Chemical modification produces species-specific changes in cucurbitacin antifeedant effect

Article abstract of DOI:10.1021/jf4002457

Cucurbitacins are secondary metabolites that mediate insect plant interactions not only as allomones against generalists but also as kairomones for specialist herbivores. This study was undertaken to identify the potential of cucurbitacin derivatives as insect antifeedant agents. The antifeedant capacity against a Cucurbitaceae specialist [Epilachna paenulata (Coleoptera: Coccinellidae)] and a polyphagous insect [Pseudaletia adultera (Lepidoptera: Noctuidae)] was evaluated in preference tests in which the insects were given a choice between food plants either treated with the cucurbitacin derivatives or treated with the solvent. The activity was found not to be related to the basic cucurbitacin skeleton, as only 15 of the 28 tested cucurbitacin derivatives were active. Only one of the tested compounds was phagostimulant to the specialist insect (the hemissuccinate of 16-oxo-dihydrocucurbitacin B derivative), while all other active derivatives were deterrent against one of the insects (13 compounds) or both of them (3 compounds). Changes in ring A of the cucurbitacins, as well as in the side chain, modified the activity. As a general trend, when chemical modifications of the basic structure produced a change in activity, the response was opposite in both insects used as biodetectors, indicating that a selective variation in the activity may be achieved by chemical modifications of the cucurbitacin skeleton.

Full text of DOI:10.1021/jf4002457

Article
pubs.acs.org/JAFC
Chemical Modification Produces Species-Specific Changes in
Cucurbitacin Antifeedant Effect
Karen Luise Lang,† Emilio Deagosto,‡ Lara Almida Zimmermann,† Vanessa Rocha Machado,†
Lílian Sibelle Campos Bernardes,† Eloir Paulo Schenkel,† Fernando Javier Duran,§ Jorge Palermo,§
and Carmen Rossini*,‡
†Laborator
Florianopolis, Santa Catarina−Brasil CEP, 88040-900 Florianop
‡Laboratorio de Ecología Química, Departamento de Química Organ
Flores 2124, CP 11800 Montevideo, Uruguay
§UMYMFOR-CONICET, Departamento de Química Organ
Aires, Ciudad Universitaria, Pabellon
́
io de Química Farmaceu
̂
tica, Departamento de Cien
̂
cias Farmaceu
olis, SC, Brazil
̂
ticas, Universidade Federal de Santa Catarina,
́
́
́
ica, Facultad de Química, Universidad de la Republica, General
́
́
ica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
2, 1428 Buenos Aires, Argentina
́
S
* Supporting Information
ABSTRACT: Cucurbitacins are secondary metabolites that mediate insect plant interactions not only as allomones against
generalists but also as kairomones for specialist herbivores. This study was undertaken to identify the potential of cucurbitacin
derivatives as insect antifeedant agents. The antifeedant capacity against a Cucurbitaceae specialist [Epilachna paenulata
(Coleoptera: Coccinellidae)] and a polyphagous insect [Pseudaletia adultera (Lepidoptera: Noctuidae)] was evaluated in
preference tests in which the insects were given a choice between food plants either treated with the cucurbitacin derivatives or
treated with the solvent. The activity was found not to be related to the basic cucurbitacin skeleton, as only 15 of the 28 tested
cucurbitacin derivatives were active. Only one of the tested compounds was phagostimulant to the specialist insect (the
hemissuccinate of 16-oxo-dihydrocucurbitacin B derivative), while all other active derivatives were deterrent against one of the
insects (13 compounds) or both of them (3 compounds). Changes in ring A of the cucurbitacins, as well as in the side chain,
modified the activity. As a general trend, when chemical modifications of the basic structure produced a change in activity, the
response was opposite in both insects used as biodetectors, indicating that a selective variation in the activity may be achieved by
chemical modifications of the cucurbitacin skeleton.
KEYWORDS: anti-insect, deterrent, triterpenoid, antiherbivory, cucurbitacins, semisynthesis
cucurbitacin-containing lures to control leaf-beetles have been
pursued in the past.20−23 Cucurbitacins have been also
intensively investigated for their cytotoxic and anti-inflamma-
tory properties.24,25 Most of these studies were performed with
the natural occurring cucurbitacins, but more recently semi-
synthetic derivatives were also described.26,27 Aiming to explore
the biological properties of this class of compounds, we
prepared a library of natural cucurbitacins and derivatives and
describe herein their activity against two insects: a specialist on
Cucurbitaceae [Epilachna paenulata Germar (Coleoptera:
Coccinellidae)], and a polyphagous herbivore [Pseudaletia
adultera Schaus (Lepidoptera: Noctuidae)] that prefers Poaceae
species as host plants.28
INTRODUCTION
■
Cucurbitacins are highly oxygenated tetracyclic triterpenes
occurring mostly, but not exclusively, in plants of the family
Cucurbitaceae.1 Because cucurbitacins are highly toxic to many
organisms including insects and vertebrates,2 they are generally
supposed to act as plant defense substances.3,4 However, some
herbivores have developed behavioral counter-adaptations to
the production of cucurbitacins as induced plant defenses,
avoiding these secondary metabolites produced as allomones
(i.e., a substance used by individuals of one species in their own
benefit that negatively affects individuals belonging to another
species).5,6 Furthermore, specialist herbivores may profit on
cucurbitacin occurrence in their host plant in such a way that
cucurbitacins, by being phagostimulants, become the token
stimuli7 that mediate host choice. Natural occurring cucurbi-
tacins have been intensively investigated not only as
kairomones8 for beetles belonging to the family Chrysomeli-
dae8−12 but also as sequestered defense compounds for
Diabroiticites.11 They have also been studied in regard to
their capacity as anti-insect agents, one mode of action being
their ability to compete with ecdysteroids for their ligand
binding site in the hormone receptors.13,14 Cucurbitacin effects
on insect feeding and oviposition4,15 have been described for
different insect orders;16−20 and some attempts to develop
MATERIALS AND METHODS
■
Cucurbutacins. The tested cucurbitacins and derivatives are
shown in Figure 1. Compounds 1 (dihydrocucurbitacin B), 3
(cucurbitacin R), 4 (25-deacetyl-dihydrocucurbitacin B), 5 (cayapono-
side A), and 6 (dihydrocucurbitacin B-2-O-glucoside) were isolated
Received: January 17, 2013
Revised: May 3, 2013
Accepted: May 6, 2013
© XXXX American Chemical Society
A
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Figure 1. Natural cucurbitacins tested for their antifeedant capacity.
Figure 2. Semisynthetic cucurbitacins25 tested for their antifeedant capacity.
from the roots of Wilbrandia ebracteata Cogn. (Cucurbitaceae), as
previously described.29,30 Compound 2 (cucurbitacin B) was isolated
from the fruits of Luffa operculata (L.) Cogn. (Cucurbitaceae).26
Compounds 7−20 (Figure 2) were synthesized from dihydrocucurbi-
tacin B or cucurbitacin B as previously described,26,27 and the synthesis
of the compounds 21−28 is described here for the first time (Schemes
1 and 2) and within the Supporting Information.
Insects. Epilachna paenulata. Germar (Coleoptera: Coccinelli-
dae). A laboratory colony was maintained on squash (Cucurbita
maxima Duchesne, Cucurbitaceae) under controlled conditions of
temperature (20 2 °C) and photophase (14L:10D). The colony was
initiated with individuals collected on squash plants in organic farms
near Montevideo, and new field-collected individuals have been added
every year.31
B
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a
Scheme 1. Synthesis of the compounds 21−25
a
Reagents and conditions: (a) 4-toluenesulfonyl chloride, DABCO, CH2Cl2, 0 °C; (b) CH3COSK, acetone; (c) PCC, BaCO3, CH2Cl2; (d) C6H5SH,
THF, NaH; (e) NaN3, DMF, 70 °C.
a
Scheme 2. Synthesis of the compounds 26−28
a
Reagents and conditions: (a) phthalic anhydride, Py, DMAP, CH2Cl2; (b) succinic anhydride, Py, CH2Cl2; DMAP.
Pseudaletia adultera Schaus (Lepidoptera: Noctuidae) were reared
RESULTS AND DISCUSSION
■
on an artificial diet32 at 24 1 °C, > 70% relative humidity, with a
The tested cucurbitacins were selected from previous
photoperiod of 16L:8D in a growth chamber. The colony was initiated
studies,26,27,30 and eight new compounds described herein
with adults collected in light-traps placed on wheat crops.
(Schemes 1 and 2) were obtained in order to explore the
Tests of Preference. The compounds were evaluated in choice-
importance of substituents at C-2, together with some
bioassays in Petri dishes (9 cm diameter) completely lined at the
modifications at C-16. In this way, five new derivatives (21−
bottom with a 0.3 mm width layer of agar (2%) to avoid leaf
25) were prepared by nucleophilic substitution25 using the
desiccation. Insects were offered four leaf pieces (1 cm2) of the
tosylate as leaving group, as shown in Scheme 1. Compounds
appropriate host plant [C. maxima for E. paenulata and Hordeum
26 and 27 (Scheme 2) were synthesized by esterification of
vulgare L. (Poaceae) for P. adultera]. Two of the leaf pieces
compounds 1 and 7, respectively, using phthalic anhydride, and
(treatment: T) were coated with 100 μg of the substance (10 μL of
compound 28 was obtained by esterification of 1, using succinic
anhydride. Spectroscopic data of these new compounds are
available in the Supporting Information.
These 28 natural and semisynthetic cucurbitacins were tested
for their antifeedant activity against a specialist on Cucurbita-
ceae (E. paenulata) and a polyphagous species (P. adultera
larvae). All active compounds were deterrents with the
exception of 28, the hemissuccinate ester of 16-oxo-
a 1% MeOH solution), and the other two (control: C) were treated
with 10 μL of MeOH. For E. paenulata, 3−4 day old adults were tested
individually (10 replicates per substance). In the case of P. adultera,
fourth-instar larvae were used (7−10 replicates per extract). Tests with
both insects were run for 180 min or until 75% of one of the options
was consumed. To measure food intake, a visual score of the
consumed area (in one-eighth intervals) was assigned for all leaf pieces
within the plate, and a feeding preference index (PI) was determined
dihydrocucurbitacin B that was phagostimulant to E. paenulata
for each replicate using the formula PI = (C − T)/(C + T), where C
and T are the consumed amounts of the control and treatment leaves
respectively.33,34 (In this manner, PI is greater than 0 when
compounds are deterrent35 and lower than 0 when phagostimulation
occurs.)
Statistical Procedures. Bioassay data were analyzed by Wilcoxon
Rank Tests. The activity on insect feeding was evaluated on the basis
of the percentage of consumed leaf treated with solvent (control)
compared to consumed leaf treated with tested substance (treat-
ment).36 When the results from two samples were compared, a
Mann−Whitney Test on PI was run.36
(Table 1). The natural occurring cucurbitacins were in general
more active as deterrents than the modified compounds. In
particular, the natural cucurbitacins cucurbitacin B (2) and
cucurbitacin R (3) were inactive against both species, and
dihydrocucurbitacin B (1) and the glycoside 5 were very active
against E. paenulata and inactive against P. adultera, while the
reverse was true for 4, a natural cucurbitacin with a terminal
vinyl group, and 6, the 2-O-glycoside of dihydrocucurbitacin B,
which were active against P. adultera and inactive against E.
paenulata. The effect of glycosylation is not clear, although it
should be considered that only two glycosylated compounds
C
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Changes in the oxidation pattern of the main skeleton also
have produced activity shifts. Oxidation of C-16 OH in
dihydrocucurbitacin B (1) yielded compound 7, which lost all
activity against E. paenulata but showed increased deterrence
against P. adultera. On the other hand, the reduction of the
carbonyls at C-3 and C-22 gave compounds with different
species-related activities. Reduction of both carbonyls in
cucurbitacin B (2) yielded compound 10, which was inactive
against E. paenulata but seemingly more active than the parent
compound against P. adultera.
The combined effect of both transformations, acylation and
oxidation, can be observed in compounds 27 and 28, which are
acyl derivatives of 7 (dihydrocucurbitacin B oxidized at C-16).
As in the case of the acyl derivatives of dihydrocucurbitacin B
(1), acylation of 7 yielded derivatives with variable activities.
The introduction of a phthalate group at C-2 (27) in
compound 7 decreased the activity (p < 0.05, Mann−Whitney
test) against E. paenulata. In the case of the dihemisuccinate, 9,
a loss of activity against E. paenulata was observed when
compared to its parent compound dihydrocucurbitacin B (1),
but at the same time an increase in its effect against P. adultera
was obtained. Compound 28 (the hemisuccinate of 7) not only
showed an increase in its deterrent effect against P. adultera but
also the degree of change toward E. paenulata was such that the
effect reverted to phagostimulation. Quite surprisingly, 28 was
the only tested compound that exhibited a phagostimulant
effect on E. paenulata, being at the same time deterrent against
P. adultera.
The derivatives with an enaminone on ring A produced some
interesting results. The enaminone of 1 (compound 15) and its
C-16 acetyl derivative, 16, had almost identical activities as the
parent compound against E. paenulata. Compound 16 was
active against P. adultera, whereas its parent compound was not.
In particular, compound 16 has an acetate group, which in
other derivatives was detrimental. For instance, the dihydrocu-
curbitacin B diacetylated derivative, 11, had lower bioactivity
against E. paenulata than dihydrocucurbitacin B (1); however,
an enaminone group at ring A, 16, instead of an acetate at C-2,
11, gives a product with almost the same activity as 1.
Oxidation of the C-16-OH group gives the enaminone 25 with
reduced bioactivity, suggesting that the enaminone by itself
does not guarantee the deterrent effect, which instead arises
from a combination of an α-hydroxyketone or an enaminone at
ring A and a hydroxyl or acetyl group at C-16. When the
enaminones derived from cucurbitacin B (2) were tested, the
results showed a different pattern. In fact, the enaminone 25
was inactive against E. paenulata while considerably active
against P. adultera.
As a whole, these results were unexpected in that
phagostimulation by natural cucurbitacins toward E. paenulata
was not detected as predictable if these chemicals were token
stimuli.7 The deterrent effect on this Cucurbitaceae specialist
may be due to a dose-related effect. To clarify this issue, more
studies will be carried out. However, it is also possible that
deterrence is an ecological significant effect as it has been
previously documented for other Epilachna species which, as a
matter of fact, perform a trenching behavior to avoid
cucurbitacins produced after plant damage as induced
defenses.5,6 On the other hand, deterrence against the
polyphagous P. adultera was found as expected. Finally, an
important finding from these results is the trend by which when
chemical modifications correlated to changes in activity, those
changes were opposite for both insects, that is, a chemical more
Table 1. Preference Indexes (PI as Means Standard Error)
of E. paenulata and P. adultera
cucurbitacin
P. adultera larvae
E. paenulata adults
a
1
0.1 0.2
0.2 0.2
0.7 0.2*
2
0.4 0.3
0.3 0.2
0.2 0.3
3
−0.2 0.2
0.2 0.1*
0.0 0.2
a
4
a
5
0.8 0.2*
a
6
0.91 0.04*
0.3 0.1*
0.1 0.2
0.2 0.3
0.3 0.3
NT
a
7
8
9
−0.1 0.2
0.65 0.06*
0.2 0.2
0.1 0.2
0.1 0.4
0.1 0.4
0.3 0.4
0.2 0.3
0.2 0.4
0.2 0.2
0.6 0.3
a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
−0.1 0.1
0.19 0.09*
0.0 0.1
a
a
b
0.32 0.09*
0.0 0.2
0.7 0.2**
0.2 0.3*
0.4 0.3
0.1 0.2
a
0.09 0.09
a
0.6 0.1*
−0.01 0.08
0.0 0.1
a
0.5 0.1*
0.4 0.3
0.1 0.3
−0.1 0.3
0.3 0.3
0.0 0.2
−0.1 0.2
0.0 0.1
0.3 0.2
a
0.6 0.1*
0.3 0.1*
06 0.1*
0.8 0.1*
a
a
0.85 0.08*
0.2 0.3
−0.6 0.3*,**
a
a
a,c
a
b
*P < 0.05 (2-tailed). **P < 0.05 (1-tailed) by Wilcoxon Rank tests.
NT: not tested. Phagostimulant.
c
were tested. Compound 6 was inactive against E. paenulata but
on the other hand was very active against P. adultera. The other
C-2 glycoside, 5, was almost as active as compound 1 against E.
paenulata, but in this case, ring A is aromatic, so it is unclear
which feature, or a combination thereof, is responsible for the
bioactivity.
Variations of the side chain of dihydrocucurbitacin B (1)
[unsaturation at C-23 (2), deacetylation at C-25-OH (3), and
unsaturation at C-25−C-26 (4)] led to a loss of activity against
the specialist E. paenulata, suggesting that the side chain of 1
should remain intact. However, in the case of the polyphagous
P. adultera the presence of the Δ25,26 bond in 4 led to a weak
increase of deterrence. In the same direction, a side chain
shortening (compounds 8 and 9) of the very active diol
derivative of cucurbitacin B (10) led to a complete loss of
activity against P. adultera. The diesterification of C-2 and C-16
hydroxyls of dihydrocucurbitacin B (1) gave results that were
variable depending on the acylating group. The diacetate 11,
the dihemisuccinate 14, and the derivatives with an acetate at
C-16 and a thiophenyl group (19) or a thioacetate at C-2 (18)
lost all the deterrent activity against E. paenulata exhibited by
the parent compound 1. Interestingly, 19 was considerably
more active than parent compound 1 against P. adultera. A
similar trend was observed with cucurbitacin B (2): the
diacetate 12 seems to be less active than the parent compound.
On the other hand, the diphtalate of dihydrocucurbitacin B
(26) was more active than 1 not only against E. paenulata but
also against P. adultera.
D
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active against the specialist becomes less active against the
generalist insect in its capacity to deter feeding.
active against P. adultera while inactive against the specialist
insect. Once again, these seemingly contrasting changes in
activity are indeed a required feature when seeking for potential
pest control agents with selectivity. These results stimulate the
preparation of additional derivatives in order to explore further
changes in the structure of ring A, as well as to investigate the
effect of glycosylation on the insect feeding activity.
Considering the compounds here investigated, the most
active were, in general, the natural cucurbitacins, but some of
the semisynthetic derivatives were as active as the parent
compounds. Another general trend arises from the fact that
more active compounds were found against the polyphagous
insect than against the specialist (12 vs 7) (Table 1). This trend
is opposite to that most generally observed, where specialists
are usually more sensitive than generalists to plant chemicals
not usually found in their normal diet due to some inability for
specialists to physiologically adapt to a different chemical
profile from the one they usually encounter.37 However,
opposite patterns have also been described previously.34,38
The results show that the cucurbitane skeleton by itself does
not ensure activity because almost half of the tested substances
were inactive. Another general observation is that the activity is
very different against both tested species and that it was
deterrent at the tested concentration in all cases with the
exception of cucurbitacin 28. The activity is also markedly
influenced by the structure modifications, as it was in our
previous study on cytotoxic activity,27 although a pattern of
correlations between both activities was not found. However,
some general trends could be observed by comparison of
structurally related compounds in this series, which can be
useful for the design of a more active compound.
ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation of compounds 21−28. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +5989242535. Fax: +5989241906. E-mail: crossini@
fq.edu.uy.
Funding
This work was jointly supported by a Fondo Clemente Estable
grant from Agencia Nacional de Investigacion e Innovacion,
́ ́
Uruguay (grant no. FCE2009_1 3107), Brazilian funding
agencies CNPq/MCTI and PRONEX/FAPESC (grant no.
2671/2012-9), and Argentinean funding agencies: UBACyT
2010−2012 (grant no. 20020090200143).
Notes
The side chain structure of dihydrocucurbitacin B (1) is a
common feature in most of the active compounds, not only
among the natural substances but also in the semisynthetic
derivatives as well. In this regard, an additional conclusion is
that the observed activity is a combination of all the structural
features of the side chain because a single modification on any
of them produces a loss of activity, indicating that the side chain
of 1 should remain intact in the design of a more active
derivative.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
́
We thank Valeria Cal (Laboratorio de Ecologia Quimica) for
help with maintenance of the P. adultera colony and Horacio
Silva (Facultad de Agronomia, Universidad de la Republica,
́
́
EEMAC, Paysandu) for field collection of insects to start this
́
colony.
Among the semisynthetic derivatives, only a few could match
dihydrocucurbitacin B in terms of bioactivity against E.
paenulata. The modifications performed on rings A and D of
the cucurbitane skeleton gave results that in some cases were
more difficult to rationalize. Diesterification at C-2 and C-16
gave interesting results: only the diphthalate 26 was active
against both insects, while the remaining diester derivatives
show a tendency by which this chemical modification decreases
the activity against E. paenulata and at the same time increases
the activity against P. adultera. At the same time, oxidation at C-
16 gave the same trend in selectivity (7, 27, 28), including
compound 28, which has an hemisuccinate group at C-2 and
drastically changed the sense of activity, becoming a
phagostimulant for E. paenulata. This kind of modification in
activity illustrates the potential of chemical modification to
produce compounds with selectivity, a fundamental goal when
developing pest control agents. The fact that compound 28 was
considerably more active than the dihydrocucurbitacin B
dihemisuccinate, 14, suggests that the observed activity may
arise by a combination of structural features at C-2 and C-16.
These results also suggest that it would be worth to test the
effect of substituents with greater lipophilicity at C-2 and C-16.
The effect of an enaminone on ring A (instead of the α-
hydroxy-ketone) is also species-dependent. In the case of
dihydrocucurbitacin B, the enaminones were active against E.
paenulata but considerably less active or definitely inactive
against P. adultera. However, the enaminone obtained from
cucurbitacin B with further oxidation at C-16 (27) was very
ABBREVIATIONS USED
■
PI, feeding preference index
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