Over the past decade, a variety of carbon monoxide releasing molecules (CORMs) have been developed and tested. Some CORMs spontaneously release CO once in solution, while others require a trigger mechanism to release the bound CO from its molecular complex. The modulation of biological systems by CORMs depends largely on the spatiotemporal release of CO, which likely differs among the different types of CORMs. In spontaneously releasing CORMs, CO is released extracellularly and crosses the cell membrane to interact with intracellular targets. Other CORMs can directly release CO intracellularly, which may be a more efficient method to modulate biological systems. In the present study, we compared the efficacy of extracellular and intracellular CO-releasing CORMs that either release CO spontaneously or require an enzymatic trigger. The efficacy of such CORMs to modulate HO-1 and VCAM-1 expression in TNF-α-stimulated human umbilical vein endothelial cells (HUVEC) was evaluated.

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).

In an attempt to develop new anti-inflammatory agents which act by co-release of carbon monoxide (CO) and a specialized pro-resolving mediator, we designed conjugates of a lipoxin A 4 analogue and an acyloxycyclohexadiene-Fe(CO) 3 complex as an esterase-triggered CO-releasing molecule (ET-CORM). After adjustment of the protecting group strategy, two of such compounds were successfully prepared by total synthesis (12 steps; 4-5 % overall yield) starting from deoxy-d-ribose and exploiting a Wittig olefination and an intermolecular Heck reaction as key CÀ C bond-forming steps. A crucial late reduction of an aryl-ketone moiety in the presence of a highly sensitive dienol ester functionality was achieved with BH 3 -SMe 2 in the presence of catalytic amounts of NaBH 4 . Both target compounds were dose-dependently toxic towards cultured human umbilical vein endothelial cells (HUVEC), with LipET-CORM 1-A being slightly more toxic. While induction of heme oxygenase 1 (HO-1) in HUVEC was observed for both compounds, they did not inhibit TNF-α-mediated VCAM-1 expression in these cells. In M2 polarized macrophages HO-1 expression was more pronounced as compared to M1 polarized macrophages. In both types of macrophages HO-1 expression was downregulated by lipopolysaccharide, but only in M2 macrophages HO-1 expression was rescued by LipET-CORM. 15-Lipoxygenase (15-LO) was only expressed in M2 macrophages and was not influenced by LipET-CORM. Collectively our data demonstrate that LipET-CORMs induce HO-1 expression in endothelial cells and M2 polarized macrophages. The role of the intra-cellular released lipoxin A 4 in resolution of inflammation, however, remains to be assessed.

In the course of an attempt to synthesize 2acyloxycyclohexa-1,3-diene-Fe(CO) 3 complexes in nonracemic form, we reinvestigated the "fully diastereoselective" Fe(CO) 3 complexation of (S,S)-2-ketopinoyloxy-1,3-cyclohexadiene, which had been described by Yeh and co-workers (Organometallics 2001, 20, 289−295). However, after cleaving off the chiral auxiliary unit, we only obtained racemic complexes, also for a related substrate. For this reason, we performed control experiments to exclude possible racemization mechanisms and confirmed the configurational integrity of the dienol-Fe(CO) 3 intermediates using stereochemically defined dihydrocarvone-derived complexes. We finally could show that the complexation described by Yeh actually proceeds without any detectable diastereoselectivity. At the end, the virtually inseparable diastereomers of the chiral complexation products could be distinguished by careful NMR and chiral HPLC analyses. Article pubs.acs.org/Organometallics

In the course of our research into enzyme-triggered CO-releasing molecules (ET-CORMs), we were interested in using 2-acetoxy-5-azido-1,3-cyclohexadiene–Fe(CO)3 (rac- 2) as a building block for further structural modification by means of Cu-catalyzed azide–alkyne cycloaddition (CuAAC click chemistry). Treatment of [2-acetoxy-cyclohexadienyl–Fe(CO)3]+[PF6]− with Zn(N3)2, TMS-N3, or NaN3 surprisingly afforded 2-acetoxy-1-azido-2,4-cyclohexadiene–Fe(CO)3 (rac- 9) as the main product. We could show that rac- 2 is primarily formed under kinetic control but undergoes a rapid isomerization to rac- 9 (as the thermodynamic product) in a formal vinylogous Winstein rearrangement under concomitant migration of the Fe(CO)3 moiety. This unprecedented reaction displays a 1st order kinetics and appears to proceed via an ionic (rather than a concerted intramolecular) mechanism as supported by crossover experiments using deuterated compounds. The CuAAC reaction of rac- 9 with propargylic alcohol afforded triazole rac- 13, which was demonstrated (by headspace-gas chromatography (GC)) to act as an ET-CORM in the presence of porcine liver esterase.