Benzylideneacetone derivative and use thereof

Abstract

The present invention relates to novel benzylideneacetone derivatives or uses thereof, more specifically, the present invention relates to a pharmaceutical composition for preventing or treating, or food composition for ameliorating a cancer or a bone disease comprising a compound defined by Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient.

Claims

1. A method for treating a bone cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Formula 1 below or the pharmaceutically acceptable salt thereof as an active ingredient: ##STR00037## wherein, R1 and R2 are different from each other, R1 and R2 are each independently selected from the group consisting of —H, —OH, —SH, C.sub.1-4 straight or branched alkyl, C.sub.1-4 straight or branched alkoxy, halogen, allyloxy, benzyloxy, aryloxy having one or more selected from the group consisting of hetero atoms and substituents, and heterocycloalkyl of 3 to 7 atoms having one or more hetero atoms, wherein the hetero atoms consist of 0, N and S; R3 is one selected from the group consisting of Ci-4 straight or branched alkyl, —NH.sub.2, —NHR4, —N(R4).sub.2, and —OH; and R4 is C.sub.1-4 straight or branched alkyl, wherein the compound defined by Formula 1 is selected from the group consisting of the following compounds; (1) (E)-4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one; (2) (E)-4-(3-hydroxy-4-methoxyphenyl)-3-buten-2-one; and (3) (E)-4-(3-fluoro-4-hydroxyphenyl)-3-buten-2-one.

2. A method for treating osteoporosis in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a compound defined by Formula 1 below or the pharmaceutically acceptable salt thereof as an active ingredient: ##STR00038## wherein, R1 and R2 are different from each other, R1 and R2 are each independently selected from the group consisting of —H, —OH, —SH, C.sub.1-4 straight or branched alkyl, C.sub.1-4 straight or branched alkoxy, halogen, allyloxy, benzyloxy, aryloxy having one or more selected from the group consisting of hetero atoms and substituents, and heterocycloalkyl of 3 to 7 atoms having one or more hetero atoms, wherein the hetero atoms consist of 0, N and S; R3 is one selected from the group consisting of Ci-4 straight or branched alkyl, —NH.sub.2, —NHR4, —N(R4).sub.2, and —OH; and R4 is C.sub.1-4 straight or branched alkyl, wherein the compound defined by Formula 1 is selected from the group consisting of the following compounds; (1) (E)-4-(3-hydroxy-4-methoxyphenyl)-3-buten-2-one; and (2) (E)-4-(3-fluoro-4-hydroxyphenyl)-3-buten-2-one.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result of NMR identification of (E)-4-(4-fluoro-3-hydroxyphenyl)but-3-en-2-one synthesized in Example 1-1.

(2) FIG. 2 shows the result of NMR identification of (E)-4-(3-hydroxy-4-methylphenyl)but-3-en-2-one synthesized in Example 1-2.

(3) FIG. 3 shows the result of NMR identification of (E)-3-(3-hydroxy-4-methylphenyl)acrylamide synthesized in Example 1-3.

(4) FIG. 4 shows the result of NMR identification of (E)-3-(4-fluoro-3-hydroxyphenyl)acrylamide synthesized in Example 1-4.

(5) FIG. 5 shows the result of NMR identification of (E)-4-(3-hydroxy-4-isopropoxyphenyl)but-3-en-2-one synthesized in Example 1-5.

(6) FIG. 6 shows the result of NMR identification of (E)-4-(4-benzyloxy-3-hydroxyphenyl)but-3-en-2-one synthesized in Example 1-6.

(7) FIG. 7 shows the result of NMR identification of (E)-4-(4-allyloxy-3-hydroxyphenyl)but-3-en-2-one synthesized in Example 1-7.

(8) FIG. 8 shows the results of measuring inhibitory effect of compounds including the compounds of the present invention, KP2 to KP8, and its positive controls, Fosamax and Osmundacetone, on osteoclast differentiation and proliferation, by TRAP assay (KP2: (E)-4-(3-hydroxy-4-methylphenyl)but-3-en-2-one, KP3: (E)-4-(4-fluoro-3-hydroxyphenyl)but-3-en-2-one, KP4: (E)-4-(4-hydroxy-3-methoxyphenyl)-3-buten-3-one, KP5: (E)-4-(3-hydroxy-4-methoxyphenyl)-3-buten-2-one, KP6: (E)-4-(3-fluoro-4-hydroxyphenyl)-3-buten-2-one, KP7: (E)-3-(3-hydroxy-4-methylphenyl)acrylamide, KP8: (E)-3-(4-fluoro-3-hydroxyphenyl)acrylamide)

MODE FOR CARRYING OUT THE INVENTION

(9) Hereinafter, the present invention will be described in detail with reference to the following examples.

(10) However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Synthesis of Novel Compounds

(11) In order to identify substances having anticancer activity and inhibitory effect on osteoclast differentiation, compounds were isolated and purified from the synthetic mixture of each compound, and the chemical structure of each compound was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS).

(12) Synthesis method of each compound and specific NMR and MS analysis results are as follows:

Example 1-1

Synthesis of (E)-4-(4-fluoro-3-hydroxyphenyl)but-3-en-2-one (KP3)

(13) TABLE-US-00001 TABLE 1

(14) Boron tribromide (1M methylene chloride solution, 10 mL) was gradually added dropwise to a methylene chloride (10 mL) solution of 3-methoxy-4-fluorobenzaldehyde (1a) (440 mg, 2.85 mmol) under ice-cooling. The reaction solution was agitated at room temperature for 5 hours after the dropping ended. The reaction solution was again cooled with ice, iced water was gradually added to the reaction solution to terminate the reaction, and further 5N hydrochloride solution was added until the pH reached 1. After condensing the reaction solution under reduced pressure, water and ethyl acetate were added to the residue, and the organic layer was separated. The obtained organic layer was washed with a saturated sodium chloride solution, and the solvent was evaporated under reduced pressure after dried over anhydrous magnesium sulfate. The obtained residue was purified by silica gel column chromatography (elution solvent:n-hexane-ethyl acetate 4:1), and 210 mg of 4-fluoro-3-hydroxybenzaldehyde (2a) was obtained.

(15) 4-fluoro-3-hydroxybenzaldehyde (2a) (140.11 mg, 1.0 mmol) and CuBr.sub.2 (223.35 mg, 1 mmol) in a pressured tube were dissolved in 5 mL acetone at room temperature. The reaction mixture was stirred at 60° C. After 6 h the mixture was cooled to room temperature, and filtered with Celite. The organic layer concentrated in vacuo, and product (E)-4-(4-fluoro-3-hydroxyphenyl)but-3-en-2-one (3a, 12%) as a brown solid was purified by flash chromatography using ethyl acetate and n-hexane (1:4) as an eluent.

(16) The results of NMR and MS are as follow (See, FIG. 1):

(17) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 7.56 (1H, d, J=16.4 Hz), 7.22 (1H, dd, J=7.6, 2.1 Hz), 7.13-7.10 (2H, m), 6.67 (1H, J=16.4 Hz), 2.38 (3H, s); Ms(ESI) m/z: 181.1 [M+H].sup.+;

Example 1-2

Synthesis of (E)-4-(3-hydroxy-4-methylphenyl)but-3-en-2-one (KP2)

(18) TABLE-US-00002 TABLE 2

(19) Boron tribromide (1M methylene chloride solution, 5 mL) was gradually added dropwise to a methylene chloride (5 mL) solution of 3-methoxy-4-methylbenzaldehyde (1b) (400 mg, 2.66 mmol) under ice-cooling. The reaction solution was agitated at room temperature for 5 hours after the dropping ended.

(20) The reaction solution was again cooled with ice, iced water was gradually added to the reaction solution to terminate the reaction, and further 5N hydrochloride solution was added until the pH reached 1. After condensing the reaction solution under reduced pressure, water and ethyl acetate were added to the residue, and the organic layer was separated. The obtained organic layer was washed with a saturated sodium chloride solution, and the solvent was evaporated under reduced pressure after dried over anhydrous magnesium sulfate. The obtained residue was purified by silica gel column chromatography (elution solvent:n-hexane-ethyl acetate 4:1), and 150 mg (41.4%) of 3-hydroxy-4-methylbenzaldehyde (2b) was obtained.

(21) 3-hydroxy-4-methylbenzaldehyde (2b) (136.1 mg, 1.0 mmol) and CuBr.sub.2 (223.35 mg, 1 mmol) in a pressured tube were dissolved in 5 mL acetone at room temperature. The reaction mixture was stirred at 60° C. After 6 h the mixture was cooled to room temperature, and filtered with Celite. The organic layer concentrated in vacuo, and product (E)-4-(3-hydroxy-4-methylphenyl)but-3-en-2-one 30 mg (3b, 17%) as a brown solid was purified by flash chromatography using ethyl acetate and n-hexane (1:4) as an eluent.

(22) The results of NMR and MS are as follow (See, FIG. 2):

(23) .sup.1H NMR (700 MHz, CD.sub.3OD): δ 7.56 (1H, d, J=16.24 Hz), 7.14 (1H, d, J=7.42 Hz), 7.03-7.02 (2H, m), 6.67 (1H, J=16.24 Hz), 2.38 (3H, s), 2.22 (3H, s); Ms(ESI) m/z: 177.1 [M+H].sup.+;

Example 1-3

Synthesis of (E)-3-(3-hydroxy-4-methylphenyl)acrylamide (KP7)

(24) TABLE-US-00003 TABLE 3 0

Step 1: Preparation of ethyl (E)-3-(3-methoxy-4-methylphenyl)acrylate

(25) Sodium hydride (60% dispersion oil prewashed in hexane, 300 mg, 7.5 mmol) was stirred in dry THF (10 mL) under an atmosphere of nitrogen. Triethyl phosphonoacetate (1.345 g, 6 mmol) in dry THF (3 mL) was added dropwise and stirred for 25 minutes. Then 3-methoxy-4-methylbenzaldehyde (1f, 751 mg, 5 mmol) in THF (3 mL) was added dropwise over a period of approximately 30 minutes.

(26) The resulting solution was stirred for 20 hours at room temperature. The reaction mixture was then quenched with water (100 mL) and was extracted with ethyl acetate (3×100 mL). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (ethyl acetate/n-hexane 1:4) afforded unsaturated ester (2f, 700 mg, 63%) as a yellow oil.

Step 2: Preparation of (E)-3-(3-methoxy-4-methylphenyl)acrylic acid

(27) ethyl (E)-3-(3-methoxy-4-methylphenyl)acrylate (2f, 220 mg, 1 mmol) dissolved in THF (5 mL) was added dropwise to sodium hydroxide (2.0 M, 5 mL) and heated to reflux for 2 hours. The mixture was quenched with water (10 mL) and acidified to pH 2 with hydrochloric acid (2.0 M). The solution was then extracted with ethyl acetate (3×100 mL), dried over sodium sulfate, filtered and concentrated in vacuo to yield (190 mg, 98.8%) as a white solid.

Step 3: Preparation of (E)-3-(3-methoxy-4-methylphenyl)acrylamide

(28) To a solution of (E)-3-(3-methoxy-4-methylphenyl)acrylic acid (3f, 100 mg, 0.52 mmol) and pyridine (0.1 mL) in dioxane (1 mL) was added di-tert-butyl dicarbonate (227 mg, 1.04 mmol) in one portion followed by ammonium bicarbonate (83 mg, 1.04 mmol) in one portion and the mixture was stirred at 60° C. for 2 hours.

(29) The reaction mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate and water. The ethyl acetate phase was washed with 5% aqueous sodium bicarbonate, 0.1 N HCl and brine. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo to (4f, 68 mg, 68.3%) as a yellow solid, used without further purification.

Step 4: Preparation of (E)-3-(3-hydroxy-4-methylphenyl)acrylamide

(30) Boron tribromide (1M methylene chloride solution, 5 mL) was gradually added dropwise to a methylene chloride (1 mL) solution of (E)-3-(3-methoxy-4-methylphenyl)acrylamide (4f, 40 mg, 0.21 mmol) under ice-cooling. The reaction solution was agitated at room temperature for 4 hours after the dropping ended.

(31) The reaction solution was again cooled with ice, iced water was gradually added to the reaction solution to terminate the reaction, and further 5N hydrochloride solution was added until the pH reached 1. After condensing the reaction solution under reduced pressure, water and ethyl acetate were added to the residue, and the organic layer was separated. The obtained organic layer was washed with a saturated sodium chloride solution, and the solvent was evaporated under reduced pressure after dried over anhydrous magnesium sulfate. The obtained residue was purified by silica gel column chromatography (elution solvent:methylene chloride-MeOH 20:1), and product (E)-3-(3-hydroxy-4-methylphenyl)acrylamide 5f (18 mg, 48.3%) as a yellow solid.

(32) The results of NMR and MS are as follow (See, FIG. 3):

(33) .sup.1H NMR (400 MHz, MeOD): δ 7.45 (1H, d, J=15.6 Hz), 7.10 (1H, d, J=7.6 Hz), 6.98-6.95 (2H, m), 6.53 (1H, J=15.6 Hz), 2.13 (3H, s); Ms(ESI) m/z: 178.1 [M+H].sup.+;

Example 1-4: Synthesis of (E)-3-(4-fluoro-3-hydroxyphenyl)acrylamide (KP8)

(34) TABLE-US-00004 TABLE 4

(35) By using 3-methoxy-4-fluorobenzaldehyde (1g) instead of 3-methoxy-4-methylbenzaldehyde in Step 1 of Example 1-3, yellow compound (E)-3-(4-fluoro-3-hydroxyphenyl)acrylamide (5g, 16 mg, 40%) was obtained in the same manner as Step 3 of Example 1-3.

(36) Specifically, 2g (680 mg, 60.6%) of yellow oil was obtained from 1g, 3g (183 mg, 93.2%) of white solid was obtained from 2g, and 4g (53 mg, 53.2%) of yellow solid was obtained from 3g, and 4g of novel compound (E)-3-(4-fluoro-3-hydroxyphenyl)acrylamide (5g, 16 mg, 40%) was obtained.

(37) The results of NMR and MS are as follow (See, FIG. 4):

(38) .sup.1H NMR (400 MHz, MeOD): δ 7.45 (1H, d, J=12.4 Hz), 7.15 (1H, dd, J=2.0, 2.0 Hz), 7.10-7.01 (2H, m), 6.52 (1H, d, J=16.0 Hz); Ms(ESI) m/z: 193.1 [M+H].sup.+

Example 1-5: Synthesis of (E)-4-(3-hydroxy-4-isopropoxyphenyl)but-3-en-2-one

(39) TABLE-US-00005 TABLE 5

Step 1: Preparation of 3-hydroxy-4-isopropoxybenzaldehyde

(40) A stirred suspension of 3,4-dihydrobenzaldehyde (250 mg, 1.81 mmol) and anhydrous potassium carbonate (250 mg, 1.81 mmol) in dry dimethylformamide (3 ml) was heated to 70° C. and 2-bromopropane (0.17 ml, 1.81 mmol) added dropwise under nitrogen during 30 min. The mixture was stirred for overnight at room temperature and then poured into ice water (50 ml). The aqueous mixture was extracted with ethyl acetate, and the combined extracts were washed with water, brine, and evaporated under vacuum to give a brown oil. The obtained residue was purified by silica gel column chromatography (ethyl acetate/n-hexane 1:4), and product (2h, 46 mg, 50%) as a brown solid.

Step 2: Preparation of (E)-4-(3-hydroxy-4-isopropoxyphenyl)but-3-en-2-one

(41) 3-hydroxy-4-isopropoxybenzaldehyde (2h) (50 mg, 0.277 mmol) and CuBr.sub.2 (62 mg, 0.277 mmol) in a pressured tube were dissolved in 3 mL acetone at room temperature. The reaction mixture was stirred at 60° C. After 6 h the mixture was cooled to room temperature, and filtered with Celite. The organic layer concentrated in vacuo, and product (E)-4-(3-hydroxy-4-isopropoxyphenyl)but-3-en-2-one 33 mg (3h, 53.3%) as a white solid was purified by flash chromatography using ethyl acetate and n-hexane (1:4) as an eluent.

(42) The results of NMR and MS are as follow (See, FIG. 5):

(43) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.44 (1H, d, J=16.0 Hz), 7.17 (1H, d, J=2.4 Hz), 7.05 (1H, dd, J=8.4 Hz, 6.0 Hz), 6.87 (1H, d, J=8.4 Hz), 6.60 (1H, d, J=16.0 Hz), 5.74 (1H, s), 4.67 (1H, hept, J=6.4 Hz), 2.37 (3H, s), 1.41 (6H, d, J=6.0 Hz)); Ms(ESI) m/z: 221.1 [M+H].sup.+

Example 1-6

Synthesis of (E)-4-(4-(benzyloxy)-3-hydroxyphenyl)but-3-en-2-one

(44) TABLE-US-00006 TABLE 6

(45) 4-(benzyloxy)-3-hydroxybenzaldehyde as the target compound (2i) was obtained by the same method as in Example 1-5, using benzyl bromide instead of 2-bromopropane in Step 1 of Example 1-5.

(46) 4-(benzyloxy)-3-hydroxybenzaldehyde (2i, 50 mg, 0.22 mmol) and CuBr.sub.2 (48.9 mg, 0.22 mmol) in a pressured tube were dissolved in 3 mL acetone at room temperature. The reaction mixture was stirred at 60° C. After 6 h the mixture was cooled to room temperature, and filtered with Celite. The organic layer concentrated in vacuo, and product (E)-4-(4-(benzyloxy)-3-hydroxyphenyl)but-3-en-2-one (3i, 37 mg, 62.6%) as a white solid was purified by flash chromatography using ethyl acetate and n-hexane (1:4) as an eluent.

(47) The results of NMR and MS are as follow (See, FIG. 6):

(48) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46-7.42 (6H, m), 7.19 (1H, d, J=2.0 Hz), 7.05 (1H, dd, J=8.4 Hz, 2.4 Hz), 6.95 (1H, d, J=8.4 Hz), 6.95 (1H, d, J=8.4 Hz), 6.61 (1H, d, J=16.0 Hz), 5.71 (1H, s), 5.17 (2H, s), 2.37 (3H, s); Ms(ESI) m/z: 269.2 [M+H].sup.+

Example 1-7

Synthesis of (E)-4-(4-(allyloxy)-3-hydroxyphenyl)but-3-en-2-one

(49) TABLE-US-00007 TABLE 7

(50) 4-(allyloxy)-3-hydroxybenzaldehyde as the target compound (2j) was obtained by the same method as in Example 1-5, using bromopropene instead of 2-bromopropane in Step 1 of Example 1-5.

(51) 4-(allyloxy)-3-hydroxybenzaldehyde (2j, 50 mg, 0.28 mmol) and CuBr.sub.2 (62.7 mg, 0.28 mmol) in a pressured tube were dissolved in 3 mL acetone at room temperature. The reaction mixture was stirred at 60° C. After 6 h the mixture was cooled to room temperature, and filtered with Celite. The organic layer concentrated in vacuo, and product (E)-4-(4-(allyloxy)-3-hydroxyphenyl)but-3-en-2-one (3j, 34 mg, 55.6%) as a white solid was purified by flash chromatography using ethyl acetate and n-hexane (1:4).

(52) The results of NMR and MS are as follow (See, FIG. 7):

(53) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.43 (1H, d, J=16.4 Hz), 7.18 (1H, d, J=2.4 Hz), 7.04 (1H, dd, J=8.4 Hz, 2.0 Hz), 6.87 (1H, d, J=8.0 Hz), 6.60 (1H, d, J=16.0 Hz), 6.11-6.04 (1H, m), 5.80 (1H, s), 5.43 (1H, dd, J=17.2 Hz, 12.8 Hz), 5.36 (1H, dd, J=10.4 Hz, 8.0 Hz), 4.67-4.65 (2H, m), 2.37 (3H, s); Ms(ESI) m/z: 219.2 [M+H].sup.+;

Example 2

(54) MTT Assay

(55) The cytotoxicity against normal cells and cancer cells was confirmed using the MTT assay for the novel compounds KP2 to KP8 prepared in Example 1.

(56) MTT assay method is as follows:

(57) Normal cells (RAW264.7 rat macrophage cell line (osteoblast progenitor cell) and NIH3T3 mouse fibroblast line) and cancer cells (AGS human gastric cancer cell line, A549 human lung cancer cell line, HepG2 human liver cancer cell line, HCT116 human colon cancer cell line, PC3 human prostate cancer Cell line, Caki-1 human kidney cancer cell line, T24 human bladder cancer cell line, HT1080 human fibrosarcoma cell line, B16F10 mouse melanoma cell line) were cultured in 5% CO2 in 96-well plates containing DMEM with 10% FBS (fetal bovine serum). After incubation, KP2 to KP8 and controls (osmundacetone and 4-hydroxybenzalacetone) were added to the cell medium and incubated for 48 hours. Thereafter, 100 μl of MTT (0.5 mg/mL phosphate buffered saline) was added, followed by incubation for 2 hours. 100 μl of DMSO was added to each well, followed by incubation for 10 minutes, and the absorbance was measured at 550 nm using a microplate reader (SPCTRA MAX 340PC, Molecular Devices, USA). Absorbance is calculated by the formula below as an indicator of the number of cells that survived, and reproducibility was confirmed by three experiments.

(58) The rate of cell proliferation (%)=OD550(sample)/OD550(control)

(59) TABLE-US-00008 TABLE 8 The compounds of Formula 1 KP2 4-(3-Hydroxy-4-methylphenyl)-3-buten-2-one KP3 4-(4-fluoro-3-hydroxyphenyl)-3-buten-2-one KP4 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one KP5 4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one KP6 4-(3-Fluoro-4-hydroxyphenyl)-3-buten-2-one KP7 4-(3-Hydroxy-4-methylphenyl)acrylamide KP8 4-(4-fluoro-3-hydroxyphenyl)acrylamide

(60) TABLE-US-00009 TABLE 9 The result of MTT assay for normal cells with the compoumds LD.sub.50 (μM) Normal cells Compound RAW264.7 NTH3T3 Osmundacetone 529 ± 41 >5000 4-Hydroxybenzalacetone 294 ± 45 201 ± 10 KP2 395 ± 22 688 ± 12 KP3 503 ± 57 >5000 KP4 399 ± 42 >5000 KP5 261 ± 79 >5000 KP6 1110 ± 48  >5000 KP7 >5000 >5000 KP8 >5000 >5000

(61) As can be seen in Table 9, the LD50 for normal cells calculated based on the change in cell proliferation rate after administration of KP2 to KP8 was similar to that of Osmundacetone's LD50. Only KP2 showed weak cytotoxicity. In the macrophage cell line RAW264.7, KP2 to KP5 showed weak cytotoxicity, suggesting that these compounds have weak immunosuppressive functions. Therefore, it can be seen that the compounds of the present invention having low cytotoxicity to normal cells as a whole can be safely used in pharmaceutical and food compositions.

(62) TABLE-US-00010 TABLE 10 The result of MTT assay for cancer cells with the compounds LD.sub.63 (μM) Cancer cell Compound AGS A549 HepG2 HCT116 PC3 Caki-1 T24 HT1080 H16F10 Osmund 56.1 ± 2.1 >5000 >5000 689 ± 27 60.6 ± 14  695 ± 14 2000 ± 250 >5000  64.9 ± 59 acetone KP2  209 ± 5.9  471 ± 24  339 ± 79 380 ± 32  309 ± 3.1  387 ± 57 1120 ± 67  256 ± 14  298 ± 27 KP3  239 ± 14 1090 ± 46  612 ± 95 389 ± 18  387 ± 48  408 ± 12 >5,000  379 ± 10  278 ± 15 KP4  417 ± 15 3010 ± 38 — 495 ± 13  223 ± 85  506 ± 13 >5000 >5,000  423 ± 39 KP5  181 ± 15 1890 ± 210 — 491 ± 72  291 ± 5.7  409 ± 11 >5000  381 ± 49  441 ± 58 KP6  336 ± 3.9  901 ± 28 — 490 ± 38  205 ± 13  449 ± 40 3610 ± 130 >5,000  421 ± 41 KP7  329 ± 14  989 ± 50  379 ± 38 315 ± 19  394 ± 140  361 ± 16 1040 ± 360  309 ± 10 1210 ± 110 KP8  569 ± 4.8  935 ± 10 1280 ± 390 >5,000 >5,000 2990 ± 58  203 ± 69 1090 ± 19 >5,000

(63) As can be seen in Table 10, the LD50 of the cancer cells of the compound calculated based on the change in cell proliferation rate after administration of KP2 to KP8 showed stronger anticancer activity in some cancers compared to the LD50 of Osmundacetone.

(64) Specifically, for A549 lung cancer cells, KP2 showed significantly better cancer cell inhibitory activity than Osmundacetone, and in HepG2 hepatocellular carcinoma cells, KP2 and KP7 showed significantly better cancer cell inhibitory activity, and in HP116 colorectal cancer cells, it was confirmed that KP2, KP3 and KP7 showed 2 times better inhibitory activity than Osmundacetone except for KP8. All other compounds except KP8 in Caki-1 kidney cancer cells were found to have superior inhibitory activity than Osmundacetone. In T24 bladder cancer cells, KP8 showed particularly better inhibitory activity. Finally, the inhibitory activity of the compounds except KP80 on HT1080 fibrosarcoma cancer cells were all superior to Osmundacetone, a positive control.

(65) Therefore, it was confirmed that the inhibitory activity of the compounds of the present invention on lung cancer, colorectal cancer, kidney cancer, bladder cancer, and fibrosarcoma cancer cells was much better than or similar to that of Osmundacetone, and thus the compounds of the present invention can be usefully used as anticancer agents.

Example 3

(66) Inhibitory Activity Against Proliferation and Differentiation of Osteoclast

(67) For the compounds prepared in Example 1, the inhibitory activity against proliferation and differentiation of osteoclast was confirmed by using a osteoclast specific staining method, TRAP assay (tartrate-resistant acid phosphatase).

(68) The specific method of the TRAP assay is as follows:

(69) 1. Bone Marrow Cell Culture

(70) The tibia and femur were aseptically excised from 6 to 8 weeks old male C57BL/6 mice, and bone marrow cells were collected aseptically with a syringe (21G, Korea Green Cross). Bone marrow cells were suspended in 500 μl of -MEM medium (Gibco BRL Co.) containing sodium bicarbonate (2.0 g/L), streptomycin (100 mg/L) and penicillin (100,000 units/mL), and aliquoted into a 48-well plate, and assay was carried out with triplicate. The progenitor cells of osteoclasts, monocytes, were isolated and treated with RANKL and M-CSF, which are differentiation promoters, to differentiate into osteoclasts within 5-7 days.

(71) 2. Measurement of Osteoclast Differentiation

(72) 1) Sample preparation: KP2 to KP8 (0.5 μM, 1 μM, 5 μM or 10 μM), Fosamax (0.5 μM, 1 μM, 5 μM, or 10 μM) and the like were dissolved in DMSO (dimethylsulfoxide) or sterile distilled water at appropriate concentrations, respectively. Compounds were prepared in the same manner as in Example 1.

(73) 2) Sample administration: The Samples were continuously administered to the medium at 1:20 (v/v; 25 μL of sample per 500 μL of medium) from the first day of culture of bone marrow cells, and the medium was replaced at 3 days intervals.

(74) 3) Osteoclast differentiation measurement: Osteoclasts were defined as TRAP-positive multinucleated cells stained with TRAP. TRAP staining solution was prepared by dissolving 5 mg of naphthol AS-MS phosphate (Sigma N-4875) and 25 mg of Fast Red Violet LB salt as a coloring reagent in about 0.5 mL of N,N-dimethylformamide, and then mixing with 0.1N NaHCO.sub.3 buffer solution (50 mL) containing 50 mM tartaric acid. The reaction reagents were stored in the refrigerator until use.

(75) Bone marrow cells were cultured in a medium containing differentiation factors for 6-7 days, and then the medium was removed, washed with PBS, and fixed in PBS containing 10% formalin for 2-5 minutes. After fixed for 1 minute with a 1:1 mixture of ethanol and acetone, and dried. The fixed cells were treated with TRAP staining solution for 60 minutes at 37° C. with light blocking, washed with PBS, and then stained with Hematoxylin.

(76) Among the TRAP-positive cells in the microscope field, cells with two or more nuclei were determined as osteoclasts and the number of cells was measured. The osteoclast differentiation inhibitory activity of the compound was calculated by IC50, 50% inhibitory concentration compared to the control.

(77) As can be seen in FIG. 8, the groups treated with KP2 to KP8 compound not only significantly inhibited the formation of giant osteoclasts, multinucleated cells, similar to the positive control groups (groups added with Osmundacetone or Fosamax to the culture medium), but also, in the case of KP2, the IC50 value was about three times better than the group added with Osmundacetone. In the case of KP5, IC50=1 μM showed strong osteoclast inhibitory activity, that is about 40 times of Fsamax (IC50=4 μM), the most widely used therapeutic agent for osteoporosis, and 80 times of osmundacetone (IC50=8 μM). In addition, the proliferation of osteoclast progenitor cells was also significantly suppressed, and the effect of inhibiting proliferation as well as the differentiation of osteoclasts was also found to be significant.

Example 4

(78) Effect on Proliferation and Differentiation of Osteoblast

(79) Effect of KP4 to KP6 on proliferation and differentiation of osteoblast were confirmed using ALP (Alkaline phosphatase) assay, a method for measuring osteoblast activity.

(80) Alkaline phosphatase (ALP), which is present in the cell membranes of osteoblasts, is known as a marker of osteoblast activity and is a regulator of inorganic phosphate transport, cell division or differentiation during calcification. Therefore, ALP activity was measured to determine the effect of the compounds of the present invention on the activity of osteoblasts. ALP activity was indirectly calculated by measuring the amount of p-nitrophenol, which is produced from hydrolysis of p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, Mo., USA) in which ALP act as a catalyst, using a microplate reader at 405 nm wavelength.

(81) Specifically, osteoblasts (mouse MC3T3-E1) were aliquoted into a 96-well plate (3×10.sup.3 cells/100 μL/well) with α-MEM medium containing ascorbic acid (50 μg/mL) and 10 mM β-glycerophosphate, a differentiation factors of osteoblast. Then, KP4 to KP6 of the present invention, and osmundacetone were added at a final concentration of 10 μM and 50 μM, followed by changing medium every 72 hours and incubating for 14 days. After 14 days, the culture medium was removed from each well, washed three times with PBS, lysed with 0.2% Nonidet P-40/10 mmol/L MgCl.sub.2, and then treated ultrasonic wave with sosonifier cell disrupter (Model W-380, Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.) for 3 minutes. The cell lysate was centrifuged at 1500 g for 10 minutes and the supernatant was collected to measure ALP activity. To account for changes in ALP activity according to cell number differences, total protein levels were measured using bicinchoninic acid (BCA) protein assay kit with bovine serum albumin as the standard protein, and enzyme activity was expressed as a percentage against the control group without the compound treatment.

(82) As shown in Table 11, KP4 and KP5 showed higher ALP activity at 10 μM and 50 μM concentrations than the control without the treatment of compounds, especially when KP4 ((E)-4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) was added at a concentration of 50 μM, the ALP activity was increased by 400% or more compared with the control group, and it was confirmed that the osteoblast activity was increased 1.5 times compared to osmundacetone treat group. These were very good results compared to the osteoblast activators used in the treatment of osteoporosis in the existing clinical practice. On the other hand, KP6 showed good activity on inhibiting osteoclast differentiation (IC50=6 μM), but there was no activity on activating osteoblast.

(83) TABLE-US-00011 TABLE 11 Measurement of ALP activity % Activation of ALP activity COMPOUNDS 10 μM 50 μM Osmundacetone 115 ± 9.4 279 ± 61  KP4 113 ± 2.9 422 ± 6.9 KP5 103 ± 1.1 175 ± 0.3 KP6 102 ± 0.2 100 ± 0.8

INDUSTRIAL APPLICABILITY

(84) Since the novel compounds according to the present invention exhibit a strong inhibitory activity on proliferation and differentiation of osteoclasts that cause bone loss in addition to cancer cell specific cytotoxicity, it can be usefully used for developing safe and effective anti-cancer agents, and therapeutic agents for preventing and treating or foods for ameliorating bone diseases such as osteoporosis, and the like.