Semisynthesis of Matrinic Acid/Alcohol/Ester Derivatives, Their Pesticidal Activities, and Investigation of Mechanisms of Action against Tetranychus cinnabarinus

Article abstract of DOI:10.1021/acs.jafc.8b04965

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)benzylmatri

Full text of DOI:10.1021/acs.jafc.8b04965

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Agricultural and Environmental Chemistry
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
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04965 • Publication Date (Web): 19 Nov 2018
Downloaded from http://pubs.acs.org on November 23, 2018
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Journal of Agricultural and Food Chemistry
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 712100, Shaanxi
Province, China
*H. Xu, Tel: 8629-8709-1952. Fax: 8629-8709-1952. E-mail: orgxuhui@nwsuaf.edu.cn.
§These authors contributed equally to this work.
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Abstract
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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 7-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 RT-PCR and qRT-PCR analysis, it showed
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.
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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 flavescens (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
anti-hepatitis 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 Based
on the above results, and in continuation of our program to discover new potential pesticidal
agents,24-26 therefore, 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 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 Bio-technology Co. Ltd.
(Baoji, China). Benzyl chloride, 4-fluorobenzyl bromide ， 4-chlorobenzyl chloride,
4-bromobenzyl
bromide,
4-methylbenzyl
chloride,
2-chlorobenzyl
chloride,
N,N-diisopropylcarbodiimide (DIC), lithium aluminium 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), caesium 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 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
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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.
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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:
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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),
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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.
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Data for Compound 4: Yield: 57%, white solid, mp 98–99 °C; [α]20D = -15 (c 2.0 mg/mL,
1
CHCl3); H 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.
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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 chromatography 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:
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Data for Compound 9: CAS: 1252598-43-8. Yield: 92%, white solid, mp 93–94 °C (lit., mp
1
95–96 °C);28 [α]20D = 1 (c 2.2 mg/mL, CHCl3); H 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.
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Data for Compound 10: CAS: 1345730-84-8. Yield: 95%, white solid, mp 85–86 °C (lit., mp
1
85–87 °C);29 [α]20D = 3 (c 2.2 mg/mL, CHCl3); H 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.
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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:
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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.
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Data for Compound 16: Yield: 85%, white solid, mp 74–75 °C; [α]20D = -12 (c 4.7 mg/mL,
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CHCl3); H 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,
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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.
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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:
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Data for Compound 27: CAS: 1331769-73-3. Yield: 93%, pale yellow solid, mp 52–54 °C
(lit., not reported);30 [α]20 = -14 (c 1.9 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ:
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D
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.
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Data for Compound 28: CAS: 1616339-15-1. Yield: 97%, pale yellow solid, mp 95–96 °C;
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[α]20D = -14 (c 2.0 mg/mL, CHCl3); H 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),
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1.34-1.46 (m, 5H). HRMS [ESI]: Calcd for C22H34FN2O ([M+H]+), 361.2649; found,
361.2663.
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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:
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Data for Compound 33: Yield: 41%, light yellow viscous liquid; [α]20D = -7 (c 2.0 mg/mL,
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CHCl3); H 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.
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Data for Compound 34: Yield: 53%, light yellow viscous liquid; [α]20D = -7 (c 2.5 mg/mL,
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CHCl3); H 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),
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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.
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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. 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 h 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
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3rd-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 cm)
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 till 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.
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-day-old 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
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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
(https://www.ncbi.nlm.nih.gov/nuccore/?term=Tetranychus+cinnabarinus+nicotinic+acetylch
oline+receptor and
Information
(NCBI)
https://www.ncbi.nlm.nih.gov/nuccore/?term=Tetranychus+cinnabarinus+voltage-gated+sensi
tive+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 (qRT-PCR)
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 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 (SP) cleavage sites were
predicted using SignalP 4.1 server ( http://www.cbs.dtu.dk/services/SignalP/).
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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 PrimerScriptTM 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 were separated in
1.8% agarose gels stained with ethidium bromide.34 Quantitative real-time PCR (qRT-PCR)
was carried out with TB GreenTM 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-ΔΔC method.35 Gene-specific primers were designed using
T
Primer Premier 5.0 and synthesized by BGI (Beijing, China). Differences were statistically
evaluated with t-test using SPSS 17.0.
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.
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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
1
(15–26 and 33–86).37 Their structures were determined by H NMR, optical rotation, HRMS
and mp. Moreover, three-dimensional 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 h 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
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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 n-decyl 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 sixteen 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 2-chlorobenzyl, and R3 as n-undecyl) exhibited > 7 folds more
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
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three different growth stages of some compounds and toosendanin (TN) against M. separata
were descripted 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. Whereas 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 monosaccharide-related 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 monosaccharide-related 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.
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
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compound 6 (FMR: 59.3%) containing 4-bromobenzyl 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 4-bromobenzyl group) was
hydrolyzed to compound 12, the FMR was decreased from 59.3% to 40.7%. Interestingly,
contrary to 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 4-methylphenyl), 51 (R1 as 4-chlorobenzyl, and R3 as
2-chlorophenyl), 56 (R1 as 4-chlorobenzyl, and R3 as 4-methoxyphenyl), 61 (R1 as
4-bromobenzyl, and R3 as 3-chlorophenyl), 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 necessary for the insecticidal activity, and other electron-withdrawing groups (e.g., NO2,
and CN) introduced on the benzyl could be investigated in the future.
Mechanisms of Action against T. cinnabarinus.
In order 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
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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, 86 and
imidacloprid were evaluated through RT-PCR and qRT-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. 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 folds,
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 folds, 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 folds, respectively; compound 80 regulated the
nAChR subunits α7 and β3 to 0.03 and 5.97 folds, respectively; compound 86 up-regulated
the nAChR subunits α1, α2, α4 and β3 to 4.87, 2.62, 4.04 and 6.13 folds, 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 folds, respectively (IMI: 3.13 folds); The gene that
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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 folds, 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 2-chlorobenzyl, and R3 as n-undecyl) were increased to 9.75
and 4.87 folds, respectively, whereas that to compound 1 was decreased to 0.40 folds, and that
to compound 80 (R1 as 2-chlorobenzyl, 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 folds,
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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 folds,
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 tetrodotoxin-sensitive (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, 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 up-regulated VGSC gene in T.
cinnabarinus to 4.93 and 3.89 folds, 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 folds more potent acaricidal activity than matrine against T.
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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 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.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI: Spectra of 1H NMR, data on 1H NMR, HRMS, optical rotation and melting points of
target compounds, and 72 h corrected mortality rates at five different concentrations of some
compounds against T.cinnabarinus.
AUTHOR INFORMATION
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Corresponding Author
*(H.X.) Phone/fax: +86-29-87091952. E-mail: orgxuhui@nwsuaf.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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).
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