Compound and pharmaceutical composition for preventing or treating obesity or metabolic syndrome comprising thereof

Abstract

The present invention provides a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof which can be effectively used for preventing or treating obesity or metabolic syndrome, and a pharmaceutical composition comprising the same. ##STR00001## in Chemical Formula 1, R.sub.1 and R.sub.2 are the same as defined in the specification.

Claims

1. A compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, ##STR00018## in Chemical Formula 1, R.sub.1 is -L.sub.1-(phenyl), R.sub.2 is hydrogen, or -L.sub.2-(phenyl), L.sub.1 is C.sub.1-5 alkylene, and L.sub.2 is C.sub.1-5 alkylene, with the proviso that not both R.sub.1 and R.sub.2 are benzyl.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.1 is CH.sub.2, CH.sub.2CH.sub.2, CH.sub.2CH.sub.2CH.sub.2, or CH.sub.2CH.sub.2CH.sub.2CH.sub.2.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.2 is CH.sub.2, CH.sub.2CH.sub.2, CH.sub.2CH.sub.2CH.sub.2, or CH.sub.2CH.sub.2CH.sub.2CH.sub.2.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is CH.sub.2-(phenyl), and R.sub.2 is hydrogen, CH.sub.2CH.sub.2-(phenyl), CH.sub.2CH.sub.2CH.sub.2-(phenyl), or CH.sub.2CH.sub.2CH.sub.2CH.sub.2-(phenyl).

5. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 and R.sub.2 are equal to each other.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein R.sub.1 and R.sub.2 are CH.sub.2CH.sub.2-(phenyl), CH.sub.2CH.sub.2CH.sub.2-(phenyl), or CH.sub.2CH.sub.2CH.sub.2CH.sub.2-(phenyl).

7. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is any one selected from the group consisting of: 1) 2-(3,4-diphenethoxybenzylamino)ethanol, 2) 2-(3,4-bis(3-phenylpropoxy)benzylamino)ethanol, 3) 2-(3,4-bis(4-phenylbutoxy)benzylamino)ethanol, 4) 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol, 5) 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol, 6) 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol, and 7) 2-(4-(benzyloxy)-3-(4-phenylbutoxy)benzylamino)ethanol.

8. A method for preventing or treating obesity or metabolic syndrome in a subject in need thereof, comprising administering to the subject the compound or a pharmaceutically acceptable salt thereof according to claim 1.

9. The method according to claim 8, wherein the metabolic syndrome is any one selected from the group consisting of myocardial infarction, arteriosclerosis, hyperlipidemia, hypertension, cerebral infarction, cerebral hemorrhage, fatty liver, and type 2 diabetes mellitus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 and FIG. 2 show results confirming the effects of decreasing the lipid droplet size in 3T3-L1 adipocytes and Hep G2 hepatocytes by the compounds of the present invention through Oil Red O staining

(2) FIG. 3 shows the results confirming the effects of decreasing the lipid droplet size and number in 3T3-L1 adipocytes by the compounds of the present invention through BODIPY staining.

(3) FIG. 4 shows the results confirming a phenomenon in which the compounds of the present invention activate the lipophagy process in 3T3-L1 adipocytes (FIG. 4A) and Hep G2 hepatocytes (FIG. 4B) and the activated lipophagy process decreases the size and number of lipid droplets accumulated in cells, through the immunofluorescence staining.

(4) FIG. 5 shows the results confirming that treatment of the compounds of the present invention and Bafilomycin as an inhibitor of the action of lipophagy together did not result in removal of lipid droplets, in order to verify that the compounds of the present invention had removed the lipid droplets through the lipophagy process.

(5) FIG. 6 shows the results confirming that in order to confirm the anti-obesity effect of the compounds of the present invention at the living body level, obesity was induced in wild-type mice via high-fat diets, and the body weight and abdominal adipose tissue size were reduced after treatment of the placebo and the compounds of the present invention.

(6) FIG. 7 shows the result recording the changes in body weight weekly by the treatment of the placebo and the compound of the present invention.

(7) FIG. 8 is a graph showing the measurement results of the weight of each tissue in order to confirm that the weight loss effect exhibited by the treatment of the compound of the present invention was caused by a decrease in the adipose tissue weight.

(8) FIG. 9 is a graph showing the results of comparison of the weights of adipose tissues reduced by the compounds of the present invention.

(9) FIG. 10 is a photograph comparing the actual appearance of each tissue extracted to confirm that the weight of the adipose tissue alone has reduced. In each photograph, the left side was one treated with the vehicle, and the right side was one treated with the compound of the present invention.

(10) FIG. 11 is a graph showing the percentage of the body weight reduced through the compounds of the present invention. In addition, the results of measurement of the body height was shown to compare the degree of development during the administration period. In order to determine whether the weight loss effect was due to a decrease in feed intake, the results of surveying feed intake were shown.

(11) FIG. 12 shows the results of the items that have examined blood samples taken from the placebo-treated mice and the compound-administered mice in order to understand that the weight loss caused by the compounds of the present invention has an effect on blood lipid components, blood glucose and pathological conditions of the tissue.

(12) FIG. 13 shows the results of observation of mouse adipose tissues through HnE staining in order to confirm the removal effect of lipid droplets accumulated in the tissues by the compounds of the present invention.

(13) FIG. 14 shows the result of observation of mouse adipose tissue through HnE staining in order to confirm that the size of adipocytes was reduced by the compound of the present invention.

(14) FIG. 15 shows the results confirming the improvement of the expression of LC3 protein as a marker of lipophagy activity through tissue immunochemical staining in order to confirm that the weight loss and the lipid droplet-reducing effects exhibited by the compounds of the present invention were caused by the action of lipophagy.

(15) FIG. 16 shows the results of observation of the expression patterns of macrophages, which are inflammatory cells, through tissue immunochemical staining in order to observe the relaxation effect of the compounds of the present invention against inflammatory symptoms in hepatic tissues caused by excessive accumulation of lipid droplets.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(16) Hereinafter, the present invention will be described in detail with reference to the following Examples and Experimental Examples. However, these Examples and Experimental Examples are presented for illustrative purposes only, and the scope of the invention is not limited thereto.

Example 1: Preparation of 2-(3,4-diphenethoxybenzylamino)ethanol Hydrochloride

Step 1) Preparation of 3,4-diphenethoxybenzaldehyde and 3-hydroxy-4-phenethoxybenzaldehyde

(17) ##STR00004##

(18) A solution of 3,4-dihydroxybenzaldehyde (0.50 g, 3.62 mmol) was diluted with DMF (10 mL) and stirred. (2-Bromoethyl) benzene (1.24 mL, 9.05 mmol) was slowly added and anhydrous K.sub.2CO.sub.3 (2.5 g, 18.1 mmol) was added. The mixture was stirred at room temperature for 2 hours, and K.sub.2CO.sub.3 (2.4 g, 17.3 mmol) was further added, heated to 70? C. for 30 minutes and then cooled to room temperature. The mixture was partitioned between water (120 mL) and ether (120 mL). The organic layer was separated and the aqueous layer was extracted with ether (3?50 mL). The extracted organic layer was washed with water (2?50 mL) and saturated with aqueous NaCl solution (50 mL). The light pale yellow extract was dried over anhydrous sodium sulfate, washed with hexane (75 mL) and then concentrated. The resulting residue was purified by silica gel column chromatography using ethyl acetate:hexane (1:4) to give 3,4-diphenethoxybenzaldehyde (6.57 g, 95%) as a cream-colored solid and 3-hydroxy-4-phenethoxybenzaldehyde as a cream-colored solid, respectively.

(19) 3-Hydroxy-4-phenethoxybenzaldehyde: .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.78 (s, 1H), 7.22-7.40 (m, 12H), 6.91 (d, 1H, J=6.0 Hz), 4.23 (td, 4H, J=3.0 and 6.0 Hz), 3.15 (td, 4H, J=3.0 and 6.0 Hz); ESI MS: m/z 243.17 [M+H].sup.+

(20) 3,4-diphenethoxybenzaldehyde: .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.84 (s, 1H), 7.26-7.43 (m, 7H), 6.96 (d, 1H, J=9.0 Hz), 5.62 (s, 1H), 4.36 (t, 2H, J=6.0 Hz), 3.17 (t, 2H, J=6.0 Hz); ESI MS: m/z 347.33 [M+H].sup.+

Step 2) Preparation of 2-(3,4-diphenethoxybenzylamino)ethanol

(21) ##STR00005##

(22) 2-Aminoethanol (27 mg (27 ?L), 0.45 mmol) was added to 3,4-diphenethoxybenzaldehyde (100 mg, 0.3 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them. The mixture was stirred at 80? C. for 12 hours and then cooled to room temperature. NaBH.sub.4 (17 mg, 0.45 mmol) was added and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The residue was purified by flash column to give 2-(3,4-diphenethoxybenzylamino)ethanol (96 mg, 85%).

(23) ESI MS: m/z 392.92 [M+H].sup.+

Step 3) Preparation of 2-(3,4-diphenethoxybenzylamino)ethanol Hydrochloride

(24) 2-(3,4-Diphenethoxybenzylamino)ethanol (1.0 g, 2.75 mmol) prepared in the previous step 2 was dissolved in methanol (25 mL) and HCl gas was introduced for 1 hour. The mixture was further stirred for 2 hours and evaporated to about 1 mL, and then hexane was added to prepare a solid, which was then filtered and dried to give the final compound 2-(3,4-diphenethoxybenzylamino)ethanol hydrochloride (720 mg, 65%).

Example 2: Preparation of 2-(3,4-bis(3-phenylpropoxy)benzylamino)ethanol hydrochloride

Step 1) Preparation of 3,4-bis(3-phenylpropoxy)benzaldehyde

(25) ##STR00006##

(26) 3,4-Dihydroxybenzaldehyde (500 mg, 3.62 mmol) was diluted with DMF (10 mL), and anhydrous K.sub.2CO.sub.3 (1.5 g, 10.86 mmol) and (3-bromopropyl)benzene (1.2 mL, 7.96 mmol) were slowly added in this order. The mixture was heated to 80? C. and stirred for 2 hours and then cooled to room temperature. The mixture was partitioned between water (50 mL) and ether (50 mL). The organic layer was separated and the water was extracted with ether (3?50 mL). The extracted organic layer was washed with water (2?50 mL) and saturated with aqueous NaCl solution (50 mL). The light pale yellow extract was dried over anhydrous sodium sulfate, washed with hexane (75 mL) and then concentrated. The resulting residue was purified by silica gel column chromatography using ethyl acetate:hexane (1:4) to give 3,4-bis(3-phenylpropoxy)benzaldehyde (1.3 g, 96%) as a cream-colored solid.

(27) .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.86 (s, 1H), 7.45 (dd, 1H, J=3.0 and 9.0 Hz), 7.41 (d, 1H, J=3.0 Hz), 7.22-7.34 (m, 10H) 6.95 (d, 1H, J=9.0 Hz), 4.12 (td, 4H, J=3.0 and 9.0 Hz), 2.87 (td, 4H, J=3.0 and 9.0 Hz), 2.17-2.28 (m, 4H).

Step 2) Preparation of 2-(3,4-bis(3-phenylpropoxy)benzylamino)ethanol

(28) ##STR00007##

(29) 2-Aminoethanol (25 mg (25 ?L), 0.40 mmol) was added to 3,4-bis(3-phenylpropoxy)benzaldehyde (100 mg, 0.27 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them, and the mixture was stirred at 60? C. for 12 hours. The reaction solution was cooled to room temperature. NaBH.sub.4 (15.2 mg, 0.40 mmol) was slowly added, and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4, and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(3,4-bis(3-phenylpropoxy)benzylamino)ethanol (96 mg, 86%).

(30) ESI MS: m/z 421.0 [M+2H].sup.+

Step 3) Preparation of 2-(3,4-bis(3-phenylpropoxy)-benzylamino)ethanol Hydrochloride

(31) 2-(3,4-bis(3-phenylpropoxy)benzylamino)ethanol (1.0 g, 2.75 mmol) prepared in the previous step 2 was dissolved in methanol (25 mL) and HCl gas was introduced for 1 hour. The mixture was further stirred for 2 hours and evaporated to about 1 mL, and then hexane was added to prepare a solid, which was then filtered and dried to give 2-(3,4-bis(3-phenylpropoxy)-benzylamino)ethanol hydrochloride (720 mg, 65%).

Example 3: Preparation of 2-(3,4-bis(4-phenylbutoxy)benzylamino)ethanol

Step 1) Preparation of 3,4-bis(4-phenylbutoxy)benzaldehyde

(32) ##STR00008##

(33) 3,4-Dihydroxybenzaldehyde (147 mg, 1.07 mmol) was diluted with DMF (10 mL), and anhydrous K.sub.2CO.sub.3 (442 mg, 3.20 mmol) and (4-bromobutyl)benzene (0.4 mL, 2.35 mmol) were slowly added in this order. The mixture was heated to 80? C. and stirred for 2 hours and then cooled to room temperature. The mixture was partitioned between water (50 mL) and ether (50 mL). The organic layer was separated and the water was extracted with ether (3?50 mL). The extracted organic layer was washed with water (2?50 mL) and saturated with aqueous NaCl solution (50 mL). The light pale yellow extract was dried over anhydrous sodium sulfate, washed with hexane (75 mL) and then concentrated. The resulting residue was purified by silica gel column chromatography using methanol (1% to 5% in DCM) to give 3,4-bis(4-phenylbutoxy)benzaldehyde (398 mg, 93%) as a cream-colored solid.

(34) .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 7.46-7.26 (m, 10H), 6.84 (d, 1H, J=9.0 Hz), 6.59 (d, 1H, J=3.0 Hz), 6.38 (dd, 1H, J=3.0 and 9.0 Hz), 5.12 (s, 1H), 5.07 (s, 1H), 4.03 (td, 1H, J=3.6 and 5.1 Hz), 3.93-3.86 (m, 2H), 3.25 (s, 2H), 2.92-2.86 (m, 2H), 2.72 (dd, 1H, J=8.1 and 12.0 Hz), 1.12 (d, 6H, J=6.3 Hz)

(35) ESI MS m/z: 496 [M+H].sup.+

Step 2) Preparation of 2-(3,4-bis(4-phenylbutoxy)benzylamino)ethanol

(36) ##STR00009##

(37) 2-Aminoethanol (106 mg, 1.74 mmol) was added to 3,4-bis(4-phenylbutoxy)benzaldehyde (0.35 g, 0.87 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them, and the mixture was stirred at 60? C. for 12 hours. The reaction solution was cooled to room temperature. NaBH.sub.4 (33 mg, 0.87 mmol) was slowly added and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4, and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(3,4-bis(4-phenylbutoxy)benzylamino)ethanol (0.33 g, 85%).

(38) ESI MS: m/z 449.0 [M+2H].sup.+

Example 4: Preparation of 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol Hydrochloride

(39) Step 1) Preparation of 4-benzyloxy-3-hydroxybenzaldehyde

(40) ##STR00010##

(41) K.sub.2CO.sub.3 (2.5 g, 18.1 mmol) was added to a solution of 3,4-dihydroxybenzaldehyde (2.5 g, 18.1 mmol) in anhydrous acetonitrile (30 mL) at room temperature under an inert gas (N.sub.2) atmosphere while stirring them, and benzyl bromide (3.44 mL, 29.0 mmol) was slowly added. The mixture was heated to reflux and stirred for 2 hours to perform the reaction. The reaction solvent was removed by evaporation under reduced pressure, and to the result was added a cold 10% NaOH solution and stirred for 10 minutes, and then ethyl acetate (100 mL) was added. The resulting biphasic mixture was separated and the aqueous layer was acidified with 4N HCl and extracted with DCM (3?300 mL). The organic layer was washed with brine, and water, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give a residue, which was purified by crystallization from ethyl acetate to give 4-benzyloxy-3-hydroxybenzaldehyde (2.90 g, 70%) as a white powder.

(42) .sup.1H NMR (300 MHz, CDCl.sub.3): ? 9.83 (s, 1H, CHO), 7.39-7.46 (m, 7H, ArH), 7.03 (d, 1H, J=9.0 Hz, ArH), 5.88 (s, 1H, OH), 5.20 (s, 2H, OCH.sub.2Ph)

(43) ESI MS: m/z 229.25 [M+H].sup.+

Step 2) Preparation of 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol

(44) ##STR00011##

(45) 2-Aminoethanol (81 mg (80 ?L), 1.32 mmol) was added to 4-(benzyloxy)-3-hydroxybenzaldehyde (200 mg, 0.88 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them, and the mixture was stirred at 60? C. for 12 hours and then cooled to room temperature. NaBH.sub.4 (50 mg, 1.32 mmol) was slowly added and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4, and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol (0.22 g, 92%).

(46) ESI MS: m/z 274.75 [M+H].sup.+

Step 3) Preparation of 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol Hydrochloride

(47) 2-(Benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol (1.0 g, 2.75 mmol) prepared in the previous step 2 was dissolved in methanol (25 mL) and HCl gas was introduced for 1 hour. The mixture was stirred for 2 hours and evaporated to about 1 mL, and then hexane was added to prepare a solid, which was then filtered and dried to give 2-(benzyloxy)-5-((2-hydroxyethylamino)methyl)phenol hydrochloride (720 mg, 65%).

Example 5: Preparation of 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol Hydrochloride

Step 1) Preparation of 4-(benzylbenzyloxy)-3-phenoxybenzaldehyde

(48) ##STR00012##

(49) 4-(Benzylbenzyloxy)-3-hydroxybenzaldehyde (0.50 g, 2.19 mmol) was diluted with DMF (10 mL), and anhydrous K.sub.2CO.sub.3 (604 mg, 4.38 mmol) and (2-bromoethyl)benzene (0.36 mL, 2.63 mmol) were slowly added in this order. The mixture was heated at 70? C. for 2 hours and then cooled to room temperature. The mixture was partitioned between water (20 mL) and ether (20 mL). The organic layer was separated and the aqueous layer was extracted with ether (3?20 mL). The extracted organic layer was washed with water (2?20 mL) and saturated with aqueous NaCl solution (20 mL). The light pale yellow extract was dried over anhydrous sodium sulfate and then concentrated. The resulting residue was purified by silica gel column chromatography using ethyl acetate:hexane (1:9) to give 4-(benzylbenzyloxy)-3-phenoxybenzaldehyde (0.66 g, 90%) as a cream-colored solid.

(50) .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.80 (s, 1H), 7.21-7.39 (m, 12H), 6.98 (d, 1H, J=6.0 Hz), 5.17 (s, 2H), 4.27 (t, 2H, J=6.0 Hz), 3.14 (t, 2H, J=6.0 Hz)

Step 2) Preparation of 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol

(51) ##STR00013##

(52) 2-Aminoethanol (22 mg (22 ?L), 0.36 mmol) was added to a solution of 4-(benzylbenzyloxy)-3-phenoxybenzaldehyde (100 mg, 0.30 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them. The reaction mixture was stirred at 60? C. for 12 hours and cooled to room temperature. NaBH.sub.4 (17.1 mg, 0.45 mmol) was slowly added while stirring, and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4 and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol (0.10 g, 90%).

(53) ESI MS: m/z 378.9 [M+H].sup.+

Step 3) Preparation of 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol Hydrochloride

(54) 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol (1.0 g, 2.75 mmol) prepared in the previous step 2 was dissolved in methanol (25 mL) and HCl gas was introduced for 1 hour. The mixture was stirred for 2 hours and evaporated to about 1 mL, and then hexane was added to prepare a solid, which was then filtered and dried to give 2-(4-(benzyloxy)-3-phenethoxybenzylamino)ethanol hydrochloride (720 mg, 65%).

Example 6: Preparation of 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol Hydrochloride

Step 1) Preparation of 4-(benzylbenzyloxy)-3-(3-phenylpropoxy)benzaldehyde

(55) ##STR00014##

(56) 4-(Benzylbenzyloxy)-3-hydroxybenzaldehyde (0.50 g, 2.19 mmol) was diluted with DMF (10 mL), and anhydrous K.sub.2CO.sub.3 (604 mg, 4.38 mmol) and (2-bromopropyl)benzene (0.4 mL, 2.63 mmol) were slowly added in this order. The mixture was heated at 70? C. for 2 hours and then cooled to room temperature. The mixture was partitioned between water (20 mL) and ether (20 mL). The organic layer was separated and the aqueous layer was extracted with ether (3?20 mL). The extracted organic layer was washed with water (2?20 mL) and saturated with aqueous NaCl solution (20 mL). The light pale yellow extract was dried over anhydrous sodium sulfate and then concentrated. The resulting residue was purified by silica gel column chromatography using ethyl acetate:hexane (1:9) to give 4-(benzylbenzyloxy)-3-(3-phenylpropoxy)benzaldehyde (0.68 g, 90%) as a cream-colored solid.

(57) .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.81 (s, 1H), 7.18-7.38 (m, 12H), 7.00 (d, 1H, J=9.0 Hz), 5.23 (s, 2H), 4.09 (t, 2H, J=6.0 Hz), 3.14 (t, 2H, J=6.0 Hz), 2.12-2.22 (m, 2H)

Step 2) Preparation of 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol

(58) ##STR00015##

(59) 2-Aminoethanol (22 mg (22 ?L), 0.36 mmol) was added to 4-(benzylbenzyloxy)-3-(3-phenylpropoxy)benzaldehyde (100 mg, 0.29 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them. The reaction mixture was stirred at 60? C. for 12 hours and then cooled to room temperature. NaBH.sub.4 (16.7 mg, 0.44 mmol) was slowly added, and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4, and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol (93 mg, 82%).

(60) ESI MS: m/z 392.92 [M+H].sup.+

Step 3) Preparation of 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol Hydrochloride

(61) 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol (1.0 g, 2.75 mmol) prepared in the previous step 2 was dissolved in methanol (25 mL) and HCl gas was introduced for 1 hour. The mixture was further stirred for 2 hours and evaporated to about 1 mL, and then hexane was added to prepare a solid, which was then filtered and dried to give 2-(4-(benzyloxy)-3-(3-phenylpropoxy)benzylamino)ethanol hydrochloride (720 mg, 65%).

Example 7: Preparation of 2-(4-(benzyloxy)-3-(4-phenylbutoxy)benzylamino)ethanol

Step 1) Preparation of 4-(benzylbenzyloxy)-3-(4-phenylbutoxy)benzaldehyde

(62) ##STR00016##

(63) 4-(Benzylbenzyloxy)-3-hydroxybenzaldehyde (0.50 g, 2.19 mmol) was diluted with DMF (10 mL), and anhydrous K.sub.2CO.sub.3 (604 mg, 4.38 mmol) and (4-bromopropyl)benzene (0.46 mL, 2.63 mmol) were slowly added in this order. The mixture was heated at 70? C. for 2 hours and then cooled to room temperature. The mixture was partitioned between water (20 mL) and ether (20 mL). The organic layer was separated and the aqueous layer was extracted with ether (3?20 mL). The extracted organic layer was washed with water (2?20 mL) and saturated with aqueous NaCl solution (20 mL). The light pale yellow extract was dried over anhydrous sodium sulfate and then concentrated. The resulting residue was purified by silica gel column chromatography using ethyl acetate:hexane (1:9) to give 4-(benzylbenzyloxy)-3-(4-phenylbutoxy)benzaldehyde (0.69 g, 88%) as a cream-colored solid.

(64) .sup.1H-NMR (CDCl.sub.3, 300 MHz): ? 9.86 (s, 1H), 7.29-7.48 (m, 8H), 7.22-7.24 (m, 3H), 7.03 (d, 1H, J=6.0 Hz), 5.25 (s, 2H), 4.14 (t, 2H, J=6.0 Hz), 2.74 (t, 2H, J=6.0 Hz), 1.87-1.94 (m, 4H)

Step 2) Preparation of 2-(4-(benzyloxy)-3-(4-phenylbutoxy)benzylamino)ethanol

(65) ##STR00017##

(66) 2-Aminoethanol (25.6 mg (25 ?L), 0.42 mmol) was added to 4-(benzylbenzyloxy)-3-(4-phenylbutoxy)benzaldehyde (100 mg, 0.28 mmol) prepared in the previous step 1 in ethanol (5 mL) while stirring them. The reaction mixture was stirred at 60? C. for 12 hours and then cooled to room temperature. NaBH.sub.4 (16 mg, 0.42 mmol) was slowly added, and further stirred for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in water and then extracted with ethyl acetate. The organic layer was combined, dried over Na.sub.2SO.sub.4 and then filtered and evaporated in vacuo. The residue was purified by flash column to give 2-(4-(benzyloxy)-3-(4-phenylbutoxy)benzylamino)ethanol (99 mg, 89%).

(67) .sup.1H NMR (CD.sub.3OD, 300 MHz): ? 7.44-7.41 (m, 2H), 7.32-7.14 (m, 8H), 7.05 (d, 1H, J=1.8 Hz), 7.00 (d, 1H, J=8.1 Hz), 6.90 (dd, 1H, J=1.8 & 8.1 Hz), 5.10 (s, 2H), 4.09-4.05 (m, 2H), 3.90 (s, 2H), 3.72 (t, 2H, J=5.4 Hz), 2.88 (t, 2H, J=5.4 Hz), 2.70-2.65 (m, 2H), 1.85-1.81 (m, 4H)

(68) LC-MS (ESI): m/z 406 (M+H).sup.+ and 345 (M?60).sup.+

Experimental Example 1: Analysis of the Effect of Decomposing Lipid Droplets in 3T3-L1 Preadipocytes by the Present Compound Through Oil Red O Staining

(69) The following experiments were conducted in order to evaluate the effect of decreasing lipid droplets accumulated in cells by the compounds of the present invention.

(70) 3T3-L1 preadipocytes were purchased from Korean Cell Line Bank, and cultured and maintained in newborn calf serum (NCS, Invitrogen Corporation, Auckland, New Zealand) and high-glucose DMEM (high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis, Mo., USA). For adipocyte differentiation, 3T3-L1 with a concentration of 100,000 cells/ml were grown to confluence with 10% fetal bovine serum (FBS) and high-glucose DMEM for 2 days, then cultured in 10% FBS/high-glucose DMEM containing 0.5 mM IBMX (3-isobytyl-1-methyl xanthine), 0.5 ?M dexamethasone and 5 ?g/ml insulin (MDI) for 2 days, and further cultured in 10% FBS/high-glucose DMEM containing only 5 ?g/ml insulin(I) for 4 days, following by again culturing in 10% FBS/high-glucose DMEM alone for another 6 days. Thus, the cells were cultured for a total of 10 days to differentiate into adipocytes. After confluent culture, the cells were treated with the compounds at a concentration of 10 ?M each time the medium was changed. On day 10 after differentiation, the cultured cells were fixed with 4% paraformaldehyde and then stained with Oil Red O staining solution. The Oil Red O staining was performed by diluting 0.5 g/200 ml isopropanol stock solution with 60% distilled water, and observed through a microscope at 400? magnification. The results are illustrated in FIG. 1.

(71) In FIG. 1, a red circular structure represents a lipid droplet accumulated in cells. As illustrated in FIG. 1, it shows that the size and number of lipid droplets were reduced by the treatment of the compounds according to the present invention. Therefore, it can be confirmed that the compound according to the present invention is effective for removing lipid droplets in 3T3-L1 preadipocytes.

Experimental Example 2: Analysis of the Effect of Decomposing Lipid Droplets in Hep G2 Cells by the Compound of the Present Invention Through Oil Red O Staining

(72) The following experiment was conducted to evaluate the decreasing effect of the present compounds on lipid droplets in cells.

(73) In detail, Hep G2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Hyclone, USA). When cells were approximately 50% confluent, the medium was replaced with DMEM medium containing 1 mM oleic acid and 0.5 mM palmitic acid to induce intracellular lipid accumulation, and the cells were cultured for 24 hours. The medium was then replaced with DMEM medium containing 10 ?M of each compound synthesized through the present invention and the cells were further cultured for 24 hours. After completion, the cells were fixed with 4% paraformaldehyde for 10 minutes and washed three times with PBS. The cells were then rinsed with 60% isopropanol and then stained with diluted Oil Red O solution (stock solution, 3 mg/mL in isopropanol, working solution, 60% Oil Red O stock solution diluted in water) for 1 hour. The Oil Red O staining was performed by diluting 0.5 g/200 ml isopropanol stock solution with 60% distilled water, and observed through a microscope at a 400? magnification. The results are illustrated in FIG. 2.

(74) In FIG. 2, a red circular structure represents a lipid droplet accumulated in cells. As illustrated in FIG. 2, it shows that the size and number of lipid droplets were reduced by the treatment of the compounds according to the present invention. Therefore, it can be confirmed that the compound according to the present invention had an effect of removing lipid droplets in Hep G2 hepatocytes.

Experimental Example 3: Analysis of the Effect of Decomposing Lipid Droplets in 3T3-L1 and Hep G2 Cells by the Compound of the Present Invention Through BODIPY Staining

(75) The following experiment was conducted to evaluate the decreasing effect of the present compounds on lipid droplets in cells by immunofluorescence staining.

(76) 3T3-L1 preadipocytes were purchased from Korean Cell Line Bank, and cultured and maintained in newborn calf serum (NCS, Invitrogen Corporation, Auckland, New Zealand) and high-glucose DMEM (high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis, Mo., USA). For adipocyte differentiation, 3T3-L1 with a concentration of 100,000 cells/ml were grown to confluence with 10% fetal bovine serum (FBS) and high-glucose DMEM for 2 days, then cultured in 10% FBS/high-glucose DMEM containing 0.5 mM IBMX (3-isobytyl-1-methyl xanthine), 0.5 ?M dexamethasone and 5 ?g/ml insulin (MDI) for 2 days, and further cultured in 10% FBS/high-glucose DMEM containing only 5 ?g/ml insulin(I) for 4 days, followed by again culturing in 10% FBS/high-glucose DMEM for another 6 days. Thus, the cells were cultured for a total of 10 days to differentiate into adipocytes. After confluent culture, the cells were treated with the compounds at a concentration of 10 ?M each time the medium was changed. On day 10 after differentiation, the cultured cells were fixed with 4% paraformaldehyde and then lipid droplets were stained using BODIPY, a marker of lipid droplets, and the nuclei in cells were labeled through DAPI staining. The results are illustrated in FIG. 3.

(77) In FIG. 3, a green circular structure represents a lipid droplet accumulated in cells, which shows that the size and number of lipid droplets were reduced by the treatment of the compounds according to the present invention. Therefore, it was confirmed that the compound of the present invention is effective for removing lipid droplets in preadipocytes.

Experimental Example 4: Analysis of the Effect of Decomposing Lipid Droplets in 3T3-L1 and Hep G2 Cells Via Lipophagy by the Compound of the Present Invention

(78) The following experiment was conducted to confirm that the lipid droplets are decomposed via lipophagy by the compound of the present invention.

(79) 3T3-L1 preadipocytes were purchased from Korean Cell Line Bank, and cultured and maintained in newborn calf serum (NCS, Invitrogen Corporation, Auckland, New Zealand) and high-glucose DMEM (high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis, Mo., USA). For adipocyte differentiation, 3T3-L1 with a concentration of 100,000 cells/ml were grown to confluence with 10% fetal bovine serum (FBS) and high glucose DMEM for 2 days, then cultured in 10% FBS/high-glucose DMEM containing 0.5 mM IBMX (3-isobytyl-1-methyl xanthine), 0.5 ?M dexamethasone and 5 ?g/ml insulin (MDI) for 2 days, and further cultured in 10% FBS/high-glucose DMEM containing only 5 ?g/ml insulin(I) for 4 days, followed by again culturing in 10% FBS/high-glucose DMEM alone for another 6 days. Thus, the cells were cultured for a total of 10 days to differentiate into adipocytes. After confluent culture, the cells were treated with the compounds at a concentration of 10 ?M each time the medium was changed.

(80) In addition, Hep G2 hepatocytes were inoculated into a 24 well cell culture plate containing a cover slip, cultured in a DMEM medium containing 1 mM of oleic acid and 0.5 mM of palmitic acid for 24 hours to induce lipid accumulation, which was then treated with 10 ?M of each compound and incubated for another 24 hours.

(81) Subsequently, each cell was then washed twice with PBS solution, fixed with 4% paraformaldehyde solution for 15 minutes, and blocked in PBS solution containing 2% BSA for 1 hour. After blocking was completed, it was subjected to a primary antibody reaction. The primary antibody reaction was performed using LC3 rabbit polyclonal antibody (1:300, Sigma-Aldrich, USA) overnight at 4? C. After completion of the primary antibody reaction, cover slips were washed twice with PBS, and a secondary antibody (1:500, goat anti rabbit Alexa flour 555, Thermo Fisher, US) was reacted at room temperature for 1 hour. After completion of the secondary antibody reaction, the cells were washed twice with PBS, and then BODIPY 493/503 was reacted at room temperature for 10 minutes and mounted on a slide glass using a mounting medium. The stained cells were photographed using a confocal microscope, and the results are illustrated in FIG. 4.

(82) In FIG. 4a, a green circular structure represents a lipid droplet accumulated in cells, and it can be confirmed that the lipid droplets were accumulated in 3T3-L1 preadipocytes during the differentiation process into the adipocytes. When treating the compounds of the present invention with the completion of differentiation into adipocytes, it can be confirmed that red stained LC3, which is a marker capable of tracking the level of lipophagy activity, was increased as compared with cell treated with placebo. In addition, comparing the degree of overlap between lipid droplets and LC3, it can be confirmed that when treated with the compounds of the present invention, the degree of overlap was significantly increased as compared with cells treated with placebo. Therefore, it could be seen that the size and number of the lipid droplets produced in the 3T3-L1 adipocytes were reduced due to the action of lipophagy increased by the compound of the present invention.

(83) Further, in FIG. 4b, the green circular structures represent lipid droplets accumulated in cells, and it can be confirmed that by treating oleic acid and palmitic acid, which are one type of fatty acids, the accumulation of lipid droplets was induced as compared with the control group treated with BSA. When treated with the compounds according to the present invention after treatment with oleic acid and palmitic acid, it can be confirmed that the red-stained LC3, which is a marker capable of tracking the level of the lipophagy activity, was increased. It is also shown that LC3, a marker of lipophagy, is located on the surface of lipid droplets through images obtained by superimposing an image of green-stained lipid droplets and an image of red-stained LC3. Thus, it could be seen that the effect of reducing the number and size of lipid droplets exhibited by the compounds of the present invention was mediated by the action of lipophagy.

(84) In addition, in order to confirm that removal of lipid droplets by the treatment of the compound according to the present invention resulted from the action of lipophagy, Bifilomycin, an inhibitor of the action of lipophagy, and 5 nM of the compound of the present invention were treated together to adipocytes differentiated from pre-adipocytes to accumulate lipid droplets, but it was confirmed that the removal effect of lipid droplets was not exhibited. The results are illustrated in FIG. 5.

(85) In FIG. 5, the red circular structures represent lipid droplets, which show that the lipid droplets reduced by the compounds of the present invention did not decrease in response to the treatment of bafilomycin. Thus, in view of the fact that the reduction effect of the number and size of lipid droplets did not appear when treated with the compound according to the present invention, it could be seen that the effect of removing lipid droplets by the present compound was caused by the action of lipophagy.

Experimental Example 5: Analysis of the Weight Loss Effect of the Compound of the Present Invention in High-Fat Diet-Fed Mice

(86) In order to induce obesity in experimental mice, wild-type C57BL6 mice (6 weeks old) were supplied from Medical Center for Experimental Animal Resource Development, Seoul National University and divided into general feed intake group (LFD) and high calorie diet intake group (HFD) through random assignment. The high-fat intake group was again divided into a group treated with placebo and a group treated with the compound of the present invention, and divided into a total of three groups to conduct obesity induction and compound treatment at the same time. As a result, it was analyzed whether the compound could effectively remove the fat inflowing excessively and thus prevent obesity.

(87) Specifically, the mice that had been supplied were allowed to freely consume water after a week of adaptation period in the rearing cage. As for diets, high-fat diets (protein: 10%, carbohydrate: 30%) in which 60% of the total calories (4.60 kcal/g) is composed of fat, and normal diets (protein: 18.8%, carbohydrate: 63.9%) in which 17.2% of the total calories (3.8 kcal/g) was composed of fat, were supplied, respectively. In order to evaluate the preventive and therapeutic effects of obesity, etc., the compound of the present invention was dissolved in DMSO at 1 ?L/mg (v/w), then diluted with PBS, and administered intraperitoneally three times per week at a concentration of 20 ?g/g body weight of mice. In the placebo-treated group, the same amount of DMSO was diluted with PBS and administered. In order to evaluate the weight gain associated with the high-fat diet intake and the weight loss suppressing effect associated with the administration of the compound of the present invention, mice body weights were measured three times a week. In order to verify the removal effect of the body fat in mice, euthanasia was induced by excessive supply of CO.sub.2 after 24 hours including an 8 hour fasting period after the final administration, and then laparotomy was performed, and changes in body fat were observed. The results of the body fat removing effect, the change in body weight, the difference in feed intake and the difference in height due to the administration of the high fat diets and the compound according to the present invention are illustrated in FIGS. 6, 7, 8, 9, 10 and 11, respectively.

(88) As illustrated in FIG. 6, when the compound of the present invention was administered, it can be confirmed that the size of the accumulated abdominal fat was significantly reduced as compared with the placebo-treated group. A significant reduction in body fat eventually resulted in weight loss, and the results are illustrated in FIG. 7.

(89) In light of the fact that a significant difference in feed intake was not exhibited, it can be confirmed from FIGS. 8 to 10 that the weight loss due to the weight reduction of white adipose tissue is not a phenomenon due to a decrease in inflow of fat into the body, but the disappearance of the white adipose tissue directly occurs in the body. The results showed that only the weight of the adipose tissue was reduced, without being affected by other tissues.

(90) Further, the percentage of weight loss, change in height, and change in feed intake through the compounds according to the present invention are illustrated in FIG. 11. These results are derived from the investigation results of the feed intake in order to determine whether the weight loss effect is due to the decrease of the feed intake, indicating that no significant differences were exhibited.

(91) Further, in order to understand that the weight loss caused by the compounds of the present invention has an effect on blood lipid components, blood glucose and pathological conditions of tissues, the results of the items that have examined blood samples taken from the placebo-treated mice and the compound-administered mice are illustrated in FIG. 12. As a result, it was confirmed that triglyceride levels in the blood were significantly reduced.

Experimental Example 6: Analysis of the Effect of the Compound of the Present Invention on the Removal of Lipid Droplets in Tissues of High-Fat Diets-Fed Mice Using HnE Staining Method

(92) In order to confirm the removal effect of lipid droplets accumulated in tissues by the compound of the present invention, the hepatic tissues of the mice of the previous Experimental Example 5 were observed through HnE staining method, and the results are illustrated in FIG. 13. In FIG. 13, the white circular structures indicated by yellow arrows represent lipid droplets, from which it can be seen that the size and number of lipid droplets accumulated in hepatocytes were decreased by the treatment of the compound of the present invention.

(93) To confirm that the size of adipocytes was reduced by the compounds of the present invention, the adipose tissue of the mouse of the previous Experimental Example 5 was observed through the HnE staining method, and the results are illustrated in FIG. 14. In FIG. 14, the structures indicated by blue arrows and black and yellow dots represent adipocytes in adipose tissue, from which it can be seen that the size of the adipocytes was reduced by the treatment of the compounds.

Experimental Example 7: Analysis of the Effect of Removing Lipid Droplets by the Action of Lipophagy Using Immunohistochemical Staining Method

(94) The mice of the previous Experimental Example 5 were dissected to analyze whether reduction or removal of lipid droplets in liver has occurred through immunohistochemical staining method. The staining solution used for immunohistochemical staining was purchased from Sigma-Aldrich. All procedures were performed on 5-?m sections of liver tissue embedded in paraffin at room temperature. Antigen regeneration was performed on formalin fixed tissue for 15 minutes at 100? C. in a water bath prior to immunohistochemical staining. Endogenous peroxidase activity was blocked by hydrogen peroxide. Primary antibodies were detected with HRP-conjugated polymer and developed by DAB. Slides were then counterstained with hematoxylin, and subjected to multi-stage alcohol dehydration and mounted with a mounting medium. The results are illustrated in FIG. 15. In FIG. 15, it can be seen that LC3 protein stained in brown color on the surface of the lipid droplets was disposed in the area indicated by the yellow square. From this, it can be seen that the removal of lipid droplets was directly caused by the action of lipophagy occurring on the surface of the lipid droplets.

(95) In addition, in order to observe the relaxation effect of the compound of the present invention in inflammatory symptoms in hepatic tissues caused by the excessive accumulation of lipid droplets, the expression pattern of macrophages, which are inflammatory cells, was observed through tissue immunochemical staining method, The results are illustrated in FIG. 16. In FIG. 16, the brown-labeled cells represent macrophages, from which it could be seen that macrophages accumulated in hepatic tissues were decreased by the treatment of the compound of the present invention. Thereby, it can be seen that macrophages accumulated in hepatic tissues were decreased by the treatment of the compound of the present invention.