Structure-Activity Relationships of Novel Salicylaldehyde Isonicotinoyl Hydrazone (SIH) Analogs: Iron Chelation, Anti-Oxidant and Cytotoxic Properties

<div><p>Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, tridentate iron chelator with marked anti-oxidant and modest cytotoxic activity against neoplastic cells. However, it has poor stability in an aqueous environment due to the rapid hydrolysis of its hydrazone bond. In this study, we synthesized a series of new SIH analogs (based on previously described aromatic ketones with improved hydrolytic stability). Their structure-activity relationships were assessed with respect to their stability in plasma, iron chelation efficacy, redox effects and cytotoxic activity against MCF-7 breast adenocarcinoma cells. Furthermore, studies assessed the cytotoxicity of these chelators and their ability to afford protection against hydrogen peroxide-induced oxidative injury in H9c2 cardiomyoblasts. The ligands with a reduced hydrazone bond, or the presence of bulky alkyl substituents near the hydrazone bond, showed severely limited biological activity. The introduction of a bromine substituent increased ligand-induced cytotoxicity to both cancer cells and H9c2 cardiomyoblasts. A similar effect was observed when the phenolic ring was exchanged with pyridine (<i>i.e.</i>, changing the ligating site from <i>O</i>, <i>N</i>, <i>O</i> to <i>N</i>, <i>N</i>, <i>O</i>), which led to pro-oxidative effects. In contrast, compounds with long, flexible alkyl chains adjacent to the hydrazone bond exhibited specific cytotoxic effects against MCF-7 breast adenocarcinoma cells and low toxicity against H9c2 cardiomyoblasts. Hence, this study highlights important structure-activity relationships and provides insight into the further development of aroylhydrazone iron chelators with more potent and selective anti-neoplastic effects.</p></div

The effect of SIH and its analogs on ⁵⁹Fe mobilization from pre-labeled MCF-7 cells (A) and on internalized ⁵⁹Fe uptake from ⁵⁹Fe₂-transferrin (Tf) by MCF-7 cells (B).

<p>(<b>A</b>) The ability of the ligands to promote ⁵⁹Fe mobilization from MCF-7 cells was performed by first prelabeling the cells with ⁵⁹Fe₂-Tf (0.75 µM) for 3 h/37°C, followed by washing and then reincubation for 3 h/37°C with either control medium alone, or control medium containing the chelator (25 µM). (<b>B</b>) Inhibition of ⁵⁹Fe uptake from ⁵⁹Fe₂-Tf by MCF-7 cells by chelators was performed by incubating cells for 3 h/37°C with ⁵⁹Fe₂-Tf (0.75 µM) in the presence or absence of the chelator (25 µM). Results are Mean ±SD (<i>n</i>≥3 experiments). Statistical significance (ANOVA): * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 compared to the control (untreated) group, and # <i>p</i><0.05, ## <i>p</i><0.01, ### <i>p</i><0.001 compared to the reference chelator, SIH.</p

Cytotoxic effects of the chelator, SIH (A), and the new analogues (B–L) against MCF-7 breast cancer cells.

<p>For the determination of their cytotoxic activity, MCF-7 breast adenocarcinoma cells were incubated with the analogs (0.01–3000 µM) for 72 h/37°C. Results are Mean ±SD (<i>n</i>≥4 experiments). Statistical significance (ANOVA): * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as compared to the control (untreated) group.</p

Protective and cytotoxic effects of the synthesized SIH derivatives and their calculated “selectivity ratios”.

<p>The EC₅₀ values (concentration that reduced the cytotoxicity induced by H₂O₂ (200 µM) to 50% of the untreated control) were calculated after a 24 h incubation with non-tumorigenic H9c2 cardiomyoblasts. The IC₅₀ values (concentration that reduced the cellular viability or proliferation to 50% of the untreated control) were calculated after a 72 h incubation with H9c2 cardiomyoblasts or MCF-7 breast cancer cells. Selectivity ratios were calculated <i>via</i> IC₅₀ H9c2 cells/IC₅₀ MCF-7 cells. Mean ± SD; <i>n</i>≥4 experiments. N/A - the EC₅₀ value was not achieved within the studied concentration range (no protection).</p><p>Protective and cytotoxic effects of the synthesized SIH derivatives and their calculated “selectivity ratios”.</p

Protective effects of the chelator, SIH (A), and the new analogues (B–L).

<p>The ability of the SIH derivatives to protect H9c2 cardiomyoblast cells against oxidative injury were evaluated using a 24 h/37°C incubation of the cells with H₂O₂ (200 µM) and the novel analogs (0.3–1000 µM). Results are Mean ±SD (<i>n</i>≥4 experiments). Statistical significance (ANOVA): * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 compared to the control (untreated) group, and # <i>p</i><0.05, ## <i>p</i><0.01, ### <i>p</i><0.001 compared to the H₂O₂ group.</p

Chromatographic conditions used for the determination of the stability of the new chelators in rabbit plasma.

<p>Chromatographic conditions used for the determination of the stability of the new chelators in rabbit plasma.</p

Stabilities of SIH and its novel analogs in rabbit plasma.

<p>SIH (<b>A</b>), redSIH (<b>B</b>), redHAPI (<b>C</b>), redHPPI (<b>D</b>), BHAPI (<b>E</b>), BHPPI (<b>F</b>), 2API (<b>G</b>), 7HII (<b>H</b>), H16 (<b>I</b>), H17 (<b>J</b>), H18 (<b>K</b>) and H28 (<b>L</b>) were incubated at 37°C in rabbit plasma and their concentrations were analyzed using HPLC every 60 min until <i>t</i> = 600 min. Results are expressed as a percentage of the concentration at <i>t</i> = 0 min (100 µM). Results are Mean ±SD (<i>n</i> = 3 experiments). Statistical significance (ANOVA): * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 compared to the concentration at <i>t</i> = 0 min (100%).</p

Effects of SIH and its analogs on iron-induced oxidation of ascorbic acid in a buffered solution (pH 7.4).

<p>Chelators were assayed at iron binding equivalents (IBE) of 0.1 (excess of Fe), 1 (iron-chelator complexes with a fully filled coordination sphere) and 3 (excess of free chelator). DFO and EDTA were used as negative and positive control chelators, respectively. The results are expressed as a percentage of the control group in the absence of chelator (100%). Results are Mean ±SD (<i>n</i>≥3 experiments). Statistical significance (ANOVA): * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as compared to the control group (iron with ascorbate).</p

Line drawings of the chemical structures of the iron chelators, SIH, HAPI and HPPI, and their novel analogs.

<p>Line drawings of the chemical structures of the iron chelators, SIH, HAPI and HPPI, and their novel analogs.</p

Iron chelation properties of the novel analogs in solution (A) and in MCF-7 cells (B).

<p>(<b>A</b>) The chelation dynamics of the new agents in solution were observed for 360 s using the calcein assay, and the agent was applied at <i>t</i> = 100 s. The fluorescence intensity of free calcein at <i>t</i> = 360 s was expressed as a percentage of that observed using the reference iron chelator, SIH. (<b>B</b>) The ability of the analogs to chelate “free” iron from the LIP in MCF-7 cells was measured using the calcein-AM assay. The fluorescence intensity of free calcein at <i>t</i> = 600 s was expressed as a percentage of that observed in the presence of SIH. Results are Mean ±SD (<i>n</i>≥3 experiments). Statistical significance (ANOVA): # <i>p</i><0.05, ## <i>p</i><0.01, ### <i>p</i><0.001 compared to the reference chelator, SIH.</p