A multimodal ferroptosis-inducing molecule and its preparation method and application

Abstract

The present invention proposes a multimodal ferroptosis-inducing molecule and its preparation method and application, relating to the technical field of drug development. The present invention provides a method for preparing a ferrocene-modified SN38 prodrug and explores the self-assembly characteristics of the prepared prodrug and its potential to induce ferroptosis to treat colorectal cancer. The ferrocene-modified SN38 prodrug is prepared by chemically coupling SN38 and ferrocenedicarboxylic acid through an esterification reaction of bis(2-hydroxyethyl) disulfide. The prodrug has GSH response characteristics, can achieve tumor microenvironment-specific response release, and can reduce toxic side effects on normal tissues while improving the tumor cell killing effect. The method for preparing a functionalized prodrug proposed in the present invention can be extended to a series of compounds containing reaction sites such as hydroxyl and amino groups (paclitaxel, doxorubicin, R848, all-trans retinoic acid and sulfasalazine, etc.), laying the foundation for optimizing the clinical application of approved drugs and improving their insufficient clinical application.

Description

Technical Field

[0001] The present invention relates to technical fields such as biology, materials science, synthesis of functional nanomaterials, regulation of assembly morphology, treatment of colorectal cancer, and drug delivery, and specifically to a multimodal ferroptosis-inducing molecule and its preparation method and application, and in particular to the preparation and application of the Fc-ss-SN38 multimodal ferroptosis-inducing molecule. Background Art

[0002] Colorectal cancer has rapidly become one of the major global health problems, and its etiology is closely related to the process of industrialization. Emerging research has positioned ferroptosis as a powerful anti-tumor modality, introducing a key approach for the management of colorectal cancer. However, compared with normal colon tissue, glutathione peroxidase 4 (GPX4) is significantly upregulated in colorectal cancer tissue, and the intrinsic resistance of colorectal cancer to ferroptosis weakens its therapeutic effect. To solve this therapeutic deadlock, it is necessary to increase the sensitivity of colorectal cancer cells to ferroptosis. Studies have described four major sensitization strategies: (1) regulating iron ion metabolism to increase the iron ion content in tumor cells; (2) regulating ferroptosis-related lipid metabolism to promote the accumulation of unsaturated lipid peroxides; (3) manipulating amino acid metabolism; and (4) strictly controlling glutathione synthesis and degradation. The first two sensitization strategies discussed above enhance the lipid peroxide generation system, while the latter two strategies impair the lipid peroxide clearance system. In this context, the key to iron metabolism is enhancing the chelation of endogenous iron and meticulously targeting exogenous iron to tumor sites, thereby amplifying ferroptosis efficacy through localized cellular iron overload. Furthermore, regulation of ferroptosis-related lipid metabolism is primarily achieved through lipid peroxidation catalyzed by enzymes such as arachidonic acid 12-lipoxygenase (ALOX12). Concurrently, regulation of amino acid metabolism, primarily through inhibition of glutaminase activity, restricts the uptake of specific amino acids, particularly cystine, thereby reducing intracellular reduced glutathione (GSH) concentrations and attenuating tumor resistance to ferroptosis. During lipid peroxidation, highly reactive lipid free radicals are generated, enhancing ferroptosis. Despite these advances, cancer cells demonstrate a remarkable ability to activate compensatory mechanisms after amino acid inhibition, aiming to restore GSH synthesis through alternative biochemical pathways. Consequently, increased ferroptosis sensitivity has shifted towards regulating GSH metabolism. This requires not only the consumption of excess reduced glutathione but also the inhibition of the regeneration and conversion of oxidized glutathione (GSSG) to reduced glutathione, collectively depleting tumor cell glutathione reserves and amplifying the ferroptotic response.

[0003] Ferroptosis is a multifactorial, synergistically regulated cell death mode, and the aforementioned pathways interactively influence ferroptosis in tumor cells. Integrating these pathways to enhance ferroptosis through single or dual approaches has been insufficient in maximizing therapeutic efficacy. Therefore, we propose an integrated multimodal strategy to enhance ferroptosis by simultaneously regulating the metabolism of iron ions, amino acids, and glutathione synthesis in tumor cells to significantly improve therapeutic indicators. A pragmatic approach to bringing this strategy closer to clinical application is to use existing clinical drugs as the primary reference for drug selection and combination therapy, namely drug repurposing, which utilizes established drugs with known safety profiles for new oncology uses. Drug repurposing has become an important and promising strategy in cancer therapy, providing important insights into drug selection and synergistic combinations. However, implementing drug repurposing in cancer therapy to achieve clinically potential multimodal ferroptosis-enhancing strategies remains a considerable challenge.

[0004] In response, in this study, we identified SN38, an active metabolite of the widely used chemotherapeutic drug irinotecan, as a candidate for multimodal ferroptosis-enhanced CRC therapy. By conjugating ferrocene to SN38 via a disulfide bond, we synthesized, for the first time, a GSH-responsive multimodal ferroptosis-inducing molecule. First, the camptothecin derivative SN38 not only inhibits topoisomerase I, hindering tumor cell proliferation and inducing apoptosis, but also regulates key proteins in ferroptosis: it downregulates the nuclear transcription factor erythroid 2-related factor 2 (Nrf2), reduces GSH production, and inhibits GPX4 activity, thereby attenuating lipid peroxide decomposition and promoting ferroptosis. Concurrently, SN38 upregulates p53 and enhances the expression of the arachidonic acid ALOX12, thereby promoting lipid peroxide accumulation and facilitating ferroptosis. This multimodal effect significantly enhances ferroptosis in colorectal cancer cells. In summary, this multimodal ferroptosis-inducing molecule represents a novel and promising approach for the development of colorectal cancer treatments by harnessing induced ferroptosis to enhance the efficacy of chemotherapy. Summary of the Invention

[0005] To address the shortcomings of current single or dual approaches to enhance ferroptosis by integrating ferroptosis pathways to maximize therapeutic efficacy, we proposed a comprehensive multimodal strategy to enhance ferroptosis by simultaneously modulating the metabolism of iron ions, amino acids, and glutathione synthesis in tumor cells. We generated a multimodal ferroptosis-inducing molecule to significantly improve therapeutic targets. A pragmatic approach to bringing this strategy closer to clinical application is to utilize existing clinical drugs as the primary guide for drug selection and combination therapy, a practice known as drug repurposing, where established drugs with known safety profiles are utilized for novel oncology applications.

[0006] The specific scheme adopted in the present invention is:

[0007] In the first aspect, the present invention seeks to protect a multimodal ferroptosis-inducing molecule, which is a ferrocene-modified SN38 prodrug, namely Fc-ss-SN38, which is obtained by chemical coupling of SN38 and ferrocene dicarboxylic acid through bis(2-hydroxyethyl) disulfide.

[0008] In a second aspect, the present invention claims a method for preparing the above-mentioned multimodal ferroptosis-inducing molecule, comprising the following steps:

[0009] (1) Bis(2-hydroxyethyl) disulfide and tert-butyldimethylsilyl chloride (TBSCl) were added to a DMF solution containing imidazole. After the reaction, the mixture was purified by column chromatography to obtain a colorless liquid compound 1;

[0010] (2) Ferrocene dicarboxylic acid was dissolved in DCM, EDC was added and stirred for 10 min, then DMAP and compound 1 were added. After the reaction was completed, column chromatography was performed to obtain compound 2 as a light yellow oil;

[0011] (3) Add triethylamine trihydrofluoride to the THF solution of compound 2, stir at room temperature, and after the reaction is completed, column chromatography is performed to obtain compound 3 as a yellow oil;

[0012] (4) p-Nitrophenyl chloroformate was added to a DCM solution of compound 3 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 4;

[0013] (5) SN38 was added to a DCM solution of compound 4 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 5, which is the multimodal ferroptosis-inducing molecule.

[0014] As a further optimization of the above preparation method, in step (1), the amount of bis(2-hydroxyethyl)disulfide is 5 mmol, the amount of imidazole is 5 mmol, the amount of tert-butyldimethylsilyl chloride is 11 mmol, the amount of DMF is 5 mL, the reaction time is 24 h, and the reaction temperature is room temperature.

[0015] As a further optimization of the above preparation method, in step (2), the ferrocenedicarboxylic acid is 1.06 mmol, EDC is 2.54 mmol, DMAP is 1 mmol, compound 1 is 2.54 mmol, DCM is 10 mL, the reaction time is 24 h, and the reaction temperature is room temperature.

[0016] As a further optimization of the above preparation method, in step (3), the compound 2 is 0.85 mmol, triethylamine trihydrofluoride is 0.85 mmol, THF is 5 mL, the reaction time is 1 h, and the reaction temperature is room temperature.

[0017] As a further optimization of the above preparation method, in step (4), compound 3 is 0.21 mmol, p-nitrophenyl chloroformate is 0.46 mmol, DIEA is 0.69 mmol, DCM is 10 mL, the reaction time is 12 h, and the reaction temperature is room temperature.

[0018] As a further optimization of the above preparation method, in step (5), compound 4 is 0.17 mmol, SN38 is 0.42 mmol, DIEA is 1.06 mmol, DCM is 10 mL, the reaction time is 72 h, and the reaction temperature is reflux.

[0019] As a further optimization of the above preparation method, compound SN38 in the multimodal ferroptosis-inducing molecule is replaced by a chemical drug modified with ferrocenedicarboxylic acid, an immune adjuvant, a vitamin, a polyunsaturated fatty acid or a sulfonamide drug.

[0020] As a further optimization of the above preparation method, the chemical drug is paclitaxel or doxorubicin, the immune adjuvant is R848 or imiquimod, the vitamin is all-trans retinoic acid or vitamin E, the polyunsaturated fatty acid is arachidonic acid, and the sulfonamide drug is sulfasalazine.

[0021] In a third aspect, the present invention seeks to protect the use of the above-mentioned multimodal ferroptosis-inducing molecule in any of the following:

[0022] (1) Multimodal induction of ferroptosis in colorectal cancer cells;

[0023] (2) Preparation of drugs for treating colorectal cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) A multimodal ferroptosis-inducing molecule that can simultaneously regulate the metabolism of iron ions and glutathione synthesis in tumor cells to enhance ferroptosis and significantly improve therapeutic indicators.

[0026] (2) The prodrug of this multimodal ferroptosis-inducing molecule is an active metabolite of the clinical drug irinotecan in vivo, has a known safety profile, and can be used for new oncology applications through drug repurposing strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The carbon and hydrogen spectra of Fc-ss-SN38 provided in Example 1.

[0028] Figure 2 This is a statistical graph showing the cytotoxicity of Fc-ss-SN38 on CT26 cells provided in Application Example 1.

[0029] Figure 3This is the statistical graph of Annexin V-FITC / PI double staining of CT26 cells by Fc-ss-SN38 provided in Application Example 1 and subsequent flow cytometry analysis.

[0030] Figure 4 This is the result of the cell scratch experiment of Fc-ss-SN38 on CT26 cells provided in Application Example 1.

[0031] Figure 5 This is the analysis result of the effect of Fc-ss-SN38 on the MDA content of CT26 cells provided in Application Example 1.

[0032] Figure 6 This is the analysis result of the effect of Fc-ss-SN38 on the Fe(II) content in CT26 cells provided in Application Example 1.

[0033] Figure 7 This is the analysis result of the effect of Fc-ss-SN38 on the GSH content in CT26 cells provided in Application Example 1.

[0034] Figure 8 This is the in vivo anti-tumor effect of Fc-ss-SN38 on CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0035] Figure 9 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on Ki-67 expression in CT26 subcutaneous tumor-bearing mice in Application Example 2.

[0036] Figure 10 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on in vivo tumor Tunel expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0037] Figure 11 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on the in vivo tumor GPX4 protein expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0038] Figure 12 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on in vivo tumor Nrf2 protein expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0039] Figure 13 This is the in vivo anti-tumor effect of Fc-ss-SN38 on CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0040] Figure 14 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on Ki-67 expression in tumors of CT26 orthotopic colon tumor-bearing mice provided in Application Example 2.

[0041] Figure 15 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on in vivo tumor Tunel expression in mice bearing CT26 orthotopic colon tumors, as provided in Application Example 2.

[0042] Figure 16 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on in vivo tumor GPX4 protein expression in CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0043] Figure 17 This is a graph showing the analysis results of the effect of Fc-ss-SN38 on in vivo tumor Nrf2 protein expression in CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0044] Figure 18 This is the synthetic route of Fc-ss-SN38. DETAILED DESCRIPTION

[0045] The present invention provides a synthesis and preparation method of a multimodal ferroptosis-inducing molecule. The raw materials for preparing the multimodal ferroptosis-inducing molecule include: SN38, bis(2-hydroxyethyl) disulfide, ferrocenedicarboxylic acid and solvents (DCM, DMF and THF).

[0046] The present invention uses bis(2-hydroxyethyl) disulfide to chemically couple SN38 with ferrocene dicarboxylic acid to prepare a multimodal ferroptosis-inducing molecule. The preparation process is simple, and the multimodal ferroptosis-inducing molecule prepared by this method has stable properties, a clear structure, and a reasonable yield. The effectiveness of the prepared multimodal ferroptosis-inducing molecule has been further determined, and it has good application potential in the treatment of colorectal cancer. It has a certain guiding role in the subsequent development and preparation of multimodal ferroptosis-inducing molecules and their use in optimizing colorectal cancer treatment plans.

[0047] The synthetic route of Fc-ss-SN38 is as follows Figure 18 shown.

[0048] The multimodal ferroptosis-inducing molecule is prepared by connecting SN38 and ferrocene via bis(2-hydroxyethyl) disulfide.

[0049] The preparation method of the present invention is simple and easy to operate, and the obtained multimodal ferroptosis-inducing molecules have stable properties. At the same time, the drug molecules can be expanded to include chemical drugs such as paclitaxel and doxorubicin, immune adjuvants such as R848 and imiquimod, vitamins such as all-trans retinoic acid and vitamin E, polyunsaturated fatty acids such as arachidonic acid, and sulfonamides such as sulfasalazine. It has universal applicability and is conducive to the development of a series of new corresponding drug molecules with application prospects.

[0050] (1) Bis(2-hydroxyethyl) disulfide and tert-butyldimethylsilyl chloride (TBSCl) were added to a DMF solution containing imidazole. After the reaction, the mixture was purified by column chromatography to obtain a colorless liquid compound 1;

[0051] (2) Ferrocene dicarboxylic acid was dissolved in DCM, EDC was added and stirred for 10 min, then DMAP and compound 1 were added. After the reaction was completed, column chromatography was performed to obtain compound 2 as a light yellow oil;

[0052] (3) Add triethylamine trihydrofluoride to the THF solution of compound 2, stir at room temperature, and after the reaction is completed, column chromatography is performed to obtain compound 3 as a yellow oil;

[0053] (4) p-Nitrophenyl chloroformate was added to a DCM solution of compound 3 containing DIPEA, and after completion of the reaction at room temperature, column chromatography was performed to obtain compound 4;

[0054] (5) SN38 was added to a DCM solution of compound 4 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 5, which is the multimodal ferroptosis-inducing molecule (Fc-ss-SN38) described in this project.

[0055] Preferably, in step (1), the bis(2-hydroxyethyl) disulfide is 1-10 mmol, for example, 1 mmol, 2 mmol, 4 mmol, 6 mmol, 8 mmol and 10 mmol, etc., preferably 5 mmol; tert-butyldimethylchlorosilane is 1-20 mmol, for example, 1 mmol, 2 mmol, 4 mmol, 8 mmol, 10 mmol, 11 mmol, 15 mmol and 20 mmol, etc., preferably 11 mmol; imidazole is 1-10 mmol, for example, 1 mmol, 2 mmol, 4 mmol, 6 mmol l, 8mmol and 10mmol, etc., preferably 5mmol; DMF is 2-25mL, for example, it can be 2mL, 5mL and 10mL, etc., preferably 5mL; the reaction temperature is 4-60℃, for example, it can be 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., preferably 25℃; the reaction time is 1-24h (for example, it can be 1h, 2h, 4h, 8h, 12h, 24h, etc.), preferably 4h. Preferably, in step (2), the ferrocene dicarboxylic acid is 0.1-4 mmol, for example, 0.53 mmol, 1.06 mmol, 2.12 mmol and 4 mmol, etc., preferably 1.06 mmol; compound 1 is 2.4-9.6 mmol, for example, 1.27 mmol, 2.54 mmol, 5.14 mmol and 9.6 mmol, etc., preferably 2.54 mmol; EDC is 2.4-9.6 mmol, for example, 1.27 mmol, 2.54 mmol, 5.14 mmol and 9.6 mmol, etc., preferably 2.54 mmol; DMAP is 0.1-4 mmol , for example, it can be 0.5mmol, 1mmol, 2mmol and 4mmol, preferably 1mmol; DCM is 2-25mL, for example, it can be 2mL, 5mL and 10mL, etc., preferably 5mL; the reaction temperature is 4-60℃, for example, it can be 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., preferably 25℃; the reaction time is 12-72h (for example, it can be 12h, 24h, 36h, 48h, 60h, 72h, etc.), preferably 24h.

[0056] Preferably, in step (3), the compound 2 is 0.1-10 mmol, for example, 0.11 mmol, 0.22 mmol, 0.44 mmol, 0.88 mmol, 1.76 mmol, 3.52 mmol and 7.04 mmol, etc., preferably 0.22 mmol; triethylamine trifluoroacetate is 0.4-40 mmol, for example, 0.44 mmol, 0.88 mmol, 1.76 mmol, 3.52 mmol, 7.04 mmol, 14.08 mmol and 28.16 mmol, etc. Preferably 0.88 mmol; THF is 2-25 mL, for example, it can be 2 mL, 5 mL and 10 mL, etc., preferably 5 mL; the reaction temperature is 4-60°C, for example, it can be 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc., preferably 25°C; the reaction time is 0.5-8h (for example, it can be 0.5h, 1h, 2h, 4h, 6h, 8h, etc.), preferably 1h.

[0057] Preferably, in step (4), p-nitrophenyl chloroformate is 0.1-10 mmol, for example, it can be 0.11 mmol, 0.23 mmol, 0.46 mmol, 0.92 mmol, 1.84 mmol, 3.68 mmol and 7.36 mmol, etc., preferably 0.46 mmol; compound 3 is 0.1-5 mmol, for example, it can be 0.1 mmol, 0.2 mmol, 0.4 mmol, 0.8 mmol, 1.6 mmol, 3.2 mmol and 6.4 mmol, preferably 0.21 mmol; DIEA is 0.1-15 mmol, for example, it can be 0.2 mmol, 0.4 mmol, 0.8 mmol, 1.6mmol, 3.2mmol, 6.4mmol and 12.8mmol, preferably 0.69mmol; DCM is 2-25mL, for example, it can be 2mL, 5mL, 10mL and 20mL, etc., preferably 10mL; the reaction temperature is 4-60℃, for example, it can be 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., preferably 25℃; the reaction time is 4-24h (for example, it can be 4h, 6h, 8h, 12h and 24h, etc.), preferably 12h.

[0058] Preferably, in step (5), SN38 is 0.1-5 mmol, for example, 0.1 mmol, 0.2 mmol, 0.4 mmol, 0.8 mmol, 1.6 mmol, 3.2 mmol and 6.4 mmol, preferably 0.42 mmol; compound 4 is 0.05-5 mmol, for example, 0.05 mmol, 0.1 mmol, 0.2 mmol, 0.4 mmol, 0.8 mmol, 1.6 mmol and 3.2 mmol, preferably 0.21 mmol; DIPEA is 0.3-15 mmol, for example, 0.3 mmol, 0.6 mmol, 1.2 mmol, The content of the reaction mixture is 2.4mmol, 4.8mmol, 6.4mmol and 12.8mmol, preferably 1.26mmol; the content of DCM is 5-25mL, for example, 5mL, 10mL and 20mL, etc., preferably 10mL; the reaction temperature is 4-60℃, for example, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., preferably 25℃; the reaction time is 24-72h (for example, 24h, 36h, 48h, 60h and 72h, etc.), preferably 72h.

[0059] The present invention also provides a use of the multimodal ferroptosis-inducing molecule described above in the treatment of colorectal cancer.

[0060] The specific application steps of the multimodal ferroptosis-inducing molecule in the treatment of colorectal cancer are as follows:

[0061] (A) Multimodal ferroptosis-inducing molecules were added to the culture system of colorectal cancer cells and incubated for 24 hours. Cytotoxicity assays, wound healing assays, flow cytometry apoptosis assays, and detection of ferroptosis-related indicators (malondialdehyde, glutathione, and iron ion content) were performed.

[0062] (B) Multimodal ferroptosis-inducing molecules were administered to tumor-bearing mice via tail vein injection for in vivo anti-colorectal cancer and drug safety evaluation.

[0063] Preferably, in step (A), the amount of the multimodal ferroptosis-inducing molecule added to the culture system of colorectal cancer cells is 0.03%-10% of the total culture system liquid, which is 1:(50-200), for example, it can be 0.03%, 0.06%, 0.12%, 0.25%, 0.5%, 1%, 5%, 10%, etc.

[0064] Preferably, in step (A), the cell culture temperature is 25° C., and the cell culture time is 24 hours.

[0065] Preferably, in step (B), the dosage for the tumor-bearing mice is 100 μL.

[0066] Preferably, in step (B), the tumor-bearing mice are of Balb / c breed and female gender, and the tumor-bearing methods are subcutaneous tumor-bearing and cecal in situ tumor-bearing.

[0067] In the present invention, the multimodal ferroptosis-inducing molecule achieves excellent anti-tumor effects in the treatment of colorectal cancer, and enhances the death of colorectal cancer cells by promoting cancer cell ferroptosis in a multimodal manner.

[0068] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the specific embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] The sources of the components in the following examples are as follows: SN38 (purity 98%, Sigma), DIPEA (purity 98%, Sigma), ferrocenedicarboxylic acid (purity 98%, Sigma), bis(2-hydroxyethyl) disulfide, DCM (analytical grade, Sigma), DSPE-PEG 2k (purity 98%, Sigma), tert-butyldimethylsilyl chloride (purity 98%, Sigma), imidazole (purity 98%, Sigma), DMF (analytical grade, Sigma), EDC (purity 98%, Sigma), DMAP (purity 98%, Sigma), triethylamine trihydrofluoride (purity 98%, Sigma), THF (analytical grade, Sigma), p-nitrophenyl chloroformate (purity 98%, Sigma), DIEA (purity 98%, Sigma).

[0070] Example 1

[0071] This embodiment provides a multimodal ferroptosis-inducing molecule (Fc-ss-SN38), which is prepared by the following preparation method:

[0072] (1) Weigh 5 mg of Fc-ss-SN38 into a 2 mL plastic centrifuge tube and add 1 mL of tetrahydrofuran. Shake for 5 min to dissolve the sample. If there is any undissolved part, sonicate for 3 min to completely dissolve it and avoid the presence of insoluble matter. Prepare a 5 mg / mL stock solution.

[0073] (2) 80 μL of Fc-ss-SN38 mother solution was placed in a 5 mL plastic centrifuge tube, 4 mL of deionized water was added, and the mixture was shaken for 3 min, and stirred at room temperature overnight to obtain the multimodal ferroptosis-inducing molecule working solution.

[0074] in, Figure 1 The carbon and hydrogen spectra of Fc-ss-SN38 provided in Example 1 are as follows: Figure 1 It can be seen that the multimodal ferroptosis-inducing molecules are spherical particles with uniform particle size.

[0075] Application Example 1

[0076] We performed MTT assay to investigate the cytotoxicity of Fc-ss-SN38 on CT26 cells. Figure 2 ). The results showed that Fc-ss-SN38 exhibited excellent cytotoxicity. In addition, Figure 3 Annexin V-FITC / PI double staining and subsequent flow cytometry analysis showed that the apoptosis induction rate of the Fc-ss-SN38-treated group was 40%, indicating that Fc-ss-SN38 can significantly enhance tumor cell death. Then, in order to observe the ability of Fc-ss-SN38 to inhibit the proliferation of CT26 cells, a cell scratch test was performed. Figure 4 As shown, the cell migration rate of the Fc-ss-SN38-treated group at 24h and 48h was lower than that of the control group, indicating that Fc-ss-SN38 exhibited significant anti-tumor cell proliferation ability. The above results show that Fc-ss-SN38 has a significant effect of inducing apoptosis and inhibiting cancer cell proliferation, indicating that Fc-ss-SN38 has superior anti-tumor potential in vitro.

[0077] Based on the significant anti-tumor effect of Fc-ss-SN38 in vitro, we further explored the ferroptosis-related molecular mechanism of Fc-ss-SN38-induced cell death in CT26 cells. The exploration project mainly includes the intracellular oxidative stress marker MDA ( Figure 5 ), ferrous ions ( Figure 6 ) and GSH( Figure 7 ) content. The MDA content in the Fc-ss-SN38-treated group was 16 times that of the control group; the Fe(II) and total iron contents in the Fc-ss-SN38-treated group were higher than those in the control group, proving that Fc-ss-SN38 significantly caused iron overload in CT26 cells; the cysteine ​​and GSH contents in the Fc-ss-SN38-treated group were lower than those in the control group. These results indicate that Fc-ss-SN38 can cascade reduce the production of GSH.

[0078] Application Example 2

[0079] To verify the antitumor effect of Fc-ss-SN38 in the subcutaneous CT26 tumor model, Fc-ss-SN38 was injected intravenously via the tail vein, and the changes in tumor volume were monitored. Figure 8As shown, the tumor volume increased the least in the Fc-ss-SN38-treated group, indicating that the use of Fc-ss-SN38 had a significant inhibitory effect on tumor growth. After the treatment was completed, the mice were humanely sacrificed and the tumors were removed for observation and measurement. Compared with the control group, the average tumor weight in the Fc-ss-SN38-treated group was the smallest, and the use of Fc-ss-SN38 once again demonstrated an excellent in vivo anti-tumor effect. In order to observe the internal conditions of the tumor more microscopically, sections were prepared from the tumor tissue, as shown in Figure 5. Figure 9 and Figure 10 As shown in the figure, the results of Ki67 and TUNEL showed that the Fc-ss-SN38-treated group had the fewest Ki-67-positive cells, while more TUNEL-positive cells than the other groups. These results indicate that Fc-ss-SN38 triggers necrosis and apoptosis and exerts an inhibitory effect, indicating its anti-tumor properties.

[0080] Based on the ability of Fc-ss-SN38 to trigger ferroptosis in monolayers of CT26 cells, the biological mechanisms of ferroptosis in animal models were further investigated by evaluating specific markers in subcutaneous CT26 tumor-bearing mice. Figure 11 The fluorescence signal of GPX4 in the Fc-ss-SN38-treated group was weaker than that in the control group, indicating that the Fc-ss-SN38-treated group had the most severe reduction in the ability to clear cellular lipid peroxides in vivo compared with the control group. In addition, the fluorescence signal of Nrf2 in the Fc-ss-SN38-treated group was the weakest compared with the control group, further indicating that Fc-ss-SN38 caused the downregulation of Nrf2 in vivo ( Figure 12 These results indicate that Fc-ss-SN38 inhibits tumor growth through robust ferroptosis in tumors, suggesting that Fc-ss-SN38 promotes tumor cell death and achieves antitumor effects through a multimodal cascade amplifying ferroptosis in a subcutaneous CT26 tumor-bearing mouse model.

[0081] Application Example 3

[0082] In order to more objectively simulate the entire process of tumor occurrence, development, and invasion in vivo, an orthotopic mouse colon cancer tumor model was constructed to more objectively evaluate the anti-tumor effect of Fc-ss-SN38.

[0083] like Figure 13 As shown in the figure, after the treatment, the mice were humanely sacrificed and the tumors were removed for observation and measurement. Compared with the control group, the average tumor weight of the Fc-ss-SN38-treated group was smaller, and the use of Fc-ss-SN38 once again demonstrated an in vivo anti-tumor effect. Figure 14 and Figure 15 As shown in Figure 2, the results of Ki67 and TUNEL showed that the Fc-ss-SN38-treated group had fewer Ki-67-positive cells and more TUNEL-positive cells than the control group. Figure 16As shown in the figure, the fluorescence signal of GPX4 in the tumor of the Fc-ss-SN38-treated group was weaker than that of the control group, revealing that the lipid peroxide scavenging ability of the cells in the Fc-ss-SN38-treated group was most severely decreased compared with the control group. Secondly, the fluorescence signal of Nrf2 in Fc-ss-SN38 was weaker than that in the control group, further indicating that Fc-ss-SN38 caused Nrf2 downregulation in the orthotopic CT26 tumor-bearing mouse model ( Figure 17 ).

[0084] The above results further illustrate the objective fact that Fc-ss-SN38 exerts its powerful anti-tumor efficacy through the cascade amplification of ferroptosis effects in the real process of tumor growth, development and invasion in orthotopic models.

[0085] The applicant declares that the present invention illustrates the multimodal ferroptosis-inducing molecule, its preparation method, and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A multimodal ferroptosis-inducing molecule, characterized in that: The multimodal ferroptosis-inducing molecule is a ferrocene-modified SN38 prodrug, namely Fc-ss-SN38, and its structural formula is as follows: 。 2. A method for preparing the multimodal ferroptosis-inducing molecule according to claim 1, characterized in that: The following steps are involved: (1) Bis(2-hydroxyethyl)disulfide and tert-butyldimethylsilyl chloride were added to a DMF solution containing imidazole. After the reaction was completed, the mixture was purified by column chromatography to obtain a colorless liquid compound 1. (2) Dissolve ferrocenedicarboxylic acid in DCM, add EDC and stir for 10 min, then add DMAP and compound 1. After the reaction is completed, column chromatography is performed to obtain compound 2 as a light yellow oil; (3) Add triethylamine trihydrofluoride to the THF solution of compound 2, stir at room temperature, and after the reaction is completed, column chromatography is performed to obtain yellow oily compound 3; (4) p-Nitrophenyl chloroformate was added to a DCM solution of compound 3 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 4; (5) SN38 was added to a DCM solution of compound 4 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 5, which is the multimodal ferroptosis-inducing molecule.

3. The preparation method according to claim 2, characterized in that In step (1), the amount of bis(2-hydroxyethyl)disulfide is 5 mmol, the amount of imidazole is 5 mmol, the amount of tert-butyldimethylsilyl chloride is 11 mmol, the amount of DMF is 5 mL, the reaction time is 24 h, and the reaction temperature is room temperature.

4. The preparation method according to claim 2, characterized in that In step (2), the amount of ferrocenedicarboxylic acid was 1.06 mmol, EDC was 2.54 mmol, DMAP was 1 mmol, compound 1 was 2.54 mmol, DCM was 10 mL, the reaction time was 24 h, and the reaction temperature was room temperature.

5. The preparation method according to claim 2, characterized in that In step (3), the compound 2 is 0.85 mmol, triethylamine trihydrofluoride is 0.85 mmol, THF is 5 mL, the reaction time is 1 h, and the reaction temperature is room temperature.

6. The preparation method according to claim 2, characterized in that In step (4), the amount of compound 3 was 0.21 mmol, p-nitrophenyl chloroformate was 0.46 mmol, DIPEA was 0.69 mmol, DCM was 10 mL, the reaction time was 12 h, and the reaction temperature was room temperature.

7. The preparation method according to claim 2, characterized in that In step (5), the amount of compound 4 was 0.17 mmol, the amount of SN38 was 0.42 mmol, the amount of DIPEA was 1.06 mmol, the amount of DCM was 10 mL, and the reaction time was 72 h.

8. Use of the multimodal ferroptosis-inducing molecule according to claim 1 in the preparation of a therapeutic drug for multimodally inducing ferroptosis in colorectal cancer cells.

9. Use of the multimodal ferroptosis-inducing molecule according to claim 1 in the preparation of a drug for treating colorectal cancer.

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