Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities

1.Introduction

The development of novel antibacterial molecules is a major need for the upcoming decades as there is an increasing emergence of multi-drug resistant bacterial strains. This is leading to an elevating mortality rate by infectious disease that can reach more than 10 billion of deaths by 2050 according to WHO. Among these strains, mycobacteria in particular M. tuberculosis, the pathogenic agent of tuberculosis, are still responsible in world wild of 10 million new cases by year and has killed almost 1.5 million patients in 2022. More alarming, the number of resistant and ultra-resistant strains 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 [1]. 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 of deadly infections, particularly in HIV-infected patients, but also in immunocompetent ones, affecting billions of patients and causing more than 1.5 million death per year [2,3,4,5].

Some plant natural 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 cancerology, dermatology, and infectiology [6,7,8]. 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 [9]. Despite some natural aurones such as cephalocerone [10,11] and hispidol [12] have demonstrated antimicrobial activity, aurones exhibiting natural substitution patterns did 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 [13]. Structurally, aurones are characterized by two 6-carbon rings (ring A and B) and a furanone-like third cycle (ring C). Overall, already described scaffold modifications mainly focused on the substitution at the B-ring scaffold, e.g., with the introduction of either ferrocene [14], 5-nitroimidazole [15] or quinolines [16] groups, with a global tendency to retain naturally present hydroxy groups at the A-ring (Figure 1).

In the present study, modifications of the A-ring were performed by substitution with an amino group combined with substitutions of the B-ring (Scheme 1), the 31 new aurone derivatives obtained (Table 1) being 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 (MIC) 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) (Table 2).

In this first screening, among the 31 aurone derivatives tested, compounds 10, 12, 15, 16, and 20 were found the more 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 MIC ranging from 3.12 to 50 µM. Similarly, compound 20 was active on all tested species with 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, compounds 12 and 15 found active on tested Gram positive bacteria and mycobacteria (with MIC ranging from 25 to 100 µM and from 50 to 100 µM, respectively) 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 with lowest MIC of 3.12, 12.5, 25 and 50 µM, respectively.

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 Table 3).

Figure 3. 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 48h 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 3. 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 48h 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).

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 found 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 lower MIC (Table 2)), compounds 10 and 20 gave the highest TI values (ranging from 54.1 to 151.4 and 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 1.6 to 18).

Based on antimicrobial activity and toxicity data, the 2 more active and safest compounds were identified as compounds 10 and 20 that were then tested on a larger panel of bacterial and fungal species in order to evaluate their spectrum of activity (Table 5).

he 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 food-born pathogens Listeria monocytogenes (MIC values of 3.12 and 6.25 µM for compounds 10 and 20, respectively), 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). 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. 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), similar MIC being obtained on two other Bacillus species, i.e. B. cereus and B. subtilis. On the opposite, C. perfringens, Enterococcus species, P. acnes, and S. pyogenes were found weakly sensitive to insensitive to compounds 10 and 20 with MIC values from 50 µM to > 100 µM. Compound 10 was found 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.

Compounds 10 and 20 were also found active on Gram negative bacteria, 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 sensitive with MIC ranging from 25 to 50 µM. Although P. aeruginosa was sensitive to compound 10 (MIC of 25 µM), it is 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 found 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 found 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 found 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 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 was found the less sensitive with 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 being found the safest in all cases.

3. Discussion

In the present study, 31 new aurone derivative compounds were synthetized and tested in term 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’, 4’ and 5’ positions. The first screening antimicrobial test performed on representative species of bacteria and fungi allowed 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 could indicate that benzyloxy substitution in 3’ is a key element in the activity of the compound. The same methodology could be used to compare compound 20 with other isopropyl compound such as 18, 19, 21 and 22. These four last mentioned compounds showed a weak or no activity against any pathogens. Similarly to compound 10, this could indicate that the 4’-isopropyl substitution is a far better alternative to other position and again that acetamido group is more effective than amino group in position 5. On the behalf of the results, it can be accepted that the acetamido groups is essential 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 4 of these compounds possess a ring substitution in position 3’ and 4’. Interestingly, benzyloxy-substituted aurones (i.e., 10 and 12) seems 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 compound 10 activity compared to 12, 15 and 16 activities, benzyloxy substitution must be considered more promising than phenyl ones. The other benzyloxy and phenyl-substituted aurones (i.e., 11, 13, 14 and 17) showed no activity. Isopropyl-substituted aurones also are an interesting option as shown by compound 20 which is similarly active as compound 10. Again only 4’-isopropyl aurones is active, 3’ and 2’ are inactive on the variety of pathogen 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, aurones 34 is also inactive and attests that the 5-acetamido substitution appears as 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 seems 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 structure similarity such as the 5-acetamido substitution. Out of all the compounds, the 3’-benzyloxy and 4’ benzyloxy were found as the most promising substitutions. Compared to previously described aurones active on various Gram positive bacteria but only two Gram negative (i.e. H. pylori or V. alginolyticus) [13], compounds 10 and 20 were found 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 [13] 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 plate using appropriate media and culture conditions (in term of temperature and aerobic/microaerobic/anaerobic condition) as previously described [13,17]. 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 [18,19]. 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-TG^OADC) 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-TG^OADC supplemented with 24 µg/mL D-panthothenate (Sigma-Aldrich). 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 [13,17,20,21]. For BSL-3 bacteria, the MIC of aurones was determined following the Clinical and Laboratory Standards Institut (CLSI) recommendations as previously described [22]. 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 [23,24]. 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 [17,21,25]. 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 11967, 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 assay, human cells grown on 25 cm² flasks were detached using trypsin-EDTA solution (from Thermofisher), 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 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 - 300MHz 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]. Combustion analyses were performed at the analysis facilities of Spectropole (https://fr-chimie.univ-amu.fr/spectropole) 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.

Figure 3. Route A and B used for the synthesis of 5-amino and 5-acetamido aurones.

Figure 3. Route A and B used for the synthesis of 5-amino and 5-acetamido aurones.

4.2.1. Synthesis route A

4.2.1.1. 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 add dropwise 6.6g of acetic anhydride (1.5 eq). When the addition is complete, heat the solution at 80° for 5h. Let the solution cool and filter it. Under pressure remove the solvent, dissolve the product in ethanol and hexane to obtain 5 g of 1a.

4.2.1.2. Synthesis of 2-chloro-1-{2-hydroxy-5-[(1-hydroxyethyl)amino]cyclohexyl}ethan-1-one (2a)

In a solution of 5g of 1a and 18 g of AlCl₃ dissolved in 30 ml of dichloromethane add dropwise at 0°, 3.5 eq of chloroacetyl chloride. When the addition is complete heat the solution up to 50° for 1h. Pour the mixture on ice and extract with ethyl acetate. Remove EtOAc under pressure to obtain 2a.

4.2.1.3. Synthesis of N-(3-oxo-2,3-dihydro-1-benzofuran-5-yl)acetamide (3a)

In a flask, add 2g of 2a and 1.5 eq of triethylamine in 20 ml of acetonitrile, let react at 25° for 12h. evaporate under pressure the solvent, extract with ethyl acetate. Remove EtOAc under pressure to obtain 3a.

4.2.1.4. Synthesis of substituted 5-acetamidoaurones

In a flask add 1mmol of 3a and 1mmol of the corresponding benzaldehyde, dissolved in 10 ml of Choline Chloride/ Urea. Ad 3 drop of 50% KOH solution. Heat at 80° for 2h. Add water and HCl, filter the precipitate, wash it with ether to obtain acetamido substituted aurones.

4.2.1.5. Synthesis of substituted 5-aminoaurone

10 mmol of acetamido aurones were added to a mixture of EtOH (20 mL) and conc. H2SO4 (5 mL). The solution was refluxed for 2h. 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

4.2.2.1. 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 add dropwise 26.5 g of acetic anhydride (3.5 eq). When the addition is complete, heat the solution at 80° for 5h. Let the solution cold and filter it. Under pressure remove the solvent, dissolve the product in ethanol and hexane to obtain 5 g of 1b.

4.2.2.2. Synthesis of N-(3-acetyl-4-hydroxyphenyl)acetamide (2b)

To a solution of 1b (5g, 26 mmol), is added 15g of AlCl₃ (113 mmol, 4 eq) and 1.9 g of KCl (26 mmol, 1eq). The mixture is then heated a 165° for 1h until a brown paste appear. Let it cool, then ad cold water (300 ml), obtention of 2g of a beige powder (2b).

4.2.2.3. 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) are dissolved in ethanol (20 ml). The mixture is heated for 2h at 90°. The solvent is then removed under pressure, cold water and HCl are added, and the precipitate is filtered to obtain the desired chalcone (3b).

4.2.2.4. Synthesis of substituted 5-acetamidoaurones

To a mixture of chosen 3b chalcone in pyridine (20 ml), is added 1 eq of mercury acetate. The solution is heated for 1h bat 110°. Water and HCl are added. The precipitate was filtrated and washed several times with cold water to get rid of the mercury. Obtention of a powder.

4.2.2.5. Synthesis of substituted 5-aminoaurone

For synthesis of 5-aminoaurone see section 2.2.1.5.

(Z)-N-(2-(2’-methoxybenzylidene)-3-oxo-2,3-dihydrobenzofuran-5-yl)acetamide (4):

Yield: 83%; mp: 234.8 °C; ¹H NMR (300MHz, DMSO-d₆): δ 10.15 (s, 1H, NH), 8.20-8.18 (dd, 1H, J=1.2;7.8 Hz, C-H_6’), 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-H_4’), 7.19 (s, 1H, C-H₁₀), 7.16-7.09 (m, 2H, C-H_3’,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 (300MHz, 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-H_2’,4’), 7.54-7.51 (d, 1H, J=8.9 Hz, C-H₇), 7.43 (dt, 1H, J=8.0 Hz, C-H_5’), 7.06-7.03 (dd, 1H, J=2.6;8.2 Hz, C-H_6’), 6.90 (s, 1H, C-H₁₀), 7.16-7.09 (m, 2H, C-H_3’,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 (300MHz, 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-H_2’,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-H_3’,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 (300MHz, DMSO-d₆): δ 8.18-8.16 (dd, 1H, J=1.6;7.8 Hz, C-H_6’), 7.43 (dt, 1H, J=1.5;8.5 Hz, C-H_4’), 7.26-7.23 (d, 1H, J=8.8 Hz, C-H₇), 7.14-7.11 (m, 2H, C-H_3’,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 (300MHz, DMSO-d₆): δ 7.59-7.57 (d, 1H, J=7.7 Hz, C-H_6’), 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-H_5’), 7.38-7.35 (dd, 1H, J=2.2;8.8 Hz, C-H₆), 7.24 (d, 1H, J=2.11 Hz, C-H_2’), 7.06-7.03 (dd, 1H, J=1.9;8.1 Hz, C-H_4’), 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 (300MHz, DMSO-d₆): δ 7.95-7.92 (d, 2H, J=8.1 Hz, C-H_2’), 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-H_3’), 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 (300MHz, 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-H_2’), 7.58-7.32 (m, 8H,), 7.14-7.11 (dd, 1H, J=7.9 Hz, C-H_4’), 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 (300MHz, 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-H_2’), 7.82-7.79 (dd, 1H, J=2.2;8.9 Hz, C-H₆), 7.50-7.34 (m, 6H, C-H_{7, bn}), 7.16-7.13 (d, 2H, J=8.8 Hz, C-H_3’), 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 (300MHz, DMSO-d₆): δ 7.61 (bs, 1H, C-H_2’), 7.55-7.53 (d, 1H, J=8.8 Hz, C-H₇), 7.50-7.48 (d, 2H, C-H_2bn), 7.41 (dt, 2H, C-H_3bn), 7.36-7.33 (m, 2H, C-H_5’,4bn), 7.11-7.09 (dd, 1H, J=7.9 Hz, C-H₆), 7.06-7.04 (dd, 1H, J=7.9 Hz, C-H_4’), 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 (300MHz, DMSO-d₆): δ 7.97-7.95 (d, 2H,C-H_2’-6’ J=8.8 Hz), 7.49-7.47 (m, 3H, C-H), 7.41-7.39 (m, 4H, C-H_bn), 7.28 (s, 1H, C-H₄), 7.17-7.15 (d, 2H, C-H_3’-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%). (r

(Z)-N-(3-oxo-2-(3-phenoxybenzylidene)-2,3-dihydrobenzofuran-5-yl)acetamide (14) :

Yield: 96%; mp: 196.4 °C; ¹H NMR (300MHz, 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-H_6’), 7.67 (bs, 1H, C-H_2’), 7.51 (dt, 1H, J=7.4 Hz, C-H_5’), 7.44-7.41 (m, 3H, C-H_{4’, 8’}), 7.20 (t, 1H, J=7.4 Hz, C-H_10’), 7.10-7.07 (d, 3H, C-H_{9’, 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 (300MHz, 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-H_2’), 7.83-7.79 (dd, 1H, J=2.2;8.9 Hz, C-H₆), 7.50-7.42 (m, 3H, C-H_{7, 7’}), 7.22 (t, 1H, J=7.4 Hz, C-H_8’), 7.12-7.09 (m, 4H, C-H_3’,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 (300MHz, DMSO-d₆): δ 7.71-7.69 (d, 1H, J=7.9 Hz, C-H_6’), 7.64 (bs, 1H, C-H_2’), 7.51-7.49 (d, 1H, J=7.9 Hz, C-H₆), 7.44 (dt, 2H, C-H_9’), 7.22-7.15 (m, 2H, C-H_4,5’), 7.09-7.04 (m, 4H, C-H_7,9’,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 (300MHz, DMSO-d₆): δ 7.99-7.96 (d, 2H, J=8.6 Hz, C-H_3’), 7.44 (dt, 2H, J=7.7 Hz, C-H_7’), 7.24-7.21 (m, 2H, C-H_7,8’), 7.10-7.02 (m, 5H, C-H_6,2’,6’), 6.83 (bs, 2H, C-H_4,10), 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 (300MHz, DMSO-d₆): δ 10.15 (s, 1H, NH), 8.21-8.19 (d, 1H, J=1.2;7.8 Hz, C-H_6’), 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-H_4’), 7.20 (s, 1H, C-H₁₀), 7.16-7.13 (d, 1H, J=8.3 Hz, C-H_3’), 7.08 (dt, 1H, J=7.6 Hz, C-H_5’), 4.75 (q, 1H, J=5.9;11.9 Hz, C-H_isop), 2.07 (s, 3H, NHCOCH₃), 1.35-1.33 (d, 6H, J=5.8 Hz, C-H3_isop). 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: 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 (300MHz, 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-H_2’,4’,7), 7.40 (dt, 1H, J=8.0 Hz, C-H_5’), 7.04-7.01 (dd, 1H, J=2.6;8.2 Hz, C-H_6’), 6.91 (s, 1H, C-H₁₀), 4.68 (q, 1H, J=5.9;11.9 Hz, C-H_isop), 2.07 (s, 3H, NHCOCH₃), 1.31-1.29 (d, 6H, J=5.8 Hz, C-H3_isop). 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: 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 (300MHz, 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-H_2’), 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-H_3’), 6.90 (s, 1H, C-H₁₀), 4.72 (q, 1H, J=5.9;11.9 Hz, C-H_isop), 2.07 (s, 3H, NHCOCH₃), 1.30-1.28 (d, 6H, J=5.8 Hz, C-H3_isop). 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: 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 (300MHz, DMSO-d₆): δ 8.19-8.17 (d, 1H, J=6.9 Hz, C-H_6’), 7.39 (t, 1H, J=7.3 Hz, C-H_4’), 7.26-7.24 (d, 1H, J=8.7 Hz, C-H₇), 7.14 (d, 1H, C-H_3’), 7.11 (bs, 2H, NH2), 7.08-7.06 (d, 1H, C-H_5’), 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-H_isop), 1.34-1.32 (d, 6H, J=5.8 Hz, C-H3_isop). 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: 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 (300MHz, DMSO-d₆): δ 7.57-7.54 (d, 1H, J=7.8 Hz, C-H_6’), 7.51 (bs , 1H, C-H_2’), 7.51-7.48 (d, 1H, J=8.6 Hz, C-H₇), 7.43-7.40 (d, 2H, J=7.8 Hz, C-H_4’), 7.41 (dt, 1H, J=7.8 Hz, C-H_5’), 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-H_isop), 1.31-1.29 (d, 6H, J=5.8 Hz, C-H3_isop). 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 (300MHz, DMSO-d₆): δ 7.97-7.95 (d, 2H, J=8.8 Hz, C-H_2’), 7.66-7.64 (m, 3H, C-H_4,6,7), 7.07-7.05 (d, 2H, J=8.8 Hz, C-H_3’), 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 (300MHz, DMSO-d₆): δ 10.17 (s, 1h, NH), 8.25 (t, 1H, J=7.8 Hz, C-H_2’), 8.12 (d, 1H, J=2.2 Hz, C-H₄), 7.85-7.82 (dd, 1H, J=2.5, 8.7 Hz, C-H_6’), 7.54-7.51 (m, 2H, C-H_4’,7), 7.41-7.39 (d, 1H, C-H_3’), 7.35 (dt, 1H, C-H_5’), 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=260Hz), 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 (300MHz, 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-H_6,2’,6’), 7.59-7.51 (m, 2H, C-H_4’,7), 7.31-7.39 (dt, 1H, J=2.1, 8.4 Hz, C-H_5’), 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 (300MHz, DMSO-d₆): δ 8.23 (dt, 1H, J=1.65, 7.8 Hz, C-H_6’), 7.51-7.47 (m, 1H, C-H_4’), 7.37 (t, 1H, C-H_3’), 7.34 (dt, 1H, C-H_5’), 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=2.4Hz, 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= 12Hz), 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 (300MHz, DMSO-d₆): δ 7.80-7.78 (m, 2H, C-H_2’,4’), 7.57-7.50 (dt, 1H, C-H_5’), 7.32-7.27 (m, 2H, C-H_7,6’), 7.10-7.07 (dd, 1H, J=2.5, 8.7 Hz, C-H₆), 6.87 (d, 1H, J=2.4Hz, 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=244Hz), 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 (300MHz, DMSO-d₆): δ 8.09-8.04 (dd, 2H, J=7.8 Hz, C-H_2’), 7.81-7. 78 (d, 1H, C-H_4’), 7.57-7.54 (d, 1H, J=8.4 Hz, C-H₇), 7.36 (t, 2H, C-H_3’), 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=250Hz), 145.91 (C-2), 137.62 (C-5), 133.73-133.62 (C-2', J=9Hz), 128.55 (C-1', J=3Hz), 124.24 (C-6), 123.95 (C-7), 120.81 (C-9), 116.26-115.97 (C-3', J=22Hz), 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 (300MHz, DMSO-d₆): δ 10.20 (bs, 1H, NH), 8.30-8.28 (m, 2H, C-H_2’,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-H_5’,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 (300MHz, DMSO-d₆): δ 8.31-8.29 (m, 2H, C-H_2’,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=250Hz), 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 (300MHz, 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-H_2’), 8.05-8.02 (d, 2H, J=8.8 Hz, C-H_3’), 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 (300MHz, 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-H_2”), 7.56 (t, 1H, J=8.16 Hz, C-H_5’), 7.42 (dt, 2H, C-H_3”), 7.20-7.17 (d, 1H, J=7.3 Hz, C-H_4’), 7.14 (s, 1H, C-H_2’), 7.09-7.06 (m, 2H, C-H_{10, 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 (300MHz, 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-H_2’), 7.40-7.38 (d, 1H, J=7.8 Hz, C-H_6’), 7.30 (t, 1H, J=7.8 Hz, C-H_5’), 6.89-6.86 (dd, 1H, C-H_4’), 6.82 (s, 1H, C-H₁₀), 2.07 (s, 3H, C-H_3’). 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 (300MHz, DMSO-d₆): δ 9.63 (bs, 1H, OH), 7.39 (d, 1H, C-H_2’), 7.37-7.34 (d, 1H, J=7.8 Hz, C-H_6’), 7.28 (t, 1H, J=7.8 Hz, C-H_5’), 7.25-7.22 (d, J=8.8 Hz, C-H₇), 7.07-7.04 (dd, 1H, J=2.2;8.5 Hz, C-H_4’), 6.86-6.83 (m, 2H, C-H_4,6), 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’, 4’ and 5’ 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 and also the safest regarding human cells. The two selected compound shared some structure similarity such as the 5-acetamido substitution. The SAR study from this work correlates the previous one of Olleik and al [13] showing that benzyloxy and isopropyloxy gives some interesting activity to aurone scaffold with substitution on A ring with amino or acetamido group improving the activity compared to 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 ring all together.

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.