NOVEL BICYCLIC COMPOUND, OPTICAL ISOMER THEREOF OR PHARMACEUTICALLY ACCEPTABLE SALT THEREOF, USE THEREOF, AND PREPARATION METHOD THEREFOR

Abstract

The present invention provides a compound represented by chemical formula 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing or treating Wnt/?-catenin signaling-associated diseases, comprising same.

Claims

1. A compound represented by the following Chemical Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00015## in Chemical Formula 1 above, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are the same as or different from each other, and are each independently a hydrogen atom or hydroxyl (OH); R.sub.7 is a substituted or unsubstituted C.sub.1-6 alkyl, a substituted or unsubstituted C.sub.2-6 alkene, a substituted or unsubstituted C.sub.3-8 heterocycloalkyl containing at least one oxygen atom (O) as a heteroatom, or a substituted or unsubstituted C.sub.3-8 heterocycloalkene containing at least one oxygen atom (O) as a heteroatom (wherein at least one H of the substituted C.sub.1-6 alkyl, the substituted C.sub.2-6 alkene, the substituted C.sub.3-8 heterocycloalkyl, or the substituted C.sub.3-8 heterocycloalkene may each independently be substituted with hydroxyl (OH), a halogen atom, C.sub.1-6 alkyl or oxo (?O)); n is an integer of either 0 or 1; 10 when n is 0, X.sub.1 and X.sub.2 are the same as or different from each other, and are each independently a hydrogen atom or hydroxyl (OH); and when n is 1, X.sub.1 and X.sub.2 are each independently a single bond, and X.sub.3 is O.

2. The compound of claim 1, wherein the compound represented by Chemical Formula 1 is a compound represented by the following Chemical Formula 2, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00016## in Chemical Formula 2 above, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are the same as or different from each other, and are each independently a hydrogen atom or hydroxyl (OH); and R.sub.7 is a substituted or unsubstituted C.sub.1-6 alkyl, a substituted or unsubstituted C.sub.2-6 alkene, a substituted or unsubstituted C.sub.3-8 heterocycloalkyl containing at least one oxygen atom (O) as a heteroatom, or a substituted or unsubstituted C.sub.3-8 heterocycloalkene containing at least one oxygen atom (O) as a heteroatom (wherein at least one H of the substituted C.sub.1-6 alkyl, the substituted C.sub.2-6 alkene, the substituted C.sub.3-8 heterocycloalkyl, or the substituted C.sub.3-8 heterocycloalkene may each independently be substituted with hydroxyl (OH), a halogen atom, C.sub.1-6 alkyl or oxo (?O)).

3. The compound of claim 1, wherein the compound represented by Chemical Formula 1 is a compound represented by the following Chemical Formula 3, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00017## in Chemical Formula 3 above, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are the same as or different from each other, and are each independently a hydrogen atom or hydroxyl (OH); and R.sub.7 is a substituted or unsubstituted C.sub.1-6 alkyl, a substituted or unsubstituted C.sub.2-6 alkene, a substituted or unsubstituted C.sub.3-8 heterocycloalkyl containing at least one oxygen atom (O) as a heteroatom, or a substituted or unsubstituted C.sub.3-8 heterocycloalkene containing at least one oxygen atom (O) as a heteroatom (wherein at least one H of the substituted C.sub.1-6 alkyl, the substituted C.sub.2-6 alkene, the substituted C.sub.3-8 heterocycloalkyl, or the substituted C.sub.3-8 heterocycloalkene may each independently be substituted with hydroxyl (OH), a halogen atom, C.sub.1-6 alkyl or oxo (?O)).

4. The compound of claim 1, wherein R.sub.1, R.sub.2, R.sub.4 and R.sub.5 are each independently hydroxyl (OH); and R.sub.3 and R.sub.6 are each independently a hydrogen atom.

5. The compound of claim 1, wherein R.sub.7 is C.sub.1-4 alkyl in which at least one H is each independently substituted with hydroxyl (OH), C.sub.2-4 alkene in which at least one H is each independently substituted with hydroxyl (OH), C.sub.3-5 heterocycloalkyl containing at least one oxygen atom (O) as a heteroatom in which at least one H is each independently substituted with C.sub.1-3 alkyl, oxo (?O), or both, or C.sub.3-5 heterocycloalkene containing at least one oxygen atom (O) as a heteroatom in which at least one H is each independently substituted with C.sub.1-3 alkyl.

6. The compound of claim 5, wherein R.sub.7 is C.sub.1-4 alkyl substituted with at least two hydroxyls (OH), C.sub.2-4 alkene substituted with at least two hydroxyls (OH), C.sub.3-5 heterocycloalkyl containing at least one oxygen atom (O) substituted with C.sub.1-3 alkyl as a heteroatom, C.sub.3-5 heterocycloalkyl containing at least one oxygen atom (O) substituted with C.sub.1-3 alkyl and oxo (?O) as a heteroatom, or C.sub.3-5 heterocycloalkene containing one oxygen atom (O) substituted with one C.sub.1-3 alkyl as a heteroatom.

7. The compound of claim 1, wherein R.sub.7 is propane, propene, oxirane, oxirene or dioxolane, wherein the propane, propene, oxirane, oxirene and dioxolane may be unsubstituted, or may each independently be substituted with hydroxyl (OH) or C.sub.1-3 alky.

8. The compound of claim 1, wherein R.sub.7 is ##STR00018##

9. The compound of claim 1, wherein the compound represented by Chemical Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of the following Chemical Formulas 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6: ##STR00019## ##STR00020## ##STR00021##

10. The compound of claim 1, wherein the compound, an optical isomer thereof, or a pharmaceutically acceptable salt thereof is isolated from Streptomyces rapamycinicus 17A011 KTCT 14890BP strain.

11. A pharmaceutical composition for preventing or treating Wnt/?-catenin signaling-associated diseases, comprising: the compound according to any one of claims 1 to 10, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition of claim 11, wherein the Wnt/?-catenin signaling-associated disease is at least one selected from the group consisting of neurodegenerative diseases, eye diseases, bone diseases, periodontal diseases, otosclerosis (ear sclerosis), wound healing, oral mucositis, gastrointestinal mucositis, craniofacial defects, hair loss diseases, and metabolic diseases.

13. The pharmaceutical composition of claim 12, wherein the bone disease is at least one selected from the group consisting of bone defect, osteoporosis, osteoarthrosis, osteogenesis imperfecta, bone defect, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalacia and resulting fractures, bone dysplasia, degenerative bone disease, oncolytic bone disease, Paget's disease, metabolic bone disease, leukemia, multiple myeloma, myeloma, fibrous osteodysplasia, aplastic bone disease, osteonecrosis, rickets, or malocclusion.

14. The pharmaceutical composition of claim 12, wherein the metabolic disease is at least one selected from the group consisting of obesity, dyslipidemia, fatty liver, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, myocardial infarction, cerebral infarction, sarcopenia, hyperinsulinemia, and myocardial infarction.

15. The pharmaceutical composition of claim 12, wherein the neurodegenerative disease is at least one selected from the group consisting of Parkinson's disease, stroke, spinal cord injury, ischemic brain disease, epilepsy, Alzheimer's disease, dementia, depression, bipolar disorder, and schizophrenia.

16. A food composition for preventing or improving Wnt/?-catenin signaling-associated diseases, comprising: the compound according to any one of claims 1 to 10, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

17. Streptomyces rapamycinicus KTCT 14890BP.

18. A method for treating Wnt/?-catenin signaling-associated diseases comprising: administering a therapeutically effective amount of the compound according to any one of claims 1 to 10, an optical isomer thereof, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

19. Use of the compound according to any one of claims 1 to 10, an optical isomer thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of Wnt/?-catenin signaling-associated diseases.

20. A pharmaceutical composition for use in the prevention or treatment of Wnt/?-catenin signaling-associated diseases, comprising: the compound according to any one of claims 1 to 10, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

Description

DESCRIPTION OF DRAWINGS

[0110] FIG. 1 shows TopFLASH luciferase assay measurement protocol.

[0111] FIG. 2 shows the Wnt activity evaluation results of the bicyclic compound represented by Chemical Formula 1 and Wnt3a of the present invention.

[0112] FIG. 3 shows cytotoxicity test results in 3T3-L1 cells for the treatment of the bicyclic compound represented by Chemical Formula 1-1 alone (L) and the combined treatment of Wnt3a and Chemical Formula 1-1 (Wnt3a) of the present invention.

[0113] FIG. 4 shows the Wnt activity evaluation (TopFLASH assay) results through treatment with the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0114] FIG. 5 shows the Wnt activity evaluation (TopFLASH assay) results through the combined treatment of the bicyclic compound represented by Chemical Formula 1-1 and Wnt3a of the present invention.

[0115] FIG. 6 shows changes in Wnt3a signaling protein for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0116] FIG. 7 quantitatively shows changes in Wnt3a signaling protein for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0117] FIG. 8 shows experimental results of the differentiation inhibitory effect on adipogenic differentiation cells (T3-L1 cells) for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0118] FIG. 9 shows experimental results of the differentiation inhibitory effect on adipogenic differentiation cells (3T3-L1 cells) for the combined treatment of the bicyclic compound represented by Chemical Formula 1-1 and wnt3a.

[0119] FIG. 10 shows experimental results of changes in differentiation-associated protein in adipogenic differentiation cells (3T3-L1 cells) for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0120] FIG. 11 shows experimental results of changes in differentiation-associated mRNA in adipogenic differentiation cells (3T3-L1 cells) for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0121] FIG. 12 shows the efficacy evaluation results of the bicyclic compound represented by Chemical Formula 1-1 of the present invention in suppressing obesity in mice using a high-calorie feed.

[0122] FIG. 13 shows measurement results for obesity and liver damage factors in mouse serum using a high-calorie feed for the bicyclic compound of Chemical Formula 1-1 of the present invention.

[0123] FIG. 14 shows cytotoxicity test results in MC3T3-E1 cells depending on the concentration of the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0124] FIG. 15 shows ALP activity results according to the treatment of the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

[0125] FIG. 16 shows the analysis results of alkaline phosphatase (ALP) activity of the bicyclic compound represented by Chemical Formula 1-1 of the present invention in MC3T3-E1 cells.

[0126] FIG. 17 shows Western blotting results of confirming the expression of bone differentiation-involved genes for the bicyclic compound represented by Chemical Formula 1-1 of the present invention.

BEST MODE

[0127] Hereinafter, exemplary embodiments will be described in detail.

[0128] However, the following Examples are only provided to illustrate the present invention, and the content of the present invention is not limited to these Examples.

EXAMPLE 1

Identification and Culture of Streptomyces rapamycinicus 17A011 Strain

[0129] (1) Deposit, Isolation and Identification of Streptomyces rapamycinicus 17A011 Strain

[0130] Soil samples were collected from Ochang, Cheongju, South Korea. As a result of 16S rRNA gene sequence analysis, the 17A011 strain was found to be most closely related to the Streptomyces rapamycinicus gene (99.79% homology, GenBank Accession No. KP209440.1). Thus, strain 17A011 was named Streptomyces rapamycinicus 17A011 and used in subsequent culture experiments.

(2) Culture of Streptomyces rapamycinicus 17A011 Strain

[0131] In order to culture the present actinomycete strain, a medium containing a source of nutrients commonly used by microorganisms was prepared. The used crude medium and fermentation medium for actinomycetes was YMG medium containing 10 g glucose, 20 g soluble starch, 5 g yeast extract, 5 g malt extract, and 0.5 g calcium carbonate per 1 L of distilled water. A 1000 ml Erlenmeyer flask containing 250 ml of the seed medium was sterilized at 121? C. for 15 minutes, then Streptomyces rapamycinicus 17A011 strain was inoculated with a platinum loop from a test tube for slant culture and cultured with shaking for 3 days to form a seed culture. Each of 40 Erlenmeyer flasks with a capacity of 1,000 ml containing 250 ml of fermentation medium (10 L in total) was inoculated with 3 ml of the seed culture media, followed by culturing with shaking for 7 days at 28? C.

EXAMPLE 2

Isolation and Purification of Bicyclic Compounds from Streptomyces rapamycinicus 17A011 Strain

[0132] The culture media (10 L) of Streptomyces rapamycinicus 17A011 strain cultured in Example 1 was centrifuged and extracted with 9 L of ethyl acetate, and the obtained extract was concentrated by evaporation under reduced pressure using a vacuum dryer. This concentrate (4.5 g) was adsorbed on ODS RP-18 and subjected to ODS RP-18 flash column chromatography, wherein a total of 10 fractions were obtained by eluting with methanol/water (8:2-10/0, v/v) as a mixed solvent while gradually increasing the methanol concentration. Here, fraction No. 7 (440 mg) containing Chemical Formulas 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6 was concentrated under reduced pressure, followed by high performance liquid chromatography (column: Cosmosil C18, length 25 mm, diameter 10 mm) to perform elution under conditions of an elution flow rate of 3 ml/min using 0.05% formic acid-containing acetonitrile and water (40:60) as a solvent, thereby preparing novel bicyclic compounds exhibiting UV absorption peaks of 210 nm and 260 nm.

EXAMPLE 3

Structural Analysis of Bicyclic Compound from Streptomyces rapamycinicus 17A011 Strain

[0133] The molecular weight and molecular formula of the bicyclic compounds according to the present invention prepared from the culture media of Streptomyces rapamycinicus 17A011 strain were determined using an electrospray ionization mass spectrometer (ESIMS). In addition, nuclear magnetic resonance (NMR) analysis (Bruker AVANCE HD 800 NMR spectrometer) was performed to obtain .sup.1H NMR, .sup.13C NMR, Correlation Spectroscopy (COSY), .sup.1H-Detected heteronuclear Multiple-Quantum Coherence (HMQC), Heteronuclear Multiple-Bond Coherence (HMBC), Distortionless Enhancement by Polarization Transfer (DEPT) and Nuclear Overhauser effect spectroscopy (NOESY) spectra, and molecular structures of the compounds were determined.

[0134] The measurement results are shown in Table 1 below. Materials isolated from the culture media of Streptomyces rapamycinicus 17A011 strain were identified as novel bicyclic compounds of the following Chemical Formulas 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6, and results thereof are shown in Table 1 below:

TABLE-US-00002 TABLE 1 Compound Structure NMR/MS data Chemical Formula 1-1 [00009] .sup.1H NMR (800 MHz, Pyr) ? 11.01 (d, J = 9.3 Hz, 1H), 9.98 (d, J = 3.8 Hz, 1H), 9.49 (d, J = 7.4 Hz, 1H), 9.41 (d, J = 9.6 Hz, 1H), 9.15 (d, J = 10.8 Hz, 2H), 8.61-8.57 (m, 1H), 8.53 (s, 1H), 8.38 (d, J = 2.9 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.02 (dd, J = 8.5, 2.0 Hz, 1H), 7.95 (s, 1H), 7.72 (dd, J = 8.1, 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 8.1, 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 6.8 Hz, 2H), 7.12 (dd, J = 8.4, 2.3 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 6.74 (d, J = 9.3 Hz, 2H), 6.58 (t, J = 7.4 Hz, 1H), 6.00 (q, J = 6.9 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.76 (d, J = 3.1 Hz, 1H), 5.71 (d, J = 3.9 Hz, 1H), 5.57 (d, J = 10.2 Hz, 1H), 5.51 (s, 1H), 5.38-5.32 (m, 4H), 5.27 (td, J = 9.3, 5.8 Hz, 3H), 4.99 (dd, J = 17.5, 8.0 Hz, 11H), 4.82 (td, J = 7.5, 4.6 Hz, 2H), 4.63 (dd, J = 8.3, 5.6 Hz, 2H), 4.48- 4.44 (m, 1H), 4.29 (d, J = 10.2 Hz, 1H), 4.13 (q, J = 6.6 Hz, 3H), 3.94 (dd, J = 17.5, 4.9 Hz, 1H), 3.78 (dd, J = 16.0, 6.8 Hz, 1H), 3.20 (qd, J = 15.7, 6.0 Hz, 3H), 2.33 (dd, J = 13.0, 6.0 Hz, 1H), 2.15 (dt, J = 15.2, 7.4 Hz, 2H), 1.88 (t, J = 9.2 Hz, 4H), 1.86 (s, 1H), 1.79 (tt, J = 13.2, 6.7 Hz, 2H), 1.74-1.70 (m, 2H), 1.70-1.64 (m, 2H), 1.64- 1.60 (m, 2H), 1.57 (dd, J = 12.7, 5.1 Hz, 1H), 1.56-1.54 (m, 4H), 1.54 (s, 4H), 1.09 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.4 Hz, 3H). .sup.13C NMR (200 MHz, Pyr) ? 174.95, 173.28, 172.92, 172.88, 172.26, 172.13, 171.87, 171.26, 171.15, 170.76, 170.49, 160.72, 149.38, 149.35, 143.57, 139.86, 134.65, 134.11, 133.58, 132.53, 130.34, 129.29, 127.49, 127.46, 127.34, 125.26, 125.10, 123.40, 121.69, 120.57, 117.68, 76.16, 74.60, 73.15, 72.55, 71.94, 70.97, 66.83, 65.40, 62.79, 62.31, 61.43, 59.15, 54.33, 52.07, 51.07, 48.07, 44.55, 44.18, 36.07, 31.15, 29.55, 25.60, 25.31, 23.35, 22.43, 21.58, 21.28, 18.51, 17.75, 15.23.; HRESIMS m/z 1338.5482 [M + Na].sup.+ (calcd for C.sub.62H.sub.61N.sub.11O.sub.21Na, 1338.5506) Chemical Formula 1-2 [00010] .sup.1H NMR (800 MHz, Pyr) ? 11.11 (d, J = 9.2 Hz, 0H), 9.96 (d, J = 4.1 Hz, 0H), 9.60 (d, J = 7.4 Hz, 0H), 9.45 (d, J = 9.6 Hz, 0H), 9.12 (s, 0H), 9.07 (d, J = 9.8 Hz, 0H), 8.89 (d, J = 15.3 Hz, 0H), 8.84 (d, J = 8.9 Hz, 0H), 8.52 (t, J = 6.1 Hz, 1H), 8.28 (d, J = 3.1 Hz, 0H), 7.96 (s, 0H), 7.81 (t, J = 8.8 Hz, 1H), 7.71 (dd, J = 8.1, 1.9 Hz, 0H), 7.56 (d, J = 2.5 Hz, 0H), 7.45 (d, J = 8.4 Hz, 0H), 7.26 (d, J = 7.7 Hz, 0H), 7.20- 7.17 (m, 1H), 7.10 (d, J = 1.8 Hz, 0H), 6.96 (d, J = 15.3 Hz, 0H), 6.91 (dd, J = 8.5, 2.4 Hz, 0H), 6.70-6.68 (m, 0H), 6.67 (d, J = 7.8 Hz, 0H), 5.98 (dd, J = 13.8, 6.9 Hz, 0H), 5.78 (d, J = 4.3 Hz, 0H), 5.76 (d, J = 7.1 Hz, 0H), 5.73 (d, J = 3.2 Hz, 1H), 5.64 (d, J = 10.2 Hz, 1H), 5.41 (t, J = 9.5 Hz, 1H), 4.98 (dd, J = 17.5, 8.0 Hz, 1H), 4.80 (td, J = 8.0, 4.1 Hz, 0H), 4.64 (dd, J = 8.4, 5.5 Hz, 0H), 4.53-4.50 (m, 0H), 4.27 (d, J = 10.2 Hz, 0H), 4.18 (dd, J = 12.7, 9.5 Hz, 1H), 3.93 (dd, J = 17.5, 4.9 Hz, 0H), 3.77 (dd, J = 16.0, 6.8 Hz, 0H), 3.25 (dd, J = 15.6, 3.8 Hz, 0H), 3.21 (dd, J = 15.5, 8.5 Hz, 0H), 2.53 (dt, J = 13.0, 6.7 Hz, 0H), 2.15 (dt, J = 19.6, 7.6 Hz, 0H), 1.91-1.87 (m, 1H), 1.86 (d, J = 6.8 Hz, 1H), 1.80 (ddd, J = 18.2, 12.5, 6.6 Hz, 1H), 1.70 (dd, J = 14.5, 6.2 Hz, 1H), 1.68 (d, J = 6.1 Hz, 1H), 1.68 (t, J = 6.5 Hz, 1H), 1.66 (dd, J = 13.0, 6.2 Hz, 0H), 1.62 (td, J = 9.8, 6.7 Hz, 1H), 1.48 (d, J = 6.8 Hz, 1H), 1.42 (d, J = 6.3 Hz, 1H), 1.17 (d, J = 6.8 Hz, 1H), 0.78 (d, J = 6.1 Hz, 1H), 0.71 (d, J = 6.0 Hz, 1H). .sup.13C NMR (200 MHz, Pyr) ? 174.93, 173.34, 173.26, 172.89, 172.72, 172.45, 172.05, 171.69, 171.14, 171.09, 170.60, 170.40, 160.69, 150.58, 150.49, 150.38, 150.24, 150.11, 149.95, 149.48, 149.39, 139.47, 139.42, 136.59, 136.17, 136.12, 136.01, 135.88, 135.76, 135.61, 134.03, 133.81, 132.34, 130.23, 129.69, 129.33, 128.73, 128.32, 127.75, 125.08, 124.27, 124.16, 124.13, 124.10, 123.99, 123.87, 123.75, 123.59, 123.47, 121.33, 120.89, 117.66, 76.54, 73.00, 72.04, 71.77, 70.91, 66.96, 65.64, 64.77, 62.88, 62.19, 61.41, 58.97, 54.28, 52.09, 51.11 50.03, 48.07, 44.48, 44.25, 41.43, 36.09, 31.15, 29.86, 25.60, 25.35, 23.23, 22.57, 22.07, 21.59, 21.02, 18.44, 15.51. HRESIMS m/z 1356.5100 [M + Na].sup.+ calcd for C.sub.62H.sub.63N.sub.11O.sub.22Na, 1356.5612) Chemical Formula 1-3 [00011] .sup.1H NMR (800 MHz, Pyr) ? 11.01 (d, J = 9.3 Hz, 1H), 9.98 (d, J = 3.8 Hz, 1H), 9.49 (d, J = 7.4 Hz, 1H), 9.41 (d, J = 9.6 Hz, 1H), 9.15 (d, J = 10.8 Hz, 2H), 8.61-8.57 (m, 1H), 8.53 (s, 1H), 8.38 (d, J = 2.9 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.02 (dd, J = 8.5, 2.0 Hz, 1H), 7.95 (s, 1H), 7.72 (dd, J = 8.1, 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 8.1, 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 6.8 Hz, 2H), 7.12 (dd, J = 8.4, 2.3 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 6.74 (d, J = 9.3 Hz, 2H), 6.58 (t, J = 7.4 Hz, 1H), 6.00 (q, J = 6.9 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.76 (d, J = 3.1 Hz, 1H), 5.71 (d, J = 3.9 Hz, 1H), 5.57 (d, J = 10.2 Hz, 1H), 5.51 (s, 1H), 5.38-5.32 (m, 4H), 5.27 (td, J = 9.3, 5.8 Hz, 3H), 4.99 (dd, J = 17.5, 8.0 Hz, 11H), 4.82 (td, J = 7.5, 4.6 Hz, 2H), 4.63 (dd, J = 8.3, 5.6 Hz, 2H), 4.48- 4.44 (m, 1H), 4.29 (d, J = 10.2 Hz, 1H), 4.13 (q, J = 6.6 Hz, 3H), 3.94 (dd, J = 17.5, 4.9 Hz, 1H), 3.78 (dd, J = 16.0, 6.8 Hz, 1H), 3.20 (qd, J = 15.7, 6.0 Hz, 3H), 2.33 (dd, J = 13.0, 6.0 Hz, 1H), 2.15 (dt, J = 15.2, 7.4 Hz, 2H), 1.88 (t, J = 9.2 Hz, 4H), 1.86 (s, 1H), 1.79 (tt, J = 13.2, 6.7 Hz, 2H), 1.74-1.70 (m, 2H), 1.70-1.64 (m, 2H), 1.64- 1.60 (m, 2H), 1.57 (dd, J = 12.7, 5.1 Hz, 1H), 1.56-1.54 (m, 4H), 1.54 (s, 4H), 1.09 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.4 Hz, 3H). .sup.13C NMR (200 MHz, Pyr) ? 174.95, 173.28, 172.92, 172.88, 172.26, 172.13, 171.87, 171.26, 171.15, 170.76, 170.49, 160.72, 156.35, 154.38, 153.35, 155.22, 143.57, 139.86, 134.65, 134.11, 133.58, 132.53, 130.34, 129.29, 127.49, 127.46, 127.34, 125.26, 125.10, 123.40, 121.69, 120.57, 117.68, 76.16, 74.60, 73.15, 72.55, 71.94, 70.97, 62.79, 62.31, 61.43, 59.15, 54.33, 52.07, 51.07, 48.07, 44.55, 44.18, 36.07, 31.15, 29.55, 25.60, 25.31, 23.35, 22.43, 21.58, 21.28, 18.51, 17.75, 15.23.; ESIMS m/z 1314.82 [M + H].sup.+ Chemical Formula 1-4 [00012] .sup.1H NMR (800 MHz, Pyr) ? 11.01 (d, J = 9.3 Hz, 1H), 9.98 (d, J = 3.8 Hz, 1H), 9.49 (d, J = 7.4 Hz, 1H), 9.41 (d, J = 9.6 Hz, 1H), 9.15 (d, J = 10.8 Hz, 2H), 8.61-8.57 (m, 1H), 8.53 (s, 1H), 8.38 (d, J = 2.9 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.02 (dd, J = 8.5, 2.0 Hz, 1H), 7.95 (s, 1H), 7.72 (dd, J = 8.1, 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 8.1, 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 6.8 Hz, 2H), 7.12 (dd, J = 8.4, 2.3 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 6.74 (d, J = 9.3 Hz, 2H), 6.58 (t, J = 7.4 Hz, 1H), 6.00 (q, J = 6.9 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.76 (d, J = 3.1 Hz, 1H), 5.71 (d, J = 3.9 Hz, 1H), 5.57 (d, J = 10.2 Hz, 1H), 5.51 (s, 1H), 5.38-5.32 (m, 4H), 5.27 (td, J = 9.3, 5.8 Hz, 3H), 4.99 (dd, J = 17.5, 8.0 Hz, 11H), 4.82 (td, J = 7.5, 4.6 Hz, 2H), 4.63 (dd, J = 8.3, 5.6 Hz, 2H), 4.48- 4.44 (m, 1H), 4.29 (d, J = 10.2 Hz, 1H), 4.13 (q, J = 6.6 Hz, 3H), 3.94 (dd, J = 17.5, 4.9 Hz, 1H), 3.78 (dd, J = 16.0, 6.8 Hz, 1H), 3.20 (qd, J = 15.7, 6.0 Hz, 3H), 2.33 (dd, J = 13.0, 6.0 Hz, 1H), 2.15 (dt, J = 15.2, 7.4 Hz, 2H), 1.88 (t, J = 9.2 Hz, 4H), 1.86 (s, 1H), 1.79 (tt, J = 13.2, 6.7 Hz, 2H), 1.74-1.70 (m, 2H), 1.70-1.64 (m, 2H), 1.64- 1.60 (m, 2H), 1.57 (dd, J = 12.7, 5.1 Hz, 1H), 1.56-1.54 (m, 4H), 1.54 (s, 4H), 1.09 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.4 Hz, 3H). .sup.13C NMR (200 MHz, Pyr) ? 174.95, 173.28, 172.92, 172.88, 172.26, 172.13, 171.87, 171.26, 171.15, 170.76, 170.49, 160.72, 149.38, 149.35, 143.57, 139.86, 134.65, 134.11, 133.58, 132.53, 130.34, 129.29, 127.49, 127.46, 127.34, 125.26, 125.10, 123.40, 121.69, 120.57, 117.68, 76.16, 74.60, 73.15, 72.55, 71.94, 70.97, 66.83, 65.40, 63.79, 63.31, 61.43, 59.15, 54.33, 52.07, 51.07, 48.07, 44.55, 44.18, 36.07, 31.15, 29.55, 25.60, 25.31, 23.35, 22.43, 21.58, 21.28, 18.51, 17.75, 15.23.; ESIMS m/z 1298.85 [M + H].sup.+ Chemical Formula 1-5 [00013] .sup.1H NMR (800 MHz, Pyr) ? 11.01 (d, J = 9.3 Hz, 1H), 9.98 (d, J = 3.8 Hz, 1H), 9.49 (d, J = 7.4 Hz, 1H), 9.41 (d, J = 9.6 Hz, 1H), 9.15 (d, J = 10.8 Hz, 2H), 8.61-8.57 (m, 1H), 8.53 (s, 1H), 8.38 (d, J = 2.9 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.02 (dd, J = 8.5, 2.0 Hz, 1H), 7.95 (s, 1H), 7.72 (dd, J = 8.1, 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 8.1, 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 6.8 Hz, 2H), 7.12 (dd, J = 8.4, 2.3 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 6.74 (d, J = 9.3 Hz, 2H), 6.58 (t, J = 7.4 Hz, 1H), 6.00 (q, J = 6.9 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.76 (d, J = 3.1 Hz, 1H), 5.71 (d, J = 3.9 Hz, 1H), 5.57 (d, J = 10.2 Hz, 1H), 5.51 (s, 1H), 5.38-5.32 (m, 4H), 5.27 (td, J = 9.3, 5.8 Hz, 3H), 4.99 (dd, J = 17.5, 8.0 Hz, 11H), 4.82 (td, J = 7.5, 4.6 Hz, 2H), 4.63 (dd, J = 8.3, 5.6 Hz, 2H), 4.48- 4.44 (m, 1H), 4.29 (d, J = 10.2 Hz, 1H), 4.13 (q, J = 6.6 Hz, 3H), 3.94 (dd, J = 17.5, 4.9 Hz, 1H), 3.78 (dd, J = 16.0, 6.8 Hz, 1H), 3.20 (qd, J = 15.7, 6.0 Hz, 3H), 2.33 (dd, J = 13.0, 6.0 Hz, 1H), 2.15 (dt, J = 15.2, 7.4 Hz, 2H), 1.88 (t, J = 9.2 Hz, 4H), 1.86 (s, 1H), 1.79 (tt, J = 13.2, 6.7 Hz, 2H), 1.74-1.70 (m, 2H), 1.70-1.64 (m, 2H), 1.64- 1.60 (m, 2H), 1.57 (dd, J = 12.7, 5.1 Hz, 1H), 1.56-1.54 (m, 4H), 1.54 (s, 4H), 1.09 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.4 Hz, 3H). .sup.13C NMR (200 MHz, Pyr) ? 174.95, 173.28, 172.92, 172.88, 172.26, 172.13, 171.87, 171.26, 171.15, 170.26, 170.49, 160.72, 156.35, 154.38, 149.38, 149.35, 143.57, 139.86, 134.65, 134.11, 133.58, 132.53, 130.34, 129.29, 127.49, 127.46, 127.34, 125.26, 125.10, 123.40, 121.69, 120.57, 117.68, 76.16, 74.60, 73.15, 72.55, 71.94, 70.97, 66.83, 65.40, 63.79, 59.15, 54.33, 52.07, 51.07, 48.07, 44.55, 44.18, 36.07, 31.15, 29.55, 25.60, 25.31, 23.35, 22.43, 21.58, 21.28, 18.51, 17.75, 15.23.; ESIMS m/z 1296.85 [M + H].sup.+ Chemical Formula 1-6 [00014] .sup.1H NMR (800 MHz, Pyr) ? 10.81 (d, J = 9.2 Hz, 1H), 9.96 (d, J = 3.8 Hz, 1H), 9.60 (d, J = 7.0 Hz, 1H), 9.33 (d, J = 9.5 Hz, 1H), 9.21 (s, 1H), 9.04 (d, J = 10.0 Hz, 1H), 8.72 (s, 3H), 8.64-8.59 (m, 1H), 8.59-8.53 (m, 2H), 8.34 (s, 1H), 7.97 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.33-7.27 (m, 2H), 7.29 (d, J = 15.4 Hz, 1H), 7.12 (s, 1H), 7.04 (s, 1H), 6.81 (t, J = 7.5 Hz, 1H), 6.76 (d, J = 9.8 Hz, 1H), 6.74 (dd, J = 16.8, 9.5 Hz, 2H), 6.73 (d, J = 9.2 Hz, 1H), 6.26 (d, J = 7.6 Hz, 1H), 6.00 (dd, J = 13.4, 6.6 Hz, 2H), 6.01-5.98 (m, 1H), 5.78 (d, J = 3.0 Hz, 1H), 5.74 (d, J = 4.1 Hz, 1H), 5.63 (d, J = 10.1 Hz, 1H), 5.52 (s, 1H), 5.45-5.41 (m, 1H), 5.45-5.41 (m, 1H), 5.35 (d, J = 9.9 Hz, 1H), 5.24 (d, J = 5.7 Hz, 1H), 5.23 (s, 2H), 5.20 (dd, J = 14.9, 9.2 Hz, 2H), 4.92 (d, J = 7.5 Hz, 2H), 4.93- 4.88 (m, 6H), 4.90 (s, 2H), 4.87-4.83 (m, 3H), 4.62-4.58 (m, 1H), 4.29 (d, J = 10.2 Hz, 1H), 4.13 (dd, J = 15.9, 7.0 Hz, 2H), 4.07 (d, J = 9.7 Hz, 1H), 3.95 (dd, J = 17.4, 4.9 Hz, 1H), 3.71 (dd, J = 15.7, 6.7 Hz, 1H), 3.63-3.57 (m, 1H), 3.23 (dd, J = 23.7, 15.8 Hz, 2H), 2.40-2.35 (m, 1H), 2.16 (dd, J = 12.4, 7.4 Hz, 2H), 2.16 (dd, J = 12.4, 7.4 Hz, 2H), 1.87 (d, J = 6.9 Hz, 3H), 1.82- 1.77 (m, 2H), 1.82-1.77 (m, 2H), 1.73- 1.70 (m, 2H), 1.67 (d, J = 6.2 Hz, 3H), 1.66-1.63 (m, 2H), 1.63-1.58 (m, 3H), 1.56 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.7 Hz, 4H), 1.11 (d, J = 6.8 Hz, 3H), 0.79 (d, J = 6.4 Hz, 4H), 0.75 (d, J = 6.3 Hz, 3H). .sup.13C NMR (200 MHz, Pyr) ? 174.96, 173.29, 173.21, 173.10, 172.82, 172.39, 171.97, 171.70, 171.17, 171.08, 170.72, 170.28, 160.72, 155.19, 150.58, 150.49, 150.38, 150.24, 150.11, 149.96, 149.54, 149.41, 137.93, 136.28, 136.17, 136.11, 136.00, 135.88, 135.76, 135.61, 134.23, 133.78, 133.45, 132.46, 130.38, 130.29, 130.24, 129.00, 127.93, 127.23, 125.18, 124.16, 124.10, 123.99, 123.87, 123.75, 123.59, 121.64, 120.70, 117.81, 81.32, 80.56, 76.52, 73.27, 71.77, 70.74, 67.10, 65.63, 63.13, 62.12, 61.46, 59.33, 53.85, 52.13, 51.13, 48.20, 44.64, 44.35, 36.12, 31.20, 29.71, 25.60, 25.34, 23.40, 22.47, 21.55, 21.36, 18.62, 18.42, 15.40.; HRESIMS m/z 1364.5276 [M + Na].sup.+ (calcd for C.sub.63H.sub.79N.sub.11O.sub.22Na, 1364.5299)

Experimental Example 1

Analysis of Wnt Activities of Chemical Formulas 1-1 to 1-6

[0135] In order to measure the level of Wnt activities of Chemical Formulas 1-1 to 1-6, TopFLASH luciferase assay was performed on the HEK293T cell lines (FIG. 1).

[0136] In order to measure the level of Wnt activities of Chemical Formulas 1-1 to 1-6, TopFLASH luciferase assay was performed on the HEK293T cell lines. After generating 7TFC virus plasmid (Addgene, Plasmid #24307), 293T cells were inserted thereinto. Cells with mCherry signals were selected under a fluorescence microscope (ZEISS, Observer Z1). RPE cells were transfected with the M50 Super 8? TOPFlash plasmid (Addgene, Plasmid #12456) using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000-015) according to the manufacturer's protocol. After incubation for 36 hours, the transfected cells were replaced with fresh media and treated with Wintamide in the presence or absence of Wnt3a-CM (10%) for 24 hours at 37? C. in a 5% CO.sub.2 incubator.

[0137] In 96 well plates (1?10.sup.4 cells/well), 293T-7TFC and M50 Super 8?-transfected RPE cells were cultured. After overnight incubation, cells were treated with the indicated concentrations of Chemical Formulas 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6 and Wnt3a-CM (10%), followed by incubation for 24 hours. Cells were treated with One-Glo? used as a detector in the TOPFlash luciferase assay according to the manufacturer's instructions. Luciferase intensity was measured using a photometer (Perkin Elmer, victor? X2).

[0138] As could be confirmed in FIG. 2, the luciferase values were found to increase significantly as the concentration increased during the treatment of Chemical Formulas 1-1 to 1-6. Thus, the compounds of the present invention were found to greatly enhance Wnt activity.

Experimental Example 2

Analysis of Toxicity and Inhibition of Adipogenesis in Various Cell lines for Chemical Formula 1-1

Experimental Example 2-1

Various Cytotoxicity Assays

[0139] To measure the cytotoxicity of the compound represented by Chemical Formula 1 (Chemical Formula 1-1), toxicity was measured in mouse preadipocyte 3T3-L1 cell line, human malignant melanoma A375 cell line, mouse malignant melanoma B16F10 cell line, human oral cancer MDA-MB-435 cell line, mouse breast cancer 4T1 cell line, human breast cancer MCF7 cell line, human brain cancer U87MG cell line, human glioblastoma U373MG cell line, human cervical cancer HeLa cell line, mouse embryonic fibroblast (MEF) cell line, human embryonic kidney HEK293T cell line (Korea Cell Line Bank). First, 1?10.sup.4 cells were divided into 200 ?l each in a 96-well plate using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 mg/ml streptomycin, and 50 U/ml penicillin, while maintaining various cells at 37r in an atmosphere of 5% CO.sub.2. The cells were treated with the compound of Chemical Formula 1-1 of the present invention at a maximum concentration of 100 ?M and cultured for 48 hours under the same culture conditions. After the incubation, the culture media were removed and the cytotoxicity test results were confirmed (Table 2 and FIG. 3 below).

[0140] As a result, as shown in Table 2 below, the compound of Chemical Formula 1-1 showed no cytotoxicity even in various cell lines.

TABLE-US-00003 TABLE 2 Cell line IC.sub.50 (?M) mouse preadipocyte 3T3-L1 cell line >100 human malignant melanoma A375 cell line >100 mouse malignant melanoma B16F10 cell line >100 human oral cancer MDA-MB-435 cell line >100 mouse breast cancer 4T1 cell line >100 human breast cancer MCF7 cell line >100 human brain cancer U87MG cell line >100 human glioblastoma U373MG cell line 89.5 human cervical cancer HeLa cell line >100 mouse embryonic fibroblast (MEF) cell line >100 human embryonic kidney HEK293T cell line >100

[0141] Next, in order to confirm the cytotoxicity in the Chemical Formula 1-1 and wnt3 culture, toxicity in 3T3-L1 cells was measured on the compound of Chemical Formula 1-1 of the present invention at a concentration of 2.5 to 10 ?M in L-conditional media or Wnt3a-containing culture media (2.5%). As a result, as shown in FIG. 3, cytotoxicity was not observed even in the Wnt3a-containing culture media.

Experimental Example 2-2

Wnt3a Activity Assay

[0142] In order to measure the level of Wnt activity of the compound of Chemical Formula 1-1, TopFLASH luciferase assay was performed on the HEK293T cell line. The TopFLASH luciferase assay was performed by creating a HEK293T-TopFLASH stable cell line with a viral vector made by combining Luciferase (reporter gene) with the TCF7 promoter. HEK293T-TopFLASH cells were cultured in a 96-well plate (1?10.sup.4 cells/well), and the next day, the cells were treated with 1-50 ?M of the compound of Chemical Formula 1-1 and confirmed by One-Glo? (modified luciferin, Pormega, USA) after 24 hours. As shown in TopFLASH assay results of FIGS. 4 and 5, as a result of treatment with the compound of Chemical Formula 1-1 alone and simultaneous treatment with the compound of Chemical Formula 1-1 and Wnt3a-conditional media (2.5%), it was confirmed that as the luciferase values greatly increased, Wnt activity greatly increased when treated with Chemical Formula 1-1.

Experimental Example 2-3

Analysis of Changes in Wnt3a Signaling Protein

[0143] To confirm the Wnt3a signaling of Chemical Formula 1-1, the protein level of ?, a Wnt3a signaling protein, was measured in the 3T3-L1 cell line. The 3T3-L1 cells were cultured in a 6-well plate (2?10.sup.5 cell/well), and the next day, the cells were replaced with normal culture media (Undifferentiated media; UND) and differentiation induction media (Differentiation media; DM), respectively, followed by treatment with the compound of Chemical Formula 1-1 at 7.5 ?M for 48 hours, and then the cells were replaced with new normal culture media (Maintaining media; MM). Here, the compound of Chemical Formula 1-1 at a concentration of 7.5 ?M was cultured for 72 hours, followed by western blotting. In FIGS. 6 and 7, it was confirmed that the protein level of ? increased in the treatment of the compound of Chemical Formula 1-1 alone and the treatment of Chemical Formula 1-1 and Wnt3a together.

Experimental Example 2-4

Adipogenesis Inhibitory Effect Test (1)

[0144] The adipogenesis inhibitory activity of Chemical Formula 1 according to the present invention was measured through Oil Red O staining in mouse 3T3-L1 cell line. First, 3T3-L1 cells were placed in a 48-well plate (1?10.sup.4 cell/well) and cultured for 24 hours in a 37? C. CO.sub.2 incubator. The next day, the cells were replaced with the normal culture media (Undifferentiated media; UND) and the differentiation induction medium (Differentiation media; DM), respectively, followed by treatment with 2.5-10 ?M of Chemical Formula 1-1 for 48 hours. Then, the cells were replaced with the normal culture media (Maintainment media; MM), and cultured with 2.5-10 ?M of Chemical Formula 1-1 for 72 hours, and then treated with Oil Red O.

[0145] As a result, as shown in FIG. 8, it was confirmed that the compound of Chemical Formula 1-1 exhibited the adipogenesis inhibitory activity in a concentration-dependent manner.

[0146] In addition, the same experiment was conducted with the compound of Chemical Formula 1-1 at a fixed concentration of 7.5 ?M and Wnt3a-conditional media at 10-2.5%.

[0147] As shown in FIG. 9, it was confirmed that the adipogenesis inhibitory activity was excellent even when treated with the compound of Chemical Formula 1-1 even under the Wnt3a concentration condition.

Experimental Example 2-5

Adipogenesis Inhibitory Effect Test (2)

[0148] Additionally, in order to verify the adipogenesis inhibitory ability of the Chemical Formula 1 according to the present invention, the level of adipocyte differentiation-associated proteins in the 3T3-L1 cell line was measured. The 3T3-L1 cells were cultured in a 6-well plate (2?10.sup.5 cell/well), and the next day, the cells were replaced with normal culture media (Undifferentiated media; UND) and differentiation induction media (Differentiation media; DM), respectively, followed by treatment with the compound of Chemical Formula 1-1 at 7.5 ?M for 48 hours, and then the cells were replaced with new normal culture media (Maintaining media; MM). When replacement with normal culture media, the cells were treated with 7.5 ?M of Chemical Formula 1-1, and cultured for 72 hours more, followed by Western blotting. FIG. 10 shows the results of lower protein levels in differentiated adipocytes in the treatment with the compound of Chemical Formula 1-1 alone and simultaneous treatment with the compound of Chemical Formula 1-1 and Wnt3a-conditional media (2.5%), and it was confirmed that the level of adipogenesis induction-associated proteins was reduced when treated with the compound of Chemical Formula 1 according to the present invention.

[0149] In addition, real-time polymerase chain reaction (Q-PCR) was used to confirm the expression of adipogenesis-associated genes. As a result, it was confirmed that the amount of adipogenesis induction-associated mRNA was reduced when treated with the compound of Chemical Formula 1 according to the present invention (FIG. 11).

Experimental Example 2-6

Verification in Animal Models

[0150] C57BL/C6 mice were used to test the anti-obesity effect in a mouse model with respect to the compound of Chemical Formula 1 of the present invention, which is an adipogenesis-inhibiting compound. For effective evaluation of the anti-obesity model, the control group (general diet), the control group (high calorie diet), and an anti-obesity control group (high calorie diet+Chemical Formula 1 (15 mg/kg)) were compared and evaluated (FIG. 12). The mice injected with the high-calorie diet rather than the general diet gained 131% in body weight, but the mice treated with the high-calorie diet and Chemical Formula 1 (15 mg/kg) together lost 115% in body weight. In addition, there was a significant decrease in the measurement of liver weight and abdominal fat weight. In summary of these results, the Chemical Formula 1 was confirmed to have excellent anti-obesity activity in a mouse animal model (FIG. 12).

[0151] Then, after the experiment was completed, the animal blood of each group was collected and centrifuged to separate plasma, and then representative obesity markers in the blood such as total cholesterol (tCHO), high density lipoprotein (HDL), and low density lipoprotein (LDL), and liver damage markers such as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic acid transaminase (GPT) were analyzed with a blood chemistry analyzer. As a result, it was confirmed that when compared to the control group with a high calorie intake, tCHO, GOT, GPT, and LDL among the obesity and liver damage markers were significantly reduced, whereas HDL increased compared to the control group with a high calorie intake (FIG. 13).

[0152] It was found from these results that the compound represented by Chemical Formula 1 according to the present invention not only inhibits obesity, but also helps improve liver damage and fatty liver formation caused by obesity.

Statistical Analysis

[0153] All values are presented as mean value ?standard error (standard error of the mean: SEM).

Experimental Example 3

Analysis of Osteogenesis Properties of Bicyclic Compound According to Chemical Formula 1 of the Present Invention

Experimental Example 3-1

Cytotoxicity Assay

[0154] In order to measure the cytotoxicity of the compound represented by Chemical Formula 1 (Chemical Formula 1-1), toxicity was measured in the MC3T3-E1 cell line (Korea Cell Line Bank), which is a pre-osteoblast related to bone formation.

[0155] Specifically, a cytotoxicity test was performed to determine the in vivo safety of Chemical Formula 1-1 above. MC3T3-E1 cells, which are mouse osteoblasts, were inoculated into a 96-well plate at a concentration of 5?10.sup.3 cell/well, and incubated overnight in growth media containing alpha-minimum essential media (alpha-MEM, JBI #008-53) supplemented with 10% fetal bovine serum (FBS, Gibco #16000, USA) and 1? antibiotics (Gibco #15240062, USA). Then, the cells were exchanged with media added with compounds at the indicated concentrations (0, 2.5, 5, 10, 25 and 50 mM) on Day 3, and treated for 7 days. After incubation, the media were removed, 90 ?l of the media and 10 ?l of WTS solution were added, and after 1 hour, the absorbance was measured at 450 nm using a microplate reader. Results thereof are shown in FIG. 14.

[0156] As could be confirmed in FIG. 14, it was confirmed that the Chemical Formula 1-1 prepared in the present invention did not exhibit cytotoxicity in a concentration range of 50 ?M or less.

Experimental Example 3-2

Analysis of Osteogenesis Effect Through ALP Activity Analysis (1)

[0157] In order to analyze the effect of the compound of Chemical Formula 1 according to the present invention on ALP activity, the morphology of MC3T3-E1 cells according to the treatment with the compound of Chemical Formula 1 according to the present invention was confirmed by alkaline phosphatase (ALP) staining of the MC3T3-E1 cell line.

[0158] In the present Example, the effect of Chemical Formula 1-1 on the activity of alkaline phosphatase (ALP), which is one of the representative markers of osteoblast differentiation, was measured. MC3T3-E1 cells, which are mouse osteoblasts, were inoculated into a 96-well plate at a concentration of 5?10.sup.3 cell/well, and incubated overnight in growth media containing alpha-minimum essential media (alpha-MEM, JBI #008-53) supplemented with 10% fetal bovine serum (FBS, Gibco #16000, USA) and 1? antibiotics (Gibco #15240062, USA). As a negative control, an alpha minimum essential medium containing 10% FBS was used. As positive controls, 100 ?g/ml of ALP-activating ascorbic acid and 10 mM of ?-glycerophosphate were mixed, and the positive controls were exchanged with media added with 5, 10, and 20 ?M of Chemical Formula 1-1 on Day 3, and treated for 7 days. After 7 days, the media were discarded, and the cells were washed with PBS, and fixed with 10% formalin for 1 minute. Then, after washing twice with PBS, the cells were stained with a BCIP/NBT solution (Sigma). When the cells were properly stained, the solution was discarded, and the cells were washed with PBS and dried. The experimental results are shown in FIG. 15.

[0159] Referring to FIG. 15, as to Chemical Formula 1-1, alkaline phosphatase staining (ALP) positive cells were strongly stained in a concentration-dependent manner. It was confirmed from these results that the compound represented by Chemical Formula 1 according to the present invention was excellent in promoting osteogenesis.

Experimental Example 3-3

Analysis of Osteogenesis Effect Through ALP Activity Analysis (2)

[0160] In order to analyze the effect of the compound of Chemical Formula 1 according to the present invention on ALP activity, MC3T3-E1 cells, which are mouse osteoblasts, were inoculated into a 96-well plate at a concentration of 5?10.sup.3 cell/well, and incubated overnight in growth media containing alpha-minimum essential media (alpha-MEM, JBI #008-53) supplemented with 10% fetal bovine serum (FBS, Gibco #16000, USA) and 1? antibiotics (Gibco #15240062, USA). As a negative control, an alpha minimum essential medium containing 10% FBS was used. As a positive control, 100 ?g/ml of ALP-activating ascorbic acid and 10 mM of ?-glycerophosphate were mixed, and the positive control was exchanged with media added with 5, 10, and 20 ?M of Chemical Formula 1-1 (6-J) on Day 3, and treated for 7 days. After 7 days, the media were removed and the cells were washed with PBS, and the cultured MC3T3-E1 cells were lysed with 0.1% TritonX-100 (in PBS) solution and centrifuged to obtain only the supernatant. A predetermined amount of supernatant and ALP were added and reacted for 30 minutes, and then the absorbance was confirmed at 405 nm. The experimental results are shown in FIG. 16.

[0161] As could be seen in FIG. 16, it was confirmed that the compound of Chemical Formula 1 according to the present invention increased the ALP activity of MC3T3-E1 cells, thereby increasing the differentiation of osteoblasts, indicating that the compound of Chemical Formula 1 according to the present invention is effective in promoting bone growth.

Experimental Example 3-4

Analysis of Expression Level Increasing Effect of Osteogenic Differentiation Promoting Gene

[0162] In order to confirm the effect of the compound of Chemical Formula 1 according to the present invention on promoting osteogenic differentiation-related genes, the expression levels of genes promoting osteogenesis were confirmed.

[0163] To confirm genetic changes during the differentiation process, total RNA was isolated on Day 7 of differentiation using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. The cultured cells were washed twice with cold PBS and lysed with 1 ml of Trizol reagent. To the resulting product, 200 ?l of chloroform was added and mixed, followed by centrifugation at 4? C. and 12000 rpm for 20 minutes to separate the supernatant. The same amount of isopropanol was added to the separated supernatant, mixed, and centrifuged again at 4? C. and 12000 rpm. The supernatant was removed, and the remaining pellet was washed three times with 70% ethanol to separate RNA. For RT-PCR, the same amount of total RNA (5 ?l) was put into the RT-PCR amplification kit and reacted at 45? C. for 60 minutes to prepare complementary DNA (cDNA). The cDNA was amplified by RT-PCR using the primers shown in Table 3 below. RT-PCR was performed on the cDNA by using AccuPower?RT/PCR PreMix (Bioneer, Korea). Briefly, 2 ?l each of 75 mM rATP, rUTP, rCTP, rGTP, enzyme mixture, and 10? reaction buffer was added to 8 ?l of the cDNA solution prepared above, followed by amplification according to the manufacturer's instructions, and the quantity and quality of the amplified mRNA was evaluated using nanodrops and 1% agarose gel electrophoresis. Results thereof are shown in FIG. 17.

TABLE-US-00004 TABLE3 Anneal- ling Temper- ature Primer Sequence (?C.) mColla1 Forward 60 5-CGAAGGCAACAG TCGCTTCA-3 (SEQIDNO:2) Reverse 5-CCCAAGTTCCG GTGTGA-3 (SEQIDNO:3) mOsteocalcin Forward 60 5-CCTGAGTCTGAC AAAGCCTTCA-3 (SEQIDNO:4) Reverse 5-GCCGGAGTCTGT TCACTACCTT-3 (SEQIDNO:5) mOsterix Forward 58 5-AGCGACCACTTG AGCAAACAT-3 (SEQIDNO:6) Reverse 5-GCGGCTGATTGG CTTCTTCT-3 (SEQIDNO:7) Mgapdh Forward 60 5-CATGGCCTC CAAAGGAGTAAGA-3 (SEQIDNO:8) Reverse 5-GAGGGAGATGCT CAGTGTTGG-3 (SEQIDNO:9)

[0164] As could be seen in FIG. 17, the treatment with the compound of Formula 1-1 of the present invention was found to increase the mRNA expression level of osteogenic differentiation-related genes (mCol1a1, mOsteocalcin, mOsterix).

[0165] Therefore, it was found that the compound represented by Chemical Formula 1-1 according to the present invention had an excellent ability to promote osteogenic differentiation.

[0166] As described above, the present invention has been described in an exemplary manner, as an example, and various modifications may be made by those skilled in the art to which the present invention pertains without departing from the essential characteristics of the present invention. Therefore, exemplary embodiments disclosed in the present specification are intended to explain the present invention rather than limiting it, and the spirit and scope of the present invention are not limited by these exemplary embodiments. The protection scope of the present invention should be construed by the following claims, and all techniques within the equivalent range should be construed as being included in the scope of the present invention.