Research Article Received: 10 February 2015
Revised: 14 May 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library: 6 July 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4049
Synthesis and insecticidal activity of 𝜷-dihydroagarofuran ether analogues Ximei Zhao,a† Zhan Hu,a† Jian Li,a Longbo Li,a Wenjun Wub and Jiwen Zhanga,b* Abstract BACKGROUND: 1𝜷, 2𝜷, 4𝜶, 6𝜶, 8𝜷, 9𝜶, 12-hepthydroxyl-𝜷-dihydroagarofuran is the main skeleton of 𝜷-dihydroagarofuran sesquiterpenoids, which exhibit excellent insecticidal activity. To study further the structure–activity relationship of 𝜷-dihydroagarofuran sesquiterpenoids towards finding novel botanical pesticides, two series of new structurally modified ether analogues were designed and synthesised, and their insecticidal activities were evaluated. RESULTS: Twenty-two ether derivatives were synthesised using 1𝜷, 2𝜷, 4𝜶, 6𝜶, 8𝜷, 9𝜶, 12-hepthydroxyl-𝜷-dihydroagarofuran as starting material. Bioassay results indicated that most of the derivatives, particularly compounds 5.1.2, 5.1.3, 5.1.7, 5.2.3, 5.2.6 and 5.2.7, exhibited significant insecticidal activity against the third-instar larvae of the oriental armyworm Mythimna separata. Most importantly, compound 5.2.7 showed the lowest LD50 value of 29.2 𝛍g g−1 among these synthesised compounds, which provides some important hints for further design, synthesis and structural modification of 𝜷-dihydroagarofuran sesquiterpenoids towards developing novel botanical insecticides. CONCLUSION: The structure–activity relationship illustrated that the moiety at the 1-position affected the insecticidal activity significantly, and that specifically the derivatives with two or three carbon atoms at the 1-position showed promising insecticidal activity, with mortality over 60%, while those with o-F-Bn and p-F-Bn at the 6-position showed similar activity. © 2015 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: dihydroagarofuran; sesquiterpenoid; insecticidal activity; structure–activity relationship
1
INTRODUCTION
754
Celastrus angulatus Maxim is a significant bioactive plant in China. Plant extracts from the Celastraceae family have been traditionally utilised as insecticides, as insect repellents and for the treatment of various diseases such as fever, cancer, rheumatoid arthritis and a wide range of gastrointestinal diseases.1 – 4 It has been reported that the main biologically active components of these extracts are various 𝛽-dihydroagarofuran sesquiterpenoids, which have been characterised by a 𝛽-dihydroagarofuran skeleton.5 – 8 Previous studies have shown that 𝛽-dihydroagarofuran sesquiterpene polyol esters extracted from Celastrus angulatus exhibit excellent insecticidal activity against the oriental armyworm Mythimna separata Walker,5,6,9 and that 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) forms the main framework of these 𝛽-dihydroagarofuran sesquiterpenoids. In our previous work, 1𝛽, 4𝛼, 6𝛼, 9𝛼-tetrahydroxyl-2𝛽, 12-ether-𝛽-dihydroagarofuran (2), together with twelve derivatives, eleven with the same substituent group at the 1- and 6-positions, and one with the same substituent group at the 6and 9-positions, has been synthesised, with 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) as the starting material. Bioassay results indicated that the derivatives with o-F-Bn and p-F-Bn moieties exhibited excellent insecticidal activity, with insect mortality far exceeding that of the positive control celangulin-V (Fig. 1).10 To study further the structure–activity Pest Manag Sci 2016; 72: 754–759
relationship of 𝛽-dihydroagarofuran sesquiterpenoids towards finding novel botanical pesticides, in this paper we describe the design, synthesis and bioactivity evaluation of 22 fluorinated ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) from 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) as the starting material.
2
MATERIALS AND METHODS
2.1 General Melting points were measured on an WRS-1B melting point apparatus (Shanghai YiCe Apparatus and Equipment Co., Ltd, Shanghai, China) and are uncorrected; specific rotations were determined on a Perkin-Elmer 241 MC automatic polarimeter (PerkinElmer, Waltham, MA) and are uncorrected; all the 1 H NMR and 13 C NMR spectra were recorded on a Bruker Advance spectrometer (Bruker,
∗
Correspondence to: Jiwen Zhang, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China, E-mail: [email protected]
† These authors contributed equally to this work. a College of Science, Northwest A&F University, Yangling, Shaanxi, China b Institute of Pesticide Science, Northwest A&F University, Yangling, Shaanxi, China
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Synthesis and insecticidal activity of 𝛽-dihydroagarofuran ether analogues
O
O
O O
O
O O
pressure and the residue was extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 1:2 ) to give 450 mg (99%) of product 3.
O O
O O OH OH Chemical structure of celangulin-¢õ
Figure 1. The chemical structure of celangulin-V.
Billerica, MA) at 500 MHz and 125 MHz in CDCl3 with TMS as the reference; distortionless enhancement by polarisation transfer (DEPT) spectra (flip angle 135∘ ) were obtained to determine the assignments of 13 C chemical shifts. The starting material 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) (purity ≥98%) was provided by the Institute of Pesticide Science, Northwest A & F University; thin-layer chromatography (TLC) analysis was carried out on GF254 silica gel plates, and the spots were coloured by an ultraviolet lamp or 5% (V/V) sulfuric acid in ethyl alcohol; the synthetic products were purified by column chromatography with silica gel (zcx II, 200–300 mesh) and mixtures of petroleum ether and ethyl acetate as the eluent. The bioassay was carried out by the leaf-disc method at a concentration of 40 mg mL−1 . Celangulin-V and acetone were used as positive and negative controls respectively. 2.2 Synthesis of 1𝜷, 4𝜶, 6𝜶, 9𝜶-tetrahydroxyl-2𝜷, 12-ether-𝜷-dihydroagarofuran (2) 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) (3.34 g, 10 mmol) was dissolved in 10 mL of anhydrous pyridine. Freshly distilled CH3 SO2 Cl (6 mL, 72 mmol) was added dropwise to the mixture under Ar, and then the reaction mixture was stirred at room temperature for 48 h. The progress of the reaction was monitored by TLC (V methanol :V ethyl acetate = 8:1) to determine the disappearance of the starting material. On completion, 2 mL of methanol was added to quench the reaction. Then the mixture was extracted by ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous Na2 SO4 . Finally, the solvent was removed to give a dark-grey solid (4.1 g). The product (400 mg) was dissolved in 5 mL of anhydrous tetrahydrofuran (THF). A quantity of 40 mg of lithium aluminium hydride (LAH) was added to the mixture at room temperature. The solution was refluxed for 24 h, and the reaction was monitored by TLC. On completion, 1 mL of water was added, and the solution was filtered and extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous Na2 SO4 to produce 370 mg of grey solid. The product was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 1:1) to give 190 mg (65%) of compound 2.
2.3.2 Synthesis of 4𝛼-hydroxyl-6𝛼-(2′ -fluorobenzyloxy)-1𝛽,9𝛼diolacetonide-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (4.1) Compound 3 (340 mg, 1 mmol) was dissolved in 25 mL of anhydrous THF, and NaH (120 mg, 5 mmol) was added. The mixture was stirred for 30 min at room temperature. Alpha-chloro-o-fluorotoluene (178.3 μL, 1.5 mmol) was added to the reaction mixture, and the resulting mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 1 mL of water was added, and the reaction mixture was extracted with CH2 Cl2 , washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 4:1) to produce 426 mg (95%) of compound 4.1. For data on compound 4.1, see the supporting information. 2.3.3 Synthesis of 6𝛼-(2′ -fluorobenzyloxy)-1𝛽,4𝛼,9𝛼-trihydroxy-2𝛽, 12-epoxymethano-𝛽-dihydroagarofuran (5.1) Compound 4.1 (400 mg, 0.89 mmol) was dissolved in 30 mL (0.74 mol) of methanol, 317.8 μL of 18.4 M H2 SO4 was added and the reaction mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). On completion, 3 mL of saturated acqueous sodium bicarbonate solution was added to adjust the reaction pH to neutral or weakly alkaline. The methanol was evaporated under reduced pressure, and the residue was extracted with ethyl acetate, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The ethyl acetate was removed under reduced pressure, and the residue was chromatographed (V petroleum ether :V ethyl acetate = 1:2) to give 327 mg (90%) of compound 5.1. For data on compound 5.1, see the supporting information. 2.3.4 Synthesis of compounds 5.1.1 to 5.1.10 2.3.4.1 Synthesis of 1𝛽-methoxy-6𝛼-(2′ -fluorobenzyloxy)-4𝛼,9𝛼dihydroxy-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (5.1.1). Compound 5.1 (30 mg, 0.07 mmol) was dissolved in 8 mL of anhydrous THF, and NaH (17.8 mg, 0.74 mmol) was added to the mixture while stirring at room temperature. After continuing stirring for 20 min at room temperature, iodomethane (0.11 mmol, 6.85 μL) was added dropwise to the reaction mixture. The solution was stirred for 10 h at room temperature, and the reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 0.5 mL of water was added, the THF was evaporated under reduced pressure and the residue was extracted with dichloromethane, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The dichloromethane was removed under reduced pressure, and the residue was chromatographed to give 25 mg (85%) of compound 5.1.1. For data on compound 5.1.1, see the supporting information. 2.3.4.2 Synthesis of compounds 5.1.2 to 5.1.10. The target compounds 5.1.2 to 5.1.10 were synthesised by methods similar to
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2.3 Synthesis of 𝜷-dihydroagarofuran ether analogues (5.1, 5.1.1 to 5.1.10) 2.3.1 Synthesis of 4𝛼,6𝛼-dihydroxy-1𝛽,9𝛼-diolacetonide-2𝛽,12epoxymethano-𝛽-dihydroagarofuran (3) Compound 2 (400 mg, 1.33 mmol) and ferric chloride (8.65 mg, 0.05 mmol) were dissolved in 15 mL of acetone. The mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). Once compound 2 had reacted completely, acetone was evaporated under reduced Pest Manag Sci 2016; 72: 754–759
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X Zhao et al.
that of compound 5.1.1, using corresponding alkylhalides, and the yields were all over 80%. For data on compounds 5.1.2 to 5.1.10, see the supporting information. 2.4 Synthesis of 𝜷-dihydroagarofuran ether analogues (5.2, 5.2.1 to 5.2.10) 2.4.1 Synthesis of 4𝛼-hydroxyl-6𝛼-(4′ -fluorobenzyloxy)-1𝛽, 9𝛼-diolacetonide-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (4.2) Compound 3 (340 mg, 1.0 mmol) was dissolved in 25 mL of anhydrous THF, and NaH (120 mg, 5 mmol) was added. The mixture was stirred for 30 min at room temperature. Alpha-chloro-p-fluorotoluene (179.7 μL, 1.5 mmol) was added to the reaction mixture, and the solution was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 1 mL of water was added, and the reaction mixture was extracted with CH2 Cl2 , washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 4:1) to produce 420 mg (94%) of compound 4.2. For data on compound 4.2, see the supporting information. 2.4.2 Synthesis of 6𝛼-(4′ -fluorobenzyloxy)-1𝛽,4𝛼,9𝛼-trihydroxy-2𝛽, 12-epoxymethano-𝛽-dihydroagarofuran (5.2) Compound 4.2 (400 mg, 0.89 mmol) was dissolved in 30 mL (0.74 mol) of methanol, 317.8 μL of 18.4 M H2 SO4 was added and the reaction mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). On completion, 3 mL of saturated aqueous sodium bicarbonate solution was added to adjust the reaction pH to neutral or weakly alkaline. The methanol was evaporated under reduced pressure, and the residue was extracted with ethyl acetate, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The ethyl acetate was removed under reduced pressure, and the residue was chromatographed (V petroleum ether :V ethyl acetate = 1:2) to give 327 mg (90%) of compound 5.2. For data on compound 5.2, see the supporting information. 2.4.3 Synthesis of compounds 5.2.1 to 5.2.10 2.4.3.1 Synthesis of 1𝛽-methoxy-6𝛼-(4′ -fluorobenzyloxy)-4𝛼,9𝛼dihydroxy-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (5.2.1). Compound 5.2 (30 mg, 0.07 mmol) was dissolved in 8 mL of anhydrous THF, and NaH (17.8 mg, 0.74 mmol) was added to the mixture while stirring at room temperature. After continuing stirring for 20 min at room temperature, iodomethane (0.11 mmol, 6.85 μL) was added dropwise to the reaction mixture. The solution was stirred for 10 h at room temperature, and the reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 0.5 mL of water was added, the THF was evaporated under reduced pressure and the residue was extracted with dichloromethane, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The dichloromethane was removed under reduced pressure, and the residue was chromatographed to give 29 mg (98%) of product 5.2.1. For data on compound 5.2.1, see the supporting information.
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2.4.3.2 Synthesis of compounds 5.2.2 to 5.2.10. The target compounds 5.2.2 to 5.2.10 were synthesised by methods similar to that of compound 5.2.1, using corresponding alkylhalides, and the yields were all over 80%.
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Figure 2. The X-ray crystal structure of compound 5.2.6.
For data on compounds 5.2.2 to 5.2.10, see the supporting information. 2.5 Insecticidal activity The insecticidal activity of the 22 ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) was determined against third-instar larvae of M. separata starved for 12 h with a leaf strip measuring 5 mm × 5 mm, to which 1.12 μL of a solution of the derivatives with a concentration of 40 mg mL−1 , with acetone as the solvent, was applied.9 In each bioassay, 30 larvae of M. separata were tested for one derivative; celangulin-V and untreated leaf strips served as positive and blank control respectively, and the mortality rates were recorded within 36 h. The toxicity was ascertained by establishing the median lethal dose (LD50 ) of compounds with insect mortality over 60%. 2.6 X-ray crystallography X-ray-quality crystal of compound 5.2.6 was obtained from the ethyl acetate solution after 3 days. The crystal structure of compound 5.2.6 (Fig. 2) was determined on a Bruker APEX-II CCD diffractometer using graphite monochromated MoK𝛼 radiation, and the atomic coordinates have been deposited at the Cambridge Crystallographic Data Centre (CCDC) with CCDC number 1035467.
3
RESULTS AND DISCUSSION
3.1 Chemistry The synthesis of the lead compound 2 and other target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) is shown in
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Pest Manag Sci 2016; 72: 754–759
Synthesis and insecticidal activity of 𝛽-dihydroagarofuran ether analogues
HO
OH OH
HO
1. MsCl/Pyr, OH 0 °C–rt
HO
8 7
2. THF/LAH O OH 1
OH
11
12
9
OH O
10 5
6
1 4
2 3
O OH 13 OH
14 15
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O (CH3)2CO FeCl3
R1=p-F-Bn
5.1
R1=o-F-Bn
5.2
R1=p-F-Bn
O
OH
O OR1
OH
4.1, 4.2
NaH,R2X OH
5.1, 5.2
4.2
O
OH OR2 O
OH O
O OR1
R1=o-F-Bn
THF
O OH 3
0.8% H2SO4/MeOH
4.1
O
O NaH,R1X
2 OH
O
5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 5.1.8 5.1.9 5.1.10
R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn
THF
O OR1
OH
5.1.1 to 5.1.10 5.2.1 to 5.2.10 R2=CH3; R2=C2H5; R2=n-C3H7; R2=n-C4H9; R2=n-C5H11 R2=C3H5; R2=C3H3; R2=Bn; R2=p-CH3-Bn; R2=p-F-Bn;
5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 5.2.9 5.2.10
R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn
R2=CH3; R2=C2H5; R2=n-C3H7; R2=n-C4H9; R2=n-C5H11 R2=C3H5; R2=C3H3; R2=Bn; R2=p-CH3-Bn; R2=o-F-Bn;
Scheme 1. Synthesis of 𝛽-dihydroagarofuran ether analogues.
Scheme 1. Specifically, the lead compound 2 was synthesised using 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran as the starting material, which first reacted with CH3 SO2 Cl in anhydrous pyridine, followed by reduction by LAH in anhydrous THF. All the target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) were synthesised from the lead compound 2. For example, the hydroxyl substituents at the 1- and 9-positions of the lead compound 2 were protected with acetonide groups, and then reacted with sodium hydride in anhydrous THF with subsequent treatment with alpha-chloro-o-fluorotoluene and alpha-chloro-p-fluorotoluene, respectively, to give the corresponding intermediates 4.1 and 4.2. Compounds 5.1 and 5.2 were synthesised by the deprotection of compounds 4.1 and 4.2. All the other target compounds (5.1.1 to 5.1.10, 5.2.1 to 5.2.10) were synthesised from compounds 5.1 and 5.2 first by reaction with sodium hydride in anhydrous THF, followed by treatment with various haloalkanes. All the synthetic target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) were purified by column chromatography on silica gel eluted in petroleum ether/ethyl acetate, and the structures were identified by melting point, optical rotation, 1 H NMR, 13 C NMR and DEPT-135∘ .
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3.2 Insecticidal activity The insecticidal activity of the 22 synthetic ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) was examined against the third-instar larvae of M. separata (Fig. 3) to study the influence of the variation of the moiety at 1- and 6-positions, respectively, with celangulin-V and acetone as the positive and negative control. The results are summarised in Table 1.
As shown in Table 1, the derivatives containing alkyl groups at the 1-position exhibited excellent insecticidal activity against the third-instar larvae of M. separata, especially those with the moiety of two or three carbon atoms at the 1-position. For example, compounds 5.1.2, 5.1.3, 5.1.7, 5.2.3, 5.2.6 and 5.2.7 exhibited 77.7, 73.3, 86.7, 62.5, 80.3 and 63.1% insect mortality. Moreover, compounds 5.1.2, 5.1.3, 5.2.6 and 5.2.7 showed much lower LD50 than celangulin-V, and, most importantly, compound 5.2.7 showed the lowest LD50 value of 29.2 μg g−1 among these synthesised compounds, whereas the compounds containing an aryl group at the 1-position showed moderate insecticidal activity. The bioassay results demonstrated that the substituted moiety at the 1-position played a significant role in the insecticidal activity of these ether analogues, while the substituted moiety at the 6-position had little influence. Specifically, when the moiety at the 1-position was an alkyl group, the insecticidal activity of the derivatives first rose and then decreased with increasing number of carbon atoms, reaching a maximum when the substituent group contained two or three carbon atoms. When the moiety at the 1-position was an aryl group, the insecticidal activity of the analogue was related to the electronic effect of the substituent group at the aryl moiety. When the substituent at the aryl moiety was an electron-donating group such as methyl, the insecticidal activity of the derivative (30% mortality). When the substituent at the aryl moiety was an electron-withdrawing group such as fluorine, the insecticidal activity of the analogue (>50% mortality) significantly exceeded that of the analogue with no substituent at the aryl moiety (
Revised: 14 May 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library: 6 July 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4049
Synthesis and insecticidal activity of 𝜷-dihydroagarofuran ether analogues Ximei Zhao,a† Zhan Hu,a† Jian Li,a Longbo Li,a Wenjun Wub and Jiwen Zhanga,b* Abstract BACKGROUND: 1𝜷, 2𝜷, 4𝜶, 6𝜶, 8𝜷, 9𝜶, 12-hepthydroxyl-𝜷-dihydroagarofuran is the main skeleton of 𝜷-dihydroagarofuran sesquiterpenoids, which exhibit excellent insecticidal activity. To study further the structure–activity relationship of 𝜷-dihydroagarofuran sesquiterpenoids towards finding novel botanical pesticides, two series of new structurally modified ether analogues were designed and synthesised, and their insecticidal activities were evaluated. RESULTS: Twenty-two ether derivatives were synthesised using 1𝜷, 2𝜷, 4𝜶, 6𝜶, 8𝜷, 9𝜶, 12-hepthydroxyl-𝜷-dihydroagarofuran as starting material. Bioassay results indicated that most of the derivatives, particularly compounds 5.1.2, 5.1.3, 5.1.7, 5.2.3, 5.2.6 and 5.2.7, exhibited significant insecticidal activity against the third-instar larvae of the oriental armyworm Mythimna separata. Most importantly, compound 5.2.7 showed the lowest LD50 value of 29.2 𝛍g g−1 among these synthesised compounds, which provides some important hints for further design, synthesis and structural modification of 𝜷-dihydroagarofuran sesquiterpenoids towards developing novel botanical insecticides. CONCLUSION: The structure–activity relationship illustrated that the moiety at the 1-position affected the insecticidal activity significantly, and that specifically the derivatives with two or three carbon atoms at the 1-position showed promising insecticidal activity, with mortality over 60%, while those with o-F-Bn and p-F-Bn at the 6-position showed similar activity. © 2015 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: dihydroagarofuran; sesquiterpenoid; insecticidal activity; structure–activity relationship
1
INTRODUCTION
754
Celastrus angulatus Maxim is a significant bioactive plant in China. Plant extracts from the Celastraceae family have been traditionally utilised as insecticides, as insect repellents and for the treatment of various diseases such as fever, cancer, rheumatoid arthritis and a wide range of gastrointestinal diseases.1 – 4 It has been reported that the main biologically active components of these extracts are various 𝛽-dihydroagarofuran sesquiterpenoids, which have been characterised by a 𝛽-dihydroagarofuran skeleton.5 – 8 Previous studies have shown that 𝛽-dihydroagarofuran sesquiterpene polyol esters extracted from Celastrus angulatus exhibit excellent insecticidal activity against the oriental armyworm Mythimna separata Walker,5,6,9 and that 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) forms the main framework of these 𝛽-dihydroagarofuran sesquiterpenoids. In our previous work, 1𝛽, 4𝛼, 6𝛼, 9𝛼-tetrahydroxyl-2𝛽, 12-ether-𝛽-dihydroagarofuran (2), together with twelve derivatives, eleven with the same substituent group at the 1- and 6-positions, and one with the same substituent group at the 6and 9-positions, has been synthesised, with 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) as the starting material. Bioassay results indicated that the derivatives with o-F-Bn and p-F-Bn moieties exhibited excellent insecticidal activity, with insect mortality far exceeding that of the positive control celangulin-V (Fig. 1).10 To study further the structure–activity Pest Manag Sci 2016; 72: 754–759
relationship of 𝛽-dihydroagarofuran sesquiterpenoids towards finding novel botanical pesticides, in this paper we describe the design, synthesis and bioactivity evaluation of 22 fluorinated ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) from 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) as the starting material.
2
MATERIALS AND METHODS
2.1 General Melting points were measured on an WRS-1B melting point apparatus (Shanghai YiCe Apparatus and Equipment Co., Ltd, Shanghai, China) and are uncorrected; specific rotations were determined on a Perkin-Elmer 241 MC automatic polarimeter (PerkinElmer, Waltham, MA) and are uncorrected; all the 1 H NMR and 13 C NMR spectra were recorded on a Bruker Advance spectrometer (Bruker,
∗
Correspondence to: Jiwen Zhang, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China, E-mail: [email protected]
† These authors contributed equally to this work. a College of Science, Northwest A&F University, Yangling, Shaanxi, China b Institute of Pesticide Science, Northwest A&F University, Yangling, Shaanxi, China
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Synthesis and insecticidal activity of 𝛽-dihydroagarofuran ether analogues
O
O
O O
O
O O
pressure and the residue was extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 1:2 ) to give 450 mg (99%) of product 3.
O O
O O OH OH Chemical structure of celangulin-¢õ
Figure 1. The chemical structure of celangulin-V.
Billerica, MA) at 500 MHz and 125 MHz in CDCl3 with TMS as the reference; distortionless enhancement by polarisation transfer (DEPT) spectra (flip angle 135∘ ) were obtained to determine the assignments of 13 C chemical shifts. The starting material 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) (purity ≥98%) was provided by the Institute of Pesticide Science, Northwest A & F University; thin-layer chromatography (TLC) analysis was carried out on GF254 silica gel plates, and the spots were coloured by an ultraviolet lamp or 5% (V/V) sulfuric acid in ethyl alcohol; the synthetic products were purified by column chromatography with silica gel (zcx II, 200–300 mesh) and mixtures of petroleum ether and ethyl acetate as the eluent. The bioassay was carried out by the leaf-disc method at a concentration of 40 mg mL−1 . Celangulin-V and acetone were used as positive and negative controls respectively. 2.2 Synthesis of 1𝜷, 4𝜶, 6𝜶, 9𝜶-tetrahydroxyl-2𝜷, 12-ether-𝜷-dihydroagarofuran (2) 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran (1) (3.34 g, 10 mmol) was dissolved in 10 mL of anhydrous pyridine. Freshly distilled CH3 SO2 Cl (6 mL, 72 mmol) was added dropwise to the mixture under Ar, and then the reaction mixture was stirred at room temperature for 48 h. The progress of the reaction was monitored by TLC (V methanol :V ethyl acetate = 8:1) to determine the disappearance of the starting material. On completion, 2 mL of methanol was added to quench the reaction. Then the mixture was extracted by ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous Na2 SO4 . Finally, the solvent was removed to give a dark-grey solid (4.1 g). The product (400 mg) was dissolved in 5 mL of anhydrous tetrahydrofuran (THF). A quantity of 40 mg of lithium aluminium hydride (LAH) was added to the mixture at room temperature. The solution was refluxed for 24 h, and the reaction was monitored by TLC. On completion, 1 mL of water was added, and the solution was filtered and extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous NaCl solution and dried with anhydrous Na2 SO4 to produce 370 mg of grey solid. The product was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 1:1) to give 190 mg (65%) of compound 2.
2.3.2 Synthesis of 4𝛼-hydroxyl-6𝛼-(2′ -fluorobenzyloxy)-1𝛽,9𝛼diolacetonide-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (4.1) Compound 3 (340 mg, 1 mmol) was dissolved in 25 mL of anhydrous THF, and NaH (120 mg, 5 mmol) was added. The mixture was stirred for 30 min at room temperature. Alpha-chloro-o-fluorotoluene (178.3 μL, 1.5 mmol) was added to the reaction mixture, and the resulting mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 1 mL of water was added, and the reaction mixture was extracted with CH2 Cl2 , washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 4:1) to produce 426 mg (95%) of compound 4.1. For data on compound 4.1, see the supporting information. 2.3.3 Synthesis of 6𝛼-(2′ -fluorobenzyloxy)-1𝛽,4𝛼,9𝛼-trihydroxy-2𝛽, 12-epoxymethano-𝛽-dihydroagarofuran (5.1) Compound 4.1 (400 mg, 0.89 mmol) was dissolved in 30 mL (0.74 mol) of methanol, 317.8 μL of 18.4 M H2 SO4 was added and the reaction mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). On completion, 3 mL of saturated acqueous sodium bicarbonate solution was added to adjust the reaction pH to neutral or weakly alkaline. The methanol was evaporated under reduced pressure, and the residue was extracted with ethyl acetate, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The ethyl acetate was removed under reduced pressure, and the residue was chromatographed (V petroleum ether :V ethyl acetate = 1:2) to give 327 mg (90%) of compound 5.1. For data on compound 5.1, see the supporting information. 2.3.4 Synthesis of compounds 5.1.1 to 5.1.10 2.3.4.1 Synthesis of 1𝛽-methoxy-6𝛼-(2′ -fluorobenzyloxy)-4𝛼,9𝛼dihydroxy-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (5.1.1). Compound 5.1 (30 mg, 0.07 mmol) was dissolved in 8 mL of anhydrous THF, and NaH (17.8 mg, 0.74 mmol) was added to the mixture while stirring at room temperature. After continuing stirring for 20 min at room temperature, iodomethane (0.11 mmol, 6.85 μL) was added dropwise to the reaction mixture. The solution was stirred for 10 h at room temperature, and the reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 0.5 mL of water was added, the THF was evaporated under reduced pressure and the residue was extracted with dichloromethane, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The dichloromethane was removed under reduced pressure, and the residue was chromatographed to give 25 mg (85%) of compound 5.1.1. For data on compound 5.1.1, see the supporting information. 2.3.4.2 Synthesis of compounds 5.1.2 to 5.1.10. The target compounds 5.1.2 to 5.1.10 were synthesised by methods similar to
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2.3 Synthesis of 𝜷-dihydroagarofuran ether analogues (5.1, 5.1.1 to 5.1.10) 2.3.1 Synthesis of 4𝛼,6𝛼-dihydroxy-1𝛽,9𝛼-diolacetonide-2𝛽,12epoxymethano-𝛽-dihydroagarofuran (3) Compound 2 (400 mg, 1.33 mmol) and ferric chloride (8.65 mg, 0.05 mmol) were dissolved in 15 mL of acetone. The mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). Once compound 2 had reacted completely, acetone was evaporated under reduced Pest Manag Sci 2016; 72: 754–759
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X Zhao et al.
that of compound 5.1.1, using corresponding alkylhalides, and the yields were all over 80%. For data on compounds 5.1.2 to 5.1.10, see the supporting information. 2.4 Synthesis of 𝜷-dihydroagarofuran ether analogues (5.2, 5.2.1 to 5.2.10) 2.4.1 Synthesis of 4𝛼-hydroxyl-6𝛼-(4′ -fluorobenzyloxy)-1𝛽, 9𝛼-diolacetonide-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (4.2) Compound 3 (340 mg, 1.0 mmol) was dissolved in 25 mL of anhydrous THF, and NaH (120 mg, 5 mmol) was added. The mixture was stirred for 30 min at room temperature. Alpha-chloro-p-fluorotoluene (179.7 μL, 1.5 mmol) was added to the reaction mixture, and the solution was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 1 mL of water was added, and the reaction mixture was extracted with CH2 Cl2 , washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (V petroleum ether :V ethyl acetate = 4:1) to produce 420 mg (94%) of compound 4.2. For data on compound 4.2, see the supporting information. 2.4.2 Synthesis of 6𝛼-(4′ -fluorobenzyloxy)-1𝛽,4𝛼,9𝛼-trihydroxy-2𝛽, 12-epoxymethano-𝛽-dihydroagarofuran (5.2) Compound 4.2 (400 mg, 0.89 mmol) was dissolved in 30 mL (0.74 mol) of methanol, 317.8 μL of 18.4 M H2 SO4 was added and the reaction mixture was stirred for 12 h at room temperature. The reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:4). On completion, 3 mL of saturated aqueous sodium bicarbonate solution was added to adjust the reaction pH to neutral or weakly alkaline. The methanol was evaporated under reduced pressure, and the residue was extracted with ethyl acetate, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The ethyl acetate was removed under reduced pressure, and the residue was chromatographed (V petroleum ether :V ethyl acetate = 1:2) to give 327 mg (90%) of compound 5.2. For data on compound 5.2, see the supporting information. 2.4.3 Synthesis of compounds 5.2.1 to 5.2.10 2.4.3.1 Synthesis of 1𝛽-methoxy-6𝛼-(4′ -fluorobenzyloxy)-4𝛼,9𝛼dihydroxy-2𝛽,12-epoxymethano-𝛽-dihydroagarofuran (5.2.1). Compound 5.2 (30 mg, 0.07 mmol) was dissolved in 8 mL of anhydrous THF, and NaH (17.8 mg, 0.74 mmol) was added to the mixture while stirring at room temperature. After continuing stirring for 20 min at room temperature, iodomethane (0.11 mmol, 6.85 μL) was added dropwise to the reaction mixture. The solution was stirred for 10 h at room temperature, and the reaction was monitored by TLC (V petroleum ether :V ethyl acetate = 1:1). On completion, 0.5 mL of water was added, the THF was evaporated under reduced pressure and the residue was extracted with dichloromethane, washed with saturated aqueous NaCl solution and dried with anhydrous NaSO4 . The dichloromethane was removed under reduced pressure, and the residue was chromatographed to give 29 mg (98%) of product 5.2.1. For data on compound 5.2.1, see the supporting information.
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2.4.3.2 Synthesis of compounds 5.2.2 to 5.2.10. The target compounds 5.2.2 to 5.2.10 were synthesised by methods similar to that of compound 5.2.1, using corresponding alkylhalides, and the yields were all over 80%.
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Figure 2. The X-ray crystal structure of compound 5.2.6.
For data on compounds 5.2.2 to 5.2.10, see the supporting information. 2.5 Insecticidal activity The insecticidal activity of the 22 ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) was determined against third-instar larvae of M. separata starved for 12 h with a leaf strip measuring 5 mm × 5 mm, to which 1.12 μL of a solution of the derivatives with a concentration of 40 mg mL−1 , with acetone as the solvent, was applied.9 In each bioassay, 30 larvae of M. separata were tested for one derivative; celangulin-V and untreated leaf strips served as positive and blank control respectively, and the mortality rates were recorded within 36 h. The toxicity was ascertained by establishing the median lethal dose (LD50 ) of compounds with insect mortality over 60%. 2.6 X-ray crystallography X-ray-quality crystal of compound 5.2.6 was obtained from the ethyl acetate solution after 3 days. The crystal structure of compound 5.2.6 (Fig. 2) was determined on a Bruker APEX-II CCD diffractometer using graphite monochromated MoK𝛼 radiation, and the atomic coordinates have been deposited at the Cambridge Crystallographic Data Centre (CCDC) with CCDC number 1035467.
3
RESULTS AND DISCUSSION
3.1 Chemistry The synthesis of the lead compound 2 and other target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) is shown in
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Synthesis and insecticidal activity of 𝛽-dihydroagarofuran ether analogues
HO
OH OH
HO
1. MsCl/Pyr, OH 0 °C–rt
HO
8 7
2. THF/LAH O OH 1
OH
11
12
9
OH O
10 5
6
1 4
2 3
O OH 13 OH
14 15
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O (CH3)2CO FeCl3
R1=p-F-Bn
5.1
R1=o-F-Bn
5.2
R1=p-F-Bn
O
OH
O OR1
OH
4.1, 4.2
NaH,R2X OH
5.1, 5.2
4.2
O
OH OR2 O
OH O
O OR1
R1=o-F-Bn
THF
O OH 3
0.8% H2SO4/MeOH
4.1
O
O NaH,R1X
2 OH
O
5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 5.1.8 5.1.9 5.1.10
R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn R1=o-F-Bn
THF
O OR1
OH
5.1.1 to 5.1.10 5.2.1 to 5.2.10 R2=CH3; R2=C2H5; R2=n-C3H7; R2=n-C4H9; R2=n-C5H11 R2=C3H5; R2=C3H3; R2=Bn; R2=p-CH3-Bn; R2=p-F-Bn;
5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 5.2.9 5.2.10
R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn R1=p-F-Bn
R2=CH3; R2=C2H5; R2=n-C3H7; R2=n-C4H9; R2=n-C5H11 R2=C3H5; R2=C3H3; R2=Bn; R2=p-CH3-Bn; R2=o-F-Bn;
Scheme 1. Synthesis of 𝛽-dihydroagarofuran ether analogues.
Scheme 1. Specifically, the lead compound 2 was synthesised using 1𝛽, 2𝛽, 4𝛼, 6𝛼, 8𝛽, 9𝛼, 12-hepthydroxyl-𝛽-dihydroagarofuran as the starting material, which first reacted with CH3 SO2 Cl in anhydrous pyridine, followed by reduction by LAH in anhydrous THF. All the target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) were synthesised from the lead compound 2. For example, the hydroxyl substituents at the 1- and 9-positions of the lead compound 2 were protected with acetonide groups, and then reacted with sodium hydride in anhydrous THF with subsequent treatment with alpha-chloro-o-fluorotoluene and alpha-chloro-p-fluorotoluene, respectively, to give the corresponding intermediates 4.1 and 4.2. Compounds 5.1 and 5.2 were synthesised by the deprotection of compounds 4.1 and 4.2. All the other target compounds (5.1.1 to 5.1.10, 5.2.1 to 5.2.10) were synthesised from compounds 5.1 and 5.2 first by reaction with sodium hydride in anhydrous THF, followed by treatment with various haloalkanes. All the synthetic target compounds (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) were purified by column chromatography on silica gel eluted in petroleum ether/ethyl acetate, and the structures were identified by melting point, optical rotation, 1 H NMR, 13 C NMR and DEPT-135∘ .
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3.2 Insecticidal activity The insecticidal activity of the 22 synthetic ether derivatives (5.1, 5.1.1 to 5.1.10, 5.2, 5.2.1 to 5.2.10) was examined against the third-instar larvae of M. separata (Fig. 3) to study the influence of the variation of the moiety at 1- and 6-positions, respectively, with celangulin-V and acetone as the positive and negative control. The results are summarised in Table 1.
As shown in Table 1, the derivatives containing alkyl groups at the 1-position exhibited excellent insecticidal activity against the third-instar larvae of M. separata, especially those with the moiety of two or three carbon atoms at the 1-position. For example, compounds 5.1.2, 5.1.3, 5.1.7, 5.2.3, 5.2.6 and 5.2.7 exhibited 77.7, 73.3, 86.7, 62.5, 80.3 and 63.1% insect mortality. Moreover, compounds 5.1.2, 5.1.3, 5.2.6 and 5.2.7 showed much lower LD50 than celangulin-V, and, most importantly, compound 5.2.7 showed the lowest LD50 value of 29.2 μg g−1 among these synthesised compounds, whereas the compounds containing an aryl group at the 1-position showed moderate insecticidal activity. The bioassay results demonstrated that the substituted moiety at the 1-position played a significant role in the insecticidal activity of these ether analogues, while the substituted moiety at the 6-position had little influence. Specifically, when the moiety at the 1-position was an alkyl group, the insecticidal activity of the derivatives first rose and then decreased with increasing number of carbon atoms, reaching a maximum when the substituent group contained two or three carbon atoms. When the moiety at the 1-position was an aryl group, the insecticidal activity of the analogue was related to the electronic effect of the substituent group at the aryl moiety. When the substituent at the aryl moiety was an electron-donating group such as methyl, the insecticidal activity of the derivative (30% mortality). When the substituent at the aryl moiety was an electron-withdrawing group such as fluorine, the insecticidal activity of the analogue (>50% mortality) significantly exceeded that of the analogue with no substituent at the aryl moiety (
