Colorectal cancer (CRC) accounts for approximately 10% of cancer diagnoses and cancer-related deaths yearly [1]. Clinical treatment is mainly surgical resection, adjuvant by chemotherapy and radiotherapy with obstacles of recurrence, metastasis, drug toxicity, and resistance [2]. Ferroptosis has shown great therapeutic potential in colorectal cancer, including proliferation inhibition [3] and resistance reversion [4]. Developing new molecules to trigger ferroptosis may provide a new approach to treat CRC.

Ferroptosis is a form of regulated cell death different from cell necrosis, autophagy, apoptosis, and pyroptosis, which is dependent on the accumulation of reactive oxygen species (ROS) caused by iron-mediated lipid peroxidation [5], involved in the pathogenesis of tumor [6], ischaemic organ injuries [7], neurodegeneration [8], liver fibrosis [9] and autoimmune diseases [10]. When ferroptosis occurs, ferrous ions react with hydrogen peroxide to produce excessive hydroxyl free radicals, eventually leading to cell membrane rupture and death [5,11]. There is increasing evidence to suggest that ferroptosis is inhibited in diverse cancer types and functions as a dynamic tumor suppressor in cancer development, indicating that the regulation of ferroptosis can be utilized as an interventional target for tumor treatment. Small molecules [12] or nanomaterials [13] that reprogram cancer cells to undergo ferroptosis are considered effective drugs for cancer therapy. Among them, GPX4 inhibitors have recently attracted wide attention due to their excellent anticancer potential. However, the vulnerability of cancer cells to GPX4 inhibition varies depending on their metabolic states [14]. Therefore, it is imperative to develop alternative mechanisms for inducing ferroptosis to provide a complementary therapeutic approach.

Ferritinophagy is a practical pathway to induce ferroptosis [15]. Sustained activation of ferritinophagy causes dysregulated intracellular iron homeostasis and severe iron overload, closely related to the sensitivity of the tumor to ferroptosis. Ferritinophagy is mediated by nuclear receptor coactivator 4 (NCOA4), an autophagic cargo receptor that recognizes ferritin heavy chain 1 (FTH1). When the iron level is low in cells, NCOA4 transports FTH1 to the autophagosome. Then the complex is delivered into the lysosome for degradation to release ferrous iron [[15], [16], [17]], thereby resulting in ferroptosis cascades. Previous studies have found that the natural products sesquiterpene lactone fraction [18], baicalein [19], huaier [20], dihydroartemisinin [21,22], and juglone [23] could reduce the expression of FTH1 protein. However, their deep mechanism still is not revealed.

The NCOA4-FTH1 protein-protein interaction (PPI) is an indispensable node of ferritinophagy [15]. It has been revealed that HERC2 [24], OGT [25], and IRP/IRE system [21] regulated the interaction of NCOA4 and FTH1 through ubiquitination modification, glycosylation modification and protein synthesis, respectively. Recently, the first NCOA4−FTH1 interaction inhibitor was reported for the development of ferroptosis inhibitors against neurological diseases [26], while the COPZ1/NCOA4/FTH1 axis was disclosed as a novel therapeutic target for the treatment of human glioblastoma [27]. These evidences suggest that the discovery of identified ferritinophagy inducers by enhancing NCOA4-FTH1 PPI is vital for tumor treatment targeting ferroptosis.

Sinomenine (SIN), the main active alkaloid of traditional Chinese medicine Sinomenium acutum (Qing Feng Teng), was clinically used to treat rheumatoid arthritis in China. Studies have shown that sinomenine and related ester derivatives exhibited promising antitumor activity by promoting ROS production [28] or arresting cell cycle [29], but the deep mechanism is yet not fully understood. Particularly, the abnormal increase of mitochondrial ROS was reported to stimulate ferritinophagy by activating the AMPK-ULK1 axis [30], indicating that ROS might contribute to the occurrence of ferritinophagy. Therefore, it is feasible to induce ferritinophagy by introducing ROS-inducing groups [31] on sinomenine.

Recently, due to the potential of ferroptosis in treating drug-resistant cancer, several FINs have been identified successively [[32], [33], [34], [35]]. Among them, RSL3, ML210, and Erastin are representative inducers. Studies of their SAR [34,36,37] have described that the electrophilic warheads, such as chloroacetamide and nitrile oxide, were the core active group [38] and associated with ROS production in some conditions [31,39]. Therefore, inspired by the excellent anti-tumor activity of sinomenine derivative SW33 [29], different types of electrophilic warheads were introduced based on small-scale SAR studies, mainly chloroacetyl and vinyl, to identify the active fragments with endogenous ROS-inducing function and optimize the moiety combination.

The present study involved the successive synthesis of a total of 28 compounds starting from sinomenine, followed by their evaluation for ROS-inducing activity and cytotoxicity in colorectal cancer cells. This led to the identification of compound D3-3, which possessed a chloro-vinyl group and exhibited high potency as the first ferritinophagy inducer.