Synthesis and Biological Evaluation of Water‐Soluble Esterase‐Activated CO‐Releasing Molecules Targeting Mitochondria

Abstract

Due to the beneficial effects of carbon monoxide as a cell-protective and anti-inflammatory agent, CO-releasing molecules (CORMs) offer some promising potential applications in medicine. In this context, we synthesized a set of acyloxy-cyclohexadiene-Fe(CO) 3 complexes, all displaying a Nmethyl-pyridinium triflate moiety in the ester side chain, as mitochondria-targeting esterase-triggered CORM prodrugs. Whereas 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 remarkably 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', which also displayed PLE-induced CO release, was assessed by using human umbilical vein endothelial cells (HUVEC). Whereas Mito-CORMs 2/3-B were not cytotoxic up to 500 μM (MTT assay), Mito-CORMs 2/3-A' caused significant toxicity at concentrations above 50 μM. The anti-inflammatory potential of both Mito-CORM variants was demonstrated by concentrationdependent down-regulation of the pro-inflammatory markers VCAM-1, ICAM-1 and CXCL1 as well as induction of HO-1 in TNFα-stimulated human umbilical vein endothelial cells (HUVECs; western blotting and qPCR). Energy phenotyping by seahorse real-time cell metabolic analysis, revealed opposing shifts of metabolic potentials in cells treated either with Mito-CORMs 2/3-B (increased mitochondrial respiration and glycolytic activity) or Mito-CORMs 2/3-A' (suppressed mitochondrial respiration and increased glycolytic activity). Thus, the Mito-CORMs represent valuable tools for the safe and targeted delivery of CO to mitochondria as a subcellular compartment to induce positive anti-inflammatory effects with only minor shifts in cellular energy metabolism. Also, due to their water solubility, these compounds provide a promising starting point for further pharmacological studies. Recently, Wang and co-workers developed organic CO prodrugs based on decarbonylation chemistry, some of which also allow triggered CO release, for example, by esterases, pH change, or oxidation (ROS).

Vinylogous Winstein Rearrangement: Unexpected Isomerization of an Azide-Substituted Cyclohexadiene–Fe(CO)3 Complex

“…Remarkably, however, is the finding that CO release from the azide-substituted complex rac- 9 was triggered only by LCR (to produce 0.9 equiv of CO after 75 h), while the triazole-substituted complex rac - 13 was selectively activated only by PLE (to give 0.6 equiv of CO after 75 h). However, in comparison to the parent complex (lacking the N -substituent) or other first-generation ET-CORMs, the rates of CO release observed for rac - 13 under the experimental conditions are smaller. This can possibly be attributed to steric hindrance by the additional substituent adjacent to the ester function.…”

“…Several research groups have focused on the development of CO-releasing molecules (CORMs) to selectively introduce CO into a cell or an organism . In the course of our own research in this field, we developed acyloxy-substituted cyclohexadiene–Fe­(CO) 3 complexes as enzyme-triggered CORMs (ET-CORMs) . To further improve the chemical and biological properties of such compounds, we were interested in synthesizing a library of triazole-substituted ET-CORMs of type rac - 1 via click chemistry, i.e., by Cu-catalyzed azide/alkyne cycloaddition (CuAAC) .…”

“…There is no simple relationship between the conversion of starting material and the amount of CO determined, as the CO release mechanism involves multiple steps (ester cleavage, oxidation, and subsequent decomposition of the dienol complex intermediate). While certain ET-CORMs release up to 3 equiv of CO (100% of theory), lower values are observed in most cases as detailed in ref …”

Metal-based carbon monoxide releasing molecules with promising cytotoxic properties

“…Complexes 71e and 71f elevated both mitochondrial respiration and glycolysis. 210 When tested on rats in vivo, the complexes have a little impact on liver function, but they do cause physiological harm to liver cells. Additionally, these complexes have a detrimental effect on both functional and physiological processes of the kidney.…”

Reactive Oxygen Species-Activated Metal-Free Carbon Monoxide Prodrugs for Targeted Cancer Treatment

“…To mitigate these limitations, tremendous efforts have been devoted to the development of CO prodrugs or CO-releasing molecules (CORMs) for controllable CO delivery, and some very exciting progress has been witnessed in this field. These CO donors can be generally divided into two categories, namely, metal-based − and metal-free CO prodrugs. − In the past decades, extensive efforts have been devoted to the development of metal-based CORMs, − and some of them have been widely employed for studying CO biology, such as CO’s anti-inflammatory effects. , In contrast, metal-free CO prodrugs are substantially underdeveloped due to the challenges to extrude CO from small organic molecules. Over the past decade, quite a few chemically diverse metal-free CO prodrugs have been elegantly devised using photoirradiation, − bioorthogonal chemistry, − ultrasound, or water , as the trigger, and some of them have been successfully applied in vivo to achieve localized CO delivery and recapitulated CO-associated pharmacological effects.…”