Hemmersbach_2022_Chemistry__

Due to the beneficial effects of carbon monoxide (CO) as a cell-protective and anti-inflammatory agent, CO-releasing molecules (CORMs) offer promising potential applications in medicine. We synthesized a set of acyloxy-cyclohexadiene-Fe(CO)3 complexes, all displaying a N-methyl-pyridinium triflate moiety in the ester side chain, as mitochondria-targeting esterase-triggered CORM prodrugs. While the compounds in which the acyloxy substituent is attached to the 2-position of the diene-Fe(CO)3 unit (A series) spontaneously release CO upon dissolution in phosphate buffer, which is partly suppressed in the presence of porcine liver esterase (PLE), the 1-substituted isomers (B series) show the expected PLE-induced release of CO (up to 3 equiv.). The biological activity of Mito-CORMs 2 / 3-B and their isophorone-derived analogs 2/3-A' was assessed using human umbilical vein endothelial cells (HUVEC). While Mito-CORMs 2/3-B were not cytotoxic up to 500 microM (MTT assay), Mito-CORMs 2/3-A' caused significant toxicity at concentrations above 50 microM. The anti-inflammatory potential of both Mito-CORM variants was demonstrated by concentration-dependent down-regulation of the pro-inflammatory markers as well as induction of HO-1 in TNFalpha stimulated HUVEC. Energy phenotyping by Seahorse Real-Time Cell Metabolic Analysis, revealed opposing shifts of metabolic potentials (mitochondrial respiration/glycolytic activity) in cells treated either with Mito-CORMs 2/3-B or Mito-CORMs 2/3-A'. Thus, the Mito-CORMs represent valuable tools for the safe and targeted delivery of CO to mitochondria as a subcellular compartment to induce anti-inflammatory effects with only minor shifts in cellular energy metabolism. Also, due to their water solubility, these compounds provide promising options for further pharmacological studies.