Anticancer Ruthenium(η⁶-p-cymene) Complexes of Nonsteroidal Anti-inflammatory Drug Derivatives

Article pubs.acs.org/Organometallics Anticancer Ruthenium(η6‑p‑cymene) Complexes of Nonsteroidal Anti-inflammatory Drug Derivatives Farhana Aman,†,‡ Muhammad Hanif,†,§ Waseeq Ahmad Siddiqui,*,‡ Adnan Ashraf,†,‡ Lukas K. Filak,† Jóhannes Reynisson,† Tilo Söhnel,† Stephen M. F. Jamieson,∥ and Christian G. Hartinger*,† † School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan § Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan ∥ Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand ‡ S Supporting Information * ABSTRACT: Oxicams are a versatile family of heterocyclic compounds, and the two representatives meloxicam and piroxicam are widely used drugs for the treatment of a variety of inflammatory and rheumatic diseases in humans. As cancerassociated inflammation is known to occur in carcinogenesis, we aimed to combine compounds carrying bioactive oxicam moieties with ruthenium(arene) fragments, known for anticancer activity. RuII(arene) complexes with methyl ester derivatives of the oxicam scaffold were prepared and characterized by standard methods and crystallographically. The organoruthenium compounds formed from RuII(η6-p-cymene) chlorido moieties and oxicam-based ligands were subjected to bioanalytical investigations to establish their physicochemical properties with regard to stability in DMSO and water as well as reactivity toward the amino acids L-histidine (His), L-methionine (Met), and L-cysteine (Cys) and the DNA model compound guanosine 5′-monophosphate (5′-GMP). The compounds hydrolyzed rapidly in water to give the respective aqua complexes, formed amino acid complexes with Met and His, but decompose with Cys, while interaction with 5′-GMP was through its phosphate residue. The anticancer activity of the complexes against the colon carcinoma HCT116 and breast cancer MDA MB 231 cancer cell lines was established using an in vitro assay. The cytotoxicity was found strongly dependent on the lipophilicity of the compound, as was shown through correlation with log kw and clog P values of the ligands. The most lipophilic compound [chlorido(methyl 4-oxido-2-benzyl-2H-1,2-benzothiazine-3-carboxylate-1,1-dioxide)(η6-p-cymene)ruthenium(II)] was the most active in the cell assays, with an IC50 of 80 μM in HCT116 cells. novel anticancer agents, often with nonclassic modes of action.6−8 RAPTA-C ([RuII(cym)(PTA)Cl2], PTA = 1,3,5triaza-7-phosphatricyclo[3.3.1.1]decane; cym = η6-p-cymene) and RM175 ([RuII(η6-biphenyl)(en)Cl]+, en = ethylenediamine) are the two lead structures that have been extensively studied. Despite only small differences in their structures, these compounds exhibit contrasting biological activities and modes of action. In preclinical tests, the anticancer activity of RM175 was similar to that of cisplatin and it was more active in cisplatin-resistant in vivo models, while RAPTA-C emerged as an antimetastatic agent in vivo. Both lead compounds were extensively modified at both the arene moiety and the mono- or bidentate ligand systems to develop structure−activity relationships and to equip them with functional groups to increase their targeted properties.9−18 In addition to these two lead structures, several other classes of compounds based on the Ru(arene) backbone have been Metallochemotherapeutics and in particular platinum-based complexes are well-established treatment options for a wide array of neoplastic diseases. The anticancer activity of platinum drugs is attributed to their ability to bind DNA.1 Their remarkable success has been marred somewhat due to intrinsic and acquired resistance of tumors and adverse side effects.2 With the aim to improve efficacy and reduce undesirable side effects, a large number of complexes of platinum and other metals were tested for their tumor-inhibiting properties. Out of those, ruthenium complexes emerged as a promising class of anticancer agents,3 and two Ru(III) complexes are currently in phase II clinical trials. NAMI-A (imidazolium trans[tetrachlorido(dimethyl sulfoxide)(1H-imidazole)ruthenate(III)]) inhibits metastasis, whereas KP1019 (indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) and also its more water-soluble analogue KP1339 (sodium trans[tetrachloridobis(1H-indazole)ruthenate(III)]) showed potent activity in a number of primary human tumor models.4,5 In recent years a half-sandwich-configured organoruthenium(arene) scaffold emerged as a versatile tool for the design of © XXXX American Chemical Society Received: August 14, 2014 A dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article Herein, we report bimodal anticancer agents based on a Ru moiety featuring a labile chlorido ligand that may allow the compound to bind covalently in a monodentate fashion to a target, such as DNA. The target molecule features on the other hand an oxicam ligand that may bind to a COX enzyme. The preparation and characterization of the complexes are complemented by cytotoxicity studies, molecular docking experiments, and binding experiments to biological molecules. investigated by replacing the PTA and en ligands with other mono-, bi-, and tridentate ligand systems.3,7 It has been shown that such modification has a major impact on the anticancer activity of organoruthenium complexes. A recent trend has been to introduce chelating ligands derived from bioactive organic compounds.3,7,19−25 Many of these examples act as O,O-bidentate ligand systems, forming five-membered rings with the metal center. It was shown that Ru complexes of pyrones undergo quick chlorido/aqua ligand exchange reactions, and such hydrolysis products were capable of forming adducts with DNA model compounds.7,26−29 Coordination of the pyrone-related flavonols to Ru centers not only enhanced the bioavailability of flavonols but also provided potent multitarget antitumor agents. In addition to covalent binding to DNA, these compounds inhibit topoisomerase IIα.20,21 Replacing pyrones with pyridone-based ligand systems resulted notably in relatively stable Ru(arene) complexes with significant cytotoxicity. In particular, dinuclear ruthenium complexes linked through pyridinone-based spacers were shown to exhibit strong anticancer properties comparable to oxaliplatin in human tumor cell lines while being able to crosslink DNA duplexes and DNA with proteins.30,31 These studies showed the potential of Ru(arene) complexes carrying O,O-bidentate ligands and prompted us to further explore this class of compounds. Turel et al. reported the Ru(arene) complexes of nalidixic acid, ofloxacin, and other antibacterial compounds.32−34 Such ligands also feature bidentate O,O-donor systems forming six-membered chelates with the metal center. This is a chelation motif that may also be obtained with ligands based on the oxicam scaffold. Oxicams such as piroxicam and meloxicam (Chart 1) belong to the class ■ RESULTS AND DISCUSSION The oxicam structure can be used as a scaffold to build a library of biologically active compounds. We synthesized a series of Ru(cym) complexes with O,O-chelating ligands derived from the oxicam backbone. The oxicam derivatives 1a−1d were prepared by N-alkylation of 4-hydroxy-1,1-dioxo-1,2-dihydro1λ6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester using a previously reported procedure.43 Ligands 1a−1d were converted into the respective Ru(cym) complexes 2a−2d by activating the oxicam ligands by deprotonation of the hydroxyl group at position 4. For this purpose, NaOMe was added to a stirred suspension of the ligand in dry MeOH. After stirring for about 30 min, the suspension changed in color to give a light yellow, clear solution. The Ru dimer [Ru(cym)Cl2]2 was added to this solution, and the reaction mixture was stirred for 4 h at room temperature, which resulted in precipitation of red (2a, 2b) or yellow (2c, 2d) solids in yields of 49−61%. When carrying out the same reaction under the same conditions but without activation of the ligands, no precipitation was observed, although the 1H NMR spectra indicated incomplete complex formation. Prolonged reaction times up to 24 h did not lead to complete conversion of the starting material, and workup involving evaporation of the solvent mixture under reduced pressure followed by extraction with DCM and diethyl ether afforded the products only in very low yields (<10%). Chart 1. Structures of the Two Nonsteroidal Antiinflammatory Drugs (NSAIDs) Piroxicam (Left) and Meloxicam (Right) Scheme 1. Synthetic Route to RuII(η6-p-cymene) Complexes of Oxicam-Derived Ligands and NMR Numbering Scheme for Complexes 2a−2d of nonsteroidal anti-inflammatory drugs (NSAIDs) and are highly efficient as treatments of rheumatologic diseases and to reduce inflammatory effects after surgery. NSAIDs exert their activity through inhibition of the cyclooxygenase (COX)mediated production of prostaglandins (PG) or COXindependent mechanisms.35 Cyclooxygenases (COXs) are known to play a role in tumor growth, progression, migration, and angiogenesis,36 which makes them interesting targets to develop anticancer agents with higher selectivity than established drugs, and several oxicam derivatives were shown to inhibit cyclooxygenases.37 In recent years several antiinflammatory drug derivatives were coordinated to metal centers to improve their biological activity. For example, Ott et al. developed cobalt-modified acetylsalicylic acid derivatives with promising antiproliferative properties.38,39 Copper and zinc complexes of the anti-inflammatory drug indomethacin40 and cobalt(II) complexes with the NSAIDs mefenamic acid, tolfenamic acid, and naproxen have also been reported.41,42 Such strategies often result in enhanced biological activity compared to the parent drugs. All the compounds were characterized by elemental analysis, ESI-MS, 1D and 2D NMR spectroscopy, and single-crystal Xray diffraction analysis (2b−2d). Complex formation was indicated by disappearance of the O−H signal of the ligands in the 1H NMR spectra at around 12 ppm and appearance of additional signals corresponding to the cym protons. An upfield shift for H12 (0.03−0.16 ppm) and NC−H (0.25−0.51 ppm) signals was observed upon complexation. This is probably due to the increased delocalization of electron density between two C−O groups at positions 4 and 11 of the ligand after formation of the coordination compound. B dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article The formation of the complexes was also confirmed by ESIMS. The mass spectra recorded in positive ion mode featured peaks at m/z values in close agreement with the expected values for [M − Cl]+ ions, after cleavage of the labile chlorido ligand from the metal complex during the ionization process. X-ray diffraction analyses of complexes 2b, 2c, and 2d revealed a piano-stool configuration, which is characteristic of metal(arene) complexes.7,27 The oxicam-derived ligands acted as O,O-chelating bidentate ligands and formed six-membered chelate rings upon binding to the RuII center (Figure 1). Notably, in the case of 2b only a single enantiomer was identified in the molecular structure, whereas the crystal structures of 2c and 2d featured two chiral-at-metal center enantiomers (Figures S1 and S2). The Ru−cymcentroid distances were 1.642, 1.653, and 1.641 Å, and the Ru__Cl bond lengths were 2.403(1), 2.420(1), and 2.396(1) Å in 2b, 2c, and 2d, respectively (Tables S2 and S3). These parameters were very similar to those observed for structurally related [Ru(cym)(3hydroxy-2-pyridone)Cl] complexes.27 The oxicam scaffold was nonplanar with regard to the heterocyclic ring. The nitrogen was significantly out of plane, and the torsion angles (C−C−SO2−N) were determined as 27.40°, 31.74°, and 37.69° for 2b, 2c, and 2d. This may be related to the substituents at the ring-nitrogen atom, especially with the benzyl residue tending to form π-stacking interactions with the oxicam phenyl ring and C···C distances as short as 3.321(5) Å (Figure S3). Coordination of the Ru(cym) fragment to the oxicam ligands impacted especially the C4− O2 bond lengths, which were found in 1b−1d44−46 to be 1.343(4), 1.344(2), and 1.339(17) Å. These bonds are significantly shorter in the respective complexes 2b−2d (1.280(4), 1.293(3), and 1.283(4) Å, respectively), whereas the C11−O1 bond was slightly elongated due to coordination to the metal center (Tables S2 and S3). In addition, the C3− C4 distances were found to be 1.392(4), 1.389(3), and 1.397(4) Å in 2b−2d, respectively, and were therefore significantly longer than in 1b−1d (1.343(5), 1.346(3), and 1.369(2) Å). This indicates more single-bond character of this bond than in the free ligand, resulting in a conjugated system involving O1, C11, C3, C4, and O2. Lipophilicity. The lipophilicity of bioactive compounds has a major impact on their accumulation in cells and especially the penetration through cell membranes. Complexes 2a−2d are well soluble in apolar organic solvents such as dichloromethane and chloroform. The solubility in polar solvents especially in water is limited. Taking the solubility of the organoruthenium compounds in aqueous solutions as a measure for lipophilicity, they follow the order 2a > 2b > 2c > 2d (0.34, 0.33, 0.30, and 0.28 mM in 1% DMSO/water, respectively). This pattern may be explained by the increasing size of the substituents at the nitrogen atom in 2a < 2b < 2c < 2d. In order to quantify this observation, calculated logarithmic octanol−water partition coefficients (clog P) of 1a−1d were obtained by using the software tool Molinspiration (http://www.molinspiration.com. Experimental capacity factors (log k′) of 1a−1d were determined by HPLC using MeOH/water mixtures containing 40−75% MeOH as eluent (Table 1). Compounds 1a−1d were chosen, as the Ru(cym)Cl moiety is present in all complexes and therefore has no significant impact on the relative values. Furthermore, the complexes were found to decompose during HPLC analysis, resulting in a series of peaks including some that were assigned to an uncoordinated ligand. In order to minimize the influence of the solvent and the column Figure 1. ORTEP diagrams of enantiomers of 2b (A), 2c (B), and 2d (C) drawn at the 50% probability level. Selected bond lengths and angles around the Ru center are given in Tables S2 and S3. conditions, the log k′ values were extrapolated to 0% organic solvent, giving log kw values.47 Increasing the size of the NC dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article Table 1. In Vitro Anticancer Activity (mean IC50 values ± standard deviations) of 2a−2d in Human Colon Carcinoma (HCT116) and Breast Cancer (MDA MB 231) Cell Lines (exposure time 72 h), Logarithmic Extrapolated Capacity Factors in Pure Water (log kw),57 and Calculated (clog P) Octanol−Water Partition Coefficients IC50 values/μM a compound HCT116 MDA MB 231 log kwa clog Pa 2a 2b 2c 2d >170 >165 109 ± 20 80 ± 5 >170 >165 >150 >140 2.40 ± 0.02 2.70 ± 0.05 3.15 ± 0.06 3.58 ± 0.07 2.0 2.4 2.9 3.4 Both log kw and clog P were determined for the ligands. hydrocarbon substituent led to an increase in lipophilicity in the order 1a < 1b < 1c < 1d, with the methyl derivate 1a being the least lipophilic (log kw = 2.40, clog P = 2.0) and the benzyl derivative 1d the most lipophilic (log kw = 3.58, clog P = 3.4). Overall, the log kw and clog P values for 1a−1d were in good agreement with each other, as well as with the solubility data obtained for complexes 2a−2d. Stability in DMSO and Aqueous Solution. Stability in solution is an important requirement for drug candidates. DMSO is the most widely employed solvent to prepare stock solutions for biophysical and biological testing. For some transition metal complexes, DMSO acts as a good ligand, coordinating to the metal center through either the sulfur or oxygen atom.48 Therefore, it is imperative to establish the stability of organometallic compounds in DMSO before performing biological experiments. For this purpose, 2a and 2b (1−2 mg) were dissolved in DMSO-d6, and the integrity was monitored by 1H NMR spectroscopy for up to 72 h. The compounds were stable in the first 3 h, and after 24 h a new species was formed as indicated by a signal at about δ = 5.8 ppm (10% relative abundance to the Ru complex), most likely due to the coordination of DMSO to the Ru center. This new signal increased further up to about 25% within 72 h for both tested compounds (Figure S4). In addition, cleavage of the cymene moiety was observed corresponding to new signals emerging at δ = 7.2 ppm after 3 h incubation time. The intensity of this signal increased significantly over 72 h, giving the major decomposition product. For hydrolytic experiments, 2a and 2b were dissolved in 20% DMSO-d6/D2O due to insufficient solubility of the compounds in D2O. Again a time course was recorded using 1H NMR spectroscopy, and data sets were collected after 0.5, 3, 24, and 48 h (Figure 2). Both compounds hydrolyzed immediately by exchange of the chlorido ligand with a water molecule to give 2aaqua and 2baqua. About 10% of 2aaqua and 2baqua underwent cleavage of the oxicam ligand to form the hydroxy-bridged dimeric species [Ru2(η6-p-cymene)2(OH)3]+ after 3 h. This observation was reported for other Ru(η6-arene) complexes with similar ligand systems.7,27−29 5′-GMP Binding. DNA is the primary target of the established platinum-based anticancer agents and was also suggested as a target for organoruthenium compounds. Therefore, 2a and 2b were incubated with the nucleotide 5′GMP, and the reaction was monitored by 1H and 31P{1H} NMR spectroscopy. In the 1H NMR spectra we observed a slight downfield shift of the H-8 proton signal of 5′-GMP from 8.15 to 8.24 ppm. However, addition of an excess of 5′-GMP did not result in additional peaks. The 31P{1H} NMR spectra, Figure 2. Stability study of 2b in 20% DMSO-d6/D2O by 1H NMR spectroscopy. The time-dependent spectra recorded after 0.5, 3, 24, and 48 h are shown. however, suggested binding of the phosphate to the Ru center, as indicated by the appearance of a second signal at 3.7 ppm in addition to the peak at 2.3 ppm, assigned to 5′-GMP. This is in contrast to most other organometallic compounds, which mostly prefer to coordinate through the N7 atom rather than the phosphate. However, there have also been reports that phosphate coordination occurs initially followed by conversion to the N7 of 5′-GMP.49 Reactivity toward Amino Acids. After administration into the bloodstream, metallodrugs encounter a number of biomolecules such as proteins. Serum proteins can either act as delivery agents for anticancer metallodrugs or deactivate them before they reach their cellular target(s).3,50−52 In order to understand reactivity and biologically relevant metabolization of organoruthenium−oxicam compounds, the reactions of 2a and 2b with the amino acids His, Met, and Cys were investigated by 1H NMR spectroscopy. The amino acids were incubated with 2a and 2b in equimolar amounts in DMSO-d6/ D2O (20%), and the reactions were monitored for 48 h. His and Met coordinate to the Ru center after release of chlorido, while the bidentate oxicam chelators still remain bound to ruthenium. In the 1H NMR spectra recorded for the reaction of His with 2a, a shift was observed for the signals assigned to the imidazole protons after coordination to the metal center (Figure 3). However, the Cys thiol exhibits a strong trans-effect and leads to decomposition of the complexes within minutes, as has been reported for related Ru(η6-arene) complexes.27 In Vitro Anticancer Activity. The cytotoxicity of Ru compounds 2a−2d was evaluated in human colon carcinoma (HCT116) and breast cancer (MDA MB 231) cell lines by the sulforhodamine B (SRB) assay (Table 1). Compounds 2c and 2d exhibited modest cytotoxicity against both cancer cell lines. The lipophilicity of the complexes appears to be the determining factor for the cytotoxicity, and the most lipophilic compound, 2d, was also the most cytotoxic, with an IC50 value of 80 μM against HCT116 cells. Even though the compounds were mostly noncytotoxic in both cell lines, this is not necessarily a negative property for an anticancer drug candidate. The mechanisms of action of ruthenium compounds are still not fully understood, and the examples of NAMI-A and organometallic RAPTA derivatives, which are noncytotoxic in vitro but exhibit high activity against metastases in vivo, demonstrate that IC50 values from medium to high μM range D dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article size-restricted. This may limit the access of larger molecules such as 2a−2d. ■ CONCLUSIONS In the search for new drugs, known bioactive compounds are now often repositioned and applied for the treatment of new indications. In our efforts to develop new metallodrugs, we use proven pharmacophores and coordinate them to metal fragments with established anticancer properties. In this study, we incorporated the oxicam backbone found in the nonsteroidal anti-inflammatory drugs meloxicam and piroxicam into an organometallic Ru(arene) compound. Oxicams are wellknown inhibitors of cyclooxygenases, overexpressed in many tumors. In order to estimate the potential of the Ru(cym) complexes of different oxicam derivatives, their anticancer activity was studied in vitro, and modest cytotoxic potential was observed in HCT116 but not in MDA MB 231 cells. The most lipophilic compound, 2d, as demonstrated by HPLC and in silico studies, was the most cytotoxic in the HCT116 cell line. As shown for structurally related complexes, this may be related to the properties of coordination compounds of O,O-chelating ligands in aqueous solution and in the presence of biomolecules. Therefore, the stability of the complexes was determined by 1H NMR spectroscopy, and they were found to undergo a rapid Cl/H2O exchange reaction in aqueous solutions. In contrast, in DMSO they were stable for up to 3 h, and only later during the course of the reaction additional species appeared featuring DMSO coordinated to the Ru center and cleavage of the p-cymene ligand. In addition, the reactivity toward GMP, His, Met, and Cys as biologically relevant ligands was studied. While His and Met behave as expected and replace the chlorido ligand of the complexes, Cys causes decomposition, and interestingly GMP did not bind to the metal center through its N7, but it appears as if the phosphate moiety interacted with the Ru center. Docking studies with COX-2 as the target of oxicam NSAIDs may explain the lack of anticancer activity of the compounds. While the studies with oxicam ligands 1a−1d support binding to COX-2, the complexes gave low or negative GoldScores, suggesting targets different from those of the ligand structures. Figure 3. Reactivity of 2a with His (1:2) in 10% DMSO-d6/D2O followed by 1H NMR spectroscopy. The His adduct signals and the aromatic cymene protons are indicated. are not a sufficient reason to discard a compound from further development.53−56 Molecular Modeling. The ligands 1a−1d and complexes 2a−2d were docked into the binding pocket of the COX-2 crystal structure. Reasonable scores are found for derivatives 1a−1d for all the scoring functions akin to meloxicam, suggesting a good binding to COX-2, i.e., 51−68 for ChemPLP, 26−34 for ASP, 25−30 for CS, and finally 49−70 for the GS function (Table 4). Ligands 1a−1d showed good overlap with GS in the binding pocket of COX-2, with the methyl derivative however slightly shifted compared to the oxicam scaffold of the other ligand structures (Figures S5 and S6). Complexes 2a−2d could only be docked with GS, and the fitness scores predicted were substantially lower, in the range of 5−11 for 2a−2c and with derivative 2d giving a negative number (−26). The highest scoring configurations of ligands 1a−1d and the respective Ru(cym) complexes showed no overlap of the oxicam backbones (see Figure 4 for 2c). Interestingly, 2a and 2b, with the smaller substituents at the heterocyclic nitrogen atom, showed good overlap, and the npropyl and benzyl derivatives were well overlapped. The binding pocket of COX-2 is situated deep in the enzyme and is ■ EXPERIMENTAL SECTION All reactions were carried out in dry solvents under an inert atmosphere unless otherwise stated. Chemicals obtained from commercial suppliers were used as received and were of analytical grade. Dichloromethane and methanol were dried using standard procedures. RuCl3·3H2O (40.4%) was purchased from Precious Metals Online; α-terpinene from Sigma-Aldrich; and L-histidine (His), Lmethionine (Met), and L-cysteine (Cys) from AK Scientific Inc. Guanosine 5′-monophosphate disodium salt (5′-GMP) was purchased from Fluka. The dimer bis[dichlorido(η6-p-cymene)ruthenium(II)]58 and the N-alkyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester59 were synthesized by adapting reported procedures. Melting points were measured using a SMP30 Stuart Scientific melting point apparatus. Elemental analyses for all compounds were performed at the Campbell Microanalytical Laboratory, The University of Otago. Highresolution mass spectra were recorded on the Bruker microTOF-Q II electrospray ionization (ESI) mass spectrometer in positive ion mode. 1 H, 31P{1H}, and 13C{1H} NMR spectra were recorded on Bruker DRX 400 MHz NMR spectrometers at ambient temperature at 400.13 (1H), 161.98 (31P{1H}), and 100.61 MHz (13C{1H}), and 2D NMR data were collected in a gradient-enhanced mode. 1H and 13C{1H} chemical shifts are reported vs SiMe4 and were determined by Figure 4. Overlay of the highest scoring docking configurations of 2c in the binding site of meloxicam in COX-2. E dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article reference to the residual 1H and 13C{1H} solvent peaks. All compounds were analyzed via multinuclear 2D (1H−1H COSY, 1 H−13C HSQC, and HMBC) NMR spectroscopic experiments, allowing unambiguous assignments of characteristic resonances. The X-ray diffraction data of crystals of 2b−2d were collected on a Bruker Smart APEX II diffractometer with graphite-monochromatized Mo Kα radiation, λMo = 0.710 73 Å at 100 K (see Table S1 for the measurement parameters). Data reduction was carried out using the SAINT program.60 Semiempirical absorption corrections were applied based on equivalent reflections using SADABS.61 The structure solution and refinements were performed with the SHELXL-2013 program package.62 General Procedure for Synthesis of Ru Complexes. Sodium methoxide (1.2 equiv) was added to a stirred suspension of methyl Nalkyl-1,1-dioxo-4-hydroxy-2H-1,2-benzothiazine-3-carboxylate (1.0 equiv) in dry methanol under an inert atmosphere. The dimer [Ru(η6-p-cymene)Cl2]2 (0.5 equiv) was added to the reaction mixture, and the reaction was further stirred at room temperature for 4 h. The reaction mixture was concentrated under reduced pressure, and the products were filtered, washed with diethyl ether (5 × 3 mL), and dried under vacuum. Chlorido(methyl 4-oxido-2-methyl-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide)(η6-p-cymene)ruthenium(II), 2a. The Ru(arene) complex 2a was synthesized following the general procedure using sodium methoxide (32 mg, 0.60 mmol), methyl 4hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (135 mg, 0.50 mmol), and [Ru(η6-p-cymene)Cl2]2 (153 mg, 0.25 mmol). Yield: 61% (165 mg, yellow precipitate). Mp: 224−226 °C (dec). Anal. Found: C, 47.06; H, 4.53; N, 2.67; S, 5.95. Calcd for C21H24ClNO5RuS: C, 46.79; H, 4.49; N, 2.60; S, 5.95. MS (ESI+): m/z [M − Cl]+ 504.0818. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ 8.03 (dd, 3J(H7,H8) = 8 Hz, 4J(H6,H8) = 2 Hz, 1H, H-8), 7.66 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz,1H, H-5), 7.57 (ddd, 3J(H5,H6)/ (H5,H7) = 8 Hz, 3 J(H5,H6)/ (H6,H7) = 8 Hz, 4J(H6,H8) = 2 Hz, 1H, H-6), 7.52 (ddd, 3 J(H6,H7)/ (H7,H8) = 8 Hz, 3J(H6,H7)/ (H7,H8) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H-7), 5.30−5.45 (m, 2H, H-14/H-14′), 5.27−5.24 (m, 2H, H-15/H15′), 3.86 (s, 3H, H-12), 2.94−2.87 (m, 1H, H-19), 2.64 (s, 3H, H21), 2.20 (s, 3H, H-17), 1.37 (d, 3J(H18,H19)/ (H19,H20) = 7 Hz, 3H, H-18/ H-20), 1.36 (d, 3J(H18,H19)/(H19,H20) = 7 Hz, 3H, H-18/H-20). 13C NMR (100.61 MHz, CDCl3, 25 °C): δ 169.4 (C-11), 168.1 (C-4), 135.7 (C9), 133.2 (C-10), 132.1 (C-8), 131.5 (C-5), 128.4 (C-6), 123.3 (C-7), 116.2 (C-3), 99.0 (C-13), 96.2 (C-16), 83.0, 82.3 (C-14/C-14′), 78.7, 79.8 (C-15/C-15′), 53.3 (C-12), 39.5 (C-21), 30.9 (C-19), 22.3, 22.4 (C-18/C-20), 17.8 (C-17). Chlorido(methyl 4-oxido-2-ethyl-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide)(η6-p-cymene)ruthenium(II), 2b. Complex 2b was synthesized following the general procedure using sodium methoxide (26 mg, 0.48 mmol), methyl 4-hydroxy-2-ethyl-2H-1,2benzothiazine-3-carboxylate 1,1-dioxide (113 mg, 0.40 mmol), and [Ru(η6-p-cymene)Cl2]2 (123 mg, 0.20 mmol). Yield: 52% (115 mg, dark red crystals). Mp: 198−200 °C (dec). Anal. Found: C, 47.89; H, 4.96 N, 2.53; S, 5.71. Calcd for C22H26ClNO5RuS: C, 47.78; H, 4.74; N, 2.53; S, 5.80. MS (ESI+): m/z [M − Cl]+ 518.0574. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ 8.08 (dd, 3J(H7,H8)= 8 Hz, 4J(H6,H8) = 2 Hz, 1H, H-8), 7.72 (dd, 3J(H5,H6) = 7 Hz, 4J(H5,H7) = 2 Hz, 1H, H-5), 7.61 (ddd, 3J(H5,H6)/ (H6,H7) = 8 Hz, 3 J(H5,H6)/ (H6,H7) = 8 Hz, 4J(H6,H8) = 2 Hz, 1H, H-6), 7.57 (ddd, 3 J(H6,H7)/ (H7,H8) = 8 Hz, 3J(H6,H7)/ (H7,H8) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H-7), 5.62−5.58 (m, 2H, H-14/H-14′), 5.34−5.31 (m, 2H, H-15/H15′), 3.91 (s, 3H, H-12), 3.37−3.28 (m, 1H, H-21α), 3.24−3.15 (m, 1H, H-21β), 3.04−2.94 (m, 1H, H-19), 2.29 (s, 3H, H-17), 1.44 (dd, 3 J(H18,H19)/(H19,H20) = 7 Hz, 4J(H18,H20) = 2 Hz, 6H, H-18/H-20), 0.72 (t, 3 J(H21,H22) = 6 Hz, 3H, H-20). 13C NMR (100.61 MHz, CDCl3, 25 °C): δ 170.3 (C-11), 169.5(C-4), 138.4 (C-9), 133.4 (C-10), 131.8 (C-8), 131.4 (C-5), 128.4 (C-6), 122.3 (C-7), 103.0 (C-3), 98.8 (C-13), 96.1 (C-16), 82.4, 83.0 (C-14/C-14′), 78.8, 79.8 (C-15/C-15′), 53.2 (C12), 47.6 (C-21), 35.5 (C-17), 22.3, 22.4 (C-18/C-20), 17.8 (C-19), 11.3 (C-22). Chlorido(methyl 4-oxido-2-n-propyl-2H-1,2-benzothiazine3-carboxylate 1,1-dioxide)(η6-p-cymene)ruthenium(II), 2c. Complex 2c was synthesized following the general procedure using sodium methoxide (26 mg, 0.48 mmol), methyl 4-hydroxy-2-n-propyl2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (119 mg, 0.40 mmol), and [Ru(η6-p-cymene)Cl2]2 (123 mg, 0.20 mmol). Yield: 56% (126 mg, dark red crystals). Mp: 192−193 °C (dec). Anal. Found: C, 49.00; H, 4.77; N, 2.55; S, 5.50. Calcd for C23H28ClNO5RuS: C, 48.72; H, 4.98; N, 2.47; S, 5.65. MS (ESI+): m/z [M − Cl]+ 532.0718. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ 8.08 (dd, 3J(H7,H8)= 7 Hz, 4J(H6,H8) = 2 Hz, 1H, H-8), 7.71 (dd, 3J(H5,H6) = 7 Hz, 4J(H5,H7) = 2 Hz, 1H, H-5), 7.61 (ddd, 3J(H5,H6) = 8 Hz, 3J(H6,H7) = 8 Hz, 4J(H6,H8) = 2 Hz, 1H, H-6), 7.57 (ddd, 3J(H6,H7)/ (H7,H8) = 8 Hz, 3 J(H6,H7)/ (H7,H8) = 8 Hz, 4J(H5,H7) = 1 Hz, 1H, H-7), 5.61−5.58 (m, 2H, H-14/H-14′), 5.33−5.31 (m, 2H, H-15/H-15′), 3.91 (s, 3H, H-12), 3.16−3.06 (m, 2H, H-21), 3.02−2.95 (m, 1H, H-19), 2.28 (s, 3H, H17), 1.44 (d, 3J(H18,H19)/(H19,H20) = 7 Hz, 3H, H-18/H-20), 1.43 (d, 3 J(H18,H19)/(H19,H20) = 7 Hz, 3H, H-18/H-20), 1.33−1.24 (m, 1H, H22α), 1.09−0.99 (m, 1H, H-22β), 0.59 (t, 3J(H22,H23) = 7 Hz 3H, H-23). 13 C NMR (100.61 MHz, CDCl3, 25 °C): δ 170.1(C-11), 169.5 (C-4), 138.1 (C-9), 133.2 (C-10), 131.8 (C-8), 131.4 (C-5), 128.4 (C-6), 122.4 (C-7), 103.8 (C-3), 98.9 (C-13), 96.1 (C-16), 83.0, 82.4 (C-14/ C-14′), 79.8, 78.8 (C-15/C-15′), 54.6 (C-12), 53.2 (C-21), 30.9 (C19), 22.3, 22.4 (C-18/C-20), 19.4 (C-22), 17.8 (C-17), 10.9 (C-23). Chlorido(methyl 4-oxido-2-benzyl-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide)(η6-p-cymene)ruthenium(II), 2d. Complex 2d was synthesized following the general procedure using sodium methoxide (26 mg, 0.48 mmol), methyl 4-hydroxy-2-benzyl-2H-1,2benzothiazine-3-carboxylate 1,1-dioxide (138 mg, 0.40 mmol), and [Ru(η6-p-cymene)Cl2]2 (123 mg, 0.20 mmol). Yield: 49% (120 mg, orange crystals). Mp: 233−234 °C (dec). Anal. Found: C, 52.80; H, 4.72 N, 2.31; S, 5.25. Calcd for C28H31ClNO5RuS: C, 52.72; H, 4.59; N, 2.28; S, 5.21. MS (ESI+): m/z [M − Cl]+ 580.0727. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ 7.71−7.68 (m, 1H, H-8), 7.55−7.53 (m, 1H, H-5), 7.39−7.36 (m, 2H, H-6/H-7), 7.11−7.09 (m, 2H, H-24/H-26), 6.92−6.90 (m, 3H, H-23, H-25, H-27), 5.60 (d, 3J(H14,H15)/(H14′,H15′) = 6 Hz, 1H, H-14/H-14′), 5.56 (d, 3J(H14,H15)/(H14′,H15′) = 6 Hz, 1H, H-14/H-14′), 5.31−5.29 (m, 2H, H-15, H-15′), 4.42 (d, 2J(H21α,H21β) = 14 Hz, 1H, H-21β), 4.15 (d, 2 J(H21α,H21β) = 14 Hz, H-21α), 3.79 (s, 3H, H-12), 2.98−2.91 (m, 1H, H-19), 1.43 (d, 3J(H18,H19)/(H19,H20) = 7 Hz, 3H, H-18/H-20), 1.42 (d, 3 J(H18,H19)/(H19,H20) = 7 Hz, 3H, H-18/H-20). 13C NMR (100.61 MHz, CDCl3, 25 °C): δ 170.8 (C-11), 169.6 (C-4), 138.1 (C-9), 133.4 (C10), 132.6 (C-22), 131.4 (C-8), 130.7 (C-5), 130.5 (C-24/C-26), 127.7 (C-6), 127.4 (C-23/C-27), 127.3 (C-25), 122.23 (C-7), 98.9 (C-3), 98.5 (C-13), 96.3 (C-16), 83.1, 82.4 (C-14/C-14′), 79.8, 78.6 (C-15/C-15′), 56.4 (C-12), 53.1 (C-21), 30.9 (C-19), 22.2, 22.4 (C18/C-20), 17.8 (C-17). Stability Studies in DMSO and Aqueous Solution. For stability in DMSO, 2a and 2b (1−2 mg/mL) were dissolved in DMSO-d6, and 1 H NMR spectra were recorded after 0.5, 3, 24, 48, and 72 h. Hydrolytic stability test was carried out by dissolving 2a and 2b (1−2 mg/mL) in DMSO-d6/D2O (20/80), and 1H NMR was measured after 0.5, 3, 24, and 48 h. Reactivity with Small Biomolecules. In order to investigate the reactivity of complexes with amino acids, a solution of 2a and 2b (1 mg/mL) in DMSO-d6/D2O (20/80) was treated with equimolar amounts of Cys, His, and Met, and the 1H NMR spectra were recorded after 30 min, 24 h, and 48 h. For the 5′-GMP binding experiment, 2a and 2b were first dissolved in DMSO-d6/D2O (20/80); then 2 equiv of 5′-GMP was added to each of the above solutions and the reaction was monitored by 1H and 31P{1H} NMR spectroscopy. Determination of clog P and log kw Values. Calculated logarithms of octanol−water partition coefficient (clog P) were obtained by using Molinspiration found at http://www.molinspiration. com. Experimental logarithmic capacity factors (log k′ values) were determined according to the OECD Guidelines for HPLC.57 A ThermoFisher Dionex UltiMate3000 HPLC system controlled via the F dx.doi.org/10.1021/om500825h | Organometallics XXXX, XXX, XXX−XXX Organometallics Article Chromeleon 7.1.2 software suite (ThermoFisher Dionex) and equipped with a diode array UV−vis detector (DAD-3000 RS, ThermoFisher Dionex) was used for the measurements. The column compartment was thermostated at 25 °C, and a C18 reverse-phase column (Hypersil Gold, 250 mm length, 4.6 mm inner diameter, pore size 5 μm, ThermoFisher) was used as stationary phase. Thiourea (100 μM) was added as an internal standard to determine the column deadtime, and 10 μL of 100 μM samples dissolved in 1:1 MeOH/H2O was injected. Isocratic runs using methanol/water mixtures with at least five different methanol contents between 40% and 75% were used in order to delineate the lipophilicity, and peaks were detected at 220 nm. Log kw′ values were determined by linear regression of the equation log k′ = S × φ + log kw, where k′ = (tR − t0)/t0 (tR equals the retention time on the column and t0 is the column dead-time, i.e., the retention time of thiourea), S is the slope, φ is the organic solvent ratio, and log kw is the logarithmic capacity factor extrapolated for pure water, i.e., φ = 0.47 Log k′ values were only considered if −0.5 < log k′ < 1.5 in order to ensure that the above-mentioned relationship was indeed in the linear range.47 Measurements for all compounds were done in triplicate, and the numbers given are mean ± standard deviation. Sulforhodamine B Assay. HCT116 and MDA MB 231 cells were supplied by ATCC and Dr. Adam Patterson, University of Manchester, UK, respectively, and were grown in αMEM (Life Technologies) supplemented with 5% fetal calf serum (Moregate Biotech). Cells were seeded at 750 (HCT116) or 10 000 (MDA MB 231) cells/well in 96well plates and left to settle for 24 h at 37 °C and 5% CO2. Compounds were added to the plates in a series of 3-fold dilutions in 0.5% DMSO or less for 72 h before the assay was terminated by addition of 10% trichloroacetic acid (Merck Millipore) at 4 °C for 1 h. Cells were stained with 0.4% sulforhodamine B (Sigma-Aldrich) in 1% acetic acid for 30 min in the dark at room temperature, then washed in 1% acetic acid to remove unbound dye. The stain was solubilized in unbuffered Tris base (10 mM; Serva) for 30 min on a plate shaker in the dark and quantitated on a BioTek EL808 microplate reader at an absorbance of 490 nm with a reference wavelength of 450 nm. The 10point IC50 values were calculated by fitting the inhibition data relative to no inhibitor controls and proliferation at the time of compound addition to a four-parameter logistic sigmoidal dose−response curve using Prism 6.03 (GraphPad). Molecular Modeling. The compounds were docked to the crystal structure of COX-2 (PDB ID: 4M11, resolution 2.45 Å),63 which was obtained from the Protein Data Bank (PDB).64,65 Scigress Ultra 7.7.0.4766 was used to prepare the crystal structure for docking; that is, hydrogen atoms were added, and the cocrystallized meloxicam was removed as well as crystallographic water molecules. The Scigress software suite was also used to build the inhibitors, and the MM267 force field was used to optimize the structures. The center of the binding pocket was defined as the position of the hydroxyl oxygen atom in meloxicam (x = 67.830, y = 15.016, z = 24.355) with 10 Å radius. Fifty docking runs were allowed for each ligand with default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be deprotonated. The GoldScore (GS),68 ChemScore (CS),69,70 ChemPLP,71 and ASP72 scoring functions were implemented to validate the predicted binding modes and relative energies of the ligands using the GOLD v5.2 software suite. Meloxicam was redocked into the binding pocket using the four scoring functions and correlated to the cocrystallized conformation. The root-mean-square deviation between the heavy atoms of the cocrystallized ligand and its docked counterparts was as follows: ChemPLP 0.31 Å, ASP 0.49 Å, CS 2.10 Å, and GS 1.56 Å. ■ CCDC 1015715−1015717 (www.ccdc.cam.ac.uk/data_ request/cif). ■ AUTHOR INFORMATION Corresponding Authors *(W. A. Siddiqui) E-mail: waseeq786@gmail.com. Tel: +92-489230811-15, ext 350. Fax: +92-48-3222121. *(C. G. Hartinger) E-mail: c.hartinger@auckland.ac.nz. Tel: +64-9-3737 599, ext 83220. Fax: +64-9-3737 599, ext 87422. Notes The authors declare no competing financial interests. ■ ACKNOWLEDGMENTS We thank the University of Auckland, the Higher Education Commission of Pakistan (research project NRPU 20-1582 and IRSIP to F.A.), Genesis Oncology Trust (GOT-1263-RPG), IRSIP (F.A.), the Austrian Science Fund (Schrö dinger fellowship to M.H.), and COST CM1105 for financial support. The authors are grateful to Tanya Groutso for collecting the Xray crystal data, Nick Lloyd for ESI-MS analyses, and Emma Richardson for in vitro anticancer activity testing. This article is dedicated to Prof. Gerard Jaouen on the occasion of his 70th birthday. ■ REFERENCES (1) Barry, N. P. E.; Sadler, P. J. Chem. Commun. 2013, 49, 5106− 5131. (2) Kelland, L. Nat. Rev. Cancer 2007, 7, 573−584. (3) Hartinger, C. G.; Metzler-Nolte, N.; Dyson, P. J. Organometallics 2012, 31, 5677−5685. (4) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. Chem. 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