Compound with c-Met inhibition and ferroptosis induction functions as well as preparation method and application of compound

Abstract

The invention discloses a compound with c-Met inhibition and ferroptosis induction functions, a preparation method and application, and belongs to the technical field of medicinal chemistry. The compound provided by the invention can activate a ferroptosis pathway, inhibit the expression of GPX4, target c-Met protein, inhibit a c-Met signal pathway and cut off two key survival pathways (HGF / c-Met proliferation promotion and GPX4 anti-oxidation defense) of tumor cells at the same time, and a signal pathway-metabolic homeostasis dual-strike mechanism is formed, so that the development of tumors is effectively inhibited.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a compound with c-Met inhibition and iron death induction, a preparation method and use. BACKGROUND

[0002] Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, and its complex pathological mechanism and drug resistance characteristics pose a severe challenge to the development of new treatment strategies. In recent years, research has shown that multi-target synergistic intervention strategies exhibit unique advantages in overcoming tumor heterogeneity and treatment resistance. Among the many potential targets, the synergistic targeting therapy of protein tyrosine kinase c-Met (Mesenchymal-epithelial transition factor) and iron death regulation pathway has attracted attention due to its complementary anti-tumor mechanisms.

[0003] Tumor genomics data shows that about 87% of GBM patients have abnormal activation of the c-Met signaling pathway, and its overexpression promotes tumor cell proliferation, invasion and angiogenesis through multiple downstream pathways such as PI3K / AKT / mTOR and RAS / MAPK. Notably, although the second-generation c-Met inhibitor (such as Capmatinib) can effectively inhibit kinase activity, its single-agent efficacy is limited in clinical trials, which may be related to the establishment of compensatory survival mechanisms by metabolic reprogramming in tumor cells. In this context, iron death, a new type of cell death, provides a new dimension for GBM treatment. Unlike the traditional apoptosis pathway, iron death triggers cell membrane system collapse through lipid peroxide accumulation, and has a specific killing effect on GBM cells with high metabolic activity and iron ion accumulation characteristics. Experiments have confirmed that activating iron death can reduce the survival rate of GBM stem cell-like cells by 62%-75%.

[0004] In-depth mechanism research has found that there is a significant pathological correlation between c-Met and the iron death regulation network. First, the c-Met / HGF axis can enhance the antioxidant defense capacity of cells by up-regulating the expression of glutathione peroxidase 4 (GPX4), which is a key negative regulator of iron death. Second, the epithelial-mesenchymal transition (EMT) process driven by c-Met induces down-regulation of ferroportin expression, leading to an increase in intracellular free iron concentration to a critical threshold, which provides the necessary conditions for iron death activation. More meaningfully, preclinical studies have shown that the combined use of c-Met inhibitors and iron death inducers (such as Erastin) can produce significant synergistic effects.

[0005] Currently, there is no c-Met / ferroptosis dual-targeting drug in the clinical trial stage in the world, and the main technical bottlenecks are: ① it is difficult for traditional inhibitors to simultaneously meet the requirements of kinase domain binding and redox regulation; ② the blood-brain barrier penetration and target delivery efficiency are insufficient; and ③ the pharmacokinetic matching problem of the dual-action mechanism. SUMMARY

[0006] In view of the above problems in the prior art, the present application provides a compound capable of inhibiting c-Met and inducing ferroptosis, a preparation method and use thereof, which can activate the ferroptosis pathway and inhibit the expression of GPX4, while targeting the c-Met protein and inhibiting the c-Met signaling pathway. The occurrence of c-Met and GPX4 is inhibited at the same time, cutting off two key survival paths of tumor cells (HGF / c-Met promotes proliferation and GPX4 antioxidant defense), forming a “signal pathway-metabolic homeostasis” dual-hit mechanism, thereby effectively inhibiting the development of tumors.

[0007] To achieve the above object, the technical scheme adopted by the present application to solve its technical problems is:

[0008] A compound capable of inhibiting c-Met and inducing ferroptosis, which has the following structural formula:

[0009]

[0010] wherein, R substituent is a triazole compound or an alkyl alcohol with less than four carbon atoms.

[0011] Further, the alkyl alcohol is iodoethanol.

[0012] Further, the triazole compound is 4-methyl-4H-1,2,4-triazole-3-carboxylic acid.

[0013] Further, the compound has the following structural formula:

[0014]

[0015]

[0016] A method for synthesizing the above compound, comprising the following steps:

[0017] (1) adding sodium carbonate, 5-fluoro-2-chloropyrimidine, (Ph3P)2PdCl2 and 3-acetylphenyl boronic acid into a mixed solvent, protecting under nitrogen, and refluxing under heating for 10-12 h to prepare a B1 compound; wherein the molar ratio of sodium carbonate, 5-fluoro-2-chloropyrimidine, (Ph3P)2PdCl2 and 3-acetylphenyl boronic acid is 6-7:3-3.5:0.05-0.08:3-3.5;

[0018] (2) adding a B1 compound and sodium hydride into a solvent, dropwise adding a solution of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate under ice bath, and then reacting at room temperature for 3-5 h to prepare a B2 compound; wherein the molar ratio of the B1 compound, the sodium hydride and the tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate is 1-1.5: 1.5-2: 1-1.5;

[0019] (3) adding a chiral ligand and dichlorobis(4-methylisopropylphenyl)ruthenium into water, cooling to room temperature after reacting for 5-8 h, then adding a solution of sodium formate and a solution of the B2 compound under ice bath in sequence, and reacting at room temperature for 10-12 h to prepare a B3 compound; wherein the molar ratio of the chiral ligand, the dichlorobis(4-methylisopropylphenyl)ruthenium, the sodium formate and the B2 compound is 0.1-0.2: 0.01-0.05: 2-2.5: 0.1-0.5;

[0020] (4) adding the B3 compound, 3-(6-oxo-1,6-dihydro-3-pyridazinyl)benzonitrile and triphenylphosphine into a solvent, dropwise adding DIAD (diisopropyl azodicarboxylate) under ice bath in nitrogen protection, and reacting at room temperature for 10-12 h to prepare a B4 compound; wherein the molar ratio of the B3 compound, the 3-(6-oxo-1,6-dihydro-3-pyridazinyl)benzonitrile, the triphenylphosphine and the DIAD is 0.1-0.5: 0.1-0.5: 0.5-1: 0.5-1;

[0021] (5) adding trifluoroacetic acid to a solution of the B4 compound under ice bath, and reacting at room temperature for 5-8 h to prepare a B5 compound;

[0022] (6) reacting the B5 compound with iodoethanol or 4-methyl-4H-1,2,4-triazole-3-carboxylic acid to prepare the compound for inducing ferroptosis.

[0023] Further, the mixed solvent used in step (1) comprises toluene, ethanol and water.

[0024] Further, the reaction temperature in step (1) is 80-85℃, and the time is 10-12 h.

[0025] Further, the reaction temperature of the chiral ligand and dichlorobis(4-methylisopropylphenyl)ruthenium in step (3) is 40-45℃.

[0026] Further, the chiral ligand is N-((1R,2R)-2-amino-1,2-diphenylethyl)-1-((1R,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonamide (N-[(1R,2R)-2-amino-1,2-diphenylethyl]-1-[(1R,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl]methanesulfonamide).

[0027] Further, the specific process of the reaction of the B5 compound with iodoethanol in step (6) is as follows:

[0028] After the B5 compound, iodoethanol and potassium carbonate are mixed and dissolved, the mixture is reacted at 80-95°C for 10-12h to prepare the iron death-inducing compound; wherein the molar ratio of the B5 compound, iodoethanol and potassium carbonate is 0.01-0.05:0.01-0.05:0.1-0.5.

[0029] Further, the specific process of the reaction of the B5 compound with 4-methyl-4H-1,2,4-triazole-3-carboxylic acid in step (6) is as follows:

[0030] After the B5 compound, HATU (2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate), triethylamine and 4-methyl-4H-1,2,4-triazole-3-carboxylic acid are mixed and dissolved, the mixture is reacted at room temperature for 12-16h to prepare the iron death-inducing compound; wherein the molar ratio of the B5 compound, HATU, triethylamine and 4-methyl-4H-1,2,4-triazole-3-carboxylic acid is 0.01-0.05:0.1-0.5:0.1-0.5:0.01-0.05.

[0031] An iron death inducer including the above-mentioned compound, or a pharmaceutically acceptable salt thereof as an effective ingredient.

[0032] A preparation for improving the sensitivity of tumor cells, including the above-mentioned compound.

[0033] The use of the above-mentioned compound, iron death inducer or preparation in the preparation of a preparation for treating glioblastoma.

[0034] Advantages of the present application:

[0035] The traditional iron death inducer has low bioavailability and is easily degraded in vivo. The present application provides a compound with c-Met inhibition and iron death induction, which has high bioavailability (99.67%) and good blood-brain barrier penetration. This dual-targeting strategy not only breaks through the limitations of single-target therapy, but also has the potential to reshape the tumor microenvironment through metabolic reprogramming, opening up new avenues for precise treatment of GBM. With the elucidation of target interaction mechanisms and the advancement of drug design techniques, innovative therapies that inhibit c-Met in conjunction with iron death activation are expected to become an important breakthrough in overcoming the bottleneck of GBM treatment.

[0036] The compound prepared in the present application has c-Met inhibition and iron death induction, which improves the sensitivity of tumor cells, and the IC 50 from 12.3±1.5μM to 1.21±0.09μM. In addition, it can activate the iron death pathway while inhibiting c-Met and GPX4, cutting off two key survival paths of tumor cells (HGF / c-Met promotes proliferation and GPX4 antioxidant defense), forming a "signal pathway-metabolic homeostasis" double hit mechanism, thereby effectively inhibiting the development of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The mechanism of action and selective detection results of compounds B18 and B20 are shown in the figure;

[0038] Figure 2 The detection results of compounds B18 and B20 inducing iron death are shown in the figure;

[0039] Figure 3 The effect of compounds B18 and B20 on cell autophagy is shown in the figure;

[0040] Figure 4 The detection results of compounds B18 and B20 in vivo anti-tumor activity are shown in the figure;

[0041] Figure 5 The mechanism of action of the compound prepared in the present application is shown in the figure. DETAILED DESCRIPTION

[0042] The specific embodiments of the present application are described below to facilitate understanding of the present application by those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that any changes within the spirit and scope of the present application as defined in the appended claims are obvious, and all applications utilizing the concept of the present application are within the scope of protection.

[0043] Synthesis of compounds in Example 1

[0044] 1. Synthesis of 1-(3-(5-fluoropyrimidin-2-yl)phenyl)ethan-1-one (B1)

[0045]

[0046] Sodium carbonate (66 mmol, 6.72 g), 5-fluoro-2-chloropyrimidine (33 mmol, 4.32 g), (Ph3P)2PdCl2(0.68 mmol, 0.48 g), 3-acetylphenylboronic acid (32 mmol, 5.25 g) were added to a mixture of toluene (65 mL), ethanol (33 mL) and water (32 mL) under nitrogen protection, and refluxed at 83 °C for 12 h. After cooling to room temperature, the reaction solution was extracted with ethyl acetate for 3 times. The organic phase was separated and concentrated, and the crude product was purified by column chromatography to obtain 5.54 g of white solid with a yield of 80.2%. 1 H NMR (400 MHz, CDC13) δ 8.96 (t, J = 1.61 Hz, 1H), 8.71 (s, 2H), 8.58 (s, 1H), 8.10 (s, 1H), 7.61 (s, 1H), 2.73 (s, 3H).

[0047] 2. Synthesis of 4-(((2-(3-acetylphenyl)pyrimidin-5-yl)oxy)methyl)piperidine-1- carboxylic acid tert-butyl ester (B2)

[0048]

[0049] B1 (10 mmol, 2.16 g), sodium hydride (20 mmol, 0.8 g) were added to DMF (30 mL), and a DMF solution of 4-(hydroxymethyl)piperidine-1-carboxylic acid tert-butyl ester (15 mmol, 3.23 g) was slowly added dropwise under ice bath, and then reacted at room temperature for 3 h. The reaction solution was extracted with ethyl acetate for 3 times. The organic phase was separated and concentrated, and the crude product was purified by column chromatography to obtain 2.88 g of white solid with a yield of 70.1%.1H NMR (400 MHz, CDC13) δ 8.56 (t, J = 1.61 Hz, 1H), 8.48 (s, 1H), 8.11 (s, 1H), 8.10 (s, 1H), 7.61 (s, 2H), 3.79 (d, J = 7.5 Hz, 2H), 3.15 - 3.03 (m, 4H), 2.73 (s, 3H), 1.93 - 1.72 (m, 5H), 1.48 - 1.38 (s, 9H).

[0050] 3. Synthesis of (R)-4-(((2-(3-(1-hydroxyethyl)phenyl)pyrimidin-5-yl)oxy)methyl)piperidine-1-carboxylic acid tert-butyl ester (B3)

[0051]

[0052] Chiral ligand N-((1R,2R)-2-amino-1,2-diphenylethyl)-1-((1R,4R)-7,7-dimethyl-2- oxobicyclo[2.2.1]heptan-1-yl)methanesulfonamide (0.1 mmol, 0.042 g), dichlorobis(4- methylisopropylphenyl)ruthenium (0.05 mmol, 30.61 g) were added to water (5 mL), nitrogen protection, reaction at 40 °C for 5 h. After cooling to room temperature, ice bath was added in turn sodium formate (12.5 mmol, 0.85 g) aqueous solution (5 mL), B2 dichloromethane solution (2.5 mmol, 1.03 g), reaction at room temperature for 12 h, the reaction liquid was concentrated, column chromatography purification to get brown solid 0.8 g, yield 77.6%. 1H NMR (400 MHz, CDC13) δ 8.66 (s, 1H), 8.45 (s, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.61 (s, 2H), 5.36 (m, 1H) 3.69 (d, J = 7.1 Hz, 2H), 3.16-3.09 (m, 4H), 2.03-1.79 (m, 5H), 1.45-1.41 (s, 9H), 0.98-0.91 (d, J = 6.5 Hz, 3H).

[0053] 4. Synthesis of tert-butyl 4-((2-(3-(1-(3-cyanophenyl)-6-oxopyridazin-1(6H)-yl)ethyl)phenyl)pyrimidin-5- yl)oxymethyl)piperidine-1-carboxylate (B4)

[0054]

[0055] B3 (2.5 mmol, 1.03 g), 3-(6-carbonyl-1,6-dihydro-3-pyridazinyl)benzonitrile (2.5 mmol, 0.49 g), triphenylphosphine (5 mmol, 1.31 g) were added to THF (30 mL), nitrogen protection, dropwise addition of DIAD (5 mmol, 1.01 g) under ice bath, reaction at room temperature for 12 h, the reaction liquid was extracted with ethyl acetate for 3 times, fast column chromatography purification to get white solid, which was directly used for the next reaction. 1H NMR (400 MHz, CDC13) δ 8.65 (s, 1H), 8.44 (s, 2H), 8.18 (d, J = 7.3 Hz, 1H), 8.21 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.61 - 7.46 (m, 3H), 7.40 (t, J = 7.3 Hz, 1H), 7.01 (d, J = 9.3 Hz, 1H), 6.54 (q, J = 7.1 Hz, 1H), 3.93 (d, J = 5.8 Hz, 2H), 2.93 (d, J = 11.5 Hz, 2H), 1.99 (d, J = 10.2 Hz, 2H), 1.91 (d, J = 7.0 Hz, 3H), 1.85 (m, 3H), 1.50 (m, 2H), 1.46 - 1.39 (s, 9H).

[0056] 5. Synthesis of (S)-3-(6-oxo-l-(l-(3-(5-(piperidin-4-ylmethoxy)pyrimidin-2- yl)phenyl)ethyl)-l,6-dihydropyridazin-3-yl)benzonitrile (B5)

[0057]

[0058] The B4 obtained from the above step was added to DCM (30 mL), trifluoroacetic acid (20 mmol, 2.02 g) was added dropwise under ice bath, and the reaction was carried out at room temperature for 5 h. The reaction solution was concentrated, and column chromatography was used for purification to obtain white solid 0.85 g with a yield of 68.8%. 1 H-NMR (600 MHz, d6-DMSO) δ 8.67 (s, 2H), 8.45 (s, 1H), 8.36 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 8.11 (d, J = 9.7 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.70 (m, 1H), 7.57 - 7.40 (m, 2H), 7.10 (d, J = 9.7 Hz, 1H), 4.67 (br s, 1H), 4.05 (d, J = 6.3 Hz, 2H), 3.14 - 3.01 (m, 4H), 2.74 - 2.55 (m, 5H), 1.47 (m, 3H).

[0059] 6. Synthesis of (S)-3-(l-(l-(3-(5-((l-(2-hydroxyethyl)piperidin-4-yl)methoxy)pyrimidin-2- yl)phenyl)ethyl)-6-oxo-l,6-dihydropyridazin-3-yl)benzonitrile (B18)

[0060]

[0061] B5 (0.25 mmol, 0.12 g), iodoethanol (0.25 mmol, 0.042 g), and potassium carbonate (0.5 mmol, 0.069 g) were added to DMF (5 mL), reacted at 80 °C for 12 h, and purified by column chromatography to obtain 0.07 g of white solid, yield 52.2%. 1 H-NMR(600MHz,d6-DMSO)δ8.65(s,2H),8.48(s,1H),8.39(s,1H),8.29(d,J=8.0Hz,1H),8.22(d,J =7.7Hz,1H),8.14(d,J=9.7Hz,1H),7.93(d,J=7.7Hz,1H),7.76–7.70(m,1H),7.55–7.44(m,2H),7. 11(d,J=9.7Hz,1H),6.38(q,J=7.0Hz,1H),4.67(brs,1H),4.05(d,J=6.3Hz,2H),3.59(t,J=12.4H z,2H),3.14–3.01(m,2H),2.74–2.55(m,2H),2.28(brs,2H),1.91–1.70(m,6H),1.48–1.38(m,2H). 13 C-NMR(101MHz,d6-DMSO)δ159.08,156.45,152.12,144.70,142.10,141.91,137.77,136.28,133.35,130.87,130.82,130.62 ,130.22,129.81,129.41,129.17,126.90,126.52,119.12,112.74,73.14,60.32,58.15,56.20,53.23,35.00,27.88,20.37.

[0062] 7. Synthesis of (S)-3-(1-(1-(3-(5-(1-(-4-methyl-1H-1,2,4-triazol-3-carbonyl)piperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)-6-oxo-1,6-dihydropyridazin-3-yl)benzylnitrile (B20)

[0063]

[0064] B5 (0.25 mmol, 0.12 g), HATU (0.5 mmol, 0.189 g), triethylamine (0.5 mmol, 0.05 g), 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid (0.25 mmol, 0.031 g) were added into DMF (5 mL), the reaction was carried out at room temperature for 16 h, the reaction solution was extracted with ethyl acetate for 3 times, the organic phase was concentrated, and column chromatography purification was carried out to obtain 0.1 g of white solid, with a yield of 66.7%. 1 H-NMR (600 MHz, d6-DMSO) δ 8.66 (s, 2H), 8.47 (s, 1H), 8.39 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 8.14 (d, J = 9.7 Hz, 1H), 8.06 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.76 - 7.71 (m, 1H), 7.54 - 7.46 (m, 2H), 7.11 (d, J = 9.7 Hz, 1H), 6.37 (q, J = 7.0 Hz, 1H), 4.53 (d, J = 12.9 Hz, 1H), 4.11 (d, J = 6.3 Hz, 3H), 3.98 - 3.90 (m, 3H), 3.21 - 3.15 (m, 1H), 2.93 - 2.88 (m, 1H), 2.17 (dt, J = 15.9, 5.5 Hz, 1H), 1.93 (d, J = 13.2 Hz, 1H), 1.87 - 1.81 (d, J = 7.1 Hz, 4H), 1.36 (ddt, J = 24.4, 12.3, 8.1 Hz, 2H). 13 C-NMR (101 MHz, d6-DMSO) δ 159.08, 158.08, 156.49, 152.09, 150.10, 148.18, 144.75, 142.10, 141.93, 137.77, 136.29, 133.34, 130.87, 130.81, 130.61, 130.21, 129.81, 129.41, 129.18, 126.90, 126.53, 119.12, 112.74, 72.86, 56.20, 46.74, 41.92, 37.25, 35.60, 29.31, 28.45, 20.37.

[0065] Identification of antitumor proliferation activity of compounds B18 and B20 in Example 2

[0066] U87MG, U251 and T98G cells in good condition and in logarithmic growth phase were collected, the cells were digested and counted, 7000 cells were inoculated in each well of a 96-well plate, and the plate was incubated at 37°C in a 5% carbon dioxide incubator for 24 h. After the cells adhered, the old culture medium was removed, and a culture medium containing 1% gibco was added, and different concentrations (50, 25, 10, 1, 0.1, 0.01 uM) of test compounds B18, B20, 1D228 were added, and three groups of duplicate wells were set for each concentration, and then the plate was incubated in a 37°C incubator for 48 h. After treatment, CCK8 was added to each well under sterile conditions (the final concentration was 10% of the total volume), and the plate was incubated at 37°C for 40 min. The absorbance was measured at 450 nm using a microplate reader. The cytotoxic effect of each compound was represented by the IC50 value, which represented the concentration of the drug required to inhibit tumor cell growth by 50%, and was calculated using GraphPad Prism Software version 5.02 (GraphPad Inc., La Jolla, CA, USA). Each group of experiments was independently repeated three times, and the final data was evaluated by taking the average value, and the results are shown in Table 1. 50 Table 1. Anti-tumor proliferation activity of compounds

[0067] As shown in Table 1, compared with the positive control c-Met inhibitor Tepotinib (IC 50 = 26.35 ± 1.41 uM) and the ferroptosis inducer Sorafenib (IC 50 = 8.12 ± 0.55 uM), the compounds B18 and B20 prepared by the present application showed significantly enhanced anti-proliferative activity on U87MG cells, with IC 50 values of 1.21 ± 0.09 uM (B18) and 2.03 ± 0.01 uM (B20), respectively. This data indicates that B18 and B20 have the potential to become high-efficiency anti-glioblastoma candidate compounds.

[0068] Table 1. Anti-tumor proliferation activity of compounds

[0069]

[0070] Example 3. Detection of the anti-tumor efficacy of compounds B18 and B20

[0071] 1. Mechanism of action and blood-brain penetration detection

[0072] RNA-seq analysis of gene expression profile changes after treatment with preferred compounds, collect glioblastoma treated and untreated, 3 samples in each group, samples after RNA extraction, purification, library construction, using second generation sequencing technology, based on Illumina HiSeq sequencing platform, double end sequencing of these libraries, then filter the original data, and align the high quality sequences obtained after filtering to the human reference genome. According to the alignment results, the expression of each gene is calculated. On this basis, further expression difference analysis, enrichment analysis and clustering analysis are carried out on the samples, and the results are shown in Figure 1 .

[0073] As shown in Figure 1 , RNA-Seq experiments show that the compound can down-regulate the expression of multiple oncogenes such as PIK3CG, FGF, TEK, HIF1A, PDK1, and KEGG results show that the compound mainly regulates PI3K-Akt, HIF-1α and ferroptosis signal pathways Figure 1 (A). Kinase profile results show that the compound has only inhibitory activity on C-Met kinase at a concentration of 500 nM, indicating that the compound has good kinase selectivity Figure 1 (B). Next, the effect of the compound on the blood-brain barrier was tested, as shown in 1C, the in vitro blood-brain barrier (BBB) model shows that the -LOG Pe of compounds B18 and B20 reaches 5.08±0.23 and 4.22±0.06 respectively, suggesting that they have the potential to treat CNS tumors Figure 1 (C). Western blot experiments show that after treatment with the compound, the level of c-Met receptor autophosphorylation (p-Met) decreases by 82.3±5.1%, and its downstream effector proteins p-AKT and p-ERK are also inhibited Figure 1 (D). The above results suggest that the compound blocks c-Met-mediated PI3K / AKT, MAPK / ERK, and other key oncogenic signal transduction pathways, thereby inhibiting tumor cell proliferation and survival.

[0074] 2. Related pathways that play an anti-tumor activity

[0075] (1) WB detects the effect of the compound on ferroptosis proteins

[0076] U87MG cells were cultured in DMEM medium containing 10% FBS in a 37℃, 5% CO2 incubator, and after 24 hours of drug treatment, 70-80 μL of RIPA lysis buffer containing protease inhibitors and phosphorylated proteinase inhibitors was added to each well, and the cells were collected by scraping with a cell scraper. The mixture was vortexed on ice for 3 times, 10 minutes each time. Then it was centrifuged at 12000 rpm at 4℃ for 15 minutes, and the supernatant was collected. BCA quantification was performed, and electrophoresis and electrotransfer were performed. The PVDF membrane was completely soaked in 5% milk, and incubated at room temperature for 1-2 hours. The blocked PVDF membrane was cut according to the molecular weight required by the experiment, and primary antibodies p-MET (1:1000; 3077T, Cell Signaling), c-MET (1:1000; 8198T, Cell Signaling), p-AKT (1:1000; AF6261, affinity), AKT (1:1000; GR100134-1, abcam), p-ERK (1:1000; AF1015, affinity), ERK (1:1000; 4695T, Cell signaling), and β-actin (1:1000; A5441, Sigma) were incubated at 4℃ overnight. After recovering the primary antibody, the strip was washed with TBST for 10 minutes each time, for a total of 3 times. Different secondary antibodies were added according to the source of the primary antibody and incubated at room temperature for 1 hour. After recovering the secondary antibody, the strip was washed with 1x TBST for 10 minutes each time, for a total of 3 times. Finally, ECL was used for development.

[0077] (2) CCK8 detection of ferroptosis inhibitor reversing compound-induced ferroptosis, the detection process is the same as in Example 2.

[0078] (3) Immunofluorescence detection of the effect of the compound on LPO (red: green:)

[0079] 2 μL of medium was added to the center of the bottom of a six-well plate, and the coverslips were sterilized with 75% alcohol and placed in the six-well plate. U87MG cells in good growth condition were collected and counted, so that 1x10 5Cells were incubated at 37°C for 24 hours. 500 μL of cell suspension was slowly pipetted into the center of the slide and incubated for 24 hours. After 24 hours of incubation, cells were treated with B18 (1, 5, 10 μM), B20 (1, 5, 10 μM), 1D228 (5 μM), and TMZ (1 nM) for 24 hours. After removing the culture medium, cells were fixed with 4% paraformaldehyde for 15 minutes and washed with PBS for 5 minutes each time, for a total of 3 times. 500 μL of 0.02% Triton X-100 prepared in PBS was added to each well, and the cells were incubated at room temperature for 10 minutes, followed by 3 washes with PBS for 5 minutes each time. 500 μL of 5% BSA was added to each well, and the cells were incubated at 37°C for 1 hour. After removing the blocking solution, 200 μL of primary antibody was added to each well, and the cells were incubated overnight at 4°C. After removing the 6-well plate, allow it to warm to room temperature for 30 minutes. Transfer the primary antibody to an antibody tube and wash the cell slides three times with PBS for 5 minutes each time. Add 200 μL of secondary antibody to each slide and incubate at room temperature for 1 hour. Then, recover the secondary antibody and wash with PBS for 5 minutes each time, for a total of three times. Add DAPI nuclear dye and incubate at room temperature for 10 minutes. Wash the slides with PBS for 5 minutes each time, for a total of three times. Remove the cell slides and invert them onto a glass slide, blotting away any remaining liquid with absorbent paper. Use a confocal fluorescence microscope to capture and analyze fluorescence images.

[0080] like Figure 2 As shown, target compounds B18 and B20 exert significant antitumor effects by specifically activating the ferroptosis signaling pathway. They inhibit glutathione peroxidase 4 (GPX4) activity and SLC3A2, leading to GSH depletion and lipid ROS accumulation, while simultaneously activating HO1, further releasing Fe... 2+ Amplified lipid peroxidation, which synergistically promotes ferroptosis. Figure 2 AB). Further research revealed that reversible compounds such as the ferroptosis inhibitor Ferrostatin-1 exhibited antitumor effects (restoration of cell viability). Figure 2 C). Immunofluorescence experiments demonstrated that the compound promoted the accumulation of lipid peroxidation (LPO) products and ferrous ion accumulation. Figure 2 D) thereby driving ferroptosis in tumor cells. This discovery confirms that ferroptosis is a key mechanism by which this compound exerts its pharmacological effects, providing experimental evidence for the development of anticancer drugs targeting ferroptosis.

[0081] 3. Induces autophagy in tumor cells

[0082] U87MG cells in good condition and in the logarithmic growth phase were collected and cultured in 6-well plates at 37°C with 5% CO2. After adhesion, the cells were treated with B18 (1, 5, 10 μM), B20 (1, 5, 10 μM), 1D228 (5 μM), and TMZ (1 nM) for 24 hours. After treatment, the cells were scraped off and collected in EP tubes. The tubes were centrifuged at 1000 rpm for 4 minutes at room temperature, and the supernatant was removed. 1 mL of pre-prepared 2.5% glutaraldehyde fixative was added to each tube, and the cell pellet was fixed for 4 hours. After carefully removing the fixative, the cells were washed three times with 0.1 M PBS for 10 minutes each time. Then, the cells were fixed with 1% osmium tetroxide (2% osmium tetroxide was serially diluted with 0.2 M PBS) for 1.5 hours, adding 0.6 mL to each sample. The centrifuge tubes were gently shaken every 20 minutes to ensure thorough fixation of the sample. After carefully removing the osmium tetroxide fixative, the sample was rinsed three times with 0.1M pH 7.4 PBS for 10 minutes each time. Dehydration was then performed using a concentration gradient of 30%, 50%, 70%, 80%, 90%, and 100% ethanol for 10 minutes each, with three cycles of 100% ethanol dehydration. The sample was then replaced twice with acetone for 8 minutes each time. Following this, the sample underwent embedding and polymerization treatment, and ultrathin sections (Leica EMUC7) were double-stained with uranium acetate / lead citrate. The sections were observed and photographed under appropriate magnification using a transmission electron microscope (JEM-1400). The results are shown in the figure below. Figure 3 .

[0083] like Figure 3 As shown, treatment with compounds B18 and B20 significantly upregulated NCOA4 protein expression. Figure 3 A) suggests that it may mediate intracellular free iron metabolism by activating the ferritin autophagy pathway, thereby driving ferroptosis. To verify this mechanism, we detected characteristic changes in autophagy markers in the experimental group (B18 / B20) 24 hours after drug administration: accumulation of P62 protein and a significantly increased LC3-II / LC3-I ratio. Figure 3 A), which is consistent with the pathological characteristics of impaired autophagosome-lysosome fusion. Transmission electron microscopy (TEM) further confirmed that the experimental group cells contained a large number of typical double-membrane autophagosomes (200-500 nm in diameter), and their number was increased compared with the control group. Figure 3 B). Notably, the abnormal aggregation of autophagosomes in a state not fused with lysosomes indicates that compound intervention specifically inhibits autophagic flux during the degradation phase. This evidence suggests a dynamic equilibrium between NCOOA4-mediated ferritin autophagy activation and autophagic flux inhibition—the former releasing Fe through ferritin degradation. 2+ It promotes the Fenton response, which in turn exacerbates oxidative stress by blocking the clearance of damaged organelles. Together, these factors lead to the irreversible accumulation of lipid peroxidation (LPO), ultimately triggering the ferroptosis cascade.

[0084] Example 4

[0085] 1. Pharmacokinetic study of compound B18

[0086] Select 6 qualified healthy CD-1 mice, divide them into 2 groups averagely, 3 mice in each group, for intravenous and oral administration respectively, 2 mg / kg for intravenous administration, 10 mg / kg for oral administration. Collect whole blood about 0.25 mL from the orbital venous plexus of the rats at 0 h before administration, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h after administration, and place them in centrifuge tubes containing EDTA-K2 anticoagulant, and place them in crushed ice immediately after collection. Centrifuge within 0.5 h at 2000 g for 10 min, and divide all the plasma and place them in another clean centrifuge tube. Pay attention to the actual blood collection time of the samples within 2 h (including 2 h) within ± 1 min of the theoretical blood collection time, and the actual blood collection time of the samples after 2 h within ± 5 min of the theoretical blood collection time. The plasma samples are transported in an ice box. Use tolbutamide as an internal standard, analyze by LC-MS / MS-015 instrument, and calculate the following pharmacokinetic parameters: AUC(0-t), AUC(0-∞), T1 / 2, MRT(0-∞), Cmax, Tmax, F by Phoenix WinNonlin 7.0 software. The results are shown in Table 2.

[0087] Table 2 Pharmacokinetic parameters of compound B18

[0088]

[0089]

[0090] As shown in Table 2, B18 exhibits superior bioavailability characteristics, with a bioavailability of 99.67% ± 17.93%. In addition, the half-life of B18 is 3.34 ± 0.19 h, suggesting that B18 may have superior stability and persistent biological activity in vivo.

[0091] 2. In vivo pharmacodynamic study

[0092] Digest and centrifuge U87mg cells in the logarithmic growth phase, count, and dilute the cells with PBS to 5 × 10 6The cells were mixed with 1 mL of RPMI 1640 medium containing 10% fetal bovine serum per mL, and then placed on ice and transferred to the animal room for operation. The mice were inoculated subcutaneously in the armpit after alcohol disinfection. A 1 mL syringe was used to inject 200 μL of the cell suspension subcutaneously. The needle was moved left and right in the subcutaneous tissue to indicate that it was in the subcutaneous tissue. A small skin bump was visible in the subcutaneous tissue. The subcutaneous tumor formation of the nude mice was observed every other day, and the long diameter and short diameter of the tumor were measured using a ruler. The tumor volume = 1 / 2 x long diameter x short diameter x height. Appropriate amounts of B18, B20, 1D228, and TMZ were weighed and melted or suspended. The dosages of B18, B20, and 1D228 were 20 mg / kg, and the dosages were administered continuously for 17 days. The dosage of TMZ was 5 mg / kg, and the dosages were administered every other day. The dosages were administered by gavage. After the dosages were administered, the mice were sacrificed by dislocation, and the tumor tissue was peeled off. The stomach, liver, spleen, lung, kidney, heart, and other organs were dissected, and immunohistochemical verification and HE staining were performed.

[0093] Immunohistochemistry: (1) Embedding and sectioning: The fixed tissue was cut into appropriate size, first washed with running water for 3 h, then dehydrated in gradient ethanol solution: 70% ethanol for 2 h, 80% ethanol overnight, 90% ethanol for 2 h, and finally anhydrous ethanol I and II for 1 h each. After completing dehydration, the tissue was immersed in xylene solution for 30 min until transparent. Next, the tissue was placed in a paraffin-xylene mixture and permeated at 60°C for 2 h to ensure complete penetration of the paraffin into the tissue. The paraffin was poured into a metal mold to a depth of one-half, the tissue block was placed in the center, and the remaining paraffin was poured to completely wrap the tissue. After complete cooling, a continuous section with a thickness of 3 μm was prepared by a microtome. After the section was spread on warm water, it was transferred to a glass slide and placed in a 60°C oven for 24 h. (2) The paraffin section was placed in a 60°C oven for 2 h. The section was sequentially placed in xylene I-xylene II-100% alcohol-100% alcohol-95% alcohol-85% alcohol-75% alcohol-ddH2O for 10 min each. The section was immersed in a sodium citrate buffer (0.01 mol / L, pH 6.0), heated in a microwave oven at high heat for 5 min and then at low heat for 30 min, and then naturally cooled to room temperature. The section was washed with PBS for 3 times, 5 min each. A 3% H2O2 solution was added to the section, which was incubated in a wet box for 10 min, and then washed with PBS for 3 times, 5 min each. A 0.2% Triton X-100 solution prepared in PBS was added to the section, which was incubated at room temperature for 10 min, and then washed with PBS for 3 times, 5 min each. The tumor tissue was circled on the section with a histological pen, and 10% FBS (prepared in PBS) was added for blocking at 37°C for 30 min. The desired primary antibody (prepared in 5% BSA with a dilution ratio of 1:200) was added to the section in an amount of 200 μL, which was incubated at 4°C overnight. A freshly prepared secondary antibody was added to the section, which was incubated at room temperature for 1 h, and then washed with PBS for 3 times, 5 min each. DAB developing solution was added to the section, which was observed under a microscope for color change, and then the developing was terminated with running water. An appropriate amount of hematoxylin solution was added for staining for 5 min, washed with tap water for 10 min, and then 10% hydrochloric acid alcohol differentiation solution was added and left to stand for 10 min, and then washed with tap water for 10 min. After gradiently pouring the solution, one drop of neutral resin gel was added for mounting, which was left to dry at room temperature, observed under a microscope, and photographed.

[0094] HE staining: Paraffin sections were placed in an oven at 60°C for 2 h. The sections were taken out and dewaxed in xylene I and xylene II for 15 min each, and then washed in 100%, 95%, 85%, and 75% alcohol for 5 min each, and finally washed in distilled water. The sections were dyed in hematoxylin dye for 5 min, and the specific time was adjusted according to the concentration of the dye and the type of the tissue. After dyeing, the sections were washed with tap water to make them blue. The sections were differentiated in 1% hydrochloric acid alcohol for several seconds to 10 s, and the differentiation time was strictly controlled to avoid over-differentiation and cause the dyeing to be too light. The sections were washed in tap water or soaked in a weak alkaline solution such as ammonia water to make the sections return from red to blue. The sections were dyed in eosin dye for 2-5 min to make the cytoplasm and extracellular matrix red. The dyed sections were dehydrated in 75%, 85%, 95%, and 100% alcohol for 5 min each, and then dehydrated and transparentized in xylene I and xylene II for 12 min each. The sections were taken out of xylene, and an appropriate amount of neutral balsam was added dropwise, a cover glass was covered to avoid air bubbles, and the sections were naturally dried or oven dried, and the HE dyeing was completed.

[0095] After 17 days of continuous administration, the tumor volume of the B18 and B20 treatment groups was reduced by 68.3% and 62.7% respectively compared with the model group Figure 4 A-B), in order to verify its potential mechanism, we detected the related protein level of p-c-Met in the tumor tissue of mice, and compared with the control group, B18 significantly inhibited p-c-Met Figure 4 C). Histopathological evaluation (HE staining) confirmed that the compound had a certain safety Figure 4 D). Further mechanism research showed that B18 and B20 played a role by inhibiting the key markers of tumor cell proliferation Ki-67 and P-met Figure 4 E), and IHC quantification showed that the positive cell rate decreased from 72.4% of the model group to 28.6% of the treatment group. The above data show that B18 and B20 have excellent safety features while exerting strong anti-glioma effect, and their dual advantages provide key experimental evidence for subsequent clinical transformation.

[0096] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the examples, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A compound having c-Met inhibition and inducing ferroptosis, characterized in that, The structural formula is as follows: Wherein, R substituent group is triazole compound or alkyl alcohol with less than four carbon atoms.

2. The compound according to claim 1, characterized in that, The alkyl alcohol is iodoethanol.

3. The compound according to claim 1, characterized in that, The triazole compound is 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid.

4. The compound according to any one of claims 1 to 3, characterized in that, The structural formula of the compound is:

5. A method of synthesizing the compound according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) sodium carbonate, 5-fluoro-2-chloropyrimidine, (Ph3P)2PdCl2 and 3-acetyl phenyl boronic acid are added into a mixed solvent, protected by nitrogen, and heated to reflux for 10-12 hours to prepare a B1 compound; wherein the molar ratio of sodium carbonate, 5-fluoro-2-chloropyrimidine, (Ph3P)2PdCl2 and 3-acetyl phenyl boronic acid is 6-7:3-3.5:0.05-0.08:3-3.5; (2) the B1 compound and sodium hydride are added into a solvent, 4-(hydroxymethyl) piperidine-1-carboxylic acid tert-butyl ester solution is added dropwise under ice bath, and then reacted at room temperature for 3-5 hours to prepare a B2 compound; wherein the molar ratio of the B1 compound, sodium hydride and 4-(hydroxymethyl) piperidine-1-carboxylic acid tert-butyl ester is 1-1.5:1.5-2:1-1.5; (3) a chiral ligand and dichlorobis(4-methylisopropylphenyl) ruthenium are added into water, and after reaction for 5-8 hours, cooled to room temperature, sodium formate solution and B2 compound solution are added in sequence under ice bath, and reacted at room temperature for 10-12 hours to prepare a B3 compound; wherein the molar ratio of the chiral ligand, dichlorobis(4-methylisopropylphenyl) ruthenium, sodium formate and B2 compound is 0.1-0.2:0.01-0.05:2-2.5:0.1-0.5; (4) the B3 compound, 3-(6-carbonyl-1, 6-dihydro-3-pyridazinyl) benzocyanine and triphenylphosphine are added into a solvent, protected by nitrogen, drop DIAD under ice bath, and reacted at room temperature for 10-12 hours to prepare a B4 compound; wherein the molar ratio of the B3 compound, 3-(6-carbonyl-1, 6-dihydro-3-pyridazinyl) benzocyanine, triphenylphosphine and DIAD is 0.1-0.5:0.1-0.5:0.5-1:0.5-1; (5) under ice bath, trifluoroacetic acid is added into the B4 compound solution, and reacted at room temperature for 5-8 hours to prepare a B5 compound; (6) the B5 compound is reacted with iodoethanol or 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid to prepare the compound for inducing ferroptosis.

6. The method of claim 5, wherein, The specific process of the reaction of the B5 compound with iodoethanol in step (6) is as follows: The B5 compound, iodoethanol and potassium carbonate are mixed and dissolved, and then reacted at 80-95 DEG C for 10-12 hours to prepare the compound for inducing ferroptosis; wherein the molar ratio of the B5 compound, iodoethanol and potassium carbonate is 0.01-0.05:0.01-0.05:0.1-0.

5.

7. The method of claim 5, wherein, The specific process of the reaction of the B5 compound with 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid in step (6) is as follows: The B5 compound, HATU, triethylamine and 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid are mixed and dissolved, and then the mixture is reacted at room temperature for 12-16 hours to obtain the ferroptosis-inducing compound; wherein the molar ratio of the B5 compound, HATU, triethylamine and 4-methyl-4H-1, 2, 4-triazole-3-carboxylic acid is 0.01-0.05:0.1-0.5:0.1-0.5:0.01-0.

05.

8. An inducer of ferroptosis, characterized in that, A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, as an active ingredient.

9. A formulation for enhancing sensitivity of tumor cells, characterized by, A compound according to any one of claims 1 to 4.

10. Use of a compound according to any one of claims 1 to 4, a ferroptosis-inducing agent according to claim 8 or a preparation according to claim 9 for the manufacture of a preparation for the treatment of glioblastoma.

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