Compounds and methods for inducing browning of white adipose tissue

Abstract

The present invention provides a compound for inducing browning of white adipose tissue in vitro and in vivo of formula I, the preparation method thereof, as well as a composition comprising the same. Further, the present invention also relates to the use of the compound and the method to treat metabolic disorders, such as obesity and diabetes. ##STR00001##

Claims

1. A compound or physiologically acceptable salt or hydrate or solvate thereof, selected from the group consisting of: 1. 3-(2-(pyridin-2-ylamino)thiazol-4-yl)benzonitrile; 2. N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)propionamide; 3. methyl 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoate; and 4. 4-(4-amino-3-methylphenyl)-N-(pyridin-2-yl)thiazol-2-amine.

2. A pharmaceutical composition comprising the compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1 and at least one pharmaceutically acceptable carrier or diluents.

3. The pharmaceutical composition of claim 2, wherein the composition is an oral composition, an injectable composition or a suppository.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Compound 1 promotes browning of adipose tissue from 16-week-old obese mice induced by high fat diets for 8-week. Hematoxylin and eosin staining (left panel) and UCP1 immunochemistry (right panel) analysis of adipose tissue in mice treated with compound 1 compared with control group, showing browning effects of white adipose tissue in the embodiment of reduced cell size and increased cell number.

EMBODIMENTS

(2) Abbreviation Definition or Explanation

(3) DCE 1,2-Dichloroethane DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide eq. equivalents EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlide HOBt N-hydroxybenzotriazole NB S N-bromosuccinimide TEA triethylamine THF tetrahydrofuran
Methods of Preparation

(4) The compounds of the present invention may be prepared by methods as those illustrated in the following scheme I to II. Starting materials are commercially available or prepared by the methods as those illustrated in the following scheme III to V. Additional methods for making selected compounds of the present invention are provided below. Solvents, temperatures, pressures and other reaction conditions may readily be selected by one of ordinary skill in the art.

(5) ##STR00005##
General Protocol for the Thiazole Compound 3 Formation

(6) To a solution of substituted thiourea 1 (1.0 eq.) in alcoholic solvent (about 0.5 M concentration) such as, but not limited to, methanol, ethanol or isopropanol was added substituted -bromoketone 2 (1.0 eq.), and the mixture was heated to reflux for 38 h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give compound 3 of the present invention in 6095% yield, such as the compounds 1-28, 31 and 33-83 listed below.

(7) ##STR00006##
Step 1 General Protocol for the 2-aminothiazole 5 Formation

(8) To a solution of thiourea 4 (1.0 eq.) in alcoholic solvent (about 0.5 M concentration) such as, but not limited to, methanol, ethanol or isopropanol was added substituted -bromoketone 2 (1.0 eq.), and the mixture was heated to reflux for 38 h until the starting material was consumed. Then the mixture was cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 2-aminothiazole 5 of scheme II in 6095% yield.

(9) Step 2 General Protocol for the Thiazole Compound 6 Formation

(10) 2-aminothiazole 5 (1.0 eq.) obtained from step 1 of scheme II was reacted with substituted acyl chloride (1.5 eq.) in the presence of a base (2.2 eq.) such as, but not limited to TEA, DIPEA, Na.sub.2CO.sub.3 in organic solvent (about 0.5 M concentration) such as, but not limited to DCM, DMF or CH.sub.3CN to form amide compound 6 of the present invention in 6095% yield, such as the compounds 29 and 30 listed below.

(11) or 2-aminothiazole 5 (1.0 eq.) obtained from step 1 of scheme II was coupled with substituted carboxylic acid (1.1 eq.) in the presence of classical coupling reagents (1.1 eq.) in amide synthesis such as, but not limited to, EDCl/HOBt/DIPEA combination in organic solvents (about 0.5 M concentration) such as, but not limited to, DCM, THF or DMF to afford amide compound 6 of present invention in 5585% yield, such as the compounds 32 listed below.

(12) General Procedure for the Preparation of Thiourea Intermediates 1 from Amines

(13) The substituted thiourea intermediates 1 of scheme I were commercially available or prepared according to procedures provided in the following scheme III.

(14) ##STR00007##

(15) Phenyl isothiocyanate (1.1 eq.) was added dropwise to a stirred solution of amine 7 (1.0 eq.) in organic solvent (about 0.1 M concentration) such as, but not limited to, acetone, THF or CH.sub.3CN at room temperature. The reaction mixture was heated to reflux for 1-3 h until the amine 7 was consumed. Then the mixture was cooled to room temperature, poured into water-ice, and stirred for an additional 15 min. The benzoyl thiourea precipitate was collected by filtration and washed with more water. This crude material was dissolved in methanol (about 0.05M concentration) and treated with aqueous 1N NaOH (2 eq.) and heated to 80 C. until the hydrolysis was completed. After cooling, the mixture was poured into water-ice and sufficient aqueous 1N HCl was added to produce a neutral (pH about 7) solution. The thiourea intermediate typically precipitates from the neutral solution and was collected by filtration and dried. This two-step procedure provides thiourea intermediates in 5095% overall yield. The thiourea intermediates can be used directly in the next step without further purification.

(16) General Procedure for the Preparation of Substituted -bromoketone Intermediates 2

(17) The substituted -bromoketone intermediates 2 of schemes I to II were commercially available or prepared according to procedures provided in the following scheme IV to V.

(18) ##STR00008##

(19) To a solution of ketone 8 (1.0 eq.) in organic solvent (about 0.5M concentration) such as, but not limited to, DCM, chloroform or ether was added bromine (1.0 eq.) dropwise at 0 C. The mixture was then warmed to room temperature or elevated temperature (5090 C.) if necessary and stirred for 26 h until the ketone was consumed. The mixture was then extracted, dried, evaporated and purified by silica gel chromatography to give compound 2 of scheme IV in 7095% yield.

(20) When ketones were commercially unavailable, -bromoketone intermediates 2 were prepared in an alternative way provided in the following scheme V.

(21) ##STR00009##
Step 1 General Procedure for the Preparation of Ethoxyethene Intermediates 10 Via Stile Coupling Reaction

(22) To a solution of halogenated compound 9 (1.0 eq.) in organic solvent (about 1M concentration) such as, but not limited to, dioxane, DCE or toluene was added tributyl (1-ethoxyvinyl)tin (1.5 eq.) and dichlorobis(triphenylphosphine)palladium (II) (0.05 eq.). The mixture was purged with nitrogen for 5 min, and stirred at elevated temperature (90120 C.) in a sealed tube for 1218 h until the compound 9 was consumed. The mixture was then cooled to room temperature, extracted, dried, evaporated and purified by silica gel chromatography to give compound 10 of scheme V in 6585% yield.

(23) Step 2 General Procedure for the Preparation of -bromoketone Intermediates 2 Via Bromination of Intermediates 10

(24) To a solution of compound 10 (1.0 eq.) in THF/H.sub.2O (about 0.5M concentration, V.sub.THF:V.sub.H2O=3:1) was added NBS (1.0 eq.). The mixture was stirred at room temperature for 1030 min, then extracted, dried, evaporated and purified by silica gel chromatography to afford -bromoketone intermediates 2 of scheme V in 5575% yield.

(25) Detailed Embodiments

(26) The present invention is further exemplified, but not limited, by the following and examples that illustrate the preparation of the compounds of the invention.

(27) The invention will now be further described by the following working examples, which are preferred embodiments of the invention. All temperatures are in degrees Celsius ( C.) unless otherwise indicated. Preparative Reverse Phase (RP) HPLC purifications were done on C18 reverse phase (RP) columns using water/methanol mixtures. All the synthesized compounds were characterized by at least NMR or LC/MS. During work up of reactions, the organic extract was dried over sodium sulfate, purified by silica gel column chromatography or (RP) HPLC, unless mentioned otherwise.

(28) These examples are illustrative rather than limiting and it is to be understood that there may be other embodiments that fall within the spirit and scope of the invention as defined by the claims appended hereto.

EXAMPLE 1

(29) The Preparation of Compound 1

(30) ##STR00010##
Step 1 Preparation of Intermediate 1-1

(31) Phenyl isothiocyanate (13.52 g, 0.1 mol) was added dropwise to a stirred solution of 2-aminopyridine (9.41 g, 0.1 mol) in 200 mL acetone at room temperature. The reaction mixture was heated to reflux for 3 h until the 2-aminopyridine was consumed. Then the mixture was cooled to room temperature, poured into water-ice, and stirred for an additional 15 min. The benzoyl thiourea precipitate was collected by filtration and washed with more water. This crude material was dissolved in 200 mL methanol and treated with 200 mL aqueous 1N NaOH and heated to 80 C. until the hydrolysis was completed. After cooling, the mixture was poured into water-ice and sufficient aqueous 1N HCl was added to produce a neutral (pH about 7) solution. The thiourea intermediate precipitate was collected by filtration and dried, providing 13.9 g of intermediate 1-1 in 91% yield. ESI-MS m/z 154.0 [M+H].

(32) Step 2 Preparation of Compound 1

(33) To a solution of intermediate 1-1 (0.153 g, 1.0 mmol) in 5 mL ethanol was added 2-bromo-1-(3,4-dimethoxyphenyl)ethanone 2 (0.259 g, 1.0 mmol), and the mixture was heated to reflux for 3 h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 0.266 g of compound 1 of the present invention in 85% yield. .sup.1H NMR (400 MHz, CDCl3) 9.84 (s, 1H), 8.34 (dd, J=5.0, 1.0 Hz, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 1H), 6.58 (d, J=8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H). ESI-MS m/z 314.3 [M+H].

EXAMPLE 2

(34) The Preparation of Compound 18

(35) ##STR00011##

(36) To a solution of intermediate 1-1 (0.153 g, 1.0 mmol) in 5 mL ethanol was added 2-bromo-1-(3,5-dimethoxyphenyl)ethanone intermediate 18-1 (0.259 g, 1.0 mmol), and the mixture was heated to reflux for 3 h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 0.261 g of compound 18 of the present invention in 84% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) 9.88 (s, 1H), 8.46-8.09 (m, 1H), 7.47-7.32 (m, 3H), 7.16 (s, 1H), 6.98 (d, J=2.4 Hz, 2H), 6.49 (d, J=2.4 Hz, 1H), 3.94 (s, 3H), 3.82 (s, 3H). ESI-MS m/z 314.3 [M+H].

EXAMPLE 3

(37) The Preparation of Compound 32

(38) ##STR00012##

(39) Step 1 Preparation of intermediate 32-1 To a solution of thiourea (7.61 g, 0.1 mol) in 200 mL ethanol was added intermediate 1-1 (25.9 g, 0.1 mol), and the mixture was heated to reflux for 38 h until the starting material was consumed. Then the mixture was cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 18.9 g of intermediate 32-1 in 85% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38-7.27 (m, 2H), 6.88 (d, J=8.2 Hz, 1H), 6.60 (s, 1H), 5.04 (s, 2H), 3.95 (s, 3H), 3.90 (s, 3H). ESI-MS m/z 237.3 [M+H].

(40) Step 2 Preparation of Compound 32

(41) To a solution of 2-picolinic acid (21.17 mg, 0.172 mmol) in 5 mL DCM were added EDCI (36.1 mg, 0.189 mmol), HOBt (25.5 mg, 0.189 mmol), DIPEA (48.9 mg, 0.378 mmol) and the mixture was stirred at room temperature for 15 min. Then intermediate 32-1 (40.6 mg, 0.172 mmol) was added. The mixture was continued to stir at room temperature for 12 h until intermediate 32-1 was consumed, then evaporated, under reduced pressure and the crude material was purified by silica gel chromatography to give 45.8 mg of compound 32 in 78% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) 11.23 (s, 1H), 8.69 (d, J=4.8 Hz, 1H), 8.31 (d, J=7.8 Hz, 1H), 7.96 (t, J=7.0 Hz, 1H), 7.55 (dd, J=7.4, 5.0 Hz, 1H), 7.47 (s, 1H), 7.46-7.41 (m, 1H), 7.11 (s, 1H), 6.93 (d, J=8.3 Hz, 1H), 3.99 (s, 3H), 3.93 (s, 3H). ESI-MS m/z 342.3 [M+14].

EXAMPLE 4

(42) The Preparation of Compound 36

(43) ##STR00013##
Step 1 Preparation of Compound 34

(44) To a solution of intermediate 1-1 (15.3 g, 0.1 mol) in 200 mL ethanol was added intermediate 36-1 (24.4 g, 0.1 mol), and the mixture was heated to reflux for 3 h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 27.98 g of compound 34 of the present invention in 94% yield. .sup.1H NMR (400 MHz, DMSO) 11.53 (s, 1H), 8.30 (dd, J=12.9, 6.7 Hz, 3H), 8.16 (d, J=8.7 Hz, 2H), 7.80 (s, 1H), 7.73 (t, J=7.1 Hz, 1H), 7.11 (d, J=8.3 Hz, 1H), 7.02-6.86 (m, 1H). ESI-MS m/z 299.3 [M+H].

(45) Step 2 Preparation of Compound 35

(46) To a solution of compound 34 (27.98 g, 93.8 mmol) in 200 mL acetic acid was added iron powder (15.5 g, 281 mmol) in portions. Upon complete addition, the mixture was carefully heated to 100 C. After 3 h, the mixture was cooled to room temperature and filtered through Celite. The Celite was washed with EtOH and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to give compound 35 of present invention in 88% yield. .sup.1H NMR (400 MHz, DMSO) 11.25 (s, 1H), 8.29 (d, J=4.0 Hz, 1H), 7.77-7.60 (m, 1H), 7.57 (d, J=8.5 Hz, 2H), 7.09 (d, J=8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.79 (m, 1H), 6.58 (d, J=8.5 Hz, 2H), 5.18 (s, 2H). ESI-MS m/z 267.3 [M+11].

(47) Step 3 Preparation of Compound 36

(48) To a solution of compound 35 (133.2 mg, 0.5 mmol) in 5 mL pyridine was added acetyl chloride (58.9 mg, 0.75 mmol) at 0 C. After addition, the mixture was warmed to room temperature and stirred for 8 h until compound 35 was consumed. The mixture was then extracted, dried, evaporated and purified by column chromatography to give 132 mg of compound 36 in 85% yield. .sup.1H NMR (400 MHz, DMSO) 11.37 (s, 1H), 9.99 (s, 1H), 8.30 (d, J=4.9 Hz, 1H), 7.82 (d, J=8.6 Hz, 2H), 7.70 (dd, J=11.2, 4.3 Hz, 1H), 7.62 (d, J=8.6 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J=8.3 Hz, 1H), 7.00-6.84 (m, 1H), 2.06 (s, 3H). ESI-MS m/z 311.3 [M+H].

(49) The following compounds were prepared using a procedure similar to that described above.

(50) TABLE-US-00001 No. Structure NMR/MS 1 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.84 (s, 1H), 8.34 (dd, J = 5.0, 1.0 Hz, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H). ESI-MS m/z 314.3 [M + H]. 2 .sup.1H NMR (400M, DMSO) 11.42 (br, 1H), 8.21 (d, J = 5.49 Hz, 1H), 7.49 (s, 1H), 7.46-7.39 (m, 1H), 7.31 (s, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.93 (s, 1H), 6.83 (d, J = 4.9 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H), 2.39 (s, 3H). ESI-MS m/z 328.3 [M + H]. 3 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.86 (s, 1H), 8.39-8.31 (m, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 2H), 6.58 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H). ESI-MS m/z 284.3 [M + H]. 4 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.89 (s, 1H), 8.27 (d, J = 5.0 Hz, 1H), 7.86-7.79 (m, 2H), 7.41-7.29 (m, 4H), 6.98 (s, 1H), 6.85-6.79 (m, 1H), 6.51 (d, J = 8.0 Hz, 1 H). ESI-MS m/z 254.3 [M + H]. 5 .sup.1H NMR (400 MHz, CDCl3) 9.87 (s, 1H), 8.38-8.30 (m, 1H), 7.49-7.39 (m, 3H), 6.98 (s, 1H), 6.90-6.86 (m, 1H), 6.83-6.79 (m, 2H), 6.59 (d, J = 8.3 Hz, 1H). ESI-MS m/z 279.3 [M + H]. 6 ESI-MS m/z 365.3 [M + H]. 7 0 ESI-MS m/z 293.3 [M + H]. 8 ESI-MS m/z 284.3 [M + H]. 9 ESI-MS m/z 255.3 [M + H]. 10 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.55 (s, 1H), 7.66-7.59 (m, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.03 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.89-6.81 (m, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 2.41 (s, 3H). ESI-MS m/z 328.3 [M + H]. 11 .sup.1H NMR (400M, DMSO) 11.39 (br, 1H), 8.18 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.49-7.40 (m, 2H), 7.34 (s, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H), 2.34 (s, 3H). ESI-MS m/z 328.3 [M + H]. 12 ESI-MS m/z 328.3 [M + H]. 13 ESI-MS m/z 348.9 [M + H]. 14 ESI-MS m/z 384.3 [M + H]. 15 ESI-MS m/z 314.3 [M + H]. 16 ESI-MS m/z 297.3 [M + H]. 17 0 ESI-MS m/z 344.3 [M + H]. 18 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.88 (s, 1H), 8.46-8.09 (m, 1H), 7.47-7.32 (m, 3H), 7.16 (s, 1H), 6.98 (d, J = 2.4 Hz, 2H), 6.49 (d, J = 2.4 Hz, 1H), 3.94 (s, 3H), 3.82 (s, 3H). ESI-MS m/z 314.3 [M + H]. 19 ESI-MS m/z 328.3 [M + H]. 20 .sup.1H NMR (400 MHz, CDCl.sub.3) 7.52 (s, 1H), 7.41 (d, J = 6.9 Hz, 2H), 7.36 (dd, J = 8.6, 5.7 Hz, 4H), 7.13-7.04 (m, 1H), 6.90 (d, J = 8.9 Hz, 1H), 6.71 (s, 1H), 3.95 (s, 3H), 3.92 (s, 3H). ESI-MS m/z 313.3 [M + H]. 21 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.11 (s, 1H), 8.33 (d, J = 4.5 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 6.1 Hz, 1H), 7.19 (d, J = 7.8 Hz, 2H), 6.99 (s, 1H), 6.82 (d, J = 5.1 Hz, 1H), 6.54 (s, 1H), 2.39 (s, 3H). ESI-MS m/z 268.3 [M + H]. 22 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.83 (s, 1H), 8.34 (d, J = 4.6 Hz, 1H), 7.85 (dd, J = 8.7, 5.5 Hz, 2H), 7.44 (t, J = 7.7 Hz, 1H), 7.07 (t, J = 8.6 Hz, 2H), 6.97 (s, 1H), 6.89-6.79 (m, 1H), 6.58 (d, J = 5.7 Hz, 1H). ESI-MS m/z 272.3 [M + H]. 23 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.19 (s, 1H), 8.33 (t, J = 7.5 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 6.87-6.76 (m, 1H), 6.47 (d, J = 8.3 Hz, 1H). ESI-MS m/z 338.3 [M + H]. 24 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.33 (s, 1H), 8.35 (d, J = 4.5 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.54-7.39 (m, 2H), 7.27 (d, J = 11.3 Hz, 1H), 7.01 (t, J = 8.0 Hz, 2H), 6.90-6.79 (m, 1H), 6.73 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H). ESI-MS m/z 283.3 [M + H]. 25 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.36 (s, 1H), 8.35 (d, J = 4.5 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.54-7.39 (m, 2H), 7.27 (d, J = 11.3 Hz, 1H), 7.01 (t, J = 8.0 Hz, 2H), 6.90-6.79 (m, 1H), 6.73 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H). ESI-MS m/z 363.3 [M + H]. 26 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.20 (s, 1H), 8.63 (d, J = 5.1 Hz, 2H), 8.37 (d, J = 3.9 Hz, 1H), 7.75 (d, J = 5.0 Hz, 2H), 7.56 (t, J = 7.2 Hz, 1H), 7.29 (s, 1H), 6.99-6.81 (m, 1H), 6.74 (d, J = 8.2 Hz, 1H). ESI-MS m/z 255.3 [M + H]. 27 0 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.44 (s, 1H), 8.63 (d, J = 4.5 Hz, 1H), 8.36 (d, J = 4.8 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 7.7 Hz 1H) 7.61 (s, 1H) 7.51 (dd, J = 11.1, 4.4 Hz, 1H), 7.22-7.14 (m, 1H), 6.93-6.83 (m, 1H), 6.72 (d, J = 8.3 Hz, 1H). ESI-MS m/z 255.3 [M + H]. 28 .sup.1H NMR (400 MHz, DMSO) 11.40 (s, 1H), 8.30 (d, J = 4.6 Hz, 1H), 8.16 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.71 (t, J = 7.4 Hz, 1H), 7.43 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 7.04-6.78 (m, 1H), 3.76 (s, 4H), 3.00 (s, 4H). ESI-MS m/z 418.3 [M + H]. 29 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.55 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.03 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.98 (s, 3H), 3.87 (s, 3H), 2.00 (s, 3H). ESI-MS m/z 279.3 [M + H]. 30 .sup.1H NMR (400 MHz, CDCl.sub.3) 7.98 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.08 (s, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H). ESI-MS m/z 341.3 [M + H]. 31 .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38 (s, 1H), 7.34 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.00 (s, 1H), 3.90 (d, J = 4.9 Hz, 6H), 2.88 (s, 3H). ESI-MS m/z 251.3 [M + H]. 32 .sup.1H NMR (400 MHz, CDCl.sub.3) 11.23 (s, 1H), 8.69 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.96 (t, J = 7.0 Hz, 1H), 7.55 (dd, J = 7.4, 5.0 Hz, 1H), 7.47 (s, 1H), 7.46-7.41 (m, 1H), 7.11 (s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.93 (s, 3H). ESI-MS m/z 342.3 [M + H]. 33 .sup.1H NMR (400 MHz, DMSO) 10.45 (s, 1H), 8.84 (d, J = 2.5 Hz, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.17 (d, J = 3.4 Hz, 1H), 7.59-7.45 (m, 2H), 7.38 (dd, J = 8.3, 4.6 Hz, 1H), 7.29 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H). ESI-MS m/z 314.3 [M + H] 34 .sup.1H NMR (400 MHz, DMSO) 11.53 (s, 1H), 8.30 (dd, J = 12.9, 6.7 Hz, 3H), 8.16 (d, J =8.7 Hz, 2H), 7.80 (s, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.02-6.86 (m, 1H). ESI-MS m/z 299.3 [M + H]. 35 .sup.1H NMR (400 MHz, DMSO) 11.25 (s, 1H), 8.29 (d, J = 4.0 Hz, 1H), 7.77-7.60 (m, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.79 (m, 1H), 6.58 (d, J = 8.5 Hz, 2H), 5.18 (s, 2H). ESI-MS m/z 267.3 [M + H]. 36 .sup.1H NMR (400 MHz, DMSO) 11.37 (s, 1H), 9.99 (s, 1H), 8.30 (d, J = 4.9 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.70 (dd, J = 11.2, 4.3 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 7.00-6.84 (m, 1H), 2.06 (s, 3H). ESI-MS m/z 311.3 [M + H]. 37 0 .sup.1H NMR (400 MHz, DMSO) 11.35 (s, 1H), 8.35 (s, 1H), 8.30 (d, J = 4.7 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.22 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.99-6.84 (m, 1H), 6.02 (d, J = 7.5 Hz, 1H), 3.76 (dd, J = 13.4, 6.7 Hz, 1H), 1.10 (d, J = 6.5 Hz, 6H). ESI-MS m/z 354.3 [M + H]. 38 .sup.1H NMR (400 MHz, DMSO) 11.37 (s, 1H), 9.92 (s, 1H), 8.30 (d, J = 4.0 Hz, 1H), 7.79 (t, J = 21.9 Hz, 2H), 7.77-7.67 (m, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.98-6.85 (m, 1H), 2.33 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H). ESI-MS m/z 325.3 [M + H]. 39 .sup.1H NMR (400 MHz, DMSO) 11.37 (s, 1H), 9.92 (s, 1H), 8.44-8.21 (m, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.75-7.67 (m, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 6.8, 5.5 Hz, 1H), 2.30 (t, J = 7.3 Hz, 2H), 1.62 (dd, J = 14.7, 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). ESI-MS m/z 339.3 [M + H]. 40 .sup.1H NMR (400 MHz, DMSO) 11.42 (s, 1H), 8.31 (dd, J = 5.1, 1.1 Hz, 1H), 7.89-7.84 (m, 2H), 7.75-7.69 (m, 1H), 7.64-7.59 (m, 2H), 7.50 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.96-6.91 (m, 1H). ESI-MS m/z 333.1 [M + H]. 41 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.63 (s, 1H), 8.35 (d, J = 4.2 Hz, 1H), 7.81 (t, J = 7.3 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.22 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.01(t, J = 6.4 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H). ESI-MS m/z 270.3 [M + H]. 42 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.34 (s, 1H), 8.30 (dd, J = 5.0, 1.1 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.3, 1.9 Hz, 1H), 7.23 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.92-6.89 (m, 1H), 4.64 (dt, J = 12.0, 6.0 Hz, 1H), 1.28 (d, J = 6.0 Hz, 6H). ESI-MS m/z 312.3 [M + H]. 43 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.34 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.2, 1.9 Hz, 1H), 7.24 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 0.8 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 1.76-1.65 (m, 2H), 1.51-1.38 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). ESI-MS m/z 326.3 [M + H]. 44 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.35 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.2, 1.9 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.9 Hz, 2H), 6.92 (dd, J = 6.7, 5.5 Hz, 1H), 4.81 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). ESI-MS m/z 356.3 [M + H]. 45 .sup.1H NMR (400 MHz, DMSO) 13.01 (s, 1H), 11.37 (s, 1H), 8.30 (d, J = 4.0 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.76-7.67 (m, 1H), 7.26 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.92 (dd, J = 6.9, 5.7 Hz, 1H), 4.71 (s, 2H). ESI-MS m/z 328.3 [M + H]. 46 .sup.1H NMR (400 MHz, DMSO) 11.35 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.9, 5.4, 1.9 Hz, 1H), 7.26 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.00-6.94 (m, 2H), 6.95-6.89 (m, 1H), 4.86 (s, 2H), 3.66-3.53 (m, 4H), 3.47 (s, 4H). ESI-MS m/z 397.4 [M + H]. 47 0 .sup.1H NMR (400 MHz, DMSO) 9.49 (s, 1H), 8.41 (d, J = 3.7 Hz, 1H), 8.26 (t, J = 5.5 Hz, 1H), 7.82 (dt, J = 8.9, 5.4 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.22 (s, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.05 (dd, J = 7.0, 5.0 Hz, 1H), 6.78 (d, J = 8.6 Hz, 2H), 5.02 (s, 2H), 3.20-3.07 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). ESI-MS m/z 355.3 [M + H]. 48 .sup.1H NMR (400 MHz, DMSO) 11.50 (s, 1H), 8.32 (d, J = 4.4 Hz, 1H), 8.04 (q, J = 8.6 Hz, 4H), 7.74 (t, J = 7.0 Hz, 1H), 7.66 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.98-6.93 (m, 1H), 3.87 (s, 3H). ESI-MS m/z 312.3 [M + H]. 49 .sup.1H NMR (400 MHz, DMSO) 11.51 (s, 1H), 8.32 (dd, J = 5.1, 1.1 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.93-7.85 (m, 2H), 7.79-7.71 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 6.96 (dd, J = 6.7, 5.5 Hz, 1H). ESI-MS m/z 279.3 [M + H]. 50 .sup.1H NMR (400 MHz, DMSO) 11.59 (s, 1H), 8.35 (d, J = 4.3 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.82-7.69 (m, 5H), 7.53 (s, 1H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.03-6.96 (m, 1H). ESI-MS m/z 330.3 [M + H]. 51 .sup.1H NMR (400 MHz, DMSO) 11.57 (s, 1H), 8.33 (dd, J = 5.1, 1.1 Hz, 1H), 8.22-8.14 (m, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.79-7.71 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 6.97 (dd, J = 6.5, 5.2 Hz, 1H), 3.24 (s, 3H). ESI-MS m/z 332.4 [M + H]. 52 .sup.1H NMR (400 MHz, DMSO) 11.43 (s, 1H), 8.32 (d, J = 4.0 Hz, 1H), 7.97 (t, J = 6.3 Hz, 2H), 7.77-7.68 (m, 1H), 7.54 (s, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 6.7, 5.5 Hz, 1H), 3.61-3.49 (m, 8H). ESI-MS m/z 367.3 [M + H]. 53 .sup.1H NMR (400 MHz, DMSO) 11.44 (s, 1H), 8.47 (t, J = 5.4 Hz, 1H), 8.32 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.76-7.69 (m, 1H), 7.58 (s, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 6.7, 5.5 Hz, 1H), 1.14 (t, J = 7.2 Hz, 3H). ESI-MS m/z 325.3 [M + H]. 54 .sup.1H NMR (400 MHz, CDCl.sub.3) 8.77 (s, 1H), 8.34 (d, J = 4.9 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.58-7.50 (m, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.01 (s, 1H), 6.90- 6.83 (m, 1H), 6.77 (d, J = 8.3 Hz, 1H), 3.78-3.69 (m, 4H), 3.57 (s, 2H), 2.51 (s, 4H). ESI-MS m/z 353.4 [M + H]. 55 .sup.1H NMR (400 MHz, DMSO) 11.25 (s, 1H), 8.28 (d, J = 4.0 Hz, 1H), 7.72-7.63 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.41-7.33 (m, 4H), 7.29-7.17 (m, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 6.90 (dd, J = 6.6, 5.6 Hz, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.38 (t, J = 6.0 Hz, 1H), 4.31 (d, J = 6.0 Hz, 2H). ESI-MS m/z 359.3 [M + H]. 56 .sup.1H NMR (400 MHz, DMSO) 11.26 (s, 1H), 8.29 (d, J = 4.1 Hz, 1H), 7.74-7.63 (m, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.84 (m, 1H), 6.66 (d, J = 8.7 Hz, 2H), 5.58 (t, J = 6.0 Hz, 1H), 2.85 (d, J = 6.0 Hz, 2H), 0.96 (s, 9H). ESI-MS m/z 339.3 [M + H]. 57 0 .sup.1H NMR (400 MHz, DMSO) 11.44 (s, 1H), 8.31 (d, J = 4.0 Hz, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.78-7.64 (m, 1H), 7.57 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.00-6.82 (m, 1H), 4.11 (dd, J = 14.0, 6.7 Hz, 1H), 1.18 (d, J = 6.6 Hz, 6H). ESI-MS m/z 339.3 [M + H]. 58 .sup.1H NMR (400 MHz, DMSO) 11.47 (s, 1H), 10.25 (s, 1H), 8.32 (d, J = 4.1 Hz, 1H), 8.09-8.02 (m, 4H), 7.80 (d, J = 8.3 Hz, 2H), 7.73 (t, J = 7.7 Hz, 1H), 7.64 (s, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.23-7.03 (m, 2H), 6.99-6.83 (m, 1H). ESI-MS m/z 373.3 [M + H]. 59 .sup.1H NMR (400 MHz, DMSO) 11.43 (s, 1H), 8.50-8.13 (m, 1H), 7.97 (d, J = 8.2 Hz, 2H), 7.81-7.64 (m, 1H), 7.51 (s, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 6.6, 5.4 Hz, 1H), 3.66-3.57 (br, 4H), 2.44-2.35 (br, 4H), 2.19 (s, 3H). ESI-MS m/z 380.3 [M + H]. 60 ESI-MS m/z 326.3 [M + H]. 61 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.36 (s, 1H), 8.30 (dd, J = 4.8, 1.0 Hz, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.75-7.66 (m, 1H), 7.35 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.92 (dd, J = 6.7, 5.5 Hz, 1H), 4.59-4.49 (m, 1H), 3.92 (d, J = 13.4 Hz, 1H), 3.13 (t, J = 11.9 Hz, 1H), 2.78 (t, J = 12.0 Hz, 1H), 2.63-2.59 (m, 1H), 2.03 (s, 3H), 1.91-1.82 (m, 2H), 1.69-1.60 (m, 1H), 1.49-1.42 (m, 1H). ESI-MS m/z 379.3 [M + H]. 62 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.44 (s, 1H), 8.51 (t, J = 5.5 Hz, 1H), 8.31 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.76-7.68 (m, 1H), 7.58 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.97-6.90 (m, 1H), 3.59-3.50 (m, 4H), 3.48-3.39 (m, 4H), 3.24 (s, 3H). ESI-MS m/z 399.4 [M + H]. 63 .sup.1H NMR (400 MHz, DMSO) 11.53 (s, 1H), 8.33 (d, J = 4.0 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.82-7.72 (m, 1H), 7.56-7.42 (m, 3H), 7.14 (d, J = 8.4 Hz, 1H), 7.04-6.89 (m, 1H). ESI-MS m/z 288.3 [M + H]. 64 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.56 (s, 1H), 9.87 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.46-8.39 (m, 2H), 7.85 (t, J = 7.9 Hz, 1H), 7.69-7.58 (m, 5H), 7.20-7.14 (m, 1H), 7.05 (s, 1H), 6.95-6.91 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H). ESI-MS m/z 374.4 [M + H]. 65 .sup.1H NMR (400 MHz, DMSO) 11.47 (s, 1H), 10.59 (s, 1H), 8.49 (d, J = 6.2 Hz, 2H), 8.32 (d, J = 4.1 Hz, 1H), 8.07 (q, J = 8.5 Hz, 4H), 7.81 (d, J = 6.3 Hz, 2H), 7.72 (dd, J = 11.3, 4.2 Hz, 1H), 7.66 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.98-6.92 (m, 1H). ESI-MS m/z 374.4 [M + H]. 66 ESI-MS m/z 353.4 [M + H]. 67 0 ESI-MS m/z 443.4 [M + H]. 68 .sup.1H NMR (400 MHz, DMSO) 11.49 (s, 1H), 9.91 (s, 1H), 8.62-8.36 (m, 1H), 7.86-7.77 (m, 3H), 7.77-7.69 (m, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.3 Hz, 1H), 6.97 (dd, J = 6.7, 5.4 Hz, 1H). ESI-MS m/z 408.0 [M + H]. 69 .sup.1H NMR (400 MHz, CDCl.sub.3) 10.56 (s, 1H), 9.87 (s, 1H), 8.59 J = 8.4 Hz, 1H), 8.46-8.39 (m, 2H), 7.85 (t, J = 7.9 Hz, 1H), 7.69-7.58 (m, 5H), 7.20-7.14 (m, 1H), 7.05 (s, 1H), 6.95-6.91 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H). ESI-MS m/z 374.4 [M + H]. 70 ESI-MS m/z 334.3 [M + H]. 71 ESI-MS m/z 406.3 [M + H]. 72 .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.40 (s, 1H), 8.31 (d, J = 4.0 Hz, 1H), 7.98 (d, J = 8.4Hz, 2H), 7.79-7.71 (m, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.52 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.01-6.95 (m, 1H), 4.26 (s, 2H), 2.89 (s, 3H), 1.70-1.36 (m, 7H). ESI-MS m/z 351.5 [M + H]. 73 .sup.1H NMR (400 MHz, DMSO) 11.56 (s, 1H), 8.33 (d, J = 5.2 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 8.00 (t J = 8.0 Hz, 2H), 7.76 (t, J = 8.4 Hz, 1H), 7.68 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.98 (t, J = 6.4 Hz, 1H), 2.60 (s, 3H). ESI-MS m/z 296.3 [M + H]. 74 .sup.1H NMR (400 MHz, DMSO) 11.43 (s, 1H), 11.22 (s, 1H), 9.01 (s, 1H), 8.31 (d, J = 4.1 Hz, 1H), 8.04-7.95 (m, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.72 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97-6.91 (m, 1H). ESI-MS m/z 313.3 [M + H]. 75 .sup.1H NMR (400 MHz, CDCl.sub.3) 9.06 (s, 1H), 8.37 (d, J = 4.0 Hz, 1H), 7.97 (t, J = 17.5 Hz, 2H), 7.58-7.50 (m, 1H), 7.46 (dd, J = 10.1, 6.4 Hz, 3H), 7.25 (s, 2H), 7.10 (s, 1H), 6.88 (dd, J = 7.0, 5.2 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H). ESI-MS m/z 323.1 [M + H]. 76 .sup.1H NMR (400 MHz, DMSO) 11.40 (s, 1H), 8.31 (d, J = 3.7 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.91 (dd, J = 2.7, 1.1 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.74- 7.69 (m, 1H), 7.65 (dd, J = 5.0, 2.9 Hz, 1H), 7.61 (dd, J = 5.0, 1.0 Hz, 1H), 7.46 (s, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 6.9, 5.3 Hz, 1H). ESI-MS m/z 336.4 [M + H]. 77 0 .sup.1H NMR (400 MHz, DMSO) 11.48 (s, 1H), 8.32 (d, J = 4.5 Hz, 1H), 8.09 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.83- 7.58 (m, 2H), 7.11 (d, J = 8.3 Hz, 1H), 7.00-6.82 (m, 1H). ESI-MS m/z 322.3 [M + H]. 78 .sup.1H NMR (400 MHz, CDCl.sub.3) 8.82 (s, 1H), 8.36 (d, J = 4.0 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.59-7.55 (m, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.07 (s, 1H), 6.89 (dd, J = 7.0, 5.2 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 3.12 (s, 1H). ESI-MS m/z 278.3 [M + H]. 79 .sup.1H NMR (400 MHz, DMSO) 11.44 (s, 1H), 8.65 (d, J = 5.8 Hz, 2H), 8.33 (d, J = 4.7 Hz, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 5.9 Hz, 2H), 7.73 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.99-6.91 (m, 1H). ESI-MS m/z 331.4 [M + H]. 80 .sup.1H NMR (400 MHz, DMSO) 11.63 (s, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.80 (s, 1H), 7.71 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.17 (d, J = 7.2 Hz, 2H), 7.00 (d, J = 6.0 Hz, 1H), 2.27 (s, 3H), 2.24 (s, 3H). ESI-MS m/z 282.3 [M + H]. 81 .sup.1H NMR (400 MHz, DMSO) 11.63 (s, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.80 (s, 2H), 7.71 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.98 (t, J = 6.0 Hz, 1H), 2.92-2.85 (m, 4H), 2.07-2.00 (m, 2H). ESI-MS m/z 294.3 [M + H]. 82 .sup.1H NMR (400 MHz, DMSO) 11.67 (s, 1H), 8.36 (d, J = 4.9 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.37 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 6.0 Hz, 1H), 6.97 (d, J = 7.9 Hz, 1H), 6.05 (s, 2H). ESI-MS m/z 298.3 [M + H]. 83 .sup.1H NMR (400 MHz, DMSO) 11.28 (s, 1H), 8.28 (d, J = 4.5 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.51 (s, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.90 (t, J = 5.6 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 4.97 (s, 2H), 2.10 (s, 3H). ESI-MS m/z 283.3 [M + H].

TEST EXAMPLE 1 UCP1 Luciferase Assay

Evaluating Compounds that Induce Ucp1 Up-Regulation

(51) Ucp1-luciferase knock-in mice will be housed in cages and fed on standard chow diets (the detailed procedures please see reference 14). The 8-week-old mice will be killed by the cervical dislocation and put into 75% alcohol for 5 minutes. The inguinal adipose tissue will be dissected out and transferred to the three 10-cm petri dishes and washed with PBS for three times. The adipose tissue will be dissected and minced on a 10-cm petri dish for several minutes and then pipetted into 20 ml 0.1% collagenase solution in 50 ml conical tubes and digested at 37 C. for 1 hour. Next, 20 ml culture medium will be added to terminate the digestion and centrifuged for 10 minutes at 1000 rpm respectively. The supernatants will be removed and the pellets re-suspended in 10 mL culture medium and plated into 10-cm plates. The cells will be left to adhere to the plates overnight and the culture medium will be changed every other day. The confluent cells will be trypsinized, counted and plated to 24-well plate (510.sup.4 cells/well). The cells will be washed and re-fed the next day (as day 2). Two days later (day 0), the cells will usually become confluent and be induced to differentiate with MDIR medium. On day 2, the medium will be changed to IR medium and re-fed every 2 days. Complete differentiation is usually achieved by day 10. On day 10, the medium will be changed to the culture medium of 10% FBS/DMEM with indicated drugs along with the positive and negative controls for 2 days. The media will be removed and the cells washed with 1 ml PBS per well three times. Then 100 l lysis buffer will be used to lyse the cells at 4 C. for 1 hour. 30 l culture medium, 30 l cell lysates and 60 l Steady-Glo Reagent were added to a well of 96-well-plate and the luminescence will be measured using a luminometer. The protein concentration will be determined using 20 l cell lysates and specific activity of luciferase will be tabulated and data analyzed.

(52) The bioactivity data of the compounds are listed in the following table.

(53) TABLE-US-00002 Ucp1 activation fold/corresponding Compound No. concentration 1 5.2/3 M 2 1.1/1 M 3 2.8/1 M 4 4.1/3 M 5 5.4/3 M 6 1.2/1 M 7 1.2/0.3 M 8 4.0/1 M 9 3.6/1 M 10 3.8/3 M 11 2.0/1 M 12 1.2/1 M 13 1.6/3 M 14 2.4/3 M 15 1.4/0.3 M 16 5.1/3 M 17 1.9/1 M 18 5.3/3 M 19 1.6/1 M 20 3.0/3 M 21 2.9/3 M 22 3.9/1 M 23 4.0/3 M 24 2.7/3 M 25 1.3/3 M 26 3.5/3 M 27 3.3/1 M 28 3.2/3 M 29 1.2/0.3 M 30 1.1/1 M 31 1.1/1 M 32 2.6/3 M 33 2.0/3 M 34 3.8/1 M 35 3.7/1 M 36 3.8/1 M 37 2.0/3 M 38 5.0/3 M 39 3.4/1 M 40 3.0/3 M 41 3.3/1 M 42 2.0/1 M 43 2.8/1 M 44 1.9/3 M 45 1.3/0.3 M 46 1.7/3 M 47 2.4/3 M 48 6.0/3 M 49 3.9/1 M 50 2.4/3 M 51 3.9/1 M 52 5.1/1 M 53 3.0/1 M 54 3.9/1 M 55 2.0/3 M 56 2.3/3 M 57 3.5/3 M 58 1.4/3 M 59 4.0/1 M 60 2.6/3 M 61 3.2/3 M 62 2.2/3 M 63 5.9/3 M 64 1.1/1 M 65 1.1/3 M 66 1.0/3 M 67 3.1/3 M 68 1.3/3 M 69 4.6/3 M 70 1.7/0.3 M 71 1.7/0.3 M 72 1.8/3 M 73 1.2/3 M 74 3.0/3 M 75 1.1/3 M 76 1.8/3 M 77 1.1/3 M 78 2.3/1 M 79 1.8/3 M 80 1.5/3 M 81 2.0/1 M 82 2.4/1 M 83 6.0/3 M

TEST EXAMPLE 2

High Fat Diet Induced Obesity Mouse Model

(54) 8-week-old male mice (57BL/6J) were housed in metabolism cages and maintained on a 12 h lightdark cycle at 23 C. and fed high fat diet (HFD) (21.9 kJ/g, 60% of energy as fat, 20% of energy as protein, 20% of energy as carbohydrate; D12492; Research Diet, New Brunswick, N.J., USA) for 8 weeks. Then, diets induced obese mice were randomized into two groups according to glucose levels and body weight and gavaged with daily indicated drugs (50 mg/kg) or vehicle. Food intake and body weight was measured weekly. All experiments with animals were approved by the Animal Care and Use Committee of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences.

TEST EXAMPLE 3

Hematoxylin and Eosin Staining

(55) Hematoxylin and eosin staining was performed as the standard protocol (see reference 15). As to the results, please see FIG. 1 (left panel) using compound 1 as the test compound.

TEST EXAMPLE 4

Immunochemistry

(56) Wash sections in dH.sub.2O three times for 5 minutes each. Incubate sections in 3% hydrogen peroxide for 10 minutes. Wash sections in dH.sub.2O twice for 5 minutes each, and then wash sections in wash buffer for 5 minutes. Block each section with 100-400 l blocking solution for 1 hour at room temperature. Remove blocking solution and add 100-400 l primary UCP1 antibody to each section. Incubate overnight at 4 C. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each. Add 100-400 l biotinylated secondary antibodies, diluted in TBST to each section. Incubate 30 minutes at room temperature. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each. Add 100-400 l DAB to each section and monitor staining closely. As soon as the sections develop, immerse slides in dH.sub.2O. Wash sections in dH.sub.2O two times for 5 minutes each. Incubate sections in 95% ethanol two times for 10 seconds each. Repeat in 100% ethanol, incubating sections two times for 10 seconds each. Repeat in xylene, incubating sections two times for 10 seconds each. Mount cover slips and take pictures. As to the results, please see FIG. 1 (right panel) using compound 1 as the test compound.

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