Organometallic half-sandwich iridium anticancer complexes.

Original citation: Liu, Z., et al. (2011). Organometallic half-sandwich iridium anticancer complexes. Journal of Medicinal Chemistry, 54(8), pp. 3011-3026.. Permanent WRAP url: http://wrap.warwick.ac.uk/50277 Copyright and reuse: The Warwick Research Archive Portal (WRAP) makes the work of researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available. Copies of full items can be used for personal research or study, educational, or not-forprofit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Publisher’s statement: This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Medicinal Chemistry, © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jm2000932 A note on versions: The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher’s version. Please see the ‘permanent WRAP url’ above for details on accessing the published version and note that access may require a subscription. For more information, please contact the WRAP Team at: wrap@warwick.ac.uk http://go.warwick.ac.uk/lib-publications Organometallic Half-sandwich Iridium Anticancer Complexes Zhe Liu,† Abraha Habtemariam,† Ana M. Pizarro, † Sally A. Fletcher,† Anna Kisova,‡ Oldrich Vrana,‡ Luca Salassa, † Pieter C. A. Bruijnincx,†,# Guy J. Clarkson,† Viktor Brabec‡ and Peter J. Sadler*† † Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K., and ‡ Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic Abstract: The low-spin 5d6 IrIII organometallic half- sandwich complexes [(η5 -Cpx )Ir(XY)Cl]0/+, Cpx = Cp*, tetramethyl(phenyl)cyclopentadienyl (Cp xph ) or tetramethyl(biphenyl)cyclopentadienyl (Cp xbiph ), XY = 1,10-phenanthroline (4, 5, 6), 2,2'-bipyridine (7, 8, 9), ethylenediamine (10, 11), or picolinate (12, 13, 14), hydrolyze rapidly. Complexes with N,N- chelating ligands readily form adducts with 9-ethylguanine but not 9-ethyladenine; picolinate complexes bind to both purines. Cytotoxic potency towards A2780 human ovarian cancer cells increases with phenyl substitution on Cp*: Cpxbiph > Cpxph > Cp*; Cpxbiph complexes 6 and 9 have submicromolar activity. Guanine residues are preferential binding sites for 4–6 on plasmid DNA. Hydrophobicity (log P), cell and nucleus accumulation of Ir correlate with cytotoxicity, 6 > 5 > 4; they distribute similarly within cells. Ability to displace DNA intercalator ethidium bromide from DNA correlates with cytotoxicity and viscosity of Ir-DNA adducts. Hydrophobicity and intercalative ability of Cpxph and Cpxbiph make a major contribution to the anticancer potency of their IrIII complexes. *To whom correspondence should be addressed. Phone: (+44) 024 7652 3818. Fax: (+44) 024 7652 3819. E-mail: p.j.sadler@warwick.ac.uk. # Present address: Utrecht University 1 a Abbreviations: Cp*, pentamethylcyclopentadienyl; Cpxph , tetramethyl(phenyl)cyclopentadienyl; Cp xbiph , tetramethyl(biphenyl)cyclopentadienyl; 9-EtG, 9-ethylguanine; 9-EtA, 9-ethyladenine; IC50 , half- maximum inhibitory concentration. 2 Introduction The clinical success of cisplatin, carboplatin and oxaliplatin1 has stimulated the search for other transition metal complexes which possess anticancer activity. New metal-based anticancer drugs may be able to widen the spectrum of treatable cancers, reduce toxic side-effects, and overcome platinum resistance. Interest in bio-organometallic chemistry and the design of organometallic complexes as anticancer agents is currently increasing. 2 Carbon-bound π-bonded arenes and cyclopentadienyl ligands can provide control of the hydrophilicity and hydrophobicity of the faces of the coordination complex (which influences cell uptake and targeting). 3 Most metallodrugs are prodrugs and control over ligand substitution is vital if the complex is to reach and react with its target site. In this respect octahedral low-spin d6 complexes are attractive for drug design since they are often kinetically- inert. Inertness increases from 1 st to 2nd to 3rd row of transition metals.4 The lifetime for exchange of an aqua ligand on [Ir(H2 O)6 ]3+, for example, is about 300 years!5 There are only a limited number of reported studies on the biological activity of iridium complexes. Early studies were concerned with non-organometallic IrI and IrIII complexes,6 and more recently a few studies of organometallic IrIII complexes have been reported.7 Iridium(III) complexes are generally thought to be too inert to possess high reactivity. Indeed, the inertness of IrIII has allowed the design of complexes which function as rigid scaffolds and inhibit kinase enzymes, for example.8 The biological inactivity of trans-[IrCl4 (DMSO)(Im)][ImH]9 and trans-[IrCl4 (Im)2 ][ImH] (ImH = imidazole)10 , IrIII analogs of the RuIII anticancer drugs NAMI-A and the imidazole analogue of the indazole complex KP1019, respectively, has been attributed to the kinetic inertness of IrIII. Organometallic RuII and OsII arene anticancer complexes of the type [(η6 -arene)(Ru/Os)(NN)Cl]+, where NN is a chelating diamine ligand, can be activated by hydrolysis of the Ru/Os–Cl bond, followed by binding to DNA.11 The arene is important in determining the anticancer activity and nature of the DNA distortions. In particular when the arene is an extended -system (e.g. biphenyl or 3 tetrahydroanthracene) direct binding to DNA bases (largely guanine) can be accompanied by arene intercalation between the bases.12 Neutral arene ligands do not stabilize IrIII. In contrast, negatively-charged pentamethylcyclopentadienyl (Cp*)a is an excellent stabilizing ligand for IrIII. In the work reported here we apply the design concepts discovered for RuII and OsII arene complexes to IrIII Cp* and functionalized Cp* complexes [(η5 -Cpx )Ir(XY)Cl]0/+ containing N,N-bound 1,10-phenanthroline (phen), 2,2′-bipyridine (bpy), ethylenediamine (en), and N,O-bound picolinate (pico) as chelating ligands. Iridium(III) Cp* complexes have attracted recent attention as catalysts, for example in hydrogen transfer reactions.13 Only a few iridium complexes containing functionalized Cp* ligands have been reported previously.14 We have studied the effect of Cp* functionalization on the rate of hydrolysis, acidity of the aqua adducts, interactions with nucleobases, hydrophobicity (octanol/water partition), cell accumulation (the net effect of uptake and efflux) and distribution, interaction with DNA, and cytotoxicity to cancer cells.15 We show that such complexes can be thermodynamically stable and yet kinetically labile towards substitution reactions and that substituents on the cyclopentadienyl ring and chelating ligand can have a dramatic effect on chemical and biological activity. This appears to be the first time that Cpxph and Cpxbiph have been used as ligands in iridium complexes. Results Che mistry Syntheses. Eleven IrIII half-sandwich complexes of the type [(η5 -Cpx )Ir(XY)Cl]0/+, where Cpx is pentamethylcyclopentadienyl, Cp*, or its phenyl Cpxph or biphenyl Cpxbiph derivatives, and XY is the N,N- chelating ligand 1,10-phenanthroline (phen, 4, 5 and 6), 2,2'-bipyridine (bpy, 7, 8 and 9), ethylenediamine16 (en, 10 and 11), or N,Ochelating picolinate (pico, 12, 13 and 14), were synthesized, characterized, and their cancer cell cytotoxicity investigated. Their structures were studied by X-ray crystallography and by DFT calculations, and their aqueous solution chemistry with 4 hydrolysis measurements, pKa determination for aqua adducts and interactions with nucleobases guanine and adenine. To elucidate the pronounced effect of the Cpx ligand on biological activity, detailed comparisons were made between the three phen complexes 4, 5, and 6 involving partition coefficients, distribution in cancer cells, DNA replication mapping, ethidium bromide displacement from DNA and effect on DNA viscosity. Chart 1. Iridium Cyclopentadienyl Complexes Studied in This Work. CpxH CpxphH Cp*H CpxbiphH _ N XY N N 0 / n+ Ir Z Y H2 N bpy phen Cpx O N Ir Cl X Cl N en Cpx Cl NH2 Cl Ir Cpx O pico Cpx dimer Cp* 1 2 3 Cpxph Cpxbiph Z=9-EtA Cp X Z=Cl Z=D2O/H2O Z=9-EtG 4 4A 4G Cp* phen 5 5A 5G Cp xph phen xbiph 6 6A 6G Cp 7 7A 7G Cp* xph XY phen bpy 8 8A 8G Cp 9 9A 9G Cp xbiph bpy 10 10A 10G Cp* 11 Cp 11G xph bpy en en 12 12A 12G 12Ad Cp* pico 13 13A 13G 13Ad Cp xph pico 14 14A 14G 14Ad Cp xbiph pico Complexes 4−14 were synthesized in moderate yields by reaction of 1,10-phenanthroline, 2,2′-bipyridine, ethylenediamine, or 2-picolinate, with the appropriate dimer [(η5 -Cpx )IrCl2 ]2 in methanol. The derivative Cpxbiph H, in which one of the ring methyls of Cp* is replaced by a biphenyl group, has not been previously reported. All the synthesized complexes were fully characterized by 1 H 5 NMR spectroscopy and CHN elemental analysis. Introduction of phenyl substituents on the Cp* ring decreased the reaction yields significantly. Complexes 4 and 7 were isolated as Cl− salts, complex 11 as a BPh4 − salt, and complexes 5, 6, 8, 9 and 10 as PF6 − salts. The complexes studied in this work are shown in Chart 1. X-ray Crystal Structures and Computation. The X-ray crystal structures of [(η5 -C5 Me4 C6 H5 )IrCl2 ]2 (2), [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 (5·PF6 ) [(η5 -C5 Me4 C6 H5 )Ir(bpy)Cl]PF6 (8·PF6 ), [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(bpy)Cl]PF6 (9·PF6 ), [(η5 -C5 Me4 C6 H5 )Ir(en)Cl]BPh4 (11·BPh4 ) and [(η5 -C5 Me4 C6 H5 )Ir(pico)Cl] (13) were determined. The complexes adopt the expected half-sandwich pseudo-octahedral “three- leg piano-stool” geometry with the iridium bound to an η5 -cyclopentadienyl ligand (Ir to ring centroid 1.747−1.789 Å), a chloride (2.384−2.415 Å) and a chelating ligand. Their structures and atom numbering schemes are shown in Figure 1 and Figure S1. Crystallographic data are shown in Table S1, and selected bond lengths and angles are listed in Table S2. In the structure of the Cpxph dimer 2, one of the chlorides (Cl1 or Cl2) acts as a bridging ligand to a symmetry-related molecule across an inversion center. The Ir−Ir bond distance is 3.7157(4) Å and the angle between mean planes through the phenyl and the cyclopentadienyl groups is 68.21°. The phenyl group is involved in a weak π-π interaction with a symmetry-related phenyl group of a neighboring ligand in the unit cell (Figure S2). The two interacting π systems are parallel, with a centroid−centroid distance of 3.956 Å. Further intermolecular ring stacking is observed in the crystal structures of complex 5·PF6 , between phen ligands in neighboring molecules (3.518 Å), Figure S3. The structures of the IrIII complexes 5·PF6 , 8·PF6 and 9·PF6 containing chelated phen or bpy are shown in Figure 1. The Ir−Cl and Ir−N bond lengths range from 2.3840(14) to 2.3891(5) Å and from 2.083(6) to 2.1001(17) Å, respectively (Table S2). Stacking between the Cpxbiph of neighboring molecules is present in crystals of complex 9·PF6 . The centroids of the three rings on independent molecules are separated by 4.447, 4.607 and 4.447 Å, at dihedral angles of 8.47, 0 and 8.47°, 6 respectively, Figure S4. The twist angle between the cyclopentadienyl and the central ring is 45.23°, and between the central and the terminal phenyl ring is 46.34°. In contrast, the planes of the terminal and bound rings are only twisted by 8.47°. C18 C116 C17 (A) (B) C19 C16 C20 C21 C15 C25 C117 C124 C125 C122 C29 C115 C118 C120 C126 C119 C114 C113 C22 C23 C123 C121 C127 C2 C26 C27 C3 C28 C24 lr1 Cl1 C102 C4 N1 lr1 C111 N101 C14 N12 C5 N112 C103 Cl4 C106 C107 C13 C11 C110 C6 C104 C8 C10 C105 C109 C108 C7 C9 (C) C14 (D) C13 C18 C15 C23 C29 C19 C16 C17 C30 C24 C28 C14 C17 C20 C21 C22 C25 C27 C32 C7 C18 C11 C13 C33 lr1 C19 C10 C9 C8 C11 Cl1 C2 N12 N1 C3 C6 lr1 C10 Cl1 N4 N1 C7 C9 C8 C4 C5 C3 C2 C22 C23 (E) C15 C12 C26 C16 C6 C5 C31 C16 C10 C17 C18 C9 C19 C11 C14 C12 C15 C21 lr1 C13 C20 C2 Cl1 N1 C3 O8 C6 C7 O7 C4 C5 Figure 1. X-ray crystal structures with atom numbering schemes for (A) [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 (5·PF6 ), (B) [(η5 -C5 Me4 C6 H5 )Ir(bpy)Cl]PF6 (8·PF6 ), (C) [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(bpy)Cl]PF6 (9·PF6 ), (D) [(η5 -C5 Me4 C6 H5 )Ir(en)Cl]BPh4 (11·BPh4 ), and (E) [(η5 -C5 Me4 C6 H5 )Ir(pico)Cl] (13), with thermal ellipsoids drawn at 50% probability. The hydrogen atoms and counterions have been omitted for clarity. 7 The propeller twist of the phenyl-tetramethylcyclopentadienyl ligand in complex 11·BPh4 is 63.7°. The Ir−Cl bond length (2.4152(12) Å) is the longest of these five X-ray structures. The geometries of complexes 4–14 were optimized using the PBE0 functional (Table S3, the description of frontier orbitals is in the Supporting Information). Selected calculated bond lengths are listed in Table S4 and are in good agreement with the experimental X-ray structures in the case of 5·PF6 , 8·PF6 , 9·PF6 and 11·BPh4 . DFT calculations show that Ir–Cl and Ir–cyclopentadienyl ring bond distances remain similar on changing Cp* to substituted Cp* groups. Figure 2. Electrostatic potential surfaces of the phen chlorido complexes 4–6 and aqua adduct 6A, and the pico chlorido complexes 12–14 and aqua adduct 14A. EPS surfaces are shown both in space (with positive and negative regions in blue and red, respectively) and mapped on electron density (isovalue 0.004) of the molecules. The electrostatic potential is represented with a color scale going from red (–0.100 au) to blue (0.150 au). Electrostatic potential surfaces (EPS) for phen chlorido complexes 4−6 and aqua adduct 6A, and the pico chlorido complexes 12−14 and aqua adduct 14A were calculated. N,N-chelating phen complexes 4−6 show more positive electrostatic potentials than the N,O-chelating pico complexes 12−14 (Figure 2). Moreover, higher electron density is present on the second phenyl ring of the Cpxbiph ligand in complexes 6 and 14. The same trend is observed in the electrostatic potential surfaces 8 of the aqua derivatives [(η5 -C5 Me4C6 H4C6 H5 )Ir(phen)(H2 O)]2+ (6A) and [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(pico)(H2 O)]+ (14A), which as expected show more positive surfaces compared to their chlorido analogues, 6 and 14. Hydrolysis Studies. Hydrolysis of M–Cl bonds can represent an activation step for transition metal anticancer complexes. 17 M–OH2 aqua complexes are often more reactive than the relevant chlorido complexes.18 The hydrolysis of compounds 4–10 and 12–14 in 5% MeOD-d4 /95% D2 O (v/v) was monitored by 1 H NMR at different temperatures from 278 to 293 K. The presence of methanol ensured the solubility of the complexes. All these IrIII complexes undergo relatively rapid hydrolysis. Complexes [(η5 -C5 Me5 )Ir(phen)Cl]+ (4), [(η5 -C5 Me5 )Ir(bpy)Cl]+ (7), [(η5 -C5 Me5 )Ir(en)Cl]+ (10) containing Cp*, and [(η5 -C5 Me5 )Ir(pico)Cl] (12), [(η5 -C5 Me4C6 H5 )Ir(pico)Cl] (13) and [(η5 -C5 Me4C6 H4 C6 H5 )Ir(pico)Cl] (14) containing picolinate hydrolyzed too rapidly for the rates to be determined by 1 H NMR spectroscopy even at 278 K: there was little change in the spectra between 5 min and 24 h, Figure S5. Attempts to observe hydrolysis of these complexes by UV-Vis at 288 K were also unsuccessful: equilibrium was reached before the first UV-Vis spectrum was acquired (< 1 min), Figure S6. To confirm the hydrolysis of these complexes, NaCl (1–4 mol equiv) was added to equilibrium solutions. With increase in NaCl concentration, 1 H NMR peaks for the chlorido adducts increased whilst peaks for the aqua form decreased in intensity, Figures S7–S9. Similarly, addition of NaCl to an aqueous solution of the aqua complex 4A gave rise to peaks for the chlorido complex 4, Figure S10. The hydrolysis of complexes [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(phen)Cl]+ (6), [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]+ (5), [(η5 -C5 Me4C6 H5 )Ir(bpy)Cl]+ (8), [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(bpy)Cl]+ (9), was slow enough to be studied by 1 H NMR at low temperature. Hydrolysis was monitored at temperatures ranging from 278 K to 293 K by observing the appearance of new 1 H NMR peaks over time. The time dependence for formation of the aqua adducts of 5, 6, 8 and 9 was fitted to pseudo 9 first-order kinetics (Figure S11), and their hydrolysis rate constants, half- lives and extent of hydrolysis were determined (Table 1). At 278 K, the half- life for hydrolysis of the biphenyl substituted Cpxbiph complex 6 was 32 min, about 1.3 times slower than that of the phenyl-Cp* complex 5 (25 min; Table 1). The half- lives and extent of hydrolysis of complexes 4−6 at 278 K increase with the size of the ring system in the order Cpxbiph > Cpxph > Cp*. This trend is also observed for complexes 7−9. The hydrolysis rate constants and half- lives of 5, 6, 8 and 9 at 310 K (body temperature) were calculated using the Arrhenius equation and are listed in Table 1. These range from 1 min for complexes 5 and 8 to 4 min for complex 6. In each series of complexes containing different chelating N,N- or N,O- ligands, the equilibrium constants (Kaq) for hydrolysis at 278 K increased with increasing phenyl substitution on the Cpx ligand, Table 1. Table 1. Hydrolysis Data for Complexes 4−10 and 12−14 at Various Temperatures k (min−1 ) t1/2 (min) Complexa 278 K 283 K 288 K Kaq (mM)b 293 K 310 Ke c 4 − 5 0.027 0.047 0.083 14.6 6 25.4 0.022 8.3 0.044 31.8 0.04 − d 15.9 d 0.65 0.06 0.065 1.1 0.18 0.29 10.7 3.8 − c 7 − 8 0.031 0.062 0.099 11.1 0.041 7.0 9 22.1 0.026 26.7 16.9 0.05 − d 0.89 0.08 0.078 0.8 0.23 1.45 8.8 3.0 −d 10 − c 0.44 12 −c 0.78 13 −c 1.11 14 − c 2.33 a The course of the hydrolysis of complex 11 was difficult to interpret from NMR spectra. b 278 K. c too fast to be measured. d not determined. e obtained from Arrhenius equation. 10 The effect of added chloride on the aqueous chemistry of 7 was investigated. 1 H NMR spectra of 1 mM 7 in 104, 23 or 4 mM NaCl in D2 O (mimicking the chloride concentrations in blood plasma, cell cytoplasm and cell nucleus, respectively)18b, 19 were recorded within 10 min of sample preparation and a fter incubation at 310 K for 24 h. On the basis of 1 H NMR peak integrals, almost no hydrolyzed complex 7 (7A) was found to be present in 104 mM [Cl] or in 23 mM [Cl], and only 5% of aqua complex 7A was observed at 4 mM [Cl] after 10 min with no further change after 24 h. pKa * Determination. The pKa of coordinated water can have a significant influence on its reactivity since M−OH bonds are often much less labile than M−OH2 bonds;11b moreover hydroxide is a good bridging ligand and can give rise to oligomeric species. Table 2. pKa* and pKa Valuesa for the Deprotonation of the Coordinated D2 O in Complexes 4A−10A, and 12A−14A Aqua Complex 5 2+ [(η -C5M e5)Ir(phen)(D 2O)] (4A) 5 [(η -C5M e4C6H5)Ir(phen)(D 2O)] 2+ 5 (5A) [(η -C5M e4C6H4C6H5)Ir(phen)(D 2O)] 2+ (6A) [(η5-C5M e5)Ir(bpy)(D 2O)]2+ (7A) [(η5-C5M e4C6H5)Ir(bpy)(D2O)]2+ (8A) 5 2+ [(η -C5M e4C6H4C6H5)Ir(bpy)(D2O)] (9A) 5 2+ [(η -C5M e5)Ir(en)(D2O)] (10A) 5 + [(η -C5M e5)Ir(pico)(D 2O)] (12A) [(η5-C5M e4C6H5)Ir(pico)(D 2O)]+ (13A) 5 + [(η -C5M e4C6H4C6H5)Ir(pico)(D2O)] (14A) a pKa * pKa 7.88 7.74 7.68 7.55 7.50 7.38 6.94 6.86 6.31 6.28 6.68 6.63 7.66 7.54 8.15 7.99 7.75 7.62 7.65 7.52 pKa values calculated from pKa* according to Krezel and Bal. 20 Changes in the 1 H NMR chemical shifts for coordinated chelating ligands in aqua complexes 4A−9A, 12A−14A, and methyl groups of Cp* in aqua complex 10A, were followed with change in pH* over a range of 2−11 (Figure S12). 1 H NMR peaks 11 assigned to aqua complexes gradually shifted to high field with increase in pH*. The resulting pH titration curves were fitted to the Henderson-Hasselbalch equation, from which the pKa* values of the coordinated water were determined. This gave rise to pKa values between 6.28 and 7.99 (Table 2), with the bpy complexes being the most acidic (pKa values 6.28−6.86) and the pico complexes the least acidic (pKa values 7.52−7.99). Inte ractions with Nucleobases. Since DNA is a potential target site for transition metal anticancer complexes,21 the binding of 9-ethylguanine (9-EtG) and 9-ethyladenine (9-EtA) to complexes 4−14, and aqua complex 5A were studied. The extent of nucleobase adduct formation by these complexes based on 1 H NMR peak integrals is shown in Table 3. Table 3. Extent of 9-EtG and 9-EtA Adduct Formation for Complexes 4−14, and 5A (ca. 1 mM) at 310 K after 24 h 4 5 Cpx XY Cp* phen(N,N-) 83 0 xph phen(N,N-) 42 0 xph phen(N,N-) 74 0 xbiph Cp 5A G adduct A adduct (%) (%) Cp 6 Cp phen(N,N-) 90 0 7 Cp* 8 bpy(N,N-) 61 0 xph bpy(N,N-) 47 0 xbiph Cp 9 Cp bpy(N,N-) 65 0 10 Cp* en(N,N-) 100 0 xph 11 Cp en(N,N-) 100 0 12 Cp* 13 14 pico(N,O-) 100 81 xph pico(N,O-) 100 76 xbiph pico(N,O-) 100 71 Cp Cp Addition of 1 mol equiv of 9-EtG to an equilibrium solution of complex 5, [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]+ (1.0 mM), in 5% MeOD-d4 /95% D2 O (v/v, pH* 7.2) at 310 K resulted in 15% of 5 reacting after 10 min, and a new 9-EtG H8 peak appearing at 7.68 ppm (species 5G, Figure S13), shifted by 0.15 ppm to high field relative to 12 that of free 9-EtG. After 24 h, 42% of 5 had reacted. The ESI-MS of an equilibrium solution (Figure S14) contained a major peak at m/z 374.6, confirming the formation of the 9-EtG adduct 5G, [(η5 -C5 Me4C6 H5 )Ir(phen)(9-EtG)]2+ (calcd m/z 374.5). However, 65% of aqua complex 5A (prepared by treating a solution of 5 with 1 mol equiv of AgNO 3 ) reacted with 9-EtG to form 5G after 10 min and 73% after 24 h. More than 80% of complexes 4 and 6 reacted with 9-EtG under the same conditions. Complexes [(η5 -C5 Me5 )Ir(en)Cl]+ (10) and [(η5 -C5 Me4 C6 H5 )Ir(en)Cl]+ (11) interestingly, showed an exceptionally high affinity for 9-EtG with 100% nucleobase adduct formation within 10 min. On addition of a solution of 1 mol equiv of 9-EtG gradually to an equilibrium solution of 10 (0.9 mM) in 5% MeOD-d4 /95% D2 O (v/v) at 310 K, Figure S15, the methyl peak for 10 and 10A decreased in intensity and eventually disappeared. A new Cp* methyl peak (for 10G) appeared at 1.65 ppm. When 9-EtG was in excess, a set of new peaks assignable to free 9-EtG was clearly visible (Figure S15). The geometry of the adduct (7G) between complex 7 and 9-EtG was optimized by DFT calculations. These gave an Ir–N7(9-EtG) distance of 2.14 Å. The Ir–N and Ir–cyclopentadienyl ring bond distances increase by 0.1–0.2 Å compared to complex 7. The HOMO of the adduct is 9-EtG-centered and the LUMO has bpy character (see Table S3). Addition of 1 mol equiv of 9-EtA to an equilibrium solution of 5 (1.0 mM) in 5% MeOD-d4 /95% D2 O (v/v) at 310 K resulted in no additional 1 H NMR peaks over a period of 24 h (Figure S16). Similarly, no reaction with 9-EtA was observed for other complexes containing N,N- chelating ligands (Table 3). In contrast, compounds 12−14 containing pico as chelating ligand formed both 9-EtG and 9-EtA adducts to the extent of 100% and more than 70% completion, respectively, after 24 h. Two adenine nucleobase adducts are formed in the reaction of complexes 12, 13 and 14 with 9-EtA, most likely through iridium binding to N1 or N7 of adenine forming 9-EtA adducts in 1:3.1, 1:3.2, and 1:3.0 ratios, respectively, Figure S17. 13 Cancer Cell and DNA Studies Cytotoxicity. The cytotoxicity of complexes 4−14 towards A2780 human ovarian cancer cells was investigated, see Table 4. The IC50 values (concentration at which 50% of the cell growth is inhibited) for Cp* complexes 4, 7, 10, 12 and the Cpxph pico complex 13 were all > 100 μM and are thus deemed as inactive. However, compounds 5, 6, 8, 9, 11 and 14, were all active. Complexes 5, 8 and 11 containing Cpxph , and 14 containing Cpxbiph showed good activity, displaying IC 50 values of 6−17 μM. Table 4. Inhibition of Growth of A2780 Human Ovarian Cancer Cells by Complexes 4−14 and Comparison with Cisplatin Complex IC50 a (μM) 5 >100 5 6.70 ±0.62 5 0.72 ±0.01 5 >100 5 15.86 ±1.49 5 0.57 ±0.09 5 >100 5 16.97 ±0.05 5 >100 5 >100 5 [(η -C5 Me4 C6 H4 C6 H5 )Ir(pico)Cl] (14) 16.30 ±0.32 Cisplatin 1.22 ±0.12 [(η -C5 Me5 )Ir(phen)Cl]Cl (4·Cl) [(η -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 (5·PF6) [(η -C5 Me4 C6 H4 C6 H5 )Ir(phen)Cl]PF 6 (6·PF6 ) [(η -C5 Me5 )Ir(bpy)Cl]Cl (7·Cl) [(η -C5 Me4 C6 H5 )Ir(bpy)Cl]PF6 (8·PF6 ) [(η -C5 Me4 C6 H4 C6 H5 )Ir(bpy)Cl]PF 6 (9·PF6 ) [(η -C5 Me5 )Ir(en)Cl]PF6 (10·PF6) [(η -C5 Me4 C6 H5 )Ir(en)Cl]BPh4 (11·BPh4 ) [(η - C5 Me5 )Ir(pico)Cl] (12) [(η -C5 Me4 C6 H5 )Ir(pico)Cl] (13) a Drug-treatment period was 24 h. Complexes 6 and 9 containing Cpxbiph exhibited potent cytotoxicity with IC 50 values of 0.7 and 0.6 µM, respectively, ca. twice as active as cisplatin in the A2780 cell line (IC50 1.2 µM). For all four series of complexes containing different chelating N,N- or N,O- ligands, the trend of increasing of activity with increasing phenyl substitution was the same: Cpxbiph > Cpxph > Cp*. Hydrophobicity (log P). The octanol/water partition coefficients (log P) for the phen complexes 4−6 were determined since lipophilicity correlates with cytotoxic potency for some reported series of metallodrugs.22 The determined values are listed in Table 5. Addition of NaCl (200 mM) was used in order to suppress hydrolysis of 14 the compounds, ensuring that log P values for the chlorido and not aqua complexes were determined. The log P values increase in the order 4 < 5 < 6. Only complex 4, containing the unsubstituted Cp* ligand, has a negative log P value (partitions preferentially into water, Table 5). Table 5. log P Values for Complexes 4−6a log P Complex mean SD 4 –0.82 0.01 5 0.48 0.03 6 1.11 0.17 a Results are the mean of three independent experiments and are expressed as mean ±SD. Cell Accumulation and DNA Binding. Since increased lipophilicity has often been linked to increased cell uptake and cytotoxicity,23 the accumulation and DNA binding of complexes 4−6 by A2780 ovarian cancer cells was determined after 24 h of exposure to 5 μM concentrations of the complexes. DNA from A2780 cells was isolated and the Ir content was determined. The Cp xbiph complex 6 gave rise to the highest level of iridium on DNA, ca. 4× that of complex 5, and 20× that of complex 4 (Table 6). Of the total Ir taken up by the cells, 7.7% for 4, 5.5% for 5, and 6.0% for 6 was bound to DNA. Table 6. Iridium Accumulation and Binding to DNA in A2780 Human Ovarian Cancer Cellsa cell accumulation a DNA binding (ng Ir/10 cells) (ng Ir/106 cells) mean SD mean SD 4 3.9 0.2 0.3 0.04 5 23.5 3.7 1.3 0.3 6 88.8 20.0 5.3 1.6 Complex 6 III Drug-treatment period was 24 h with 5 μM Ir complexes. Each value represents the mean ± SD for two independent experiments done in triplicate. 15 Distribution of Iridium in Cell Fractions. The iridium content of the nucleus, cytosol, membrane and cytoskeleton fractions isolated from A2780 cells after 24 h of exposure to the phen complexes 4−6 was determined, and the results are shown in Table S5 and Figure 3. The extent of accumulation of the three complexes into the different cell fractions was similar to that observed for whole cell accumulation: 6 > 5 > 4. The highest concentration of iridium was in the cell membrane/particulate fraction, accounting for 54% (6), 74% (5) and 59% (4) of the total Ir in the cell. For all complexes, the next highest concentration of Ir was in the cytosol, accounting for 24% (6), 15% (5) and 17% (4). For the two remaining fractions, the Ir concentration dropped significantly. For complexes 4 and 6, the cytoskeleton was the next major compartment for Ir accumulation. The amount of Ir in the nucleus was significant and similar for all three IrIII complexes: 7.2% of the total Ir for 6, 5.8% for 5, and 6.4% for 4, and similar in proportion to that bound to DNA. Nucleus Cytoskeleton Cytosol Membrane Complex Figure 3. Iridium content of the nucleus, cytosol, membrane and cytoskeleton fractions (ng Ir/106 cells) of A2780 cells after 24 h of exposure to 5 μM 4−6. Results are the mean of two independent experiments in triplicate and are expressed as mean ± SD. 16 Replication Mapping of Iridium-DNA Adducts. This procedure involved the extension by VentR(exo-) DNA polymerase of the 3'-end of the primer up to the metal adduct on the template strand of pSP73KB DNA linearized by HpaI restriction endonuclease. The products of the synthesis were then examined on DNA sequencing gels, and the sequence specificity of iridium adduct formation was determined to the exact base pair. In vitro DNA synthesis on DNA templates containing the adducts of the phen complexes 4−6 generated a population of DNA fragments, indicating that the adducts of these complexes effectively terminated DNA synthesis (Figure 4A, lanes 4−6). Complexes 4–6 exhibit a sequence dependence of the inhibition clearly different from that of cisplatin. The Ir compounds form more blocks on DNA for DNA polymerase than cisplatin and some of them occur at different sequences. These results are consistent with a less regular sequence specificity of complexes 4–6 in comparison with cisplatin. Identical patterns of blocks on DNA were observed if the template DNA was incubated with the metal complex for 8, 24 or 72 h (data not shown). 17 transPt cisPt Control A T GC 6 5 4 (A) (B) Figure 4. Replication mapping of Ir–DNA adducts. (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by VentR DNA polymerase on the pSP73KB plasmid DNA linearized by HpaI restriction enzyme and subsequently modified by IrIII complexes, cisplatin or transplatin. Lanes: control, unmodified template; A, T, G and C, chain-terminated marker DNAs (note that these dideoxy sequencing lanes give the sequence complementary to the template strand); 4−6, DNA modified by complexes 4−6 at r b = 0.01, respectively; cisPt, DNA modified by cisplatin at r b = 0.01; transPt, DNA modified by transplatin at r b = 0.01. The numbers correspond to the nucleotide sequence in Figure 4B. (B) Schematic diagram showing a portion of the sequence used to monitor inhibition of DNA synthesis on the template containing adducts of 18 IrIII complexes. The arrow indicates the direction of the synthesis. ○, major stop signals from Figure 4A, lanes 4−6. The numbering of the nucleotides in this scheme corresponds to the numbering of the nucleotides in the pSP73KB nucleotide sequence map. Ethidium Bromide (EtBr) Displacement. The ability of the complexes to displace the DNA intercalator EtBr from CT DNA was probed by monitoring the relative fluorescence of EtBr bound to DNA after treating the DNA with varying concentrations of 4−6. Figure 5 shows a plot of relative fluorescence vs rb for complexes 4−6, cisplatin and monofunctional chloridobis(2-aminoethyl)amineplatinum(II) chloride ([Pt(dien)Cl]Cl). Figure 5. Plots showing the dependence of EtBr fluorescence on rb for calf thymus DNA modified by IrIII complexes 4−6, cisplatin, and [Pt(dien)Cl]Cl in 10 mM NaClO 4 at 310 K for 24 h: (), complex 4; (), complex 5; (○), complex 6; (▲), cisplatin; (■), [Pt(dien)Cl]Cl. Data points measured in triplicate varied on average ±3% from their mean. The adducts of monofunctional IrIII complexes competitively replaced intercalated EtBr more effectively than the adducts of monofunctional [Pt(dien)Cl]Cl, but slightly less than the adducts of bifunctional cisplatin. Notably, the trend in ability to displace DNA intercalator EtBr from CT DNA was 6 > 5 > 4, which correlates with their 19 cytotoxicity (Table 4). Viscometry. The effects of complexes 4−6 on the viscosity of rod- like CT DNA (0.15 mg/mL or 0.47 mM in phosphorus content) are shown in Figure 6. On increasing the amounts of 4−6 bound to DNA (in the range of rb values of 0.005−0.04), the relative viscosity of CT DNA increased steadily; the effect follows the order 6 > 5 > 4, which correlates with their cytotoxicity (Table 4) as well as with their ability to displace DNA intercalator EtBr from DNA (Figure 5). Figure 6. Plots showing the dependence of relative viscosity on r b for calf thymus DNA modified by IrIII complexes 4−6. The viscosity was measured in 10 mM NaClO 4 , pH 6 at 310 K. (), complex 4; (), complex 5; (○), complex 6. Discussion The clinical success of cisplatin and related platinum anticancer drugs 1 has led to the search for active complexes of other transition metals, especially to combat the limited spectrum of activity of platinum, drug resistance and side-effects. Can the design features found in platinum drugs be introduced into other transition metal complexes so as to rationalize the design process? From studies of organometallic half-sandwich arene complexes of the type [(η6 -arene)M(diamine)Cl]+ (M = RuII or OsII), there is evidence that they can. 2c, 24 These organometallic complexes contain an M−Cl bond which can be activated by hydrolysis and a neighboring NH group can stabilize binding to the nucleobase guanine via NH−C6O H-bonding. Unlike cisplatin, which contains two labile Pt−Cl bonds and can crosslink bases on DNA, 1b these 20 complexes are monofunctional, but nevertheless can cause replication stop sites when bound to DNA, unlike monofunctional PtII adducts, such as [Pt(dien)Cl]Cl.25 Their strong selectivity for G versus A can be reversed by changing the NH group on the chelated ligand (e.g. ethylenediamine) to an H-bond acceptor oxygen such as in acetylacetonate.26 The interaction of RuII and OsII arene complexes with DNA and their potency towards cancer cells can be increased by incorporating an extended arene which can intercalate between base pairs neighboring the metal coordination site.25, 27 In the present work we sought to apply these design concepts to Ir III complexes since the biological activity of iridium complexes has received relatively little attention.6-7 The low-spin d6 metal ion IrIII is often described in the literature as totally inert, perhaps because water exchange on [Ir(H2 O)6 ]3+ ions has long been known to take hundreds of years.5a On the other hand it is also known that water exchange in [(η5 -Cp*)Ir(H2 O)3 ]2+ is ca. 1014 times faster.28 In general the dependence of ligand exchange reactions on the nature of the ligands in IrIII complexes has been little studied. We have investigated IrIII complexes containing Cp* ligands since they form highly stable Ir–C bonds, unlike arenes. The catalytic properties of IrIII Cp* complexes have recently been explored. 13, 29 We have prepared the Cp* 1,10-phenanthroline complex [(η5 -Cp*)Ir(phen)Cl]+ (4, a complex recently reported to be inactive towards MCF-7 breast and HT-29 colon cancer cells),30 and compared its aqueous chemistry (hydrolysis and acidity of aqua adduct), nucleobase binding, transcription mapping, cytotoxicity towards A2780 human ovarian cancer cells, hydrophobicity (octanol/water partition), cellular distribution and DNA intercalation with that of related novel phen complexes containing phenyl- and biphenyl-substituted Cp* ligands (5 and 6), and with analogous complexes containing bipyridine (bpy, 7−9), or ethylenediamine (en, 10 and 11) as the N,N-chelating ligand, or picolinate (pico, 12−14) as an N,O-chelating ligand. Structures of the Complexes. A search of the Cambridge Database revealed that no structure of metal complexes containing the ligand Cpxbiph has been reported. 21 Complex [(η5 -C5 Me4 C6 H4C6 H5 )Ir(bpy)Cl]PF6 (9·PF6) therefore appears to be the first such structure. The crystal [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 structures (5·PF6 ), of [(η5 -C5 Me4 C6 H5 )IrCl2 ]2 [(η5 -C5 Me4 C6 H5 )Ir(bpy)Cl]PF6 (2), (8·PF6 ), [(η5 -C5 Me4 C6 H5 )Ir(en)Cl]BPh4 (11·BPh4 ) and [(η5 -C5 Me4C6 H5 )Ir(pico)Cl] (13) are the first reported with (η5 -C5 Me4 C6 H5 ) (Cpxph ) coordinated to iridium. The bond distances and angles in the dimer [(η5 -C5 Me4 C6 H5 )IrCl2 ]2 (2) compare well to those found for the corresponding Cp* analogue [(η5 -C5 Me5 )IrCl2 ]2 (1).31 The Ir–Cl(bridging)–Ir and Cl(bridging)–Ir–Cl(bridging) angles in 2 are 1.2° more acute and obtuse, respectively, than those of the Cp* analogue. The Ir–Cl bond lengths in complexes 8·PF6 and 9·PF6 (2.3859(19) and 2.3840(14) Å, respectively, Table S2) are almost the same, however, the Ir–Cl bond length in complex 7 is slightly longer (2.404(2) Å)32 . The twist angles in complex 9·PF6 are similar to those angles in RuII terphenyl arene complexes.27c The Ir–Cl bond length in complex 13 (2.3860(10) Å) is similar to that in complex 12 [(η5 -C5 Me5 )Ir(pico)Cl] (2.3997(15) Å)33 . The distance between IrIII [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 and (5·PF6) the (1.783 centroid of Å) similar is Cp ring in to that in [(η5 -C5 Me5 )Ir(phen)Cl]CF3 SO 3 (1.780 Å)34 , and the Ir−Cl bond length in 5·PF6 (2.3891(5) Å) is similar to that in [(η5 -C5 Me5 )Ir(phen)Cl]CF3 SO3 (2.395 Å)34 . On changing Cp* to substituted Cp* ligands, no significant change is observed by DFT calculations for Ir–Cl and Ir–cyclopentadienyl ring bond distances. These results suggest that the introduction of phenyl substituent on the Cp* ring does not give rise to significant change in structure. Hydrolysis and pKa of Aqua Adducts. There are only a few previous studies of the aquation of organometallic IrIII complexes.5a, 35 In general, all the complexes studied in this work hydrolyze rapidly. Complexes 4, 7 and 10 containing Cp*, and 12–14 containing picolinate hydrolyzed too rapidly to be observed by conventional UV-Vis at 288 K (t1/2 < 1 min). Even for omplex [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(phen)Cl]+ (6), which hydrolyzed the slowest of these complexes, had a calculated half life at 310 K of < 4 min (Table 1). These results illustrate that IrIII complexes are not always inert 22 and can be quite labile. The hydrolysis of Ir–Cl bonds in iridium complexes is strongly dependent on the coordinated ligands. These Cpx IrIII complexes undergo even faster hydrolysis than low-spin d6 arene RuII and OsII phen complexes,11a,36 more than 2 orders of magnitude faster than OsII for example. The electron-donor methyl groups on the Cp ring may contribute to the fast hydrolysis. These increase the effective charge on Ir and facilitate chloride loss. This behavior is consistent with that of hexamethylbenzene RuII complexes.37 Previous studies on the hydrolysis rates of OsII arene compounds of the type [(η6 -arene)Os(XY)Cl]n+ have shown that the aqueous reactivity of these complexes is highly dependent on the nature of the chelating ligand.38 In particular the negatively-charged electron-donating picolinate ligand increases the rate of hydrolysis compared to complexes with diamine ligands, as seen here for complexes 12–14. The presence of bpy as a π-acceptor in complex [(η6 -bip)Ru(bpy)Cl]PF6 , where bip = biphenyl, decreases the rate of hydrolysis by a factor of two compared to the en analogue.36, 39 The π-acceptor ligands bpy and phen can withdraw electron density from a metal center, increasing the positive charge on the metal, making it less favorable for Cl− to leave, slowing down the hydrolysis. As a result, complexes 5, 6, 8 and 9 containing bpy or phen as the chelating ligand hydrolyzed much more slowly than the en complex 10 and pico analogues, complexes 13 and 14. However, despite the electron withdrawing ability of bpy and phen, the hydrolysis rates of complexes 4 and 7 are still relatively fast and appear to be controlled by the powerful electron donor Cp*. Our previous work has shown that the interaction of [(η6 -bip)Ru(en)Cl]+ with amino acids,40 proteins,40 peptides,41 and DNA bases11b involves aquation (substitution of Cl by H2 O) as the first step. The anticancer drug cisplatin also undergoes aquation prior to platination of the target site, DNA.18b, 42 The equilibrium constants at 278 K for hydrolysis of complexes 4−6, 7−9, and 12−14, decrease in the order 6 > 5 > 4, 9 > 8 > 7, and 14 > 12, 13 (Table 1), which parallels their cytotoxicity (Table 4), perhaps indicating that activation by aquation is important for the 23 mechanism of their cytotoxic action. At chloride concentrations typical of blood plasma (104 mM) and cell cytoplasm (23 mM), the inactive complex [(η5 -C5 Me5 )Ir(bpy)Cl]+ (7) (IC50 > 100 M, Table 4) was found to be almost all present in solution as the intact chlorido species, which is relatively unreactive compared to the aqua complex 7A. At a chloride concentration of 4 mM, close to that of the cell nucleus, only about 5% of 7 was present as the reactive aqua species. When the pKa values of the aqua complexes (Table 2) are compared, it is evident that the presence of phenyl or biphenyl substitutent lowers the pKa value by ca. 0.4 units consistent with withdrawal of electron density from the Ir center. Replacement of the π-acceptor ligand bpy in aqua complex 7A by the chelating diamine donor en in 10A, leads to a significant increase in pKa by ca. 0.7 units, consistent with an increased electron density on the metal center. Similarly, the replacement of the neutral chelated bpy ligand by the anionic pico ligand raises the pKa by 1.1 units. There appears to be no correlation between the pKa values of the aqua adducts and the cytotoxicity of these complexes. The pKa values of the pico, en and phen aqua complexes 4A, 6A, 10A, 12A−14A suggest that they will be present largely as the reactive aqua adducts as opposed to the less reactive hydroxo adducts at physiological pH (7.4), whereas the pKa values of the bpy complexes 7A−9A are significantly lower, especially 8A, and therefore most of the hydrolyzed bpy complexes would be present as the hydroxido form (at pH 7.4). Despite this, complexes 8 and 9 exhibit good activity. Their reactivity would be aided by the small lowering of pH which is thought to occur in tumors.43 For RuII complexes which contain biphenyl as the arene and bpy as chelating ligand, loss of the arene is observed in aqueous solution. 36 In contrast no loss of any Cpx ligands was observed for any of the IrIII complexes studied in this work. Inte ractions with Nucleobases. DNA is often a target for cytotoxic transition metal anticancer complexes.21 There is little reported work on interactions of iridium complexes with nucleobases.44 In the present study, the reactions of complexes 4−14, 24 and aqua complex 5A with 9-ethylguanine and 9-ethyladenine were investigated. Complexes 4−11 and 5A containing a neutral N,N- chelating ligand all bind selectively to 9-EtG compared to 9-EtA, with which no reaction was observed after 24 h. This result is consistent with the replication mapping experiments (Figure 4) which show that G residues are the preferential binding sites on polymeric DNA modified with complexes 4−6. The less regular sequence specificity of complexes 4−6 in comparison with cisplatin might arise from the faster DNA binding of 4−6. The selectivity in nucleobase binding can be rationalized in terms of H-bonding, non-bonding repulsive interactions between the chelating ligand and nucleobase substituents, and the electronic properties o f the various nucleobase coordination sites.38a Our previous studies of Ru−N7 guanine adducts have revealed a strong H-bonding interaction between one en NH and G C6O.11b This may explain the strong affinity of 9-EtG for the IrIII en complexes 10 and 11. A phenanthroline ligand cannot provide a donor NH group, but instead the interaction with G C6O may be stabilized by a C−H H-bond similar to that observed in a bipyridine complex of RuII.36 This possibility was indicated by the DFT optimized structure of the 9-EtG adduct of 6 (Figure S18). Complexes 4−11 containing an N,N-chelating ligand did not react with 9-EtA, most likely due to the steric hindrance of the NH2 group on the 6-position of the adenine ring. Compared to complexes containing an N,N-chelating ligand, compounds 12−14 which contain the N,O- chelating ligand pico, bind significantly (70−100%) to both nucleobases, see Table 3. These picolinate IrIII adducts of complexes 12, 13 and 14 with 9-EtA (12Ad, 13Ad, and 14Ad, respectively) can be stabilized by hydrogen bonding between the NH2 group of adenine and a carboxylate oxygen of the picolinate ligand.45 As expected, the aqua complex 5A reacted to a greater extent with 9-EtG compared to the chlorido complex 5, see Table 3, consistent with the increased reactivity of aqua adducts compared to their chlorido forms,18a but the selectivity for G versus A was the same. 25 Hydrophobicity (log P) and Cell Accumulation. log P values for octanol/water partition provide a measure of hydrophobicity which is often a factor relevant for cell uptake and anticancer activity. For several classes of metallo-anticancer complexes, a correlation between increased hydrophobicity and increased cytotoxic activity has been reported.22-23 In this study, as expected, the log P values (Table 5) and hydrophobicity increase with increasing size of the substituted Cp* ligand. Additionally, the hydrophobicity, cancer cell activity, and cell accumulation correlate significantly, following the order 6 > 5 > 4. Complex [(η5 -C5 Me5 )Ir(phen)Cl]+ (4) is the least hydrophobic, the least cytotoxic, and the least taken up by the cells, whereas complex [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(phen)Cl]+ (6) displays the highest hydrophobicity, is the most cytotoxic, and the most taken up by the cells. These data suggest that in the ovarian A2780 cancer cell line, the log P value is a useful parameter for predicting the cytotoxicity of this class of iridium complexes. These data also show that using more extended coordinated Cpx ligands such as tetramethyl(biphenyl)cyclopentadienyl (Cpxbiph ) gives rise to in increased hydrophobicity leading to higher cellular uptake and higher cytotoxicity. For OsII arene complexes this range of log P values (–0.51 and 0.86) is also accompanied by promising anticancer activity.46 Such complexes are hydrophobic enough to partition efficiently into cells and yet hydrophilic enough to exhibit reasonable aqueous solubility. Distribution of Iridium in Cells. The accumulation of the three phen complexes 4–6 into the different cell fractions was studied. A significant proportion of the total iridium (54−74%) was in the cell membrane fraction, see Figure 3. This may be related not only to Ir being transported into the cytoplasm but also to Ir being exported by cells. For all complexes, the next highest concentration of iridium was found in the cytosol showing that passage through the outer membrane readily occurs. Although the lowest proportion of iridium was found in the nucleus, especially for complexes 4 and 6, it is notable that there is a correlation between nucleus accumulation and cytotoxicity of the complexes, both of which follow the order 6 > 26 5 > 4, suggesting that penetrating the nucleus and binding to nuclear DNA may provide an important contribution to the mechanism of cytotoxicity. A similar relationship between nucleus accumulation and cytotoxicity was observed for OsII arene complexes.46 DNA Binding in A2780 Carcinoma Cells. The amount of iridium found on the DNA of A2780 cells (Table 6) incubated with the complexes for 24 h follows the order 6 > 5 > 4, which correlates with their cytotoxicity, hydrophobicity (log P), and cellular accumulation (Table 6), and is similar to the total accumulation by cell nuclei. The extent of iridium binding of 4−6 to DNA, 5.5−7.7% of the total iridium taken up by the cells, is higher than that reported for cisplatin (~1%) 47 and OsII arene complexes.46 DNA may therefore be a potential target for these cytotoxic iridium complexes, although we cannot rule out the possibility that nuclear DNA may not be the only target.48 EtBr Displacement and Viscometry. The fluorescent probe EtBr can be used to distinguish between intercalating and nonintercalating ligands. 25, 49 Viscosity measurements are also useful for probing the nature of DNA interactions since viscosity is sensitive to alterations in DNA length. For instance, complexes or ligands that intercalate cause an increase in overall DNA contour length due to the increase in separation of base pairs at the intercalation sites, which leads to an increase in viscosity of DNA solutions. On the other hand, drug molecules which bind in DNA grooves cause less pronounced changes in the viscosity of DNA solutions.50 Modification of CT DNA by complexes 4−6 resulted in a decrease of EtBr fluorescence intensity (Figure 5) and an increase in the relative viscosity of CT DNA (Figure 6) in the same order 6 > 5 > 4, which correlates with their cytotoxicity (IC 50 values). These results indicate that addition of phenyl substituents to the Cp* ring in these iridium complexes enhances the intercalative ability into DNA. Dual- mode intercalation/G N7 coordination DNA binding may therefore play an important role in the cytotoxicity of these IrIII complexes. This observation parallels that of RuII and OsII arene complexes for which extended arenes can also intercalate and increase the 27 potency of the complexes.12a, 24c For example, the RuII anticancer complex [(η6 -p-terp)Ru(en)Cl]+ (where p-terp = para-terphenyl, a similar arene ligand to Cpxbiph ) also exhibits combined intercalative and monofunctional (coordination) binding to DNA.27c The DFT-computed electrostatic potential surfaces of the phen complexes 4–6 show more positive values than those of pico complexes 12–14 (same trend for the aqua derivatives 6A and 14A). This may favor interaction with negatively charged DNA in the case of phen complexes. The higher electron density on the second phenyl ring of the Cpxbiph ligands (Figure 2), together with the lower steric hindrance of the terminal phenyl ring, can explain the better intercalation properties of 6. Complex 6 is likely to be present in the nucleus largely as the aqua complex 6A which has an even higher positive charge distributed over its surface (Figure 2). Cytotoxicity. [(η5 -C5 Me5 )Ir(bpy)Cl]Cl Complexes (7·Cl), [(η5 -C5 Me5 )Ir(phen)Cl]Cl [(η5 -C5 Me5 )Ir(en)Cl]PF6 (10·PF6 ) (4·Cl), and [(η5 -C5 Me5 )Ir(pico)] (12) containing Cp* were non-toxic (IC50 > 100 μM) toward the human ovarian A2780 cancer cell line (Table 4). The cytotoxicity of complexes [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 (5·PF6 ) and [(η5 -C5 Me4C6 H4C6 H5 )Ir(phen)Cl]PF6 (6·PF6 ) containing phenyl and biphenyl substituents, respectively, on the tetramethylcyclopentadienyl ring increases dramatically compared to the parent Cp* complex 4. Complex 6 containing Cpxbiph is ca. twice as potent towards A2780 human ovarian cancer cells as the anticancer drug cisplatin (Table 4). The introduction of phenyl and biphenyl substituents also resulted in significant increases in activity for complexes 7–9, 10–11 and 12–14, suggesting that these phenyl groups play a crucial role in the mechanism of action. This is consistent with our previous observations that the cytotoxicity of η6 -arene RuII compounds increases with the size of the arene ring system in the order benzene < p-cymene < biphenyl < dihydroanthracene < tetrahydroanthracene.3c The increase in potency on addition of phenyl substituents to the Cp ring, by about an order of magnitude for each of the additions (from 4 to 5 to 6) is more dramatic than in the case of ruthenium arene ethylenediamine complexes. 27c, 28 51 For these iridium complexes, the phenyl substitutents not only enhance lipophilicity and cell accumulation, but also introduce an additional mode of DNA interaction (intercalation). Both complexes [(η5 -C5 Me4C6 H5 )Ir(bpy)Cl]+ (8) and [(η5 -C5 Me4 C6 H5 )Ir(pico)Cl] (13) contain Cpxph , however, 8 exhibits activity towards A2780 cancer cells whereas 13 is inactive, which suggests that the chelating ligand also plays a role. Replacement of neutral bpy by anionic pico as the chelating ligand increases the rate and extent of hydrolysis, the pKa of the aqua complex (from 6.31 to 7.75 for ring = Cpxph , Table 2), and changes the nucleobase specificity. For complexes 7−9 containing neutral bpy, there is exclusive binding to 9-EtG. In contrast, complexes 12−14 containing anionic pico bind strongly to both 9-EtG and 9-EtA. These results are in agreement with previous work on RuII and OsII complexes in which neutral en is replaced by negatively charged acetylacetonate (acac). 26, 38a The rapid hydrolysis, low acidity of aqua adducts and strong binding to biomolecules may account for the low cytotoxicity of the picolinate complexes. The highly reactive aqua species may interact with other cellular biomolecules before they reach target sites, thus effectively being deactivated. For example, the amino acids (methionine or cysteine) or tripeptides can form S-bound adducts with IrIII Cp* complexes.44a Interestingly some ruthenium27a and osmium11a arene complexes containing phen or bpy derivatives show poor or no activity against A2780 cells. The inactivity of phen complex 4 and bpy complex 7 may be correlated with their low extent of hydrolysis and poor cellular accumulation. Although complex 7 may reach the cell nucleus, its hydrolysis is largely suppressed (only 5% hydrolyzed in the presence of 4 mM NaCl), and the complex therefore remains predominately as the less-reactive intact chlorido species. These factors may explain the inactivity of 4 and 7, and also the inactivity of 10. Conclusions The goal of the present study was to explore the rational design of monofunctional 29 organometallic half-sandwich IrIII anticancer complexes based on knowledge of the features which contribute to the activity of half-sandwich RuII and OsII arene complexes.2, 24a, 52 The biological and medicinal chemistry of iridium complexes has been little explored previously,6-7 perhaps because it is often assumed that low-spin 5d6 IrIII complexes are highly kinetically inert.5, 9-10 Our data show that this is not always the case. Cyclopentadienyl ligands, whilst stabilizing Ir III, can confer kinetic lability on trans monodentate ligands such as chloride. Moreover phenyl substituents on the Cp* ring as in Cpxph and Cpxbiph can have a major effect on the chemical and biological behavior of [(η5 -Cpx )Ir(XY)Cl]0/+ complexes. This appears to be the first time that Cpxph and Cpxbiph ligands have been used in iridium complexes. The crystal structure of complex [(η5 -C5 Me4 C6 H4 C6 H5 )Ir(bpy)Cl]PF6 (9·PF6 ) appears to be the first reported structure containing the ligand Cpxbiph . The crystal structures of [(η5 -C5 Me4 C6 H5 )IrCl2 ]2 (2), [(η5 -C5 Me4 C6 H5 )Ir(phen)Cl]PF6 (5·PF6 ), [(η5 -C5 Me4 C6 H5 )Ir(bpy)Cl]PF6 (8·PF6 ), [(η5 -C5 Me4 C6 H5 )Ir(en)Cl]BPh4 (11·BPh4 ) and [(η5 -C5 Me4 C6 H5 )Ir(pico)Cl] (13) are the first to be reported with η5 -C5 Me4 C6 H5 coordinated to iridium. Figure 7 provides an overview of the relationships between cancer cell cytotoxicity, intercalative ability, cellular accumulation, hydrophobicity, and rates and extents of hydrolysis for the phen complexes 4–6. Figure 8 shows how the chelating and Cp x ligands affect the anticancer activity, nucleobase binding, and aqueous chemistry of the bpy complexes 7–9 and pico complexes 12–14. These [(η5 -Cpx )Ir(XY)Cl]0/+ complexes hydrolyze rapidly (the slowest half- life < 4 min at 310 K), and the nature of the cyclopentadienyl and chelating ligands significantly influence their aqueous chemistry. In general, the introduction of phenyl and biphenyl Cp* ring substituents slows down the hydrolysis rate, increases the extent of hydrolysis, and increases the acidity of the respective aqua species. The chelating ligand appears to determine the selectivity of nucleobase binding. The complexes containing N,N- chelating ligands discriminate strongly between the purine nucleobases guanine and adenine, showing little binding to the latter for either for Cp*, Cpxph or Cpxbiph complexes. In contrast, 30 complexes 12−14 containing the N,O- chelating ligand picolinate bind both to 9-EtG and 9-EtA. 150 100 100 >100 IC50 50 (μM)50 Activity 6.7 00 1.12 1.12 0.72 1 2 Viscosity 1.106 3 1.056 1.05 1.05 1.032 0.98 0.98 100 100 1 Ir ng/106 cells η/η0 Series1 2 88.8 3 Cellular Accumulation 50 50 23.5 1.500 3.9 1 2 11 Hydrophobicity 0.5 0.5 0.48 Log P 00 1.11 3 -0.82 -0.5 -0.5 0.29 -1 0.3 0.3 0.2 0.2 Kaq 0.1 0.1 00 45 45 Hydrolysis Equilibrium Constant 0.04 1 2 Hydrolysis Rate 30 (288 K) t1/2 30 (min) 15 15 00 0.06 3 31.8 S… 25.4 Series1 <1 1 2 3 Figure 7. Bar charts illustrating the relationship between cytotoxicity toward human cancer cells, intercalative ability, cellular accumulation, hydrophobicity, and rates (288 K) and equilibrium constants (278 K) of hydrolysis for iridium complexes [(η5 -Cpx )Ir(phen)Cl]+ containing Cpx = Cp* (4), Cpxph (5), and Cpxbiph (6). 31 pico (N,O-) bpy(N,N-) >100 >100 >100 IC50 / μM G/A adducts (%) G G G A A A <5 <5 <5 12 13 14 G G G A A A pKa t1/2 (min) (278K) <5 7 8 9 Cp* Cpxph Cpxbiph Cp* Cpxph Cpxbiph Figure 8. Bar charts illustrating the influence of N,N- and N,O-chelating ligands on the cytotoxicity, nucleobase binding, hydrolysis and pKa of the aqua adducts of the bpy complexes 7–9, and pico complexes 12–14. The introduction of a phenyl substituent into the Cp* ring switches on cancer cell cytotoxicity. For example, the tetramethyl(phenyl)cyclopentadienyl complex 5 is more than one order of magnitude more potent than the pentamethylcyclopentadienyl complex 4, and the biphenyl complex 6 is more than 2 orders of magnitude more potent than complex 4, and twice as potent as cisplatin in the same cell line. This increase in activity parallels the increase in hydrophobicity, increase in cell accumulation and DNA binding. Complexes 5 and 6 can exhibit dual mode binding to DNA: iridium binding to G N7 accompanied by intercalation of the phenyl substituents on the Cp* ring. Significant increases in activity for complexes 7–9, 10–11 and 12–14 are also observed. On the other hand, the chelating ligand can also 32 play an important role in the anticancer activity. For example, the pico complex 13 shows no activity toward the A2780 cell line while the bpy analogue complex 8 shows good activity, and the bpy complex 9 is more than one order of magnitude more potent than the pico analogue, complex 14. The work reported here demonstrates that rational chemical design can be applied to IrIII complexes to achieve potent cancer cell cytotoxicity. It is notable that Cp ring substituents can also play a major role in controlling the chemical and biological properties of ferrocenyl and titanocenyl anticancer complexes.53 In general, organometallic complexes offer much promise for the design of novel therapeutic agents.2, 24a, 52a Experime ntal Section Details of the materials used and the synthesis and characterization of complexes are in the Supporting Information. The general synthetic route to the iridium complexes involved reaction of 1,10-phenanthroline, 2,2′-bipyridine, ethylenediamine, or 2-picolinate, with the appropriate dimer [(η5 -Cpx )IrCl2 ]2 in methanol. CHN elemental analyses were carried out for all complexes and synthesized ligands, by which their purity were confirmed to be in excess of 97.0%. Methods and Instrumentation. X-ray crystallography. All diffraction data were obtained on an Oxford Diffraction Gemini four-circle system with a Ruby CCD area detector using Mo Kα radiation. Absorption corrections were applied using ABSPACK. 54 The crystals were mounted in oil and held at 100(2) K with the Oxford Cryosystem Cobra. The structures were solved by direct methods using SHELXS (TREF) 55 with additional light atoms found by Fourier methods. Complexes were refined against F2 using SHELXL56 , and hydrogen atoms were added at calculated positions and refined riding on their parent atoms. X-ray crystallographic data for complexes 2, 5·PF6 , 8·PF6 , 9·PF6 , 11·BPh4 and 13 are available as Supporting Information and have been deposited in the Cambridge 33 Crystallographic Data Centre under the accession numbers CCDC 802289, 802288, 802287, 802291, 802290, and 802286, respectively. NMR Spectroscopy. 1 H NMR spectra were acquired in 5 mm NMR tubes at 298 K (unless stated otherwise) on either a Bruker DPX 400 (1 H = 400.03 MHz) or AVA 600 (1 H = 600.13 MHz) spectrometers. 1 H NMR chemical shifts were internally referenced to (CHD2 )(CD3 )SO (2.50 ppm) for DMSO-d6 , CHCl3 (7.26 ppm) for chloroform-d1 or to 1,4-dioxane (3.75 ppm) for aqueous solutions. All data processing was carried out using XWIN-NMR version 3.6 (Bruker UK Ltd.). Mass Spectrometry. Electrospray ionization mass spectra (ESI-MS) were obtained by preparing the samples in 50% CH3 CN and 50% H2 O (v/v) and infusing into the mass spectrometer (Bruker Esquire 2000). The mass spectra were recorded with a scan range of m/z 50–1000 for positive ions. Ele mental Analysis. CHN elemental analyses were carried out on a CE-440 elemental analyzer by Exeter Analytical (UK) Ltd. pH Measurement. pH* values (pH meter reading without correction for effects of deuterium on glass electrode) of NMR samples in D2 O were measured at ca. 298 K directly in the NMR tube, before and after recording NMR spectra, using a Corning 240 pH meter equipped with a micro combination electrode calibrated with Aldrich buffer solutions of pH 4, 7 and 10. UV-Vis Spectroscopy. A Cary 300 UV-Vis recording spectrophotometer was used with 1 cm path-length quartz cuvettes (0.5 mL) and a PTP1 Peltier temperature controller. Spectra were processed using UVWinlab software. Experiments were carried out at 288 K unless otherwise stated. Kinetics of Hydrolysis. Solutions of complexes 4–10 and 12–14 with final concentrations of 0.2–0.7 mM in 5% MeOD-d4 /95% D2 O (v/v) were prepared by dissolution of the complexes in MeOD-d4 followed by rapid dilution with D2 O. 1 H NMR spectra were recorded after various time intervals. The rates of hydrolysis were determined by fitting plots of concentrations (determined from 1 H NMR peak integrals) versus time to a first-order rate equation using ORIGIN version 8.1. The 34 hydrolysis of complexes 4, 7, 10, 12, 13 and 14 was monitored by 1 H NMR at 278 K (to slow down the rate and avoid freezing the samples), and for complexes 5, 6, 8 and 9 at 278, 283, 288 and 293 K. The Arrhenius equation ln(k) = ln(A) – Ea/RT was used to calculate the hydrolysis rate constants and half- lives of 5, 6, 8 and 9 at 310 K. Determination of pKa Values. To generate the aqua complexes, chlorido complexes were dissolved in D2 O and 0.98 mol equiv of AgNO3 were added. The solution was stirred for 24 h at 298 K, and AgCl was removed by filtration. For determinations of pKa* values (pKa values for solutions in D2 O) the pH* values of solutions of the aqua complexes in this study in D2 O were varied from ca. pH* 2 to 11 by the addition of dilute NaOD and DClO 4 , and 1 H NMR spectra were recorded. The chemical shifts of the chelating ligand protons and/or methyl protons of Cpx were plotted against pH*. The pH* titration curves were fitted to the Henderson-Hasselbalch equation, with the assumption that the observed chemical shifts are weighted averages according to the populations of the protonated and deprotonated species. These pKa* values can be converted to pKa values by use of the equation pKa = 0.929pKa* + 0.42 as suggested by Krezel and Bal20 for comparison with related values in the literature. Computational Details. The Gaussian 03 package57 was employed for all calculations. Geometry optimization calculations for complexes 4–14, aqua complexes 6A and 14A, and 9-ethylguanine (9-EtG) adduct 7G were performed in the gas phase with the gradient-corrected correlation functional PBE0.58 The LanL2DZ basis set and effective core potential59 were used for the Ir atom and the 6-31G** basis set was used for all other atoms.60 The nature of all stationary points was confirmed by performing a normal- mode analysis. Electrostatic potential surfaces (EPS) for complexes 4–6, 12–14, 6A and 14A were calculated and mapped on electron density (isovalue 0.004) of the molecules. The electrostatic potential is represented with a color scale ranging from red (–0.100 au) to blue (0.150 au). Inte ractions with Nucleobases. The reaction of complexes 4–14 and 5A (ca. 1 mM) with nucleobases typically involved addition of a solution containing one mol 35 equiv of nucleobase in D2 O to an equilibrium solution of complexes 4–14 in 5% MeOD-d4 /95% D2 O (v/v), or to a solution of the aqua complex 5A (prepared by the addition of 1 mol equiv of AgNO 3 to a solution of 5 and removal of AgCl by filtration). 1 H NMR spectra of these solutions were recorded at 310 K after various time intervals. ICP-MS Instrumentation and Calibration. All ICP-MS analyses were carried out on an Agilent Technologies 7500 series ICP-MS instrument. The water used for ICP-MS analysis was doubly deionized (DDW) using a Millipore Milli-Q water purification system and a USF Elga UHQ water deionizer. The iridium Specpure plasma standard (Alfa Aesar, 1000 ppm in 10% HCl) was diluted with 3% HNO 3 DDW to freshly prepare calibrants at concentrations 1000, 800, 400, 200, 100, 50, 10, 1 and 0.1 ppb. The ICP-MS instrument was set to detect 193 Ir with typical detection limits of ca. 8 ppt using no gas mode. Cell Culture and Cytotoxicity. The A2780 ovarian cell line was obtained from the ECACC (European Collection of Animal Cell Culture, Salisbury, UK). The cells were maintained in RPMI 1640 media which was supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin. All cells were grown at 310 K in a humidified atmosphere containing 5% CO 2 . Stock solutions of the IrIII complexes were firstly prepared in DMSO to assist dissolution, and then diluted into 0.9% saline and medium (1:1). After plating 5000 A2780 cells per well on day 1, IrIII complexes were added to the cancer cells on day 3 at concentrations ranging from 0.05 μM to 100 μM, depending on the preliminary activity data obtained in screening assays. Cells were exposed to the complexes for 24 h, washed with PBS, supplied with fresh medium, allowed to grow for three doubling times (72 h), and then the protein content (proportional to cell survival) measured using the sulforhodamine B (SRB) assay. 61 The standard errors are based on two independent experiments of three replicates each. log P Determination. Octanol-saturated water (OSW) and water-saturated octanol (WSO) were prepared using analytical grade octanol and 0.2 M aqueous NaCl 36 solution (to suppress hydrolysis of the chlorido complexes). Aliquots of stock solutions of iridium complexes in OSW were added to equal volumes of WSO and shaken in an IKA Vibrax VXC basic shaker for 4 h at 500 g/min to allow partition at ambient temperature (∼298 K). The aqueous layer was carefully separated from the octanol layer for iridium analysis. 193 Ir was quantified from aliquots taken from the octanol-saturated aqueous samples before and after partition. Partition coefficients of IrIII complexes were calculated using the equation log P = log ([Ir]WSO/[Ir]OSW ), where [Ir]WSO was obtained by subtraction of the Ir content of the aqueous layer after partition from the Ir content of the aqueous layer before partition. Cell Accumulation, Cellular Distribution, and DNA Binding in A2780 Human Carcinoma Cells. A2780 cells were plated at a density of 5×106 cells/100 mm Petri dish in 9 mL of culture medium on day 1 (three dishes were prepared per compound tested, and three untreated control dishes, in two independent experiments). On day 2 cells were exposed to the IrIII complexes 46. Stock solutions of the iridium compounds were prepared fresh in DMSO and diluted in 0.9% saline and medium (1:1; 0.5% v/v DMSO final concentration) to a final concentration of Ir on the plates of 5 μM. After 24 h of drug exposure at 310 K on a 5% CO 2 incubator, the drug-containing medium was removed and the cells were washed, trypsinized, and counted using a haemocytometer. One-third of the cells were centrifuged, quickly washed with PBS, and stored at 253 K for determination of total cell accumulation (the net effect of uptake and efflux) of iridium. Another third of the samples was used for cytosol, nucleus, membrane/particulate and cytoskeleton fractionation, using a FractionPREP™ cell fractionation kit from BioVision (Mountain View, CA). The last third of the samples was used for quantification of Ir bound to DNA using the Nucleon genomic DNA extraction kit (GE healthcare, Amersham, UK; BACC-1 protocol). All the cell pellets and solid cell fractions were digested in freshly distilled 72% HNO 3 in Wheaton V-Vials with a PTFE-faced rubber lined cap (Sigma-Aldrich) for 16 h at 373 K. After cooling, the samples were diluted with DDW to a maximum final concentration of 7.2% HNO 3 (suitable for ICP-MS analysis) prior quantification 37 of iridium. Sequence Preference of DNA Adducts. The primer extension footprinting assay was used to evaluate the sequence selectivity of DNA modification by complexes 4–6. A fragment of pSP73KB DNA linearized by HpaI (2464 bp) was incubated with IrIII complexes in 10 mM NaClO 4 for 24 h at 310 K to obtain rb = 0.01 (bound Ir/base). The excess of drug was removed by ethanol precipitation. Circum VentTM Thermal Cycle Sequencing Kit with Vent(exo -) DNA polymerase was used along with the protocol for thermal cycle DNA sequencing with 5′ end- labeled 20-mer SP6 primer recommended by the manufacturer with small modifications. 62 The synthesis products were separated by electrophoresis on a denaturing polyacrylamide (PAA) gel [6% polyacrylamide (PAA)/8M urea]; sequence ladders were obtained in parallel using untreated control DNA fragment. Fluorescence Measurements. These measurements were performed on a Shimadzu RF 40 spectrofluorophotometer using a 1 cm quartz cell. Fluorescence measurements of CT DNA modified by IrIII complexes, cisplatin or [Pt(dien)Cl]Cl (dien = diethylenetriamine), in the presence of EtBr, were performed at an excitation wavelength of 546 nm, and the emitted fluorescence was analyzed at 590 nm. The fluorescence intensity was measured at 298 K in 0.4 M NaCl to avoid secondary binding of EtBr to DNA.63 The concentrations were 0.01 mg/mL for DNA and 0.04 mg/mL for EtBr, which corresponded to the saturation of all intercalation sites for EtBr in DNA.63a Viscometry. The relative viscosity of the solutions of CT DNA nonmodified or modified by complexes 4–6 at the concentration of 150 µg/mL was measured by microviscometry (AMVn Automated Micro Viscometer, Anton Paar GmbH, Austria) using a 1.6- mm capillary tube at 310 K. The densities of the solutions were measured using a Density Meter DMA 4500 instrument (Anton Paar GmbH, Austria). Acknowledge ments. Z.L. was supported by the University of Warwick Research Scholarship. L.S. by 38 ERC BIOINCMED and MC-IEF (L.S. 220281 PHOTORUACD), P.C.A.B. by the Netherlands Organisation for Scientific Research through a Rubicon Scholarship, and V.B. by the Czech National Foundation (Grant nos. P303/11/P047 and P301/10/0598). We thank the ERC (grant no. 247450 for P.J.S.), EPSRC, ORSAS, ERDF and AWM for Science City funding, and members of COST Action D39 for stimulating discussions. Supporting Information Available: Details of the materials used, synthesis and characterization of ligands and complexes, crystallographic data (Tables S1, S2, and Figures S1–S4), calculations (Tables S3, S4, S6 and S18), aqueous chemistry (Figures S5–S12), nucleobase studies (Figures S13–S17), distribution in cell fractions (Table S5). This material is available free of charge via the Internet at http://pubs.acs.org. X-ray crystallographic data in CIF format are available from the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/). 39 References (1) (a) Kelland, L. The Resurgence of Platinum- Based Cancer Chemotherapy. Nat. Rev. Cancer 2007, 7, 573-584. (b) Wang, D.; Lippard, S. J. Cellular Processing of Platinum Anticancer Drugs. Nat. Rev. Drug. Discov. 2005, 4, 307-320. (2) (a) Halpern, J. Organometallic Chemistry at the Threshold of a New Millennium. Retrospect and Prospect. Pure Appl. Chem. 2001, 73, 209-220. (b) Fish, R. H.; Jaouen, G. Bioorganometallic Chemistry: Structural Diversity of Organometallic Complexes with Bioligands and Molecular Recognition Studies of Several Supramolecular Hosts with Biomolecules, Alkali- Metal Ions, and Organometallic Pharmaceuticals. Organometallics 2003, 22, 2166-2177. (c) Jaouen, G.; Metzler-Nolte, N., Eds. Medicinal Organometallic Chemistry (Topics in Organometallic Chemistry), 1st ed.; Springer-Verlag: Heidelberg, Germany, 2010; Vol. 32. (d) Hartinger, C. G.; Dyson, P. J. Bioorganometallic Chemistry—from Teaching Paradigms to Medicinal Applications. Chem. Soc. Rev. 2009, 38, 391-401. (e) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Organometallic Chemistry, Biology and Medicine: Ruthenium Arene Anticancer Complexes. Chem. Commun. 2005, 4764-4776. (f) Suss-Fink, G. Arene Ruthenium Complexes as Anticancer Agents. Dalton Trans. 2010, 39, 1673-1688. (3) (a) Mendoza-Ferri, M. G.; Hartinger, C. G.; Mendoza, M. A.; Groessl, M.; Egger, A. E.; Eichinger, R. E.; Mangrum, J. B.; Farrell, N. P.; Maruszak, M.; Bednarski, P. J.; Klein, F.; Jakupec, M. A.; Nazarov, A. A.; Severin, K.; Keppler, B. K. Transferring the Concept of Multinuclearity to Ruthenium Complexes for Improvement of Anticancer Activity. J. Med. Chem. 2009, 52, 916-925. (b) Loughrey, B. T.; Healy, P. C.; Parsons, P. G.; Williams, M. L. Selective Cytotoxic Ru(II) Arene Cp* Complex Salts [R-PhRuCp*]+X− for X = BF4 −, PF6 −, and BPh4 −. Inorg. Chem. 2008, 47, 8589-8591. (c) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris, R. E.; Chen, H.; Sadler, P. J.; Jodrell, D. I. In Vitro and in Vivo Activity and Cross Resistance Profiles of Novel Ruthenium (II) 40 Organometallic Arene Complexes in Human Ovarian Cancer. Br. J. Cancer 2002, 86, 1652-1657. (4) (a) Tobe, M. L.; Burgess, J. Inorganic Reaction Mechanisms; Addison Wesley Longman Inc.: Essex, 1999. (b) Lay, P. A.; Harman, W. D. In Advances in Inorganic Chemistry; Sykes, A. G., Ed.; Academic Press: 1991; Vol. 37, pp 219-379. (5) (a) Helm, L.; Merbach, A. E. Water Exchange on Metal Ions: Experiments and Simulations. Coord. Chem. Rev. 1999, 187, 151-181. (b) Richens, D. T. Ligand Substitution Reactions at Inorganic Centers. Chem. Rev. 2005, 105, 1961-2002. (6) (a) Cleare, M. J. Transition Metal Complexes in Cancer Chemotherapy. Coord. Chem. Rev. 1974, 12, 349-405. (b) Sava, G.; Giraldi, T.; Mestroni, G.; Zassinovich, G. Antitumor Effects of Rhodium(I), Iridium(I) and Ruthenium(II) Complexes in Comparison with cis-dichlorodiammino Platinum(II) in Mice Bearing Lewis Lung Carcinoma. Chemico-Biological Interactions 1983, 45, 1-6. (c) Sava, G.; Zorzet, S.; Perissin, L.; Mestroni, G.; Zassinovich, G.; Bontempi, A. Coordination Metal Complexes of Rh(I), Ir(I) and Ru(II): Recent Advances on Antimetastatic Activity on Solid Mouse Tumors. Inorg. Chim. Acta 1987, 137, 69-71. (d) Giraldi, T.; Sava, G.; Mestroni, G.; Zassinovich, G.; Stolfa, D. Antitumour Action of Rhodium (I) and Iridium (I) Complexes. Chemico-Biological Interactions 1978, 22, 231-238. (e) Köpf-Maier, P. Complexes of Metals Other Than Platinum as Antitumour Agents. Eur. J. Clin. Pharmacol. 1994, 47, 1-16. (7) (a) Casini, A.; Edafe, F.; Erlandsson, M.; Gonsalvi, L.; Ciancetta, A.; Re, N.; Ienco, A.; Messori, L.; Peruzzini, M.; Dyson, P. J. Rationalization of The Inhibition Activity of Structurally Related Organometallic Compounds Against the Drug Target Cathepsin B by DFT. Dalton Trans. 2010, 39, 5556-5563. (b) Amouri, H.; Moussa, J.; Renfrew, A. K.; Dyson, P. J.; Rager, M. N.; Chamoreau, L.-M. Discovery, Structure, and Anticancer Activity of an Iridium Complex of Diselenobenzoquinone. Angew. Chem., Int. Ed. 2010, 49, 7530-7533. (c) Ali Nazif, M.; Bangert, J.-A.; Ott, I.; Gust, R.; Stoll, R.; Sheldrick, W. S. Dinuclear 41 Organoiridium(III) mono- and bis- intercalators with Rigid Bridging Ligands: Synthesis, Cytotoxicity and DNA Binding. J. Inorg. Biochem. 2009, 103, 1405-1414. (d) Wirth, S.; Rohbogner, C.; Cieslak, M.; Kazmierczak-Baranska, J.; Donevski, S.; Nawrot, B.; Lorenz, I.-P. Rhodium(III) and Iridium(III) Complexes with 1,2-naphthoquinone-1-oximate as a Bidentate Ligand: Synthesis, Structure, and Biological Activity. J. Biol. Inorg. Chem. 2010, 15, 429-440. (e) Gras, M.; Therrien, B.; Süss-Fink, G.; Casini, A.; Edafe, F.; Dyson, P. J. Anticancer Activity of New Organo-Ruthenium, Rhodium and Iridium Complexes Containing the 2-(pyridine-2-yl)thiazole N,N-chelating Ligand. J. Organomet. Chem. 2010, 695, 1119-1125. (f) Schäfer, S.; Sheldrick, W. S. Coligand Tuning of the DNA Binding Properties of Half-Sandwich Organometallic Intercalators: Influence of Polypyridyl (pp) and Monodentate Ligands (L = Cl, (NH2 )2 CS, (NMe2 )2 CS) on the (η5 -pentamethylcyclopentadienyl)- iridium(III)- Intercalation of dipyridoquinoxaline and -dipyridophenazine Complexes. J. Organomet. Chem. 2007, 692, 1300-1309. (g) Kokoschka, M.; Bangert, J. A.; Stoll, R.; Sheldrick, W. S. Sequence-Selective Organoiridium DNA bis- intercalators with Flexible Dithiaalkane Linker Chains. Eur. J. Inorg. Chem. 2010, 1507-1515. (h) Hartinger, C. G. Trapping Unstable Benzoquinone Analogues by Coordination to a [(η5 -C5 Me5 )Ir] Fragment and the Anticancer Activity of the Resulting Complexes. Angew. Chem., Int. Ed. 2010, 49, 8304-8305. (i) Leung, S.-K.; Kwok, K. Y.; Zhang, K. Y.; Lo, K. K.-W. Design of Luminescent Biotinylation Reagents Derived from Cyclometalated Iridium(III) and Rhodium(III) Bis(pyridylbenzaldehyde) Complexes. Inorg. Chem. 2010, 49, 4984-4995. (j) Shao, F.; Barton, J. K. Long-Range Electron and Hole Transport through DNA with Tethered Cyclometalated Iridium(III) Complexes. J. Am. Chem. Soc. 2007, 129, 14733-14738. (k) Sliwinska, U.; Pruchnik, F. P.; Ulaszewski, S.; Latocha, M.; Nawrocka-Musial, D. Properties of η5 -pentamethylcyclopentadienyl Rhodium(III) and Iridium(III) Complexes with Quinolin-8-ol and Their Cytostatic Activity. Polyhedron 2010, 29, 1653-1659. (8) Wilbuer, A.; Vlecken, D. H.; Schmitz, D. J.; Kräling, K.; Harms, K.; Bagowski, C. 42 P.; Meggers, E. Iridium Complex with Antiangiogenic Properties. Angew. Chem., Int. Ed. 2010, 49, 3839-3842. (9) Messori, L.; Marcon, G.; Orioli, P.; Fontani, M.; Zanello, P.; Bergamo, A.; Sava, G.; Mura, P. Molecular Structure, Solution Chemistry and Biological Properties of the Novel [ImH][trans-IrCl4 (Im)(DMSO)], (I) and of the Orange Form of [(DMSO)2 H][trans-IrCl4 (DMSO)2 ], (II), Complexes. J. Inorg. Biochem. 2003, 95, 37-46. (10) Marcon, G.; Casini, A.; Mura, P.; Messori, L.; Bergamo, A.; Orioli, P. Biological Properties of IRIM, the Iridium(III) Analogue of (Imidazolium (Bisimidazole) Tetrachlororuthenate) (ICR). Metal-Based Drugs 2000, 7, 195-200. (11) (a) Peacock, A. F. A.; Habtemariam, A.; Moggach, S. A.; Prescimone, A.; Parsons, S.; Sadler, P. J. Chloro Half-Sandwich Osmium(II) Complexes: Influence of Chelated N,N-Ligands on Hydrolysis, Guanine Binding, and Cytotoxicity. Inorg. Chem. 2007, 46, 4049-4059. (b) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. Highly Selective Binding of Organometallic Ruthenium Ethylenediamine Complexes to Nucleic Acids: Novel Recognition Mechanisms. J. Am. Chem. Soc. 2003, 125, 173-186. (12) (a) Liu, H.-K.; Berners-Price, S. J.; Wang, F.; Parkinson, J. A.; Xu, J.; Bella, J.; Sadler, P. J. Diversity in Guanine-Selective DNA Binding Modes for an Organometallic Ruthenium Arene Complex. Angew. Chem., Int. Ed. 2006, 45, 8153-8156. (b) Liu, H.-K.; Parkinson, J. A.; Bella, J.; Wang, F.; Sadler, P. J. Penetrative DNA Intercalation and G-base Selectivity of an Organometallic Tetrahydroanthracene RuII Anticancer Complex. Chem. Sci., 2010, 258-270. (13) Liu, S.; Rebros, M.; Stephens, G.; Marr, A. C. Adding Value to Renewables: a One Pot Process Combining Microbial Cells and Hydrogen Transfer Catalysis to Utilise Waste Glycerol from Biodiesel Production. Chem. Commun. 2009, 2308-2310. (14) (a) Hanasaka, F.; Fujita, K.-i.; Yamaguchi, R. Synthesis of New Iridium N-Heterocyclic Carbene Complexes Bearing a Functionalized Cp* Ligand and Their High Catalytic Activities in the Oppenauer-Type Oxidation of Alcohol. 43 Organometallics 2006, 25, 4643-4647. (b) Ito, M.; Endo, Y.; Tejima, N.; Ikariya, T. Bifunctional Triflylamide-Tethered Cp′Rh and Cp′Ir Complexes: A New Entry for Asymmetric Hydrogenation Catalysts. Organometallics 2010, 29, 2397-2399. (c) Kohl, G.; Pritzkow, H.; Enders, M. Rhodium(III) and Iridium(III) Complexes with Quinolyl-Functionalized Cp Ligands: Synthesis and Catalytic Hydrogenation Activity. Eur. J. Inorg. Chem. 2008, 4230-4235. (d) Pontes da Costa, A.; Viciano, M.; Sanaú, M.; Merino, S.; Tejeda, J.; Peris, E.; Royo, B. First Cp*-Functionalized N-Heterocyclic Carbene and Its Coordination to Iridium. Study of the Catalytic Properties. Organometallics 2008, 27, 1305-1309. (15) Liu, Z.; Habtemariam, A.; Pizarro, A. M.; Fletcher, S.; Clarkson, G.; Sadler, P. J. ISBOMC´ 10, 5th International Symposium on Bioorganometallic Chemistry ; Ruhr-University: Bochum, Germany, 2010; p 32. (16) Synthesis of the IrIII complex containing Cpxbiph and ethylenediamine proved to be difficult and we are not able to report on this complex. (17) Pizarro, A. M.; Habtemariam, A.; Sadler, P. J. In Medicinal Organometallic Chemistry (Topics in Organometallic Chemistry), 1st ed.; Jaouen, G., Metzler-Nolte, N., Ed.; Springer-Verlag: Heidelberg, Germany, 2010; Vol. 32, pp 21-56. (18) (a) Hohmann, H.; Hellquist, B.; Van Eldik, R. Kinetics and Mechanism of the Complex Formation Reactions of Diaqua(ethylenediamine)- and Diaqua(tetraethylethylenediamine)palladium(II) with the Purine Nucleosides Adenosine and Inosine. Inorg. Chem. 1992, 31, 345-351. (b) Martin, R. B. In Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug ; Lippert, B., Ed.; VHCA & Wiley-VCH: Zürich, Switzerland, 1999; Vol., pp 181-205. (19) Jennerwein, M.; Andrews, P. A. Effect of Intracellular Chloride on the Cellular Pharmacodynamics of cis-diamminedichloroplatinum(II). Drug Metab. Dispos. 1995, 23, 178-184. (20) Krezel, A.; Bal, W. A Formula for Correlating pKa Values Determined in D2 O and H2 O. J. Inorg. Biochem. 2004, 98, 161-166. (21) (a) Zhang, C. X.; Lippard, S. J. New Metal Complexes as Potential Therapeutics. 44 Curr. Opin. Chem. Biol. 2003, 7, 481-489. (b) Deubel, D. V.; Lau, J. K.-C. In Silico Evolution of Substrate Selectivity: Comparison of Organometallic Ruthenium Complexes with the Anticancer Drug Cisplatin. Chem. Commun. 2006, 2451-2453. (22) (a) Mendoza-Ferri, M.-G.; Hartinger, C. G.; Eichinger, R. E.; Stolyarova, N.; Severin, K.; Jakupec, M. A.; Nazarov, A. A.; Keppler, B. K. Influence of the Spacer Length on the in Vitro Anticancer Activity of Dinuclear Ruthenium−Arene Compounds. Organometallics 2008, 27, 2405-2407. (b) Gramatica, P.; Papa, E.; Luini, M.; Monti, E.; Gariboldi, M.; Ravera, M.; Gabano, E.; Gaviglio, L.; Osella, D. Antiproliferative Pt(IV) Complexes: Synthesis, Biological Activity, and Quantitative Structure–Activity Relationship Modeling. J. Biol. Inorg. Chem. 2010, 15, 1157-1169. (23) (a) Loh, S. Y.; Mistry, P.; Kelland, L. R.; Abel, G.; Harrap, K. R. Reduced Drug Accumulation as a Major Mechanism of Acquired Resistance to Cisplatin in a Human Ovarian Carcinoma Cell line: Circumvention Studies Using Novel Platinum (II) and (IV) Ammine/amine Complexes. Br. J. Cancer 1992, 66, 1109-1115. (b) Oldfield, S. P.; Hall, M. D.; Platts, J. A. Calculation of Lipophilicity of a Large, Diverse Dataset of Anticancer Platinum Complexes and the Relation to Cellular Uptake. J. Med. Chem. 2007, 50, 5227-5237. (24) (a) Jaouen, G., Eds. Bioorganometallics: Biomolecules, Labeling, Medicine; Wiley-VCH: Weinheim, Germany, 2006. (b) Chen, H.; Parkinson, J. A.; Parsons, S.; Coxall, R. A.; Gould, R. O.; Sadler, P. J. Organometallic Ruthenium(II) Diamine Anticancer Complexes: Arene-Nucleobase Stacking and Stereospecific Hydrogen-Bonding in Guanine Adducts. J. Am. Chem. Soc. 2002, 124, 3064-3082. (c) Kostrhunova, H.; Florian, J.; Novakova, O.; Peacock, A. F. A.; Sadler, P. J.; Brabec, V. DNA Interactions of Monofunctional Organometallic Osmium(II) Antitumor Complexes in Cell-Free Media. J. Med. Chem. 2008, 51, 3635-3643. (25) Novakova, O.; Chen, H.; Vrana, O.; Rodger, A.; Sadler, P. J.; Brabec, V. DNA Interactions of Monofunctional Organometallic Ruthenium(II) Antitumor 45 Complexes in Cell-Free Media. Biochemistry 2003, 42, 11544-11554. (26) Fernández, R.; Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler, P. J. Use of Chelating Ligands to Tune the Reactive Ruthenium(II)–Arene Anticancer Complexes. Site of Chem.-Eur. Half-Sandwich J. 2004, 10, 5173-5179. (27) (a) Habtemariam, A.; Melchart, M.; Fernández, R.; Parsons, S.; Oswald, I. D. H.; Parkin, A.; Fabbiani, F. P. A.; Davidson, J. E.; Dawson, A.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. Structure−Activity Relationships for Cytotoxic Ruthenium(II) Arene Complexes Containing N,N-, N,O-, and O,O-Chelating Ligands. J. Med. Chem. 2006, 49, 6858-6868. (b) The parallel for Pt is the incorporation of intercalating side-arms into square-planar PtII complexes, for example, Sundquist, W. J.; Bancroft, D. P.; Lippard, S. J. Synthesis, Characterization, and Biological Activity of cis-diammineplatinum(II) Complexes of the DNA Intercalators 9-aminoacridine and Chloroquine. J. Am. Chem. Soc. 1990, 112, 1590-1596; Ma, Z.; Choudhury, J. R.; Wright, M. W.; Day, C. S.; Saluta, G.; Kucera, G. L.; Bierbach, U. A. A Non-Cross-Linking Platinum- Acridine Agent with Potent Activity in Non-Small-Cell Lung Cancer. J. Med. Chem. 2008, 51, 7574-75 . (c) Bugarcic, T.; Nov kov , . i.; .; al mikov , A.; erz nkov , .; r na, Ka p rkov , J.; Habtemariam, A.; Parsons, S.; Sadler, P. J.; Brabec, V. Cytotoxicity, Cellular Uptake, and DNA Interactions of New Monodentate Ruthenium(II) Complexes Containing Terphenyl Arenes. J. Med. Chem. 2008, 51, 5310-5319. (28) Cayemittes, S.; Poth, T.; Fernandez, M. J.; Lye, P. G.; Becker, M.; Elias, H.; Merbach, A. E. Mechanistic Investigation on the Water Substitution in the η5 -Organometallic Complexes Cp*Ir(H2 O)3 2+ and Cp*Rh(H2 O)3 2+. Inorg. Chem. 1999, 38, 4309-4316. (29) (a) Dobereiner, G. E.; Crabtree, R. H. Dehydrogenation as a Substrate-Activating Strategy in Homogeneous Transition-Metal Catalysis. Chem. Rev. 2009, 110, 681-703. (b) Oro, L. A.; Claver, C., Eds. Iridium Complexes in Organic Synthesis; Wiley-VCH Verlag GmbH & Co. KgaA: Weinheim, Germany, 2009. 46 (30) Schäfer, S.; Ott, I.; Gust, R.; Sheldrick, W. S. Influence of the Polypyridyl (pp) Ligand Size on the DNA Binding Properties, Cytotoxicity and Cellular Uptake of rganoruthenium(II) Complexes of the Type [(η6 -C6 Me6 )Ru(L)(pp)]n+ [L = Cl, n = 1; L = (NH2 )2 CS, n = 2]. Eur. J. Inorg. Chem. 2007, 3034-3046 recently reported the inactivity of the phenanthroline complex 4 towards MCF-7 breast and HT-29 colon cancer cells; incorporation of strongly intercalating dipyridoquinoxaline and dipyridophenazine ligands gave rise to cytotoxic complexes. The intercalating dipyridyl ligands can lead to initial DNA recognition with the possibility of a switch to nucleobase binding: Schäfer, S.; Sheldrick, W. S. Coligand Tuning of the DNA Binding Properties of Half-Sandwich Organometallic Intercalators: Influence of Polypyridyl (pp) and Monodentate Ligands (L = Cl, (NH2 )2 CS, (NMe2 )2CS) on the Intercalation of (η5 -pentamethylcyclopentadienyl)- iridium(III)- dipyridoquinoxaline and -dipyridophenazine Complexes. J. Organomet. Chem. 2007, 692, 1300-1309. In contrast in the present report dual mode DNA base binding and intercalation is possible for complexes 5 and 6. (31) Churchill, M. R.; Julis, S. A. Crystal Structure and Molecular Geometry of Homogeneous Hydrogenation Catalyst [(η5 -C5 Me5 )IrCl]2 (μ-H)(μ-Cl) and of Its Precursor [(η5 -C5 Me5 )IrCl]2 (μ-Cl)2 . A Direct Comparison of Ir(μ-H)(μ-Cl)Ir and Ir(μ-Cl)2 Ir Bridging Systems. Inorg. Chem. 1977, 16, 1488-1494. (32) Youinou, M.-T.; Ziessel, R. Synthesis and Molecular Structure of a New Family of Iridium(III) and Rhodium(III) Complexes: [(η5 -Me5 C5 )Ir(LL)X]+ and [(η5 -Me5 C5 )Rh(LL)Cl]+ ; LL = 2,2'-bipyridine or 1,10-phenanthroline; X = Cl or . Single Crystal Structures of [(η5 -Me5 C5 )Ir(bpy)Cl]Cl and [(η5 -Me5 C5 )Rh(phen)Cl]ClO 4 . J. Organomet. Chem. 1989, 363, 197-208. (33) Gorol, M.; Roesky, H. W.; Noltemeyer, M.; Schmidt, H.-G. (η5 -Pentamethylcyclopentadienyl)iridium(III) Complexes with η2 -N, and η2 -P,S Ligands. Eur. J. Inorg. Chem. 2005, 4840-4844. (34) Scharwitz, M.; Schäfer, S.; Almsick, T. v.; Sheldrick, W. S. Chlorido(η 47 5 -pentamethylcyclopentadienyl)(1,10-phenanthroline- κ 2 N,N ′ )iridium(III) Trifluoromethanesulfonate. Acta Crystallogr. 2007, E63, m1111- m1113. (35) Koelle, U. Organometallic Aqua Ions of the Transition Metals. Coord. Chem. Rev. 1994, 135-136, 623-650. (36) Bugarcic, T.; Habtemariam, A.; Stepankova, J.; Heringova, P.; Kasparkova, J.; Deeth, R. J.; Johnstone, R. D. L.; Prescimone, A.; Parkin, A.; Parsons, S.; Brabec, V.; Sadler, P. J. The Contrasting Chemistry and Cancer Cell Cytotoxicity of Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes. Inorg. Chem. 2008, 47, 11470-11486. (37) Wang, F.; Habtemariam, A.; van der Geer, E. P. L.; Fernández, R.; Melchart, M.; Deeth, R. J.; Aird, R.; Guichard, S.; Fabbiani, F. P. A.; Lozano-Casal, P.; Oswald, I. D. H.; Jodrell, D. I.; Parsons, S.; Sadler, P. J. Controlling Ligand Substitution Reactions of Organometallic Complexes: Tuning Cancer Cell Cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18269-18274. (38) (a) Peacock, A. F. A.; Habtemariam, A.; Fernández, R.; Walland, V.; Fabbiani, F. P. A.; Parsons, S.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene Complexes under Physiological Conditions. J. Am. Chem. Soc. 2006, 128, 1739-1748. (b) Peacock, A. F. A.; Parsons, S.; Sadler, P. J. Tuning the Hydrolytic Aqueous Chemistry of Osmium Arene Complexes with N,O-Chelating Ligands to Achieve Cancer Cell Cytotoxicity. J. Am. Chem. Soc. 2007, 129, 3348-3357. (39) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.; Sadler, P. J. Kinetics of Aquation and Anation of Ruthenium(II) Arene Anticancer Complexes, Acidity and X-ray Structures of Aqua Adducts. Chem.-Eur. J. 2003, 9, 5810-5820. (40) Wang, F.; Bella, J.; Parkinson, J. A.; Sadler, P. J. Competitive Reactions of a Ruthenium Arene Anticancer Complex with Histidine, Cytochrome c and an Oligonucleotide. J. Biol. Inorg. Chem. 2005, 10, 147-155. (41) Wang, F.; Xu, J.; Habtemariam, A.; Bella, J.; Sadler, P. J. Competition between Glutathione and Guanine for a Ruthenium(II) Arene Anticancer Complex: 48 Detection of a Sulfenato Intermediate. J. Am. Chem. Soc. 2005, 127, 17734-17743. (42) Reedijk, J. Bioinorganic Chemistry Special Feature: New Clues for Platinum Antitumor Chemistry: Kinetically Controlled Metal Binding to DNA Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3611-3616. (43) Fukamachi, T.; Chiba, Y.; Wang, X.; Saito, H.; Tagawa, M.; Kobayashi, H. Tumor Specific Low pH Environments Enhance the Cytotoxicity of Lovastatin and Cantharidin. Cancer Lett. 2010, 297, 182-189. (44) (a) Herebian, D.; Sheldrick, W. S. Synthesis and DNA Binding Properties of Bioorganometallic (η5 -pentamethylcyclopentadienyl)iridium(III) Complexes of the Type [(η5 -C5 Me5 )Ir(Aa)(dppz)]+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine, n = 1-3), with S-coordinated Amino Acids (Aa) or Peptide. J. Chem. Soc., Dalton Trans. 2002, 966-974. (b) Annen, P.; Schildberg, S.; Sheldrick, W. S. (η5 -Pentamethylcyclopentadienyl)iridium(III) Complexes of Purine Nucleobases and Nucleotides: a Comparison with (η6 -arene)ruthenium(II) and (η5 -pentamethylcyclopentadienyl)rhodium(III) Species. Inorg. Chim. Acta 2000, 307, 115-124. (c) Yamanari, K.; Ito, R.; Yamamoto, S.; Konno, T.; Fuyuhiro, A.; Kobayashi, M.; Arakawa, R. Diastereomeric Separations and Crystal Structures of Rhodium(III) and Iridium(III) Complexes Containing Adenosine and Related Nucleosides. Dalton Trans. 2003, 380-386. (d) Yamanari, K.; Ito, R.; Yamamoto, S.; Konno, T.; Fuyuhiro, A.; Fujioka, K.; Arakawa, R. Cyclic Tetramers Composed of Rhodium(III), Iridium(III), or Ruthenium(II) Half-Sandwich and 6-Purinethiones. Inorg. Chem. 2002, 41, 6824-6830. (45) van Rijt, S. H.; Peacock, A. F. A.; Johnstone, R. D. L.; Parsons, S.; Sadler, P. J. Organometallic Osmium(II) Arene Anticancer Complexes Containing Picolinate Derivatives. Inorg. Chem. 2009, 48, 1753-1762. (46) van Rijt, S. H.; Mukherjee, A.; Pizarro, A. M.; Sadler, P. J. Cytotoxicity, Hydrophobicity, Uptake, and Distribution of Osmium(II) Anticancer Complexes in Ovarian Cancer Cells. J. Med. Chem. 2010, 53, 840-849. (47) Jung, Y.; Lippard, S. J. Direct Cellular Responses to Platinum-Induced DNA 49 Damage. Chem. Rev. 2007, 107, 1387-1407. (48) In separate experiments we have shown that complex 6 is significantly more active towards wild-type CHO-K1 cells compared to its mutant cell line MMC-2 which carries an ERCC3/XPB mutation (ERCC3 is an ATP-dependent DNA helicase that plays an essential role in nucleotide excision repair). Thus, cells which are deficient in DNA repair are more sensitive to killing by complex 6. This suggests that the cell must remove or bypass DNA lesions if it is to survive treatment and implies that unrepaired DNA damage contributes to its cytotoxicity. These data will be reported elsewhere. (49) (a) Keck, M. V.; Lippard, S. J. Unwinding of Supercoiled DNA by Platinum- Ethidium and Related Complexes. J. Am. Chem. Soc. 1992, 114, 3386-3390. (b) Kasparkova, J.; Marini, V.; Najajreh, Y.; Gibson, D.; Brabec, V. DNA Binding Mode of the Cis and Trans Geometries of New Antitumor Nonclassical Platinum Complexes Containing Piperidine, Piperazine, or 4-Picoline Ligand in Cell-Free Media. Relations to Their Activity in Cancer Cell Lines. Biochemistry 2003, 42, 6321-6332. (5 ) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Neither Δ- nor Λ-tris(phenanthroline)ruthenium(II) Binds to DNA by Classical Intercalation. Biochemistry 1992, 31, 9319-9324. (51) Morris, R. E.; Aird, R. E.; del Socorro Murdoch, P.; Chen, H.; Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler, P. J. Inhibition of Cancer Cell Growth by Ruthenium(II) Arene Complexes. J. Med. Chem. 2001, 44, 3616-3621. (52) (a) Barry, N. P. E.; Edafe, F.; Dyson, P. J.; Therrien, B. Anticancer Activity of Osmium Metalla-Rectangles. Dalton Trans. 2010, 39, 2816-2820. (b) Peacock, A. F. A.; Sadler, P. J. Medicinal Organometallic Chemistry: Designing Metal Arene Complexes as Anticancer Agents. Chem. Asian J. 2008, 3, 1890-1899. (53) (a) Allen, O. R.; Gott, A. L.; Hartley, J. A.; Hartley, J. M.; Knox, R. J.; McGowan, P. C. Functionalised Cyclopentadienyl Titanium Compounds as Potential Anticancer Drugs. Dalton Trans. 2007, 5082-5090. (b) Tan, Y. L. K.; 50 Pigeon, P.; Hillard, E. A.; Top, S.; Plamont, M.-A.; Vessieres, A.; McGlinchey, M. J.; Muller-Bunz, H.; Jaouen, G. Synthesis, Oxidation Chemistry and Cytotoxicity Studies on Ferrocene Derivatives of Diethylstilbestrol. Dalton Trans. 2009, 10871-10881. (54) CrysAlis PRO; Oxford Diffraction Ltd.: Abington, Oxfordshire, U. K., 2007. (55) Sheldrick, G. M. Phase Annealing in SHELX-90: Direct Methods for Larger Structures. Acta Crystallogr. 1990, A46, 467-473. (56) Sheldrick, G. M. SHELXL-97; University of Göttingen: Göttingen, Germany, 1997. (57) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision D.01; Gaussian, Inc.: Wallingford, CT, USA, 2003. (58) Adamo, C.; Barone, V. Toward Reliable Density Functional Methods without Adjustable Parameters: The PBE0 Model. J. Chem. Phys. 1999, 110, 6158-6170. (59) Hay, P. J.; Wadt, W. R. Ab Initio Effective Core Potentials For Molecular Calculations. Potentials for the Transition Metal Atoms Sc to Hg. J. Chem. Phys. 1985, 82, 270-283. 51 (60) McLean, A. D.; Chandler, G. S. Contracted Gaussian Basis Sets for Molecular Calculations. I. Second Row Atoms, Z=11-18. J. Chem. Phys. 1980, 72, 5639-5648. (61) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. J. Natl. Cancer Inst. 1990, 82, 1107-1112. (62) Novakova, O.; Kasparkova, J.; Vrana, O.; van Vliet, P. M.; Reedijk, J.; Brabec, V. Correlation between Cytotoxicity and DNA Binding of Polypyridyl Ruthenium Complexes. Biochemistry 1995, 34, 12369-12378. (63) (a) Butour, J.-L.; Macquet, J.-P. Differentiation of DNA ·Platinum Complexes by Fluorescence. Eur. J. Biochem 1977, 78, 455-463. (b) Butour, J.- L.; Alvinerie, P.; Souchard, J.-P.; Colson, P.; Houssier, C.; Johnson, N. P. Effect of the Amine Non-Leaving Group on the Structure and Stability of DNA Complexes with cis-[Pt(R-NH2 )2 (NO3 )2 ]. Eur. J. Biochem 1991, 202, 975-980. 52 Table of Contents (TOC) graphic 53