Design, synthesis and biological evaluation of novel urolithin derivatives targeting liver cancer cells.

Introduction
Cancer is becoming an urgent medical issue globally due to the annual increasing incidence and mortality rates1. It is imperative to develop new cancer treatment drugs.
Natural plants contain numerous natural active ingredients that are readily accessible and have good activity. Natural medicines or foods, including pomegranate, raspberry, and round-leaf grapes, are rich in ellagitannins, which have antioxidant2, antitumor3,4, antibacterial5, and antiviral properties6; however, they are poorly bioavailable in living organisms and are primarily metabolised by intestinal flora in the body to urolithins, which can accumulate in the body at concentrations of 0.2–20 μM7. They are eventually excreted in the urine and faeces8,9. Urolithins are named urolithins A–D according to the number of hydroxyl groups and substitution positions10. Urolithins are uncommon and predominant in sulphated, glycosylated, and methylated forms. They are widely distributed in the urine, faeces, and bile of animals11,12.
Previous studies have reported that urolithin has various biological activities, including antioxidant13, anti-inflammatory14–16, neuroprotective17–19, and anti-cancer. The anti-cancer activity is one of the most extensively researched areas, and it has different degrees of anti-cancer effects on bladder20,21,24,25, lung22, liver23–25, colorectal26–29, prostate30–33, breast34–37, pancreatic ductal adenocarcinoma38, and endometrial cancer39–41. However, urolithin is a dibenzopyran-6-one derivative with a rigid skeletal structure, which results in poor water solubility and low bioavailability, and there is a need to improve its pharmacological activity, which affects the drug-forming properties. Therefore, the structure was designed and modified herein to rectify the defective physicochemical properties of urolithins. Based on the drug collocation principle, alkylation and amination reactions were used to synthesise a series of derivatives with the structure of mUA-spacer group-cyclic amine group, with mUA as the parent compound and cyclic amine group as the terminal structure and brominated alkanes as the flexible carbon chain connecting mUA and cyclic amine group. We screened the anti-cancer activity of the derivatives and preliminary investigated the anti-cancer mechanism to obtain anti-cancer drugs with high anti-cancer activity, good solubility, high bioavailability, low toxic side effects, and new structures.

Materials and methods

General remarks
All the chemical reagents and solvents were obtained from commercial companies (Wuhan Xinshenshi Chemical Technology Co., Ltd., Hubei, China) and were used without further purification. The 1H NMR and 13C NMR of all urolithin derivatives (UD-2a, UD-3a to UD-3g, UD-4a to UD-4g, UD-5a to UD-5g) were determined using Brucker NMR spectrometer (400, 101, and 151 MHz) with chemical shifts (δ values) in parts per million of TMS units. Mass spectra were recorded using a high-resolution ESI on a Thermo Fisher Scientific ultra-high-resolution mass spectrometer at Wuhan University Medical Department. The purity was analytically determined using HPLC and was > 95%.
HepG2(Catalogue number:SCSP-510), T24(Catalogue number:SCSP-536), DU145(Catalogue number:TCHu222), and HK-2(Catalogue number:SCSP-511) cells were procured from the Shanghai Cell Bank of the Chinese Academy of Sciences. HepG2 and HK-2 cells were cultured in DMEM medium, T24 cells in McCoys’ 5 A medium, and DU145 cells in MEM medium (containing NEAA). Unless otherwise stated, all media contained 10% foetal bovine serum, 1% 100 U/mL penicillin, and 100 mg/mL streptomycin. All cells were cultured in an incubator at 37 °C with 5% CO2.

Synthesis

Synthesis of compound mUA
2-Bromo-5-methoxybenzoic acid (0.13 mol, 30 g), sodium hydroxide (0.28 mol, 11.25 g), and water (135 ml) were added into a round bottom flask and stirred until the solution became clear. Resorcinol (0.27 mol, 30 g) was added and reacted for 90 min in an oil bath at 70 °C. Additionally, 5% of CuSO4 (54 ml) solution was added dropwise and reacted in an oil bath at 80 °C. The reaction was monitored using TLC and completed after 2 h. The reaction solution was subsequently washed with water and filtered, the solid was dried, the resulting solid was recrystallized with isopropanol, and the precipitated solid was filtered and dried again to obtain 12.2 g of yellow-white solid powder.

3-hydroxy-8-methoxy-6H-benzo[c]chromen-6-one(mUA)
Light yellow solid, yield 38.79%.1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.18 (d, J = 8.9 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 2.8 Hz, 1H), 7.47 (dd, J = 8.9, 2.8 Hz, 1H), 6.81 (dd, J = 8.7, 2.4 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 160.97, 159.40, 158.91, 151.60, 128.96, 124.57, 124.37, 123.98, 120.45, 113.53, 111.19, 109.94, 103.28, 55.99. U-HR-MS (ESI) m/z: Calcd. for C14H9O4 [M－H]－: 241.0506. Found: 241.0505.

Synthesis of compound UD-2a
Methyl urolithin A (2.06 mmol, 500 mg) and 4–(2-chloroethyl)morpholine hydrochloride (4.12 mmol, 770 mg) were added into a round-bottom flask. Subsequently, potassium carbonate (4.12 mmol, 570 mg) and acetone (60 ml) were added. The reaction was stirred using a magnetic stirrer at 62 °C in an oil bath. The reaction process was monitored using TLC and completed after 24 h. Subsequently, the acetone was evaporated using a rotary evaporator, and the sample was purified with dichloromethane and silica gel, followed by silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator with a gradient elution with petroleum ether and ethyl acetate as the mobile phases, and 644.8 mg of white solid powder was obtained.

8-methoxy-3–(2-morpholinoethoxy)-6H-benzo[c]chromen-6-one(UD-2a)
White solid, yield 87.90%. 1H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 9.0 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.50 (dd, J = 8.8, 2.8 Hz, 1H), 7.08 − 6.93 (m, 2H), 4.19 (t, J = 5.7 Hz, 2H), 3.90 (s, 3H), 3.60 (t, J = 4.6 Hz, 4H), 3.35 (s, 2H), 2.62 (d, J = 85.6 Hz, 4H). 13C NMR (151 MHz, DMSO-d6) δ 160.42, 159.57, 158.84, 151.09, 128.13, 124.08, 123.94, 123.92, 120.39, 112.72, 110.93, 110.83, 101.98, 66.12, 65.83, 56.83, 55.62, 53.56. U-HR-MS (ESI) m/z: Calcd. for C20H22NO5 [M + H]+: 356.1492. Found: 356.1485.

Synthesis of compound UD-3a∼UD-3g
Methyl urolithin A (82.57 mmol, 20 g), 1,3-dibromopropane (165.14 mmol, 33.4 g), potassium carbonate (165.14 mmol, 22.8 g), and acetone (240 ml) were added into a round-bottom flask, and the reaction was stirred at 62 °C in an oil bath. The reaction was monitored using TLC and completed after 24 h. The reaction was subsequently evaporated using a rotary evaporator. The acetone was extracted, and dichloromethane and silica gel were added to purify the sample. Subsequently, silica gel column chromatography was used to separate the target product, and the eluate containing the target product was evaporated using a rotary evaporator with petroleum ether and ethyl acetate as the mobile phase gradient to obtain 5.95 g of urolithin intermediate (UI) UI-1
UI-1 (0.55 mmol, 200 mg), potassium carbonate (cyclic amine group: 1.1 mmol, 152 mg; open chain amine group: 1.65 mmol, 228 mg), acetonitrile (30 ml), and a small amount of potassium iodide were added into a round bottom flask. Subsequently, the amine group was added to the reaction at a molar ratio of UI-1: cyclic amine group terminal of 1:1.1. The reaction was performed in an oil bath at 82 °C. The reaction was monitored using TLC and the iodine cylinder colour development principle, and the process was completed after 24 h. The acetonitrile was subsequently evaporated using a rotary evaporator. Dichloromethane and silica gel were added to purify the sample. The sample was subjected to silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator with petroleum ether and ethyl acetate or dichloromethane and methanol as the mobile phase gradient. The resulting solid was dissolved in dichloromethane and filtered. The resulting solid was dissolved in dichloromethane and filtered, and the filtrate was evaporated and dried to obtain the derivatives UD-3a ∼ UD-3g.

3–(3-bromopropoxy)-8-methoxy-6H-benzo[c]chromen-6-one(UI-1)
White solid, yield 19.84%. 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 8.9 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.50 (dd, J = 8.9, 2.8 Hz, 1H), 7.01 (s, 2H), 6.99 (d, J = 2.6 Hz, 1H), 4.19 (t, J = 5.9 Hz, 2H), 3.90 (s, 3H), 3.70 (t, J = 6.5 Hz, 2H), 2.29 (p, J = 6.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.88, 159.89, 159.31, 151.54, 128.55, 124.64, 124.42, 120.88, 113.06, 111.45, 111.35, 102.45, 66.34, 56.09, 32.14, 31.62. U-HR-MS (ESI) m/z: Calcd. for C17H16BrO4 [M + H]+: 363.0226. Found: 363.0226.

8-methoxy-3–(3-(pyrrolidin-1-yl)propoxy)-6H-benzo[c]chromen-6-one(UD-3a)
Yellow-white solid, yield 72.92%. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.0 Hz, 1H), 8.22 − 8.14 (m, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.51 (dd, J = 8.9, 2.9 Hz, 1H), 7.05 − 6.92 (m, 2H), 4.14 (t, J = 6.2 Hz, 2H), 3.90 (s, 3H), 2.83 (d, J = 23.2 Hz, 6H), 2.01 (p, J = 6.4 Hz, 2H), 1.80 (p, J = 3.3 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 160.87, 160.02, 159.27, 151.52, 128.55, 124.57, 124.40, 124.37, 120.81, 113.04, 111.33, 111.28, 102.34, 66.48, 56.08, 54.02, 52.30, 27.48, 23.38. U-HR-MS (ESI) m/z: Calcd. for C21H24NO4 [M + H]+: 354.1700. Found: 354.1700.

8-methoxy-3–(3-(piperidin-1-yl)propoxy)-6H-benzo[c]chromen-6-one(UD-3b)
Yellow-white solid, yield 62.45%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.36 (dd, J = 8.8, 2.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.6 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.92 (s, 3H), 2.50 (dd, J = 8.4, 6.6 Hz, 2H), 2.42 (s, 4H), 2.08 − 1.96 (m, 2H), 1.60 (q, J = 5.6 Hz, 4H), 1.46 (d, J = 6.9 Hz, 2H). 13C NMR (151 MHz, Chloroform-d) δ 161.83, 160.37, 159.34, 151.84, 128.94, 124.65, 123.27, 122.99, 121.18, 112.95, 111.40, 111.20, 102.42, 67.16, 56.02, 55.94, 54.85, 26.81, 26.10, 24.57. U-HR-MS (ESI) m/z: Calcd. for C22H26NO4 [M + H]+: 368.1856. Found: 368.1856.

3–(3-(Azepan-1-yl)propoxy)-8-methoxy-6H-benzo[c]chromen-6-one(UD-3c)
Yellow-white solid, yield 62.95%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.90 (dd, J = 8.7, 2.5 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 2.69 (td, J = 6.4, 5.8, 3.0 Hz, 6H), 2.06–1.95 (m, 2H), 1.72–1.57 (m, 8H). 13C NMR (151 MHz, Chloroform-d) δ 161.86, 160.42, 159.35, 151.86, 128.96, 124.67, 123.28, 123.00, 121.19, 112.97, 111.40, 111.21, 102.43, 67.04, 55.96, 55.71, 54.78, 28.09, 27.46, 27.22. U-HR-MS (ESI) m/z: Calcd. for C23H28NO4 [M + H]+: 382.2013. Found: 382.2013.

8-methoxy-3–(3-(4-methylpiperidin-1-yl)propoxy)-6H-benzo[c]chromen-6-one(UD-3d)
Yellow solid, yield 51.75%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.09 (t, J = 6.2 Hz, 2H), 3.93 (s, 3H), 3.08 (d, J = 10.7 Hz, 2H), 2.73 − 2.64 (m, 2H), 2.21 − 2.09 (m, 4H), 1.72 (s, 1H), 1.49 − 1.37 (m, 4H), 0.96 (d, J = 5.5 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 161.60, 159.90, 159.13, 151.54, 128.62, 124.44, 123.13, 122.81, 120.93, 112.55, 111.30, 110.94, 102.24, 66.63, 55.77, 55.38, 53.87, 33.46, 30.42, 26.20, 21.68. U-HR-MS (ESI) m/z: Calcd. for C23H28NO4 [M + H]+: 382.2013. Found: 382.2013.

8-methoxy-3–(3-morpholinopropoxy)-6H-benzo[c]chromen-6-one(UD-3e)
White solid, yield 79.17%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.6 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 4.09 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.74 (t, J = 4.7 Hz, 4H), 2.55 (t, J = 7.3 Hz, 2H), 2.49 (t, J = 4.7 Hz, 4H), 2.01 (p, J = 6.6 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.60, 160.04, 159.11, 151.58, 128.64, 124.45, 123.10, 122.78, 120.92, 112.72, 111.22, 110.93, 102.10, 66.99, 66.55, 55.74, 55.41, 53.76, 26.27. U-HR-MS (ESI) m/z: Calcd. for C21H24NO5 [M + H]+: 370.1649. Found: 370.1649.

8-methoxy-3–(3-(4-methylpiperazin-1-yl)propoxy)-6H-benzo[c]chromen-6-one(UD-3f)
Yellow-white solid, yield 66.29%. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.0 Hz, 1H), 8.22 − 8.15 (m, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.52 (dd, J = 8.9, 2.8 Hz, 1H), 6.98 (d, J = 7.5 Hz, 2H), 4.12 (t, J = 6.3 Hz, 2H), 3.91 (s, 3H), 2.64 (d, J = 69.9 Hz, 10H), 2.44 (s, 3H), 1.93 (p, J = 6.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.90, 160.14, 159.28, 151.55, 128.59, 124.59, 124.44, 124.40, 120.82, 113.09, 111.35, 111.24, 102.31, 66.69, 56.09, 54.20, 53.92, 51.50, 26.20. U-HR-MS (ESI) m/z: Calcd. for C22H27N2O4 [M + H]+: 383.1965. Found: 383.1960.

8-methoxy-3–(3–(4-phenylpiperazin-1-yl)propoxy)-6H-benzo[c]chromen-6-one(UD-3g)
White solid, yield 44.81%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 7.32 − 7.23 (m, 2H), 6.99 − 6.82 (m, 5H), 4.11 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.28 − 3.21 (m, 4H), 2.67 (t, J = 5.0 Hz, 4H), 2.63 (t, J = 7.3 Hz, 2H), 2.07 (p, J = 6.6 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.63, 160.08, 159.14, 151.62, 151.29, 129.14, 128.68, 124.49, 123.13, 122.82, 120.96, 119.75, 116.09, 112.73, 111.26, 110.95, 102.17, 66.67, 55.77, 55.07, 53.32, 49.14, 26.60. U-HR-MS (ESI) m/z: Calcd. for C27H29N2O4 [M + H]+: 445.2122. Found: 445.2121.

Synthesis of compound UD-4a∼UD-4g
Methyl urolithin A (82.57 mmol, 20 g), 1,4-dibromobutane (165.14 mmol, 35.7 g), potassium carbonate (165.14 mmol, 22.8 g), and acetone (240 ml) were added into a round-bottom flask. The reaction was stirred at 62 °C in an oil bath. The reaction process was monitored using TLC and completed after 24 h. The acetone was removed by evaporation using a rotary evaporator. Dichloromethane and silica gel were added to purify the sample, followed by silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator with petroleum ether and ethyl acetate as mobile phases in gradient elution to obtain 5.48 g of intermediate UI-2.
UI-2 (0.53 mmol, 200 mg), potassium carbonate (cyclic amine group: 1.06 mmol, 147 mg; open chain amine group: 1.59 mmol, 220 mg), acetonitrile (30 ml), and a small amount of potassium iodide were added into a round bottom flask. Subsequently, the amine group terminals were added to the reaction at a molar ratio of UI-2: cyclic amine group terminal of 1:1.1. The reaction was magnetically stirred in an oil bath at 82 °C. The reaction was monitored using TLC and the iodine cylinder colour development principle, and the process was completed after 24 h. Subsequently, the acetonitrile was removed by evaporation using a rotary evaporator, and dichloromethane and silica gel were added to purify the sample, followed by silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator with petroleum ether and ethyl acetate or dichloromethane and methanol as the mobile phase gradient. The resulting solid was dissolved in dichloromethane and filtered, and the filtrate was evaporated and dried to obtain the derivatives UD-4a ∼ UD-4g.

3–(4-bromobutoxy)-8-methoxy-6H-benzo[c]chromen-6-one(UI-2)
White solid, yield 17.59%. 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 9.0 Hz, 1H), 8.18 − 8.11 (m, 1H), 7.59 (d, J = 2.8 Hz, 1H), 7.49 (dd, J = 8.9, 2.9 Hz, 1H), 6.96 (d, J = 7.9 Hz, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.89 (s, 3H), 3.63 (t, J = 6.6 Hz, 2H), 2.04 − 1.91 (m, 2H), 1.94 − 1.78 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.87, 160.08, 159.23, 151.52, 128.57, 124.51, 124.36, 124.33, 120.78, 113.05, 111.29, 111.20, 102.27, 67.65, 56.06, 35.30, 29.48, 27.70. U-HR-MS (ESI) m/z: Calcd. for C18H18BrO4 [M + H]+: 377.0383. Found: 377.0383.

8-methoxy-3–(4-(pyrrolidin-1-yl)butoxy)-6H-benzo[c]chromen-6-one(UD-4a)
Yellow solid, yield 91.37%. 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.9 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.52 (dd, J = 8.9, 2.9 Hz, 1H), 7.04 − 6.96 (m, 2H), 4.13 (d, J = 5.6 Hz, 2H), 3.91 (s, 3H), 3.22 (t, J = 7.5 Hz, 2H), 3.21 (s, 2H), 2.02 − 1.77 (m, 10H). 13C NMR (151 MHz, DMSO-d6) δ 160.42, 159.59, 158.88, 151.11, 128.12, 124.16, 123.99, 123.97, 120.40, 112.66, 110.97, 110.88, 101.97, 67.39, 55.65, 53.66, 53.21, 40.05, 25.60, 22.59, 22.20. U-HR-MS (ESI) m/z: Calcd. for C22H26NO4 [M + H]+: 368.1856. Found: 368.1851.

8-methoxy-3–(4-(piperidin-1-yl)butoxy)-6H-benzo[c]chromen-6-one(UD-4b)
Yellow-white solid, yield 77.13%. 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 9.0 Hz, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.52 (dd, J = 8.9, 2.8 Hz, 1H), 7.03 − 6.95 (m, 2H), 4.14 − 4.07 (m, 2H), 3.90 (s, 3H), 2.85 (s, 6H), 1.88 − 1.40 (m, 10H). 13C NMR (101 MHz, DMSO-d6) δ 160.89, 160.08, 159.27, 151.54, 128.57, 124.57, 124.39, 120.80, 113.08, 111.33, 111.24, 102.32, 68.03, 56.79, 56.07, 53.16, 26.41, 23.99, 22.71, 21.53. U-HR-MS (ESI) m/z: Calcd. for C23H28NO4 [M + H]+: 382.2013. Found: 382.2007.

3–(4-(Azepan-1-yl)butoxy)-8-methoxy-6H-benzo[c]chromen-6-one(UD-4c)
Yellow solid, yield 50.07%. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.0 Hz, 1H), 8.22 − 8.14 (m, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.51 (dd, J = 8.9, 2.9 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.90 (s, 3H), 2.76 (d, J = 35.0 Hz, 6H), 1.86 − 1.50 (m, 12H). 13C NMR (101 MHz, DMSO-d6) δ 160.88, 160.16, 159.23, 151.53, 128.59, 124.52, 124.38, 124.33, 120.77, 113.06, 111.30, 111.14, 102.24, 68.28, 57.22, 56.07, 54.94, 26.80, 26.63, 23.25. U-HR-MS (ESI) m/z: Calcd. for C24H30NO4 [M + H]+: 396.2169. Found: 396.2170.

8-methoxy-3–(4-(4-methylpiperidin-1-yl)butoxy)-6H-benzo[c]chromen-6-one(UD-4d)
Yellow-white solid, yield 70.72%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.7 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.92 − 6.82 (m, 2H), 4.03 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 2.93 (d, J = 10.4 Hz, 2H), 2.44 − 2.36 (m, 2H), 1.99 − 1.78 (m, 5H), 1.77 − 1.60 (m, 4H), 1.27 (qd, J = 11.9, 3.6 Hz, 2H), 0.93 (d, J = 6.1 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 161.65, 160.14, 159.08, 151.60, 128.72, 124.46, 123.07, 122.78, 120.90, 112.77, 111.11, 110.91, 102.04, 68.23, 58.66, 55.74, 54.08, 34.32, 30.85, 27.25, 23.61, 21.94. U-HR-MS (ESI) m/z: Calcd. for C24H30NO4 [M + H]+: 396.2169. Found: 396.2164.

8-methoxy-3–(4-morpholinobutoxy)-6H-benzo[c]chromen-6-one(UD-4e)
Light yellow solid, yield 84.12%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.7, 2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 4.05 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.73 (t, J = 4.7 Hz, 4H), 2.48 (s, 4H), 2.46 − 2.39 (m, 2H), 1.86 (dt, J = 8.6, 6.4 Hz, 2H), 1.71 (ddd, J = 14.8, 9.0, 6.1 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.63, 160.07, 159.11, 151.60, 128.68, 124.48, 123.11, 122.79, 120.92, 112.75, 111.18, 110.93, 102.02, 68.11, 66.98, 58.58, 55.75, 53.72, 27.02, 23.05. U-HR-MS (ESI) m/z: Calcd. for C22H26NO5 [M + H]+: 384.1805. Found: 384.1802.

8-methoxy-3–(4-(4-methylpiperazin-1-yl)butoxy)-6H-benzo[c]chromen-6-one(UD-4f)
White solid, yield 90.39%. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.52 (dd, J = 8.9, 2.9 Hz, 1H), 7.04 − 6.94 (m, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.90 (s, 3H), 3.78 − 2.63 (m, 10H), 2.57 (s, 3H), 1.77 (p, J = 6.4 Hz, 2H), 1.64 (d, J = 12.5 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.63, 160.07, 159.11, 151.60, 128.68, 124.48, 123.11, 122.79, 120.92, 112.75, 111.18, 110.93, 102.02, 68.11, 66.98, 58.58, 55.75, 53.72, 27.02, 23.05. U-HR-MS (ESI) m/z: Calcd. for C23H29N2O4 [M + H]+: 397.2122. Found: 397.2128.

8-methoxy-3-(4-(4-phenylpiperazin-1-yl)butoxy)-6H-benzo[c]chromen-6-one(UD-4g)
White solid, yield 81.03%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 7.27 (d, J = 15.8 Hz, 1H), 6.97 − 6.81 (m, 6H), 4.06 (t, J = 6.2 Hz, 2H), 3.93 (s, 3H), 3.22 (t, J = 5.0 Hz, 4H), 2.68 − 2.61 (m, 4H), 2.54 − 2.45 (m, 2H), 1.94 − 1.83 (m, 2H), 1.81 − 1.69 (m, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.65, 160.11, 159.12, 151.62, 151.32, 129.13, 128.70, 124.49, 123.13, 122.81, 120.94, 119.71, 116.05, 112.78, 111.19, 110.94, 102.06, 68.17, 58.22, 55.76, 53.29, 49.15, 27.13, 23.43. U-HR-MS (ESI) m/z: Calcd. for C28H31N2O4 [M + H]+: 459.2278. Found:459.2276.

Synthesis of compound UD-5a∼UD-5g
Methyl urushiol A (82.57 mmol, 20 g), 1,5-dibromobutane (165.14 mmol, 38 g), potassium carbonate (165.14 mmol, 22.8 g), and acetone (240 ml) were added into a round-bottom flask. The reaction was stirred in an oil bath at 62 °C. The reaction was monitored using TLC and completed after 24 h. The acetone was evaporated using a rotary evaporator. Subsequently, the sample was purified with dichloromethane and silica gel, followed by silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator to obtain 5.79 g of intermediate UI-3.
UI-3 (0.51 mmol, 200 mg), potassium carbonate (cyclic amine group: 1.02 mmol, 141 mg; open chain amine group: 1.53 mmol, 212 mg), acetonitrile (30 ml), and a small amount of potassium iodide were added into a round bottom flask, and the amine group terminals were added into the reaction at a molar ratio of UI-3:amine group terminals of 1:1.1. The oil bath was stirred magnetically at 82 °C, and the reaction was monitored using TLC and the iodine cylinder colour development principle and completed after 24 h. Subsequently, the acetonitrile evaporated using a rotary evaporator. Subsequently, dichloromethane and silica gel were added to purify the sample, followed by silica gel column chromatography to separate the target product. The eluate containing the target product was evaporated using a rotary evaporator with petroleum ether and ethyl acetate or dichloromethane and methanol as the mobile phase gradient. The resulting solid was dissolved with dichloromethane and filtered, and the filtrate was evaporated and dried to obtain the derivatives UD-5a ∼ UD-5g.

3-((5-bromopentyl)oxy)-8-methoxy-6H-benzo[c]chromen-6-one(Intermediate3)
White solid, yield 17.92%. 1H NMR (400 MHz, DMSO-d6) δ 8.23 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 9.6 Hz, 1H), 7.59 (d, J = 2.8 Hz, 1H), 7.48 (dd, J = 8.8, 2.8 Hz, 1H), 6.99 − 6.91 (m, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.88 (s, 3H), 3.57 (t, J = 6.7 Hz, 2H), 1.88 (dt, J = 14.6, 6.8 Hz, 2H), 1.76 (p, J = 6.6 Hz, 2H), 1.61 − 1.45 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.86, 160.24, 160.16, 159.19, 151.51, 128.57, 124.47, 124.33, 124.29, 120.75, 113.02, 111.27, 111.12, 102.21, 68.33, 56.02, 35.54, 32.41, 28.13, 24.72. U-HR-MS (ESI) m/z: Calcd. for C19H20BrO4 [M + H]+: 391.0539. Found: 391.0539.

8-methoxy-3-((5-(pyrrolidin-1-yl)pentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5a)
White solid, yield 67.03%. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.0 Hz, 1H), 8.21 − 8.14 (m, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.51 (dd, J = 8.9, 2.9 Hz, 1H), 6.98 (d, J = 8.2 Hz, 2H), 4.08 (t, J = 6.4 Hz, 2H), 3.90 (s, 3H), 2.95 (s, 4H), 2.87 (t, J = 7.8 Hz, 2H), 1.84 (p, 4H), 1.77 (q, J = 7.0 Hz, 2H), 1.63 (p, J = 15.4, 7.6 Hz, 2H), 1.53 − 1.40 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.90, 160.19, 159.26, 151.55, 128.59, 124.56, 124.42, 124.37, 120.79, 113.08, 111.33, 111.17, 102.27, 68.31, 56.08, 54.88, 53.79, 28.58, 26.48, 23.42, 23.20. U-HR-MS (ESI) m/z: Calcd. for C23H28NO4 [M + H]+: 382.2013. Found: 382.2008.

8-methoxy-3-((5-(piperidin-1-yl)pentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5b)
Yellow-white solid, yield 96.95%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 3.92 (s, 3H), 2.65 (s, 4H), 2.60 − 2.51 (m, 2H), 1.86 (dt, J = 14.5, 6.5 Hz, 2H), 1.77 (dq, J = 13.1, 7.7, 6.8 Hz, 6H), 1.58 − 1.46 (m, 4H). 13C NMR (101 MHz, Chloroform-d) δ 161.64, 160.09, 159.08, 151.56, 128.68, 124.44, 123.12, 122.80, 120.88, 112.67, 111.15, 110.92, 102.08, 68.14, 58.90, 55.75, 54.32, 28.89, 25.88, 25.09, 24.03, 23.85. U-HR-MS (ESI) m/z: Calcd. for C24H30NO4 [M + H]+: 396.2169. Found: 396.2164.

3-((5-(Azepan-1-yl)pentyl)oxy)-8-methoxy-6H-benzo[c]chromen-6-one(UD-5c)
Yellow solid, yield 92.05%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.75 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.82 (d, J = 2.5 Hz, 1H), 4.02 (t, J = 6.2 Hz, 2H), 3.92 (s, 3H), 3.03 − 2.96 (m, 4H), 2.85 − 2.77 (m, 2H), 1.86 (q, J = 7.1 Hz, 8H), 1.69 (p, J = 2.7 Hz, 4H), 1.54 (ddd, J = 15.2, 9.2, 6.3 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.63, 160.01, 159.10, 151.53, 128.64, 124.43, 123.16, 122.83, 120.87, 112.61, 111.20, 110.94, 102.12, 68.02, 57.78, 55.76, 55.03, 28.72, 26.96, 25.46, 23.80. U-HR-MS (ESI) m/z: Calcd. for C25H32NO4 [M + H]+: 410.2326. Found: 410.2320.

8-methoxy-3-((5–(4-methylpiperidin-1-yl)pentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5d)
Light yellow solid, yield 86.80%. 1H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J = 8.9 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 2.8 Hz, 1H), 7.39 (dd, J = 8.8, 2.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.09 − 3.01 (m, 2H), 2.53 − 2.44 (m, 2H), 2.08 (t, J = 10.9 Hz, 2H), 1.93 − 1.81 (m, 2H), 1.70 (dt, J = 10.6, 3.8 Hz, 4H), 1.59 − 1.47 (m, 2H), 1.42 (td, J = 14.1, 11.6, 6.6 Hz, 3H), 0.96 (d, J = 5.3 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 161.67, 160.12, 159.10, 151.59, 128.72, 124.48, 123.12, 122.81, 120.91, 112.70, 111.16, 110.93, 102.09, 68.18, 58.67, 55.77, 53.87, 33.56, 30.50, 28.93, 26.25, 24.08, 21.72. U-HR-MS (ESI) m/z: Calcd. for C25H32NO4 [M + H]+: 410.2326. Found: 410.2320.

8-methoxy-3-((5-morpholinopentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5e)
White solid, yield 85.64%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.73 (t, J = 4.7 Hz, 4H), 2.46 (d, J = 5.2 Hz, 4H), 2.43–2.34 (m, 2H), 1.85 (p, J = 6.7 Hz, 2H), 1.64–1.46 (m, 4H). 13C NMR (151 MHz, Chloroform-d) δ 161.85, 160.37, 159.37, 151.87, 128.94, 124.69, 123.31, 123.00, 121.19, 112.99, 111.40, 111.21, 102.29, 68.46, 67.16, 59.14, 55.96, 53.98, 29.19, 26.46, 24.17. U-HR-MS (ESI) m/z: Calcd for C23H28NO5 [M + H]+: 398.1962. Found: 398.1958.

8-methoxy-3-((5–(4-methylpiperazin-1-yl)pentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5f)
Yellow solid, yield 62.52%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 4.02 (t, J = 6.2 Hz, 2H), 3.93 (s, 3H), 2.99 (s, 8H), 2.71 − 2.63 (m, 2H), 2.57 (s, 3H), 1.91 − 1.80 (m, 2H), 1.80 − 1.70 (m, 2H), 1.54 (tt, J = 10.0, 6.2 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 161.64, 160.00, 159.12, 151.54, 128.64, 124.46, 123.19, 122.84, 120.88, 112.69, 111.23, 110.96, 102.08, 68.03, 57.47, 55.77, 53.23, 50.97, 44.68, 28.73, 25.44, 23.70.U-HR-MS (ESI) m/z: Calcd. for C24H31N2O4 [M + H]+: 411.2278. Found: 411.2278.

8-methoxy-3-((5–(4-phenylpiperazin-1-yl)pentyl)oxy)-6H-benzo[c]chromen-6-one(UD-5g)
White solid, yield 85.69%. 1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.37 (dd, J = 8.8, 2.8 Hz, 1H), 7.26 (d, J = 15.8 Hz, 1H), 6.97 − 6.81 (m, 6H), 4.03 (t, J = 6.4 Hz, 2H), 3.92 (s, 3H), 3.22 (t, J = 5.0 Hz, 4H), 2.63 (t, J = 5.0 Hz, 4H), 2.49 − 2.41 (m, 2H), 1.87 (p, J = 6.7 Hz, 2H), 1.70 − 1.52 (m, 4H). 13C NMR (101 MHz, Chloroform-d) δ 161.66, 160.15, 159.11, 151.62, 151.32, 129.12, 128.72, 124.49, 123.12, 122.80, 120.93, 119.70, 116.05, 112.78, 111.16, 110.93, 102.04, 68.25, 58.58, 55.77, 53.34, 49.13, 29.03, 26.66, 24.08. U-HR-MS (ESI) m/z: Calcd. for C29H33N2O4 [M + H]+: 473.2435. Found: 473.2429.

Cell viability assay
The CCK-8 assay was used to determine cell viability. The density of DU145 and T24 cells was adjusted to 1 × 104 cells/mL, and the density of HepG2 cells was adjusted to 8,000 cells/mL using the fresh complete medium. Subsequently, 100 μL of cell suspension were added into 96-well plates and incubated at 37 °C with 5% CO2 for 24 h. Furthermore, 100 μL of compound solution with different concentrations was re-added to each well and incubated for an additional 48 h. The drug solution was aspirated, and the wells were rinsed with 100 μL of basal medium containing 10% CCK-8 reagent. The plate was placed in an electrically heated incubator at 37 °C for incubation. After 30 min, the absorbance at 450 nm was measured using a multi-functional imaging microplate reader. The IC50 was calculated using GraphPad Prism software (version 8.0), and each concentration was repeated at least thrice.

Colony assay
HepG2 cells were placed into 6-well plates at 5 × 103 cells per well and incubated for 24 h. Subsequently, the cells were treated with compound UD-4c at concentrations of 0, 2, 4, and 10 μM for 48 h. The 6-well plates were replaced every two days for six days, after which the cells were fixed with 4% paraformaldehyde for 40 min. Subsequently, 1 ml of 1% crystalline violet staining solution was added for 30 min at room temperature and photographed and recorded.

Hoechst 33,258 staining
HepG2 cells were placed into 6-well plates at 1.8 × 105 cells per well and incubated for 24 h. Subsequently, the cells were treated with compound UD-4c at concentrations of 0, 2, 4, and 10 μM for 48 h. After removal and rinsing, 1 ml of Hoechst 33258 staining solution was added to each well. The samples were incubated at 37 °C for 30 min to avoid light staining. Subsequently, the staining solution was aspirated and rinsed twice. The samples were photographed using an inverted fluorescence microscope.

Cell apoptosis assay
HepG2 cells were placed into 6-well plates at 2.5 × 105 cells per well and incubated for 24 h. Subsequently, the cells were treated with compound UD-4c at concentrations of 0, 4, 10, and 20 μM for 48 h, followed by wetting and digestion, and all cells were collected. We added 100 μL of pre-chilled 1 × Annexin V Binding Buffer to each group, and the cells were resuspended. Each group was added with 5 μL each of Annexin V-FITC and PI staining solution, vortexed and mixed, and stained at room temperature for 15 min. Subsequently, 400 μL of pre-chilled 1 × Annexin V Binding Buffer working solution was added and detected using a flow cytometer.

Cell cycle assay
HepG2 cells were placed into 6-well plates at 2.0 × 105 cells per well and incubated for 24 h. Subsequently, the cells were treated with compound UD-4c at concentrations of 0, 4, 10, and 20 μM for 48 h, washed, digested, and centrifuged. Subsequently, 1 ml of PBS was added, and the cells were resuspended. The suspended cell was added to 3 ml of pre-chilled anhydrous ethanol at −20 °C several times. The cells were centrifuged, and 500 μL of PI/Rnase A staining solution was added, vortexed, mixed, and stained for 40 min at room temperature, protected from light, and detected using a flow cytometer.

Result and discussion

Chemistry
Schemes 1–5 depict the overall synthetic routes of urolithin derivatives. We synthesised mUA from resorcinol and 2-bromo-5-methoxybenzoic acid, which were first reacted to form an ester under the action of NaOH. Subsequently, mUA was synthesised in a reaction catalysed by Cu2+, resulting in a 38.79% yield (Scheme 1). In Scheme 2, mUA was synthesised by an alkylation reaction with 4–(2-chloroethyl)morpholine hydrochloride in the presence of K2CO3 to synthesise the derivative UD-2a in an 87.90% yield. In Schemes 3, 4, and 5, mUA was alkylated with 1,3-dibromopropane, 1,4-dibromobutane, and 1,5-dibromopentane, respectively, catalysed by K2CO3 to generate urolithin derivative intermediates UI-1, UI-2, and UI-3, and intermediates UI-1, UI-2, and UI-3 were alkylated with a series of cyclic amine group terminals, respectively, catalysed by K2CO3 and KI to generate a series of derivatives, UD-1, UI-2, and UI-3. The amination reaction produced a series of derivatives, UD-3a ∼ UD-3g, UD-4a ∼ UD∼4g, and UD-5a ∼ UD-5g, with yields ranging from 44.81%–81.52%, 35.25%–91.37%, and 49.99%–97.42%, respectively.

In vitro cytotoxicity
The Cell Counting Kit-8 (CCK-8 assay) assay was used to assess the effects of all urolithin derivatives on HepG2 (human hepatocellular carcinoma cells), T24 (human bladder metastatic cell carcinoma cells), and DU145 (human bladder carcinoma cells) proliferation. The clinical drug sorafenib was used as a positive control for HepG2 cells, while cisplatin was used as a positive control for T24 and DU145 cells. The cytotoxicity of the derivatives is expressed as half-inhibitory concentration IC50 in Table 1, and IC50 was calculated using linear regression analysis of the concentration-inhibition curves for each derivative. The results revealed that most derivatives inhibited DU145, T24, and HepG2 cell proliferation to varying degrees, and the activity was superior to that of the parent compound mUA.
For DU145 cells, the IC50 after 48 h of mUA treatment was 55.84 ± 0.51 μM. Table 1 reveals that the IC50 of all derivatives except derivatives UD-2a, UD-3d, UD-3g, UD-4e, UD-4g, UD-5e, and UD-5g was < 55.84 ± 0.51 μM, and the anti-proliferative activity was better than that of mUA, among which UD-5b exhibited the best anti-proliferative activity with an IC50 of 10.13 ± 0.36 μM. However, the IC50 of all derivatives was greater than that of the positive drug cisplatin (IC50 = 2.95 ± 0.22 μM), and the anti-proliferative activity was weaker than that of cisplatin.
For T24 cells, the IC50 after 48 h of mUA treatment was 49.76 ± 0.44 μM. Table 1 reveals that the IC50 of all derivatives except derivatives UD-2a, UD-3d, UD-3e, UD-3g, UD-4e, UD-4g, and UD-5e, UD-5g were < 49.76 ± 0.44 μM, and the anti-proliferative activity was better than that of mUA. However, the IC50 of all derivatives was higher than that of the cisplatin (IC50 = 14.49 ± 0.13 μM). The proliferative activity of UD-3a was the best, with IC50 of 17.03 ± 0.26 μM.
For HepG2 cells, the IC50 of mUA treatment for 48 h was 11.36 ± 0.92 μM. Table 1 reveals the IC50 of all derivatives was < 11.36 ± 0.92 μM, except for derivatives UD-2a, UD-3e, UD-3f, UD-3g, UD-3h, UD-4e, UD-4g, UD-5e, UD-5g, and the anti-proliferative activity was better than that of mUA. The IC50 of most derivatives was < 10 μM after 48 h treatment of HepG2 cells. Compared with the positive drug sorafenib (IC50 = 7.76 ± 0.12 μM), the derivatives UD-3a (IC50 = 7.17 ± 0.26 μM), UD-3d (IC50 = 7.06 ± 0.34 μM), UD-4b (IC50 = 6.60 ± 0.11 μM), and UD-5a (IC50 = 6.38 ± 0.24 μM) inhibited HepG2 cell proliferation slightly better than Sorafenib. The derivatives UD-3b (IC50 = 5.57 ± 0.16 μM), UD-4c (IC50 = 4.66 ± 0.12 μM), UD-5b (IC50 = 5.10 ± 0.02 μM), and UD-5c (IC50 = 4.95 ± 0.03 μM) inhibited the HepG2 cell proliferation significantly better than Sorafenib, while derivative UD-4c exhibited the best anti-proliferative activity.
Derivatives UD-3g, UD-4g, and UD-5g with N-phenylpiperazine at the amino-terminal demonstrated no anti-proliferative activity against DU145, T24, and HepG2, while derivatives UD-3f, UD-4f, and UD-5f with N-methyl piperazine at the amino-terminal demonstrated better anti-proliferative activity against the above three cancer cells because the spatial site resistance of phenyl in N-phenylpiperazine is higher than that of methyl in N-methyl piperazine. Therefore, the steric hindrance of the amino-terminal should be moderate. UD-2a demonstrated no anti-proliferative activity against DU145, T24, and HepG2. However, UD-3e, UD-4e, and UD-5e demonstrated better anti-proliferative activity than UD-2a, while the amino-terminals of UD-2a, UD-3e, UD-4e, and UD-5e are all morpholine. The only difference lies in the number of carbon atoms in the flexible carbon chain, which are two, three, four, and five, respectively. This indicates that the flexible carbon chain should be moderate, with the carbon atoms more than two.
Table 2 and Figure 1. Illustrate the toxicity of the derivative UD-4c on human renal cortical proximal tubule epithelial cells (HK-2). Compared with HepG2 cells, UD-4c inhibited HepG2 growth much more than HK-2 and was less toxic to normal human cells.
The anti-proliferative activity of urolithin derivatives against HepG2 cells was significantly better than that of DU145 and T24 cells, and the derivative UD-4c exhibited the best activity. We subsequently used HepG2 cells to investigate the potential antitumor mechanism of derivative UD-4c based on the significant anti-proliferative activity and high selectivity of UD-4c against human hepatocellular carcinoma HepG2 cells.

Effect of compound UD-4c on growth inhibition in human hepatoma cancer cell lines HepG2
We used CCK-8 assay to measure the inhibition rate of derivative UD-4c acting on HepG2 cells for 24, 48, and 72 h. Figure 2 depicts the results. Derivative UD-4c significantly inhibited HepG2 cell proliferation dose- and time-dependently, and to further evaluate the anti-proliferative activity of derivative UD-4c on HepG2 cells, a plate clone formation assay was performed. Figure 3 depicts the results. Compared with the control group, the number of clone-forming cell colonies and cells per colony decreased as the derivative UD-4c concentration increased, and UD-4c significantly reduced HepG2 cell colony formation, especially in the high-dose group.

Effect of compound UD-4c on apoptosis in HepG2 cells
We performed apoptosis-related experiments to investigate whether the derivative UD-4c could induce apoptosis in HepG2 cells. Initially, we used Hoechst 33258 staining to qualitatively detect the ability of derivative UD-4c to promote apoptosis of HepG2 cells. The results are illustrated in Figure 4. Normal cells in the control group were stained blue, exhibited normal morphological structure, and were round or oval. More cells were apoptotic after UD-4c treatment for 48 h. The nuclei of apoptotic cells were stained bright blue and concentrated in the middle or side of the cells. Some were distributed in fragments inside the cells. The percentage of bright blue cells increased with concentration. Subsequently, we used the Annexin V-FITC&PI double staining method to quantify the apoptosis rate of derivative UD-4c treated HepG2 cells after 48 h. The results are depicted in Figures 5 and 6, where the upper right part is the late apoptotic or necrotic cells, and the lower right part is the early or middle apoptotic cells. Compared with the control group, the apoptosis rate of late apoptotic or necrotic cells and early or mid-stage apoptotic cells was higher than that of the control group, and the apoptosis rate increased with concentration. The qualitative and quantitative analyses revealed that the derivative UD-4c induced apoptosis in HepG2 cells dose-dependently.

Effect of compound UD-4c on cell cycle in HepG2 cells
Cell cycle analysis was used to investigate the anti-proliferative activity of the derivative UD-4c to confirm if it correlated with HepG2 cell cycle arrest. HepG2 cells were treated with the indicated concentrations of UD-4c for 48 h, stained with PI(Propidium Iodide) staining solution, and assayed using a flow cytometer. Figures 7 and 8 depict the result. Compared with the control group, the percentage of HepG2 cells in the G2/M phase gradually increased, from 12.73% to 16.36%, 20.77%, and 32.4%, respectively, while the cells in the S phase gradually decreased from 34.45% to 30.61%, 27.26%, and 18.16%, respectively. The change of cells in the G1/G0 phase was nonobvious; therefore, the derivative UD-4c induced HepG2 cells in the G2/M phase arrest dose-dependently, thus inhibiting HepG2 cell proliferation.

Conclusions
We synthesised 22 structurally new urolithin derivatives, evaluated their in vitro antitumor activity, and preliminary investigated the potential mechanism by which the derivative UD-4c inhibits hepatocellular carcinoma cell proliferation. In vitro, anti-proliferative experiments revealed that most derivatives exhibited good anti-proliferative activity against DU145, T24, and HepG2 cells. Additionally, they were more effective than the parent compound mUA and exhibited highly selective cytotoxicity against HepG2 cells. The derivative UD-4c exhibited the most potent proliferation inhibitory effect on HepG2 cells, significantly better than the first-line drug sorafenib for hepatocellular carcinoma treatment. The introduction of the amine structure significantly improved the anti-cancer activity. The preliminary investigation of the mechanism revealed that the derivative UD-4c could induce cell cycle delay, block HepG2 cells in the G2/M phase, and dose-dependently induce apoptosis of HepG2 cells, thus inhibiting HepG2 cell proliferation and exerting therapeutic effects on cancer while the toxicity of UD-4c to human normal cells HK-2 was much less than that of human hepatocellular carcinoma HepG2 cells.
This research broadens the prospects of study on drugs for the treatment of liver cancer. In the future, we will further study the cytotoxicity of the derivative UD-4c on other hepatocellular carcinoma cells. Meanwhile, we hypothesise that the anti-liver cancer effect of UD-4c may be related to its induction of oxidative stress, leading to mitochondrial apoptosis, and the regulation of apoptosis by affecting the ROS/AKT/FoxO3a axis, thus promoting the apoptosis of liver cancer cells. Meanwhile, we will explore the anti-liver cancer action targets and signalling pathways of UD-4c by combining experimental techniques such as molecular docking and western-blot, and conduct in - vivo verification using mice. We aim to deeply elucidate the anti-liver cancer mechanism of UD-4c and strive to provide potential therapeutic drugs for clinical practice.