The ruthenium (II) polypyridyl complexes (RPCs) Δ-[(phen)2Ru(tatpp)]Cl2 (Δ-[3]Cl2) and ΔΔ-[(phen)2Ru(tatpp)Ru(phen)2]Cl4 (ΔΔ-[4]Cl4) are a new generation of metal-based anti-tumor agents. These RPCs bind DNA via intercalation of the tatpp ligand which itself is redox-active and easily reduced at biologically relevant potentials. We have previously shown that RPC 44+ cleaves DNA when reduced by glutathione to a radical species, and that this DNA cleavage is potentiated under hypoxic conditions in vitro. Here we show that 32+ also exhibits free-radical mediated DNA cleavage in vitro, and that 32+ and 44+ both exhibit selective cytotoxicity towards cultured malignant cell lines, and marked inhibition of tumor growth in vivo. The murine acute toxicity of RPCs 32+ and 44+ (maximum tolerable doses (MTD’s) ~ 65 µmol/kg) is comparable with that for cisplatin (LD50 ~57 µmol/kg) but unlike cisplatin, RPC’s are generally cleared from the body unchanged via renal excretion without appreciable metabolism or nephrotoxic side effects. RPCs 32+ and 44+ are demonstrated to suppress growth of human non-small cell lung carcinoma (~83%), show potentiated cytotoxicity in vitro under hypoxic conditions, and induce apoptosis through both intrinsic and extrinsic pathways. The novel hypoxia-enhanced DNA cleavage activity and biological activity suggest a promising new anti-cancer pharmacophore based on metal complexes with aromatic ligands that are easily reduced at biologically accessible potentials.
In the absence of O 2 , the cationic complex, [(phen) 2 Ru(tatpp)Ru(phen) 2 ] 4+ (P 4+ ), undergoes in situ reduction by glutathione (GSH) to form a species that induces DNA cleavage. Exposure to air strongly attenuates the cleavage activity, even in the presence of a large excess of reducing agent (e.g., 40 equiv GSH per P 4+ ) suggesting the complex may be useful in targeting cells with a low oxygen microenvironment (hypoxia) for destruction via DNA cleavage. The active species is identified as the doubly reduced, doubly protonated complex H 2 P 4+ and a carbon-based radical species is implicated in the cleavage action.. We postulate that the pO 2 regulates the degree to which carbon radical forms and thus regulates the DNA cleavage activity.The use of transitions metal complexes in medicine has enjoyed extensive attention given the tremendous success of cisplatin as a chemotherapeutic agent 1 and the ability of many metal complexes to interact with and damage cellular structures, particularly DNA. 2-7 A large number of DNA cleaving metal complexes function via the activation of dioxygen to generate reactive oxygen species (ROS), such as hydroxyl radical and superoxide radical. 8,9 These ROS are ultimately responsible for the DNA cleavage. Others, including cisplatin and certain photoactivated, 10-14 oxidizing 15,16 or hydrolyzing complexes, 8 do not require O 2 to function, but they are also insensitive to the cellular [O 2 ]. Compounds that show enhanced cleavage activity under a low oxygen microenvironment (hypoxia) are rare 17-21 but offer a unique mechanism to target tumor cells under such conditions. These hypoxic tumor cells are often the most resistant to radiotherapy 22,23 and chemotherapy 24,25 (tatpp = 9,11,20,22 -tetraazatetrapyrido[3,2-a: 2′,3′-c: 3″,2″-1: 2‴,3‴-n]-pentacene and phen = 1,10-phenanthroline) shown above (water soluble as the chloride salt) not only induces DNA cleavage in the presence of mild reducing agents but shows enhanced activity under anaerobic conditions. The fact that exposure to air attenuates the cleavage activity suggests that ROS are not responsible for the observed cleavage and that such a complex might be useful in targeting cells under hypoxic conditions. Complex P 4+ is known to intercalate and bind DNA tightly (K b 1.1×10 7 M −1 at 25 mM NaCl). 28,29 The strong interaction with DNA is not unusual for this class of cationic complexes and it has a number of structural similarities to many known metallointercalators 13,14,30-33 including those that are know to thread their way through the DNA double-helix. 34The ability of P 4+ to cut DNA was examined by following the conversion of supercoiled plasmid DNA (form I) to the circular form (form II) or linear form (form III) using agarose gel electrophoresis to separate the products (experimental details given in ESI). As shown in Figure 1, P 4+ alone does not cause appreciable DNA cleavage (lane 2), however, addition of a mild reducing agent such as glutathione (GSH) leads to cleavage activity (lanes 4 &5). ...
Four mononuclear [(L-L) Ru(tatpp)] and two dinuclear [(L-L) Ru(tatpp)Ru(L-L) ] ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me phen) or 4,7-diphenyl-1,10-phenanthroline (Ph phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs.
Chemical speciation modeling (equilibrium simulation program, MINTEQA2) was used to understand the chemistry behind titrimetric analysis and to monitor changes in experimental conditions in chemical titrations quickly and easily. Determination of magnesium and both magnesium and calcium in a mixture by EDTA titrations can be done at pH 10 in the presence of Eriochrom black T indicator. The end point is detected by the color change from red to blue. Simulation results of these titrations highlighted the fact that over the pH range 7-11, complexation of magnesium and calcium by EDTA is independent of pH while indicator speciation is highly dependent on pH. Thus the quantitative determination of magnesium and total magnesium and calcium by EDTA titrations using Eriochrom black T should be done at pH values of 9-10. By using computer simulation programs it is easy to calculate the chemical speciation at each step in a titration and to gain a better understanding of why a particular procedure does or does not work under particular experimental conditions.
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