Synthesis, Structure, and Anticancer Activity of Arene–Ruthenium(II

Nov 7, 2016 - Influence of Functionalized η 6 -Arene Rings on Ruthenium(II) .... Zegke , Thomas Swift , Amr A. A. Attia , Rianne M. Lord , Nicolas P...
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Synthesis, Structure, and Anticancer Activity of Arene−Ruthenium(II) Complexes with Acylpyrazolones Bearing Aliphatic Groups in the Acyl Moiety Jessica Palmucci,† Fabio Marchetti,*,# Riccardo Pettinari,*,† Claudio Pettinari,† Rosario Scopelliti,‡ Tina Riedel,‡ Bruno Therrien,§ Agustin Galindo,∥ and Paul J. Dyson‡ †

School of Pharmacy and #School of Science and Technology, University of Camerino, via S. Agostino 1, 62032 Camerino MC, Italy Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland § Institute of Chemistry, University of Neuchatel, Ave de Bellevaux 51, Neuchatel, CH 2000, Switzerland ∥ Departamento de Química Inorgánica, Facultad de Química, Universidad de Sevilla, Aptdo 1203, 41071 Sevilla, Spain ‡

S Supporting Information *

ABSTRACT: A series of neutral ruthenium(II) arene complexes [(arene)Ru(QR)Cl] (arene = p-cymene (cym) or hexamethylbenzene (hmb)) containing 4-acyl-5-pyrazolonate QR ligands with different electronic and steric substituents (R = 4-cyclohexyl, 4-stearoyl, or 4-adamantyl) and related ionic complexes [(arene)Ru(QR)(PTA)][PF6] (PTA = 1,3,5-triaza-7phosphaadamantane) were synthesized and characterized by spectroscopy (IR, UV−vis, ESI-MS, and 1H and 13C NMR), elemental analysis, X-ray crystallography, and density functional theory studies. The cytotoxicity of the proligands and metal complexes was evaluated in vitro against human ovarian carcinoma cells (A2780 and A2780cisR), as well as against nontumorous human embryonic kidney (HEK293) cells. In general the cationic PTA-containing complexes are more cytotoxic than their neutral precursors with a chloride ligand in place of the PTA. Moreover, the complexes do not show cross-resistance and are essentially equally cytotoxic to both the A2780 and A2780cisR cell lines, although they only show limited selectivity toward the cancer cell lines.



INTRODUCTION

Ru(II) scaffold and the consequent anticancer activity of the complexes.32−35 Acylpyrazolones represent a well-known family of chelators, extensively investigated from their different coordination chemistry with respect to classic β-diketones.36,37 Previously we demonstrated that a methyl group bonded to N1 of the pyrazole ring leads to water-soluble acylpyrazolone ligands and (arene)Ru(II) complexes, which display good selectivity and cytotoxic activity on certain cancer cell lines.35 We also described successful docking features of one derivative onto a guanosine-containing DNA fragment.35 The introduction of a planar aromatic diphenyl group on the acyl moiety of the acylpyrazolone compounds influences the biological activity of the (arene)Ru(II) complexes, yielding dose- and cancer cell line-dependent cytotoxicity, exerted by an apoptotic mechanism. 34 Dinuclear (arene)Ru(II) complexes with bis(acylpyrazolone) ligands were found to be cytotoxic toward cancer cell lines to a comparable extent to cisplatin but with higher capacity to discriminate between nontumorigenic and

Platinum-based chemotherapeutics are currently used in a wide number of anticancer treatment regimens, despite problems associated with drug toxicity and resistance.1−4 Therefore, new metal-based anticancer complexes should ideally exhibit minimal side effects while providing selectivity and cytotoxicity toward tumor cell lines including chemo-resistant types.5−9 In the past decade half-sandwich piano-stool Ru(II) complexes have emerged as potential alternatives to platinum-based drugs due to a number of interesting features such as redox-accessible oxidation states, biocompatible ligand exchange rates, covalent binding with DNA and/or proteins, and low toxicity.10−18 In this respect, the RAPTA family of compounds [Ru(η6-arene) (PTA)Cl2] (PTA = 1,3,5-triaza-7-phosphaadamantane) represents an exciting metallodrug scaffold massively investigated, as some of them have shown to possess antimetastatic,19,20 antiangiogenic,21,22 and anticancer23 properties in vivo.24 We recently reported several studies on arene-Ru(II) complexes with O,O- and O,N-chelating ligands displaying potent in vitro cytotoxic activity.25−31 In particular, we started to explore the synthesis of some 4-acyl-5-pyrazolone ligands to the (arene)© XXXX American Chemical Society

Received: August 1, 2016

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DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry cancer cells.33 Herein we described further investigations in this area to find delineate structure−activity relationships. We report the synthesis of new neutral and ionic (arene)Ru(II) complexes with diverse HQR acylpyrazolones bearing hydrophobic R groups in the acyl moiety (Scheme 1).

the solid state and in solution; they are highly soluble in most organic solvents but exhibit poor solubility in water. The complexes were characterized by elemental analysis, infrared, UV−visible, 1H and 13C NMR spectroscopies, and electrospray ionization (ESI) mass spectrometry (MS). The conductivity measurements performed in dimethyl sulfoxide (DMSO), values in the range of 3−16 S cm2 mol−1, indicate partial chloride dissociation at room temperature.38 The extent of chloride loss increases slightly with temperature. The IR spectra of 1−5 show the typical shift of the ν(CO) vibrations to lower wavenumbers upon coordination of the acylpyrazolone ligand to the metal ion in the O,O′-bidentate chelating mode.39 Moreover, new medium-to-strong absorption bands appear upon coordination, which may be assigned to ν(Ru−O) and ν(Ru−Cl) vibrations.40,41 The ESI mass spectra of 1−5 in positive ion mode, performed in acetonitrile, provide parent peaks corresponding to a cation fragments [Ru(arene)(QR)]+ and [Ru(arene)(QR)(CH3CN)]+ generated by the loss of the chloride ligand and acetonitrile coordination (Figures S1−S3). The 1H and 13C NMR spectra of 1−5 recorded in CDCl3 display the expected distinct changes in frequency for the resonances of the acylpyrazolone protons and carbon atoms in comparison to the free proligands. The asymmetry of the complexes induces significant modifications to the 1H and 13C NMR signals of the p-cymene moiety in 1, 3, and 5. The 1H NMR spectra of 1 and 3 exhibit a doublet of doublet for the methyl groups of the isopropyl moiety, and the proton resonances attributable to the p-cymene ring are in the range of 5.25−5.53 ppm, which is typical of ruthenium−arene systems.42,43 In the 13C NMR spectrum of 3 six different pcymene ring carbon peaks are observed in the range of 79.1− 99.6 ppm, together with peaks corresponding to two different methyl groups of the isopropyl moiety in the range of 22.4− 22.8 ppm. A similar pattern was observed in the 1H NMR and 13 C NMR spectra of 1 and 5, where, however, two sets of signals are observed. They are presumably due to the presence of two conformers in solution differing in the orientation of the moiety group in the ligand. The 13C NMR spectra of 1 and 5 further confirm the presence of two conformational isomers in solution, with eight different p-cymene ring carbon peaks being observed in the range of 79.2−99.8 ppm (four for each isomers), together with the presence of four different resonances in the range of 20.7−22.7 ppm attributable to the methyl group carbon atoms of the isopropyl moiety. 1H NMR spectra of 1 and 3 were also recorded in d6-DMSO, which showed the appearance of two sets of resonances within 96 h, thus confirming the partial dissociation of the chloride, as suggested by the conductivity values (Figures S4−S7). The UV−vis spectra of proligands and complexes 1−5 were recorded in ethanol and all absorbance values are reported in Table 1. As for previous investigations on other HQR molecules37 the electronic spectra of our proligands display the typical absorptions due to n→π* and π→π* transitions centered at ca. 200, 220, and 262 nm, respectively (Figure S8). Upon coordination to the arene-Ru fragment, essentially the same spectral behavior for the acylpyrazolonato moieties is observed, with an additional absorption arising above 300 nm, attributed to a Ru(4d6)→π* charge transfer transition (Figure S9).27 The chloride ligand in derivatives 1−5 can be easily removed by reaction with AgPF6 in acetone and replaced by the watersoluble phosphine, 1,3,5-triaza-7-phosphaadamantane, affording [Ru(cym)(Qcy)(PTA)][PF6] (6), [Ru(hmb)(Qcy)(PTA)]-

Scheme 1. Proligands HQR Employed in This Work

The main reason to choose these hydrophobic substituents was based on previous findings that complexes bearing a hexamethylbenzene on ruthenium, with respect to those with benzene or cymene, displayed a superior ability to stimulate caspase activity, with consequent DNA fragmentation, accumulation of pro-apoptotic proteins, and down-regulation of antiapoptotic proteins, leading ultimately to cancer cell death.34 Herein, we investigate also the effect of a PTA ligand, in place of a chloride ligand, on the cytotoxicity of the complexes.



RESULTS AND DISCUSSION The proligands (Scheme 1) 3-methyl-1-phenyl-4-cyclohexyl-5pyrazolone (HQcy), 3-methyl-1-phenyl-4-stearoyl-5-pyrazolone (HQC17), and 3-methyl-1-phenyl-4-adamantyl-5-pyrazolone (HQAD) were prepared as previously described.36,37 Complexes 1−5 were prepared in high yield by reacting [Ru(arene)Cl2]2 (arene = cym; hmb) and the appropriate deprotonated ligand in methanol (Scheme 2). All complexes are air-stable in both Scheme 2. Synthesis of Complexes 1−5

B

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Table 1. Wavelengthsa of Maximum Absorbance and Extinction Coefficients for Proligands HQR and (Arene)Ru(II) Complexes 1−10 in Ethanol λ, nm (∈, mol−1 L cm−1) compound Cy

HQ HQC17 HQAd 1 2 3 4 5 6 7 8 9 10 a

IL π→π*

IL n→π* 200 203 201 200 202 201 204 207 203 207 202 203 205

(24 395) (9839) (26 965) (24 976) (27 396) (12 328) (22 550) (29 776) (19 684) (29 682) (14 304) (10 896) (21 781)

233 (14 415) 233 (4543) 234 (15 471)

232 220 220 220 220 220

(23 698) (14 607) (25 402) (10 580) (7869) (17 239)

Ru(4d6)→π* 264 262 266 247 258 256 258 259 257 256 254 255 255

(16 809) (8882) (14 661) (10 804) (11 662) (6640) (11 721) (21 292) (8515) (15 367) (6288) (4414) (9788)

280 294 296 296 298 303 305 303 313 303

(6611) (5584) (3186) (5524) (12 358) (3902) (5855) (2806) (1617) (4339)

IL ≅ intraligand.

[PF6] (7), [Ru(cym)(QC17)(PTA)][PF6] (8), [Ru(hmb)(QC17)(PTA)][PF6] (9), and [Ru(cym)(QAD)(PTA)][PF6] (10), as depicted in Scheme 3.

difference after chloride displacement by PTA being a slight shift of the π→π* transition from ca. 260 to 255 nm (Figure S13). All spectra remain unchanged after a week, thus confirming that complexes 1−10 are stable in ethanol. In D2O−d6-DMSO (80−20% for 6 and 50−50% for 8, due to its lower solubility in water) the 31P NMR spectra of 6 and 8 remain unchanged after 96 h (Figures S14−S15). The stability of 6 and 8 was also determined under pseudopharmacological conditions in 5 mM NaCl solution (simulating the low intracellular NaCl concentration in cells) and in 100 mM NaCl solution (approximating the higher NaCl levels in blood plasma). Solutions of 6 and 8 (c = 2.0 mM) in aqueous NaCl (c = 5 mM or 100 mM in D2O containing 20% d6-DMSO for 6 and 50% d6-DMSO for 8) were prepared and maintained at 37 °C for 96 h and monitored by 31P NMR spectroscopy. Compounds 6 and 8 are stable in 5 mM NaCl solution in the monitored period (Figure 1a,b), whereas in 100 mM aqueous NaCl solution a new peak at −34.4 emerges, corresponding to [Ru(cym)(PTA)Cl2].47 Additional peaks in the range from −13 to −19 ppm appear after 72 h for 6 and 48 h for 8, due to PTA dissociation from the starting complexes. In particular, the resonance at −19 ppm becomes the most intense in the case of 8, probably due to adduct formation between PTA and DMSO.48 To get a better idea of the possible other species observed in solution, we recorded time-evolved ESI-MS spectra for complexes 6 and 8 in a 30:70 mixture of methanol/aqueous NaCl (c = 100 mM) at regular intervals over 4 d. Differently to what was previously observed by Hartinger for analogous species,49,50 here all the spectra remain unchanged within this period, displaying only the intact cationic fragments [Ru(cym)(QR)(PTA)]+ (Figures S16−S19). The molecular structures of 1−7 and 10 were confirmed by single-crystal X-ray structure analysis (see Experimental Section and Supporting Information for details of the data collections and structure refinements in Table S1a,b). The molecular structures of 1−3, 5−7, and 10 are shown in Figure 2, and the structure of 4 is presented in Figure 3. All complexes show a typical piano-stool geometry with central chirality at the metal, and despite having in some cases enantiomerically pure crystals, no attempt to separate and isolate the two enantiomers was performed. From Figure 2 it is apparent that the acylpyrazolone ligands (QR) are always O,O′ coordinated to the ruthenium ion, forming a six-membered

Scheme 3. Synthesis of Complexes 6−10

Complexes 6−10 are soluble in alcohols, acetone, acetonitrile, DMSO, and chlorinated solvents. In DMSO, they display conductivity values within the range typical of 1:1 electrolytes.38 The IR spectra of the ionic derivatives contain a strong, sharp absorption at 832 and 834 cm−1 due to the PF6− counteranion.44,45 The 1H NMR spectra in CDCl3 of 6−10 show the expected signals due to the coordinated arene ring, acylpyrazolone, and PTA ligands, two types of methylene protons being present for the coordinated PTA ligand carbon atoms. The 31P NMR spectra in CDCl3 of 6, 8, and 10, containing the p-cymene moiety, display a singlet at ca. −30 ppm, whereas 7 and 9 with the hexamethyl moiety show a resonance at ca. −37 ppm, in the range typical of related compounds and in accordance with the existence of only one species in solution.46 Moreover, the 31P NMR spectra of 6−10 contain a septet at ca. −143 ppm, characteristic of the PF6− anion. In the positive ESI mass spectra of 6−10 performed in acetonitrile, peaks arising from the cationic fragment [(arene)Ru(QR)(PTA)]+ are observed as the predominant, high mass species (Figures S10−S12). The UV−visible spectra of 6−10 also display similar features to those of 1−5, the main C

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Figure 1. 31P NMR spectra of 6 (a) and 8 (b) in 5 mM (left) and in 100 mM (right) aqueous NaCl solution.

metallacycle. Delocalization within the metallacycle is observed, with the Ru−O and O−C bond lengths being almost equivalent (see Table 2). Ranging from 1.259(5) to 1.284(5) Å these C and O bond lengths are intermediate between those of CO single and CO double bonds. Replacement of the chloride ligand (compounds 1−5) with PTA (compounds 6, 7, and 10) slightly increases (+ 0.05 Å) the distance between the centroid of the arene ligand and the ruthenium ion. Overall, the geometrical parameters are typical of such compounds.34 The crystals of 2 and 4 contain two independent molecules (named A and B) per unit cell. The main geometrical difference in these molecules involves the angle between the plane of the pyrazolone moiety and the plane of the phenyl ring (see Table 2). These two planes can be almost coplanar, such as in molecule A of compound 4, where an angle of only 1.51° is observed. However, it can also exceed 30°, as in compounds 5, 6, and 10, thus suggesting the possibility of free rotation of the phenyl ring in solution. In the crystal packing of complexes 1− 5, the chloride interacts weakly with neighboring complexes, forming several C−H···Cl interactions. Similar weak C−H···F interactions are observed in the salts 6, 7, and 10. Interestingly, on the one hand, in the p-cymene derivatives, complexes 1, 3, 5, 6, and 10, π-stacking interactions take place between the pcymene and the phenyl ring of the acylpyrazolone ligand. On the other hand, in the hexamethylbenzene derivatives (2, 4, 7), the arene shows π-stacking interaction with a symmetry-related

Figure 2. Molecular structures of compounds 1−3, 5−7, and 10.

Figure 3. Dimeric structure observed in the crystal packing of 4.

hexamethylbenzene unit. In compound 4, as well as in compound 3, the long alkyl chain of the complex interacts with the alkyl chain of a neighboring complex to form a headto-tail dimeric structure in the solid state (see Figure 3). To gain further insights into the arene−ruthenium− acylpyrazolone system, compounds 1c−7c and 10c (those with X-ray determination; label c was used for calculated structures; coordinates of the optimized compounds are reported in Table S2) were analyzed using density functional theory (DFT). Additionally, [Ru(arene)(acac)(Cl)] and [Ru(arene)(acac)(PTA)]+ (arene = cym, hmb) were also computed for comparative purposes. Geometry optimizations were performed with the actual compounds, without symmetry D

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Inorganic Chemistry Table 2. Selected Geometrical Parameters of 1−7 and 10 compound distances (Å) Ru-centroid Ru−O1 Ru−O2 Ru−Cl Ru−P N1−N2 O1−C O2−C angles (deg) planepyrazoloneplanephenyl O1−Ru−O2 Cl−Ru−O1 Cl−Ru−O2 P−Ru−O1 P−Ru−O2 a

1

2a

4a

3 A

B

A

B

5

6

7

10

1.699 2.088(2) 2.105(2)

1.703 2.096(2) 2.111(2)

1.707 2.080(7) 2.085(6)

1.650 2.092(1) 2.096(1) 2.421(1)

1.653 2.089(3) 2.102(3) 2.426(1)

1.654 2.093(3) 2.103(3) 2.425(1)

1.650 2.089(4) 2.106(4) 2.416(2)

1.649 2.099(3) 2.109(3) 2.418(1)

1.646 2.096(3) 2.111(3) 2.419(1)

1.644 2.078(6) 2.087(7) 2.406(2)

1.403(2) 1.276(2) 1.267(2)

1.400(5) 1.265(5) 1.259(5)

1.398(5) 1.283(5) 1.279(5)

1.416(7) 1.278(7) 1.267(7)

1.401(5) 1.276(5) 1.260(5)

1.402(5) 1.284(5) 1.267(5)

1.393(11) 1.274(11) 1.271(11)

2.3182(6) 1.403(3) 1.280(3) 1.275(3)

2.3245(8) 1.403(3) 1.281(3) 1.270(3)

2.344(3) 1.405(10) 1.271(9) 1.274(10)

5.60

15.06

9.01

16.88

1.51

5.19

36.70

34.53

14.95

30.06

88.27(5) 85.78(5) 82.41(4)

87.65(12) 86.20(8) 85.00(10)

87.50(13) 85.86(9) 86.84(8)

88.50(16) 84.41(12) 84.55(12)

87.50(11) 85.10(9) 87.72(9)

88.02(12) 84.55(9) 87.41(9)

84.9(3) 86.9(2) 84.6(2)

88.56(6)

87.79(7)

85.6(2)

87.02(5) 86.20(5)

83.14(6) 87.50(6)

89.32(19) 84.9(2)

The crystals of 2 and 4 contain two independent molecules per unit cell.

The selected combination of method and basis sets provides a good structural description of the ruthenium complexes based on a comparison of the structural parameters of the optimized structures with those experimentally determined by X-ray diffraction (see Table S3 for the comparison of selected complexes). The bond distances and angles fit within 0.02 Å and 3°, respectively, with the Ru-arene centroid bond distance being slightly overestimated. The overall structural conformations of the arene ligand and the acylpyrazolone moiety are also well-described. The same occurred with the optimized [Ru(arene)(acac)(Cl)] and [Ru(arene)(acac)(PTA)]+. Their structural parameters fit quite well with respect to the X-ray characterized [Ru(cym)(acac)(Cl)]42 and [Ru(arene)(acac)(PTA)]X (X = BPh4, BF4) compounds.51 In compounds 1c−7c and 10c the d orbitals were mainly found in the highest occupied molecular orbital (HOMO−2) to lowest unoccupied molecular orbital (LUMO+1), in agreement with the general d6-(arene)ML3 formulation. Concerning the energies of the frontier orbitals, only small differences were found for complexes 1c−7c and 10c (see Table S4), although some general trends can be deduced. For example, the substitution of chloride by PTA produces a HOMO stabilization of ca. 0.1 eV with a minor diminution of the HOMO−LUMO gap of ∼0.01 eV. In addition, a slight HOMO stabilization (∼0.01 eV) is observed by the formal substitution of hexamethylbenzene by the p-cymene ligand, with a concomitant diminution of the HOMO−LUMO gap. In relation to the three different R groups at the 4 position of the acylpyrazolone (QR), no substantial changes were observed in the frontier orbitals energies, in agreement with the similar alkyl nature of the R substituent. The HOMO of all the complexes (1c−7c and 10c, Figure S24) has an important contribution of the π part of the acylpyrazolonate ligand, as previously described for related arene−Ru−acylpyrazolonate derivatives.17 This π contribution displays at the HOMO an antibonding combination with the dz2 ruthenium orbital. Additionally, the HOMO shows a π donor antibonding combination from the chloride ligand for 1c−4c, whereas no contribution to the HOMO from the phosphorus atom is observed in the PTA derivatives, 5c−7c, and 10c. Some minor differences were

restrictions, starting from the X-ray structure parameters for 1c−7c and 10c. The computed optimized structures of these compounds are shown in Figure 4 (those of the acac derivatives are collected in the Supporting Information as Figures S20− S23).

Figure 4. Optimized structures of 1c−7c and 10c. E

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•••

•••

ν(C O), ν(CN), ν(C C), and ν(C N) vibrational modes of the QR ligands. To rationalize the observed stability trends of these ruthenium complexes, the bond dissociation energies (BDEs) corresponding to the Ru−Cl and Ru−P bonds were calculated for complexes 1c−7c, 10c, and the related acac derivatives. The BDE for the Ru−Cl bond is higher than the Ru−P one in all the cases (see computational details and Table S6), and this trend is also followed by the computed Mayer bond indexes of Ru−Cl and Ru−P bonds, which show, respectively, values of 0.91 and 0.75 (see Table S7). These gas-phase BDEs are of the same order of magnitude as those reported for related complexes.52 In spite of these BDE values, the hydrolysis of chloride ligand has been previously observed in [Ru(benzene)(PTA)(Cl)2] complex without any evidence of PTA dissociation.53,54 The behavior of 6 and 8 in aqueous NaCl solution (Figure 1) resembles that previously observed for [Ru(cym)(acac)(PTA)]BF 4 complex, in which acac dissociation occurred.51 In fact, the Ru−O Mayer bond indexes for acac and QR ligands are similar (mean values of 0.50 and 0.48, respectively, see Table S7), and they are lower than the Ru−P one (mean value of 0.75, Table S7). Therefore, the formation of [Ru(cym)(PTA)Cl2], detected by 31P NMR, from 6 and 8 in aqueous NaCl solution, could be explained through a stepwise process in which there is a QR ligand dissociation, as occurred with acac,51 and then chloride coordination from the NaCl solution.

detected in the HOMO composition of the neutral [Ru(arene)(QR)(Cl)] compounds, 1c−5c, with respect to the cationic complexes, [Ru(arene)(QR)(PTA)]+ 6c, 7c, and 10c. For this reason, we decided to analyze the frontier orbitals of the acylpyrazolone ligands by optimizing the HQR compounds without symmetry restrictions at the same level of theory. The optimized structures are shown in Figure S25, and selected MOs of HQCy are depicted in Figure 5 along with the HOMOs

Figure 5. Comparison of selected MOs of the HQCy ligand with the HOMOs of 1c and 10c (isovalue = 0.03).

of complexes 1c and 10c containing the QCy ligand. From an inspection of these orbitals it is evident that the π contribution of the acylpyrazolonate in the HOMO of the cation 10c comes almost exclusively from the HOMO of the HQCy, whereas for neutral 1c, both the HOMO and HOMO−1 of HQ Cy contributes to the acylpyrazolonate π part of the HOMO. Additional evidence for the appropriateness of the selected method-basis set combination stems from a comparison of the calculated and the experimental IR spectra. In the range of 1650−1350 cm−1, where the acylpyrazolonate ligands give strong IR absorptions, the computed IR bands fit very well with the experimental ones. Table S5 collects the IR absorptions for 1, as an example, and the corresponding assignments to the



CYTOTOXICITY STUDIES The ligands and complexes were tested for their cytotoxicity to human ovarian A2780 carcinoma cells and the A2780cisR variant with acquired resistance to cisplatin as well as against noncancerous human embryonic kidney (HEK293) cells. IC50 values of the compounds were determined after exposure of the cells to the compounds for 72 h using the MTT assay (see Table 3). All complexes tested induced a dose- and cell-dependent decrease in cell viability, with the parent ligands exerting only

Table 3. Cytotoxicity (IC50, μM) of the Ligands and Complexes 1−10 Following Exposure for 72 h to Nontumorigenic Human Embryonic Kidney (HEK293) Cells and Ovarian Carcinoma A2780 and A2780cisR (Cisplatin Resistant) Cell Linesa compound

HEK293

A2780

A2780cisR

HqCy HqC17 HqAd 1 2 3 4 5 6 7 8 9 10 [Ru(cym) (acac)Cl]55 [Ru(cym) (acac) (Pta)]Bf451 Rapta-C46 cisplatin

>100 >100 89 ± 18 123 ± 3 81 ± 7 54 ± 11 4.9 ± 1.0 85 ± 6 42 ± 2 45 ± 5 1.56 ± 0.21 1.25 ± 0.15 92 ± 5

69.6 ± 5.5 >100 52 ± 5 87 ± 10 31 ± 4 29 ± 4 >5b 36 ± 2 32 ± 1 34 ± 4 1.02 ± 0.09 0.82 ± 0.09 41 ± 2 19 15 230 1.0 ± 0.2

>100 >100 53 ± 12 97 ± 3 36 ± 4 32 ± 4 >5b 34 ± 5 42 ± 3 35 ± 3 0.94 ± 0.13 0.64 ± 0.05 109 ± 18

>1000 7.3 ± 0.6

270 25 ± 1

a RAPTA-C, cisplatin, and related Ru complexes are included for comparison. bThe IC50 is above the limit of solubility of the compound in the final culture medium.

F

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minor effects. Compared to analogous mononuclear arene Ru(II) acylpyrazolonate complexes previously reported,20 all compounds, except complexes 4, 8, and 9, which carry the hydrophobic HQC17 ligand, display a similar activity in the medium micromolar range against A2780 and A2780cisR cancer cell lines with modest selectivities for cancer cells over the model-healthy (HEK293) cell line. The cytotoxicities of the complexes are correlated to the hydrophobicity of the HQcy, HQAD, and HQC17 ligands; that is, the complexes with the HQC17-derived ligand are the most cytotoxic. On the basis of calculated values for the ligands,56 the log P values of the HQcy, HQAD, and HQC17 ligands are 2.64, 3.00, and 8.37, respectively; the most cytotoxic compounds in each series corresponds to those with the HQC17 ligand, that is, 3 and 4 and 8 and 9. The difference in hydrophobicity of the arene ligands, log P(cym) = 3.19 and log P(hmb) = 3.72, is comparatively small, and yet, those with the more hydrophobic hmb ligand in each pair are the most cytotoxic. Moreover, precipitation of 4 was even encountered during the cell tests illustrating the highly hydrophobic nature of this complex. Exchanging the cymene ligand by the hmb ligand leads to an activity increase for the Cl complexes, however, not for the PTA complexes. Notably, the combination of the HQC17 and PTA ligands resulted in the most cytotoxic compounds (8 and 9) for all cell lines tested with a comparable potency as cisplatin for A2780 cells, equally cytotoxic to both the A2780 and the cisplatin-resistant A2780cisR cancer cell lines and comparable to related ruthenium(II) arene complexes bearing both a PTA ligand and a long hydrophobic chain.54 The strong cytotoxicity of 8 and 9 is particularly remarkable, as they can be considered as a prodrug that generates the same species as RAPTA-C inside a cell, and RAPTA-C is essentially not cytotoxic.57 The comparatively slow hydrolysis of 8 and 9 relative to RAPTAC and the presence of the long hydrophobic chain presumably both contribute to the high cytotoxicity observed. Moreover, the high cytotoxicity cannot be attributed to the liberated, the free acylpyrazolonate ligand, which displays only a modest activity. In contrast, 1−5 do not generate the same type of active species as RAPTA-C and 6−10 and therefore probably operate via a different mechanism.

Article

EXPERIMENTAL SECTION

Materials and Methods. The dimers [Ru(arene)Cl2]2 (arene = cym or hmb) were purchased from Aldrich. The acylpyrazolone ligands HQcy, HQC17, and HQAD were synthesized using literature methods.17 All other materials were obtained from commercial sources and were used as received. IR spectra were recorded from 4000 to 30 cm−1 on a PerkinElmer Frontier FT-IR instrument. 1H, 13C, and 31P NMR spectra were recorded on a 400 Mercury Plus Varian instrument operating at room temperature (400 MHz for 1H, 100 MHz for 13C, and 161 MHz for 31P). Referencing is relative to tetramethylsilane (1H and 13C) and 85% H3PO4 (31P). Positive and negative ion electrospray mass spectra were obtained on a Series 1100 MSI detector HP spectrometer using methanol as the mobile phase. Solutions (3 mg/ mL) for ESI-MS were prepared using reagent-grade acetonitrile. Masses and intensities were compared to those calculated using IsoPro Isotopic Abundance Simulator, version 2.1.28. Melting points are uncorrected and were recorded on an STMP3 Stuart scientific instrument and on a capillary apparatus. Samples for microanalysis were dried in vacuo to constant weight (20 °C, ca. 0.1 torr) and analyzed on a Fisons Instruments 1108 CHNS-O elemental analyzer. Electrical conductivity measurements (ΛM, reported as S cm2 mol−1) of DMSO solutions of the complexes were recorded using a Eutech Instruments conductimeter CON2700 at room temperature and at 37 °C. X-ray Crystallography. Diffraction data of 1, 2, 3, 4, 5, 6, and 7 were measured at low temperature [120(2) K] using Mo Kα radiation on a Bruker APEX II CCD diffractometer equipped with a κ geometry goniometer. The data sets were reduced by EvalCCD58 and then corrected for absorption.59 The solutions and refinements were performed by SHELX.60 The crystal structures were refined using fullmatrix least-squares based on F2 with all non-hydrogen atoms anisotropically defined. Hydrogen atoms were placed in calculated positions by means of the “riding” model. A crystal of 10 was mounted on a Stoe Image Plate Diffraction system equipped with a ϕ circle goniometer, using Mo Kα graphite monochromatic radiation (λ = 0.710 73 Å) with ϕ range of 0−180°. The structure was solved by direct methods using the program SHELXS-97, while the refinement and all further calculations were performed using SHELXL-97.60 The H atoms were included in calculated positions and treated as riding atoms using the SHELXL default parameters. The non-H atoms were refined anisotropically, using weighted full-matrix least-squares on F2. Additional crystallographic information is available in the Supporting Information. Cell Culture and Cytotoxicity Determinations. Human A2780 and A2780cisR ovarian carcinoma cells were obtained from the European Centre of Cell Cultures (ECACC, Salisbury, U.K.) and HEK-293 cells were obtained from ATCC (Sigma, Switzerland). A2780 and A2780cisR cells were routinely grown in RPMI 1640 medium with GlutaMAX containing 5% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37 °C and 5% CO2. HEK-293 cells were grown in DMEM GlutaMax medium containing 5% FBS and 1% antibiotics (penicillin and streptomycin) at 37 °C and 5% CO2. To keep the A2780cisR cells resistant to cisplatin the cells were monthly treated by adding 2 μM cisplatin to the culture medium for one passage. Cytotoxicity was determined using the PrestoBlue Cell Viability Assay. The cells were seeded in 96-well plates (10 000 cells per well) and grown for 24 h in complete medium. For each testing, compounds were freshly prepared as DMSO stock solution, then dissolved in the culture medium and immediately serially diluted to the appropriate concentration, to give a final DMSO concentration of 0.5% v/v. A 100 μL portion of drug solution was added to each well, and the plates were incubated at 37 °C for 72 h. Following drug exposure, 20 μL of PrestoBlue Cell Viability Reagent (Thermo Fisher Scientific, Switzerland) was added to the cells and incubated for at least 2 h. The fluorescence of each well (96-well plates) was monitored at ex/em 560/590 nm using a multiwell plate reader (Molecular Devices, U.K.), and the percentage of surviving cells was calculated from the ratio of fluorescence of treated to untreated cells. The IC50 values for the inhibition of cell growth were determined by fitting the



CONCLUSIONS Ruthenium(II)−arene complexes containing acylpyrazolones bearing aliphatic groups in the acyl moiety were prepared to evaluate the influence of aliphatic substituents on their in vitro anticancer activity. The crystal structures of neutral [Ru(arene)(QR)Cl] and cationic [Ru(arene)(QR)(PTA)]+ complexes display a typical piano-stool geometry with acylpyrazolone ligands always O,O′ coordinated to the ruthenium ion. Replacement of the chloride ligand with PTA slightly increases the distance between the centroid of the arene ligand and the ruthenium ion. From DFT studies, substitution of chloride by the PTA ligand to give the corresponding cations affords a HOMO stabilization with a minor diminution of the HOMO− LUMO gap. Similarly, a slight HOMO stabilization is observed by the formal substitution of hexamethylbenzene by the cymene moiety, with a concomitant diminution of the HOMO−LUMO gap. The proligands were not cytotoxic; nevertheless, some of the complexes are reasonably cytotoxic, and, interestingly, the combination of the HQC17 and PTA ligands resulted in the most cytotoxic compounds (8 and 9) for all cell lines tested with a comparable potency to cisplatin for A2780 cells. G

DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

H of Qcy), 7.18t, 7.37t, 7.90d (5H, Harom of Qcy). 13C NMR (CDCl3, 298 K): δ 15.5s (C6(CH3)6), 17.2s (C3-CH3), 26.0s, 26.2s, 26.3s, 30.1s, 31.0s, 45.5s (6C, Calif of Qcy), 89.8s (C6(CH3)6), 104.6s (C4), 120.7s, 121.0s, 124.9s, 128.4s, 129.2s (6C, Carom of Qcy), 147.7s (C3CH3), 196.8s (CO). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 547 [100] [Ru(hmb)(Q cy )] + , 588 [45] [Ru(hmb)(Qcy ) (CH3CN)]+. [Ru(cym)(QC17)Cl] (3). The synthesis was performed as for 1 using HQC17. 3 is soluble in acetone, DMSO, and chlorinated solvents. mp 88−90 °C. Anal. Calcd for C38H57ClN2O2Ru: C, 64.25; H, 8.09; N, 3.94. Found: C, 64.30; H, 8.14; N, 3.78%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 8 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 10 S cm2 mol−1. IR (cm−1): 3041w, 2957m, 2919s 2852s ν(Carom−H), 1605s, 1588vs,1578vs ν(CO), 1536m, 1499w, 1468s, 1439m ν(C C, CN, C−N), 1371m, 1074s, 805m, 752vs, 623s, 660m, 512m, 388m ν(Ru−O), 283m ν(Ru−Cl). 1H NMR (CDCl3, 298 K): δ 0.88t (3H, H of QC17, 3J = 6.8 Hz), 1.25m and 1.71m (30H, H of QC17), 1.35dd (6H, CH3−C6H4−CH(CH3)2, 3J = 2.4 Hz), 2.25s (C3−CH3), 2.32s (3H, CH3-C6H4−CH(CH3)2), 2.70m (2H, H of QC17), 2.95 sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.8 Hz), 5.28m (2H, CH3−C6H4− CH(CH3)2), 5.53m (2H, CH3−C6H4−CH(CH3)2), 7.17t, 7.36t 7.88d, (5H, Harom of QC17). 13C NMR (CDCl3, 298 K): δ 17.3s (C3-CH3), 18.1s (CH3−C6H4−CH(CH3)2), 22.5s (CH3−C6H4−CH(CH3)2), 22.8s (CH3−C6H4−CH(CH3)2), 14.2s, 22.4s, 26.1s, 29.5s, 29.6s, 29.7s, 29.7s, 29.8s, 32.0s, 38.5s (17C, Calif of QC17), 30.9s (CH3−C6H4CH(CH3)2), 79.1s, 79.3s, 82.5s, 82.s, 96.6s, 99.6s (CH3-C6H4− CH(CH3)2), 105.7s (C4), 121.1s, 125.2s, 128.5s, 138.9s (6C, Carom of QC17), 148.2s (C3-CH3), 162.6s and 193.6s (CO). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 675 [100] [Ru(cym)(QC17)]+, 716 [45] [Ru(cym)(QC17) (CH3CN)]+. [Ru(hmb)(QC17)Cl] (4). The synthesis was performed as for 3 using [Ru(hmb)Cl2]2. 4 is soluble in acetone, DMSO, and chlorinated solvents. mp 188−189 °C. Anal. Calcd for C40H61ClN2O2Ru: C, 65.06; H, 8.33; N, 3.79. Found: C, 65.13; H, 8.42; N, 3.76%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 3 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 4 S cm2 mol−1. IR (cm−1): 2919vs, 2850s ν(Calif− H), 1603s, 1590s, 1577s ν(CO), 1533s, 1477s, 1439s, 1396w, 1369s, 1396s ν(CC, CN, C−N), 1172w, 1075vs, 913w, 804w, 765vs, 511m, 388w ν(Ru−O), 285w ν(Ru−Cl). 1H NMR (CDCl3, 298 K): δ 0.87t (3H, H of QC17, 3J = 6.8 Hz), 1.26m, 1.78m, 2.74m (32H, H of QC17), 2.14s (18H, C6(CH3)6), 2.47s (C3−CH3), 7.21t, 7.40t, 7.90d (5H, Harom of QC17). 13C NMR (CDCl3, 298 K): δ 15.6s (6C, C6(CH3)6), 16.0s (C3-CH3), 14.3s, 15.9s, 16.1s, 17.6s, 22.9s, 24.9s, 26.2s, 29.6s, 29.6s, 29.6s, 29.7s, 29.7s, 29.8s, 29.9s, 32.1s, 38.7s, 39.1s (17C, Calif of QC17), 89.8s (6C, C6(CH3)6), 121.0s, 125.0s, 126.7s, 128.5s, 129.3s, 139.1s (6C, Carom of QC17), 148.3s (C3-CH3), 193.3s (CO). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 703 [100] [Ru(hmb)(QC17)]+, 744 [25] [Ru(hmb)(QC17) (CH3CN)]+. [Ru(cym)(QAD)Cl] (5). The synthesis was performed as for 1 using HQAD. 5 is soluble in acetone, acetonitrile, DMSO, and chlorinated solvents. mp 141−143 °C. Anal. Calcd for C31H37ClN2O2Ru: C, 61.42, H, 6.15, N, 4.62. Found: C; 61.63, H; 6.22, N, 4.30%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 12 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 12 S cm2 mol−1. IR (cm−1): 2905m, 2849m ν(Carom−H), 1591s, 1565vs ν(CO), 1495vs, 1455m, 1412s ν(CC, CN, C− N), 1336m, 1308s, 1028vs, 984s, 876m, 765vs, 695s, 622s, 515m, 387m ν(Ru−O), 286m ν(Ru−Cl). 1H NMR (CDCl3, 298 K): δ 1.36d (6H, CH3−C6H4−CH(CH3)2, 3J = 6.8 Hz), 1.76−2.09m (15H, H of QAD), 2.26s (C3−CH3), 2.58s (3H, CH3-C6H4−CH(CH3)2), 2.95sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.9 Hz), 5.27m, (2H, CH3− C6H4−CH(CH3)2), 5.49m (2H, CH3−C6H4−CH(CH3)2), 7.18t, 7.36t,7.82d, (5H, Harom of QAD). 13C NMR (CDCl3, 298 K): δ 18.5s (C3-CH3), 19.1s (CH3−C6H4−CH(CH3)2), 22.3s, 22.5s, 22.7s (CH3−C6H4−CH(CH3)2), 30.8s (CH3−C6H4-CH(CH3)2), 20.7, 22.2, 28.2, 28.7s, 31.0s, 36.7s, 36.7s, 37.8s, 38.8s, 45.6s (10C, Calif of QAD), 79.3s, 79.6s, 80.7s, 81.5s, 82.3s, 82.4s, 96.5s, 99.8s (CH3-C6H4− CH(CH3)2), 121.29s, 121.9s, 125.5s, 126.8s, 128.5s, 129.2s (6C, Carom of QAD), 145.7s (C3-CH3), 165.3s and 200.8s (CO). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 571 [80] [Ru(cym)(QAD)]+, 612 [100] [Ru(cym)(QAD) (CH3CN)]+.

plot of the logarithmic percentage of surviving cells against the logarithm of the drug concentration using a linear regression function. Mean values and standard deviations computed from three independent experiments, each comprising four microcultures per concentration level, are reported in Table 1. Log P Value Determination. The log P values were calculated using a method available at http://www.cheminfo.org. This predicting engine distinguishes 368 atom types, which are composed of various properties of the atom itself (atomic number and ring membership) and its direct neighbors (bond type, aromaticity state, and encoded atomic number). Computational Details. The electronic structure and geometries of [Ru(arene)(QR)(Cl)], 1c−5c, and [Ru(arene)(acac)(Cl)], cations [Ru(arene)(QR)(PTA)]+, 6c, 7c, 10c, [Ru(arene)(acac) (PTA)]+ (arene = cym, hmb), and proligands HQR (R = Cy, C17, AD) were computed using DFT at the B3LYP level.61,62 The Ru atom was described with the LANL2DZ basis set,63,64 while the 6-31G* basis set was used for the other atoms. Molecular geometries of the ruthenium complexes 1c−7c, 10c were optimized starting from the crystallographic coordinates. Frequency calculations were performed at the same level of theory to identify all of the stationary points as minima (zero imaginary frequencies). The computed IR spectra were scaled by a factor of 0.96.65,66 Bond dissociation energies were calculated from the difference in the optimized energies of the ground states of the unsaturated species ([Ru(arene)(QR)]+ or [Ru(arene)(acac)]+) and the dissociated ligands (Cl− and PTA) and those of the corresponding ruthenium complexes. The DFT calculations were performed using the Gaussian 09 suite of programs.67 The Cartesian coordinates of all optimized compounds and the frontier orbital energies of ruthenium complexes are reported in the Supporting Information. Syntheses and Characterization. [Ru(cym)(Qcy)Cl] (1). To the proligand HQcy (185.0 mg, 0.65 mmol) dissolved in methanol (20 mL), KOH (36.6 mg, 0.65 mmol) was added. The mixture was stirred for 1 h at room temperature, and then [Ru(cym)Cl2]2 (200.0 mg, 0.33 mmol) was added. The resulting solution was stirred under reflux for 24 h. The solvent was removed under reduced pressure, and dichloromethane (10 mL) was added; the mixture was filtered to remove potassium chloride. The solution was concentrated to ca. 2 mL and stored at 4 °C affording red crystals (174.0 mg, 0.31 mmol, yield 96%) that are soluble in alcohols, acetone, acetonitrile, DMSO, and chlorinated solvents. mp 214−216 °C. Anal. Calcd for C27H33N2RuClO2: C, 58.53; H, 6.00; N, 5.06. Found: C, 58.32; H, 5.99; N, 4.68%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 11 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 12 S cm2 mol−1. IR (cm−1): 2917m, 2852m ν(Carom−H), 1601s ν(CO), 1589s, 1578s, 1538m, 1455m ν(CC, CN, C−N), 1373s, 1079s, 752vs, 628s, 516m, 468m ν(Ru−O), 274m ν(Ru−Cl). 1H NMR (CDCl3, 298 K): δ 1.36dd (6H, CH3−C6H4−CH(CH3)2, 3J = 1.7 Hz), 1.77m (10H, H of Qcy), 2.25s (C3−CH3), 2.36s (3H, CH3-C6H4−CH(CH3)2), 2.85m (1H, H of Qcy), 2.94sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.9 Hz), 5.25m (2H, CH3−C6H4−CH(CH3)2, 3J = 5.6 Hz), 5.51m (2H, CH3− C6H4−CH(CH3)2, 3J = 5.6 Hz), 7.17t, 7.35t, 7.87d (5H, Harom of Qcy). 13 C NMR (CDCl3, 298 K): δ 17.2s (C3-CH3), 18.2s (CH3−C6H4− CH(CH3)2), 22.1s, 22.4s, 22.7s (CH3−C6H4−CH(CH3)2), 26.0s, 26.2s, 29.1s, 30.0s, 30.4s, 45.6s (6C, Calif of Qcy), 31.0s (CH3−C6H4CH(CH3)2), 79.2s, 79.2s, 82.5s, 82.7s, 86.1s, 87.2s, 96.5s, 99.4s (CH3C6H4−CH(CH3)2), 120.7s, 121.1s, 125.2s, 128.6s, 129.2s, 138.8s (6C, Carom of Qcy), 147.9s (C3-CH3), 197.2s (CO). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 519 [100] [Ru(cym)(Qcy)]+. [Ru(hmb)(Qcy)Cl] (2). The synthesis was performed as for 1 using [Ru(hmb)Cl2]2. 2 is soluble in alcohols, acetone, acetonitrile, DMSO, and chlorinated solvents. mp 245−246 °C. Anal. Calcd for C29H37ClN2O2Ru: C, 59.83; H, 6.41; N, 4.81. Found: C, 59.41; H, 6.42; N, 4.73%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 13 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 16 S cm2 mol−1. IR (cm−1): 3322w, 2919m, 2855w ν(Calif−H), 1604s, 1592s, 1579s ν(CO), 1531m, 1478s, 1457s, 1440s, 1398m, 1373s ν(CC, CN, C−N), 1252w, 1077m, 953w, 815m, 755s, 659s, 468w, 380w ν(Ru−O), 286w ν(Ru−Cl). 1H NMR (CDCl3, 298 K): δ 1.30m, 1.59m, 1.83m (10H, Halif of Qcy), 2.09sbr (18H, C6(CH3)6), 2.39s (C3−CH3), 2.81m (1H, H

DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry [Ru(cym)(Qcy)(PTA)][PF6] (6). AgPF6 (91.3 mg, 0.36 mmol) was added to a solution of 1 (200.0 mg, 0.36 mmol) in acetone. The reaction mixture was stirred for 1 h at room temperature, and PTA (56.7 mg, 0.36 mmol) was added. After 24 h, the mixture was filtered to remove AgCl, the solution was concentrated to ca. 2 mL and stored at 4 °C affording yellow crystals (6, 269.3 mg, 0.33 mmol, yield 91%) that are soluble in alcohols, acetone, acetonitrile, DMSO, and chlorinated solvents. mp > 350 °C. Anal. Calcd for C33H45F6N5O2P2Ru: C, 48.29; H, 5.53; N, 8.53. Found: C, 48.18; H, 5.36; N, 8.16%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 36 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 38 S cm2 mol−1. IR (cm−1): 2934w ν(Carom−H), 1604m, 1593s, 1577s ν(CO), 1533m, 1503m, 1479s, 1459w ν(CC, CN, C−N), 1361s, 1383s, 1242s, 974s, 833vs ν(PF6), 555m, 451m, 387w ν(Ru−O). 1H NMR (CDCl3, 298 K): δ 1.16m (6H, CH3−C6H4−CH(CH3)2), 1.42−1.90m (10H, H of Qcy), 1.93s (C3−CH3), 2.40s (3H, CH3-C6H4−CH(CH3)2), 2.57sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.8 Hz), 2.97m (1H, H of Qcy), 4.12s (6H, PCHAHBN, PTA), 4.49m (6H, NCHAHBN, PTA), 5.72m (2H, CH3−C6H4−CH(CH3)2), 5.86m (2H, CH3−C6H4− CH(CH3)2), 7.23d, 7.39t, 7.72t (5H, Harom of Qcy). 13C NMR (CDCl3, 298 K): δ 17.3s (C3-CH3), 17.5s (CH3−C6H4−CH(CH3)2), 22.2s (CH3−C6H4−CH(CH3)2), 22.3s (CH3−C6H4−CH(CH3)2), 30.9s (CH3−C6H4-CH(CH3)2), 25.8, 26.0, 30.4, 30.5, 30.9, 46.3 (s, 6C, Calif of Qcy), 50.8d (PCH2N, PTA, JCP = 12.9 Hz), 72.9d (NCH2N, PTA, JCP = 6.8 Hz), 86.2s, 88.0s, 88.9s, 90.3s, 97.2s, 103.8s (CH3C6H4−CH(CH3)2), 106.7s (C4), 120.50s, 120.8s, 126.5s, 129.2s, 129.3s, 137.9s (6C, Carom of Qcy), 148.2s (C3), 162.0s and 200.3s (CO). 31P NMR (CDCl3, 298 K): δ −29.38s (PTA), −142.86sept (PF6). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 677 [100] [Ru(cym)(Qcy) (PTA)]+. [Ru(hmb)(Qcy)(PTA)][PF6] (7). The synthesis was performed as for 6 using [Ru(hmb)Cl2]2. 7 is soluble in methanol, acetone, acetonitrile, and DMSO. mp 250 °C. Anal. Calcd for C35H49F6N5O2P2Ru: C, 49.53; H, 5.82; N, 8.25. Found: C, 49.71; H, 5.93; N, 8.17%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 35 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 38 S cm2 mol−1. IR (cm−1): 2927w, 2857w ν(Calif−H), 1600m, 1591m, 1572s ν(CO), 1533m, 1475m, 1442m, 1380m ν(CC, CN, C−N), 1240w, 1103w, 1012m, 977m, 834vs ν(PF6), 692m, 556s. 1H NMR (CD3CN, 298 K): δ 0.91t, 1.30−1.94m (10H, Halif of Qcy), 2.04s (18H, C6(CH3)6), 2.46s (C3−CH3), 3.09m (1H, H of HQcy), 4.07s (6H, PCHAHBN, PTA), 4.45m (6H, NCHAHBN, PTA), 7.31t, 7.49t, 7.91d (5H, Harom of Qcy). 13C NMR (CD3CN, 298 K): δ 15.5s (6C, C6(CH3)6), 16.6s (C3-CH3), 22.6s, 25.8s, 25.9s, 30.4s, 30.6s, 45.8s (6C, Calif of Qcy), 49.1d (PCH2N, PTA, JCP = 12.1 Hz), 72.4d (NCH2N, PTA, JCP = 6.8 Hz), 97.5s (6C, C6(CH3)6), 106.0s (C4), 117.5s, 120.4s, 125.9s, 129.1s, 138.4s (6C, Carom of Qcy), 148.6s (C3-CH3), 162.2 and 200.5s (CO). 31P NMR (CD3CN, 298 K): δ −37.07s (PTA), −143.53sept (PF6). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 704 [100] [Ru(hmb)(Qcy)PTA]+. [Ru(cym)(QC17)(PTA)][PF6] (8). The synthesis was performed as for 6 using HQC17. 8 is soluble in acetone, acetonitrile, DMSO, and chlorinated solvents. mp 161−163 °C. Anal. Calcd for C44H69F6N5O2P2Ru: C, 54.09; H, 7.12; N, 7.17. Found: C, 54.22; H, 7.18; N, 7.04%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 39 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 40 S cm2 mol−1. IR (cm−1): 2923s, 2851s ν(C−H), 1592s, 1575vs, ν(CO), 1537s, 1482s, 1448m, 1421w ν(CC, CN, C−N), 1377m, 1245w, 1077s, 1014s, 974s, 947vs, 832vs ν(PF6), 740m, 556m, 450w, 389w ν(Ru− O). 1H NMR (CDCl3, 298 K): δ 0.89t (3H, H of QC17, 3J = 6.4 Hz), 1.23−1.39m (32H, H of QC17), 1.81s (6H, CH3−C6H4−CH(CH3)2), 1.99s (C3−CH3), 2.43s (3H, CH3-C6H4−CH(CH3)2), 2.55 sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.9 Hz), 4.23s (6H, PCHAHBN, PTA), 4.64m (6H, NCHAHBN, PTA), 5.87m (2H, CH3−C6H4−CH(CH3)2), 5.94m (2H, CH3−C6H4−CH(CH3)2), 7.26t, 7.43t, 7.78d (5H, Harom of QC17). 13C NMR (CDCl3, 298 K): δ 14.1s (C3-CH3), 17.3s (CH3− C6H4−CH(CH3)2), 22.2s (CH3−C6H4−CH(CH3)2), 22.8s (CH3− C6H4−CH(CH3)2), 22.1s, 26.9s, 29.8sbr, 32.0s, 38.7s (17C, Calif of QC17), 30.9s (CH3−C6H4-CH(CH3)2), 51.2d (PCH2N, PTA, 3J = 12.9 Hz), 73.0d (NCH2N, PTA, 3J = 6.9 Hz), 86.8s, 88.3s, 89.1s, 97.7s,

103.9s (CH3-C6H4−CH(CH3)2), 107.5s (C4), 120.5s, 126.5s, 129.1s, 137.9s (6C, Carom of QC17), 148.5s (C3-CH3), 161.8s and 197.1s (CO). 31 P NMR (CDCl3, 298 K): δ −25.79s (PTA), −142.87sept (PF6). ESIMS (+) (CH3CN) (m/z, relative intensity %): 832 [100] [Ru(cym)(QC17) (PTA)]+. [Ru(hmb)(QC17)(PTA)][PF6] (9). The synthesis was performed as for 8 using [Ru(hmb)Cl2]2. 9 is soluble in methanol, acetone, acetonitrile, DMSO, and chlorinated solvents. mp 214−217 °C. Anal. Calcd for C46H73F6N5O2P2Ru: C, 54.97; H, 7.32; N, 6.97. Found: C, 55.21; H, 7.23; N, 6.72%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 38 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 40 S cm2 mol−1. IR (cm−1): 2920m, 2850m ν(Calif−H), 1600w, 1591w, 1573s ν(CO), 1535m, 1478m, 1459w, 1378m ν(CC, CN, C−N), 1282w, 1101w, 947m, 899w, 833vs ν(PF6), 742ws, 556s. 1H NMR (CH3CN, 298 K): δ 0.90t (3H, H of QC17, 3J = 6.8 Hz), 1.29m, 1.74m (30H, H of QC17), 2.17sbr (18H, C6(CH3)6), 2.42s (C3−CH3), 2.83m (2H, H of QC17), 4.06s (6H, PCHAHBN, PTA), 4.44s (6H, N−CHAHBN, PTA), 7.31t, 7.49t, 7.92d (5H, Harom of QC17). 13C NMR (CD3CN, 298 K): δ 13.6s (Calif of QC17), 15.4s (6C, C6(CH3)6), 16.8s (C3-CH3), 22.6s, 25.4s, 29.3s, 29.4s, 29.6m, 31.8s, 38.9s (Calif of Q17), 49.2d (PCH2N, PTA, 3JCP = 13.0 Hz), 72.4d (NCH2N, PTA, 3JCP = 6.9 Hz), 97.6s (6C, C6(CH3)6), 120.3s, 125.9s, 129.4s (Carom of QC17), 149.0s (C3-CH3). 31P NMR (CD3CN, 298 K): δ −36.92s (PTA), −143.52sept (PF6). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 703 [20] [Ru(hmb)(QC17)]+, 861 [100] [Ru(QC17) (hmb) (PTA)]+. [Ru(cym)(QAD)(PTA)][PF6] (10). The synthesis was performed as for 8 using HQAD. 10 is soluble in methanol, acetone, acetonitrile, DMSO, and chlorinated solvents. mp 214−215 °C. Anal. Calcd for C37H49F6N5O2P2Ru: C, 50.91; H, 5.66; N, 8.02. Found: C, 50.73; H, 5.59; N, 8.07%. Λm (DMSO, 298 K, 1 × 10−4 mol/L): 40 S cm2 mol−1. Λm (DMSO, 310 K, 1 × 10−4 mol/L): 40 S cm2 mol−1. IR (cm−1): 2907m, 2857w ν(Calif−H), 1597w, 1555s, 1522m ν(CO), 1492m, 1457m, 1404s ν(CC, CN, C−N), 1379w, 1243s, 1102m, 1014s, 973s, 946s, 915w, 834vs ν(PF6), 764m, 757m, 739s, 692s. 1H NMR (CDCl3, 298 K): δ 1.18d (6H, CH3−C6H4−CH(CH3)2, 3J = 6.8 Hz), 1.79−2.00m (15H, H of QAD), 2.15s (3H, CH3-C6H4− CH(CH3)2), 2.45sept (1H, CH3−C6H4−CH(CH3)2, 3J = 6.9 Hz), 2.69s (C3−CH3), 4.19s (6H, PCHAHBN, PTA), 4.57m (6H, NCHAHBN, PTA), 5.74d (2H, CH3−C6H4−CH(CH3)2, 3J = 6.2 Hz), 5.82d (1H, CH3−C6H4−CH(CH3)2, 3J = 6.2 Hz), 5.88d (1H, CH3−C6H4−CH(CH3)2, 3J = 6.2 Hz), 7.25t, 7.42t, 7.17d, (5H, Harom of QAD). 13C NMR (CDCl3, 298 K): δ 17.8s (C3-CH3), 18.6s (CH3− C6H4−CH(CH3)2), 22.3s (CH3−C6H4−CH(CH3)2), 22.4s (CH3− C6H4−CH(CH3)2), 28.2sbr, 31.0s, 36.4s, 36.7s, 37.9s, 39.2s, 46.7s, 58.6s (10C, Calif of QAD), 51.2d (PCH2N, PTA, 3J = 13.8 Hz), 73.0d (NCH2N, PTA, 3J = 6.8 Hz), 85.6s, 87.6s, 88.8s, 89.8s, 97.6s (CH3C6H4−CH(CH3)2), 121.2s, 126.8s, 129.1s, 129.2s, 137.7s (6C, Carom of QAD), 145.9s (C3-CH3), 164.3s and 204.9s (CO). 31P NMR (CDCl3, 298 K): δ −31.07s (PTA), −142.87sept (PF6). ESI-MS (+) (CH3CN) (m/z, relative intensity %): 669 [100] [Ru(cym)(QAD) (PTA)]+.



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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01861. Mayer index values, bond dissociation energies, experimental and calculated IR absorption values, illustrated optimized structures, illustrated HOMOs, HOMO− LUMO gaps, MO energies, calculated and experimental structural parameters, coordinates of optimized compounds, illustrated calculated optimized structure with calculated structural parameters, crystallographic and structure refinement parameters, positive ESI-MS, NMR, and UV/vis spectra (PDF) Graphical and textual abstract (PDF) X-ray crystallographic information (CIF) I

DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX

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(11) Hartinger, C. G.; Dyson, P. J. Bioorganometallic chemistry-from teaching paradigms to medicinal applications. Chem. Soc. Rev. 2009, 38, 391−401. (12) Singh, A. K.; Pandey, D. S.; Xu, Q.; Braunstein, P. Recent advances in supramolecular and biological aspects of arene ruthenium (II) complexes. Coord. Chem. Rev. 2014, 270, 31−56. (13) Wang, F.; Xu, J.; Wu, K.; Weidt, S. K.; MacKay, C. L.; Langridge-Smith, P. R. R.; Sadler, P. J. Competition between glutathione and DNA oligonucleotides for ruthenium(II) arene anticancer complexes. Dalton Trans. 2013, 42, 3188−3195. (14) Liu, H. K.; Sadler, P. J. Metal Complexes as DNA Intercalators. Acc. Chem. Res. 2011, 44, 349−359. (15) Singh, S. K.; Pandey, D. S. Multifaceted half-sandwich areneruthenium complexes: Interactions with biomolecules, photoactivation, and multinuclearity approach. RSC Adv. 2014, 4, 1819−1840. (16) Kumar, P.; Gupta, R. K.; Pandey, D. S. Half-sandwich arene ruthenium complexes: synthetic strategies and relevance in catalysis. Chem. Soc. Rev. 2014, 43, 707−733. (17) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.; Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H. F.; Ang, W. H.; Dröge, P.; et al. Ligand substitutions between ruthenium−cymene compounds can control protein versus DNA targeting and anticancer activity. Nat. Commun. 2014, 5, 3462. (18) Babak, M. V.; Meier, S. M.; Huber, K. V. M; Reynisson, J.; Legin, A. A.; Jakupec, M. A.; Roller, A.; Stukalov, A.; Gridling, M.; Bennett, K. L.; Colinge, J.; Berger, W.; Dyson, P. J.; Superti-Furga, G.; Keppler, B. K.; Hartinger, C. G. Target profiling of an antimetastatic RAPTA agent by chemical proteomics: Relevance to the mode of action. Chem. Sci. 2015, 6, 2449−2456. (19) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In vitro and in vivo evaluation of ruthenium (II)-arene PTA complexes. J. Med. Chem. 2005, 48, 4161−4171. (20) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Modulation of the metastatic progression of breast cancer with an organometallic ruthenium compound. Int. J. Oncol. 1992, 33, 1281. (21) Nowak-Sliwinska, P.; van Beijnum, J. R.; Casini, A.; Nazarov, A. A.; Wagnieres, G.; van den Bergh, H.; Dyson, P. J.; Griffioen, A. W. Organometallic ruthenium(II) arene compounds with antiangiogenic activity. J. Med. Chem. 2011, 54, 3895−3902. (22) Weiss, A.; Ding, X.; Van Beijnum, J. R.; Wong, I.; Wong, T. J.; Berndsen, R. H.; Dormond, O.; Dallinga, M.; Shen, L.; Schlingemann, R. O.; et al. Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer. Angiogenesis 2015, 18, 233−244. (23) Weiss, A.; Berndsen, R. H.; Dubois, M.; Müller, C.; Schibli, R.; Griffioen, A. W.; Dyson, P. J.; Nowak-Sliwinska, P. In vivo anti-tumor activity of the organometallic ruthenium (II)-arene complex [Ru(η6-pcymene)Cl2(pta)](RAPTA-C) in human ovarian and colorectal carcinomas. Chem. Sci. 2014, 5, 4742−4748. (24) Murray, B. S.; Babak, M. V.; Hartinger, C. G.; Dyson, P. J. The development of RAPTA compounds for the treatment of tumors. Coord. Chem. Rev. 2016, 306, 86−114. (25) Pettinari, R.; Marchetti, F.; Pettinari, C.; Condello, F.; Petrini, A.; Scopelliti, R.; Riedel, T.; Dyson, P. J. Organometallic rhodium(III) and iridium(III) cyclopentadienyl complexes with curcumin and bisdemethoxycurcumin co-ligands. Dalton Trans. 2015, 44, 20523− 20531. (26) Pettinari, R.; Marchetti, F.; Pettinari, C.; Petrini, A.; Scopelliti, R.; Clavel, C. M.; Dyson, P. J. Synthesis, Structure, and Antiproliferative Activity of Ruthenium(II) Arene Complexes with N,O-Chelating Pyrazolone-Based β-Ketoamine Ligands. Inorg. Chem. 2014, 53, 13105−13111. (27) Pettinari, R.; Marchetti, F.; Condello, F.; Pettinari, C.; Lupidi, G.; Scopelliti, R.; Mukhopadhyay, S.; Riedel, T.; Dyson, P. J. Ruthenium(II)−Arene RAPTA Type Complexes Containing Curcumin and Bisdemethoxycurcumin Display Potent and Selective Anticancer Activity. Organometallics 2014, 33, 3709−3715. (28) Antonyan, A.; De, A.; Vitali, L. A.; Pettinari, R.; Marchetti, F.; Gigliobianco, M. R.; Pettinari, C.; Camaioni, E.; Lupidi, G. Evaluation

(CIF) (CIF) (CIF) (CIF) (CIF) (CIF) (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +39 0737402217. (F.M.) *E-mail: [email protected]. Phone: +39 0737402338. (R.P.) 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. CCDC-1490088 (1), 1490089 (2), 1490090 (3), 1490091 (4), 1490092 (5), 1490093 (6), 1490094 (7), and 1490095 (10) contain the supplementary crystallographic data for this paper. These data 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, U.K.; fax: (international) + 44−1223/336−033; E-mail: [email protected]].



ACKNOWLEDGMENTS We thank the Swiss National Science Foundation, EPFL, the Junta de Andalucia,́ and the Univ. of Camerino for financial support. A.G. thanks the Centro de Servicios de Informática y Redes de Comunicaciones, Univ. de Granada, for providing the computing time.



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DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.6b01861 Inorg. Chem. XXXX, XXX, XXX−XXX