Ester Derivatives, Their

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Cite This: J. Agric. Food Chem. 2018, 66, 12898−12910

Semisynthesis of Matrinic Acid/Alcohol/Ester Derivatives, Their Pesticidal Activities, and Investigation of Mechanisms of Action against Tetranychus cinnabarinus Bingchuan Zhang,†,§ Zhiqiang Sun,†,§ Min Lv,†,§ and Hui Xu*,†,‡ †

Research Institute of Pesticidal Design & Synthesis, College of Plant Protection and ‡College of Chemistry and Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, China

J. Agric. Food Chem. 2018.66:12898-12910. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 12/19/18. For personal use only.

S Supporting Information *

ABSTRACT: To discover new natural-product-based potential pesticides, 85 matrinic acid/alcohol/ester derivatives were synthesized by structural modifications of a quinolizidine alkaloid matrine. N-(4-Methyl)benzylmatrinyl n-decylate (76) and N(2-chloro)benzylmatrinyl n-undecylate (86) exhibited greater than seven-fold more pronounced acaricidal activity than matrine against Tetranychus cinnabarinus; N-(2-chloro)benzylmatrinyl benzoate (80) showed the most promising insecticidal activity against Mythimna separata. The carboxyl group of matrinic acids and introduction of n-decyl/n-undecylcarbonyl into matrinic alcohols were important for the acaricidal activity; introduction of alkyloxy into the carboxyl of matrinic acids and introduction of the electron-withdrawing groups on the N-benzyl of matrinic esters were necessary for the insecticidal activity. Through RTPCR and qRT-PCR analysis, it was shown that the lactam ring of matrine was vital for action on VGSC; opening the lactam ring of matrine and the alkylcarbonyl of side-chain were two important factors for acting with α1, α2, and α4 nAChR subunits; α1, α2, α4, and β3 subunits may be the target of action of compound 86 against T. cinnabarinus. KEYWORDS: matrine, structural modification, acaricidal activity, growth inhibitory activity, mechanism of action, natural product

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INTRODUCTION Matrine (1, Figure 1) (another name: matridin-15-one, and sophocarpidin), a well-known quinolizidine alkaloid, is the main component of the traditional Chinese medical herb Sophora f lavescens (Kushen). It could be also isolated from the roots of Sophora tonkinensis and Sophora alopecuroides (Kudouzi).1 Compound 1 and its derivatives exhibited lots of pharmacological effects such as relieving itching property,2 stimulating F508del-cystic fibrosis transmembrane conductance regulator activity,3 anticancer activity,4−6 anti-inflammatory property,7 antihepatitis B virus activity,8−10 and analgesic activity.11 On the other hand, it was reported that the compound 1-based extraction could be used as a botanical pesticide for controlling agricultural pests.12,13 More recently, to improve its pesticidal activities, structural modifications of compound 1 as a lead compound, focused on the C-14 and C-15 positions of its lactam ring, have been conducted in Wang’s and our groups.14−16 Although some derivatives showed promising pesticidal activities, there are still considerable differences in magnitude for activities between them and popular pesticides. Recently, structural modifications of natural products as lead compounds for the discovery and development of pesticidal candidates have received much attention.17−23 On the basis of the above results, and in continuation of our program to discover new potential pesticidal agents,24−26 in this paper we wanted to prepare a series of target compounds, matrinic ester derivatives (Figure 1), by opening the lactam ring of compound 1, followed by introduction of other functional groups. Their insecticidal and acaricidal activities were evaluated against two typically crop-threatening pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval. Moreover, mechanisms of action © 2018 American Chemical Society

of these derivatives against T. cinnabarinus were also investigated.

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MATERIALS AND METHODS

Chemicals. All reagents and solvents were of reagent grade or purified according to standard methods before use. Matrine (1) was purchased from Baoji Haoxiang Biotechnology Co. Ltd. (Baoji, China). Benzyl chloride, 4-fluorobenzyl bromide,4-chlorobenzyl chloride, 4bromobenzyl bromide, 4-methylbenzyl chloride, 2-chlorobenzyl chloride, N,N-diisopropylcarbodiimide (DIC), lithium aluminum hydride (LiAlH4), and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Ethyl acetate (EA), petroleum ether (PE), dichloromethane (DCM), ethanol (EtOH), and methanol (MeOH) were analytical grade and purchased from Kelong Chemical Reagent Co., Ltd. (Chengdu, China). Tetrahydrofuran (THF) was performed from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Anhydrous sodium sulfate (Na2SO4), cesium carbonate (Cs2CO3), sodium chloride (NaCl), sodium hydroxide (NaOH), and sodium bicarbonate (NaHCO3) were purchased from Guangzhou Jinhuada Chemical Reagent Co., Ltd. (Guangzhou, China). Analytical thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were performed with silica gel plates using silica gel 60 GF254 from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China). Silica gel column chromatography was performed with silica gel 200−300 mesh from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China). Instruments. Melting point (mp) was determined using the XT-4 digital melting point apparatus (Beijing Tech Instrument Ltd., Beijing, China). Optical rotation was measured using an Autopol III automatic Received: Revised: Accepted: Published: 12898

September 11, 2018 November 14, 2018 November 19, 2018 November 19, 2018 DOI: 10.1021/acs.jafc.8b04965 J. Agric. Food Chem. 2018, 66, 12898−12910

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of matrine (1) and target compounds. polarimeter (Rudolph Research Analytical, NJ). Proton nuclear magnetic resonance spectra were carried out with the Avance III 500 MHz equipment (Bruker, Karlsruhe, Germany). High resolution mass spectra (HRMS) were carried out with LTQ FT Ultra instrument (Thermo Fisher Scientific Inc., MA). X-ray crystallography was recorded on a SMART APEX II equipment (Bruker, Karlsruhe, Germany). Synthesis of Compound 2. A solution of matrine (1, 8.0 mmol) in 6 M aq. HCl (10 mL) was refluxed. After 6 h, when the reaction was complete checked by TLC analysis, MeOH (10 mL) was added to the mixture, which was stirred at room temperature for 3 h. Then the mixture was concentrated, and purified by silica gel column chromatography eluting with DCM/MeOH (5:1, v/v) to afford 2 in 97% yield. Data for Compound 2. CAS: 109616−04−8. White solid, mp 116− 117 °C (lit., not reported);27 [α]20D = −3 (c 2.4 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 3.67 (s, 3H, −COOCH3), 3.43−3.48 (m, 1H), 3.10 (dd, J = 4.0, 12.0 Hz, 1H), 2.79−2.85 (m, 2H), 2.35−2.45 (m, 3H), 2.21−2.27 (m, 1H), 1.90−2.03 (m, 7H), 1.79−1.83 (m, 1H), 1.53−1.68 (m, 5H), 1.42−1.47 (m, 3H). HRMS [ESI]: Calcd for C16H29N2O2 ([M + H]+), 281.2223; found, 281.2210. General Procedure for Synthesis of Compounds 3−8. A mixture of compound 2 (4.0 mmol), substituted benzyl bromide/ chloride (4.8 mmol), and Cs2CO3 (4.8 mmol) in dry DCM (10 mL) was stirred at room temperature, and the reaction process was checked by TLC analysis. After 6−12 h, the mixture was filtered, and the

solution was concentrated and purified by PTLC eluting with PE/EA (1:1 or 1:4, v/v) to give compounds 3−8 in 32−57% yields. Exemplary data for compounds 3 and 4 are as follows. Data for Compound 3. CAS: 1616292−80−8. Yield: 41%, pale yellow solid, mp 66−67 °C (lit., mp 72−73 °C);28 [α]20D = −15 (c 2.0 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.27−7.34 (m, 4H, Ar−H), 7.21−7.22 (m, 1H, Ar−H), 4.05 (d, J = 13.0 Hz, 1H), 3.62 (s, 3H, −COOCH3), 3.10 (d, J = 13.0 Hz, 1H), 2.83−2.86 (m, 2H), 2.77 (d, J = 11.5 Hz, 1H), 2.58−2.62 (m, 1H), 2.34 (d, J = 11.5 Hz, 1H), 2.26−2.28 (m, 2H), 2.03 (s, 1H), 1.85−1.96 (m, 3H), 1.65−1.80 (m, 7H), 1.55−1.60 (m, 1H), 1.31−1.42 (m, 5H). HRMS [ESI]: Calcd for C23H35N2O2 ([M + H]+), 371.2693; found, 371.2685. Data for Compound 4. Yield: 57%, white solid, mp 98−99 °C; [α]20D = −15 (c 2.0 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.26−7.28 (m, 2H, Ar−H), 6.96−7.11 (m, 2H, Ar−H), 4.05 (d, J = 13.5 Hz, 1H), 3.63 (s, 3H, OCH3), 3.06 (d, J = 13.5 Hz, 1H), 2.82−2.86 (m, 2H), 2.77 (d, J = 10.5 Hz, 1H), 2.56−2.60 (m, 1H), 2.26−2.29 (m, 3H), 2.03 (s, 1H), 1.85−1.95 (m, 3H), 1.67−1.76 (m, 7H), 1.56−1.60 (m, 1H), 1.34−1.42 (m, 5H). HRMS [ESI]: Calcd for C23H34FN2O2 ([M + H]+), 389.2598; found, 389.2583. General Procedure for Synthesis of Compounds 9−14. A mixture of compounds 3−8 (2.0 mmol) in a saturated solution of NaOH in MeOH (5 mL) was refluxed, and the reaction process was checked by TLC analysis. After 2−5 h, the pH value of the mixture was adjusted to 7. The mixture was concentrated, dissolved in DCM, filtered, concentrated, and purified by silica gel column chromatog12899

DOI: 10.1021/acs.jafc.8b04965 J. Agric. Food Chem. 2018, 66, 12898−12910

Article


HRMS [ESI]: Calcd for C22H34FN2O ([M + H]+), 361.2649; found, 361.2663. General Procedure for Synthesis of Compounds 33−86. A mixture of compounds 27−32 (0.5 mmol), substituted carboxylic acids (0.6 mmol), DIC (0.6 mmol), and DMAP (0.1 mmol) in DCM (5 mL) was stirred at room temperature, and the reaction process was checked by TLC analysis. After 2−6 h, the mixture was filtered, concentrated, and purified by silica gel column chromatography eluting with DCM/ MeOH (15:1, v/v) to afford compounds 33−86 in 40−85% yields. Exemplary data for compounds 33 and 34 are as follows. Data for Compound 33. Yield: 41%, light yellow viscous liquid; [α]20D = −7 (c 2.0 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.77 (d, J = 7.5 Hz, 1H, Ar−H), 7.37−7.43 (m, 2H, Ar−H), 7.32 (d, J = 7.0 Hz, 2H, Ar−H), 7.24−7.26 (m, 3H, Ar−H), 7.21 (d, J = 6.5 Hz, 1H, Ar−H), 4.34 (t, J = 6.0 Hz, 2H), 4.08 (d, J = 13.5 Hz, 1H), 3.12 (d, J = 13.5 Hz, 1H), 2.83−2.89 (m, 2H), 2.78 (d, J = 11.0 Hz, 1H), 2.64 (t, J = 12.0 Hz, 1H), 2.34 (d, J = 10.5, 1H), 2.04 (s, 1H), 1.85−1.96 (m, 3H), 1.62−1.78 (m, 9H), 1.53−1.57 (m, 1H), 1.34−1.42 (m, 5H). HRMS [ESI]: Calcd for C29H38ClN2O2 ([M + H]+), 481.2616; found, 481.2600. Data for Compound 34. Yield: 53%, light yellow viscous liquid; [α]20D = −7 (c 2.5 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.98 (s, 1H, Ar−H), 7.87 (d, J = 7.5 Hz, 1H, Ar−H), 7.51 (d, J = 8.5 Hz, 1H, Ar−H), 7.30−7.34 (m, 3H, Ar−H), 7.24−7.27 (m, 2H, Ar−H), 7.18−7.20 (m, 1H, Ar−H), 4.30−4.32 (m, 2H), 4.07 (d, J = 13.5 Hz, 1H), 3.13 (d, J = 13.5 Hz, 1H), 2.83−2.90 (m, 2H), 2.77 (d, J = 11.5 Hz, 1H), 2.66 (t, J = 12.0 Hz, 1H), 2.35 (dd, J = 3.0, 11.0 Hz, 1H), 2.04 (s, 1H), 1.85−1.95 (m, 3H), 1.70−1.79 (m, 6H), 1.65−1.67 (m, 1H), 1.60−1.63 (m, 3H), 1.50−1.53 (m, 1H), 1.39−1.42 (m, 2H), 1.34− 1.36 (m, 2H). HRMS [ESI]: Calcd for C29H38ClN2O2 ([M + H]+), 481.2616; found, 481.2600. Biological Assay. Acaricidal Activity of Compounds 1−86 against Tetranychus cinnabarinus.31,32 The acaricidal activity of compounds 1−86 against the female adults of Tetranychus cinnabarinus was assessed by slide-dipping method. Spirodiclofen (a commercial acaricidal agent) was used as a positive control. The solutions of compounds 1−86 and spirodiclofen were prepared in Tween-80 in water (0.1 g/L) at 0.5 mg/mL. For each compound, 90−120 healthy and size-consistency female adults of spider mites (30−40 mites per group) were selected. Then 30−40 spider mites were adfixed dorsally in two lines to a strip of double-coated masking tape on a microscope slide by using a small brush. Then the slides were dipped into the corresponding solution for 5 s and taken out. Excess solutions on the slides were removed by filter paper. The slides treated with Tween-80 in water (0.1 g/L) alone were used as a blank control group (CK). The experiment was carried out at 26 ± 1 °C and 60−80% relative humidity (RH) and on 14 h/10 h (light/dark) photoperiod. The results were checked by binocular dissecting microscope. Their mortalities were recorded at 48 and 72 h after treatment. Their corrected mortality rate values were calculated as follows: corrected mortality rate (%) = (T − C) × 100/ (100% − C); C is the mortality rate of CK, and T is the mortality rate of the treated T. cinnabarinus. Finally, the linear regressions of 72 h mortality rates (%) versus five concentrations of some potent compounds and spirodiclofen were obtained (Table S1), and the LC50 values were calculated. Growth Inhibitory Activity of Compounds 1−86 against Mythimna separata.18 Thirty early third-instar larvae of M. separata were chosen as the tested insects for each compound. The solutions of compounds 1−86 and toosendanin (a positive control) were prepared in acetone at 1 mg/mL. After dipped into the corresponding solution for 3 s, wheat leaf discs (1 × 1 cm2) were taken out and dried. Wheat leaf discs were treated by acetone alone as the blank control group (CK). Some above discs were added to each culture dish (ten insects per dish). Once the discs were consumed, additional ones were added. After 48 h, the rest of compound-soaked discs was cleaned out, and the untreated ones were added until the end of pupae (temperature, 25 ± 2 °C; RH, 65−80%; photoperiod, light/dark = 12 h/12 h). Their corrected mortality rate values were calculated as follows: corrected mortality rate (%) = (T − C) × 100/(100 − C); C is the mortality rate of CK, and T is the mortality rate of the treated M. separata.

raphy eluting with DCM/MeOH (5:1, v/v) to afford compounds 9−14 in 92−95% yields. Exemplary data for compounds 9 and 10 are as follows. Data for Compound 9. CAS: 1252598−43−8. Yield: 92%, white solid, mp 93−94 °C (lit., mp 95−96 °C);28 [α]20D = 1 (c 2.2 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.45 (d, J = 6.5 Hz, 2H, Ar− H), 7.33−7.34 (m, 3H, Ar−H), 4.23 (d, J = 9.5 Hz, 1H), 3.87 (d, J = 12.5 Hz, 1H), 3.59−3.61 (m, 1H), 3.15−3.20 (m, 1H), 2.93−3.00 (m, 2H), 2.54−2.60 (m, 2H), 2.20−2.36 (m, 3H), 2.04−2.11 (m, 3H), 1.79−1.93 (m, 5H), 1.67−1.71 (m, 2H), 1.34−1.54 (m, 5H). HRMS [ESI]: Calcd for C22H33N2O2 ([M + H]+), 357.2536; found, 357.2538. Data for Compound 10. CAS: 1345730−84−8. Yield: 95%, white solid, mp 85−86 °C (lit., mp 85−87 °C);29 [α]20D = 3 (c 2.2 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.20−7.23 (m, 2H, Ar−H), 6.90−6.93 (m, 2H, Ar−H), 3.93 (d, J = 12.5 Hz, 1H), 2.91−2.94 (m, 1H), 2.66−2.77 (m, 3H), 2.44−2.46 (m, 1H), 2.03−2.10 (m, 3H), 1.74−1.83 (m, 4H), 1.54−1.67 (m, 6H), 1.39−1.46 (m, 2H), 1.24− 1.30 (m, 3H), 1.15−1.16 (m, 2H). HRMS [ESI]: Calcd for C22H32FN2O2 ([M + H]+), 375.2442; found, 375.2443. General Procedure for Synthesis of Compounds 15−26. A mixture of compounds 9−14 (0.5 mmol), absolute ethanol (or isopentanol, 0.6 mmol), DIC (0.5 mmol), and DMAP (0.1 mmol) in DCM (5 mL) was stirred at room temperature, and the reaction process was checked by TLC analysis. After 6−20 h, the mixture was filtered, concentrated, and purified by silica gel column chromatography eluting with DCM/MeOH (15:1, v/v) to afford compounds 15−26 in 60− 85% yields. Exemplary data for compounds 15 and 16 are as follows. Data for Compound 15. CAS: 1572038−35−7. Yield: 60%, pale yellow solid, mp 90−91 °C (lit., mp 97−98 °C);28 [α]20D = −13 (c 4.2 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.34 (d, J = 7.0 Hz, 2H, Ar−H), 7.27−7.30 (m, 2H, Ar−H), 7.19−7.22 (m, 1H, Ar−H), 4.07−4.09 (m, 3H), 3.10 (d, J = 13.5 Hz, 1H), 2.87 (t, J = 10.5 Hz, 2H), 2.77 (d, J = 11.0 Hz, 1H), 2.63 (t, J = 12.0 Hz, 1H), 2.25−2.34 (m, 3H), 2.03 (s, 1H), 1.84−1.95 (m, 3H), 1.68−1.81 (m, 7H), 1.56−1.60 (m, 1H), 1.31−1.42 (m, 5H), 1.22 (t, J = 6.5 Hz, 3H, −COOCH2CH3). HRMS [ESI]: Calcd for C24H37N2O2 ([M + H]+), 385.2849; found, 385.2857. Data for Compound 16. Yield: 85%, white solid, mp 74−75 °C; [α]20D = −12 (c 4.7 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.29 (d, J = 13.5 Hz, 2H, Ar−H), 6.96−6.99 (m, 2H, Ar−H), 4.03−4.10 (m, 3H), 3.08 (d, J = 13.0 Hz, 1H), 2.83−2.86 (m, 2H), 2.78 (d, J = 9.5 Hz, 1H), 2.57−2.61 (m, 1H), 2.27−2.30 (m, 3H), 2.04 (s, 1H), 1.85− 1.96 (m, 3H), 1.67−1.77 (m, 7H), 1.54−1.60 (m, 1H), 1.35−1.46 (m, 5H), 1.22 (t, J = 6.5 Hz, 3H, −COOCH2CH3). HRMS [ESI]: Calcd for C24H36FN2O2 ([M + H]+), 403.2755; found, 403.2747. General Procedure for Synthesis of Compounds 27−32. A solution of compounds 3−8 (2.0 mmol) in THF (2 mL) was added dropwise to a solution of LiAlH4 (2.2 mmol) in THF (4 mL) at 0 °C, and the mixture was stirred at 0 °C for 15 min. Then the reaction was quenched by one drop of water. The mixture was filtered and washed by THF. Finally, the filtrate was concentrated to afford compounds 27−32 in 93−97% yields. Exemplary data for compounds 27 and 28 are as follows: Data for Compound 27. CAS: 1331769−73−3. Yield: 93%, pale yellow solid, mp 52−54 °C (lit., not reported);30 [α]20D = −14 (c 1.9 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.28−7.34 (m, 4H, Ar−H), 7.21−7.23 (m, 1H, Ar−H), 4.06 (d, J = 13.0 Hz, 1H), 3.63 (s, 2H), 3.16 (d, J = 13.5 Hz, 1H), 2.90−2.92 (m, 1H), 2.84 (d, J = 11.0 Hz, 1H), 2.77 (d, J = 10.5 Hz, 1H), 2.68 (t, J = 12.5 Hz, 1H), 2.35 (d, J = 11.5 Hz, 1H), 2.03 (s, 1H), 1.81−1.95 (m, 4H), 1.68−1.73 (m, 4H), 1.50−1.57 (m, 4H), 1.31−1.45 (m, 6H). HRMS [ESI]: Calcd for C22H35N2O ([M + H]+), 343.2743; found, 343.2733. Data for Compound 28. CAS: 1616339−15−1. Yield: 97%, pale yellow solid, mp 95−96 °C; [α]20D = −14 (c 2.0 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.28 (d, J = 6.0 Hz, 2H, Ar−H), 6.99 (m, 2H, Ar−H), 4.01 (d, J = 13.0 Hz, 1H), 3.64 (s, 2H), 3.12 (d, J = 12.5 Hz, 1H), 2.89−2.91 (m, 1H), 2.75−2.84 (m, 2H), 2.66 (t, J = 11.0 Hz, 1H), 2.29 (d, J = 10.0 Hz, 1H), 2.03 (s, 1H), 1.87−1.95 (m, 3H), 1.79−1.81 (m, 1H), 1.67−1.72 (m, 4H), 1.54−1.59 (m, 5H), 1.34−1.46 (m, 5H). 12900


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Figure 2. Synthesis of matrinic acid/alcohol/ester derivatives (2−86). Mechanisms of Action against T. cinnabarinus. Sample Preparation.33 The same-day eggs of T. cinnabarinus were placed on the back of leaves of Cowpea, which were maintained at 26 ± 1 °C and 60−80% RH, and on 14 h/10 h (light/dark) photoperiod. After 12 days, for each compound, 450 healthy and size-consistency three-dayold female adults of spider mites (150 mites per group) were selected out and placed on the back of fresh leaves of Cowpea. The solutions of compounds 1 (72 h LC50: 1.45 mg/mL), 59 (72 h LC50: 0.54 mg/mL), 80 (72 h LC50: 0.68 mg/mL), and 86 (72 h LC50: 0.19 mg/mL) were prepared in aq. Tween-80 (0.1 g/L) at 0.25 mg/mL. The solutions of two positive controls, imidacloprid and fenpropathrin, were prepared in aq. Tween-80 (0.1 g/L) at 0.055 and 0.017 mg/mL, respectively. Then the leaves of Cowpea were dipped into the corresponding solutions for 5 s and taken out. Excess solutions on the slides were removed by filter paper. The leaves treated with aq. Tween-80 (0.1 g/L) alone were used as a blank control group (CK). After 72 h, 180 alive spider mites of each treatment were selected out for the next experiments. Identification and Sequence Analysis of T. cinnabarinus Nicotinic Acetylcholine Receptor (nAChR) Subunits and Voltage-Gated

Sodium Channel (VGSC) Genes. The putative nAChRs and VGSC mRNAs from T. cinnabarinus were obtained from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/ nuccore/?term= Tetranychus+cinnabarinus+nicotinic+acetylcholine+receptor and https://www.ncbi.nlm.nih.gov/nuccore/?term= Tetranychus+cinnabarinus+voltagegated+sensitive+sodium+channel+mRNA). We obtained six different nAChR subunits, such as α1 (accession number: KP694226.1), α2 (accession number: KP694227.1), α4 (accession number: KP694228.1), α5 (accession number: KP761743.1), α7 (accession number: KP761744.1), and β3 (accession number: KP761745.1); and one VGSC (accession number: JX290514.1) in T. cinnabarinus. The primers for quantitative real-time polymerase chain reaction (qRTPCR) were designed using primer 3 online (http://primer3.ut.ee/). The prediction of transmembrane regions of the nAChR subunits was performed using TMHMMv.2.0 (http://www.cbs.dtu.dk/services/ TMHMM-2.0/). The SMART program provided by EMBL (http:// smart.embl-heidelberg.de/) was employed for the identification of 12901


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Figure 3. Five X-ray crystal structures of matrinic ester derivatives (36, top left; 43, top right; 52, middle; 70, bottom left; 71, bottom right).

modular domains. The neurotransmitter-gated ion-channel ligand binding domain (NC-LBD) was predicted by NCBI conserved domain

search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and UniProt (https://www.uniprot.org/). The N-terminal signal-peptide 12902


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Journal of Agricultural and Food Chemistry Table 1. Acaricidal Activity of Compounds 1−86 against T. cinnabarinus Treated at Concentration of 0.5 mg/mL corrected mortality rate (mean ± SE, %)

corrected mortality rate (mean ± SE, %)

compound

48 h

72 h

compound

48 h

72 h

spirodiclofen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

73.7 ± 1.5 11.2 ± 1.6 36.9 ± 2.1 34.0 ± 1.4 28.7 ± 1.3 37.4 ± 4.7 15.0 ± 1.2 39.1 ± 2.6 38.9 ± 2.3 18.1 ± 1.1 20.1 ± 3.2 18.1 ± 4.0 22.0 ± 1.4 17.4 ± 2.2 20.1 ± 2.7 2.1 ± 1.3 4.7 ± 1.0 6.0 ± 0.9 3.3 ± 1.1 4.9 ± 0.9 7.5 ± 0.9 4.8 ± 1.0 24.1 ± 0.8 3.3 ± 0.1 9.6 ± 0.6 6.3 ± 0.4 2.7 ± 1.1 42.4 ± 2.9 15.1 ± 2.0 49.6 ± 3.9 20.0 ± 2.1 27.7 ± 3.0 34.6 ± 2.3 18.5 ± 0.7 18.0 ± 0.8 30.8 ± 3.8 17.6 ± 1.3 30.0 ± 1.9 26.2 ± 1.3 34.9 ± 1.0 24.5 ± 1.8 34.6 ± 0.7 20.5 ± 1.7 30.6 ± 1.8

89.8 ± 0.9 27.9 ± 3.0 60.2 ± 2.1 54.4 ± 3.7 43.7 ± 2.0 56.3 ± 2.2 37.9 ± 1.7 51.1 ± 2.8 65.6 ± 4.8 81.0 ± 1.2 84.2 ± 1.6 80.1 ± 2.1 77.7 ± 1.8 76.2 ± 1.9 79.7 ± 1.6 30.3 ± 2.5 35.2 ± 0.8 18.8 ± 1.3 16.9 ± 3.0 55.1 ± 1.8 46.6 ± 1.5 45.2 ± 1.7 43.8 ± 1.4 29.9 ± 2.3 63.9 ± 1.7 38.6 ± 1.8 40.8 ± 7.2 67.0 ± 1.5 41.5 ± 1.6 83.0 ± 1.3 33.4 ± 3.9 42.9 ± 1.0 75.7 ± 3.5 24.3 ± 3.6 20.9 ± 1.3 55.8 ± 1.6 40.8 ± 1.7 54.7 ± 2.0 37.2 ± 2.4 56.6 ± 2.8 61.1 ± 2.0 67.5 ± 2.7 45.0 ± 5.1 55.7 ± 2.5

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 /

27.9 ± 2.1 33.3 ± 3.2 38.1 ± 2.7 32.3 ± 5.4 21.2 ± 3.2 46.1 ± 1.5 54.2 ± 2.3 33.1 ± 1.0 33.4 ± 2.2 39.8 ± 1.2 41.6 ± 1.8 34.0 ± 1.4 11.3 ± 3.5 37.5 ± 1.7 30.1 ± 1.7 25.2 ± 2.7 25.6 ± 1.0 29.3 ± 1.6 45.5 ± 0.8 8.7 ± 1.9 16.5 ± 0.4 27.1 ± 1.0 26.2 ± 3.3 47.5 ± 2.8 45.1 ± 2.7 21.5 ± 1.2 43.9 ± 1.5 26.7 ± 2.9 29.4 ± 2.8 30.4 ± 1.8 40.5 ± 2.8 22.6 ± 1.0 51.4 ± 2.7 38.6 ± 1.1 34.1 ± 2.2 21.1 ± 3.9 37.0 ± 1.4 35.0 ± 1.5 39.2 ± 3.7 33.3 ± 3.8 41.0 ± 1.3 65.9 ± 1.6 63.2 ± 0.8 /

65.2 ± 4.7 34.4 ± 4.3 54.0 ± 2.2 68.9 ± 0.8 43.1 ± 3.9 84.1 ± 1.1 85.6 ± 1.4 40.1 ± 2.9 57.8 ± 1.9 64.0 ± 1.0 67.5 ± 1.0 84.5 ± 1.5 49.0 ± 1.1 62.6 ± 3.1 49.4 ± 1.9 49.4 ± 1.0 56.6 ± 1.1 32.0 ± 3.3 61.4 ± 2.6 41.9 ± 2.6 39.7 ± 0.8 55.8 ± 3.1 30.1 ± 1.1 66.6 ± 1.2 67.6 ± 1.3 53.3 ± 3.2 80.7 ± 1.1 52.6 ± 3.9 32.5 ± 2.0 48.8 ± 1.5 59.1 ± 2.4 52.0 ± 2.3 84.7 ± 1.4 75.4 ± 3.3 56.9 ± 4.8 46.5 ± 1.5 41.8 ± 0.5 63.3 ± 1.6 67.4 ± 2.1 68.3 ± 1.4 66.5 ± 2.3 81.7 ± 1.6 89.5 ± 1.9 /

were separated in 1.8% agarose gels stained with ethidium bromide.34

(SP) cleavage sites were predicted using SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP/). Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Real-Time PCR (qRT-PCR) Analysis. Total RNA was extracted from treated or susceptible female adults of T. cinnabarinus using TRIzol reagent (invitrogen, Carlsbad, CA, USA). RNA was treated using RNase-free water and the concentration was measured using Nanodrop (Thermo Scientific Nanodrop 2000). cDNA was synthesized using the PrimerScript reverse transcription polymerase chain reaction (RT-PCR) kit (Takara, Japan). RT-PCR was performed in a 50 μL reaction containing 5 μL of 10 × TaKaRa Ex Taq buffer, 0.5 μL of TaKaRa Ex Taq (Takara, Japan), 1 μL of cDNA template, 4 μL of dNTP mixture, 2 μL of each primer (10 μmol/L), and 35.5 μL of sterile water. The PCR cycling parameters were as follows: 94 °C for 3 min, followed by 35 cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 1 min, with a final extension for 10 min at 72 °C. The RT-PCR products

Quantitative real-time PCR (qRT-PCR) was carried out with TB Green Advantage qPCR Primix (Takara, Japan) and an ABI 7500 Real Time PCR System (Applied Biosystems). The following cycling parameters were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 40 s. The relative expressed level of the translation β-actin was quantified to normalize the cDNA templates. Three biological replicates were measured. Relative quantification was performed via the comparative 2−ΔΔCT method.35 Gene-specific primers were designed using Primer Premier 5.0 and synthesized by BGI (Beijing, China). Differences were statistically evaluated with t-test using SPSS 17.0. 12903


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■

Table 2. LC50 Values of Some Compounds at 72 h against T. cinnabarinus

RESULTS AND DISCUSSION

Synthesis. The matrinic acid/alcohol/ester derivatives (2− 86) were prepared as shown in Figure 2. First, matrinic methyl ester (2) was obtained by reaction of matrine (1) with 6 M aq. HCl, followed by MeOH.27 Second, compound 2 reacted with substituted benzyl bromide/chloride in the presence of Cs2CO3 to give N-benzylmatrinic methyl ester derivatives (3−8).28 Third, hydrolysis of compounds 3−8 in the presence of NaOH afforded N-benzylmatrinic acid derivatives (9−14).29 Fourth, reduction of compounds 3−8 in the presence of LiAlH4 produced N-benzylmatrinic alcohol derivatives (27−32).36 Finally, compounds 9−14 reacting with ethanol/isopentanol, or compounds 27−32 reacting with carboxylic acids, in the presence of DIC and DMAP afforded matrinic ester derivatives (15−26 and 33−86).37 Their structures were determined by 1H NMR, optical rotation, HRMS, and mp. Moreover, threedimensional structures of compounds 36, 43, 52, 70, and 71 were unambiguously confirmed by single-crystal X-ray diffraction (Figure 3). Crystallographic data (excluding structure factors) of compounds 36, 43, 52, 70, and 71 have been deposited at the Cambridge Crystallographic Data Centre (CCDC) with deposition numbers of 1859864, 1859867, 1859868, 1859866, and 1859865, respectively. Pesticidal Activities. The 48 and 72 h acaricidal results of compounds 1−86 at 0.5 mg/mL against the female adults of T. cinnabarinus were shown in Table 1. Among all derivatives, compounds 9−14, 29, 32, 49, 50, 55, 70, 76, 77, 85, and 86 exhibited the pronounced acaricidal activity with 72 h mortality rates (MRs) greater than 70%; for example, 72 h MRs of compounds 9−14, 29, 32, 49, 50, 55, 70, 76, 77, 85, and 86 were between 75.4% and 89.5%, whereas the 72 h MR of compound 1 against T. cinnabarinus was only 27.9%. Notably, the matrinic acid/alcohol/ester derivatives showed more promising acaricidal activity than andrographolide-related esters and quinolinomatrine derivatives.16,32 The lactam ring of compound 1 was not absolutely essential to the acaricidal activity: opening the lactam ring of compound 1 led to a more potent compound 2, matrinic methyl ester (e.g., 60.2% (72 h MR of 2) vs 27.9% (72 h MR of 1)). Although introduction of different substituted benzyl groups on the nitrogen atom of compound 2 could not produce promising derivatives, hydrolysis of compounds 3−8 resulted in promising derivatives 9−14. Interestingly, once carboxylic acids 9−14 were turned into ester derivatives 15−26, the acaricidal activity of which was all decreased. It demonstrated that the carboxyl group of compounds 9−14 was necessary for the acaricidal activity. When the methyl ester of compounds 3−8 was changed to the methylene alcohol, two corresponding products 29 (72 h MR: 83.0%) and 32 (72 h MR: 75.7%) displayed the potent acaricidal activity. When the methylene alcohol group of compounds 27−32 was turned into ester groups to produce derivatives 33−86, compounds 49, 50, 55, 70, 76, 77, 85, and 86 showed the pronounced acaricidal activity. Among compounds 33−50 and 60−86, when R3 was ndecyl or n-undecyl, the corresponding derivatives all exhibited the potent acaricidal activity. It suggested that R3 as other alkyl groups could be further investigated. As described in Table 2, the 72 h LC50 values of 16 potent derivatives against T. cinnabarinus were calculated. The LC50 values of 9−14, 29, 32, 49, 50, 55, 70, 76, 77, 85, and 86 were between 0.19 and 0.37 mg/mL. Especially compounds 76 (R1 as 4-methylbenzyl, and R3 as n-decyl) and 86 (R1 as 2chlorobenzyl, and R3 as n-undecyl) exhibited >7-fold more

compound

LC50 (mg/mL)

regression equation

r

1 9 10 11 12 13 14 29 32 49 50 55 70 76 77 85 86 spirodiclofen

1.45 0.30 0.30 0.33 0.34 0.33 0.32 0.23 0.37 0.26 0.22 0.27 0.28 0.20 0.21 0.25 0.19 0.09

Y = −0.0522 + 1.5974X Y = −1.8594 + 2.7626X Y = −3.0455 + 3.2538X Y = −2.3878 + 2.9384X Y = −1.8899 + 2.7133X Y = −1.9138 + 2.7423X Y = −1.7021 + 2.6782X Y = −0.9989 + 2.5354X Y = −2.1491 + 2.7810X Y = −1.6468 + 2.7616X Y = −0.3053 + 2.2708X Y = −1.6626 + 2.7381X Y = −0.2285 + 2.1375X Y = −0.0612 + 2.1904X Y = 0.3887 + 1.9835X Y = −0.6271 + 2.3541X Y = −1.0193 + 2.6366X Y = 2.2129 + 1.4286X

0.9474 0.9910 0.9667 0.9921 0.9894 0.9938 0.9915 0.9971 0.9906 0.9961 0.9910 0.9934 0.9866 0.9953 0.9977 0.9957 0.9937 0.9871

potent acaricidal activity than compound 1 (72 h LC50 value: 1.45 mg/mL). As shown in Table 3, compounds 1−86 were tested their growth inhibitory activity against M. separata at 1 mg/mL. Compounds 6, 7, 10, 11, 14−23, 25, 26, 32, 33, 37, 39, 41, 45, 47, 48, 50, 51, 56, 57, 61, 62, 66, 73, 75, 76, and 80 displayed the promising activity with the final mortality rates (FMRs) greater than 50%. Among them, especially compound 80 displayed the most pronounced activity with the FMR of 70.4%. The percentages of FMRs at three different growth stages of some compounds and toosendanin (TN) against M. separata were described in Figure 4. The percentages of FMRs at the larval stage of compounds 1, 6, 33, 39, 45, 48, 50, 51, 56, 66, and 80 were larger than 42%, and these results were the same with those of TN, esters of fraxinellone C4/10-oxime, and some matrine ethers;15,18 especially the percentages of FMRs at the larval stage of compounds 1 and 51 were 78.6% and 70.0%, respectively. However, the percentages of FMRs at the larval stage of compounds 15, 17−19, 25, 26, 57, and 61 were less than 27%. Additionally, the largest partition of percentages of FMRs of compounds 17 and 19 was at the pupation stage, and these results were the same with those of some monosacchariderelated esters;32 the largest partition of percentages of FMRs of compounds 15, 18, 25, 26, 57, 61, and 76 was at the adult stage. It demonstrated that compounds 1, 6, 33, 39, 45, 48, 50, 51, 56, 66, and 80 may exhibit the same mechanism of action with TN against M. separata; similarly, compounds 17 and 19 may show the same mechanism of action with some monosacchariderelated esters against M. separata. Moreover, it further suggested that although some matrinic ester derivatives showed the potent insecticidal activity, the mechanism of action of matrinic esters, such as 15, 17−19, 25, and 26, might be different from that of esters 33, 39, 45, 48, 50, 51, 56, 66, and 80. The symptoms of the treated M. separata during the larval, pupation, and adult periods were expressed as the same as previously described.18,25 As shown in Figures S1−S3, the larvae with thin and wrinkled bodies died at the larval stage, some malformed and dead pupae appeared at the pupation period, and some malformed moths emerged at the adult emergence stage. 12904


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Table 3. Growth Inhibitory Activity of Compounds 1−86 against M. separata on Leaves Treated at Concentration of 1 mg/mL corrected mortality rate (%)a compound toosendanin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

10 days 31.0 34.5 20.7 20.7 27.6 6.9 24.1 10.4 3.4 13.8 6.9 13.8 6.9 10.4 17.2 3.4 6.9 10.4 6.9 13.8 13.8 3.4 13.8 6.9 6.9 13.8 10.4 13.8 10.4 13.8 17.2 24.1 34.5 24.1 10.4 24.1 17.2 31.0 24.1 37.9 3.4 27.6 20.7 24.1 24.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.4 3.4 3.4 3.4 0 0 3.4 3.4 3.4 3.4 0 3.4 0 3.4 0 3.4 0 3.4 0 3.4 3.4 3.4 3.4 0 0 3.4 3.4 3.4 3.4 3.4 0 3.4 3.4 3.4 3.4 3.4 0 3.4 3.4 0 3.4 0 3.4 3.4 3.4

25 days 39.3 35.7 32.1 25.0 25.0 39.3 35.7 14.3 3.6 42.9 25.0 35.7 32.1 35.7 39.3 25.0 10.7 10.7 14.3 39.3 17.9 21.4 25.0 39.3 32.1 35.7 7.1 17.9 7.1 14.3 17.9 25.0 35.7 42.9 14.3 32.1 25.0 32.1 28.6 46.4 28.6 35.7 21.4 25.0 32.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.6 0 3.6 0 0 3.6 0 0 0 3.6 0 0 3.6 0 3.6 0 3.6 3.6 0 3.6 3.6 3.6 0 3.6 3.6 0 3.6 3.6 3.6 0 3.6 6.2 0 7.1 0 7.1 6.2 7.1 3.6 6.2 3.6 0 7.1 6.2 3.6

corrected mortality rate (%)a 35 days

44.4 40.7 37.0 48.2 37.0 48.2 59.3 51.9 40.7 44.4 51.9 55.6 40.7 44.4 51.9 59.3 55.6 63.0 59.3 59.3 51.9 55.6 51.9 51.9 48.2 63.0 59.3 29.6 18.5 29.6 33.3 40.7 51.9 59.3 40.7 40.7 29.6 51.9 44.4 59.3 33.3 51.9 25.9 48.2 40.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

compound

0 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 0 3.7 0 3.7 0 3.7 3.7 0 3.7 3.7 3.7 3.7 0 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 6.4 3.7 3.7 3.7 3.7 3.7 3.7 3.7 6.4 3.7 0 3.7 3.7 3.7 3.7

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

a

10 days 34.5 34.5 24.1 27.6 10.4 27.6 44.8 20.7 20.7 10.4 20.7 31.0 10.4 20.7 10.4 17.2 13.8 27.6 24.1 17.2 24.1 24.1 24.1 20.7 27.6 27.6 6.9 6.9 17.2 34.5 10.4 20.7 20.7 17.2 24.1 44.8 13.8 20.7 24.1 13.8 17.2 17.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.4 3.4 3.4 0 3.4 6.0 3.4 3.4 3.4 3.4 3.4 3.4 3.4 6.9 3.4 6.0 3.4 0 3.4 0 6.9 3.4 3.4 3.4 6.0 0 0 0 0 3.4 3.4 3.4 3.4 0 3.4 3.4 3.4 3.4 3.4 6.9 6.0 6.0

25 days 39.3 32.1 42.9 35.7 7.1 32.1 53.6 25.0 25.0 17.9 25.0 46.4 25.0 32.1 21.4 14.3 32.1 46.4 32.1 21.4 28.6 32.1 25.0 17.9 35.7 28.6 21.4 14.3 28.6 35.7 10.7 28.6 17.9 21.4 25.0 53.6 28.6 25.0 28.6 32.1 14.3 17.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.6 3.6 3.6 0 3.6 3.6 3.6 0 6.2 3.6 6.2 6.2 6.2 3.6 3.6 6.2 3.6 6.2 3.6 3.6 7.1 3.6 0 3.6 0 3.6 3.6 6.2 7.1 6.2 7.1 3.6 3.6 3.6 0 3.6 3.6 6.2 3.6 3.6 6.2 7.1

35 days 63.0 40.7 51.9 59.3 29.6 59.3 63.0 29.6 25.9 29.6 40.7 66.7 59.3 37.0 25.9 18.5 63.0 51.9 37.0 40.7 29.6 59.3 48.2 29.6 48.2 40.7 37.0 33.3 51.9 44.4 51.9 59.3 48.2 40.7 29.6 70.4 29.6 25.9 37.0 37.0 29.6 37.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 6.4 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 6.4 3.7 0 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7

Values are the mean ± SE of three replicates.

acaricidal activity, all esters 15−26 showed potent insecticidal activity (FMRs: from 48.2% to 63.0%), and it demonstrated that the ester group was important for the insecticidal activity. Reduction of compounds 3−7 gave alcohols 27−31, the corresponding FMRs of which were all decreased sharply. To esters 33−86, compounds 45 (R1 as 4-fluorobenzyl, and R3 as 4methylphenyl), 51 (R1 as 4-chlorobenzyl, and R3 as 2chlorophenyl), 56 (R1 as 4-chlorobenzyl, and R3 as 4methoxyphenyl), 61 (R1 as 4-bromobenzyl, and R3 as 3chlorophenyl), and 80 (R1 as 2-chlorobenzyl, and R3 as phenyl) exhibited the pronounced insecticidal activity. It suggested that introduction of electron-withdrawing groups such as the fluorine, chlorine, and bromine atoms on the benzyl was

As described in Table 3, although opening the lactam ring of compound 1 cannot lead to a potent compound 2, introduction of substituted benzyl groups on the nitrogen atom of compound 2 afforded some promising derivatives (e.g., compounds 3, and 5−7). Especially compound 6 (FMR: 59.3%) containing 4bromobenzyl group exhibited more potent insecticidal activity than those containing 4-fluoro, 4-chloro, and 4-methylbenzyl groups. Hydrolysis of compounds 4 (containing 4-fluorobenzyl group), 5 (containing 4-chlorobenzyl group), and 8 (containing 2-chlorobenzyl group) resulted in promising derivatives 10, 11, and 14, respectively; however, once compound 6 (containing 4bromobenzyl group) was hydrolyzed to compound 12, the FMR was decreased from 59.3% to 40.7%. Interestingly, contrary to 12905


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Figure 4. Percentages of final mortality rates (FMRs) at three different growth stages of some compounds and toosendanin (TN) against M. separata.

Table 4. Primers of nAChR Subunits and VGSC of T. cinnabarinus Used for qRT-PCR

necessary for the insecticidal activity, and other electronwithdrawing groups (e.g., NO2, and CN) introduced on the benzyl could be investigated in the future. Mechanisms of Action against T. cinnabarinus. To compare the influence of different substituents to mechanisms of action against T. cinnabarinus, besides the most potent compound 86 (R1 as 2-chlorobenzyl, and R3 as n-undecyl), derivatives 59 (R1 as 4-chlorobenzyl, and R3 as n-undecyl) and 80 (R1 as 2-chlorobenzyl, and R3 as phenyl) were selected. Imidacloprid (IMI) was used as a positive control. The 72 h LC50 values of compounds 1, 59, 80, and 86 against T. cinnabarinus were 1.45, 0.54, 0.68, and 0.19 mg/mL, respectively. As shown in Table 4, the primers for qRT-PCR were designed using primer 3 online (http://primer3.ut.ee/). Previously, it was reported that matrine (1) exhibited more than 50% inhibition of [3H]IMI binding at 10 μM in membrane of Periplaneta americana nerve cords, and it indicated that compound 1 interacted with P. americana nAChRs.38 Therefore, we investigated compound 1 and its derivatives targeting on which of nAChR subunits of T. cinnabarinus. The schematic diagram of nAChR subunits such as α1, α2, α4, α5, α7, and β3 in T. cinnabarinus was described in Figure 5a. Changes of six nAChR subunits of T. cinnabarinus against compounds 1, 59, 80,

gene

sequence (5′−3′)b

β-actin

F-gtttggatttggctggtcgt R-tgctcaaagtcaagggcaac F-tgtctctccttcgcctcttg R-ctcggtgagtcaacattggc F-tggtgacttgttccgttgtg R-ggcggtttcatgagcagaat F-caacatcccttgcagttccc R-tgagtcgatggtgaacggaa F-gtcgttgcctgttcagttgt R-ttgaacttggtgaggcttgc F-ccagccactttcaccacaaa R-ggcaacaagagcaaacctga F-gcccatcatctaacaaaccca R-agccgtaaaagtagagccca F-tgacctgggctcgtaacaat R-aggtgttcttgggcgtctaa

α1 α2 α4 α5 α7 β3 VGSCa

annealing temperature (°C)

product size (bp)

60

145

60

158

60

136

60

121

60

181

60

119

60

170

60

85

a

VGSC: voltage-gated sodium channel. bF, forward primer; R, reverse primer.

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Figure 5. Expression patterns of nAChR subunits in female adults of T. cinnabarinus collected 72 h post-treatment with 0.25 mg/mL of each compound (imidacloprid: 0.055 mg/mL). (a) Schematic diagram of nAChR subunits in T. cinnabarinus. (b−f) nAChR subunits were evaluated through reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR). The mRNA expressions of nAChR subunits were normalized to β-actin expression (mean ± SD, n = 3). Asterisks indicate significant differences (P < 0.05) compared with CK. CK: blank control group. Red squares represent the N-terminal signal peptides (SP), blue squares represent the neurotransmitter-gated ion-channel ligand binding domain (NC-LBD), and green squares represent the transmembrane regions (TM).

86 and imidacloprid were evaluated through RT-PCR and qRT-

It showed that transcript abundance of nAChR subunits in female adults of T. cinnabarinus were different. As shown in Figure 5f, to imidacloprid (IMI), nAChR subunits α1, α2, α4, α5, and β3 of T. cinnabarinus were significantly up-regulated to 10.71-, 5.69-, 4.13-, 4.55-, and 3.13-

PCR (Figure 5b−f). The nAChR subunit α1 of CK was expressed at a high level, whereas subunits α4 and β3 of CK were expressed at low levels. 12907


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Journal of Agricultural and Food Chemistry fold, respectively. Because of IMI acting as an agonist for insects nAChR α or β subunits,39−45 nAChR subunits α1, α2, α4, α5, and β3 of T. cinnabarinus may interact with IMI. As shown in Figure 5b−e, compound 1 regulated the nAChR subunits α1, α5, α7, and β3 to 0.40-, 0.43-, 3.64-, and 44.81-fold, respectively; compound 59 up-regulated the nAChR subunits α1, α2, α4, α7, and β3 to 9.75-, 2.71-, 1.87-, 3.54-, and 14.08-fold, respectively; compound 80 regulated the nAChR subunits α7 and β3 to 0.03and 5.97-fold, respectively; compound 86 up-regulated the nAChR subunits α1, α2, α4, and β3 to 4.87-, 2.62-, 4.04-, and 6.13-fold, respectively. Interestingly, compounds 1, 59, 80, and 86 significantly increased the expression level of subunit β3 to 44.81-, 14.08-, 5.97-, and 6.13-fold, respectively (IMI: 3.13fold); the gene that encodes the nAChR subunit β3 has been reported as a modulator to regulate gene expression and nicotine intake behaviors, and mice lacking the subunit β3 showed reduced voluntary nicotine consumption compared that of wild type ones.46 N-Terminal extracellular domains in nAChR subunit β3 could enhance nAChR α6 gene expression in mouse, and truncated transcripts of subunit gene α6 are associated with spinosad resistance in Bactrocera dorsalis.47,48 For compounds 1, 59, 80, and 86 all highly up-regulated nAChR β3 subunit in female adults of T. cinnabarinus, the β3 subunit may be one of their targets of action in T. cinnabarinus. On the other hand, in Ctenocephalides felis and Drosophila melanogaster, nAChR α1 subunit in recombinant hybrid receptors conferred higher sensitivity to nicotinoids ((2S)nicotine) and neonicotinoid insecticides (e.g., IMI) than α2 subunit by the patch-clamp technique.49 In C. felis, α1 and α3 subunits contributed to nAChR populations that were sensitive to IMI.50 Mutations in nAChR α1 or α2 subunit can confer resistance to nitenpyram, IMI, and thiamethoxam in D. melanogaster.51 For compounds 59 and 86 increased the expression level of α1 subunit to 9.75- and 4.87-fold, respectively (IMI: 10.71 folds), the α1 subunit may be another of their targets of action in T. cinnabarinus. It is noteworthy that after structural modifications of compound 1, the mRNA expressions of nAChR subunits were different to compound 1 with its derivatives 59, 80, and 86 (Figure 5b−e). The expressions of α1 subunit to compounds 59 (R1 as 4-chlorobenzyl, and R3 as n-undecyl) and 86 (R1 as 2chlorobenzyl, and R3 as n-undecyl) were increased to 9.75- and 4.87-fold, respectively, whereas that to compound 1 was decreased to 0.40-fold, and that to compound 80 (R1 as 2chlorobenzyl, and R3 as phenyl) was no obvious change; the expressions of α2 subunit to compounds 59 and 86 were increased to 2.71- and 2.62-fold, respectively; however, those to compounds 1 and 80 were no obvious change; similarly, the expressions of α4 subunit to compounds 59 and 86 were increased to 1.87- and 4.04-fold, respectively, but those to compounds 1 and 80 were not obvious. It demonstrated that opening the lactam ring of compound 1 and the esters functional group of side-chain were important for acting with α1, α2, and α4 subunits. Additionally, because compound 1 could inhibit tetrodotoxinsensitive (TTX-S) sodium currency of Helicoverpa armigera in a concentation-dependent manner using patch-clamp analysis,52 the possible mechanism of action of compound 1 and its derivatives against voltage-gated sodium channel (VGSC) in female adults of T. cinnabarinus was also studied. It was reported that a mutation (F1538I), identified from the sodium channel gene of fenpropathrin (FN)-resistant CSM, played an important role in FN resistance in T. cinnabarinus.53 In citrus red mite,

Figure 6. Expression patterns of voltage-gated sodium channel (VGSC) in female adults of T. cinnabarinus collected 72 h posttreatment with 0.25 mg/mL of each compound (fenpropathrin: 0.017 mg/mL). (a) Schematic diagram of the VGSC in T. cinnabarinus. (b) VGSC was evaluated through RT-PCR and qRT-PCR. The mRNA expression of VGSC was normalized to β-actin expression (mean ± SD, n = 3). Asterisks indicate significant differences (P < 0.05) compared with CK. CK: blank control group. FN: fenpropathrin. Red squares represent the N-terminal signal peptides (SP), and green squares represent the transmembrane regions (TM).

Panonychus citri, F1538I mutation was also vital to FN resistance.54 FN was used as a positive control. The schematic diagram of the VGSC in T. cinnabarinus was shown in Figure 6a. The mRNA expressions of VGSC to compounds 1, 59, 80, and 86 were described in Figure 6b. Compound 1 and FN upregulated VGSC gene in T. cinnabarinus to 4.93- and 3.89-fold, respectively, whereas its derivatives 59, 80, and 86 did not affect VGSC expressions at all. It suggested that the lactam ring of compound 1 was very necessary for action on VGSC. In conclusion, a series of matrinic acid/alcohol/ester derivatives were prepared from matrine. Their structures were characterized by proton nuclear magnetic resonance spectra, optical rotation, and high-resolution mass spectra. Especially steric configurations of five compounds were confirmed by single-crystal X-ray diffraction. Among them, compounds 76 and 86 exhibited >7-fold more potent acaricidal activity than matrine against T. cinnabarinus. Compound 80 showed the most promising insecticidal activity against M. separata. It was found that, for the acaricidal activity, the lactam ring of matrine was not necessary; the carboxyl group of matrinic acids was important; introduction of n-decyl/n-undecylcarbonyl into matrinic alcohols was vital. In addition, for the insecticidal activity, introduction of alkyloxy into the carboxyl of matrinic acids was necessary, and introduction of the electron-withdrawing groups on the N-benzyl of matrinic esters was important. It is noteworthy that the lactam ring of matrine was necessary for action on VGSC in T. cinnabarinus, and opening the lactam ring of matrine and the functional group such as alkylcarbonyl of 12908


Article

Journal of Agricultural and Food Chemistry side-chain were two important factors for acting with α1, α2, and α4 nAChR subunits of T. cinnabarinus. Compound 86 significantly induced the nAChR subunits α1, α2, α4, and β3 expression level of T. cinnabarinus, and α1, α2, α4, and β3 subunits may be the target of action of compound 86 to T. cinnabarinus. To confirm whether compound 86 targets on nAChR subunits of T. cinnabarinus, the related experiments about using RNAi of nAChR subuints and voltage clamp technique will be conducted in our group. These results will pave the way for future structural modifications and application of matrine derivatives as pesticides for agriculture.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04965.

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Spectra of 1H NMR; data on 1H NMR, HRMS, optical rotation, and melting points of target compounds; 72 h corrected mortality rates at five different concentrations of some compounds against T. cinnabarinus (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 8629-8709-1952. Fax: 8629-8709-1952. ORCID

Hui Xu: 0000-0002-3152-9922 Author Contributions §

These authors contributed equally to this work.

Funding

The present research was supported by National Natural Science Foundation of China (Project No. 21877090), Key R&D Program of Shaanxi Province (No. 2018NY-153), and Special Funds of Central Colleges Basic Scientific Research Operating Expenses, Northwest A&F University (Project No. 2452015096). Notes

The authors declare no competing financial interest.

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