Semisynthesis and quantitative structure-activity relationship (QSAR) study of novel aromatic esters of 4-demethyl-4-deoxypodophyllotoxin as lnsecticidal agents

Article abstract of DOI:10.1021/jf9020812

By using podophyllotoxin as a phytoinsecticidal lead compound, 15 novel aromatic esters of 4'demethyl-4-deoxypodophyllotoxin were semisynthesized and preliminarily tested for their insecticidal activity against the pre-third-instar larvae of Mythimna separata Walker in vivo for the first time. Among all of the tested compounds, especially two compounds, 4m and 4o, containing a pyridinyl group, for which final corrected mortality rates against M. separata at 1 mg/mL were 62.9 and 59.2%, respectively, showed the most promising and pronounced insecticidal activity as compared with toosendanin, a commercial insecticide derived from Melia azedarach. The quantitative structure-activity relationships (QSAR) of compounds 4a-4o showed that the relative number of benzene rings and final heat of formation were very important descriptors to their insecticidal activity.

Full text of DOI:10.1021/jf9020812

J. Agric. Food Chem. 2009, 57, 7919–7923 7919
DOI:10.1021/jf9020812
Semisynthesis and Quantitative Structure-Activity
Relationship (QSAR) Study of Novel Aromatic Esters of
40-Demethyl-4-deoxypodophyllotoxin as Insecticidal Agents
§
,
H
UI
X
U
,*,† JUANJUAN
IAOJUN
W
ANG,† HUIJUN
AO,*,§ AND
S
UN,§ MIN
L
V
,† XUAN
T
IAN
†
X
Y
X
ING
Z
HANG
†Laboratory of Pharmaceutical Design and Synthesis, College of Life Sciences, Northwest A&F
University, 22 Xinong Road, Yangling 712100, Shannxi Province, People’s Republic of China, and
§Department of Chemistry, Lanzhou University, Gansu Province, People’s Republic of China
By using podophyllotoxin as a phytoinsecticidal lead compound, 15 novel aromatic esters of 40-
demethyl-4-deoxypodophyllotoxin were semisynthesized and preliminarily tested for their insecticidal
activity against the pre-third-instar larvae of Mythimna separata Walker in vivo for the first time.
Among all of the tested compounds, especially two compounds, 4m and 4o, containing a pyridinyl
group, for which final corrected mortality rates against M. separata at 1 mg/mL were 62.9 and
59.2%, respectively, showed the most promising and pronounced insecticidal activity as compared
with toosendanin, a commercial insecticide derived from Melia azedarach. The quantitative struc-
ture-activity relationships (QSAR) of compounds 4a-4o showed that the relative number of
benzene rings and final heat of formation were very important descriptors to their insecticidal
activity.
KEYWORDS: Podophyllotoxin; synthesis; insecticidal activity; structure-activity relationship
INTRODUCTION
against pre-third-instar larvae of M. separata Walker in vivo for
the first time. The quantitative structure-activity relationships
(QSAR) of these compounds were also studied.
In the course of our screening for novel naturally occurring
phytoinsecticides from the plants in northwestern China, podo-
phyllotoxin (1) (Figure 1) was isolated as an insecticidal compo-
nent by bioassay-guided fractionation from Juniperus sabina
Linnaeus (1). It is well-known that structural modification is an
effective method to optimize natural bioactive compounds.
Recently, to obtain more potent insecticidal podophyllotoxin
analogues, compound 1 has been used as a lead compound for
structural modification, and some derivatives exhibited more
potent insecticidal activity than 1 against the larvae of Drosophila
melanogaster Meigen, Pieris rapae Linnaeus, or Mythimna
separata Walker in vivo (2-6). Obviously, the recent structural
modification of 1 as an insecticidal agent has been mainly focused
on the C-4 position of 1. To the best of our knowledge, however,
little attention has been paid to structural modification on the
C-40 position of 1. In continuation of our program aimed at the
discovery and development of bioactive molecules (3-7), we
want to investigate the effect of substituents on the C-40 position
of podophyllotoxin analogues to the insecticidal activity. In the
meantime, You et al. reported that some alkyl and carboxylalkyl
esters of 40-demethyl-4-deoxypodophyllotoxin exhibited good
antitumor activity (8). Therefore, in this paper 15 novel aromatic
esters of 40-demethyl-4-deoxypodophyllotoxin (4a-4o) were
semisynthesized from 1 and tested for their insecticidal activity
MATERIALS AND METHODS
Synthetic 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.). Melting points were
determined on a digital melting-point apparatus and were uncorrected.
Infrared spectra (IR) were recorded on a Thermo Nicolet Nexus FTIR-
8700 spectrometer. Proton nuclear magnetic resonance spectra (1H NMR)
were recorded on a Bruker Avance DMX 400 MHz instrument using TMS
as internal standard and CDCl3 as solvent. High-resolution mass spectra
(HR-MS) and electron ionization mass spectrometry (EI-MS) or electro-
spray iontrap mass spectrometry (ESI-TRAP-MS) were carried out with
APEX II Bruker 4.7T AS and Thermo DSQ GC-MS or Bruker ESI-
TRAP Esquire 3000 plus mass spectrometry instruments, respectively.
4-Deoxypodophyllotoxin (2). A mixture of 10% palladium/carbon
(12.0 g) and podophyllotoxin (1, 16.0 g, 38.6 mmol) in acetic acid solution
(150 mL) was stirred at 95 °C under 2 atm of hydrogen for 5 h. After
filtration to remove the catalyst and evaporation of the solvent, the residue
was purified by silica gel column chromatography (diethyl ether/dichloro-
methane, 6:1) to give the crude product, which was further purified by
recrystallization from methanol to afford 9.8 g (66%) of 2 as a white solid:
mp 165-167 °C [lit., 166-168 °C (9)]; [R]20D=-116° (c 1 mg/mL, CHCl3);
IR cm-1 2892, 2831, 1763, 1587, 1482, 1457, 1223, 1120, 925, 768; 1H
NMR (400 MHz, CDCl3) δ 6.67 (s, 1H, H-5), 6.52 (s, 1H, H-8), 6.34 (s, 2H,
H-20,60), 5.93 (d, J=8.0 Hz, 2H, OCH2O), 4.60 (s, 1H, H-1), 4.43 (m, 1H,
*Author to whom correspondence should be addressed [(H.X.)
telephone þ86(0)29-87091952; fax þ86(0)29-87091952; e-mail orgxuhui@
nwsuaf.edu.cn. (X.Y.) e-mail xjyao@lzu.edu.cn].
© 2009 American Chemical Society
Published on Web 08/18/2009
pubs.acs.org/JAFC

7920 J. Agric. Food Chem., Vol. 57, No. 17, 2009
Xu et al.
Scheme 1
H-11), 3.89 (m, 1H, H-11), 3.79 (s, 3H, 40-OCH3), 3.75 (s, 6H, 30,50-OCH3),
3.06 (m, 1H, H-4), 2.71 (m, 3H, H-2,3,4); MS (EI), m/z (%) 398.1
(Mþ, 100).
([M þ Na]þ, 39). HRMS: Anal. Calcd for C28H28NO8 ([M þ NH4]þ),
506.1809; found, 506.1802.
Biological Assay. The insecticidal activity of 4a-4o against the pre-
third-instar larvae of M. separata Walker was assessed by leaf-dipping
method as described previously (5). For each compound, 30 larvae
(10 larvae per group) were used. Acetone solutions of 4a-4o and
toosendanin (used as a positive control) were prepared at the concentra-
tion of 1 mg/mL. Fresh corn 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 control group. Several treated leaves were
kept in each dish, where every 10 larvae were raised. If the treated leaves
were consumed, corresponding ones were added to the dish. After 48 h,
untreated fresh leaves were added to all dishes until the adult emergence.
The experiment was carried out at 25 ( 2 °C and relative humidity (RH)
65-80% on 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
40-Demethyl-4-deoxypodophyllotoxin (3). 2 (2 g, 50.2 mmol) was
suspended in 1,2-dichloroethane (25 mL) and diethyl ether (2.5 mL) at
0 °C. A flow of dry hydrobromic acid was passed in the above solution.
When the reaction was complete according to TLC analysis, water
(25 mL), acetone (25 mL), and a little amount of BaCO3 were added to
the reaction mixture, which continued to stir for 0.5 h and extracted by
EtOAc (30 mL ꢀ 4). Subsequently, the combined organic phase was
washed by brine (50 mL ꢀ 2), dried over anhydrous Na2SO4, filtered,
concentrated in vacuo, and purified by silica gel column chromatography
(chloroform/ethyl acetate, 5:1) to give 1.16 g (60%) of 3 as a khaki solid:
mp 246-248 °C [lit., 244-249 °C (10)]; [R]20D =-130° (c 0.4 mg/mL,
CHCl3); IR cm-1 2899, 2824, 1757, 1608, 1478, 1458, 1214, 1105, 922, 769;
1H NMR (400 MHz, CDCl3) δ 6.66 (s, 1H, H-5), 6.51 (s, 1H, H-8), 6.35 (s,
2H, H-20,60), 5.92 (m, 2H, OCH2O), 5.39 (s, 1H, 40-OH), 4.59 (d, J=2.4 Hz,
1H, H-1), 4.42 (m, 1H, H-11), 3.88 (m, 1H, H-11), 3.78(s, 6H, 30,50-OCH3),
3.05 (m, 1H, H-4), 2.71 (m, 3H, H-2,3,4); MS (EI), m/z (%) 383.9
(Mþ, 100).
General Synthetic Procedure for Aromatic Esters of 40-De-
methyl-4-deoxypodophyllotoxin (4a-4o). A mixture of the corre-
sponding acid (0.3 mmol), diisopropylcarbodiimide (DIC, 0.3 mmol), 4-
dimethylaminopyridine (DMAP, 0.1 mmol), and 3 (0.25 mmol) in dried
dichloromethane (10 mL) was stirred at 0 °C. When the reaction was
complete according to TLC analysis, the resulting suspension was filtered,
and water (25 mL) was added to the solution of the above mixture, which
was extracted with dichloromethane (30 mL ꢀ 4). Then the organic phase
were combined, dried over anhydrous Na2SO4, concentrated in vacuo, and
purified by PTLC to give the pure target products in 38-93% yields. The
example data of 4a and 4b are shown as follows, whereas data for 4c-4o
can be found in the Supporting Information.
corrected mortality rate ð%Þ ¼ ðT -CÞ ꢀ 100=ð1 -CÞ
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. As shown in Scheme 1, 4-deoxypodophyllotoxin (2)
was first obtained in a 66% yield by catalytic hydrogenolysis of 1
in the presence of 10% palladium/carbon (9). Then regioselective
40-demethylation of 2 with dry hydrobromide, followed by the
mixed solvent system (water/acetone) and BaCO3, could proceed
readily to give the 40-demethyl-4-deoxypodophyllotoxin (3) in a
60% yield (10). Finally, 15 esters of 40-demethyl-4-deoxypodo-
phyllotoxin (4a-4o) were obtained by the reaction of 3 with the
corresponding acids containing aromatic cycles in the presence of
DIC and DMAP. The structures of the target compounds were
Data for 4a: Rf=0.52 (petroleum ether/ethyl acetate, 1:1); yield=93%;
white solid, mp 223-225 °C; [R]20D=-79° (c 0.29 mg/mL, CHCl3); IR
cm-1 2921, 2849, 1779, 1759, 1597, 1482, 1459, 1224, 1122, 928, 774, 731;
1H NMR (400 MHz, CDCl3) δ 7.38 (d, J=7.2 Hz, 2H, H-200,600), 7.32 (m,
2H, H-300,500), 7.28 (d, J=7.6 Hz, 1H, H-400), 6.65 (s, 1H, H-5), 6.51 (s, 1H,
H-8), 6.35 (s, 2H, H-20,60), 5.92 (dd, J=1.2 Hz, J=8.0 Hz, 2H, OCH2O),
4.61 (d, J=4.4 Hz, 1H, H-1), 4.42 (m, 1H, H-11), 3.88 (m, 3H, H-11 and
CH2C6H5), 3.62 (s, 6H, 30,50-OCH3), 3.03 (m, 1H, H-4), 2.69 (m, 3H, H-2,
3, 4); MS (ESI-TRAP), m/z (%) 525 ([M þ Na]þ, 25). HRMS: Anal. Calcd
for C29H30NO8 ([M þ NH4]þ), 520.1966; found, 520.1960.
1
well characterized by H NMR, HRMS, MS, optical rotation,
and IR.
Biological Activity. The insecticidal activity of 4a-4o against
the pre-third-instar larvae of M. separata Walker in vivo was
investigated by the leaf-dipping method at the concentration of
1 mg/mL. Toosendanin, a commercial insecticide derived from
M. azedarach, was used as a positive control. As shown in Table 1,
the corresponding corrected mortality rates caused by these
compounds after 36 days were far higher than those after 12
and 24 days. For example, the corrected mortality rate of 4m
against M. separata after 12 days was only 3.5%; after 24 days,
the corresponding mortality rate was increased to 10.7%, but
after 36 days the corresponding mortality rate was sharply
Data for 4b: Rf =0.7 (dichloromethane/acetone, 50:1); yield=71%;
white solid, mp 256-258 °C; [R]20D=-80° (c 0.26 mg/mL, CHCl3); IR
cm-1 2921, 2849, 1761, 1738, 1597, 1486, 1454, 1230, 1128, 928, 771, 706;
1H NMR (400 MHz, CDCl3) δ 8.19 (d, J=7.6 Hz, 2H, H-200,600), 7.59(t, J=
7.6 Hz, 1H, H-400), 7.46 (t, J=7.6 Hz, 2H, H-300,500), 6.68 (s, 1H, H-5), 6.59
(s, 1H, H-8), 6.43 (s, 2H, H-20,60), 5.94 (d, J=7.6 Hz, 2H, OCH2O), 4.67 (s,
1H, H-1), 4.46 (m, 1H, H-11), 3.89 (m, 1H, H-11), 3.69(s, 6H, 30,50-OCH3),
3.07 (m, 1H, H-4), 2.76 (m, 3H, H-2,3,4); MS (ESI-TRAP), m/z (%) 511

Article
J. Agric. Food Chem., Vol. 57, No. 17, 2009 7921
Table 1. Insecticidal Activity of Novel Aromatic Esters of 40-Demethyl-4-deoxypodophyllotoxin (4a-4o) against Mythimna separata Walker in Vivo
increased to 62.9%, which was nearly 18 times the mortality rate
after 12 days. That is, these compounds showed delayed insecti-
cidal activity (5), which is different from the conventional
neurotoxic insecticides, such as organophosphates, carbamates,
and pyrethroids. Meanwhile, the symptoms of the tested
M. separata were also characterized as the same as our previous
reports (5). Additionally, the pupation of the larvae and the adult
emergence of M. separata were inhibited by these compounds;
therefore, the stage from the larvae to adulthood of M. separata
was prolonged as compared to the control group. Moreover,
many larvae of the treated groups were unable to reach adulthood
and died during the stage of pupation. Among all of the tested

7922 J. Agric. Food Chem., Vol. 57, No. 17, 2009
Xu et al.
Table 2. BMLR Model and Its Statistical Parameters
descriptor
Xa
DXb
t test valuec
intercept
relative number of benzene rings
1.9193 ꢀ 102
-1.2144 ꢀ 103
6.4032 ꢀ 10-1
1.1577 ꢀ 101
2.3448 ꢀ 101
2.2330 ꢀ 102
1.1081 ꢀ 10-1
2.5599 ꢀ 10
8.1853
-5.4385
5.7785
final heat of formation
RNCS relative negative charged SA (SAMNEG*RNCG) [quantum-chemical PC]
4.5225
a X, regression coefficient of the descriptor. b DX, standard errors of the regression coefficient X. c t test value, t statistics of the regression coefficient X.
compounds, especially compounds 4m and 4o containing a
pyridinyl group, for which corrected mortality rates against
M. separata after 36 days were 62.9 and 59.2%, respectively, exhi-
bited the most promising and best insecticidal activity as com-
pared to toosendanin (40.7%).
Table 3. Information of Predicted Corrected Mortality Rate, Experimental
Corrected Mortality Rate, Errors, and Detail Information of the Selected
Descriptors
selected descriptors
no.
exptl (%)
pred (%)
error (%)
D1a
D2b
D3c
Qualitative Structure-Activity Relationships (SAR). As shown
in Table 1, when the methyl group was introduced at the meta
position on the phenyl ring of 4b to give 4d, the insecticidal
activity of 4d was more potent than the one having a p-methyl
group on the phenyl ring of 4b (4d vs 4c). The same results were
also found for compounds 4i and 4j; for example, when the nitro
group was introduced at the meta position on the phenyl ring of
4b to give 4i, the insecticidal activity after 36 days of 4i was
increased as compared with 4b (48.1% for 4b and 51.8% for 4i).
However, when the nitro group was introduced at the para
position on the phenyl ring of 4b to give 4j, the insecticidal
activity after 36 days of 4j was decreased as compared with 4b
(48.1% for 4b and 40.7% for 4j). Interestingly, if two nitro groups
were stimultaneously introduced at the meta position on the
phenyl ring of 4b to give 4k, the insecticidal activity after 36 days
of 4k was decreased sharply as compared with 4b (48.1% for 4b
and 22.2% for 4k); that is, 4k exhibited >2-fold less potency
than 4b. Moreover, introducing one electron-withdrawing group
(e.g., nitro group) on the phenyl ring of 4b will usually give more
active compounds than the one having an electron-donating
group (e.g., methyl, methoxy, and chloro) on the phenyl ring of
4b (4i and 4j vs 4c-4g). It was noteworthy that especially when
the pyridinyl ring was substituted for the phenyl ring of 4b, the
corrected mortality rates after 36 days of the corresponding
compounds 4m and 4o were increased to 62.9 and 59.2%,
respectively. Therefore, some aromatic esters of 40-demethyl-4-
deoxypodophyllotoxin containing other heterocyclic rings will be
synthesized and studied in our laboratory.
4a
4b
4c
4d
4e
4f
29.6
48.1
33.3
40.7
25.9
37.0
29.6
37.0
51.8
40.7
22.2
48.1
62.9
40.7
59.2
31.1
44.7
39.6
41.4
27.4
33.2
28.9
36.1
45.7
35.6
26.7
56.9
58.0
41.4
60.3
1.5
-3.4
6.3
0.0476
0.0500
0.0476
0.0476
0.0469
0.0500
0.0500
0.0580
0.0484
0.0484
0.0469
0.0469
0.0339
0.0411
0.0339
-217.089
-208.910
-218.458
-218.454
-247.513
-213.294
-215.347
-199.103
-217.155
-218.012
-220.094
-197.082
-201.476
-209.582
-201.747
3.1080
4.0793
3.9182
4.0721
4.3925
3.3296
3.0755
3.6353
4.4526
3.6289
2.8180
4.1559
3.1336
2.8962
3.3438
0.7
1.5
-3.8
-0.7
-0.9
-6.1
-5.1
4.5
4g
4h
4i
4j
4k
4l
8.8
4m
4n
4o
-4.9
0.7
1.1
a D1, relative number of benzene rings. b D2, final heat of formation. c D3, RNCS
relative negative charged SA (SAMNEG*RNCG) [quantum-chemical PC].
Figure 1. Structure of podophyllotoxin (1).
Quantitative Structure-Activity Relationships (QSAR). The
structures of 15 molecules 4a-4o were drawn by using Sybyl
6.9 (11). All structures were preoptimized by the molecular
mechanics force field (MMþ) (12) and then optimized using the
semiempirical method PM3 implemented in Hyperchem and
MOPAC (13).
corrected mortality rate, the experimental corrected mortality
rate, the errors, and the values of the selected descriptors are given
in Table 3.
The three selected descriptors belonged to constitutional and
quantum-chemical descriptors. They were relative number of
benzene rings, final heat of formation, and RNCS relative
negative charged SA (SAMNEG*RNCG) [quantum-chemical
PC]. According to the t test value, the relative number of benzene
rings and final heat of formation were very important descriptors.
A negative coefficient before the relative number of benzene rings
in this model indicated that an increase of this value led to a
decrease in the mortality rate of the compounds due to the
reduction in the solubility of the compound. The final heat of
formation was proportionally interrelated with the Gibbs free
energy. The plot of the predicted corrected mortality rate of the
compounds versus their experimental values is shown in Figure 2.
In conclusion, 15 novel aromatic esters of 40-demethyl-4-deox-
ypodophyllotoxin 4a-4o were semisynthesized and tested for their
insecticidal activity against pre-third-instar larvae of M. separata in
vivo at the concentration of 1 mg/mL. Among all of the tested
compounds, especially compounds 4m and 4o showed the most
promising and best insecticidal activity as compared to toosendanin.
Comprehensive Descriptors for Structural and Statistical
Analysis (CODESSA) is a comprehensive program for develop-
ing quantitative structure-activity/property relationships (QS-
AR/QSPR) by integrating all necessary mathematical and com-
putational tools. The output files from MOPAC were used by the
CODESSA program to calculate molecular descriptors (14). The
Best Multilinear Regression method (BMLR) was used to select
the significant descriptors and develop the QSAR model. Four
hundred and seventy-three descriptors were calculated for each
molecule. After using the BMLR method, three descriptors
correlated with the final corrected mortality rate (%) were
selected for the 15 40-demethyl-4-deoxypodophyllotoxin deriva-
tives 4a-4o. The BMLR model and its statistical parameters are
shown in Table 2. This model gave a squared correlation
coefficient (R2) of 0.868, a squared standard deviation (s2) of
23.543, and a cross-validated squared correlation coefficient
(R2cv) of 0.749. Detailed information about the predicted

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Received June 19, 2009. Revised manuscript received July 29, 2009.
Accepted July 30, 2009. This work was supported by the program for
New Century Excellent University Talents, State Education Ministry of
China (NCET-06-0868), the Key Project of Chinese Ministry of
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