Comparative study on the larvicidal activity of drimane sesquiterpenes and nordrimane compounds against drosophila melanogaster til-til

Article abstract of DOI:10.3390/molecules18044192

Natural compounds from Drimys winteri Forst and derivatives exhibited larvicidal effects against Drosophila melanogaster til-til. The most active compound was isodrimenin (4). The highest lethal concentration to the larvae of D. melanogaster was 4.5 ± 0.8 mg/L. At very low concentrations drimenol (1), confertifolin (3), and drimanol (5) displayed antifeedant and larvae growth regulatory activity. The antifeedant results of nordrimanic and drimanic compounds were better in first instar larvae. The EC₅₀ value of polygodial (2) was 60.0 ± 4.2 mg/L; of diol 15 45.0 ± 2.8 mg/L, and of diol 17 36.9 ± 3.7 mg/L, while the new nordrimane compound 12 presented a value of 83.2 ± 3.5 mg/L.

Full text of DOI:10.3390/molecules18044192

Molecules 2013, 18, 4192-4208; doi:10.3390/molecules18044192
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Comparative Study on the Larvicidal Activity of Drimane
Sesquiterpenes and Nordrimane Compounds against
Drosophila melanogaster til-til
1
,
2
2,
1
1
Ivan Montenegro *, Luis Pino , Enrique Werner *, Alejandro Madrid , Luis Espinoza ,
2
3
4,
Luis Moreno , Joan Villena and Mauricio Cuellar *
1
Departamento de Química, Universidad Técnica Federico Santa María, Av. España N° 1680,
Valparaíso 2340000, Chile; E-Mails: alejandro.madrid@usm.cl (A.M.); luis.espinozac@usm.cl (L.E.)
2
Departamento de Ciencias Básicas, Universidad del Biobío, Campus Fernando May,
Avda. Andrés Bello s/n casilla 447, Chillán 3780000, Chile;
E-Mails: luisandrespinocastro@hotmail.com (L.P.); lmoreno@ubiobio.cl (L.M.)
3
Escuela de Medicina, Centro de Investigaciones Biomédicas (CIB), Universidad de Valparaíso,
Av. Hontaneda N° 2664, Valparaíso 234000, Chile; E-Mail: juan.villena@uv.cl
4
Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña N° 1093,
Valparaíso 234000, Chile
*
Authors to whom correspondence should be addressed;
E-Mails: ivan.montenegro@postgrado.usm.cl (I.M.); ewerner@ubiobio.cl (E.W.);
mauricio.cuellar@uv.cl (M.C.); Tel.: +56-032-250-8193 (I.M).
Received: 16 February 2013; in revised form: 7 March 2013 / Accepted: 1 April 2013 /
Published: 10 April 2013
Abstract: Natural compounds from Drimys winteri Forst and derivatives exhibited
larvicidal effects against Drosophila melanogaster til-til. The most active compound was
isodrimenin (4). The highest lethal concentration to the larvae of D. melanogaster was
4.5 ± 0.8 mg/L. At very low concentrations drimenol (1), confertifolin (3), and drimanol (5)
displayed antifeedant and larvae growth regulatory activity. The antifeedant results of
nordrimanic and drimanic compounds were better in first instar larvae. The EC value of
50
polygodial (2) was 60.0 ± 4.2 mg/L; of diol 15 45.0 ± 2.8 mg/L, and of diol 17 36.9 ± 3.7 mg/L,
while the new nordrimane compound 12 presented a value of 83.2 ± 3.5 mg/L.
Keywords: antifeedant activity; Drosophila melanogaster til-til; nordrimanes; drimanes;
sesquiterpenes

Molecules 2013, 18
. Introduction
Droshophila melanogaster is a yellow-red dipterous (with two wings) 3–5 mm in size insect,
4193
1
usually with red eyes, also known as the vinegar fly or fruit fly. Its development cycle is very fast, and
is favored by access to sugary liquids. The entire life cycle lasts between 9 and 20 days, and in summer
it is approximately 7 to 8 days. The females come to lay 700 to 800 eggs during their lifetime. The
egg-laying takes place on wounded fruits, and liquids like juices, vinegar, wine, beer and soft drinks.
The larvae are tiny worms, whitish in appearance, that can be found inside the damaged fruit. The
main damage caused by this insect is due to its ability to transmit diseases such as sour rot,
recognizable by the strong vinegar smell emit by contaminated items. The til-til strain proved to be a
new species of Drosophila belonging to the funebris group [1–3].
Since the discovery of drimenol (1), the first drimane sesquiterpenoid, in Drimys winteri f. bark by
Appel on 1948 [4], hemisynthetic derivatives of this natural compound have been prepared. At a
biological level, drimenol has a similar function to the heteroauxin indoleacetic acid, a growth
promoting hormone occurring in some plants [5]. However, its most important feature from the
organic synthesis viewpoint is its utility as a starting compound for the preparation of biologically
active drimanes and nordrimanes. Among the biological activities of drimanic compounds are their
antimicrobial, antifungal, growth inhibitors, molluscicide and gastroprotective properties [6–9]. For
example, ugandensidial is a strong antifungal and warbunganal, another drimane dialdehyde naturally
found in Warburgia ugandensis, is used in Africa for its various properties, which include antiallergy,
antibacterial, antifungal and antifeedant activities, and can be synthesized using drimenol (1) as
starting material [5,10,11].
2
. Results and Discussion
2
.1. Chemistry
The primary defence compound in P. colorata leaves is known—it is the sesquiterpene dialdehyde
polygodial (2) which imparts a pungent taste and has potent insect antifeedant properties [12]. In vitro
studies have shown polygodial was an effective antifeedant at concentrations of 3 mg/g [13]. In this
paper, we report the synthesis of two new nordrimanes and five known nordrimanes from drimenol (1)
which were then compared with eleven sesquiterpene compounds for activity against Drosophila
melanogaster til-til. The compounds 5–10 and 13–14 were synthesized from drimenol (1) using
standard methods. The new epoxides 11 and 12 were synthesized from 6 and 7, respectively. This was
performed through the m-CPBA epoxidation protocol producing a 50% yield for 11 and a 70% yield
for 12, respectively (Scheme 1).
The catalytic hydrogenation of the double endocyclic bond of compound 8 was carried out using H
2
and Pd/C as previously described for other alkenes [14]. This takes place side via the less sterically
hindered α-face, to give 13 in 90% yield (Scheme 1). The methyl group is positioned on the β face. By
treating 13 with sodium methoxide in methanol, an epimerization of the CH
3
group occurs, as
evidenced by the fact that the axial position changed to equatorial giving ketone 14. By comparing the
13
C-NMR spectra of 13 and 14, it can be seen that carbonyl signal shift changed from 220.5 to 216.7 ppm,
respectively. All the compounds were characterized by their IR, NMR and MS spectral data.

Molecules 2013, 18
Scheme 1. Synthesis of drimane and nordrimane derivatives of drimenol (1).
4194
OH
H
OAc
5
O
14
a
OH
1
9
6
11
e
2
3
10
8
11
b
5
15
f
4
O
1
9
6
12
O
H
H
2
3
10
8
H
12
13
5
7
6
11
10
4
OH
e
c
H
O
13
14
1
e
O
d
OAc
OAc
H
H
H
9
7
12
O
O
O
g
h
H
H
H
8
13
14
2 2 2 2 2
Reagents and Conditions: (a) H , Pd/C N , r.t, 12 h, 5; 75%. (b) PCC, CH Cl , N , r.t, 2 h, 6; 75%.
(
c) Pb(OAc) , benzene, reflux,7; 60%. (d) Jones’ reagent, 0 °C, 1 h, 8; 55%, 15; 55.1%. (e) m-CPBA,
4
CH Cl , r.t, 1.5 h, 9; 70%, 11; 50% and 12; 70%. (f) Ac O, DMAP, CH Cl , r.t, 2 h, 10; 95%.
2
2
2
2
2
(
g) H (30 psi), Pd/C, N , r.t, 12 h, 13; 90%. (h) MeONa, MeOH, N , r.t, 2 h, 14; 93%.
2 2 2
The identities of the other compounds 5–10 and 13,14 were established by comparison with the
previously reported spectral data for the same compounds [15–21]. Epoxidation of 6 with
m-chloroperbenzoic acid produced a single epoxide 11 in 70% yield. This reaction was performed
1
under standard epoxidation conditions. The structure was determined by the H-NMR spectrum, where
a shift in the H-9 signal from δ = 6.22 to δ = 3.35 ppm was observed [22].
The assignment of the configuration in C-8 and C-9 for compound 11 was proposed on the basis of
1
NOE-spectra observations. From a H-1D sel. gs-NOESY experiment, when CH
3
-11 was selectively
irradiated only long-range interactions with H-9, H-1 and H-5 were observed. In addition, no long-range
interactions between H-9 with the CH -14 were observed. Thereby this NMR experiment confirms the
spatial orientation of the epoxide ring and the stereochemistry of C-8 and C-9, according to Figure 1.
3


Molecules 2013, 18
4195
1
1
Figure 1. Bottom: H-NMR spectrum of compound 11. Top: 1D H sel. gs NOESY
experiment (selnogp pulse program from Bruker Library) showing the main long-range
interactions between CH -11 and H-5, H-1 and H-9.
3
On the other hand, from the 1H-1D sel. gs-NOESY experiment for compound 12, when CH
3
-11
was selectively irradiated long-range interactions with H-9 and CH CO were observed. Additionally
3
due to the conformation of ring B in the molecule, a long-range interaction, probably a three spin
system with H-6 and H-7, was also observed (see Figure 2). In compound 12 H-9 has a NOE
interaction with H-11 of the methyl group but it did not presented a NOE interaction with the H-7
which is placed in the α direction, which confirms the β direction of the methyl group on C-11 and the
α stereochemistry of the epoxide group. This stereochemistry is explained by considering el steric
hindrance caused by the acetate group, redirecting below the epoxidation reactions. Additionally shifts
of C-8 and C-9 in compound 12 were compared with a compound of similar structure [23].
The compounds 15–18 were synthesized via the common starting material polygodial (2) as
detailed below (Scheme 2). Their structures were established by comparison with the previously
reported spectral data for the same compounds [24,25].

Molecules 2013, 18
4196
1
1
Figure 2. Bottom: H-NMR spectrum of compound 12. Top: 1D H sel. gs NOESY
experiment (selnogp pulse program from Bruker Library) showing the main long-range
3 3
interactions between CH -11 and H-9, and CH CO-9. Three spins NOE effect was also
observed between CH -11 with H-7 and H-6 (in negative phase).
3
Scheme 2. Synthesis of drimane derivatives of polygodial (2).
OH
OH
OH
b
OH
O
H
H
17
18
c
OH
OH
CHO
CHO
OH
OH
a
b
O
H
H
H
2
15
16
4 2 2
Reagents and Conditions: (a) NaBH , MeOH, r.t, 2 h, 15; 90%. (b) m-CPBA, CH Cl , r.t, 1.5 h, 16;
6
0.1% and 18; 75%. (c) (i) NaOH, MeOH, r.t, 20 min; (ii) NaBH , MeOH, r.t, 2 h, 17; 50.3%.
4

Molecules 2013, 18
.2. Biological Results
In our program designed to discover more active compounds from Drimys winteri and derivatives it
4197
2
was found that the compounds showed larvicidal activity. Based on this information and knowing that
this tree has metabolites with a high resistance to insect and pathogen attack we have carried out a
larvae growth regulatory study. The compounds were first used in a bioassay trial, and subsequently, in
order to obtain more satisfactory data, they were used in as low concentrations as possible to establish the
larvicidal activity, ergo there was no effect due to the toxicity of the samples to many targets that may
not be specific to the insect but general or broad spectrum for plants, insects and humans.
2
.3. Antifeedant Activity No-Choice Test
Some of the compounds showed a feeding dissuasive activity against D. melanogaster larvae in
choice assays. The larvae treated with compounds 1, 3, 4, 15–17 showed moderate levels of feeding
inhibition compared to control, showing a decrease in the weight at low concentrations of drimane
compound (see Table 1). It is considered that significant feeding inhibition occurs when the antifeedant
index (AI) is superior to 75% and moderate when it is between 50 and 75%. All samples were
evaluated at 100, 50, 25, and 5 mg/L. The most active compound against D. melanogaster was 17,
which showed high antifeedant effects (see Table 1), even at concentrations of 5 ppm (77.51%), while
the effect was reduced to moderate on the next concentration at 25 ppm (59.41%). Isodrimenin (4)
showed moderate effects, as well, with values between 5.0 and 25.0 ppm. Polygodial (2) has no
significant antifeedant activity against the tested strain of Drosophila melanogaster, and although
previous studies established it as a good insect antifeedant, we did not get a strong effect on larvae.
Polygodial (2) had significative antifeedant activity as reported by Messchendorp et al. [26], who
stated that drimanes are inactive at concentrations of less than 5 mM. We assayed a range from 21.4 µM to
2
.14 mM. Only drimane compounds possessing a lactone group inhibited feeding in the no-choice
tests. An exception is warburganal. In a previous work [27] Messchendorp et al. reported that the
drimanes with a lactone group on the B-ring appear to be the most potent antifeedants at 5 mM (they
tested a range from 1 mM to 5 mM). We observed antifeedant activity with isodrimenine (4) at 5 ppm
which is equivalent to 21.2 µM, as shown in Table 1. It has been established that for polygodial (2) and
other drimanic aldehydes, the variation in the antifeedant activity of these compounds is consistent
with the proposed mode of action for antifeedant drimanes, that is, adduct formation with amino
groups on the insects’ molecular targets [24,28,29]. On the other hand, this is the first report of activity
of those compounds on Drosophila melanogaster, and it is not possible to compare different kinds or
species of insects, i.e., Spodoptera frugiperda [24] with the fruit fly since phylogenetically they are
very distant and furthermore Drosophila possess strong CytP450 activity [30]. For example,
overexpression of a single P450 gene, Cyp6g1, is sufficient to confer DDT resistance in Drosophila [31].
This emphasis on adaptive responses to xenobiotics arises from the importance of insecticide
resistance, which remains the main impediment for effective crop protection and the control of insect-borne
diseases [32]. The Nrf2 ortholog cap ‘n’ collar isoform-C (CncC) has been identified as a central
regulator of xenobiotic responses in Drosophila. Nrf2 plays an important role in regulating cellular

Molecules 2013, 18
4198
defenses against oxidative and electrophilic stress (Nguyen et al. [33], Sykiotis and Bohmann [34]). The
remaining compounds have no significant antifeedant activity.
Table 1. Results obtained in antifeedant tests, using different concentrations of compounds
on D. melanogaster larvae.
Concentration
ppm)
AI **
(Mean ± SD)
0.0
1
3
4
15
16
17
(
Control solvent
5
51.82 ± 6.7 52.25 ± 5.4 53.00 ± 0.9 44.79 ± 3.3 53.98 ± 3.6
45.18 ± 3.4 43.77 ± 6.2 31.55 ± 4.2 37.68 ± 3.6 29.46 ± 3.9
77.51 ± 2.7
59.41 ± 1.7
41.63 ± 4.2
31.32 ± 2.8
2
5
5
0
34.27 ± 3.0 32.63 ± 2.3
18.17 ± 1.2 16.40 ± 1.9
32.63 ± 5.3 24.98 ± 6.3
16.50 ±2.8 20.80 ± 2.2
1
00
Safrole 5
Azadirachtin 5
*
33.21 ± 0.8
53.26 ± 1.6
*
The values followed by the same letter are not significantly different. The significance level p < 0.05.
* AI (%) = antifeedant index; AI = [(C−T)/(C+T)] × 100. * are (+)-ve controls.
*
2
.4. Larvicidal Activity against D. melanogaster
The mortality at 168 h and EC50 values during the first 6 days for compounds tested in no-choice
experiments against first instar larvae of D. melanogaster, are shown in Table 2, respectively. The
compounds 2, 4, 7, 12, 15–17 present larvicidal activity at 48 h (data not shown). Surviving larvae
pupate later and undergo eclosion, giving rise to normal insects. The larvicidal assay was evaluated
over a 6 day period because the biological cycle from larva to pupa in D. melanogaster til-til requires 7
days. Polygodial (2), had an EC50 of 60.0 ± 4.2 mg/L, exhibiting 50% mortality at 168 h. Isodrimenin (4)
showed a 100% mortality and the lowest EC50 (4.5 ± 0.8 mg/L), but its isomeric form confertifolin (3) had
a lower mortality, only 4.1%. Compound 7 showed 33.3% mortality at 168 h. The new nordrimanic
compound 12 presented a moderate activity (12.5% mortality and EC50 of 83.2 ± 3.5 mg/L). The diol 15
and 17 prepared from 2 caused a similar mortality effect, but the EC50 values were not similar, because
1
7 presented a high 70.9 ± 3.7 mg/L values in comparison with compound 15 (45.0 ± 2.8 mg/L). This
is the first time that the larvicidal activity of drimanic derivatives was measured. Previous studies have
reported that molecules having sesquiterpenic skeletons with an α,β-unsaturated systems showed
strong insecticidal activity on larvae of Drosophila melanogaster [35].
Table 2. Mortality percentage of D. melanogaster larvae, after 168 h of the application of
the compounds at different concentrations in larvae’s diet D. melanogaster til-til.
Compounds
% Mortality
15.0 ± 0.3
50.0 ± 0.4
4.10 ± 0.9
100 ± 0.2
0.0
EC50 (mg/L)
>100
1
2
3
4
5
6
60.0 ± 4.2
>100
4.5 ± 0.8
>100
4.2 ± 1.2
>100

Molecules 2013, 18
4199
Table 2. Cont.
Compounds
% Mortality
33.3 ± 1.1
0.0
EC50 (mg/L)
57.8 ± 2.3
>100
7
8
9
0.0
>100
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
0.0
>100
0.0
>100
12.5 ± 1.2
0.0
83.2 ± 3.5
>100
0.0
>100
46.2 ± 1.3
35.3 ± 0.2
35.0 ± 1.1
0.0
45.0 ± 2.8
70.0 ± 2.6
70.9 ± 3.7
>100
Safrole *
20.0 ± 1.6
36.7 ± 0.5
18.0 ± 1.5
15.0 ± 1.1
Azadirachtin *
*
(+)-ve controls.
2.5. Growth Inhibition and Relative Growth Index for D. melanogaster til-til
The growth index (GI or number of surviving larvae/total larvae used) and relative growth index
RGI or GItreated/GIcontrol) (Table 3) shown that the strongest effects correspond to compound 4
(
between 5.0 and 25.0 ppm (RGI 0.26 at 5 ppm and RGI 0.12 at 25 ppm). Compound 4 caused a strong
decrease in the number of larvae that reach pupation (45% at 5.0 ppm). The percentage of larvae that
reached pupation also decreased drastically with 15 (see Table 4) but the active concentration is
different with compared to 4 (100, 250 and 500 ppm). Other compounds assayed exhibited a moderate
activity, but the percentage of emergence of adults from the pupae was also drastically affected by
increasing the concentration (see Table 4)
Table 3. GI and RGI of D. melanogaster til-til as a function of increased concentrations of
most active compounds from D. winteri and derivates.
Concentration
x
z
Compounds
GI RGI
(
ppm)
0.0
Control solvent
Safrole *1
0.95 ± 0.03 x
1.0
0.47
0.20
0.97
0.91
0.83
0.64
0.34
0.26
0.12
-
5.0
0.40 ± 0.02 x
0.19 ± 0.03 x
0.92 ± 0.04 y
0.86 ± 0.05 y
0.79 ± 0.08 y
0.61 ± 0.19 y
0.32 ± 0.09 y
0.25 ± 0.03 y
0.11 ± 0.09 y
-
Azadirachtin *2
5.0
2
5
5.0
0.0
2
4
100.0
2
5
50.0
00.0
5
.0
25.0
0.0 *
5

Molecules 2013, 18
4200
Table 3. Cont.
Concentration
ppm)
x
z
Compounds
GI RGI
(
2
5
5.0
0.0
0.95 ± 0.12 y
0.90 ± 0.13 y
0.73 ± 0.10 y
0.62 ± 0.09 y
0.29 ± 0.06 y
0.63 ± 0.07 y
0.50 ± 0.09 y
0.35 ± 0.12 y
0.15 ± 0.04 y
0.09 ± 0.05 y
0.56 ± 0.12 y
0.48 ± 0.10 y
0.31 ± 0.06 y
-
1.0
0.95
0.77
0.65
0.31
0.66
0.53
0.37
0.16
0.09
0.59
0.50
0.33
-
7
100.0
2
5
50.0
00.0
2
5.0
0.0
5
1
5
7
100.0
2
5
50.0
00.0
2
5.0
0.0
5
1
1
00.0
x Mean of three replicates. y Means followed by the same letter within a column after ± standard error values
are not significantly different in a Student–Newman–Keuls (SNK) test at p < 0.05 (treatments are compared
by concentration to control), 95% confidence limits. z RGItreatment = GItreated/GIcontrol (for details please
see the Experimental Section). * this concentration is more toxic to larvae.* are (+)-ve controls.
Table 4. Activities of compounds isolated from Drimys winteri F., compared with
derivative compounds in this paper.
Doses
Mean weight
Pupation
Emergence Mortality
d
Treatment
%
c
e
f
(
mg/L)
gained (mg)
(SP)
(%)
100
100
(%)
Control solvent
0.0
5
0.813 ± 0.15 a
0.270 ± 0.16 a
0.433 ± 0.13 a
0.258 ± 0.14 a
0.398 ± 0.17 a
0.563 ± 0.18 a
0.59 ± 0.16 a
0.647 ± 0.14 a
0.698 ±0.12 a
0.255 ± 0.13 a
0.413 ± 0.14 a
0.587 ± 0.11 a
0.250 ± 0.10 a
0
100
33.2
53.3
32.0
49.0
69.0
72.6
79.6
85.9
31.4
50.8
72.2
30.7
0
100
100
47.5
70.0
78.3
80.6
72.7
80.0
86.0
80.0
85.0
80.0
45.0
0
0
Safrole *
20.0
36.7
5
63.3
78.0
76.3
77.4
98.0
97.0
98.0
70.0
85.0
80.0
46.7
0
Azadirachtin *
5
5
0
1
2
15.0
1
00
5
5
0.0
5
0
00
5
1
3
4
6
50
4.1
100
4.2
1
00
5
50
1
00
5
0
0
0
0
0.810 ± 0.10 a
0.797 ± 0.13 a
0.800 ± 0.12 a
0.801 ± 0.08 a
99.6
98.0
98.4
98.5
80.0
80.0
85.0
91.6
78.3
93.5
86.7
95.0
50
1
00
5

Molecules 2013, 18
4201
Table 4. Cont.
Doses
Mean weight
Pupation
Emergence
Mortality
(%)
d
Treatment
%
c
e
f
(
mg/L)
gained (mg)
(SP)
(%)
7
50
0.763 ± 0.09 a
0.750 ± 0.09 a
0.803 ± 0.09 a
0.793 ± 0.10 b
0.769 ± 0.12 a
0.310± 0.08 a
0.413± 0.08 a
0.587 ± 0.06 a
0.243 ± 0.12 a
0.443 ± 0.08 b
0.533 ± 0.08 a
0.103 ± 0.11 a
0.207 ± 0.12 a
0.480 ± 0.15 a
93.8
92.3
98.8
97.5
94.6
38.3
50.8
72.2
29.8
54.5
65.5
12.6
25.4
59.0
95.0
73.3
100
100
100
80.0
70.0
95
95.0
97.8
98.3
91.7
91.7
80.0
80.0
80.0
60.0
90.0
50.5
93.5
85.0
91.5
33.3
1
00
5
1
1
1
1
2
5
6
7
50
12.5
46.2
35.3
35.0
1
00
5
50
1
00
5
60.0
91.6
58.3
93.3
85.0
65.0
50
1
00
5
50
1
00
*
(+)-ve controls. (a) The values for growth bioassay were from weight, values taken at 8 ± 1 days before pupation, the
criteria followed was to account larvae that formed pupae, the larvae that not formed pupae were counted as died larvae.
b) Values taken after pupation. (c) Means followed by the same letter within a column after ± standard error values are
(
not significantly different in a Student–Newman–Keuls (SNK) test at p < 0.05 (treatments are compared by concentration
to control), 95% confidence limits. Mean of three replicates. (d) Percentage with respect to control. (e) SP: Survival Pupation =
number of surviving pupa e × 100/total larvae for pupation. (f)% = number of adults emerged × 100/total number of pupae.
(g) As control was used a normal diet with solvent only.
In some compounds (1, 3 and 15) we can observe a decrease in the effect upon increasing the dose,
which has been consistent in all replicates, requiring further studies to elucidate the possible
mechanisms, both chemical and physiological. Xenobiotic metabolizing systems for a given xenobiotic
chemical are very complex, and a number of nuclear receptors, transcription factors, genes, and
enzymes may be involved in its metabolism and detoxification [36,37]. A single xenobiotic molecule
may be modified by multiple enzymes on multiple functional groups, producing multiple metabolites [36].
We postulate that the activation of xenosensors that induce metabolizing enzyme expression at low
stressor doses, may overreact to some extent, resulting in a net response that transforms the added
compound into a highly toxic substance and at higher doses, the compensatory mechanism is
overwhelmed, leading to a reversal of the response [38].
3. Experimental
3.1. General
Unless otherwise stated, all purchased chemical reagents (Merck or Aldrich) were of the highest
commercially available purity and were used without previous purification. Safrole and azadirachtin
was obtained commercially from Sigma-Aldrich. IR spectra were recorded as thin films on a Nicolet
−1
6
700 FT-IR spectrometer and frequencies are reported in cm . ESI-MS/MS data was collected using a
high resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass spectrometer

Molecules 2013, 18
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(Micromass Q-Tof, Manchester, UK) with constant nebulizer temperature of 80 °C. The ESI source and the
mass spectrometer were operated in a negative ion mode, and the cone and extractor potentials ware of
1
0 eV, with a scan range of m/z 100–500. The band infused into the ESI source at flow rates of
−1
5
μL min was dissolved in acetonitrile ion-induced dissociation (CID) with argon in the collision
chamber. The values expressed are average mass and correspond to the [M-H] (University of Talca).
1
13
13
1
H, C, C DEPT-135, sel. gs 1D H NOESY, gs 2D HSQC and gs 2D HMBC spectra were recorded
in CDCl solutions and are referenced to the residual peaks of CHCl at δ = 7.26 ppm and δ = 77.0 ppm
3
3
1
13
for H and C, respectively, on a Bruker Avance 400 Digital NMR spectrometer, operating at
1
3
4
00.1 MHz for 1H and 100.6 MHz for C. Silica gel (Merck 200–300 mesh) was used for column
chromatography (C.C.) and HF-254 silica gel plates were used for TLC. TLC spots were detected by
heating after spraying with 25% H SO in H O.
2
4
2
3
.2. Plant Material
The stem bark of D. winteri adult trees was collected at the Malleco Province (Chile), in February
2
010. A voucher specimen (N° Dw-10112) was deposited at the Herbarium of the Natural Products
Laboratory, “Dr. Herbert Appel A.”, Department of Chemistry, Universidad Técnica Federico Santa
María, Valparaíso, Chile. Fresh bark was carefully washed with abundant distilled water to remove any
residue. Afterwards, it was dried in an oven at 35 °C to a constant weight. Once dried, it was stored in
hermetically sealed plastic containers at 4 °C.
3
.3. Obtainment of Natural Compounds 1–4
The natural drimanes drimenol (1), polygodial (2), confertifolin (3) and isodrimenin (4) (Figure 3)
were isolated from the dichloromethane extract of D. winteri (Winteraceae) bark. The extraction
methodology and isolation of pure compounds was performed according to reported procedures [24,39].
1
Compounds 1–4 were identified by melting point, optical rotation and spectroscopic data including H-
1
3
and C-NMR and comparisons with data reported in the literature [39–41].
Figure 3. Compounds characterized from the D. winteri dichloromethane extract.
O
OH
O
O
CHO
CHO
O
H
H
H
H
1
2
3
4
3
.4. Preparation of Drimenol Derivatives 5–14
Compounds 5–10 and 14 were synthesized by treating drimenol (1) using different protocols
reported in the literature [15,42,43]. The synthesis of compounds 11–13 is described below:
Synthesis of (5S,8R,9R,10S)-4,4,8α,10β-tetramethyloctahydronaphtho[8,9-β]oxiren-7)-one (11) from 6.
The enone 6 (1.0 g, 4.8 mmol) was treated in CH
2
Cl
2
(30 mL) with m-CPBA (0.94 g, 5.4 mmol). Then the
Cl
mixture was stirred for 1 h at room temperature. After this, the solution was taken up in CH
2
2

Molecules 2013, 18
4203
(
20 mL) and washed with saturated NaHCO solution. Later, the organic layer was dried over MgSO ,
3
4
filtered and evaporated. Then it was absorbed on silica and subjected to C.C. eluting with mixtures of
petroleum ether/EtOAc of increasing polarity (20.0:0.0→18.0:2.0) to give an colorless oil identified as
2
7
−1
1
epoxide 11 (0.26 g, 24.8%); [α]D = +0.5° (c = 14.58, CHCl ); IR (cm ): 1699; 1452; 1251. H-NMR:
3
3
1
5
.35 (s, 1H, H-9); 2.27 (m, 2H, H-6); 1.84 (m, 2H, H-1); 1.52 (m, 3H, H-2 and H-5); 1.41 (s, 3H, H-11);
1
3
.14 (m, 2H, H-3); 1.01 (s, 3H, H-14); 0.92 (s, 3H, H-12); 0.89 (s, 3H, H-13). C-NMR: 208.2 (C-7);
9.5 (C-9); 56.2 (C-8); 46.1 (C-10); 41.4 (C-3); 39.0 (C-5); 33.2 (C-4); 32.7 (C-2); 32.6 (C-13); 22.5
(
C-12); 22.0 (C-1); 17.8 (C-6); 16.9 (C-14); 16.8 (C-11). HREIMS: M+H ion m/z 223.1619 (calcd. For
C H O : 222.3286).
1
4
22
2
Synthesis of (5S,7S,8R,9S,10S)-4,4,8β,10β-tetramethyldecahydronaphtho[8,9-α]oxiren-7-yl acetate
12) from 7. Compound 7 (1.0 g, 3.99 mmol) was treated in CH Cl (30 mL) with m-CPBA (0.94 g,
.4 mmol). Then the mixture was stirred for 1 hour at room temperature. After this, the solution was
(
2
2
5
taken up in CH Cl (20 mL) and then washed with saturated NaHCO solution. Later, the organic layer
2
2
3
was dried over MgSO
4
, filtered, and evaporated. Then it was absorbed on silica, and subjected to C.C.
eluting with mixtures of petroleum ether/EtOAc of increasing polarity (20.0:0.0→18.0:2.0) to give an
2
7
white solid identified as epoxide 12 (0.74 g, 68.0%.); p.f.: 68.5–70.0 °C. [α]
= +0.36° (c = 14.58,
); IR (cm ): 1779; 1452; 1254. H-NMR: 5.05 (t,1 H, J = 8.8 Hz, H-7); 2.52 (s, 1H, H-9); 2.09
s, 3H, CH ); 1.88 (m, 1H, H-6α); 1.60 (m, 2H, H-4 and H-6 β); 1.47 (m, 4H, H-1 and H-2); 1.24 (s,
D
−1
1
CHCl
3
(
3
1
3
3
1
3
H, H-11); 1.18 (m, 2H, H-3); 1.06 (s, 3H, H-14); 0.84 (s, 3H, H-12); 0.79 (s, 3H, H-13). C-NMR:
70.4.2 (CH CO); 71.5 (C-7); 70.7 (C-9); 58.9 (C-8); 4154 (C-5); 39.9 (C-3); 36.3 (C-1); 34.5 (C-5);
3.0 (C-10); 32.5 (C-13); 25.0 (C-12); (CH CO); 21.1 (C-2); 19.5 (C-14); 18.2 (C-11). HREIMS:
: 266.3819).
3
3
26 3
M+H ion m/z 267.1882 (calcd. For C16H O
3
.5. Preparation of Polygodial Derivatives 15–18
Compounds 15–18 were synthesized by treating polygodial (2) using different protocols reported in
the literature [25].
3.6. Fly Strains
Wild Drosophila melanogaster til-til, captured in the town of Til-Til metropolitan region, were
grown in a Burdick culture medium, with propanoic acid as antifungal. Cultures were observed daily
with a ZEISS Stemi DV4 binocular loupe to analyze the developmental stages of eggs and first instar
larvae. Later, adult flies were used to deposit their eggs in medium and grown in a MEMMERT Model
UM-500 incubation chamber at 25 °C for 4 days.
3
.7. No-Choice Test (Insecticidal Bioassay) against D. melanogaster
The bioassay for insecticidal activity against larvae of D. melanogaster was carried out as follows:
six concentrations (10.0, 25.0, 50.0, 100.0, 250.0 and 500.0 ppm of sample) were used for determining
EC50 values. For the bioassay test compounds were dissolved in 50 µL of acetone and mixed in 1 mL
of artificial diet containing (for 1 kg of diet): sterile water (800 mL), agar (10.0 g), soy meal (50.0 g),
corn meal (96.0 g), yeast extract (40.0 g), wheat germ (4.0 g), sorbic acid (2.0 g), choline chloride (2.0 g),

Molecules 2013, 18
4204
ascorbic acid (4.0 g), p-hydroxybenzoic acid methyl ester (2.5 g), Wesson salt mixture (7.0 mL),
Vanderzant vitamin mixture for insects (15.0 mL), formaldehyde (2.5 mL), streptomycin (0.1 unit),
aureomycin (5.0 g), and milled corn grain ears (20.0 g), which was prepared by the procedure
described earlier (Mihm [44]). Safrole and azadirachtin was used as a positive control at 5 ppm. A
control diet was treated with 50 µL of acetone only. About 100 adults of D. melanogaster were
introduced into a culture bottle with artificial diet, were subsequently transferred into a Petri dish, and
allowed to oviposit at 25 °C and relative humidity > 60% for 3 h. The diet was taken out of the bottle,
and 20 new eggs were collected and transplanted onto each diet (1 mL) in glass tubes and reared at 25 °C
and relative humidity >90% for 8 days. One day after transplantation, the larvae were hatched and fed
each test compound with the artificial diet [45]. In each instance, the developmental stage was
observed, and the numbers of pupae were recorded and compared with those of a control. Ten new
eggs were used in each of three replicates. The EC , median effective concentrations, was determined
5
0
by log-PROBIT analysis.
.8. Relative Growth Index and Growth Index
The relative growth index (RGI) and growth index (GI) were calculated according to the literature [46],
3
where: RGI = GItreated/GIcontrol, GItreated = number of surviving larvae after treatment, GIcontrol = total
larvae used.
3
.8.1. Antifeedant Test against D. melanogaster
To determine the amount of larvae feeding, tests were carried out as follows a four concentrations
(
100, 50, 25, and 5 ppm of sample) and two controls were taken, one with acetone and the other
without any treatment as a check. Twenty larvae previously weighed about at the transition between
the second and third terms were placed on the diet (the same diet of the larvicidal assay) in each
container for each assay. Based on the amount of food consumed, the absolute deterrence coefficient
was calculated using the formula according to Kielczewski et al. [47]:
coefficient AI* = (C − T): (C + T) × 100
(1)
where T is weight of food eaten by larvae in the experimental variant with compound and C is the
weight of food consumed in the control variant. The changes in the body weight of tested larvae and
the amount of eaten food per mg of body weight increase were also noted. After 48 h, the larvae
received fresh diet every 24 h [48]. The first control group was treated with acetone and the second
control group without any treatment for the first 48 h and then with fresh diet every 24 h, and finally
dead larvae were removed [49].
3
.8.2. Statistical Analyses
Data shown in tables are average results obtained by means of three or 10 replicates and are
presented as average ± standard errors of the mean (SEM). Data were subjected to analysis of variance
ANOVA) with significant differences between means identified by GLM procedures. Results are
(
given in the text as probability values, with p < 0.05 adopted as the criterion of significance.
Differences between treatment means were established with a Student–Newman–Keuls (SNK) test.

Molecules 2013, 18
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The EC50 values for each activity were calculated by PROBIT analysis based on percentage of
mortality obtained at each concentration of the samples. EC50 is half maximal effective concentration.
Complete statistical analysis was performed by means of the MicroCal Origin 6.0 statistics and graphs
PC program.
4
. Conclusions
Three new nordrimanes were synthetized from drimenol (1) and were compared with 11
sesquiterpenes and four nordrimanes. The new epoxide 12 presented an EC value of 83.2 ± 3.5 mg/L
5
0
against Drosophila melanogaster til-til. In this work polygodial (2) did not show effective antifeedant
activity against that biological target. The most active compound was isodrimenin (4) which presented
a higher larvicidal activity of 4.5 ± 0.8 mg/L.
Acknowledgments
The authors thank and DGIP of Universidad Técnica Federico Santa María (PAC 2013 and PIIC 2013)
for financial support.
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©
2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(