Sonochemical Synthesis of 2’-Hydroxy-Chalcone Derivatives with Potential Anti-Oomycete Activity

This work reports on the synthesis of eight new 2′-hydroxy-chalcones with potential anti-phytopathogenic applications in agroindustry, AMONG others, via Claisen–Schmidt condensation and ultrasound assisted reaction. Assays showed three chalcones with allyl moieties strongly inhibited growth of phytopathogenic oomycete Phytophthora infestans; moreover, compound 8a had a half maximal effective concentration (EC50) value (32.5 µg/mL) similar to that of metalaxyl (28.6 µg/mL). A software-aided quantitative structure–activity relationship (QSAR) analysis of the whole series suggests that the structural features of these new chalcones—namely, the fluoride, hydroxyl, and amine groups over the carbon 3′ of the chalcone skeleton—increase anti-oomycete activity.


Introduction
In recent years, worldwide per capita food consumption has increased while growth rates in global agricultural production and crop yields have declined [1]; this latter is largely due to phytosanitary problems like pests, fungi, bacteria, and, mainly in the post-harvest phase, diseases produced by oomycetes [2][3][4]. Of the diseases caused by oomycetes, root rot and fruit blight [5] are estimated to render up to 30% of the harvested vegetables and fruits inedible during postharvest handling [6]. As the main causal agent of stem, root, and fruit rot in commercial cultivars, genus Phytophthora [7] is present scattered in almost all producing regions worldwide, causing heavy economic losses and disastrous consequences for natural ecosystems [8].
Phytophthora [7] is present scattered in almost all producing regions worldwide, causing heavy economic losses and disastrous consequences for natural ecosystems [8].
Specifically, oomycete Phytophthora infestans is renowned as the most significant pathogen of the genus, and is responsible for the grave potato and tomato disease known as late blight or potato blight [9]. While there are several fungicides presently used as pre-and post-harvest treatments for control of P. infestans [10], their use leaves residues on food and in cultivar soils, increases pathogen resistance, and damages human health and the environment [11]. The prevalence of P. infestans and the downsides to chemical controls have stimulated modern agriculture research into alternative microorganism control methods. In addressing these shortcomings, natural product derivatives appear to be a viable approach to reducing late blight disease [12].
Chalcones are well documented as biologically active natural products [13], with potential use in diseases caused by Phytophthora spp. In this context, several studies have shown that natural and synthetic chalcones with -OH and -O-alkyl chains linked to different positions of the A and B rings are known to have bactericidal, antifungal, anthelmintic, antiviral, and anti-oomycetes activities [14][15][16][17][18]. These reports illustrate that antimicrobial activity depends on the number, type, and length of side chain, and position of these substituents in the aromatic ring. Due to the importance of hydroxyl groups and alkyl side chains, this work reports on a facile sonochemical synthesis of a series of 2′hydroxy-chalcone derivatives, in which oxyalkyl chains were attached to the A aromatic ring in different positions and variable lengths. The anti-oomycete activities of these chalcone derivatives were tested against P. infestans to evaluate the effect of the 2′ position of the hydroxyl group as well as the position and length of the alkyl chain.

Results and Discussion
This study synthesized twelve derivatives of 2′-hydroxy-chalcone (7a-d, 8a-d, and 9a-d), characterized by spectroscopy. The base structure was manipulated to include different oxyalkyl chains-allyl, 2-methyl-3propenyl, crotyl, and prenyl-in the 4′, 5′, and 6′ positions, as shown in Scheme 1.   As shown Scheme 1, alkoxy acetophenone (4a-d, 5a-d, and 6a-d) synthesis had good yield for the alkylation of the corresponding acetophenone, with the desired alkyl halide in the presence of K 2 CO 3 under ultrasound irradiation. Alkoxy acetophenones obtained were characterized by nuclear magnetic resonance (NMR) spectra analysis. This stage produced five molecules never before described, which, due mainly to the use of ultrasound irradiation, were synthesized nearly stoichiometric. This was notably true for those derived from 2 ,6 -dihydroxyacetophenone, whose double hydrogen bridge, under normal synthesis conditions, is obtained inefficiently [19]. From here, 2 -hydroxy-chalcone derivatives were prepared by Claisen-Schmidt condensation of the different alkoxy acetophenones with benzaldehyde under ultrasound irradiation [20], resulting in a known series (7a-d) and two new series of molecules (8a-d and 9a-d). The mild reaction conditions, shorter reaction time, and high yields make this reaction more efficient than the classic reflux agitation method used for chalcone synthesis [21].

Compound
Furthermore, all compounds tested showed strong anti-oomycete activity and were superior to difenoconazole. Among them, compound 8a was the most potent derivative of the series, with activity similar to that of metalaxyl.
A membrane damage experiment-based on the direct action of the compounds on the formation of sterol in the cells-was performed to establish the possible death pathway of P. infestans strains. Compound effects were compared to 2% sodium dodecyl sulfate (SDS), an anionic surfactant that produces 100% cell lysis. Percentage of membrane lysis of P. infestans strains are summarized in Table 2.
This type of test is based on the direct action of the compounds on the formation of sterol in the cells of the oomycete membranes' paper anterior.
The membrane damage test showed antifungal effectiveness of compound 8a against P. infestans similar to a chaotropic agent, like SDS. These results are consistent with previous reports that short oxyalkylated chains significantly enhance and improve antimicrobial activities [22].
Additionally, some structure-activity relationship studies suggest that the antimicrobial effect of chalcones is mainly attributable to the presence of hydroxyl phenolic groups (and their high affinity for membrane proteins), especially in the 2 position of the A ring [23,24]. Furthermore, alkyl group substitution of the A ring, especially O-alkyl, is thought to increase lipophilicity, and consequently, enhance antimicrobial activity [25][26][27]. Table 2. Percentage of membrane lysis of the synthetic compounds 7a-d, 8a-d, and 9a-d.

Compounds
% Membrane Lysis a The assay was performed in triplicate.
To this end, quantitative structure-activity relationship (QSAR) analysis (i.e., a series of multivariate linear regressions between biological activity (pEC 50 , dependent variable) and various descriptors (physicochemical, steric, or other, independent variables)) was performed following [28][29][30], in order to better describe activity and aid in the design and prediction of new compounds [31].
The multivariate equations obtained in the gaseous and condensed phase (Equations (1) and (2), respectively) showed that the inhibitory activity of 2 -hydroxy-chalcone derivatives on P. infestans depends on the molecular area as well as on the atomic charge of carbon C 3 . Indeed, the atomic charge of chalcone derivatives has previously been discussed for antifungal activity against B. cinerea and M. fructicola [28,32]. Moreover, the atomic charge of 2-allylphenol derivatives has been related to inhibition activity against other Phytopthora strains [32]. Furthermore, while the molecular surface (MS) has been used to explain lipophilicity [7], this descriptor has not been reported as an important feature of P. infestans inhibition activity. The QSAR model obtained in the condensed phase was more robust than that of the gaseous phase (Equation (2) greater values of r, r 2 , F, and q 2 than Equation (1)). Equation (2) suggests that the growth inhibition activity of 2 -hydroxy-chalcone derivatives against P. infestans decreases when molecular surface increases (negative slope MS). However, the most important descriptor is the atomic charge on C 3 (~79-folds more than MS). Thus, the atomic charge on C 3 very likely provides more active compounds against P. infestans.
The most active compounds of the series are 7a, 8a, and 9a; while these compounds have a similar molecular surface, the atomic charge on C 3 is different due to the substitution pattern (see Table S2). Thus, new compounds with substitution on C 3 were calculated using the compounds 7a, 8a, and 9a as core (see Tables S3-S5). These new compounds differed in calculated pIC 50 , depending on the stereo-electronic properties. For example, using compounds 7a, 8a, and 9a as the core caused the electronegative substituent -F, -OH, and NH 2 bonded to C 3 to increase in positive atomic charge, allowing for increased inhibition growth activity against P. infestans (see Tables S3-S5). Interestingly, the derivatives calculated from 7a showed less activity than derivatives from cores 8a and 9a. This phenomenon is likely related to substituent proximity in the 7a derivatives, which causes stereo-electronic repulsions (see Tables S3-S5). Thus, the most active compound against P. infestans (8a, see Table 1), the most promising substituents for the inhibition activity of 2-hydroxychalcones against P. infestans bonded to C 3 were -F and -OH, which allow for~1.7-folds.
In sum, QSAR analysis yielded important structural information as well as allowed for calculations of other compounds (see Tables S3-S5) summarized in Figure 1.
Antibiotics 2020, 9, x FOR PEER REVIEW 5 of 12 electronic repulsions (see Tables S3-S5). Thus, the most active compound against P. infestans (8a, see Table 1), the most promising substituents for the inhibition activity of 2-hydroxychalcones against P. infestans bonded to C3′ were -F and -OH, which allow for ~1.7-folds. In sum, QSAR analysis yielded important structural information as well as allowed for calculations of other compounds (see Tables S3-S5) summarized in Figure 1.

Chemicals and Reagents
All chemicals were purchased from Sigma-Aldrich Co.

Alkoxy Acetophenones
A mixture of the respective dihydroxyacetophenone, alkyl bromide, and anhydrous potassium carbonate at a ratio of 1:1.2:1 in 2 mL of acetone and a ratio of 1:2:4 in 2 mL of acetonitrile for monoalkylated and dialkylated chalcones, respectively. The reaction was irradiated in a water bath of an ultrasonic cleaner (Elmasonic S 10 H, Elma Schmidbauer GmbH, Sigen, Germany) with frequency of 37 KHz and a nominal power of 240 W at 45-50 °C for 20-30 min. After the reaction time the extraction, separation and identification protocols was performed according to the reported procedures [33]. This procedure made it possible to obtain known compounds 4a-d, 5a, 6a, and 6d [34][35][36]. NMR data for 4a-d, 5a, 6a, and 6d were consistent with those previously reported. In addition, new compounds such as 5b-d and 6b-d were obtained. The structure of these compounds was established by NMR data.

Chemicals and Reagents
All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), GIBCO BRL Life Technologies (Grand Island, NY, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Spectra were recorded in CDCl 3 solutions and were referenced to the residual peaks of CHCl 3 , δ = 7.26 ppm, and δ = 77.0 ppm for 1 H and 13 C, respectively, on a Bruker Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany), operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. Chemical shifts were reported in δ ppm and coupling constants (J) were given in Hz. HRMS were measured on Termo Finnigan MAT95XL mass spectrometers. Silica gel (Merck 200-300 mesh) was used for column chromatography.

Alkoxy Acetophenones
A mixture of the respective dihydroxyacetophenone, alkyl bromide, and anhydrous potassium carbonate at a ratio of 1:1.2:1 in 2 mL of acetone and a ratio of 1:2:4 in 2 mL of acetonitrile for monoalkylated and dialkylated chalcones, respectively. The reaction was irradiated in a water bath of an ultrasonic cleaner (Elmasonic S 10 H, Elma Schmidbauer GmbH, Sigen, Germany) with frequency of 37 KHz and a nominal power of 240 W at 45-50 • C for 20-30 min. After the reaction time the extraction, separation and identification protocols was performed according to the reported procedures [33]. This procedure made it possible to obtain known compounds 4a-d, 5a, 6a, and 6d [34][35][36]. NMR data for 4a-d, 5a, 6a, and 6d were consistent with those previously reported. In addition, new compounds such as 5b-d and 6b-d were obtained. The structure of these compounds was established by NMR data.

Oxyalkyl Chalcones
To a solution of benzaldehyde (1.5 mmol) and the corresponding oxyalkyl acetophenone (1.5 mmol) in ethanol (3 mL) taken in a flask (25 mL), a catalytic quantity of sodium hydroxide (0.75 mmol) was added and the reaction mixture was irradiated in the water bath of an ultrasonic cleaner with a frequency of 37 KHz and a nominal power of 240 W at 30-35 • C for 3 h, after which 10% HCl solution was added until pH~7 to end the reaction, and the mixture was extracted with EtOAc (3 × 30 mL). The organic layer was dried with Na 2 CO 3 , filtered, and separated with column chromatography on silica gel.
This procedure made it possible to obtain four known chalcones, 7a, 7b, 7c, and 7d, with high percentages of yield 9%, 85.3%, 86.0%, and 81.1%, respectively. NMR data for 7a-7d were consistent with those previously reported [37]. The structure of the new compounds obtained was established by NMR and mass data, as detailed below:

Anti-Oomycete Activity
All oxyalkyl chalcones 7a-d, 8a-d, and 9a-d were subjected to antifungal assays against Phytophthora infestans, obtained from the collection maintained in our Laboratory of Biochemistry and Environmental Microbiology in Chillan, Chile.
Mycelial discs (4 mm in diameter) of test fungi grown on rye B agar [38] were cut from the margins of the colony and placed on the same medium containing different concentrations of the test oxyalkyl chalcones (12.5-400 µg/mL). After incubation at 20 • C for seven days, radial mycelial growth was measured and was compared with the controls [39]. Control plates were treated with difenoconazole and metalaxil. Activity was expressed as EC 50 (the concentration inhibiting growth by 50%), MIC (the minimum concentration inhibition of mycelial growth), and MOC was defined as the lowest concentration of the chemicals that prevented visible growth or germination of mycelium. The experiments were repeated three times with three replicates.

Membrane Damage
P. infestans strains were cultured by shaking at 20 • C and then washed twice and diluted to approximately (3 × 10 4 zoospores/mL with cold 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, pH 6.0. Cells were aliquoted to tubes, and 7a-d, 8a-d, and 9a-d was added at a final concentration of 100 µg/mL. SDS (2%) was used as the reference compound, which produced 100% cellular oomycete leakage. P. infestans was incubated at 20 • C, and samples were taken at time intervals (6,12,24, and 48 h) and spun at 3500 rpm for 7 min in microcentrifugetubes. The supernatants were collected for absorbance analysis at 260 nm in a Beckman DU-600 spectrophotometer [37]. Results are the means of values from at least two independent assays.

Computational Details
All compounds (7a-d, 8a-d, and 9a-d) were optimized using DFT-B3LYP-6-31G (d,p) level of theory calculations, and the optimized structures were verified by frequency calculations (obtaining no imaginary frequencies) in the gas phase and using the integral equation formalism (IEFPCM) (water) model as the solvent phase according to a previous report [27]. The descriptors obtained from quantum mechanical calculations such as the dipolar moment (DM), atomic charge from the electrostatic potential (C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , Cα, Cβ, CO), highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) were obtained directly from the output file, while the chemical potential (µ), hardness (η), softness (S), and electrophilic global index (ω) values were calculated using the following equations.