Materials and methods
Chemistry
Synthesis of compound 4. To a solution of 3 (500 mg, 2.74 mmol) and TBSCl (455 mg, 3.0 mmol) in CH2Cl2 was added imidazole (204 mg, 3.0 mmol) at 0 °C. The mixture was stirred overnight, quenched with NH4Cl, and extracted with CH2Cl2. The CH2Cl2 layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide 4 (731 mg, 2.47 mmol, yield 90%).
Synthesis of compound 5. To a solution of trimethyl phosphonoacetate (1.48 mL, 9.17 mmol) in anhydrous THF (20 mL) was added LiHMDS (1.0 M, 9.17 mL, 9.17 mmol) at − 20 °C. After 1.5 h, the mixture was cooled to − 78 °C. Compound 4 (1.36 g, 4.59 mmol) in THF was added to the mixture. The reaction mixture was quenched with saturated NaCl and extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude oil. The resulting oil was dissolved in THF, which was added TBAF in THF (5 mL, 1 N). After 12 h, the reaction was quenched with NH4Cl, extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give an oil, which was purified with silica gel column chromatography to provide a white solid 5 (915 mg, yield 84%). 1H NMR (400 MHz, MeOD-d4) δ 8.19 (d, J = 16.2 Hz, 1H), 6.79 (d, J = 16.2 Hz, 1H), 6.20 (s, 2H), 3.95 (s, 6H), 3.86 (s, 3H). 13C NMR (100 MHz, MeOD-d4) δ 171.3, 162.4, 162.3, 137.4, 115.7, 104.9, 92.5, 55.9, 51.7. HRMS (ESI) cald for C12H15O5 [M + H]+ 239.0914, found 239.0910.
Synthesis of compounds 6a-6e. A mixture of 5 (100 mg, 0.42 mmol, 1 eq), K2CO3 (232.1 mg, 1.68 mmol, 4 eq) and different alkynyl bromide (1.47 mmol, 3.5 eq) in 4 mL anhydrous DMF was heated to 40 °C for 4 h. The reaction was quenched with NaCl and extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4 to give a crude oil, which was purified with silica gel column chromatography to provide white solid 6a-6e in yields of 67%–88%.
Synthesis of compound 8a. A mixture of 6a (110 mg, 0.4 mmol), LiOH•H2O (336 mg, 8 mmol, 20 eq) and THF–H2O (1:1) (4 mL) was stirred at 40 °C for 12 h. The pH of the mixture adjusted to 2–3 with 2 N HCl. The resulting mixture was extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude oil. The mixture of the crude oil, 7 (211 mg, 0.8 mmol, 2 eq), EDCI (155 mg, 0.8 mmol, 2 eq), DMAP (1.2 mg, 0.01 mmol), and Et3N (110.9 μL, 0.8 mmol, 2 eq) in 1 mL CH2Cl2 was stirred at room temperature for 12 h and quenched with sat. NaHCO3. The resulting mixture was extracted with CH2Cl2. The CH2Cl2 layer was dried over anhydrous Na2SO4 to give a crude oil, which was purified by silica gel column chromatography to provide a white solid 8a (109 mg, 0.22 mmol, 54% for two steps). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 16.2 Hz, 1H), 6.71 (d, J = 16.2 Hz, 1H), 6.20 (d, J = 5.2 Hz, 3H), 5.74 (t, J = 8.1 Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.73 (q, J = 4.9 Hz, 3H), 4.60 (d, J = 12.4 Hz, 1H), 3.95–3.75 (m, 7H), 3.20–3.04 (m, 1H), 2.94 (d, J = 9.4 Hz, 1H), 2.58 (t, J = 2.3 Hz, 1H), 2.54–2.13 (m, 6H), 1.65 (dd, J = 19.5, 6.7 Hz, 1H), 1.60–1.54 (m, 3H), 1.14 (t, J = 12.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.7, 168.8, 161.4, 161.0, 138.8, 136.4, 135.6, 131.0, 120.6, 116.9, 106.4, 100.1, 91.4, 81.2, 78.0, 76.3, 67.1, 63.5, 60.1, 56.0, 55.9, 42.9, 36.8, 26.3, 25.3, 24.0. HRMS (ESI) cald for C29H32NaO8 [M + Na]+ 531.1989, found 531.1992.
8b (white solid, 30%) was synthesized following the similar procedure for 8a. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 16.2 Hz, 1H), 6.70 (d, J = 16.2 Hz, 1H), 6.20 (d, J = 3.2 Hz, 1H), 6.12 (s, 2H), 5.74 (t, J = 8.1 Hz, 1H), 5.53 (d, J = 2.8 Hz, 1H), 4.73 (d, J = 12.5 Hz, 1H), 4.60 (d, J = 12.3 Hz, 1H), 4.13 (t, J = 7.0 Hz, 2H), 3.93–3.77 (m, 7H), 3.13 (t, J = 9.6 Hz, 1H), 2.94 (d, J = 9.4 Hz, 1H), 2.77–2.63 (m, 2H), 2.55–2.13 (m, 6H), 2.07 (s, 1H), 1.70–1.63 (m, 1H), 1.25 (s, 3H), 1.14 (t, J = 12.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.7, 168.8, 162.0, 161.5, 138.9, 136.5, 135.7, 131.1, 131.0, 129.0, 120.5, 116.6, 91.1, 81.3, 70.3, 67.1, 66.2, 63.5, 60.1, 55.9, 43.0, 36.8, 26.4, 25.3, 24.1, 19.7, 18.2. HRMS (ESI) cald for C30H34NaO8 [M + Na]+ 545.2146, found 545.2148.
8c (white solid, 44%) was synthesized following the similar procedure for 8a. 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 16.2 Hz, 1H), 6.68 (d, J = 16.2 Hz, 1H), 6.18 (s, 1H), 6.10 (s, 2H), 5.72 (t, J = 8.0 Hz, 1H), 5.52 (s, 1H), 4.71 (d, J = 12.4 Hz, 1H), 4.59 (d, J = 12.4 Hz, 1H), 4.10 (t, J = 5.9 Hz, 2H), 3.85 (s, 7H), 3.10 (t, J = 9.4 Hz, 1H), 2.92 (d, J = 9.4 Hz, 1H), 2.53–2.10 (m, 8H), 2.01 (dd, J = 12.0, 5.4 Hz, 3H), 1.68–1.60 (m, 1H), 1.54 (s, 3H), 1.12 (t, J = 12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.6, 168.8, 162.5, 161.5, 138.8, 136.5, 135.6, 130.8, 120.4, 116.3, 105.7, 91.0, 83.3, 81.2, 69.2, 67.0, 66.3, 63.4, 60.1, 55.8, 42.9, 36.8, 28.1, 26.3, 25.2, 24.0, 18.1, 15.2. HRMS (ESI) cald for C31H36NaO8 [M + Na]+ 559.2302, found 559.2305.
8d (white solid, 64%) was synthesized following the similar procedure for 8a. 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 16.2 Hz, 1H), 6.68 (d, J = 16.2 Hz, 1H), 6.19 (d, J = 3.4 Hz, 1H), 6.09 (s, 2H), 5.73 (t, J = 8.1 Hz, 1H), 5.52 (d, J = 3.0 Hz, 1H), 4.72 (d, J = 12.4 Hz, 1H), 4.59 (d, J = 12.4 Hz, 1H), 4.02 (t, J = 6.2 Hz, 2H), 3.86 (d, J = 13.4 Hz, 7H), 3.18–3.04 (m, 1H), 2.93 (d, J = 9.4 Hz, 1H), 2.55–2.12 (m, 8H), 1.97 (t, J = 2.3 Hz, 1H), 1.96–1.88 (m, 2H), 1.72 (dt, J = 14.7, 7.4 Hz, 2H), 1.64 (dd, J = 18.3, 6.8 Hz, 1H), 1.55 (s, 3H), 1.13 (t, J = 12.7 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.7, 168.8, 162.6, 161.5, 138.9, 136.6, 135.7, 130.9, 120.5, 116.3, 105.6, 91.0, 84.0, 81.2, 68.9, 67.6, 67.0, 63.5, 60.1, 55.9, 42.9, 36.8, 28.3, 26.3, 25.3, 25.1, 24.0, 18.3, 18.2. HRMS (ESI) cald C32H38NaO8 [M + Na]+ for 573.2459, found 573.2462.
8e (white solid, 42%) was synthesized following the similar procedure for 8a. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 16.1 Hz, 1H), 6.69 (d, J = 16.2 Hz, 1H), 6.20 (s, 1H), 6.09 (s, 2H), 5.74 (t, J = 7.8 Hz, 1H), 5.53 (s, 1H), 4.72 (d, J = 12.4 Hz, 1H), 4.60 (d, J = 12.5 Hz, 1H), 4.00 (t, J = 6.1 Hz, 2H), 3.87 (d, J = 13.5 Hz, 6H), 3.12 (t, J = 9.3 Hz, 1H), 2.94 (d, J = 9.6 Hz, 1H), 2.50–2.12 (m, 8H), 1.96 (s, 1H), 1.89–1.75 (m, 2H), 1.64 (m, 6H), 1.55 (s, 3H), 1.14 (t, J = 12.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.7, 168.9, 162.7, 161.5, 138.9, 136.7, 135.7, 130.9, 120.5, 116.2, 105.6, 91.0, 84.4, 81.2, 68.6, 68.0, 67.0, 63.5, 60.1, 55.9, 42.9, 36.8, 28.8, 28.3, 26.3, 25.3, 25.3, 24.0, 18.5, 18.2. HRMS (ESI) cald for C33H40NaO8 [M + Na]+ 587.2615, found 587.2618.
Synthesis of 10a
A mixture of azide (80 mg, 0.097 mmol) and 8a (60 mg, 0.118 mmol), CuSO4 (0.014 mmol), sodium ascorbate (0.014 mmol), tert-butanol (1 mL) and water (0.5 mL) was stirred overnight at room temperature. Then, 3 mL water was added and the reaction mixture was extracted (3 × 10 mL) with EtOAc. The combined organic layers were washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to give crude product, which was purified on a silica gel column to give compound 10a.
1H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 8.06 (d, J = 16.2 Hz, 1H), 7.77 (s, 2H), 7.69 (s, 1H), 7.51 (s, 1H), 7.26 (d, J = 9.3 Hz, 2H), 7.05 (dd, J = 9.6, 2.4 Hz, 2H), 6.97–6.91 (m, 2H), 6.66 (d, J = 16.2 Hz, 1H), 6.33 (s, 2H), 5.70 (t, J = 8.3 Hz, 1H), 5.59 (d, J = 3.2 Hz, 1H), 5.24 (s, 2H), 4.73–4.61 (m, 2H), 4.62–4.57 (m, 2H), 3.85 (s, 6H), 3.67 (m, 10H), 3.62–3.56 (m, 4H), 3.54 (d, J = 10.5 Hz, 7H), 3.46 (t, J = 5.5 Hz, 4H), 3.34 (s, 7H), 2.92 (d, J = 9.5 Hz, 1H), 2.53 (s, 2H), 2.41 (d, J = 6.9 Hz, 3H), 2.37–2.25 (m, 2H), 2.23–2.08 (m, 3H), 1.55 (s, 4H), 1.29–1.28 (m, 15H).
Synthesis of compound 12 in two methods
Method A: A solution of 7 (1.1 g, 4.16 mmol) in dimethylamine (20.8 mL, 41.6 mmol, 2 N in THF) was stirred for 1 h at 0 °C. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2: MeOH = 50:1) to afford the desired product 11 (1.2 g, 93%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 5.58 (t, J = 7.9 Hz, 1H), 4.10 (dd, J = 30.7, 13.1 Hz, 2H), 3.85 (t, J = 9.2 Hz, 1H), 3.38 (s, 1H), 2.81 (d, J = 9.4 Hz, 1H), 2.73 (dd, J = 12.9, 5.1 Hz, 1H), 2.61 (dd, J = 12.9, 5.4 Hz, 1H), 2.51–2.38 (m, 4H), 2.30–2.18 (m, 8H), 2.17–2.06 (m, 2H), 1.67–1.55 (m, 1H), 1.52 (s, 3H), 1.12–1.02 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 177.0, 141.2, 127.5, 81.7, 66.3, 64.2, 60.0, 57.6, 45.8, 44.3, 42.1, 37.2, 27.7, 26.0, 23.9, 18.1. HRMS (ESI) calcd for C17H28N2O4 [M + H]+ 310.2013, found 310.2015.
To a solution of 2,6-dimethoxylcinnamic acid (712 mg, 3.42 mmol), compound 11 (705 mg, 2.28 mmol), EDCI (655.6 mg, 3.42 mmol) and DMAP (27.8 mg, 0.228 mmol) in 25 mL CH2Cl2 was added TEA (0.45 mL, 3.42 mmol) at 0 °C. The mixture was stirred for 8 h at room temperature. The reaction was quenched with saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 75 mL). The combined organic layers were washed with saturated brine, dried over Na2SO4, and concentrated to give an oily crude product, which was purified on a silica gel column [DCM:MeOH = 50:1] to yield compound 12 (987.2 mg, 86%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 16.3 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 6.89 (d, J = 16.3 Hz, 1H), 6.56 (d, J = 8.4 Hz, 2H), 5.67 (t, J = 7.8 Hz, 1H), 4.84 (d, J = 12.8 Hz, 1H), 4.66 (d, J = 12.8 Hz, 1H), 3.97–3.80 (m, 7H), 2.84 (d, J = 9.3 Hz, 1H), 2.73 (d, J = 4.3 Hz, 1H), 2.65 (d, J = 5.9 Hz, 1H), 2.58–2.26 (m, 6H), 2.23 (s, 6H), 2.15 (d, J = 12.5 Hz, 2H), 1.58 (d, J = 20.6 Hz, 4H), 1.10 (t, J = 12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 177.2, 168.3, 160.2, 136.3, 136.1, 131.6, 128.5, 120.2, 112.2, 103.8, 81.3, 66.2, 64.0, 60.0, 58.5, 55.9, 45.9, 44.7, 43.3, 37.1, 27.2, 25.0, 23.9, 18.1. HRMS (ESI) calcd for C28H38NO7 [M + H]+ 500.2643, found 500.2644.
Method B. Synthesis of compound 2. To a mixture of 7 (53 mg, 0.2 mmol), EDCI (115 mg, 0.6 mmol), DMAP (1.2 mg, 0.01 mmol) and 2,6-dimethoxylcinnamic acid (0.3 mmol, 1.5 eq) in CH2Cl2 2 mL was added triethylamine (83.4 μL, 0.6 mmol). After 12 h, saturated NaHCO3 was added. The resulting mixture was extracted with CH2Cl2. The CH2Cl2 layer was dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude product, which was purified by silica gel column chromatography to afford compound 2 (yield 83%). 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 16.3 Hz, 1H, H-18), 7.30–7.22 (m, 1H, overlap with CHCl3, H-4′), 6.83 (d, J = 16.3 Hz, 1H, H-17), 6.54 (d, J = 8.4 Hz, 2H, H-3′, H-5′), 6.19 (d, J = 3.5 Hz, 1H, H-13), 5.73 (t, J = 8.1 Hz, 1H, H-1), 5.53 (d, J = 3.2 Hz, 1H, H-13), 4.74 (d, J = 12.4 Hz, 1H, H-14), 4.60 (d, J = 12.4 Hz, 1H, H-14), 3.96–3.75 (m, 7H, H-6, H-19, H-18), 3.14–3.02 (m, 1H, H-7), 2.91 (d, J = 9.4 Hz, 1H, H-5), 2.49–2.11 (m, 6H, H-2, H-3, H-8, H-9), 1.71–1.60 (m, 1H, H-8), 1.54 (s, 3H, H-15), 1.12 (t, J = 12.6 Hz, 1H, H-3). 13C NMR (100 MHz, CDCl3) δ 169.6(C-12), 168.4(C-16), 160.2(C-2′, C-6′), 138.8(C-11), 136.5(C-18), 135.5(C-10), 131.7(C-4′), 130.9(C-1), 120.5(C-13), 119.5(C-17), 111.9(C-1′), 103.7(C-3′, 5′), 81.2(C-6), 67.1(C-14), 63.4(C-5), 60.1(C-4), 55.9(C-19, C-20), 42.8(C-7), 36.7(C-3), 26.2(C-8), 25.1(C-9), 24.0(C-2), 18.1(C-15). HRMS (ESI) cald for C26H34NO7 [M + NH4]+ 472.2330, found 472.2329.
A solution of compound 2 (930 mg, 2.05 mmol) and dimethylamine (10.5 mL, 21.0 mmol, 2 N in THF) at 0 °C was stirred 1 h at 0 °C. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2: MeOH = 50:1) to afford the desired product 12 (985 mg, 96%) as a white solid.
Synthesis of compound 13. To a solution of compound 12 (1.15 g, 2.3 mmol) in methanol (23 mL), fumaric acid (267 mg, 2.3 mmol) was added. The mixture was stirred for 6.5 h and concentrated under vacuum to afford compound 13 as a white amorphous solid (1.3 g, yield 92%). 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 2H), 8.00 (d, J = 16.3 Hz, 1H), 7.37 (t, J = 8.4 Hz, 1H), 6.78 (d, J = 16.3 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H), 6.61 (s, 2H), 5.59 (t, J = 7.6 Hz, 1H), 4.79 (d, J = 12.7 Hz, 1H), 4.57 (d, J = 12.6 Hz, 1H), 4.03 (t, J = 9.5 Hz, 1H), 3.86 (s, 6H), 2.77–2.54 (m, 4H), 2.39–2.23 (m, 4H), 2.18 (s, 6H), 2.10 (dd, J = 24.6, 10.8 Hz, 3H), 1.65 (t, J = 11.4 Hz, 1H), 1.48 (s, 3H), 0.94 (t, J = 12.3 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 177.1, 167.2, 166.2, 159.6, 135.8, 135.2, 134.2, 132.2, 128.1, 119.4, 110.7, 104.1, 80.4, 66.0, 63.2, 59.8, 57.9, 56.0, 45.2, 43.3, 42.6, 36.6, 25.6, 24.2, 23.1, 17.5. HRMS (ESI) calcd for C28H38NO7 [M + H]+ 500.2643, found 500.2645.
Cell culture
Human triple negative breast cancer cell lines MDA-MB-231, SUM159, BT574, and mouse breast cancer cell 4T1 were purchased from ATCC and were cultured in 1640 medium supplement with 10% FBS under a 5% CO2 humidified atmosphere at 37 °C. Hs578T and MDA-MB-468 breast cancer cells were cultured in DMEM medium supplement with 10% FBS under a 5% CO2 humidified atmosphere at 37 °C.
MTT assay
Human triple negative breast cancer cells were seeded into 96 well plate at the density of 4000 cells/200µL/well. After the adherent cell growth, DMOCPTL was added with a series concentration for 72 h. Then, 20 µL thiazolyl blue tetrazolium bromide (MTT, 5 mg/mL) was added and incubated for additional 4–6 h. The supernatant was discarded and the precipitate was dissolved with DMSO. Then, the absorbance at 570 nm was measured.
Breast cancer cells MDA-MB-231 and 4T1 were seeded into 6 well plate with a concentration of 500 cells/1 mL/well. After 8–12 h, compounds at different concentration were added and incubated for 10 days. Then, the cells were fixed with 4% polyoxymethylene at room temperature for 15 min. After being washed with PBS, the cells were stained with crystal violet solution at room temperature for 15 min. The excess crystal violet was removed and washed with water for 3 times. The number of colonies was counted.
Cell death manner assay
To investigate the mechanism of DMOCPTL in breast cancer proliferation inhibition, the manners of cells death were analyzed. The cell apoptosis inhibitor Z-VAD-FMK, cell autophagy inhibitor 3-methyladenine, cell necrosis inhibitor necrostatin-1, and ferroptosis inhibitors N-Acetyl-L-cysteine, deferoxamine and Ferrostatin-1 were purchased and stored at –20 °C. MDA-MB-231 cells were seeded with a density of at 5000 cells/200µL/well into 96 well plate. After 8–12 h, DMOCPTL was added at different concentrations. Following that 10 µM Z-VAD-FMK, 5 mM 3-methyladenine, 10 µM necrostatin-1, 3 mM N-acetyl-L-cysteine, 200 µM deferoxamine and 10 µM Ferrostatin-1 were added and co-incubated with DMOCPTL, respectively, for 72 h. Then, 20 µL thiazolyl blue tetrazolium bromide (MTT, 5 mg/mL) was added and incubated at 37 °C for additional 4 h. Then, supernatant was discarded and 200 μL DMSO was added to dissolve the precipitate. After 15 min, the absorbance was measured at 570 nm. The percentage of inhibition was calculated.
Cell apoptosis assay
MDA-MB-231 and SUM159 cells were collected and washed with cold PBS. Then, the cells were suspended with 100 μL of binding buffer; 5 μL of Annexin V-APC and 5 μL PI were added and incubated for 15 min in dark at room temperature. Then, the cells were analyzed by flow cytometry within 1 h.
Flow cytometry with reactive O2 species assay
MDA-MB-231 and SUM159 cells in logarithmic growth period were digested and divided into control group, DMOCPTL-treated group. The cells were washed with PBS for 3 times and DMOCPTL was added. Then, 10 µM DCF-DA or BODIPY 581/591 C11 were added and co-incubated with DMOCPTL in an incubator at 37 °C. The cells were collected by centrifugation and analyzed by flow cytometry within 1 h.
Fe2+ intensity assay
MDA-MB-231 was collected and washed with cold PBS buffer. Then, cells were incubated with PGSK at 10 µM at 37 °C for 30 min in dark. Then, cells were collected, washed and analyzed by flow cytometry within 1 h.
RNA interference
Breast cancer cell at about 40–50% confluence was transfected with siRNA using lipofectamine™ 2000 according to the manufacturer's instructions. After 48 h, the efficacy of siRNA was confirmed by western blot assay.
Immunofluorescence assay
MDA-MB-231 cells were cultured on gelatin-coated glass coverslips for 24 h and treated with DMOCPTL for 48 h. Then, the cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with Triton X-100 for 15 min. After blocked with horse serum for 30 min, the cells were incubated with GPX4 antibody over night at 4 °C. After the incubation, the cells were washed five times with PBST and incubated with the anti-rabbit IgG-FITC secondary antibody for 1 h at room temperature. Then, the cell nucleus was stained with DAPI and analyzed using fluorescence microscopy. As to flow cytometry assay, MDA-MB-231 cells with DMOCPTL treatment for 48 h were collected, fixed with 4% paraformaldehyde for 20 min, permeabilized with Triton X-100 for 15 min, incubated with GPX4 antibody overnight, then incubated with corresponding FITC-conjunct second antibody. Fluorescence intensity of GPX4 expression was analyzed by flow cytometry.
Western blot assay
MDA-MB-231 and SUM159 cells were collected and lysed with RIPA buffer for 30 min on ice. Then, the protein (50 μg) from each sample were separated by 12% tris-acrylamide gel electrophoresis and transferred onto PVDF membrane. After blocking with 5% skim milk for 1 h at room temperature, Primary antibodies against GPX-4, EGR1, Bax, Bcl-2, Bcl-xl, cytochrome C, caspase 3, caspase 9 and PARP was used and incubated at 4 °C overnight on rotary shaker. After washing with PBST for 5 times, the membrane was probed with goat anti-rabbit IgG highly cross-adsorbed secondary antibody (1:10,000) for 2 h at room temperature. Then, the membrane was washed for 5 times and developed with ECL reagent.
Probe pull-down assay
The pull-down experiments were performed using the probe of DMOCPTL. In vitro pull-down assay, MDA-MB-231 and SUM159 cells were collected and lysed, respectively, in RIPA buffer supplemented with protease inhibitors. Then, probe 10 was added and incubated for 2 h at room temperature with different concentrations. Then, excessive pre-cooled methanol was added and incubated at − 80 °C for 30 min to precipitate the protein. After centrifuged at 14,000×g for 15 min, the precipitated proteins were dissolved and incubated with streptavidin conjunct agarose A + G beads at 4 °C overnight. Then, the streptavidin beads were washed three times with PBS buffer, and the bead-bound proteins were collected and detected by western blot experiments.
As to in situ pull-down assay, MDA-MB-231 and SUM159 cells were seeded into 6 well plate. After 12 h, probe 8a was added at different concentrations and incubated for additional 6 h. Then, the cells were collected and lysed, respectively, in RIPA buffer supplemented with protease inhibitors. After the centrifugation, the supernatant was collected and a mix of TBTA (0.1 mM), TCEP (1 mM), biotin-N3 (100 µM) and CuSO4 (1 mM) was added to conjunct biotin on probe 8a. Then, excessive pre-cooled methanol was added and incubated at − 80 °C for 30 min to precipitate the protein. After centrifuged at 14,000×g for 15 min, the precipitated proteins were dissolved and incubated with streptavidin conjunct agarose A + G beads at 4 °C overnight. Then, the streptavidin beads were washed three times with PBS buffer, and the bead-bound proteins were collected and detected by western blot experiments or sliver staining.
Co-localization assay
MDA-MB-231 cells were cultured on gelatin-coated glass coverslips for 24 h and treated with probe 10a for 6 h at 2 µM. The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with Triton X-100 for 15 min. After being blocked with horse serum for 30 min, the cells were incubated with anti-GPX4 antibody (rabbit) and anti-Ubiquitin (mouse) over night at 4 °C. After the incubation, the cells were washed five times with PBST and incubated with the anti-rabbit IgG-FITC secondary antibody and anti-mouse IgG-AF647 secondary antibody for 2 h at room temperature. After being washed for 3 times, the cell nucleus was stained with DAPI and analyzed using fluorescence microscopy.
Docking simulations
Molecular Docking simulations were performed with the software of AutoDock. The crystal structure of the published GPX4 (PDB code: 5H5S) was retrieved from the RCSB Protein Data Bank. The solvent molecules within the protein structure were removed in the docking calculations, and the best ligand pose was chosen according to the dock score.
In vitro ubiquitination assay
MDA-MB-231 cells in logarithmic growth period were plated into 6-well plate with the density at 1 × 105 cells/mL/well. Then, DMOCPTL at the concentration of 0.05 µM, 0.1 µM, 0.2 µM, 0.5 µM and 1 µM were added and incubated for 24 h. Then, 10 µM MG-132 was added to inhibit the protein degradation for 4 h. After that the cells were collected by centrifugation and washed with PBS, subsequently suspended with RIPA buffer for 30 min on ice. After centrifugation, the GPX4 antibody was added with 1:100 dilution for 2 h on ice, then 10 µL agarose G was added and co-incubated at 4 °C overnight on rotary shaker. The samples were collected and washed with PBS for 3 times, then the SDS-PAGE protein electrophoresis was performed and the protein was transferred onto PVDF membrane. Following that the membrane was blocked in 5% skim milk for 1 h at room temperature, and subsequently was incubated with ubiquitin antibody (P4D1) at 4 °C overnight on rotary shaker.
Immuno-coprecipitation assay
MDA-MB-231 and SUM159 cells were collected and lysed with RIPA buffer for 30 min on ice. The indicated protein in the cell lysates was immunoprecipitated using ubiquitin antibody. After incubated with ubiquitin antibody at 4 °C for 6 h, 10 µL agarose A + G was added and co-incubated at 4 °C overnight on rotary shaker. The samples were washed with PBS and visualized using an ECL detection system.
Pharmacodynamics
Compound 13 was injected into three male SD rats by intravenous administration at a dose of 1 mg/kg. Then, the blood samples from jugular sinus of rats were collected at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h and 8 h after administration. After being centrifugated at 12000 rpm for 1 min, the supernatant was collected, respectively. Then, equal amount of acetonitrile was added into the sample, which was vortexed for 2 min, centrifugated at 12000 rpm for 5 min, and then the supernatant was collected and analyzed by LC/MS. The pharmacodynamic of 13 on SD rats was calculated by DAS 3.3.
Acute toxicity assay in Bar b/c mice
To verify the toxicity of 13, Bar b/c mice were administrated by intravenous administration at a dose of 50 mg/kg or oral administration with 13 at a dose of 500 mg/kg or vehicle control. During the experiment, their behavior was observed, and the body weight was recorded every day. The level of GPT, GOT and Cr in serum was detected by ELISA assay and the major organs of mice including liver, spleen, lung, kidney, heart, and brain were weighted, fixed with 4% paraformaldehyde and sectioned to 4 µm slides. After dewaxing and hydration, HE staining was performed. The histology and morphology were observed under microscope.
In vivo anti-tumor activity assay
The experimental procedures of the animal experiments were permitted by the Animal Care and Use Committee at Nankai University. 4T1 triple negative breast cancer cells (1 × 105) were injected on the Bar b/c mouse's breast fat pad. Then, compound 13 was administrated through intravenous injection (7.5 mg/kg) according to body weight. Body weights and tumor size were measured every other day. The tumor growth inhibition was calculated and the tumor weight was recorded when the mice were sacrificed. The tumor was collected and crushed with cell lysate buffer. Then, the expression of GPX4 and EGR1 were analyzed in vehicle and 13-treated group by western blot assay. Furthermore, to analysis the effect of 13 on the overall survival compound 13 was administrated orally (50 mg/kg) according to body weight on tumor animal model. After administration for 6 times every other day the overall survival and body weight were recorded and analyzed.
Immunohistochemical assay
The tumors isolated from the mice were fixed with 4% paraformaldehyde and sectioned to 4 µm slides. As to IHC assay, after dewaxing, hydration and antigen retrieval, the slides were incubated with primary antibodies at 4 °C overnight. After washed with PBST for 3 times, the slides were incubated with biotinylated secondary antibody for 1 h. Then, the slides were developed by DAB.