Antifungal activity of eugenol derivatives against Botrytis cinerea

Article abstract of DOI:10.3390/molecules24071239

Botrytis cinerea is a worldwide spread fungus that causes the grey mold disease, which is considered the most important factor in postharvest losses in fresh fruit crops. Consequently, the control of gray mold is a matter of current and relevant interest

Full text of DOI:10.3390/molecules24071239

molecules
Article
Antifungal Activity of Eugenol Derivatives against
Botrytis Cinerea
Andrés F. Olea 1 , Angelica Bravo 2, Rolando Martínez 2 , Mario Thomas 2, Claudia Sedan 2,
Luis Espinoza 3 , Elisabeth Zambrano 4, Denisse Carvajal 4, Evelyn Silva-Moreno 4,
*
and Héctor Carrasco 1,
*
1
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile,
San Miguel, Santiago 8900000, Chile; andres.olea@uautonoma.cl
Departamento de Ciencias Químicas, Universidad Andrés Bello, Viña del Mar 2520000, Chile;
angebravor@gmail.com (A.B.); rmartinez@unab.cl (R.M.); m.thomaselgueda@gmail.com (M.T.);
claudiasedan@hotmail.com (C.S.)
Departamento de Química, Universidad Técnica Federico Santa María, Valparaíso 2340000, Chile;
Luis.espinozac@usm.cl
Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile,
San Miguel, Santiago 8900000, Chile; e.zambrano01@ufromail.cl (E.Z.); denisse.carvajals@gmail.com (D.C.)
Correspondence: hector.carrasco@uautonoma.cl (H.C.); evelyn.silva@uautonoma.cl (E.S.-M.);
Tel.: +56-223-036-665 (H.C.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Antonio Evidente
Received: 4 March 2019; Accepted: 26 March 2019; Published: 29 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Botrytis cinerea is a worldwide spread fungus that causes the grey mold disease, which is
considered the most important factor in postharvest losses in fresh fruit crops. Consequently,
the control of gray mold is a matter of current and relevant interest for agricultural industries.
In this work, a series of phenylpropanoids derived from eugenol were synthesized and characterized.
Their effects on the mycelial growth of a virulent and multi-resistant isolate of B. cinerea (PN2)
have been evaluated and IC50 values for the most active compounds range between 31–95 ppm.
The antifungal activity exhibited by these compounds is strongly related to their chemical structure,
i.e., increasing activity has been obtained by isomerization of the double bond or introduction of
a nitro group on the aromatic ring. Based on the relationship between the fungicide activities and
chemical structure, a mechanism of action is proposed. Finally, the activity of these compounds is
higher than that reported for the commercial fungicide BC-1000 that is currently employed to combat
this disease. Thus, our results suggest that these compounds are potential candidates to be used in
the design of new and effective control with inspired natural compounds of this pathogen.
Keywords: Botrytis cinerea; eugenol; mycelial growth; resistant isolate; reactive oxygen species;
chemical fungicides
1. Introduction
B. cinerea is a serious worldwide problem because it causes high losses in pre- and postharvest
fresh fruit crops. The high economic losses associated with Botrytis infection represents a growing
burden for the agricultural industry [
1]. To reduce this effect, a series of control mechanisms have
been developed, the application of chemical fungicides being the most spread and more used [
2].
Currently, the fungicides available to control B. cinerea on grapevines are hydroxianilides (fenhexamid),
anilinopyrimidines (cyprodinil and pyrimethanil), dicarboximide (iprodione), carboxamides (boscalid),
strobilurin, phenylpirroles (fludioxonil) and some inhibitors of ergosterol synthesis [
3]. However,
despite the variety of action mechanisms of these chemical fungicides their indiscriminate use has led
Molecules 2019, 24, 1239; doi:10.3390/molecules24071239
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1239
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to the appearance of resistant isolates. This undesirable outcome has prompted the search of new and
effective fungicide agents, mainly from natural resources [4–7].
Eugenol (4-allyl-2-methoxyphenol) (
the Eugenia caryoplyllata. Much attention has been dedicated to this molecule because it exhibits
many different biological activities, such as analgesic [ ], antimicrobial [10 11], antifungal [12 13],
1) is a major component of essential oil isolated from
8
,9
,
,
antioxidant [14], anti-inflammatory [15], anti-carcinogenic [16], anti-mutagenic and even repellent
properties [17,18].
Various studies of the antifungal activity of eugenol and some analogues (Figure 1) against
phytopathogenic and human fungi, have been reported [19–25].
Figure 1. Chemical structure of eugenol (1), isoeugenol (2) and safrole (3).
Zemek et al. [19] reported that
and Aspergillus niger (MICs around 3000 ppm), while
same fungi with MICs ranging between 100 and 250 ppm in both dilution methods. On the other
hand, Kubo et al. [20] reported that possess moderate activity against S. cerevisiae, Candida subtilis,
Pityrosporum ovale, and Penicillum chrysogenum, with MICs between 100 to 800 ppm in both dilution
methods with shaking, P. ovale being the most sensitive fungus; and exhibit moderate activity
against C. albicans, i.e., measured MICs were 800 and 200 ppm, respectively [21]; is active against S.
1
is almost inactive against Saccharomyces cerevisiae, Candida albicans
2
exhibits a moderate inhibitory effect on the
1
1
3
3
cerevisiae at 200 ppm [22]. Eugenol has also shown activity against Alternaria sp. and P. chrysogenum,
but is inactive against A. niger, Botryosphaeria rhodina or Rhizoctonia sp. in agar diffusion assays [23].
Recently, it has been found that
1 exhibits effective antifungal activity against Aspergillus,
Penicillium, Emericella and Fusarium spp., at concentrations between 100 to 150 ppm. This activity has
been attributed mainly to the phenolic group [24].
In previous work, we have assessed the antifungal properties of a series of phenylpropanoids
including eugenol, safrole and some synthetic derivatives against a panel of opportunistic pathogenic
fungi humans. One of these derivatives, 4-allyl-2-methoxy-5-nitrophenol, possesses a high activity
on C. albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes. Additionally,
it was shown that this molecule did not bind to the main sterol of the fungal membrane up to
250 ppm [25].
However, the antifungal activity of eugenol against B. cinerea has been barely studied. The effect
of eugenol and its essential oils against four apple pathogens, including B. cinerea, have been evaluated
in vitro and in vivo
medium, was 2000 ppm, whereas in the saturated atmosphere of volatile eugenol a complete inhibition
of mycelial growth of four pathogens was obtained at 150 L of volatile eugenol per liter of air volume.
[26]. The MIC value determined for eugenol, incorporated in malt extract agar
µ
No effect on the germination of all tested pathogens at room temperature was found for eugenol [26].
Recently, the in vitro activity of eugenol on B. cinerea has been studied. The IC50 value measured
on mycelial radial growth of B. cinerea was 38.6 ppm; and no bioactivity against conidia germination
was observed. Eugenol induces the generation of H2O2 and increases a free Ca2+ concentration in
the cytoplasm. These results strongly support the idea that the antifungal activity of eugenol is due
to membrane binding and permeability alteration, leading to destabilization and disruption of the
plasma membrane [27].

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Herein, we report the synthesis of a series of eugenol derivatives (Figure 2), six of them
new compounds ( 13), and their evaluation as inhibitors of mycelial growth of a virulent and
8–
multidrug-resistant native isolate (PN2). A relationship between the chemical structures of these
compounds and their activities is discussed.
Figure 2. Chemical structures of synthesized eugenol derivatives.
2. Results
A series of eugenol derivatives have been synthesized and their antifungal properties have
been evaluated (see Figure 2). The synthesis and spectroscopic characterization of compounds
and 14 17 have been previously described [25 26]. In this work, new compounds 13 have been
synthesized and a full spectroscopic characterization is given. Eugenol derivatives 10 and 11 were
obtained from 4-allyl-2-methoxy-6-nitrophenol ( ) with high reaction yields (55–80%). The reduction of
aromatic nitro group in and 11 gives anilines and 12 with 70 and 45 % of reaction yield, respectively.
Finally, the acetylation of and 12 leads to and 13 with 70% and 53% reaction yield, respectively.
4–7,
–
,
8–
8,
7
7
8
8
9
All these syntheses were carried out following standard procedures.
The antifungal activity of eugenol and its derivatives was studied using PN2, a resistant
Botrytis cinerea native isolate. This isolate was obtained from cherry fruit and presents resistance
to fenhexamid, iprodione, pyrimethanil and boscalid. The virulence of this isolate was assessed using
a tomato and the results are shown in Figure 3.

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Figure 3. Virulence assay of damage produced by Botrytis on tomato after inoculating fruit with 10
µL
of conidia suspension (2.5
×
106 conidia/mL) and incubated at 22 ◦C. Gamborg medium was used
as negative control, whereas 7AC was used as positive control. Acronyms: B05.10, corresponds to a
model isolate; PN2, corresponds to a multi-resistant native isolate; 7AC, corresponds to a native isolate
resistant to fenhexamid (gift gently provided by Diagnofruit S.A. Santiago, Chile); dpi means days
post inoculation.
Results shown in Figure 3 indicate that an important injury is produced on tomato fruits after
four days of inoculation with all tested isolates of B. cinerea. The damage is proportional to the
post-inoculation time and therefore we may conclude that PN2 is a virulent and resistant native isolate.
Evaluation of Antifungal Activity of Eugenol and Eugenol Derivatives on Botrytis cinerea
The antifungal activity of eugenol and its derivatives was evaluated in radial growth
measurements in malt-yeast medium. The results shown in Figure 4 indicate that the eugenol
derivatives affect mycelial growth of B. cinerea in a concentration-dependent manner. The percentage
of growth inhibition was determined by measuring the mycelial growth in the absence and presence
of different concentrations of tested compounds (see Experimental section). Results obtained for the
most active compounds are shown in Figure 5.
Figure 4. Effect of eugenol derivatives on hyphae growth of Botrytis cinerea in solid media. Photographs
of hyphal growth of B. cinerea in the absence and presence of different concentrations of 11, 14 and 15
after 48 h of incubation.

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Figure 5. Effect of eugenol derivatives on mycelial growth of Botrytis cinerea in solid media. Percentage
of inhibition of mycelial growth determined after 48 h of incubation at different concentrations: ( 14
15, ( 11, ( ) BC-1000 commercial compound used as control. Each bar represents the average of
at least three independent experiments standard deviation. ( ) indicates significant differences
between each compound concentration, evaluated by Tukey’s test; p < 0.05.
A
)
,
(
B
)
C)
D
±
a–f
3. Discussion
The antifungal activity was evaluated by the measurement growth inhibition. The fitting of
percentage of inhibition to a dose-response curve allows the obtention of IC50 values. The calculated
values of IC50 for all assayed compounds are given in Table 1 and range from 31 to 440 ppm.
Table 1. IC50 values of eugenol and its synthetic derivatives on in vitro mycelial growth of B. cinerea.
These values were estimated by measuring the colony diameter after 48 h of incubation.
Eugenol Derivatives (from Figure 2)
IC50 (ppm)
1
4
5
149
109
95
6
7
80
62
8
9
10
11
I*
440
221
45
12
13
14
D*
103
31
15
16
17
39
108
311
140
BC-1000 (positive control)
*I: inactive; *D: decompose.

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The results indicate that the chemical modification of eugenol induces important changes on
the antifungal activity of this compound. The antifungal activity of eugenol has been attributed to
disruption of the fungal membrane structure, mainly by accumulation of eugenol in the phospholipid
bilayer due to its lipophilic character. This interaction alters fluidity and permeability of fungal
membranes and affects the function of important membrane-bound enzyme or protein [27].
In this work, the chemical modification of eugenol includes changes in the existing functional
groups (Figure 2, compounds
(Figure 2, compounds 17). In the first group, isomerization of the double bond (compare chemical
structures of compounds and , Figure 2) has been carried out, and there is almost no effect on eugenol
4–6) and the addition of nitrogen-containing group on the aromatic ring
7
–
1
4
lipophilicity. However, this small structural modification reduces the IC50 value from 149 to 109 ppm.
In other words, an increase of growth inhibition is induced in eugenol derivatives by conjugation of
the side chain double bond with the aromatic system. As lipophilicity is quite similar, the observed
increase on the antifungal activity must be explained in terms of an additional mechanism by which
molecules 4–6 acts on fungus. A possible explanation is the enhanced Michael-type reaction that could
occur between the side propenyl chain and biological nucleophiles. The reaction mechanism would be
like that proposed for menadione, a lipid-soluble molecule that causes changes in plasma membrane
permeability. Briefly, menadione and compounds 4–6 might react with nucleophilic molecules through
a Michael-type reaction generating an intermediate reactive species that later is stabilized by reaction
with an acid. A comparison of this mechanism, for menadione and isoeugenol, is depicted in Scheme 1.
Scheme 1. Reaction mechanism of menadione and isoeugenol with biological nucleophiles.
Further reductions are reached by changing the hydroxyl group to acetyl (
groups, i.e., by decreasing the polarity of isoeugenol.
5) or methoxy (6)
On the other hand, the addition of nitrogen-containing group on the aromatic ring (Figure 2,
compounds 17) produces different effects on the antifungal activity. Results shown in Table 1
indicate that introduction of nitro group decreases the IC50 value in a factor of 2.5–4.5 (compounds
14 and 15 in Table 1). However, this effect disappears when the nitro group is replaced by amine or
acetamide groups (compare IC50 values of and , and 11 and 12, respectively). Considering that the
7–
7,
7
8
polarity of nitro compounds is higher than that of related eugenol derivatives, the antifungal activity
of these compounds, associated to lipophilic concentration in the membrane, should be lower than
that shown by eugenol. Therefore, additional mechanisms must be considered to explain the lowest
values of IC50 obtained for these compounds. The nitro group is very strong electron withdrawing
and might react directly with double bonds substituted with other electron withdrawing groups.
In addition, it has been shown that aromatic nitro compounds produce reactive oxygen species (ROS)
in an enzymatic-mediated process [28]. Both processes can lead to a chemical disruption of the
membrane palisade. The different activities shown by the nitro eugenol derivatives might be attributed
to the location of these compounds in the membrane or electronic effect affecting the efficiency of both
reactions. Figure 6 depicts a schematic representation of different locations of bioactive compounds
into the fungus cell wall.
The compound-position and orientation in the membrane is determined by the chemical structure
of the adsorbed molecule. Eugenol and derivatives must orientate themselves with the side alkyl chain
parallel to the membrane chains, and the hydroxyl group anchored to the polar surface. Consequently,
the nitro group in compound
7 is located near the surface, whereas in compound 14 the nitro group is
buried into the palisade. In the latter, the spatial arrangement could enhance the nucleophilic attack of

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double bonds on the electrophilic nitro groups, affecting the membrane permeability and probably the
transport of electrons. A similar configuration should be adopted by 15. This mechanism of action
reduces IC50 values from 149 (eugenol) to 62, 31 and 39 ppm for 7, 14 and 15, respectively. The presence
of other groups in the aromatic ring may affect the reactivity of nitro compounds either by modifying
the electronic distribution or the location in the palisade. For example, the replacement of the hydroxyl
group by acetyl decreases strongly the activity of nitroeugenol derivatives, i.e., compares IC50 values
of
7 and 10, 14 and 16, 15 and 17. These results can be attributed to the highest electronic attraction of
the hydroxyl group, which enhances the nucleophilic reaction with double bonds.
Figure 6. Schematic representation of nitro compounds 7 and 14 location in cell membrane.
The compound-position and orientation in the membrane is determined by the chemical structure
of the adsorbed molecule. Eugenol and derivatives must orientate themselves with the side alkyl chain
parallel to the membrane chains, and the hydroxyl group anchored to the polar surface. Consequently,
the nitro group in compound
7 is located near the surface, whereas in compound 14 the nitro group is
buried into the palisade. In the latter, the spatial arrangement could enhance the nucleophilic attack of
double bonds on the electrophilic nitro groups, affecting the membrane permeability and probably the
transport of electrons. A similar configuration should be adopted by 15. This mechanism of action
reduces IC50 values from 149 (eugenol) to 62, 31 and 39 ppm for 7, 14 and 15, respectively. The presence
of other groups in the aromatic ring may affect the reactivity of nitro compounds either by modifying
the electronic distribution or the location in the palisade. For example, the replacement of the hydroxyl
group by acetyl decreases strongly the activity of nitroeugenol derivatives, i.e. compares IC50 values
of
7 and 10, 14 and 16, 15 and 17. These results can be attributed to the highest electronic attraction of
the hydroxyl group, which enhances the nucleophilic reaction with double bonds.
On the other hand, the chemical reactivity of the nitro group decreases by substitution of the
ortho hydroxy group by a methoxy group (compounds 7 and 11) due to its lower positive inductive
effect, but it takes the molecule deeper in the membrane. These two opposite factors induce a slight
decrease on the IC50 value.
Thus, our results indicate that the growth inhibition observed for eugenol derivatives evaluated
in this work could be a consequence of two different and parallel mechanisms: (i) accumulation in
fungal membrane by lipophilic interaction and (ii) Michael-type reactions between eugenol derivatives
and membrane components or ROS production by enzymatic reduction of nitro compounds.
The second mechanism seems to be more important and probably these chemical reactions induce
the production of reactive oxygen species. It is known that ROS are generated in cells by a variety
of processes associated with the normal function of cells or induced by the presence of exogenous
cytotoxic substrates. In addition, plants produce high amounts of ROS to avoid fungus infection,
and therefore the inability of pathogen to reduce the ROS level may be the cause of fungicide effect [29].
To elucidate this hypothesis, the effect of the most active nitroeugenol derivatives on ROS production
has been assessed. Figure 7 shows the luminescence yields measured in the absence and presence
of nitro compounds and menadione that is used as a positive control. Luminescence is a measure of
H2O2 concentration or ROS production.

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Figure 7. Effect of compounds 7, 11, 14 and 15 on production of reactive oxygen species by
Botrytis cinerea. Compounds concentrations were equal to their IC50 values, whereas menadione used
as positive control was applied at 10 ppm final concentration (a, and b indicate statistical differences
with respect to the negative control).
The results shown in Figure 7 indicate that nitroeugenol derivatives induce ROS production
in conidia in an amount equal or larger than that generated by menadione (positive control),
which is a model cytotoxic compound that induces ROS production inside the cell by mitochondria
uncoupling [30]. Interestingly, the magnitude of ROS production follows the same order of IC50 values,
i.e., 15 > 11 > 7. In other words, the formation of ROS correlates quite well with the inhibition activity
suggesting that nitro compounds are acting on the fungus membrane by generating reactive oxygen
species and by alteration of membrane in a similar way to that reported for eugenol both in fungi and
bacteria [31].
4. Materials and Methods
4.1. Chemistry
Unless otherwise stated, all chemical reagents were purchased with the highest commercially
available purity (Merck, Darmstadt, Germany or Aldrich, St. Louis, MO, USA) and were used without
previous purification. Melting points were measured on a Stuart-Scientific SMP3 apparatus and are
uncorrected. IR spectra were recorded as thin film or KBr pellets in a Nicolet Impact 420 spectrometer
1
(Thermo Scientific, San Jose, CA, USA).
ν
values are expressed in cm−1. H- and 13C-NMR spectra
max
were recorded in CDCl3 solutions and referenced to the residual peak of CHCl3 at δ = 7.26 ppm and
δ = 77.00 ppm for 1H and 13C, respectively, on a Bruker Avance 400 Digital NMR spectrometer (Bruker,
Rheinstetten, Germany), operating at 400.1 MHz for 1H and 100.6 MHz for 13C. Chemical shifts are
reported in
δ ppm and coupling constants (J) are given in Hz. Silica gel (Merck 200–300 mesh) was
used for column chromatography (CC) and silica gel plates and HF-254 for thin layer chromatography
(TLC). Spots were detected on TLC by heating after spraying with 25% H2SO4 in H2O.

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4.2. Synthesis
Eugenol was isolated from cloves essence, according to the standard procedure [32]. Synthesis of
new eugenol derivatives are shown in Scheme 2.
H3CO
O
NO2
O
10
c
H3CO
HO
H3CO
HO
H3CO
HO
H3CO
a
b
c
O
NO2
NH2
HN
O
1
7
8
9
O
d
H3CO
H3CO
H3CO
H3CO
c
b
H3CO
H3CO
HN
NO2
NH2
12
O
13
11
Scheme 2. Synthesis of new eugenol derivatives. Conditions: (
a) NaNO3/KHSO4, SiO2/H2O (1:1),
r.t. 5.5 h; ( ) Zn, HCOONH4, MeOH, r.t. 5 h; ( ) Ac2O, DMAP, CH2Cl2 r.t 2.5 h; (d
b
c
) (CH3)2CO,
dimethylsulphate, K2CO3, reflux overnight.
General Procedures
Reduction of nitroeugenol derivatives to anilines. Nitroeugenol derivatives
transformed to the respective aniline ( and 12, respectively) by the following procedure. Zn in
powder (100 mg, previously treated with HCl 5%) and ammonium formate (1 mL) were added to a
7 and 11 were
8
−4
−4
stirred solution of nitroeugenols (
7
, 200 mg, 9.6
×
10 mol; 11, 100 mg, 4.48
×
10 mol) in methanol
(5.0–10 mL). The solution was stirred at room temperature for 5 h, and complete disappearance of
the starting product was confirmed by TLC (AcOEt:n-hexane, 1:3). The reacted mixture was filtered,
and the solvent evaporated in vacuum. The crude reaction product was dissolved in AcOEt (10 mL)
and the organic phase was washed with brine (3
evaporated. The pure product was obtained by CC.
Acetylation reaction. Phenols and aniline 12 were reacted with acetic anhydride to give
, and 13, respectively, by the following synthetic procedure. To a stirred solution of phenol
(100 mg, 5.6
10−4 mol) or aniline (100 mg, 4.48 10−4 mol) in dichloromethane (15 mL) was added
4-N,N-dimethylaminopyridine (DMAP) (10 mg, 8.2
10−5 mol). Acetic anhydride (0.25 mL) was
×
10 mL), dried with anhydrous Na2SO4 and vacuum
7, 8
10, 9
×
×
×
added and the reaction was left to continue at room temperature (1.5–2 h for phenols; 1.5 h for aniline).
After this period, complete disappearance of the starting product was confirmed by means of the
TLC (ethyl acetate: n-hexane 1:3). Aqueous potassium hydrogen sulphate solution (10%, 20 mL) was
added, and the organic phase was extracted with dichloromethane. The extract was washed with
water (3 × 20 mL), dried with anhydrous Na2SO4 and vacuum evaporated. Pure compounds were
obtained by CC.

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4-allyl-2-amino-6-methoxyphenol (
derivative
in hexane), as white crystal (94.5 mg, 55% yield); melting point: 107–108◦C. IR (film)
8
): Compound
8
was synthesized from the nitroeugenol
7
by reduction of the nitro group. Pure compound
8
was obtained by CC (1:8–1:7, AcOEt
max/cm−1
υ
:
3308 (OH), 3372 (NH), 3308 (C=CH Ar), 3072 (CH=CH2), 1608 (C=C), 1221 (C-N), 1190 (C-O), 1132
1
(C-O), 1080, 896. H NMR:
δ
3.24 (2H, d, J = 6.7 Hz, H-10); 3.69 (2H, b. s, NH2); 3.84 (3H, s, OCH3);
5.05 (2H, m, H-30); 5.33 (1H, s, OH); 5.93 (1H, m, H-20); 6.19 (1H, s, H-3); 6.25 (1H, s, H-5). 13C NMR:
δ
40.1(C-10); 55.9 (OCH3); 101.8 (C-5); 109.4 (C-30); 115.3 (C-3); 131.1 (C-4); 131.6 (C-6); 138.0 (C-1); 134.0
(C-20); 137.9 (C-1); 146.6 (C-2).
2-acetamide-4-allyl-6-methoxyphenyl acetate (9): Compound 9 was synthesized by acetylation of
8
. Pure compound 9 was obtained by CC (1:9–1:5, ethyl acetate in hexane) as white crystal (103 mg,
70%); melting point: 146–149 ◦C; IR (film) υmax/cm−1: 3320 (N-H), 3060-2800 (C=C Ar), 1750 (C=O),
1
1250 (C-CO-O), 1120 (C-O), 900-730 (C-H Ar). H NMR:
δ 2.15 (3H, s, C-CH3); 2.35 (3H, s, C-CH3); 3.36
(2H, d, J = 6.5, H-10); 3.80 (3H, s, OCH3); 5.10 (2H, m, H-30); 5.95 (1H, m, H-20); 6.56 (1H, s, H-3); 7.14
(1H, b.s, -NH); 7.70 (1H, s, H-5). 13C NMR:
δ 20.5 (CH3CO2); 24.7 (C
H3CO2); 40.4 (C-10); 56.0 (OCH3);
108.0 (C-5); 113.9 (C-30); 116.3 (C-3); 127.9 (C-4); 130.9 (C-6); 136.8 (C-20); 138.9 (C-1); 150.7 (C-2); 168.0
(CH3CO2); 168.4 (CH3CO2).
4-allyl-2-methoxy-6-nitrophenyl acetate (10): Compound 10 was synthesized by acetylation of
7
. Pure compound 10 was obtained by CC (1:9–1:7, ethyl acetate in hexane) as yellow crystal (0.45 g,
75%); melting point: 60–61 ◦C; IR (film)
υ
max/cm−1: 3100-2800 (C=C Ar), 1750 (C=O), 1550 (
−
NO2),
1
1250 (C-CO-O), 1250-1050 (C-O), 900–730 (C-H Ar). H NMR:
δ
2.36 (30H, s, CO-CH3); 3.43 (2H, d,
0
0
J = 6.5 Hz. H-1 ); 3.87 (3H, s, -O-CH3); 5.17 (2H, m, H-3 ); 5.92 (1H, m, H-2 ); 7.03 (1H, s, H-3); 7.43 (1H,
s, H-5). 13C NMR:
δ 20.2 (CH3CO2); 39.5 (C-1 ); 56.5 (OCH3); 116.0 (C-5); 117.1 (C-3 ); 117.4 (C-3); 132.1
0
0
(C-4); 135.2 (C-6); 139.1 (C-20); 142.4 (C-1); 152.6 (C-2); 167.9 (CH3CO2).
5-allyl-1,2-dimethoxy-3-nitrobenzene (11): Potassium carbonate (1.0 g, 7.25
×
10−3 mol) was
added to a stirred solution of 7 (600 mg, 2.87
sulphate (1.2 mL, 1.27
reflux. Complete disappearance of the starting product was confirmed by TLC (1:3 ethyl acetate:
hexane). The crude reaction product is diluted in acetone (30 mL), water (50 mL), and extracted with
×
10−3 mol) in dry acetone (60 mL). Then, dimethyl
×
10−2 mol) was added and the reaction was left to continue over night under
dichloromethane (3
×
50 mL). The extract is dried with anhydrous Na2SO4 and vacuum evaporated.
Pure product (513 mg, 80%) was obtained by CC (1:9–1:7, ethyl acetate in hexane as marron oil.
Compound 11: IR (film)
υ
max/cm−1: 3100-2800 (0C=C Ar), 1550 (-NO2), 1400-1000 (C-O), 1000-650
1
(H2C=CH2). H RMN:
δ 3.38 (2H, d, J = 6.4 Hz, H-1 ); 3.90 (3H, s, OCH3); 3.95 (3H, s, OCH3); 5.14 (2H,
m, H-30); 5.91 (1H, m, H-20); 6.92 (1H, s, H-3); 7.16 (1H, s, H-5). 13C RMN: 39.6 (C-10); 56.4 (OCH3);
δ
61.9 (OCH3); 115.6 (C-6); 116.3 (C-30); 117.3 (C-3); 135.7 (C-4); 136.3 (C-20); 141.1 (C-5); 144.7 (C-1);
153.9 (C-2).
5-allyl-2,3-dimethoxyaniline (12): Aniline 12 was synthesized from the nitroeugenol derivative 11
by reduction of the nitro group. Pure compound 12 was obtained by CC (1:8–1:5, AcOEt in hexane)
as marron oil (45.7 mg, 46% yield). Compound 12: IR (film)
υ
max/cm−1: 3500-3300 (N0H2), 3100-2800
1
(C=C Ar), 1400-1000 (C-O), 1000-650 (H2C=CH2). H NMR:
δ 3.25 (2H, d, J = 6.4 Hz, H1 ); 3.69 (2H, b.s,
NH2); 3.79 (3H, s, OCH3); 3.82 (3H, s, OCH3); 5.07 (2H, m, H-30); 5.95 (1H, m, H-20); 6.17 (1H, s, H-3);
6.23 (1H, s, H-5). 13C
δ
NMR: 40.3 (C-10); 55.6 (OCH3); 59.9 (OCH3); 102.7 (C-6); 108.8 (C-30); 115.7
(C-3); 134.3 (C-4); 136.2 (C-20); 137.5 (C-5); 140.2 (C-1); 152.8 (C-2).
N-(5-allyl-2,3-dimethoxyphenyl) acetamide (13): Compound 13 was synthesized by acetylation of
12. Pure compound 13 was obtained by CC (1:5-1:3, ethyl acetate in hexane) as white crystal (64 mg,
◦
53%); melting point: 78–79 C; IR (film)
υ
1
max/cm−1: 3300 (N-H), 3100-2800 (C=C Ar), 1715 (R-(CO)-R’),
0
1400-1000 (C-O), 1000-650 (H2C=CH2). H NMR:
δ
2.18 (3H, s, CH3CON); 3.31 (2H, d, J = 6.4 Hz, H-1 );
3.82 (3H, s, OCH3); 3.82 (3H, s, OCH3); 5.07 (2H, m, H-30); 5.92 (1H, m, H-20); 6.47 (1H, s, H-3); 7.81
(1H, s, H-5); 7.83 (-HN). 13C NMR:
0
δ 24.7 (CH3CON); 40.3 (C-1 ); 55.6 (OCH3); 60.5 (OCH3); 107.6 (C-6);
112.4 (C-30); 115.7 (C-3); 131.7 (C-4); 135.4 (C-20); 136.2 (C-5); 137.1 (C-1); 151.7 (C-2); 168.2 (CH3CON).

Molecules 2019, 24, 1239
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4.3. Biological Assays
4.3.1. B. cinerea Isolates
PN2 is a native resistant isolate of B. cinerea obtained from cherry. It was maintained and grown
under conditions previously described [33]. Its sensibility to current fungicides was tested following
a described method [34]. Briefly, to test the single fungicide resistance spectra, of collected Botrytis
isolates, discriminatory fungicide concentrations of the following fungicides were used: carbendazim
5
µg/mL; cyprodinil 5
µg/mL; fenhexamid 5
µg/mL; iprodion 5
µg/mL; tebuconazole 7 µg/mL;
pyrimethanil 10 g/mL; boscalid 5
µ
µg/mL and tolnaftate 5 µg/mL. These concentrations were chosen
according to IC50 values of single fungicide resistant strains [35]. In addition, the virulence of PN2
was evaluated by evaluating the damage produced by Botrytis isolate on a tomato. Tomatoes fr◦uit
were inoculated with 10
µ
L of conidia suspension (2.5
×
106 conidia/mL) and incubated at 22 C.
The photographical recording was made after 1–4 days post-inoculation to evaluate fruit damage.
The Gamborg medium and 7AC isolate (gently provided by Diagnofruit S.A. Santiago, Chile) were
used as negative and positive controls, respectively.
4.3.2. Effect of Eugenol Derivatives on Mycelial Growth of B. cinerea in Solid Media
Fungitoxicity of eugenol and synthetic derivatives, and of commercial fungicide BC-1000, was
assessed using the radial growth test on malt-yeast extract agar [36]. All the compounds, except
BC-1000, were applied in dichloromethane solution at different final concentrations (20, 40, 80,
160 ppm). Eugenol and its synthetic derivatives were completely soluble at all tested concentrations.
An aliquot of these solutions (200 µL) was added to 7 mL of malt-yeast extract agar. The amount of
added dichloromethane was identical in controls and treatment assays. The medium with presence
or absence of synthetized compounds as well as BC-1000 was poured into 6 cm diameter Petri
dishes. Dishes were left open in a biosecurity hood for 40 min to remove solvent. After solvent
evaporation, Petri dishes were inoculated with 5 mm diameter agar discs with thin mycelium of B.
cinerea. Cultures were incubated in the dark at 22 ◦C during several days. Mycelial growth diameters
were measured daily, and inhibition percentages were calculated using the following equation
(dC − d0) − (dS − d0)
Growth inhibition (%) =
× 100
(1)
(dC − d0)
where dC, d0 and dS represent diameters (in mm) of blank control fungus, fungus agar disc,
and compound-treated fungus. Inhibition percentages were plotted as a function of fungicide
concentration using a dose-response equation, for all tested compounds. The IC50 values were
obtained by fitting of data to this curve. Plotting of the data, fitting, and IC50 calculation were carried
out with Origin v8.0 (OriginLab, Northhampton, MA, USA). Significant differences were evaluated
with a two-way analysis of variance (Tukey’s test; p < 0.05).
4.3.3. Effect of Eugenol and its Derivatives on ROS Production by Botrytis cinerea
The production of reactive oxygen species was measured following a described procedure [36,37].
Menadione was used as a positive control. This molecule has been described as an inducer of ROS
generation, since it is capable of both redox cycling and arylating nucleophilic substrates by Michael
addition [37
WI, USA) [37]. Briefly, conidia (79
conidiam L−1/well. Later, each well was incubated in presence of each compound at their IC50
concentration, 20 L of buffer substrate H2O2 and cultured for 2 h at 21
1 ◦C. After this period of
incubation, 100 L of ROS-GLOtm reagent was added to each well and incubated for 20 min at room
temperature. The ROS production was measured using a luminometer (Tecan infinite m200pro; Tecan
,
38]. The ROS was evaluated using ROS-GLOtm H2O2 assay kit (Promega, Madison,
µL) were plated in a 96-well plate at a concentration of 1
×
105
µ
±
µ

Molecules 2019, 24, 1239
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Group Ltd., Hombrechtikon, Switzerland). Mean values with at least significant difference (p < 0.05)
were considered.
5. Conclusions
A series of eugenol derivatives has been synthesized and their antifungal activity on mycelial
growth of B. cinerea has been evaluated. The growth inhibition activity depends on the chemical
structure of eugenol derivatives. An analysis of the structure-activity relationship suggests that these
compounds act on fungus by two mechanisms, i.e., accumulation on the fungus membrane and
chemical reactions with unsaturated chains or enzymatic-mediated reduction. The first is controlled
by the lipophilic character of these molecules and the second is due to the presence of strong electron
withdrawing groups in the aromatic ring. This action leads to the disruption of fungus membranes
and ROS production. Our results show that some eugenol derivatives have a much higher antifungal
activity or, at least, comparable to the commercial fungicide BC-1000. Finally, the high activity values
presented by the nitro compounds make them potential antifungal agents for the chemical control
of B. cinerea.
Author Contributions: A.B., C.S., M.T., H.C. and R.M. collaborated in the synthesis and purification of
2-allylphenolderivatives; H.C. and A.F.O. collaborated in the discussion of structure-activity relationship,
and writing of the manuscript; E.Z. and D.C. performed the bioassays; E.S.-M. supervised the biological work
and wrote the biological component of this manuscript; H.C. and A.F.O. collaborated in the discussion and
interpretation of the results, manuscript redaction, and corrections.
Funding: This research was funded by FONDECYT grant number 1170706 and 1130742.
Acknowledgments: The authors are grateful to FONDECYT for financial support of this work under Grant
1170706 and 1130742.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8–13 are available from the authors.
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2019 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).