Hydroxychalcones as Herbicides

Abstract

Efforts to develop weed management alternatives are urgently required due to various challenges, such as declining crop yields, rising production costs, and the growing prevalence of herbicide-resistant weed species. Chalcones occur in nature and have phytotoxic potential and concise synthesis; additionally, they are multifunctional, with diverse biomolecular targets and a broad spectrum of biological activities. This study sought to assess the herbicidal potential of 3′-hydroxychalcones against weed species under laboratory conditions. Their effects were investigated using germination bioassays, early growth measurements, and the seedling vigor index, all prepared with a concentration of 1 × 10⁻³ mol L⁻¹ 3′-hydroxychalcones. 3′-Hydroxy-4-pyridyl-chalcone caused the greatest inhibition (81%) of the seedling length in Urochloa decumbens. Other 3′-hydroxychalcones also caused large initial growth reductions, such as 3′-hydroxy-4-pyridyl-chalcone (75%) and 3′-hydroxy-4-nitrochalcone (68%) in Digitaria insularis and 3′-hydroxy-4-bromochalcone (73%) in Raphanus raphanistrum. The greatest reduction in the seedling vigor index was 81% in D. insularis treated with the 3′-hydroxy-4-bromochalcone. The same 3′-hydroxychalcone caused an 80% reduction in Amaranthus viridis. In conclusion, 3′-hydroxychalcones exhibit herbicidal activity, suggesting they could serve as a solution for future weed management strategies.

1. Introduction

There is a growing emphasis on developing weed management alternatives due to several pressing challenges. These factors encompass reduced crop yields, escalating production expenses [1], the spread of herbicide-resistant weed species [2], and heightened concerns regarding the long-term impacts of herbicides on both human health and the environment [3]. Additionally, stricter registration procedures in various countries [4] underscore the urgent need for more sustainable, eco-conscious approaches to weed control. The most effective weed management strategies at present involve mechanical or manual weeding, alongside the use of synthetic herbicides [5]. Over the past several decades, synthetic herbicides have been applied extensively, particularly in intensive agriculture [6], contributing to the rise of herbicide-resistant weed populations.

In this regard, nature offers a wealth of chemical diversity that should not be overlooked, supplying bioactive compounds with potential for pest control, including weeds [7]. Thus, even if conventional herbicides cannot be eliminated, their use could be reduced with natural products [8] and with synthetic ones with a chemical structure based on natural products. Furthermore, these substances may present a more ecologically sustainable option for weed control, offering an environmentally friendly alternative to conventional herbicides [9,10].

Potential phytotoxic agents found in plants are derived from their variety of biologically active metabolites. Prior research has characterized some of these substances as good inhibitors [8]. For instance, Chotsaeng et al. [11] reported that 37 different aldehydes exhibited inhibitory effects on Amaranthus tricolor (L.) and Echinochloa crus-galli (L.) Beauv. Numerous plant-derived compounds negatively affect the metabolism of other plants in a manner similar to herbicides [12]. The chemical modification of these natural products should clarify the bioactive subunit of the molecule and help to develop more active and selective commercial herbicides [13] or to discover effective biochemical routes of action [14], as the natural herbicides currently available on the market have little or no selectivity and need to be applied in large quantities [4].

Chalcones (1,3-diaryl-2-propen-1-ones) occur in nature and have good phytotoxic potential and concise synthesis [15]. In addition, they are multifunctional, with diverse biomolecular targets and a broad spectrum of biological activities, presenting insecticidal, fungicidal, bactericidal, anthelmintic, and antiviral actions [6]. Chalcones are flavonoid-derived secondary metabolites that play crucial roles in plant development and defense against pathogens [3,16]. They are structurally defined by two aromatic rings connected via a three-carbon α,β-unsaturated ketone (Figure 1) [16]. While chalcones are primarily found as pigments in flower petals, they have also been identified in other plant tissues, including heartwood, bark, leaves, fruits, and roots [17]. Additionally, they occur in various edible sources, such as wheat products, hops, strawberries, berries, tomatoes, pears, apples, and citrus fruits [3].

Modifying chalcones by introducing specific functional groups can enhance their biological activity [3]. However, because plants produce these compounds in limited amounts, their extraction from natural sources remains challenging [3]. To date, only a few chalcones have been identified as inhibitors of photosystem II [18] or plant growth regulators [19]. This suggests that many unexplored chalcones hold the potential for development as herbicidal agents in crop protection [8].

3′-hydroxychalcones have moderate water solubility, with a Log S of −4.38, which can lead to reduced mobility in the soil and, consequently, a lower risk of environmental contamination. Their lipophilicity is relatively high, with a log P_O/W of 3.45, suggesting a strong capacity to diffuse across lipid membranes. This property enhances their absorption and translocation within plant tissues. In general, herbicides with high log P_O/W values tend to be more effective, as they readily enter plant cells and disrupt key physiological processes, such as photosynthesis and cell division [20].

In this context, exploring the phytotoxic potential of 3′-hydroxychalcones is relevant for developing new and more sustainable herbicides. This research is driven by the urgent need for eco-friendly substances that are less harmful to the environment and human health while effectively managing weeds. Sustainable alternatives, like these, are vital in addressing the growing demand for safer, more responsible weed control solutions.

This research sought to assess the herbicidal potential of 3′-hydroxychalcones by examining their effects on the germination and early growth of various weed species under laboratory conditions.

2. Materials and Methods

2.1. Study Seeds

In the germination and initial growth screening, lettuce (Lactuca sativa L.) was used, due to its abundant and rapid germination and good sensitivity to substances with phytotoxic potential [4]. The four most potent inhibitory compounds were selected for testing on weed species, including Digitaria insularis (L.) Fedde (sourgrass), Rottboellia cochinchinensis (Lour.) Clayton (itchgrass), Urochloa decumbens (Stapf) R.D. Webster (signalgrass), Amaranthus viridis L. (slender amaranth), Bidens pilosa L. (hairy beggartick), and Raphanus raphanistrum L. (wild radish). Among these, the first three are monocotyledons, while the remaining species are eudicotyledons. The surface of all seeds was disinfected before the bioassays. The seeds were immersed in a 1% sodium hypochlorite solution and left to soak for 5 min with occasional agitation. Finally, they were rinsed three times with sterile distilled water to remove any disinfectant residue. The lettuce seeds were sourced from Isla^®, while the weed seeds were commercially acquired from Agro Cosmos, based in Engenheiro Coelho, São Paulo state, Brazil.

2.2. Test Solutions

All 3′-hydroxychalcones used in this study (Figure 1) were diluted in ethanol to prepare 1 × 10⁻³ mol L⁻¹ concentration solutions. This concentration was selected because natural herbicides typically have significant inhibitory effects within the range of 10⁻² to 10⁻³ mol L⁻¹ [21].

Positive controls, namely tebuthiuron (the active substance of Combine^® herbicide, widely used for pre-emergent weed control) and glyphosate (the active substance of Roundup^® herbicide, used for post-emergence control) were also prepared in ethanol absolute at 1 × 10⁻³ mol L⁻¹ concentration. All 3′-hydroxychalcones were synthesized and supplied by Dr. Luis Octavio Regasini from the Laboratory of Antibiotics and Chemotherapeutics at São Paulo State University, São José do Rio Preto campus. All positive controls were obtained commercially from Sigma-Aldrich^®, Darmstadt, Germany.

2.3. Germination Experiments

The 3′-hydroxychalcones HC-01, HC-02, HC-05, and HC-15 were selected for the weed seed germination test as they exhibited the highest germination inhibition rates in the initial screening with lettuce. In the germination assays, 4 replicates of 25 seeds of L. sativa or each weed species were arranged on a Whatman No. 1 filter paper sheet inside 9 cm Petri dishes, which were then moistened with 5 mL of either absolute ethanol (negative control) or the test solutions. Once the ethanol had evaporated, 5 mL of distilled water was introduced into the Petri dishes of all treatments. The pH (6.5) of the distilled water was controlled. Tebuthiuron, at a 1 × 10⁻³ mol L⁻¹ concentration, was utilized as the positive control. The germination chamber (Eletrolab, São Paulo, Brazil) was programmed for a 12 h photoperiod, with a photosynthetic photon flux density of 60 μmol m⁻² s⁻¹, maintained at a temperature of 25 ± 1 °C. Germination was observed daily for 30 days. Seeds were classified as germinated once the radicle reached a length of 1 mm. The mean germination time (MGT) was determined using the following formula: MGT = ∑ (n × d)/N, where n represents the number of seeds that germinated each day, d is the number of days elapsed since the test began, and N is the total number of seeds that had germinated by the conclusion of the experiment. The germination percentage (GP) was determined using the following formula: GP = (number of germinated seeds/total number of seeds × 100).

2.4. Initial Growth Experiments

The 3′-hydroxychalcones HC-12, HC-13, HC-14, and HC-15 were chosen for the weed seedling test due to their strong growth inhibition effects observed in the lettuce screening. In all experiments, seeds of all species (4 replicates with 25 seeds each) were previously germinated to 1 mm of radicle protrusion. The germinated seeds were arranged on a sheet of Whatman No. 1 filter paper inside 9 cm Petri dishes, which were moistened with either 5 mL of absolute ethanol (serving as the negative control) or 1 × 10⁻³ mol L⁻¹ test solutions. Once the ethanol had evaporated, 5 mL of distilled water was added to the Petri dishes for all treatments. The pH (6.5) of the distilled water was controlled. Glyphosate served as positive control, applied at 1 × 10⁻³ mol L⁻¹ concentration. The Petri dishes were kept in a germination chamber (Eletrolab, São Paulo, Brazil) with a 12 h light period, exposed to a photosynthetic photon flux density of 60 μmol m⁻² s⁻¹, and maintained at 25 ± 1 °C. Finally, 60 seedlings were selected from the Petri dishes (15 randomly chosen seedlings per dish) and blotted dry using a filter, and then their root and shoot lengths were measured. Initial growth was assessed once the control seedlings reached an approximate height of 5 cm.

2.5. Evaluation of Seedling Vigor Index

The seedling vigor index (SVI) was determined using the following formula: SVI = [mean shoot length (mm) + mean root length (mm)] × mean germination percentage. The seedling vigor index was only calculated for 3′-hydroxychalcone HC-15 because it was the only compound selected for testing on weeds for germination and growth.

2.6. Statistical Analysis

The data were assessed for normal distribution using the Shapiro–Wilk test (p > 0.05) and for homogeneity of variances using Levene’s test (p > 0.05). If the dataset satisfied these assumptions, the experiments were analyzed using a one-way ANOVA followed by Tukey’s post hoc test. Data that did not meet these criteria were subjected to the Kruskal–Wallis test followed by Dunn’s post hoc test (p ≤ 0.05). The Benjamini–Hochberg procedure was performed, adjusting the p values to correct the type I error rate in multiple comparisons, allowing the false discovery rate (FDR) to be controlled. Data analysis was performed using BioEstat software (Version 5.0) and R software (Version 4.2.2).

3. Results

3.1. 3′-Hydroxychalcones’ Effects on Germination

Four 3′-hydroxychalcones decreased the germination percentage of lettuce in the initial screening compared to the negative control. The highest germination reduction (44%) rate was achieved for seeds germinated with the HC-01 solution (

Table 1. Effect of 3′-hydroxychalcones at 1 × 10⁻³ mol L⁻¹ on lettuce seed germination.

Treatment	Germination (%) 1	MGT 2 (days) 1
Negative control	95 ± 6	1.3 ± 0.2
Tebuthiuron	51 ± 17 *	2.3 ± 0.8 *
HC-01	53 ± 13 *	2.5 ± 0.7
HC-02	57 ± 14 *	1.6 ± 0.4
HC-03	68 ± 24	1.6 ± 0.1
HC-04	71 ± 29	1.6 ± 0.3
HC-05	55 ± 4 *	1.6 ± 0.3
HC-06	84 ± 6	3.9 ± 0.6 *
HC-07	88 ± 12	4.1 ± 0.6 *
HC-08	86 ± 11	3.8 ± 0.3 *
HC-09	93 ± 7	3.8 ± 0.5 *
HC-10	81 ± 13	3.9 ± 0.8 *
HC-11	86 ± 8	3.6 ± 0.2 *
HC-12	89 ± 8	4.0 ± 0.6 *
HC-13	97 ± 4	3.7 ± 0.3 *
HC-14	91 ± 9	3.7 ± 0.5 *
HC-15	64 ± 15 *	4.1 ± 1.2 *

The negative control was distilled water and tebuthiuron was the positive control. ¹ Significant differences from the negative control are marked with *, while significant differences from the positive control are highlighted in bold. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s test for parametric data, and a Kruskal–Wallis test followed by Dunn’s test for non-parametric data (p ≤ 0.05, 25 seeds per replicate); mean values are presented with standard error (±); (n = 4). ² Abbreviation: MGT, mean germination time.

). In addition, HC-05, HC-02, and HC-15 decreased the lettuce germination percentage by 42, 40, and 33%, respectively (Table 1).

The MGT of lettuce increased for ten 3′-hydroxychalcones compared to the negative control and for six compared to the positive control. The highlight was HC-07, which increased the MGT by 207% compared to the negative control and by 79% compared to the positive control (Table 1).

The four selected 3′-hydroxychalcones decreased the germination percentage of two weed species compared to the negative control. The highest germination reduction (47%) was achieved in A. viridis seeds treated with a solution of HC-05 when compared to the negative control (

Table 2. Effect of the most active 3′-hydroxychalcones at 1 × 10⁻³ mol L⁻¹ on weed seed germination.

Treatment	Germination (%) 1	MGT 2 (Days) 1
A. viridis
Negative control	53 ± 13 ab	2.7 ± 0.4 ab
Tebuthiuron	60 ± 20 a	4.6 ± 0.5 ab
HC-01	30 ± 11 b	7.7 ± 2.9 a
HC-02	47 ± 14 ab	4.0 ± 0.3 ab
HC-05	28 ± 7 b	2.5 ± 0.4 b
HC-15	37 ± 5 ab	3.8 ± 0.8 ab
B. pilosa
Negative control	73 ± 10 a	3.0 ± 0.4 a
Tebuthiuron	50 ± 10 ab	9.4 ± 1.60 b
HC-01	50 ± 14 ab	10.0 ± 3.0 b
HC-02	46 ± 10 b	5.5 ± 0.7 ab
HC-05	61 ± 13 ab	5.3 ± 0.8 ab
HC-15	57 ± 2 ab	6.5 ± 1.8 ab
D. insularis
Negative control	27 ± 15 a	4.5 ± 0.9 a
Tebuthiuron	20 ± 9 a	8.2 ± 1.7 b
HC-01	7 ± 9 a	5.2 ± 0.7 a
HC-02	8 ± 7 a	4.7 ± 1.4 a
HC-05	20 ± 3 a	4.6 ± 1.3 a
HC-15	15 ± 7 a	3.7 ± 0.9 a
R. raphanistrum
Negative control	43 ± 8 ab	2.3 ± 0.4 a
Tebuthiuron	35 ± 11 a	3.9 ± 1.9 a
HC-01	51 ± 13 ab	3.1 ± 0.5 a
HC-02	59 ± 4 b	2.3 ± 0.4 a
HC-05	41 ± 9 ab	3.0 ± 0.9 a
HC-15	50 ± 12 ab	2.4 ± 0.2 a
R. cochinchinensis
Negative control	19 ± 7 a	3.9 ± 0.6 a
Tebuthiuron	7 ± 4 a	4.3 ± 0.5 a
HC-01	5 ± 2 a	3.5 ± 0.6 a
HC-02	13 ± 4 a	4.1 ± 0.4 a
HC-05	23 ± 13 a	3.7 ± 0.6 a
HC-15	21 ± 12 a	3.9 ± 0.2 a
U. decumbens
Negative control	70 ± 8 a	3.2 ± 0.3 ab
Tebuthiuron	58 ± 17 a	4.6 ± 1.2 a
HC-01	60 ± 7 a	3.5 ± 0.7 ab
HC-02	66 ± 11 a	3.4 ± 0.4 ab
HC-05	68 ± 14 a	3.0 ± 0.2 b
HC-15	75 ± 21 a	3.2 ± 0.5 ab

The negative control was distilled water, and tebuthiuron was the positive control. ¹ Significant differences within each column are represented by different letter(s), as determined by a one-way ANOVA followed by Tukey’s test for parametric data, and a Kruskal–Wallis test followed by Dunn’s test for non-parametric data (p ≤ 0.05); mean values are presented with standard error (±); (n = 4, 25 seeds per replicate). ² Abbreviation: MGT, mean germination time.

); when compared to the positive control, it decreased by 53%.

When treated with HC-01, the reduction was 50% compared to the positive control (Table 2). MGT increased only for B. pilosa seeds, which had a 233% increase with HC-01 (Table 2).

3.2. 3′-Hydroxychalcones’ Effects on Initial Growth

All 3′-hydroxychalcones inhibited at least one parameter in the initial growth of lettuce in the screening when compared to the negative control (

Table 3. Effects of 3′-hydroxychalcones at 1 × 10⁻³ mol L⁻¹ on the initial growth of lettuce.

Treatment	Root Measurement (mm) 1	Shoot Measurement (mm) 1	Root + Shoot Measurement (mm) 1
Negative control	25.2 ± 11.3	7.3 ± 3.5	32.5 ± 12.2
Glyphosate	8.2 ± 2.2 *	6.1 ± 2.6	14.3 ± 3.6 *
HC-01	18.8 ± 14.2 *	5.8 ± 3.2 *	24.6 ± 15.9 *
HC-02	22.0 ± 15.2 *	3.8 ± 2.3 *	25.8 ± 16.2 *
HC-03	31.3 ± 15.7	5.5 ± 2.3 *	36.8 ± 16.9
HC-04	26.4 ± 14.6	5.5 ± 2.4 *	32.0 ± 16.0
HC-05	29.5 ± 16.7	5.4 ± 3.4 *	34.9 ± 19.0
HC-06	24.0 ± 13.9	5.1 ± 2.0 *	29.1 ± 15.1 *
HC-07	27.7 ± 15.7	5.3 ± 3.1 *	33.1 ± 17.6
HC-08	20.3 ± 13.5 *	5.8 ± 3.8 *	26.0 ± 15.5 *
HC-09	24.0 ± 14.9	5.8 ± 3.2 *	29.8 ± 16.8 *
HC-10	25.9 ± 14.7	4.7 ± 3.3 *	30.6 ± 16.8
HC-11	29.2 ± 12.6	5.9 ± 2.4 *	35.1 ± 13.6
HC-12	10.5 ± 11.9 *	4.4 ± 4.3 *	14.8 ± 15.6 *
HC-13	3.3 ± 3.6 *	1.6 ± 1.9 *	4.9 ± 5.3 *
HC-14	7.1 ± 10.3 *	3.0 ± 3.1 *	10.1 ± 13.0 *
HC-15	5.3 ± 7.5 *	1.9 ± 1.8 *	7.1 ± 9.0 *

The negative control was distilled water, and glyphosate was the positive control. ¹ Significant differences from the negative control are marked with *, while significant differences from the positive control are highlighted in bold, according to a Kruskal–Wallis test followed by Dunn’s post hoc test (p ≤ 0.05); standard error (±); (n = 60 seedlings).

). HC-13 exhibited the strongest inhibitory effect, reducing root and shoot length by 85% (Table 3). Significant reductions in initial growth were also observed with other 3′-hydroxychalcones, including HC-15 (78%), HC-14 (69%), and HC-12 (54%) (Table 3). In most cases, shoot length exhibited a slightly greater reduction than root length following treatment with 3′-hydroxychalcones (Table 3).

The growth of all weeds was inhibited by the selected 3′-hydroxychalcones (

Table 4. Effects of the most active 3′-hydroxychalcones at 1 × 10⁻³ mol L⁻¹ on the initial growth of weeds.

Treatment	Root Measurement (mm)1	Shoot Measurement (mm) 1	Root + Shoot Measurement (mm) 1
A. viridis
Negative control	31.2 ± 5.3 a	16.4 ± 3.0 a	47.6 ± 6.9 a
Glyphosate	13.2 ± 3.5 b	10.9 ± 3.7 b	24.1 ± 6.6 b
HC-12	7.8 ± 3.3 c	5.5 ± 2.6 c	13.3 ± 4.9 c
HC-13	7.2 ± 3.1 c	5.5 ± 2.2 c	12.7 ± 4.6 c
HC-14	15.3 ± 6.6 b	11.7 ± 5.8 b	27.0 ± 10.3 b
HC-15	8.6 ± 3.3 c	5.3 ± 2.5 c	14.0 ± 5.1 c
B. pilosa
Negative control	35.7 ± 12.1 a	25.8 ± 6.4 a	61.5 ± 15.9 a
Glyphosate	18.4 ± 5.2 b	23.1 ± 6.0 b	41.5 ± 10.0 bd
HC-12	13.7 ± 7.4 c	14.9 ± 6.6 c	28.6 ± 12.9 c
HC-13	15.2 ± 8.4 bc	14.2 ± 6.2 c	29.4 ± 13.2 c
HC-14	38.2 ± 18.4 ad	18.0 ± 6.4 d	56.2 ± 23.0 ad
HC-15	31.9 ± 18.3 d	15.2 ± 5.6 c	47.1 ± 22.4 d
D. insularis
Negative control	33.9 ± 6.2 a	3.4 ± 0.8 a	37.3 ± 6.5 a
Glyphosate	27.9 ± 7.2 a	3.0 ± 1.0 a	31.0 ± 7.6 a
HC-12	8.0 ± 4.3 bc	1.2 ± 0.5 b	9.2 ± 4.4 bc
HC-13	7.1 ± 4.4 c	1.1 ± 0.3 b	8.2 ± 4.4 c
HC-14	10.7 ± 5.6 b	1.3 ± 0.6 b	12.0 ± 5.7 b
HC-15	11.3 ± 6.6 b	1.4 ± 0.6 b	12.6 ± 6.9 b
R. raphanistrum
Negative control	30.3 ± 26.0 a	16.3 ± 9.5 a	46.6 ± 31.8 a
Glyphosate	9.6 ± 3.4 b	10.9 ± 5.6 bd	20.5 ± 7.1 b
HC-12	22.9 ± 18.4 a	13.3 ± 7.7 ad	36.2 ± 22.5 ad
HC-13	13.4 ± 13.7 b	12.8 ± 7.0 ad	26.3 ± 16.4 bd
HC-14	14.8 ± 16.8 b	11.8 ± 7.8 bd	26.7 ± 22.3 b
HC-15	6.4 ± 10.8 c	6.2 ± 8.7 c	12.6 ± 18.3 c
R. cochinchinensis
Negative control	10.7 ± 2.9 a	10.7 ± 4.6 a	21.5 ± 7.0 a
Glyphosate	9.5 ± 2.1 a	8.1 ± 3.5 a	17.5 ± 5.0 a
HC-12	4.7 ± 1.2 b	1.5 ± 2.1 b	6.2 ± 2.8 b
HC-13	5.3 ± 2.3 b	1.9 ± 3.3 b	7.3 ± 5.3 b
HC-14	4.9 ± 2.4 b	2.4 ± 4.1 b	7.3 ± 6.2 b
HC-15	5.1 ± 2.3 b	2.1 ± 3.8 b	7.3 ± 6.0 b
U. decumbens
Negative control	47.2 ± 23.8 a	5.5 ± 2.6 a	52.7 ± 25.9 a
Glyphosate	24.0 ± 5.9 b	4.1 ± 1.4 a	28.0 ± 6.9 b
HC-12	15.4 ± 10.6 c	2.7 ± 1.4 b	18.0 ± 6.9 c
HC-13	8.2 ± 6.5 d	1.6 ± 1.4 c	9.8 ± 7.4 d
HC-14	23.2 ± 12.4 be	2.4 ± 1.5 b	25.5 ± 13.3 be
HC-15	19.6 ± 11.7 ce	2.2 ± 1.3 bc	21.8 ± 12.6 ce

The negative control was distilled water and glyphosate was the positive control. ¹ Significant differences within each column are denoted by a different letter(s), determined by a one-way ANOVA followed by Tukey’s test for parametric data, and a Kruskal–Wallis test followed by Dunn’s test for non-parametric data (p ≤ 0.05); standard error (±); (n = 60 seedlings).

). HC-13 exhibited the greatest inhibitory effect on root + shoot length, reducing it by 81% in U. decumbens relative to the negative control and by 74% in D. insularis compared to the positive control (Table 4).

Other 3′-hydroxychalcones also caused large initial growth reductions, such as HC-12 (75%) and HC-14 (68%) in D. insularis and HC-15 (73%) in R. raphanistrum (Table 4). In most cases, root length exhibited a slightly greater reduction than shoot length following treatment with the 3′-hydroxychalcones (Table 4).

3.3. Evaluation of Seedling Vigor Index

HC-15 resulted in the most significant decrease in the seedling vigor index of lettuce, having an 85.1% reduction when compared to the negative control (Figure 2). Other 3′-hydroxychalcones also caused large seedling vigor index reductions, such as HC-13 (84.6%) and HC-14 (70.2%) (Figure 2).

Considering the studied weeds, the greatest reduction in seedling vigor index was seen in D. insularis treated with HC-15, showing a reduction of 81% towards the negative control (Figure 3).

The same 3′-hydroxychalcone caused an 80% reduction in seedling vigor index in A. viridis (Figure 3).

4. Discussion

4.1. 3′-Hydroxychalcones’ Effects on Germination

The 3′-hydroxychalcones caused less of a reduction in the seed germination of lettuce than in the weed species, except for B. pilosa. Seed germination is a critical phase in plant establishment, preceding successful growth and development [22]. HC-05 had some pre-emergence weed control of A. viridis.

Studies have suggested that chalcones inhibit the germination of certain weeds, such as trans-chalcone, which inhibited the germination of Plantago lanceolata [15]. Other study has demonstrated that 2,4′-dimethoxy-chalcone inhibited the germination of Mimosa pudica and Senna obtusifolia seeds by 58% and 48%, respectively, at a concentration of 1.1 × 10⁻³ mol L⁻¹ [23]. In this study, there was a 37% reduction in the germination of B. pilosa treated with HC-02 at 1 × 10⁻³ mol L⁻¹. The inhibition of germination in various weeds is influenced by the specific chemical substituent groups and concentrations of chalcones, highlighting the significance of studies on the differential sensitivity of species.

In general, herbicidal activity was most evident when compared with the positive control; HC-05 and HC-01 decreased the germination of A. viridis by 53% and 50% over the tebuthiuron inhibition, respectively. Therefore, 3′-hydroxychalcones may potentially be more efficient than tebuthiuron in some of these assays.

Díaz-Tielas et al. [15] observed that increasing the concentration of trans-chalcone caused a reduction in the germination speed for most weeds and kept it constant for crops. In the current study, MGT only increased for B. pilosa seeds treated with HC-01, with no significant differences observed for the other treatments. The reduction in germination and the delayed mean germination time observed in B. pilosa is beneficial from a management perspective, as it would diminish the weed species’ competitive edge over the crops [24].

Regarding the structure–activity relationship, 3′-hydroxychalcones with halogen substituents at position 4 of the B ring (HC-02, HC-05, and HC-15) exhibited the strongest inhibitory effects on lettuce germination, with the exception of HC-01, which lacks substituents at this position. However, HC-11, which also has a 4-chloro substituent along with a 6-chloro substituent, does not exhibit germination inhibition, making it an exception to the other results. In the case of weed germination, the greatest inhibitions were also caused by 3′-hydroxychalcones with halogen substituents in position 4 of the B ring (HC-02 and HC-05), more specifically, 4-chloro. In the production of biocidal agents, it is very common to replace the para-hydrogens of the chalcone benzene rings with halogens at the outermost position of the chemical structure [25,26,27]. Typically, organisms face challenges in metabolizing molecules containing toxic substituents in this position due to the distinct spatial and electronic arrangement of these substituents. This configuration has the potential to disrupt the capacity of metabolic enzymes to recognize and bind to chalcones during the metabolic process [25,26,27]. In this sense, the position 4 substituted by halogen is strategic in the synthesis of more effective chalcones.

The greatest increases in mean germination time (MGT) were caused by heterocyclic chalcones (HC-06; HC-07, and HC-12). The present work confirms the findings of Macías et al. [28], in which the herbicidal activity of 3′-hydroxychalcones was related to differences in the number and positions of substituent groups.

4.2. 3′-Hydroxychalcones Effects’ on Initial Growth

All 3′-hydroxychalcones inhibited at least one parameter in the initial growth of lettuce in the screening experiment. This inhibition of lettuce growth corroborates the results obtained by Chen et al. [29] for this species, studying another chalcone (1,3-diphenyl-2-propen-1-one).

All species had their initial growth inhibited by 3′-hydroxychalcones, except for two species that had no inhibition for one of the substances. HC-13 induced the most significant inhibition in root and shoot length of both lettuce and U. decumbens. Other 3′-hydroxychalcones caused large initial growth reductions, such as HC-12 and HC-14 in D. insularis and HC-15 in R. raphanistrum.

Gomes et al. [30] investigated the herbicidal effects of 13 synthetic chalcones on the early growth of Sesamum indicum. One of the chalcones studied by them was HC-14, also studied in the present work. In the study by Gomes et al. [30], this chalcone did not affect the initial growth of S. indicum, instead having a minor stimulation of root growth at a concentration of 1.5 × 10⁻³ mol L⁻¹. Here, HC-14 inhibited the initial growth of roots and shoots of all weeds, except for B. pilosa. The greatest reduction in total initial growth caused by this chalcone was 68% in D. insularis. Again, these findings suggest that the inhibition of the initial growth of plants is species-dependent.

Also, in the work by Gomes et al. [30], two chalcones inhibited the early growth of two study species, with comparable inhibition to glyphosate. In the present study, for D. insularis and R. cochinchinensis, the inhibition of total initial growth by HC-14 was greater than glyphosate, and for other species the reduction was similar. All species were inhibited by HC-13, showing greater inhibition than glyphosate, except in R. raphanistrum where the inhibition was similar.

In most species, the reduction in root length was slightly more pronounced than in the shoot when the 3′-hydroxychalcones were tested on weeds. HC-13 affected both root and shoot development. The study by Gomes et al. [30] reported divergences related to the most sensitive part of the plant; for the eudicotyledonous S. indicum, the inhibition was higher in the shoot, while the monocotyledonous U. decumbens presented greater inhibition in the root, as in the present study.

Díaz-Tielas et al. [15] observed that trans-chalcone at 0.4 × 10⁻³ mol L⁻¹ was harmful for the initial growth of the weeds’ root; for Plantago lanceolata the reduction was 67%. Díaz-Tielas et al. [31] demonstrated that at a concentration of 1.2 × 10⁻³ mol L⁻¹, the same trans-chalcone inhibited the root growth of Arabidopsis thaliana by 94%, which is 20% higher than the concentration used in the present study. In this study, the greatest inhibition (83%) of root length was caused by HC-13 with 1 × 10⁻³ mol L⁻¹ in U. decumbens.

If we consider the structure–activity relationship, we can infer that the most active 3′-hydroxychalcones are the heterocyclic ones that contain nitrogen (HC-12 and HC-13) and those substituted in position 4 of the B ring (HC-14 and HC-15). The greatest inhibitions of root, shoot, and total growth in lettuce and weeds are directly related to these structural characteristics. Therefore, there appears to be a structure–activity relationship in the herbicidal activity of chalcones. Other studies have also concluded that chalcone structures, in terms of the presence, number, and position of substituent groups, are key factors in determining their biological activity [2,6], influencing their potency and selectivity both in vitro and ex vitro [2].

4.3. Seedling Vigor Index

All 3′-hydroxychalcones demonstrated a significant reduction in the seedling vigor index during the lettuce screening. Among them, HC-15 exhibited the strongest effect, followed closely by HC-13 and HC-14. The high inhibition rates observed indicate that these compounds interfere with essential physiological processes required for seedling establishment, such as water uptake, cell division, and elongation.

In the weed bioassay, HC-15 maintained its potency, causing a reduction in D. insularis and A. viridis. The consistency of HC-15’s strong inhibitory effects across different plant species suggests that its mechanism of action is not species-specific, highlighting its potential as a broad-spectrum herbicidal agent. Moreover, the substantial reduction in seedling vigor of both monocotyledonous and eudicotyledonous weeds reinforces the idea that 3′-hydroxychalcones could serve as effective alternatives to conventional herbicides.

The superior performance of HC-15 compared to the other tested compounds may be attributed to the presence of a halogen at position 4 of ring B. Halogenation is known to enhance the bioactivity of various phytotoxins by increasing their stability, lipophilicity, and interaction with biological targets, although exceptions may exist. This structural feature likely contributes to improved absorption and translocation within plant tissues, leading to more pronounced phytotoxic effects.

5. Conclusions

The study demonstrated that the investigated 3′-hydroxychalcones exhibit significant phytotoxic activity, capable of inhibiting both germination and, more prominently, the initial growth of monocotyledonous and eudicotyledonous weed species. This suggests that 3′-hydroxychalcones interfere with essential physiological processes required for seedling establishment. Their effectiveness across different plant groups highlights their broad-spectrum potential as herbicidal agents. Furthermore, their observed activity under laboratory conditions provides a strong foundation for future investigations into their mechanisms of action, selectivity, and environmental behavior. These findings open new avenues for the development of hydroxychalcone-based herbicides, which could contribute to more sustainable weed management strategies in agriculture.

6. Patents

There are patents resulting from the work reported in this manuscript. A provisional application was filed in the United States under number 63/688,403 and entitled “Chal-cone-Related Herbicides”. A provisional application was also filed in Brazil under number BR 10 2024 026838 5 and entitled “Herbicida de Chalconas”.