Synthesis of the title compounds was accomplished as outlined in Figure 2.

Figure 2. Way of general synthesis of compounds.

All of the chemicals used in the synthesis were purchased from Sigma-Aldrich and were used as such. Thin layer chromatography was used to monitor the progress of the reactions. Melting points were determined in capillary tube using an MPD Mitamura Riken Kogyo (Japan) electrothermal melting point apparatus and are uncorrected. IR spectra in the range 4000 - 400 cm⁻¹ were obtained on a Bruker-Vector FTIR spectrophotometer, with samples investigated as thin film from CDCl₃ solution. ¹H NMR spectra were recorded on a Bruker-Avance-300 spectrometer, operating at 300 MHz. Mass spectra were recorded on a TOF LCT Premier (WATERS) spectrometer coupled to an HPLC Alliance 2695 chain.

4-nitrobenzaldehyde (0.8 mmol) and hexane-1,6-diamine (0.4 mmol) were dissolved in ether (30 ml). At room temperature, the mixture was stirred for three days to give a white precipitate. The precipitate obtained was filtered and recrystallized in methanol. Rf: 0.81 in hexane/acetone (50:50), yield: 86.26%; IR (thin film from CDCl₃ solution, cm⁻¹): 2853; 2819; 1643; 1535; 850; 1H NMR (300 MHz, MeOD): 8.50 (s, 2H), 7.98-8.30 (m, 8H), 1.39-3.64 (m, 12H); 13C NMR (75 MHz, MeOD): 155.92, 146.44, 139.27, 126.14, 129.06, 121.33, 59.33, 28.12, 24.65; ESI-HR-MS: peak at m/z383.1741 [M + H]+ corresponding to C₂₀H₂₂N₄O₄.

In a 250 mL flask, 0.8 mmol of 4-nitrobenzaldehyde and 0.4 mmol of benzene-1,3-diamine were introduced in 30 mL of ether. At room temperature, the mixture was stirred for three days to give a burgundy-red precipitate. The resulting precipitate was washed and recrystallized in ethanol Rf: 0.74 in benzene/acetone (50; 50), yield: 80.25%; IR (Thin film from CDCl₃ solution, cm⁻¹): 2987; 2903; 1660; 1550; ¹H NMR (300 MHz, DMSO): 8.90 (s, 2H), 7.30 (dd, 2H), 7.53 (dd, 1H), 7.35 (t, 1H), 8.25 (m, 8H); ¹³C NMR (75 MHz, DMSO): 159.67, 151.63, 148.93, 141.40, 130.16, 129.73, 124.07, 120.13, 113.44; ESI-HR-MS: peak at m/z383.1741 [M + H]+ corresponding to C₂₀H₂₀N₄O₄.

4-Nitrobenzaldehyde (0.8 mmol) and cyclohexane-1,2-diamine (0.4 mmol) were dissolved in diethyl ether (30 mL). At room temperature, the mixture was stirred for three days to give a pale yellow precipitate. The resulting precipitate was filtered, washed, and recrystallized in ethanol. Rf: 0.75 in hexane/acetone (50:50), yield: 83%; IR (Thin film from CDCl₃ solution, cm⁻¹): 2939; 2851; 1642; ¹H NMR (300 MHz, CDCl₃): 8.46 (s, 2H), 8.15 (d, 4H), 7.85 (d, 4H), 3.69 (m, 1H), 3.52 (m, 1H), 1.78 (m, 2H), 1.50 (m, 2H), 2.05 (m, 2H), 1.63 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): 158.57; 148.9; 141.60; 130.5; 124.25; 74.01; 30.89; 24.22 ; ESI-HR-MS: peak at m/z381.1522 [M + H]+ corresponding to C₂₀H₂₀N₄O₄.

We evaluated the antimicrobial activity of our tested compounds on seven microbial strains, and bacteriological tests were carried out at the laboratory of the Swiss Center for Scientific Research (CSRS) in Abidjan.

Stock solutions of the compound to be tested were prepared in dimethyl sulfoxide (DMSO) at a concentration of 30 mg/mL. These solutions were then diluted to a concentration of 1500 μg/mL to form stock solutions, which were stored in a refrigerator at 4˚C until use.

Furthermore, a stock solution of the control antibiotics was prepared at a concentration of 1 mg/mL from gentamicin and tetracycline powders. This solution was then diluted to obtain a final concentration of 25 μg/mL, used as a positive control in the antimicrobial activity tests.

After 18 hours of incubation, a well-isolated bacterial colony was collected using a Pasteur pipette and suspended in a tube containing 10 mL of sterile distilled water. Subsequently, 5 to 6 drops of this pre-culture were transferred to a second tube also containing 10 mL of distilled water to obtain the appropriate dilution. This suspension allows for a concentration estimated at approximately 10⁶ CFU/mL, corresponding to standard inoculation conditions. This suspension constitutes the bacterial inoculum used for the tests, corresponding to a 1:100 dilution [15].

Our compounds evaluated for their antimicrobial activity were dissolved in DMSO to prepare stock solutions at an initial concentration of 30 mg/mL. The solutions were then diluted to 1500 µg/mL and stored at 4˚C until use in microbiological testing.

To determine the activity of the pure compounds against the tested microbial strains, the well diffusion method on agar was used, in accordance with the protocols described by Bakkiyaraj and Pandiyaraj (2011) [16].

This method allowed us to screen the antibacterial activity of our pure compounds using a standardized device.

Petri dishes containing nutrient agar (or Sabouraud agar for Candida albicans) were surface-inoculated with bacterial inoculum standardized to 10⁶ CFU/mL.

Six-mm diameter wells were then made in the agar using a sterile punch and filled with a precise volume of the solution of the compound to be tested. The plates were incubated at 37˚C for 18 to 24 hours for bacteria and at 30˚C for yeasts.

Antimicrobial activity was assessed by measuring the diameter of the microbial growth inhibition zone around the wells, compared to those obtained with the control antibiotics (gentamicin and tetracycline at 25 µg/mL).

3.5.1. Principle

This method relies on the diffusion of the antimicrobial product in a solid medium (agar) contained in a Petri dish. After a certain contact time, a concentration gradient forms around the well containing the compound to be tested. The product gradually diffuses through the agar, interacting with the target microorganism inoculated onto the surface of the medium.

The efficacy of an antimicrobial compound is assessed by formation of a zone of microbial growth inhibition around the well. The diameter of this zone (expressed in millimeters) is measured after incubation and determines susceptibility of the microbial strain to the tested compound. Based on the size of this zone, the strain is classified as resistant, susceptible, highly susceptible, or extremely susceptible.

3.5.2. Experimental Protocol

Petri dishes containing Mueller-Hinton agar were used for antibacterial assays. Each dish was uniformly flood-inoculated with a suspension of microbial inoculum diluted 1:100 (approximately 10⁶ CFU/mL). After a few moments, the excess suspension was removed using a Pasteur pipette fitted with a bulb, and inoculated plates were left to dry at room temperature for 15 minutes. Under sterile conditions, wells were then made in the agar using a Pasteur pipette. A 50 μL sample of each substance to be tested was added to each well. The plates were incubated at 37˚C for 18 hours for bacterial strains in an incubator. This procedure was repeated three times to ensure reproducibility of the results. Gentamicin (25 μg/mL) was used as a positive control for bacteria, while amphotericin B was used for fungal strains (Candida albicans).

The results were read by measuring the diameter of the inhibition zone (in millimeters) around each well using a ruler. The results are expressed as the size of this inhibition zone [17].

In accordance with the criteria defined by Ponce et al. (2003) [17], the interpretation of the results is based on the following classification:

Non-sensitive or resistant: diameter < 8 mm.

Sensitive: 9 mm ≤ diameter ≤ 14 mm.

Very sensitive: 15 mm ≤ diameter ≤ 19 mm.

Extremely sensitive: diameter > 20 mm.

For MIC determination, the inoculum is prepared by taking 0.3 mL of an 18-hour culture of Staphylococcus aureus in broth and subculturing it in 10 mL of Mueller-Hinton broth. These new suspensions are incubated at 37˚C for 3 to 5 hours, until slight opalescence appears, equivalent to an optical density of 0.5 on the MacFarland scale. For the final inoculum, 1 mL of these cultures is added to 10 mL of Mueller-Hinton broth [18].

Each microplate contains 96 wells arranged in 8 rows of 12 columns each. The first and second columns of each microplate contain 100 µL of broth; these are used to monitor for potential contamination of the culture medium. The third column, containing 50 µL of broth, was used after inoculation to monitor the quality and growth of the bacterial strain. It is unaffected by the dilution of the pure compounds. The following 8 columns receive 50 µL of broth. The twelfth column is reserved for the pure compounds and the control antibiotics (gentamicin and tetracycline). For each pure compound (1500 µg/mL) and each antibiotic (25 µg/mL), 100 µL of solution are loaded in a double row. Next, using a precision multipipette, successive dilutions are performed applying a geometric progression with a ratio of 2, starting from the column containing the solutions to be tested. Finally, 50 µL of the inoculum are added to the contents of each well from the third to the twelfth column. The final volume (culture medium, inoculum and drugs) of all wells is 100 µL. The number of bacteria is estimated at 5.10⁴ per well, except in the first two columns, which were not inoculated. The experiment was repeated twice for each pure compound. The microplates were incubated at 37˚C for 18 hours, followed by a visual reading of 100 µL. The bacterial count was estimated at 5 × 10⁴ per well, except in the first two columns, which were not inoculated. The experiment was repeated twice for each pure compound. The microplates were incubated at 37˚C for 18 hours, followed by a visual reading.

3.7.1. Test with DiPhenyl-1-PicrylHydrazyl (DPPH)

2,2-diphenyl-1-picrylhydrazyl was one of the first free radicals used to study the structure-antioxidant activity relationship of phenolic compounds [19]-[21].

3.7.2. Principle

Reduction of free radical DPPH by an antioxidant can be followed by UV-Visible spectrometry, by measuring the decrease in absorbance at 517 nm caused by antioxidants [22]. In the presence of free radical traps, purple-colored DPPH is reduced to yellow 2,2-diphenyl-1-picrylhydrazine [23].

3.7.3. Dosage

DPPH radical trapping activity was measured according to the protocol described by Lopes-Lutz et al. [24] and Athamena et al. [22] 100 μL of each methanolic solution of the pure compound at different concentrations (3.125 - 100 mg/mL) was added to 2.5 mL of methanolic solution of DPPH (0.025 g/L). In parallel, a negative control was prepared by mixing 100 μL of methanol with 2.5 mL of methanolic solution of DPPH. Absorbance reading was made against a blank prepared for each concentration at 517 nm after 30 minutes of incubation in the dark and at room temperature. Positive control was represented by a solution of a standard antioxidant, ascorbic acid, whose absorbance was measured under the same conditions as the samples and for each concentration [25].

The results were expressed in inhibition percentages (I%) of free radicals using the following formula:

I% = [(Abs of con neg − Abs sample)/Abs of con neg] × 100

I%: Percentage of DPPH inhibition.

Abs Sample: Absorbance of the sample.

Abs of con neg: Absorbance of the negative control.

3.7.4. Statistical Analysis of the Results

For data analysis, STATISTICA 7.1 software [26].

-. was used. The influence of the activities of the different compounds was studied by comparing means using one-way analysis of variance (ANOVA).

When there is a significant difference between activities, multiple comparisons are performed using the smallest significant difference (SSD) test. This test allows us to identify the compound(s) that differ from each other. The significance of the test is determined by comparing the probability P associated with the Fisher F-test statistic at the theoretical threshold α = 5% [27].

Thus, when P > 5%, we conclude that there is no significant difference between the means, and when P < 5%, there is a significant difference between the means.

TheP values are read from a reference table after calculating the chi-square (χ²) value. χ² = Σ((o − e)²/e), where “o” corresponds to observed or actual data, while “e” corresponds to expected or theoretical data.