Delong Wang‡
,
Min Li‡,
Jing Li,
Yali Fang* and
Zhijia Zhang*
Department of Pharmaceutical Engineering, College of Plant Protection, Shanxi Key Laboratory of Integrated Pest Management in Agriculture, Shanxi Agricultural University, Taiyuan 030031, China. E-mail: fang_august@163.com; Zhang770504@126.com
First published on 3rd April 2023
In an effort to exploit the bioactive natural scaffold 3,4-dihydroisoquinolin-1(2H)-one for plant disease management, 59 derivatives of this scaffold were synthesized using the Castagnoli–Cushman reaction. The results of bioassay indicated that their antioomycete activity against Pythium recalcitrans was superior to the antifungal activity against the other 6 phytopathogens. Compound I23 showed the highest in vitro potency against P. recalcitrans with an EC50 value of 14 μM, which was higher than that of the commercial hymexazol (37.7 μM). Moreover, I23 exhibited in vivo preventive efficacy of 75.4% at the dose of 2.0 mg/pot, which did not show significant differences compared with those of hymexazol treatments (63.9%). When the dose was 5.0 mg per pot, I23 achieved a preventive efficacy of 96.5%. The results of the physiological and biochemical analysis, the ultrastructural observation and lipidomics analysis suggested that the mode of action of I23 might be the disruption of the biological membrane systems of P. recalcitrans. In addition, the established CoMFA and CoMSIA models with reasonable statistics in the three-dimensional quantitative structure–activity relationship (3D-QSAR) study revealed the necessity of the C4-carboxyl group and other structural requirements for activity. Overall, the above results would help us to better understand the mode of action and the SAR of these derivatives, and provide crucial information for further design and development of more potent 3,4-dihydroisoquinolin-1(2H)-one derivatives as antioomycete agents against P. recalcitrans.
The fragment of 3,4-dihydroisoquinolin-1(2H)-one (1) (Fig. 1) is prevalently encountered in numerous NPs with various biological activities.7,8 Consequently, it has been utilized as a privileged scaffold in the discovery of synthetically accessible drug molecules, such as antitumor,9–13 antimicrobial,14–18 antiviral,19 and antifungal16 agents. Among the many synthetic approaches explored, the Castagnoli–Cushman reaction (CCR) between homophthalic anhydride and inimes is especially appealing because the reaction not only produces the desired products in high yields, but also offers a remarkably facile and often diastereoselective entry to derivatives of this scaffold that are 2,3-disubstituted and occur a carboxylic acid function at the C4 site.20,21 By simply drawing from pertinent pools of substrates, this robust, streamlined, straightforward, and flexible approach could enable independent change of substituents around the core scaffold in question, generating diverse chemical libraries for use in the screening of valuable molecules. However, as revealed by thorough literature retrieval, the derivatives of scaffold 1 regarding the bioactivity against phytopathogens have received far less attention. Nevertheless, despite the dearth of research, compounds 2–5 (Fig. 1), four isoquinolin-1(2H)-one derivatives sharing the same carbon skeleton with scaffold 1, showed moderate in vivo control efficacies against Blumeria graminis (DC.) Speer, Puccinia recondite, Botrytis cinerea, and Plasmopara viticola,22 indicating its potential as a starting point for the development of crop protection agents in the plant disease management.
In the present study, we synthesized 59 derivatives of scaffold 1 by employing the CCR and esterification reaction (Scheme 1 and 2), and then we evaluated their antifungal or antioomycete activities. The obtained compounds exhibited high in vitro inhibition activity against the phytopathogen Pythium recalcitrans, and their antioomycete activity was optimized via sequential incorporation of various substituents to the N2, C3, and C4 sites. The three-dimensional quantitative structure–activity relationship (3D-QSAR) was further studied. Finally, the control efficacy of the most potent compound and its antioomycete effects on the physiological and morphological changes of P. recalcitrans were investigated as well.
2-Butyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I1): white powder, yield 508 mg (52.4%), mp 206–208 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.15 (s, 1H), 7.93–7.91 (m, 1H), 7.40–7.36 (m, 2H), 7.26–7.17 (m, 4H), 7.07–7.06 (m, 2H), 5.34 (s, 1H), 4.13 (s, 1H), 4.04–3.97 (m, 1H), 2.77–2.70 (m, 1H), 1.54–1.50 (m, 2H), 1.29–1.23 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.6, 163.4, 140.0, 134.1, 132.1, 130.0, 129.6, 129.0, 128.2, 127.9, 127.2, 126.5, 61.1, 51.1, 46.0, 30.0, 20.1, 14.3. HRMS (ESI) m/z: [M + H]+, calcd for C20H22NO3: 324.1600, found: 324.1596.
2-Isobutyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I2): white powder, yield 553 mg (57%), mp 176–178 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.18 (s, 1H), 7.98–7.96 (m, 1H), 7.41–7.38 (m, 2H), 7.27–7.18 (m, 4H), 7.08–7.06 (m, 2H), 5.36 (s, 1H), 4.18 (s, 1H), 3.96–3.91 (m, 1H), 2.49–2.45 (m, 1H), 2.06–1.99 (m, 1H), 0.89 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.3, 163.6, 139.4, 133.6, 131.7, 129.6, 129.1, 128.6, 127.8, 127.4, 126.9, 126.0, 61.1, 52.8, 50.7, 26.9, 20.3, 20.2. HRMS (ESI) m/z: [M + H]+, calcd for C20H22NO3: 324.1600, found: 324.1596.
2-Octyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I3): white powder, yield 603 mg (53%), mp 212–214 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 7.92–7.91 (m, 1H), 7.41–7.36 (m, 2H), 7.25–7.16 (m, 4H), 7.07–7.05 (m, 2H), 5.33 (s, 1H), 4.11 (s, 1H), 3.99–3.93 (m, 1H), 2.77–2.72 (m, 1H), 1.54–1.52 (m, 2H), 1.22 (m, 10H), 0.85 (t, J = 5.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.6, 163.4, 140.1, 134.1, 132.1, 130.0, 129.7, 129.0, 128.2, 127.9, 127.2, 126.5, 61.2, 51.2, 46.4, 31.7, 29.3, 29.1, 27.8, 26.9, 22.6, 14.4. HRMS (ESI) m/z: [M + H]+, calcd for C24H30NO3: 380.2226, found: 380.2224.
2-Hexadecyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I4): white powder, yield 833 mg (56.5%), mp 224–226 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.02 (s, 1H), 8.03–8.02 (m, 1H), 7.54–7.48 (m, 2H), 7.44–7.41 (m, 1H), 7.21–7.18 (m, 3H), 7.01–6.99 (m, 2H), 5.09 (d, J = 4.8 Hz, 1H), 4.70 (d, J = 4.4 Hz, 1H), 3.84–3.78 (m, 1H), 2.86–2.81 (m, 1H), 1.53–1.52 (m, 1H), 1.44–1.43 (m, 1H), 1.20 (m, 26H), 0.84 (t, J = 5.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.8, 163.1, 137.9, 133.9, 132.2, 129.6, 128.6, 128.5, 128.3, 128.2, 127.8, 127.7, 61.7, 48.7, 46.2, 31.8, 29.6, 29.5, 29.4, 29.2, 27.8, 26.9, 22.6, 14.4. HRMS (ESI) m/z: [M + H]+, calcd for C32H46NO3: 492.3478, found: 492.3484.
2-(3-Butoxypropyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I5): white powder, yield 789 mg (69%), mp 236–238 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.06 (s, 1H), 7.94–7.92 (m, 1H), 7.40–7.37 (m, 2H), 7.26–7.18 (m, 4H), 7.07–7.06 (m, 2H), 5.33 (s, 1H), 4.13 (s, 1H), 4.04–4.00 (m, 1H), 3.40–3.33 (m, 4H), 2.84–2.79 (m, 1H), 1.83–1.74 (m, 2H), 1.47–1.44 (m, 2H), 1.32–1.27 (m, 2H), 0.86 (t, J = 6.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.1, 163.0, 139.5, 133.5, 131.7, 129.6, 129.1, 128.6, 127.8, 127.5, 126.8, 126.0, 69.7, 67.6, 60.9, 50.6, 43.6, 31.4, 27.9, 18.9, 13.8. HRMS (ESI) m/z: [M + H]+, calcd for C23H28NO4: 382.2018, found: 382.2022.
2-(Furan-2-ylmethyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I6): white powder, yield 781 mg (75%), mp 238–239 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.99 (s, 1H), 8.03–8.01 (m, 1H), 7.48 (s, 1H), 7.42–7.40 (m, 2H), 7.21–7.15 (m, 4H), 7.04–7.02 (m, 2H), 6.31 (s, 2H), 5.39 (s, 1H), 5.12 (d, J = 12.4 Hz, 1H), 4.23 (d, J = 12.4 Hz, 1H), 4.14 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.0, 163.0, 150.0, 142.6, 139.0, 133.7, 132.1, 129.6, 128.9, 128.5, 128.0, 127.4, 127.1, 125.9, 110.4, 108.9, 60.7, 50.8, 42.0. HRMS (ESI) m/z: [M + H]+, calcd for C21H18NO4: 348.1236, found: 348.1229.
2-(3-Morpholinopropyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I7): white powder, yield 869 mg (73.5%), mp 221–223 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.00 (s, 1H), 7.92–7.91 (m, 1H), 7.40–7.34 (m, 2H), 7.26–7.18 (m, 4H), 7.07–7.05 (m, 2H), 5.36 (s, 1H), 4.06 (s, 1H), 4.03–3.98 (m, 1H), 3.57 (t, J = 1.6 Hz, 4H), 2.81–2.76 (m, 1H), 2.40–2.35 (m, 6H), 1.80–1.71 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.3, 163.1, 139.8, 134.1, 131.6, 129.5, 129.1, 128.6, 127.6, 127.4, 126.7, 126.0, 65.9, 61.1, 55.2, 52.9, 51.1, 44.2, 24.1. HRMS (ESI) m/z: [M + H]+, calcd for C23H27N2O4: 395.1971, found: 395.1967.
2-Benzyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I8): white powder, yield 772 mg (72%), mp 171–173 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.07–8.06 (m, 1H), 7.44–7.42 (m, 2H), 7.30–7.19 (m, 9H), 7.08–7.07 (m, 2H), 5.35 (s, 1H), 5.32 (d, J = 12.0 Hz, 1H), 4.14 (s, 1H), 3.91 (d, J = 12.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.1, 163.5, 139.2, 137.2, 133.6, 132.0, 129.6, 129.0, 128.7, 128.2, 128.0, 128.0, 127.6, 127.1, 127.0, 126.1, 61.3, 50.9, 49.4. HRMS (ESI) m/z: [M + H]+, calcd for C23H20NO3: 358.1443, found: 358.1433.
1-Oxo-2-phenethyl-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I9): white powder, yield 855 mg (76.8%), mp 168–170 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.07 (s, 1H), 7.96–7.94 (m, 1H), 7.43–7.38 (m, 2H), 7.29–7.18 (m, 9H), 7.12–7.10 (m, 2H), 5.54 (s, 1H), 4.19 (s, 1H), 4.18–4.12 (m, 1H), 3.05–2.92 (m, 2H), 2.80–2.74 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.1, 162.8, 139.5, 138.9, 133.7, 131.8, 129.6, 129.1, 128.6, 128.4, 128.2, 127.8, 127.4, 126.8, 126.2, 126.1, 60.8, 50.6, 48.0, 33.6. HRMS (ESI) m/z: [M + H]+, calcd for C24H22NO3: 372.1600, found: 372.1589.
1-Oxo-3-phenyl-2-(3-phenylpropyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I10): white powder, yield 811 mg (70.2%), mp 164–166 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.09 (s, 1H), 7.97–7.95 (m, 1H), 7.42–7.37 (m, 2H), 7.28–7.17 (m, 9H), 7.09–7.07 (m, 2H), 5.39 (s, 1H), 4.16 (s, 1H), 4.12–4.06 (m, 1H), 2.83–2.78 (m, 1H), 2.61–2.56 (m, 2H), 1.88 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.2, 163.0, 141.9, 139.5, 133.5, 131.8, 129.6, 129.1, 128.6, 128.3, 127.8, 127.5, 126.8, 126.1, 125.7, 60.6, 50.6, 45.7, 32.7, 29.5. HRMS (ESI) m/z: [M + H]+, calcd for C25H24NO3: 386.1756, found: 386.1754.
2-Cyclopropyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I11): white powder, yield 733 mg (79.5%), mp 155–157 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.01 (s, 1H), 7.94–7.92 (m, 1H), 7.40–7.36 (m, 2H), 7.27–7.21 (m, 3H), 7.19–7.17 (m, 1H), 7.12–7.11 (m, 2H), 5.30 (s, 1H), 4.17 (s, 1H), 2.80–2.77 (m, 1H), 0.92–0.90 (m, 1H), 0.77–0.74 (m, 1H), 0.67–0.56 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.0, 164.6, 139.8, 133.5, 131.9, 129.7, 129.1, 128.6, 127.7, 127.3, 126.8, 125.8, 61.6, 50.7, 29.7, 8.2, 5.3. HRMS (ESI) m/z: [M + H]+, calcd for C19H18NO3: 308.1287, found: 308.1283.
2-Cyclopentyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I12): white powder, yield 784 mg (78%), mp 172–175 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 7.93–7.91 (m, 1H), 7.37–7.35 (m, 2H), 7.22–7.19 (m, 2H), 7.15–7.14 (m, 2H), 7.09–7.08 (m, 2H), 5.32 (s, 1H), 4.90–4.83 (m, 1H), 4.10 (s, 1H), 1.78–1.74 (m, 2H), 1.66–1.63 (m, 1H), 1.57–1.50 (m, 2H), 1.45–1.37 (m, 2H), 1.27–1.18 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 171.9, 163.1, 141.0, 133.1, 131.5, 130.1, 129.2, 128.4, 127.7, 127.1, 126.8, 125.9, 57.1, 55.2, 51.3, 28.8, 28.0, 23.3, 22.7. HRMS (ESI) m/z: [M + H]+, calcd for C21H22NO3: 336.1600, found: 336.1591.
2-Cyclohexyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I13): white powder, yield 852 mg (81.3%), mp 188–190 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 7.95–7.93 (m, 1H), 7.37–7.36 (m, 2H), 7.20–7.07 (m, 6H), 5.40 (s, 1H), 4.49–4.46 (m, 1H), 4.03 (s, 1H), 1.76–1.74 (m, 1H), 1.66–1.52 (m, 4H), 1.34–1.31 (m, 2H), 1.21–1.16 (m, 1H), 1.08–0.99 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 171.9, 162.5, 141.3, 133.0, 131.5, 130.3, 129.2, 128.2, 127.8, 127.1, 126.9, 126.1, 56.8, 53.2, 51.7, 30.1, 29.9, 25.5, 25.4, 24.9. HRMS (ESI) m/z: [M + H]+, calcd for C22H24NO3: 350.1756, found: 350.1750.
2-Cyclooctyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I14): white powder, yield 877 mg (77.5%), mp 185–188 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.08 (s, 1H), 7.96–7.94 (m, 1H), 7.38–7.35 (m, 2H), 7.20–7.07 (m, 6H), 5.35 (s, 1H), 3.99 (s, 1H), 1.97–1.93 (m, 1H), 1.71–1.44 (m, 14H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.2, 162.2, 141.3, 133.0, 131.4, 130.5, 129.1, 128.3, 127.8, 127.2, 126.9, 126.2, 58.6, 55.1, 51.8, 31.3, 30.3, 26.1, 26.0, 25.5, 24.8, 24.2. HRMS (ESI) m/z: [M + H]+, calcd for C24H28NO3: 378.2069, found: 378.2067.
1-Oxo-2,3-diphenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I15): white powder, yield 690 mg (67%), mp 202–203 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.09–8.07 (m, 1H), 7.62–7.57 (m, 2H), 7.52–7.48 (m, 1H), 7.33–7.29 (m, 2H), 7.20–7.16 (m, 6H), 7.05–7.03 (m, 2H), 5.50 (d, J = 5.6 Hz, 1H), 4.96 (d, J = 5.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.9, 141.7, 137.3, 134.3, 132.3, 129.2, 128.6, 128.2, 128.0, 127.9, 127.8, 127.6, 127.4, 126.6, 64.4, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C22H18NO3: 344.1287, found: 344.1277.
2-(4-Butylphenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I16): white powder, yield 650 mg (64.3%), mp 186–188 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.10–8.08 (m, 1H), 7.62–7.53 (m, 2H), 7.48–7.45 (m, 1H), 7.15–7.04 (m, 9H), 5.46 (d, J = 4.0 Hz, 1H), 4.97 (d, J = 4.0 Hz, 1H), 2.50 (t, J = 6.0 Hz, 2H), 1.51–1.48 (m, 2H), 1.28–1.24 (m, 2H), 0.86 (t, J = 5.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 162.9, 140.7, 139.4, 137.3, 134.1, 132.2, 129.3, 128.4, 128.1, 128.0, 127.8, 127.8, 127.6, 127.1, 64.6, 49.0, 34.4, 33.0, 21.8, 13.8. HRMS (ESI) m/z: [M + H]+, calcd for C26H26NO3: 400.1913, found: 400.1903.
2-(3,5-Dimethylphenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I17): white powder, yield 702 mg (63%), mp 189–191 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.09 (s, 1H), 8.12–8.11 (m, 1H), 7.66–7.47 (m, 3H), 7.18–7.17 (m, 3H), 7.06–6.85 (m, 5H), 5.47 (d, J = 4.8 Hz, 1H), 5.02 (d, J = 4.8 Hz, 1H), 2.18 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.8, 141.8, 137.8, 137.3, 134.1, 132.3, 129.4, 128.3, 128.2, 128.1, 127.9, 127.9, 127.8, 127.6, 125.1, 64.7, 48.9, 20.8. HRMS (ESI) m/z: [M + H]+, calcd for C24H22NO3: 372.1600, found: 372.1589.
2-(4-Ethoxyphenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I18): white powder, yield 872 mg (75.1%), mp 207–209 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 8.09–8.07 (m, 1H), 7.62–7.55 (m, 2H), 7.50–7.47 (m, 1H), 7.18–7.16 (m, 3H), 7.09–7.03 (m, 2H), 7.03–7.01 (m, 2H), 6.85–6.82 (m, 2H), 5.42 (d, J = 5.6 Hz, 1H), 4.97 (d, J = 5.6 Hz, 1H), 3.96 (q, J = 6.8 Hz, 2H), 1.29 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.9, 163.4, 157.4, 137.8, 134.8, 134.7, 132.7, 129.7, 129.0, 128.6, 128.4, 128.4, 128.3, 128.2, 128.0, 114.7, 65.2, 63.6, 49.4, 15.1. HRMS (ESI) m/z: [M + H]+, calcd for C24H22NO4: 388.1549, found: 372.1549.
2-(Benzo[d][1,3]dioxol-5-yl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I19): white powder, yield 779 mg (67.1%), mp 217–219 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.09–8.07 (m, 1H), 7.61–7.56 (m, 2H), 7.50–7.47 (m, 1H), 7.19–7.18 (m, 3H), 7.05–7.04 (m, 2H), 6.84–6.78 (m, 2H), 6.63–6.61 (m, 1H), 6.00 (s, 1H), 5.98 (s, 1H), 5.42 (d, J = 4.8 Hz, 1H), 4.95 (d, J = 4.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.1, 147.1, 145.8, 137.2, 135.6, 134.3, 132.3, 129.2, 128.2, 128.1, 128.0, 127.8, 127.6, 120.8, 118.1, 108.9, 107.8, 101.5, 64.8, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C23H18NO5: 388.1185, found: 388.1195.
2-(Naphthalen-2-yl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I20): white powder, yield 866 mg (73.4%), mp 195–198 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.08 (s, 1H), 8.15–8.13 (m, 1H), 7.87–7.79 (m, 4H), 7.66–7.60 (m, 3H), 7.54–7.51 (m, 1H), 7.49–7.47 (m, 2H), 7.39–7.37 (m, 1H), 7.15–7.11 (m, 4H), 5.70 (d, J = 4.8 Hz, 1H), 5.01 (d, J = 4.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.2, 139.3, 137.2, 134.4, 132.9, 132.4, 131.4, 129.2, 128.2, 128.0, 128.0, 127.9, 127.9, 127.7, 127.7, 127.5, 126.3, 126.2, 125.5, 64.5, 49.2. HRMS (ESI) m/z: [M + H]+, calcd for C26H20NO3: 394.1443, found: 394.1444.
2-([1,1′-Biphenyl]-4-yl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I21): white powder, yield 864 mg (68.7%), mp 191–193 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.06 (s, 1H), 8.16–8.14 (m, 1H), 7.66–7.58 (m, 7H), 7.51–7.50 (m, 1H), 7.45–7.42 (m, 2H), 7.36–7.32 (m, 3H), 7.20–7.19 (m, 2H), 7.12–7.11 (m, 2H), 5.59 (d, J = 4.4 Hz, 1H), 5.03 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 141.1, 139.4, 138.3, 137.3, 134.2, 132.4, 129.2, 129.0, 128.3, 128.1, 127.9, 127.8, 127.7, 127.6, 126.9, 126.7, 64.4, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C28H22NO3: 420.1600, found: 420.1602.
2-(4-Isopropylphenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I22): white powder, yield 811 mg (70.2%), mp 214–216 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.18 (s, 1H), 8.02–8.0 (m, 1H), 7.48–7.42 (m, 3H), 7.27–7.22 (m, 8H), 7.18–7.17 (m, 1H), 5.69 (s, 1H), 4.25 (s, 1H), 2.89–2.84 (m, 1H), 1.18 (d, J = 5.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.1, 162.7, 146.7, 140.0, 139.3, 133.7, 132.3, 129.7, 129.2, 128.6, 128.0, 127.4, 127.3, 126.6, 126.2, 64.3, 51.1, 33.0, 23.8, 23.8. HRMS (ESI) m/z: [M + H]+, calcd for C25H24NO3: 386.1756, found: 386.1754.
2-(4-(4-Chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I23): white powder, yield 1.17 g (83%), mp 236–238 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.01 (s, 1H), 8.09–8.07 (m, 1H), 7.61–7.58 (m, 3H), 7.51–7.48 (m, 1H), 7.43–7.41 (m, 2H), 7.21–7.19 (m, 4H), 7.06–7.05 (m, 2H), 6.99–6.95 (m, 4H), 5.51 (d, J = 4.8 Hz, 1H), 4.93 (d, J = 4.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.4, 137.3, 137.2, 134.3, 132.3, 129.9, 129.1, 128.2, 128.0, 128.0, 127.8, 127.8, 127.6, 127.3, 120.3, 118.7, 64.4, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C28H21ClNO4: 470.1159, found: 470.1147.
2-(4-Fluorophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I24): white powder, yield 821 mg (75.8%), mp 193–195 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.97 (s, 1H), 8.10–8.09 (m, 1H), 7.62–7.57 (m, 2H), 7.52–7.49 (m, 1H), 7.24–7.21 (m, 2H), 7.19–7.18 (m, 3H), 7.15–7.11 (m, 2H), 7.07–7.05 (m, 2H), 5.51 (d, J = 4.8 Hz, 1H), 4.93 (d, J = 4.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 163.1, 160.2 (d, 1JC–F = 245.5 Hz), 137.8 (d, 4JC–F = 2.5 Hz), 137.1, 134.4, 132.3, 129.5 (d, 3JC–F = 8.8 Hz), 129.0, 128.1, 128.0, 128.0, 127.8, 127.7, 127.6, 115.3 (d, 2JC–F = 23.0 Hz), 64.4, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C22H17FNO3: 362.1192, found: 362.1189.
2-(4-Chlorophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I25): white powder, yield 752 mg (66.4%), mp 200–201 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.08–8.06 (m, 1H), 7.60–7.59 (m, 2H), 7.52–7.48 (m, 2H), 7.38–7.35 (m, 2H), 7.23–7.18 (m, 4H), 7.06–7.04 (m, 2H), 5.54 (d, J = 5.6 Hz, 1H), 4.88 (d, J = 5.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.1, 140.4, 137.1, 134.5, 132.5, 130.8, 129.3, 128.9, 128.6, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 64.1, 49.1. HRMS (ESI) m/z: [M + H]+ calcd for C22H17ClNO3: 378.0897, found: 378.0888.
2-(4-Bromophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I26): white powder, yield 920 mg (72.7%), mp 211–213 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.99 (s, 1H), 8.09–8.08 (m, 1H), 7.60–7.49 (m, 4H), 7.19–7.15 (m, 6H), 7.07–7.05 (m, 2H), 5.55 (d, J = 4.8 Hz, 1H), 4.89 (d, J = 4.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 163.0, 140.8, 137.0, 134.5, 132.5, 131.5, 129.6, 128.9, 128.0, 127.9, 127.9, 127.7, 127.7, 119.2, 64.0, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C22H17BrNO3: 422.0392, found: 422.0387.
2-(4-Iodophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I27): white powder, yield 1.13 g (80.3%), mp 208–210 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.99 (s, 1H), 8.08–8.07 (m, 1H), 7.66–7.59 (m, 4H), 7.51–7.48 (m, 1H), 7.18–7.17 (m, 3H), 7.05–7.01 (m, 4H), 5.53 (d, J = 4.4 Hz, 1H), 4.89 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.9, 163.4, 141.8, 137.9, 137.5, 134.9, 133.0, 130.3, 129.4, 128.7, 128.6, 128.4, 128.3, 128.2, 125.8, 92.6, 64.5, 49.6. HRMS (ESI) m/z: [M + H]+, calcd for C22H17INO3: 470.0253, found: 470.0246.
1-Oxo-3-phenyl-2-(4-(trifluoromethoxy)phenyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I28): white powder, yield 956 mg (74.6%), mp 211–213 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.10 (m, 1H), 7.61–7.60 (m, 2H), 7.52–7.49 (m, 1H), 7.35–7.30 (m, 4H), 7.19–7.18 (m, 3H), 7.09–7.07 (m, 2H), 5.58 (d, J = 4.4 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.1, 146.3, 140.6, 137.0, 134.5, 132.5, 129.3, 128.9, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 121.1 (q, 1JC–F = 256.2 Hz), 64.2, 49.2. HRMS (ESI) m/z: [M + H]+, calcd for C23H17F3NO4: 428.1110, found: 428.1100.
2-(4-(Ethoxycarbonyl)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I29): white powder, yield 869 mg (69.8%), mp 187–189 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.11–8.10 (m, 1H), 7.89–7.87 (m, 2H), 7.61–7.60 (m, 2H), 7.53–7.50 (m, 1H), 7.39–7.37 (m, 2H), 7.18–7.17 (m, 3H), 7.10–7.08 (m, 2H), 5.67 (d, J = 4.4 Hz, 1H), 4.87 (d, J = 4.4 Hz, 1H), 4.28 (q, J = 5.6 Hz, 2H), 1.28 (t, J = 5.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 165.2, 163.1, 145.7, 137.0, 134.5, 132.6, 129.4, 128.9, 128.2, 128.0, 128.0, 127.9, 127.8, 127.7, 127.5, 127.4, 63.8, 60.7, 49.3, 14.1. HRMS (ESI) m/z: [M + H]+, calcd for C25H22NO5: 416.1498, found: 416.1496.
2-(4-Chloro-3-(trifluoromethyl)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoqui-noline-4-carboxylic acid (I30): white powder, yield 1.01 g (76.1%), mp 221–223 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.01 (s, 1H), 8.10–8.08 (m, 1H), 7.74–7.73 (m, 1H), 7.65–7.58 (m, 3H), 7.53–7.50 (m, 1H), 7.46–7.44 (m, 1H), 7.21–7.19 (m, 3H), 7.11–7.09 (m, 2H), 5.69 (d, J = 4.4 Hz, 1H), 4.86 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.4, 140.6, 136.8, 134.8, 133.1, 132.8, 131.8, 128.6, 128.1, 128.0, 127.9, 127.8, 127.0, 126.6 (q, 2JC–F = 31.4 Hz), 122.5 (q, 1JC–F = 273.2 Hz), 63.7, 49.2. HRMS (ESI) m/z: [M + H]+, calcd for C23H16ClF3NO3: 446.0771, found: 446.0760.
2-(3-Cyano-4-fluorophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I31): white powder, yield 759 mg (65.5%), mp 230–233 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.10–8.09 (m, 1H), 7.87–7.85 (m, 1H), 7.62–7.56 (m, 3H), 7.53–7.50 (m, 1H), 7.46–7.42 (m, 1H), 7.21–7.19 (m, 3H), 7.12–7.10 (m, 2H), 5.67 (d, J = 4.4 Hz, 1H), 4.84 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.4, 160.4 (d, 1JC–F = 255.2 Hz), 138.2 (d, 4JC–F = 3.1 Hz), 136.6, 135.7 (d, 3JC–F = 9.0 Hz), 134.8, 132.8, 128.5, 128.3, 128.2, 128.1, 127.9, 127.8, 116.7 (d, 2JC–F = 20.5 Hz), 113.5, 100.2 (d, 3JC–F = 16.3 Hz), 63.8, 49.3. HRMS (ESI) m/z: [M + H]+, calcd for C23H16FN2O3: 387.1145, found: 387.1134.
2-(3-Fluoro-4-morpholinophenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquino-line-4-carboxylic acid (I32): white powder, yield 843 mg (63%), mp 239–241 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.05 (s, 1H), 8.11–8.10 (m, 1H), 7.63–7.56 (m, 2H), 7.50–7.47 (m, 1H), 7.20–7.18 (m, 3H), 7.10–7.06 (m, 3H), 6.94–6.92 (m, 2H), 5.50 (d, J = 4.4 Hz, 1H), 4.97 (d, J = 4.4 Hz, 1H), 3.71 (t, J = 3.6 Hz, 4H), 2.96 (t, J = 3.6 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 163.0, 154.0 (d, 1JC–F = 244.7 Hz), 138.2 (d, 3JC–F = 8.3 Hz), 137.2, 135.8 (d, 3JC–F = 9.7 Hz), 134.2, 132.4, 129.1, 128.2, 128.1, 127.9, 127.6, 123.6 (d, 4JC–F = 2.7 Hz), 118.4 (d, 4JC–F = 3.7 Hz), 115.5 (d, 2JC–F = 22.2 Hz), 66.2, 64.4, 50.4, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C26H24FN2O4: 447.1720, found: 447.1711.
2-(4-(Difluoromethoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I33): white powder, yield 859 mg (70%), mp 205–208 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.01 (s, 1H), 8.10–8.09 (m, 1H), 7.62–7.59 (m, 2H), 7.52–7.49 (m, 1H), 7.27–7.25 (m, 5H), 7.21–7.06 (m, 5H), 5.52 (d, J = 4.4 Hz, 1H), 4.95 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.1, 149.1, 138.5, 137.1, 134.4, 132.4, 129.1, 129.0, 128.2, 128.1, 127.9, 127.9, 127.8, 127.7, 118.8, 116.3 (t, 1JC–F = 257.2 Hz), 64.4, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C23H18F2NO4: 410.1204, found: 410.1192.
2-(3-(Ethoxycarbonyl)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I34): white powder, yield 908 mg (72.9%), mp 196–198 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.11–8.09 (m, 1H), 7.84–7.77 (m, 2H), 7.61–7.60 (m, 2H), 7.53–7.50 (m, 1H), 7.44–7.43 (m, 2H), 7.19–7.18 (m, 3H), 7.08–7.06 (m, 2H), 5.60 (d, J = 4.4 Hz, 1H), 4.96 (d, J = 4.4 Hz, 1H), 4.28 (q, J = 5.6 Hz, 2H), 1.28 (t, J = 5.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 165.2, 163.1, 137.1, 134.5, 132.5, 132.1, 130.3, 129.0, 128.9, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.2, 64.1, 60.9, 49.1, 14.1. HRMS (ESI) m/z: [M + H]+, calcd for C25H22NO5: 416.1498, found: 416.1496.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-isopropylphenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinoline-4-carboxylic acid (II1): white powder, yield 1.15 g (75.3%), mp 225–227 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.00 (s, 1H), 8.09–8.08 (m, 1H), 7.64–7.62 (m, 1H), 7.59–7.47 (m, 2H), 7.42–7.21 (m, 4H), 7.06–7.05 (m, 2H), 7.00–6.96 (m, 6H), 5.46 (d, J = 4.4 Hz, 1H), 4.96 (d, J = 4.4 Hz, 1H), 2.80–2.75 (m, 1H), 1.11 (d, J = 5.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 162.9, 155.5, 154.4, 147.9, 137.4, 134.4, 134.3, 132.3, 129.9, 129.2, 129.0, 127.8, 127.5, 127.3, 126.1, 120.2, 118.8, 64.3, 48.9, 32.8, 23.6. HRMS (ESI) m/z: [M + H]+, calcd for C31H27ClNO4: 512.1629, found: 512.1626.
3-(4-(tert-Butyl)phenyl)-2-(4-(4-chlorophenoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II2): white powder, yield 1.14 g (72.7%), mp. 219–221 °C 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 8.11–8.09 (m, 1H), 7.65–7.64 (m, 1H), 7.59–7.56 (m, 1H), 7.50–7.47 (m, 1H), 7.41–7.39 (m, 2H), 7.24–7.19 (m, 4H), 6.99–6.95 (m, 6H), 5.47 (d, J = 4.4 Hz, 1H), 4.98 (d, J = 4.4 Hz, 1H), 1.18 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.4, 162.9, 155.5, 154.4, 150.3, 137.5, 134.3, 134.1, 132.3, 129.9, 129.2, 128.9, 127.9, 127.6, 127.5, 127.4, 125.0, 120.3, 118.7, 118.0, 64.3, 48.8, 34.2, 31.0, 31.0. HRMS (ESI) m/z: [M + H]+, calcd for C32H29ClNO4: 526.1785, found: 526.1786.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-isobutylphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II3): white powder, yield 1.09 g (69.1%), mp 234–236 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.99 (s, 1H), 8.10–8.09 (m, 1H), 7.62–7.56 (m, 2H), 7.50–7.47 (m, 1H), 7.41–7.19 (m, 4H), 6.98–6.93 (m, 8H), 5.48 (d, J = 4.0 Hz, 1H), 4.90 (d, J = 4.0 Hz, 1H), 2.33 (d, J = 5.6 Hz, 2H), 1.76–1.70 (m, 1H), 0.77 (d, J = 5.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.4, 140.9, 137.4, 134.5, 134.5, 132.3, 129.9, 129.1, 128.7, 127.8, 127.8, 127.6, 127.3, 120.2, 118.7, 64.3, 49.1, 44.1, 29.4, 22.1, 22.0. HRMS (ESI) m/z: [M + H]+, calcd for C32H29ClNO4: 526.1785, found: 526.1796.
3-([1,1′-Biphenyl]-4-yl)-2-(4-(4-chlorophenoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II4): white powder, yield 1.15 g (70.7%), mp 241–243 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.07 (s, 1H), 8.12–8.10 (m, 1H), 7.64–7.59 (m, 4H), 7.52–7.50 (m, 2H), 7.43–7.33 (m, 4H), 7.26–6.96 (m, 10H), 5.57 (d, J = 4.0 Hz, 1H), 4.98 (d, J = 4.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.4, 139.5, 139.1, 137.4, 136.4, 134.4, 132.4, 129.9, 129.1, 128.9, 128.6, 127.9, 127.8, 127.7, 127.6, 127.3, 126.9, 126.5, 126.3, 120.2, 118.8, 64.2, 49.0. HRMS (ESI) m/z: [M + H]+ calcd for C34H25ClNO4: 546.1472, found: 546.1467.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-methoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II5): white powder, yield 1.25 g (83.7%), mp 228–230 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.93 (s, 1H), 8.09–8.07 (m, 1H), 7.63–7.57 (m, 2H), 7.50–7.48 (m, 1H), 7.43–7.19 (m, 4H), 7.00–6.73 (m, 8H), 5.44 (d, J = 4.4 Hz, 1H), 4.90 (d, J = 4.4 Hz, 1H), 3.66 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.9, 158.8, 155.5, 154.4, 137.4, 134.4, 132.3, 129.9, 129.1, 129.0, 127.8, 127.8, 127.6, 127.3, 120.3, 118.8, 113.5, 64.0, 54.9, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C29H23ClNO5: 500.1265, found: 500.1265.
2-(4-(4-Chlorophenoxy)phenyl)-1-oxo-3-(4-(pentyloxy)phenyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II6): white powder, yield 1.31 g (79%), mp 244–246 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.97 (s, 1H), 8.08–8.07 (m, 1H), 7.63–7.56 (m, 2H), 7.50–7.47 (m, 1H), 7.42–7.19 (m, 4H), 6.99–6.71 (m, 8H), 5.44 (d, J = 4.4 Hz, 1H), 4.88 (d, J = 4.4 Hz, 1H), 3.84 (t, J = 5.2 Hz, 2H), 1.67–1.62 (m, 2H), 1.36–1.28 (m, 4H), 0.86 (t, J = 5.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.9, 158.3, 155.6, 154.3, 137.4, 134.4, 132.2, 129.9, 129.8, 129.1, 128.8, 127.8, 127.8, 127.5, 127.2, 118.8, 113.9, 67.2, 63.9, 49.1, 28.3, 27.7, 21.8, 13.8. HRMS (ESI) m/z: [M + H]+, calcd for C33H31ClNO5: 556.1891, found: 556.1890.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-isopropoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II7): white powder, yield 1.14 g (72.4%), mp 238–240 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.93 (s, 1H), 8.08–8.06 (m, 1H), 7.62–7.57 (m, 2H), 7.50–7.40 (m, 3H), 7.20–6.69 (m, 10H), 5.42 (d, J = 4.4 Hz, 1H), 4.87 (d, J = 4.4 Hz, 1H), 4.51–4.47 (m, 1H), 1.18 (d, J = 4.8 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.9, 157.1, 155.6, 154.3, 137.4, 134.5, 132.2, 129.8, 129.2, 129.1, 128.7, 127.8, 127.6, 127.3, 120.2, 118.8, 114.9, 69.0, 64.0, 49.1, 21.8, 21.7. HRMS (ESI) m/z: [M + H]+, calcd for C31H27ClNO5, 528.1578, found: 528.1577.
2-(4-(4-Chlorophenoxy)phenyl)-1-oxo-3-(4-phenoxyphenyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II8): white powder, yield 1.29 g (76.7%), mp 246–248 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.10–8.08 (m, 1H), 7.63–7.59 (m, 2H), 7.51–7.49 (m, 1H), 7.42–7.10 (m, 6H), 7.07–6.97 (m, 7H), 6.91–6.80 (m, 4H), 5.52 (d, J = 4.4 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.0, 156.3, 156.2, 155.5, 154.5, 137.3, 134.4, 132.4, 132.1, 130.0, 129.9, 129.7, 129.3, 129.1, 127.9, 127.7, 127.4, 123.7, 120.3, 118.7, 118.0, 63.9, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C34H25ClNO5: 562.1421, found: 562.1416.
2-(4-(4-Chlorophenoxy)phenyl)-1-oxo-3-(4-(trifluoromethoxy)phenyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II9): white powder, yield 1.31 g (79.4%), mp 249–251 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.99 (s, 1H), 8.10–8.08 (m, 1H), 7.62–7.59 (m, 2H), 7.42–7.39 (m, 3H), 7.22–7.19 (m, 6H), 6.99–6.96 (m, 4H), 5.61 (d, J = 4.4 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.5, 137.0, 136.6, 134.2, 132.5, 130.0, 129.9, 129.3, 128.9, 128.3, 128.3, 128.0, 127.8, 127.4, 121.0, 120.5, 120.3, 118.8, 63.5, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C29H20ClF3NO5: 554.0982, found: 554.0982.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-(difluoromethoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II10): white powder, yield 1.19 g (74.2%), mp 248–250 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 8.09–8.08 (m, 1H), 7.60–7.50 (m, 3H), 7.43–7.41 (m, 2H), 7.23–7.09 (m, 2H), 7.01–6.97 (m, 9H), 5.54 (d, J = 4.4 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.5, 150.5, 137.2, 134.2, 133.9, 132.4, 129.9, 129.7, 129.2, 129.0, 127.9, 127.8, 127.7, 127.4, 120.3, 118.8, 117.1 (t, 1JC–F = 260.1 Hz), 63.7, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C29H21ClF2NO5: 536.1076, found: 536.1077.
2-(4-(4-Chlorophenoxy)phenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II11): white powder, yield 1.11 g (69.9%), mp 251–253C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.06–8.04 (m, 1H), 7.64–7.62 (m, 1H), 7.60–7.57 (m, 1H), 7.50–7.43 (m, 3H), 7.21–7.20 (m, 2H), 7.02–6.99 (m, 4H), 6.67–6.48 (m, 3H), 5.36 (d, J = 3.6 Hz, 1H), 4.91 (d, J = 3.6 Hz, 1H), 4.14 (s, 2H), 4.13 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 162.9, 155.6, 154.4, 143.1, 142.7, 137.4, 134.4, 132.3, 130.1, 129.9, 129.1, 129.0, 127.9, 127.8, 127.6, 127.3, 120.9, 120.3, 118.8, 116.7, 116.5, 63.9, 63.9, 48.9. HRMS (ESI) m/z: [M + H]+, calcd for C30H23ClNO6: 528.1214, found: 528.1211.
2-(4-(4-Chlorophenoxy)phenyl)-3-(2,3-dihydrobenzofuran-5-yl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II12): white powder, yield 1.05 g (68.3%), mp 252–254 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.04 (s, 1H), 8.01–8.00 (m, 1H), 7.49–7.46 (m, 1H), 7.44–7.35 (m, 2H), 7.32–7.04 (m, 3H), 7.07–7.03 (m, 6H), 6.89–6.59 (m, 2H), 5.60 (s, 1H), 4.42 (t, J = 7.2 Hz, 2H), 4.18 (s, 1H), 3.04 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 172.2, 162.6, 159.2, 155.5, 154.4, 138.0, 133.9, 132.4, 130.9, 129.9, 129.8, 129.1, 128.2, 128.0, 127.8, 127.4, 127.4, 125.8, 123.0, 120.4, 118.9, 108.7, 71.0, 64.1, 51.4, 28.9. HRMS (ESI) m/z: [M + H]+, calcd for C30H23ClNO5: 512.1265, found: 512.1273.
3-(4-Chloro-3-fluorophenyl)-2-(4-(4-chlorophenoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II13): white powder, yield 1.19 g (76%), mp 243–245 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.97 (s, 1H), 8.10–8.08 (m, 1H), 7.60–7.51 (m, 3H), 7.45–7.39 (m, 4H), 7.25–7.24 (m, 2H), 7.07–6.97 (m, 5H), 5.63 (d, J = 4.4 Hz, 1H), 4.92 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 156.6 (d, 1JC–F = 247.0 Hz), 155.5, 154.6, 139.1 (d, 3JC–F = 6.0 Hz), 136.9, 134.1, 132.6, 130.5, 130.0, 129.9, 129.3, 128.9, 128.3, 128.0, 128.0, 127.7, 127.4, 125.4 (d, 4JC–F = 3.0 Hz), 120.5, 120.3, 119.1 (d, 3JC–F = 17.3 Hz), 118.9, 118.8, 116.6 (d, 2JC–F = 21.6 Hz), 63.1, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C28H19Cl2FNO4: 522.0675, found: 522.0676.
2-(4-(4-Chlorophenoxy)phenyl)-3-(3,4-dimethoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II14): white powder, yield 1.25 g (78.6%), mp 238–240 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 12.98 (s, 1H), 8.08–8.07 (m, 1H), 7.64–7.48 (m, 5H), 7.42–7.40 (m, 2H), 7.24–6.96 (m, 4H), 6.76–6.60 (m, 3H), 5.47 (d, J = 4.0 Hz, 1H), 4.84 (d, J = 4.0 Hz, 1H), 3.66 (s, 3H), 3.51 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.7, 163.1, 155.7, 154.3, 148.3, 147.9, 137.4, 134.6, 132.3, 129.9, 129.3, 127.8, 127.6, 127.3, 120.4, 120.1, 118.8, 111.6, 111.1, 64.0, 55.3, 55.0, 49.4. HRMS (ESI) m/z: [M + H]+, calcd for C30H25ClNO6: 530.1370, found: 530.1379.
2-(4-(4-Chlorophenoxy)phenyl)-3-(3-fluoro-4-methoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II15): white powder, yield 1.14 g (73.3%), mp 241–244 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.02 (s, 1H), 8.08–8.07 (m, 1H), 7.59–7.41 (m, 5H), 7.22–6.81 (m, 9H), 5.49 (d, J = 3.6 Hz, 1H), 4.88 (d, J = 3.6 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.0, 155.5, 154.5, 150.6 (d, 1JC–F = 243.9 Hz), 146.7 (d, 3JC–F = 10.5 Hz), 137.1, 134.3, 132.5, 129.9, 129.2, 129.0, 127.9, 127.8 (d, 4JC–F = 2.7 Hz), 127.4, 124.4 (d, 4JC–F = 2.6 Hz), 120.3, 118.2, 115.5 (d, 2JC–F = 19.1 Hz), 113.3, 63.3, 55.8, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C29H22ClFNO5: 518.1171, found: 518.1174.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-fluoro-3-phenoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II16): white powder, yield 1.29 g (74.1%), mp 253–255 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.07 (s, 1H), 8.00–7.99 (m, 1H), 7.60–7.58 (m, 2H), 7.48–7.41 (m, 3H), 7.32–7.20 (m, 5H), 7.18–6.97 (m, 6H), 6.87–6.72 (m, 3H), 5.60 (d, J = 4.4 Hz, 1H), 4.84 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.6, 163.0, 156.4, 155.5, 154.5, 154.0, 152.0, 142.1, 142.0, 136.9, 134.8, 134.8, 134.4, 132.5, 130.0, 129.9, 129.4, 128.9, 127.9, 127.8, 127.6, 125.3, 125.2, 123.3, 121.9, 120.3, 118.7, 117.0, 116.9, 116.5, 63.2, 49.2. HRMS (ESI) m/z: [M + H]+, calcd for C34H24ClFNO5: 580.1327, found: 580.1322.
3-(4-Bromophenyl)-2-(4-(4-chlorophenoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II17): white powder, yield 1.29 g (78.4%), mp 249–251 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.03 (s, 1H), 8.09–8.07 (m, 1H), 7.59–7.58 (m, 2H), 7.43–7.39 (m, 5H), 7.22–7.21 (m, 2H), 7.02–6.96 (m, 6H), 5.54 (d, J = 4.4 Hz, 1H), 4.92 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.5, 137.1, 136.7, 134.2, 132.5, 131.1, 130.2, 129.9, 129.9, 129.2, 128.9, 128.6, 128.2, 127.9, 127.8, 127.4, 121.3, 120.4, 120.3, 118.8, 63.7, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C28H20BrClNO4: 548.0264, found: 548.0254.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-fluorophenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II18): white powder, yield 1.20 g (82.1%), mp 246–248 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.02 (s, 1H), 8.09–8.08 (m, 1H), 7.60–7.59 (m, 2H), 7.52–7.49 (m, 1H), 7.43–7.41 (m, 2H), 7.21–6.96 (m, 10H), 5.55 (d, J = 4.4 Hz, 1H), 4.91 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 162.6, 160.7, 155.5, 154.5, 137.1, 134.3, 133.5, 132.4, 130.1, 130.0, 129.9, 129.3, 129.0, 127.9, 127.8, 127.7, 127.4, 120.3, 118.8, 115.1, 114.9, 63.6, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C28H20ClFNO4: 488.1065, found: 488.1063.
2-(4-(4-Chlorophenoxy)phenyl)-3-(4-chlorophenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II19): white powder, yield 1.22 g (80.9%), mp 248–250 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.09 (s, 1H), 8.10–8.08 (m, 1H), 7.58–7.40 (m, 5H), 7.27–7.21 (s, 4H), 7.09–6.96 (m, 6H), 5.56 (d, J = 4.4 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 155.5, 154.5, 137.1, 136.3, 134.2, 132.7, 132.5, 129.9, 129.9, 129.2, 129.0, 128.6, 128.2, 128.2, 127.9, 127.8, 127.5, 127.4, 120.3, 118.8, 63.6, 49.0. HRMS (ESI) m/z: [M + H]+, calcd for C28H20Cl2NO4: 504.0769, found: 504.0770.
3-(4-Bromo-3-fluorophenyl)-2-(4-(4-chlorophenoxy)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (II20): white powder, yield 1.32 g (77.6%), mp 253–255 °C. 1H NMR (400 MHz, DMSO-d6) δH (ppm): 13.10 (s, 1H), 8.09–8.07 (m, 1H), 7.61–7.51 (m, 4H), 7.42–7.24 (m, 3H), 7.23–7.05 (m, 2H), 7.00–6.87 (m, 5H), 5.61 (d, J = 4.4 Hz, 1H), 4.91 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δC (ppm): 170.5, 163.0, 158.6, 156.7, 155.5, 154.6, 136.9, 134.2, 133.3, 132.6, 130.0, 129.9, 129.3, 128.9, 128.9, 128.3, 128.2, 128.0, 128.0, 127.7, 127.4, 125.8, 116.6, 116.4, 107.7, 107.5, 63.1, 49.1. HRMS (ESI) m/z: [M + H]+, calcd for C28H19BrClFNO4: 566.0170, found: 566.0167.
Methyl 2-(4-(4-chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (III1): white powder, yield 406 mg (83.9%), mp 146–148 °C. 1H NMR (400 MHz, CDCl3) δH (ppm): 8.25–8.22 (m, 1H), 7.46–7.44 (m, 2H), 7.31–7.27 (m, 3H), 7.26–7.18 (m, 5H), 7.16–7.14 (m, 2H), 6.95–6.93 (m, 4H), 5.61 (s, 1H), 4.02 (d, J = 0.8 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, CDCl3) δC (ppm): 171.5, 163.8, 155.9, 155.9, 132.8, 132.5, 130.1, 129.8, 129.7, 129.2, 129.0, 128.8, 128.8, 128.6, 128.4, 126.7, 120.7, 119.4, 65.3, 53.3, 52.0. HRMS (ESI) m/z: [M + H]+, calcd for C29H23ClNO4: 484.1316, found: 484.1303.
Phenyl 2-(4-(4-chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (III2): white powder, yield 465 mg (85.2%), mp 167–169 °C. 1H NMR (400 MHz, CDCl3) δH (ppm): 8.30–8.28 (m, 1H), 7.51–7.49 (m, 2H), 7.36–7.31 (m, 5H), 7.27–7.21 (m, 8H), 7.00–6.98 (m, 2H), 6.94–6.91 (m, 4H), 5.69 (s, 1H), 4.28 (d, J = 0.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δC (ppm): 169.4, 163.4, 155.7, 155.6, 150.5, 138.8, 137.7, 132.7, 131.8, 129.8, 129.6, 129.5, 129.0, 128.9, 128.7, 128.5, 128.4, 128.2, 126.5, 126.4, 121.2, 120.3, 119.1, 64.9, 51.8. HRMS (ESI) m/z: [M + H]+, calcd for C34H25ClNO4: 546.1472, found: 546.1472.
Benzyl 2-(4-(4-chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (III3): white powder, yield 457 mg (81.6%), mp 172–174 °C. 1H NMR (400 MHz, CDCl3) δH (ppm): 8.26–8.24 (m, 1H), 7.45–7.43 (m, 2H), 7.31–7.30 (m, 3H), 7.27–7.25 (m, 2H), 7.22–7.19 (m, 6H), 7.16–7.14 (m, 4H), 6.93–6.92 (m, 2H), 6.87–6.85 (m, 2H), 5.58 (s, 1H), 5.22 (d, J = 8.4 Hz, 1H), 5.16 (d, J = 8.4 Hz, 1H), 4.05 (d, J = 0.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δC (ppm): 170.8, 163.8, 155.9, 155.8, 139.4, 138.0, 135.5, 132.8, 132.5, 130.1, 129.8, 129.8, 129.2, 129.0, 129.0, 128.8, 128.8, 128.80, 128.5, 128.5, 128.4, 126.7, 120.6, 119.4, 67.9, 65.3, 52.2. HRMS (ESI) m/z: [M + H]+, calcd for C35H27ClNO4: 560.1629, found: 560.1628.
Phenethyl 2-(4-(4-chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (III4): light yellow oily liquid, yield 478 mg (83.3%). 1H NMR (400 MHz, CDCl3) δH (ppm): 8.24–8.23 (m, 1H), 7.42–7.40 (m, 2H), 7.27–7.22 (m, 4H), 7.22–7.18 (m, 6H), 7.13–7.10 (m, 3H), 7.02–7.01 (m, 2H), 6.94–6.91 (m, 4H), 5.56 (s, 1H), 4.38–4.33 (m, 2H), 3.96 (d, J = 0.8 Hz, 1H), 2.86 (t, J = 5.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δC (ppm): 170.8, 163.8, 155.9, 155.8, 139.4, 138.1, 137.7, 132.8, 132.5, 130.1, 129.9, 129.7, 129.1, 128.9, 128.9, 128.8, 128.8, 128.7, 128.5, 128.3, 127.0, 126.7, 120.6, 119.4, 66.7, 65.2, 52.1, 35.2. HRMS (ESI) m/z: [M + H]+, calcd for C36H29ClNO4: 574.1785, found: 574.1784.
Cyclohexyl 2-(4-(4-chlorophenoxy)phenyl)-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (III5): white powder, yield 456 mg (82.7%), mp 158–160 °C. 1H NMR (400 MHz, CDCl3) δH (ppm): 8.24–8.23 (m, 1H), 7.45–7.43 (m, 2H), 7.32–7.30 (m, 2H), 7.27–7.26 (m, 2H), 7.23–7.16 (m, 6H), 6.94–6.92 (m, 4H), 5.61 (s, 1H), 4.86–4.84 (m, 1H), 3.99 (d, J = 0.8 Hz, 1H), 1.77–1.76 (m, 1H), 1.66–1.65 (m, 1H), 1.55–1.45 (m, 1H), 1.44–1.41 (m, 3H), 1.36–1.24 (m, 4H). 13C NMR (100 MHz, CDCl3) δC (ppm): 170.4, 163.8, 156.0, 155.7, 133.0, 132.6, 130.0, 129.7, 129.6, 129.1, 128.8, 128.7, 128.7, 128.5, 128.3, 126.8, 120.6, 119.4, 74.4, 65.3, 52.5, 31.6, 31.4, 25.5, 23.5, 23.4. HRMS (ESI) m/z: [M + H]+ calcd for C34H31ClNO4: 552.1942, found: 552.1939.
Compd. | Inhibition rate (%) | ||||||
---|---|---|---|---|---|---|---|
Bo. (6 d) | Fu. (6 d) | Co. (7 d) | Tr. (7 d) | Al. (7 d) | Rh. (3 d) | Py. (2 d) | |
a Bo.: Botrytis cinerea; Fu.: Fusarium oxysporum f. sp. niveum; Co.: Colletotrichum gloeosporioides; Tr.: Trichothecium roseum; Al.: Alternaria mali; Rh.: Rhizoctonia solani; Py.: Pythium recalcitrans. The number of days in each bracket is the culture duration of the strains on PDA plates before measuring the diameters of mycelial colonies. The data in the table are expressed as the mean ± standard error of mean (SEM) (n = 3). | |||||||
I1 | 71.9 ± 2.2 | 86.7 ± 2.1 | 86.3 ± 1.0 | 28.6 ± 1.3 | 69.2 ± 1.6 | 96.5 ± 3.5 | 99.2 ± 0.4 |
I2 | 69.6 ± 0.9 | 70.3 ± 1.4 | 82.3 ± 0.7 | 27.8 ± 1.5 | 66.0 ± 0.9 | 81.2 ± 1.6 | 98.4 ± 0.3 |
I3 | 70.2 ± 1.0 | 81.5 ± 0.3 | 88.3 ± 0.5 | 39.6 ± 3.3 | 73.0 ± 1.0 | 88.4 ± 0.7 | 99.7 ± 0.3 |
I4 | 65.3 ± 0.6 | 55.1 ± 0.7 | 76.1 ± 1.1 | 33.3 ± 3.6 | 55.3 ± 1.8 | 55.8 ± 2.1 | 97.2 ± 0.1 |
I5 | 72.2 ± 1.2 | 82.6 ± 1.2 | 78.3 ± 0.8 | 44.9 ± 1.3 | 71.3 ± 0.5 | 87.9 ± 2.1 | 98.9 ± 0.2 |
I6 | 72.6 ± 0.7 | 73.9 ± 2.4 | 80.4 ± 0.5 | 39.6 ± 0.7 | 64.0 ± 3.9 | 89.8 ± 3.5 | 98.4 ± 0.1 |
I7 | 68.6 ± 1.7 | 67.5 ± 1.4 | 79.1 ± 0.4 | 37.6 ± 0.8 | 61.7 ± 1.0 | 93.3 ± 3.4 | 96.7 ± 0.5 |
I8 | 69.6 ± 1.4 | 64.7 ± 0.4 | 85.7 ± 0.1 | 44.4 ± 1.2 | 71.0 ± 1.2 | 80.3 ± 2.7 | 98.3 ± 0.4 |
I9 | 72.6 ± 1.3 | 53.5 ± 2.9 | 86.5 ± 0.4 | 41.9 ± 0.5 | 73.0 ± 1.3 | 71.5 ± 1.1 | 98.2 ± 0.2 |
I10 | 73.4 ± 1.5 | 80.6 ± 1.0 | 86.8 ± 0.3 | 36.3 ± 1.6 | 74.5 ± 0.8 | 79.8 ± 1.2 | 99.1 ± 0.3 |
I11 | 74.5 ± 0.7 | 72.5 ± 0.7 | 78.8 ± 0.5 | 38.3 ± 1.3 | 65.7 ± 0.3 | 66.9 ± 1.6 | 94.7 ± 0.7 |
I12 | 71.6 ± 2.4 | 74.4 ± 1.0 | 83.8 ± 0.7 | 39.1 ± 3.0 | 64.9 ± 0.8 | 81.4 ± 1.7 | 96.7 ± 0.2 |
I13 | 65.3 ± 1.2 | 67.0 ± 0.5 | 84.3 ± 0.4 | 35.6 ± 1.0 | 65.4 ± 1.5 | 79.3 ± 0.3 | 97.2 ± 0.1 |
I14 | 73.5 ± 0.9 | 73.1 ± 0.9 | 90.2 ± 0.3 | 41.4 ± 2.3 | 71.8 ± 0.8 | 82.8 ± 1.2 | 97.3 ± 0.1 |
I15 | 68.9 ± 2.0 | 82.3 ± 1.2 | 87.5 ± 0.5 | 44.0 ± 0.4 | 68.4 ± 1.2 | 82.2 ± 0.8 | 98.9 ± 0.3 |
I16 | 76.2 ± 0.6 | 86.8 ± 1.3 | 92.5 ± 0.1 | 45.9 ± 1.2 | 75.0 ± 0.8 | 81.7 ± 1.5 | 100.0 |
I17 | 74.5 ± 1.7 | 85.5 ± 0.7 | 90.3 ± 0.4 | 44.4 ± 0.8 | 75.6 ± 3.1 | 78.7 ± 1.0 | 99.6 ± 0.1 |
I18 | 80.8 ± 0.9 | 84.8 ± 1.4 | 89.7 ± 0.5 | 49.1 ± 1.8 | 70.7 ± 0.6 | 90.6 ± 0.7 | 99.6 ± 0.1 |
I19 | 79.2 ± 2.1 | 90.8 ± 1.0 | 92.9 ± 0.4 | 50.1 ± 0.5 | 74.2 ± 0.3 | 93.8 ± 1.2 | 100.0 |
I20 | 82.1 ± 0.6 | 87.6 ± 0.5 | 93.3 ± 0.8 | 43.0 ± 1.2 | 78.5 ± 1.6 | 73.1 ± 1.0 | 100.0 |
I21 | 74.0 ± 0.4 | 86.3 ± 0.3 | 90.3 ± 0.1 | 39.3 ± 2.8 | 80.5 ± 1.5 | 85.7 ± 2.8 | 100.0 |
I22 | 74.5 ± 0.9 | 78.3 ± 0.3 | 90.3 ± 0.4 | 36.5 ± 1.9 | 71.0 ± 1.8 | 100.0 | 100.0 |
I23 | 76.4 ± 3.1 | 87.2 ± 0.4 | 88.5 ± 0.3 | 46.9 ± 1.0 | 81.4 ± 0.8 | 100.0 | 100.0 |
I24 | 56.4 ± 1.5 | 37.9 ± 2.6 | 68.1 ± 1.1 | 42.3 ± 1.8 | 66.3 ± 1.3 | 93.7 ± 10.1 | 100.0 |
I25 | 63.6 ± 2.9 | 42.7 ± 1.0 | 73.1 ± 1.5 | 45.9 ± 3.7 | 69.5 ± 1.0 | 77.2 ± 7.8 | 100.0 |
I26 | 59.7 ± 1.2 | 39.4 ± 0.4 | 74.6 ± 1.5 | 42.3 ± 2.1 | 72.4 ± 0.3 | 78.6 ± 10.0 | 100.0 |
I27 | 67.6 ± 0.3 | 39.3 ± 1.7 | 73.6 ± 2.2 | 50.0 ± 7.6 | 74.7 ± 1.3 | 68.2 ± 4.7 | 100.0 |
I28 | 61.6 ± 0.3 | 42.3 ± 2.2 | 65.6 ± 1.6 | 49.5 ± 2.2 | 71.8 ± 2.8 | 76.2 ± 0.6 | 100.0 |
I29 | 55.0 ± 1.2 | 41.8 ± 0.6 | 63.4 ± 0.8 | 46.4 ± 5.8 | 63.7 ± 2.8 | 62.5 ± 3.9 | 97.0 ± 0.4 |
I30 | 65.9 ± 1.8 | 51.4 ± 0.7 | 77.1 ± 1.0 | 57.1 ± 6.3 | 76.2 ± 1.1 | 69.4 ± 2.8 | 100.0 |
I31 | 60.2 ± 1.6 | 41.4 ± 2.1 | 64.6 ± 0.3 | 52.2 ± 6.3 | 70.7 ± 1.1 | 83.2 ± 10.1 | 98.8 ± 0.7 |
I32 | 57.7 ± 1.4 | 42.7 ± 0.7 | 64.1 ± 0.1 | 53.3 ± 4.0 | 66.6 ± 0.6 | 67.7 ± 0.6 | 100.0 |
I33 | 63.6 ± 1.6 | 41.4 ± 1.3 | 68.2 ± 0.2 | 41.6 ± 0.5 | 67.5 ± 1.1 | 94.5 ± 1.4 | 98.3 ± 0.3 |
I34 | 60.6 ± 1.2 | 40.4 ± 0.5 | 68.9 ± 1.6 | 47.9 ± 1.8 | 64.0 ± 1.8 | 85.7 ± 5.8 | 95.9 ± 0.1 |
To obtain the toxicity of the compounds, 2 mg, 5 mg, 10 mg, 20 mg, 40 mg, 100 mg and 200 mg of each test compound were dissolved in 800 μL of DMSO containing 5% OA-12, respectively. The following mix of stock solution (20 μL) with PDA medium (50 mL) rendered the concentrations of 1 μg mL−1, 2 μg mL−1, 5 μg mL−1, 10 μg mL−1, 20 μg mL−1, 50 μg mL−1 and 100 μg mL−1. The following operations were performed by following the same procedures described above. The inhibition rate of each concentration was calculated. The concentration was log-transformed value (y) and the inhibition rate was transformed into probit value (x). A linear regression equation was obtained by fitting y against x using Microsoft Excel 2013 and the median effective concentration value (EC50) was calculated from the obtained equation. The experiment was replicated three times and the result was presented as the mean value of three independent replicates ± SD.
Compd. | EC50 (μM) | Compd. | EC50 (μM) |
---|---|---|---|
a The data in the table are presented as the mean ± standard derivation (SD) of three repeated experiments. | |||
I1 | 46.6 ± 2.9 | I32 | 26.9 ± 0.3 |
I2 | 45.8 ± 2.3 | I33 | 43.1 ± 0.8 |
I3 | 29.5 ± 2.0 | I34 | 40.9 ± 2.3 |
I4 | 45.4 ± 2.8 | II1 | 18.2 ± 0.2 |
I5 | 53.7 ± 1.0 | II2 | 27.7 ± 0.1 |
I6 | 46.8 ± 2.6 | II3 | 32.0 ± 0.5 |
I7 | 43.6 ± 0.5 | II4 | 38.5 ± 1.8 |
I8 | 59.8 ± 0.6 | II5 | 45.3 ± 2.6 |
I9 | 57.4 ± 0.5 | II6 | 43.0 ± 2.0 |
I10 | 54.0 ± 0.7 | II7 | 31.5 ± 5.4 |
I11 | 46.5 ± 2.1 | II8 | 47.1 ± 1.5 |
I12 | 67.8 ± 0.1 | II9 | 31.3 ± 2.3 |
I13 | 65.1 ± 0.1 | II10 | 41.3 ± 0.2 |
I14 | 40.6 ± 1.5 | II11 | 16.9 ± 0.4 |
I15 | 42.8 ± 0.7 | II12 | 35.8 ± 3.2 |
I16 | 20.9 ± 0.2 | II13 | 29.6 ± 0.4 |
I17 | 23.2 ± 1.1 | II14 | 47.8 ± 1.1 |
I18 | 25.1 ± 1.7 | II15 | 31.3 ± 0.1 |
I19 | 21.5 ± 0.5 | II16 | 48.7 ± 0.9 |
I20 | 23.2 ± 1.0 | II17 | 30.3 ± 0.3 |
I21 | 14.7 ± 0.3 | II18 | 33.1 ± 0.1 |
I22 | 20.8 ± 0.1 | II19 | 32.2 ± 0.1 |
I23 | 14.0 ± 0.3 | II20 | 34.0 ± 2.8 |
I24 | 38.9 ± 4.0 | III1 | 951.4 ± 41.4 |
I25 | 33.2 ± 1.1 | III2 | 1258.1 ± 76.3 |
I26 | 29.6 ± 1.9 | III3 | 874.3 ± 65.2 |
I27 | 26.0 ± 0.1 | III4 | 895.7 ± 58.7 |
I28 | 29.4 ± 1.2 | III5 | 1055.2 ± 80.1 |
I29 | 41.7 ± 3.5 | Hymexazol | 37.7 ± 3.8 |
I30 | 26.0 ± 0.6 | Dimethomorph | >258.4 |
I31 | 36.3 ± 2.7 |
For CoMFA studies, a cubic lattice with a grid spacing of 4.0 Å was generated to calculate steric and electrostatic fields using the sp3 hybridized carbon as the probe atom with a +1.0 charge. Cut-off values for both fields were set to 30.0 kcal mol−1. For CoMSIA studies, the same lattice with the parameters (probe atom with a +1.0 charge and attenuation factor of 0.3) was used to calculate steric and electrostatic, hydrophobic, and hydrogen bond donor and acceptor fields.
For the partial least squares (PLS) analysis, the leave-one-out (LOO) method was used to carry out a cross-validation analysis, giving the square of the cross-validation coefficient (qcv2) and the optimum number of components (Nopt). Using Nopt, a final model was generated with the non-cross-validated correlation coefficient (rncv2), standard error of estimation (SEEncv), and F-test value (F) using Sybyl software.
For the test set, the predictive correlation coefficient (rtest2) was calculated using the following formula:
The lipid absolute quantification analysis was performed using liquid chromatography with tandem mass spectrometry (LC-MS-MS) method. Reverse phase chromatography was used for LC separation (charged surface hybrid C18 column: pore size 130 Å, particle size 1.7 μm, 2.1 mm × 100 mm, Waters). The eluting solution A was comprised of CH3CN/H2O (6:4, v/v) with 0.1% formic acid and 0.1 Mm ammonium formate and B was comprised of CH3CN/isopropanol (1:9, v/v) with 0.1% formic acid and 0.1 Mm ammonium formate. In brief, the lipid extract solutions (in 200 μL 90% isopropanol/CH3CN) were centrifuged at 14000 g for 15 min at 10 °C. An aliquot of 3.0 μL of sample was injected into the chromatographic instrument. The initial eluting solvent system was 30% B in A at a flow rate of 300 μL min−1. It was kept for 2 min, and then linearly increased to 100% solvent B in 23 min, followed by equilibrating at 5% solvent B for 10 min. MS was carried out by Q-Exactive Plus in positive and negative mode, respectively. ESI parameters were optimized and preset for all measurements as follows: source temperature, 300 °C; capillary temperature, 350 °C; the ion spray voltage, 3000 V; S-Lens RF level, 50%; and the scan range, m/z 200–1800.
Lipid Search, a search engine, was used for the identification of lipid species based on MS/MS math. Lipid Search contains more than 30 lipid classes and more than 1500000 fragment ions in the database. Both mass tolerance for precursor and fragment were set to 5 ppm.
Generally, the CCR generates racemic products. The relative configuration assignment was initially performed on the basis of the chemical shifts of H-4 and the coupling constants between H-3 and H-4. The high field signals (<4.30 ppm) were found for the H-4 signals of 3-phenyl trans-diastereomers, while the relative low field signals (>4.50 ppm) occurred for the cis-isomers.24,26–29 According to the reported molecular models,24,26–28 this might be ascribed to the shielded effects of the aromatic cloud on H-4 of trans-isomers. Meanwhile, a relative larger H-3/H-4 coupling constant of cis-diastereomers usually occurred higher than 4.0 Hz (Fig. 2). Conversely, trans-configured products generally show H-3/H-4 coupling constant of less than 2.5 Hz. The relative configuration analysis of trans-I6 was exemplified via its single-crystal X-ray results (Fig. 2 and Table S1†). Thereby, compounds I4, I5–I21, I23, I24, I26–I34, II1–II3, II5, II7, II8, II11, II14–II16, and II18 were obtained predominantly as a single and racemic cis-isomer. However, the only trans-isomer was formed for compounds I1–I3, I5–I8, I10–I14, I22, II12, and III1–III5, since their H-3/H-4 signals appeared singlet (or unresolved doublets). Collectively, the relative configuration ratios of the other compounds were shown in Table S2.† Particularly notable is the configuration inversion of the trans-esters III1–III5 from cis-I23. It has been demonstrated that the base could promote totally isomerization of cis- to trans-configuration when cis-products of CCR were exposed to aqueous NaOH solution30 or the organic base N,N′-carbonyldiimidazole (CDI) was used in the trans-ester synthesis from the cis-precursor.21 The cis-products of CCR are conceived as the kinetic products and they were removed from the reaction system by precipitation at room temperature and the isomerization to more stable trans-configured counterpart might occur under some conditions, such as the existence of bases. In the present study, we found the general coupling reagent DCC could lead to the configuration inversion as well.
Fig. 2 The reported δH4 values and H-3/H-4 coupling constants of cis- and trans-diastereomers of 3-phenyl tetrahydroisoquinolone-4-carboxylic acid compounds (a); and the δH4 value and H-3/H-4 coupling constant of cis-I6 and its single-crystal structure (CCDC Deposition No. 2180985†) (b). |
It is noteworthy that the oomycete P. recalcitrans is a soil-borne phytopathogen with a wide host-range, such as tobacco,31 soybean,32 corn,33 alfalfa,34 grape, beet35 and carrot,36 causing seed decay, seedling collapse and root rot, cavity spot, and significantly reducing crop yield. Due to its easy recovery profile from flooding soil37 and insensitivity to many commercial fungicides,33,36 there is a need for exploring effective antioomycete agents to control this pathogen. The toxicity of compounds I1–I34,II1–II20 and III1–III5 against mycelial growth of P. recalcitrans was further determined and the results were shown in Table 2. I23 stood out as the most potent activity with the EC50 value of 14.0 μM (Fig. S2†), which was superior over hymexazol (EC50 = 37.7 μM) and dimethomorph (EC50 > 258.4 μM). Generally, the EC50 values of 4-carboxyl derivatives I1–I34 and II1–II20 ranged from 14.0 μM to 67.8 μM. Nevertheless, loss of a free carboxyl group (III1–III5) triggered a significant decrease in toxicity, indicating the necessity of unsubstituted carboxyl group for the activity.
Parameters | CoMFA | CoMSIA |
---|---|---|
a “—” indicates that the data has not been determined. | ||
qcv2 | 0.609 | 0.711 |
Nopt | 4 | 5 |
rncv2 | 0.940 | 0.945 |
SEEncv | 0.111 | 0.107 |
F-value | 171.652 | 147.469 |
rtest2 | 0.989 | 0.989 |
SEPtest | 0.130 | 0.131 |
Steric | 0.581 | 0.110 |
Electrostatic | 0.419 | 0.187 |
Hydrophobic | — | 0.178 |
H-bond donor | — | 0.135 |
H-bond acceptor | — | 0.391 |
The internal validation parameters revealed that these two models were statistically significant of predicating the activity. As shown in Fig. S3,† the actual and predicated pEC50 values of the training and test set molecules (Table S3†) were strongly correlated in a linear fashion. Meanwhile, the external validation parameters of the test set reflect the good predicative capacity of the models. The rtest2 (and SEPtest) of the CoMFA and CoMSIA models were 0.989 (0.130) and 0.989 (0.131), respectively.
In the steric contour maps of CoMFA model (Fig. 3a), it can be observed that a big green contour was near the N2 site, indicating that bulky groups at that position might be more favorable for the activity. This was identical with the following experimental results: I21 (R1 = 4-C6H5C6H4) > I16 (4-CH3(CH2)3C6H4) > I15 (C6H5) > I1 ((CH2)3CH3) ≈ I11 (cyclopropyl), I28 (4-CF3OC6H4) > I33 (4-OCHF2C6H4), II13 (3-F,4-ClC6H3) > II19 (4-ClC6H4). The other two big yellow contour regions around the phenyl group at C3 site and the carboxyl group at C4 site suggest that bulky groups at those sites furnish negative effects to the bioactivity. This could be exemplified by the comparison of compounds I23 and II1–II20. Meanwhile, the above analysis have confirmed that the substitution of carboxyl group at C4 site significantly decreased the activity. It is found that a blue contour map overlaid on the para-position of the C-3 phenyl group in the electrostatic contour maps (Fig. 3b), signifying that the superior substituents at that site should cause fairly lesser electron density. The situation can be proved by the activity order: II1 (R2 = 4(CH3)2CHC6H4) > II2 (4-(CH3)3CC6H4) > II17 (4-BrC6H4). The presence of a red block near the meta-position of the C-3 phenyl group denotes that the electron-rich substituents attached there are harmful to activity, for instances, II15 (R2 = 3-F,4-CH3OC6H3) > II14 (3,4-diCH3OC6H3) and II13 (3-F,4-ClC6H3) > II19 (4-ClC6H4).
As shown in Fig. 3c, the steric and electrostatic contour maps of the CoMSIA model are similar to those of the CoMFA model, showing the similar trends of the influences of these two fields on activity in the CoMSIA model. For the hydrophobic field (Fig. 3d), most of the yellow contour is covered around R1 and R2 groups, revealing that the general hydrophobic substituents are advantageous. Besides, it is well known that the hydrophobicity would enhance with the increase of the alkane chain length, which is consistent with the sequences of activity: I3 (R1 = (CH2)7CH3) > I1 ((CH2)3CH3) and I10 ((CH2)3C6H5) > I9 ((CH2)2C6H5) > I8 (CH2C6H5). This may also explain that compounds with hydrophobic substituent such as –X (I24–I27, II13, and II17–II20), alkyl (I16, I17, I21, I22, and II1–II3) and ether group (I18 and II7) at these positions possess the desirable activity.
The hydrogen bond donor contour map (Fig. 3e) identifies a purple contour at the N2 position, implying that the R2 groups with H-bond donor are not welcome. Simultaneously, a magenta region near the same sites in hydrogen bond acceptor contour map (Fig. 3e) suggests that compounds with H-bond acceptor can ameliorate the activity, which is basically in line with the circumstance reflected in the H-bond donor field. The potency of compound II15 (R2 = 3-F,4-CH3OC6H3) with an H-bond acceptor at this site was raised compared with compound II5 (4-CH3OC6H4). On the contrary, the red polyhedron is nearest to the C4 position, suggesting that compounds I and II series with H-bond donor (R3 = H) have fantastic activity than esterified compounds.
Notely, when P. recalcitrans was exposed to the EC50 and EC70, their relative electrical conductivities were significantly greater than those of the EC30 and the control. Therefore, the EC50 was chosen to detect the effect of I23 on the ultrastructural changes of P. recalcitrans by TEM. The untreated control remained intactness of cellular ultrastructures, including dense and even cell walls, clear and complete plasma membranes, mitochondria with visible cristae, distinct electron-dense vesicles (EDVs) and organelles (Fig. 5a–c). After treatment with I23 at the EC50, moderate to extreme ultrastructural alternations occurred in the cells of P. recalcitrans. The density of the cell walls decreased and the membranous organelles became disorganized, with abnormal and indiscernable morphologies. Conspicuously, mitochondrial size was reduced and the incidence of empty vacuoles increased compared with those of cells from the untreated control (Fig. 5d and e). Even worse, the organelles degraded severely, the cell walls were not intact, and the inner wall layer of the original hyphae was retracted into cell lumen (Fig. 5f). Considering the ultrastructural changes of the cellular membranous systems, it is supposed that I23 had profound impacts on the biological membrane.
These impacts could be exemplified by the FM4-64 staining and mitochondrial membrane potential vanishing by JC-1 staining as well. The lipophilic vital styryl dye FM4-64 primarily stains the plasma membrane, endosomes and vesicles in the untreated fine cells of P. recalcitrans (Fig. 6Aa and b). In contrast, after P. recalcitrans was treated with I23 at the EC50 for 12 h, no definitive staining pattern was observed either at the plasma membrane or in the cytosol, up to 60 min after exposure to the dye (Fig. 6Ac and d). Further JC-1 staining showed that the decreasing and vanishing status of mitochondrial membrane potential occurred as early as 6 h post-treatment (Fig. 6B), indicating the mitochondria damaged or dead states. As a matter of fact, it has been found that organic acids could serve as uncouplers that generally dissipate pH and electrical gradients across biological membranes.41 Taken together, it has been speculated that I23 might exert the antioomycete activity against P. recalcitrans by interfering with cytoplasmic membrane structure and membrane proteins or membrane uncoupling capabilities.
Fig. 6 The fluorescence microscopy photos of S. sclerotiorum mycelia stained by FM4-64 (A) and JC-1 dyes (B). |
The results of the lipid absolute quantification analysis (Fig. S5†) showed that I23 could lead to the increase of the total lipid content but this difference did not reach significance. Notwithstanding, among the cell membrane components with the contents higher than 1%, Cer class, and PI and PC classes showed significant increase and decreases, respectively. To have a specific overview of how changes in the three lipid classes, the carbon chain length and the degree of unsaturation of the corresponding lipid species were further analyzed. As depicted in Fig. S6,† the Cer content enhanced at the different chain length and the degree of unsaturation levels, while PI and PC decreased. The alternation of Cer, PI and PC confirmed the instability and the integrity changes of the cell membrane induced by I23, which might be responsible for the malfunction and the increased permeability of cellular membrane P. recalcitrans. Besides, the 3D-QSAR analysis indicated that the hydrophobic R1 and R2 groups are favorable, suggesting the necessity of the hydrophobicity of the potent compounds. It has been demonstrated that the small hydrophobic molecules can easily enter into the cellular membranes, which might induce detrimental actions of I23 on cellular membranes.
Footnotes |
† Electronic supplementary information (ESI) available. CCDC 2180985. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3ra00855j |
‡ These authors contributed equally to this work and share the first authorship. |
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