Maintaining the homeostasis of anions is crucial to cellular processes that are tightly controlled in physiological systems [1]. As the most abundant cellular anions, chloride anions play a pivotal role in regulating intracellular pH balance. For instance, to ensure the effective function of lysosomes in the degradation of cellular waste and foreign substances by lysosomal enzymes, the lumen of lysosomes maintains an acidic environment (pH 4.5 − 5.0) and an elevated level of chloride anions (∼80 mM). In contrast, cytosol maintains a level of chloride anions ranging from 5 to 20 mM and a nearly neutral pH of 7.2 − 7.4 [2]. In addition, lysosomal pH in cancer cells often decreases to approximately 4 or even lower [3]. The occurrence of lysosomal hyper-acidification under hypoxic intertumoral conditions may lead to cancer progression and multidrug resistance [4]. Thus, modulating pH balance in tumors may shed light on new therapeutic approaches against cancers [5].

It is reported that some agents have shown promise in modulating pH dynamics and hold high potentials in cancer treatment [6]. For example, chloroquine (CQ, 1, Fig. 1A) changes lysosomal pH by acting as a weak base. As a result, it impedes autophagy flux [7]. However, cancer cells may acquire resistant mechanisms to CQ at acidic pH [8]. This resistance poses a significant challenge to the efficacy of CQ [9]. Previous reports have shown that synthetic anion transporters (e.g., 2, Fig. 1A) can facilitate the transport of anions across lipid bilayers, cause perturbation of anion homeostasis and dissipate pH gradient [10]. The consequential increase in pH ultimately culminates in lysosomal dysfunction and inhibition of autophagy flux [11]. These studies have provided solid evidences in support of the proposition that anion transporters endow the feature of disrupting the homeostasis of chloride anions, consequently leading to the dissipation of pH gradients in acidic organelles [12], [13], [14]. In addition, an increase in the concentration of intracellular chloride anions induced by anion transporters may disrupt mitochondrial function [15]. These perturbation in both pH balance and mitochondrial function ultimately culminates in the demise of cancer cells [16].

On the other hand, iron homeostasis plays a critical role in cancer development and progression [17]. Alteration of iron homeostasis by cancer cells leads to the increased uptake and storage of iron and then may contribute to the growth and survival of cancer cells [18]. Several studies have shown that iron chelators (e.g., 3, Fig. 1A) can remove excess iron, inhibit cancer growth and promote cancer cell death [19]. Aliphatic hydroxamic acids (e.g., 4, Fig. 1A) possess the ability to efficiently sequester and capture iron [20]. This capability plays a pivotal role in the regulation of both iron homeostasis and bioactivity. It has been reported that iron chelators can enhance the efficacy of conventional cancer treatments, such as chemotherapy and radiation therapy [21]. Moreover, iron chelators can significantly impair mitochondrial function and exacerbate oxidative stress [22]. However, the clinical efficacy of iron chelation therapy for cancer treatment is still under investigation [23]. In addition, autophagy plays a crucial role in maintaining cellular iron balance by facilitating the degradation of iron-storage proteins [24], [25]. When the levels of cellular iron are reduced by an iron chelator, cells tend to increase autophagy [26]. The up-regulation of autophagy effectively promotes the release of iron that is stored in ferritin within lysosomes [27]. This process serves as a counteraction against the effects of iron chelators. Though autophagy is triggered by iron deficiency in cancer cells, it can also have cytoprotective effects that may contribute to drug resistance [26], [28].

Literature reports have clearly shown that combination of a lysosomal pH modulator (i.e., CQ) with an iron chelator (i.e., DFO) was synthetically lethal in a mouse pancreatic cancer model [29]. However, drug combination therapies are often hindered by some factors, such as poor patient compliance, toxic effects, and potential drug–drug interactions. These challenges could be addressed by dual-functional agents that are able to act on dual target concurrently. Such an approach holds the potential to achieve synergistic effect and reduce toxic effect. The synergistic effects of lysosomal pH inhibitors and iron chelators in combination treatment strongly suggest that dual functional modulators for lysosomal pH and iron homeostasis may offer new opportunities to enhance therapeutic efficacy.

In our previous studies, we have shown that compounds 5–7 (Fig. 1A) act as potent anion transporters and effectively elevate lysosomal pH [30], [31]. Thus, we reasoned that equipping an anion transport-active moiety (e.g., squaramide) with an iron-chelating element (e.g., hydroxamic acid) may afford dual-functional conjugates that are capable of disrupting the homeostasis of anions and iron for cancer treatment. In these conjugates, the anion transport-active moieties mediate the transport of chloride anions and hinder lysosomal function, while the iron chelating unit sequesters and captures excess iron. Both of these actions may lead to the inhibition of autophagy. Moreover, fluctuations in the concentrations of anions and iron could alter mitochondrial function [15], [32], which may lead to increased production of reactive oxygen species (ROS) and thus trigger apoptosis [33].

Herein, we utilized pharmacophore fusion based on compounds 2 and 4 to design squaramide-hydroxamic acid conjugates 8–20 (Fig. 1B) in which hydroxamic acid, a potent iron-chelating group, was linked to a squaramido scaffold for synergistic modulation of anion and iron homeostasis. We evaluated the antiproliferative activity of compounds 8–20 toward several cancer cells, and systematically studied the effect of the optimal compound 16 on the anionophoric activity, lysosomal pH, cellular iron, mitochondrial membrane potential, ROS levels, cell cycle arrest and in vivo antitumor activity. We explored the probable mechanism of action of compound 16 for inducing apoptosis and inhibiting autophagy. For comparison, we also prepared compound 21 bearing an ethyl subunit. This compound displayed similar LogP values with the optimized compound 16 (cLogP: 2.40 for compound 16 and 2.23 for compound 21).