Composition for Prevention, Amelioration, or Treatment of Cancer

Abstract

A composition according to the present disclosure may be very effectively used not only to prevent, ameliorate or treat cancer, but also to inhibit metastasis of cancer, by inhibiting the growth of cancer cells and very effectively inhibiting the metastasis of cancer cells to other tissues.

Claims

1-14. (canceled)

15. A method for preventing or treating cancer comprising administering to a target individual an effective amount of a compound represented by the following Formula 1: ##STR00014## wherein: L.sub.1 and L.sub.2 are each independently selected from the group consisting of C.sub.3 to C.sub.40 cycloalkylene, C.sub.6 to C.sub.60 arylene, and a heteroarylene having 5 to 60 nuclear atoms; X and Y are each independently selected from the group consisting of deuterium, a halogen, cyano, nitro, sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, azide, hydroxy, C.sub.1 to C.sub.40 alkyl, C.sub.2 to C.sub.40 alkenyl, C.sub.1 to C.sub.40 alkoxy, unsubstituted or substituted C.sub.6 to C.sub.60 aryloxy, unsubstituted or substituted C.sub.3 to C.sub.40 cycloalkyl, an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms, unsubstituted or substituted C.sub.6 to C.sub.60 aryl, an unsubstituted or substituted heteroaryl having 5 to 60 nuclear atoms, and —NR′R″; R′ and R″ are each independently selected from the group consisting of hydrogen, C.sub.1 to C.sub.10 alkyl, C.sub.6 to C.sub.60 aryl, C.sub.3 to C.sub.40 cycloalkyl, C.sub.6 to C.sub.60 arylsulfonyl, and a heteroaryl having 5 to 60 nuclear atoms; n and m are each independently an integer ranging from 0 to 5, provided that n and m are not 0 at the same time; when X or Y is plural, the plurality of X or Y are the same as or different from each other; and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one substituent selected from the group consisting of deuterium, halogen, and nitro.

16. The method of claim 15, wherein: L.sub.1 and L.sub.2 are each phenylene; a and m are each independently an integer of 1 or 2; X and Y are each independently selected from the group consisting of sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, C.sub.1 to C.sub.40 alkoxy, —NR′R″, hydroxy, C.sub.6 to C.sub.60 aryloxy, and an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms; R′ and R″ are each independently hydrogen or C.sub.6 to C.sub.60 arylsufonyl, and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one halogen.

17. The method of claim 16, wherein X is —NR′R″ or a substituent represented by the following Formula 3: ##STR00015## and R′ and R″ are each independently hydrogen or a substituent represented by the following Formula 4: ##STR00016## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, a halogen, hydroxy, C.sub.1 to C.sub.40 alkyl, and C.sub.2 to C.sub.40 alkenyl; and R.sub.2 is selected from the group consisting of hydrogen, deuterium, a halogen, and nitro.

18. The method of claim 16, wherein Y is hydroxy, C.sub.1 to C.sub.6 alkoxy, or a substituent represented by the following Formula 5: ##STR00017##

19. The method of claim 15, wherein the compound is selected from the following compounds: ##STR00018## ##STR00019## ##STR00020##

20. The method of claim 15, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, chorionic carcinoma, skin cancer, ovarian cancer, thyroid cancer, brain cancer, blood cancer, head and neck cancer, malignant melanoma, and lymphoma.

21. A method for preventing or treating cancer comprising administering to a target individual an effective amount of a compound represented by the following Formula 1: ##STR00021## and an anticancer drug, wherein: L.sub.1 and L.sub.2 are each independently selected from the group consisting of C.sub.3 to C.sub.40 cycloalkylene, C.sub.6 to C.sub.60 arylene, and a heteroarylene having 5 to 60 nuclear atoms; X and Y are each independently selected from the group consisting of deuterium, a halogen, cyano, nitro, sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, azide, hydroxy, C.sub.1 to C.sub.40 alkyl, C.sub.2 to C.sub.40 alkenyl, C.sub.1 to C.sub.40 alkoxy, unsubstituted or substituted C.sub.6 to C.sub.60 aryloxy, unsubstituted or substituted C.sub.3 to C.sub.40 cycloalkyl, an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms, unsubstituted or substituted C.sub.6 to C.sub.60 aryl, an unsubstituted or substituted heteroaryl having 5 to 60 nuclear atoms, and —NR′R″; R′ and R″ are each independently selected from the group consisting of hydrogen, C.sub.1 to C.sub.10 alkyl, C.sub.6 to C.sub.60 aryl, C.sub.3 to C.sub.40 cycloalkyl, C.sub.6 to C.sub.60 arylsulfonyl, and a heteroaryl having 5 to 60 nuclear atoms; n and m are each independently an integer ranging from 0 to 5, provided that n and m are not 0 at the same time; when X or Y is plural, the plurality of X or Y are the same as or different from each other; and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one substituent selected from the group consisting of deuterium, halogen, and nitro.

22. A method for inhibiting metastasis of cancer comprising administering to a target individual an effective amount of a compound represented by the following Formula 1: ##STR00022## wherein: L.sub.1 and L.sub.2 are each independently selected from the group consisting of C.sub.3 to C.sub.40 cycloalkylene, C.sub.6 to C.sub.60 arylene, and a heteroarylene having 5 to 60 nuclear atoms; X and Y are each independently selected from the group consisting of deuterium, a halogen, cyano, nitro, sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, azide, hydroxy, C.sub.1 to C.sub.40 alkyl, C.sub.2 to C.sub.40 alkenyl, C.sub.1 to C.sub.40 alkoxy, unsubstituted or substituted C.sub.6 to C.sub.60 aryloxy, unsubstituted or substituted C.sub.3 to C.sub.40 cycloalkyl, an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms, unsubstituted or substituted C.sub.6 to C.sub.60 aryl, an unsubstituted or substituted heteroaryl having 5 to 60 nuclear atoms, and —NR′R″; R′ and R″ are each independently selected from the group consisting of hydrogen, C.sub.1 to C.sub.10 alkyl, C.sub.6 to C.sub.60 aryl, C.sub.3 to C.sub.40 cycloalkyl, C.sub.6 to C.sub.60 arylsulfonyl, and a heteroaryl having 5 to 60 nuclear atoms; n and m are each independently an integer ranging from 0 to 5, provided that n and m are not 0 at the same time; when X or Y is plural, the plurality of X or Y are the same as or different from each other; and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one substituent selected from the group consisting of deuterium, halogen, and nitro.

23. The method of claim 22, wherein: L.sub.1 and L.sub.2 are each phenylene; n and m are each independently an integer of 1 or 2; X and Y are each independently selected from the group consisting of sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, C.sub.1 to C.sub.40 alkoxy, —NR′R″, hydroxy, C.sub.6 to C.sub.60 aryloxy, and an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms; R′ and R″ are each independently hydrogen or C.sub.6 to C.sub.60 arylsufonyl, and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one halogen.

24. The method of claim 23, wherein X is —NR′R″ or a substituent represented by the following Formula 3: ##STR00023## and R′ and R″ are each independently hydrogen or a substituent represented by the following Formula 4: ##STR00024## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, a halogen, hydroxy, C.sub.1 to C.sub.40 alkyl, and C.sub.2 to C.sub.40 alkenyl; and R.sub.2 is selected from the group consisting of hydrogen, deuterium, a halogen, and nitro.

25. The method of claim 23, wherein Y is hydroxy, C.sub.1 to C.sub.6 alkoxy, or a substituent represented by the following Formula 5: ##STR00025##

26. The method of claim 22, wherein the compound is selected from the following compounds: ##STR00026## ##STR00027## ##STR00028##

27. The method of claim 22, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, chorionic carcinoma, skin cancer, ovarian cancer, thyroid cancer, brain cancer, blood cancer, head and neck cancer, malignant melanoma, and lymphoma.

28. A method for inhibiting metastasis of cancer comprising administering to a target individual an effective amount of a compound represented by the following Formula 1: ##STR00029## and an anticancer drug, wherein: L.sub.1 and L.sub.2 are each independently selected from the group consisting of C.sub.3 to C.sub.40 cycloalkylene, C.sub.6 to C.sub.60 arylene, and a heteroarylene having 5 to 60 nuclear atoms; X and Y are each independently selected from the group consisting of deuterium, a halogen, cyano, nitro, sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, azide, hydroxy, C.sub.1 to C.sub.40 alkyl, C.sub.2 to C.sub.40 alkenyl, C.sub.1 to C.sub.40 alkoxy, unsubstituted or substituted C.sub.6 to C.sub.60 aryloxy, unsubstituted or substituted C.sub.3 to C.sub.40 cycloalkyl, an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms, unsubstituted or substituted C.sub.6 to C.sub.60 aryl, an unsubstituted or substituted heteroaryl having 5 to 60 nuclear atoms, and —NR′R″; R′ and R″ are each independently selected from the group consisting of hydrogen, C.sub.1 to C.sub.10 alkyl, C.sub.6 to C.sub.60 aryl, C.sub.3 to C.sub.40 cycloalkyl, C.sub.6 to C.sub.60 arylsulfonyl, and a heteroaryl having 5 to 60 nuclear atoms; n and m are each independently an integer ranging from 0 to 5, provided that n and m are not 0 at the same time; when X or Y is plural, the plurality of X or Y are the same as or different from each other; and the arylsulfonyl of each of R′ and R″ is unsubstituted or substituted with at least one substituent selected from the group consisting of deuterium, halogen, and nitro.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0106] FIG. 1 shows the results of evaluating, by Western blot analysis, the effect of increasing the stability of AMPK protein in one example of the present disclosure.

[0107] FIGS. 2A and 2B show the results of evaluating, by cell viability assay (a) and clonogenic assay (b), the effect of inhibiting the survival and growth of cancer cell lines in one example of the present disclosure.

[0108] FIG. 3A shows the results of evaluating, by cell viability assay, the effect of inhibiting the survival and growth of cancer cell lines in one example of the present disclosure.

[0109] FIG. 3B shows the results of evaluating, by the expression level of p-AMPK (Thr172), the ability of activating AMPK in cancer cells in one example of the present disclosure.

[0110] FIGS. 4A, 4B and 4C show the results of evaluating the viability inhibitory effects and AMPK activation abilities of an AMPK activator, a compound of Production Example 6 and a compound of Production Example 8 having a similar structure against gastric cancer, brain cancer and pancreatic cancer cell lines in one example of the present disclosure.

[0111] FIG. 5 shows the results of evaluating, by flow cytometry assay, the effect of inducing apoptosis of cancer cell lines in one example of the present disclosure.

[0112] FIG. 6 shows the results of evaluating, by Western blot analysis, the effect of inducing apoptosis of cancer cell lines in one example of the present disclosure.

[0113] FIGS. 7A and 7B show the results of evaluating, by Western blot analysis, the effect of inhibiting EMI of cancer cell lines in one example of the present disclosure.

[0114] FIGS. 8A, 8B, 8C and 8D show the results of evaluating tumor regression effects in animal models in one example of the present disclosure.

[0115] FIGS. 9A and 9B show the results of evaluating the effect of inhibiting tumor metastasis in animal models in one example of the present disclosure.

BEST MODE

[0116] An embodiment of the present disclosure is directed to a composition for preventing, ameliorating or treating cancer, the composition containing, as an active ingredient, a compound represented by Formula 1 below.

[0117] Another embodiment of the present disclosure is directed to a composition for inhibiting metastasis of cancer, the composition containing, as an active ingredient, a compound represented by Formula 1 below.

[0118] Still another embodiment of the present disclosure is directed to a method for preventing or treating cancer, the method including administering an effective amount of a compound represented by Formula 1 below to a subject in need of administration.

[0119] Yet another embodiment of the present disclosure is directed to a method for inhibiting metastasis of cancer, the method including administering an effective amount of a compound represented by Formula 1 below to a subject in need of administration.

##STR00013##

[0120] wherein:

[0121] L.sub.1 and L.sub.2 are each independently selected from the group consisting of C.sub.3 to C.sub.40 cycloalkylene, C.sub.6 to C.sub.60 arylene, and a heteroarylene having 5 to 60 nuclear atoms;

[0122] X and Y are each independently selected from the group consisting of deuterium, a halogen, cyano, nitro, sulfonyl, C.sub.1 to C.sub.10 alkylsulfonyl, azide, hydroxy, C.sub.1 to C.sub.40 alkyl, C.sub.2 to C.sub.40 alkenyl, C.sub.1 to C.sub.40 alkoxy, unsubstituted or substituted C.sub.6 to C.sub.60 aryloxy, unsubstituted or substituted C.sub.3 to C.sub.40 cycloalkyl, an unsubstituted or substituted heterocycloalkyl having 3 to 20 nuclear atoms, unsubstituted or substituted C.sub.6 to C.sub.60 aryl, an unsubstituted or substituted heteroaryl having 5 to 60 nuclear atoms, and —NR′R″;

[0123] R′ and R″ are each independently selected from the group consisting of hydrogen, C.sub.1 to C.sub.10 alkyl, C.sub.6 to C.sub.60 aryl, C.sub.3 to C.sub.40 cycloalkyl, C.sub.6 to C.sub.60 arylsulfonyl, and a heteroaryl having 5 to 60 nuclear atoms;

[0124] n and m are each independently an integer ranging from 0 to 5, provided that n and m are not 0 at the same time;

[0125] when X or Y is plural, the plurality of X or Y may be the same as or different from each other; and

[0126] the arylsulfonyl of each of R′ and R″ may be unsubstituted or substituted with at least one substituent selected from the group consisting of deuterium, halogen, and nitro.

MODE FOR INVENTION

[0127] Hereinafter, the present disclosure will be described in more detail with reference to examples. It will be apparent to those of ordinary skill in the art that these examples are only for explaining the present disclosure in more detail, and the scope of the present disclosure according to the subject matter of the present disclosure is not limited by these examples.

EXAMPLES

[Production Examples 1 to 15] Synthesis of Candidate Compounds

[0128] The compounds of Production Examples 1 to 15 shown in Table 1 below were produced by Method 1 or Method 2.

TABLE-US-00001 TABLE 1 Production Example Compound Production (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en- Example 1 1-one (YE-01) Production (E)-1-(4-aminophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en- Example 2 1-one (YE-02) Production (E)-1-(3-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one Example 3 (YE-03) Production (E)-1-(3-aminophenyl)-3-(4-hydroxy-2-methoxyphenyl)prop- Example 4 2-en-1-one (YE-04) Production (E)-1-(3-aminophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en- Example 5 1-one (YE-05) Production (E)-4-chloro-N-(4-(3-(2,5- Example 6 dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-06) Production (E)-4-chloro-N-(3-(3-(4- Example 7 methoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-07) Production (E)-4-chloro-N-(3-(3-(2,5- Example 8 dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-08) Production (E)-4-chloro-N-((4-chlorophenyl)sulfonyl)-N-(3-(3-(2,5- Example 9 dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-09) Production (E)-1-(4-aminophenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2- Example 10 en-1-one (YE-10) Production (E)-1-(3-aminophenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2- Example 11 en-1-one (YE-11) Production (E)-1-(4-hydroxyphenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2- Example 12 en-1-one (YE-12) Production (E)-1-(2,4-dihydroxyphenyl)-3-(4-(piperidin-1- Example 13 yl)phenyl)prop-2-en-1-one (YE-13) Production (E)-3-(4-hydroxy-2-methoxyphenyl)-1-(4-(piperazin- Example 14 1-yl)phenyl)prop-2-en-1-one (YE-14) Production (E)-3-(4-hydroxyphenyl)-1-(4-(4-methylpiperazin-1- Example 15 yl)phenyl)prop-2-en-1-one (YE-15)

[0129] [Method 1]

[0130] Method including steps of: adding an acetophenone derivative (1 equivalent), a benzaldehyde derivative (1 equivalent) and NaOH (1 equivalent) to an ethanol solvent, followed by stirring at room temperature; after completion of the reaction, adding water to the reaction mixture, followed by extraction with ethyl acetate; and collecting the organic solvent layer, washing the collected material once with water, drying the washed material with anhydrous MgSO.sub.4, removing the solvent by distillation under reduced pressure, and purifying the residue by silica gel chromatography.

[0131] [Method 2]

[0132] Method including steps of: adding an acetophenone derivative (1 equivalent), a 4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde derivative (1 equivalent) and NaOH (1 equivalent) to an ethanol solvent, followed by stirring at room temperature; after completion of the reaction, adding 4 M HCl to the mixture, followed by stirring for 20 minutes, addition of water, and extraction with ethyl acetate; and collecting the organic solvent layer, washing the collected material once with water, drying the washed material with anhydrous MgSO.sub.4, removing the solvent by distillation under reduced pressure, and purifying the residue by silica gel chromatography.

[Production Example 1] (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (YE-01)

[0133] According to Method 1 above, 4-aminoacetophenone (0.30 g, 2.22 mmol), 2,4-dimethoxybenzaldehyde (0.37 g, 2.22 mmol) and NaOH (0.09 g, 2.22 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:2.fwdarw.1:1) to obtain the compound of Production Example 1 (0.15 g, 23.0% yield) as a yellow solid. R.sub.f 0.33 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.85 (s, 3H), 3.89 (s, 3H), 6.47 (d, J=2.0 Hz, 1H), 6.52 (dd, J=8.4, 2.4 Hz, 1H), 6.69 (d, J=8.4 Hz, 2H), 7.55 (d, J=15.6 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.92 (d, J=8.4 Hz, 2H), 8.02 (d, J=15.6 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 55.6, 55.7, 98.7, 105.5, 114.1, 117.8, 120.7, 129.4, 130.8, 131.1, 139.0, 150.9, 160.4, 162.8, 189.1 ppm.

[Production Example 2] (E)-1-(4-aminophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en-1-one (YE-02)

[0134] According to Method 1 above, 4-aminoacetophenone (0.50 g, 3.70 mmol), 2,5-dimethoxybenzaldehyde (0.62 g, 3.70 mmol) and NaOH (0.15 g, 3.70 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing 25 solvent:ethyl acetate/n-hexane=1:2.fwdarw.1:1) to obtain the compound of Production Example 2 (0.66 g, 62.5% yield) as a yellow solid. R.sub.f 0.36 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.81 (s, 3H), 3.85 (s, 3H), 4.19 (br s, 2H), 6.69 (d, J=8.8 Hz, 2H), 6.86 (d, J=8.8 Hz, 1H), 6.91 (dd, J=8.8, 2.8 Hz, 1H), 7.16 (d, J=2.8 Hz, 1H), 7.58 (d, J=15.6 Hz, 1H), 7.92 (d, J=8.8 Hz, 2H), 8.04 (d, J=15.6 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 56.0, 56.3, 112.7, 113.9, 114.1, 116.8, 123.3, 125.2, 128.9, 131.3, 138.6, 151.2, 153.4, 153.7, 188.8 ppm.

[Production Example 3] (E)-1-(3-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (YE-03)

[0135] According to Method 1 above, 3-aminoacetophenone (1.00 g, 7.40 mmol), 4-methoxybenzaldehyde (1.00 g, 7.40 mmol) and NaOH (0.30 g, 7.40 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:(ethyl acetate/n-hexane=1:2.fwdarw.1:1) to obtain the compound of Production Example 3 (0.83 g, 62.5% yield) as an orange solid. R.sub.f 0.40 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.83 (br s, 2H), 3.85 (s, 3H), 6.88 (ddd, J=8.0, 2.4, 0.8 Hz, 1H), 6.93 (d, J=8.8 Hz, 2H), 7.27 (dd, J=8.0, 7.6 Hz, 1H), 7.31 (dd, J=2.0, 1.6 Hz, 1H), 7.36 (d, J=15.6 Hz, 1H), 7.38 (ddd, J=7.6, 1.6, 0.8 Hz, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.76 (d, J=15.6 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 56.0, 56.3, 112.7, 113.9, 114.1, 116.8, 123.3, 125.2, 128.9, 131.3, 138.6, 151.2, 153.4, 153.7, 188.8 ppm.

[Production Example 4] (E)-1-(3-aminophenyl)-3-(4-hydroxy-2-methoxyphenyl)prop-2-en-1-one (YE-04)

[0136] According to Method 2 above, 3-aminoacetophenone (0.40 g, 2.96 mmol), 2-methoxy-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (0.70 g, 2.96 mmol) and NaOH (0.12 g, 2.96 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:1) to obtain the compound of Production Example 4 (0.28 g, 35.1% yield) as an orange solid. R.sub.f 0.25 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.71 (s, 3H), 6.30 (d, J=2.4 Hz, 1H), 6.34 (dd, J=8.4, 2.0 Hz, 1H), 6.72 (ddd, J=8.0, 2.4, 0.8 Hz, 1H), 7.08 (dd, J=8.4, 8.0 Hz, 1H), 7.13 (d, J=2.4 Hz, 1H), 7.16 (ddd, J=7.6, 7.6, 0.8 Hz, 1H), 7.29 (d, J=15.6 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.85 (d, J=15.6 Hz, 1H), 9.35 (br s, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 55.3, 99.1, 108.2, 114.1, 115.3, 118.1, 118.7, 119.3, 129.1, 130.6, 139.8, 140.3, 147.0, 60.5, 161.4, 191.1 ppm.

[Production Example 5] (E)-1-(3-Aminophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en-1-one (YE-05)

[0137] According to Method 1 above, 4-aminoacetophenone (0.50 g, 3.70 mmol), 2,5-dimethoxybenzaldehyde (0.62 g, 3.70 mmol) and NaOH (0.15 g, 3.70 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:3) to obtain the compound of Production Example 5 (0.27 g, 25.4% yield) as a yellow solid. R.sub.f 0.62 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.81 (s, 3H), 3.86 (s, 3H), 6.87 (d, J=8.8 Hz, 1H), 6.88 (ddd, J=8.4, 2.0, 0.8 Hz, 1H), 6.94 (dd, J=8.8, 2.8 Hz, 1H), 7.16 (d, J=2.8 Hz, 1H), 7.27 (dd, J=8.0, 8.0 Hz, 1H), 7.31 (dd, J=2.8, 2.8 Hz, 1H), 7.38 (ddd, J=8.0, 2.0, 1.6 Hz, 1H), 7.53 (d, J=15.6 Hz, 1H), 8.06 (d, J=15.6 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 56.1, 56.3, 112.7, 113.9, 114.7, 117.4, 119.1, 119.4, 123.6, 124.8, 129.6, 139.8, 140.0, 147.0, 153.5, 153.7, 191.4 ppm.

[Production Example 6] (E)-4-chloro-N-(4-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-06)

[0138] To a solution of the compound of Production Example 2 (0.67 g, 2.36 mmol) and triethylamine) (TEA, 0.26 g, 2.60 mmol) in CH.sub.2Cl.sub.2, 4-chlorobenzenesulfonyl chloride (0.75 g, 3.54 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Water was added to the reaction mixture which was then extracted with ethyl acetate. The organic solvent layer was collected, washed with saturated NaHCO.sub.3, and dried with anhydrous MgSO.sub.4. Then, the solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:2) to obtain the compound of Production Example 6 (0.55 g, 50.9% yellow) as a yellow solid. R.sub.f 0.18 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, DMSO-d6) δ 3.74 (s, 3H), 3.80 (s, 3H), 6.84 (d, J=8.8 Hz, 1H), 6.88 (dd, J=8.8, 2.4 Hz, 1H), 7.16 (d, J=2.4 Hz, 1H), 7.20 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.54 (d, J=15.6 Hz, 1H), 7.74 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.4 Hz, 2H), 7.91 (d, J=15.6 Hz, 1H), 10.58 (s, 1H); .sup.13C-NMR (100 MHz, DMSO-d6) δ5.2, 55.6, 112.1, 112.7, 117.0, 118.0, 121.6, 123.5, 128.0, 128.1, 128.7, 129.4, 132.9, 137.8, 138.3, 141.5, 152.5, 152.9, 187.9 ppm.

[Production Example 7] (E)-4-chloro-N-(3-(3-(4-methoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-07)

[0139] N-(3-acetylphenyl)-4-chlorobenzenesulfonamide (0.10 g, 0.32 mmol), 4-methoxybenzaldehyde (0.04 g, 0.32 mmol) and NaOH (0.03 g, 0.80 mmol) were added to an ethanol solvent, followed by stirring at room temperature for 72 hours. A dilute aqueous hydrochloric acid solution was added to the reaction mixture which was then extracted with ethyl acetate. The organic solvent layer was collected, washed with water, and dried with anhydrous MgSO.sub.4. The solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:3.fwdarw.1:1) to obtain the compound of Production Example 7 (0.01 g, 6.5% yield) as a yellow solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.84 (s, 3H), 6.92 (d, J=8.8 Hz, 2H), 7.28 (d, J=15.6 Hz, 1H), 7.33 (dd, J=8.0, 7.6 Hz, 1H), 7.36 (d, J=8.8 Hz, 2H), 7.41 (ddd, J=8.0, 2.0, 1.2 Hz, 1H), 7.57 (d, J=8.8 Hz, 2H), 7.68 (ddd, J=7.6, 1.6, 1.2 Hz, 1H), 7.71-7.78 (m, 4H), 9.25 (s, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 55.6, 114.6, 119.5, 121.1, 124.9, 125.0, 127.6, 128.8, 129.4, 129.7, 130.5, 137.

[Production Example 8] (E)-4-chloro-N-(3-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-08)

[0140] To a solution of the compound of Production Example 5 (0.12 g, 0.43 mmol) and triethylamine (0.03 g, 2.60 mmol) in CH.sub.2Cl.sub.2, 4-chlorobenzenesulfonyl chloride (0.09 g, 0.43 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Water was added to the reaction mixture which was then extracted with ethyl acetate. The organic solvent layer was collected, washed with saturated NaHCO.sub.3, and dried with anhydrous MgSO.sub.4. Then, the solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:3) to obtain the compound of Production Example 8 (0.08 g, 37.3% yield) as a yellow solid. R.sub.f 0.33 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.82 (s, 3H), 3.88 (s, 3H), 6.88 (d, J=8.8 Hz, 1H), 6.96 (dd, J=8.4, 2.8 Hz, 1H), 7.15 (d, J=2.8 Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.41-7.43 (m, 2H), 7.55 (d, J=16.0 Hz, 1H), 7.73 (d, J=8.8 Hz, 2H), 7.71-7.72 (m, 1H), 7.76-7.79 (m, 1H), 8.08 (d, J=16.0 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 56.1, 56.3, 112.7, 114.5, 117.9, 121.6, 122.8, 124.4, 125.5, 125.8, 128.9, 129.7, 130.0, 137.1, 137.7, 139.9, 140.0, 141.6, 153.7, 153.8, 190.3 ppm.

[Production Example 9] (E)-4-chloro-N-((4-chlorophenyl)sulfonyl)-N-(3-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)benzenesulfonamide (YE-09)

[0141] To a solution of the compound of Production Example 5 (0.22 g, 0.78 mmol) and trimethylamine (0.22 g, 2.12 mmol) in CH.sub.2Cl.sub.2, 4-chlorobenzenesulfonyl chloride (0.25 g, 1.17 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Water was added to the reaction mixture which was then extracted with ethyl acetate. The organic solvent layer was collected, washed with saturated NaHCO.sub.3, and dried with anhydrous MgSO.sub.4. Then, the solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:4) to obtain the compound of Production Example 8 (0.20 g, 39.8% yield) as a yellow solid. R.sub.f 0.77 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 3.83 (s, 3H), 3.86 (s, 3H), 6.89 (d, J=9.2 Hz, 1H), 6.97 (dd, J=9.2, 3.2 Hz, 1H), 7.13 (d, J=2.8 Hz, 1H), 7.19 (dd, J=8.4, 2.0 Hz, 1H), 7.42 (d, J=16.0 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 7.54 (d, J=8.8 Hz, 4H), 7.66 (dd, J=1.6, 1.6 Hz, 1H), 7.89 (d, J=8.8 Hz, 4H), 8.05 (d, J=16.0 Hz, 1H), 8.09 (d, J=8.0 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 56.1, 56.3, 112.7, 114.3, 117.9, 122.9, 124.3, 129.8, 129.9, 130.3, 130.6, 131.6, 134.6, 135.2, 137.8, 140.2, 141.4, 141.8, 153.7, 153.8, 189.9 ppm.

[Production Example 10] (E)-1-(4-aminophenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (YE-10)

[0142] According to Method 1 above, 4-aminoacetophenone (0.40 g, 2.96 mmol), 4-(piperidin-1-yl)benzaldehyde (0.56 g, 2.96 mmol) and NaOH (0.12 g, 2.96 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent: MeOH:CHCl.sub.3=1:19) to obtain the compound of Production Example 10 (0.28 g, 30.9% yield) as an orange solid. R.sub.f 00.43 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 1.62-1.71 (m, 6H), 3.29 (t, J=5.2 Hz, 4H), 4.10 (br s, 2H), 6.69 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 7.37 (d, J=15.6 Hz, 1H), 7.53 (d, J=8.8 Hz, 2H), 7.75 (d, J=15.6 Hz, 1H), 7.95 (d, J=8.8 Hz, 2H).

[Production Example 11] (E)-1-(3-aminophenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (YE-11)

[0143] According to Method 1 above, 3-aminoacetophenone (0.40 g, 2.96 mmol), 4-(piperidin-1-yl)benzaldehyde (0.56 g, 2.96 mmol) and NaOH (0.12 g, 2.96 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent: MeOH:CHCl.sub.3=1:19) to obtain the compound of Production Example 11 (0.45 g, 49.6% yield) as an orange solid. R.sub.f 0.47 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 1.61-1.72 (m, 6H), 3.31 (t, J=5.6 Hz, 4H), 3.80 (br s, 2H), 6.86 (ddd, J=8.0, 2.4, 0.8 Hz, 1H), 6.89 (d, J=8.8 Hz, 2H), 7.28 (dd, J=8.0, 8.0 Hz, 1H), 7.30 (d, J=15.6 Hz, 1H), 7.31 (dd, J=2.0, 2.0 Hz, 1H), 7.37 (ddd, J=8.8, 1.2, 1.2 Hz, 1H), 7.53 (d, J=8.8 Hz, 2H), 7.75 (d, J=15.6 Hz, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 24.6, 25.7, 49.3, 114.7, 115.0, 118.4, 119.0, 119.1, 124.7, 129.5, 130.4, 140.3, 145.4, 146.9, 153.4, 191.1 ppm.

[Production Example 12] (E)-1-(4-Hydroxyphenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (YE-12)

[0144] According to Method 2 above, 1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one (0.50 g, 2.27 mmol), 4-(piperidin-1-yl)benzaldehyde (0.43 g, 2.27 mmol) and NaOH (0.09 g, 2.27 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:3.fwdarw.1:1) to obtain the compound of Production Example 12 (0.24 g, 33.7% yield) as an orange solid. R.sub.f 0.17 (ethyl acetate/n-hexane=1:3); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 1.58-1.65 (m, 6H), 3.25 (t, J=5.6 Hz, 4H), 6.84 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 7.32 (d, J=15.6 Hz, 1H), 7.48 (d, J=8.8 Hz, 2H), 7.68 (d, J=15.6 Hz, 1H), 7.90 (d, J=8.8 Hz, 2H), 9.34 (s, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 24.4, 25.5, 49.2, 114.9, 115.6, 117.8, 124.8, 130.1, 130.6, 130.9, 144.2, 153.1, 161.7, 189.0 ppm.

[Production Example 13] (E)-1-(2,4-Dihydroxyphenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (YE-13)

[0145] According to Method 2 above, 1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one (0.50 g, 2.12 mmol), 4-(piperidin-1-yl)benzaldehyde (0.40 g, 2.12 mmol) and Ba(OH).sub.2.8H.sub.2O (0.73 g, 2.33 mmol) were used as starting materials. The residue was purified by silica gel chromatography (developing solvent:ethyl acetate/n-hexane=1:1) to obtain the compound of Production Example 13 (0.12 g, 16.8% yield) as an orange solid. R.sub.f 0.66 (ethyl acetate/n-hexane=1:1); .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 1.58-1.65 (m, 6H), 3.27 (t, J=5.6 Hz, 4H), 6.37-6.40 (m, 2H), 6.84 (d, J=8.8 Hz, 2H), 7.34 (d, J=15.2 Hz, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.73 (d, J=8.4 Hz, 1H), 7.76 (d, J=15.2 Hz, 1H), 9.64 (s, 1H), 13.6 (s, 1H).

[Production Example 14] (E)-3-(4-Hydroxy-2-methoxyphenyl)-1-(4-(piperazin-1-yl)phenyl)prop-2-en-1-one (YE-14)

[0146] According to Method 2 above, 1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one (0.50 g, 2.45 mmol), 2-methoxy-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (0.58 g, 2.45 mmol) and NaOH (0.20 g, 4.90 mmol) were used as starting materials. After removal of the solvent, the resulting solid was treated with a mixed solvent of ethyl acetate/n-hexane. The resulting solid was filtered and dried in a vacuum to obtain the compound of Production Example 14 (0.28 g, 33.8% yield) as an orange solid. .sup.1H-NMR (400 MHz, DMSO-d6) δ 2.89 (dd, J=7.2, 3.2 Hz, 4H), 3.26 (dd, J=7.2, 4.0 Hz, 4H), 3.82 (s, 3H), 6.40 (dd, J=8.8, 2.0 Hz, 1H), 6.41 (d, J=1.6 Hz, 1H), 6.89 (d, J=8.8 Hz, 2H), 7.51 (d, J=15.6 Hz, 1H), 7.55 (d, J=8.8 Hz, 1H), 7.87 (d, J=15.6 Hz, 1H), 7.90 (d, J=8.8 Hz, 2H); .sup.13C-NMR (100 MHz, DMSO-d6) 45.1, 47.5, 55.1, 98.7, 107.9, 112.9, 114.7, 118.0, 127.7, 129.7, 129.8, 137.7, 153.7, 159.7, 161.2, 186.8 ppm.

[Production Example 15] (E)-3-(4-Hydroxyphenyl)-1-(4-(4-methylpiperazin-1-yl)phenyl)prop-2-en-1-one (YE-15)

[0147] According to Method 2 above, 1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one (0.50 g, 2.29 mmol), 4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (0.47 g, 2.29 mmol) and NaOH (0.09 g, 2.29 mmol) were used as starting materials. After removal of the solvent, the resulting solid was treated with a mixed solvent of ethyl acetate/n-hexane. The resulting solid was filtered and dried in a vacuum to obtain the compound of Production Example 15 (0.70 g, 94.8% yield) as an orange solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 2.33 (s, 3H), 2.55 (t, J=5.2 Hz, 4H), 3.37 (t, J=5.2 Hz, 4H), 6.86 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 7.38 (d, J=15.6 Hz, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.72 (d, J=15.6 Hz, 1H), 7.95 (d, J=8.8 Hz, 2H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 46.2, 47.4, 54.8, 113.7, 116.2, 118.8, 126.8, 128.7, 130.3, 130.6, 143.7, 154.0, 159.7, 188.4 ppm.

EXPERIMENTAL METHODS

[Experimental Method 1] Test Cell Lines and Cell Line Culture Method

[0148] Human cell lines (VERO, HFL-1, L929, NIH 3T3 and CHO-K1) were used to evaluate the cytotoxicity of the compound of Production Example 6 according to the present disclosure. In addition, in order to evaluate the abilities of the compound of Production Example 6 according to the present disclosure and 13 compounds, including the compounds of Production Examples 1 to 5 and 8 to 15 having a similar structure (YE-01, 02, 03, 04, 05, 08, 09, 10, 11, 12, 13, 14 and 15), to inhibit the growth of cancer cell lines, the following cell lines were used: human gastric cancer cell lines (NCI-N87, and SNU-216), human brain cancer cell lines (U-87 MG, and Hs 683), human pancreatic cell lines (AsPC-1, and MIA PaCa-2), human breast cancer cell lines (BT549, MDA-MB-231 and Hs578t), and a mouse breast cancer cell line 4T1 or 4T1-luc (a cell line constructed so that luciferase can be expressed in 4T1 cell line and the location of the cell can be visually confirmed by simple pretreatment).

[0149] Each of the cell lines was provided from ATCC (American Type Culture Collection), JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank) or Korea Cell Line Bank (KCRB). Each of the cell lines was cultured according to the culture medium and culture conditions described on the ATCC official website. In addition, in order to prevent mycoplasma infection which may affect gene expression in the cell line, whether or not mycoplasma infection would occur was regularly checked during culture of the cell line, and when mycoplasma infection was confirmed, it was treated with an anti-mycoplasma antibiotic for 1 to 2 weeks. Here, the 4T1-luc cell line was used for the construction of xenogeneic/orthotopic animal models.

[Experimental Method 2] Conditions for Treating Cell Lines with Compound of Production Example 6

[0150] The compound of Production Example 6 was dissolved in DMSO at a concentration of 1 to 50 μM. Thereafter, for each breast cancer cell line of Experimental Method 1, the concentration and time at which the compound of Preparation Example 6 showed optimal activity and a clear tendency were examined. At this time, a control group for treatment with the compound of Preparation Example 6 was treated with only DMSO. In order not to cause interference such as inhibition of cell activity, treatment with DMSO was performed such that DMSO did not exceed 5% of the total medium.

[Experimental Method 3] Method for Measuring the Ability to Stabilize and Activate AMPK (AMP-Activated Protein Kinase)

[0151] In order to examine whether or not the compound of Production Example 6 and 13 compounds, including the compounds of Production Examples 1 to 5 and 8 to 15 having a similar structure (YE-01, 02, 03, 04, 05, 08, 09, 10, 11, 12, 13, 14 and 15), bind to the AMP binding site of AMPK, cellular thermal shift assay (CTSA) was performed. Specifically, the cultured human breast cancer-derived cell line (BT549 or MDA-MB-231 cell line) was detached from the culture dish using trypsin. The detached cell line was diluted in PBS, and the same amount of the cells were dispensed in each tube for PCR and heated for 3 minutes at a temperature of room temperature to 65° C. After completion of the heating, protein was isolated from the cell line using liquid nitrogen, and Western blot analysis was performed to confirm the temperature at which AMPK would be denatured by heat.

[0152] [3-1] The breast cancer cell line of Experimental Method 1 above was treated with the compound of Production Example 6 at a concentration of 0 to 30 μM, and then cultured for 24 hours. Thereafter, the cells were harvested and lysed in RIPA buffer, and the protein was electrophoresed by SDS-PAGE, and then the expression level of the protein was analyzed using antibodies specific for AMPK and phospho-AMPK (Thr172). GAPDH or β-actin was used as a loading control for protein quantification.

[0153] [3-2] The three breast cancer cell lines (4T1, BT549, and MDA-MB-231) of Experimental Method 1 were treated with each of the compound of Production Example 6 and 13 compounds, including the compounds of Production Examples 1 to 5 and 8 to 15 having a similar structure (YE-01, 02, 03, 04, 05, 08, 09, 10, 11, 12, 13, 14 and 15), at a concentration of 10 μM, and then cultured for 24 hours. In addition, each of two types of gastric cancer cell lines, brain cancer cell lines and pancreatic cancer cell lines was treated with the compound of Production Example 6 or the compound of Production Example 8 having a similar structure at a concentration of 10 μM and then cultured for 12 hours. Thereafter, the cells were harvested and lysed in RIPA buffer, and the protein was electrophoresed by SDS-PAGE, and then the expression level of the protein was analyzed using antibodies specific for AMPK and phospho-AMPK (Thr172). GAPDH or 3-actin was used as a loading control for protein quantification.

[Experimental Method 4] Methods for Measuring Growth Inhibition and Apoptosis Levels of Cancer Cell Lines

[0154] [4-1] Cell Viability Assay

[0155] [4-1-1] The breast cancer cell line of Experimental Method 1 was treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (which is not the compound of Production Example 6) or the compound of Production Example 6 at a concentration of 0 to 30 μM, and then cultured for 24 hours, 48 hours and 72 hours. Thereafter, the cultured cells were treated with WST-1 reagent, and then the absorbance at a wavelength of 450 nm was measured. Here, the WST-1 reagent allows the cell viability to be measured by changing the color of the culture medium depending on the level of the mitochondrial dehydrogenase present in the cell.

[0156] [4-1-2] The breast cancer cell line 4T1, BT549 or MDA-MB-231 of Experimental Method 1 was treated with each of the compound of Production Example 6 and 13 compounds, including the compounds of Production Examples 1 to 5 and 8 to 15 having a similar structure (YE-01, 02, 03, 04, 05, 08, 09, 10, 11, 12, 13, 14 and 15), at a concentration of 0 to 50 μM, and then cultured for 24 hours. Each of three types of gastric cancer cell lines, brain cancer cell lines and pancreatic cancer cell lines was treated with the compound of Production Example 6 or the compound of Production Example 8 having a similar structure at a concentration of 0 to 50 μM, and then cultured for 24 hours. Thereafter, the cultured cells were treated with WST-1 reagent according to the manufacturer's instruction, and after a certain time, the absorbance at a wavelength of 450 nm was measured. Here, the WST-1 reagent allows the cell viability to be measured by changing the color of the culture medium depending on the level of the mitochondrial dehydrogenase present in the cell.

[0157] [4-2] Clonogenic Assay

[0158] The breast cancer cell line of Experimental Method 1 was dispensed into a 6-well plate at a density of 1 to 2×10.sup.3 cells/well. After the cells were sufficiently attached, they were treated with the compound of Production Example 6 at a concentration of 0 to M and cultured for 10 days to 14 days. Next, the cultured cells were fixed with 4% formaldehyde and stained with 1% crystal violet, and the colonies were counted under a microscope.

[0159] [4-3] Flow Cytometry Assay

[0160] The breast cancer cell line of Experimental Method 1 was treated with the compound of Production Example 6 at a concentration of 0 to 30 μM and cultured for 24 hours. Thereafter, the cultured cells were detached by trypsin, and then double-stained with Annexin V; recognizing apoptosis) and PI (propidium iodide; recognizing cell necrosis), and then the pattern and level of apoptosis were measured using flow cytometry.

[Experimental Method 5] Method for Measurement of Epithelial-Mesenchymal Transition (EMT) in Cancer Cell Line

[0161] The culture medium of the breast cancer cell line of Experimental Method 1 was replaced with a serum-free medium. Then, the cells were treated with 2 ng/mL of TGF-β1 and the compound of Preparation Example 6 and cultured for 20 to 24 hours. Thereafter, as described in Experimental Method 3, the expression level of the protein was measured using an antibody specific for the EMT-related protein.

[Experimental Method 6] Construction of Breast Cancer Cell Line Xenograft/Orthotopic Animal Models

[0162] After 4-week-old athymic nude mice (female) were purchased, only healthy mice were selected while the mice were adapted to the environment of the animal breeding room for 7 days. For identification of each mouse, the ears were marked using an ear punch. In addition, the breeding box was identified by attaching a mouse identification card describing the test number, test substance name, test item, receipt date, test date, test content, and the person in charge of the test. The environment of the animal breeding room was maintained at a temperature of 22±3° C., a relative humidity of 50±20%, 10 to 15 ventilations/hour, a 12-hour light (8:30 to 20:30)/dark cycle, and an illuminance of 150 to 300 lux. In addition, the animals were fed feed and drinking water during the breeding period, and quarantined during the acclimatization and testing periods.

[0163] The nude mice were anesthetized by inhalation with 2.5% isoflurane, and then injected with the mouse breast cancer cell line 4T1-luc of Experimental Method 1 by a sterile syringe. The cells were diluted in 100 μl of PBS (phosphate buffer saline) so that 1 to 2.5×10.sup.6 cells could be injected per nude mouse, and then the dilution was injected into the flank or mammary fat pad. The constitution of the xenograft animal model obtained by injecting the breast cancer cell line into the flank is shown in Table 2 below. In addition, the constitution of the orthotopic animal model obtained by injecting the breast cancer cell line into the mammary gland pad is shown in Table 3 below.

TABLE-US-00002 TABLE 2 Treatment content Efficacy test Drug administration Number of Experimental for each group substance route mice purpose Control group 4T1-luc xenograft Saline Tail vein injection 4 Confirmation of (flank, s.c.) (IV) tumor regression ability Experimental 4T1-luc xenograft Production Tail vein injection 4 Confirmation of group (flank, s.c.) Example 6 (IV) tumor regression (4 mg/kg b.w.) ability

TABLE-US-00003 TABLE 3 Treatment content Efficacy test Drug administration Number of Experimental for each group substance route mice purpose Control group 4T1-luc orthotopic Saline Intmperitoneal 3 Confirmation of graft (fat pad, s.c.) injection (IP) cancer metastasis inhibitory ability Experimental 4T1-luc orthotopic Production Intraperitoneal 3 Confirmation of group graft (fat pad, s.c.) Example 6 (20 injection (IP) cancer metastasis mg/kg b.w.) inhibitory ability Control group 4T1-luc orthotopic Saline Intraperitoneal 14 Confirmation of graft (fat pad, s.c.) injection (IP) survival rate Experimental 4T1-luc orthotopic Production Intraperitoneal 14 Confirmation of group graft (fat pad, s.c.) Example 6 (20 injection (IP) survival rate mg/kg b.w.)

[0164] Meanwhile, after mice with abnormalities, mice that did not gain weight normally, and mice that were not healthy were excluded during the animal acclimatization period, mice were grouped so that the average body weight and tumor size could be uniform.

[Experimental Method 7] Method for Evaluating Effect of Compound of Production Example 6 in Animal Model

[0165] [7-1] Methods for Evaluating Effect in Xenograft Animal Model

[0166] During a period ranging from the day the mouse breast cancer cell line 4T1-luc was injected to the end of the experiment (dissection date), the tumor volume was measured every day. When the volume of the tumor reached about 80 to 100 mm.sup.3, the compound of Production Example 6 was injected into the tail vein of the animal model at a concentration of 4 mg/kg once every 3 days a total of 6 times. In order to confirm the tumor regression effect, the volume of the tumor was measured before administration of the compound of Production Example 6, and at day 28 after the first administration of the compound of Production Example 6, the animals were euthanized, and the primary tumors were excised and their volumes were compared.

[0167] [7-2] Method for Evaluating Effect of Improving Survival Rate in Orthotopic Animal Model

[0168] During a period ranging from the day the mouse breast cancer cell line 4T1-luc was injected to the end of the experiment (the date of death), the volume of the tumor was measured once every 3 days and the survival rate of the animal model was checked every day. When the volume of the tumor reached about 80 to 100 mm.sup.3, the compound of Production Example 6 was injected into the abdominal cavity of the animal model at a concentration of 20 mg/kg once every 3 days a total of 6 times. Meanwhile, for the dignity of the animal model, the experiment was terminated at the time point (day 32) when the volume of the primary tumor of the control group reached 3,000 mm.sup.3, and the animal model was euthanized.

[0169] [7-3] Method for Evaluating Effect in Orthotopic Animal Model

[0170] During a period ranging from the day the mouse breast cancer cell line 4T1-luc cell line was injected to the end of the experiment (dissection date), lung metastasis of the cancer cell line was checked at intervals of 1 to 2 days. After the animal model was anesthetized by inhalation with 2.5% isoflurane, luminescence intensity was measured using an in-vivo imaging system (IVIS). Lung metastasis was checked in real time by measuring the location and amount of the 4T1-luc cell line that metastasized to the lung. During a period ranging from the day of the first administration of the compound of Production Example 6 to the end of the experiment, 20 mg/kg of the compound of Production Example 6 was intraperitoneally injected continuously at 2-day intervals.

[0171] [Results]

[Example 1] Evaluation of Effect of Increasing Stability of AMPK Protein

[0172] Whether the compound of Production Example 6 binds to the AMP binding site of AMPK was examined through the CTSA method described in Experimental Method 3, and the results are shown in FIG. 1.

[0173] As shown in FIG. 1, when the AMPK protein was present alone, the level of the protein was reduced from 50° C. (T.sub.m value was 50° C.), whereas when the cells were treated with the compound of Production Example 6, the level of the AMPK protein maintained up to 55° C. (T.sub.m value was 55° C.).

[0174] From the above results, it can be seen that the compound according to the present disclosure can increase the heat stability of the AMPK protein by binding to the AMP binding site of AMPK.

[Example 2] Evaluation of Cancer Cell Line Survival and Growth Inhibitory Effects and AMPK Activation Ability

[0175] [2-1] Evaluation of Cancer Cell Line Survival and Growth Inhibitory Effects

[0176] The effects of reducing the survival and inhibiting the growth of cancer cell lines were evaluated by the cell viability assay (a) and clonogenic assay (b) described in Experimental Method 4, and the results are shown in FIGS. 2A and 2B.

[0177] As shown in FIGS. 2A and 2B, when the cell lines were treated with the positive control AICAR, the cell viability was inhibited by 50% or more at 1 mM, whereas when both the 4T1 and BT549 cell lines were treated with 10 μM of the compound of Production Example 6, the cell viability was inhibited by 50% or more (see FIG. 2A). Furthermore, when the cell lines were treated with 10 μM of the compound of Production Example 6, colony formation was significantly reduced in all of the 4T1, BT549 and MDA-MB-231 cell lines (see FIG. 2B).

[0178] From the above results, it can be seen that the compound according to the present disclosure can reduce the viability of cancer cells and inhibit the growth of cancer cells.

[0179] [2-2] Evaluation of Effect of Inhibiting Growth of Cancer Cell Lines

[0180] The effect of inhibiting the growth of cancer cells was evaluated through the cell viability assay described in Experimental Method 4-1, and the results are shown in FIGS. 3A, 4A, 4B and 4C. As shown in FIG. 3A, it was confirmed that, when the breast cancer cell lines 4T1, BT549 and MDA-MB-231 were treated with various concentrations of each of a total of 14 compounds, including the compound of Production Example 6 and the compounds of Production Examples 1 to 5 and 8 to 15 (YE-01, 02, 03, 04, 05, 08, 09, 10, 11, 12, 13, 14 and 15) having a similar structure, for 24 hours, the compound that inhibited cancer cell viability by 50% or more in all the three breast cancer cell lines at a concentration of 10 μM was the compound of Production Example 6 or the compound of Production Example 8. In addition, it was confirmed that, each of three types of gastric cancer, brain cancer and pancreatic cancer cell lines was treated with various concentrations of the compound of Preparation Example 6 or the compound of Preparation Example 8 for 24 hours, the cancer cell viability was effectively inhibited by 50% or more (see FIGS. 4A, 4B and 4C). From the above results, it can be seen that the compound of Production Example 6 and the compound of Production Example 8 having a similar structure according to the present disclosure can more effectively reduce not only the viability of breast cancer cells, but also the viability of gastric cancer, brain cancer and pancreatic cancer cells.

[0181] [2-3] Evaluation of AMPK Activation Ability in Cancer Cell Lines

[0182] According to the method described in Experimental Method 3 above, the AMPK activation ability in the cancer cell line was evaluated by the expression level of p-AMPK (Thr172), and the results are shown in FIG. 3B. Here, the ratio of p-AMPK/AMPK was graphed by calculating a value normalized to the loading control (GAPDH) of each blot. As shown in FIG. 3B, it was confirmed that, when the 4T1, BT549 and MDA-MB-231 cell lines were treated with each of the compound of Production Example 6 and the compound of Production Example 8, the compound that effectively increased the p-AMPK level in all the three cell lines was particularly the compound of Production Example 6 and the compound of Production Example 8. In addition, it can be confirmed that, when each of three types of gastric cancer, brain cancer or pancreatic cancer cell lines was treated with each of the compound of Production Example 6, the compound of Production Example 8 and AICAR at a concentration of 10 μM for 12 hours, both the compound of Production Example 6 and the compound of Production Example 8 increased the expression of p-AMPK compared to the untreated group (control) (4A, 4B and 4C). Here, it can be seen that AICAR used as a positive control had an insignificant effect compared to the compound of Production Example 6 and the compound of Production Example 8, whereas the compound of Production Example 6 and the compound of Production Example 8 having a similar structure according to the present disclosure can more effectively induce AMPK activation in gastric cancer, brain cancer and pancreatic cancer cells in addition to breast cancer cells, compared to the AMPK activator AICAR.

[Example 3] Evaluation of Effect of Inducing Apoptosis of Cancer Cell Lines

[0183] The effect of inducing apoptosis of cancer cells was evaluated by the flow cytometry assay described in Experimental Method 4 and the Western blot assay described in Experimental Method 3, and the results are shown in FIGS. 5 and 6.

[0184] As shown in FIG. 5, when both the 4T1 cell line and the MDA-MB-231 cell line were treated with the compound of Production Example 6, the level of the apoptosis marker cleaved-PARP (poly(ADP-ribose) polymerase) protein increased as the phospho-AMPK protein increased in a concentration-dependent manner, and the levels of Bcl-2 and Cyclin D1 proteins decreased.

[0185] As shown in FIG. 6, when both the 4T1 cell line and the MDA-MB-231 cell line were treated with the compound of Production Example 6, the cells stained with annexin V or PI increased in a concentration-dependent manner.

[0186] From the above results, it can be seen that the compound according to the present disclosure can induce apoptosis of cancer cells.

[Example 4] Evaluation of Effect of Inhibiting EMT of Cancer Cell Lines

[0187] EMT of the cancer cell line was measured according to the method described in Experimental Method 5, and the results are shown in FIGS. 7A and 7B. Here, MMP2 (matrix metalloproteinase 2) and MMP 9 (matrix metalloproteinase 9) are enzymes that degrade type IV collagen, which is an important component of the basement membrane, and are most directly related to cancer migration and metastasis. Thus, the levels of the secreted MMP2 and secreted MMP9 proteins were measured.

[0188] As shown in FIG. 7A, the EMT protein markers α-SMA, Vimentin, ZEB-1 and Slug proteins, which increased when all of BT549, MDA-MB-231 and 4T1 cell lines were treated with TGF-01, decreased when the cell lines were treated with TGF-β1 together with the compound of Production Example 6.

[0189] As shown in FIG. 7B, the levels of secreted MMP2 and MMP9, which are EMT protein markers present in the BT549 cell line when treated with TGF-01, were significantly decreased by treatment with the compound of Production Example 6.

[0190] From the above results, it can be seen that the compound according to the present disclosure can very effectively inhibit cancer cell invasion and cancer metastasis by inhibiting EMT of cancer cells.

[Example 5] Evaluation of Tumor Regression Effect in Animal Model

[0191] The tumor regression effect of the compound of Production Example 6 was evaluated according to Method 7-1 of Experimental Method 7, and the results are shown in FIGS. 8A, 8B and 8C. In addition, the survival rate improvement effect of the compound of Production Example 6 was evaluated according to Method 7-2 of Experimental Method 7, and the results are shown in FIG. 8D.

[0192] As shown in FIGS. 8a to 8c, compared to the case where the compound of Production Example 6 was not administered (Vehicle), the volume of the tumor in the group (YE-06) to which the compound of Production Example 6 was administered decreased in a manner depending on the breeding time of the animal model (FIGS. 8A and 8B). In addition, it was observed that there was no weight loss depending on the breeding time in both the case where the compound of Production Example 6 was not administered (Vehicle) and the case where the compound of Production Example 6 was administered (YE-06) (FIG. 8C).

[0193] In addition, as shown in FIG. 8D, it was confirmed that the survival rate was significantly improved in the group (YE-06) to which the compound of Production Example 6 was administered, compared to the case where the compound of Production Example 6 was not administered (Vehicle).

[0194] From the above results, it can be seen that the compound according to the present disclosure has no toxicity even in the animal model, and administration of this compound can very effectively inhibit tumor growth. Furthermore, it can be seen that the compound according to the present disclosure can significantly increase the overall survival rate of individuals with cancer by effectively inhibiting tumor growth and metastasis in the animal model.

[Example 6] Evaluation of Tumor Metastasis Inhibitory Effect in Animal Model

[0195] According to Method 7-3 of Experimental Method 7, the tumor metastasis inhibitory effect of the compound of Production Example 6 was evaluated, and the results are shown in FIGS. 9A and 9B.

[0196] As shown in FIGS. 9A and 9B, it was confirmed that 4T1-luc metastasis was observed only in the lung among lung, liver, spleen and kidney, and in the case in which the compound of Production Example 6 was not administered (Vehicle), a very high luminescence intensity was observed in the lung of the orthotopic animal model, whereas in the case in which the compound of Production Example 6 was administered (YE-06), the luminescence intensity significantly decreased in the lung of the orthotopic animal model.

[0197] From the above results, it can be seen that the compound according to the present disclosure can very effectively inhibit tumor metastasis, particularly lung metastasis, in the animal model.

[0198] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0199] The composition according to the present disclosure may be very effectively used not only to prevent, ameliorate or treat cancer, but also to inhibit metastasis of cancer, by inhibiting the growth of cancer cells and very effectively inhibiting the metastasis of cancer cells to other tissues.