Half-Sandwich Ir(III) Complex of <i>N</i>1-Pyridyl-7-azaindole Exceeds Cytotoxicity of Cisplatin at Various Human Cancer Cells and 3D Multicellular Tumor Spheroids

Article Cite This: Organometallics XXXX, XXX, XXX−XXX pubs.acs.org/Organometallics Half-Sandwich Ir(III) Complex of N1‑Pyridyl-7-azaindole Exceeds Cytotoxicity of Cisplatin at Various Human Cancer Cells and 3D Multicellular Tumor Spheroids Pavel Š tarha,*,† Zdeněk Trávníček,† Hana Crlíková,‡ Ján Vančo,† Jana Kašpárková,‡ and Zdeněk Dvořaḱ † Downloaded via EASTERN KENTUCKY UNIV on August 15, 2018 at 20:29:20 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. † Division of Biologically Active Complexes and Molecular Magnets, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, Š lechtitelů 27, 783 71 Olomouc, Czech Republic ‡ Department of Biophysics, Faculty of Science, Palacký University, 17. listopadu 12, 771 46 Olomouc, Czech Republic S Supporting Information * ABSTRACT: The half-sandwich iridium(III) complexes [Ir(η5-Cpx)(phaza−)Cl] (1, 2), [Ir(η5-Cpx)(thaza−)Cl] (3, 4), and [Ir(η5-Cpx)(pyaza)Cl]PF6 (5, 6) containing deprotonated 1-phenyl-7-azaindole (phaza−) and 1-(thiophen-2-yl)-7-azaindole (thaza−) and electroneutral 1-(pyridin-2-yl)-7-azaindole (pyaza), were prepared; Cpx = pentamethylcyclopentadienyl (Cp*; for 1, 3, and 5) or 1,2,3,4-tetramethyl-5-phenylcyclopentadienyl (Cpph; for 2, 4, and 6). The complexes were thoroughly characterized, including a single-crystal X-ray analysis of complexes 1, 5, and 6. All of the complexes were screened for their in vitro cytotoxicity at the A2780 human ovarian carcinoma cell line and its A2780R cisplatin-resistant variant (2D culture cells). The best-performing complex 6 was further studied against the human DU-145 prostatic carcinoma, A549 lung carcinoma, HCT116 colon carcinoma, HeLa cervix adenocarcinoma, and MCF7 breast adenocarcinoma cell lines (2D culture cells). Complex 6 showed a cytotoxic proﬁle diﬀerent from that of cisplatin at the used cells, with the highest activity detected at the A2780, MCF7, and HCT116 cells (IC50 = 3.1, 6.9, and 10.4 μM, respectively). Complex 6 exhibited relevant selectivity toward cancer cells (IC50 = 3.1−13.0 μM) over the MRC-5 human noncancerous lung ﬁbroblast cells (IC50 > 50.0 μM). Complex 6 was markedly more accumulated by the A2780 cells in comparison to cisplatin after 24 h exposure. Flow cytometry studies showed that the cell cycle of the A2780 cells treated by complex 6 is modiﬁed diﬀerently (G0/G1 arrest) in comparison to cisplatin (G2/M arrest). Additionally to the monolayer (2D) cancer cell cultures, the cytotoxicity of complex 6 was for the ﬁrst time among half-sandwich iridium(III) complexes also assessed at spheroid (3D) MCF7 cells, where its potency (IC50 = 22.9 μM for complex 6) remained signiﬁcantly better than that for the reference drug cisplatin (IC50 = 35.4 μM). ■ Cpph)(bpy)Cl]PF6 (IC50 = 15.9 μM) are markedly less potent than the electroneutral analogues [Ir(η5-Cp*)(phpy)Cl] (IC50 = 10.8 μM) and [Ir(η5-Cpph)(phpy)Cl] (IC50 = 2.1 μM), containing isoelectronic anionic C,N-coordinated ligands6 (bpy = 2,2′-bipyridine, Hphpy = 2-phenylpyridine, Cp* = pentamethylcyclopentadienyl, Cpph = 1,2,3,4-tetramethyl-5phenylcyclopentadienyl); all of the cited IC50 values are associated with the A2780 human ovarian carcinoma cell line. Further, complexes with extended cyclopentadienyl ligands (e.g., Cpph) showed considerably higher potency in comparison to their Cp* analogues.3b,5c,7 Both approaches (i.e., C,Nvs N,N-ligands and Cp* vs Cpph complexes) were applied in this work. INTRODUCTION Because of the known drawbacks (limited number of treatable tumors, intrinsic and acquired resistance, negative side eﬀects, intravenous application) of the conventional platinum-based anticancer chemotherapeutics (e.g., cisplatin),1 the ﬁeld of medicinal chemistry faces a continual demand for substances overcoming these limitations. One of the most prospective groups of compounds includes organometallic complexes of non-platinum precious metals.2 Among these compounds, iridium(III) half-sandwich cyclopentadienyl complexes were recently reported to be highly cytotoxic,3 acting through a redox-mediated mechanism of action (MoA) diﬀerent from the platinum-based drugs used in clinics.3b,4 Concerning the structure−activity relationship of cytotoxic cyclopentadienyl iridium(III) complexes,3−5 it was reported that ionic complexes containing electroneutral N,N-donor ligands such as [Ir(η5-Cp*)(bpy)Cl]PF6 (IC50 > 100 μM) and [Ir(η5© XXXX American Chemical Society Received: June 15, 2018 A DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics diﬀerently to the Ir(III) metal center, in particular as C,N-, N,N-, and N,S-ligands, respectively. In the case of phaza and pyaza derivatives, the electroneutral complexes [Ir(η5-Cp*)(phaza−)Cl] (1) and [Ir(η5-Cpph)(phaza−)Cl] (2), containing the deprotonated C,N-coordinated phaza− ligand, and the cationic complexes [Ir(η5-Cp*)(pyaza)Cl]PF6 (5) and [Ir(η5Cpph)(pyaza)Cl]PF6 (6), containing pyaza as the electroneutral bidentate N,N-donor ligand (Figure 1), were successfully prepared by syntheses using the dimeric complexes [Ir(μCl)(η5-Cp*)Cl]2 and [Ir(μ-Cl)(η5-Cpph)Cl]2 as the starting Ir(III) compounds (Figure S2). Concerning the thaza derivative, despite the fact that no base was added to the reaction mixtures, the electroneutral complexes [Ir(η5-Cp*)(thaza−)Cl] (3) and [Ir(η5-Cpph)(thaza−)Cl] (4) were prepared, containing thaza− as a deprotonated ligand coordinated through the N7 atom of the 7-azaindole moiety and C12 atom of the pendant thiophenyl group (see the Supporting Information for the atom-numbering scheme; Figure 1). Recently, it was described that the pendant thiophenyl ring of 3-(diphenylphosphane)thiophene (P,C-ligand at alkaline pH) changes a P,Ccoordination mode to a P,S-mode after the addition of triﬂuoroacetic acid (Htfa), which is accompanied by protonation of the appropriate carbon atom.16 Indeed, subsequent addition of a base led to the reversion from P,Sto P,C-coordination mode. A similar phenomenon was reported for an iridium complex containing C,N-coordinated 2-(thiophene)ethaneimine, which reverted to the C,S-ligand with the addition of Htfa.17 In the case of complexes 3 and 4 containing the C,N-coordinated thaza− ligand, a similar change from the observed C,N-coordination mode to the anticipated N,S-coordination mode was not detected by 1H NMR even at pH ∼1 (the addition of Htfa). General Characterization. The HPLC-MS results showed the purity of complexes 1−6 in methanolic solutions as 95.3− 99.9% (Table S1) with a dominant chromatographic peak assigned to [Ir(η5-Cpx)(naza−)]+ for electroneutral complexes 1−4 (naza− = phaza− or thaza−), and to [Ir(η5-Cpx)(pyaza)Cl]+ for cationic complexes 5 and 6. A content of the dominant species detected by HPLC changed negligibly after 72 h of standing in MeOH at ambient temperature, where only complex 4 showed lower than 95% purity. The ESI+ mass spectra (using direct injection without HPLC) of complexes 1−4 contained only one dominant peak (100% relative intensity) assignable to the dechlorinated species [Ir(Cpx)(naza−)]+ (naza− = phaza− or thaza−; Figure S3). In contrast, the ESI+ mass spectra of the cationic complexes 5 and 6 showed two peaks assignable to the [Ir(Cpx)(pyaza)Cl]+ and {[Ir(Cpx)(pyaza)] − H}+ species. All of the hydrogen atoms of cationic complexes 5 and 6 containing the electroneutral pyaza ligand were detected by 1H NMR spectroscopy (Figures S4−S6). In contrast, the 1H NMR spectra of electroneutral complexes 1−4, whose structures involve the deprotonated phaza− and thaza− ligands, showed the appropriate 1H NMR integral intensity decrease due to the mentioned deprotonation of the carbon coordination sites (i.e., C9 for phaza− involved in complexes 1 and 2, and C12 for thaza− involved in complexes 3 and 4) during the complex formation (Figure 2). Regarding the 13C and 15N NMR spectra, all of the carbon and nitrogen atoms were detected for complexes 1−6. A comparison of the spectra of the starting 7azaindole derivatives with complexes 1−6 indirectly indicated the coordination sites of the used naza ligands. The 15N NMR 1H-Pyrrolo[2,3-b]pyridine (7-azaindole) is a biologically very interesting heterocyclic compound, whose derivatives8 or platinum(II) or gold(I) complexes9 were recently reported as highly active against diverse diseases by various research groups. Our recent research also proved that monodentately coordinated 7-azaindole C derivatives release from halfsandwich Ru(II) dichlorido complexes in water-containing solutions.10 This is why we decided to prepare the N1substituted 7-azaindole derivatives, in particular 1-phenyl-7azaindole (phaza),11 1-(pyridin-2-yl)-7-azaindole (pyaza),12 and 1-(thiophen-2-yl)-7-azaindole (thaza),13 as prospective bidentate ligands for anticancer half-sandwich complexes (Figure S1). Regarding the transition-metal complexes containing these ligands, no complexes containing phaza and thaza have been reported to date. As for pyaza, several complexes were reported as containing this ligand bidentately coordinated to the metal center through the nitrogen atoms of both its pyridines (numbered N7 and N9 in this work). In particular, [Cu(pyaza)(PPh3)2]BPh4,14 [Zn(pyaza)(ac)2], [Zn(pyaza)(mba)2],12 [Pt(pyaza)(Ph)2], and [Pt(pyaza)(Me)2]15 were previously reported in the literature, but none of them were studied in connection with any kind of biological activity (ac = acetate anion, mba = 2-methylbutyrate anion, Ph = phenyl, and Me = methyl). This work deals with the preparation, thorough characterization, and studies of in vitro cytotoxicity of a series of the neutral [Ir(η5-Cpx)(naza−)Cl] (naza = phaza for 1 and 2 or thaza for 3 and 4) and cationic [Ir(η5-Cpx)(pyaza)Cl]PF6 (5 and 6) iridium(III) complexes containing the aforementioned 7-azaindole derivatives as bidentate C,N (phaza−, thaza−)- or N,N-donor ligands (pyaza) (Cpx = Cp* for 1, 3, and 5, and Cpph for 2, 4, and 6) (Figure 1). The best-performing complex, Figure 1. Structural formulas and speciﬁcation of composition of complexes 1−6. 6, was, as the ﬁrst half-sandwich iridium(III) complex, studied for its in vitro cytotoxicity at the spheroid (3D) cell culture, representing an advanced model for biocharacterization of pharmacologically prospective cytotoxic agents. ■ RESULTS AND DISCUSSION Synthesis. A series of phaza, pyaza, and thaza 7-azaindole derivatives (Figure S1) was designed to obtain complexes containing bidentate 7-azaindole-based ligands coordinated B DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Table 1. Selected Bond Lengths (Å) and Angles (deg) Determined by a Single-Crystal X-ray Analysis for Complexes 1, 5, and 6 1 Ir−N(7) Ir−C(9) Ir−N(9) Ir−Cl Ir−C(20) Ir−C(21) Ir−C(22) Ir−C(23) Ir−C(24) Ir−Cga N(7)−Ir−C(9) N(7)−Ir−N(9) N(7)−Ir−Cl N(7)−Ir−Cga C(9)−Ir−Cl N(9)−Ir−Cl C(9)−Ir−Cga N(9)−Ir−Cga Cl−Ir−Cga Figure 2. 1H NMR spectra of free thaza (in red) and complex [Ir(η5Cp*)(thaza−)Cl] (3), given with the general assignment of the detected aromatic hydrogen signals (all of the depicted signals have an integral intensity of ∼1). coordination shifts (Δδ) of N1, N7, and N9 of pyaza are −1.9, −94.2, and −93.8 ppm for complex 5 and −2.9, −97.5, and −96.4 ppm for complex 6. Similar 15N NMR coordination shifts of N1 and N7 were detected for C,N-ligands phaza− and thaza− contained in complexes 1 (−4.2 and −105.2 ppm), 2 (−11.8 and −104.5 ppm), 3 (−0.8 and −110.8 ppm), and 4 (−2.4 and −113.1 ppm) on N7-coordination of the mentioned naza− ligands. A coordination of phaza− and thaza− through the deprotonated C9 (for phaza− of 1 and 2) and C12 (for thaza− of 3 and 4) carbon atoms was proved by the considerable 13C NMR upﬁeld shifts of the aforementioned atoms with Δδ values ranging from −35.4 to −10.2 ppm. The characteristic peaks of the vibrations of the Cpx ring were found in the FT-IR spectra of complexes 1−6 at ca. 2910−2970 cm−1 for νs(C−H) and νas(C−H) and at ca. 1450 and 800 cm−1 for νas(C−C) and νas(C−CH3).18 The spectra of the cationic complexes 5 and 6 contained a set of peaks of the ν(P−F) vibrations at 825, 770, and 555 cm−1.19 X-ray Structures of Complexes 1, 5, and 6. The crystallographically characterized complexes 1, 5, and 6 adopt a piano-stool arrangement with a coordinated cyclopentadienyl ring, chlorido ligand, and a bidentate-bonded naza ligand (Figure 3, Table 1, and Table S2). The phaza− ligand of complex 1 is coordinated through the N7 atom of the pyridine ring of the 7-azaindole moiety and the C9 atom of the pendant phenyl N1-substituent. Regarding 5 Bond Lengths (Å) 2.087(3) 2.028(3) 2.054(4) 2.158(3) 2.4107(9) 2.4995(9) 2.150(3) 2.314(4) 2.157(4) 2.341(4) 2.233(3) 2.209(4) 2.256(4) 2.278(4) 2.165(3) 2.306(4) 1.8207(2) 1.9710(3) Bond Angles (deg) 86.77(14) 78.48(11) 88.91(9) 75.97(8) 126.03(8) 132.26(8) 89.03(11) 82.33(8) 127.78(9) 129.91(8) 125.68(2) 135.98(2) 6 2.087(4) 2.122(4) 2.3824(12) 2.171(5) 2.169(5) 2.188(5) 2.157(5) 2.176(4) 1.7886(5) 82.5(2) 87.06(11) 127.30(12) 88.01(11) 128.89(12) 128.11(4) a Cg = centroid of the cyclopentadienyl ring of the Cp* (for 1 and 5) and Cpph (for 6) ligands. complexes 5 and 6, the N7 (7-azaindole moiety) and N9 (pendant pyridine substituent) atoms were detected as the coordination sites of the pyaza chelating ligand. In contrast to the electroneutral complex 1, the molecular structures of the cationic complexes 5 and 6 further contain the PF6− anion, with the shortest Ir−P distance of 6.4795(11), and 6.1421(13) Å, respectively. The dihedral angles formed by the cyclopentadienyl and 7-azaindole rings equal 24.80(11), 17.04(12), and 23.3(2)° for complexes 1, 5, and 6, respectively. The intraligand dihedral angles between the 7azaindole moiety and pendant N1-substituent were found to be 17.29(9), 36.67(10), and 31.4(2)° for complexes 1, 5, and 6, respectively. The N(1)−C(8)−X(9)−Ir torsion angles (X = C for 1 and N for 5 and 6) equal 10.9(5), 12.3(4), and −21.7(7)° for complexes 1, 5, and 6, respectively, while the N(1)−C(7A)−N(7)−Ir torsion angles were −21.9(5), −28.3(5), and 17.3(7)° for complexes 1, 5, and 6, respectively. The crystal structures of complexes 1, 5, and 6 are stabilized by C−H···Cl, C−H···C, and C···C types of noncovalent contacts, while the C−H···F contacts were also found in addition to those mentioned in the crystal structure of the cationic complexes 5 and 6 containing the PF6− counterion (Table S3). Concerning the half-sandwich iridium(III) complexes deposited in the Cambridge Crystallographic Data Centre (CCDC; version 5.39, updated November 2017),20 only complexes [Ir(η5-Cp*)(L1)Cl] (CCDC ﬁle 938957)21 and [Ir(η5-Cp*)(L2)Cl] (CCDC ﬁle 1033927)22 can be seen as having compositions similar to the herein reported complexes 1−6 (represented by crystallographically characterized complexes 1, 5, and 6) (HL1 = 3-phenylimidazo[1,2-a][1,8]naphthyridine, HL 2 = 2-(2,3-dihydro-1H-inden-1-yl)pyrimidine). In particular, complexes [Ir(η5-Cp*)(L1)Cl] and [Ir(η5-Cp*)(L2)Cl] both contain, analogously with complexes Figure 3. Molecular structures of complex 1 (left) and cations of complexes 5 (middle) and 6 (right). Hydrogen atoms (for 1, 5, and 6) and PF6− anions (for 5 and 6) are omitted for clarity and the nonhydrogen atoms are drawn as thermal ellipsoids at 50% probability. C DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Table 2. In Vitro Cytotoxicity (IC50 ± SD; μM) of Complexes 1−6 and Cisplatin against Cisplatin-Sensitive Ovarian Carcinoma Cells (A2780) and Cisplatin-Resistant Ovarian Carcinoma Cells (A2780R)a A2780 A2780R 1 2 3 6 cisplatin 25.4 ± 2.0 >50.0 >50.0 20.3 ± 2.3 20.2 ± 3.2 >25.0 3.1 ± 0.2 17.4 ± 1.3 7.1 ± 1.3 20.6 ± 1.9 a MTT assay, 24 h exposure followed by 72 h recovery. Complexes 4 and 5 were inactive up to the highest tested concentration at both cells (IC50 > 50.0 μM). 1−6, a chelating ligand based on fused six- and ﬁve-membered rings with a pendant aromatic substituent, giving together the deprotonated bidentate C,N-donor ligands. Further for the CCDC deposited X-ray structures, the Cp*-containing halfsandwich iridium(III) complexes are numerous in the literature,3 while crystallographically characterized Cpph analogues (complex 6 in this work) are quite rare.6c,23 In Vitro Cytotoxicity. The in vitro cytotoxicity of complexes 1−6 was screened by an MTT assay against two human cancer cell lines: namely, ovarian carcinoma cells naturally sensitive to cisplatin (A2780) and its variant with the acquired resistance to cisplatin (A2780R). The platinum-based drug cisplatin was used as the standard in this study. The obtained in vitro cytotoxicity results are given in Table 2. Complex 6 had signiﬁcantly lower IC50 values (p < 0.05) against the A2780 ovarian carcinoma cells in comparison to cisplatin. However, the obtained results did not show the ability of complex 6 to overcome the acquired resistance of the A2780 cells toward the therapeutic eﬀect of cisplatin, because the calculated resistance factor (RF = IC50(A2780R)/ IC50(A2780)) of complex 6 (RF ≈ 5.6) was even higher than for cisplatin (RF ≈ 2.9). In accordance with the literature data reported for similar ionic half-sandwich Ir(III) complexes,3b,6c,7 the Cpph complex 6 exceeds the potency of its inactive Cp* analogue 5. Surprisingly, the same trend was not observed for the group of electroneutral complexes 1−4, where the Cp* complexes exceeded the cytotoxicity of their Cpph analogues against the A2780 cells. An unusual cytotoxic proﬁle was observed for complex 2, which showed moderate activity against the resistant A2780R cells, although it was inactive against the A2780 cells. The representative complexes 1, 3 and 6, which were potent against the A2780 cells, were also studied for their toxicity against noncancerous human cells (MRC-5) up to 50.0 μM concentration (Figure 4). Only complex 1 reached the IC50 value (28.3 ± 4.7 μM) within the tested concentration range, indicating low selectivity toward the cancer cells (i.e., A2780) over the noncancerous cells (i.e., MRC-5). Complexes 3 and 6 were markedly less toxic against the MRC-5 cells (IC50 > 50.0 μM) in comparison to cisplatin (IC50 = 7.2 ± 1.2 μM), suggesting their promising selectivity, especially for the best-performing complex 6. With respect to the discussed results of cytotoxicity (A2780 and A2780R cells) and toxicity (MRC-5 cells) screening (vide supra), only the leading complex 6 was selected for additional cytotoxicity experiments focusing on its ability to eﬀectively kill cancer cells other than those of ovarian origin. Thus, the cytotoxic eﬀect of complex 6 was tested against a panel of ﬁve diﬀerent human cancer cell lines: namely, prostatic carcinoma cell DU145, lung carcinoma cells A549, colon carcinoma cells HCT116, cervix adenocarcinoma HeLa, and breast adenocarcinoma MCF7. The cytotoxic eﬃciency of complex 6 in these cell lines was compared to that of clinically used cisplatin. In this experiment, a colorimetric assay based on neutral red uptake was used for in vitro sensitivity testing of cell lines. The MTT assay, commonly used for cytotoxicity testing (and also used in the previous experiment with ovarian cancer cells), requires mitochondrial metabolic activity (measures mitochondria dehydrogenase activity as a marker of cell viability) to convert the colorless tetrazolium to the purple-colored formazan dye. However, a large number of Ir complexes have been shown to interfere with mitochondrial activity24 so that the metabolization of MTT can reﬂect an eﬀect of the Ir complexes on the mitochondrial metabolism rather than the viability of cells. Conversely, the neutral red uptake assay is based on the abilities of viable cells to incorporate and bind the dye in lysosomes, so that it is not aﬀected by changes in the mitochondrial metabolism. Under experimental conditions used in this experiment, complex 6 showed signiﬁcantly better activity toward colon HCT116 and breast MCF7 cancer cells in comparison to cisplatin (Table 3). However, the sensitivity of other cancer cell lines to complex 6 was either less than (Du-145) or comparable to (HeLa, A549) the sensitivity toward cisplatin. These results indicate that the cytotoxic eﬀect of complex 6 might be selective to certain types of cancer. In Vitro Cytotoxicity in Spheroid (3D) Culture. Despite the fact that 2D monolayer cell cultures are frequently used for testing of cytotoxic activity of various compounds, including metallodrugs, their utilization is not ideal because 2D cell monolayers represent an environment distinctly diﬀerent from that in native tumors, in which most of the tissues are 3D with a speciﬁc organization and architecture. Hence, three-dimensional (3D) growth of cell cultures is regarded as a more stringent and representative model for in vitro drug screening.25 Cells growing in 3D cultures possess several in vivo features of tumors such as cell−cell interaction, hypoxia, drug penetration, response, and resistance, and production/ deposition of extracellular matrix.26 Hence, it is now a common opinion that in vitro 3D cultures could ﬁll the gap between conventional 2D in vitro testing and animal models, and the use of 3D cell cultures in drug screening programs is Figure 4. MRC-5 cell viability curves obtained for complexes 1, 3, and 6, given with the reference drug cisplatin. D DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Table 3. In Vitro Cytotoxicity (IC50 ± SD; μM) of Complex 6 and Cisplatin in a Panel of Five Human Cell Linesa 6 cisplatin HCT116 HeLa MCF7 DU-145 A549 10.4 ± 1.4 23.1 ± 1.2 10.5 ± 0.7 11.9 ± 1.1 6.9 ± 0.1 21.7 ± 2.1 13.0 ± 1.4 5.0 ± 0.3 9.1 ± 0.7 6.0 ± 0.5 a The cells were treated for 72 h, and cell viability was evaluated by using an assay based on neutral red. The data represent mean ± SD from at least three independent experiments, each made in quadruplicate. or Pt were measured after 24 h exposure of the A2780 cells to complex 6 and cisplatin at their IC50 concentrations. The cellular levels of a particular metal were quantiﬁed by means of inductively coupled plasma mass spectrometry (ICP-MS) analysis. The accumulation of Ir from lipophilic complex 6 in the A2780 cells after 24 h of incubation (22159 ± 1501 pmol Ir/106 cells) was ca. 250-fold greater than the amount of Pt associated with cells treated with hydrophilic cisplatin (86 ± 4 pmol Ir/106 cells). Thus, the hydrophobicity (log P) and cell accumulation correlate signiﬁcantly. However, the cytotoxicity of complex 6 in A2780 cells was only ca. 2-fold higher than that of cisplatin (Table 2), so that the cytotoxic activity of complex 6 does not fully reﬂect elevated uptake and accumulation. Cell Cycle Analysis. Complex 6 induced the relevant changes of the A2780 cell cycle (Figure 6). In particular, its recommended as support for conventional 2D monolayer studies and before activation of animal protocols.27 Therefore, in this study, the eﬀect of complex 6 and cisplatin also on 3D cultures of MCF7 cells was tested to provide more relevant data on its antitumor activity. Under the experimental conditions used in this experiment, the cytotoxicity of complex 6 was ca. 1.5-fold better than that of cisplatin (IC50 values determined from two independent experiments were 22.9 ± 2.8 and 35.4 ± 1.0 μM for complex 6 and cisplatin, respectively) (Figure 5). Thus, these results Figure 5. Comparison of cytotoxicities of complex 6 (and cisplatin for comparative purposes) against MCF7 human breast adenocarcinoma cells, as observed at the 2D (adherent cells) and 3D (spheroids) cultures, both studied at a 72 h exposure time. RA stands for relative activity calculated as IC50(cisplatin)/IC50(complex 6). Figure 6. Results (% cells) of the ﬂow cytometry studies (PI/RNase staining) of the A2780 cells treated with complex 6 and with the reference drug cisplatin, given as arithmetic mean from three independent experiments. The signiﬁcant diﬀerences between the obtained results of complex 6 vs the negative control and cisplatin are given as * for p < 0.05 and *** for p < 0.005. conﬁrm the trend found for monolayer MCF7 cells (Table 3); however, the diﬀerence between cytotoxicities of complex 6 and cisplatin is less pronounced, which may reﬂect properties typical for 3D but not for 2D cultures, such as diﬀerent penetration to the tissue of spheroids. Hydrophobicity and Cellular Accumulation. Hydrophobicity (lipophilicity) is generally accepted as an important and worthy of study feature of newly developed biologically active compounds, including anticancer transition-metal complexes, mainly because it has been shown as being consistent with their potency.3b,5d,28 The log P values determined for complex 6 by an octanol/water partition (NaCl was added to water to suppress hydrolysis) equaled 0.43 ± 0.01, implying markedly higher lipophilicity than for cisplatin (log P = −2.19).29 An eﬃcient cellular uptake through the cellular membrane and accumulation in cells represent key factors essential for the biological activity of drugs. In order to assess a possible eﬀect of these processes on the in vitro cytotoxicity of complex 6, the cellular accumulation of iridium from complex 6 was determined and compared with the cellular accumulation of platinum from the clinically used cisplatin. Cellular levels of Ir application to the A2780 cells induced higher cell populations in the G0/G1-cell cycle phase (77.2 ± 5.2%) than in the case of the untreated cells (60.0 ± 2.8%) employed as the negative control. The observed G0/G1-cell cycle phase arrest induced by complex 6 was connected with the reduction of the G2/M-cell cycle phase population of the cells treated by complex 6 (12.0 ± 0.9%) in comparison to that observed for the negative control (23.5 ± 2.1%). The cell cycle perturbation provoked by complex 6 at the used A2780 cells (i.e., G0/G1 arrest) was comparable with similar half-sandwich Ir(III) chlorido complexes, such as the apoptosis-inducing complex [Ir(η5Cpbph)(phen)Cl]PF6.3b,30 On the other hand, complex 6 induced diﬀerent cell cycle perturbation in the used A2780 cells in comparison to the conventional platinum-based drug cisplatin (Figure 6), which is indicative of diﬀerent mechanisms of action of the studied agents (i.e., 6 vs cisplatin) aﬀecting the cell cycle progression diﬀerently. E DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Studies of Hydrolysis and Interactions with Biomolecules in Vitro. For further interaction studies using mass spectrometry, the cationic complex 6 was selected due to its promising biological activities proﬁle (vide supra). At the start of the experiment, in which the hydrolytic properties of complex 6 should have been shown, we obtained a mass spectrum similar to that discussed above (Figure S3), containing the signals (at m/z 425.2, 584.3, and 620.0, corresponding to the [Ir(Cpph)Cl]+, {[Ir(Cpph)(pyaza)] − H}+, and [Ir(Cpph)(pyaza)Cl]+, species, respectively) of the intact complex only (Figure S7). After 24 h, the spectrum was enriched by a new peak at m/z 292.7, assignable to the dicationic species [Ir(Cpph)(pyaza)]2+, and after 72 h one new peak with low intensity arose at m/z 602.2, corresponding to the [Ir(Cpph)(pyaza)(OH)]+ species. In contrast to platinum(II) complexes used in anticancer therapy (e.g., cisplatin) having DNA as the dominant molecular target, the molecular targets of the complexes containing iridium are quite variable. Some of these targets are cytosolic or membrane proteins. Due to this fact, interactions with model proteins cytochrome c (Cyt c) and hen egg white lysozyme (HEWL) were studied in mixtures containing complex 6 on a 72 h time scale. The intensity of these species, corresponding to the intact protein electrostatically bonding one PF6− residue (Δmass = 145 Da), increased to ca. 20% and 30% of the intensity of the intact protein for Cyt c and HEWL, respectively after 72 h (see Figure S8). Another molecular target, very often identiﬁed in the context of anticancer activity, is reduced glutathione (GSH) and other antioxidants, depletion of which can upset the metabolic balance in the target cells and induce the process of apoptosis (usually via a mitochondrial pathway).31 For this reason, the ability of complex 6 to interact with a mixture of two sulfurcontaining biomolecules (GSH and L-cysteine (CySH)) applied at normal serum concentrations was investigated by mass spectrometry. As can be seen from Figure 7, the mass spectrum of a mixture containing complex 6 (at 10 μM concentration), GSH (at 6 μM concentration), and CySH (at 290 μM concentration), measured after 72 h of standing at laboratory temperature, contained, in addition to the peaks corresponding to complex 6 (at m/z 425.3, 584.4, and 620.2) and those originating from native and oxidized forms of CySH and GSH (at m/z 122.2, 241.1, and 308.2), also two lowintensity peaks corresponding to the interaction intermediates [Ir(Cpph)(CyS)]+ at m/z 510.2 and {[Ir(Cpph)(pyaza)(CyS)]+H2O}+ at m/z 722.0. These results conﬁrmed very slow kinetics of the ligand’s exchange in the studied complex 6. The low interaction rate with the biomolecules used may be attributed to the high hydrolytic stability of complex 6. Figure 7. ESI+ mass spectra of a mixture of CySH and GSH after 72 h (top), complex 6 in MeOH/H2O (1/1, v/v) measured immediately after preparation (middle), and a mixture of complex 6 (at 10 μM concentration), GSH (at 6 μM concentration), and CySH (at 290 μM concentration) measured after 72 h of standing at laboratory temperature (bottom). The speciﬁc peak positions and corresponding ionic species are noted. and some relevant diﬀerences within the processes connected with its mechanism of action (higher cellular accumulation and diﬀerent cancer cell cycle perturbation in comparison with conventional cisplatin). Complex 6 was, as the ﬁrst halfsandwich iridium(III) complex, studied for its cytotoxicity at the spheroid cancer cell culture (MCF7 cells), where it showed higher cytotoxicity in comparison to the platinum-based drug cisplatin. ■ EXPERIMENTAL SECTION Materials. IrCl3·nH2O (99%) was purchased from Precious Metals Online, and 1,2,3,4,5-pentamethylcyclopentadiene (95%), 2,3,4,5tetramethyl-2-cyclopentenone (95%), phenylmagnesium bromide (3.0 M in diethyl ether), 7-azaindole (98%), CuI (≥95%), LiCl (99%), K2CO3 (≥99%), bromobenzene (99%), 2-bromopyridine (99%), 2-bromothiophene (98%), NH 4 Cl (≥99.5%), MgSO 4 (≥99.5%), CH3COONa (≥99%), NH4PF6 (≥98%), KCl (≥99%), L-glutathione reduced (GSH), L-cysteine hydrochloride (CySH·HCl), Cyt c from bovine heart, HEWL, and cisplatin (99.9%) were purchased from Sigma-Aldrich and Alfa Aesar Ltd. Solvents of laboratory grade were purchased from Sigma-Aldrich, FisherScientiﬁc, and Litolab and used without further puriﬁcation, except THF that was dried using 4 Å molecular sieves. Deuterated solvents (DMSO-d6, MeOD-d4, D2O) for NMR experiments and MeCN of HPLC grade were purchased from Sigma-Aldrich. Roswell Park Memorial Institute (RPMI-1640) medium, fetal bovine serum, glutamine, penicillin/streptomycin mixture, trypsin, and phosphatebuﬀered saline (PBS) were purchased from Sigma-Aldrich and FisherScientiﬁc. Synthesis of the Starting Compounds. The starting N1substituted 7-azaindole derivatives 1-phenyl-7-azaindole (phaza),11 1(pyridin-2-yl)-7-azaindole (pyaza),12 and 1-(thiophen-2-yl)-7-azaindole (thaza)13 were prepared using modiﬁcations of the previously ■ CONCLUSIONS Six new half-sandwich iridium(III) complexes were prepared and thoroughly characterized. Complexes contain various N1derivatives of 7-azaindole (phaza, thaza, pyaza) and various cyclopentadienyl rings (Cp*, Cpph). The crystallographic studies of complexes 1, 5, and 6 proved the 7-azaindolebased ligands to be bidentately coordinated through the N7 atom of the 7-azaindole ring and through either carbon (for phenyl derivative phaza− of 1) or nitrogen (for pyridyl derivative pyaza of 5 and 6) atom of the pendant N1substituent. The best-performing complex 6 showed high cytotoxic activity against several of the used human cancer cell lines, high selectivity toward cancer cells over the normal cells, F DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics z): 589.2 (calcd 589.1 for [Ir(Cpph)(thaza−)]+; 100%). Anal. Calcd for IrC26H24N2SCl (624.22): C, 50.03; H, 3.88; N, 4.49. Found: 49.60; H, 3.83; N, 4.12. [Ir(η5-Cp*)(pyaza)Cl]PF6 (5). The starting iridium(III) dimer [Ir(μCl)(η5-Cp*)Cl]2 (0.10 mmol) was poured into a solution of pyaza (0.25 mmol) in 2 mL of methanol. The yellow reaction mixture was ﬁltered after 30 min of stirring at ambient temperature, and an excess of NH4PF6 (0.50 mmol) was added. The mixture was stirred for 10 min and then concentrated by nitrogen gas until a yellow microcrystalline product formed. The product was collected by ﬁltration, washed with methanol (1 × 0.5 mL) and diethyl ether (3 × 1 mL), and dried under vacuum (15 min). Yield: 85% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSO-d6, 300 K): δ 8.72 (d, 1H, J = 5.5 Hz), 8.57 (d, 1H, J = 4.6 Hz), 8.47 (d, 1H, J = 5.5 Hz), 8.38 (d, 1H, J = 8.3 Hz), 8.28 (m, 1H), 8.12 (d, 1H, J = 8.3 Hz), 7.59 (m, 1H), 7.54 (m, 1H), 7.18 (d, 1H, J = 4.6 Hz), 1.32 (s, 15H) ppm. 13C NMR (600 MHz, DMSO-d6, 300 K): δ 154.8, 148.2, 147.2, 144.0, 142.4, 133.6, 128.6, 124.4, 123.6, 122.2, 115.5, 108.2, 88.5, 7.9 ppm. 15N NMR (600 MHz, DMSO-d6, 300 K): δ 188.4, 173.0, 154.3 ppm. ESI+ MS (MeOH; m/z): 558.0 (calcd 558.1 for [Ir(Cp*)(pyaza)Cl]+; 50%), 522.2 (calcd 522.2 for {[Ir(Cp*)(pyaza)] − H}+; 100%). Anal. Calcd for IrC22H24N3ClPF6 (703.08): C, 37.58; H, 3.44; N, 5.98. Found: 37.42; H, 3.36; N, 6.07. Crystals of complex 2 suitable for a single-crystal X-ray analysis were obtained by slow evaporation (ca. 24 h) of its methanolic solution at ambient temperature. [Ir(η5-Cpph)(pyaza)Cl]PF6 (6). The synthesis was performed as for complex 5 using the reaction of pyaza (0.35 mmol), [Ir(μ-Cl)(η5Cpph)Cl]2 (0.10 mmol), and NH4PF6 (0.50 mmol). Yield: 80% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSOd6, 300 K): δ 8.72 (d, 1H, J = 5.5 Hz), 8.59 (d, 1H, J = 4.6 Hz), 8.39 (d, 1H, J = 5.5 Hz), 8.36 (d, 1H, J = 8.3 Hz), 8.27 (t, 1H, J = 7.3 Hz), 8.12 (d, 1H, J = 8.3 Hz), 7.46 (m, 7H), 7.19 (d, 1H, J = 3.7 Hz), 1.47 (s, 3H), 1.42 (s, 3H), 1.39 (s, 3H), 1.23 (s, 3H) ppm. 13C NMR (600 MHz, DMSO-d6, 300 K): δ 154.8, 148.2, 147.2, 144.0, 142.4, 133.6, 129.4−128.6, 124.4, 123.6, 122.2, 115.5, 108.2, 97.1, 96.6, 87.1, 81.3, 9.1, 8.1 ppm. 15N NMR (600 MHz, DMSO-d6, 300 K): δ 185.8, 169.7, 153.3 ppm. ESI+ MS (MeOH; m/z): 620.0 (calcd 620.1 for [Ir(Cpph)(pyaza)Cl]+; 85%), 584.2 (calcd 584.2 for {[Ir(Cpph)(pyaza)] − H}+; 100%). Anal. Calcd for IrC27H26N3ClPF6 (765.15): C, 42.38; H, 3.42; N, 5.49. Found: 42.03; H, 3.05; N, 5.11. General Methods. Elemental analyses were carried out using a Flash 2000 CHNS Elemental Analyzer (Thermo Scientiﬁc). Electrospray ionization mass spectrometry (ESI-MS) was performed with an LCQ Fleet ion trap spectrometer (Thermo Scientiﬁc; QualBrowser software, version 2.0.7) in positive ionization mode (ESI+) on methanolic solutions of the studied complexes. 1H, 13C, 1H−1H gsCOSY, 1H−13C gs-HMQC, 1H−13C gs-HMBC and 1H−15N gsHMBC spectra were recorded using a JEOL JNM-ECA 600II device on DMSO-d6 solutions of the starting compounds and the studied complexes at 300 K (gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence). 1H and 13C NMR spectra were calibrated against the residual signal of the solvent found at 2.50 ppm (1H) and 39.5 ppm (13C),33 while 1H−15N gs-HMBC spectra were calibrated externally against the signals of DMF found at 8.03 ppm (1H) and 104.7 ppm (15N). The splitting of proton resonances in the reported 1H spectra is deﬁned as s = singlet, d = doublet, t = triplet, br = broad band, m = multiplet. Coordination shifts (Δδ; ppm) were calculated as δcomplex − δligand. Infrared spectra (400−4000 cm−1, ATR technique) were acquired with a Nexus 670 FT-IR instrument (Thermo Nicolet). Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to positive electrospray ionization mode mass spectrometry (ESI+ MS) was carried out using a UHPLC-MS instrument (Dionex/Thermo Fisher Scientiﬁc) equipped with an ReproSil-Pur Basic C18 column (5 μm pore size, 200 × 4.6 mm), with MeCN used as the mobile phase (the detection wavelength was 254 nm). X-ray Crystallography. The X-ray diﬀraction data of complexes 1, 5, and 6 were collected with a Bruker D8 QUEST diﬀractometer reported protocols (see the Supporting Information). The iridium(III) dimeric complex [Ir(μ-Cl)(η5-Cp*)Cl]2 was prepared following the reported procedure,32 while the reported synthesis of complex [Ir(μ-Cl)(η5-Cpph)Cl]223a was slightly modiﬁed (Supporting Information). All of the named starting compounds were synthesized using a Monowave 300 microwave reaction system (Anton Paar). Synthesis of Complexes 1−6. [Ir(η5-Cp*)(phaza−)Cl] (1). The 7-azaindole derivative phaza (0.55 mmol) was dissolved in 2 mL of methanol, and [Ir(μ-Cl)(η5-Cp*)Cl]2 (0.25 mmol) and CH3COONa (1.00 mmol) were added. The reaction mixture was stirred at ambient temperature for 30 min. After that, the reaction mixture was ﬁltered and the bright yellow ﬁltrate obtained was concentrated by nitrogen gas until a yellow microcrystalline solid of complex 1 formed. The product was collected by ﬁltration, washed with methanol (1 × 0.5 mL) and diethyl ether (3 × 1 mL), and dried under vacuum (15 min). Yield: 90% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSO-d6, 300 K): δ 8.54 (d, 1H, J = 4.1 Hz), 8.46 (d, 1H, J = 5.5 Hz), 8.34 (d, 1H, J = 6.9 Hz), 7.70 (d, 1H, J = 8.2 Hz), 7.60 (m, 1H), 7.34 (m, 1H), 7.18 (m, 1H), 7.02 (m, 1H), 6.94 (d, 1H, J = 3.4 Hz), 1.37 (s, 15H) ppm. 13C NMR (600 MHz, DMSO-d6, 300 K): δ 150.7, 142.1, 136.2, 133.5, 128.5, 126.8, 126.6, 124.9, 124.5, 120.3, 116.7, 104.8, 96.5, 87.8, 8.3 ppm. 15N NMR (600 MHz, DMSO-d6, 300 K): δ 161.0, 141.9 ppm. ESI+ MS (MeOH; m/z): 521.2 (calcd 521.2 for [Ir(Cp*)(phaza−)]+; 100%). Anal. Calcd for IrC23H24N2Cl (556.12): C, 49.67; H, 4.35; N, 5.04. Found: 49.66; H, 4.48; N, 4.91. Crystals suitable for a single-crystal X-ray analysis were obtained by slow evaporation (ca. 48 h) of a methanolic solution of complex 1 at ambient temperature. [Ir(η5-Cpph)(phaza−)Cl] (2). The synthesis was performed as for complex 1 using the overnight reaction of phaza (0.25 mmol), [Ir(μCl)(η5-Cpph)Cl]2 (0.10 mmol), and sodium acetate (0.40 mmol) in 2 mL of methanol. Yield: 32% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSO-d6, 300 K): δ 8.41 (d, 1H, J = 3.7 Hz), 8.40 (d, 1H, J = 4.6 Hz), 8.10 (d, 1H, J = 7.3 Hz), 7.53 (m, 1H), 7.41 (d, 1H, J = 8.3 Hz), 7.27 (m, 5H), 7.14 (dd, 1H, J = 8.3, 5.5 Hz), 6.91 (t, 1H, J = 8.3 Hz), 6.82 (d, 1H, J = 3.7 Hz) 6.79 (t, 1H, J = 6.5 Hz), 1.44 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H), 1.20 (s, 3H) ppm. 13C NMR (600 MHz, DMSO-d6, 300 K): δ 149.1, 142.9, 136.1, 132.6, 130.7, 129.8, 128.2, 127.1, 124.6, 123.4, 118.6, 114.2, 103.4, 97.0, 86.4, 85.0, 9.7, 8.9 ppm. 15N NMR (600 MHz, DMSO-d6, 300 K): δ 161.7, 134.3 ppm. ESI+ MS (MeOH; m/z): 583.2 (calcd 583.2 for [Ir(Cpph)(phaza−)]+; 100%). Anal. Calcd for IrC28H26N2Cl (618.19): C, 54.40; H, 4.24; N, 4.53. Found: 54.25; H, 4.23; N, 4.47. [Ir(η5-Cp*)(thaza−)Cl] (3). 1-(Thiophen-2-yl)-7-azaindole (thaza; 0.25 mmol) reacted at ambient temperature in methanol (2 mL) for 30 min with [Ir(μ-Cl)(η5-Cp*)Cl]2 (0.10 mmol). The reaction mixture was ﬁltered, and the yellow ﬁltrate was concentrated using nitrogen gas. The obtained product was collected by ﬁltration, washed with methanol (1 × 0.5 mL) and diethyl ether (3 × 1 mL), and dried under vacuum (15 min). Yield: 80% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSO-d6, 300 K): 1H NMR (600 MHz, DMSO-d6, 300 K): δ 8.52 (d, 1H, J = 5.5 Hz), 8.42 (d, 1H, J = 7.3 Hz), 8.10 (d, 1H, J = 3.7 Hz), 7.40 (m, 1H), 7.35 (d, 1H, J = 5.5 Hz), 6.95 (d, 1H, J = 5.5 Hz), 6.92 (d, 1H, J = 3.7 Hz), 1.54 (s, 15H) ppm. 13C NMR (600 MHz, DMSO-d6, 300 K): δ 150.7, 138.9, 135.7, 133.8, 129.0, 128.5, 123.2, 120.0, 119.1, 111.8, 103.8, 96.0, 8.3 ppm. 15 N NMR (600 MHz, DMSO-d6, 300 K): δ 156.7, 143.6 ppm. ESI+ MS (MeOH; m/z): 527.2 (calcd 527.1 for [Ir(Cp*)(thaza−)]+; 100%). Anal. Calcd for IrC21H22N2SCl (562.15): C, 44.87; H, 3.94; N, 4.98. Found: 44.62; H, 3.76; N, 4.82. [Ir(η5-Cpph)(thaza−)Cl] (4). The synthesis was performed as for 3 using the reaction (3 h) of thaza (0.35 mmol) and [Ir(μ-Cl)(η5Cpph)Cl]2 (0.10 mmol) in 2 mL of methanol. Yield: 62% (related to the starting Ir(III) dimer). 1H NMR (600 MHz, DMSO-d6, 300 K): δ 8.47 (d, 1H, J = 5.5 Hz), 8.33 (d, 1H, J = 8.3 Hz), 8.16 (d, 1H, J = 3.7 Hz), 7.35 (m, 1H), 7.21 (m, 4H), 6.97 (m, 4H), 2.05 (s, 3H), 1.78 (s, 3H), 1.68 (s, 3H), 1.38 (s, 3H) ppm. 13C NMR (600 MHz, DMSOd6, 300 K): δ 151.3, 139.0, 136.4, 134.5, 130−128.8, 123.9, 120.0, 111.6, 105.4, 103.0, 98.6, 93.7, 89.0, 10.2, 9.1 ppm. 15N NMR (600 MHz, DMSO-d6, 300 K): δ 154.4, 142.0 ppm. ESI+ MS (MeOH; m/ G DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics (Mo Kα radiation) equipped with a PHOTON 100 CMOS detector. The obtained data were processed and reduced by the APEX3 software package,34 and the molecular structures of complexes 1, 5, and 6 were solved by direct methods (SHELXS) and reﬁned by a fullmatrix least-squares procedure (SHELXL).35 Hydrogen atoms of all the structures were found in the diﬀerence Fourier maps and reﬁned using a riding model with C−H = 0.95 Å for CHaromatic and 0.98 Å for CH3, and with Uiso(H) = 1.2 Ueq(CH) and 1.5 Ueq(CH3). The F2, F4, and F5 atoms of the hexaﬂuorophosphate anion in complex 5 were reﬁned as disordered over two positions with a 0.53/0.47 combination of occupancy factors. X-ray crystallographic data for complexes 1, 5, and 6 have been deposited with the Cambridge Crystallographic Data Centre under the CCDC numbers 1844497, 1844498, and 1844499, respectively. The crystal data and structure reﬁnement details are given in Table S2. The graphics were drawn and additional structural calculations were performed by DIAMOND36 and Mercury37 software. Cell Culture. The human ovarian carcinoma (A2780), cisplatinresistant ovarian carcinoma (A2780R), and human normal lung ﬁbroblast cell lines (MRC-5) (supplied by European Collection of Cell Cultures, ECACC) were cultured, according to the ECACC instructions, in the RPMI-1640 medium supplemented with 10% of fetal calf serum, 1% of 2 mM glutamine, and 1% penicillin/ streptomycin. Human prostatic carcinoma DU-145, human lung carcinoma cells A549, human colon carcinoma cells HCT116, human cervix adenocarcinoma HeLa, and human breast adenocarcinoma MCF7 were cultured in DMEM high glucose supplemented with heat-inactivated FBS (10%) and antibiotics (streptomycin 100 μg mL−1, penicillin 100 U mL−1). All cell lines were grown as adherent monolayers at 37 °C and 5% CO2 under a humidiﬁed atmosphere. In Vitro Cytotoxicity Testing. An appropriate amount of complexes 1−6, cisplatin, or oxaliplatin was dissolved in 500 μL of DMF to give stock solutions of 50 mM concentration. The stock solutions were diluted by RPMI-1640 medium to concentrations of 0.01−25.0 μM (for 3) and 0.01−50.0 μM (for 1, 2, 4−6, and cisplatin). The A2780 and A2780R cells were seeded to 96-well culture plates, preincubated in drug-free media at 37 °C for 24 h, and treated with various concentrations of complexes 1−6 and standards. After 24 h of drug exposure, the supernatants were removed and the cells were washed with drug-free PBS followed by 72 h recovery in drug-free medium at 37 °C. In parallel, the cells were also treated with 0.1% DMF and 1% Triton X-100 to assess the minimal and maximal cell damage, respectively. The MTT assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to determine the cell viability. The concentration of the formed dye was evaluated spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC). The same experiments were performed at the MRC-5 cells, but only for complexes 1, 3, and 6 (and cisplatin for comparative purposes). The DU-145, A549, HCT116, HeLa, and MCF7 cells were seeded in 96-well tissue culture plates at a density of 10000 cells/well. After overnight incubation, the cells were treated with the investigated compounds and kept for 72 h under cultivation conditions. Stock solutions of complex 6 and cisplatin were always freshly prepared in DMF before use, and concentrations of Ir and Pt in cultivation media were always checked by atomic absorption spectrometry (Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer). After the treatment period, 20 μL of a 0.33% solution of neutral red in culture medium was added to each well with adherent cells and the plate was incubated at 37 °C under a humidiﬁed 5% CO2 atmosphere for 2 h. Afterward, the dye was carefully removed and the cells were quickly rinsed with PBS. The incorporated dye was then solubilized in 200 μL of 1% acetic acid in 50% ethanol, plates were allowed to stand for 10 min at room temperature, and the absorbance was measured at 540 nm (absorbance reader Synergy Mx, Biotek, USA). The data from the cancer cells were acquired from three independent experiments (conducted in triplicate) using cells from diﬀerent passages. The data were expressed as the percentage of viability, and the resulting IC50 values (μM), calculated from viability curves, are given as arithmetic mean ± SD. The signiﬁcance of the diﬀerences between the obtained results (p < 0.05, p < 0.01, and p < 0.005 considered to be signiﬁcant) was assessed by an ANOVA analysis (QC Expert 3.2, Statistical software, TriloByte Ltd.). Cytotoxicity Testing in Spheroid Culture (3D Culture) of MCF7 Cells. The human Caucasian breast adenocarcinoma cells (derived from the pleural eﬀusion MCF7 cells) were transferred as single cells to 96-well ultralow attachment plates (ULA; Corning, NY, USA), cultivated for 144 h in DMEM medium supplemented with 2% B27 (Thermo Fisher Scientiﬁc Inc., MA, USA), 20 ng mL−1 epidermal growth factor (EGF; Sigma-Aldrich, Darmstadt, Germany), and 0.15% (w/v) human serum albumin (HSA; Sigma-Aldrich, Darmstadt, Germany). Then, the cells were treated with various concentrations of the investigated compounds and incubated for another 72 h. After the treatment period, the wells were ﬁlled with an equal amount of CellTiter-Glo 3D Reagent (100 μL) and the plates were vigorously mixed for 5 min followed by 25 min of incubation at room temperature. Cell viability was evaluated by measuring the luminescence using an Inﬁnite 200 PRO NanoQuant luminescence reader (Tecan). IC50 values were calculated from curves constructed by plotting cell survival (%) versus drug concentration (μM). All experiments were done in triplicate. The reading values were converted to the percentage of control (% cell survival). Hydrophobicity Studies (log P). Octanol-saturated water (OSW) and water-saturated octanol (WSO) were prepared from octanol and a 0.2 M water solution of NaCl by overnight shaking (Vibramax 100, Heidolph Instruments). The stock solution was prepared by dissolving 3 μmol of complex 6 in 20 mL of OSW (ultrasonicated for 30 min). The mixture was centrifuged (5 min, 11000 rpm), and the supernatant was collected. A 5 mL portion of this solution was added to 5 mL of WSO and shaken for 2 h at ambient temperature. After that, the mixture was centrifuged, the aqueous layer was separated carefully, and the Ir concentration was determined by ICP-MS (the obtained value was corrected for adsorption eﬀects). The equation log P = log([Ir]WSO/[Ir]OSWa) was used for the partition coeﬃcient calculation; [Ir]OSWb and [Ir]OSWa stand for the Ir concentration before and after partition, respectively, and [Ir]WSO = [Ir]OSWb − [Ir]OSWa. The experiment was conducted in triplicate, and the results are presented as arithmetic mean ± SD. Cellular Accumulation. The A2780 cells were seeded in six-well culture plates (1 × 106 cells per well) and cultured for 24 h at 37 °C and 5% CO2 humidiﬁed atmosphere in complete growth RPMI-1640 medium. Subsequently, the medium was removed, the cells were washed with PBS, and fresh medium was added. The cells were treated with complex 6 and cisplatin at their IC50 concentrations for 24 h. Stock solutions of metal complexes were always freshly prepared in DMF and diluted to the cultivation media so that the ﬁnal concentration of DMF in cell culture medium did not exceed 0.2% (v/v). After an incubation period, cells were harvested by trypsinization, thoroughly washed with PBS, and collected by centrifugation. Cell pellets were digested in 500 μL of nitric acid (3 min, 150 °C) using a Monowave 300 instrument to give a fully homogenized solution, which was diluted with 4.5 mL of water, and the ﬁnal iridium or platinum content was determined by ICP-MS (Agilent 7700x, Agilent, Japan). The obtained values were corrected for adsorption eﬀects. The experiments were conducted in triplicate, and the results are presented as arithmetic mean ± SD. Cell Cycle Analysis. The A2780 cells (1.0 × 106 per well) were preincubated in a six-well plate for 24 h as described above. Complex 6 (cisplatin was involved in the study for comparative purposes) was added at its IC50 concentration (determined at the used cell line). After 24 h, the drug-containing medium was removed and the attached cells were harvested using trypsin/EDTA in PBS. The cells were washed twice with PBS and ﬁxed in 70% ethanol. Cells were resuspended in PBS, and DNA staining was achieved by a solution of propidium iodide (PI) supplemented with RNaseA (30 min, 25 °C, in the dark). After that, the cells were resuspended and DNA content was measured using ﬂow cytometry (CytoFlex, Beckman Coulter), H DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Organometallics ■ detecting the emission of DNA-bound PI (maximum at 617 nm) after excitation at 535 nm. The data were analyzed using CytExpert software (Beckman Coulter). Studies of Hydrolysis and Interactions with Biomolecules. The ESI+ MS studies of interactions of complex 6 with sulfurcontaining GSH and CySH were performed as follows: complex 6 was dissolved in MeOH (500 μL, 10 μM ﬁnal concentration), and 500 μL of the mixture of normal serum concentrations38 of GSH (6 μM ﬁnal concentration) and CySH (290 μM ﬁnal concentration) in H2O was added. The obtained solutions were mixed properly, and the ESI+ mass spectra were recorded immediately after preparation and after 24 h and 72 h of standing at laboratory temperature. The methanol solution of complex 6 and solution of complex 6 in an MeOH/H2O mixture (1/1, v/v) were used as reference solutions in this experiment. The water-containing solution should provide a clue about the hydrolytic behavior of complex 6. Additionally, the interactions of complex 6 with the model proteins HEWL and Cyt c were studied by means of ESI+ mass spectrometry. The samples contained mixtures of complex 6 (at the 10 μM ﬁnal concentration) with HEWL or Cyt c (at ca. 3 μM ﬁnal concentration) in an MeOH/H2O mixture (1/1, v/v). The ESI+ mass spectra were recorded immediately after preparation and then after 24 and 72 h of standing at laboratory temperature. The speciﬁc experimental conditions were as follows: the sample solutions were introduced into the mass spectrometer by an HPLC (Dionex UltiMate 3000; Thermo Scientiﬁc; Waltham, MA, USA) autosampler in 50 μL spikes into a continual ﬂow of methanol (0.2 mL/min ﬂow rate), and the ionization source was set to 5.3 kV spray voltage and 110 V and 275 °C capillary voltage and temperature. The ESI+ MS spectra were acquired in the range of m/z 50−2000, and the raw spectra of proteins and mixtures thereof were deconvoluted using Promass for Xcalibur ver. 3.0, rev. 10 software (Novatia LLC, Newtown, PA, USA) producing the neutral mass spectra, representing the interacting intermediates. ■ ACKNOWLEDGMENTS The authors gratefully thank the Ministry of Education, Youth and Sports of the Czech Republic (projects LO1305 and CZ.1.05/2.1.00/19.0377) and the Czech Science Foundation (GAČ R 17-08512Y) for ﬁnancial support. The authors also thank Ms. Eva Mácǎ lová for her help with syntheses of complexes 1−6, Ms. Kateřina Kubešová for performing part of the cell-based studies, Dr. Peter Antal for recording the NMR spectra, Dr. Bohuslav Drahoš for recording the ESI-MS and HPLC data, Mrs. Pavla Richterová for performing elemental analysis, and Dr. Alena Klanicová for recording the FTIR spectra. The work of H.C. and J.K. was supported by the student project of the Palacky University in Olomouc (IGA_PrF_2018_022). ■ REFERENCES (1) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584. (2) Gasser, G.; Ott, I.; Metzler-Nolte, N. Organometallic anticancer compounds. J. Med. Chem. 2011, 54, 3−25. (3) (a) Leung, C. H.; Zhong, H. J.; Chan, D. S. H.; Ma, D. L. Bioactive iridium and rhodium complexes as therapeutic agents. Coord. Chem. Rev. 2013, 257, 1764−1776. (b) Liu, Z.; Sadler, P. J. Organoiridium complexes: Anticancer agents and catalysts. Acc. Chem. 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(b) Liu, Z.; Habtemariam, A.; ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.8b00415. Details of synthesis and characterization of phaza, pyaza, thaza, and [Ir(μ-Cl)(η5-Cpph)Cl]2, FT-IR spectral data for complexes 1−6, structural formulas of the starting compounds\, ESI+ mass spectra and 1H NMR spectra, results of ESI-MS studies of hydrolysis and interaction with model protein cytochrome c, results of HPLC-MS analysis, crystal data and structure reﬁnement details, and parameters of the noncovalent contacts (PDF) Accession Codes CCDC 1844497−1844499 contain 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 Author *E-mail for P.S.: pavel.starha@upol.cz. ORCID Pavel Š tarha: 0000-0003-0422-045X Zdeněk Trávníček: 0000-0002-5890-7874 Jana Kašpárková: 0000-0002-5279-5381 Notes The authors declare no competing ﬁnancial interest. I DOI: 10.1021/acs.organomet.8b00415 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Pizarro, A.; Clarkson, G. J.; Sadler, P. J. Organometallic iridium(III) cyclopentadienyl anticancer complexes containing C,N-chelating ligands. Organometallics 2011, 30, 4702−4710. (c) 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. 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Half-Sandwich Ir(III) Complex of N1-Pyridyl-7-azaindole Exceeds Cytotoxicity of Cisplatin at Various Human Cancer Cells and 3D Multicellular Tumor Spheroids