Synthesis and Antiviral and Fungicidal Activity Evaluation of β

Article pubs.acs.org/JAFC

Synthesis and Antiviral and Fungicidal Activity Evaluation of β‑Carboline, Dihydro-β-carboline, Tetrahydro-β-carboline Alkaloids, and Their Derivatives Hongjian Song, Yongxian Liu, Yuxiu Liu, Lizhong Wang, and Qingmin Wang* State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, People’s Republic of China ABSTRACT: Six known β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline alkaloids and a series of their derivatives were designed, synthesized, and evaluated for their anti-tobacco mosaic virus (TMV) and fungicidal activities for the first time. All of the alkaloids and some of their derivatives (compounds 3, 4, 14, and 19) exhibited higher anti-TMV activity than the commercial antiviral agent Ribavirin both in vitro and in vivo. Especially, the inactivation, curative, and protection activities of alkaloids Harmalan (62.3, 55.1, and 60.3% at 500 μg/mL) and tetrahydroharmane (64.2, 57.2, and 59.5% at 500 μg/mL) in vivo were much higher than those of Ribavirin (37.4, 36.2, and 38.5% at 500 μg/mL). A new derivative, 14, with optimized physicochemical properties, obviously exhibited higher activities in vivo (50.4, 43.9, and 47.9% at 500 μg/mL) than Ribavirin and other derivatives; therefore, 14 can be used as a new lead structure for the development of anti-TMV drugs. Moreover, most of these compounds exhibited good fungicidal activity against 14 kinds of fungi, especially compounds 4, 7, and 11. KEYWORDS: β-carboline, dihydro-β-carboline, tetrahydro-β-carboline, alkaloids, anti-TMV, fungicidal activity, structure−activity relationship

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reversible inhibitor of MAO-A.7 The current research on the activity of β-carboline alkaloids and their analogues has mainly focused on antitumor,8,9 inhibition of monoamine oxidase,10 and antiparasitic activities,11,12 but few studies have focused on antiviral13−15 and fungicidal activities. In this study, six known β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline alkaloids were synthesized, and their anti-TMV and fungicidal activities were systematically evaluated for the first time. To study the role of the substituents played in the biological activities, β-carbolines containing different substituents were synthesized, and their anti-TMV and fungicidal activities were also systematically evaluated (Figure 3).

INTRODUCTION Tobacco mosaic virus (TMV) may cause heavy losses to modern agriculture (Figure 1), but its control remains a challenge.1

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Figure 1. Leaves after infection.

Many plant viral inhibitors (Ribavirin, Ningnanmycin, BTH, etc.) (Figure 2) are used to prevent TMV disease; however, these chemical pesticides cannot completely cure the infected plant tissues or fully protect plants from TMV infection under field conditions. Plant pathogens also lead to great harm to crops; they can either infect the whole crops or infect crop organs. Therefore, novel and more practical antiviral and fungicidal reagents still need to be developed. Natural products can be used as ideal lead structures to develop agrochemicals. β-Carboline and its saturated analogue are common structural motifs in natural products and pharmaceuticals.2 Harmine and tetrahydroharmine, which are β-carboline and tetrahydro-β-carboline alkaloids, respectively, are two representative harmala alkaloids. Harmine was originally isolated from P. harmala L.3 and found to exhibit a cytotoxic effect on HL60 and K562 leukemic cell lines.4 Tetrahydroharmine is a fluorescent indole alkaloid present in the tropical liana species Banisteriopsis caapi,5 and it weakly inhibits serotonin reuptake.6 Harmaline is a central nervous system stimulant and a © 2014 American Chemical Society

MATERIALS AND METHODS

Instruments. 1H NMR spectra were obtained at 400 MHz using a Bruker AV400 spectrometer in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal standard. HRMS data were obtained on an FTICR-MS instrument (Ionspec 7.0 T). The melting points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and are uncorrected. General Synthesis. All anhydrous solvents were dried and purified by using standard techniques. The synthetic routes are given in Schemes 1−5. Synthesis of Harmalan (1-Methyl-4,9-dihydro-3H-pyrido[3,4-b]indole).16 To a solution of tryptamine (0.50 g, 3.13 mmol) and triethylamine (2 mL) in dichloromethane was added dropwise acetyl chloride (0.27 g, 3.44 mmol) in dichloromethane (5 mL) at room temperature, and the mixture was stirred at room temperature Received: Revised: Accepted: Published: 1010

November 6, 2013 January 3, 2014 January 15, 2014 January 24, 2014 dx.doi.org/10.1021/jf404840x | J. Agric. Food Chem. 2014, 62, 1010−1018

Journal of Agricultural and Food Chemistry

Article

Figure 2. Virus inhibitors.

Figure 3. Design of target compounds.

Scheme 1. Synthesis of Harmalan, Tetrahydroharmane, and Harmane

175 °C (lit.19 mp = 177−178 °C); 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H, NH), 7.35 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.27 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 6.98−7.02 (m, 1H, Ar−H), 6.91−6.95 (m, 1H, Ar−H), 3.99−4.04 (m, 1H, CHNH), 3.33 (s, 1H, CHNH), 3.14−3.19 (m, 1H, CH2NH), 2.81−2.87 (m, 1H, CH2NH), 2.51−2.62 (m, 2H, CH2CH2), 1.36 (d, 3JHH = 6.8 Hz, 3H, CH3CH); HRMS (ESI) calcd for C12H15N2 (M + H)+ 187.1230, found 187.1231. Synthesis of Harmane (1-Methyl-9H-pyrido[3,4-b]indole).20 To a stirred solution of maleic acid (0.53 g, 4.57 mmol) in water at room temperature was added 1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (0.85 g, 4.57 mmol), and the mixture was stirred until the solid dissolved in water (120 mL); then Pd/C (0.85 g) was added, and the mixture was heated at reflux for 8 h. Then the mixture was cooled and filtered, and the filtered solid was washed carefully with water. The combined filtrates were adjusted to pH 9 with aqueous NaOH and then extracted with dichloromethane (3 × 50 mL), and the combined organic phases were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give harmane as a white solid (0.50 g, 60%): mp = 244−245 °C (lit.21 mp = 235−238 °C); 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H, NH), 8.37 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 8.12 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.83 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.52−7.57 (m, 2H, Ar−H), 7.26− 7.32 (m, 1H, Ar−H), 2.84 (s, 3H, CH3); HRMS (ESI) calcd for C12H11N2 (M + H)+ 183.0917, found 183.0915. Synthesis of Quaternary Ammonium Salts 1. To a solution of harmine (0.50 g, 2.36 mmol) in ethyl acetate (120 mL) was added benzyl bromide (0.48 g, 2.83 mmol), and the mixture was heated at reflux for 12 h. Then the mixture was cooled and filtered, and the filter cake was washed with ethyl acetate and dried to give 1 as a white solid (0.67 g, 74%): mp = 276−277 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H, NH), 8.74 (d, 3JHH = 6.4 Hz, 1H, Ar−H), 8.58 (d, 3JHH = 6.8 Hz, 1H, Ar−H), 8.37 (d, 3JHH = 8.8 Hz, 1H, Ar−H), 7.35−7.44

for 5 h. Then the mixture was washed with a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, and filtered, and the solvent was removed under reduced pressure. The crude was dissolved in toluene (20 mL) and chloroform (20 mL), phosphorus oxychloride (3 mL) was added dropwise to the above solution, and then the mixture was heated at reflux for 7 h. The mixture was adjusted to pH 9 with a saturated aqueous solution of Na2CO3 after it was cooled to room temperature. The resulting solution was extracted with dichloromethane (3 × 20 mL), the combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 10:1) as eluent to give harmalan as a brown solid (0.35 g, 60%): mp = 180−183 °C (lit.17 mp = 182−183 °C); 1H NMR (400 MHz, CDCl3) δ 9.47 (s, 1H, NH), 7.60 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.48 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 7.31 (t, 3JHH = 8.0 Hz, 1H, Ar−H), 7.16 (t, 3JHH = 8.0 Hz, 1H, Ar−H), 3.88 (t, 3JHH = 8.4 Hz, 2H, CH2), 2.95 (t, 3JHH = 8.8 Hz, 2H, CH2), 2.53 (s, 3H, CH3); HRMS (ESI) calcd for C12H13N2 (M + H)+ 185.1073, found 185.1077. Synthesis of Tetrahydroharmane (1-Methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole).18 To a solution of 40% aqueous solution of acetaldehyde (8.1 mL, 43.75 mmol) in water (250 mL) was added concentrated H2SO4 (5 drops), the mixture was stirred at room temperature for 0.5 h. Then tryptamine (3.50 g, 21.88 mmol) was added, and the mixture was heated at reflux for 7 h. The mixture was adjusted to pH 10 with an aqueous solution of NaOH after it was cooled to room temperature. The resulting solution was extracted with dichloromethane (3 × 100 mL), and the combined organic phase was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and then purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 5:1) as eluent to give tetrahydroharmane as a brown solid (2.53 g, 62%): mp = 173− 1011

dx.doi.org/10.1021/jf404840x | J. Agric. Food Chem. 2014, 62, 1010−1018

Journal of Agricultural and Food Chemistry

Article

(m, 3H, Ar−H), 7.23 (d, 3JHH = 7.2 Hz, 2H, Ar−H), 7.12 (d, 4JHH = 1.0 Hz, 1H, Ar−H), 7.08 (dd, 3JHH = 8.8 Hz, 4JHH = 1.0 Hz, 1H, Ar−H), 5.98 (s, 2H, CH2), 3.95 (s, 3H, OCH3), 2.98 (s, 3H, CH3). Synthesis of 2-Benzyl-7-methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (2). To a solution of 1 (0.67 g, 1.75 mmol) in methanol (150 mL) was added dropwise sodium borohydride (0.53 g, 14.00 mmol) in methanol (30 mL), and then the mixture was heated at reflux for 15 h. The solvent was distilled off, and the residue was diluted with dichloromethane (2 × 50 mL) and water (50 mL); the combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 5:1) as eluent to give 2 as a brown oil (0.47 g, 88%): 1H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H, NH), 7.50−7.52 (m, 2H, Ar−H), 7.43−7.47 (m, 3H, Ar−H), 7.36−7.40 (m, 1H, Ar−H), 6.90 (dd, 3JHH = 8.8 Hz, 4JHH = 2.4 Hz, 1H, Ar−H), 6.83 (d, 4 JHH = 2.4 Hz, 1H, Ar−H), 3.97 (d, 2JHH = 13.6 Hz, 1H, CH2C6H5), 3.81 (q, 3JHH = 6.8 Hz, 1H, CHCH3), 3.77 (d, 2JHH = 13.6 Hz, 1H, CH2C6H5), 3.22−3.29 (m, 1H, CH2CH2), 2.85−2.95 (m, 2H, CH2CH2), 2.68−2.74 (m, 1H, CH2CH2), 1.61 (d, 3JHH = 6.8 Hz, 1H, CHCH3). Synthesis of 7-Methoxy-1-methyl-2,3,4,4a,9,9a-hexahydro1H-pyrido[3,4-b]indole (3).22 To a solution of 2 (0.70 g, 2.29 mmol) in 2,2,2-trifluoroethanol (120 mL) was added Pd/C (0.70 g), and then the reaction system was evacuated and purged with hydrogen three times; the mixture was stirred in a hydrogen atmosphere overnight at room temperature. The mixture was concentrated in vacuo and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 10:1) as eluent to give 3 as a pale yellow solid (0.37 g, 74%): mp = 158−160 °C; 1H NMR (400 MHz, CDCl3) δ 6.93 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 6.31 (dd, 3 JHH = 8.0 Hz, 4JHH = 2.0 Hz, 1H, Ar−H), 6.26 (d, 4JHH = 2.0 Hz, 1H, Ar−H), 3.76 (s, 3H, OCH3), 3.49 (s, 1H, NH), 3.39−3.45 (m, 1H, CHCH3), 3.35 (t, 3JHH = 8.4 Hz, 1H, CH), 2.97−3.02 (m, 1H, CH), 2.69−2.72 (m, 1H, CH2), 2.53−2.60 (m, 1H, CH2), 2.08−2.20 (m, 2H, CH2NH), 1.26 (s, 1H, NH), 1.23 (d, 3JHH = 6.4 Hz, 3H, CH3); HRMS (ESI) calcd for C13H19N2O (M + H)+ 219.1492, found 219.1494. Data for 7-Methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4b]indole (Tetrahydroharmine). The compound was synthesized using a similar procedure as for compound 3; the mixture was stirred for 3 h to give tetrahydroharmine as a white solid (0.56 g, 80%): mp = 195− 197 °C (lit.23 mp = 198−200 °C); 1H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H, NH), 7.35 (d, 3JHH = 8.8 Hz, 1H, Ar−H), 6.85 (d, 4JHH = 2.0 Hz, 1H, Ar−H), 6.77 (dd, 3JHH = 8.4 Hz, 4JHH = 2.4 Hz, 1H, Ar−H), 4.14−4.19 (m, 1H, CHCH3), 3.84 (s, 3H, OCH3), 3.33−3.39 (m, 1H, CH2CH2), 3.01−3.07 (m, 1H, CH2CH2), 2.66−2.78 (m, 2H, CH2CH2), 1.65 (s, 1H, NH), 1.44 (d, 3JHH = 6.8 Hz, 3H, CH3); HRMS (ESI) calcd for C13H17N2O (M + H)+ 217.1335, found 217.1337. Synthesis of Compounds 4 and 5.24 To a solution of harmane (0.20 g, 1.10 mmol) in glacial acetic acid (10 mL) was added NBS (0.20 g, 1.10 mmol) at room temperature, and the mixture was stirred at room temperature for 6 h. Then the mixture was concentrated under reduced pressure and adjusted to pH 10 with an aqueous solution of NaHCO3, and the aqueous solution was extracted with dichloromethane (20 mL × 4). The combined organic layer was dried over anhydrous Na2SO4 and then was concentrated in vacuo and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 40:1→20:1) as eluent to give 4 (0.24 g) and 5 (0.05 g). 6-Bromo-1-methyl-9H-pyrido[3,4-b]indole (4). This compound was obtained as a white solid in 83% yield: mp = 256−257 °C; 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H, NH), 8.39 (d, 3JHH = 5.6 Hz, 1H, Ar−H), 8.24 (d, 4JHH = 2.0 Hz, 1H, Ar−H), 7.78 (d, 3JHH = 5.6 Hz, 1H, Ar−H), 7.63 (dd, 3JHH = 8.4 Hz, 4JHH = 2.0 Hz, 1H, Ar−H), 7.42 (d, 3JHH = 8.8 Hz, 1H, Ar−H), 2.83 (s, 3H, CH3); HRMS (ESI) calcd for C12H10BrN2 (M + H)+ 261.0022, found 261.0026. 8-Bromo-1-methyl-9H-pyrido[3,4-b]indole (5). This compound was obtained as a yellow solid in 17% yield: mp = 201−204 °C; 1H NMR (400 MHz, CDCl3) δ 8.36−8.49 (m, 2H, NH and Ar−H),

8.05 (d, 3JHH = 6.4 Hz, 1H, Ar−H), 7.80 (s, 1H, Ar−H), 7.70 (d, 3 JHH = 6.4 Hz, 1H, Ar−H), 7.18 (t, 3JHH = 6.4 Hz, 1H, Ar−H), 2.88 (s, 3H, CH3); HRMS (ESI) calcd for C12H10BrN2 (M + H)+ 261.0022, found 261.0025. Synthesis of Compounds 6 and 7.25 To a mixture of harmane (0.40 g, 2.20 mmol) and sodium nitrate (0.93 g, 10.99 mmol) was added dropwise trifluoroacetic acid (20 mmol) at 0 °C, and then the mixture was stirred overnight at room temperature. The mixture was adjusted to pH 10−11 with an aqueous solution of NaHCO3 in an ice bath and then was extracted by dichloromethane (20 mL × 3). The combined organic phase was concentrated in vacuo and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 20:1) as eluent to give 6 (0.24 g) and 7 (0.06 g) . 1-Methyl-6-nitro-9H-pyrido[3,4-b]indole (6). This compound was obtained as a pale yellow solid in 72% yield: mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 9.30 (d, 4JHH = 2.0 Hz, 1H, Ar−H), 8.41 (dd, 3JHH = 8.8 Hz, 4JHH = 3.0 Hz, 1H, Ar−H), 8.33 (d, 3JHH = 5.6 Hz, 1H, Ar−H), 8.20 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.73 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 2.79 (s, 3H, CH3); HRMS (ESI) calcd for C12H10N3O2 (M + H)+ 228.0768, found 228.0767. 1-Methyl-8-nitro-9H-pyrido[3,4-b]indole (7). This compound was obtained as a yellow solid in 12% yield: mp = 207−210 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H, NH), 8.77 (d, 3JHH = 7.6 Hz, 1H, Ar−H), 8.50 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 8.38 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 8.11 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.48 (t, 3JHH = 7.6 Hz, 1H, Ar−H), 2.92 (s, 3H, CH3); HRMS (ESI) calcd for C12H10N3O2 (M + H)+ 228.0768, found 228.0772. Synthesis of Harmol (1-Methyl-9H-pyrido[3,4-b]indol-7-ol). A solution of harmine (0.50 g, 2.36 mmol) in glacial acetic acid (18 mL) and 40% hydrobromic acid solution (18 mL) was stirred at room temperature for 1 h, and then the mixture was heated at reflux for 10 h. After cooling to room temperature, the mixture was adjusted to pH 8 with a saturated aqueous solution of NaHCO3. The yellow slurry was filtered, and the cake was washed with water to afford harmol as a yellow solid (0.46 g, 98%): mp > 300 °C (lit.26 mp = 304−307 °C); 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, NH), 9.72 (s, 1H, OH), 8.11 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.94 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.75 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 6.90 (d, 4JHH = 1.2 Hz, 1H, Ar−H), 6.69 (dd, 3JHH = 8.4 Hz, 4JHH = 1.6 Hz, 1H, Ar−H), 2.69 (s, 3H, CH3); HRMS (ESI) calcd for C12H11N2O (M + H)+ 199.0866, found 199.0867. Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl isopropylcarbamate (8).27 To a solution of harmol (0.50 g, 2.53 mmol) in DMF (50 mL) was added isopropyl isocyanate via syringe over 5 min, the mixture was stirred at room temperature for 2 h, then triethylamine (0.08 g, 0.758 mmol) was added, and the mixture was stirred overnight at room temperature. Then brine (100 mL) was added, the aqueous solution was extracted with ethyl acetate (20 mL × 4), and the combined organic layer was dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 20:1) as eluent to give 8 as a pale white solid (0.50 g, 70%): mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H, NH), 8.20 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 8.16 (d, 3JHH = 8.8 Hz, 1H, Ar−H), 7.90 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.76 (d, 3JHH = 8.0 Hz, 1H, NHCO), 7.27 (d, 4JHH = 1.6 Hz, 1H, Ar−H), 6.95 (dd, 3JHH = 8.4 Hz, 4JHH = 2.0 Hz, 1H, Ar− H), 3.65−3.73 (m, 1H, CH), 2.75 (s, 3H, CH3), 1.16 (d, 3JHH = 6.4 Hz, 6H, (CH3)2CH); HRMS (ESI) calcd for C16H18N3O2 (M + H)+ 284.1394, found 284.1399. Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl dimethylcarbamate (9). To a solution of harmol (0.40 g, 2.02 mmol) in THF (150 mL) was added triethylamine (0.31 g, 3.03 mmol), and the mixture was stirred for 0.5 h. Then dimethylcarbamic chloride was added dropwise, and the mixture was stirred overnight at room temperature. Then water (20 mL) and dichloromethane (20 mL) were added. The separated organic layer was dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and then purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 10:1) as eluent to give 9 as a pale white solid (0.48 g, 89%): mp = 225− 227 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H, NH), 1012

dx.doi.org/10.1021/jf404840x | J. Agric. Food Chem. 2014, 62, 1010−1018

Journal of Agricultural and Food Chemistry

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8.20 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 8.17 (d, 3JHH = 8.4 Hz, 1H, Ar− H), 7.91 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.30 (d, 4JHH = 2.0 Hz, 1H, Ar−H), 6.97 (dd, 3JHH = 8.4 Hz, 4JHH = 2.0 Hz, 1H, Ar−H), 3.10 (s, 3H, CH3), 2.94 (s, 3H, CH3), 2.75 (s, 3H, CH3); HRMS (ESI) calcd for C15H16N3O2 (M + H)+ 270.1237, found 270.1240. Compounds 10 and 11 were synthesized using a procedure similar to that used for compound 9. Data for 1-Methyl-9H-pyrido[3,4-b]indol-7-yl acetate (10). This compound was obtained as a white solid in 50% yield: mp = 237− 240 °C; 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H, NH), 8.23 (d, 3 JHH = 2.4 Hz, 1H, Ar−H), 7.78 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 7.70 (d, 3JHH = 2.0 Hz, 1H, Ar−H), 7.16 (s, 1H, Ar−H), 6.93 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 2.76 (s, 3H, CH3), 2.42 (s, 3H, CH3CO); HRMS (ESI) calcd for C14H13N2O2 (M + H)+ 241.2647, found 241.2650. Data for Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl pivalate (11). This compound was obtained as a white solid in 85% yield: mp = 221−222 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H, NH), 8.30 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.75 (d, 3JHH = 8.4 Hz, 1H, Ar− H), 7.54 (d, 3JHH = 4.8 Hz, 1H, Ar−H), 7.09 (s, 1H, Ar−H), 6.86 (d, 3 JHH = 8.0 Hz, 1H, Ar−H), 2.73 (s, 3H, CH3), 1.45 (s, 9H, C(CH3)3); HRMS (ESI) calcd for C17H19N2O2 (M + H)+ 283.1441, found 283.1446. Synthesis of (S)-1-Methyl-9H-pyrido[3,4-b]indol-7-yl-2-(benzyloxycarbonylamino)-3-methylbutanoate (12). To a solution of amino acid (0.80 g, 3.03 mmol) in dichloromethane (150 mL) was added triethylamine (0.41 g, 4.04 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (0.76 g, 4.04 mmol), and DMAP (0.50 g, 4.04 mmol), and then the mixture was stirred overnight at room temperature. The mixture was washed with a saturated aqueous solution of NH4Cl (50 mL × 2) and a saturated aqueous solution of NaHCO3 (50 mL) successively, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and then purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 20:1) as eluent to give 12 as a pale white solid (0.80 g, 90%): mp = 69−71 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H, NH), 8.34 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.92 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 7.66 (d, 3JHH = 4.8 Hz, 1H, Ar−H), 7.35−7.38 (m, 5H, Ar−H), 7.19 (s, 1H, Ar−H), 6.94 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 5.39 (d, 3JHH = 8.4 Hz, 1H, NHCO), 5.17 (s, 2H, CH2), 4.54−4.66 (m, 1H, CHNH), 2.77 (s, 3H, CH3), 2.38−2.50 (m, 1H, CH(CH3)2), 1.14 (d, 3JHH = 6.8 Hz, 3H, CH(CH3)2), 1.09 (d, 3JHH = 6.8 Hz, 3H, CH(CH3)2); HRMS (ESI) calcd for C25H26N3O4 (M + H)+ 432.1918, found 432.1920. Synthesis of (1S,3S)-1-Methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid (13). 28 To a solution of L-tryptophan (20.00 g, 98.0 mmol) in water (500 mL) were added concentrated H2SO4 (2 mL) and a 40% aqueous solution of acetaldehyde (20 mL) successively, and the mixture was stirred overnight at room temperature. Then the mixture was adjusted to pH 6−7 with ammonia solution (25−28%, w/w), and a white solid precipitated. The white slurry was filtered, and the cake was washed with water to afford 13 as a white solid (16.7 g, 74%): mp = 278−280 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H, COOH), 7.45 (d, 3JHH = 7.8 Hz, 1H, Ar−H), 7.34 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.09 (t, 3JHH = 7.6 Hz, 1H, Ar−H), 7.00 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 4.52 (q, 3JHH = 6.4 Hz, 1H, CH), 3.61 (dd, 3JHH = 11.6 Hz, 3JHH = 4.4 Hz, 1H, CH), 3.16 (dd, 2 JHH = 16.0 Hz, 3JHH = 4.0 Hz, 1H, CH2), 2.74−2.81 (m, 1H, CH2), 1.62 (d, 3JHH = 6.4 Hz, 3H, CH3); (dr = 11.7:1); HRMS (ESI) calcd for C13H15N2O2 (M + H)+ 231.1128, found 231.1132. Synthesis of Ethyl (1S,3S)-1-Methyl-2,3,4,9-tetrahydro-1Hpyrido[3,4-b]indole-3-carboxylate (14). To a solution of acid 13 (16.0 g, 69.0 mmol) in ethanol (500 mL) was added dropwise thionyl chloride in an ice bath, and then the mixture was heated at reflux for 5 h. The solvent was distilled off, the residue was dissolved in water (250 mL), and then the mixture was adjusted to pH 9 with an aqueous solution of NaHCO3. The aqueous was extracted by ethyl acetate (150 mL × 3), the combined organic phase was washed with brine and dried over anhydrous Na2SO4, and then the mixture was concentrated in vacuo to give 14 as a light yellow solid (16.40 g, 92%): mp = 136−137 °C; 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H, NH), 7.49 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.33 (d, 3JHH = 8.0 Hz, 1H, Ar−H),

7.17 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 7.11 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 4.26−4.31 (m, 3H, CH and OCH2), 3.81 (dd, 3JHH = 11.2 Hz, 3JHH = 4.4 Hz, 1H, CH), 3.13 (dd, 2JHH = 15.2 Hz, 3JHH = 4.0 Hz, 1H, CH2), 2.79−2.86 (m, 1H, CH2), 1.52 (d, 3JHH = 6.4 Hz, 3H, CH3), 1.35 (t, 3 JHH = 7.2 Hz, 3H, OCH2CH3); HRMS (ESI) calcd for C15H19N2O2 (M + H)+ 259.1441, found 259.1443. Synthesis of Ethyl 1-Methyl-9H-pyrido[3,4-b]indole-3-carboxylate (15).29 A mixture of 14 (12.4 g, 47.7 mmol) and sulfur (3.10 g, 95.4 mmol) in xylene (150 mL) was heated at reflux for 12 h. Then the mixture was cooled to 0 °C and kept for 1 h, dichloromethane (10 mL) was added to the mixture, and pink solid was precipitated. The mixture was filtered, and the cake was washed with toluene to afford 15 as a brown solid (8.40 g, 69%): mp = 217− 219 °C; 1H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H, NH), 8.79 (s, 1H, Ar−H), 8.18(d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.54−7.60 (m, 2H, Ar− H), 7.32−7.36 (m, 1H, Ar−H), 4.50 (d, 3JHH = 6.8 Hz, 2H, OCH2), 2.79 (s, 3H, CH3), 1.41 (t, 3JHH = 7.2 Hz, 3H, OCH2CH3); HRMS (ESI) calcd for C15H15N2O2 (M + H)+ 255.1128, found 255.1131. Synthesis of 1-Methyl-9H-pyrido[3,4-b]indole-3-carboxylic acid (16). To a solution of ester 15 (2.00 g, 7.87 mmol) in alcohol (60 mL) was added NaOH (0.47 g, 11.81 mmol) in portions, and then the mixture was heated at reflux for 6 h. Then the mixture was concentrated under reduced pressure, the residue was dissolved in water (50 mL), and the aqueous solution was extracted by diethyl ether (50 mL × 2). Then the solution was adjusted to pH 5−6 with an aqueous solution of diluted hydrochloric acid (3 M), the slight yellow slurry was filtered, and the cake was washed with water to afford 16 as a yellow solid (1.46 g, 82%): mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.04 (s, 1H, COOH), 8.77 (s, 1H, Ar−H), 8.36 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.66 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.60 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 7.30 (d, 3JHH = 7.2 Hz, 1H, Ar−H), 7.31 (d, 3JHH = 7.2 Hz, 1H, Ar−H), 2.82 (s, 3H, CH3); HRMS (ESI) calcd for C13H11N2O2 (M + H)+ 227.0815, found 227.0811. Synthesis of (1-Methyl-9H-pyrido[3,4-b]indol-3-yl)methanol (17). To a solution of ester 15 (2.00 g, 7.40 mmol) in THF (300 mL) was added LiAlH4 (0.60 g, 15.7 mmol) in portions at 0 °C, and then the mixture was stirred overnight at room temperature. Then the reaction was quenched by water, and methanol (100 mL) was added. After the mixture had been stirred at room temperature for 2 h, the slurry was filtered, the cake was washed with dichloromethane, the filtrate was concentrated to afford 17 as a yellow solid (1.58 g, 95%): mp = 195−197 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H, NH), 8.19 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.95 (s, 1H, Ar−H), 7.56 (d, 3 JHH = 8.0 Hz, 1H, Ar−H), 7.49−7.53 (m, 1H, Ar−H), 7.18−7.22 (m, 1H, Ar−H), 5.30 (t, 3JHH = 6.0 Hz, 1H, OH), 4.67 (d, 3JHH = 6.0 Hz, 2H, CH2OH), 2.73 (s, 3H, CH3). Synthesis of 1-Methyl-9H-pyrido[3,4-b]indole-3-carbaldehyde (18). The mixture of alcohol 17 (1.16 g, 5.47 mmol) and IBX (3.04 g, 10.93 mmol) in DMSO (60 mL) was stirred overnight at room temperature. After water (200 mL) and dichloromethane (50 mL × 3) were added, the separated organic layer was dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and purified by flash chromatography on silica gel using dichloromethane and methanol (v/v = 10:1) as eluent to give 18 as a white solid (0.46 g, 40%): mp = 194−196 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H, NH), 10.07 (s, 1H, CHO), 8.68 (s, 1H, Ar−H), 8.38 (d, 3JHH = 7.6 Hz, 1H, Ar−H), 7.68 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.61 (t, 3JHH = 7.6 Hz, 1H, Ar−H), 7.33 (t, 3JHH = 7.6 Hz, 1H, Ar−H), 2.87 (s, 3H, CH3). Synthesis of (E)-3-(1-Methyl-9H-pyrido[3,4-b]indol-3-yl)acrylic acid (19). To a solution of aldehyde 18 (0.45 g, 2.14 mmol) and piperidine (3 drops) in pyridine was added malonic acid (0.33 g, 3.21 mmol), and the reaction mixture was heated at 120 °C for 4 h. Then the mixture was concentrated in vacuo, water was added (50 mL), the pH was adjusted to 11−12 with an aqueous solution of NaOH, and then the mixture was extracted with diethyl ether. The aqueous phase was adjusted to pH 5−6 with dilute hydrochloric acid (3 M), the yellow slurry was filtered, and the cake was washed with water to afford 19 as a yellow solid (0.51 g, 94%): mp = 220−223 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H, NH), 11.85 (s, 1H, COOH), 8.31 (s, 1H, 1013

dx.doi.org/10.1021/jf404840x | J. Agric. Food Chem. 2014, 62, 1010−1018

Journal of Agricultural and Food Chemistry

Article

Scheme 2. Synthesis of Tetrahydroharmine and 3

Biological Assay. The anti-TMV and fungicidal activities of the synthesized compounds were tested using our previously reported methods.30,31

Scheme 3. Synthesis of Compounds 4−7

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RESULTS AND DISCUSSION

Synthesis. Harmalan and tetrahydroharmane were obtained from tryptamine by Bischler−Napieralski and Pictet−Spengler reaction, respectively. Tetrahydroharmane was oxidized using the Pd/C−maleic acid system in water to give harmane in 60% yield (Scheme 1). In the synthesis of Tetrahydroharmine, harmine was used as starting material. First, harmine was reacted with benzyl bromide to give a quaternary ammonium salt 1 in 74% yield, then it was reduced by sodium borohydride to give tetrahydrocarboline 2 in 88% yield, and finally tetrahydroharmine was obtained from 2 by debenzylation using palladium−carbon and hydrogen. The reaction time of debenzylation should be controlled at