Cancer is one of the most intractable diseases posing a threat to human health. Chemotherapy is one of the main methods for cancer treatment; however, in the battle against cancer, chemotherapy often “kills a thousand enemies while causing self-harm of 800”, accompanied by severe toxic side effects. Therefore, it is of great significance to develop highly selective anti-cancer drugs based on new strategies to achieve precise therapy [1]. In contrast to normal cells, cancer cells demonstrate aberrant redox homeostasis; specifically, they not only demand higher levels of reactive oxygen species (ROS) to maintain their malignant phenotype but also upregulate antioxidant levels cope with the augmented ROS [2], [3]. In response to the abnormal redox homeostasis of cancer cells, a pro-oxidative anticancer strategy has been devised to eradicate cancer cells by promoting the generation of ROS [2]. Nevertheless, a central challenge in pro-oxidative anticancer research lies in achieving selective and efficient generation of ROS within cancer cells to maximize therapeutic efficacy.

In addition to the abnormal redox homeostasis, the reprogramming of energy metabolism constitutes another remarkable characteristic of cancer cells, namely, a shift in the generation of adenosine triphosphate (ATP) from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis, which fulfills their augmented demands for ATP generation and macromolecular biosynthesis [4]. The energy metabolic reprogramming has thus emerged as an attractive target for cancer therapy [5], [6], [7], [8]. However, achieving precise and effective intervention in reprogramming remains a challenging task, because of the metabolic heterogeneity [4], [6] and plasticity [6], [8] of cancer cells.

Mitochondria, being a crucial organelle within eukaryotic cells, serve as the main site for the generation of ROS. They are also the location where nutrients like sugars, fats, and proteins generate ATP through processes such as the tricarboxylic acid cycle, electron transport, and oxidative phosphorylation [9]. Furthermore, cancer cells possess a strikingly higher negative mitochondrial inner membrane potential than normal cells (Δψm = −220 mV as opposed to Δψm = −140 mV in normal cells [10]), in an effort to sustain their malignant phenotypes, thereby presenting an opportunity to selectively target the mitochondria of cancer cells for more optimal therapy [10], [11], [12]. This initiates a series of elegant researches on the development of promising anticancer agents that target mitochondria by grafting a lipophilic triphenylphosphonium (TPP) cation [13], [14], [15], [16], [17], [18], [19], based on its electrostatic interaction with the negatively charged mitochondrial inner membrane.

Previously, we engineered a curcumin analogue (MitoCur-1) that targets mitochondria through the integration of a TPP unit [20]. It was found that MitoCur-1 can selectively and efficiently produce ROS via the inhibition of mitochondrial thioredoxin reductase 2 (TrxR2) in cancer cells (Scheme 1 A). As a result, it induces a ROS-dependent energy crisis in a dual-effect inhibition mode against both mitochondrial and glycolytic metabolisms, effectively addressing the metabolic plasticity of cancer cells. Inspired by this work, we shifted our focus from a Michael acceptor-type ROS-generating agent (exemplified by MitoCur-1) to a catechol-type ROS-generating agent. The catechol-type molecules are currently considered for the following reasons. Firstly, the catechol unit is prevalently found in nature and serves as a crucial component for constructing dietary natural products, such as green tea polyphenols, piceatannol, quercetin, caffeic acid, hydroxytyrosol and so forth [21], [22]. Exploring the role of the catechol unit in mitochondria holds great significance for further understanding the cancer chemopreventive mechanism of dietary natural products possessing such a unit. Secondly, as an alternative type of ROS-generating agents distinct from Michael acceptor-type ROS-generating agents, we aim to employ catechol-type molecules to explore the universality of a ROS-generating strategy by targeting mitochondria in effectively inducing an energy crisis within cancer cells (Scheme 1 A). Thirdly and most significantly, the mitochondrial matrix exhibits a remarkable alkalinity (pH ≈ 8) [23]. Additionally, catechol-type molecules are more acidic than phenols due to the intramolecular hydrogen bond interaction in either catechol-type molecules or their deprotonated o-hydroxyphenoxides. These facts facilitate the deprotonation of catechol-type molecules, leading to the formation of o-hydroxyphenoxides (Scheme 1B). These o-hydroxyphenoxides are more potent electron donors compared to the parent catechols. As a result, the accelerated generation of o-hydroxyphenoxyl radicals through the first single electron transfer with molecular oxygen becomes feasible [24]. The acidity dissociation constant of the o-hydroxyphenoxyl radical is considerably lower (pKa1 = 4.1) [25] than that of catechol (pKa1 = 9.25) [26]. Thus, the o-hydroxyphenoxyl radical is anticipated to dissociate and form o-semiquinone radical anions. The o-semiquinone radical anion is more prone to undergo further oxidation to form the final product, o-quinone, via the second single electron transfer [27]. Overall, the two-step single electron transfer processes that occur during the auto-oxidation of catechol-type molecules facilitate the generation of ROS. Fourthly, despite our previous affirmation that catechol-type diphenylpolyenes can effectively target mitochondria without the introduction of additional TPP [27], we postulate that grafting TPP onto the catechol-type diphenylpolyene framework would expedite its accumulation in mitochondria within cancer cells. This, in turn, will facilitate its auto-oxidation and the efficient and selective generation of ROS. Fifthly, catechol-type diphenylpolyenes are capable of emitting blue fluorescence [27], which simplifies the monitoring of their subcellular localization. Correspondingly, in this study, we designed several mitochondria-targeted catechol-based diphenylpolyenes (diphenylethylene, diphenylbutadiene, and diphenylhextriene) by inserting diverse quantities of double bonds between two aromatic rings to elongate the conjugated links, and coupling with a TPP unit. These include Mito-DHS ((E)-(4-(4-(3,4-dihydroxystyryl)phenoxy)butyl)triphenylphosphonium), Mito-DHB ((4-(4-((1E,3E)-4-(3,4-dihydroxyphenyl)buta-1,3-dien-1-yl)phenoxy)butyl)triphenylphosphonium), and Mito-DHH ((4-(4-((1E,3E,5E)-6-(3,4-dihydroxyphenyl)hexa-1,3,5-trien-1-yl)phenoxy)butyl)triphenylphosphonium), together with their parent molecules, namely DHS (3,4-dihydroxy-trans-stilbene), DHB (4-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)benzene-1,2-diol), and DHH (4-((1E,3E,5E)-6-phenylhexa-1,3,5-trien-1-yl)benzene-1,2-diol), as well as the control molecule DHH-Br (4-((1E,3E,5E)-6-(4-(4-bromobutoxy)phenyl)hexa-1,3,5-trien-1-yl)benzene-1,2-diol). The control molecule is unable to rapidly localize in mitochondria due to the lack of a TPP unit (Scheme 2).