Introducing the 4-Phenyl-1,2,3-Triazole Moiety as a Versatile Scaffold for the Development of Cytotoxic Ruthenium(II) and Osmium(II) Arene Cyclometalates.

Article pubs.acs.org/IC Introducing the 4‑Phenyl-1,2,3-Triazole Moiety as a Versatile Scaﬀold for the Development of Cytotoxic Ruthenium(II) and Osmium(II) Arene Cyclometalates Christoph A. Riedl,†,‡ Lea S. Flocke,† Michaela Hejl,† Alexander Roller,† Matthias H. M. Klose,†,‡ Michael A. Jakupec,†,‡ Wolfgang Kandioller,*,†,‡ and Bernhard K. Keppler†,‡ † Institute of Inorganic Chemistry, Faculty of Chemistry, and ‡Research Platform “Translational Cancer Therapy Research”, University of Vienna, Waehringer Strasse 42, 1090 Vienna, Austria S Supporting Information * ABSTRACT: Herein we report the synthesis, anticancer potency in vitro, biomolecule interaction, and preliminary mode of action studies of a series of cyclometalated 1,2,3triazole-derived ruthenium(II) (2a−e) and osmium(II) (3a− e) organometallics of the general form [(η6-p-cym)RuCl(κ2C^N-L)] with varying substituents in postion 1 of the 1,2,3triazole moiety. These cyclometalates were characterized by standard analytical methods and their structures unambiguously assigned by single crystal X-ray crystallography. The anticancer activity of these novel compounds was tested in the human tumor cell lines A549 (non-small cell lung cancer), SW480 (colon adenocarcinoma), and CH1/PA-1 (ovarian teratocarcinoma), and preliminary structure−activity relationships were derived from the obtained data sets. Various representatives exhibit promising antineoplastic eﬀects with IC50 values down to the low micromolar range. The compounds readily formed stable DMSO adducts after aquation in DMSO-containing solution, but employing DMSO as solubilizer in cytotoxicity assays had no pronounced eﬀect on the cytotoxicity, compared to analogous experiments with DMF for most compounds. We isolated and characterized selected DMSO adducts as triﬂate salts and found that they show activities in the same range as the parent chlorido metalacycles in MTT assays with the use of DMSO. Osmium(II) cyclometalates exhibited higher antiproliferative activities than their ruthenium(II) counterparts. The IC50 values within each metal series decreased with increasing lipophilicity, which was attributed to higher cellular accumulation. Investigations on their mode of action revealed that the prepared organometallics were unable to inhibit topoisomerase IIα. Still, the most cytotoxic representatives 2b and 3b showed pronounced eﬀects on cell cycle distribution. ■ general form [Ru(η6-arene)(en)X]+ (X = halide) developed by Sadler and co-workers4 and RAPTA compounds of the type [Ru(η6-arene)(PTA)X2] (PTA = 1,3,5-triaza-7-phosphaadamantane) established by the Dyson group.5 Their investigations aﬀorded the two promising anticancer agents RM175 and RAPTA-C, which are currently at an advanced preclinical stage (Figure 1). Additionally, coordination of biologically active ligand systems to exploit synergistic eﬀects has been a promising approach in recent years.6 Both the scope of employed ligand systems, such as paullones,7 ﬂavonoids,8 and naphtoquinones,9 as well as the high antiproliferative activity of the derived complexes are impressive and continue to stimulate further research. Most ruthenium(II) arene compounds feature a halido leaving group and a bidentate N,N-, N,O-, O,O-, or S,Ochelating ligand. Replacement of the halido leaving group by a INTRODUCTION Over the last decades, ruthenium-based metallodrugs have emerged as promising therapeutic agents in anticancer therapy. The ﬁrst-in-class ruthenium(III) complex indazolium trans[tetrachlorido-bis(1H-indazole)ruthenate(III)] (KP1019) has shown preclinical anticancer activity and a mild toxicity proﬁle in a clinical phase I trial while eliciting disease stabilizations.1 NKP-1339 or IT-139 (Figure 1), the sodium analog of KP1019, oﬀers superior aqueous solubility and has successfully ﬁnished a clinical phase I trial with promising activity, especially in neuroendocrine tumors and non-small cell lung cancer.2 The activation of Ru(III) agents by reduction to Ru(II) in the hypoxic tumor tissue is assumed to be essential in their mechanism of action.3 Ruthenium(II) arene compounds oﬀer a versatile framework for the development of novel anticancer agents, whose physicochemical, biological, and pharmacological properties can be readily modiﬁed by variation of the ligand sphere. Promising ruthenium(II) arene-based compound classes include 1,2-diaminoethane (en) complexes of the © XXXX American Chemical Society Received: October 5, 2016 A DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry In order to expand the library of cyclometalated ruthenium(II) and osmium(II) arene compounds, we turned to the highly versatile, robust, and extensively studied 1,2,3-triazole moiety. The discovery of the copper-catalyzed azide−alkyne cycloaddition (CuAAC) of an azide dipole and an alkyne dipolarophile for the regioselective synthesis of 1,4-disubstituted 1,2,3-triazoles independently by both Sharpless26 and Meldal27 in 2002 has led to a phenomenal rise in triazolerelated research. The reaction is outstanding due to mild reaction conditions, high yield, facile workup, wide scope, and insensitivity to functional groups.28 Convenient for medicinal applications, 1,2,3-triazoles possess high stability to metabolic degradation and hydrogen bonding capabilities to facilitate interaction with biological targets29 while providing high variability by introduction of diﬀerent substituents at positions 1 and 4. Through the straightforward variation of alkyne and azide substrates, the CuAAC reaction provides a robust pathway for the design of extensive ligand libraries and synthesis of diverse triazole-based coordination compounds. Complexation of 1,4-disubstituted 1,2,3-triazoles to metal ions can occur at the N2- and N3-positions, of which the N3 atom possesses a higher Lewis basicity.30 In contrast, alkylation at position N3 and subsequent deprotonation of the acidic C5− H proton allows monodentate coordination of the triazole ring as an abnormal mesoionic N-heterocyclic carbene (NHC).31−33 Besides monofunctional coordination compounds, the facile introduction of donor functionalities through modiﬁed azide and alkyne CuAAC substrates has encouraged the development of bi- and multidentate triazole ligand systems. Nitrogen pendant donor functions such as 2-pyridyl residues34 at position 4 or 2-picolyl substituents35 at position 1 provide N,N-chelating moieties that readily form stable 5- or 6membered chelate rings, respectively. Especially pyridylsubsitituted triazoles (pytz), as well as the symmetrical 4,4′bitriazolyl ligands (btz), have attracted considerable attention as bipyridine analogues for the design of photoactive complexes of ruthenium,36−38 rhenium,39 and iridium.36,40 Similarly, cyclometalation of the aryl-1,2,3-triazole scaﬀold has also produced conveniently tunable photoactive complexes of ruthenium(II)41 and iridium(III).42 Furthermore, complexes featuring CNHC,Nor CNHC,CPh-coordination motifs have been explored for their catalytic activities.43−46 In a recent investigation, organosulfuror selenium-donor side chains allowed the synthesis of S,N- and Se,N-coordinated ruthenium(II) and palladium(II) complexes.47 Figure 1. Promising ruthenium-based anticancer agents IT-139 (left), RAPTA-C (right, top), and RM175 (right, bottom). water molecule is thought to be essential for the mechanism of action,10 which is referred to as the activation-by-aquation hypothesis. However, poor stability of the bidentate coordination motif oftentimes hampers application of the respective organometallics as anticancer therapeutics,11−13 which emphasizes the need to explore other coordination modes with altered properties. Over the last years, especially ruthenium(II) arene complexes bearing C,N-coordinating ligand systems have attracted considerable attention, and promising anticancer activities of cyclometalated 2-aryldiazole,14 2-phenylindole,15 2-phenylpyridine,16 and benzimidazole17,18 scaﬀolds have been reported. Ruthena(II)cycles may be prepared by transmetalation from toxic organomercurated precursors, or more conveniently by carboxylate-assisted C−H activation.19,20 While numerous examples of cytotoxic cyclometalated ruthenium(II) arene compounds can be found in the literature, osma(II)cycles seem to have been largely neglected. Ryabov and co-workers have reported and characterized osma(II)cycles of the form [Os(p-cym)(2-PhPyC∧N)L] (L = Cl, MeCN; 2-PhPy = 2phenylpyridine) as intermediates in the synthesis of [Os(II)(chel)3] metalacycles as electron carriers for active sites of oxidoreductases,21−23 and N-pyridyl 1,2,3-triazolylidene ligandbearing Os(II) metalacycles were explored for their applicability in transfer hydrogenation catalysis.24 However, the biological properties and particularly the cytotoxicity of osma(II)cycles of the form [Os(arene)LC∧NCl] remain nearly unexplored and were only touched upon by Pfeﬀer and co-workers, who reported the promising cytotoxicity of [Os(p-cym)(2PhPyC∧N)Cl] in the low micromolar range.25 Scheme 1. Synthesis of 1-Substituted 4-Phenyltriazole Ligands 1a−e, and Ruthenium(II) and Osmium(II) Arene Metalacycles 2a−e and 3a−e B DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry to form biaryl derivatives.52 Subsequently, the transitional acetate complex [Ru(p-cym)(trzC∧N)(OAc)] undergoes a ligand exchange to the more favorable chlorido complex,19 which precipitates from the reaction solution. Acceptable reaction times are achieved only with high concentrations of triazole ligands and metal dimer in the reaction solution. However, neither increased reaction temperatures, microwave irradiation, higher acetate catalyst, nor additional acetic acid cocatalyst loadings could accelerate the reaction rate. The precipitated product was collected by ﬁltration, washed with methanol to remove impurities, then redissolved in dichloromethane and ﬁltered to remove inorganic salts. Evaporation to dryness aﬀorded the desired metalacyles in elemental analysis purity and moderate to good yields (52−81%). Recently, a similar procedure was published by the Dixneuf group, which has to rely on chromatographic puriﬁcation.53 Characterization. The isolated compounds were characterized by standard analytical methods such as 1H, 13C and 2D NMR, high-resolution ESI-mass spectrometry, X-ray crystallography, and elemental analysis. Upon metal coordination, the rotational symmetry of the 4phenyl ring is broken, which aﬀords the characteristic coordination motif of two doublets and two doubletic doublets in 1H NMR spectra, which were utilized to conﬁrm the conversion. The aryl proton ortho to the metalated carbon is signiﬁcantly shifted downﬁeld and found around 8.15 ppm (RuII) and 8.01 ppm (OsII) due to the electron withdrawing eﬀect of metal coordination. Additionally, coordination of the triazole ligand hinders inversion at the metal center in aprotic CDCl3. As a consequence, the arene protons of p-cymene can be observed as four non-equivalent doublets, while the diastereotopic methylene proton of the triazole substituents in position 1 are split into two distinct doublets with a germinal coupling constant between 15 and 18 Hz. The high-resolution ESI mass spectra of the ligands 1a−e are dominated by the monomeric and dimeric sodium adducts [M +Na]+ and [2M+Na]+, while the proton adducts [M+H]+ are observed as the least intense peak. The metal complexes 2a−e and 3a−e are found both as sodium adduct [M+Na]+, as well as chlorido abstracted species [M−Cl]+. Additionally, the osmium(II) complexes are observed as dimeric sodium adducts [2M+Na]+.The intensity of these peaks varies greatly between compounds and is seemingly uncorrelated to their structures. This can be explained by a varying degree of chlorido leaving group hydrolysis due to diﬀerent times between sample preparation and measurement in the chosen solvent system (MeCN:MeOH = 1:1, +1% H2O). The structures of the synthesized metalacycles were unambiguously conﬁrmed by X-ray crystallography. Single crystals of 2a−e and 3a−e suitable for X-ray diﬀraction analysis were obtained by slow diﬀusion of n-hexane or diethyl ether into dichloromethane or chloroform complex solutions (Figure S5−S14). For detailed crystal data, data collection parameters, structure reﬁnement details, and CCDC codes, please refer to the Supporting Information. Selected bond lengths, bond angles, and torsion angles are listed in Table S3−S6. These half sandwich complexes adopt the pseudotetrahedral “piano-stool” conﬁguration, with the π-bonded p-cymene ligand acting as “seat”, while the cyclometalated triazole ligand and chlorido leaving group formally act as “legs” (Table S2, Figure S1). The benzyl (2a, 2b, 3b) and ester (2e, 3e) derivatives are representatives of the triclinic space group P1̅, while complexes Intrigued by the possibilities for the implementation of bioactive functional groups and straightforward synthetic derivatization to explore structure−activity relationships, we aim to establish the cyclometalation of 1-substituted 4-phenyl 1,2,3-triazoles as a versatile motif for the development of novel ruthenium(II) and osmium(II) arene metallodrugs. In this work, we report the straightforward synthesis, characterization, and preliminary biological evaluation of a model series comprised of ruthena(II) and osma(II)cycles. The impact of the substituents at position 1 of the triazole ring, the diﬀerence between ruthenium(II) and osmium(II) metal centers, and the choice of solubilizer for biological evaluations on the behavior in aqueous solution, interaction with biomolecules, antiproliferative activity, cellular accumulation, and cell cycle eﬀects will be discussed. ■ RESULTS AND DISCUSSION Synthesis. A series of ﬁve diﬀerent 1-substituted 4-phenyl 1,2,3-triazoles (1a−e) featuring benzylic, aliphatic, or methyl ester residues R was synthesized via the CuAAC reaction. The required organic azides were prepared in situ by reaction of the respective organohalides with sodium azide in DMSO.48 In order to avoid isolation of potentially explosive short-chained azides,49 a one-pot CuAAC protocol was employed. Herein, the cycloaddition of organic azides in a mixture of water and DMSO (1:1) with phenyl acetylene is catalyzed by copper(I) generated in situ from copper(II) sulfate and sodium ascorbate.50 After a reaction time of up to 24 h, the desired products 1a−e were precipitated by addition of water and obtained in high purity and good to excellent (53−93%) yields (Scheme 1). Subsequently, a reliable cyclometalation procedure based on previous research was established. Kinetic investigations on the arylation of functional arenes by Jutand and coworkers19,20 led to the formulation of a plausible mechanism for the carboxylate-assisted C−H activation. First, the weakly bound monocoordinated acetate in the 18e− complex [Ru(p-cym) (OAc)2] is cleaved oﬀ, yielding a coordinatively unsaturated species, which enables coordination of the heterocyclic nitrogen. Subsequent formation of the C,Ncoordinated metalacycle can be explained by deprotonation of the ortho C−H bond by uncoordinated acetate via an SE3 mechanism.51 The formed acetic acid accelerates the formation of the metalacycle by favoring the dissociation of acetate from [Ru(p-cym) (OAc)2]; therefore, the reaction proceeds through an intermolecular deprotonation favored by carboxylate via an autocatalytic process. In their eﬀorts to isolate the intermediate [Ru(p-cym)(2-PhPyC∧N)(OAc)] by reaction of [Ru(p-cym)(OAc)2] with 2-phenylpyridine, Jutand and co-workers instead observed conversion to the chlorido complex. An authentic sample of [Ru(p-cym)Cl(2-PhPyC∧N)] was obtained by reaction of [Ru(p-cym)Cl2]2 with 2-PhPy in the presence of KOAc in acetonitrile at room temperature.19 Based on this research, we reﬁned the complexation procedure for 1,2,3-triazole ligand systems. The target compounds were conveniently obtained by equimolar reaction of the respective ligand with the dimeric ruthenium(II) or osmium(II) arene metal precursor in the presence of sodium acetate (2.0 equiv) in anhydrous methanol. During the comparatively long reaction time of up to 48 h at room temperature, the product precipitates as a microcrystalline yellow solid. Under the chosen reaction conditions, no suitable substrate or sacriﬁcial oxidant is present to exploit the activated metal−C(sp2) bond, e.g. for direct arylation or homocoupling C DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry The metal−chlorido bond lengths were found to be between 2.4165(17) and 2.4320(14) Å, a typical range for organometallic ruthenium55,56 and osmium57,58 complexes. The metal−N and metal−C bonds range from 2.069(6) to 2.088(4) Å, and 2.057(6) to 2.096(5) Å, respectively. Slightly shorter metal−arene distances were found in all osmium complexes, except 3e, compared to their ruthenium counterparts. Metal coordination distorts the phenyl ring, thereby signiﬁcantly shortening bonds emanating from the metalated carbon, and elongating the remaining C−C bonds of the phenyl ring, which can be attributed to the electron withdrawing eﬀect of coordination. Additionally, the phenyl ring is bent toward the triazole N2, as can be seen in the decreased CPh−Ctrz‑4−Ntrz‑3 bond angle from 115.11(10) to 113.8(7)° in the complexes compared to the free ligand with 122.8° (Table S5). Stability in aqueous solution. All biological investigations were performed with the help of a solubilizing agent such as DMSO or DMF in order to compensate the limited aqueous solubilities of the developed compounds. UV−vis spectra of the complexes (20 μM, 1% solubilizer/67 mM phosphate buﬀer pH 7.4) were recorded over 24 h to determine the stability of the synthesized metalacycles under pseudophysiological conditions (Figure S15−S24). In 1% DMF, the initially clear solutions of ruthenium(II) and osmium(II) complexes undergo slight precipitation over time, which results in an overall decrease of absorption without the emergence of new peaks, shift of peak maxima, or occurrence of isosbestic points. In contrast, most compound spectra in 1% DMSO undergo a small shift of the peak maxima after the ﬁrst measurement, yet afterward, the absorption over the whole wavelength range remains constant without the microprecipitation observed in DMF. In order to rule out interaction of the employed phosphate buﬀer with the organometallics, 1H and 31P NMR spectra of 2e and 3e in 10% d6-DMSO or d7-DMF and phosphate-buﬀered D2O were recorded over 24 h. No spectral change or emergence of new peaks in 31P spectra compared to reference spectra without cyclometalates was observed over the experiment time (Figure S25−S28). Additionally, the respective 1H spectra showed the same changes over time as unbuﬀered samples, suggesting that interaction of phosphate buﬀer with the metal compounds does not cause the change in UV−vis spectra. Recently, Gasser and co-workers presented a systematic study of the in-solution displacement of κN-monodentate bound ligands by DMSO in stock solutions of RuII organometallic complexes of the form [Ru(η6-arene)(X)(Y)(Z)].59 It was proposed that coordination and dissociation of the monodentate organic ligand occurs as an equilibrium reaction and is mainly inﬂuenced by the properties of the organic moiety. Complexes featuring ligands prone to dissociation in DMSO stock solutions self-evidently exhibit altered properties in biological screening when tested from DMSO stock solutions. Consequently, DMSO-mediated complex dissociation is essential to be investigated prior to the biological experiments of the bidentate triazole-derived metalacycles presented in this work. Due to the suspected greater stability of bidentate coordination motifs, we propose exchange of the chlorido ligand with DMSO to be the cause of the spectral change in UV−vis investigations of 2a−e and 3a−e. To further investigate this behavior, 5 μM complex solutions in 1% DMSO/400 μM ammonium acetate solution (5 μM, pH 7.4) were incubated at 37 °C; samples were taken after 0, 1, 3, 6, and 24 h and analyzed via ESI-MS after dilution with featuring either propyl (2c, 3c) or butyl (2d, 3d) side chains crystallize in the tetragonal space group I41/a. In contrast, the benzyl derivative 3a crystallizes in the monoclinic P21/n space group, supposedly due to incorporation of a dichloromethane solvent molecule into the crystal lattice, which also leads to increased ellipsoids through thermal oscillation. The crystal lattices of 2b−e and 3a−e are populated with dimeric building blocks containing one (R) and one (S)conﬁgured isomer of these chiral-at-metal metalacycles. As a result of cyclometalation, the phenyl and triazole rings are locked in plane, as can be seen from the low torsion angles between 0.6(6)° to 4.4(8)° along the triazole−phenyl bond. The coplanar triazole and phenyl rings of the dimeric pairs are arranged in parallel layers, with the chlorido ligands pointing toward the interaction partner, while the bulky isopropyl group of the arene ring residing at position 1 is rotated toward the backside of the plane (Figure 2) due to hydrophobic interactions. Figure 2. Interaction pair of 2e as an example of dimeric subunits. The driving force for the parallel assembly is likely π−π stacking between the 4-phenyl triazole rings. The observed centroid−centroid distances range from 3.6 to 4.0 Å with a displacement angle from 23 to 31°. Typical parallel-displaced π stacking interaction is reported to occur in centroid distances up to 3.8 Å and angles around 20°.54 The slightly increased oﬀset in the 4-phenyl triazole-based organometallics can be explained by intermolecular πtrz−σtrz‑H attraction and in some cases πPh−σCH2 attraction between side chain protons and the phenyl ring (Figure S3, Table S7−S9). The far order of the dimeric subunits is determined by the side chains. The tetragonal alkyl derivatives 2c, 2d, 3c, and 3d arrange themselves in orthogonal layers (Figure S2, left) in order to minimize energy by aggregation of lipophilic alkyl chains and comparatively polar metal centers. In most benzyl and ester derivatives (2b, 2e, 3a, 3b, 3e), the triazole and phenyl rings are locked in parallel plains (Figure S2, right). In crystals of 2a the individual molecules arrange themselves in long parallel chains rather than the dimeric interaction pairs discussed above, facilitated by alternating π-interactions between the triazole and phenyl rings of consecutive molecules (πtrz(a)−πPh(b), πtrz(b)−πPh(c), etc.; Figure S4). D DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Solutions of all organometallics 2a−e and 3a−e in d7-DMF and d6-DMSO containing adventitious water showed no sign of decomposition, adduct formation, or hydrolysis in 1H NMR over several days, which allowed easy handling of stock solutions for biological assays. Even upon addition of 10% D2O, only slight formation of the aquated complex (DMF) and the DMSO adduct (DMSO) was observed (<5%, 2e) overnight. Formation of the DMSO adducts in 10% DMSO/water proceeded with the same rate in pure water and physiological chloride concentrations (0.9% NaCl). Furthermore, no set of signals corresponding to the aquated species was observable in 1 H NMR, which suggests that aquation is the rate-determining step for DMSO adduct formation. Amino acid interaction. The reactivity of the synthesized metalacycles toward amino acids was investigated by incubating 5 μM solutions containing of 2a−e and 3a−e with equimolar amounts of L-histidine (His), L-methionine (Met), and Lcysteine (Cys) in 1% DMF or DMSO/ammonium acetate solution (400 μM, pH 7.4). After 0, 1, 3, 6, and 24 h, aliquots were taken and analyzed by ESI-MS. In 1% DMF the aquated metalacycles readily formed amino acid adducts, preferably with Met, to a lesser degree with His, and barely detectable with Cys (Figure 4). After 6 h, only methanol. Over the whole time span, only DMSO adducts [M−Cl+DMSO]+ were observed, conﬁrming the proposed leaving group exchange (Figure S29). On the contrary, in 1% DMF/ammonium acetate solution only the molecular ion [M−Cl] was observed, whose peak height decreased over time by precipitation from solution, but also by decomposition yielding the mixed hydroxido/ methoxido ruthenium dimers [(p-cym)Ru(OH)m(OMe)nRu(p-cym)] (m + n = 3, Figure 3). Figure 3. ESI-MS spectra of 2a (5 μM) in 1% DMF/ammonium acetate solution (400 μM, pH 7.4) measured after 0, 1, 3, 6, and 24 h of incubation time at 37 °C. Aquation−anation equilibria. The aquation−anation equilibria of 2e and 3e were studied by recording the 1H NMR spectra of 500 μM compound solutions in 10% d7-DMF/ D2O at 25 °C, both without NaCl and in the presence of 5 mM or 154 mM NaCl as model conditions for intra- and extracellular chloride concentrations. The chlorido species and the derived aquated complex reach equilibrium concentrations after approximately 30 min (2e, Figure S31) and 90 min (3e, Figure S32), respectively. The equilibrium concentrations are inﬂuenced by the chloride concentration in solution (Table 1), with almost Figure 4. (A) ESI-MS spectrum of 2a (5 μM) after 24 h of incubation with equimolar amounts of His, Met, and Cys; (B) calculated isotopic distributions of amino-acid adducts of 2a: [M−Cl+Cys]+, [M−Cl +Met]+, and [M−Cl+His]+. Table 1. Equilibrium Aquation−Anation Ratio Equilibrium aquation [%] Compound 0 mM NaCl 5 mM NaCl 154 mM NaCl 2e 3e 95 98 85 78 25 9 amino acid adduct peaks were observed in mass spectra of all ruthena(II) and osma(II) cycles. In 1% DMSO-containing solutions, the only observable metal-containing peak corresponded to the DMSO adduct [M−Cl+DMSO]+ (Figure S30), attesting to the strength of the DMSO-Ru bond. Cytotoxicity. The antiproliferative activity of the ligands 1a−e and the respective ruthenium 2a−e and osmium 3a−e complexes in human non-small cell lung cancer (A549), colon adenocarcinoma (SW480), and human ovarian carcinoma (CH1/PA-1) cell lines were determined by means of the colorimetric MTT assay. As could be seen from studies on interactions with small biomolecules, the in situ formed DMSO adducts showed no interactions with nucleophiles in contrast to the aqua complex. As the reactivity of the aqua species toward nucleophiles is postulated to be essential for the cytotoxic eﬀect (activation-by-aquation hypothesis),62 DMSO adduct formation should have an impact on anticancer potency. However, prescreens did not reveal major diﬀerences in activities with the use of DMSO or DMF as a solubilizer. Therefore, we were compelled to investigate the antiproliferative activity of the complete hydrolysis in pure water, slightly decreased hydrolysis in intracellular chloride concentrations (5 mM NaCl), and signiﬁcantly hindered aquation under extracellular conditions (154 mM NaCl). This behavior suggests that a larger portion of the compound may enter the cell intact and subsequently undergo aquation in close proximity to the cellular targets. As to be expected, the rate of aquation is signiﬁcantly slower in osmium(II) complexes than their ruthenium(II) counterparts, which may inﬂuence their biological eﬀects.60,61 It has to be noted that slight precipitation of 2e in 154 mM NaCl solution, and of 3e under all NaCl concentrations was observed. Addition of sodium chloride to a completely hydrolyzed sample of 2e and 3e shifted the reversible aquation−anation equilibrium back toward the chlorido complex. However, a strong excess of sodium chloride induced quantitative precipitation (Figure S33), as previously reported for structurally related metalacycles.17 E DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry series, the Os(II)(cym) complexes 3a and 3b bearing a pendant benzyl group at the N1 of the triazole backbone were found to be the most active of the series. The presented cytotoxicity data for the osmium(II) arene cyclometalates 3a−e enable the ﬁrst broader comparison of Os(II) arene complexes bearing C,Ncoordinated 4-phenyl 1,2,3-triazoles to their ruthenium(II) congeners. Organometallics derived from benzyl-substituted triazole ligands show the highest cytotoxic potency, especially in the chemosensitive CH1/PA-1 cells. Methyl ester-bearing triazole organometallics 2e and 3e exhibit the lowest cytotoxicity over all tested cell lines, with a more than 3- to 6-fold decreased activity depending on the cell line, compared to the most active representatives. Introduction of a methoxy group in benzyl derivatives and elongation of the alkyl chain only have a minor eﬀect on IC50 values. In order to elucidate the reason for the divergent IC50 values within the ruthenium(II) and osmium(II) metalacycle series, the cellular accumulation of selected ruthenium(II) compounds was measured in SW480 cells. Cellular accumulation is highest for the comparatively lipophilic benzyl residue compounds 2a and 2b, slightly lower for the alkyl derivative 2c, and lowest for the metalacycle 2e, featuring the least lipophilic methyl esterbearing ligand 1e (Table 4). prepared organometallics in DMSO- and DMF-containing cytotoxicity assays. The free triazole ligands 1a−e were tested from DMSO stocks with eﬀective maximum contents of 0.5% v/v DMSO in the test plates, but IC50 values were not reached with soluble concentrations (1a, 1b) or with concentrations of up to 200 μM (1c−e), except for 1a in CH1/PA-1 (Table 2). Table 2. 50% Inhibitory Concentrations (means ± standard deviations) of the Free Triazole Ligands 1a−e with DMSO as a Solubilizer in Three Human Cancer Cell Lines IC50 (μM) Compound A549 SW480 CH1/PA-1 1a 1b 1c 1d 1e >80 >50 >200 >200 >200 >80 >50 >200 >200 >200 62 ± 2 >50 >200 >200 >200 The prepared complexes 2a−e represent the ﬁrst examples of RuII(cym)triazoles with a CPh,N3-chelation motif. In contrast to the free ligands, the ruthenacycles 2a−c exhibited considerable cytotoxic eﬀects with IC50 values down to the low micromolar range for complexes 2a and 2b. In general, the complexes were most active in the chemosensitive CH1/PA-1 cell line. The antiproliferative potential of the investigated ruthenium complexes was found in the same range as the most active reported ruthenacycles of the general formula [RuII(arene)(LC∧N)], such as cyclometalated 2-aryldiazole (10−150 μM),14 2-phenylindole (0.7−5 μM),15 2-phenylpyridine (3−100 μM),16 and benzimidazole (1−150 μM)17,18 scaﬀolds. However, comparison of the cytotoxicities determined here with literature reports of other C,N-cyclometalates measured in diﬀerent cell lines is not straightforward, and the observed IC50 values can therefore only give an estimate of the cytotoxicity relationships within each compound series. In contrast to several reports on the anticancer potential of [RuII(arene)(LC^N)] ruthenacycles, the respective Os(II) analogs were rather sparse in the literature. In this study, the prepared Os(II) complexes 3a−e were found to be signiﬁcantly more cytotoxic than the corresponding ruthenated complexes of the same ligand (Table 3) by factors of around 2−4 depending on the cell line and ligand. Like in the ruthenium Table 4. Cellular Accumulation in SW480 Cancer Cells (0.5% DMSO, 50 μM compound), and Calculated LogP Values of the Corresponding Free Ligands (Molinspiration) Compound Cell uptake (fg Ru/cell) cLogP (free ligand) 2a 2b 2c 2e 240 ± 46 306 ± 1 80 ± 28 34 ± 3 3.12 3.18 2.40 1.39 Increased cellular accumulation correlates well with lower IC50 values (Figure 5), which suggests that chain variation mainly impacts the antiproliferative abilities by modulation of the lipophilicity and presumably does not contribute additional interactions. As far as the diﬀerences associated with the use of diﬀerent solubilizers (DMSO vs DMF) are concerned, the benzyl and alkyl residues bearing ruthena(II)- and osma(II)cycles mostly exhibit antiproliferative activities in a comparable range. The Table 3. 50% Inhibitory Concentrations (means ± standard deviations) of the Ruthenium and Osmium Arene Organometallics 2a−e and 3a−e in Three Human Cancer Cell Lines Solubilizer: DMF a Solubilizer: DMSO Compound A549 SW480 CH1/PA-1 A549 2a 2b 2c 2d 2e 3a 3b 3c 3d 3e cisplatin63,a 16 ± 2 13 ± 1 >50 >12.5 109 ± 10 6.0 ± 0.4 5.5 ± 0.6 17 ± 1 7.7 ± 0.2 34 ± 2 6.4 ± 0.4 6.8 ± 0.1 6.6 ± 0.1 30 ± 4 >12.5 37 ± 3 3.6 ± 0.1 3.6 ± 0.1 11 ± 1 6.8 ± 0.5 20 ± 1 3.3 ± 0.2 5.7 ± 0.1 4.6 ± 0.7 15 ± 3 10 ± 2 16 ± 3 0.98 ± 0.20 1.2 ± 0.2 2.0 ± 0.2 1.5 ± 0.2 3.3 ± 0.7 0.077 ± 0.006 13.7 ± 0.3 13.7 ± 0.3 23.2 ± 0.3 >12.5 >200 12 ± 2 9.8 ± 2.0 43 ± 2 >50 6.4 ± 0.4 SW480 CH1/PA-1 7.3 ± 0.4 7.1 ± 0.1 19 ± 1 11.0 ± 0.2 >200 7.7 ± 0.8 6.0 ± 1.0 20 ± 2 Insuﬃcient solubility >50 3.3 ± 0.2 4.3 ± 0.9 4.0 ± 0.6 8.8 ± 0.7 6.1 ± 1.3 61 ± 14 1.4 ± 0.3 1.20 ± 0.01 4.1 ± 1.3 33 ± 13 0.077 ± 0.006 Tested without the use of a solubilizing agent. F DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry discussed, the stability of the metal−DMSO bond may be diﬀerent under extra- and intracellular conditions, thereby allowing for the formation of reactive species and a reaction with amino acids and other cellular targets. As mentioned above, the use of either DMSO or DMF as a solubilizer has only a minor eﬀect on the IC50 values of benzyl and alkyl derivatives 2a−d and 3a−d, while the methyl ester compounds 2e and 3e experience a large decrease in activity due to DMSO coordination. Through replacement of the chlorido ligand by DMSO, charged metal complexes with increased hydrophilicity are formed. It can be hypothesized that the already comparatively hydrophilic metalacycles 2e and 3e are unable to enter the cells to a suﬃcient degree upon DMSO coordination, which should diminish the antiproliferative activity. On the other hand, the DMSO adducts of more apolar metal complexes may still possess suﬃcient lipophilicity to pass through cell membranes. Additionally, ester hydrolysis might contribute to the eﬀect observed in the MTT assay, but was not detected in the stability investigations. Topoisomerase IIα inhibition. Several metal(II)−arene complexes have been shown to be potent inhibitors of topoisomerase IIα, an enzyme which is essential for DNA replication, transcription, and recombination, and a wellestablished target for cancer chemotherapy. Complexation to ruthenium(II)−arene fragments has been reported to enhance the inhibitory capacity of known topoisomerase IIα inhibitors (e.g., ﬂavonoids8,64) or confer topoisomerase inhibitory capabilities to otherwise inactive free ligands (e.g., 1,3dioxoindan-2-carboxamides,56 2-aryl-4-thiopyrones65). Consequently, the topoisomerase IIα inhibitory activity of highly cytotoxic triazole organometallics 2b and 3b featuring 4methoxybenzyl residues, the less active methyl ester derivatives 2e and 3e, as well as their corresponding free ligands 1b and 1e was tested. Topoisomerase IIα inhibition is detected by decreased uncoiling of a plasmid DNA substrate from the supercoiled conformation to the relaxed conformation. The six selected substances were incubated with the enzyme and plasmid DNA, revealing that none is able to inhibit the function of topoisomerase IIα at 25 μM or to act as a topoisomerase poison (Figure S34). Considering that the tested organometallics 2b and 3b show cytotoxic activity at comparable or lower concentrations in MTT assays, it is unlikely that inhibition of topoisomerase IIα contributes to their mechanism of action. Cell cycle analysis. The cell cycle distribution of SW480 cells in response to treatment with the organometallics 2b, 3b, 2e, and 3e, and their respective free ligands 1b and 1e was determined at various concentrations after 48 h (Figure 6). Figure 5. IC50 values (hatched) and cellular accumulation (plain) of 2a−d in SW480 cells. largest decrease in activity by the use of DMSO is found for methyl ester functionalized compounds 2e and 3e. To investigate this behavior, we developed a procedure to isolate selected DMSO adducts 2a′, 2e′, and 3e′ as triﬂate salts based on chlorido ligand abstraction with silver triﬂate and reaction with DMSO in THF, and determined their cytotoxic activity (Table 5). Table 5. 50% Inhibitory Concentration (mean ± standard deviation) of Cancer Cell Growth in Three Cell Lines Obtained by the MTT Assay with either DMSO (I) or DMF (II) as Solubilizer IC50 (μM) Compound A549 SW480 CH1/PA-1 2aI 2eI 3eI 2aII 2eII 3eII 2a′II 2e′II 3e′II 13.7 ± 0.3 >200 >50 16 ± 2 109 ± 10 34 ± 2 14 ± 1 >200 >200 7.3 ± 0.4 >200 >50 6.8 ± 0.1 37 ± 3 20 ± 1 8.4 ± 0.3 140 ± 9 >200 4.3 ± 0.9 61 ± 14 33 ± 13 5.7 ± 0.1 16 ± 3 3.3 ± 0.7 3.8 ± 0.5 43 ± 12 117 ± 5 The antiproliferative activity of the isolated DMSO adducts 2a′, 2e′, and 3e′ is remarkably close to those of the parent chlorido-bearing metal complexes 2a, 2e, and 3e tested from DMSO stocks (Table 5). This suggests that the DMSO adducts may contribute to the antiproliferative eﬀect measured in MTT assays. While no biomolecule interaction of the in situ formed DMSO adducts could be observed via ESI-MS methods as Figure 6. Cell cycle distributions (means of three independent experiments) in SW480 cells exposed to 1b (left), 2b (center), and 3b (right) for 48 h compared to untreated control (0 μM) at diﬀerent concentrations. Application of higher concentrations than those indicated was impossible due to limited solubility in most cases. G DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Elemental analyses were performed at the Microanalytical Laboratory of the University of Vienna, using a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer for CHN analyses and a Eurovector EA3000 Elemental Analyzer for CHNS analyses. pH values were measured with a EcoScan pH 6 pH meter equipped with a Eutech Instruments Ag/AgCl pH electrode calibrated with Alfa Aesar Specpure standard buﬀer solutions at pH 4.00, 7.00, and 10.00. Analytical grade solvents were purchased from commercial suppliers and used without further puriﬁcation. Methanol for complexation reactions was distilled from Mg/I2 and stored over molecular sieve (3 Å). Millipore water (Milli-Q Advantage A10, 18.2 MΩ/25 °C, 2 ppb TOC) and methanol (HPLC grade, Fisher) and were used for mass spectrometry measurements. Benzyl bromide (98%, Acros), 4methoxybenzyl chloride (98%, Acros), 1-bromopropane (99%, Aldrich), 1-bromobutane (99%, Sigma), methyl bromoacetate (99%, Acros), sodium azide (≥99.0%, Fluka), copper(II)sulfate pentahydrate (≥99.0%, Fluka), phenylacetylene (98%, Aldrich), L-(+)-ascorbic acid sodium salt (99.0%, Fluka), silver triﬂuoromethanesulfonate (99+%, Acros), dimethyl sulfoxide (anal. grade, Fisher), ruthenium(III) chloride hydrate (Johnson Matthey), osmium tetroxide (Johnson Matthey), hydrazine dihydrochloride (≥98.0%, Sigma), α-terpinene (90%, Acros), sodium acetate anhydrous (≥98.5%, Fluka), molecular sieve (3 Å, beads, 4−8 mesh), phosphate buﬀered saline (pH 7.4, 10x, gibco), L-cysteine (Fluka), L-histidine (Merck) and L-methionine (Merck) were used without further puriﬁcation. The dimeric metal precursors dichlorido(p-cymene)ruthenium(II)67 [Ru(p-cym)Cl2]2 and dichlorido(p-cymene)osmium(II)68 [Os(p-cym)Cl2]2 were prepared according to literature procedures. Synthesis and characterization of the ligands 1a−f can be found in the Supporting Information. General procedure for the synthesis of ruthenium(II) and osmium(II) arene complexes 2a−e and 3a−e. Ligand 1a−f and anhydrous sodium acetate were suspended in methanol abs. and stirred for 15 min under argon atmosphere. Subsequently, the respective metal dimer [Ru(cym)Cl2]2 or [Os(cym)Cl2]2 was added and the reaction mixture was stirred under argon atmosphere at room temperature for 16 h to 2 days, during which precipitation of the product occurred. The formed solid was ﬁltered oﬀ, washed with cold methanol, redissolved in dichloromethane, ﬁltered and evaporated to dryness to aﬀord the desired organometallic complexes 2a−e and 3a− e in moderate to good yields. [Chlorido(1-benzyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)ruthenium(II)] (2a). The reaction was performed according to the general complexation procedure, using 1a (154 mg, 0.65 mmol), [Ru(p-cym)Cl2]2 (200 mg, 0.33 mmol) and anhydrous sodium acetate (59 mg, 0.72 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 263 mg, 80%. ESI-HR-MS+ m/z Found (Calculated): [M− Cl]+ 470.1184 (470.1171), [M+Na]+ 528.0767 (528.0754). Elemental Anal. Calc. for C25H26ClN3Ru: C 59.46, H 5.19, N 8.32; Found: C 59.45, H 4.94, N 8.35. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.15 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.47 (s, 1H, ArHTrz‑5); 7.35−7.30 (m, 3H, ArHBn‑3″/5″, ArHBn‑4″); 7.24−7.19 (m, 3H, ArHBn‑2″/6″, ArHPh‑6′); 7.10 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 6.97 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑5′); 5.55 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.49 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.45 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.35 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 5.22 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.18 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 2.38 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.02 (s, 3H, CH3, Cym‑a); 0.92 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g); 0.92 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g); 0.84 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 298.6 K): δ = 176.3 (CPh‑2′); 155.5 (CTrz‑4); 139.7 (CHPh‑3′); 135.3 (CPh‑1′); 134.6 (CBn‑1″); 129.1 (CHBn‑3″/5″); 128.9 (CHBn‑4″); 128.2 (CHBn‑2″/6″); 127.7 (CHPh‑4′); 122.8 (CHPh‑5′); 122.4 (CHPh‑6′); 117.5 (CHTrz‑5); 99.5 (CCym‑e); 98.7 (CCym‑b); 89.1 (CHCym‑c); 87.3 (CHCym‑d); 85.6 (CHCym‑d); 83.5 (CHCym‑c); 54.9 (CH2, Bn); 30.8 (CHCym‑f); 22.3 (CH3, Cym‑g); 22.2 (CH3, Cym‑g); 18.9 (CH3, Cym‑a) ppm. [Chlorido(1-(4″-methoxybenzyl)-4-(2′-κC)-phenyl-(3-κN)-1,2,3triazolato)(η6-p-cymene)ruthenium(II)] (2b). The reaction was performed according to the general complexation procedure, using After staining with propidium iodide, the relative DNA content of the cells was determined by ﬂow cytometry. The two ligands 1b and 1e show no marked eﬀects on the cell cycle distribution at concentrations of up to 50 μM (Figure 6, Figure S35). The two ruthenium complexes 2b and 2e show mild eﬀects. 2b induces an increase of cells in the S phase at a concentration of 25 μM, whereas 2e requires higher concentrations for comparable eﬀects. The osmium arene complex 3b shown to exhibit the highest antiproliferative eﬀect almost causes an elimination of the S phase fraction (Figure 6). In contrast, the rather inactive compound 3e shows almost no eﬀect on the cell cycle distribution (Figure S35). These results suggest that the cytotoxic eﬀect of the osmium(II) derivatives is primarily exerted in the DNA synthesis (S) phase, while no deﬁnitive conclusion can be drawn from the behavior of tested ruthenium(II) compounds. ■ CONCLUSION In this work, we have established the 1,2,3-triazole backbone as a promising C,N-ligand system for cyclometalated Ru(II) and Os(II) arene complexes. Crystal structures of all prepared organometallics were obtained and allowed the description of interesting side chain dependent crystallization behaviors. The stability and aquation of the complexes in aqueous solution containing 1% DMF, as well as anation of DMSO to replace the chlorido leaving group in the presence of 1% DMSO was described via UV−vis, ESI-MS, and NMR techniques. It could be shown that aquation is dependent on external chloride concentrations and, therefore, is considerably hindered under extracellular conditions but favored in the intracellular milieu. This behavior allows for activation of the prepared organometallics close to their biological targets. The cytotoxicity of all organometallics and their corresponding free ligands was determined in three human cancer cell lines (A549, SW480, CH1/PA-1). While most ligands barely show any cytotoxic eﬀect in the tested concentration range, the respective organometallics exhibit IC50 values down to the low micromolar range. Cytotoxicity could be correlated to cellular accumulation and seems to be mainly inﬂuenced by the lipophilicity of the ligands. The DMSO adducts of three ruthenium(II) complexes were isolated as trifalte salts and exhibited activities in a range comparable to the respective parent compounds featuring chlorido leaving groups when tested in MTT assays with the use of DMSO as solubilizer. Selected compounds were found unable to inhibit topsoisomerase IIα, but the most cytotoxic compounds have a pronounced eﬀect on cell cycle distribution, which stimulates further mode-of-action studies. The facile synthesis, modiﬁability, stability under physiological conditions, and high activity in vitro of the prepared complexes encourage future development of this promising compound class. ■ EXPERIMENTAL SECTION Materials and Methods. 1H, 13C (APT), 31P, and 2D NMR spectra were recorded on a Bruker FT-NMR spectrometer Avance III 500 MHz in deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) and referenced to residual solvent signals66 (1H, 13 C) or 85% phosphoric acid (31P). High resolution ESI mass spectra were recorded at the Core Facility for Mass Spectrometry of the University of Vienna (Faculty of Chemistry) on a Bruker Maxis UHR qTOF Mass Spectrometer by direct infusion. Electrospray ionization mass spectra were recorded on a Bruker AmaZon SL ion trap mass spectrometer by direct infusion. Data ﬁles were analyzed using Bruker data analysis software ESI Compass 1.3 and Data Analysis 4.0. H DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry (CH3, Cym‑g); 22.1 (CH3, Cym‑g); 19.7 (CH2, But‑3″); 18.8 (CH3, Cym‑a); 13.5 (CH3, But‑4″) ppm. [Chlorido(methyl-2″-(4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazol-1ylato)acetato)(η6-p-cymene)ruthenium(II)] (2e). The reaction was performed according to the general complexation procedure, using 1e (177 mg, 0.82 mmol), [Ru(p-cym)Cl2]2 (250 mg, 0.41 mmol) and anhydrous sodium acetate (74 mg, 0.90 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 243 mg, 61%. ESI-HR-MS+ m/z Found (Calculated): [M−Cl]+ 452.0914 (452.0912), [M+Na]+ 510.0494 (510.0495). Elemental Anal. Calc. for C21H24ClN3O2Ru · 0.5H2O: C 50.86, H 5.08, N 8.47, S 0.00; Found: C 51.04, H 4.94, N 8.55, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 299.9 K): δ = 8.16 (d, 3 JH,H = 7 Hz, 1H, ArHPh‑3′); 7.74 (s, 1H, ArHTrz‑5); 7.17 (dd, 3JH,H = 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑6′); 7.14 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 7.01 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑5′); 5.51 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.47 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.44 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.20 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 4.61 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 4.53 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 3.67 (s, 3H, OCH3, Ac); 2.35 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 1.98 (s, 3H, CH3, Cym‑a); 0.91 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g); 0.87 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 300.4 K): δ = 176.2 (CPh‑2′); 166.9 (COAc); 154.9 (CTrz‑4); 139.9 (CHPh‑3′); 135.3 (CPh‑1′); 127.7 (CHPh‑4′); 122.9 (CHPh‑5′); 122.4 (CHPh‑6′); 119.8 (CHTrz‑5); 99.5 (CCym‑e); 99.1 (CCym‑b); 88.9 (CHCym‑c); 88.0 (CHCym‑d); 85.5 (CHCym‑d); 83.3 (CHCym‑c); 52.9 (OCH3, Ac); 50.9 (CH2, Ac); 30.8 (CHCym‑f); 22.3 (CH3, Cym‑g); 22.1 (CH3, Cym‑g); 18.7 (CH3, Cym‑a) ppm. [Chlorido(1-benzyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)osmium(II)] (3a). The reaction was performed according to the general complexation procedure, using 1a (119 mg, 0.51 mmol), [Os(p-cym)Cl2]2 (200 mg, 0.25 mmol) and anhydrous sodium acetate (46 mg, 0.56 mmol) in methanol abs. (5 mL) for a reaction time of 48 h. Yield: 192 mg, 64%. ESI-HR-MS+ m/z Found (Calculated): [M− Cl]+ 560.1748 (560.1737), [M+Na]+ 618.1325 (618.1312), [2M +Na] + 1211.2710 (1211.2710). Elemental Anal. Calc. for C25H26ClN3Os: C 50.54, H 4.41, N 7.07, S 0.00; Found: C 50.45, H 4.48, N 6.81, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.01 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.46 (s, 1H, ArHTrz‑5); 7.41− 7.37. (m, 3H, ArHBn‑3″/5″, ArHBn‑4″); 7.32 (dd, 3JH,H = 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑6′); 7.30−7.24 (m, 2H, ArHBn‑2″/6″); 7.05 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 6.94 (ddd, 3JH,H = 7 Hz, 7 Hz, 4 JH,H = 1 Hz, 1H, ArHPh‑5′); 5.65−5.59 (m, 2H, ArHCym‑d, CH2, Bn); 5.55−5.52 (m, 2H, ArHCym‑c, ArHCym‑d); 5.47 (d, 2JH,H = 15 Hz, CH2, Bn); 5.35 (d, 3JH,H = 5 Hz, 1H, ArHCym‑c); 2.39 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.18 (s, 3H, CH3, Cym‑a); 0.96−0.90 (m, 6H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 299.3 K): δ = 162.6 (CPh‑2′); 157.4 (CTrz‑4); 139.5 (CHPh‑3′); 136.1 (CPh‑1′); 134.5 (CBn‑1″); 128.2 (CHBn‑3″/5″); 128.9 (CHBn‑4″); 128.4 (CH Ph‑4′); 128.2 (CHBn‑2″/6″); 122.7 (CHPh‑5′); 122.1 (CHPh‑6′); 117.7 (CHTrz‑5); 91.7 (CCym‑b); 90.3 (CCym‑e); 79.3 (CHCym‑c); 78.2 (CHCym‑d); 76.6 (CHCym‑d); 73.3 (CHCym‑c); 55.0 (CH2, Bn); 31.1 (CHCym‑f); 22.5 (CH3, Cym‑g); 22.7 (CH3, Cym‑g); 18.7 (CH3, Cym‑a) ppm. [Chlorido(1-(4″-methoxybenzyl)-4-(2′-κC)-phenyl-(3-κN)-1,2,3triazolato)(η6-p-cymene)osmium(II)] (3b). The reaction was performed according to the general complexation procedure, using 1b (134 mg, 0.51 mmol), [Os(p-cym)Cl2]2 (200 mg, 0.25 mmol) and anhydrous sodium acetate (46 mg, 0.56 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 248 mg, 78%. ESI-HR-MS+ m/z Found (Calculated): [M−Cl]+ 590.1856 (590.1843), [M+Na]+ 648.1432 (648.1418), [2M+Na]+ 1271.2969 (1271.2922). Elemental Anal. Calc. for C26H28ClN3OOs: C 50.03, H 4.52, N 6.73, S 0.00; Found: C 49.89, H 4.61, N 6.52, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 297.3 K): δ = 8.01 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.46 (s, 1H, ArHTrz‑5); 7.28 (dd, 3JH,H = 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑6′); 7.24− 7.20 (m, 2H, ArHBn‑2″/6″); 7.04 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 6.94 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑5′); 6.90−6.87 (m, 2H, ArHBn‑3″/5″); 5.63 (d, 3JH,H = 5 Hz, 1H, ArHCym‑d); 5.53−5.50 (m, 2H, ArHCym‑c, ArHCym‑d); 5.42 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 5.37 (d, 3JH,H = 5 Hz, 1H, ArHCym‑c); 5.28 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 3.80 (s, 3H, OCH3, Bn); 2.37 (sept, 3JH,H = 7 Hz, 1b (217 mg, 0.82 mmol), [Ru(p-cym)Cl2]2 (250 mg, 0.41 mmol) and anhydrous sodium acetate (74 mg, 0.90 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 300 mg, 69%. ESI-HR-MS+ m/z Found (Calculated): [M−Cl]+ 500.1292 (500.1277), [M+Na]+ 558.0874 (558.0860). Elemental Anal. Calc. for C26H28ClN3ORu: C 58.37, H 5.27, N 7.85, S 0.00; Found: C 58.32, H 5.35, N 8.18, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.14 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.47 (s, 1H, ArHTrz‑5); 7.22−7.18 (m, 3H, ArHPh‑6′, ArHBn‑2″/6″); 7.09 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 6.98 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑5′); 6.88−6.84 (m, 2H, ArHBn‑3″/5″); 5.54 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.49−5.45 (m, 2H, ArHCym‑d); 5.32 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 5.22 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.15 (d, 2JH,H = 15 Hz, 1H, CH2, Bn); 3.79 (s, 3H, OCH3, Bn); 2.40 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.02 (s, 3H, CH3, Cym‑a); 0.92 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g); 0.86−0.84 (m, 3H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 298.6 K): δ = 176.3 (CPh‑2′); 160.1 (CBn‑4″); 155.4 (CTrz‑4); 139.6 (CHPh‑3′); 135.3 (CPh‑1′); 129.9 (CHBn‑2″/6″); 127.7 (CHPh‑4′); 126.4 (CBn‑1″); 122.7 (CHPh‑5′); 122.4 (CHPh‑6′); 117.1 (CHTrz‑5); 114.5 (CHBn‑3″/5″); 99.6 (CCym‑e); 98.7 (CCym‑b); 89.0 (CHCym‑c); 87.4 (CHCym‑d); 85.4 (CHCym‑d); 83.5 (CHCym‑c); 55.5 (OCH3, Bn); 54.6 (CH2, Bn); 30.8 (CHCym‑f); 22.3 (CH3, Cym‑g); 22.2 (CH3, Cym‑g); 18.9 (CH3, Cym‑a) ppm. [Chlorido(1-propyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)ruthenium(II)] (2c). The reaction was performed according to the general complexation procedure, using 1c (153 mg, 0.82 mmol), [Ru(p-cym)Cl2]2 (250 mg, 0.41 mmol) and anhydrous sodium acetate (74 mg, 0.90 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 213 mg, 57%. ESI-HR-MS+ m/z Found (Calculated): [M− Cl]+ 422.1176 (422.1170), [M+Na]+ 480.0758 (480.0753). Elemental Anal. Calc. for C21H26ClN3Ru · 0.75H2O: C 53.61, H 5.89, N 8.93; Found: C 53.67, H 5.65, N 8.99. 1H NMR (CDCl3, 500.32 MHz, 300.5 K): δ = 8.15 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.55 (s, 1H, ArHTrz‑5); 7.24 (d, 3JH,H = 7 Hz, 1H, ArHPh‑6′); 7.10 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑4′); 6.97 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑5′); 5.54 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.49 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.45 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.20 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 4.27−4.20 (m, 1H, CH2, Prop‑1″); 4.11−4.03 (m, 1H, CH2, Prop‑1″); 2.42 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.03 (s, 3H, CH3, Cym‑a); 1.94−1.83 (m, 2H, CH2, Prop‑2″); 0.95−0.88 (m, 9H, CH3, Prop‑3″, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.81 MHz, 300.6 K): δ = 176.2 (CPh‑2′); 155.2 (CTrz‑4); 139.6 (CHPh‑3′); 135.4 (CPh‑1′); 127.7 (CHPh‑4′); 122.7 (CHPh‑5′); 122.3 (CHPh‑6′); 117.2 (CHTrz‑5); 99.4 (CCym‑e); 99.0 (CCym‑b); 88.8 (CHCym‑c); 87.8 (CHCym‑d); 85.2 (CHCym‑d); 83.3 (CHCym‑c); 53.1 (CH2, Prop‑1″); 30.9 (CHCym‑f); 23.6 (CH2, Prop‑2″); 22.4 (CH3, Cym‑g); 22.1 (CH3, Cym‑g); 18.9 (CH3, Cym‑a); 11.0 (CH3, Prop‑3″) ppm. [Chlorido(1-butyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)ruthenium(II)] (2d). The reaction was performed according to the general complexation procedure, using 1d (132 mg, 0.65 mmol), [Ru(p-cym)Cl2]2 (200 mg, 0.33 mmol) and anhydrous sodium acetate (59 mg, 0.72 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 250 mg, 81%. ESI-HR-MS+ m/z Found (Calculated): [M−Cl]+ 436.1326 (436.1327), [M+Na]+ 494.0905 (494.0910). Elemental Anal. Calc. for C22H28ClN3Ru · 0.5H2O: C 55.05, H 6.09, N 8.75, S 0.00; Found: C 54.94, H 5.85, N 8.54, S < 0.02. 1H NMR (CDCl3, 500.32 MHz, 299:8 K): δ = 8.14 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.55 (s, 1H, ArHTrz‑5); 7.22 (d, 3JH,H = 7 Hz, 1H, ArHPh‑6′); 7.09 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑4′); 6.97 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑5′); 5.53 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.48 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.45 (d, 3JH,H = 6 Hz, 1H, ArHCym‑d); 5.20 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 4.26−4.19 (m, 1H, CH2, But‑1″); 4.08−4.01 (m, 1H, CH2, But‑1″); 2.41 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.02 (s, 3H, CH3, Cym‑a); 1.83−1.78 (m, 2H, CH2, But‑2″); 1.34−1.21 (m, 2H, CH2, But‑3″); 0.96−0.87 (m, 9H, CH3, But‑4″, CH3, Cym‑g) ppm. 13 C NMR (CDCl3, 125.81 MHz, 301.0K): δ = 176.2 (CPh‑2′); 155.2 (CTrz‑4); 139.6 (CHPh‑3′); 135.4 (CPh‑1′); 127.6 (CHPh‑4′); 122.7 (CHPh‑5′); 122.3 (CHPh‑6′); 117.3 (CHTrz‑5); 99.4 (CCym‑e); 99.0 (CCym‑b); 88.8 (CHCym‑c); 87.7 (CHCym‑d); 85.3 (CHCym‑d); 83.3 (CHCym‑c); 51.2 (CH2, But‑1″); 32.1 (CH2, But‑2″); 30.9 (CHCym‑f); 22.4 I DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry (d, 3JH,H = 5 Hz, 1H, ArHCym‑d); 5.51−5.48 (m, 2H, ArHCym‑c, ArHCym‑d); 5.36 (d, 3JH,H = 5 Hz, 1H, ArHCym‑c); 4.67 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 4.52 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 3.68 (s, 3H, OCH3, Ac); 2.30 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.14 (s, 3H, CH3, Cym‑a); 0.91−0.87 (m, 6H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 299.1 K): δ = 166.8 (COAc); 162.5 (CPh‑2′); 156.8 (CTrz‑4); 139.7 (CHPh‑3′); 136.1 (CPh‑1′); 128.4 (CHPh‑4′); 122.9 (CHPh‑5′); 122.1 (CHPh‑6′); 120.2 (CHTrz‑5); 92.3 (CCym‑b); 90.2 (CCym‑e); 79.1 (CHCym‑c); 78.7 (CHCym‑d); 76.6 (CHCym‑d); 73.0 (CHCym‑c); 52.9 (OCH3, Ac); 51.0 (CH2, Ac); 31.0 (CHCym‑f); 22.7 (CH3, Cym‑g); 22.3 (CH3, Cym‑g); 18.6 (CH3, Cym‑a) ppm. General procedure for the synthesis of DMSO adducts 2a′, 2e′, 3e′. The respective chlorido metal complex (1.0 equiv) and DMSO (2.0 equiv) were suspended in THF (10 mL). Silver triﬂuoromethanesulfonate (2.0 equiv) was added, after which the reaction mixture was stirred for 30 min in darkness at room temperature. The solvent was evaporated in vacuo, the residue redissolved in DCM, ﬁltered, diluted with DCM to a volume of 20 mL and washed three times with H2Otridest. (10 mL). The organic layer was separated, evaporated to dryness, puriﬁed by ﬂash chromatography on silica (0 to 5% MeOH in DCM) and dried in vacuo to aﬀord the desired products as pale yellow solids. [(κS-Dimethyl sulfoxide)(1-benzyl-4-(2′-κC)-phenyl-1,2,3-(3-κN)triazolato)(η6-p-cymene)ruthenium(II)] triﬂuoromethanesulfonate (2a′). The reaction was performed according to the general leaving group exchange procedure, using 2a (150 mg, 0.30 mmol), DMSO (42 μL, 0.60 mmol, 2.0 equiv) and AgOTf (153 mg, 0.60 mmol). Yield: 158 mg, 76%. ESI-HR-MS+ m/z Found (Calculated): [M−DMSO]+ 470.1175 (470.1171), [M]+ 548.1316 (548.1311). Elemental Anal. Calc. for C28H32F3N3O4RuS2: C 48.27, H 4.63, N 6.03, S: 9.18; Found: C 48.14, H 4.63, N 6.02, S 9.12. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.28 (s, 1H, ArHTrz‑5); 7.91 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.51−7.45 (m, 3H, ArHPh‑6′, ArHBn‑2″/6″); 7.42−7.34 (m, 3H, ArHBn‑3″/5″, ArHBn‑4″); 7.18 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 2 Hz, 1H, ArHPh‑4′); 7.14 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz); 4.01−5.97 (m, 1H, ArHCym-c); 5.83−5.79 (m, 2H, ArHCym‑d); 5.78−5.70 (m, 2H, CH2, Bn); 5.63−5.59 (m, 1H, ArHCym‑c); 2.62 (sept, 3JH,H = 7 Hz, 1H, ArHCym‑f); 2.62 (s, 3H, CH3, DMSO); 2.39 (s, 3H, CH3, DMSO); 2.23 (s, 3H, CH3, Cym‑a); 1.00 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g); 0.94 (d, 3 JH,H = 7 Hz, 3H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 298.5 K): δ = 167.1 (CPh‑3′); 156.2 (CTrz‑4); 140.7 (CHPh‑3′); 136.0 (CPh‑1′); 134.1 (CBn‑1″); 129.4 (CHBn‑3″/5″); 129.3 (CHPh‑4′*, CHBn‑4″*); 129.2 (CHPh 4′*, CHBn 4″*); 129.0 (CHBn‑2″/6″); 125.4 (CHPh‑5′); 124.3 (CHPh‑6′); 120.6 (CHTrz‑5); 116.8 (CCym‑e); 108.3 (CCym‑b); 93.6 (CHCym‑c); 91.6 (CHCym‑d); 91.1 (CHCym‑c); 90.2 (CHCym‑d); 56.4 (CH2, Bn); 46.6 (CH3, DMSO); 46.0 (CH3, DMSO); 31.0 (CHCym‑f); 22.7 (CH3, Cym‑g); 21.7 (CH3, Cym‑g); 18.9 (CH3, Cym‑a) ppm. (*): indistinguishable. [(κS-Dimethyl sulfoxide) (methyl-2″-(4-(2′-κC)-phenyl-1,2,3-(3κN)-triazol-1-ylato)acetato)(η6-p-cymene)ruthenium(II)] triﬂuoromethanesulfonate (2e′). The reaction was performed according to the general procedure for the synthesis of DMSO adducts, using 2e (100 mg, 0.21 mmol), DMSO (30 μL, 0.42 mmol, 2.0 equiv) and AgOTf (106 mg, 0.42 mmol). Yield: 107 mg, 77%. ESI-HR-MS+ m/z Found (Calculated): [M−DMSO]+ 452.0915 (452.0912), [M]+ 530.1058 (530.1051). Elemental Anal. Calc. for C24H30F3N3O6RuS2: C 42.47, H 4.46, N 6.19, S 9.45; Found: C 42.19, H 4.38, N 6.08, S 9.22. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.42 (s, 1H, ArHTrz‑5); 7.94−7.90 (m, 1H, ArHPh‑3′); 7.55 (dd, 3JH,H = 7 Hz,4H,H = 2 Hz, 1H, Ar3JH,H = 7 Hz3JH,H = 7 Hz3JH,H = 7 Hz3JH,H = 7 Hz2JH,H = 18 Hz2JH,H = 18 Hz3JH,H = 7 Hz3JH,H = 7 Hzd, 3JH,H = 7 Hz13C NMR (CDCl3, 125.75 MHz, 298.5 K): δ = 167.1 (COAc*, CPh‑2′*); 156.0 (CTrz‑4); 140.6 (CHPh‑3′); 135.9 (CPh‑1′); 129.4 (CHPh‑4′); 125.6 (CHPh‑5′); 124.4 (CHPh‑6′); 122.6 (CHTrz‑5); 116.5 (CCym‑e); 109.2 (CCym‑b); 93.3 (CHCym‑d); 91.4 (CHCym‑c); 91.0 (CHCym‑c); 90.8 (CHCym‑d); 53.4 (OCH3, Ac); 52.7 (CH2, Ac); 46.4 (CH3, DMSO); 46.0 (CH3, DMSO); 31.0 (CHCym‑f); 22.5 (CH3, Cym‑g); 22.1 (CH3, Cym‑g); 18.9 (CH3, Cym‑a) ppm. (*) indistinguishable. [(κS-Dimethyl sulfoxide) (methyl-2″-(4-(2′-κC)-phenyl-1,2,3-(3κN)-triazol-1-ylato)acetato)(η6-p-cymene)osmium(II)] triﬂuorome- 1H, CHCym‑f); 2.17 (s, 3H, CH3, Cym‑a); 0.94−0.88 (m, 6H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 299.3 K): δ = 162.6 (CPh‑2′); 160.2 (CBn‑4″); 157.5 (CTrz‑4); 139.5 (CPh‑3′); 136.1 (CPh‑1′); 129.9 (CHBn‑2″/6″); 128.5 (CHPh‑4′); 126.0 (CBn‑1″); 122.7 (CHPh‑5′); 122.0 (CHPh‑6′); 117.1 (CHTrz‑5); 114.7 (CHBn‑3″/5″); 91.8 (CCym‑b); 90.4 (CCym‑e); 79.2 (CHCym‑c); 78.3 (CHCym‑d); 76.3 (CHCym‑d); 73.2 (CHCym‑c); 55.5 (OCH3, Bn); 54.8 (CH2, Bn); 31.1 (CHCym‑f); 22.7 (CH3, Cym‑g); 22.5 (CH3, Cym‑g); 18.7 (CH3, Cym‑a) ppm. [Chlorido(1-propyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)osmium(II)] (3c). The reaction was performed according to the general complexation procedure, using 1c (95 mg, 0.51 mmol), [Os(p-cym)Cl2]2 (200 mg, 0.25 mmol) and anhydrous sodium acetate (46 mg, 0.56 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 196 mg, 71%. ESI-HR-MS+ m/z Found (Calculated): [M− Cl]+ 512.1748 (512.1737), [M+Na]+ 570.1323 (570.1312), [2M +Na] + 1115.2728 (1115.2708). Elemental Anal. Calc. for C21H26ClN3Os: C 46.18, H 4.80, N 7.69, S 0.00; Found: C 45.91, H 4.88, N 7.35, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 297.8 K): δ = 8.01 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.58 (s, 1H, ArHTrz‑5); 7.31 (d, 3 JH,H = 7 Hz, 1H, ArHPh‑6′); 7.05 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑4′); 6.95 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑5′); 5.61 (d, 3JH,H = 5 Hz, 1H, ArHCym‑c); 5.54−5.50 (m, 2H, ArHCym‑c, ArHCym‑d); 5.36 (d, 3 JH,H = 5 Hz, 1H, ArHCym‑c); 4.27−4.20 (m, 1H, CH2, Prop‑1″); 4.11− 4.04 (m, 1H, CH2, Prop‑1″); 2.37 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.17 (s, 3H, CH3, Cym‑a); 1.94−1.82 (m, 2H, CH2, Prop‑2″); 0.94−0.88 (m, 9H, CH3, Prop‑3″, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 299.0 K): δ = 162.5 (CPh‑2′); 157.1 (CTrz‑4); 139.4 (CHPh‑3′); 136.2 (CPh‑1′); 128.3 (CHPh‑4′); 122.7 (CHPh‑5′); 122.0 (CHPh‑6′); 117.5 (CHTrz‑5); 92.0 (CCym‑b); 90.1 (CCym‑e); 78.9 (CHCym‑c); 78.5 (CHCym‑d); 76.2 (CHCym‑d); 73.0 (CHCym‑c); 53.1 (CH2, Prop‑1″); 31.1 (CHCym‑f); 23.6 (CH2, Prop‑2″); 22.8 (CH3, Cym‑g); 22.4 (CH3, Cym‑g); 18.7 (CH3, Cym‑a); 11.0 (CH3, Prop‑3″) ppm. [Chlorido(1-butyl-4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazolato)(η6-pcymene)osmium(II)] (3d). The reaction was performed according to the general complexation procedure, using 1d (102 mg, 0.51 mmol), [Os(p-cym)Cl2]2 (200 mg, 0.25 mmol) and anhydrous sodium acetate (46 mg, 0.56 mmol) in methanol abs. (5 mL) for a reaction time of 48 h. Yield: 222 mg, 78%. ESI-HR-MS+ m/z Found (Calculated): [M− Cl]+ 526.1891 (526.1893), [M+Na]+ 584.1473 (584.1469), [2M +Na] + 1143.3113 (1143.3022). Elemental Anal. Calc. for C22H28ClN3Os · 0.5H2O: C 46.42, H 5.14, N 7.38; Found: C 46.44, H 5.04, N 7.31. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.01 (d, 3 JH,H = 7 Hz, 1H, ArHPh‑3′); 7.58 (s, 1H, ArHTrz‑5); 7.31 (dd; 3JH,H = 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑6′); 7.05 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑4′); 6.95 (ddd, 3JH,H = 7 Hz, 7 Hz, 4JH,H = 1 Hz, 1H, ArHPh‑5′); 5.61 (d, 3JH,H = 5 Hz, 1H, ArHCym‑d); 5.53−5.49 (m, 2H, ArHCym‑c, ArHCym‑d); 5.36 (d, 3JH,H = 5 Hz, 1H, ArHCym‑c); 4.33−4.26 (m, 1H, CH2, But‑1″); 4.17−4.10 (m, 1H, CH2, But‑1″); 2.37 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.16 (s, 3H, CH3, Cym‑a); 1.85 (tt, 3JH,H = 7 Hz, 7 Hz, 2H, CH2, But‑2″); 1.36−1.25 (m, 2H, CH2, But‑3″); 0.96−0.91 (m, 9H, CH3, But‑4″, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 298.2 K): δ = 162.5 (CPh‑2′); 157.1 (CTrz‑4); 139.4 (CHPh‑3′); 136.2 (CPh‑1′); 128.4 (CHPh‑4′); 122.7 (CHPh‑5′); 122.0 (CHPh‑6′); 117.4 (CHTrz‑5); 91.9 (CCym‑b); 90.1 (CCym‑e); 78.9 (CHCym‑c); 78.5 (CHCym‑d); 76.3 (CHCym‑d); 73.1 (CHCym‑c); 51.3 (CH2, But‑1″); 32.1 (CH2, But‑2″); 31.1 (CHCym‑f); 22.8 (CH3, Cym‑g); 22.4 (CH3, Cym‑g); 19.7 (CH2, But‑3″); 18.7 (CH3, Cym‑a); 13.5 (CH3, But‑4″) ppm. [Chlorido(methyl-2″-(4-(2′-κC)-phenyl-(3-κN)-1,2,3-triazol-1ylato)acetato)(η6-p-cymene) osmium(II)] (3e). The reaction was performed according to the general complexation procedure, using 1e (110 mg, 0.51 mmol), [Os(p-cym)Cl2]2 (200 mg, 0.25 mmol) and anhydrous sodium acetate (46 mg, 0.56 mmol) in methanol abs. (3 mL) for a reaction time of 48 h. Yield: 152 mg, 52%. ESI-HR-MS+ m/z Found (Calculated): [M−Cl]+ 542.1494 (542.1478), [M+Na]+ 600.1069 (600.1054), [2M+Na]+ 1175.2267 (1175.2192). Elemental Anal. Calc. for C21H24ClN3O2Os: C 43.78, H 4.20, N 7.29, S 0.00, Found: C 43.80, H 4.28, N 7.06, S < 0.02. 1H NMR (CDCl3, 500.10 MHz, 297.2K): δ = 8.03 (d, 3JH,H = 7 Hz, 1H, ArHPh‑3′); 7.79 (s, 1H, ArHTrz‑5); 7.23 (d, 3JH,H = 7 Hz, 1H, ArHPh‑6′); 7.09 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑4′); 6.98 (dd, 3JH,H = 7 Hz, 7 Hz, 1H, ArHPh‑5′); 5.60 J DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry OLEX2.74 Experimental parameters can be found in Table S1. Crystal data, data collection parameters, and structure reﬁnement details are given in Tables S10−S29. Molecular Structures in “Ortep View” are given in Figure S5−S14. A tabular overview for half sandwich structure parameters is given in Table S2. The parameters for π−π and σ−π interactions and their graphical representation can be found in Table S7−S9 and Figure S4 respectively. Disordered moieties are excluded from detailed analysis in all tables listing structural parameters, due to constraints and restraints. cLogP calculation. The octanol−water partition coeﬃcient LogP was calculated for the free ligands with Molinspiration (v2014.11). The cLogP values allow the comparison of relative lipophilicities within a series of osmium(II)- or ruthenium(II)-arene metalacycles, as the metal-arene fragments remain unchanged and should add a constant contribution to the overall lipophilicity, while the ligands are varied. Cell culture. CH1/PA-1 (kindly provided by Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK; identiﬁed through STR proﬁling as PA-1 ovarian teratocarcinoma cells76 by Multiplexion, Heidelberg, Germany), SW480 (colon carcinoma; from ATCC) and A549 (non-small cell lung cancer; from ATCC) cells were grown as adherent monolayer cultures in 75 cm2 culture ﬂasks (Starlab, UK) in minimal essential medium (MEM; Sigma-Aldrich) supplemented with 10% heatinactivated fetal bovine serum (Invitrogen or Biowest), 1 mM sodium pyruvate, 4 mM L-glutamine, and 1% non-essential amino acids from 100× ready-to-use stock solution (all purchased from Sigma-Aldrich). Cell cultures were incubated at 37 °C in a moist atmosphere containing 5% CO2 in air. MTT assay. The cytotoxicity of the compounds was determined by the colorimetric MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide). For this purpose, cells were harvested from culture ﬂasks by trypsinization, seeded in 100 μL aliquots into 96-well microculture plates (Starlab, UK) in densities of 1 × 103 (CH1/PA-1), 2 × 103 (SW480) and 3 × 103 (A549) cells per well, and incubated for 24 h prior to exposure to the test compounds. Stock solutions of test compounds were prepared in DMSO or DMF, which were then diluted in MEM (not to exceed a ﬁnal content of 0.5% v/v of organic solvent in the test plates), and serial dilutions were added in aliquots of 100 μL per well. After continuous exposure for 96 h, drug solutions were replaced with 100 μL medium/MTT mixtures [6 parts of RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum and 2 mM L-glutamine; 1 part of MTT solution in phosphate-buﬀered saline (5 mg/mL)]. After incubation for 4 h, the medium/MTT mixtures were removed, and the produced formazan crystals were dissolved in 150 μL DMSO per well. Optical densities at 550 nm were measured spectrophotometrically (ELx808 Absorbance Microplate Reader, Bio-Tek, USA) by using a reference wavelength of 690 nm to correct for unspeciﬁc absorption. 50% inhibitory concentrations (IC50) were calculated from concentration−eﬀect curves by interpolation based on at least three independent experiments, each comprising triplicates per concentration level. Cellular accumulation. Cellular accumulation of the compounds was studied based on a method described previously77 with minor modiﬁcations. 1.2 × 105 SW480 cells per well were seeded into six-well plates in aliquots of 2.5 mL complete MEM (see above) and incubated at 37 °C for 24 h. Then, cells were exposed for 2 h at 37 °C to 50 μM solutions of the test compounds (containing 0.5% DMSO) in fresh 2.5 mL of complete MEM upon exchange of the medium. Afterward, cells were washed three times with PBS, and lysed with 0.4 mL subboiled HNO3 per well for 1 h at room temperature. Ruthenium content was quantiﬁed by inductively coupled plasma mass spectrometry (ICPMS) using an ICP-quadrupole MS Agilent 7500ce instrument (Agilent Technologies, Waldbronn, Germany) equipped with a CETAX ASX520 autosampler (Nebraska, USA) and a MicroMist nebulizer. Ruthenium and indium standards were obtained from CPI International (Amsterdam, The Netherlands). The instrument was equipped with nickel cones and operated at an RF power of 1560 W, with argon as the plasma gas (15 L min−1), carrier gas (1.0−1.1 L min−1) and thanesulfonate (3e′). The reaction was performed according to the general leaving group exchange procedure, using 3e (100 mg, 0.17 mmol), DMSO (25 μL, 0.35 mmol) and AgOTf (90 mg, 0.35 mmol). Yield: 98 mg, 73%. ESI-HR-MS+ m/z Found (Calculated): [M− DMSO]+ 542.1479 (542.1478), [M]+ 620.1620 (620.1616). Elemental Anal. Calc. for C24H30F3N3O6OsS2: C 37.54, H 3.94, N 5.47, S 8.35; Found: C37.46, H 3.96, N 5.38, S 8.28. 1H NMR (CDCl3, 500.10 MHz, 298.2 K): δ = 8.48 (s, 1H, ArHTrz‑5); 7.86−7.83 (m, 1H, ArHPh‑3′); 7.60−7.56 (m, 1H, ArHPh‑6′); 7.20−7.13 (m, 2H, ArHPh‑4′, ArHPh‑5′); 5.90−5.86 (m, 2H, ArHCym‑c, ArHCym‑d); 5.70 (d, 3JH,H = 6 Hz, 1H ArHCym‑d); 5.60 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 5.56 (d, 3JH,H = 6 Hz, 1H, ArHCym‑c); 5.53 (d, 2JH,H = 18 Hz, 1H, CH2, Ac); 3.83 (s, 3H, OCH3, Ac); 2.77 (sept, 3JH,H = 7 Hz, 1H, CHCym‑f); 2.52 (s, 3H, CH3, DMSO); 2.49 (s, 3H, CH3, DMSO); 2.33 (s, 3H, CH3, DMSO); 1.12 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g), 1.03 (d, 3JH,H = 7 Hz, 3H, CH3, Cym‑g) ppm. 13C NMR (CDCl3, 125.75 MHz, 298.5 K): δ = 167.1 (COAc); 158.0 (CTrz‑4); 152.3 (CPh‑2′); 140.5 (CHPh‑3′); 136.3 (CPh‑1′); 130.0 (CHPh‑4′); 125.4 (CHPh‑5′); 124.0 (CHPh‑6′); 122.9 (CHTrz‑5); 110.5 (CCym‑e); 103.2 (CCym‑b); 83.3 (CHCym‑c); 83.0 (CHCym‑d); 82.4 (CHCym‑c); 82.0 (CHCym‑d); 53.4 (OCH3, Ac); 52.7 (CH2, Ac); 47.1 (CH3, DMSO); 44.2 (CH3, DMSO); 30.7 (CHCym‑f); 22.8 (CH3, Cym‑g); 22.2 (CH3, Cym‑g); 18.7 (CH3, Cym‑a) ppm. Stability in aqueous solution by UV−vis spectroscopy. Stock solutions (2 mM) of complexes in DMSO or DMF were prepared, diluted with phosphate buﬀer (67 mM, pH 7.4) to a concentration of 20 μM in 1% DMSO or DMF/buﬀer, ﬁltered, and UV−vis spectra recorded in 15 min intervals for 24 h at 25 °C on a PerkinElmer Lambda 35 UV−vis spectrophotometer. Stability in aqueous solution and amino acid binding studies by ESI-MS. Stock solutions of complexes in DMSO or DMF were prepared and diluted to concentration of 5 μM complex in 1% DMSO or DMF in 400 μM ammonium acetate solution (pH 7.4) or water. For competitive amino acid binding experiments, complex stock solutions were incubated with 400 μM ammonium acetate solution (pH 7.4) containing 5 μM L-cysteine, L-histidine, and L-methionine. Aliquots were taken after 0, 1, 3, 6, and 24 h of incubation at 37 °C and stored at −20 °C until analysis. Electrospray ionization mass spectra of the collected samples were recorded on a Bruker AmaZon SL ion trap mass spectrometer by direct infusion after dilution with methanol (1:1). Aquation-anation equilibrium by NMR spectroscopy. Stock solutions of 2e and 3e in d7-DMF (5 mM) were diluted to a ﬁnal concentration of 500 μM with pure D2O, or D2O containing 5 mM and 154 mM NaCl respectively, whereupon NMR spectra acquisition was started as fast as possible (∼3.5 min after sample preparation). To test the reversibility of aquation, a 500 μM solution of 2e prepared as described above was allowed to fully hydrolyze for 30 min, then appropriate amounts of solid NaCl were added to an overall concentrations of 5 mM, 154 mM and 1 M and NMR spectra measured. Stability toward aqueous phosphate buﬀers by NMR spectroscopy. Stock solutions (5 mM) of 2e and 3e in d6-DMSO and d7-DMF respectively were diluted with D2O containing a 12 mM phosphate buﬀer at pD 7.9 (equals pH 7.4)69 to 10% DMF/DMSO in D2O. Subsequently, 1H and 31P NMR spectra were recorded over 24 h. Single crystal X-ray diﬀraction analysis. The X-ray intensity data was measured on a Bruker D8-Venture or a Bruker X8-APEX2 diﬀractometer equipped with multilayer monochromators, Mo K/a INCOATEC micro focus sealed tubes (λMo = 0.71073 Å) and Kryoﬂex cooling devices. All samples were uploaded to the CCDC, for CCDC codes see Table S1. The structures were solved by either direct methods or charge ﬂipping, and reﬁned by full-matrix least-squares techniques. Non-hydrogen atoms were reﬁned with anisotropic displacement parameters. Hydrogen atoms were inserted in calculated positions and reﬁned with a riding model as rotating systems. The following software was used: Frame integration - Bruker SAINT sof tware package70 using a narrow-frame algorithm; Absorption correction - SADABS;71 structure solution - SHELXS-97;72 reﬁnement - SHELXL-2014,73 OLEX2,74 SHELXLE;75 molecular diagrams K DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry make up gas (0.1−0.2 L min−1). The Agilent MassHunter software package (Workstation Software, Version B.01.01, Build 123.11, Patch 4, 2012) was used for data processing. Adsorption/desorption blank data were subtracted from the data for the corresponding accumulation sample, and the ruthenium content is given relative to the cell number. Results are based on three independent experiments, each comprising triplicate samples. Monitored isotopes were 101Ru, 102 Ru and 115In, the latter is being used as internal standard. The ICPMS was tuned on a daily basis to achieve maximum sensitivity. Topoisomerase IIα assay. Topoisomerase IIα inhibition experiments were conducted with a topoisomerase II drug screening kit from Enzo Life Sciences according to the manufacturer guidelines. First, two 1% agarose gels were prepared, one containing 0.75 μg/mL EtBr and the other without EtBr. All test compounds were dissolved and diluted in DMF so that a maximum of 0.625% DMF was loaded onto the gel, while 2b was additionally dissolved and diluted in DMSO to a ﬁnal concentration of 0.08% DMSO in the gel. 800 μM etoposide (VP-16) was used as a positive control. Linear DNA was used as a marker and 0.625% DMF was used as a solvent control. DMSO was previously shown to not inhibit topoisomerase IIα in the used concentrations.65 Two mastermixes were prepared, the ﬁrst containing 10 μL water and 1 μL DNA per reaction and the second containing 1 μL topoisomerase IIα and 2 μL buﬀer A and 2 μL buﬀer B per reaction. Reaction tubes were incubated at 37 °C for 30 min. The compound was added in 4 μL of water, the ﬁnal reaction volume was 20 μL. Reaction was stopped by using 2 μL 10% SDS. Afterward, proteinase K was added to digest the enzyme at 37 °C for 15 min. Then loading dye was added and each sample split in two. Half of the sample was loaded onto the EtBr containing gel, the other half was loaded onto the other agarose gel. Gels were run with 60 V until they reached 30% of the gel and then voltage was increased to 80 V until the staining front reached the edge of the gel. The EtBr containing gel was washed for 20 min in running buﬀer. The other gel was stained for 20 min in running buﬀer containing 0.75 μg/mL EtBr. Pictures were taken by using a Fusion FX7 system (Vilber Lourmat, Germany). Two independent experiments were performed. Cell cycle analysis. 6 ×104 cells (SW480 cell line) were seeded into 24-well plates and allowed to adhere for 24 h at 37 °C under 5% CO2 before treatment. Cells were treated with indicated concentrations for 48 h at 37 °C under 5% CO2. For this purpose, the test substances were dissolved in DMF and diluted to diﬀerent concentrations in MEM. Positive controls were 0.01 μM and 0.05 μM gemcitabine. After treatment the cells were viable and 80−90% conﬂuent. Cells were washed once with PBS, detached by using 150 μL trypsin-EDTA. After trypsinization was stopped by adding 850 μL of MEM, cells were collected in microreaction tubes and centrifuged at 300g at RT for 3 min. Supernatants were discarded and cells washed once with 1 mL PBS. Cells were then resuspended in 300 μL PI/HSF solution (50 μg/mL) and stained overnight. Measurement was performed on a Guava easyCyte ﬂow cytometer (Millipore). Cells were sorted by forward and side scatter and the red ﬂuorescence measured dependent on the cell number. At least three independent experiments were performed. Analysis was performed by using the cell cycle function in FlowJo software (TreeStar, Inc., Ashland, OR, USA). ■ ■ with 1e, 2e, and 3e; 1H and 13C NMR spectra of 2a−e, 3a−e, 2a′, 2e′, and 3e′ (PDF) Crystallographic data for 2a−e and 3a−e (CIF) AUTHOR INFORMATION Corresponding Author *E-mail: wolfgang.kandioller@univie.ac.at; Tel: +43-1-427752609; Fax: +43-1-4277-9526. ORCID Wolfgang Kandioller: 0000-0002-5630-712X Notes The authors declare no competing ﬁnancial interest. ■ ■ ACKNOWLEDGMENTS We thank the University of Vienna for ﬁnancial support. REFERENCES (1) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. From Bench to Bedside − Preclinical and Early Clinical Development of the Anticancer Agent Indazolium Trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J. Inorg. Biochem. 2006, 100, 891−904. (2) Thompson, D. S.; Weiss, G. J.; Jones, S. F.; Burris, H. A.; Ramanathan, R. K.; Infante, J. R.; Bendell, J. C.; Ogden, A.; Von Hoﬀ, D. D. NKP1339 Phase I Trial. J. Clin. Oncol. 2012, 30 (Suppl.), Abstr. 3033. (3) Jakupec, M. A.; Reisner, E.; Eichinger, A.; Pongratz, M.; Arion, V. B.; Galanski, M.; Hartinger, C. G.; Keppler, B. K. Redox-Active Antineoplastic Ruthenium Complexes with Indazole: Correlation of in Vitro Potency and Reduction Potential. J. Med. Chem. 2005, 48, 2831− 2837. 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(11) Kandioller, W.; Hartinger, C. G.; Nazarov, A. A.; Kuznetsov, M. L.; John, R. O.; Bartel, C.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. From Pyrone to Thiopyrone Ligands−Rendering Maltol-Derived Ruthenium(II)−Arene Complexes That Are Anticancer Active in Vitro. Organometallics 2009, 28, 4249−4251. ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02430. Synthesis and characterization of ligands 1a−e; X-ray diﬀraction experimental and acquisition parameters; CCDC codes; geometry parameters; crystal structures in ORTEP view; sample and crystal data; data collection and reﬁnement parameters; UV−vis stability spectra; ESI-MS stability and amino acid interaction spectra of 2a; aquation−anation curve of 3e; topoisomerase IIα screening assay; cell cycle distribution after treatment L DOI: 10.1021/acs.inorgchem.6b02430 Inorg. Chem. 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