Use of caffeic acid ester derivatives in the preparation of drugs for preventing and treating gastric cancer

Abstract

The application belongs to the field of biological medicine and medicinal chemistry, and discloses application of caffeic acid ester derivatives in preparation of drugs for preventing and treating gastric cancer. In view of the key mechanism that ZEB1 transcription factor target point drives energy metabolism reprogramming and mesothelial-mesenchymal transformation of peritoneal mesothelial cells, the application provides a powerful antagonist Phellinsin which specifically targets ZEB1. Experiments prove that the binding affinity and stability of Phellinsin to ZEB1 protein are significantly better than those of ethyl caffeic acid ester, and Phellinsin can effectively block ZEB1 and intervene in the energy metabolism and mesothelial-mesenchymal transformation process of peritoneal mesothelial cells. In-vivo and in-vitro studies show that the compound can significantly destroy the tumor metastasis microenvironment, inhibit peritoneal adhesion of gastric cancer cells, reduce metastatic nodules and ascites formation, and has significant drug treatment potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and medicinal chemistry, specifically relating to the application of caffeic acid ester derivatives in the preparation of drugs for the prevention and treatment of gastric cancer. Background Technology

[0002] Peritoneal metastasis is a major cause of poor prognosis in the progression of gastric cancer. Studies have shown that even in the early stages of gastric cancer before obvious macroscopic metastasis, the peritoneal microenvironment may have already changed. The formation of this "pre-metastatic niche" involves intercellular interactions and microenvironmental remodeling. The colonization and growth of gastric cancer cells in the peritoneum is a complex process, in which changes in the state of peritoneal mesothelial cells play a crucial role. Mesothelial-to-mesenchymal transition (MMT) is considered an important mechanism for peritoneal fibrosis and the formation of the tumor metastatic microenvironment. Although current treatments include systemic chemotherapy and intraperitoneal hyperthermic chemotherapy, the efficacy of treatment for unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis remains limited due to the peritoneal blood barrier and the lack of specific intervention targets, necessitating the development of new treatment strategies.

[0003] Primary gastric cancer that has spread to the peritoneum / implanted metastases falls under the category of advanced, metastatic, or recurrent gastric cancer. The pathological type of recurrent or metastatic gastric cancer with peritoneal metastasis can be described as a clinical indication / target patient population. "With peritoneal metastasis" further limits the disease status and lesion location, essentially belonging to a specific subtype or specific metastatic site population under the broad indication of "unresectable, recurrent, or metastatic gastric cancer".

[0004] In the development, progression, and metastasis of tumors, zinc finger E-box binding homeodomain 1 (ZEB1) has been identified as a key transcription factor. ZEB1 is not only abnormally highly expressed in various tumors, including cervical cancer, promoting cell growth, invasion, and epithelial-mesenchymal transition (EMT), but also plays a crucial role in regulating cellular energy metabolism reprogramming. Recent studies indicate that ZEB1 can regulate the gene expression of glycolytic enzymes such as hexokinase-II [HK2], platelet phosphofructokinase (PFKP), and pyruvate kinase M2 isoform (PKM2), thereby promoting aerobic glycolysis. This metabolic reprogramming provides the energy and material basis for rapid cell proliferation and fibrosis. In studies of renal fibrosis models, inhibiting ZEB1 expression significantly reduced the rate of glycolysis, thus improving the fibrosis process, suggesting that targeting ZEB1-mediated metabolic regulation is a potential strategy for treating fibrosis and related pathological processes.

[0005] Caffeic acid and its derivatives have attracted attention due to their broad range of biological activities, such as antitumor, anti-inflammatory, antibacterial, and antioxidant effects (Reference 1: Discovery and Development of Caffeic Acid Analogs as Versatile Therapeutic Agents. Pharmaceuticals 2024, 17, 1403. https: / / doi.org / 10.3390 / ph17101403). Among them, ethyl caffeate (EC), an ester derivative of caffeic acid, has been found to possess significant pharmacological activity. Studies have shown that ethyl caffeate can act as a ligand for ZEB1, directly binding to and inhibiting its expression, thereby blocking ZEB1-mediated glycolytic reprogramming and fibrosis. Specifically, ethyl caffeate treatment can reduce the levels of glycolytic enzymes and key metabolites (lactic acid, pyruvate), reversing cellular metabolic abnormalities. Furthermore, caffeic acid amide derivatives and other analogs have also been shown to protect endothelial cells from oxidative damage and regulate cellular metabolism.

[0006] Although caffeic acid and some of its derivatives have been reported in the literature to possess anti-inflammatory, antioxidant, and antitumor biological activities, the pharmacological effects of these compounds are highly dependent on specific substitution structures, esterification / salt formation forms, routes of administration, dosages, and the disease microenvironment. Therefore, it is impossible to directly deduce that a specific caffeic acid ester derivative can be used for peritoneal metastatic gastric cancer. For example, publicly available data shows that ethyl caffeic acid ester has poor water solubility, with a predicted solubility of approximately 0.81 g / L. Since oral medications need to be fully dissolved in gastrointestinal fluids before absorption and distribution to peritoneal metastatic lesions, the poor water solubility, in vivo effective exposure, and lesion accessibility of ethyl caffeic acid ester significantly limit its effective absorption by peritoneal metastatic lesions and its ability to exert a therapeutic effect after administration.

[0007] Given ZEB1's central role in regulating cellular energy metabolism and its crucial function in mesothelial / epithelial cell transformation, regulating ZEB1 to intervene in the metabolic state of peritoneal mesothelial cells holds promise for disrupting the microenvironment of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. Although ethyl caffeate has shown some potential, more potent and targeted drug molecules are still needed to address this specific pathological environment of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. Summary of the Invention

[0008] Unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis is one of the leading causes of death in gastric cancer patients, and currently, there is a lack of effective targeted therapies. Existing research indicates that the remodeling of the peritoneal microenvironment, particularly the mesothelial-mesenchymal transition (MMT) of peritoneal mesothelial cells and the accompanying energy metabolism reprogramming, provides fertile ground for cancer cell colonization. Zinc finger E-box binding homeobox factor 1 (ZEB1), as a key transcription factor, not only drives the EMT / MMT process but also promotes aerobic glycolysis by upregulating hexokinase-II (HK2), platelet phosphofructokinase (PFKP), and pyruvate kinase M2 isoform (PKM2). Although ethyl caffeate (EC) in Reference 1 has been found to act as a ligand for ZEB1, inhibiting its activity, its binding affinity and metabolic regulatory efficacy still need improvement. This invention aims to provide a caffeate derivative with stronger binding affinity and higher metabolic regulatory activity for effectively blocking unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis.

[0009] This invention provides the application of caffeic acid ester derivatives in the preparation of drugs for the prevention and treatment of gastric cancer. The caffeic acid ester derivative pharmaceutical composition includes an effective amount of the active ingredient and pharmaceutically acceptable excipients, carriers, or excipients; wherein the active ingredient is Phellinsin, and Phellinsin has the following chemical structural formula: .

[0010] Phellinsin (CAS No.: 307494-43-5) specifically binds to the ZEB1 protein, and its binding affinity is significantly superior to that of ethyl caffeic acid ester. The mechanism of action of the drug is as follows: by targeting and inhibiting the expression or activity of ZEB1, it specifically targets and inhibits the expression of GLS2 and glutamine catabolism in peritoneal mesothelial cells, thereby reversing the glycolytic metabolic reprogramming and MMT process of peritoneal mesothelial cells.

[0011] Furthermore, the caffeic acid ester derivative pharmaceutical composition comprises phyllinin or a pharmaceutically acceptable salt of phyllinin.

[0012] Furthermore, the pharmaceutically acceptable salt of Phellinsin is Phellinsin sodium salt.

[0013] Furthermore, it also includes the pharmaceutically acceptable excipients.

[0014] Furthermore, the excipient is one or more of a cosolvent, an isotonic regulator, or a pH-adjusting buffer.

[0015] Furthermore, the co-solvent is polysorbate 80.

[0016] Furthermore, the isotonic regulator is mannitol.

[0017] Furthermore, the pH adjustment buffer is a phosphate buffer.

[0018] Furthermore, the caffeic acid ester derivative pharmaceutical composition is formulated into a clinically acceptable injectable formulation.

[0019] Furthermore, according to the CSCO Guidelines for the Diagnosis and Treatment of Gastric Cancer and the WHO Classification of Digestive System Tumors, the gastric cancer described is diffuse gastric cancer.

[0020] Compared with ethyl caffeic acid ester in the prior art, the present invention has the following significant beneficial effects: DARTS experiments have confirmed that the caffeic acid ester derivative Phellinsin binds to ZEB1 protein much less strongly than ethyl caffeic acid ester (binding is tighter), and can more effectively protect ZEB1 protein from enzymatic degradation, showing its potential as a potent ZEB1 antagonist; in unresectable, recurrent, or metastatic gastric cancer models with peritoneal metastasis, the caffeic acid ester derivative significantly reduces the number of peritoneal metastatic nodules and ascites by inhibiting the MMT process of peritoneal mesothelial cells (downregulating α-SMA and Fibronectin) and remodeling the metabolic microenvironment.

[0021] Phellinsin, a caffeic acid ester derivative, can act as an antagonist of zinc finger E-box binding homeobox factor 1 (ZEB1). By targeting and inhibiting the expression or activity of ZEB1, it intervenes in the energy metabolism reprogramming and mesothelial-mesenchymal transition (MMT) process of peritoneal mesothelial cells, thereby disrupting the microenvironment of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. It can be used to prepare pharmaceutical compositions for the prevention or treatment of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The bar chart shows the average binding energy and predicted binding constant of Phellinsin and ZEB1 protein molecules, provided in the embodiments of the present invention.

[0023] Figure 2 The figure shows the experimental results of Phellinsin and ZEB1 protein resisting protease degradation provided in the embodiments of the present invention.

[0024] Figure 3The experimental results of the effect of Phellinsin on the expression of TGF-β-induced mesothelial markers (E-cadherin, Calretinin) and mesothelial-mesenchymal markers (α-SMA, Vimentin, Fibronectin, Collagen I) provided in the embodiments of the present invention are shown in the figure (blank group: untreated original cell group; model group: control group with lesions (fibrosis / mesenchymal transformation) induced by TGF-β; EC group: positive control drug or comparative experimental group; Phellinsin group: experimental drug group of the present invention).

[0025] Figure 4 The figure shows the evaluation results of the ability of Phellinsin to inhibit the adhesion of gastric cancer cells to the surface of mesothelial cells provided in the embodiments of the present invention (the figure shows the statistical results of the number of gastric cancer cells adhering under each high magnification field after Phellinsin treatment compared with ethyl caffeic acid ester treatment).

[0026] Figure 5 The figure shows the experimental results of the inhibitory effect of Phellinin sodium salt injection on ascites formation provided in the embodiments of the present invention.

[0027] Figure 6 The figure shows the experimental results of the effect of Phellinsin sodium injection on the number of metastatic nodules provided in the embodiments of the present invention.

[0028] Figure 7 The figure shows the experimental results of the effect of Phellinin sodium salt injection on tumor weight provided in the embodiments of the present invention.

[0029] Figure 8 The image shows the tumor inhibition rate of nude mice treated with Phellinsin sodium salt injection for gastric cancer, as provided in an embodiment of the present invention.

[0030] Figure 9 The figure shows the results of the correlation analysis between GLS2-mediated glutamine metabolism and peritoneal fibrosis under Phellinsin intervention conditions provided in this embodiment of the invention. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0032] It should be noted that, in this document, the terms "comprising," "including," and any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Specific examples have been used in this document to illustrate the principles and implementation methods of the present invention. These examples are merely for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be pointed out that, due to the limitations of written expression and the objective existence of infinite specific structures, those skilled in the art can make several improvements, modifications, or variations without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, variations, or combinations, or the direct application of the concept and technical solution of the present invention to other situations without modification, should all be considered within the scope of protection of the present invention.

[0033] Example 1: Verification of the binding affinity between Phellinsin and ZEB1 protein This embodiment aims to evaluate the binding affinity of the caffeic acid ester derivative Phellinsin described in this invention to the ZEB1 protein, a key target of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis, using computer-simulated molecular docking technology, and to compare it with ethyl caffeate (EC) to verify the excellent targeting binding ability of the compound of this invention. The following experimental materials and software platform were used for molecular docking research in this embodiment: AutoDockVina 1.1.2 was used as the core software for molecular docking simulation to calculate the binding energy and conformation of the ligand and receptor; protein structure preprocessing (including removal of water molecules, removal of irrelevant ligands, and addition of hydrogen atoms) and visualization analysis of the final docking results were all performed using PyMol 2.4 software; the PDBQT format files required for docking simulation were prepared using AutoDock Tools 1.5.6. The receptor protein used in the experiment was ZEB1 (Uniprot ID: P37275), and the control ligand molecule in Reference 1 was ethyl caffeate (PubChem CID: 5317238). The ZEB1 protein structure was preprocessed using PyMol 2.4 software, including the removal of water molecules, the removal of extraneous ligands, and the addition of hydrogen atoms. The preprocessed protein and ligand molecules were then converted to the PDBQT file format required for docking simulation using AutoDock Tools 1.5.6, keeping other parameters at default settings, and AutoDock Vina was run. Ten optimal docking sites were output, and the conformation with the lowest binding energy and the highest clustering frequency was selected as the most probable ligand-protein binding mode.

[0034] Conclusion: Figure 1 Molecular docking calculations showed that the absolute value of the average binding energy |ΔG| between phyllinin and ZEB1 protein was 7.8 kcal / mol, significantly higher than the 5.4 kcal / mol of the control drug ethyl caffeic acid ester (EC). The predicted binding constant (Kd) of phyllinin was only 1.9 nM, while the Kd value of EC was 115 nM. A smaller Kd value indicates higher affinity; the affinity of phyllinin was approximately 60 times higher than that of EC. The calculation results indicate that the binding affinity of phyllinin to ZEB1 protein is significantly higher than that of ethyl caffeic acid ester. This suggests that specific modifications to the caffeic acid ester structure (such as the unique structure of phyllinin) significantly enhance the interaction between the drug molecule and key amino acid residues of the ZEB1 protein.

[0035] Example 2: Affinity Response and Target Stability Experiment of Phellinsin to ZEB1 Protein The ability of the compound Phellinsin to resist protease hydrolysis after binding to ZEB1 protein was detected by the Drug Affinity Responsive Target Stability (DARTS) experiment. Ethyl caffeic acid ester (EC) was used as a positive control to verify the tightness and stability of the binding between Phellinsin and the target ZEB1. Human peritoneal mesothelial cells (HMrSV5) were used as the cell line in this experiment. Cells were lysed and collected on ice using the M-PER mammalian protein extraction reagent (Thermo Scientific) containing a mixture of protease inhibitors. The supernatant was collected by centrifugation, and protein concentration was measured. Subsequently, drug incubation was performed. The cell lysates were divided into three groups, with blank control (DMSO), positive control ethyl caffeate ester (EC, 100 μM), and the compound of this invention, Phellinsin (100 μM), added respectively. Incubation was carried out at room temperature for 1 hour to allow the drugs to fully bind to the target proteins. Next, limiting enzymatic digestion was performed. Streptomycin (Roche) was added to each group of samples, controlling the protease to total protein ratio at 1:1000 or 1:500, and the reaction was carried out at room temperature for 20 minutes. After the reaction, SDS loading buffer was added, and the mixture was boiled for 5 minutes to terminate the enzymatic digestion. Western blotting was then performed. After SDS-PAGE electrophoresis, transfer to a membrane, and blocking, samples were analyzed using a specific ZEB1 antibody (purchased from Shanghai Xuanya Biotechnology). The sample was incubated overnight at 4°C, and then incubated with secondary antibody before ECL staining. Finally, data analysis was performed. The gray values ​​of the ZEB1 protein band were quantified using ImageJ software, and normalization was performed using GAPDH antibody as an internal control to calculate the percentage of protein residue relative to the unenzyme-added sample.

[0036] Conclusion: Figure 2 The DARTS experimental results showed that, under the same protease digestion conditions, the ZEB1 protein residue rate in the Phellinsin group (89%) was significantly higher than that in the ethyl caffeate group (57%) in Reference 1. This confirms that Phellinsin has a stronger affinity for the ZEB1 protein, and can more effectively stabilize the target protein structure and protect it from enzymatic degradation. This stronger physical binding ability is the molecular basis for its superior glycolysis inhibitory activity and efficacy against unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis in subsequent experiments.

[0037] Example 3: Inhibitory effect of Phellinin on TGF-β1-induced adhesion of peritoneal mesothelial cells (MMT) and gastric cancer cells. 1. Experimental objective: To verify whether the compound Phellinsin of this invention can reverse the mesothelial-mesenchymal transition (MMT) phenotype of peritoneal mesothelial cells and weaken the adhesion ability of gastric cancer cells on peritoneal mesothelial cells by regulating energy metabolism and ZEB1 activity. Ethyl caffeic acid ester (EC) was set as a positive control drug in the experiment.

[0038] 2. Experimental Materials: Human peritoneal mesothelial cell line (HMrSV5); human gastric cancer cell line (MGC-803); recombinant human TGF-β1 (PeproTech, used to induce MMT); DID cell membrane red fluorescent dye (Invitrogen); Matrigel; RIPA lysis buffer; BCA protein quantification kit; E-cadherin, Calretinin (mesothelial markers); α-SMA, Vimentin, Fibronectin, Collagen I (mesenchymal markers); GAPDH (internal control); Phellinsin (CAS No.: 307494-43-5, purchased from MedChemExpress, catalog number HY-N15048, Batch No. 148293), ethyl caffeic acid ester (purchased from Aladdin, catalog number E189374, Batch No. I2315021).

[0039] 3. Experimental methods: Experimental grouping: HMrSV5 cells were seeded in culture plates and, after cell adhesion, were divided into the following 4 groups: Blank control group (Control): an equal volume of DMSO solvent was added; Model group (TGF-β1): TGF-β1 (10 ng / mL) was added and induced for 48 hours to construct the MMT model; Control group (TGF-β1+EC): TGF-β1 (10 ng / mL) and ethyl caffeate (20 μM) were added and co-treated for 48 hours; Invention group (TGF-β1+Phellinsin): TGF-β1 (10 ng / mL) and Phellinsin (20 μM) were added and co-treated for 48 hours.

[0040] MMT biomarker detection: Total protein was extracted from cells in each group, separated by SDS-PAGE electrophoresis, and transferred to a membrane. The cells were then incubated with the corresponding primary antibodies (E-cadherin, Calretinin, α-SMA, Vimentin, Fibronectin, CollagenI), with GAPDH as an internal control. ImageJ software was used to perform grayscale scanning of the protein bands, and the relative expression levels (target protein grayscale / internal control grayscale) were calculated. The results were statistically analyzed using a blank control group as a baseline (normalized to 1.0).

[0041] Tumor cell-mesothelial cell adhesion assay: Human peritoneal mesothelial cells (HMrSV5) treated with different drugs for 48 hours were seeded in 12-well plates and cultured until a dense monolayer formed. Simultaneously, MGC-803 gastric cancer cells in logarithmic growth phase were collected and labeled with DID fluorescent dye (1:100 dilution) at 37°C for 40 minutes. Unbound dye was removed by washing with PBS. Subsequently, the labeled MGC-803 cells were divided into groups of 8 × 10⁶ cells per well. 4 A density of cells was added to a pre-prepared HMrSV5 monolayer and incubated at 37°C for 1 hour. After the reaction, the cells were gently washed three times with PBS to remove unattached cancer cells. Five high-power fields (200×) were randomly selected under an inverted fluorescence microscope for imaging, and the number of attached red fluorescent cancer cells was counted to evaluate the adhesion ability of gastric cancer cells to peritoneal mesothelial cells.

[0042] 4. Results and Conclusions The regulatory effect of phellinsin on the expression of MMT-related proteins was shown in Western blotting grayscale analysis (data used for bar chart presentation, see below). Figure 3 Following TGF-β1 induction, mesothelial cells significantly lost epithelial characteristics (decreased E-cadherin and Calretinin) and acquired mesenchymal characteristics (increased α-SMA, FN, and ColI). Compared with the model group, the phyllin group showed a significantly stronger reversal effect on MMT than the EC group. After phyllin treatment, the levels of fibrosis markers α-SMA and ColagenI decreased to 35% and 38% of those in the model group, respectively, while they only decreased to 65% and 70% in the EC group.

[0043] The compound Phellinsin of this invention significantly inhibits TGF-β1-induced transformation of peritoneal mesothelial cells into mesenchymal cells (MMT). Specifically, it upregulates epithelial markers such as E-cadherin and significantly downregulates fibrosis and mesenchymal markers such as α-SMA and Fibronectin. More importantly, this phenotypic reversal directly translates into a significant inhibition of gastric cancer cell adhesion (Statistical analysis of the number of MGC-803 gastric cancer cells adhering to HMrSV5 mesothelial cells, such as...). Figure 4 (As shown). Compared with ethyl caffeic acid ester, Phellinsin exhibits superior pharmacological activity, providing a strong candidate for blocking the early implantation process of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis.

[0044] Example 4: Phellinsin blocks ZEB1-mediated GLS2 / glutamine metabolic reprogramming and peritoneal mesothelial cell MMT Experimental Objective: To verify the dual regulatory effect of ZEB1 on glycolysis and glutamine metabolism (GLS2 pathway) in peritoneal mesothelial cells; and to confirm the molecular mechanism by which Phellinsin targets and inhibits ZEB1, blocks the "GLS2-glutamate-α-ketoglutarate" metabolic axis, thereby reversing peritoneal mesothelial thrombolysis (MMT) and reducing peritoneal adhesion ability of gastric cancer cells.

[0045] Experimental Methods: 1. Cell Culture and Model Construction: Human peritoneal mesothelial cells (HMrSV5) were routinely cultured in DMEM / F12 medium containing 10% fetal bovine serum and antibiotics. HMrSV5 cells were treated with TGF-β1 (10 ng / mL) combined with 30% serum-free conditioned medium containing MGC-803 gastric cancer cells (the supernatant was collected after centrifugation and filtration after 24 h of culture) for 48 hours to establish a peritoneal metastasis microenvironment-induced mesothelial metastasis (MMT) model. 2. Grouping and Intervention: The experiment was divided into 9 core groups (see Table 1 for details). Phellinsin and positive control ethyl caffeic acid ester (EC) were dissolved in DMSO. ZEB1 siRNA, negative control siRNA, and each overexpression plasmid were transfected using liposomes. After 24 hours of transfection, the modeling conditions and corresponding drugs were added for 48 hours of continued intervention. 3. Detection indicators: After treatment, samples were extracted for RT-qPCR and Western blot to detect the expression of core genes; glutamine (Gln) consumption, glutamate (Glu), α-ketoglutarate (α-KG) and lactate levels were detected using a metabolic kit; in addition, ChIP-qPCR and dual-luciferase reporter gene assays were used to verify the direct transcriptional regulation of GLS2 promoter activity by ZEB1. 4. Experimental Results: The comprehensive detection results of gene expression, metabolic markers and some transcriptional activities in each group are shown in Table 1.

[0046] Table 1. Effects of Phellinsin on gene expression, metabolic markers, and promoter activity in MMT model cells:

[0047] Conclusions: As shown in Table 1, the expression of metabolic enzymes such as ZEB1, GLS2, and PKM2 in mesothelial cells increased sharply after modeling, accompanied by typical MMT characteristics (increased α-SMA and decreased E-cadherin). Although the positive control drug EC could reduce glycolysis (PKM2), its ability to intervene in GLS2 expression and the Gln-Glu-α-KG metabolic axis was limited; while Phellinsin (20 μM) not only completely inhibited glycolysis but also deeply blocked GLS2-mediated glutamine catabolism, and its effect in reversing MMT was significantly better than EC. In the target complementation experiment, ZEB1 overexpression reversed the protective effect of Phellinsin; while GLS2 overexpression alone, although it could not restore ZEB1 levels, was sufficient to reactivate glutamine metabolism and induce MMT (α-SMA rose back to 3.01, and E-cadherin decreased to 0.52), confirming the core role of GLS2 in metabolic reprogramming. ChIP-qPCR experiments confirmed that in the model group, the enrichment folds of ZEB1 in the two E-box regions of the GLS2 promoter surged to 5.60±0.48 and 4.90±0.42, respectively, while Phellinsin (20 μM) and ZEB1 siRNA could reduce them to baseline levels (1.1–1.4 fold). Further reporter gene experiments showed that overexpression of ZEB1 alone could increase the activity of the wild-type GLS2 promoter to 5.10±0.45, but had no effect on the E-box mutant promoter (activity was only 1.24±0.15). ZEB1 drives glutamine metabolism reprogramming by directly binding to the E-box-like region of the GLS2 promoter, thereby providing metabolic support for peritoneal mesothelial cell MMT. Phellinsin, as a potent ZEB1 inhibitor, can simultaneously block the dual metabolic network of "glycolysis" and "GLS2-glutamine breakdown", thereby disrupting the fibrotic microenvironment required for peritoneal metastasis of gastric cancer, showing significant pharmacological advantages over traditional single-pathway metabolic inhibitors (such as ethyl caffeate).

[0048] Example 5: Therapeutic effect and pharmacodynamic verification of phyllin in mouse models of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. At the whole animal level, a nude mouse model of unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis was established to evaluate the inhibitory effect of the compound Phellinsin of this invention on the growth of peritoneal metastases and the formation of ascites. Ethyl caffeate (EC) from Reference 1 was used as a positive control to verify the superior efficacy of Phellinsin in treating unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis by regulating the energy metabolism and microenvironment of mesothelial cells.

[0049] 1. Preparation of Phellinin Sodium Salt and Animal Preparations 1.0 g of Phellinsin technical grade was suspended in 20 mL of anhydrous ethanol. An equivalent amount of sodium hydroxide ethanol solution was slowly added dropwise under ice bath conditions. Through an acid-base reaction, the carboxyl groups in the molecule were converted to a more hydrophilic ionic state. After stirring for 2 hours until the solid was completely dissolved and the solution became clear, the solvent was removed by vacuum distillation. An appropriate amount of ethyl acetate was added for recrystallization, filtration, and vacuum drying to finally obtain solid Phellinsin sodium salt. This salt-forming technique significantly improved the solubility of Phellinsin in physiological saline from extremely low solubility to over 10 mg / mL, effectively overcoming the bottleneck of poor solubility of the technical grade drug. This fully meets the requirements for high-concentration injection administration in in vivo efficacy evaluation experiments in animals such as mice and rats. The formulation composition for the animal preparation is shown in Table 2.

[0050] Table 2. Composition of animal preparation formulations:

[0051] 2. Experimental materials: Experimental animals: BALB / c-nu / nu female nude mice, 4-6 weeks old, weighing 18-22g, purchased from Vital River, and raised in a specific pathogen-free (SPF) environment; human gastric cancer cell line (MGC-803) and human peritoneal mesothelial cell line (HMrSV5); positive control drug: ethyl caffeic acid ester (EC); animal preparations from Example 5 (derived from 1. Phellinsin sodium salt and animal preparations in Example 5); Matrigel matrix gel and PBS buffer.

[0052] 3. Experimental Methods Log-phase MGC-803 gastric cancer cells and HMrSV5 mesothelial cells were collected and mixed at a 1:1 ratio (5 × 10⁶ cells per cell type). 5 10, total cell count 1×10 6(The following is a list of unresectable, recurrent, or metastatic gastric cancer models with peritoneal metastasis, which are established by intraperitoneal injection of ethyl caffeate ester (EC)) suspended in 200 μL PBS (with the addition of appropriate Matrigel to aid tumor formation). On day 7 post-inoculation (as reported in the literature, small nodules are visible on day 8), the mice were randomly divided into four groups (n=6-8 / group) to begin treatment. The administration regimen was based on the effective dose range (25-100 mg / kg) of EC as described in the literature: model control group (Vehicle, intraperitoneal injection of equal volume of solvent), control group (EC, intraperitoneal injection of 50 mg / kg), low-dose group of this invention (low-dose Phellinsin sodium, intraperitoneal injection of 25 mg / kg), and high-dose group of this invention (Phellinsin-H, intraperitoneal injection of 50 mg / kg). The animal was given a dose of mg / kg once daily for 3-4 weeks, with its weight measured every 3 days to monitor its health. At the end of the experiment (days 28-30), the animals were euthanized. Before opening the abdominal cavity, ascites was aspirated with a syringe, and the volume (mL) was accurately measured and the appearance of the ascites was observed and recorded as bloody or clear / pale yellow. Subsequently, the tumor nodules on the peritoneum, greater omentum, mesentery, liver surface, and diaphragm surface were carefully examined. The number of visible metastatic nodules was counted, and all metastatic tumor tissue in the abdominal cavity was removed and weighed wet (g). The PCI score was performed according to the modified Sugarbaker peritoneal carcinoma index to evaluate the treatment effect.

[0053] 4. Results and Conclusions Conclusion: Figures 5-8 As shown, in vivo pharmacodynamic experiments confirmed that the compound Phellinsin of this invention can significantly inhibit the implantation and metastasis of gastric cancer cells in the peritoneal cavity of nude mice. Compared with the existing ZEB1 inhibitor ethyl caffeate (EC), Phellinsin exhibits significant technical advantages in reducing the number of peritoneal nodules, alleviating tumor burden, and controlling malignant ascites. In particular, its ability to change ascites from "bloody" to "clear" and significantly reduce its volume suggests that Phellinsin effectively blocks the deterioration of the tumor microenvironment and vascular / lymphatic leakage caused by mesothelial cell metabolic reprogramming.

[0054] Example 6: A Study on the Differential Regulation of Glutamine Metabolism in Peritoneal Mesothelial Cells by Phellininin and Ethyl Caffeate Example 5 demonstrates that the sodium phenelzine preparation is significantly more effective than ethyl caffeate ester (EC) in inhibiting unresectable, recurrent, or metastatic gastric cancer with peritoneal metastasis. The inventors have noted recent research advancements: peritoneal dialysis and high-glucose environments can induce epithelial-mesenchymal transdifferentiation (EMT) in peritoneal mesothelial cells, and glutaminase 2 (GLS2) plays a crucial regulatory role in this process. GLS2 catalyzes the conversion of glutamine to glutamate, which in turn generates α-ketoglutarate and enters the tricarboxylic acid cycle, providing energy and material basis for the transdifferentiation and fibrosis of mesothelial cells (Reference 2: Li Shan, Zhang Lijie, Xu Xinxin, Liu Jiahan, Jiang Tianxin, Zhao Zhanzheng, Xiao Jing. The role of glutaminase 2 in high glucose-induced epithelial-mesenchymal transdifferentiation of peritoneal mesothelial cells [J]. Chinese Journal of Nephrology, 2025, 41(12):989-996. DOI:10.3760 / cma.j.cn441217-20250514-00531). In order to explore its deeper mechanism, the inventors used peritoneal tissue samples from mice in each group in Example 4 to compare and analyze the differences in the regulation of GLS2 expression and glutamine metabolic pathway in peritoneal mesothelial cells by Phellinsin and EC.

[0055] 1. Experimental Materials Peritoneal tissue from the abdominal wall of nude mice was collected at the experimental endpoint (day 30) of Example 5. Groups included: a model control group, a control group (EC 50 mg / kg), and the high-dose group of this invention (Phellinsin 50 mg / kg). Normal nude mice were also included as a blank control group. Reagents: collagenase, trypsin (for cell isolation); anti-GLS2 antibody, anti-E-cadherin antibody, anti-α-SMA antibody; glutamate detection kit (for detecting glutamine degradation products).

[0056] 2. Experimental Methods Peritoneal tissue was harvested from mice under aseptic conditions, and primary peritoneal mesothelial cells were isolated using trypsin / EDTA digestion. Proteins and total RNA were extracted from the primary cells of each group, and the expression levels of GLS2 (a key enzyme in glutamine degradation), E-cadherin (an epithelial marker), and α-SMA (a mesenchymal / fibrotic marker) were detected. Since GLS2 catalyzes the conversion of glutamine to glutamate, the glutamate content in the cell lysates of each group was detected using a colorimetric assay kit, directly reflecting the enzyme activity of GLS2 and the rate of glutamine degradation.

[0057] 3. Results and Conclusions like Figure 9The results showed that, compared with the normal group, the expression levels of GLS2 protein and mRNA in peritoneal mesothelial cells of mice in the model group were significantly increased (approximately 3.5-fold), accompanied by significant downregulation of E-cadherin and significant upregulation of α-SMA, suggesting that the tumor microenvironment induced high levels of glutamine catabolic metabolism and EMT. Although EC showed some tumor-suppressing effect in Example 5 (mainly likely due to inhibition of ZEB1), in this experiment, the expression level of GLS2 in mesothelial cells derived from the EC group did not decrease significantly (no statistical difference compared with the model group, P>0.05), and the intracellular glutamate content remained at a high level. This indicates that ethyl caffeate cannot block GLS2-mediated glutamine metabolic reprogramming. In mesothelial cells derived from the high-dose Phellinsin group, such as Figure 9 As shown, intracellular glutamate levels decreased significantly, returning to near-normal levels. Along with the blockade of metabolic pathways, α-SMA expression was significantly reduced.

[0058] Phellinsin specifically targets and inhibits GLS2 expression and glutamine catabolism in peritoneal mesothelial cells, a function that ethyl caffeate does not possess. By blocking the glutamine metabolic energy supply (GLS2-glutamate axis) required for mesothelial cell transdifferentiation, Phellinsin fundamentally reverses the peritoneal fibrotic microenvironment (EMT), thereby producing a significantly superior anti-peritoneal metastasis effect compared to ethyl caffeate.

[0059] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of caffeic acid ester derivatives in the preparation of drugs for the prevention and treatment of gastric cancer, characterized in that, The caffeic acid ester derivative pharmaceutical composition comprises an effective amount of an active ingredient and pharmaceutically acceptable excipients, carriers, or excipients; wherein the active ingredient is phyllinin, and phyllinin has the following chemical structural formula: 。 2. The application according to claim 1, characterized in that: The caffeic acid ester derivative pharmaceutical composition comprises phyllinin or a pharmaceutically acceptable salt of phyllinin.

3. The application according to claim 2, characterized in that: The pharmaceutically acceptable salt of Phellinsin is Phellinsin sodium salt.

4. The application according to claim 3, characterized in that: It also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that: The excipient is one or more of a cosolvent, isotonic regulator, or pH-adjusting buffer.

6. The application according to claim 5, characterized in that: The cosolvent is polysorbate 80.

7. The application according to claim 5, characterized in that: The isotonic regulator is mannitol.

8. The application according to claim 5, characterized in that: The pH adjustment buffer is a phosphate buffer.

9. The application according to claim 1, characterized in that: The caffeic acid ester derivative pharmaceutical composition is formulated into a clinically acceptable injectable.

10. The application according to claim 1, characterized in that: The gastric cancer mentioned is diffuse gastric cancer.

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