Fungicidal Potential of 3-Acyl-6-bromoindole Derivatives: Synthesis, In Vitro Activity, and Molecular Docking Against Botrytis cinerea and Monilinia fructicola

Abstract

Gray mold (Botrytis cinerea) and brown rot (Monilinia fructicola) are devastating diseases in stone fruit production. In this work, a series of 3-acyl-6-bromoindoles, including six new compounds, were synthesized via green and efficient microwave-assisted methodology to evaluate their antifungal potential. Bioassays revealed a remarkable duality in the compounds’ mechanism of action. The starting material, 6-bromoindole (I), proved to be a highly potent inhibitor of mycelial growth, with a remarkable EC₅₀ of 11.62 µg/mL against B. cinerea and 18.84 µg/mL against M. fructicola, surpassing BC-1000^® and Captan^® and comparable to Mystic^®. Conversely, compound I was ineffective at inhibiting conidial germination. In stark contrast, the simple acetylated derivative 3-acetyl-6-bromoindole (II) emerged as a formidable inhibitor of spore germination, achieving 100% inhibition for B. cinerea and 96% for M. fructicola, outperforming several controls. This suggests that, while compound I is effective against established infections, compound II acts as a powerful preventative agent against initial spore-based propagation. The antifungal activity of the compounds directly correlates with their ability to bind to the enzymes SDH and MfCat2, the latter being the main mechanism of action. These findings highlight the potential of developing specialized fungicides from the 6-bromoindole scaffold, targeting different stages of fungal development.

1. Introduction

Stone fruits, such as peaches, nectarines, plums, and cherries, are of significant economic and nutritional importance worldwide [1]. However, their post-harvest lifespan is severely threatened by fungal diseases. The filamentous fungi Botrytis cinerea and Monilinia fructicola are two of the most devastating pathogens in the post-harvest stage of these fruits, capable of causing substantial economic losses that can exceed 80% of production under favorable conditions [2,3].

Botrytis cinerea, the causative agent of gray mold, is a necrotrophic fungus that affects a wide variety of crops [4]. Meanwhile, M. fructicola, responsible for brown rot, is considered one of the most aggressive pathogens of stone fruits [5]. The presence of M. fructicola is widespread across North, Central, and South America [1]. Both fungi can infect the fruit in the field, remaining latent until the ripening and post-harvest storage conditions favor their development, which is characterized by the appearance of brown spots and subsequent sporulation that deteriorates the fruit [5,6].

The economic losses caused by these pathogens are substantial. Globally, it is estimated that B. cinerea alone can generate losses exceeding $100 billion USD [7]. In the Americas, post-harvest diseases are a major concern for stone fruit producers [8]. For instance, in Chile, gray mold caused by B. cinerea is a significant issue for various fruit crops, leading to major economic losses both before and after harvest. The disease is the primary reason for the rejection and losses of Chilean table grapes in international markets [9,10]. For stone fruits, rots caused by B. cinerea and Monilinia spp. are among the leading causes of losses in the production chain [11].

Traditionally, the control of these diseases has relied on the application of synthetic fungicides [12,13]. However, growing concerns about pesticide residues in food, the development of resistance by pathogens, and the environmental impact of these products have driven the search for safer and more sustainable alternatives.

In this context, natural products are emerging as a promising alternative. Specifically, indole and its derivatives—both natural compounds isolated from plants and versions obtained through synthesis—have been shown to possess effective antifungal activity [14,15,16]. To enhance this activity, various synthetic strategies are employed, with halogenation being one of the most effective, particularly through the introduction of bromine into the structure. In fact, bromoindoles have demonstrated significantly greater antifungal activity than their non-halogenated counterparts, where the position and number of bromine atoms allow for the precise tuning of their biological properties [17,18,19].

In parallel, acylation at the C3 position of the ring is another key modification. For example, Xu and collaborators (2011) synthesized a series of 3-acylindole analogs, showing that many exhibited potent fungicidal activity, some even surpassing that of commercial reference fungicides [20]. The combination of these two strategies, bromination and acylation, has resulted in hybrid derivatives such as 3-(5-bromo-1H-indol-3-yl)-5-bromo-3-hydroxyindolin-2-one, compounds that exhibit remarkable broad-spectrum activity against various phytopathogenic fungi [21].

Considering that the research field on 3-acyl-bromoindole derivatives with broad-spectrum antifungal activity remains active, and taking advantage of advances in synthesis strategies, the objective of this study is to synthesize a series of eleven 3-acyl-6-bromoindole derivatives, six of which are previously unreported, through regioselective, microwave-assisted Friedel–Crafts acylation under solvent-free conditions. The use of microwave irradiation is proposed in this study because this methodology drastically reduces reaction time. As a result, an increase in the yield of the desired product is achieved while minimizing the formation of by-products [22]. Subsequently, their antifungal capacity against B. cinerea and M. fructicola was evaluated, and their interactions with the succinate dehydrogenase enzyme and catalase 2 from M. fructicola (MfCat2) were analyzed via molecular docking in order to validate them as potential fungicides for the control of post-harvest diseases.

2. Materials and Methods

All solvents, chemicals, and reagents were obtained commercially from AK Scientific Inc. (Union City, CA, USA) and Sigma-Aldrich Co. (St. Louis, MO, USA) and were used without purification.

2.1. Synthesis of 6-Bromo-3-acylindoles

The target molecules were synthesized in a CEM Discover 2.0 microwave synthesizer (CEM Corporation, Matthews, NC, USA) by adapting a method from the literature [23], with conditions adjusted according to the linear or aromatic nature of the anhydride used. Briefly, we charged a 10 mL microwave vessel with 6-bromoindole (1.0 mmol), acid anhydride (linear or aromatic, 1.0 mmol), a metal triflate (Y(OTf)₃, 0.01 mmol), and an ionic liquid ([BMI]BF₄, 1 mmol). We sealed the container with a Teflon lid and irradiated the mixture under various temperature and time settings. The reactions were carried out in standard mode, with constant high-intensity stirring and the output power set to 150 W. Once the reaction was complete, the container was cooled to 50 °C using a rapid compressed air cooling system (included in the Discover 2.0) and then left to cool to room temperature and then extracted the mixture with Et₂O (5 × 10 mL). We washed the combined ether layers with water (2 × 10 mL), saturated aqueous NaHCO₃ (2 × 20 mL), and brine (2 × 10 mL). After drying the organic layer over MgSO₄, we filtered it with Whatman No. 1 filter paper and then removed the solvent using a rotary evaporator. Finally, we purified the crude product by crystallization in the appropriate solvent and determined the isolated yield.

The known linear acylated derivatives 1-(6-bromo-1H-indol-3-yl)ethan-1-one (II) and 1-(6-bromo-1H-indol-3-yl)-2-methylpropan-1-one (V) were isolated as a white solid (87.5% yield; m.p.:233–234 °C) and a yellow solid (82.3% yield; m.p.: 175–176 °C), respectively. Spectroscopic data of compounds II and V were consistent with those reported in the literature [24,25] and were corroborated with their mass spectra (see Supplementary Materials). The new linear acylated derivatives are described below:

1-(6-bromo-1H-indol-3-yl)propan-1-one (III): The compound was isolated as a light brown solid with a yield of 75.9%. m.p.: 233–234 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.24 (d, J = 8.5 Hz, 1H, H-4); 8.23 (s, 1H, H-7); 7.69 (s, 1H, H-2); 7.33 (dd, J = 1.8 and 8.5 Hz, 1H, H-5); 2.88 (dd, J = 7.4 and 14.8 Hz, 2H, H-2′); 1.15 (m, 3H, H-3′). ¹³C NMR (100 MHz, CD₃COCD₃): δ 195.5 (C-1′); 137.8 (C-8); 133.8 (C-2); 125.0 (C-5); 124.7 (C-9); 123.8 (C-4); 116.9 (C-3); 115.8 (C-7); 114.7 (C-6); 32.0 (C-2′); 8.2 (C-3′). EI-MS, m/z (%): 252 (4) M⁺, 251 (20), 224 (94), 222 (100), 194 (12), 143 (15), 115 (24).

1-(6-bromo-1H-indol-3-yl)butan-1-one (IV): The compound was isolated as a light brown solid with a yield of 70.9%. m.p.: 219–220 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.26 (d, J = 7.4 Hz, 1H, H-4); 8.25 (s, 1H, H-7); 7.69 (s, 1H, H-2); 7.33 (dd, J = 1.8 and 8.5 Hz, 1H, H-5); 2.85 (m, 2H, H-2′); 1.73 (m, 2H, H-3′). 0.96 (t, J = 7.4 Hz, 3H, H-4′). ¹³C NMR (100 MHz, CD₃COCD₃): δ 195.0 (C-1′); 138.2 (C-8); 133.2 (C-2); 124.9 (C-5); 124.7 (C-9); 123.5 (C-4); 117.4 (C-3); 115.9 (C-7); 114.7 (C-6); 41.0 (C-2′); 18.2 (C-3′); 13.4 (C-4′). EI-MS, m/z (%): 266 (4) M⁺, 265 (20), 224 (94), 222 (100), 194 (10), 143 (11), 115 (22).

1-(6-bromo-1H-indol-3-yl)pentan-1-one (VI): The compound was isolated as a light pink solid with a yield of 65.2%. m.p.: 179–180 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.26 (d, J = 7.6 Hz, 1H, H-4); 8.24 (s, 1H, H-7); 7.69 (s, 1H, H-2); 7.33 (dd, J = 1.8 and 8.5 Hz, 1H, H-5); 2.87 (t, J = 7.4 Hz, 2H, H-2′); 1.69 (m, 2H, H-3′), 1.39 (m, 2H, H-4′); 0.96 (s, 3H, H-5′). ¹³C NMR (100 MHz, CD₃COCD₃): δ 195.1 (C-1′); 137.8 (C-8); 133.1 (C-2); 125.0 (C-5); 124.7 (C-9); 123.5 (C-4); 117.3 (C-3); 115.9 (C-7); 114.7 (C-6); 38.8 (C-2′); 27.1 (C-3′); 22.3 (C-4′); 13.4 (C-5′). EI-MS, m/z (%): 280 (2) M⁺, 279 (10), 237 (36), 224 (90), 222 (100), 194 (12), 143 (12), 115 (26).

1-(6-bromo-1H-indol-3-yl)-3-methylbutan-1-one (VII): The compound was isolated as a light brown solid with a yield of 65.3%. m.p.: 210–211 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.27 (d, J = 8.2 Hz, 1H, H-4); 8.25 (s, 1H, H-7); 7.69 (s, 1H, H-2); 7.33 (dd, J = 1.8 and 8.5 Hz, 1H, H-5); 2.73 (d, J = 7.0 Hz, 2H, H-2′); 2.27 (m, 1H, H-3′), 0.96 (d, J = 6.8 Hz, 6H, H-4′ and H-5′). ¹³C NMR (100 MHz, CD₃COCD₃): δ 194.8 (C-1′); 137.8 (C-8); 133.3 (C-2); 125.0 (C-5); 124.7 (C-9); 123.6 (C-4); 117.8 (C-3); 115.9 (C-7); 114.7 (C-6); 48.1 (C-2′); 25.5 (C-3′); 22.2 (C-4′ and C-5′). EI- EI-MS, m/z (%): 280 (4) M⁺, 279 (18), 265 (6), 237 (15), 224 (94), 222 (100), 196 (10), 143 (8), 115 (24).

The synthesis of the known acylated aromatic derivatives (6-bromo-1H-indol-3-yl)(phenyl)methanone (VIII), (6-bromo-1H-indol-3-yl)(4-methylphenyl)methanone (IX), and (6-bromo-1H-indol-3-yl)(4-methoxyphenyl)methanone (X) yielded a white solid (f.p.: 262–263 °C), light brown solid (m.p.: 255–256 °C), and a yellow solid (m.p.: 258–259 °C) in 59.9%, 62.5%, and 50.0% yields, respectively. The spectroscopic data obtained for these derivatives are consistent with those reported in previous studies [26,27,28] and were corroborated with their mass spectra (see Supplementary Materials). The synthesis and characterization of the new derivatives are described below:

(6-bromo-1H-indol-3-yl)(4-fluorophenyl)methanone (XI): The compound was isolated as a light yellow solid with a yield of 33.5%. m.p.: 252–253 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.28 (d, 1H, J = 8.4 Hz, H-4); 8.00 (s, 1H, H-2); 7.93 (m, 2H, H-2′ and H-6′); 7.76 (d, J = 1.8 Hz, 1H, H-7); 7.41 (dd, J = 1.8 and 8.5 Hz, 1H, H-5); 7.29 (m, 2H, H-3′ and H-5′). ¹³C NMR (100 MHz, CD₃COCD₃): δ 189.0 (C-10); 165.1(C-4′); 137.7 (C-8); 136.0 (C-1′); 131.7 (C-2′ and C-6′); 126.3 (C-9); 125.6 (C-2); 124.1 (C-5); 116.8(C-4); 116.4 (C-7); 115.8 (C-3); 115.6 (C-3′ and C-5′); 115.4 (C-6). EI-MS, m/z (%): 318 (8) M⁺, 317 (62), 238 (56), 224 (96), 222 (100), 182 (14), 143 (12), 115 (24), 95 (22).

(6-bromo-1H-indol-3-yl)(4-chlorophenyl)methanone (XII): The compound was isolated as a light brown solid with a yield of 28.8%. m.p.: 215–216 °C. ¹H NMR (400 MHz, CD₃COCD₃): δ 8.28 (d, 1H, J = 8.4 Hz, H-4); 8.00 (s, 1H, H-2); 7.86 (d, J = 8.4 Hz, 2H, H-2′ and H-6′); 7.76 (s, 1H, H-7); 7.56 (d, J = 8.3 Hz, 2H, H-3′ and H-5′); 7.41 (d, J = 8.4 Hz, 1H, H-5). ¹³C NMR (100 MHz, CD₃COCD₃): δ 189.2 (C-10); 139.8 (C-4′); 137.2 (C-1′); 136.2 (C-8); 130.8 (C-3′ and C-5′); 129.0 (C-2′ and C-6′); 126.2 (C-9); 125.7 (C-2); 124.1 (C-5); 116.9 (C-4); 116.3 (C-7); 115.5 (C-3 and C-6). EI-MS, m/z (%): 335 (52) M⁺, 333 (40), 224 (92), 222 (100), 207 (24).

2.2. Antifungal Activity

2.2.1. Strains

The fungal strains B. cinerea (UK-1) and M. fructicola (S1), used throughout this study, were obtained from the culture collection of the Laboratorio de Pruebas Biológicas at Universidad Técnica Federico Santa María, Chile. Strain UK-1 was originally isolated from a naturally infected grape (Vitis vinifera) and strain S1 from a nectarine (Prunus persica). Stock cultures were maintained on potato dextrose agar (PDA; Difco, Detroit, MI, USA) at 4 °C. For the bioassays, inoculum was prepared by culturing the fungi on fresh PDA plates at 23 °C for 5 days under a 16 h light/8 h dark photoperiod.

2.2.2. Inhibition of Mycelial Growth Assay

The antifungal activity of the compounds I–XII and three commercial fungicides (BC-1000^®, Mystic^® 520 SC, and Captan^® 13W) was evaluated using a radial growth inhibition assay on potato dextrose agar (PDA). Briefly, the PDA medium was amended with each test compound to achieve final concentrations of 10–250 µg/mL. Control plates contained 1% ethanol, the same amount used as the solvent for the compounds. Each plate was centrally inoculated with a 4 mm mycelial disc. Plates inoculated with B. cinerea were incubated for 3 days, while those with M. fructicola were incubated for 5 days, all at 23 °C in complete darkness. All treatments were performed in triplicate. After the incubation period, mycelial growth diameters were measured, and the percentage of growth inhibition was calculated relative to the control. The inhibition percentages for each concentration were used to obtain the half-maximal effective concentration (EC₅₀), defined as the concentration required to inhibit 50% of mycelial growth, which was determined by regression analysis of the dose–response data using OriginPro v.8 software. Significant differences were determined using a one-way analysis of variance, followed by a pairwise comparison of means (LSD test; p < 0.05).

2.2.3. Conidial Germination Inhibition Assay

The inhibition of conidial germination (ICG) was assessed by adapting the method of Ferreira et al. (2025) [29]. A spore suspension (40 µL, 1 × 10⁵ conidia/mL) from either M. fructicola (9-day culture) or B. cinerea (6-day culture) was spread onto PDA plates containing the compounds at concentrations from 10 to 250 µg/mL. After incubation in darkness (23 °C, 16 h), the percentage of germinated conidia was determined by microscopic examination of 100 spores per replicate. Conidia were considered germinated if the germ tube was at least twice the spore width. The ICG% was calculated, and all assays were performed in triplicate.

2.3. In Silico Assay

2.3.1. Ligand Preparation

Three-dimensional models of the ligands were constructed using Avogadro v1.2.0n. Molecular geometries were optimized and their energies minimized using the MMFF94 force field. The optimized structures were visualized and exported with BIOVIA Discovery Studio Visualizer v17.2.0.16349 (Dassault Systèmes, Paris, France).

2.3.2. Three-Dimensional Modeling of MfCat2

The amino acid sequence of catalase 2 from Monilinia fructicola (MfCat2) was retrieved from the NCBI database (Accession: KAA8570148.1) in FASTA format. This protein, encoded by the gene EYC84_002478, is annotated as a hypothetical peroxisomal catalase. The three-dimensional model was generated by homology modeling using the SWISS-MODEL server (https://swissmodel.expasy.org, accessed on 20 July 2025), with the catalase from Botryotinia fuckeliana (M7TYR4, chain A) as the template. The sequence identity was 93.62%, and the GMQE score was 0.95, indicating high structural reliability. The final model, consisting of 522 residues, was exported in PDB format for further analysis.

2.3.3. Active Site Prediction of MfCat2

Binding site prediction was performed using the PrankWeb server (https://prankweb.cz, accessed on 20 July 2025), which applies machine learning methods. A primary pocket (Pocket #1) comprising 22 residues was identified, with a score of 34.37 and a probability of 0.938. The pocket center was located at coordinates (X: 6.73, Y: 3.24, Z: −10.26 Å) and was considered the putative active site.

2.3.4. Structural Validation by Alignment

To validate the location of the catalytic site, the MfCat2 model was structurally aligned with the crystallized catalase from Helicobacter pylori (PDB ID: 1QQW, resolution 2.0 Å). Both models were superimposed using PyMOL v2.5 with the align command. Catalytic residues from 1QQW (His75, Asp141, Arg354, Tyr358) were compared to those within Pocket #1 of MfCat2, revealing structural and topological conservation in the catalytic region.

2.3.5. Preparation of the SDH Receptor

The crystal structure of succinate dehydrogenase (SDH) from Gallus gallus was obtained from the Protein Data Bank (PDB ID: 2FBW, resolution 2.06 Å), (http://www.rcsb.org, accessed on 20 July 2025). Although this structure is derived from avian SDH, it was chosen because the residues that form the binding pocket for ligands are highly conserved between avian and fungal SDH. The structure contains the co-crystallized ligand flavin-adenine dinucleotide, which was used as a positive control in docking studies.

2.3.6. Molecular Docking

Molecular docking of ligands with SDH and MfCat2 was performed using AutoDock 4.2, employing the Lamarckian genetic algorithm. The macromolecule was treated as rigid, while ligands were allowed full flexibility. Search parameters included 50 runs per ligand with a maximum of 25 million energy evaluations. Clustering of results used an RMSD threshold of <0.5 Å. The SDH active site was defined using a grid box centered at X: 20.46 Å, Y: 54.98 Å, Z: 25.31 Å, with dimensions of 20 × 20 × 25 grid points. Protein–ligand complexes were selected according to the most populated cluster with the lowest binding free energy (ΔG). To validate the protocol, the co-crystallized ligand CBE was re-docked under identical conditions, yielding an RMSD of 2.49 Å, confirming the reproducibility and accuracy of the method.

3. Results

3.1. Synthesis of Compounds II–XII

The introduction of an acyl group at the C3 position of the indole nucleus is a synthetic transformation of great importance, since 3-acylindoles are key precursors in the synthesis of numerous compounds with important biological activities. Historically, Friedel–Crafts acylation has been the most direct method for achieving this functionalization. However, classic acylation protocols at room temperature or under reflux present significant drawbacks, such as competition from indole nitrogen (N-acylation), which reduces regioselectivity. In addition, the need to employ protection and deprotection strategies decreases the overall efficiency of the process [30,31]. To overcome these limitations, a modern, efficient, and sustainable methodology was chosen, which uses microwaves for the synthesis of 3-acyl-6-bromoindole derivatives (II–XII). Using 6-bromo-indole (I) and a series of linear and aromatic anhydrides as starting materials (Scheme 1), we followed conditions similar to those reported in the seminal work of Tran et al. [23], albeit with slight modifications in reaction times and temperatures depending on the nature of each anhydride.

Using this protocol, 3-acyl-6-bromoindole derivatives II–XII were obtained in moderate to good yields (28.8–87.5%). The lower yields observed in the acylation of 6-bromoindole, compared to unsubstituted indole, are mainly due to the electronic influence of the bromine atom. This effect deactivates the indole ring, making it less reactive toward the electrophile, which translates into a lower reaction rate or a longer reaction time to achieve acylation at the 3-position [32]. Despite this deactivating effect, the regioselectivity of the reaction is maintained, as the 3-position remains the preferred site for acylation due to the stability of its reaction intermediate [33].

Furthermore, this trend is significantly impacted by the nature of the anhydride used. The decrease in yield with the chlorinated anhydride, which may seem counter-intuitive, is explained by the fact that electrophile activation can be a dual effect. Although the chlorine atom makes the anhydride a stronger electrophile, this increased reactivity may also raise the rate of undesirable side reactions, such as anhydride hydrolysis by trace water or deactivation of the Lewis acid catalyst. Consequently, the chlorinated anhydride, despite being more reactive, may be less efficient in practice. This contrasts with the unsubstituted benzoic anhydride, which, being less reactive, is also more stable under the reaction conditions, thus minimizing side reactions and resulting in a higher overall yield [34].

3.2. Antifungal Activities of Compounds I–XII

The in vitro antifungal activity was evaluated for a series of 3-acyl-6-bromoindole derivatives against two relevant phytopathogenic fungi, B. cinerea and M. fructicola. The unmodified starting indole I was included in the assays, thus allowing for a comparative evaluation of the efficacy of its synthetic derivatives II–XII. The evaluation against B. cinerea, for which the radial growth results are presented in Figure 1, demonstrated differentiated activity among the compounds. It was observed that the starting indole I as well as derivatives II and V inhibited the mycelial growth of this pathogen. It is important to note that this effect was concentration-dependent, with inhibition intensifying as the compound’s dose increased. However, the other tested derivatives did not show effective antifungal activity against this species at the evaluated concentrations.

Similarly, the evaluation against M. fructicola, illustrated in Figure 2, also revealed differentiated activity. In this case, it was identified that only compound I and derivatives II, VII, and XII were capable of inhibiting the fungus’s growth. For these four active compounds, the antifungal efficacy was clearly concentration-dependent, with compound I showing the highest activity. Conversely, the remaining derivatives did not exhibit inhibitory activity against this pathogen under the assay conditions.

To quantify their antifungal potency, the half-maximal effective concentration (EC₅₀) values and the percentage of conidial germination inhibition were determined for each compound. The results of this evaluation are summarized in

Table 1. In vitro Antifungal Activity of compound I and 3-acyl-6-bromoindole derivatives II–XII.

Compound	B. cinerea		M. fructicola
	EC50 (µg/mL)	ICG (%)	EC50 (µg/mL)	ICG (%)
I	11.62 ± 0.05	7	18.84 ± 0.23	0
II	98.42 ± 0.68	100	31.78 ± 0.40	96
III	>250	NI	>250	NI
IV	>250	NI	>250	NI
V	179.00 ± 0.81	39	>250	NI
VI	>250	NI	>250	NI
VII	>250	NI	113.97 ± 0.71	37
VIII	>250	NI	>250	NI
IX	>250	NI	>250	NI
X	>250	NI	>250	NI
XI	>250	NI	>250	NI
XII	>250	NI	182.23 ± 0.81	0
BC-1000®	95.13 ± 0.67	100	<10	84
Captan®	82.89 ± 0.69	100	-	-
Mystic® 520 SC	-	-	<10	85

Values are the mean ± standard deviation (SD) of three replicates. “-” means no experiment is carried out. NI: no inhibitory effect.

.

The analysis of the antifungal activity of 6-bromoindole and its acylated derivatives revealed an interesting spectrum of efficacy. In the fight against the mycelial growth of B. cinerea, compound I emerged as the most potent agent, with a remarkable EC₅₀ of 11.62 µg/mL that surpassed not only its derivatives (being 8.5 times more active than compound II and 15.4 times more than V) but also the commercial controls. This superiority was maintained against M. fructicola, where compound I again led with an EC₅₀ of 18.84 µg/mL. However, a different pattern emerged when examining the inhibition of conidial germination. Surprisingly, compound I, so effective against mycelium, inhibited germination in B. cinerea by only 7% and failed to inhibit that of M. fructicola. In stark contrast, compound II, despite having more moderate mycelial activity, was revealed to be an exceptionally potent germination inhibitor, achieving 100% inhibition of B. cinerea conidia, matching the controls, and an impressive 96% on M. fructicola conidia, surpassing the fungicides BC-1000^® and Mystic^® 520 SC by 16% and 15%, respectively.

The results presented herein reveal a fascinating duality in the potential of 3-acyl-bromo indoles as fungicides, where compound I acts as a potent suppressor of established mycelial growth, and compound II proves to be a formidable inhibitor of the initial infection and spread via spores. This selective mode of action, which focuses on the inhibition of spore germination, represents a crucial and differentiating strategy compared to conventional fungicides that often target mycelial growth [35]. This selectivity may stem from the interference with specific metabolic pathways and proteins critical to the germination process, thus positioning 3-acyl-bromo indoles as exceptional candidates for the development of new agrochemical agents. The role of the bromo group in the chemical structure of these compounds is pivotal. As documented in the literature, the halogenation of active ingredients is a key strategy in agrochemical design, as it enhances biological activity through steric and electronic effects, thereby optimizing the compound’s efficacy and stability [36]. This structural modification is a determining factor in the potency demonstrated by these compounds. The relevance of this class of molecules is further supported by previous research on the antifungal activity of halogenated indoles. For example, the marine bisindole alkaloid 2,2-bis(6-bromo-3-indolyl)ethylamine has shown significant activity against various fungi, with successful applications in preventing fungal growth on building materials [37]. Similarly, other halogenated indoles have been shown to be effective in inhibiting the formation of biofilms and hyphae, a critical virulence factor in pathogens such as Candida albicans [38]. The selective targeting of spore germination may also serve to minimize the risk of developing fungal resistance. This is a growing concern, as exemplified by resistance to benzimidazoles [39]. The development of compounds with novel modes of action is vital for overcoming this challenge. By requiring lower concentrations for effective protection, these compounds could also reduce environmental impact, cementing their promise as a sustainable alternative for managing fungal diseases in agriculture.

3.3. Molecular Docking

In this study, in addition to evaluating the antifungal activity of 6-bromoindole derivatives against B. cinerea and M. fructicola, their binding affinity was investigated through molecular docking to two potential fungal targets: succinate dehydrogenase (SDH) and catalase MfCat2.

SDH is a key mitochondrial enzyme in the tricarboxylic acid (TCA) cycle and the electron transport chain, catalyzing the oxidation of succinate to fumarate and contributing to ATP generation [40]. In pathogenic fungi such as B. cinerea and Monilinia spp., SDH is essential for the energetic metabolism required for spore germination, development, and pathogenicity. Its inhibition decreases energy production, limiting fungal growth and infectivity. Therefore, SDH-inhibiting fungicides, such as fluopyram and boscalid, are effective in controlling a wide range of fungal diseases [29]. Importantly, docking simulations were performed using the structure of avian SDH (PDB ID: 2FBW), as the crystal structure of fungal SDH is currently not available in the Protein Data Bank. However, this receptor has been widely adopted in SDH inhibitor (SDHI) research because the residues forming the ubiquinone-binding pocket are highly conserved across eukaryotes, including fungi. Approximately 70% sequence identity has been shown between the relevant subunits of Mycosphaerella graminicola SDH and avian SDH [41], making it a suitable template for exploring potential interactions.

The enzyme MfCat2, a specific catalase of M. fructicola, plays a crucial role in detoxifying hydrogen peroxide (H₂O₂) and maintaining redox homeostasis during conidial germination and sporulation. Chen et al. (2024) [42] reported that deletion of the MfCat2 gene led to a marked reduction in spore production and germination capacity, accompanied by an accumulation of reactive oxygen species (ROS) and increased sensitivity to oxidative stress, highlighting its importance in fungal survival and early development [42]. Thus, MfCat2 inhibition constitutes a strategic molecular target for controlling M. fructicola dispersion and establishment.

The acetic derivative (II) was the most active compound, completely inhibiting conidial germination in B. cinerea (100%) and showing 96% inhibition in M. fructicola. In contrast, 6-bromoindole displayed low biological activity and virtually no germination inhibition, suggesting a different mode of action. Derivatives with bulky or branched aliphatic chains generally exhibited low or no activity, except for the isobutyric derivative (V), which showed moderate activity against B. cinerea, and the isovaleric derivative (VII), which exhibited selectivity towards M. fructicola. Halogenated, methoxylated, and benzoic derivatives displayed neither relevant antifungal activity nor conidial inhibition capacity.

Molecular docking studies (

Table 2. Binding Energies to SDH and MfCat2 of Series 6-bromoindole Derivatives.

Compound	Binding Energy on SDH kcal/mol	Binding Energy on MfCat2 kcal/mol
I	−8.960	−8.789
II	−8.808	−9.722
III	−8.031	−8.481
IV	−8.086	−8.023
V	−8.076	−8.575
VI	−8.722	−9.578
VII	−8.540	−9.313
VIII	−7.789	−8.799
IX	−8.089	−8.308
X	−8.037	−8.589
XI	−8.090	−8.533
XII	−8.008	−8.683

) revealed that the most active compounds, particularly the acetic and isobutyric derivatives, exhibited high binding affinity for MfCat2, with docking scores of −9.722 and −9.578 kcal/mol, respectively.

Interestingly, despite its poor in vitro conidial inhibitory capacity, 6-bromoindole showed one of the best docking scores for SDH (−8.960 kcal/mol), suggesting a possible interaction with the mitochondrial respiratory chain.

Figure 3 and Figure 4 illustrate the specific interactions between the evaluated compounds and key binding-site residues in both enzymes. In SDH, the compound II formed three hydrogen bonds with Gly206, Glu388, and Ala203 (2.74, 1.63, and 2.74 Å, respectively), in addition to a carbon–hydrogen bond with Gly205 (3.85 Å) and two π–stacking interactions with Thr204 (4.16 and 3.89 Å). Among the most active compounds, the compound V established a hydrogen bond with Glu388 (1.57 Å) and a carbon–hydrogen bond with His47 (3.31 Å). The compound VII formed two hydrogen bonds with Ala17 and Gly16 (2.00 and 2.95 Å, respectively). The parent compound I, 6-bromoindole, exhibited good affinity for SDH, forming a hydrogen bond with His355 (2.79 Å), four π–cation interactions (two with Arg288 and two with Arg399), and several alkyl-type interactions.

For MfCat2, the compound II established a hydrogen bond with Tyr380 (2.06 Å) and two T-shaped π–π interactions with the same residue (4.92 and 5.20 Å). The compound V generated a hydrogen bond with Arg90 (2.30 Å) and two T-shaped π–π interactions with His93 (4.35 and 5.90 Å). The compound VII showed a hydrogen bond with Tyr380 (2.59 Å) and several alkyl interactions.

Docking results reveal a clear correlation between antifungal activity and the ability of the compounds to establish specific interactions with functional residues of SDH and MfCat2. The compound II, the most active compound, displayed multiple hydrogen bonds and π–stacking interactions with catalytic residues of SDH (Gly206, Glu388, and Thr204), indicating strong active-site affinity, consistent with its inhibitory effect [43]. In MfCat2, the most active compounds shared frequent interactions with Tyr380 through hydrogen bonding and T-shaped π–π stacking. This residue lies within a highly conserved region near the catalytic center, according to structural predictions (PrankWeb, Score 34.37; probability 0.938), suggesting functional relevance. Previous studies have reported that tyrosine residues located in homologous regions of other fungal catalases play essential roles in catalysis and ligand binding [44].

These findings indicate that antifungal activity largely depends on the nature of the substituent group. The high activity and MfCat2 affinity of the compound II support the hypothesis that inhibition of this antioxidant catalase may represent the main mechanism of action, consistent with studies linking catalase loss of function to reduced fungal virulence due to ROS accumulation [45]. Conversely, the affinity of 6-bromoindole for SDH suggests a potential effect on mitochondrial metabolism, which is essential for ATP production and fungal growth [46].

Finally, the low activity of derivatives with long or highly branched aliphatic chains may be explained by reduced cell permeability or lower enzyme target affinity. Nevertheless, the compound V retained moderate activity and MfCat2 affinity, suggesting that small branching does not entirely hinder molecular interaction. The lack of activity in halogenated, methoxylated, and benzoic derivatives could be attributed to poor capacity to effectively interact with the enzymes or to cross fungal cell barriers.

4. Conclusions

This study reveals a remarkable functional duality in the 6-bromoindole scaffold for fungal control. The parent compound, 6-bromoindole (I), proved to be a potent inhibitor of mycelial growth, ideal for treating established infections but ineffective against spore germination. In stark contrast, its acetylated derivative, 3-acetyl-6-bromoindole (II), is a formidable inhibitor of conidial germination, positioning it as an excellent preventive agent. The molecular docking results support this duality by revealing preferential interactions of compound I with succinate dehydrogenase, a key enzyme in energy metabolism. Compound II, on the other hand, showed a stronger affinity for MfCat2 catalase, a critical enzyme in antioxidant defense. These complementary binding profiles, although theoretical, provide a mechanistic basis for the antifungal activities observed at each stage and justify future experimental validations. This finding opens a promising strategy for developing specialized fungicides, where simple chemical modifications allow for targeting different stages of the fungal life cycle.