Novel Anthraquinone Compounds Inhibit Colon Cancer Cell Proliferation via the Reactive Oxygen Species/JNK Pathway.

A series of amide anthraquinone derivatives, an important component of some traditional Chinese medicines, were structurally modified and the resulting antitumor activities were evaluated. The compounds showed potent anti-proliferative activities against eight human cancer cell lines, with no noticeable cytotoxicity towards normal cells. Among the candidate compounds, 1-nitro-2-acyl anthraquinone-leucine (8a) showed the greatest inhibition of HCT116 cell activity with an IC50 of 17.80 μg/mL. In addition, a correlation model was established in a three-dimensional quantitative structure-activity relationship (3D-QSAR) study using Comparative Molecular Field Analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). Moreover, compound 8a effectively killed tumor cells by reactive oxygen species (ROS)-JNK activation, causing an increase in ROS levels, JNK phosphorylation, and mitochondrial stress. Cytochrome c was then released into cytoplasm, which, in turn activated the cysteine protease pathway and ultimately induced tumor cell apoptosis, suggesting a potential use of this compound for colon cancer treatment.

100 mL of 98% concentrated sulfuric acid was then added under cooling in an ice bath, as the mixture was allowed to below 5 °C . 10 g of fine powder potassium nitrate powder was then added in one hour. After the addition was completed, the reaction was stirred at room temperature and the progress of the reaction was checked by TLC. After the reaction was completed, the solution was poured into 500 mL of ice water with vigorous stirring, then vacuum-filtered. The filter cake was washed first with acid water, then with hot water, and dried to obtain a crude product. This was then recrystallized from acetic acid to give a pale yellow solid of higher purity.

Synthesis of 1-amino-2-methylanthraquinone (3)
10.4 g (39 mmol) of compound 2 (2) was weighed into a 250 mL three-necked flask. 100 mL of 50% concentrated DMF was then added. 1.5 g (39.5 mmol) of NaBH4 was then added under stirring. After the addition was completed, the reaction was stirred at room temperature and the progress of the reaction was checked by TLC. After the reaction was completed, the solution was poured into 500 mL of ice water with vigorous stirring, then vacuum-filtered. The filter cake was dried to obtain a crude product. Column chromatography was used for purification with neutral

Synthesis of compound (4)
0.45 g (1.9 mmol) of 1-amino-2-methylanthraquinone was dissolved in 50 mL of anhydrous THF, and added to a 100 mL three-necked flask, 0.7 mL (5.2 mmol) of triethylamine was added. 26 mmol of acetyl chloride or chloroacetyl chloride was added dropwise in an ice bath, and the reaction was stirred at room temperature for 4 hours, and the basic reaction was completed by TLC. After the completion of the reaction, 200 mL of ethyl acetate was added, and the mixture was stirred, filtered, and the filtrate was washed twice with saturated brine and then washed with  44.5, 124.4, 125.4, 125.9, 131.3, 133.2, 133.5, 134.7, 135.2, 142.7, 164.0, 181.0, 182.7; UV λmax (THF) (nm): 262,335.

Synthesis of 1-nitro-2-carboxy anthraquinone (5)
5.0 g (18.7 mmol) of 1-nitro-2-methylanthraquinone (2)and 15.7 g (60.0 mmol) of Na2Cr2O7 were weighed into a 250 mL three-necked flask. 10 mL of water was added and the mixture was mixed as40 mL of 98% concentrated sulfuric acid was added under ice-water cooling. Thereaction was carried out at 60 °C for 5 hours before the temperature was raised to 85-90 °C for another 6 hours. TLC was used to detect the completion of the reaction. After cooling, the mixture was poured into a large amount of ice water while being stirred and filtered. The filter cake was then redissolved in 30 mL ofa 5% NaOH aqueous solution at 90 °C . After dissolving for 1 hour and undergoing another round of vacuum-filtration, the filtrate was acidified with 6 mol/L hydrochloric acid, vacuum filtered, and the filter cake was dried to obtain 3.2 g of a solid crystal. Synthesis of 1-nitro-2-benzoyl anthraquinone (6) [S2] 3.5 g (11.8 mmol) of 1-nitro-2-carboxy anthraquinone (3) was weighed into a 100 mL three-necked flask. 40 mL of chlorobenzene was added as a solvent. 5 mL of resteamed SOCl2 at room temperature was then added. The mixture was heated to 120 °C , and refluxed for 4 hours.
After the reaction was completed, it was cooled to room temperature and vacuum-filtered. The filter cake was washed with a small amount of anhydrous diethyl ether and dried under an infrared lamp to obtain 3.2 g of beige solid. The yield was 57.5%. The product was directly subjected to the next reaction without further purification. Melting point: 286 °C .

Synthesis of 1-amino-2-carboxy anthraquinone (7)
6.0 g (20.2mmol) of 1-nitro-2-carboxy anthraquinone (5) was weighed into a 500mL single-necked flask, add 400mL of 1% ammonia solution as solvent, and slowly add 10mL of 0.7g (18.5mmol) of NaBH4 aqueous solution at room temperature. After the addition is completed, the reaction solution is allowed to stand at room temperature. After stirring for 4 hours, TLC followed the progress of the reaction. After completion of the reaction, the reaction solution was diluted with 100 mL of distilled water, acidified with dilute hydrochloric acid, suction filtered, and the filter cake was washed three times with water and dried to give a product of 5.1 g. The yield was 99%.

General synthesis of anthraquinone-amino acid compounds (8)
(8a-8i) About 7.5 mmol of each amino acid was weighed into a 100 mL three-necked flask. 25 mL of an aqueous solution containing about 0.3 g of NaOH was added, the mixture was stirred and cooled to 0 °C with ice water, and 1.6 g of 1-nitro-2-benzoyl anthraquinone (4) (5 mmol) in THF was added along with a 5 mL of an NaOH solution after 20 min. A dropping funnel was used to maintain a pH of 8-10 in the vigorously stirred solution. After completion of the dropwise addition, the reaction continued under ice water for 1 hour, and the reaction was allowed to proceed overnight at room temperature. The following day, the reaction mixture was poured into 200 mL of ice water, acidified with dilute hydrochloric acid (pH 1-2), stirred, vacuum-filtered, and the filtrate was washed with a small amount of water several times to obtain a solid product. This product was then separated and purified by column chromatography.