Application of 1,3,5-tris(4-boronic acid phenyl)benzene in the preparation of pyroptosis inducers

Abstract

This invention relates to the field of medicinal chemistry, disclosing the application of 1,3,5-tris(4-boronic acid phenyl)benzene in the preparation of pyroptosis inducers. This invention is the first to discover that 1,3,5-tris(4-boronic acid phenyl)benzene possesses the characteristic of inducing pyroptosis, and its preparation is simple and low-cost, making it promising for large-scale industrial production. Furthermore, its borate group has significant potential for modification and can be widely applied in drug development, demonstrating its value for widespread application.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of 1,3,5-tris(4-boronic acid phenyl)benzene in the preparation of pyroptosis inducers. Background Technology

[0002] With the continuous development of the medical field, the diversity and complexity of programmed cell death have been extensively studied. Pyroptosis, as a recently discovered mode of programmed cell death, has attracted widespread attention due to its unique cell death process and its role in the innate immune response. Pyroptosis is characterized by a significant expansion of cell volume, eventually leading to cell membrane rupture and the release of cell contents.

[0003] Since 2015, Academician Shao Feng and his team have made groundbreaking progress in the field of pyroptosis. Their research discovered that caspase-1 and caspase-11 / 4 / 5 trigger pyroptosis by cleaving the Gasdermin-D (GSDMD) protein (Shi et al., Nature 2015; Ding et al., Nature 2016). After being cleaved by the corresponding caspase, the N-terminal domain of the GSDMD protein is released and binds to cell membrane lipids, forming pores in the cell membrane. This process leads to changes in intracellular osmotic pressure, causing continuous cell expansion until the cell membrane ruptures.

[0004] Pyroptosis plays a crucial role in the body's innate immune response, particularly in combating infection and responding to endogenous danger signals. In the field of cancer treatment, effectively inducing pyroptosis in tumor cells can not only eliminate tumor cells but also activate local autoimmune responses through the released contents, thus providing a new strategy for cancer immunotherapy. However, the types of reagents currently available for inducing pyroptosis are limited, and their high cost significantly restricts their application and development in clinical treatment.

[0005] To overcome the aforementioned problems, researchers are dedicated to developing new compounds or drugs capable of inducing pyroptosis. Against this backdrop, Chinese patent CN116730976A discloses the preparation and application of a class of photocontrolled pyroptosis inducers (PyPSs) targeting the endoplasmic reticulum. It demonstrates a novel method for inducing GSDME-mediated pyroptosis using a novel photocontrolled material. Through an endoplasmic reticulum-targeted pyroptosis inducer, under light irradiation, various cancer cells produce ROS, which then activates caspase-3. Activated caspase-3 cleaves GSDME to form the N-terminal fragment (GSDME-N). GSDME-N causes cell membrane perforation, cell swelling, and the release of inflammatory components, ultimately leading to pyroptosis. This class of pyroptosis inducers can induce pyroptosis in various cancer cells under both normoxic and hypoxic conditions, showing broad application prospects in the field of tumor therapy.

[0006] Furthermore, patent CN 112939951A discloses a pyroptosis drug prodrug, its preparation method, and the pyroptosis drug itself. This pyroptosis drug prodrug targets mitochondria, causing mitochondrial destruction, releasing cytochrome c, activating Caspase 3 to cleave GSDME, and inducing pyroptosis. This prodrug (NCyNH2) is selective for tumor cells, reduces damage to normal cells, and its activation can be detected by the recovery of its autofluorescence. After intratumoral administration, this prodrug can effectively regulate the tumor immune microenvironment and activate T cell-mediated anti-tumor immune responses. This molecule has great application potential in mitochondrial targeting, inhibiting cellular respiration, inducing pyroptosis, improving the tumor immune microenvironment, and activating T cell-mediated anti-tumor immune responses.

[0007] Despite the immense potential and importance of pyroptosis in innate immune responses and cancer therapy, several key challenges remain in its practical application. In particular, the variety of reagents currently available for inducing pyroptosis is relatively limited, and their production costs are generally high. This severely restricts the widespread clinical application of pyroptosis-related therapies. Furthermore, the high cost of reagents significantly limits the pace of clinical and preclinical research in this field. Therefore, developing new, more cost-effective pyroptosis inducers to broaden the range of reagents and reduce treatment costs has become an urgent need in this research area. Summary of the Invention

[0008] This invention addresses the urgent need for pyroptosis inducers by providing 1,3,5-tris(4-boronic acid phenyl)benzene as a pyroptosis inducer. It has been found that this compound induces pyroptosis and can be used to prepare therapeutic drugs for malignant tumors, autoimmune diseases, infectious diseases, and neurodegenerative diseases. By inducing pyroptosis in cancer cells or diseased cells, it can alleviate or treat related diseases, which has significant implications for advancing clinical research.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention reveals that 1,3,5-tris(4-boronic acid phenyl)benzene has the effect of inducing pyroptosis and can be used in the preparation of pyroptosis inducers. The structure of 1,3,5-tris(4-boronic acid phenyl)benzene is as follows:

[0011]

[0012] Preferably, the pyroptosis inducer is used to prepare drugs for the prevention or treatment of neoplastic diseases, autoimmune diseases, infectious diseases, or neurodegenerative diseases.

[0013] Preferably, the pyroptosis inducer comprises 1,3,5-tris(4-boronic acid phenyl)benzene and other active ingredients.

[0014] Preferably, the other active ingredients include one or more of the following: pyroptosis-inducing drug active ingredients other than 1,3,5-tris(4-boronicophenyl)benzene, active ingredients that promote bodily functions, drugs that improve disease symptoms, and pharmaceutically acceptable excipients.

[0015] Preferably, the active pharmaceutical ingredient that induces pyroptosis, which is different from 1,3,5-tris(4-boronic acid phenyl)benzene, includes one or more of Paris polyphylla saponin VI, α-ketoglutarate, trichlorobenzazole, punicea tannin, and nigramycin.

[0016] Preferably, the active ingredients that promote bodily functions include one or more of glucose, fermented cordyceps powder, wolfberry, ginseng, astragalus, ganoderma, polygonatum, atractylodes, codonopsis, salvia miltiorrhiza, angelica, and folic acid.

[0017] Preferably, the drug for improving disease symptoms includes one or more of the following: immunotherapy drugs such as PD-L1 monoclonal antibodies, chemotherapy drugs such as doxorubicin, and radiosensitizers such as paraoxonase. Preferably, the pharmaceutically acceptable excipients include one or more of the following: dispersants, solubilizers, stabilizers, preservatives, sweeteners, flavoring agents, anti-caking agents, lubricants, disintegrants, and adsorbents.

[0018] Preferably, the neoplastic disease includes one or more of pancreatic cancer, lung cancer, liver cancer, colorectal cancer, breast cancer, stomach cancer, esophageal cancer, brain cancer, osteosarcoma, lymphoma, multiple myeloma, leukemia, and melanoma.

[0019] The present invention also provides the application of the aforementioned pyroptosis inducer in the construction of a pyroptosis model.

[0020] The present invention also provides a medicament for the prevention or treatment of neoplastic diseases, autoimmune diseases, infectious diseases or neurodegenerative diseases, wherein the main active ingredient of the medicament is 1,3,5-tris(4-boronic acid phenyl)benzene.

[0021] The 1,3,5-tris(4-boronic acid phenyl)benzene has the effect of inducing pyroptosis.

[0022] The cells include human cancer cells or cells of mammals, such as those of dogs, cats, horses, donkeys, sheep, cattle, or pigs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) This invention first discovered that 1,3,5-tris(4-boronic acid phenyl)benzene has the effect of inducing pyroptosis. This compound has been produced on a large scale in industry at a low cost.

[0025] (2) The 1,3,5-tris(4-boronic acid phenyl)benzene in this invention can be further synthesized into a multifunctional responsive pyroptosis inducer with stimulus-responsive capabilities (such as reactive oxygen species response and pH response) through its boric acid group. This derivatization capability can overcome the side effects and biotoxicity of pyroptosis inducers in the prior art. Attached Figure Description

[0026] Figure 1 Microscopic images showing the pyroptosis behavior of pancreatic cancer cells induced by the drug-free group, 1,3,5-tris(4-boronicophenyl)benzene, and 1,3,5-tris(4-phenylboronicophenyl)pinacol ester in Example 1.

[0027] Figure 2 This is a bar chart showing the release of LDH from pancreatic cancer cells induced by 1,3,5-tris(4-boronic acid phenyl)benzene and 1,3,5-tris(4-phenylboronic acid pinacol ester)benzene at different drug concentrations in Example 2.

[0028] Figure 3 This is a bar chart comparing the effects of 1,3,5-tris(4-boronic acid phenyl)benzene and 1,3,5-tris(4-phenylboronic acid pinacol ester)benzene on the proliferation of pancreatic cancer cells in Example 3.

[0029] Figure 4This is a heatmap showing the effects of 1,3,5-tris(4-boronic acid phenyl)benzene on genes related to the pyroptosis pathway in pancreatic cancer cells, as described in Example 4. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0031] All raw materials used in the following specific implementation methods were purchased from the market.

[0032] Example 1

[0033] To verify that the pyrode activity of 1,3,5-tris(4-boronic acid phenyl)benzene originates from the boric acid group, 1,3,5-tris(4-phenylboronic acid pinacol ester)benzene (CAS No.: 1017967-97-3) was selected as a control, and its structure is shown below:

[0034]

[0035] In this embodiment, pancreatic cancer cells Bxpc-3 were selected for cell microscopy imaging experiments: When the pancreatic cancer cells Bxpc-3 reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. They were then seeded into 24-well cell culture plates at a density of 20,000 cells per well. After adhesion for 24 hours, 200 μL of fresh cell culture medium containing 20 μM 1,3,5-tris(4-phenylboronic acid)phenyl (P3B3), 20 μM 1,3,5-tris(4-phenylboronic acid)pinacol ester benzene (P3B3-PIN), and drug-free fresh cells were added. After culturing for 1 day, cell micrographs were taken.

[0036] The results are as follows Figure 1 As shown, (a) is a photograph of pancreatic cancer cells in the drug-free group; (b) is a photograph of pancreatic cancer cells after incubation with 20 μM 1,3,5-tris(4-phenylboronic acid)pinacol ester; and (c) is a photograph of pancreatic cancer cells after incubation with 20 μM 1,3,5-tris(4-phenylboronic acid)pinacol ester. It can be seen that at a low concentration of 20 μM, 1,3,5-tris(4-phenylboronic acid)pinacol ester can effectively induce pyroptosis in pancreatic cancer cells. The cells in the image show obvious cell swelling (indicated by the black arrow), and under the microscope, the proportion of swollen cells exceeds 70%. However, the same concentration of 1,3,5-tris(4-phenylboronic acid)pinacol ester has no significant effect on pancreatic cancer cells, and their cell morphology is basically the same as that of the control group.

[0037] Example 2

[0038] To investigate the ability of 1,3,5-tris(4-boronic acid phenyl)benzene to induce the release of lactate dehydrogenase (LDH, a typical marker of pyroptosis), pancreatic cancer cells Bxpc-3 were selected for LDH release experiments in this embodiment. When the pancreatic cancer cells Bxpc-3 reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. They were then seeded into 96-well cell culture plates at a density of 8000 cells per well. After adhesion for 24 hours, 200 μL of fresh cell culture medium containing 5 μM, 10 μM, and 20 μM 1,3,5-tris(4-phenylboronic acid phenyl)benzene and 1,3,5-tris(4-phenylboronic acid pinacol ester)benzene were added. After culturing for 24 hours, the cells were tested for LDH release.

[0039] See results Figure 2 1,3,5-Tris(4-phenylboronic acid)benzene (P3B3) significantly induced LDH release, with an average LDH release exceeding 40% at a dose of 20 μM, showing a concentration-dependent effect. It can be expected that higher concentrations of 1,3,5-tris(4-phenylboronic acid)benzene will induce even greater LDH release. However, the same dose of 1,3,5-tris(4-phenylboronic acid)pinacol ester (P3B3-PIN) did not induce LDH release.

[0040] Example 3

[0041] To investigate the effect of 1,3,5-tris(4-boronic acid phenyl)benzene on the growth of pancreatic cancer cells, this example selected pancreatic cancer cells Bxpc-3 for cell proliferation behavior testing: When the pancreatic cancer cells Bxpc-3 reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. They were then seeded into 96-well cell culture plates at a density of 5000 cells per well. After 24 hours of adhesion, 200 μL of fresh cell culture medium containing 5 μM, 10 μM, and 20 μM 1,3,5-tris(4-phenylboronic acid phenyl)benzene and 1,3,5-tris(4-phenylboronic acid pinacol ester)benzene were added. After 24 hours of culture, the cell density was statistically analyzed.

[0042] The results are as follows Figure 3 As shown, 1,3,5-tris(4-phenylboronic acid)benzene effectively inhibits the proliferation of pancreatic cancer cells, especially at doses above 20 μM, where the cell survival rate is approximately 20%. In contrast, the same dose of 1,3,5-tris(4-phenylboronic acid)pinacol ester showed no significant toxicity to cells, with a cell survival rate greater than 90%.

[0043] Example 4

[0044] To investigate the effects of 1,3,5-tris(4-boronic acid phenyl)benzene on genes related to the pyroptosis pathway, this study selected pancreatic cancer cells Bxpc-3 for transcriptome sequencing (RNA-seq). When the pancreatic cancer cells Bxpc-3 reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. They were then seeded into 6-well cell culture plates at a density of 200,000 cells per well. After 24 hours of adhesion, 200 μL of fresh cell culture medium containing 5 μM, 10 μM, and 20 μM 1,3,5-tris(4-boronic acid phenyl)benzene was added. After 24 hours of culture, the cells were collected for RNA-seq experiments.

[0045] The results are as follows Figure 4 As shown, pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database (KEGG) revealed that P3B3 significantly affects cellular metabolism of multiple amino acids and upregulates the gene expression of pyroptosis-related pathways such as PI3K-Akt and MAPK. This further confirms that P3B3 can significantly induce pyroptosis and thus programmed cell death.

Claims

1. 1,3,5-Tris(4-boronic acid phenyl)benzene in the preparation of pyroptosis inducers, characterized in that, The pyroptosis inducer is used to prepare drugs for the prevention or treatment of neoplastic diseases; The neoplastic diseases include one or more of pancreatic cancer and liver cancer.

2. The use of the pyroptosis inducer according to claim 1 in the preparation of a medicament for the prevention or treatment of neoplastic diseases; The neoplastic diseases include one or more of pancreatic cancer and liver cancer.

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