Allelochemical Activity of Eugenol-Derived Coumarins on Lactuca sativa L.

Coumarins are widely distributed substances in plant species that promote phytotoxic effects, allowing them to be exploited as herbicides less harmful to the environment, since many invasive species have demonstrated resistance to commercially available products. The derived coumarins used in this study had not been tested in plant models and their effect on plants was unknown. The objective of this study was to evaluate the phytotoxic action of these coumarins in bioassays with Lactuca sativa L., in order to select the most responsive substance whose toxicity was best elucidated by chromosomal complement and enzymatic antioxidant metabolism studies. From the phytotoxicity assays, coumarin 8-methoxy-2-oxo-6-(prop-2-en-1-yl)-2H-chromene-3-carboxylic acid (A1), reported here for the first time, was selected as the most responsive and caused a reduction in the following parameters: number of normal seedlings, fresh biomass, root length and shoot length. Subsequent studies demonstrated that this coumarin is cytogenotoxic due to damage caused to the cell cycle and the occurrence of chromosomal abnormalities. However, it did not interfere with antioxidant enzyme activity and did not cause lipid peroxidation. The changes caused by coumarin A1 described herein can contribute to better understanding the allelochemical actions of coumarins and the potential use of these substances in the production of natural herbicides.

: Antioxidant enzyme activity and lipid peroxidation. (a) Superoxide dismutase (SOD) activity in Lactuca sativa seedlings exposed to the different concentrations of coumarin A1. (b) Catalase (CAT) activity in Lactuca sativa seedlings exposed to the different concentrations of coumarin A1. (c) Quantification of lipid peroxidation in seedlings of Lactuca sativa exposed to the different concentrations of coumarin A1. Columns followed by the same letter do not differ statistically by the Scott-Knott test at 5% significance. Bar: standard error.

Generalities
All required chemicals were purchased from Sigma-Aldrich (Sao Paulo, Brazil) and used without further purification. Thin-layer chromatography (TLC) on sílica gel TLC plates (ALUGRAMR Xtra Sil G/UV254, Macherey-Nagel) was used to check purity of the obtained compounds and to monitor the reactions progress. The spots were detected by exposure to the UV light at 254 nm. For column chromatography, column grade (0.040-0.063-mm mesh size) silica gel was employed (Sorbiline R) and the mobile phases are described in each experiment. Melting points of the compounds were obtained on a PFM-II melting point apparatus (MS Tecnopon, Piracicaba, Brazil) and are uncorrected. IR spectra were recorded on a FT-IR-Affinity-1 spectrometer with an ATR dispositive (ShimadzuR, Kyoto, Japan). NMR spectra were recorded on a Bruker AC-300 spectrometer (Rheinstetten, Germany) operating at 300 MHz for 1 H-NMR and 75 MHz for 13 C-NMR spectra. Chemical shifts are expressed as δ (ppm) relative to TMS as the internal standard. The coupling constants (J) are given in Hertz. Multiplets are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), sex (sextet), and m (multiplet).

Synthesis, purification and characterization data for Coumarins A1-A6
1.2.1. Synthesis of the intermediate formyleugenol Hexamine (5 eq) was solubilized in glacial acetic acid (40 mL) and this solution was stirred at 125 °C for 10 min, then eugenol (1 eq) was added in one portion. Reaction was maintained at 125 °C for 5 h under stirring. Following, 2 M HCl (10 mL) was added and the reaction mixture was maintained as such for additional 30 min. Then, this mixture was cooled down to 25 °C, washed with aqueous NaHCO3, and extracted with dichloromethane. The organic phase solvente was evaporated in a rotary evaporator and the oil purified by column chromatography (hexanes : ethyl acetate, 9:1, v/v).

General procedure for the synthesis and purification of coumarins A1-A4
The specific β-ketoester or malonic acid (1 eq.), formyleugenol (1 eq.) and piperidine (0.1 eq) were added to ethanol, mixtured and heated to 80 °C in a glycerol bath until TLC indicated the end of the reaction (hexane : ethyl acetate; 8:2, v/v). The remaining slurry solid was purified by filtration and trituration with diethyl ether until a fine powder was formed, which was again isolated by simple filtration.

Synthesis of 3-(4-aminobenzoyl)-8-methoxy-6-(prop-2-en-1-yl)-2H-chromen-2-one (A5)
Derivative A4 (1 eq) and tin chloride (II) dihydrate (5 eq) were added to ethanol (20 mL) and the mixture was stirred under reflux. After 2 h, TLC analysis (hexanes : ethyl acetate, 7:3, v/v) showed the reaction completion and the mixture was cooled to room temperature. Aqueous NaHCO3 was then added to raise pH to eight and the mixture was extracted with ethyl acetate. The resulting organic phase was washed with brine, dried with anhydrous sodium sulfate, and the solvent evaporated in a rotary evaporator, leading to the pure product.

IR AND NMR SPECTRA
IR spectrum of formyleugenol.
1 H NMR spectrum of formyleugenol (CDCl3, 300 MHz). Figure S1: Antioxidant enzyme activity and lipid peroxidation. (a) Superoxide dismutase (SOD) activity in Lactuca sativa seedlings exposed to the different concentrations of coumarin A1. (b) Catalase (CAT) activity in Lactuca sativa seedlings exposed to the different concentrations of coumarin A1. (c) Quantification of lipid peroxidation in seedlings of Lactuca sativa exposed to the different concentrations of coumarin A1. Columns followed by the same letter do not differ statistically by the Scott-Knott test at 5% significance. Bar: standard error.