Kathleen E. Prosser

CF3 Derivatives of the Anticancer Ru(III) Complexes KP1019, NKP-1339, and Their Imidazole and Pyridine Analogues Show Enhanced Lipophilicity, Albumin Interactions, and Cytotoxicity.

The Ru(III) complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (NKP-1339) are leading candidates for the next generation of metal-based chemotherapeutics. Trifluoromethyl derivatives of these compounds and their imidazole and pyridine analogues were synthesized to probe the effect of ligand lipophilicity on the pharmacological properties of these types of complexes. Addition of CF3 groups also provided a spectroscopic handle for (19)F NMR studies of ligand exchange processes and protein interactions. The lipophilicities of the CF3-functionalized compounds and their unsubstituted parent complexes were quantified by the shake-flask method to give the distribution coefficient D at pH 7.4 (log D7.4). The solution behavior of the CF3-functionalized complexes was characterized in phosphate-buffered saline (PBS) using (19)F NMR, electron paramagnetic resonance (EPR), and UV-vis spectroscopies. These techniques, along with fluorescence competition experiments, were also used to characterize interactions with human serum albumin (HSA). From these studies it was determined that increased lipophilicity correlates with reduced solubility in PBS but enhancement of noncoordinate interactions with hydrophobic domains of HSA. These protein interactions improve the solubility of the complexes and inhibit the formation of oligomeric species. EPR measurements also demonstrated the formation of HSA-coordinated species with longer incubation. (19)F NMR spectra show that the trifluoromethyl complexes release axial ligands in PBS and in the presence of HSA. In vitro testing showed that the most lipophilic complexes had the greatest cytotoxic activity. Addition of CF3 groups enhances the activity of the indazole complex against A549 nonsmall cell lung carcinoma cells. Furthermore, in the case of the pyridine complexes, the parent compound was inactive against the HT-29 human colon carcinoma cell line but showed strong cytotoxicity with CF3 functionalization. Overall, these studies demonstrate that lipophilicity may be a determining factor in the anticancer activity and pharmacological behavior of these types of Ru(III) complexes.

Anticancer copper pyridine benzimidazole complexes: ROS generation, biomolecule interactions, and cytotoxicity

The Cu(II) complex CuCl2(pbzH), pbzH=2-(2-pyridyl)benzimidazole, and derivatives modified at the non-coordinated nitrogen of the benzimidazole fragment, have been studied as anticancer agents. These compounds show promising cytotoxicity against A549 adenocarcinomic alveolar basal epithelial cells with IC50 values in the range of 5-10μM. Importantly, this activity is higher than either CuCl2·2H2O or the individual ligands, demonstrating that ligand coordination to the Cu(II) centres of the complexes is required for full activity. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopies were used to characterize the solution behaviour of the complexes. These studies demonstrate: (i) two types of solvated species in buffer, (ii) both coordinate and non-coordinate interactions with albumin, and (iii) weak interactions with DNA. Further DNA studies using agarose gel electrophoresis demonstrate strand cleavage by the complexes in the presence of ascorbate, which is mediated by reactive oxygen species (ROS). Through a fluorescence-based in vitro assay, intracellular ROS generation in the A549 cell line was observed; indicating that damage by ROS is responsible for the observed activity of the complexes.

Electronic Structure Description of a Doubly Oxidized Bimetallic Cobalt Complex with Proradical Ligands

The geometric and electronic structure of a doubly oxidized bimetallic Co complex containing two redox-active salen moieties connected via a 1,2-phenylene linker was investigated and compared to an oxidized monomeric analogue. Both complexes, namely, CoL(1) and Co2L(2), are oxidized to the mono- and dications, respectively, with AgSbF6 and characterized by X-ray crystallography for the monomer and by vis-NIR (NIR = near-infrared) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, superconducting quantum interference device (SQUID) magnetometry, and density functional theory (DFT) calculations for both the monomer and dimer. Both complexes exhibit a water molecule coordinated in the apical position upon oxidation. [CoL(1)-H2O](+) displays a broad NIR band at 8500 cm(-1) (8400 M(-1) cm(-1)), which is consistent with recent reports on oxidized Co salen complexes (Kochem, A. et al., Inorg. Chem., 2012, 51, 10557-10571 and Kurahashi, T. et al., Inorg. Chem., 2013, 52, 3908-3919). DFT calculations predict a triplet ground state with significant ligand and metal contributions to the singularly occupied molecular orbitals. The majority (∼75%) of the total spin density is localized on the metal, highlighting both high-spin Co(III) and Co(II)L(•) character in the electronic ground state. Further oxidation of CoL(1) to the dication affords a low-spin Co(III) phenoxyl radical species. The NIR features for [Co2L(2)-2H2O](2+) at 8600 cm(-1) (17 800 M(-1) cm(-1)) are doubly intense in comparison to [CoL(1)-H2O](+) owing to the description of [Co2L(2)-2H2O](2+) as two non-interacting oxidized Co salen complexes bound via the central phenylene linker. Interestingly, TD-DFT calculations predict two electronic transitions that are 353 cm(-1) apart. The NIR spectrum of the analogous Ni complex, [Ni2L(2)](2+), exhibits two intense transitions (4890 cm(-1)/26 500 M(-1) cm(-1) and 4200 cm(-1)/21 200 M(-1) cm(-1)) due to exciton coupling in the excited state. Only one broad band is observed in the NIR spectrum for [Co2L(2)-2H2O](2+) as a result of the contracted donor and acceptor orbitals and overall CT character.

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

Abstract 1319: A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models

CX-5461 is a RNA polymerase I inhibitor currently in Phase I clinical trial in Australia for patients with advanced hematologic malignancies. In the pre-clinical setting, CX-5461 is efficacious in a wide range of hematologic and solid tumor models when given orally or intraperitoneally. Currently, the compound is given intravenously (iv) in the first-in-human clinical trial. CX-5461 is solubilized in low pH (3.5) 50 mM sodium phosphate as it is sparingly soluble in water ( Citation Format: Ada W.Y. Leung, Malathi Anantha, Kathleen E. Prosser, Mohamed Wehbe, Charles J. Walsby, Marcel B. Bally. A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1319.

A stable open-shell redox active ditopic ligand.

Herein we describe the synthesis, structure and electronic properties of an unusual redox-active ditopic ligand with a stable open-shell configuration. This stable phenoxyl radical features intense and very low energy electronic transitions in the near infrared (NIR) part of the spectrum and is structurally set up to strongly spin couple coordinated transition metal ions in [2 × 2] grid-type structures.

Transition Metal Ions Promote the Bioavailability of Hydrophobic Therapeutics: Cu and Zn Interactions with RNA Polymerase I Inhibitor CX5461

Low aqueous solubility is a major barrier to the clinical application of otherwise promising drug candidates. We demonstrate that this issue can be resolved in medicinal molecules containing potential ligating groups, through the addition of labile transition-metal ions. Incubation of the chemotherapeutic CX5461 with Cu2+ or Zn2+ enables solubilization at neutral pH but does not affect intrinsic cytotoxicity. Spectroscopic and computational studies demonstrate that this arises from coordination to the pyrazine functionality of CX5461 and may involve bidentate coordination at physiological pH.

Combining a Ru(II)-arene complex with a NO-releasing nitrate-ester ligand generates cytotoxic activity

A Ru(ii) arene complex with a NO-releasing 4-nitrooxymethyl-pyridine ligand shows increased cytotoxicity against the non-small cell lung cancer cell line A549 as compared to either the free ligand or the unfunctionalized complex. EPR spin-trapping studies show that NO release is selective, being limited in phosphate buffered saline or human serum, but promoted by glutathione.