KR102007282B1 - Antioxidant composition comprising marliolide derivatives - Google Patents

Abstract

The present invention relates to a marliolide derivative and an antioxidant composition containing the same as an active component, wherein the marliolide derivative increases the expression of a transcription factor Nrf2 that regulates the expression of an antioxidant-related protein without cytotoxicity, thereby increasing the expression of HO-1 and NQO1, which are antioxidant-related proteins. In addition, since the marliolide derivative is confirmed to inhibit the oxidation of DNA and lipids induced by TPA that induces oxidative stress, a composition containing the marliolide derivative can be provided as the antioxidant composition.

Description

Antioxidant composition containing marolilide derivatives as an active ingredient

It relates to a maleilide derivative and an antioxidant composition containing the same as an active ingredient.

Our body protects itself from external physical, chemical, and biological stimuli by constantly maintaining homeostasis, removing risks, and discharging.

Among them, oxidative stress caused by free radical molecules such as O ₂ ^- and H ₂ O ₂ can cause aging, and the continuous exposure to these free radical molecules can lead to various diseases including cancer. Preventing exposure to molecules and discharging them out of the body is very important for antioxidants, anti-aging and anticancer.

Nrf2 is a contrasting transcription factor that regulates the expression of genes related to antioxidants. In general, Nrf2 binds to a dimer of a protein called Keap1 through two domains, and is constantly ubiquitinated by the Keap1-Cul3 E3 ligase system. And decomposes.

However, exposure to oxidative stress alters Keap1-Cul3 structure, which inhibits the ubiquitination of Nrf2, thereby disrupting Nrf2 degradation and translocating to the nucleus of cells to regulate the expression of antioxidant proteins HO-1 and NQO1. To induce removal and release of oxidative stress.

Heme oxygenase (HO) is an enzyme that removes heme from cells and blood by biliverdin, and HO is two types of isoforms, HO-1 and HO- Unlike HO-2, which is present at 2 and is always expressed, HO-1 is significantly increased for skin protection when exposed to oxidative stress.

Since HO-1 is known to promote wound healing as well as antioxidant activity in the skin, a substance capable of modulating HO-1 enzyme activity may be developed as an excellent antioxidant.

Republic of Korea Patent Publication No. 2002-0073776 (published Sep. 28, 2002)

The present invention is to provide a composition containing the malolilide derivative as an active ingredient as an antioxidant composition by identifying a maleoliolide derivative that can exhibit excellent antioxidant activity by controlling HO-1 enzyme activity.

The present invention provides a maleolilide derivative represented by the formula (1).

[Formula 1]

In another aspect, the present invention provides a cosmetic composition for antioxidant containing a malolilide derivative represented by the formula (1) as an active ingredient.

[Formula 1]

According to the present invention, the maleolilide derivatives increased expression of the antioxidant factors HO-1 and NQO1 by increasing the expression of the transcription factor Nrf2, which regulates the expression of antioxidant-related proteins without cytotoxicity, and increased oxidative stress. As it has been found to inhibit the oxidation of DNA and lipids induced by TPA, a composition containing a malolilide derivative as an active ingredient may be provided as an antioxidant composition.

1 is a result of confirming cytotoxicity in HaCaT cells, MTT assay results confirmed the cytotoxicity after treatment with the maleolilide derivative compound 1 at concentrations of 0, 0.1, 1 and 10 μM for 24 hours.
Figure 2 shows the luciferase activity in the HaCaT-ARE-Luc-reporter cell line as a result, the luciferase activity in the dual-glo system every 12 and 24 hours after treatment with the compound 1 and resveratrol, a maleilide derivative at a concentration of 10 μM This is the result of checking.
Figure 3 is a result of confirming the HO-1, NQO1 and Nrf2 protein expression level in HaCaT cells, Western blot analysis to determine the protein expression level at each time point after treatment with a concentration of 10 μM compound 1 and resveratrol, a malolilide derivative As a result, actin was used as a loading control.
4 is a result of confirming the HO-1 and NQO1 mRNA expression level in HaCaT cells, it is a real-time PCR analysis result to confirm the relative mRNA expression level for each time point after the treatment with malolilide derivative compound 1 10 μM concentration.
5 is a result of confirming the TPA-induced DNA oxidation inhibitory effect in HaCaT cells, after 4 hours of treatment with TPA 100 nM alone or in combination with 10 gM of the maleilide derivative compound 1 confirmed the inhibitory effect on DNA oxidation Immunofluorescence analysis results.
6 is a result confirming the TPA-induced lipid oxidation inhibitory effect in HaCaT cells, after 4 hours of treatment with TPA 100 nM alone or in combination with 10 mg of the malolilide derivative compound 1 confirmed the inhibitory effect on lipid oxidation Immunofluorescence analysis results.

Hereinafter, the present invention will be described in more detail.

The present invention can provide a maleolilide derivative represented by the formula (1).

[Formula 1]

The maleolilide derivative represented by Formula 1 may increase the expression of antioxidant proteins Nrf2, HO-1 and NQO1.

The maleolilide derivative represented by Chemical Formula 1 may inhibit DNA oxidation and lipid oxidation.

According to an embodiment of the present invention, HaCaT cells were inoculated at 1 × 10 ⁶ in a 100π plate in order to compare the expression level change of the antioxidant-related proteins Nrf2, HO-1 and NQO1 with resveratrol following the compound 1 treatment in HaCaT cells. And incubated for 24 hours. Thereafter, the compound 1 and resveratrol were each treated at a predetermined time of 10 μM, and then Western blot was performed. As a result, the expression duration of the antioxidant proteins by FIG. 3 and compound 1 was longer than that of resveratrol.

To determine whether increased expression of antioxidant-related proteins by Compound 1 confirmed in previous experiments could inhibit oxidative stress, TPA (100 nM), a representative chemical that causes oxidative stress in HaCaT cells, alone or in combination After treatment with 1 (10 μM) and incubated for 4 and 8 hours and confirmed the fluorescence image, the DNA oxidation and lipid oxidation increased as shown in Figs. 5 and 6, respectively 8-OHdG and 4- oxidation markers While increased expression of HNE was confirmed, the increase in expression of the 8-OHdG and 4-HNE was inhibited in the experimental group treated with Compound 1, so that Compound 1 effectively inhibited DNA oxidation and lipid oxidation. there was.

Accordingly, the present invention can provide an anti-oxidant cosmetic composition containing a maleilide derivative represented by the formula (1) as an active ingredient.

[Formula 1]

The cosmetic composition comprises a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. Can have

The cosmetic composition may include conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavors, and a carrier, in addition to the maleilide derivative which is an active ingredient.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, oils, powder foundations, emulsion foundations, It may be formulated as a wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a sun cream, a flexible lotion, a convergent lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a pack, a spray or a powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide may be used as a carrier component. .

If the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray additionally chlorofluorohydrocarbon, propane / butane Or propellants such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizer or emulsion is used as carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 Fatty acid esters of butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose as carrier components , Aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

< Synthesis Example > Maliolide  Derivative Compound 1 Synthesis

Scheme 1

750 kg (500 mol, 1.0 equiv) of D-ribose was dissolved in 207 L of water at room temperature, then cooled to 10-15 ° C. and 82.5 kg (597 mol, 1.2 equiiv) of solid potassium carbonate were added and the temperature was 10 It was kept at -15 ° C.

The reaction solution was cooled to 0-5 ° C. and then 87.4 kg (547 mol, 1.1 equiiv) of bromin was added, followed by stirring for 3-4 hours. After the bromine addition was complete, the reaction mixture was stirred at rt overnight.

Thereafter, 88% formic acid (25 L) was added thereto to adjust the pH to 2-3, and the acidic solution was titrated at 505 DEG C under vacuum to obtain a brown solid.

The brown solid was dissolved in 500L of acetone (1185kg, 20403mol, 41equiv) and 7.5L of sulfuric acid (Sulfuric acid) was heated to a boiling point under nitrogen condition for 4 hours. Thereafter, the reaction mixture was cooled to 20-30 ° C. and maintained at temperature for 8 hours.

The pH of the reaction mixture was adjusted to 5.5-6.0 using sodium hydroxide, filtered, and titrated at 40 ° C. in vacuo to give a solid product.

The crude solid product was dissolved by stirring at 250 ° C. with 250 L of ethyl acetate, then the solution was filtered and titrated in vacuo by 50% of the initial volume.

The solution was then cooled to -5 ° C to give solid compound 9, which was purified by filtration and drying in 35 ° C vacuum.

To 50 kg (266 mol, 1 equiiv) of the solution of Compound 9, 750 L of dichloromethane and 29.8 kg (41 L, 294 mol, 1.1 equiiv) of triethylamine were added under nitrogen.

After stirring at room temperature until the solution becomes clear, it is cooled to -20 ° C, and 33.3 kg (22.5L, 291mol, 1.1equiv) of methanesulfonyl chloride is slowly added to the solution at -20 ° C. After stirring for an hour, the temperature of the solution was raised to room temperature and maintained for 8 hours.

400L of water was added to the solution to stop the reaction, the organic layer was separated, and the aqueous layer was extracted with 400L of dichloromethane.

400 L of water was added to the separated organic layer, washed, and titrated under vacuum at 30 ° C. to obtain compound 10 in a semi-solid mesylate state.

36.5 kg (651 mol, 2.44 equiv) / water 245 L of potassium hydroxide was added to the crude mesylate and maintained at 30 ° C. or lower.

After stirring the solution at the same temperature for 4 hours, the pH was adjusted to 2.5-3.0 with 33.5 L of 3M hydrochloric acid, and the acidic solution was titrated at 45 ° C. under vacuum.

The solid thus obtained was dissolved in 300 L of acetone, heated to boiling point, acetone was removed, dried over 35 kg of sodium sulfate, and filtered.

The filtrate filtered in the above process was titrated at 35 ° C. or lower in vacuo to confirm the yield of the crude product, and white crystals (3) were obtained from 100 L 2-propanol.

Compound 9 NMR: IR (KBr): 3455, 1762 cm-1. 1 H NMR (300 MHz, CDCl 3): δ 1.36 (s, 3H, CH 3), 1.41 (s, 3H, CH 3), 2.95 (brs, 1H, OH), 3.75-3.80 (dd, 1H, C-5 ′ H), 3.95-3.99 (dd, 1H, C-5H), 4.60-4.62 (m, 1H, C-4H), 4.74-4.78 (d, 1H, C-3H), 4.80-4.83 (m, 1H, C-2H).

Compound 3 NMR: IR (KBr): 3423, 1778 cm-1. 1 H NMR (300 MHz, CDCl 3): δ 1.36 (s, 3H, CH 3), 1.44 (s, 3H, CH 3), 2.72 (brs, 1H, OH), 3.75-3.77 (d, 1H, C-5 ′ H), 3.91-4.01 (m, 2H, C-5H and C-5′H), 4.60-4.64 (m, 1H, C-4H), 4.83-4.89 (m, 2H, C-3H and C-2H ). Anal. Calcd for C 8 H 12 O 5: C, 51.06; H, 6.38. Found: C, 50.96; H, 6.44.

Scheme 2

250 mg (1.33 mmol) of Compound 3 was dissolved in 5 ml of dry toluene, mixed with 280 mg (1.54 mmol) of phenyl selenocyanate, and left under N ₂ inert conditions.

The mixture was cooled to −20 ° C., and 5 ml of toluene, in which 0.38 ml (1.54 mmol) of tributylphosphine was dissolved, was added dropwise, followed by stirring at −20 ° C. overnight.

The mixture was stirred at 0 ° C. again for 4 h and then the solvent was evaporated.

The crude product obtained in the above procedure was purified by silica gel column chromatography to obtain compound 4.

Compound 4 NMR: ¹ H NMR (400 MHz, CDCl 3), δ 7.58 (m, 2H), 7.30-7.26 (m, 3H), 4.89 (t, J = 5.2 Hz, 1H), 4.78 (d, J ¼ 5.2 Hz, 1H), 4.57 (m, 1H), 3.27 (d, J = 6.4 Hz, 2H), 1.46 (s, 3H), 1.34, (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 173.8, 133.3, 129.3, 128.5, 127.8, 114.1, 78.4, 76.3, 76.0, 26.8, 25.9, 24.7; IR (neat) 1781.15, 1374.94, 1178.29, 1085.72 cm ⁻¹ ; LCMS (ESI) m / z calcd for C ₁₄ H ₁₆ O ₄ Se [M + H] ⁺ : 329.02, found: 329.10.

Scheme 3

At room temperature, 140 mg (0.426 mmol) of Compound 4 were dissolved in 5 ml of dry toluene and nitrogen was removed for 15 minutes.

Then, 7 mg (0.0426 mmol) of azobisisobutyronitrile and 0.34 ml (1.278 mmol) of tri-n-butyltin hydride were added thereto, and the mixture was stirred at 130 ° C. for 5 hours. The reaction was carried out.

The crude product obtained through the above procedure was cooled to room temperature and subjected to silica gel column chromatography without evaporating toluene to obtain compound 5.

Compound 5 NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.82 (d, J = 5.2 Hz, 1H), 4.70 (t, J = 4.0 Hz 1H), 4.61 (m, 1H), 1.49 (d, J = 6.8 Hz, 6H), 1.40 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 174.0, 114.0, 26.7, 25.9, 14.4; IR (neat) 1771.55, 1180.4, 1086.27, 913.72 cm_1; LCMS (ESI) m / z calcd for C ₈ H ₁₂ O ₄ [M + H] ⁺ : 173.07, found: 173.25.

Scheme 4

240 mg (1.394 mmol) of the compound 5 were dissolved in 2.5 ml of 1 M HCl, and then stirred at room temperature for 5 hours.

The solvent in the solution was evaporated in vacuo and the crude product was then evaporated 2-3 times with toluene to remove the residue to give a white solid compound 6.

Compound 6 NMR: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 5.73 (d, J = 7.6 Hz, 1H), 5.30 (d, J = 4.0 Hz, 1H), 4.48-4.41 (m, 2H), 4.04 (m, 1 H), 1.24 (d, J = 6.4 Hz, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 176.8, 76.4, 71.4, 70.9, 14.4; IR (neat) 3440.41, 3295.40, 1749.97, 1159.12, 1031.55, 972.87 cm ⁻¹ ; LCMS (ESI) m / z calcd for C ₅ H ₈ O ₄ [M + H] ⁺ : 133.04, found: 133.25.

Scheme 5

500 mg (3.78 mmol) of Compound 6 and 20 ml of CH ₂ Cl ₂ were mixed and cooled to 0 ° C., followed by 0.6 ml (4.33 mmol) of phenylchlorothinoformate and 1.2 ml (14.8 mmol) of pyridine. Was added and stirred at room temperature for 3 hours to react.

The reaction mixture was diluted with CH ₂ Cl ₂ and washed with water, the organic layer was dried over MgSO ₄ , filtered and evaporated.

Azobisisobutyronitrile 30 mg (0.18 mmol) was added to the thocarboate ester product in 20 ml of dry toluene, nitrogen was removed for 15 minutes, and then tri-n-butyltin hydride was added. ) 1.4 ml (5.29 mmol) was added and heated to 90 ° C. for 5 h.

The crude product obtained in the above procedure was cooled to room temperature and subjected to silica gel column chromatography to give yellow syrup product 2.

Compound 2 NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.50 (m, 1H), 4.39 (m, 1H), 2.77-2.71 (dd, J = 5.2, 12.4 Hz, 1H), 2.52 (d, J = 17.6 Hz, 1H), 1.87 (bs, 1H), 1.38 (d, J = 6.4 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 176.1, 81.1, 69.5, 39.4, 13.7; IR (neat) 3423.27, 1760.57, 1736.93, 1166.25, 1053.54, 940.29 cm ⁻¹ ; LCMS (ESI) m / z calcd for C ₅ H ₈ O ₃ [M + H] ⁺ : 117.05, found: 117.05.

Scheme 6

500 mg (4.31 mmol) of the product 2 was dissolved in 4 ml of dry THF, and then 2 ml of dry THF in which 10 ml (2.0 M in heptane 20.4 mmol) of lithium diisopropylamide was dissolved was added at -78 ° C. and nitrogen for 2 hours. After stirring, 4 ml of dry THF and 5 ml of HMPA in which 900 mg (4.24 mmol) of myristyl aldehyde was dissolved were added dropwise by using a syringe. The solution was warmed at -30 [deg.] C. for 3 hours and after 3 hours sat. The reaction was stopped by the addition of 20 ml NH ₄ Cl.

Thereafter, the aqueous layer was repeatedly extracted with ethyl acetate, and the organic layers extracted together were sat. After washing with Na _H CO3 and evaporated and dried by filtration with MgSO _4.

Compound 7 was obtained by performing silica gel column chromatography on the crude product obtained in the above procedure.

Compound 7 NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.77-4.70 (m, 1H), 4.69-4.65 (m, 0.2H), 4.49 (t, J = 6.4 Hz, 0.8H), 4.13 (s , 0.2 H), 3.97 (q, J = 7.6 Hz, 0.8H), 2.64 (m, 1H), 2.54 (bs, 0.8H), 2.17 (bs, 0.8H), 1.72 (q, J = 7.2 Hz, 2H), 1.54 (m, 1H), 1.40 (d, J = 6.4 Hz, 3H), 1.36-1.20 (bs, 22H), 0.89 (t, J = 5.6 Hz, 3H); IR (neat) 2918, 2849.45, 1739.13, 1204.25, 1053.55 cm ⁻¹ ; LCMS (ESI) m / z calcd for C ₁₉ H ₃₆ O ⁴ [M + H] ⁺ : 329.26, found: 329.26.

Scheme 7

120 mg (0.365 mmol) / 2 ml of DMF) was added to 50 mg (0.730 mmol) of imidazole and 66 mg of tert-butyldimethylchlorosilane at room temperature and then stirred overnight at room temperature. It was.

Sat. To the reaction product. The reaction was stopped by addition of Na _H CO 3 solution, extracted with ethyl acetate, and the extracted organic layer was dried over MgSO ₄ , filtered and evaporated.

The crude product obtained in the above procedure was purified by silica gel column chromatography to obtain Compound 8 in a 4: 1 stereoisomeric mixture.

Compound 8 NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.69-4.62 (m, 1H), 4.55 (t, J = 4.4 Hz, 0.2H), 4.40 (t, J = 5.2 Hz, 0.8H), 4.08 (s, 0.2H), 3.84 (m, 0.8H), 2.60 (t, J = 4.0 Hz, 0.2H), 2.55 (t, J = 4.8 Hz, 0.8H), 2.22 (d, J = 4.8 Hz , 0.8H), 2.04 (d, J = 5.2 Hz, 0.2H), 1.71 (q, J = 7.2 Hz, 1.6H), 1.47 (m, 1H), 1.33 (d, J = 6.4 Hz, 3H), 1.30-1.22 (bs, 22H), 0.9 (bs, 9H), 0.88 (t, J = 7.2 Hz, 3H), 0.1 (m, 6H); IR (neat) 2922.02, 2852.50, 1764.37, 1252.62, 1063.81, 834.67 cm ⁻¹ ; HRMS (ESI) m / z calcd for C ₂₅ H ₅₀ O ₄ Si [M + H] ⁺ : 443.3555, found: 443.3555.

Scheme 8

After adding 0.33 ml of trimethylamine and 0.06 ml of methanesulfonyl chloride to ₂ ml of CH ₂ Cl _{2 in} which 70 mg (0.158 mmol) of Compound 8 at 0 ° C was dissolved, 0.06 ml (0.790 mmol) of methanesulfonyl chloride was added. The mixture was stirred at the same temperature for 3 hours.

Excess 0.33 ml (2.37 mmol) of triethylamine was added and the reaction mixture was then slowly heated to 45 ° C. and maintained overnight.

The reaction mixture was diluted with CH ₂ Cl ₂ and then sat. After the reaction was stopped by addition of NaHCO ₃ , the organic layer was dried over MgSO ₄ , filtered and evaporated.

The crude product obtained in the above procedure was purified by silica gel column chromatography to obtain 9a, a white solid E-isomer, and 9b, a Z-isomer in a melting solid form.

Compound 9a NMR: ¹ H NMR (400 MHz, CDCl 3) δ 6.77 (t, J = 7.2 Hz, 1H), 4.83 (d, J = 4.4 Hz, 1H), 4.43 (m, 1H), 2.34 (m, 2H ), 1.49 (m, 2H), 1.37 (d, J = 6.4 Hz, 3H), 1.34-1.22 (bs, 20H), 0.89 (bs, 9H), 0.88 (t, J = 7.2 Hz, 3H), 0.11 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 174.8,148.7,134.6, 83.4, 73.6, 36.3, 34.5e33.8, 32.9, 30.1, 27.1, 22.6, 19.4,18.5, 0.3, 0.0; IR (neat) 2923.57, 2853.38, 1759.72, 1463.03, 1024.32, 775.35 cm ⁻¹ ; HRMS (ESI) m / z calcd for C ₂₅ H ₄₈ O ₃ Si [M + H] ⁺ : 425.3448, found: 425.3448.

Compound 9b NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.29 (t, J = 8.0 Hz, 1H), 4.68 (d, J = 5.2 Hz, 1H), 4.49 (m, 1H), 2.78-2.62 ( m, 2H), 1.44 (m, 3H), 1.30 (d, J = 6.8 Hz, 3H), 1.25-1.20 (bs, 20H), 0.90 (bs, 9H), 0.88 (t, J = 6.8 Hz, 3H ), 0.09 (s, 6H); ¹³ C NMR (100 MHz, CDCl 3) δ 173.2, 151.3, 133.7, 82.7, 76.7, 36.5, 34.3-33.8, 32.2, 30.3, 27.3, 22.7, 19.4, 18.7, 0.3, 0.0; IR (neat) 2923.97, 2853.68, 1760.6, 1462.12, 1024.53, 835.30 cm_1; LCMS (ESI) m / z calcd for C ₂₅ H ₄₈ O ₃ Si [M + H] ⁺ : 425.34, found: 425.40.

Scheme 9

Methanol (5 ml) in which 150 mg (0.35 mmol) of the compound 9a was dissolved was added with 0.3 ml of 1 M HCl. The reaction mixture was heated to 60 ° C. and maintained overnight, and the reaction was completed by monitoring with TLC, and the reaction mixture was cooled at room temperature. I was.

The residue obtained by evaporation of the cooled reaction mixture in high vacuum was dissolved in ethyl acetate, dried over MgSO ₄ , filtered and evaporated. The crude product obtained in the above procedure was purified by silica gel column chromatography to obtain a white solid marliolide 1.

Marliolide 1 NMR: ¹ H NMR (400 MHz, CDCl 3) δ 6.95 (t, J = 8.0 Hz, 1H), 4.82 (t, J = 5.2 Hz, 1H), 4.53 (m, 1H), 2.40 (m, 2H), 1.65-1.59 (m, 1H), 1.56-1.50 (m, 2H), 1.45 (d, J = 6.8 Hz, 3H), 1.39-1.25 (bs, 20H), 0.88 (t, J = 6.4 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) d 169.9, 147.8, 130.5, 78.7, 67.8, 31.9, 29.9e29.4, 28.4, 22.7, 14.1, 14.0; IR (neat) 3383.66, 2920.30, 2846.80, 1745.52, 1692.72, 1212.58, 1050.76 cm ⁻¹ ; HRMS (ESI) m / z calcd for C ₁₉ H ₃₄ O ₃ [M + H] ⁺ : 311.2583, found: 311.2583; Anal. calculated for C ₁₉ H ₃₄ O ₃ : C, 73.50; H, 11.04. Found: C, 73.48; H, 11.03.

Scheme 10

Ag ₂ O (10 mg, 0.06 mmol) and MeI (45 mg, 0.32 mmol) were added and stirred to a solution of malolilide 1 (10 mg, 0.03 mmol) dissolved in ACN (1 ml).

After stirring under nitrogen at room temperature, the resulting solution was filtered through a celite filter, dried, filtered and evaporated to purify the residue by silica gel column chromatography (Heaxne / ethylacetate = 9: 1 v / v). To obtain the final compound of the off-white compound 1, the yield of the compound 1 was confirmed to be 38%.

Compound 1 Compound NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.98 (t, J = 7.6 Hz, 1H), 4.52-4.48 (m, 1H), 4.39 (d, J = 4.8 Hz, 1H ), 3.34 (s, 3H), 2.38-2.31 (m, 2H), 1.51-1.47 (m, 2H), 1.45 (d, J = 6.4 Hz, 3H), 1.3-1.26 (bs, 20 H), 0.88 (t, J = 6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 170, 146.7, 128, 78.2, 75.7, 56.7, 31.8, 30.2, 29.6-28.4, 22.6, 14, 13.9; IR (neat) 2924, 2848, 1754, 1681, 1463, 1211, 1076 cm ⁻¹ ; HRMS (ESI) m / z calcd for C ₂₀ H ₃₇ 0 ₃ [M + H] ⁺ : 325.2743, found: 325.2740.

< Experimental Example  1> Reagent

RPMI, fetal bovine serum (FBS) and penicillin / streptomycin (PS) were purchased from WELGENE (Daegu, Korea). TPA (phorbol-12-myristate-13-acetate) and MTT reagents were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA) and used for NRF2, actin, and 8-OH-G antibodies as Santa Cruz biotechnology. (Santa Cruz, CA, USA) purchased and used. HO-1 antibody was purchased from Enzo Life Science (Farmingdale, NY, USA) and used. 4-HNE, NQO1 antibody was purchased from Abcam (Cambridge, MA, USA).

< Experimental Example  2> statistics

All experiments were repeated three times, with mean and standard deviation values. Significance was carried out by Student's t-test, and * P <0.05, ** P <0.01, *** P <0.001 according to the significance.

< Experimental Example  3> Cell Culture

HaCaT cells were purchased from Korea Cell Line Bank (Seoul, Korea).

HaCaT cells were cultured in an incubator at 37 ° C., 5% CO ₂ , using medium with 10% FBS and penicillin / streptomycin (100 U / ml) added to RPMI. HaCaT-ARE-Luc-receptor cells were also cultured under the same conditions.

< Example  1> Confirmation of Cytotoxicity of Compound 1

Before examining the antioxidant effect of Compound 1, it was first confirmed by MTT assay whether it has cytotoxicity in itself.

HaCaT cells were dispensed at 30,000 cells / well in 96 well plates and incubated for 24 hours. Compound 1 was then treated with 0.1, 1 and 10 μM concentrations and further incubated for 24 hours.

After incubation, washed 2-3 times with PBS and incubated for 4 hours by the addition of 200 μl MTT solution (5 mg / ml in media). After 4 hours, the cells were washed once with PBS, and 100 μl DMSO was added to dissolve the cell crystals at 37 ° C. for 30 minutes, and the absorbance was measured at 560 nm.

As a result, as shown in FIG. 1, the compound 1 was treated in HaCaT cells from 0.1 μM to 10 μM in 10-fold increments to confirm that cytotoxicity was not observed.

< Example  2> ARE- Luciferase  Activity analysis

Regulation of antioxidant-related proteins of Nrf2 occurs primarily through binding to the ARE sequence.

Therefore, permanent transfection was performed to express the ARE-Luc-reporter gene in HaCaT cells, and 10 μM of resveratrol was treated as a positive control with Compound 1 to confirm luciferase activity.

HaCaT-ARE-Luc-reporter cells were permanently inoculated at 1 × 10 ⁵ cells / well in 24 well plates and incubated for 24 hours. Compound 1 and resveratrol were treated at 10 μM concentration and then incubated for 12 and 24 hours.

The cells were then washed 2-3 times with PBS and 100 μl of luciferase lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA) was added to the cells. Dissolve at 4 ° C. for 30 minutes.

Luciferase activity was measured in lysed cells using the GLOMAX Multi-system (Promega, Madison, Wis., USA), and the measured luciferase values were normalized to protein concentration.

As a result, as shown in Fig. 2, both Compound 1 and resveratrol showed 4 to 6 times higher luciferase activity than the negative control group, but Compound 1 showed higher luciferase activity than resveratrol in both 12 and 24 hour treatment groups. It became.

< Example  3> Confirmation of changes in expression of antioxidant regulators

Changes in the expression levels of antioxidant-related proteins Nrf2, HO-1 and NQO1 following compound 1 treatment in HaCaT cells were compared with resveratrol.

HaCaT cells were seeded at 100 × plates at 1 × 10 ⁶ and incubated for 24 hours. Compound 1 and resveratrol were then treated each time at 10 μM concentrations. Cells were then washed 2-3 times with PBS and cell pellets were obtained with a scraper.

Centrifugation of the pellet followed by RIPA lysis buffer [50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4, 1 mM 200 μl of dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)] was added and incubated at 4 ° C. for 1 hour to extract the protein and centrifuge.

Supernatants obtained after centrifugation were measured for protein concentration using BCA Protein Assay Kits (Thermo-Fisher Scientific, Waltham, Mass., USA). The same amount of protein was transferred to a PVDF membrane after electrophoresis on 12% SDS-PAGE, and a blocking buffer (5% skin milk in 1x PBS-T) was added to the membrane and incubated at 4 ° C for 1 hour.

Thereafter, after washing 2-3 times with 1 × PBS-T, PBS mixed with a primary antibody in a 1: 1000 ratio was added to the membrane and incubated at 4 ° C. for 8 hours. Then, washed 2-3 times with 1 × PBS-T and incubated at 4 ° C. for 1 hour with blocking buffer containing HRP-bound secondary antibody in a 1: 5000 ratio, followed by 2- × 1 with PBS-T. After washing three times, the protein expression was confirmed using an ECL detection system, and actin (Actin) was used to confirm the protein content.

As a result, it was confirmed that the expression duration of the antioxidant proteins by FIG. 3 and Compound 1 lasted longer than resveratrol.

< Example  4> HO-1 and Of NQO1 mRNA  Confirmation of expression change

It was confirmed that the increase in HO-1 and NQO1 protein induced by compound 1 was due to the increase in HO-1 and NQO1 mRNA expression induced by Nrf2. Nrf2 was regulated by ubiquitination, so no mRNA change was measured. Compound 1 was treated and mRNA change over time was measured by real-time PCR.

HaCaT cells were seeded in 6 well culture plates at 5 × 10 ⁵ / well and incubated for 24 hours. Compound 1 was then treated for 4, 8 and 12 hours at 10 μM concentration and washed 2-3 times with PBS to obtain pellet with scraper.

Total RNA was extracted from the cell pellet using a Hybrid-R RNA extraction kit (GeneAll, Seoul, Korea). The concentration of the extracted total RNA was measured to synthesize cDNA using 1 μg of RNA using PrimeScript RT-PCR kit (TAKARA Korea, Seoul, Korea).

PCR was performed after mixing SYBR green mix, Forward primer, reverse primer and dH2O to the synthesized cDNA. PCR was performed at 95 ° C. for 5 minutes and 1 cycle, then at 95 ° C. for 10 seconds, at 59 ° C. for 10 seconds, at 72 ° C. for 30 seconds, and at 40 ° C. for 5 minutes and 1 cycle. Each mRNA was corrected by GAPDH house keeping gene.

As a result, as compared to the negative control group as shown in Figure 4 HO-1 was increased 3.3 times, 3.7 times and 2.6 times in the 4, 8 and 12 hours treatment group, respectively, NQO1 3.2 times in the 4 and 8 hours treatment group, respectively And 2.5-fold increase.

< Example  5> 8- by TPA With OHdG  4- Of HNE  Increased expression and confirmation of inhibitory effect by Compound 1

In the previous experiments, the increase in the expression of antioxidant-related proteins was confirmed by Compound 1, and it was confirmed whether these phenomena could suppress oxidative stress.

HaCaT cells were inoculated at 50,000 cells / well in a 24-well plate with a round cover glass and incubated for 24 hours. TPA (100 nM) was then treated alone or with Compound 1 (10 μM) and then incubated for 4 and 8 hours.

After incubation, the cells were washed 2-3 times with PBS, the cells were fixed with 250 μl of 4% para-formaldehyde for 15 minutes, and washed with 500 μl of Immuno-CytoChemistry (ICC) washing buffer for 30 minutes.

The primary antibody was then incubated at 4 ° C. for 8 hours, washed 2-3 times with ICC wash buffer, and the rabbit secondary antibody (Jackson-Immuno) conjugated with DAPI and fluorescein isothiocyanate (FITC). Research, West Grove, PA, USA) was added to the ICC wash buffer in a ratio of 1: 1000 and incubated at 4 ° C. under light blocking for 4 hours. Fluorescence images were taken using a C2 confocal microscope (Nikon Korea, Seoul, Korea).

TPA is a representative chemical that induces oxidative stress, and as a result of treating TPA at 100 nM concentration, DNA oxidation and lipid oxidation were increased as shown in FIGS. 5 and 6, and 8-OHdG and 4-HNE, respectively, were markers of oxidation. Increased expression of was also confirmed.

On the other hand, in the experimental group treated with Compound 1, it was confirmed that increased expression of 8-OHdG and 4-HNE was suppressed, and that Compound 1 effectively inhibited the oxidation of DNA and lipids.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

Marliolide derivative represented by the formula (1).
[Formula 1]

The method of claim 1, wherein the malolilide derivative represented by Formula 1 is a maleolilide derivative, characterized in that to increase the expression of antioxidant proteins Nrf2, HO-1 and NQO1. The maleolilide derivative according to claim 1, wherein the maleolilide derivative represented by Chemical Formula 1 inhibits DNA oxidation and lipid oxidation. Antioxidant cosmetic composition containing a maleilide derivative represented by the formula (1) as an active ingredient.
[Formula 1]

The group of claim 4 wherein the cosmetic composition comprises a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray Cosmetic composition for antioxidant, characterized in that it has a formulation selected from.

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