Among the medicinal chemistry strategies for developing new molecules, the concept of molecular juxtaposition is currently one of the most rapidly growing strategies. It involves combining two or more biologically active entities to obtain new molecules with high anti-infective potential. The application of this concept has led to the development of numerous drug molecules such as trioxaquines (antimalarials), vancomycins (antibiotics), etc. This so-called “dual-shot” strategy was developed with the aim of reducing the emergence of drug-resistant pathogens [12][13]. Having proven its effectiveness, we have adopted this research method in our work to discover new molecules with anti-infective properties. Thus, we have designed a hybrid chemical profile resulting from the judicious combination of the benzimidazole heterocycle and the styryl functional chain. The choice of benzimidazole is justified by its role as the pharmacophore vector in several drugs used in therapy. Furthermore, it possesses numerous anti-infective activities, including anthelmintic, antibacterial, antiviral, and antifungal properties [14]-[17]. As for the styryl chain or aryl α,β-ethylene group, it is a functional chain that acts as a modulator of biological activities, particularly anti-infective ones (antibacterial, antifungal, antiparasitic, and antiviral) [18]-[21]. It is characterized by the ability of the π electrons of the ethylene bond to conjugate with those of the aryl ring, especially when the latter bears electron-withdrawing or electron-donating groups. This is why we have undertaken our research work around the benzimidazole nucleus and the styryl chain, due to their multiple pharmacological potentials, particularly antifungal. Thus, by applying the medicinal chemistry concepts of juxtaposing bioactive entities, it seemed logical to us to design a new chemical profile combining the styryl and benzimidazole groups. These styryl derivatives could themselves possess potential antifungal activities, provided they are judiciously attached to the benzimidazole pharmacophore vector of Chlormidazole [22] (Figure 1).

Figure 1. Design of 2-styrylbenzimidazoles.

The synthesis of the 2-styrylbenzimidazoles was carried out in two main steps: the construction of 2-methylbenzimidazole and its condensation with aldehydes to yield the 2-styrylbenzimidazoles. The construction of 2-methylbenzimidazole required the prior synthesis of ortho-phenylenediamine via the reduction of 2-nitroaniline. This reduction was performed according to the method described by Ramaiah and colleagues [23]. It involves treating 2-nitroaniline with zinc powder in methanol in the presence of ammonium chloride. After cooling, the reaction mixture is filtered to remove insoluble impurities, and the filtrate is evaporated to dryness using a rotary evaporator. The crude ortho-phenylenediamine obtained from the reduction was used without purification in a condensation reaction with acetic acid in the presence of diluted hydrochloric acid (4N) under reflux for 5 hours. After cooling, the reaction mixture was neutralized with an ammonium hydroxide (NH₄OH) solution. The 2-methylbenzimidazole was isolated and purified by recrystallization in water. This compound was then subjected to a solvent-free condensation reaction with benzaldehyde or its derivatives, following a Knoevenagel-type reaction [24]. The reaction mixture was heated to between 170 and 200˚C for 3 hours. After cooling, the resulting solid was dissolved in 2-propanol, and the target 2-styrylbenzimidazole was obtained by adding an oxalic acid solution followed by neutralization with ammonia.

Synthesis Procedure forortho-Phenylenediamine

In a round-bottom flask containing 2-nitroaniline (a) (7.2 mmol), zinc powder (144.9 mmol), ammonium chloride (36.23 mmol), and 20 ml of methanol were added (Figure 2). The reaction mixture was heated under reflux for 6 hours. After cooling, the mixture was filtered, and the filtrate was evaporated to dryness using a rotary evaporator. The resulting ortho-phenylenediamine (b) was used without further purification in the subsequent reaction step.

Figure 2. Reduction of 2-nitroaniline to ortho-phenylenediamine.

Procedure for the synthesis of 2-methylbenzimidazole

The ortho-phenylenediamine (b) (46 mmol) obtained from the previous synthesis was dissolved in 50 mL of diluted (4N) HCl in a 150 mL round-bottom flask. Acetic acid (c) (64 mmol) was added to this solution. The reaction mixture was brought to reflux for 5 hours (Figure 3). Subsequently, the mixture was cooled, and the pH of the solution was adjusted to 8 using ammonia. The resulting solid was filtered under vacuum and recrystallized in 50 mL of distilled water to yield the expected 2-methylbenzimidazole (d) with an 87% yield.

Figure 3. Synthesis of 2-methylbenzimidazole.

Procedure for the synthesis of 2-styrylbenzimidazoles

In a round-bottom flask, 0.5 g of 2-methylbenzimidazole (d) (3.8 mmol) and 3.8 mmol of benzaldehyde or its derivative (e) were added. The reaction mixture was heated without solvent at a temperature between 170˚C and 200˚C for 3 hours (Figure 4). After cooling, the resulting brown mass was dissolved in 10 mL of isopropanol. To this solution, 0.56 g of oxalic acid in 5 mL of isopropanol was added while stirring. The precipitated solid was filtered under vacuum and washed with ice-cold water. This precipitate was then suspended in ice-cold water, and the resulting suspension was treated with aqueous ammonia (NH₄OH) to adjust the pH to between 8 and 10. The solid obtained was filtered, washed with hexane, and dried to yield the expected 2-styrylbenzimidazole derivatives (1-10).

Figure 4. Synthesis of 2-styrylbenzimidazoles.

The antifungal activity of 2-styrylbenzimidazoles was evaluated using the microplate dilution method against the clinical strain Candida glabrata (U3538) of vaginal origin from the University Hospital Center (CHU) of Angré (Ivory Coast).

The antifungal susceptibility test performed on the clinical strain of Candida glabrata showed sensitivity to fluconazole, nystatin, and amphotericin B, and resistance to econazole and miconazole. As fluconazole is the most commonly used molecule in the treatment of fungal infections in our country, we chose it as the reference molecule in our study. The microplate dilution method involves exposing a Candida inoculum to serial dilutions of the test compound in 96-well microplates (12 rows of 8 wells). The inhibition of fungal growth is assessed by colorimetric detection of dehydrogenase activity in the yeast cells using Methyl Thiazolyl Tetrazolium chloride (MTT). This (purple) coloration originates from the activity of mitochondria in viable, metabolically active cells. The MIC (Minimum Inhibitory Concentration) is determined as the lowest concentration that prevents the change from the yellow color of MTT [25]-[30].

Preparation of the inoculum

The preparation of the inoculum began by subculturing the Candida strain on Sabouraud glucose agar (Sabouraud 4% glucose agar, Fluka®) in a Petri dish. After 24 hours of incubation at 37˚C, one to three colonies were inoculated into 50 mL of Tryptone Soya Broth (OXOID®) and left at room temperature for 12 - 24 hours to yield broth1. Then, 5 mL of broth1 was transferred into 25 mL of Tryptone Soya Broth (OXOID®) and placed under agitation for 6 hours (the time required to reach exponential growth of Candida) to yield broth2. At the time of the test, 2.5 mL of broth2 was added to 25 mL of Tryptone Soya Broth (OXOID®) to obtain an inoculum containing approximately 10⁵ cells/mL [25]-[30]. The colony-forming unit count in the inoculum was confirmed using a Malassez counting chamber.

Determination ofminimum inhibitory concentrations

The test was performed using 96-well microplates. Stock solutions of the different compounds were prepared in Dimethyl Sulfoxide (DMSO) at a concentration of 1 mg/mL and then diluted with Tryptone Soya Broth (OXOID®) to obtain solutions concentrated at 100 µg/mL. 100 µL of this dilution was deposited into the wells of the first row. Then, 50 µL of Tryptone Soya Broth (OXOID®) was distributed into all other wells. A serial dilution was performed using 50 µL from the first row’s wells. 50 µL of broth3 containing the inoculum was distributed into all wells. The plates were incubated at 30˚C for 48 hours. For the revelation of the prepared microplates, 40 µL of an aqueous solution of Methyl Thiazolyl Tetrazolium chloride (MTT) at a concentration of 2.5 mg/mL was distributed into the wells and incubated for an additional 2 hours at room temperature. The aqueous MTT solution is yellow in color. Wells containing still viable cells turn purple due to the dehydrogenase activity of the mitochondria. The MIC determination, which is the lowest concentration at which no color change was observed, was made visually. [25]-[30]. All samples were tested in duplicate, and the tests were repeated twice. The minimum inhibitory concentrations, initially expressed in µg/mL (mass/volume), were converted to µM (number of molecules/volume). This conversion is justified because two molecules with the same MIC in µg/mL can exhibit very different biological activities if their molar masses differ. Conversion to µM normalizes the data in terms of the number of active molecules, providing a more accurate relative potency for each compound.

Chemistry

The synthesis of the 2-styrylbenzimidazole derivatives was carried out using established methods, yielding two intermediates and ten final compounds. The characterization of the chemical structures of the various synthesized derivatives has been described in a previous article [31].

Antifungalactivity

The Minimum Inhibitory Concentrations of the 2-styrylbenzimidazole derivatives against the clinical strain of Candida glabrata are summarized in Table 1 below.

Table 1. Activity of 2-styrylbenzimidazoles against the clinical strain of Candida glabrata.

These results indicate that the 2-styrylbenzimidazoles exhibit anticandidal activity against the clinical strain of Candida glabrata used. Compound 1 (unsubstituted 2-styrylbenzimidazole) and compound 8 (2-nitro derivative) are the least active derivatives against Candida glabrata, with MICs of 56.74 µM and 47.12 µM, respectively. Compounds 2 (benzenic ring para-methyl derivative), 3 (para-hydroxy), 4 (para-dimethylamino), 5 (para-fluoro), 7 (2,4-dichloro), 9 (2-cyano), and 10 (pyridinyl derivative) induced antifungal activities close to that of Fluconazole, with MICs between 20 µM and 30 µM. Compound 6 (2-chloro derivative) demonstrated the best anticandidal efficacy, with an MIC of 12.26 µM.

Following the evaluation of the antifungal activities of our 2-styrylbenzimidazole derivatives against Candida glabrata, we undertook a structure-activity relationship (SAR) discussion. The objective is to establish a correlation between the structure and antifungal activities in order to identify the structural elements that contribute to inducing, or even enhancing, the expected antifungal effects. In this discussion, we use the minimum inhibitory concentration (MIC) to quantify the anticandidal efficacy of each compound; that is, a compound is considered more effective if its MIC is lower. According to the results, it is evident that 2-styrylbenzimidazoles can inhibit Candida strains, particularly Candida glabrata. These results once again confirm the antifungal properties of the benzimidazole nucleus, as reported by Wolley in 1944 [22]. Furthermore, the results reveal that the efficacy of these 2-styrylbenzimidazole derivatives varies depending on the nature of the substituent on the benzenic ring. Therefore, our discussion aims to compare the antifungal efficacy of the different molecules based on the nature of the substituent on the benzenic ring. Analysis of the obtained results established that linking the benzimidazole heterocycle to the styryl functional group at position 2 yields compound 1, which possesses anticandidal activity against C. glabrata with an MIC of 56.74 µM. The presence of the styryl group at position 2 of the benzimidazole appears favorable for the emergence and maintenance of antifungal activities. Given this result, various structural modifications were undertaken around the benzenic ring of compound 1 to improve its activity. These modifications allowed us to establish that: Introducing a small alkyl radical like a methyl group at position 4 (compound 2) leads to an improvement in antifungal activity, with an MIC of 26.67 µM. This performance is twice that of compound 1 (MIC = 56.74 µM) and is on the same order of magnitude as the reference drug, Fluconazole (MIC = 20.40 µM). This result with the methyl group could be partially explained by the greater lipophilicity imparted by the methyl group compared to the hydrogen atom. Replacing the methyl with a hydroxyl group (compound 3) maintains the anticandidal efficacy, with an MIC of 26.45 µM. This efficacy, equivalent to compound 2, could be explained by the ability of the hydroxyl group to form additional hydrogen bonds with the biological target. Furthermore, the hydroxyl group at position 4 was replaced by a more electron-donating dimethylamino group (compound 4) to verify the impact of this effect. The antifungal result of compound 4 (MIC = 23.73 µM) shows that the dimethylamino group leads to a slight improvement in antifungal activity, thus confirming the benefit of electron-donating groups in enhancing antifungal activities. Such modulation of antifungal activities by electron-donating groups appears favorable for maintaining these anticandidal activities. Replacing the hydroxyl group with a halogen atom at position 4, specifically fluorine (compound 5), results in the same efficacy observed with compounds 3 and 4, with an MIC of 26.23 µM. The presence of the fluorine atom, considered a modulator of the anticandidal performance of Fluconazole in the conazole series [32], does not appear to be relevant in the 2-styrylbenzimidazole series. In contrast, introducing a chlorine atom at position 2 (compound 6) leads to potent anticandidal activity with an MIC of 12.26 µM. This 2-chloro derivative exhibited the best anticandidal performance in the chemical series of 2-styrylbenzimidazoles, with efficacy superior to the reference drug, Fluconazole (MIC = 20.40 µM). Such performance confirms the ability of chlorine atoms to improve anticandidal activities, as reported with first-generation conazoles [32]. Duplicating the chlorine atom at positions 2 and 4 (compound 7) did not improve the anticandidal performance. Indeed, compound 7, with an MIC of 21.61 µM, proved less effective than its monochloro analogue; however, this performance is comparable to that of the reference drug, Fluconazole (MIC = 20.40 µM). Ultimately, the modulation of antifungal performance by halogen atoms proves more judicious with a chlorine atom at position 2 of the benzenic ring of the 2-styrylbenzimidazoles. Replacing the chlorine atom at position 2 with an electron-withdrawing nitro group yields compound 8. The latter, with an MIC of 47.12 µM, exhibited efficacy nearly equivalent to the unsubstituted compound 1 and lower than that of the other derivatives obtained by chemical modulation of the benzenic ring. The nitro group, known in medicinal chemistry as a modulator of anti-infective activities in the 5-nitroimidazole series and analogues [33], does not appear conducive to enhancing the expected antifungal activities. Substituting the nitro group at position 2 with another electron-withdrawing group, a nitrile (compound 9), leads to a slight improvement in antifungal activities, with an MIC of 25.48 µM. This performance is superior to that of the unsubstituted compound 1 and comparable to that of the 4-methyl, 4-hydroxy, and 4-fluoro derivatives. Replacing the benzenic ring of the unsubstituted compound 1 with a hexagonal heteroaryl group like 2-pyridinyl (compound 10) maintains the expected antifungal activities, with an MIC of 28.24 µM. While this is slightly lower than that of the reference drug, Fluconazole (MIC = 20.40 µM), it remains twice as high as that obtained with the unsubstituted compound 1 (MIC = 56.74 µM). Such structural variation using heterocycles appears favorable for a slight increase in anticandidal activities.