Growth-inhibitory activities of benzofuran and chromene derivatives toward Tenebrio molitor

Article abstract of DOI:10.1021/np9800248

Growth-inhibitory activities of selected natural benzofurans (4-9), trans-cinnamic acid derivatives (10-13), chromene compounds (14 and 16), and some semisynthetic derivatives were determined in last instar larvae of Tenebrio molitor via topical administr

Full text of DOI:10.1021/np9800248

J . Nat. Prod. 1998, 61, 1209-1211
1209
Gr ow th -In h ibitor y Activities of Ben zofu r a n a n d Ch r om en e Der iva tives tow a r d
Ten ebr io m olitor
†
‡
†
§
†
Roberto Carrizo F., Marta E. Sosa, Laura S. Favier, Fabricio Penna, Eduardo Guerreiro,
†
,†
Oscar S. Giordano, and Carlos E. Tonn*
Facultad de Qu ´ı mica, Bioqu ı´ mica y Farmacia, Universidad Nacional de San Luis,
Chacabuco y Pedernera, 5700, San Luis, Argentina
Received J anuary 29, 1998
Growth-inhibitory activities of selected natural benzofurans (4-9), trans-cinnamic acid derivatives (10-
3), chromene compounds (14 and 16), and some semisynthetic derivatives were determined in last instar
larvae of Tenebrio molitor via topical administration in Me CO. The most inhibitory of the tested
1
2
compounds were 3-γ,γ-dimethylallyl-p-coumaric acid (10) and the benzofuran derivative 12-(p-cumaroy-
loxy)-tremetone (5), the former compound acting on the pupae and the latter on the last instar larvae.
Several developmental deficiencies were observed, and some structure-activity relationships are discussed.
Benzopyrans, benzofurans, prenylated phenols, and ac-
etophenone derivatives are present in many species of
higher plants, particularly in the Asteraceae.1 In this
regard, precocene I (1) and precocene II (2), a special type
of chromene lacking the methyl ketone group, were isolated
Ta ble 1. Growth Inhibition Activity of Compounds 4-18
toward T. molitor Last Instar Larvae
percent successfully
duration ofc
pupal stage
compounda
pupating
b
d
10.5 (2.1)e
4
5
6
7
8
9
1
1
12
1
1
1
16
1
1
83.0 (3.1)
26.0 (0.8)
75.0 (6.5)
75.0 (7.8)
73.3 (2.1)
73.0 (8.1)
63.0 (3.4)
50.0 (8.2)
87.0 (3.4)
92.0 (5.6)
77.0 (6.4)
87.0 (7.1)
66.6 (1.7)
87.0 (1.3)
75.0 (3.8)
f
10.5 (1.0)e
3
0 years ago, and their influence on the metamorphosis of
f
g
9.8 (2.2)
8.6 (0.5)
11.2 (2.3)
11.6 (2.1)
certain sensitive insect species, which results in precocious
molting, has been intensively studied.2 The acetyl-
chromene encecalin (3) has been reported as the principal
chemical defense of the genus Encelia (Asteraceae) against
herbivorous insects3 and over the past 10 years, complete
investigations of the metabolic fate of encecalin adminis-
tered to insects have been carried out.6
f
g
f
f
f
f
f
f
0
1
13.0 (1.3)
f
e
10.2 (1.3)
-5
g
10.0 (2.1)e
g
g
3
4
5
8.7 (1.9)
f
10.2 (0.9)e
-8
g
g
7.0 (1.4)
11.5 (2.1)
12.1 (2.4)
11.4 (2.1)
f
f
Recently, it has been reported that the chromene homo-
logue methyl 4-hydroxy-3-(3′-methyl-2′-butenyl) benzoate
from Piper guanacastensis (Piperaceae) is an important
active constituent toward Aedes atropalpus L. (Diptera:
Culicidae).9
The yellow mealworm Tenebrio molitor L. (Coleoptera:
Tenebrionidae) has a high reproductive capacity and at-
tacks several classes of stored grains and flour, making this
insect a major pest. Identification of new natural products
active against insects enhances our understanding of the
chemical defense mechanisms of plants. As part of a study
g
f
7
8
f
f
control
93.0 (6.1)
8.6 (1.6)
a
b
Dose: 120 µg/larvae; topically applied. The values are mean
(SE); n ) 60. Data treated by ø2 test. c The values, in days, are
d,e,f
mean (SE); n ) 60. Data treated by Dunnet test.
Difference
significant (p < 0.05, p < 0.01 and 0.001, respectively) from control
2
g
by ø test or Dunnet test. No significant difference.
of the interaction between plant secondary metabolites and
this insect at the larvae stage,1
0-13
we report here the
effects of selected natural compounds, previously isolated
from species that grow in the central-western semiarid area
of Argentina, and semisynthetic aromatic derivatives of
these.
T. molitor has a variable number of larval instars and,
therefore, selection of larvae by weight is necessary. It has
been reported that larvae weighing between 120 and 140
mg belong to the last instar,14 and for our experiments we
used larvae weighing between 113 and 160 mg. In normal
1
4
development, the pupal stage takes about 9 days, and this
period was determined to be 8.6 ( 1.6 days in our controls
F igu r e 1. Regression graph of dose/response for compound 5.
(see Table 1).
To analyze structure-activity relationships of the as-
Insects were treated topically, via the abdomen, with the
test compounds dissolved in acetone. A single dose of 120
µg/larva was applied. The dosage value was chosen taking
into account preliminary results obtained using 5. This
tremetone cumarate showed both inhibition of successful
pupation as well as a significant increase in the pupal stage
duration at this dosage (Table 1). Results of the dosage
increase of 5 are summarized in Figure 1. The percentage
of larvae that reached the pupae instar diminished in
sayed compounds they were divided into three main groups;
namely, benzofuran derivatives (4-9), trans-cinnamic acid
derivatives (10-13), and benzopyran derivatives (14-17).
*
To whom correspondence should be addressed. Tel.: 54 652 23789.
Fax: 54 652 30224. E-mail: ctonn@unsl.edu.ar.
†
INTEQUI-CONICET.
C a´ tedra de Entomolog ´ı a.
Bioestad ´ı stica.
‡
§
1
0.1021/np9800248 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/19/1998

1
210 J ournal of Natural Products, 1998, Vol. 61, No. 10
Carrizo et al.
dependence to log dose in the regression analysis (n ) 20
larvae each point), and from these data the DE50 ) 63.5
µg/larva was calculated. To ensure reaching the toxic dose,
we used nearly two-fold the DE50 of the higher molecular
weight compound 5 for all the assayed compounds. In the
group of benzofuran derivatives (4-9), all of the compounds
showed significant effects on the ratio of larvae pupating
at the end-point of the experiment (30 days), but the most
important activity was observed for compound 5. In this
case, only 26% of the treated larvae reached the pupal
stage, with the rest typically dead in the course of the first
distal parts of the abdomen, and there were deformities of
the elytra. Finally, compounds 4, 5, 8, and 9 showed
significant differences from controls in the duration of the
pupal instar with prolongation of this stage.
It has been reported that benzofurans are not toxic to
other insects species; however, they antagonize the toxicity
of the chromene encecalin (3).7 In our study using similar
dose:insect weight ratios (0.33 µmol/insect for compound
5), the benzofurans were toxic by themselves toward T.
molitor. Recently it was noted that 2-(1′-oxo-5′-methyl-4′-
hexenyl)benzofuran derivatives show toxicity to spruce
budworm, Christoneura fumiferana Clem., larvae at the
level of 0.8 µmol per insect.15 Presumably, as in compound
1
0 days of the test.
5
, the bulky group at C-2 is important for the biological
response.
In the second group of compounds (trans-cinnamic acid
derivatives), 10-13, the simple and ubiquitous acids 12
and 13 were essentially inactive; on the other hand, the
3
-γ,γ-dimethylallyl-p-coumaric acid (10) showed both ac-
tivities, toxic on the last instar larvae as well as being the
most active on the pupal stage. Of particular interest in
this set of products was the change in activity of 10 when
hydrogenated to 11. The hydrogenated acid-phenolic 11
weakly affected the duration of the pupal instar but showed
enhanced larval toxicity.
Bearing in mind the activity of 10, we then prepared
derivatives 14 and 15. The first was obtained from 10 by
a cyclization reaction with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ)16 and the second via a halo-cyclization
reaction to give the 3-bromine derivative of 15 (epimeric
mixture), by way of the thermodynamically preferred ring
closure, followed by hydride reduction.17 This series of
reactions was adopted in order to preserve the carboxylic
unsaturated side chain. The semisynthetic compounds 14
and 15 belong in the benzopyran series (14-17).
In this group of products, both acetylbenzopyrans (16 and
1
1
7) were active at the pupal stage, but only the chromene
6 (demethylencecalin) was significantly toxic to the larvae.
It is noteworthy that a C-3-C-4 double bond in the pyrane
7
ring is essential for the toxic or antihormonal activity. This
is the site of attack for mixed function oxidases that
epoxidize the double bond leading to a more soluble trans-
3
,4-diol. On the other hand, the highly reactive benzylic
position of the oxirane ring can be attacked by nucleophilic
functional groups, for example, the thiol function of en-
zymes quenching the enzyme activity.8 The absence of
heterocycle unsaturation in the chroman 15 could be the
reason for lack of activity.
In about 3% of the larvae treated with compounds 16-
8 we found multiple morphological abnormalities. There
1
were adultoid insects showing tarsi and antennae partly
unsegmented (16); severe deficiencies in the process of new
cuticle deposition (17), and adultoid insects enclosed in
pupal exuviae but showing mobile appendages (18). None
of the control larvae showed any of these macroscopic
defects in their development.
This report extends previous studies demonstrating that
T. molitor L. is an insect sensitive to plant natural
products.1
0-12
In this case some acetophenone derivatives
The presence of a bulky aromatic ester group on the side
chain at C-2 could be the key functionality for activity. In
compounds 6 and 7, where the p-cumaroyloxy group had
been replaced by an acetoxyl or hydroxyl group, respec-
tively, this dramatic effect was not observed; however, 25%
of the larvae died without pupation. Similar results were
achieved with the 6-hydroxy analogues 8 and 9 and
euparine (4). Another interesting observation made with
compound 8 was that with two replicates, two morphologi-
cally normal adults had fragments of pupal exuviae on the
and compounds with the chromene skeleton were active
as insect-growth regulators. It would be desirable to
extend this investigation to other insect species before
drawing more general conclusions.
Exp er im en ta l Section
1
Gen er a l Exp er im en ta l P r oced u r es. H NMR spectra
(ppm, δ) were recorded in CDCl3 at 200.13 MHz, 13C NMR
spectra were obtained at 50.23 MHz; COSY, XH-CORR, and

Growth-Inhibitory Activity of Benzofurans
J ournal of Natural Products, 1998, Vol. 61, No. 10 1211
COLOC experiments were obtained using standard Bruker
software. Column chromatography was performed on Si gel
G 70-230 mesh and Kieselgel 60 H using mixtures of n-hex-
ane-EtOAc, in increasing polarities, as solvent. TLC was
carried out on Si gel 60 F254 0.2-thick plates using C H -1,4-
6 6
dioxane-AcOH, 30:5:1 (v:v:v) as solvent.
(C-12), 114.5 (C-8), 75.0 (C-2), 51.5 (OMe), 32.5 (C-3), 26.8 (C-
14 and C-15), 22.3 (C-4).
Bioa ssa ys. Bioassays were performed with last instar
larvae of T. molitor based on live weight (113-160 mg). For
each compound, test solutions (in Me
applied to ventral abdominal segments with a microsyringe
2 µL/larva; equivalent to 120 µg of the assayed compound).
2
CO) were topically
In sect. A stock culture of Tenebrio molitor L. (Coleoptera:
(
Tenebrionidae) larvae was maintained on wheat bran in plastic
Controls were treated with the solvent alone. For each
individual compound there were two replicates of 10 larvae
each, and the study was repeated three times. After treatment
insects were placed individually in Petri dishes (5 cm diameter)
1
0
boxes at 24 ( 1° with a 16:8 (L:D) photoperiod. A voucher
specimen is deposited at the C a´ tedra de Entomolog ´ı a, Facultad
de Ciencias Agrarias, Universidad Nacional de Cuyo, Mendoza,
Argentina.
and held at 24 ( 1° with a 16:8 (L:D) photoperiod.
A
Test Com p ou n d s. Euparin (4) was isolated from leaves
of Flourensia oolepis (Compositae), as previously reported.18
From the aerial parts of Parastrephya lepidophylla (Wedd)
Cabr., 12-(p-cumaroyloxy)-tremetone (5) and 12-acetyloxy-
moistened piece of cotton was used to preserve humidity.
Insects were not fed during this assay. The number of larvae
that successfully pupated as well as the duration of the pupal
stage (in days) were recorded every 24 h for 30 days (end-point
of the experiment). Results are shown in Table 1.
1
9
tremetone (6) were isolated. The derivative 7 was prepared
from 6 by KOH-MeOH hydrolysis followed by acidification
with dilute HCl and extraction with Et
2
O. The known
Sta tistica l An a lysis. Data of the percent of larvae suc-
2
compounds 8, 9, and 16-18 were isolated from aerial parts of
cessfully pupating was treated by ø . Prior to statistical
2
0
Ophryosporus axilliflorus (Griseb) Hieron. From Baccharis
grisebachii Hyeronimus, 3-γ,γ-dimethylallyl-p-coumaric acid
analysis of the duration of pupal stage, it was proven, on the
basis of the Shapiro-Wilk test, that the patterns come from a
population with approximately normal distribution. The
homogeneity of variances was determined by the Bartlett test.
The mean length of pupal stage (in days) for the different
2
1
(
10) and 12 were obtained. Compounds 11, 14, and 15 were
prepared from 10 as described below. Compound 13 was a
commercial sample.
P r ep a r a tion of 11 fr om 10. A solution of 10 (100 mg, 0.42
23
treatments was analyzed by using the Dunnet test.
mmol) in EtOAc (30 mL) was stirred under H
2
at 30 °C
overnight in the presence of Pd 5% on activated carbon. The
catalyst was removed by filtering through Celite, and the
filtrate was evaporated to give 95 mg of the pure 11 as an oil:
Ack n ow led gm en t. We thank CONICET (PIP 5030) and
UNSL (Project 7301) for financial support. This paper is a part
of the Doctoral thesis of M.E.S.
1
H NMR (CDCl
1H,d, J ) 10.8 Hz, H-5), 6.67 (1H, dd, J ) 10.8, 2.5 Hz, H-6),
.90 (2H, t, J ) 6.0 Hz, H-13), 2.61 (4H, m, H-7 and H-12),
.58 (2H, m, H-8), 1.50 (1H, m, H-9), 0.93 (6H, d, J ) 5.4 Hz,
, 50.23 MHz) δ 179.1 (C-14),
51.8 (C-4), 132.1 (C-1), 129.8 (C-2), 129.1 (C-3), 126.4 (C-6),
15.3 (C-5), 38.9 (C-13), 35.9 (C-7), 29.8 (C-12), 27.8 (C-9), 27.7
3
, 200.13 MHz) δ 6.94 (1H, br s, H-2), 6.88
(
2
1
Refer en ces a n d Notes
1
3
H-10 and H-11); C NMR (CDCl
1
1
3
(1) Proksch, P.; Rodriguez, E. Phytochemistry 1983, 22, 2335-2348.
(2) Bowers, W. S. In Insecticide Mode of Action; Coats, J . R., Ed.;
Academic: New York, 1982; pp 403-427.
3) Isman, M. B.; Proksch, P. Phytochemistry 1985, 24, 1949-1951.
4) Proksch, P.; Rodriguez, E. Biochem. Syst. Ecol. 1984, 12, 179-181.
(
(
(C-8), 22.4 (C-10 and C-11).
P r ep a r a tion of 14 fr om 10. To a stirring solution of
(5) Isman, M. B.; Proksch, P.; Clark, C. In Biochemistry of the Mevalonic
Acid Pathway of Terpenoids; Towers, G. N. H., Stafford, H. A., Eds.;
Plenum: New York, 1990; pp 249-264.
compound 10 (100 mg, 0.42 mmol) in 10 mL of 1,4-dioxane-
(1:1) at room temperature, under N , was added DDQ
290 mg, 1.26 mmol). After 1 day in darkness, the solvent
was evaporated under reduced pressure and the residue
dissolved in Et O. The organic solution was washed with brine
until the aqueous phase became colorless, dried (Na SO ), and
C
(
6
H
6
2
(
6) Isman, M. B.; Proksch, P.; Witte, L. Arch. Insect. Biochem. Physiol.
987, 6, 109-120.
1
6
1
(
7) Isman, M. B. In Insecticides of Plant Origin; Arnason, J . T., Philog e` ne,
B. J . R., Morand, P., Eds.; ACS Symposium Series 387: Washington,
DC, 1989; pp 44-58.
2
2
4
(
8) Kunze, A.; Aregulin, M.; Rodriguez, E.; Proksch, P. J . Chem. Ecol.
concentrated under reduced pressure. After column chroma-
1
996, 22, 491-498.
tography purification, 80 mg of 14, identical in all respects
(
9) Pereda-Miranda, R.; Bernard, C. B.; Durst, T.; Arnason, J . T.;
S a´ nchez-Vindas, P.; Poveda, L.; San Rom a´ n, L. J . Nat. Prod. 1997,
60, 282-284.
2
2
with previous reports, were recovered.
P r ep a r a tion of 15 fr om 10. The halo-cyclization reaction
was carried out under N . The methyl ester of 10, 400 mg
1.6 mmol), was prepared in the usual way with CH -Et
(10) Sosa, M. E.; Tonn, C. E.; Giordano, O. S. J . Nat. Prod. 1994, 57, 1262-
265.
2
1
(
2
N
2
2
O
(
11) Enriz, R. D.; Baldoni, H. A.; J a´ uregui, E. A.; Sosa, M. E.; Tonn, C.
E.; Giordano, O. S. J . Agric. Food Chem. 1994, 42, 2958-2963.
12) Sosa, M. E.; Tonn, C. E.; Guerreiro, E.; Giordano, O. S. Rev. Soc.
Entomol. Argentina 1995, 54, 45-48.
(13) Ce n˜ al, J . P.; Giordano, O. S.; Rossomando, P. C.; Tonn, C. E. J . Nat.
Prod. 1997, 60, 490-492.
and dissolved with stirring in 5 mL of dry MeCN at -20 °C.
After 15 min, 660 mg (1.6 mmol) of 2,4,4,6-tetrabromo-2,5-
cyclohexadienone, suspended in 10 mL of the same solvent,
(
1
7
was added. The heterogeneous reaction was stirred for 3 h,
until starting material was not detectable by TLC, and was
allowed to warm to 30 °C. The reaction was quenched by
(
(
14) Quennedey, A.; Quennedey, B. Tissue Cell 1993, 25, 219-236.
15) Findlay, J . A.; Buthelezi, S.; Li, S.; Sevech, M.; Miller, J . D. J . Nat.
Prod. 1997, 60, 1214-1215.
2
addition of brine and extracted twice with Et O. The combined
organic layers were washed, dried, and concentrated. After
flash chromatographic purification, 350 mg of the epimeric
(
16) Schmitt, A.; Telikepalli, H.; Mitscher, L. Phytochemistry 1991, 30,
3
569-3570.
3
-bromine derivative of 15 was obtained. The dehalogenation
reaction was carried out by refluxing the mixture in C with
addition of n-Bu SnH (2.6 eq) and a catalytic amount of 2,2′-
(17) Tonn, C. E.; Palaz o´ n, J . M.; Ruiz-P e´ rez, C.; Rodriguez, M. L.; Mart ı´ n,
6
H
6
V. S. Tetrahedron Lett. 1988, 29, 3149-3152.
18) Guerreiro, E.; Kavka, J .; Giordano, O. S.; Gros, E. G. Phytochemistry
(
(
(
3
1
979, 18, 1235-1237.
19) Nieto, M.; Gianello, J . C.; Tonn, C. E. Anales Asoc. Qu ı´ m. Argentina
994, 82, 105-109.
20) Favier, L. S.; Guerreiro, E.; Tonn, C. E. Unpublished results.
azobis(2-methylpropionitrile) for 3 h. After the usual work up
and separation by column chromatography, 120 mg of 15 was
obtained (oil): IR νmax 1710, 1632, 1601, 1440, 1250, 1068, 620
1
-
1
1
cm ; H NMR (CDCl
Hz, H-11), 7.32 (1H, dd, J ) 9.0, 2.2 Hz, H-7), 7.25 (1H, d, J
2.2 Hz, H-5), 6.79 (1H, d, J ) 9.0 Hz, H-8), 6.29 (1H, d, J )
5.5 Hz, H-12), 3.80 (3H, s, OMe), 2.79 (2H, t, J ) 6.6 Hz,
3
, 200.13 MHz) δ 7.62 (1H, d, J ) 15.5
(21) Gianello, J . C.; Giordano, O. S. Anales Asoc. Qu ı´ m. Argentina 1987,
75, 1-3.
22) Labbe, C.; Rovirosa, J .; Faini, F.; Mahu, N.; San Mart ´ı n, A.; Castillo,
M. J . Nat. Prod. 1986, 49, 517-518.
23) Sokal, R. R.; Rohlf, F. J . In Biometr ı´ a; H. Blume: Madrid, 1979; pp
(
)
1
(
H-4), 1.82 (2H, t, J ) 6.6 Hz, H-3), 1.34 (6H, s, H-14 and H-15);
C NMR (CDCl
C-11), 129.9 (C-7), 127.3 (C-5), 126.1 (C-10), 121.2 (C-6), 117.8
1
69, 273, 408.
1
3
3
, 50.23 MHz) δ 173.0 (C-13), 156.3 (C-9), 144.9
(
NP9800248