From synthesis to biological impact of palladium bis(benzimidazol-2-ylidene) complexes: Preparation, characterization, and antimicrobial and scavenging activity

2.1 Preparation of benzimidazolium (2a–d) salts

Benzimidazole salts (2a–d) were prepared via the two-step N-alkylation process, as depicted in Scheme 1. 1H-Benzo[d]imidazole-(4-(tert-butyl)benzyl) (1a) was prepared by refluxing benzimidazole with 1-(bromomethyl)-4-(tert-butyl)benzene in EtOH for 16 h. The benzimidazolium salts (2a–d) were synthesized by quaternization of 1H-Benzo[d]imidazole-(4-(tert-butyl)benzyl) (1a), with the corresponding benzylbromide derivatives for 48 h at 70°C in degassed dimethylformamide (DMF) to produce the respective benzimidazolium salts (2a–d) with yields in the range of 50–89%. The target salts were obtained as white solids. These salts are stable in solid state and solution against air and moisture. The salts are soluble in chlorinated solvents, alcohols, and water. The reaction has been monitored following thin-layer chromatography, and after this time, the formation of salts (2a–d) has been observed for every target compound. The benzimidazolium salts (2a–d) were air- and moisture-stable both in the solid state and in solution. The FTIR spectroscopy, ¹H NMR and ¹³C{1H} NMR spectroscopy, and elemental analysis data of the title compounds confirm the proposed structures. The synthesis of NHC precursors and their respective ligands is described in Scheme 1.

Scheme 1

General preparation of benzimidazolium salts (2a–d).

NMR spectra of all the compounds were analyzed in d-CDCl₃. In the ¹H NMR spectra, acidic protons (NCHN) for benzimidazolium salts (2a–d) were seen at 11.17, 11.63, 11.64, and 11.58 ppm, respectively, as a characteristic sharp singlet. Aliphatic carbons appeared from 20.7 to 35 ppm, while aromatic carbons were from 113.38 and 152.82 ppm. Whereas methyl protons showed up as singlets between 1.25 and 2.25 ppm, the aromatic protons showed up as a multiplet between 6.9 and 7.63 ppm. In the ¹³C{1H} NMR spectra of benzimidazole salts (2a–d), the NCHN carbon was detected as typical singlets at 142.95, 163.06, 162.9, and 152.8 ppm, respectively. These values are consistent with related literature (Hu et al., 2004; Doǧan et al., 2001; Ozdemir et al., 2010; Iqbal et al., 2013). In the IR spectra, the ʋ(C═N) bands for salts (2a–d) were observed at 1,548, 1,672, 1,670, and 1,565 cm⁻¹, respectively.

Bis(benzimidazol-2-ylidene)palladium complexes (3a–d) were prepared by reaction of salts (2a–d) with PdCl₂ in high yields of 75–97% (Scheme 2). The reaction was carried out at reflux in anhydrous tetrahydrofuran (THF) in the presence of potassium carbonate (K₂CO₃) as a base for 24 h. Complex 3 is an air-stable solid, soluble in acetone, chloroform, dichloromethane, DMF, dimethyl sulfoxide (DMSO), ethanol, and acetonitrile but insoluble in hexane, ether, and benzene. The structural characterization of the palladium complex was confirmed by NMR, FT-IR, and elemental analysis techniques. In the ¹H NMR spectra of complexes 3a–3d, the characteristic down-field signals for the acidic C(2)–H protons of the benzimidazolium salts 2a–d disappeared in the ¹H NMR spectra of palladium complex confirming the formation of the NHC-Pd bonds. In the ¹³C NMR spectrum of complexes 3a–3d, characteristic signal of C(2)-carbon of imidazolinium salts 3a–3d between δ = 142.9–162.9 ppm were completely disappeared, and the characteristic Pd-C(carbene) bond signals of the complexes 3a–3d were observed as singlet. In the ¹³C NMR spectra, the carbene signals of the Pd-PEPPSI-NHC complexes 3a–3e were observed at δ = 180.58, 180.81, 180.87, and 180.83 ppm, respectively. The Fourier-transform infrared spectroscopy of the palladium complexes 3a–3d unveiled a characteristic ν(CN) band, which was observed at 1,608, 1,610, 1,606, and 1,612 cm⁻¹, respectively. The stretching frequency of v(CN) was lower compared to the salt, which can be attributed to the electron flow from the carbene ligand toward the palladium center, causing a weak C═N bond. Moreover, the data from the elemental analysis of the palladium complexes agreed with the suggested structure.

Scheme 2

Preparation of palladium bis(benzimidazol-2-ylidene) complexes 3a–d.

Numerous attempts were made to obtain a suitable crystal of palladium bis(benzimidazol-2-ylidene) complexes for single-crystal X-ray diffraction study in order to provide a more detailed explanation of the structures of our complexes. The solvent diffusion method was employed using various solvent systems, including dichloromethane/diethyl ether CH₂Cl₂/Et₂O, ethanol/ether EtOH/Et₂O, and others. However, despite all attempts, a suitable single crystal for X-ray analysis could not be obtained for the palladium bis(benzimidazol-2-ylidene) complexes. All spectroscopic and analytical data acquired were consistent with those of previously reported similar palladium complexes of the same type (Slimani et al., 2022).

Both optimized cis and trans isomer structures of the 3a complex are shown in Figure 1, taking into account the spin multiplicity of palladium(ii). The obtained geometrical parameters are summarized in Table 1. Computed electronic energies relative to the lowest-energy spin multiplicity state S 2 , Δ E (in kcal·mol⁻¹), ε LUMO , ε HOMO , and energy gap Δ ε L − H in (eV) of complex 3a isomers as functions of 2 S + 1 multiplicities at B3LYP/6-311 G(d,p)/LANL2DZ theory level are summarized in Table 2.

Figure 1

Structures of trans and cis isomers of complex 3a.

According to the data listed in Table 1, we can conclude that complex 3a crystallizes in square planar geometry. The trans isomer for a singlet spin multiplicity is more stable than the cis one with relative energy equal to 12.4 kcal · mol ⁻¹. The computed Br 5 − Pd 4 ^ − Br 6 , 2 − Pd 4 ^ − C 7 , Br 6 − Pd 4 ^ − C 2 , and C 7 − Pd 4 ^ − Br 5 bond angles in the case of trans isomer are equal to 90°. Whereas, in the cis isomer, a distorted square planar geometry was found (Table 1). This deformation may be explained by the steric hindrance of aromatic rings.

2.2 Optical property analysis

Complex 3a showed three absorbance bands at 248 and 288 attributed to π−π^* a minor one at 374 nm assigned to the metal−ligand CT (MLCT). The assignment provided is supported by our previous study (Slimani et al., 2022) on a similar PdCl₂–NHC complex, where we examined the performance of three functionals (M06-2X, WB97X-d, and B3LYP). In that study, we observed that the TD-DFT spectra obtained using these functionals closely matched the experimental spectra. Additionally, the confirmation of the CT band assignment was achieved through excited-state CT calculations (Abumelha et al., 2020; Al-Dawood and Al-Hazmi, 2019; Al-Fahemi et al., 2018, 2020; Arslanoğlu and Hamuryudan, 2007; Bayazeed et al., 2020; Boman and Kaletta, 1957; Edwards and Hahn, 2011; El-Daly et al., 1996; El-Metwaly et al., 2019, 2021; Gaber et al., 2017; Garzon et al., 1987; Gwang-Hyeon et al., 2002; Katouah et al., 2019, 2020; Kelly et al., 2018; Keppler and Rupp, 1986; Li-Hua et al., 2013; Richert et al., 2019; Shinde et al., 1999 and Sigel et al., 2018) (Table 3).

2.2.1 CT analysis

A detailed analysis of the CTs that occur between the palladium and NHC fragment was reported since the UV absorption spectrum showed a MLCT transition. We have considered two main fragments, as shown in Figure 2, and the results are displayed in Table 4. An energy diagram showing the molecular orbital interactions and localizations of frontier molecular orbitals is presented in Figure 3.

Figure 2

Selected fragments in complex (3a) (blue: fragment 1 [carbene derivative], red: fragment 2: PCl₂).

Table 4

Computed charge population (before the complexation q 1 , after the complexation q 2 ) and the amount of the transferred charge ( q CT ) in (e) of selected sites within complex 3a at B₃LYP/6-311 + G(d,p)/LANL₂DZ level of theory

Site	N1	C2	N3	Pd4	Cl5	Cl6
q 1	−0.466	0.149	−0.465	−0.037	0.018	0.018
q 2	−0.420	0.366	−0.422	0.070	−0.479	−0.479
q CT	−0.046	−0.217	−0.043	−0.107	0.497	0.497

Figure 3

An interaction diagram for complex 3a of symmetry C₁.

To evaluate the CT taking place, an analysis of selected sites was performed using NBO analysis (Esrafili and Hadipour, 2011; Karthikeyan et al., 2011; Kepler et al., 2019). This analysis allowed for the determination of the electronic transitions and the extent of charge redistribution between these specific sites. By utilizing NBO analysis, insights into the nature and magnitude of the CT occurring in the system were obtained, enabling a better understanding of the electronic interactions.

According to the data summarized in Table 2, for carbine. This unit is the most efficient donor fragment because it has the highest charge transfer q CT value. Accordingly, the organic moiety (C₂₆H₂₈N₂) is the donor, while the PdCl₂ moiety is the acceptor. An important CT amount occurred from carbene to Pd(ii) was obtained. This transferred charge amount was mostly attracted by both chlorides since the latters exhibit strong electron affinity. The CT was confirmed by second-order perturbation theory analysis of the Fock matrix on an NBO basis, as shown in Table 5. The larger the E(2) value, the more intensive the interaction between electron donors and electron acceptors, i.e., the more the donating tendency from electron donors to electron acceptors, the greater the extent of conjugation of the whole system (Sebastian and Sundaraganesan, 2010). The significantly higher stabilization energy value E(2) is observed in the case of intramolecular interaction for the 3a complex, resulting from the orbital overlap between the carbene derivative (organic base) acting as an electron donor and PdCl₂ as an electron acceptor (BD (C7–N8) and LP* (Pd4)).

Table 5

Second-order perturbation theory analysis of fock matrix in NBO basis of 3a complex

Donor NBO (i)	Acceptor NBO (j)	E(2) (kcal·mol−1)	E(j)–E(i) (a.u.)	F(i,j) (a.u.)
BD(C7–N8)	LP * (Pd4)	4.51	0.85	0.056
BD (C7–N8)	BD * (Pd4–Cl5)	0.81	0.77	0.023
BD (C7–N8)	BD * (Pd4–Cl6)	1.07	0.36	0.018
BD (C2–N1)	BD * (Pd4–Cl6)	0.71	0.88	0.023
BD (C2–N1)	BD * (Pd4–Cl6)	0.84	0.46	0.018

4.1 Antioxidant activity

The scavenging activities have been investigated to support the biological potential of the palladium bis(benzimidazol-2-ylidene) complexes 3a–d (Jomova et al., 2012; Meyerstein, 2013). The palladium bis(benzimidazol-2-ylidene) complexes 3a–d were used to examine the decrease in absorbance or scavenging action of a stable-free DPPH (Taha et al., 2011; Trivedi et al., 2012). Comparing the DPPH absorption at 517 nm to the Pd(ii) complexes’ percentage scavenging activity was done in a concentration-dependent manner (Li et al., 2006; Suh and Chaires, 1995). For Pd(ii) complexes, the absorbance of the DPPH free radical at 517 nm with DMSO was 0.906. Complexes have expressed a decrease in absorption (Figure 8).

The IC₅₀ (concentration in grams’ mL⁻¹ with 50% effect) was used. Compounds 2d, 2a, 3b, and 3a from Table 6 show notable DPPH radical scavenging activity. These substances’ respective IC₅₀ values were 50.55, 46.88, 47.17, and 48.07 g·mL⁻¹. In terms of ABTS free radical activity, the IC₅₀ of the produced compounds 2a–d and 3a–d ranged from 20.45 to 33.17 g·mL⁻¹ (Figure 8). Complexes 3a (30.22 g·mL⁻¹), 3c (29.55 g·mL⁻¹), 2d (33.17 g·mL⁻¹), and 2b (20.45 g·mL⁻¹ g·mL⁻¹) had the highest activity in the ABTS assay (Figure 7). In the ABTS free radical experiment, the control butylated hydroxytoluene (BHT) (a potent antioxidant molecule) had an IC₅₀ value of g·mL⁻¹ (Figure 8). Due to their antioxidant qualities, Pd compounds have medical and material relevance.

Figure 7

Best position and the interactions of the 3a complex within the 5JQ9 (Yersinia pestis).

Figure 8

The antioxidative activity of compounds 2a–d and 3a–d synthesized was assessed by DPPH and ABTS techniques and expressed as IC₅₀ in g·mL⁻¹. The BHT was used as a control.

General methods

All manipulations were carried out under argon using standard Schlenk line techniques in accordance with our earlier work (Slimani et al., 2021). Chemicals and solvents were purchased from Sigma-Aldrich Co. (Poole, Dorset, UK). The solvents used were purified by distillation over the drying agents indicated and were transferred under argon. Melting points were measured in open capillary tubes using an Electrothermal-9200 melting points apparatus. IR spectra were recorded on an ATR unit in the range of 400–4,000 cm⁻¹ with a Perkin Elmer Spectrum 100 spectrophotometer. ¹H NMR and ¹³C NMR spectra were recorded using Bruker Avance AMX and Bruker Avance III spectrometer operating at 400 MHz (¹H NMR) and 100 MHz (¹³C NMR) in CDCl₃ with TMS added. NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, hept = heptet, and m = multiplet signal. The NMR studies were carried out in high-quality 5 mm NMR tubes. The chemical shifts (d) are reported in ppm relative to CDCl₃. Coupling constants (J values) are given in hertz. Elemental analyses were performed by the LECO CHNS-932 elementary chemical analyzer. The chemical shifts (d) are reported in ppm relative to CDCl₃. Coupling constants (J values) are given in hertz.

Synthesis of 1-(4-(tert-butyl)benzyl)-1H-benzo[d]imidazole (1a)

This compound was synthesized according to our previous work (Slimani et al., 2022).

Synthesis of benzimidazolium salts (2a–2d)

The synthesis is carried out by reacting 1.0 g (3.42 mmol) of 1-(4-tert-butylbenzyl) benzimidazole (1a) with an equivalent molar amount of the appropriate aryl bromide or chloride and then dissolving in 2 mL of dried N,N-DMF. The solution was stirred at 70°C for 24–48 h. As a result, a product as a white powder was obtained.

Synthesis of palladium-bis(NHC) complexes 3(a–d)

The complexes considered in this work are prepared by mixing a solution of 5,6-dimethylbenzimidazolium salts (1 mmol), PdCl₂ (0.5 mmol; 0.09 g), and K₂CO₃ (0.6 g, 4.3 mmol). Next, the anhydrous THF (25 mL) was added, and the mixture was heated at reflux for 24 h at a temperature of about 100°C. The obtained solid was solved in DCM (5 mL) and then purified by flash column chromatography.

Computational details

The ground state S 0 of the complex was optimized using DFT with hybrid B3LYP (Becke, 1993; Stephens et al., 1994) functional, combined with the 6-311G(d) basis set for the non-metal atoms and a double-ζ quality basis set LANL2DZ (Chiodo et al., 2006) for palladium. Singlet and triplet spin multiplicities were taken into account. Vibrational frequency analyses were performed to confirm that the optimized structures were a true minimum. The electronic populations were analyzed by the NBO population in order to investigate the CT.

The Gaussian16A program package (Gaussian 16, Revision B.01 et al., 2016) was used to perform all calculations. We opted for the B3LYP functional due to its demonstrated effectiveness in optimizing metal-NHC complexes, as established in our recent research (Hassen et al., 2022). NBO analysis (Glendening et al., 2012) was utilized to determine the charge populations at specific sites and subsequently quantify the CT that took place between the relevant fragments in the complex.

The second-order Fock matrix was carried out to evaluate the donor–acceptor interactions in the NBO analysis. The interaction result is a loss of occupancy from the localized NBO of the idealized Lewis structure into an empty non-Lewis orbital. For each donor (i) and acceptor (j), the stabilization energy E(2) associated with the delocalization i – j is estimated as follows (Dindar et al., 2022):

UV-Vis electronic absorption spectrum and fluorescence were recorded on a UV-Vis Shimadzu 1650 spectrophotometer, as shown in Figure 9. Quartz cells of 10 mm in length were used. Absorption was measured in the spectrum range of 200–800 nm. The fluorescence spectrum was taken on a Jazco, FP-8200 spectrofluorometer, excitation bandwidth 5 nm, emission bandwidth 5 nm, with an Xe lamp Light source.

Figure 9

Experimental ultraviolet (UV) absorption and emission ( λ exc = 370 nm) spectra of 2.5 × 10⁻⁴ M 3a in CHCl₃.

Fluorescence quantum yield in liquid was determined using the optically dilute solution relative method with either 9,10-diphenyleanthracene or quinine sulfate solutions, depending on the emission wavelength range. Light intensity was determined using ferrioxalate actinometry (Guven et al., 2017). Eq. 1 was applied to calculate the fluorescence quantum yields:

The integrals denote the corrected fluorescence peak areas, A denotes the absorbance at the excitation wavelength, and n denotes the solvent’s refractive index. The subscripts s and r indicate sample and reference, respectively.

Antibacterial activity