English

• 1. Introduction

  Signal transducer and activator of transcription (STAT) is cytoplasmic transcription factors family which is involved in transducing extracellular growth factor and cytokine signals and activating gene transcription, and it comprises seven members, including STAT1–4, STAT5a, STAT5b and STAT6 [1]. Of which, STAT3 participates in several fundamental cell functions, including cell proliferation, apoptosis, differentiation, immunity, inflammatory response, and mitochondrial oxidative stress [2]. STAT3 is abnormally up-regulated and constitutively activated in numerous cancers through upstream signaling molecules Janus kinases (JAKs) as well as non-receptor tyrosine kinases like sarcoma (Src) and Abelson (Abl), and further regulates the gene levels associated with the cellular cycle, anti-apoptosis, angiogenesis, migration, and invasion [3]. The activation of STAT3 is mainly caused by the phosphorylation of two key amino acid residues, including tyrosine 705 (Tyr705) and serine 727 (Ser727), and STAT3 is highly expressed in several types of cancers and significantly correlated to poor prognosis [4,5]. The constitutive activation mediated by STAT3 phosphorylation is one of non-negligible causes of tumor formation, progression, metastasis and recurrence. Hence, designing and discovering drugs to target STAT3 protein and inhibit its phosphorylation represent a promising direction for effective therapies of those related tumors [6-8].

  The STAT3 signaling pathway could be suppressed by directly targeting the druggable sites of STAT3, blocking the activation of upstream kinases in the STAT3-dependent signaling pathway, and inhibiting STAT3 phosphorylation, etc. [9,10]. Although numerous small molecule inhibitors (SMIs) targeting the upstream kinases have shown potential anti-tumor effects via reducing STAT3 activity, they also inhibit all STAT family proteins with broad-spectrum inhibitory properties, which maybe lead to unfavorable off target effects [11]. Like, apart from inhibiting the phosphorylation of JAK2, the JAK2 inhibitor also simultaneously suppressed the phosphorylation of the downstream proteins STAT3 and STAT5 [12]. Therefore, discovering SMIs which can specifically target the STAT3 protein may be a more promising strategy for developing safe and potent anti-cancer therapeutics. In recent decades, several potent SMIs that directly inhibited STAT3 phosphorylation and activation have been developed, including chemically synthetic, semi-synthetic and natural inhibitors [13-15]. Meanwhile, these inhibitors can be categorized according to the specific domains that inhibit STAT3. In addition, STAT3 inhibitors have both non-covalent and covalent types depending on their binding interaction modes [7].

  So far, most STAT3 SMIs have interacted with the Src homology 2 (SH2) domain through blocking its dimerization, or inhibiting phosphorylation of Tyr705 located in the transactivation domain (TAD), or targeting the DNA-binding domain (DBD) and then inhibited its interaction with DNA. However, most of STAT3 inhibitors suffered from several drawbacks, such as poor suppression, lack of selectivity, high effective concentrations, numerous adverse reactions, and occurrence of chemoresistance. In recent years, with the development and successful application of allosteric inhibitors, a promising way to allosterically regulate STAT3 or its upstream proteins and partially surmount these obstacles has been provided [16,17]. Additionally, STAT3 based dual-target inhibitors could concurrently target multiple kinase sites besides STAT3, exerting cooperative anti-tumor effects and enhancing therapeutic efficacy [18]. Moreover, connecting STAT3 inhibitors with cereblon (CRBN) or other E3 ligands via different linkers to develop proteolysis-targeting chimeras (PROTACs) can selectively and rapidly degrade STAT3 protein in vitro and in vivo, which displayed favorable treatment results for diverse neoplasms [13].

  Several previous reported reviews have summarized the STAT3 inhibitors development journey year by year. For example, Neamati et al. summarized the STAT3 SMIs reported from 2006 to 2012 [19]. Villa et al. reviewed SMIs, natural derivatives, and oligonucleotides targeting different domains of STAT3 over the past decade in 2019 [20]. Wang et al. briefly introduced the role of STAT3 in various cancers and immunotherapy, and systematically reviewed the discovery of non-covalent and covalent STAT3 inhibitors over the past five years in 2019 [7]. Li et al. comprehensively sum up the STAT3 inhibitors discovered in the decade from 2010 to 2021 [21]. Qin et al. reviewed the discovery, structure classification, and optimization process of direct STAT3 SMIs or degraders reported from 2015 to early 2021 [13]. In addition, some natural STAT3 inhibitors have been summarized, such as Qin et al. summarized natural product inhibitors targeting upstream of the STAT3 pathway and directly binding to STAT3 [22], Xie et al. summarized the discovery and chemical synthesis of natural STAT3 inhibitors [14], and Rangappa et al. reviewed that selected natural inhibitors of STAT3 have anti-tumor activity [23]. In this review, we summarized the recent advances of STAT3 SMIs and degraders in different chemical structure types over the last three years and provided insights into the mode of action and pharmacological activities of these compounds, which provide reference and inspiration for further exploitation of anti-cancer drugs through targeting STAT3.

  2. The structure and functions of STAT3

  STAT3 is a signaling molecule expressed in diverse organizations and an important component of the STAT protein family, which regulates the processes of cell proliferation, angiogenesis, differentiation, immune response, inflammation, apoptosis, and drug resistance [24]. STAT3 is composed of six domains with different functions. As shown in Fig. S1 (Supporting information), these domains are named N-terminal domain (NTD), coiled-coil domain (CCD), DBD, linker domain (LD), SH2 domain, and TAD [7]. SH2 as the most conservative domain within the STAT family, is mainly responsible for recruiting a variety of phosphorylated receptors during STAT3 phosphorylation, which contributes to the dimerization of phosphorylated STAT3 [25].

  Fig. 1 summarizes the current mechanisms by which STAT3 may be activated in numerous tumors by cytokines and growth factors such as interleukin-6 (IL-6), epidermal growth factor (EGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF). Associated upstream kinase activities are increased, including JAKs and non-receptor tyrosine kinases (Src and Abl), which in turn activate STAT3 by phosphorylating Tyr705 or Ser727 residues [12,26]. Meanwhile, endogenous negative regulators including protein inhibitor of activated STAT (PIAS), protein tyrosine phosphatase receptor T (PTPRT), and suppressor of cytokine signaling (SOCS) are inactivated or decreased in expression, leading to a loss of negative regulation of STAT3. In addition to being canonically regulated by Tyr705 phosphorylation, STAT3 is also non-canonically regulated by Ser727 phosphorylation of certain members of mitogen-activated protein kinase (MAPK) [27]. Specifically, Ser727 phosphorylation of STAT3 occurs predominantly in response to the serine kinases extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), or MAPK, and then enters mitochondria through interactions with gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), or heat-shock protein 22 (HSP22) [28]. Later, it strengthens oxidative phosphorylation (OXPHOS), which increases adenosine 5′-triphosphate (ATP) production and decreases mitochondrial reactive oxygen species (ROS) production. Furthermore, phosphorylation of STAT3 at Ser727 contributes to maximizing nuclear transcription of STAT3 [29-31].

  Figure 1

  

  Figure 1.  STAT3 signaling pathway. Canonically, STAT3 is translocated to nucleus, which in turn regulates specific gene transcription. Non-canonically, STAT3 can be imported into mitochondria through interaction with GRIM-19 and HSP22. This figure was created using Figdraw (www.figdraw.com).

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  Moreover, mitochondrial STAT3 phosphorylation at Ser727 also promotes Ras-dependent oncogenic transformation [32]. Ras induced activation of the MAPK signaling pathway, which in turn induced STAT3 phosphorylation at Ser727 via the Raf1-MEK-ERK-dependent pathway and the Rac1-p38/JNK pathways, respectively [33,34]. The phosphorylation of Tyr705 or Ser727 ultimately leads to the expression of STAT3 downstream target genes, which then regulate tumor cell proliferation and anti-apoptosis, migration and invasion, chemoresistance, angiogenesis, and immune suppression [10,13]. Meanwhile, the high expression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in hematopoietic tumors promotes STAT3 Ser727 phosphorylation and contributes to the development of B-cell acute lymphoblastic leukemia (B-ALL), and the repression of DYRK1A helped to reduce phosphorylation, decrease mitochondrial ROS and promote B-ALL cell death [35-37]. In addition to phosphorylation, acetylation and methylation also regulate STAT3 signaling through various mechanisms, providing new options to inhibit STAT3 signaling [38]. For instance, acetylation of STAT3 at Lys685 stabilized STAT3 dimers and initiated canonical transactivation of STAT3 downstream genes [39].

  3. The pivotal role of STAT3 in tumorigenesis and development

  3.1 STAT3 in tumorigenesis

  STAT3 is instantaneously activated under normal physiological conditions, however, persistent activation and aberrant expression of STAT3 are often occurred in tumor cells and then intimately linked to tumorigenesis and evolution [40]. Activated STAT3 promoted aerobic glycolysis process in cancer cells, facilitated OXPHOS process in mitochondria, regulated the level of ROS generation, increased lipid uptake in tumor cells, and promoted glutamine expression, thus promoting tumor development [1]. The activation of STAT3 could lead to the regulation of oncogene expression [41]. Abnormal expression or overactivation of STAT3 often occurs in nearly 70% of solid tumors and hematologic malignancies, including breast, brain, liver, ovarian, pancreas, lung, kidney, prostate and gastric cancer, and diverse myeloma, as well as acute myeloid leukemia (AML) [42-44]. Highly active STAT3 can affect the expression of cyclin, thus promoting proliferation of tumor cells. Cyclin D1 (CCND1) and p-STAT3 are overexpressed and accumulated within stomach cancer and oral cancerous cells, and CCND1 gene was upregulated by STAT3, which promoted the stomach cancer cell multiplication [45].

  In addition, the STAT3 pathway promotes cell survival and inhibits apoptosis by regulating apoptosis regulatory proteins, including the B-cell lymphoma 2 (Bcl-2) family [46,47]. Therefore, the activation of STAT3 increases the amount of anti-apoptotic proteins and cycle, facilitating proliferation and repressing apoptosis. STAT3 expression within aging cancer-associated fibroblasts (CAFs) also increases cancer cell viability, and STAT3 was persistently activated in CAFs and formed a feedback axis with platelet-activating factor receptor (PAFR), which promotes malignancy progression through IL-6 and IL-11 with signal communication with tumor cells [8]. Inhibition of STAT3 and PAFR will synergistically and effectively block tumor growth in esophageal phosphoribocytic carcinoma. Meanwhile, Rho-associated coiled-coil kinase 2 (ROCK2) binds to phosphorylated STAT3 and regulates inflammatory responses, and specific inhibition of ROCK2 could modulate STAT3 and attenuate glycolysis, which is critical for tumor cell energy metabolism [48,49]. Some STAT3 inhibitors, such as napabucasin, C188–9, and YY201, have achieved efficacy in a variety of human cancers by directly targeting STAT3 and inhibiting STAT3 activation, effectively suppressing tumorigenesis and progression, and even entering clinical trials (Table 1).

  Table 1

  

  Table 1.  Overview of STAT3 inhibitors and degrader in clinical trials.

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  Drug	Mechanism	Disease	Phase	Identifier	Status
  TTI-101 (also known as C188–9)	STAT3 SH2 domain	Colorectal cancer, breast cancer, hepatocellular cancer, head and neck squamous cell carcinoma, melanoma, gastric adenocarcinoma, NSCLC, advanced cancer	Ⅰ	NCT03195699	Active, not recruiting
  		Hepatocellular carcinoma	Ⅰ/Ⅱ	NCT05440708	Recruiting
  		Breast cancer	Ⅰ/Ⅱ	NCT05384119	Completed
  		Squamous cell carcinoma of head and neck	Ⅰ	NCT05845307	Not yet recruiting
  		Head and neck squamous cell carcinoma	Ⅰ	NCT05668949	Not yet recruiting
  		Pancreatic cancer	Ⅰ/Ⅱ	NCT06141031	Recruiting
  WP1066	STAT3/JAK2	Brain tumor, medulloblastoma, brain Metastases	Ⅰ	NCT04334863	Completed
  		Glioblastoma	Ⅱ	NCT05879250	Recruiting
  		Metastatic melanoma, glioblastoma	Ⅰ	NCT01904123	Completed
  WP1220	STAT3	Cutaneous T-cell lymphoma	Ⅰ	NCT04702503	Completed
  Napabucasin (also known as BBI608 or GB201)	STAT3 SH2 domain	Metastatic pancreatic cancer	Ⅱ/Ⅲ	NCT03721744	Recruiting
  		Metastatic colorectal cancer	Ⅲ	NCT03522649	Recruiting
  OPB-111077	STAT3 SH2 domain	Diffuse large B-cell lymphoma	Ⅰ	NCT04049825	Active, not recruiting
  		Solid tumor	Ⅰ	NCT02250170	Completed
  OPB-31121	STAT3 SH2 domain	Advanced solid tumor	Ⅰ	NCT00955812	Completed
  OPB-51602	STAT3 SH2 domain	Malignant solid tumor	Ⅰ	NCT01184807	Completed
  		Advanced cancer	Ⅰ	NCT01423903	Completed
  DSP-0337	STAT3 SH2 domain	Neoplasms	Ⅰ	NCT03416816	Terminated
  SC-43	STAT3/SHP-1	NSCLC, biliary tract cancer	Ⅰ/Ⅱ	NCT04733521	Unknown
  AZD9150 (also known as Danvatirsen)	Antisense oligonucleotide	NSCLC	Ⅱ	NCT03334617	Active, not recruiting
  		Advanced solid tumors	Ⅰ	NCT03421353	Active, not recruiting
  		Head and neck squamous cell carcinoma	Ⅱ	NCT05814666	Recruiting
  		Colorectal carcinoma, NSCLC, pancreatic cancer	Ⅱ	NCT02983578	Active, not recruiting
  VVD-130850	STAT3	Advanced solid tumors	Ⅰ	NCT06188208	Recruiting
  Silibinin	STAT3 SH2 domain	NSCLC, breast cancer	Not applicable	NCT05689619	Recruiting
  YY201 (also known as YY002)	STAT3 SH2 domain	Advanced solid tumor	Ⅰ	NCT06225856	Recruiting
  KT-333	STAT3 degrader	Lymphoma, solid tumors	Ⅰ	NCT05225584	Recruiting

  3.2 STAT3 in tumor immunology

  STAT3 is not only abnormally activated in tumor cells, but also abnormally expressed in immune cells and CAFs in the tumor microenvironment (TME). Over-activated STAT3 signaling regulates the expression of immune suppressors (e.g., IL-6 and epidermal growth factor receptor (EGFR)) and recruits immunosuppressive cells (e.g., regulatory T (Treg) cells), and polarizes M2-like macrophagocytes to form a tolerant TME [50], which plays a critical function in promoting tumor progression, notably by mediating the emergence of drug resistance in tumor immunotherapy, forcing tumor cells to undergo immune escape [51]. Specifically, STAT3 is a predominant promoter of increasing immune checkpoint factor (e.g., programmed cell death 1 ligand 1 (PD-L1)) expression among immunosuppressed cells. STAT3 blocks T-lymphocyte immune responses through upregulation of PD-L1 within prostate cancer cells [52]. Stimulation of STAT3 signaling by IL-6 induces the expression of PD-L1 in immunosuppressive myeloid cells, induces apoptosis of tumor cells, and represses the immune function of T cells [53]. Activation on STAT3 signaling upregulates PD-L1, which induces T cell apoptosis in colorectal cancer cells [54]. As an upstream regulator of hypoxia-inducible factor 1α (HIF-1α), STAT3 can stimulate HIF-1α upregulation, enhance the expression of immunosuppressive cells such as Treg cells and myeloid-derived suppressor cells (MDSCs), inhibit the immune response, and contribute to the immune escape of tumor cells [55]. Furthermore, STAT3 also inhibits anti-tumor capacity of dendritic cells by suppressing their ripening, activation, and antigen rendering [56]. STAT3 not only suppresses tumor growth directly, but also augments the anti-cancer immune response and rescues the depressed immune microenvironment within tumors [57]. Hence, targeting STAT3 signaling to develop cancer immunotherapeutic drugs is also very promising.

  Combination cancer immunotherapy is considered a promising direction to enhance the effectiveness of cancer treatment and decrease drug resistance. Niclosamide could block STAT3 and the transcription of the immune checkpoint PD-L1, thereby enhancing the therapeutic effect of anti-programmed death 1 (PD-1)/PD-L1 antibodies in non-small cell lung cancer (NSCLC) [58]. ACT001 inhibited phosphorylation of STAT3 in glioblastoma by targeting STAT3, thereby blocking the expression of PD-L1 [59]. The combination of two natural anti-tumor molecules, celastrol and glycyrrhetinic acid, in polymer micelles can inhibit JAK1 and STAT3 phosphorylation, thereby modulating tumor immune differentiation and potently ameliorating the immunosuppressive microenvironment [60]. In addition, the combination of STAT3 inhibitors and chimeric antigen receptor T-Cell immunotherapy (CAR-T) cell therapy will improve durability, anti-tumor effects and reduce toxicities, and inhibition of STAT3 improves the efficacy of CAR-T in liver cancer treatment [61]. C-X-C motif chemokine receptor 4 (CXCR4)-modified CAR-T cells inhibited STAT3 activation and reduced the release of inflammatory factors and stromal cell-derived factor-1α (SDF-1α), thereby reducing the migration of myeloid-derived suppressor cells and promoting therapeutic efficacy against pancreatic cancer [62]. Use of IL6-STAT3/protein kinase B (AKT) pathway and PD-L1 inhibitors sensitizes cancer cells to CAR-T cell therapy and restores the therapeutic effect of adriamycin in breast cancer [63]. Integration of STAT3 and IL-2 binding sites in 5^th generation CAR-T will promote CAR-T cell proliferation [64]. The developed tLyp1 peptide-conjugated hybrid nanoparticles can target Treg cells and inhibit STAT3 and STAT5 phosphorylation, leading to ameliorated TME and anti-tumor immunotherapy [65]. Thus, combination therapy of STAT3 inhibitors with immune checkpoint inhibitors and CAR-T may open new possibilities for long-term and multiple tumor control. This highlighted the important value of developing inhibitors with STAT3 inhibitory activity for the treatment of cancer.

  4. Recent advances in STAT3 inhibitors

  4.1 Benzo[b]thiophene 1,1-dioxide analogs

  The benzo[b]thiophene 1,1-dioxide (BTP) fragment has an effect of inhibiting the STAT3 phosphorylation at Tyr705, which resulted in suppressing tumor cell multiplication and promoting cell death by facilitating the ROS production. Presently, numerous STAT3 inhibitors based on BTP fragments have been reported to perform remarkable STAT3 suppressive effects [13]. Based on BTP scaffold, Zhang et al. spliced a tetramethylpyrazine to obtain 1 (Fig. 2) [66], which had good inhibitory activities against hepatocellular carcinoma, breast cancer and colon cancer cell lines with half maximal inhibitory concentration (IC₅₀) values of 1.50–5.94 µmol/L and blocked the phosphorylation of STAT3. Meanwhile, 1 decreased intracellular ROS content and caused the scavenging of mitochondrial membrane potential. Compound 2 could inhibit STAT3 phosphorylation as well as attenuate transforming growth factor β1 (TGFβ1)-induced proliferation and differentiation of lung fibroblasts, and reverse the epithelial-mesenchymal transition (EMT) process [67]. It potently repressed the NIH-3T3 cell proliferation (IC₅₀ = 0.47 µmol/L), and could prevent and cure pulmonary fibrosis by enhancing the immune microenvironment and restraining inflammation.

  Figure 2

  

  Figure 2.  Structures of BTP analogs 1–4 targeting the SH2 domain.

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  In addition, linking 2-arylimidazo[1,2-a]pyridine to BTP, Wang et al. synthesized 3, which selectively inhibited STAT3 Tyr705 phosphorylation, impaired nuclear translocation and ability to bind DNA, and inhibited STAT3 dimerization within intact cells [68]. Also, it slightly and selectively inhibited the phosphorylation of Ser727 in MDA-MB-231 cells and had potent anti-proliferative effect against human tumor cell lines with IC₅₀s of 1.10–1.60 µmol/L. Subsequently, Wang et al. proceeded to optimize the structure and obtained WZ-2–033 (4) [69]. Despite the reduced binding affinity to the STAT3 SH2 domain tested by surface plasmon resonance (SPR), its anti-proliferative activities against a wide range of cancer cells, including breast and gastric cancers, was somewhat enhanced. At administration of 5 and 15 mg/kg, WZ-2–033 (4) showed more than 45% tumor growth inhibition than compound 3.

  4.2 Sulfonamide analogs

  Sulfonamide analogs hold an essential seat among reported STAT3 inhibitors, and a series of sulfonamide moiety containing STAT3 inhibitors generally exhibited outstanding anti-tumor activities and selective targeting of STAT3 [13,21]. Most of the sulfonamide analogs have a carboxylic acid moiety, which renders the compounds defective in terms of high polarity and low cell permeability, thus exhibiting poor cellular potency. Using phenylboronic acid instead of benzoic acid, W1046 (5, Fig. 3) showed better in vivo anti-tumor characteristics (IC₅₀ = 1.54 µmol/L) and pharmacokinetic properties [70]. W1046 (5) was able to made reversible covalent borates, and showed sustained STAT3-targeted binding and anti-proliferative effects after compound elution. Furthermore, W1046 (5) was able to reduce the expression of V-domain Ig suppressor of T cell activation (VISTA) in vivo and enhance the therapeutic capacity of VISTA monoclonal antibody, as well as significantly inhibit AML by association [71].

  Figure 3

  

  Figure 3.  Structures of sulfonamide analogs 5–7 targeting the SH2 domain.

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  LLL12B (6) is a prodrug molecule remodeled from the LLL12, which selectively inhibited IL-6/STAT3 signaling, and had better inhibition effects on triple-negative breast cancer (TNBC) cell migration and proliferation than C188–9 [72]. Moreover, administered 5 mg/kg of LLL12B (6) had a significant inhibitory effect on the growth of medulloblastoma and ovarian cancer [73,74]. DL14 (7) is a modified inhibitor based on tubulin inhibitor ABT-751 and STAT3 inhibitor C188–9 with the ability to inhibit both STAT3 and microtubule activities [75]. It exhibited excellent anti-tumor effects in vivo, inhibiting 83% of xenograft tumor growth at 30 mg/kg.

  4.3 Quinone analogs

  The majority of quinone analogs with STAT3 inhibitory activities are 1,4-naphthoquinone derivatives, which is also an important scaffold used in the design of anti-cancer drugs. Napabucasin (BBI608) was reported to apply for orphan drug eligibility for the treatment of gastroesophageal junction (GEJ) and pancreatic cancer in 2016, but it was not approved by the US Food and Drug Administration (FDA) for orphan indication (https://www.accessdata.fda.gov/scripts/opdlisting/oopd/listResult.cfm). Currently, it is in the clinical research for the treatment of more cancers [76]. However, the combination with paclitaxel for advanced gastric cancer is in clinical phase Ⅲ, and the results showed that the combination therapy did not improve the survival prognosis of pretreated advanced gastric cancer [77]. Therefore, napabucasin-based STAT3 inhibitors need to be further enhanced their activities. SIL-14 (8, Fig. 4) have been synthesized by fragment-based drug design (FBDD) through introducing the dominant naphthoquinone fragment [78]. SIL-14 (8) exhibited good anti-proliferative and colonization inhibition activities on gastric cancer cell lines, also reduced p-STAT3 levels at 2 µmol/L.

  Figure 4

  

  Figure 4.  Structures of quinone analogs 8–14 targeting the SH2 domain.

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  Current work has developed a variety of new napabucasin-coupled melatonin affixes that can act as useful STAT3 suppressors [79]. The introduced melatonin moiety successfully occupies the missing side pocket in the SH2 domain, allowing the 9 to fit perfectly into the indicated cavity with improved binding affinity (IC₅₀ = 12.95 µmol/L vs. > 100 µmol/L of napabucasin). Compound 9 achieved equivalent anti-tumor levels when administered at 20 mg/kg compared to napabucasin. The isonapabucasin was used as a lead compound to design new derivative 10, and the carbonyl group formed an additional hydrogen bond to the Asp566 residue, enhancing the interaction between STAT3 [80]. In Molm-16 cells, 10 inhibited STAT3 phosphorylation with an IC₅₀ of 4.30 µmol/L. Zhang et al. introduced an oxime moiety and an aromatic amide into the 1,4-naphthoquinone structure to obtain 11, which targeted both indoleamine-2,3-dioxygenase 1 (IDO1) (K_D = 0.08 µmol/L) and STAT3 (K_D = 0.53 µmol/L) [81]. It had the potent anti-proliferative activity with IC₅₀s = 12–37 nmol/L and significantly reduced tumor growth in both immunoreactive mice and nude mice, suggesting dual anti-cancer and immunomodulatory activities.

  Compound 12 was synthesized bearing a sulfonamide moiety, which showed the significant anti-proliferative activities towards NSCLC and breast cancer cells [82]. Besides, Yang et al. utilized a scaffold hopping strategy to replace the phenyl with an isoxazole, and then obtained 13 [83]. It inhibited multiple colorectal cancer cells and was preferable to napabucasin, especially the anti-proliferative activity against HT29 cells up to 5.07 nmol/L. 13 inhibited the phosphorylation of both Tyr705 and Ser727 and had no effect on the phosphorylation of STAT1, STAT5 and JAK2. Similarly, Yang et al. obtained SZ6 (14) by replacing the furan with a thiazole, which effectively bound to STAT3 (K_D = 0.97 µmol/L), almost entirely inhibited Tyr705 phosphorylation at 0.20 µmol/L and inhibited the growth of transplanted tumor at 10 mg/kg [84].

  4.4 Oxadiazole analogs

  Oxadiazole moiety is a key molecular feature for STAT3 inhibitors. Our group reported two polyaromatic heterocyclic compounds HP590 (16) and 18 both containing 1,2,4-oxadiazole (Figs. 5A and B), which have the ability to simultaneously inhibit both Tyr705 and Ser727 phosphorylation [85,86]. Initially, an in-house library was screened and hit 15 was found, which exhibited luciferase inhibition at IC₅₀ of 2.80 µmol/L, and reduced ATP production with IC₅₀ of 3.50 µmol/L [85]. Also, its proliferation inhibitory activities on gastric cancer cells were at low micromolar level (IC₅₀s = 0.90–2.70 µmol/L). To undertake structure activity relationship (SAR) research and improve anti-tumor activity, HP590 (16) was obtained and it specifically targeted STAT3 (K_D = 76.40 nmol/L) and its SH2 domain (K_D = 111.40 nmol/L). It finally boosted the anti-proliferative potency against gastric cancer cells by 50–300-fold (IC₅₀s = 8.70–13.50 nmol/L) compared to hit 15.

  Figure 5

  

  Figure 5.  (A–C) Structures of oxadiazole analogs 15–20 targeting the SH2 domain. The structures of compounds 19 and 20 are not yet published and "Ar" is used to represent their aryl groups. (D) Binding mode and surface binding site of compound 17 with STAT3 (PDB: 1BG1). (E) Binding mode and surface binding site of compound 18 with STAT3 (PDB: 1BG1).

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  Similarly, hit 17 with 1,2,4-oxadiazole and a tetra-aromatic heterocycle skeleton was found (Fig. 5B), which inhibited STAT3 luciferase (IC₅₀ = 1.10 µmol/L) and reduced ATP production (IC₅₀ = 1.60 µmol/L) [86]. Also, it exhibited inhibitory activities with IC₅₀s around 1 µmol/L against two pancreatic cancer cells, BxPC-3 and Capan-2. Subsequently, 18 was found to have enhanced potency, and it had highly potent phosphorylation inhibition activity at both Tyr705 and Ser727 with IC₅₀s of 5.30 and 4.20 nmol/L. Furthermore, 18 successfully blocked the STAT3 nucleus transcription in pancreatic carcinoma cells and inhibited the mitochondrial OXPHOS process. Also, 18 significantly repressed the BxPC-3 xenograft tumor growth in low dosage of 10 mg/kg, with a therapeutic effect greater than positive control BBI608. The interactions between 17 and 18 with the SH2 domains are shown in Figs. 5D and E.

  Furthermore, further structure optimization yielded WB737 (19, structure not published, Fig. 5C), which potently induced natural killer/T-cell lymphoma regression in vivo through concurrently repressing phosphorylation at both Tyr705 and Ser727, and inhibited their growth at concentrations less than 80 nmol/L [87]. Meanwhile, YY002 (20, YY201, structure not published) exhibited significant anti-proliferative activities against diverse pancreatic cancer cells with high levels of Tyr705 and Ser727 phosphorylation, and is currently undergoing phase Ⅰ clinical studies [30]. YY002 (20) had a good oral bioavailability (31.30%, T_1/2 = 14.30 h), and growth inhibition of PANC-1 pancreatic cancer tumor in vivo by 93.56% at a dose of 20 mg/kg.

  4.5 Diazole analogs

  Several compounds containing a diazole or benzodiazole exhibit excellent STAT3 suppression effects. Compounds 21–24 are novel diazole analogs with STAT3 inhibitory effects and they are illustrated in Fig. S2 (Supporting information). As well, W1131 (25, Fig. 6A), comprising an imidazo[1,2-a]pyridine backbone, potently inhibited STAT3 Tyr705 phosphorylation, which revealed remarkable curative efficacies in vivo in both MGC-803 and patient-transfected gastric cancer mice with a low dose of 10 mg/kg [88]. Nevertheless, W1131 (25) had a dramatically shorter half-life (T_1/2 = 12.20 min) and could not be considered as the drug candidate. Subsequently, the authors used a ligand-based drug design (LBDD) strategy for optimization to obtain 26, which showed remarkable results in repressing the cell growth and invasion in both AGS and MGC-803 cells [89]. Its metabolic stabilization was dramatically enlarged (T_1/2 = 41.20 min). Notably, in MGC-803 transplanted mice, 26 showed excellent potency in a low dose at 15 mg/kg.

  Figure 6

  

  Figure 6.  (A, B) Structures of diazole analogs 25–28 targeting the SH2 domain. (C) Binding mode of compound 27 with STAT3 (PDB: 1BG1). (D) Binding mode of compound 28 with STAT3 (PDB: 1BG1).

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  The hit 27 (Fig. 6B) bearing a 1,3,4-thiadiazole was screened to have good IL-6/JAK/STAT3 suppressing activity (IC₅₀ = 4.26 µmol/L) [90]. Then 28 was obtained by further optimization, which exhibited the potent repressive active against the IL-6/JAK/STAT3 pathway (IC₅₀ = 0.65 µmol/L). Meanwhile, 28 achieved 65.30% growth suppression of tumors in DU145 transplanted mice at 50 mg/kg. The (4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol group occupied the pY+1 subpocket of the SH2 domain, but none of them interacted well with the pY and pY-X active sites (Figs. 6C and D).

  4.6 Triazole analogs

  Many triazoles analogs have extensive medicinal properties, including anti-carcinogenic, anti-microbial, and anti-inflammatory. In view of the favorable STAT3 inhibition potential of 1,2,4-triazole derivatives, Abulkhair et al. synthesized 29 (Fig. 7) [91], which showed good potency against multidrug-resistant human mammary carcinoma cell line MDA-MB-231 having an IC₅₀ at 3.61 µmol/L. Kothayer et al. optimized a series of triazole and oxadiazole derivatives and found that 30 showed the most potent anti-proliferative activity in MCF-7 cells among the investigated ones (IC₅₀ = 1.50 µmol/L) [92]. At a concentration of 0.02 µmol/L, the inhibitory activity of STAT3 protein up to 71%, it occupied simultaneously three active pockets (pY, pY+1 and pY-X) of the SH2 domain.

  Figure 7

  

  Figure 7.  Structures of triazole analogs 29 and 30 targeting the SH2 domain.

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  4.7 Indole analogs

  Indole structures are widely distributed in anti-tumor drugs, but most compounds with inhibitory activities against STAT3 are melatonin derivatives. Wang et al. coupled salicylic acid and melatonin to explore their inhibitory effects on inflammation-related cancers [93], and 31 (Fig. 8) reduced the emission of the pro-cancer cell factors tumor necrosis factor-α (TNF-α) and IL-6 in RAW 264.7 immune cells, decreased STAT3 expression and inhibited STAT3 activation in MGC-803 and HCT-116 cells. And then, the authors combined N-phenylanthranilic acid with melatonin to synthesize NP16 (32), which had therapeutic effects on glioblastoma and suppressed the reproduction of U251 glioma cells at the IC₅₀ as 1.87 µmol/L, while inhibiting hCOX-2 (IC₅₀ = 8.29 µmol/L) [94]. Strikingly, NP16 (32) diminished the content of p-JAK2 and p-STAT3 in glial cells, prevented STAT3 transfer to the nucleus.

  Figure 8

  

  Figure 8.  Structures of indole analogs 31–33 targeting the SH2 domain.

  DownLoad: Full-Size Img PowerPoint

  Recently, Wang et al. further optimized the substituents on the phenyl ring of melatonin and found that the 6-dimethylamino compound (structure not shown), which significantly elevated the anti-neuroinflammatory activity and inhibited the activation of microglia [95]. In addition, Hong et al. connected β-carboline alkaloid through a linker to obtain marinacarboline analog MC0704 (33), which had good cytotoxicity against breast cancer MDA-MB-231 cell in IC₅₀ at 3.11 µmol/L [96]. Meanwhile, it effectively down-regulated STAT3 pathway in docetaxel-resistant cell line in IC₅₀ at 2.13 µmol/L and inhibited the invasiveness and metastasis of TNBC cells, as well as restrained the STAT3-mediated EMT process.

  In addition, flavonoid analogs 34 and 35 (Fig. S3A) [97], pyrimidine analogs 36 and 37 (Fig. S3B) [98,99], other structure types of compounds 38–46 (Fig. S4 in Supporting information) [100-108], natural STAT3 inhibitors 47–50 (Fig. S5 in Supporting information) [109-112], compounds that target DBD domain 51–55 (Fig. S6 in Supporting information) [113-116], target LD domain 56 and 57 (Fig. S7A) [117,118] and target NTD domain 58 and 59 (Fig. S7B) [119] can be found in Supporting information.

  5. STAT3 degraders

  Achieving STAT3 protein degradation utilizing PROTAC is a very prospective strategy for oncotherapy. As shown in Fig. 9A, the PROTAC molecule acts as a bifunctional molecule that forms a ternary complex through binding to the STAT3 protein and recruiting the E3 ubiquitin ligase, which is then degraded to fragments by the proteasome [120]. PROTACs can bind to any site of the target protein regardless of high binding affinity [121], providing a new strategy for undruggable targets like STAT3 [122]. Wang et al. firstly reported STAT3 PROTAC SD-36 (61, Fig. 9B) in 2019 that can efficiently induce STAT3 degradation at low nanomolar concentrations [123,124]. It is developed based on SI-109, a SMI targeting the STAT3 SH2 domain. As illustrated by the co-crystal structure of SI-109 with the STAT3 protein in Fig. S8 (Supporting information), the amide bond provided the linker connection site. However, SD-36 (61) was unstable in some cases, and the difluoromethylene was hydrolyzed, then converted to a ketone moiety to obtain SD-91 (62) [125]. SD-91 (62) not only showed the similar binding affinity to STAT3 (K_i = 5.50 nmol/L), but also indicated a bit improved ability to degrade STAT3 protein.

  Figure 9

  

  Figure 9.  (A) The mechanism of action of PROTACs. (B) Structures of STAT3 degraders 61–64. Fig. 9A was created using Figdraw (www.figdraw.com).

  DownLoad: Full-Size Img PowerPoint

  Subsequently, Kymera Therapeutics, Inc. further optimized the linker and CRBN ligand to obtain I-174 (63), which possessed significant STAT3 degrading activity, although the binding activity was weak (WO2020206424A1). Meanwhile, they announced that KT-333 (64) with STAT3 degrading activity in 2022, and has entered phase 1 clinical (NCT05225584) for the treatment of T-cell malignant lymphomas and solid tumors [126]. KT-333 (64) resulted in growth arrest and increased cell death in ALK-positive anaplastic large cell lymphoma (ALCL) in vitro and in vivo, as well as in NK/T-cell lymphomas harboring STAT3 mutants, and in ALK-negative ALCL cell lines. Intermittent administration of KT-333 (64) resulted in 90% or more STAT3 degradation within 48 h, while inhibiting and eliminating lymphoma growth in mice.

  Furthermore, as shown in Fig. S9 (Supporting information), TSM-1 (65, Fig. S9A) based on Toosendanin had potent binding affinity for STAT3 (K_D = 308 nmol/L) and exhibited good anti-proliferative efficacies towards diverse epithelial carcinoma cells at IC₅₀s in 0.29–5.78 µmol/L, and efficiently degraded STAT3 protein [127]. Despite these advances, the development of STAT3 degraders remains challenging, and PROTACs developed based on STAT3 SMIs are inefficient or off-target, such as S3D1 (66, Fig. S9B) based on sulfonamide analogs (CN117720443A), Ⅲ-4 (67, Fig. S9C) based on quinone analogs (CN118026997A), and SDL-1 (68, Fig. S9D) based on S3I-201 [128]. Although they exhibited certain STAT3 degrading activities, their degradation and anti-proliferative effects were poor and still need to be improved. In addition, not all PROTACs developed based on STAT3 inhibitors achieved STAT3 degradation, e.g., XD2–149 developed based on napabucasin did not show STAT3 degradation activity but degraded the ZFP91 protein [129].

  Presently, some emerging protein degradation technologies (TPD) are being accelerated and applied to degrade non-druggable targets, such as molecular glue, autophagy targeting chimeras (AUTAC), lysosome targeting chimeras (LYTAC) and hydrophobic tag tethering degrader (HyTTD) [130-132]. Wang et al. used chaperone-mediated autophagy targeting chimera (CMATAC) to achieve accurate targeting and delivery to tumor cells. STAT3 protein in HCC827 and A547 tumor cells was rapidly and entirely degraded at 50 µmol/L. Zhong et al. developed liposome-encapsulated STAT3 PROTAC prodrug, which had a chemical reprogramming effect on hepatocarcinoma-associated cancer stem cells, and effectively inhibited the growth of hepa1–6-luc tumors in vivo [133]. Further, Kortylewski et al. coupled Decoy-oligodeoxynucleotides (D-ODNs) to acquire a C-STAT3D^PROTAC, which could selectively target myeloid/B cells and successfully degraded STAT3 protein in myeloid cells (decreased 85%, 2 µmol/L) [134]. However, the degradation potency of these emerging TPD strategies still needs to be improved, and they tend to have larger volume or a large number of negative states, poor pharmacokinetic, which pose a challenge for drug delivery and further development.

  6. Clinical trial advances in STAT3 inhibitors and degrader

  Drug development efforts targeting STAT3 protein have been ongoing for nearly three decades, unfortunately, no drugs targeting STAT3 are currently authorized by the FDA to market for cancer treatment. Presently, several STAT3 inhibitors and one degrader are undergoing clinical trials to be administered singly or combined with chemotherapy or targeted drugs (Table 1), several of them have preliminarily shown favorable trial results (http://clinicaltrials.gov/). Orally administered WP1066 has indicated promising therapeutic effects for patients with recurrent glioblastoma at phase Ⅰ clinical trial (NCT01904123) and received a rare pediatric disease designation from the FDA for the treatment of pediatric brain tumor patients (NCT04334863) [114]. Although the phase Ⅲ clinical trial targeting NSCLC declared a failure (NCT02826161), napabucasin has indicated promising clinical results in colorectal and pancreatic cancers, and is also undergoing a phase Ⅲ clinical trial (NCT03721744, NCT03522649). A phase Ⅰ clinical trial of napabucasin's prodrug DSP-0337 against advanced solid tumors was terminated.

  The highly selective TTI-101 is still in clinical phase Ⅰ and the trials are either enrolling or not yet enrolled. Several clinical studies have evaluated the security, tolerance, pharmacokinetic and primary anti-tumor efficacy of AZD9150 in terminal/metastatic cancer patients, and also currently in phase Ⅱ trials against a variety of solid tumors. In addition, YY201 has entered phase Ⅰ clinical trials as the world's first highly active dual phosphorylation inhibitor. Meanwhile, KT-333, the first STAT3 degrader to enter the clinic, is currently in phase Ⅰ and recruiting patients to further explore its clinical safety, pharmacokinetics and other clinical activities against lymphoma and a variety of solid tumors (Table 1).

  7. Summary and perspective

  The direct inhibition of constitutive STAT3 activation has been shown to be a prospective way to cancer therapies. A large number of inhibitors and degraders directly targeting STAT3 have been developed over the past three decades, unfortunately only a few STAT3 inhibitors and one degrader are presently in clinical studies [13,21]. The direct approach focuses on acting upon the SH2 domain to repress STAT3 phosphorylation, dimerization, and nuclear translocation [135]. The development of STAT3 inhibitors that can repress both Tyr705 and Ser727 phosphorylation of STAT3 has also been demonstrated as a promising tactic for STAT3-related drug discovery [85,86]. However, direct STAT3 inhibitors may be characterized by potential off-target effects, low membrane permeability, clinical toxicities, as well as bad oral bioavailability, which further obstruct STAT3-targeted drug development and clinical investigations, and novel tactics are warranted to overcome the above molecular limitations and provide more potent STAT3 inhibitors [7].

  To date, although numerous STAT3 degraders have been designed and developed based on PROTAC technology, just one of them is being evaluated as a drug candidate in clinical trial phase. Considering the possible reasons, on the one hand, they have a variety of PROTAC's own advantages, such as (1) more potent and highly selective, which can override the restricted clinical effectiveness, adverse reactions, and virulence brought by orthosteric or allosteric drugs of STAT3, (2) only catalytic amount of PROTAC molecules is required to achieve degradation, and (3) high binding affinity is not required. However, since the PROTAC molecule is a ternary complex, it usually has a large molecular weight, poor permeability, poor in vivo metabolic stability, and low oral bioavailability, which are unavoidable hurdles on the way to becoming a real drug [136]. Meanwhile, several emerging protein degradation technologies are also swarming into this track of STAT3 protein degradation. Although the FDA has not authorized any STAT3 inhibitors for marketing so far, the better results of the preliminary clinical trials are inspiring us to explore more effective STAT3 inhibitors or degraders. Drug repurposing, natural product-based, drug combinations, and the development of dual-target or multi-target inhibitors that simultaneously target multiple domains of STAT3 or other upstream and downstream targets or other signaling pathway proteins will facilitate the development of STAT3 inhibitors for cancer therapy.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Zhaoyong Kang: Writing – original draft, Investigation. Shen Li: Writing – original draft, Investigation. Yan Li: Writing – original draft, Investigation. Jingfeng Song: Writing – original draft. Yangrui Peng: Writing – original draft, Investigation. Yihua Chen: Writing – review & editing, Project administration, Funding acquisition.

  Acknowledgments

  This study was supported by grants from the National Key Research and Development Program of China (No. 2023YFA1800403), The Science and Technology Commission of Shanghai Municipality (No. 21S11907800), and Key Research and Development Program of Ningxia (No. 2023BEG02010).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110447.

  
  
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  Figure 1  STAT3 signaling pathway. Canonically, STAT3 is translocated to nucleus, which in turn regulates specific gene transcription. Non-canonically, STAT3 can be imported into mitochondria through interaction with GRIM-19 and HSP22. This figure was created using Figdraw (www.figdraw.com).

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  Figure 2  Structures of BTP analogs 1–4 targeting the SH2 domain.

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  Figure 3  Structures of sulfonamide analogs 5–7 targeting the SH2 domain.

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  Figure 4  Structures of quinone analogs 8–14 targeting the SH2 domain.

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  Figure 5  (A–C) Structures of oxadiazole analogs 15–20 targeting the SH2 domain. The structures of compounds 19 and 20 are not yet published and "Ar" is used to represent their aryl groups. (D) Binding mode and surface binding site of compound 17 with STAT3 (PDB: 1BG1). (E) Binding mode and surface binding site of compound 18 with STAT3 (PDB: 1BG1).

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  Figure 6  (A, B) Structures of diazole analogs 25–28 targeting the SH2 domain. (C) Binding mode of compound 27 with STAT3 (PDB: 1BG1). (D) Binding mode of compound 28 with STAT3 (PDB: 1BG1).

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  Figure 7  Structures of triazole analogs 29 and 30 targeting the SH2 domain.

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  Figure 8  Structures of indole analogs 31–33 targeting the SH2 domain.

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  Figure 9  (A) The mechanism of action of PROTACs. (B) Structures of STAT3 degraders 61–64. Fig. 9A was created using Figdraw (www.figdraw.com).

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  Table 1.  Overview of STAT3 inhibitors and degrader in clinical trials.

  Drug	Mechanism	Disease	Phase	Identifier	Status
  TTI-101 (also known as C188–9)	STAT3 SH2 domain	Colorectal cancer, breast cancer, hepatocellular cancer, head and neck squamous cell carcinoma, melanoma, gastric adenocarcinoma, NSCLC, advanced cancer	Ⅰ	NCT03195699	Active, not recruiting
  		Hepatocellular carcinoma	Ⅰ/Ⅱ	NCT05440708	Recruiting
  		Breast cancer	Ⅰ/Ⅱ	NCT05384119	Completed
  		Squamous cell carcinoma of head and neck	Ⅰ	NCT05845307	Not yet recruiting
  		Head and neck squamous cell carcinoma	Ⅰ	NCT05668949	Not yet recruiting
  		Pancreatic cancer	Ⅰ/Ⅱ	NCT06141031	Recruiting
  WP1066	STAT3/JAK2	Brain tumor, medulloblastoma, brain Metastases	Ⅰ	NCT04334863	Completed
  		Glioblastoma	Ⅱ	NCT05879250	Recruiting
  		Metastatic melanoma, glioblastoma	Ⅰ	NCT01904123	Completed
  WP1220	STAT3	Cutaneous T-cell lymphoma	Ⅰ	NCT04702503	Completed
  Napabucasin (also known as BBI608 or GB201)	STAT3 SH2 domain	Metastatic pancreatic cancer	Ⅱ/Ⅲ	NCT03721744	Recruiting
  		Metastatic colorectal cancer	Ⅲ	NCT03522649	Recruiting
  OPB-111077	STAT3 SH2 domain	Diffuse large B-cell lymphoma	Ⅰ	NCT04049825	Active, not recruiting
  		Solid tumor	Ⅰ	NCT02250170	Completed
  OPB-31121	STAT3 SH2 domain	Advanced solid tumor	Ⅰ	NCT00955812	Completed
  OPB-51602	STAT3 SH2 domain	Malignant solid tumor	Ⅰ	NCT01184807	Completed
  		Advanced cancer	Ⅰ	NCT01423903	Completed
  DSP-0337	STAT3 SH2 domain	Neoplasms	Ⅰ	NCT03416816	Terminated
  SC-43	STAT3/SHP-1	NSCLC, biliary tract cancer	Ⅰ/Ⅱ	NCT04733521	Unknown
  AZD9150 (also known as Danvatirsen)	Antisense oligonucleotide	NSCLC	Ⅱ	NCT03334617	Active, not recruiting
  		Advanced solid tumors	Ⅰ	NCT03421353	Active, not recruiting
  		Head and neck squamous cell carcinoma	Ⅱ	NCT05814666	Recruiting
  		Colorectal carcinoma, NSCLC, pancreatic cancer	Ⅱ	NCT02983578	Active, not recruiting
  VVD-130850	STAT3	Advanced solid tumors	Ⅰ	NCT06188208	Recruiting
  Silibinin	STAT3 SH2 domain	NSCLC, breast cancer	Not applicable	NCT05689619	Recruiting
  YY201 (also known as YY002)	STAT3 SH2 domain	Advanced solid tumor	Ⅰ	NCT06225856	Recruiting
  KT-333	STAT3 degrader	Lymphoma, solid tumors	Ⅰ	NCT05225584	Recruiting

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