Compound goniomine and its preparation method and application

Abstract

This invention relates to a method for preparing the compound goniomine, the structural formula of which is: [structural formula would be inserted here]. This invention also relates to the compound goniomine obtained by this method and its application in the preparation of antitumor drugs. Compound 1, goniomine, can significantly induce pyroptosis in PC-9 cells, as well as PANC-1 and MIA PaCa-2 cells, exhibiting good anti-non-small cell lung cancer and anti-pancreatic cancer activity. Given that pyroptosis is an inflammatory programmed cell death mechanism, this process is expected to further activate the body's antitumor immune response and improve the tumor immune microenvironment, providing new candidate drugs and research directions for the prevention and treatment of related tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a compound called goniomine, its preparation method, and its applications. Background Technology

[0002] Gonioma Malagasy It is a plant belonging to the Apocynaceae family, distributed in Madagascar. Screening of alkaloid extracts from this plant revealed antimalarial or cytotoxic activity against chloroquine-resistant Plasmodium falciparum FcB1 strain. Early phytochemical studies identified several structurally unique indole alkaloids from this plant, including pleiomutinine, goniomitine, and goniomine.

[0003] Goniomine was first isolated by Husson et al. in 1980, and its structure was confirmed by X-ray single-crystal diffraction. Its chemical structural formula is as follows: This compound is considered a rearranged indole monoterpene alkaloid with a unique benzotetrahydro-1H-1-azapyrrolizoid skeleton, exhibiting high complexity and steric crowding. Studies suggest its biosynthetic pathway may be related to indole alkaloids of the Strychnos family.

[0004] In 2019, melocochine A and melocochine B, natural products structurally similar to goniomine, were isolated and found to promote lysosome formation, providing new insights for the treatment of lysosome-related diseases. This further suggests that alkaloids like goniomine, with their rare azadratic skeletons, may also possess undiscovered biological activities worthy of in-depth exploration. However, to date, no studies on the chemical synthesis and biological activity of goniomine have been reported.

[0005] Pyroptosis is a pro-inflammatory programmed cell death mediated by Gasdermin (GSDM) family proteins and dependent on caspase activation. It differs from the non-inflammatory characteristics of apoptosis and the passive damage of necrosis, and is a core pathway in the body's innate immunity and inflammation regulation. Pyroptosis is characterized by rapid cell membrane perforation, cell swelling and rupture, and the massive release of pro-inflammatory factors such as IL-1β / IL-18 and damage-associated molecular patterns (DAMPs). It participates in physiological and pathological processes such as pathogen clearance, damage repair, and tumor surveillance by initiating local and systemic inflammatory responses.

[0006] In the classical pyroptosis pathway, pattern recognition receptors (such as NLRP3) assemble inflammasomes, activating Caspase-1 / 4 / 5 / 11, cleaving GSDMD and releasing its N-terminal pore-forming domain. This domain oligomerizes on the plasma membrane, forming pores and triggering osmotic imbalance and cell lysis. Recent studies have further revealed non-classical activation pathways of family members such as GSDME, GSDMC, and GSDMB, which can be regulated by upstream signals such as Caspase-3 / 8 and granzymes. This allows pyroptosis to broadly encompass various cell types, including immune cells, tumor cells, and epithelial cells, forming a multi-layered, multi-node regulatory network.

[0007] Disruptions in pyroptosis homeostasis are closely related to human diseases: overactivation drives sepsis, cytokine storms, neuroinflammation, autoimmune diseases, metabolic disorders, and the progression of fibrosis; while inhibition of pyroptosis leads to pathogen escape, weakened immune surveillance, and tumor drug resistance. In the field of oncology, inducing pyroptosis in tumor cells can directly kill malignant cells and transform "cold tumors" into "hot tumors" by enhancing immunogenicity. This, combined with immune checkpoint inhibitors, improves efficacy and has become a cutting-edge direction in the development of anti-tumor drugs.

[0008] Non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) are both highly malignant and difficult-to-treat solid tumors. NSCLC accounts for approximately 85% of all lung cancers, with a five-year survival rate of less than 20% for advanced-stage patients. PDAC has an even worse prognosis, with a five-year survival rate of less than 10%, and is projected to become the second leading cause of cancer-related death by 2030. Both face similar bottlenecks in clinical treatment: low surgical resection rates, widespread primary or acquired resistance to chemotherapy, particularly platinum-based and gemcitabine-based drugs, and both are immunosuppressive "cold tumors" with low response rates to PD-1 / PD-L1 immune checkpoint inhibitors.

[0009] From a molecular perspective, approximately 50% of NSCLC cases have TP53 mutations, and 20%-30% have LKB1 mutations, leading to defects in the apoptosis pathway and low levels of tumor immune infiltration. In PDAC cases, over 90% have KRAS mutations, and 50%-75% have TP53 mutations. Combined with highly dense fibrotic stroma, this further exacerbates drug delivery barriers and immune rejection. These molecular pathological features collectively constitute multiple resistances to traditional treatments.

[0010] Activating the pyroptosis pathway offers a new approach to overcoming the aforementioned therapeutic bottlenecks. Inducing GSDME or NLRP3 / GSDMD-mediated pyroptosis can directly kill apoptosis-resistant tumor cells and recruit and activate effector T cells by releasing damage-related molecular patterns and inflammatory factors, thereby reshaping the tumor immune microenvironment and transforming "cold" tumors into "hot" tumors, thus sensitizing chemotherapy and immunotherapy. However, currently only a few molecules, such as high-dose ascorbic acid and iridium(III) complexes, have been reported to induce pyroptosis, and these generally suffer from low induction efficiency and poor specificity, lacking highly efficient and specific pyroptosis enhancers.

[0011] Therefore, developing novel compounds that can specifically enhance pyroptosis in tumor cells is of great scientific significance and clinical application potential in the treatment of non-small cell lung cancer and pancreatic cancer, as it is crucial for overcoming apoptosis resistance, reversing immunosuppression, and breaking through existing treatment bottlenecks. Summary of the Invention

[0012] The purpose of this invention is to overcome the defects in the prior art described above. This invention provides a compound goniomine, its preparation method, and its applications.

[0013] To achieve the above objectives, a first aspect of the present invention provides a method for preparing the compound goniomine, characterized in that the compound goniomine has the following structural formula: .

[0014] The preparation method includes the following steps: (1) Indoleacetic acid ester compound 1-3 was synthesized by coupling reaction of tetrahydropyridinol compound 1-1 and indoleacetic acid 1-2; (2) Indole acetate compounds 1-3 were rearranged under Ireland-Claisen conditions and decarboxylated under acidic conditions to synthesize indoleene compounds 1-4; (3) Indolediol compounds 1-5 were synthesized by reacting indoleene compounds 1-4 with a dihydroxylating agent; (4) Tetracyclic epoxy compounds 1-6 were synthesized by tandem Polonovski–Potier / Friedel–Crafts reaction of indole diol compounds 1-5 under acidic conditions and epoxidation. (5) Tetracyclic thioacetal compounds 1-8 are generated by benzyl deprotection of tetracyclic epoxides 1-6 and reductive amination with thioacetals 1-7. (6) Pentyl-p-toluene sulfide compounds 1-9 were synthesized by cyclization of tetracyclic thioacetal compounds 1-8 with dimethyl(methylthio)sulfonium tetrafluoroborate under alkaline conditions; (7) Compound 1 goniomine was generated by deprotection of pentacyclic p-toluene sulfide compounds 1-9 and reaction with Eschenmoser salt on silica gel.

[0015] Preferably, in step (1), the coupling reaction is carried out in a dichloromethane (DCM) solution, initially at a low temperature of 0±5 °C. o The reaction was carried out at C, and then the temperature was raised to room temperature; the condensing agent was selected from DCC, DIC and EDCI. In step (2), the rearrangement reaction is carried out in a tetrahydrofuran (THF) solution, initially at a low temperature of -78±5°C. o The reaction is carried out at C, and then brought to room temperature. Hydrogen removal can be performed using a base commonly used in the art, with the base selected from lithium bis(trimethylsilylamino) and lithium diisopropylamino. The decondensation reaction proceeds spontaneously under acidic conditions, with a solution pH range of 1-3.

[0016] Preferably, in step (3), the dihydroxylation reaction is carried out in a mixed solution of THF / tert-butanol / water at a reaction temperature of 0±5 °C. o C; The oxidant is selected from NMO / potassium osmium tetroxide, osmium tetroxide aqueous solution, and potassium hexacyanoferrate / potassium osmium tetroxide.

[0017] Preferably, in step (4), the cyclization reaction is carried out in a dichloromethane (DCM) solution, and the oxidation reaction is carried out at a low temperature of 0±5 °C. o C, the oxidant is selected from m-chloroperoxybenzoic acid and hydrogen peroxide aqueous solution; the Polonovski–Potier reaction is first carried out at a low temperature of 0±5℃. o The reaction was carried out at C, then raised to room temperature. The reagents used in the reaction were selected from trifluoroacetic anhydride and trifluoromethanesulfonic anhydride. The epoxidation reaction was first carried out at a low temperature of 0±5℃. o The reaction was carried out at C and then brought to room temperature. The reagents were selected from methanesulfonic anhydride / triethylamine and trifluoromethanesulfonic acid chloride / pyridine.

[0018] Preferably, in step (5), the hydrogen debenzylation reaction is carried out in a trifluoroethanol (TFE) solution at room temperature, and the solvent is selected from methanol, ethanol, and trifluoroethanol; the reductive amination reaction is carried out in a tetrahydrofuran solution and a methanol solution, first at a low temperature of 0±5℃. o The reaction is carried out at C, and then the temperature is raised to room temperature. The reducing agent is selected from sodium cyanoborohydride and sodium triacetoxyborohydride.

[0019] Preferably, in step (6), the cyclization reaction is carried out in an acetonitrile solution at a temperature of -10±5 °C. oC, the reagent used in the reaction is selected from dimethyl(methylthio)sulfonium tetrafluoroborate, dimethyl(methylthio)sulfonium tetrafluoroborate / 2,6-dimethylaminopyridine.

[0020] Preferably, in step (7), the phenylthio group removal reaction is carried out in an acetone solution at room temperature; a conventional reducing agent in the art can be used to reduce the phenylthio group to hydrogen, and the reducing agent is selected from Raney nickel, lithium / liquid ammonia, sodium / naphthalene; the dimethylamino reaction is carried out in a dichloromethane solution, and the reaction is initially carried out at a low temperature of 0±5℃. o The reaction was carried out at C, and then the temperature was raised to room temperature. The reagent combination was selected from tetramethylmethanediamine / trifluoroacetic anhydride and N,N-dimethylmethylene ammonium iodide.

[0021] A second aspect of the invention provides a compound called Goniomine prepared by the method described above.

[0022] A third aspect of the present invention provides the use of the compound goniomine or its hydrate, solvate, stereoisomer, racemate or pharmaceutically acceptable salt thereof in the preparation of antitumor drugs, characterized in that the compound goniomine has the following structural formula: .

[0023] Preferably, the antitumor drug is an anti-non-small cell lung cancer and anti-pancreatic cancer drug. Attached Figure Description

[0024] Figure 1 This is a graph showing the cytotoxicity results of compound 1 on tumor cells in Example 2.

[0025] Figure 2A The image shows an optical microscope image of DMSO, 20 µM compound 1, 40 µM compound 1, 60 µM compound 1 and human non-small cell lung cancer PC-9 cells after culture in Example 3.

[0026] Figure 2B This is an optical microscope image of DMSO, 40 µM compound 1, and human pancreatic cancer PANC-1 cells cultured in Example 3.

[0027] Figure 2C This is an optical microscope image of human pancreatic cancer MIA PaCa-2 cells cultured with DMSO, 40 µM compound 1, and compound 1 in Example 3.

[0028] Figure 3 The image shows the results of Annexin V-FITC cell death detection for compound 1 in Example 4.

[0029] Figure 4The graph shows the LDH release detection results of compound 1 in Example 5.

[0030] Figure 5 The figure shows the results of compound 1 in Example 6 upregulating the expression level of pyroptosis-related proteins. Detailed Implementation

[0031] To better understand the technical content of this invention, the specific implementation method of this invention will be further described below.

[0032] Unless otherwise specified, all reactions were carried out under an anhydrous, argon atmosphere. Tetrahydrofuran was refluxed using sodium sand / benzophenone; dichloromethane was refluxed using calcium hydride (CaH2); methanol was refluxed using magnesium shavings; other reagents were treated according to the methods described in *Purification of Laboratory Chemicals* (Armarego, WLF; Perrin, DDButterworth, Heinemann, 1997). Deuterated chloroform was purchased directly from Merck and used directly. Solvents used for column chromatography purification were provided by Sinopharm Chemical Reagent Company and the Exploration Platform.

[0033] The reaction was detected by thin-layer chromatography (TLC) using S-2 0.25 mm E. Merck silica gelplates (60F-254). The chromogenic reagent was CAM or alkaline KMnO4 solution. Column chromatography purification used E. Merck silica gel (60, particle size 0.040–0.063 μm). NMR data were provided by a Bruker AV-400 and an Agilent 500. 1 Chemical shifts in H NMR are expressed in ppm, with the undeuterated residual peak of CDCl3 at 7.26 ppm. Coupling constants are expressed in Hz. Splitting abbreviations are: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad peak. 13 The chemical shifts of the 10⁻⁶ C NMR spectra are expressed in ppm, with the solvent peak of CDCl₃ at 77.16 ppm. IR spectra were determined using an FTS-185 and a Nicolet 380 infrared spectrometer; mass spectra were determined using a Finnigan 4021, HP 5989A, and Finnigan FTMS-2000 instrument; high-resolution mass spectra were obtained using a Finnigan MAT and a Kratos Concept instrument. 1 Measured using an H-type mass spectrometer; specific rotation was measured using a Jasco P-1030 automatic polarimeter.

[0034] Example 1 Preparation of Compound 1 Compound 1-1 has a published structure; its preparation method can be found in: Asymmetry. 1997, 8 , 935-948. Indoleacetic acid compounds 1-3: At 0 °C, DCC (29.2 g, 141 mmol, 1.2 equivalent) and DMAP (1.44 g, 11.8 mmol, 0.1 equivalent) were added sequentially to a solution of tetrahydropyridinol 1-1 (25.6 g, 118 mmol, 1.0 equivalent) in dichloromethane (200 mL), followed by the slow addition of indole-2-acetic acid (22.7 g, 130 mmol, 1.1 equivalent) at the same temperature. The resulting mixture was heated to 22 °C and stirred for 50 min, and the reaction was quenched by adding 5% aqueous acetic acid solution (50 mL). After neutralization with saturated sodium bicarbonate solution (150 mL), the mixture was filtered through a diatomaceous earth short column. The filtrate was extracted with dichloromethane (3 × 200 mL), and the combined organic phases were washed with saturated brine (300 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:4 → 2:1) as the eluent to give a yellow oily indoleacetic acid 1-3 (39.7 g, 90%).

[0035] Indoleacetic acid ester compounds 1-3: R f = 0.25 (silica gel, EtOAc / petroleum ether =1:1); [α]25 D = +34.6 ( c = 1.0 in CHCl3); IR (film): ν max = 2977, 2925, 1730,1454, 1371, 1240, 1151, 1043, 744, 698 cm −1 ; 1 H NMR (400 MHz, CDCl3) δ = 8.70(s, 1H), 7.62 – 7.52 (m, 1H), 7.38 – 7.23 (m, 6H), 7.20 – 7.13 (m, 1H), 7.12– 7.05 (m, 1H), 6.39 – 6.30 (m, 1H), 5.84 – 5.74 (m, 1H), 5.35 (q,J = 6.6 Hz,1H), 3.80 (s, 2H), 3.54 (dd, J = 13.1, 5.4 Hz, 2H), 3.00 – 2.88 (m, 2H), 2.60 –2.50 (m, 1H), 2.49 – 2.39 (m, 1H), 2.25 – 2.07 (m, 2H), 1.34 (d, J = 6.6 Hz,3H) ppm. 13 C NMR (126 MHz, CDCl3) δ = 170.0, 138.2, 136.5, 135.5, 130.8, 129.3,128.4, 128.3, 127.3, 123.1, 121.8, 120.2, 119.9, 110.9, 102.0, 73.7, 62.7,52.1, 49.3, 34.3, 25.7, 18.9 ppm; HRMS ( m / z ): [M + H] + calcd for C 24 H 27 N2O2 + 375.2067, found 375.2067. Indole compounds 1-4: To a THF (500 mL) solution of indole acetate compounds 1-3 (19.3 g, 51.5 mmol, 1.0 equivalent), LiHMDS (113 mL, 1.0 M THF solution, 113 mmol, 2.0 equivalent), TMSCl (47.0 mL, 40.1 g, 369 mmol, 7.0 equivalent), and Et3N (62.6 g, 86.0 mL, 619 mmol, 12 equivalent) were added sequentially. The solution was then heated to 22 °C and stirred for 8 hours, followed by quenching with 1 M, 400 mL hydrochloric acid solution at 0 °C. The pH of the mixture was adjusted to approximately 3.0 by continuous dropwise addition of hydrochloric acid aqueous solution (1.0 M), followed by stirring at 22 °C for 12 hours, and then quenched with saturated sodium bicarbonate aqueous solution (500 mL). The mixture was extracted with ethyl acetate (3 × 400 mL), and the combined organic phases were washed with saturated brine (500 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:4 → 2:1) as the eluent to give a yellow oily indoleene compound 1-4 (14.6 g, 76%).

[0036] Indole compounds 1-4: R f = 0.34 (silica gel, EtOAc / petroleum ether 1:1;[α]25 D = +8.28 ( c = 1.0 in CHCl3); IR (film): ν max = 3404, 2920, 1454, 1415,1342, 1286, 1014, 785, 764, 698 cm −1 ; 1 H NMR (400 MHz, CDCl3): δ = 7.88 (s, 1H), 7.57 – 7.49 (m, 1H), 7.38 – 7.26 (m, 6H), 7.16 – 7.02 (m, 2H), 6.30 – 6.19(m, 1H), 5.35 (q, J = 6.6, Hz, 1H), 3.68 – 3.51 (m, 2H), 3.40 (d, J= 12.2 Hz,1H), 3.14 – 3.00 (m, 1H), 2.83 – 2.74 (m, 2H), 2.74 – 2.66 (m, 1H), 2.47 –2.38 (m, 1H), 2.38 – 2.28 (m, 1H), 1.82 – 1.72 (m, 1H), 1.60 (d, J = 6.6, Hz,3H), 1.49 – 1.38 (m, 1H) ppm. 13 C NMR (151 MHz, CDCl3) δ = 138.3, 138.2, 137.8,136.0, 129.4, 128.9, 128.3, 127.2, 121.1, 119.9, 119.8, 117.5, 110.4, 100.9,63.1, 52.6, 52.5, 42.1, 31.9, 31.2, 13.0 ppm.; HRMS ( m / z ): [M + H] + calcd forC 23 H 27 N2 + 331.2169, found 331.2170. Indolediol compounds 1-5: Potassium osmium tetroxide dihydrate (K₂O₅sO₄·2H₂O, 278 mg, 0.76 mmol, 0.1 equivalent) was added to a THF / tert-butanol / water (75 mL / 75 mL / 75 mL) solution of N-methylmorpholine-N-oxide (NMO, 2.66 g, 22.7 mmol, 3.0 equivalent) at 22°C. The resulting mixture was stirred at this temperature for 5 hours and then cooled to 0°C for later use.

[0037] At 22°C, triethylamine (Et3N, 2.30 g, 3.2 mL, 22.7 mmol, 3.0 equivalent), DMAP (92.4 mg, 0.76 mmol, 0.1 equivalent), and di-tert-butyl dicarbonate (Boc2O, 2.47 g, 2.6 mL, 11.3 mmol, 1.5 equivalent) were added sequentially to a THF (75 mL) solution of indolene compounds 1-4 (2.50 g, 7.56 mmol, 1.0 equivalent). The solution was stirred at this temperature for 3 hours and then cooled to 0°C for later use.

[0038] A solution of indoleene was added to a pre-prepared mixture of potassium osmium tetroxide at 0 °C. The resulting mixture was stirred at 0 °C for 24 hours, and the reaction was quenched with sodium dithionite solid (Na₂S₂O₆, 13.2 g, 75.6 mmol, 10 equivalences). The mixture was then extracted with ethyl acetate (3 × 400 mL), and the combined organic phases were washed with saturated brine (400 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:2 → 1:0) as eluent to give pale yellow foamy indole diol compounds 1-5 (2.28 g, 65%).

[0039] Indolediol compounds 1-5: [α]25 D = +67.1 ( c = 1.0 in CHCl3); R f = 0.29(silica gel, EtOAc / petroleum ether 2:1); IR (film): ν max =3516, 2933, 1714,1454, 1371, 1327, 1157, 1118, 1085, 700 cm −1 ; 1 H NMR (600 MHz, DMSO- d 6, 80 °C): δ = 7.99 – 7.95 (m, 1H), 7.49 – 7.45 (m, 1H), 7.34 – 7.27 (m, 5H), 7.26 –7.22 (m, 1H), 7.22 – 7.18 (m, 1H), 7.17 – 7.14 (m, 1H), 6.45 (s, 1H), 4.09(s, 1H), 3.96 (q, J = 6.3 Hz, 1H), 3.87 (s, 1H), 3.54 – 3.49 (m, 1H), 3.48 (d, J = 13.2 Hz, 1H), 3.41 (d, J= 13.2 Hz, 1H), 3.09 – 3.03 (m, 1H), 3.04 – 3.00 (m,1H), 2.71 – 2.67 (m, 1H), 2.66 – 2.60 (m, 1H), 2.11 – 2.04 (m, 1H), 2.01 –1.93 (m, 2H), 1.67 (s, 9H), 1.52 – 1.42 (m, 1H), 1.08 (d, J = 6.3 Hz, 3H)ppm. 13 C NMR (151 MHz, DMSO- d 6) δ = 149.7, 141.3, 137.9, 135.8, 128.5, 128.4,127.5, 126.4, 122.5, 121.9, 119.2, 114.3, 106.8, 83.6, 72.4, 67.8, 61.9,61.0, 51.7, 43.9, 28.2, 27.4, 26.6, 17.6 ppm. HRMS ( m / z ): [M + H] + calcd forC 28 H 37 N2O4 + 465.2748, found 465.2753. Tetracyclic epoxy compounds 1-6: At 0 °C, m-chloroperoxybenzoic acid was added to a solution of indolediol compounds 1-5 (2.28 g, 4.91 mmol, 1.0 equivalent) in dichloromethane (24 mL). mCPBA (1.10 g, 85%, 5.41 mmol, 1.1 equivalents) was added. The resulting solution was stirred at this temperature for 30 minutes, followed by the addition of trifluoroacetic anhydride (TFAA, 7.22 g, 4.8 mL, 34.3 mmol, 7.0 equivalents). The reaction mixture was heated to 22 °C and stirred for 30 minutes, then trifluoroacetic acid (TFA, 6 mL) was added, and stirring continued for 3 hours. The reaction was quenched with saturated sodium bicarbonate aqueous solution (100 mL), and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product containing the target cyclized product was dissolved in dichloromethane (50 mL), and triethylamine (Et3N, 1.49 g, 2.0 mL, 14.7 mmol, 3.0 equivalent), DMAP (60.0 mg, 0.491 mmol), and methanesulfonic anhydride (Ms2O, 1.71 g, 9.82 mmol) were added sequentially at 0 °C. The resulting solution was heated to 22 °C and stirred for 8 hours, then quenched with saturated sodium bicarbonate aqueous solution (100 mL). The mixture was extracted with ethyl acetate (3 × 100 mL), and the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:8 → 1:1) as eluent to give a white foamy tetracyclic epoxy compound: 1-6 (1.01 g, 60% for the two steps).

[0040] Tetracyclic epoxy compounds 1-6: R f = 0.24 (silica gel, EtOAc / petroleum ether 1:4);[α]25 D = −81.6 ( c = 1.0 in CHCl3); IR (film): ν max = 3307, 2926, 1460, 1429,1215, 1010, 981, 902, 827, 700 cm −1 ; 1H NMR (400 MHz, CDCl3): δ = 8.00 (s, 1H),7.49 – 7.42 (m, 2H), 7.41 – 7.28 (m, 4H), 7.25 – 7.19 (m, 1H), 7.19 – 7.13(m, 1H), 7.12 – 7.06 (m, 1H), 3.88 (d, J = 14.0 Hz, 1H), 3.67 (s, 1H), 3.37 (d, J = 14.0 Hz, 1H), 3.19 (dd, J = 17.0, 7.1 Hz, 1H), 2.99 (q, J = 5.5 Hz, 1H), 2.87(d, J = 17.0 Hz, 1H), 2.60 – 2.48 (m, 1H), 2.38 – 2.22 (m, 2H), 2.17 – 2.08 (m,1H), 1.69 – 1.64 (m, 1H), 1.29 (d, J = 5.5 Hz, 3H) ppm. 13 C NMR (151 MHz, CDCl3)δ = 139.7, 136.5, 136.0, 128.7, 128.4, 128.3, 126.8, 121.5, 120.0, 118.7,110.6, 107.0, 65.2, 59.9, 59.2, 58.5, 43.9, 30.8, 29.6, 29.1, 13.2 ppm. HRMS( m / z ): [M + H] + calcd for C 23 H 25 N2O + 345.1961, found 345.1969. Tetracyclic thioacetal compounds 1-8: Pd(OH)₂ / C (20 wt% Pd, 202 mg, 0.382 mmol, 1.3 equivalent) was added to a TFE (60 mL) solution of tetracyclic epoxy compounds 1-6 (1.01 g, 2.93 mmol, 1.0 equivalent) at 22 °C. The resulting mixture was stirred under a hydrogen atmosphere for 3 hours, followed by purging with argon. The mixture was mixed with diatomaceous earth, filtered through a small section of diatomaceous earth, and washed with ethyl acetate (60 mL). The combined filtrates were concentrated under reduced pressure, and the crude product containing the target secondary amine was dissolved in THF (30 mL). Bis(p-tolylthio)acetal compounds 1-7 (1.27 g, 4.41 mmol, 1.5 equivalent) were added to this secondary amine solution at 22 °C. The resulting solution was stirred at this temperature for 24 hours, and then MeOH (30 mL) and NaBH3CN (369 mg, 5.88 mmol, 2.0 equivalent) were added sequentially at 0 °C. The solution was heated to 22 °C, 1–2 mg of bromocresol green indicator was added, and the pH of the reaction was maintained by dropwise addition of HOAc (the solution color changed from blue to dark green and remained thereafter). The mixture was stirred at this temperature for 2 hours, and then quenched with saturated sodium bicarbonate aqueous solution (50 mL). The resulting mixture was extracted with ethyl acetate (3 × 50 mL), the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:10 → 1:2) as eluent to give white foamy tetracyclic thioacetal compounds 1–8 (1.02 g, 66% for the two steps).

[0041] Tetracyclic thioacetal compounds 1-8: R f = 0.35 (silica gel, EtOAc / petroleum ether 1:6); [α]25 D = +0.56 ( c = 1.0 in CHCl3); IR (film): ν max = 3300, 2924, 1490,1460,1327, 1259, 1101, 1045, 1016, 810 cm −1 ; 1 H NMR (400 MHz, CDCl3) δ = 8.05 (s,1H), 7.47 (d,J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H),7.19 – 7.06 (m, 6H), 7.06 – 6.99 (m, 1H), 4.46 (d, J = 6.8 Hz, 1H), 3.52 (s,1H), 3.15 (dd, J = 17.1, 7.0 Hz, 1H), 3.04 (dd, J = 13.7, 7.7 Hz, 1H), 2.98 (q, J =5.5 Hz, 1H), 2.83 (d, J = 17.1 Hz, 1H), 2.75 – 2.67 (m, 1H), 2.63 (dd, J = 13.7,7.7 Hz, 1H), 2.36 (s, 3H), 2.33 (s, 3H), 2.32 – 2.24 (m, 1H), 2.11 – 2.05 (m,1H), 1.65 – 1.58 (m, 1H), 1.27 (d, J = 5.5 Hz, 3H) ppm. 13 C NMR (151 MHz, CDCl3)δ = 137.9, 137.8, 136.6, 135.8, 133.9, 133.9, 131.0, 130.5, 129.7, 129.6,127.5, 121.5, 120.0, 118.6, 110.6, 107.3, 65.0, 60.8, 59.7, 58.9, 58.7, 44.2,30.5, 29.4, 28.9, 21.3, 21.3, 13.1 ppm. HRMS ( m / z ): [M + H] + calcd forC 32 H 35 N2OS2 + 527.2185, found 527.2185. Pentacyclic p-toluene thioether compounds 1-9: At 22 °C, 2,6-dimethylpyridine (40.7 mg, 44 μL, 0.380 mmol, 1.0 equivalent) and 4 Å molecular sieve (300 mg) were added sequentially to a solution of tetracyclic thioacetal compounds 1-8 (100 mg, 0.190 mmol, 1.0 equivalent) in acetonitrile. The resulting mixture was stirred at this temperature for 1 hour and then cooled to -10 °C for further use. At 22 °C, 4 Å molecular sieve (200 mg) was added to a solution of DMTSF (74.4 mg, 0.380 mmol, 2.0 equivalent) in acetonitrile (1.0 mL) and stirred at this temperature for 1 hour. At -10 °C, the mixture of DMTSF was added to the stirred mixture of tetracyclic thioacetal compounds 1-8. The mixture was stirred at this temperature for 12 hours, then quenched with a saturated aqueous sodium bicarbonate solution (10 mL). The resulting mixture was extracted with EtOAc (3 × 10 mL), and the combined organic phases were washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:1 → 1:0) as eluent to give white, foamy pentacyclic p-toluene thioether compounds 1-9 (46.9 mg, 60%).

[0042] Pentacyclic p-toluene sulfide compounds 1-9: R f = 0.39 (silica gel, EtOAc / petroleum ether1:0); [α]25 D = +59.9 ( c = 1.0 in CHCl3); IR (film): ν max = 2924, 2862, 1570,1454, 1261, 1095, 1012, 846, 804, 495 cm −1 ; 1 H NMR (400 MHz, CDCl3): δ = 7.42(d, J = 7.6 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.32 – 7.27 (m, 1H), 7.19 – 7.14(m, 1H), 6.94 – 6.82 (m, 4H), 4.25 (dd, J= 11.2, 5.9 Hz, 1H), 3.46 (dd, J =12.3, 5.9 Hz, 1H), 3.34 – 3.27 (m, 1H), 3.23 (d, J = 2.1 Hz, 1H), 3.18 – 3.12(m, 1H), 3.12 – 3.05 (m, 1H), 3.03 – 2.93 (m, 1H), 2.87 – 2.79 (m, 1H), 2.20(s, 3H), 2.19 – 2.13 (m, 1H), 2.08 (q, J = 5.5 Hz, 1H), 2.00 (d, J = 11.0 Hz, 1H), 1.81 (d, J = 14.0 Hz, 1H), 0.98 (d, J = 5.5 Hz, 3H) ppm. 13 C NMR (126 MHz, CDCl3) δ = 185.6, 155.6, 139.6, 137.7, 132.3, 131.0, 129.8, 128.8, 124.8,124.4, 120.3, 76.7, 69.7, 65.5, 60.2, 57.3, 50.0, 45.1, 30.1, 30.0, 27.5,21.2, 13.4 ppm. HRMS ( m / z ): [M + H] + calcd for C 25 H 27 N2OS + 403.1839, found 403.1837. Compound 1: At 22 °C, deactivated Raney nickel (200 mg) was added to an acetone (5 mL) solution of pentacyclic p-toluene sulfides 1-9 (46.9 mg, 0.113 mmol, 1.0 equivalent). The resulting mixture was stirred vigorously for 3 hours, and then diatomaceous earth was added. The mixture was filtered through a small section of diatomaceous earth and washed with ethyl acetate (10 mL) and methanol (10 mL). The combined filtrates were concentrated under reduced pressure, and the crude product containing the target imine was dissolved in DCM (0.5 mL) for further use.

[0043] At 0 °C, TFAA (189 mg, 0.13 mL, 0.904 mmol, 9.0 equivalents) was added to a DCM (2 mL) solution of tetramethylmethanediamine (115 mg, 0.15 mL, 1.13 mmol, 10 equivalents). The resulting mixture was stirred for 10 min, and then the imine solution from the previous step was added dropwise. The resulting mixture was heated to 22 °C and stirred for 20 min, then quenched with a saturated sodium bicarbonate aqueous solution. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (3 × 5.0 mL). The combined organic phases were washed with saturated brine (5.0 mL), dried over anhydrous magnesium sulfate, and filtered. The combined filtrate was concentrated under reduced pressure, and the crude product containing the target product was dissolved in THF / H2O (0.50 mL: 0.10 mL). At 22 °C, silica gel (500 mg) was added to this product solution. The mixture was allowed to stand for 8 hours, and then purified by silica gel column chromatography using ethyl acetate / triethylamine (1:0 → 4:1) as eluent to give a white powdery solid compound, 1goniomine (9.81 mg, 28% for the two steps). Compound 1: [α]25 D = −136 ( c = 0.5 in CHCl3); IR (film): ν max = 3238,2934, 2360, 2341, 1675, 1590, 1456, 1066, 1053 cm −1 ; 1 H NMR (400 MHz, Chloroform- d ) δ 7.45 (d, J = 7.8 Hz, 1H), 7.11 (td, J = 7.5, 1.4 Hz, 1H), 7.02(td, J = 7.6, 1.4 Hz, 1H), 6.77 (dd, J = 7.6, 1.4 Hz, 1H), 6.00 (d, J = 1.7 Hz, 1H), 5.15 (d, J = 1.5 Hz, 1H), 3.68 (d, J = 19.8 Hz, 1H), 3.15 (q, J = 3.1 Hz, 1H), 3.03 (q, J= 6.7 Hz, 1H), 2.99 – 2.89 (m, 2H), 2.84 (s, 1H), 2.79 – 2.71 (m,1H), 2.61 (dtd, J = 15.2, 8.2, 2.8 Hz, 2H), 2.47 (ddd, J = 13.7, 11.7, 3.8 Hz,1H), 1.60 – 1.47 (m, 1H), 1.38 (d, J = 6.7 Hz, 3H). HRMS ( m / z ): [M + H] + calcdfor C 19 H 23 N2O2 + 311.1754, found 311.1762.

[0044] Example 2 Cytotoxicity screening The cell viability of compound 1 (compound 1:goniomine, prepared by the research group of Li Ang at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, as described in Example 1) in different tumor cells (cells sourced from the Cell Bank of the Chinese Academy of Sciences) was detected using the CCK8 assay. The steps are as follows: Select cells in the logarithmic growth phase, remove the culture medium, add 3 mL of PBS to rinse the cells, then add trypsin to digest for 1-2 min, add 5 mL of fresh culture medium to stop the digestion, and slowly pipette the cells to completely detach them from the culture dish.

[0045] To achieve 100,000 cells per milliliter, the cell counting procedure is as follows: Sterilize the counting chamber and slides by spraying them with 75% ethanol, then dry them thoroughly. Place half the volume of cells into the loading well, then add the remaining amount of fresh culture medium needed for plating. Mix the cells thoroughly by pipetting. Take 10 μL of cells and culture medium from the loading well and add it along the gaps between the counting chamber and the slide. Observe using a 20× or 40× microscope and count the cells. The counting principle is "take the top, not the bottom; take the left, not the right." Count the total number of cells in four 16-square grids. The total number of cells divided by 4 gives the number of cells per milliliter (tens of thousands). The specific cell count should be determined based on the actual experimental setup. If there are insufficient cells, more can be drawn from the stock solution; if there are too many cells, fresh culture medium needs to be added to dilute to the required cell count.

[0046] Use a 100 μL pipette to aspirate cells into a 96-well plate, and add 100 μL of PBS to the edge of the 96-well plate.

[0047] After approximately 12 hours of cell adhesion, blank control, control group, and compound 1 treatment group were set up. The compound 1 treatment group consisted of 10, 20, 40, and 60 µM solutions; the control group was treated with the corresponding concentration of DMSO, and each concentration was replicated in triplicate.

[0048] During the incubation process, it is necessary to observe and record the cell morphology and death status at different time points after drug treatment.

[0049] After the cells in the compound 1 concentration treatment group died (observed under a microscope as the cells became round and detached), 10 μL of CCK8 solution was added to each well, and the cells were incubated in a cell culture incubator in the dark for 1-4 h (depending on the number of cells).

[0050] Place the 96-well plate into a microplate reader and read and record the OD value at a wavelength of 450 nm.

[0051] The cell viability was calculated and plotted using Graphpad Prism software, following the formula: Cell viability = (OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells) × 100%.

[0052] As shown in Figure 1, compound 1 exhibits significant cytotoxicity in human non-small cell lung cancer PC-9 and A549 cells, as well as human pancreatic cancer PANC-1 and MIA PaCa-2 cells.

[0053] Example 3 Compound 1-mediated pyroptosis-like morphological changes Human non-small cell lung cancer PC-9 cell culture and processing PC-9 was divided into a control group (DMSO) and compound 1 treatment groups (20, 40, and 60 µM), and seeded in 24-well plates. The plates were incubated at 37°C with 5% CO2 in DMEM medium. Results were analyzed after 24 hours of incubation.

[0054] Culture and treatment of human pancreatic cancer PANC-1 and MIA PaCa-2 cells PANC-1 and MIA PaCa-2 were divided into a control group (DMSO) and a compound 1 treatment group (40 µM), respectively, and seeded in 24-well plates. The plates were incubated at 37°C with 5% CO2 using 1640 medium. Results were analyzed after 24 hours of incubation.

[0055] Image capture using an optical microscope: After processing, bright-field images were captured using an optical microscope with a 20× objective lens.

[0056] As shown in Figures 2A to 2C, the cells in the compound 1 treatment group exhibited cell swelling and the formation of numerous bubble-like protrusions, indicating that it induced cell death characterized by cell membrane rupture, which is a typical pyroptosis-like morphological change, suggesting that the cells may undergo pyroptosis.

[0057] Example 4 Detection of Annexin V-FITC cell death pathways in compound 1 The Beyotime Annexin V-FITC apoptosis detection kit (catalog number: C1062) was used, and the specific procedures are as follows: Cell treatment: PC-9 cells were divided into four groups: blank group, Annexin V single-stained group, PI single-stained group, control group (DMSO), and compound 1 treatment group (10, 20 µM). Cells were seeded in 6-well plates and cultured in DMEM medium at 37 °C and 5% CO2. Subsequent procedures were performed after 24 h of culture.

[0058] Cell collection and washing: Remove the culture plate, collect the cell culture medium, digest the cells of each group with trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash the cells twice with pre-cooled PBS buffer to remove residual culture medium.

[0059] Staining reaction: Following the kit instructions, gently resuspend the cells in the cell pellet by adding 195 μL of Annexin V-FITC binding buffer. Then add 5 μL of Annexin V-FITC reagent and 10 μL of PI reagent, mix gently, and incubate at room temperature (20-25 ℃) in the dark for 10-20 minutes, followed by placing in an ice bath. Aluminum foil can be used for light protection. Resuspending the cells 2-3 times during incubation can improve staining results.

[0060] Detection and analysis: Flow cytometry was used for detection. Annexin V was used as the x-axis and PI was used as the y-axis for two-parameter analysis. The proportion of cells in each of the four quadrants was recorded. Three replicates were set for each concentration group.

[0061] The results are shown in Figure 3. Compound 1 increased the number of positive quadrants (i.e., the upper right quadrant) in a concentration-dependent manner. ⁺) Cell ratio, compared to classical apoptosis, often shows early phosphatidylserine eversion ( The results showed that the proportion of PI-positive cells was significantly increased, indicating a rapid loss of cell membrane integrity and a large release of cellular contents. This suggests that compound 1 induced cell death characterized by cell membrane rupture. Combined with pyroptosis-like morphological changes, this result is more consistent with the typical phenotype of cell membrane perforation and rupture associated with pyroptosis, further suggesting that compound 1 may induce cell death by inducing pyroptosis.

[0062] Example 5 LDH release detection of compound 1 Take cells in the logarithmic growth phase, to Cells were seeded at a density of cells / well in 96-well cell culture plates. The culture wells were divided into the following groups: cell-free culture medium wells (blank control wells), untreated control cell wells (negative control wells), untreated cell wells for subsequent lysis (maximum enzyme activity control wells / positive control wells), and compound-treated cell wells (test wells), and labeled accordingly.

[0063] After cells adhered and stabilized, they were treated with 10, 20, 40, and 60 µM compound 1, with an equal volume of solvent added to the negative control wells. 45 min before the detection time point, 10 μL of the kit-supplied lysis buffer was added to the positive control wells to fully lyse the cells.

[0064] Once the detection time is reached, take 50 μL of supernatant from all control and test wells and transfer it to a new 96-well plate. Add 50 μL of LDH detection working solution (CytoTox 96® reagent) to each well, wrap with aluminum foil to protect from light, and incubate at room temperature for 30 min.

[0065] After incubation, 50 μL of stop solution was added to each well, and large air bubbles in the well were punctured with a syringe needle. The absorbance at 490 nm was recorded within 1 hour after the stop solution was added.

[0066] The cell mortality rate was calculated and plotted using Graphpad Prism software. The formula for calculating the cell mortality rate is: (absorbance of experimental wells - absorbance of blank control wells / (absorbance of positive control wells - absorbance of blank control wells) × 100%.

[0067] As shown in Figure 4, compound 1 promotes LDH release in a concentration-dependent manner, further indicating that compound 1 can disrupt cell membrane integrity and lead to a large amount of intracellular contents leakage. The combined results of pyroptosis-like morphological changes, increased Annexin V / PI double positivity rate, and increased LDH release indicate that it induces cell death characterized by cell membrane rupture, strongly suggesting that compound 1 can induce pyroptosis in PC-9 cells, as well as PANC-1 and MIA PaCa-2 cells.

[0068] Example 6 Compound 1 upregulates the expression levels of pyroptosis-related proteins. The experimental steps of Western blotting are as follows: SDS-PAGE preparation: Prepare the SDS-PAGE gel one day in advance or before the experiment (the concentration of SDS-PAGE can be determined according to the molecular weight of the protein to be analyzed; the lower the molecular weight, the higher the gel concentration). If preparing one day in advance, simply soak the gel in ultrapure water and store it at 4 °C.

[0069] Electrophoresis: Open the metal bath beforehand, remove the sample from the -20 °C sample box, and heat in the metal bath for 5-10 minutes. During this time, remove the pre-prepared SDS-PAGE gel and mount it on the electrophoresis tank. Use 1× gel to check for leaks between the two gels. Remove the protein marker from 4 °C (adding protein markers to the air pockets on both sides of the sample helps indicate the location of the target protein. Different amounts of protein markers on both sides help determine the loading direction). Add 3 μL of protein marker to the first well, and then add a certain mass of protein sample according to the BCA results. Note that the sample loading should not be too fast, otherwise it may cause the sample to overflow from the gel well, leading to inaccurate results. After loading, add 1× gel to the outside of the electrophoresis tank, ensuring the liquid level covers the metal wire. Perform electrophoresis at a voltage of 80 V for the stacking gel and 120 V for the separating gel.

[0070] Transfer: Near the end of electrophoresis, prepare the necessary items for transfer: electrophoresis tank, transfer plate, anhydrous methanol, PVDF membrane, tweezers, gel cutter, 1× transfer buffer (pre-cooled at 4 °C), sandwich clips, and filter paper. Remove the gel plate from the transfer tank. Place the sandwich clips in the transfer plate, and place two layers of filter paper on each side of the clips. Pour in 1× transfer buffer to cover the sandwich clips and moisten the filter paper. Use the gel cutter to pry open the gel plate and, according to the protein marker, cut off the gel side containing the target protein. Following the "black gel, white membrane" principle, place it on the black side of the sandwich filter paper. Select a PVDF membrane of appropriate size according to the molecular weight of the target protein, cut it to a size similar to the cut gel side, activate it in anhydrous methanol for 1 min, then equilibrate it in 1× transfer buffer for 30 seconds. Carefully place it on the gel side using tweezers, using the gel cutter to carefully remove air bubbles between the gel side and the PVDF membrane. Then, place the white side of the sandwich clip on the PVDF membrane and carefully attach the sandwich clip. Keep the gel moist throughout the process. Place the sandwich clip into the transfer tank, following the "black to black, white to white" rule, and pour in 1× transfer buffer. The transfer conditions are a constant current of 230 mA. The transfer time depends on the protein molecular weight, generally "1 min 1 Kd". Because heat generated during transfer may cause excessive temperature and damage to the protein bands, ice bath transfer is recommended.

[0071] Blocking: After the transfer is complete, prepare a sealing box and pour in a certain amount of 5% skim milk. Remove the sandwich clamp from the transfer tank, carefully open it, use tweezers to grasp the protein marker on the PVDF membrane, transfer it to the sealing box, and incubate it on a shaker at room temperature for 1-2 hours.

[0072] Primary antibody incubation: Remove the antibody for the target protein from the antibody box at -20 °C and dilute it with 5% skim milk according to the dilution ratio recommended in the antibody instructions. The required antibody dilution solution depends on the size of the protein band. For example, to prepare 1 mL of antibody dilution solution, if the recommended Western blotting (WB) dilution ratio is 1:1000, add 1 μL of antibody to 5% skim milk and mix well. Label the protein name on the plastic bag for antibody incubation. Place the corresponding protein PVDF membrane into the plastic bag, seal one side with a plastic sealer, add the prepared primary antibody dilution solution, carefully remove air bubbles, and seal with a plastic sealer. Place the band on a silent mixer and rotate for incubation. Incubation can be done at room temperature for 3-4 hours or overnight at 4 °C.

[0073] Primary antibody washing: Label the protein name on the antibody incubation cassette and pour in a certain amount of TBST. After primary antibody incubation, place the band into the antibody incubation cassette and wash on a shaker for ten minutes. Repeat three times.

[0074] Secondary antibody incubation: Prepare the corresponding secondary antibody according to the resistance of the target protein antibody. The dilution ratio of the secondary antibody is 1:10000. For example, to prepare 10 mL of secondary antibody dilution solution, add 1 μL of secondary antibody to 10 mL of 5% skim milk. After primary antibody washing, place the protein band in a plastic bag, add the corresponding secondary antibody dilution solution, and seal with a plastic sealer. Place the band on a silent mixer and incubate at room temperature for 1 hour.

[0075] Secondary antibody washing: Label the protein name on the antibody incubation cassette and pour in a certain amount of TBST. After secondary antibody incubation, place the band into the antibody incubation cassette and wash on a shaker for ten minutes. Repeat three times.

[0076] Color development and exposure: Turn on the chemiluminescence analyzer and software. Prepare the color development solution by mixing solution A and solution B in a 1:1 ratio. Remove the PVDF membrane from the antibody incubation chamber and place it in the color development area. Add the developing solution evenly, and the color development can begin.

[0077] The results of Western blot experiments are shown in Figure 5. The concentration gradient of compound 1 upregulated the expression levels of pyroptosis-related proteins such as cleaved-caspase 3 and NT-GSDME, verifying the occurrence of pyroptosis.

[0078] The above-described studies of this invention demonstrate that compound 1 can induce typical pyroptosis-like morphological changes in human non-small cell lung cancer PC-9 cells, with a concentration-dependent increase. The proportion of double-positive cells significantly increased LDH release and upregulated the expression of pyroptosis-related proteins. Furthermore, compound 1 exhibited strong cytotoxicity against pancreatic cancer PANC-1 and MIA PaCa-2 cells, induced classic pyroptosis-like morphological changes, and significantly enhanced LDH release.

[0079] In summary, compound 1 can significantly induce pyroptosis in PC-9 cells, as well as PANC-1 and MIA PaCa-2 cells, exhibiting good anti-non-small cell lung cancer and anti-pancreatic cancer activity. Given that pyroptosis is an inflammatory programmed cell death process, this process is expected to further activate the body's anti-tumor immune response and improve the tumor immune microenvironment, providing new candidate drugs and research directions for the prevention and treatment of related tumors.

[0080] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, this specification should be considered illustrative rather than restrictive.

Claims

1. A method for preparing the compound goniomine, characterized in that, The structural formula of the compound goniomine is as follows: The preparation method includes the following steps: (1) Indoleacetic acid ester compound 1-3 was synthesized by coupling reaction of tetrahydropyridinol compound 1-1 and indoleacetic acid 1-2; (2) Indole acetate compounds 1-3 were rearranged under Ireland-Claisen conditions and decarboxylated under acidic conditions to synthesize indoleene compounds 1-4; (3) Indolediol compounds 1-5 were synthesized by reacting indoleene compounds 1-4 with a dihydroxylating agent; (4) Tetracyclic epoxy compounds 1-6 were synthesized by tandem Polonovski–Potier / Friedel–Crafts reaction of indole diol compounds 1-5 under acidic conditions and epoxidation. (5) Tetracyclic thioacetal compounds 1-8 are generated by benzyl deprotection of tetracyclic epoxides 1-6 and reductive amination with thioacetals 1-7. (6) Pentyl-p-toluene sulfide compounds 1-9 were synthesized by cyclization of tetracyclic thioacetal compounds 1-8 with dimethyl(methylthio)sulfonium tetrafluoroborate under alkaline conditions; (7) Compound 1 goniomine was generated by deprotection of pentacyclic p-toluene sulfide compounds 1-9 and reaction with Eschenmoser salt on silica gel.

2. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (1), the coupling reaction is carried out in a dichloromethane solution, first at a low temperature of 0±5 °C. o The reaction was carried out at C, and then the temperature was raised to room temperature; the condensing agent was selected from DCC, DIC and EDCI. In step (2), the rearrangement reaction is carried out in a tetrahydrofuran solution, first at a low temperature of -78±5 °C. o The reaction is carried out at C, and then brought to room temperature; the base used for hydrogen removal is selected from lithium bis(trimethylsilylamino) and lithium diisopropylamino; the decondensation reaction proceeds spontaneously under acidic conditions, with a solution pH range of 1-3.

3. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (3), the dihydroxylation reaction is carried out in a mixed solution of THF / tert-butanol / water at a temperature of 0±5 °C. o C; The oxidant is selected from NMO / potassium osmium tetroxide, osmium tetroxide aqueous solution, and potassium hexacyanoferrate / potassium osmium tetroxide.

4. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (4), the cyclization reaction is carried out in a dichloromethane solution, and the oxidation reaction is carried out at a low temperature of 0±5℃. o C, the oxidant is selected from m-chloroperoxybenzoic acid and hydrogen peroxide aqueous solution; the Polonovski–Potier reaction is first carried out at a low temperature of 0±5℃. o The reaction was carried out at C, then raised to room temperature. The reagents used in the reaction were selected from trifluoroacetic anhydride and trifluoromethanesulfonic anhydride. The epoxidation reaction was first carried out at a low temperature of 0±5℃. o The reaction was carried out at C and then brought to room temperature. The reagents were selected from methanesulfonic anhydride / triethylamine and trifluoromethanesulfonic acid chloride / pyridine.

5. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (5), the hydrogen debenzylation reaction is carried out in a trifluoroethanol solution at room temperature, with the solvent selected from methanol, ethanol, and trifluoroethanol; the reductive amination reaction is carried out in a tetrahydrofuran solution and a methanol solution, first at a low temperature of 0±5℃. o The reaction is carried out at C, and then the temperature is raised to room temperature. The reducing agent is selected from sodium cyanoborohydride and sodium triacetoxyborohydride.

6. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (6), the cyclization reaction is carried out in an acetonitrile solution at a temperature of -10±5 °C. o C, the reagent used in the reaction is selected from dimethyl(methylthio)sulfonium tetrafluoroborate, dimethyl(methylthio)sulfonium tetrafluoroborate / 2,6-dimethylaminopyridine.

7. The method for preparing the compound goniomine according to claim 1, characterized in that, In step (7), the phenylthio group removal reaction is carried out in acetone solution at room temperature; the reducing agent is selected from Raney nickel, lithium / liquid ammonia, and sodium / naphthalene; the dimethylamino reaction is carried out in dichloromethane solution, and the reaction is initially carried out at a low temperature of 0±5℃. o The reaction was carried out at C, and then the temperature was raised to room temperature. The reagent combination was selected from tetramethylmethanediamine / trifluoroacetic anhydride and N,N-dimethylmethylene ammonium iodide.

8. The compound goniomine prepared by the method of any one of claims 1 to 7, characterized in that, The structural formula of the compound goniomine is as follows: 。 9. The use of compound goniomine or its hydrate, solvate, stereoisomer, racemate or pharmaceutically acceptable salt in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The anti-tumor drugs mentioned are drugs for treating non-small cell lung cancer and pancreatic cancer.

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