α-Diimines as Versatile, Derivatizable Ligands in Ruthenium(II) p-Cymene Anticancer Complexes.

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/IC α‑Diimines as Versatile, Derivatizable Ligands in Ruthenium(II) p‑Cymene Anticancer Complexes Lorenzo Biancalana,† Lucinda K. Batchelor,‡ Tiziana Funaioli,† Stefano Zacchini,§ Marco Bortoluzzi,∥ Guido Pampaloni,† Paul J. Dyson,‡ and Fabio Marchetti*,† † Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via G. Moruzzi 13, I-56124 Pisa, Italy Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland § Dipartimento di Chimica Industriale “Toso Montanari”, Università di Bologna, Viale Risorgimento 4, I-40136 Bologna, Italy ∥ Dipartimento di Scienze Molecolari e Nanosistemi, Università Ca’ Foscari Venezia, Via Torino 155, I-30170 Mestre, Venice, Italy ‡ S Supporting Information * ABSTRACT: α-Diimines are among the most robust and versatile ligands available in synthetic coordination chemistry, possessing finely tunable steric and electronic properties. A series of novel cationic ruthenium(II) p-cymene complexes bearing simple α-diimine ligands, [(η6-p-cymene)RuCl{κ2N-(HCNR)2}]NO3 (R = Cy, [1]NO3; R = 4-C6H10OH, [2]NO3; R = 4C6H4OH, [3]NO3), were prepared in near-quantitative yields as their nitrate salts. [2]NO3 displays high water solubility. The potential of the α-diimine ligand in [3]NO3 as a carrier of bioactive molecules was investigated via esterification reactions with the hydroxyl groups. Thus, the double-functionalized derivatives [(η6-p-cymene)RuCl{κ2N-(HCN(4-C6H4OCO-R))2}]NO3 (R = aspirinate, [5]NO3; valproate, [6]NO3) and also [4]Cl (R = Me) were obtained in good-to-high yields. UV−vis and multinuclear NMR spectroscopy and cyclic voltammetric studies in aqueous solution revealed only minor ruthenium chloride hydrolytic cleavage, biologically accessible reduction potentials, and pH-dependent behavior of [3]NO3. Density functional theory analysis was performed in order to compare the Ru−Cl bond strength in [1]+ with the analogous ethylenediamine complex, showing that the higher stability observed in the former is related to the electron-withdrawing properties of the αdiimine ligand. In vitro cytotoxicity studies were performed against tumorigenic (A2780 and A2780cisR) and nontumorigenic (HEK-293) cell lines, with the complexes bearing simple α-diimine ligands ranging from inactive to IC50 values in the low micromolar range. The complexes functionalized with bioactive components, i.e., [5]NO3 and [6]NO3, exhibited a marked increase in the cytotoxicity with respect to the precursor [3]NO3. ■ INTRODUCTION ruthenium(III) compounds, i.e., [imidazoleH][trans-Ru(κNimidazole)(κS-DMSO)Cl4] (NAMI-A) and [indazoleH][transRu(κN-indazole)2Cl4] (KP1019; Figure 1), and a sodium salt, Na[trans-Ru(κN-indazole)2Cl4] (NKP-1339/IT139), have entered phase I/II clinical trials.3 These ruthenium(III) species are believed to act as prodrugs and are reduced to their more active ruthenium(II) counterparts in the hypoxic tumor environment. 4 Bypassing the prodrug characteristics, ruthenium(II) complexes, especially those based on the The introduction of cisplatin into clinics over 40 years ago generated widespread interest in platinum complexes as viable anticancer drugs. However, despite the efficacy of cisplatin and other platinum complexes toward many types of cancers, problems are associated with their use including severe side effects and the progressive acquisition of drug resistance. Consequently, considerable efforts have been invested in the development of alternative metal-based anticancer drugs that overcome the limitations of platinum chemotherapics.1 Ruthenium compounds are regarded as promising candidates for the next generation of metal anticancer drugs.1b−e,2 Two © XXXX American Chemical Society Received: April 2, 2018 A DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 1. Relevant ruthenium compounds exhibiting anticancer activity. Scheme 1. Structures and General Synthetic Procedure for α-Diimine Ligands L1−L3 Scheme 2. Synthesis of Ruthenium(II) p-Cymene Complexes with α-Diimine Ligands, [1−3]NO3 ruthenium(II) η6-arene scaffold, have attracted considerable attention.5 In particular, the RAPTA series, bearing a 1,3,5triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) ligand,6 and RAED complexes, possessing an ethylene-1,2-diamine (en) ligand,7 are considered to be prominent species exhibiting significant antitumor activity in vivo (Figure 1). A wide range of ruthenium η6-arene complexes with modified ligands were developed with the aim of improving their anticancer properties. In the case of the RAED complexes, the en ligand has been replaced with substituted diamines8 and many N,N-chelating ligands,9 and the resulting compounds were tested against various cancer cell lines. α-Diimines (also called 1,4-diaza-1,3-dienes) are among the most robust and versatile ligands available in synthetic coordination chemistry and possess finely tunable steric and electronic properties.10 Although some ruthenium η6-arene complexes with α-diimine ligands, i.e., (R)NC(R′)C(R′) N(R) (R = alkyl or aryl; R′ = H and Me) were reported, they did not undergo biological evaluation.11 A promising strategy used to optimize the anticancer activity of metal complexes involves the inclusion of bioactive organic fragments. This approach is designed to enhance the interaction of the resulting compounds with specific targets overexpressed or uniquely expressed in cancer cells.12 Thus, a range of organic groups with known biological functions have been directly coordinated to the ruthenium(II) center,13 tethered to the arene,14 or introduced via nitrogen and phosphorus ligands.15 Herein, we describe the synthesis and characterization of stable, significantly water (H2O)-soluble ruthenium(II) pcymene-α-diimine complexes, demonstrating that a specific αdiimine ligand with phenolic substituents undergoes clean functionalization with bioactive carboxylic acids. The cytotoxicity of the compounds was evaluated and is discussed with respect to the spectroscopic and electrochemical properties of the complexes in an aqueous solution. ■ RESULTS AND DISCUSSION Synthesis and Characterization of the α-Diimine Ligands and Their Ruthenium(II) p-Cymene Complexes. α-Diimine ligands, of the general formula (R)NCHCH N(R) (R = C6H11 = Cy, L1;16 R = 4-C6H10OH, L2; R = 4C 6H4OH, L317), were prepared via the acid-catalyzed condensation of glyoxal with the appropriate primary amine in alcohols (Scheme 1). The synthesis of the unprecedented L2 (47% yield) and that of L3 (82% yield) were optimized with varying solvent and temperature. The salient IR, NMR, and UV−vis data of L1−L3 are compiled in Table S1. The preparation of the ruthenium α-diimine compounds [(η6-p-cymene)RuCl{κ2N-(HCNR)2}]NO3 (R = Cy, [1]NO3; R = 4-C6H10OH, [2]NO3; R = 4-C6H4OH, [3]NO3) was achieved via the reaction of the dimer [(p-cymene)RuCl2]2 with AgNO3, followed by the addition of the α-diimine (ruthnium/silver/α-diimine ratio 1:1:1; Scheme 2). At variance with the literature,10 AgNO3 was selected as an unusual but B DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry convenient chloride abstractor because it does not require inert and anhydrous conditions. The choice of solvent [acetonitrile (MeCN) or methanol (MeOH)] and reaction temperature proved crucial to achieving full conversion and high selectivity. The products were isolated as air-stable orange ([1,2]NO3) or dark-red/brown ([3]NO3) solids in high yields (93−98%). Complexes [1−3]NO3 are soluble in H2O (a detailed discussion of their solubility and stability in an aqueous medium is given below). Compound [1]NO3 is also soluble in common organic solvents excluding diethyl ether (Et2O) and hydrocarbons, whereas [2,3]NO3, bearing hydroxyl groups on the N-substituents, are insoluble in chlorinated solvents but soluble in alcohols and dimethyl sulfoxide (DMSO). The products were fully characterized using analytical techniques (CHN analysis, mass spectrometry, and conductivity) and NMR, IR, and UV−vis spectroscopy (selected data are compiled in Table S1). The 1H and 13C NMR spectra of [1]NO3, recorded in deuterated chloroform (CDCl3), and [2,3]NO3, in CD3OD or DMSO-d6, contain a single set of resonances for the p-cymene and α-diimine ligands. A singlet, corresponding to the two imine protons of the coordinated ligand, was observed at 8.3−8.4 ppm, i.e., slightly deshielded (ΔδH ≤ 0.4 ppm) with respect to uncoordinated L1−L3. The imine carbon experiences a more significant downfield shift (ΔδC = 4−9 ppm) with a resonance around 165 ppm in the 13C NMR spectra of [1−3]NO3, compared to 156.0−160.2 ppm for the free α-diimine. Signals relating to the hydroxyl group protons of [2]+ and [3]+ are observed in the 1H NMR spectra at 4.7 and 10.4 ppm in DMSO-d6, respectively, and remain at a value comparable to that of the corresponding ligands L2 and L3. In DMSO-d6, the 1H resonances of [3]NO3 are broad, and negligible changes are observed with increasing temperature from 25 to 60 °C. This suggests that a strong NO3−/[3]+ interaction (and related association phenomena11a,b) takes place in DMSO-d6 and presumably involves hydrogen bonding with the phenolic groups. Well-resolved 1H NMR spectra were recorded for [3]NO3 in CD3OD and for [2]NO3 in both DMSO-d6 and CD3OD solutions, which indicated that the ions are well solvated and separated. The UV−vis spectra of [1−3]NO3 in CH2Cl2 or MeOH feature metal-to-ligand charge-transfer (MLCT) bands around 270−280 and 420−450 nm.10c Increased band intensity (ε) and an additional absorption at ca. 550 nm were observed for [3]NO3, which contains an extended π system in the α-diimine ligand. The solid-state IR spectra of [1−3]NO3 show strong absorptions due to the [NO3]− ion (ca. 1320 cm−1) and medium/weak CN stretching vibrations in the region 1530− 1630 cm−1. The relative intensity of this CN absorption is lower in the complexes compared to the respective uncoordinated α-diimines, and a considerable decrease in the wavenumber (ca. −85 cm−1) is observed upon coordination of L1 and L2.18 The solid-state structure of [1]NO3 was determined by single-crystal X-ray diffraction (Figure 2), and relevant parameters are presented in Table 1. The cation [1]+ possesses the expected three-legged piano-stool geometry typical of other ruthenium(II) arene compounds,19 and the bonding parameters around the ruthenium(II) center are similar to those reported for related [(p-cymene)RuCl(α-diimine)]+ structures.11a Moreover, C−C and C−N distances within the main skeleton of the L1 ligand are comparable to those previously observed in RuCl2(L1)2.20 Figure 2. Molecular structure of [1+] within [1]NO3. Displacement ellipsoids are at the 50% probability level. Derivatization of the Complexes with Bioactive Groups. In principle, the OH groups belonging to the αdiimines L2 and L3 could be exploited for functionalization; in particular, esterification reactions may represent a strategy to incorporate bioactive molecules featuring −CO2H groups within (p-cymene)RuII complexes via α-diimine linkers. Our first attempts, using various synthetic protocols,21 to perform the esterification of L2 and L3 with different carboxylic acids afforded mixtures of products, and the α-diimine moiety did not tolerate the conditions used. This is in alignment with literature reports, indicating that functionalization of the arene ring in N-aryl-α-diimine systems is usually achieved prior to generation of the imine skeleton.22 Upon further investigation, we noticed that two esterification reactions of L3, coordinated in transition-metal complexes, were previously described.23 Therefore, we focused on the direct esterification of L2 and L3 coordinated to the (η6-p-cymene)Ru frame in the corresponding complexes [2,3]NO3. After several attempts, we found that the addition of an excess of acetyl chloride, as a model reactant, to a refluxing trichloromethane (CHCl3) solution of [3]NO3 treated with 1,5-diazabicyclo[5.4.0]undec-7-ene (DBU) resulted in the formation of the diester [4]Cl, which was isolated in 68% yield (Scheme 3a). On the other hand, [2]NO3 was largely unreactive. Then, aspirin and valproic acid were selected as viable candidates to functionalize [3]NO3. These bioactive molecules contain carboxylic acid groups and are known to possess anticancer properties.24,25 It was previously demonstrated that the incorporation of aspirin and valproic acid into metal complexes, including (p-cymene)RuII complexes,15c can provide a synergism resulting in enhanced cytotoxicity.26,27 Because the conditions employed for the synthesis of [4]Cl were not suitable to aspirin and valproic acid, N-[3(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDCI·HCl) and 4-(dimethylamino)pyridine (DMAP) were used as the coupling agent and base catalyst, respectively (Steglich protocol28). An excess of the bioactive component was reacted with [3]NO3 to yield the bis-functionalized complexes [5]NO3 and [6]NO3 in 92 and 69% yield, respectively (Scheme 3b). This synthetic route overcomes two common problems usually experienced in this chemistry, i.e., harsh conditions intolerable to the α-diimine backbone and C DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Table 1. Selected Bond Distances (Å) and Angles (deg) for [1]+ Ru(1)−(η6-p-cymene)av Ru(1)−N(1) N(1)−C(11) N(1)−C(13) C(11)−C(12) 2.20(2) 2.080(6) 1.279(10) 1.484(10) 1.436(11) Ru(1)−Cl(1) Ru(1)−N(2) N(2)−C(12) N(2)−C(19) 2.384(2) 2.071(6) 1.269(10) 1.491(10) N(1)−Ru(1)−N(2) N(1)−C(11)−C(12) C(12)−N(2)−Ru(1) 76.5(2) 116.4(7) 115.7(5) Ru(1)−N(1)−C(11) C(11)−C(12)−N(2) 114.9(5) 116.4(7) Scheme 3. Esterification of [3]NO3: (a) Preparation of [4]Cl with DBU/MeCOCl and (b) EDCI/DMAP-Mediated Reaction with Aspirin (asp-CO2H) and Valproic Acid (vp-CO2H), Affording Compounds [5,6]NO3 nature of the nucleus, with the exception of highly symmetric species such as [NH4]+ or [NO3]−.31 For instance, NaNO3 in a D2O or CD3OD solution displayed narrow 14N NMR signals at −5.0 or −2.7 ppm, respectively (see the Experimental Section). Consistent with this observation, a signal around −3 ppm was present in the 14N NMR spectra of [5,6]NO3 in MeOH, whereas no signal was observed in the spectrum of [4]Cl. Solubility, Stability, and Speciation of the Complexes in an Aqueous Solution. Compounds [1−3]NO3 readily dissolve in H2O, affording yellow/red solutions, and the 1H NMR and UV−vis spectra recorded in D2O resembled those recorded in organic solvents. The solubility of [1−3]NO3 was evaluated in saturated D2O solutions at 21 °C using 1H NMR spectroscopy with dimethyl sulfone (Me2SO2), employed as an internal standard.32 The solubility is 5.6 × 10−3 M for [3]NO3 and 1.0 × 10−2 M for [1]NO3, reaching 0.1 M for [2]NO3 (Table 2). Considering that α-diimines L1−L3 are insoluble in H2O, it appears that the H2O solubility of [1−3]NO3 is most likely favored by the [NO3]− anion rather than a hydrophilic ligand. Conversely, the H2O solubility of [(η6-p-cymene)RuCl 2 (PTA)] (RAPTA-C) and [(η 6 -p-cymene)RuCl(NH2CH2CH2NH2)]+ (RAED-C) complexes is achieved by the coordination of a H2O-soluble ligand (Figure 1). The solutions of [1−3]NO3 were then maintained at 37 °C for 72 h, the temperature under which the cell studies were performed, and monitored by 1H and 35Cl NMR, UV−vis, and conductivity measurements. Because of limited solubility in H2O, evaluation of the H2O stability of [4−6]X (X = Cl and NO3) had to be carried out in DMSO/H2O (9:1) mixtures. In general, the complexes were stable, with 80−99% of the the need to protect the ruthenium center during the peripheral esterification reaction.6c,15c It is noteworthy that two bioactive fragments are associated here with a single ruthenium center; recently, the introduction of two bioactive compounds to a single ruthenium or osmium complex via (bi)pyridine ligands resulted in a significant cytotoxic effect.15a,29 Compounds [4−6]X (X = Cl and NO3) are air-stable darkred/brown solids that possess good solubility in chlorinated solvents, acetone, and DMSO but have low solubility in MeOH and H2O. The lack of H2O solubility correlates with the substantial increase in the hydrophobicity of the cation upon going from [3]+ to [4−6]+. The NMR spectroscopic data (CD3OD solution;30 Table S1) related to the imine CH units show a small deshielding with respect to the precursor [3]NO3 (ΔδH = +0.2 ppm; ΔδC = +3.5 ppm). Conversely, the 13C NMR resonance of the carbonyl carbon undergoes a marked upfield shift following the introduction of bioactive molecules relative to the ruthenium(II) α-diiminodiester (ΔδC ≈ −6−7 ppm). The IR spectra of [4−6]X (X = Cl and NO3) display a strong absorption in the 1750−1760 cm−1 region due to the ν(CO) stretching of the newly formed ester moiety as well as a medium-weak absorption around 1600 cm−1 due to the ν(CN) stretching. The presence of chloride or nitrate counterions in [4−6]X was ascertained using an X−/[BF4]− metathesis assay (see the Supporting Information, SI), IR spectroscopy (the presence or absence of intense [NO3]− absorptions around 1335 cm−1), and 14N NMR spectroscopy. Broad resonances are generally observed in 14N NMR spectra because of the quadrupolar D DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry [(p‐cymene)Ru(H 2O)(α ‐diimine)]2 + (aq) Table 2. Solubility of [1−3]NO3 in D2O and the Fraction of [1−6]X (X = NO3 and Cl) after 72 h at 37 °C in D2O or DMSO-d6/D2O (9:1, v/v) Solutionsa compound solvent solubility (21 °C)/mol·L−1 [1]NO3 [2]NO3 [3]NO3 4[Cl] H2O H2O H2O 9:1 DMSO/ H2O 9:1 DMSO/ H2O 9:1 DMSO/ H2O 1.0 × 10−2 1.0 × 10−1 5.6 × 10−3 [5]NO3 [6]NO3 → [(p‐cymene)Ru(OH)(α ‐diimine)]+ (aq) + H+(aq) (2) % complex remaining (72 h, 37 °C) + 35,35 In contrast to [1−3] , both RAED and RAPTA compounds36 (Figure 1) are known to undergo rapid and extensive hydrolysis in H2O, a feature that is considered to activate the complexes. Even the addition of 1 equiv of AgNO3 to [1−3]NO3 (in 9:1 H2O/MeOH; see the SI) caused no change in their UV−vis and 1H NMR spectra, and AgCl precipitation was not observed. In comparison, the structurally related RAED compounds with 1,2-diamine ligands (Figure 1) undergo quantitative chloride/H2O displacement upon the addition of Ag+, which is only partially reversed in a 0.1 M NaCl medium.37 Unlike [1,2]NO3, a freshly prepared solution of [3]NO3 in H2O has a rather low pH value (5.7), and the UV−vis spectrum quickly and reversibly changes when small volumes of NaOH(aq) or HCl(aq) are added in sequence. Consequently, an isosbestic point was detected at 437 nm (Figure S27) and could be related to the possible deprotonation of one phenolic group, resulting in the green-colored 3B (Scheme 4a). A parallel 1 H NMR spectroscopic study revealed chemical shift variations related to the aromatic o-CH (ΔδH = −0.37 ppm) and the imine HCN (ΔδH = −0.24 ppm) groups, suggesting that 3B experiences some delocalization of the negative charge over the π system of the L3 ligand (Table S9 and Figure S29). A value of pKa = 7.7 ± 0.1 was determined for [3]+ in a 0.1 M NaCl solution using a spectrophotometric method (see the Experimental Section), and therefore similar quantities of [3]+ and its conjugate base 3B are expected to be present in solution at physiological pH (≈7.4). Basic solutions containing [3]+/3B were not stable at room temperature, as indicated by changes in the UV−vis spectra and the appearance of a second set of signals in the 1H NMR spectra (Figure S28 and Tables S10 and S11). These variations become increasingly evident with increasing pH and are thus attributed to presumable Cl−/OH− substitution, affording [(η6p-cymene)Ru(OH){κ 2 N,N′-(HCN) 2 (4-C 6 H 4 OH)(4C6H4O)}] (3BW; Scheme 4b). In fact, ca. 70% of 3BW was observed at pH ≈ 12 after a few minutes, accompanied by a sharp 35Cl NMR signal at ca. 0 ppm; conversely, red solutions containing [3]NO3 at pH = 1.5−7 were stable for several days at room temperature, and no trace of 3BW was detected by 1H NMR spectroscopy. Density Functional Theory (DFT) Study. The observed stability of the metal−chloride bond in [1−6]+ seems exceptional in the context of ruthenium(II) arene compounds, and potential anticancer compounds belonging to this family 91 97 99 81 86 80 a All values are based on 1H NMR spectroscopy (Me2SO2 as an internal standard). complex unmodified after 72 h (Table 2). Minor degradation in the DMSO/H2O medium was associated with the release of pcymene from [4−6]+ and of acetic acid from [4]+ and [5]+ because of cleavage of the ester linkages. Interestingly, the αdiimine ligand and the bioactive fragment were not released from [5]+ and [6]+ (see the SI for details). The initial molar conductivity values of [1−3]NO3 in H2O (Λm ≈ 125 S cm2 mol−1) are in the range of a 1:1 electrolyte33 and are in accordance with the presence of intact cationic complexes [1−3]+. Accordingly, the 35Cl NMR spectra, recorded on freshly prepared solutions in H2O/MeOH (9:1, v/v; to increase the solubility of [3]NO3), showed no evidence of free Cl− ions,34 and the addition of NaCl (0.11 M) had no effect on the 1H NMR spectra. The quantity of [1]+ and [2]+ decreased progressively with time due to the formation of a secondary (p-cymene)Ru(α-diimine) species (less than 10%; 1W and 2W; Figures S19 and S20), as indicated by the appearance of a new set of 1H resonances and a signal around 0 ppm in the 35Cl NMR spectra. In addition, a decrease in the pH (from 6.5 to 5.9−6.0) and an increase in the molar conductivity (Λm ≈ 160 S cm2 mol−1) were detected, which is consistent with the occurrence of minor chloride/H2O replacement (eq 1), possibly followed by proton release from the H2O ligand in the resulting dicationic complex (eq 2).35 [(p‐cymene)RuCl(α ‐diimine)]+ (aq) + H 2O → [(p‐cymene)Ru(H 2O)(α ‐diimine)]2 + (aq) + Cl−(aq) (1) Scheme 4. Speciation of [3]+ in H2O: Acid−Base Equilibrium with the Formation of 3B (Resonance Structures Are Shown in Figure S29) and Subsequent Ru−Cl Hydrolysis with the Formation of 3BW E DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 3. HOMO−2 molecular orbitals of [(η6-benzene)Ru(NHCHCHNH)Cl]+ and [(benzene)Ru(en)Cl]+ (surface isovalue = 0.035 au). Inset: Bond energy variation (kcal mol−1) versus Ru−Cl distance for [1]+ and [(p-cymene)Ru(en)Cl]+. C-PCM/ωB97X calculations and H2O as the continuous medium. Previous electrochemical studies on [(η6-C6H6)RuCl{κ2N(HCNiPr)2}]11c allowed the identification of both a reduction process (ca. −0.67 V) and an oxidation process (ca. +1.8 V) in a CH3CN solution, presumably favored by solvent coordination and by the redox noninnocent character of the α-diimine ligand.40 The redox properties of [1−3]NO3 were assessed by cyclic voltammetry (CV) with a glassy carbon (GC) electrode in an aqueous medium, and a parallel study was conducted on L1 and [1]NO3 in a CH2Cl2/[Bu4N]PF6 solution. The peak potentials for the observed electron transfer are compiled in Table 3, and selected CV profiles are shown in Figures S30− S33. Compounds [1−3]NO3 in a phosphate buffer (PB) solution (pH = 7.3) displayed two independent redox processes, associated with reduction and oxidation processes of the complexes. The oxidation of [1−3]NO3 occurs at potentials between +0.8 and +1.4 V; however, it is not followed by a reverse peak, indicating an irreversible process and leading to degradation of the compounds. During the cathodic scan, peaks were observed at −0.30, −0.34, and −0.20 V, which are associated with the reduction of [1]+, [2]+, and [3]+, respectively. For [1]NO3 and [3]NO3, this process was followed by a very intense reoxidation peak at ca. +0.14 V on the reverse scan. The high current peak intensity indicates an accumulation of the electrogenerated species on the surface of the electrode during the reduction step. In contrast, [2]+ displays a return peak of comparable current intensity occurring at a much lower potential (− 0.16 V; ΔE = 206 mV), and consequently the reduction process can be considered as quasi- are usually believed to be readily activated by hydrolytic Ru−Cl cleavage (see above). To rationalize this unusual stability, we carried out a DFT study to compare the Ru−Cl bond strength in [1]+, as a representative compound, with that in the structurally related RAED cation [(η6-p-cymene)Ru(κ2N-en)Cl]+ (Figure 1). Chloride dissociation from the latter complex is more favorable with respect to [1]+ by 2.1 kcal mol−1 [Gibbs free energy, conductor-like polarizable continuum model (C-PCM)/ωB97X calculations, and H2O as the continuous medium]. The higher stability of the Ru−Cl bond in [1]+ is apparent from the plot of the bond energy upon variation of the Ru−Cl distance (see the inset in Figure 3). A comparative analysis of the occupied orbitals in the model compounds [(η6-benzene)Ru(κ2N-NH CHCHNH)Cl]+ and [(η6-benzene)Ru(κ2NNHyCHyCHyNH2)Cl]+ suggests that the different strength of the Ru−Cl bond is due to delocalization of the electron density on the lowest-energy π* orbital of the α-diimine ligand. This feature is highlighted in particular by the HOMO−2 molecular orbitals of the two compounds (Figure 3), wherein ethylenediamine behaves as a σ donor, while α-diimine exhibits π acidic behavior. Electrochemical Studies.38 Because the cytotoxicity of ruthenium compounds has often been associated with the occurrence of redox processes,4 the electrochemical behavior of [1−3]NO3 was investigated. Although H2O is the most obvious solvent with respect to biological studies, there is a paucity of electrochemical studies in aqueous media concerning (η6arene)Ru compounds evaluated for their possible anticancer activity.39 F DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Table 3. Peak Potentials versus Normal Hydrogen Electrode for L1 and [1−3]NO3 in Aqueous Media and a CH2Cl2 Solution reduction processb a compound electrolyte L1 [1]NO3 CH2Cl2/[Bu4N]PF6 CH2Cl2/[Bu4N]PF6 PB/H2O (pH = 7.3) PB/H2O (pH = 7.3) + 0.1 M NaCl PB/H2O (pH = 7.3) PB/H2O (pH = 7.3) + 0.1 M NaCl PB/H2O (pH = 7.3) AB/H2O (pH = 4.5) [2]NO3 [3]NO3 Table 4. IC50 Values (μM) of Ligands L1−L3, Complexes [1−6]X (X = Cl and NO3), Bioactive Molecules (asp-CO2H and vp-CO2H), and Cisplatin Used as a Control against A2780, A2780cisR, and HEK-293 Cell Lines after 72 h of Exposurea oxidation processb Epc/V Epa/V (return peak) Epa/V n.d. −0.707 −0.301 −0.277 n.d. n.d. +0.142c +0.177c n.d. +1.355 +1.385 −0.362 −0.342 −0.156 −0.142 +1.43d n.d. −0.200 −0.198 +0.148c −0.031 +0.864 +1.035 a PB = phosphate buffer; AB = acetate buffer. bCathodic (Epc) and anodic (Epa) peak potentials measured at 0.1 V s−1. cHigh peak current intensity. dThe wave occurred at the upper limit of the potential window provided by the electrolyte. n.d. = not decteted. a compound A2780 A2780cisR HEK-293 L1 L2 L3 [1]NO3 [2]NO3 [3]NO3 [4]Cl [5]NO3 [6]NO3 vp-CO2H43 asp-CO2H15c cisplatin >200 >200 19 ± 1 2.6 ± 0.6 >200 >200 30 ± 3 21 ± 2 2.2 ± 0.3 >1000 >200 2.0 ± 0.4 >200 >200 25 ± 2 3.9 ± 0.5 >200 >200 54 ± 2 18 ± 2 3.7 ± 0.9 >1000 >200 24 ± 3 >200 >200 48 ± 1 3.9 ± 0.5 >200 >200 56 ± 3 21 ± 3 2.5 ± 0.9 >200 8.9 ± 1.3 Values are given as the mean ± standard deviation. cell lines (IC50 = ca. 2−4 μM), but no selectivity toward the cancer cell lines was observed. Complexes [2]NO3 and [3]NO3, containing L2 and L3, respectively, were inactive against all cell lines, despite the cytotoxicity displayed by uncoordinated L3. Acetylation of the phenolic functions of [3]NO3 significantly increases the cytotoxicity of the resulting complex [4]Cl; i.e., IC50 goes from >200 μM to 30 ± 3 μM against the A2780 cell line. A stronger effect is obtained when aspirin and valproic acid, which are both inactive against the three cell lines in their free (carboxylic acid) form, are tethered to the [3]+ scaffold; in particular, a marked increase in the cytotoxicity was observed for the valproate derivative [6]NO3, compared to the parent [3]NO3. The octanol/H2O partition coefficients (log Pow; Table 5) of the complexes were determined spectrophotometrically using reversible.41 The addition of NaCl (0.1 M) to the solutions of [1]NO3 and [2]NO3 led to small variations in the respective CV profiles and peak potentials. Because of the acid−base properties of [3]NO3, CV was also conducted in an acetate buffer (AB) solution (pH = 4.5), where the complex remains mostly undissociated ([3]+; pKa = 7.7). The peak potential during the cathodic scan at pH = 4.5 is identical with that at pH = 7.3, thus in both cases related to the reduction of [3]+ (not 3B). However, the reduction process assumed a quasi-reversible behavior at pH = 4.5 because the current intensity of the reverse peak is comparable, and the peak-to-peak separation (ΔE) is 150 mV (vs 360 mV in a PB solution). Therefore, the different electrochemical behavior of [3]NO3 in PB and AB solutions may be related to acid−base equilibria involving the reduced species [3•]. From these data, the oxidation of [1−3]NO3 in an aqueous solution at pH = 7.3 lies outside a biologically relevant range of potentials (−0.40 V < E < +0.80 V;42 see the SI). Conversely, the reduction of [1−3]NO3 falls within such an electrochemical window. In comparison, CV of [1]NO3 in CH2Cl2 showed an irreversible reduction process at a much lower potential (− 0.71 V), comparable to that reported for [(η6-C6H6)RuCl{κ2N(HCNiPr)2}] in MeCN (see above).11c Therefore, the aqueous medium plays a key role in making the reduction of [1]+ (and presumably of [2]+ and [3]+) more accessible (than in organic solvents). The complexes could potentially undergo a reduction in the physiological environment, favored by the peculiar electronic properties of the α-diimine ligand, which could contribute to their biological action. Cytotoxicity Studies. The cytotoxicity of [1−6]X (X = Cl and NO3), ligands L1−L3, and control compounds cisplatin, aspirin, and valproic acid was assessed against human ovarian carcinoma (A2780), human ovarian carcinoma with acquired cisplatin resistance (A2780cisR), and human embryonic kidney (HEK-293) cell lines (Table 4). L1 and L2 are inactive against all three cell lines, whereas L3 is active in the mid-to-low micromolar range, with 2-fold selectivity observed toward the A2780 cell line. Interestingly, [1]NO3, comprising inactive ligand L1, showed cytotoxicity in the low micromolar against all Table 5. Partition Coefficients (log Pow) of Ruthenium Compounds compound solvent system partition coefficient (log Pow) [1]NO3 [2]NO3 [3]NO3 1-octanol/H2O 1-octanol/H2O 1-octanol/H2O 1-octanol/PB solution 1-octanol/H2O 1-octanol/H2O 1-octanol/H2O −0.78 <−2.5 −0.91 −0.64 −0.26 >2.5 >2.5 [4]Cl [5]NO3 [6]NO3 the shlake-flask method (see the Experimental Section and SI), and the values tend to correlate with the observed cytotoxicities. Indeed, the least lipophilic complexes, [2]NO3 and [3]NO3, are also the least active of the series with IC50 values of >200 μM. As the hydrophilicity decreases, the cytotoxicity of the complexes generally increases; complex [1]NO3 is an outlier, with IC50 values in the low micromolar range and a log Pow value of −0.78. The most hydrophobic complexes, [5]NO3 and [6]NO3, possessing log Pow values of >2.5, present IC50 values in the mid-to-low micromolar range. This indicates that the aspirinate and valproate moieties might contribute to the cytotoxicity of the respective complexes mainly because of the lipophilicity that they provide. G DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry MICRO cube instrument (Elementar). Mass spectrometry (MS) spectra were obtained on a LTQ Orbitrap Elite (Thermo Fischer) in positive-ion mode. Melting points/decomposition temperatures were determined on a STMP3 Stuart scientific instrument with a capillary apparatus. pH measurements were performed with an Orion pH meter equipped with a Hamilton glass pH electrode, routinely calibrated with pH = 4.0 and 7.0 buffer solutions (Sigma-Aldrich). Conductivity measurements were carried out at 21 °C using an XS COND 8 instrument (cell constant = 1.0 cm−1).49 The NMR (14N, 35Cl) and molar conductivity data for reference compounds are given. NaNO3. Λm (MeOH, c = 1.7 × 10−3 M) = 119 S·cm2·mol−1. 14N NMR (D2O): δ −5.0 (Δν1/2 = 7 Hz). 14N NMR (CH3OD): δ −2.7 (Δν1/2 = 14 Hz). NaCl. Λm (MeOH, c = 3 × 10−3 mol·L−1) = 85 S·cm2·mol−1. 35Cl NMR (CD3OD, acq. time 1 min): δ −28.3 (Δν1/2 = 1.7 × 102 Hz). NH4Cl. 14N NMR (D2O): δ −360.7 (Δν1/2 = 2 Hz). 14N NMR (CH3OH/C6D6 capillary): δ −366.1 (Δν1/2 = 5 Hz). [Et3NH]Cl. 35Cl NMR (CD3OD, acq. time 1 min): δ −22.7 (Δν1/2 = 2.3 × 102 Hz). Synthesis and Characterization of Compounds. N,N′-Bis(cyclohexyl)ethylenediimine (L1). Compound L1 (Chart 1) was Accordingly, the ester linkages connecting the bioactive fragments to the metal fragment appear quite stable in aqueous media (see above), possibly favoring uptake of the complexes into the cells. ■ CONCLUSIONS Ruthenium(II) arene complexes have been intensively investigated as future anticancer drugs, and promising results were previously obtained, among the others, with some derivatives containing N,N-bidentate ligands. Despite α-diimines have been widely employed as robust N,N-bidentate ligands in coordination chemistry, ruthenium(II) arene-α-diimine complexes were not considered to date for biological evaluation. Herein, we have reported a series of new, H2O-soluble, cationic (p-cymene)RuII complexes with simple α-diimines, unusually obtained as nitrate salts. The appropriate coordinated α-diimine can be directly modified via esterification reactions, at the expense of the H2O solubility, without the need for protection/deprotection steps of the ruthenium(II) center. In general, the cytotoxicity of the complexes strongly depends on the nature of the α-diimine N-substituents, including bioactive molecules tethered to it, resulting in some cases in IC50 values in the low micromolar range. As a consequence of the peculiar electron-withdrawing properties of the α-diimine ligands, the complexes undergo only minor ruthenium chloride hydrolytic cleavage (rationalized by DFT calculations), and their electrochemical reduction in H2O falls within a biologically relevant range of potentials. In contrast, the prototype classes of anticancer compounds RAPTA and RAED are known to undergo rapid and extensive hydrolysis in H2O, which is believed to activate the complexes. ■ Chart 1. Structure of L1a a The numbering refers to carbon atoms. prepared according to the literature:16 pale-yellow solid, soluble in Et2O, poorly soluble in DMSO. Anal. Calcd for C14H24N2: C, 76.31; H, 10.98; N, 12.72. Found: C, 76.11; H, 10.76; N, 12.90. IR (solid state, cm−1): 2922s, 2852s, 2793w, 2756w, 2657w, 1622s (νCN), 1468w, 1449m, 1443m-sh, 1435w-sh, 1371m, 1347m, 1321w, 1287m, 1260w-sh, 1252w, 1236w, 1182w, 1151w, 1063m, 1031w, 962m, 951s, 918w, 899w, 886s, 844m, 801w, 786w. UV−vis [CH2Cl2, c = 1.0 × 10−3 M; λmax/nm (ε/M−1·cm−1)]: 279 (8.2 × 102). 1H NMR (CDCl3): δ 7.93 (s, 2H, C1−H), 3.15 (tt, 3JHH = 10.5 and 4.0 Hz, 2H, C2−H), 1.84−1.77 (m, 4H), 1.75−1.63 (m, 6H), 1.50 (ddd, J = 15.1, 12.7, and 3.1 Hz, 4H), 1.34 (qt, J = 12.4 and 3.2 Hz, 4H), 1.24 (tt, J = 12.1 and 3.2 Hz, 2H). 13C{1H} NMR (CDCl3): δ 160.2 (C1), 69.6 (C2), 34.1 (C3), 25.7 (C4), 24.8 (C5). N,N′-Bis(4-hydroxycyclohexyl)ethylenediimine (L2). Glyoxal (40% w/w in H2O, 0.40 mL, 3.5 mmol) and AcOH (50 μL, 0.87 mmol) were added to a suspension of trans-4-aminocyclohexanol (520 mg, 4.51 mmol) in iPrOH (5 mL). The resulting pale-pink suspension was stirred at room temperature for 14 h. Therefore, the suspension was filtered, and the colorless solid was washed with iPrOH (2 × 2 mL) and Et2O and then dried under vacuum (40 °C) over P2O5. Yield: 266 mg, 47%. Alternative conditions: MeOH, room temperature, 0% yield (no precipitation); MeOH, 50 °C, 27% yield; MeOH, reflux, 0% yield (dec); EtOH, room temperature, 40% yield; iPrOH, room temperature, 2.5 h, 32%; THF, room temperature, 51% yield but product contains traces of trans-4-aminocyclohexanol. Compound L2 (Chart 2) is soluble in MeOH and hot DMSO, poorly soluble in MeCN, EtOH, and iPrOH, and insoluble in chlorinated solvents and H2O. EXPERIMENTAL SECTION General Experimental Details. All reagents and solvents were obtained from Alfa Aesar, Sigma-Aldrich, or TCI Europe and used without further purification. The following reagents were stored under N2 as received: 4-aminophenol (4 °C, in the dark), aspirin (aspCO2H), valproic acid (vp-CO2H), acetyl chloride, triethylamine (over 4 Å molecular sieves), 1,5-diazabicyclo[5.4.0]undec-7-ene (DBU; over 4 Å molecular sieves), and ethyl(diisopropylamino)carboxydiimide hydrochloride (EDCI·HCl; −20 °C). Glyoxal (40% w/w in H2O) was stored at 4 °C. Compounds [(η6-p-cymene)RuCl2]244 and N,N′bis(cyclohexyl)ethylenediimine (L1)16 were prepared according to literature methods. The synthesis of [4−6]X (X = Cl and NO3) was carried out under a N2 atmosphere using standard Schlenk techniques and solvents distilled from the appropriate drying agents. All of the other operations were carried out in air with common laboratory glassware. Once isolated, all of the complexes were obtained as airstable solids. NMR spectra were recorded on a Bruker Avance II DRX400 instrument equipped with a BBFO broad-band probe at 25 °C, unless otherwise specified. Chemical shifts (expressed in parts per million) are referenced to the residual solvent peaks45 (1H and 13C) or to external standards (14N to CH3NO2 and 35Cl to 1 M NaCl in D2O).46 In mixed solvents, chemical shifts were referenced to the residual peak of the major component as the pure solvent (δH = 2.50 ppm for DMSO in 9:1 DMSO-d6/D2O and δH = 4.79 ppm for HDO in 9:1 D2O/CD3OD). Spectra were assigned with the assistance of DEPT-135 spectra and 1H−1H (COSY) and 1H−13C (gs-HSQC and gs-HMBC) correlation experiments.47 IR spectra of solid samples were recorded on a PerkinElmer Spectrum One FT-IR spectrometer, equipped with a UATR sampling accessory. IR spectra of solutions were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer with a CaF2 liquid transmission cell (4000−1000 cm−1 range). UV−vis spectra were recorded on an Ultraspec 2100 Pro spectrophotometer. IR and UV−vis spectra were processed with Spectragryph software.48 Carbon, hydrogen, and nitrogen analyses were performed on a Vario Chart 2. Structure of L2a a H The numbering refers to carbon atoms. DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Chart 4. Structure of [1]NO3a Anal. Calcd for C14H24N2O2: C, 66.63; H, 9.59; N, 11.10. Found: C, 66.30; H, 9.70; N, 11.22. IR (solid state, cm−1): 3399m (νO−H), 3332m (νO−H), 2958m, 2946m, 2928m, 2903m, 2882m, 2859s, 1625s (νCN), 1454m-sh, 1444m, 1414w, 1372m, 1355m, 1334m, 1303m, 1289m, 1248w, 1218m, 1199w, 1124m, 1078s-sh, 1069s, 1036s, 1006m, 944s, 933s-sh, 901s, 886m. UV−vis [MeOH, c = 3.6 × 10−3 M; λmax/nm (ε/ M−1·cm−1)]: 267 (1.0 × 103). 1H NMR (CH3OD): δ 7.91 (s, C1−H). 1 H NMR (DMSO-d6, 40 °C): δ 7.88 (s, 2H, C1−H), 4.49 (d, 3JHH = 4.4 Hz, 2H, OH), 3.49−3.37 (m, 2H, C5−H), 3.15 (ddd, J = 14.3, 10.4, and 3.9 Hz, 2H, C2−H), 1.85 (dd, J = 12.2 and 2.4 Hz, 4H, C4− H), 1.61 (dd, J = 13.0 and 2.3 Hz, 4H, C3−H), 1.46 (dq, J = 13.1 and 3.0 Hz, 4H, C3−H′), 1.26 (dq, J = 12.9 and 3.1 Hz, 4H, C4−H′). 13 C{1H} NMR (DMSO-d6, 40 °C): δ 156.0 (C1), 68.0 (C5), 67.6 (C2), 33.2 (C4), 31.6 (C3). N,N′-Bis(4-hydroxyphenyl)ethylenediimine (L3). Compound L3 (Chart 3) was prepared according to a modified literature procedure.17 a 6.81; N, 7.75. ESI-MS(+). Found: m/z 491.1773 ([M]+). Calcd for C24H38ClN2Ru+: m/z 491.1770. Tm = 112−115 °C (dec). IR (solid state, cm−1): 3600−3300w-br, 3056w, 3040w, 2962w-sh, 2929m, 2856m, 1633w-br, 1537w (νCN), 1505w, 1470w-sh, 1452m, 1354ssh, 1324s-br (νNO3), 1264m-sh, 1190w, 1160w, 1145w, 1090w, 1076m, 1054w, 1034w,1013w, 926w, 873m, 828w, 803w, 774w, 730w, 669w. UV−vis [CH2Cl2, c = 1.0 × 10−3 M; λmax/nm (ε/M−1·cm−1)]: 285 (3.2 × 103), 368 (2.3 × 103), 427 (2.9 × 103). Λm [c = (1.0−2.0) × 10−3 M] = 18 S·cm2·mol−1 (CH2Cl2) and 113 S·cm2·mol−1 (MeOH). 1 H NMR (CDCl3): δ 8.34 (s, 2H, C8−H), 5.87 (d, 3JHH = 5.6 Hz, 2H, C4−H), 5.70 (d, 3JHH = 5.6 Hz, 2H, C3−H), 4.35 (t, 3JHH = 10.8 Hz, 2H, C9−H), 2.81 (hept, 3JHH = 6.7 Hz, 1H, C6−H), 2.51 (d, 3JHH = 11.3 Hz, 2H, C10−H), 2.34 (d, 3JHH = 11.1 Hz, 2H, C10′−H), 2.28 (s, 3H, C1−H), 1.96 (d, 3JHH = 13.0 Hz, 2H, C11−H*), 1.92−1.84 (m, 4H, C11−H*), 1.79−1.65 (m, 4H, C10′−H′ + C11−H*), 1.58−1.38 (m, 4H, C12−H), 1.29−1.22 (m, 4H, C10−H′ + C11−H*), 1.20 (d, 3 JHH = 6.8 Hz, 6H, C7−H). Asterisks refer collectively to a proton attached to C11 or C11′. No change in the 1H NMR spectrum was observed after 14 days at room temperature. 13C{1H} NMR (CDCl3): δ 163.9 (C8), 108.9 (C5), 104.3 (C2), 87.3 (C4), 86.7 (C3), 75.9 (C9), 35.3 (C10), 33.4 (C10′), 31.8 (C6), 26.0 (C11/C11′), 25.6 (C11/C11′), 25.4 (C12), 22.4 (C7), 19.1 (C1). [(η6-p-Cymene)RuCl(κ2N-{HCN(4-C6H10OH))2}]NO3 ([2]NO3). A brick-red suspension of [(η6-p-cymene)RuCl2]2 (104 mg, 0.170 mmol) and AgNO3 (58 mg, 0.34 mmol) in MeCN (3 mL) was stirred at room temperature for 1 h under protection from light. The resulting suspension (yellow-orange solution + colorless AgCl precipitate) was filtered over Celite and the solid washed with MeCN. Compound L2 (86 mg, 0.34 mmol) was added to the orange filtrate solution, and the mixture was stirred at reflux temperature for 3.5 h. Therefore, the red solution was cooled to room temperature, and volatiles were removed under vacuum. The residue was suspended in Et2O (20 mL) and then filtered. The resulting orange-brown solid was washed with Et2O and dried under vacuum (40 °C) over P2O5. Yield: 191 mg, 96%. On the other hand, a mixture of ruthenium compounds containing [2]NO3 (Chart 5) was obtained when the Chart 3. Structure of L3a a The numbering refers to carbon atoms. The numbering refers to carbon atoms. Glyoxal (40% w/w in H2O, 0.65 mL, 5.7 mmol) and AcOH (0.10 mL, 1.7 mmol) were added to a suspension of 4-aminophenol (1.002 g, 9.18 mmol) in iPrOH (14 mL). The resulting yellow suspension was stirred at 40 °C for 2.5 h under protection from light and then filtered. The resulting yellow solid was washed with iPrOH (2 × 2 mL) and Et2O, dried under vacuum (40 °C) over P2O5, and stored in the dark. Yield: 908 mg, 82%. Alternative conditions: MeOH, room temperature, 22 h, 63% yield; MeOH, reflux, 2 h, 83% yield; EtOH, room temperature, 22 h, 69% yield; iPrOH, room temperature, 22 h, 72% yield. Compound L3 is soluble in DMSO and MeOH, poorly soluble in acetone and MeCN, and insoluble in chlorinated solvents and H2O. Anal. Calcd for C14H12N2O2: C, 69.99; H, 5.03; N, 11.66. Found: C, 69.60; H, 4.98; N, 11.80. IR (solid state, cm−1): 3300−3000w-br (νO−H), 3019m, 2988m, 2954m, 2902m, 2812m, 2743m, 2684m, 2603m, 2541w, 2513w, 2476w, 1888w, 1607s (νCN), 1592m-sh, 1574s, 1504s, 1453s, 1382m, 1330w, 1301w, 1269s, 1237s, 1200s, 1172s, 1158s, 1116m, 1104m, 1008w, 952w, 928w, 830s, 813s-sh, 794m-sh, 776s, 715m-sh. UV−vis [MeOH, c = 3.0 × 10−4 M; λmax/nm (ε/M−1·cm−1)]: 242 (4.1 × 104), 296 (2.5 × 104), 380 (7.6 × 104). 1H NMR (CH3OD): δ 8.36 (s, 2H, C1−H), 7.26 (d, 3JHH = 8.7 Hz, 4H, C3−H), 6.81 (d, 3JHH = 8.7 Hz, 4H, C4−H). 13C{1H} NMR (CH3OD): δ 157.5 (C1), 124.3 (C3), 117.1 (C4). 1H NMR (DMSOd6): δ 9.79 (s-br, 2H, OH), 8.40 (s, 2H, C1−H), 7.32 (d, 3JHH = 8.6 Hz, 4H, C3−H), 6.82 (d, 3JHH = 8.6 Hz, 4H, C4−H). 13C{1H} NMR (DMSO-d6): δ 157.8 (C5), 156.4 (C1), 141.2 (C2), 123.4 (C3), 115.9 (C4). 1H NMR (CD3CN): δ 8.36 (s, 2H, C1−H), 7.29 (d, 3JHH = 8.4 Hz, 4H, C3−H), 7.20 (s, 2H, OH), 6.87 (d, 3JHH = 8.6 Hz, 4H, C4− H). [(η6-p-Cymene)RuCl{κ2N-(HCN(C6H11))2}]NO3 ([1]NO3). A suspension of [(η6-p-cymene)RuCl2]2 (201 mg, 0.328 mmol), AgNO3 (113 mg, 0.665 mmol), and L1 (147 mg, 0.667 mmol) in MeOH (6 mL) was stirred at room temperature for 5 h under protection from light. The resulting suspension (orange-red solution + colorless AgCl precipitate) was filtered over Celite. Volatiles were removed under vacuum from the filtrate solution, and the orange residue was suspended in Et2O (20 mL). The suspension was filtered, and the resulting crystalline orange solid was washed with Et2O and then dried under vacuum (40 °C). Yield: 337 mg, 93%. Compound [1]NO3 (Chart 4) is soluble in H2O, MeOH, acetone, and chlorinated solvents and insoluble in Et2O and hexane. Crystals suitable for X-ray diffraction were obtained from CH2Cl2 solutions of [1]NO3 layered with heptane or hexane and settled aside at −20 °C. Anal. Calcd for C24H38ClN3O3Ru: C, 52.12; H, 6.92; N, 7.60. Found: C, 52.04; H, Chart 5. Structure of [2]NO3a a The numbering refers to carbon atoms. reaction was carried out in MeOH at room temperature. Compound [2]NO3 is soluble in H2O, MeOH, and EtOH, less soluble in acetone, poorly soluble in CH2Cl2, and insoluble in Et2O. Anal. Calcd for C24H38ClN3O5Ru: C, 49.27; H, 6.55; N, 7.18. Found: C, 49.04; H, 6.41; N, 7.23. ESI-MS(+). Found: m/z 523.1667 ([M]+). Calcd for C24H38ClN2O2Ru+: 523.1665. IR (solid state, cm−1): 3380m-br (νO−H), 3054w, 2958m-sh, 2935m, 2905w, 2862m, 1648w-br, 1538w (νCN), 1504w, 1469m-sh, 1454m, 1377s, 1363s, 1321s-br (νNO3), 1305s, 1252m, 1229m, 1204m, 1161w, 1145w, 1121w, 1082s, 1062s, I DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry 125.4 (C10), 117.0 (C11), 108.8 (C5), 107.8 (C2), 90.1 (C4), 89.3 (C3), 32.5 (C6), 22.2 (C7), 19.0 (C1). [(η6-p-Cymene)RuCl{κ2N-(HCN(4-C6H4OCOCH3))2}]Cl ([4]Cl). In a 25 mL Schlenk tube, DBU (0.10 mL, 0.67 mmol) was added to a suspension of [3]NO3 (94 mg, 0.16 mmol) in CHCl3 (10 mL). The dark-green reaction mixture was heated under reflux for 1 h, and then CH3COCl (50 μL, 0.70 mmol) was introduced. The resulting dark purple-red mixture was heated under reflux for 14 h and then allowed to cool to room temperature. The mixture was extracted with H2O (3 × 20 mL), and then volatiles were removed under vacuum from the organic phase. The residue was dissolved in a small volume of Me2CO, and petroleum ether was added under stirring, causing precipitation of the title compound as a dark-brown solid. The suspension was filtered, and the solid was washed with Et2O and then dried under vacuum (40 °C). Yield: 71 mg, 68%. Compound [4]Cl (Chart 7) is soluble in 1040m-sh, 996w, 965m, 901w, 877m, 828m, 804w, 787w, 679w. UV− vis [MeOH, c = 9.9 × 10−4 M; λmax/nm (ε/M−1·cm−1)]: 277 (2.9 × 103), 372sh (1.7 × 103), 426 (2.8 × 103). Λm (MeOH, c = 9.9 × 10−4 M) = 108 S·cm−2·mol−1. 1H NMR (DMSO-d6): δ 8.38 (s, 2H, C8− H), 6.31 (d, 3JHH = 6.0 Hz, 2H, C4−H), 5.91 (d, 3JHH = 6.0 Hz, 1H, C3−H), 4.73 (d, 3JHH = 3.3 Hz, 2H, OH), 4.43 (t, 3JHH = 9.9 Hz, 2H, C9−H), 3.53−3.41 (m, 2H, C12−H), 2.69 (hept, 3JHH = 6.4 Hz, 1H, C6−H), 2.30 (d, J = 12.4 Hz, 2H, C10−H), 2.16 (s, 3H, C1−H), 2.10 (d, J = 12.3 Hz, 2H, C10′−H), 1.93 (d, J = 10.8 Hz, 2H, C11′−H), 1.89−1.77 (m, 4H, C11−H + C10′−H′), 1.61−1.38 (m, 4H, C11−H′ + C11−H′), 1.27 (q, J = 10.8 Hz, 2H, C10−H′), 1.05 (d, 3JHH = 6.6 Hz, 6H, C7−H). 1H NMR (CD3OD): δ 8.33 (s, 2H, C8−H), 6.18 (d, 3 JHH = 6.0 Hz, 2H, C4−H), 5.81 (d, 3JHH = 5.9 Hz, 2H, C3−H), 4.49 (t, 3JHH = 11.2 Hz, 2H, C9−H), 3.64 (t, 3JHH = 10.5 Hz, 2H, C12−H), 2.77 (hept, 3JHH = 6.7 Hz, 1H, C6−H), 2.52 (d, J = 12.9 Hz, 2H, C10−H), 2.32−2.27 (m, 2H, C10′−H), 2.27 (s, 3H, C1−H), 2.13 (d, J = 12.0 Hz, 2H, C11′−H), 2.04 (d, J = 13.4 Hz, 2H, C11−H), 1.91 (q, J = 11.4 Hz, 2H, C10′−H′), 1.62 (q, J = 10.1 Hz, 2H, C11′−H′), 1.54 (q, J = 10.1 Hz, 2H, C11−H′), 1.37 (q, J = 11.2 Hz, 2H, C10− H′), 1.16 (d, 3JHH = 6.7 Hz, 6H, C7−H). No change in the 1H NMR spectrum was observed after 5 days at room temperature. 13C{1H} NMR (CD3OD): δ 165.3 (C8), 109.2 (C5), 108.4 (C2), 89.5 (C4), 87.3 (C3), 75.7 (C9), 70.1 (C12), 34.9 (C11), 34.5 (C11′), 34.0 (C10), 33.1 (C6), 32.1 (C10′), 22.7 (C7), 19.4 (C1). [(η6-p-Cymene)RuCl{κ2N-(HCN(4-C6H4OH))2}]NO3 ([3]NO3). The first step of the synthesis was performed as described for [2]NO3, using [(η6-p-cymene)RuCl2]2 (460 mg, 0.751 mmol), AgNO3 (255 mg, 1.50 mmol), and MeOH (5 mL). Therefore, the suspension was filtered over Celite, and compound L3 (360 mg, 1.50 mmol) was added to the orange filtrate solution, causing immediate darkening of the mixture. The solution was stirred at room temperature for 2 h, then volatiles were removed under vacuum. The residue was suspended in Et2O and then filtered. The resulting dark-red-brown solid was washed with Et2O and dried under vacuum (40 °C) over P2O5. Yield: 846 mg, 98%. Compound [3]NO3 (Chart 6) is soluble in Chart 7. Structure of [4]Cla a DMSO, acetone, CHCl3, poorly soluble in MeOH, and insoluble in Et2O, hexane, and H2O. Anal. Calcd for C28H30ClN3O7Ru: C, 51.18; H, 4.60; N, 6.40. Found: C, 51.18; H, 4.66; N, 6.48. ESI-MS(+). Found: m/z 595.0941 ([M]+). Calcd for C28H30ClN2O4Ru+: m/z 595.0942. IR (solid state, cm−1): 3500w-br, 3060w, 2962w, 2933w, 2872w, 1890m-sh, 1860m, 1756s (νCO), 1622w, 1599w (νCN), 1494s, 1367s, 1269w, 1210s-sh, 1183s, 1161s, 1104m, 1041w, 1012s, 909m, 874w, 842m. IR (CH2Cl2, cm−1): 1874w, 1765m (νCO), 1622w-sh, 1602m (νCN), 1497s, 1371m, 1215s-sh, 1194s, 1164m, 1015m. UV−vis [CH2Cl2, c = 9.6 × 10−4 M; λmax/nm (ε/M−1·cm−1)]: 355 (7.6 × 103), 440 (4.5 × 103), 550−650br (1.8 × 103). Λm (c = 9.6 × 10−4 M) = 6.1 S·cm2·mol−1 (CH2Cl2), 60 S·cm2·mol−1 (MeOH). 1H NMR [CDCl3 or 1:1 (v/v) CDCl3/CD3OD)]: broad resonances. 1H NMR (CD3OD): δ 8.55 (s, 2H, C8−H), 7.85 (d, 3JHH = 8.6 Hz, 4H, C10−H), 7.40 (d, 3JHH = 8.6 Hz, 4H, C11−H), 5.58 (d, 3JHH = 6.2 Hz, 2H, C4−H), 5.48 (d, 3JHH = 7.0 Hz, 2H, C3−H), 2.43 (hept, 3JHH = 6.6 Hz, 1H, C6−H), 2.36 (s, 6H, C14−H), 2.27 (s, 3H, C1−H), 1.06 (d, 3JHH = 6.9 Hz, 6H, C7−H). Minor variations were observed in the 1 H NMR spectrum of the solution maintained at room temperature for 2 months. 13C{1H} NMR (CD3OD): δ 171.0 (C13), 166.7 (C8), 153.9 (C12), 151.3 (C9), 124.8 (C10), 124.3 (C11), 110.3 (C5), 108.9 (C2), 90.4 (C4), 89.4 (C3), 32.6 (C6), 22.3 (C7), 20.9 (C14), 19.1 (C1). 14N NMR (CD3OD; acq. time 14 h): no signal. [(η6-p-Cymene)RuCl{κ2N-(HCN(4-C6H4OCO-asp))2}]NO3 ([5]NO3). In a 25 mL Schlenk tube, [3]NO3 (110 mg, 0.192 mmol), DMAP (4 mg, 0.03 mmol), asp-CO2H (85 mg, 0.47 mmol), CH2Cl2 (7 mL), and EDCI·HCl (93 mg, 0.49 mmol) were introduced in this order, and the resulting dark-red suspension was stirred at room temperature. After 14 h, the reaction mixture was extracted with a H2O/NaNO3 0.1 M solution (4 × 10 mL) and volatiles were removed under vacuum from the organic phase. The residue was suspended in Et2O and filtered; the resulting dark-brown solid was washed with Et2O and dried under vacuum (40 °C). Yield: 158 mg, 92%. The title compound could not be alternatively obtained via acyl chloride-mediated esterification: compound [3]NO3 was completely unreactive toward asp-COCl/Et3N in a refluxing CHCl3 or THF solution. Compound [5]NO3 (Chart 8) is soluble in DMSO, acetone, and CHCl3, less soluble in MeOH, and insoluble in Et2O, hexane, and H2O. Anal. Calcd for C42H38ClN3O11Ru: C, 56.21; H, 4.27; N, 4.68. Found: C, 56.01; H, 4.13; N, 4.70. ESI-MS(+). Found: m/z 835.1375 ([M]+). Calcd for C42H38ClN2O8Ru+: m/z 835.1369. IR (solid state, cm−1): 3061w, 3036w, 2968w, 2934w, 2875w, 1762m-sh (νC13O), 1741s (νC20O), 1605m (νCN), 1580w, 1538w, 1494m, 1485m, 1452m, Chart 6. Structure of [3]NO3a a The numbering refers to carbon atoms. The numbering refers to carbon atoms. H2O, DMSO, MeOH, and acetone and insoluble in chlorinated solvents, Et2O, and hexane. Anal. Calcd for C24H26ClN3O5Ru: C, 50.31; H, 4.57; N, 7.33. Found: C, 50.12; H, 4.68; N, 7.20. ESI-MS(+). Found: m/z 511.0735 ([M]+). Calcd for C24H26ClN2O2Ru+: m/z 511.0730. Tm = 152−155 °C (dec). IR (solid state, cm−1): 3560− 3000m-br (νO−H), 3064m, 2965m, 2879w, 2813w, 2692w, 2593w-br, 1604m (νC=N), 1591m-sh, 1564m, 1504s, 1453m, 1370s, 1317s-br (νNO3), 1274s, 1227s, 1164s, 1107m, 1055w, 1035w, 1010w, 956w, 878w, 837s, 823m-sh, 805m-sh, 722w, 670w. UV−vis [MeOHc = 1.0 × 10−3 M; λmax/nm (ε/M−1·cm−1)]: 268 (1.3 × 104), 422 (1.5 × 104), 550−575br (3.4 × 103). Λm (MeOH, c = 1.0 × 10−3 M) = 113 S·cm2· mol−1. 1H NMR (DMSO-d6, 25 and 60 °C): broad resonances, δ 10.3 (br, 2H, OH), 8.47 (s-br, 2H, C8−H), 7.66 (s-br, 4H, C10−H), 6.98 (s-br, 4H, C11−H), 5.50 (s-br, 4H, C3−H + C4−H), 2.25* (s-br, C1−H), 0.95 (s-br, 6H, C7−H). Asterisks indicate peaks partially overlapped with the DMSO signal. 1H NMR (CD3OD): δ 8.36 (s, 2H, C8−H), 7.69 (d, 3JHH = 8.3 Hz, 4H, C10−H), 6.99 (d, 3JHH = 8.3 Hz, 4H, C11−H), 5.47 (d, 3JHH = 6.1 Hz, 2H, C4−H), 5.42 (d, 3JHH = 6.1 Hz, 2H, C3−H), 2.39 (hept, 3JHH = 6.8 Hz, 1H, C6−H), 2.30 (s, 3H, C1−H), 1.05 (d, 3JHH = 6.9 Hz, 6H, C7−H). No variations in the 1H NMR spectrum were observed after 17 days at room temperature. 13 C{1H} NMR (CD3OD): δ 163.2 (C8), 161.8 (C12), 146.2 (C9), J DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry 360 (1.0 × 104), 440 (3.8 × 103), 550−600br (1.4 × 103). Λm (c = 9.2 × 10−4 M) = 7.8 S·cm2·mol−1 (CH2Cl2) and 90 S·cm2·mol−1 (MeOH). 1H NMR (CD3OD): δ 8.52 (s, 2H, C8−H), 7.86 (d, 3JHH = 8.5 Hz, 4H, C10−H), 7.34 (d, 3JHH = 8.5 Hz, 4H, C11−H), 5.56 (d, 3 JHH = 6.2 Hz, 2H, C4−H), 5.46 (d, 3JHH = 6.2 Hz, 2H, C3−H), 2.71 (m, J = 14.1, 9.1, and 5.2 Hz, 2H, C14−H), 2.43 (hept, 3JHH = 6.8 Hz, 1H, C6−H), 2.24 (s, 3H, C1−H) 1.84−1.73 (m, 4H, C15−H), 1.68− 1.59 (m, 4H, C15−H′), 1.49 (sex, 3JHH = 7.2 Hz, 8H, C16−H), 1.05 (d, 3JHH = 6.9 Hz, 6H, C7−H), 1.02 (t, 3JHH = 7.3 Hz, 12H, C17−H). 13 C{1H} NMR (CD3OD): δ 176.2 (C13), 166.6 (C8), 153.8 (C12), 151.3 (C9), 124.9 (C10), 124.1 (C11), 110.3 (C5), 108.6 (C2), 90.2 (C4), 89.4 (C3), 46.5 (C14), 35.7 (C15), 32.5 (C6), 22.3 (C7), 21.7 (C16), 19.1 (C1), 14.4 (C17). 14N NMR (CD3OD): δ −2.9 (Δν1/2 = 19 Hz, NO3−). X-ray Crystallography. Crystal data and collection details for [1]NO3 are reported in Table 6. Data were recorded on a Bruker Chart 8. Structure of [5]NO3a a The numbering refers to carbon atoms. 1367m, 1338m (νNO3), 1287m, 1244s, 1185s, 1161s, 1125m, 1109m, 1072m, 1047s, 1036s, 1011s, 962w, 915m, 875m, 832w, 807m, 751m, 700m. UV−vis [CH2Cl2, c = 8.5 × 10−4 M; λmax/nm (ε/M−1·cm−1)]: 273sh (1.6 × 104), 357 (1.0 × 104), 442 (3.3 × 103), 550−600br (1.1 × 103). Λm (c = 8.5 × 10−4 M) = 8.4 S·cm2·mol−1 (CH2Cl2), 67 S·cm2· mol−1 (MeOH). 1H NMR (CDCl3): broad resonances. 1H NMR (CD3OD): δ 8.58 (s, 2H, C8−H), 8.26 (dd, 3JHH = 7.8 Hz, 4JHH = 1.3 Hz, 2H, C15−H), 7.92 (d, 3JHH = 8.7 Hz, 4H, C10−H), 7.76 (dt, 3JHH = 7.8 Hz, 4JHH = 1.4 Hz, 2H, C17−H), 7.50 (d, 3JHH = 8.9 Hz, 4H, C11−H), 7.50−7.46 (m, 2H, C16−H), 7.29 (d, 3JHH = 8.1 Hz, 2H, C18−H), 5.62 (d, 3JHH = 6.4 Hz, 2H, C4−H), 5.52 (d, 3JHH = 6.4 Hz, 2H, C3−H), 2.47 (hept, 3JHH = 6.8 Hz, 1H, C6−H), 2.31 (s, 6H, C21−H), 2.29 (s, 3H, C1−H), 1.09 (d, 3JHH = 6.9 Hz, 6H, C7−H). Minor variations were observed in the 1H NMR spectrum of the solution maintained at room temperature for 3 days. 13C{1H} NMR (CD3OD): δ 171.2 (C20), 166.8 (C8), 164.3 (C13), 153.7 (C12), 152.7 (C19), 151.6 (C9), 136.4 (C17), 133.1 (C15), 127.6 (C16), 125.3 (C18), 125.0 (C11), 124.4 (C10), 123.4 (C14), 110.3 (C5), 109.0 (C2), 90.4 (C4), 89.4 (C3), 32.6 (C6), 22.3 (C7), 21.0 (C21), 19.2 (C1). 14N NMR (CD3OD): δ −3.1 (Δν1/2 = 15 Hz, NO3−). 35Cl NMR (CD3OD, acq. time 30′): no signal. [(η6-p-Cymene)RuCl{κ2N-(HCN(4-C6H4OCO-vp))2}]NO3 ([6]NO3). The reaction was performed as described for [5]NO3, using [3]NO3 (97 mg, 0.17 mmol), DMAP (5 mg, 0.04 mmol), vp-CO2H (86 μL, 0.54 mmol), CH2Cl2 (7 mL), and EDCI·HCl (102 mg, 0.532 mmol). After 5 h, H2O (10 mL) was added to the dark-red suspension with vigorous stirring. The organic phase was then separated, and volatiles were removed under vacuum. The residue was dissolved in EtOAc/ Et2O (1:1, v/v) and extracted with a 0.1 M H2O/NaNO3 solution (3 × 10 mL). Volatiles were removed under vacuum from the organic phase, and the residue was dissolved in a small volume of Me2CO. The addition of petroleum ether under stirring caused precipitation of the title compound as a dark red-brown solid. The suspension was filtered; the solid was washed with petroleum ether and dried under vacuum (40 °C). Yield: 96 mg, 69%. Compound [6]NO3 (Chart 9) is soluble Table 6. Crystal Data and Measurement Details for [1]NO3 formula fw T, K λ, Å cryst syst space group a, Å b, Å c, Å β, deg cell volume, Å3 Z Dc, g·cm−3 μ, mm−1 F(000) cryst size, mm θ limits, deg reflns collected indep reflns data/restraints/param GOF on F2 R1 [I > 2σ(I)] wR2 (all data) largest diff peak/hole, e·Å−3 APEX II diffractometer equipped with a Photon 100 detector using Mo Kα radiation. Data were corrected for Lorentz polarization and absorption effects (empirical absorption correction SADABS).50 The structure was solved by direct methods and refined by full-matrix least squares based on all data using F2.51 Hydrogen atoms were fixed at calculated positions and refined by a riding model. All non-hydrogen atoms were refined with anisotropic displacement parameters. Solubility and Stability Studies. All measurements (pH, UV− vis, NMR, and conductivity) were performed at room temperature. The molar conductivity (Λm) and molar absorption coefficients (ε) were calculated with respect to the starting material. Molar percent values of the compounds in solution are based on 1H NMR spectroscopy and refer to identified compounds only (indicated as % NMR) or to Me2SO2 used as an internal standard (indicated as % NMR vs internal standard).32 NMR signals in braces indicate superimpositions with other species. The solubility (S/M, at 21 °C) was calculated on saturated D2O solutions by 1H NMR with respect to Me2SO2 (internal standard). Data are reported for each compound (Tables S2−S8 and Figures S19−S25); selected data are compiled in Table 2. General Procedure (D2O Solution). The compound ([1−3]NO3, 0.10 mmol) was suspended in a D2O solution (1.0 mL) containing Me2SO2 [c = 7.1 × 10−3 mol·L−1; δ 3.13 (s, 6H) in D2O] and stirred at Chart 9. Structure of [6]NO3a a C24H38ClN3O3Ru 553.09 100(2) 0.71073 monoclinic P21/c 11.6812(7) 11.5991(7) 18.3969(10) 100.709(2) 2449.2(2) 4 1.500 0.780 1152 0.15 × 0.13 × 0.09 1.774−25.050 28912 4327 [Rint = 0.1359] 4327/228/290 1.171 0.0850 0.1648 1.709/−2.360 The numbering refers to carbon atoms. in MeCN (with decomposition), DMSO, EtOAc, and CHCl3, poorly soluble in Et2O, and insoluble in hexane and H2O. Anal. Calcd for C40H54ClN3O7Ru: C, 58.21; H, 6.59; N, 5.09. Found: C, 58.20; H, 6.70; N, 5.11. ESI-MS(+). Found: m/z 763.2836 ([M − Cl]+). Calcd for C40H54ClN2O4Ru+: m/z 763.2810. IR (solid state, cm−1): 3061w, 2958m, 2933m, 2872m, 1752s (νCO), 1654w, 1622w, 1599w (νCN), 1537w, 1494s, 1464m, 1371m-sh, 1336s (νNO3), 1190s, 1161s, 1147msh, 1124s-sh, 1101s, 1068m-sh, 1051m-sh, 1014m, 974w, 923w, 873m, 827m. UV−vis [CH2Cl2, c = 9.2 × 10−4 M; λmax/nm (ε/M−1·cm−1)]: K DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry 21 °C for 24 h. An aliquot (0.50 mL) of the resulting saturated solution was transferred to an NMR tube and analyzed by 1H NMR spectroscopy. The solution was kept at 37 °C for 72 h and at 21 °C for at least 6 days. After each period, the solution was analyzed by 1H and 35 Cl NMR spectroscopy. Finally, NaCl (50 μL of a 1.0 M solution in D2O, cNaCl = 0.1 M) was added and the 1H spectrum repeated. Parallel experiments were carried out on dilute solutions (cRu = 1.0 × 10−3 M), which were kept at 37 °C for 72 h. The molar conductivity (Λm), pH, and UV−vis spectra were recorded immediately after dissolution and at the end of this period. See Figures S19−S21 and Tables S2−S4 for details. General Procedure (DMSO-d6/D2O Solution). The compound ([3−6]X, where X = Cl and NO3) was dissolved in a 9:1 (v/v) DMSO-d6/D2O solution (1.0 mL; [Ru] = 1.5 × 10−2 mol·L−1) containing Me2SO2 [c = 5.5 × 10−3 mol·L−1; δ 2.97 (s, 6H) in 9:1 (v/ v) DMSO-d6/D2O]. An aliquot of the resulting solution (0.60 mL) was transferred to a NMR tube, maintained at 37 °C for 72 h, and analyzed by 1H NMR spectroscopy as a function of time (35Cl NMR analysis was performed at the end of the experiment). The remaining solution was diluted up to 4.0 mL with 9:1 (v/v) DMSO/H2O (final [Ru] = 1.5 × 10−3 mol·L−1), maintained at 37 °C for 72 h and analyzed by conductivity and UV−vis spectroscopy as a function of time. All measurements (NMR, conductivity, and UV−vis) were performed upon a brief cooling to ambient temperature, and then Rucontaining solutions were heated again at 37 °C. See Figures S22−S25 and Tables S5−S8 for details. Speciation of [3]NO3 in H2O at Different pH Values: Acid Dissociation and Hydrolysis. All operations were carried out at room temperature. NaOH and HCl solutions in H2O were prepared from a 1.0 M Normex solution (Carlo Erba) and standardized by titration before use. UV−Vis Measurements. To a graduated 5 mL flask were added [3]NO3 (2.50 mL of a 1.75 × 10−3 M solution in H2O) and NaCl (0.50 mL of a 1.0 M solution in H2O), followed by the appropriate amount of NaOH (3.37 × 10−3 or 1.0 M) or HCl (1 × 10−2 or 1.0 M) and then H2O up to a constant volume (5.0 mL). The resulting red/ green solutions (cRu = 8.75 × 10−4 M; I = 0.1 M)52 were stirred for 10 min at room temperature, then the pH value was measured (pH = 1.57−12.2), and their UV−vis spectra were recorded. The solutions were maintained at room temperature for 20 h, and their UV−vis spectrum and pH measurement were repeated. NMR Measurements. Progressively increasing amounts of KOH (0−0.80 μL; 0.19 M solution in D2O) were added to a solution of [3]NO3 in D2O (4.0 mL; cRu = 4.4 × 10−3 M). After each addition, the pH* of the solution was measured, and an aliquot (0.50 mL) of the same was transferred to an NMR tube. The pD was calculated according to the equation pD = pH* + 0.4,53 where pH* is the reading of the H2O-calibrated pH meter (pD = 7.17−11.8). Within 30 min from the addition of KOH, 1H and 35Cl NMR spectra of each solution were recorded. Therefore, the solutions were maintained at room temperature for 62 h, and a new 1H NMR spectrum was registered. NMR and UV−Vis Characterization of [3]+ and 3B. Solutions of [3]NO3 in H2O underwent instantaneous and reversible UV−vis variations upon the addition of NaOH or HCl in the pH range 1−10. Furthermore, a single set of pH-dependent 1H resonances were observed in the 1H NMR spectra (except for the most basic solutions, see onward). These features are consistent with an acid−base equilibrium (acid, [3]+; base, 3B; see Scheme 4). Plots of UV−vis spectra for [3]NO3/H2O solutions at different pH values are given in Figures S27 and S28. 1H NMR resonances of [3+]/3B at different pH values are compiled in Table S9. Selected UV−vis and NMR data are reported below (the same atom numbering as [3]NO3 is used; see Chart 6). pH = 5.25: red solution, major species [3]+. UV−vis [λmax/nm (ε/ M−1·cm−1)]: 266 (8.3 × 103), 415 (1.3 × 104), 540−560 (2.6 × 103). pH = 9.27: green solution, major species 3B. UV−vis [λmax/nm (ε/ M−1·cm−1)]: 240sh (1.5 × 104), 283 (9.0 × 103), 364 (4.8 × 103), 468 (1.2 × 104), 610sh (1.3 × 104), 638 (1.4 × 104). pD = 11.8: dark-green solution. 1H NMR (D2O, 3B): δ 8.17 (s, 2H, C8−H), 7.59 (d, 3JHH = 9.0 Hz, 4H, C10−H), 6.70 (d, 3JHH = 8.7 Hz, 4H, C11−H), 5.59 (d, 3 JHH = 6.9 Hz, 2H, C4−H), 5.51 (d, 3JHH = 6.2 Hz, 2H, C3−H), 2.33 (s, 3H, C1−H), 0.99 (d, 3JHH = 6.8 Hz, 6H, C7−H). Determination of pKa.54 UV−vis spectra of freshly prepared [3]NO3/H2O solutions with pH in the range 5.25−10.80 displayed an isosbestic point (λ = 437 nm; Figure S27) and were used for the determination of pKa of [3]+, according to a previously published method.55 Briefly, values of y = log[(A − Aacid)/(Abasic − A)] were calculated, where A is the absorbance of the selected solution at a given wavelength and Aacid and Abasic represent the absorbance at the lowest (5.25) and highest (10.80) pH values at the same wavelength. Leastsquares linear regression of (pH; y) data gave an equation of the type y = apH + b; therefore, pKa could be calculated as pKa = −b/a. The procedure was repeated at four different wavelengths (two before and two after the isosbestic point), and the results were averaged, affording pKa = 7.7 ± 0.1. pH-Dependent Behavior of [3]NO3 and the Formation of [(η6-pCymene)Ru(OH){κ2N,N′-(HCN)2(4-C6H4OH)(4-C6H4O)}] (3BW) in Basic Solutions. The stability of [3]NO3/H2O solutions at room temperature for 20 or 62 h at different pH/pD values was evaluated by UV− vis and 1H NMR spectroscopy. Variations in the UV−vis spectra as well as the appearance of a second set of 1H signals were attributed to the formation of 3BW from 3B, upon Ru−Cl hydrolysis (Scheme 4). Data and observations are compiled in Tables S10 and S11. The amount of 3BW in solution (as percent molar ratio with respect to [3]+ + 3B) as a function of time was calculated from the 1H NMR spectrum. The NMR data for 3BW are given below (pD = 11.8; the same atom numbering as [3]NO3 is used; see Chart 6). 1H NMR (D2O): δ 8.22 (s, 2H, C8−H), 7.54 (d, 3JHH = 8.9 Hz, 4H, C10−H), 6.71 (d, 3JHH = 8.7 Hz, 4H, C11−H), 5.47 (d, 3JHH = 6.1 Hz, 2H, C4−H), 5.38 (d, 3 JHH = 6.0 Hz, 2H, C3−H), 2.32 (s, 3H, C1−H), 0.90 (d, 3JHH = 6.8 Hz, 6H, C7−H). 35Cl NMR (D2O, acq. time 10 min): δ 0.18 (Δν1/2 = 9 Hz, Cl−). Determination of Partition Coefficients (log Pow). Partition coefficients (Pow; IUPAC, KD partition constant56), defined as Pow = corg/caq, where corg and caq are the molar concentrations of the selected compound in the organic and aqueous phase, respectively, were determined by the shake-flask method57 and UV−vis measurements. Values of log Pow for compounds [1−5]NO3 and [6]Cl are compiled in Table 5. All of the operations were carried out at 21 ± 1 °C. Deionized H2O and 1-octanol were mixed and vigorously stirred for 24 h at room temperature to allow saturation of both phases, then separated by centrifugation, and used for the following experiments. 1Octanol-saturated PB solution (Na2HPO4/KH2PO4, ∑cPO4 = 50 mM, pH = 7.3) was prepared analogously. A solution of the selected ruthenium compound ([1−3]NO3) in the aqueous phase (V = 20 mL) was prepared, and its UV−vis spectrum was recorded. An aliquot of the solution (Vaq = 3.0 mL) was then transferred into a test tube, and the organic phase (Vorg = 3.0 mL) was added. The mixture was vigorously stirred for 2 h, and the resulting emulsion was centrifuged (2000 rpm, 15 min) to separate the phases. Hence, the UV−vis spectrum of the aqueous phase was recorded. The partition coefficient was then calculated following the method described in the Supporting Information. An analogous procedure was followed for compounds [4] Cl and [5−6]NO3, which were initially dissolved in the organic phase (V = 20 mL). UV−vis measurements were carried out using 1 cm quartz cuvettes. The wavelength of the maximum absorption of each compound was used for UV−vis quantification (λmax = 420 nm for [1− 3]NO3 and 360 nm for [4]Cl and [5−6]NO3). Solutions of the ruthenium compound in the aqueous or organic phase ([Ru] ≈ 5 × 10−4 M for [1,2]NO3; [Ru] ≈ 1.5 × 10−4 M for [3−6]X, where X = Cl and NO3) were prepared so as to give absorbance values of around 1.2−1.5 at λmax. Computational Studies. The electronic structures of the compounds were optimized using the range-separated ωB97X DFT functional58 in combination with Ahlrichs’ split-valence-polarized basis set, with an effective core potential on the ruthenium center.59 The CPCM implicit solvation model was added to ωB97X calculations, considering H2O as a continuous medium.60 The stationary points were characterized by IR simulations (harmonic approximation), from L DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry The cytotoxicity of the compounds was determined using 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.67 Cells were seeded in flat-bottomed 96-well plates as a suspension in a medium containing 10% heat-inactivated FBS and a 1% penicillin/streptomycin solution (100 μL and approximately 4300 cells·well−1) and incubated for 24 h. Stock solutions of compounds were prepared in DMSO, or Milli-Q water in the case of [2]NO3 and [3]NO3, and were rapidly diluted in the medium. The solutions were sequentially diluted (final DMSO concentration of 0.5%) to give a compound concentration range (0−500 μM). Cisplatin was included as a positive control (0−100 μM). The compounds were added to the preincubated 96-well plates in 100 μL aliquots, and the plates were incubated for 72 h. MTT (20 μL, 5 mg/mL Dulbecco’s phosphatebuffered saline) was added to the cells, and the plates were incubated for a further 4 h. The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO (100 μL·well−1). The absorbance of the resulting solutions, directly proportional to the number of surviving cells, was quantified at 590 nm using a SpectroMax M5e multimode microplate reader (using Sof tMax Pro software, version 6.2.2). The percentage of surviving cells was calculated from the absorbance of wells corresponding to the untreated control cells. The reported IC50 values (Table 1) are based on the means from two independent experiments, each comprising four testings per concentration level. which zero-point vibrational energies and thermal corrections (T = 25 °C) were obtained.61 The software used was Gaussian 09.62 Electrochemistry. CV measurements were performed at 24 ± 1 °C with a PalmSens4 instrument interfaced to a computer employing PSTrace5 electrochemical software. All potentials are reported versus normal hydrogen electrode (NHE). Current sign convention adopted: negative currents/cathodic process; positive currents/anodic process. Peak potentials for compounds [1−3]NO3 and L1 are compiled in Table 3, and cyclic voltammograms are given in Figures S30−S33. Experiments in Aqueous Media. PB (Na2HPO4/KH2PO4, ∑cPO4 = 50 mM, pH = 7.3) and AB (NaOAc/AcOH, ∑cAcO = 0.21 M, pH = 4.5) solutions were prepared in ultrapure H2O and used as supporting electrolytes. The three-electrode home-built cell was equipped with a platinum sheet counter electrode, a Teflon-encapsulated GC working electrode (BASi, diameter 3 mm), and a leak-free 3.4 M Ag/AgCl/KCl reference electrode (eDAQ). The supporting electrolyte (5.0 mL) was introduced into the cell and deareated by argon bubbling for some minutes. The working electrode was cycled several times between the cathodic and anodic limits (−1.15/+1.55 and −1.05/+1.71 V for PB and AB solutions, respectively) until there was no change in the charging current. The analyte was then introduced (c ≈ 7 × 10−4 M), and voltammograms were recorded (scan rate: 0.1 V·s−1). NaCl (30 mg, 0.10 M) was then added to the solution, and the voltammograms were repeated. The reference electrode was calibrated against a HydroFlex hydrogen reference electrode (eDAQ) placed in a 1.00 M HCl solution (thus acting as a NHE).63 Prior to measurements, the GC working electrode was polished by the following procedure:64 manual rubbing with a 0.3 μM Al2O3 slurry in H2O (eDAQ) for 2 min, then sonication in ultrapure H2O for 10 min, manual rubbing with a 0.05 μM Al2O3 slurry in H2O (eDAQ) for 2 min, and then sonication in ultrapure H2O for 10 min. The three-electrode cell was routinely checked by measuring E1/2 and ΔE of the Fe(CN)63−/Fe(CN)64− couple in the PB solution.65 Experiments in Dichloromethane. HPLC-grade dichloromethane (Sigma-Aldrich) was stored under argon over 3 Å molecular sieves. [nBu4N][PF6] (Fluka, electrochemical grade) and Cp2Fe (Fluka) were used without further purification. CV measurements were carried out under argon using 0.2 M [nBu4N][PF6] in CH2Cl2 as the supporting electrolyte. The working and counter electrodes consisted of a platinum disk and a platinum gauze, respectively. A platinum quasireference electrode was employed as a reference. The three-electrode home-built cell was predried by heating under vacuum and filled with argon. The Schlenk-type construction of the cell maintained anhydrous and anaerobic conditions. The solution of supporting electrolyte was introduced into the cell, and the working electrode was cycled several times between the cathodic and anodic limits (−2.88/ +1.71 V vs NHE, respectively) until there was no change in the charging current. The analyte was then introduced (c ≈ 7 × 10−4 M), and the voltammograms were recorded (scan rate: 0.1 V·s−1); then a small amount of ferrocene was added, and the voltammograms were repeated. The potentials were determined by placing E1/2 = +0.39 V versus saturated calomel electrode (SCE) for the Cp2Fe+/Cp2Fe couple (as experimentally determined for our home-built cell)66 and then referenced to NHE (ESCE = +0.241 V vs NHE).42 Cell Culture and Cytotoxicity Studies. Human ovarian carcinoma (A2780 and A2780cisR) cell lines were obtained from the European Collection of Cell Cultures (ECACC, U.K.). The nontumoral human embryonic kidney (HEK-293) cell line was obtained from ATCC (Sigma, Switzerland). RPMI-1640 GlutaMAX and DMEM GlutaMAX media were obtained from Life Technologies (Switzerland), fetal bovine serum (FBS) was obtained from Sigma, a penicillin/streptomycin solution was obtained from Life Technologies, and cisplatin was obtained from TCI. The cells were routinely cultured in RPMI-1640 GlutaMAX (A2780 and A2780cisR) and DMEM GlutaMAX (HEK-293) media containing 10% heat-inactivated FBS and a 1% penicillin/streptomycin solution at 37 °C and CO2 (5%). The A2780cisR cell line were routinely treated with cisplatin (2 μM) in the medium. ■ ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00882. Figures S1−S33 and Tables S1−S12, which include IR, NMR, and UV−vis spectra of compounds, solubility/ stability studies in H2O, chloride/solvent exchange experiments, cyclic voltammograms, and other information (PDF) Cartesian coordinates of the DFT-optimized structures (XYZ) Accession Codes CCDC 1816209 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ AUTHOR INFORMATION Corresponding Author *E-mail: fabio.marchetti1974@unipi.it. Webpage: https:// people.unipi.it/fabio_marchetti1974/. ORCID Stefano Zacchini: 0000-0003-0739-0518 Guido Pampaloni: 0000-0002-6375-4411 Paul J. Dyson: 0000-0003-3117-3249 Fabio Marchetti: 0000-0002-3683-8708 Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS We thank the University of Pisa (PRA 2017: “Composti di metalli di transizione come possibili agenti antitumorali”) and the Swiss National Science Foundation for financial support. M DOI: 10.1021/acs.inorgchem.8b00882 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry ■ Conformational control of anticancer activity: the application of arenelinked dinuclear ruthenium(II) organometallics. Chem. Sci. 2014, 5, 2536−2545. (d) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.; Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H. F.; Ang, W. H.; Dröge, P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. 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