Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic-Acid-Modified Pyridine and Triphenylphosphine Ligands.

Article pubs.acs.org/IC Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic-Acid-Modiﬁed Pyridine and Triphenylphosphine Ligands Gabriele Agonigi,† Tina Riedel,‡ Stefano Zacchini,§ Emilia Păunescu,‡ Guido Pampaloni,† Niccolò Bartalucci,† Paul J. Dyson,*,‡ and Fabio Marchetti*,† † Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via G. Moruzzi 13, I-56124 Pisa, Italy Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland § Dipartimento di Chimica Industriale “Toso Montanari”, Università di Bologna, Viale Risorgimento 4, I-40136 Bologna, Italy ‡ S Supporting Information * ABSTRACT: Pyridine- and phosphine-based ligands modiﬁed with ethacrynic acid (a broad acting glutathione transferase inhibitor) were prepared and coordinated to ruthenium(II)−arene complexes and to a ruthenium(III) NAMI-A type complex. All the compounds (ligands and complexes) were fully characterized by analytical and spectroscopic methods and, in one case, by single-crystal Xray diﬀraction. The in vitro anticancer activity of the compounds was studied, with the compounds displaying moderate cytotoxicity toward the human ovarian cancer cell lines. All the complexes led to similar levels of residual GST activity in the diﬀerent cell lines, irrespective of the stability of the Ru−ligand bond. ■ INTRODUCTION There are currently considerable ongoing research eﬀorts to develop new, eﬃcient metal-based anticancer agents that overcome the limitations associated with platinum-based drugs.1 In this context, ruthenium-based complexes have aroused great interest, and two of them, i.e., [indazoleH][trans-Ru(N-indazole)2Cl4] (KP1019) and [imidazoleH][transRu(N-imidazole)(S-DMSO)Cl4] (NAMI-A), have entered phase II of clinical trials (Figure 1).2 It is believed that KP1019 and NAMI-A are pro-drugs, being converted into more active Ru(II) species in the tumor environment.3 In part, this feature has triggered studies on ruthenium(II) complexes, especially those based on the [Ru(η6-arene)Cl2] frame.4 In this respect, complexes containing 1,3,5-triaza-7phosphatricyclo[3.3.1.1]decane (PTA, aﬀording RAPTA complexes − Figure 1) and ethylene-1,2-diamine as ligands have emerged as among the most promising species, showing relevant antitumor properties in vivo.5 In addition to these nontargeted compounds, i.e., compounds that were not designed to interfere with speciﬁc targets overexpressed or uniquely expressed in cancer cells, the synthesis of metal complexes containing organic fragments with known biological functions can lead to enhanced anticancer activities.6 In this context, the ruthenium(II)− arene structure has been modiﬁed with various bioactive groups, usually via the inclusion of a functional ligand or via modiﬁcation of the η6-coordinated arene ligand with an © XXXX American Chemical Society Figure 1. Structures of ruthenium complexes with known antitumor activity. appropriate functional moiety.7 For example, 3-hydroxyﬂavones,8 lapachol,9 paullones,10 and lonidamine,11 each with a well-characterized biological (anticancer) property, have been directly coordinated to the ruthenium(II)−arene fragment. Of Received: April 9, 2015 A DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Scheme 1. Synthesis of N- and P-Donor Ligands from Ethacrynic Acid (EA-CO2H) Scheme 2. Preparation of the Ruthenium(II)-p-cymene Complexes 1−3 Containing Ethacrynic-Acid-Functionalized Ligands complexes have been shown to inactivate GSTs and induce apoptosis even in cisplatin resistant cell lines.15 In the present study, new EA-CO2H-functionalized N- and P-donor ligands are described, together with their coordination to ruthenium(II)−arene and ruthenium(III) NAMI-A-like complexes. The antiproliferative properties of all the compounds were explored. the diﬀerent biologically active groups used, ethacrynic acid (EA-CO2H), an eﬀective inhibitor of glutathione transferases (GSTs), which comprises a family of cytosolic detoxiﬁcation enzymes associated with drug resistance in primary and metastatic tumors,12 has been tethered to the ruthenium(II)− arene unit via both the arene ligand12 and via imidazolemodiﬁed coligands.13 Furthermore, platinum(IV) complexes have also been modiﬁed with ethacrynic acid.14 These B DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry ■ RESULTS AND DISCUSSION Three ethacrynic-acid-functionalized ligands were prepared from the reaction of EA-CO2H with 4-(diphenylphosphino)benzoic acid, 4-pyridinemethanol, or nicotinic acid (Scheme 1). Thus, LN1 was obtained in 77% yield from EA-CO2H and 4pyridinemethanol via a EDCI/DMAP-mediated coupling reaction. The synthesis of LN2 and LP required initial derivatization of ethacrynic acid into the alcohol EA-(C O)O(CH2)2OH. This underwent EDCI/DMAP-mediated coupling reactions with nicotinic acid and 4(diphenylphosphino)benzoic acid to give LN2 (81% yield) and LP (62% yield), respectively. EA-(CO)O(CH2)2OH, LN1, LN2, and LP were puriﬁed by silica chromatography, isolated as solid materials and then characterized by means of analytical and spectroscopic techniques. EA-(CO)O(CH2)2OH, LP, and LN2 were found to be room temperature stable, whereas LN1 required low temperature storage. The reaction of [(η6-p-cymene)RuCl2]2 with LP in chloroform under reﬂux aﬀorded 1, which was isolated in 60% yield after workup. Complexes 2−3 were obtained in ca. 75% yield by reaction of [(η6-p-cymene)RuCl2]2 with the appropriate pyridine ligand, LN1 or LN2, in dichloromethane at room temperature (Scheme 2). Complexes 1−3 are air-stable in the solid state and in solution and are soluble in common organic solvents, but insoluble in water. The IR spectra of 1−3 (similar to the spectra of LP, LN1, and LN2) display absorptions in the range 1764−1661 cm−1, attributable to the stretching vibrations of the carbonyl and alkene moieties. The 1H and 13C NMR spectra of 1−3 in CDCl3 display the resonances due to EACO2H very close in value to those previously reported for the same fragment.13 However, as a consequence of N-coordination, the 1H and 13C resonances related to the adjacent nuclei within the pyridyl moiety of 2−3 are shifted signiﬁcantly downﬁeld. More precisely, for the 3−LN2 pair a diﬀerence of ΔδH ≈ 0.4 ppm in the 1H spectrum and ΔδC ≈ 5 ppm in the 13 C spectrum was observed. Analogously, the 31P resonance of LP (δP = −4.9 ppm) undergoes strong downﬁeld shift in 1 (δP = 25.3 ppm), as result of coordination to the ruthenium center. The proposed structures were also conﬁrmed by mass spectrometry analyses. Single crystals of 2 suitable for X-ray diﬀraction analysis were obtained from a THF/hexane solution settled at −30 °C. The molecular structure of 2 is shown in Figure 2 with key geometric parameters given in Table 1. Compound 2 adopts the characteristic three-leg piano-stool geometry previously found in several Ru(II)−arene complexes.16 The bonding parameters around the Ru(II) center are similar to those previously found in related complexes.13,17 The bonding parameters of the EA-CO2H unit are not signiﬁcantly diﬀerent with respect to other reported structures.12,13,18 In principle, the synthesis of the NAMI-A-like compounds incorporating LN1 or LN2 involves the reaction of the NAMIA precursor, [(DMSO)2H][trans-RuCl4(DMSO)2],24 with LN1 or LN2. ESI-MS analysis of the residue generated from the reaction of [(DMSO)2H][trans-RuCl4(DMSO)2] with LN2 in acetone at room temperature revealed the formation of a mixture of products. In contrast, [HLN1][transRuCl4(DMSO)(LN1)], 4, was obtained in good yield under the same experimental conditions (Scheme 3). Conductivity measurements and NMR spectroscopy were used to assess the stability of 1−4 in DMSO/H2O mixture at Figure 2. Molecular structure of Ru(η6-p-cymene)Cl2(LN1), 2. Displacement ellipsoids are at the 50% probability level. Table 1. Selected Bond Distances (Å) and Angles (deg) for 2 Ru(1)−C(21) Ru(1)−C(23) Ru(1)−C(25) Ru(1)−Cl(1) Ru(1)−N(1) N(1)−C(5) C(6)−O(1) C(7)−O(2) C(8)−O(3) Cl(1)−Ru(1)−Cl(2) Cl(2)−Ru(1)−N(1) C(6)−O(1)−C(7) O(1)−C(7)−C(8) C(7)−C(8)−O(3) 2.180(4) 2.139(5) 2.177(4) 2.4111(10) 2.133(3) 1.341(5) 1.441(5) 1.183(5) 1.429(9) 89.90(9) 86.24(9) 118.4(3) 107.6(6) 108.6(9) Ru(1)−C(22) Ru(1)−C(24) Ru(1)−C(26) Ru(1)−Cl(2) N(1)−C(1) C(3)−C(6) O(1)−C(7) C(7)−C(8) O(3)−C(9) Cl(1)−Ru(1)−N(1) C(3)−C(6)−O(1) O(1)−C(7)−O(2) O(2)−C(7)−C(8) C(8)−O(3)−C(9) 2.135(5) 2.188(5) 2.163(4) 2.4047(11) 1.338(5) 1.505(5) 1.324(5) 1.513(9) 1.344(7) 85.89(9) 107.4(3) 125.0(4) 127.3(6) 119.7(10) 37 °C. These experiments suggest that, in the cases of 2 and 3, rapid chloride release takes place to aﬀord aquated, ionic species. Furthermore, the NMR spectra indicate that progressive dissociation of LN1 from 2 (approximately 30% after 17 h and 75% after 72 h), LN2 from 3 (38% after 17 h and ca. 75% after 72 h), and LN1 from 4 (approximately 90% after 17 h, almost complete after 72 h) takes place. Complex 1 appears to be more inert toward ligands displacement, and only 20% LP was found not to be coordinated to the ruthenium(II) center after 72 h. Biological Studies. The ability of the compounds, except LN1 due to solubility problems, to inhibit cell growth was evaluated against the cisplatin-sensitive A2780 and the cisplatinresistant A2780cisR human ovarian carcinoma cell lines and nontumoral HEK-293 (immortalized human embryonic kidney) cells (Table 2). On the two cancer cell lines all the tested ligands and complexes are considerably more cytotoxic than RAPTA-C. Even the ligands, LP and LN2, are more cytotoxic than EA-CO2H, and there is little diﬀerence in cytotoxicity between the ligands and complexes 1−4. The IC50 values obtained for all the compounds are signiﬁcantly lower than the corresponding ones referred to as NAMI-A.19 Conversely, the IC50 values of 1−4 are similar to those previously reported for ruthenium(II)−arene complexes modiﬁed with an EA-CO2H unit, attached either via the η6arene ligand or via an imidazole coligand.12,13 On the basis of these observations, it seems plausible that the EA-CO2H C DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Scheme 3. Synthesis of 4, a NAMI-A-like Complex Containing the EA Skeleton Table 2. IC50 Values (μM) Determined for RAPTA-C,21 EACO2H, Cisplatin, LP, LN2, and 1−4 on Human Ovarian (A2780 and A2780cisR) Cancer Cells and Human Embryonic Kidney (HEK-293) Cells at 72 ha a compd A2780 A2780cisR HEK-293 RAPTA-C EA-CO2H LP LN1 LN2 1 2 3 4 cisplatin NAMI-A 230 40 ± 3 9±1 270 53 ± 5 40 ± 4 >1000 13 ± 2 11 ± 2 21 ± 2 13 ± 3 31 ± 8 0.9 ± 0.1 >50019 11 ± 1 15 ± 4 41 ± 6 26 ± 2 27 ± 2 25 ± 3 >50019 7.0 ± 0.5 13 ± 4 22 ± 1 16 ± 3 21 ± 3 9±2 >500 38 ± 9 Figure 3. Residual GST activity in A2780, A2780cisR, and HEK cell lines, expressed as % of control. The changes in ﬂuorescence over a 40 min period were used to calculate GST activities. Values are given as the mean ± SD. NAMI-like complexes. The antiproliferative activity of all of the compounds was determined, with little diﬀerence between the ligands and the complexes discerned, although all compounds are markedly more cytotoxic than RAPTA-C, NAMI-A, and EA-CO2H. The Ru−N bond within N-donor-based complexes is labile in aqueous−DMSO solutions and is expected to partially dissociate prior to cellular uptake. In contrast, the Ru− P bond is signiﬁcantly more stable, and dissociation of the EACO2H fragment presumably takes place following uptake of the related complex into the cell. Despite these diﬀerences, the activity of all the compounds is similar in the in vitro studies (cytotoxicity and residual GST activity); nevertheless, in vivo, the simultaneous delivery of the two active components to the tumor, i.e., the ruthenium species and the EA-CO2H unit, is likely to be important. fragment contributes to the anticancer eﬀect via the inhibition of GSTs, which sensitizes the cancer cells toward the ruthenium fragments. Esterases may be implicated in separating the EACO2H fragment from the ligand/complex.20 Such a hypothesis has been proposed previously.15 The compounds are not endowed with cancer cell selectivity; i.e., they are also cytotoxic to the HEK-293 cells. Complexes 1− 3 have similar IC50 values on both the A2780 and HEK-293 cells and are less active against A2780cisR cells that are crossresistant to cisplatin. It has been shown that one mechanism of cisplatin resistance is due to overexpression of GSTs,22 which could reduce the eﬃcacy of these complexes, and an excess of EA-CO2H would be needed to overcome resistance. In order to investigate this point, residual intracellular GST activities after incubation with complexes 1−4 were determined by ﬂuorescence spectroscopy (Figure 3). Since the 50% growthinhibitory concentration of 1−4 diﬀered between the A2780, A2780cisR, and HEK cell lines (Table 2), GST activity in each cell line was measured at the IC50 concentration, but over a shorted incubation period. Incubation with 1−4 led to a decrease in GST activity by 10−30% in A2780cisR cells, whereas in the other cell lines no activity decrease was observed. These results indicate that inhibition of the GSH/ GST detoxiﬁcation system in A2780cisR contributes to the relatively high cytotoxicities (IC50 values comparable to IC50 values in A2780 cells). ■ EXPERIMENTAL SECTION RuCl3·3H2O (99.9%) was purchased from Strem, and the organic reactants were obtained from Alfa Aesar or Apollo Sci. and were of the highest purity available. Solvents were purchased from Sigma-Aldrich. Complexes [(η 6 -p-cymene)RuCl2 ] 2 23 and [(DMSO) 2 H][transRuCl4(DMSO)2]24 were prepared according to literature methods. The synthesis of EA-(CO)O(CH2)2OH, LP, LN1, and LN2 was performed under nitrogen atmosphere using solvents distilled over P4O10. NMR spectra were recorded at 298 K on a Bruker Avance DRX400 instrument equipped with a BBFO broadband probe. The chemical shifts for 1H and 13C NMR spectra were referenced to a nondeuterated aliquot of the solvent, and spectra were assigned with the assistance of 1H,13C correlation spectroscopy using gs-HSQC and gs-HMBC techniques.25 Infrared spectra were recorded at 298 K on a PerkinElmer FT IR spectrometer equipped with UATR sampling accessory. Carbon, hydrogen, and nitrogen analysis was performed on ■ CONCLUSIONS Novel P- and N-donor ligands modiﬁed with ethacrynic acid were prepared and coordinated to ruthenium(II)-p-cymene and D DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry a Carlo Erba model 1106 instrument. Mass spectra were obtained on a ThermFinnigan LCQ Deca XP Plus Quadrupole ion-trap instrument in the positive ion mode. Melting points are uncorrected and were recorded on a STMP3 Stuart scientiﬁc instrument with a capillary apparatus. Conductivity measurements were carried out using an Eutech Con 700 Instrument (cell constant = 1.0 cm−1).26 Synthesis of 2-Hydroxyethyl-2-(2,3-dichloro-4-(2methylenebutanoyl)phenoxy)acetate, EA-(CO)O(CH2)2OH. A mixture of EA-CO2H (200 mg, 0.660 mmol), Chart 1, and ethylene (t, 3 H, 3JHH = 7.4 Hz, C1−H). 13C{1H} NMR (CDCl3): δ = 195.7 (C5); 167.6 (C13); 166.1 (C16); 155.4 (C9); 150.1 (C3); 144.8 (d, 1 JCP = 14.3 Hz, P-C); 136.1 (C20); 134.0, 133.2 (d, 2JCP = 19 Hz, C19 + C19′), 129.4−129.2 (C18 + C18′ + Ph); 132.3 (C8); 131.5 (C7); 130.5 (C17); 128.7 (C4); 126.8 (C11); 110.9 (C10); 123.4 (C6); 66.1 (C12); 63.3, 62.4 (C14 + C15); 23.4 (C2); 12.4 ppm (C1). 31P NMR (CDCl3): δ = −4.9 ppm. Synthesis of Pyridin-4-yl-methyl-2-(2,3-dichloro-4-(2methylenebutanoyl)phenoxy)acetate, LN1. LN1 (Chart 3) was Chart 1. EA-C(O)CH2CH2OH (Numbering Refers to Carbon Atoms) Chart 3. LN1 (Numbering Refers to Carbon Atoms) prepared by the same procedure described for the synthesis of EA(CO)O(CH2)2OH, from EA-CO2H (428 mg, 1.41 mmol), 4pyridinemethanol (140 mg, 1.28 mmol), EDCI (245 mg, 1.28 mmol), and DMAP (23 mg, 0.188 mmol). Chromatography: hexane/Et2O (progressively decreasing v/v ratio). Colorless solid stored at −30 °C, yield 430 mg (77%). Anal. Calcd for C19H17Cl2NO4: C, 57.81; H, 4.39; N, 3.21. Found: C, 57.90; H, 4.26; N, 3.12. IR (solid state): υ = 2969w, 2935w, 2376m, 2279w, 1762s, 1662s, 1633m, 1584s, 1508w, 1468m, 1436s, 1383m, 1293m, 1258m, 1185vs, 1168vs, 1122m, 1077vs, 1001m, 940w, 893w, 809s, 767m, 731w, 706w, 665w cm−1. 1H NMR (CDCl3): δ = 8.54 (d, 2 H, 3JHH = 4.3 Hz, C17−H + C17′-H); 7.46 (d, 2 H, 3JHH = 4.6 Hz, C16−H + C16′-H); 7.13 (d, 1 H, 3JHH = 8.3 Hz, C11−H); 6.83 (d, 1 H, 3JHH = 8.3 Hz, C10−H); 5.95, 5.57 (m, 2 H, C4−H); 5.25 (s, 2 H, C14−H); 4.86 (s, 2 H, C12−H); 2.45 (q, 2 H, 3JHH = 7 Hz, C2−H); 1.16 ppm (t, 3 H, 3JHH = 7 Hz, C1−H). 13 C{1H} NMR (CDCl3): δ = 195.6 (C5); 167.1 (C13); 155.1 (C9); 150.1 (C3); 147.9 (C15); 147.6 (C17); 134.2 (C8); 131.6 (C7); 128.9 (C4); 126.8 (C11); 123.4 (C6); 123.3 (C16); 110.9 (C10); 66.1 (C12); 64.2 (C14); 23.4 (C2); 12.4 ppm (C1). Synthesis of 2-(2-(2,3-Dichloro-4-(2-methylenebutanoyl)phenoxy)acetoxy)ethyl-nicotinate, LN2. LN2 (Chart 4) was glycol (0.15 mL, 2.69 mmol) in CH2Cl2 (20 mL) was treated with EDCI (127 mg, 0.662 mmol) and then with DMAP (12 mg, 0.098 mmol). The resulting mixture was allowed to stir at room temperature overnight. The resulting colorless solution was charged on a silica column. A minor fraction corresponding to EA-(CO)O(CH2)2O(CO)-EA27 was collected by using CH2Cl2/Et2O (3:1 v/v ratio) as eluent. Then, EA-(CO)O(CH2)2OH was eluted with CH2Cl2/Et2O (1:1 v/v ratio). The product was isolated as a colorless powder upon removal of the solvent. Yield 171 mg, 75%. Anal. Calcd for C15H16Cl2O5: C, 51.89; H, 4.65. Found: C, 51.75; H, 4.76. Mp: 92 °C. IR (solid state): υ = 3511m, 2974w, 2920w, 2880w, 1736s, 1661s, 1586m-s, 1471m, 1444w, 1382m, 1357w, 1291m-s, 1257m, 1229vs, 1204vs, 1123m, 1075vs, 1012m, 1000s, 943m-s, 891m, 840m, 809m, 768m, 735w cm−1. 1H NMR (CDCl3): δ = 7.16 (d, 1 H, 3JHH = 8.48 Hz, C11−H); 6.82 (d, 1 H, 3JHH = 8.48 Hz, C10−H); 5.93, 5.59 (d, 2 H, 2JHH = 1.46 Hz, C4−H); 4.79 (s, 2 H, C12−H); 4.31, 3.82 (m, 4 H, C14−H + C15−H); 2.44 (q, 2 H, 3JHH = 7.4 Hz, C2−H); 1.85 (m, 1 H, OH); 1.12 ppm (t, 3 H, 3JHH = 7.4 Hz, C1−H). 13C{1H} NMR (CDCl3): δ = 195.9 (C5); 168.0 (C13); 155.4 (C9); 150.1 (C3); 133.8 (C8); 131.4 (C7); 128.9 (C4); 126.9 (C11); 110.9 (C10); 123.2 (C6); 66.9 (C14); 66.2 (C12); 60.6 (C15); 23.4 (C2); 12.4 ppm (C1). Synthesis of 2-((4-(Diphenylphosphanyl)benzyl)oxy)ethyl-2(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetate, LP. LP (Chart 2) was prepared by the same procedure described for the Chart 4. LN2 (Numbering Refers to Carbon Atoms) Chart 2. LP (Numbering Refers to Carbon Atoms) prepared by the same procedure described for the synthesis of EA(CO)O(CH2)2OH, from EA-(CO)O(CH2)2OH (220 mg, 0.634 mmol), nicotinic acid (94 mg, 0.761 mmol), EDCI (122 mg, 0.636 mmol), and DMAP (11 mg, 0.090 mmol). Chromatography: Et2O. Colorless solid, yield 232 mg (81%). Anal. Calcd for C21H19Cl2NO6: C, 55.77; H, 4.23; N, 3.10. Found: C, 55.90; H, 4.35; N, 3.13. ESIMS(+): m/z found 452.067 [M + H]+, calcd for C21H20Cl2NO6+ 452.067; the isotopic pattern ﬁts well the calculated one. Mp: 87−89 °C. IR (solid state): υ = 3063w, 2966w, 2919w, 1759m, 1731m-br, 1664m, 1583s, 1573s, 1505vs, 1483vs, 1455m, 1447w-m, 1435w, 1400s, 1386vs, 1369s, 1287s, 1269s, 1246m, 1234s, 1192vs, 1166m, 1139m, 1116m, 1079s, 1070m-sh, 1058m, 1027m, 1013vs, 996s, 975wm, 961w, 936vs, 916w, 908w, 833s, 828w, 801m, 766m-sh, 756vs, 748vs, 699vs, 687s, 681s cm−1. 1H NMR (CDCl3): δ = 9.23 (s, 1 H, C18−H); 8.80 (d, 1 H, 3JHH = 4.89 Hz, C19−H); 8.28 (m, 1 H, C21− H); 7.43 (m, 1 H, C20−H); 7.09 (d, 1 H, 3JHH = 8.5 Hz, C11−H); 6.81 (d, 1 H, 3JHH = 8.5 Hz, C10−H); 5.93, 5.58 (m, 2 H, C4−H); 4.81 (s, 2 H, C12−H); 4.60 (m, 4 H, C14 + C15); 2.46 (q, 2 H, 3JHH = 7.34 Hz, C2−H); 1.15 ppm (t, 3 H, 3JHH = 7.34 Hz, C1−H). 13C{1H} NMR (CDCl3): δ = 195.7 (C5); 167.6 (C13); 165.0 (C16); 155.3 (C9); 153.7 (C19); 150.9 (C18); 150.1 (C3); 137.3 (C21); 134.0 (C8); 131.5 (C7); 128.6 (C4); 126.8 (C11); 125.5 (C17); 123.5 synthesis of EA-(CO)O(CH2)2OH, from EA-(CO)O(CH2)2OH (400 mg, 1.15 mmol), 4-(diphenylphosphino)benzoic acid (387 mg, 1.27 mmol), EDCI (220 mg, 1.15 mmol), and DMAP (20 mg, 0.164 mmol). Chromatography: hexane/Et2O (1:4 v/v ratio). Colorless solid, yield 453 mg (62%). Anal. Calcd for C34H29Cl2O6P: C, 64.26; H, 4.60. Found: C, 64.12; H, 4.67. ESI-MS(+): m/z found 635.117 [M + H]+, calcd for C34H30Cl2O6P+ 635.116; the isotopic pattern ﬁts well the calculated one. Mp: 72−74 °C. IR (solid state): υ = 3071w-br, 2964w-sh, 2121w-br, 1763m, 1746m-sh, 1718m-s, 1665m, 1584m, 1468w-m, 1434m, 1394w-m, 1384w-m, 1338w, 1266vs, 1193s, 1181s, 1121m, 1107m, 1080vs, 1017m, 999m, 941w-m, 895w, 850w, 802w-m, 761m-s, 743s, 718w-m, 694vs cm−1. 1H NMR (CDCl3): δ = 7.98 (8 H, C18−H + C18′−H + C19−H + C19′−H + Ph); 7.36 (6 H, Ph); 7.09 (d, 1 H, 3JHH = 8.5 Hz, C11−H); 6.82 (d, 1 H, 3JHH = 8.5 Hz, C10− H); 5.93, 5.58 (m, 2 H, C4−H); 4.79 (s, 2 H, C12−H); 4.56 (m, 4 H, C14−H + C15−H); 2.47 (q, 2 H, 3JHH = 7.4 Hz, C2−H); 1.15 ppm E DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry MS(+): m/z found 665.045 [M − Cl]+, calcd for C29H31Cl3NO4Ru+ 665.044; the isotopic pattern ﬁts well the calculated one. Mp: 166− 168 °C. IR (solid state): υ = 3075w, 2966w, 2935w, 2874w, 1764vs, 1692w, 1661m, 1616w, 1584s, 1499w, 1470m, 1446m, 1426m, 1390m, 1383m, 1364w, 1338w, 1328w, 1301m, 1244s, 1228m, 1191vs, 1120w, 1074vs, 1065vs, 1030m, 999m, 959w, 878w, 813s, 760w, 734w, 716w, 666w cm−1. 1H NMR (CDCl3): δ = 8.88 (d, 2 H, 3JHH = 5 Hz, C17−H + C17′−H); 7.16 (d, 2 H, 3JHH = 5 Hz, C16−H + C16′−H); 7.06 (d, 1 H, 3JHH = 8 Hz, C11−H); 6.84 (d, 1 H, 3JHH = 8 Hz, C10−H); 5.88, 5.52 (m, 2 H, C4−H); 5.38, 5.17 (d, 4 H, 3JHH = 5 Hz, arom CHcymene); 5.14 (s, 2 H, C14−H); 4.84 (s, 2 H, C12−H); 2.87 (m, 1 H, CHMe2); 2.38 (q, 2 H, 3JHH = 7.2 Hz, C2−H); 2.00 (s, 3 H, MeC6H4); 1.22 (d, 6 H, 3JHH = 6.8 Hz, CHMe2); 1.06 (t, 3 H, 3JHH = 7.4 Hz, C1−H) ppm. 13C NMR (CDCl3): δ = 195.7 (C5); 167.2 (C13); 155.1 (C9); 154.8 (C17 + C17′); 149.9 (C3); 146.4 (C15); 133.8 (C8); 131.1 (C7); 129.0 (C4); 127.1 (C11); 122.9 (C6); 122.6 (C16 + C16′); 111.3 (C10); 103.3 (CCHMe2); 97.2 (arom CMe); 88.8, 82.1 (arom CHcymene); 66.2 (C12); 64.2 (C14); 30.6 (CHMe2); 23.3 (C2); 22.2 (CHMe2); 18.2 (MeC6H4); 12.4 (C1) ppm. Synthesis of Ru(η6-p-cymene)Cl2(2-(2-(2,3-dichloro-4-(2methylenebutanoyl)phenoxy)acetoxy)ethyl nicotinate), 3. This compound was prepared by the same procedure described for the synthesis of 2, from [(η6-p-cymene)RuCl2]2 (0.105 g, 0.171 mmol) and LN2 (185 mg, 0.409 mmol). Dark-orange solid, yield 200 mg (77%). Compound 3 (Chart 7) is soluble in chlorinated solvents and (C20); 123.4 (C6); 110.8 (C10); 66.1 (C12); 63.1, 62.8 (C14, C15); 23.4 (C2); 12.4 ppm (C1). Synthesis of Ru(η6-p-cymene)Cl2(2-((4(diphenylphosphanyl)benzyl)oxy)ethyl-2-(2,3-dichloro-4-(2methylenebutanoyl)phenoxy)acetate), 1. [Ru(η6-p-cymene)Cl2]2 (0.140 g, 0.229 mmol) was added to a solution of LP (330 mg, 0.519 mmol) in CHCl3 (20 mL). The resulting mixture was heated at reﬂux for 18 h. The resulting red solution was cooled to room temperature, and the solvent was removed under reduced pressure. The dark-red residue was washed with Et2O (3 × 20 mL) and then dried under vacuum. The product, 1 (Chart 5), was obtained as a dark-red solid. Chart 5. Ru(η6-p-cymene)Cl2(LP), 1 (Numbering Refers to Carbon Atoms) Yield 256 mg, 60%. Compound 1 is soluble in chlorinated solvents and DMSO and insoluble in H2O. Anal. Calcd for C44H43Cl4O6PRu: C, 56.12; H, 4.60. Found: C, 56.24; H, 4.73. ESI-MS(+): m/z found 906.091 [M − Cl]+, calcd for C44H43Cl3O6PRu+ 906.098; the isotopic pattern ﬁts well with the calculated one. Mp: 117−118 °C. IR (solid state) υ: 3058w-br, 2965w-m-sh, 1760m, 1717m-s, 1663m, 1584m, 1469m, 1435m, 1384m, 1338w, 1263s, 1187s, 1110m-s, 1079vs, 1017m-s, 1000m, 942w-m, 895w-m, 854w-m, 800m, 762m, 748m, 721m, 696vs cm−1. 1H NMR (CDCl3): δ = 7.93, 7.84, 7.43 (14 H, C18−H + C18′−H + C19−H + C19′−H + Ph); 7.11 (m, 1 H, C11− H); 6.82 (m, 1 H, C10−H); 5.94, 5.60 (m, 2 H, C4−H); 5.25, 5.00 (m, 4 H, arom CHcymene); 4.79 (s, 2 H, C12−H); 4.53 (m, 4 H, C14− H + C15−H); 2.88 (m, 1 H, CHMe2); 2.46 (m, 2 H, C2−H); 1.88 (s, 3 H, MeC6H4); 1.35−1.13 ppm (m, 9 H, C1−H + CHMe2). 13C{1H} NMR (CDCl3): δ = 195.8 (C5); 167.5 (C13); 165.8 (C16); 155.3 (C9); 150.1 (C3); 139.8, 139.3, 136.7 (C20 + P-C); 134.5, 134.4, 128.7, 128.3 (C18 + C18′ + C19 + C19′ + arom CHPh); 133.3 (C8); 131.5 (C7); 132.4 (C17); 129.5 (C4); 126.9 (C11); 111.0 (C10); 123.3 (C6); 111.6 (CCHMe2); 96.3 (arom CMe); 88.8, 87.4 (arom CHcymene); 66.0 (C12); 63.2, 62.3 (C14 + C15); 30.3 (CHMe2); 23.4 (C2); 21.8 (CHMe2); 17.8 (MeC6H4); 12.4 ppm (C1). 31P NMR (CDCl3): δ = 25.3 ppm. Synthesis of Ru(η6-p-cymene)Cl2(pyridin-4-yl-methyl-2-(2,3dichloro-4-(2-ethylenebutanoyl)phenoxy)acetate), 2. [Ru(η6-pcymene)Cl2]2 (0.120 g, 0.196 mmol) was added to a solution of LN1 (185 mg, 0.469 mmol) in CH2Cl2 (20 mL). The resulting mixture was allowed to stir at room temperature for 18 h, and the solvent was removed under reduced pressure. CHCl3 (2 mL) and then Et2O (50 mL) were added. The resulting precipitate was washed with Et2O (2 × 20 mL) and then dried under vacuum. The product, 2 (Chart 6), was obtained as a red solid. Yield 203 mg, 74%. Red crystals suitable for Xray analysis were collected from a THF/hexane mixture set aside at −30 °C for 2 weeks. Compound 2 is soluble in chlorinated solvents and DMSO and insoluble in H2O. Anal. Calcd for C29H31Cl4NO4Ru: C, 49.73; H, 4.46; N, 2.00. Found: C, 49.85; H, 4.55; N, 1.92. ESI- Chart 7. (η6-p-cymene)RuCl2(LN2), 3 (Numbering Refers to Carbon Atoms) DMSO and insoluble in H2O. Anal. Calcd for C31H33Cl4NO6Ru: C, 49.09; H, 4.39; N, 1.85. Found: C, 48.95; H, 4.39; N, 1.88. ESIMS(+): m/z found 723.042 [M − Cl]+, calcd for C31H33Cl3NO6Ru+ 723.049; the isotopic pattern ﬁts well the calculated one. Mp: 69−70 °C. IR (solid state): υ = 3071w-br, 2963w, 2930w, 2874w, 1760m-sh, 1729s, 1662m, 1604w, 1585s, 1501w, 1469m, 1434m, 1408w, 1384m, 1363w, 1286vs, 1259s, 1195vs, 1138m, 1115s, 1079vs, 1056vs, 1031s, 1003s, 941w-br, 913w, 895w, 872w-br, 803s, 769w, 744s, 728s, 689m cm−1. 1H NMR (CDCl3): δ = 9.59 (s, 1 H, C18−H); 9.23 (d, 1 H, 3 JHH = 4.89 Hz, C19−H); 8.30 (d, 1 H, 3JHH = 7.34 Hz, C21−H); 7.45 (m, 1 H, C20−H); 7.12 (d, 1 H, 3JHH = 8.5 Hz, C11−H); 6.94 (d, 2 H, 3 JHH = 8.5 Hz, C10−H); 5.93, 5.58 (s, 2 H, C4−H); 5.49, 5.30 (d, 4 H, 3 JHH = 5.38 Hz, arom CHcymene); 4.95 (s, 2 H, C12−H); 4.60 (m, 4 H, C14 + C15); 2.94 (m, 1 H, CHMe2); 2.45 (q, 2 H, 3JHH = 7.82 Hz, C2−H); 2.07 (s, 3 H, MeC6H4); 1.30 (d, 6 H, 3JHH = 6.85 Hz, CHMe2); 1.14 ppm (t, 3 H, 3JHH = 7.82 Hz, C1−H). 13C{1H} NMR (CDCl3): δ = 195.8 (C5); 167.9 (C13); 163.3 (C16); 158.3 (C19); 156.1 (C18); 155.3 (C9); 150.1 (C3); 138.9 (C21); 133.8 (C8); 131.3 (C7); 128.9 (C4); 126.7 (C11); 111.1 (C10); 127.1 (C17); 124.4 (C20); 123.0 (C6); 111.6 (CCHMe2); 97.5 (arom CMe); 83.1, 82.1 (arom CHcymene); 66.2 (C12); 63.4, 62.6 (C14, C15); 30.8 (CHMe2); 23.4 (C2); 22.3 (CHMe2); 18.3 (MeC6H4); 12.4 ppm (C1). Synthesis of [HLN1]+[trans-RuCl4(DMSO)(LN1)]−, 4. LN1 (760 mg, 1.93 mmol) was added to a suspension of freshly prepared [(DMSO)2H][trans-RuCl4(DMSO)2] (505 g, 0.908 mmol) in acetone (20 mL). The mixture was stirred at room temperature, in the dark. After 2 h an orange solution had formed. The solvent was removed under reduced pressure, and the resulting solid residue was washed with Et2O (3 × 20 mL) and then dried under vacuum. Compound 4 (see Chart 8) was obtained as an orange microcrystalline powder. Yield 836 mg, 83%. Compound 4 is soluble in DMSO and methanol Chart 6. (η6-p-cymene)RuCl2(LN1), 2 (Numbering Refers to Carbon Atoms) F DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry II diﬀractometer equipped with a CCD detector using Mo Kα radiation. Data were corrected for Lorentz polarization and absorption eﬀects (empirical absorption correction SADABS).28 The structure was solved by direct methods and reﬁned by full-matrix least-squares based on all data using F2.29 Hydrogen atoms were ﬁxed at calculated positions and reﬁned by a riding model. All non-hydrogen atoms in 2 were reﬁned with anisotropic displacement parameters, whereas the disordered THF molecule was treated isotropically. Part of the LN1 ligand and the isopropyl group of p-cymene in 2 and the THF molecule are disordered and, therefore, have been split into two positions and reﬁned isotropically using one occupancy parameter per disordered group. Similar U restraints were applied to the C, N, and O atoms of the LN1 and p-cymene ligands (SIMU line in SHELXL, s.u. 0.01). Some atoms of the disordered groups have been restrained to isotropic behavior (ISOR line in SHELXL, s.u. 0.02). The disordered groups have been restrained to have similar geometries (SAME line in SHELXL, s.u. 0.02). The Ph-rings of the disordered LN1 ligand were constrained to ﬁt regular hexagons (AFIX 66 line in SHELXL). Restraints to bond distances were applied as follows (s.u. 0.02): 1.43 Å for C−O and 1.53 Å for C−C in THF. Cell Culture. Human A2780 and A2780cisR ovarian carcinoma cells were obtained from the European Centre of Cell Cultures (ECACC, U.K.). Nontumorigenic HEK-293 cells were provided by the Institute of Pathology, CHUV, Lausanne, Switzerland. A2780 and A2780cisR were routinely grown in RPMI 1640 medium supplemented with GlutaMAX (Gibco), while HEK-293 cells were grown in DMEM medium, both containing heat-inactivated fetal calf serum (FCS, Sigma) (10%) and antibiotics (penicillin/streptomycin, 1%) at 37 °C and CO2 (5%). Determination of Antiproliferative Activity. Cytotoxicity was determined using the MTT assay (MTT = 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Cells were seeded in 96-well plates as monolayers with 100 μL of cell solution per well and preincubated for 24 h in the cell medium. Compounds were prepared as DMSO solutions that were rapidly dissolved in the culture medium and serially diluted to the appropriate concentration to give a ﬁnal DMSO concentration of 0.5%. A 100 μL portion of the drug solution was added to each well, and the plates were incubated for another 72 h. Subsequently, MTT (5 mg/mL solution) was added to the cells and the plates were incubated for further 4 h. The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, directly proportional to the number of surviving cells, was quantiﬁed at 540 nm using a multiwell plate reader, and the fraction of surviving cells was calculated from the absorbance of untreated control cells. Evaluation is based on means from two independent experiments, each comprising three microcultures per concentration level. GST Activity Assay. A2780, A2780cisR, and HEK cells were plated in six well plates and incubated for 24 h in a CO2 incubator at 37 °C. The cells were then exposed to compounds 1−4 at ﬁnal concentrations ranging from 10 to 40 μM, according to the IC50 concentration for each respective cell line, for 6 h. The cells were then washed with ice-cold PBS, harvested, lysed by a repetitive freeze−thaw cycle, and centrifuged at 10 000g for 15 min at 4 °C. The supernatants were used for the analysis of GST activity according to a ﬂuorometric GST detection kit (Abnova). The changes in ﬂuorescent intensities at Ex/Em 380/460 nm were recorded in a kinetic mode, every 5 min over 60 min, on a microplate reader (Molecular Devices). The GST activities were measured in duplicate and expressed as U/min/mL per mg protein and then converted to % of control. The GST activity of the test samples was calculated by applying the equation ΔRFU = RFU2 − RFU1 to the GST standard curve to get B [mU] during the reaction time (ΔT = T2 − T1). The sample GST activity is calculated by the following formula: sample GST activity = B/(ΔT × V) × dilution factor [mU/min/mL], where B is sample GST activity from the GST standard curve [mU], ΔT is the reaction time (min), V is the sample volume added into the reaction well [mL]. Protein Determination. The protein concentration was determined by a Bradford assay (Bio-Rad) using BSA as a standard. Chart 8. NAMI-A-like Complex Containing EA and insoluble in H2O. Anal. Calcd for C40H41Cl8N2O9RuS: C, 43.26; H, 3.72; N, 2.52. Found: C, 43.11; H, 3.89; N, 2.38. ESI-MS(+): m/z found 394.061 [MHLN1]+, calcd for C19H18Cl2NO4+ 394.061; the isotopic pattern ﬁts well the calculated one. ESI-MS(−): m/z found 715.858 [M[trans‑RuCl4(DMSO)(LN1)]]−, calcd for C21H23Cl6NO5RuS− 715.248; the isotopic pattern ﬁts well the calculated one. Mp: decomp at 85 °C. IR (solid state): ν = 3210w, 3068w, 2968w, 2933w, 2870w, 1759m, 1691w, 1662m, 1643w, 1620w, 1583s, 1507w, 1468m, 1428m, 1383m, 1340w, 1293m, 1256m, 1221m, 1185vs, 1117m, 1074vs, 1020s, 1001s, 938w, 907w, 892w, 806s, 768m, 721w, 684w cm−1. Stability Studies. (A) The general procedure for NMR spectroscopy follows. Complexes 1−4 (0.050 mmol) were dissolved in 0.55 mL of DMSO-d6/H2O mixture (v/v ratio 9:1), and CDCl3 (0.50 mmol) was added as a reference. The NMR tubes were sealed, maintained at 37 °C, and analyzed by NMR spectroscopy as a function of time. (B) The general procedure for conductivity follows. Complexes 1−3 (ca. 0.05 mmol) were dissolved in 5 mL of DMSO/H2O mixture (v/v ratio 9:1) and kept at 37 °C. Measurements were recorded as a function of time. Complex 1: 3 min, ΛM = 0.30 S × cm2 × mol−1; 90 min, ΛM = 0.40 S × cm2 × mol−1; 72 h, ΛM = 0.83 S × cm2 × mol−1. Complex 2: 3 min, ΛM = 4.3 S × cm2 × mol−1; 90 min, ΛM = 5.9 S × cm2 × mol−1; 72 h, ΛM = 6.2 S × cm2 × mol−1. Complex 3: 3 min, ΛM = 1.5 S × cm2 × mol−1; 90 min, ΛM = 2.5 S × cm2 × mol−1; 72 h, ΛM = 2.9 S × cm2 × mol−1. X-ray Crystallography. Crystal data and collection details for 2· THF are reported in Table 3. Data were recorded on a Bruker APEX Table 3. Crystal Data and Measurement Details for 2·THF formula fw T, K λ, Å cryst syst space group a, Å b, Å c, Å β, deg cell volume, Å3 Z Dc, g cm−3 μ, mm−1 F(000) cryst size, mm3 θ limits, deg reﬂns collected indep reﬂns data/restraints/params GOF on F2 R1 (I > 2σ(I)) wR2 (all data) largest diﬀ peak and hole, e Å−3 C33H39Cl4NO5Ru 772.52 293(2) 0.710 73 monoclinic P21/c 14.2454(11) 10.6848(8) 23.3046(18) 98.5820(10) 3507.5(5) 4 1.463 0.791 1584 0.21 × 0.16 × 0.12 1.45−27.00 37 831 7469 [Rint = 0.0290] 7469/834/506 1.039 0.02479 0.1574 0.958/−0.610 G DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry ■ 2011, 11, 2688−2702. (d) Levina, A.; Mitra, A.; Lay, P. A. Metallomics 2009, 1, 458−470. (e) Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med. Chem. 2011, 54, 3−25. (8) (a) Kurzwernhart, A.; Kandioller, W.; Bächler, S.; Bartel, C.; Martic, S.; Buczkowska, M.; Mühlgassner, G.; Jakupec, M. A.; Kraatz, H.-B.; Bednarski, P. J. J. Med. 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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b00802. CCDC reference number 1056723 (2) containing the supplementary crystallographic data for the X-ray study reported in this paper, which can be obtained free of charge at www.ccdc.cam.ac.uk/ conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax (int) +44-1223/336-033; e-mail deposit@ccdc.cam.ac.uk). ■ AUTHOR INFORMATION Corresponding Authors *E-mail: paul.dyson@epﬂ.ch. *E-mail: fabio.marchetti1974@unipi.it. Notes The authors declare no competing ﬁnancial interest. ■ ■ ACKNOWLEDGMENTS We thank the Swiss National Science Foundation and the University of Pisa for ﬁnancial support. REFERENCES (1) (a) van Rijt, S. H.; Sadler, P. J. Drug Discovery Today 2009, 14, 1089−1097. (b) Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med. Chem. 2011, 54, 3−25. (c) Ronconi, L.; Sadler, P. J. Coord. Chem. 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Found: C, 53.67; H, 4.57. 1H NMR (CDCl3): δ = 7.17, 6.82 (d, 4 H, 3JHH = 8.48 Hz); 5.96, 5.62 (d, 4 H, 2JHH = 1.17 Hz); 4.79 (s, 4 H); 4.49 (s, 4 H); 2.50 (q, 2 H, 3JHH = 7.4 Hz); 1.17 ppm (t, 3 H, 3JHH = 7.4 Hz). 13C{1H} NMR (CDCl3): δ = 195.8, 167.6, 155.3, 150.1, 134.1, 131.5, 128.8, 126.8, 110.9, 123.4, 66.0, 62.9, 23.4, 12.4 ppm. (28) Sheldrick, G. M. SADABS, Program for Empirical Absorption Correction; University of Göttingen: Göttingen, Germany, 1996. (29) Sheldrick, G. M. SHELX97, Program for Crystal Structure Determination; University of Göttingen: Göttingen, Germany, 1997. I DOI: 10.1021/acs.inorgchem.5b00802 Inorg. Chem. XXXX, XXX, XXX−XXX