Stereoselective synthesis of 2α-chloropicropodophyllotoxins and insecticidal activity of their esters against oriental armyworm, mythimna separata walker

Article abstract of DOI:10.1021/jf405316w

As part of ongoing efforts to discover new natural-product-based insecticidal agents, in the present study, an efficient method for the stereoselective α-chlorination at the C-2 position of 2′(2′, 6′)-(di)halogenopodophyllotoxin derivatives was first developed. Subsequently, a series of novel esters of 2α-chloro-2′(2′, 6′)-(di)halogenopicropodophyllotoxin with modified C, D, and E rings of podophyllotoxin were smoothly obtained. Finally, all of the title compounds were tested against the pre-third-instar larvae of oriental armyworm (Mythimna separata Walker) at 1 mg/mL. It was found that besides their 2′-halogen-substituted E ring, the stereoselective α-chlorination at the C-2 position of 2′(2′,6′)-(di)halogenopodophyllotoxins was also related to the chlorination reagents. Especially 2α-chloro- 4α-(benzoyl)oxy-2′-chloropicropodophyllotoxin (6e) and 2α-chloro-4α-(2-chlorophenylacyl)oxy-2′- bromopicropodophyllotoxin (8f) showed the most potent insecticidal activities, with final mortality rates of >60%. For 4α-(alkylacyl)oxy derivatives of 2α-chloro-2′(2′,6′)-(di)halogenopicropodophyllotoxin, the effect of the length of their side chain at the C-4 position of podophyllotoxin skeleton on the insecticidal activity was not very obvious. For 4α-(arylacyl)oxy derivatives of 2α-chloro-2′-chloro/ bromopicropodophyllotoxin, an electronic effect of the substituents on their phenyl ring at the C-4 position of podophyllotoxin skeleton on the insecticidal activity was observed.

Full text of DOI:10.1021/jf405316w

Article
pubs.acs.org/JAFC
Stereoselective Synthesis of 2α-Chloropicropodophyllotoxins and
Insecticidal Activity of Their Esters against Oriental Armyworm,
Mythimna separata Walker
Lingling Fan,†,§,∥ Yong Guo,†,∥ Xiaoyan Zhi,† Xiang Yu,† and Hui Xu*,†
†Laboratory of Pharmaceutical Design and Synthesis, College of Sciences, Northwest A&F University, Yangling 712100, Shaanxi
Province People’s Republic of China
§School of Pharmacy, Guiyang Medical University, Guiyang 550004, Guizhou Province, People’s Republic of China
S
* Supporting Information
ABSTRACT: As part of ongoing efforts to discover new natural-product-based insecticidal agents, in the present study, an
efficient method for the stereoselective α-chlorination at the C-2 position of 2′(2′,6′)-(di)halogenopodophyllotoxin derivatives
was first developed. Subsequently, a series of novel esters of 2α-chloro-2′(2′,6′)-(di)halogenopicropodophyllotoxin with
modified C, D, and E rings of podophyllotoxin were smoothly obtained. Finally, all of the title compounds were tested against the
pre-third-instar larvae of oriental armyworm (Mythimna separata Walker) at 1 mg/mL. It was found that besides their 2′-halogen-
substituted E ring, the stereoselective α-chlorination at the C-2 position of 2′(2′,6′)-(di)halogenopodophyllotoxins was also
related to the chlorination reagents. Especially 2α-chloro-4α-(benzoyl)oxy-2′-chloropicropodophyllotoxin (6e) and 2α-chloro-
4α-(2-chlorophenylacyl)oxy-2′-bromopicropodophyllotoxin (8f) showed the most potent insecticidal activities, with final
mortality rates of >60%. For 4α-(alkylacyl)oxy derivatives of 2α-chloro-2′(2′,6′)-(di)halogenopicropodophyllotoxin, the effect of
the length of their side chain at the C-4 position of podophyllotoxin skeleton on the insecticidal activity was not very obvious.
For 4α-(arylacyl)oxy derivatives of 2α-chloro-2′-chloro/bromopicropodophyllotoxin, an electronic effect of the substituents on
their phenyl ring at the C-4 position of podophyllotoxin skeleton on the insecticidal activity was observed.
KEYWORDS: podophyllotoxin, mortality rate, insecticidal activity, chemical modification, stereoselective chlorination,
natural-product-based insecticide
insecticidal and antifungal activities.16,17 Therefore, total
synthesis18−23 and structural modifications24−27 of 1 and its
INTRODUCTION
■
The larvae of many lepidopteran species are major insect pests
analogues have been carried out in many research groups.
Recently, we have prepared a series of 2β-chloropodophyllo-
toxin28,29 (I, Figure 1) and 4α-acyloxy-2′(2′,6′)-(di)-
halogenopodophyllotoxin derivatives30 (II, Figure 1) as
insecticidal agents and found that some compounds exhibited
more potent insecticidal activity than toosendanin, a
commercial botanical insecticide isolated from Melia azedarach.
More importantly, it was observed that introduction of a
chlorine atom at the C-2, C-2′, or C-2′,6′ position of
podophyllotoxin was important for obtaining potent deriva-
tives. Therefore, as shown in Figure 1, we herein designed
another series of 2α/β-chloro-2′(2′,6′)-(di)-
halogenopodophyllotoxin derivatives (III) by simultaneous
introduction of halogen atoms at the C-2 and C-2′ (or C-
2′,6′) positions of 1. Their insecticidal activity was tested
against oriental armyworm (Mythimna separata Walker), a
typical lepidopteran pest.
in agriculture, and their outbreaks can lead to a widespread
incidence and complete crop loss.1 Therefore, many synthetic
agrochemicals have been applied in agriculture to control
lepidopteran pests during the past decades. Of course, these
chemical pesticides have played an important role in crop
protection. However, resistance in lepidopteran pest popula-
tions and environmental problems have also occurred because
of the repeated and increasing use of agrochemicals for a long
time.2−6 Meanwhile, due to plant secondary metabolites
originating from the interaction between the plants and the
environment (life and nonlife) during the long period of plant
evolution, pesticides produced from plant secondary metabo-
lites may cause less or slower resistance development and lower
environmental pollution.7 Consequently, the discovery of new
pesticides directly or indirectly from plant secondary
metabolites has recently been crucial in the research and
development of agrochemicals.8−14
Podophyllotoxin (1, Figure 1), extracted and isolated from
the roots and rhizomes of some Podophyllum and Juniperus
species, has already been used as the lead compound for
chemical modifications in the field of medicinal sciences.15 For
example, some antitumor drugs originating from compound 1,
for example, etoposide (VP-16), teniposide (VM-26), and
etoposide phosphate, have been successfully applied in the
clinic. On the other hand, compound 1 displayed interesting
Received: November 25, 2013
Revised: March 19, 2014
Accepted: April 13, 2014
Published: April 14, 2014
© 2014 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Chemical structures of podophyllotoxin (1) and its derivatives (I−III).
CDCl3) δ 172.3, 152.1, 149.7, 148.7, 148.2, 142.5, 132.3, 129.7, 128.5,
121.5, 109.1, 107.4, 101.5, 73.5, 73.0, 67.0, 61.1, 61.0, 56.3, 50.7, 45.2;
HRMS m/z calcd for C22H20O8Cl2Na ([M + Na]+) 505.0427, found
505.0428.
MATERIALS AND METHODS
■
General. All chemical reagents were commercially available, and
solvents were purified with standard methods31 before use. Analytical
thin-layer chromatography (TLC) and preparative thin-layer chroma-
tography (PTLC) were performed with silica gel plates using silica gel
60 GF254 (Qingdao Haiyang Chemical Co., Ltd. Qingdao, China), and
the solvent system was a mixture of ethyl acetate and petroleum ether.
Melting points were carried out on an XT-4 digital melting-point
apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and
were uncorrected. Proton nuclear magnetic resonance spectra (1H
NMR) and carbon nuclear magnetic resonance spectra (13C NMR)
were recorded in CDCl3 on a Bruker Avance 400 or 500 MHz
instrument using tetramethylsilane (TMS) as the internal standard.
High-resolution mass spectra (HR-MS) were carried out with an
IonSpec 4.7 T FTMS instrument. 2′-Chloropodophyllotoxin (2a, 85%
yield), 2′,6′-dichloropodophyllotoxin (2b, 90% yield), and 2′-
bromopodophyllotoxin (2c, 85% yield) were prepared in the same
way as in our previous paper.30
Data for 5b: white solid; mp = 92−93 °C; [α]20 = −104 (c 3.9
D
1
mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.04 (s, 1H, H-5),
6.13 (s, 1H, H-8), 5.99 (s, 1H, H-1), 5.90 (dd, J = 7.2, 1.2 Hz, 2H,
OCH2O), 4.82 (dd, J = 8.8, 3.6 Hz, 1H, H-11), 4.62 (d, J = 9.2 Hz,
1H, H-11), 4.43 (d, J = 9.2 Hz, 1H, H-4), 3.98 (s, 3H, 3′-OCHH3),
3.96 (s, 3H, 5′-OCHH3), 3.82 (s, 3H, 4′-OCHH3), 3.16 (dd, J = 9.6,
3.6 Hz, 1H, H-3), 1.58 (s, 1H, OH); 13C NMR (100 MHz, CDCl3) δ
173.3, 149.4, 148.9, 147.7, 147.5, 147.3, 134.2, 129.6, 127.4, 127.2,
125.9, 107.6, 104.7, 101.3, 70.8, 69.0, 65.6, 61.3, 61.2, 61.1, 50.8, 44.1;
HRMS m/z calcd for C22H19O8Cl3Na ([M + Na]+) 539.0037, found
538.0051.
Data for 5c: white solid; mp = 170−172 °C; [α]20 = −94 (c 3.4
D
1
mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.10 (s, 1H, H-5),
6.74 (s, 1H, H-8), 6.64 (s, 1H, H-6′), 5.91 (dd, J = 5.2, 1.2 Hz, 2H,
OCH2O), 5.56 (s, 1H, H-1), 4.89 (d, J = 2.8 Hz, 1H, H-4), 4.81 (dd, J
= 9.2, 4.8 Hz, 1H, H-11), 4.47 (d, J = 9.2 Hz, 1H, H-11), 3.92 (s, 3H,
3′-OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.72 (s, 3H, 4′-OCHH3), 3.14
(t, J = 4.0 Hz, 1H, H-3), 2.53 (s, 1H, OH); 13C NMR (100 MHz,
CDCl3) δ 172.3, 152.8, 150.7, 148.6, 148.2, 142.4, 134.1, 129.8, 128.4,
113.1, 109.4, 108.9, 107.5, 101.5, 73.4, 73.1, 67.1, 61.0, 61.0, 56.4, 50.7,
47.8; HRMS m/z calcd for C22H20O8ClBrNa ([M + Na]+) 548.9922,
found 548.9906.
Stereoselective Synthesis of 2α-Chloro-2′(2′,6′)-(di)-
halogenopicropodophyllotoxin Derivatives (5a−c). Two drops
of phosphorus oxychloride were added to a solution of 2a, 2b, or 2c
(1.8 mmol) in dihydropyran (10 mL). Then the mixture was stirred at
room temperature. After 2 h, the mixture was poured into ice-cold
petroleum ether (100 mL) and vigorously stirred. The resulting solids
were collected and dissolved in CH2Cl2 (50 mL). The corresponding
solution was washed by water (25 mL × 2), dried over anhydrous
Na2SO4, concentrated in vacuo, and purified by silica gel column
chromatography to give products 3a (89% yield), 3b (78% yield), or
3c (85% yield). To a solution of lithium diisopropylamide (LDA, 0.6
mmol) in dry tetrahydrofuran (THF, 5 mL) at −78 °C under N2 was
then added dropwise a solution of 3a, 3b, or 3c (0.5 mmol) in dry
THF (5 mL) for 10 min. After the addition, the reaction mixture was
stirred for 15 min, and a solution of hexachloroethane (C2Cl6, 0.7
mmol) in dry THF (5 mL) was added dropwise for 10 min.
Subsequently, the solution was allowed to warm slowly from −78 °C
to the ambient temperature. When the reaction was complete
according to TLC analysis, the solvent was removed and the residue
was diluted by CH2Cl2 (50 mL). The corresponding solution was
washed by water (25 mL), 0.5 N aqueous HCl (25 mL), and water (25
mL), dried over anhydrous Na2SO4, and concentrated under reduced
pressure to give the residue, which was used directly for the next step
without further purification. A solution of the above residue in
concentrated HCl/THF (1:9, v/v, 10 mL) was stirred for 2 h and then
adjusted a pH value of 5 by using 5% aqueous Na2CO3. After removal
of THF, the mixture was extracted with CH2Cl2 (40 mL × 3), and the
combined organic phase was washed by 5% aqueous Na2CO3 (20 mL)
and brine (20 mL), dried over anhydrous Na2SO4, concentrated in
vacuo, and purified by silica gel column chromatography to give 5a
(68% yield), 5b (62% yield), or 5c (71% yield).
General Procedure for the Synthesis of 2α-Chloro-2′(2′,6′)-
(di)halogenopicropodophyllotoxin Esters (6a−k, 7a−i, and
8a−h). A solution of compound 5a, 5b, or 5c (0.15 mmol), the
corresponding carboxylic acids RCO2H (0.18 mmol), N,N′-
dicyclohexylcarbodiimide (DCC, 0.18 mmol), and 4-N,N-dimethyla-
minopyridine (DMAP, 0.03 mmol) in dry CH2Cl2 (10 mL) was stirred
at room temperature. When the reaction was complete as checked by
TLC analysis, the solution of the mixture was diluted with CH2Cl2 (40
mL). Subsequently, the solution was washed by water (15 mL), 0.1 N
aqueous HCl (25 mL), 5% aqueous Na2CO3 (25 mL), and brine (25
mL), dried over anhydrous Na2SO4, concentrated in vacuo, and
purified by PTLC to give the target products 6a−k, 7a−i, and 8a−h.
The example data of 6a−c, 7a−c, and 8a−c are shown as follows,
whereas data of 6d−k, 7d−i, and 8d−h can be found in the
Supporting Information.
Data for 6a: yield 82%; white solid; mp = 150−151 °C; [α]20
=
D
1
−78 (c 3.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 6.68 (s,
1H, H-5), 6.64 (s, 1H, H-8), 6.58 (s, 1H, H-6′), 5.93−5.95 (m, 3H, H-
4, OCH2O), 5.52 (s, 1H, H-1), 4.80−4.81 (m, 2H, H-11), 3.94 (s, 3H,
3′−OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.76 (s, 3H, 4′-OCHH3),
2.97−2.98 (m, 1H, H-3), 2.16 (s, 3H, COCH3); 13C NMR (125 MHz,
CDCl3) δ 171.9, 170.6, 152.0, 149.9, 149.2, 148.2, 142.6, 132.1, 130.8,
123.9, 122.0, 108.8, 108.6, 108.3, 101.7, 75.1, 73.2, 66.8, 61.1, 61.1,
56.1, 49.3, 44.7, 21.1; HRMS m/z calcd for C24H22O9Cl2Na ([M +
Na]+) 547.0533, found 547.0529.
Data for 5a: white solid; mp = 183−184 °C; [α]20 = −83 (c 3.1
D
1
mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.07 (s, 1H, H-5),
Data for 6b: yield 78%; white solid; mp = 179−180 °C; [α]20
=
6.74 (s, 1H, H-8), 6.62 (s, 1H, H-6′), 5.92 (dd, J = 5.2, 1.2 Hz, 2H,
OCH2O), 5.48 (s, 1H, H-1), 4.90 (d, J = 2.8 Hz, 1H, H-4), 4.81 (dd, J
= 9.2, 4.8 Hz, 1H, H-11), 4.47 (d, J = 9.2 Hz, 1H, H-11), 3.93 (s, 3H,
3′-OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.73 (s, 3H, 4′-OCHH3),
3.14−3.16 (m, 1H, H-3), 2.47 (s, 1H, OH); 13C NMR (100 MHz,
D
1
−80 (c 2.9 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 6.67 (s,
1H, H-5), 6.62 (s, 1H, H-8), 6.57 (s, 1H, H-6′), 5.93−5.94 (m, 3H,
OCH2O, H-4), 5.51 (s, 1H, H-1), 4.80−4.81 (m, 2H, H-11), 3.93 (s,
3H, 3′-OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.75 (s, 3H, 4′-OCHH3),
3727
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Journal of Agricultural and Food Chemistry
Article
Scheme 1. Synthetic Approach for 2α-Chloropicropodophyllotoxin Derivatives (5a−c) and 2β-Chloropodophyllotoxin
Derivatives (5a′−c′)
2.96−2.97 (m, 1H, H-3), 2.35−2.42 (m, 2H, CH3CH2), 1.20 (t, J = 7.6
Hz, 3H, CH3CH2); 13C NMR (100 MHz, CDCl3) δ 174.2, 171.9,
152.1, 150.0, 149.2, 148.2, 142.8, 132.2, 130.7, 124.2, 122.1, 108.8,
108.6, 108.5, 101.7, 74.9, 73.1, 66.8, 61.1, 61.1, 56.2, 49.4, 44.7, 27.5,
9.0; HRMS m/z calcd for C25H24O9Cl2Na ([M + Na]+) 561.0689,
found 561.0691.
OCHH3), 3.76 (s, 2H, PhCH2), 3.31 (dd, J = 7.2, 3.6 Hz, 1H, H-3);
13C NMR (100 MHz, CDCl3) δ 172.7, 171.6, 149.2, 148.9, 148.1,
147.6, 147.3, 133.2, 133.1, 129.2, 128.8, 127.6, 127.5, 127.5, 125.8,
125.5, 107.7, 105.8, 101.4, 71.6, 71.1, 64.9, 61.3, 61.2, 61.1, 49.0, 44.1,
41.5; HRMS m/z calcd for C30H25O9Cl3Na ([M + Na]+) 657.0456,
found 657.0424.
Data for 6c: yield 77%; white solid; mp = 79−80 °C; [α]19D = −66
Data for 8a: yield 95%; white solid; mp = 176−178 °C; [α]20
=
D
1
1
(c 3.6 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.27−7.39
−94 (c 3.4 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 6.71 (s,
1H, H-5), 6.63 (s, 1H, H-8), 6.60 (s, 1H, H-6′), 5.93−5.95 (m, 3H,
OCH2O, H-4), 5.59 (s, 1H, H-1), 4.80−4.81 (m, 2H, H-11), 3.92 (s,
3H, 3′-OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.76 (s, 3H, 4′-OCHH3),
2.97 (dd, J = 5.2, 2.8 Hz, 1H, H-3), 2.15 (s, 3H, COCH3); 13C NMR
(100 MHz, CDCl3) δ 171.9, 170.6, 152.7, 150.9, 149.2, 148.2, 142.6,
133.9, 130.9, 123.9, 113.7, 108.8, 108.7, 108.6, 101.7, 75.1, 73.1, 66.8,
61.1, 61.0, 56.2, 49.4, 47.3, 21.0; HRMS m/z calcd for
C24H22O9ClBrNa ([M + Na]+) 591.0028, found 591.0028.
(m, 5H), 6.67 (s, 1H, H-5), 6.57 (s, 1H, H-8), 6.51 (s, 1H, H-6′), 5.93
(d, J = 1.2 Hz, 2H, OCH2O), 5.89 (d, J = 3.2 Hz, 1H, H-4), 5.50 (s,
1H, H-1), 4.77−4.78 (m, 2H, H-11), 3.93 (s, 3H, 3′-OCHH3), 3.89 (s,
3H, 5′-OCHH3), 3.75 (s, 3H, 4′-OCHH3), 3.67 (d, J = 6.8 Hz, 2H,
PhCH2), 2.99−3.00 (m, 1H, H-3); 13C NMR (100 MHz, CDCl3) δ
171.8, 171.6, 152.0, 150.0, 149.2, 148.2, 142.8, 132.6, 132.1, 130.6,
129.0, 128.9, 127.7, 123.9, 122.1, 108.7, 108.6, 108.4, 101.7, 75.5, 72.9,
66.7, 61.1, 61.1, 56.3, 49.2, 44.6, 41.1; HRMS m/z calcd for
C30H26O9Cl2Na ([M + Na]+) 623.0846, found 623.0849.
Data for 8b: yield 93%; white solid; mp = 169−170 °C; [α]20
=
D
Data for 7a: yield 90%; white solid; mp = 92−93 °C; [α]20D = −86
1
−83 (c 3.2 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 6.71 (s,
1H, H-5), 6.62 (s, 1H, H-8), 6.60 (s, 1H, H-6′), 5.93−5.94 (m, 3H,
OCH2O, H-4), 5.59 (s, 1H, H-1), 4.80−4.81 (m, 2H, H-11), 3.92 (s,
3H, 3′-OCHH3), 3.88 (s, 3H, 5′-OCHH3), 3.75 (s, 3H, 4′-OCHH3),
2.96−2.99 (m, 1H, H-3), 2.35 (q, J = 7.6 Hz, 2H, COCH2CH3), 1.20
(t, J = 7.6 Hz, 3H, COCH2CH3); 13C NMR (100 MHz, CDCl3) δ
174.2, 171.9, 152.7, 150.9, 149.2, 148.2, 142.7, 134.0, 130.8, 124.1,
113.7, 108.9, 108.7, 108.5, 101.7, 74.8, 73.1, 66.8, 61.1, 61.0, 56.2, 49.4,
47.2, 27.5, 9.0; HRMS m/z calcd for C25H24O9ClBrNa ([M + Na]+)
605.0184, found 605.0169.
1
(c 3.3 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 6.51 (s, 1H,
H-5), 6.23 (s, 1H, H-8), 6.07 (s, 1H, H-1), 5.92−5.93 (m, 3H,
OCH2O, H-4), 4.76−4.78 (m, 1H, H-11), 4.57−4.59 (m, 1H, H-11),
3.98 (s, 3H, 3′-OCHH3), 3.96 (s, 3H, 5′-OCHH3), 3.83 (s, 3H, 4′-
OCHH3), 3.39−3.41 (m, 1H, H-3), 2.24 (s, 3H, COCH3); 13C NMR
(100 MHz, CDCl3) δ 172.8, 171.1, 149.3, 148.9, 148.1, 147.6, 147.4,
133.3, 127.5, 125.9, 125.5, 107.8, 105.7, 101.5, 71.2, 70.9, 64.9, 61.3,
61.3, 61.2, 48.9, 44.1, 21.1; HRMS m/z calcd for C24H21O9Cl3Na ([M
+ Na]+) 581.0143, found 581.0150.
Data for 7b: yield 61%; white solid; mp = 84−86 °C; [α]20
=
D
Data for 8c: yield 98%; white solid; mp = 88−90 °C; [α]20D = −68
−107 (c 2.8 mg/mL, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.49 (s,
1H, H-5), 6.23 (s, 1H, H-8), 6.07 (s, 1H, H-1), 5.90−5.93 (m, 3H,
OCH2O, H-4), 4.75 (dd, J = 7.2, 3.2 Hz, 1H, H-11), 4.58 (d, J = 7.6
Hz, 1H, H-11), 3.98 (s, 3H, 3′-OCHH3), 3.96 (s, 3H, 5′-OCHH3),
3.83 (s, 3H, 4′-OCHH3), 3.38 (dd, J = 5.6, 2.8 Hz, 1H, H-3), 2.49−
1
(c 3.6 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.28−7.39
(m, 5H), 6.70 (s, 1H, H-5), 6.59 (s, 1H, H-8), 6.51 (s, 1H, H-6′), 5.94
(d, J = 0.8 Hz, 2H, OCH2O), 5.88 (d, J = 3.2 Hz, 1H, H-4), 5.58 (s,
1H, H-1), 4.78−4.79 (m, 2H, H-11), 3.92 (s, 3H, 3′-OCHH3), 3.88 (s,
3H, 5′-OCHH3), 3.75 (s, 3H, 4′-OCHH3), 3.67 (d, J = 7.2 Hz, 2H,
PhCH2), 2.99 (dd, J = 5.2, 3.2 Hz, 1H, H-3); 13C NMR (100 MHz,
CDCl3) δ 171.8, 171.6, 152.7, 151.0, 149.2, 148.2, 142.7, 133.9, 132.6,
130.8, 129.0, 128.9, 127.7, 123.8, 113.8, 108.9, 108.6, 108.3, 101.7,
75.4, 72.9, 66.8, 61.09, 61.01, 56.3, 49.3, 47.2, 41.1; HRMS m/z calcd
for C30H26O9ClBrNa ([M + Na]+) 667.0341, found 667.0320.
Biological Assay. On the basis of the leaf-dipping method,30 the
b i o l o g i c a l a c t i v i t y o f 2 α - c h l o r o - 2 ′ ( 2 ′ , 6 ′ ) - ( d i ) -
halogenopicropodophyllotoxin esters (6a−k, 7a−i, and 8a−h) was
evaluated as the mortality rate values against Mythimna separata
Walker. For each compound, 30 pre-third-instar larvae (10 larvae per
2.52 (m, 2H, COCH2CH3), 1.23 (t, J = 6.0 Hz, 3H, COCH2CH3); 13
C
NMR (100 MHz, CDCl3) δ 174.4, 172.8, 149.3, 149.0, 148.1, 147.6,
147.4, 133.3, 127.5, 126.1, 125.5, 107.8, 105.8, 101.4, 71.3, 70.9, 65.0,
61.3, 61.2, 61.1, 49.1, 44.1, 27.7, 9.1; HRMS m/z calcd for
C25H23O9Cl3Na ([M + Na]+) 595.0299, found 595.0300.
Data for 7c: yield 71%; white solid; mp = 78−80 °C; [α]20D = −95
1
(c 2.8 mg/mL, CHCl3); H NMR (400 MHz, CDCl3) δ 7.31−7.38
(m, 5H), 6.34 (s, 1H, H-5), 6.21 (s, 1H, H-8), 6.05 (s, 1H, H-1), 5.88
(dd, J = 10.0, 1.2 Hz, 2H, OCH2O), 5.86 (d, J = 7.2 Hz, 1H, H-4),
4.65 (dd, J = 9.2, 4.0 Hz, 1H, H-11), 4.46 (d, J = 9.6 Hz, 1H, H-11),
3.97 (s, 3H, 3′-OCHH3), 3.95 (s, 3H, 5′-OCHH3), 3.83 (s, 3H, 4′-
3728
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Table 1. Investigation of 3a−c Reacting with Different Chlorination Reagents (La−d)
entry
compd
chlorination reagent
T (°C)
t (h)
isolated yield (%) of 5
1
3a
3a
3a
3a
3b
3b
3b
3b
3c
3c
3c
3c
La
Lb
Lc
Ld
La
Lb
Lc
Ld
La
Lb
Lc
Ld
−78 to 23
−78 to −18
−78 to −39
−78 to 27
−78 to 26
−78 to −23
−78 to −40
−78 to 27
−78 to 17
−78 to −21
−78 to −33
−78 to 27
16
3.5
2
5a (68)
2
5a (56)
3
5a (66)
4
24
18
3
5a (0)
5
5b (62)
a
a
6
5b (45) + 5b′ (14)
5b (49) + 5b′ (21)
5b (0)
7
2
8
24
12
3
9
5c (71)
a
10
11
12
5c (13) + 5c′ (44)
5c (45) + 5c′ (20)
5c (0)
a
2.5
24
a
1
Determined by H NMR spectra.
group) were used. Acetone solutions of compounds 6a−k, 7a−i, and
8a−h were prepared at the concentration of 1 mg/mL. Toosendanin
was used as the positive control 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. Several treated leaves were kept in each dish (10
larvae were raised in each dish, and three dishes were used per
treatment), which was then placed in a conditioned room (25 2 °C,
65−80% relative humidity (RH), 12 h/12 h (light/dark) photo-
period). If the treated leaves were consumed, additional treated leaves
were added to the dish. Forty-eight hours later, untreated fresh leaves
were added to all dishes until adult emergence. The corrected
mortality rate was assessed by the formula
with four different chlorination reagents, such as hexachloro-
ethane (C2Cl6, La), NCS (Lb), 1,3-dichloro-5,5-dimethylhy-
dantoin (Lc), and 1,3,5-trichloroisocyanuric acid (Ld), were
investigated. The chlorination of 3a−c with La stereoselectively
afforded 2α-chloropicropodophyllotoxins (5a−c) in 62−71%
yields (entries 1, 5, and 9), and the chlorination of 3a with La−
c stereoselectively gave 5a in 56−68% yields (entries 1−3).
Although chlorination of 3a with Lb or Lc stereoselectively
produced 5a, chlorination of 3b and 3c with Lb or Lc gave
mixtures of two isomers, 5b/5b′ (2β-chloro-2′,6′-dichloropo-
dophyllotoxin) and 5c/5c′ (2β-chloro-2′-bromopodophyllotox-
in) (entries 6, 7, 10, and 11), respectively. The ratio of 5b/5b′
1
or 5c/5c′ was determined by the corresponding H NMR
corrected mortality rate (%) = (T − C) × 100/(100% − C)
spectra (see the Supporting Information). However, chlorina-
tion of 3a−c with Ld did not afford any products except the
starting materials, even if the reaction time was prolonged to 24
h (entries 4, 8, and 12). Obviously, chlorination reagent La
could be used to stereoselectively prepare 5a−c. In our
previous paper, we found when 1 reacted with La in the
presence of LDA, 2β-chloropodophyllotoxin was stereo-
selectively obtained.28,29 According to the above results, this
suggested that besides their 2′-halogen-substituted E ring,
stereoselective α-chlorination at the C-2 position of 3a−c was
also related to the chlorination reagents. The structures of 5a−c
were well characterized by 1H NMR, 13C NMR, HRMS, optical
rotation, and mp. Moreover, the precise three-dimensional
steric structures of 5b and 5c were further ascertained by single-
crystal X-ray diffraction (Figures 2 and 3). This obviously
demonstrated that the C-2 chlorine atoms of 5b and 5c were
clearly present in α configuration. The configuration of 5a was
where T is the mortality rate in the tested compounds group and C is
the mortality rate in the blank control group (T and C were expressed
as percentages).
RESULTS AND DISCUSSION
■
Synthesis. As shown in Scheme 1, first, three halogenation
products of 1 such as 2′-chloropodophyllotoxin (2a), 2′,6′-
dichloropodophyllotoxin (2b), and 2′-bromopodophyllotoxin
(2c) were smoothly prepared by the reaction of 1 with N-
chlorosuccinimide (NCS) or N-bromosuccinimide (NBS).30
Second, the 4-hydroxy group of 2a−c was protected by a
tetrahydropyranyl (THP) group in the presence of phosphorus
oxychloride (POCl3) and dihydropyran (DHP) to produce 3a−
c. Subsequently, as described in Table 1, compounds 3a−c
reacted with lithium diisopropylamide (LDA) at −78 °C via the
intermediates 4a−c (lithium enolates), followed by reaction
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Figure 4. X-ray crystal structure of 6a.
Scheme 2. Route for Synthesis of 2α-Chloro-2′(2′,6′)-
(di)halogenopicropodophyllotoxin Esters (6a−k, 7a−i, and
8a−h)
Figure 2. X-ray crystal structure of 5b.
Figure 3. X-ray crystal structure of 5c.
confirmed on the basis of the X-ray crystallography structure of
its ester 6a (see Figure 4), and the C-2 chlorine atom of 6a
adopted an α configuration. Therefore, the C-2 chlorine atom
of 5a should also adopt an α configuration. Finally, 2α-chloro-
2′(2′,6′)-(di)halogenopicropodophyllotoxin esters (6a−k, 7a−
i, and 8a−h) were afforded by reaction of 5a, 5b, or 5c with the
corresponding carboxylic acids (9) in the presence of N,N′-
dicyclohexylcarbodiimide (DCC) and 4-N,N-dimethylamino-
pyridine (DMAP) (Scheme 2). The structures of the title
compounds were identified by H NMR, 13C NMR, HRMS,
1
optical rotation, and mp. Crystallographic data (excluding
structure factors) for the structures of three compounds, 5b, 5c,
and 6a, have been deposited at the Cambridge Crystallographic
Data Centre with Data CCDC 918889, 918888, and 918696,
respectively. These data can be obtained free of charge from the
CCDC [fax +44 (0)1223 336033 or e-mail deposit@ccdc.cam.
ac.uk].
Insecticidal Activity. The insecticidal activity of novel 2α-
chloro-2′(2′,6′)-(di)halogenopicropodophyllotoxin esters (6a−
k, 7a−i, and 8a−h) against the pre-third-instar larvae of M.
separata was tested by the leaf-dipping method at 1 mg/mL.
Toosendanin was used as the positive control at the
concentration of 1 mg/mL. In general, we found that the
mortality rates of M. separata caused by these derivatives after
35 days were higher than those after 10 and 20 days in the same
way as in our previous studies,28−30,32 As described in Table 2,
foe 6e, for instance, the corrected mortality rates against M.
separata after 10 and 20 days were 10 and 43.3%, respectively,
but after 35 days, the mortality rate was increased to 66.7%,
which was >6 times that after 10 days. Meanwhile, the
3730
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Table 2. Insecticidal Activity of 2α-Chloro-2′(2′,6′)-
(di)halogenopicropodophyllotoxin Esters (6a−k, 7a−i, and
8a−h) against the Pre-third-Instar Larvae of M. separata on
Leaves Treated with a Concentration of 1 mg/mL
corrected mortality rate (%)
compd
10 days
20 days
35 days
1
6.7 3.3
30.0
0
37.0 3.3
2a
2b
2c
5a
5b
5c
6a
6b
6c
6d
6e
6f
10.0
0
36.7 3.3
36.7 3.3
20.0 5.8
33.3 3.3
23.3 3.3
23.3 3.3
13.3 3.3
33.3 6.7
23.3 3.3
51.9
55.6
0
0
10.0 5.8
0
0
48.1 3.3
48.1 3.3
33.3 5.8
51.9 3.3
37.0 3.3
40.7 3.3
25.9 3.3
44.4 5.8
66.7 5.8
16.7 3.3
10.0
Figure 5. Representative abnormal larvae pictures of 6f, 7c, 7g, 8f, and
8h during the larval period (CK, blank control group).
0
10.0 5.8
6.7 3.3
10.0 5.8
13.3 3.3
16.7 3.3
10.0 5.8
3.3 4.7
3.3 3.3
6.7 3.3
10.0 5.8
13.3 3.3
30.0
0
43.3 3.3
40.0
20.0
0
0
55.6
0
6g
6h
6i
48.1 3.3
37.0 3.3
44.4 5.8
48.1 3.3
40.7 3.3
33.3 5.8
37.0 3.3
55.6 5.8
37.0 3.3
44.4 5.8
26.7 3.3
30.0
0
6j
40.0 5.8
26.7 3.3
6k
7a
7b
7c
7d
7e
7f
0
0
6.7 3.3
20.0
20.0
0
0
Figure 6. Representative malformed pupae pictures of 6g, 7e, 7i, 8a,
and 8e during the pupation period (CK, blank control group).
0
0
10.0 5.8
10.0 8.2
10.0 5.8
6.7 6.7
36.7 3.3
26.7 3.3
33.3 3.3
23.3 3.3
36.7 3.3
33.3 3.3
36.7 3.3
40.0 5.8
26.7 3.3
26.7 3.3
44.4
0
7g
7h
7i
0
0
51.9 3.3
40.7 3.3
40.7 3.3
16.7 3.3
6.7 3.3
8a
8b
8c
8d
8e
8f
10.0
0
44.4
44.4
0
0
6.7 3.3
10.0
0
13.3 6.7
3.3 3.3
6.7 3.3
13.3 3.3
6.7 3.3
6.7 3.3
33.3 5.8
40.7 3.3
44.4 5.8
63.0 3.3
48.1 3.3
51.9 3.3
51.9 3.3
30.0
40.0
0
0
53.3 3.3
30.0
Figure 7. Representative malformed moth pictures of 6e,6f,6k, 7g, and
8e during the emergence period (CK, blank control group).
8g
8h
0
23.3 3.3
36.7 3.3
a
toosendanin
a
Toosendanin was used as a positive control at 1 mg/mL.
5b. For 4α-(alkylacyl)oxy derivatives of 5a−c, the effect of the
length of their side chain at the C-4 position of the
podophyllotoxin skeleton on the insecticidal activity was not
very obvious (e.g., 37% for 6a, 40.7% for 6b, and 40.7% for 6k;
33.3% for 7a and 37% for 7b; 44.4% for 8a and 44.4% for 8b).
However, for 4α-(arylacyl)oxy derivatives of 5a, introduction of
the electron-withdrawing groups on their phenyl ring at the C-4
position of podophyllotoxin skeleton could lead to the potent
compounds. For example, the final mortality rates of 6e (R =
Ph), 6f (R = 2-ClPh), and 6g (R = 3-ClPh) were 66.7, 55.6, and
48.1%, respectively, whereas the final mortality rate of 6h (R =
3-MePh) was only 37%. For 4α-(arylacyl)oxy derivatives of 5c,
introduction of the electron-withdrawing or electron-denoting
substituents on their phenyl ring at the C-4 position of
podophyllotoxin skeleton could lead to the more potent
compounds. For example, the final mortality rates of 8f (R = 2-
ClPh), 8g (R = 3-ClPh), and 8h (R = 3-MePh) were 63, 48.1,
and 51.9%, respectively, whereas the final mortality rate of 8e
(R = Ph) was 44.4%. However, introduction of the
(heterocyclylacyl)oxy group at the C-4 position of 5a or 5b
symptoms of M. separata in the treated groups were
characterized; for example, at the larval stage some larvae
with slim and wrinkled bodies were dying due to feeding too
much on treated leaves during the first 48 h (Figure 5), some
larvae were molting to malformed pupae or dying during the
pupation period (Figure 6), and malformed moths were
appearing with imperfect wings (Figure 7).28−30,32 According
to the above symptoms, the tested compounds probably
exhibited the antimolting hormone effect. As shown in Table 2,
nine compounds, 2a, 2b, 5c, 6e, 6f, 7c, 7g, 8f, and 8h, displayed
insecticidal activity equal to or higher than that of toosendanin.
Among all of the derivatives, especially compounds 6e and 8f
exhibited the most potent insecticidal activity with final
mortality rates >60%. As in our previous studies,29,30
introduction of a chlorine or bromine atom at the C-2′ or C-
2′ and C-6′ position on the E ring or at the C-2 position of
podophyllotoxin could lead to the more potent compounds
2a−c, 5a, and 5c than their precursor podophyllotoxin, except
3731
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could not afford the more potent compounds (e.g., 44.4% for 6i
and 48.1% for 6j; 40.7% for 7i). In our previous paper, we
found that introduction of a (1-naphthylacetyl)oxy group at the
C-4 position of the corresponding podophyllotoxin derivatives
led to the more potent compounds,29,30 but herein introduction
of a (1-naphthylacetyl)oxy group at the C-4 position of 5a−c
did not result in the potent compounds. For example, the final
mortality rates of 6d, 7d, and 8d were only 44.4, 37, and 40.7%,
respectively, so the insecticidal activity of 6d, 7d, and 8d was
also related to the chemical structures of their precursors.
In summary, a series of novel esters of 2α-chloro-2′(2′,6′)-
(di)halogenopicropodophyllotoxin with modified C, D, and E
rings of podophyllotoxin were prepared. In the meantime, an
efficient method for the stereoselective α-chlorination at the C-
2 position of 2′(2′,6′)-(di)halogenopodophyllotoxin derivatives
in the presence of hexachloroethane (C2Cl6) was developed.
Three steric structures of compounds 5b, 5c, and 6a were
determined by single-crystal X-ray diffraction. Their insecticidal
activity was evaluated against pre-third-instar larvae of the
oriental armyworm, M. separata (Walker), in vivo at a
concentration of 1 mg/mL. This demonstrated that besides
their 2′-halogen-substituted E ring, the stereoselective α-
chlorination at the C-2 position of 2′(2′,6′)-(di)-
halogenopodophyllotoxins was also related to the chlorination
reagents. Among all of the derivatives, 2α-chloro-4α-(benzoyl)-
oxy-2′-chloropicropodophyllotoxin (6e) and 2α-chloro-4α-(2-
chlorophenylacyl)oxy-2′-bromopicropodophyllotoxin (8f)
showed the most potent insecticidal activities with final
mortality rates of >60%. For 4α-(alkylacyl)oxy derivatives of
2α-chloro-2′(2′,6′)-(di)halogenopicropodophyllotoxin, the ef-
fect of the length of their side chain at the C-4 position of
podophyllotoxin skeleton on the insecticidal activity was not
very obvious. For 4α-(arylacyl)oxy derivatives of 2α-chloro-2′-
chloro/bromopicropodophyllotoxin, the electronic effect of the
substituents on their phenyl ring at the C-4 position of
podophyllotoxin skeleton on the insecticidal activity was
observed.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Data on H NMR, 13C NMR, optical rotation, and melting
point for the target compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(H.X.) Phone: +86(0)29-87091952. Fax: +86(0)29-
87091952. E-mail orgxuhui@nwsuaf.edu.cn.
■
Author Contributions
∥L.F. and Y.G. contributed equally to this work.
Funding
The present research was supported by the National Natural
Science Foundation of China (No. 31071737,31171896) and
Special Funds of Central Colleges Basic Scientific Research
Operating Expenses (YQ2013008) to H.X.
Notes
The authors declare no competing financial interest.
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