Fluorinated nitroimidazoles and their ruthenium complexes : potential hypoxia-imaging agents (2009)

With the goal of synthesizing nitroimidazole-based hypoxia imaging agents for the detection of cancerous tumours, the chemistry of halogen incorporation, especially fluorine, into the NI side-chain of nitroimidazoles was investigated. The coordination of these nitroimidazoles and other methyl (Me)-substituted imidazoles to Ru(II) and Ru(III) centres was also investigated. The general objective was to combine the hypoxia-selective nature of the nitroimidazole moiety and the DNA-binding properties of Ru to deliver the active species selectively to its target. The nitroimidazole compounds and Ru complexes were characterized in general by a combination of NMR, IR and UV-Visible spectroscopies, as well as mass spectrometry, cyclic voltammetry, conductivity and elemental analysis; six nitroimidazoles and eleven Ru complexes were also characterized by X-ray crystallography. The halogenated nitroimidazoles were synthesized using a standard amide coupling reaction; this was used for the synthesis of EF5 [2-(2-nitro-l-H-imidazol-l-yl)-N- (2,2,3,3,3-pentafluoropropyl) acetamide], a known, sensitive probe for quantifying the amount of hypoxia within cells. Treatment of a desired acid with N-methylmorpholine and zso-butylchloroformate, followed by addition of the appropriate amine (either H₂NCH₂CX₃ or H₂NCH₂CX₂CX₃, where X= H, F, CI and/or Br) led to formation of the halogenated nitroimidazole; 2-nitro- (2N₂), 2-methyl-5-nitro- (2Me₅N0₂) and 2-methyl- 4-nitro- (2Me₄N0₂) imidazole compounds were isolated in yields of 15 to 83 %. The synthesis of EF5 (typical yield 45 %) was improved by coupling iodoacetic acid with H₂NCH₂CF₂CF₃ to give ICH₂C(0)NHCH₂CF₂CF₃ (TF5) which was subsequently reacted with 2N02Im and Cs₂C0₃ to yield the final product (78 %); this 2-step method circumvented the previously required 5-step synthesis. Other analogous compounds were less stable than IF5, especially those species containing a C-Br moiety, and they often decomposed in the presence of heat and/or light. Alteration of the associated side-chain on EF5 by 'reversal' of its amide linkage was performed; however, the biological activity of the derivative was significantly less than that of EF5. A nitroimidazole (N0₂lm) containing side-chain with a highly reactive terminal group, with the potential to exchange fluoride, was also sought. Attempts to isolate a tosylated derivative of SR2508 [2-(2-mtro-l-H-irnidazol-l-yl)-N-(2-hydroxyethyl) acetamide] proved to be futile. The reaction with tosyl chloride led to the formation of a monochloro analogue of SR2508, presumably via the reactive tosylate intermediate, while reaction with triflic anhydride led to an intramolecular cyclization of the side-chain to give a compound whose X-ray structure was determined. The focus was then shifted to F-for-Br exchange; however, attempts to perform such a task revealed that the C-N0₂ bond of the Im ring was more labile than the C-Br bond, and this resulted in the formation of a 2-fluoroimidazole species. Further efforts to incorporate an F-atom into a nitroimidazole side-chain were unsuccessful. [Ru(II)(L)₆]²⁺ complexes were synthesized from [Ru(DMF)₆][CF₃S0₃]₃; DMF = dimethylformamide, L = imidazole (Im), N-methylimidazole (NMelm) and 5-methylimidazole (5MeIm). The 2-methylimidazole complex fram-[Ru(CO)(DMF)(2MeIm)₄] [CF₃S0₃]₂ was synthesized via a reaction involving abstraction of CO from DMF; this complex loses CO reversibly at ambient temperature to form [Ru(DMF)(2MeIm)₄] [CF₃S0₃]₂ , and the DMF can be removed to generate [Ru(CF₃S0₃)x(2MeIm) ₄][CF₃S0₃]y (x = 2, y = 0, or x = 1 = y). The X-ray structures of [Ru(Im)₆][CF₃S0₃]₂, [Ru(NMeIm)₆][CF₃S0₃]₂ and [Ru(5MeIm)6][CF₃S0₃]₂ were obtained. Analogous [Ru(II)(L)x] ₂+ nitroimidazole complexes (L = 2N0₂Im, x = 6; L = 4N0₂Im, x = 6; L = 2Me5N0₂Im, x = 5) were isolated from reaction with [Ru(DMF)6][CF₃S0₃]₃ . The reaction with EF5 and SR2508 in EtOH yielded the bis-substituted complexes [Ru(DMF)₂ (EF5)₂ (EtOH)₂][CF₃S0₃]₃ and [Ru(DMF)₄(SR2508)₂][CF₃SO₃]₃, respectively. Ru(III) complexes of composition RuCl₃L₃ (L = 2N0₂Im, 4N0₂Im, 2Me₅N0₂Im, metro) and RuCl₃L₂(EtOH) (L= EF5, SR2508) were synthesized directly from RuCl₃»3H₂0. Their *H NMR spectra were typically broad and sometimes signals were not observed; however, their paramagnetic, d5 low-spin composition was confirmed using the Evans method. Some new Ru(II) and Ru(III) bis-β -diketonate (acac = acetylacetonate; hfac = 1,1,1,5,5,5-hexafluoroacetylacetonate) Im and N0₂lm complexes were synthesized. Reaction of two equiv. of an imidazole with c/s-[Ru(acac)₂ (MeCN)₂][CF₃S0₃] (synthesized from Ru(acac) ₃ and CF₃S0₃H in MeCN) yielded Ru(III) complexes with composition [Ru(acac)₂ (L) ₂][CF₃S0₃] (L = Im, NMelm, 2MeIm, 5MeIm, 2N0₂Im, metro, EF5 and SR2508), the first four being structurally characterized by X-ray crystallography. The analogous Ru(II) hfac complexes were isolated from a reaction of cz'5-Ru(hfac) ₂(MeCN) ₂ (71) (synthesized from either Ru(hfac)₃ or Na[Ru(hfac)₃] and CF₃S0₃H in MeCN) with two equiv. of imidazole to yield Ru(hfac)₂ (L)₂ (L = Im, NMelm, 2MeIm, 4(5)MeIm, 2N0₂Im, EF5 and SR2508). X-ray structures of 71 and Ru(hfac)₃ were obtained. Reaction of 71 with neat NMelm gave [Ru(hfac)CNMeIm)₄][hfac]. The mixed ligand complex c/5-Ru(hfac)(acac)(MeCN)₂ (X-ray) was synthesized from the new species Ru(hfac)₂ (acac), which was isolated from a reaction of 71 with Hacac. In vitro assays for toxicity and monoclonal antibody (MoAb) binding with SCCVLT cells were used to evaluate the potential of selected nitroimidazoles as hypoxia-selective imaging agents. The toxicity, MoAb binding, cell accumulation and DNA-binding assays were used to test the utility of selected Ru complexes for transporting and/or localizing the coordinated nitroimidazoles within the cell. A preliminary radiosensitization study was also performed with two Ru complexes. In general, the nitroimidazoles (N0₂lms) and Ru complexes were non-toxic under both oxic and hypoxic conditions. The accumulation of the N0₂lms within the cell required hypoxic conditions, while the amount of N0₂ lm bound within the hypoxic cells correlated with its one-electron reduction potential, the N0₂lms with the more positive reduction potentials giving the higher concentrations. The interaction of the fluorescently labeled MoAbs (ELK3-51 and ELK5-A8) with the N0₂lms depended on the length, size and composition of the halogenated side-chains. The Ru-N0₂lm complexes displayed relatively high cell accumulation and DNA-binding levels when compared to literature data for other Ru complexes. The most interesting result came from the MoAb assay in which the cells treated with [Ru(acac)₂ (EF5)₂] [CF₃S0₃] afforded a fluorescence signal four times greater than that seen for EF5 itself.