Chalcone-related herbicides and methods thereof

Abstract

The present disclosure provides herbicides developed from 3'-hydroxychalcone (T3), 3'hydroxy-3,4-dichlorochalcone (Li-9), 3'-hydroxy-4-chlorochalcone (Li-6), 3'-hydroxy-3-pyridyl-chalcone (Li-45), 3'-hydroxy-4-pyridyl-chalcone (Li-46), 3'-hydroxy-4-nitrochalcone (Li-58), 3'-hydroxy-4-bromochalcone (LR-72), 4'-hydroxy-3'-methoxychalcone (CH1), 3,4-dimethoxychalcone (CH2) or cinnamaldehyde (P1) for weed control.

Description

91392-428425- 1 -CHALCONE-RELATED HERBICIDES AND METHODS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 688,403, filed on August 29, 2024. the entire disclosure of which is incorporated herein by reference.TECHNICAL FIELD

[0002] This disclosure relates to the herbicidal substances, for instance compositions and methods including 3 ’-hydroxy chai cone (T3), 3’hydroxy-3,4-dichlorochalcone (Li-9), 3’- hydroxy-4-chlorochalcone (Li-6), 3’ -hydroxy -3-pyridyl-chalcone (Li-45), 3’-hydroxy-4- pyridyl-chalcone (Li-46), 3’-hydroxy-4-nitrochalcone (Li-58), 3’-hydroxy-4-bromochalcone (LR-72), 4’-hydroxy-3’-methoxychalcone (CHI). 3,4-dimethoxychalcone (CH2), and cinnamaldehyde (Pl) for controlling weed plants.BACKGROUND AND SUMMARY

[0003] Large-scale use of various herbicides available on the market, which target the same mechanism of action for weed control, promotes the emergence of resistant weeds over time. Herbicide resistance is a common result of natural selection. Worldwide, more than 200 weed species have already developed resistance to one or more herbicides. Currently, herbicides already encounter resistance in 21 of the 25 known molecular targets for weed control.

[0004] Weed control is essential for achieving good yields in global food production (CHOTSAENG et al, 2018; GARRIDO et al. 2023). Weeds have caused more severe economic losses than any other agricultural pest (DIN et al, 2017; NGUYEN et al, 2016).

[0005] In recent decades, the development of new phytotoxic agents with different molecular pathways has become the primary and constant objective of weed control research (DAYAN et al, 2009; DIAZ-TIELAS et al, 2012; GOMES et al, 2018), in addition to changes in management programs (HEAP, 2014). Natural product-based pesticides are generally water- soluble and have relatively short half-lives (BHOWMIK; INDERJIT, 2003), since their chemical structures lack “non-naturaf’ rings and contain few halogen substituents (DAYAN et al, 2009). Because of these characteristics, they are considered safe from an environmental toxicology standpoint (BHOWMIK; INDERJIT, 2003). Additionally, they could be obtained from natural extracts or synthesized in the laboratory (COPPING; DUKE, 2007). Many chemical molecules produced by plants act similarly to herbicides in the metabolism of other91392-428425- 2 - plants. and botanical studies characterize some of these substances as good inhibitory agents (BITENCOURT et al, 2007). For example, 2, 4'-dimethoxy chaicone reduced seed germination of Mimosa pudica L. and Senna obtiisifolia L. by 58 and 48%, respectively (BITENCOURT et al, 2007).

[0006] Chaicones are multifunctional molecules with various molecular targets and a broad spectrum of biological activities, exhibiting bactericidal, antifungal, antihelminthic, insecticidal, and antiviral actions (DIAZ-TIELAS et al, 2016). They also have good phytotoxic potential and are relatively easy to synthesize.

[0007] The present disclosure provides advancement of the development of herbicides originating from 3 '-hydroxy chaicone (T3), 3’-hydroxy-3,4-dichlorochalcone (Li-9), 3’- hydroxy-4-chlorochalcone (Li-6), 3’-hydroxy-3-pyridyl-chalcone (Li-45), 3’-hydroxy-4- pyridyl-chalcone (Li-46), 3’-hydroxy-4-nitrochalcone (Li-58), 3’-hydroxy-4-bromochalcone (LR-72), 4’ -hydroxy-3 ’-methoxy chaicone (CHI). 3,4-dimethoxychalcone (CH2), or cinnamaldehyde (Pl). As provided herein, the chaicones are sy nthetic and have a structure similar to that of natural products. These substances serve as alternatives for weed control compared to existing market herbicides, especially since many species have already developed resistance to them or may do so in the future.

[0008] Various aspects of the disclosure are described more fully below. However, different aspects of the disclosure may be implemented in many ways and should not be construed as limited to the aspects set forth herein. The following detailed description is, therefore, not to be taken in a limiting sense.BRIEF DESCRIPTION OF THE FIGURES

[0009] Figures 1 A-1C show the chemical structures of methoxy chaicones and cinnamaldehyde. Figure 1A shows the chemical structure of substance 4 ‘-hydroxy -3’- methoxy chaicone (CHI), Figure IB shows the chemical structure of substance 3,4- dimethoxychalcone (CH2), and Figure 1C shows the chemical structure of cinnamaldehyde (Pl).

[0010] Figures 2A-2F show Arabidopsis thaliana seedlings growing on 0.44% macro and micronutrients, with 1% sucrose on 1% agar. Negative controls (0.1% ethanol) (Figures 2A, 2B, and 2C) and substances at 100 pmol L1(Figure 2D: cinnamaldehyde), (Figure 2E: 3,4- dimethoxychalcone), and (Figure 2F: 4’-hydroxy-3 ’-methoxy chaicone); grid spacing is 1 cm.91392-428425- 3 -

[0011] Figures 3A-3D are graphs showing the dose-response curve of 4'-hydroxy-3'- methoxy chaicone (CHI) (Figure 3A), 3,4-dimethoxy chaicone (CH2) (Figure 3B), cinnamaldehyde (Pl) (Figure 3C), and all overlapping substances (Figure 3D).

[0012] Figure 4 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3.4-dimethoxy chaicone (CH2) on phenylalanine ammonia-lyase (PAL) activity. The negative control included enzyme, assay buffer, and 0.5% ethanol. The positive control (PC) included enzyme, assay buffer, and perchloric acid. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0013] Figure 5 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxychalcone (CHI), and 3,4-dimethoxy chaicone (CH2) on electrolyte extravasation. The negative control (NC) included assay buffer and 1% DMSO. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0014] Figure 6 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3,4-dimethoxy chaicone (CH2) on photosynthetic electron transport. The negative control (NC) included assay buffer and 1% DMSO. The positive control (PC) included assay buffer and diuron diluted in 1% DMSO. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0015] Figure 7 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3,4-dimethoxy chaicone (CH2) on the photosynthetic evolution of oxygen. The negative control (NC) included assay buffer and 1% DMSO. The positive control (PC) included assay buffer and diuron diluted in 1% DMSO. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0016] Figures 8A-8F are representative optical micrographs of onion root apex cells at different phases of mitosis in the negative control and of abnormal cells in treatments (interphase 8A, prophase 8B, metaphase 8C, anaphase 8D, telophase 8E and abnormal 8F).

[0017] Figure 9 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3,4-dimethoxy chaicone (CH2) on cell division. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.91392-428425- 4 -

[0018] Figure 10 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3, 4-dimethoxy chaicone (CH2) on the interphase. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0019] Figure 11 is a graph showing the effect of cinnamaldehyde (Pl), 4'-hydroxy-3'- methoxy chaicone (CHI), and 3, 4-dimethoxy chaicone (CH2) on the mitotic index. Different letters at the top of the bars indicate significant differences in treatments in the one-way ANOVA test followed by Tukey’s test (p < 0.05); values are means ± standard deviation.

[0020] Figure 12 are chemical structures of hydroxy chaicones and positive controls used in the present disclosure.DETAILED DESCRIPTION

[0021] Various embodiments are described herein as follows. In an illustrative aspect, a composition comprising one or more chaicones with herbicidal activity is provided. The one or more chaicones is selected from the group consisting of 3'-hydroxychalcone (T3), 3'-hydroxy-4- chlorochalcone (Li-6), 3'-hydroxy-4-methylchalcone (Li-7), 3'-hydroxy-4-methoxy chaicone (Li-8), 3'hydroxy-3,4-dichlorochalcone (Li-9), 3‘-hydroxy-2-furanyl-chalcone (Li-15), 3'- hydroxy-3-thienyl-chalcone (Li-16), 3 ’-hydroxy- 1 -naphthyl-chalcone (Li-21), 3'-hydroxy-2- naphthyl-chalcone (Li-22), 3'-hydroxy-3-chlorochalcone (Li-40), 3’-hydroxy-2,4- dichlorochalcone (Li-42), 3'-hydroxy-3-pyridyl-chalcone (Li-45), 3’-hydroxy-4-pyridyl- chalcone (Li-46), 3'-hydroxy-4-nitrochalcone (Li-58), 3'-hydroxy-4-bromochalcone (LR-72), 3, 4-dimethoxy chaicone (CH2), and cinnamaldehyde (Pl).

[0022] In an embodiment.

[0023] In an illustrative aspect, a second composition comprising one or more chaicones with herbicidal activity is provided. The one or more chaicones is selected from the group consisting of 3 ’-hydroxy chaicone (T3). 3’hydroxy-3,4-dichlorochalcone (Li-9), 3’-hydroxy-4- chlorochalcone (Li-6). 3’-hydroxy-3-pyridyl-chalcone (Li-45), 3 ’-hydroxy -4-pyridyl-chalcone (Li-46), 3’ -hydroxy -4-nitrochalcone (Li-58), 3’ -hydroxy -4-bromochalcone (LR-72), 4’- hydroxy-3’ -methoxy chaicone (CHI), 3, 4-dimethoxy chaicone (CH2), and cinnamaldehyde (Pl).

[0024] In an illustrative aspect, a method of treating a plant is provided. The method comprises a step of administering a composition comprising one or more chaicones with herbicidal activity to the plant. In various embodiments, the composition comprising one or more chaicones with herbicidal activity can be any of the compsitions described herein. In an91392-428425- 5 - embodiemnt, the method decreases growth of a weed. In an embodiment, the method decreases germination of a weed seed.

[0025] The following numbered embodiments are contemplated and are non-limiting: .1. A composition comprising one or more chaicones with herbicidal activity, wherein the one or more chaicones is selected from the group consisting of 3'-hydroxy chaicone (T3), 3'- hydroxy-4-chlorochalcone (Li-6), 3'-hydroxy-4-methylchalcone (Li-7), 3'-hydroxy-4- methoxy chaicone (Li-8), 3'hydroxy-3,4-dichlorochalcone (Li-9), 3 ‘ -hydroxy -2-furanyl- chalcone (Li-15), 3'-hydroxy-3-thienyl-chalcone (Li-16), 3' -hy droxy- 1-naphthyl-chalcone (Li- 21), 3'-hydroxy-2-naphthyl-chalcone (Li-22), 3'-hydroxy-3-chlorochalcone (Li-40), 3’-hydroxy-2.4-dichlorochalcone (Li-42), 3'-hydroxy-3-pyridyl-chalcone (Li-45), 3’ -hydroxy -4-pyridyl- chalcone (Li-46), 3'-hydroxy-4-nitrochalcone (Li-58), 3'-hydroxy-4-bromochalcone (LR-72),3.4-dimethoxy chaicone (CH2), and cinnamaldehyde (Pl).2. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy chaicone (T3).3. The composition of clause 1 , any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-4-chlorochalcone (Li-6).4. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-4-methylchalcone (Li-7).5. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-4-methoxychalcone (Li-8).6. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'hydroxy-3,4-dichlorochalcone (Li-9).7. The composition of clause 1. any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3‘-hydroxy-2-furanyl-chalcone (Li-15).8. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3 -hydroxy-3-thienyl-chalcone (Li-16).9. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3 ’-hydroxy- 1-naphthyl-chalcone (Li-21).10. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3 -hydroxy -2 -naphthyl-chal cone (Li-22).11. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-3-chlorochalcone (Li-40).12. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3’-hydroxy-2,4-dichlorochalcone (Li-42).91392-428425- 6 -13. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-3-pyridyl-chalcone (Li-45).14. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3’-hydroxy-4-pyridyl-chalcone (Li-46).15. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3'-hydroxy-4-nitrochalcone (Li-58).16. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3 -hydroxy-4-bromochalcone (LR-72).17. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is 3, 4-dimethoxy chaicone (CH2).18. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the chaicone is or cinnamaldehyde (Pl).19. A composition comprising one or more chaicones with herbicidal activity, wherein the one or more chaicones is selected from the group consisting of 3 ’-hydroxy chaicone (T3), 3’hydroxy-3,4-dichlorochalcone (Li-9), 3 ’-hydroxy -4-chlorochalcone (Li-6), 3’-hydroxy-3- pyridyl-chalcone (Li-45), 3’-hydroxy-4-pyridyl-chalcone (Li-46), 3’-hydroxy-4-nitrochalcone (Li-58), 3’ -hydroxy -4-bromochalcone (LR-72), 4’-hydroxy-3’-methoxychalcone (CHI), 3,4- dimethoxy chalcone (CH2), and cinnamaldehyde (Pl).20. A method of treating a plant, the method comprising a step of administering a composition comprising one or more chaicones with herbicidal activity to the plant.21. The method of clause 20, any other suitable clause, or any combination of suitable clauses, wherein the composition comprising one or more chaicones with herbicidal activity is the composition of any one of clauses 1 to 19.22. The method of clause 20, any other suitable clause, or any combination of suitable clauses, wherein the method decreases growth of a w eed.23. The method of clause 20, any other suitable clause, or any combination of suitable clauses, wherein the method decreases germination of a weed seed.EXAMPLES

[0026] The 18 methoxy chai cones employed in the previous herbicidal activity study included: 4'-hydroxy-3'-methoxychalcone (CHI); 2’-methoxychalcone (BC-02); 3'-methoxy chalcone (BC- 03); 4'-methoxychalcone (BC-04); 2',4'-dimethoxychalcone (BC-06); 2',5'-dimethoxychalcone (BC-07); 3',4'-dimethoxychalcone (BC-08); 2',4',5'-trimethoxychalcone (BC-09); 3',4',5'- trimethoxychalcone (BC-10); 4-methoxy chalcone (BC-11); 4-hydroxy-3-methoxychalcone (BC-91392-428425- 7 -12); 2 -methoxy chaicone (BC-13); 2, 3-dimethoxy chaicone (BC-15); 2.4-dimethoxy chaicone (BC-16); 2,5-dimethoxychalcone (BC-17); 3, 4-dimethoxy chaicone (CH2); 2,4,5- trimethoxychalcone (BC-19) and 3,4,5-trimethoxychalcone (BC-20). The synthesis of methoxy chaicones was performed via the Claisen-Schmidt reaction (SANTOS et al., 2017; PASSALACQUA et al., 2015). Appropriate acetophenone derivatives and their benzaldehyde derivatives were solubilized in ethanol and NaOH ethanolic solution. All crude chaicones were purified using successive silica gel columns with mixtures of hexane and ethyl acetate as the mobile phase. Nuclear magnetic resonance (NMR) spectra were recorded using three spectrometers: Bruker® Avance III 600 MHz (14.1 T), Bruker® Avance III 400 MHz (9.4 T). and Bruker® Fourier 300 MHz (7.1 T). The two parietal CHI chaicone substances employed in the previous herbicidal activity study were curcumin and cinnamaldehyde (Pl). After the previous studies, substances 4’-hydroxy-3’-methoxychalcone (CHI), 3,4-dimethoxychalcone (CH2) and cinnamaldehyde (Pl) proceeded to the next step. Seeds of Arabidopsls lhaliana (L.) Heynh. of the Columbia (Col-0) ecotype were sterilized with chlorine gas (Ch) and stored at 4 °C for 72 h for stratification. Sterile square Petri dishes (100 x 15 mm) were prepared with 0.44% macro and micronutrients (w / v; Murashige-Skoog, Sigma- Aldrich, St Louis, MO) + 1% sucrose (pH adjusted to 6.0 using KOH) in 1% agar (30 mL / plate; Phy toagar, Life Technologies) one day prior to sowing. Twenty seeds were sown per dish, which were then sealed with adhesive tape (3M Micropore) and placed in a growth chamber at 25 °C and light / dark photoperiod of 16 / 8 h under light intensity of 120 pmol m2s '. A dose-response curve was conducted with 4'-hydroxy- 3'-methoxychalcone (CHI) and 3,4-dimethoxychalcone (CH2) chaicones and with cinnamaldehyde (Pl) (Figure 1) to calculate IC50 and ICso concentrations. Agar solutions containing 4'-hydroxy-3'-methoxychalcone (CHI), 3.4-dimethoxychalcone (CH2) and cinnamaldehyde (Pl) were prepared at concentrations of 0, 5, 10, 25, 50 and 100 pmol L1in 0.1% Ethanol (four replicates per concentration). The dishes were placed at a 45° angle in a growth chamber for 21 days. After this time, the root length w as measured, and the IC50 and ICso were calculated. Nonlinear regression was used to analyze the dose-response curve data and calculate the IC50 and ICso values using the drc package in R Studio (DI AZ-TIELAS et al, 2019).

[0027] For Phenylalanine Ammonia-Lyase (PAL) activity, the extraction buffer included 50 mmol L1Tris-HCl (pH = 7.8), 20 mmol L’1P-mercaptoethanol, 0.1% isoascorbic acid, 20% glycerol, and 10% w / v polyvinylpyrrolidone (insoluble, molecular weight: 40 kDa). Frozen spinach leaves (10 g) were milled with mortar and pestle in liquid nitrogen until a fine powder was obtained. Spinach is commonly used in the phenylalanine ammonia-lyase (PAL) activity test because it is a good source of phenolic compounds, including phenylalanine, the PAL substrate.91392-428425- 8 -The powder was added to 20 mL of ice-cooled extraction buffer. It was then homogenized for 10 min in cold blender. The homogenate was centrifuged at 12,000 g for 20 min at 4°C. Solid ammonium sulfate was slowly added to the supernatant to the required level (30 or 80%) and stirred for an additional 15 min. The solution was centrifuged at 12,000 g for 20 min at 4°C. The fraction that was soluble at 30% ammonium sulfate but became insoluble at 80% ammonium sulfate, was collected and resuspended by adding 2.5 mL of extraction buffer. The resuspended solution was desalted using a PD-10 column containing Superdex-G25, which was equilibrated with the same buffer. The desalted solution was stored at -20°C for the enzyme activity' assay.

[0028] The PAL assay reaction was initiated by adding 30 pg of protein in 40 pL of the appropriate buffer to a solution of 20 mmol L1of L-phenylalanine (80 pL) and 50 mmol L1of Tris-PLSO i (pH = 8.8, 160 pL). The mixture was incubated for 1 h at 40°C. The reaction was stopped by adding 60 pL of 2N perchloric acid, and the protein was removed by centrifugation at 12,000 g for 20 min. Cinnamic acid, as a reaction product in the supernatant, was determined by measuring absorption at 290 nm in a spectrophotometer. The negative control was comprised of enzy me, assay buffer, and 0.5% ethanol. The positive control was comprised of enzyme, assay buffer, and perchloric acid. The remaining treatments were composed of 4'-hydroxy-3'- methoxy chaicone (CHI) and 3, 4-dimethoxy chaicone (CH2) chai cones and cinnamaldehyde (Pl) prepared in 0.5% ethanol. Results were analyzed using a one-way ANOVA test followed by Tukey’s test (p < 0.05). Data were analyzed using the BioStat statistical package (version 5.0). Results were expressed as % activity relative to the negative control. For the electrolyte extravasation test, cucumber plants were grown in a greenhouse for 10 days until cotyledons were fully expanded. Twenty-five discs with 4 mm cotyledons were placed in 5 mL of assay medium (2% sucrose in 1 mM MES) which was adjusted to pH 6.5 at room temperature in 60 x 15 mm disposable Petri dishes. The test substances were prepared as lOOx stocks and added, in 50 pL aliquots, to the assay medium. Control samples were exposed to the same volume of solvent (DMSO) in the absence of test substances. Dishes were incubated in the dark for 16 h prior to exposure to 1000 pmol m'2s'1photosynthetically active radiation (PAR) in an incubator. Assay medium conductivity was measured using a conductivity meter immediately after the incubation period in the dark and after 8 hours of incubation in the light. Results were analyzed using a oneway ANOVA test followed by Tukey’s test (p < 0.05). Data were analyzed using the BioStat statistical package (version 5.0).

[0029] Chlorophyll fluorescence was measured non-destructively on 4 discs of cucumber leaves floating in 5 mL of assay medium (2% sucrose 1 mmol L'1MES, pH adjusted to 6.5 at room temperature) with inhibitor concentrations of 100 pmol L’1in 60 X 15 mm disposable Petri91392-428425- 9 - dishes. In kinetic mode, the instrument was calibrated to ensure that the initial instantaneous fluorescence (Ft) signal value for the control samples was around 210. The gain of the instrument detector was set between 75 and 85. The quantum yield was determined by the following light treatment: each cycle included a 0.8 s saturating light pulse generated with an actinic laser diode source to saturate photosystem II (PSII), followed by a 4 s far-red light pulse used to reoxidize PSII, and a 10 s delay pulse to allow PSII to recover from steady-state conditions. Seven cycles were performed for each sample. Diuron was used as a positive control. Results were analyzed using a one-way ANOVA test followed by Tukey’s test (p < 0.05). Data were analyzed using the BioStat statistical package (version 5.0).

[0030] Oxygen evolution was monitored from a preparation containing intact chloroplasts. Spinach (Spinacia oleracea / ..) was used because it produces excellent preparations of chloroplasts. Fresh tissue (50 g) and 250 mL of extraction buffer (1.65 mmol L1sorbitol, 50 mmol L’1HEPES, 25 mmol L1cysteine, 5 mmol L1MgCh, 5 mmol L'1EDTA, pH adjusted to 7.7) were homogenized in a blender. The homogenate was filtered through a layer of Miracloth (Merck) lined by two layers of gauze and collected in a cold beaker. The filtrate was centrifuged for 20 min at 6000 X g and 4 °C. The sediment containing chloroplasts was resuspended in 0.5 mL buffer (1.65 mmol L'1sorbitol, 50 mmol L1HEPES, 5 mmol L1dithiothreitol, 5 mmol L'1MgCb, 5 mmol L1EDTA, pH adjusted to 7.7). The tubes were rinsed with 0.5 mL of additional buffer in series, combined with the chloroplast extract. Measurement of oxygen evolution was performed by means of polarography using an oxygen probe. These experiments were conducted under saturating light conditions (2400 pmol m'2s'1PAR). We used a fiber optic light source with two sources that provided 1,300 lumens (Schott-Foster, LLC, Southbridge, MA 01550 USA), and the measurements were conducted via a Hansatech OXY-GRAPH PLUS oxygen electrode system (PP System, Amesbury. MA 01913 USA). All analyses were performed at 30°C using a water bath. The chaicones and cinnamaldehyde were diluted in DMSO; both treatments and controls received the same concentration of solvent (less than 1% v / v). Thylakoid membranes of chloroplasts were incubated with test substances (100 pmol L’1) on ice for 20 minutes prior to the assay. The analysis was initiated by adding the thylakoid membranes to the reaction assay buffer (500 mmol L1HEPES, 5 mmol L'1EDTA, pH adjusted to 7.5), and the rate of oxygen evolution was measured over 120 seconds in the linear portion of the curve. The negative control included assay buffer and 1% DMSO, while the positive control was assay buffer and diuron diluted in 1% DMSO. Results were subjected to one-way analysis of variance (ANOVA), followed by Tukey’s test (p < 0.05). All analyses were performed using the BioStat statistical package (version 5.0), and the data were expressed as relative activity.91392-428425- 10 -

[0031] For the mitotic index, onion seeds (Allium cepa L.) were germinated for seven days in Petri dishes on wet filter paper, under 25 °C and for a 14-hour photoperiod in the presence of chaicones and cinnamaldehyde. The substances tested were prepared as xlOO stock solutions (10 mmol L’1) in 50% ethanol. Control samples received the same amount of solvent as treated samples. Twenty root tips (1 cm sections) were collected for each treatment, one per plant, and were fixed in glacial acetic acid: absolute ethanol (1 :3 v / v) for 30 minutes. The segments were hydrolyzed in 5 N HC1 at room temperature for 1 hour and washed several times with distilled water. Subsequently, the segments were stained with Schiff s reagent for 45 minutes in the dark at room temperature. Root segments were transferred to a drop of 45% acetic acid on top of a microscope slide using forceps. Once on the slide, a razor was used to cut the tips and remove the remaining root segments. Then, a coverslip was carefully placed over the root tips and gently pressed with light and constant pressure directly on the tissues. The edge of the coverslip was sealed with enamel to delay evaporation of the acetic acid. The mitotic index was calculated by counting cells at various stages of mitosis using an Olympus BX60 microscope. At least 1000 cells per slide, and in triplicate (3000 cells per treatment), were counted for statistical analysis. Cells with abnormal mitotic configurations were counted as a separate class. Results were analyzed using a one-way ANOVA test followed by Tukey ’s test (p < 0.05). Data were analyzed using the BioStat statistical package (version 5.0). The values shown in the graphs are expressed as percentages.

[0032] Compared to the negative control, cinnamaldehyde (Pl) caused an 83% inhibition in root length, while 3,4-dimethoxychalcone (CH2) caused a 79% inhibition, and 4'-hydroxy-3'- methoxy chaicone (CHI) caused a 96% inhibition in A. thaliana root length (Figure 2).

[0033] 4'-hydroxy-3'-methoxychalcone (CHI) was the most potent chaicone in inhibiting root growth, with an IC50 of 17.3 pmol L'1(Figure 3). Cinnamaldehyde (Pl) and 3,4- dimethoxychalcone (CH2) were less active, with IC50 values of 72.3 pmol L'1and 31.4 pmol L" respectively.

[0034] The substances cinnamaldehyde (Pl) and 3,4-dimethoxychalcone (CH2) exhibited ICso near 100 pmol L’1, while 4'-hydroxy-3'-methoxychalcone (CHI) exhibited ICso near 56.1 pmol L'1(Figure 3). Treatments using chaicones and cinnamaldehyde demonstrated a significant increase compared to controls. Notably, 4’-hydroxy-3’-methoxychalcone (CHI) and cinnamaldehyde (Pl) exhibited an even greater increase in enzy matic activity’ when compared to 3,4-dimethoxychalcone (CH2) (Figure 4). The cells subjected to treatment with the substances exhibited distinct characteristics compared to the negative control. The substances induced the elongation of the formed cells, while the negative control exhibited cells at various phases of91392-428425- 1 1 - mitosis. The promotion of the formation of abnormal, notably elongated cells was observed in the treated samples (Figure 8). In the cinnamaldehyde (Pl) treatment, 18.2% of the formed cells exhibited abnormalities, while in the 4'-hydroxy-3'-methoxychalcone (CHI) and 3, 4- dimethoxy chaicone (CH2) treatments, these percentages were 23.3% and 26%, respectively (Figure 9). All treatments showed significant differences compared to the vehicle control in the interphase phase, with the exception of cinnamaldehyde (Pl) (Figure 10). All substances showed differences compared to the vehicle control, exhibiting a lower proportion of cells in mitosis phases, except anaphase (Figure 11). Cinnamaldehyde (Pl) caused the greatest reduction in the prophase, while 4'-hydroxy-3'-methoxychalcone (CHI) mainly contributed to the decrease in metaphase and telophase phases (Figure 1 1).

[0035] In another aspect, the potential of hydroxychaicones for herbicide development was examined. Evaluations have been performed in several species, including: Digitaria insularis (L.) Fedde (sourgrass), Rottboellia cochinchinensis (Lour.) Clayton (itchgrass), Urochloa decumbens (Stapf) R.D. Webster (signal grass). Amaranthus viridis L. (slender amaranth), Bidens pilosa L. (black-jack) and Raphanus rapha nisi rum L. (wild radish). The first three are monocotyledons, while the others are eudi cotyledons. For germination and emergence screening, all substances were evaluated in Lactuca sativa L. (lettuce), chosen due to its rapid germination and sensitivity to substances with potential phytotoxic effect. The four substances with the highest herbicidal activity were subsequently tested in weeds.

[0036] The 15 hydroxy chai cones employed in this study (Figure 12) were: 3 ’-hydroxy chaicone (T3); 3’ -hydroxy -4-chlorochalcone (Li-6); 3'-hydroxy-4-methylchalcone (Li-7); 3 ’-hydroxy -4- methoxy chaicone (Li-8); 3’hydroxy-3,4-dichlorochalcone (Li-9); 3’-hydroxy-2-furanyl- chalcone (Li-15); 3’-hydroxy-3-thienyl-chalcone (Li-16); 3’ -hydroxy- 1 -naphthyl-chalcone (Li- 21); 3’-hydroxy-2-naphthyl-chalcone (Li-22); 3 ’-hydroxy -3-chlorochalcone (Li-40); 3’- hydroxy-2,4-dichlorochalcone (Li-42); 3’-hydroxy-3-pyridyl-chalcone (Li-45); 3’-hydroxy-4- pyridyl-chalcone (Li-46); 3’ -hydroxy -4-nitrochalcone (Li-58); 3’-hydroxy-4-bromochalcone (LR-72). Hydroxychaicones were prepared by Claisen-Schmidt condensation using 3- hydroxyacetophenone and benzaldehyde derivatives as starting materials, with sodium hydroxide and ethanol as the catalyst and solvent, respectively (SANTOS, 2017; TREIN, 2019). The chaicone derivatives were purified by precipitation, crystallization, and chromatographic methods such as Normal Phase Column Chromatography (NPCC) and Gel Permeation Chromatography (GPC, LH-20, Sephadex®). The obtaining of the chaicones was confirmed through the analysis of their Hydrogen Nuclear Magnetic Resonance t'H NRM) and Carbon- 13 Nuclear Magnetic Resonance (1?C NMR) spectra. All hydroxy chaicones were properly diluted in91392-428425- 12 - absolute ethanol to create solutions with a concentration of 1 x 10'3mol L’1. The choice of this concentration was based on the observation that natural herbicides generally exhibit significant inhibitor)' activity at concentrations between 10’2and 10'3mol L1(MACIAS et al, 2000). In addition, positive controls tebuthiuron (the active substance in Combine® herbicide, which is widely used for pre-emergent weed control) and glyphosate (the active substance in Roundup® herbicide, which is employed for post-emergent weed control) were also prepared in absolute ethanol at a concentration of 1 x 10'3mol L'1. All hydroxy chaicones used were synthesized by the research group, while positive controls were commercially acquired from Sigma- Aldrich.

[0037] In the germination assessment, four 25 seed replicates were placed on a Whatman #1 filter paper sheet in 9 cm Petri dishes. The filter paper was moistened with 5 mL distilled water (negative control treatment) or test solutions. After evaporation of the solvent, 5 mL of distilled water was added to maintain the original concentration. Tebuthiuron was used as a positive control at the same concentration as hydroxy chaicones. Tebuthiuron is one of the most commonly used herbicides for pre-emergent weed control. The germination chamber was set to a 12-hour photoperiod with photosynthetic photon flux density of 60 pmol m2s1at 25 ± 1°C. Distilled water was added whenever necessary to keep the filter paper moist throughout the experiment. Germination was monitored daily for 30 days (d). The seeds were considered germinated when the radicle emerged by 1 mm. Mean germination time (GMT) was calculated using the following equation: MGT = (n * d) / N, where n = number of germinated seeds on each day, d = number of days from the start of the test, and N = total number of germinated seeds at the end of the experiment. The percentage of germination (PG) was calculated using the following equation: PG = germinated seeds / total seeds x 100.

[0038] At the initial growth assessment, seeds from all species were previously germinated up to 1 mm of radicle protrusion. Four replicates with 25 germinated seeds were placed on a Whatman #1 filter paper sheet in 9 cm Petri dishes. The filter paper was moistened with 5 mL distilled water (negative control treatment) or 1 x 10'3mol L'1test solution. Filter paper was kept wet throughout the experiment, with the addition of distilled water or test solutions. Petri dishes were placed in a germination chamber and subjected to a 12-hour photoperiod with photosynthetic photon flux density of 60 pmol m'2s'1at 25 ± 1 °C. In the end, the seedlings were removed from the Petri dishes and rinsed with the aid of filter paper before measuring the length of the root and the shoot. The initial growth assessment was conducted when the control group seedlings reached approximately 5 cm in length.

[0039] The Seedling Vigor Index (SVI) w as calculated as SVI = [average length of shoot (mm) + average length of root (mm)] x average germination percentage.91392-428425- 13 -

[0040] Four hydroxy chaicones significantly decreased the percentage of lettuce germination in the initial screening compared to the negative control. The greatest reduction in germination (44%) was achieved for seeds germinated with a 3 ’-hydroxy chaicone (T3) solution (Table 1). In addition, 3'hydroxy-3,4-dichlorochalcone (Li-9). 3'-hydroxy-4-chlorochalcone (Li-6), and 3'- hydroxy-4-bromochalcone (LR-72) decreased the percentage of lettuce germination by 42%. 40%, and 33%, respectively (Table 1).Table 1 - Effect of hydroxychaicones at a concentration of 1 x 10'3mol L'1on the germination of lettuce seeds.91392-428425- 14 -

[0041] The negative control was distilled water and the positive control was tebuthiuron. Significant differences from the negative control are indicated by * and significant differences from the positive control are indicated by bold according to a one-way ANOVA test followed by Tukey’s test for parametric data and Kruskal- Wallis test followed by Dunn’s test for nonparametric data (p < 0.05, 25 seeds per replicate); standard error (±); (n = 4). Abbreviation: MGT, Mean Germination Time.

[0042] The MGT of lettuce significantly increased to 10 hydroxy chaicones compared to the negative control, and to six compared to the positive control. Notably, 3’-hydroxy-3-thienyl- chalcone (Li-16) stood out, as it increased the MGT by 207% relative to the negative control, and 79% relative to the positive control (Table 1).

[0043] Relative to weeds, selected hydroxychaicones significantly decreased the germination percentage of two of them compared to the negative control. 3'hydroxy-3,4-dichlorochalcone (Li- 9) provided the greatest reduction in germination of Amaranthus viridis seeds, reaching 47%, compared to the negative control (Table 2). Relative to the positive control, the reduction was 53%. When treated with 3 ’-hydroxy chaicone (T3) the reduction was 50% relative to the positive control (Table 2). MGT increased significantly only for Bidens pilosa seeds, which increased by 233% with 3’-hydroxychalcone (T3) (Table 2).Table 2 - Effect of the most active hydroxy chaicones at a concentration of 1 x 10'3mol L’1on the germination of weed seeds.91392-428425- 15 -91392-428425- 16 -

[0044] The negative control was distilled water and the positive control was tebuthiuron. 1 Significant differences are indicated in each column by different letters according to a one-way ANOVA test followed by Tukey’s test for parametric data and Kruskal-Wallis test followed by Dunn’s test for non-parametric data (p < 0.05); standard error (±); (n = 4, 25 seeds per replicate). * Data obtained from replicate with more than one germinated seed and on different days. ** Data obtained from two replicates. 2 Abbreviation: MGT, Mean Germination Time.

[0045] All hydroxychaicones inhibited at least one parameter in early lettuce growth at screening when compared to the negative control (Table 3). 3’ -hydroxy -4-pyridyl-chalcone (Li- 46) caused the greatest inhibition (85%) in the length of the root + shoot (Table 3). Other91392-428425- 17 - hydroxychalcones also caused large initial growth reductions, including 3'-hydroxy-4- bromochalcone (LR-72) 78%, 3'-hydroxy-4-nitrochalcone (Li-58) 69%, and 3'-hydroxy-3- pyridyl-chalcone (Li-45) 54% (Table 3). Overall, it was observed that the shoots exhibited a slightly greater reduction in length than that of the roots treated with hydroxy chaicones (Table 3).

[0046] The selected hydroxy chaicones inhibited the growth of all weeds evaluated (Table 4). 3'-hydroxy-4-pyridyl-chalcone (Li-46) caused greater inhibition (81%) in the length of the root + shoot in Urochloa decumbens compared to the negative control and 74% in Digitaria insularis compared to the positive control (Table 4).Table 3 - Effects of hydroxychaicones at a concentration of 1 x 10'3mol L'1on early lettuce growth.91392-428425- 18 -

[0047] The negative control was distilled water and the positive control was glyphosate. 1 Significant differences from the negative control are indicated by * and significant differences from the positive control are indicated in bold according to a Kruskal-Wallis test followed by Dunn’s test (p < 0.05, 25 seeds per replicate); standard error (±); (n = 4).

[0048] Other hydroxy chaicones also caused large initial growth reductions, such as 3'-hydroxy- 4-pyridyl-chalcone (Li-45) 75% and 3'-hydroxy-4-nitrochalcone (Li-58) 68% in Digitaria insularis'. and 3'-hydroxy-4-bromochalcone (LR-72) 73% in Raphanus raphanistrum (Table 4). In most cases, roots were found to have a slightly more significant length reduction than shoots when treated with hydroxy chaicones (Table 4).Table 4 - Effects of most active hydroxy chaicones at a concentration of 1 x 10'3mol L'1on early weed growth.91392-428425- 19-91392-428425- 20 -

[0049] The negative control was distilled water and the positive control was glyphosate. 1 Significant differences are indicated in each column by different letters according to a one-way ANOVA test followed by Tukey’s for parametric data and Kruskal- Wallis test followed by Dunn’s for non-parametric data (p < 0.05, 25 seeds per replicate); standard error (±); (n = 4).91392-428425- 21 -

[0050] The greatest reduction in lettuce seedling vigor index was caused by 3 ’-hydroxy -4- bromochalcone (LR-72); the treated [group] was 85.1% lower than the negative control (Table 5). Other hydroxy chaicones also caused large reductions in seedling vigor index such as 3’- hydroxy-4-pyridyl-chalcone (Li-46) 84.6% and 3'-hydroxy-4-nitrochalcone (Li-58) 70.2% (Table 5).Table 5 - Effect of hydroxy chaicones on lettuce seedling vigor index.

[0051] The negative control was distilled water. 1 Seedling Vigor Index = [average length of shoot (mm) + average length of root (mm)] x average germination percentage; standard error (±).

[0052] Considering the weeds analyzed, the largest reduction in the seedling vigor index was in Digitaria insularis, with 3’-hydroxy-4-bromochalcone (LR-72), 81% lower than the negative control (Table 6). The same hydroxychaicone caused an 80% reduction in the seedling vigor index in Amaranthus viridis (Table 6).Table 6 - Effect of 3’-hydroxy-4-bromochalcone (LR-72) on weed seedling vigor index.91392-428425- 22 -

[0053] The negative control was distilled water. 1 Seedling Vigor Index = [average length of shoot (mm) + average length of root (mm)] x average germination percentage; standard error (±).

[0055] Herbicidal activity results were generally superior when compared to the positive control; 3'-hydroxy-3,4-dichlorochalcone (Li-9) and 3'-hydroxychalcone (T3) reduced germination by 53% and 50%, respectively, in Amaranthus viridis. compared to tebuthiuron. Thus, hydroxy chaicones have the potential to be more effective than tebuthiuron in combating some weed species.

Claims

91392-428425- 23 -WHAT IS CLAIMED IS:

1. A composition comprising one or more chai cones with herbicidal activity, wherein the one or more chaicones is selected from the group consisting of 3'- hydroxy chaicone (T3), 3'-hydroxy-4-chlorochalcone (Li-6), 3'-hydroxy-4-methylchalcone (Li- 7), 3'-hydroxy-4-methoxy chaicone (Li-8), 3'hydroxy-3,4-dichlorochalcone (Li-9), 3‘-hydroxy- 2-furanyl-chalcone (Li-15), 3'-hydroxy-3-thienyl-chalcone (Li-16), 3’-hydroxy-l-naphthyl- chalcone (Li-21), 3'-hydroxy-2-naphthyl-chalcone (Li-22), 3'-hydroxy-3-chlorochalcone (Li- 40), 3'-hydroxy-2,4-dichlorochalcone (Li-42), 3'-hydroxy-3-pyridyl-chalcone (Li-45), 3’- hydroxy-4-pyridyl-chalcone (Li-46), 3'-hydroxy-4-nitrochalcone (Li-58), 3'-hydroxy-4- bromochalcone (LR-72), 3,4-dimethoxychalcone (CH2), and cinnamaldehyde (Pl).

2. The composition of claim 1, wherein the chaicone is 3'-hydroxy chaicone (T3).

3. The composition of claim 1, wherein the chaicone is 3'-hydroxy-4- chlorochalcone (Li-6).

4. The composition of claim 1, wherein the chaicone is 3'-hydroxy-4- methylchalcone (Li-7).

5. The composition of claim 1, wherein the chaicone is 3'-hydroxy-4- methoxychalcone (Li-8).

6. The composition of claim 1, wherein the chaicone is 3'hydroxy-3,4- dichlorochalcone (Li-9).

7. The composition of claim 1, wherein the chaicone is 3 -hydroxy-2- furanyl-chalcone (Li- 15).

8. The composition of claim 1, wherein the chaicone is 3'-hydroxy-3- thienyl-chalcone (Li- 16).

9. The composition of claim 1, wherein the chaicone is 3’-hydroxy-l- naphthyl-chalcone (Li-21).

10. The composition of claim 1, wherein the chaicone is 3'-hydroxy-2- naphthyl-chalcone (Li-22).

11. The composition of claim 1, wherein the chaicone is 3'-hydroxy-3- chlorochalcone (Li-40).

12. The composition of claim 1, wherein the chaicone is 3 ’ -hydroxy-2, 4- dichlorochalcone (Li-42).91392-428425- 24 -13. The composition of claim 1, wherein the chaicone is 3'-hydroxy-3- pyridyl-chalcone (Li-45).

14. The composition of claim 1, wherein the chaicone is 3’-hydroxy-4- pyridyl-chalcone (Li-46).

15. The composition of claim 1, wherein the chaicone is 3'-hydroxy-4- nitrochalcone (Li-58).

16. The composition of claim 1, wherein the chaicone is 3'-hydroxy-4- bromochalcone (LR-72).

17. The composition of claim 1, wherein the chaicone is 3,4- dimethoxy chaicone (CH2).

18. The composition of claim 1, wherein the chaicone is or cinnamaldehyde (Pl).

19. A composition comprising one or more chai cones with herbicidal activity, wherein the one or more chaicones is selected from the group consisting of 3’- hydroxy chaicone (T3), 3’hydroxy-3,4-dichlorochalcone (Li-9), 3’-hydroxy-4-chlorochalcone (Li-6), 3’-hydroxy-3-pyridyl-chalcone (Li-45), 3’-hydroxy-4-pyridyl-chalcone (Li-46), 3’- hydroxy-4-nitrochalcone (Li-58), 3'-hydroxy-4-bromochalcone (LR-72), 4’-hydroxy-3’- methoxy chaicone (CHI), 3, 4-dimethoxy chaicone (CH2), and cinnamaldehyde (Pl).

20. A method of treating a plant, the method comprising a step of administering a composition comprising one or more chaicones with herbicidal activity to the plant.

21. The method of claim 20, wherein the composition comprising one or more chaicones with herbicidal activity is the composition of any one of claims 1 to 19.

22. The method of claim 20, wherein the method decreases growth of a weed.

23. The method of claim 20, wherein the method decreases germination of a weed seed.

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