In Vitro Nematicidal Activity of Aryl Hydrazones and Comparative GC

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In vitro nematicidal activity of aryl hydrazones and comparative GC-MS metabolomics analysis Kodjo Eloh, Monica Demurtas, Alessandro Deplano, Alvine Ngoutane Mfopa, Antonio Murgia, Andrea Maxia, Valentina Onnis, and Pierluigi Caboni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04815 • Publication Date (Web): 03 Nov 2015 Downloaded from http://pubs.acs.org on November 8, 2015

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Journal of Agricultural and Food Chemistry

In vitro nematicidal activity of aryl hydrazones and comparative GC-MS metabolomics analysis Kodjo Eloh, Monica Demurtas, Alessandro Deplano, Alvine Ngoutane Mfopa, Antonio Murgia, Andrea Maxia, Valentina Onnis, and Pierluigi Caboni* Department of Life and Environmental Sciences, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy

Corresponding Author * Phone: +39 070 6758617. Fax: +39 070 6758612 E-mail: [email protected] Running Title: Nematicidal activity of arylhydrazones

1 ACS Paragon Plus Environment


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ABSTRACT

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A series of aryl hydrazones were synthesized and in vitro assayed for their activity on the root-

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knot nematode Meloidogyne incognita. The phenylhydrazones of thiophene-2-carboxyaldehyde

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5, 3-methyl-2-thiophenecarboxyaldehyde 6 and salicylaldehyde 2, were the most potent with

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EC50/48h values of 16.6 ± 2.2, 23.2 ± 2.7, and 24.3 ± 1.4 mg/L, respectively. A GC-MS

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metabolomics analysis, after in vitro nematode treatment with hydrazone 6 at 100 mg/L for 12 h,

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revealed elevated levels of fatty acids such as lauric acid, stearic acid, 2-octenoic acid and

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palmitic acid. While control samples showed highest levels of monoacylglycerols such us

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monostearin and 2-monostearin. Surprisingly, after 2h treatment with hydrazone 6, nematodes

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excreted three times the levels of ammonia eliminated in the same conditions by controls. Thus,

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phenylhydrazones may represent a good scaffold in the discovery and synthesis of new

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nematicidal compounds while a metabolomics approach may be helpful in understanding their

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mechanisms of toxicity and mode of action.

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KEYWORDS: M. incognita, ammonia, fatty acid, monoacylglycerols, 2-octenoic acid

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INTRODUCTION

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Root-knot nematodes are soil-born plant parasites which second-stage juveniles (J2) penetrate

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crop roots to establish a permanent feeding site. Subsequently, infected hosts undergo formation

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of knots in root tissues thus affecting the plant uptake of nutrients and water. In the case of

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tubers, the infection makes long-term storage impossible because taproots begin to rot due to

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fungal infection associated with nematode gall degradation.1 Nematode infestation is responsible

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of annual yield losses estimated to roughly $100 billion USD worldwide.2 Different methods are

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used in field to control nematodes among which crop rotation, conventional chemical, botanical

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nematicides3 and biological controls, soil solarization and the use of resistant crop varieties. Crop

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rotation is a very difficult method for controlling Meloidogyne spp. because of the wide host

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range of this genera.4 While synthetic methyl bromide, a highly efficacious fumigant used for

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decades on more than 100 crops, to control soil borne plant pathogens was listed as a

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stratospheric ozone depletory compound making use of methyl bromide alternatives a necessity.5

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On the other hand, planting cultivars that are highly resistant to these organisms places extensive

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selection pressure on the target species and affects nontarget species as well. Problems

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encountered with long-term planting of cultivars resistant to nematodes include shifts in

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nematode races or species and the occurrence of multiple species of nematodes within the same

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field. Moreover, nematicides in conjunction with resistant cultivars may be used to limit damage

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by multiple nematode species.6 For all these reasons, it is mandatory the search for new

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nematicides lead by optimizing a target-diverse approach.7,8

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Arylhydrazones (R1R2C=NNHAr) are key compounds for drug design, possible ligands for metal

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complexes, organocatalysis and preparation of heterocyclic rings.9 Carbonyl cyanide



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phenylhydrazones have been known as protonophore and inhibitor of oxidative phosphorylation

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for many years and used as insecticides.10,11 Although these compounds are potent mitochondria

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uncouplers of the oxidative phosphorylation for various organisms including mammals, plants

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and insects,12 they have not found any real agrochemical application. Interestingly, the

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replacement of malononitrile with an isoxazolone group, led to a well-known class of fungicide

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(drazoxolon) used for the control of powdery mildews.13,14

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Metabolomics is a dynamic and growing field of research that involves qualitative and

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quantitative measurement of metabolic response of a living organism to a genetic modification,

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external stimulus, and/or stressor. GC-MS-based metabolomics methods enable detection,

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identification and quantification of metabolic changes in organisms.15,16 A metabolomics

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approach to study Eisenia fetida earthworm after exposure with sub lethal nanoparticle was

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reported by Lankadurai et al. where different amino acids and sugars were indicated as potential

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bio indicators of exposure to C60 nanoparticles.17 Similarly, Ratnasekhar, Ch H et al. reported a

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metabolomics study on Caenorhabditis elegans exposed to titanium oxide nanoparticles.18 In

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addition, Copes et al. recently reported a study on the proteome and metabolome changes with

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aging in a C. elegans model.19

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In this study, we report the: 1) chemical synthesis of substituted aryl hydrazones, 2) in vitro

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nematicidal activity of hydrazones on M. incognita, 3) in vitro GC-MS metabolomics analysis of

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nematodes after treatment with hydrazone 6.

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MATERIALS AND METHODS

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General Methods. Melting points were determined on a Stuart Scientific Melting point SMP1

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and are uncorrected. Proton NMR spectra were recorded on a Varian Inova 500 spectrometer.

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The chemical shift are reported in part per million (δ, ppm) downfield from tetramethylsilane

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(TMS), which was used as internal standard. Infrared spectra were obtained with a Bruker Vector

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22 spectrophotometer. The purity of tested compounds was determined by combustion elemental

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analyses conducted with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested

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compounds yielded data consistent with a purity of at least 95% as compared with the theoretical

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values. Reaction courses and product mixtures were routinely monitored by thin layer

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chromatography (TLC) on E. Merck TLC plates coated with silica gel 60 F254 (0.25 mm layer

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thickness). TLC visualization was carried out using an UV lamp.

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Materials. All synthetic precursors and solvents were purchased from Sigma Aldrich (Milan,

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Italy). Hydrazones

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available or obtained as previously described.

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General procedure for the synthesis of arylhydrazones. A mixture of arylhydrazine

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hydrochloride (1 mmol), triethylamine (0.1 mL, 1.1 mmol) and the appropriate aldehyde (1

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mmol) in EtOH (10 mL) was refluxed for 4 h. After cooling the formed precipitate was filtered

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off and purified by crystallization from the adequate solvent to give the hydrazone derivatives.

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(E)-1-(4-methylbenzylidene)-2-phenylhydrazine (4) Yield 66%. mp 148-149 °C (EtOH). 1H

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NMR (DMSO-d6): δ 6.69-7.13 (m, 3H, Ar) 7.51 (d, 1H, J = 14.9 Hz, CH), 7.66 (m, 1H, Ar), 8.15

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(d, J = 8.2 Hz, 2H, Ar), 8.03 (d, 1H, J = 14.9 Hz, CH), 8.22 (d, J = 8.5 Hz, 2H, Ar), 10.26 (s, 1H,

1,20 2,21 3,22 7,23 8,24 9,25 10,26 11,2713,28 and 1729 were commercially



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OH). IR (Nujol) 1603, 1515 cm-1. Anal. (C14H14N2) 210.27. Calcd. C, 79.97; H, 6.71; N, 13.32.

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Found C, 80.03; H, 6.70; N, 13.35.

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(E)-1-phenyl-2-(thiophen-2-ylmethylene)hydrazine (5) Yield 40%. mp 126-127 °C (2-PrOH). 1H

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NMR (DMSO-d6): δ 6.71 (t, J=7.5 Hz, 1H, Ar), 6.96 (d, J=8.0 Hz, 2H, Ar), 7.01-7.19 (m, 4H,

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Ar), 7.42 (s, 1H, Ar), 8.02 (s, 1H, CH), 10.24 (s, 1H, NH). IR (Nujol) 3325, 1603, 1536 cm-1.

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Anal. (C11H10N2S) 202.28 Calcd. C, 65.32; H, 4.98; N, 13.85. Found C, 65.26; H, 5.00; N,

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13.88.

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(E)-1-((3-methylthiophen-2-yl)methylene)-2-phenylhydrazine (6) Yield 70%. mp 110-112 °C (lit

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113-114 °C 26). 1H NMR (DMSO-d6): δ 2.27 (s, 3H, CH3), 6.702-7.21 (m, 6H, Ar), 7.35 (s, 1H,

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Ar), 8.07 (s, 1H, CH), 10.23 (s, 1H, NH). IR (Nujol) 3291, 1600, 1508 cm-1. Anal. (C12H12N2S)

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216.30 Calcd. C, 66.63; H, 5.59; N, 12.95. Found C, 66.58; H, 5.61; N, 12.91.

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(E)-1-benzylidene-2-(4-nitrophenyl)hydrazine (10) Yield 50%. mp 186-188 °C (EtOH). 1H NMR

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(DMSO-d6): δ 7.17 (d, J=8.0 Hz, 2H, Ar), 7.37 (t, J=7.5 Hz, 1H, Ar), 7.41 (d, J=8.0 Hz, 1H, Ar),

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7.43 (d, J=7.5Hz, 1H, Ar), 7.72 (d, J=8.5Hz, 2H, Ar), 8.04 (s, 1H, CH), 8.13 (d, J=8.5 Hz, 2H,

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Ar). IR (Nujol) 3259, 1587, 1544 cm-1. Anal. (C13H11N3O2) 241.25 Calcd. C, 64.72; H, 4.60; N,

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17.42. Found C, 64.78; H, 4.58; N, 17.45.

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(E)-2-((2-(o-tolyl)hydrazono)methyl)phenol (12) Yield 66%. mp 106-108 °C (lit 110-111 °C19).

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1

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7.11-7.19 (m, 4H, Ar), 7.50 (d, J=7.5Hz, 1H, Ar), 8.41 (s, 1H, CH), 9.59 (s, 1H, NH), 10.60 (s,

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1H, OH). IR (Nujol) 3343, 1619, 1598, 1586, 1567 cm-1. Anal. (C14H14N2O) 226.27 Calcd. C,

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74.31; H, 6.24; N, 12.38. Found C, 74.26; H, 6.26; N, 12.35.

H NMR (DMSO-d6): δ 2.21 (s, 3H, CH3), 6.71 (t, J=7.5Hz, 1H, Ar), 6.85-6.89 (m, 2H, Ar)


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(E)-2-((2-(2-ethylphenyl)hydrazono)methyl)phenol (14) Yield 42%. mp 45-47 °C (n-hexane). 1H

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NMR (DMSO-d6): δ 1.15 (d, J=8Hz, 3H, CH3), 2.62 (t, J=8Hz, 2H, CH2) 6.75-6.78 (m, 1H, Ar),

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6.85-6.88 (m, 2H, Ar) 7.11-7.21 (m, 4H, Ar), 7.50 (m, 1H, Ar), 8.42 (s, 1H, CH), 9.64 (s, 1H,

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NH), 10.60 (s, 1H, OH). IR (Nujol) 3346, 1622, 1604, 1587 cm-1. Anal. (C15H16N2O) 240.30

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Calcd. C, 74.97; H, 6.71; N, 11.66. Found C, 75.06; H, 6.74; N, 11.62.

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(E)-2-((2-(2-(trifluoromethyl)phenyl)hydrazono)methyl)phenol (15) Yield 50%. mp 106-108 °C

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(2-PrOH). 1H NMR (DMSO-d6): δ 6.85-6.94 (m, 3H, Ar), 7.19 (t, J=7.5Hz, 1H, Ar), 7.51-755

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(m, 2H, Ar), 7.59-7.63 (m, 2H, Ar), 8.63 (s, 1H, CH), 9.78 (s, 1H, NH), 10.22 (s, 1H, OH). IR

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(Nujol) 3398, 1610, 1591, 1532 cm-1. Anal. (C14H11F3N2O) 280.25 Calcd. C, 60.00; H, 3.96; N,

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10.00. Found C, 60.06; H, 3.95; N, 10.02.

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(E)-2-((2-(2-fluorophenyl)hydrazono)methyl)phenol (16) Yield 50%. mp 112-114 °C (lit. 126

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°C27). 1H NMR (DMSO-d6): δ 6.85-6.94 (d, J=5Hz, 1H, Ar), 6.83 (t, J=7Hz, 1H, Ar), 6.90 (d,

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J=8Hz, 1H, Ar), 7.08-7.17 (m, 3H, Ar), 7.31 (t, J=8Hz, 1H, Ar), 7.55 (d, J=7.5Hz, 1H, Ar), 8.40

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(s, 1H, CH), 10.28 (s, 1H, NH), 10.62 (s, 1H, OH). IR (Nujol) 3309, 2605, 2499, 1629, 1540 cm-

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1

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12.22.

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Nematode population. A population of M. incognita race30 was reared on susceptible tomato

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plants (Solanum lycopersicum L.) (cv. Rutgers) in a greenhouse in Cagliari, Italy, for two months

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at 25 ± 2 °C. Infested plants were uprooted and roots with numerous large galls and egg masses

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were gently washed free of adhering soil. Then roots were cut into 2 cm pieces and egg masses

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handy picked from them. Each batch containing 20,000 eggs were placed on 2 cm diameter

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sieves (215 µm) and each sieve was put in a 3.5 cm diameter Petri dish. Two mL of distilled

. Anal. (C13H11FN2O) 230.24 Calcd. C, 67.82; H, 4.82; N, 12.17. Found C, 67.77; H, 4.84; N,



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water, natural hatching agent, sufficient to cover egg masses, were then added to the batches to

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allow eggs hatch. The dishes were incubated in a growth cabinet at 25 °C.31 All second-stage

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juveniles (J2) hatching in the first 3 days were discarded after 24 h or more J2s were collected

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and used in the experiments.

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Nematicidal assay. The nematicidal activity of the hydrazones, in terms of nematode juveniles’

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motility suppression, was tested, and the EC50 values were calculated. Stock solutions of pure

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compounds were prepared by dilution with DMSO, whereas working solutions were obtained by

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dilution with distilled water containing the polysorbate surfactant 20 (Tween-20). Final

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concentrations of DMSO and Tween-20 in each well never exceeded 2 and 0.3% v/v,

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respectively, because preliminary trials showed that the motility of nematodes exposed at those

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concentration levels was similar to the motility of nematodes maintained in distilled water.

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Distilled water, as well as a mixture of water with DMSO and Tween-20 at concentrations

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equivalent to those in the treatment wells, were used as controls. Thirty juveniles were used per

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each treatment well in Cellstar 96-well plates (Greiner bio-one). The plates were covered to

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prevent evaporation and kept in dark conditions at 25°C. Moreover, at that point, nematodes

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were moved to plain water after washing in tap water through a 20 µm pore screen to remove

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excess test compound. Juveniles were separated into two distinct categories, motile or immotile,

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under an inverted microscope (Zeiss, West Germany) at 40× after 48h. Assessments were made

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by pricking the juvenile body with a needle, and they were counted. Paralyzed nematodes that

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never regained movement after transfer in water and pricking were considered to be dead. Every

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compound was replicated six times and the experiment repeated at least twice.


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Sample extraction. Nematodes polar metabolites extraction for the metabolomics analysis was

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performed after in vitro treating 250 J2 of M. incognita with 100 mg/L of hydrazone 6 with a

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total volume of 200 µL. A negative control consisting of nematodes treated with 1% DMSO was

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prepared. After 12 h, test solutions were transferred to 1.5 mL Eppendorf tubes and

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ultrasonicated with a Vibracell cell disruptor (Labotal Scientific Equipment, Abu Ghosh, Israel)

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for the nematode cuticle lysis. Ultrasonication was made twice with three times pulse of 20

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seconds each at 60% of amplitude (130 Watt, 20 KHz). Finally, 800 µL of tert-butylmethylether

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were added and vortexed for 1 min. Samples were centrifuged at 18,000 rpm at 25 °C for 15 min

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and the liquid supernatant was taken and dried overnight in vials under gentle nitrogen steam.

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Dried extracts were derivatized for GC-MS analysis with a solution of methoxamine chloride

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dissolves in pyridine at 10 mg/mL. After 17 h, 80 µL of N-methyl-N-(trimethylsilyl)

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trifluoroacetamide (MSTFA) were added. After 1 hour, 50 µL of hexane containing 20 mg/L of

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2,2,3,3-d4-succinic acid- as internal standard were added. Four replications were done for every

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sample and the experiment was repeated twice at different times. The chromatographic

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separation of the metabolites for component identification purposes was performed on an Agilent

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Technologies 6850 gas chromatograph coupled with a mass detector 5973 and a 7683B Series

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Injector autosampler, and the injection was performed in splitless mode. The resulting data was

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elaborated using MSD ChemStation. The column was 5% phenylmethylpolysyloxane (30 m x

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0.25 mm; film thickness 0.25 µm). Injector temperature was kept at 250 °C. The oven

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temperature was programmed as follows: from 50 to 230 °C (5 °C/min in 36 min) and kept at

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this temperature for 2 min. The carrier gas was helium with a flow of 1 mL/ min; and 1 µL of the

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sample was injected. The mass detector settings were as follows: ionization voltage, 70 eV; scan

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rate, 2.91 scan/s; mass range, 50-550; transfer line, 230 °C. The components of the samples were 9 ACS Paragon Plus Environment


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identified by (a) comparison of their relative retention times and mass fragmentation with those

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of authentic standards and (b) computer matching against NIST98, as well as retention indices as

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calculated according to Kovats, for alkanes C9-C24 compared with those reported-by Adams.32

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Quantitative analysis of each component was carried out with an external standard method when

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available.

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Statistical Analysis. The percentages of dead J2 were corrected by eliminating the natural death

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in the water Tween 20 0,3%/DMSO (2:98 v/v) control (5% of total number of J2) according to

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the Schneider Orellis formula:33

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corrected % =

% % %

× 100 (1)

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and they were analyzed (ANOVA) after being combined over time. Since ANOVA indicated no

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significant treatment by time interaction, means were averaged over experiments. Corrected

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percentages of death J2 treated with tested compounds were subjected to nonlinear regression

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analysis using the loglogistic equation proposed by Seefeldt et al.34

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Y=C+

! "#$ %&"#$ '()*

(2)

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where C = the lower limit, D = the upper limit, b = the slope at the EC50, and EC50 = the test

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compounds concentration required for 50% death/immotility of nematodes after elimination of

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the control (natural death/immotility). In the regression equation, the test compounds

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concentration (% w/v) was the independent variable (x) and the immotile J2 (percentage increase

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over water control) was the dependent variable (y). The mean value of the six replicates per

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compound concentration and immersion period was used to calculate the EC50 value.


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Multivariate analysis. From the data obtained through the GC-MS analysis a matrix of 8 X 28

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data composed of the analyzed samples (4 controls and 4 treated samples) and from the areas of

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the chromatographic peaks (28 variables). The normalization for the internal standard was used.

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When a variable showed an abnormal distribution, was adjusted using a logarithmic

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transformation validated from the Skewness correction using SIMCA-P software (version 14.0,

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Umetrics, Umea, Sweden). Prior to analysis, data set were subjected to the unit variance

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centering. The matrices obtained were subjected to multivariate analysis. Using the software

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SIMCA-P principal components analysis (PCA) and a discriminant analysis OPLS-DA was

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performed. The quality of the model had been validated on the basis of the parameters R2X

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(change in X explained by the model), R2Y (the total of Y explained) and Q2 sum parameter in

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cross-validation. The investigation of the discriminant variables was performed using the

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loadings analysis.

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RESULTS AND DISCUSSION

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Seventeen aryl hydrazones were synthesized and their nematicidal activity was in vitro tested on

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juveniles of the root-knot nematode M. incognita. With the aim of exploring the potency of

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synthetic compounds, the percent nematicidal activity of hydrazones was in vitro determined at

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100 mg/L after 48h of treatment (Table 1). The most active compounds were 1, 2, 3, 5, and 6

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further tested for the nematicidal paralysis experiments. EC50/48h values for tested compounds are

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reported in Table 2. The benzaldehyde phenylhydrazone 1 showed an EC50/48h of 36 ± 11 mg/L

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while the corresponding salicylaldehyde phenylhydrazone 2 displayed a comparable activity

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(EC50/48h = 24 ± 7 mg/L). On the contrary, the 4-nitrobenzaldehyde phenylhydrazone 3 showed

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reduced activity (EC50/48h =53 ± 16 mg/L). The isosteric replacement of aldehyde aryl ring with a

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thiophene ring led to the most effective 2-thiophenecarboxaldehyde phenylhydrazone 5 (EC50/48h

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= 16 ± 11 mg/L) and 3-methyl-2-thiophenecarboxaldehyde phenylhydrazone 6 (EC50/48h = 23 ±

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13 mg/L). Interestingly, the logP range for the optimum nematicidal activity of arylhydrazones

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was comprised between 3 and 4 (Table 2). Noteworthy, the nematicidal activity dropped to 33 ±

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8% of mortality after 48h at 100 mg/L when 4-methylbenzaldehyde was used to afford the

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hydrazone 4 (Table 1). The naphthalylaldehydes phenyl hydrazone 8 and 9 are completely

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inactive on nematodes. Furthermore, the introduction of electron donating or electro withdrawing

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substituent on the arylhydrazino moiety of phenylhydrazone failed to enhance the nematicidal

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activity of compounds 11, 12, 14, 15, 16, and 17-19. Nematicidal activity was compared with

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fosthiazate and abamectin on previously reported data (EC50 of 0.4 ± 0.3 and 0.9 ± 1.6 mg/L

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respectively)35,

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previously reported with an in vivo nematicidal activity on Bursaphelenchus xylophilus with the

36

N-arylsulfonyl-3-acylindole arylcarbonyl hydrazone derivatives were


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most active compounds having EC50 values of 1.0969 and 1.2632 mg/L.36 Samaritoni et al.

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reported the nematicidal activity on M. incognita of the tosylhydrazone N-(4-chloro-3-methyl-5-

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isothiazolyl)-2-[p-[(α,α,α-trifluoro-ptolyl)oxy]phenyl]glyoxylamide-2-[(p-tolylsulfonyl)

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hydrazine.37

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Metabolomics easily allows the analysis of hundreds of metabolites in biological samples and

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recently is gaining visibility throughout scientists to address toxicological challenges such as in

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vitro cell system studies for understanding drug effects.37 With the GC-MS metabolomics

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analysis, we were able to detect small nematode endogenous compounds such as carbohydrates,

229

amino acids, fatty acids, and monoacylglycerols. From the multivariate analysis of the matrices

230

of GC-MS data, we succeeded to separate samples treated with hydrazone 6 and the control

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(Figure 1). Q2, R2X and R2Y values found were 0.79, 0.72 and 0.98 respectively. From the

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loadings analysis upregulated compounds for nematodes treated with hydrazone 6 were lauric

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acid, stearic acid, 2-octenoic acid, palmitic acid, while upregulated metabolites for control

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samples were 1- monostearin and 2–monostearin (Figure 2). Discriminant metabolites were

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found in the vinegar eelworm, Turbatrix aceti.38 Altered levels of fatty acids are probably related

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to the uncoupling of the oxidative phosphorylation by hydrazone 6. Same results were elegantly

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described by Cope et al in a model of C. elegans aging model.19 Fatty acid metabolism is

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involved in nematode adaptation to temperature changes.40 Furthermore, Horikawa et al found

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that fatty-acid metabolism is involved in osmotic-stress resistance while unsaturated fatty acids

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are readily oxidized by intercellular reactive oxygen species suggesting that unsaturated fatty

241

acid can act as intracellular scavengers.40 The upregulated levels of free fatty acids in the

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nematodes treated with hydrazone 6 can be also due to the decreased beta oxidation resulting



mitochondrial

malfunctioning

and

to

the

increase

243

from

244

monoacylglycerols as confirmed by elevated level of the latter in the control samples.

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Surprisingly, when we treated nematodes with hydrazone 6 at 100 mg/L at 2 h, the nematode

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excrete 3 times the amount of ammonia ions if compared with the control samples (data not

247

shown). On the other hand, we did not observed any differences in the excreted levels of

248

chloride, fluoride, sulfate, nitrate, nitrite and phosphate. Ammonia is a major compound excreted

249

of most aquatic and few terrestrial animals.41 Recently, Adlimoghaddam et al 45 reported that the

250

soil dwelling nematode C. elegans excreted ammonia as end-product of the cell metabolism not

251

only by apical ammonia trapping but also via vesicular transport and exocytosis. The excretion

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of ammonia model mechanism is reported by Larsen et al46 and involves the participation of the

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V-type H+ ATPase, carbonic anhydrase, Na+/K+ ATPase and a functional microtubule network.

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In fact Schmidtea mediterranea, a fresh water planarian, excretes ammonia across epidermis and

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this fact seems to be dependent on the environmental pH through a mechanism where fluid

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ammonia is pumped as NH4+ across the basolateral membrane by Na+/K+ ATPase into the

257

cytoplasm.47 Protons from cytoplasmatic carbonic anhydrase are then excreted apically via the

258

V-ATPase and the cation/proton exchanger (NHE). The low pH protonates apical ammonia into

259

its ionic form and thereby creates a transcellular PNH3 gradient.48 The role and significance of the

260

elevated excretion levels of ammonia in the M. incognita model should be further studied.

261

Thus considering the mode of action of aryl hydrazones i.e uncoupling of the oxidative

262

phosphorylation, these compounds, with low EC50 values, can be used as a scaffold for exploring

263

new active ingredients with a selective mode of action and lower toxicity to be used in the


rate

Page 14 of 29

of

hydrolysis

from

Page 15 of 29


264

integrated pest management protocols. Furthermore, the one-step chemical synthesis of these

265

compounds from raw materials could be a significant advantage for the industry.

266

We cannot conclude if upregulated metabolites can be ascribed to a specific mode of action or

267

are a secondary response to toxicity.

268

269

AUTHOR INFORMATION

270

Corresponding Author

271

* Phone: +39 070 6758617. Fax: +39 070 6758612

272

E-mail: [email protected]

273

ACKNOWLEDGMENTS

274

We are grateful to the DREAM project of Porto University. The authours also thank Alessandra

275

Porcu and Martina Demuru for assistance.

276

ABBREVIATIONS USED

277

GC-MS, gas chromatography-mass spetrometry; S.D, standard deviation, TLC, thin layer

278

chromatography.

279



280

Page 16 of 29

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FIGURES CAPTIONS

418

Figure 1: OPLS-DA score plot of GC-MS data, t1 versus t2, showing the positions of the

419

samples from treated nematodes vs. controls. Treated nematode samples with arylhydrazone 6

420

(gray circles) are separated from the controls (black circles) in the multivariate space. Validation

421

parameters were: respectively; R2Y=0.98 and Q2Y= 0.79)

422

Figure 2: Comparison between most discriminant metabolites of treated (T) and control

423

nematodes (C) The box is drawn from the first to the third percentiles in the distribution of

424

intensities. The median, or 50th percentile, is drawn as a darker gray horizontal line inside the

425

box. The first percentile was deleted for a better view of the plot. The third percentile is drawn as

426

a lighter gray line inside the box. The whiskers describe the error bars of data within the first and

427

the third percentiles.

428



429

Page 24 of 29

Table 1: Percent of paralyzed juveniles M. incognita tested at 100 mg/L after 48h (n = 6). Compound

Formula

Paralysis (%)±SD

1

87 ± 7

2

100 ± 0

3

94 ± 6

4

33 ± 8

5

93 ± 4

6

96± 3

7

NA

8

NA

9

NA


Page 25 of 29

430


10

20 ± 8

11

NA

12

NA

13

4±2

14

NA

15

NA

16

15 ± 6

17

5±2

*NA = Not active

431



432 433

Table 2: EC50/48h of the most actives hydrazones (EC50/48h

In Vitro Nematicidal Activity of Aryl Hydrazones and Comparative GC

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