Aminopyrimidine SSAO inhibitor and its preparation method

Abstract

This application relates to an amino pyrimidine SSAO inhibitor and its preparation method. The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein n and R1 are as defined herein, a method for treating liver diseases in a patient, and a method for preparing said compound.

Description

[0001] This application is a divisional application of the invention patent application with the application date of August 4, 2017, application number 201780006868.4, and title "Aminopyrimidine SSAO Inhibitors". Technical Field

[0002] The present invention relates to aminopyrimidine compounds, pharmaceutically acceptable salts of said compounds, and therapeutic uses of said compounds and salts. Background Art

[0003] Semicarbazide-sensitive amine oxidase / vascular adhesion protein-1 (SSAO) / VAP-1) is a member of the semicarbazide-sensitive amine oxidase family. SSAO / VAP-1 is also known as VAP-1 or SSAO. SSAO / VAP-1 is an enzyme that exists in two forms, a membrane-bound isoform and a soluble isoform; it is mainly expressed on the surface of endothelial cells, vascular smooth muscle, and adipocytes. SSAO / VAP-1 is involved in many cellular processes, including glucose disposal, inflammatory responses, and leukocyte recruitment. High levels of activity of this enzyme are associated with diabetes, atherosclerosis, stroke, chronic kidney disease, and Alzheimer's disease, as well as other conditions. Recently, SSAO / VAP-1 has been implicated in the pathogenesis of liver diseases, such as fatty liver disease. (Weston C.J. et al., Journal of Neural Transmission, 2011, 118, 1055 - 1064.) Fatty liver disease (FLD) encompasses a range of disease conditions in which fat accumulates in excess in the liver and is accompanied by inflammation. FLD can give rise to non-alcoholic fatty liver disease (NAFLD) characterized by insulin resistance. Unchecked NAFLD progresses to persistent inflammatory responses or non-alcoholic steatohepatitis (NASH), progressive liver fibrosis, and ultimately cirrhosis. There is a current need to provide alternative therapeutic regimens for liver diseases, such as NAFLD and / or NASH. Summary of the Invention

[0004] It is believed that SSAO / VAP-1 inhibitors will reduce liver inflammation and fibrosis and thereby provide a treatment for liver diseases, particularly for NAFLD and / or NASH. The present invention provides compounds that inhibit the SSAO / VAP-1 enzyme and can address one or more of these needs.

[0005] The present invention provides compounds of formula 1:

[0006]

[0007] wherein n is 1 or 2; and R1 is H or -CH3; or a pharmaceutically acceptable salt thereof. The description is The bond to fluorine indicates that the fluorine atom and the methoxypyrimidine group can be Z (cis, together) or E (trans, opposite) relative to each other (Brecher J., et al., "Graphical Representation of Stereochemical Configuration", Pure and Appl. Chem, 2006, 78(10) 1897, page 1959). The structures described by Formula 1 include compounds having a Z stereochemical configuration, an E stereochemical configuration, or a mixture of compounds in the Z or E stereochemical configuration. Preferred compounds of the present invention have an E stereochemical configuration.

[0008] In one form, the present invention provides a compound of Formula 1 in free base form. In other forms, the present invention provides a compound of Formula 1 in the form of an acid addition salt (such as a mono- or dihydrochloride addition salt or a sulfonate salt, preferably 4-methylbenzenesulfonate (tosylate)).

[0009] In one form, the present invention provides a compound of Formula 2:

[0010]

[0011] wherein n is 1 or 2; and R1 is H or -CH3; or a pharmaceutically acceptable salt thereof.

[0012] In another form, the present invention provides a compound of Formula 3:

[0013]

[0014] wherein n is 1 or 2; and R1 is H or -CH3; or a pharmaceutically acceptable salt thereof.

[0015] In one embodiment, the present invention provides a compound according to one of Formulas 1, 2 and 3, wherein n is 1 or a pharmaceutically acceptable salt thereof. In another embodiment, the present invention provides a compound according to one of Formulas 1, 2 and 3, wherein n is 2 or a pharmaceutically acceptable salt thereof.

[0016] In another embodiment, the present invention provides a compound according to one of Formulas 1, 2 and 3, wherein R1 is H or a pharmaceutically acceptable salt thereof. In yet another embodiment, the present invention provides a compound according to one of Formulas 1, 2 and 3, wherein R1 is -CH3 or a pharmaceutically acceptable salt thereof.

[0017] In another form, the present invention provides a compound of formula 4

[0018]

[0019] wherein R1 is H or -CH3, or a pharmaceutically acceptable salt thereof.

[0020] In another form, the present invention provides a compound according to formula 5:

[0021]

[0022] or a pharmaceutically acceptable salt thereof. In one embodiment, the compound of formula 5 is provided in the form of an acid addition salt. Preferably, the acid addition salt is a mono- or dihydrochloride addition salt or a sulfonate salt, such as a methanesulfonic acid or 4-methylbenzenesulfonic acid addition salt to provide a mesylate or a 4-methylbenzenesulfonate (tosylate).

[0023] In another form, the present invention provides a pharmaceutical composition comprising a compound according to any one of formulas 1 to 5, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the pharmaceutical composition comprises a compound according to formula 5, or a pharmaceutically acceptable salt thereof. Preferably, the pharmaceutically acceptable anion of the salt is a mono- or dichloride ion, a mesylate or a 4-methylbenzenesulfonate (tosylate).

[0024] In another form, the present invention provides a method of treating a liver disorder in a patient in need thereof. The method comprises administering to the patient an effective amount of a pharmaceutical composition comprising a compound according to any one of formulas 1 to 5 or a pharmaceutically acceptable salt thereof.

[0025] In another form, the present invention provides a method of treating a liver disorder in a patient in need thereof. The method comprises administering to the patient an effective amount of a compound according to any one of formulas 1 to 5 or a pharmaceutically acceptable salt thereof. Examples of liver disorders include liver inflammation, fibrosis and steatohepatitis. In certain embodiments, the method comprises treating a liver disorder in a patient in need thereof, wherein the liver disorder is selected from: liver fibrosis, alcohol-induced fibrosis, alcoholic steatosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In a particularly preferred embodiment, the method comprises treating a patient in need thereof with non-alcoholic steatohepatitis (NASH).

[0026] In another form, the present invention provides a compound of formula 6, which is (2E)-3-fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate (1:1).

[0027]

[0028] In one embodiment, (2E)-3-fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate (1:1) is provided in crystalline form, characterized by an X-ray powder diffraction pattern obtained from a CuKα source having peaks at: a) 2θ of 18.6, 19.1, 21.0, 21.9 and 22.4 ± 0.2°, or b) 2θ of 17.6, 11.0, 16.8, 18.6, 19.1, 21.0, 21.9, 22.4 and 26.1 ± 0.2°.

[0029] In another form, the present invention provides a compound according to any one of formulas 1 to 6, or a pharmaceutically acceptable salt thereof, for use in therapy. In a preferred embodiment, the therapy is for liver disorders. Preferably, the therapy is for liver disorders selected from: liver fibrosis, alcohol-induced fibrosis, alcoholic steatosis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. In one embodiment, the therapy is for treating liver fibrosis. In another embodiment, the therapy is for non-alcoholic fatty liver disease. In yet another embodiment, the therapy is for non-alcoholic steatohepatitis.

[0030] In another form, the present invention provides a compound according to any one of formulas 1 to 6, or a pharmaceutically acceptable salt thereof, for treating liver disorders. In one embodiment, the liver disorder is selected from: liver fibrosis, alcohol-induced fibrosis, alcoholic steatosis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. In another embodiment, the liver disorder is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. In a particularly preferred embodiment, the liver disorder is non-alcoholic steatohepatitis (NASH).

[0031] In yet another embodiment, the present invention provides the use of a compound according to any one of formulas 1 to 6 in the manufacture of a medicament for treating liver disorders. In a preferred embodiment, the liver disorder is selected from: liver fibrosis, alcohol-induced fibrosis, alcoholic steatosis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0032] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention that are considered clinically and / or veterinarily acceptable. Examples of pharmaceutically acceptable salts and their common preparation methods can be found in "Handbook of Pharmaceutical Salts: Properties, Selection and Use" by P. Stahl et al., 2nd Revised Edition, Wiley-VCH, 2011 and S.M. Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19.

[0033] The pharmaceutical compositions for use in the present invention can be prepared using pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" for a pharmaceutical composition refers to one or more carriers, diluents, and excipients that are compatible with the other additives of the composition or formulation and are not harmful to the patient. Examples of pharmaceutical compositions and their preparation methods can be found in "Remington: The Science and Practice of Pharmacy", edited by Loyd, V. et al., 22nd Edition, Mack Publishing Co., 2012. Non-limiting examples of pharmaceutically acceptable carriers, diluents, and excipients include the following: saline, water, starches, sugars, mannitol, and silica derivatives; binders (such as carboxymethyl cellulose and other cellulose derivatives), alginates, gelatin, and polyvinyl-pyrrolidone; kaolin and bentonite; polyethylene glycol.

[0034] As used herein, the term "effective amount" refers to an amount, as a dose, that is effective in treating a condition, such as a liver disease including liver inflammation, fibrosis, and steatohepatitis. A caregiver, as a person skilled in the art, can readily determine the effective amount by using conventional techniques and by observing results obtained in similar circumstances. When determining the effective amount or dose of a compound, many factors are considered, including but not limited to: whether the compound or its salt will be administered; co-administration of other agents (if used); the species of mammal; its size, age, and general health; the extent or severity of the condition involved; the response of the individual patient; the mode of administration; the bioavailability characteristics of the formulation administered; the dosing regimen selected; the use of other concomitant medicaments; and other relevant circumstances.

[0035] As used herein, the term "treating / to treat / treatment" includes slowing, reducing, or reversing the progression or severity of an existing symptom, condition, state, or disease, which may include treating liver diseases such as liver inflammation, fibrosis, and steatohepatitis.

[0036] As used herein, the term "patient" refers to a mammal, poultry, or fish. Preferably, the patient is a human or a companion mammal such as a dog or a cat, or other domesticated mammals such as cows, pigs, horses, sheep, rabbits, and goats.

[0037] A treating physician, veterinarian, or other medical personnel will be able to determine an effective amount of the compound for treating a patient in need. Preferred pharmaceutical compositions can be formulated as tablets or capsules for oral administration, solutions for oral administration, or injectable solutions. The tablets, capsules, or solutions may include the compound of the present invention in an amount effective to treat the patient in need.

[0038] The abbreviations used herein are defined according to Daub G.H. et al., "The Use of Acronyms in Organic Chemistry", Aldrichimica Acta, 1984, 17(1), 6-23. Other abbreviations are defined as follows: "ACN" refers to acetonitrile; "AUC" refers to area under the curve; "Boc" represents tert-butoxycarbonyl; "DCM" refers to dichloromethane; "DIPEA" refers to N,N-diisopropylethylamine; "DMF" refers to dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA" refers to ethylene glycol tetraacetic acid; "ES / MS" refers to electrospray mass spectrometry; "EtOAc" refers to ethyl acetate; "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; "HPLC" refers to high performance liquid chromatography; "hr or hrs" refers to hour / hours; "HRP" refers to horseradish peroxidase; "IC 50 " refers to the reagent concentration that produces 50% of the maximum inhibitory response of the said reagent (relative to IC 50 ), or the reagent concentration that produces 50% inhibition of the target activity compared to a placebo control (absolute IC 50 ); "MAOa and MAOb" refer to monoamine oxidase a and b isoforms, respectively; "MeOH" refers to methyl alcohol or methanol; "min" refers to minute; "MS" refers to mass spectrometry; "PE" refers to petroleum ether; "R t " refers to retention time; "SSAO" refers to semicarbazide-sensitive amine oxidase; and "hSSAO" refers to human SSAO.

[0039] The compounds of the present invention or salts thereof can be prepared by a variety of procedures, some of which are illustrated in the preparations and examples below. The products of each step in the procedures below can be recovered by conventional methods, including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization. Unless otherwise noted, the reagents and starting materials are readily available.

[0040] In the preparations described herein, hydroxy and amino functional groups can be protected to facilitate the synthesis of the compounds described herein. Examples of protecting functional groups can be found in "Greene's Protective Groups in Organic Synthesis," Wuts P.G.M. et al., 5th Edition, John Wiley and Sons, 2014. Other functional groups that can be readily converted to hydroxy or amino can be used. Such functional groups, their preparations, and the conversions of these groups can be found in "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" by Larock R.C., Wiley VCH, 1999 and "March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure," Smith M.B., 7th Edition, Wiley-Interscience, 2013. Detailed Description

[0041] Chemical Synthesis Section

[0042] The following preparations and examples further illustrate the present invention and represent typical syntheses of the compounds of the present invention.

[0043] Preparation 1

[0044] (3S)-tert-Butyl 3-methoxypyrrolidine-1-carboxylate

[0045] Iodomethane (0.398 g, 2.80 mmol) was added to a mixture of (3S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (0.500 g, 2.67 mmol) and sodium hydride (60% by mass in mineral oil) (0.160 g, 4.01 mmol) in DMF (5 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated aqueous NH4Cl (30 mL) and extracted with EtOAc (3 × 30 mL). The aqueous layer was discarded. The organic extracts were combined, washed with brine, dried over Na2SO4, filtered and the filtrate was evaporated to dryness to give the title compound (475 mg, 0.475 g, 88.4%). The crude material could be used in the next step without further purification. ES / MS (m / z): 224.2 (M+Na).

[0046] Preparation 2

[0047] (3S)-3-Methoxypyrrolidine

[0048] A solution of (3S)-tert-butyl 3-methoxypyrrolidine-1-carboxylate (475 mg, 2.36 mmol) and trifluoroacetic acid (1 mL, 13.23 mmol) in DCM (3 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo to give the title compound (240 mg, 2.35 mmol, 99.5%), which could be used in the next step without further purification.

[0049] Preparation 3

[0050] 1-(5-Benzyloxypyrimidin-2-yl)piperidin-4-ol

[0051]

[0052] A mixture of 5-benzyloxy-2-chloropyrimidine (2.30 g, 9.90 mmol), piperidin-4-ol (1.23 g, 11.9 mmol) and DIPEA (3.88 mL, 29.7 mmol) in DMF (30 mL, 388 mmol) was stirred at 100 °C under N2 atmosphere for 17 h. The mixture was diluted with water (200 mL) and extracted with EtOAc (3 × 50 mL). The organic extracts were combined; washed with brine (3 × 50 mL), dried over Na2SO4, filtered and the filtrate was concentrated. The residue was subjected to silica gel flash column chromatography eluting with a mixture of 60% EtOAc and 40% PE to give the title compound as a white solid (2.00 g, 6.31 mmol, 63.7%). 11H NMR (400 MHz, CDCl3) δ 1.40 - 1.60 (m, 2H), 1.90 - 1.99 (m, 2H), 3.17 - 3.29 (m, 2H), 3.85 - 3.95 (m, 1H), 4.25 - 4.39 (m, 2H), 5.09 (s, 2H), 7.38 - 7.52 (m, 5H), 8.14 (s, 2H).

[0053] Preparation 4

[0054] 2-(4-Hydroxy-1-piperidinyl)pyrimidin-5-ol

[0055]

[0056] Palladium (10 wt% on carbon, 600 mg, 0.564 mmol) was added to 1-(5-benzyloxypyrimidin-2-yl)piperidin-4-ol (2.00 g, 6.31 mmol) in MeOH (40 mL, 989 mmol). The resulting mixture was stirred at 15 °C under a hydrogen atmosphere (103 kPa) for 2 h. The resulting mixture was filtered through celite. The filtrate was concentrated to afford the title compound as a yellow solid (0.70 g, 3.59 mmol, 56.8%). 1 1H NMR (400 MHz, CDCl3) δ 1.23 - 1.38 (m, 2H), 1.60 - 1.77 (m, 2H), 2.65 - 2.90 (m, 2H), 3.00 - 3.25 (m, 2H), 3.50 - 3.70 (m, 2H), 4.05 - 4.16 (m, 1H), 7.90 (s, 2H).

[0057] Preparation 5

[0058] tert-Butyl N-[(E)-2-[(2-chloropyrimidin-5-yl)oxymethyl]-3-fluoroallyl]carbamate

[0059]

[0060] K2CO3 (5.71 g, 41.3 mmol) was added to a solution of 2-chloropyrimidin-5-ol (5.01 g, 38.3 mmol) and tert-butyl N-[(E)-2-(bromomethyl)-3-fluoroallyl]carbamate (3.67 g, 13.7 mmol) in DMF (25 mL). The resulting solution was stirred at room temperature for 12 h. The reaction was quenched by the addition of water (80 mL) and EtOAc (100 mL). The organic and aqueous phases were separated. The aqueous phase was extracted with EtOAc (3 × 100 mL). All the organic extracts were combined. The combined organic extracts were dried over Na2SO4, filtered and the filtrate was concentrated in vacuo to give a residue. The residue was subjected to flash chromatography on silica gel eluting with a mixture of 30% EtOAc in hexanes to afford the title compound as a white solid (4.15 g, 13.1 mmol, 96% yield). ES / MS (m / z): 340 (M+Na).

[0061] Preparation 6

[0062] tert-Butyl N-[(Z)-3-fluoro-2-[[2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]allyl]carbamate

[0063]

[0064] A mixture of 2-(4-hydroxypiperidin-1-yl)pyrimidin-5-ol (400 mg, 2.05 mmol), tert-butyl N-[(Z)-2-(bromomethyl)-3-fluoroallyl]carbamate (1.10 g, 4.02 mmol) and K2CO3 (0.858 g, 6.15 mmol) in DMF (10 mL) was stirred at 60 °C for 3 h. The resulting mixture was diluted with EtOAc (50 mL). The mixture was washed successively with water (100 mL) then brine (2 × 50 mL). The organic phase was dried over Na2SO4, filtered and concentrated to give a residue. The residue was subjected to flash column chromatography on silica gel eluting with a gradient of 65 - 75% EtOAc in PE to afford the title compound as a yellow gum (558 mg, 1.46 mmol, 71.2%). 1 1H NMR (400 MHz, CDCl3) δ 1.25 (s, 9H), 1.40 - 1.65 (m, 4H), 1.89 - 1.98 (m, 2H), 3.23 - 3.30 (m, 2H), 3.91 (s, 1H), 3.89 - 3.97 (m, 1H), 4.36 - 4.40 (m, 2H), 4.64 (s, 2H), 4.76 (br, 1H), 6.62 (d, J = 84.0 Hz, 1H) 8.10 (s, 2H).

[0065] Preparation 7

[0066] tert-Butyl N-[(E)-3-fluoro-2-[[2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]allyl]carbamate

[0067]

[0068] tert-Butyl N-[(E)-2-[(2-chloropyrimidin-5-yl)oxymethyl]-3-fluoroallyl]carbamate (21.3 g, 67.0 mmol), piperidin-4-ol (25.0 g, 235 mmol) and DIPEA (41 mL, 235 mmol) were combined in 1,4-dioxane (200 mL). The resulting mixture was heated to 105 °C for 7 h under a N2 atmosphere. The mixture was concentrated and partitioned between water (100 mL) and EtOAc (150 mL), and then the phases were separated. The aqueous phase was extracted with EtOAc (100 mL). All the organic phases were combined. The combined organic phases were washed with brine (100 mL), dried over Na2SO4, filtered and the filtrate was concentrated to give a residue. The residue was dissolved in EtOAc (21 mL), and then heptane (126 mL) was added dropwise. The resulting mixture was stirred at room temperature for 20 h. The mixture was filtered to collect the solid, and the solid was rinsed with EtOAc / heptane (5:1, 21 mL). The solid was dried in vacuo to give the title compound (23.5 g, 61.5 mmol, 91.7%). ES / MS (m / z): 383 (M+H).

[0069] Preparation 8

[0070] tert-Butyl N-[(E)-3-fluoro-2-[[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]allyl]carbamate

[0071]

[0072] tert-Butyl N-[(E)-2-[(2-chloropyrimidin-5-yl)oxymethyl]-3-fluoroallyl]carbamate (4.10 g, 12.9 mmol) was divided into two equal portions (2.05 g + 2.05 g) and each was placed in a separate microwave vial (20 mL). 4-Methoxypiperidine (4.31 g, 37.3 mmol), 1,4-dioxane (30 mL, 15 mL) and DIPEA (6 mL, 34.4 mmol, 3 mL) were added to each vial. The vials were flushed with N2 gas, sealed and heated to 120 °C by microwave for 12 h. The two reaction mixtures were combined and concentrated in vacuo to give a residue. The residue was subjected to flash chromatography on silica gel eluting with a 30% mixture of EtOAc in hexanes to afford the title compound as a yellow oil (4.24 g, 10.2 mmol, 79%). ES / MS (m / z): 397 (M+H).

[0073] Preparation 9

[0074] tert-Butyl N-[(E)-3-fluoro-2-[[2-[(3S)-3-methoxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]allyl]carbamate

[0075]

[0076] tert-Butyl N-[(E)-2-[(2-chloropyrimidin-5-yl)oxymethyl]-3-fluoroallyl]carbamate (400 mg, 1.26 mmol) was added to a mixture of (3S)-3-methoxypyrrolidine (240 mg, 2.37 mmol) and K2CO3 (0.696 g, 5.04 mmol) in 1,4-dioxane (5 mL). The resulting mixture was stirred at 120 °C under microwave conditions for 12 h. The mixture was concentrated in vacuo to give the title compound as a crude material which could be used in the next step without further purification (481 mg, 1.26 mmol, 99.9%). ES / MS (m / z): 383.2 (M+H).

[0077] Preparation 10

[0078] tert-Butyl N-[(E)-3-fluoro-2-[[2-[(3S)-3-hydroxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]allyl]carbamate

[0079]

[0080] tert-Butyl N-[(E)-2-[(2-chloropyrimidin-5-yl)oxymethyl]-3-fluoroallyl]carbamate (594.3 mg, 1.87 mmol) and (3S)-pyrrolidin-3-ol (488.9 mg, 5.61 mmol) were dissolved in 1,4-dioxane (15 mL) and DIPEA (3 mL, 17.2 mmol). The solution was flushed with N2 gas, the container was sealed, and the mixture was heated to 120 °C for 12 h by microwave. The resulting mixture was concentrated in vacuo to give a residue. The residue was subjected to silica gel flash chromatography eluting with a gradient of 80 - 90% EtOAc in hexanes to afford the title compound as a yellow foam (608.8 mg, 1.57 mmol, 84%). ES / MS (m / z): 369 (M+H).

[0081] Example 1

[0082] 1-[5-[(Z)-2-(Aminomethyl)-3-fluoroallyloxy]pyrimidin-2-yl]piperidin-4-ol hydrochloride

[0083]

[0084] tert-Butyl N-[(Z)-3-fluoro-2-[[2-(4-hydroxy-1-piperidinyl)pyrimidin-5-yl]oxymethyl]allyl]carbamate (558 mg, 1.46 mmol) was added to HCl (4 mol / L) and MeOH (10 mL). The resulting mixture was stirred at 10 °C for 1.5 h. The mixture was concentrated under reduced pressure to give a residue. The residue was subjected to a preparative HPLC column: (Phenomenex Synergi C18 150 30 mm, 4 μm) eluting with a gradient of 10 to 15% 0.05% aqueous HCl in ACN; flow rate: 25 mL / min, R t : 5.95 min to afford the title compound as a yellow solid with a Z:E ratio greater than 20:1 (456 mg, 1.43 mmol, 98.0%). ES / MS (m / z): 283.1 (M+H), 1 1H NMR (400 MHz, d4-MeOD) δ 1.56 - 1.78 (m, 2H), 1.92 - 2.13 (m, 2H), 3.61 - 3.72 (m, 2H), 3.75 (s, 2H), 3.95 - 4.06 (m, 1H), 4.11 - 4.25 (m, 2H), 4.89 - 4.93 (m, 2H), 7.19 (d, J = 80.4 Hz, 1H), 8.47 (s, 2H).

[0085] Example 2

[0086] 1-[5-[(E)-2-(Aminomethyl)-3-fluoro-allyloxy]pyrimidin-2-yl]piperidin-4-ol dihydrochloride

[0087]

[0088] tert-Butyl N-[(E)-3-fluoro-2-[[2-(4-hydroxy-1-piperidinyl)pyrimidin-5-yl]oxymethyl]allyl]carbamate (31 g, 81.07 mmol), MeOH (20 mL, 494 mmol), and HCl were combined in MeOH (120 mL, 4 mol / L, 486.4 mmol). The resulting mixture was stirred for 30 h at room temperature under a N2 atmosphere. EtOAc (250 mL) was added dropwise and the mixture was stirred for 30 min. The mixture was filtered to collect the solid, the solid was rinsed with EtOAc (20 mL), and the solid was dried in vacuo to afford the title compound (26.4 g, 72.8 mmol, 89.8%) with an E:Z ratio greater than 20:1. ES / MS (m / z): 283 (M+H), 1 H NMR (500 MHz, d6-DMSO) δ 1.28 - 1.36 (m, 2H), 1.73 - 1.76 (m, 2H), 3.18 - 3.25 (m, 2H), 3.55 - 3.60 (m, 2H), 3.68 - 3.75 (m, 1H), 4.18 (dt, J = 13.5, 4.5 Hz, 2H), 4.65 (d, J = 3.0 Hz, 2H), 6.55 - 7.10 (br, 2H), 7.27 (d, J = 82.0 Hz, 1H), 8.27 (s, 2H), 8.32 - 8.45 (br, 3H), 19 F NMR (500 MHz, d6-DMSO) δ 122.2 (s).

[0089] Example 3

[0090] (E)-3-Fluoro-2-[[2-(4-methoxy-1-piperidinyl)pyrimidin-5-yl]oxymethyl]prop-2-en-1-amine, dihydrochloride

[0091]

[0092] Dissolve tert-butyl N-[(E)-3-fluoro-2-[[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]allyl]carbamate (4.2374 g, 10.69 mmol) in MeOH (100 mL, 50 mmol, 0.5 mol / L). Heat the resulting clear solution to 60 °C for 4 h. Concentrate the mixture in vacuo to give a residue (3.95 g). Suspend the residue in MeOH (7 mL) and reflux the mixture to give a clear solution. Cool the solution to room temperature to give needle crystals, and then cool the mixture to -20 °C. Filter the mixture to collect the solid, wash the solid with cold MeOH to give the title compound as pale yellow crystals (2.73 g, 7.01 mmol, 66%). The resulting yellow crystals can be further purified by recrystallization via the same procedure described above to give the title compound as a colorless crystalline substance with an E:Z ratio greater than 20:1. ES / MS (m / z): 297 (M+H), 1 H NMR (500 MHz, d6-DMSO) δ 1.33 - 1.40 (m, 2H), 1.84 - 1.89 (m, 2H), 3.26 (dt, J = 13.5, 9.5 Hz, 2H), 3.27 (s, 3H), 3.40 - 3.45 (m, 1H), 3.56 - 3.62 (m, 2H), 4.11 (dt, J = 13.5, 5.0 Hz, 2H), 4.62 (d, J = 3.0 Hz, 2H), 5.26 - 5.94 (br, 1H), 7.27 (d, J = 82.0 Hz, 1H), 8.26 (s, 2H), 8.21 - 8.31 (br, 3H), 19 F NMR (500 MHz, d6-DMSO) δ 122.1 (s).

[0093] Example 4

[0094] (E)-3-fluoro-2-[[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]prop-2-en-1-amine

[0095]

[0096] Dissolve N-[(E)-3-fluoro-2-[[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxymethyl]allyl]carbamate (167.0 mg, 0.42 mmol) in a 0.95 M HCl solution in 18 mL of EtOAc / MeOH (10:1 v / v). Stir the mixture overnight. Concentrate the resulting suspension in vacuo and dissolve the residue in water. Subject the residue to preparative HPLC; LC column: C18 30×150 mm 5 μm; gradient elution with 14 to 24% 10 mM NH4HCO3 aqueous solution in ACN for 0 - 11 minutes; column temperature: room temperature; flow rate: 35 mL / min, R t = 7.8 minutes, stop after 17 minutes by UV monitoring. Collect and concentrate the appropriate eluate fractions to obtain a residue as an oil. Dissolve the residue in water and lyophilize to obtain the title compound as a white solid with an E:Z ratio greater than 20:1 (97 mg, 0.31 mmol, 74%, 95% purity). ES / MS (m / z): 297 (M+H), 1 1H NMR (500 MHz, d6-DMSO) δ 1.33 - 1.41 (m, 2H), 1.52 - 1.69 (br, 1H), 1.83 - 1.89 (m, 2H), 3.23 - 3.29 (m, 4H), 3.27 (s, 3H), 3.30 - 3.35 (br, 1H), 3.38 - 3.44 (m, 1H), 4.11 (dt, J = 13.5, 4.5 Hz, 2H), 4.55 (d, J = 4.5 Hz, 2H), 6.93 (d, J = 85.0 Hz, 1H), 8.22 (s, 2H), 19 19F NMR (500 MHz, d6-DMSO) δ 131.8 (s).

[0097] Example 4a

[0098] (2E)-3-Fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate (1:1)

[0099]

[0100] Dissolve (E)-3-fluoro-2-[[2-(4-methoxy-1-piperidinyl)pyrimidin-5-yl]oxymethyl]prop-2-en-1-amine (2.316 g, 7.81 mmol) in methyl acetate (3 mL) and stir at 1000 rpm at room temperature to obtain a pale yellow solution. Add 4-toluenesulfonic acid monohydrate (1.62 g, 8.43 mmol) to a solution of methyl acetate (4 mL). The mixture becomes turbid and rapidly forms a viscous yellow slurry. Filter the solid by vacuum filtration through a filter paper. Wash the filter cake with methyl acetate (4 mL) to obtain a white filter cake. Dry the solid under a vacuum air stream for 10 minutes and then dry overnight in a vacuum oven at room temperature to obtain the title compound (3.00 g, 81.8%).

[0101] X-ray powder diffraction of Example 4a

[0102] The X-ray powder diffraction (XRD) pattern of crystalline (2E)-3-fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate was obtained on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKa source and a Vantec detector, operating at 35 kV and 50 mA. The sample was scanned between 4 and 40° 2θ with a step size of 0.009° 2θ and a scan rate of 0.5 seconds / step, and with 0.6 mm divergence, 5.28 fixed anti-scatter, and 9.5 mm detector slit. The dry powder was filled in a quartz sample holder and a smooth surface was obtained using a glass slide. The diffraction pattern of the crystal form was collected at ambient temperature and relative humidity. It is well known in the art of crystallography that for any given crystal form, the relative intensities of the diffraction peaks can vary due to preferred orientation resulting from factors such as crystal morphology and habit. In the presence of the effect of preferred orientation, the peak intensities vary, but the characteristic peak positions of the polymorph remain unchanged. See, for example, The United States Pharmacopeia, 23rd Edition, The National Formulary, 18th Edition, pages 1843 - 1844, 1995. In addition, it is also well known in the art of crystallography that for any given crystal form, the angular peak positions can vary slightly. For example, the peak positions may shift due to the temperature or humidity at which the sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, peak position variations of ± 0.2 2θ will be taken into account for these possible variations without preventing the clear identification of the indicated crystal form. Confirmation of the crystal form can be based on any unique combination of distinguishing peaks (in ° 2θ), usually the more prominent peaks. The diffraction pattern of the crystal form was collected at ambient temperature and relative humidity and adjusted based on the NIST 675 standard peaks at 8.853 and 26.774° 2θ

[0103] The sample of prepared (2E)-3-fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate is characterized by an XRD pattern using CuKa radiation and having the diffraction peaks (2θ values) described in Table 1 below, and in particular having a combination of a peak at 18.6 with one or more of the peaks selected from 22.4, 19.1, and 21.0; having a tolerance of diffraction angle of 2θ of + / - 0.2°, or the salt may be characterized by an XRD pattern having one or more peaks at 2θ of 18.6, 19.1, 21.0, 21.9, and 22.4 + / - 0.2°, or 2θ of 17.6, 11.0, 16.8, 18.6, 19.1, 21.0, 21.9, 22.4, and 26.1 + / - 0.2°

[0104] Table 1

[0105] X-ray powder diffraction peaks of (2E)-3-fluoro-2-({[2-(4-methoxypiperidin-1-yl)pyrimidin-5-yl]oxy}methyl)prop-2-en-1-amine 4-methylbenzenesulfonate

[0106] Peak Angle (°2θ) + / - 0.2° Relative intensity (% of the strongest peak) 1 7.4 17.6％ 2 11.0 23.4％ 3 12.7 5.2％ 4 16.8 10.1％ 5 18.6 100.0％ 6 19.1 42.2％ 7 21.0 41.9％ 8 21.9 31.6％ 9 22.4 77.5％ 10 26.2 18.6％

[0107] Example 5

[0108] (E)-3-fluoro-2-[[2-[(3S)-3-methoxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]prop-2-en-1-amine

[0109]

[0110] A solution of tert-butyl N-[(E)-3-fluoro-2-[[2-[(3S)-3-methoxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]allyl]carbamate (481 mg, 1.26 mmol) and trifluoroacetic acid (1 mL, 13.23 mmol) in DCM (3 mL) was stirred at room temperature for 1 h. The mixture was concentrated in vacuo. The residue was subjected to preparative HPLC: LC column: C18 30×150 mm 5 μm; eluted with 5% 10 mM aqueous NH4HCO3 in ACN for 0 - 2 min, followed by a gradient of 6 - 11% 10 mM aqueous NH4HCO3 in ACN over 2 - 12 min; stopped at 18 min; column temperature: room temperature; flow rate: 35 mL / min, R t = 10.6 min; monitored by UV to give the title compound as a white solid (226 mg, 61.7%) with an E:Z ratio greater than 20:1. ES / MS (m / z): 283.1 (M+H), 1 1H NMR (500 MHz, CDCl3) δ 1.12 - 1.78 (br, 2H), 2.07 - 2.16 (m, 2H), 3.37 (s, 3H), 3.53 - 3.68 (m, 6H), 4.07 (m, 1H), 4.44 (s, 2H), 6.57 (d, J = 83.0 Hz, 1H), 8.12 (s, 2H).

[0111] Example 6

[0112] (3S)-1-[5-[(E)-2-(aminomethyl)-3-fluoro-allyloxy]pyrimidin-2-yl]pyrrolidin-3-ol; dihydrochloride

[0113]

[0114] Dissolve tert-butyl N-[(E)-3-fluoro-2-[[2-[(3S)-3-hydroxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]allyl]carbamate (605.9 mg, 1.65 mmol) in a mixture of HCl in MeOH (30 mL, 15 mmol, 0.5 mol / L) and HCl in water (5 mL, 60 mmol, 12 mol / L). Stir the resulting clear solution overnight. Concentrate the reaction mixture under vacuum to give a residue. Subject the residue to preparative HPLC: LC column: C18 30×150 mm 5 μm; H2O 10 mM NH4HCO3; at room temperature; elute with 2% ACN for 0 - 2 minutes, followed by a gradient of 2 - 10% ACN over 2 - 10 minutes; flow rate at: 35 mL / min, R t = 8.0 minutes (monitored via UV detection); stop at 16 minutes. Collect the appropriate eluate fractions and concentrate to give the free base of the title compound. Dissolve the free base compound in 0.5 M HCl in MeOH (15 mL). Concentrate the solution, add water, and then lyophilize to give the title product as a pale yellow solid with an E:Z ratio greater than 20:1 (352.7 mg, 0.982 mmol, 59%). ES / MS (m / z): 269 (M+H), 1 1H NMR (500 MHz, d6-DMSO) δ 1.87 - 1.94 (m, 1H), 1.98 - 2.05 (m, 1H), 3.44 (d, J = 11.5 Hz 1H), 3.51 - 3.61 (m, 5H), 4.39 - 4.42 (m, 1H), 4.66 (d, J = 3.0 Hz, 2H), 5.33 - 5.90 (br, 2H), 7.28 (d, J = 82.0 Hz, 1H), 8.35 (s, 2H), 8.35 - 8.44 (br, 3H), 19 19F NMR (500 MHz, d6-DMSO) δ 121.9 (s).

[0115] Alternative preparation of Example 6

[0116] tert-Butyl N-[(E)-3-fluoro-2-[[2-[(3S)-3-hydroxypyrrolidin-1-yl]pyrimidin-5-yl]oxymethyl]allyl]carbamate (1.1601 g, 3.15 mmol) was dissolved in a solution of HCl in EtOAc (50 mL, 50 mmol, 1.0 mol / L) (pre-mixed with 0.5 mol / L HCl in MeOH, 5 mL). The resulting solution was stirred overnight. The white suspension was concentrated in vacuo to give a white powder. The white powder was dissolved in water and the solution was lyophilized to give the title compound as a pale yellow solid (860.7 mg, 2.42 mmol, 77%).

[0117] The material prepared as in Example 6 was dissolved in water (5 mL) and combined with the material of Alternative Example 6 in water (5 mL). The mixture was lyophilized to give the title compound with an E:Z ratio greater than 20:1 (1.151 g, 3.27 mmol). ES / MS m / z: 269 (M+H), 1 H NMR (500 MHz, d6-DMSO) δ 1.87 - 1.94 (m, 1H), 1.98 - 2.05 (m, 1H), 3.44 (d, J = 11.5 Hz, 1H), 3.51 - 3.61 (m, 5H), 4.39 - 4.42 (m, 1H), 4.66 (d, J = 3.0 Hz, 2H), 5.33 - 5.90 (br, 2H), 7.28 (d, J = 82.0 Hz, 1H), 8.35 (s, 2H), 8.35 - 8.44 (br, 3H), 19 F NMR (500 MHz, d6-DMSO) δ 121.9 (s).

[0118] Bioanalysis

[0119] SSAO / VAP-1 in vitro activity

[0120] The amine oxidase activities of recombinant SSAO, MAOa, and MAOb isoforms were measured using the MAO-Glo TM assay kit from Promega (V1402). The test compounds (using DMSO as a vehicle, 0.5% v / v for SSAO) and the enzymes were incubated for 10 minutes at room temperature, followed by the addition of the luminescent substrate. The substrate concentration for human recombinant SSAO was 10 μM. The assay was performed in a microplate in pH 7.4 buffer (50 mM HEPES, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.4 mM MgCl2, 0.001% Tween-20). Substrate oxidation was carried out for 2 hours, followed by the addition of the detection reagent according to the manufacturer's protocol. The IC 50Values were calculated by fitting the dose - response curve using a 4 - parameter non - linear regression program. The IC 50 values of the compounds of Examples 3, 5, and 6 are listed in Table 2.

[0121] Table 2

[0122] Example <![CDATA[hSSAO inhibition relative to IC 50 (nM)]]> 3 12±1,n＝5 5 32±7,n＝4 6 52±5,n＝7

[0123] Data are presented as the mean ± SEM (SEM = standard error of the mean) of the analyzed number (n).

[0124] The compounds of the examples showed an IC 50 of hSSAO less than 60 nM. The compounds of the examples showed an IC 50 of hMAOa and hMAOb greater than 50 μM and 200 μM, respectively, indicating that the compounds of the examples are selective for hSSAO over hMAOa or hMAOb.

[0125] SSAO target engagement

[0126] Using MAO - Glo from Promega (V1402) TMThe assay kit measures SSAO activity in rat plasma and liver tissues. The residual SSAO activity in rats after compound treatment was estimated by measuring the total amine oxidase activity in plasma or liver lysates that was insensitive to the presence of the MAO inhibitors clorgyline and pargyline. The compound of Example 2 was administered to rats at doses of 15, 3, 0.6, 0.12, 0.025, 0.005 mg / kg. The control group was administered the same volume (2 ml / kg) of the dosing vehicle (1% w / v hydroxyethylcellulose, 0.25% Tween 80). Plasma and liver were collected 2 or 24 hours after compound treatment and stored at -78 °C until analysis. Tissue lysates were prepared by homogenization in lysis buffer (20 mM HEPES, pH 7.4; 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and 1× Roche Complete protease inhibitor tablet). Tissue pellets were removed by centrifugation at 12,000 rpm for 30 minutes at 4 °C. 40 μl of plasma or liver lysate was incubated with clorgyline (10 μM) and pargyline (10 μM) for 20 minutes at room temperature, after which the luminescent substrate (50 μM) was added for 60 minutes. The resulting product was quantified according to the manufacturer's procedure. The activity fraction insensitive to the presence of the MAO inhibitor was used as a surrogate for residual SSAO activity. The compound of Example 2 administered at various doses was evaluated in a protocol substantially as described above. The results are presented in Table 3.

[0127] Table 3

[0128] SSAO target engagement of Example 2

[0129]

[0130] Data are presented as mean ± SEM, n = 6

[0131] The results showed that the compound of Example 2 inhibited SSAO activity in a dose-dependent manner in rat plasma and liver.

[0132] Mouse model of 3H diet-induced NASH and fibrosis

[0133] Male C57BL / 6N mice were fed a diet of D09100301 (research diet, 40% fat, 2% cholesterol, 24% fructose, (high-fat, high-cholesterol and high-fructose, "3H diet")) for 150 days. Subsequently, each mouse was housed individually after a 5-day adaptation period. Plasma alanine aminotransferase (ALT) and cytokeratin 18 (CK18) were measured. After one week of recovery, the mice were randomly divided into 5 groups based on their ALT values, CK18 values and body weights. Each group of animals was administered a vehicle (0.5% methylcellulose (MC) + 0.25% Tween 80 in distilled water) or the compound of Example 6 (at doses of 0.06, 2, 6 and 20 mg / kg) once daily, at a volume of 5 ml / kg, for 11 weeks.

[0134] Blood was collected from the mice treated with the compound of Example 6 for 76 days 2 hours after the last dose. The compound content in the plasma was analyzed by mass spectrometry. The results are listed in Table 4 below. The treated mice showed a dose-dependent increase in plasma compound content. All groups of mice treated with Example 6 showed a significant decrease in ALT, indicating reduced liver lesions in those animals. Animals treated with the compound of Example 6 at 6 and 20 mg / kg also showed reduced triglyceride content in the blood.

[0135] At the end of the study, the animals were sacrificed and their livers excised. Two sections from the left and right lobes were fixed in neutral buffered 10% formalin. Liver tissue slides were stained with hematoxylin and eosin (H&E), Sirius red and Masson's Trichrome to prepare slides for pathological analysis. All specimens were examined microscopically and scored according to the modified Brunt score NASH Activity Score. The score was based on the grading scheme and endpoints described by Brunt E.M. et al., "Histopathology of nonalcoholic fatty liver disease," World J. of Gastroenterol, 2010, 16(42), 5286 - 5296. Group means were then calculated for each individual endpoint. The following endpoints were used to characterize the fast food model of NASH in mice modified from the NASH endpoints (see Brunt E.M. "Histopathology of nonalcoholic fatty liver disease," Clin Liver Dis., 2009, 13, 533 - 544 and Brunt E.M. et al., "Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions," Am J Gastroenterology, 1999, 94(9), 2467 - 2474).

[0136] The histopathological analysis of the livers from mice treated with the compound of Example 6 is provided in Table 4. The results showed a significant reduction in hepatitis, macrovesicular vacuolization, and perisinusoidal fibrosis in mice treated with 20 mg / kg of the compound of Example 6.

[0137] Table 4

[0138] Efficacy results of the compound of Example 6

[0139] Dose (mg / kg) Number of animals Pharmaceutical compound content (ng / ml) <![CDATA[ALT(IU / L) 1 > <![CDATA[Plasma TG (mg / dL) 1 > Vehicle 12 - 713±43 69±4 0.6 13 66.±5 530±45* 55±6 2 13 163±21 483±44** 70±11 6 11 560±29 495±25* 46±4* 20 11 2117±219 466±37*** 45±5*

[0140] 1The MIXED model was applied to compare the fold change from baseline adjusted for baseline between the compound treatment group and the vehicle group (see "Generalized, Linear, and Mixed Models," McCulloch C.E. and Searle S.R., eds., John Wiley and Sons, 2000 and "Mixed-Effects Models in S and S-PLUS," Pinheiro J.C. and Bates D.M., eds., Springer, 2000). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

[0141] Table 5

[0142] Histopathological analysis of the liver of the compound of Example 6

[0143]

[0144]

[0145] 1 Non-parametric tests were applied to compare the scores between the compound treatment group and the vehicle group. The scores on the left and right sides were compared separately. Data are presented as mean ± SEM.

[0146] *p < 0.05; **p < 0.01; ***p < 0.001.

Claims

1. A compound of formula .

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