2.1 General procedures

¹H NMR (300 MHz) and ¹³C NMR (75 MHz) spectra were recorded on a Varian VXR 300 instrument at 293 K in CDCl₃ or DMSO- d₆. Spectra were internally referenced to TMS. Peaks are reported in ppm downfield of TMS. The melting points of compounds were determined in open glass capillaries in a paraffin bath and are uncorrected. Elemental microanalysis was obtained by the microanalytical laboratory services, university of Rennes 1 in France.

3-Acetyl-4-hydroxycoumarin (1) was synthesized using our previous work (Al-Ayed and Hamdi, 2014).

Preparation of coumarins derivatives 3 Fig. 1 (Supplementary material, SS1).

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Fig. 1

Synthesis of coumarins derivatives carbonitriles 3 catalyzed by NH₄OAc.

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2.2 Antibacterial activity tests

Antibacterial activities were tested against selected food-borne pathogens and clinical Gram-positive and Gram-negative bacteria strains obtained from local culture collection and International Culture Collections (ATCC). We have selected for this study Staphylococcus aureus (S. aureus) ATCC 6538, Micrococcus luteus (M. luteus) LB14110, Listeria monocytogenes (L. monocytogenes) ATCC 19117 Salmonella typhimurium (S. typhimurium) ATCC 14028 and Agrobacterium tumifaciens (A. tumifaciens).

Analysis of the activities of compounds against these selected bacteria was conducted using the Agar well diffusion method according to Guven et al. (2005) with some modification as reported by Al-Ayed and Hamdi (2014). Minimum inhibitory concentration (MIC) is defined as the lowest concentration of the tested compound for which the bacteria did not growth in the optimal condition of incubation. The procedures for MIC tests of the newly synthesized products were conducted in accordance with NCCLS guideline M7-A6 and M38-P (National Committee for Clinical Laboratory Standard, 2003) and detailed in Al-Ayed and Hamdi (2014).

2.3 Acetylcholinesterase inhibitory potential (AChEI)

AChEI was performed following the spectrophotometric procedures previous reported by Ellman et al. (1961). All obtained compounds were tested at concentration of 100 mg/ml. We have followed the same protocol reported in Al-Ayed and Hamdi (2014).

For all biological teste we have expressed the measurements in average ± standard deviation after triplicate assays.

3.1 Chemistry

Reaction between 4-hydroxycoumarin and phosphorus oxychloride (POCl₃) in glacial acetic acid, under reflux for 24 h afforded 3-acetyl-4-hydroxycoumarine 1 in excellent yield (90%) (Hamdi et al., 2011).

The IR spectra of compound 1 revealed a strong band at 3185 cm⁻¹ confirming the presence of OH group and showed band in the region of 1700 cm⁻¹ which is the characteristic for C⚌O of coumarin. The ¹H NMR data of compound 1 revealed signal at 2.72 ppm attributed to the methylic protons, the aromatic protons resoned between 7.1 and 7.98 pp. While hydroxylic proton (OH) resonated at δ 17.69 ppm.

The reaction of 3-acetyl-4-hydroxycoumarine 1 with ethyl cyanoacetate and benzaldehyde in was explored using different solvents (DMC, DEC, ethanol, H₂O, tetrahydrofuran (THF), dichloromethane and toluene). (Table 1, entries 1–7) in the presence of a catalytic amount of ammonium acetate. The results are summarized in Table 1.

From the obtained results we can say that solvents affected the yields (Table 1, entries 1–7), and the best result was obtained in excellent yield and high purity with DMC.

Then, we performed the reaction with different amounts of catalyst at ambient temperature (5 mol%, 10 mol%, 15 mol%, and 20 mol%) in order to optimize the ammonium acetate loading. The results are given in Table 2.

When we increase the percentage of the catalyst the obtained yields were also increased up, but after 20 mol% there is no improvement of the yield of the reaction.

In order to explore more this method, the cyclocondensation reaction with different series of substituted aromatic aldehydes was achieved. The results are given in (Table 3).

The scope of the method was assayed with a series of substituted aromatic aldehydes after optimization of the reaction conditions. The results are summarized in Table 3. As shown in Table 3, the aromatic aldehydes bearing both the electron withdrawing (inputs 2,3) and electron donor groups (1,5) were subjected to successful condensation with ethyl cyanoacetate and 4-hydroxycoumarin. In the presence of a catalytic amount of ammonium acetate in the refluxing DMC to give the corresponding products in convenient yields. It appears that the electronic effects and the nature of the substituents on the arylaldehyde ring have a small effect on the reaction yield and on the time required to complete the reaction. Electron donor groups somewhat increased the reactivity and provided higher yields compared to the electron-withdrawing groups. Remarkably, the reactions were clean, and all products were obtained after only filtration and simple washing with water and ethanol. Thus, a simple treatment gives the title products without need of chromatographic purification.

The structure of the new compounds 3 was identified by spectroscopic data and either by their elemental analysis. ¹³C NMR showed the amide (NH–CO) signal at δ 177.8 ppm, the C_1′ signal with a chemical shift of δ = 61.9 ppm, while the C₄ signal was assigned further upfield at δ = 96.47 ppm. However, the C₂ signal was further upfield, δ = 180.13 ppm, than was the C_4′ signal was assigned as δ = 134.48 ppm.

The proposed mechanism of compound 3 is given by the reaction sequence in Fig. 2. First, condensation of ammonium acetate with ethyl cyanoacetate is proposed to give intermediate (i), then 3-acetyl-4-hydroxycoumarine react with arylaldehydes to give intermediate (ii). The Michael addition of intermediate (i) with intermediate (ii) occurs to provide the intermediate (iii) which undergoes aromatization to form the target coumarins carbonitriles 3.

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Fig. 2

Proposed mechanism for the synthesis of compound 3.

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3.2 Biological activities