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Article

Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities

by
Attilio Di Maio
1,†,
Hamza Olleik
2,†,
Elise Courvoisier-Dezord
2,
Sophie Guillier
3,
Fabienne Neulat-Ripoll
3,
Romain Haudecoeur
4,
Jean-Michel Bolla
3,
Magali Casanova
5,
Jean-François Cavalier
5,
Stéphane Canaan
5,
Valérie Pique
1,
Yolande Charmasson
2,
Elias Baydoun
6,
Akram Hijazi
7,
Josette Perrier
2,
Marc Maresca
2,* and
Maxime Robin
1,*
1
Aix Marseille University, University Avignon, CNRS, IRD, IMBE, 13013 Marseille, France
2
Aix Marseille University, CNRS, Centrale Marseille, iSm2, 13013 Marseille, France
3
Aix Marseille University, INSERM, SSA, MCT, 13385 Marseille, France
4
University Grenoble Alpes, CNRS, DPM, 38000 Grenoble, France
5
Aix-Marseille University, CNRS, LISM UMR7255, IMM FR3479, 13009 Marseille, France
6
Department of Biology, American University of Beirut, Beirut 1107, Lebanon
7
Plateforme de Recherche et D’analyse en Sciences de L’environnement (EDST-PRASE), Beirut 1107, Lebanon
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2024, 13(4), 300; https://doi.org/10.3390/antibiotics13040300
Submission received: 23 February 2024 / Revised: 19 March 2024 / Accepted: 25 March 2024 / Published: 26 March 2024
(This article belongs to the Special Issue Rise of Antibiotic Resistance: Mechanisms Involved and Solutions to Tackle It)
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Abstract

The development of new and effective antimicrobial compounds is urgent due to the emergence of resistant bacteria. Natural plant flavonoids are known to be effective molecules, but their activity and selectivity have to be increased. Based on previous aurone potency, we designed new aurone derivatives bearing acetamido and amino groups at the position 5 of the A ring and managing various monosubstitutions at the B ring. A series of 31 new aurone derivatives were first evaluated for their antimicrobial activity with five derivatives being the most active (compounds 10, 12, 15, 16, and 20). The evaluation of their cytotoxicity on human cells and of their therapeutic index (TI) showed that compounds 10 and 20 had the highest TI. Finally, screening against a large panel of pathogens confirmed that compounds 10 and 20 possess large spectrum antimicrobial activity, including on bioweapon BSL3 strains, with MIC values as low as 0.78 µM. These results demonstrate that 5-acetamidoaurones are far more active and safer compared with 5-aminoaurones, and that benzyloxy and isopropyl substitutions at the B ring are the most promising strategy in the exploration of new antimicrobial aurones.

1. Introduction

The development of novel antibacterial molecules is a major necessity in the upcoming decades, due to the increasing emergence of multi-drug resistant bacterial strains. This is leading to an elevating mortality rate by infectious disease that may reach more than 10 billion deaths by 2050, according to World Health Organization (WHO) [1]. Among these strains, mycobacteria, in particular M. tuberculosis, the pathogenic agent of tuberculosis, are still responsible for 10 million new cases every year worldwide and killed almost 1.5 million patients in 2022. More alarmingly, the number of strains resistant and ultra-resistant to cocktails of antibiotics currently used to treat infection is constantly rising. One of the major concerns is about methicillin resistant Staphylococcus aureus (MRSA), but also other Gram-positive and Gram-negative species such as Acinetobacter baumannii, Pseudomonas aeruginosa or Klebsiella pneumoniae [2]. In addition to bacteria, fungi (filamentous fungi such as Aspergillus fumigatus, as well as yeasts such as Candida species and Cryptococcus neoformans) are also responsible for deadly infections, particularly in HIV-infected patients, but also in immunocompetent ones, affecting billions of patients and causing more than 1.5 million deaths per year [3,4,5,6].
Some natural plant molecules and/or their derivatives, including flavonoids, have been reported to possess strong antimicrobial activity. For decades, the biological effect of flavonoids has been studied, focusing on the major subclasses such as flavones, flavonols, flavanones and chalcones. However, in the past ten years, the aurone subclass has been demonstrated to display strong biological effects in diverse fields, such as oncology, dermatology, and infectiology [7,8,9]. The natural occurrences of aurones is limited to a limited number of advanced plant species where they play a variety of key roles as flower pigments, antioxidants and as nectar guides [10]. Although some natural aurones such as cephalocerone [11,12] and hispidol [13] have demonstrated antimicrobial activity, aurones exhibiting natural substitution patterns do not generally lead to the most effective antimicrobial agents. On the other hand, a series of synthetical aurone derivatives showed a strong effect against Gram-positive bacteria [14]. Structurally, aurones are characterized by a benzofuran moiety bearing in position 2 a benzylidene type substituent (rings A, B, and C) (Figure 1). Overall, research on scaffold modifications has mainly focused on the substitution at the B-ring scaffold, e.g., with the introduction of either ferrocene [15], 5-nitroimidazole [16] or quinoline [17] groups, with a global tendency to retain naturally present hydroxy groups at the A-ring.
In the present study, modifications of the A-ring were performed by substitution with an amino group combined with substitutions of the B-ring (Figure 1). The 31 new aurone derivatives obtained (Table 1) were tested in terms of antibacterial and antifungal activities.

2. Results

Antimicrobial effect of the aurone derivatives was first evaluated by the determination of their Minimum Inhibitory Concentrations (MICs) on five different bacterial strains representative of Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), mycobacteria (Mycobacterium smegmatis), and one fungal strain (Candida albicans) (µM) (Table 2).
In this first screening, among the 31 aurone derivatives tested, compounds 10, 12, 15, 16, and 20 were the most active (Figure 2). Compound 10 gave the lowest MIC values on all micro-organisms tested in Table 2 (i.e., Gram-positive and -negative bacteria, mycobacteria and fungi), with MICs ranging from 3.12 to 50 µM. Similarly, compound 20 was active on all tested species (MIC ranging from 12.5 to 50 µM), except P. aeruginosa for which MIC was superior to 100 µM. Compound 16, although active on B. subtilis, S. aureus, E. coli, and M. smegmatis (MIC ranging from 25 to 50 µM), was however inactive on P. aeruginosa and C. albicans. Finally, although compounds 12 and 15 were active on tested Gram-positive bacteria and mycobacteria (MIC ranging from 25 to 100 µM and from 50 to 100 µM, respectively), they were inactive on tested Gram-negative bacteria and C. albicans. Based on MIC values on this first screening, the observed order of antimicrobial activities is as follows: compound 10 > 20 > 12 = 16 > 15.
The safety of the five most active derivatives (i.e., compounds 10, 12, 15, 16, and 20) was then evaluated using different human cell types (Figure 3 and Figure 4, Table 3).
Overall, compounds 10, 12, 15, 16, and 20 were found to be safe with most of their CC50 values higher than their MIC ones. Compounds 10 and 20 were the safest molecules with mean CC50 of 321.5 and 305.1 µM (ranging from 169.0 to 472.4 µM and from 125.9 to >500 µM for compounds 10 and 20, respectively). Compounds 12, 15, and 16 were more toxic, with mean CC50 of 129.3, 130.6, and 218.0 µM, respectively. The highest safety of compounds 10 and 20 was further demonstrated when comparing the therapeutic indexes (TI) of the five aurones. Indeed, when calculating the TI of each aurone (by dividing their CC50 on human cells (Table 3) by their MIC on S. aureus (Table 2)), compounds 10 and 20 gave the highest TI values (ranging from 13.5 to 37.7 and from 10.0 to >40, for compounds 10 and 20, respectively) compared to the TI values of compounds 12, 15, and 16 (Table 4) (ranging from 0.4 to 18.0).
Based on antimicrobial activity and toxicity data, the two most active and safest compounds were identified as compounds 10 and 20, which were then tested on a larger panel of bacterial and fungal species in order to evaluate their spectrum of activity (Table 5).
The results of this second screening confirmed that compounds 10 and 20 are primarily active on Gram-positive bacteria, with MIC values as low as 0.78 and 3.12 µM for compounds 10 and 20, respectively. Compounds 10 and 20 were particularly active on the various foodborne pathogens. For example, MIC values of 3.12 and 6.25 µM were obtained on Listeria monocytogenes for compounds 10 and 20, respectively. Compounds 10 and 20 gave good activity against Clostridi such as C. difficile (MIC values of 12.5 and 3.12 µM for compounds 10 and 20, respectively) and C. botulinum (MIC values of 0.78 and 3.12 µM for compounds 10 and 20, respectively). In addition, good activities were also observed on the WHO group 3 pathogen Bacillus anthracis, responsible for anthrax disease and used as biological weapon (MIC values of 12.5 and 6.25 µM for compounds 10 and 20, respectively). These MIC values were consistent with the ones obtained on two other Bacillus species, i.e., B. cereus and B. subtilis. On the other hand, C. perfringens, Enterococcus species, P. acnes, and S. pyogenes were found to be weakly sensitive to insensitive to compounds 10 and 20 with MIC values from 50 µM to >100 µM. Overall, compound 10 was more active than compound 20 on most tested Gram-positive bacterial strains, except C. difficile, E. faecium, and B. anthracis, for which compound 20 was more active. S. aureus and methicillin-resistant S. aureus (MRSA) showed good sensitivity with MIC of 12.5–25 µM, showing that resistance to methicillin did not affect the activity of compounds 10 and 20.
Compounds 10 and 20 were also active on Gram-negative bacteria, with A. baumannii, E. coli, and H. pylori being the more sensitive strains (MIC values as low as 12.5 and 25 µM for compounds 10 and 20, respectively). S. enterica, S. flexneri, and V. alginolyticus were also found to be sensitive, with MICs ranging from 25 to 50 µM. Although P. aeruginosa was sensitive to compound 10 (MIC of 25 µM), it was insensitive to compound 20 (MIC > 100 µM). Good activities were also obtained on the WHO group 3 pathogens Brucella melitensis, Francisella tularensis, and Yersinia pestis (MIC values as low as 12.5 µM). E. cloacae and K. pneumoniae were insensitive to compounds 10 and 20 (MIC > 100 µM). Compound 10 was more active than compound 20 on most Gram-negative strains tested, except for B. melitensis and F. tularensis, for which compound 20 gave lower MIC values.
Although compounds 10 and 20 were active on M. smegmatis (Table 2), they were inactive on tested pathogenic mycobacteria, i.e., M. abscessus and M. tuberculosis.
Finally, in term of antifungal effect, compounds 10 and 20 were active on the filamentous fungi F. oxysporum (MIC of 25 µM) but inactive on another important human pathogen A. fumigatus. Antifungal activity was also observed on yeasts, including various Candida species (C. albicans, C. auris, C. glabrata, and C. tropicalis) and Cryptococcus neoformans with MIC values as low as 25 and 12.5 µM for compounds 10 and 20, respectively. In most cases, compounds 10 and 20 gave the same MIC values except for C. auris, for which compound 20 was more active than compound 10 (MIC of 12.5 and 50 µM, respectively). C. tropicalis had a less sensitive MIC value of 100 µM.
The therapeutic indexes (TI) values of compounds 10 and 20 were calculated using the MIC values reported in Table 5 and the CC50 on human cells reported in Table 3 (Table 6 and Table 7).
TI values ranged from 216.6 to 605.6 and from 40.3 to >160.2 for compounds 10 and 20, respectively, confirming that these two aurone derivatives possess good therapeutic values. Compound 10 was the safest in all cases.

3. Discussion

In the present study, 31 new aurone derivatives were synthetized (Figure 5, see Supplementary Materials) and tested in terms of antimicrobial activity against various bacteria and fungi. These new compounds were obtained by the substitution of the aurone scaffold at position 5 by amino and acetamido groups, and through various substitutions at the 2′, 3′ and 4′ positions. The first screening antimicrobial test performed on representative species of bacteria and fungi allowed us to identify compounds 10, 12, 15, 16, and 20 as the more active aurones. Comparisons between active and inactive structures afforded insightful information to identify the most interesting substitution. Compound 10 can be compared to compound 11, 12 and 13. All these compounds are substituted in position 3′ or 4′ by a benzyloxy group. However, only compound 10 and 12 showed an interesting activity. This suggests that benzyloxy substitution in 3′ may be a key element in the activity of the compound. The same methodology could be used to compare compound 20 with other isopropyloxy compounds such as 18, 19, 21 and 22. These four last mentioned compounds showed a weak or no activity against tested micro-organisms. Similarly to compound 10, this could indicate that the 4′-isopropyl substitution may be a far better alternative to the other position and again that the acetamido group is more effective than the amino group in position 5. From the results, it can be suggested that the acetamido group is important for the antibacterial capacity of the aurones. Thus, compounds 12 and 16 are both 5-aminoaurones; compounds 15 and especially compounds 10 and 20 are 5-acetamidoaurones and showed far better antibacterial activity. Moreover, four of these compounds possess a ring substitution in position 3′ and 4′. Interestingly, benzyloxy-substituted aurones (i.e., 10 and 12) seem to only be active in positions 3′ when phenyl-substituted aurones (i.e., 15 and 16) showed activity when substituted both in 3′ and 4′ position. However, considering the activity of compound 10 compared to 12, 15 and 16, benzyloxy substitution must be considered more promising than phenyl substitution. The other benzyloxy- and phenyl-substituted aurones (i.e., 11, 13, 14 and 17) showed no activity. Isopropyloxy-substituted aurones also are an interesting option as shown by compound 20, which is similar in activity to compound 10. Again only 4′-isopropyl aurones were active; 3′ and 2′ were inactive on the varieties of pathogens tested. Methyl (i.e., 4–9), fluoro (i.e., 24–28), carboxy (i.e., 31), trifluoromethyl (i.e., 29 and 30) and hydroxy (i.e., 32 and 33) substituted aurones showed no activity and thus should not be considered as privileged substitution in the development of antibacterial aurones. Finally, aurone 34 is also inactive and shows that the 5-acetamido substitution is not enough to produce an antibacterial activity to aurones and that a substitution on the B-ring is mandatory. These five aurones were then tested in term of toxicity against various human cell types. The toxicity data demonstrated that compounds 10 and 20 were the safest ones compared to compounds 12, 15, and 16. Again, 5-acetamido aurones seem to be more interesting as they are safer on human cells than 5-aminoaurones. Compounds 10 and 20 were then further tested on a larger panel of pathogens, including Gram-positive and Gram-negative bacteria. In this second screening, these compounds showed an interesting activity as antimicrobials against Gram-positive strains such as S. aureus, methicillin-resistant S. aureus, L. monocytogenes, B. subtilis and C. difficile and Gram-negative strains such as E. coli, A. baumannii and H. pylori. The two selected compounds shared some structural similarities, such as the 5-acetamido substitution. Out of all the compounds, 3′-benzyloxy and 4′-isopropyloxy were the most promising substitutions. Compared to previously described aurones active on various Gram-positive bacteria but only two Gram-negative strains (i.e., H. pylori or V. alginolyticus) [14], compounds 10 and 20 were active on a large number of Gram-positive and -negative bacteria as well as fungi. Structurally, the most active compound synthetized by Olleik et al. [14] was a 5,7-dihydroxyaurone substituted in 4′ by a benzyloxy group and in 3′ by a methoxy group. Again, this shows the promising nature of the benzyloxy substitution and that hydroxy aurones, vastly found in nature, are far less active than synthetic aurones such as amino and acetamidoaurones.

4. Materials and Methods

4.1. Biology

4.1.1. Microorganism Strains and Growth Conditions

Bacterial and fungi strains used in this study, except when mentioned, were obtained from either the American Type Culture Collection (ATCC), the German Leibniz Institute (DSMZ), or the French Pasteur Institute (CIP) and correspond to reference strains. They were maintained on agar plates using appropriate media and culture conditions (in terms of temperature and aerobic/microaerobic/anaerobic condition) as previously described [14,18]. Regarding BSL-3 strains, Bacillus anthracis, Francisella tularensis, and Brucella melitensis strains were maintained on Chocolate agar PolyViteX (Biomerieux) agar at 37 °C, and at 26 °C for Yersinia pestis [19,20]. Regarding mycobacteria, M. smegmatis mc2155 (ATCC700084) was grown in Middlebrook 7H9 complete medium containing 0.05% Tween-80 and 0.2% Glycerol (7H9-TG) and M. abscessus (CIP104536T) S and R morphotypes, were cultured in 7H9-TG containing 10% BBL™ Middlebrook OADC Enrichment (7H9-TGOADC) at 37 °C under stirring (200 rpm). M. tuberculosis mc26230, a derivative of H37Rv which contains a deletion of the RD1 region and panCD, resulting in a pan(−) phenotype, was grown in 7H9-TGOADC supplemented with 24 µg/mL D-panthothenate (Sigma-Aldrich, Lyon, France). Cultures were kept at 37 °C without shaking.

4.1.2. Antimicrobial Activity Assay

The antimicrobial activity of aurones on BSL2 bacteria and fungi was evaluated through determination of the Minimum Inhibitory Concentration (MIC) using two-fold serial dilutions in liquid media following the National Committee of Clinical Laboratory Standards (NCCLS, 1997) as previously described [14,18,21,22]. For BSL-3 bacteria, the MIC of aurones was determined following the Clinical and Laboratory Standards Institute (CLSI) recommendations as previously described [23]. For determining the antimycobacterial activity of the different aurones, the microdilution method was used in sterile 96-well flat-bottom Greiner Bio-One polypropylene microplates with lid (Thermo Fisher Scientific) using the resazurin microtiter assay (REMA) as previously described [24,25]. The concentration of aurones leading to 90% inhibition of mycobacteria growth was defined as the MIC. All experiments were performed independently at least three times.

4.1.3. Cytotoxic Assays

The impact of aurones on the viability of human cells were evaluated as previously described [18,22,26]. Human cells used were kidney epithelial cell line A498 (ATCC® HTB-44), normal lung epithelial cells BEAS-2B (ATCC® CRL-9609), intestinal cell line Caco-2 (ATCC® HTB-37), normal epidermal keratinocytes (HaCaT) (from Creative Bioarray, Shirley, NY, USA), liver cell line HepG2 (ATCC® HB-8065), and normal lung fibroblasts IMR-90 (ATCC® CCL186). Cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 1% l-glutamine, and 1% antibiotics (all from Invitrogen (Carlsbad, CA, USA). Cells were routinely grown on 25 cm2 flasks and maintained in a 5% CO2 incubator at 37 °C. For toxicity assays, human cells grown on 25 cm2 flasks were detached using trypsin-EDTA solution (from Thermofisher, Waltham, MA, USA), counted using Malassez counting chamber, diluted in appropriate culture media, and seeded into 96-well cell culture plates (Greiner bio-one, Paris, France) at approximately 104 cells per well. The cells were left to grow for 48–72 h at 37 °C in a 5% CO2 incubator until confluence. Media from wells was then aspirated and cells were treated with 100 µL of appropriate culture media containing increasing concentrations of tested aurones (from 0 to 400 µM, 1:2 serial dilutions). Volume of DMSO corresponding to 400 µM of aurones was used as negative control and was found not toxic. The plates were then incubated at 37 °C for 48 h. Resazurin-based in vitro toxicity assay kit (from Sigma-Aldrich, Lyon, France) was then used to assess the viability of the cells following manufacturer’s instructions. Briefly, resazurin stock solution was diluted 1:10 in sterile PBS containing calcium and magnesium (PBS++, pH 7.4). Plates were aspirated and 100 µL of the diluted solution was added per well. After 2 h incubation at 37 °C, fluorescence intensity was measured using a microplate reader (Biotek, Synergy Mx, Colmar, France) (excitation wavelength of 530 nm/emission wavelength of 590 nm). The fluorescence values were normalized by the controls (DMSO treated cells) and expressed as percentage of cell viability. The CC50 values of aurones (i.e., the concentrations causing a reduction of 50% of the cell viability) were calculated using GraphPad® Prism 7 software (San Diego, CA, USA). Experiments were conducted in triplicate (n = 3).

4.2. Chemistry

1H and 13C NMR spectra were recorded on a Bruker Avance III nanobay—300 MHz instrument (Bruker, Bremen, Germany, 300 MHz for 1H, 75 MHz for 13C). Chemical shifts are reported in ppm relative to the solvent in which the spectrum was recorded [1H: δ (d6-DMSO) = 2.50 ppm, δ (CDCl3) = 7.27 ppm; 13C: δ (d6-DMSO) = 39.52 ppm, δ (CDCl3) = 77.16 ppm], full spectra are presented in Supplementary Materials. Combustion analyses were performed at the analysis facilities of Spectropole (https://fr-chimie.univ-amu.fr/spectropole, accessed on 20 December 2023) with a Thermo Finnigan (San Jose, CA, USA) EA 1112 apparatus; all compounds had purity higher than 95%. Microwave-assisted reactions were performed in a CEM Discover microwave reactor with a focused field (CEM Corporation, Matthews, NC, USA). Silica gel F-254 plates (0.25 mm; Merck, Darmstadt, Germany) were used for thin-layer chromatography (TLC), and silica gel 60 (200–400 mesh; Merck) was used for flash chromatography. Unless otherwise stated, reagents were obtained from commercial sources and were used without further purification.

4.2.1. Synthesis Route A

Synthesis of N-(4-methoxyphenyl)acetamide (1a)
In a solution of 8 g of m-anisidine and 2.5 eq. of NaOH in 50 mL of ethyl acetate, 6.6 g of acetic anhydride (1.5 eq.) was added dropwise. When the addition was completed, the mixture was heated at 80 °C for 5 h. The solution was cooled and filtered. Under pressure, the solvent was removed, the obtained product was dissolved in ethanol and hexane to precipitate, to obtain 5 g of 1a.
Synthesis of 2-Chloro-1-{2-hydroxy-5-[(1-hydroxyethyl)amino]cyclohexyl}ethan-1-one (2a)
A total of 5 g of 1a and 18 g of AlCl3 were dissolved in 30 mL of dichloromethane then at 0 °C, 3.5 eq. of chloroacetyl chloride was added dropwise. When the addition was completed, the solution was heated up to 50 °C for 1 h. The mixture was poured on ice and extracted with ethyl acetate. EtOAc was removed under pressure to obtain 2a.
Synthesis of N-(3-oxo-2,3-dihydro-1-benzofuran-5-yl)acetamide (3a)
In a flask, 2 g of 2a and 1.5 eq. of triethylamine were added to 20 mL of acetonitrile; the solution reacted at 25 °C for 12 h. Solvent was removed under pressure. The left-over mixture was dissolved in EtOAc, washed several times with water then extracted. EtOAc was removed under pressure to obtain 3a.
Synthesis of Substituted 5-Acetamidoaurones
In a flask, 1 mmol of 3a and 1 mmol of the corresponding benzaldehyde were dissolved in 10 mL of choline chloride/urea. Three drops of 50% KOH solution were added. The mixture was heated at 80 °C for 2 h. Water and HCl were added, then the precipitate was filtered and washed several times with ether to obtain acetamido substituted aurones.
Synthesis of Substituted 5-Aminoaurone
A total of 10 mmol of acetamido aurones was added to a mixture of EtOH (20 mL) and 0.5 M HCl (5 mL). The solution was refluxed for 2 h. Upon cooling, the solvent was removed under vacuum and the residue obtained was poured onto iced water (100 mL). The resulting solution was neutralized with NH4OH 16% until pH = 7. The precipitate formed was collected by filtration and washed with excess cold water.

4.2.2. Synthesis Route B

Synthesis of 4-Acetamidophenyl Acetate (1b)
To a solution of 8 g of 4-aminophenol and 2.5 eq. of NaOH in 50 mL of ethyl acetate, 26.5 g of acetic anhydride (3.5 eq.) was added dropwise. When the addition was completed, the solution was heated at 80 °C for 5 h. Upon cooling, the mixture was filtered. Solvent was removed under pressure; the product was recrystallized in ethanol and hexane to obtain 5 g of 1b.
Synthesis of N-(3-acetyl-4-hydroxyphenyl)acetamide (2b)
To a solution of 1b (5 g, 26 mmol), 15 g of AlCl3 (113 mmol, 4 eq.) and 1.9 g of KCl (26 mmol, 1 eq.) were added. The mixture was then heated a 165 °C for 1 h until a brown paste appeared. Upon cooling, ice cold water was added (300 mL) and the mixture was filtered to obtain 2 g of a beige powder (2b).
Synthesis of Substituted of 5-Acetamidochalcones (3b)
In a flask, 193 mg of 2b (0.001 mmol), 1 eq. of chosen benzaldehyde and 188 mg of LiOH (16 eq., 0.016 mmol) were dissolved in ethanol (20 mL). The mixture was heated for 2 h at 90 °C. The solvent was then removed under pressure, cold water and HCl were added, and the precipitate was filtered to obtain the desired chalcone (3b).
Synthesis of Substituted 5-Acetamidoaurones
To a mixture of chosen 3b chalcone in pyridine (20 mL), 1 eq. of mercury acetate was added. The solution was heated for 1 h at 110 °C. Water and HCl were added. The precipitate was filtered and washed several times with ice cold water to get rid of the mercury. Obtention of a powder red-yellow powder depended the substitution.
Synthesis of Substituted 5-Aminoaurone
For synthesis of 5-aminoaurone see Section Synthesis of Substituted 5-Aminoaurone.
Yield: 83%; mp: 234.8 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 8.20–8.18 (dd, 1H, J = 1.2;7.8 Hz, C-H6′), 8.10 (d, 1H, J = 1.9 Hz, C-H4), 7.83–7.80 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.52–7.49 (d, 1H, J = 8.9 Hz, C-H7), 7.46 (dt, 1H, J = 7.1 Hz, C-H4′), 7.19 (s, 1H, C-H10), 7.16–7.09 (m, 2H, C-H3′,5′), 3.91 (s, 3H, OCH3), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.53(C-3), 168.41(CO), 161.21(C-8), 158.32(C-2′), 146.75(C-2) 135.53(C-5), 131.99(C-4′), 131.11(C-6′), 128.71(C-6), 120.89(C-1′), 120.68(C-5′), 120.08(C-9), 113.31(C-3′), 113.15(C-7), 111.57(C-10), 105.47(C-4), 55.84(OCH3), 23.83(CH3). Elemental analysis calcd (%) for C18H15NO4: C, 69.89; H, 4.89; N, 4.53; found C, 69.87; H, 4.91; N, 4.52. m/z: 309.1001 (100.0%).
(Z)-N-(2-(3-methoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (5):
Yield: 71%; mp: 204.2 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.16 (s, 1H, NH), 8.10 (d, 1H, J = 2 Hz, C-H4), 7.83–7.80 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.60–7.55 (m, 2H, C-H2′,4′), 7.54–7.51 (d, 1H, J = 8.9 Hz, C-H7), 7.43 (dt, 1H, J = 8.0 Hz, C-H5′), 7.06–7.03 (dd, 1H, J = 2.6;8.2 Hz, C-H6′), 6.90 (s, 1H, C-H10), 7.16–7.09 (m, 2H, C-H3′,5′), 3.82 (s, 3H, OCH3), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.68(C-3), 168.42(CO), 161.33(C-8), 159.42(C-3′), 146.88(C-2), 135.58(C-5), 133.08(C-6′), 130(C-2′), 128.82(C-6), 123.74(C-1′), 120.59(C-9), 116.57(C-6′), 115.76(C-4′), 113.35(C-7), 113.15(C-4), 112.07(C-10), 55.15(OCH3), 23.83(CH3). Elemental analysis calcd (%) for C18H15NO4: C, 69.89; H, 4.89; N, 4.53; found C, 69.84; H, 4.88; N, 4.53. m/z: 309.1001 (100.0%).
(Z)-N-(2-(4-methoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (6):
Yield: 91%; mp: 252 °C [1]; 1H NMR (300 MHz, DMSO-d6): δ 10.17 (s, 1H, NH), 8.10 (d, 1H, J = 2 Hz, C-H4), 7.96–7.93 (d, 2H, J = 7.9 Hz, C-H2′,6′), 7.81–7.78 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.50–7.47 (d, 1H, J = 8.9 Hz, C-H7), 7.08–7.06 (d, 2H, J = 8.0 Hz, C-H3′,5′), 6.91 (s, 1H, C-H10), 3.83 (s, 3H, OCH3), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.37(C-3), 168.46(CO), 161.06(C-8), 161.00(C-4′), 145.75(C-2), 135.47(C-5), 133.40(C-2′,6′), 128.51(C-6), 124.47(C-1′), 120.98(C-9), 114.71(C-3′-5′), 113.30(C-7), 113.07(C-4), 112.74(C-10), 55.40(OCH3), 23.91(CH3). Elemental analysis calcd (%) for C18H15NO4: C, 69.89; H, 4.89; N, 4.53; found C, 69.78; H, 4.87; N, 4.48. m/z: 309.1001 (100.0%).
(Z)-5-amino-2-(2-methoxybenzylidene)benzofuran-3(2H)-one (7):
Yield: 22%; mp: 189.3 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.18–8.16 (dd, 1H, J = 1.6;7.8 Hz, C-H6′), 7.43 (dt, 1H, J = 1.5;8.5 Hz, C-H4′), 7.26–7.23 (d, 1H, J = 8.8 Hz, C-H7), 7.14–7.11 (m, 2H, C-H3′,5′), 7.10 (s, 1H, C-H10), 7.06–7.03 (dd, 1H, J = 2.5;8.8 Hz, C-H6), 6.84 (d, 1H, J = 2.4 Hz, C-H4), 5.23 (bs, 2H, NH2), 3.90 (s, 3H, OCH3). 13C NMR (75 MHz, DMSO-d6): δ 184.09(C-3), 158.13(C-2′), 158.00(C-8), 147.05(C-2), 145.56(C-5), 131.55(C-4′), 130.98(C-6′), 124.55(C-6), 121.01(C-9, 120.84(C-5′), 120.41(C-1′), 113.14(C-7), 111.47(C-10), 105.44(C-3′), 104.18(C-4), 55.79(OCH3). Elemental analysis calcd (%) for C16H13NO3: C, 71.90; H, 4.90; N, 5.24; found C, 71.85; H, 4.95; N, 5.21. m/z: 267.0895 (100.0%).
(Z)-5-amino-2-(3-methoxybenzylidene)benzofuran-3(2H)-one (8):
Yield: 50%; mp: 190 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.59–7.57 (d, 1H, J = 7.7 Hz, C-H6′), 7.54 (d, 1H, J = 2.4 Hz, C-H4), 7.49–7.46 (d, 1H, J = 8.8 Hz, C-H7), 7.42 (dt, 1H, J = 8.2 Hz, C-H5′), 7.38–7.35 (dd, 1H, J = 2.2;8.8 Hz, C-H6), 7.24 (d, 1H, J = 2.11 Hz, C-H2′), 7.06–7.03 (dd, 1H, J = 1.9;8.1 Hz, C-H4′), 6.88 (s, 1H, C-H10), 3.82 (s, 3H, OCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.6 (C-3), 160.79 (C-8), 159.42 (C-3′), 146.96 (C-2), 137.91 (C-5′), 133.13 (C-1′), 129.99 (C-5′), 127.97(C-6), 123.71 (C-7), 121.18 (C-9), 116.54 (C-6′), 115.72 (C-4′), 113.87 (C-4), 111.88 (C-2′), 111.18 (C-10), 55.16 (OCH3). Elemental analysis calcd (%) for C16H13NO3: C, 71.90; H, 4.90; N, 5.24; found C, 71.92; H, 4.92; N, 5.28. m/z: 267.0895 (100.0%).
(Z)-5-amino-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (9):
Yield: 86%; mp: 110.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.95–7.92 (d, 2H, J = 8.1 Hz, C-H2′), 7.31–7.28 (d, 1H, J = 8.8 Hz, C-H7), 7.13–7.10 (dd, 1H, J = 2.2;8.8 Hz, C-H6), 7.09–7.06 (d, 2H, J = 8.1 Hz, C-H3′), 6.93 (d, 1H, J = 2.4 Hz, C-H4), 6.83 (s, 1H, C-H10), 3.82 (s, 3H, OCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.77 (C-4), 160.61 (C-4′), 158.51 (C-8), 145.94 (C-5), 143.60 (C-2), 133.16 (C-2′), 125.22 (C-1′), 124.69 (C-6), 121.35 (C-9), 114.65 (C-3′), 113.30 (C-4), 111.68 (C-10), 106.77 (C-7), 55.37 (OCH3). Elemental analysis calcd (%) for C16H13NO3: C, 71.90; H, 4.90; N, 5.24; found C, 71.88; H, 4.97; N, 5.30. m/z: 267.0895 (100.0%).
(Z)-N-(2-(3-(benzyloxy)benzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (10):
Yield: 80%; mp: 204.5 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.16 (s, 1H, NH), 8.10 (d, 1H, J = 2 Hz, C-H4), 7.84–7.80 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.63 (bs, 1H, C-H2′), 7.58–7.32 (m, 8H,), 7.14–7.11 (dd, 1H, J = 7.9 Hz, C-H4′), 6.89 (s, 1H, C-H10), 5.18 (m, 2H, CH2), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.74 (C-4), 168.51 (CO), 161.36 (C-8), 158.53 (C-3′), 146.92 (C-2), 136.87 (C-1bn), 135.64 (C-5), 133.14 (C-5′), 130.1 (C-6′), 128.87 (C-6), 128.48 (C-3bn), 127.94 (C-4bn), 127.83 (C-2bn), 124.14 (C-1′), 120.62 (C-9), 117.27 (C-4′), 116.75 (C-2′), 113.45 (C-7), 113.17 (C-4), 112.09 (C-10), 69.34 (CH2), 23.91 (CH3). Elemental analysis calcd (%) for C24H19NO4: C, 74.79; H, 4.97; N, 3.63; found C, 74.74; H, 5.01; N, 3.60. m/z: 385.13141 (100.0%).
(Z)-N-(2-(4-(benzyloxy)benzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (11):
Yield: 72%; mp: 212.8 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.14 (s, 1H, NH), 8.09 (d, 1H, J = 2.02 Hz, C-H4), 7.96–7.93 (d, 2H, J = 8.8 Hz, C-H2′), 7.82–7.79 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.50–7.34 (m, 6H, C-H7, bn), 7.16–7.13 (d, 2H, J = 8.8 Hz, C-H3′), 6.90 (s, 1H, C-H10), 5.18 (m, 2H, CH2), 2.07 (s, 3H, NHCOCH3).
13C NMR (75 MHz, DMSO-d6): δ 183.37 (C-3), 168.48 (CO), 161.06 (C-8), 159.93 (C-4′), 145.75 (C-2), 136.58 (C-1bn), 135.45 (C-5), 133.39 (C-2′), 128.56 (C-1′), 128.49 (C-3bn), 128 (C-4bn), 127.84 (C-2bn), 124.64 (C-6), 120.95 (C-9), 115.52 (C-3′), 113.33 (C-7), 113.08 (C-4), 112.68 (C-10), 69.44 (CH2), 23.9 (CH3). Elemental analysis calcd (%) for C24H19NO4: C, 74.79; H, 4.97; N, 3.63; found C, 74.77; H, 4.96; N, 3.61. m/z: 385.13141 (100.0%).
(Z)-5-amino-2-(3-(benzyloxy)benzylidene)benzofuran-3(2H)-one (12):
Yield: 80%; mp: 132.6 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.61 (bs, 1H, C-H2′), 7.55–7.53 (d, 1H, J = 8.8 Hz, C-H7), 7.50–7.48 (d, 2H, C-H2bn), 7.41 (dt, 2H, C-H3bn), 7.36–7.33 (m, 2H, C-H5′,4bn), 7.11–7.09 (dd, 1H, J = 7.9 Hz, C-H6), 7.06–7.04 (dd, 1H, J = 7.9 Hz, C-H4′), 6.83 (d, 1H, J = 2 Hz, C-H4), 6.77 (s, 1H, C-H10), 5.26 (bs, 2H, NH2), 5.17 (m, 2H, CH2). 13C NMR (75 MHz, DMSO-d6): δ 184.24 (C-3), 158.48 (C-3′), 158.14 (C-8), 147.21 (C-2), 145.63 (C-5), 136.87 (C-1bn), 133.43 (C-1′), 129.97 (C-5′), 128.42 (C-3bn), 127.86 (C-4bn), 127.75 (C-2bn), 124.71 (C-6), 123.89 (C-7), 120.91 (C-9), 117.04 (C-4′), 116.4 (C-2′), 113.21 (C-6′), 110.73 (C-10), 105.45 (C-4), 69.31 (CH2). Elemental analysis calcd (%) for C22H17NO3: C, 76.95; H, 4.99; N, 4.08; found C, 76.88; H, 5.01; N, 4.04. m/z: 343.12084 (100.0%).
(Z)-5-amino-2-(4-(benzyloxy)benzylidene)benzofuran-3(2H)-one (13):
Yield: 80%; mp: 141.6 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.97–7.95 (d, 2H, C-H2′-6′ J = 8.8 Hz), 7.49–7.47 (m, 3H, C-H), 7.41–7.39 (m, 4H, C-Hbn), 7.28 (s, 1H, C-H4), 7.17–7.15 (d, 2H, C-H3′-5, J = 8.8 Hz), 6.92 (s, 1H, C-H10), 5.20 (s, 2H, CH2). 13C NMR (75 MHz, DMSO-d6): δ 183.16 (C-3), 160.82 (C-4′), 159.91 (C-8), 145.76 (C-2), 136, 55 (C-5), 133.36 (C-1′), 128.46 (C-3bn, C-5bn), 128.20 (C-1bn), 127.92 (C-4bn), 127.81 (C-2bn, C-6bn), 124.64 (C-6), 121.55 (C-9), 115.5 (C-2′, C-6′), 113.90 (C-7),112.58 (C-4), 111.79 (C-10), 69.42 (CH2). (CH3). Elemental analysis calcd (%) for C22H17NO3: C, 76.95; H, 4.99; N, 4.08; found C, 76.77; H, 4.98; N, 4.06. m/z: 343.13141 (100.0%).
(Z)-N-(3-oxo-2-(3-phenoxybenzylidene)-2,3-dihydrobenzofuran-5-yl)acetamide (14):
Yield: 96%; mp: 196.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.16 (s, 1H, NH), 8.10 (d, 1H, J = 2 Hz, C-H4), 7.83–7.79 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.75–7.72 (d, 1H, J = 7.9 Hz, C-H6′), 7.67 (bs, 1H, C-H2′), 7.51 (dt, 1H, J = 7.4 Hz, C-H5′), 7.44–7.41 (m, 3H, C-H4′,8′), 7.20 (t, 1H, J = 7.4 Hz, C-H10′), 7.10–7.07 (d, 3H, C-H9′, 7), 6.93 (s, 1H, C-H10), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.72 (C-3), 168.52 (CO), 161.30 (C-8), 157.14 (C-7′), 156.19 (C-3′), 147.11 (C-2), 135.68 (C-5), 133.78 (C-1′), 130.60 (C-6), 130.17 (C-9′), 128.92 (C-5′), 126.54 (C-9), 123.88 (C-4), 120.62 (C-6′), 120.57 (C-10′), 119.97 (C-4′), 118.99 (C-8′), 113.30 (C-2′), 113.21 (C-7), 111.41 (C-10), 23.91 (CH3). Elemental analysis calcd (%) for C23H17NO4: C, 74.38; H, 4.61; N, 3.77; found C, 74.33; H, 4.63; N, 3.71. m/z: 371.11576 (100.0%).
(Z)-N-(3-oxo-2-(4-phenoxybenzylidene)-2,3-dihydrobenzofuran-5-yl)acetamide (15):
Yield: 67%; mp: 213.5 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 8.11 (d, 1H, J = 2 Hz, C-H4), 8.03–8.00 (d, 2H, J = 7.9 Hz, C-H2′), 7.83–7.79 (dd, 1H, J = 2.2;8.9 Hz, C-H6), 7.50–7.42 (m, 3H, C-H7,7′), 7.22 (t, 1H, J = 7.4 Hz, C-H8′), 7.12–7.09 (m, 4H, C-H3′,6′), 6.94 (s, 1H, C-H10), 2.07 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.43 (C-3), 168.38 (CO), 161.14 (C-8), 158.41 (C-1″), 155.43 (C-4′), 146.18 (C-2), 135.5 (C-5), 133.43 (C-3″), 130.15 (C-2′), 128.67 (C-6), 126.79 (C-1′), 124.27 (C-4″), 120.76 (C-9), 119.41 (C-3′), 118.28 (C-2″), 113.24 (C-7), 113.11 (C-4), 111.83 (C-10), 23.82 (CH3). Elemental analysis calcd (%) for C23H17NO4: C, 74.38; H, 4.61; N, 3.77; found C, 74.35; H, 4.67; N, 3.73. m/z: 371.11576 (100.0%).
(Z)-5-amino-2-(3-phenoxybenzylidene)benzofuran-3(2H)-one (16):
Yield: 51%; mp: 145.6 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.71–7.69 (d, 1H, J = 7.9 Hz, C-H6′), 7.64 (bs, 1H, C-H2′), 7.51–7.49 (d, 1H, J = 7.9 Hz, C-H6), 7.44 (dt, 2H, C-H9′), 7.22–7.15 (m, 2H, C-H4,5′), 7.09–7.04 (m, 4H, C-H7,9′,10′), 6.83 (d, 1H, J = 2.02 Hz, C-H4), 6.77 (s, 1H, C-H10), 5.41 (bs, 2H, NH2). 13C NMR (75 MHz, DMSO-d6): δ 184.24(C-3), 158.13(C-7′), 157.1(C-3′), 156.21(C-8), 147.41(C-2), 145.56(C-5), 134.09(C-1′), 130.52(C-5′), 130.15(C-9′), 126.34(C-6), 124.82(C-9), 123.81(C-6′), 120.88(C-10′), 120.43(C-4′), 119.65(C-4), 118.95(C-8′), 113.13(C-8), 110.13(C-10), 105.61(C-7). Elemental analysis calcd (%) for C21H15NO3: C, 76.58; H, 4.59; N, 4.25; found C, 76.56; H, 4.61; N, 4.22. m/z: 329.10519 (100.0%).
(Z)-5-amino-2-(4-phenoxybenzylidene)benzofuran-3(2H)-one (17):
Yield: 76%; mp: 170.6 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.99–7.96 (d, 2H, J = 8.6 Hz, C-H3′), 7.44 (dt, 2H, J = 7.7 Hz, C-H7′), 7.24–7.21 (m, 2H, C-H7,8′), 7.10–7.02 (m, 5H, C-H6,2′,6′), 6.83 (bs, 2H, C-H4,10), 5.25 (bs, 2H, NH2). 13C NMR (75 MHz, DMSO-d6): δ 184.11 (C-3), 158.16 (C-5′), 158.02 (C-8), 155.58 (C-4′), 146.56 (C-2), 145.6 (C-5), 133.27 (C-7′), 130.24 (C-2′), 127.21 (C-1′), 124.62 (C-6), 124.28 (C-8′), 121.15 (C-9), 119.41 (C-3′), 118.39 (C-6′), 113.17 (C-4), 110.62 (C-10), 105.45 (C-7). Elemental analysis calcd (%) for C21H15NO3: C, 76.58; H, 4.59; N, 4.25; found C, 76.43; H, 4.64; N, 4.54. m/z: 329.10519 (100.0%).
(Z)-N-(2-(2-isopropoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (18):
Yield: 93%; mp: 230 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 8.21–8.19 (d, 1H, J = 1.2;7.8 Hz, C-H6′), 8.12 (bs, 1H, C-H4), 7.81–7.78 (d, 1H, J = 8.7 Hz, C-H6), 7.52–7.49 (d, 1H, J = 8.9 Hz, C-H7), 7.42 (dt, 1H, J = 7.7 Hz, C-H4′), 7.20 (s, 1H, C-H10), 7.16–7.13 (d, 1H, J = 8.3 Hz, C-H3′), 7.08 (dt, 1H, J = 7.6 Hz, C-H5′), 4.75 (q, 1H, J = 5.9;11.9 Hz, C-Hisop), 2.07 (s, 3H, NHCOCH3), 1.35–1.33 (d, 6H, J = 5.8 Hz, C-H3isop). 13C NMR (75 MHz, DMSO-d6): δ 183.52 (C-3), 168.39 (CO), 161.15 (C-8), 156.76 (C-2′), 146.69 (C-2), 135.5 (C-5), 131.85 (C-6′), 131.38 (C-4′), 128.64 (C-6), 121.03 (C-9), 120.73 (C-1′), 120.69 (C-5′), 113.85 (C-7), 113.28 (C-4), 113.11 (C-10), 105.92 (C-3′), 70.49 (CHiPr), 23.83 (CH3), 21.74 (CH3iPr). Elemental analysis calcd (%) for C20H19NO4: C, 71.20; H, 5.68; N, 4.15; found C, 71.14; H, 5.67; N, 4.12. m/z: 337.13 (100.0%).
(Z)-N-(2-(3-isopropoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (19):
Yield: 91%; mp: 167 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.19 (s, 1H, NH), 8.12 (d, 1H, J = 2 Hz, C-H4), 7.83–7.80 (dd, 1H, J = 2.2; 8.9 Hz, C-H6), 7.57–7.52 (m, 3H, C-H2′,4′,7), 7.40 (dt, 1H, J = 8.0 Hz, C-H5′), 7.04–7.01 (dd, 1H, J = 2.6;8.2 Hz, C-H6′), 6.91 (s, 1H, C-H10), 4.68 (q, 1H, J = 5.9;11.9 Hz, C-Hisop), 2.07 (s, 3H, NHCOCH3), 1.31–1.29 (d, 6H, J = 5.8 Hz, C-H3isop). 13C NMR (75 MHz, DMSO-d6): δ 183.73 (C-3), 168.49 (CO), 161.35 (C-8), 157.66 (C-3′), 146.87 (C-2), 135.62 (C-5), 133.17 (C-1′), 130.1 (C-5′), 128.86 (C-6), 123.56 (C-6′), 120.64 (C-9), 118.25 (C-2′), 117.32 (C-3′), 113.41 (C-7), 113.17 (C-4), 112.28 (C-10), 69.35 (CHiPr), 23.88 (CH3), 21.77 (CH3iPr). Elemental analysis calcd (%) for C20H19NO4: C, 71.20; H, 5.68; N, 4.15; found C, 71.18; H, 5.66; N, 4.16. m/z: 337.13 (100.0%).
(Z)-N-(2-(4-isopropoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (20):
Yield: 93%; mp: 200.2 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.14 (s, 1H, NH), 8.10 (d, 1H, J = 1.9 Hz, C-H4), 7.94–7.91 (d, 2H, J = 8.8 Hz, C-H2′), 7.82–7.78 (dd, 1H, J = 2.2;8.8 Hz, C-H6), 7.51–7.48 (d, 1H, J = 8.89 Hz, C-H7), 7.06–7.03 (d, 2H, J = 8.8 Hz, C-H3′), 6.90 (s, 1H, C-H10), 4.72 (q, 1H, J = 5.9;11.9 Hz, C-Hisop), 2.07 (s, 3H, NHCOCH3), 1.30–1.28 (d, 6H, J = 5.8 Hz, C-H3isop). 13C NMR (75 MHz, DMSO-d6): δ 183.25 (C-3), 168.39 (CO), 160.98 (C-8), 159.16 (C-4′), 145.6 (C-2), 135.39 (C-5), 133.41 (C-2′), 128.48 (C-6), 124.02 (C-1′), 120.94 (C-9), 115.94 (C-3′), 113.2 (C-7), 113.06 (C-4), 112.77 (C-10), 69.5 (CHiPr), 23.83 (CH3), 21.68 (CH3iPr). Elemental analysis calcd (%) for C20H19NO4: C, 71.20; H, 5.68; N, 4.15; found C, 71.18; H, 5.69; N, 4.12. m/z: 337.13 (100.0%).
(Z)-5-amino-2-(2-isopropoxybenzylidene)benzofuran-3(2H)-one (21):
Yield: 68%; mp: 126.7 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.19–8.17 (d, 1H, J = 6.9 Hz, C-H6′), 7.39 (t, 1H, J = 7.3 Hz, C-H4′), 7.26–7.24 (d, 1H, J = 8.7 Hz, C-H7), 7.14 (d, 1H, C-H3′), 7.11 (bs, 2H, NH2), 7.08–7.06 (d, 1H, C-H5′), 7.06–7.04 (dd, 1H, J = 2.2;7.7 Hz, C-H6), 6.95 (s, 1H, C-H10), 6.85 (d, 1H, J = 2.2 Hz, C-H4), 4.73 (q, 1H, J = 5.9;11.9 Hz, C-Hisop), 1.34–1.32 (d, 6H, J = 5.8 Hz, C-H3isop). 13C NMR (75 MHz, DMSO-d6): 184.16 (CO), 158.14 (C-8), 156.62 (C-2′), 147.02 (C-2), 145.24 (C-5), 131.55 (C-6′), 131.33 (C-4′), 124.76 (C-6), 121.4 (C-9), 121.11 (C-5′), 120.72 (C-1′), 113.85 (C-7), 113.23 (C-10), 105.77 (C-3′), 104.8 (C-4), 70.46 (CHiPr), 21.82 (CH3iPr). Elemental analysis calcd (%) for C20H19NO4: C, 71.20; H, 5.68; N, 4.15; found C, 71.24; H, 5.74; N, 4.18. m/z: 337.13 (100.0%).
(Z)-5-amino-2-(3-isopropoxybenzylidene)benzofuran-3(2H)-one (22):
Yield: 51%; mp: 198.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.57–7.54 (d, 1H, J = 7.8 Hz, C-H6′), 7.51 (bs, 1H, C-H2′), 7.51–7.48 (d, 1H, J = 8.6 Hz, C-H7), 7.43–7.40 (d, 2H, J = 7.8 Hz, C-H4′), 7.41 (dt, 1H, J = 7.8 Hz, C-H5′), 7.27 (d, 1H, J = 1.9 Hz, C-H4), 7.04–7.01 (dd, 1H, J = 2.0;8.0 Hz, C-H6), 6.89 (s, 1H, C-H10), 4.68 (q, 1H, J = 5.9;11.9 Hz, C-Hisop), 1.31–1.29 (d, 6H, J = 5.8 Hz, C-H3isop). 13C NMR (75 MHz, DMSO-d6): δ 183.65 (C-3), 160.93 (C-8), 157.67 (C-3′), 146.95 (C-2), 137.64 (C-5), 133.24 (C-1′), 130.11 (C-5′), 128.17 (C-6), 123.56 (C-6′), 121.25 (C-9), 118.24 (C-2′), 117.28 (C-3′), 113.96 (C-7), 112.14 (C-4), 111.46 (C-10), 69.35 (CHiPr), 21.79 (CH3iPr). Elemental analysis calcd (%) for C10H17NO3: C, 71.20; H, 5.68; N, 4.15; found C, 71.18; H, 5.65; N, 4.12. m/z: 295.12 (100.0%).
(Z)-5-amino-2-(4-isopropoxybenzylidene)benzofuran-3(2H)-one (23):
Yield: 50%; mp: >350 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.97–7.95 (d, 2H, J = 8.8 Hz, C-H2′), 7.66–7.64 (m, 3H, C-H4,6,7), 7.07–7.05 (d, 2H, J = 8.8 Hz, C-H3′), 6.98 (s, 1H, C-H10). 13C NMR (75 MHz, DMSO-d6): δ 183.22 (C-3), 160.22 (C-8), 159.12 (C-4′), 145.7 (C-2), 133.38 (C-2′), 138.33 (C-5), 127.35 (C-7), 124.1 (C-1′), 121.52 (C-9), 115.94 (C-3′), 113.69 (C-6), 112.51 (C-4), 110.58 (C-10), 69.49 (CH2iPr), 21.68 (CH3iPr). Elemental analysis calcd (%) for C10H17NO3: C, 71.20; H, 5.68; N, 4.15; found C, 71.15; H, 5.66; N, 4.18. m/z: 295.12 (100.0%).
(Z)-N-(2-(2-fluorobenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (24):
Yield: 68%; mp: 226 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.17 (s, 1h, NH), 8.25 (t, 1H, J = 7.8 Hz, C-H2′), 8.12 (d, 1H, J = 2.2 Hz, C-H4), 7.85–7.82 (dd, 1H, J = 2.5, 8.7 Hz, C-H6′), 7.54–7.51 (m, 2H, C-H4′,7), 7.41–7.39 (d, 1H, C-H3′), 7.35 (dt, 1H, C-H5′), 6.90 (s, 1H, C-H10), 2.08 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 183.5 (C-3), 168.44 (CO), 164.9–161.44 (C-2′, J = 260 Hz), 161.41 (C-8), 147.71 (C-2), 135.78 (C-5), 132.24–132.12 (C-4′, J = 8.8 Hz), 131.31 (C-6′), 129.02 (C-6), 125.11 (C-5′, J = 3.3 Hz), 120.38 (C-9), 119.67–119.52 (C-1′, J = 11 Hz), 115.93–115.64 (C-3′, J = 22 Hz), 113.38 (C-7), 113.25 (C-4), 102.1–102.0 (C-10, J = 7.7 Hz), 23.83 (CH3). Elemental analysis calcd (%) for C17H12FNO3: C, 68.68; H, 4.07; N, 4.71; found C, 68.65; H, 4.01; N, 4.65. m/z: 297.08 (100.0%).
(Z)-N-(2-(3-fluorobenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (25):
Yield: 82%; mp: 243.6 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.22 (s, 1h, NH), 8.12 (d, 1H, J = 2.1 Hz, C-H4), 7.83–7.79 (m, 3H, C-H6,2′,6′), 7.59–7.51 (m, 2H, C-H4′,7), 7.31–7.39 (dt, 1H, J = 2.1, 8.4 Hz, C-H5′), 6.59 (s, 1H, C-H10), 2.07 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 183.83(C-3), 168.6(CO), 163.85–160.62 (C-3′, J = 244 Hz), 161.48 (C-8), 147.37 (C-2), 135.77 (C-5), 134.3–134.19 (C-1′, J = 8.25 Hz), 131.08–130.97 (C-5′, J = 8.25 Hz), 129.05 (C-6), 127.62–127.58 (C-6′, J = 2.75 Hz), 120.53 (C-9), 117.45–117.15 (C-4′, J = 22.56 Hz), 117.02–116.73 (C-2′, J = 21.5 Hz), 113.53 (C-7), 113.25 (C-4), 110.69–110.66 (C-10, J = 2.75 Hz), 23.92 (CH3). Elemental analysis calcd (%) for C17H12FNO3: C, 68.68; H, 4.07; N, 4.71; found C, 68.58; H, 4.12; N, 4.73. m/z: 297.08 (100.0%).
(Z)-5-amino-2-(2-fluorobenzylidene)benzofuran-3(2H)-one (26):
Yield: 62%; mp: 161.3 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.23 (dt, 1H, J = 1.65, 7.8 Hz, C-H6′), 7.51–7.47 (m, 1H, C-H4′), 7.37 (t, 1H, C-H3′), 7.34 (dt, 1H, C-H5′), 7.28–7.25 (d, 1H, J = 8.8 Hz, C-H7), 7.09–7.05 (dd, 1H, J = 2.5, 8.7 Hz, C-H6), 6.86 (d, 1H, J = 2.4 Hz, C-H4), 6.81 (s, 1H, C-H10), 5.28 (bs, 2H, NH2). 13C NMR (75 MHz, DMSO-d6): δ 184.05 (C-3), 164.75–161.71 (C-2′, J = 260 Hz), 158.19 (C-8), 148.04 (C-2), 145.83 (C-5), 131.84–131.73 (C-4′, J = 8 Hz), 131.2 (C-6′), 125.11–125.06 (C-5′, J = 3 Hz), 124.84 (C-6), 120.69 (C-9), 119.97–119.81 (C-1′, J = 12 Hz), 115.85–115.56 (C-3′), 113.22 (C-7), 105.54 (C-4), 100.82–100.72 (C-10).
Elemental analysis calcd (%) for C15H10FNO2: C, 70.58; H, 3.95; N, 5.49; found C, 70.44; H, 3.99; N, 5.32. m/z: 255.07 (100.0%).
(Z)-5-amino-2-(3-fluorobenzylidene)benzofuran-3(2H)-one (27):
Yield: 85%; mp: 159.7 °C; 1H NMR (300 MHz, DMSO-d6): δ 7.80–7.78 (m, 2H, C-H2′,4′), 7.57–7.50 (dt, 1H, C-H5′), 7.32–7.27 (m, 2H, C-H7,6′), 7.10–7.07 (dd, 1H, J = 2.5, 8.7 Hz, C-H6), 6.87 (d, 1H, J = 2.4 Hz, C-H4), 6.85 (s, 1H, C-H10). 13C NMR (75 MHz, DMSO-d6): δ 184.26(C-3), 163.81–160.58 (C-3′, J = 244 Hz), 158.49 (C-8), 147.63 (C-2), 145.01 (C-5), 134.58–134.47 (C-1′, J = 8.25 Hz), 130.85 (C-5′), 127.34 (C-6), 125.18 (C-6′), 120.84(C-9), 117.22–116.92 (C-4′, J = 22.5 Hz), 116.64–116.36 (C-2′), 113.38(C-7), 109.49–109.45 (C-10, J = 2.75 Hz), 106.08 (C-4). Elemental analysis calcd (%) for C15H10FNO2: C, 70.58; H, 3.95; N, 5.49; found C, 70.66; H, 4.08; N, 5.31. m/z: 255.07 (100.0%).
(Z)-5-amino-2-(4-fluorobenzylidene)benzofuran-3(2H)-one (28):
Yield: 82%; mp: 164.4 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.09–8.04 (dd, 2H, J = 7.8 Hz, C-H2′), 7.81–7. 78 (d, 1H, C-H4′), 7.57–7.54 (d, 1H, J = 8.4 Hz, C-H7), 7.36 (t, 2H, C-H3′), 7.33 (d, 1H, C-H4), 6.98 (s, 1H, C-H10). 13C NMR (75 MHz, DMSO-d6): δ 183.5 (C-4), 165.39 (C-8), 164.38–161.07 (C-4′, J = 250 Hz), 145.91 (C-2), 137.62 (C-5), 133.73–133.62 (C-2′, J = 9 Hz), 128.55 (C-1′, J = 3 Hz), 124.24 (C-6), 123.95 (C-7), 120.81 (C-9), 116.26–115.97 (C-3′, J = 22 Hz), 113.14 (C-4), 111.04 (C-10). Elemental analysis calcd (%) for C15H10FNO2: C, 70.58; H, 3.95; N, 5.49; found C, 70.52; H, 4.07; N, 5.23. m/z: 255.06956 (100.0%).
(Z)-N-(3-oxo-2-(3-(trifluoromethyl)benzylidene)-2,3-dihydrobenzofuran-5-yl)acetamide (29):
Yield: 82%; mp: 252.1 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.20 (bs, 1H, NH), 8.30–8.28 (m, 2H, C-H2′,4′), 8.13 (d, 1H, J = 2.1 Hz, C-H4), 7.85–7.81 (dd, 1H, J = 2.3, 8.8 Hz, C-H6), 7.79–7.72 (m, 2H, C-H5′,6′), 7.56–7.53 (d, 1H, J = 8.9 Hz, C-H7), 7.06 (s, 1H, C-H10), 2.07 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 183.81 (C-3), 168.54 (CO), 161.44 (C-8), 147.58 (C-2), 135.82 (C-5), 134.74 (C-1′), 133.09 (C-6), 130.15 (C-6′), 129.94 (C-5′), 129.63–129.03 (C-3′, J = 31.7 Hz), 127.46 (C-4′), 126.17 (C-2′), 125.32–122.61 (CF3, J = 270 Hz), 120.48 (C-9), 113.5 (C-7), 113.23 (C-4), 110.18 (C-10), 23.91 (CH3). Elemental analysis calcd (%) for C18H12F3NO3: C, 62.25; H, 3.48; N, 4.03; found C, 62.09; H, 3.54; N, 3.98. m/z: 347.07693 (100.0%).
(Z)-5-amino-2-(3-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one (30):
Yield: 82%; mp: >350 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.31–8.29 (m, 2H, C-H2′,4′), 7.80–7.73 (m, 2H, C-H-6′,5′), 7.52–7.49 (d, 1H, J = 8.9 Hz, C-H7), 7.40–7.36 (dd, 1H, J = 2.3, 8.9 Hz, C-H7), 7.26 (s, 1H, C-H4), 7.05 (s, 1H, C-H10). 13C NMR (75 MHz, DMSO-d6): δ 183.7 (C-3), 161.09 (C-8), 147.65 (C-2), 134.75 (C-1′), 133.13 (C-5), 130.16 (C-6′), 130.01–129.59 (C-3′, J = 31 Hz), 128.44 (C-7), 127.48 (C-4′), 127.43 (C-2′), 126.11–122.78 (CF3, J = 250 Hz), 121.1 (C-9), 114.05 (C-6), 111.68 (C-4), 110.05 (C-10). Elemental analysis calcd (%) for C16H10F3NO2: C, 62.96; H, 3.30; N, 4.59; found C, 63.11; H, 3.35; N, 4.55. m/z: 305.06636 (100.0%).
(Z)-4-((5-acetamido-3-oxobenzofuran-2(3H)-ylidene)methyl)benzoic acid (31):
Yield: 71%; mp: 165.3 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.28 (s, 1H, NH), 8.14 (d, 1H, J = 1.9 Hz, C-H4), 8.10–8.07 (d, 2H, J = 8.8 Hz, C-H2′), 8.05–8.02 (d, 2H, J = 8.8 Hz, C-H3′), 7.87–7.83 (dd, 1H, J = 2.2;8.8 Hz, C-H6), 7.55–7.53 (d, 1H, J = 8.89 Hz, C-H7), 6.98 (s, 1H, C-H10), 2.08 (s, 3H, NHCOCH3). 13C NMR (75 MHz, DMSO-d6): δ 183.85 (C-3), 168.54 (CO), 166.77 (COOH), 161.47 (C-8), 147.69 (C-2), 136.08 (C-1′), 135.82 (C-5), 131.35 (C-4′), 131.23 (C-3′), 129.74 (C-2′), 129.04 (C-6), 120.47 (C-9), 113.44 (C-7), 113.25 (C-4), 110.61 (C-10), 23.89 (CH3). Elemental analysis calcd (%) for C18H13NO5: C, 66.87; H, 4.05; N, 4.33; found C, 66.85; H, 4.12; N, 4.27. m/z: 323.07937 (100.0%).
(Z)-N-(7-nitro-3-oxo-2-(3-phenoxybenzylidene)-2,3-dihydrobenzofuran-5-yl)acetamide (32):
Yield: 91%; mp: 230.3 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.51 (bs, 1H, NH), 8.71 (d, 1H, J = 2.2 Hz, C-H6), 8.35 (d, 1H, J = 2.2 Hz, C-H4), 7.89–7.86 (d, 2H, J = 7.8 Hz, C-H2″), 7.56 (t, 1H, J = 8.16 Hz, C-H5′), 7.42 (dt, 2H, C-H3″), 7.20–7.17 (d, 1H, J = 7.3 Hz, C-H4′), 7.14 (s, 1H, C-H2′), 7.09–7.06 (m, 2H, C-H10, 6′), 2.11 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 178.66 (C-3), 169.01 (CO), 157.07 (C-1″), 156.36 (C-3′), 153.21 (C-8), 146.13 (C-2), 143.98 (C-7), 135.31 (C-5), 133.21 (C-1′), 130.64 (C-5′), 130.09 (C-3″), 127.01 (C-6), 124.60 (C-9), 123.69 (C-4″), 121.59 (C-6′), 120.86 (C-4), 119.76 (C-4′), 119.35 (C-2′), 118.66 (C-2″), 113.73 (C-10), 23.86 (CH3). Elemental analysis calcd (%) for C23H16N2O6: C, 66.34; H, 3.87; N, 6.73; found C, 66.21; H, 3.74; N, 6.71. m/z: 416.10084 (100.0%).
(Z)-N-(2-(3-hydroxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (33):
Yield: 75%; mp: >350 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 9.67 (bs, 1H, OH), 8.11–8.10 (d, 1H, J = 2.2 Hz, C-H4), 7.83–7.80 (dd, 1H, J = 2.2;8.8 Hz, C-H6), 7.50–7.47 (d, 1H, J = 8.9 Hz, C-H7), 7.42 (d, 1H, C-H2′), 7.40–7.38 (d, 1H, J = 7.8 Hz, C-H6′), 7.30 (t, 1H, J = 7.8 Hz, C-H5′), 6.89–6.86 (dd, 1H, C-H4′), 6.82 (s, 1H, C-H10), 2.07 (s, 3H, C-H3′). 13C NMR (75 MHz, DMSO-d6): δ 183.65 (C-3), 168.41 (CO), 161.27 (C-8), 157.58 (C-3′), 146.69 (C-2), 135.54 (C-5), 132.9 (C-1′), 129.89 (C-5′), 128.8 (C-6), 122.64 (C-6′), 120.65 (C-9), 117.53 (C-3′), 117.46 (C-2′), 113.21 (C-7), 113.16 (C-4), 112.47 (C-10), 23.83 (CH3). Elemental analysis calcd (%) for C17H13NO4: C, 69.15; H, 4.44; N, 4.74; found C, 69.01; H, 4.48; N, 4.69. m/z: 295.08 (100.0%).
(Z)-5-amino-2-(3-hydroxybenzylidene)benzofuran-3(2H)-one (34):
Yield: 95%; mp: 225.2 °C; 1H NMR (300 MHz, DMSO-d6): δ 9.63 (bs, 1H, OH), 7.39 (d, 1H, C-H2′), 7.37–7.34 (d, 1H, J = 7.8 Hz, C-H6′), 7.28 (t, 1H, J = 7.8 Hz, C-H5′), 7.25–7.22 (d, J = 8.8 Hz, C-H7), 7.07–7.04 (dd, 1H, J = 2.2;8.5 Hz, C-H4′), 6.86–6.83 (m, 2H, C-H4,6), 6.70 (s, 1H, C-H10), 5.24 (bs, 2H, NH2). 13C NMR (75 MHz, DMSO-d6): δ 184.22 (C-3), 158.08 (C-8), 157.53 (C-3′), 146.99 (C-2), 145.57 (C-5), 133.21 (C-1′), 129.82 (C-5′), 124.66 (C-6), 122.42 (C-6′), 120.98 (C-9), 117.34 (C-3′), 117.1 (C-2′), 113.06 (C-7), 111.17 (C-4), 105.46 (C-10). Elemental analysis calcd (%) for C15H11NO3: C, 71.14; H, 4.38; N, 5.53; found C, 71.21; H, 4.34; N, 5.49. m/z: 253.07 (100.0%).

5. Conclusions

In the present study, 31 new aurone derivative compounds were synthetized. These new compounds were obtained by the substitution of the aurone scaffold at position 5 by amino and acetamido groups, and through various substitutions at the 2′, 3′ and 4′ positions. Antimicrobial testing identified two of these compounds, i.e., 10 and 20, as the most active on both Gram-positive and -negative bacteria with MIC values as low as 0.78 µM. These were also the safest regarding human cells. The two selected compounds shared some structural similarity with the 5-acetamido substitution. The SAR study from this work correlates with the results previously obtained by Olleik et al. [14] showing that benzyloxy and isopropyloxy lead to interesting activities in aurone scaffolds with substitution on the A ring with amino or acetamido groups, improving the activity compared to the natural OH group. Taken together, these results confirm that the aurone scaffold is a promising structure that could be the starting point for the design of new antibacterial agents by diversifying the substitution pattern on A and B rings altogether.

6. Patents

Aurone derivatives and uses thereof for controlling bacteria and/or fungi. PCT/EP2021/069047. BOLLA Jean Michel., MARESCA Marc, NEULAT-RIPOLL Fabienne, OLLEIK Hamza, PERRIER-VIRET Josette, PIQUE Valérie. ROBIN Maxime.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics13040300/s1, File S1: NMR Spectra of compounds 4–34.

Author Contributions

Conceptualization, M.R.; methodology, M.R., J.-M.B. and M.M.; validation, M.R., J.-M.B. and M.M.; formal analysis, A.D.M., H.O., E.C.-D., S.G., F.N.-R., R.H., M.C., J.-F.C., S.C., V.P., Y.C., M.M. and M.R.; investigation, A.D.M., H.O., E.C.-D., S.G., F.N.-R., R.H., M.C., J.-F.C., S.C., V.P., Y.C., M.M. and M.R.; data curation, M.R., S.C., J.-M.B. and M.M.; writing—original draft preparation, A.D.M., M.R. and M.M.; writing—review and editing, A.D.M., H.O., E.C.-D., S.G., F.N.-R., R.H., M.C., J.-F.C., S.C., V.P., Y.C., E.B., A.H., J.P., M.M. and M.R.; supervision, E.B., A.H., J.P., M.R. and M.M.; project administration, M.M. and M.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Région PACA EJD no. 2021-04403 for A.D.M. PhD fellowship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated for this study are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Antibiotics 13 00300 g001
Figure 1. Structure of the aurone scaffold (top left), natural antimicrobial aurones, and examples of synthetic, heavily modified analogues.
Figure 1. Structure of the aurone scaffold (top left), natural antimicrobial aurones, and examples of synthetic, heavily modified analogues.
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Antibiotics 13 00300 g002
Figure 2. Synthetic route of the aurone derivatives. A: benzofuranone (1) 1 eq. and various benzaldehydes 1 eq. in Choline chloride/Urea (1/2), 80 °C, 2 h. B: (2) 1 eq. and various benzaldehydes 1 eq. in EtOH, LiOH 3 eq., 90 °C, 2 h. (3 a-o) 1 eq. and Mercuric acetate 1 eq. in pyridine, 110 °C, 1 h. Acetamido Aurone (4–6, 10, 11, 14, 15, 18–20, 24, 25, 29, 31, 32, 34) are converted to their amino analogues (7–9, 12, 13, 16, 17, 21–23, 26–28, 30, 33) in EtOH, 0.5 M HCl, 100 °C, 2 h.
Figure 2. Synthetic route of the aurone derivatives. A: benzofuranone (1) 1 eq. and various benzaldehydes 1 eq. in Choline chloride/Urea (1/2), 80 °C, 2 h. B: (2) 1 eq. and various benzaldehydes 1 eq. in EtOH, LiOH 3 eq., 90 °C, 2 h. (3 a-o) 1 eq. and Mercuric acetate 1 eq. in pyridine, 110 °C, 1 h. Acetamido Aurone (4–6, 10, 11, 14, 15, 18–20, 24, 25, 29, 31, 32, 34) are converted to their amino analogues (7–9, 12, 13, 16, 17, 21–23, 26–28, 30, 33) in EtOH, 0.5 M HCl, 100 °C, 2 h.
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Antibiotics 13 00300 g003
Figure 3. Structures of the more active aurone derivatives identified during the first screening (compounds 10, 12, 15, 16, and 20).
Figure 3. Structures of the more active aurone derivatives identified during the first screening (compounds 10, 12, 15, 16, and 20).
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Antibiotics 13 00300 g004
Figure 4. Evaluation of the toxicity of compounds 10, 12, 15, 16, and 20 on human cells. Human cells corresponding to kidney epithelial cells (A498), lung epithelial cells (BEAS-2B), intestinal epithelial cells (Caco-2), skin cells (HaCaT), liver cells (HepG2), or fibroblasts (IMR-90) were exposed to increasing concentrations of aurones for 48 h before measurement of the cell viability using resazurin. Results are expressed as % of cell viability, DMSO alone being used as negative control giving 100% viability. Data were plotted using GraphPad Prism 7 (means +/− S.D, n = 3).
Figure 4. Evaluation of the toxicity of compounds 10, 12, 15, 16, and 20 on human cells. Human cells corresponding to kidney epithelial cells (A498), lung epithelial cells (BEAS-2B), intestinal epithelial cells (Caco-2), skin cells (HaCaT), liver cells (HepG2), or fibroblasts (IMR-90) were exposed to increasing concentrations of aurones for 48 h before measurement of the cell viability using resazurin. Results are expressed as % of cell viability, DMSO alone being used as negative control giving 100% viability. Data were plotted using GraphPad Prism 7 (means +/− S.D, n = 3).
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Antibiotics 13 00300 g005
Figure 5. Route A and B used for the synthesis of 5-amino and 5-acetamido aurones.
Figure 5. Route A and B used for the synthesis of 5-amino and 5-acetamido aurones.
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Table 1. List of the synthetized aurones and their respective substitution on each position (OBn: Obenzyl, OPh: Ophenyl, OiPr: Oisopropyl).
Table 1. List of the synthetized aurones and their respective substitution on each position (OBn: Obenzyl, OPh: Ophenyl, OiPr: Oisopropyl).
Compound52′3′4′
4NHCOCH3OCH3HH
5NHCOCH3HOCH3H
6NHCOCH3HHOCH3
7NH2OCH3HH
8NH2HOCH3H
9NH2HHOCH3
10NHCOCH3HOBnH
11NHCOCH3HHOBn
12NH2HOBnH
13NH2HHOBn
14NHCOCH3HOPhH
15NHCOCH3HHOPh
16NH2HOPhH
17NH2HHOPh
18NHCOCH3OiPrHH
19NHCOCH3HOiPrH
20NHCOCH3HHOiPr
21NH2OiPrHH
22NH2HOiPrH
23NH2HHOiPr
24NHCOCH3FHH
25NHCOCH3HFH
26NH2FHH
27NH2HFH
28NH2HHF
29NHCOCH3HCF3H
30NH2HCF3H
31NHCOCH3HHCOOH
32NHCOCH3HOHH
33NH2HOHH
34NHCOCH3HHH
Table 2. Evaluation of the antimicrobial activities of the 31 newly synthetized aurone derivatives. The antimicrobial activities were determined using a MIC assay on species representative of Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (E. coli, P. aeruginosa), mycobacteria (M. smegmatis), and fungi (C. albicans). Amphotericin B and gemifloxacin were used as control antimicrobials for fungi and bacteria, respectively. The MIC values are given in µM (n = 2–3).
Table 2. Evaluation of the antimicrobial activities of the 31 newly synthetized aurone derivatives. The antimicrobial activities were determined using a MIC assay on species representative of Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (E. coli, P. aeruginosa), mycobacteria (M. smegmatis), and fungi (C. albicans). Amphotericin B and gemifloxacin were used as control antimicrobials for fungi and bacteria, respectively. The MIC values are given in µM (n = 2–3).
Gram-PositiveGram-NegativeMycobacteriaFungi
B. subtilis
ATCC6633		S. aureus
ATCC6538P		E. coli
ATCC8739		P. aeruginosa
ATCC9027		M. smegmatis
ATCC700084		C. albicans
DSM10697
Gemifloxacin0.030.060.060.252-
Amphotericin B-----0.62
450100>100>100>100>100
5>100100>100>100100>100
6>100>100>100>100>100>100
7>100>100>100>100>100>100
8>100>100>100>100>100>100
9>100>100>100>100>100>100
103.1212.512.5255050
11>100>100>100>100>100>100
1225100>100>10050>100
13100>100>100>100>100>100
14>100>100>100>100>100>100
1550100>100>10050>100
16252525>10050>100
17>100>100>100>100>100>100
18>100>100>100>100>100>100
19>100>100>10025>100>100
202512.525>1005050
21>100>100>100>100>100>100
22>100>100>100>100>100>100
23>100>100>100>100>100>100
24>100>100>100>100>100>100
2550>100>100>100100>100
2650>100>100>100>100>100
27>100>100>100>100>100>100
28>100>100>100>100>100>100
29>100>100>100>100>100>100
30>100>100>100>100>100>100
3150100>100>100100>100
32>100>100>100>100>100>100
33>100>100>100>100>100>100
3450>100>100>100>100>100
Table 3. Determination of the cytotoxic concentrations of compounds 10, 12, 15, 16, and 20 on human cells. The cytotoxic concentrations 50 (CC50, in µM) (i.e., the concentrations of aurones causing 50% reduction of the cell viability after 48 h exposure) were calculated from Figure 3 using GraphPad Prism 7. Results are expressed as means +/− S.D (µM) (n = 3).
Table 3. Determination of the cytotoxic concentrations of compounds 10, 12, 15, 16, and 20 on human cells. The cytotoxic concentrations 50 (CC50, in µM) (i.e., the concentrations of aurones causing 50% reduction of the cell viability after 48 h exposure) were calculated from Figure 3 using GraphPad Prism 7. Results are expressed as means +/− S.D (µM) (n = 3).
Compound1012151620
A498398.2 +/− 164.6152.4 +/− 31.4145.7 +/− 17.3452.3 +/− 147.9453.0 +/− 46.4
BEAS-2B169.0 +/− 28.374.6 +/− 12.1109.5 +/− 17.4129.6 +/− 18.3125.9 +/− 17.2
Caco-2199.6 +/− 33.5111.7 +/− 15.0136.8 +/− 7.2131.7 +/− 18.0186.5 +/− 27.8
HaCaT268.5 +/− 51.651.3 +/− 9.5>50080.4 +/− 17.8322.9 +/− 73.2
HepG2472.4 +/− 145.9343.4 +/− 68.0>500397.8 +/− 94.1>500
IMR-90421.4 +/− 119.342.4 +/− 9.6>500116.5 +/− 29.4437.6 +/− 128
Mean CC50321.5129.3130.6218.0305.1
Table 4. Determination of the therapeutic indexes of compounds 10, 12, 15, 16, and 20. The therapeutic indexes (TI) of each aurone was calculated by dividing their CC50 on human cells (Table 3) by their MIC values on S. aureus from Table 2. MIC and CC50 are expressed in µM.
Table 4. Determination of the therapeutic indexes of compounds 10, 12, 15, 16, and 20. The therapeutic indexes (TI) of each aurone was calculated by dividing their CC50 on human cells (Table 3) by their MIC values on S. aureus from Table 2. MIC and CC50 are expressed in µM.
Compound1012151620
MIC on S. aureus12.51001002512.5
Lowest CC50169.042.4109.580.4125.9
Highest CC50472.4343.4>500452.3>500
Lowest TI13.50.41.03.210.0
Highest TI37.73.4>518.0>40
Table 5. Antimicrobial activities of compounds 10 and 20 on various bacterial and fungal species. The antimicrobial activities were determined using MIC assay as described in Section 4. The MIC values are given in µM (n = 2–3).
Table 5. Antimicrobial activities of compounds 10 and 20 on various bacterial and fungal species. The antimicrobial activities were determined using MIC assay as described in Section 4. The MIC values are given in µM (n = 2–3).
1020
Gram-positive
Bacillus anthracis (CNR-charbon_04022)12.56.25
Bacillus cereus (DSM31)12.525
Bacillus subtilis (ATCC6633)3.1225
Clostridium botulinum (DSM1985)0.783.12
Clostridium difficile (DSM1296)12.53.12
Clostridium perfringens (ATCC13124)>100>100
Enterococcus faecalis (DSM2570)50100
Enterococcus faecium (DSM20477)10025
Listeria monocytogenes (DSM20600)3.126.25
Propionibacterium acnes (ATCC6919)100>100
Staphylococcus aureus (ATCC6538P)12.512.5
MRSA (ATCCBAA-1717)12.525
Streptococcus pyogenes (DSM20565)5050
Gram-negative
Acinetobacter baumannii (DSM30007)12.525
Brucella melitensis (NR-256)2512.5
Enterobacter cloacae (DSM30054)>100>100
Escherichia coli (ATCC8739)12.525
Francisella tularensis (NR-643)5012.5
Helicobacter pylori (ATCC43504)12.512.5
Klebsiella pneumonia (DSM26371)>100>100
Pseudomonas aeruginosa (ATCC9027)25>100
Salmonella enterica (CIP80.39)2550
Shigella flexneri (ATCC12022)2550
Vibrio alginolyticus (DSM2171)2550
Yersinia pestis (NR-641)12.512.5
Mycobacteria
Mycobacterium abscessus S (CIP 104536T)>100>100
Mycobacterium abscessus R(CIP 104536T)>100>100
Mycobacterium smegmatis (ATCC700084)5050
Mycobacterium tuberculosis H37Rv (mc26230)>100>100
Filamentous fungi
Aspergillus fumigatus (DSM819)>100>100
Fusarium oxysporum (DSM62316)2525
Yeasts
Candida albicans (DSM10697)5050
Candida auris (DSM21092)5012.5
Candida glabrata (DSM11226)5050
Candida tropicalis (DSM9419)100100
Cryptococcus neoformans (DSM11959)2525
Table 6. Safety evaluation for compound 10. Therapeutic indexes (TI) values were calculated by dividing CC50 values by the lowest MIC value (obtained on C. botulinum, i.e., 0.78 µM) for compound 10 (Table 5).
Table 6. Safety evaluation for compound 10. Therapeutic indexes (TI) values were calculated by dividing CC50 values by the lowest MIC value (obtained on C. botulinum, i.e., 0.78 µM) for compound 10 (Table 5).
A498BEAS-2BCaco-2HaCaTHepG-2IMR-90
CC50 (µM)398.2169.0199.6268.5472.4421.4
TI with MIC of 0.78 µM510.5216.6255.8344.2605.6540.2
Table 7. Safety evaluation for compound 20. Therapeutic indexes (TI) values were calculated by dividing CC50 values by the lowest MIC value (obtained on C. botulinum, i.e., 3.12 µM) for compound 20 (Table 5).
Table 7. Safety evaluation for compound 20. Therapeutic indexes (TI) values were calculated by dividing CC50 values by the lowest MIC value (obtained on C. botulinum, i.e., 3.12 µM) for compound 20 (Table 5).
A498BEAS-2BCaco-2HaCaTHepG-2IMR-90
IC50 (µM)453.0125.9186.5322.9>500437.6
TI with MIC of 3.12 µM145.140.359.7103.4>160.2140.2
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Di Maio, A.; Olleik, H.; Courvoisier-Dezord, E.; Guillier, S.; Neulat-Ripoll, F.; Haudecoeur, R.; Bolla, J.-M.; Casanova, M.; Cavalier, J.-F.; Canaan, S.; et al. Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities. Antibiotics 2024, 13, 300. https://doi.org/10.3390/antibiotics13040300

AMA Style

Di Maio A, Olleik H, Courvoisier-Dezord E, Guillier S, Neulat-Ripoll F, Haudecoeur R, Bolla J-M, Casanova M, Cavalier J-F, Canaan S, et al. Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities. Antibiotics. 2024; 13(4):300. https://doi.org/10.3390/antibiotics13040300

Chicago/Turabian Style

Di Maio, Attilio, Hamza Olleik, Elise Courvoisier-Dezord, Sophie Guillier, Fabienne Neulat-Ripoll, Romain Haudecoeur, Jean-Michel Bolla, Magali Casanova, Jean-François Cavalier, Stéphane Canaan, and et al. 2024. "Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities" Antibiotics 13, no. 4: 300. https://doi.org/10.3390/antibiotics13040300

APA Style

Di Maio, A., Olleik, H., Courvoisier-Dezord, E., Guillier, S., Neulat-Ripoll, F., Haudecoeur, R., Bolla, J.-M., Casanova, M., Cavalier, J.-F., Canaan, S., Pique, V., Charmasson, Y., Baydoun, E., Hijazi, A., Perrier, J., Maresca, M., & Robin, M. (2024). Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities. Antibiotics, 13(4), 300. https://doi.org/10.3390/antibiotics13040300

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