichloromethane and heptane (202 ml). The mixture is stirred at room temperature for 30 minutes and then filtered. The filtered solid is dried under vacuum at 40 Ό for 2 hours to give the title compound (18.0 g, 70%). MS m / z 189.0 (M + H).
    Alternative synthesis of N- (4-fluor-5-formyl-thiazol-2-yl) acetamide (Method BL [0039] Add dichloromethane (1325 g, 15.6 mol) to 2-amino-4 chlorothiazole-5-carbaldehyde ( 100 g, 0.61 mol) and pyridine (194.6 g, 2.46 moles) and cool to 0-5 Ό. Add anhydride to skeptic (188.4 g, 1.85 mol) dropwise , keeping the temperature at 0-5 Ό. After the addition is complete, adjust the temperature to 20-25 Ό and stir for 41 hours. Concentrate under reduced pressure, followed by the addition of 35% aqueous HCI (200 mL) and water (1.5 L), keeping the temperature at less than 40 Ό. Cool to 20-25 Ό and stir for 18 hours. Filter the mixture and wash the collected solid with water. Dry the solids at 6065Ό for 24 h to provide a N- (4-chloro-5-form-ylthiazole-2yl) acetamide (75 g, 0.4 mol).
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    16/32 [0040] Under an inert atmosphere, add sulfolane (1000 ml) to N- (4-chloro-5-formylthiazol-2-yl) acetamide (50 g, 0.244 mol, prepared directly above), tetramethylammonium chloride (107.1 g, 0.977 mol) and cesium fluoride (370.6 g, 2.44 mmol). Heat to 130 Ό and stir for 23 h. HPLC analysis shows a 75% conversion with an in situ yield of 45% of the title compound.
    Alternative synthesis of N- (4-fluor-5-formyl-thiazol-2-yl) acetamide (Method QL [0041] Add 2-propanol (150 mL) to tetramethylammonium fluoride.tetrahydrate (10.2 g, 109.0 mmols) and concentrate the mixture to 2-3 volumes, under vacuum, with the internal temperature maintained at 70 para to remove the water. Add 2-propanol (200 mL) and concentrate the mixture to 2-3 volumes, under vacuum. Repeat two more times. Add to DMF (200 mL) and concentrate to 2-3 volumes, under vacuum. Add THF (200 mL) and concentrate to 2-3 volumes. Repeat two more times. Load N- (4-chloro-5-formylthiazol-2-yl) acetamide (1.22 g, 5.96 mmols, prepared above in Method B) and DMF (12 mL). Heat to 110 Ό and stir for 12 h. the reaction mixture up to 25 Ό Add 2-methyltetrahydrofuran (40 ml) and water (40 ml). The layers are separated and the aqueous layer was extracted with 2-methyltetrahydrofuran (40 ml). The layers were separated and the layers combined organic were washed with water (20 mL) The layers were separated and the organic layer was concentrated. Add ethyl acetate (20 ml) and water (5 ml). The layers were separated and the organic layer concentrated to remove the solvent. Add ethyl acetate (2 ml) and heptane (2 ml) and filter. The filtered solid is dried under vacuum at 55 Ό for 18 hours to give the title compound as a 93% mixture with N- (4-chloro-5-formylthiazol-2yl) acetamide.
    Preparation 3
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    17/32
    Synthesis of tert-butyl (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylate.
    OH [0042] To a flask are added tert-butyl (2S) -2-methyl-4-oxo-piperidine-1-carboxylate (50 g, 234.44 mmols) and tetrahydrofuran (500 ml). The mixture is cooled to -65 Ό, under a nitrogen atmosphere, and lithium tri (sec-butyl) borohydride (304.77 mL, 304.77 mmols; 1 M in tetrahydrofuran) is added dropwise drop for 45 minutes, maintaining an internal temperature below -60 Ό. The reaction mixture is stirred at room temperature for 1 hour, then cooled to -30 Ό. A mixture of water (25.34 ml) and tetrahydrofuran (100.16 ml) is added to the reaction mixture, maintaining an internal temperature below -20 Ό. An aqueous solution of hydrogen peroxide (118.88 mL, 1.17 mol, 30% w / w) in water (126.70 mL) is added dropwise over 1 hour, maintaining an internal temperature below 10 Ό. To the mixture are added an aqueous solution of hydrogen chloride (46.89 mL, 234.44 mmols, 5 M) and methyl butyl ether (1.00 L) and the mixture is warmed to room temperature. The layers are separated and the organic phase is stirred with a solution of sodium metabisulfite (222.84 g, 1.17 mol) in water (500 mL), for 10 minutes, at room temperature. The layers are separated and the organic phase is dried over magnesium sulfate and concentrated. The residue is purified by flash chromatography (0-50% methyl butyl ether / isohexane, silica gel) and the fractions containing the product are combined and concentrated to the title compound (40.4 g, 78%). ES / MS (m / e) 238 (M + Na).
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    18/32
    Alternative synthesis of tert-butyl (2S, 4S) -4-hydroxy-2-methyl-oioeridine-1-carboxylate.
    OH [0043] To a glass-lined reactor containing deionized water (460 L) and potassium dihydrogen phosphate (6.5 kg, 0.41 equiv.), At 20 Q C, DMSO (27.4 kg, 1.0 vol) and D - (+) - glucose monohydrate (28.9 kg 1.25 equiv.). The internal temperature is adjusted to 30 Ό and the pH of the reaction is adjusted to 6.9 by the addition of aqueous sodium hydroxide (8%, 15 L, 0.28 equiv). The reactor is charged with tert-butyl (2S) 2-methyl-4-oxo-piperidine-1-carboxylate (24.9 kg, 1.0 equiv (99.1% ee)) and the mixture is stirred at 30 Ό for 15 mi η. Ketorreductase (KRED-130, 250 g, 1% w / w), glucose dehydrogenase (GDH-101,250 g, 1% w / w) and the sodium NADP salt (63 g, 0.25% w / w) they are loaded directly into the reaction mixture through an open door. The mixture is maintained at a temperature of 30 Ό and pH 7.0 ± 0.2 through the addition of 8% aqueous NaHCOa. After stirring for 16.5 h (99.5% conversion), the reaction is charged with Celite® (12.5 kg, 50% w / w) and toluene (125 L, 5 vol). After stirring for 30 min at 30 Ό, the mixture is transferred to another 2000 L reactor through an in-line GAF filter (4 sock) for a period of 1 h. The mixture is left to stand for 30 min without stirring, the layers are separated and the aqueous layer is extracted again with toluene (2 x 125 L). The combined organic layers are filtered (in-line GAF filter) and the toluene mixture is washed with 25% aqueous sodium chloride solution (25%, 125 L, 5 vol). The resulting toluene solution is azeotropically dried (partial vacuum, internal temperature <60 Ό) with 0.10% w / w water
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    19/32 cooled to 20 Ό. The mixture is filtered from the reactor through a cartridge filter for clean drums, under positive pressure of nitrogen. The reaction mixture is then transferred from the drums to a 500 L glass lined vessel and concentrated in vacuo (<60 Ό) to a residual target volume of 56 L (2.25 vol). The n-heptane (169 kg, 10 vol) is charged at 40 Ό and the mixture is seeded with 25 g of tert-butyl (2S, 4S) -4hydroxy-2-methyl-piperidine-1-carboxylate. The resulting thick paste is diluted with additional n-heptane (25 L, 1 vol) and cooled to 16 Ό for 4 h. The product is isolated by centrifugation, washing with nheptane (25 L per spin; 4 required turns), producing 20.3 kg (81%;> 99.9% ee) after drying for 11 h in a tray dryer, to 30 Ό. ES / MS (m / e) 238 (M + Na).
    Preparation 4
    Synthesis of tert-butyl (2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4-oxadiazol-3yl) methoxy] piperidine-1-carboxylate.
    [0044] 3- (chloromethyl) -5-methyl-1,2,4-oxadiazole (43.5 g, 301 mmol) is added to a solution of (2S, 4S) -4-hydroxy-2-methyl- tert-butyl piperidine-1-carboxylate (29.5 g, 137 mmols) in acetonitrile (590 ml), at room temperature. The reaction mixture is stirred in an ice bath and sodium tert-butoxide (54.3 g, 548 mmols) is added in portions for 10 minutes, maintaining an internal temperature below 10 Ό. The reaction mixture is stirred in a bath with ice, at an internal temperature of 5 Ό, for 9 hours,
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    20/32 then it is slowly warmed to room temperature and stirred overnight. The reaction mixture is cooled in a water bath with ice and a saturated aqueous solution of ammonium chloride (200 mL) is added for 5 minutes, maintaining an internal temperature below 10 Ό during the addition. The mixture is then diluted with water (100 ml) and heated to room temperature. The mixture is extracted with methyl tert-butyl ether (2 x 300 ml) and the combined organics are washed with brine (300 ml). The combined organics are dried over sodium sulfate, filtered and concentrated to give a residue. The residue is passed quickly through a silica gel filling (300 g) eluting with tert-butyl methyl ether (1 L) and the filtrate is concentrated to give the title compound (46.5 g, 109%). MS m / z 334.0 (M + Na).
    Alternative synthesis of tert-butyl (2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4-oxadiazol-3yl) methoxyDioeridine-1-carboxylate.

    [0045] To a solution of tert-butyl (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylate (0.25 g, 1.16 mmol) and 3- (chloromethyl) -5-methyl-
    1,2,4-oxadiazole (0.308 g, 2.32 mmol), in N, N-dimethylformamide (3 mL), under nitrogen, at 0 Q C, sodium tert-butoxide ( 0.35 g, 3.5 mmols) for 5 min. The reaction mixture is stirred at rt for 10 min, then at 40 Q C for 12 h. The reaction mixture is cooled to room temperature, then finished with water (10 ml). The layers are separated and the aqueous phase is extracted with methyl tert ether
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    21/32 butyl (2 x 10 mL). The combined organic extracts are washed with an aqueous solution of lithium chloride (5%), dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the title compound (0.49 g, 0.7 mmol, 81% yield, 60% purity) as a brown oil. MS m / z 334.0 (M + Na).
    Preparation 5
    Synthesis of 5-methyl-3 - [[(2S, 4S) -2-methyl-4-piperidyl] oxymethyl] - hydrochloride
    1,2,4-oxadiazole.
    H [0046] A vial containing tert-butyl (2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4oxadiazol-3-yl) methoxy] piperidine-1-carboxylate (4, 03 g, 12.9 mmols) is immersed in a water bath with ice. To this flask is added a 4 M solution of hydrochloric acid in 1,4-dioxane (25.9 mL, 104 mmols), dropwise, for 5 minutes, with agitation, maintaining an internal temperature below 20 Ό during the addition. The reaction mixture is stirred at room temperature for 1 hour, then it is concentrated to give the title compound (3.56 g, 92% yield based on 83% purity measured by 1 H NMR, MS m / z 212 , 0 (M + H) Alternative synthesis of 5-methyl-3 - [[((2S, 4S) -2-methyl-4DIDDeridilloximetill-1,2,4-oxadiazole hydrochloride · [0047] Add methanol (50 mL ) to (2S, 4S) -2-methyl-4 - [(5-methyl-
    1,2,4-oxadiazol-3-yl) methoxy] tert-butyl piperidine-1-carboxylate (12.9 g 0.041 mol). The mixture is cooled to 0 Ό. A solution of 4 M hydrochloric acid in methanol (80 ml) is added dropwise to the cooled mixture, maintaining an internal temperature below 20 Ό. The mixture
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    22/32 reaction is then stirred at room temperature for 18 hours. The mixture is then concentrated to remove the solvent. Acetone (10 ml) is added and the mixture is stirred for 20 min. Tetrahydrofuran (40 ml) is added and the mixture is stirred for 3 hours. The solid is collected by filtration under nitrogen and the filtered solid cake is rinsed with tetrahydrofuran. The filtered solid is then dried under vacuum at 45 ° for 2 hours to give the title compound as 90% pure. Recrystallization using acetone can increase the purity of the title compound to 95%.
    Preparation 6
    Synthesis of 5-methyl-3 - [[(2S, 4S) -2-methyl-4-piperidyl] oxymethyl] -1,2,4oxadiazole.

    [0048] To a solution of tert-butyl (2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4oxadiazol-3-yl) methoxy] piperidine-1-carboxylate (0.49 g, 1.6 mmols) in dichloromethane (10 ml), under nitrogen, trifluoroacetic acid (1.8 ml, 23 mmols) is added. The mixture is stirred at room temperature for 3 h. The mixture is concentrated under reduced pressure to provide a yellow oil. The residue is dissolved in methanol (5 ml) and poured into a cation exchange cartridge, eluted with methanol (2x10 ml), then with a 2 M ammonia solution in methanol (10 ml). The filtrate is concentrated under reduced pressure to give the title compound (0.3 g, 1.4 mmol, 91%). MS m / z 212.0 (M + H).
    Example 1
    Synthesis of N- [4-fluor-5 - [[((2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4-oxadiazol-3yl) methoxy] -1-piperidyl] methyl] thiazol-2-yl] acetamide.
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    [0049] N- (4-fluor-5-formyl-thiazol-2-yl) acetamide (28.3 g, 150 mmol) is added to 5-methyl-3 hydrochloride - [[(2S, 4S) - 2-methyl-4piperidyl] oxymethyl] -1,2,4-oxadiazole (48.7 g, 185 mmols, 94% purity), in ethyl acetate (707 mL), at room temperature. The reaction mixture is stirred at room temperature and N, N-diisopropylethylamine (34.1 ml, 195 mmols) is added dropwise over 1 minute, then sodium triacetoxyborohydride (98.5 g 451 mmols) is added in one portion. The reaction mixture is stirred in a heating block at 31 Ό overnight with an internal temperature of 30 Ό, then cooled in an ice water bath to an internal temperature of 5 Ό. To the mixture is added a 2M aqueous hydrochloric acid solution (226 mL) for 15 minutes, maintaining an internal temperature below 10 Ό. To the mixture is added water (250 ml) and the mixture is stirred at room temperature for 5 minutes. The layers are separated and the organic layer is extracted with a mixture of 2M aqueous hydrochloric acid solution (28 ml) in water (50 ml). The first aqueous layer is stirred in an ice water bath and the 50% aqueous sodium hydroxide solution (25.7 mL) is added dropwise over 10 minutes, maintaining an internal temperature below 10 Ό. The mixture is diluted with saturated aqueous sodium bicarbonate solution (100 ml), then it is stirred at room temperature for 10 minutes and then it is extracted with ethyl acetate (3 χ 400 ml). The combined organics are dried over
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    24/32 sodium sulfate, filtered and concentrated to give a residue. The second aqueous layer of the extraction with aqueous hydrochloric acid is diluted with 2-methyltetrahydrofuran (200 ml) and the mixture is passed through a short pad of diatomaceous earth. The filtrate is transferred to a separating funnel and the layers are separated. The aqueous layer is stirred in an ice water bath and a 50% aqueous sodium hydroxide solution (3.15 mL) is added dropwise over 5 minutes, maintaining an internal temperature below 10 Ό. The mixture is diluted with saturated aqueous sodium bicarbonate solution (10 mL), then stirred at room temperature for 5 minutes and then extracted with ethyl acetate (3 x 40 mL) and 10% isopropanol in ethyl acetate ( 100ml). The combined organics are dried over sodium sulfate, filtered and concentrated to give a residue, which is combined with the residue from the first part of the production. The combined residue is passed through a filling of silica gel (350 g) eluting with ethyl acetate (3.5 L) and the filtrate is concentrated to give a residue (45.8 g).
    [0050] The residue (47.5 g of combined batches, 123.9 mmols) is purified by flash chromatography, eluting with 50-100% ethyl acetate in heptane. The product-containing fractions are concentrated to a residue, which is suspended in a 1: 1 mixture of methyl tert-butyl ether and heptane (448 ml). The mixture is stirred in a heating block at 46 Ό for 30 minutes, at an internal temperature of 45 Ό, then cooled to room temperature for 2 hours, with stirring. The mixture is filtered, washing the solid with a 1: 1 mixture of methyl tert-butyl ether and heptane (30 ml). The filtered solid is vacuum dried at 40 Ό overnight to give the title compound (28.5 g). MS m / z 384.0 (M + H);
    [0051] [a] D 20 = + 33.4 ° (C = 0.26, methanol).
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    Alternative synthesis of N- [4-fluor-5 - [[((2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4oxadiazol-3-yl) methoxy1-1-DIDididyl] methyl1thiazole- 2-yl1acetamide · [0052] To a solution of N- (4-fluor-5-formyl-thiazol-2-yl) acetamide (0.05 g, 0.28 mmol) and 5-methyl-3 - [[( 2S, 4S) -2-methyl-4-piperidyl] oxymethyl] -
    1,2,4-oxadiazole (0.04 g, 0.19 mmol) in dichloromethane (10 mL), under nitrogen, are added to N, N diisopropylethylamine (0.1 mL, 0.57 mmol) and the sodium triacetoxyborohydride (0.12 g, 0.57 mmol). The reaction mixture is stirred at room temperature for 12 h. The reaction mixture is poured into a saturated aqueous solution of sodium bicarbonate (10 mL). The layers are separated and the aqueous phase is extracted with dichloromethane (2x10 ml). The combined organic extracts are dried over magnesium sulfate, filtered and concentrated under reduced pressure to provide an orange oil.
    [0053] The residue is absorbed in methanol (up to a total volume of 9.8 ml), filtered and purified by prep-HPLC (Phenomenex Gemini-NX 10 Micron 50 * 150 mm C-18) (CH3CN and Water with bicarbonate of 10 mM ammonium adjusted to pH 9 with ammonium hydroxide, 15% to 100% CH3CN for 10 min, at 110 ml / min) (1 injection) (271/204 nm), to give the title compound (0, 02 g, 0.05 mmol, 28%). MS m / z 384.2 (M + H).
    Example 1A
    N- [4-Fluorine-5 - [[(2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4-oxadiazol-3-yl) methoxy] -1piperidyl] methyl] thiazole- 2-yl] crystalline acetamide [0054] Suspend N- [4-fluorine-5 - [[(2S, 4S) -2-methyl-4 - [(5-methyl-1,2,4oxadiazol-3-yl) methoxy] -1-piperidyl] methyl] thiazol-2-yl] acetamide crude (29.9 g) in 448 mL of 50% methyl tert-butyl ether in heptane, at 46 Ό, for 30 minutes. Stir the mixture and cool to 19 Ό for two hours before filtering, followed by a wash of 30 ml of 50% methyl tert butyl ether in heptane, to provide the title compound (28.5 g, 95% yield ).
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    Powder X-Ray Diffraction (XRPD) of Example 1A [0055] XRPD standards for crystalline solids are obtained on a Bruker D4 Endeavor powder X-ray diffractometer, equipped with a CuKa source (λ = 1.54060 Â) and a Vantec detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40 ° at 20 °, with a phase size of 0.0087 ° at 20 ° and a scan rate of 0.5 second / phase and with 0.6 mm deviation, fixed anti-dispersion of 5, 28 mm and 9.5 mm detector slots. The dry powder is placed on a quartz sample holder and a smooth surface is obtained using a glass slide. It is well known in the crystallography technique that, for any given crystal form, the relative intensities of the diffraction peaks can vary due to the preferred orientation resulting from factors such as the morphology and the habit of the crystal. Where the effects of the preferred orientation are present, the peak intensities are changed, but the characteristic positions of the polymorph peak are unchanged. (See, eg, The U. S. Pharmacopeia 38 - National Formulary 35 Chapter 941 Characterization of crystalline and partially crystalline solids by X-ray powder diffraction (XRPD) Official May 1, 2015). Furthermore, it is also well known in the crystallography technique that, for any given crystal form, the positions of the angular peaks can vary slightly. For example, peak positions may change due to a change in temperature or humidity, in which a sample is analyzed, the displacement of the sample or the presence or absence of an internal pattern. In the present case, a peak position variability of ± 0.2 in 20 will take these potential variations into account, without preventing the unambiguous identification of the indicated crystal shape. Confirmation of a crystal shape can be made based on any unique combination of characteristic peaks (in units of 20 °), typically the most prominent peaks. The diffraction patterns of the crystal form, collected at room temperature and at
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    27/32 relative humidity, are adjusted based on the standard peaks of NIST 675 to 8.85 and 26.77 degrees 2-theta.
    [0056] A prepared sample of N- [4-fluorine-5 - [[((2S, 4S) -2-methyl-4 [(5-methyl-1,2,4-oxadiazol-3-yl) methoxy] - 1-piperidyl] methyl] thiazol-2yl] crystalline acetamide is characterized by an XRPD pattern, using CuKa radiation, as having diffraction peaks (2-theta values) as described in Table 1 below. Specifically, the pattern contains a peak at 12.1 ° in combination with one or more peaks selected from the group consisting of 15.3 °, 21.6 °, 22.2 °, 22.7 °, 2 3.5 ° , 24.3 ° and 26.8 °, with a 0.2 degree tolerance for diffraction angles.
    Table 1: X-ray powder diffraction peaks of N- [4-fluorine-5 - [[((2S, 4S) -2methyl-4 - [(5-methyl-1,2,4-oxadiazol-3-yl ) methoxy] -1-piperidyl] methyl] thiazol-2yl] crystalline acetamide, Example 1A.
    Peak Angle (° 2-Theta) +/- 0.2 ° Relative intensity (% of the most intense peak) 1 7.7 9 2 10.1 9 3 12.1 100 4 15.3 50 5 18.3 11 6 19.3 13 7 21.6 16 8 22.2 16 9 22.7 16 10 23.5 30 11 24.3 35 12 26.8 27
    In vitro human OGA enzyme assay
    Generation of QGA proteins
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    28/32 [0057] The nucleotide sequence encoding the human-sized human O-GlcNAc-β-Νacetylglycosaminidase (NM012215) is inserted into the vector pFastBad (Invitrogen) with a N terminal polyhistidine (HIS) tag. The generation of baculovirus is performed according to the protocol of the Baculovirus Expression system Bacto-Bac (Invitrogen). Sf9 cells are infected with 1.5 x 10 6 cells / ml using 10 ml of P1 viruses per liter of culture, and incubated at 28 Q C for 48 h. The cells are centrifuged, rinsed with PBS and the pellet stored at -80 Q C above OGA protein (His-OGA) is purified as follows: 4 L cells are lysed in 200 ml of buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM Imidazole, 1 mM Dithiothreitol (DTT), 0.1% Triton® X-100, 4 protease inhibitor tablets (EDTA-Free complete , Roche) for 45 min, at 4 Q C. This cell lysate is then centrifuged for 40 min at 16500 rpm, at 4 Q C, and the supernatant incubated with 6 mL of Ni-NTA resin (nitrilotriacetic nickel acid) for 2 hours. , at 4 Q C.
    [0058] The resin is then packed on the column and washed with 50 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM Imidazole, 0.1% Triton® X-100, 1 mM DTT, followed by 50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM Imidazole, 10% glycerol, 1 mM DTT. The proteins are eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 300 mM Imidazole, 10% glycerol, 1 mM DTT. The combined His-OGA fractions are concentrated to 6 mL and loaded into Superdex75 (16/60). The protein is eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM DTT. The fractions containing His-OGA are combined and the protein concentration is measured with the BCA (Bradford Colorimetric Assay).
    OGA enzyme assay [0059] The OGA enzyme catalyzes the removal of O-GIcNAc from nucleocytoplasmic proteins. To measure this activity, the
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    Fluorescein di-N-acetyl-pN-acetyl-D-glycosaminide (FD-GIcNAc, Kim, Eun Ju; Kang, Dae Ook; Love, Dona C .; Hanover, John A. Carbohydrate Research (2006), 341 (8) , 971 -982) is used as a substrate at a final concentration of 10 μΜ (in the 96-well test format) or 6.7 μΜ (in the 384-well test format). This fluorogenic substrate becomes fluorescent after dividing by OGA, so that the enzymatic activity can be measured by the increase in fluorescence detected at 535 nm (excitation at 485 nm).
    [0060] The assay buffer is prepared to give a final concentration of 50 mM HzNaPOa-HNazPOa, 0.01% bovine serum albumin and 0.01% Triton® X-100 in water, at pH 7. The concentration enzyme endpoint is 3 nM (in the 96-well assay format) or 3.24 nM (in the 384-well assay format). Both test formats produce essentially equivalent results.
    [0061] The compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using ten-point concentration response curves. The maximum concentration of the compound in the reaction mixture is 30 μΜ. The compounds in the appropriate concentration are pre-incubated with the OGA enzyme, for 30 minutes, before the reaction is initiated by the addition of the substrate. The reactions are allowed to run for 60 minutes at room temperature. Then, without stopping the reaction, the fluorescence is read. IC50 values are calculated by plotting the normalized vs. 0 logging the compound and adjusting the data using a four parameter logistic equation.
    [0062] The compound of Example 1 was tested essentially as described above and exhibited an IC 50 of 2.36 nM ± 0.786 (n = 8). This data demonstrates that the compound of Example 1 inhibits the activity of the OGA enzyme in vitro.
    Whole cell assay to measure inhibition of OGA enzyme activity
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    Preparation of Plate Cells:
    [0063] Using standard conditions known in the art, TRex-293 cells, modified for the inducible expression of the P301S-1N4R form of the tau protein associated with microtubules, are generated and maintained in growth medium, consisting of DMEM High Glucose (Sigma No. D5796), supplemented with 10% Fetal Bovine Serum without Tetracycline (FBS, Sigma F2442), 20 mM HEPES, 5 pg / mL Blasticidine (Life Technologies No. A11139-03) and 200 pg / mL Zeocin ( Life Technologies No. R250-01). For the experiments, cells are prepared in growth medium, 10,000-14,000 cells per well, in a 384-well Corning Biocoat plate (356663) coated with poly-D-Lysine and incubated 20-24 h in a cell incubator 37 Q C / 5% CO2. The experiments are carried out without inducing the expression of Tau.
    Compound treatment:
    [0064] The compounds to be tested are diluted in series to 1/3, in pure DMSO, using the ten point concentration response curves, and additionally diluted in growth medium. 20-24 h after preparation on the plate, cells are treated with the test compound in growth medium; the maximum concentration of the compound is 15 pM (0.15% DMSO). Maximum inhibition is defined by repeated measurements of 15 µM Thiamet G and minimum inhibition is defined by repeated measurements of treatment with 0.15% DMSO. Cells are returned to incubator at 37 Q C / 5% CO2 for 20 to 24 hours. The compounds are tested in duplicates within each plate. Immunocoloration:
    [0065] After 20-24 hours of treatment with the compound, the medium is removed from the assay plate and 25 µl of 3.7% Formaldehyde solution (Sigma no. F1635) in DPBS (Sigma no. D8537) is added each well and incubated for 30 minutes. The cells are then washed
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    31/32 once with DPBS and then permeabilized with 0.1% Triton® X-100 (Sigma no. T9284). After 30 minutes, the cells are washed twice with DPBS and then a blocking solution (1% BSA / 0.1% DPBS / Triton® X-100) is added to each well and incubated for 60 minutes. The blocking solution is removed and a 0.400.33 pg / mL antibody to the O-GIcNAc protein (clone RL2, Thermo, MA1072) in the blocking solution is added to the cells and left to stand overnight at 2 -8 ° C. The next day, the cells are washed twice with DPBS and the secondary antibody, goat anti-mouse IgG Alexa Fluor 488 (Life Technologies no. A11001), at 2 µg / mL in DPBS, is added to each well and left to rest in the room temperature for 90 min. The secondary antibody is removed, the cells washed twice with DPBS and a solution of DAPI (Sigma no. D9564) and RNase (Sigma, R6513) in DPBS, at a concentration of 1 and 50 µg / mL, respectively, is added to each well. The plate is sealed, incubated for one hour and analyzed on an Acumen eX3 hei (TTP Labtech). All incubations and washing steps described above are done at room temperature, except for the primary antibody.
    Analysis and Results:
    [0066] The plates are analyzed on an Acumen eX3 instrument using lasers in excitation of 488 and 405 nm and two emission filters FL2 (500-530 nm) and FL1 (420-490 nm). The FL2 filter is the signal corresponding to the antibody to the O-GIcNAc Protein (clone RL2) and the FL1 filter is the signal corresponding to the cell nuclei (DAPI). The ratio FL2 Total / FL1 Total (Total fluorescence of each well, without selection of object or population) is used for data analysis. The data is normalized to a maximum inhibition as referred by a treatment of Thiamet G at 15 μ mínima and a minimum inhibition as achieved by a treatment with 0.15% DMSO. The data is adjusted with a
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    32/32 application of adjustment of the non-linear curve (logistic equation of 4 parameters) and the IC50 values are calculated and reported.
    [0067] The compound of Example 1 was tested essentially as described above and exhibited an IC 50 of 21.9 nM ± 7.3 (n = 5). This data demonstrates that the compound of Example 1 inhibits the activity of the OGA enzyme in a cell assay.
    权利要求:
    Claims (16)
    [1]
    1. Compound, characterized by the fact that it presents the formula:

    [2]
    2. Compound according to claim 1, characterized by the fact that the methyl, in position 2, is in the cis configuration in relation to oxygen, in position 4 on the piperidine ring:

    [3]
    3. Compound according to claim 1 or 2, characterized by the fact that it is N- [4-fluorine-5 - [[((2S, 4S) -2-methyl-4 - [(5methyl-1,2 , 4-oxadiazol-3-yl) methoxy] -1-piperidyl] methyl] thiazol-2-yl] acetamide, or a pharmaceutically acceptable salt thereof.
    [4]
    4. Compound according to claim 3, characterized by the fact that it is N- [4-fluorine-5 - [[(2S, 4S) -2-methyl-4 - [(5-methyl-1,2, 4oxadiazol-3-yl) methoxy] -1-piperidyl] methyl] thiazol-2-yl] acetamide.
    [5]
    5. Compound according to claim 4, characterized by the fact that it is crystalline.
    [6]
    6. Compound according to claim 5, characterized by the fact that it is defined by a peak in the powder X-ray diffraction spectrum, at the 12.1 ° 2-theta diffraction angle in combination with one or more peaks selected from the group consisting of 15.3 °, 21.6 °,
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    2/3
    22.2 ° 22.7 ° 23.5 ° 24.3 ° and 26.8 ° with a 0.2 degree tolerance for diffraction angles.
    [7]
    Use of a compound, as defined in any of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for the manufacture of a medicament for the treatment of Alzheimer's disease.
    [8]
    8. Use of a compound, as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for the manufacture of a medicament to prevent the progression from moderate cognitive impairment to Alzheimer's.
    [9]
    Use of a compound, as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for the manufacture of a medicament for the treatment of progressive supranuclear palsy.
    [10]
    A compound or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, characterized in that it is for use in therapy.
    [11]
    A compound or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, characterized in that it is for use in the treatment of Alzheimer's disease.
    [12]
    A compound or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, characterized by the fact that it is for use in preventing the progression of moderate cognitive impairment to Alzheimer's disease.
    [13]
    13. A compound or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, characterized in that it is for use in the treatment of progressive supranuclear palsy.
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    [14]
    Pharmaceutical composition, characterized in that it comprises a compound or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 6, with one or more pharmaceutically acceptable vehicles, diluents or excipients.
    [15]
    15. Process for the preparation of a pharmaceutical composition, characterized in that it comprises mixing a compound or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 6, with one or more pharmaceutically acceptable vehicles, diluents or excipients.
    [16]
    16. Invention, characterized by the fact that it is in any of its modalities or any categories of applicable claims, for example, product, process or use included by the subject initially described, disclosed or illustrated in the patent application.
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    法律状态:
    2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2021-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO EM FUNCAO DA REVOGACAO DO ART. 229-C DA LEI NO 9.279, DE 1996, POR FORCA DA LEI NO 14.195, DE 2021 |
    2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|
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