4.1 Chemistry experimental materials

Melting points were obtained and uncorrected on a Haineng melting-point apparatus. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a Bruker Avance 400 and Bruker Avance 600 spectrometers at 400 MHz and 600 MHz. Carbon-13 nuclear magnetic resonance (13C-NMR) was recorded on Bruker Avance 400 spectrometer at 100 MHz. Carbon-13 nuclear magnetic resonance (13C-NMR) was recorded on Bruker Avance 600 spectrometer at 150 MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane (TMS) for all recorded NMR spectra. High Resolution Mass spectra were taken on Thermo Fisher LC-MSO/TOR mass spectrometer. Silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qingdao Marine Chemical Company (China). All air- or moisture- sensitive reactions were conducted under an argon atmosphere. Starting materials and reagents used in reactions were obtained commercially from TCI, Adamas, Aladdin, Bidepharm and were used without purification, unless otherwise indicated. Purity of all compounds was determined by high-performance liquid chromatography (HPLC) using an Agilent instrument.

4.2 Synthesis of compounds a2-a224.2.1 Synthesis of compounds a2

Compound a1 (0.10 g, 0.36 mmol) was dissolved in 20 mL of analytical-grade acetic acid in a 50 mL microwave tube. Ammonium acetate (0.11 g, 1.45 mmol) was added, followed by the dropwise addition of 3-pyridinecarbaldehyde (0.051 mL, 0.54 mmol) at room temperature. The mixture was stirred for 10 min, then heated to 100 °C in a microwave reactor and maintained for 1 h. After cooling to room temperature, the solution was transferred to a 100 mL round-bottom flask and neutralized to pH 7 using saturated aqueous NaOH. The neutralized mixture was extracted with ethyl acetate (3 × 30 mL), and the aqueous phase was further extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, eluent: dichloromethane/ethyl acetate = 10:1 → 5:1, v/v, containing 1% (v/v) formic acid) to afford a2 as yellow powder in 76% yield. (Experimental findings & Structural elucidation of a2 are included in the Supporting information)

4.2.1.1 1,6-dimethyl-11-(pyridin-3-yl)-12H-furo[2',3':1,2]phenanthro[3,4-d] imidazole (a2). Yield 76%. Yellow powder, m.p. 288–289 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.74 (d, J = 9.0 Hz, 1H), 9.58 (d, J = 1.8 Hz, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.62 (dd, J = 4.8, 1.2 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 1.8 Hz, 1H), 7.63 (dd, J = 8.4, 6.6 Hz, 1H), 7.57 (dd, J = 8.4, 4.8 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 2.70 (s, 3H), 2.60 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 150.0, 149.0, 148.2, 146.8, 146.7, 142.1, 136.7, 134.5, 134.0, 130.6, 130.3, 127.2, 126.8, 126.6, 126.5, 123.9, 122.1, 119.2, 119.0, 115.9, 115.6, 112.9, 20.0, 10.0 ppm. HRMS (ESI-TOF) m/z Calcd for C24H18N3O [M + H]+ 364.1444, found 364.1447. HPLC purity: 98.6%.

4.2.2 Synthesis of compounds a3-a22

Compound a2 (0.05 g, 0.14 mmol) was charged into a 50 mL round-bottom flask and dissolved in ultra-dry acetonitrile (20 mL). The mixture was heated under reflux at 100 °C for 12 h until complete dissolution of a2 was achieved. Subsequently, various bromides (5.0 equiv relative to a2) were introduced sequentially, each dissolved in ultra-dry acetonitrile (20 mL per 50 mg substrate). The reaction mixtures were refluxed with stirring for an additional 24–48 h and monitored by TLC. Upon completion, solvents were removed under reduced pressure, and the precipitated solid was collected by filtration, thoroughly washed with ethyl acetate (3 × 60 mL), and dried under vacuum at 60 °C to afford tanshinone I-pyridinium derivatives a3-a22 as yellow powder in 36–97% yields. (Structural determination for representative derivative a4 is detailed in the Supporting information)

4.2.2.1 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(2-oxo-2-phenylethyl) pyridin-1-ium bromide (a3). Yield 85%. Yellow powder, m.p. 287–288 °C. IR νmax (cm−1): 3429, 3048, 2959, 1703, 1624, 1449, 1369, 1341, 1319, 1218, 1087, 798, 780, 704, 685, 590. 1H NMR (400 MHz, DMSO-d6) δ 10.67 (d, J = 8.8 Hz, 1H), 9.96 (s, 1H), 9.49 (d, J = 8.4 Hz, 1H), 9.07 (d, J = 6.0 Hz, 1H), 8.49 (dd, J = 8.0, 6.0 Hz, 1H), 8.34 (d, J = 9.2 Hz, 1H), 8.20—8.13 (m, 3H), 8.04 (d, J = 1.2 Hz, 1H), 7.89—7.82 (m, 1H), 7.75 – 7.66 (m, 3H), 7.51 (d, J = 6.8 Hz, 1H), 6.71 (s, 2H), 2.76 (s, 3H), 2.61 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (100 MHz, DMSO-d6) δ 190.8, 149.6, 145.4, 144.4, 142.4, 142.4, 142.3, 136.7, 135.0, 134.1, 133.6 130.6, 130.2, 130.0, 129.2, 128.4, 127.8, 127.3, 126.9, 126.8, 126.7, 122.7, 119.2, 118.8, 116.4, 115.4, 112.5, 66.9, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C32H24N3O2 [M]+ 482.1863, found 482.1867. HPLC purity: 97.7%.

4.2.2.2 1-(2-(4-bromophenyl)-2-oxoethyl)-3-(1,6-dimethyl-12H-furo[2',3':1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a4). Yield 76%. Yellow powder, m.p. 268–269 °C. IR νmax (cm−1): 3429,3040, 2951, 1702, 1584, 1396, 1336, 1216, 1098, 998, 821, 727, 705, 688, 591. 1H NMR (600 MHz, DMSO-d6) δ 13.48 (s, 1H), 10.68 (d, J = 8.4 Hz, 1H), 9.96 (s, 1H), 9.52 (d, J = 8.4 Hz, 1H), 9.06 (d, J = 6.0 Hz, 1H), 8.50 (dd, J = 8.4, 6.0 Hz, 1H), 8.36 (d, J = 9.0 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 1.2 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.69 (dd, J = 8.4, 7.2 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 6.69 (s, 2H), 2.77 (s, 3H), 2.63 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 149.6, 145.4, 144.4, 142.5, 142.4, 142.4, 136.7, 134.1, 132.7, 132.4, 130.6, 130.4, 130.3, 130.0, 129.0, 127.8, 127.3, 127.0, 126.7, 126.6, 122.7, 119.2, 118.8, 116.4, 115.4, 112.5, 66.8, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C32H23O2N3Br [M]+ 560.0968, found 560.0973. HPLC purity: 98.1%.

4.2.2.3 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(2-(4-methoxyphenyl)-2-oxoethyl) pyridin-1-ium bromide (a5). Yield 90%. Yellow powder, m.p. 326–327 °C. IR νmax (cm−1): 3419, 3048, 2943, 1688, 1623, 1598, 1263, 1174, 1022, 838, 704, 678, 593. 1H NMR (600 MHz, DMSO-d6) δ 13.53 (s, 1H), 10.71 (d, J = 9.0 Hz, 1H), 9.97 (s, 1H), 9.54 (d, J = 8.4 Hz, 1H), 9.08 (d, J = 6.0 Hz, 1H), 8.50 (dd, J = 7.8, 6.0 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 1.8 Hz, 1H), 7.71—7.67 (m, 1H), 7.53 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 6.65 (s, 2H), 3.93 (s, 3H), 2.78 (s, 3H), 2.64 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 189.0, 164.4, 149.6, 145.5, 144.5, 142.6, 142.5, 142.3, 142.1, 136.7, 134.2, 130.9, 130.6, 130.3, 130.0, 127.8, 127.3, 127.0, 126.8, 126.6, 126.4, 126.4, 122.7, 119.2, 118.8, 116.5, 115.4, 114.5, 112.6, 66.7, 55.9, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C33H26N3O3 [M]+ 512.1969, found 512.1965. HPLC purity: 96.1%.

4.2.2.4 3-(1,6-dimethyl-12H-furo[2',3':1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(2-(naphthalen-2-yl)-2-oxoethyl) pyridin-1-ium bromide (a6). Yield 36%. Yellow powder, m.p. 292–293 °C. IR νmax (cm−1): 3423, 3051, 2922, 1698, 1624, 1467, 1357, 1316, 1278, 1176, 1146, 866, 821, 705, 677. 1H NMR (600 MHz, DMSO-d6) δ 13.63 (s, 1H), 10.74 (d, J = 9.0 Hz, 1H), 10.05 (s, 1H), 9.61 (d, J = 7.8 Hz, 1H), 9.14 (d, J = 6.0 Hz, 1H), 8.94 (d, J = 1.8 Hz, 1H), 8.56 (dd, J = 8.4, 6.0 Hz, 1H), 8.40 (d, J = 9.0 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.21 (d, J = 9.0 Hz, 2H), 8.14—8.11 (m, 3H), 7.81—7.78 (m, 1H), 7.76—7.73 (m, 1H), 7.68 (dd, J = 8.4, 7.2 Hz, 1H), 7.53 (d, J = 7.2 Hz, 1H), 6.83 (s, 2H), 2.78 (s, 3H), 2.68 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 191.1, 150.1, 146.0, 145.1, 143.2, 143.0, 137.3, 136.2, 134.7, 132.5, 131.4, 131.3, 131.1, 130.9, 130.5, 130.3, 130.1, 129.4, 128.5, 128.4, 128.1, 127.8, 127.5, 127.2, 127.0, 123.8, 123.2, 119.7, 119.3, 117.0, 115.9, 113.1, 67.4, 20.3, 10.2 ppm. HRMS (ESI-TOF) m/z Calcd for C36H26N3O2 [M]+ 532.2020, found 532.2020. HPLC purity: 95.9%.

4.2.2.5 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(3-methylbenzyl) pyridin-1-ium bromide (a7). Yield 67%. Yellow powder, m.p. 280–281 °C. IR νmax (cm−1): 3377, 3006, 1590, 1397, 1302, 1161, 819, 712, 593. 1H NMR (600 MHz, DMSO-d6) δ 10.57 (s, 1H), 10.06 (s, 1H), 9.30 (s, 1H), 9.21 (s, 1H), 8.34 (d, J = 7.2 Hz, 1H), 8.27 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.96 (s, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.53 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 6.6 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 6.0 Hz, 1H), 6.05 (s, 2H), 2.74 (s, 3H), 2.57 (s, 3H), 2.38 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO) δ 149.8, 144.0, 142.9, 142.6, 142.1, 139.2, 136.9, 134.4, 134.4, 131.2, 130.9, 130.6, 130.3, 130.1, 129.7, 128.7, 127.6, 127.0, 126.9, 126.7, 122.8, 119.4, 119.2, 116.7, 115.8, 113.0, 64.0, 21.4, 20.3, 10.3. ppm; HRMS (ESI-TOF) m/z Calcd for C32H26N3O [M]+ 468.2070, found 468.2074. HPLC purity: 97.6%.

4.2.2.6 1-(2-cyanobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a8). Yield 84%. Yellow powder, m.p. 285–286 °C. IR νmax (cm−1): 3426, 3069, 3028, 2961, 2919, 2854, 2223, 1878, 1624, 1489, 1456, 1314, 1146, 816, 773, 702, 686. 1H NMR (600 MHz, DMSO-d6) δ 13.54 (s, 1H), 10.68 (d, J = 8.4 Hz, 1H), 10.06 (d, J = 1.8 Hz, 1H), 9.51 (d, J = 7.8 Hz, 1H), 9.22 (d, J = 6.0 Hz, 1H), 8.47 (dd, J = 7.8, 6.0 Hz, 1H), 8.36 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 8.07 (d, J = 1.8 Hz, 1H), 7.88 (td, J = 7.8, 1.2 Hz, 1H), 7.74—7.67 (m, 3H), 7.55 (d, J = 7.2 Hz, 1H), 6.37 (s, 2H), 2.78 (s, 3H), 2.63 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 144.2, 143.6, 142.5, 142.4, 142.2, 137.0, 136.7, 134.3, 134.1, 134.0, 130.9, 130.6, 130.2, 130.0, 129.8, 128.7, 127.3, 127.0, 126.7, 126.6, 122.8, 119.1, 118.8, 117.1, 116.5, 115.4, 112.6, 111.5, 61.9, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C32H23N4O [M]+ 479.1866, found 479.1870. HPLC purity: 97.9%.

4.2.2.7 1-(2-bromobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a9). Yield 69%. Yellow powder, m.p. 260–261 °C. IR νmax (cm−1): 3423, 3083, 3006, 2933, 2860, 1805, 1627, 1491, 1473, 1371, 1165, 1048, 1027, 812, 757, 679. 1H NMR (600 MHz, DMSO-d6) δ 13.55 (s, 1H), 10.62 (d, J = 8.4 Hz, 1H), 9.98 (s, 1H), 9.51 (d, J = 8.4 Hz, 1H), 9.19 (d, J = 6.0 Hz, 1H), 8.46 (t, J = 7.2 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.08 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.63—7.55 (m, 3H), 7.51 (d, J = 7.2 Hz, 1H), 6.19 (s, 2H), 2.79 (s, 3H), 2.64 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 144.2, 143.1, 142.5, 142.4, 142.1, 136.7, 134.2, 133.6, 133.1, 131.8, 131.6, 130.7, 130.6, 129.9, 128.9, 128.6, 127.4, 127.0, 126.7, 126.5, 123.6, 122.8, 119.1, 118.8, 116.5, 115.4, 112.6, 63.7, 19.9, 9.7 ppm. HRMS (ESI-TOF) m/z Calcd for C31H23N3OBr [M]+ 532.1019, found 532.1024. HPLC purity: 98.9%.

4.2.2.8 1-(4-bromobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a10). Yield 89%. Yellow powder, m.p. 285–286 °C. IR νmax (cm−1): 3382, 3091, 3037, 2993, 2927, 1904, 1584, 1484, 1442, 1351, 1169, 1071, 838, 814, 649. 1H NMR (400 MHz, DMSO-d6) δ 10.57 (d, J = 8.4 Hz, 1H), 10.07 (s, 1H), 9.30 (d, J = 8.0 Hz, 1H), 9.21 (d, J = 6.0 Hz, 1H), 8.35 (dd, J = 8.4, 6.0 Hz, 1H), 8.27 (d, J = 9.2 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H), 8.00—7.94 (m, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.68 (dd, J = 8.8, 7.2 Hz, 1H), 7.49 (d, J = 6.8 Hz, 1H), 6.09 (s, 2H), 2.74 (s, 3H), 2.56 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (100 MHz, DMSO-d6) δ 149.5, 143.7, 142.7, 142.2, 141.7, 136.5, 134.0, 133.3, 132.3, 131.6, 130.6, 130.5, 129.9, 128.4, 127.2, 126.7, 126.6, 123.2, 122.5, 119.0, 118.7, 116.3, 115.3, 112.4, 62.8, 19.9, 9.9 ppm. HRMS (ESI-TOF) m/z Calcd for C31H23N3OBr [M]+ 532.1019, found 532.1022. HPLC purity: 98.0%.

4.2.2.9 1-(4-chlorobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a11). Yield 92%. Yellow powder, m.p. 261–262 °C. IR νmax (cm−1): 3422, 3092, 3038, 2923, 2853, 1629, 1594, 1489, 1399, 1283, 1143, 1088, 842, 776, 700, 677. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (d, J = 8.4 Hz, 1H), 10.08 (s, 1H), 9.32 (d, J = 8.4 Hz, 1H), 9.21 (d, J = 6.0 Hz, 1H), 8.36 (dd, J = 8.0, 2.0 Hz, 1H), 8.28 (d, J = 9.2 Hz, 1H), 8.10 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.83—7.76 (m, 2H), 7.71—7.66 (m, 1H), 7.66—7.62 (m, 2H), 7.50 (d, J = 6.8 Hz, 1H), 6.11 (s, 2H), 2.75 (s, 3H), 2.58 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (101 MHz, DMSO-d6) δ 149.5, 143.7, 142.7, 142.3, 141.8, 136.5, 134. 5, 134.0, 132.9, 131.4, 130.6, 130.5, 129.9, 129.4, 128.4, 127.2, 126.8, 126.6, 122.6, 119.0, 118.7, 116.4, 115.3, 112.5, 62.7, 19.9, 9.9 ppm. HRMS (ESI-TOF) m/z Calcd for C31H23ON3Cl [M]+ 488.1524, found 488.1526. HPLC purity: 96.2%.

4.2.2.10 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(4-fluorobenzyl) pyridin-1-ium bromide (a12). Yield 97%. Yellow powder, m.p. 276–277 °C. IR νmax (cm−1): 3426, 3009, 2941, 1627, 1591, 1526, 1318, 1228, 1160, 1144, 818, 773, 707, 684. 1H NMR (400 MHz, DMSO-d6) δ 10.63 (d, J = 8.4 Hz, 1H), 10.08 (s, 1H), 9.38 (d, J = 8.4 Hz, 1H), 9.22 (d, J = 5.6 Hz, 1H), 8.38 (dd, J = 8.4, 6.0 Hz, 1H), 8.33 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 9.2 Hz, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.87—7.81 (m, 2H), 7.71 (dd, J = 8.8, 6.8 Hz, 1H), 7.54 (d, J = 6.8 Hz, 1H), 7.45—7.39 (m, 2H), 6.09 (s, 2H), 2.77 (s, 3H), 2.63—2.60 (m, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (100 MHz, DMSO-d6) δ 162.79 (d, 1JC-F = 244.9 Hz), 149.6, 143.7, 142.7, 142.44, 142.38, 141.8, 136.6, 134.1, 132.0 (d, 3JC-F = 8.5 Hz), 130.6, 130.60, 130.2 (d, 4JC-F = 3.1 Hz), 130.0, 128.4, 127.3, 126.8, 126.6, 122.7, 119.1, 118.8, 116.3 (d, 2JC-F = 21.8 Hz), 115.4, 112.5, 62.8, 19.9, 9.9 ppm. HRMS (ESI-TOF) m/z Calcd for C31H23ON3F [M]+ 472.1820, found 472.1818. HPLC purity: 98.6%.

4.2.2.11 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(4-(trifluoromethyl) benzyl)pyridin-1-ium bromide (a13). Yield 85%. Yellow powder, m.p. 273–274 ℃. 1H NMR (600 MHz, DMSO-d6) δ 13.55 (s, 1H), 10.70 (d, J = 8.4 Hz, 1H), 10.16 (s, 1H), 9.48 (d, J = 8.4 Hz, 1H), 9.23 (d, J = 6.0 Hz, 1H), 8.43 (dd, J = 8.4, 6.0 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.95—7.88 (m, 4H), 7.72 (dd, J = 8.4, 6.6 Hz, 1H), 7.56 (d, J = 6.6 Hz, 1H), 6.22 (s, 2H), 2.79 (s, 3H), 2.65 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 144.1, 143.3, 142.6, 142.5, 142.2, 138.6, 136.7, 134.2, 130.6 (q, 2JC-F = 35.9 Hz), 129.7, 130.0, 129.9, 128.6, 127.4, 126.9, 126.7 (q, 3JC-F = 9.15 Hz), 126.2 (q, 4JC-F = 3.9 Hz), 124.0 (q, 1JC-F = 270.8 Hz), 122.7, 119.2, 118.8, 116.5, 115.4, 112.6, 62.9, 19.9, 9.8 ppm; HRMS (ESI-TOF) m/z Calcd for C32H23N3OF3 [M]+ 522.1788, found 522.1788. HPLC purity: 97.0%.

4.2.2.12 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(4-formylbenzyl) pyridin-1-ium bromide (a14). Yield 74%. Yellow powder, m.p. 243–244 °C. IR νmax (cm−1): 3424, 3076, 2926, 2851, 1696, 1624, 1455, 1211, 1170, 814, 778, 702, 682, 594. 1H NMR (600 MHz, DMSO-d6) δ 13.52 (s, 1H), 10.68 (d, J = 9.0 Hz, 1H), 10.15 (s, 1H), 10.08 (s, 1H), 9.47 (d, J = 8.4 Hz, 1H), 9.24 (d, J = 6.0 Hz, 1H), 8.43 (dd, J = 8.4, 6.0 Hz, 1H), 8.36 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 8.09—8.03 (m, 3H), 7.88 (d, J = 7.8 Hz, 2H), 7.71 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 6.23 (s, 2H), 2.78 (s, 3H), 2.68—2.59 (m, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 192.9, 149.6, 144.1, 143.3, 142.5, 142.4, 142.2, 140.2, 136.7, 136.6, 134.2, 130.9, 130.6, 130.3, 130.0, 129.6, 128.6, 127.3, 126.9, 126.7, 126.6, 122.7, 119.6, 118.8, 116.5, 115.4, 112.6, 63.2, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C32H24N3O2 [M]+ 482.1863, found 482.1866.

4.2.2.13 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(4-methylbenzyl) pyridin-1-ium bromide (a15). Yield 60%. Yellow powder, m.p. 250–251 °C. IR νmax (cm−1): 3421, 3011, 2921, 1628, 1589, 1443, 1303, 1143, 1068, 816, 774, 678, 609, 595. 1H NMR (600 MHz, DMSO-d6) δ 13.59 (s, 1H), 10.70 (d, J = 8.4 Hz, 1H), 10.07 (s, 1H), 9.46 (d, J = 8.4, 1H), 9.19 (d, J = 6.0 Hz, 1H), 8.43—8.37 (m, 2H), 8.21 (d, J = 9.0 Hz, 1H), 8.10 (d, J = 1.8 Hz, 1H), 7.73 (dd, J = 8.4, 6.6 Hz, 1H), 7.61—7.57 (m, 3H), 7.36 (d, J = 7.8 Hz, 2H), 6.04 (s, 2H), 2.80 (s, 3H), 2.66 (d, J = 1.2 Hz, 3H), 2.35 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 143.8, 142.8, 142.7, 142.5, 142.0, 139.2, 136.7, 134.2, 131.0, 130.7, 130.7, 130.0, 129.9, 129.3, 129.2, 128.5, 128.4, 127.4, 127.0, 126. 7, 126.6, 122.8, 119.2, 118.8, 116.5, 115.4, 112.6, 63.6, 20.8, 19.9, 9.7 ppm. HRMS (ESI-TOF) m/z Calcd for C32H26N3O [M]+ 468.2070, found 468.2071. HPLC purity: 95.6%.

4.2.2.14 1-(3,4-dichlorobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)pyridin-1-ium bromide (a16). Yield 79%. Yellow powder, m.p. 297–298 °C. IR νmax (cm−1): 3427, 3007, 1626, 1587, 1491, 1468, 1397, 1146, 817, 773, 707, 687. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (d, J = 8.4 Hz, 1H), 10.11 (s, 1H), 9.32 (d, J = 8.0 Hz, 1H), 9.22 (d, J = 6.0 Hz, 1H), 8.36 (dd, J = 8.0, 6.0 Hz, 1H), 8.28 (d, J = 9.2 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 8.4, 2.0 Hz, 1H), 7.72—7.67 (m, 1H), 7.51 (d, J = 6.8 Hz, 1H), 6.11 (s, 2H), 2.75 (s, 3H), 2.58 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (100 MHz, DMSO-d6) δ 149.5, 143.7, 143.0, 142.3, 142.3, 141.9, 136.5, 134.7, 134.0, 132.5, 131.9, 131.7, 131.5, 130.7, 130.5, 129.9, 129.9, 128.4, 127.2, 126.7, 126.7, 126.6, 122.6, 119.1, 118.7, 116.4, 115.3, 112.4, 62.1, 19.9, 9.9 ppm. HRMS (ESI-TOF) m/z Calcd for C31H22N3OCl2 [M]+ 522.1134, found 522.1139. HPLC purity: 98.7%.

4.2.2.15 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(3-fluoro-4-nitrobenzyl) pyridin-1-ium bromide (a17). Yield 61%. Yellow powder, m.p. 241–242 °C. IR νmax (cm−1): 3417, 3015, 2922, 1603, 1528, 1348, 1277, 1090, 841, 818, 746, 711, 677, 538. 1H NMR (600 MHz, DMSO-d6) δ 13.62 (s, 1H), 10.76 (d, J = 8.4 Hz, 1H), 10.18 (s, 1H), 9.53 (dt, J = 7.8, 1.8 Hz, 1H), 9.21 (d, J = 6.0 Hz, 1H), 8.46 (dd, J = 7.8, 6.0 Hz, 1H), 8.40 (d, J = 9.0 Hz, 1H), 8.32 (t, J = 8.4 Hz, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 1.2 Hz, 1H), 7.98 (dd, J = 12.0, 1.8 Hz, 1H), 7.76—7.71 (m, 2H), 7.58 (d, J = 7.2 Hz, 1H), 6.23 (s, 2H), 2.80 (s, 3H), 2.68 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 154.6 (d, 1JC-F = 260.9 Hz), 149.6, 144.2, 143.8, 142.7, 142.6, 142.5 (d, 3JC-F = 8.1 Hz), 137.2, 137.2, 136.8, 134.3, 131.9 (d, 2JC-F = 54.7 Hz), 130.0, 128.7, 127.4, 127.1, 127.0, 126.7 (d, 3JC-F = 9.0 Hz), 125.7 (d, 4JC-F = 3.9 Hz), 122.8, 119.2, 119.1, 118.9 (d, 2JC-F = 24.9 Hz), 116.5, 115.4, 112.6, 62.2, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C31H22N4O3F [M]+ 517.1670, found 517.1670. HPLC purity: 96.3%.

4.2.2.16 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(3-methyl-4-nitrobenzyl) pyridin-1-ium bromide (a18). Yield 47%. Yellow powder, m.p. 275–276 °C. IR νmax (cm−1): 3423, 3014, 2926, 1615, 1522, 1345, 1281, 1144, 967, 842, 820, 775, 679, 595. 1H NMR (600 MHz, DMSO-d6) δ 13.60 (s, 1H), 10.74 (d, J = 8.4 Hz, 1H), 10.16 (s, 1H), 9.51 (d, J = 8.4 Hz, 1H), 9.21 (d, J = 6.0 Hz, 1H), 8.44 (dd, J = 7.8, 6.0 Hz, 1H), 8.39 (d, J = 9.6 Hz, 1H), 8.21 (d, J = 9.0 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 1.2 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H), 7.76—7.71 (m, 2H), 7.57 (d, J = 6.6 Hz, 1H), 6.18 (s, 2H), 2.80 (s, 3H), 2.67 (d, J = 1.2 Hz, 3H), 2.55 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 149.3, 144.1, 143.5, 142.6, 142.5, 142.3, 139.1, 136.6, 134.2, 133.5, 133.1, 131.0, 130.7, 130.0, 128.7, 127.8, 127.4, 127.0, 126.7, 126.6, 125.2, 122.8, 119.2, 118.8, 116.5, 115.4, 112.6, 62.7, 19.9, 19.4, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C32H25N4O3 [M]+ 513.1921, found 513.1917. HPLC purity: 96.9%.

4.2.2.17 1-(2-bromo-4-nitrobenzyl)-3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl) pyridin-1-ium bromide (a19). Yield 40%. Yellow powder, m.p. 266–267 °C. IR νmax (cm−1): 3423, 3037, 1623, 1587, 1523, 1346, 1146, 1024, 840, 816, 795, 678. 1H NMR (600 MHz, DMSO-d6) δ 13.58 (s, 1H), 10.69 (d, J = 9.0 Hz, 1H), 10.11 (s, 1H), 9.58 (d, J = 8.4 Hz, 1H), 9.19 (d, J = 6.0 Hz, 1H), 8.65 (d, J = 2.4 Hz, 1H), 8.50 (dd, J = 8.4, 6.0 Hz, 1H), 8.38 (d, J = 9.0 Hz, 1H), 8.34 (dd, J = 8.4, 2.4 Hz, 1H), 8.20 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.71 (dd, J = 8.4, 7.2 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 6.32 (s, 2H). 2.79 (s, 3H), 2.66 (d, J = 1.2 Hz, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.7, 148.4, 144.5, 143.9, 142.6, 142.5, 142.4, 140.5, 136.7, 134.2, 131.2, 131.0, 130.7, 130.0, 128.7, 127.9, 127.4, 127.0, 126.7, 126.5, 123.4, 123.4, 122.8, 119.2, 118.8, 116.5, 115.4, 112.6, 63.3, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C31H22N4O3Br [M]+577.0870, found 577.0876. HPLC purity: 96.9%.

4.2.2.18 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(naphthalen-1-ylmethyl) pyridin-1-ium bromide (a20). Yield 87%. Yellow powder, m.p. 266–267 °C. IR νmax (cm−1): 3428, 3192, 3064, 3008, 2931, 2861, 1868, 1626, 1491, 1452, 1398, 1173, 814, 796, 774, 677. 1H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 10.33 (d, J = 8.4 Hz, 1H), 9.85 (s, 1H), 9.23 (d, J = 8.0 Hz, 1H), 9.19 (d, J = 6.0 Hz, 1H), 8.35—8.25 (m, 2H), 8.17—8.10 (m, 2H), 8.06 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.84 (s, 1H), 7.71—7.66 (m, 3H), 7.64—7.54 (m, 2H), 7.42 (d, J = 7.2 Hz, 1H), 6.55 (s, 2H), 2.66 (s, 3H), 2.44 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (100 MHz, DMSO-d6) δ 149.4, 143.7, 142.3, 142.2, 142.0, 141.6, 136.44, 134.0, 133.7, 130.6, 130.5, 130.5, 130.4, 129.8, 129.2, 129.2, 129.0, 128.4, 127.7, 127.2, 126.8, 126.7, 126.5, 126.4, 125.8, 123.1, 122.5, 118.9, 118.7, 116.3, 115.3, 112.3, 61.4, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C35H26N3O [M]+ 504.2070, found 504.2074. HPLC purity: 98.0%.

4.2.2.19 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(naphthalen-2-ylmethyl) pyridin-1-ium bromide (a21). Yield 65%. Yellow powder, m.p. 288–289 °C. IR νmax (cm−1): 3420, 3041, 3004, 2918, 1805, 1589, 1393, 1370, 1282, 1160, 851, 816, 773, 678. 1H NMR (600 MHz, DMSO-d6) δ 13.57 (s, 1H), 10.65 (d, J = 8.4 Hz, 1H), 10.13 (s, 1H), 9.48 (d, J = 8.4 Hz, 1H), 9.27 (d, J = 6.0 Hz, 1H), 8.45—8.40 (m, 1H), 8.37 (d, J = 9.0 Hz, 1H), 8.26 (s, 1H), 8.18 (d, J = 9.0 Hz, 1H), 8.12—8.06 (m, 2H), 8.04—8.00 (m, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.64—7.58 (m, 3H), 7.55 (d, J = 7.2 Hz, 1H), 6.27 (s, 2H), 2.78 (s, 3H), 2.65 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 144.0, 143.0, 142.6, 142.5, 142.0, 136.7, 134.2, 133.1, 132.8, 131.4, 130.8, 130.6, 129.9, 129.2, 128.9, 128.5, 128.2, 127.8, 127.3, 127.2, 127.0, 126.9, 126.6, 126.5, 126.1, 122.8, 119.1, 118.8, 116.5, 115.4, 112.6, 63.9, 19.9, 9.8 ppm. HRMS (ESI-TOF) m/z Calcd for C35H26N3O [M]+ 504.2070, found 504.2069. HPLC purity: 96.4%.

4.2.2.20 3-(1,6-dimethyl-12H-furo[2′,3′:1,2]phenanthro[3,4-d]imidazol-11-yl)-1-(2-methylallyl) pyridin-1-ium bromide (a22). Yield 73%. Yellow powder, m.p. 290–291 °C. IR νmax (cm−1): 3421, 2922, 1625, 1490, 1283, 1161, 1104, 1068, 815, 773, 680, 592. 1H NMR (600 MHz, DMSO-d6) δ 13.48 (s, 1H), 10.68 (d, J = 8.4 Hz, 1H), 9.96 (s, 1H), 9.47 (d, J = 7.8 Hz, 1H), 9.09 (d, J = 6.0 Hz, 1H), 8.41 (t, J = 7.2 Hz, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.05 (s, 1H), 7.71 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 6.6 Hz, 1H), 5.47 (s, 2H), 5.26 (s, 1H), 5.00 (s, 1H), 2.77 (s, 3H), 2.63 (s, 3H), 1.86 (s, 3H) ppm (one resonance was not observed due to active N–H of imidazole); 13C NMR (150 MHz, DMSO-d6) δ 149.6, 144.0, 142.9, 142.5, 142.4, 142.2, 139.6, 136.7, 134.2, 130.7, 130.6, 130.0, 128.4, 127.3, 126. 9, 126.7, 126.6, 122.7, 119.2, 118.8, 116.6, 116.4, 115.4, 112.6, 65.8, 19.9, 19.7, 9.9 ppm. HRMS (ESI-TOF) m/z Calcd for C28H24N3O [M]+ 418.1914, found 418.1917.

4.3 Biological4.3.1 Materials and cell culture

The human cancer cell lines, including breast cancer (MDA-MB-231), hepatocellular carcinoma (HepG2), and prostate cancer (22RV1) were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 mg/mL streptomycin (HyClone, Logan, UT, USA). All the cells were incubated at 37 °C, 5% CO2 in a humidified atmosphere.

4.3.2 Cytotoxicity assay

Cytotoxic activities were evaluated by the MTS assay. All the compounds tested were absolutely dissolved to 10 mM in dimethyl sulfoxide (DMSO) in stock. Cells (5 × 103 cells/well) were plated into 96-well plates and cultured for 12 h before treatment and continuously exposed to 0.032, 0.16, 0.8, 4 and 20 µM test compounds for 48 h. Then MTS reagent (Promega, Madison, WI, USA) was added to each well, and cells were incubated at 37 °C for an additional 1–4 h and the optical density (OD) was measured at 492 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The IC50 values were calculated from dose–response curves.

4.3.3 Cell cycle analysis

For cell cycle analysis, cells were harvested and washed twice with phosphate-buffered saline (PBS), then fixed overnight at 4 °C in 70% ethanol. After fixation, cells were washed again and incubated with propidium iodide (PI, 50 µg/mL) in the presence of RNase A (50 µg/mL) at room temperature for 30 min. Cell cycle distribution was analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA). Data analysis was performed using FlowJo software (version 10.9.0).

4.3.4 Apoptosis analysis

Cell apoptosis was analyzed by flow cytometry using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's instructions. Briefly, cells were seeded into 6-well plates at 3 × 105 cells/well, and treated with indicated concentrations of test compounds for 48 h. Cells were harvested, washed twice with cold PBS, and resuspended in binding buffer containing Annexin V-FITC and PI. Following incubation at room temperature in the dark for 15 min, fluorescence intensity was quantified using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

4.3.5 Scratch wound healing assay

Cell migration rates following 24 h incubation with a4 was quantitatively assessed using ImageJ software by measuring the wound closure area between the opposing edges.

4.3.6 PI3K activity assay

The inhibitory activity of a4 on PI3Kα was evaluated as previously based on the production of PIP3 with the PI3-Kinase Activity ELISA kit (K-1000 s, Echelon) [25].

4.3.7 Western blot analysis

The cells (Breast cancer, MDA-MB-231) were treated with compounds for indicated time and subjected to western blot analysis as previously [25]. The antibodies against AKT (#4691), phosphor-AKT (Ser473) (#9271), S6K (#2708), phosphor-S6K (Thr389) (#9234), S6 (#9202), phospho-S6 (Ser240/244) (#88441) and PD-L1 (#13684) were all from the Cell signaling technology, and the antibody for GAPDH (MAB374) was provided by Sigma.

4.3.8 Statistical analysis

All data were expressed as means ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.5 software. Pairwise comparisons among groups were analyzed using Tukey's test. Differences were considered statistically significant at p < 0.05.