Potent Nematicidal Activity of Maleimide Derivatives on Meloidogyne incognita

Article abstract of DOI:10.1021/acs.jafc.6b02250

Different maleimide derivatives were synthesized and assayed for their in vitro activity on the soil inhabiting, plant-parasitic nematode Meloidogyne incognita, also known as root-knot nematode. The compounds maleimide, N-ethylmaleimide, N-isopropylmaleimide, and N-isobutylmaleimide showed the strongest nematicidal activity on the second stage juveniles of the root-knot nematode with EC_50/72h values of 2.6 ± 1.3, 5.1 ± 3.4, 16.2 ± 5.4, and 19.0 ± 9.0 mg/L, respectively. We also determined the nematicidal activity of copper sulfate, finding an EC₅₀ value of 48.6 ± 29.8 mg/L. When maleimide at 1 mg/L was tested in combination with copper sulfate at 50 mg/L, we observed 100% mortality of the nematodes. We performed a GC-MS metabolomics analysis after treating nematodes with maleimide at 8 mg/L for 24 h. This analysis revealed altered fatty acids and diglyceride metabolites such as oleic acid, palmitic acid, and 1-monopalmitin. Our results suggest that maleimide may be used as a new interesting building block for developing new nematicides in combination with copper salts.

Full text of DOI:10.1021/acs.jafc.6b02250

Article
pubs.acs.org/JAFC
Potent Nematicidal Activity of Maleimide Derivatives on Meloidogyne
incognita
Kodjo Eloh,† Monica Demurtas,† Manuel Giacomo Mura,† Alessandro Deplano,† Valentina Onnis,†
Nicola Sasanelli,‡ Andrea Maxia,† and Pierluigi Caboni*,†
†Department of Life and Environmental Sciences, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy
‡Institute for Sustainable Plant Protection, CNR, via G. Amendola 122/D, 70126 Bari, Italy
ABSTRACT: Different maleimide derivatives were synthesized and assayed for their in vitro activity on the soil inhabiting,
plant-parasitic nematode Meloidogyne incognita, also known as root-knot nematode. The compounds maleimide, N-
ethylmaleimide, N-isopropylmaleimide, and N-isobutylmaleimide showed the strongest nematicidal activity on the second
stage juveniles of the root-knot nematode with EC50/72h values of 2.6 1.3, 5.1 3.4, 16.2 5.4, and 19.0 9.0 mg/L,
respectively. We also determined the nematicidal activity of copper sulfate, finding an EC50 value of 48.6 29.8 mg/L. When
maleimide at 1 mg/L was tested in combination with copper sulfate at 50 mg/L, we observed 100% mortality of the nematodes.
We performed a GC-MS metabolomics analysis after treating nematodes with maleimide at 8 mg/L for 24 h. This analysis
revealed altered fatty acids and diglyceride metabolites such as oleic acid, palmitic acid, and 1-monopalmitin. Our results suggest
that maleimide may be used as a new interesting building block for developing new nematicides in combination with copper salts.
KEYWORDS: V-ATPase, metabolomics, oxidative stress, redox-active metals, nematicide
environmentally relevant concentrations in a nontarget species,
leading to a novel mechanistic hypothesis.16
INTRODUCTION
■
Nematodes are small soil worms; some of them are plant
parasites and play a significant role in the susceptibility of the
host plant to the attack by other pathogens.1 Worldwide crop
losses due to plant-parasitic nematode infections are estimated
at USD 157 billion per year.2 Root-knot nematodes (RKN),
Meloidogyne spp., are widely distributed, polyphagous parasites
infesting a wide range of host plants including horticultural
crops such as cucumber, aubergine, melon, pepper, tomato, and
fruit trees making them one of the major agricultural pests in
the world.3,4 Plant symptoms include formation of galls on root
systems, due to hyperplasia and hypertrophy as reaction to the
trophic activity of the nematode juveniles, retarded develop-
ment and growth of hosts as well as reduced quality and
quantity of the harvest. In the case of severe nematode attack,
plants can eventually die. Although chemical nematicides are
the most reliable means of controlling RKN, concerns for the
environment have led to the progressive withdrawal of many
synthetic nematicides and to the search of alternative strategies
or novel and specific biochemical targets.5 Recently, we
reported on the nematicidal activity of arylhydrazones,6
acetophenones, and chalcones on Meloidogyne incognita.7
Current research is also focused on the potential use of natural
plant extracts for the development of new nematicides.8−14
Modern toxicology employs new analytical methods to study
toxicological effects. Metabolomics is a dynamic method of
research that involves qualitative and quantitative measure-
ments of metabolic responses in a living organism due to a
genetic modification, external stimulus, and/or stressor. The
field of metabolomics has grown rapidly over the past decade
and has been proven to be a powerful omics methodology in
predicting and explaining complex phenotypes in diverse
biological systems.15 When metabolomics was used, it revealed
that imidacloprid induced metabolic changes at low and
Maleimide (1) is a five-membered ring compound belonging
to the cyclic imides class. Maleimide derivatives are emerging as
potent pharmacophores showing different biological activ-
ities.17,18 When investigated on Escherichia coli and Staph-
ylococcus aureus, maleimides were approximately 30 times more
active than succinimides.19 When tested on Sclerotinia
sclorotiorum, different synthesized maleimide derivatives
showed an interesting fungicidal activity.20 Moreover, by acting
like thiol reagents, maleimides like N-ethylmaleimide and
related N-arylcyclic imides show herbicidal activity.21 At low
concentrations (1−2 μM), N-ethylmaleimide (2), a covalently
binding cysteinyl reagent,22,23 is considered an inhibitor of the
eukaryotic vacuolar (V-type) ATPases, while at higher
concentrations (0.1−1 mM), it inhibits the P-type ATPases,
F-type ATPases that are resistant to inhibition by 2.24,25 In the
present study, starting with the hypothesis of the involvement
of maleimide in the inhibition of the nematode V-ATPase
activity, we prepared various maleimides and succinimides with
different substituents and assessed their nematicidal activity in
vitro. The synergistic activity of maleimide with copper and
iron sulfates was also tested. Furthermore, we performed an in
vitro GC-MS metabolomics analysis on nematodes, after their
treatment with maleimide, by measuring the levels of ammonia
excretion.
Received: May 18, 2016
Revised: May 30, 2016
Accepted: June 1, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
7.21 (d, J = 8.5 Hz, 1H, Ar), 7.16 (s, 2H, CH), 7.10 (d, J = 8.5 Hz, 1H,
Ar), 3.88 (s, 3H, CH3). IR (Nujol) 1770, 1712, 1685, 1560 cm−1.
HRMS (ESI-TOF) (m/z) [M + H]+ calcd for (C11H8ClNO3)
238.0265, found 238.0262. Calcd: C, 55.60; H, 3.39; Cl, 14.92; N,
5.82. Found: C, 55.62; H, 3.36; Cl, 14.90; N, 5.84.
1-(Naphthalen-2-yl)-1H-pyrrole-2,5-dione (25). Yield 72%, mp
105 °C. 1H NMR (DMSO-d6): δ 7.87−8.03 (m, 3H, Ar), 7.78 (s, 1H,
Ar), 7.47−7.63 (m, 3H, Ar), 7.16 (s, 2H, CH). IR (Nujol) 1787, 1764,
1711, 1675 cm−1. HRMS (ESI-TOF) (m/z) [M + H]+ calcd for
(C14H9NO2) 224.0706, found 224.0705. Calcd: C, 75.33; H, 4.06; N,
6.27. Found: C, 75.31; H, 4.08; N, 6.30.
MATERIALS AND METHODS
■
General Methods. Uncorrected melting points were measured on
an SMP1 apparatus melting point apparatus (Stuart Scientific, Stone,
UK). Proton NMR spectra were acquired on Inova 500 spectrometer
(Varian, Palo Alto, CA). The chemical shifts are reported in parts per
million (δ, ppm) downfield from tetramethylsilane (TMS) used as
internal standard. Infrared spectra were obtained with a Vector 22
spectrophotometer (Bruker, Bremen, Germany). Purity of synthesized
chemicals was determined by an elemental analyzer (Yanagimoto,
Kyoto, Japan) and was greater than 95% when compared with the
theoretical values. High resolution mass spectra of synthesized
compounds were acquired using an Agilent LC-QTOF 6520 (Agilent
Technologies, Palo Alto, CA). Thin layer chromatography (TLC) on
E. Merck TLC plates coated with silica gel 60 F254 (0.25 mm layer
thickness) was used to monitor reaction courses. TLC visualization
was carried out using a UV lamp at 254 nm.
Materials. All synthetic precursors and solvents were purchased
from Sigma-Aldrich (Milan, Italy). Maleimides and succinimides 1, 2,
3,26 4,20 5,27 8,27 9, 10,27 11,28 12,29 13,29 15,30 16,29 17,31 18,20 19,31
21,28 24,29 26,29 27,32 and 2827 were commercially available or
obtained as previously reported.
General Procedure for the Synthesis of Maleimides. A
mixture of maleic anhydride (0.196 g, 2 mmol) and the appropriate
amine (2 mmol) in CH2Cl2 (5 mL) was stirred at room temperature
for 1 h. The solvent was removed in vacuo, and the residue, maleamic
acid, was then dissolved in 3 mL of acetic anhydride. To this solution
we added sodium acetate (0.10 g, 1.22 mmol), and the mixture was
then heated for 2 h at 100 °C and cooled with water afterward. The
aqueous solution was extracted with Et2O (3 × 10 mL), and the
organic layers were collected, dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo to give N-substituted maleimides 2−8 and
11−28.
1-Benzylpyrrolidine-2,5-dione (10). A mixture of benzylamine
(0.21 g, 2 mmol), succinic anhydride (0.2 g, 2 mmol), and anhydrous
triethylamine (1 mL, 7.2 mmol) in 25 mL of toluene was refluxed with
a Dean−Stark apparatus for 36 h. The reaction mixture was cooled
then concentrated under reduced pressure. The residue was dissolved
in ethyl acetate (25 mL) and washed with saturated aqueous NaHCO3
solution (3 × 10 mL) and then with water (3 × 10 mL). The organic
layer was dried over anhydrous Na2SO4 and concentrated under
vacuum to give the title compound in 80% yield. Analytical and
spectroscopic data were in accordance with literature values.27
Nematode Population. A M. incognita33 population was reared
for two months at 25 2 °C on susceptible tomato plants (Solanum
lycopersicum L.; cv. Rutgers) in a greenhouse located in Bari, Italy.
Infested plants were uprooted, and roots showing galls and egg masses
were washed to remove soil. The roots were then cut into 2 cm pieces
and the egg masses hand-picked. Batches of 20 egg masses of similar
size (averaging 20000 eggs) were placed on 2 cm diameter sieves (215
μm), and each sieve was put in a 3.5 cm diameter plastic Petri dish; we
then added 3 mL of distilled water (natural hatching agent for
Meloidogyne spp.), sufficient to cover the egg masses, to allow the eggs
to hatch. The dishes were incubated in a growth cabinet at 25 °C.34 All
second-stage juveniles (J2) which hatched in the first 3 d were
eliminated, and only the J2s hatched after 24 h or more were collected
and used to verify the in vitro nematicidal effect of the selected
maleimides.
Nematicidal Assay. Maleimides and succinimides were tested for
the nematicidal activity on the second stage juveniles and the
corresponding EC50 values were calculated. Stock solutions of
synthetic compounds were prepared using water, methanol, or
DMSO as solvent, whereas working solutions were made by dilution
with tap water. The final concentrations of the organic solvent in each
well never exceeded 1% because preliminary experiments showed that
the motility of nematodes exposed to those concentrations was similar
to the motility of the controls. Tap water was used with the controls.
Treatments were performed in 96-well plates using 30 juveniles in
each replication. To avoid solvent evaporation, plates were covered
and kept in the dark at 28 °C. After treatment, juveniles were moved
to plain water and grouped into two distinct categories, motile or
immotile, with the use of an inverted microscope at 40× after pricking
the juvenile body with a needle. Nematodes that never regained
movement after being moved to tap water and pricked were
considered to be dead. Six replications were made, and the experiment
repeated at least twice.
1-(Heptan-2-yl)-1H-pyrrole-2,5-dione 96). Yield 82%, colorless oil.
1H NMR (DMSO-d6): δ 6.95 (s, 2H, CH), 4.00−4.03 (m, 1H, CH),
1.79−1.86 (m, 1H, CH2), 1.52−1.59 (m, 1H, CH2), 1.27 (d, J = 7.0
Hz, 3H, CH3), 1.09−1.23 (m, 6H, CH2), 0.81 (t, J = 7.0 Hz, 3H,
CH3). IR (neat) 1710, 1457, 1405, 1369 cm−1. HRMS (ESI-TOF)
(m/z) [M + H]+ calcd for (C11H17NO2) 196.1332, found 196.1335.
Calcd: C, 67.66; H, 8.78; N, 7.17. Found: C, 67.67; H, 8.76; N, 7.18.
1-(6-Methylheptan-2-yl)-1H-pyrrole-2,5-dione (7). Yield 79%,
1
colorless oil. H NMR (DMSO-d6): δ 6.95 (s, 2H, CH), 4.00−4.03
(m, 1H, CH), 1.78−1.84 (m, 1H, CH2), 1.51−1.55 (m, 1H,CH2)
1.42−1.47 (m, 1H, CH2), 1.27 (d, J = 6.5 Hz, 3H, CH3), 1.06−1.15
(m, 4H, CH2), 0.80 (t, J = 7.0 Hz, 6H, CH3). IR (neat) 1704, 1460,
1405, 1367 cm−1. HRMS (ESI-TOF) (m/z) [M + H]+ calcd for
(C12H19NO2) 210.1489, found 210.1487. Calcd: C, 68.87; H, 9.15; N,
6.69. Found: C, 68.89; H, 9.13; N, 6.70.
1-(4-Fluoro-3-nitrophenyl)-1H-pyrrole-2,5-dione (14). Yield 82%,
1
mp 115−118 °C. H NMR (DMSO-d6): δ 7.24 (s, 2H, CH), 7.72−
7.83 (m, 2H, Ar), 7.85 (s, 1H, Ar). IR (Nujol) 1714, 1594, 1538, 1503
cm−1. HRMS (ESI-TOF) (m/z) [M + H]+ calcd for (C10H5FN2O4)
237.0306, found 237.0302. Calcd: C, 50.86; H, 2.13; F, 8.04; N, 11.86.
Found: C, 50.87; H, 2.15; F, 8.03; N, 11.88.
1-(4-(Methylthio)phenyl)-1H-pyrrole-2,5-dione (20). Yield 63%,
mp 76−79 °C. 1H NMR (DMSO-d6): δ 7.31 (s, 2H, CH), 7.35 (d, J =
8.5 Hz, 2H, Ar), 7.26 (d, J = 8.5 Hz, 2H, Ar), 2.49 (s, 3H, CH3). IR
(Nujol) 1793, 1768, 1706, 1665 cm−1. HRMS (ESI-TOF) (m/z) [M +
H]+ calcd for (C11H9NO2S) 220.0427, found 220.0429. Calcd: C,
60.26; H, 4.14; N, 6.39; S, 14.62. Found: C, 60.25; H, 4.12; N, 6.36; S,
14.63.
Synergistic activity of maleimide with metal salts was also studied.
By assuming the linearity of the nematicidal assay, we used the method
reviewed by Berenbaum.35 The combination of two agents A and B is
termed (da and db), where da and db are the concentrations of A and B,
respectively. The effect is treated as a mathematical function E; thus
E(da,db) is the combined effect and E(da) and E(db) are the effects of
the individual agents A and B, respectively. Zero interactive
combination is observed when the summation of the effects of the
individual agents is not significantly different from the combination
effect, i.e., E(da,db) = E(da) + E(db). If the combination effect is smaller
or greater than the summation of the individual agents effects, the
combination is antagonistic or synergistic, respectively. The compar-
ison was made at two concentration levels, of maleimide and 1, 1 and 2
mg/L, in combination with copper and iron sulfates at different
concentration levels, 50 and 75 and 125 and 200 mg/L, respectively.
The experiment was conducted at 24 and 72 h and repeated twice. The
1-(4-Ethoxyphenyl)-1H-pyrrole-2,5-dione (22). Yield 75%, mp 78−
1
80 °C. H NMR (DMSO-d6): δ 7.38 (d, J = 8.5 Hz, 2H, Ar), 7.18 (s,
2H, CH), 6.98 (d, J = 8.5 Hz, 2H, Ar), 4.05 (q, J = 7.0 Hz, 2H, CH2),
1.32 (t, J = 7.0, 3H, CH3). IR (Nujol) 1793, 1763, 1713, 1666 cm−1.
HRMS (ESI-TOF) (m/z) [M + H]+ calcd for (C12H11NO3) 218.0811,
found 218.0810. Calcd: C, 66.85; H, 5.10; N, 6.45. Found: C, 66.83;
H, 5.12; N, 6.46.
1-(3-Chloro-4-methoxyphenyl)-1H-pyrrole-2,5-dione (23). Yield
1
56%, mp 132−135 °C. H NMR (DMSO-d6): δ 7.49 (s, 1H, Ar),
B
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
sum of dead J2s obtained from bioassays performed using solutions of
each test compound separately (expected) was compared with the
number of dead J2s observed and caused by immersion of M. incognita
J2 in the respective combinations.8
was performed by the SIMCA software, version 14.0 (Umetrics, Umea,
Sweden). Prior to analysis, data arrays were subjected to centering
through the centering unit variance. The matrices obtained were
subjected to multivariate analysis. Using software SIMCA-P, the
following analyses were made: principal components analysis (PCA)
and a discriminant regression method (PLS-DA) and its orthogonal
extension (OPLS-DA). The quality of the model has been validated on
the basis of the parameters R2X (change in X explained by the model),
R2Y (the total of Y explained), and Q2 (sum parameter in cross-
validation).38 A scatter plot of possible discriminant metabolites was
obtained using an S-plot, which combines covariance and correlation
loading profiles.
Sample Extraction. For the metabolomics analysis, nematode
polar metabolites extraction was performed after treating 300 juveniles
of M. incognita J2 in vitro with 8 mg/L of maleimide (1) in a total
volume of 200 μL. A negative control consisting of nematodes
immersed in tap water was prepared. After 24 h, test solutions were
transferred to 1.5 mL Eppendorf tubes and ultrasonicated with a
Vibracell cell disruptor (Labotal Scientific Equipment, Abu Ghosh,
Israel) for the nematode cuticle lysis. The ultrasonication was
performed twice with 20 s pulses at 60% amplitude (130 W, 20
kHz). Finally, 800 μL of tert-butyl methyl ether were added to the
samples, which were then vortexed for 1 min. After centrifugation at
18000 rpm at 25 °C for 15 min, the supernatant liquid was removed
and dried overnight in glass vials. Extracts were submitted to a
derivatization step for GC-MS analysis using a solution of methox-
amine chloride dissolved in pyridine (10 mg/mL). Then 80 μL of N-
methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) were added
after 17 h from the treatment. After 1 h, we added 50 μL of hexane
containing the internal standard 2,2,3,3-d4-succinic acid (2 mg/L).
Eight replications were made for every sample. The chromatographic
separation was performed on a Agilent 6850 gas chromatograph
coupled with a mass detector 5973 and a 7683B series injector
autosampler (Agilent Technologies, Palo Alto, CA). The resulting data
was analyzed using MSD ChemStation. The column was a 30 m ×
0.25 mm, 0.25 μm DB5-MS (Agilent Technologies, Palo Alto, CA),
and the injector temperature was set at 250 °C. The oven temperature
program was as follows: from 50 to 230 °C at 5 °C/min for 36 min
and kept at this temperature for 2 min. Helium was the carrier gas, at a
constant flow rate of 1 mL/min, and 1 μL of the sample was injected
in the splitless mode. The detector settings were as follows: ionization
voltage, 70 eV; scan rate, 2.91 scan/s; mass range, 50−550; transfer
line, 230 °C. Metabolites were identified by (a) comparison of their
relative retention times and mass fragmentation with those of
authentic standards and (b) computer matching against NIST98, as
well as retention indices as calculated according to Kovats, for the
series of alkanes C9−C24.
RESULTS AND DISCUSSION
■
We report for the first time the nematicidal activity of
maleimide derivatives. A series of maleimide and succinimide
derivatives were synthesized through the one-pot two-step
procedure described in Figure 1, and their nematicidal activity
Statistical Analysis. According to the Schneider Orellis formula,
we corrected the percentages of dead juveniles by eliminating the
natural death control samples (5% of total number of J2):36
corrected % = ((mortality % in treatment
− mortality % in control)
Figure 1. Synthetic route to maleimides. Reagents and conditions: (i)
CH2Cl2, 1 h, rt; (ii) (CH3CO)2O, NaOCOCH3, 2 h, 100 °C; (iii)
TEA, toluene, 36 h, reflux.
/(100 − mortality % in control)) × 100
(1)
and they were analyzed (ANOVA) after being combined over time
and means were averaged over experiments. Afterward, corrected
percentages of dead J2s were submitted to a nonlinear regression
analysis according to Seefeldt et al.37
was tested in vitro on second stage juveniles of the root-knot
nematode M. incognita. Initially, we explored the effectiveness of
the maleimide derivatives testing each compound at the
concentration of 100 mg/L after 72 h treatments (Table 1).
Compounds showing a paralyzing activity greater than 90%
were then subjected to EC50/72h determination (Table 2). For
comparison, abamectin and fosthiazate were used as chemical
eq 2
D − C
Y = C +
1 + eb log x − log EC
50
(2)
control, with EC50/72h of 2.1
respectively.
1.2 and 0.6
0.2 mg/L,
where the EC50 is the test compound concentration required for 50%
death/immotility of nematodes compared to natural death/immobility
in the control, C = the lower limit, D = the upper limit, and b = the
slope at the EC50. In the regression equation, the test compounds
concentration (% w/v) was the independent variable (x) and the
immotile J2 (percentage increase over water control) was the
dependent variable (y). The mean value of the six replicates was
used to the EC50 evaluation.
Multivariate Analysis. From the data obtained through the GC-
MS analysis, a matrix of 16 × 28 data composed of the samples
analyzed (eight controls and eight treated samples) and from the areas
of the chromatographic peaks (28 variables) was built. When a variable
showed an abnormal distribution, it was canceled using a logarithmic
transformation validated from the Skewness test; statistical analysis
The maleimide, 1, showed an EC50/72h of 2.6 1.3 mg/L
while the succinimide, 9, was not active. The N-ethylmaleimide,
2, N-isopropylmaleimide, 3, and N-isobutylmaleimide, 4,
showed 100% mortality in the primary screening experiment
and EC50/72h values in the 2.6−19 mg/L range (5.1 3.4, 5.1
3.4, and 19.0
9.0, respectively). On the contrary, reduced
activity is produced by the n-butyl derivative, 5, as well as by the
introduction of a long chain as in compounds, 6−8. Among N-
arylmaleimides, the 4-nitrophenyl, 11, the 4-chlorophenyl, 12,
and the 4-carboxyethylphenyl, 28, were the most effective
(EC50/72h = 23.8
3.0, 53.4
3.0, and 60.9
20.6,
C
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
position of a hydroxyl group produced maleimide 26, which
also showed a reduced nematicidal activity.
Table 1. Percentage Nematicidal Activity of Tested
Maleimide and Succinimide Derivatives at 100 mg/L after
72 h (n = 6)
Metabolomics is one of the latest omics sciences which
allows the unsupervised or supervised determination of low
molecular mass metabolites in biological samples. Recently, we
reported on the use of metabolomics in detecting upregulated
or downregulated metabolites in M. incognita treated with
different nematicidal arylhydrazones.6 Using a GC-MS
metabolomics approach, we detected different endogenous
metabolites such as carbohydrates, amino acids, fatty acids, and
monoacylglycerols. Statistical significant polar metabolites were
studied using the software SIMCA-P (Umetrics, Umea,
Sweden, ver. 14.0.0.1359). The first step was to perform a
PCA to examine interrelation between groups, clustering, and
outlier diagnostics among the samples. In the second step, a
PLS-DA was performed to maximize the difference of
metabolic profiles between treated and control samples and
allowing metabolite recognition. The final step of the statistical
analysis was to perform a supervised OPLS-DA with the goal to
separate samples in two clusters and identify potential
biomarkers among nematodes treated with maleimide at 8
mg/L for 24 h and the control group. Statistical parameters for
the OPLS-DA model were: Q2 = 0.63, R2X = 0.60, and R2Y =
0.80 (Figure 2). The permutation test parameters relative to
PLS-DA were: R2 = 0.61 and Q2 = −0.47, respectively.
Upregulated metabolites for M. incognita nematodes treated
with maleimide were in decreasing order oleic acid and palmitic
acid, while the downregulated metabolite was 1-monopalmitin.
Altered levels of fatty acids are probably related to the
monounsaturated fatty acids which are essential components of
membrane and storage lipids. Their synthesis depends on the
conversion of saturated fatty acids to unsaturated fatty acids by
Δ9-desaturases.39 Biosynthesis of oleic acid starting from
palmitic acid in our inhibition experiment with maleimide
may be a scavenger strategy of the nematodes to cope with
reactive oxygen species.40,41 Considering the small pool of
metabolites identified in this work, further metabolomic studies,
using different analytical platforms and different extraction
procedures, are needed to generate a full nematode
metabolome and contribute with a mechanistic assessment.
Moreover, considering that vma yeast mutants, lacking V-
ATPase subunits, are hypersensitive to multiple forms of
oxidative stress,42 suggesting an antioxidant role of V-ATPase,
we treated nematodes with two redox active metals, i.e., copper
and iron as sulfates known to be able to produce superoxide
radicals.43 In our experimental conditions, both metal salts were
highly toxic to M. incognita, with EC50s of 48.6 29.8 and 126
48 mg/L for copper and iron sulfate, respectively (Table 2).
compd
mortality (%) SD
compd
mortality (%) SD
maleimide (1)
100 0.0
100 0.0
100 0.0
100 0.0
28.0 8.2
35.0 6.6
40.0 7.1
15.7 9.7
15
16
17
18
19
20
21
22
23
24
25
26
27
28
6.9 5.0
12.0 4.0
7.7 3.9
2
3
4
11.0 9.7
8.9 3.0
5
6
7.5 2.9
7
18.8 4.8
20.6 10.0
11.0 5.4
22.8 7.5
11.3 2.5
40.5 8.0
8
a
succimide (9)
NA
a
10
11
12
13
14
NA
100 0.0
100 0.0
10.8 2.3
6.4 0.8
a
NA
92.3 3.3
a
NA = not active.
Table 2. EC50/72h of the Most Active Maleimide Derivatives
and Copper and Iron Sulfate Solutions
compd
EC50/72h SD (mg/L)
1
2.6 1.3
5.15.1 3.4
16.2 5.4
19.0 9.0
23.8 3.0
53.4 3.0
60.9 20.6
48.6 29.8
126 48
2
3
4
11
12
28
copper sulfate
iron sulfate
fosthiazate
abamectin
0.6 0.2
2.1 1.2
respectively). The shift of chlorine to the 2-position, 16, or the
introduction of a further chlorine atom, as in maleimides, 17−
19, led to a reduction in activity. The nematicidal activity
dropped to 6−20% of mortality after 72 h at 100 mg/L when a
4-nitro or a 4-chlorine group was replaced by a fluorine, 13,
trifluoromethyl, 15, ether, 21−22, or thioether, 20, groups.
Furthermore, the introduction of a nitro, 14, or a chlorine, 23,
group on the aryl ring failed to enhance the nematicidal activity
of maleimides, 13 and 21, respectively. The replacement of the
ester moiety of maleimide, 28, with a carboxyl group produced
the inactive derivative, 27. The introduction in the same
Figure 2. Score plot of OPLS-DA of nematodes treated with maleimide 1 (black circles) vs controls (white circles).
D
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Table 3. Synergistic Activity of Maleimide 1 in Combination with Cu2+ and Fe3+ after 24 and 72 h
mortality (%) SD
24 h
72 h
combination
concentration (mg/L)
expected
observed
expected
observed
1/Cu2+
1/50
1/75
2/50
2/75
2.2 2.6
25.7 5.5
11.4 9.5
35.2 9.0
84.6 8.7
61.7 4.2
93.5 5.8
100
8.8 2.5
100
100
100
100
100
100
100
1/Fe3+
1/125
1/200
2/125
2/200
3.6 2.7
34.6 5.9
12.9 6.2
100
35.1 6.5
33.2 7.0
95.6 3.5
100
7.2 2.1
89.3 7.3
30.7 3.4
90.0 5.6
78.2 3.7
93.9 5.2
100
100
Furthermore, we measured the synergistic activity of maleimide,
1, with copper and iron sulfate. After 72 h, we observed a
synergistic nematicidal activity with a strength increased by a
factor of 10 (Table 3). Consistent with our previous
experiments with (E)-1-((3-methylthiophen-2-yl)methylene)-
2-phenylhydrazine,6 when nematodes where treated with the V-
ATPase inhibitor 1 at 2 and 6 h, we measured a 3.5-fold
increase in the excretion of ammonium (Table 4). On the other
REFERENCES
■
(1) Sikora, R.; Carter, W., Nematode interactions with fungal and
bacterial plant pathogens: fact or fantasy? In istas on Nematology: A
Commemoration of the Twenty-Fifth Anniversary of the Society of
Nematologists; Veech, J. A., Dickson, D. W., Eds.; Society of
Nematologists: Hyattsville, MD, 1987; pp 307−312.
(2) Sasser, J.; Freckman, D., World perspective on nematology: the
role of the society. In Vistas on Nematology: A Commemoration of the
Twenty-Fifth Anniversary of the Society of Nematologists; Veech, J. A.,
Dickson, D. W., Eds.; Society of Nematologists: Hyattsville, MD, 1987.
Table 4. Excretion Levels of Some Cations when Nematodes
were Treated with Maleimide 1 at 8 mg/L after 2 and 6 h
(3) Lourenco̧ -Tessutti, I. T.; Souza Junior, J. D. A.; Martins-de-Sa, D.;
Viana, A. A. B.; Carneiro, R. M. D. G.; Togawa, R. C.; de Almeida-
Engler, J.; Batista, J. A. N.; Silva, M. C. M.; Fragoso, R. R.; Grossi-de-
Sa, M. F. Knock-down of heat-shock protein 90 and isocitrate lyase
gene expression reduced root-knot nematode reproduction. Phytopa-
thology 2015, 105, 628−637.
concentration (mg/L)
cation
Na+
time (h)
1
control
2
6
2
6
2
6
2
6
2
6
2.9 0.3
2.5 0.1
2.6 0.1
2.6 0.2
(4) Abad, P.; Gouzy, J.; Aury, J.-M.; Castagnone-Sereno, P.; Danchin,
E. G. J.; Deleury, E.; Perfus-Barbeoch, L.; Anthouard, V.; Artiguenave,
F.; Blok, V. C.; Caillaud, M.-C.; Coutinho, P. M.; Dasilva, C.; De Luca,
F.; Deau, F.; Esquibet, M.; Flutre, T.; Goldstone, J. V.; Hamamouch,
N.; Hewezi, T.; Jaillon, O.; Jubin, C.; Leonetti, P.; Magliano, M.;
Maier, T. R.; Markov, G. V.; McVeigh, P.; Pesole, G.; Poulain, J.;
Robinson-Rechavi, M.; Sallet, E.; Segurens, B.; Steinbach, D.; Tytgat,
T.; Ugarte, E.; van Ghelder, C.; Veronico, P.; Baum, T. J.; Blaxter, M.;
Bleve-Zacheo, T.; Davis, E. L.; Ewbank, J. J.; Favery, B.; Grenier, E.;
Henrissat, B.; Jones, J. T.; Laudet, V.; Maule, A. G.; Quesneville, H.;
Rosso, M.-N.; Schiex, T.; Smant, G.; Weissenbach, J.; Wincker, P.
Genome sequence of the metazoan plant-parasitic nematode
Meloidogyne incognita. Nat. Biotechnol. 2008, 26, 909−915.
(5) D’Addabbo, T.; Carbonara, T.; Leonetti, P.; Radicci, V.; Tava, A.;
Avato, P. Control of plant parasitic nematodes with active saponins
and biomass from Medicago sativa. Phytochem. Rev. 2011, 10, 503−519.
(6) Eloh, K.; Demurtas, M.; Deplano, A.; Ngoutane Mfopa, A.;
Murgia, A.; Maxia, A.; Onnis, V.; Caboni, P. In vitro nematicidal
activity of aryl hydrazones and comparative GC-MS metabolomics
analysis. J. Agric. Food Chem. 2015, 63, 9970−9976.
(7) Caboni, P.; Aissani, N.; Demurtas, M.; Ntalli, N.; Onnis, V.
Nematicidal activity of acetophenones and chalcones against
Meloidogyne incognita and structure−activity considerations. Pest
Manage. Sci. 2016, 72, 125.
(8) Ntalli, N.; Oplos, C.; Michailidis, M.; Thanasenaris, A.; Kontea,
D.; Caboni, P.; Tsiropoulos, N. G.; Menkissoglu-Spiroudi, U.;
Adamski, Z. Strong synergistic activity and egg hatch inhibition by
(E,E)-2,4-decadienal and (E)-2-decenal in Meloidogyne species. J. Pest
Sci. 2016, 89, 1−15.
+
NH4
0.057 0.007
0.076 0.012
1.1 0.5
0.016 0.004
0.020 0.003
0.7 0.1
K+
0.6 0.0
0.6 0.1
Mg2+
Ca2+
1.9 0.1
1,9 0.1
1.8 0.2
1,7 0.1
6.9 0.8
7.0 0.5
6.2 0.5
6.7 0.3
hand, levels of sodium, potassium, magnesium, and calcium
were not altered. Thus, assuming that maleimide derivatives
were able to inhibit the nematode V-ATPase at low EC50 values
and considering that their strength is greatly augmented when
used in combination with redox metals such as copper and iron,
these compounds may potentially be used to develop new
active ingredients with nematicidal properties or be used in the
control of root knot nematodes in the field.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 070 6758617. Fax: +39 070 6758612. E-mail:
caboni@unica.it.
■
Notes
The authors declare no competing financial interest.
(9) Aissani, N.; Urgeghe, P. P.; Oplos, C.; Saba, M.; Tocco, G.;
Petretto, G. L.; Eloh, K.; Menkissoglu-Spiroudi, U.; Ntalli, N.; Caboni,
P. Nematicidal activity of the volatilome of Eruca sativa on Meloidogyne
incognita. J. Agric. Food Chem. 2015, 63, 6120−6125.
(10) Jang, J. Y.; Le Dang, Q.; Choi, Y. H.; Choi, G. J.; Jang, K. S.;
Cha, B.; Luu, N. H.; Kim, J. C. Nematicidal activities of 4-quinolone
ACKNOWLEDGMENTS
■
We thank Dr. Mariano Medda and Dr. Giorgia Sarais for
helpful suggestions. This study was supported by the Regione
Autonoma della Sardegna (Project L.R. 7/2007, No.
2012_CRP-59473).
E
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
alkaloids isolated from the aerial part of Triumfetta grandidens against
Meloidogyne incognita. J. Agric. Food Chem. 2015, 63, 68−74.
(11) Ntalli, N. G.; Vargiu, S.; Menkissoglu-Spiroudi, U.; Caboni, P.
Nematicidal carboxylic acids and aldehydes from Melia azedarach
fruits. J. Agric. Food Chem. 2010, 58, 11390−11394.
(12) Caboni, P.; Saba, M.; Tocco, G.; Casu, L.; Murgia, A.; Maxia, A.;
Menkissoglu-Spiroudi, U.; Ntalli, N. Nematicidal activity of mint
aqueous extracts against the root-knot nematode Meloidogyne incognita.
J. Agric. Food Chem. 2013, 61, 9784−9788.
(13) Aissani, N.; Tedeschi, P.; Maietti, A.; Brandolini, V.; Garau, V.
L.; Caboni, P. Nematicidal activity of allylisothiocyanate from
horseradish (Armoracia rusticana) roots against Meloidogyne incognita.
J. Agric. Food Chem. 2013, 61, 4723−4727.
(14) Cavoski, I.; Chami, Z. A.; Bouzebboudja, F.; Sasanelli, N.;
Simeone, V.; Mondelli, D.; Miano, T.; Sarais, G.; Ntalli, N.; Caboni, P.
Melia azedarach controls Meloidogyne incognita and triggers plant
defense mechanisms on cucumber. Crop Prot. 2012, 35, 85−90.
(15) Putri, S. P.; Nakayama, Y.; Matsuda, F.; Uchikata, T.; Kobayashi,
S.; Matsubara, A.; Fukusaki, E. Current metabolomics: Practical
applications. J. Biosci. Bioeng. 2013, 115, 579−589.
(16) Tufi, S.; Stel, J. M.; de Boer, J.; Lamoree, M. H.; Leonards, P. E.
G. Metabolomics to explore imidacloprid-induced toxicity in the
central nervous system of the freshwater snail Lymnaea stagnalis.
Environ. Sci. Technol. 2015, 49, 14529−14536.
(17) Coghlan, M. P.; Culbert, A. A.; Cross, D. A. E.; Corcoran, S. L.;
Yates, J. W.; Pearce, N. J.; Rausch, O. L.; Murphy, G. J.; Carter, P. S.;
Roxbee Cox, L.; Mills, D.; Brown, M. J.; Haigh, D.; Ward, R. W.;
Smith, D. G.; Murray, K. J.; Reith, A. D.; Holder, J. C. Selective small
molecule inhibitors of glycogen synthase kinase-3 modulate glycogen
metabolism and gene transcription. Chem. Biol. 2000, 7, 793−803.
(18) Warnecke, A.; Fichtner, I.; Garmann, D.; Jaehde, U.; Kratz, F.
Synthesis and biological activity of water-soluble maleimide derivatives
of the anticancer drug carboplatin designed as albumin-binding
prodrugs. Bioconjugate Chem. 2004, 15, 1349−1359.
(19) Cechinel Filho, V.; Pinheiro, T.; Nunes, R. J.; Yunes, R. A.;
Cruz, A. B.; Moretto, E. Antibacterial activity of N-phenylmaleimides,
N-phenylsuccinimides and related compounds. Structure-activity
relationships. Farmaco 1994, 49, 675−677.
(20) Chen, X. L.; Zhang, L. J.; Li, F. G.; Fan, Y. X.; Wang, W. P.; Li,
B. J.; Shen, Y. C. Synthesis and antifungal evaluation of a series of
maleimides. Pest Manage. Sci. 2015, 71, 433−440.
derivatives as selective monoglyceride lipase inhibitors. J. Med. Chem.
2009, 52, 7410−7420.
(30) Miller, C. W.; Jonsson, E. S.; Hoyle, C. E.; Viswanathan, K.;
̈
Valente, E. J. Evaluation of N-aromatic maleimides as free radical
photoinitiators: a photophysical and photopolymerization character-
ization. J. Phys. Chem. B 2001, 105, 2707−2717.
(31) Budke, B.; Kalin, J. H.; Pawlowski, M.; Zelivianskaia, A. S.; Wu,
M.; Kozikowski, A. P.; Connell, P. P. An optimized RAD51 inhibitor
that disrupts homologous recombination without requiring Michael
acceptor reactivity. J. Med. Chem. 2013, 56, 254−263.
(32) Hamelin-Morrissette, J.; Cloutier, S.; Girouard, J.; Belgorosky,
D.; Eijan, A. M.; Legault, J.; Reyes-Moreno, C.; Berube, G.
́
Identification of an anti-inflammatory derivative with anti-cancer
potential: The impact of each of its structural components on
inflammatory responses in macrophages and bladder cancer cells. Eur.
J. Med. Chem. 2015, 96, 259−268.
(33) Taylor, A.; Sasser, J. Biology, Identification and Control of Root-
Knot Nematodes (Meloidogyne Species); NC State University Graphics:
Raleigh, NC, 1978.
(34) Ekanayake, H.; Di Vito, M. Influence of root leachates and
temperatures on egg hatch of Meloidogyne species. Nematol. Mediterr.
1984, 12, 119−127.
(35) Berenbaum, M. C. What is synergy? Pharmacol. Rev. 1989, 41,
93−141.
(36) Puntener, W.; Zahner, O. Manual for Field Trials in Plant
̈
Protection; Ciba-Geigy: Basel, Switzerland, 1981.
(37) Seefeldt, S. S.; Jensen, J. E.; Fuerst, E. P. Log-logistic analysis of
herbicide dose−response relationships. Weed Technol. 1995, 218−227.
(38) Veselkov, K. A.; Vingara, L. K.; Masson, P.; Robinette, S. L.;
Want, E.; Li, J. V.; Barton, R. H.; Boursier-Neyret, C.; Walther, B.;
Ebbels, T. M.; Pelczer, I.; Holmes, E.; Lindon, J. C.; Nicholson, J. K.
Optimized preprocessing of ultra-performance liquid chromatogra-
phy/mass spectrometry urinary metabolic profiles for improved
information recovery. Anal. Chem. 2011, 83, 5864−5872.
(39) Brock, T. J.; Browse, J.; Watts, J. L. Fatty acid desaturation and
the regulation of adiposity in Caenorhabditis elegans. Genetics 2007,
176, 865−875.
(40) Guerzoni, M. E.; Lanciotti, R.; Cocconcelli, P. S. Alteration in
cellular fatty acid composition as a response to salt, acid, oxidative and
thermal stresses in Lactobacillus helveticus. Microbiology 2001, 147,
2255−2264.
(41) Horikawa, M.; Sakamoto, K. Fatty-acid metabolism is involved
in stress-resistance mechanisms of Caenorhabditis elegans. Biochem.
Biophys. Res. Commun. 2009, 390, 1402−1407.
(21) Kirino, O.; Rubin, B.; Casida, J. E. N-Phenyl-maleimides,-
isomaleimides and-maleamic acids as selective herbicide antidotes.
Agric. Biol. Chem. 1985, 49, 267−268.
(22) Griffith, C. J.; Rea, P. A.; Blumwald, E.; Poole, R. J. Mechanism
of stimulation and inhibition of tonoplast H+-ATPase of Beta vulgaris
by chloride and nitrate. Plant Physiol. 1986, 81, 120−125.
(23) Randall, S. K.; Sze, H. Probing the catalytic subunit of the
tonoplast H+-ATPase from oat roots. Binding of 7-chloro-4-nitro-
benzo-2-oxa-1, 3,-diazole to the 72-kilodalton polypeptide. J. Biol.
Chem. 1987, 262, 7135−7141.
(42) Kane, P. M. The long physiological reach of the yeast vacuolar
H+-ATPase. J. Bioenerg. Biomembr. 2007, 39, 415−421.
(43) Halliwell, B.; Gutteridge, J. Oxygen toxicity, oxygen radicals,
transition metals and disease. Biochem. J. 1984, 219, 1.
(24) Feng, Y.; Forgac, M. Inhibition of vacuolar H (+)-ATPase by
disulfide bond formation between cysteine 254 and cysteine 532 in
subunit A. J. Biol. Chem. 1994, 269, 13224−13230.
(25) Hunt, I. E.; Sanders, D. The kinetics of N-ethylmaleimide
inhibition of a vacuolar H+-ATPase and determination of nucleotide
dissociation constants. Plant Physiol. 1996, 110, 97−103.
(26) Akalay, D.; Durner, G.; Bats, J. W.; Gobel, M. W. C2-symmetric
̈
̈
bisamidines: Chiral Brønsted bases catalysing the Diels-Alder reaction
of anthrones. Beilstein J. Org. Chem. 2008, 4, 28.
(27) Reddy, P. Y.; Kondo, S.; Toru, T.; Ueno, Y. Lewis acid and
hexamethyldisilazane-promoted efficient synthesis of N-alkyl-and N-
arylimide derivatives. J. Org. Chem. 1997, 62, 2652−2654.
(28) Haval, K. P.; Mhaske, S. B.; Argade, N. P. Cyanuric chloride:
decent dehydrating agent for an exclusive and efficient synthesis of
kinetically controlled isomaleimides. Tetrahedron 2006, 62, 937−942.
(29) Matuszak, N.; Muccioli, G. G.; Labar, G.; Lambert, D. M.
Synthesis and in vitro evaluation of N-substituted maleimide
F
DOI: 10.1021/acs.jafc.6b02250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX