Acute Larvicidal Activity against Mosquitoes and Oxygen Consumption

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Acute larvicidal activity against mosquitoes and oxygen consumption inhibitory activity of dihydroguaiaretic acid derivatives Hisashi Nishiwaki, Yoshimi Tabara, Taro Kishida, Kosuke Nishi, Yoshihiro Shuto, Takuya Sugahara, and Satoshi Yamauchi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504816a • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 16, 2015

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Journal of Agricultural and Food Chemistry

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Acute larvicidal activity against mosquitoes and oxygen consumption inhibitory activity

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of dihydroguaiaretic acid derivatives

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Hisashi Nishiwaki,† Yoshimi Tabara,† Taro Kishida,†,‡ Kosuke Nishi,† Yoshihiro Shuto,†

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Takuya Sugahara†,‡ and Satoshi Yamauchi†,‡

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†

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Japan

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‡

Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566,

South Ehime Fisheries Research Center, 1289-1 Funakoshi, Ainan, Ehime 798-4292, Japan

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Corresponding should be addressed

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Satoshi Yamauchi, Ph. D.

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TEL: +81-89-946-9846

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FAX: +81-89-977-4364

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT:

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(−)-Dihydroguaiaretic acid (DGA) and its derivatives having 3-hydroxyphenyl (3-OH-DGA)

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and variously substituted phenyl groups instead of 3-hydroxy-4-methoxyphenyl groups were

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synthesized to measure their larvicidal activity against the mosquito Culex pipiens Linnaeus,

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1758 (Diptera: Culicidae). Compared with DGA and 3-OH-DGA (LC50 (M), 3.52 × 10−5 and

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4.57 × 10−5, respectively), (8R, 8’R)-lignan-3-ol (3), and its 3-Me (10), 2-OH (12), 3-OH (13),

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2-OMe (15) derivatives showed low potency (ca. 6–8 × 10-5 M). The 4-Me derivative (11)

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showed the lowest potency (12.1 × 10−5 M), and the 2-F derivative (4) the highest (2.01 ×

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10−5 M). All of the synthesized compounds induced acute toxic symptom against mosquito

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larvae, with potency varying with the type and position of the substituents. The 4-F derivative

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(6), which killed larvae almost completely within 45 min, suppressed the O2 consumption of

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the mitochondrial fraction, demonstrating that this compound inhibited mitochondrial O2

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consumption contributing to a respiratory inhibitory activity.

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Key words: dihydroguaiaretic acid; lignans; larvicides; Culex pipiens; acute toxicity

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INTRODUCTION

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Mosquitoes are vectors of parasites and viruses inducing severe infectious diseases such as

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malaria and West Nile fever. Several vector-control insecticides and repellents have been

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developed, and novel chemicals killing mosquitoes are still explored constantly. Since it is

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only adult mosquitoes that carry the disease-inducing parasites and viruses between hosts,

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compounds killing the larvae before metamorphosis into adults should be desired. As an

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insecticide for controlling mosquito larvae, methoprene, an analog of the juvenile hormone of

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insects, is employed.1 Since methoprene is one of the insect growth regulators and its lethal

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effects are based on the disruption of the endocrine system mediating developmental

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processes such as metamorphosis, it does not exert the acute larvicidal effects within several

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hours after application unlike central nervous system toxicants. And methoprene-resistant

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populations have been reported.2,3 Another example of larvicides is microbial biopesticides

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such as Bacillus thuringiensis and Lysinibacillus sphaericus, and their proteinaceous toxins

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such as δ-endotoxin (B. thuringiensis), Mtx-1 and 2 (L. sphaericus) reportedly damage the

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midgut of the mosquito larvae.4-6 Various vector control reagents are successful on the

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markets, but compounds inducing acute toxic symptom against the mosquito larvae should be

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explored, and it is desirable that these compounds be developed based on structures of natural

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compounds.

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In our previous studies,7,8 dihydroguaiaretic acid (DGA, 1 in Figure 1), one of the

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natural lignans, was reported to show insecticidal activity against larvae of the mosquito

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Culex pipiens, as well as adult houseflies, Musca domestica. In particular, its derivative with

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3-hydroxyphenyl group instead of the 3-hydroxy-4-methoxyphenyl group (3-OH-DGA, 2 in

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Figure 1) induced acute toxic symptom in mosquito larvae within 1 h. Modification of the 3



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lignan derivative as a lead compound should suggest novel structures for effective larvicides.

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In the present study, we synthesized compounds with variously substituted phenyl

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groups instead of 3-hydroxy-4-methoxyphenyl groups at the 7 and 7′ positions (compounds

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3–20 in Figure 1) to evaluate their larvicidal activity against C. pipiens 24 h after application

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as well as to monitor their acute toxic symptom within 2 h. To investigate the mode of action

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of the potent compounds, the O2 consumption inhibitory activity of potent compounds was

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evaluated.

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MATERIALS AND METHODS Chemicals

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Reagents used for the syntheses were purchased from Wako Pure Chemical

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Industries, Ltd (Osaka, Japan), Nacalai Tesque, Inc (Kyoto, Japan), Tokyo Chemical Industry

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Co, Ltd (Tokyo, Japan), and Aldrich Chemical Co (Milwaukee, WI, USA). Melting points of

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the compounds were measured with a Yanaco melting point apparatus (Kyoto, Japan) and

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were uncorrected. Optical rotations were measured on a SEPA-200 instrument (Horiba Ltd.,

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Kyoto, Japan). NMR data were obtained using an ECS400 spectrometer (JEOL

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RESONANCE Inc., Tokyo, Japan). EIMS data were measured with a JMS-MS700V

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spectrometer (JEOL Ltd., Tokyo, Japan). Silica gel column chromatography was performed

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using Wakogel C-300 (Wako, 200-300 mesh). The numbering of atoms follows nomenclature

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of lignans.9 The synthetic scheme was shown in Figure 2, which was fundamentally the same

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as the earlier report.7 The compounds 4-18 were obtained from

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(R)-3-(3-benzyloxybenzyl)-4-butanolide and the corresponding substituted-benzyl bromide

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(preparation for hydroxybenzyl derivatives 12-14 needs a benzyloxybenzyl bromide to be 4


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deprotected in the final step) by employing same method described for the synthesis of 3. The

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compound 20 was synthesized using the preparation procedure of compound 19. The

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compounds 21 and 22 were our stock samples.

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Synthesis of derivatives

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(8R, 8’R)-Lignan-3-ol 3

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To a solution of lithium diisopropylamide (2.10 mmol) in tetrahydrofuran (THF, 10 mL) was

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added a solution of (R)-3-(3-benzyloxybenzyl)-4-butanolide (0.50 g, 1.77 mmol; See the

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preparation method in the Supporting Information) in THF (10 mL), and then the resulting

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solution was stirred at -70 oC for 30 min before addition of a solution of benzyl bromide

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(0.30 g, 1.75 mmol) in THF (5 mL). After the reaction solution was stirred at room

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temperature for 1 h, sat. aq NH4Cl solution and ethyl acetate (EtOAc) were added. The

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organic solution was separated, washed with brine, dried (Na2SO4), and concentrated. The

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residue was applied to silica gel column chromatography (EtOAc/hexane = 1/3). To an

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ice-cooled suspension of LiAlH4 (14 mg, 0.37 mmol) in THF (5 mL) was added a solution of

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the obtained compound in THF (5 mL). The resulting reaction mixture was stirred at room

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temperature for 30 min, and then sat. aq. MgSO4 and K2CO3 were added. The mixture was

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stirred at room temperature for 30 min. After filtration of the mixture, the filtrate was

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concentrated. To an ice-cooled solution of the residue and Et3N (0.54 mL, 3.87 mmol) in

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CH2Cl2 (5 mL) was added MsCl (0.30 mL, 3.88 mmol). After the reaction mixture was

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stirred at room temperature for 30 min, H2O and CH2Cl2 were added. The organic solution

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was separated, dried (Na2SO4), and concentrated. A solution of the residue and NaBH4 (90

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mg, 2.38 mmol) in hexamethylphosphoric triamide (HMPA, 5 mL) was heated at 60 oC for 5



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30 min before additions of sat. aq. NH4Cl solution and EtOAc. The organic solution was

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separated, washed with brine, dried (Na2SO4), and concentrated. A reaction mixture of the

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residue and 5% Pd/C (0.20 g) in EtOAc (10 mL) was stirred under H2 gas at the ambient

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temperature for 2 h before filtration. The filtrate was concentrated, and then the residue was

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applied to silica gel column chromatography (EtOAc/hexane = 1/5) to give 3 (40 mg, 0.16

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mmol, 9% in 5 steps) as a colorless oil, [α]20D = -29 (c 0.5, CHCl3) ; 1H NMR (400 MHz,

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CDCl3) δ: 0.82 (3H, d , J = 6.4 Hz), 0.84 (3H, d, J = 6.4 Hz), 1.78-1.82 (2H, m), 2.38 (1H, dd,

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J = 13.3, 8.0 Hz), 2.43 (1H, dd, J = 12.5, 8.2 Hz), 2.60 (1H, dd, J = 12.5, 6.2 Hz), 2.64 (1H,

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dd, J = 13.3, 6.5 Hz), 4.77 (1H, s), 6.51 (1H, m), 6.62-6.67 (2H, m), 7.09-7.13 (2H, m), 7.13

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(1H, d, J = 7.2 Hz), 7.17 (1H, m), 7.24-7.27 (2H, m). 13C NMR (100 MHz, CDCl3) δ: 14.0,

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37.8, 38.1, 41.2, 41.4, 112.5, 115.8, 121.6, 125.6, 128.1, 129.1, 129.2, 141.6, 143.6, 155.3.

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EIMS m/z (%): 254 (M+, 62), 108 (100), 91 (73).

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254.1672, found 254.1665.

HRMS (EI) m/z calcd for C18H22O

14 15

(8R, 8’R)-2’-Fluorolignan-3-ol 4.

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31% yield through 5 steps as a colorless oil, [α]20D = -33 (c 0.8, CHCl3) ; 1H NMR (400 MHz,

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CDCl3) δ: 0.84 (3H, d , J = 6.7 Hz), 0.85 (3H, d, J = 6.8 Hz), 1.69-1.82 (2H, m), 2.37 (1H, dd,

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dd, J = 13.4, 6.0 Hz), 4.72 (1H, s), 6.54 (1H, m), 6.63 (1H, dd, J = 8.0, 2.7 Hz), 6.67 (1H, d, J

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= 7.6 Hz), 6.96-7.04 (2H, m), 7.06-7.17 (3H, m). 13C NMR (100 MHz, CDCl3) δ: 14.0, 34.1,

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37.4, 38.2, 41.1, 112.5, 115.1 (J = 22.8 Hz), 115.8, 121.6, 123.7 (J = 3.1 Hz), 127.3 (d, J =

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3.1 Hz), 128.4 (d, J = 15.4 Hz), 129.3, 131.3 (d, J = 5.1 Hz), 143.6, 155.3, 161.3 (d, J = 244.3

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Hz,).

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F NMR (376 MHz, CDCl3) δ: -119.1.

EIMS m/z (%): 272 (M+, 15), 109 (100).

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HRMS (EI) m/z calcd for C18H21O F 272.1576, found 272.1571.

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(8R, 8’R)-3’-Fluorolignan-3-ol 5

4


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CDCl3) δ: 0.82 (6H, d, J = 6.3 Hz), 1.76-1.80 (2H, m), 2.38 (1H, dd, J = 13.7, 8.3 Hz), 2.41

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(1H, dd, J = 13.7, 8.3 Hz), 2.58 (1H, dd, J = 13.6, 6.0 Hz), 2.62 (1H, dd, J = 13.6, 6.1 Hz),

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4.88 (1H, s), 6.55 (1H, m), 6.63-6.67 (2H, m), 6.79 (1H, d, J = 10.0 Hz), 6.83-6.90 (2H, m),

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7.11 (1H, dd, J= 7.8, 7.7 Hz), 7.19 (1H, m); 13C NMR (100 MHz, CDCl3) δ: 13.9, 14.0, 37.86,

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37.90, 41.07, 41.14, 112.5, (d, J = 21.1 Hz), 112.6, 115.6, 115.8, 121.6, 124.7 (d, J = 2.6 Hz),

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129.3, 129.4 (d, J = 8.3 Hz), 143.4, 144.2, (d, J = 7.3 Hz), 155.3, 162.8 (d, J = 244.9 Hz); 19F

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NMR (376 MHz, CDCl3) δ: -114.1. EIMS m/z (%): 272 (M+, 61), 108 (100). HRMS (EI)

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m/z calcd for C18H21O F 272.1576, found 272.1575.

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(8R, 8’R)-4’-Fluorolignan-3-ol 6

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CDCl3) δ: 0.81 (3H, d, J = 6.7 Hz), 0.83 (3H, d, J = 6.6 Hz), 1.72-1.79 (2H, m), 2.37 (1H, dd,

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dd, J = 13.4, 6.6 Hz), 4.79 (1H, s), 6.55 (1H, m), 6.63-6.67 (2H, m), 6.90-6.96 (2H, m),

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7.00-7.08 (2H, m), 7.11 (1H, dd, J = 7.8, 7.7 Hz); 13C NMR (100 MHz, CDCl3) δ: 13.87,

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13.93, 37.8, 38.2, 40.5, 41.2, 112.6, 114.8 (d, J = 21.0 Hz), 115.8, 121.6, 129.3, 130.2 (d, J =

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7.7 Hz), 137.1, (d, J = 3.0 Hz), 143.5, 155.3, 161.1 (d, J = 242.6 Hz);

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CDCl3) δ: -119.0. EIMS m/z (%): 272 (M+, 24), 109 (100). HRMS (EI) m/z calcd for

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C18H21O F, 272.1576, found 272.1573. 7


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F NMR (376 MHz,


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(8R, 8’R)-3’, 4’-Difuorolignan-3-ol 7

3


4


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(1H, dd, J = 13.3, 8.2 Hz), 2.57 (1H, dd, J = 13.3, 6.4 Hz), 2.58 (1H, dd, J = 13.6, 6.4 Hz),

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4.78 (1H, s), 6.56 (1H, m), 6.64-6.66 (2H, m), 6.77 (1H, m), 6.86 (1H, m) , 7.02 (1H, ddd, J

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= 10.3, 8.3, 8.3 Hz), 7.12 (1H, dd, J = 7.81, 7.78 Hz). 13C NMR (CDCl3) δ: 13.8, 13.9, 37.8,

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37.9, 40.5, 41.2, 112.7, 115.8, 116.7 (d, J = 16.6 Hz), 117.5 (d, J = 16.5 Hz), 121.5, 124.7 (dd,

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J = 5.7, 3.5 Hz), 129.3, 138.5 (J = 5.1, 4.2 Hz), 143.4, 148.6 (dd, J = 245.1, 12.6 Hz), 150.0 19

F NMR (376 MHz, CDCl3) δ: -139.8, -143.5. EIMS m/z

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(dd, J = 248.6, 14.9 Hz), 155.3.

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(%): 290 (M+, 44), 127 (66), 108 (100). HRMS (EI) m/z calcd for C18H20O F2 290.1481,

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found 290.1476.

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(8R, 8’R)-3’,4’,5’-Trifuluorolignan-3-ol 8

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29% yield through 5 steps as a colorless oil, [α]20D = -16 (c 0.5, CHCl3); 1H NMR (400 MHz,

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CDCl3) δ: 0.821 (3H, d, J = 6.7 Hz), 0.824 (3H, d, J = 6.6 Hz), 1.68-1.76 (2H, m), 2.35 (1H,

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dd, J = 13.7, 8.2 Hz), 2.39 (1H, dd, J = 13.4, 8.1 Hz), 2.56 (1H, dd, J = 13.7, 6.3 Hz), 2.57

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(1H, dd, J = 13.4, 6.4 Hz), 4.80 (1H, s), 6.57 (1H, m), 6.65-6.69 (4H, m), 7.13 (1H, dd, J =

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7.83, 7.75 Hz); 13C NMR (100 MHz, CDCl3) δ: 13.7, 13.9, 37.6, 37.8, 40.7, 41.1, 112.7,

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115.8, 121.5 129.4, 137.2 (d, J = 110.1 Hz), 138.5 (d, J = 133.6 Hz), 143.2, 150.8 (d, J =

21

252.8 Hz), 150.9 (d, J = 244.4 Hz), 155.4.

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-165.8. EIMS m/z (%): 308 (M+, 40), 145 (58), 108 (100). HRMS (EI) m/z calcd for

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C18H19O F3 308.1386, found 308.1381.

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F NMR (376 MHz, CDCl3) δ: -136.5, -165.7,

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(8R, 8’R)-2’-Methyllignan-3-ol 9

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CDCl3) δ: 0.82 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J = 6.7 Hz), 1.76 (1H, m), 1.84 (1H, m), 2.19

5

(3H, s), 2.39 (1H, br d, J = 13.5 Hz), 2.42 (1H, br d, J = 13.5 Hz), 2.58 (1H, dd, J = 13.6, 7.5

6

Hz), 2.63 (1H, dd, J = 13.6, 5.8 Hz), 4.73 (1H, s), 6.48 (1H, m), 6.62 (1H, dd, J = 8.1, 2.3

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Hz), 6.66 (1H, d, J = 7.6 Hz), 7.03 (1H, m), 7.08-7.09 (4H, m); 13C NMR (100 MHz, CDCl3)

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δ: 13.8, 14.3, 19.4, 36.3, 38.3, 38.8, 41.0, 112.5, 115.7, 121.5, 125.5, 125.7, 129.3, 130.0,

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130.2, 136.3, 139.8, 143.6, 155.3; EIMS m/z (%): 268 (M+, 97), 121 (28), 108 (100).

10

HRMS (EI) m/z calcd for C19H24O 268.1828, found 268.1830.

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(8R, 8’R)-3’-Methyllignan-3-ol 10.

13


14


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s), 2.37 (1H, dd, J = 13.5, 5.2 Hz), 2.39 (1H, dd, J = 13.5, 5.1 Hz), 2.59 (1H, br d, J = 13.5

16

Hz), 2.61 (1H, br d, J = 13.5 Hz), 4.80 (1H, br s), 6.53 (1H, m), 6.63 (1H, dd, J = 8.1, 2.1 Hz),

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6.67 (1H, d, J = 7.6 Hz), 6.89-6.90 (2H, m), 6.99 (1H, d, J = 7.5 Hz), 7.11-7.14 (2H, m). 13C

18

NMR (100 MHz, CDCl3) δ: 14.0, 14.1, 21.4, 37.8, 38.0, 41.16, 41.23, 112.5, 115.8, 121.6,

19

126.1, 126.3, 128.0, 129.2, 129.8, 137.6, 141.6, 143.6, 155.3; EIMS m/z (%): 268 (M+, 47),

20

108 (100). HRMS (EI) m/z calcd for C19H24O 268.1828, found 268.1823.

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(8R, 8’R)-4’-Methyllignan-3-ol 11

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8% yield through 5 steps as a colorless oil, [α]20D = -23 (c 0.6, CHCl3) ; 1H NMR (400 MHz, 9



1

CDCl3) δ: 0.81 (3H, d, J = 6.6 Hz), 0.82 (3H, d, J = 6.7 Hz), 1.74-1.83 (2H, m), 2.31 (3H, s),

2

2.35 (1H, dd, J = 13.6, 6.0 Hz), 2.38 (1H, dd, J = 13.6, 6.0 Hz), 2.60 (1H, dd, J = 13.6, 6.1

3


4

Hz), 6.66 (1H, d, J = 7.6 Hz), 6.97 (2H, d, J = 7.9 Hz), 7.05 (2H, d, J = 7.9 Hz), 7.09 (1H, dd,

5

J = 7.83, 7.78 Hz); 13C NMR (CDCl3) δ: 13.97, 13.99, 21.0, 37.8, 38.2, 40.9, 41.2, 112.5,

6

115.8, 121.6, 128.8, 128.9, 129.2, 135.0, 138.5, 143.6, 155.3; EIMS m/z (%): 268 (M+, 38),

7

107 (74), 105 (100); HRMS (EI) m/z calcd for C19H24O 268.1828, found 268.1827.

8 9

(8R, 8’R)-Lignane-2',3-diol 12

10


11


12


13

dd, J = 13.6, 6.1 Hz), 4.91 (1H, s), 5.04 (1H, s), 6.52 (1H, m), 6.63 (1H, dd, J = 8.0, 2.3 Hz),

14

6.67 (1H, d, J = 7.5 Hz), 6.71 (1H, d, J = 7.8 Hz), 6.83 (1H, dd, J = 7.8, 7.5 Hz), 7.02-7.06

15

(2H, m), 7.04 (1H, dd, J = 7.8, 7.7 Hz); 13C NMR (100 MHz, CDCl3) δ: 14.0, 14.1, 35.5, 36.3,

16

38.0, 41.1, 112.5, 115.3, 115.7, 120.6, 121.7, 127.0, 127.6, 129.3, 131.2, 143.6, 153.6, 155.2;

17

EIMS m/z (%): 270 (M+, 73), 107 (100); HRMS (EI) m/z calcd for C18H22O2 270.1621, found

18

270.1624.

19 20

(8R, 8’R)-Lignane-3,3'-diol 13

21

25% yield through 5 steps as colorless crystals, mp 67-68 oC, [α]20D = -14 (c 0.8, CHCl3); 1H

22

NMR (400 MHz, CDCl3) δ: 0.81 (6H, d, J = 6.7 Hz), 1.77 (2H, m), 2.40 (2H, dd, J = 13.6,

23

7.4 Hz), 2.53 (2H, dd, J = 13.6, 7.1 Hz), 5.49 (2H, s), 6.53 (2H, m), 6.65-6.68 (4H, m), 7.11 10


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(2H, dd, J = 7.8, 7.7 Hz); 13C NMR (100 MHz, CDCl3) δ: 13.9, 36.8, 41.1, 112.7, 115.8,

2

121.8, 129.3, 143.5, 155.2; EIMS m/z (%): 270 (M+, 76), 107 (100); HRMS (EI) m/z calcd

3

for C18H22O2 270.1621, found 270.1623.

4 5

(8R, 8’R)-Lignane-3,4'-diol 14

6

10% yield through 5 steps as a colorless oil, [α]20D = -38.0 (c 0.4, CHCl3) ; 1H NMR (400

7

MHz, CDCl3) δ: 0.81 (6H, d, J = 6.7 Hz), 1.68-1.79 (2H, m), 2.36 (1H, dd, J = 13.5, 7.9 Hz),

8


9

Hz), 5.40 (1H, s), 5.41 (1H, s), 6.50 (1H, m), 6.63-6.67 (2H, m), 6.73 (2H, m), 6.93 (2H, m),

10

7.10 (1H, dd, J = 7.78, 7.81 Hz); 13C NMR (CDCl3) δ: 13.85, 13.93, 37.3, 38.0, 40.4, 41.2,

11

112.6, 115.0, 115.7, 121.6, 129.2, 130.0, 133.8, 143.6, 153.3, 155.3; EIMS m/z (%): 270 (M+,

12

87), 107 (100); HRMS (EI) m/z calcd for C18H22O2 270.1621, found 270.1618.

13 14

(8R, 8’R)-2’-Methoxylignan-3-ol 15

15


16


17


18

dd, J = 13.1, 6.1 Hz), 3.72 (3H, s), 4.86 (1H, s), 6.50 (1H, m), 6.62 (1H, dd, J = 8.1, 2.2 Hz),

19

6.66 (1H, d, J = 7.5 Hz), 6.81 (1H, d, J = 8.1 Hz), 6.85 (1H, dd, J = 7.5, 7.4 Hz), 7.04 (1H, dd,

20

J = 7.3, 1.8 Hz), 7.09 (1H, dd, J = 7.9, 7.8 Hz), 7.16 (1H, ddd, J = 7.7, 7.7, 1.8 Hz). 13C NMR

21

(CDCl3) δ: 14.1, 14.2, 35.7, 36.1, 38.0, 41.1, 55.1, 110.3, 112.4, 115.7, 120.1, 121.6, 126.8,

22

129.1, 130.2, 130.7, 143.9, 155.3, 157.7; EIMS m/z (%): 284 (M+, 99), 121 (100), 107 (53);

23

HRMS (EI) m/z calcd for C19H24O2 284.1777, found 284.1771. 11



1 2


3


4


5

(1H, dd, J = 13.8, 8.0 Hz), 2.55-2.63 (2H, m), 3.78 (3H, s), 4.98 (1H, s), 6.52 (1H, m),

6

6.63-6.65 (2H, m), 6.68 (1H, d, J = 7.8 Hz), 6.71-6.75 (2H, m), 7.10 (1H, dd, J = 7.81, 7.78

7

Hz), 7.17 (1H, dd, J = 7.9, 7.7 Hz); 13C NMR (100 MHz, CDCl3) δ: 13.9, 14.0, 37.4, 37.6,

8

41.1, 41.4, 55.2, 110.9, 112.6, 114.8, 115.8, 121.6, 121.7, 129.0, 129.2, 143.3, 143.5, 155.4,

9

159.3; EIMS m/z (%): 284 (M+, 99), 121 (100), 107 (53); HRMS (EI) m/z calcd for C19H24O2

10

284.1777, found 284.1773.

11 12


13


14


15

br d, J = 13.5 Hz), 2.38 (1H, br d, J = 13.5), 2.55-2.61 (2H, m), 3.79 (3H, s), 4.87 (1H, s),

16

6.53 (1H, m), 6.63 (1H, dd, J = 8.4, 2.6 Hz), 6.66 (1H, d, J = 7.0 Hz), 6.81 (2H, m), 7.00 (2H,

17

m), 7.11 (1H, dd, J = 7.81, 7.78 Hz); 13C NMR (CDCl3) δ: 13.9, 14.0, 35.7, 38.2, 40.4, 41.2,

18

55.3, 112.5, 113.5, 115.8, 121.6, 129.2, 129.9, 133.7, 143.6, 155.3, 157.5; EIMS m/z (%): 284

19

(M+, 46), 121 (100); HRMS (EI) m/z calcd for C19H24O2 284.1777, found 284.1778.

20 21

(8R, 8’R)-2’-Chlorolignan-3-ol 18

22


23

CDCl3) δ: 0.83 (3H, d , J = 6.9 Hz), 0.88 (3H, d, J = 6.7 Hz), 1.84 (1H, m), 1.91 (1H, m), 12


Page 12 of 29

Page 13 of 29


1


2


3

Hz), 6.67 (1H, d, J = 7.6 Hz), 7.09-7.15 (4H, m), 7.31 (1H, m); 13C NMR (CDCl3)δ: 13.8,

4

14.2, 36.6, 38.4, 38.8, 41.0, 112.5, 115.8, 121.5, 126.3, 127.1, 129.2, 129.4, 131.3, 134.3,

5

139.2, 143.5, 155.3; EIMS m/z (%): 288 (M+, 37), 125 (55), 108 (100); HRMS (EI) m/z calcd

6

for C18H21OCl 288.1282, found 288.1288.

7 8


9

(8R, 8’R)-3-Benzyloxy-3’-chlorolignan was obtained from

10

(R)-3-(3-benzyloxybenzyl)-4-butanolide and 3-chlorobenzyl bromide by employing same

11

method described for the synthesis of 3. To a solution of (8R,

12

8’R)-3-benzyloxy-3’-chlorolignan (0.10 g, 0.35 mmol) in CH2Cl2 (5 mL) was added BBr3

13

(0.049 mL, 0.52 mmol) at -10 oC, and then the reaction solution was stirred at -10 oC for 30

14

min before addition of sat. aq. NaHCO3 solution. The organic solution was separated, washed

15

with brine, and dried (Na2SO4). Concentration followed by silica gel column chromatography

16

(EtOAc/hexane = 1/4) gave title compound (69 mg, 0.24 mmol, 32% in 5 steps) as a colorless

17

oil, [α]20D = -32 (c 0.9, CHCl3); 1H NMR (400 MHz, CDCl3) δ: 0.819 (3H, d, J = 6.6 Hz),

18

0.824 (3H, d, J = 6.6 Hz), 1.74-1.82 (2H, m), 2.37 (1H, dd, J = 13.6, 7.9 Hz), 2.39 (1H, dd, J

19

= 13.6, 8.0 Hz), 2.59 (1H, dd, J = 13.6, 8.0 Hz), 2.60 (1H, dd, J = 13.6, 8.6 Hz), 4.85 (1H, s),

20

6.55 (1H, m), 6.65 (1H, dd, J = 7.6, 2.8 Hz), 6.66 (1H, d, J = 7.2 Hz), 6.96 (1H, m), 7.08 (1H,

21

m), 7.10-7.19 (3H, m); 13C NMR (CDCl3) δ: 13.9, 14.0, 37.78, 37.82, 41.0, 41.1, 112.6, 115.8,

22

121.6, 125.8, 127.2, 129.0, 129.32, 129.34, 133.8, 143.4, 143.7, 155.3; EIMS m/z (%): 288

23

(M+, 25), 125 (29), 108 (100); HRMS (EI) m/z calcd for C18H21OCl 288.1282, found 13



1

288.1283.

2 3


4


5

CDCl3) δ: 0.81 (3H, d , J = 6.7 Hz), 0.82 (3H, d , J = 6.7 Hz), 1.73-1.79 (2H, m), 2.37 (1H,

6

dd, J = 13.6, 5.5 Hz), 2.39 (1H, dd, J = 13.6, 5.7 Hz), 2.58 (1H, dd, J = 13.4, 6.6 Hz), 2.59

7

(1H, dd, J = 13.4, 6.7 Hz), 4.72 (1H, s), 6.54 (1H, m), 6.64 (1H, dd, J = 7.9, 2.6 Hz), 6.65

8

(1H, d, J = 7.3 Hz), 6.96 (2H, d, J = 8.3 Hz), 7.11 (1H, dd, J = 7.8, 7.8 Hz), 7.21 (2H, m); 13C

9

NMR (CDCl3) δ: 13.89, 13.94, 37.8, 38.0, 40.7, 41.2, 112.6, 115.8, 121.6, 128.2, 129.3, 130.3,

10

131.3, 140.0, 143.4, 155.3; EIMS m/z (%): 288 (M+, 17), 125 (27), 108 (100); HRMS (EI)

11

m/z calcd for C18H21OCl 288.1282, found 288.1285.

12 13

Insects

14

Eggs of the common house mosquito Culex pipiens were purchased from Sumika

15

Techno Service, Co. Ltd. (Hyogo, Japan) and reared. Larvae were maintained at 25 °C under

16

a photoperiod of 12 h of light and 12 h of darkness.

17 18 19

Evaluation of the larvicidal activity The larvicidal activity of the test compounds was evaluated according to our earlier

20

report.7 Briefly, twenty larvae (10 larvae released in 1 mL of water × 2 tubes) were prepared

21

for each concentration, and the lethal concentration for inducing death in 50% of mosquito

22

larvae [LC50 (M)] 24 h after applying the compound was calculated by probit analysis.10

23

Acute toxic symptom at the concentration of 2.5 x 10-4 M was also monitored within 2 h 14


Page 14 of 29

Page 15 of 29


1

using the same procedure. The insecticidal assay was performed in triplicate. DMSO at the

2

concentration of 0.5% demonstrated no adverse effect on mosquitoes. Imidacloprid was

3

employed as a positive control.

4 5

Evaluation of the inhibitory activity of the chemicals on the O2 consumption of the

6

mitochondrial fraction

7

For the compound 6 inducing the highest acute toxic symptom in the present study

8

as well as the reference compounds 1 ((-)-DGA) and 2 (3-OH-DGA), the O2 consumption

9

inhibitory activity was measured. To compare 1, 2, and 6, the inhibitory activity of

10

3-methoxy-DGA derivatives (3-OMe-DGA) and (-)-secoisolariciresinol ((-)-SECO)

11

(compounds 21 and 22 in Figure 1), whose LC50 values were 4.73 x 10-5 and > 45 x 10-5 M

12

(mortality is 0% at this concentration), respectively,7 was also evaluated. In 10 mL of the ice

13

cold buffer A (3 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA),

14

2.8 g of rat liver was homogenized, followed by the centrifugation (500 x g, 10 min, 4 °C).

15

The resultant pellet was suspended in the buffer A, and the solution was centrifuged (8,000 x

16

g, 10 min, 4 °C; two times) to get the mitochondrial fraction (resuspended in buffer A). After

17

the protein concentration of the fraction was measured by the Bradford Reagent (Sigma) with

18

bovine serum albumin as the standard, it was adjusted to 5 mg/mL using buffer A. The

19

prepared mitochondrial fraction was kept at -80 °C before use.

20

In each well of the 96-well microplate (Nunc F96 MicroWell black polystyrene plate,

21

Thermo Fisher Scientific K. K., Yokohama, Japan), 1 µL of DMSO solution containing each

22

test sample, 50 µL of buffer B (3 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose, 1

23

mM EDTA, 125 mM succinic acid disodium salt), 50 µL of buffer C (3 mM Tris-HCl buffer 15



1

(pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 8.25 mM ADP), 100 µL of MitoXpress

2

probe solution (Luxcel Biosciences Ltd., Cork, Ireland; 0.1 µM of the solution diluted by

3

buffer A) and 50 µL of the mitochondrial fraction. The DMSO solutions without any samples

4

and 160 µM NaCN were employed as the negative and positive controls, respectively. After

5

addition of 100 µL of the mineral oil into each well, the fluorescence intensity was recorded

6

by using the fluorescence microplate reader (SH-8000Lab, Corona electric Co., Ltd.,

7

Hitachinaka, Japan) in the time-dependent manner (time-resolved mode; 30ºC, 3.3 min

8

intervals for 99 min, excitation 380 nm, emission 650 nm, and 100 µs as the delay and gate

9

times.). The final concentrations of the test samples other than SECO 22 were set to 4, 40,

10

120, and 200 µM. SECO was applied at the concentration of 3.98 x 10-3 M. The slope of the

11

intensity over time for each curve was calculated, and the concentration, which induced the

12

mean value of the slope values of the positive and negative controls, was defined as the EC50

13

value (M). The assay was performed in triplicate or more (n = 3 - 5).

14 15

RESULTS AND DISCUSSION

16

LC50 values of the synthesized lignan derivatives are shown in Table 1. Compared

17

with the activity of (−)-DGA and SECO (LC50 values were ca. 3.5–4.5 × 10−5 M), that of the

18

unsubstituted compound 3 was somewhat low (7 × 10−5 M). The fluorinated compounds 4–8

19

showed a higher activity than the unsubstituted compound 3. The 2-F derivative 4 showed the

20

highest activity, 3.5-fold higher than that of compound 3. Differing potency among the

21

positions of the substituents was not observed (compounds 4–6), and the potency of multiple

22

substituted derivatives (compounds 7 and 8) did not differ from that of the monofluorinated

23

compounds. Among the methylated compounds 9–11, 3-Me and 4-Me derivatives 16


Page 16 of 29

Page 17 of 29


1

(compounds 10 and 11) were 2- and 4-fold less potent, respectively, than 2-Me derivative

2

(compound 9), which was almost equipotent to the fluorinated compounds. The 4-OH

3

derivative 14 showed higher potency than the other OH derivatives 12 and 13, and the

4

3-OMe derivative 16 showed higher potency than that of the other positioned OMe

5

derivatives 15 and 17. The replacement of a fluorine atom of compounds 4–6 with a chlorine

6

atom retained the high activity. The ratio of the highest activity (2-F 4, 2.01 × 10−5 M) to the

7

lowest (4-Me 11, 12.1 × 10−5 M) was 6, demonstrating that the effect of the substituent at the

8

7′ phenyl moiety on activity was not as large when a compound had the 3-OH phenyl ring at

9

the 7 position, regardless of the physicochemical properties of substituents, such as

10

electron-withdrawing/donating and hydrophilic/hydrophobic features.

11

For all of the synthesized compounds, their acute toxic symptom against the larvae

12

of the mosquitoes was observed (Figure 3). The unsubstituted derivative 3 was equipotent to

13

3-OH DGA 2 (Figure 3A). Among the derivatives with an ortho-substituted phenyl group

14

(Figure 3B), the 2-F derivative 4 induced complete death of the larvae within 1 h, whereas

15

the 2-OH derivatives 18 induced a mortality of only 60% in 2 h [62 ± 12% (n = 3)]. The other

16

ortho-substituted compounds (2-Me, 2-OMe, and 2-Cl) showed almost the same curves as

17

one another. Among the meta-substituted compounds (Figure 3C), the 3-F derivative 5 killed

18

all of the mosquitoes at 75 min, showing some delay compared with the 2-F derivative. The

19

3-OMe derivative 16 did not completely kill the mosquitoes even after 120 min [mortality, 77

20

± 9% (n = 3)]. Among the para-substituted compounds (Figure 3D), 4-F derivative 6 killed

21

almost all larvae within 45 min, showing the fastest larvicidal activity among all of the

22

synthesized compounds. The 4-OH derivative 14 killed 70% of mosquitoes at 120 min

23

[mortality, 70 ± 10% (n = 3)]. Compound 2, inducing high mortality within 1 h (Figure 3A), 17



1

has 3-OMe as well as 4-OH groups, although the compound having either the 3-OMe or

2

4-OH group took much more time to kill the mosquito larvae. The reason why compound 2

3

induced acute toxic symptom remains unclear, but the multiple substituents may exert a

4

synergistic effect. The multifluorine-substituted derivatives 7 and 8 killed most of the

5

mosquitoes within 1 h, the same effectiveness was observed for the monofluorinated

6

derivatives. Replacement of a fluorine atom of compounds 4–6 with a chlorine atom

7

(compounds 18–20) reduced the speed of killing of the mosquito larvae (for example, ca.

8

100% mortality induced by the 4-Cl derivative 20 was observed 90–105 min after

9

application), suggesting that electron-withdrawing groups do not always induce rapid toxicity.

10

Ultimately, the unsubstituted derivative 3 and fluorinated compounds 4–8 were found to

11

exhibit higher toxicity than the others, suggesting that smaller substituents better promote

12

acute toxicity. The 3-OMe derivative 16, which did not kill mosquitoes perfectly within 2 h,

13

showed higher insecticidal activity (Table 1), suggesting that acute toxicity is not the primary

14

factor for inducing high insecticidal activity.

15

In search of clues to the mode of action of the active lignan derivatives, it was

16

investigated whether or not compounds 1, 2, 6 (selected for most acute toxicity), 21 (selected

17

for exerting almost the same insecticidal activity as compounds 2 and 6, but not acute

18

toxicity) and 22 (selected for exerting neither insecticidal activity nor acute toxicity) were

19

able to suppress the O2 consumption of the mitochondrial fraction, associated with the

20

respiratory system (Table 2). All of the insecticidal compounds tested in the O2 consumption

21

assay were found to show suppressive activity at almost the same concentration, whether

22

they induced acute toxic symptom or not. (−)-SECO 22 did not suppress the O2 consumption

23

even at the 50-fold higher concentration. Although the good correlation between the 18


Page 18 of 29

Page 19 of 29


1

insecticidal (LC50) and O2 consumption (IC50) activities was not observed (data not shown),

2

the insecticidal lignan derivatives suppressed the O2 consumption of the mitochondrial

3

fraction in vitro, suggesting that their insecticidal activity was associated, at least in part, with

4

the inhibition of the respiratory system. Compounds 1 and 21, which did not kill mosquito

5

larvae within 2 h, may take more time to reach target sites in insects, probably owing to their

6

hydrophobicity. As insecticides and acaricides, mitochondrial electron transport inhibitors,

7

such as tebufenpyrad and fenpyroximate (targeting complex I), have been developed,11 and

8

investigation of the novel structure of complex I inhibitors has been reported.12 In the present

9

study, it remains unclear how the synthesized lignan derivatives suppressed the O2

10

consumption in the mitochondrial fraction; however, it has been reported that a partially

11

demethylated DGA (2,3-dimethyl-1-(3,4-dihydroxyphenyl)-4-(4-hydroxy-3-methoxyphenyl)

12

butane) showed inhibitory activity toward mitochondrial succinoxidase and the

13

NADH-oxidase enzyme system,13 suggesting that the present lignan derivatives also inhibit

14

mitochondrial electron transport.

15

In summary, we identified 3-OH-DGA derivatives, in particular, monofluorinated

16

3-OH-DGA derivatives, showing insecticidal activity as well as acute toxicity against the

17

larvae of the mosquito, C. pipiens. Some of the compounds were found to inhibit the O2

18

consumption of the mitochondrial fraction, suggesting that they inhibit the respiratory system.

19

The insecticidal activity of these natural lignan-based compounds was lower than that of

20

imidacloprid, one of the most famous commercial neuroactive insecticides (LC50 value of

21

imidacloprid was 2.66 ± 0.52 x 10-7 M (n=3)). But the simple structure of the compounds

22

inducing acute larvicidal activity and structure-activity relationship elucidated in the present

23

study should be available for further structural modification leading to novel and useful 19



1

vector-control agents.

2 3 4 5

ACKNOWLEDGEMENTS Part of this study was performed at the INCS (Johoku station) of Ehime University. We are grateful to Marutomo Co. for financial support.

20


Page 20 of 29

Page 21 of 29


1

REFERENCES

2

1. Henrick, C.A. Methoprene. J. Am. Mosq. Control Assoc. 2007, 23, 225-239.

3

2. Dame, D.A.; Wichterman, G.J.; Hornby, J.A. Mosquito (Aedes taeniorhynchus)

4

resistance to methorprene in an isolated habitat. J. Am. Mosq. Control Assoc. 1998, 14,

5

200-203.

6

3. Cornel, A.J.; Stanich, M.A.; McAbee, R.D.; Mulligan, F.S. High level methoprene

7

resistance in the mosquito Ochlerotatus nigromaculis (Ludlow) in Central California. Pest

8

Manag. Sci. 2002, 58, 791-798.

9

4. Baumann, P.; Clark, M.A.; Baumann, L.; Broadwell, A.H. Bacillus sphaericus as a

10

mosquito pathogen: properties of the organism and its toxins. Microbiol. Rev. 1991, 55,

11

425-436.

12

5. Charles, J.F.; Nielson-LeRoux, C.; Delecluse, A. Bacillus sphaericus toxins: molecular

13

biology and mode of action. Annu. Rev. Entomol. 1996, 41, 451-472.

14

6. el-Bendary, M.A. Bacillus thuringiensis and Bacillus sphaericus biopesticides

15

production. J. Basic Microbiol. 2006, 46, 158-170.

16

7. Nishiwaki, H.; Hasebe, A.; Kawaguchi, Y.; Akamatsu, M.; Shuto, Y.; Yamauchi, S.

17

Larvicidal activity of (-)-dihydroguaiaretic acid derivatives against Culex pipiens. Biosci.

18

Biotechnol. Biochem. 2011, 75, 1735-1739.

19

8. Wukirsari, T.; Nishiwaki, H.; Hasebe, A.; Shuto, Y.; Yamauchi, S. First discovery of

20

insecticidal activity of 9,9′-epoxylignane and dihydroguaiaretic acid against houseflies and

21

the structure−activity relationship. J. Agric. Food Chem. 2013, 61, 4318-4325.

22

9. Moss, G.P. Nomenclature of lignans and neolignans (IUPAC recommendations 2000).

23

Pure Appl. Chem. 2000, 72, 1493-1523. 21



1

10. Finney, D.J. Probit analysis, 2 nd ed., Cambridge University Press, London. 1952.

2

11. Schuler, F.; Casida, J.E. The insecticide target in the PSST subunit of complex I. Pest

3

Manag. Sci. 2001, 57, 932–940.

4

12. Song, H.; Liu, Y.; Xiong, L.; Li, Y.; Yang, N.; Wang, Q. Design, synthesis, and

5

insecticidal evaluation of new pyrazole derivatives containing imine, oxime ether, oxime

6

ester, and dihydroisoxazoline groups based on the inhibitor binding pocket of respiratiory

7

complex I. J. Agric. Food Chem. 2013, 61, 8730-8736.

8

13. Heiser, R.W.; Cheng, C.C.; Pardini, R.S. Inhibition of mitochondrial electron transport

9

by partially demethylated dihydroguaiaretic acid. Pharmacol. Res. Commun. 1977, 9,

10

917-925.

11

22


Page 22 of 29

Page 23 of 29


1

Figure captions

2

Figure 1 Structures of (-)-dihydroguaiaretic acid, 1, its 3-OH derivatives, 2 - 20,

3

3-OMe-DGA derivative 21 and secoisolariciresinol 22.

4 5

Figure 2 Synthetic scheme of the compounds 3 - 20.

6 7

Figure 3 Time-dependent curves of the acute larvicidal activity against Culex pipiens of the

8

test chemicals within 2 h. No adverse effect on mosquitoes was observed in the control

9

within 2 h. A, 3-OH-DGA 2 and the unsubstituted 3, B, ortho-substituted phenyl derivatives

10

4, 9, 12, 15, and 18, C, meta-substituted phenyl derivatives 5, 10, 13, 16, and 19, D,

11

para-substituted phenyl derivatives 6, 11, 14, 17, 20, and multi-fluorinated derivatives 7 and

12

8.

13

23



1

Table 1 Larvicidal activity against Culex pipiens of 3-OH DGA derivatives 24 h after the

2

sample application (LC50 (M), mean ± SEM) Insecticidal activity No. 1

(−)-DGA

2 3-OH-DGA

Insecticidal activity

LC50 (x 10-5 M)

No.

LC50 (x 10-5 M)

3.52 ±

0.56 a

11

4.57 ±

1.27 a

12 2-OH

7.94 ± 0.15 ab

4-Me

12.1

±

0.44 b

3

H

7.12 ±

1.39 ab

13 3-OH

8.24 ± 2.24 ab

4

2-F

2.01 ±

0.49 a

14 4-OH

4.97

5

3-F

2.56 ±

0.50 a

15 2-OMe

5.93 ± 1.99 ab

6

4-F

3.66 ±

1.08 a

16 3-OMe

2.74 ± 0.58 a

7

3,4-F

2.50 ±

0.24 a

17 4-OMe

4.63 ± 1.29 a

8

3,4,5-F

2.37 ±

0.26 a

18

2-Cl

2.52 ± 0.26 a

9

2-Me

3.03 ±

0.95 a

19

3-Cl

2.23 ± 0.16 a

10

3-Me

6.13 ±

2.99 ab

20

4-Cl

2.56

± 0.69 a

± 0.33 a

3

a, b

4

from each other when labeled with the same letters (one-way ANOVA, Tukey; P < 0.05;

5

PRISM software 5.0). The LC50 value of imidacloprid (positive control) was 2.66 ± 0.52 x

6

10-7 M (n=3).

Data are represented as mean and standard error of mean (n=3). Not significantly different

7

24


Page 24 of 29

Page 25 of 29


1

Table 2 Oxygen consumption inhibitory activity of (-)-DGA derivatives (IC50 (M), mean ±

2

SEM) Oxygen consumption

Insecticidal

inhibitory activity a

activity

IC50 (x 10-5 M)

LC50 at 24 h

Mortality

(x 10-5 M)

at 45 min (%)

No.

Acute toxicity

1

(−)-DGA

6.89

± 2.69 (n =4)

3.52 ± 0.56 b

0

2

3-OH-DGA

6.78

± 2.41 (n =5)

4.57 ± 1.27 b

83 ± 4.4

6

4-F

4.03

± 0.83 (n =3)

3.66

21 3-OMe-DGA 5.08

± 1.52 (n =4)

4.73 ± 1.46 b

22

(−)-SECO

± 1.08 c

> 45 b

> 398 (n =3)

98

± 1.7 0 0

3

a

The IC50 value of NaCN (positive control) was 3.66 ± 0.02 x 10-6 M (n=3).

4

b

Cited from reference 7.

5

c

Cited from Table 1.

6

25



Page 26 of 29

1 2

Figure 1 H3C

H3C

O

O

OH

OH

CH3

CH3 HO

O H3C

CH3

CH3 HO (-)-Dihydroguiaretic acid (DGA), 1

3-OH-DGA, 2

CH3 HO R CH3

R = H (3), 2-F (4), 3-F (5), 4-F (6), 3,4-F (7), 3,4,5-F (8), 2-Me (9), 3-Me (10), 4-Me (11), 2-OH (12), 3-OH (13), 4-OH (14), 2-OMe (15), 3-OMe (16), 4-OMe (17), 2-Cl (18), 3-Cl (19), 4-Cl (20)

H3C

H3C O

O OH

OH

CH3 O

O

H3C

H3C CH3 HO 3-MeO-DGA, 21

HO (-)-Secoisolariciresinol (-)-SECO, 22

3 4 5 6

26


OH

Page 27 of 29


1 2

3 4 5 6

27



Page 28 of 29

1 2

Figure 3 A

B 100

2, 3-OH-DGA 3, H

80 60 40 20

Mortality (%)

Mortality (%)

100

0

60 40

4, 9, 12, 15, 18,

2-F 2-Me 2-OH 2-OMe 2-Cl

6, 7, 8, 11, 14, 17, 20,

4-F 3,4-F 3,4,5-F 4-Me 4-OH 4-OMe 4-Cl

20 0

0

30

60

90

120

0

Time (min)

C

30

60

90

120

Time (min)

D

100

100

5, 10, 13, 16, 19,

80 60 40

3-F 3-Me 3-OH 3-OMe 3-Cl

Mortality (%)

Mortality (%)

80

20 0

80 60 40 20 0

0

30

60

90

120

0

Time (min)

30

60

90

Time (min)

3 4 5 6

28


120

Page 29 of 29


1 2

3

TOC graphics

4 5

Larvae of Culex pipiens Mortality (%)

100

4-F

80 60 40 20 0 0

30

60

90

Time (min) 6 7

29


120

Acute Larvicidal Activity against Mosquitoes and Oxygen Consumption

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