**1. Introduction**

Increasing evidence has proven 1,8-naphthalimides as promising candidates for the treatment of cancer, with several such derivatives (e.g., amonafide and mitonafide) being tested in clinical trials against various solid and soft tumors [1]. Despite their potent anti-cancer activity, the clinical application of most of these compounds is hampered due to the toxic side effects [1,2]. Accordingly, several strategies have been developed to modify the naphthalimide ring in order to improve the anti-tumor effects and lower its toxicity [2]. This has led to the synthesis of various naphthalimide-based conjugates, such as metal complexes with naphthalimide ligands [2]. In this regard, naphthalimide-gold(I) phosphine complexes, whose synthesis was inspired by pervious

observations on the lead compound auranofin, have shown an increase in the overall cellular uptake and in the nuclear accumulation of gold(I) as compared with the naphthalimide-free analogues [3]. Other interesting examples are the naphthalimide-based ruthenium(II) arene complexes, showing enhanced cancer cell selectivity, which is possibly achieved by the simultaneous action of naphthalimide as a DNA intercalator along with the ability of ruthenium(II) in binding proteins [4]. More recently, 1,8-naphthalimides containing a metal *N*-heterocyclic carbene (NHC) moiety have been synthesized [5,6]. These conjugates are shown to act via both interaction with DNA—related to the naphthalimide structure—as well as metal-based mechanisms, such as the inhibition of the thioredoxin reductase (TrxR) [5,6].

Among the molecular mechanisms that are involved in the anti-cancer efficacy of organometallic drugs is the mitogen activated protein kinase (MAPK) pathway. MAPKs encompass three signaling cascades; (i) extracellular signal-related kinases (ERKs), (ii) the c-Jun N-terminal kinase (JNK), and (iii) p38 MAPK, all of which have key roles in cellular proliferation and survival [7]. p38 MAPK has been repeatedly implicated in cancer therapy and its activation is shown to be necessary for cancer cell death triggered by a number of chemotherapeutic agents [8]. We and others have shown a determinant role for the activation of p38 signaling in the pro-apoptotic effects of metal-based drugs, such as cisplatin [9–11], auranofin [12] as well as gold-containing NHC complexes [13]. However, reports regarding the influence of naphthalimide derivatives on MAPKs are sparse. As an example, a novel amonafide analogue has been shown to down-regulate ERK1/2 and p38 via TAK1 inhibition, leading to its anti-inflammatory effects [14].

In continuation to the aforementioned studies, a series of 4-ethylthio-1,8-naphthalimide conjugates has been recently synthesized, carrying rhodium(I)- and ruthenium(II) NHC fragments as metal units [15]. The compounds were found to interact with DNA via an intercalation mechanism and they were able to trigger strong anti-proliferative effects in MCF-7 breast cancer and HT-29 colon carcinoma cells [15]. In this article, we describe more detailed analyses on the molecular mechanisms underlying the anti-tumor effects of Rh(I)- and Ru(II) naphthalimide-NHC compounds, as well as the metal-free ligand, designated as MC7, MC6, and MC5, respectively. All of the complexes showed potent anti-proliferative effects against various breast- and colorectal cancer (Colorectal cancer (CRC)) cell lines, but exhibited mild toxicity in human foreskin fibroblasts (HFFs). Using HCT116 CRC cells, we have assessed the involvement of reactive oxygen species (ROS) and p38 MAPK signaling in the mode of action of these molecules. We observed elevated intracellular- and mitochondrial ROS production, and a remarkable activation of p38 MAPK in response to naphthalimide-NHC analogues, with no clear regulation of other members of the MAPK family. Additionally, the modulation of ROS and p38α by anti-oxidants and either chemical inhibitors or siRNA, respectively, led to a significant reduction in the pro-apoptotic and growth inhibitory functions of the Ru(II) derivative. Our findings propose p38 signaling as a novel anti-cancer target of organometallic complexes with naphthalimide ligands.
