Design, Synthesis, and Antitobacco Mosaic Virus Activity of Water

Jan 22, 2018 - Phone: +86-(0)22-23503952. ... The bioassay results indicated that most of the designed structural analogues showed good in vivo anti-T...
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Design, Synthesis, and Antitobacco Mosaic Virus Activity of WaterSoluble Chiral Quaternary Ammonium Salts of Phenanthroindolizidines Alkaloids Guifang Han, Linwei Chen, Qiang Wang, Meng Wu, Yuxiu Liu, and Qingmin Wang* State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China ABSTRACT: To study the influence of the substituent at the N-10 position on antiviral activity, the chiral quaternary ammonium salt derivatives of R- and S-tylophorine were designed, synthesized, and evaluated for antiviral activity against tobacco mosaic virus (TMV). The bioassay results indicated that most of the designed structural analogues showed good in vivo antiTMV activity, among which propargyl quaternary ammonium salt compound S-7b showed the best anti-TMV activities (80.5%, 77.6%, 76.6%, 82.1%) at 500 μg/mL both in vitro and in vivo in the laboratory. In the field trials of antiviral efficacy against TMV, S-7b as well exhibited better activities than control plant virus inhibitors. The stability of compound S-7b was obviously increased, and its solubility was more than 500-times higher than that of S-tylophorine. Therefore, chiral quaternary ammonium salt S-7b was expected to be developed as a promising candidate as an inhibitor of plant virus. KEYWORDS: phenanthroindolizidines alkaloids, quaternary ammonium salts, derivatives, anti-TMV, structure−activity relationship, stability, solubility

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amoebic,10 and anti-inflammatory effects,11 as well as a most notable antitumor activity.12−19 Our group first found that R-antofine, 2 (Figure 1), displayed excellent antiviral activity against TMV.20 The further antiviral mechanism studies revealed that antofine-based alkaloids have a favorable interaction with the origin of TMV RNA (oriRNA), likely exerting its virus inhibition by binding to oriRNA and interfering with virus assembly initiation.21 Although a structure−activity relationship (SAR) study on phenanthroindolizidine alkaloids has been extensively investigated,22−24 work was mainly focused on: (1) the position and number of methoxy groups on the phenanthrene ring, (2) derivatization at the C-14 position, (3) derivatation at the C-13a position, and (4) D ring opened derivatives. However, phenanthroindolizidine alkaloids and their derivatives have drawbacks such as low stability, low water-solubility, high ability to penetrate the blood−brain barrier, and obvious CNS toxicity.25,26 All of these limited their application in plant protection. Because of the presence of nitrogen at the 10-position, the phenanthroindolizidines alkaloids easily undergo racemization and oxidation to the iminium ion or enamine.27,28 To improve the stability and study the influence of the substituent on nitrogen and optimize phenanthroindolizidine alkaloids as antiviral agent against TMV, the new optically active N-10 substituted tylophorine analogues targeting TMV RNA were designed, synthesized, and evaluated for antiviral activity against TMV. The stability, water solubility, and SAR study of the optically active N-substituted tylophorine analogues against TMV are discussed. Meanwhile, field trial evaluations were also conducted for the compound

INTRODUCTION Plant viruses are pathogenic to plants, affect more than 700 plants,1 and cause an estimated US $60 billion loss in crop yields worldwide each year. Tobacco mosaic virus (TMV) has been voted as the most important plant virus in molecular plant pathology based on their perceived importance, scientifically or economically.2 Like other plant pathogenic viruses, TMV has a very wide host range and has different effects depending on the host being infected. It is known to infect members of nine plant families and at least 125 individual species including tobaccos, tomatoes, peppers (all members of the useful Solanaceae), cucumbers, and a number of ornamental flowers.3 To date there are no efficient chemical treatments that protect plant parts from virus infection. Additionally, there are no known chemical treatments under field conditions that can completely eliminate viral infections from plant tissues once they do occur. The existing commercial antiviral agents such as ribavirin and ningnanmyc (Figure 1) are in the low level of antiviral activities (30−60% at 150 g/hm2). Because of the great economic loss caused by TMV and the unsatisfactory inhibitory effects of these antiviral agents, methods to develop highly efficient and environmentally benign antiviral agents are urgently needed. Owing to easy decomposition, environmental friendliness, specification to targeted species, and unique mode of action, natural productbased antiviral agents can be used as ideal lead structures to develop agrochemicals.4,5 Phenanthroindolizidine alkaloids, a small family of natural products, are isolated mainly from Cynanchum, Pergularia, Tylophora, and some other genera of the Asclepiadaceae family.6 Since the first isolation of (R)(+)-tylophorine, 1 (Figure 1), in 1935 from Tylophora indica,7 to date more than 80 alkaloids with the same skeleton have been isolated. This class of natural products has potent biological activities, such as antiarthritis,8 antilupus,9 anti© 2018 American Chemical Society

Received: Revised: Accepted: Published: 780

July 25, 2017 October 9, 2017 October 10, 2017 January 22, 2018 DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of ningnanmycin, ribavirin, and (R)-(+)-tylophorine.

Figure 2. Synthetic route for products R/S-1 to R/S-12. General Procedure for Synthesis of Quaternary Ammonium Salts Derivatives R(S)-1 to R(S)-12. To a solution of (R)- or (S)tylophorine (1.5 mmol) in CHCl3 was added α-bromo compounds ethyl 2-bromoacetate, 2-bromoacetonitrile, 2-bromoacetamide, 2bromo-N-methylacetamide, 2-bromo-N,N-dimethylacetamide, 2bromo-1-phenyl- ethanone, 3-bromoprop-1-yne, 1-bromobut-2-yne, 3-bromoprop-1-ene, (bromomethyl)benzene, 1-(bromomethyl)-4(tert-butyl)benzene), or 1-(bromomethyl)-4-(trifluoromethyl)benzene (3 mmol) in CHCl3. The solution was stirred and refluxed for 24−48 h. The solvent was evaporated to give crude product of two isomers, which were separated by column chromatography, using CH2Cl2/ MeOH (20:1, v/v) as eluent to give the products R/S-1 to R/S-12. (10R,13aS)-10-(2-Ethoxy-2-oxoethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-1a). White solid, Mp: 173−175 °C. Yield: 47%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.09 (s, 1H), 7.47 (s, 1H), 7.34 (s, 1H), 5.28 (d, J = 16.0 Hz, 1H), 5.07 (d, J = 16.0 Hz, 1H), 4.57−4.43 (m, 3H), 4.19 (q, J = 6.8 Hz, 2H), 4.06 (s, 3H), 4.06 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H), 3.99−3.98 (m, 2H), 3.74−3.68 (m, 1H), 2.43−2.38 (m, 1H), 2.27−2.14 (m, 2H), 1.80− 1.68 (m, 1H), 1.19 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSOd6) δ 165.3, 149.5, 149.1, 149.0, 124.2, 123.7, 123.6, 123.5, 122.7,

with high anti-TMV activity to assess its potential as an antivirus candidate.

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MATERIALS AND METHODS

Instruments. 1H, 13C, 2D-HMQC, and 2D-NOE nuclear magnetic resonance (NMR) spectra were obtained at 400 MHz using a Bruker AC-P 400 (Tianjin, China). Chemical shift values (δ) were given in parts per million (ppm) and were downfield from internal tetramethylsilane. High resolution mass spectra (HRMS) were recorded on an FT-ICR MS (Ionspec, 7.0 T) (Tianjin, China). Melting points were determined on an X-4 binocular microscope melting point apparatus and were uncorrected (Tianjin, China). Reagents were purchased from commercial sources and were used as received. All anhydrous solvents were dried and purified according to standard techniques just before use. Conversion was monitored by thin layer chromatography (TLC). Flash column chromatography was performed over silica gel (200−300 mesh). General Synthesis. The phenanthroindolizidine alkaloids (Figure 2) were prepared as described previously.29,30 The synthetic route of derivatives R(S)-1 to R(S)-12 is shown in Figure 2. 781

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry

(s, 1H), 7.87 (s, 1H), 7.72 (s, 1H), 7.41 (s, 1H), 7.16 (s, 1H), 5.96 (d, J = 16.0 Hz, 1H), 4.96 (d, J = 16.0 Hz, 1H), 4.57 (br, 1H), 4.23−4.15 (m, 1H), 4.06 (s, 6H), 3.98 (s, 3H), 3.95 (s, 3H), 3.98−3.95 (m, 1H), 3.81−3.69 (m, 3H), 3.24−3.16 (m, 1H), 2.57−2.53 (m, 1H), 2.35− 2.28 (m, 2H), 2.14−2.03 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 165.6, 149.4, 149.1, 148.9, 148.9, 123.9, 123.9, 123.8, 123.4, 122.5, 118.8, 104.5, 104.4, 104.1, 103.0, 71.1, 62.2, 59.1, 56.0, 55.8, 55.6, 48.6, 26.6, 25.3, 18.8. HRMS (ESI) calcd for C26H31N2O5+ [M-Br−] 451.2227, found 451.2231. Data for compound R-3b: pale yellow solid. Mp: 230−232 °C. Yield: 39%. 1H NMR, 13C NMR, and HRMS (ESI) are the same our for compound S-3b. (10R,13aS)-2,3,6,7-Tetramethoxy-10-(2-(methylamino)-2-oxoethyl)-10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-4a). Yellow solid. Mp 205−209 °C. Yield: 35%. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J = 4.4 Hz, 1H), 8.10 (s, 1H), 8.09 (s, 1H), 7.46 (s, 1H), 7.33 (s, 1H), 5.42 (d, J = 16.0 Hz, 1H), 4.94 (d, J = 16.0 Hz, 1H), 4.21 (br, 1H), 4.24−4.14 (m, 3H), 4.06 (s, 6H), 3.99 (s, 3H), 3.98 (s, 3H), 3.69−3.56 (m, 2H), 2.62 (d, J = 4.4 Hz, 3H), 2.45−2.38 (m, 1H), 2.28−2.19 (m, 2H), 1.87− 1.81 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.8, 149.5, 149.1, 148.9, 148.9, 124.2, 123.7, 123.5, 122.7, 122.6, 118.2, 104.5, 104.3, 103.5, 68.7, 63.6, 59.5, 56.0, 55.8, 55.6, 53.5, 27.8, 25.5, 25.2, 19.6. HRMS (ESI) calcd for C27H33N2O5+ [M-Br−] 465.2384, found 465.2378. Data for compound R-4a: yellow solid. Mp: 217−219 °C. yield: 33%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-4a. (10S,13aS)-2,3,6,7-Tetramethoxy-10-(2-(methylamino)-2-oxoethyl)-10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-4b). White solid. Mp: 232−234 °C. Yield: 35%. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 4.4 Hz, 1H), 8.10 (s, 1H), 8.09 (s, 1H), 7.41(s, 1H), 7.18 (s, 1H), 6.00 (d, J = 15.6 Hz, 1H), 4.95 (d, J = 15.6 Hz, 1H), 4.49−4.11 (m, 1H), 4.24− 4.14 (m, 1H), 4.06 (s, 6H), 3.99 (s, 3H), 3.96 (s, 3H), 3.81−3.67 (m, 3H), 3.25−3.18 (m, 1H), 2.58 (d, J = 4.4 Hz, 3H), 2.58 (m, 1H), 2.33−2.25 (m, 2H), 2.13−2.01 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.8, 149.4, 149.1, 149.0, 148.9, 123.9, 123.9, 123.8, 123.5, 122.6, 118.8, 104.5, 104.4, 104.2, 103.0, 71.0, 62.1, 59.2, 56.0, 55.8, 55.6, 49.1, 26.5, 25.6, 25.3, 18.8. HRMS (ESI) calcd for C27H33N2O5+ [M-Br−] 465.2384, found 465.2388. Data for compound R-4b: white solid. Mp: 232−235 °C. Yield: 38%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-4b. (10R,13aS)-10-(2-(Dimethylamino)-2-oxoethyl)-2,3,6,7-tetramethoxy-10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2b]isoquinolin-10-ium bromide (S-5a). Yellow solid. Mp: 190−194 °C. Yield: 23.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 8.08 (s, 1H),7.47(s, 1H), 7.27 (s, 1H), 5.53 (d, J = 16.0 Hz, 1H), 5.02 (d, J = 16.0 Hz, 1H), 4.62−4.57 (m, 2H), 4.53 (br, 1H), 4.05 (s, 6H), 3.98 (s, 3H), 3.97 (s, 3H), 4.00−3.97 (m, 1H), 3.89−3.65 (m, 3H), 2.87 (s, 3H), 2.86 (s, 3H), 2.37−2.35 (m, 1H), 2.20−2.16 (m, 2H), 1.78−1.70 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 164.1, 149.4, 149.0, 148.9, 124.1, 123.7, 123.6, 123.4, 122.7, 118.5, 104.5, 104.2, 103.2, 70.1, 63.8, 59.2, 56.0, 55.7, 55.6, 52.6, 36.1, 35.2, 27.8, 25.3, 19.8. HRMS (ESI) calcd for C28H35N2O5+ [M-Br−] 479.2540, found 479.2541. Data for compound R-5a: yellow solid. Mp: 191−193 °C. Yield: 25%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-5a. (10S,13aS)-10-(2-(Dimethylamino)-2-oxoethyl)-2,3,6,7-tetramethoxy-10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2b]isoquinolin-10-ium bromide (S-5b). Pale yellow solid. Mp: 195− 197 °C. Yield: 40%. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 8.03 (s, 1H), 7.36 (s, 1H), 7.09 (s, 1H), 6.09 (d, J = 16.0 Hz, 1H), 5.00 (d, J = 16.0 Hz, 1H), 4.66 (br, 1H), 4.16−4.12 (m, 3H), 4.05 (s, 6H), 4.02 (s, 6H), 3.98 (s, 3H), 3.95 (s, 3H), 3.69−3.51 (m, 2H), 3.51− 3.49 (m, 1H), 2.78 (s, 6H), 2.51 (br, 1H), 2.27 (br, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.4, 149.3, 149.1, 148.9, 148.8, 123.9, 123.8, 123.6, 122.5, 118.7, 104.5, 104.0, 102.8, 72.0, 62.7, 59.4, 56.0,

118.3, 104.5, 104.2, 103.4, 69.8, 64.0, 62.0, 59.4, 56.0, 55.8, 55.6, 53.4, 28.2, 25.3, 20.0, 13.7. HRMS (ESI) calcd for C28H34NO6+ [M-Br−] 480.2381, found 480.2384. Data for compound R-1a: white solid. Mp: 175−176 °C. Yield: 42%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-1a. (10S,13aS)-10-(2-Ethoxy-2-oxoethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-1b). White solid. Mp: 174−177 °C. Yield: 40%. 1H NMR (400 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.84 (s, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 5.52 (d, J = 17.2 Hz, 1H), 4.86 (d, J = 16.0 Hz, 1H), 4.30−4.27 (m, 1H), 4.05−3.99 (m, 2H), 3.90 (q, J = 7.2 Hz, 2H), 3.90−3.78 (m, 7H), 3.75−3.73 (m, 7H), 3.66−3.44 (m, 2H), 2.28 (br, 1H), 2.13−1.91 (m, 3H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.1, 149.4, 149.1, 148.9, 148.8, 123.9, 123.7, 123.4, 122.5, 118.6, 104.5, 104.1, 103.1, 71.8, 62.5, 62.1, 59.4, 56.0, 55.9, 55.6, 48.9, 26.2, 24.9, 18.8, 13.6. HRMS (ESI) calcd for C28H34NO6+ [M-Br−] 480.2381, found 480.2387. Data for compound R-1b: white solid. Mp: 169−171 °C. Yield: 46%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-1b. (10R,13aS)-10-(Cyanomethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-2a). Pale yellow solid. Mp: 212−215 °C. Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 8.10 (s, 1H), 7.49 (s, 1H), 7.44 (s, 1H), 5.32 (d, J = 16.0 Hz, 1H), 5.15 (d, J = 16.0 Hz, 1H), 5.07 (s, 2H), 4.40−4.38 (m, 1H), 4.07 (s, 6H), 4.02 (s, 3H), 4.00 (s, 3H), 3.74−3.63 (m, 2H), 2.48−2.45 (m, 1H), 2.31− 2.27 (m, 2H), 1.94−1.84 (m, 1H).13C NMR (100 MHz, DMSO-d6) δ 149.6, 149.1, 149.0, 149.0, 124.4, 123.7, 123.5, 123.4, 122.5, 117.8, 112.5, 104.5, 104.3, 103.6, 69.7, 65.0, 56.0, 55.9, 55.6, 55.0, 48.7, 28.6, 25.3, 20.1. HRMS (ESI) calcd for C26H29N2O4+ [M-Br−] 433.2122, found 433.2127. Data for compound R-2a: pale yellow solid. Mp: 212−214 °C. Yield: 44%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-2a. (10S,13aS)-10-(Cyanomethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-2b). Pale yellow solid, Mp: 217−219 °C. Yield: 28%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.06 (s, 1H), 7.37 (s, 1H), 7.31 (s, 1H), 5.54 (d, J = 16.0 Hz, 1H), 5.21 (d, J = 16.0 Hz, 1H), 4.98 (d, J = 16.8 Hz, 1H), 4.81 (d, J = 16.8 Hz, 1H), 4.44−4.36 (m, 1H), 4.29−4.22 (m, 1H), 4.06 (s, 3H), 4.05 (s, 3H), 4.00 (s, 3H), 3.98 (s, 3H), 4.01−3.95 (m, 1H), 3.88−3.83 (m, 1H), 3.24−3.19 (m, 1H), 2.58−2.55 (m, 1H), 2.43−2.31 (m, 2H), 2.23− 2.13 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 149.5, 149.2, 149.0, 148.9, 124.0, 123.9, 123.7, 123.3, 122.4, 118.1, 112.5, 104.5, 104.1, 103.4, 70.5, 64.1, 60.8, 56.0, 55.6, 41.1, 26.2, 25.1, 18.8. HRMS (ESI) calcd for C26H29N2O4+ [M-Br−] 433.2122, found 433.2127. Data for compound R-2b: pale yellow solid. Mp: 216−220 °C. Yield: 30%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-2b. (10R,13aS)-10-(2-Amino-2-oxoethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-3a). Pale yellow solid. Mp: 211−215 °C. Yield: 40%. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 8.10 (s, 1H), 7.83 (s, 1H), 7.71 (s, 1H), 7.46 (s, 1H), 7.31 (s, 1H), 5.38 (d, J = 16.4 Hz, 1H), 4.90 (d, J = 16.4 Hz, 1H), 4.30 (br, 1H), 4.17−4.12 (m, 10H), 3.98 (s, 3H), 3.97 (s, 6H), 3.68−3.56 (m, 2H), 2.45−2.39 (m, 1H), 2.28−2.19 (m, 2H), 1.88−1.78 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 165.6, 149.5, 149.1, 149.0, 148.9, 124.2, 123.7, 123.53, 122.8, 122.6, 118.2, 104.5, 104.3, 103.4, 68.8, 63.6, 59.1, 56.0, 55.7, 55.6, 53.3, 27.7, 25.3, 19.7. HRMS (ESI) calcd for C26H31N2O5+ [M-Br−] 451.2227, found 451.2236. Data for compound R-3a: pale yellow solid. Mp: 211−213 °C. Yield: 40%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-3a. (10S,13aS)-10-(2-Amino-2-oxoethyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-3b). Pale yellow solid. Mp: 228−231 °C. Yield: 37.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.08 782

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry

isoquinolin-10-ium bromide (S-8a). White solid. Mp: 220−224 °C. Yield: 24%. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.14 (s, 1H), 7.51 (s, 1H), 7.45 (s, 1H), 5.26 (d, J = 16.0 Hz, 1H), 5.03 (d, J = 16.0 Hz, 1H), 4.53−4.29 (m, 3H), 4.11 (s, 6H), 4.05 (s, 3H), 4.03 (s, 3H), 3.96−3.78 (m, 2H), 3.65 (br, 2H), 2.50−2.40 (m, 1H), 2.31− 2.22 (m, 2H), 2.00 (s, 3H), 1.94−1.80 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 149.9, 149.5, 149.5, 149.4, 124.6, 124.1, 124.0, 123.7, 123.1, 118.8, 105.0, 104.7, 104.1, 88.6, 68.7, 67.3, 63.2, 56.5, 56.5, 56.4, 56.1, 55.4, 53.6, 51.6, 28.9, 25.7, 20.1, 3.9; HRMS (ESI) calcd for C28H32NO4+ [M-Br−] 446.2326, found 446.2331. Data for compound R-8a: white solid. Mp: 222−223 °C. Yield: 22%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-8a. (10R,13aS)-10-(But-2-yn-1-yl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-8b). White solid. Mp: 240−242 °C. Yield: 24%. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.14 (s, 1H), 7.43 (s, 1H), 7.34 (s, 1H), 5.57 (d, J = 16.0 Hz, 1H), 4.99 (d, J = 16.0 Hz, 1H), 4.38−4.35 (m, 4H), 4.12 (s, 6H), 4.04 (s, 3H), 4.03 (s, 3H), 3.88−3.60 (m, 2H), 3.40−3.26 (m, 1H), 2.50 (br, 1H), 2.34− 2.24 (m, 2H), 2.20−2.10 (m, 1H), 1.98 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 149.4, 149.1, 149.0, 148.8, 123.8, 123.8, 123.7, 123.6, 122.5, 118.5, 104.5, 104.0, 103.3, 88.7, 68.6, 67.5, 61.4, 58.5, 56.0, 56.0, 55.6, 55.0, 42.2, 26.2, 25.3, 18.6, 3.4; HRMS (ESI) calcd for C27H30NO4+ [M-Br−] 432.2169, found 432.2177. Data for compound R-8b: white solid. Mp: 240−243 °C. Yield: 30%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-8b. (10S,13aS)-10-Allyl-2,3,6,7-tetramethoxy-10,11,12,13,13a,14hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-9a). Pale yellow solid. Mp: 180−185 °C. Yield: 20%. 1H NMR (400 MHz, DMSO) δ 8.10 (s, 1H), 8.08 (s, 1H), 7.44 (s, 1H), 7.36 (s, 1H), 6.29−6.10 (m, 1H), 5.58 (d, J = 10.4 Hz, 1H), 5.53 (d, J = 15.6 Hz, 1H), 5.07 (d, J = 16.0 Hz, 1H), 4.79 (t, J = 16.0 Hz, 1H), 4.12−4.08 (m, 1H), 4.06 (m, 1H), 4.06 (s, 6H), 3.98 (s, 3H), 3.97 (s, 3H), 3.87−3.79 (m, 2H), 3.76−3.68 (m, 2H), 3.58−3.50 (m, 1H), 2.53−2.50 (m, 1H), 2.29−2.10 (m, 2H), 1.90−1.75 (m, 1H). HRMS (ESI) calcd for C27H32NO4+ [M-Br−] 434.2326, found 434.2328. (10R,13aS)-10-Allyl-2,3,6,7-tetramethoxy-10,11,12,13,13a,14hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-9b). Pale yellow solid. Mp: 200−204 °C. Yield: 60%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.09 (s, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.25 (s, 1H), 6.23−6.13 (m, 1H), 5.53 (d, J = 11.2 Hz, 1H), 5.41−5.36 (m, 1H), 4.79 (d, J = 16.0 Hz, 1H), 4.26−4.14 (m, 1H), 4.12−4.08 (m, 1H), 4.06 (m, 1H), 4.06 (s, 6H), 3.98 (s, 3H), 3.97 (s, 3H), 3.87−3.79 (m, 2H), 3.76−3.68 (m, 2H), 3.58−3.50 (m, 1H), 2.53−2.50 (m, 1H), 2.29−2.14 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.4, 149.1, 149.0, 148.9, 127.0, 125.9, 123.9, 123.8, 123.8, 122.5, 118.8, 104.5, 104.2, 103.4, 69.4, 60.2, 57.7, 56.0, 56.0, 55.9, 55.6, 51.6, 26.2, 25.2, 18.5. HRMS (ESI) calcd for C27H32NO4+ [M-Br−] 434.2326, found 434.2328 (10S,13aS)-10-Benzyl-2,3,6,7-tetramethoxy-10,11,12,13,13a,14hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-10a). White solid. Mp: 178−181 °C. Yield: 30%. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 2H), 7.49 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz, 1H), 7.37 (t, J = 7.2 Hz, 2H), 7.28 (s, 1H), 7.22 (s, 1H), 5.30 (d, J = 12.8 Hz, 1H), 5.15 (d, J = 16.0 Hz, 1H), 5.07−4.96 (m, 1H), 4.77−4.67 (m, 2H), 4.61 (dd, J = 19.6, 9.6 Hz, 1H), 4.16 (s, 3H), 4.14 (s, 3H), 4.09 (s, 3H), 4.07 (s, 3H), 3.75 (dd, J = 18.0, 6.8 Hz, 2H), 3.55 (d, J = 18.0 Hz, 1H), 2.62−2.43 (m, 1H), 2.32−2.24 (m, 1H), 2.19−2.15 (m, 1H), 1.93−1.83 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 149.9, 149.7, 149.5, 149.5, 132.8, 130.7, 129.3, 127.9, 124.7, 124.2, 123.8, 122.7, 121.3, 117.1, 103.6, 103.5, 103.4, 103.0, 66.2, 62.3, 61.7, 57.0, 56.2, 56.2, 56.1, 51.3, 28.2, 25.5, 18.9. HRMS (ESI) calcd for C31H34NO4+ [M-Br−] 484.2482, found 484.2480. (10R,13aS)-10-Benzyl-2,3,6,7-tetramethoxy-10,11,12,13,13a,14hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-10b). White solid. Mp: 192−194 °C. Yield: 58%. 1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.86 (s, 1H), 7.42 (t, J = 7.4 Hz, 1H), 7.30 (t, J = 7.7 Hz, 2H), 7.11 (s, 1H), 6.96 (s, 1H), 6.92 (d, J = 7.3 Hz, 2H), 5.38 (d, J = 16.0 Hz, 1H), 4.96 (d, J = 16.0 Hz, 1H),

55.8, 55.5, 47.2, 36.4, 35.4, 26.1, 24.9, 18.7. HRMS (ESI) calcd for C28H35N2O5+ [M-Br−] 479.2540, found 479.2540. Data for compound R-5b: pale yellow solid. Mp: 197−199 °C. Yield: 40%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-5b. (10R,13aS)-2,3,6,7-Tetramethoxy-10-(2-oxo-2-phenylethyl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-6a). White solid. Mp: 179−180 °C. Yield: 33%. 1H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 2H), 8.02 (d, J = 7.6 Hz, 2H), 7.70 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 7.50 (s, 1H), 7.27 (s, 1H), 5.42 (s, 2H), 5.34 (d, J = 16.0 Hz, 1H), 5.13 (d, J = 16.0 Hz, 1H), 4.56−4.48 (m, 1H), 4.19−4.13 (m, 1H), 4.06 (s, 3H), 4.03 (s, 3H), 4.00 (s, 3H), 3.97 (s, 3H), 3.95−3.86 (m, 2H), 3.69 (d, J = 16.8 Hz, 1H), 2.43−2.36 (m, 1H), 2.25−2.20 (m, 2H), 1.80−1.70 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 191.8, 149.4, 149.0, 148.9, 148.9, 134.5, 134.3, 128.7, 128.2, 124.1, 123.8, 123.6, 123.2, 122.8, 118.6, 104.5, 104.3, 103.4, 70.4, 64.5, 63.9, 56.0, 55.7, 55.6, 53.0, 27.7, 25.0, 20.0. HRMS (ESI) calcd for C32H34NO5+ [M-Br−] 512.2431, found 512.2435 Data for compound R-6a: white solid. Mp: 175−178 °C. Yield: 33%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-6a. (10S,13aS)-2,3,6,7-Tetramethoxy-10-(2-oxo-2-phenylethyl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-6b). White solid. Mp:182−183 °C. Yield: 42%. 1H NMR (400 MHz, DMSO-d6) δ 8.04−8.01 (m, 4H), 7.63 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.39 (s, 1H), 7.12 (s, 1H), 5.89 (d, J = 16.0 Hz, 1H), 5.16 (d, J = 16.0 Hz, 1H), 5.05 (d, J = 18.0 Hz, 1H), 4.82 (d, J = 18.0 Hz, 1H), 4.68−4.64 (m, 1H), 4.20− 4.16 (m, 1H), 4.03 (s, 3H), 4.02 (s, 3H), 4.01 (s, 3H), 3.97 (s, 3H), 3.92−3.89 (m, 1H), 3.77−3.64 (m, 2H), 2.42−2.27 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 191.5, 149.3, 149.0, 148.8, 148.7, 134.5, 134.4, 128.5, 128.4, 123.9, 123.8, 123.5, 122.6, 118.9, 104.6, 104.5, 104.0, 103.0, 72.6, 62.8, 59.7, 56.0, 55.8, 55.5, 52.5, 26.0, 24.8, 18.9. HRMS (ESI) calcd for C32H34NO5+ [M-Br−] 512.2431, found 512.2435. Data for compound R-6b: white solid. Mp: 180−181 °C. Yield: 40%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-6b. (10S,13aS)-2,3,6,7-Tetramethoxy-10-(prop-2-yn-1-yl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-7a). Pale yellow solid, Mp: 216−217 °C. Yield 20%. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.10 (s, 1H), 7.47 (s, 1H), 7.39 (s, 1H), 5.20 (d, J = 16.0 Hz, 1H), 4.97 (d, J = 16.0 Hz, 1H), 4.52−4.39 (m, 2H), 4.30 (br, 1H), 4.07 (s, 6H), 4.00 (s, 3H), 3.99 (s, 3H), 3.88−3.86 (m, 2H), 3.70−3.55 (m, 2H), 3.12− 3.11 (m, 1H), 2.44−2.42 (m, 1H), 2.29−2.19 (m, 2H), 1.94−1.78 (m, 1H); 13C (100 MHz, DMSO-d6) δ 149.5, 149.1, 149.0, 149.0, 124.2, 123.7, 123.5, 123.3, 122.6, 118.1, 104.5, 104.3, 103.6, 82.5, 72.8, 67.5, 63.1, 56.0, 55.8, 55.6, 50.5, 45.6, 28.4, 25.2, 19.7; HRMS (ESI) calcd for C27H30NO4+ [M-Br−] 432.2169, found 432.2178. Data for compound R-7a: pale yellow solid. Mp: 212−214 °C. Yield: 22%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-7a. (10R,13aS)-2,3,6,7-Tetramethoxy-10-(prop-2-yn-1-yl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-7b). Pale yellow solid. Mp: 237−239 °C. Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.10 (s, 1H), 7.39 (s, 1H), 7.30 (s, 1H), 5.51 (d, J = 16.0 Hz, 1H), 4.97 (d, J = 16.0 Hz, 1H), 4.34−4.32 (m, 3H), 4.19 (br, 1H), 4.06 (s, 6H), 3.98 (s, 6H), 3.88−3.64 (m, 2H), 3.35−3.26 (m, 2H), 2.50 (br, 1H), 2.35− 2.24 (m, 2H), 2.20−2.10 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 149.4, 149.2, 149.1, 148.9, 123.9, 123.9, 123.8, 123.6, 122.5, 118.4, 104.5, 104.5, 104.1, 103.2, 83.1, 72.3, 68.9, 61.8, 58.7, 56.0, 55.6, 41.7, 26.2, 25.3, 18.6; HRMS (ESI) calcd for C28H32NO4+ [M-Br−] 446.2326, found 446.2333. Data for compound R-7b: pale yellow solid. Mp: 235−238 °C. Yield: 40%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-7b. (10S,13aS)-10-(But-2-yn-1-yl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]783

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry

Antiviral Biological Assay. The procedure of purifying TMV and the method to test the anti-TMV activity of the synthesized compounds were the same with our previously reported method in the literature.24,31 The field trials were carried out in the trial area of the Institute of Agricultural Environment and Resource, Yunnan Academy of Agricultural Sciences, Kunming City, China, between June and July 2014. The solution of compound S-7b (1% emulsifiable concentrates (EC)) was prepared in our research laboratory, which was diluted to 10, 50, or 100 g(ai)/ha (weight (gram) of active ingredient/hectare) before use. The methods to test the anti-TMV activity of the compound S-7b and control sample moroxydine hydrochloride−cupric acetate and aminooligosaccharins were the same with our previously reported method.32

4.65−4.52 (m, 1H), 4.36 (dd, J = 20.4, 10.0 Hz, 1H), 4.19 (s, 3H), 4.19 (s, 3H), 4.14−4.03 (m, 5H), 3.98 (s, 3H), 3.94−3.86 (m, 1H), 3.53 (dd, J = 17.5, 4.8 Hz, 1H), 3.29 (dd, J = 17.4, 12.1 Hz, 1H), 2.78− 2.68 (m, 1H), 2.64−2.53 (m, 1H), 2.43−2.26 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 148.9, 148.7, 148.6, 148.4, 131.5, 130.1, 128.7, 128.5, 125.2, 123.5, 123.5, 122.9, 122.1, 121.4, 117.3, 102.7, 102.5, 102.4, 101.5, 99.0, 69.1, 57.9, 56.2, 55.6, 55.3, 55.2, 51.3, 28.7, 26.6, 25.4, 21.7, 18.2, 13.1. HRMS (ESI) calcd for C31H34NO4+ [M-Br−] 484.2482, found 484.2486. (10S,13aS)-10-(4-(tert-Butyl)benzyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-11a). White solid. Mp: 185−188 °C. Yield: 35%. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 2H), 7.36 (br, 4H), 7.28 (s, 1H), 7.22 (s, 1H), 5.22−5.15 (m, 2H), 5.04−5.00 (m, 1H), 4.71−4.64 (m, 2H), 4.61−4.53 (m, 1H), 4.16 (s, 3H), 4.15 (s, 3H), 4.09 (s, 3H), 4.08 (s, 3H), 3.86−3.68 (m, 2H), 3.54 (d, J = 18 Hz, 1H), 2.57−2.50 (m, 1H), 2.36−2.26 (m, 1H), 2.22−2.11 (m, 1H), 1.96−1.85 (m, 1H), 1.28 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 154.2, 149.9, 149.8, 149.6, 149.5, 132.5, 126.2, 124.7, 124.3, 123.9, 122.7, 121.1, 117.1, 103.6, 103.6, 103.5, 103.0, 65.8, 62.2, 61.7, 56.9, 56.2, 56.2, 56.1, 51.1, 34.8, 31.1, 28.2, 25.4, 18.8. HRMS (ESI) calcd for C35H42NO4+ [M-Br−] 540.3108, found 540.3108. (10R,13aS)-10-(4-(tert-Butyl)benzyl)-2,3,6,7-tetramethoxy10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-11b). White solid. Mp: 193−197 °C. Yield: 45% 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.86 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 6.98 (s, 1H), 6.82 (d, J = 8.4 Hz, 2H), 5.35 (d, J = 15.2 Hz, 1H), 4.94 (d, J = 15.2 Hz, 1H), 4.43 (br, 1H), 4.35−4.25 (m, 1H), 4.20 (s, 3H), 4.19 (s, 3H), 4.09−4.05 (m, 4H), 4.01−3.97 (m, 4H), 3.94−3.87 (m, 1H), 3.52−3.44 (m, 1H), 3.33−3.21 (m, 1H), 2.70 (br, 1H), 2.56 (br, 1H), 2.45−2.22 (m, 2H), 1.77 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 154.5, 149.9, 149.6, 149.5, 149.4, 132.2, 126.4, 124.4, 124.3, 123.9, 123.3, 123.2, 122.4, 118.3, 103.7, 103.3, 102.7, 70.1, 59.0, 57.5, 56.7, 56.4, 56.2, 52.0, 34.8, 31.0, 27.5, 26.3, 19.1. HRMS (ESI) calcd for C35H42NO4+ [M-Br−] 540.3108, found 540.3110. (10S,13aS)-2,3,6,7-Tetramethoxy-10-(4-(trifluoromethyl)benzyl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-12a). Pale yellow solid. Mp: 175− 178 °C. Yield: 35.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.13 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.45 (s, 1H), 7.17 (s, 1H), 4.87(d, J = 17.2 Hz, 1H), 4.71 (s, 2H), 4.60 (d, J = 17.2 Hz, 1H), 4.36−4.30 (m, 1H), 4.08 (s, 6H), 4.06−3.97 (m, 4H), 4.89 (s, 3H), 3.82−3.67 (m, 3H), 2.51−2.50 (m, 1H), 2.20 (br, 2H), 2.02−1.97 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 149.5, 149.1, 149.0, 148.9, 133.5, 133.0, 130.5 (q, J = 31.8 Hz), 125.8, 125.8, 124.1, 123.9 (q, J = 271.0 Hz), 123.9, 123.8, 122.2, 121.2, 117.0, 104.6, 104.3, 103.4, 66.6, 61.5, 60.8, 56.1, 56.1, 55.7, 55.6, 50.2, 27.2, 24.3, 18.1. HRMS (ESI) calcd for C32H33F3NO4+ [M-Br−] 552.2356, found 552.2363. Compound R-12a: pale yellow solid. Mp: 174−176 °C. Yield: 35.6%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S-12a. (10R,13aS)-2,3,6,7-Tetramethoxy-10-(4-(trifluoromethyl)benzyl)10,11,12,13,13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (S-12b). White solid. Mp: 181−183 °C. Yield: 53.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 8.11 (s, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.44 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.10 (s, 1H), 5.13 (d, J = 16.4 Hz, 1H), 4.60 (d, J = 16.4 Hz, 1H), 4.49 (d, J = 13.2 Hz, 1H), 4.36 (d, J = 13.2 Hz, 1H), 4.32−4.31 (m, 1H), 4.09 (s, 3H), 4.09 (s, 3H), 4.00 (s, 3H), 3.94−3.89 (m, 1H), 3.84− 3.82 (m, 4H), 3.55−3.46 (m, 2H), 2.70−2.53 (m, 2H), 2.40−2.25 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 149.5, 149.2, 149.2, 148.9, 133.8, 132.3, 130.4 (q, J = 31.8 Hz), 125.8, 125.7, 124.1, 124.1, 124.0, 123.9, 123.8 (q, J = 270.7 Hz), 122.2, 118.5, 104.6, 104.5, 104.2, 103.0, 70.4, 59.6, 57.2, 56.1, 56.0, 55.9, 55.6, 50.9, 26.5, 25.4, 18.6. HRMS (ESI) calcd for C32H33F3NO4+ [M-Br−] 552.2356, found 552.2367. Compound R-12b: white solid. Mp: 183−184 °C. Yield: 53.3%. 1H NMR, 13C NMR, and HRMS (ESI) are the same as our compound S12b.

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RESULTS AND DISCUSSION Chemistry Synthesis. The phenanthroindolizidine alkaloids (Figure 2) were prepared as described previously.27,28 The

Figure 3. Observed NOE Effect for S-7a and S-7b.

Table 1. Relevant 1H NMR Data of H-9 for Products R/S-1 to R/S-12 compound R/S-1a R/S-1b R/S-3a R/S-3b R/S-5a R/S-5b R/S-7a R/S-7b R/S-9a R/S-9b R/S-11a R/S-11b

δ (H-9)/ppm J/Hz 5.28, 5.52, 5.38, 5.96, 5.53, 6.09, 5.20, 5.51, 5.07, 5.38, 5.17, 5.34,

5.07; 16.0 Hz 4.86; 16.0 Hz 4.90; 16.4 Hz 4.96; 16.0 Hz 5.02; 16.0 Hz 5.00; 16.0 Hz 4.97; 16.0 Hz 4.97; 16.0 Hz 4.79; 16.0 Hz 4.79, 16.0 Hz 4.66; 16.0 Hz 4.94; 15.2 Hz

compound R/S-2a R/S-2b R/S-4a R/S-4b R/S-6a R/S-6b R/S-8a R/S-8b R/S-10a R/S-10b R/S-12a R/S-12b

δ (H-9)/ppm J/Hz 5.32, 5.54, 5.42, 6.00, 5.34, 5.89, 5.26, 5.57, 5.15, 5.38, 4.87, 5.13,

5.15; 5.21; 4.94; 4.95; 5.13; 5.16; 5.03; 4.99; 4.73; 4.96; 4.60; 4.60;

16.0 16.0 16.0 15.6 16.0 16.0 16.0 16.0 16.0 16.0 17.2 16.4

Hz Hz Hz Hz Hz Hz Hz Hz Hz Hz Hz Hz

quaternization of tylophorine with a variety of alkyl halides were carried out according to Figure 2. Generally, the reactions proceeded in refluxed CHCl3 for 24−48 h to give the quaternary ammonium salts as a pair of diastereoisomers in good to excellent yields, but there were also a number of reactions that failed. For example, tylophorine did not undergo quaternization with the alkyl halides without an activating group such as bromoethane and 2-iodopropane. The two isomers were separated by column chromatography (CH2Cl2/ MeOH = 20:1 as eluent) and characterized through melting point, 1H NMR, 13C NMR, high-resolution mass spectrometry. Configuration. As mentioned above, the prepared quaternary ammonium salt of R- or S-tylophorine is a pair of diastereoisomers, owing to the substituents on N-10 being unable to undergo inversion so the N-10 displays as a chiral center. Owing to the different reactivity and steric hindrance of halides, the ratio of the two isomers was different (cis/trans 784

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry Table 2. In Vitro and in Vivo Antiviral Activities of Compounds 1−12 against TMV compound

configuration (cis/trans)

S-1a

cis

S-1b

trans

S-2a

cis

S-2b

trans

S-3a

cis

S-3b

trans

S-4a

cis

S-4b

trans

S-5a

cis

S-5b

trans

S-6a

cis

S-6b

trans

S-7a

cis

S-7b

trans

S-8a

cis

S-8b

trans

S-9b

trans

S-10a

cis

S-10b

trans

S-11a

cis

S-11b

trans

S-12a

cis

S-12b

trans

S-tylophorine

S

R-1a

cis

R-1b

trans

R-2a

cis

R-2b

trans

R-3a

cis

R-3b

trans

R-4a

cis

concentration (μg/mL)

in vitro effect

protection effect

inactivation effect

curative effect

500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500

25.7 0 35.2 5.4 23.5 0 45.3 12.2 17.7 0 42.6 18.3 33.3 0 28.5 0 36.1 0 55.3 24.1 42.3 18.9 44.5 11.8 54.9 20.9 80.5 42.3 40.9 16.4 48.9 17.6 59.3 27.6 55.9 30 59.7 12.8 65.2 24.3 72.3 27.6 58.6 22.2 55.6 25.8 49.6 22.3 40 8.9 42.5 10.8 43.2 20.8 40 15.7 29.2 15.9 48.6 20 44.4

35 8.9 34.8 0 27.8 0 40.5 9.7 23.5 0 41.2 12.4 30.8 0 36.5 7.2 30.7 0 61.2 18.2 37.6 10.7 50.2 18.6 54.8 27.1 77.6 40.2 46.8 20.6 55.4 25.6 61.3 33.4 58 25.7 54.3 27.8 63.4 32.1 70.2 34.8 56.8 21.4 53.4 20 45 17.1 36.8 10.5 43.4 20.4 48.3 22.4 42.3 11.5 35.6 17.6 46.5 12.8 46

33.4 0 37.9 12.2 26.7 0 43.6 10.8 22.4 0 38.3 16.9 32.7 0 36 0 32.3 12.7 63.8 22.9 39.2 21.2 42.1 17.9 55.3 22.6 76.6 39.8 43.7 23.2 53 19.1 68.5 36.7 62.1 31.6 61.2 20.1 68.4 28.9 65.8 37.2 53.7 23.9 50 17.4 46.7 15.4 37.4 14.1 45.7 14.3 48.9 14.2 42.5 14.2 33.7 10.3 44.1 16.9 49.5

28.9 0 37.5 0 29.4 0 42.1 17.1 18.6 0 36.4 10 26.5 0 32.8 0 36.8 18.9 60 33.3 40.3 16.4 43.7 12.2 57.2 25.9 82.1 43.1 41.8 18 58.2 20.7 65.3 30.9 65.3 29.2 51.7 23.9 70 30.7 75.9 31.4 57.2 15.5 51.5 22.5 50.9 24 46.7 12.4 39.7 16.2 47.4 19.3 38.8 16.3 36.2 12.1 47.3 18.4 49.3

785

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry Table 2. continued compound

configuration (cis/trans)

R-4b

trans

R-5a

cis

R-5b

trans

R-6a

cis

R-6b

trans

R-7a

cis

R-7b

trans

R-8a

cis

R-8b

trans

R-9b

trans

R-12a

cis

R-12b

trans

R-tylophorine

R

concentration (μg/mL)

in vitro effect

protection effect

inactivation effect

curative effect

100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100

15.1 48.6 17.4 26.9 0 34.2 0 47.3 18.6 46.3 16 52.1 21.4 62.2 30 50.3 11.2 46.8 6.6 42.8 9.8 51.2 17.7 32.2 0 52.2 20.7 41.3 12.6

8.5 47.3 18.2 30.7 0 36.4 8.8 48.2 15.7 49.3 14.2 56.2 30 65.2 28.7 47.2 17.6 43.8 13.7 40.8 12.5 47.1 17.1 28.1 5.9 56.1 18.9 38.9 15.6

16.5 42.1 16.5 22.8 0 37.9 5.2 43.5 17.3 50 21.2 57.1 28.1 59.8 25.3 55.9 26.1 41.5 18.3 36.6 10 46.2 12.8 34.1 0 55.9 21.5 37.1 14.6

13.3 45.1 8.9 24.4 0 35.2 0 41.4 16.6 47.5 18.7 53.9 25.4 57.3 27.5 53.7 24.0 40.9 11.5 43.4 16.6 48.8 21.4 36.6 0 52.2 23.8 38.9 13.5

ribavirin

cis configuration, so the absolute configuration of S-7a was (10S, 13aS). In comparison, in the 1H NMR spectra of compound S-7b the methylene on the propargyl were observed at δ 4.34 and 4.17 ppm as a pair of doublets, and the H-13a were observed at δ 4.26−4.34 ppm as multiplet. In the H, HNOESY experiment of S-7b, the NOE effect between the methylene on the propargyl and H-13a were not observed, but some other intense NOE effects could be found: (1) between Ha-14 and Ha-13, (2) between Ha-9 and Ha-13, (3) the methylene on the propargyl and Hb-14. These information suggested the propargyl group and H-13a was in trans form, so the absolute configuration of S-7b was (10R, 13aS). Compared with each pair of diastereoisomers, the differences in the 1H NMR spectra could easily be seen: the chemical shift of H-9 in trans configuration (all named as b compound) was 0.20−0.60 ppm higher than that in the cis configuration (all named as a compound) due to respective chemical environment (Table 1). Bioassays. To explore the impact of the substituent group at the N-10 position and the chirality of the C-13a and N-10, the in vitro (half-leaf method using picked leaves) and in vivo (protection, inactivation, and curative effect assays using whole plants) antiviral activities against TMV of the quaternary salts of tylophorine compounds R(S)-1−12 were tested. R- and Stylophorine and ribavirin were used as the control. The compounds were tested at both 500 and 100 μg/mL. The results are listed in Table 2. Generally, all of the tylophorine derivatives exhibited good antiviral activity against TMV both in vitro and in vivo, and some of them showed even higher activity than ribavirin. These

Table 3. Results of Anti-TMV Activities of Field Trial in 2014 after 10 Days of Application of S-7b, Amino Oligosaccharins, and Moroxydine Hydrochloride-cupric Acetate compounda S-7b (1% ME)

blank control amino oligosaccharins (5% aqueous solution) moroxydine hydrochloridecupric acetate (20% WP)

concentration (g ai/ha)

I′eqb

Ieqc

Ed (%)

0.00 1.66 7.93 16. 86 5.42

100 90.17 53.33

100

0.00 0.96 2.30 2.81 3.33

600

1.23

5.72

66.57

100 50 10

65.70

a

ME, microemulsion; WP, wettable powder. bI′eq: Average disease index of the spraying area before spraying. cIeq: Average disease index of the spraying area 10 days after the third time spraying. dE: Average control effect 10 days after the third time spraying.

from 1:1 to 1:2). The absolute stereochemistry was assigned by analyzing 1H NMR, C−H HMQC, and H,H-NOE data. The propargyl quaternary ammonium salt S-7 was used as an example. In 1H NMR spectra of compound S-7a, the methylene on the propargyl was observed at δ 4.46 ppm as a singlet, and the H-13a was observed at δ 4.24−4.34 ppm as multiplet. In the H,H-NOESY experiment, intense NOE effects could be seen: (1) between the methylene on the propargyl and H-13a, (2) between the methylene on the propargyl and Ha-9, (3) between the methylene on the propargyl and Ha-13 (Figure 3). This information suggested the propargyl group and H-13a was in a 786

DOI: 10.1021/acs.jafc.7b03418 J. Agric. Food Chem. 2018, 66, 780−788

Article

Journal of Agricultural and Food Chemistry observations indicated that the unshared pair of electrons of N10 was not essential for the anti-TMV activity. On the whole, the structure−activity relationships showed the same tendency in the four test modes (in vitro activity, protection, inactivation, and curative activities). Interestingly, tylophorine with an Rconfiguration at C-13a showed greater potencies than its enantiomer at the higher concentration (500 μg/mL), but tylophorine derivatives with an S-configuration at C-13a showed greater potencies than their enantiomers. Therefore, the stereochemistry at C-13a plays a fundamental role in the biological activity. For example, compounds S-5b, S-7b, S-12a, and S-12b showed higher activity than R-5b, R-7b, R-12a, and R-12b at 500 μg/mL, respectively. For S-tylophorine derivatives, the compounds with the trans configuration showed higher activity than those with cis configuration generally. For example, compounds S-2b, S-3b, S-5b, S-7b, S-8b, and S-11b, showed higher activity than S-2a, S-3a, S-5a, S-7a, S-8a, and S-11a at 500 μg/mL, respectively. Specifically, when electron-withdrawing group (ester group, cyano group, acylamino, or ketone group) (S-1−6 except S-6b) was introduced at the N-10 position, the antiviral activity distinctly decreased in all four test modes, whereas when propargyl group (S-7), allyl group (S-9), and benzyl group (S10−12) were introduced, the antiviral activity distinctly increased. Compound S-7b showed excellent antiviral activity (inactivation activity, 80.5%; curative activity, 77.6%; protection activity, 76.6%; curative effect, 82.1%) at 500 μg/mL, which are much higher than ribavirin and S-tylophorine, while compound S-7b showed good antiviral activity at 100 μg/mL. For Rtylophorine analogues, the antiviral activities are basically retained or slightly decrease except for compound R-7b. These observations indicated that activities varied significantly depending upon C-13 and N-10 chirality. Generally, these stereoisomers followed this sequence: C-13 S > R, and trans > cis. Stability and Aqueous Solubility of Compound S-7b. To investigate whether our structural modifications improved the application potency in plant protection, the stability and aqueous solubility properties of compound S-7b were investigated and compared with its parent alkaloid Stylophorine. Tylophorine was unstable in organic solution, especially in CHCl3, because tylophorine can decompose and racemize promptly, through oxidation to the iminium ion or enamine, and the 1H NMR spectrum become greatly complex.28 The stability of S-7b was investigated through 1H NMR in CDCl3. There was no impurity was found in 1H NMR spectrum even after 6 days. The stability was obviously increased, owing to that the unshared electrons of N-10 in tylophorine were protected as quaternary ammonium salt form. The aqueous solubilities of S-tylophorine and its quaternary ammonium salts derivatives were determined. In our actual solubility experiments, we found that S-7b displayed much better solubility (5.0 mg/mL) than S-tylophorine (