Synthesis of novel 4α-(Acyloxy)-2′(2′,6′)-(di) halogenopodophyllotoxin derivatives as insecticidal agents

Article abstract of DOI:10.1021/jf4025079

In continuation of our program aimed at the discovery and development of natural-product-based insecticidal agents, we have prepared three series of novel 4α-(acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin derivatives modified in the C and E rings of podophyllotoxin, which is a naturally occurring aryltetralin lignan isolated from the roots and rhizomes of Podophyllum hexandrum. Their structures were well characterized by ¹H NMR, HRMS, ESI-MS, optical rotation, and mp. The stereochemical configurations of compounds 5s, 6b, 6d, and 7q were unambiguously confirmed by single-crystal X-ray diffraction. Their insecticidal activity was evaluated against the pre-third-instar larvae of oriental armyworm, Mythimna separata Walker, in vivo at a concentration of 1 mg/mL. These derivatives likely displayed the antimolting hormone effect. Among all the derivatives, especially compounds 5a, 5n, 7f, 7n, and 7w exhibited the most potent insecticidal activity with final mortality rates of 70% or so. This suggested that a chlorine or bromine atom introduced at the C2′ or C2′ and C6′ positions on the E ring of podophyllotoxin was necessary for obtaining the potent compounds. This will pave the way for further design, structural modification, and development of podophyllotoxin derivatives as insecticidal agents.

Full text of DOI:10.1021/jf4025079

Article
pubs.acs.org/JAFC
Synthesis of Novel 4α-(Acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin
Derivatives as Insecticidal Agents
Zhiping Che,† Xiang Yu,† Xiaoyan Zhi,† Lingling Fan,† Xiaojun Yao,‡ and Hui Xu*,†
†Laboratory of Pharmaceutical Design & Synthesis, College of Sciences, Northwest A&F University, Yangling 712100, Shaanxi
Province, People’s Republic of China
‡Department of Chemistry, Lanzhou University, Lanzhou 730000, Gansu Province, People’s Republic of China
S
* Supporting Information
ABSTRACT: In continuation of our program aimed at the discovery and development of natural-product-based insecticidal
agents, we have prepared three series of novel 4α-(acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin derivatives modified in the C
and E rings of podophyllotoxin, which is a naturally occurring aryltetralin lignan isolated from the roots and rhizomes of
1
Podophyllum hexandrum. Their structures were well characterized by H NMR, HRMS, ESI-MS, optical rotation, and mp. The
stereochemical configurations of compounds 5s, 6b, 6d, and 7q were unambiguously confirmed by single-crystal X-ray
diffraction. Their insecticidal activity was evaluated against the pre-third-instar larvae of oriental armyworm, Mythimna separata
Walker, in vivo at a concentration of 1 mg/mL. These derivatives likely displayed the antimolting hormone effect. Among all the
derivatives, especially compounds 5a, 5n, 7f, 7n, and 7w exhibited the most potent insecticidal activity with final mortality rates
of 70% or so. This suggested that a chlorine or bromine atom introduced at the C2′ or C2′ and C6′ positions on the E ring of
podophyllotoxin was necessary for obtaining the potent compounds. This will pave the way for further design, structural
modification, and development of podophyllotoxin derivatives as insecticidal agents.
KEYWORDS: podophyllotoxin, acyloxy, halogenation, botanical insecticide, insecticidal activity, Mythimna separata Walker
compound 1 has also received much research attention for its
interesting insecticidal and antifungal activities.18−23 More recently,
we have investigated the insecticidal activity of a series of 2β-
chloropodophyllotoxin (I; Figure 1) and 2α/β-bromopodophyllo-
toxin (II and III; Figure 1) derivatives with modified C and D rings
of 124−28 and 4-(acyloxy)podophyllotoxin derivatives (IV; Figure 1)
with modified A and C rings of 1.29 Some compounds showed
more potent insecticidal activity than toosendanin, a commercial
botanical insecticide isolated from Melia azedarach. Encouraged by
the above-mentioned interesting results, and to find novel natural-
product-based insecticidal agents to control the lepidopteran pests,
we herein designed and synthesized three series of novel 4α-
(acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin derivatives (V;
Figure 1) by introduction of the halogen atom at the C2′ or C2′
and C6′ positions on the E ring as insecticidal agents against the
pre-third-instar larvae of oriental armyworm, M. separata Walker, in
vivo. Additionally, their structure−activity relationship (SAR) studies
were also described.
INTRODUCTION
■
Lepidoptera is one of the most widespread and widely
recognizable insect orders in the world. The larvae of many
lepidopteran species are major pests in agriculture and can
cause extensive damage to certain crops, especially new plants.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
result in widespread incidence and complete crop loss.2
Although a wide variety of synthetic insecticides were used to
control lepidopteran pests, repeat application of those agro-
chemicals over years has led to the development of resistance in
lepidopteran pest populations and environmental problems.3−6
On the other hand, plant secondary metabolites result from the
interaction between plants and the environment (life and
nonlife) during the long period of evolution in plants, and
pesticides produced from plant secondary metabolites may result in
less or slower resistance development in pest populations and lower
environmental pollution.7 Consequently, the discovery and
development of new insecticidal compounds directly from plant
secondary metabolites, or by using them as lead compounds for
further structural modifications, have recently been an important
area of research and development of new pesticides.8−11 Nowadays,
some botanical insecticides, such as nicotine, pyrethrum, and neem
extracts, are made by plants as defenses against insect pests.12
Podophyllotoxin (1; Figure 1), a naturally occurring
aryltetralin lignan, is isolated from the roots and rhizomes of
Podophyllum hexandrum and some Juniperus species.13 Besides
its use as the lead compound for the preparation of potent
anticancer drugs such as etoposide, teniposide, and etopophos,14−17
MATERIALS AND METHODS
■
General Procedures. 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 thin-layer chromatography (TLC) and preparative thin-layer
chromatography (PTLC) were performed with silica gel plates using
silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd.). Silica gel
Received: June 7, 2013
Revised: August 5, 2013
Accepted: August 5, 2013
Published: August 5, 2013
© 2013 American Chemical Society
8148
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Figure 1. Chemical structures of podophyllotoxin (1) and its derivatives (I−V).
159−160 °C; [α]2D0 = −30 (c 3.3 mg/mL, CHCl3); H NMR (500
MHz, CDCl3) δ 7.15 (s, 1H, H-5), 6.31 (s, 1H, H-8), 5.94 (d, J = 0.5
Hz, 2H, OCH2O), 5.52 (d, J = 8.5 Hz, 1H, H-1), 4.77 (d, J = 10.0 Hz,
1H, H-4), 4.66 (dd, J = 8.5 Hz, 7.0 Hz, 1H, H-11), 4.08 (dd, J = 10.5,
9.0 Hz, 1H, H-11), 3.95 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 3.49−3.53 (m, 1H, H-3), 3.01 (dd, J = 15, 8.5 Hz, 1H, H-
2); 13C NMR (125 MHz, CDCl3) δ 172.8, 149.4, 149.0, 147.8, 147.4,
147.3, 132.6, 130.9, 130.2, 129.1, 124.2, 107.8, 105.8, 101.4, 72.5, 70.9,
61.3, 61.2, 61.1, 43.9, 43.1, 39.3; MS (ESI) m/z (rel intens) 505 ([M +
Na]+, 100), 507 ([M + Na]+, 73), 509 ([M + Na]+, 22); HRMS m/z
calcd for C22H24NO8Cl2 ([M + NH4]+) 500.0873, found 500.0868.
Synthesis of 2′-Bromopodophyllotoxin (4). To a solution of 1
(83 mg, 0.2 mmol) in dry DMF (2 mL) at 0 °C was added dropwise
for 10 min a solution of N-bromosuccinimide (NBS) (41.6 mg, 0.23
mmol) in dry DMF (2 mL). During 1 h, the solution was allowed to
warm slowly from 0 to 16 °C. When the reaction was complete,
checked by TLC analysis, the reaction mixture was diluted with water
(15 mL) and extracted with ethyl acetate (30 mL × 3). Subsequently,
the combined organic phase was washed by saturated aqueous
Na2CO3 (30 mL × 3) and brine (30 mL), dried over anhydrous
Na2SO4, concentrated in vacuo, and purified by silica gel column
chromatography eluting with petroleum ether/ethyl acetate (1:1, v/v)
to afford 4: CAS no. 40456-15-3; white solid; yield 88%; mp 140−141 °C
1
column chromatography was performed with silica gel 200−300 mesh
(Qingdao Haiyang Chemical Co., Ltd.). Melting points were
determined on a digital melting point apparatus and were uncorrected.
Proton nuclear magnetic resonance (1H NMR) spectra and carbon
nuclear magnetic resonance (13C NMR) spectra were recorded on a
Bruker Avance III 500 MHz instrument in CDCl3 using
tetramethylsilane (TMS) as the internal standard. High-resolution
mass spectrometry (HR-MS) and electrospray ion trap mass
spectrometry (ESI-MS) were carried out with an IonSpec 4.7 T
FTMS instrument and a Bruker ESI-TRAP Esquire 6000 plus mass
spectrometry instrument, respectively.
Synthesis of 2′-Chloropodophyllotoxin (2). To a solution of 1
(0.4 mmol) in dry DMF (4 mL) at 0 °C was added dropwise for
10 min a solution of N-chlorosuccinimide (NCS; 62.6 mg, 0.46 mmol)
in dry DMF (4 mL). During 1 h, the solution was allowed to warm
slowly from 0 to 28 °C. When the reaction was complete, checked by
TLC analysis, the reaction mixture was diluted with water (15 mL)
and extracted with ethyl acetate (30 mL × 3). Subsequently, the
combined organic phase was washed by saturated aqueous Na2CO3
(30 mL × 3) and brine (30 mL), dried over anhydrous Na2SO4,
concentrated in vacuo, and purified by silica gel column chromatog-
raphy eluting with petroleum ether/ethyl acetate (1:2, v/v) to afford 2:
white solid; yield 79%; mp 116−117 °C; [α]2D0 = −63 (c 3.8 mg/mL,
1
(lit.30 mp not reported); [α]D20 = −42 (c 3.5 mg/mL, CHCl3); H
1
CHCl3); H NMR (500 MHz, CDCl3) δ 7.08 (s, 1H, H-5), 6.37 (s,
1H, H-8), 6.19 (s, 1H, H-6′), 5.94 (s, 2H, OCH2O), 5.20 (d, J = 4.0
Hz, 1H, H-1), 4.79 (d, J = 8.5 Hz, 1H, H-4), 4.66 (dd, J = 8.5, 6.5 Hz,
1H, H-11), 4.10 (t, J = 9.0 Hz, 1H, H-11), 3.91 (s, 3H, OCH3), 3.86
NMR (500 MHz, CDCl3) δ 7.08 (s, 1H, H-5), 6.36 (s, 1H, H-8), 6.20
(s, 1H, H-6′), 5.94 (s, 2H, OCH2O), 5.27 (s, 1H, H-1), 4.78 (d, J =
9.0 Hz, 1H, H-4), 4.63−4.66 (m, 1H, H-11), 4.10 (t, J = 9.0 Hz, 1H,
H-11), 3.91 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.64 (s, 3H, OCH3),
2.91−2.99 (m, 2H, H-2, 3), 2.52 (s, 1H, OH); 13C NMR (125 MHz,
CDCl3) δ 173.1, 152.2, 150.9, 147.9, 147.6, 142.6, 135.6, 132.4, 131.5,
114.5, 110.5, 109.6, 105.6, 101.5, 71.8, 70.9, 61.0, 56.4, 44.1, 41.8, 41.7;
MS (ESI) m/z (rel intens) 515 ([M + Na]+, 100), 517 ([M + Na]+, 98).
General Procedure for the Synthesis of 5a−o,s,w,a′,b′,
6b,d−g,i,k,l, 7a−d,f,j−w,a′, and 8b,d,k,l. A mixture of 2, 3, 4, or
1 (0.2 mmol), the corresponding acids RCO2H (0.24 mmol), N,N′-
dicyclohexylcarbodiimide (DCC; 0.24 mmol), and 4-(N,N-
dimethylamino)pyridine (DMAP; 0.04 mmol) in dry CH2Cl2 (10
mL) was stirred at 28 °C. When the reaction was complete according
to TLC analysis, the mixture was filtered, and the filtrate was collected
and diluted by CH2Cl2 (40 mL). Subsequently, the mixture was
washed by 0.1 N HCl (25 mL), saturated aqueous NaHCO3 (25 mL),
and brine (25 mL), dried over anhydrous Na2SO4, concentrated in
vacuo, and purified by PTLC to give the pure target products 5a−
o,s,w,a′,b′, 6b,d−g,i,k,l, 7a−d,f,j−w,a′, and 8b,d,k,l. The example data
(s, 3H, OCH3), 3.65 (s, 3H, OCH3), 2.93−2.96 (m, 2H, H-2, 3); 13
C
NMR (125 MHz, CDCl3) δ 173.0, 151.6, 150.1, 147.9, 147.6, 142.8,
133.6, 132.6, 131.4, 110.6, 109.7, 105.6, 101.5, 72.0, 70.9, 61.1, 61.1,
56.5, 44.2, 41.9; MS (ESI) m/z (rel intens) 466 ([M + NH4]+, 100);
HRMS m/z calcd for C22H25NO8Cl ([M + NH4]+) 466.1263, found
466.1270.
Synthesis of 2′,6′-Dichloropodophyllotoxin (3). To a solution
of 1 (0.4 mmol) in dry DMF (4 mL) at 0 °C was added dropwise for
10 min a solution of NCS (109 mg, 0.8 mmol) in dry DMF (4 mL).
During 1 h, the solution was allowed to warm slowly from 0 to 28 °C.
When the reaction was complete, checked by TLC analysis, the
reaction mixture was diluted with water (15 mL) and extracted with
ethyl acetate (30 mL × 3). Subsequently, the combined organic phase
was washed by saturated aqueous Na2CO3 (30 mL × 3) and brine
(30 mL), dried over anhydrous Na2SO4, concentrated in vacuo, and
purified by silica gel column chromatography eluting with petroleum
ether/ethyl acetate (1:2, v/v) to afford 3: white solid; yield 90%; mp
8149
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Scheme 1. Route for the Synthesis of 4α-(Acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin Derivatives 5a−o,s,w,a′,b, 6b,d-
g,i,k,l, and 7a−d,f,j-w,a′
for 5a,b, 6b,d, 7a,b, and 8b,d are shown as follows, whereas data for
5c−o,s,w,a′,b′, 6e−g,i,k,l, 7c,d,f,j−w,a′, and 8k,l can be found in the
Supporting Information.
(d, J = 8.5 Hz, 1H, H-1), 4.41 (t, J = 8.0 Hz, 1H, H-11), 4.13−4.17 (m,
1H, H-11), 3.95 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.60−3.64 (m, 1H, H-3), 3.11 (dd, J = 15.0, 8.5 Hz, 1H, H-2),
2.23 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ 172.1, 171.5,
149.4, 149.0, 148.0, 147.4, 147.1, 130.8, 130.7, 129.1, 127.9, 124.2,
107.8, 106.1, 101.4, 73.0, 70.6, 61.2, 61.1, 61.0, 43.0, 41.6, 39.0, 21.1;
HRMS m/z calcd for C24H26NO9Cl2 ([M + NH4]+) 542.0979, found
542.0971.
Data for 5a: yield 85%; white solid; mp 94−95 °C; [α]2D0 = +48 (c
1
3.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H,
CHO), 6.77 (s, 1H, H-5), 6.42 (s, 1H, H-8), 6.21 (s, 1H, H-6′), 6.06
(d, J = 9.5 Hz, 1H, H-4), 5.96 (s, 2H, OCH2O), 5.25 (d, J = 5.0 Hz,
1H, H-1), 4.46 (dd, J = 9.0, 7.0 Hz, 1H, H-11), 4.18−4.22 (m, 1H, H-
11), 3.92 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.66 (s, 3H, OCH3),
3.13−3.16 (m, 1H, H-3), 3.01−3.05 (m, 1H, H-2); 13C NMR (125
MHz, CDCl3) δ 171.9, 160.9, 151.5, 150.2, 148.4, 147.6, 142.8, 132.8,
132.5, 126.9, 122.7, 110.4, 109.7, 106.2, 101.6, 72.7, 70.6, 61.1, 61.0,
56.2, 44.3, 39.3; MS (ESI-TRAP) m/z (rel intens) 499 ([M + Na]+,
100), 501 ([M + Na]+, 43); HRMS m/z calcd for C23H25NO9Cl
([M + NH4]+) 494.1212, found 494.1222.
Data for 6d: yield 78%; white solid; mp 190−191 °C; [α]2D0 = −27
1
(c 3.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 6.74 (s, 1H,
H-5), 6.34 (s, 1H, H-8), 5.94−5.97 (m, 3H, H-4 and OCH2O), 5.54
(d, J = 8.0 Hz, 1H, H-1), 4.36−4.39 (m, 1H, H-11), 4.17 (t, J = 10.0
Hz, 1H, H-11), 3.95 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.59−3.63 (m, 1H, H-3), 3.11 (dd, J = 15.0, 8.5 Hz, 1H, H-2),
2.47−2.53 (m, 2H, CH2CH3), 1.26 (t, J = 7.5 Hz, 3H, CH2CH3); 13
C
Data for 5b: yield 89%; white solid; mp 246−247 °C; [α]2D0 = +59
NMR (125 MHz, CDCl3) δ 174.9, 172.2, 149.4, 149.0, 147.9, 147.4,
147.0, 130.8, 130.7, 129.1, 128.0, 124.2, 107.8, 106.1, 101.4, 72.8, 70.7,
61.2, 61.1, 61.0, 43.1, 41.6, 39.0, 27.7, 9.2; HRMS m/z calcd for
C25H28NO9Cl2 ([M + NH4]+) 556.1136, found 556.1130.
1
(c 3.3 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 6.74 (s, 1H,
H-5), 6.41 (s, 1H, H-8), 6.21 (s, 1H, H-6′), 5.95 (d, J = 2.5 Hz, 2H,
OCH2O), 5.92 (d, J = 9.5 Hz, 1H, H-4), 5.23 (d, J = 4.5 Hz, 1H, H-1),
4.44 (dd, J = 9.0, 6.5 Hz, 1H, H-11), 4.17−4.21 (m, 1H, H-11), 3.92
(s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 2.99−3.07
(m, 2H, H-2, 3), 2.23 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ
172.1, 171.2, 151.4, 150.1, 148.2, 147.5, 142.8, 133.0, 132.3, 127.7,
122.7, 110.5, 109.7, 106.2, 101.6, 73.0, 70.7, 61.1, 61.0, 56.3, 44.4, 39.5,
21.1; HRMS m/z calcd for C24H27NO9Cl ([M + NH4]+) 508.1369,
found 508.1361.
Data for 7a: yield 78%; white solid; mp 98−99 °C; [α]2D0 = −52 (c
1
4.1 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H,
CHO), 6.77 (s, 1H, H-5), 6.42 (s, 1H, H-8), 6.23 (s, 1H, H-6′), 6.05
(d, J = 9.5 Hz, 1H, H-4), 5.96 (s, 2H, OCH2O), 5.32 (s, 1H, H-1),
4.46 (dd, J = 9.0, 7.0 Hz, 1H, H-11), 4.21 (t, J = 9.5 Hz, 1H, H-11),
3.91 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 3.14−
3.18 (m, 1H, H-3), 3.05 (dd, J = 15.0, 6.0 Hz, 1H, H-2); HRMS m/z
calcd for C23H21O9Br ([M]+) 520.0363, found 520.0366.
Data for 6b: yield 88%; white solid; mp 191−192 °C; [α]2D0 = −26
1
(c 4.5 mg/mL, CHCl3); H NMR (500 MHz, CDCl3) δ 6.76 (s, 1H,
Data for 7b: yield 73%; white solid; mp 234−235 °C; [α]2D0 = −48
(c 3.6 mg/mL, CHCl3); 1H NMR (500 MHz, CDCl3) δ 6.74 (s, 1H, H-5),
H-5), 6.34 (s, 1H, H-8), 5.94−5.96 (m, 3H, H-4 and OCH2O), 5.54
8150
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1
Figure 2. Partial H NMR spectra of 5l, 6l, and 7l.
6.41 (s, 1H, H-8), 6.23 (s, 1H, H-6′), 5.95 (d, J = 2.5 Hz, 2H, OCH2O),
5.91 (d, J = 9.5 Hz, 1H, H-4), 5.30 (d, J = 4.0 Hz, 1H, H-1), 4.44 (dd, J =
9.0, 7.0 Hz, 1H, H-11), 4.20 (t, J = 10.0 Hz, 1H, H-11), 3.91 (s, 3H,
OCH3), 3.87 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.07−3.12 (m, 1H,
H-3), 3.03 (dd, J = 15.0, 6.0 Hz, 1H, H-2), 2.23 (s, 3H, CH3); HRMS
m/z calcd for C24H23O9Br ([M]+) 534.0520, found 534.0523.
(t, J = 10.0 Hz, 1H, H-11), 3.81 (s, 3H, 4′-OCH3), 3.76 (s, 6H, 3′, 5′-
OCH3), 2.94 (dd, J = 14.5, 4.0 Hz, 1H, H-2), 2.81−2.84 (m, 1H, H-3),
2.44−2.49 (m, 2H, CH2CH3), 1.21 (t, J = 7.5 Hz, 3H, CH2CH3); 13
C
NMR (125 MHz, CDCl3) δ 174.8, 173.6, 152.6, 148.1, 147.6, 137.2,
134.8, 132.3, 128.4, 109.7, 108.1, 106.9, 101.5, 73.4, 71.3, 60.7,
56.1, 45.6, 43.7, 38.7, 27.7, 9.1; MS (ESI) m/z (rel intens) 488
Data for 8b: yield 89%; white solid; mp 210−212 °C (lit.31 mp
+
([M + NH4] , 100).
210−211 °C); [α]2D0 = −149 (c 4.4 mg/mL, CHCl3); H NMR (500
Biological Assay. The insecticidal activity of 4α-(acyloxy)-
2′(2′,6′)-(di)halogenopodophyllotoxin derivatives 5a−o,s,w,a′,b′,
6b,d−g,i,k,l, and 7a−d,f,j−w,a′ against the pre-third-instar larvae of M.
separata was assessed by the leaf-dipping method as described
previously.32,33 For each compound, 30 pre-third-instar larvae (10 larvae
per group) were used. Acetone solutions of compounds 5a−o,s,w,a′,b′,
6b,d−g,i,k,l, and 7a−d,f,j−w,a′ were prepared at a concentration of 1 mg/
mL. Compounds 8b,d,k,l and toosendanin were used as the positive
control at a concentration of 1 mg/mL. Fresh wheat leaves were dipped
into the corresponding solution for 3 s and 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, in which 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
dishes until adult emergence. The experiment was carried out at 25 2 °C
1
MHz, CDCl3) δ 6.77 (s, 1H, H-5), 6.54 (s, 1H, H-8), 6.39 (s, 2H, H-
2′, 6′), 5.99 (d, J = 5.5 Hz, 2H, OCH2O), 5.89 (d, J = 9.0 Hz, 1H, H-
4), 4.60 (d, J = 4.0 Hz, 1H, H-1), 4.36−4.40 (m, 1H, H-11), 4.18−4.22
(m, 1H, H-11), 3.81 (s, 3H, 4′-OCH3), 3.76 (s, 6H, 3′, 5′-OCH3),
2.94 (dd, J = 14.5, 4.5 Hz, 1H, H-2), 2.80−2.87 (m, 1H, H-3), 2.18 (s,
3H, CH3); 13C NMR (125 MHz, CDCl3) δ 173.6, 171.3, 152.6, 148.1,
147.6, 137.2, 134.8, 132.3, 128.3, 109.7, 108.1, 107.0, 101.6, 73.6, 71.3,
60.7, 56.1, 45.6, 43.7, 38.7, 21.0; MS (ESI) m/z (rel intens) 479 ([M +
Na]+, 100).
Data for 8d: yield 82%; white solid; mp 137−138 °C (lit.31 mp
136−138 °C);31 [α]D20 = −138 (c 3.6 mg/mL, CHCl3); 1H NMR (500
MHz, CDCl3) δ 6.76 (s, 1H, H-5), 6.54 (s, 1H, H-8), 6.39 (s, 2H,
H-2′, 6′), 5.99 (d, J = 5.5 Hz, 2H, OCH2O), 5.90 (d, J = 9.0 Hz, 1H,
H-4), 4.61 (d, J = 4.0 Hz, 1H, H-1), 4.35−4.39 (m, 1H, H-11), 4.22
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and a relative humidity (RH) of 65−80% and in a 12 h/12 h (light/dark)
photoperiod. The insecticidal activity of the tested compounds against the
pre-third-instar larvae of M. separata was calculated by the formula
corrected mortality rate (%) = [(T − C) × 100]/(100 − 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.
RESULTS AND DISCUSSION
■
Synthesis. Three series of novel 4α-(acyloxy)-2′(2′,6′)-
(di)halogenopodophyllotoxin derivatives (5a−o,s,w,a′,b′,
6b,d−g,i,k,l, and 7a−d,f,j−w,a′) modified in the C and E
rings were prepared as shown in Scheme 1. First, three
intermediates (2−4) were smoothly synthesized by reaction of
1 with NCS or NBS. Then compounds 5a−o,s,w,a′,b′, 6b,d−
g,i,k,l, 7a−d,f,j−w,a′, and 8b,d,k,l (used as the control for the
next bioassay) were obtained by reaction of 2, 3, 4, or 1 with
the corresponding carboxylic acids 9 in the presence of DCC
and DMAP. The structures of the target compounds were well
1
characterized by H NMR, HRMS, ESI-MS, optical rotation,
and mp. The assignment of the configuration at the C-4
position of the above derivatives was based on J3.4 coupling
Figure 3. X-ray crystal structure of compound 5s.
constants: The C-4β-substituted compounds have a J3.4
4.0 Hz due to a cis relationship between H-3 and H-4. J3.4
10.0 Hz indicates that H-3 and H-4 are in a trans relationship,
and the substituent at the C-4 position of podophyllotoxin is in
the α configuration.34 For example, as described in Figure 2, the
≈
≥
J3.4 values of H-4 of 5l, 6l, and 7l were all 9.5 Hz; therefore, the
4-pyridyloyloxy groups at the C-4 position of 5l, 6l, and 7l were
all in the α configuration. The precise three-dimensional
structural information on 5s, 6b, 6d, and 7q was further
determined by single-crystal X-ray diffraction as illustrated in
Figures 3−6. It obviously suggested that the substituents on the
C-4 position of 5s, 6b, 6d, and 7q were all in the α
configuration, and the chlorine atom on the E ring of 5s and the
bromine atom on the E ring of 7q were at the C-2′ position.
Crystallographic data (excluding structure factors) for the structures
of 5s, 6b, 6d, and 7q have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication
numbers CCDC 894364, 894557, 894575, and 941537,
respectively. Copies of the data can be obtained, free of charge,
on application to the CCDC, 12 Union Rd., Cambridge CB2 1EZ,
U.K. [fax +44 (0)1223 336033 or e-mail deposit@ccdc.cam.ac.uk].
Insecticidal Activity. The insecticidal activity of novel 4α-
(acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin derivatives
5a−o,s,w,a′,b′, 6b,d−g,i,k,l, and 7a−d,f,j−w,a′) against the
pre-third-instar larvae of M. separata was tested by the leaf-
dipping method at a concentration of 1 mg/mL. Compounds
8b,d,k,l and toosendanin were used as the positive control at a
concentration of 1 mg/mL. As shown in Table 1, the corresponding
mortality rates caused by these compounds after 30 days were
generally higher than those after 10 and 20 days. For example, the
corrected mortality rate of 7f against M. separata after 10 days was
only 6.7%, and after 20 days, the corresponding mortality rate was
increased to 48.3%. However, after 30 days, the corresponding
mortality rate was sharply increased to 74.1%, which was nearly 11-
fold greater than that after 10 days. That is, these compounds, in a
time-dependent manner, different from other conventional neuro-
toxic insecticides such as organophosphates, carbamates, and
pyrethroids, showed delayed insecticidal activity. On the other
hand, the symptoms of the tested M. separata were also
characterized in the same way as in our previous reports:23,29,32
Figure 4. X-ray crystal structure of compound 6b.
(a) due to feeding too many treated leaves during the first 48 h,
some larvae died slowly with slim and wrinkled bodies during the
larval period (Figure 7); (b) many larvae of the treated groups
molted to malformed pupae and died during the pupation stage
(Figure 8); (c) malformed moths with imperfect wings also
appeared in the treated groups (Figure 9). According to the above-
mentioned symptoms, the prepared derivatives likely exhibited the
antimolting hormone effect. As described in Table 1, many
compounds exhibited insecticidal activity equal to or higher than
that of toosendanin. Among all the derivatives, the final mortality
rates of compounds 2, 3, 5a, 5f, 5j, 5m−o, 6g, 7f, 7n, 7w, and 7z
were all greater than 60%. Especially compounds 5a, 5n, 7f, 7n,
and 7w exhibited the most potent insecticidal activity with final
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4α-(acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin derivatives
(e.g., 5b,d,k,l, 6b,d,k,l, and 7b,d,k,l) was compared with that of
the corresponding 4α-(acyloxy)podophyllotoxin derivatives (e.g.,
8b,d,k,l). This further demonstrated that a chlorine or bromine
atom introduced at the C2′ or C2′ and C6′ positions on the E ring
of podophyllotoxin was important for obtaining the potent
derivatives. For example, the final mortality rates of 5b,d,k,l,
6b,d,k,l, and 7b,d,k,l were 57.7%/57.7%/57.7%/38.5%, 57.7%/
57.7%/53.8%/53.8%, and 55.6%/51.9%/59.3%/51.9%, respectively;
however, the final mortality rates of 8b,d,k,l were 38.5%, 34.6%,
38.5%, and 34.6%, respectively. For 4α-(alkylacyl)oxy derivatives,
the effect of the length of their side chain at the C-4 position on the
insecticidal activity was not very obvious. However, introduction of
the (chloroacetyl)oxy group at the C-4 position of 2 or 4 resulted
in less potent compounds (e.g., 42.3% for 5c and 33.3% for 7c).
Similarly, for 4α-(arylacyl)oxy derivatives, the electronic effect of the
substituents on their phenyl ring at the C-4 position on the
insecticidal activity was generally not very clear. Interestingly,
introduction of a (1-naphthylacetyl)oxy group at the C-4 position of
2 or 4 could also lead to more potent compounds (e.g., 69.2% for
5n and 70.4% for 7n) in the same way as described in our previous
papers.24,26,29 As compared with 2β-chloropodophyllotoxin deriva-
tives (I), 2′(2′,6′)-(di)chloropodophyllotoxin derivatives 5 and 6
generally exhibited more potent insecticidal activity.24,26 For
example, the final mortality rate of 2β-chloropodophyllotoxin was
48.1%; whereas the final mortality rates of 2′-chloropodophyllotoxin
(2) and 2′,6′-dichloropodophyllotoxin (3) were 65.4% and 61.5%,
respectively. Similarly, the final mortality rates of 4α-(acetyloxy)-2β-
chloropodophyllotoxin and 4α-(propanoyloxy)-2β-chloropodophyl-
lotoxin were 40.7%, but the final mortality rates of 4α-(acetyloxy)-
2′-chloropodophyllotoxin (5b) and 4α-(propanoyloxy)-2′,6′-di-
chloropodophyllotoxin (5d) were 57.7%. In general, 2′-bromopo-
dophyllotoxin derivatives (7) displayed more promising insecticidal
activity than the 2α/β-bromopodophyllotoxin derivatives (II and
III).27,28 For example, the final mortality rates of 2α-bromo-
podophyllotoxin and 2β-bromopodophyllotoxin were 41.7%, and
45.8%, respectively; whereas the final mortality rate of 2′-
bromopodophyllotoxin (4) was 55.6%. Likewise the final mortality
rates of 4α-(acetyloxy)-2α-bromopodophyllotoxin, 4α-[(m-nitro-
benzoyl)oxy]-2α-bromopodophyllotoxin, and 4α-[(1-naphthyl-
acetyl)oxy]-2α-bromopodophyllotoxin were 37.5%, 58.3%, and
50%, respectively; however, the final mortality rates of 4α-(acetyloxy)-
2′-bromopodophyllotoxin (7a), 4α-[(m-nitrobenzoyl)oxy]-2′-bromo-
podophyllotoxin (7n), and 4α-[(1-naphthylacetyl)oxy]-2′-bromopo-
dophyllotoxin (7w) were 59.3%, 70.4%, and 70.4%, respectively.
In summary, three series of novel 4α-(acyloxy)-2′(2′,6′)-
(di)halogenopodophyllotoxin derivatives modified in the C and
E rings of podophyllotoxin were synthesized and evaluated for
their insecticidal activity against the pre-third-instar larvae of
Figure 5. X-ray crystal structure of compound 6d.
Figure 6. X-ray crystal structure of compound 7q.
mortality rates of approximately 70%. It is noteworthy that
introduction of a chlorine or bromine atom at the C2′ or C2′ and
C6′ positions on the E ring of podophyllotoxin could result in more
potent compounds compared to their precursor podophyllotoxin.
For example, the final mortality rate of compound 1 was only 37%,
whereas the final mortality rates of the E ring halogenation products
of podophyllotoxin (2−4) were 65.4%, 61.5%, and 55.6%,
respectively. In addition, the insecticidal activity of some
Figure 7. Representative abnormal larvae pictures of 2, 5a, 6f, 7n, and
7y during the larval period (CK = blank control group).
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Table 1. Insecticidal Activity of Three Series of 4α-(Acyloxy)-2′(2′,6′)-(di)halogenopodophyllotoxin Derivatives (5a−
o,s,w,a′,b′, 6b,d−g,i,k,l, and 7a−d,f,j−w,a′) at 1 mg/mL against M. separata
corrected mortality rate (%)
corrected mortality rate (%)
compd
10 days
20 days
30 days
compd
10 days
20 days 30 days
a
1
13.3 3.3
26.7 3.3
23.3 3.3
13.3 3.3
56.7 3.3
46.7 3.3
43.3 3.3
46.7 3.3
23.3 3.3
53.3 3.3
27.6
0
37.0 3.3
65.4
61.5 6.7
55.6
6l
50.0
0
3.3 3.3
10.0 5.8
6.7 3.3
13.3 3.3
6.7 3.3
46.4
0
53.8 5.8
59.3 3.3
2
39.3 6.7
42.9 3.3
41.4 3.3
60.7 3.3
53.6 6.7
39.3 3.3
53.6 3.3
34.5 3.3
57.1 5.8
41.4 3.3
44.8 3.3
46.4 5.8
35.7 5.8
53.6 6.7
32.1 3.3
32.1 3.3
0
7a
7b
7c
7d
7f
34.5 3.3
31.0 3.3
3
55.6
33.3
0
0
4
0
27.6
44.8 3.3
48.3
24.1 3.3
37.9
0
5a
5b
5c
5d
5e
5f
69.2 3.3
57.7 3.3
42.3 5.8
57.7 3.3
50.0 3.3
61.5 3.3
53.6 3.3
50.0 3.3
51.9 3.3
74.1 3.3
37.0 3.3
59.3 3.3
51.9 3.3
59.3 3.3
70.4 3.3
51.9 3.3
48.1 3.3
0
7j
10.0
20.0
10.0
20.0
10.0
0
0
0
0
0
7k
7l
0
34.5 3.3
44.8 3.3
7m
7n
7o
7p
7q
7r
5g
5h
5i
30.0
0
37.9
27.6
27.6
0
0
0
26.7 3.3
33.3 3.3
20.0 5.8
16.7 3.3
10.0
53.8
0
0
5j
61.5 6.7
57.7 3.3
38.5 3.3
61.5 3.3
69.2 3.3
61.5 3.3
50.0 3.3
20.0 5.8
16.7 3.3
13.3 3.3
3.3 3.3
27.6 5.8
27.6
55.6
59.3 3.3
33.3
0
5k
5l
50.0
0
0
36.7 3.3
26.7 3.3
43.3 3.3
7s
7t
24.1 3.3
20.7 3.3
20.7 3.3
24.1 3.3
0
5m
5n
5o
5s
5w
5a′
5b′
6b
6d
6e
6f
48.1 3.3
40.7 3.3
48.1 3.3
70.4 3.3
48.1 3.3
51.9 3.3
63.0 3.3
46.4
0
7u
7v
7w
7x
7y
7z
7a′
8b
8d
8k
8l
10.0 5.8
13.3 3.3
50.0
0
53.6 3.3
28.6 3.3
50.0 6.7
50.0 3.3
28.6 3.3
39.3 6.7
46.4 5.8
48.3 8.2
50.8 3.3
26.7 3.3
46.7 3.3
10.0
0
48.3
41.4 3.3
37.9
0
53.8
0
23.3 6.7
20.0 5.8
23.3 3.3
13.3 6.7
30.0
0
57.7 3.3
38.5 3.3
57.7 3.3
57.7 3.3
51.9 4.7
59.3 3.3
64.3 3.3
53.8 5.8
0
26.7 3.3
23.3 3.3
36.7 3.3
30.0 8.2
41.6 3.3
23.3 3.3
46.7 3.3
44.8 3.3
44.8 3.3
55.6
0
a
a
a
a
a
30.0
0
35.7
35.7
0
0
38.5 3.3
34.6 3.3
38.5 3.3
34.6 3.3
51.9 3.3
33.3 3.3
33.3 3.3
35.7 5.8
28.6 3.3
31.0 3.3
6g
6i
48.3
0
30.0
20.0
0
0
b
46.4 5.8
46.4 5.8
toosendanin
6k
20.0
0
53.8
0
a
b
After 33 days. Toosendanin was used as a positive control at 1 mg/mL.
M. separata in vivo at a concentration of 1 mg/mL. These
derivatives likely displayed the antimolting hormone effect.
Among all the derivatives, especially compounds 5a, 5n, 7f, 7n,
and 7w exhibited the most potent insecticidal activity with final
mortality rates of 70% or so. This suggested that a chlorine or
bromine atom introduced at the C2′ or C2′ and C6′ positions
on the E ring of podophyllotoxin was necessary for obtaining
the potent compounds. This will pave the way for further
design, structural modification, and development of podophyl-
lotoxin derivatives as insecticidal agents.
Figure 8. Representative malformed pupae pictures of 2, 5i, 6g, 7f, and
7w during the pupation period (CK = blank control group).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, HRMS, optical rotation, and melting point data for
the target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86-029-87091952. Fax: +86-029-87091952. E-mail:
orgxuhui@nwsuaf.edu.cn.
Funding
The present research was supported by the National Natural
Science Foundation of China (Grant No. 31071737) and
Special Funds of Central Colleges Basic Scientific Research
Figure 9. Representative malformed moth pictures of 3, 5j, 5n, 7f, and
7n during the emergence period (CK = blank control group).
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Operating Expenses (Grant YQ2013008), Northwest A&F
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Notes
The authors declare no competing financial interest.
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