# Synthesis, antimicrobial and antibiofilm activities of silver(I) complexes with N-alkylbenzimidazole derivatives and their protein interaction modelling study

Neslihan Şahin, Mohamed Ali Mosrati, Abderrahmen Merghni, İsmail Özdemir, Hayet Sellami, Kawther Bedchiche, Salim Krayiem, Sami Aifa, Dorra Abdelmalek, David Sémeril

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Neslihan Şahin, Mohamed Ali Mosrati, Abderrahmen Merghni, İsmail Özdemir, Hayet Sellami, et al. Synthesis, antimicrobial and antibiofilm activities of silver(I) complexes with N-alkylbenzimidazole derivatives and their protein interaction modelling study. Journal of Molecular Structure, 2025, 1322, pp.140440. 10.1016/j.molstruc.2024.140440. hal-05336543

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# Synthesis, antimicrobial and antibiofilm activities of silver(I) complexes with N-alkylbenzimidazole derivatives and their protein interaction modelling study

Neslihan Şahin<sup>a,b</sup>, Mohamed Ali Mosrati<sup>c</sup>, Abderrahmen Merghni<sup>d</sup>, İsmail Özdemir<sup>e</sup>,
Hayet Sellami<sup>f,g</sup>, Kawther Bedchiche<sup>c</sup>, Salim Krayiem<sup>c</sup>, Sami Aifa<sup>c</sup>, Dorra Abdelmalek<sup>c,*</sup>,
David Sémeril<sup>b,*</sup>

## ARTICLE INFO

### Keywords:

- Silver complex

- Antibacterial

- Antibiofilm

- Molecular docking

- Protein interaction model

## ABSTRACT

This study investigates the antimicrobial and antibiofilm potential of three silver(I) complexes derived from N-alkylbenzimidazole derivatives: bis[(Z)-1-styryl-benzimidazole]silver(I) nitrate (4), bis[(E)-1-styryl-benzimidazole]silver(I) nitrate (5), and bis(1-cinnamyl-benzimidazole)silver(I) nitrate (6). Detailed synthesis and characterization of these complexes are followed by assessments of their efficacy against *Staphylococcus aureus*, *Enterococcus faecalis*, *Pseudomonas aeruginosa*, *Escherichia coli*, and *Candida albicans*. Among the tested compounds, complex 6 exhibited the highest antimicrobial activity, with a minimal inhibitory concentration of 6 µmol/L against *P. aeruginosa*, and significant antibiofilm activity, achieving 67.72 % inhibition at a concentration of 3 µmol/L. These interesting findings led us to conduct a molecular docking study to understand the mechanisms of action by investigating the interactions between the silver(I) complexes and some key protein targets involved in bacterial and fungal biofilm formation, including (p)ppGpp synthetase/hydrolases, FtsZ proteins, and pyruvate kinases. This comprehensive approach, combining experimental and computational analyses derived from N-alkylbenzimidazole derivatives, particularly complex 6, which exhibits remarkable efficacy against various pathogens, reveals promising therapeutic applications of these silver(I) complexes and advances our understanding of their potential mode of action against biofilm-associated infections.

## 1. Introduction

Although there are many antibacterial drugs currently available, microbial resistance to antibiotics becomes a major problem [1-3]. As example, *Pseudomonas aeruginosa*, which is a bacterial species resistant to a large number of antibiotics, is particularly dangerous for patients in intensive care who need respiratory assistance. This Gram-negative strain forms bacterial biofilms that are difficult to clean on endotra-cheal incubation probes inserted into the respiratory tract, causing pneumonia in about 25 % of patients [4-6]. Therefore, the development

of new antibiotics against resistant bacteria is a necessity.

In this context, silver is particularly interesting, its natural antibacterial action being known since antiquity [7-8]. Although many silver complexes, especially N-heterocyclic carbene-silver(I) complexes [9], with antimicrobial properties have been developed, their antibiofilm activities have been underdeveloped. With the exception of silver nanoparticles [10-14], only a few studies have been realized such as the work of Youngs and co-workers who studied the antibiofilm properties of the carbine-silver(I) complex A (Fig. 1). The authors demonstrated that complex A was more efficient than currently used clinical

<footnote><sup>a</sup> Department of Science Education, Faculty of Education, Cumhuriyet University, 58040 Sivas, Turkey</footnote>

<footnote><sup>b</sup> Synthèse Organométallique et Catalyse, UMR-CNRS 7177, Strasbourg University, 67008 Strasbourg, France</footnote>

<footnote><sup>c</sup> Laboratory of Molecular and Cellular Screening Processes, Centre of Biotechnology of Sfax, University of Sfax, 3041 Sfax, Tunisia</footnote>

<footnote><sup>d</sup> Laboratory of Antimicrobial Resistance LR99ES09, Faculty of Medicine of Tunis, University of Tunis El Manar, 1007 Tunis, Tunisia</footnote>

<footnote><sup>e</sup> Drug Application and Research Center, İnönü University, 44280 Malatya, Turkey</footnote>

<footnote><sup>f</sup> Parasitology and Mycology Laboratory, Faculty of Medicine of Sfax, University of Sfax, 3041 Sfax, Tunisia</footnote>

<footnote><sup>g</sup> Drosophila research unit UR22ES03 - Faculty of Medicine of Sfax, University of Sfax, 3041 Sfax, Tunisia</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: dorra.abdelmalek@cbs.rnrt.tn (D. Abdelmalek), dsemeril@unsitra.fr (D. Sémeril).</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. Example of N-heterocyclic carbene-silver(I) complexes A-F exhibiting antibiofilm activities.</figcaption></figure>

antibiotics ciprofloxacin and doxycycline against biofilm organisms of *Bacillus anthracis*. Similar or slightly better efficiency was observed when silver(I) complex was employed instead of the aminoglycoside antibiotic gentamicin against biofilms of *B. anthracis* and methicillin resistant *Staphylococcus aureus* [15].

Activities of several N-heterocyclic carbene-silver(I) complexes against biofilm formation of pathogenic bacteria (*Listeria monocytogenes*, *Staphylococcus aureus*, *Staphylococcus epidermis*, *Pseudomonas aeruginosa* and *Escherichia coli*) were reported by Roland and co-workers using the high-throughput screening methodology. The tests realized at a concentration of 1 mg/mL revealed that complexes bearing lipophilic N-heterocyclic carbene ligands with bulky aromatic groups on the nitrogen atom for example complex B of Fig. 1) were the most efficient in inhibiting biofilm formation [16].

Recently, Şahin and co-workers reported four families of N-heterocyclic carbene-silver(I) complexes whose most effective drugs in each set are chloro[1-allyl-3-(2,4,6-trimethylbenzyl)-5,6-dimethyl-benzimidazole-2-ylidene]silver(I) (C) [17], chloro[1-methallyl-3-benzyl-5, 6-dimethyl-benzimidazole-2-ylidene]silver(I) (D) [18], bromo[1-allyl-3-(4-tert-butylbenzyl)-5,6-di-methylbenzimidazole-2-ylidene] silver(I) (E) [19] and chloro[1-methallyl-3-(anthracen-9-yl-methyl) benzimidazol-2-ylidene]silver(I) (F) [20] (Fig. 1). These complexes have been tested as antibiofilms of Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Biofilm inhibitions in the range of 59 to 76 % were measured with complexes C and D at a concentration of 3.9 and 9.5 µg/mL, respectively. The two other complexes (E and F) were more efficient and displayed biofilm inhibitions higher than 90 % at a concentration of 1.9 µg/mL. Only molecular docking studies with deoxyribonucleic acid gyrase [21], Quorum-Quenching N-Acyl Homoserine Lactone Lactonase [22] and SarA [23] have been carried out, the mechanisms of action of these complexes remain unknown.

On the other hand, many compounds containing the aromatic benzimidazole moiety [24-29] or a transition metal center [30] have displayed effectiveness in the inhibition of biofilms. Therefore, in continuation of our previous reports [31-32], we now report the synthesis of cationic bis-(N-alkylbenzimidazole)-silver(I) complexes, their ability to inhibit bacterial biofilm formation and a rigorous in silico investigation to elucidate the intricacies of the protein interaction model

with the specifically targeted proteins, thereby providing deeper insights into the underlying mechanisms governing the antifungal and antibacterial activities.

## 2. Materials and methods

### 2.1. General

The manipulations were carried out under dry argon with dried solvents. Routine ¹H and ¹³C{¹H} spectra were recorded with AC 500 Bruker FT instrument. Chemical shifts and coupling constants are reported in ppm and Hz, respectively. ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ and they referenced to residual protonated solvent (δ = 2.50 and 39.52 ppm, respectively). Mass spectra were recorded on a Bruker MicroTOF spectrometer (ESI-TOF). Infrared spectra were recorded on a Bruker FT-IR Alpha-P spectrometer. The molar conductance was determined for 10⁻³ M solution of complexes in DMSO at 20.8 °C with a five Easy Mettler Toledo LE703 conductometer. Elemental analyses were carried out by the Service de Microanalyse, Institut de Chimie, Université de Strasbourg. (Z)-1-Styryl-benzimidazole (1) [33], (E)-1-styryl-benzimidazole (2) [34] and 1-cinnamyl-benzimidazole (3) [35] were prepared according to adapted published procedures.

### 2.2. General procedure for the synthesis of bis(1-alkyl/alkenyl-benzimidazole)silver(I) nitrate complexes

The silver(I) complexes were synthesized according to the reported procedure [31]. Under an inert atmosphere of argon, in a Schlenk tube wrapped in aluminum foil, AgNO₃ (0.07 mmol) was added to a stirring solution of 1-alkyl/alkenyl-benzimidazole (1-3; 0.14 mmol) in ethanol (5 mL). After 4 h at room temperature, the formed precipitate was filtered, washed with diethyl ether (3 × 10 mL) and dried under vacuum to give silver(I) complexes 4-6.

Bis[(Z)-1-styryl-benzimidazole]silver(I) nitrate (4): Yield: 89 %; white solid; Molar conductance 37.4 Ω⁻¹ cm² mol⁻¹; FT-IR: ν(NO₃) 1318 and ν(CN) 1502 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.34 (s, 2H, NCHN), 7.80 (dt, 2H, arom CH, ³JHH = 8.0 Hz, ⁴JHH = 1.2 Hz), 7.42-7.39 (m, 2H, CH arom), 7.36-7.30 (m, 4H, CH arom), 7.24-7.22
<figure><img src="image_4.png" /><figcaption>Scheme 1. Synthesis of the silver(I) complexes 4–6.</figcaption></figure>

(m, 6H, CH arom), 7.20 (d, 2H, NCH=CHPh, ³JHH = 8.0 Hz), 7.02–7.00 (m, 4H, CH arom), 6.88 (d, 2H, NCH=CHPh, ³JHH = 8.0 Hz); ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 144.04 (s, NCHN), 141.64, 133.62, 132.16, 128.94, 128.63, 128.44, 124.32, 123.52, 119.59, 111.65 (10 s, arom Cs), 127.50 (s, NCH=CHPh), 120.93 (s, NCH=CHPh) ppm. MS (ESI-TOF): *m/z* = 547.10 [M - NO₃]⁺ (expected isotopic profile). Elemental analysis (%): calcd for C₃₀H₂₄AgN₅O₃ (610.42): C: 59.03; H: 3.96; N: 11.47; found C: 58.95; H: 3.87 N: 11.38.

Bis[(E)-1-styryl-benzimidazole]silver(I) nitrate (5): Yield: 86%; beige solid; Molar conductance 37.8 Ω⁻¹ cm² mol⁻¹; FT-IR: ν(NO₃) 1316 and ν(CN) 1506 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.93 (s, 2H, NCHN), 8.10 (d, 2H, NCH=CHPh, ³JHH = 15.0 Hz), 8.08 (d, 2H, arom CH, ³JHH = 8.0 Hz), 7.84 (d, 2H, arom CH, ³JHH = 8.0 Hz), 7.68 (d, 4H, arom CH, ³JHH = 7.0 Hz), 7.47–7.38 (m, 8H, CH arom), 7.33 (d, 2H, arom CH, ³JHH = 8.0 Hz), 7.31 (d, 2H, NCH=CHPh, ³JHH = 15.0 Hz); ¹³C {¹H} NMR (126 MHz, DMSO-d₆): δ = 142.77 (s, NCHN), 142.46, 135.03, 132.06, 128.99, 128.03, 126.62, 124.44, 123.68, 119.80, 112.05 (10 s, arom Cs), 122.00 (s, NCH=CHPh), 119.62 (s, NCH=CHPh) ppm. MS (ESI-TOF): *m/z* = 547.10 [M - NO₃]⁺ (expected isotopic profile). Elemental analysis (%): calcd for C₃₀H₂₄AgN₅O₃ (610.42): C: 59.03; H: 3.96; N: 11.47; found C: 58.99; H: 3.93 N: 11.41.

Bis(1-cinnamyl-benzimidazole)silver(I) nitrate (6): Yield: 86%; white solid; Molar conductance 34.9 Ω⁻¹ cm² mol⁻¹; FT-IR: ν(NO₃) 1325 and ν(CN) 1508 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.64 (s, 2H, NCHN), 7.86–7.84 (m, 2H, CH arom), 7.77–7.75 (m, 2H, CH arom), 7.44–7.42 (m, 4H, CH arom), 7.40–7.35 (m, 4H, CH arom), 7.35–7.31 (m, 4H, CH arom), 7.26 (tt, 2H, CH arom, ³JHH = 7.2 Hz, ⁴JHH = 1.5 Hz), 6.72 (d, 2H, NCH₂CH=CHPh, ³JHH = 16.0 Hz), 6.52 (dt, 2H, NCH₂CH=CHPh, ³JHH = 16.0 Hz, ³JHH = 6.5 Hz), 5.18 (d, 4H, NCH₂CH=CHPh, ³JHH = 6.5 Hz); ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ

= 145.46 (s, NCHN), 141.85, 135.86, 133.00, 128.88, 128.29, 126.66, 123.85, 123.17, 119.37, 111.67 (10 s, arom Cs), 133.40 (s, NCH₂CH=CHPh), 124.15 (s, NCH₂CH=CHPh), 46.91 (s, NCH₂CH=CHPh) ppm. MS (ESI-TOF): *m/z* = 575.14 [M - NO₃]⁺ (expected isotopic profile). Elemental analysis (%): calcd for C₃₂H₂₈AgN₅O₃ (638.47): C: 60.20; H: 4.42; N: 10.97; found C: 60.28; H: 4.47 N: 10.93.

## 2.3. X-Ray crystal structure analysis

Single crystals of silver(I) complexes 4 and 6, suitable for X-ray analysis, were obtained by slow diffusion of Et₂O into a CH₂Cl₂ solution of the complex. The samples were studied on a Bruker APEX-II CCD, using Mo-Kα radiation (λ = 0.71073 Å) at T = 173(2) K. The structures were solved with SHELXT-2018/2 [36], which revealed the non-hydrogen atoms of the molecule. After anisotropic refinement, all of the hydrogen atoms were found with a Fourier difference map. The structure was refined with SHELXL-2019/3 [37] by the full-matrix least-square techniques (use of *F* square magnitude; x, y, z, βᵢⱼ for C, Ag, N and O atoms; x, y, z in riding mode for H atoms) (see Table S1 in Supplementary Materials).

## 2.4. Antimicrobial activity

The antimicrobial activities of the tested compounds were assessed by the determination of the minimal inhibitory concentration (MIC), the minimal bactericidal concentration (MBC) and the minimal fungicidal concentration (MFC) values, according to the recommendations of the CLSI [38]. Two Gram-positive strains, namely *S. aureus* (ATCC 43,300) and *E. faecalis* (ATCC 29,212), two Gram-negative strains, *E. coli* (ATCC 25,922) and *P. aeruginosa* (ATCC 27,853) as well as fungal strain
<figure><img src="image_5.png" /><figcaption>Fig. 2. ¹H NMR spectra of 1-styryl-benzimidazole (1) (top) and its corresponding bis[(Z)-1-styryl-benzimidazole]silver(I) nitrate (4) complex (bottom) in DMSO-d₆.</figcaption></figure>

C. albicans (ATCC 2091) were used for the antimicrobial activities. Stock solutions of each compound were prepared aseptically in DMSO 10% (v/v), and transferred to sterile 96-well microliter plates containing the Mueller-Hinton broth (MHB). Each compound was then subjected to a series of cascade dilutions to achieve a final concentration ranging from 0.97 to 500 µg/mL. The final volume in each microplate well was fixed to 200 µl, containing a specific concentration of each compound, the growth medium (MH) and the microbial inoculum (0.5 McF). The negative control, which served as a reference and did not contain any compound, was also included. Plates were then placed in an incubator at a temperature of 37 °C for 24 h. MIC was determined as the lowest concentration of each compound that inhibits the growth of test microorganisms.

## 2.5. Antibiofilm effect

The antibiofilm activity of each compound was evaluated by the crystal violet (CV) staining test. Mature biofilms of 48 from each bacterial strain tested are established in sterile 96-well microplates as previously described [39]. Following incubation, non-adherent bacterial cells were removed and the established biofilms were treated with half MIC concentrations (MIC/2) of the tested compounds prepared in DMSO and Brain Hear Infusion (BHI) broth for each bacterial strain and Sabouraud's dextrose broth for the fungal strain [40-41]. After 24 h of incubation, biofilms were stained with 100 µl of CV for 30 min, then pates were washed, air dried and biofilm biomasses were measured using a microplate reader, untreated biofilms represent the negative control. The formula [42] below calculates the percentage of biofilm inhibition: %Biofilm Eradication: [(OD growth control-OD sample)/OD growth control] × 100

## 2.6. Molecular docking investigation

Predicted 3D structures of the target proteins and crystal structures were downloaded from AlphaFold [43] and PDB [44] databases, respectively. Molecular Operating Environment 2019.0102 (MOE) (2019) was used for sequence alignment, homology modeling and structural visualization. During the docking process, we introduced counterion $NO_3^-$ as a co-ligand. The models were stereochemically evaluated by the program PROCHECK [45]. The complex structures were subjected to energy minimization (geometry optimization) using MOE program.

## 3. Results and discussion

In the present study, three silver(I) complexes will be considered, namely bis[(Z)-1-styryl-benzimidazole]silver(I) nitrate (4), bis[(E)-1-styryl-benzimidazole]silver(I) nitrate (5) and bis(1-cinnamyl-benzimidazole)silver(I) nitrate (6). The structure of these complexes will allow us to observe the influence of the stereochemistry of the double bond (cis or trans) and its position regarding to the benzimidazole (presence of a $CH_2$ linker) on their bactericidal properties.

### 3.1. Synthesis of the silver(I) complexes

The three targeted cationic bis-(N-alkylbenzimidazole)-silver(I) complexes 4–6 were obtained by mixing two equivalents of N-alkyl-benzimidazole ligands 1–3 with one equivalent of $AgNO_3$ salt in ethanol. After 4 h at room temperature, the reaction mixture, which was protected from light with an aluminum foil, was filtered and the silver(I) complexes were isolated in 86–89% yield (Scheme 1).

The complexes are sensitive to light but stable to air and moisture in the solid state. The stability of these complexes in solution (DMSO-d6) was confirmed by recording ¹H NMR spectra every 2 days for 10 days. The solution was kept in the dark during the measurements. According to the recorded spectra, no change in position and intensity of the signals was observed, which confirms the stability of the complexes (see Figures S13-S15 in Supplementary Materials). They were fully characterized by infrared spectroscopy (FT-IR), multi-nuclear magnetic resonance spectroscopy (¹H and ¹³C NMR), mass spectroscopy and elemental analysis (see Section 2 and Figures S1-S12 in Supplementary Materials).

In comparison with the ligands 1–3, the FT-IR spectra of the complexes 4–6 displayed a redshifted band for the C = N vibration (1502–1508 cm⁻¹) from 12 to 20 cm⁻¹, which is characteristic of an azole coordinated to a silver cation [31]. The band observed in the range 1316–1325 cm⁻¹ confirmed the presence of the $NO_3^-$ anion. NMR analysis realized on the silver(I) complexes revealed downfield singlets, in the range of 8.34–8.64 ppm, for the NCHN protons with regard to the ligands (Fig. 2). The stereochemistry of the external carbon-carbon double bonds was determined by using the coupling constants between olefinic protons, $^3J_{HH}$ of 8.0 Hz for the cis-double bond in complex 4 and $^3J_{HH}$ of 15.0 and 16.0 Hz for the trans-double bonds in complexes 5 and 6, respectively.

The presence of two coordinated N-alkylbenzimidazole to the silver cation was unambiguously confirmed by the elemental analysis, which fitted with the formation of $[AgL_2]^{+}NO_3^-$ complexes (L = 1–3) and by the mass spectra of complexes, which revealed intense peaks at m/z = 547.10, for complexes 4 and 5, and at m/z = 575.14, for complex 6,
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** ORTEP drawing of the silver(I) complex **4** (50 % probability thermal ellipsoids). Important bond lengths (Å) and angles (°): Ag1-N1 2.179(2), Ag1-N3 2.165(2), Ag1-O1 2.612(3), N1-C1 1.321(4), N1-C2 1.402(3), N2-C1 1.353(3), N3-C16 1.312(4), N3-C22 1.394(4), N4-C16 1.365(3), N1-Ag1-N3 152.86(9), N1-Ag1-O1 95.54(9), O1-Ag1-N3 111.50(9), C1-N1-Ag1 124.06 (19), Ag1-N1-C2 130.0(2), C1-N1-C2 105.3(2), C16-N3-C22 105.2(2), Ag1-N3-C16 125.8(2), Ag1-N3-C22 126.6(2).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** ORTEP drawing of the silver(I) complex **6** (50 % probability thermal ellipsoids). Important bond lengths (Å) and angles (°): Ag1-N1 2.124(4), Ag1-N3 2.120(4), Ag1-Ag1 3.179(2), Ag1-O1 2.912(3), Ag1-O3 3.091(3), C1-N1 1.322(6), C1-N2 1.355(5), C17-N3 1.309(5), C17-N4 1.350(5), N1-Ag1-N3 168.19(13), N1-Ag1-Ag1 83.62(11), N3-Ag1-Ag1 104.71(11), Ag1-N1-C1 127.7(3), Ag1-N1-C7 126.5(3), N1-C1-N2 112.7(4), Ag1-N3-C17 124.0(3), Ag1-N3-C23 130.2(3), N3-C17-N4 113.6(4).</figcaption></figure>

corresponding [M - NO₃]⁺ cations with the expected isotopic profiles.

## 3.2. Single-crystal X-ray diffraction studies

Formation of cationic bis-(N-alkylbenzimidazole)-silver(I) complexes was confirmed by two single-crystal X-ray diffraction studies realized on complexes **4** and **6**. Suitable crystals were obtained by slow diffusion of diethyl ether into a dichloromethane solution of silver(I) complexes. Both complexes crystallized in the triclinic space group P-1.

In the complex **4** (Fig. 3), the silver atom adopts a triangular geometry [46-48]. Two nitrogen atoms of the two benzimidazole moieties

(Ag1-N1 2.179(2) Å and Ag1-N3 2.165(2) Å, in agreement for a cationic bis-benzimidazole silver(I) complex [49]) and on the oxygen atom of the anion (Ag1-O1 2.612(3) Å) are coordinated to the metal center. The two benzimidazole rings are almost parallel with a dihedral angle of 3.56°. The phenyl groups are oblique to the azole with dihedral angles of 64.62° and 61.07°, respectively.

It is evident that this Ag-O bond, despite being smaller than the sum of the van der Waals radii of two atoms, is longer than an Ag-O covalent bond (2.4 Å), which demonstrates the weakness of this bond. The NO₃⁻ counter anion is coordinated with the silver(I) atom in the crystals used for this crystallographic study. However, in the non-crystalline state and solution, the Ag-O bond no longer exists. This is evident from the infrared spectra, which displays the ν(NO₃) band at 1318 cm⁻¹. When NO₃⁻ is bonded to a silver(I) atom, this frequency shifts to 1354–1384 cm⁻¹ [50-51]. The uncoordination of the NO₃⁻ anion is confirmed by electrical molar conductances carried out in DMSO at room temperature for the three silver(I) complexes. The values in the range 34.9–37.8 Ω⁻¹ cm² mol⁻¹ clearly indicate that the silver(I) complexes **4-6** have a 1:1 ionic nature [52].

Interestingly, in the solid state, the silver(I) complex **6** dimerized (Fig. 4). The two molecules are linked via an argonophilic interaction [53] with Ag1-Ag1 distance of 3.179(2) Å, which is shorter than the sum of the van der Waals radii of two silver atoms (3.44 Å) [54]. The silver cations formed a T-shaped arrangement [55-56] with N1-Ag1-N3 and N1-Ag1-Ag1 angles of 168.19(13)° and 83.62(11)°, respectively. The two coordinate benzimidazole moieties (Ag1-N1 2.124(4) Å and Ag1-N3 2.120(4) Å) on the same silver atom are slightly distorted with dihedral angles of 14.04° and 14.40°, respectively, but two by two parallel with the coordinated aromatic rings of the second silver atom (dihedral angles of 0.0° and 0.39°, respectively). Note that the two anions are localized close to the silver atoms with Ag1-O1 and Ag1-O3 bonds of 2.912(3) Å and 3.091(3) Å, respectively.

## 3.3. Antimicrobial and antibiofilm activities

The bactericidal potential of the three silver(I) complexes **4-6** were evaluated against two Gram-positive bacteria (*E. faecalis* ATCC 29,212 and *S. aureus* ATCC 43,300, which is resistant to methicillin and oxacillin), two Gram-negative bacteria (*E. coli* ATCC 25,922 and *P. aeruginosa* ATCC 27,853) and one fungus (*C. albicans* ATCC 2091).

Minimum inhibitory concentrations (MIC) reported in Table 1 clearly demonstrated the benefit role played by the silver(I) atom. As observed, when N-alkylbenzimidazoles **1-3** were in contact with the bacterial strains and the fungus, the measured MIC values were in the range of 533 to 2270 µmol/L (Table 1, entries 1-3). With the silver(I) drugs, lower MIC values were measured (Table 1, entries 4-6). In the case of the Gram-positive bacteria, the complex **6** displayed the lower MICs, 49 and 98 µmol/L against *E. faecalis* and *S. aureus*, respectively. For the Gram-negative bacteria The cis-olefinic complex **4** led to low MIC values, 26 and 51 µmol/L against *P. aeruginosa* and *E. coli*, respectively. Nevertheless, complex **6** displayed the greatest antimicrobial activity with MIC value as low as 6 µmol/L against *P. aeruginosa*. It would seem that this value is approximately six times lower than those

<table><caption>**Table 1**
Minimum inhibitory concentrations (MICs) of N-alkylbenzimidazoles **1-3** and silver(I) complexes **4-6** (MICs in µmol/L).</caption><thead><tr><th>Entry</th><th>Compound</th><th>S. Aureus</th><th>E. Faecalis</th><th>E. Coli</th><th>P. Aeruginosa</th><th>C. Albicans</th></tr></thead><tbody><tr><td>1</td><td>1</td><td>2270</td><td>2270</td><td>2270</td><td>1135</td><td>2270</td></tr><tr><td>2</td><td>2</td><td>2270</td><td>2270</td><td>2270</td><td>2270</td><td>2270</td></tr><tr><td>3</td><td>3</td><td>2134</td><td>2134</td><td>2134</td><td>2134</td><td>533</td></tr><tr><td>4</td><td>4</td><td>102</td><td>102</td><td>51</td><td>26</td><td>6</td></tr><tr><td>5</td><td>5</td><td>102</td><td>204</td><td>102</td><td>102</td><td>13</td></tr><tr><td>6</td><td>6</td><td>98</td><td>49</td><td>98</td><td>6</td><td>12</td></tr><tr><td>7 [31]</td><td>Ampicillin</td><td>4</td><td>4</td><td>9</td><td></td><td></td></tr><tr><td>8 [31]</td><td>Amikacin</td><td></td><td></td><td></td><td>3</td><td></td></tr><tr><td>9 [31]</td><td>Fluconazole</td><td></td><td></td><td></td><td></td><td>20</td></tr></tbody></table>
<caption>**Table 2**</caption>

Minimum bactericidal concentration (MBSs) and minimum fungicidal concentration (MFCs) of N-alkylbenzimidazoles 1–3 and silver(I) complexes 4–6 (MBSs and MFCs in µmol/L).

<table><caption>**Table 3**</caption><thead><tr><th>Entry</th><th>Compound</th><th>S. Aureus</th><th>E. Faecalis</th><th>E. Coli</th><th>P. Aeruginosa</th><th>C. Albicans</th></tr></thead><tbody><tr><td>1</td><td>1</td><td>> 2270</td><td>> 2270</td><td>> 2270</td><td>> 2270</td><td>2270</td></tr><tr><td>2</td><td>2</td><td>> 2270</td><td>> 2270</td><td>> 2270</td><td>> 2270</td><td>2270</td></tr><tr><td>3</td><td>3</td><td>> 2134</td><td>> 2134</td><td>> 2134</td><td>> 2134</td><td>2134</td></tr><tr><td>4</td><td>4</td><td>205</td><td>819</td><td>205</td><td>410</td><td>51</td></tr><tr><td>5</td><td>5</td><td>205</td><td>819</td><td>410</td><td>205</td><td>26</td></tr><tr><td>6</td><td>6</td><td>196</td><td>783</td><td>392</td><td>98</td><td>25</td></tr></tbody></table>

Biofilm inhibition (%) of silver(I) complexes 4–6 measured at 0.5 MIC (µmol/L).

<table><thead><tr><th>Entry</th><th>Biofilm</th><th>Silver(I) complex</th><th>Inhibition (%)</th><th>0.5 MIC (µmol/L)</th></tr></thead><tbody><tr><td>1</td><td>S. aureus</td><td>4</td><td>26.55</td><td>51</td></tr><tr><td>2</td><td></td><td>5</td><td>41.79</td><td>51</td></tr><tr><td>3</td><td></td><td>6</td><td>24.57</td><td>49</td></tr><tr><td>4</td><td>E. faecalis</td><td>4</td><td>7.68</td><td>51</td></tr><tr><td>5</td><td></td><td>5</td><td>9.90</td><td>102</td></tr><tr><td>6</td><td></td><td>6</td><td>71.37</td><td>24</td></tr><tr><td>7</td><td>E. coli</td><td>4</td><td>8.53</td><td>26</td></tr><tr><td>8</td><td></td><td>5</td><td>17.23</td><td>51</td></tr><tr><td>9</td><td></td><td>6</td><td>9.63</td><td>49</td></tr><tr><td>10</td><td>P. aeruginosa</td><td>4</td><td>56.85</td><td>13</td></tr><tr><td>11</td><td></td><td>5</td><td>24.06</td><td>51</td></tr><tr><td>12</td><td></td><td>6</td><td>67.72</td><td>3</td></tr><tr><td>13</td><td>C. albicans</td><td>4</td><td>17.51</td><td>3</td></tr><tr><td>14</td><td></td><td>5</td><td>0.88</td><td>6</td></tr><tr><td>15</td><td></td><td>6</td><td>3.96</td><td>6</td></tr></tbody></table>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Anti-adherent activity of complexes 4, 5 and 6 as evaluated with crystal violet quantification Assay against S. aureus form MIC and MIC/2 in triplicates.</figcaption></figure>

measured with the bis(1-(2-morpholinoethyl)-5,6-dimethylbenzimidazole)silver(I) nitrate complex (36 µmol/L) [57] and only twice than that the measured MIC with the commercially available drug Amikacin (3 µmol/L [31]; Table 1, entry 8).

We can also observe, that the fungicidal activities of silver(I) complexes are remarkable with MICs of 13 and 12 µmol/L for complexes 5 and 6, respectively. The most efficient drug is the cis-olefinic substituted complex 4 with a MIC value of 6 µmol/L. For comparison, the commercially available antifungal Fluconazole exhibits a MIC value of 20 µmol/L [31] (Table 1, entry 9), about three times the value

<caption>**Table 4**</caption>

The docking results for interaction of silver(I) complexes 4–6 with (p)ppGpp synthetases/hydrolases from the three bacterial strains are presented as docking scores (kcal/mol).

<table><thead><tr><th>Entry</th><th>Complex</th><th>S. aureus Rel (AF)</th><th>E. faecalis Rel (AF)</th><th>P. aeruginosa Rel (AF)</th><th>E. coli RelA (AF)</th></tr></thead><tbody><tr><td>1</td><td>4</td><td>-10.593</td><td>-5.965</td><td>-6.443</td><td>-6.803</td></tr><tr><td>2</td><td>5</td><td>-7.741</td><td>-7.645</td><td>-6.374</td><td>-6.246</td></tr><tr><td>3</td><td>6</td><td>-10.659</td><td>-7.746</td><td>-6.192</td><td>-7.184</td></tr></tbody></table>

determinate with the complex 4.

Minimum bactericidal concentration (MBSs) and minimum fungicidal concentration (MFCs), which are the minimum concentration of drug killing 99.9 % of the microorganisms, were determined (Table 2). As attempt, the N-substituted benzimidazoles 1–3 displayed high MBSs and MFCs, higher than 2134 µmol/L (Table 2, entries 1–3).

Surprisingly, moderate MBS values in the range to of 196 to 205 µmol/L were measured for the three complexes 4–6 against S. aureus, and high concentrations were observed for the second Gram-positive bacterium E. faecalis (783–819 µmol/L). Regarding the Gram-negative bacteria, the lower MBS values were obtained either with the cis-olefinic complex 4 for E. coli (205 µmol/L; Table 2, entry 4) or with the complex bearing the cinnamyl substituent 6 for P. aeruginosa (98 µmol/L). The latter complex was also the most efficient against the C. albicans fungus with a MCF value of 25 µmol/L (Table 2, entry 6).

Finally the ability of the three silver(I) complexes 4–6 to reduce the challenging biofilm formation of pathogenic bacteria was reported in Table 3. The measurements were realized at concentrations of 0.5 MIC.

The three complexes 4–6 displayed low antibiofilm activities (0.88 to 17.51 %) towards E. coli and C. albicans. Moderate inhibition (41.79 %) was obtained when biofilm of S. aureus was treated with the trans-olefinic complex 5 at a concentration of 51 µmol/L (Fig. 5). The higher inhibition (71.37 % at 24 µmol/L) was observed when biofilm of E. faecalis was in contact with the complex bearing the cinnamyl substituent 6, the two other complexes displayed low activities. The latter complex 6 was also especially efficient in reducing biofilm of P. aeruginosa, an inhibition of 67.72 % was measured at a concentration as low as 3 µmol/L. For comparison, the use of the metal precursor AgNO₃ inhibits 63 % of the biofilm of P. aeruginosa at a concentration of 11 µmol/L. This result clearly shows that N-alkylbenzimidazole substituents are essential for the antibiofilm activity of silver(I) complexes 4–6.

### 3.4. In silico studies of the reactivity of the silver(I) complexes

Several mechanisms of action of antibiofilm agents involving numerous biomolecular targets have been reported [58-59]. Based on their modes of action [60-63], we focused on three enzymes, namely the (p)ppGpp synthetase/hydrolases [64-67] (inhibition of the alarmone system to avoid the bacterial stringent response), Filamenting temperature-sensitive mutant Z (FtsZ) proteins [68-69] (fundamental role in scissiparity) and pyruvate kinases [70-72] (catalyst in the formation of adenosine triphosphate). In the present study, in silico molecular docking calculations have been employed to investigate these three possible mechanisms of action of silver(I) complexes 4–6 against S. aureus, E. faecalis (two Gram-positive bacteria), E. coli (a Gram-negative bacterium) P. aeruginosa (a high antibiotic resistance strain) and C. albicans (one fungus).

First, we performed a virtual screening between (p)ppGpp synthetase/hydrolases and the three bacteria S. aureus Rel (AF), E. faecalis Rel (AF), P. aeruginosa Rel (AF) and E. coli RelA (AF) whose predicted 3D structures, obtained from the AlphaFold database [43], are P0A0F0, A0A1J6XYM9, A0A485ILR5 and P0AG20, respectively. The active site of the synthetase domain was determined from the crystal structure RelSeq (1VJ7) with the co-crystallized GDP substrate. In fact, there are
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Interaction details of silver(I) complex 6 with (p)ppGpp SYNTHETASE/HYDROLASES of (a) *S. aureus* Rel (AF), (b) *E. faecalis* Rel (AF), (c) *P. aeruginosa* Rel (AF) and (d) *E. coli* RelA (AF).</figcaption></figure>

several molecular mechanisms involved in bacterial persistence and tolerance to antibiotics, reviewed by Trastoy and co-workers [73], which include the (p)ppGpp network, toxin-antitoxin (TA) system, the quorum sensing (QS) system, drug efflux pumps, reactive oxygen species (ROS), the SOS response, and RpoS (sigma factor of stationary phase). (p)ppGpp orchestrates the stringent response (SR) in bacteria; thus, it is produced during nutrient stress (such as amino acid or fatty acid starvation) by proteins belonging to the RelA/SpoT homolog family (RSH) [74]. As interfered from Table 4, the results of the *in silico* analysis revealed that the three silver(I) complexes 4–6 are able to bind to the active sites of (p)ppGpp synthetases. The silver(I) complexes displayed average binding energies of –9.664, –7.119, –6.336 and –6.744 kcal/mol for *S. aureus* Rel, *E. faecalis* Rel, *P. aeruginosa* Rel and *E. coli* RelA, respectively. In these three cases, the complex 6 showed the most favorable binding energy of –10.659 kcal/mol with *S. aureus* Rel creating all three types of interactions. The docking interactions described in the present work between complex 6 and *S. aureus* Rel are crucial for understanding the molecular basis of their binding and potential therapeutic implications. The favorable binding energy of –10.659 kcal/mol indicates a strong interaction between complex 6 and *S. aureus* Rel. This suggests a high affinity and stability of the complex at the binding site on the target protein. Key residues involved are mainly **MET**$^{309}$, **TYR**$^{408}$, and **ASP**$^{400}$ (Fig. 6a). **MET**$^{309}$ interaction pattern, likely forms hydrophobic interactions with specific moieties of complex 6. Hydrophobic interactions play a crucial role in stabilizing protein-ligand complexes by minimizing the exposure of hydrophobic residues to water. Then, Tyrosine **TYR**$^{408}$ might participate in hydrogen bonding or π-π stacking interactions with complex 6. Hydrogen bonds and π-π interactions contribute significantly to the specificity and strength of ligand binding to proteins. Finally, Aspartic acid (**ASP**$^{400}$) could be involved in electrostatic interactions or hydrogen bonding with complex 6.

<caption>**Table 5**</caption>

The docking results for interaction of silver(I) complexes 4–6 with FtsZ proteins from the three bacterial strains are presented as docking scores (kcal/mol).

<table><thead><tr><th>Entry</th><th>Complex</th><th>S. aureus<br>(AF)</th><th>E. faecalis<br>(AF)</th><th>P. aeruginosa<br>Rel (AF)</th><th>E. coli<br>(AF)</th></tr></thead><tbody><tr><td>1</td><td>4</td><td>-5.458</td><td>-6.646</td><td>-6.4738</td><td>-7.725</td></tr><tr><td>2</td><td>5</td><td>-6.213</td><td>-6.568</td><td>-6.2041</td><td>-7.878</td></tr><tr><td>3</td><td>6</td><td>-6.369</td><td>-7.607</td><td>-6.6457</td><td>-7.918</td></tr></tbody></table>

Electrostatic interactions, especially salt bridges, can significantly contribute to the overall stability of protein-ligand complexes. It's noteworthy that the salt bridge formed by the counterion $NO_3^-$, which is the ion that accompanies a positively charged silver(I) ion, present in all molecules, exhibits a remarkable increase in binding energy. This phenomenon underscores the importance of understanding specific molecular interactions in protein-ligand binding, as it can have significant implications for drug design and molecular engineering strategies aimed at enhancing binding affinity and specificity.

Notably, significant results have been found with *E. faecalis* Rel (–7.746 kcal/mol; Fig. 6b) and with *E. coli* RelA (–7.184 kcal/mol; Fig. 6d) (Table 4, entry 3).

In a second step, we realized the molecular docking on the essential proteins FtsZ from *S. aureus*, *E. faecalis* and *E. coli*, whose predicted 3D structures, obtained from the AlphaFold database [43], are P0A029, A0A 3N3ZGTO and A0A5B9AMC7, respectively, for *P. aeruginosa* 3D strcuture was obtained from pdb (1OFU). As observed by the group of Mendagralo [29], with the silver(I) complexes 4–6, no formation of inclusion complexes was observed with the inhibitor PC190723 (cystalline structure PDB 4DXD). Conversely, interactions between the silver (I) complexes and the active site of the *S. aureus* FtsZ structure PDB 3WGN could be highlighted. Table 5 summarizes the observed
<figure><img src="image_10.png" /><figcaption>Fig. 7. Interaction details of silver(I) complex 6 with FTSZ PROTEINS of (a) *S. aureus* FtsZ, (b) *E. faecalis* FtsZ, (c) *P. aeruginosa* FtsZ and (d) *E. coli* FtsZ.</figcaption></figure>

<caption>**Table 6**</caption>

The docking results for interaction of silver(I) complexes 4–6 with pyruvate kinases from the three bacterial strains and the fungus are presented as docking scores (kcal/mol).

<table><thead><tr><th>Entry</th><th>Complex</th><th><i>S. aureus</i><br>(PDB 3T0T)</th><th><i>E. faecalis</i><br>(AF)</th><th><i>E. coli</i><br>(PDB 1PKY)</th><th><i>P.aeruginosa</i><br>(PDB7001)</th><th><i>C. albicans</i><br>(AF)</th></tr></thead><tbody><tr><td>1</td><td>4</td><td>-7.931</td><td>-7.734</td><td>-7.484</td><td>-8.740</td><td>-7.585</td></tr><tr><td>2</td><td>5</td><td>-9.114</td><td>-8.063</td><td>-7.379</td><td>-9.637</td><td>-7.711</td></tr><tr><td>3</td><td>6</td><td>-8.738</td><td>-8.286</td><td>-8.142</td><td>-8.407</td><td>-7.798</td></tr></tbody></table>

interactions between the silver(I) complexes 4–6 with the GTP binding site of FtsZ proteins for the three bacterial strains. The organometallic compounds displayed an average binding energy of -6.013, -6.946 and -7.840 kcal/mol with *S. aureus* FtsZ, *E. faecalis* FtsZ, *P. aeruginosa* FtsZ and *E. coli* FtsZ, respectively. Once again, the complex 6 exhibited the strongest interactions with the three sites (Table 5, entry 3), the calculated energies are -6.369, -7.607, -6.646 and -7.918 kcal/mol with *S. aureus* FtsZ, *E. faecalis* FtsZ, *P. aeruginosa* FtsZ and *E. coli* FtsZ, respectively (Fig. 7).

Interestingly in *silico* interactions between the silver(I) complexes and the pyruvate kinase enzyme, present in both bacteria and fungi, were examined in detail. The molecular docking study utilized the crystal structures of pyruvate kinases from *S. aureus* (PDB 3T0T), *P. aeruginosa* (PDB7001) and *E. coli* (PDB 1PKY), sourced from the AlphaFold database. For *E. faecalis* and *C. albicans*, pyruvate kinase structures were modeled through structural alignment of AlphaFold-predicted homologous pyruvate kinases from *Mycobacterium tuberculosis* and *S. aureus* (PDB 3T0T), respectively. Docking simulations revealed notable binding affinities between the silver(I) complexes and the active sites of pyruvate kinases. The interactions were quantified by calculating the binding energies, with the most significant results summarized in Table 6. For the four bacterial strains, the strongest interactions were observed with silver(I) complex 5 and the pyruvate kinase of *S. aureus*, and *P. aeruginosa* showing a binding energy of -9.114 and -9.637 kcal/mol, respectively, and with silver(I) complex 6 and the pyruvate kinases of *E. faecalis*, and *E. coli*, showing binding

energies of -8.286 and -8.142 kcal/mol, respectively. These interactions are visually represented in Fig. 8, highlighting the specific binding sites and the nature of the interactions at the molecular level. The strong binding affinities suggest a potential mechanism through which these silver(I) complexes exert their antimicrobial and antibiofilm effects, providing insights into their therapeutic potential against bacterial and fungal infections.

Regarding the inclusion complexes between the three silver(I) complexes and the kynase pyruvate of *C. albicans*, the binding energies are of the same order of magnitude between -7.585 and -7.798 kcal/mol (Fig. 9). If we compare these results with the antibiofilm tests for which the greatest inhibition was observed with complex 4 (17.51 % for a concentration of 3 µmol/L; Table 4, entry 1), the other complexes being almost inactive, we can conclude that the mode of action of the silver(I) complexes 4–6 does not involve the pyruvate kinase enzyme of the fungus.

While the pyruvate kinase of *C. albicans* may not be the appropriate protein target for testing with these studied molecules, there is a significant agreement between *in silico* predictions and *in vitro* results for the bacteria *S. aureus*, *E. faecalis*, and *E. coli*.

In fact, the antibiofilm tests revealed that the measured inhibitions were most pronounced when complexes 5 and 6 were in contact with *S. aureus* (Fig. 10) and *E. faecalis*, respectively (Table 4). These two complexes also exhibited the highest binding energies with the pyruvate kinases of the corresponding bacteria (Table 6). It is noteworthy that this could be explained by the positioning of the *trans*-styryl substituent
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Interaction details of silver(I) complex 5 with PYRUVATE KINASES of (a) *S. aureus* (PDB 3T0T) and silver(I) complex 6 with (b) *E. faecalis* (AF), (c) *P. aeruginosa* (PDB7001) and (d) *E. coli* (PDB 1PKY).</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Interaction details of silver(I) complex 6 with PYRUVATE KINASES of *C. albicans* (AF).</figcaption></figure>

relative to the benzimidazole moiety between the three complexes 4–6. For instance, the *trans*-styryl substituent as it is directly grafted to the nitrogen atom of the aromatic cycle is interacting with **VAL**<sup>356</sup>, **ALA**<sup>573</sup> and **ARG**<sup>484</sup> with electrostatic π-π interactions for *S. aureus* (Fig. 8a). These specific interactions play a fascinating role as it facilitates the precise positioning and stabilization of the host within the binding pocket of the protein. By forming stable interactions with aromatic residues, such as arginine, phenylalanine and valine, optimal binding

orientations could be established. Whereas the presence of a methylene bridge (complex 6) has created mixed π/alkyl hydrophobic interactions (Fig. 8b), especially with ASP<sup>133</sup> and ASP<sup>134</sup>, these data suggest a role of aspartic acid in inducing and stabilizing the active conformation of the complex at the catalytic pocket of pyruvate kinase protein from *E. faecalis*.

This study investigated also the intricate mechanisms underlying bacterial cell division, with a particular focus on the protein target FtsZ. Its dynamic assembly and disassembly are tightly regulated to achieve proper spatial and temporal control over cell division. The conducted in vitro and *in silico* studies, specifically on the Gram-negative bacterium *E. coli*, highlighted the efficacy of complexes 4 and 5 as potent anti-biofilm agents. These complexes exhibited notable binding energies with FtsZ proteins, forming numerous hydrogen bond interactions with amino acids such as GLY<sup>20</sup> and ALA<sup>48</sup> (Fig. 7d). Moreover, electrostatic π-charges played a significant role in these interactions, further enhancing the binding affinity. Interestingly, the configuration of the styryl substituent (*cis* or *trans*) showed a nuanced impact on the antibiofilm activities of the silver(I) complexes against *E. coli*. This observation hints at a potential mode of action involving FtsZ proteins, where the specific structural features of the complexes influence their ability to disrupt biofilm formation.

Overall, these findings shed light on the molecular interactions between silver(I) complexes and three main target proteins, providing valuable insights into their antibiofilm mechanisms and opening avenues for the development of novel antibacterial agents targeting bacterial cell division processes. Additionally, molecular dynamic work should be undertaken in the future to further elucidate the dynamic behavior and stability of these complexes in biological environments.
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** Interaction diagrams (2D) of *S. aureus* strain protein profiling: (a) Pyruvate Kinase with silver(I) complex 5, (b) Synthase hydrolase with silver(I) complex 6 and (c) Protein FtsZ: Filamenting temperature-sensitive mutant Z with silver(I) complex 6. Schematic details of interactions between inorganic complex atoms and protein residues.</figcaption></figure>

**4. Conclusion**

In this study, we synthesized three bis[N-alkylbenzimidazole]silver(I) nitrate complexes and evaluated their antimicrobial and antibiofilm activities against *S. aureus*, *P. aeruginosa*, *E. faecalis*, *E. coli*, and *C. albicans* using both experimental and computational approaches. Among these, the bis(1-cinnamyl-benzimidazole)silver(I) nitrate complex (6) demonstrated the most significant antibacterial and antibiofilm properties, with a minimum inhibitory concentration comparable to that of amikacin against *P. aeruginosa*.

To elucidate the mode of action of these silver(I) complexes, molecular docking simulations were conducted to investigate their interactions with key proteins involved in bacterial and fungal biofilm

formation, including (p)ppGpp synthetase/hydrolases, FtsZ proteins, and pyruvate kinases. The *in silico* studies revealed notable binding affinities between the silver(I) complexes and the active sites of these proteins. The most favored interactions were observed for bis(1-cinnamyl-benzimidazole)silver(I) nitrate (6) with *S. aureus* Rel (binding energy of -10.659 kcal/mol) and for bis[(Z)-1-styryl-benzimidazole]silver(I) nitrate (4) or bis[(E)-1-styryl-benzimidazole]silver(I) nitrate (5) with FtsZ proteins of *E. coli* (binding energies of -7.725 and -7.878 kcal/mol, respectively).

These findings underscore the importance of understanding specific molecular interactions in protein-ligand binding, as they have significant implications for drug design and molecular engineering strategies aimed at enhancing binding affinity and specificity. By leveraging this
knowledge, researchers can tailor ligands to target proteins with greater precision and efficacy, potentially leading to the development of more potent therapeutic agents.

## Funding

This research received no external funding.

## CRediT authorship contribution statement

**Neslihan Şahin:** Validation, Methodology, Investigation, Formal analysis, Conceptualization.
**Mohamed Ali Mosrati:** Software. **Abderrahmen Merghni:** Validation, Resources, Methodology, Investigation, Formal analysis, Data curation.
**İsmail Özdemir:** Supervision. **Hayet Sellami:** Formal analysis. **Kawther Bedchiche:** Data curation.
**Salim Krayiem:** Data curation.
**Sami Aifa:** Formal analysis.
**Dorra Abdelmalek:** Writing – review & editing, Writing – original draft, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**David Sémeril:** Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

## Acknowledgements

N.Ş. thanks the Scientific and Technological Research of Turkey (TÜBITAK - 2219 - International Postdoctoral Research Scholarship Program) for a research fellowship.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.molstruc.2024.140440.

## Data availability

Data will be made available on request. The data presented in this study are available upon request from the corresponding authors.

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