Hetero-Bis-Conjugation of Bioactive Molecules to Half-Sandwich Ruthenium(II) and Iridium(III) Complexes Provides Synergic Effects in Cancer Cell Cytotoxicity.

pubs.acs.org/IC Article Hetero-Bis-Conjugation of Bioactive Molecules to Half-Sandwich Ruthenium(II) and Iridium(III) Complexes Provides Synergic Eﬀects in Cancer Cell Cytotoxicity Lorenzo Biancalana,* Hana Kostrhunova, Lucinda K. Batchelor, Mouna Hadiji, Ilaria Degano, Guido Pampaloni, Stefano Zacchini, Paul J. Dyson, Viktor Brabec,* and Fabio Marchetti Downloaded via UNIV OF GLASGOW on August 11, 2021 at 20:46:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: Inorg. Chem. 2021, 60, 9529−9541 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Four bipyridine-type ligands variably derivatized with two bioactive groups (taken from ethacrynic acid, ﬂurbiprofen, biotin, and benzylpenicillin) were prepared via sequential esteriﬁcation steps from commercial 2,2′-bipyridine-4,4′-dicarboxylic acid and subsequently coordinated to ruthenium(II) p-cymene and iridium(III) pentamethylcyclopentadienyl scaﬀolds. The resulting complexes were isolated as nitrate salts in high yields and fully characterized by analytical and spectroscopic methods. NMR and MS studies in aqueous solution and in cell culture medium highlighted a substantial stability of ligand coordination and a slow release of the bioactive fragments in the latter case. The complexes were assessed for their antiproliferative activity on four cancer cell lines, showing cytotoxicity to the low micromolar level (equipotent with cisplatin). Additional biological experiments revealed a multimodal mechanism of action of the investigated compounds, involving DNA metalation and enzyme inhibition. Synergic eﬀects provided by speciﬁc combinations of metal and bioactive fragments were identiﬁed, pointing toward an optimal ethacrynic acid/ﬂurbiprofen combination for both Ru(II) and Ir(III) complexes. ■ antineoplastic agents.4 From a synthetic standpoint, these complexes are obtained by sequential derivatization of trans (axial) Pt−OH groups with ester or carbamate linkages from common Pt(IV) precursors (Scheme 1a). The anticancer activity of such multitargeted/multiaction derivatives has been extensively investigated; some of these were able to outperform reference platinum drugs in terms of cancer cell cytotoxicity and also in 3D cell cultures and activity against primary tumors in vivo.4b−d4h Inspired by the same principle, we recently reported a series of platinum(II) complexes containing (hetero)bis-functionalized bipyridine ligands (Scheme 1b).5 Complexes carrying ethacrynic acid and/or ﬂurbiprofen (vide infra) displayed marked cytotoxicity with respect to cisplatin in retinoblastoma and ovarian cancer cell lines, along with improved cancer cell selectivity. INTRODUCTION The conjugation of biologically active metal scaﬀolds and organic molecules is a well-documented strategy to enhance the therapeutic eﬀects of the resulting compound and is widely applied to cytotoxic metal complexes investigated as potential anticancer agents.1 In general, “bioactive molecules” are both those possessing a pharmacological function on their own (providing a multiaction compound) and those behaving as “vectors” for increasing the localization of a drug (providing a targeted compound). This approach to combine bioactive molecules with metal complexes met with success in several cases, where considerable synergic eﬀects have been observed for the bioconjugates with respect to nonfunctionalized metal complexes and/or coadministration of the two individual components.2 The mechanism of action of such derivatives is likely multimodal and often largely unknown, with respect to the interplay of the metal center and the bioactive ligands. In this regard, the incorporation of multiple bioactive organic molecules on a monometallic structure is expected to further improve anticancer activity, particularly if the former are endowed with diﬀerent (complementary) modes of action and cellular targets.3 In the past few years, several platinum(IV) compounds of this kind have been prepared, featuring various combinations of fragments targeting speciﬁc enzymes, receptors, and cellular compartments as well as organic © 2021 American Chemical Society Received: March 3, 2021 Published: June 22, 2021 9529 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article Scheme 1. General Structure of Platinum(IV) (a) and Platinum(II) (b) Complexes Conjugated with Two Diﬀerent Bioactive Molecules Reported in the Literature and Bioactive Carboxylic Acids Employed in This Work (c) ﬂurbiprofen (LEF) or biotin (LEB), according to the recently reported procedure (Scheme 2a).5 A diﬀerent strategy was adopted for the functionalization with benzylpenicillin, since a clean conversion of the commercially available potassium salt into the carboxylic acid was not eﬀective. Thus, the bipyridine diacid was ﬁrst converted into its 2-chloroethyl diester and the latter was allowed to react with an excess of potassium benzylpenicillin in DMF at 85 °C, to aﬀord the desired compound (LPP; Scheme 2b). Subsequently, treatment of [RuCl2(η6-p-cymene)]2 or [IrCl2(η5-C5Me5)]2 with AgNO3 in MeCN followed by reaction with the appropriate bipyridine ligand at reﬂux aﬀorded the complexes [RuCl(η6-p-cymene)(κ2N-L)]+ and [IrCl(η5-C5Me5)(κ2N-L)]+, respectively (LEF, [1,2]+; LEB, [3,4]+; LPP, [5]+; Scheme 2c). The reaction of bipyridine ligands (LEF, LEB) with the iridium dimer in chloroform under reﬂux and subsequent Cl−/NO3− exchange was also eﬀective (Scheme 2d). Dimethyl[2,2′-bipyridine]4,4′-dicarboxylate (LMeMe) was synthesized according to a literature method16 and used to obtain [6]+ and [7]+ as reference compounds. All reactions were carried out under stoichiometric conditions, and the Ru/Ir complexes were isolated as their nitrate salts in 83−95% yield, without the need for puriﬁcation. The air- and moisture-stable ocher yellow (Ru) and orange (Ir) solids are soluble in polar organic solvents (e.g., DMSO, MeCN, CH2Cl2) but poorly soluble in water. To the best of our knowledge, the series of complexes [1−5]+ includes the ﬁrst cases of iridium and ruthenium compounds derivatized with ﬂurbiprofen or benzylpenicillin17 and also the ﬁrst iridium−ethacrynic acid conjugate. The novel compounds were characterized by analytical (CNH analysis), spectroscopic (solid-state IR, multinuclear and bidimensional NMR), and high-resolution MS spectrometry techniques (see Figures S1−S33 in the Supporting Information). In addition, the crystal structure of [6]BF4 (obtained by [6]NO3 + NaBF4 metathesis) was conﬁrmed by X-ray diﬀraction (see Figure S34 and Table S1).18 The IR spectra of [1−5]NO3 (in the solid state) display strong bands in the region 1760−1730 cm−1 accounting for the ester groups, along with other nearby CO stretching absorptions. For instance, the bis-penicillin ester LPP and the corresponding Ru(II) derivative [5]NO3 show an additional intense band at 1780 cm−1 due to the β-lactam ring. The NMR spectra of [1−4]NO3 contain two sets of signals, often overlapping; this eﬀect is especially noticeable for 1H/13C In light of these promising results, we decided to coordinate the bis-functionalized bipyridines to Ru(II) p-cymene and Ir(III) pentamethylcyclopentadienyl structures, which have been widely investigated for their anticancer potential.6 Note that double functionalization with two diﬀerent biofragments is quite rare for these metal scaﬀolds.7 Four bioactive carboxylic acids were selected for the present work: ethacrynic acid (ECO2H), f lurbiprofen (F-CO2H), biotin (B-CO2H), and benzylpenicillin (P-CO2H) (Scheme 1c). Ethacrynic acid is an inhibitor of glutathione S-transferases (GST), enzymes that are implicated in resistance mechanisms in various cancer cell lines.2a,8 Ruthenium arene complexes derivatized with ethacrynic acid typically are cytotoxic also in cisplatin-resistant cell lines,9 whereas iridium−ethacrynic acid conjugates have not yet been reported. Cyclooxygenase enzymes (COX, especially COX-2) represent another important drug target, as they are upregulated in several human cancers.10 Thus, a variety of nonsteroidal anti-inﬂammatory drugsnot including ﬂurbiprofenhas been tethered to half-sandwich ruthenium and iridium complexes (mostly Ru) to investigate their anticancer activity.11 Biotin (vitamin B12) has been frequently introduced in the structure of metal complexes, aiming to assist their cellular uptake, taking advantage of the overexpression of vitamin receptors.7b,12 In this regard, several biotinylated Ir(η5Cp*) derivatives have been investigated within the ﬁeld of artiﬁcial metalloenzymes, but not in view of their possible anticancer activity.13 Benzylpenicillin was considered with a view to expand the panel of bioactive fragments of interest in the design of anticancer metal complexes. As a matter of fact, transitionmetal conjugates with β-lactam antibiotics (ampicillin, penicillin, and related species) are sparse and have been mostly studied for their antibacterial activity.14 The cytotoxicity of herein reported hetero(bis)conjugated Ru and Ir complexes was investigated against ovarian, breast, and cervical cancer cell lines, these cancers being currently treated using combination chemotherapy involving platinum and/or organic drugs.15 ■ RESULTS AND DISCUSSION Synthesis and Characterization. Derivatization of 2,2′bipyridine-4,4′-dicarboxylic acid with ethylene glycol followed by Steglich esteriﬁcation/silica chromatography steps aﬀorded the heterofunctionalized ligands containing ethacrynic acid and 9530 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article Scheme 2. Structures and Synthetic Routes for Bipyridine Ligands Derivatized with Ethacrynic Acid (E-CO2H), Flurbiprofen (F-CO2H), biotin (B-CO2H), and Benzylpenicillin (P-CO2H) and the Respective Ruthenium(II) p-Cymene and Iridium(III) Pentamethylcyclopentadienyl Complexesa a RT = room temperature. NMR signals related to the bipyridine rings as well as in 19F NMR spectra of [1,2]NO3. Indeed, the asymmetry of bipyridine ligands LEB and LEF results in a chiral metal center and therefore complexes [1,2]+ were obtained as a (racemic) mixture of diastereomers. On the other hand, [5]NO3 exists as a single diastereomer, in which the doubling of NMR signals is due to diastereotopic atoms (e.g., the CH protons of the pcymene ligand). Accordingly, [6,7]NO3 display symmetric, achiral bipyridine ligands, giving rise to a single set of signals. The nitrate anion in [1−7]NO3 manifests itself with a strong IR absorption around 1330 cm−1 and a 14N NMR resonance occurring in the −1.5 to +3.5 ppm range. Behavior in Aqueous Solution and Cell Culture Medium. A preliminary assessment of the stability of ruthenium and iridium complexes in an aqueous medium19 was carried out on 10 mM solutions in DMSO-d6/D2O 5/1 v/ v at 37 °C by 1H NMR. Under these conditions, all compounds were completely inert over 72 h (≥96% stability; Figures S35−S39). The release of organic fragments, resulting from ligand dissociation and/or ester hydrolysis, was ruled out by comparison with the respective 1H NMR spectrum. Next, in order to check for metal chloride solvolysis, a stoichiometric amount of Ag(CF3SO3) was added to [6]+ and [7]+. Partial or complete conversion of the starting material required heating at 50 °C for 3−5 h (Figures S40 and S41); a spectral comparison before and after reaction with Ag(CF3SO3) allowed unambiguous assignment of the set of signals to the parent chloro ([6]+, [7]+) and solvato ([6S]2+, [7S]2+) complexes (Scheme S1). Nevertheless, hydrolysis of metal− chloride bonds on half-sandwich Ru(II) and Ir(III) 2,2′bipyridine complexes should be facilitated on increasing the dilution and water content of the medium.20 In contrast, transesteriﬁcation processes are triggered upon dissolution in methanol at room temperature, as ascertained by NMR spectroscopy and MS spectrometry (see the Experimental Section and the Supporting Information). Such 9531 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC reactivity was previously observed for the bipyridines LEF and LEB and their Pt(II) complexes.5 In the case of [1−4]+, methanolysis occurs predominantly at the bipyridine-bound carboxyl, leading to the release of the 2-hydroxyethyl esters of ethacrynic acid, ﬂurbiprofen, and biotin. For comparison, the LMeMe derivatives [6,7]+ are much more inert, indicating that the steric bulk of bioconjugated 2,2′-bipyridine ligands favors ester cleavage. Next, the stability of ruthenium and iridium complexes under physiologically relevant conditions was investigated. Therefore, 40 μM solutions of [1−5]NO3 in RPMI cell culture medium (1% DMSO; pH ∼7.4) were incubated at 37 °C for variable times (0/24/48 h) and then analyzed by HPLC-MS. The parent organometallic cation was observed in solution in each case, along with fragments derived from the hydrolysis of ester bonds. In this respect, the release of benzylpenicillin and the four 2-hydroxyethyl esters could not be quantiﬁed, due to matrix eﬀects that have not been clariﬁed. Conversely, the percentage amounts of ethacrynic acid, biotin, and ﬂurbiprofen (carboxylates) released over time are reported in Table 1. Complexes [1]+ and [2]+ readily produce a minor amount (6− 12%) of ethacrynic acid, which decreases after 48 h, possibly due to its degradation. No ﬂurbiprofen was found in solution above the quantitation limit (0.4 μM) at any incubation time. A consistent amount of ethacrynic acid quickly detaches from both [3]+ and [4]+ (35−40%), and more than half of the biotin was found in solution after 48 h. In conclusion, the stability experiments highlighted that Ru(II) and Ir(III) complexes [1−5]+ possess a robust ligand set around the metal center but are susceptible to ester hydrolysis in the cell culture medium, leading to the progressive release of their bioactive organic cargo (in the form of carboxylates or 2-hydroxyethyl esters), thus behaving as prodrugs. Indeed, the aforementioned 2-hydroxyethyl esters might be biologically active themselves (e.g., in terms of enzyme inhibition or anticancer activity21) or undergo a subsequent hydrolysis to release the bioactive carboxylate. The timing of ester cleavage may aﬀect their biological activity (vide infra). Cytotoxicity, Cellular Uptake, DNA Metalation, and Enzyme Inhibition. The antiproliferative activity of complexes [1−7]+ was determined in four human cancer cell lines (A2780, A2780cisR ovarian ± cisplatin-resistant, MCF-7 breast, and HeLa cervical) and nontumorigenic human embryonic kidney cells (HEK293), following 72 h incubation. IC50 data are compiled in Table 2 with cisplatin, RAPTA-C, bipyridine ligands (LEF, LEB, LPP), and the bioactive carboxylic acids as reference compounds. Ethacrynic acid derivatized compounds [1−4]+ are potent cytotoxic agents in the panel of cell lines, with IC50 values ranging from 2.8 to 40 μM. The compounds are particularly eﬀective on MCF-7 cells (in comparison to cisplatin) and on the cisplatin-resistant A2780cisR cell line, with resistance factors below 2.2 (vs 7 for cisplatin). Conversely, benzylpenicillin conjugates [5]NO3 and LPP are essentially nontoxic in all cell lines. Interestingly, some cytotoxicity trends on varying the nature of the metal center (Ru/Ir) and the organic fragments can be delineated. First, each iridium complex ([2]+/[4]+/[6]+) is Table 1. Percentages of Ethacrynic Acid, Biotin, and Flurbiprofen Carboxylates Released from Solutions of [1− 4]+ in RPMI Cell Culture Medium (ca. 40 μM, 1% DMSO, pH ∼7.4) at 37 °C for Diﬀerent Incubation Times (0−48 h) % E-CO2H releaseda % F-CO2H or B-CO2H releaseda,b compound 0h 24 h 48 h 0h 24 h 48 h [1]NO3 [2]NO3 [3]NO3 [4]NO3 6 12 34 39 4 14 33 28 3 8 28 26 0 0 9 10 0 0 29 40 0 0 51 64 Article a Starting concentrations of the complexes were in the range 33−41 μM. The analytical blank did not contain any relevant amount of the analytes. The CV% was below 10%. b% F-CO2H for [1,2]+; % BCO2H for [3,4]+. Table 2. IC50 values (μM) on Human Ovarian (A2780 and A2780cisR), Breast (MCF-7), and Cervical (HeLa) Cancer Cells and Human Embryonic Kidney (HEK-293) Cells after 72 h Incubationa compound(s) metal/bioactive fragments A2780 A2780cisR MCF-7 HeLa HEK293 [1]NO3 [2]NO3 [3]NO3 [4]NO3 [5]NO3 [6]NO3 [7]NO3 LEF LEB LPP ethacrynic acidb ﬂurbiprofen benzylpenicillinc [6]NO3 + E-CO2H + F-CO2Hd [7]NO3 + E-CO2H + F-CO2Hd cisplatin RAPTA-Ce Ru/ethacrynic acid/ﬂurbiprofen Ir/ethacrynic acid/ﬂurbiprofen Ru/ethacrynic acid/biotin Ir/ethacrynic acid/biotin Ru/benzylpenicillin (×2) Ir/− Ru/− −/ethacrynic acid/ﬂurbiprofen −/ethacrynic acid/biotin −/benzylpenicillin (×2) 5.6 ± 0.6 2.8 ± 0.4 11 ± 3 7.0 ± 0.8 >200 >100 >100 >200 12 ± 1 >200 40 ± 3 >200 >200 34 ± 2 27 ± 3 2.1 ± 0.4 >200 8.0 ± 0.7 6.4 ± 0.5 11 ± 2 8.5 ± 0.5 >200 63 ± 6 >100 >200 16 ± 1 >200 53 ± 5 >200 >200 35 ± 2 39 ± 3 15 ± 1 >200 10.6 ± 0.8 8.0 ± 0.9 22 ± 1 9±1 270 ± 30 140 ± 30 310 ± 40 >200 17 ± 2 >200 47 ± 4 190 ± 30 >200 14.8 ± 0.9 - 28 ± 4 17.1 ± 0.6 40 ± 6 24 ± 5 190 ± 10 190 ± 9 220 ± 20 >200 14 ± 2 >200 56 ± 9 200 ± 50 >200 15 ± 3 - 7.3 ± 0.9 10.3 ± 0.6 9 ± 4f 12.5 ± 0.8 >200f >100f >100 >200 25 ± 2 >200 39 ± 1 >200 >200 37 ± 3 36 ± 2 5.3 ± 0.7 >200 Ir/ethacrynic acid/ﬂurbiprofen Ru/ethacrynic acid/ﬂurbiprofen Values are given as mean ± SD. bData for A2780/A2780cisR and HEK293 taken from ref 9c. cAs the K+ salt. dCoadministration of the compounds in a 1:1:1 molar ratio. e[RuCl2(η6-p-cymene)(κP-1,3,5-triaza-7-phosphaadamantane)]. fTested on HEK-293T cells. a 9532 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article Table 3. Cellular Uptake of Ru or Ir in A2780 Cells Treated with 5 or 10 μM of [1−7]NO3 for 8 or 24 h and DNA Metalation in A2780 Cells Treated with 10 μM of [1−7]NO3 for 24 h cellular uptake (pmol Ru(Ir)/106 cells) compound metal/bioactive fragments 10 μM/8 h 5 μM/24 h 10 μM/24 h DNA metalation (fmol Ru(Ir)/μg DNA) [1]NO3 [2]NO3 [3]NO3 [4]NO3 [5]NO3 [6]NO3 [7]NO3 Ru/ethacrynic acid/ﬂurbiprofen Ir/ethacrynic acid/ﬂurbiprofen Ru/ethacrynic acid/biotin Ir/ethacrynic acid/biotin Ru/benzylpenicillin (×2) Ir/− Ru/− 88 ± 7 61 ± 7 9±1 11 ± 2 4.6 ± 0.9 5±1 2.1 ± 0.3 65 ± 9 46 ± 6 7±2 8±2 3.2 ± 0.9 5±1 4±1 130 ± 9 100 ± 10 13 ± 4 17 ± 4 7±1 9.0 ± 0.7 6.4 ± 0.9 52 ± 9 (21 ± 7)a 36 ± 3 (16 ± 3)a 5.0 ± 0.9 12 ± 2 4.8 ± 0.6 8±2 4.2 ± 0.7 DNA metalation with 5 μM [1]NO3 and [2]NO3 for 24 h is given in parentheses. a cytotoxicity, which is the highest for compounds better internalized by the cells (i.e., [1]+ and [2]+). To further explore whether DNA metalation plays a role in the cell-killing eﬀect of the investigated compounds, we measured their antiproliferative activity in a pair of Chinese hamster ovary cell lines, CHO-K1 (wild-type) and its mutant cell line MMC-2. The latter is deﬁcient in the DNA nucleotide excision repair (NER); thus, the DNA damage in MMC-2 cells is more harmful than in the NER-proﬁcient CHO-K1 cells. The degree of DNA damage involvement in the mechanism of action can be estimated from the ratio of IC50 values estimated for CHO-K1 and MMC-2 cells (factor F in Table 4). The four slightly more cytotoxic than the respective ruthenium analogue ([1]+/[3]+/[7]+). Second, ﬂurbiprofen/ethacrynic acid conjugates ([1,2]+) are more cytotoxic than their biotin/ ethacrynic acid counterparts ([3,4]+), while the opposite behavior is observed for the bipyridine ligands (LEF being substantially noncytotoxic while LEB shows IC50 values comparable to those of its complexes). Third, cancer cell selectivity (i.e., IC50 found for nontumorigenic HEK293/IC50 found for the cancer cell line) increases on going from [3]+/ [4]+ to [1]+/[2]+, peaking at ca. 4 for [2]NO3 in A2780 cells. These trends hold for all of the tested cell lines, indicating that the cytotoxic eﬀect of [1−4]+ is heavily inﬂuenced by the selection of metal center and bioactive fragments and evidencing iridium/ethacrynic acid/ﬂurbiprofen as the best combination. Notably, ruthenium and iridium bipyridine with simple methyl esters on the bipyridine ligand ([6]+/[7]+) are considerably less cytotoxic than the bis-conjugated derivatives [1−4]+, suggesting that synergic eﬀects are responsible for the biological activity of the latter. Note that incubation of a 1:1:1 mixture of ﬂurbiprofen, ethacrynic acid, and the Ru/Ir complex lacking bioactive fragments ([6]+ and [7]+) resulted in a 5−12fold lower cytotoxicity with respect to the administration of a single compound ([1]+ and [2]+). The overall better performance of ﬂurbiprofen/ethacrynic acid derivatives [1,2]+, with respect to the biotin/ethacrynic acid analogues [3,4]+ may in part be related to a more controlled ester hydrolysis in the cellular environment (see above). It is noteworthy that biotinylated derivatives were not particularly eﬀective also among the bis-functionalized platinum(IV) complexes (Scheme 1a).4a−g In order to gain insights into the mode of action of the compounds, A2780 cells were incubated with 10 μM of [1− 7]+ for 8 and 24 h and 5 μM of [1−7]+ for 24 h, and the metal content was determined by ICP-MS following established protocols (Table 3).22 The cellular uptake for all tested compounds increases with incubation time and with concentration. Treatment with ethacrynic acid/ﬂurbiprofen conjugates [1,2]+ resulted in a substantially higher metal accumulation with respect to the nonfunctionalized reference complexes [6,7]+. In contrast, biotin-functionalized complexes [3,4]+ showed only a modest increase in cellular internalization, and the ruthenium content determined for the bispenicillin [5]+ and the bis-methyl [7]+ esters was not signiﬁcantly diﬀerent. The DNA metalation level, following incubation with 10 μM of [1−7]+ for 24 h, decreases in the sequence [1]+ ≫ [3]+ ≈ [5]+ ≈ [7]+ and [2]+ ≫ [4]+ > [6]+ for Ru and Ir complexes, respectively, reﬂecting the amount of compound taken up by the cells in an almost linear fashion (Figure S45). Overall, these data correlate well with A2780 Table 4. Antiproliferative Data (IC50/μM)a for CHO-K1 (Wild-Type) and MMC-2 (NER-Deﬁcient) Cells Treated with [1−4]NO3 and Cisplatinb compound CHO-K1 MMC-2 Fc [1]NO3 [2]NO3 [3]NO3 [4]NO3 cisplatin 23 ± 4 18.9 ± 0.6 29 ± 2 25.7 ± 0.8 29 ± 1 12.9 ± 0.3 10.1 ± 0.2 9.1 ± 0.7 8.9 ± 0.2 3.3 ± 0.1 1.8 1.9 3.2 2.9 8.8 a Determined by MTT assay after 72 h treatment. bValues are given as mean ± SD. cDeﬁned as IC50(NER eﬃcient)/IC50(NER deﬁcient). Ru/Ir bioconjugated complexes [1−4]+ are slightly more eﬃcient in the NER-deﬁcient cell line MMC-2 than in the wild-type CHO-K1; this eﬀect is more pronounced for the biotin derivatives [3,4]+ in comparison to the ﬂurbiprofen derivatives [1,2]+ (Figure S46). The prominent involvement of DNA damage in cells treated with cisplatin is apparent, the compound being 9 times more active in the NER-deﬁcient cells. Next, we performed cell viability studies of [1−4]+ and cisplatin in A2780 cells using the MTT assay with variable treatment times (24/48/72 h; Table 5). The eﬀect of DNA metalation resulting in DNA damage is usually related to the inhibition of DNA replication or transcription, which only becomes apparent after a longer time of the treatment (after at least one cell cycle is completed). Cisplatin is a typical example of this behavior, whose mechanism of action is primarily related to its ability to modify DNA, and its cytotoxicity is strongly time-dependent. Indeed, the IC50 values obtained for cisplatin after 24 h were markedly higher (9 times) than those determined after 72 h treatment. Conversely, we observed almost no variation of the IC50 values along the treatment times (except for [3]NO3 at 24 h). Thus, it seems reasonable to suggest that the cytotoxic eﬀects of [1−4]+ might contribute 9533 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC caused a stronger inhibition, reducing the enzyme activity to 68 and 62%, respectively. No signiﬁcant inhibition of COX activity was observed, except for [2]NO3. In conclusion, the mechanism of action of the investigated compounds [1−4]+ is likely multimodal, involving DNA metalation as well as enzyme inhibition. However, the cytotoxic eﬀects associated with the enzyme inhibition activity possibly outweigh the eﬀects caused by DNA metalation. Enzyme inhibition by [1]+ and [2]+ could be enacted by metal-based species (e.g., the organometallic cations) or by the bioactive organic fragments that are released upon entering the cell. Judging from the stability studies in cell culture medium and the metal uptake experiments (vide supra), it is apparent that both the internalization of the compounds and the release of the bioactive groups from the respective Ir or Ru complexes are time-dependent. Nevertheless, the fact that cytotoxicity does not increase from 24 to 72 h (Table 5) points out that these compounds mostly exert their biological activity within the ﬁrst 24 h of the treatment. Table 5. Antiproliferative Data (IC50/μM) for [1−4]NO3 and Cisplatin in A2780 Cells with Variable Treatment Timesa IC50 (μM) compound 24 h 48 h 72 h [1]NO3 [2]NO3 [3]NO3 [4]NO3 cisplatin 6.7 ± 0.2 3.0 ± 0.1 20 ± 2 8±1 34 ± 4 6.1 ± 0.3 2.9 ± 0.1 12.7 ± 0.2 7.6 ± 0.8 4.9 ± 0.9 5.6 ± 0.3 2.8 ± 0.1 11.1 ± 0.4 7.0 ± 0.8 3.6 ± 0.7 Article Values are given as mean ± SD. a to the overall cell growth inhibition more than their antiproliferative eﬀects. In accordance with the antiproliferative activity in cell lines proﬁcient and deﬁcient in NER (Table 4), these results support the view that DNA metalation might be involved in the mechanism of action of [1−4]+, although other routes are likely to play a more signiﬁcant role. In this respect, we performed experiments to assess whether the ethacrynic acid/ﬂurbiprofen derivatized complexes [1,2]+ are able to aﬀect glutathione S-transferase (GST) and cyclooxygenase (COX) activity. Thus, lysates of the HeLa cells treated for 18 h with [1]NO3 and [2]NO3 at concentrations corresponding to their respective IC50 values (determined with MTT after 72 h; Table 2) were prepared and analyzed with either Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen) or Cyclooxygenase Activity Kit (Fluorimetric) (Abcam). Similarly, combinations of the nonfunctionalized complexes [6]+/[7]+ with the reference enzyme inhibitors (F-CO2H/E-CO2H) or the bipyridine ligand LEF at identical concentrations were tested. The results are shown in Figure 1 and Figure S47. The GST activity was signiﬁcantly reduced in the cells treated with both ethacrynic acid carrying compounds [1]NO3 and [2]NO3, as well as in the cells treated with the mixtures containing E-CO2H. Nevertheless, [1]NO3 and [2]NO3 ■ CONCLUSIONS Ruthenium(II) arene and iridium(III) cyclopentadienyl complexes are among the most investigated transition-metal compounds in the landscape of the development of new anticancer drugs able to overcome the limitations associated with the use of platinum chemotherapeutics. In addition, the conjugation with suitable bioactive fragments is an intriguing and well-established strategy aimed to improve the anticancer activity of a metal complex. Remarkably, the bis-functionalization of metal complexes with two diﬀerent bioactive molecules appears to be an even more promising approach, which has been successfully investigated on platinum(IV) complexes, but its applicability to other metal structures remains undeveloped, possibly due to the lack of adequate synthetic routes. Here, we have employed a commercial bipyridine as a convenient starting material to obtain new half-sandwich Ru(II) and Ir(III) complexes doubly functionalized with diﬀerent Figure 1. Eﬀect of the tested compounds on GST (A) and COX (B) activity in HeLa cells. The cells were incubated with [1]NO3 and [2]NO3 at the concentrations corresponding to their respective IC50 values (72 h; MTT) for 18 h. For comparison, the cells were also treated with combinations of [7]NO3 and [6]NO3 with E-CO2H (A) and F-CO2H (B) or with LEF at concentrations identical with those of [1]NO3 or [2]NO3, respectively. The enzyme activities are expressed in percent of enzyme activities in nontreated control cells. The GST activity was determined with Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen). The COX activity was determined with Cyclooxygenase Activity Assay Kit (Fluorimetric) (Abcam). Statistical analysis was performed using Student’s t test. Statistically signiﬁcant diﬀerences from nontreated control are shown above the individual bars; statistically, signiﬁcant diﬀerences between samples are given above the brackets (*p ≤ 0.05; **p ≤ 0.01, and ***p ≤ 0.001). 9534 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC 20 μg/g. Analytical and spectroscopic and spectrometric characterizations of compounds are given in the Supporting Information. Synthesis of Compounds. Bis(2-chloroethyl)[2,2′-bipyridine]4,4′-dicarboxylate (Chart 1). In a 50 mL Schlenk tube under N2, bioactive carboxylic acids, including heterofunctionalized derivatives. The complexes manifest a stable coordination environment and a variable tendency to release the bioactive molecules in aqueous media. Biological experiments on ovarian, breast, and cervical cancer cell lines revealed an improved cytotoxicity proﬁle for both Ru(II) and Ir(III) complexes, with ethacrynic acid/ﬂurbiprofen as the most eﬀective combination. The activity of the compounds is associated with an increased cellular uptake, DNA metalation, and COX/GST enzyme inhibition. The last activity is possibly related to the bioactive carboxylic acids (or their derivatives) that are released by hydrolysis. Synergic eﬀects on cytotoxicity and enzyme inhibition were clearly observed by control combination experiments. The eﬃcacy of the proposed method strongly encourages further studies aimed at identifying other combinations of bioactive molecules and may be easily extended to other metal scaﬀolds investigated for pharmacological activity. ■ Article Chart 1. Structure of Bis(2-chloroethyl)[2,2′-bipyridine]4,4′-dicarboxylate 2,2′-bipyridine-4,4′-dicarboxylic acid (529 mg, 2.17 mmol), DMAP (65 mg, 0.57 mmol), EDCI·HCl (992 mg, 5.17 mmol), THF (12 mL), and 2-chloroethanol (0.30 mL, 4.47 mmol) were introduced in that order. The milky colorless suspension was stirred at room temperature overnight, aﬀording a colorless solution and a pink solid. Volatiles were removed under vacuum; the residue was suspended in CH2Cl2 and moved on top of a silica column (height 4 cm, diameter 3.5 cm). Impurities were eluted with CH2Cl2 (40 mL), and the title compound was eluted with a CH2Cl2/hexane/acetone 10/5/2 v/v/v mixture (85 mL). Following removal of the volatiles under vacuum (40 °C), the resulting colorless solid was dried under vacuum (RT over P2O5) and then stored under N2. Yield: 465 mg, 58%. Bis(2-benzylpenicillinyloxyethyl)[2,2′-bipyridine]-4,4′-dicarboxylate (LPP) (Chart 2). In a 50 mL Schlenk tube under N2, bis(2chloroethyl)[2,2′-bipyridine]-4,4′-dicarboxylate (202 mg, 0.546 mmol), K[P-CO2] (544 mg, 1.46 mmol), and DMF (8 mL) were introduced. The suspension (colorless solution + solid) was stirred at 85 °C for 18 h. The conversion was checked by 1H NMR (CDCl3), and then the brown suspension was cooled to room temperature and volatiles were removed under vacuum. The residue was suspended in acetone and ﬁltered over a short silica pad. The ﬁltrate was taken to dryness under vacuum; the residue was dissolved in CH2Cl2 and moved on top of a silica column (height 6 cm, diameter 2.5 cm). Impurities were eluted with CH2Cl2, and the title compound was eluted with CH2Cl2/acetone (7/1 to 3/1 v/v gradient). Following removal of the volatiles under vacuum, the resulting pale yellow foamy solid was dried under vacuum (RT over P2O5) and then stored at 4 °C. Yield: 201 mg, 37%. Synthesis of Ru and Ir Compounds. Method A. A solution of [RuCl2(η6-p-cymene)]2 or [IrCl2(η5-C5Me5)]2 (20−50 mg) in MeCN (2 mL) was treated with AgNO3 (2.0 equiv) and stirred at room temperature in the dark. After 1 h, the suspension (yellow/orange solution + colorless solid) was ﬁltered over a Celite pad. The selected bipyridine ligand (2.0 equiv) was added to the ﬁltrate, and the mixture was stirred at reﬂux for 1−2 h. Next, the conversion was checked by NMR (1H CDCl3) and the volatiles were removed under vacuum. The residue was dissolved in CH2Cl2 and the solution was ﬁltered through Celite. The ﬁltrate was dried under vacuum; the resulting ocher yellow (Ru) or orange (Ir) solid was washed with Et2O and dried under vacuum (40 °C). Method B. In a 25 mL Schlenk tube, a solution of [IrCl2(η5C5Me5)]2 (ca. 25 mg) and the selected bipyridine (2.0 equiv) in CHCl3 (8 mL) was stirred at reﬂux for 14 h. The resulting orange solution was taken to dryness under vacuum, and the residue was triturated in Et2O. The suspension was ﬁltered; the orange solid, namely [IrCl(η5-C5Me5)(bipyridine)]Cl, was washed with Et2O and dried under vacuum (40 °C). Subsequently, a fraction of the Ir compound (ca. 60 mg) was dissolved in MeCN (2 mL) and treated with AgNO3 (1.0 equiv). The suspension was stirred at room temperature in the dark for 4 h. Next, the mixture (orange solution + colorless solid) was ﬁltered over a Celite pad and the ﬁltrate was taken to dryness under vacuum. The residue was dissolved in CH2Cl2 and ﬁltered again through Celite. The ﬁltrate was dried under vacuum; the resulting orange solid was washed with Et2O and dried under vacuum (40 °C). EXPERIMENTAL SECTION General Experimental Details. RuCl3·xH2O (41.9% Ru; Chimet S.p.A), IrCl3·xH2O (59.9% Ir; Merck), and other chemicals (≥99% purity) were purchased from Alfa Aesar, Merck, Apollo Scientiﬁc, or TCI Chemicals. Ethacrynic acid (E-CO2H),23 ﬂurbiprofen (FCO2H),23 biotin (B-CO2H),23 potassium benzylpenicillin (K[PCO 2 ]), 23 2,2′-bipyridine-4,4′-dicarboxylic acid, and ethyl(diisopropylamino)carboxydiimide hydrochloride (EDCI·HCl; at −20 °C) were stored under N2 as received. Ethylene glycol was dried under high vacuum over P2O5 and stored under N2. Column chromatography was carried out with silica gel (70−230 mesh). [RuCl2(η6-p-cymene)]2,24 [IrCl2(η5-C5Me5)]2,25 and bipyridine ligands5 LEF and LEB were synthesized according to literature methods. All operations were carried out in air with common laboratory glassware, except for the syntheses of the bioconjugated bipyridine ligands [2]Cl and [4]Cl, which were carried out under N2 using standard Schlenk techniques and solvents distilled from appropriate drying agents (DMF from BaO, THF from CaH2, CH2Cl2 and CHCl3 from P2O5). Once isolated, organic bipyridine esters were stored at 4 °C; all Ru and Ir compounds were air- and moisture-stable in the solid state at room temperature. NMR spectra were recorded at 25 °C on a Bruker Avance II DRX400 instrument equipped with a BBFO broad-band probe. Chemical shifts (in ppm) are referenced to the residual solvent peaks26 (1H, 13C) or to external standards27 (14N to CH3NO2, 19F to CFCl3, 35Cl to 1 M NaCl in D2O). 1H and 13C spectra were assigned with the aid of 13C DEPT 135, 1H−1H COSY, 1H−13C gs-HSQC and 1H−13C gs-HMBC experiments.28 CDCl3 for NMR analysis was stored in the dark over Na2CO3. IR spectra (650−4000 cm−1) were recorded on a PerkinElmer Spectrum One FT-IR spectrometer, equipped with a UATR sampling accessory and processed with Spectragryph software.29 CHNS analyses were performed on a Vario MICRO cube instrument (Elementar). ESI-Q/ToF ﬂow injection analyses (FIA) were conducted on a 1200 Inﬁnity HPLC coupled to a Jet Stream ESI interface with a 6530 Inﬁnity Q-TOF quadrupole time of ﬂight tandem mass spectrometer (Agilent Technologies, USA). HPLC-MS grade acetonitrile was used as the mobile phase (Carlo Erba, Italy). The ﬂow rate was 0.2 mL/min (total run time 3 min), and the injection volume was 3 μL. ESI operating conditions: drying gas (N2, purity >98%), 350 °C and 10 L/min; capillary voltage 4.5 kV; nozzle voltage 1 kV; nebulizer gas 35 psig; sheath gas (N2, purity >98%) 375 °C and 11 L/min. The fragmentor was kept at 50 V, the skimmer at 65 V, and the OCT 1 RF at 750 V. High-resolution MS and MS/MS spectra were obtained in the positive mode in the range 100−1700 m/z; the mass axis was routinely calibrated using the tuning mix HP0321 (Agilent Technologies) prepared in acetonitrile and water. Prior to injection, each sample was properly diluted to ca. 9535 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article Chart 2. Structure of LPP Chart 3. Structure of [1]+ Chart 4. Structure of [2]+ Chart 5. Structure of [3]+ Chart 6. Structure of [4]+ mg, 0.088 mmol), and LEF (76 mg, 0.089 mmol), according to method A. Yield: 104 mg, 93%. An alternative preparation from [2]Cl (79 mg, 0.064 mmol) and AgNO3 (11 mg, 0.064 mmol) followed method B. Yield: 67 mg, 83%. [RuCl(η6-p-cymene)(κ2N-LEB)]NO3 ([3]NO3) (Chart 5). The complex was prepared using [RuCl2(η6-p-cymene)]2 (30 mg, 0.05 mmol), AgNO3 (17 mg, 0.10 mmol), and LEB (84 mg, 0.10 mmol), according to method A. Yield: 109 mg, 95%. [IrCl(η5-C5Me5)(κ2N-LEB)]+ ([4]+) (Chart 6). [4]Cl was prepared using [IrCl2(η5-C5Me5)]2 (27 mg, 0.034 mmol), and LEB (58 mg, 0.067 mmol), according to method B. Yield: 78 mg, 94%. [4]NO3 was Note: the use of methanol as an alternative solvent for these procedure is NOT recommended, due to transesteriﬁcation processes (vide inf ra).30 [RuCl(η6-p-cymene)(κ2N-LEF)]NO3 ([1]NO3) (Chart 3). The complex was prepared using [RuCl2(η6-p-cymene)]2 (30 mg, 0.05 mmol), AgNO3 (17 mg, 0.10 mmol) and LEF (86 mg, 0.10 mmol), according to method A. Yield: 102 mg, 88%. [IrCl(η5-C5Me5)(κ2N-LEF)]+ ([2]+) (Chart 4). [2]Cl was prepared using [IrCl2(η5-C5Me5)]2 (26 mg, 0.033 mmol) and LEF (58 mg, 0.069 mmol), according to method B. Yield: 79 mg, 98%. [2]NO3 was prepared using [IrCl2(η5-C5Me5)]2 (35 mg, 0.045 mmol), AgNO3 (15 9536 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article Chart 7. Structure of [5]+ dried under vacuum (40 °C). Yield: 47 mg, 89%. X-ray-quality crystals of [6]BF4 were obtained from a MeCN solution layered with Et2O and set aside at −20 °C (Table S1 and Figure S34). [RuCl(η6-p-cymene)(κ2N-LMeMe)]NO3 ([7]NO3) (Chart 10). The complex was prepared using [RuCl2(η6-p-cymene)]2 (57 mg, 0.093 mmol), AgNO3 (32 mg, 0.19 mmol), and LMeMe (51 mg, 0.19 mmol), according to method A. Yield: 107 mg, 96%. prepared using [IrCl2(η5-C5Me5)]2 (36 mg, 0.045 mmol), AgNO3 (16 mg, 0.091 mmol), and LEB (77 mg, 0.091 mmol), according to method A. Yield: 110 mg, 95%. An a lternative preparation from [4] Cl (60 mg, 0.048 mmol) and AgNO3 (9 mg, 0.053 mmol) followed method B. Yield: 51 mg, 83%. [RuCl(η6-p-cymene)(κ2N-LPP)]NO3 ([5]NO3) (Chart 7). The complex was prepared using [RuCl2(η6-p-cymene)]2 (17 mg, 0.028 mmol), AgNO3 (10 mg, 0.059 mmol), and LPP (55 mg, 0.057 mmol), according to method A. Yield: 68 mg, 94%. Dimethyl[2,2′-bipyridine]-4,4′-dicarboxylate (LMeMe) (Chart 8). The title compound was prepared according to a modiﬁed literature Chart 10. Structure of [7]+ Chart 8. Structure of LMeMe procedure.16 In a 100 mL round-bottom ﬂask, 98% H2SO4 (1.6 mL, 30 mmol) was added dropwise to a suspension of 2,2′-bipyridine-4,4′dicarboxylic acid (502 mg, 2.06 mmol) in MeOH (30 mL). The mixture was stirred at reﬂux for 29 h. The resulting pink solution was cooled to room temperature, and NaOH (1 M in water, 30 mL, 30 mmol) was added dropwise. Subsequently, MeOH was removed under reduced pressure and the mixture (pink aqueous solution + colorless precipitate) was extracted with CH2Cl2 (3 × 40 mL). The combined organic extracts were ﬁltered through a Celite pad and dried under vacuum. The resulting colorless solid was washed with Et2O and dried under vacuum (40 °C). Yield: 509 mg, 91%. [IrCl(η5-C5Me5)(κ2N-LMeMe)]X ([6]X; X = NO3, BF4) (Chart 9). The synthesis of the related compound [6]Cl from [IrCl2(η5-C5Me5)]2/ X-ray Crystallography. XRD experiments were performed using Mo Kα radiation on a Bruker APEX II diﬀractometer equipped with a PHOTON2 detector. Data were corrected for Lorentz−polarization and absorption eﬀects (empirical absorption correction SADABS).31 The structure was solved by direct methods and reﬁned by full-matrix least squares on the basis of all data using F2.32 H atoms were ﬁxed at calculated positions and reﬁned by a riding model. Non-hydrogen atoms were reﬁned with anisotropic displacement parameters. Crystal data and collection details for [6]BF4 are reported in Table S1. Solution Stability Studies. Stability in DMSO/Water. The selected Ru or Ir compound (1.0 × 10−2 M for [1−5]NO3; 2 × 10−2 M for [6,7]NO3) was dissolved in a DMSO-d6/D2O 5/1 v/v solution (0.6 mL) containing dimethyl sulfone (Me2SO2, 5 × 10−3 M) as an internal standard for 1H NMR33 (δ 2.96 ppm (s, 6H) in DMSO-d6/ D2O 5/1). A single set of 1H and 19F{1H} NMR signals was observed for the freshly prepared yellow (Ir) or orange (Ru) solution, attributed to the starting material. Next, the solution was heated to 37 °C for 72 h and NMR analyses were repeated (Figures S35−S41). The percent amount of starting material in solution with respect to the initial spectrum, falling in the 96−98% range for all compounds, was calculated by 1H NMR integration with respect to Me2SO2 as an internal standard. NMR data for the tested compounds are reported in the Supporting Information (DMSO-d6/D2O 5/1 v/v solutions); chemical shifts are referenced to the residual peak as in pure DMSOd6 (δ/ppm 2.50). Chloride/Solvent Exchange in DMSO/Water. A DMSO-d6/D2O 5/1 v/v solution of [6]NO3 or [7]NO3 (0.5 mL; 2 × 10−2 M) was maintained at 37 °C for up to 86 h and then analyzed by 14N and 35Cl NMR (2000 scans).34 Subsequently, Ag(CF3SO3) was added (0.10 mL of a 0.12 M solution in DMSO-d6/D2O 2/1 v/v; 1.2 equiv) and the NMR tube was kept in the dark at 50 °C for 5 h, aﬀording an orange (Ru) or yellow (Ir) solution and a colorless precipitate (AgCl). The solid was separated, and 1H, 14N, and 19F{1H} NMR spectra were recorded. Complete ([6]+) or partial ([7]+, ∼50%) conversion of the starting material was observed in the 1H NMR spectrum (Figures S40 and S41). The new set of signals, shifted to Chart 9. Structure of [6]+ LMeMe was previously reported.18 [6]NO3 was prepared using [IrCl2(η5-C5Me5)]2 (36 mg, 0.069 mmol), AgNO3 (24 mg, 0.14 mmol), and LMeMe (38 mg, 0.14 mmol), according to method A. Yield: 85 mg, 83%. [6]BF4 was prepared by dissolution of compound [6]NO3 (51 mg, 0.073 mmol) in acetone (5 mL), and the solution was treated with NaBF4 (11 mg, 0.10 mmol). The mixture was heated at reﬂux for 4 h and then taken to dryness under vacuum. The residue was suspended in CH2Cl2 and ﬁltered through a Celite pad, in order to remove NaNO3. The ﬁltrate was taken to dryness under vacuum and dissolved in MeOH (ca. 3 mL). The solution was heated to reﬂux then cooled to room temperature. The resulting bright orange crystalline solid was collected by ﬁltration, washed with Et2O, and 9537 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC low ﬁeld with respect to the starting metal chlorido species, was attributed to the solvato species [Ir(η5-C5Me5)(LMeMe)(solv)]2+ ([6S]2+) and [Ru(η6-p-cymene)(LMeMe)(solv)]2+ ([7S]2+) (solv = D2O, DMSO-d6; Scheme S1). No solvolysis of metal−chloride bonds occurred before Ag(CF3SO3) addition, as indicated by the absence of a 35Cl NMR signal and comparison with 1H NMR data for the solvato species [6S]2+ and [7S]2+. Stability in Methanol. A freshly prepared solution of the selected compound in CD3OD (0.6 mL; 5 × 10−3 M) was analyzed by 1H and 19 1 F{ H} NMR. Next the orange (Ru) or yellow/orange (Ir) solution was maintained at room temperature (∼25 °C) and periodically analyzed by 1H and 19F{1H} NMR. Once equilibrium was reached (no further change observed in NMR spectra), 1D and 2D NMR experiments (1H, 13C, 19F, 14N, 13C-DEPT 135, 1H−1H COSY, 1 H−13C gs-HSQC, 1H−13C gs-HMBC) were performed for a complete spectral assignment. The identity of compounds in solution (Schemes S2−S7) was checked by comparison with NMR data of the pure compounds and by ESI-MS(+) spectrometry on CH3OH solutions in the case of [3]+ and [4]+. The relative amount of compounds in solution (%) was calculated by 1H NMR integration. Experimental details and MS and NMR data (CH3OH and CD3OD, respectively) for the tested compounds are reported in Figures S42− S44 and Tables S2−S4 in the Supporting Information. Stability in Cell Culture Medium. A stock solution of the selected Ru/Ir compound (ca. 3 × 10−3 mmol/g) was prepared in DMSO and stored at room temperature. An RPMI 1640 cell culture medium (Sigma-Aldrich) was treated with NaH2PO4/Na2HPO4 (cPO4 = 190 mM, pH 7.35) and then stored at 4 °C under N2. In three 2 mL vials, the stock solution (20 μL) was added to the cell culture medium (2.0 mL) and the ﬁnal mixtures (Ru/Ir ca. 45 μmol/g, 1% DMSO) were heated at 37 °C for diﬀerent times (0/24/48 h) with stirring. A fourth reference solution was prepared by adding the stock solution (20 μL) to HPLC water (2.0 mL). Next, the solutions were ﬁltered on PTFE syringe ﬁlters (0.45 μm) and analyzed by HPLC/ESI(+)-MS. LC separation was conducted on an analytical reversed-phase Poroshell 120 EC-C18 column (3.0 × 75 mm, particle size 2.7 μm; Agilent Technologies) with a Zorbax precolumn (4.6 × 12.5 mm, particle size 5 μm; Agilent Technologies) at 30 °C. The separation was achieved using a gradient of formic acid (FA) 0.1% v/v in H2O (eluent A) and FA 0.1% v/v in CH3CN (eluent B), both LC-MS grade; the ﬂow rate was 0.4 mL/min, and the injection volume was 4 μL. The elution program started from 95% A, hold for 2.6 min, then a linear gradient to 50% B in 13 min, then to 70% B in 5 min, and then to 100% B in 6 min, held for 5 min. Re-equilibration took 5 min. ESI operating conditions: drying gas (N2, purity >98%) 350 °C and 10 L/min; capillary voltage 4.5 kV; nozzle voltage 1 kV; nebulizer gas 35 psig; sheath gas (N2, purity >98%) 375 °C and 11 L/min. The fragmentor was at 175 V, the skimmer at 65 V, and the OCT 1 RF at 750 V. High-resolution MS spectra were achieved from 5 to 30 min in positive mode in the range 100−1700 m/z; the mass axis was calibrated daily using the Agilent tuning mix HP0321 (Agilent Technologies) prepared in acetonitrile and water. Calibration curves for E-CO2H, B-CO2H, and F-CO2H were derived in the 0.4−10 μM range from stock solutions containing the three analytes ca. 80 μmol/ g in water, appropriately diluted in HPLC-grade water. Biological Studies. Cytotoxicity. Human cell lines A2780 and A2780cisR (ovarian carcinoma) were obtained from the European Collection of Cell Cultures, HEK-293 (embryonic kidney) was obtained from the ATCC (Merck, Buchs, Switzerland), and MCF-7 (breast carcinoma) and HeLa (cervical carcinoma) were obtained from the ECACC (European Collection of Authenticated Cell Cultures, Salisbury). HEK-293T cells were provided by the biological screening facility (EPFL, Switzerland). CHO-K1 and MMC-2 (derived from CHO-K1) Chinese hamster ovary cell lines were provided by Miroslav Pirsel (Institute of Experimental Oncology SAS, Bratislava). Cell culture media RPMI 1640 GlutaMAX and DMEM GlutaMAX and penicillin/streptomycin were purchased from Life Technologies, and fetal bovine serum (FBS) was obtained from Merck. Cancer cells were cultured in RPMI 1640 GlutaMAX;HEK- Article 293 and HEK-293T cells were cultured in DMEM GlutaMAX. Both media were supplemented with 10% FBS and 1% penicillin/ streptomycin. The cells were cultured at 37 °C with CO2 (5%). The resistance in A2780cisR cells was maintained by routine additions of cisplatin (2 μM) to the medium. The cytotoxicity was determined using the MTT assay.35 Cells were seeded in ﬂat-bottomed 96-well plates as suspensions in a prepared medium at a density of 4300 cells/ well in 100 μL. Stock solutions of metal complexes were prepared in DMSO and sequentially diluted in the medium up to the desired concentration (0−200 μM range) and 0.5% DMSO. The cytotoxic eﬀects of RAPTA-C and cisplatin were also assessed as negative and positive control experiments. The compounds were added to the cells following a 24 h preincubation in 100 μL aliquots, and the plates were incubated for an additional 72 h. MTT (20 μL, 5 mg/mL in Dulbecco’s phosphate buﬀered saline) was then added to the cells, and the plates were incubated for another 4 h. The medium was removed, and the purple formazan products, formed by the mitochondrial dehydrogenase activity of vital cells, were dissolved in DMSO (100 μL/well). The absorbance directly proportional to the number of surviving cells was read at 590 nm using a SpectroMax M5e multimode microplate reader (using SoftMax Pro software, version 6.2.2). The percentage of surviving cells in treated wells vs untreated control wells was calculated; the resulting IC50 values are reported as means ± standard deviation from two independent experiments, each comprising four tests per concentration level. Cellular Uptake. A2780 cells were seeded at a density of 2 × 106 cells/Petri dish and incubated overnight. The cells were treated with the tested compounds at the concentrations and times given in the respective table. Following the treatment, the cells were harvested (trypsin), washed, and pelleted. The pellets were digested with HCl using a microwave digestion system (CEM Mars). The Ru/Ir amount was determined with ICP-MS. DNA Metalation. After the treatment (see above), the cells were lyzed using DNAzol (DNAzol, MRC) reagent, following the manufacturer’s protocol. The DNA content was determined spectrophotometrically; the samples were lyzed in 30% HCl (Suprapur, Merck Millipore), and the Ru/Ir content was quantiﬁed by ICP-MS. Enzyme Inhibition Assays. Preparation of Cell Lysates for Analysis of Enzyme Activities. HeLa cells were seeded at a density of 3 × 105 cells/dish and incubated at 37 °C. The next day, the cells were treated with the investigated compounds or mixtures of their respective components at concentrations corresponding to the IC50 values previously determined with MTT assays after 72 h. Following a further 18 h treatment, the cells were scraped, washed with PBS, and counted. Identical cell counts were pelleted and lyzed in PBS-based lysis buﬀer (1% NP40, 1 mM PMSF, cocktail of protease inhibitors) on ice for 15 min. The supernatant was cleared and immediately subjected to an enzyme activity analysis. Analysis of Glutathione S-Transferase activity. The enzyme activity was evaluated with a Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen) following the manufacturer’s protocol. Brieﬂy, the cell lysates were plated in several dilutions in the Assay Buﬀer. Master mix containing Detection Reagent and Glutathione was added to the wells. The ﬂuorescence was read in a kinetic mode at 460 nm with excitation at 390 nm. After blank subtraction, the data were plotted as ΔRFU in the initial 5 min of the reaction. Data from three to ﬁve measurements were normalized to the data of nontreated control cells. Analysis of Cyclooxygenase (COX) Activity. The enzyme activity was evaluated with a Cyclooxygenase Activity Assay Kit (Abcam) following the manufacturer’s protocol. Brieﬂy, the cell lysates were plated in several dilutions in Assay Buﬀer. Then the Reaction Mix containing COX Probe and COX Cofactor was added to each well. The reaction was initiated with the addition of an arachidonic acid/ NaOH solution. Fluorescence data were read in a kinetic mode at 587 nm with excitation at 535 nm. After blank subtraction, the data were plotted as ΔRFU in the initial 5 min of the reaction. Data from three to ﬁve measurements were normalized to the data of nontreated control cells. 9538 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry ■ ■ pubs.acs.org/IC ASSOCIATED CONTENT REFERENCES (1) (a) Š tarha, P.; Trávníček, Z. Non-platinum complexes containing releasable biologically active ligands. Coord. Chem. Rev. 2019, 395, 130−145. (b) Kenny, R. G.; Marmion, C. J. Toward Multi-Targeted Platinum and Ruthenium Drugs-A New Paradigm in Cancer Drug Treatment Regimens? Chem. Rev. 2019, 119, 1058−1137. (c) Khoury, A.; Deo, K. M.; Aldrich-Wright, J. R. Recent advances in platinumbased chemotherapeutics that exhibit inhibitory and targeted mechanisms of action. J. Inorg. Biochem. 2020, 207, 111070. (d) Boros, E.; Dyson, P. J.; Gasser, G. Classification of Metal-based Drugs According to Their Mechanisms of Action. Chem. 2020, 6, 41− 60. (2) Selected references: (a) Ang, W. H.; Khalaila, I.; Allardyce, C. S.; Juillerat-Jeanneret, L.; Dyson, P. J. Rational design of platinum(IV) compounds to overcome glutathione-S-transferase mediated drug resistance. J. Am. Chem. Soc. 2005, 127, 1382−1383. (b) Cheng, Q.; Shi, H.; Wang, H.; Wang, J.; Liu, Y. Asplatin enhances drug efficacy by altering the cellular response. Metallomics 2016, 8, 672−678. (c) Nazarov, A. A.; Meier, S. M.; Zava, O.; Nosova, Y. N.; Milaeva, E. R.; Hartinger, C. G.; Dyson, P. J. Protein ruthenation and DNA alkylation: chlorambucil-functionalized RAPTA complexes and their anticancer activity. Dalton Trans. 2015, 44, 3614−3623. (d) Yang, J.; Sun, X.; Mao, W.; Sui, M.; Tang, J.; Shen, Y. Conjugate of Pt(IV)− Histone Deacetylase Inhibitor as a Prodrug for Cancer Chemotherapy. Mol. Pharmaceutics 2012, 9, 2793−2800. (e) Dhar, S.; Lippard, S. J. Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22199− 22204. (f) Petruzzella, E.; Phillip Braude, J.; Aldrich-Wright, J. R.; Gandin, V.; Gibson, D. A Quadruple-Action Platinum(IV) Prodrug with Anticancer Activity Against KRAS Mutated Cancer Cell Lines. Angew. Chem., Int. Ed. 2017, 56, 11539−11544. (3) Gibson, D. Platinum(IV) anticancer agents; are we en route to the holy grail or to a dead end? J. Inorg. Biochem. 2021, 217, 111353. (4) (a) Hu, W.; Fang, L.; Hua, W.; Gou, S. Biotin-Pt (IV)indomethacin hybrid: A targeting anticancer prodrug providing enhanced cancer cellular uptake and reversing cisplatin resistance. J. Inorg. Biochem. 2017, 175, 47−57. (b) Petruzzella, E.; Sirota, R.; Solazzo, I.; Gandin, V.; Gibson, D. Triple action Pt(IV) derivatives of cisplatin: a new class of potent anticancer agents that overcome resistance. Chem. Sci. 2018, 9, 4299. (c) Babak, M. V.; Zhi, Y.; Czarny, B.; Boon Toh, T.; Hooi, L.; Kai-Hua Chow, E.; Ang, W. H.; Gibson, D.; Pastorin, G. Dual-Targeting Dual-Action Platinum(IV) Platform for Enhanced Anticancer Activity and Reduced Nephrotoxicity. Angew. Chem., Int. Ed. 2019, 58, 8109−8114. (d) Karmakar, S.; Poetsch, C.; Kowol, R.; Heffeter, P.; Gibson, D. Synthesis and Cytotoxicity of Water-Soluble Dual- and Triple-Action Satraplatin Derivatives: Replacement of Equatorial Chlorides of Satraplatin by Acetates. Inorg. Chem. 2019, 58, 16676−16688. (e) Shi, H.; Imberti, C.; Huang, H.; Hands-Portman, I.; Sadler, P. J. Biotinylated photoactive Pt(IV) anticancer complexes. Chem. Commun. 2020, 56, 2320−2323. (f) Babu, T.; Sarkar, A.; Karmakar, S.; Schmidt, C.; Gibson, D. Multiaction Pt(IV) Carbamate Complexes Can Codeliver Pt(II) Drugs and Amine Containing Bioactive Molecules. Inorg. Chem. 2020, 59, 5182−5193. (g) Muhammad, N.; Tan, C.-P.; Nawaz, U.; Wang, J.; Wang, F.-X.; Nasreen, S.; Ji, L.-N.; Mao, Z.-W. Multiaction Platinum(IV) Prodrug Containing Thymidylate Synthase Inhibitor and Metabolic Modifier against Triple-Negative Breast Cancer. Inorg. Chem. 2020, 59, 12632−12642. (h) Karmakar, S.; Kostrhunova, H.; Ctvrtlikova, T.; Novohradsky, V.; Gibson, D.; Brabec, V. Platinum(IV)-Estramustine Multiaction Prodrugs Are Effective Antiproliferative Agents against Prostate Cancer Cells. J. Med. Chem. 2020, 63, 13861−13877. (5) Biancalana, L.; Batchelor, L. K.; Pereira, S. A. P.; Tseng, P.-J.; Zacchini, S.; Pampaloni, G.; Saraiva, L. M. F. S.; Dyson, P. J.; Marchetti, F. Bis-conjugation of Bioactive Molecules to Cisplatin-like Complexes through (2,2′-Bipyridine)-4,4′-Dicarboxylic Acid with Optimal Cytotoxicity Profile Provided by the Combination Ethacrynic Acid/Flurbiprofen. Chem. - Eur. J. 2020, 26, 17525− 17535. sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c00641. Characterization of compounds, including IR, NMR and MS spectra, X-ray diﬀraction data, and solution stability studies (NMR data). Additional biological data (cytotoxicity, enzyme inhibition assays) (PDF) Accession Codes CCDC 2063385 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ Article AUTHOR INFORMATION Corresponding Authors Lorenzo Biancalana − Dipartimento di Chimica e Chimica Industriale, Università di Pisa, I-56124 Pisa, Italy; orcid.org/0000-0002-9276-0095; Email: lorenzo.biancalana@unipi.it Viktor Brabec − Czech Academy of Sciences, Institute of Biophysics, CZ-61265 Brno, Czech Republic; orcid.org/ 0000-0002-8233-1393; Email: brabec@ibp.cz Authors Hana Kostrhunova − Czech Academy of Sciences, Institute of Biophysics, CZ-61265 Brno, Czech Republic Lucinda K. Batchelor − Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Mouna Hadiji − Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Ilaria Degano − Dipartimento di Chimica e Chimica Industriale, Università di Pisa, I-56124 Pisa, Italy; orcid.org/0000-0002-3585-8555 Guido Pampaloni − Dipartimento di Chimica e Chimica Industriale, Università di Pisa, I-56124 Pisa, Italy Stefano Zacchini − Dipartimento di Chimica Industriale “Toso Montanari”, Università di Bologna, I-40136 Bologna, Italy; orcid.org/0000-0003-0739-0518 Paul J. Dyson − Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland; orcid.org/0000-0003-3117-3249 Fabio Marchetti − Dipartimento di Chimica e Chimica Industriale, Università di Pisa, I-56124 Pisa, Italy; orcid.org/0000-0002-3683-8708 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.1c00641 Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS We gratefully thank the University of Pisa (Fondi di Ateneo 2020 and PRA_2020_39), the Czech Science Foundation (Grant 20-14082J), and the Swiss National Science Foundation for ﬁnancial support. 9539 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC (6) (a) Nazarov, A. A.; Hartinger, C. G.; Dyson, P. J. Opening the lid on piano-stool complexes: an account of ruthenium (II)−arene complexes with medicinal applications. J. Organomet. Chem. 2014, 751, 251−260. (b) Murray, B. S.; Babak, M. V.; Hartinger, C. G.; Dyson, P. J. The development of RAPTA compounds for the treatment of tumors. Coord. Chem. Rev. 2016, 306, 86−114. (c) Anthony, E. J.; Bolitho, E. M.; Bridgewater, H. E.; Carter, O. W. L.; Donnelly, J. M.; Imberti, C.; Lant, E. C.; Lermyte, F.; Needham, R. J.; Palau, M.; Sadler, P. J.; Shi, H.; Wang, F.-X.; Zhang, W.-Y.; Zhang, Z. Metallodrugs are unique: opportunities and challenges of discovery and development. Chem. Sci. 2020, 11, 12888−12917. (d) Meier-Menches, S. M.; Gerner, C.; Berger, W.; Hartinger, C. G.; Keppler, B. K. Structure−activity relationships for ruthenium and osmium anticancer agents − towards clinical development. Chem. Soc. Rev. 2018, 47, 909−928. (e) SoldevilaBarreda, J. J.; Metzler-Nolte, N. Intracellular Catalysis with Selected Metal Complexes and Metallic Nanoparticles: Advances toward the Development of Catalytic Metallodrugs. Chem. Rev. 2019, 119, 829− 869. (7) (a) Movassaghi, S.; Leung, E.; Hanif, M.; Lee, B. Y. T.; Holtkamp, H. U.; Tu, J. K. Y.; Söhnel, T.; Jamieson, S. M. F.; Hartinger, C. G. A Bioactive L-Phenylalanine-Derived Arene in Multitargeted Organoruthenium Compounds: Impact on the Antiproliferative Activity and Mode of Action. Inorg. Chem. 2018, 57, 8521−8529. (b) Biancalana, L.; Gruchała, M.; Batchelor, L. K.; Błauż, A.; Monti, A.; Pampaloni, G.; Rychlik, B.; Dyson, P. J.; Marchetti, F. Conjugating Biotin to Ruthenium(II) Arene Units via Phosphine Ligand Functionalization. Eur. J. Inorg. Chem. 2020, 2020, 1061−1072. (8) (a) Hayes, J. D.; Flanagan, J. U.; Jowsey, I. R. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 51−88. (b) Townsend, D. M.; Findlay, V. L.; Tew, K. D. Glutathione STransferases as Regulators of Kinase Pathways and Anticancer Drug Targets. Methods Enzymol. 2005, 401, 287−307. (9) (a) Ang, W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Rational design of an organometallic glutathione transferase inhibitor. Angew. Chem., Int. Ed. 2009, 48, 3854−3857. (b) Agonigi, G.; Riedel, T.; Zacchini, S.; Paunescu, E.; Pampaloni, G.; Bartalucci, N.; Dyson, P. J.; Marchetti, F. Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic Acid-Modified Pyridine- and Triphenylphosphine-Ligands. Inorg. Chem. 2015, 54, 6504−6512. (c) Păunescu, E.; Soudani, M.; Martin, P.; Scopelliti, R.; Lo Bello, M.; Dyson, P. J. Organometallic Glutathione S-Transferase Inhibitors. Organometallics 2017, 36, 3313−3321. (10) (a) Knights, K. M.; Mangoni, A. A.; Miners, J. O. Defining the COX inhibitor selectivity of NSAIDs: implications for understanding toxicity. Expert Rev. Clin. Pharmacol. 2010, 3, 769−776. (b) Regulski, M.; Regulska, K.; Prukała, W.; Piotrowska, H.; Stanisz, B.; Murias, M. COX-2 inhibitors: a novel strategy in the management of breast cancer. Drug Discovery Today 2016, 21, 598−615. (c) Tan, J.; Li, C.; Wang, Q.; Li, S.; Chen, S.; Zhang, J.; Wang, P. C.; Ren, L.; Liang, X.-J. A Carrier-Free Nanostructure Based on Platinum(IV) Prodrug Enhances Cellular Uptake and Cytotoxicity. Mol. Pharmaceutics 2018, 15, 1724−1728. (11) (a) Raja, M. U.; Tauchman, J.; Therrien, B.; Süss-Fink, G.; Riedel, T.; Dyson, P. J. Arene ruthenium and pentamethylcyclopentadienyl rhodium and iridium complexes containing N,O-chelating ligands derived from piroxicam: Synthesis, molecular structure and cytotoxicity. Inorg. Chim. Acta 2014, 409, 479−483. (b) Paunescu, E.; McArthur, S.; Soudani, M.; Scopelliti, R.; Dyson, P. J. Nonsteroidal Anti-inflammatory Organometallic Anticancer Compounds. Inorg. Chem. 2016, 55, 1788−1808. (c) Biancalana, L.; Batchelor, L. K.; De Palo, A.; Zacchini, S.; Pampaloni, G.; Dyson, P. J.; Marchetti, F. A general strategy to add diversity to ruthenium arene complexes with bioactive organic compounds via a coordinated (4-hydroxyphenyl)diphenylphosphine ligand. Dalton Trans. 2017, 46, 12001−12004. (d) Ashraf, A.; Aman, F.; Movassaghi, S.; Zafar, A.; Kubanik, M.; Siddiqui, W. A.; Reynisson, J. H.; Söhnel, T.; Jamieson, S. M.; Hanif, Article M. Structural Modifications of the Antiinflammatory Oxicam Scaffold and Preparation of Anticancer Organometallic Compounds. Organometallics 2019, 38, 361−374. (12) (a) Maiti, S.; Paira, P. Biotin conjugated organic molecules and proteins for cancer therapy: A review. Eur. J. Med. Chem. 2018, 145, 206−223. (b) Bertrand, B.; O’Connell, M. A.; Waller, Z. A. E.; Bochmann, M. A Gold(III) Pincer Ligand Scaffold for the Synthesis of Binuclear and Bioconjugated Complexes: Synthesis and Anticancer Potential. Chem. - Eur. J. 2018, 24, 3613−3622. (c) Côrte-Real, L.; Karas, B.; Bras, A. R.; Pilon, A.; Avecilla, F.; Marques, F.; Preto, A.; Buckley, B. T.; Cooper, K. R.; Doherty, C.; Garcia, M. H.; Valente, A. Ruthenium−Cyclopentadienyl Bipyridine−Biotin Based Compounds: Synthesis and Biological Effect. Inorg. Chem. 2019, 58, 9135−9149. (d) Plazuk, D.; Zakrzewski, J.; Salmain, M.; Błauz, A.; Rychlik, B.; Strzelczyk, P.ł; Bujacz, A.; Bujacz, G. Ferrocene−Biotin Conjugates Targeting Cancer Cells: Synthesis, Interaction with Avidin, Cytotoxic Properties and the Crystal Structure of the Complex of Avidin with a Biotin−Linker−Ferrocene Conjugate. Organometallics 2013, 32, 5774−5783. (13) (a) Wu, S.; Zhou, Y.; Rebelein, J. G.; Kuhn, M.; Mallin, H.; Zhao, J.; Igareta, N. V.; Ward, T. R. Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes. J. Am. Chem. Soc. 2019, 141, 15869−15878. (b) Okamoto, Y.; Köhler, V.; Paul, C. E.; Hollmann, F.; Ward, T. R. Efficient In Situ Regeneration of NADH Mimics by an Artificial Metalloenzyme. ACS Catal. 2016, 6, 3553−3557. (c) Muñoz Robles, V.; Dürrenberger, M.; Heinisch, T.; Lledós, A.; Schirmer, T.; Ward, T. R.; Maréchal, J.-D. Structural, Kinetic, and Docking Studies of Artificial Imine Reductases Based on Biotin−Streptavidin Technology: An Induced Lock-andKey Hypothesis. J. Am. Chem. Soc. 2014, 136, 15676−15683. (14) (a) Lewandowski, E. M.; Skiba, J.; Torelli, N. J.; Rajnisz, A.; Solecka, J.; Kowalski, K.; Chen, Y. Antibacterial properties and atomic resolution X-ray complex crystal structure of a ruthenocene conjugated β-lactam antibiotic. Chem. Commun. 2015, 51, 6186− 6189. (b) Skiba, J.; Rajnisz, A.; Navakoski de Oliveira, K.; Ott, I.; Solecka, J.; Kowalski, K. Ferrocenyl bioconjugates of ampicillin and 6aminopenicillinic acid–synthesis, electrochemistry and biological activity. Eur. J. Med. Chem. 2012, 57, 234−239. (c) Ketikidis, J.; Banti, C. N.; Kourkoumelis, N.; Tsiafoulis, C. G.; Papachristodoulou, C.; Kalampounias, A. G.; Hadjikakou, S. K. Conjugation of PenicillinG with Silver(I) Ions Expands Its Antimicrobial Activity against Gram Negative Bacteria. Antibiotics 2020, 9, 25. (15) (a) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 2010, 39, 8113−8127. (b) Yu, C.; Wang, Z.; Sun, Z.; Zhang, L.; Zhang, W.; Xu, Y.; Zhang, J.-J. Platinum-Based Combination Therapy: Molecular Rationale, Current Clinical Uses, and Future Perspectives. J. Med. Chem. 2020, 63, 13397−13412. (16) (a) Zhang, D.; Dufek, E. J.; Clennan, E. L. Syntheses, Characterizations, and Properties of Electronically Perturbed 1,1′Dimethyl-2,2′-bipyridinium Tetrafluoroborates. J. Org. Chem. 2006, 71, 315−319. (b) Miyoshi, D.; Karimata, H.; Wang, Z.-M.; Koumoto, K.; Sugimoto, N. Artificial G-Wire Switch with 2,2′-Bipyridine Units Responsive to Divalent Metal Ions. J. Am. Chem. Soc. 2007, 129, 5919−5925. (17) To the best of our knowledge, only a few transition-metal benzylpenicillin complexes have been reported in the literature: (a) Grochowski, T.; Samochoka, K. Structural Characterization of the Platinum(II)-Penicillin Complexes. Polyhedron 1991, 10, 1473−1477. Refat, M. S.; Al-Saif, F. A. Spectroscopic and thermal investigations of transition and non-transition metal complexes of penicillin G as potential biological active species. Russ. J. Gen. Chem. 2014, 84, 143− 156 (characterization limited to IR spectroscopy and magnetic analysis). (18) The cation [6]+ was previously reported as a chloride salt: Brewster, T. P.; Miller, A. J. M.; Heinekey, D. M.; Goldberg, K. I. Hydrogenation of Carboxylic Acids Catalyzed by Half-Sandwich Complexes of Iridium and Rhodium. J. Am. Chem. Soc. 2013, 135, 16022−16025. 9540 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541 Inorganic Chemistry pubs.acs.org/IC Article (30) A solution of [IrCl2(η5-C5Me5)]2 (29 mg, 0.036 mmol) in MeOH (5 mL) was treated with AgNO3 (12 mg, 0.073 mmol) and stirred at room temperature in the dark. After 1 h, the mixture was ﬁltered over a celite pad and LEB (66 mg, 0.078 mmol) was added to the yellow ﬁltrate solution. The suspension was stirred at reﬂux for 15 h, aﬀording an orange solution. NMR analysis (1H, CDCl3) revealed a mixture of [6]+, B-CO2(CH2)2OH, E-CO2(CH2)2OH, and E-CO2H (1:1:0.8:0.2 molar ratio). (31) Sheldrick, G. M. SADABS-2008/1 - Bruker AXS Area Detector Scaling and Absorption Correction; Bruker AXS: Madison, WI, USA, 2008. (32) Sheldrick, G. M. Crystal Structure Refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 3−8. (33) Rundlöf, T.; Mathiasson, M.; Bekiroglu, S.; Hakkarainen, B.; Bowden, T.; Arvidsson, T. Survey and qualification of internal standards for quantification by 1H NMR spectroscopy. J. Pharm. Biomed. Anal. 2010, 52, 645−651. (34) 14N and 35Cl NMR reference data. All data refer to the pure compounds dissolved in the selected solvent. NaCl: 35Cl NMR (8.8 × 10−2 M, DMSO/H2O 5/1, few scans): δ/ppm 44.6 (Δν1/2 = 545 Hz). KCl: 35Cl NMR (1.9 × 10−2 M, DMSO/H2O 5/1, few scans): δ/ppm 41.2 (Δν1/2 = 447 Hz). NaNO3: 14N NMR (DMSO/H2O 5/1): δ/ ppm −5.1 (Δν1/2 = 23 Hz). (35) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (19) The maximum amount of water in these experimentsand thus the DMSO/water ratiois limited by the solubility of the compounds and the millimolar concentration required for a goodquality 1H NMR spectrum. (20) (a) Bugarcic, T.; Habtemariam, A.; Stepankova, J.; Heringova, P.; Kasparkova, J.; Deeth, R. J.; Johnstone, R. D. L.; Prescimone, A.; Parkin, A.; Parsons, S.; Brabec, V.; Sadler, P. J. The Contrasting Chemistry and Cancer Cell Cytotoxicity of Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes. Inorg. Chem. 2008, 47, 11470−11486. (b) Liu, Z.; Habtemariam, A.; Pizarro, A. M.; Fletcher, S. A.; Kisova, A.; Vrana, O.; Salassa, L.; Bruijnincx, P. C. A.; Clarkson, G. J.; Brabec, V.; Sadler, P. J. Organometallic Half-Sandwich Iridium Anticancer Complexes. J. Med. Chem. 2011, 54, 3011−3026. (21) For ethacrynic acid: (a) Wang, R.; Li, C.; Song, D.; Zhao, G.; Zhao, L.; Jing, Y. Ethacrynic Acid Butyl-Ester Induces Apoptosis in Leukemia Cells through a Hydrogen Peroxide−Mediated Pathway Independent of Glutathione S-Transferase P1−1 Inhibition. Cancer Res. 2007, 67, 7856−7564. (b) Agonigi, G.; Riedel, T.; Pilar Gay, M.; Biancalana, L.; Oñate, E.; Dyson, P. J.; Pampaloni, G.; Paunescu, E.; Esteruelas, M. A.; Marchetti, F. Arene Osmium Complexes with Ethacrynic Acid-Modified Ligands: Synthesis, Characterization, and Evaluation of Intracellular Glutathione S-Transferase Inhibition and Antiproliferative Activity. Organometallics 2016, 35, 1046−1056. (c) Mignani, S.; El Brahmi, N.; El Kazzouli, S.; Eloy, L.; Courilleau, D.; Caron, J.; Bousmina, M. M.; Caminade, A.-M.; Cresteil, T.; Majoral, J.-P. A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action. Eur. J. Med. Chem. 2016, 122, 656−673. (22) (a) Schmitt, F.; Kasparkova, J.; Brabec, V.; Begemann, G.; Schobert, R.; Biersack, B. New (arene)ruthenium(II) complexes of 4aryl-4H-naphthopyrans with anticancer and anti-vascular activities. J. Inorg. Biochem. 2018, 184, 69. (b) Novohradsky, V.; Zerzankova, L.; Stepankova, J.; Kisova, A.; Kostrhunova, H.; Liu, Z.; Sadler, P. J.; Kasparkova, J.; Brabec, V. A dual-targeting, apoptosis-inducing organometallic half-sandwich iridium anticancer complex. Metallomics 2014, 6, 1491−501. (23) Ethacrynic acid (CAS 58-54-8): 2,3-dichloro-4-(2-methylene-1oxobutyl)phenoxyacetic acid. Flurbiprofen (CAS 5104-49-4): (±)-2ﬂuoro-α-methyl-(1,1′-biphenyl)-4-acetic acid (racemic mixture). Biotin (CAS 58-85-5): 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4d]imidazol-4-yl]pentanoic acid. Potassium benzylpenicillin (CAS 11398-4): potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl) amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate. (24) (a) Bennett, M. A.; Smith, A. K. Arene ruthenium(II) complexes formed by dehydrogenation of cyclohexadienes with ruthenium(III) trichloride. J. Chem. Soc., Dalton Trans. 1974, 233− 241. (b) Optimized procedure: Biancalana, L.; Zacchini, S.; Ferri, N.; Lupo, M. G.; Pampaloni, G.; Marchetti, F. Tuning the cytotoxicity of ruthenium(II) para-cymene complexes by mono-substitution at a triphenylphosphine/phenoxydiphenylphosphine ligand. Dalton Trans. 2017, 46, 16589−16604. (25) White, C.; Yates, A.; Maitlis, P. M. (η5pentamethylcyclopentadienyl)rhodium and -iridium compounds. Inorg. Synth. 2007, 29, 228−234. (26) Fulmer, G. R.; Miller, A. J. M.; Sherden, N. H.; Gottlieb, H. E.; Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg, K. I. NMR Chemical Shifts of Trace Impurities: Common Laboratory Solvents, Organics, and Gases in Deuterated Solvents Relevant to the Organometallic Chemist. Organometallics 2010, 29, 2176−2179. (27) Harris, R. K.; Becker, E. D.; Cabral De Menezes, S. M.; Goodfellow, R.; Granger, P. NMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC Recommendations 2001). Pure Appl. Chem. 2001, 73, 1795−1818. (28) Willker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W. Gradient selection in inverse heteronuclear correlation spectroscopy. Magn. Reson. Chem. 1993, 31, 287−292. (29) Menges, F. Spectragryph-optical spectroscopy software, Ver. 1.2.14d, 2016−2020; http://www.eﬀemm2.de/spectragryph. 9541 https://doi.org/10.1021/acs.inorgchem.1c00641 Inorg. Chem. 2021, 60, 9529−9541