AROMATIC COMPOUND, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF
20240083860 ยท 2024-03-14
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K31/53
HUMAN NECESSITIES
C07C271/64
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
C07D403/12
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
Abstract
The present invention discloses an aromatic compound, a method of preparation thereof and applications thereof. The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof. The compounds of the present invention have demonstrated good safety, tolerability and reduction of liver fat in mice and potential efficacy in the treatment of NASH in animal experiments in NASH disease models.
##STR00001##
Claims
1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, characterized in that the structure is as follows: ##STR00045## wherein A is O or CH.sub.2; M is ##STR00046## X and Y are independently chlorine, bromine, iodine, isotope .sup.124I or .sup.131I of I, or C.sub.1 to C.sub.6 alkyl; R.sup.1 is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, one or more fluorine-substituted C.sub.1-C.sub.6 alkyl, one or more fluorine-substituted C.sub.2-C.sub.6 alkenyl, one or more deuterium-substituted C.sub.1-C.sub.6 alkyl, or one or more deuterium-substituted C.sub.2-C.sub.6 alkenyl; R.sup.2 is C.sub.2-C.sub.6 alkenyl, one or more fluorine-substituted C.sub.1-C.sub.6 alkyl, one or more fluorine-substituted C.sub.2-C.sub.6 alkenyl, one or more deuterium-substituted C.sub.1-C.sub.6 alkyl, or one or more deuterium-substituted C.sub.2-C.sub.6 alkenyl.
2. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that X and Y are independently chlorine, bromine, iodine or CH.sub.3; and/or, R.sup.1 is C.sub.2 to C.sub.6 alkenyl or one or more fluorine substituted C.sub.1 to C.sub.6 alkyl; and/or, R.sup.2 is one or more fluorine-substituted C.sub.1 to C.sub.6 alkyl.
3. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, when R.sup.1 is C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00047## and/or, when R.sup.1 is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; and/or, when R.sup.1 is one or more fluorine-substituted C.sub.1 to C.sub.6 alkyl, the C.sub.1 to C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; said one or more being 1 or 3; and/or, when R.sup.1 is one or more deuterium substituted C.sub.1 to C.sub.6 alkyl, the C.sub.1 to C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; said one or more being 1 or 3; and/or, when R.sup.1 is one or more fluorine substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00048## and/or, when R.sup.1 is one or more deuterium substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00049## said one or more being 1 or 3; and/or, when R.sup.2 is C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00050## and/or, when R.sup.2 is one or more fluorine-substituted C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl-of or tert-butyl; said one or more being 1 or 3; and/or, when R.sup.2 is one or more deuterium-substituted C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; said one or more being 1 or 3 and/or, when R.sup.2 is one or more fluorine-substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00051## said one or more being 1 or 3; and/or, when R.sup.2 is one or more deuterium substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00052## said one or more being 1 or 3; and/or, when X is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; and/or, when Y is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.
4. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that it is defined as any one of the following schemes: Scheme 1, wherein A is O or CH.sub.2; X and Y are independently chlorine, bromine, iodine, or C.sub.1 to C.sub.6 alkyl; M is ##STR00053## R.sup.1 is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, one or more fluorine-substituted C.sub.1-C.sub.6 alkyl, one or more fluorine-substituted C.sub.2-C.sub.6 alkenyl, one or more deuterium-substituted C.sub.1-C.sub.6 alkyl, or one or more deuterium-substituted C.sub.2-C.sub.6 alkenyl, or, M is ##STR00054## R.sup.2 is one or more fluorine-substituted C.sub.1 to C.sub.6 alkyl; Scheme 2 wherein A is O; X and Y are independently chlorine, bromine, or iodine; M is ##STR00055## ; R.sup.1 is C.sub.2 to C.sub.6 alkenyl or one or more fluorine substituted C.sub.1 to C.sub.6 alkyl; Scheme 3 wherein A is O, X and Y are independently chlorine or bromine; M is ##STR00056## R.sup.1 is C.sub.2-C.sub.6 alkenyl or C.sub.1-C.sub.6 alkyl substituted by one fluorine.
5. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that R.sup.1 is ##STR00057## and/or, ##STR00058##
6. A preparation method of a compound represented by formula I, characterized in that the method comprises the following steps: in the solvent, and in the presence of alkali, the compound represented by formula II-a is subjected to the following ring-closing reaction, to obtain the compound represented by formula I; wherein the definition of M, X, Y and A is according to claim 1, and R.sup.6 is C.sub.1-C.sub.6 alkyl; ##STR00059##
7. A compound represented by formula II-a, characterized in that M, A, X and Y are defined according to claim 1, and R.sup.6 is C.sub.1-C.sub.6 alkyl; ##STR00060##
8. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises substance A and one or more pharmaceutically acceptable carriers; the substance A is the compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1.
9. An application of substance B in preparing a THR- agonist agent, characterized in that the substance B is the compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1.
10. Use of a substance B in the manufacture of a medicament for the treatment and/or prophylaxis of diseases associated with THR-, characterized in that the substance B is the compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1.
11. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, when R.sup.1 is C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00061## and/or, when R.sup.1 is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is isopropyl; and/or, when R.sup.1 is one or more fluorine-substituted C.sub.1 to C.sub.6 alkyl, the C.sub.1 to C.sub.6 alkyl is ##STR00062## said one or more being 1 or 3; and/or, when R.sup.1 is one or more deuterium substituted C.sub.1 to C.sub.6 alkyl, the C.sub.1 to C.sub.6 alkyl is ##STR00063## said one or more being 1 or 3; and/or, when R.sup.1 is one or more fluorine substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00064## and/or, when R.sup.1 is one or more deuterium substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00065## said one or more being 1 or 3; and/or, when R.sup.2 is C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00066## and/or, when R.sup.2 is one or more fluorine-substituted C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is ##STR00067## said one or more being 1 or 3; and/or, when R.sup.2 is one or more deuterium-substituted C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is ##STR00068## said one or more being 1 or 3; and/or, when R.sup.2 is one or more fluorine-substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00069## said one or more being 1 or 3; and/or, when R.sup.2 is one or more deuterium substituted C.sub.2 to C.sub.6 alkenyl, the C.sub.2 to C.sub.6 alkenyl is ##STR00070## said one or more being 1 or 3; and/or, when X is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl; and/or, when Y is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl.
12. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that R.sup.1 is ##STR00071## and/or, ##STR00072## wherein the compound represented by formula I is any one of the following compounds: ##STR00073## ##STR00074## ##STR00075## ##STR00076##
13. A compound represented by formula II-a, characterized in that M, A, X and Y are defined according to claim 1, and R.sup.6 is C.sub.1-C.sub.6 alkyl; ##STR00077## wherein the compound represented by formula II-a is any one of the following compounds: ##STR00078## ##STR00079##
14. An application of substance B in preparing a THR- agonist agent, characterized in that the substance B is the pharmaceutical composition according to claim 8.
15. Use of a substance B in the manufacture of a medicament for the treatment and/or prophylaxis of diseases associated with THR-, characterized in that the substance B is the pharmaceutical composition according to claim 8.
16. Use of a substance B in the manufacture of a medicament for the treatment and/or prophylaxis of diseases associated with THR- according to claim 10; wherein the disease is selected from one or more of non-alcoholic fatty liver disease, obesity, liver fibrosis, type-2 diabetes and primary hypercholesterolemia.
17. Use of a substance B in the manufacture of a medicament for the treatment and/or prophylaxis of diseases associated with THR- according to claim 15; wherein the disease is selected from one or more of non-alcoholic fatty liver disease, obesity, liver fibrosis, type-2 diabetes and primary hypercholesterolemia.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0081] The present invention will be further described by way of example, but this is not intended to limit the present invention to the scope of the embodiments described. In the following examples, the experimental methods of specific conditions are not specified, the compound are made according to the conventional methods and conditions, or according to the specification of the article.
EXAMPLE 1
2(3,5-dichloro-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 1)
[0082] ##STR00033##
[0083] Step 1: 2-isopropenyl benzene-1,4-diol
[0084] To a solution of 2-bromobenzene-1,4-diol (5.0 g, 226.5 mmol) in 1,4-dioxane (100 mL), potassium carbonate aqueous solution(11.0 g, 79.4 mol, 20 mL), isopropenylboronic acid pinacol ester (6.67 g, 39.7 mmol) and Pd (dppf) Cl.sub.2 (0.97 g, 1.32 mmol) was added under nitrogen atmosphere. The mixture was heated to 105 C. After stirring at 105 C. for 5 hours, the reaction mixture was cooled down to room temperature and concentrated under reduced pressure to remove 1,4-dioxane. Water (50 ml) and ethyl acetate (50 ml) was added into the mixture and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phase was combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 ml). The organic phase was dried with anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=10:1-5:1) to obtain 2.5 g of the target product in 63% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 6.80 (d, J=9.2 Hz, 1H), 6.66-6.64 (m, 2H), 5.39 (t, J=1.6 Hz, 1H), 5.30 (s, 1H), 5.14 (s, 1H), 4.50 (s, 1H), 2.09 (s, 3H).
[0085] Step 2: 4-(2,6-dichloro-4-nitrophenoxy)-2-(isopropenyl)phenol
[0086] To a solution of 2-isopropenylbenzene-1,4-diol (3.00 g, 20 mmol) in acetonitrile (50 ml), sodium carbonate (7.57 g, 71.4 mmol), and 1,3-dichloro-2-fluoro-5-nitrobenzene (3 g, 14.3 mmol) were added. The mixture was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction mixture was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 ml) and ethyl acetate (50 ml) were added into the solution and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phase was combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 ml). The organic phase was dried with anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 2.85 g of the target product in 59% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.29 (s, 2H), 6.85 (d, J=8.8 Hz, 1H), 6.67 (d, J=3.2 Hz, 1H), 6.60 (dd, J=8.8, 3.2 Hz, 1H), 5.46 (s, 1 H), 5.41 (t, J=1.2 Hz, 1H), 5.17 (s, 1H), 2.08 (s, 3H).
[0087] Step 3: 4-(4-amino-2,6-dichloro-phenoxy)-2-(isopropenyl)phenol
[0088] To a solution of 4-(2,6-dichloro-4-nitrophenoxy)-2-(isopropenyl)phenol (2.85 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL) aqueous ammonium chloride (4.48 g, 83.8 mmol) (20 mL) and iron powder (2.82 g, 50.3 mmol) were added in a reaction flask. The mixture was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction mixture was cooled down to room temperature. Water (50 ml), saturated aqueous sodium bicarbonate solution (30 ml) and ethyl acetate (50 ml) were added into the solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (30 mL3) and organic phase was combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 ml), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.42 g of product in 93% yield. [M+H].sup.+ 310.1. .sup.1H NMR (400 MHz, CDCl.sub.3): 6.81 (d, J=8.8 Hz, 1H), 6.68-6.67 (m, 3H), 6.59 (dd, J=8.8, 3.2 Hz, 1H), 5.38 (t, J=1.2 Hz, 1H), 5.36 (s, 1H), 5.15 (s, 1H), 3.72 (br s, 2H), 2.08 (s, 3H).
[0089] Step 4: (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-isopropenyl-phenoxy)phenyl)-hydrazino) acetyl)carbamic acid ethyl ester
[0090] To a solution of 6M aqueous hydrochloric acid (40 ml) in anhydrous ethanol (30 ml) was added 4-(4-amino-2,6-dichlorophenoxy)-2-(isopropenyl)phenol (2.40 g, 7.74 mmol). The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 ml) was added dropwise to the mixture. After the reaction was complete, water (30 ml) was added into the solution. During filtering, the filter cake was drenched with water (10 ml3). The filter cake was dried under reduced pressure to give 2.7 g of product in 73% yield. [M+H].sup.+: 477.2.
[0091] Step 5: 2-(3,5-dichloro-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0092] To a solution of 2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-isopropenyl phenoxy)phenyl)hydrazone)acetyl)ethyl carbamate (2.70 g, 5.66 mmol) in acetic acid (30 mL) sodium acetate (928 mg, 11.3 mmol) was added. The mixture was stirred at 118 C. for 3 hours. After the reaction was complete, the reaction mixture was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.05 g of product in 22% yield. [MH].sup.: 428.8. .sup.1HNMR (400 MHz, DMSO-d.sub.6): 13.29 (br s, 1H), 9.34 (s, 1H), 7.78 (s, 2H), 6.77 (d, J=8.8 Hz, 1H), 6.66 (d, J=3.2 Hz, 1H), 6.54 (dd, J=8.8, 3.2 Hz, 1H), 5.10 (s, 2 H), 2.05 (s, 3H).
EXAMPLE 2
2-(3,5-dichloro-4-((5-(2-fluoropropane-2-yl)-6-oxo-1,6-dihydro-pyridazin-3-yl)oxy)phenyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 2)
[0093] ##STR00034##
[0094] Step 1: 3,6-dichloro-4-(2-fluoro-propane-2-yl)-pyridazine
[0095] To a solution of 3,6-dichloropyridazine (17.6 g, 118 mmol) in concentrated sulfuric acid (17.4 g, 177 mmol) and water (110 mL) 2-fluoro-2-methylpropanoic acid (25 g, 236 mmol) was added. After stirring at 40 C. for 5 minutes, silver nitrate (2 g, 11.8 mmol) was added into the solution and the reaction was heated to 62 C. A mixed solution of ammonium persulfate (45.8 g, 210 mmol) and water (220 mL) was added dropwise at 62 C. After the reaction was stirred at 80 C. for an hour, the reaction solution was cooled down to 0-15 C. Ammonium hydroxide was added dropwise into the solution to adjust the pH of the system to 9. The reaction was extracted with diisopropyl ether (200 mL) and the organic phase was combined. The combined organic phase was washed with sodium bisulfite (100 mL). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=10:1) to obtain 15.9 g of the target product in 64.9% yield. MS (ESI) m/z: 209.0 [M+H].sup.+.
[0096] Step 2: 3,5-dichloro-4-((6-chloro-5-(2-fluoropropane-2-yl)-pyridazin-3-yl)oxy)aniline
[0097] To a solution of 3,6-dichloro-4-(2-fluoropropan-2-yl)pyridazine (7.8 g, 37.5 mmol) in DMSO (78 mL) was added 4-amino-2,6-dichlorophenol (6.67 g, 27.5 mmol), potassium carbonate (20.7 g, 150 mmol), and CuI (4.27 g, 22.5 mmol). The reaction was stirred at 90 C. for 17 h. After the reaction was completed, the reaction was cooled to room temperature. Water (500 ml) was added into the mixture. The mixture was extracted with ethyl acetate (30 mL3) and the organic phase was combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 ml). The organic phase was dried with anhydrous magnesium sulfate and filtered. The filtrate was concentrated to obtain the crude 3,5-dichloro-4-((6-chloro-5-(2-fluoropropan-2-yl)pyridazin-3-yl)oxy)aniline (12.5 g). MS (ESI) m/z: 350.0 [M+H].sup.+.
[0098] Step 3: N-(3,5-dichloro-4-((5-(2-fluoropropane-2-yl)-6-oxo-1,6-dihydro-pyridazin-3-yl)oxy)phenyl)acetamide
[0099] To a solution of 3,5-Dichloro-4-((6-chloro-5-(2-fluoropropan-2-yl)pyridazin-3-yl)oxy)aniline (12.4 g, 35.8 mL) in glacial acetic acid, sodium acetate (10.3 g, 125 mmol) was added. The reaction was stirred at 100 C. for 16 h. After the reaction was completed, the reaction solution was cooled down to room temperature and the pH of the system was adjusted to 8-9 with 1M sodium hydroxide aqueous solution. The mixture was extracted with ethyl acetate (100 mL) and the organic phase was combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 ml). The organic phase was dried with anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=5:1-2:1) to obtain N-(3,5-dichloro-4-((5-(2-fluoropropan-2-yl)-6-oxo-1,6-dihydropyridazin-3-yl)oxy).-yl)oxy)phenyl)acetamide (2.28 g, 16.3% yield). MS (ESI) m/z: 373.1 [M+H].sup.+.
[0100] Step 4: 6-(4-amino-2,6-dichloro-phenoxy)-4-(2-fluoro-propane-2-yl)-pyridazin-3(2H)-one
[0101] To a solution of N-(3,5-dichloro-4-((5-(2-fluoropropan-2-yl)-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)acetamide (1.0 g, 2.7 mmol) in ethanol (20 mL), 6N hydrochloric acid (24 mL) was added. The reaction mixture was stirred at 70 C. for 2.5 h. After the reaction was completed, the reaction solution was cooled to room temperature. Then the reaction was filtered, the filter cake was washed with water, and the solid was dried under reduced pressure to give 6-(4-amino-2,6-dichlorophenoxy)-4-(2-fluoropropan-2-yl)pyridazin-3(2H)-one (749 mg, yield: 84%). MS (ESI) m/z: 331.1 [M+H].sup.+.
[0102] Step 5: (2-cyano-2-(2-(3,5-dichloro-4-((5-(2-fluoro-propyl-2-yl)-6-oxo-1,6-dihydro-pyridazin-3-yl)oxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0103] To a solution of 6-(4-amino-2,6-dichlorophenoxy)-4-(2-fluoropropan-2-yl)pyridazin-3(2H)-one (500 mg, 1.51 mmol) in 4N aqueous hydrochloric acid (22.5 mL), 0.26N aqueous sodium nitrite (7.5 mL) was added dropwise at 0 C. After the dropwise addition, the reaction was stirred at 0 C. for 2 h. Then the reaction mixture was filtered. The filtrate was added dropwise to a mixed solution of ethyl cyanoacetylcarbamate (236 mg, 1.51 mmol), pyridine (9.2 mL), and water (30 mL). The reaction was stirred at 0 C. for 1.5 h. After the reaction was completed, the mixture was filtered. The filter cake was washed with water (10 mL) and petroleum ether (10 mL), and the solid was dried under reduced pressure to obtain (2-cyano-2-(2-(3-(3,5-dichloro-4-((5-(2-fluoropropyl-2-yl)-6-oxo-1,6- dihydropyridazin-3-yl)oxy)phenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (218 mg, yield 29%). MS (ESI) m/z: 498.1 [M+H].sup.+.
[0104] Step 6: 2-(3,5-dichloro-4-((5-(2-fluoropropane-2-yl)-6-oxo-1,6-dihydro-pyridazin-3-yl)oxy)phenyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6- carbonitrile
[0105] To a solution of (2-cyano-2-(2-(3,5-dichloro-4-((5-(2-fluoropropan-2-yl)-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)hydrazinyl)acetyl)carbamate (254 mg, 0.5 mmol) in glacial acetic acid (4.8 mL), sodium acetate (208 mg, 2.5 mmol) was added. The reaction mixture was stirred 120 C. for 2 hours. Then the reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification on a C18 column (mobile phase: acetonitrile: aqueous solution=20%-95%) to give 55 mg of product in 24.2% yield. MS (ESI) m/z: 452.0 [M+H].sup.+. H NMR (400 MHz, DMSO-d.sub.6): 7.79 (s, 2H), 7.50 (s, 1H), 1.74 (s, 3H), 1.68 (s, 3H).
EXAMPLE 3
2-(3,5-dichloro-4-(3-(2-fluoro-propane-2-yl)-4-hydroxy-phenoxy)-phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 3)
[0106] ##STR00035##
[0107] Step 1: 4-(2,6-dichloro-4-nitrophenoxy)-2-(2-fluoropropane-2-yl)phenol
[0108] To a solution of 2-(2-fluoropropan-2-yl)benzene-1,4-diol (3.00 g, 17.6 mmol) in acetonitrile (50 mL) sodium carbonate (6.66 g, 63 mmol), and 1,3-dichloro-2-fluoro-5-nitrobenzene (2.65 g, 12.6 mmol) were added. The reaction was stirred at 50 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 mL) and ethyl acetate (50 mL) were added into the solution, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrated was concentrated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=20:1-15:1) to give 3.2 g, yield 70%. MS (ESI) m/z: 361.0 [M+H].sup.+.
[0109] Step 2: 4-(4-amino-2,6-dichloro-phenoxy)-2-(2-fluoro-propane-2-yl)phenol
[0110] To a solution of 4-(2,6-dichloro-4-nitrophenoxy)-2-(2-fluoropropan-2-yl)phenol (3.0 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride (4.48 g, 83.8 mmol) aqueous solution (20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.35 g of product in 85% yield. MS (ESI) m/z: 331.1 [M+H].sup.+.
[0111] Step 3: (2-cyano-2-(2-(3,5-dichloro-4-(3-(2-fluoropropane-2-yl)-4-hydroxy-phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0112] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dichlorophenoxy)-2-(2-fluoropropan-2-yl)phenol (2.30 g, 6.96 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture was continued to stir for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.55 g of product in 72% yield. MS (ESI) m/z: 497.4 [M+H].sup.+.
[0113] Step 4: 2-(3,5-dichloro-4-(3-(2-fluoropropane-2-yl)-4-hydroxy-phenoxy)-phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0114] To a solution of ethyl (2-cyano-2-(2-(3,5-dichloro-4-(3-(2-fluoropropan-2-yl)-4-hydroxyphenoxy)phenyl)hydrazinyl)acetyl)carbamate (2.50 g, 5.03 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.02 g of product in 45% yield. MS (ESI) m/z: 452.3 [M+H].sup.+. .sup.1HNMR (400 MHz, DMSO-d.sub.6): 13.24 (br s, 1H), 7.79 (s, 2H), 7.08 (d, 1H), 7.05 (d, 1H), 6.83 (dd, 1H), 1.70 (d, 3H), 1.68 (d, 3H)
EXAMPLE 4
2-(3,5-Diiodo-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 4)
[0115] ##STR00036##
[0116] Step 1: 4-(2,6-diiodo-4-nitrophenoxy)-2-(isopropenyl)phenol
[0117] To a solution of 2-isopropenylbenzene-1,4-diol (3.00 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol), and 1,3-diiodo-2-fluoro-5 nitrobenzene (5.62 g, 14.3 mmol) were added. The reaction was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. To the concentrate, water (50 mL) and ethyl acetate (50 mL) were added, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 4.86 g of the target product in 65% yield. MS (ESI) m/z: 524.1 [M+H].sup.+.
[0118] Step 2: 4-(4-amino-2,6-diiodo-phenoxy)-2-(isopropenyl)phenol
[0119] To a solution of 4-(2,6-diiodo-4-nitrophenoxy)-2-(isopropenyl)phenol (4.38 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution (4.48 g, 83.8 mmo, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 3.72 g of product in 90% yield. MS (ESI) m/z: 494.1 [M+H].sup.+.
[0120] Step 3: (ethyl 2-cyano-2-(2-(3,5-diiodo-4-(4-hydroxy-3-isopropenylphenoxy)phenyl)hydrazinylidene)acetyl)carbamate
[0121] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-diiodophenoxy)-2-(isopropenyl)phenol (3.82 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture continued to be stirred for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL), and ethyl cyanoacetylcarbamate (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 3.47 g of product in 68% yield. MS (ESI) m/z: 661.2 [M+H].sup.+.
[0122] Step 4: 2-(3,5-diiodo-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl) -3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0123] To a solution of ethyl (2-cyano-2-(2-(3,5-diiodo-4-(4-hydroxy-3-isopropenylphenoxy)phenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (3.74 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.91 g of product in 55% yield. MS (ESI) m/z: 615.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 13.10 (br s, 1H), 9.50 (s, 1H), 7.93 (s, 2H), 7.15 (m, 2H), 6.90 (s, 1H), 5.10 (d, 2H), 2.43 (s, 1H).
EXAMPLE 5
2-(3,5-dichloro-4-(4-hydroxy-3-(trifluoromethyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 5)
[0124] ##STR00037##
[0125] Step 1: 4-(2,6-dichloro-4-nitrophenoxy)-2-(trifluoromethyl)phenol
[0126] To a solution of 2-trifluoromethylbenzene-1,4-diol (3.56 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol) and 1,3-dichloro-2-fluoro-5 nitrobenzene (3.00 g, 14.3 mmol) were added. The reaction was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 mL) and ethyl acetate (50 mL) were added to the concentrate, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 3.32 g of the target product in 62% yield. MS (ESI) m/z: 369.1 [M+H].sup.+.
[0127] Step 2: 4-(4-amino-2,6-dichloro-phenoxy)-2-(trifluoromethyl)phenol
[0128] To a solution of 4-(2,6-dichloro-4-nitrophenoxy)-2-(trifluoromethyl)phenol (3.08 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution(4.48 g, 83.8 mmo, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.55 g of product in 90% yield. MS (ESI) m/z: 339.1 [M+H].sup.+.
[0129] Step 3: (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-trifluoromethyl-phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0130] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dichlorophenoxy)-2-(trifluoromethyl)phenol (2.62 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture continued to be stirred for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL), and ethyl (2-cyanoacetyl)carbamate (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.93 g of product in 75% yield. MS (ESI) m/z: 506.3 [M+H].sup.+.
[0131] Step 4: 2-(3,5-dichloro-4-(4-hydroxy-3-(trifluoromethyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0132] To a solution of (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-trifluoromethylphenoxy)phenyphydrazinylidene)acetyl)carbamic acid ethyl ester (2.70 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.3 g of product in 50% yield. MS (ESI) m/z: 460.1 [M+H].sup.+. .sup.1HNMR (400 MHz, DMSO-d.sub.6): 13.0 (br s, 1H), 9.70 (s, 1H),7.80 (s, 2H), 7.38 (d, 1H), 7.26 (d, 1H), 6.84 (d, 1H).
EXAMPLE 6
2-(3,5-Dichloro-4-(4-hydroxy-3-(deuterated methyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-carbonitrile (Compound 6)
[0133] ##STR00038##
[0134] Step 1: 4-(2,6-dichloro-4-nitrophenoxy)-2-(deuterated methyl)phenol
[0135] To a solution of 2-deuteromethylbenzene-1,4-diol (2.55 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol), and 1,3-dichloro-2-fluoro-5 nitrobenzene (3.00 g, 14.3 mmol) were added. The reaction was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 mL) and ethyl acetate (50 mL) were added to the concentrate, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent:petroleum: ethyl acetate=20:1-15:1) to obtain 2.95 g of the target product in 65% yield. MS (ESI) m/z: 318.1 [M+H].sup.+.
[0136] Step 2: 4-(4-amino-2,6-dichloro-phenoxy)-2-(deuterated methyl)phenol
[0137] To a solution of 4-(2,6-dichloro-4-nitrophenoxy)-2-(deuteromethyl)phenol (2.66 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution(4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.24 g of product in 93% yield. MS (ESI) m/z: 288.2 [M+H].sup.+.
[0138] Step 3: (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-deuterated methyl phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0139] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dichlorophenoxy)-2-(deuteromethyl)phenol (2.22 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture was continued to stir for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL), and ethyl (2-cyanoacetyl)carbamate (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.6 g of product in 75% yield. MS (ESI) m/z: 455.3 [M+H].sup.+.
[0140] Step 4: 2-(3,5-dichloro-4-(4-hydroxy-3-(deuterated methyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0141] To a solution of (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-deuteromethylphenoxy)phenyphydrazinylidene)acetyl)carbamic acid ethyl ester (2.70 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.04 g of product in 45% yield. MS (ESI) m/z: 409.2 [M+H].sup.+. .sup.1 H NMR (400 MHz, DMSO-d.sub.6): 13.50 (br s, 1H), 9.41 (s, 1H), 7.84 (s, 2H), 7.07 (d, 1H), 6.99 (s, 1H), 6.79 (d, 1H).
EXAMPLE 7
2-(3,5-dimethyl-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-carbonitrile (Compound 7)
[0142] ##STR00039##
[0143] Step 1: 4-(2,6-dimethyl-4-nitrophenoxy)-2-(isopropenyl)phenol
[0144] To a solution of 2-isopropenylbenzene-1,4-diol (3.00 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol), and 1,3-dimethyl-2-fluoro-5 nitrobenzene (2.42 g, 14.3 mmol) were added. The reaction was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 mL) and ethyl acetate (50 mL) were added to the concentrate, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 2.99 g of the target product in 69.8% yield. MS (ESI) m/z: 300.3 [M+H].sup.+.
[0145] Step 2: 4-(4-amino-2,6-dimethyl-phenoxy)-2-(isopropenyl)phenol
[0146] To a solution of 4-(2,6-dimethyl-4-nitrophenoxy)-2-(isopropenyl)phenol (2.85 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution (4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.14 g of product in 95% yield. MS (ESI) m/z: 270.3 [M+H].sup.+.
[0147] Step 3: (2-cyano-2-(2-(3,5-dimethyl-4-(4-hydroxy-3-isopropenyl phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0148] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL) was added 4-(4-amino-2,6-dimethylphenoxy)-2-(isopropenyl)phenol (2.08 g, 7.74 mmol). The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture was continued to stir for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL), and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.53 g of product in 75% yield. MS (ESI) m/z: 437.5 [M+H].sup.+.
[0149] Step 4: 2-(3,5-dimethyl-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0150] To a solution of (2-cyano-2-(2-(2-(3,5-dimethyl-4-(4-hydroxy-3-isopropenylphenoxy)phenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (2.47 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.05 g of product in 22% yield. MS (ESI) m/z: 391.4 [M+H].sup.+. .sup.1 H NMR (400 MHz, DMSO-d.sub.6): 13.40 (br s, 1H), 9.38 (s, 1H), 7.50 (s, 2H), 7.16 (m, 2H), 6.91 (s, 1H), 5.15 (s, 2H), 2.30 (s, 3H), 2.15 (s, 6H).
EXAMPLE 8
2-(3,5-dibromo-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 8)
[0151] ##STR00040##
[0152] Step 1: 4-(2,6-4-nitrophenoxy)-2-(isopropenyl)phenol
[0153] To a solution of 2-isopropenylbenzene-1,4-diol (3.00 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol) and 1 1,3-dibromo-2-fluoro-5 nitrobenzene (4.28 g, 14.3 mmol) were added. The reaction was stirred at 48 C. for 8 hours. After the reaction was complete, the reaction solution was cooled down to room temperature and concentrated under reduced pressure to remove the acetonitrile. Water (50 mL) and ethyl acetate (50 mL) were added to the concentrate, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 3.68 g of the target product in 60% yield. MS (ESI) m/z: 430.1 [M+H].sup.+.
[0154] Step 2: 4-(4-amino-2,6-bromo-phenoxy)-2-(isopropenyl)phenol
[0155] To a solution of 4-(2,6-dibromo-4-nitrophenoxy)-2-(isopropenyl)phenol (3.59 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution(4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 3.18 g of product in 95% yield. MS (ESI) m/z: 400.1 [M+H].sup.+.
[0156] Step 3: (2-cyano-2-(2-(3,5-dibromo-4-(4-hydroxy-3-isopropenyl phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0157] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dibromophenoxy)-2-(isopropenyl)phenol (3.09 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture continued to be stirred for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 3.24 g of product in 74% yield. MS (ESI) m/z: 567.2 [M+H].sup.+.
[0158] Step 4: 2-(3,5-dibromo-4-(4-hydroxy-3-(isopropenyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0159] To a solution of (2-cyano-2-(2-(3,5-dibromo-4-(4-hydroxy-3-isopropenylphenoxy)phenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (3.20 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.32 g of product in 45% yield. MS (ESI) m/z: 521.1 [M +H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 13.35 (br s, 1H), 9.45 (s, 1H), 7.77 (s, 2H), 7.14 (m, 2H), 6.91 (s, 1H), 5.10 (s, 2H), 2.35 (s, 3H).
EXAMPLE 9
2-(4-(4-hydroxy-3-(trifluoromethyl)benzyl)-3,5-dimethylphenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-carbonitrile (Compound 9)
[0160] ##STR00041##
[0161] Step 1: (2,6-dimethyl-4-nitrophenyl) (4-methoxy-3-(trifluoromethyl)phenyl)methanol
[0162] To a solution of 2-bromo-1,3-dimethyl-5-nitrobenzene (2.83 g, 12.3 mmol) in tetrahydrofuran (80 mL), n-butyllithium (10 mL, 24.5 mmol) was added dropwise at 70 C.
[0163] After being stirred at 70 C. for 20 min, 4-methoxy-3-(trifluoromethyl)benzaldehyde (3.0 g, 14.7 mmol) in tetrahydrofuran (20 mL) was added dropwise into the above reaction solution, and the reaction was stirred at 70 C. for 60 min. After the reaction was completed, the reaction solution was poured into saturated aqueous ammonium chloride solution (300 mL). The mixture was extracted with ethyl acetate (150mL2) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (200 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 10-95% (% B)) to give 2.84 g of product in 65% yield. MS (ESI) m/z: 336.3 [M+H].sup.+.
[0164] Step 2: 2-(4-methoxy-3-(trifluoromethyl)benzyl)-1,3-dimethyl-5-nitrobenzene
[0165] To a solution of (2,6-dimethyl-4-nitrophenyl)(4-methoxy-3-(trifluoromethyl)phenyl)methanol (2.75 g, 7.74 mmol) in dichloromethane (40 mL), trifluoroacetic acid (10 mL) and triethylsilane (20 mL) were added dropwise at room temperature. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution and extracted by dichloromethane (300 mL2). The organic phases were combined and washed with saturated aqueous sodium chloride solution (250 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-85% (% B)) to give 1.58 g of product in 60% yield. MS (ESI) m/z: 340.3 [M+H].sup.+.
[0166] Step 3: 4-(2,6-dimethyl-4-nitro-benzyl)-2-(trifluoromethyl)phenol
[0167] 2-(4-methoxy-3-(trifluoromethyl)benzyl)-1,3-dimethyl-5-nitrobenzene (1.41 g, 4.15 mmol) and pyridine hydrochloride (17.0 g) were stirred at 160 C. for 16 h. After the reaction was completed, the temperature was lowered to room temperature. Ethyl acetate (200 mL) and water (200 mL) were added into the solution, and the reaction was stirred for 30 min. The liquid was partitioned, and the aqueous phase was extracted with ethyl acetate (100 mL). The organic phases were combined and washed with saturated aqueous sodium chloride (200 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-75% (% B)) to give 945 mg of product in 70% yield. MS (ESI) m/z: 326.3 [M+H].sup.+.
[0168] Step 4: 4-(4-amino-2,6-dimethyl-benzyl)-2-(trifluoromethyl)phenol
[0169] To a solution of 4-(2,6-dimethyl-4-nitrobenzyl)-2-(trifluoromethyl)phenol (2.73 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution(4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.30 g of product in 93% yield. MS (ESI) m/z: 296.3 [M+H].sup.+.
[0170] Step 5: (2-cyano-2-(2-(4-(4-hydroxy-3-(trifluoromethyl)benzyl)-3,5-dimethyl-phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0171] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dimethylbenzyl)-2-(trifluoromethyl)phenol (2.29 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture was continued to stir for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) of n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.68 g of product in 75% yield. MS (ESI) m/z: 463.4 [M+H].sup.+.
[0172] Step 6: 2-(4-(4-hydroxy-3-(trifluoromethyl)benzyl)-3,5-dimethyl-phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0173] To a solution of (2-cyano-2-(2-(2-(4-(4-hydroxy-3-(trifluoromethyl)benzyl)-3,5-dimethylphenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (2.62 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.18 g of product in 50% yield. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.95 (br s, 1H), 9.50 (s, 1H), 7.42 (s, 2H), 7.32 (d, 1H), 7.08 (d, 1H), 6.76 (d, 1H), 3.96 (s, 2H), 2.18 (s, 6H).
EXAMPLE 10
2-(4-(4-hydroxy-3-(deuterated methyl)benzyl)-3,5-dimethylphenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-carbonitrile (Compound 10)
[0174] ##STR00042##
[0175] Step 1: (2,6-dimethyl-4-nitrophenyl)-(4-methoxy-3-(deuterated methyl)phenyl)methanol
[0176] To a solution of 2-bromo-1,3-dimethyl-5-nitrobenzene (2.83 g, 12.3 mmol) in tetrahydrofuran (80 mL) was added n-butyllithium (10 mL, 24.5 mmol) dropwise at 70 C.
[0177] After stirred at 70 C for 20 min, 4-methoxy-3-(deuteromethyl)benzaldehyde (2.25 g, 14.7 mmol) in tetrahydrofuran (20 mL) was added dropwise into the above reaction solution, and the reaction was stirred at 70 C. for 60 min. After the reaction was completed, the reaction solution was poured into saturated aqueous ammonium chloride solution (300 mL). The mixture was extracted with ethyl acetate (150 mL2) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (200 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 10-95% (% B)) to give 2.55 g of product in 68% yield. MS (ESI) m/z: 305.4 [M+H].sup.+.
[0178] Step 2: 2-(4-methoxy-3-(deuterated methyl)benzyl)-1,3-dimethyl-5-nitrobenzene
[0179] To a solution of (2,6-dimethyl-4-nitrophenyl) (4-methoxy-3-(deuteromethyl)phenyl)methanol (2.36 g, 7.74 mmol) in dichloromethane (40 mL), trifluoroacetic acid (10 mL) and triethylsilane (20 mL) were added dropwise at room temperature. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution and extracted by dichloromethane (300 mL2).The organic phases were combined and washed with saturated aqueous sodium chloride solution (250 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-85% (% B)) to give 1.45 g of product in 65% yield. MS (ESI) m/z: 289.4 [M+H].sup.+.
[0180] Step 3: 4-(2,6-dimethyl-4-nitro-benzyl)-2-(deuterated methyl)phenol
[0181] 2-(4-methoxy-3-(deuteromethyl)benzyl)-1,3-dimethyl-5-nitrobenzene (1.20 g, 4.15 mmol) and pyridine hydrochloride (17.0 g) were stirred at 160 C. for 16 h. After the reaction was completed, the temperature was lowered to room temperature. Ethyl acetate (200 mL) and water (200 mL) were added into the solution, and the reaction was stirred for 30 min. The liquid was partitioned, and the aqueous phase was extracted with ethyl acetate (100 mL). The organic phases were combined and washed with saturated aqueous sodium chloride (200 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-75% (% B)) to give 910 mg of product in 80% yield. MS (ESI) m/z: 275.3 [M+H].sup.+.
[0182] Step 4: 4-(4-amino-2,6-dimethyl-benzyl)-2-(deuterated methyl)phenol
[0183] To a solution of 4-(2,6-dimethyl-4-nitrobenzyl)-2-(deuteromethyl)phenol (2.3 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution (4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 1.95 g of product in 95% yield. MS (ESI) m/z: 245.3 [M+H].sup.+.
[0184] Step 5: (2-cyano-2-(2-(4-(4-hydroxy-3-(deuterated methyl)benzyl)-3,5-dimethyl-phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0185] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dimethylbenzyl)-2-(deuteromethyl)phenol (1.90 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture was continued to stir for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.42 g of product in 76% yield. MS (ESI) m/z: 412.5[M+H].sup.+.
[0186] Step 6: 2-(4-(4-hydroxy-3-(deuterated methyl)benzyl)-3,5-dimethyl-phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0187] To a solution of (2-cyano-2-(2-(2-(4-(4-hydroxy-3-(deuteromethyl)benzyl)-3,5-dimethylphenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (2.33 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.1 g of product in 53% yield. MS (ESI) m/z: 366.4 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.87 (br s, 1H), 9.26 (s, 1H), 7.42 (s, 2H), 6.93 (d, 1H), 6.87 (d, 1H), 6.71(d, 1H), 3.96(s, 2H), 2.10 (s, 6H).
EXAMPLE 11
2-(3,5-dichloro-4-(4-hydroxy-3-(allyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 11)
[0188] ##STR00043##
[0189] Step 1: 4-(2,6-dichloro-4-nitrophenoxy)-2-(allyl)-phenol
[0190] To a solution of 2-allylbenzene-1,4-diol (3.00 g, 20.0 mmol) in acetonitrile (50 mL), sodium carbonate (7.57 g, 71.4 mmol) and 1,3-dichloro-2-fluoro-5 nitrobenzene (3.00 g, 14.3 mmol) were added. The reaction mixture was stirred at 48 C. for 8 hours. Water (50 mL) and ethyl acetate (50 mL) were added to the concentrate, and the pH was adjusted to 2-3 with 10% aqueous HCl. The mixture was extracted with ethyl acetate (30 mL3) and the organic phases were combined. The organic phase was washed with saturated aqueous sodium chloride solution (10 mL), then dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum: ethyl acetate=20:1-15:1) to obtain 2.85 g of the target product in 59% yield. [M+H].sup.+: 340.0.
[0191] Step 2: 4-(4-amino-2,6-dichloro-phenoxy)-2-(allyl)-phenol
[0192] To a solution of 4-(2,6-dichloro-4-nitrophenoxy)-2-(allyl)phenol (2.85 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution(4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.42 g of product in 93% yield. [M+H].sup.+: 310.1.
[0193] Step 3: (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-allyl-phenoxy)phenyl)-hydrazino)acetyl)carbamic acid ethyl ester
[0194] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dichlorophenoxy)-2-(allyl)phenol (2.40 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture continued to be stirred for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.7 g of product in 73% yield. [M+H].sup.+: 477.1.
[0195] Step 4: 2-(3,5-dichloro-4-(4-hydroxy-3-(allyl)phenoxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0196] To a solution of (2-cyano-2-(2-(3,5-dichloro-4-(4-hydroxy-3-allylphenoxy)phenyphydrazone)acetyl)carbamic acid ethyl ester (2.70 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate=20%-80%) to give 1.05 g of product in 43% yield. MS (ESI) m/z: 431.0 [MH].sup..
EXAMPLE 12
2-(4-(4-hydroxy-3-(allyl)benzyl)-3,5-dimethylphenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-carbonitrile (Compound 12)
[0197] ##STR00044##
[0198] Step 1: (2,6-dimethyl-4-nitrophenyl)(4-allyloxy-phenyl)methanol
[0199] To a solution of 2-bromo-1,3-dimethyl-5-nitrobenzene (2.83 g, 12.3 mmol) in tetrahydrofuran (80 mL), n-butyllithium (10 mL, 24.5 mmol) was added dropwise at 70 C. After being stirred at 70 C. for 20 min, 4-allyloxy-benzaldehyde (3.0 g, 14.7 mmol) in tetrahydrofuran (20mL) was added dropwise into the above reaction solution, and the reaction was stirred at 70 C. for 60 min. After the reaction was completed, the reaction solution was poured into saturated aqueous ammonium chloride solution (300 mL). The mixture was extracted with ethyl acetate (150mL2) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (200 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 10-95% (% B)) to give 2.84 g of product in 65% yield. MS (ESI) m/z: 314.1 [M+H].sup.+.
[0200] Step 2: 2-(4-allyloxy)-benzyl-1,3-dimethyl-5-nitrobenzene
[0201] To a solution of (2,6-dimethyl-4-nitrophenyl)(4-allyloxyphenyl)methanol (2.75 g, 7.74 mmol) in dichloromethane (40 mL), trifluoroacetic acid (10 mL) and triethylsilane (20 mL) were added dropwise at room temperature. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution and extracted by dichloromethane (300 mL2). The organic phases were combined and washed with saturated aqueous sodium chloride solution (250 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-85% (% B)) to give 1.58 g of product in 60% yield. MS (ESI) m/z: 298.2 [M+H].sup.+.
[0202] Step 3: 4-(2,6-dimethyl-4-nitro-benzyl)-2-(allyl)-phenol
[0203] To a solution of 2-(4-allyloxy)benzyl-1,3-dimethyl-5-nitrobenzene (1.41 g, 4.15 mmol) in dichloromethane (20 mL) was added diethylaluminium chloride (1.2 eq). The reaction mixture was stirred at room temperature for 3 h. The mixture was quenched by adding water (5 mL), and stirred for 30 min. The liquid was partitioned, and the aqueous phase was extracted by dichloromethane (100 mL). The organic phases were combined and washed by saturated aqueous sodium chloride solution (200 mL). The combined organic phase was dried with anhydrous magnesium sulfate, then filtered. The filtrate was concentrated and purified by reversed-phase preparative liquid phase purification (mobile phase A: water, mobile phase B: acetonitrile, gradient: 5-75% (% B)) to give 945 mg of product in 70% yield. MS (ESI) m/z: 298.2 [M+H].sup.+.
[0204] Step 4: 4-(4-amino-2,6-dimethyl-benzyl)-2-(allyl)-phenol
[0205] To a solution of 4-(2,6-dimethyl-4-nitrobenzyl)-2-(trifluoromethyl)phenol (2.73 g, 8.38 mmol) in tetrahydrofuran (20 mL) and anhydrous methanol (20 mL), ammonium chloride aqueous solution (4.48 g, 83.8 mmol, 20 mL) and iron powder (2.82 g, 50.3 mmol) were added. The reaction was stirred at 70 C. for 3 hours. After the reaction was complete, the reaction solution was cooled down to room temperature. Water (50 mL), saturated aqueous sodium bicarbonate solution (30 mL), and ethyl acetate (50 mL) were added to the reaction solution, stirred for 10 minutes, and then filtered. The filtrate was extracted with ethyl acetate (20 mL3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give 2.30 g of product in 93% yield. MS (ESI) m/z: 268.2 [M+H].sup.+.
[0206] Step 5: (2-cyano-2-(2-(4-(4-hydroxy-3-(allyl)benzyl)-3,5-dimethyl-phenyl)-hydrazono)acetyl)carbamic acid ethyl ester
[0207] To a solution of 6M aqueous hydrochloric acid (40 mL) in anhydrous ethanol (30 mL), 4-(4-amino-2,6-dimethylbenzyl)-2-(allyl)phenol (2.29 g, 7.74 mmol) was added. The mixture was cooled down to 5 C. and an aqueous solution of sodium nitrite (534 mg, 7.74 mmol) (3 mL) was added dropwise to the mixture. After dropwise addition, the mixture continued to be stirred for 30 min at 0-5 C. A solution of pyridine (30 mL), water (40 mL) and n-cyanoacetylurethane (1.12 g, 7.74 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 5 C. for 2 h. After the reaction was complete, water (30 mL) was added to the reaction solution. During filtering, the filter cake was drenched with water (10 mL3). The filter cake was dried under reduced pressure to give 2.68 g of product in 75% yield. MS (ESI) m/z: 435.2 [M+H].sup.+.
[0208] Step 6: 2-(4-(4-hydroxy-3-(allyl)benzyl)-3,5-dimethyl-phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
[0209] To a solution of (2-cyano-2-(2-(2-(4-(4-hydroxy-3-(allyl)benzyl)-3,5-dimethylphenyl)hydrazinylidene)acetyl)carbamic acid ethyl ester (2.62 g, 5.66 mmol) in glacial acetic acid (30 mL), sodium acetate (928 mg, 11.3 mmol) was added. The mixture was heated to 118 C. and stirred at 118 C. for 3 hours. The reaction solution was cooled down to 60 C. and concentrated to dryness under reduced pressure. The concentrate was purified by preparative liquid phase purification over a C18 column (mobile phase: acetonitrile: 0.1% aqueous ammonium bicarbonate =20%-80%) to give 1.18 g of product in 50% yield. MS (ESI) m/z: 389.2 [M+H].sup.+.
[0210] Effect Testing
[0211] Effect Testing 1: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Thyroid Receptor Co-Activation Test
[0212] Experimental Procedure: [0213] (1) The complete TR-FRET CotBuffer C was prepared by the addition of 1MDTT (dithiothreitol) to TR-FRET CofferBuffer C (purchased in Thermofisher), and the final concentration is 5 mM DTT. [0214] (2) 100 nL of the compound to be tested with the concentration of 200 is added to each hole. If it is control group, then 100 nL DMSO is added; [0215] (3) adding 10 L of complete TR-FRET Coffin Buffer C in each hole; [0216] (4) preparing the TR-LBD (thyroid hormone ligand binding domain) of 4 by using the pre-cooled Complete TR-fret Comm Buffer C; [0217] (5) adding 5 L of 4 TR-LBD into the experiment plate; preparing complete TR-FRET Comm BufferC at room temperature with a solution containing 0.4 M of the fluorescent enzyme-SRC2-2 (purchased from Thermofisher Corp.) (4 times) and 8 nM of Tbanti-GST (4-fold). 5 L of 4peptide/4antibody solution (purchased in Thermofisher Corporation) was added to the experimental plate. [0218] (6) lightly mixing the 384-hole plate on the plate shaking machine, avoiding light incubation 2 h at room temperature. using instrument set, the wavelength is 520 nm and 495 nm, and the specific parameter is as follows:
TABLE-US-00001 Excitation Wavelength 340 nm Bandpass Filter (30 nm bandwidth) Emission Wavelength 520 nm Bandpass Filter (25 nm bandwidth) Emission Wavelength 490 nm or 495 nm Bandpass Filter (10 nm bandwidth) Lag Time 100 s Integration Time 200 s
[0219] Test compounds: Transthyretin T3 (positive control), MGL3196 (positive compound), the compound of the embodiment of the present invention.
[0220] Experimental Results:
TABLE-US-00002 TR TR-FRET Coactivator TR TR-FRET Coactivator Assay Assay EC.sub.50 (nM) % MAX of T3 EC.sub.50 (nM) % MAX of T3 T3 0.12 100.00 0.16 100.00 MGL3196 602 32.24 60.7 48.46 compound 1 4.27 75.04 0.86 90.73 compound 2 868 14.88 169 27.30 compound 3 2.62 89.73 4.47 90.63
[0221] The results showed that compounds 1 and 3 of the present invention showed agonistic activities on both TR and TR, which were already close to the positive control T3, and the agonistic effect on TR had reached more than 90% of the agonistic effect of T3. Both compounds also showed significantly higher EC50 values and agonistic effects on TR and TR than compound MGL3196.
[0222] Effect Testing 2: Fluorescence enzyme functional test based on HEK293/TR-luc cell [0223] (1) Composite plates were prepared and 125 nL of HEK293/TR-luc cells were added to each well of the assay plate. The cell density was determined by counting and the cell suspension was diluted with complete medium at a cell density of 410.sup.5/mL. [0224] (2) adding 25 L cell to each hole in the detection plate containing the compound to be tested, incubating for 24 h at 37 degrees centigrade and 5% CO.sub.2 environment; [0225] (3) adding 25 L of Steady-Glo (Promega) in each hole; [0226] (4) centrifuging for 2 minutes at 2000 RPM to eliminate bubbles; [0227] (5) incubating at room temperature for 10 minutes, using an Envision (Envision 2105 model, PerkinElmer company) reading board.
[0228] Test compounds: Transthyretin T3 (positive control), MGL3196 (positive control), test example compound.
[0229] Experimental Results:
TABLE-US-00003 HEK293/TR cells HEK293/TR Selectivity EC50 (nM) cells EC50 (nM) Ratio T3 4.56 1.914 2.4 MGL3196 26890 3356 8.0 compound 1 1859 83.8 22.2 compound 3 334 74.14 4.5
[0230] The results display that the compound 1 of the invention 1 display the agonist activity to TR and TR, the EC50 value is significantly higher than the compound MGL3196, the selectivity for the TR cell is 22.2 times of the TR cell, and also significantly higher than MGL3196.
[0231] Effect Testing 3: NASH Animal Model Effect and Safety Test
[0232] C57BL/6J male mice, starting from 6 weeks old, are continuously fed with a CDAA-HFD diet (choline deficiency levorotatory amino acid high fat diet), and after 6 weeks (42 days), the moulding is successful. The model through VLDL (very low density lipoprotein) damages liver triglyceride secretion, mouse serum ALT and AST increases, and in 3 weeks there is fatty degeneration and inflammation, and hepatic fibrosis occurs at 5-6 weeks. The model is developed into liver cirrhosis in 24 weeks, and portal vein high pressure and liver failure. CDAA-HFD diet can induce liver steatosis and fibrosis in a short time, and has no obesity, hyperglycemia and hypertriglyceridemia, and so on, strong reference meaning for NASH disease research project. The invention uses the CDAA-HFD model to simulate the pathological process and physiological state of NASH, and the test compound for the early stage NASH treatment effect.
[0233] Specific implementation schemes are as follows:
TABLE-US-00004 Dosage 1. Grouping and dosing animal (mg/kg, Administration regimens: Group number mpk) method Normal control group 12 PBS p.o. q.d, 42 days (conventional feed) Model group (CDAA-HFD) 16 PBS p.o. q.d, 42 days Fenobert 12 100 p.o. q.d, 42 days MGL-3196 6 3.0 p.o. q.d, 42 days compound 1 group 6 3.0 p.o. q.d, 42 days Note: p.o means oral (intragastric) administration; and q.d means once daily
[0234] The self-molding was successfully grouped for 42 days, and then administered 42 days in accordance with the dosing regimen.
[0235] Drug preparation: the sample powder was weighed for each administration group, then put in a 5 ml centrifugal tube, and a proper amount of 0.5% MC was added, then uniformly mixing via vortex oscillation, and preparing into a solution with a corresponding concentration of the existing preparation.
[0236] The state of the animal every time was observed and recorded. If the animal was killed, the animal was generally dissected, and the visceral abnormalities were observed by the naked eye, and recorded. During the experiment period, the weight of the animal was measured twice per week. The animal body weight curve of administration for 42 days is shown in
[0237] 2. The Pathology Scoring
[0238] After weighing all mice at the corresponding time point, carbon dioxide was used with increased concentration to perform euthanason. Then cardiac puncture, taking blood, 7000 rpm for 10 min, taking plasma, immediately putting on dry ice, rotating and storing at 80 degrees centigrade. Subsequently the blood biochemical index was determined, comprising TCHO (total cholesterol), LDL (low density lipoprotein) and so on.
[0239] Also the liver was taken, and its weight was measured. Part of the liver was cut off (the same position of each animal) and fixed in 4% of paraformaldehyde, for histopathological analysis (H-E staining and Tianwolf star red staining): liver fat and inflammatory cell infiltration degree, fibrosis and NAS score (NAS scoring system reference Chinese medical institute of non-alcoholic fatty liver disease diagnosis and treatment guide (2010 year revision), Ishak scoring system reference Journal of Hepatology 47 (2007) 598-607, Grading and III.).).
[0240] 3. Statistical Analysis
[0241] The data is expressed by its average value+SEM. The statistical analysis of the difference between groups adopts single factor variance analysis (ANOVA), then using SPSS statistics software for a Dunnett test, where a P value less than 0.05 represents a statistical significance between two groups of data. In the scheme chart, * represents P<0.05; ** represents P<0.01; *** represents P<0.001.
[0242] 4. Results of Experiment
[0243] 4.1 Compound 1 of Lipid-Lowering Effect
[0244] TCHO (total cholesterol) as shown in
[0245] 4.2 Compound 1 of Hepatocyte Gas Ball Sample Change and Inflammation Score
[0246] The change of the hepatocyte balloon can indicate the severity of the liver fat accumulation. As shown in
[0247] 4.3 Compound 1 of Fibrosis and NAS Score
[0248] The liver fibrosis score as shown in
[0249] The same method was used to test compound 2 for a lipid-lowering effect, by comparing compound 2 of the normal diet group, model group and fenofibrate group are significantly reduced, confirming that compound 2 as THR-beta inhibitor can effectively reduce liver fat. The same method was used to test compound 2 for the hepatocyte gas ball sample change and inflammation score. This shows that compound 2 also shows good effect of inhibiting and reversing liver inflammation.
[0250] Effect Testing 4: The Maximum Tolerated Dose Test
[0251] C57BL/6J mice, 24, half male and half female, single administration through mouth and stomach of 30, 100, 300 mg/kg of compound 1, the solvent is 0.1% tween80+0.5% MC water solution. After administration, clinical symptoms and weight was recorded, once a day, continuously for 3 days. On the fourth day, euthanasia was performed, and blood was collected for hematology, and blood biochemical detection.
[0252] The weight results are shown in
[0253] Effect Testing 5: hERG Test
[0254] Full automatic patch clamp QPatch technology was used, testing compound 1 to inhibit the CHO cell hERG potassium current.
[0255] Specific implementation schemes are as follows:
[0256] 1. Preparation of Cell
[0257] CHO-hERG cell culture is added in a 175 cm.sup.2 culture bottle, the cell density grew to 60 to 80%, then the culture solution was removed, 7 mL PBS (Phosphate Buffered Saline phosphate buffer solution) was used for washing once, then 3 mL Detachin was added for digestion. After complete digestion, 7 mL culture liquid was added and then centrifuged, so the the supernatant was absorbed, then 5 mL culture liquid was added to re-suspend, to ensure the cell density was 2-510.sup.6/mL.
[0258] 2. The Electrophysiological Recording Process
[0259] Single cell high impedance sealing and the whole cell model process is automatically finished by the Qpatch instrument, the full cell model is obtained, and cell clamp is 80 millivolt, before a 5 seconds+40 millivolt depolarization stimulation is carried out, firstly applying a 50 millisecond pre-voltage of 50 millivolts, then re-polarizing to 50 millivolt for 5 seconds, and then returning to 80 millivolt. The voltage stimulation is applied every 15 seconds, recorded for 2 minutes, the extracellular fluid is recorded for 5 minutes, then the administration process is started. The highest test concentration of compound 1 is 40.00 M, and sequentially it is 40.00, 13.33, 4.44, 1.48, 0.49, 0.16 M. The DMSO content in the final test concentration is not more than 0.2%. The concentration of DMSO does not affect the hERG potassium channel. Starting the compound concentration from the lowest test concentration, each test concentration is administered for 2.5 minutes, after continuously feeding all the concentrations, the positive control compound Cisapride is given. Each concentration at least tests 3 cells (n is more than or equal to 3).
[0260] 3. Data Analysis
[0261] The experimental data is analyzed by GraphPad Prism 5.0 software. 4. Results of Experiment
TABLE-US-00005 maximum maximum test concentration Compound concentration (M) inhibition rate (%) IC50 (M) Cisapride 3 98.9 0.023 Compound 1 13.3 20.7 >13.3 Note: Compound 1 can not maintain normal sealing when the concentration of 40 M, the data of this concentration is not included in the statistics.
The results demonstrate that compound 1 exhibits minimal impact on CHO cell hERG potassium currents, suggesting a lower likelihood of potential cardiac safety concerns.