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Application of sustainable natural resources in crop protection: Podophyllotoxin-based botanical pesticides derived from Podophyllum hexandrum for controlling crop-threatening insect pests
Industrial Crops & Products 107 (2017) 45–53
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Application of sustainable natural resources in crop protection: Podophyllotoxin-based botanical pesticides derived from Podophyllum hexandrum for controlling crop-threatening insect pests Ruige Yanga,1, Xiaobo Huanga,1, Zhiping Chea,1, Yuanyuan Zhanga, Hui Xua,b,
MARK
⁎
a Research Institute of Pesticidal Design & Synthesis, College of Chemistry and Pharmacy/Plant Protection, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China b Shaanxi Key Laboratory of Natural Products & Chemical Biology, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Podophyllum hexandrum Podophyllotoxin Botanical pesticide Natural bioresource
Podophyllotoxin is isolated as a sustainable natural bioresource from the roots and rhizomes of Podophyllum hexandrum and Juniperus Sabina. In continuation of our program aimed at the discovery of biorenewable natural product-based pesticides, a series of 4α-acyloxy derivatives of 2'-chloro-5-bromopicropodophyllotoxin or 2',5dibromopicropodophyllotoxin were synthesized by structural modification of podophyllotoxin, and evaluated as insecticidal and acaricidal agents against two serious agricultural pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval. Among all derivatives, compounds 6a and 7a exhibited more promising insecticidal and acaricidal activities than podophyllotoxin, and the final mortality rates of 6a, 7a and 1 against M. separata (at 1 mg/mL) and T. cinnabarinus (at 0.5 mg/mL) were 50%/53.3%/36.7%, and 33%/41.8%/1.6%, respectively. This suggested that introduction of the acetoxy moiety at the C-4 position of 2'-chloro-5bromopicropodophyllotoxin/2',5-dibromopicropodophyllotoxin was necessary for the insecticidal and acaricidal activities.
1. Introduction Mythimna separata Walker (Oriental armyworm) and Tetranychus cinnabarinus Boisduval (Spider mite) are two crop-threatening insect pests, and their infestations are extremely hard to prevent and control (Jiang et al., 2011). In 2012, the intermittent outbreaks of M. separata widely occurred in China, and about 4 million hectares of crops were complete loss (Zeng et al., 2013). Additionally, the long-term and unreasonable use of synthetic agrochemicals to cope with these serious insect pests has led to insect resistance and environmental problems (Sun et al., 2012; Guillette and Iguchi, 2012). With the drive toward increasing demand for organic food, botanical pesticides, due to their less or slower resistance development and lower pollution, have received much more attention in the agricultural field when compared with conventional chemical pesticides (Isman, 2006; Seiber et al., 2014). It was reported that about 69 botanical pesticides were registered and commercialized in the United States at the end of 2013 (Zhang et al., 2015). Podophyllotoxin (1, Fig. 1) contains four consecutive chiral centers
(labeled C-1–C-4), and is isolated as a sustainable natural bioresource from the roots and rhizomes of Podophyllum hexandrum and Juniperus Sabina (Xu et al., 2009). In addition to preparation of potent anticancer drugs such as etoposide, teniposide and etoposide phosphate as a lead compound (Desbene and Giorgi-Renault, 2002; Gordaliza et al., 2004; Lv and Xu, 2011; You, 2005), compound 1 also showed a variety of interesting insecticidal (Miyazawa et al., 1999; Gao et al., 2004; Zhi et al., 2013, 2014), antifungal (Kumar et al., 2007), antiviral (Castro et al., 2003; MacRae et al., 1989; Saitoh et al., 2008; Zhu et al., 2004), anti-inflammatory (Guerrero et al., 2013), immunosuppressive (Gordaliza et al., 2001), antirheumatic (Carlstrom et al., 2000), and hypolipidemic activities (Iwasaki et al., 1995). More recently, halogenation of E-ring of podophyllotoxin has been investigated in our group, and some 2′(2′,6′)-(di)halogen-substituted podophyllotoxin derivatives (I and II, Fig. 1) showed more pronounced insecticidal activity than toosendanin against pre-third-instar larvae of Mythimna separata (Che et al., 2013a; 2013b; Fan et al., 2015). Meanwhile, two unexpected intermediates, 2'-chloro-5-bromopodophyllotoxin (2, Fig. 1) and 2',5-dibromopodophyllotoxin (3, Fig. 1), were produced (Che et al.,
⁎ Corresponding author at: Research Institute of Pesticidal Design & Synthesis, College of Chemistry and Pharmacy/Plant Protection, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China. E-mail address: [email protected] (H. Xu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.indcrop.2017.05.033 Received 20 February 2017; Received in revised form 7 May 2017; Accepted 17 May 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of podophyllotoxin (1), 2'-chloro-5-bromopodophyllotoxin (2), 2',5-dibromopodophyllotoxin (3), 2' −chloro-5-bromo-4α-propionyloxypicropodophyllotoxin (4), and its derivatives (I−III).
2013a). Interestingly, when compound 2 reacted with propionic acid in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and 4-N,N'dimethylaminopyridine (DMAP), 2'-chloro-5-bromo-4α-propionyloxypicropodophyllotoxin (4, Fig. 1) was obtained, and the configuration of its lactone was cis, which was determined by the X-ray crystallography (Che et al., 2013a). Moreover, compound 4 exhibited the potent insecticidal activity against pre-third-instar larvae of M. separata (Che et al., 2013a). Encouraged by the above results, and in continuation of our program aimed at the discovery of biorenewable natural product-based pesticides, herein some 4α-acyloxy derivatives of 2 and 3 were further prepared. Their insecticidal and acaricidal activities were tested against two major worldwide agricultural pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval in vivo.
2.2. General procedure for synthesis of 4α-acyloxy derivatives of 2'-chloro/ bromo 5-bromopicropodophyllotoxins (6a,c,d,f,g and 7a–g) A mixture of 2 or 3 (0.05 mmol), the corresponding acids (0.6 mmol), DCC (0.6 mmol), and DMAP (0.01 mmol) in dry CH2Cl2 (2 mL) was stirred at 28 °C for 20−72 h. Then the mixture was diluted by CH2Cl2 (18 mL). Subsequently, the mixture was washed by 0.1 N aqueous HCl (10 mL × 2), saturated aqueous NaHCO3 (10 mL × 2) and brine (10 mL × 2), dried over anhydrous Na2SO4, concentrated in vacuo, and purified by preparative thin-layer chromatography to give the target products 6a,c,d,f,g and 7a–g in 20%−73% yields. Data for 6a: Yield = 68%, white solid, m.p. 122−123 °C; 1 [α]20 D = −73 (c 2.1 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.47 (s, 1H, H-8), 6.34 (s, 1H, H-6′), 6.03-6.08 (m, 3H, OCH2O and H4), 5.23 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.53 (m, 1H, H-11), 4.24-4.28 (m, 1H, H-11), 3.91 (s, 3H, 3′-OCH3), 3.88 (s, 3H, 5′-OCH3), 3.71 (s, 3H, 4′-OCH3), 3.07-3.12 (m, 1H, H-3), 2.96 (dd, J = 15.0 Hz, 4.5 Hz, 1H, H2), 2.15 (s, 3H, CH3); HRMS (ESI): Calcd for C24H23O9BrCl ([M + H]+), 569.0208; found, 569.0202. Data for 6c: Yield = 72%, white solid, m.p. 144−146 °C; 1 [α]20 D = −64 (c 2.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.90 (d, J = 6.0 Hz, 2H), 8.13 (d, J = 5.5 Hz, 2H), 6.55 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.31 (d, J = 7.5 Hz, 1H, H-4), 6.08-6.12 (m, 2H, OCH2O), 5.24 (s, 1H, H-1), 4.58-4.61 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.86 (s, 3H, 5′-OCH3), 3.66 (s, 3H, 4′OCH3), 3.22-3.30 (m, 1H, H-3), 3.07 (dd, J = 5.0 Hz, 15.0 Hz, 1H, H-
2. Materials and methods 2.1. General N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Ethyl acetate, petroleum ether, and dichloromethane were obtained from Bodi Chemical Industrial Inc. (Tianjin, China). Two intermediates, 2'-chloro-5-bromopodophyllotoxin (2) and 2',5-dibromopodophyllotoxin (3), were prepared from compound 1 accroding to our previously reported methods (Che et al., 2013a). 46
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Scheme 1. The synthetic route for the preparation of 4α-acyloxy derivatives of 2'-chloro/bromo-5-bromopicropodophyllotoxins (6a,c,d,f,g and 7a−g).
2); HRMS (ESI): Calcd for C28H24O9NBrCl ([M + H]+), 632.0317; found, 632.0304. Data for 6d: Yield = 20%, White solid, m.p. 133−135 °C; 1 [α]20 D = −63 (c 1.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.02 (d, J = 7.0 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 6.53 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.25 (d, J = 8.0 Hz, 1H, H-4), 6.09 (d, J = 15.0 Hz, 2H, OCH2O), 5.29 (d, J = 4.0 Hz, 1H, H-1), 4.624.66 (m, 1H, H-11), 4.38-4.42 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.60 (s, 3H, 4′-OCH3), 3.20-3.28 (m, 1H, H-3), 3.05 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H25O9BrCl ([M + H]+), 631.0365; found, 631.0358. Data for 6f: Yield = 45%, White solid, m.p. 140−142 °C; 1 [α]20 D = −62 (c 2.4 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.80-7.82 (m, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.53 (s, 1H, H-8), 6.44 (s, 1H, H-6′), 6.26 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 10.5 Hz, 2H, OCH2O), 5.29 (d, J = 4.5 Hz, 1H, H-1), 4.61-4.64 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.91 (s, 3H, 3′OCH3), 3.85 (s, 3H, 5′-OCH3), 3.62 (s, 3H, 4′-OCH3), 3.19-3.27 (m, 1H, H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C30H27O9BrCl ([M + H]+), 645.0521; found, 645.0506. Data for 6g: Yield = 40%, White solid, m.p. 129−130 °C; 1 [α]20 D = −84 (c 1.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.85-7.86 (m, 1H), 7.45-7.49 (m, 2H), 7.31-7.35 (m, 1H), 6.50 (s, 1H, H-8), 6.38 (s, 1H, H-6′), 6.35 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 24.0 Hz, 2H, OCH2O), 5.28 (d, J = 4.0 Hz, H-1), 4.66-4.69 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s,
3H, 5′-OCH3), 3.50 (s, 3H, 4′-OCH3), 3.25-3.33 (m, 1H, H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H23O9BrCl2 ([M]+), 663.9897; found, 663.9876. Data for 7a: Yield = 70%, white solid, m.p. 108−109 °C; 1 [α]20 D = −83 (c 4.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.48 (s, 1H, H-8), 6.36 (s, 1H, H-6′), 6.01-6.08 (m, 3H, OCH2O and H4), 5.31 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.54 (m, 1H, H-11), 4.23-4.27 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.87 (s, 3H, 5′-OCH3), 3.71 (s, 3H, 4′-OCH3), 3.07-3.16 (m, 1H, H-3), 2.95 (dd, J = 15.0 Hz, 4.5 Hz, 1H, H2), 2.15 (s, 3H, CH3); HRMS (ESI): Calcd for C24H23O9Br2 ([M + H]+), 612.9703; found, 612.9691. Data for 7b: Yield = 67%, white solid, m.p. 112−113 °C; 1 [α]20 D = −99 (c 3.7 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.48 (s, 1H, H-8), 6.36 (s, 1H, H-6′), 6.03-6.08 (m, 3H, H-4 and OCH2O), 5.31 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.53 (m, 1H, H-11), 4.244.28 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.87 (s, 3H, 5′-OCH3), 3.70 (s, 3H, 4′-OCH3), 3.07-3.15 (m, 1H, H-3), 2.96 (dd, J = 15.5 Hz, 5.0 Hz, 1H, H-2), 2.33-2.52 (m, 2H, CH2CH3), 1.22 (t, J = 7.5 Hz, 3H, CH2CH3); HRMS (ESI): Calcd for C25H25O9Br2 ([M + H]+), 626.9860; found, 626.9857. Data for 7c: Yield = 49%, white solid, m.p. 145−146 °C; 1 [α]20 D = −65 (c 3.8 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.82 (d, J = 4.5 Hz, 2H), 7.83 (d, J = 5.5 Hz, 2H), 6.54 (s, 1H, H-8), 6.41 (s, 1H, H-6′), 6.27 (d, J = 8.0 Hz, 1H, H-4), 6.11 (dd, J = 17.5 Hz, 1.0 Hz, 2H, OCH2O), 5.37 (s, 1H, H-1), 4.59-4.63 (m, 1H, H-11), 4.364.40 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.61 47
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Fig. 2. Comparison of partial 1H NMR spectra of 4 with those of 6a,c,d,f,g and 7a−g.
8.01-8.02 (m, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 6.53 (s, 1H, H-8), 6.45 (s, 1H, H-6′), 6.25 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 0.5 Hz, 15.5 Hz, 2H, OCH2O), 5.37 (d, J = 3.0 Hz, 1H, H-1), 4.63-4.66 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′OCH3), 3.84 (s, 3H, 5′-OCH3), 3.59 (s, 3H, 4′-OCH3), 3.22-3.30 (m, 1H,
(s, 3H, 4′-OCH3), 3.22-3.30 (m, 1H, H-3), 3.05 (dd, J = 15.5 Hz, 5.0 Hz, 1H, H-2); HRMS (ESI): Calcd for C28H24O9NBr2 ([M + H]+), 675.9812; found, 675.9794. Data for 7d: Yield = 37%, White solid, m.p. 130−132 °C; 1 [α]20 D = −64 (c 2.1 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 48
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Fig. 2. (continued)
J = 8.0 Hz, 1H), 6.55 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.33 (d, J = 8.0 Hz, 1H, H-4), 6.11 (dd, J = 1.0 Hz, 11.0 Hz, 2H, OCH2O), 5.38 (s, 1H, H-1), 4.58-4.61 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.91 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.68 (s, 3H, 4′-OCH3), 3.25-3.33 (m, 1H, H-3), 3.06 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS
H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H25O9Br2 ([M + H]+), 674.9860; found, 674.9848. Data for 7e: Yield = 73%, White solid, m.p. 158−160 °C; 1 [α]20 D = −55 (c 2.6 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.77-8.78 (m, 1H), 8.47-8.49 (m, 1H), 8.41 (d, J = 7.5 Hz, 1H), 7.74 (t, 49
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Fig. 3. The representative abnormal larvae pictures of 6d (YRG-60), 6c (YRG-61) and 7f (YRG-63) during the larval period (CK: blank control group).
Fig. 4. The representative malformed pupae pictures of 6d (YRG-60), 7f (YRG-63), 6c (YRG-61), 7 g (YRG-64) and 6f (YRG-65) during the pupation period (CK: blank control group).
(ESI): Calcd for C29H24O11NBr2 ([M + H]+), 719.9711; found, 719.9690. Data for 7f: Yield = 53%, White solid, m.p. 143−144 °C; 1 [α]20 D = −63 (c 2.2 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.80-7.82 (m, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.53 (s, 1H, H-8), 6.45 (s, 1H, H-6′), 6.26 (d, J = 8.0 Hz, 1H, H-4), 6.09 (d, J = 11.0 Hz, 2H, OCH2O), 5.37 (d, J = 4.0 Hz, 1H, H-1), 4.61-4.65 (m, 1H, H-11), 4.36-4.40 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.84 (s, 3H, 5′-OCH3), 3.61 (s, 3H, 4′-OCH3), 3.21-3.30 (m, 1H, H-3), 3.03 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2), 2.40 (s, 3H); HRMS (ESI): Calcd for C30H27O9Br2 ([M + H]+), 689.0016; found, 688.9993. Data for 7g: Yield = 46%, White solid, m.p. 138−140 °C; 1 [α]20 D = −80 (c 3.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ:
7.86 (d, J = 7.5 Hz, 1H), 7.45-7.50 (m, 2H), 7.31-7.35 (m, 1H), 6.51 (s, 1H, H-8), 6.40 (s, 1H, H-6′), 6.35 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 25.0 Hz, 2H, OCH2O), 5.36 (d, J = 4.0 Hz, 1H, H-1), 4.674.70 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.89 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.49 (s, 3H, 4′-OCH3), 3.27-3.35 (m, 1H, H-3), 3.03 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H24O9Br2Cl ([M-H]+), 708.9470; found, 708.9458. 2.3. Biological assay 2.3.1. The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a−g against the pre-third-instar larvae of Mythimna separata The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a−g 50
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Fig. 5. The representative malformed moth pictures of 6d (YRG-60), 6c (YRG-61), 7f (YRG-63), 6f (YRG-65), 6 g (YRG-68), 7b (C-08), and 6a (C-29) during the stage of adult emergence (CK: blank control group). Table 1 Insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the pre-third-instar larvae of M. separata on leaves treated with a concentration of 1 mg/mL. Compound
Corrected mortality rate (%)a 15 days
1 6a 6c 6d 6f 6g 7a 7b 7c 7d 7e 7f 7g toosendanin
Table 2 Final mortality rates of compounds 6a, 6f, 7a–c and 7f at three different growth stages of M. separata.
20.0 abcd 13.3cd 10.0 d 23.3 abc 10.0 d 13.3cd 26.7 ab 23.3 abc 16.7 bcd 23.3 abc 20.0 abcd 26.7 ab 13.3 cd 23.3 abc
b
Compound
25 days
35 days
30.0 33.3 23.3 26.7 13.3 13.3 30.0 36.7 36.7 26.7 23.3 33.3 23.3 36.7
36.7 50.0 30.0 40.0 50.0 23.3 53.3 50.0 53.3 36.7 30.0 60.0 33.7 46.7
ab ab bc ab c c ab a a ab bc ab bc a
Percentage of FMR at three different stages (%)a larvae
6a 6f 7a 7b 7c 7f toosendanin
cde abc de bcd abc e ab abc ab cde de a de abc
a
53.3 20.0 50.0 46.7 43.8 50.0 57.1
± ± ± ± ± ± ±
3.3 0 3.3 3.3 3.3 5.8 3.3
pupae
adult
26.6 ± 0 6.7 ± 3.3 31.3 ± 3.3 33.3 ± 0 37.5 ± 3.3 30.5 ± 0 24.1 ± 0
20.0 73.3 18.8 20.0 18.8 19.5 18.7
± ± ± ± ± ± ±
0 5.8 3.3 0 3.3 0 3.3
Values are means ± S.D. of three replicate.
photoperiod: L/D = 12/12 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.
a
Values are means of three replicate. Multiple range test using Duncan’s test (p < 0.05). The same letters denote treatments not significantly different from each other. b
2.3.2. The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus was assessed by slide-dipping method (Chang and Knowles, 1977; Shi et al., 2015) as follows: Spirodiclofen was used as a positive control. The solutions of 1; 6a,c,d,f,g; 7a–g; and spirodiclofen were prepared in acetone/deionized water (v/v = 1/1) 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 acetone/deionized water (v/v = 1/1) alone were used as a blank control group (CK). The
against the pre-third-instar larvae of Mythimna separata was assessed by leaf-dipping method (Zhi et al., 2013; Yang et al., 2015; Li et al., 2016) as follows: Toosendanin, isolated from Melia azedarach, was used as a positive control. Thirty early pre-third-instar larvae of M. separata were selected to each compound. Acetone solutions of the tested compounds and toosendanin were prepared at 1 mg/mL. Fresh wheat leaves were dipped into the corresponding solution for 3 s, then taken out, and dried in a room. Leaves treated with acetone alone were used as a blank control group (CK). Several treated leaves were kept in each dish, where every 10 larvae were raised. If the treated leaves were consumed, additional treated leaves were added to the dish. After 48 h, compoundsoaked leaves were removed, and the larvae were fed with untreated ones till the end of pupae (temperature: 25 ± 2 °C; RH: 65–80%; 51
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the pre-third-instar larvae of Mythimna separata was evaluated by leafdipping method at a concentration of 1 mg/mL. Toosendanin, a commercial botanical insecticide isolated from Melia azedarach, was used as a positive control at 1 mg/mL. Leaves treated with acetone alone were used as a blank control group. Analysis of variance (ANOVA) was followed by Duncan’s post test, which was conducted using SPSS 20 for Windows 7. The symptoms of M. separata treated with the above compounds during three insect developmental stages (larval, pupation, and adult emergence) were in the same way as in our previous reports (Che et al., 2013a, 2013b; Fan et al., 2015; Zhi et al., 2013; Li et al., 2016): some larvae with the wrinkled bodies died at the larval stage (Fig. 3); some larvae molted to malformed pupae or died during the pupation period (Fig. 4); malformed moths with imperfect wings also appeared (Fig. 5). It suggested that these podophyllotoxin derivatives might exhibit the anti-molting hormone effect. As shown in Table 1, compounds 6a, 6f, 7a–c, and 7f showed the insecticidal activity equal to, or higher than, that of toosendanin. For example, the final mortality rates (FMRs) of 6a, 6f, 7a–c, and 7f were 50.0%, 50.0%, 53.3%, 50.0%, 53.5%, and 60.0%, respectively; whereas the FMRs of 1 and toosendanin were 36.7% and 46.7%, respectively. To compounds 6a,c,d,f,g, introduction of pyrid-4-ylcarbonyloxy, benzoyloxy or 2-chlorobenzoyloxy moiety at the C-4 position of compound 2 gave less active derivatives 6c,d,g. To compounds 7a–g, introduction of the benzoyloxy, 3-nitrobenzoyloxy or 2-chlorobenzoyloxy group at the C-4 position of compound 3 gave less potent derivatives 7d,e,g; whereas introduction of the acetoxy, pyrid-4-ylcarbonyloxy, or 3methylbenzoyloxy moiety at the C-4 position of 3 led to more potent compounds (e.g., 7a, 7c and 7f). In addition, the percentages of FMRs at three different growth periods of potent compounds 6a, 6f, 7a–c, and 7f were described in Table 2. The large parts of FMRs for compounds 6a, 7a–c, and 7f were at the larval stage; whereas the percentage of FMR at the larval stage of 6f was only 20%, and the percentage of its FMR at the adult stage was 73.3%. It was noteworthy that these results were different with those of 2′(2′,6′)-(di)chloropicropodophyllotoxins (more than half of their FMRs were generally at the pupation stage) (Wang et al., 2015). The acaricidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus was tested by slidedipping method at a concentration of 0.5 mg/mL. Spirodiclofen was used as a positive control at 0.5 mg/mL. As described in Table 3, compound 1 almost had no acaricidal activity against T. cinnabarinus; however, after structural modifications of 1, derivatives 6a, 6g, 7a, and 7c displayed the potent acaricidal activity; especially compound 7a showed the most promising acaricidal activity. For example, the mortality rates at 72 h of compounds 6a and 7a were 33.0% and 41.8%, respectively; whereas the corresponding mortality rate of 1 was only 1.6%. That is, the mortality rates at 72 h of 6a and 7a were greater than 20-fold of that of 1. Interestingly, introduction of the acetoxy moiety at the C-4 position of 2/3 could afford more potent compounds 6a and 7a. All in all, here compounds 6a and 7a (containing the 4-acetyloxy group) showed potent insecticidal and acaricidal activities against M. separata and T. cinnabarinus, respectively. This result was consistent with that in our previous reports, where it was found that introduction of acetyloxy group at the C-4 position of the corresponding podophyllotoxin derivatives could lead to more promising compounds (Che et al., 2013b; Huang et al., 2017).
Table 3 Acaricidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of T. cinnabarinus treated at a concentration of 0.5 mg/mL. Compound
Corrected mortality rate (%)a 48 h
1 6a 6c 6d 6f 6g 7a 7b 7c 7d 7e 7f 7g spirodiclofen
72 h b
1.4 f 20.1 ab 8.4 d 7.9 d 3.5 ef 21.3 ab 19.0 b 13.4 c 15.4 c 6.8 de 14.0 c 6.0 de 5.7 de 22.9 a
1.6 j 33.0 c 17.4 ef 8.4 gh 3.7 ij 25.3 d 41.8 b 14.2 f 23.0 d 11.0 g 14.5 f 6.4 hi 19.6 e 59.7 a
a
Values are means of three replicate. Multiple range test using Duncan’s test (p < 0.05). The same letters denote treatments not significantly different from each other. b
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. 3. Results and discussion 3.1. Chemistry As depicted in Scheme 1, podophyllotoxin (1) first reacted with 1 equiv. of N-chlorosuccinimide (NCS) to give 2'-chloropodophyllotoxin (2′), which further reacted with 1 equiv. of N-bromosuccinimide (NBS) to afford 2'-chloro-5-bromopodophyllotoxin (2); 2',5-dibromopodophyllotoxin (3) were obtained by reaction of podophyllotoxin (1) with 2 equiv. of NBS (Che et al., 2013a). Finally, in the presence of DCC and DMAP, target compounds 6a,c,d,f,g and 7a–g were smoothly obtained by reaction of 2/3 with the corresponding carboxylic acids (5). The chemical structures of all compounds were well characterized by 1 HNMR, HRMS, optical rotation, and mp. For the cis configuration of the lactone of 4 was determined by the X-ray crystallography (Che et al., 2013a), assignment of the configuration at the C-2 position of 6a,c,d,f,g and 7a–g was based on their coupling constants (J) of H-2, which were compared with those of 4. Comparison of partial 1H NMR spectra of 4 with those of 6a,c,d,f,g and 7a–g was depicted in Fig. 2. The coupling constants (J) of H-2 of 4 were 15.5 and 5.0 Hz, respectively; the coupling constants (J) of H-2 of 6a,c,d,f,g and 7a–g was 15.5 (or 15.0) and 5.0 (or 4.5) Hz, respectively; this indicates that the configuration of the lactones of 6a,c,d,f,g and 7a–g was the same of that of 4. So the configuration of their lactones was all converted from trans into cis. However, in our previous papers, when 2′(2′,6′)-(di) halogenopodophyllotoxins reacted with carboxylic acids in the same reaction conditions, the configuration of the lactones of the corresponding products was all trans and was not changed (Che et al., 2013b; Huang et al., 2017). It demonstrated that when 2/3 reacted with carboxylic acids in the presence of DCC and DMAP, their C-5 bromine atom may affect the configuration of the lactone.
4. Conclusion In summary, a series of 4α-acyloxy derivatives of 2'-chloro/bromo5-bromopodophyllotoxins were prepared by structural modification of podophyllotoxin as a sustainable natural resource, and evaluated for their insecticidal and acaricidal activities against M. separata and T. cinnabarinus. Among all derivatives, compounds 6a and 7a all exhibited
3.2. Pesticidal activity The pesticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against 52
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Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51, 45–66. Iwasaki, T., Kondo, K., Nishitani, T., Kuroda, T., Hirakoso, K., Ohtani, A., Takashima, K., 1995. Arylnaphthalene lignans as novel series of hypolipidemic agents raising highdensity lipoprotein level. Chem. Pharm. Bull. 43, 1701–1705. Jiang, X.F., Luo, L.Z., Zhang, L., Sappington, T.W., Hu, Y., 2011. Regulation of migration in Mythimna separata (Walker) in China: a review integrating environmental, physiological, hormonal, genetic, and molecular factors. Environ. Entomol. 40, 516–533. Kumar, K.A., Singh, S.K., Kumar, B.S., Doble, M., 2007. Synthesis, anti-fungal activity evaluation and QSAR studies on podophyllotoxin derivatives. Cent. Eur. J. Chem. 5, 880–897. Li, Q., Huang, X., Li, S., Ma, J., Lv, M., Xu, H., 2016. ,Semisynthesis of esters of fraxinellone C4/10-oxime and their pesticidal activities. J. Agric. Food Chem. 64, 5472–5478. Lv, M., Xu, H., 2011. Recent advances in semisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: an update (2008–2010). Mini-Rev. Med. Chem. 11, 901–909. MacRae, W.D., Hudson, J.B., Towers, G.H.N., 1989. The antiviral action of lignans. Planta Med. 55, 531–535. Miyazawa, M., Fukuyama, M., Yoshio, K., Kato, T., Ishikawa, Y., 1999. Biologically active components against Drosophila melanogaster from Podophyllum hexandrum. J. Agric. Food Chem. 47, 5108–5110. Saitoh, T., Kuramochi, K., Imai, T., Takata, K., Takehara, M., Kobayashi, S., Sakaguchia, K., Sugawaraa, F., 2008. Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro. Bioorg. Med. Chem. 16, 5815–5825. Seiber, J.N., Coats, J., Duke, S.O., Gross, A.D., 2014. Biopesticides: state of the art and future opportunities. J. Agric. Food Chem. 62, 11613–11619. Shi, L., Zhang, J., Shen, G., Xu, Z., Wei, P., Zhang, Y., Xu, Q., He, L., 2015. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval). Sci. Rep. 5, 15581. Sun, J.Y., Liang, P., Gao, X.W., 2012. Cross-resistance patterns and fitness in fufenozideresistant diamondback moth, Plutella xylostella (Lepidoptera: plutellidae). Pest Manag. Sci. 68, 285–289. Wang, R., Zhi, X.Y., Li, J., Xu, H., 2015. Synthesis of novel oxime sulfonate derivatives of 2′(2′, 6′)-(di)chloropicropodophyllotoxins as insecticidal agents. J. Agric. Food Chem. 63, 6668–6674. Xu, H., Lv, M., Tian, X., 2009. A review on hemisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: 2003–2007. Curr. Med. Chem. 16, 327–349. Yang, C., Zhi, X., Li, J., Zha, J., Xu, H., 2015. Natural products-based insecticidal agents 20. Design, synthesis and insecticidal activity of novel honokiol/magnolol azo derivatives. Ind. Crop Prod. 76, 761–767. You, Y.J., 2005. Podophyllotoxin derivatives: current synthetic approaches for new anticancer agents. Curr. Pharm. Des. 11, 1695–1717. Zeng, J., Jiang, Y., Liu, J., 2013. Analysis of the armyworm outbreak in 2012 and suggestions of monitoring and forecasting. Plant Prot. 39, 117–121. Zhang, X., Ma, Z.Q., Feng, J.T., Han, L.R., 2015. Review on research and developmentof botanical pesticides. Chin. J. Biol. Control 5, 685–698. Zhi, X.Y., Yang, C., Zhang, R., Hu, Y., Ke, Y., Xu, H., 2013. Natural productsbasedinsecticidal agents 13. Semisynthesis and insecticidal activity of novelphenazine derivatives of 4-acyloxypodophyllotoxin modified in the Eringagainst Mythimna separata Walker in vivo. Ind. Crop Prod. 42, 520–526. Zhi, X.Y., Yang, C., Yu, X., Xu, H., 2014. Synthesis and insecticidal activity of new oxime derivatives of podophyllotoxin-based phenazines against Mythimna separata Walker. Bioorg. Med. Chem. Lett. 24, 5679–5682. Zhu, X.K., Guan, J., Xiao, Z.Y., Cosentino, L.M., Lee, K.H., 2004. Anti-AIDS agents. Part 61: Anti-HIV activity of new podophyllotoxin derivatives. Bioorg. Med. Chem. 12, 4267–4273.
more promising insecticidal and acaricidal activities against M. separata and T. cinnabarinus, respectively. It suggested that introduction of the acetoxy moiety at the C-4 position of 2/3 was necessary for the insecticidal and acaricidal activities. This will pave the way for further design and structural modifications of podophyllotoxin as potentially botanical acaricidal and insecticidal agents in crop protection. Financial support The present research was partly supported by National Natural Science Foundation of China (No. 31672071), and Special Funds of Central Colleges Basic Scientific Research Operating Expenses (No.2452015096) to H. X. References Carlstrom, K., Hedin, P.J., Jonsson, L., Lerndal, T., Lien, J., Weitoft, T., Axelson, M., 2000. Endocrine effects of the podophyllotoxine derivative drug CPH 82 (Reumacon) in patients with rheumatoid arthritis. Scand. J. Rheumatol. 29, 89–94. Castro, M.A., del Corral, J.M.M., Gordaliza, M., Gomez-Zurita, M.A., de la Puente, M.L., Betancur-Galvis, L.A., Sierra, J., Feliciano, A.S., 2003. Synthesis, cytotoxicity and antiviral activity of podophyllotoxin analogues modified in the E-ring. Eur. J. Med. Chem. 38, 899–911. Chang, K.M., Knowles, C.O., 1977. Formamidine acaricides. Toxicity and metabolism studies with two spotted spider mites, Tetranychus urticae Koch. J. Agric. Food Chem. 25, 493–501. Che, Z.P., Yu, X., Fan, L.L., Xu, H., 2013a. Insight into dihalogenation of E-ring of podophyllotoxins, and their acyloxyation derivatives at the C4 position as insecticidal agents. Bioorg. Med. Chem. Lett. 23, 5592–5598. Che, Z.P., Yu, X., Zhi, X.Y., Fan, L.L., Xu, H., 2013b. Synthesis of novel 4α-(acyloxy)-2′(2′, 6′)-(di)halogenopodophyllotoxin derivatives as insecticidal agents. J. Agric. Food Chem. 61, 8148–8155. Desbene, S., Giorgi-Renault, S., 2002. Drugs that inhibit tubulin polymerization: the particular case of podophyllotoxin and analogues. Curr. Med. Chem. Anti-Cancer Agents 2, 71–90. Fan, L.L., Zhi, X.Y., Che, Z.P., Xu, H., 2015. Insight into 2α-chloro-2′(2′, 6′)-(di) halogenopicropodophyllotoxins reacting with carboxylic acids mediated by BF3∙Et2O. Sci. Rep. 5, 16285. Gao, R., Gao, C., Tian, X., Yu, X., Di, X., Xiao, H., Zhang, X., 2004. Insecticidal activity of deoxypodophyllotoxin, isolated from Juniperus sabina L, and related lignans against larvae of Pieris rapae L. Pest Manag. Sci. 60, 1131–1136. Gordaliza, M., del Corral, J.M.M., Castro, M.A., Garcia-Garcia, P.A., San Feliciano, A., 2001. Cytotoxic cyclolignans related to podophyllotoxin. Farmaco 56, 297–304. Gordaliza, M., Garcia, P.A., Miguel Del Corral, J.M., Castro, M.A., Gomez-Zurita, M.A., 2004. Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives. Toxicon 44, 441–459. Guerrero, E., Abad, A., Montenegro, G., del Olmo, E., López-Pérez, J.L., Feliciano, A.S., 2013. Analgesic and anti-inflammatory activity of podophyllotoxin derivatives. Pharm. Biol. 51, 566–572. Guillette Jr., L.J., Iguchi, T., 2012. Life in a contaminated world. Science 337, 1614–1615. Huang, J.L., Xu, M., Li, S.C., He, J.N., Xu, H., 2017. Synthesis of some ester derivatives of 4′-demethoxyepipodophyllotoxin/2′-chloro-4′-demethoxyepipodophyllotoxin as insecticidal agents against oriental armyworm, Mythimna separata Walker. Bioorg. Med. Chem. Lett. 27, 511–517.
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Application of sustainable natural resources in crop protection: Podophyllotoxin-based botanical pesticides derived from Podophyllum hexandrum for controlling crop-threatening insect pests Ruige Yanga,1, Xiaobo Huanga,1, Zhiping Chea,1, Yuanyuan Zhanga, Hui Xua,b,
MARK
⁎
a Research Institute of Pesticidal Design & Synthesis, College of Chemistry and Pharmacy/Plant Protection, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China b Shaanxi Key Laboratory of Natural Products & Chemical Biology, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Podophyllum hexandrum Podophyllotoxin Botanical pesticide Natural bioresource
Podophyllotoxin is isolated as a sustainable natural bioresource from the roots and rhizomes of Podophyllum hexandrum and Juniperus Sabina. In continuation of our program aimed at the discovery of biorenewable natural product-based pesticides, a series of 4α-acyloxy derivatives of 2'-chloro-5-bromopicropodophyllotoxin or 2',5dibromopicropodophyllotoxin were synthesized by structural modification of podophyllotoxin, and evaluated as insecticidal and acaricidal agents against two serious agricultural pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval. Among all derivatives, compounds 6a and 7a exhibited more promising insecticidal and acaricidal activities than podophyllotoxin, and the final mortality rates of 6a, 7a and 1 against M. separata (at 1 mg/mL) and T. cinnabarinus (at 0.5 mg/mL) were 50%/53.3%/36.7%, and 33%/41.8%/1.6%, respectively. This suggested that introduction of the acetoxy moiety at the C-4 position of 2'-chloro-5bromopicropodophyllotoxin/2',5-dibromopicropodophyllotoxin was necessary for the insecticidal and acaricidal activities.
1. Introduction Mythimna separata Walker (Oriental armyworm) and Tetranychus cinnabarinus Boisduval (Spider mite) are two crop-threatening insect pests, and their infestations are extremely hard to prevent and control (Jiang et al., 2011). In 2012, the intermittent outbreaks of M. separata widely occurred in China, and about 4 million hectares of crops were complete loss (Zeng et al., 2013). Additionally, the long-term and unreasonable use of synthetic agrochemicals to cope with these serious insect pests has led to insect resistance and environmental problems (Sun et al., 2012; Guillette and Iguchi, 2012). With the drive toward increasing demand for organic food, botanical pesticides, due to their less or slower resistance development and lower pollution, have received much more attention in the agricultural field when compared with conventional chemical pesticides (Isman, 2006; Seiber et al., 2014). It was reported that about 69 botanical pesticides were registered and commercialized in the United States at the end of 2013 (Zhang et al., 2015). Podophyllotoxin (1, Fig. 1) contains four consecutive chiral centers
(labeled C-1–C-4), and is isolated as a sustainable natural bioresource from the roots and rhizomes of Podophyllum hexandrum and Juniperus Sabina (Xu et al., 2009). In addition to preparation of potent anticancer drugs such as etoposide, teniposide and etoposide phosphate as a lead compound (Desbene and Giorgi-Renault, 2002; Gordaliza et al., 2004; Lv and Xu, 2011; You, 2005), compound 1 also showed a variety of interesting insecticidal (Miyazawa et al., 1999; Gao et al., 2004; Zhi et al., 2013, 2014), antifungal (Kumar et al., 2007), antiviral (Castro et al., 2003; MacRae et al., 1989; Saitoh et al., 2008; Zhu et al., 2004), anti-inflammatory (Guerrero et al., 2013), immunosuppressive (Gordaliza et al., 2001), antirheumatic (Carlstrom et al., 2000), and hypolipidemic activities (Iwasaki et al., 1995). More recently, halogenation of E-ring of podophyllotoxin has been investigated in our group, and some 2′(2′,6′)-(di)halogen-substituted podophyllotoxin derivatives (I and II, Fig. 1) showed more pronounced insecticidal activity than toosendanin against pre-third-instar larvae of Mythimna separata (Che et al., 2013a; 2013b; Fan et al., 2015). Meanwhile, two unexpected intermediates, 2'-chloro-5-bromopodophyllotoxin (2, Fig. 1) and 2',5-dibromopodophyllotoxin (3, Fig. 1), were produced (Che et al.,
⁎ Corresponding author at: Research Institute of Pesticidal Design & Synthesis, College of Chemistry and Pharmacy/Plant Protection, Northwest A & F University, Yangling 712100, Shaanxi Province, PR China. E-mail address: [email protected] (H. Xu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.indcrop.2017.05.033 Received 20 February 2017; Received in revised form 7 May 2017; Accepted 17 May 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of podophyllotoxin (1), 2'-chloro-5-bromopodophyllotoxin (2), 2',5-dibromopodophyllotoxin (3), 2' −chloro-5-bromo-4α-propionyloxypicropodophyllotoxin (4), and its derivatives (I−III).
2013a). Interestingly, when compound 2 reacted with propionic acid in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and 4-N,N'dimethylaminopyridine (DMAP), 2'-chloro-5-bromo-4α-propionyloxypicropodophyllotoxin (4, Fig. 1) was obtained, and the configuration of its lactone was cis, which was determined by the X-ray crystallography (Che et al., 2013a). Moreover, compound 4 exhibited the potent insecticidal activity against pre-third-instar larvae of M. separata (Che et al., 2013a). Encouraged by the above results, and in continuation of our program aimed at the discovery of biorenewable natural product-based pesticides, herein some 4α-acyloxy derivatives of 2 and 3 were further prepared. Their insecticidal and acaricidal activities were tested against two major worldwide agricultural pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval in vivo.
2.2. General procedure for synthesis of 4α-acyloxy derivatives of 2'-chloro/ bromo 5-bromopicropodophyllotoxins (6a,c,d,f,g and 7a–g) A mixture of 2 or 3 (0.05 mmol), the corresponding acids (0.6 mmol), DCC (0.6 mmol), and DMAP (0.01 mmol) in dry CH2Cl2 (2 mL) was stirred at 28 °C for 20−72 h. Then the mixture was diluted by CH2Cl2 (18 mL). Subsequently, the mixture was washed by 0.1 N aqueous HCl (10 mL × 2), saturated aqueous NaHCO3 (10 mL × 2) and brine (10 mL × 2), dried over anhydrous Na2SO4, concentrated in vacuo, and purified by preparative thin-layer chromatography to give the target products 6a,c,d,f,g and 7a–g in 20%−73% yields. Data for 6a: Yield = 68%, white solid, m.p. 122−123 °C; 1 [α]20 D = −73 (c 2.1 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.47 (s, 1H, H-8), 6.34 (s, 1H, H-6′), 6.03-6.08 (m, 3H, OCH2O and H4), 5.23 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.53 (m, 1H, H-11), 4.24-4.28 (m, 1H, H-11), 3.91 (s, 3H, 3′-OCH3), 3.88 (s, 3H, 5′-OCH3), 3.71 (s, 3H, 4′-OCH3), 3.07-3.12 (m, 1H, H-3), 2.96 (dd, J = 15.0 Hz, 4.5 Hz, 1H, H2), 2.15 (s, 3H, CH3); HRMS (ESI): Calcd for C24H23O9BrCl ([M + H]+), 569.0208; found, 569.0202. Data for 6c: Yield = 72%, white solid, m.p. 144−146 °C; 1 [α]20 D = −64 (c 2.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.90 (d, J = 6.0 Hz, 2H), 8.13 (d, J = 5.5 Hz, 2H), 6.55 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.31 (d, J = 7.5 Hz, 1H, H-4), 6.08-6.12 (m, 2H, OCH2O), 5.24 (s, 1H, H-1), 4.58-4.61 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.86 (s, 3H, 5′-OCH3), 3.66 (s, 3H, 4′OCH3), 3.22-3.30 (m, 1H, H-3), 3.07 (dd, J = 5.0 Hz, 15.0 Hz, 1H, H-
2. Materials and methods 2.1. General N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Ethyl acetate, petroleum ether, and dichloromethane were obtained from Bodi Chemical Industrial Inc. (Tianjin, China). Two intermediates, 2'-chloro-5-bromopodophyllotoxin (2) and 2',5-dibromopodophyllotoxin (3), were prepared from compound 1 accroding to our previously reported methods (Che et al., 2013a). 46
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Scheme 1. The synthetic route for the preparation of 4α-acyloxy derivatives of 2'-chloro/bromo-5-bromopicropodophyllotoxins (6a,c,d,f,g and 7a−g).
2); HRMS (ESI): Calcd for C28H24O9NBrCl ([M + H]+), 632.0317; found, 632.0304. Data for 6d: Yield = 20%, White solid, m.p. 133−135 °C; 1 [α]20 D = −63 (c 1.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.02 (d, J = 7.0 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 6.53 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.25 (d, J = 8.0 Hz, 1H, H-4), 6.09 (d, J = 15.0 Hz, 2H, OCH2O), 5.29 (d, J = 4.0 Hz, 1H, H-1), 4.624.66 (m, 1H, H-11), 4.38-4.42 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.60 (s, 3H, 4′-OCH3), 3.20-3.28 (m, 1H, H-3), 3.05 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H25O9BrCl ([M + H]+), 631.0365; found, 631.0358. Data for 6f: Yield = 45%, White solid, m.p. 140−142 °C; 1 [α]20 D = −62 (c 2.4 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.80-7.82 (m, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.53 (s, 1H, H-8), 6.44 (s, 1H, H-6′), 6.26 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 10.5 Hz, 2H, OCH2O), 5.29 (d, J = 4.5 Hz, 1H, H-1), 4.61-4.64 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.91 (s, 3H, 3′OCH3), 3.85 (s, 3H, 5′-OCH3), 3.62 (s, 3H, 4′-OCH3), 3.19-3.27 (m, 1H, H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C30H27O9BrCl ([M + H]+), 645.0521; found, 645.0506. Data for 6g: Yield = 40%, White solid, m.p. 129−130 °C; 1 [α]20 D = −84 (c 1.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.85-7.86 (m, 1H), 7.45-7.49 (m, 2H), 7.31-7.35 (m, 1H), 6.50 (s, 1H, H-8), 6.38 (s, 1H, H-6′), 6.35 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 24.0 Hz, 2H, OCH2O), 5.28 (d, J = 4.0 Hz, H-1), 4.66-4.69 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s,
3H, 5′-OCH3), 3.50 (s, 3H, 4′-OCH3), 3.25-3.33 (m, 1H, H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H23O9BrCl2 ([M]+), 663.9897; found, 663.9876. Data for 7a: Yield = 70%, white solid, m.p. 108−109 °C; 1 [α]20 D = −83 (c 4.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.48 (s, 1H, H-8), 6.36 (s, 1H, H-6′), 6.01-6.08 (m, 3H, OCH2O and H4), 5.31 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.54 (m, 1H, H-11), 4.23-4.27 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.87 (s, 3H, 5′-OCH3), 3.71 (s, 3H, 4′-OCH3), 3.07-3.16 (m, 1H, H-3), 2.95 (dd, J = 15.0 Hz, 4.5 Hz, 1H, H2), 2.15 (s, 3H, CH3); HRMS (ESI): Calcd for C24H23O9Br2 ([M + H]+), 612.9703; found, 612.9691. Data for 7b: Yield = 67%, white solid, m.p. 112−113 °C; 1 [α]20 D = −99 (c 3.7 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 6.48 (s, 1H, H-8), 6.36 (s, 1H, H-6′), 6.03-6.08 (m, 3H, H-4 and OCH2O), 5.31 (d, J = 4.0 Hz, 1H, H-1), 4.50-4.53 (m, 1H, H-11), 4.244.28 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.87 (s, 3H, 5′-OCH3), 3.70 (s, 3H, 4′-OCH3), 3.07-3.15 (m, 1H, H-3), 2.96 (dd, J = 15.5 Hz, 5.0 Hz, 1H, H-2), 2.33-2.52 (m, 2H, CH2CH3), 1.22 (t, J = 7.5 Hz, 3H, CH2CH3); HRMS (ESI): Calcd for C25H25O9Br2 ([M + H]+), 626.9860; found, 626.9857. Data for 7c: Yield = 49%, white solid, m.p. 145−146 °C; 1 [α]20 D = −65 (c 3.8 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.82 (d, J = 4.5 Hz, 2H), 7.83 (d, J = 5.5 Hz, 2H), 6.54 (s, 1H, H-8), 6.41 (s, 1H, H-6′), 6.27 (d, J = 8.0 Hz, 1H, H-4), 6.11 (dd, J = 17.5 Hz, 1.0 Hz, 2H, OCH2O), 5.37 (s, 1H, H-1), 4.59-4.63 (m, 1H, H-11), 4.364.40 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.61 47
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Fig. 2. Comparison of partial 1H NMR spectra of 4 with those of 6a,c,d,f,g and 7a−g.
8.01-8.02 (m, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 6.53 (s, 1H, H-8), 6.45 (s, 1H, H-6′), 6.25 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 0.5 Hz, 15.5 Hz, 2H, OCH2O), 5.37 (d, J = 3.0 Hz, 1H, H-1), 4.63-4.66 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.90 (s, 3H, 3′OCH3), 3.84 (s, 3H, 5′-OCH3), 3.59 (s, 3H, 4′-OCH3), 3.22-3.30 (m, 1H,
(s, 3H, 4′-OCH3), 3.22-3.30 (m, 1H, H-3), 3.05 (dd, J = 15.5 Hz, 5.0 Hz, 1H, H-2); HRMS (ESI): Calcd for C28H24O9NBr2 ([M + H]+), 675.9812; found, 675.9794. Data for 7d: Yield = 37%, White solid, m.p. 130−132 °C; 1 [α]20 D = −64 (c 2.1 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 48
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Fig. 2. (continued)
J = 8.0 Hz, 1H), 6.55 (s, 1H, H-8), 6.43 (s, 1H, H-6′), 6.33 (d, J = 8.0 Hz, 1H, H-4), 6.11 (dd, J = 1.0 Hz, 11.0 Hz, 2H, OCH2O), 5.38 (s, 1H, H-1), 4.58-4.61 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.91 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.68 (s, 3H, 4′-OCH3), 3.25-3.33 (m, 1H, H-3), 3.06 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS
H-3), 3.04 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H25O9Br2 ([M + H]+), 674.9860; found, 674.9848. Data for 7e: Yield = 73%, White solid, m.p. 158−160 °C; 1 [α]20 D = −55 (c 2.6 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 8.77-8.78 (m, 1H), 8.47-8.49 (m, 1H), 8.41 (d, J = 7.5 Hz, 1H), 7.74 (t, 49
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Fig. 3. The representative abnormal larvae pictures of 6d (YRG-60), 6c (YRG-61) and 7f (YRG-63) during the larval period (CK: blank control group).
Fig. 4. The representative malformed pupae pictures of 6d (YRG-60), 7f (YRG-63), 6c (YRG-61), 7 g (YRG-64) and 6f (YRG-65) during the pupation period (CK: blank control group).
(ESI): Calcd for C29H24O11NBr2 ([M + H]+), 719.9711; found, 719.9690. Data for 7f: Yield = 53%, White solid, m.p. 143−144 °C; 1 [α]20 D = −63 (c 2.2 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.80-7.82 (m, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.53 (s, 1H, H-8), 6.45 (s, 1H, H-6′), 6.26 (d, J = 8.0 Hz, 1H, H-4), 6.09 (d, J = 11.0 Hz, 2H, OCH2O), 5.37 (d, J = 4.0 Hz, 1H, H-1), 4.61-4.65 (m, 1H, H-11), 4.36-4.40 (m, 1H, H-11), 3.90 (s, 3H, 3′-OCH3), 3.84 (s, 3H, 5′-OCH3), 3.61 (s, 3H, 4′-OCH3), 3.21-3.30 (m, 1H, H-3), 3.03 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2), 2.40 (s, 3H); HRMS (ESI): Calcd for C30H27O9Br2 ([M + H]+), 689.0016; found, 688.9993. Data for 7g: Yield = 46%, White solid, m.p. 138−140 °C; 1 [α]20 D = −80 (c 3.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ:
7.86 (d, J = 7.5 Hz, 1H), 7.45-7.50 (m, 2H), 7.31-7.35 (m, 1H), 6.51 (s, 1H, H-8), 6.40 (s, 1H, H-6′), 6.35 (d, J = 8.0 Hz, 1H, H-4), 6.09 (dd, J = 1.0 Hz, 25.0 Hz, 2H, OCH2O), 5.36 (d, J = 4.0 Hz, 1H, H-1), 4.674.70 (m, 1H, H-11), 4.37-4.41 (m, 1H, H-11), 3.89 (s, 3H, 3′-OCH3), 3.85 (s, 3H, 5′-OCH3), 3.49 (s, 3H, 4′-OCH3), 3.27-3.35 (m, 1H, H-3), 3.03 (dd, J = 5.0 Hz, 15.5 Hz, 1H, H-2); HRMS (ESI): Calcd for C29H24O9Br2Cl ([M-H]+), 708.9470; found, 708.9458. 2.3. Biological assay 2.3.1. The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a−g against the pre-third-instar larvae of Mythimna separata The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a−g 50
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Fig. 5. The representative malformed moth pictures of 6d (YRG-60), 6c (YRG-61), 7f (YRG-63), 6f (YRG-65), 6 g (YRG-68), 7b (C-08), and 6a (C-29) during the stage of adult emergence (CK: blank control group). Table 1 Insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the pre-third-instar larvae of M. separata on leaves treated with a concentration of 1 mg/mL. Compound
Corrected mortality rate (%)a 15 days
1 6a 6c 6d 6f 6g 7a 7b 7c 7d 7e 7f 7g toosendanin
Table 2 Final mortality rates of compounds 6a, 6f, 7a–c and 7f at three different growth stages of M. separata.
20.0 abcd 13.3cd 10.0 d 23.3 abc 10.0 d 13.3cd 26.7 ab 23.3 abc 16.7 bcd 23.3 abc 20.0 abcd 26.7 ab 13.3 cd 23.3 abc
b
Compound
25 days
35 days
30.0 33.3 23.3 26.7 13.3 13.3 30.0 36.7 36.7 26.7 23.3 33.3 23.3 36.7
36.7 50.0 30.0 40.0 50.0 23.3 53.3 50.0 53.3 36.7 30.0 60.0 33.7 46.7
ab ab bc ab c c ab a a ab bc ab bc a
Percentage of FMR at three different stages (%)a larvae
6a 6f 7a 7b 7c 7f toosendanin
cde abc de bcd abc e ab abc ab cde de a de abc
a
53.3 20.0 50.0 46.7 43.8 50.0 57.1
± ± ± ± ± ± ±
3.3 0 3.3 3.3 3.3 5.8 3.3
pupae
adult
26.6 ± 0 6.7 ± 3.3 31.3 ± 3.3 33.3 ± 0 37.5 ± 3.3 30.5 ± 0 24.1 ± 0
20.0 73.3 18.8 20.0 18.8 19.5 18.7
± ± ± ± ± ± ±
0 5.8 3.3 0 3.3 0 3.3
Values are means ± S.D. of three replicate.
photoperiod: L/D = 12/12 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.
a
Values are means of three replicate. Multiple range test using Duncan’s test (p < 0.05). The same letters denote treatments not significantly different from each other. b
2.3.2. The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus The insecticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus was assessed by slide-dipping method (Chang and Knowles, 1977; Shi et al., 2015) as follows: Spirodiclofen was used as a positive control. The solutions of 1; 6a,c,d,f,g; 7a–g; and spirodiclofen were prepared in acetone/deionized water (v/v = 1/1) 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 acetone/deionized water (v/v = 1/1) alone were used as a blank control group (CK). The
against the pre-third-instar larvae of Mythimna separata was assessed by leaf-dipping method (Zhi et al., 2013; Yang et al., 2015; Li et al., 2016) as follows: Toosendanin, isolated from Melia azedarach, was used as a positive control. Thirty early pre-third-instar larvae of M. separata were selected to each compound. Acetone solutions of the tested compounds and toosendanin were prepared at 1 mg/mL. Fresh wheat leaves were dipped into the corresponding solution for 3 s, then taken out, and dried in a room. Leaves treated with acetone alone were used as a blank control group (CK). Several treated leaves were kept in each dish, where every 10 larvae were raised. If the treated leaves were consumed, additional treated leaves were added to the dish. After 48 h, compoundsoaked leaves were removed, and the larvae were fed with untreated ones till the end of pupae (temperature: 25 ± 2 °C; RH: 65–80%; 51
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the pre-third-instar larvae of Mythimna separata was evaluated by leafdipping method at a concentration of 1 mg/mL. Toosendanin, a commercial botanical insecticide isolated from Melia azedarach, was used as a positive control at 1 mg/mL. Leaves treated with acetone alone were used as a blank control group. Analysis of variance (ANOVA) was followed by Duncan’s post test, which was conducted using SPSS 20 for Windows 7. The symptoms of M. separata treated with the above compounds during three insect developmental stages (larval, pupation, and adult emergence) were in the same way as in our previous reports (Che et al., 2013a, 2013b; Fan et al., 2015; Zhi et al., 2013; Li et al., 2016): some larvae with the wrinkled bodies died at the larval stage (Fig. 3); some larvae molted to malformed pupae or died during the pupation period (Fig. 4); malformed moths with imperfect wings also appeared (Fig. 5). It suggested that these podophyllotoxin derivatives might exhibit the anti-molting hormone effect. As shown in Table 1, compounds 6a, 6f, 7a–c, and 7f showed the insecticidal activity equal to, or higher than, that of toosendanin. For example, the final mortality rates (FMRs) of 6a, 6f, 7a–c, and 7f were 50.0%, 50.0%, 53.3%, 50.0%, 53.5%, and 60.0%, respectively; whereas the FMRs of 1 and toosendanin were 36.7% and 46.7%, respectively. To compounds 6a,c,d,f,g, introduction of pyrid-4-ylcarbonyloxy, benzoyloxy or 2-chlorobenzoyloxy moiety at the C-4 position of compound 2 gave less active derivatives 6c,d,g. To compounds 7a–g, introduction of the benzoyloxy, 3-nitrobenzoyloxy or 2-chlorobenzoyloxy group at the C-4 position of compound 3 gave less potent derivatives 7d,e,g; whereas introduction of the acetoxy, pyrid-4-ylcarbonyloxy, or 3methylbenzoyloxy moiety at the C-4 position of 3 led to more potent compounds (e.g., 7a, 7c and 7f). In addition, the percentages of FMRs at three different growth periods of potent compounds 6a, 6f, 7a–c, and 7f were described in Table 2. The large parts of FMRs for compounds 6a, 7a–c, and 7f were at the larval stage; whereas the percentage of FMR at the larval stage of 6f was only 20%, and the percentage of its FMR at the adult stage was 73.3%. It was noteworthy that these results were different with those of 2′(2′,6′)-(di)chloropicropodophyllotoxins (more than half of their FMRs were generally at the pupation stage) (Wang et al., 2015). The acaricidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of Tetranychus cinnabarinus was tested by slidedipping method at a concentration of 0.5 mg/mL. Spirodiclofen was used as a positive control at 0.5 mg/mL. As described in Table 3, compound 1 almost had no acaricidal activity against T. cinnabarinus; however, after structural modifications of 1, derivatives 6a, 6g, 7a, and 7c displayed the potent acaricidal activity; especially compound 7a showed the most promising acaricidal activity. For example, the mortality rates at 72 h of compounds 6a and 7a were 33.0% and 41.8%, respectively; whereas the corresponding mortality rate of 1 was only 1.6%. That is, the mortality rates at 72 h of 6a and 7a were greater than 20-fold of that of 1. Interestingly, introduction of the acetoxy moiety at the C-4 position of 2/3 could afford more potent compounds 6a and 7a. All in all, here compounds 6a and 7a (containing the 4-acetyloxy group) showed potent insecticidal and acaricidal activities against M. separata and T. cinnabarinus, respectively. This result was consistent with that in our previous reports, where it was found that introduction of acetyloxy group at the C-4 position of the corresponding podophyllotoxin derivatives could lead to more promising compounds (Che et al., 2013b; Huang et al., 2017).
Table 3 Acaricidal activity of compounds 1; 6a,c,d,f,g and 7a–g against the female adults of T. cinnabarinus treated at a concentration of 0.5 mg/mL. Compound
Corrected mortality rate (%)a 48 h
1 6a 6c 6d 6f 6g 7a 7b 7c 7d 7e 7f 7g spirodiclofen
72 h b
1.4 f 20.1 ab 8.4 d 7.9 d 3.5 ef 21.3 ab 19.0 b 13.4 c 15.4 c 6.8 de 14.0 c 6.0 de 5.7 de 22.9 a
1.6 j 33.0 c 17.4 ef 8.4 gh 3.7 ij 25.3 d 41.8 b 14.2 f 23.0 d 11.0 g 14.5 f 6.4 hi 19.6 e 59.7 a
a
Values are means of three replicate. Multiple range test using Duncan’s test (p < 0.05). The same letters denote treatments not significantly different from each other. b
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. 3. Results and discussion 3.1. Chemistry As depicted in Scheme 1, podophyllotoxin (1) first reacted with 1 equiv. of N-chlorosuccinimide (NCS) to give 2'-chloropodophyllotoxin (2′), which further reacted with 1 equiv. of N-bromosuccinimide (NBS) to afford 2'-chloro-5-bromopodophyllotoxin (2); 2',5-dibromopodophyllotoxin (3) were obtained by reaction of podophyllotoxin (1) with 2 equiv. of NBS (Che et al., 2013a). Finally, in the presence of DCC and DMAP, target compounds 6a,c,d,f,g and 7a–g were smoothly obtained by reaction of 2/3 with the corresponding carboxylic acids (5). The chemical structures of all compounds were well characterized by 1 HNMR, HRMS, optical rotation, and mp. For the cis configuration of the lactone of 4 was determined by the X-ray crystallography (Che et al., 2013a), assignment of the configuration at the C-2 position of 6a,c,d,f,g and 7a–g was based on their coupling constants (J) of H-2, which were compared with those of 4. Comparison of partial 1H NMR spectra of 4 with those of 6a,c,d,f,g and 7a–g was depicted in Fig. 2. The coupling constants (J) of H-2 of 4 were 15.5 and 5.0 Hz, respectively; the coupling constants (J) of H-2 of 6a,c,d,f,g and 7a–g was 15.5 (or 15.0) and 5.0 (or 4.5) Hz, respectively; this indicates that the configuration of the lactones of 6a,c,d,f,g and 7a–g was the same of that of 4. So the configuration of their lactones was all converted from trans into cis. However, in our previous papers, when 2′(2′,6′)-(di) halogenopodophyllotoxins reacted with carboxylic acids in the same reaction conditions, the configuration of the lactones of the corresponding products was all trans and was not changed (Che et al., 2013b; Huang et al., 2017). It demonstrated that when 2/3 reacted with carboxylic acids in the presence of DCC and DMAP, their C-5 bromine atom may affect the configuration of the lactone.
4. Conclusion In summary, a series of 4α-acyloxy derivatives of 2'-chloro/bromo5-bromopodophyllotoxins were prepared by structural modification of podophyllotoxin as a sustainable natural resource, and evaluated for their insecticidal and acaricidal activities against M. separata and T. cinnabarinus. Among all derivatives, compounds 6a and 7a all exhibited
3.2. Pesticidal activity The pesticidal activity of compounds 1; 6a,c,d,f,g and 7a–g against 52
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