Combined Effect of Caspase-Dependent and Caspase-Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives

pharmaceuticals Article Combined Effect of Caspase-Dependent and Caspase- Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives Lara Rouco 1,Ángeles Sánchez-González 2,*, Rebeca Alvariño 3,* , Amparo Alfonso 3, Ezequiel M. Vázquez-López 4, Emilia García-Martínez 4and Marcelino Maneiro 1,*   Citation:Rouco, L.; Sánchez-González, Á.; Alvariño, R.; Alfonso, A.; Vázquez- López, E.M.; García-Martínez, E.; Maneiro,M.CombinedEffectofCaspase- Dependent and Caspase-Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives. Pharmaceuticals 2021,14, 10. https://dx.doi.org/10.3390/ph1401 0010 Received: 24 November 2020 Accepted: 21 December 2020 Published: 24 December 2020 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This articleisan open accessarticledistributed under the terms and conditions of the CreativeCommonsAttribution(CCBY) license(https://creativecommons.org/ licenses/by/4.0/). 1Departamento de Química Inorgánica, Facultade de Ciencias, Universidade de Santiago de Compostela, 27002 Lugo, Spain; [email protected] 2Departamento de Química Inorgánica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 3Departamento de Farmacología, Facultade de Veterinaria, Campus Terra, Universidade de Santiago de Compostela, 27002 Lugo, Spain; [email protected] 4Departamento de Química Inorgánica, Facultade de Química, Campus Universitario Lagoas-Marcosende, Universidade de Vigo, 36310 Vigo, Spain; [email protected] (E.M.V.-L.); [email protected] (E.G.-M.) *Correspondence: [email protected] (Á.S.-G.); [email protected] (R.A.); marcelino.maneir[email protected] (M.M.); Tel.: +34-982-824-106 (M.M.) Abstract: Since the potential anticancer activity of auranofin was discovered, gold compounds have attracted interest with a view to developing anticancer agents that follow cytotoxic mechanisms other than cisplatin. Two benzimidazole gold(I) derivatives containing triphenylphosphine (Au(pben)(PPh 3 )) ( 1 ) or triethylphosphine (Au(pben)(PEt 3 )) ( 2 ) were prepared and characterized by standard techniques. X-ray crystal structures for 1 and 2 were solved. The cytotoxicity of 1 and 2 was tested in human neuroblastoma SH-SY5Y cells. Cells were incubated with compounds for 24 h with concentrations ranging from 10 µ M to 1 nM, and the half-maximal inhibitory concentration (IC 50 ) was determined. 1 and 2 showed an IC 50 of 2.7 and 1.6 µ M, respectively. In order to better understand the type of cell death induced by compounds, neuroblastoma cells were stained with Annexin-FITC and propidium iodide. The fluorescence analysis revealed that compounds were inducing apoptosis; however, pre-treatment with the caspase inhibitor Z-VAD did not reduce cell death. Analysis of compound effects on caspase-3 activity and reactive oxygen species (ROS) production in SH-SY5Y cells revealed an antiproliferative ability mediated through oxidative stress and both caspase-dependent and caspase-independent mechanisms. Keywords: gold(I) compounds; cytotoxic activity; caspase; apoptosis; neuroblastoma SH-SY5Y 1. Introduction The pharmacological use of gold compounds for the treatment of different diseases has been documented since ancient times [ 1 , 2 ]. More recently, in the twentieth century, the antiarthritic properties for Au(I) thiolates were observed [ 2 ], but their adverse effects led to the development of gold complexes with phosphine ligands, which are more lipophilic and remain in circulation for a longer time [ 3 ]. Auranofin, 2,3,4,6-tetra-O-acetyl- L-thio- β -D-glyco-pyranosato-S-(triethylphosphine) gold(I) is an example of these Au(I) thiolates-phosphine derivatives (see Scheme 1A), which contain a S-Au-P fragment, used as disease-modifying anti-rheumatic drugs [ 3 – 5 ] but also investigated for other potential therapeutic applications, from neurodegenerative disorders [ 6 ] or bacterial infections [ 7 ] to the inhibition of the novel SARS-COV-2 coronavirus [8]. Pharmaceuticals 2021,14, 10. https://dx.doi.org/10.3390/ph14010010 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021,14, 10 2 of 17 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 18 carcinoma [12], colorectal cancer [13], cervical epithelial carcinoma [14], and lung cancer cells [15]. The mechanism associated with this therapeutic activity has not yet been totally established, but results point to the ability of the Au(I) complexes to interact with biological targets such as proteins or nucleic acids [16,17] and, particularly, with thiol-containing proteins like the thioredoxin reductase (TrxR) [18,19], an enzyme involved both in the defense against oxidative damage and in the redox signaling, being relevant for cell growth and development [20]. Other studies have clearly shown the proteasome as a primary target for these complexes [21] or the inhibition of other enzymes such as glutathione reductase or peroxidase [22]. The anticancer activity of these gold compounds seems to follow different mechanisms than cisplatin or other Pt(II)/Pd(II) compounds, which mainly act by binding to DNA and inhibiting its replication. Gold compounds also exhibit cytotoxic activity against cisplatin-resistant cell lines [9,10]. The increasing interest in developing different types of S-Au-P gold-based anticancer candidates led to new synthetic drugs rather than the former pyranosidic derivatives. In this sense, gold complexes incorporating ligands such as thiosemicarbazone [14,23,24], thiolate [25,26], dithiocarbamate [27], and sulfanylcarboxylates [28] have been probed to inhibit thioredoxin reductase by binding the selenocysteines or the cysteinyl thiols of the enzyme. The replacement of the S-donor ligands by N-donor ligands produced a new family gold complexes that have revealed interesting anticancer activities. These N-donor ligands include imidazoles [29], naphthalimides [30], cytosines [31], bipyridines [32], phenanthrolines [33], or benzimidazoles [34–36]. The biochemical mechanism of action involved in the anticancer activity of the gold complexes with N-donor ligands is presumably the same as that of auranofin, although it is a matter of study interest due to the diversity of results obtained in different investigations regarding biological targets for auranofin [18–22], and due to the different complex stabilization by the replacement of the thiolate ligand by the N-donor ligand. In the present study, we propose to use 2-(2′-pyridyl)benzimidazole (Hpben) as a ligand with potentially three N-donor atoms (see Scheme 1B), which would lead to different coordinations with the gold atom [35]. Benzimidazoles are very versatile ligands that show various pharmacological properties such as antibacterial, antiviral, and anti-inflam- matory [37,38]. Our aim is to obtain N-Au-P complexes, for which Hpben and two different phosphines are used: triphenylphosphine and triethylphosphine. Human neuroblastoma SH-SY5Y is the cell line chosen to study the cytotoxicity, the type of cell death in the anticancer activity, and the mechanism of antiproliferative action of these gold complexes. (A) (B) Scheme 1. (A) Auranofin: 2,3,4,6-tetra-O-acetyl-L-thio-β-D-glyco-pyranosato-S-(triethylphosphine) gold(I); (B) Numbering scheme of 2-(2′-pyridyl)benzimidazole (Hpben), which is the N-donor ligand used in this work to coordinate gold atom instead of the S-glycoside ligand of auranofin. Scheme 1. ( A ) Auranofin: 2,3,4,6-tetra-O-acetyl-L-thio- β -D-glyco-pyranosato-S-(triethylphosphine) gold(I); ( B ) Numbering scheme of 2-(2 0 -pyridyl)benzimidazole (Hpben), which is the N-donor ligand used in this work to coordinate gold atom instead of the S-glycoside ligand of auranofin. The anticancer activity of this type of compound has also attracted interest due to its cytotoxic activity against cells from several tumor cell lines [ 9 – 11 ]. Among them, ovarian carcinoma [ 12 ], colorectal cancer [ 13 ], cervical epithelial carcinoma [ 14 ], and lung cancer cells [ 15 ]. The mechanism associated with this therapeutic activity has not yet been totally established, but results point to the ability of the Au(I) complexes to interact with biological targets such as proteins or nucleic acids [ 16 , 17 ] and, particularly, with thiol-containing proteins like the thioredoxin reductase (TrxR) [ 18 , 19 ], an enzyme involved both in the defense against oxidative damage and in the redox signaling, being relevant for cell growth and development [ 20 ]. Other studies have clearly shown the proteasome as a primary target for these complexes [ 21 ] or the inhibition of other enzymes such as glutathione reductase or peroxidase [ 22 ]. The anticancer activity of these gold compounds seems to follow different mechanisms than cisplatin or other Pt(II)/Pd(II) compounds, which mainly act by binding to DNA and inhibiting its replication. Gold compounds also exhibit cytotoxic activity against cisplatin-resistant cell lines [9,10]. The increasing interest in developing different types of S-Au-P gold-based anticancer candidates led to new synthetic drugs rather than the former pyranosidic derivatives. In this sense, gold complexes incorporating ligands such as thiosemicarbazone [ 14 , 23 , 24 ], thiolate [ 25 , 26 ], dithiocarbamate [ 27 ], and sulfanylcarboxylates [ 28 ] have been probed to inhibit thioredoxin reductase by binding the selenocysteines or the cysteinyl thiols of the enzyme. The replacement of the S-donor ligands by N-donor ligands produced a new family gold complexes that have revealed interesting anticancer activities. These N-donor ligands include imidazoles [ 29 ], naphthalimides [ 30 ], cytosines [ 31 ], bipyridines [ 32 ], phenanthrolines [ 33 ], or benzimidazoles [ 34 – 36 ]. The biochemical mechanism of action involved in the anticancer activity of the gold complexes with N-donor ligands is presumably the same as that of auranofin, although it is a matter of study interest due to the diversity of results obtained in different investigations regarding biological targets for auranofin [ 18 – 22 ], and due to the different complex stabilization by the replacement of the thiolate ligand by the N-donor ligand. In the present study, we propose to use 2-(2 0 -pyridyl)benzimidazole (Hpben) as a ligand with potentially three N-donor atoms (see Scheme 1B), which would lead to different coordinations with the gold atom [ 35 ]. Benzimidazoles are very versatile ligands that show various pharmacological properties such as antibacterial, antiviral, and antiinflammatory [ 37 , 38 ]. Our aim is to obtain N-Au-P complexes, for which Hpben and two different phosphines are used: triphenylphosphine and triethylphosphine. Human neuroblastoma SH-SY5Y is the cell line chosen to study the cytotoxicity, the type of cell death in the anticancer activity, and the mechanism of antiproliferative action of these gold complexes. Pharmaceuticals 2021,14, 10 3 of 17 2. Results 2.1. Analytical and Spectroscopic Characterization of 1and 2 Complexes 1 and 2 were obtained in high yield as detailed in the experimental section (Scheme 2). Their structures were fully characterized using 1 H, 13 C and 31 P NMR spectroscopy, IR and UV spectroscopy, electrospray ionization mass spectroscopy (ESIMS), and single-crystal X-ray crystallography. Elemental analyses establish a formula (Au(pben)(PR 3 )), being R=Ph for 1 and R=Et for 2 . ESI-MS spectra confirmed these formulas for the neutral complexes by showing molecular ion peaks at m/z644.14 for 1 and m/z510.13 for 2 (Figures S1 and S2). UV-Vis spectra showed in DMSO a common absorption band at 313 nm assigned to π – π * transitions located in the heteroaromatic benzimidazole rings (Figures S3 and S4). Complex 1 displays an additional band at 325 nm, which has also previously been found in gold(I) complexes containing the ancillary PPh3ligand [34]. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 18 2. Results 2.1. Analytical and Spectroscopic Characterization of 1 and 2 Complexes 1 and 2 were obtained in high yield as detailed in the experimental section (Scheme 2). Their structures were fully characterized using 1H, 13C and 31P NMR spectroscopy, IR and UV spectroscopy, electrospray ionization mass spectroscopy (ESI-MS), and single-crystal X-ray crystallography. Elemental analyses establish a formula (Au(pben)(PR3)), being R=Ph for 1 and R=Et for 2. ESI-MS spectra confirmed these formulas for the neutral complexes by showing molecular ion peaks at m/z 644.14 for 1 and m/z 510.13 for 2 (Figures S1 and S2). UV-Vis spectra showed in DMSO a common absorption band at 313 nm assigned to π–π* transitions located in the heteroaromatic benzimidazole rings (Figures S3 and S4). Complex 1 displays an additional band at 325 nm, which has also previously been found in gold(I) complexes containing the ancillary PPh3 ligand [34]. Scheme 2. Synthesis of complexes 1 and 2. The IR spectra of these complexes (Figures S5 and S6), when compared with the corresponding spectrum of its free ligand, show the absence of the ν(N–H) band, which is present in the spectrum of the Hbpen between 2990–2700 cm−1, consistent with the deprotonation of this group and the N-coordination to the Au atom. In both complexes, the aromatic tension bands of the pben ligand do not undergo significant changes when coordination occurs, although rigorous assignment is made difficult by the great intensity showed by the bands from the phosphines. The 1H NMR spectra of 1 and 2 showed each one as well-resolved set of signals (Figures S7 and S8); the H6′ proton at δ 8.57/8.52 was the most deshielded and downfield shifted of 0.13–0.18 ppm with respect to the free ligand (1H NMR and 13C NMR spectra for free ligand are collected in Figures S9–S12), while the resonance of the H3′ proton was shifted at about 0.18 ppm, which suggests some involvement of the pyridinic nitrogen in the Au-pben bonding. In both complexes, the N–H signal of the free ligand disappeared, confirming the deprotonation of Hbpen. In the spectrum of 1, the signals corresponding to the co-ligand PPh3 overlap with those of pben-, so the assignment is tentative. Aromatic carbons from pben- and triphenylphosphine ligands are registered in the 13C NMR spectrum of 1 (Figure S13). 31P NMR spectrum of 1 shows a single signal at 30.9 ppm, which is evidence that the gold complex is not altered in solution (Figure S14). In the case of 2, the identification and assignment of PEt3 signals were unambiguous with the –CH3 signals as a doublet of triplets at 1.31 ppm due to the coupling with 31P (coupling constant 3J(H–P) = Scheme 2. Synthesis of complexes 1and 2. The IR spectra of these complexes (Figures S5 and S6), when compared with the corresponding spectrum of its free ligand, show the absence of the ν (N–H) band, which is present in the spectrum of the Hbpen between 2990–2700 cm −1 , consistent with the deprotonation of this group and the N-coordination to the Au atom. In both complexes, the aromatic tension bands of the pben ligand do not undergo significant changes when coordination occurs, although rigorous assignment is made difficult by the great intensity showed by the bands from the phosphines. The 1 H NMR spectra of 1 and 2 showed each one as well-resolved set of signals (Figures S7 and S8); the H6 0 proton at δ 8.57/8.52 was the most deshielded and downfield shifted of 0.13–0.18 ppm with respect to the free ligand ( 1 H NMR and 13 C NMR spectra for free ligand are collected in Figures S9–S12), while the resonance of the H3 0 proton was shifted at about 0.18 ppm, which suggests some involvement of the pyridinic nitrogen in the Au-pben bonding. In both complexes, the N–H signal of the free ligand disappeared, confirming the deprotonation of Hbpen. In the spectrum of 1 , the signals corresponding to the co-ligand PPh 3 overlap with those of pben − , so the assignment is tentative. Aromatic carbons from pben − and triphenylphosphine ligands are registered in the 13 C NMR spectrum of 1 (Figure S13). 31 P NMR spectrum of 1 shows a single signal at 30.9 ppm, which is evidence that the gold complex is not altered in solution (Figure S14). In the case of 2 , the identification and assignment of PEt 3 signals were unambiguous with the –CH 3 signals as a doublet of triplets at 1.31 ppm due to the coupling with 31 P (coupling constant 3 J(H–P) Pharmaceuticals 2021,14, 10 4 of 17 = 18.5 Hz, slightly higher than the corresponding to the free PEt 3 , 14 Hz). The 13 C NMR spectrum of 2 showed two signals (Figure S15) corresponding to the phosphine at 9.2 (CH 3 ) and 18.5 (d, CH 2 ,J(C–P) = 37.3 Hz) ppm, δ and J values which are like those found for other Au(I)-PEt 3 complexes [ 14 , 28 ], and that contrast with the values of non-coordinated PEt3.31P NMR spectrum of 2showed a single signal at 26.9 ppm (Figure S16). The electrochemical behavior of compounds 1 , 2 was investigated in DMSO- tetraethy- lammonium perchlorate 0.1 M solvent system through cyclic voltammetry. Figure 1shows the voltammograms at different scan rates. The Au(I) gold complexes undergo one oxidation process (at −0.594 for 1and at −0.597 V for 2) to form the Au(III) species. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 18 18,5 Hz, slightly higher than the corresponding to the free PEt3, 14 Hz). The 13C NMR spectrum of 2 showed two signals (Figure S15) corresponding to the phosphine at 9.2 (CH3) and 18.5 (d, CH2, J(C–P) = 37.3 Hz) ppm, δ and J values which are like those found for other Au(I)-PEt3 complexes [14,28], and that contrast with the values of non-coordi- nated PEt3. 31P NMR spectrum of 2 showed a single signal at 26.9 ppm (Figure S16). The electrochemical behavior of compounds 1,2 was investigated in DMSO-tetrae- thylammonium perchlorate 0.1 M solvent system through cyclic voltammetry. Figure 1 shows the voltammograms at different scan rates. The Au(I) gold complexes undergo one oxidation process (at −0.594 for 1 and at −0.597 V for 2) to form the Au(III) species. (A) (B) Figure 1. Cyclic voltammograms for (A) 1 and (B) 2 at different scan rates: 0.02 V s−1 (blue), 0.05 V s−1 (green) and 0.09 V s−1 (red). The redox properties of the two gold(I) complexes showed grossly similar behavior consisting of a quasi-reversible Au(I)/Au(III) oxidation. The criteria of reversibility were checked by observing the constancy of peak-peak separation (ΔEp = Epa – Epc) of 188 mV for 1 and 214 mV for 2 at 0.02 V s−1. The reversible character decreased as scan rates increased. Thus, ΔEp are 281 mV for 1 and 259 mV for 2 at 0.05 V s−1, and even 344 mV for 1 and 297 mV for 2 at 0.09 V s−1. As is predictable, the intensity of the current also increased at higher scan rates. Cyclic voltammograms of 1,2 endorse the purity and stability of both complexes in solution. 2.2. Crystal Structures of Complexes 1 and 2 Single crystals of complex 1 suitable for X-ray diffraction studies were obtained by slow evaporation of the methanolic solution at room temperature. The molecular structure is shown in Figure 2 and Figure S17, and the main bond distances and angles are shown in Table 1. The asymmetric unit contained two molecules of 1, (Au(pben)PPh3), with slightly different bond distances and angles values and one disordered methanol molecule. Since it did not establish any relevant interaction with (Au(pben)PPh3), it was removed using the SQUEEZE program [39]. In the two molecules of the complex, the metal ion is coordinated to the phosphorous atom of triphenylphosphine and to the nitrogen atom of the benzimidazolate ring with an almost linear disposition. An additional contact was established between the nitrogen atom of the pyridine and the gold ion, giving rise to a 2 + 1 coordination number. Figure 1. Cyclic voltammograms for ( A ) 1 and ( B ) 2 at different scan rates: 0.02 V s −1 (blue), 0.05 V s −1 (green) and 0.09 V s−1(red). The redox properties of the two gold(I) complexes showed grossly similar behavior consisting of a quasi-reversible Au(I)/Au(III) oxidation. The criteria of reversibility were checked by observing the constancy of peak-peak separation ( ∆ E p = E pa – E pc ) of 188 mV for 1 and 214 mV for 2 at 0.02 V s −1 . The reversible character decreased as scan rates increased. Thus, ∆ E p are 281 mV for 1 and 259 mV for 2 at 0.05 V s −1 , and even 344 mV for 1 and 297 mV for 2 at 0.09 V s −1 . As is predictable, the intensity of the current also increased at higher scan rates. Cyclic voltammograms of 1 , 2 endorse the purity and stability of both complexes in solution. 2.2. Crystal Structures of Complexes 1and 2 Single crystals of complex 1 suitable for X-ray diffraction studies were obtained by slow evaporation of the methanolic solution at room temperature. The molecular structure is shown in Figure 2and Figure S17, and the main bond distances and angles are shown in Table 1. Pharmaceuticals 2021,14, 10 5 of 17 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 18 Figure 2. ORTEP representation of one of the molecules presents in the crystal structure of compound 1 (hydrogen atoms have been omitted for clarity). The Au-P Bond lengths (2.230(2) y 2.235(2) Å) were similar to those found in the cationic complex (Au(Hpben)(PPh 3 ))ClO 4 [40]. However, the Au-N bond distances (2.058(6) and 2.060(6)) were slightly shorter than those found in the (Au(Hpben)(PPh 3 )) + cation (2.075(4) Å), consistent with a stronger interaction between the metal ion and the deprotonated ligand. The bond length between the gold and the pyridine nitrogen (Au(1)···N(3) = 2,729(7) Å and Au(2)···N(6) = 2,782(7) Å) was shorter than the sum of the Van der Waals radii (3,25 Å), and shorter than that observed in (Au(Hpben)(PPh 3 ))ClO 4 (2,930 Å) [40]. Bond lengths and angles found in the triphenylphosphine ligands (not collected in Table 2) showed normal values and similar to those reported for complexes of Au(I) and PPh 3 [40–42]. Table 1. Selected bond lengths (Å) and angles (°) for 1. Bond Lengths (Å) Bond Angles (°) Au(1)-N(1) 2.058(6) Au(2)-N(4) 2.060(6) Au(1)-P(1) 2.230(2) Au(2)-P(2) 2.235(2) Au(1)···N(3) 2.792(7) Au(2)···N(6) 2.782(7) N(1)-Au(1)-P(1) 176.40(18) N(4)-Au(2)-P(2) 174.75(18) N(1)-Au(1)-N(3) 68.5(2) N(4)-Au(2)-N(6) 68.8(2) P(1)-Au(1)-N(3) 113.52(17) P(2)-Au(2)-N(6) 114.48(16) C(13)-P(1)-Au(1) 110.5(3) C(43)-P(2)-Au(2) 113.9(3) C(25)-P(1)-Au(1) 115.1(3) C(49)-P(2)-Au(2) 108.0(3) C(19)-P(1)-Au(1) 112.9(3) C(55)-P(2)-Au(2) 114.9(3) C(3)-N(1)-Au(1) 129.6(6) C(31)-N(4)-Au(2) 126.3(5) C(1)-N(1)-Au(1) 125.5(5) C(33)-N(4)-Au(2) 128.7(5) Figure 2. ORTEP representation of one of the molecules presents in the crystal structure of compound 1(hydrogen atoms have been omitted for clarity). Table 1. Selected bond lengths (Å) and angles (◦) for 1. Bond Lengths (Å) Bond Angles (◦) Au(1)-N(1) 2.058(6) Au(2)-N(4) 2.060(6) Au(1)-P(1) 2.230(2) Au(2)-P(2) 2.235(2) Au(1)···N(3) 2.792(7) Au(2)···N(6) 2.782(7) N(1)-Au(1)-P(1) 176.40(18) N(4)-Au(2)-P(2) 174.75(18) N(1)-Au(1)-N(3) 68.5(2) N(4)-Au(2)-N(6) 68.8(2) P(1)-Au(1)-N(3) 113.52(17) P(2)-Au(2)-N(6) 114.48(16) C(13)-P(1)-Au(1) 110.5(3) C(43)-P(2)-Au(2) 113.9(3) C(25)-P(1)-Au(1) 115.1(3) C(49)-P(2)-Au(2) 108.0(3) C(19)-P(1)-Au(1) 112.9(3) C(55)-P(2)-Au(2) 114.9(3) C(3)-N(1)-Au(1) 129.6(6) C(31)-N(4)-Au(2) 126.3(5) C(1)-N(1)-Au(1) 125.5(5) C(33)-N(4)-Au(2) 128.7(5) C(8)-N(3)-Au(1) 106.2(5) C(38)-N(6)-Au(2) 104.7(5) C(9)-N(3)-Au(1) 134.2(7) C(39)-N(6)-Au(2) 135.3(6) The asymmetric unit contained two molecules of 1 , (Au(pben)PPh 3 ), with slightly different bond distances and angles values and one disordered methanol molecule. Since it did not establish any relevant interaction with (Au(pben)PPh 3 ), it was removed using the SQUEEZE program [ 39 ]. In the two molecules of the complex, the metal ion is coordinated to the phosphorous atom of triphenylphosphine and to the nitrogen atom of the benzimidazolate ring with an almost linear disposition. An additional contact was established between the nitrogen atom of the pyridine and the gold ion, giving rise to a 2 + 1 coordination number. The Au-P Bond lengths (2.230(2) y 2.235(2) Å) were similar to those found in the cationic complex (Au(Hpben)(PPh 3 ))ClO 4 [ 40 ]. However, the Au-N bond distances (2.058(6) and 2.060(6)) were slightly shorter than those found in the (Au(Hpben)(PPh 3 )) + cation (2.075(4) Å), consistent with a stronger interaction between the metal ion and the deprotonated ligand. The bond length between the gold and the pyridine nitrogen (Au(1) ··· N(3) = 2.729(7) Å and Au(2) ··· N(6) = 2.782(7) Å) was shorter than the sum of the Van der Waals radii (3.25 Å), and shorter than that observed in (Au(Hpben)(PPh 3 ))ClO 4 (2.930 Å) [ 40 ]. Bond lengths and angles found in the triphenylphosphine ligands (not collected in Table 2) showed normal values and similar to those reported for complexes of Au(I) and PPh3[40–42]. Pharmaceuticals 2021,14, 10 6 of 17 Table 2. Selected bond lengths (Å) and angles (◦) for 2. Bond Lengths (Å) Bond Angles (◦) Au(1)-N(1) 2.050(6) Au(2)-N(4) 2.058(5) Au(1)-P(1) 2.2288(18) Au(2)-P(2) 2.224(2) Au(1)···N(3) 2.975(6) Au(2)···N(6) 2.849(6) N(1)-Au(1)-P(1) 172.59(16) N(4)-Au(2)-P(2) 174.37(17) N(1)-Au(1)-N(3) 66.34(19) N(4)-Au(2)-N(6) 67.38(19) P(1)-Au(1)-N(3) 120.75(12) P(2)-Au(2)-N(6) 116.88(13) Single crystals of 2 were obtained as detailed in the experimental section; a perspective view is shown in Figure 3and Figure S18, and selected bond parameters are collected in Table 2. The asymmetric unit of the crystal structure of 2 also consists of two chemically equivalent molecules in which the benzimidazolate unit is acting as a monodentate ligand through the N(1) nitrogen atom. The gold(I) ion was also arranged in the usual almost linear coordination geometry, coordinated to pben and to the phosphorous atom of triethylphosphine. In the same way as in 1 , an additional contact was established between the nitrogen atom of the pyridine and the gold ion, giving rise to a 2 + 1 coordination number. Au-P Bond lengths (2.2288(18) and 2.224(2) Å) and Au-N bond distances (2.050(6) and 2.058(5)) are values very close to those found in 1. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 18 C(8)-N(3)-Au(1) 106.2(5) C(38)-N(6)-Au(2) 104.7(5) C(9)-N(3)-Au(1) 134.2(7) C(39)-N(6)-Au(2) 135.3(6) Single crystals of 2 were obtained as detailed in the experimental section; a perspective view is shown in Figure 3 and Figure S18, and selected bond parameters are collected in Table 2. The asymmetric unit of the crystal structure of 2 also consists of two chemically equivalent molecules in which the benzimidazolate unit is acting as a monodentate ligand through the N(1) nitrogen atom. The gold(I) ion was also arranged in the usual almost linear coordination geometry, coordinated to pben and to the phosphorous atom of triethylphosphine. In the same way as in 1, an additional contact was established between the nitrogen atom of the pyridine and the gold ion, giving rise to a 2 +1 coordination number. Au-P Bond lengths (2.2288(18) and 2.224(2) Å) and Au-N bond distances (2.050 (6) and 2.058 (5)) are values very close to those found in 1. Figure 3. ORTEP representation of one of the molecules presents in the crystal structure of compound 2 (hydrogen atoms have been omitted for clarity). Table 2. Selected bond lengths (Å) and angles (°) for 2. Bond Lengths (Å) Bond Angles (°) Au(1)-N(1) 2.050(6) Au(2)-N(4) 2.058(5) Au(1)-P(1) 2.2288(18) Au(2)-P(2) 2.224(2) Au(1)···N(3) 2.975(6) Au(2)···N(6) 2.849(6) N(1)-Au(1)-P(1) 172.59(16) N(4)-Au(2)-P(2) 174.37(17) N(1)-Au(1)-N(3) 66.34(19) N(4)-Au(2)-N(6) 67.38(19) P(1)-Au(1)-N(3) 120.75(12) P(2)-Au(2)-N(6) 116.88(13) 2.3. Cytotoxicity Studies and Determination of Cell Death Type SH-SY5Y neuroblastoma cells were used to study the toxic effect of the Au(I) complexes. Cells were treated with different concentrations of 1 and 2 at two incubation times, Figure 3. ORTEP representation of one of the molecules presents in the crystal structure of compound 2(hydrogen atoms have been omitted for clarity). 2.3. Cytotoxicity Studies and Determination of Cell Death Type SH-SY5Y neuroblastoma cells were used to study the toxic effect of the Au(I) complexes. Cells were treated with different concentrations of 1 and 2 at two incubation times, 6 and 24 h. Table 3shows IC 50 values calculated for compounds (see also Figure S19). Complex 2 was more cytotoxic, presenting lower IC 50 values than complex 1 at both times. Pharmaceuticals 2021,14, 10 7 of 17 Table 3. IC 50 ( µ M) of gold compounds in SH-SY5Y cells determined with a 3-(4,5-dimethyl thiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. IC 50 (half maximal inhibitory concentration), R2(coefficient of determination which gives the goodness-of-fit for the regression model). Compound Incubation Time (h) IC50 (µM) R295% Confidence Interval 16 6.2 0.97 3.851 to 8.30 24 2.7 0.94 1.534 to 4.684 26 3.1 0.93 1.340 to 5.015 24 1.6 0.91 0.8094 to 3.018 Next, to determine the type of cell death produced by gold complexes, their effects on neuroblastoma cells were evaluated using the fluorescent dyes Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI), which allow the detection of necrotic and apoptotic cell populations by flow cytometry. Three populations were discriminated: viable cells (Annexin V-FITC and PI negative cells), apoptotic cells (including Annexin V- FITC positive and PI negative cells, and Annexin V-FITC and PI-positive cells), and necrotic cells (Annexin V-FITC negative and PI-positive cells). In this assay, SH-SY5Y cells were treated with compounds at IC 50 concentrations for 6 and 24 h. Furthermore, to check if the complexes 1 and 2 were inducing caspase-dependent apoptosis, cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl- [O-methyl]-fluoromethylketone) at 40 µ M for 24 h, followed by treatment with compounds at IC 50 concentrations for 6 and 24 h. In order to validate the model, staurosporine (STS) was used as cell death control (Figure 4). Pharmaceuticals 2021,14, 10 8 of 17 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 18 6 and 24 h. Table 3 shows IC50 values calculated for compounds (see also Figure S19). Complex 2 was more cytotoxic, presenting lower IC50 values than complex 1 at both times. Table 3. IC50 (µM) of gold compounds in SH-SY5Y cells determined with a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. IC50 (half maximal inhibitory concentration), R2 (coefficient of determination which gives the goodness-of-fit for the regression model). Compound Incubation Time (h) IC50 (μM) R2 95% Confidence Interval 1 6 6.2 0.97 3.851 to 8.30 24 2.7 0.94 1.534 to 4.684 2 6 3.1 0.93 1.340 to 5.015 24 1.6 0.91 0.8094 to 3.018 Next, to determine the type of cell death produced by gold complexes, their effects on neuroblastoma cells were evaluated using the fluorescent dyes Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI), which allow the detection of necrotic and apoptotic cell populations by flow cytometry. Three populations were discriminated: viable cells (Annexin V-FITC and PI negative cells), apoptotic cells (including Annexin V- FITC positive and PI negative cells, and Annexin V-FITC and PI-positive cells), and necrotic cells (Annexin V-FITC negative and PI-positive cells). In this assay, SH-SY5Y cells were treated with compounds at IC50 concentrations for 6 and 24 h. Furthermore, to check if the complexes 1 and 2 were inducing caspase-dependent apoptosis, cells were pre-in- cubated with the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspar- tyl-[O-methyl]-fluoromethylketone) at 40 µM for 24 h, followed by treatment with compounds at IC50 concentrations for 6 and 24 h. In order to validate the model, staurosporine (STS) was used as cell death control (Figure 4). Figure 4. Determination of cell death type produced by 1 and 2 on SH-SY5Y cells. Human neuroblastoma cells were treated with compounds for 6 (A) and 24 h (B) with and without pre-incubation with 40 µM Z-VAD. Staurosporine (STS) was used as a positive control. Cell death type was determined with Annexin V-FITC/PI staining by flow cytometry. Values are mean ± SEM of three independent replicates expressed as a percentage of untreated control cells. Statistical differences were determined by Student’s t test. * p < 0.05, comparison of compound treatment and control cells. # p < 0.05, comparison between treatments with compounds alone and co-treatment with compounds and Z-VAD. Figure 4. Determination of cell death type produced by 1 and 2 on SH-SY5Y cells. Human neuroblastoma cells were treated with compounds for 6 ( A ) and 24 h ( B ) with and without pre-incubation with 40 µ M Z-VAD. Staurosporine (STS) was used as a positive control. Cell death type was determined with Annexin V-FITC/PI staining by flow cytometry. Values are mean ± SEM of three independent replicates expressed as a percentage of untreated control cells. Statistical differences were determined by Student’s ttest. * p< 0.05, comparison of compound treatment and control cells. # p < 0.05, comparison between treatments with compounds alone and co-treatment with compounds and Z-VAD. Treatment with compounds 1 and 2 for 6 h produced a significant decrease in viable cells, with levels of 70.6 ± 3.0% and 74.1 ± 1.4%, respectively (p< 0.05) (Figure 4A). In this assay, gold compounds produced a lower decrease in cell viability than expected, which could be due to the differences between the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Annexin/PI staining. IC 50 values were determined with MTT, which quantifies cell viability based on mitochondrial activity, so it was more sensitive than the Annexin/PI staining, which detects changes in the plasmatic membrane [ 43 – 45 ]. Both complexes produced an increase in apoptotic cells (15.0 ± 1.2% for 1 and 13.7 ± 1.3% for 2 ). Moreover, compound 1 significantly augmented necrotic cells (14.4 ± 1.0%) compared to control cells. As expected, the addition of 0.1 µ M STS, a known apoptotic inducer, generated an increase of apoptotic cells (29.3 ±3.1%). On the other hand, cells co-treated with Z-VAD and compounds 1 and 2 for 6 h presented a significant decrease in viable cells (71.6 ± 2.7% and 74.3 ± 2.1%, respectively). Interestingly, the percentages of apoptotic cells after pre-treatment with Z-VAD ( 14.1 ±0.7% and 13.0 ± 0.9%, respectively) were similar to those obtained with the compounds alone. Otherwise, the apoptotic death produced by 0.1 µ M STS alone was significantly reduced when neuroblastoma cells were pre-treated with the caspase inhibitor (18.5 ± 1.1%, p< 0.05 ). Regarding necrosis, its levels were comparable to those observed with gold complexes alone, with percentages among 12.7–14.3%. In the case of STS, necrotic cells were significantly reduced by pre-treatment with Z-VAD. The results obtained with STS validated the model since its cytotoxic effects were reduced when the caspase inhibitor was added to SH-SY5Y cells. However, Z-VAD was unable to reduce the cell death produced by gold compounds. This assay was repeated after a longer incubation time, 24 h (Figure 4B). A reduction in cell survival was observed when cells were treated with complexes 1 and 2 ( 68.1 ±0.9% and 59.3 ± 2.0%, respectively). Once again, compounds produced a significant augmentation of apoptotic cells, with levels of 22.8 ± 0.6% after the addition of complex 1 , and 27.0 ±0.5% when cells were treated with 2 . In order to further confirm the results obtained at 6 h, neuroblastoma cells were pre-treated with 40 µ M Z-VAD, followed by the addition of complexes 1 and 2 for 24 h. In this assay, both compounds produced a significant decrease in viable cells (67.5 ± 2.1% and 66.2 ± 3.3%, respectively). In addition, the levels of Pharmaceuticals 2021,14, 10 9 of 17 apoptotic cells were between 19.9–23.2%, confirming the inability of Z-VAD to inhibit the death of neuroblastoma cells. With respect to STS, pre-treatment with Z-VAD did not produce a significant reduction in the apoptotic death generated by the compound after 24 h. This effect has been previously described and is related to the ability of STS to produce caspase-dependent and caspase-independent apoptosis. The first one happens at shorter incubation times, whilst the second one occurs more slowly and can be observed at longer incubation times [ 46 , 47 ]. In our model, Z-VAD reduced the apoptosis produced by STS after 6 h of incubation, but no effect was found at 24 h, agreeing with these previous results. In the case of gold complexes, previous studies have reported that this class of compounds can also induce apoptosis through both caspase-dependent and caspaseindependent mechanisms [ 48 , 49 ]. In this context, the effect of complexes 1 and 2 on caspase-3 activity was analyzed. SH-SY5Y cells were treated with compounds at IC 50 concentrations for 6 and 24 h, and the enzymatic activity was determined (Figure 5). Pharmaceuticals 2021, 14, x FOR PEER REVIEW 9 of 18 Figure 5. Caspase-3 activity in human neuroblastoma cells treated with gold compounds 1 and 2. Compounds were added to the cells for 6 h and 24 h. STS was used as a positive control. Data are mean ± SEM of three experiments performed in triplicate, expressed as a percentage of control cells, and compared control cells by Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001. At 6 h, STS produced an augmentation in caspase-3 activity of 242.5 ± 7.2% (p < 0.001), agreeing with its previously reported effect on caspase-dependent apoptosis at short incubation times [46,47]. With regard to gold complexes, only 2 showed a significant increase in enzyme activity (190 ± 10.3%; p < 0.001). The assay was repeated for 24 h and both complexes augmented caspase-3 activity, 129.0 ± 10.5% (p < 0.05) and 140.8 ± 12.3% (p < 0.01), respectively. At this time, STS also produced a significant increase in caspase-3 activity (170.5 ± 6.8%, p < 0.001). This rise was smaller than the observed at 6 h, confirming that the involvement of caspase-3 in STS-induced death is lesser at 24 h. With regard to gold complexes, compound 2 presented a similar behavior to STS, with a lower increase in caspase-3 activity at 24 h, whereas complex 1 produced a slight augmentation of caspase-3 activity. These results could explain the lack of effect of pre-treatment with Z- VAD in our previous assays, pointing to a combined effect of the compound on caspasedependent and caspase-independent apoptosis. Previously published studies [48,49] stated that Au(I) and Au(III) complexes produce cell death through the induction of oxidative stress. Therefore, the effect of compounds on ROS production was analyzed. Cells were co-treated with 1 and 2 at nine concentrations (0.01–10 µM) for 24 h, and the levels of these damaging molecules were evaluated with a fluorometric assay (Figure 6). Treatment with complex 1 significantly augmented ROS production, reaching levels of 122.4 ± 11.9% (p < 0.05) at 5 µM compared to control cells. Compound 2 also produced a significant increase in ROS release at three concentrations (0.01, 0.5, and 7.5 µM), being the greater augmentation at 7.5 µM (129.7 ± 13.8%, p < 0.05). The increase generated by gold complexes was comparable to the rise produced by the known pro-oxidant tert-butyl hydroperoxide (TBHP) at 75 µM (134.9 ± 7.1%, p < 0.001). Figure 5. Caspase-3 activity in human neuroblastoma cells treated with gold compounds 1 and 2 . Compounds were added to the cells for 6 h and 24 h. STS was used as a positive control. Data are mean ± SEM of three experiments performed in triplicate, expressed as a percentage of control cells, and compared control cells by Student’s ttest. * p< 0.05, ** p< 0.01, *** p< 0.001. At 6 h, STS produced an augmentation in caspase-3 activity of 242.5 ± 7.2% ( p< 0.001 ), agreeing with its previously reported effect on caspase-dependent apoptosis at short incubation times [ 46 , 47 ]. With regard to gold complexes, only 2 showed a significant increase in enzyme activity (190 ± 10.3%; p< 0.001). The assay was repeated for 24 h and both complexes augmented caspase-3 activity, 129.0 ± 10.5% (p< 0.05) and 140.8 ± 12.3% (p< 0.01), respectively. At this time, STS also produced a significant increase in caspase-3 activity (170.5 ± 6.8%, p< 0.001). This rise was smaller than the observed at 6 h, confirming that the involvement of caspase-3 in STS-induced death is lesser at 24 h. With regard to gold complexes, compound 2 presented a similar behavior to STS, with a lower increase in caspase-3 activity at 24 h, whereas complex 1 produced a slight augmentation of caspase-3 activity. These results could explain the lack of effect of pre-treatment with Z-VAD in our previous assays, pointing to a combined effect of the compound on caspase-dependent and caspase-independent apoptosis. Previously published studies [ 48 , 49 ] stated that Au(I) and Au(III) complexes produce cell death through the induction of oxidative stress. Therefore, the effect of compounds on ROS production was analyzed. Cells were co-treated with 1 and 2 at nine concentrations (0.01–10 µ M) for 24 h, and the levels of these damaging molecules were evaluated with a fluorometric assay (Figure 6). Treatment with complex 1 significantly augmented ROS Pharmaceuticals 2021,14, 10 16 of 17 12. Marzo, T.; Massai, L.; Pratesi, A.; Stefanini, M.; Cirri, D.; Magherini, F. Replacement of the Thiosugar of Auranofin with Iodide Enhances the Anticancer Potency in a Mouse Model of Ovarian Cancer. ACS Med. Chem. Lett. 2019,10, 656–660. [CrossRef] 13. Marzo, T.; Cirri, D.; Gabbiani, C.; Gamberi, T.; Magherini, F.; Pratesi, A. Auranofin, Et3PAuCl, and Et3PAuI Are Highly Cytotoxic on Colorectal Cancer Cells: A Chemical and Biological Study. ACS Med. Chem. Lett. 2017,8, 997–1001. [CrossRef] [PubMed] 14. Gonzalez-Barcia, L.M.; Fernandez-Fariña, S.; Rodríguez-Silva, L.; Bermejo, M.R.; González-Noya, A.M.; Pedrido, R. Comparative study of the antitumoral activity of phosphine-thiosemicarbazone gold(I) complexes obtained by different methodologies. J. Inorg. Biochem. 2020,203, 110931. [CrossRef] [PubMed] 15. Yoo, M.-H.; Xu, X.-M.; Carlson, B.A.; Gladyshev, V.N.; Hatfield, D.L. Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J. Biol. Chem. 2006,281, 13005–13008. [CrossRef] [PubMed] 16. Casini, A.; Hartinger, C.; Gabbiani, C.; Mini, E.; Dyson, P.J.; Keppler, B.K.; Messori, L. Gold (III) compounds as anticancer agents: Relevance of gold–protein interactions for their mechanism of action. J. Inorg. Biochem. 2008 ,102, 564–575. [CrossRef] [PubMed] 17. Nobili, S.; Mini, E.; Landini, I.; Gabbiani, C.; Casini, A.; Messori, L. Gold compounds as anticancer agents: Chemistry, cellular pharmacology, and preclinical studies. Med. Res. Rev. 2010,30, 550–580. [CrossRef] 18. Ott, I. On the medicinal chemistry of gold complexes as anticancer drugs. J. Coord. Chem. Rev. 2009,253, 1670–1681. [CrossRef] 19. Hassan, F.; Al-Aridhi, D.T. Antitumor effect of 4-(N, N-dimethyl)-3-(3-Mercapto-5-Phenyl [1,2,4] triazol-4yl)-thiazolidin-4-one in liver carcinoma cell lines Hep G2 by (HCS) technique. Int. J. Pharm. Sci. 2015,5, 1317–1322. 20. Zhong, L.; Arnér, E.S.J.; Holmgren, A. Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide. Proc. Natl. Acad. Sci. USA 2007,104, 12288–12293. 21. Zhang, X.; Frezza, M.; Milacic, V.; Ronconi, L.; Fan, Y.; Bi, C.; Fregona, D.; Dou, Q.P. Inhibition of tumor proteasome activity by gold-dithiocarbamato complexes via both redox-dependent and -independent processes. J. Cell. Biochem. 2010 ,109, 162–172. [CrossRef] 22. Barnard, P.; Berners-Price, S.J. Targeting the mitochondrial cell death pathway with gold compounds. Coord. Chem. Rev. 2007 ,251, 1889–1902. [CrossRef] 23. González-Barcia, L.M.; Romero, M.J.; González-Noya, A.M.; Bermejo, M.R.; Maneiro, M.; Zaragoza, G.; Pedrido, R. “The Golden Method”: Electrochemical synthesis is an efficient route to gold complexes. Inorg. Chem. 2016 ,55, 7823–7825. [CrossRef] [PubMed] 24. Rodríguez-Fanjul, V.; López-Torres, E.; Mendiola, M.A.; Pizarro, A.M. Gold (III) bis (thiosemicarbazonate) compounds in breast cancer cells: Cytotoxicity and thioredoxin reductase targeting. Eur. J. Med. Chem. 2018,148, 372–383. [CrossRef] 25. Ortego, L.; Cardoso, F.; Manrins, S.; Fillat, M.F.; Laguna, A.; Meireles, M.; Villacampa, M.D.; Gimeno, M.C. Strong inhibition of thioredoxin reductase by highly cytotoxic gold (I) complexes. DNA binding studies. J. Inorg. Biochem. 2014 ,130, 32–37. [CrossRef] 26. Serebryanskaya, T.V.; Lyakhov, A.S.; Ivashkevich, L.S.; Schur, J.; Frias, C.; Prokop, L. Gold (I) thiotetrazolates as thioredoxin reductase inhibitors and antiproliferative agents. Dalton Trans. 2015,44, 1161–1169. [CrossRef] 27. Saggioro, D.; Rigobello, M.P.; Paloschi, L.; Folda, A.; Moggach, S.A.; Parsons, S.; Ronconi, L.; Fregona, D.; Bindoli, A. Gold (III)-dithiocarbamato complexes induce cancer cell death triggered by thioredoxin redox system inhibition and activation of ERK pathway. Chem. Biol. 2007,14, 1128–1139. [CrossRef] 28. Barreiro, E.; Casas, J.S.; Couce, M.D.; Sánchez, A.; Sánchez-González, A.; Sordo, J.; Vázquez-López, E. Mono and dinuclear phosphinegold(I) sulfanylcarboxylates: Influence of nuclearity and substitution of PPh3 for Pet3 on cytotoxicity. J. Inorg. Biochem. 2014,138, 89–98. [CrossRef] 29. Bian, M.; Fan, R.; Jiang, G.; Wang, Y.; Lu, Y.; Liu, W. Halo and pseudohalo gold(I)-NHC complexes derived from 4,5- diarylimidazoles with excellent in vitro and in vivo anticancer activities against HCC. J. Med. Chem. 2020 ,63, 9197–9211. [CrossRef] 30. Meyer, A.; Oehninger, L.; Geldmacher, Y.; Alborzinia, H.; Wölfl, S.; Sheldrick, W.S.; Ott, I. Gold(I) N-heterocyclic carbene complexes with naphthalimide ligands as combined thioredoxin reductase inhibitors and DNA intercalators. ChemMedChem 2014,9, 1794–1800. [CrossRef] [PubMed] 31. Terrón, A.; Buils, J.; Mooibroek, T.J.; Barceló-Oliver, M.; García-Raso, A.; Fiol, J.J.; Frontera, A. Synthesis, X-ray characterization and regium bonding interactions of a trichlorido(1-hexylcytosine)gold(III) complex. Chem. Commun. 2020 ,56, 3524–3527. [CrossRef] 32. Casini, A.; Cinellu, M.A.; Minghetti, G.; Gabbiani, C.; Coronnello, M.; Mini, E.; Messori, L. Structural and solution chemistry, antiproliferative effects, and DNA and protein binding properties of a series of dinuclear gold(III) compounds with bypyridyl ligands. J. Med. Chem. 2006,49, 5524–5531. [CrossRef] [PubMed] 33. Olsen, P.M.; Ruiz, C.; Lussier, D.; Le, B.K.; Angel, N.; Smith, M.; Hwang, C.; Khatib, R.; Jenkins, J.; Adams, K.; et al. Synthesis, characterization, and antitumor activity of unusual pseudo five coordinate gold(III) complexes: Distinct cytotoxic mechanism or expensive ligand delivery systems? J. Inorg. Biochem. 2014,141, 121–131. [CrossRef] [PubMed] 34. Serratrice, M.; Cinellu, M.A.; Maiore, L.; Pilo, M.; Zucca, A.; Gabbiani, C.; Guerri, A.; Landini, I.; Nobili, S.; Mini, E.; et al. Synthesis, structural characterization, solution behavior, and in vitro antiproliferative properties of a series of gold complexes with 2-(2 0 -pyridyl)benzimidazole as ligand: Comparisons of gold(III) versus gold(I) and mononuclear versus binuclear derivatives. Inorg. Chem. 2012,51, 3161–3171. [CrossRef] [PubMed] 35. Maiore, L.; Aragoni, M.C.; Deiana, C.; Cinellu, M.A.; Isaia, F.; Lippolis, V.; Pintus, A.; Serratrice, M.; Arca, M. Structure-activity relationships in cytotoxic AuI/AuIII complexes derived from 2-(2 0 -pyridyl)benzimidazole. Inorg. Chem. 2014 ,53, 4068–4080. [CrossRef] [PubMed] Pharmaceuticals 2021,14, 10 17 of 17 36. Serratrice, M.; Maiore, L.; Zucca, A.; Stoccoro, S.; Landini, I.; Mini, E.; Massai, L.; Ferraro, G.; Merlino, A.; Messori, L.; et al. Cytotoxic properties of a new organometallic platinum(II) complex and its gold(I) heterobimetallic derivatives. Dalton Trans. 2016,45, 579–590. [CrossRef] [PubMed] 37. Jin, I.S.; Jo, M.J.; Park, C.-W.; Chung, Y.B.; Kim, J.-S.; Shin, D.H. Physicochemical, pharmacokinetic, and toxicity evaluation of Soluplus polymeric micelles encapsulating fenbendazole. Pharmaceutics 2020,12, 1000. [CrossRef] [PubMed] 38. Vasava, M.S.; Bhoi, M.N.; Rathwa, S.K.; Jethava, D.J.; Acharya, P.T.; Patel, D.B.; Patel, H.D. Benzimidazole: A milestone in the field of medicinal chemistry. Mini-Rev. Med. Chem. 2020,20, 532–565. [CrossRef] [PubMed] 39. Spek, A.L. PLATON, an Integrated Tool for the Analysis of the Results of a Single Crystal Structure Determination. Acta Crystallogr. 1990,46, C34. 40. Munakata, M.; Yan, S.G.; Maekawa, M.; Akiyama, M.; Kitagawa, S. Solid and solution structures of ternary gold(I) complexes with triphenylphosphine and nitrogen-containing ligands. J. Chem. Soc. Dalton Trans. 1997, 4257–4262. [CrossRef] 41. Nomiya, K.; Noguchi, R.; Ohsawa, K.; Tsuela, K. Synthesis and crystal structure of gold(I) complexes with triazole and triphenylphosphine ligands: Monomeric complex [Au(1,2,3-L)(PPh 3 )] and dimeric complex [Au(1,2,4-L)(PPh3)]2 (HL = triazole) through an Au–Au bond in the solid state. J. Chem. Soc. Dalton trans. 1998, 4101–4108. [CrossRef] 42. Barreiro, E.; Casas, J.S.; Couce, M.D.; Sánchez, A.; Sánchez-González, A.; Sordo, J. Dinuclear triphenylphosphinegold(I) sulfanylcarboxylates: Synthesis, structure and cytotoxic activity against cancer cell lines. J. Inorg. Biochem. 2010 ,104, 551–559. [CrossRef] 43. Van Tonder, A.; Joubert, A.M.; Cromarty, A.D. Limitations of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res. Notes 2015,8, 47. [CrossRef] 44. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988,48, 4827–4833. 45. Stepanenko, A.A.; Dmitrenko, V.V. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene 2015,574, 193–203. [CrossRef] 46. Belmokhtar, C.A.; Hillion, J.; Ségal-Bendirdjian, E. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene 2001,20, 3354–3362. [CrossRef] [PubMed] 47. Déas, O.; Dumont, C.; MacFarlane, M.; Rouleau, M.; Hebib, C.; Harper, F.; Senik, A. Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. J. Immunol. 1998,161, 3375–3383. 48. Van Le, H.; Babak, M.V.; Ali Ehsan, M.; Altaf, M.; Reichert, L.; Gushchin, A.L.; Han Ang, W.; Isab, A.A. Highly cytotoxic gold(I)- phosphane dithiocarbamate complexes trigger an ER stress-dependent immune response in ovarian cancer cells. Dalton Trans. 2020,49, 7355–7363. [CrossRef] 49. Wang, Y.; He, Q.Y.; Sun, R.W.Y.; Che, C.M.; Chiu, J.F. Gold (III) porphyrin 1a induced apoptosis by mitochondrial death pathways related to reactive oxygen species. Cancer Res. 2005,65, 11553–11564. [CrossRef] [PubMed] 50. Donzelli, E.; Carfi, M.; Miloso, M.; Strada, A.; Galbiati, S.; Bayssas, M.; Griffon-Etienne, G.; Cavaletti, G.; Petruccioli, M.G.; Tredici, G. Neurotoxicity of platinum compounds: Comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. J. Neurooncol. 2004,67, 65–73. [CrossRef] [PubMed] 51. Bindoli, A.; Rigobello, M.P.; Scutari, G.; Gabbiani, C.; Casini, A.; Messori, L. Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs. Coord. Chem. Rev. 2009,253, 1692–1707. [CrossRef] 52. Scalcon, V.; Bindoli, A.; Rigobello, M.P. Significance of the mitochondrial thioredoxin reductase in cancer cells: An update on role, targets and inhibitors. Free Rad. Biol. Med. 2018,127, 62–79. [CrossRef] 53. You, B.R.; Park, W.H. Auranofin induces mesothelioma cell death through oxidative stress and GSH depletion. Oncol. Rep. 2016 , 35, 546–551. [CrossRef] [PubMed] 54. McCord, J.M.; Fridovich, I. Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 1969 ,244, 6049–6055. [PubMed] 55. Vega-Avila, E.; Pugsley, M.K. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proc. West. Pharmacol. Soc. 2011,54, 10–14. 56. Alvariño, R.; Alonso, E.; Bornancin, L.; Bonnard, I.; Inguimbert, N.; Banaigs, B.; Botana, L.M. Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells. Mar. Drugs 2020,18, 364. [CrossRef] 57. Tetz, L.M.; Kamau, P.W.; Cheng, A.A.; Meeker, J.D.; Loch-Caruso, R. Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species. J. Pharmacol. Toxicol. Methods 2013 , 67, 56–60. [CrossRef] [PubMed] 58. Sheldrick, G.M. SADABS. Program for Absorption Correction; University of Göttingen: Göttingen, Germany, 1996. 59. Sheldrick, G.M. SHELX97. An Integrated System for Solving and Refining Crystal Structures from Diffraction Data; University of Göttingen: Göttingen, Germany, 1997. 60. Ibers, J.A.; Hamilton, W.C. (Eds.) International Tables for X-ray Crystallography; Kynoch Press: Birmingham, UK; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1974; Volume IV. 61. Burnett, M.N.; Johnson, C.K. ORTEPIII, ORNL-6895; Oak Ridge National Laboratory: Oak Ridge, TN, USA, 1996. 62. Macrae, C.F.; Edgington, P.R.; McCabe, P.; Shields, G.P.; Taylor, R.; Towler, M.; van de Streek, J. Mercury: Visualization and analysis of crystal structures. J. Appl. Crystallogr. 2006,39, 453–457. [CrossRef]