Article pubs.acs.org/JAFC
Small Changes Result in Large Differences: Discovery of (−)-Incrustoporin Derivatives as Novel Antiviral and Antifungal Agents Aidang Lu,*,† Jinjin Wang,‡ Tengjiao Liu,‡ Jian Han,† Yinhui Li,† Min Su,† Jianxin Chen,*,† Hui Zhang,§ Lizhong Wang,§ and Qingmin Wang*,§ †
School of Marine Science and Engineering and ‡School of Chemical Engineering, Hebei University of Technology, Tianjin 300130, China § State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China S Supporting Information *
ABSTRACT: On the basis of the structure of natural product (−)-incrustoporin (1), a series of lactone compounds 4a−i and 5a−i were designed and synthesized from nitroolefin. The antiviral and antifungal activities of these compounds were evaluated in vitro and in vivo. The small changes between 4 and 5 at the 3,4-position result in large differences in bioactivities. Compounds 4 exhibited significantly higher antiviral activity against tobacco mosaic virus (TMV) than dehydro compounds 5. However, the antifungal activity of 4 is relatively lower than that of 5. Compounds 4a, 4c, and 4i with excellent in vivo anti-TMV activity emerged as new antiviral lead compounds. Compounds 5d−g showed superiority over the commercial fungicides chlorothalonil and carbendazim against Cercospora arachidicola Hor at 50 mg kg−1. The present study provides fundamental support for the development and optimization of (−)-incrustoporin derivatives as potential inhibitors of plant virus and pathogenic fungi. KEYWORDS: 2,5-dihydrofuran-2-ones, (−)-incrustoporin derivatives, anti-TMV, antifungal activity
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INTRODUCTION Plant diseases are caused primarily by fungal and bacterial pathogens, leading to severe losses for agriculture and horticulture crop production worldwide, and constitute a great threat to global food security.1 The yield of major food and cash crops was reduced nearly 20% by pathogenic fungi.2 Plant viral diseases, known as “plant cancer”, are serious threats after fungal diseases. They cause a variety of detrimental effects, and usually the infected plants are more susceptible to damage by pests and pathogens. Tobacco mosaic virus (TMV), one of the most well-studied plant viruses, is known to infect more than 400 plant species belonging to 36 families, such as tobacco, tomato, potato, and cucumber. It is found that in certain fields 90−100% of the plants show mosaic or leaf necrosis by harvesting time.3 Many of the currently available antifungal and antiviral agents have several drawbacks, such as pesticide related toxicity, severe pesticide resistance, and serious pesticide interactions.4−7 Therefore, there is a growing need to develop new antifungal agents to effectively control these agricultural diseases. Using natural products as lead compounds to develop new pesticides with novel structures and mechanisms is one of the most effective pesticide design methods.8,9 (−)-Incrustoporin (1, Figure 1), isolated from Incrustoporia carneola (Bres.) Ryv. (Basidiomycetes) by Zapf in 1995,10 is found to possess antifungal activity against a wide array of pathogenic fungi (Candida albicans strains, Candida krusei, Candida glabrata, and so on).11−17 Biological screening has revealed that the endocyclic double bond is necessary for the antifungal effect, and the substitution of the C(3)-aryl ring with halogens could © XXXX American Chemical Society
Figure 1. Design of (−)-incrustoporin derivatives 4a−i and 5a−i.
boost their antifungal activity. The preparation of (−)-incrustoporin and its derivatives has been reported by Mori,18 Rossi,19 Wu,20 Fontes,21 Oh,22 and Fernandes23 successively. Until now, most of the studies about (−)-incrustoporin and its derivatives were focused on antifungal activity against a panel of human pathogenic fungi; there have been no reports about the antiviral activity against TMV and only a few reports about antifungal activity against plant pathogenic fungi.10 In the process of developing new potent plant virus inhibitors, phenanthroindolizidine alkaloids,24 phenanthroquinolizidine alkaloids,25 and β-carboline alkaloids26 have been found to have good anti-TMV activity. As a continued work, 3aryl-5-methyl-γ-butyrolactones 4 and 3-aryl 5-methyl-2,5dihydrofuran-2-ones 5 were designed, synthesized from nitroolefin on the basis of the structure of the natural product (−)-incrustoporin (1). Their antiviral activity against TMV and Received: June 27, 2014 Revised: August 2, 2014 Accepted: August 13, 2014
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dx.doi.org/10.1021/jf503060k | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
(±)-4-(4-Bromophenyl)-5-nitropentan-2-one (3f): white solid; 92% yield; mp 78−80 °C (lit.31 103.4−106.5 °C); 1H NMR δ 2.13 (s, 3 H), 2.89 (d, J = 6.8 Hz, 2 H), 3.98 (quintet, J = 6.8 Hz, 1 H), 4.58 (dd, J = 8.0 and 12.4 Hz, 1 H), 4.67 (dd, J = 6.8 and 12.4 Hz, 1 H), 7.10 (d, J = 7.6 Hz, 2 H), 7.45 (d, J = 7.6 Hz, 2 H); 13C NMR δ 205.0, 137.8, 132.1, 129.1, 121.8, 79.0, 45.8, 38.4, 30.3. (±)-5-Nitro-4-(4-(trifluoromethyl)phenyl)pentan-2-one (3g): yellow oil; 96% yield; 1H NMR δ 2.14 (s, 3 H), 2.93 (d, J = 7.2 Hz, 2 H), 4.09 (quintet, J = 7.2 Hz, 1 H), 4.63 (dd, J = 8.0 and 12.8 Hz, 1 H), 4.71 (dd, J = 6.4 and 12.4 Hz, 1 H), 7.35 (d, J = 8.0 Hz, 2 H), 7.59 (d, J = 8.0 Hz, 1 H); 13C NMR δ 204.7, 143.0, 130.4, and 130.1 (JC−F = 32.6 Hz), 127.9, 126.1 (JC−F = 3.4 Hz), 125.2 and 122.5 (JC−F = 270.4 Hz), 78.9, 45.8, 38.7, 30.3. (±)-4-(Furan-2-yl)-5-nitropentan-2-one (3h): brown solid; 88% yield; mp 47−49 °C (lit.30 48−49 °C); 1H NMR δ 2.15 (s, 3 H), 2.84−2.99 (m, 2 H), 4.08 (quintet, J = 6.8 Hz, 1 H), 4.61−4.70 (m, 2 H), 6.12 (d, 1 H, J = 3.2 Hz), 6.27 (m, 1 H), 7.32 (s, 1 H); 13C NMR δ 205.1, 151.7, 142.3, 110.5, 107.1, 77.0, 43.5, 32.9, 30.2. (±)-5-Nitro-4-(thiophen-2-yl)pentan-2-one (3i): brown solid; 88% yield; mp 35−37 °C (lit.30 37−38 °C); 1H NMR δ 2.17 (s, 3 H), 2.98 (d, J = 7.2 Hz, 2 H), 4.32 (quintet, J = 6.8 Hz, 1 H), 4.63 (dd, J = 7.2 and 12.4 Hz, 1 H), 4.70 (dd, J = 6.4 and 12.4 Hz, 1 H), 6.91−6.95 (m, 2 H), 7.20 (dd, J = 1.2 and 5.2 Hz, 1 H); 13C NMR δ 205.0, 141.5, 127.1, 125.6, 124.7, 79.7, 46.8, 34.5, 30.3. General Synthetic Procedures for Intermediate 4a−i (Diastereomers of 4a−g Shown as A and B, Respectively).32 NaBH4 (0.19 g, 5 mmol) was added to a solution of 3a−i (5 mmol) in MeOH/ CH2Cl2 (v/v = 1:1, 10 mL) at 0 °C. The mixture was stirred for 15 min at the same temperature, and H2O (10 mL) was added. After evaporation of the methanol under reduced pressure, HCl (aq) 1 M (10 mL) was added, and the aqueous phase was extracted with CH2Cl2 (3 × 15 mL). The combined organic layers were dried over MgSO4 and evaporated to afford the expected nitroalcohol, which was redissolved in DMSO (8.5 mL). Sodium nitrite (0.80 g, 12.75 mmol) and acetic acid (1.7 mL, 42.5 mmol) were then added, and the solution was stirred at room temperature overnight and at 40 °C for 6 h. The mixture was allowed to cool, and HCl (aq) 1 M (50 mL) was added. The aqueous phase was extracted with CH2Cl2 (3 × 50 mL). The organic layers were combined, dried over MgSO4, and evaporated under vacuum to afford the γ-butyrolactones 4a−i, which were then purified by flash column chromatography on silica gel (100−200 mesh, petroleum ether/ethyl acetate = 12:1). 3-Phenyl-5-methyltetrahydrofuran-2-one (4a): colorless oil (lit.13 colorless oil); yield 60%; 1H NMR A/B = 1.7:1, A δ 1.43 (d, J = 6.4 Hz, 3 H), 2.28−2.35 (m, 1 H), 2.47−2.54 (m, 1 H), 3.90 (dd, J = 7.2 and 9.2 Hz, 1 H), 4.73−4.81 (m, 1 H), 7.25−7.29 (m, 5 H); B δ 1.46 (d, J = 6.4 Hz, 3 H), 1.93−2.02 (m, 1 H), 2.71−2.77 (m, 1 H), 3.86 (dd, J = 8.8 and 12.8 Hz, 1 H), 4.56−4.61 (m, 1 H), 7.32−7.36 (m, 5 H); 13C NMR A δ 177.3, 137.2, 129.0, 128.2, 127.7, 75.0, 45.7, 37.9, 21.0; B δ 177.0, 136.7, 128.8, 128.2, 127.6, 75.0, 47.7, 39.8, 20.8. 3-(4-Methylphenyl)-5-methyltetrahydrofuran-2-one (4b): white crystals; yield 60%; mp 46−48 °C (lit.13 colorless oil); 1H NMR A/ B = 1.5:1, A δ 1.45 (d, J = 6.4 Hz, 3 H), 2.29−2.36 (m, 1 H), 2.48− 2.55 (m, 1 H), 3.89 (dd, J = 7.6 and 9.2 Hz, 1 H), 4.75−4.83 (m, 1 H), 7.16 (m, 4 H); B δ 1.49 (d, J = 6.0 Hz, 3 H), 1.96−2.04 (m, 1 H), 2.72−2.79 (m, 1 H), 3.85 (dd, J = 7.2 and 11.2 Hz, 1 H), 4.57−4.65 (m, 1 H), 717 (m, 4 H); 13C NMR A δ 177.5, 137.3, 134.0, 129.7, 127.5, 75.2, 45.3, 38.0, 21.1, 21.1; B δ 177.2, 137.3, 133.5, 129.5, 128.0, 75.0, 47.4, 39.9, 21.1, 20.9. 3-(4-Fluorophenyl)-5-methyltetrahydrofuran-2-one (4c): yellow solid; yield 53%; mp 36−38 °C (lit.13 33−35 °C); 1H NMR A/B = 3.7:1, A δ 1.46 (d, J = 6.4 Hz, 3 H), 2.33−2.39 (m, 1 H), 2.48−2.55 (m, 1 H), 3.92 (t, J = 8.8 Hz, 1 H), 4.78−4.83 (m, 1 H), 7.05 (m, 2 H), 7.26 (m, 2 H); B δ 1.51 (d, J = 6.0 Hz, 3 H), 1.95−2.04 (m, 1 H), 2.75−2.82 (m, 1 H), 3.89 (dd, J = 8.8 and 12.4 Hz, 1 H), 4.61−4.66 (m, 1 H), 7.15 (m, 2 H), 7.26 (m, 2 H); 13C NMR A δ 176.9, 160.9, and 163.3 (JC−F = 244.9 Hz), 132.6 and 132.7 (JC−F = 3.2 Hz), 129.2 and 129.3 (JC−F = 8.0 Hz), 115.7 and 115.9 (JC−F = 21.5 Hz), 75.0, 44.7, 37.7, 20.9; B δ 176.6, 160.9, and 163.3 (JC−F = 241.4 Hz), 132.15
antifungal activity against plant pathogenic fungi were systematically investigated for the first time.
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MATERIALS AND METHODS
Instruments. The melting points of the products were determined on an X-4 binocular microscope (Gongyi Yuhua Instrument Co., China) and were not corrected. NMR spectra were acquired with a Bruker 400 MHz (100 MHz for 13C) instrument at room temperature. Chemical shifts were measured relative to residual solvent peaks of CDCl3 as internal standards (1H, δ = 7.26 ppm; 13C, δ = 77.0 ppm). The following abbreviations were used to designate chemical shift multiplicities: s, singlet; d, doublet; t, triplet; m, multiplet; and bs, broad singlet. All first-order splitting patterns were assigned on the basis of multiplet appearance. Splitting patterns that could not be easily interpreted were designated multiplet (m) or broad (br). Enantiomeric excesses were determined with an HP1-100 instrument (chiral column; mobile phase of hexane/2-propanol). HRMS data were recorded with a Varian QFT-ESI instrument. Analytical TLC was performed on silica gel GF 254. Column chromatographic purification was performed using silica gel. All reagents were of analytical reagent grade or chemically pure and purified prior to use when necessary. The β-nitrostyrene 2a−i were prepared according to the literature.27 (S)-3d were prepared according to our previously reported procedure.28 General Experimental Procedures. General Procedures for the Preparation of Intermediate 3a−i.29 To a mixture of CHCl3 (10 mL), Et3N (20−30 mol %), and acetone (40.0 mmol, 3 mL) was added the corresponding nitroolefin (4.0 mmol) followed by (±)-proline (20−30 mol %), and the resulting mixture was stirred at room temperature until TLC indicated the reaction was complete. The reaction mixture was evaporated under vacuum, and the residue was dissolved in CH2Cl2 (30 mL). The mixture was treated with 1 M HCl (20 mL) solution, the layers were separated, and the aqueous layer was extracted several times with CH2Cl2, dried with anhydrous MgSO4, and evaporated. The pure products 3a−g were obtained by recrystallization from methanol. 3h and 3i were purified by column chromatography on silica gel (100−200 mesh, petroleum ether/ethyl acetate = 5:1). (±)-5-Nitro-4-phenylpentan-2-one (3a): white solid; 92% yield; mp 110−112 °C (lit.30 110−112 °C); 1H NMR δ 2.12 (s, 3 H), 2.92 (d, J = 6.8 Hz, 2 H), 4.01 (quintet, J = 7.2 Hz, 1 H), 4.60 (dd, J = 8.0 and 12.4 Hz, 1 H), 4.70 (dd, J = 6.8 and 12.4 Hz, 1 H), 7.22 (d, J = 7.6 Hz, 2 H), 7.27−7.35 (m, 3 H); 13C NMR δ 205.4, 138.8, 129.0, 127.9, 127.3, 79.4, 46.1, 40.0, 30.4. (±)-5-Nitro-4-(p-tolyl)pentan-2-one (3b): white solid; 93% yield; mp 68−70 °C (lit.30 70−72 °C); 1H NMR δ 2.11 (s, 3 H), 2.31 (s, 3 H), 2.89 (d, J = 6.8 Hz, 2 H), 3.97 (quintet, J = 7.2 Hz, 1 H), 4.58 (dd, J = 7.6 and 12.4 Hz, 1 H), 4.66 (dd, J = 7.2 and 12.4 Hz, 1 H), 7.09 (d, J = 8.0 Hz, 2 H), 7.13 (d, J = 8.0 Hz, 2 H); 13C NMR δ 205.5, 137.6, 135.7, 129.7, 127.2, 79.6, 46.2, 38.7, 30.4, 21.0. (±)-4-(4-Fluorophenyl)-5-nitropentan-2-one (3c): white solid; 97% yield; mp 100−103 °C (lit.31 102.0−104.9 °C); 1H NMR δ 2.12 (s, 3 H), 2.89 (d, J = 6.8 Hz, 2 H), 3.40 (quintet, J = 7.2 Hz, 1 H), 4.58 (dd, J = 7.6 and 12.0 Hz, 1 H), 4.66 (dd, J = 6.4 and 12.4 Hz, 1 H), 6.99−7.03 (m, 2 H), 7.17−7.20 (m, 1 H); 13C NMR δ 205.1, 163.4, and 161.0 (JC−F = 245.2 Hz), 134.6 and 134.6 (JC−F = 3.2 Hz), 129.1 and 129.0 (JC−F = 7.9 Hz), 116.1 and 115.9 (JC−F = 21.5 Hz), 79.4, 46.2, 38.3, 30.4. (±)-4-(4-Chlorophenyl)-5-nitropentan-2-one (3d): white solid; 93% yield; mp 75−77 °C (lit.30 89−91 °C); 1H NMR δ 2.13 (s, 3 H), 2.89 (d, J = 7.2 Hz, 2 H), 3.99 (quintet, J = 7.2 Hz, 1 H), 4.58 (dd, J = 8.0 and 12.4 Hz, 1 H), 4.66 (dd, J = 6.8 and 12.4 Hz, 1 H), 7.16 (d, J = 8.4 Hz, 2 H), 7.30 (d, J = 8.4 Hz, 2 H); 13C NMR δ 205.0, 137.3, 133.8, 129.3, 128.8, 79.2, 46.0, 38.4, 30.4. (±)-4-(2,4-Dichlorophenyl)-5-nitropentan-2-one (3e): white solid; 83% yield; mp 52−54 °C (lit.30 48−50 °C); 1H NMR δ 2.17 (s, 3 H), 2.91−3.08 (m, 2 H), 4.40 (quintet, J = 6.4 Hz, 1 H), 4.73 (d, J = 6.4 Hz, 2 H), 7.14 (d, J = 8.0 Hz, 1 H), 7.22 (d, J = 8.0 Hz, 1 H), 7.42 (s, 1 H); 13C NMR δ 205.0, 134.6, 134.4, 134.2, 130.2, 129.3, 127.7, 77.1, 44.2, 35.3, 30.2. B
dx.doi.org/10.1021/jf503060k | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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lactone (4a−i) (10.0 mmol) in THF (5 mL) was added. The reaction temperature was maintained at −60 °C for 30 min, and a solution of phenylselenenyl chloride (15.0 mmol) in THF (10 mL) was added. The resultant mixture was slowly warmed to room temperature, diluted with ethyl acetate (50 mL), washed with saturated aqueous NH4Cl, dried over Na2SO4, and concentrated. The crude phenylselanyl derivative was rapidly purified by column chromatography and dissolved in CHCl3 (20 mL), and then m-CPBA (15.0 mmol) was added to the solution in several portions at 0 °C. The reaction mixture was then stirred for 2 h at room temperature or until TLC indicated the reaction was complete. A further portion of CHCl3 (10 mL) was added along with 5% aqueous Na2CO3 (20 mL). The layers were separated, and the organic phase was washed with 5% aqueous Na2CO3 (20 mL), dried over Na2SO4, and evaporated. Products 5a−i were purified by column chromatography on silica gel (100−200 mesh, petroleum ether/ethyl acetate = 15:1) and were obtained with overall yields in the range 31−51% over two steps. 3-Phenyl-5-methyl-2,5-dihydrofuran-2-one (5a): white solid; yield 40%; mp 49−51 °C (lit.13 45−46 °C); 1H NMR δ 1.52 (d, J = 6.8 Hz, 3 H), 5.15 (qd, J = 6.4 and 13.2 Hz, 1 H), 7.40−7.43 (m, 3 H), 7.55 (s, 1 H), 7.85 (d, J = 6.4 Hz, 2 H); 13C NMR δ 171.8, 149.2, 131.3, 129.5, 129.3, 128.7, 127.1, 76.8, 19.2; HRMS (ESI) calcd for C11H10O2 (M + H)+ 175.0754, found 175.0756. 3-(4-Methylphenyl)-5-methyl-2,5-dihydrofuran-2-one (5b): white solid; yield 39%; mp 53−54 °C (lit.13 55−57 °C); 1H NMR δ 1.50 (d, J = 6.8 Hz, 3 H), 2.37 (s, 3 H), 5.14 (qd, J = 6.8 and 13.6 Hz, 1 H), 7.22 (d, J = 8.0 Hz, 2 H), 7.49 (s, J = 2.0 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H); 13C NMR δ 171.9, 148.1, 139.4, 131.2, 129.4, 126.9, 126.7, 76.7, 21.4, 19.2; HRMS (ESI) calcd for C12H12O2 (M + H)+ 189.0910, found 189.0912. 3-(4-Fluorophenyl)-5-methyl-2,5-dihydrofuran-2-one (5c): white solid; yield 32%; mp 71−73 °C (lit.13 81−83 °C); 1H NMR δ 1.52 (d, 3 H, J = 6.8 Hz), 5.16 (m, 1 H), 7.08−7.13 (m, 2 H), 7.51 (d, 1 H, J = 2.0 Hz), 7.85−7.88 (m, 2 H); 13C NMR δ 171.6, 164.5, and 162.1 (JC−F = 248.1 Hz), 148.5, 130.4, 129.1, and 129.0 (JC−F = 8.3 Hz), 125.69 and 125.66 (JC−F = 2.9 Hz), 115.8 and 115.6 (JC−F = 21.6 Hz), 76.7, 19.1; HRMS (ESI) calcd for C11H9FO2 (M + H)+ 193.0659, found 193.0661. 3-(4-Chlorophenyl)-5-methyl-2,5-dihydrofuran-2-one (5d): white solid; yield 31%; mp 101−102 °C (lit.13 99−101 °C); 1H NMR δ 1.51 (d, J = 6.8 Hz, 3 H), 5.15 (qd, J = 6.4 and 13.2 Hz, 1 H), 7.36 (d, J = 8.0 Hz, 2 H), 7.56 (s, 1 H), 7.80 (d, J = 8.4 Hz, 1 H); 13C NMR δ 171.5, 149.5, 135.26, 130.1, 128.9, 128.4, 128.0, 76.9, 19.1; HRMS (ESI) calcd for C11H9ClO2 (M + H)+ 209.0364, found 209.0362. (R)-3-(4-Chlorophenyl)-5-methyl-2,5-dihydrofuran-2-one (5d): white solid; yield 51%; [α]20 D = −15.5° (c 1.0, CHCl3); mp 38−40 °C; 1H NMR δ 1.51 (d, J = 6.4 Hz, 3 H), 5.15 (qd, J = 6.4 and 12.8 Hz, 1 H), 7.38 (d, J = 7.6 Hz, 2 H), 7.55 (s, 1 H), 7.81 (d, J = 7.6 Hz, 1 H); 13C NMR δ 171.4, 149.2, 135.4, 130.3, 128.9, 128.4, 127.9, 76.8, 19.1; HRMS (ESI) calcd for C11H9ClO2 (M + H)+ 209.0364, found 209.0367; HPLC analysis (Chiralpak OJ-H column, hexane/2propanol = 98:2, flow rate = 1.0 mL/min, wavelength = 254 nm) tR = 57.23 (minor) and 68.66 min (major), 56% ee. 3-(2,4-Dichlorophenyl)-5-methyl-2,5-dihydrofuran-2-one (5e): yellow oil; yield 42%; 47−49 °C; 1H NMR δ 1.55 (d, J = 7.2 Hz, 3 H), 5.23 (qd, J = 6.8 and 13.6 Hz, 1 H), 7.32 (dd, J = 6.4 and 8.4 Hz, 1 H), 7.47 (d, J = 2.0 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 1 H), 7.78 (d, J = 1.2 Hz, 1 H); 13C NMR δ 171.4, 154.9, 135.3, 133.9, 131.6, 130.0, 128.3, 127.3, 126.9, 77.4, 19.0; HRMS (ESI) calcd for C11H8Cl2O2 (M + H)+ 242.9974, found 242.9975. 3-(4-Bromophenyl)-5-methyl-2,5-dihydrofuran-2-one (5f): white solid; yield 44%; mp 103−104 °C (lit.13 105−106 °C); 1H NMR δ 1.52 (d, J = 6.8 Hz, 3 H), 5.15 (qd, J = 6.8 and 13.6 Hz, 1 H), 7.54− 7.56 (m, 3 H), 7.75 (d, J = 8.4 Hz, 2 H); 13C NMR δ 171.4, 149.4, 131.9, 130.4, 128.6, 128.4, 123.7, 76.9, 19.1; HRMS (ESI) calcd for C11H9BrO2 (M + H)+ 252.9859, found 252.9854. 3-(4-Trifluoromethylphenyl)-5-methyl-2,5-dihydrofuran-2-one (5g): white solid; yield 43%; mp 88−90 °C (lit.13 91−93 °C); 1H NMR δ 1.55 (d, J = 6.8 Hz, 3 H), 5.20 (qd, J = 6.8 and 13.6 Hz, 1 H), 7.66 (s, 1 H), 7.67 (d, J = 8.8 Hz, 2 H), 8.00 (d, J = 8.1 Hz, 2 H); 13C
and 132.18 (JC−F = 3.1 Hz), 129.6 and 129.7 (JC−F = 8.1 Hz), 115.5 and 115.8 (JC−F = 21.5 Hz), 74.9, 46.9, 36.7, 20.7. 3-(4-Chlorophenyl)-5-methyltetrahydrofuran-2-one (4d): white crystals; yield 68%; mp 40−42 °C (lit.13 38−40 °C); 1H NMR A/B = 1:1.1, A δ 1.46 (d, J = 6.4 Hz, 3 H), 2.32−2.39 (m, 1 H), 2.47− 2.54(m, 1 H), 3.91 (t, J = 8.4 Hz, 1 H), 4.76−4.83 (m, 1 H), 7.21 (d, J = 3.6 Hz, 2 H), 7.33 (d, J = 8.4 Hz, 2 H); B δ 1.50 (d, J = 6.0 Hz, 3 H), 1.94−2.03 (m, 1 H), 2.74−2.81 (m, 1 H), 3.87 (dd, J = 8.4 and 12.4 Hz, 1 H), 4.58−4.67 (m, 1 H), 7.23 (d, J = 3.6 Hz, 2 H), 7.33 (d, J = 8.4 Hz, 2 H); 13C NMR A δ 177.4, 136.1, 134.3, 129.8, 129.8, 75.8, 45.6, 38.4, 21.7; B δ 177.1, 135.6, 134.3, 130.2, 129.7, 75.7, 47.7, 40.3, 21.5. (3S,5R)-3-(4-Chlorophenyl)-5-methyltetrahydrofuran-2-one ((3S,5R)-4d): white crystals; yield 55%; mp 38−40 °C; 1H NMR δ 1.47 (d, J = 6.4 Hz, 3 H), 2.32−2.39 (m, 1 H), 2.48−2.55 (m, 1 H), 3.91 (t, J = 9.2 Hz, 1 H), 4.76−4.84 (m, 1 H), 7.22 (d, J = 8.4 Hz, 2 H), 7.33 (d, J = 8.4 Hz, 2 H); 13C NMR δ 176.6, 135.4, 133.6, 129.1, 129.1, 75.1, 44.9, 37.7, 21.0. (3S,5S)-3-(4-Chlorophenyl)-5-methyltetrahydrofuran-2-one ((3S,5S)-4d): white crystals; yield 63%; mp 38−40 °C; 1H NMR δ 1.51 (d, J = 6.4 Hz, 3 H), 1.94−2.04 (m, 1 H), 2.75−2.81 (m, 1 H), 3.88 (dd, J = 8.4 and 12.8 Hz, 1 H), 4.59−4.68 (m, 1 H), 7.23 (d, J = 8.4 Hz, 2 H), 7.34 (d, J = 8.4 Hz, 2 H); 13C NMR δ 176.4, 134.9, 133.6, 129.5, 129.0, 75.0, 47.0, 39.6, 20.8. 3-(2,4-Dichlorophenyl)-5-methyltetrahydrofuran-2-one (4e): yellow solid; yield 64%; mp 41−43 °C (lit.13 45−47 °C); 1H NMR A/B = 3:1, A δ 1.48 (d, J = 6.4 Hz, 3 H), 2.39−2.43 (m, 2 H), 4.27 (t, J = 9.2 Hz, 1 H), 4.76−4.84 (m, 1 H), 7.19 (d, J = 8.4 Hz, 1 H), 7.27 (d, J = 7.6 Hz, 1 H), 7.43 (s, 1 H); B δ 1.51 (d, J = 6.4 Hz, 3 H), 1.87−1.96 (m, 1 H), 2.81−2.87 (m, 1 H), 4.26 (t, J = 9.2 Hz, 1 H), 4.66−4.71 (m, 1 H), 7.23−7.28 (m, 2 H), 7.42 (s, 1 H); 13C NMR A δ 176.2, 134.7, 134.2, 134.0, 130.1, 129.8, 127.7, 75.1, 43.4, 37.1, 21.1; B δ 175.7, 134.8, 134.2, 133.4, 130.6, 129.7, 127.8, 75.3, 45.5, 38.9, 20.8. 3-(4-Bromophenyl)-5-methyltetrahydrofuran-2-one (4f): yellow solid; yield 52%; mp 47−49 °C (lit.13 52−53 °C); 1H NMR A/B = 1:1.3, A δ 1.44 (d, J = 6.4 Hz, 3 H), 2.30−2.37 (m, 1 H), 2.45−2.52 (m, 1 H), 3.88 (t, J = 8.8 Hz, 1 H), 4.73−4.81 (m, 1 H), 7.14 (d, J = 2.8 Hz, 2 H), 7.47 (d, J = 8.4 Hz, 2 H); B δ 1.48 (d, J = 6.0 Hz, 3 H), 1.91−2.00 (m, 1 H), 2.72−2.78 (m, 1 H), 3.84 (dd, J = 8.4 and 12.4 Hz, 1 H), 4.56−4.61 (m, 1 H), 7.17 (d, J = 2.8 Hz, 2 H), 7.47 (d, J = 8.4 Hz, 2 H); 13C NMR A δ 176.6, 136.0, 132.0, 129.4, 121.6, 75.1, 44.9, 37.6, 21.0; B δ 176.3, 135.5, 131.9, 129.8, 121.6, 75.0, 47.1, 39.5, 20.8. 3-(4-Trifluoromethylphenyl)-5-methyltetrahydrofuran-2-one (4g): white crystals; yield 65%; mp 75−77 °C (lit.13 88−89 °C); 1H NMR A/B = 1:5.5, A δ 1.49 (d, J = 6.4 Hz, 3 H), 2.37−2.46 (m, 1 H), 2.53−2.60 (m, 1 H), 4.00 (dd, J = 8.8 and 12.8 Hz, 1 H), 4.81−4.85 (m, 1 H), 7.42−7.44 (m, 2 H), 7.62−7.64 (m, 2 H); B δ 1.53 (d, J = 6.0 Hz, 3 H), 1.99−2.08 (m, 1 H), 2.79−2.85 (m, 1 H), 3.97 (dd, J = 8.8 and 12.8 Hz, 1 H), 4.63−4.71 (m, 1 H), 7.42−7.44 (m, 2 H), 7.62−7.64 (m, 2 H); 13C NMR A δ 176.4, 140.9, 130.5, and 129.5 (JC−F = 109.1 Hz), 128.2, 126.0 (JC−F = 3.4 Hz), 125.3, 75.2, 45.3, 37.5, 21.0; B δ 176.1, 140.4, 130.1, and 129.8 (JC−F = 32.5 Hz), 128.6, 125.8 (JC−F = 3.3 and JC−F = 7.3 Hz), 125.4 and 122.7 (JC−F = 270.7 Hz), 75.2, 47.4, 39.5, 20.8. 3-(Furan-2-yl)-5-methyltetrahydrofuran-2-one (4h): yellow oil; yield 43%; 1H NMR δ 1.45 (d, J = 6.4 Hz, 3 H), 2.20−2.27 (m, 1 H), 2.60−2.66 (m, 1 H), 3.99 (dd, J = 6.0 and 9.2 Hz, 1 H), 4.80−4.88 (m, 1 H), 6.27 (d, J = 3.2 Hz, 1 H), 6.34 (dd, J = 1.6 and 3.2 Hz, 1 H), 7.37 (d, 1 H, J = 1.6 Hz); 13C NMR δ 174.7, 149.4, 142.6, 110.6, 107.5, 76.0, 40.4, 35.4, 20.9. 3-(Thiophen-2-yl)-5-methyltetrahydrofuran-2-one (4i): yellow oil; yield 52%; 1H NMR δ 1.49 (d, J = 6.0 Hz, 3 H), 2.05−2.14 (m, 1 H), 2.84−2.91 (m, 1 H), 4.14 (dd, J = 8.8 and 12.8 Hz, 1 H), 4.60−4.68 (m, 1 H), 6.99−7.02 (m, 2 H), 7.25−7.27 (m, 1 H); 13C NMR δ 175.5, 138.2, 127.0, 125.8, 125.1, 75.2, 42.8, 39.6, 20.8. General Procedures for the Preparation of Furanones 5a−5i.13 Butyllithium (11.0 mmol) was added to a solution of diisopropylamine (10.6 mmol) in dry THF (30 mL) at 0 °C under N2. After 30 min, the LDA solution was cooled to −60 °C, and a solution of a saturated C
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NMR δ 171.14, 150.96, 132.91, 131.3, and 130.9 (d, JC−F = 32.4 Hz), 130.3, 127.4, 125.6 (q, JC−F = 3.2 and JC−F = 7.1 Hz), 125.3, 77.0, 19.0; HRMS (ESI) calcd for C12H9F3O2 (M + H)+ 243.0627, found 243.0624. 3-(Furan-2-yl)-5-methyl-2,5-dihydrofuran-2-one (5h): colorless oil; yield 36%; 1H NMR δ 1.49 (d, J = 6.4 Hz, 3 H), 5.18 (qd, J = 6.4 and 12.8 Hz, 1 H), 6.47 (s, 1 H), 7.13 (s, 1 H), 7.41(s, 1 H), 7.47 (s, 1 H); 13C NMR δ 170.1, 145.3, 144.2, 143.6, 123.0, 111.8, 111.1, 77.1, 19.2; HRMS (ESI) calcd for C9H8O3 (M + H)+ 165.0546, found 165.0542. 3-(Thiophen-2-yl)-5-methyl-2,5-dihydrofuran-2-one (5i): colorless oil; yield 38%; 1H NMR δ 1.51 (d, J = 6.8 Hz, 3 H), 5.17 (qd, J = 6.8 and 13.6 Hz, 1 H), 7.09 (t, J = 3.6 Hz, 1 H), 7.38 (d, J = 5.2 Hz, 1 H), 7.75 (d, J = 3.2 Hz, 1 H); 13C NMR δ 170.9, 145.2, 131.5, 127.8, 127.5, 127.2, 126.3, 77.4, 19.2; HRMS (ESI) calcd for C9H8SO2 (M + H)+ 181.0318, found 181.0316. Biological Assay. All bioassays were performed on representative test organisms reared in the laboratory. Percentage mortalities were evaluated according to a percentage scale of 0−100, in which 0 indicates no activity and 100 indicates total kill. 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 those reported previously in the literature.24,25 Antifungal Biological Assay. The detailed assay method to test the antifungal activity of the synthesized compounds was described in the literature.33
Figure 3. ORTEP drawing of compound (3S,5R)-4d (CCDC 1009910).
conditions from (3S,5R)-4d, and (R)-5d was obtained with enantioselectivities (16% ee) from (3S,5S)-4d. Phytotoxic Activity. Compounds 4a−i and 5a−i were first tested for their phytotoxic activity against the test plant. The results indicated that compounds 4a−i showed no phytotoxic activity at 500 μg mL−1. However, dehydro compounds 5a−g displayed phytotoxic activity to tobacco (Nicotiana tabacum var. Xanthi nc), so the antiviral activity of 5a−g was not tested at 500 μg mL−1. Antiviral Activity. The anti-TMV activity of compounds 4a−i and 5a−i was tested. The commercial plant virucide ribavirin was used as the control. In Vitro Anti-TMV Activity. The in vitro antiviral assay results of all of the synthesized compounds are listed in Table 1. As the control, ribavirin exhibited a 41% inhibitory effect at 500 μg mL−1, whereas most of the synthesized compounds 4a−i exhibited higher antiviral activity than ribavirin; especially compound 4i with 61% inhibitory effect emerged as a new lead compound for anti-TMV research. When the concentration was lowered to 100 μg mL−1, compounds 4c, (3S,5R)-4d, 4i, and 5i displayed nearly equal or higher inhibitory effect than that of ribavirin. To investigate the influence of chirality at C(5) and C(3) on the anti-TMV activity, compounds (3S,5R)-4d, (3S,5S)-4d, and (3RS, 5RS)-4d (or 4d for short) were tested, which exhibited 48, 43, and 40% inhibitory effect at 500 μg mL−1, respectively. The bioassay results also showed that the (3S, 5R) configuration is the preferred antiviral configuration for 3-aryl-5-methyl-γ-butyrolactone [(3S,5R)-4d > (3S,5S)-4d > 4d]. As shown in Table 1, most of the compounds 4a−i displayed good in vitro activity against TMV; however, almost all of the compounds 5a−i displayed phytotoxic activity against the test plant at 500 μg mL−1. Therefore, compounds 4a−i were bioassayed further to investigate their antiviral activity in vivo. In Vivo Anti-TMV Activity. As shown in Table 2, most of the compounds also displayed higher in vivo activity than that of the ribavirin. 4i showed the best in vivo anti-TMV activity (inactivation activity = 52% at 500 μg mL−1 and 20% at 100 μg mL−1; curative activity = 57% at 500 μg mL−1 and 30% at 100 μg mL−1; and protection activity = 56% at 500 μg mL−1 and 28% at 100 μg mL−1), which is significantly higher than that of ribavirin (inactivation activity = 40% at 500 μg mL−1 and 12% at 100 μg mL−1; curative activity = 37% at 500 μg mL−1 and 14% at 100 μg mL−1; and protection activity = 40% at 500 μg mL−1 and 17% at 100 μg mL−1). Unlike in vitro antiviral assay results, 4d displayed higher in vivo activity than that of (3S,5R)-4d and (3S,5S)-4d (in vivo activity: 4d > (3S,5R)-4d > (3S,5S)-4d), except for the protection effect of (3S,5R)-4d.
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RESULTS AND DISCUSSION Chemistry. Synthesis of Compounds 5a−i. Compounds 5a−i were synthesized according to procedures in Figure 2.
Figure 2. Synthesis of (−)-incrustoporin derivatives 4a−i and 5a−i.
Under the catalysis of proline, acetone reacted with different nitroolefins to give corresponding adducts 3a−i in high yield (83−97%). Via reactions of reduction, oxidation, and intramolecular esterification successively, 3a−i were converted to corresponding 3-aryl-5-methyl-γ-butyrolactone 4a−i.32 Finally, the double bond was introduced by enolization of the lactones 4a−i followed by treatment with phenylselenenyl chloride, oxidation, and spontaneous selenoxide elimination to yield the target compounds 5a−i.13 Synthesis of the Compound (R)-5d. To explore the effect of chirality at C(5) on biological activity, (R)-5d was prepared from (S)-3d by using the method shown in Figure 2. (3S,5RS)4, obtained via reactions of reduction, oxidation, and intramolecular esterification of (S)-3d, were separated by column chromatography to give (3S,5R)-4d and (3S,5S)-4d with excellent diastereoselective (trans/cis = 23:1 and 1:15, respectively) determined by 1H NMR analysis. Their absolute configurations were confirmed by spectroscopic data (1H NMR, 13C NMR) and single-crystal X-ray diffraction analysis (Figure 3). Unfortunately, (R)-5d was obtained with moderate enantioselectivities (56% ee) under the above reaction D
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Table 1. In Vitro Antiviral Activity of Compounds 4a−i and 5a−i against TMV concn (μg mL−1)
compd
500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100
4a 4b 4c 4d (3S,5R)-4d (3S,5S)-4d 4e 4f 4g 4h 4i a
inhibition ratea (%) 46 8 38 0 53 16 40 5 48 20 43 0 45 0 46 0 43 0 35 0 61 27
compd
±1 ±1 ±2 ± ± ± ± ± ± ±
5a 5b
1 2 2 1 2 1 2
5e
±1
5f
±2
5g
±1
5h
±2
5i
±1 ±1
ribavirin
5c 5d (R)-5d
inhibition ratea (%)
500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100
NT 0 NT 5±1 NT 9±1 NT 0 NT 6±1 NT 0 NT 0 NT 0 37 ± 1 0 38 ± 2 10 ± 1 41 ± 2 10 ± 1
Average of three replicates. All results are expressed as mean ± SD. NT, not tested.
4a. However, 4-fluorophenyl-substituted compound 4c gave relatively higher activity (48, 57, and 60% at 500 μg mL−1). Compound 4h containing a furan ring at C(3) exhibited a lower activity than the others, whereas 4i containing a thiofuran ring was the optimal compound for anti-TMV research. Fungicidal Activity. The fungicidal activities of compounds 4a−i and 5a−i were investigated. The commercial fungicides chlorothalonil, carbendazim, and azoxystrobin were used as the controls. In Vitro Fungicidal Activity. The target compounds 5 and 4 were evaluated for their in vitro antifungal activity on 14 kinds of plant fungi at 50 mg kg−1 (Table 3). Unlike the antiviral activity, saturated lactones 4 displayed moderate fungicidal activity, whereas most of the unsaturated lactones 5 exhibited excellent fungicidal activity against the tested fungi, which indicated that the small changes between 4 and 5 at the 3,4position result in large differences in bioactivities. Compound 5g, containing a trifluoromethyl group at the 4-position of phenyl ring, exhibited broad-spectrum and good fungicidal activity against 13 kinds of fungi. Compounds 5d−g exhibited higher fungicidal activity than chlorothalonil and carbendazim against C.H (Cercospora arachidicola Hori) at 50 mg kg−1. Some of these compounds selectively exhibited fungicidal activity to certain fungi. For example, the inhibitory effect of compound 5b (against Rhizoctonia solani), compound 5c−d (against Sclerotinia sclerotiorum), and 5d−g (against Cercospora arachidicola Hori, Rhizoctonia cerealis, Rhizoctonia solani, Botrytis cinerea) were >90%, which is much higher than that for other fungi. The fungicidal activities of compound 5b (against Rhizoctonia solani) and compounds 5d−g (against Cercospora arach-idicola Hori, Rhizoctonia cerealis, Rhizoctonia solani, Botrytis cinerea) were also excellent at 25 mg kg−1 (>70%, except for 5g against Rhizoctonia cerealis). Compounds (R)-5d and 5g exhibited >50% inhibition rate against Rhizoctonia solani at 6.25 mg kg−1 (Table 4).
Table 2. In Vivo Antiviral Activity of Compounds 4a−i against TMV compd 4a 4b 4c 4d (3S,5R)-4d (3S,5S)-4d 4e 4f 4g 4h 4i ribavirin a
concn (μg mL−1)
concn (μg mL−1)
inactivation effecta (%)
curative effecta (%)
protection effecta (%)
500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100
50 ± 1 19 ± 3 35 ± 1 0 48 ± 2 25 ± 2 43 ± 1 15 ± 1 42 ± 1 13 ± 1 30 ± 2 0 38 ± 2 0 46 ± 1 14 ± 2 42 ± 1 0 31 ± 2 0 52 ± 2 20 ± 1 40 ± 2 12 ± 1
55 ± 2 20 ± 1 33 ± 3 0 57 ± 1 27 ± 1 51 ± 2 19 ± 1 40 ± 3 9±1 41 ± 1 0 34 ± 2 0 43 ± 1 9±2 35 ± 1 0 39 ± 2 11 ± 2 57 ± 1 30 ± 2 37 ± 1 14 ± 1
58 ± 1 22 ± 1 47 ± 1 0 60 ± 2 21 ± 1 47 ± 1 11 ± 1 50 ± 1 18 ± 1 46 ± 2 10 ± 1 44 ± 1 0 34 ± 1 0 39 ± 2 16 ± 1 47 ± 1 0 56 ± 2 28 ± 1 40 ± 2 17 ± 1
Average of three replicates. All results are expressed as mean ± SD.
Introduction of a methyl or trifluoromethyl group at the 4position of the phenyl ring dramatically decreased inhibition effect (in vivo activity: 4a > 4g > 4b). Compounds 4d−f containing halogen atoms such as chlorine and bromine on the phenyl ring also exhibited lower inhibitory effect than that of E
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F
8 15 23 23 23 23 31 46 8 8 8 31 46 54 85 100 100 92 100 0 8 73
