Synthesis, Characterization, and Biological Properties of Steroidal Ruthenium(II) and Iridium(III) Complexes Based on the Androst-16-en-3-ol Framework.

Synthesis, Characterization, and Biological Properties of Steroidal Ruthenium(II) and Iridium(III) Complexes Based on the Androst-16en-3-ol Framework Vanessa Koch,† Anna Meschkov,†,‡ Wolfram Feuerstein,§ Juliana Pfeifer,‡ Olaf Fuhr,∥ Martin Nieger,⊥ Ute Schepers,†,‡ and Stefan Bräse*,†,# † Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Fritz Haber Weg 6, 76131 Karlsruhe, Germany Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, 76344 Eggenstein Leopoldshafen, Germany § Institute of Inorganic Chemistry, Division Molecular Chemistry, Karlsruhe Institute of Technology (KIT), Engesserstr. 15, 76131 Karlsruhe, Germany ∥ Institute for Nanotechnology (INT) and Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, 76344 Eggenstein Leopoldshafen, Germany ⊥ Department of Chemistry, University of Helsinki, P.O. Box 55, 00014 Helsinki, Finland # Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, D 76344 Eggenstein Leopoldshafen, Germany ‡ ABSTRACT: A range of novel cyclometalated ruthenium(II) and iridium(III) complexes with a steroidal backbone based on androsterone were synthesized and characterized by NMR spectroscopy and X ray crystallography. Their cytotoxic properties in RT112 and RT112 cP (cisplatin resistant) cell lines as well as in MCF7 and somatic ﬁbroblasts were compared with those of the corresponding nonsteroidal complexes and the noncyclometalated pyridyl complexes as well as with cisplatin as reference. All steroidal complexes were more active in RT112 cP cells than cisplatin, whereby the cyclometalated pyridinylphenyl complexes based on 5c showed high cytotoxicity while maintaining low resistant factors of 0.33 and 0.50. ■ INTRODUCTION Over the past 60 years, the interest in steroid bearing transition metal complexes has increased continuously.1 In the late 1970s, the biological application of these complexes was discovered, enabling new perspectives for metal containing approaches, e.g., in the treatment of cancer.1 Although platinum based anticancer complexes2 such as cisplatin3,4 or oxaliplatin5,6 are worldwide recognized for the treatment of cancer, still some drawbacks remain. Diverse side eﬀects as well as multifactorial drug resistance mechanisms, including, e.g., enhanced DNA repair, increased drug eﬄux, and detoxiﬁca tion, cause severe limitations in therapeutic use.7,8 In order to circumvent intrinsic and acquired resistance and to reduce side eﬀects, other transition metal based anticancer agents have been explored.9 On the basis of ruthenium(III), KP101910 and NAMI A11 (Figure 1) were developed, whereby both drugs already passed phase I of the clinical trials.12−14 Ruthenium (III) probably serves as a pro drug and is reduced within the cell to the active ruthenium(II) complex. Noteworthy, ruthenium complexes of the type [Ru(η6 arene)Cl2(PTA)] (PTA = 1,3,5 triaza 7 phosphatricyclo[3.3.1.1]decane) also show promising anticancer properties and are therefore broadly investigated.15−17 By connecting the metal center to a steroidal backbone as shown in complexes 1 and 2 (Figure 1), biological properties can be tuned. Since the steroidal framework enables the binding to steroid receptors, cell penetration can be improved. It has been also demonstrated that by the incorporation of a C 3 modiﬁed cholesterol ruthenium(III) complex 2 into a liposome bilayer, the ruthenium moiety was protected from degradation and the cellular uptake was favored. When integrated into a biomimetic membrane, the complex was found to be 6 fold times more active against the MCF 7 cell line (breast cancer) than the corresponding nonsteroidal complex.18 Moreover, Jaouen et al.19,20 and later Hannon et al.21−24 could show that a suﬃcient Figure 1. Currently investigated Ru(III) and Ru(II) anticancer drugs and ruthenium complexes based on an androgen (1) and a cholesterol (2) framework. Scheme 1. Synthesis of the Steroidal Pyridine Containing Ligands 5 Starting from epi Androsterone (3) via a Two Step Procedure31 recognition of steroidal receptors is retained if the organo metallic site is attached at the end of a rigid spacer such as an ethynyl group at C 17 of an estradiol or testosterone derivative. In this context, Ruiz et al. found that the androgen containing ruthenium(II) complex 1 was 8 fold more active than cisplatin in T47D cell lines (breast cancer).25,26 Lv et al. also reported an enhancement of the antiproliferative activity of an N heterocyclic carbene ruthenium complex in MCF 7 cells by conjugating a 17α ethynyl testosterone via a disulﬁde linkage.27 Compared to the number of studies on the anticancer activity of ruthenium complexes, only a few reports regarding the anticancer activity of iridium(III) complexes have been published.25,28−30 Nevertheless, Sadler et al. showed that the biological activity of pentamethylcyclopentadienyl (Cp*)Ir (III) complexes was increased by the incorporation of phenyl substituents. This resulted in an enhanced cellular accumu lation due to the higher hydrophobicity of these complexes. Furthermore, the substitution of N,N ligands by C,N chelating ligands was shown to improve antiproliferative activity. Motivated by these results, we envisioned to investigate the chemical, spectroscopic, and biological properties of novel ruthenium(II) and iridium(III) complexes based on epi androsterone with the metal center located closer to the steroidal backbone compared to previous examples.25,26 Scheme 2. Synthesis of Novel Cycloruthenated Ruthenium(II) Complexes 6, 7, and 8 Figure 2. Molecular structure of the pyridylphenyl ruthenium(II) complex 7 (displacement parameters are drawn at 50% probability level). Characteristic bond lengths: Ru−N 2.097(3) Å; Ru−C 2.047(3) Å; Ru−Cl 2.4253(7) Å; Ru−Ccymene 2.154(3)−2.290(3) Å; Ru−Ccymene/centroid 1.706(3) Å. Selected bond angles: N−Ru−C 77.72(12)°, N−Ru−Cl 86.98(3)°, Cl−Ru−C 85.76(9)°, N−Ru−cymenecentroid 132.3(1)°, Cl−Ru− cymenecentroid 126.6(1)°, C−Ru−cymenecentroid 130.3(1)°. ■ DESIGN AND SYNTHESIS OF THE NEW RUTHENIUM COMPLEXES In order to bring the steroidal backbone in close proximity to the metal center, we aimed to modify C 17 of epi androsterone (3) in such a manner that the complexation of ruthenium(II) and the iridium(III) is feasible either by an N pyridine moiety or by κ2 N,C cyclometalation. Therefore, diﬀerent pyridine substituted androsterone derivatives (5a, 5b) and a 4′ (2 pyridinyl)phenyl derivative (5c) were synthesized. As previously shown by our group, pyridine containing sub stituents are best introduced by the Stille cross coupling reaction.31 Starting from epi androsterone (3), the desired ligands were easily accessible by a two step procedure (Scheme 1). Hence, epi androsterone (3) was treated with hydrazine to form the hydrazone giving either the alkenyl iodide 4a by adding iodine in the presence of triethylamine or the alkenyl bromide 4b by adding NBS with pyridine as a base. The following palladium catalyzed Stille cross coupling reac tion aﬀorded the 2′ pyridinyl derivative 5a, 3′ pyridinyl derivative 5b, or 4 (pyridin 2′ yl)phenyl derivative 5c in good yields ranging from 60−74%. By washing the obtained products 5 with n hexane, traces of remaining stannanes could be removed, which was crucial with regard to biological tests. Since numerous procedures exist in the literature for the synthesis of cyclic ruthenium(II) complexes of 2 phenyl pyridines,32−34 we tried analogous reaction conditions with 2 (4 bromophenyl)pyridine. With one equivalent of dimeric ruthenium precursor [Ru(η6 para cymene)Cl2]2 and two equivalents of the ligand in the presence of four equivalents of KOAc in MeOH, the Ru(II) complex was formed after stirring at room temperature for 24 h. After ﬂash column chromatography on silica gel, the ruthenium(II) complex 6 was isolated with 67% yield (Scheme 2). Applying the same reaction conditions, the synthesis of the phenylpyridinyl ruthenium(II) complex 7 and the pyridinyl ruthenium(II) complex 8 succeeded in moderate yields starting from their ligands 5c or 5a (Scheme 2). Table 1. Experimental Proton and Carbon Resonances of the Diastereomers (R Ru) 8 and (S Ru) 8 and Their Experimental Diﬀerences of the Chemical Shifts Δδ = δS−δR As Well As Their Diﬀerences calΔδ= δ(S)−δ(R) Calculated on the TPSSh/def2 TZVPP Level of Theory Using bp86/def2 TZVPP Structuresa (R)-diastereomer (S)-diastereomer 13 1 C 16-Cq Ru 17-Cq Pyr 13-Cq 18-CH3 15α-CH2 15β-CH2 14-CH H δR [ppm] Δδ(S R)[ppm] δS [ppm] 208.7 150.4 44.8 16.6 44.8 +1.0 +0.3 0.5 +0.8 0.5 209.7 150.7 44.3 17.4 44.3 +0.6 +0.2 0.2 +1.7 0.3 58.5 0.2 58.3 +1.1 cal Δδ[ppm] δR [ppm] Δδ[ppm] δS[ppm] 0.84 3.11 2.50 n.d. +0.09 0.13 +0.34 n.d. 0.93 2.98 2.84 n.d. cal Δδ[ppm] +0.12 0.25 +0.33 0.01 a By comparing the calculated with experimental diﬀerences in chemical shifts, the diastereomers (R Ru) 8 and (S Ru) 8 were assigned. n.d. = not determined. See text for computational details. Figure 3. 1H NMR spectra (CDCl3, 500 MHz, r.t.) of the diastereomers (R Ru) 8 (top) and (S Ru) 8 (bottom). Signiﬁcantly diﬀerent shifts of the two diastereomers are highlighted (blue, 15 CH2; yellow, cymene; green, 18 CH3). By recording 1H and 13C NMR, IR, and FAB mass spectra the complexes 6, 7, and 8 were successfully characterized. The Ru metal takes a pseudotetrahedral “piano stool” coordination geometry generating a new stereogenic center. Hence, most of the NMR resonances of the pyridinylphenyl ruthenium(II) complex 7 in d1 chloroform were duplicated (see Supporting Information). DFT calculations on the BP8635,36/def2 TZVPP37 level reveal the two diastereomers to diﬀer only 5.6 kJ/mol in Gibbs free energy. Accordingly, both diastereomers were formed in comparable amounts as evidenced by NMR signal intensities showing that no diastereomeric induction for cycloruthenation occurred. Furthermore, a single crystal suitable for X ray crystallography was obtained conﬁrming the stated molecular structure for the R diastereomer as depicted in Figure 2. The coordination geometry of the Ru(II) center shows the expected pseudote trahedral geometry. The N−Ru−C angle of 77.72(12)° is signiﬁcantly smaller than the N−Ru−Cl (86.98(3)°) and the Cl−Ru−C (85.76(9)°) angles, which is in agreement with reported nonsteroidal cycloruthenated 2 phenylpyridinyl com plexes.38 In comparison with nonsteroidal complexes reported in the literature,38,39 ruthenium(II) complexes 7 show similar bond angles and bond lengths, whereby Ru−X bond lengths (X = N, Cl, C) are slightly longer and bond angles Y−Ru−Z (Y≠ Z = N, Cl, C) slightly smaller. Unfortunately, we were not able to assign the crystal structure to one of the NMR signal sets, since the NMR chemical shifts of the diastereomers are too similar, as predicted by NMR shielding calculations (see Supporting Information). Fortunately, in the case of the 2 pyridinyl ruthenium(II) complex 8, the diastereomers could be separated by column chromatography on silica, whereby both diastereomers (R Ru) 8 and (S Ru) 8 were formed in equal amounts according to the integration of the crude 1H NMR spectrum and could be isolated in comparable amounts. As for complex 7, by optimizing the molecular structures of both diastereomers at the BP8635,36/def2 TZVPP37 level of theory, we could show that none of the two diastereomers was noticeably thermodynamically favored standing in line with the nearly equimolar ratio of the isolated product. Both diastereomers showed nearly the same Gibbs free energies diﬀering only by 3.2 kJ/mol in favor of the (S) diastereomer. In addition, we calculated 1H and 13C NMR shifts employing diﬀerent density functionals using the optimized structures of both diaster eomers to be compared with the experimental resonances. The TPSSh functional40 turned out to yield the best accordance with the experimental NMR shift diﬀerences between (R Ru) 8 and (S Ru) 8 (see Supporting Information). This allowed for the assignment of the obtained NMR spectra to the two diastereomers (Table 1). The corresponding 1H NMR spectra and the relevant assignments are depicted in Figure 3. The stacked NMR spectra show no diﬀerences in chemical shifts for the proton resonances of the pyridinyl moiety. The aromatic and aliphatic proton resonances of the cymene ligand on the other hand, were clearly shifted similar to the three isopropyl resonances of (S Ru) 8 that appear at lower chemical shifts compared to the ones of (R Ru) 8. Also, the resonances of the steroidal backbone close to the ruthenium center are aﬀected by the diﬀerent electronic environments of the two diastereomers. For example, in case of the diastereomer (R Ru) 8, the methyl group and the chlorido substituent were located on the same side, resulting in a chemical shift of δ = 0.94 ppm, while the resonances of the diastereomer (S Ru) 8 are shifted upﬁeld to δ = 0.84 ppm. Furthermore, the two diastereomeric 15 CH2 resonances were inﬂuenced by the coordination to the pseudotetrahedral ruthenium center, whereby both signal sets were shifted downﬁeld compared to those of the steroidal ligand 5b. NOESY experiments and the evaluation of the coupling constants allowed the assignment of the more shielded signals to the 15β CH2 protons, while the signals that arise more downﬁeld belong to the 15α CH2 protons, being in accordance with our calculations of the chemical shielding. It is noteworthy that for diastereomer (S Ru) 8, the individual 15 CH2 resonances were closer to each other than those for the diastereomer (R Ru) 8, which was predicted by our chemical shielding calculations as well. This behavior is caused by the chlorido substituent: The nonbonding electrons lead to a shielding of spatially close protons by n−σ* interactions.41 Hence, the β proton of diastereomer (R Ru) 8 is shifted to higher ﬁelds compared to its (S Ru) 8 counterpart. The same held true for the α proton of (S Ru) 8, which is, however, less pronounced. We were able to obtain a crystal structure of the (R) diastereomer of 8 (Figure 4) conﬁrming the molecular Figure 4. Molecular structure of the 2 pyridinyl ruthenium(II) complex (R Ru) 8 (displacement parameters are drawn at 50% probability level). Characteristic bond lengths: Ru−N 2.078(10) Å; Ru−C 1.931 Å; Ru−Cl 2.415(3) Å; Ru−cymene 2.130(16)− 2.341(14) Å; Ru−cymene/centroid 1.729 Å. Selected bond angles: N−Ru−C 76.3(4)°, N−Ru−Cl 85.2(2)°, Cl−Ru−C 86.4(3)°, N− Ru−cymenecentroid 132.33°, Cl−Ru−cymenecentroid 126.60°, C−Ru− cymenecentroid 130.34°. structure and the correct assignments of the diastereomers based on the calculated chemical shifts. In contrast to the phenylpyridinyl ruthenium(II) complex 6 and its steroidal counterpart 7, the Ru−C bond of (R Ru) 8 is signiﬁcantly shorter (1.931 Å vs 2.062 Å of 639/2.047 Å of 7). This is an indication for the inferior electron donating ability of the cyclopentenido moiety of the steroidal D ring compared to phenyl. To the best of our knowledge, the herein presented ruthenium complex 8 is the ﬁrst example of a cyclopentenido pyridinyl ruthenium(II) complex.42,43 Furthermore, comparable iridium(III) complexes were synthesized by applying similar reaction conditions and [IrCp*Cl2]2 as a metal precursor. The three cyclometalated Ir(III) complexes 9, 10, and 11 (Scheme 3) were synthesized in overall good yields, whereby the diastereomers of 10 and 11 were formed in equal amounts giving two sets of signals in the 1 H and 13C NMR spectra. Unfortunately, a separation of the diastereomers 11 via column chromatography was not successful. For comparison with the cycloruthenated complexes 7 and 8 as well as for the analogous iridium(III) complexes 10 and 11, (17 (3′ pyridinyl)androsten)dichloride ruthenium(II) com plex 12 and the corresponding iridium(III) complex 13 were synthesized by stirring two equivalents of the ligand 5b and one equivalent of the dimeric metal precursor in dichloro methane (Scheme 4). After precipitation with n hexane, the metal complexes 12 and 13 were obtained in good yields. We were able to obtain crystal structures suitable for crystal structure analysis for both 3 pyridyl complexes 12 and 13 Scheme 3. Synthesis of Novel Cyclometalated Iridium(III) Complexes 9, 10, and 11 Scheme 4. Synthesis of the (17 (3′ Pyridinyl)androstenido)dichloride Ruthenium(II) Complex 12 and the Corresponding Iridium(III) Complex 13 Figure 5. Molecular structure of the 3 pyridyl ruthenium(II) complex 12 (left) and the 3 pyridyl iridium(III) 13 (right). The cymene ligand of ruthenium(II) complex 12 is disordered. Selected bond lengths for 12/13: Ru−N 2.125(3) Å; Ru−Ccymene 2.159(10)−2.231(10) Å; Ru−Cl(1) 2.419 Å; Ru−Cl(2) 2.404 Å/Ir−Cl(1) 2.395(5) Å; Ir−Cl(2) 2.408(6) Å; Ir−N 2.090(5) Å; Ir−CCp* 2.11(2)−2.26(2) Å; Ir−CCp*/centroid 1.788 Å. Selected bond angles for 12/13: Cl(1)−Ir−Cl(2) 89.2(2)°; N−Ir−Cl(1) 85.9(5)°; N−Ir−Cl(2) 85.6(5)°; N−Ir−CCp*/centroid 125.7(2)°; Cl(1)−Ir− CCp*/centroid 127.4(8)°; Cl(2)−Ir−CCp*/centroid 128.9(5)°/Cl(1)−Ru−Cl(2) 87.4(7)°; N−Ru−Cl(1) 85.2(1)°; N−Ir−Cl(2) 84.9(0)°. (Figure 5), conﬁrming their molecular structure and the pseudotetrahedral coordination geometry around the metal. Interestingly, in the solid state, the nitrogen atom of the pyridyl moiety points toward the 18 methyl group and the metal atoms were located on the upper side of the steroidal framework. It is noteworthy that the ruthenium(II) complex 12 shows almost no distortion (6.81°) in contrast to the free ligand 5b (see Supporting Information for the crystal structure) and the iridium(III) complex 13 whose pyridine units are twisted to the D ring plane with a torsion of 32.9(3)° or 29.1(8)°. ■ BIOLOGICAL ACTIVITY AND CYTOTOXICITY STUDIES To evaluate the cytotoxicity of the compounds, an in vitro MTT assay was performed (Table 2). The MTT (3 (4,5 Table 2. IC50 Values and Resistance Factors (RF, IC50(resistant)/IC50(sensitive)) of the Ru(II) and Ir(III) Complexes, Free Ligands, and Cisplatin (μM) entry compound RT112 RT112 cP (RF) MCF-7 NHDF 1 (4-bromophenyl) pyridine Ru(II) complex 6 Ir(III) complex 9 ligand 5a Ru(II) complex 8 (S)/ (R) 1:1 (S-Ru) complex 8 (R-Ru) complex 8 Ir(III) complex 11 ligand 5b Ru(II) complex 12 Ir(III) complex 13 ligand 5c Ru(II) complex 7 Ir(III) complex 10 cisplatin >50.0 >50.0 >50.0 >50.0 5.0 7.5 2.5 3.5 5.0 (1.00) 8.0 (1.07) 2.0 (0.80) 6.5 (1.86) 5.5 40.0 3.3 6.3 5.5 35.0 8.0 9.1 2.5 3.9 4.5 7.5 9.0 9.0 >50.0 3.0 2.0 3.5 5.5 (2.20) 6.0 (1.54) 2.5 (0.56) 7.0 (0.93) 7.5 (0.83) 11.0 (1.22) >50.0 1.0 (0.33) 1.0 (0.50) >50.0 6.0 5.5 5.7 9.0 7.0 8.5 6.6 2.5 8.5 22.5 10.5 7.0 6.9 >50.0 >50.0 >50.0 >50.0 5.5 17.5 >50.0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide) re agent can be reduced to blue purple formazan by the mitochondrial enzymes of living cells. The amount of resulting formazan can be determined photometrically and correlates directly with the cell viability, since this reaction can only take place in metabolically active cells. The cytotoxicity was tested in diﬀerent tumor and somatic cells including bladder cancer cells, which show resistance to cisplatin and breast cancer cells (MCF7), which usually show hormone dependency. In order to test a potential application in cisplatin resistant cells, the human bladder carcinoma cell line RT112 and its cisplatin resistant counterpart RT112 cP were cultivated with varying concentrations (0.5−50 μM) of the ruthenium(II) (6, 7, 8, and 12) and iridium(III) complexes (9, 10, 11, and 13), and the cell viability was monitored after 72 h of incubation. In addition, the cytotoxicity of the free ligands ((4 bromophenyl)pyridine, 5a−5c) and cisplatin was evaluated for comparison. There are some reports on the expression of various steroid receptors in bladder cancer cells,44,45 assuming that steroidal ligands could have an eﬀect. All complexes demonstrated higher cytotoxicity against RT112 cP cells compared to cisplatin (IC50 values 1−11 μM and >50 μM, respectively). The cytotoxicity of the complexes 8 (both (S) and (R) diastereomers), 11, 12, and 13 was in the same order of magnitude as that of the corresponding free steroidal ligands (5a and 5b), with IC50 values in the range between 2.5 and 7.5 μM for both cell lines. However, the steroidal ligand 5c demonstrated high biocompatibility (IC50 > 50 μM for both cell lines), whereas the corresponding Ru(II) (7) and Ir(III) (10) complexes showed promising antiproliferative eﬀects in both cell lines. This indicates that the cisplatin resistance was successfully overcome with IC50 values of 1 μM for RT112 cP cells and very low resistance factors of 0.33 and 0.5, respectively. Although the nonsteroidal complexes 6 and 9 were also more toxic (IC50 5−8 μM) compared to the free nonsteroidal (4‑bromophenyl)pyridine ligand (IC50 > 50 μM), the steroidal complexes 7 and 10 were signiﬁcantly more eﬀective with considerably lower IC50 values and resistance factors. For the MCF 7 cells, all the steroid complexes demonstrated a high cytotoxic activity with IC50 values in the range of 2.5−8.5 μM, signiﬁcantly more eﬀective as cisplatin with an IC50 value of 22.5 μM. The Ru(II) complex 7 was slightly more active in comparison to the corresponding ligand 5c (IC50 values 2.5 and 6.6 μM, respectively). Moreover, the cytotoxic activity of the majority of the compounds was lower for NHDF compared to the cancer cell lines, with especially high IC50 values for the complexes 12 and 13 (>50 μM), suggesting a therapeutic window with a selectivity for cancer cells. ■ CONCLUSION In conclusion, a set of Ru(II) and Ir(III) complexes with diﬀerent N containing ligands based on a steroidal backbone was synthesized and characterized by NMR spectroscopy and X ray crystallography. All evaluated complexes showed high cytotoxicity in the tested cancer cell lines and were more active in RT112 cP and MCF 7 cells than cisplatin. Remarkable is the very low resistant factor of the complexes in the range between 0.33 and 1.22, indicating successful overcoming of the cisplatin resistance. Especially promising results were obtained for the complexes 7 and 10 with the steroidal ligand 5c, since the advantageous high biocompatibility of 5c (IC50 > 50 μM) was combined with a pronounced antiproliferative eﬀect of the complexes 7 (IC50 3 μM for RT112 and 1 μM for RT112 cP) and 10 (IC502 μM for RT112 and 1 μM for RT112 cP) with resistant factors 0.33 and 0.5, respectively. In breast cancer cells, which show a proliferation dependency on hormone expression, all complexes showed an eﬀective cytotoxicity including the complex 7 (IC50 = 2.5 μM). This antiproliferative activity was signiﬁcantly higher than for the well established ruthenium complexes RAPTA T and NAMI A, with IC50 values >200 μM and 800 μM (both MCF 7), reported by Nazarov et al.46 and Pluim et al.,47 respectively. The cytotoxic eﬀect of the compounds was also higher than that of the mentioned cholesterol ruthenium(III) complex incorporated into a liposome bilayer, demonstrated by Simeone et al. (IC50 of about 70 μM),18 and was in the range of the testosterone− ruthenium conjugate described by Lv et al. (IC50 of about 4.5 μM).27 Furthermore, the complexes showed a promising selectivity for the cancer cells, exhibiting lower cytotoxicity against normal ﬁbroblasts compared to the tested cancer cell lines. ■ EXPERIMENTAL SECTION Instrumental Measurements. ATR IR spectra were performed on Bruker alpha p and a FT IR IFS 88 spectrometer. 1H and 13C NMR spectra were recorded on diﬀerent types of Bruker Avance 400, Bruker Avance III HD, or Bruker Avance 600 spectrometer with residual proton signals of the deuterated solvent as the internal standard. EI and FAB mass spectra (positive mode) were measured on a Finnigan MAT95. Further information is given in the Supporting Information. General Procedure for Metallacyclization Reactions with Ruthenium(II) and Iridium(III). Under an argon atmosphere, the ligand (2.00 equiv), [RuCl2(p cymene)]2 (1.00 equiv) or [IrCp*Cl2]2 (1.00 equiv), and KOAc (4.00 equiv) were dissolved in dry MeOH or CH2Cl2 and stirred at room temperature for 24 h. The suspension was concentrated, and the residue was puriﬁed by ﬂash column chromatography on silica gel to obtain the cyclometalated complexes as yellow to orange solids. The reactions based on a steroidal ligand were performed on a 30−120 μmol scale. Crystal Structure Determinations. The single crystal X ray diﬀraction study of 5b31 and 7 was carried out on a Bruker D8 Venture diﬀractometer with a Photon100 detector at 123(2) K using Cu−Kα radiation (λ = 1.54178 Å). Direct Methods (SHELXS 97)48 was used for structure solution, and reﬁnement was carried out using SHELXL 2014 (full matrix least squares on F2).49 Hydrogen atoms were localized by diﬀerence electron density determination and reﬁned using a riding model (H(O) free). Semiempirical absorption corrections were applied. The absolute conﬁguration was determined by reﬁnement of Parsons’ x parameter.50 The single crystal X ray diﬀraction study of (R Ru) 8, 12, and 13 was performed on a Stoe StadiVari diﬀractometer using Ga Kα radiation (λ = 1.34143 Å) generated by an Metaljet X ray source. The crystals were kept at 180.15 K during data collection. Using Olex2,51 the structures were solved with the ShelXS48 structure solution program using Direct Methods and reﬁned with the ShelXL49 reﬁnement package using Least Squares minimization. Non hydrogen atoms were reﬁned with anisotropic displacement parameters; hydrogen atoms were modeled on idealized positions. CCDC 1521243 (5b), 1859054 (7), 1944097 ((R Ru) 8), 1944098 (12), and 1944099 (13) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data request/cif. Computational Details. Structure optimizations were done on the BP8635,36/def2 TZVPP37 level of theory using the TURBO MOLE 7.1 program package.52 Solvent eﬀects of chloroform were taken into account with the COSMO solvation model.53 The RI approximation was used throughout.54 Stationary points were veriﬁed to be minimum energy structures by numerically calculating the molecular Hessian and analyzing the so obtained vibrational frequencies. The numerical frequencies were used to calculate thermodynamic properties at 298.15 K and 1 bar in harmonic and ideal gas approximations. NMR chemical shifts were calculated on the basis of Gauge Including Atomic Orbitals (GIAO).55 Cell Culture. RT112 (human bladder carcinoma cell line), RT112 cP (cisplatin resistant), NHDF (normal human dermal ﬁbroblasts), and MCF 7 (breast cancer cell line) were cultured in RPMI (Roswell Park Memorial Istitute) medium (Gibco, for RT112 and RT112 cP) or DMEM (Dulbecco’s modiﬁed eagle medium, Gibco, for NHDF and MCF 7) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptavidin (Gibco) at 37 °C, 5% CO2, and a humid atmosphere. For all in vitro experiments, cells were trypsinized (0.05% trypsin EDTA, Gibco) and seeded in 96 well plates (toxicity assay) at the required densities. Incubation was performed under the culture conditions as described above. Cytotoxicity Assay. RT112 (human bladder carcinoma cell line), RT112 cP (cisplatin resistant), NHDF (normal human dermal ﬁbroblasts), and MCF 7 (breast cancer cell line) were seeded in the 96 well plates at a density of 1 × 104 cells/well in RPMI (RT112 and RT112 cP) medium or DMEM (NHDF and MCF 7) supplemented with 10% FCS and 1% penicillin/streptomycin. After 24 h of incubation at 37 °C and 5% CO2, the medium was removed and the cells were treated with various concentrations of the compounds in the corresponding culture medium and incubated for 72 h at 37 °C and 5% CO2. The stock solutions of the compounds were prepared in DMSO and the end concentration of the solvent in the test dilutions was held under 0.5%. Since the cytotoxic activity of cisplatin was shown to be aﬀected by DMSO,56 a stable 5 mM stock solution of cisplatin in DPBS−/− (Gibco) was prepared. As a negative control, the cell culture medium was exchanged without addition of the compounds. Thereafter, 15 μL of the MTT reagent (Promega) was given in each well. For the positive control, Triton X 100 (1%) was added in some wells before treating them with the MTT reagent. After 3 h of incubation, the cells were lysed using the Stop Solution (Promega) to release the blue purple formazan. The cell viability was determined by measuring the absorbance of the resulting formazan at 595 nm using a multiwell plate reader (SpectraMax ID3, Molecular Devices, USA) and calculated in relation to the negative control. ■ ASSOCIATED CONTENT * Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorg chem.9b02402. Additional spectroscopic data, experimental and compu tational details, and characterization data (PDF) Accession Codes CCDC 1859054 and 1944097−1944099 contain the supple mentary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data request/ cif, or by emailing data request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ AUTHOR INFORMATION Corresponding Author *E mail: braese@kit.edu. ORCID Vanessa Koch: 0000 0002 2115 3124 Stefan Bräse: 0000 0003 4845 3191 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS We acknowledge the DFG Core Facility MOLECULE ARCHIVE (grant numbers: BR1750/40 1, JU2909/5 1) for the management and provision of the compounds for screening. V.K. gratefully acknowledges the Studienstiftung des Deutschen Volkes and W.F., the Carl Zeiss Stiftung for ﬁnancial support. The work was supported by the Deutsche Forschungsgemeinschaft (DFG), within the Research Training Group 2039 (A.M., U.S. S.B.) and the Helmholtz Program Biointerfaces in Technology and Medicine (BIFTM; U.S., A.M., S.B.). We thank Prof. Dr. Frank Breher for supporting this cooperation, Dr. Beate Köberle for providing cell lines, and Magdalena Winklhofer for assistance in in vitro experiments. ■ REFERENCES (1) Le Bideau, F.; Dagorne, S. Synthesis of Transition Metal Steroid Derivatives. Chem. Rev. 2013, 113 (10), 7793−7850. (2) Wilson, J. J.; Lippard, S. J. Synthetic Methods for the Preparation of Platinum Anticancer Complexes. Chem. Rev. 2014, 114 (8), 4470− 4495. (3) Rosenberg, B.; Van Camp, L.; Krigas, T. Inhibition of Cell Division in Escherichia coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205, 698. (4) Rosenberg, B. V. C.; Grimley, L.; Eugene, B.; Thomson, A. J. 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