Biological Activities of NHC–Pd(II) Complexes Based on Benzimidazolylidene N-heterocyclic Carbene (NHC) Ligands Bearing Aryl Substituents

: N-heterocyclic carbene (NHC) precursors ( 2a–i ), their pyridine-enhanced precatalyst preparation stabilization and initiation (PEPPSI)-themed palladium N-heterocyclic carbene complexes ( 3a–i ) and palladium N-heterocyclic triphenylphosphines complexes ( 4a–i ) were synthesized and characterized by elemental analysis and 1 H NMR, 13 C NMR, IR, and LC–MS spectroscopic techniques. The (NHC)Pd(II) complexes 3–4 were tested against MCF7 and MDA-MB-231 cancer cells, Escherichia coli , methicillin-resistant Staphylococcus aureus (MRSA), Candida albicans microorganisms, Leishmania major promastigotes and amastigotes, Toxoplasma gondii parasites, and Vero cells in vitro. The biological assays indicated that all compounds are highly active against cancer cells, with an IC 50 < 1.5 µ g mL − 1 . Eight compounds proved antibacterial and antileishmanial activities, while only three compounds had strong antifungal activities against C. albicans . In our conclusion, compounds 3 ( b , f , g , and h ) and 4b are the most suitable drug candidates for anticancer, antimicrobial, and antiparasitical.

Based on the structural correlations between palladium and platinum complexes, Pd(II)-based complexes have become a group of antitumor compounds of interest with the same activities as Pt(II)based compounds for metallotherapeutical uses [31]. However, despite their potential activity as antitumor agents, only a few numbers of Pd(II)-NHC compounds have been mentioned previously, but their antitumor activities were found to be more efficient [32][33][34]. There is similarity in the mode of action for both Pd(II) and Pt(II)-NHC by affecting directly the organelles of cancer cells [35]. In our recent results, we found the structure of Ag(I)-NHC compounds and respective benzimidazolium salt to be of potent antitumor property [36,37].
The aim of this work was to study the activities against cancer cells, Escherichia coli, methicillinresistant Staphylococcus aureus (MRSA), Candida albicans, Leishmania major, Toxoplasma gondii of novel benzimidazolium salts 2a-i, PEPPSI-type N-functionalized N-heterocyclic carbene complexes 3 and palladium N-heterocyclic triphenylphosphine complexes 4. In addition, their cytotoxicity was tested using Vero cells.

Synthesis and Characterization
N-heterocyclic carbene ligands have proven to be very useful for designing new metal complexes for catalysis. [38]. All of the benzimidazolium salts used as NHC precursors were prepared similarly by using the published procedures [39,40]. As shown in Scheme 1, benzimidazole salts 2a-2i were synthesized in good yields by quaternization of compound 1 in DMF at 70°C for 3 days with the corresponding arylchlorides or bromides. The benzimidazolium salts 2a-2i are stable in air and moisture, both in the solid-state and in solution. They were characterized by 1 H-NMR, 13 C{1H} NMR, IR, and elemental analysis techniques. The structures of the benzimidazole salts 2 can be easily confirmed by the spectroscopic data of 1 HNMR. The characteristic carbonic protons (NCHN) are located at 10.56, 11.06, 11.40, 11.24, 10. [41][42][43][44]. As expected, the absence of pro-carbenic protons can be observed upon coordination of the benzimidazole salts with the palladium (II), confirming the formation of the NHC-Pd(II) complexes 3-4. In the 13 [45]. The Pd(II)-N-heterocyclic carbene (NHC) complexes 3 were synthesized by treatment of the benzimidazolium salts 2 with the precursor PdCl 2 in pyridine in the presence of an excess of potassium carbonate. These metal(II) complexes were obtained as colored solids in 75%-88% yield. Complexes 4 were obtained by substitution of the pyridine by the triphenylphosphine, with moderate yields (40%-49%) (Scheme 2).  [45]. The Pd(II)-N-heterocyclic carbene (NHC) complexes 3 were synthesized by treatment of the benzimidazolium salts 2 with the precursor PdCl2 in pyridine in the presence of an excess of potassium carbonate. These metal(II) complexes were obtained as colored solids in 75%-88% yield. Complexes 4 were obtained by substitution of the pyridine by the triphenylphosphine, with moderate yields (40%-49%) (Scheme 2).

Scheme 2. Protocol synthesis of Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4.
The elemental analysis data of the Pd(II)-N-heterocyclic carbene (NHC) complex 3c is in agreement with the theoretical values for the synthesized complexes. The benzylic -CH2-proton signals H1′ and H1′′ for complex 3c as representatives were observed at 5.03 and 5.99 ppm, respectively, and the aromatic protons appeared at δ between 6.86 and 7.48 ppm whilst the pyridine protons were detected as three signals at 7.28, 7.70 and 8.95 ppm.
The carbene carbon signals of Pd(II)-N-heterocyclic carbene (NHC) complex 3c were observed at δ 163.37 ppm in the 13 C NMR spectrum, while the C1′ and C1′′ carbon signals were at δ 48.76 and 53.31 ppm, respectively. The mass spectrum of the same complex gave the most prominent peak at m/z = 295.2.
The 1 H NMR spectra of the Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 showed less intense and downfield shifted signals of benzimidazoles compared to the free ligands. In the 13 C NMR spectra of the complexes, a downfield shift in C=N resonance of the ligands upon complexation indicates the binding of benzimidazoles to palladium through the NHC carbene atom. The aromatic The elemental analysis data of the Pd(II)-N-heterocyclic carbene (NHC) complex 3c is in agreement with the theoretical values for the synthesized complexes. The benzylic -CH 2 -proton signals H 1 and H 1 for complex 3c as representatives were observed at 5.03 and 5.99 ppm, respectively, and the aromatic protons appeared at δ between 6.86 and 7.48 ppm whilst the pyridine protons were detected as three signals at 7.28, 7.70 and 8.95 ppm.
The carbene carbon signals of Pd(II)-N-heterocyclic carbene (NHC) complex 3c were observed at δ 163.37 ppm in the 13 C NMR spectrum, while the C 1 and C 1 carbon signals were at δ 48.76 and 53.31 ppm, respectively. The mass spectrum of the same complex gave the most prominent peak at m/z = 295.2.
The 1 H NMR spectra of the Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 showed less intense and downfield shifted signals of benzimidazoles compared to the free ligands. In the 13 C NMR spectra of the complexes, a downfield shift in C=N resonance of the ligands upon complexation indicates the binding of benzimidazoles to palladium through the NHC carbene atom. The aromatic carbons of the benzene ring resonate between 112 and 152 ppm. The methyl peak in the Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 is observed approximately between 16 and 34 ppm. These results are in agreement with the data of other such complexes [46][47][48][49]. Table 1 indicates that all of the compounds were highly efficient and active against the two types of cancer cells investigated in this study. Their IC 50 were in the range of 1.4 to 0.3 µg mL −1 . Regarding MCF7, 3g and 3f were the most active with IC 50 = 0.518 and 0.675 µM, respectively.  Table 2 indicates that 8 compounds had antibacterial activity against E. coli better than the reference drug, but 3g and 4f were the most potent with an inhibition zone (IZ) of 26.3 mm. The compounds 3f, 4f, and 4c were more active compounds than the reference drug against MRSA with IZ of 28.5, 28.0, and 27.0 mm, respectively. Compounds 3b, 3g, and 4e had the best antifungal activity against C. albicans with IZ of 32.0, 29.5, and 29.0 mm, respectively. Table 2 NHC metals, particularly silver synthesized compounds as well as copper derivatives, have been previously found to have potent antibacterial activities [50,51]. Our findings support the previous results. 18  Table 3 shows that all of the compounds except 3e, 4g, and 4h possess antileishmanial activity against both L. major amastigotes and promastigotes in vitro with an IC 50 less than 7 µg mL −1 . Eight compounds had an IC 50 less than 1.0 µg mL −1 against the two stages. Nine compounds had an IC 50 less than 1.0 µg mL −1 against L. major amastigotes, namely, 3 (a-d, f, and h) and 4 (a, b, and i). In addition, 11 compounds showed an IC 50 less than 1.0 µg mL −1 against L. major promastigotes, namely, 3 (a-d, f) and 4 (a-d, f, and i). The SI values of all active compounds were in the range of 6-46.6, which indicates the safety threshold of these compounds. Compound 4b was the most active and strongest among all of them with an IC 50 less than 0.2 and 0.4 µg mL −1 against L. major amastigotes and promastigotes, respectively, with SI values greater than 24 and 12, respectively, better than the results of the amphotericin B (AmB) reference drug. In recent conducted investigations, NHC gold complexes showed promising antileishmanial activities against L. infantum promastigotes and amastigotes in vitro [52]. These results support our finding here for Pd(II)-NHC complexes 3-4 against L. major promastigotes and amastigotes in vitro. Table 4 indicates that only 7 compounds possess good antitoxoplasmal activity against T. gondii in vitro with an IC 50 less than 5 µg mL −1 . These compounds are 3a, 3b, 3c, 3h, 4a, 4b, and 4c with IC 50 of 4.2, 3.9, 4.6, 1.2, 4.8, 3.6, and 3.9 µg mL −1 , respectively. However, their SI values were found to be less than 2. Although NHC carbene metal complexes with silver and gold derivatives were found in previous studies to show good antiparasitical activities against apicomplexan protozoa such as Plasmodium spp. [53], these findings are not in agreement with our results for (NHC) palladium metallic complexes against T. gondii. Table 3. Antileishmanial activity of Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 against L. major promastigotes and amastigotes. 15.9 ± 3.0 8.5 ± 1.7 1.9 4a

General Methods
All manipulations were carried out under argon using standard Schlenk line techniques. Chemicals and solvents were purchased from Sigma-Aldrich Co. (Poole, Dorset, UK). The solvents used were purified by distillation and were transferred under argon. DMAc analytical grade (99%) was not distilled before use. KOAc (99%) was employed. Benzimidazoles salts 1-2, palladium PEPPSI complexes 3, palladium triphenylphosphine 4, and biological assays were done according to our previous work [40,52] and they are given in supplementary materials. Elemental analyses were performed by ElementarVario EL III Carlo Erba 1108 (Malatya, Turkey). The melting points of the complexes and NHC precursors were determined using Stuart automatic melting point apparatus (SMP-40) (Malatya, Turkey). IR spectra were recorded on ATR unit in the range of 400-4000 cm −1 with Perkin Elmer Spectrum 100 Gladi ATR FT/IR Spectrophotometer (Malatya, Turkey). 1 H NMR and 13 C NMR spectra were recorded using a Bruker Avance III HD spectrometer operating at 400 MHz ( 1 H NMR) and at 100 MHz ( 13 C NMR) in CDCl 3 or DMSO-d 6 (Malatya, Turkey). 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 tetramethylsilane for 1 H, 13 C NMR spectra as standard. Coupling constants (J values) are given in hertz. The HRMS (ESI) electrospray ionization mass spectra were recorded on a Shimadzu LCMS-IT-Toff spectrometer in CH 3 CN/CHCl 3 . (Malatya, Turkey) Column chromatography was performed using silica gel 60 (70-230 mesh).

Conclusions
In this work, Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 have been already synthesized and characterized starting from benzimidazolium salts (2a-i). The molecular structures of the benzimidazolium salts (2a-i) and the Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 have been characterized by elemental analysis and 1 H-and 13 C-NMR spectra. The present results indicate that all of the synthesized Pd(II)-N-heterocyclic carbene (NHC) complexes 3-4 had potent anticancer activity, particularly 3g, 3f, 3h, and 4i. The compounds 3f, 3g, and 4c are the most active antibacterial drugs, while 3b, 3g, and 4e proved to be very strong antifungals. In this investigation, 8 compounds were found to be most active against both L. major promastigotes and amastigotes with high SI values. Compound 4b had the most potent activity against L. major. These candidates need more investigations of their mode of action and drug standardization.