Synthesis and quantitative structure-activity relationship (QSAR) study of novel 4-acyloxypodophyllotoxin derivatives modified in the A and C rings as insecticidal agents

Article abstract of DOI:10.1021/jf305011n

In continuation of our program aimed at the discovery and development of natural-product-based insecticidal agents, we have synthesized three series of novel 4-acyloxy compounds derived from podophyllotoxin modified in the A and C rings, which is isolated as the main secondary metabolite from the roots and rhizomes of Podophyllum hexandrum. Their insecticidal activity was preliminarily evaluated against the pre-third-instar larvae of Mythimna separata in vivo. Compound 9g displayed the best promising insecticidal activity. It revealed that cleavage of the 6,7-methylenedioxy group of podophyllotoxin will lead to a less active compound and that the C-4 position of podophyllotoxin was the important modification location. A quantitative structure-activity relationship (QSAR) model was developed by genetic algorithm combined with multiple linear regression (GA-MLR). For this model, the squared correlation coefficient (R ²) is 0.914, the leave-one-out cross-validation correlation coefficient (Q²_LOO) is 0.881, and the root-mean-square error (RMSE) is 0.024. Five descriptors, BEHm2, Mor14v, Wap, G1v, and RDF020e, are likely to influence the biological activity of these compounds. Among them, two important ones are BEHm2 and Mor14v. This study will pave the way for further design, structural modification, and development of podophyllotoxin derivatives as insecticidal agents.

Full text of DOI:10.1021/jf305011n

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Article
Synthesis and Quantitative Structure-Activity Relationship
(QSAR) Study of Novel 4-Acyloxypodophyllotoxin Derivatives
Modified in the A and C rings as Insecticidal Agents
Shu-zhen He, Yonghua Shao, Lingling Fan, Zhiping Che, Hui
Xu, Xiaoyan Zhi, Juan Juan Wang, Xiaojun Yao, and Huan Qu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf305011n • Publication Date (Web): 01 Jan 2013
Downloaded from http://pubs.acs.org on January 6, 2013
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Synthesis and Quantitative Structure-Activity Relationship (QSAR) Study
of Novel 4-Acyloxypodophyllotoxin Derivatives Modified in the A and C
rings as Insecticidal Agents
Running Title: 4-Acyloxypodophyllotoxin Derivatives Modified in the A and
C rings as Insecticidal Agents
§
Shuzhen He,†, Yonghua Shao,‡ Lingling Fan,† Zhiping Che,† Hui Xu,*,† Xiaoyan Zhi,†
Juanjuan Wang,† Xiaojun Yao,*,‡ and Huan Qu†
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†Laboratory of Pharmaceutical Design & Synthesis, College of Sciences, Northwest A&F
University, Yangling 712100, Shannxi Province, People′s Republic of China
‡Department of Chemistry, Lanzhou University, Lanzhou 730000, Gansu Province, People′s
Republic of China
§Spice and Beverage Research Institute, Chinese Academy of Tropical Agriculture Science,
Wanning 571533, Hainan Province, People′s Republic of China
* Author to whom correspondence should be addressed [(H.X.) telephone
+86(0)29ꢀ87091952; fax: +86(0)29ꢀ87091952; eꢀmail orgxuhui@nwsuaf.edu.cn;
xjyao@lzu.edu.cn].
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Abstract
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In continuation of our program aimed at the discovery and development of natural
ꢀproductꢀbased insecticidal agents, we have synthesized three series of novel 4ꢀacyloxy
compounds derived from podophyllotoxin modified in the A and C rings, which is isolated as
the main secondary metabolite from the roots and rhizomes of Podophyllum hexandrum.
Their insecticidal activity was preliminarily evaluated against the preꢀthirdꢀinstar larvae of
Mythimna separata in vivo. Compound 9g displayed the best promising insecticidal activity.
It revealed that cleavage of 6,7ꢀmethylenedioxy group of podophyllotoxin will lead to the less
active compound and the Cꢀ4 position of podophyllotoxin was the important modification
location. Quantitative structureꢀactivity relationship (QSAR) model was developed by genetic
algorithm combined with multiple linear regression (GAꢀMLR). For this model, the square
correlation coefficient (R2) is 0.914, the leaveꢀoneꢀout crossꢀvalidation correlation coefficient
(Q2Loo) is 0.881 and root mean square error (RMSE) is 0.024. Five descriptors such as
BEHm2, Mor14v, Wap, G1v and RDF020e, are likely to influence the biological activity of
these compounds. Among them, two important ones are the BEHm2 and Mor14v. It will pave
the way for further design, structural modification and development of podophyllotoxin
derivatives as insecticidal agents.
KEYWORDS: Podophyllotoxin; Acyloxy; A and C rings modification; Botanical insecticide;
Insecticidal activity; QSAR; Mythimna separata Walker
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INTRODUCTION
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Lepidoptera are the most diverse pest insect order. The larvae of many lepidopteran
species are major pests in agriculture and can cause extensive damage to certain crops.1 For
example, the oriental armyworm (Mythimna separata Walker), a typical lepidopteran pest, is
widely distributed in China, Japan, Southeast Asia, India, Eastern Australia, New Zealand
and some Pacific Islands, and sometimes its outbreaks could result in widespread incidence
and complete crop loss.2 Although a wide variety of synthetic insecticides were introduced to
control lepidopteran pests, the extensive application of synthetic agrochemicals over the years
has resulted in the development of resistance in lepidopteran pests populations.3ꢀ5
Development of new effective, selective and safe pesticides, therefore, is still a challenging
task. As plant secondary metabolites result from the interaction between plants and
environment (life and nonꢀlife) during the long period of evolution in plants, and pesticides
produced from plant secondary metabolites may result in less or slower resistance
development and lower pollution,6 consequently, the discovery and development of new
insecticidal compounds directly from plant secondary metabolites, or by using them as the
leadꢀcompounds for further modifications has recently been one of the important ways for
research and development of new pesticides.7ꢀ9
Podophyllotoxin (1, Figure 1), a naturally occurring aryltetralin lignan, besides its use as
the lead compound for the preparation of potent anticancer drugs such as etoposide (VPꢀ16, 2,
Figure 1), teniposide (VMꢀ26, 3, Figure 1) and etopophos (etoposide phosphate, 4, Figure
1),10ꢀ12 has also received much research attention for its interesting insecticidal and antifungal
activities.13ꢀ18 Recently, we have studied the insecticidal activity of a series of
2βꢀchloropodophyllotoxin and 2α/βꢀbromopodophyllotoxin derivatives with modified C and
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D rings of 1, and found some compounds showed more potent insecticidal activity than
toosendanin, a commercial botanical insecticide extracted from Melia azedarach.19ꢀ22
Encouraged by the aboveꢀmentioned interesting results, and to find novel natural
productsꢀbased insecticidal agents to control the lepidopteran pests, we herein designed and
synthesized three series of 4ꢀacyloxypodophyllotoxin derivatives modified in the A and C
rings as insecticidal agents against the preꢀthirdꢀinstar larvae of Mythimna separata, and
wanted to investigate the influence of A and C rings and the configuration of acyloxy at the
Cꢀ4 position on the insecticidal activity. Meanwhile, quantitative structureꢀactivity
relationship (QSAR) studies were also described.
MATERIALS AND METHODS
General: Podophyllotoxin was purchased from Gansu Gerui Medicinal Materials Co., Ltd.
All reagents and solvents were of reagent grade or purified according to standard methods
before use. Analytical thinꢀlayer chromatography (TLC) and preparative thinꢀlayer
chromatography (PTLC) were performed with silica gel plates using silica gel 60 GF254
(Qingdao Haiyang Chemical Co., Ltd.). Melting points were determined on a digital
meltingꢀpoint apparatus and were uncorrected. Infrared spectra (IR) were recorded on a
Bruker TENSOR 27 spectrometer. Proton nuclear magnetic resonance spectra (1H NMR)
were recorded on a Bruker Avance III 500 MHz instrument in CDCl3 using TMS
(tetramethylsilane) as the internal standard. Highꢀresolution mass spectra (HRꢀMS) were
carried out with IonSpec 4.7 Tesla FTMS instrument. The purities of the tested compounds
were determined by reverse phase high performance liquid chromatography (RPꢀHPLC),
which were recorded on a Shimadzu LCꢀ15C liquid chromatograph (SPDꢀ15C UVꢀVIS
spectrophotometric detector (190ꢀ700 nm)) using a flow rate of 1.0 mL/min (MeOH/H2O =
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5/1), and a Hypersil ODS C18 column (5 ꢀm, 4.6×150 mm) as the stationary phase.
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Synthesis of 6,7ꢁO,OꢁDemethylenepodophyllotoxin (5): To a solution of boron trichloride in
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dichloromethane (1 M, 1mL) precooled at ꢀ70 C was added dropwise podophyllotoxin (1)
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(0.1 g, 0.25 mmol) in dichloromethane (5 mL) over 15 min. After the mixture was stirred at
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ꢀ70 C for an additional 2 h, the mixture was poured into 20 mL of iceꢀwater, and extracted
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with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine until the
pH was 6ꢀ7, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated in
vacuo to give a white solid, which was put into a mixture of acetoneꢀwaterꢀcalcium carbonate
(1 mL, 1 mL, 0.3 g) and refluxed for 1 h. The white suspension was filtered off, and the
filtrate was acidified by aq. HCl (2 M) until pH was 2ꢀ3 and extracted by ethyl acetate (5 × 30
mL). The combined organic layers were dried over anhydrous sodium sulfate, and evaporated
in vacuo to afford 5 in a 72% yield as a white solid.
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Synthesis of 6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdimethylpodophyllotoxin (6): To a mixture of 5
(0.05 g, 0.125 mmol) and K2CO3 (0.276 g, 2 mmol) in acetone (5 mL) at 0 oC, methyl iodide
(0.071 g, 0.5 mmol) was added. Then the mixture was stirred at room temperature. When the
reaction was complete according to TLC analysis, the mixture was filtered. The filtrate was
evaporated in vacuo and purified by silica gel column chromatography to give 6 in a 86%
yield as a white solid.
Synthesis of 6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdimethylepipodophyllotoxin (7): To a mixture of
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6 (430 mg, 1 mmol) and NaI (299 mg, 2 mmol) in CH3CN (10 mL) at 0 C, a solution of
BF3ꢁEt2O (0.378 mL, 1.4 mmol) in CH3CN (3 mL) was added dropwise. After adding, the
mixture was stirred at room temperature. When the reaction was complete according to TLC
analysis, the mixture was evaporated in vacuo to afford the residue. To the above residue,
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acetoneꢀwater (5 mL, 10 mL) and BaCO3 (395 mg, 2 mmol) was added. Then the mixture
was reacted at room temperature. When the reaction was complete according to TLC analysis,
the mixture was extracted with CH2Cl2 (3 × 30 mL). The combined organic layers were
washed with water and aq. Na2S2O3 (10%, 25 mL), dried over anhydrous Na2SO4,
concentrated in vacuo, and purified by silica gel column chromatography to give 7 in a 69%
yield as a white solid.
Synthesis of 6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdibenzylpodophyllotoxin (8): To a mixture of 5
(0.1 g, 0.25 mmol), Cs2CO3 (0.195 g, 0.6 mmol), and KI (12 mg, 0.075 mmol) in acetone (10
mL), benzyl bromide (0.103 g, 0.6 mmol) was added. Then the mixture was stirred at room
temperature. When the reaction was complete according to TLC analysis, the mixture was
filtered. The filtrate was evaporated in vacuo to give the residue, which was diluted with
CH2Cl2 (30 mL), washed by water, dried over anhydrous sodium sulfate, and purified by
PTLC eluting with petroleum ether/ethyl acetate (1:2, v/v) to give 8 in a 36% yield as a white
solid.
General
Procedure
for
the
Synthesis
of
6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdimethylꢁ4αꢁacyloxypodophyllotoxin Derivatives (9a-m),
6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdimethylꢁ4βꢁacyloxypodophyllotoxin Derivatives (10c, d, f, g
and l), and 6,7ꢁO,OꢁDemethyleneꢁ6,7ꢁO,Oꢁdibenzylꢁ4αꢁacyloxypodophyllotoxin Derivatives
(11d, f, g and l): A mixture of the corresponding acids (0.32 mmol), diisopropylcarbodiimide
(DIC, 0.32 mmol), 4ꢀdimethylaminopyridine (DMAP, 0.045 mmol), and 6, 7 or 8 (0.23 mmol)
in dry CH2Cl2 (10 mL) was stirred at room temperature. When the reaction was complete
according to TLC analysis, the mixture was diluted by CH2Cl2 (30 mL), washed by aq. HCl
(0.1 M, 20 mL), aq. NaHCO3 (5%, 20 mL) and brine (20 mL), dried over anhydrous Na2SO4,
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concentrated in vacuo, and purified by PTLC to give the pure target products 9aꢀm, 10c, d, f,
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g and l, and 11d, f, g and l. Their structures were well characterized by H NMR, HRMS,
optical rotation, IR, and mp. The example data of 9a, 9b, 10c, 10d, 11d and 11f are shown as
follows, whereas those of other compounds can be found in the Supporting Information.
Data for 9a: Yield = 69%, white solid, mp = 70ꢀ72 C. [α]20D = ꢀ2 (c 3.2 mg/mL, CHCl3). IR
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(cmꢀ1): 2959, 2921, 2837, 1772, 1726, 1588, 1514, 1465, 1236, 1125, 1008, 762. H NMR
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(500 MHz, CDCl3) δ: 6.76 (s, 1H, Hꢀ5), 6.54 (s, 1H, Hꢀ8), 6.38 (s, 2H, Hꢀ2′, 6′), 5.91 (d, J =
9.0 Hz, 1H, Hꢀ4), 4.64 (s, 1H, Hꢀ1), 4.37ꢀ4.40 (m, 1H, Hꢀ11), 4.19ꢀ4.23 (m, 1H, Hꢀ11), 3.90
(s, 3H), 3.80 (s, 6H), 3.74 (s, 6H), 2.91ꢀ2.94 (m, 1H, Hꢀ2), 2.77ꢀ2.86 (m, 1H, Hꢀ3), 2.20 (s,
3H). HRMS: calcd for C25H28O9 ([M]+), 472.1728; found, 472.1739.
Data for 9b: Yield = 34%, white solid, mp = 191ꢀ193 oC. [α]20D = ꢀ72 (c 3.2 mg/mL, CHCl3).
IR (cmꢀ1): 2955, 2921, 2837, 1780, 1752, 1590, 1518, 1420, 1263, 1243, 1226, 1193, 1169,
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1125, 1104, 995, 866. H NMR (500 MHz, CDCl3) δ: 6.80 (s, 1H, Hꢀ5), 6.56 (s, 1H, Hꢀ8),
6.38 (s, 2H, Hꢀ2′, 6′), 5.99 (d, J = 9.0 Hz, 1H, Hꢀ4), 4.66 (d, J = 3.5 Hz, 1H, Hꢀ1), 4.40ꢀ4.43
(m, 1H, Hꢀ11), 4.21ꢀ4.26 (m, 1H, Hꢀ11), 4.16 (d, J = 8.0 Hz, 2H, CH2Cl), 3.90 (s, 3H), 3.82
(s, 6H), 3.74 (s, 6H), 2.85ꢀ2.98 (m, 2H, Hꢀ2, 3). HRMS: calcd for C25H27O9Cl ([M]+),
506.1338; found, 506.1345.
Data for 10c: Yield = 59%, white solid, mp = 76ꢀ78 C. [α]20 = ꢀ92 (c 3.5 mg/mL, CHCl3).
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IR (cmꢀ1): 2963, 2936, 2837, 1777, 1729, 1589, 1515, 1461, 1249, 1172, 1127, 1110, 1047,
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997, 853, 745. H NMR (500 MHz, CDCl3) δ: 6.90 (s, 1H, Hꢀ5), 6.56 (s, 1H, Hꢀ8), 6.27 (s,
2H, Hꢀ2′, 6′), 6.20 (d, J = 2.5 Hz, 1H, Hꢀ4), 4.70 (d, J = 5.0 Hz, 1H, Hꢀ1), 4.34ꢀ4.38 (m, 1H,
Hꢀ11), 3.91ꢀ3.95 (m, 1H, Hꢀ11), 3.89 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (s, 6H), 3.24 (dd,
J = 14.0, 4.5 Hz, 1H, Hꢀ2), 2.97ꢀ3.01 (m, 1H, Hꢀ3), 2.33 (t, J = 7.5 Hz, 2H, CH2CH2CH3),
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1.64ꢀ1.71 (m, 2H, CH2CH2CH3), 0.94 (t, J = 7.5 Hz, 3H, CH2CH2CH3). HRMS: calcd for
C27H32O9 ([M]+), 500.2041; found, 500.2052.
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Data for 10d: Yield = 84%, white solid, mp = 66ꢀ68 C. [α]20D = ꢀ80 (c 3.2 mg/mL, CHCl3).
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IR (cmꢀ1): 2955, 2836, 1779, 1728, 1589, 1515, 1463, 1248, 1127, 1110, 997, 856, 747. H
NMR (500 MHz, CDCl3) δ: 6.90 (s, 1H, Hꢀ5), 6.56 (s, 1H, Hꢀ8), 6.27 (s, 2H, Hꢀ2′, 6′), 6.19
(d, J = 3.0 Hz, 1H, Hꢀ4), 4.70 (d, J = 4.5 Hz, 1H, Hꢀ1), 4.34ꢀ4.37 (m, 1H, Hꢀ11), 3.91ꢀ3.95
(m, 1H, Hꢀ11), 3.89 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (s, 6H), 3.24 (dd, J = 14.5, 4.5 Hz,
1H, Hꢀ2), 2.95ꢀ3.00 (m, 1H, Hꢀ3), 2.34 (t, J = 7.5 Hz, 2H, CH2(CH2)3CH3), 1.61ꢀ1.67 (m, 2H,
CH2CH2(CH2)2CH3), 1.25ꢀ1.31 (m, 4H, CH2CH2(CH2)2CH3), 0.87 (t, J = 7.0 Hz, 3H,
CH2(CH2)3CH3). HRMS: calcd for C29H36O9 ([M]+), 528.2354; found, 528.2358.
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Data for 11d: Yield = 23%, white solid, mp = 50ꢀ52 C. [α]20 = ꢀ49 (c 3.2 mg/mL, CHCl3).
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IR (cmꢀ1): 2928, 2859, 1778, 1728, 1587, 1510, 1454, 1331, 1244, 1167, 1127, 1002, 997,
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739. H NMR (500 MHz, CDCl3) δ: 7.27ꢀ7.43 (m, 10H, 2×OCH2C6H5), 6.82 (s, 1H, Hꢀ5),
6.64 (s, 1H, Hꢀ8), 6.31 (s, 2H, Hꢀ2′, 6′), 5.85 (d, J = 9.0 Hz, 1H, Hꢀ4), 5.07ꢀ5.16 (m, 4H,
2×OCH2C6H5), 4.58 (d, J = 4.0 Hz, 1H, Hꢀ1), 4.33ꢀ4.36 (m, 1H, Hꢀ11), 4.16ꢀ4.20 (m, 1H,
Hꢀ11), 3.82 (s, 3H), 3.70 (s, 6H), 2.88 (dd, J = 14.5, 4.0 Hz, 1H, Hꢀ2), 2.71ꢀ2.80 (m, 1H, Hꢀ3),
2.33 (t, J = 7.5 Hz, 2H, CH2(CH2)3CH3), 1.62ꢀ1.68 (m, 2H, CH2CH2(CH2)2CH3), 1.25ꢀ1.34
(m, 4H, CH2CH2(CH2)2CH3), 0.90 (t, J = 6.0 Hz, 3H, CH2(CH2)3CH3). HRMS: calcd for
C41H44O9 ([M]+), 680.2980; found, 680.2989.
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Data for 11f: Yield = 21%, white solid, mp = 69ꢀ70 C. [α]20 = ꢀ55 (c 3.6 mg/mL, CHCl3).
D
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IR (cmꢀ1): 2924, 2881, 1777, 1732, 1508, 1455, 1245, 1126, 992, 742. H NMR (500 MHz,
CDCl3) δ: 7.27ꢀ7.40 (m, 15H, 2×OCH2C6H5, CH2C6H5), 6.66 (s, 1H, Hꢀ5), 6.61 (s, 1H, Hꢀ8),
6.29 (s, 2H, Hꢀ2′, 6′), 5.83 (d, J = 9.0 Hz, 1H, Hꢀ4), 5.05 (s, 2H, OCH2C6H5), 4.96 (s, 2H,
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OCH2C6H5), 4.56 (d, J = 4.0 Hz, 1H, Hꢀ1), 4.24ꢀ4.27 (m, 1H, Hꢀ11), 4.12ꢀ4.16 (m, 1H, Hꢀ11),
3.82 (s, 3H), 3.69 (s, 6H), 3.67 (s, 2H, CH2C6H5), 2.86 (dd, J = 14.5, 4.5 Hz, 1H, Hꢀ2),
2.70ꢀ2.78 (m, 1H, Hꢀ3). HRMS: calcd for C43H40O9 ([M]+), 700.2667; found, 700.2674.
Biological Assay. The insecticidal activity of three series of 4ꢀacyloxypodophyllotoxin
derivatives (9aꢀm, 10c, d, f, g and l, and 11d, f, g and l) against the preꢀthirdꢀinstar larvae of
M. separata was assessed by leafꢀdipping method, as described previously.23 For each
compound, 30 preꢀthirdꢀinstar larvae (10 larvae per group) were used. Acetone solutions of
compounds 9aꢀm, 10c, d, f, g and l, 11d, f, g and l, and toosendanin (used as a positive
control) were prepared at the concentration of 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. 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, untreated fresh leaves were added to all
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dishes until adult emergence. The experiment was carried out at 25 ± 2 C and relative
humidity (RH) 65ꢀ80%, and on 12 h/12 h (light/dark) photoperiod. The insecticidal activity
of the tested compounds against the preꢀthirdꢀinstar larvae of M. separata was calculated by
the following formula:
corrected mortality rate (%) = (T − C) × 100/ (1 − C)
Where T is the mortality rate in the treated group expressed as a percentage and C is the
mortality rate in the untreated group expressed as a percentage.
QSAR Model Development.
Data Set. The experimental data used in this study contained 27 compounds (1, 5-8, 9aꢀm,
10c, d, f, g and l, and 11d, f, g and l). The biological activity of 27 compounds was expressed
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by final mortality rate values and used as dependent variable in the following analyses.
Molecular Descriptor Calculation and Filtering. The 2D structures of 27 compounds were
drawn into HyperChem24 software and preꢀoptimized by using MM+ molecular mechanics
force field. The final lowest energy conformation of the compound was obtained by using the
semiꢀempirical AM1 method.25 The theoretical molecular descriptors for the optimized
geometries were calculated using DRAGON5.4 software.26 In DRAGON, a total of 1664
0Dꢀ3D molecular descriptors were calculated to describe structural features for each
compound. The types of descriptors include (a) 0D molecular descriptors (constitutional
descriptors), (b) 1D molecular descriptors (functional groups counts and atomꢀcentered
fragments), (c) 2D molecular descriptors (topological descriptors, walk and path counts,
connectivity indices, information indices, 2D autocorrelations, edge adjacency indices,
Burden eigenvalues, topological charge index and eigenvalueꢀbased index), (d) 3D molecular
descriptors (3DꢀRandic molecular profiles, geometrical descriptors, RDF descriptors,
3DꢀMoRSE descriptors, WHIM descriptors27 and GETAWAY descriptors28), and (e) other
molecular descriptors (charge descriptors and molecular properties). The list and meanings of
different types of descriptors could be found in the references of the DRAGON package. The
detailed calculation procedure could be found in the Handbook of Molecular Descriptors.29
Constant values and descriptors found to be pairwise correlated with a correlation coefficient
greater than 0.99 were excluded to reduce the redundant information. Thus, 605 molecular
descriptors were left after pretreatment step. The quadratic term of the descriptors was also
added to the descriptors pool; thus, a total of 1210 descriptors would subject to the
descriptors selection procedure.
Descriptors Selection, Model Development and Validation. The genetic algorithm30 (GA)
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method was used for descriptors selection. Multiple linear regression (MLR) was used for the
QSAR model development based on the descriptors selected by GA. The genetic algorithm
was applied to the pool of 1210 molecular descriptors to extract the molecular descriptors
relevant to the studied biological activity. In general, the genetic algorithm selects variable by
a mechanism of selection, crossover and mutation operation by mimicking the biological
population evolution theory. The three operations were repeated until the stopping conditions
are achieved. In this paper, GAꢀMLR calculation was performed by using the Moby Digs
software.31 The fitness function was correlation coefficient of leaveꢀoneꢀout (LOO) cross
validation (Q2LOO). The parameters used in the GA process were as follows: population size
100, maximum allowed descriptors in a model 5 and reproduction/mutation tradeꢀoff 0.5. The
default values of the other parameters in the Moby Digs software were used. Several different
parameters were applied to assess the performance of the developed QSAR model such as
square correlation coefficient R2, leaveꢀoneꢀout crossꢀvalidation (Q2LOO) and root mean
square error (RMSE).
Applicability Domain of the QSAR Model. The analysis of the applicability domain (AD) of
the developed QSAR model allows to verify the prediction reliability and to identify the
possible outliers in the QSAR model. Williams plot32 (the plot of crossꢀvalidated standardized
residuals versus hat values) was used to describe the applicability domain of the QSAR
model. When the compound with a high hat value (h) (h>h*, the waning hat being h* = 3p’/n,
where p’ is the number of model parameters plus one, and n is number of the chemical used to
build model), it should keep in mind that the prediction for the compound is extrapolated and
may be considered less reliable. A compound is considered as a Y outlier if the compound
with cross validated standardized residuals greater than three standard deviation units.
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RESULTS AND DISCUSSION
Synthesis. Three series of novel 4ꢀacyloxypodophyllotoxin derivatives (9aꢀm, 10c, d,
f, g and l, and 11d, f, g and l) modified in the A and C rings were synthesized as shown in
Scheme 1. First, the intermediate 5 was prepared via selective removing the
6,7ꢀmethylenedioxy group of 1 by treating with boron trichloride (BCl3), followed by weak
basic hydrolysis with calcium carbonate (CaCO3).33 Methylation and benzylation of 5 were
then achieved by using methyl iodide and benzyl bromide in the presence of K2CO3 or
Cs2CO3 to afford 6 and 8, respectively.34 Compound 6 reacted with NaI/BF3 Et2O to give 7.35
.
A series of 6,7ꢀO,Oꢀdemethyleneꢀ6,7ꢀO,Oꢀdimethylꢀ4αꢀacyloxypodophyllotoxin derivatives
(9aꢀm) were obtained by reaction of 6 with the corresponding carboxylic acids (12a-m) in the
presence of N,N'ꢀdiisopropylcarbodiimide (DIC) and 4ꢀN,Nꢀdimethylaminopyridine
(DMAP).19
Similarly,
a
series
of
6,7ꢀO,Oꢀdemethyleneꢀ6,7ꢀO,Oꢀdimethylꢀ4βꢀacyloxypodophyllotoxin derivatives (10c, d, f, g
and l) were produced by reaction of 7 with 12c, d, f, g and l in the presence of DIC and
DMAP.
Finally,
a
series
of
6,7ꢀO,Oꢀdemethyleneꢀ6,7ꢀO,O
ꢀdibenzylꢀ4αꢀacyloxypodophyllotoxin derivatives (11d, f, g and l) were afforded by reaction
of 8 with 12d, f, g and l in the presence of DIC and DMAP.
The assignment of configuration at the Cꢀ4 position of the above three series of
4ꢀacyloxypodophyllotoxin derivatives was based on J3.4 coupling constants: the
Cꢀ4βꢀsubstituted compounds have a J3.4 ≈ 4.0 Hz due to a cis relationship between Hꢀ3 and
Hꢀ4; if J3.4 ≧ 10.0 Hz, it indicates that Hꢀ3 and Hꢀ4 is trans relationship, and the substituent
at the Cꢀ4 position of podophyllotoxin is α configuration.36 For example, as shown in Figure
2, the J3.4 values of Hꢀ4 of 9f and 10f were 9.0 and 3.5 Hz, respectively, therefore, the
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298
phenylacetyloxy groups at the Cꢀ4 position of 9f and 10f were α and β configuration,
respectively. The relationships of the configuration at the Cꢀ4 position of three series of
4ꢀacyloxypodophyllotoxins and their J3.4 coupling constants were described in Table 1.
Insecticidal Activity.
The insecticidal activity of three series of
4ꢀacyloxypodophyllotoxin derivatives (9aꢀm, 10c, d, f, g and l, and 11d, f, g and l) against
the preꢀthirdꢀinstar larvae of M. separata was tested by the leafꢀdipping method at the
concentration of 1 mg/mL. The purities of all target compounds were all greater than 95%
measured with reverse phase high performance liquid chromatography (RPꢀHPLC) (see
Supporting Information).
As indicated in Table 2, the corresponding mortality rates caused by these compounds
after 35 days were usually higher than those after 10 and 20 days. For example, the corrected
mortality rate of 9g against M. separata after 10 days was only 10%, after 20 days the
corresponding mortality rate was increased to 26.7%, but after 35 days the corresponding
mortality rate was sharply increased to 60%, which was 6ꢀfold of that after 10 days. That is,
these compounds, in a timeꢀdependent manner, different from other conventional neurotoxic
insecticides such as organophosphates, carbamates and pyrethroids, showed delayed
insecticidal activity. Meanwhile, the symptoms of the tested M. separata were also
characterized in the same way as our previous reports.23 Due to feeding too much treated
leaves during the first 48 h, some larvae died slowly with the slim and wrinkled bodies during
the larval period. In the meantime, many larvae of the treated groups moulted to malformed
pupae, and died during the stage of pupation. Malformed moths with imperfect wings were
also appeared in the treated groups. Compounds 9e, 9g, 9j, and 10g exhibited equal or higher
insecticidal activity than toosendanin. Especially 9g, bearing αꢀnaphthylacetyloxy at the Cꢀ4
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321
position, displayed the best promising insecticidal activity with the final mortality rate of
60%. In general, cleavage of 6,7ꢀmethylenedioxy group of 1 will lead to the less active
compound 5. For example, when compound 1, whose final mortality rate was 40%, was
selectively removed the 6,7ꢀO,Oꢀmethylene group to give 5, the final mortality rate of 5 was
decreased to 23.3%. However, introduction of methyloxy or benzyloxy group at the Cꢀ6 and
Cꢀ7 positions of 5 afforded 6 and 8, respectively, the corresponding mortality rates of 6
(36.7%) and 8 (33.3%) were increased as compared with that of 5. The Cꢀ4 position of
podophyllotoxin was the important modification location; that is, introduction of appropriate
substituents at the Cꢀ4 position of 6 could give the more active derivatives (e.g., 9e, 9g, 9j,
and 10g) than 6. However, the effect of the configuration of acyloxy at the Cꢀ4 position on
the insecticidal activity was not very obvious. For example, to 4αꢀacyloxypodophyllotoxin
series, the final mortality rates of 9c, 9d, 9f, 9g and 9l were 33.3%, 36.7%, 26.7%, 60.0% and
36.7%, respectively; to the corresponding 4βꢀacyloxypodophyllotoxin series, the final
mortality rates of 10c, 10d, 10f, 10g and 10l were 36.3%, 43.3%, 33.3%, 50.0% and 36.7%,
respectively. Interestingly, when the 6,7ꢀdimethoxy group of 8g was substituted by the
6,7ꢀdibenzyloxy one to afford 12g, the corresponding mortality rates of 12g was sharply
decreased to 33.3%. To alkylacyloxy series of 9a-e, introduction of nꢀheptylacyloxy at the
Cꢀ4 position of 6 resulted in the more potent compound 9e. For example, the final mortality
rates of 9a (R = Me), 9b (R = CH2Cl), 9c (R = nꢀPr) and 9d (R = nꢀpentyl) were 36.7%,
26.7%, 33.3% and 36.7%, respectively; whereas the final mortality rate of 9e (R = nꢀheptyl)
was 53.3%. Introduction of αꢀnaphthylacetyloxy at the Cꢀ4 position of 6 could lead to the
more potent compound 9g by the same way as described in our previous paper.19,21
QSAR Model. In order to select the most relevant descriptors responsible for the
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323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
biological activity, a total of 1210 molecular descriptors were subjected to the GA selection
procedure. When adding another descriptor did not improve the statistics parameter (Q2
)
LOO
of a model significantly, it is means that the best descriptors combination has been achieved.
On the basis of this principle, the 5ꢀdescriptor model was selected, whose regression equation
and the statistical items were as follows:
Y = 0.776BEHm2 + 0.338RDF020e – (0.720Wap)2 + 0.741(Mor14v)2 + 0.471(G1v)2 – 4.806
(1)
Where Y is the final mortality rate.
ndataset = 27, R2 = 0.914, RMSE = 0.024, Q2LOO = 0.881, RMSELOO = 0.028
From the above statistical parameters, it can be seen that the model was stable and robust.
The predicted biological activity values by this model were listed in Table 3. Figure 3 shows
the graph of the experimental versus predicted values of 27 compounds by GAꢀMLR model.
By a deep analysis of the descriptors in derived QSAR model, it allows us to find some
factors that are likely to influence the biological activity of these compounds. From the Eq.
(1), we can see that five descriptors were involved. The relative importance was determined
by their standardized coefficient values. The most important descriptor is the BEHm2
(highest eigenvalue n.2 of Burden matrix / weighted by atomic masses) encoding the Burden
eigenvalues descriptor, which is calculated based on hydrogenꢀincluded molecular graph
weighted by atomic masses. Another important descriptor is Mor14v, which belongs to
3DꢀMoRSE descriptors (3DꢀMolecule Representation of Structure based on Electron
diffraction) were obtained based on the idea of combination of the 3D atomic coordinates of
the molecular and chemical atomic information. This descriptor represents the 3DꢀMoRSE
signal 14 / weighted by atomic van der Waals volumes. Wap is a topological descriptor based
on allꢀpath Wiener index, and the Wiener index is calculated as the halfꢀsum of all topological
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345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
distances collected in the distance matrix. The topological descriptors are based on the graph
representation of the molecule, which are sensitive to one or more structural features of the
molecule such as size, shape, symmetry, branching and cyclicity and can also encode
chemical information concerning atom type and bond multiplicity. G1v is a WHIM descriptor,
which represents 1st component symmetry directional WHIM index/weighted by atomic van
der Waals volumes. WHIM descriptors are built in such a way as to capture relevant
molecular 3D information regarding molecular size, shape, symmetry, and atom distribution
with respect to invariant reference frames. RDF020e is a RDF descriptor representing Radial
Distribution Function – 2.0/weighted by atomic Sanderson electronegativities. The
applicability domain of the derived model described by Williams plot (the crossꢀvalidated
standardized residual versus hat values) was shown in Figure 4. It obviously suggested that
there is no Y outlier, and only compound 11g (No.26) is outlier from molecular structure
(with the hat value higher than the warning h* value of 0.667).
In summary, three series of novel 4ꢀacyloxypodophyllotoxin derivatives modified in the
A and C rings were synthesized and evaluated for their insecticidal activity against the
preꢀthirdꢀinstar larvae of M. separata in vivo at the concentration of 1 mg/mL. Especially 9g
exhibited the more promising and pronounced insecticidal activity than toosendanin. It
suggested that cleavage of 6,7ꢀmethylenedioxy group of podophyllotoxin will lead to the less
active compound and the Cꢀ4 position of podophyllotoxin was the important modification
location. QSAR model demonstrated that five descriptors, such as BEHm2, Mor14v, Wap,
G1v and RDF020e, are likely to influence the biological activity of these compounds. Among
them, two important ones are the BEHm2 and Mor14v. It will pave the way for further design,
structural modification and development of podophyllotoxin derivatives as insecticidal agents.
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370
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374
375
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377
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380
381
382
ASSOCIATED CONTENT
Supporting Information
1H NMR, HRMS, optical rotation, melting point and IR data, and HPLC spectra for the target
compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone:
+86ꢀ029ꢀ87091952.
Fax:
+86ꢀ029ꢀ87091952.
Email:
orgxuhui@nwsuaf.edu.cn; xjyao@lzu.edu.cn
Funding
The present research was supported by National Natural Science Foundation of China
(No.31071737), the Fok Ying Tong Education Foundation for Young Talents (No.
121032), and Special Funds of Central Colleges Basic Scientific Research Operating
Expenses (QN2009045).
Notes
The authors declare no competing financial interest.
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Figure 1. Chemical structures of podophyllotoxin (1), etoposide (2), teniposide (3) and
etopophos (4).
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Hꢀ4,J=9.0 Hz
9f
Figure 2. The partial 1HNMR spectra of 8f, 9f and 11f.
Hꢀ4,J=3.5 Hz
10f
Figure 2. The partial 1HNMR spectra of 9f and 10f.
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Dataset
0.6
0.5
0.4
0.3
0.2
0.2
0.3
0.4
0.5
0.6
Experimental values
Figure 3. Plot of experimental and predicted biological activity values of 27 compounds by
GAꢀMLR model.
4
Dataset
3
2
1
0
26
-1
-2
-3
-4
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Hat
Figure 4. Williams plot for the GAꢀMLR model with five descriptors.
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OCOR
OCOR
OH
MeO
MeO
O
MeO
MeO
MeO
MeO
O
12c,d,f,g,l
DIC/DMAP
O
O
O
O
r.t./4-10 h
59-92%
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OMe
10c,d,f,g,l
OMe
9a-m
7
12a-m
DIC/DMAP
r.t./1-11 h
34-90%
.
1) NaI/BF3 Et2O
2) acetone-H2O-BaCO3
reflux/2 h
MeCN/r.t./1 h
69%
OH
OH
MeO
HO
O
O
1) BCl3/DCM/-70 oC/2 h
MeI/K2CO3/acetone
MeO
HO
1
O
O
r.t./12 h
86%
2) acetone-H2O-CaCO3
reflux/1 h
72%
MeO
OMe
MeO
OMe
OMe
OMe
5
6
BnBr/Cs2CO3/KI/acetone
r.t./10 h
36%
OCOR
OH
BnO
BnO
12d,f,g,l
DIC/DMAP
O
BnO
BnO
O
r.t./4-12 h
21-29%
O
O
MeO
OMe
MeO
OMe
OMe
OMe
8
11d,f,g,l
O
a
b
c
d
e
R= Me; R= CH2Cl; R= n-Pr; R= n-pentyl; R= n-heptyl;
f R= PhCH2; g R= 1-naphthylmethylene; h R= Ph; i R= (p-Me)Ph; j R= (m-Cl)Ph;
OH
12a-m
R
k
l
m
R= (p-NO2)Ph; R= (m-NO2)Ph; R= phenylethenyl.
Scheme 1. The route for the synthesis of three series of 4ꢀacyloxypodophyllotoxin
derivatives (9aꢀm, 10c, d, f, g and l, and 11d, f, g and l).
27
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Journal of Agricultural and Food Chemistry
Page 28 of 31
Table 1. The Relationships of the Configuration of C-4 Position of Three Series of
4-Acyloxypodophyllotoxin Derivatives with Their J3.4 Coupling Constants.
compound
δHꢀ4 (ppm)
4.80
4.90
5.92
6.00
5.95
5.94
5.94
5.90
5.89
6.16
6.14
6.17
6.20
6.23
6.07
6.20
6.20
6.14
6.13
6.49
J
3.4 (Hz)
9.5
3.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
8.5
8.5
8.0
8.5
8.0
9.0
2.5
3.0
3.5
3.0
3.0
configuration
6
7
α
β
α
α
α
α
α
α
α
α
α
α
α
α
α
β
β
β
β
β
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
10c
10d
10f
10g
10l
11d
5.86
9.0
α
11f
11g
11l
5.84
5.83
6.17
9.0
9.5
8.5
α
α
α
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Page 29 of 31
Journal of Agricultural and Food Chemistry
Table 2. Insecticidal Activity of Three Series of 4-Acyloxypodophyllotoxin Derivatives
(9a-m, 10c, d, f, g and l, and 11d, f, g and l) at 1 mg/mL against M. separata
corrected mortality rate (%)
compound
10 days
13.3 ± 3.3
0 ± 0
20 days
35 days
23.3 ± 6.7
6.7 ± 6.7
40.0 ± 5.8
23.3 ± 3.3
36.7 ± 3.3
40.0 ± 5.8
33.3 ± 6.7
36.7 ± 3.3
26.7 ± 3.3
33.3 ± 3.3
36.7 ± 3.3
53.3 ± 3.3
26.7 ± 3.3
60.0 ± 0
36.7 ± 3.3
33.3 ± 3.3
50.0 ± 5.8
46.7 ± 3.3
36.7 ± 3.3
33.3 ± 3.3
36.7 ± 3.3
43.3 ± 3.3
33.3 ± 3.3
50.0 ± 5.8
36.7 ± 3.3
33.3 ± 3.3
40.0 ± 5.8
33.3 ± 3.3
33.3 ± 3.3
50.0 ± 5.8
1
5
6
0 ± 0
23.3 ± 3.3
23.3 ± 3.3
33.3 ± 6.7
23.3 ± 6.7
13.3 ± 6.7
20.0 ± 5.8
23.3 ± 3.3
30.0 ± 5.8
13.3 ± 3.3
26.7 ± 3.3
26.7 ± 3.3
16.7 ± 3.3
23.3 ± 3.3
13.3 ± 3.3
16.7 ± 3.3
23.3 ± 6.7
10.0 ± 5.8
26.7 ± 6.7
16.7 ± 6.7
20.0 ± 5.8
26.7 ± 3.3
13.3 ± 3.3
16.7 ± 6.7
26.7 ± 3.3
6.7 ± 3.3
16.7 ± 3.3
10.0 ± 5.8
10.0 ± 5.8
0 ± 0
7
8
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
6.7 ± 3.3
6.7 ± 3.3
10.0 ± 0
3.3 ± 3.3
10.0 ± 5.8
10.0 ± 0
6.7 ± 6.7
3.3 ± 3.3
3.3 ± 3.3
3.3 ± 3.3
6.7 ± 3.3
3.3 ± 3.3
23.3 ± 3.3
10.0 ± 0
13.3 ± 6.7
10.0 ± 5.8
10.0 ± 0
3.3 ± 3.3
3.3 ± 3.3
3.3 ± 3.3
10.0 ± 0
9k
9l
9m
10c
10d
10f
10g
10l
11d
11f
11g
11l
toosendanin
20.0 ± 0
29
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