The compound was minimized using an OPLS_2005 force field with a dielectric constant value 80
The compound was minimized using an OPLS_2005 force field with a dielectric constant value 80.0 in Macro Model. (d, = 8.1 Hz, 1H), 7.30 (s, 1H), 6.41 (d, = 10.5 Hz, 1H), 2.29 (s, 3H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.9, 184.2, 175.3, 160.5, 150.7, 142.0, 139.2, 132.3, 132.3, 128.6, 128.5, 125.3, 123.5, 121.5, 120.6, 47.9, 27.6, 8.8. MS (ESI, [M + Na]+) 329.3. HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, CORO1A 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.6, 183.3, 175.5, 161.9, 151.6, 141.4, 138.8, 132.8, 132.5, 129.0, 128.0, 126.1, 125.2, 123.8, 120.1, 55.2, 48.1, 27.6. MS (ESI, [M + Na]+) 345.2. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). The reaction mixture was stirred at rt for 1 h and then evaporated the solvent, diluted with H2O, and extracted with EtOAc (50 mL 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was further purified by silica gel column, and elution with 1%C2% MeOH in CH2Cl2 afforded the desired product 10 (27 mg, 90%) as a red solid. m.p. 185.2-188.5 C. 1H-NMR (400 MHz, CDCl3) 7.87 (d, = 10.2 Hz, 1H), 7.64C7.55 (m, 2H), 7.24 (s, 1H), 6.39 (dd, = 10.2, 4.4 Hz, 1H), 4.08 (d, = 4.5 Hz, 1H), 2.26 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H). 13C-NMR (126 MHz, CDCl3) 184.2, 175.6, 161.2, 147.1, 141.5, 135.8, 135.5, 131.5, 127.7, 125.0, 123.6, 122.1, 121.2, 120.2, 72.5, 40.0, 26.3, 21.9, 8.8. Synthesis of 1 1,6,6-trimethyl-7,8-dihydrophenanthro[1,2-b]furan-9,10,11(6= 8.2 Hz, 1H), 7.57 (d, = 8.2 Hz, 1H), 7.26 (d, = 1.4 Hz, 1H), 2.92 (t, = 7.2 Hz, 2H), 2.26 (d, = 1.3 Hz, 3H), 2.07 (t, = 7.2 Hz, 2H), 1.34 (s, 6H). Compound 14: m.p. 158.3C160.8 C.1H-NMR (400 MHz, CDCl3) 7.81 (d, = 8.3 Hz, 1H), 7.72 (d, = 8.3 Hz, 1H), 7.27 (s, 1H), 6.80 (d, = 10.2 Hz, 1H), 6.48 (d, = 10.2 Hz, 1H), 2.26 (s, 3H), 1.50 (s, 6H). 13C-NMR (126 MHz, CDCl3) 185.8, 183.4, 179.3, 159.6, 154.6, 151.8, 141.9, 135.3, 132.8, 131.6, 128.9, 127.4, 124.5, 121.3, 120.9, 38.3, 29.4, 8.7. MS (ESI, [M + H]+) 307.4. HRMS (ESI) calcd for C19H15O4, 307.0965; found, 307.0967. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-9,10,11(6= 8.3 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.45 (s, 1H), 6.82 (d, = 10.3 Hz, 1H), 6.47 (d, = 10.2 Hz, 1H), 4.69 (s, 2H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.7, 183.3, 179.4, 161.1, 154.6, 152.5, 141.2, 135.7, 132.8, 131.8, 128.5, 127.4, 126.1, 124.7, 120.5, 55.2, 38.4, 29.5. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found, 323.0901. 3.2. Biology hIDO-1 enzymatic assay. The hIDO-1 enzymatic assay was performed as described previously [36]. Briefly, a standard reaction mixture (30 L) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mmol/L ascorbic acid and 0.01% Triton X-100, 200 g/mL catalase, 20 mol/L methylene blue, and 0.05 M rhIDO-1 was added to the solution (60 L) containing the substrate l-tryptophan (250 mol/L) and the test sample at a determined concentration. The reaction was carried out at 37 C for 30 min and stopped by adding 45 L of 30% (w/v) trichloroacetic acid. After being heated at 65 C for 15 min, the reaction mixture was centrifuged at 12,000 rpm for 10 min. The supernatant (100 L) was transferred into a well of a 96-well microplate and mixed with 100 L of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow pigment derived from kynurenine was measured at 492 nm using a Spectra Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA). IC50 values were calculated by using Graph Pad Prism 6 software (version 6.00, Graph Pad Software, San Diego, CA, USA). Cell-based assay of IDO-1 activity. The cellular activity of IDO-1 was detected as described previously [36]. HEK 293 cells were seeded in a 6-well culture plate at a density.HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). 6H). 13C-NMR (126 MHz, CDCl3) 201.9, 184.2, 175.3, 160.5, 150.7, 142.0, 139.2, 132.3, 132.3, 128.6, 128.5, 125.3, 123.5, 121.5, 120.6, 47.9, 27.6, 8.8. MS (ESI, [M + Na]+) 329.3. HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.6, 183.3, 175.5, 161.9, 151.6, 141.4, 138.8, 132.8, 132.5, 129.0, 128.0, 126.1, 125.2, 123.8, 120.1, 55.2, 48.1, 27.6. MS (ESI, [M + Na]+) 345.2. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). The reaction mixture was stirred at rt for 1 h and then evaporated the solvent, diluted with H2O, and extracted with EtOAc (50 mL 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was further purified by silica gel column, and elution with 1%C2% MeOH in CH2Cl2 afforded the desired product 10 (27 mg, 90%) as a red solid. m.p. 185.2-188.5 C. 1H-NMR (400 MHz, CDCl3) 7.87 (d, = 10.2 Hz, 1H), 7.64C7.55 (m, 2H), 7.24 (s, 1H), 6.39 (dd, = 10.2, 4.4 Hz, 1H), 4.08 (d, = 4.5 Hz, 1H), 2.26 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H). 13C-NMR (126 MHz, CDCl3) 184.2, 175.6, 161.2, 147.1, 141.5, 135.8, 135.5, 131.5, 127.7, 125.0, 123.6, 122.1, 121.2, 120.2, 72.5, 40.0, 26.3, 21.9, 8.8. Synthesis of 1 1,6,6-trimethyl-7,8-dihydrophenanthro[1,2-b]furan-9,10,11(6= 8.2 Hz, 1H), 7.57 (d, = 8.2 Hz, 1H), 7.26 (d, = 1.4 Hz, 1H), 2.92 (t, = 7.2 Hz, 2H), 2.26 (d, = 1.3 Hz, 3H), 2.07 (t, = 7.2 Hz, 2H), 1.34 (s, 6H). Compound 14: m.p. 158.3C160.8 C.1H-NMR (400 MHz, CDCl3) 7.81 (d, = 8.3 Hz, 1H), 7.72 (d, = 8.3 Hz, 1H), 7.27 (s, 1H), 6.80 (d, = 10.2 Hz, 1H), 6.48 (d, = 10.2 Hz, 1H), 2.26 (s, 3H), 1.50 (s, 6H). 13C-NMR (126 MHz, CDCl3) 185.8, 183.4, 179.3, 159.6, 154.6, 151.8, 141.9, 135.3, 132.8, 131.6, 128.9, 127.4, 124.5, 121.3, 120.9, 38.3, 29.4, 8.7. MS (ESI, [M + H]+) 307.4. HRMS (ESI) calcd for C19H15O4, 307.0965; found, 307.0967. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-9,10,11(6= 8.3 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.45 (s, 1H), 6.82 (d, = 10.3 Hz, 1H), 6.47 (d, = 10.2 Hz, 1H), 4.69 (s, 2H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.7, 183.3, 179.4, 161.1, 154.6, 152.5, 141.2, 135.7, 132.8, 131.8, 128.5, 127.4, 126.1, 124.7, 120.5, 55.2, 38.4, 29.5. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found, 323.0901. 3.2. Biology hIDO-1 enzymatic assay. The hIDO-1 enzymatic assay was performed as described previously [36]. Briefly, a standard reaction mixture (30 L) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mmol/L ascorbic acid and 0.01% Triton X-100, 200 g/mL catalase, 20 mol/L methylene blue, and 0.05 M rhIDO-1 was added to the solution (60 L) containing the substrate l-tryptophan (250 mol/L) and the test sample at a determined concentration. The reaction was carried out at 37 C for 30 min and stopped by adding 45 L of 30% (w/v) trichloroacetic acid. After being heated at 65 C for 15 min, the reaction mixture was centrifuged at 12,000 rpm for 10 min. The supernatant (100 L) was transferred into a well of a 96-well microplate and mixed with 100 L of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow pigment derived from kynurenine was measured at 492 nm using a Spectra Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA). IC50 values were calculated by using Graph Pad Prism 6 software (version 6.00, Graph Pad Software, San Diego, CA, USA). Cell-based assay of IDO-1 activity. The cellular activity of IDO-1 was detected as described previously [36]. HEK 293 cells were seeded in a 6-well culture plate at a denseness of 5 105 cells/well and cultured over night. After 24 h, HEK 293 cells.Assisting from National Science & Technology Major Project Major New Drug Creation and Manufacturing System, China (Quantity: 2018ZX09711002-010-001), as well as give from your Customized MedicinesMolecular Signature-based Drug Discovery and Development, Strategic Priority Research Program of the Chinese Academy of Sciences (Give No. 3H), 1.30 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.4, 175.9, 161.5, 148.5, 141.3, 137.4, 134.0, 130.3, 127.3, 124.5, 123.1, 121.5, 121.1, 120.0, 37.9, 34.2, 28.4, 8.8. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.79 (d, = 8.2 Hz, 1H), 7.72 (d, = 8.1 Hz, 1H), 7.30 (s, 1H), 6.41 (d, = 10.5 Hz, 1H), 2.29 (s, 3H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.9, 184.2, 175.3, 160.5, 150.7, 142.0, 139.2, 132.3, 132.3, 128.6, 128.5, 125.3, 123.5, 121.5, 120.6, 47.9, 27.6, 8.8. MS (ESI, [M + Na]+) 329.3. HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found out, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.6, 183.3, 175.5, 161.9, 151.6, 141.4, 138.8, 132.8, 132.5, 129.0, 128.0, 126.1, 125.2, 123.8, 120.1, 55.2, 48.1, 27.6. MS (ESI, [M + Na]+) 345.2. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) LGX 818 (Encorafenib) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). The reaction combination was stirred at rt for 1 h and then evaporated the solvent, diluted with H2O, and extracted with EtOAc (50 mL 3). The LGX 818 (Encorafenib) combined organic coating was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was further purified by silica gel column, and elution with 1%C2% MeOH in CH2Cl2 afforded the desired product 10 (27 mg, 90%) like a reddish solid. m.p. 185.2-188.5 C. 1H-NMR (400 MHz, CDCl3) 7.87 (d, = 10.2 Hz, 1H), 7.64C7.55 (m, 2H), 7.24 (s, 1H), 6.39 (dd, = 10.2, 4.4 Hz, 1H), 4.08 (d, = 4.5 Hz, 1H), 2.26 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H). 13C-NMR (126 MHz, CDCl3) 184.2, 175.6, 161.2, 147.1, 141.5, 135.8, 135.5, 131.5, 127.7, 125.0, 123.6, 122.1, 121.2, 120.2, 72.5, 40.0, 26.3, 21.9, 8.8. Synthesis of 1 1,6,6-trimethyl-7,8-dihydrophenanthro[1,2-b]furan-9,10,11(6= 8.2 Hz, 1H), 7.57 (d, LGX 818 (Encorafenib) = 8.2 Hz, 1H), 7.26 (d, = 1.4 Hz, 1H), 2.92 (t, = 7.2 Hz, 2H), 2.26 (d, = 1.3 Hz, 3H), 2.07 (t, = 7.2 Hz, 2H), 1.34 (s, LGX 818 (Encorafenib) 6H). Compound 14: m.p. 158.3C160.8 C.1H-NMR (400 MHz, CDCl3) 7.81 (d, = 8.3 Hz, 1H), 7.72 (d, = 8.3 Hz, 1H), 7.27 (s, 1H), 6.80 (d, = 10.2 Hz, 1H), 6.48 (d, = 10.2 Hz, 1H), 2.26 (s, 3H), 1.50 (s, 6H). 13C-NMR (126 MHz, CDCl3) 185.8, 183.4, 179.3, 159.6, 154.6, 151.8, 141.9, 135.3, 132.8, 131.6, 128.9, 127.4, 124.5, 121.3, 120.9, 38.3, 29.4, 8.7. MS (ESI, [M + H]+) 307.4. HRMS (ESI) calcd for C19H15O4, 307.0965; found out, 307.0967. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-9,10,11(6= 8.3 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.45 (s, 1H), 6.82 (d, = 10.3 Hz, 1H), 6.47 (d, = 10.2 Hz, 1H), 4.69 (s, 2H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.7, 183.3, 179.4, 161.1, 154.6, 152.5, 141.2, 135.7, 132.8, 131.8, 128.5, 127.4, 126.1, 124.7, 120.5, 55.2, 38.4, 29.5. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0901. 3.2. Biology hIDO-1 enzymatic assay. The hIDO-1 LGX 818 (Encorafenib) enzymatic assay was performed as explained previously [36]. Briefly, a standard reaction combination (30 L) comprising 100 mM potassium phosphate buffer (pH 6.5), 40 mmol/L ascorbic acid and 0.01% Triton X-100, 200 g/mL catalase, 20 mol/L methylene blue, and 0.05 M rhIDO-1 was added to the perfect solution is (60 L) containing the substrate l-tryptophan (250 mol/L) and the test sample at a determined concentration. The reaction was carried out at 37 C for 30 min and halted by adding 45 L of 30% (w/v) trichloroacetic acid. After being heated at 65 C for 15 min, the reaction combination was centrifuged at 12,000 rpm for 10 min. The supernatant (100 L) was transferred into a well of a 96-well microplate and mixed with 100 L of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow pigment derived from kynurenine was measured at 492 nm using a Spectra Maximum Plus 384 microplate reader (Molecular Products, Sunnyvale, CA, USA). IC50 ideals were calculated by using Graph Pad Prism 6 software (version 6.00, Graph Pad Software,.HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found out, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 8.8. MS (ESI, [M + Na]+) 329.3. HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found out, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.6, 183.3, 175.5, 161.9, 151.6, 141.4, 138.8, 132.8, 132.5, 129.0, 128.0, 126.1, 125.2, 123.8, 120.1, 55.2, 48.1, 27.6. MS (ESI, [M + Na]+) 345.2. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). The reaction combination was stirred at rt for 1 h and then evaporated the solvent, diluted with H2O, and extracted with EtOAc (50 mL 3). The combined organic coating was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was further purified by silica gel column, and elution with 1%C2% MeOH in CH2Cl2 afforded the desired product 10 (27 mg, 90%) like a reddish solid. m.p. 185.2-188.5 C. 1H-NMR (400 MHz, CDCl3) 7.87 (d, = 10.2 Hz, 1H), 7.64C7.55 (m, 2H), 7.24 (s, 1H), 6.39 (dd, = 10.2, 4.4 Hz, 1H), 4.08 (d, = 4.5 Hz, 1H), 2.26 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H). 13C-NMR (126 MHz, CDCl3) 184.2, 175.6, 161.2, 147.1, 141.5, 135.8, 135.5, 131.5, 127.7, 125.0, 123.6, 122.1, 121.2, 120.2, 72.5, 40.0, 26.3, 21.9, 8.8. Synthesis of 1 1,6,6-trimethyl-7,8-dihydrophenanthro[1,2-b]furan-9,10,11(6= 8.2 Hz, 1H), 7.57 (d, = 8.2 Hz, 1H), 7.26 (d, = 1.4 Hz, 1H), 2.92 (t, = 7.2 Hz, 2H), 2.26 (d, = 1.3 Hz, 3H), 2.07 (t, = 7.2 Hz, 2H), 1.34 (s, 6H). Compound 14: m.p. 158.3C160.8 C.1H-NMR (400 MHz, CDCl3) 7.81 (d, = 8.3 Hz, 1H), 7.72 (d, = 8.3 Hz, 1H), 7.27 (s, 1H), 6.80 (d, = 10.2 Hz, 1H), 6.48 (d, = 10.2 Hz, 1H), 2.26 (s, 3H), 1.50 (s, 6H). 13C-NMR (126 MHz, CDCl3) 185.8, 183.4, 179.3, 159.6, 154.6, 151.8, 141.9, 135.3, 132.8, 131.6, 128.9, 127.4, 124.5, 121.3, 120.9, 38.3, 29.4, 8.7. MS (ESI, [M + H]+) 307.4. HRMS (ESI) calcd for C19H15O4, 307.0965; found out, 307.0967. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-9,10,11(6= 8.3 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.45 (s, 1H), 6.82 (d, = 10.3 Hz, 1H), 6.47 (d, = 10.2 Hz, 1H), 4.69 (s, 2H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.7, 183.3, 179.4, 161.1, 154.6, 152.5, 141.2, 135.7, 132.8, 131.8, 128.5, 127.4, 126.1, 124.7, 120.5, 55.2, 38.4, 29.5. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0901. 3.2. Biology hIDO-1 enzymatic assay. The hIDO-1 enzymatic assay was performed as explained previously [36]. Briefly, a standard reaction combination (30 L) comprising 100 mM potassium phosphate buffer (pH 6.5), 40 mmol/L ascorbic acid and 0.01% Triton X-100, 200 g/mL catalase, 20 mol/L methylene blue, and 0.05 M rhIDO-1 was added to the perfect solution is (60 L) containing the substrate l-tryptophan (250 mol/L) and the test sample at a determined concentration. The reaction was carried out at 37 C for 30 min and halted by adding 45 L of 30% (w/v) trichloroacetic acid. After being heated at 65 C for 15 min, the reaction combination was centrifuged at 12,000 rpm for 10 min. The supernatant (100 L) was transferred into a well of a 96-well microplate and mixed with 100 L of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow pigment derived from kynurenine was measured at 492 nm using a Spectra Maximum Plus 384 microplate reader (Molecular Products, Sunnyvale, CA, USA). IC50 ideals were calculated by using Graph Pad Prism 6 software (version 6.00, Graph Pad Software, San Diego, CA, USA). Cell-based assay of IDO-1 activity. The cellular activity.MS (ESI, [M + Na]+) 329.3. 1H), 2.27 (d, = 1.3 Hz, 3H), 1.30 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.4, 175.9, 161.5, 148.5, 141.3, 137.4, 134.0, 130.3, 127.3, 124.5, 123.1, 121.5, 121.1, 120.0, 37.9, 34.2, 28.4, 8.8. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.79 (d, = 8.2 Hz, 1H), 7.72 (d, = 8.1 Hz, 1H), 7.30 (s, 1H), 6.41 (d, = 10.5 Hz, 1H), 2.29 (s, 3H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.9, 184.2, 175.3, 160.5, 150.7, 142.0, 139.2, 132.3, 132.3, 128.6, 128.5, 125.3, 123.5, 121.5, 120.6, 47.9, 27.6, 8.8. MS (ESI, [M + Na]+) 329.3. HRMS (ESI, [M + H]+) calcd for C19H15O4, 307.0965; found out, 307.0973. Synthesis of 1-(hydroxymethyl)-6,6-dimethylphenanthro[1,2-b]furan-7,10,11(6= 10.5 Hz, 1H), 7.83 (d, = 8.1 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.48 (s, 1H), 6.44 (d, = 10.5 Hz, 1H), 4.71 (s, 2H), 1.52 (s, 6H). 13C-NMR (126 MHz, CDCl3) 201.6, 183.3, 175.5, 161.9, 151.6, 141.4, 138.8, 132.8, 132.5, 129.0, 128.0, 126.1, 125.2, 123.8, 120.1, 55.2, 48.1, 27.6. MS (ESI, [M + Na]+) 345.2. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0915. Synthesis of 7-hydroxy-1,6,6-trimethyl-6,7-dihydrophenanthro[1,2-b]furan-10,11-dione (10) [33] To a solution of 11 (30 mg, 0.098 mmol) in MeOH (2 mL) was added NaBH4 (11 mg, 0.300 mmol). The reaction combination was stirred at rt for 1 h and then evaporated the solvent, diluted with H2O, and extracted with EtOAc (50 mL 3). The combined organic coating was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was further purified by silica gel column, and elution with 1%C2% MeOH in CH2Cl2 afforded the desired product 10 (27 mg, 90%) like a reddish solid. m.p. 185.2-188.5 C. 1H-NMR (400 MHz, CDCl3) 7.87 (d, = 10.2 Hz, 1H), 7.64C7.55 (m, 2H), 7.24 (s, 1H), 6.39 (dd, = 10.2, 4.4 Hz, 1H), 4.08 (d, = 4.5 Hz, 1H), 2.26 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H). 13C-NMR (126 MHz, CDCl3) 184.2, 175.6, 161.2, 147.1, 141.5, 135.8, 135.5, 131.5, 127.7, 125.0, 123.6, 122.1, 121.2, 120.2, 72.5, 40.0, 26.3, 21.9, 8.8. Synthesis of 1 1,6,6-trimethyl-7,8-dihydrophenanthro[1,2-b]furan-9,10,11(6= 8.2 Hz, 1H), 7.57 (d, = 8.2 Hz, 1H), 7.26 (d, = 1.4 Hz, 1H), 2.92 (t, = 7.2 Hz, 2H), 2.26 (d, = 1.3 Hz, 3H), 2.07 (t, = 7.2 Hz, 2H), 1.34 (s, 6H). Compound 14: m.p. 158.3C160.8 C.1H-NMR (400 MHz, CDCl3) 7.81 (d, = 8.3 Hz, 1H), 7.72 (d, = 8.3 Hz, 1H), 7.27 (s, 1H), 6.80 (d, = 10.2 Hz, 1H), 6.48 (d, = 10.2 Hz, 1H), 2.26 (s, 3H), 1.50 (s, 6H). 13C-NMR (126 MHz, CDCl3) 185.8, 183.4, 179.3, 159.6, 154.6, 151.8, 141.9, 135.3, 132.8, 131.6, 128.9, 127.4, 124.5, 121.3, 120.9, 38.3, 29.4, 8.7. MS (ESI, [M + H]+) 307.4. HRMS (ESI) calcd for C19H15O4, 307.0965; found out, 307.0967. Synthesis of 1 1,6,6-trimethylphenanthro[1,2-b]furan-9,10,11(6= 8.3 Hz, 1H), 7.75 (d, = 8.3 Hz, 1H), 7.45 (s, 1H), 6.82 (d, = 10.3 Hz, 1H), 6.47 (d, = 10.2 Hz, 1H), 4.69 (s, 2H), 1.51 (s, 6H). 13C-NMR (126 MHz, CDCl3) 184.7, 183.3, 179.4, 161.1, 154.6, 152.5, 141.2, 135.7, 132.8, 131.8, 128.5, 127.4, 126.1, 124.7, 120.5, 55.2, 38.4, 29.5. HRMS (ESI, [M + H]+) calcd for C19H15O5, 323.0914; found out, 323.0901. 3.2. Biology hIDO-1 enzymatic assay. The hIDO-1 enzymatic assay was performed as explained previously [36]. Briefly, a standard reaction combination (30 L) comprising 100 mM potassium phosphate buffer (pH 6.5), 40 mmol/L ascorbic acid and 0.01% Triton X-100, 200 g/mL catalase, 20 mol/L methylene blue, and 0.05 M rhIDO-1 was added to the perfect solution is (60 L) containing the substrate l-tryptophan (250 mol/L) and the test sample at a determined concentration. The reaction was carried out at 37 C for 30 min.