**1. Introduction**

CO-releasing molecules (CORMs) can release carbon monoxide (CO) either spontaneously, enzymatically, or triggered by an external stimulus [1]. Their therapeutic potential relies on the release of a limited amount of CO. Along with NO and H2S, CO is the third small signaling molecule and is produced endogenously by enzymes of the Heme Oxygenase (HO) class through heme oxidative degradation. The expression of the HO inducible isoform (HO-1) is triggered by cell responses toward oxidative stress and inflammation and results in cyto- and tissue protection. HO-1 metabolites, including CO, are important in restoring redox homeostasis and resolution of inflammation, and it has been widely demonstrated that the HO-1/CO axis can help to prevent cellular and tissue damage. Therefore, the manipulation of the HO-1/CO system is an attractive strategy to treat conditions linked to oxidative-stress-induced inflammation, such as lung hyper-inflammation in cystic fibrosis, sepsis and modulation of chronic pain [2–7]. The chemistry of CO is unique: unlike NO and H2S that react indiscriminately with intracellular targets, CO offers the advantage of binding only to transition metals in a low oxidation state. Such preferential reactivity,

**Citation:** Appetecchia, F.; Consalvi, S.; Berrino, E.; Gallorini, M.; Granese, A.; Campestre, C.; Carradori, S.; Biava, M.; Poce, G. A Novel Class of Dual-Acting DCH-CORMs Counteracts Oxidative Stress-Induced Inflammation in Human Primary Tenocytes. *Antioxidants* **2021**, *10*, 1828. https://doi.org/10.3390/ antiox10111828

Academic Editors: Elias Lianos and Maria G. Detsika

Received: 14 October 2021 Accepted: 16 November 2021 Published: 18 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

along with its greater stability, makes it a more versatile candidate for the development of gaseous-based pharmaceuticals [8]. Indeed, gaseous CO has great potential as a therapeutic tool and has been found beneficial in the treatment of several inflammatory, cardiovascular, and neurological diseases [9–12]. For low-dose CO inhalation, the feasibility of the first clinical trials has been recently assessed [13]. However, the accurate delivery of gaseous CO to its molecular targets through inhalation is challenging, and inhalation therapy is hampered by CO low bioavailability and high affinity to hemoglobin, with consequent toxicity [14]. In this scenario, CORMs have emerged as a safer and attractive therapeutic strategy to deliver a controlled amount of CO to cells. To date, most of the developed CORMs are metal carbonyl complexes (MCCs) [15,16]. Indeed, considering the preferential reactivity of CO for transition metals in a low oxidation state, organometallic complexes have emerged as suitable models to safely deliver CO in vivo and generate innovative therapeutic agents with reasonable pharmacological properties. These molecules have an octahedral shape with six ligands around a central metal and can release CO spontaneously, mainly through hydrolysis in biological buffers. Romão and co-workers [17] introduced a conceptual model to rationalize and improve the design of MCCs with appropriate pharmaceutical properties. This model comprises three portions: (i) a metal core, which accounts for toxicity and the main properties of the MCC; (ii) a coordination-sphere, which influences the electronic density around the metal, tuning the stability and the chemical behavior of the whole complex and triggering CO release under specific conditions; and (iii) a drug-sphere, obtained through modulation of the distal sites of the metal complexes and accounting for pharmacological properties and drug-likeness.

The choice of the transition metal is crucial to design metal-based CORMs. CORMs containing an atom of cobalt (dicobalt(0)hexacarbonyl complexes, DCH) are innovative COreleasing agents with interesting biological features and good CO-release kinetics [18–25]. The DCH metal core is a hexacarbonyl dicobalt moiety (Co2CO6) coordinated through an alkyne bond, which is in turn linked to the drug sphere. One of the main advantages of DCH-CORMs is their synthetic accessibility. Indeed, this highly versatile chemical scaffold is easy to synthesize, facilitating the chemical manipulation of the drug sphere. A series of dual acting DCH-CORMs-carbonic anhydrase inhibitors (CAI-CORMs) have very recently shown promising anti-inflammatory properties under oxidative-stress conditions in different oxidative-based disease models [22,23]. Interestingly, Gallorini et al. [26] demonstrated that some of these compounds were able to differentially modulate inflammation and counteract the H2O2-induced stress in rotator-cuff-derived human tenocytes, which activate the nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2)/HO-1/CO pathway to mitigate oxidative stress. It has also been reported that sustained oxidative stress causes aberrant cytokine secretion in a model of rotator cuff disease (RCD) in vitro [27]. Oxidative stress endurance and, consequently, inflammation occurrence, are considered the major factors causing the failure of tendon healing in clinical practice and can lead to chronic pain and disability. Moreover, the benefits of non-steroidal anti-inflammatory drug (NSAID)-based therapy in the acute phase are broadly accepted, but their use in chronic tendon-related diseases is still controversial [28]. Therefore, an innovative therapeutic approach for the treatment of tendon-derived diseases is urgently needed, as their therapeutic management remains a challenge.

In this light, we synthesized a small set of 1,5-diarylpyrrole and 1,5-diarylpyrazolebased DCH-CORMs linked through a propargylic chain (compounds **1**–**9**, Figure 1). According to the Romão model, the first aim of this study was to analyze the influence of different electronic and steric properties of the drug sphere on the CO release rate. Five selected compounds (**1**–**5**) were then tested on human primary tendon-derived cells stimulated with a low concentration of hydrogen peroxide (H2O2), using the NSAID Meloxicam as a reference compound. The present work aims to assess their efficacy in restoring cell redox homeostasis and counteracting inflammation in terms of PGE2 secretion and at investigating their potential use in vitro to manage musculoskeletal diseases.

**Figure 1.** Chemical structures and conceptual model of compounds **1**–**9**.
