METHOD FOR TREATING LIVER DISEASE BY ADMINISTERING PHARMACEUTICAL COMPOSITION COMPRISING CATECHOL DERIVATIVE

Abstract

The present invention relates to a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof. The compound according to the present invention can be usefully used for the prevention or treatment of autophagy-related diseases.

Claims

1. A method of treating liver diseases in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound represented by Chemical Formula 1 below, or a pharmaceutically acceptable salt thereof: ##STR00023## wherein, in Chemical Formula 1, R.sub.1 and R.sub.2 are each independently C.sub.1-4 alkyl, or R.sub.1 and R.sub.2 are connected together to form C.sub.1-4 alkylene, R.sub.3 is C.sub.1-4 alkyl, C.sub.1-4 alkyl substituted with C.sub.5-10 heteroaryl, C.sub.5-10 aryl, or C.sub.5-10 heteroaryl, X is CH, or N, and Y is NH, or O.

2. The method according to claim 1, wherein R.sub.1 and R.sub.2 are methyl, or R.sub.1 and R.sub.2 are connected together to form methylene or ethylene.

3. The method according to claim 1, wherein R.sub.3 is methyl, ethyl, isopropyl, isobutyl, pyridinylmethyl, phenyl, or pyridine.

4. The method according to claim 1, wherein R.sub.1 and R.sub.2 are each independently C.sub.1-4 alkyl, or R.sub.1 and R.sub.2 are connected together to form C.sub.1-4 alkylene, R.sub.3 is C.sub.1-4 alkyl, C.sub.1-4 alkyl substituted with C.sub.5-10 heteroaryl, C.sub.5-10 aryl, or C.sub.5-10 heteroaryl, X is CH, and Y is NH.

5. The method according to claim 1, wherein R.sub.1 and R.sub.2 are connected together to form C.sub.1-4 alkylene, R.sub.3 is C.sub.1-4 alkyl, or C.sub.6-10 aryl, X is N, and Y is NH.

6. The method according to claim 1, wherein R.sub.1 and R.sub.2 are methyl, or R.sub.1 and R.sub.2 are connected together to form methylene, R.sub.3 is isopropyl, isobutyl, or phenyl, X is CH, and Y is NH.

7. The method according to claim 1, wherein the compound is any one selected from the group consisting of: 1) 5-(3,4-dimethoxyphenyl)-N-isopropylthiophene-2-carboxamide, 2) 5-(benzo[d][1,3]dioxol-5-yl)-N-isopropylthiophene-2-carboxamide, 3) 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isopropylthiophene-2-carboxamide, 4) 5-(3,4-dimethoxyphenyl)-N-isobutylthiophene-2-carboxamide, 5) 5-(benzo[d][1,3]dioxol-5-yl)-N-isobutylthiophene-2-carboxamide, 6) 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isobutylthiophene-2-carboxamide, 7) 5-(3,4-dimethoxyphenyl)-N-phenylthiophene-2-carboxamide, 8) 5-(benzo[d][1,3]dioxol-5-yl)-N-phenylthiophene-2-carboxamide, 9) 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-phenylthiophene-2-carboxamide, 10) 5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)thiophene-2-carboxamide, 11) 5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide, 12) 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide, 13) 2-(benzo[d][1,3]dioxol-5-yl)-N-isopropylthiazole-5-carboxamide, 14) 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isopropylthiazole-5-carboxamide, 15) 2-(benzo[d][1,3]dioxol-5-yl)-N-isobutylthiazole-5-carboxamide, 16) 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isobutylthiazole-5-carboxamide, 17) 2-(benzo[d][1,3]dioxol-5-yl)-N-phenylthiazole-5-carboxamide, 18) 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-phenylthiazole-5-carboxamide, 19) methyl 5-(benzo[d][1,3]dioxol-5-yl)thiophene-2-carboxylate, and 20) ethyl 2-(benzo[d][1,3]dioxol-5-yl)thiazole-5-carboxylate.

8. The method according to claim 1, wherein the liver diseases are liver fibrosis, liver cirrhosis, hepatitis, alcoholic liver disease, fatty liver, or non-alcoholic steatohepatitis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 and FIG. 2 show the results of confirming by an increase in LC3-II that autophagy is significantly increased by the treatment of the compound prepared in one example of the present invention in ischemia-reperfusion model using primary hepatocytes.

[0058] FIG. 3 shows that hepatocyte inhibits mTOR activation by the treatment of the compound prepared in one example of the present invention in ischemia-reperfusion model, which shows the results confirming that the phosphorylation of mTOR at 2448 position is significantly inhibited.

[0059] FIG. 4 shows the results of histologically confirming the acute liver injury induced by carbon tetrachloride and the therapeutic effect of the compound prepared in one example of the present invention through H & E staining. The arrows in FIG. 4 represents a portal vein.

[0060] FIG. 5 shows the results of observing morphological abnormalities of liver during autopsy after induction of acute liver injury.

[0061] FIG. 6 shows the results of observing morphological abnormalities of liver during autopsy after administration of thioacetamide for inducing liver fibrosis and the compound prepared in one example of the present invention.

[0062] FIG. 7 shows the results of confirming, through Masson's Trichrome staining, the collagen deposition of liver tissue sections removed after administration of thioacetamide and the compound prepared in one example of the present invention.

[0063] FIG. 8 shows the results of histologically confirming infiltration of inflammatory cells in liver tissue damaged due to thioacetamide through H & E staining.

[0064] FIG. 9 shows the results of observing morphological abnormalities of liver during autopsy after administration of carbon tetrachloride for inducing liver fibrosis and the compound prepared in one example of the present invention.

[0065] FIG. 10 shows the results of confirming, through Masson's Trichrome staining, the collagen deposition in liver tissue sections removed after administration of carbon tetrachloride and the compound prepared in one example of the present invention.

[0066] FIG. 11 shows the results of observing morphological abnormalities of liver during autopsy after bile duct ligation surgery is performed to induce liver fibrosis.

[0067] FIG. 12 shows the results of confirming, through Masson's Trichrome staining, the collagen deposition in liver tissue sections removed after bile duct ligation surgery.

[0068] FIG. 13 shows the results of quantitative experiment of hydroxyproline using liver tissue removed after bile duct ligation surgery.

DETAILED DESCRIPTION OF THE INVENTION

[0069] Below, the present invention will be described in more detail by way of examples. However, these examples are provided for illustrative purposes only, and should not be construed as limiting the scope of the present invention to these examples.

Example 1: Preparation of 5-(3,4-dimethoxyphenyl)-N-isopropylthiophene-2-carboxamide

[0070] ##STR00003##

[0071] 5-Chloro-N-isopropylthiophene-2-carboxamide (2.015 mmol), 3,4-dimethoxyphenylboronic acid (3.022 mmol), sodium carbonate (1281.4 mg, 12.09 mmol) and palladium (II) acetate (27.56 mg, 0.05 mmol) were added to 1,2-dimethoxyethane (8 mL) and double distilled water (2 mL), and the mixture was stirred at 120 C. for 200 minutes together with ultrasonic treatment and then extracted three times with dichloromethane. The extracted solution was dried over anhydrous magnesium sulfate. The organic solvent was concentrated under reduced pressure, purified by flash column chromatography (n-Hx:EtOAc=3:1) and then dried by a vacuum pump to give the title compound as a white solid (yield: 66.70%).

[0072] .sup.1H NMR (400 MHz, DMSO-d6) 8.18 (d, 1H, J=7.8 Hz), 7.73 (d, 1H, J=3.9 Hz), 7.42 (d, 1H, J=3.9 Hz), 7.24-7.17 (m, 2H), 6.98 (d, 1H, J=8.3 Hz), 4.10-3.97 (m, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 1.15 (d, 6H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 160.5, 149.6, 149.5, 147.9, 138.6, 129.2, 126.5, 123.5, 118.6, 112.6, 109.6, 56.0, 56.0, 41.4, 22.8, 22.8; ESI (m/z) 306 (MH.sup.+)

Example 2: Preparation of 5-(benzo[d][1,3]dioxol-5-yl)-N-isopropylthiophene-2-carboxamide

[0073] ##STR00004##

[0074] 5-Chloro-N-isopropylthiophene-2-carboxamide (2.015 mmol), benzo[d][1,3]dioxol-5-ylboronic acid (3.022 mmol), sodium carbonate (1281.4 mg, 12.09 mmol) and palladium (II) acetate (27.56 mg, 0.05 mmol) were added to 1,2-dimethoxyethane (8 mL) and double distilled water (2 mL), and the mixture was stirred at 120 C. for 200 minutes together with ultrasonic treatment and then extracted three times with dichloromethane. The extracted solution was dried over anhydrous magnesium sulfate. The organic solvent was concentrated under reduced pressure, purified by flash column chromatography (n-Hx:EtOAc=3:1) and then dried by a vacuum pump to give the title compound as a brown solid (yield: 64.41%).

[0075] .sup.1H NMR (400 MHz, DMSO-d6) 8.19 (d, 1H, J=7.7 Hz), 7.70 (d, 1H, J=3.9 Hz), 7.38 (d, 1H, J=3.9 Hz, 1H), 7.27 (d, 1H, J=1.7 Hz), 7.16 (dd, 1H, J.sub.A=8.1 Hz, J.sub.B=1.7 Hz), 6.94 (d, 1H, J=8.1 Hz), 6.05 (s, 2H), 4.07-3.99 (m, 1H), 1.14 (d, 6H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 160.5, 148.5, 147.9, 147.5, 138.9, 129.1, 127.9, 123.8, 120.0, 109.2, 106.4, 101.8, 41.4, 22.8, 22.8; ESI (m/z) 290 (MH.sup.+)

Example 3: Preparation of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isopropylthiophene-2-carboxamide

[0076] ##STR00005##

[0077] 5-Chloro-N-isopropylthiophene-2-carboxamide (2.015 mmol), 2,3-dihydrobenzo[b][1,4]dioxin-6-yldiboric acid (3.022 mmol), sodium carbonate (1281.4 mg, 12.09 mmol) and palladium (II) acetate (27.56 mg, 0.05 mmol) were added to 1,2-dimethoxyethane (8 mL) and double distilled water (2 mL), and the mixture was stirred at 120 C. for 200 minutes together with ultrasonic treatment and then extracted three times with dichloromethane. The extracted solution was dried over anhydrous magnesium sulfate. The organic solvent was concentrated under reduced pressure, purified by flash column chromatography (n-Hx:EtOAc=3:1) and then dried by a vacuum pump to give the title compound as a white solid (yield: 45.52%).

[0078] .sup.1H NMR (400 MHz, DMSO-d6) 8.18 (d, 1H, J=7.7 Hz), 7.70 (d, 1H, J=3.9 Hz), 7.36 (d, 1H, J=3.9 Hz), 7.18-7.09 (m, 2H), 6.89 (d, 1H, J=8.3 Hz), 4.25 (s, 4H), 4.10-3.97 (m, 1H), 1.14 (d, 5H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 160.5, 147.3, 144.2, 144.1, 138.8, 129.2, 127.1, 123.6, 119.2, 118.1, 114.5, 64.6, 64.5, 41.47, 22.8, 22.8; ESI (m/z) 304 (MH.sup.+)

Example 4: Preparation of 5-(3,4-dimethoxyphenyl)-N-isobutylthiophene-2-carboxamide

[0079] ##STR00006##

[0080] The title compound (yield: 55.01%) was obtained as a yellow solid in the same manner as in Example 1, except that 5-chloro-N-isobutylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0081] .sup.1H NMR (400 MHz, DMSO-d6) 8.42 (t, 1H, J=5.8 Hz), 7.72 (d, 1H, J=3.9 Hz), 7.42 (d, 1H, J=3.9 Hz), 7.23-7.17 (m, 2H), 6.98 (d, 1H, J=8.1 Hz), 3.82 (s, 3H), 3.77 (s, 3H), 3.04 (t, 2H, J=6.4 Hz), 1.87-1.73 (m, 1H), 0.87 (d, 6H, J=6.7 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 161.4, 149.6, 149.5, 147.9, 138.5, 129.2, 126.5, 123.6, 118.7, 112.6, 109.7, 56.0, 56.0, 47.0, 28.6, 20.6, 20.6; ESI (m/z) 320 (MH.sup.+)

Example 5: Preparation of 5-(benzo[d][1,3]dioxol-5-yl)-N-isobutylthiophene-2-carboxamide

[0082] ##STR00007##

[0083] The title compound (yield: 37.68%) was obtained as a white solid in the same manner as in Example 2, except that 5-chloro-N-isobutylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0084] .sup.1H NMR (400 MHz, DMSO-d6) 8.43 (t, 1H, J=5.7 Hz), 7.70 (d, 1H, J=3.8 Hz), 7.38 (d, 1H, J=3.8 Hz), 7.27 (s, 1H), 7.15 (d, 1H, J=8.0 Hz), 6.95 (d, 1H, J=8.1 Hz), 6.05 (s, 2H), 3.04 (t, 2H, J=6.4 Hz), 1.91-1.74 (m, 1H), 0.87 (d, 6H, J=6.7 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 161.4, 148.5, 147.9, 147.5, 138.7, 129.1, 127.9, 123.9, 120.1, 109.2, 106.4, 101.8, 47.0, 28.6, 20.6, 20.6; ESI (m/z) 304 (MH.sup.+)

Example 6: Preparation of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isobutylthiophene-2-carboxamide

[0085] ##STR00008##

[0086] The title compound (yield: 37.11%) was obtained as a white solid in the same manner as in Example 3, except that 5-chloro-N-isobutylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0087] .sup.1H NMR (400 MHz, DMSO-d6) 8.42 (t, 1H, J=5.6 Hz), 7.70 (d, 1H, J=3.8 Hz), 7.36 (d, 1H, J=3.8 Hz), 7.16 (s, 1H), 7.13 (d, 1H, J=8.4 Hz), 6.89 (d, 1H, J=8.3 Hz), 4.25 (s, 4H), 3.04 (t, 2H, J=6.3 Hz), 1.89-1.72 (m, 1H), 0.87 (d, 6H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 161.4, 147.3, 144.2, 144.1, 138.7, 129.2, 127.1, 123.7, 119.3, 118.1, 114.5, 64.6, 64.5, 47.0, 28.6, 20.6, 20.6; ESI (m/z) 318 (MH.sup.+)

Example 7: Preparation of 5-(3,4-dimethoxyphenyl)-N-phenylthiophene-2-carboxamide

[0088] ##STR00009##

[0089] The title compound (yield: 66.53%) was obtained as a yellow solid in the same manner as in Example 1, except that 5-chloro-N-phenylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0090] .sup.1H NMR (400 MHz, DMSO-d6) 10.18 (s, 1H), 7.99 (d, 1H, J=3.9 Hz), 7.73 (d, 2H, J=8.0 Hz), 7.53 (d, 1H, J=3.9 Hz), 7.34 (t, 2H, J=7.8 Hz), 7.28-7.22 (m, 2H), 7.08 (t, 1H, J=7.3 Hz), 7.00 (d, 1H, J=8.3 Hz), 3.84 (s, 3H), 3.78 (s, 3H); .sup.13C NMR (100 MHz, DMSO-d6) 160.1, 149.8, 149.5, 149.3, 139.2, 138.1, 130.6, 129.1, 129.1, 126.3, 124.1, 123.8, 120.7, 120.7, 118.8, 112.5, 109.7, 56.0, 56.0; ESI (m/z) 340 (MH.sup.+), 362 (MNa.sup.+)

Example 8: Preparation of 5-(benzo[d][1,3]dioxol-5-yl)-N-phenylthiophene-2-carboxamide

[0091] ##STR00010##

[0092] The title compound (yield: 15.66%) was obtained as a white solid in the same manner as in Example 2, except that 5-chloro-N-phenylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0093] .sup.1H NMR (400 MHz, DMSO-d6) 10.18 (s, 1H), 7.97 (d, 1H, J=3.9 Hz), 7.72 (d, 2H, J=7.8 Hz), 7.49 (d, 1H, J=3.9 Hz), 7.40-7.28 (m, 3H), 7.22 (dd, 1H, J.sub.A=8.1 Hz, J.sub.B=1.6 Hz), 7.09 (t, 1H, J=7.3 Hz), 6.97 (d, 1H, J=8.1 Hz), 6.07 (s, 2H); ESI (m/z) 346 (MNa.sup.+)

Example 9: Preparation of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-phenylthiophene-2-carboxamide

[0094] ##STR00011##

[0095] The title compound (yield: 44.53%) was obtained as a white solid in the same manner as in Example 3, except that 5-chloro-N-phenylthiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0096] .sup.1H NMR (400 MHz, DMSO-d6) 10.18 (s, 1H), 7.97 (d, 1H, J=3.7 Hz), 7.72 (d, 2H, J=8.0 Hz), 7.47 (d, 1H, J=3.7 Hz), 7.34 (t, 2H, J=7.7 Hz), 7.22 (s, 1H), 7.19 (d, 1H, J=8.4 Hz), 7.08 (t, 1H, J=7.2 Hz), 6.91 (d, 1H, J=8.3 Hz), 4.26 (s, 4H); .sup.13C NMR (100 MHz, DMSO-d6) 160.1, 148.7, 144.4, 144.1, 139.1, 138.3, 130.6, 129.1, 129.1, 126.8, 124.1, 123.9, 120.7, 120.7, 119.4, 118.2, 114.7, 64.6, 64.5; ESI (m/z) 338 (MH.sup.+), 360 (MNa.sup.+)

Example 10: Preparation of 5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)thiophene-2-carboxamide

[0097] ##STR00012##

[0098] The title compound (yield: 13.54%) was obtained as a yellow solid in the same manner as in Example 2, except that 5-chloro-N-(pyridin-2-yl)thiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0099] .sup.1H NMR (400 MHz, DMSO-d6) 10.86 (s, 1H), 8.37 (d, 1H, J=4.6 Hz), 8.18 (d, 1H, J=4.0 Hz), 8.13 (d, 1H, J=8.4 Hz), 7.81 (t, 1H, J=7.8 Hz), 7.47 (d, 1H, J=3.9 Hz), 7.34 (s, 1H), 7.22 (d, 1H, J=8.1 Hz), 7.14 (t, 1H, J=7.6 Hz), 6.97 (d, 1H, J=8.1 Hz), 6.07 (s, 2H); .sup.13C NMR (100 MHz, DMSO-d6) 160.6, 152.3, 149.6, 148.5, 148.3, 148.2, 138.6, 137.7, 131.6, 127.6, 124.4, 120.4, 120.1, 115.0, 109.3, 106.5, 101.9; ESI (m/z) 325 (MH.sup.+), 347 (MNa.sup.+)

Example 11: Preparation of 5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide

[0100] ##STR00013##

[0101] The title compound (yield: 24.12%) was obtained as a white solid in the same manner as in Example 2, except that 5-chloro-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0102] .sup.1H NMR (400 MHz, DMSO-d6) 9.12 (t, 1H, J=5.9 Hz), 8.50 (d, 1H, J=4.2 Hz), 7.78 (d, 1H, J=3.9 Hz), 7.75 (td, 1H, J.sub.A=7.8 Hz, J.sub.B=1.7 Hz), 7.42 (d, 1H, J=3.9 Hz), 7.35-7.29 (m, 2H), 7.25 (dd, 1H, J.sub.A=7.0 Hz, J.sub.B=5.2 Hz), 7.18 (dd, 1H, J.sub.A=8.1 Hz, J.sub.B=1.7 Hz), 6.96 (d, 1H, J=8.1 Hz), 6.06 (s, 2H), 4.53 (d, 2H, J=6.0 Hz); .sup.13C NMR (400 MHz, DMSO-d6) 161.6, 159.0, 149.3, 148.5, 148.0, 148.0, 138.0, 137.2, 129.7, 127.8, 124.1, 122.6, 121.5, 120.2, 109.3, 106.5, 101.9, 44.9; ESI (m/z) 339 (MH.sup.+)

Example 12: Preparation of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide

[0103] ##STR00014##

[0104] The title compound (yield: 59.09%) was obtained as a white solid in the same manner as in Example 3, except that 5-chloro-N-(pyridin-2-ylmethyl)thiophene-2-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0105] .sup.1H NMR (400 MHz, DMSO-d6) 9.11 (t, 1H, J=6.0 Hz), 8.50 (d, 1H, J=4.2 Hz), 7.78 (d, 1H, J=3.9 Hz), 7.75 (td, 1H, J.sub.A=7.8 Hz, J.sub.B=1.7 Hz), 7.40 (d, 1H, J=3.9 Hz), 7.31 (d, 1H, J=7.8 Hz), 7.25 (dd, 1H, J.sub.A=7.1 Hz, J.sub.B=5.1 Hz), 7.19 (d, 1H, J=2.1 Hz), 7.15 (dd, 1H, J.sub.A=8.4 Hz, J.sub.B=2.2 Hz), 6.90 (d, 1H, J=8.4 Hz), 4.53 (d, 2H, J=6.0 Hz), 4.26 (s, 4H); .sup.13C NMR (100 MHz, DMSO-d6) 161.6, 159.0, 149.3, 147.8, 144.3, 144.1, 138.0, 137.2, 129.7, 127.0, 123.9, 122.6, 121.5, 119.3, 118.2, 114.6, 64.6, 64.5, 44.9; ESI (m/z) 353 (MH.sup.+)

Example 13: Preparation of 2-(benzo[d][1,3]dioxol-5-yl)-N-isopropylthiazole-5-carboxamide

[0106] ##STR00015##

[0107] The title compound (yield: 66.33%) was obtained as a yellow solid in the same manner as in Example 2, except that 2-chloro-N-isopropylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0108] .sup.1H NMR (400 MHz, DMSO-d6) 8.41 (d, 1H, J=7.6 Hz), 8.35 (s, 1H), 7.51-7.44 (m, 2H), 7.01 (d, 1H, J=8.1 Hz), 6.10 (s, 2H), 4.10-3.96 (m, 1H), 1.15 (d, 6H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 169.9, 159.2, 150.0, 148.5, 143.8, 135.5, 127.4, 121.8, 109.3, 106.5, 102.3, 41.6, 22.7, 22.7; ESI (m/z) 291 (MH.sup.+)

Example 14: Preparation of 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isopropylthiazole-5-carboxamide

[0109] ##STR00016##

[0110] The title compound (yield: 65.40%) was obtained as a white solid in the same manner as in Example 3, except that 2-chloro-N-isopropylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0111] .sup.1H NMR (400 MHz, DMSO-d6) 8.41 (d, 1H, J=7.6 Hz), 8.35 (s, 1H), 7.44-7.40 (m, 2H), 6.95 (d, 1H, J=7.9 Hz), 4.28 (s, 4H), 4.13-3.94 (m, 1H), 1.15 (d, 6H, J=6.6 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 169.8, 159.3, 146.3, 144.1, 143.9, 135.5, 126.5, 120.3, 118.2, 115.2, 64.8, 64.5, 41.6, 22.7, 22.7; ESI (m/z) 305 (MH.sup.+)

Example 15: Preparation of 2-(benzo[d][1,3]dioxol-5-yl)-N-isobutylthiazole-5-carboxamide

[0112] ##STR00017##

[0113] The title compound (yield: 58.92%) was obtained as a white solid in the same manner as in Example 2, except that 2-chloro-N-isopropylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0114] .sup.1H NMR (400 MHz, DMSO-d6) 8.64 (t, 1H, J=5.8 Hz), 8.36 (s, 1H), 7.53-7.45 (m, 2H), 7.01 (d, 1H, J=8.1 Hz), 6.10 (s, 2H), 3.05 (t, 2H, J=6.4 Hz), 1.88-1.70 (m, 1H), 0.87 (d, 6H, J=6.7 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 170.0, 160.1, 150.0, 148.5, 143.8, 135.3, 127.4, 121.8, 109.3, 106.5, 102.3, 47.0, 28.6, 20.6, 20.6; ESI (m/z) 305 (MH.sup.+)

Example 16: Preparation of 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-isobutylthiazole-5-carboxamide

[0115] ##STR00018##

[0116] The title compound (yield: 65.87%) was obtained as a white solid in the same manner as in Example 3, except that 2-chloro-N-isopropylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0117] .sup.1H NMR (400 MHz, DMSO-d6) 8.63 (t, 1H, J=5.8 Hz), 8.36 (s, 1H), 7.45-7.38 (m, 2H), 6.95 (d, 1H, J=8.6 Hz), 4.28 (s, 4H), 3.05 (t, 2H, J=6.4 Hz), 1.86-1.73 (m, 1H), 0.87 (d, 6H, J=6.7 Hz); .sup.13C NMR (100 MHz, DMSO-d6) 169.9, 160.1, 146.3, 144.1, 143.9, 135.3, 126.5, 120.3, 118.3, 115.2, 64.8, 64.5, 47.0, 28.6, 20.6, 20.6; ESI (m/z) 319 (MH.sup.+)

Example 17: Preparation of 2-(benzo[d][1,3]dioxol-5-yl)-N-phenylthiazole-5-carboxamide

[0118] ##STR00019##

[0119] The title compound (yield: 75.82%) was obtained as a yellow solid in the same manner as in Example 2, except that 2-chloro-N-phenylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0120] .sup.1H NMR (400 MHz, DMSO-d6) 10.39 (s, 1H), 8.59 (s, 1H), 7.70 (d, 2H, J=7.8 Hz), 7.55 (dd, 1H, J.sub.A=8.1, J.sub.B=1.7 Hz), 7.52 (d, 1H, J=1.5 Hz), 7.35 (t, 2H, J=7.9 Hz), 7.11 (t, 1H, J=7.4 Hz), 7.03 (d, 1H, J=8.1 Hz), 6.12 (s, 2H); .sup.13C NMR (100 MHz, DMSO-d6) 170.9, 158.9, 150.2, 148.6, 144.9, 138.8, 135.2, 129.1, 129.1, 127.2, 124.4, 122.0, 120.8, 120.8, 109.3, 106.6, 102.3; ESI (m/z) 325 (MH.sup.+)

Example 18: Preparation of 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-phenylthiazole-5-carboxamide

[0121] ##STR00020##

[0122] The title compound (yield: 45.13%) was obtained as a yellow solid in the same manner as in Example 3, except that 2-chloro-N-phenylthiazole-5-carboxamide was used instead of 5-chloro-N-isopropylthiophene-2-carboxamide.

[0123] .sup.1H NMR (400 MHz, DMSO-d6) 10.39 (s, 1H), 8.59 (s, 1H), 7.70 (d, 2H, J=7.9 Hz), 7.51-7.45 (m, 2H), 7.35 (t, 2H, J=7.8 Hz), 7.11 (t, 1H, J=7.3 Hz), 6.98 (d, 1H, J=8.1 Hz), 4.29 (s, 4H); .sup.13C NMR (100 MHz, DMSO-d6) 170.8, 158.9, 146.5, 145.0, 144.2, 138.8, 135.2, 129.1, 129.1, 126.4, 124.4, 120.8, 120.8, 120.4, 118.3, 115.3, 64.8, 64.5; ESI (m/z) 339 (MH.sup.+), 337 (MH.sup.)

Example 19: Preparation of methyl 5-(benzo[d][1,3]dioxol-5-yl)thiophene-2-carboxylate

[0124] ##STR00021##

[0125] Methyl 5-chlorothiophene-2-carboxylate (176 mg, 0.996 mmol), benzo[d][1,3]dioxol-5-ylboronic acid (248.03 mg, 1.495 mmol), sodium carbonate (633.70 mg, 5.979 mmol) and palladium (II) acetate (34.97 mg, 0.05 mmol) were added to 1,2-dimethoxyethane (4 mL) and double distilled water (1 mL), and the mixture was stirred at 120 C. for 200 minutes together with ultrasonic treatment and then extracted three times with dichloromethane. The extracted solution was dried over anhydrous magnesium sulfate. The organic solvent was concentrated under reduced pressure, purified by flash column chromatography (n-Hx:EtOAc=3:1) and then dried by a vacuum pump to give the title compound as a white solid (yield: 27.47%).

[0126] .sup.1H NMR (400 MHz, DMSO-d6) 7.74 (d, 1H, J=5.2 Hz), 7.48 (d, 1H, J=3.2 Hz), 7.35 (s, 1H), 7.23 (d, 1H, J=8.0 Hz), 6.97 (d, 1H, J=8.0 Hz), 6.07 (s, 2H), 3.81 (s, 3H)

Example 20: Preparation of ethyl 2-(benzo[d][1,3]dioxol-5-yl)thiazole-5-carboxylate

[0127] ##STR00022##

[0128] Ethyl 2-bromothiazole-5-carboxylate (200 mg, 0.847 mmol), benzo[d][1,3]dioxol-5-ylboronic acid (210.86 mg, 1.271 mmol), sodium carbonate (538.73 mg, 5.083 mmol) and palladium (II) acetate (27.73 mg, 0.042 mmol) were added to 1,2-dimethoxyethane (4 mL) and double distilled water (1 mL), and the mixture was stirred at 120 C. for 200 minutes together with ultrasonic treatment and then extracted three times with dichloromethane. The extracted solution was dried over anhydrous magnesium sulfate. The organic solvent was concentrated under reduced pressure, purified by flash column chromatography (n-Hx:EtOAc=3:1) and then dried by a vacuum pump to give the title compound as a white solid (yield: 8.56%).

[0129] .sup.1H NMR (400 MHz, DMSO-d6) 8.40 (s, 1H), 7.57 (dd, 1H, J.sub.A=8.1 Hz, J.sub.B=1.5 Hz), 7.52 (d, 1H, J=1.4 Hz), 7.04 (d, 1H, J=8.1 Hz), 6.12 (s, 2H), 4.31 (q, 2H, J=7.1 Hz), 1.29 (t, 3H, J=7.1 Hz); ESI (m/z) 278 (MH.sup.+)

Experimental Example 1: Effect of Activation of Autophagy

[0130] 1) Method for Autophagy Activation

[0131] Primary hepatocyte was isolated from male C57BL/6 mice by a collagenase perfusion method which is a known method (Gastroenterology 2011, 141, 2188). Survival rate of the isolated cells was measured by trypan blue exclusion method. Autophagy was induced by ischemia-reperfusion method (Gastroenterology 2011, 141, 2188).

[0132] Specifically, in order to induce ischemia, hepatocytes were incubated at 37 C. in Krebs-Ringer-HEPES (KRH) buffer (pH 6.2) in an anaerobic chamber for 2 hours. The respective hepatocytes were treated with DMSO, the compound prepared in Examples described above, or Rapamycin as a control substance, and then KRH buffer solution at pH 7.4 was treated under aerobic conditions for 1 hour to induce reperfusion. Subsequently, hepatocytes were lysed with an extraction buffer containing protease inhibitor cocktail (PBS solution containing 0.5% Triton X-100, 1 mM Na3VO4, etc.), and then fragmentation of DNA was performed by sonication. The amount of protein was measured with bovine serum albumin as a protein standard using Bio-Rad Protein Assay Kit (Cat No. 500-0002). After electrophoresis of 10-50 g of total cell protein in 8-12% (w/v) polyacrylamide gel containing 0.1% SDS, and then the proteins present in a gel were transferred to PVDF membrane by electroblotting. Then, in order to block non-specific binding, the PVDF membrane was placed in a TBS-tween (tris-buffered saline-tween, Sigma Co.) solution containing 5% nonfat dry milk and reacted at room temperature for 1 hour. The filter was placed in a TBS-tween solution containing an antibody against LC3 protein (Cell Signaling), allowed to stand at 4 C. for 12 hours, and then labeled with a secondary antibody labeled with HRP (Horseradish Peroxidase, Sigma Co., Cat No. P0889). Then, bands were measured using ECL (Enhanced Chemiluminescence, Thermo Scientific, Cat No. 34080).

[0133] 2) Results of Measurement of Autophagy Activation

[0134] The induction of autophagy can be confirmed by an increase in specific LC3-II molecules. LC3 precursors are usually scattered in the cytoplasm, subjected to proteolytic cleavage, and present in the form of LC3-I. When autophagy is activated, the C-terminal glycine is modified to form LC3-II, which migrates to autophagosome and is distributed in puncta form.

[0135] The measurement results are shown in Table 1 below. The amount of LC3-II increased by the compound treatment was quantified by measuring the band density, and then compared with the amount of LC3-II increased by the treatment of Rapamycin used as a control substance, and the comparison values are shown in Table 1. That is, the case having the same autophagy-inducing capacity as that of Rapamycin used as a control substance is represented by a value of 1; the case having superior autophagy-inducing capacity to that of Rapamycin is represented by a value greater than 1; and the case having weaker autophagy-inducing capacity than that of Rapamycin is represented by a value less than 1.

TABLE-US-00001 TABLE 1 Example No. Autophagy-inducing capacity 1 2.02 2 1.92 3 0.88 4 1.33 5 2.05 6 1.11 7 1.52 8 1.24 9 1.39 10 0.65 11 0.88 12 0.54 13 0.73 14 0.57 15 0.29 16 0.78 17 1.39 18 1.27 19 0.77 20 0.79 Rapamycin 1

[0136] As shown in Table 1 above, it can be confirmed by an increase in LC3-II that hepatocytes induce autophagy by the treatment of Rapamycin used as a control substance in ischemia-reperfusion model. Also, the compound according to the present invention showed an increase in LC3-II by more than twice as compared with Rapamycin used as a control substance. Therefore, it can be seen that the compounds according to the present invention had superior autophagy-inducing capacity to rapamycin.

[0137] In addition, FIG. 1 and FIG. 2 show the results of Western immunoblotting of some compounds having excellent autophagy-inducing capacity. As shown in FIG. 1 and FIG. 2, it can be confirmed by an increase in LC3-II that hepatocytes exhibited a significant increase in autophagy induction by treatment of the compounds of Examples 1 and 5 in ischemia-reperfusion model.

Experimental Example 2: Effect of Inhibition of mTOR Enzyme Activity

[0138] 1) Method of Measurement of mTOR Enzyme Activity

[0139] Western immunoblotting was performed using the cell-disrupted liquid obtained in Experimental Example 1. The PVDF membrane obtained from electrophoresis and electroblotting was placed in a TBS-tween solution containing an antibody against mTOR or p-mTOR (Ser 2448) proteins (Cell Signaling), allowed to stand at 4 C. for 12 hours, and then labeled with a secondary antibody labeled with HRP (Horseradish Peroxidase, Sigma Co., Cat No. P0889). Then, bands were measured using ECL (Enhanced Chemiluminescence, Thermo Scientific, Cat No. 34080).

[0140] 2) Results of Measurement of mTOR Enzyme Activity

[0141] The measurement results are shown in FIG. 3. mTOR is known to be one of the major proteins regulating autophagy. mTOR serves to inhibit autophagy at a quiescent state, whereas phosphorylation at serine 2448-position inhibits the function of mTOR, resulting in activation of autophagy. As shown in FIG. 3, it was confirmed that hepatocytes significantly inhibited the phosphorylation of mTOR at serine 2448 position by the treatment of the compound of Example 1 in ischemia-reperfusion model

Experimental Example 3: Effect in a Model of Induced Acute Liver Injury

[0142] Attempts were made to confirm the inhibitory effect of autophagy activator on liver injury in an animal model of induced liver fibrosis due to acute liver injury. In order to confirm the effect of inhibiting acute liver injury, carbon tetrachloride (CCl.sub.4) was administered to C57BL/6 mice to induce acute liver injury, and then the compound according to the present invention was administered. It was then confirmed whether liver injuries were inhibited. [0143] 1) Experimental Method

[0144] Five-week-old female C57BL/6 mice were introduced and acclimated for one week, and then divided into six groups respectively; a normal control group, a group to which carbon tetrachloride was administered, and groups to which the compounds according to the present invention (Examples 1, 2, 5, and 7) were administered. Each of the compounds according to the present invention was dissolved in corn oil and administered orally three times at a dose of 50 mg/kg (body weight) every 12 hours. 30 Minutes after final oral administration, carbon tetrachloride was intraperitoneally administered at a dose of 5 mL/kg (body weight) and then autopsied 24 hours later top remove a sample of liver tissue under autopsy and fix it, thereby preparing a tissue specimen. After preparation of tissue specimen slides, H & E staining was performed and liver injuries were confirmed with a microscope.

[0145] 2) Experimental Result

[0146] It can be observed that acute liver injury induced by carbon tetrachloride is characterized by necrosis of hepatocytes at the adjacent site of the portal vein and that fat granules are accumulated in damaged liver cells. After administration of carbon tetrachloride and four compounds according to the present invention in experimental animals, liver tissue specimens were prepared by autopsy. Liver injury as well as inhibitory effect of autophagy activator on liver injury were confirmed through H & E staining (FIG. 4). As shown in FIG. 4, in the group to which carbon tetrachloride alone was administered, necrosis of hepatocytes located in the vicinity of the portal vein was confirmed, and fat granules of injured hepatocytes were observed, indicating that acute liver injury was induced by carbon tetrachloride. In the groups to which Examples 1 and 5 according to the present invention were administered, it was observed that the liver injury was clearly recovered. In the groups to which Examples 2 and 7 were administered, the recovery effect of liver injury was relatively small. In addition, the presence or absence of liver injury during autopsy was confirmed with naked eyes, and no morphological abnormalities were observed in the liver of all the groups (FIG. 5).

Experimental Example 4: Effect in a Model of Induced Liver Fibrosis

[0147] In order to confirm the effect of inhibiting liver fibrosis caused by chronic liver injury, thioacetamide (TAA) was administered to C57BL/6 mice to induce chronic liver injury, thereby inducing liver fibrosis, and then the compounds according to the present invention were administered. It was then confirmed whether chronic liver injuries were inhibited.

[0148] 1) Experimental Method

[0149] Five-week-old female C57BL/6 mice were introduced and acclimated for one week, and then divided into six groups respectively; a normal control group, a group to which thioacetamide was administered, and groups to which the compounds according to the present invention (Examples 2, 5 and 7) were administered. Thioacetamide was dissolved in phosphate-buffered physiological saline and administered intraperitoneally at a dose of 200 mg/kg (body weight) three times a week, for a total of 6 weeks. Three compounds according to the present invention was dissolved in corn oil at a final 6 weeks and administered orally at a dose of 50 mg/kg (body weight) once a day for a total of 8 days. Twenty-four hours after the final oral administration, an autopsy was performed to remove a sample of liver tissue and fix it, thus preparing a tissue specimen. After preparation of tissue specimen slides, H & E staining was performed, and liver tissue injury as well as inflammatory cell infiltration in liver tissue were confirmed by microscopy. In addition, Masson's Trichrome staining was performed, and collagen deposition in the liver tissue was confirmed by a microscope.

[0150] 2) Experimental Result

[0151] Histologically, liver fibrosis deposits collagen in the capillary network of liver tissue, which is deposited in the form of lobules surrounding the central vein of the liver. This can be observed using Masson's Trichrome staining. After the administration of thioacetamide and the compounds according to the present invention, the presence or absence of liver injuries during autopsy was confirmed with naked eyes.

[0152] Morphologically, in the liver of the control group, the surface was smooth and no abnormalities were observed, whereas in all groups in which liver injuries were induced by thioacetamide, fine nodules were observed on the surface of the liver and the liver sizes were all similar (FIG. 6). It was confirmed by microscopic observation with Masson's Trichrome staining that liver fibrosis was induced by the administration of thioacetamide for 6 weeks. It was also confirmed that fibrosis was alleviated by the administration of the compounds according to the present invention (FIG. 7). In particular, it was observed that fibrosis was clearly alleviated by Examples 2 and 5 according to the present invention (FIG. 7). In addition, it was observed that the infiltration of inflammatory cells, which play an important role in fibrosis, was reduced by treating the compounds according to the present invention (FIG. 8).

Experimental Example 5: Effect in a Model of Induced Liver Fibrosis

[0153] In order to confirm the effect of inhibiting liver fibrosis caused by chronic liver injury, carbon tetrachloride was administered to C57BL/6 mice to induce chronic liver injury, thereby inducing liver fibrosis, and then the compounds according to the present invention were administered. It was then confirmed whether chronic liver injuries were inhibited.

[0154] 1) Experimental Method

[0155] Five-week-old female C57BL/6 mice were introduced and acclimated for one week, and then divided into six groups respectively; a normal control group, a group to which carbon tetrachloride was administered, and groups to which the compounds according to the present invention (Examples 1 and 5) were administered. Carbon tetrachloride was dissolved in corn oil and administered intraperitoneally at a dose of 0.8 ml/kg (body weight) twice a day, for a total of 8 days. Twenty-four hours after the final oral administration, an autopsy was performed to remove a sample of liver tissue and fix it, thus preparing a tissue specimen. After preparation of tissue specimen slides, H & E staining was performed, and liver tissue injury as well as inflammatory cell infiltration in liver tissue were confirmed by microscopy. In addition, Masson's Trichrome staining was performed, and collagen deposition in the liver tissue was confirmed by microscope.

[0156] 2) Experimental Result

[0157] After the administration of carbon tetrachloride and autophagy activator, the presence or absence of liver injuries during autopsy was confirmed with naked eyes. Morphologically, in the liver of the control group, the surface was smooth and no abnormalities were observed, whereas in all groups in which liver injuries were induced by carbon tetrachloride, fine nodules were observed on the surface of the liver, and the liver sizes were all similar (FIG. 9). It was confirmed by Masson's Trichrome staining in liver tissue slide sections that liver fibrosis was induced by the administration of carbon tetrachloride for 8 weeks. It was also confirmed that fibrosis was alleviated by the administration of the compounds (Examples 1 and 5) according to the present invention (FIG. 10). In addition, it was confirmed that the effect of Example 1 was relatively greater in light of the fact that collagen deposition was less in the group treated with Example 1 than in the group treated with Example 5.

Experimental Example 6: Effect in a Model of Induced Liver Fibrosis

[0158] In order to confirm the effect of inhibiting liver fibrosis caused by chronic liver injury, bile duct ligation (BDL) was performed in Sprague Dawley (SD) rats to induce chronic liver injury, thereby inducing liver fibrosis, and then the compounds according to the present invention were administered. It was then confirmed whether chronic liver injuries were inhibited.

[0159] 1) Experimental Method

[0160] Seven-week-old female SD rats were introduced and acclimated for one week, and then divided into five groups respectively; a control group, a bile duct ligation surgery group, and groups to which the compound according to the present invention (Example 15) was administered at three doses (12.5, 25 and 50 mg/kg body weight). In the control group, the bile duct was not exposed or ligated after laparotomy, and the other groups performed bile duct ligation surgery. One week after bile duct ligation, the compound according to the present invention (Example 1) was dissolved in corn oil and administered orally for each dose once a day for a total of 14 days. Twenty-four hours after the final administration, an autopsy was performed to remove a sample of liver tissue and fix it, thus preparing a tissue specimen. After preparation of tissue specimen slides, H & E staining was performed, and collagen deposition in liver tissue were confirmed by microscopy. In addition, a part of the removed liver (10 mg) was decomposed to perform a quantitative experiment of hydroxyproline which is a major component of collagen, and the amount of collagen deposited was quantified, thereby confirming the effects of the compound according to the present invention.

[0161] 2) Experimental Result

[0162] After the bile duct ligation surgery and the administration of the compounds according to the present invention, the presence or absence of liver injuries during autopsy was confirmed with naked eyes. Morphologically, in the liver of the control group, the surface was smooth and no abnormalities were observed, whereas in all groups in which liver injuries were induced by bile duct ligation, fine nodules were observed on the surface of the liver and the liver sizes were larger than that of the control group (FIG. 11). There was no significant difference between the group treated only with the bile duct ligation and the groups treated with the bile duct ligation surgery and the compounds according to the present invention (FIG. 11). It was confirmed by Masson's Trichrome staining in liver tissue slide sections that liver injury and fibrosis were induced by bile duct ligation. It was confirmed that liver fibrosis progressed in the vicinity of the central vein was alleviated by the administration of the compounds according to the present invention (FIG. 12).

[0163] Similarly, a quantitative experiment of hydroxyproline for quantitatively determining the amount of collagen deposited in the liver tissue was carried out. As a result, it was found that the larger the quantity of the compound according to the present invention (Example 1), the smaller the collagen deposition (FIG. 13). In addition, in all groups treated with the compounds according to the present invention, the number of injured hepatocytes adjacent to the portal vein was markedly reduced as compared to the untreated group, confirming that liver injuries were alleviated histologically (FIG. 11).