(1R,3R,3S)-3-(4-Bromobenzoyl)-1-hydroxy-5,7-dinitro-3,4-dihydro-1H-spiro[indoline-3,2-naphthalen]-2-1 (4o) White good, 88% produce (33.2?mg, 0.06?mmol), 0.10 in CH2Cl2), m.p.>220?C. as apoptotic amounts using Annexin V-FITC/PI dual staining (Keygen, Nanjing, China). The chemical substance induced significant apoptosis and cell routine arrest in G1 stage both in cell lines (Fig.?4C and D). Furthermore, assays, we treated U251 glioblastoma xenografts in mice with anti-cancer aftereffect of (rat, 15?mg/kg)0.10 in CH2Cl2), m.p.>220?C. 1H NMR (400?MHz, DMSO-10.43 (s, 1H, NH), 8.68 (s, 1H, ArH), 8.46 (s, 1H, ArH), 7.31 (d, 178.4, 171.2, 148.3, 146.0, 145.5, 144.1, 138.9, 131.3, 128.9, 124.2, 123.2, 121.6, 118.3, 109.5, 71.6, 60.5, 55.0, 46.4, 25.7, 13.7; HR-MS Ascomycin (ESI): Calcd. for C20H17N3O8Na [M+Na]+: 450.0913, Ascomycin Found 450.0916. HPLC evaluation: MeOH/H2O (60:40), 11.27?min, HPLC purity 99.7%. The substances 4bC4q were ready based on the synthetic approach to 4a. 4.1.1.1. Ethyl (1R,3R,3S)-5-fluoro-1-hydroxy-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4b) White solid, 85% produce (30.8?mg, 0.07?mmol), 0.10 in CH2Cl2), Rabbit Polyclonal to FSHR m.p.>220?C. 1H NMR (600?MHz, DMSO-10.60 (s, 1H, NH), 8.72 (s, 1H, ArH), 8.43 (s, 1H, ArH), 7.07 (td, 176.4, 171.3, 157.3 (d, Calcd. for C20H16N3O8FNa [M+Na]+: 468.0819, Found 468.0821. HPLC evaluation: MeOH/H2O (60:40), 12.80?min, HPLC purity 97.5%. 4.1.1.2. Ethyl (1R,3R,3S)-7-fluoro-1-hydroxy-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4c) White solid, 82% produce (29.5?mg, 0.07?mmol), 0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-11.23 (s, 1H, NH), 8.77 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.19 (s, 1H, CHOH), 7.09 (t, 178.8, 163.8, 147.6, 146.7, 146.3 (d, Calcd. for C20H16N3O8FNa [M+Na]+: 468.0819, Found 468.0822. HPLC evaluation: MeOH/H2O (60:40), 14.20?min, HPLC purity 99.3%. 4.1.1.3. Ethyl (1R,3R,3S)-5-chloro-1-hydroxy-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4d) White solid, 87% produce (31.6?mg, 0.07?mmol), ee 99%, 0.10 in CH2Cl2), 10.48 (s, 1H, NH), 8.67 (s, 1H, ArH), 8.53 (s, 1H, ArH), 7.60 (d, 176.3, 171.1, 148.3, 146.2, 144.3, 142.9, 137.4, 133.2, 128.6, 125.7, 125.4, 124.2, 118.6, 110.7, 72.1, 60.8, 52.8, 44.6, 26.3, 13.9; HR-MS (ESI): Calcd. for C20H16N3O8ClNa [M+Na]+: 484.0524, Found out 484.0526. HPLC evaluation: MeOH/H2O (60:40), 9.80?min, HPLC purity 99.7%. (0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-10.73 (s, 1H, NH), 8.72 (s, 1H, ArH), 8.42 (s, 1H, ArH), 7.08 (d, 176.7, 171.4, 148.1, 145.5, 144.0, 142.3, 137.4, 132.8, 128.6, 127.6, 127.4, 120.7, 118.9, 109.1, 68.8, 60.5, 51.7, 41.5, 25.7, 13.5; HR-MS (ESI): Calcd. for C20H16N3O8ClNa [M+Na]+: 484.0524, Found out 484.0523. HPLC evaluation: MeOH/H2O (60:40), 14.07?min, HPLC purity 99.4%. 4.1.1.5. Ethyl (1R,3R,3S)-4-bromo-1-hydroxy-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4f) White solid, 89% produce (33.2?mg, 0.07?mmol), 0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-10.65 (s, 1H, NH), 8.66 (s, 1H, ArH), 8.54 (s, 1H, ArH), 7.20?7.15 (m, 2H, ArH), 6.81 (t, 174.9, 170.4, 147.6, 145.8, 145.5, 143.4, 137.2, 130.6, 127.0, 125.3, 124.9, 118.0, 117.9, 108.5, 67.9, 60.3, 54.3, 41.6, 25.3, 13.2; HR-MS (ESI): Calcd. for C20H16N3O8BrNa [M+Na]+: 528.0018, Found 528.0020. HPLC evaluation: MeOH/H2O (60:40), 16.87?min, HPLC purity 98.6%. 4.1.1.6. Ethyl (1R,3R,3S)-5-bromo-1-hydroxy-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4g) White solid, 88% produce (32.8?mg, 0.06?mmol), ee 95%, 0.10 in CH2Cl2), 10.48 (s, 1H, NH), 8.67 (s, 1H, ArH), 8.52 (s, 1H, ArH), 7.72 (d, 176.2, 171.1, 148.3, 146.2, 144.3, 143.2, Ascomycin 137.4, 133.5, 131.4, 126.9, 125.4, 118.5, 113.3, 111.2, 72.1, 60.8, 52.8, 44.6, 26.2, 13.9; HR-MS (ESI): Calcd. for C20H16N3O8BrNa [M+Na]+: 528.0018, Found 528.0019. HPLC evaluation: MeOH/H2O (60:40), 10.73?min, HPLC purity 98.5%. (0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-10.87 (s, 1H, NH), 8.77 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.20 (s, 1H, CHOH), 6.98 (d, 178.8, 163.8, 147.5, 146.8, 144.7, 143.0, 136.7, 130.3, 129.8, 124.9, 123.4, 121.6, 119.5, 112.3, 71.8, 61.5, 55.6, 30.9, 22.0, 13.6; HR-MS (ESI): Calcd. for C20H16N3O8BrNa [M+Na]+: 528.0018, Found 528.0020. Ascomycin HPLC?evaluation: MeOH/H2O (60:40), 32.13?min, HPLC purity 98.9%. 4.1.1.8. Ethyl (1R,3R,3S)-1-hydroxy-5,5,7-trinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4i) White solid, 84% produce (30.8?mg, 0.06?mmol), 0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-11.15 (s, 1H, NH), 8.69 (s, 1H, ArH), 8.55 (d, 176.5, 170.5, 150.0, 147.6, 145.6, 143.1, 141.7, 136.5, 131.6, 125.8, 124.7, 119.3, 118.1, 108.8, 71.3, 60.4, 52.1, 43.5, 25.5, 13.2; HR-MS (ESI): Calcd. for C20H16N4O10Na [M+Na]+: 495.0764, Found out 495.0763. HPLC evaluation: MeOH/H2O (60:40), 6.80?min, HPLC purity 97.9%. 4.1.1.9. Ethyl (1R,3R,3S)-1-hydroxy-5-methyl-5,7-dinitro-2-oxo-3,4-dihydro-1H-spiro[indoline-3,2-naphthalene]-3-carboxylate (4j) White solid, 83% produce (29.8?mg, 0.07?mmol), 0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-10.25 (s, 1H, NH), 8.66 (s, 1H, ArH), 8.52 (s, 1H, ArH), 7.24 (s, 1H, ArH), 7.01 (d, 179.1, 164.0, 147.5, 146.7, 143.5, 140.6, 137.7, 130.0, 129.9, 129.7, 129.3, 125.7, 123.7, 123.5, 119.6, 109.4, 72.0, 61.4, 56.0, 20.5, 13.6; HR-MS (ESI): Calcd. for C21H19N3O8Na [M+Na]+: 464.1070, Found 464.1071. HPLC evaluation: MeOH/H2O (60:40), 8.73?min, HPLC purity 98.1%. 4.1.1.10. (1R,3R,3S)-3-Benzoyl-1-hydroxy-5,7-dinitro-3,4-dihydro-1H-spiro[indoline-3,2-naphthalen]-2-one (4k) White colored solid, 87% produce (31.2?mg, 0.07?mmol), 0.10 in CH2Cl2), m.p.>220?C. 1H NMR (600?MHz, DMSO-10.52 (s, 1H, NH), 8.72 (s, 1H, ArH), 8.48 (s, 1H, ArH), 7.86 (d, 199.7, 177.1, 147.9, 145.4, 142.5, 142.4, 137.8, 135.6, 133.7, 129.7, 128.8 (2C), 128.2 (2C), 128.0, 125.5, 120.6, 118.6, 108.9, 69.2, 51.3, 41.8, 26.7; HR-MS (ESI): Calcd. for C24H17N3O7Na [M+Na]+: 482.0964, Found out 482.0966. HPLC evaluation: MeOH/H2O.