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3 March 2026

Synthesis, Characterization, and Bioactivity Investigation of Novel Benzimidazole Derivatives as Potential Antibacterial and Antifungal Agents

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1
Department of Chemistry, College of Science, Sultan Qaboos University, Al Khod P.O. Box 36, 123 Muscat, Oman
2
Department of Biology, College of Science, Sultan Qaboos University, Al Khod P.O. Box 36, 123 Muscat, Oman
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Author to whom correspondence should be addressed.
Molecules2026, 31(5), 844;https://doi.org/10.3390/molecules31050844
This article belongs to the Section Organic Chemistry
Version Notes

Abstract

A series of benzimidazole derivatives 6a–j was designed and synthesized via the condensation of the corresponding o-phenylenediamine intermediates with formic acid. Antibacterial activity was evaluated in vitro using the agar well diffusion method against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae, with nitrofurantoin (300 µg/mL) as the positive control. Antifungal screening was performed against Aspergillus flavus, Penicillium duclauxii, and P. italicum at 20 and 50 µg/mL, with Amphotericin B as the reference drug at the same concentrations. Most compounds exhibited moderate to good antimicrobial activity. MIC determination identified 6h as the most active antibacterial agent (MIC = 5.0 µg/mL). The SEM analysis of bacteria treated with 6h revealed marked morphological damage, including cell deformation and membrane disruption, supporting a bactericidal mode of action. Collectively, these results highlight benzimidazole derivatives as promising scaffolds for the development of broad-spectrum antibacterial and antifungal agents.

1. Introduction

Heterocyclic scaffolds remain a cornerstone to medicinal chemistry because they provide diverse functionality and well-defined three-dimensional structures that can be systematically optimized for potency and drug-like behavior [1,2,3]. Numerous active pharmaceutical ingredients incorporate one or more heterocycle moiety, which indicates the ability of nitrogen-containing heterocycles to engage with biological targets through hydrogen bonding, ionic interactions, π-stacking, and/or metal coordination. In parallel, the continued emergence of difficult-to-treat infectious diseases and the rapid growth of antimicrobial resistance have intensified the demand for new drugs with novel mechanisms and improved pharmacokinetic profiles [2,3,4,5].
Among privileged heterocycles, benzimidazole is the lead choice as a natural pharmacophore in drug discovery. Benzimidazole derivatives have been repeatedly validated as bioactive motifs through a broad spectrum of antimicrobial, antiparasitic, anti-inflammatory, and anticancer agents [6,7].
A major strategy to improve the potency of benzimidazoles is by carefully incorporating fluorine as a substituent. Fluorine has a special and unique interest in medicinal chemistry because it may lead to a powerful structural modification in heterocyclic systems. Its high ability to withdraw electron density and the polarized C–F bond can change how electrons are arranged locally, which may bring a significant enhancement of the bioassay of the molecules [8].
Another important design feature that helps improve the potency of drugs is the use of piperazine as a core moiety, which is often seen as a valuable part in drug design because it has several interesting features. It acts as a flexible linker that can connect different parts of the molecule, making it easier to position active parts of the drug. Moreover, it may also make changes to the drug’s properties like how it is absorbed, distributed, metabolized, and excreted [9,10].
The introduction of both a fluorine and piperazine moiety into heterocycles has been shown to be a successful strategy to enhance the potency of quinolone drugs [11,12,13]. The quinolone-like heterocycles are expected to be more effective and work against a wider range of bacteria [14,15]. We have reported several heterocyclic scaffolds bearing fluorine and piperazine substituents, and our findings consistently demonstrate that the biological activities are markedly enhanced when both motifs are present [16,17,18,19,20,21].
In parallel, the benzimidazole scaffold has remained a central focus, owing to its well-reported biological activities [6,22,23,24,25] and its significance as a naturally occurring motif, notably as the benzimidazole is an integral part of vitamin B12 [26,27,28,29].
These design tactics are very important in the development of new antibiotics, where resistance is the main challenge causing existing drugs to be less effective. Because of this, it is still very valuable to create new types of small molecules that can fight a wide range of microbes and work well against important disease-causing organisms. Benzimidazoles have several modes of actions, e.g., inhibiting enzymes [7,30,31], affecting DNA processes [32,33], or interfering with cell membranes [34,35].
Guided by these considerations, the present study aims at the synthesis, characterization and bioassay of some novel 5-fluoro-6-(4-substituted-piperazinyl) benzimidazoles (Figure 1).
Figure 1. The general structure of the current work.

2. Results and Discussion

2.1. Chemical Synthesis

The synthetic route banks on the use of commercially available 3-chloro-4-fluoroaniline SM and proceeds through a multi-step sequence (Scheme 1) including acylation, regioselective nitration, deacylation, piperazinylation, and a final reduction to afford the corresponding o-phenylenediamine intermediates 5a–j. The subsequent condensation of 5a–j with formic acid furnished the target benzimidazole derivatives 6a–j in moderate to good yields. 3-chloro-4-fluoroaniline SM was converted to the corresponding N-acetylated derivative 1 at a high yield using acetic anhydride at room temperature; the product was isolated and purified through a recrystallization from absolute ethanol. The regioselective nitration of N-(3-chloro-4-fluorophenyl) acetamide 1 was then carried out using nitric acid in the presence of concentrated sulfuric acid at 0 °C to afford N-(5-chloro-4-fluoro-2-nitrophenyl) acetamide 2 in good yield, which was purified through a recrystallization from absolute ethanol. It is worth mentioning that, if the temperature of the reaction mixture exceeded 5 °C during the addition of nitric acid, the yield will dramatically decrease. The subsequent deacetylation of N-(5-chloro-4-fluoro-2-nitrophenyl) acetamide 2 under reflux in absolute ethanol with concentrated hydrochloric acid gives 5-chloro-4-fluoro-2-nitroaniline 3. The nucleophilic aromatic substitution of intermediate 5-chloro-4-fluoro-2-nitroaniline 3 with the corresponding piperazine or other N-substituted nitrogen heterocycles was performed under reflux in DMSO to give the corresponding 4-fluoro-5-(4-N-substitutedheterocycle-1-yl)-2-nitroaniline 4a–j in good yields. The reduction of the nitro group in 4a–j using tin (II) chloride in concentrated hydrochloric acid cleanly provided the o-phenylenediamine intermediates 5a–j. Finally, the condensation of 5a–j with formic acid under reflux afforded the corresponding benzimidazoles 6a–j.
Scheme 1. General synthetic route to benzimidazole derivatives. Reagents and conditions: (i) Ac2O, RT, 1.5 h (ii) c. H2SO4, HNO3, 0 °C, 1 h (iii) c. HCl, EtOH, reflux 78 °C, 2 h (iv) DMSO, R, reflux 140 °C, 2 h (v) c. HCl, SnCl2, RT, H2O, 1 h, 40% NaOH (vi) 98% HCOOH, reflux 100 °C, 2 h, 10% NaOH.

2.2. Chemistry

All intermediates and final products were fully characterized using standard spectroscopic and spectrometric methods. The FT-IR spectra showed a N–H stretching frequency in the range of 3381–3418 cm−1 (secondary amine N–H [36], 3300–3500 cm−1) and a characteristic C=N stretching band near 1632–1636 cm−1, consistent with benzimidazole ring formation (aromatic heterocyclic C=N [37], 1620–1550 cm−1). Moreover, a strong broad C–F stretching band was observed around 1021–1237 cm−1 across the series (aromatic heterocyclic C–F [38], 1000–1360 cm−1). In the 1H NMR spectra, the benzimidazole C2–H proton (Figure 1), which is located at the sp2-hybridized carbon located between the two nitrogen atoms in the five-membered imidazole ring, appeared as a singlet at approximately δ 8.12 ppm. In line with this task, the relevant 13C NMR signal for C2 resonance was observed around δ 152 ppm in the 13C NMR, confirming the successful cyclization and construction of the benzimidazole ring. Aromatic protons exhibited the expected coupling to fluorine, the ortho-coupled doublet around δ 7.40 ppm (J ≈ 12.0 Hz) and a meta-coupled doublet around δ 7.16 ppm (J ≈ 8.2 Hz), which were consistent throughout the series. Finally, mass spectrometric analyses provided molecular ion peaks in agreement with the calculated molecular masses, further supporting the assigned structures and indicating the high purity of the synthesized compounds.

2.3. Biological Activities

2.3.1. Antibacterial Activity

The agar diffusion method was used to test the synthesized compounds in vitro as antimicrobial agents against Gram-positive bacteria (B. cereus/S. aureus) and Gram-negative bacteria (E. coli/K. pneumoniae). The inhibition zone in mm of the studied compounds was compared with Nitrofurantoin (300 µg/mL) as a reference for all bacteria strains, while DMSO was used as a negative control. The antibacterial screening results of compounds are summarized in Table 1 and Figure 2 and Figure 3. All the synthesized derivatives exhibited moderate to good antibacterial activity, with a variation in potency depending on the nature of the substituent at the N-1 position of the benzimidazole nucleus. At a concentration of 10 µg/mL, compounds 6e, 6f, 6h, and 6j showed comparatively larger inhibitory zones, ranging between 13 and 16 mm, particularly against B. cereus, S. aureus, and E. coli. Among these, compound 6f displayed a broad-spectrum activity, with inhibition zones of 15 mm against S. aureus and B. cereus and 11 mm against E. coli. Similarly, 6e showed potent inhibition (15 mm) against K. pneumoniae and 13 mm against B. cereus and E. coli, whereas 6h exhibited remarkable activity (E. coli, 13 mm; K. pneumoniae, 12 mm) and 15 mm against S. aureus and B. cereus. In contrast, compounds 6a–6d and 6i demonstrated relatively lower antibacterial effects, with inhibition zones below 13 mm, indicating a reduced efficiency possibly due to electronic factors.
Table 1. Antibacterial activity of synthesized benzimidazole derivatives 6a–j presented as diameter of inhibition zones (mm) against S. aureus, B. cereus, E. coli, and K. pneumoniae at 10 µg/mL.
Figure 2. Antibacterial activity of the synthesized benzimidazole derivatives 6a–j evaluated at a concentration of 10 µg/mL against S. aureus, B. cereus, E. coli, and K. pneumoniae. The chart represents the diameter of inhibition zones (mm) measured for each compound compared with the standard drug nitrofurantoin (300 µg/mL) and the negative control (DMSO). Compounds 6f, 6h and 6j showed the highest antibacterial potencies toward the tested strains, showing inhibition profiles comparable to or exceeding the reference drug against selected microorganisms.
Figure 3. (a) Antibacterial activity of benzimidazole derivatives against B. cereus for compounds 6g, 6h, 6i and 6j. (b) Antibacterial activity against K. pneumoniae for compounds 6d, 6e and 6f. In both activities, the well diffusion method was used. In both assays, the mentioned compounds exhibited distinct zones of inhibition, indicating significant antimicrobial efficacy.
In the case of Gram-positive bacteria, compounds 6a, 6b, 6c, 6d, and 6e at a concentration of (10 µg/mL) showed a moderate activity (12–13 mm) against B. cereus, which is comparable and nearly equivalent to the activity of Nitrofurantoin (13 mm). Meanwhile, compounds 6f, 6g and 6h showed a high activity (15 mm), which is greater than Nitrofurantoin (13 mm). Compounds 6f and 6h showed moderate activity against S. aureus (15 mm) compared to the reference antibiotic (16 mm). Other compounds did not show any antibacterial activity against S. aureus. Additionally, compound 6j showed poor efficacy (9–10 mm) compared to the reference (13–16 mm) against B. cereus and S. aureus bacteria. In the case of Gram-negative bacteria, compounds 6e and 6j showed the highest antibacterial activity (15–16 mm) against E. coli and K. pneumoniae compared to Nitrofurantoin (14 mm). Other compounds 6a, 6b, 6c, 6f, and 6g showed a moderate activity (11–12 mm) against K. pneumoniae. Moreover, compounds 6h and 6i showed a lower activity (10–12 mm) against K. pneumonia compared to the reference antimicrobial agent. Meanwhile, compounds 6a, 6b, 6c, 6d and 6i did not show any antimicrobial activities against the tested Gram-positive bacteria or Gram-negative bacteria. These findings indicate that selected compounds (especially 6e, 6f, 6h, and 6j) possess a promising antibacterial potential, particularly against B. cereus and K. pneumonia [39,40,41].
  • Minimum inhibitory concentration (MIC)
MIC was measured for the most active compound 6h using serial dilution in nutrient broth against B. cereus, K. pneumoniae and S. aureus. Compound 6h exhibited MIC values of 5.00 µg/mL, confirming its strong antibacterial potency (Table 2 and Figure 4). MIC testing was performed using concentrations of 5.00, 2.50, 1.25, 0.625, 0.031 and 0.00 µg/mL, confirming a complete bacterial growth inhibition at 5.00 µg/mL. The tubes were then examined for growth using spectrophotometry. Therefore, compound 6h was selected for further morphological analysis using scanning electron microscopy (SEM) analysis due to its potent activity (Figure 4).
Table 2. MIC and percentage growth inhibition of the synthesized benzimidazole derivative 6h against the tested bacterial strains at different concentrations (5.00–0.00 µg/mL). Percentage inhibition values were calculated relative to untreated controls (0.00 µg/mL) using the formula % growth inhibition = (ODControl − ODSample)/ODControl) × 100, whereas ODControl = the average optical density of control and ODSample = the average optical density of sample. Optical density (OD) values represent the mean of three independent replicates (n = 3). Higher inhibition values at lower OD readings indicate strong antibacterial activity of 6h, particularly against B. cereus and K. pneumoniae. The control containing no compound exhibited maximal growth (0.00% inhibition).
Figure 4. MIC profile of compound 6h against B. cereus, S. aureus and K. pneumoniae. The chart shows the relationship between 6h concentration (5.00–0.00 µg/mL), and the calculated percentage of growth inhibition for each bacterium. A gradual increase in % growth inhibition with increasing concentration reveals enhanced antimicrobial activity. Compound 6h exhibited complete inhibition (100%) of bacterial growth at the highest concentration (5.00 µg/mL) for all tested strains, whereas partial inhibition was observed at intermediate concentrations, and no inhibition at the control (0.00 µg/mL). These results confirm the strong dose-dependent antibacterial activity of 6h.
Compound 6h displayed a clear concentration-dependent antibacterial effect, with ≥90% growth inhibition at low µg/mL concentrations against all tested bacteria (B. cereus, K. pneumoniae and S. aureus), indicating a broad-spectrum minimum inhibitory concentration in the low µg/mL range. The observed high activity can be accounted for by the structure of 6h, which integrates a 6-fluoro-benzimidazole nucleus with a piperidin-1-yl substituent. The benzimidazole core is known to mimic the purine structure and to employ hydrogen-bonding interactions and π–π stacking with key enzymes and microbial DNA, which is a common characteristic of antimicrobial benzimidazoles [7]. The presence of an electron-withdrawing fluorine atom on the aromatic ring raises the electron deficiency of the heterocycle, improves membrane permeation and reinforces lipophilicity, and numerous recent research studies have provided evidence of that fluoro-substituted benzimidazole derivatives exhibit outstanding antibacterial and antifungal activities compared with their non-fluorinated analogs [42]. In addition, the basic piperidine side chain in 6h is protonated at physiological pH, providing a cationic center that can interact with negatively charged bacterial and fungal cell surfaces, promote binding to intracellular targets, and facilitate uptake; similar SAR trends have been recorded for piperidinyl- and piperazinyl-benzimidazole series, where the incorporation of a tertiary amine ring considerably enhances the activity against K. pneumoniae, S. aureus and various fungal species [43]. Moreover, it should be emphasized that several SAR analyses of benzimidazole libraries have proven that halogen (Cl, F) or nitro substituents on the phenyl/benzimidazole ring, joined with a basic heterocycle, are optimal for antibacterial and antifungal potency, especially against Gram-positive bacteria and filamentous fungi, in excellent agreement with the manner of compound 6h in the current study [44]. In general, the MIC information for 6h subsequently aligns well with published SAR for benzimidazole antimicrobials and suggests that the integration between the protonatable piperidine moiety and the electron-poor 6-fluorobenzimidazole scaffold is responsible for the powerful inhibitory effect observed against both bacterial and fungal pathogens.
  • SEM Analysis
The SEM analysis was performed to visualize the morphological modifications in the bacterial cells subjected to compound 6h (2.50 µg/mL) compared with untreated controls. The SEM visualization obviously confirms that fluoro-substituted benzimidazole derivatives have important antibacterial activities (Figure 5 and Figure 6).
Figure 5. (a) SEM image of K. pneumoniae treated with compound 6h (2.50 µg/mL) in nutrient broth at 20 kV and magnifications of ×8000 and ×16,000, showing a significantly reduced number of bacterial cells with evident membrane damage, shrinkage, and surface collapse, indicating strong bactericidal activity. (b) SEM image of K. pneumoniae grown in nutrient broth without compound 6h at 20 kV and magnifications of ×8000 and ×35,000, showing intact and numerous rod-shaped cells with smooth surfaces and normal morphology.
Figure 6. (a) SEM image of S. aureus treated with compound 6h (2.50 µg/mL) in nutrient broth at 20 kV and magnifications of ×8000 and ×16,000, showing a significant reduction in bacterial cells with evident membrane damage, shrinkage, and surface collapse, indicating potent bactericidal activity. (b) SEM image of S. aureus cultured in nutrient broth without compound 6h at 20 kV and magnifications of ×8000 and ×16,000, showing intact, numerous, typical spherical cells with healthy surfaces and normal morphology.
The compound 6h exhibited potent bactericidal activity through membrane-targeting mechanisms, lately compromising cellular integrity due to the structural characteristics of 6h, a benzimidazole derivative bearing a fluorinated aromatic system and a piperidine moiety, both of which are linked to intracellular penetration and the reinforced microbial membrane affinity. The electron-withdrawing fluorine atom raises the electronic polarization and lipophilicity of the benzimidazole ring, helping deeper membrane insertion and assisting interactions with negatively charged cell envelopes [45]. The observed structure–activity relationship trends in the SEM images support earlier discoveries that benzimidazoles work by targeting key microbial cellular structures, mostly causing rupture, membrane inhibition of DNA synthesis, and interference with essential enzymatic pathways. Numerous new studies concerning fluorinated benzimidazole analogs have revealed similar destructive impacts on Gram-negative and Gram-positive bacteria, including cytoplasmic leakage, membrane pitting, and cell deformation [46,47].

2.3.2. Antifungal Activity

The antifungal evaluation results for the same series of compounds against A. flavus, P. duclauxii, and P. italicum at concentrations of 20 µg/mL and 50 µg/mL are presented in (Table 3 and Figure 7). Amphotericin B at the same concentrations served as the standard antifungal reference drug, while DMSO served as a negative control. In general, most compounds exhibited concentration-dependent activity, with a greater inhibition observed at 50 µg/mL. Among all compounds, 6e, 6f, 6h, and 6j displayed an excellent antifungal potency compared to Amphotericin B at the same concentrations, especially against P. italicum and P. duclauxii, showing inhibition zones between 20 and 25 mm at 50 µg/mL. Notably, compounds 6f and 6e demonstrated the highest antifungal efficiency, exhibiting 25 mm zones against P. italicum and P. duclauxii, respectively. Compound 6h also showed a substantial antifungal response (25 mm) against P. duclauxii. At a concentration of 50 µg/mL, compounds 6a, 6b, 6d, and 6h showed a particularly high activity against A. flavus, producing inhibition zones ranging from 20 to 28 mm, which were markedly greater than that observed for Amphotericin B under the same conditions. Notably, 6a, 6b, and 6h each produced an inhibition zone of 28 mm against A. flavus. Meanwhile, other compounds, including 6c, 6e, 6f, 6g and 6i, exhibited a moderate antifungal activity, with zone diameters ranging between 15 and 18 mm against A. flavus. Against P. italicum, compounds 6d, 6e, 6f, 6g, and 6h demonstrated strong antifungal effects (23–25 mm) compared to the reference antifungal (19 mm). Other compounds, 6a, 6b, 6c and 6i, showed lower antifungal activities, with inhibition zone values ranging from 12 to 17 mm. Meanwhile, compounds such as 6h and 6j represent a broad spectrum against all tested fungi. Compound 6h showed an inhibition zone of 25 mm against both A. flavus and P. duclauxii and 23 mm against P. italicum. Similarly, compound 6j showed a high inhibition zone (24 mm) and 20 mm against both A. flavus and P. italicum, highlighting its potential as a broad-spectrum antifungal agent (Figure 8).
Table 3. Antifungal activity of the synthesized benzimidazole derivatives 6a–j against A. flavus, P. duclauxii, and P. italicum at 20 and 50 µg/mL, expressed as the diameter of the inhibition zone (mm).
Figure 7. Antifungal activity of benzimidazole derivatives 6a–j against A. flavus, P. duclauxii, and P. italicum at concentrations of 20 and 50 µg/mL. The chart shows the diameter of inhibition zones (mm) generated by each synthesized compound in comparison with positive control (Amphotericin B) and negative control (DMSO). The derivatives 6e, 6f, 6h, and 6j exhibited strong and broad-spectrum antifungal potency, with inhibitory zones approaching or surpassing those of Amphotericin B at one or both tested concentrations. Activity generally increased with higher concentration (50 µg/mL), confirming a clear concentration-dependent antifungal response.
Figure 8. (a) Antifungal activity of benzimidazole derivatives against P. italicum for compounds 6g, 6h, 6i and 6j at 50 µg/mL. (b) Antifungal activity against A. flavus for compounds 6g, 6h, 6i and 6j at 50 µg/mL. In both assays, the well diffusion method was used. Compounds 6g, 6h and 6j exhibited distinct zones of inhibition in both assays, indicating significant antifungal efficacy.
This observed increase might be gained from the presence of electron-withdrawing substituents that increase the interaction of the benzimidazole nucleus with cell wall components or fungal enzymes. On the contrary, 6a–c and 6i exhibited a moderate to weak inhibition (≤15 mm), suggesting that substituents with reduced hydrophobic or electronic interactions decrease antifungal effectiveness. The DMSO control displayed no inhibition, confirming that the shown activity was caused by the synthesized compounds. Ref. [48] reported that several 1-alkyl-1H-benzimidazole derivatives showed distinguished antifungal impacts against different species of Candida and Aspergillus. In the study by Karaburun [49], a series of benzimidazole–1,3,4-oxadiazole derivatives were synthesized and examined for antifungal activity. Among those synthesized compounds, two of them exhibited the strongest inhibition of fungal growth. The elevated activity exhibited by the synthesized benzimidazole derivatives might be connected to the fact that the benzimidazole derivative notably inhibits ergosterol biosynthesis and results in the accumulation of unusual sterol intermediates in fungi, indicating a disruption of the normal sterol synthesis pathway [50].

2.3.3. Structure Activity Relationship (SAR)

The benzimidazole nucleus represents a widely recognized privileged structure within antimicrobial drug discovery; Its combined benzene–imidazole structure can imitate purine bases and participate in π–π stacking and hydrogen bonding with microbial enzymes and nucleic acids, which demonstrates its repeated appearance in efficient antibacterial and antifungal agents [51]. In the 6a–j series, all compounds contain a 6-fluoro-1H-benzo[d]imidazole core connected at the 5-position to a basic nitrogen heterocycle (piperazine, pyrrolidine, piperidine or morpholine). The presence of the strongly electronegative fluorine at C-6 increases lipophilicity and the electron deficiency of the aromatic system, which is known to increase penetration through bacterial membranes and to reinforce binding to hydrophobic pockets of biological targets, similar to the 6-fluoro substitution that is important for the broad-spectrum activity of fluoroquinolones [52]. The second essential pharmacophoric motif is represented by the terminal cyclic amine. Piperazine and related rings are protonated at physiological pH, giving a cationic center that can interact with negatively charged bacterial cell walls and fungal membranes, disrupt membrane integrity and facilitate uptake; analogous behavior has been noted for cationic amphiphilic agents and other piperazine-containing benzimidazoles [53].
In this shared framework, the diversity in the N-substituent on the piperazine ring or the exchange for pyrrolidine, morpholine or piperidine adjusts the relationship between the polarity and lipophilicity and thus relates to the detected changes in activity. The most active constituents of the series 6e, 6f, 6h and 6j join the 6-fluorobenzimidazole core with moderately hydrophobic, conformationally flexible side chains: a butyl group in 6e, a phenylpiperazine in 6f, a piperidinyl fragment in 6h and a hydroxyethylpiperazine in 6j. These substituents improve the capability of the molecule to be let into and across fungal and bacterial membranes, while continuing to exhibit a sufficient hydrogen-bonding capacity and basicity for significant interactions with intracellular targets. Analogous SAR patterns have been reported for 2-(substituted piperazin-1-yl)phenyl-benzimidazoles, where the inclusion of lipophilic aryl or alkyl groups on the terminal nitrogen considerably reinforced activity against S. aureus, E. coli, P. aeruginosa and several fungal strains [54]. Conversely, derivatives bearing more polar or shorter substituents on the side chain (e.g., 6a–c) exhibit weaker inhibition regions, in agreement with the decreased membrane partitioning and less favorable hydrophobic contacts at the binding site. The morpholine analog in compound 6i, which is more polar and less basic, shows an intermediate activity, consistent with the literature reports where the replacement of piperazine by morpholine in benzimidazole hybrids frequently results in a reduced antimicrobial potency [55].
Our SAR is in strong alignment with recent research on antimicrobial benzimidazole derivatives. Çevik et al. documented that 5-fluoro-substituted benzimidazoles incorporating basic side chains show a pronounced action against S. aureus, E. coli and Pseudomonas, emphasizing the collaborative impact of protonatable amine and halogenation substituents [56]. Mendogralo et al. likewise reported that benzimidazole derivatives holding electron-withdrawing substituents on the ring and terminal basic heterocycles show an increased antifungal and antibacterial activity, typically analogous to or better than standard drugs against Gram-negative and Gram-positive bacteria for Candida or Aspergillus species [57]. Furthermore, benzimidazole–piperazine scaffolds and bis-heterocyclic systems joined via piperazine have lately been recorded as having broad-spectrum antimicrobial and powerful activities, with mechanistic investigations indicating membrane damage and the blockage of essential enzymes as main mechanisms of action [58]. Combined together, these data from the literature provide support for our results that the incorporation of a 6-fluorobenzimidazole nucleus linked to appropriately substituted piperidine, piperazine or related rings is a favorable method for designing molecules active against the tested bacterial strains (S. aureus, B. cereus, E. coli, K. pneumoniae) and thread-like fungi (A. flavus, P. duclauxii, P. italicum). The excellent activity of 6e, 6f, 6h and 6j can thus be justified to an optimal combination of a π-rich benzimidazole core, an electron-deficient target affinity and a fluorine atom enhancing lipophilicity, and a cationic, moderately hydrophobic side chain that reinforces cellular uptake, membrane interaction, and strong binding to microbial targets.
Overall, the observed SAR denotes that electron-withdrawing substituents (e.g., halogens such as –F) promote antibacterial activity. The enhanced activity of 6h can be associated with the increased lipophilicity and improved cell membrane permeability gained by its substituent (Table 4).
Table 4. SAR and literature comparison of synthesized benzimidazole derivatives (6a–6j) supporting the substituted nitrogen heterocycle motif.

3. Materials and Methods

3.1. Experimental

3.1.1. Chemicals and Reagents

All reagents and solvents used throughout the study were of analytical grade and used as such without further purification. Chloroform, dichloromethane (DCM), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), 3-chloro-4-fluoroaniline, piperazine derivatives or N-substituted nitrogen heterocycles (R) and tin (II) chloride were purchased from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (HCl), formic acid (HCOOH), sulfuric acid (H2SO4), nitric acid (HNO3), acetic anhydride (Ac2O), sodium hydroxide (NaOH), and sodium sulfate (Na2SO4) were obtained from BDH (Poole, UK). Absolute ethanol (EtOH) was purchased from Merck (Darmstadt, Germany).

3.1.2. Instruments

The synthesized compounds were analyzed using 1H-NMR, 13C-NMR, LC–MS or GC–MS, and FT-IR spectroscopy. 1H-NMR spectra were recorded on the Bruker Biospin Avance HD III 700 MHz (Bruker BioSpin GmbH, Rheinstetten, Germany) or JEOL ECZ-400 NMR (400 MHz) spectrometer (JEOL Ltd., Tokyo, Japan) instrument with CDCl3 or DMSO-d6 as a solvent, using Tetramethylsilane (TMS) as an internal standard, and the chemical shifts are reported in parts per million (δ ppm) and the coupling constant (J) values are given in Hertz (Hz). The 1H-NMR and 13C-NMR spectra were processed and analyzed using MestReNova (Mnova) v6.0.2-5475 (Mestrelab Research S.L.U., Santiago de Compostela, Spain). The mass spectra were measured on a Liquid Chromatography/Mass Spectrometry (LC-MS) Agilent 6530B LC Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), Gas Chromatography/Mass Spectrometry (GC-MS) Agilent 7890B GC mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), Advion-CMS single Quad mass spectrometer (Advion Interchim Scientific, Ithaca, NY, USA) or high-resolution mass spectrometer Agilent 6530B LC Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The FT-IR spectra were recorded on a PerkinElmer ALPHA II Platinum ATR FT-IR spectrometer (PerkinElmer, Inc., Waltham, MA, USA). The melting points were measured using a Stuart Scientific SMP10 apparatus (Bibby Scientific Ltd., Stone, UK). Thin-layer chromatography (TLC) was performed using silica gel 60 F254 aluminum sheets (20 × 20 cm, Merck, Darmstadt, Germany) using various solvent systems, and spots were visualized under a handheld UV lamp (UVP UVGL-58, Upland, CA, USA) equipped with 6 W, dual-wavelength 254/365 nm.

3.1.3. Synthesis of Compounds (1–3)

  • Synthesis of N-(3-chloro-4-fluorophenyl) acetamide (1)
The starting material, 3-chloro-4-fluoroaniline (SM, 10.346 g, 71.080 mmol), was added to acetic anhydride (20.0 mL) portion-wise at room temperature over 30.0 min. Following the addition, the reaction mixture was stirred for an hour then placed into an ice/water bath. The target compound was obtained through suction filtration, washing with water and drying under vacuum overnight to give (1) as white crystals. Yield 11.98 g, 93.90%, M.P.: 119–120 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3294, 3266, 3202, 3134, 3080, 2913, 1665, 1607, 1547, 1490, 1389, 1249, 1020, 748. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.14 (s, 3H), 7.03 (dd, J = 8.8 Hz, 1H), 7.28 (dd, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.2, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 168.87, 154.15, 134.58, 122.37, 121.08, 119.77, 116.60, 24.48. GC–MS (EI, single quadrupole): m/z calculated for C8H7ClFNO [M]•+ = 187.6, found [M]•+ = 187.0 (Figures S1–S4).
  • Synthesis of N-(5-chloro-4-fluoro-2-nitrophenyl) acetamide (2)
N-(3-chloro-4-fluorophenyl) acetamide (1) (10.100 g, 53.850 mmol) was dissolved in concentrated sulfuric acid (30.0 mL) with stirring. The reaction mixture was cooled in an ice–salt bath until the temperature reached 0 °C. Concentrated nitric acid (28.0 mL) was then added dropwise to the chilled mixture at a rate maintaining the reaction mixture below 5 °C. The reaction mixture was then stirred for another one hour at 0–5 °C and then poured into an ice/water bath with stirring. The yellow precipitate formed was then filtered, dried, and recrystallized from absolute ethanol to offer the target compound. Yield 12.23 g, 97.64%, M.P.: 112–113 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3377, 2924, 2363, 2334, 1700, 1582, 1492, 1341, 1263, 1152, 1022, 993, 859. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.30 (s, 3H), 8.03 (d, J = 8.7 Hz, 1H), 9.00 (d, J = 7.0 Hz, 1H), 10.27 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 169.11, 153.23, 151.86, 134.39, 131.88, 123.86, 113.18, 25.68. GC–MS (EI, single quadrupole): m/z calculated for C8H6ClFN2O3 [M]•+ = 232.6, found [M]•+ = 232.0 (Figures S5–S8).
  • Synthesis of 5-chloro-4-fluoro-2-nitroaniline (3)
N-(5-chloro-4-fluoro-2-nitrophenyl) acetamide (2) (5.039 g, 21.595 mmol) was dissolved in concentrated acid HCl (10.0 mL) and absolute ethanol (40.0 mL). The reaction mixture was refluxed for two hours. After cooling, the reaction mixture was then poured into ice/water (200.0 mL). The resulting orange-yellow solid was collected through suction filtration, washed several times with water (200.0 mL) and recrystallized using absolute ethanol to give yellow crystals. Yield 4.00 g, 98.09%, M.P.: 144–145 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3488, 3375, 3158, 3105, 3057, 1638, 1570, 1496, 1337, 1238, 1001, 876, 783. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 6.04 (s, 2H), 6.91 (d, J = 7.0 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 149.62, 148.24, 141.60, 130.84, 119.56, 112.83. GC–MS (EI, single quadrupole): m/z calculated for C6H4ClFN2O2 [M + H]+ = 191.6, found [M + H]+ = 191.0 (Figures S9–S14).

3.1.4. General Procedures for the Synthesis of 4-Fluoro-5-(4-N-substitutedheterocycle-1-yl)-2-nitroaniline (4a–j)

4-fluoro-5-(4-substitutedpiperazin-1-yl)-2-nitroaniline (4a–j) was prepared by dissolving 5-Chloro-4-fluoro-2-nitroaniline (3) (5.00 g, 26.24 mmol) in 15 mL DMSO; then, N-substituted piperazine or N-substituted nitrogen heterocycle derivative (15 g, 174.14 mmol) was added. The reaction mixture was refluxed at 140 °C for two hours. After cooling, the reaction mixture was then transferred into a beaker with water added. A precipitate was then produced, filtered, and dried under vacuum. The precipitate was recrystallized with hot absolute ethanol, and fine orange crystals formed.
  • 4-fluoro-5-(4-piperazin-1-yl)-2-nitroaniline (4a)
Solid orange crystals; molecular formula: C10H13FN4O2. Yield 4.72 g, 88.57%, M.P.: 187–188 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3462, 3425, 3358, 3331, 2946, 2913, 2846, 2823, 1630, 1572, 1504, 1473, 1244, 1196, 904, 862, 832. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.66 (s, 1H), 3.02 (dd, J = 6.0, 4.0 Hz, 4H), 3.20 (dd, J = 6.0, 3.9 Hz, 4H), 6.04 (d, J = 7.5 Hz, 1H), 6.08 (s, 2H), 7.77 (d, J = 14.0 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.17, 145.80, 143.59, 124.08, 112.66, 103.95, 50.84, 45.98. GC–MS (EI, single quadrupole): m/z calculated for C10H13FN4O2 [M]•+ = 240.2342, found [M]•+ = 240.000. LC–MS (ESI, APCI, positive/negative): m/z calculated for C10H13FN4O2 [M + H]+ = 241.2342, [M − H]− = 239.2342 found [M + H]+ = 241.3000, [M − H]− = 238.9000 (Figures S15–S19).
  • 4-fluoro-5-(4-methylpiperazin-1-yl)-2-nitroaniline (4b)
Solid orange crystals; molecular formula: C11H15FN4O2. Yield 6.64 g, 99.10%, M.P.: 135–137 °C ([61], 136–137 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3498, 3428, 3381, 3278, 2938, 2800, 1634, 1572, 1504, 1479, 1391, 1246, 1195, 1141, 1001, 913. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.26 (s, J = 6.86 Hz, 3H), 2.50 (t, J = 4.83 Hz, 4H), 4.83 (t, J = 4.97 Hz, 4H), 5.99 (d, J = 7.63 Hz, 1H), 6.06 (s, 1H), 7.70 (d, J = 14.0 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.70, 145.63, 143.74, 125.85, 112.84, 103.99, 54.66, 54.66, 49.40, 49.40, 46.06. GC–MS (EI, single quadrupole): m/z calcd for C11H15FN4O2 [M]•+ = 254.2608, found 254.1000 (Figures S20–S23).
  • 4-fluoro-5-(4-ethylpiperazin-1-yl)-2-nitroaniline (4c)
Solid orange crystals; molecular formula: C12H17FN4O2. Yield 6.73 g, 94.91%, M.P.: 143–145 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3473, 3346, 2973, 2834, 1633, 1589, 1482, 1406, 1237, 1084, 1020, 874, 781. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.02 (s, 3H), 2.50 (s, 4H), 2.38 (q, J = 7.2 Hz, 2H), 3.17 (s, 4H), 5.96 (d, J = 7.6 Hz, 1H), 6.04 (s, 2H), 7.65 (d, J = 14.0 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.85, 145.75, 143.72, 123.98, 112.73, 104.00, 52.55, 52.41, 52.41, 49.58, 49.58, 12.06. GC–MS (EI, single quadrupole): m/z calcd for C12H17FN4O2 [M]•+ = 268.2874, found 268.1000 (Figures S24–S27).
  • 4-fluoro-5-(4-isopropylpiperazin-1-yl)-2-nitroaniline (4d)
Solid orange crystals; molecular formula: C13H19FN4O2. Yield 2.28 g, 61.62%, M.P.: 121–123 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3467, 3338, 2965, 2829, 1632, 1574, 1500, 1475, 1382, 1242, 872. 1H NMR (700 MHz, δ/ppm, CDCl3): 1.11–0.97 (m, 6H), 2.66 (s, 4H), 2.72 (s, 1H), 3.25 (s, 4H), 6.04 (d, J = 7.6 Hz, 1H), 6.15 (s, 2H), 7.73 (d, J = 11.2 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.88, 145.85, 143.73, 123.80, 112.45, 103.89, 54.65, 49.79, 48.41, 18.54. LC–MS (ESI, APCI, positive/negative): m/z calcd for C13H19FN4O2 [M + H]+ = 283.3140, [M − H]− = 281.3140 found [M + H]+ = 283.2500, [M − H]− = 281.1000 (Figures S28–S32).
  • 4-fluoro-5-(4-butylpiperazin-1-yl)-2-nitroaniline (4e)
Solid orange crystals; molecular formula: C14H21FN4O2. Yield 3.57 g, 91.8%, M.P.: 109–111 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3570, 3538, 3437, 3294, 3181, 2957, 2930, 2825, 1630, 1574, 1502, 1220, 991. 1H NMR (700 MHz, δ/ppm, CDCl3): 0.92 (t, J = 7.4 Hz, 3H), 1.37–1.30 (m, 2H), 1.49 (dt, J = 15.3, 7.6 Hz, 2H), 2.41–2.33 (m, 2H), 2.61–2.55 (m, 4H), 3.28–3.22 (m, 4H), 6.04 (d, J = 7.6 Hz, 1H), 6.12 (s, 2H), 7.75 (d, J = 14.0 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.77, 145.72, 143.68, 123.93, 112.68, 103.93, 58.47, 52.94, 49.54, 29.01, 20.82, 14.17. LC–MS (ESI, APCI, positive/negative): m/z calcd for C14H21FN4O2 [M + H]+ = 297.3405, [M − H]− = 295.3405 found [M + H]+ = 297.3000, [M − H]− = 295.1000 (Figures S33–S37).
  • 4-fluoro-5-(4-phenylpiperazin-1-yl)-2-nitrobenzenamine (4f)
Solid orange crystals; molecular formula: C16H17FN4O2. Yield 4.02 g, 94.37%, M.P.: 183–185 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3465, 3339, 2826, 1643, 1600, 1497, 1383, 1315, 1220, 1029, 936, 757, 691. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 3.32–3.35 (m, 4H), 3.39–3.42 (m, 4H), 6.11 (d, J = 7.5 Hz, 1H), 6.14 (s, 2H), 6.92 (t, J = 6.3 Hz, 2H), 6.97 (d, J = 8.8 Hz, 4H), 7.29–7.34 (m, 4H), 7.80 (d, J = 13.9 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 150.99, 147.64, 145.71, 143.60, 129.38, 129.20, 120.59, 116.54, 112.84, 104.18, 49.60, 49.28. GC–MS (EI, single quadrupole): m/z calcd for C16H17FN4O2 [M]•+ = 316.3302, found 316.2000 (Figures S38–S41).
  • 4-fluoro-2-nitro-5-(pyrrolidin-1-yl) aniline (4g)
Solid orange crystals; molecular formula: C10H12FN3O2. Yield 5.61 g, 94.94%, M.P.: 187–189 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3479, 3363, 2968, 2891, 2851, 1633, 1583, 1534, 1402, 1294, 1254, 1202, 931, 860, 793. 1H NMR (700 MHz, δ/ppm, CDCl3): 1.99–1.96 (m, 4H), 3.50 (td, J = 6.4, 2.9 Hz, 4H), 5.64 (d, J = 7.8 Hz, 1H), 6.11 (s, 2H), 7.71 (d, J = 14.9 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 144.63, 144.17, 143.35, 120.98, 112.52, 98.05, 50.35, 25.48. LC–MS (ESI, APCI, positive/negative): m/z calcd for C10H12FN3O2 [M + H]+ = 226.2196, [M − H]− = 224.2196 found [M + H]+ = 225.9000, [M − H]− = 223.7000 (Figures S42–S46).
  • 4-fluoro-2-nitro-5-(piperidin-1-yl) aniline (4h)
Orange crystals; molecular formula: C11H14FN3O2. Yield 2.88 g, 91.8%, M.P.: 133–135 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3472, 3346, 2961, 2932, 2800, 1636, 1593, 1504, 1477, 1308, 1211, 869. 1H NMR (700 MHz, δ/ppm, CDCl3): 1.62 (s, 2H), 1.70 (s, 4H), 3.20 (s, 4H), 6.03 (d, J = 7.7 Hz, 1H), 6.11 (s, 2H), 7.72 (d, J = 14.1 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.59, 145.69, 143.67, 123.47, 112.59, 103.67, 50.93, 25.81, 24.21, LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H14FN3O2 [M + H]+ = 240.2462, [M − H]− = 238.2462 found [M + H]+ = 240.0000, [M − H]− = 237.9000 (Figures S47–S51).
  • 4-fluoro-2-nitro-5-(morpholin-1-yl) aniline (4i)
Solid orange crystals; molecular formula: C10H12FN3O3. Yield 2.88 g, 90.0%, M.P.: 186–188 °C (literature [61], 188–190 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3443, 3325, 3196, 2963, 2864, 1634, 1473, 1242, 1115, 917, 872. 1H NMR (700 MHz, δ/ppm, CDCl3): 3.44–3.06 (m, 4H), 4.13–3.67 (m, 4H), 6.05 (d, J = 7.5 Hz, 1H), 6.08 (s, 2H), 7.79 (d, J = 13.9 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.10, 145.74, 143.47, 124.22, 112.95, 104.17, 49.90, 66.66. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H14FN3O2 [M + H]+ = 241.2190, [M − H]− = 240.2190 found [M + H]+ = 241.9000, [M − H]− = 240.0000 (Figures S52–S56).
  • 2-(4-(5-amino-2-fluoro-4-nitrophenyl)piperazin-1-yl)ethanol (4j)
Solid orange crystals; molecular formula: C12H17FN4O3. Yield 3.130 g, 83.9%, M.P.: 150–151 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3538, 3449, 3327, 3142, 2954, 2839, 1632, 1570, 1506, 1387, 1246, 1003, 921. 1H NMR (700 MHz, δ/ppm, CDCl3): 2.64–2.60 (m, 2H), 2.70–2.65 (m, 4H), 3.29–3.24 (m, 4H), 3.68–3.64 (m, 2H), 6.05 (d, J = 7.5 Hz, 1H), 6.09 (s, 2H), 7.78 (d, J = 13.9 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 147.69, 145.74, 143.53, 124.20, 112.76, 104.14, 59.47, 57.85, 52.62, 49.66. LC–MS (ESI, APCI, positive/negative): m/z calcd for C12H17FN4O3 [M + H]+ = 284.2868, [M − H]− = 284.2868 found [M + H]+ = 285.0500, [M − H]− = 283.0500 (Figures S57–S61).

3.1.5. General Procedure for the Synthesis of 4-Fluoro-5-(4-substitutedheterocycle-1-yl) benzene-1,2-diamine (5a–j)

4-fluoro-5-(4-substitutedheterocycle-1-yl)-2-nitrobenzenamine (4a–j) (1.0342 g, 4.112 mmol) was dissolved in concentrated hydrochloric acid (10 mL). After that, tin (II) chloride (5.2133 g, 26.93 mmol) was added to the solution with stirring at room temperature to give a white suspension. Water was added until a clear solution was formed, with continued stirring for a further 30 min. The cooled colorless solution was treated with cooled 40% sodium hydroxide to a pH of 13 and extracted with dichloromethane (6 × 30 mL). The organic extract was dried using Na2SO4, and then the dichloromethane evaporated using a rotatory evaporator and white foamy solid was formed.
  • 4-fluoro-5-(4-piperazin-1-yl) benzene-1,2-diamine (5a)
Solid cream crystals; molecular formula: C10H15FN4. Yield 0.92 g, 77.54%, M.P.: 111–113 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3418, 3381, 3286, 3222, 2936, 2808, 1671, 1632, 1514, 1450, 1372, 1284, 1191, 1146, 1010. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.05 (s, 1H), 2.97–2.91 (m, 4H), 3.04–3.02 (m, 3H), 6.27 (s, 4H), 6.38 (d, J = 8.2 Hz, 1H), 6.46 (d, J = 12.7 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 150.16, 133.04, 129.99, 130.79, 108.93, 105.38, 53.00, 46.54. LC–MS (ESI, APCI, positive/negative): m/z calcd for C10H15FN4 [M + H]+ = 210.2513, [M − H]− = 209.2513 found [M + H]+ = 210.9000, [M − H]− = 209.0000 (Figures S62–S66).
  • 4-fluoro-5-(4-methylpiperazin-1-yl) benzene-1,2-diamine (5b)
Solid cream crystals; molecular formula: C11H17FN4. Yield 0.92 g, 77.54%, M.P.: 79–81 °C (literature [61], 78–80 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3381, 3213, 2936, 2813, 1672, 1631, 1516, 1453, 1282, 1192, 1145, 1009. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.33 (s, 3H), 2.59 (bs, 4H), 2.99 (bs, 4H), 6.37 (d, J = 7.4 Hz, 1H), 6.45 (d, J = 13.7 Hz, 1H), 6.67 (s, 4H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 151.55, 132.67, 130.40, 129.92, 108.45, 105.64, 55.38, 51.14, 45.97. GC–MS (EI, single quadrupole): m/z calcd for C11H17FN4 [M]•+ = 224.2779, found M]•+ = 224.1000. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H17FN4 [M + H]+ = 225.2779, [M − H]− = 223.2779 found [M + H]+ = 225.0000, [M − H]− = 222.8000 (Figures S67–S72).
  • Synthesis of 4-fluoro-5-(4-ethylpiperazin-1-yl) benzene-1,2-diamine (5c)
Solid cream crystals; molecular formula: C12H19FN4. Yield 0.76 g, 85.60%, M.P.: 94–96 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3410, 3379, 3313, 3223, 2975, 2950, 2817, 1674, 1631, 1518, 1448, 1311, 1188, 1122, 946, 776. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.09 (t, J = 7.3 Hz, 3H), 2.45 (q, J = 7.2 Hz, 2H), 2.59 (bs, 4H), 2.98 (bs, 4H), 6.34 (d, J = 8.2 Hz, 1H), 6.42 (d, J = 12.8 Hz, 1H), 6.67 (s, 4H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 149.61, 132.49, 129.97, 129.91, 108.39, 105.50, 53.03, 52.42, 51.26, 12.10. GC–MS (EI, single quadrupole): m/z calcd for C12H19FN4 [M]•+ = 238.3045, found M]•+ = 238.1000. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H17FN4 [M + H]+ = 238.3045, [M − H]− = 238.3045 found [M + H]+ = 239.0000, [M − H]− = 237.1000 (Figures S73–S78).
  • 4-fluoro-5-(4-isopropylpiperazin-1-yl) benzene-1,2-diamine (5d)
Solid cream crystals; molecular formula: C13H21FN4. Yield 0.97 g, 99.5%, M.P.: 127–129 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3781, 3706, 3377, 3235, 2975, 2821, 2365, 1675, 1632, 1523, 1267, 1189, 1141,972. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.09 (s, 3H), 1.10 (s, 3H), 2.77–2.71 (m, 2H), 3.01 (s, 4H), 3.25 (s, 4H), 6.27 (s, 4H), 6.37 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 12.8 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 151.04, 149.64, 132.65, 130.44, 108.50, 105.41, 54.78, 51.53, 48.95, 18.67. LC–MS (ESI, APCI, positive/negative): m/z calcd for C16H19FN4 [M + H]+ = 252.3310, [M − H]− = 251.3310 found [M + H]+ = 252.9000, [M − H]− = 251.0000 (Figures S79–S83).
  • 4-fluoro-5-(4-butylpiperazin-1-yl) benzene-1,2-diamine (5e)
Solid cream crystals. Molecular formula: C14H23FN4. Yield 0.96 g, 99.5%, M.P.: 119–121 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3424, 3381, 3317, 3233, 2969, 2821, 1673, 1630, 1525, 1446, 1273, 1195, 972. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 0.92 (t, J = 7.4 Hz, 3H), 1.33 (s, 2H), 1.50 (s, 2H), 2.39 (s, 2H), 2.62 (s, 4H), 3.00 (s, 4H), 6.36 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 12.8 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 150.97, 132.63, 130.44, 129.95, 108.47, 105.55, 58.66, 53.52, 51.27, 29.04, 20.93, 14.20. LC–MS (ESI, APCI, positive/negative): m/z calcd for C14H23FN4 [M + H]+ = 266.3576, [M − H]− = 265.3576 found [M + H]+ = 266.9000, [M − H]− = 265.2000 (Figures S84–S88).
  • 4-fluoro-5-(4-phenylpiperazin-1-yl) benzene-1,2-diamine (5f)
Solid cream crystals; molecular formula: C16H19FN4. Yield 0.95 g, 99.4%, M.P.: 281–283 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3410, 3375, 3295, 3221, 2823, 1664, 1631, 1598, 1494, 1227, 1143, 940, 757, 685. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.49 (bs, 4H), 2.70 (bs, 4H), 5.77 (d, J = 8.2 Hz, 1H), 5.84 (d, J = 12.7 Hz, 1H), 6.28 (d, J = 26.9 Hz, 1H), 6.35 (d, J = 7.9 Hz, 2H), 6.67 (s, 2H), 6.67 (s, 4H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 151.30, 149.61, 132.31, 130.45, 130.16, 129.14, 119.95, 116.30, 108.49, 105.38, 51.42, 49.56. LC–MS (ESI, APCI, positive/negative): m/z calcd for C16H19FN4 [M + H]+ = 286.3473, [M − H]− = 286.3473 found [M + H]+ = 287.0000, [M − H]− = 285.2000 (Figures S89–S93).
  • 4-fluoro-5-(piperidin-1-yl)benzene-1,2-diamine (5h)
Solid cream crystals. Molecular formula: C11H16fN3. Yield 0.97 g, 99.5%, M.P.: 128–130 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3426, 3319, 3204, 2934, 2851, 2814, 1632, 1593, 1514, 1240, 1195, 661. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.52 (dt, J = 11.8, 6.0 Hz, 2H), 1.71 (s, 4H), 2.88 (s, 4H), 3.09 (s, 4H), 6.38 (d, J = 8.3 Hz, 1H), 6.44 (d, J = 12.7 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 149.75, 133.99, 130.34, 129.66, 108.88, 105.42, 53.03, 26.41, 24.33. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H16fN3 [M + H]+ = 209.2632, [M − H]− = 208.2632 found [M + H]+ = 209.9000, [M − H]− = 208.1000 (Figures S94–S98).
  • 4-fluoro-5-(morpholin-1-yl) benzene-1,2-diamine (5i)
Solid cream crystals; molecular formula: C10H14FN3O. Yield 0.92 g, 99.4%, M.P.: 127–128 °C (literature [61], 126–127 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3435, 3356, 3224, 2957, 2868, 2833, 1630, 1520, 1265, 1191, 1110, 919, 847. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.97–2.94 (m, 4H), 3.86–3.83 (m, 4H), 6.08 (s, 4H), 6.36 (d, J = 8.2 Hz, 1H), 6.47 (d, J = 12.7 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 149.69, 132.47, 130.52, 130.25, 108.37, 105.51, 67.26, 51.80. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H16fN3 [M + H]+ = 211.2361, [M − H]− = 210.2361 found [M + H]+ = 212.1000, [M − H]− = 210.0000 (Figures S99–S103).
  • 2-(4-(4,5-diamino-2-fluorophenyl)piperazin-1-yl)ethanol (5j)
Solid cream crystals; molecular formula: C12H19FN4O. Yield 0.8 g, 89.4, M.P.: 115–117 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3505, 3373, 3228, 2948, 2825, 1638, 1523, 1436, 1277, 1195. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.69 (bs, 1H), 2.63–2.58 (t, 2H), 2.68 (bs, 4H), 2.99 (bs, 4H), 3.27 (bs, 4H), 3.66–3.63 (m, 2H), 6.37 (d, J = 8.2 Hz, 1H), 6.46 (d, J = 12.8 Hz, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 149.68, 132.53, 130.48, 130.13, 108.57, 105.61, 59.37, 57.81, 53.16, 51.47. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H16fN3 [M + H]+ = 254.3039, [M − H]− = 253.3039 found [M + H]+ = 254.9500, [M − H]− = 253.2500 (Figures S104–S108).

3.1.6. General Procedure for the Synthesis of 6-Fluoro-5-(4-substitutedheterocycle-1-yl)-1H-benzo[d]imidazole (6a–j)

6-fluoro-5-(4-substitutedheterocycle-1-yl)-1H-benzo[d]imidazole (6a–j) was prepared by dissolving 4-fluoro-5-(4-substitutedheterocycle-1-yl) benzene-1,2-diamine (5a–j) (1.00 g, 4.46 mmol) in 4.0 mL of 90% formic acid. The reaction mixture was refluxed at 100 °C for two hours. After cooling, 10% sodium hydroxide solution was added slowly with constant stirring until the mixture was just alkaline (pH = 7.5) to litmus. After that, the mixture was extracted using ethyl acetate (6 × 30 mL). The organic extract was dried using anhydrous Na2SO4, and then the ethyl acetate was evaporated using a rotary evaporator and orange solid crystals were formed.
  • 6-fluoro-5-(4-piperazin-1-yl)-1H-benzo[d]imidazole (6a)
Solid orange crystals; molecular formula: C11H13FN4. Yield 0.84 g, 80.46%, M.P.: 234–236 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3248, 3084, 2944, 2795, 2490, 1881, 1474, 1251, 1165, 897. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.91 (s, 1H), 2.86 (bs, 4H), 2.88 (bs, 4H), 5.00 (s, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 12.5 Hz, 1H), 8.12 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.44, 152.08, 142.16, 137.29, 137.66, 103.69, 103.24, 52.51, 45.64. GC–MS (EI, single quadrupole): m/z calcd for C11H13FN4 [M]•+ = 220.2461, found 220.0000. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C11H13FN4 [M + H]+ = 221.2461; found 221.1000 (Figures S109–S113).
  • 6-fluoro-5-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazole (6b)
Solid orange crystals; molecular formula: C12H15FN4. Yield 0.85 g, 80.45%, M.P.: 118–120 °C (literature [61], 120–122 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3292, 3103, 2939, 2848, 2808, 1665, 1599, 1455, 1410, 1143, 830. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.22 (s, 3H), 2.97 (bs, 4H), 3.42 (bs, 4H), 7.16 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 12.4 Hz, 1H), 8.12 (s, 1H), 12.38 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.38, 152.03, 142.53, 142.22, 136.67, 104.85, 101.38, 54.90, 51.01, 45.85. GC–MS (EI, single quadrupole): m/z calcd for: C12H15FN4 [M]•+ = 234.2727, found [M]•+ = 234.1000. HRMS (ESI+, TOF): m/z calcd for: C12H15FN4 [M + H]+ = 235.2727; found [M + H]+ = 235.13520. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C12H15FN4 [M + H]+ = 235.2727; found [M + H]+ = 235.13520 (Figures S114–S119).
  • 6-fluoro-5-(4-ethylpiperazin-1-yl)-1H-benzo[d]imidazole (6c)
Solid orange crystals; molecular formula: C13H17FN4. Yield 0.85 g, 81.7%, M.P.: 138–139 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3254, 3097, 2968, 2939, 2845, 2815, 2293, 1694, 1601, 1473, 1142, 1024, 831. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.02 (s, 3H), 2.37 (s, 2H), 2.51 (bs, 4H), 2.97 (s, 1H), 3.40 (bs, 4H), 7.15 (s, 1H), 7.36 (d, J = 11.5 Hz, 1H), 8.12 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 152.14, 151.86, 142.20, 136.74, 128.97, 104.86, 101.01, 51.70, 51.15, 12.09. GC–MS (EI, single quadrupole): m/z calcd for: C13H17FN4 [M]•+ = 248.2993, found [M]•+ = 248.0000. HRMS (ESI+, TOF): m/z calcd for: C13H17FN4 [M + H]+ = 249.2993; found [M + H]+ = 249.15130. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C13H17FN4 [M + H]+ = 249.2993; found [M + H]+ = 249.1000 (Figures S120–S125).
  • 6-fluoro-5-(4-isopropylpiperazin-1-yl)-1H-benzo[d]imidazole (6d)
Solid orange crystals. Molecular formula: C14H19FN4. Yield 0.85 g, 81.2%, M.P.: 147–149 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3774, 3715, 3101, 2975, 2821, 2359, 1696, 1605, 1467, 1267, 1141, 867. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 0.84 (d, J = 6.6 Hz, 6H), 2.43 (s, 4H), 2.80 (s, 1H), 3.25 (s, 4H), 6.93 (bs, 1H), 7.21 (bs, 1H), 7.96 (s, 1H), 12.19 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 151.85, 142.15, 139.37, 136.99, 129.77, 105.18, 101.01, 53.74, 51.52, 48.26, 18.28. GC–MS (EI, single quadrupole): m/z calcd for: C14H19FN4 [M]•+ = 262.3259, found [M]•+ = 262.2000 (Figures S126–S129).
  • 6-fluoro-5-(4-butylpiperazin-1-yl)-1H-benzo[d]imidazole (6e)
Solid orange crystals; molecular formula: C15H21FN4. Yield 0.93 g, 89.8%, M.P.: 140–142 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3261, 3095, 2932, 2814, 1702, 1599, 1471, 1263, 1143, 834. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 0.89 (s, 3H), 1.31 (s, 2H), 1.43 (s, 2H), 2.31 (s, 2H), 2.97 (s, 4H), 3.38 (s, 4H), 7.16 (s, 1H), 7.36 (d, J = 12.4 Hz, 1H), 8.12 (s, 1H), 12.36 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.39, 152.04, 142.73, 142.17, 136.69, 104.26, 102.26, 57.63, 53.05, 51.16, 28.56, 20.18, 14.01. GC–MS (EI, single quadrupole): m/z calcd for: C15H21FN4 [M]•+ = 276.3524, found [M]•+ = 276.2000 (Figures S130–S133).
  • 6-fluoro-5-(4-phenylpiperazin-1-yl)-1H-benzo[d]imidazole (6f)
Solid orange crystals; molecular formula: C17H17FN4. Yield 0.84 g, 81.1%, M.P.: 130–132 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3099, 3053, 3020, 2968, 2944, 2815, 1664, 1598, 1476, 1229, 1140, 937, 761. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 3.13 (bs, 4H), 3.30 (bs, 4H), 6.67 (s, 1H), 6.81 (t, J = 7.3 Hz, 1H), 7.00 (d, J = 7.9 Hz, 2H), 7.24 (dd, J = 6.9, 1.7 Hz, 2H), 7.25 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 12.4 Hz, 1H), 8.14 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 151.23, 142.38, 142.17, 136.45, 129.05, 129.19, 119.07, 115.49, 116.60, 103.60, 103.11, 51.20, 48.61. GC–MS (EI, single quadrupole): m/z calcd for: C17H17FN4 [M]•+ = 296.3421, found [M]•+ = 296.1000. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C17H17FN4 [M + H]+ = 297.3421 found [M + H]+ = 297.2000 (Figures S134–S138).
  • 6-fluoro-5-(4-pyrrolidin-1-yl)-1H-benzo[d]imidazole (6g)
6-fluoro-5-(4-pyrrolidin-1-yl)-1H-benzo[d]imidazole (6g) was prepared by charging 4-fluoro-2-nitro-5-(pyrrolidin-1-yl) aniline (1.00 g, 4.440 mmol), iron powder 325 mesh (2.234 g, 10.0 mmol), NH4Cl (2.139 g, 10.0 mmol), and a magnetic stir bar in a 50 mL round bottom flask. Then, 2-PrOH (20.0 mL) and formic acid (20.0 mL) were added, the reaction mixture was refluxed at 80 °C for 4.0 h, and progress was monitored using TLC, which showed complete conversion to the product. The reaction mixture was diluted with 2-PrOH (40 mL) and filtered to remove insoluble materials. The filtrate was concentrated to dryness, and the resulting residue was partitioned between CH2Cl2 (20 mL) and (10 mL) sat. aq NaHCO3 to pH 8.0. The aqueous layer was extracted with additional CH2Cl2 (5 × 20 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated to yield the pure product as solid orange crystals. Solid orange crystals. Molecular formula: C11H12FN3. Yield 0.86 g, 94.4%, M.P.: 190–192 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3099, 2971, 2880, 2812, 1634, 1580, 1467, 1413, 1135, 948, 758. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.95 (m, 4H), 6.86 (d, J = 7.7 Hz), 3.30 (td, J = 6.4, 2.9 Hz, 4H), 7.30 (d, J = 14.9 Hz, 1H), 7.93 (s, 2H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.15, 150.36, 139.95, 135.88, 133.04, 103.40, 99.49, 50.67, 25.11. GC–MS (EI, single quadrupole): m/z calcd for: C11H12FN3 [M]•+ = 205.2315, found [M]•+ = 205.1000. LC–MS (ESI, APCI, positive/negative): m/z calcd for C11H12FN3 [M + H]+ = 206.2315, [M − H]− = 204.2315 found [M + H]+ = 206.3000, [M − H]− = 204.2000 (Figures S139–S144).
  • 6-fluoro-5-(4-piperidin-1-yl)-1H-benzo[d]imidazole (6h)
Solid orange crystals. Molecular formula: C12H14FN3. Yield 0.96 g, 95.4%, M.P.: 168–170 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3099, 2938, 2858, 2804, 1640, 1582, 1471, 1271, 1133, 851. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 1.51 (dt, J = 11.8, 6.0 Hz, 2H), 1.66 (s, 4H), 2.91 (s, 4H), 3.38 (s, 1H), 7.15 (s, 1H), 7.34 (d, J = 12.3 Hz, 1H), 8.11 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.49, 152.14, 142.08, 137.75, 130.26, 104.54, 101.93, 52.58, 26.03, 23.82. GC–MS (EI, single quadrupole): m/z calcd for: C12H14FN3 [M]•+ = 219.2581, found [M]•+ = 219.2000. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C12H14FN3 [M + H]+ = 220.2581; found [M + H]+ = 220.2000 (Figures S145–S149).
  • 6-fluoro-5-(4-morpholine-1-yl)-1H-benzo[d]imidazole (6i)
Solid orange crystals; molecular formula: C11H12FN3O. Yield 0.81 g, 82.8%, M.P.: 209–211 °C, (literature [61], 208–210 °C). FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3128, 3088, 2959, 2862, 1601, 1473, 1259, 1160, 1123, 911, 867. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.97 (m, 4H), 3.38 (s, 1H), 3.76 (m, 4H), 7.18 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 12.5 Hz, 1H), 8.14 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.38, 152.03, 142.35, 136.47, 136.40, 103.60, 103.04, 66.37, 51.56. GC–MS (EI, single quadrupole): m/z calcd for: C12H14FN3 [M]•+ = 221.2309, found [M]•+ = 221.1000. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C12H14FN3 [M + H]+ = 221.2309; found [M + H]+ = 222.1000 (Figures S150–S154).
  • 2-(4-(6-fluoro-1H-benzo[d]imidazol-5-yl)piperazin-1-yl)ethanol (6j)
Solid orange crystals; molecular formula: C13H17FN4O. Yield 0.87 g, 83.71%, M.P.: 120–122 °C. FT-IR (Platinum-ATR, diamond interface, υ/cm−1): 3255, 2983, 2915, 2810, 1582, 1473, 1329, 1137, 937, 701. 1H NMR (700 MHz, δ/ppm, CDCl3): δ 2.55 (t, 2H), 2.70 (s, 4H), 3.01 (s, 4H), 3.54 (bs, 1H), 3.57 (t, 2H), 4.66 (s, 1H), 7.17 (bs, 1H), 7.36 (d, J = 12.4 Hz, 1H), 8.12 (s, 1H). 13C NMR (176 MHz, δ/ppm, CDCl3): δ 153.43, 152.08, 142.34, 142.31, 136.53, 104.34, 101.73, 60.01, 58.13, 53.22, 50.74. LC–MS (ESI, APCI, positive/negative): m/z calcd for C13H17FN4O [M + H]+ = 264.2987, [M − H]− = 263.2987 found [M + H]+ = 265.3000, [M − H]− = 263.2000. MS (ESI+/APCI+ Advion Expression CMS Single Quadrupole): m/z calcd for: C12H14FN3 [M + H]+ = 264.2987; found [M + H]+ = 265.2000 (Figures S155–S160).

3.1.7. Antibacterial Activity

The novel titled compounds were screened for their antibacterial activity using the agar well diffusion method according to Magaladi [62]. Antibacterial activity was evaluated against Gram-positive (Staphylococcus aureus, Bacillus cereus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria. The bacterial strains were provided by the Biology Department, Sultan Qaboos University, Oman. Nutrient agar (NA) was used as a nutrient medium. Nutrient agar medium (OXOID, CM0003, Basingstoke, UK; pH 7.4 ± 0.2 at 25 °C) was used as a culture medium and prepared as per the manufacturer’s protocol and sterilized at 121 °C for 15 min before use in bacterial culture and antimicrobial assays. The culture plates that contain the bacterial strains were incubated and grown on nutrient agar at 37 °C for 24 h. Antibacterial activity was determined by measuring the diameter (mm) of the inhibition zone and recorded. Dimethyl sulfoxide (DMSO) served as the negative control, and all test samples were suspended using DMSO as the solvent. Nitrofurantoin 300 μg/mL was used as the positive control for all tested bacteria.

3.1.8. Minimum Inhibition Concentration (MIC) Determination

Minimum inhibitory concentrations (MICs) were determined using glass sterilized tubes of nutrient broth (OXOID, Basingstoke, UK), which was used as a culture medium. The synthesized benzimidazole 6h was tested for MIC against the selected bacterial strains B. cereus, K. pneumoniae and S. aureus. The tubes were incubated at 37 °C for 24 h, and the optical density was measured at 600 nm using a Thermo Spectronic Helios E spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The nutrient broth was diluted from different concentrations of the synthesized benzimidazole 6h at 5.00, 2.50, 1.25, 0.625, and 0.031 µg/mL. For sample preparation, each of the test compounds and standards was dissolved in DMSO, at a concentration of 5.00 μg/mL. Further dilutions of the compounds and standards in the test medium were prepared at the required quantities of 5.00, 2.50, 1.25, 0.625, and 0.031 µg/mL.

3.1.9. Scanning Electron Microscopy (SEM) Analysis

Bacterial cells treated with compound 6h (2.5 µg/mL) were examined using scanning electron microscopy (SEM) to reveal structural changes, and the findings were compared with untreated controls. SEM was completed using a JEOL JSM-5600LV scanning electron microscope (JEOL Ltd., Tokyo, Japan). Images were obtained at an accelerating voltage of 20 kV using a secondary electron (SE) detector. Samples were prepared under a vacuum of 1 × 10−3 Pa and coated with a 10 nm gold layer prior to imaging. Micrographic images were captured at ×8000 and ×16,000 magnification levels. For the SEM experiment, the samples were prepared following the protocol proposed by Bozzola and Russell [63] with some modifications. For every sample and corresponding culture plate, a 2 cm × 2 cm section containing bacterial colonies was moved to a processing basket, and then transferred into a 20 mL processing vial that contained EM fixative (2.5% glutaraldehyde) left on a mixer for 2 h. Afterward, the samples were rinsed twice using a cacodylate buffer (pH 7.2–7.4) for 5 min each, then post-fixed using osmium tetroxide for one hour. After that, the samples were dehydrated in a series of ethanol, starting with distilled water and advancing through increasing concentrations of ethanol, with 25% ethanol, 75% ethanol, 95% ethanol, and 99.9% ethanol, each for 10 min. The samples were mixed with hexamethyldisilazane and absolute ethanol in a 1:1 ratio for 30 min, then the ratio was increased to 1:3 for 10 min. They were next transferred into pure hexamethyldisilazane (HMDS) for 30 min, after which they were allowed to air-dry. Afterwards, samples were attached to aluminum stubs measuring 10 mm in diameter and subsequently coated with gold using BioRad SEM coating devices (Bio-Rad, Oxfordshire, UK). A JEOL JSM 4500LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) operating at 20 KV accelerating voltage was used to screen the samples. The last set of micrographs was obtained.

3.1.10. Antifungal Activity

The antifungal activity was recorded against fungal strains Aspergillus flavus, Penicillium duclauxii, P. italicum at 20 and 50 μg/mL levels for the synthesized benzimidazole derivatives. The fungal strains were provided by the Biology Department, Sultan Qaboos University, Oman. The fungal strains were incubated and grown on Potato Dextrose Agar (PDA) (HiMedia Laboratories GmbH—Einhausen, Germany) at 25 °C. The wells were made with a sterile cork borer (8 mm diameter). The culture plates were incubated at 25 °C for 3–7 days. Antifungal activity was determined by measuring the diameter (mm) of the inhibition zone. Amphotericin B at 20 μg/mL and 50 μg/mL was used as a positive control drug. Dimethyl sulfoxide (DMSO) was used as a negative solvent control, which did not show any zone of inhibition.

4. Conclusions

A series of fluoro-substituted benzimidazole derivatives 6a–j was synthesized and structurally confirmed using FT-IR, 1H-NMR, 13C-NMR, and LC–MS analyses. All compounds were examined for their antibacterial and antifungal activities, revealing that the biological activity was considerably influenced by the effects of lipophilicity and by the nature of the substituents on the benzimidazole ring, electronic distribution across the benzimidazole scaffold, and cell penetration. Among the entire set of derivatives, compounds 6e, 6f, 6h and 6j showed the highest antimicrobial effectiveness, exhibiting inhibition zones analogous to standard reference drugs. The existence of electron-withdrawing groups like fluorine considerably enhances the activity, and it is proposed that electron-withdrawing substituents increase both the spectrum of activity and potency. Minimum inhibitory concentration (MIC) studies proved the efficient antibacterial impacts of 6h, with an MIC value of 5.00 µg/mL. However, MIC, minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) studies should be performed in future investigations as part of an extended pharmacological study. The SEM analysis of compound 6h against S. aureus and K. pneumoniae showed pronounced morphological modifications, providing indirect evidence of membrane-associated damage. On the whole, the findings reveal that fluoro-substituted benzimidazoles exhibit a significant antibacterial and antifungal potential, and the inclusion of fluorine substituents substantially enhances their activity. These results propose that such derivatives can function as precious lead compounds for the improvement of new broad-spectrum antimicrobial agents.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31050844/s1. Figures S1–S160 are available as Supplementary Materials.

Author Contributions

Conceptualization, R.J.A.-J.; methodology, S.A., R.J.A.-J., N.S. and S.H.A.H.; software, R.J.A.-J., N.S. and S.H.A.H.; validation, R.J.A.-J., N.S. and S.H.A.H.; formal analysis, S.A., R.J.A.-J., N.S. and S.H.A.H.; investigation, S.A., R.J.A.-J., N.S. and S.H.A.H.; resources, R.J.A.-J., N.S. and S.H.A.H.; Data curation, S.A. and R.J.A.-J.; writing—original draft preparation, S.A., R.J.A.-J., N.S. and S.H.A.H.; writing—review and editing; S.A., R.J.A.-J., N.S. and S.H.A.H.; visualization, R.J.A.-J., N.S. and S.H.A.H.; supervision, R.J.A.-J., N.S. and S.H.A.H.; project administration, R.J.A.-J.; funding acquisition, R.J.A.-J. All authors have read and agreed to the published version of the manuscript.

Funding

Sultan Qaboos University is gratefully acknowledged for its financial support (S.A., PhD; Bench Fees).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors would like to thank the Central Analytical and Applied Research Unit (CAARU) for its FT-IR, LC-MS, GC-MS and NMR spectroscopic measurements (Zahra Al-Mamari, Samuel Premkumar and Shareef Alhashemi). The authors would like to thank the college of medicine and health sciences for scanning electron microscopy (Mohammad Al Kindi). The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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