Curcumin analogues, methods of synthesis and use thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a curcumin analogue, a synthetic method and application thereof. The present application takes curcumin as a lead compound, introduces cyclopropylmethyl, n-butyl and isobutyl in acetylacetone, introduces 4-acetylamino, 3,4-dimethoxy, 2-fluoro and 3-trifluoromethyl on a benzene ring, and designs and synthesizes a series of curcumin analogues. In vitro cytotoxicity experiments show that the killing effect of the newly synthesized curcumin analogue on prostate cancer cells is stronger than that of dimethyl curcumin, and the androgen receptor inhibition effect is also stronger than that of dimethyl curcumin.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a curcumin analogue, its synthesis method, and its application. Background Technology

[0002] Curcumin is one of the most representative active ingredients in the curcumin family, mainly extracted and isolated from the rhizome of turmeric (Curcuma longa L.). It is currently the most widely studied natural polyphenol compound. Studies have confirmed that curcumin possesses various pharmacological activities, including antitumor, anti-inflammatory, antioxidant, antibacterial, and antiviral effects. It can inhibit tumor cell proliferation, invasion, and metastasis by regulating multiple signaling pathways such as NF-κB, STAT3, and WNT / β-catenin, and induce tumor cell apoptosis through the mitochondrial pathway, demonstrating great potential in the prevention and treatment of malignant tumors. However, natural curcumin itself has poor water solubility, rapid in vivo metabolism, and low oral absorption efficiency, resulting in poor bioavailability.

[0003] Dimethylcurcumin (DMC) can selectively promote the degradation of androgen receptors (AR) and effectively inhibit the activation of the AR signaling pathway. It has a significant inhibitory effect on the proliferation of castration-resistant prostate cancer cells, but its anti-tumor activity is limited, which restricts its clinical application. Summary of the Invention

[0004] The purpose of this invention is to synthesize novel curcumin analogues with anti-prostate cancer and androgen receptor inhibitory effects by modifying the β-diketone structure or substituents on the benzene ring of curcumin. The curcumin analogues designed and synthesized in this invention have the following general chemical formula:

[0005]

[0006] Wherein, R1 is cyclopropylmethyl, n-butyl, or isobutyl; and R2 is 4-acetamido, 3,4-dimethoxy, 2-fluoro, or 3-trifluoromethyl.

[0007] The chemical names and chemical structural formulas of the newly synthesized curcumin analogues are as follows:

[0008] A: N,N'-(((1E,6E)-4-butyl-3,5-dioxohep-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine

[0009]

[0010] B: N,N'-(((1E,6E)-4-isobutyl-3,5-dioxoheptane-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine

[0011]

[0012] C: (1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-isobutylhept-1,6-diene-3,5-dione

[0013]

[0014] D: (1E,6E)-4-(cyclopropylmethyl)-1,7-di(2-fluorophenyl)hept-1,6-diene-3,5-dione

[0015]

[0016] E: (1E,6E)-4-isobutyl-1,7-bis(3-trifluoromethylphenyl)hept-1,6-diene-3,5-dione

[0017] .

[0018] The preparation method and synthetic route of the novel curcumin analogue are as follows:

[0019]

[0020] (1) Dissolve acetylacetone, brominated product and potassium carbonate in N,N-dimethylformamide, heat and stir, remove solvent under reduced pressure after reaction, add distilled water to dissolve residue, extract twice with ethyl acetate and combine organic phases, wash with saturated sodium chloride and dry with anhydrous magnesium sulfate, filter, take filtrate and evaporate to dryness, remove solvent by reduced pressure distillation to obtain a pale yellow transparent liquid.

[0021] The brominated derivatives are bromomethylcyclopropane, bromobutane, and bromoisobutane; the molar ratio of acetylacetone, brominated derivatives, and potassium carbonate is 1:1~1.2:2~3; the temperature for heating and stirring the reaction is 40~60℃; and the reaction time is 6~10 hours.

[0022] (2) The acetylacetone analog, boric acid, tributyl borate, n-butylamine and benzaldehyde with different substituents prepared in step (1) were added to the solvent, heated and reacted, 20% acetic acid was added, and the mixture was stirred overnight. After cooling to room temperature, the solid was obtained by filtration. The solid was recrystallized from methanol to obtain the target product in the form of yellow powder.

[0023] The benzaldehydes substituted with different substituents are: p-acetaminobenzaldehyde, 3,4-dimethoxybenzaldehyde, 2-fluorobenzaldehyde, and 3-trifluoromethylbenzaldehyde; the solvent is N,N-dimethylformamide; the molar ratio of acetylacetone analog, benzaldehydes substituted with different substituents, boric acid, tributyl borate, and n-butylamine is 1:2~2.5:0.5~1:2~2.5:1~1.5; the temperature for heating and stirring the reaction is 60~80℃, and the reaction time is 2~4 hours.

[0024] The curcumin analogues of this invention are used to prepare therapeutic drugs for prostate cancer or androgen receptor-related diseases.

[0025] The beneficial effects of this invention are:

[0026] By introducing cyclopropylmethyl, n-butyl, and isobutyl groups into acetylacetone, and simultaneously introducing 4-acetamido, 3,4-dimethoxy, 2-fluoro, and 3-trifluoromethyl groups onto the benzene ring, a new compound was obtained that exhibited superior anti-prostate cancer cell activity and androgen receptor inhibitory activity compared to dimethylcurcumin. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described in detail below through examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] Example 1 Synthesis of Compound A

[0029] (1) Preparation of 3-Butylpentane-2,4-dione

[0030] Acetylacetone (5 g, 49.94 mmol), n-butane bromide (8.09 g, 59.93 mmol), and potassium carbonate (13.8 g, 99.98 mmol) were dissolved in 50 mL of N,N-dimethylformamide. The mixture was stirred at 60 °C for 6 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was dissolved in distilled water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride and dried over anhydrous magnesium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness. The solvent was removed by vacuum distillation to obtain a pale yellow transparent liquid (5.5 g, yield 71.4%).

[0031] (2) Preparation of N,N'-(((1E,6E)-4-butyl-3,5-dioxohep-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine

[0032] 3-Butylpentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and p-acetaminobenzaldehyde (1.04 g, 6.4 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain a yellow solid powder of 921.3 mg, with a yield of 64.1%. 1H NMR (400 MHz, DMSO) δ 10.20(s, 2H), 7.93 – 7.84 (m, 1H), 7.76 – 7.59 (m, 9H), 7.25 (dd, J = 31.6, 15.4Hz, 1H), 6.94 (d, J = 16.0 Hz, 1H), 2.75 (d, J = 7.6 Hz, 1H), 2.08 (t, J =5.0 Hz, 6H), 1.85 (q, J = 7.3 Hz, 1H), 1.49 – 1.19 (m, 4H), 1.00 – 0.82 (m,3H).13C NMR (101 MHz, DMSO) δ 195.90, 183.08, 177.78, 169.29, 169.09, 143.57,141.68, 141.26, 130.11, 129.82, 129.68, 124.21, 119.40, 119.34, 112.36,60.83, HRMS: m / z of [M+H]+ calculated forC27H32N2O4 447.2284, found 447.2289.

[0033] Example 2 Synthesis of Compound B

[0034] (1) Preparation of 3-isobutylpentane-2,4-dione

[0035] Acetylacetone (5 g, 49.94 mmol), isobutane bromo (8.09 g, 59.93 mmol), and potassium carbonate (13.8 g, 99.98 mmol) were dissolved in 50 mL of N,N-dimethylformamide. The mixture was stirred at 60 °C for 7 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was dissolved in distilled water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride and dried over anhydrous magnesium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness. The solvent was removed by vacuum distillation to obtain a pale yellow transparent liquid (5.8 g, yield 75.3%).

[0036] (2) N,N'-(((1E,6E)-4-isobutyl-3,5-dioxohep-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine

[0037] 3-Isobutylpentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and p-acetaminobenzaldehyde (1.04 g, 6.4 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain a yellow solid powder of 941.2 mg, with a yield of 65.6%. 1H NMR (400 MHz, DMSO) δ10.20 (d, J = 2.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 7.70 – 7.64 (m, 7H),7.62 (d, J = 6.1 Hz, 1H), 7.22 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 16.0 Hz,1H), 2.58 (d, J = 7.1 Hz, 1H), 2.08 (d, J = 4.3 Hz, 6H), 1.77 (t, J = 6.9 Hz,1H), 1.57 (ddt, J = 43.1, 13.3, 6.7 Hz, 1H), 0.94 (dd, J = 22.3, 6.5 Hz, 6H).13C NMR (101 MHz, DMSO) δ 195.92, 183.57, 169.55, 169.48, 143.63, 142.03,141.45, 141.01, 130.22, 130.10, 129.75, 129.19, 124.13, 119.86, 119.57,119.48, 111.44, 60.69, 40.49, 40.21, 40.00, 39.79, 39.58, 39.37, 39.17,38.96, 37.44, 33.31, 31.39, 26.53, 24.45, 22.89, 22.11.HRMS: m / z of [M+H]+calculated for C27H32N2O4 447.2284, found 447.2284.

[0038] Example 3 Synthesis of Compound C

[0039] (1) The preparation of 3-isobutylpentane-2,4-dione is the same as step (1) in Example 2.

[0040] (2) Preparation of (1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-isobutylhept-1,6-diene-3,5-dione

[0041] 3-Isobutylpentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and 3,4-dimethoxybenzaldehyde (1.06 g, 6.4 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain a yellow solid powder of 936.6 mg, with a yield of 64.7%. 1H NMR (600 MHz, DMSO) δ7.63 (d, J = 15.4 Hz, 2H), 7.37 – 7.31 (m, 4H), 7.18 (d, J = 15.4 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 4.62 (t, J = 6.9 Hz, 1H), 3.83 (d, J = 14.4 Hz, 12H), 2.61 (d, J = 7.0 Hz, 2H), 1.57 (dtt, J = 59.5, 13.4, 6.7 Hz, 1H), 0.96 (d, J = 6.6 Hz, 6H).13C NMR (101 MHz, DMSO) δ 195.51, 183.63, 151.83, 151.34,149.43, 144.21, 141.50, 128.40, 127.38, 124.10, 123.85, 123.09, 119.51,112.21, 112.06, 111.54, 110.93, 60.60, 56.12, 56.05, 56.03, 40.58, 40.37,40.16, 39.95, 39.74, 39.53, 39.33, 37.39, 31.59, 26.57, 23.02, 22.22. HRMS:m / z of [M+H]+ calculated for C27H32O6 453.2277, found 453.2287.

[0042] Example 4 Synthesis of Compound D

[0043] (1) Preparation of 3-(cyclopropylmethyl)pentane-2,4-dione

[0044] Acetylacetone (5 g, 49.94 mmol), cyclopropylmethyl bromide (7.31 g, 54.93 mmol), and potassium carbonate (17.2 g, 124.85 mmol) were dissolved in 50 mL of N,N-dimethylformamide. The mixture was stirred at 70 °C for 8 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was dissolved in distilled water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride and dried over anhydrous magnesium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness. The solvent was removed by vacuum distillation to obtain a pale yellow transparent liquid (6.1 g, yield 79.2%).

[0045] (2) Preparation of (1E,6E)-4-(cyclopropylmethyl)-1,7-di(2-fluorophenyl)hept-1,6-diene-3,5-dione

[0046] 3-(cyclopropylmethyl)pentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and 2-fluorobenzaldehyde (794.3 mg, 6.4 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain 856.6 mg of yellow solid powder, with a yield of 73.1%.1H NMR (400 MHz, DMSO) δ7.98 (t, J = 7.8 Hz, 1H), 7.88 (t, J = 7.7 Hz, 1H), 7.76 (t, J = 16.5 Hz, 2H), 7.55 – 7.40 (m, 3H), 7.29 (td, J = 11.3, 6.2 Hz, 4H), 7.14 (d, J = 16.2Hz, 1H), 4.76 (t, J = 6.9 Hz, 1H), 2.71 (d, J = 6.2 Hz, 1H), 1.79 (t, J = 7.0Hz, 1H), 0.92 – 0.65 (m, 1H), 0.41 (dt, J = 12.8, 6.5 Hz, 2H), 0.22 (t, J =4.9 Hz, 1H), 0.11 (t, J = 4.9 Hz, 1H).13C NMR (101 MHz, DMSO) δ 196.24,183.43, 162.60, 162.40, 160.09, 159.91, 135.43, 135.40, 133.38, 133.35,133.26, 132.75, 132.66, 129.90, 129.87, 129.84, 128.30, 128.25, 125.55,125.51, 125.48, 123.97, 123.92, 123.12, 116.75, 116.67, 116.53, 116.46,112.59, 112.57, 62.67, 40.59, 40.39, 40.18, 39.97, 39.76, 39.55, 39.34,33.51, 27.90, 13.43, 9.61, 5.31, 4.50.HRMS: m / z of [M+H]+ calculated forC23H20F2O2 367.1510, found 367.1511..

[0047] Example 5 Synthesis of Compound E

[0048] (1) The preparation method of 3-isobutylpentane-2,4-dione is the same as step (1) in Example 1.

[0049] (2) Preparation of (1E,6E)-4-isobutyl-1,7-bis(3-trifluoromethylphenyl)hept-1,6-diene-3,5-dione

[0050] 3-Isobutylpentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and 3-trifluoromethylbenzaldehyde (1.11 g, 6.4 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain a yellow solid powder of 989.4 mg, with a yield of 66.3%. 1H NMR (400 MHz, DMSO) δ 8.19– 8.11 (m, 3H), 8.07 (d, J = 7.9 Hz, 1H), 7.80 (dd, J = 16.4, 9.5 Hz, 4H), 7.69 (q, J = 7.2 Hz, 2H), 7.51 (d, J = 15.6 Hz, 1H), 7.32 (d, J = 16.1 Hz,1H), 4.75 (t, J = 6.9 Hz, 1H), 2.69 (d, J = 7.0 Hz, 1H), 1.82 (t, J = 6.9 Hz,1H), 1.57 (dp, J = 20.6, 6.7 Hz, 1H), 0.94 (dd, J = 10.5, 6.6 Hz, 6H).13C NMR(101 MHz, DMSO) δ 196.08, 183.16, 141.88, 140.03, 136.59, 135.85, 132.81,132.58, 130.77, 130.50, 130.46, 130.40, 130.14, 129.82, 128.56, 127.93,127.34, 126.79, 125.85, 125.76, 125.63, 125.59, 125.56, 125.53, 123.42,123.14, 123.05, 113.69, 62.32, 40.60, 40.39, 40.18, 39.97, 39.77, 39.56,39.35, 34.99, 29.82, 28.07, 24.33, 22.63, 21.77, 14.31, 14.23. HRMS: m / z of[M+H]+ calculated for C25H22F6O2 469.1602, found 469.1602.

[0051] Example 6 Synthesis of Compound E

[0052] (1) Preparation of 3-isobutylpentane-2,4-dione

[0053] Acetylacetone (5 g, 49.94 mmol), bromoisobutane (6.84 g, 49.94 mmol), and potassium carbonate (13.8 g, 99.98 mmol) were dissolved in 50 mL of N,N-dimethylformamide. The mixture was stirred at 60 °C for 7 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was dissolved in distilled water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride and dried over anhydrous magnesium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness. The solvent was removed by vacuum distillation to obtain a pale yellow transparent liquid (5.2 g, yield 66.6%).

[0054] (2) Preparation of (1E,6E)-4-isobutyl-1,7-bis(3-trifluoromethylphenyl)hept-1,6-diene-3,5-dione

[0055] 3-Isobutylpentane-2,4-dione (0.5 g, 3.2 mmol), boric acid (0.098 g, 1.6 mmol), and N,N-dimethylformamide (5 mL) were heated to 80 °C and reacted for 15 minutes. Then, tributyl borate (1.47 g, 6.4 mmol) and 3-trifluoromethylbenzaldehyde (1.39 g, 8 mmol) were added, and the mixture was stirred for about 15 minutes. Then, n-butylamine (0.117 g, 1.6 mmol) was added dropwise, and the mixture was reacted at 80 °C for 2 hours. Then, 20% acetic acid (30 mL) was added, and the mixture was stirred for 1 hour. The mixture was cooled to room temperature, filtered to obtain a solid, and the solid was recrystallized from methanol and dried to obtain a yellow solid powder of 991.6 mg, with a yield of 66.4%.

[0056] Synthesis of compound A2 (Comparative Example 1)

[0057] (1) Preparation of 3-(cyclobutylmethyl)pentane-2,4-dione

[0058] 2 g (20.0 mmol) of acetylacetone and 5.52 g (40.0 mmol) of potassium carbonate were dissolved in 15 mL of DMF. 3.55 g (24.0 mmol) of bromomethylcyclobutyl was added, and the mixture was stirred at 60 °C for 5 hours. After cooling, reaction solution A was dissolved in 150 mL of distilled water, extracted with ethyl acetate (EA), and the organic phase was collected and dried over anhydrous sodium sulfate. The solid was removed by filtration, and the solvent was removed by rotary evaporation. The solution was then purified by vacuum distillation to obtain a yellow liquid.

[0059] (2) Preparation of N,N'-(((1E,6E)-4-(cyclobutylmethyl)-3,5-dioxa-1,6-diene-1,7-diyl)bis(3,1-phenyl))dimethylsulfonamide

[0060] 500 mg (2.97 mmol) of 3-(cyclobutylmethyl)pentane-2,4-dione was dissolved in 3 mL of DMF. 1.19 g (5.95 mmol) of 3-methanesulfonamide benzaldehyde, 91.9 mg (1.49 mmol) of boric acid, 1.37 g (5.95 mmol) of tributyl borate, and 108.8 mg (1.49 mmol) of n-butylamine were added. The mixture was stirred at 80 °C for 3 hours. 30 mL of 20% acetic acid aqueous solution was added, and the mixture was stirred for another 3 hours. The mixture was filtered, and the residue was collected. Recrystallization from methanol and drying yielded 1138.4 mg of an orange-yellow solid powder, with a yield of 72.3%.

[0061] Comparative Example 2: Synthesis of Compound I

[0062] Boron trioxide (1.4 g, 20 mmol), 2,4-pentanedione (4.0 g, 40 mmol), and acetone dimethyl acetal (1.04 g, 10 mmol) were dispersed in 40 mL of N,N-dimethylformamide and reacted in a 160 W microwave reactor for 20 minutes. Then, 3-methanesulfonamide benzaldehyde (15.92 g, 80 mmol) and 1,2,3,4-tetrahydroquinoline (2.66 g, 20 mmol) were added, and the reaction was continued in the microwave reactor for 2 hours. After the reaction was completed, 180 mL of 20% acetic acid aqueous solution was added and stirred overnight. A precipitate was formed, which was collected by filtration and recrystallized from methanol to give 17.6 g, with a yield of 80%.

[0063] Experimental Example 1: Cell Growth Inhibition Activity

[0064] The methods and results for studying the cell proliferation activity of curcumin analogues AE, A2, and I are as follows:

[0065] Preparation of experimental drugs: The curcumin analogs prepared in Examples 1-5 and Comparative Examples 1-2 of this invention were dissolved in dimethyl sulfoxide to prepare solutions with a concentration of 8 mmol / L. The curcumin analogs were then diluted to 1.25, 2.5, 5, 10, 20, and 40 μmol / L using culture medium. Dimethylcurcumin was used as a control.

[0066] Cell lines: human prostate cancer (22Rv1) and human sebaceous gland cells (SZ95). Cells were routinely cultured in 1640 and DMEM medium (10% fetal bovine serum) at 37°C in a 5% CO2 incubator, with the medium changed every 24 hours. When cells reached saturation, they were passaged using 0.25% trypsin, with passages every 2-3 days. Cells in the logarithmic growth phase were used in the experiments.

[0067] Cell viability assay: The MTT assay was used to determine the effect of curcumin analogues on the proliferation activity of human prostate cancer cells 22Rv1 and human sebaceous gland cells SZ95. 22Rv1 cells and SZ95 cells were cultured at 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells per well in 96-well plates, with four replicates for each concentration. Dimethyl curcumin and curcumin analogs prepared in Examples 1-5 and Comparative Examples 1-2 were diluted with complete culture medium to a series of concentrations (1.25, 2.5, 5, 10, 20, and 40 μM) and added to 96-well plates. The plates were co-incubated with 22Rv1 cells for 24 h. The culture medium was then aspirated, and MTT solution was added to each well. After incubation for 4 h, the culture medium was aspirated, and 200 μL of dimethyl sulfoxide was added to each well to fully dissolve the formazan generated by MTT reduction. The absorbance at 490 nm was measured using a multi-functional microplate reader. The cell viability of each group was calculated with the blank control group as 100% viability. The maximum half-inhibitory concentration (IC50) was calculated based on the cell viability at different concentrations.

[0068] As shown in Table 1, the curcumin analogs prepared in Examples 1-5 of this invention all exhibited strong inhibitory effects on the proliferation of 22RV1 cells. Compared with dimethylcurcumin, compounds A, B, C, D, and E showed significantly enhanced cytotoxicity against 22RV1 cells. Compound A2 showed only slightly higher cytotoxicity against 22RV1 cells than dimethylcurcumin. Compound I also showed significantly higher cytotoxicity against 22RV1 cells than dimethylcurcumin. Compounds A, B, C, D, and E all showed higher cytotoxicity against 22RV1 cells than compound A2. Compounds A and B showed higher cytotoxicity against 22RV1 cells than compound I, while compounds D and E showed cytotoxicity against 22RV1 cells comparable to compound I.

[0069] Table 1. Inhibitory activity of curcumin analogues against 22Rv1 cells (24h)

[0070]

[0071] The cytotoxicity of compounds that showed good inhibitory effects on 22Rv1 cells was further tested on human sebaceous gland SZ95 cells. As shown in Table 2, compounds A, B, and E showed higher selectivity against prostate cancer cells compared to dimethylcurcumin. The selectivity of compound A was significantly higher than that of compound A2, while the selectivity of compounds B and E was comparable to that of compound A2. The selectivity of compounds A, B, and E against prostate cancer cells was higher than that of compound I.

[0072] Table 2. Inhibitory activity of curcumin analogues on SZ95 cells (24h)

[0073]

[0074] Experimental Example 2: Androgen Receptor Protein Inhibitory Activity

[0075] Preparation of experimental drugs: The curcumin analogs prepared in Examples 1-5 and Comparative Examples 1-2 of this invention were dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 8 mmol / L. The curcumin analogs were then diluted to 2 μmol / L using culture medium, with dimethyl curcumin as a control.

[0076] Cell line: Human prostate cancer (22Rv1) cells were routinely cultured in 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0077] Androgen receptor protein inhibition experiment:

[0078] (1) AR protein extraction and quantification: 22Rv1 cells were extracted and quantified at a density of 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured overnight. Cells incubated in drug-free medium served as the control group, while cells containing the curcumin analogue prepared in Examples 1-5 were diluted in medium and used as the experimental group. These cells were co-incubated with the control group for 24 hours. After incubation, the old medium was removed, and the cells were washed three times with 1 mL PBS. 100 μl of RIPA lysis buffer was added to each well, and the cells were lysed in an ice-water bath for 5 minutes. The cell lysate was collected in 1.5 mL centrifuge tubes, and lysis was continued in an ice-water bath for 30 minutes. The tubes were then centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. Protein concentration was determined using a BCA kit, and the protein samples were adjusted to the same concentration using lysis buffer. After adding protein loading buffer, the protein was denatured in a boiling water bath for 5 minutes and then stored at -80°C for later use.

[0079] (2) SDS-PAGE and Western blot: 8% SDS-PAGE solution was prepared for electrophoresis, with 10 μL of sample loaded into each well. Electrophoresis was performed at a constant voltage of 100V until bromophenol blue reached the bottom of the separating gel. Wet transfer was used to transfer the protein to a PVDF membrane at 100V for 90 min. The PVDF membrane was then cut according to the protein marker. The membrane was blocked with 5% skim milk at room temperature for 2 h. 5 mL of AR antibody and β-actin antibody were incubated with different PVDF membranes at 4℃ overnight. After incubation, the primary antibody was recovered, and the PVDF membrane was washed with TBST buffer on a shaker for 10 min, repeated three times. The PVDF membrane was then incubated with the secondary antibody at room temperature for 1.5 h. After incubation, the membrane was washed three times with TBST buffer, and then analyzed and imaged using a gel imaging system. The grayscale value of the target protein band was detected using ImageJ software. β-actin was used as an internal control protein to quantitatively analyze the relative expression level of the target protein in each group.

[0080] The results are shown in Table 3. The curcumin analogs prepared in Examples 1-5 of this invention have degradation effects on both the full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7). The inhibitory effect on AR-FL is significantly stronger than that of dimethyl curcumin. Compounds A, C, and E also have significantly stronger inhibitory effects on AR-V7 than dimethyl curcumin. The inhibitory effect of compound A2 on AR-FL and AR-V7 is comparable to that of dimethyl curcumin. The degradation effect of compound I on AR-FL and AR-V7 is better than that of dimethyl curcumin. The degradation effects of the compounds prepared in Examples 1-5 of this invention on AR-FL and AR-V7 are all better than those of compound A2. The degradation abilities of compounds A, C, and E on AR-FL and AR-V7 are higher than those of compound I.

[0081] Table 3. Inhibition rate of curcumin analogues on androgen receptor protein in 22Rv1 cells

[0082]

Claims

1. A curcumin analogue, characterized in that, The structure of the curcumin analogue is shown in the following formula: , Wherein, R1 is cyclopropylmethyl, n-butyl, or isobutyl; and R2 is 4-acetamido, 3,4-dimethoxy, 2-fluoro, or 3-trifluoromethyl.

2. The curcumin analogue according to claim 1, characterized in that, The chemical names and chemical structural formulas of the curcumin analogues are as follows: A: N,N'-(((1E,6E)-4-butyl-3,5-dioxohep-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine , B: N,N'-(((1E,6E)-4-isobutyl-3,5-dioxoheptane-1,6-diene-1,7-diyl)bis(4,1-phenylene))diacetamine , C: (1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-isobutylhept-1,6-diene-3,5-dione , D: (1E,6E)-4-(cyclopropylmethyl)-1,7-di(2-fluorophenyl)hept-1,6-diene-3,5-dione , E: (1E,6E)-4-isobutyl-1,7-bis(3-trifluoromethylphenyl)hept-1,6-diene-3,5-dione 。 3. A method for preparing a curcumin analog according to claim 1, characterized in that, The preparation method steps are as follows: (1) Preparation of acetylacetone analogues: Acetylacetone, brominated derivative and potassium carbonate were dissolved in N,N-dimethylformamide, heated and stirred. After the reaction was completed, the solvent was removed by vacuum distillation, the residue was dissolved by distilled water, extracted twice with ethyl acetate and the organic phases were combined. After washing with saturated sodium chloride, the mixture was dried with anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness. After vacuum distillation, a pale yellow transparent liquid was obtained. (2) Preparation of curcumin analogue: The acetylacetone analogue prepared in step (1), boric acid, tributyl borate, n-butylamine and benzaldehyde with different substituents were added to the solvent, heated and stirred, 20% acetic acid was added, and stirring was continued overnight. After cooling to room temperature, the solid was obtained by filtration. The solid was recrystallized from methanol to obtain the target product in powder form.

4. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (1), the bromine derivatives are: bromomethylcyclopropane, bromobutane, and bromoisobutane.

5. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (1), the molar ratio of acetylacetone, brominated product and potassium carbonate is 1:1~1.2:2~3.

6. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (1), the temperature for heating and stirring the reaction is 40~60℃; the reaction time is 6~10 hours.

7. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (2), the benzaldehydes substituted with different substituents are: p-acetaminobenzaldehyde, 3,4-dimethoxybenzaldehyde, 2-fluorobenzaldehyde, and 3-trifluoromethylbenzaldehyde; the solvent is N,N-dimethylformamide.

8. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (2), the molar ratio of acetylacetone analog, benzaldehyde with different substituents, boric acid, tributyl borate and n-butylamine is 1:2~2.5:0.5~1:2~2.5:1~1.

5.

9. The method for preparing curcumin analogues according to claim 3, characterized in that, In step (2), the temperature for heating and stirring the reaction is 60~80℃, and the reaction time is 2~4 hours.

10. An application of the curcumin analogue according to claim 1, characterized in that, The curcumin analogues are used to prepare therapeutic drugs for prostate cancer or androgen receptor-related diseases.

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