First Discovery of Polycarpine, Polycarpaurines A and C, and Their

May 19, 2016 - ... Key Laboratory of Inorganic−Organic Hybrid Functional Materials Chemistry (Tianjin Normal University), Ministry of Education, Col...
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First Discovery of Polycarpine, Polycarpaurines A and C, and Their Derivatives as Novel Antiviral and Antiphytopathogenic Fungus Agents Pengbin Guo,† Ziwen Wang,*,‡ Gang Li,† Yuxiu Liu,† Yunfu Xie,‡ and Qingmin Wang*,† ‡

Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Key Laboratory of Inorganic−Organic Hybrid Functional Materials Chemistry (Tianjin Normal University), Ministry of Education, College of Chemistry, Tianjin Normal University, Tianjin 300387, China † State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Marine natural products polycarpine, polycarpaurines A and C, and their derivatives were designed, synthesized, and characterized on the basis of 1H NMR and mass spectroscopy. The antiviral and antiphytopathogenic fungus activities of these alkaloids were first evaluated. Polycarpine derivative 1g displayed excellent in vivo antiviral activity against TMV (inactivation inhibitory effect, 57%/500 μg mL−1 and 19%/100 μg mL−1; curative inhibitory effect, 62%/500 μg mL−1 and 23%/100 μg mL−1; and protection inhibitory effect, 56%/500 μg mL−1 and 29%/100 μg mL−1), which is evidently higher than the activity of ribavirin (inactivation inhibitory effect, 37%/500 μg mL−1 and 9%/100 μg mL−1; curative inhibitory effect, 36%/500 μg mL−1 and 13%/100 μg mL−1; and protection inhibitory effect, 39%/500 μg mL−1 and 17%/100 μg mL−1), thus emerging as a new lead compound for antiviral research against TMV. Fungicidal testing in vitro showed that most of the compounds displayed good fungicidal activity against plant pathogenic fungi. Further in vivo fungicidal testing indicated that compounds 6a, 6f, and 8a−c displayed good fungicidal activity. Current results provide support for the development of polycarpine alkaloids as novel agrochemicals. KEYWORDS: marine natural products, polycarpine, anti-TMV, antifungal activity

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INTRODUCTION The global population will exceed 9 billion people by 2050 from the present 7 billion, which is associated with the demand for increasing food production.1,2 Plant diseases, caused by fungal and bacterial pathogens and viruses, are known as “plant cancer”. These diseases can lead to severe losses to agriculture and horticulture crops. Thus, new tools to control these diseases are critically needed. On the other hand, many pesticides are losing their effectiveness due to the evolution of pathogen resistance over the past decade. As such, more novel agrochemicals are needed.3 Marine natural products have played a very important role in the development of new drugs. The search for leads or scaffolds from marine natural products started from the middle of the last century.4 Nereistoxin, from the polychaeta worm Lumbrinereis sp., is the first marine natural product with industrial use as a highly effective, low toxicity, and low residue pesticide.5,6 With the improvement of elucidation techniques for molecules and chemical synthesis, more and more bioactive natural products have been characterized from marine organisms. Polycarpine (1a, Figure 1) was isolated from the ascidian Polycarpa aurata.7 As a promising marine natural product, polycarpine was previously found to have antitumor activity.8,9 Polycarpaurines A and C (2 and 3, Figure 1) are another two interesting marine natural products, isolated from Indonesian ascidian.9 Tobacco Mosaic Virus (TMV) can infect more than 400 plant species, such as tobacco, potato, tomato, and cucumber. As a well-studied plant virus, TMV can also be used as template test virus to discover antiviral agents against other viruses. © 2016 American Chemical Society

Figure 1. Structures of alkaloids polycarpine (1a) and polycarpaurines A (2) and C (3).

For example, phenanthroindolizidine alkaloids with anti-TMV activity also displayed good anti-HIV activity.10 In the discovery process of antiviral agents from natural products using TMV as template test virus, phenanthroindolizidine alkaloids,11 phenanthroquinolizidine alkaloids,12 and beta-carboline alkaloids13 have been discovered as antiviral agents against TMV. In current work, polycarpine, polycarpaurines A and C, and their derivatives were systematically investigated for their antiviral and antiphytopathogenic fungus activities.

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MATERIALS AND METHODS

Instruments. The melting points of the synthesized compounds were determined on an X-4 binocular microscope (Beijing Tech Instruments Co., Beijing, China). NMR spectra were obtained by using

Received: Revised: Accepted: Published: 4264

March 27, 2016 May 2, 2016 May 9, 2016 May 19, 2016 DOI: 10.1021/acs.jafc.6b01415 J. Agric. Food Chem. 2016, 64, 4264−4272

Article

Journal of Agricultural and Food Chemistry

3.04 mmol), and the mixture was stirred at room temperature for 1 h. The mixture was concentrated, and the residue was recrystallized with acetone (50 mL) to provide polycarpaurine C (3, 0.5 g, 72%) as a yellow solid: mp 184 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H, NH), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 7.72 (s, 2H, NH2), 7.04 (d, J = 8.9 Hz, 2H, Ar−H), 3.81 (s, 3H, O−CH3), 3.50 (s, 3H, N−CH3). General Procedures for the Preparation of Intermediates 4a−g. To a solution of imidazole amine 14 (1.0 g, 4.92 mmol) and NEt3 (1.7 mL, 11.81 mmol) in dichloromethane (125 mL) was added a solution of corresponding acyl chlorides (11.81 mmol) in dichloromethane (10 mL) drop by drop. The mixture was stirred for 30 min at room temperature, washed with NaHCO3 solution (100 mL) and brine (100 mL), dried over anhydrous MgSO4, and then filtered and concentrated in vacuo. After purification by column chromatography on silica gel, compound 4a was obtained. Compounds 4b−g were used directly for the next step without purification. N-Acetyl-N-(4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2yl)acetamide (4a). White powder: yield 78%; mp 133−136 °C; 1 H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 8.9 Hz, 2H, Ar−H), 7.14 (s, 1H, CH), 6.91 (d, J = 8.9 Hz, 2H, Ar−H), 3.82 (s, 3H, O−CH3), 3.49 (s, 3H, N−CH3), 2.34 (s, 6H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 172.3, 158.9, 140.1, 138.9, 126.3, 126.0, 115.7, 114.0, 55.3, 32.3, 25.9; HRMS (ESI) calcd for C15H18N3O3 [M + H]+ 288.1343, found 288.1348. General Procedures for the Preparation of Intermediates 5a−g. A solution of the corresponding compounds 4a−g (2.78 mmol), TBAF (3.6 g, 13.90 mmol) and pyridine (1.1 g, 13.90 mmol) in THF (100 mL) was stirred at room temperature for 3.5 h and concentrated. The residue was taken into CH2Cl2 (150 mL), washed successively with H2O (100 mL) and brine (100 mL), and then dried over anhydrous MgSO4. After filtration and concentration, the residue was recrystallized from acetone to give the corresponding compounds 5a−g. N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)acetamide (5a). White powder: yield 58%; mp 200−203 °C; 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 6.7 Hz, 2H, Ar−H), 6.97 (s, 1H, CH), 6.93 (d, J = 7.8 Hz, 2H, Ar−H), 3.85 (s, 3H, O−CH3), 3.60 (s, 3H, N−CH3), 2.16 (s, 3H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 171.9, 158.7, 141.0, 137.4, 126.4, 125.8, 114.4, 114.1, 55.3, 33.3, 23.1; HRMS (ESI) calcd for C13H16N3O2+ [M + H]+ 246.1237, found 246.1242. N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)methanesulfonamide (5b). Light gray powder: yield 5%; mp 110− 113 °C; 1H NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 7.30 (d, J = 8.8 Hz, 2H, Ar−H), 6.93 (d, J = 8.8 Hz, 2H, Ar−H), 6.52 (s, 1H, CH), 3.83 (s, 3H, O−CH3), 3.42 (s, 3H, N−CH3), 3.03 (s, 3H, CH3SO2); 13 C NMR (100 MHz, CDCl3) δ 159.7, 147.3, 125.6, 125.0, 120.0, 114.6, 109.6, 55.4, 42.4, 31.6; HRMS (ESI) calcd for C12H16N3O3S+ [M + H]+ 282.0907, found 282.0907. N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)pivalamide (5c). White powder: yield 53%; mp 225−226 °C; 1H NMR (400 MHz, CDCl3) δ 9.71 (s, 1H, NH), 7.50 (d, J = 8.7 Hz, 2H, Ar−H), 6.89 (s, 1H, CH), 6.88 (d, J = 8.7 Hz, 2H, Ar−H), 3.81 (s, 3H, O−CH3), 3.51 (s, 3H, N−CH3), 1.31 (s, 9H, C−CH3); 13C NMR (100 MHz, CDCl3) δ 158.7, 125.8, 114.0, 55.3, 39.6, 32.8, 27.7; HRMS (ESI) calcd for C16H22N3O2+ [M + H]+ 288.1707, found 288.1705. N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)benzamide (5d). White powder: yield 47%; mp 127−127 °C; 1 H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 6.4 Hz, 2H, Ar−H), 7.48−7.41 (m, 5H, Ar−H), 6.95 (d, J = 8.7 Hz, 2H, Ar−H), 6.71 (s, 1H, CH), 3.85 (s, 3H, O−CH3), 3.68 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 159.6, 137.8, 130.9, 128.7, 128.0, 125.6, 114.5, 110.0, 55.4, 31.8; HRMS (ESI) calcd for C18H18N3O2+ [M + H]+ 308.1394, found 308.1399. N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)benzenesulfonamide (5e). Light gray powder: yield 8%; mp 188− 192 °C; 1H NMR (400 MHz, CDCl3) δ 10.13 (s, 1H, NH), 7.96 (d, J = 7.7 Hz, 2H, Ar−H), 7.52−7.41 (m, 3H, Ar−H), 7.30 (d, J = 7.9 Hz, 2H, Ar−H), 6.94 (d, J = 7.9 Hz, 2H, Ar−H), 6.52 (s, 1H, CH), 3.84 (s, 3H, O−CH3), 3.42 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 159.9, 147.2, 144.3, 131.3, 128.6, 125.8, 125.6, 125.1, 120.0, 114.7, 109.6, 55.4, 31.8; HRMS (ESI) calcd for C17H18N3O3S+ [M + H]+ 344.1063, found 344.1068.

Bruker AV 400/300 spectrometer. Chemical shifts (δ) are given in parts per million (ppm) and were measured downfield from internal tetramethylsilane. High-resolution mass spectra were obtained with an FT-ICR MS spectrometer (Ionspec, 7.0 T). Synthesis of 1-(4-Methoxyphenyl)ethanone (10). A mixture of 1-(4-hydroxyphenyl)ethanone (9, 25.0 g, 0.18 mol), K2CO3 (51.1 g, 0.37 mol), CH3I (28.4 g, 0.20 mol), and acetone (500 mL) was stirred and heated to reflux for 2 h. Then the mixture was filtered and evaporated. The residue was taken into CH2Cl2 (300 mL) and H2O (300 mL) and separated. The aqueous layer was extracted with CH2Cl2 (60 mL × 2), and then the organic layer was dried over anhydrous MgSO4 and evaporated. After purification by column chromatography on silica gel, 1-(4-methoxyphenyl)ethanone (10, 24.6 g, 89%) was obtained as a white solid: mp 33−35 °C; 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.8 Hz, 2H, Ar−H), 6.94 (d, J = 8.9 Hz, 2H, Ar−H), 3.87 (s, 3H, O−CH3), 2.56 (s, 3H, CH3CO). Synthesis of 2-Bromo-1-(4-methoxyphenyl)ethanone (11). To a solution of 1-(4-methoxyphenyl)ethanone (10, 24.0 g, 0.16 mol) and concentrated HCl (0.2 mL) in MeOH (100 mL) was added a solution of Br2 (9.8 mL, 0.19 mol) in MeOH (80 mL) drop by drop at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and then warmed to room temperature. After stirring for another 2 h at room temperature, the mixture was concentrated in vacuo to 40 mL, kept at 0 °C for 3 h, and then filtered to give 2-bromo-1-(4-methoxyphenyl)ethanone (11, 31.0 g, 85%) as a white solid: mp 70−73 °C; 1H NMR (300 MHz, CDCl3) δ 7.97 (d, J = 8.9 Hz, 2H, Ar−H), 6.96 (d, J = 8.9 Hz, 2H, Ar−H), 4.40 (s, 2H, CH2CO), 3.88 (s, 3H, O−CH3). Synthesis of 1-(4-Methoxyphenyl)-2-(methylamino)ethanone Hydrochloride (12). To a solution of methylamine (30% solution in absolute ethanol, 104 mL, 0.68 mol) in acetonitrile (200 mL) was added dropwise a solution of 2-bromo-1-(4methoxyphenyl)ethanone (11, 31.0 g, 0.14 mol) in acetonitrile (150 mL) and dichloromethane (50 mL) at −30 °C, and then the mixture was stirred for 10 min. The mixture was deliquated with dichloromethane (350 mL), washed successively with NaHCO3 solution (200 mL) and H2O (200 mL), then dried over anhydrous MgSO4, and filtered. To the filtrate was added a solution of HCl in ethyl acetate (239 mL, 1.125 M, 0.27 mol). The resulting mixture was stirred for 5 min at room temperature and evaporated. The residue was recrystallized from acetone (100 mL) to give compound 12 (17.1 g, 59%) as a white solid: mp 196−199 °C; 1H NMR (400 MHz, CD3OD) δ 8.01 (d, J = 9.0 Hz, 2H, Ar−H), 7.08 (d, J = 9.0 Hz, 2H, Ar−H), 4.69 (s, 2H, CH2CO), 3.90 (s, 3H, O−CH3), 2.83 (s, 3H, CH3NH). Synthesis of compound 14. To a solution of compound 12 (25.3 g, 0.12 mol) and 2-ethylisothiourea hydroiodide 13 (40.6 g, 0.18 mol)14 in H2O (350 mL) was added a solution of NaOH (16.4 g, 0.41 mol) in H2O (150 mL). The resulting mixture was stirred for 24 h at room temperature and filtered to give compound 14 (18.2 g, 76%) as a yellow powder: mp 143−145 °C; 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.8 Hz, 2H, Ar−H), 6.88 (d, J = 8.8 Hz, 2H, Ar−H), 6.71 (s, 1H, CH), 4.01 (s, 2H, NH2), 3.81 (s, 3H, O−CH3), 3.45 (s, 3H, N−CH3). Synthesis of Polycarpine (1a). To a solution of compound 14 (4.0 g, 19.68 mmol) in acetic acid (200 mL) was added S2Cl2 (1.6 g, 11.81 mmol) and stirred for 10 h at room temperature. The mixture was concentrated, and the residue was recrystallized with acetone (50 mL) to obtain 1a (3.4 g, 63%) as a yellow powder: mp 206−208 °C (lit.8 mp 209−211 °C); 1H NMR (400 MHz, DMSO-d6) δ 13.43 (s, 2 H, HCl), 7.67 (s, 4 H, NH2), 7.46 (d, J = 8.4 Hz, 4 H, Ar−H), 7.01 (d, J = 8.9 Hz, 4 H, Ar−H), 3.89 (s, 3 H, OMe), 3.19 (s, 3 H, NMe). Synthesis of Polycarpaurine A (2). To a solution of compound 14 (4.0 g, 19.68 mmol) in acetic acid (200 mL) was added SCl2 (1.2 g, 11.81 mmol), and the mixture was stirred at room temperature for 10 h. Then the mixture was concentrated, and the residue was recrystallized with acetone (50 mL) to obtain polycarpaurine A (2, 2.1 g, 42%) as an orange red powder: mp 180 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 2H, HCl), 7.81 (s, 4H, NH2), 7.69 (d, J = 8.8 Hz, 4H, Ar−H), 7.11 (d, J = 8.8 Hz, 4H, Ar−H), 3.84 (s, 6H, O−CH3), 2.90 (s, 6H, N−CH3). Synthesis of Polycarpaurine C (3). To a solution of polycarpine (1a, 0.5 g, 0.90 mmol) in H2O (50 mL) was added NaHSO3 (0.3 g, 4265

DOI: 10.1021/acs.jafc.6b01415 J. Agric. Food Chem. 2016, 64, 4264−4272

Article

Journal of Agricultural and Food Chemistry N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)hexanamide (5f). White powder: yield 47%; mp 134−135 °C; 1 H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.6 Hz, 2H, Ar−H), 6.93 (s, 1H, CH), 6.89 (d, J = 8.6 Hz, 2H, Ar−H), 3.82 (s, 3H, O−CH3), 3.58 (s, 3H, N−CH3), 2.39 (t, J = 7.6 Hz, 2H, CH2CO), 1.71−1.59 (m, 2H, COCH2CH2), 1.31−1.23 (m, 4H, CH2CH2CH3), 0.86 (t, J = 6.8 Hz, 3H, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 175.2, 158.8, 141.2, 136.8, 126.1, 125.9, 114.1, 114.1, 55.3, 36.4, 33.4, 31.4, 25.3, 22.4, 13.9; HRMS (ESI) calcd for C17H24N3O2+ [M + H]+ 302.1863, found 302.1871. Ethyl 2-((4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2-yl)amino)-2-oxoacetate (5g). White powder: yield 47%; mp 111− 113 °C; 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.8 Hz, 2H, Ar−H), 6.96 (d, J = 8.8 Hz, 2H, Ar−H), 6.76 (s, 1H, CH), 4.36 (q, J = 7.1 Hz, 2H, CH2CH3), 3.84 (s, 3H, O−CH3), 3.67 (s, 3H, N−CH3), 1.40 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 166.0, 163.8, 160.0, 149.9, 126.4, 125.7, 120.1, 114.7, 110.4, 61.9, 55.4, 32.2, 14.2; HRMS (ESI) calcd for C15H18N3O4+ [M + H]+ 304.1292, found 304.1297. General Procedures for the Preparation of Compounds 6a−g. To a solution of the corresponding compounds 5a−g (3.67 mmol) in acetic acid (50 mL) was added S2Cl2 (2.20 mmol). The mixture was stirred at room temperature for 10 h and then concentrated. The residue was dissolved in CH2Cl2 (150 mL), washed successively with saturated aqueous NaHCO3 solution (100 mL) and brine (100 mL), and then dried over anhydrous MgSO4. After filtration and concentration, the residue was recrystallized from acetone (20 mL) to provide the corresponding compounds 6a−g. (N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))diacetamide) Dihydrochloride (6a). Yellow powder: yield 55%; mp 139−142 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 2H, HCl), 7.40 (d, J = 8.8 Hz, 4H, Ar−H), 6.95 (d, J = 8.8 Hz, 4H, Ar−H), 3.81 (s, 6H, O−CH3), 3.41 (s, 6H, N−CH3), 2.20 (s, 6H, CH3CO); 13C NMR (100 MHz, DMSO-d6) δ 170.0, 160.4, 141.5, 129.2, 114.3, 55.8, 31.8, 23.4; HRMS (ESI) calcd for C26H29N6O4S2+ [M + H − 2HCl]+ 553.1686, found 553.1697. N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))dimethanesulfonamide Dihydrochloride (6b). Yellow powder: yield 20%; mp 218−220 °C; 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.4 Hz, 4H, Ar−H), 6.97 (d, J = 8.7 Hz, 4H, Ar−H), 3.92 (s, 6H, O−CH3), 3.22 (s, 6H, N−CH3), 2.93 (s, 6H); 13 C NMR (100 MHz, CDCl3) δ 161.2, 146.5, 135.5, 127.3, 117.5, 114.8, 55.6, 42.6, 28.9; HRMS (ESI) calcd for C24H29N6O6S4+ [M + H − 2HCl]+ 625.1026, found 625.1014. N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))bis(2,2-dimethylpropanamide) Dihydrochloride (6c). Yellow powder: yield 52%; mp 150−152 °C; 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 2H, HCl), 7.64 (d, J = 8.6 Hz, 4H, Ar−H), 6.97 (d, J = 8.7 Hz, 4H, Ar−H), 6.53 (s, 2H, NH), 3.78 (s, 6H, O−CH3), 3.28 (s, 6H, N−CH3), 1.28 (s, 18H, C(CH3)3); 13 C NMR (100 MHz, DMSO-d6) δ 178.0, 159.6, 141.7, 128.5, 122.4, 115.3, 114.0, 55.1, 39.2, 30.9, 26.8; HRMS (ESI) calcd for C32H41N6O4S2+ [M + H − 2HCl]+ 637.2625, found 637.2634. N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))dibenzamide Dihydrochloride (6d). Yellow powder: yield 22%; mp 186−189 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 2H, HCl), 8.09 (d, J = 7.4 Hz, 4H, Ar−H), 7.71−7.57 (m, 12H, Ar−H and NH), 7.01 (d, J = 8.6 Hz, 4H),

3.76 (s, 6H, O−CH3), 3.44 (s, 6H, N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 166.5, 159.7, 132.9, 132.2, 128.6, 128.4, 122.3, 114.0, 55.1, 31.1; HRMS (ESI) calcd for C36H33N6O4S2+ [M + H − 2HCl]+ 677.1999, found 677.2003. N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))dibenzenesulfonamide Dihydrochloride (6e). Yellow powder: yield 26%; mp 220−224 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 2H, HCl), 7.90 (d, J = 6.8 Hz, 4H, Ar−H), 7.53−7.46 (m, 6H, Ar−H), 7.16 (d, J = 8.1 Hz, 4H, Ar−H), 6.92 (d, J = 8.6 Hz, 4H, Ar−H), 3.84 (s, 6H, O−CH3), 2.89 (s, 6H, N−CH3); 13 C NMR (100 MHz, DMSO-d6) δ 160.6, 146.5, 145.0, 131.7, 130.1, 129.1, 126.1, 118.6, 114.4, 114.1, 55.7, 28.9; HRMS (ESI) calcd for C34H33N6O6S4+ [M + H − 2HCl]+ 749.1339, found 749.1333. N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))dihexanamide Dihydrochloride (6f). Yellow powder: yield 41%; mp 123−126 °C; 1H NMR (400 MHz, CD3OD) δ 7.39 (d, J = 7.7 Hz, 4H, Ar−H), 6.93 (d, J = 7.7 Hz, 4H, Ar−H), 3.79 (s, 6H, O−CH3), 3.48 (s, 6H, N−CH3), 2.51 (t, J = 6.8 Hz, 4H, CH2CO), 1.69 (brs, 4H, COCH2CH2), 1.34 (brs, 8H, CH2CH2CH3), 0.87 (brs, 6H, CH2CH3); 13C NMR (100 MHz, CD3OD) δ 173.8, 162.8, 141.7, 139.1, 130.5, 118.2, 115.5, 56.3, 36.9, 32.4, 31.7, 25.2, 23.5, 14.3; HRMS (ESI) calcd for C34H45N6O4S2+ [M + H − 2HCl]+ 665.2938, found 665.2952. 2,2′-((5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl1H-imidazole-5,2-diyl))bis(azanediyl))bis(2-oxoacetate) Dihydrochloride (6g). Yellow powder: yield 55%; mp 200−204 °C; 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 2H, HCl), 7.61 (brs, 4H, Ar−H), 6.91 (d, J = 8.3 Hz, 4H, Ar−H), 4.32 (q, J = 6.6 Hz, 4H, CH2CH3), 3.77 (s, 6H, O−CH3), 3.33 (s, 6H, N−CH3), 1.33 (t, J = 6.6 Hz, 6H, CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ 159.7, 128.6, 114.2, 62.9, 55.5, 30.8, 14.3; HRMS (ESI) calcd for C30H33N6O8S2+ [M + H − 2HCl]+ 669.1796, found 669.1795. General Procedures for the Preparation of Intermediates 7a−c. A mixture of compound 14 (2.46 mmol), K2CO3 (2.46 mmol) and the corresponding aldehydes (9.84 mmol) in ethanol (60 mL) and toluene (20 mL) was stirred at reflux for 8 h. Then the mixture was filtered and evaporated in vacuo. The residue was taken into CH2Cl2 (200 mL), washed with brine (200 mL), dried over anhydrous MgSO4, filtered, and evaporated. The residue was recrystallized from ethyl acetate (10 mL) to obtain the corresponding intermediates 15a−c, which was used for the next step directly. A mixture of 15a−c and NaBH4 (2.95 mmol) was stirred for 2 h at 65 °C. After cooling to room temperature, H2O (100 mL) and CH2Cl2 (200 mL) was added. The mixture was separated and extracted with CH2Cl2 (60 mL × 2), then dried over anhydrous MgSO4, and evaporated. After purification by column chromatography on silica gel, the corresponding compounds 7a−c were obtained. N-Benzyl-4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2amine (7a). Yellow powder: yield 63%; mp 125−128 °C; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.8 Hz, 2H, Ar−H), 7.46 (d, J = 7.2 Hz, 2H, Ar−H), 7.39−7.28 (m, 3H, Ar−H), 6.89 (d, J = 8.8 Hz, 2H, Ar−H), 6.69 (s, 1H, CH), 4.63 (d, J = 5.8 Hz, 2H, CH2), 4.23 (brs, 1H, NH), 3.81 (s, 3H, O−CH3), 3.39 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 158.1, 149.6, 139.5, 136.9, 128.6, 128.3, 127.8, 127.5, 125.6, 113.9, 111.1, 55.3, 48.7, 31.1; HRMS (ESI) calcd for C18H20N3O+ [M + H]+ 294.1601, found 294.1607. N-(4-Methoxybenzyl)-4-(4-methoxyphenyl)-1-methyl-1Himidazol-2-amine (7b). Yellow powder: yield 49%; mp 144−147 °C;

Scheme 1. Synthesis of Compound 14

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Journal of Agricultural and Food Chemistry Scheme 2. Synthesis of Polycarpine (1a) and Polycarpaurine A (2)

111.1, 55.32, 55.29, 48.2, 31.1; HRMS (ESI) calcd for C19H22N3O2+ [M + H]+ 324.1707, found 324.1709. 4-(4-Methoxyphenyl)-1-methyl-N-(4-(trifluoromethyl)benzyl)-1H-imidazol-2-amine (7c). Yellow powder: yield 50%; mp 113−116 °C; 1H NMR (400 MHz, CDCl3) δ 7.67−7.53 (m, 6H, Ar−H), 6.88 (d, J = 8.8 Hz, 2H, Ar−H), 6.74 (s, 1H, CH), 4.66 (d, J = 6.0 Hz, 2H, CH2), 3.81 (s, 3H, O−CH3), 3.79 (s, 1H, NH), 3.40 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 158.2, 149.2, 143.7, 136.8, 129.4, 128.3, 126.4 (q, J = 210 Hz), 125.6, 125.4 (q, J = 3.8 Hz), 122.8, 113.9, 111.3, 55.3, 48.0, 31.1; HRMS (ESI) calcd for C19H19F3N3O+ [M + H]+ 362.1475, found 362.1482. General Procedures for the Preparation of Compounds 8a−c. The solution of corresponding compounds 7a−c (8.44 mmol) and S2Cl2 (4.22 mmol) in AcOH (120 mL) was stirred at room temperature for 10 h, then concentrated. The residue was taken into CH2Cl2 (200 mL), washed successively with saturated aqueous NaHCO3 solution (200 mL) and brine (200 mL), then dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was recrystallized from acetone (30 mL) to provide the corresponding compounds 8a−c.

Scheme 3. Synthesis of Polycarpaurine C (3)

1 H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 7.4 Hz, 2H, Ar−H), 7.37 (d, J = 7.4 Hz, 2H, Ar−H), 6.89 (d, J = 8.2 Hz, 4H, Ar−H), 6.72 (s, 1H, CH), 4.51 (d, J = 5.1 Hz, 2H, CH2), 3.81 (s, 6H, O−CH3), 3.59 (s, 1H, NH), 3.36 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 159.0, 158.1, 149.6, 136.8, 131.6, 129.6, 127.8, 125.6, 114.0, 113.9,

Scheme 4. Synthesis of Compounds 4a−g, 5a−g, and 6a−g

Scheme 5. Synthesis of Compounds 15a−c, 7a−c, and 8a−c

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Journal of Agricultural and Food Chemistry Scheme 6. Synthesis of Compounds 1b−g

5,5′-Disulfanediylbis(N-benzyl-4-(4-methoxyphenyl)-1methyl-1H-imidazol-2-amine) Dihydrochloride (8a). Brick red powder: yield 58%; mp 152−154 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.20 (s, 2H, HCl), 8.69 (s, 2H, NH), 7.62 (d, J = 8.6 Hz, 4H, Ar−H), 7.44−7.33 (m, 8H, Ar−H), 7.30 (t, J = 6.7 Hz, 2H, Ar−H), 7.04 (d, J = 8.6 Hz, 4H, Ar−H), 4.96 (d, J = 15.0 Hz, 2H, CH2), 4.34 (d, J = 15.0 Hz, 2H, CH2), 3.89 (s, 6H, O−CH3), 3.26 (s, 6H, N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 160.4, 146.8, 137.2, 129.0, 128.3, 127.4, 127.4, 117.2, 113.6, 110.2, 55.5, 45.8, 29.5; HRMS (ESI) calcd for C36H37N6O2S2+ [M + H − 2HCl]+ 649.2414, found 649.2428. 5,5′-Disulfanediylbis(N-(4-methoxybenzyl)-4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2-amine) Dihydrochloride (8b). Yellow powder: yield 34%; mp 152−154 °C; 1H NMR (400 MHz, CD3OD) δ 7.61 (d, J = 8.8 Hz, 4H, Ar−H), 7.34 (d, J = 8.6 Hz, 4H, Ar−H), 7.15 (d, J = 8.8 Hz, 4H, Ar−H), 6.98 (d, J = 8.6 Hz, 4H, Ar−H), 4.54 (d, J = 13.6 Hz, 2H, CH2), 4.33 (d, J = 14.3 Hz, 2H, CH2), 3.95 (s, 6H, O−CH3), 3.82 (s, 6H, O−CH3), 3.35 (s, 6H, N−CH3); 13C NMR (100 MHz, CD3OD) δ 161.3, 159.8, 146.9, 136.9, 128.9, 128.7, 127.4, 117.4, 113.9, 54.9, 54.4, 46.4, 28.5; HRMS (ESI) calcd for C38H41N6O4S2+ [M + H − 2HCl]+ 709.2625, found 709.2613. 5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-N-(4(trifluoromethyl)benzyl)-1H-imidazol-2-amine) Dihydrochloride (8c). Gray powder: yield 27%; mp 153−155 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 2H, HCl), 8.28 (s, 2H, NH), 7.56 (d, J = 7.1 Hz, 4H, Ar−H), 7.42 (d, J = 6.3 Hz, 4H, Ar−H), 7.33 (d, J = 7.3 Hz, 4H, Ar−H), 6.82 (d, J = 7.6 Hz, 4H, Ar−H), 4.84 (d, J = 17.2 Hz, 2H, CH2), 4.66 (d, J = 17.3 Hz, 2H, CH2), 3.71 (s, 6H, O−CH3), 3.27 (s, 6H, N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.0, 157.1, 139.9, 128.4, 128.3, 127.8, 127.5, 125.8, 124.6 (q, J = 3.6 Hz), 113.7, 88.4, 55.1, 43.4, 27.0; HRMS (ESI) calcd for C38H33F6N6O2S2+ [M − H − 2HCl]+ 783.2005, found 783.2000. General Procedures for the Preparation of Compounds 1b−g. A solution of polycarpine (1a, 0.5 g, 0.92 mmol) in MeOH (5 mL), saturated aqueous NaHCO3 solution (30 mL), and CH2Cl2 (100 mL) was stirred for 30 min at room temperature. The mixture was separated and extracted with CH2Cl2 (30 mL), then dried over anhydrous Na2SO4, and filtered. To the filtrate was added dropwise a solution of the corresponding acids (1.84 mmol) in methanol (100 mL). The reaction mixture was stirred for 2 h at 40 °C, concentrated to 80 mL, and then filtered to obtained the corresponding compounds 1b−g. For 1b: red powder; yield 63%; mp 136−138 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.15 (brs, 2H, COOH), 9.59 (brs, 2H, OH), 7.54 (brs, 4H, NH2), 7.49 (d, J = 15.9 Hz, 2H, CH), 7.28 (s, 2H, Ar−H), 7.08 (d, J = 8.0 Hz, 2H, Ar−H), 6.83 (brs, 4H, Ar−H), 6.79 (d, J = 8.0 Hz, 2H, Ar−H), 6.37 (d, J = 15.9 Hz, 2H, CH), 6.13 (brs, 4H, Ar−H), 3.81 (s, 6H, O−CH3), 3.75 (brs, 6H, O−CH3), 3.17 (brs, 6H, N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 168.7, 149.5, 148.4, 144.9, 126.3, 123.3, 116.3, 116.0, 111.6, 56.1, 55.5, 21.6.

Table 1. In Vitro Antiviral Activity of Compounds Ribavirin and the Compounds 1a−g, 2, 3, 4a, 5a−g, 6a−g, 7a−c, 8a−c, and 14 against TMV compd 1a 1b 1c 1d 1e 1f 1g 2 3 4a 5a 5b 5c 5d 5e 5f a

concn (μg/mL)

nhibn rate (%)a

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

53 ± 2 14 ± 3 32 ± 2 12 ± 1 29 ± 1 18 ± 2 38 ± 3 17 ± 2 47 ± 1 20 ± 2 58 ± 1 19 ± 2 53 ± 2 12 ± 1 14 ± 2 0 23 ± 2 0 12 ± 3 0 0 0 31 ± 2 8±1 28 ± 2 0 10 ± 1 0 31 ± 2 0 15 ± 2 0

compd 5g 6a 6b 6c 6d 6e 6f 6g 7a 7b 7c 8a 8b 8c 14 ribavirin

concn (μg/mL)

inhibn rate (%)a

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

36 ± 2 0 0 0 19 ± 1 0 10 ± 2 0 27 ± 3 0 18 ± 2 0 32 ± 2 0 15 ± 3 0 12 ± 2 0 37 ± 2 10 ± 1 22 ± 2 0 29 ± 1 0 41 ± 2 0 27 ± 1 0 0 0 41 ± 2 10 ± 1

Average of three replicates. All results are expressed as mean ± SD.

For 1c: yellow powder; yield 67%; mp 193 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 4H, NH2), 6.81 (s, 4H, Ar−H), 6.23 (s, 4H, Ar−H), 4.26−4.18 (m, 1H, CH), 3.77 (s, 6H, O−CH3), 3.17 (s, 6H, N−CH3), 2.60 (dd, J = 15.6, 5.5 Hz, 1H, CH2), 2.42 (dd, J = 15.6, 7.3 Hz, 1H, CH2). 4268

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Journal of Agricultural and Food Chemistry

Table 2. In Vivo Antiviral Activity of Compounds Ribavirin and the Compounds 1a−g, 2, 3, 4a, 5a−g, 6a−g, 7a−c, 8a−c, and 14 against TMV effect (%)a compd

concn (μg/mL)

inactivation

curative

protection

1a

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

51 ± 2 26 ± 1 36 ± 2 0 49 ± 2 8±2 46 ± 1 0 42 ± 1 8±2 56 ± 2 13 ± 2 57 ± 2 19 ± 1 28 ± 2 0 30 ± 2 0 18 ± 2 0 16 ± 1 0 47 ± 2 20 ± 2 9±2 0 23 ± 1 0 34 ± 2 5±2 24 ± 2 0

47 ± 1 17 ± 2 44 ± 3 5±2 39 ± 1 10 ± 2 50 ± 2 14 ± 2 35 ± 3 0 52 ± 1 11 ± 2 62 ± 2 23 ± 2 33 ± 2 0 15 ± 1 0 27 ± 3 0 13 ± 2 0 40 ± 2 16 ± 2 12 ± 1 0 16 ± 2 0 26 ± 3 0 17 ± 2 0

45 ± 2 18 ± 2 45 ± 2 9±2 47 ± 2 21 ± 1 43 ± 2 6±2 33 ± 2 0 49 ± 2 22 ± 2 56 ± 2 29 ± 1 24 ± 2 0 34 ± 2 0 15 ± 2 0 25 ± 2 0 43 ± 2 10 ± 2 19 ± 2 0 10 ± 2 0 19 ± 2 0 26 ± 2 0

1b 1c 1d 1e 1f 1g 2 3 4a 5a 5b 5c 5d 5e 5f a

effect (%)a compd 5g 6a 6b 6c 6d 6e 6f 6g 7a 7b 7c 8a 8b 8c 14 ribavirin

concn (μg/mL)

inactivation

curative

protection

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

26 ± 1 0 9±2 0 7±1 0 11 ± 2 0 30 ± 3 0 27 ± 1 0 22 ± 2 0 10 ± 1 0 17 ± 2 0 42 ± 1 16 ± 2 32 ± 2 0 20 ± 1 0 35 ± 2 0 30 ± 2 0 32 ± 2 0 37 ± 2 9±1

22 ± 2 0 0 0 13 ± 2 0 22 ± 2 0 21 ± 2 0 11 ± 1 0 30 ± 2 0 18 ± 3 0 32 ± 2 0 46 ± 2 10 ± 3 28 ± 2 0 34 ± 2 0 22 ± 2 0 39 ± 2 0 24 ± 1 0 36 ± 2 13 ± 2

17 ± 2 0 12 ± 1 0 8±2 0 19 ± 2 0 23 ± 1 0 14 ± 2 0 36 ± 2 0 6±2 0 33 ± 2 0 49 ± 2 25 ± 2 37 ± 2 0 31 ± 1 0 33 ± 2 0 26 ± 2 0 29 ± 2 0 39 ± 1 17 ± 1

Average of three replicates. All results are expressed as mean ± SD.

For 1d: yellow powder; yield 48%; mp 187 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 4H, NH2), 6.81 (s, 4H, Ar−H), 6.24 (s, 4H, Ar−H), 4.26−4.13 (m, 1H, CH), 3.76 (s, 6H, O−CH3), 3.15 (s, 6H, N−CH3), 2.60 (dd, J = 15.6, 5.4 Hz, 1H, CH2), 2.42 (dd, J = 15.6, 7.2 Hz, 1H, CH2). For 1e: yellow powder; yield 55%; mp 214 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 7.50 (brs, 4H, NH2), 6.80 (brs, 4H, Ar−H), 6.62 (s, 2H, CH2), 6.33 (brs, 4H, Ar−H), 3.77 (s, 6H, O−CH3), 3.15 (s, 6H, N−CH3). For 1f: yellow powder; yield 74%; mp 240−243 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 2H), 7.78 (s, 4H, NH2), 7.56 (d, J = 8.1 Hz, 4H, Ar−H), 7.42 (d, J = 8.6 Hz, 4H, Ar−H), 7.18 (d, J = 7.9 Hz, 4H, Ar−H), 6.98 (d, J = 8.9 Hz, 4H, Ar−H), 3.85 (s, 6H, O−CH3), 3.15 (s, 6H, N−CH3), 2.30 (s, 6H, CH3). For 1g: yellow powder; yield 70%; mp 220 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 13.72 (s, 2H, COOH), 7.90 (s, 4H, NH2), 7.42 (d, J = 8.5 Hz, 4H, Ar−H), 6.94 (d, J = 8.5 Hz, 4H, Ar−H), 3.86 (s, 6H, O−CH3), 3.16 (s, 6H, N−CH3). Biological Assay. The activity of synthesized compounds was tested on representative test organisms. Each bioassay was replicated three times at 25 ± 1 °C. Percentage of mortalities was calculated according to a percentage scale of 0−100 (0 means no activity, and 100 means total kill). Antiviral Biological Assay. The synthesized compounds were tested for their antiviral activity using TMV as template test virus on tobacco (Nicotiana tabacum var. Xanthi nc). The test procedures and method have been reported in our previous work.11,12

Antifungal Biological Assay. The test method of antifungal activity of the synthesized compounds was described in previous work.15

■

RESULTS AND DISCUSSION Chemistry. Although Novikov and coauthour have reported the synthesis of polycarpine,8 the experimental details were not given. As shown in Scheme 1, methylation and bromination of acetophenone 9 gave bromide 11. The introduction of methylamine group is a critical step as it is difficult to obtain compound 12 as a pure solid. After condition optimization, compound 12 was given with 59% yield over two steps using acetone as a solvent for recrystallization. The detailed procedure is given in Materials and Methods. Condensation of 12 with 13 gave the key intermediate 14. Oxidative coupling of 14 with S2Cl2 gave polycarpine (1a, Scheme 2). As shown in Scheme 2, using SCl2 in place of S2Cl2 gave polycarpaurine A (2) in 42% yield. Polycarpaurine C (3) can be obtained from polycarpine (1a) in the presence of NaHSO3 (Scheme 3). In order to investigate structure−activity relationship (SAR), the derivatives 4−8 were designed and synthesized. As depicted in Scheme 4, acetylation of 2-aminoimidazole 14 with corresponding acyl chlorides gave amides 4. As the impact of imidazole ring, the acetylation only gave a mixture of amides 4 and 5 even at low temperature or with excess acyl chloride. 4269

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19 ± 1 7±2 14 ± 2 10 ± 2 12 ± 2 12 ± 3 21 ± 2 10 ± 1 2±2 14 ± 2 5±2 14 ± 1 14 ± 2 19 ± 1 26 ± 2 19 ± 2 11 ± 2 26 ± 3 21 ± 2 29 ± 1 26 ± 2 29 ± 3 29 ± 2 31 ± 3 5±2 2±1 14 ± 2 0 10 ± 1 17 ± 2 18 ± 2 100