Identification and Biological Evaluation of Juglone-Derived STAT3 Inhibitors against Colorectal Cancer

Abstract

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) drives colorectal cancer progression by promoting cell proliferation, inhibiting apoptosis, and facilitating angiogenesis, making STAT3 a highly promising therapeutic target. In this study, a series of juglone-based quinone derivatives were designed, synthesized, and systematically evaluated as STAT3 inhibitors. Among them, compound YZZ-24 exhibited the strongest STAT3 binding affinity (K _i = 0.26 μM), which was significantly superior to juglone (K _i = 17.11 μM). Mechanistic studies revealed that YZZ-24 directly binds to STAT3 and effectively inhibits STAT3 phosphorylation at tyrosine 705 (p-STAT3^Tyr705) and STAT3 phosphorylation at serine 727 (p-STAT3^Ser727), thereby blocking downstream oncogenic signaling with minimal effects on upstream kinases. Cellular functional assays demonstrated that YZZ-24 exerts potent antitumor activity against colorectal cancer cells (IC₅₀ = 1.03 μM) and possesses an acceptable safety window (SI = 10.82). Collectively, YZZ-24 is a promising STAT3 inhibitor for the treatment of colorectal cancer.

Introduction

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide. , Despite advances in surgery, chemotherapy, and targeted therapies, the clinical management of CRC is frequently limited by metastasis, recurrence, and therapeutic resistance, highlighting the need for new molecularly targeted strategies. As a point of convergence for multiple oncogenic signaling pathways, STAT3 is constitutively activated in a broad spectrum of human cancers, including CRC. , Upon phosphorylation at Tyr705, STAT3 undergoes dimerization through its Src homology 2 (SH2) domain and subsequently translocates to the nucleus to regulate genes associated with cell proliferation, survival, angiogenesis, and immune evasion. − Persistent STAT3 activation has also been closely associated with chemoresistance in CRC, underscoring STAT3 as an attractive therapeutic target.

The central role of the SH2 domain in mediating both the dimerization and the recruitment of STAT3 to activated receptor complexes makes it an attractive site for therapeutic intervention. Small molecules that disrupt SH2-mediated protein–protein interactions (PPIs) can effectively inhibit STAT3 signaling. Although several STAT3 inhibitors, such as Napabucasin (BBI-608), TTI-101, and WP1066, have entered clinical trials, none have yet received regulatory approval. A major challenge in developing STAT3 inhibitors lies in designing small molecules capable of competing with the high-affinity phosphotyrosine interactions within the SH2 domain while maintaining favorable drug-like properties. Structural analysis of the STAT3 SH2 domain (PDB: 6NJS) reveals a binding interface composed of the phosphotyrosine (pTyr) subsite and adjacent hydrophobic subsite (pTyr+1 and pTyr+X), which collectively determine ligand affinity and selectivity. Accordingly, structure-based drug design (SBDD) strategies that simultaneously engage multiple subpockets within the SH2 domain have emerged as a promising approach for developing potent STAT3 inhibitors. Consistent with this strategy, several reported inhibitors, including SI-109, BP-1–102, and S3I-201, achieve enhanced binding affinity by occupying multiple subpockets within the SH2 domain. Among them, the phosphopeptidomimetic inhibitor SI-109, developed by Wang et al., exhibits strong binding affinity for STAT3. Crystallographic studies reveal that the difluoromethylphosphonate group of SI-109 occupies the pTyr pocket, while its indole and phenyl moieties interact with the hydrophobic pTyr+1 region, providing key structural insights for the rational design of STAT3 inhibitors. However, phosphopeptidomimetic inhibitors often exhibit suboptimal drug-like properties, and SI-109 shows only limited efficacy in certain tumor types. These limitations underscore the need to develop nonpeptidic small-molecule STAT3 inhibitors with improved drug-like properties and enhanced binding affinity toward the SH2 domain.

Juglone (Figure C), a natural naphthoquinone scaffold, has been reported to exhibit anticancer activity and to directly interact with the STAT3 SH2 domain, thereby disrupting STAT3 dimerization. However, its relatively weak binding affinity (K _i = 17.11 μM) and modest antitumor activity limit its direct therapeutic application. Notably, the compact quinone scaffold of juglone contains multiple modifiable positions, making it an attractive starting point for structure-based optimization toward improved SH2 binding. In this study, we employed this dual-site targeting strategy using the natural naphthoquinone juglone as a novel core scaffold. Through rational design and systematic optimization, we developed a series of juglone-derived STAT3 inhibitors. This effort led to the identification of YZZ-24, a potent and selective STAT3 inhibitor. Mechanistic studies demonstrate that YZZ-24 directly binds to the STAT3 SH2 domain and effectively suppresses STAT3 phosphorylation and downstream signaling pathways. Furthermore, YZZ-24 exhibits significant antiproliferative activity in colorectal cancer models. These findings establish the juglone scaffold as a valuable lead for the development of next-generation STAT3-targeted therapies.

1.

Representative STAT3 inhibitors and characterization of juglone. (A) Chemical structures of reported STAT3 inhibitors BBI608, TTI101, and WP1066. (B) Co-crystal structure of the STAT3 SH2 domain in complex with the phosphopeptide SI-109 (PDB ID: 6NJS), showing the binding interface. (C) Chemical structure of juglone, a natural product inhibitor of STAT3. (D) Dose–response curve for the inhibition of STAT3 by juglone, as determined by a fluorescence polarization (FP) assay. Data points represent the mean ± SD from three independent experiments.

Results and Discussion

Docking studies indicated that juglone binds to the STAT3 SH2 domain by occupying the pTyr site. Considering the presence of an adjacent hydrophobic pTyr+X subpocket, a dual-site targeting strategy was adopted to improve binding affinity and selectivity. Accordingly, hydrophobic substituents were introduced onto the 5-hydroxy-1,4-naphthoquinone core to enable simultaneous interactions with both the pTyr site and the hydrophobic subpocket (Figure ). The synthetic routes to the target compounds YZZ-1–25, YZZ-A, and YZZ-B are outlined in Scheme . Juglone was prepared via oxidation of 1,5-dihydroxynaphthalene, followed by bromination to afford intermediate HTK-Br. Subsequent nucleophilic substitution with substituted phenols furnished YZZ-A and YZZ-1–25, while Cu­(OAc)₂-catalyzed coupling with aniline yielded YZZ-B.

2.

Rational design of novel STAT3 inhibitors based on the juglone scaffold. The design starts with the lead compound, juglone. A molecular docking analysis predicted its binding mode within the STAT3 SH2 domain (PDB: 1BG1), showing that the juglone scaffold occupies the phosphotyrosine (pTyr) subsite. Target compounds were designed by introducing various hydrophobic fragments onto the juglone core to exploit this pocket and potentially enhance inhibitory activity.

1. Reagents and Conditions: (a) H₂O₂, Ac₂O, MeOH, 40–60 °C, 5 h; (b) Br₂, CH₃COOH, EtOH, 95 °C; (c) R-ArOH, K₂CO₃, DMF, r.t., 3 h; 5 h; (d) Aniline, Cu­(OAc)₂·H₂O, AcOH, 73 °C, Overnight.

In this study, fluorescence polarization (FP) technology was used to detect the interaction between the STAT3 protein and the fluorescence labeled peptide 5-FAM-SpYLPQTV. This labeled peptide can specifically bind to the SH2 domain of STAT3. When the peptide binds to the STAT3 protein to form a complex, the increased molecular weight leads to a decreased rotational rate, which is reflected as an increased polarization value. When a test compound binds to the SH2 domain, it displaces the fluorescence labeled peptide from the complex. The free peptide, due to its small molecular weight and fast rotational rate, exhibits a lower polarization value. By detecting changes in the polarization value, the binding affinity of compounds to the STAT3 SH2 domain can be indirectly evaluated, providing direct experimental evidence for the interaction between the compounds and the STAT3 SH2 domain. The K _i was calculated using the Cheng-Prusoff equation.

The inhibitory activities of the synthesized compounds were evaluated using a FP assay. Initial screening showed that the oxygen-linked derivative (YZZ-B) exhibited higher activity than the nitrogen-linked analogue (YZZ-A), indicating a linker-dependent effect on STAT3 inhibition. Further structure–activity relationship (SAR) analysis revealed that the introduction of electron-rich alicyclic amines (e.g., piperazine and morpholine) significantly improved inhibitory potency (Table S1). Compounds YZZ-22-YZZ-25 displayed inhibition rates above 90% at 50 μM and were selected for further evaluation. These compounds exhibited K _i values of 0.47–0.26 μM, representing approximately 30–54-fold improvements in binding affinity relative to juglone (K _i = 17.11 μM) (Table ). Notably, YZZ-24 and YZZ-25 exhibited markedly stronger binding affinity than the classical inhibitor Stattic (K _i = 43.79 μM), and representative compounds were therefore selected for further evaluation of their antitumor activity and underlying mechanisms. To further evaluate the physicochemical properties of the compounds, ADMETlab 3.0 was used to predict the physicochemical parameters of the representative compounds and the peptidomimetic SI-109, including cLogP, logS, and TPSA (Table ). Results showed that YZZ-24 and YZZ-25 comply with Lipinski’s Rule of Five and possess superior drug-likeness potential compared to the peptidomimetic SI-109 described above.

1. Determination Results of the Inhibition Constants (K _i) of Related Juglone Derivatives.

^aThe inhibition constant (K _i), a measure of binding affinity, was calculated using the equation K _i = IC₅₀/(1 + [C]/K _d ), where [C] is the protein concentration. A lower K _i value signifies a higher affinity. IC₅₀ is defined as the compound concentration that halves the binding signal of the fluorescent substrate to the protein. Values represent the mean ± SD from three independent experiments.

2. Predicted Physicochemical Characteristics of Representative Compounds .

Compd.	MW(g/mol)	cLogP	logS	TPSA (Å 2 )
SI-109	835.29	1.45	–3.49	244.33
juglone	174.03	1.87	–2.73	54.37
YZZ-A	266.06	2.62	–4.14	63.6
YZZ-24	350.13	1.64	–2.83	78.87
YZZ-25	351.11	2.31	–3.99	76.07

^aAll data were predicted by ADMETlab 3.0.

To explore the structural contributions of the piperazine and morpholine rings to the improvement of compound activity, molecular docking analyses of YZZ-24 and YZZ-25 with the STAT3 protein were performed in this study (Figure A and B). Results showed that both compounds could occupy the pTyr and pTyr+X subsites of the STAT3 SH2 domain and form stable binding conformations. For YZZ-24, the carbonyl oxygen of its naphthoquinone core forms a stable hydrogen bond with the key residue Arg⁶⁰⁹ in the pTyr pocket. The terminal piperazine group interacts with Lys⁵⁹¹ in the pTyr+X pocket via hydrogen bonding. The benzene ring on the side chain forms a π-cation interaction with Lys⁵⁹¹, which further enhances the binding stability between the side chain and the pocket, with a docking binding energy of −6.63 kcal/mol.YZZ-25 exhibited an overall binding mode to STAT3 highly consistent with YZZ-24. The main difference lies in the heterocyclic structure of the side chain: the morpholine ring of YZZ-25 fails to form a direct hydrogen bond with Lys⁵⁹¹, resulting in slightly weaker activity than YZZ-24. Its docking binding energy is −6.62 kcal/mol. Furthermore, ADMETlab 3.0 predictions (Table ) showed that compared with YZZ-A, which contains only a benzene ring, YZZ-24 and YZZ-25 exhibited higher logS values and TPSA through the introduction of piperazine and morpholine rings, indicating enhanced molecular polarity and improved water solubility. The improved water solubility may contribute to their superior activity.

3.

Binding Modes of Juglone Derivatives. (A) Binding modes of YZZ-24 and (B) YZZ-25 with the STAT3 SH2 domain (PDB: 1BG1). (C) Binding modes of YZZ-24 with the STAT1 SH2 domain (PDB: 1BF5) and (D) with the STAT5 SH2 domain (PDB: 1Y1U). Hydrogen bonds are shown in green, and π-cation interactions in orange. Their docking energies are marked in red.

To further explore the selectivity of compound YZZ-24, molecular docking analyses of YZZ-24 with STAT1 and STAT5 were performed (Figure C and D). Results demonstrated that the docking binding energy of YZZ-24 with STAT1 was −5.72 kcal/mol, indicating a moderate binding level and poor stability of the formed complex. Although the carbonyl oxygen of the naphthoquinone core of YZZ-24 could forms a stable hydrogen bond with the key residue Arg⁶⁰² in the pTyr pocket, its side chain failed to insert into the pTyr+X subsite, lacking key site interactions, resulting in weaker affinity for STAT1 than for STAT3.The docking binding energy of YZZ-24 with STAT5 was −5.59 kcal/mol, also indicating poor stability of the complex. While the naphthoquinone core of YZZ-24 could simultaneously form hydrogen bonds with Arg⁶¹⁸ and Ser⁶²² in the pTyr subsite, the compound only formed a single hydrogen bond with Asn⁶⁴² in the pTyr+X subsite, failing to reproduce the characteristic π–cation interaction observed in the STAT3 complex. Due to the lack of strong stabilizing forces in the pTyr+X subsite, the overall binding interaction was weakened, leading to significantly lower affinity of YZZ-24 for STAT5 compared to STAT3.

The antiproliferative activities of the juglone-derived STAT3 inhibitors were evaluated in a panel of human cancer cell lines using the CCK-8 assay. STAT3 is a pan-cancer target that exhibits aberrant and sustained activation in diverse tumor cells. As summarized in Table , YZZ-24 exhibited broad-spectrum antiproliferative activity across multiple cancer cell lines, including pancreatic (PANC-1, BxPC-3, AsPC-1), breast (MDA-MB-231, MCF-7, BT549), and colorectal cancer cells (HT29, HCT116, SW480), with IC₅₀ values predominantly in the low micromolar range. In contrast, YZZ-25 showed more variable activity, with significantly reduced potency in certain cell lines (e.g., BxPC-3, AsPC-1, and HCT116), indicating that subtle structural differences markedly influence cellular activity. Notably, YZZ-24 consistently demonstrated superior antiproliferative activity compared to the classical STAT3 inhibitor Stattic across most tested cancer cell lines, highlighting its enhanced cellular efficacy. In particular, YZZ-24 exhibited potent growth inhibitory effects against SW480 and MDA-MB-231 cells, with IC₅₀ values of 1.03 μM and 1.04 μM, respectively. This is approximately 2-fold more potent than Stattic (IC₅₀ = 2.17 μM). Although YZZ-24 showed similar antiproliferative activity against colorectal cancer cells (SW480) and breast cancer cells (MDA-MB-231), it is worth noting that the safety window of YZZ-24 against normal intestinal epithelial cells (HIEC6) (SI = 10.82) was superior to that of normal mammary epithelial cells (MCF-10A) (SI = 8.63), indicating that this compound exhibits better selectivity in the colorectal cancer model.

3. Broad-Spectrum Screening of Compounds on Proliferation Inhibition Activity of Different Cell Lines.

Cell		YZZ-24 IC50 (μM)	YZZ-25 IC50 (μM)	Stattic IC50 (μM)
Cancer cell	PANC-11	1.60 ± 0.19	2.70 ± 0.11	3.53 ± 0.19
	BxPC-3	3.67 ± 0.12	8.42 ± 0.99	3.45 ± 0.34
	AsPC-1	1.75 ± 0.09	8.15 ± 3.20	2.47 ± 0.07
	MDA-MB-231	1.04 ± 0.01	2.05 ± 0.11	2.26 ± 0.06
	BT549	3.58 ± 0.54	>10.0	3.88 ± 0.23
	MCF-7	1.17 ± 0.14	1.53 ± 0.48	5.09 ± 0.69
	HT29	2.67 ± 0.17	7.76 ± 0.49	3.87 ± 0.35
	HCT116	1.99 ± 0.11	>10.0	3.58 ± 0.17
	SW480	1.03 ± 0.10	2.09 ± 0.15	2.17 ± 0.11
Normal cell	HpDe6	6.29 ± 0.99	25.12 ± 2.77	9.80 ± 0.19
	MCF-10A	8.98 ± 0.90	23.22 ± 1.34	4.03 ± 0.54
	HIEC6	11.15 ± 1.05	N.T.	3.70 ± 0.77

^aIC₅₀ values, defined as the half-maximal inhibitory concentration against cell proliferation, are expressed as the mean ± standard deviation (SD) from three independent experiments. The Selectivity Index (SI) was calculated as the ratio of the half-maximal cytotoxic concentration in normal cells (HIEC-6) to that in cancer cells (SW480) to assess selective toxicity. N.T. denotes compounds not tested.

In summary, YZZ-24 exhibits broad-spectrum anticancer activity while demonstrating favorable selectivity toward colorectal cancer cells, highlighting its potential as a lead compound for the treatment of colorectal cancer. Therefore, subsequent mechanistic studies and pharmacological evaluations were all focused on colorectal cancer.

To elucidate the role of the STAT family in colon cancer, we first analyzed the dependency of a panel of human colon cancer cell lines on STAT family members using publicly available genome-wide CRISPR and RNAi screening data sets. The analysis revealed a pronounced dependency on STAT3 across the majority of the examined cell lines, implicating the STAT3 signaling pathway as a critical mediator of colon cancer cell survival and proliferation (Figure A and B). Consistently, analysis of clinical data sets indicated that elevated STAT3 expression is significantly associated with poor overall survival in patients with colorectal cancer, supporting its potential as a therapeutic target (Figure C).

4.

Mechanistic characterization of YZZ-24 targeting STAT3 in colorectal cancer. (A, B) STAT3 dependency scores derived from CRISPR (A) and RNAi (B) data sets obtained from the Cancer Dependency Map (DepMap) portal. (C) Kaplan–Meier survival analysis showing the association between STAT3 expression and overall survival in patients with colorectal cancer. (D) Protein thermal shift assay (PTSA) demonstrating the interaction between YZZ-24 and STAT3. (E) Western blot analysis of total and phosphorylated STAT family proteins in SW480 cells treated with YZZ-24 for 48 h. (F, G) Densitometric quantification of STAT protein expression and phosphorylation levels. (H) Western blot analysis of phosphorylated upstream kinases (JAK2, Src, and ERK) in SW480 cells following YZZ-24 treatment. (I) Quantitative analysis of phosphorylated upstream kinases. (J) Western blot analysis of STAT3 downstream effector proteins (Cyclin D1, BCL-2, BCL-xL, and Survivin) in SW480 cells treated with YZZ-24. (K) Quantitative analysis of downstream effector protein expression. Data are presented as mean ± SD. Statistical significance was determined relative to the control group (*P < 0.05, ****P < 0.0001).

To further validate STAT3 as the molecular target of YZZ-24, the direct interaction between YZZ-24 and STAT3 was investigated using protein thermal shift assays (PTSA). YZZ-24 markedly enhanced the thermal stability of STAT3, consistent with a direct binding event; notably, apo-STAT3 exhibited thermal denaturation at approximately 54 °C, whereas the presence of YZZ-24 maintained protein stability up to 62.4 °C (Figure D). Subsequent mechanistic investigations revealed that YZZ-24 dose-dependently suppresses p-STAT3^Tyr705 and p-STAT3^Ser727 (Figure E and F, P < 0.0001). In contrast, no significant effects were observed on the phosphorylation of STAT1 or STAT5, nor on the phosphorylation of upstream kinases, including JAK2, Src, and ERK (Figure E and G–I, all p > 0.05). These findings indicate that YZZ-24 selectively targets STAT3 signaling. Functionally, inhibition of STAT3 signaling by YZZ-24 resulted in the downregulation of key downstream effectors, including the cell cycle regulator Cyclin D1 and antiapoptotic proteins BCL-2, BCL-xL, and Survivin, thereby contributing to its antiproliferative effects (Figure J and K, P < 0.05 and P < 0.0001). Collectively, these results demonstrate that YZZ-24 directly engages STAT3, selectively inhibits its phosphorylation, and disrupts downstream oncogenic signaling, thereby exerting antitumor activity in colorectal cancer cells.

The antitumor activity of YZZ-24 was further evaluated in human colon cancer SW480 cells. Wound healing assays showed that YZZ-24 significantly inhibited the migratory capacity of SW480 cells at concentrations of 4 and 6 μM, with inhibition rates of 66.79% and 96.01%, respectively. These results indicate a concentration-dependent inhibition of cell migration. Colony formation assays further demonstrated that YZZ-24 markedly reduced the clonogenic capacity of SW480 cells in a concentration-dependent manner over the range of 0.2–0.8 μM, with inhibition rates of 18.53%, 73.49%, 98.23%, and 99.20%, respectively (Figure C and D, P < 0.0001). These findings support the strong antiproliferative effect of YZZ-24. Hoechst 33342 staining revealed condensed and fragmented nuclei in SW480 cells following YZZ-24 treatment, indicative of apoptotic cell death (Figure E). Consistently, flow cytometric analysis using Annexin V-FITC/7-AAD double staining demonstrated that YZZ-24 significantly increased both early and late apoptotic cell populations in a concentration-dependent manner (Figure F and G, P < 0.05).

5.

Effects of YZZ-24 on migration, proliferation, and apoptosis in SW480 cells. (A) Representative images of wound healing assays following treatment with YZZ-24. (B) Quantitative analysis of cell migration. (C) Representative images of colony formation assays. (D) Quantification of colony formation. (E) Hoechst 33342 staining showing nuclear morphological changes. (F) Flow cytometry analysis of apoptosis using Annexin V-FITC/7-AAD staining. (G) Quantification of apoptotic cell populations. Data are presented as mean ± SD. Statistical significance was determined relative to the control group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Collectively, these results demonstrate that YZZ-24 exerts antitumor effects in colon cancer cells by inhibiting cell migration and proliferation and inducing apoptosis.

Conclusions and Discussion

Colorectal cancer remains a major global health burden due to its high incidence and unfavorable clinical outcomes. STAT3, a key signaling mediator implicated in the initiation and progression of colorectal cancer, has emerged as a compelling therapeutic target; however, no STAT3-targeted therapies have been approved for clinical use to date, largely due to the challenge of disrupting high-affinity phosphotyrosine interactions in the SH2 domain while maintaining drug-like properties. Juglone, a natural product, has been reported to exhibit STAT3 inhibitory activity and antitumor effects, making it a promising scaffold for the development of novel STAT3-targeted agents.

In this study, we employed a dual-site targeting strategy to simultaneously engage the pTyr and adjacent hydrophobic pTyr+X subpockets of the STAT3 SH2 domain. A series of juglone-derived small molecules were synthesized and evaluated, leading to the identification of YZZ-24 as a highly potent and selective STAT3 inhibitor (K _i = 0.26 μM), representing a ∼54-fold enhancement over juglone (K _i = 17.11 μM) and superior potency relative to the classical inhibitor Stattic. This structure-guided optimization demonstrates the effectiveness of dual-site engagement in enhancing both binding affinity and cellular efficacy. Although juglone belongs to the PAINS structure, its derivative YZZ-24 selectively inhibits STAT3 activity, while having no significant effects to STAT1, STAT5, and upstream kinases. Furthermore, all compound concentrations used in the activity evaluations of this study were below their CC₅₀ values against normal cells, which further excludes the possibility of false-positive results.

In cellular assays, YZZ-24 exhibited broad-spectrum antiproliferative activity, with particularly strong activity against colorectal cancer SW480 cells (IC₅₀ = 1.03 μM) and a favorable selectivity index (SI = 10.82), underscoring its cancer-selective cytotoxicity. Mechanistic studies confirmed that YZZ-24 directly binds the STAT3 SH2 domain and selectively inhibits p-STAT3^Tyr705 and p-STAT3^Ser727, without affecting STAT1, STAT5, or the phosphorylation of upstream kinases (JAK2, Src, ERK), indicating high pathway specificity. Downstream signaling analysis revealed effective suppression of STAT3-regulated oncogenic proteins, including Cyclin D1, BCL-2, BCL-xL, and Survivin. Functionally, YZZ-24 inhibited colony formation, migration, and induced apoptosis in colorectal cancer cells.

Collectively, these findings highlight the juglone scaffold as a versatile framework for developing novel STAT3-targeted therapeutics. The potent, selective, and mechanism-based activity of YZZ-24 provides proof-of-concept for dual-site SH2 engagement as a rational strategy to overcome the historical challenges of STAT3 druggability. Future studies will focus on in vivo efficacy, pharmacokinetics, and potential combination strategies to further validate the translational potential of YZZ-24.

Glossary

Abbreviations

STAT3

signal transducer and activator of transcription 3

SH2

Src homology domain 2

K _i

Inhibition constant

CCK-8

Cell Counting Kit-8.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00161.

• Supporting Information: Docking system construction and molecular docking methods; Preliminary screening results of inhibitory activity of juglone derivatives on STAT3 (Table S1); Antibody information (Table S2); synthesis and characterization of target compounds; HPLC, ¹H and ¹³C NMR spectra, and HRMS spectra (PDF)

§.

J.X.Z., X.Q.L., and Z.J.Z. contributed equally to this work. W.Y.Y. and Y.X.L. supervised the project and participated in the design of the study. J.X.Z., X.Q.L, J.Y.W., T.D., and Y.C.Z. performed the experiments and statistical analysis as well as the writing of the paper. Z.J.Z. and Z.D. guided the design and modified the manuscript. All authors have given approval to the final version of the manuscript.

No unexpected or unusually high safety hazards were encountered.

The authors declare no competing financial interest.