Chirality influence on the cytotoxic properties of anionic chiral bis(N-heterocyclic carbene)silver complexes

Journal of Inorganic Biochemistry 235 (2022) 111924 Available online 9 July 2022 0162-0134/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Chirality influence on the cytotoxic properties of anionic chiral bis (N-heterocyclic carbene)silver complexes Carlos J. Carrasco a , Francisco Montilla a , * , Eleuterio ´ Alvarez b , Jos´ e Manuel Calder´ on-Monta˜ no c , Miguel L´ opez-L´ azaro c , Agustín Galindo a , * a Departamento de Química Inorg´ anica, Facultad de Química, Universidad de Sevilla, Aptdo 1203, 41071 Sevilla, Spain b Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Avda. Am´ erico Vespucio 49, 41092 Sevilla, Spain c Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain ARTICLE INFO Keywords: NHC-dicarboxylate Chiral Silver Anticancer X-ray ABSTRACT Complexes Na 3 [Ag(NHC R ) 2 ], 2a-e and 2b’-c’, where NHC R is a N-heterocyclic carbene of the 2,2′-(1H-2λ 3 ,3λ 4 - imidazole-1,3-diyl)dicarboxylate type, were prepared by treatment of compounds HL R , 1a-e and 1b’-c’ (2-(1- (carboxyalkyl)-1H-imidazol-3-ium-3-yl)carboxylate), with silver oxide in the presence of aqueous sodium hydroxide. They were characterized by analytical, spectroscopic (infrared, IR, 1 H and 13 C nuclear magnetic resonance, NMR, and circular dichroism) and X-ray methods (2a). In the solid state, the anionic part of complex 2a, [Ag(NHC H ) 2 ] 3− , shows a linear disposition of C carbene -Ag-C carbene atoms and an eclipsed conformation of the two NHC ligands. The proposed bis(NHC) nature of the silver complexes was maintained in solution according to NMR and density functional theory (DFT) calculations. The cytotoxic activity of compounds 2 was evaluated against four cancer cell lines and one non-cancerous cell line and several structure-activity correlations were found for these complexes. For instance, the activity decreased when the bulkiness of the R alkyl group in Na 3 [Ag (NHC R ) 2 ] increased. More interesting is the detected chirality-anticancer relationship, where complexes Na 3 [Ag {(S,S)-NHC R } 2 ] (R =Me, 2b; i Pr, 2c) showed better anticancer activity than those of their enantiomeric derivatives Na 3 [Ag{(R,R)-NHC R } 2 ] (R =Me, 2b’; i Pr, 2c’). 1. Introduction Transition metal complexes with N-Heterocyclic Carbene (NHC) scaffolds have become effective molecules that have gained enormous interest in recent decades due to the numerous applications they have demonstrated in several disciplines [1–4]. An area in which NHC ligands have shown great relevance, due to their robust coordination to metals, is their applications in medicinal chemistry. In particular, transition metal NHCs are the subject of a growing field of research as potential drugs against cancer disease [5–8]. For example, gold-NHC complexes have a well-recognized efficient anticancer activity [9,10], while studies on the anticancer effectiveness of Ag-NHC derivatives are in continuous progress [11–24]. In this area, the development of chiral and potentially water-soluble NHC ligands is of particular importance [25–27]. In fact, concerning chirality in organometallic anticancer complexes Romero and Sadler indicated years ago “the comparative cytotoxicity between stereoisomers has hardly been explored” [28]. From that statement to now, specific research about the anticancer-chirality relationship have been mainly focused in platinum derivatives [29–31] and, to our knowledge no related studies exist for chiral silver complexes. Following our interest in the investigation of complexes containing amino acid-derived ligands [32–36] and their medicinal applications [37,38], we decided to explore the preparation of new silver complexes containing chiral NHC R ligands, where NHC R is a N-heterocyclic carbene of 2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)dicarboxylate type (Scheme 1). Here, we present the synthesis and characterization of new watersoluble silver complexes Na 3 [Ag(NHC R ) 2 ] (R =H, 2a), Na 3 [Ag{(S,S)- NHC R } 2 ] (R =Me, 2b; i Pr, 2c; i Bu, 2d; s Bu, 2e) and Na 3 [Ag{(R,R)- NHC R } 2 ] (R =Me, 2b’; i Pr, 2c’). The cytotoxic activity of these complexes versus four human cancer cell lines (melanoma cells MeWo, lung adenocarcinoma A549, bladder cancer cells T24, and gastric cancer cells KATO III) was evaluated and compared with a human non-malignant cell line (skin cells, HaCaT). The study of the cytotoxic activity of enantiomerically related complexes 2b/2b’ and 2c/2c’ is unprecedented for NHC‑silver complexes and from the comparison of their activities a chiral-anticancer relationship was observed. * Corresponding author. E-mail address: [email protected] (A. Galindo). Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio https://doi.org/10.1016/j.jinorgbio.2022.111924 Received 30 May 2022; Received in revised form 20 June 2022; Accepted 4 July 2022 Journal of Inorganic Biochemistry 235 (2022) 111924 2 2. Results and discussion 2.1. Synthesis and characterization of Na 3 [Ag(NHC R ) 2 ] (2a-e and 2b’- c’) complexes Initially, we planned the synthesis of bis(carbene) silver complexes 2 by reaction of compounds HL R (1, 2-(1-(carboxyalkyl)-1H-imidazol-3- ium-3-yl)carboxylate) with Ag 2 O in methanol. Surprisingly, dicarboxylate silver compounds, [Ag(L R )], were instead obtained, which were characterized as coordination polymeric species in the solid-state [37]. In solution, the C 2 – H group of imidazolium in [Ag(L R )] complexes experienced a fast H – D exchange with D 2 O, which was explained on the basis of the formation of a transient carbene intermediate according to DFT studies [37]. This prompted us to explore the reactivity of [Ag(L R )] with a base like aqueous sodium hydroxide in a NMR tube. The NMR spectrum of the crude reaction showed the disappearance of the C 2 – H signals at ca. 8.9 ( 1 H) and 137 ( 13 C) ppm that suggests the formation of such a carbene species. In fact, the best synthetic procedure for the preparation of complexes 2 was by treating compounds HL R , 1, with Ag 2 O in the presence of aqueous sodium hydroxide (Scheme 2). They were obtained as colorless crystals or solids in good yields, which were soluble in water or methanol but insoluble in organic solvents. Other source of silver(I) cations can be employed in the synthesis (silver nitrate or silver acetate), but silver oxide gave better results. An intense and slightly broad band centered around 1600 cm −1 characterized the IR spectra of compounds 2 (Fig. S1, Supplementary data). This absorption was assigned to the antisymmetric COO vibrations of the carboxylate groups, while the symmetric COO vibrations appeared within the 1387–1371 cm −1 range. The ν as (COO) band appeared at lower wavenumber than those of the parent compounds 1 (around 1625 cm −1 ) and this fact suggests a major delocalization of the carboxylate group, in agreement with the observed solid-state structure (see below). 1 H and 13 C{ 1 H} NMR spectra of 2 (Figs. S2 and S3) displayed the expected signals of the corresponding alkyl groups and the typical pattern due to the imidazolium ring. For example, the equivalent CH groups at 4 and 5 ring positions give signals at ca. 7.5 and 120 ppm, in 1 H and 13 C{ 1 H} NMR, respectively. Carboxylate carbon atoms were identified in the 13 C{ 1 H} NMR spectra by singlets in the 170–175 ppm range, which are analogous to those recorded for related NHC- carboxylate silver complexes [38]. Although carbene 13 C resonances are often difficult to observe due to their long T 1 relaxation times or possible dynamic processes [39], 13 C{ 1 H} NMR spectra of complexes 2 showed the characteristic low field resonance for the C 2 atom. This signal appeared at around 180 ppm, but only for complexes 2c and 2e is well resolved showing the well-known pattern of two doublets due to coupling constants of 1 J CAg =185 and 213Hz. These values of 1 J CAg are in agreement with an homoleptic binding mode of NHC ligands, C-Ag-C, in agreement with the proposed formulation, because larger coupling constants are often observed for heteroleptic binding modes, C-Ag-X or C-Ag-N [40,41]. Additionally, enantiomeric complexes Na 3 [Ag{(S,S)- NHC R } 2 ] (R =Me, 2b; i Pr, 2c) and Na 3 [Ag{(R,R)-NHC R } 2 ] (R =Me, 2b’; i Pr, 2c’) were spectroscopically characterized by circular dichroism (CD). Compounds 2b/2b’ showed a maximum at 271 nm, which obviously have different sign of θ obs , while the maximum of 2c/2c’ appeared at 261 nm (see Fig. S4). 2.2. Structural characterization of complex 2a in the solid-state Suitable crystals of complex Na 3 [Ag(NHC H ) 2 ], 2a, were obtained by cooling to 0 ◦C a concentrated aqueous solution of 2a. The anionic part of the complex, [Ag(NHC H ) 2 ] 3− , is shown in Fig. 1, while selected structural data are collected in Table S1. Anions are composed of the silver center and two bonded NHC H ligands with Ag – C bond lengths of 2.077(7) Å. The typical linear arrangement of NHC H groups was found with a C carbene -Ag-C carbene angle of 173.1(3)◦. They also display an eclipsed conformation of the two carbene ligands with a N1-C1-C1-N2 torsion angle of ca. 164◦. These structural data agree well with those reported for silver mononuclear bis(carbene) complexes according to a CSD search (mean values of 2.088 Å and 177◦for Ag – C and C-Ag-C, respectively, for 179 hits with R factor <0.075 and restrictions ‘no errors’, ‘non-disordered’ and ‘not polymeric’) [42]. The C – O distances in the four carboxylate groups are similar (mean value of 1.249 Å). These carboxylate groups have two singular features, they are oriented to the same molecular side showing a C5-C4-C6-C7 torsion angle of ca. 0◦and the angle between the planes defined by the COO atoms is only of ca. 19◦. This arrangement allows the formation of a complex hydrogen bonding network with the hydrated sodium cations. These cations are placed in a zigzag arrangement along a axis with bridging water molecules of hydration that form discrete [Na 5 (H 2 O) 18 ] 5+ units (Fig. S5). These sodium units are maintained along this axis by forming hydrogen bonds with [Ag(NHC H ) 2 ] 3− anions and two additional water molecules of hydration (Fig. 2). The resulting three-dimensional crystal packing is controlled by these hydrogen bonds, showing along a axis the approximately linear assemblage of sodium ions surrounded by the [Ag (NHC H ) 2 ] 3− anions (Fig. S6). The structure of 2a resembles to those found by Flores, de Jesús and coworkers in related bis(sulfonated NHC) silver compounds (refcodes SIFVEQ and SIFVOU) [43]. These complexes and 2a are some of the few examples of anionic bis(NHC) silver complexes reported in the literature [44–47]. 2.3. DFT analysis of complexes 2 The solid-state structure of complexes 2 was proposed to be analogous to that of 2a, determined by X-ray diffraction methods. According to the NMR data, this structure is maintained in solution, resulting in complete dissociation of 2 in [Ag(NHC R ) 2 ] 3− anions and solvated sodium cations. To further verify this hypothesis, DFT calculations for the anions 2a-e, [Ag{(S,S)-NHC R } 2 ] 3− , and 2b’-c’, [Ag{(R,R)-NHC R } 2 ] 3− , were performed at the B3LYP/LANL2DZ/6–311++G** level of theory. The resulting optimized structures of these complexes are shown in Fig. S7. The selected combination of the method and basis sets provides a good structural description of these complexes according to the satisfactory comparison of the calculated and experimental structural parameters of complex 2a. In this case, the conformation of the NHC ligands in the optimized complex is not eclipsed but alternated with an angle between NHC planes of 88.2◦. Different angles between these Scheme 1. C.J. Carrasco et al. Journal of Inorganic Biochemistry 235 (2022) 111924 3 planes were found for the other anions with staggered conformations (2b, 53.4◦; 2c, 48.4◦; 2d, 61.9◦), except for 2e where the angle is only 15.1◦. The conformer of 2a in which the carbene ligands are completely eclipsed is optimized as a transition state. This is destabilized by 7.0 kcal⋅mol −1 (ΔG) with respect to the staggered species and is characterized by one imaginary frequency with a very small absolute value. Scheme 2. Fig. 1. Structure of the anionic part, [Ag(NHC H ) 2 ] 3− , of complex 2a. Fig. 2. Hydrogen bonding network between [Ag(NHC H ) 2 ] 3− anions and sodium hydrated cations in complex 2a. C.J. Carrasco et al. Journal of Inorganic Biochemistry 235 (2022) 111924 4 This situation is completely similar to that first described by Frenking and coworkers in the theoretical analysis of bis(NHC) complexes of Group 11 metals [48]. The solid-state eclipsed conformation of NHC ligands can be explained on the basis of the extensive hydrogen bonding network between the carboxylate groups and the water hydration molecules of sodium cations, which counterbalances this energy difference. The 1 H and 13 C NMR chemical shifts were calculated for the optimized [Ag(NHC R ) 2 ] 3− anions of complexes 2a-c. Excellent correlations between the calculated and experimental chemical shifts values were found (Fig. S8) confirming the formulation of these derivatives as bis (NHC) silver species and supporting their existence in solution. 2.4. Cytotoxic activity The cytotoxicity of complexes 2 was evaluated in vitro by determining the half Inhibitory Concentrations (IC 50 ) against human BRAF wild-type melanoma MeWo cells, human lung adenocarcinoma A549, human bladder cancer cells T24, and human gastric cancer KATO III, as well as against human non-cancerous HaCaT cells. IC 50 values were determined after 72 h of drug exposure to the cells using the resazurin assay. The results of viability are shown in Fig. 3 for enantiomeric complexes 2b/2b’ and 2c/2c’, while for other 2 complexes are collected in Fig. S9. All results are also summarized in Table 1 and Table S2 (Selectivity Index values). Cisplatin and gemcitabine, two anticancer drugs used in clinics, were evaluated under the same experimental conditions (Fig. S9). Complexes 2 show cytotoxic activity against all Fig. 3. Effect of complexes 2b/2b’ and 2c/2c’ on the viability of human non-malignant cells (HaCaT) and human cancer cells (A549, MeWo, T24 and KATO III). Cells were exposed to several concentrations of compounds for 72 h and cell viability was measured using the resazurin assay. Data represent mean ±SEM from at least two independent experiments. Table 1 IC 50 values of compounds tested against human cell lines. After 72 h of treatment, cell viability was measured using the resazurin assay. a Complex IC 50 (Mean ±SEM, μ M) HaCaT A549 MeWo T24 KATO III 2a 17.8 ± 0.2 33.0 ± 4.5 27.9 ± 7.0 17.6 ± 0.4 34.4 ± 8.7 2b 17.5 ± 0.3 33.8 ± 4.7 29.2 ± 5.6 18.3 ± 0.3 35.9 ± 9.4 2b’ 19.1 ± 1.1 39.8 ± 4.2 33.6 ± 3.6 22.2 ± 3.3 38.3 ± 7.8 2c 18.4 ± 0.3 38.3 ± 3.8 31.6 ± 3.7 18.3 ± 0.1 35.2 ± 8.6 2c’ 35.0 ± 5.8 48.0 ± 1.6 43.3 ± 3.4 48.2 ± 5.3 57.1 ± 3.0 2d 52.4 ± 1.5 69.9 ± 11.4 61.2 ± 15.6 52.6 ± 3.1 71.2 ± 8.7 2e 23.2 ± 4.1 44.2 ± 3.8 35.6 ± 4.1 36.7 ± 10.7 52.1 ± 1.7 Cisplatin 2.7 ±0.2 2.7 ±0.4 3.6 ±0.5 1.6 ±0.1 16.2 ± 0.9 Gemcitabine 0.0254 ± 0.0108 0.0038 ± 0.0004 0.0065 ± 0.0008 0.0027 ± 0.0005 0.0060 ± 0.0006 a Cell lines: HaCaT: Skin non-malignant; A549: Lung adenocarcinoma; MeWo: Melanoma BRAF WT; T24: Bladder cancer; KATO III: Gastric cancer. μ M values are given as the mean value obtained from at least two independent experiments ±standard error of the mean (SEM) (see Experimental for details). C.J. Carrasco et al. Journal of Inorganic Biochemistry 235 (2022) 111924 5 cancer cell lines, within the range 17.6–71.2 μ M, but all of them are less active than cisplatin and, by far, than gemcitabine (Table 1). Complexes 2 have activities that are in general slightly lower, or in some cases comparable, to those reported by Youngs et al. [19] and Tacke et al. [18] in several silver acetate NHCs, although with different cancerous cell lines. They described a better behavior against cancer cells of heteroleptic silver NHC compounds than those of homoleptic bis(NHC) derivatives. In fact, any of the IC 50 values for our complexes 2 are higher than those reported for several silver chloride NHCs (for example, results of Roland [17] and Gautier [7] research groups). Given the anionic nature of 2, we looked for an example of anionic bis(NHC) silver complex with a reported cytotoxic study to compare the cytotoxic activities of closely related compounds. Santini and coworkers published several complexes of this type [24,45], and they were evaluated for the same cell line, lung adenocarcinoma A549, which was also tested for 2. Table S3 collects the IC 50 values reported by Santini et al. and our data, which were ordered from the lowest to highest value. Complex Na 4 [AgCl (Im PrSO3 ) 2 ] [24] has the lowest value, 9.8 ±2.3 μ M, while complexes 2 with a mean IC 50 value of 43.9 μ M are between Na 4 [AgBr(Bzim’) 2 ] and Na 4 [AgCl(Im’) 2 ] derivatives (IC 50 values of 25.0 and 53.1 μ M, respectively [45]). Concerning complexes 2, the best activity was found versus Bladder cancer T24 cell line and the worst against gastric cancer KATO III. The analysis of IC 50 values of 2a–e and 2b’-c’ reveals a clear structur- e–anticancer effect relationship. The activity decreases when the steric properties of the R alkyl group in Na 3 [Ag(NHC R ) 2 ] complexes increase. In fact, complex Na 3 [Ag(NHC H ) 2 ], 2a, displays the lowest IC 50 value for each cell line. A similar relationship was recently described by us for related silver {Ag[NHC Mes,R ]} n complexes, where the antimicrobial activity versus Gram-negative bacteria E. coli and P. aeruginosa decreases when the bulkiness of the R alkyl group increases [38]. On the contrary, the inverse effect was described for NHC ruthenium complexes where the anticancer activity correlates with the complex lipophilicity [49]. A possible explanation of the reverse effect in 2 may be related with the presence of four hydrophilic carboxylate substituents in the two NHC ligands of the anions. An interesting result is the different anticancer behavior of enantiomerically related complexes 2b / 2b’ and 2c / 2c’. Complexes 2b’ and 2c’ were prepared using the precursors 1b’ and 1c’ from the non-proteinaceous amino acids D-alanine and D-valine, respectively, while 2b and 2c derives from natural amino acids. Complex 2b shows slightly better anticancer activity than that of their enantiomeric derivative 2b’, but the activity of 2c is clearly better than that of 2c’ for all tested cancer cell lines. These data suggest a chiralityanticancer correlation. An analogous tendency was recently observed by us in the antimicrobial study of amino acid based carboxylate silver complexes, where a slight different behavior was observed for enantiomerically related complexes [37]. Most available anticancer drugs usually do not cure the disease as a result of their limited selectivity toward cancer cells. They are toxic to both tumor and normal cells, especially normal cells with high proliferation rate, causing severe side effects such as skin ulcer, neurotoxicity, immunosuppression, etc. For this reason, the cytotoxic effect of complexes 2 was also evaluated against the human skin HaCaT cells, a noncancerous cell line with a high proliferation rate. Complexes 2 showed IC 50 values for human non-cancerous HaCaT cells (17.5 to 52.4 μ M range) that are lower than those observed for cancerous cell lines (Fig. 3 and Table 1). However, it is worth mentioning that cisplatin was also more toxic to HaCaT cells than to most cancer cells tested. Therefore, the potential anticancer activity of 2 cannot be ruled out and more studies are needed to evaluate the selective anticancer activity of these complexes. 3. Experimental 3.1. General All preparations and other operations were carried out under anaerobic conditions. Solvents were purified appropriately prior to use, using standard procedures. Cisplatin and Resazurin were purchased from Acros Organics and Sigma, respectively, while Gemcitabine was obtained from Pfizer. Other chemicals were obtained from several commercial sources and used as supplied. Zwitterionic imidazolium dicarboxylate compounds HL R were prepared according to the literature procedures [50,33]. Infrared spectra were recorded using the ATR technique on Perkin-Elmer FT-IR Spectrum Two. NMR spectra were recorded at the Centro de Investigaciones, Tecnología e Innovaci´ on (CITIUS) of the University of Sevilla by using Avance III spectrometers. 1 H and 13 C{ 1 H} NMR shifts were referenced to residual signals from deuterated solvents. All data are reported in ppm downfield from Si (CH 3 ) 4 . Electronic circular dichroism (CD) spectra were recorded in a Biologic Mos-450 spectropolarimeter (Barcelona, Spain). Elemental analyses (C, H, N) were conducted by the CITIUS of the University of Sevilla on an Elemental LECO CHNS 93 analyzer. High-resolution mass spectra were obtained on a QExactive Hybrid Quadrupole-Orbitrap mass spectrometer from Thermo Scientific (CITIUS of the University of Sevilla). 3.2. Cell lines HaCaT cells (human keratinocytes [51]), A549 (human non-small cell lung cancer cells), KATO III (human gastric cancer cells), T24 (human bladder cancer cells), and MeWo (human BRAF wild-type melanoma cells) were purchased from Cell Lines Service (CLS). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) that contained high glucose levels and L-glutamine. The medium was supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μ g/mL streptomycin. All cells were maintained at 37 ◦C with 5% CO 2 in a humidified incubator. 3.3. Synthesis 3.3.1. Sodium bis(2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)diacetate)argentate (I), Na 3 [Ag(NHC H ) 2 ] (2a) Compounds HL H , 1a, (0.184 g, 1.00 mmol) and Ag 2 O (0.058 g, 0.25 mmol) were mixed in a Schlenk flask and dissolved in deoxygenated H 2 O (5 mL) under a nitrogen atmosphere. Then, NaOH (0.060 g, 1.5 mmol) was added and a dark brown solid was observed. The mixture was stirred for 16 h at room temperature in the dark. After that, the mixture was centrifuged, filtered and the filtrate was concentrated to a quarter of the volume using an intermediate trap and cooled to 0 ◦C. Uncolored crystals of compound 2a were obtained (0.211 g, 73% yield). IR (ATR, cm −1 ): 3167 (m), 3134 (m), 3108 (m), 2976 (w), 2941 (w), 1707 (w), 1610 (vs), 1563 (s), 1451 (m), 1419 (m), 1382 (vs), 1302 (vs), 1248 (m), 1137 (w), 1177 (m), 1109 (w), 1048 (w), 967 (w), 924 (w), 814 (m), 760 (m), 693 (s), 664 (s), 654 (vs), 579 (s), 470 (w), 422 (m). 1 H NMR (D 2 O, 500 MHz): δ 4.71 (s, 8H, CH 2 ), 7.14 (s, 4H, CH, H 4 /H 5 ), 13 C{ 1 H} NMR (D 2 O, 125.78 MHz): δ 54.5 (s, CH 2 ), 122.5 (s, CH, C 4 /C 5 ), 175.5 (s, COO), 181.9 (br s, C carbene). HR-MS (negative mode), found: m/z = 472.9866, calculated for C 14 H 14 N 4 O 8 Ag ([Ag(NHC H ) 2 ] 3− +2H), 472.9868. Elemental Anal. Calc. for C 14 H 16 N 4 O 10 AgNa 3 (2a⋅2H 2 O): C, 29.14; H, 2.79; N, 9.71. Found: C, 29.83; H, 2.83, N, 9.83%. 3.3.2. Sodium bis(2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)dipropionate) argentate(I), Na 3 [Ag{(S,S)-NHC Me } 2 ] (2b) and Na 3 [Ag{(R,R)-NHC Me } 2 ] (2b’) Compounds (S,S)-HL Me , 1b, (0.212 g, 1.00 mmol), Ag 2 O (0.058 g, 0.25 mmol) and NaOH (0.060 g, 1.5 mmol) were reacted following the same procedure described above. The resulting filtrate was concentrated C.J. Carrasco et al. Journal of Inorganic Biochemistry 235 (2022) 111924 6 to dryness using an intermediate trap to afford compound 2b as a white solid (0.309 g, 88% yield). IR (ATR, cm −1 ): 3137 (w), 3096 (w), 2983 (w), 2940 (w), 2818 (w), 1733 (vw), 1587 (vs), 1455 (m), 1387 (s), 1356 (s), 1324 (m), 1298 (m), 1253 (m), 1169 (s), 1112 (w), 1079 (w), 1027 (m), 973 (w), 874 (m), 848 (w), 764 (w), 740 (w), 667 (m), 535 (w), 429 (w). 1 H NMR (CD 3 OD, 500 Hz): δ 1.73 (d, 3 J HH =7 Hz, 12H, CHCH 3 ), 5.05 (c, 3 J HH =7 Hz, 4H, CHCH 3 ), 7.36 (s, 2H, CH, H 4 /H 5 ). 13 C{ 1 H} NMR (CD 3 OD, 75.47 MHz): δ 18.9 (s, CHCH 3 ), 60.5 (s, CHCH 3 ), 119.0 (s, CH, C 4 /C 5 ), 176.2 (s, COO), 180.4 (br s, C carbene). HR-MS (negative mode), found: m/z =529.0485, calculated for C 18 H 22 N 4 O 8 Ag ([Ag (NHC Me ) 2 ] 3− +2H), 529.0489. Elemental Anal. Calc. for C 18 H 32 N 4 O 14 AgNa 3 (2b⋅6H 2 O): C, 30.65; H, 4.57; N, 7.94. Found: C, 30.89; H, 4.51; N, 7.23%. Following the same experimental method was prepared the complex Na 3 [Ag{(R,R)-NHC Me } 2 ], 2b’, (0.288 g, 94% yield). Compound 2b’ presented identical IR, 1 H and 13 C{ 1 H} NMR and HR-MS spectra that its enantiomer 2b. Elemental Anal. Calc. for C 18 H 22 N 4 O 9 AgNa 3 (2b’⋅H 2 O): C, 35.14; H, 3.60; N, 9.11. Found: C, 35.52; H, 4.28; N, 8.84%. 3.3.3. Sodium bis(2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)bis(3- methylbutanoate))argentate(I), Na 3 [Ag{(S,S)-NHC iPr } 2 ] (2c) and Na 3 [Ag {(R,R)-NHC iPr } 2 ] (2c’) Compounds (S,S)-HL iPr , 1c, (0.268 g, 1.00 mmol), Ag 2 O (0.058 g, 0.25 mmol) and NaOH (0.060 g, 1.5 mmol) were reacted following the same procedure described above. The resulting filtrate was concentrated to dryness using an intermediate trap to afford compound 2c as a white solid (0.276 g, 72% yield). IR (ATR, cm −1 ): 2960 (m), 2931 (m), 2873 (w), 1740 (vw), 1598 (vs), 1562 (m), 1466 (w), 1371 (vs), 1260 (m), 1240 (w), 1195 (w), 1166 (w), 1137 (w), 1105 (w), 1086 (w), 1041 (w), 980 (w), 950 (w), 918 (w), 870 (w), 841 (w), 825 (w), 749 (s), 638 (w), 603 (w), 503 (w), 439 (w). 1 H NMR (CD 3 OD, 500 MHz): δ 0.85, 1.12 (d, 3 J HH =7.0 Hz, 12H, CH(CH 3 ) 2 ), 2.52 (o, 3 J HH =7.0 Hz, 4H, CH(CH 3 ) 2 ), 4.65 (d, 3 J HH =9 Hz, 4H, CH i Pr), 7.53 (s, 4H, CH, H 4 /H 5 ). 13 C{ 1 H} NMR (CD 3 OD, 125.78 MHz): δ 18.2, 19.2 (s, CH(CH 3 ) 2 ), 31.9 (s, CH(CH 3 ) 2 ), 74.5 (s, CH i Pr), 120.0 (s, CH, C 4 /C 5 ), 174.9 (s, COO), 180.6 (dd, 1 J ( 107 Ag – 13 C) =185.3 Hz, 1 J( 109 Ag – 13 C) =213.6 Hz, NCN). HR-MS (negative mode), found: m/z =641.1732, calculated for C 26 H 38 N 4 O 8 Ag ([Ag(NHC iPr ) 2 ] 3− +2H), 641.1741. Elemental Anal. Calc. for C 26 H 42 N 4 O 11 AgNa 3 (2c⋅3H 2 O): C, 40.90; H, 5.55; N, 7.34. Found: C, 40.37; H, 6.04; N, 7.17%. Following the same experimental method was prepared the complex Na 3 [Ag{(R,R)-NHC iPr } 2 ], 2c’, (0.330 g, 79% yield). Compound 2c’ presented identical IR, 1 H and 13 C{ 1 H} NMR and HR-MS spectra that its enantiomer 2c. Elemental Anal. Calc. for C 26 H 50 N 4 O 15 AgNa 3 (2c’⋅7H 2 O): C, 37.38; H, 6.03; N, 6.71. Found: C, 37.71; H, 6.23; N, 6.70%. 3.3.4. Sodium bis(2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)bis(4- methylpentanoate))argentate(I), Na 3 [Ag{(S,S)-NHC iBu } 2 ] (2d) Compounds (S,S)-HL iBu (1d) (0.299 g, 1.00 mmol), Ag 2 O (0.058 g, 0.25 mmol) and NaOH (0.060 g, 1.5 mmol) were reacted following the same procedure described above. The resulting filtrate was concentrated to dryness using an intermediate trap to afford compound 2d as a brown microcrystalline solid (0.320 g, 78% yield). IR (ATR, cm −1 ): 3383 (vb), 3134 (m), 3095 (w), 2956 (m), 2928 (m), 2871 (m), 2154 (w), 1608 (vs), 1467 (m), 1382 (vs), 1368 (vs), 1289 (m), 1243 (w), 1224 (w), 1195 (w), 1169 (m), 1122 (w), 1047 (w), 1008 (w), 964 (w), 939 (w), 918 (w), 898 (w), 880 (w), 830 (w), 738 (m), 697 (m), 636 (w), 513 (w), 440 (w). 1 H NMR (CD 3 OD, 500 MHz): δ 0.96, 0.99 (t, 3 J HH =7.0 Hz, 12H, CH 2 CH (CH 3 ) 2 ), 1.42 (m, 3 J HH =7.0 Hz, 4H, CH 2 CH(CH 3 ) 2 ), 2.06 (m, 3 J HH = 7.0 Hz, 8H, CH 2 CH(CH 3 ) 2 ), 4.86 (dd, 3 J HH =10.9 Hz, 3 J HH =5.6 Hz, 4H, CH i Bu), 7.64 (s, 4H, CH, H 4 /H 5 ). 13 C{ 1 H} NMR (CD 3 OD, 125.78 MHz): δ 20.2, 21.8 (s, CH 2 CH(CH 3 ) 2 ), 24.8 (s, CH 2 CH(CH 3 ) 2 ), 41.6 (s, CH 2 CH (CH 3 ) 2 ), 63.9 (s, CH i Bu), 121.2 (s, CH, C 4 /C 5 ), 172.9 (s, COO), 182.4 (br s, C carbene). HR-MS (negative mode), found: m/z =697.2352, calculated for C 30 H 46 N 4 O 8 Ag ([Ag(NHC iBu ) 2 ] 3− +2H), 697.2372. Elemental Anal. Calc. for C 30 H 50 N 4 O 11 AgNa 3 (2d⋅3H 2 O): C, 43.96; H, 6.15; N, 6.84. Found: C, 43.57; H, 6.28; N, 6.60%. 3.3.5. Sodium bis(2,2′-(1H-2λ 3 ,3λ 4 -imidazole-1,3-diyl)bis(3- methylpentanoate))argentate(I), Na 3 [Ag{(S,S)-NHC sBu } 2 ] (2e) Compounds (S,S)-HL sBu , 1e, (0.299 g, 1.00 mmol), Ag 2 O (0.058 g, 0.25 mmol) and NaOH (0.060 g, 1.5 mmol) were reacted following the same procedure described above. The resulting filtrate was concentrated to dryness using an intermediate trap to afford compound 2e as a brown microcrystalline solid (0.355 g, 89% yield). IR (ATR, cm −1 ): 3371 (w), 2963 (m), 2932 (m), 2877 (w), 1601 (vs), 1562 (m), 1453 (w), 1403 (s), 1373 (vs), 1258 (m), 1193 (w), 1156 (w), 1089 (w), 1050 (w), 976 (w), 915 (w), 880 (w), 851 (w), 835 (w), 742 (m), 642 (w), 616 (w), 558 (w), 428 (w). 1 H NMR (CD 3 OD, 500 MHz): δ 0.85 (t, 3 J HH =7.0 Hz, 12H, CH 2 CH 3 ), 1.03 (m, 3 J HH =7.0 Hz, 4H, CH 2 CH 3 ), 1.05 (d, 3 J HH =7.0 Hz, 12H, CHCH 3 ), 1,25 (m, 3 J HH =7.0 Hz, 4H, CH 2 CH 3 ), 2.24 (m, 3 J HH = 7.0 Hz, 4H, CHCH 3 ), 4.67 (d, 3 J HH =10 Hz, 4H, CH s Bu), 7.49 (s, 4H, CH, H 4 /H 5 ). 13 C{ 1 H} NMR (CD 3 OD, 125.78 MHz): δ 10.4 (s, CH 2 CH 3 ), 15.3 (s, CHCH 3 ), 25.0 (s, CH 2 CH 3 ), 38.1 (s, CHCH 3 ), 73.4 (s, CH s Bu), 119.9 (s, CH, C 4 /C 5 ), 174.9 (s, COO), 180.7 (dd, 1 J( 107 Ag – 13 C) =184.7 Hz, 1 J ( 109 Ag – 13 C) =213.2 Hz, NCN). HR-MS (negative mode), found: m/z = 697.2369, calculated for C 30 H 46 N 4 O 8 Ag ([Ag(NHC sBu ) 2 ] 3− +2H), 697.2372. Elemental Anal. Calc. for C 30 H 48 N 4 O 10 AgNa 3 (2e⋅2H 2 O): C, 44.95; H, 6.04; N, 6.99. Found: C, 45.15; H, 6.32; N, 7.03%. 3.3.6. Cell viability assays Exponentially growing cells (3000–6000 cells per well) were seeded in 96-well plates and allowed to grow for 24 h. Cells were then exposed to several concentrations of the new compounds or the anticancer drugs cisplatin and gemcitabine. After a 72-h treatment period, cell viability was estimated with the resazurin assay. This assay is a colorimetric technique based on the capability of viable cells to reduce the blue reagent resazurin to the pink soluble product resorufin. The amount of resorufin produced is generally proportional to the number of living cells. After the treatment period, cells were washed once with phosphate buffered saline (PBS), and 150 μ L resazurin (20 μ g/mL in medium) was added to each well. Because KATO III cells grow as a mixture of adherent and suspension cells, 50 μ L resazurin (60 μ g/mL in medium) was added directly without prior washing. The plates were incubated for 4–5 h at 37 ◦C, 5% CO2, and finally optical densities were measured at 540 and 620 nm with a multiwell plate spectrophotometer reader (Multiskan EX Labsystems). Cell viability was calculated as a percentage in relation to nontreated cells. Results were expressed as the means ±standard error of the mean (SEM). Data for complexes 2 are at least from four independent experiments. For statistical analysis, the t-test (paired, twotailed) was used. A p value >0.05 is not considered statistically significant and is not represented by any symbol. A p value <0.05 is considered statistically significant and is indicated with an asterisk (*) and a p value <0.01 is indicated with a double asterisk (**). The statistical analysis was carried out to compare the cytotoxicity of a particular concentration of the compound between non-malignant HaCaT cells and a particular cancer cell line. The selectivity index (S.I.) values (Table S2) are useful in analyzing the selective anticancer activity of new compounds. S.I. values were calculated over each cancer cell line as the mean of the IC 50 value in the non-cancerous cell line (HaCaT) by the IC 50 in the respective cancer cell line obtained in each independent experiment. 3.3.7. Computational details The electronic structure and geometries of the [Ag(NHC R ) 2 ] 3− anions of 2a-e were investigated by using density functional theory at the B3LYP level [52,53]. For the optimization the Ag atom was described with the LANL2DZ basis set [54], while 6–311++G(d,p) was used for the other atoms. Molecular geometries were optimized without symmetry restrictions. Frequency calculations were carried out at the same level of theory to identify the stationary points as minima (zero imaginary frequencies) and to provide the thermal correction to free energies at 298.15 K and 1 atm. The GIAO method was used for the NMR C.J. Carrasco et al. Journal of Inorganic Biochemistry 235 (2022) 111924 7 calculations ( 1 H and 13 C NMR isotropic shielding tensors), which were carried out at the 6–311 +G(2d,p) level of theory. The DFT calculations were executed using the Gaussian 09 program package [55]. Coordinates of optimized compounds are collected in the Supplementary data (Table S4). 3.3.8. Single-crystal X-ray crystallography A summary of the crystallographic data and the structure refinement results for compound 2a is given in Table 2. Crystals of a suitable size for X-ray diffraction analysis were coated with dry perfluoropolyether and mounted on glass fibers and fixed in a cold nitrogen stream (T =193 K) to the goniometer head. Data collection was carried out on a Bruker- AXS, D8 QUEST ECO, PHOTON II area detector diffractometer, using monochromatic radiation λ(Mo K α ) =0.71073 Å, by means of ω and φ scans with a width of 0.50◦. The data were reduced (SAINT [56]) and corrected for absorption effects by the multi-scan method (SADABS) [57]. The structure was solved using direct methods (SIR-2002 [58]) and refined against all F 2 data by full-matrix least-squares techniques (SHELXTL-2018/3 [59]) minimizing w[F o 2 -F c 2 ] 2 . All non‑hydrogen atoms were refined anisotropically. The hydrogen atoms were included from calculated positions and refined riding on their respective carbon atoms with isotropic displacement parameters. Since there are seven positive charges due to the metal cations (two silver and five sodium) and eight possible negative charges on the carboxylate anions, by symmetry in the crystal structure, a proton was added between the carboxylate groups for charge balance. A search for solvent accessible voids for this crystal structure using SQUEEZE [60] showed a small volume of potential solvent of 68 Å 3 for each (39 electron count), whose solvent content could not be identified or refined with the most severe restrictions, but due to the volume and the electrons present, it would coincide with four molecules of disordered water. The corresponding CIF data represent SQUEEZE treated structures with the solvent molecules handling as a diffuse contribution to the overall scattering, without specific atom position and excluded from the structural model. The SQUEEZE results were appended to the CIF. The corresponding crystallographic data were deposited with the Cambridge Crystallographic Data Centre as supplementary publications. CCDC 2169477 (2a) contain the supplementary crystallographic data for this paper. The data can be obtained free of charge via: https://www.ccdc.cam.ac.uk/structures/. 4. Conclusions Complexes Na 3 [Ag(NHC R ) 2 ] (R =H, 2a), Na 3 [Ag{(S,S)-NHC R } 2 ] (R =Me, 2b; i Pr, 2c; i Bu, 2d; s Bu, 2e) and Na 3 [Ag{(R,R)-NHC R } 2 ] (R =Me, 2b’; i Pr, 2c’) were obtained by reaction of the appropriate compound HL R , 1, (2-(1-(carboxyalkyl)-1H-imidazol-3-ium-3-yl)carboxylate) with Ag 2 O in the presence of aqueous sodium hydroxide. They were spectroscopically characterized as anionic bis(NHC) silver complexes and, additionally, complex Na 3 [Ag(NHC H ) 2 ], 2a, was structurally identified. The anion [Ag(NHC H ) 2 ] 3− showed the expected linear arrangement of NHC H groups and an eclipsed conformation of these carbene ligands. The cytotoxic behavior of these complexes was evaluated against five human cell lines: lung adenocarcinoma A549, Melanoma BRAF WT MeWo, Bladder cancer T24, gastric cancer KATO III and skin nonmalignant HaCaT. Although the anticancer activity of 2 was lower than cisplatin and gemcitabine, two structure-activity correlations were found for these compounds. First, a decrease in the activity of the Na 3 [Ag(NHC R ) 2 ] species was observed when the bulkiness of the R alkyl group increased. Second, an interesting chirality-anticancer relationship was detected when comparing the activities of enantiomerically related complexes. Actually, complexes Na 3 [Ag{(S,S)-NHC R } 2 ] (R =Me, 2b; i Pr, 2c) showed better cytotoxic activity than those of their enantiomeric derivatives Na 3 [Ag{(R,R)-NHC R } 2 ] (R =Me, 2b’; i Pr, 2c’), which were synthesized with precursors 1b’ and 1c’ obtained from the nonproteinaceous amino acids D-alanine and D-valine, respectively. Although the IC 50 values for non-cancerous cells HaCaT were lower than those observed for 2, related behavior was observed for cisplatin in most cancer cells tested. For this reason, the potential anticancer activity of complexes 2 cannot be completely excluded and further studies are in progress to evaluate and improve the selective anticancer activity of these complexes. Author contributions Conceptualization, A. G. and F. M.; Synthesis and characterizations; C.J. C. and E. A.; Biological Studies: M. L.-L. and J.M. C.-M.; Investigation: C.J. C., F. M., E. A., J.M. C.-M., M. L.-L. and A. G.; resources: A. G., E. A. and M. L.-L.; writing—original draft preparation: A. G.; writing—review and editing: C.J. C., F. M., E. A., J.M. C.-M., M. L.-L. and A. G.; supervision: F- M., A. G. and M. L.-L.; project administration: A. G. and M. L.-L.; funding acquisition: A. G and M. L.-L. 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 paper. Data availability Data will be made available on request. Acknowledgements Financial support from the Spanish Ministerio de Ciencia e Innovaci´ on (PGC2018-093443-B-I00) is gratefully acknowledged. C. J. C. thanks a research contract from PAIDI 2020, supported by the European Social Fund and the Junta de Andalucía. This research was partially funded by the University of Sevilla through the “VI Plan Propio de Investigaci´ on y Transferencia” (grant number VIPPIT-2021-I.5). The authors thank to Centro de Investigaciones, Tecnología e Innovaci´ on Table 2 Crystal data and structure refinement for compound 2a. Empirical formula C 28 H 69 Ag 2 N 8 Na 5 O 38 Formula weight 1456.60 Temperature 193(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group C 2/m a 15.9802(7) Å b 15.4531(7) Å c 12.8627(5) Å α 90◦ β 102.5720(10)◦ γ 90◦ Volume 3100.2(2) Å 3 Z 2 Density (calculated) 1.561 Mg/m 3 Absorption coefficient 0.765 mm −1 F(000) 1492 Crystal size 0.500 ×0.300 ×0.200 mm 3 Theta range for data collection 2.270 to 25.248◦ Index ranges −19 ≤h ≤19, −18 ≤k ≤18, −15 ≤l ≤15 Reflections collected 82,594 Independent reflections 2898 [R(int) =0.0339] Completeness to theta =25.242◦98.8% Absorption correction Semi-empirical from equivalents Max. and min. Transmission 0.7461 and 0.6241 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 2898 / 69 / 209 Goodness-of-fit on F 2 1.109 Final R indices [I >2 σ (I)] R 1 =0.0813, wR 2 =0.2215 R indices (all data) R 1 =0.0814, wR 2 =0.2215 Extinction coefficient n/a Largest diff. Peak and hole 4.857 and −0.610 e⋅Å −3 C.J. 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