Breast cancers are malignant tumors that originate from the excessive proliferation of breast epithelial cells. Among them, triple-negative breast cancer (TNBC), accounting for 15 %–20 % of newly diagnosed breast cancer cases [1,2]. Conventional chemotherapy is currently the predominant strategy for TNBC treatment, but the efficacy of anthracycline, paclitaxel and platinum is limited and frequently concomitant with significant toxicities [[3], [4], [5], [6]]. These facts emphasize the urgent need to develop more effective treatments for TNBC.

Signal transducer and activator of transcription 3 (STAT3), a critical member of the STAT family [7,8], regulates fundamental cellular processes including cell transformation, apoptosis, differentiation, and growth [9]. Aberrant activation of STAT3 has been strongly implicated in tumorigenesis, promoting progression of diverse malignancies such as breast [10,11], head and neck [12], non-small cell lung [13], and colorectal cancers [14], as well as hematologic diseases [15,16]. This oncogenic versatility establishes STAT3 as a compelling therapeutic target. STAT3 is often over-activated in breast cancer cells relative to normal breast epithelial cells and is highly expressed in primary breast cancer tissues. In addition, the expression and activation levels of STAT3 are positively correlated with the recurrence rate of breast cancer patients. Studies have demonstrated that the overexpression and persistent activation of STAT3 in TNBC are closely associated with its tumorigenesis, progression, invasion, metastasis, and drug resistance [17]. Thus, STAT3 is a potential target for TNBC treatment [[18], [19], [20], [21]].

Mechanistically, STAT3 is activated in the cytoplasm of mammary cells by phosphorylation of upstream kinases, such as Janus kinases (JAKs), and forms a dimer, which enters the nucleus and exerts its transcription factor function [[22], [23], [24]]. The Src homology 2 (SH2) domain is an important region for STAT3 activation, and phosphorylating activation of STAT3 relies on the proximity to and recognition of the SH2 region [[25], [26], [27]]. Precisely targeting this domain has emerged as a prominent strategy for STAT3 inhibition, with reported small molecule inhibitors including napabucasin [28], stattic [29], S3I-201 [30], STA-21 [31], TTI-101 [32] and STX-0119 [33] (Fig. 1). However, the challenges of inadequate efficacy and unsatisfactory physicochemical properties of STAT3 inhibitors have hindered the development and clinical progress of STAT3-targeted drugs [25]. As a result, no FDA-approved STAT3 inhibitors are currently available for TNBC.

Napabucasin, a natural naphthoquinone isolated from Newbouldia laevis, Ekmanianthe longiflora, and Handroanthus impetiginosus, is a small molecule with extensive anti-cancer activities [34]. It was identified as a cancer stemness inhibitor by suppressing STAT3 [35], and is undergoing clinical trials for cancer treatment [36]. However, it faces challenges of high toxicity, insufficient STAT3 inhibition, and low oral bioavailability in clinical applications. To the best of our knowledge, previous structural modifications of napabucasin have mainly focused on converting quinones to phenols, which are then transformed into various types of ester prodrugs to improve solubility, as well as structural modification of the acetyl group at the 2-position of the furan ring [[37], [38], [39], [40], [41]], which lacks structural diversity. In particular, we previously reported two quinone derivatives, B20 [42] and 15e [43] (Fig. 1), which can significantly inhibit STAT3 phosphorylation and showed promising efficacy in psoriasis treatment. Here, considering the excellent STAT3 inhibiting ability of 15e, we firstly evaluated the antiproliferative activity of 15e and its thieno[3′,2':4,5]benzo[1,2-d]isoxazole-4,8-dione derivatives on TNBC cells. In order to develop more potent and low-toxic STAT3 inhibitors for TNBC treatment, a series of novel furano[3′,2':4,5]benzo[1,2-d]isoxazole-4,8-dione and pyrrolo[3′,2':4,5]benzo[1,2-d]isoxazole-4,8-dione derivatives were designed, synthesized and evaluated for antiproliferative activity against TNBC cells. The most potent analogue, B16, was further subjected to intensive in vitro and in vivo mechanistic and pharmacodynamic studies, which proved to be a potent STAT3 inhibitor and a promising candidate for TNBC treatment.