Small molecule compounds targeting egfr and uses thereof

Abstract

The present application belongs to the technical field of tumor targeted therapy, and particularly relates to a small-molecule compound targeting EGFR and application thereof. The present application provides a small-molecule compound targeting EGFR or a pharmaceutically acceptable salt thereof. The spiro alkaloid compound provided by the present application contains an arylated four-membered stereocenter heterocyclic framework, including a helical heterocycle, a distributed heterocycle, a fused heterocycle and a bridged heterocycle, which can be applied to preparation of an EGFR-targeted inhibitory drug.

Description

Technical Field

[0001] This invention belongs to the field of tumor targeted therapy technology, specifically involving small molecule compounds that target EGFR and their applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, often accompanied by metastasis and recurrence, treatment failure, and poor prognosis. Clinically, most HCCs are already in advanced stages, and treatment options are limited at the time of initial diagnosis. In recent years, significant advances in molecular targeted therapy and immunotherapy have brought new hope to patients with advanced HCC. Among these, sorafenib, lenvatinib, and the combination of azolamb / bevacizumab have been approved as first-line treatments. Lenvatinib is the second authorized first-line drug for advanced HCC after sorafenib. It is an oral multi-target tyrosine kinase receptor inhibitor that inhibits VEGFR 1 / 2 / 3, FGFR 1 / 2 / 3 / 4, KIT, and RET. Studies have reported that lenvatinib treatment activates the JAK-STAT and EGFR-pak2-erk5 signaling axes, leading to drug resistance in patients. Therefore, new treatment strategies are urgently needed to overcome drug resistance.

[0003] EGFR, also known as ERBB1 or HER1, belongs to a subfamily of RTKs. EGFR homoligands include EGF and TGFα, which induce EGFR homodimerization or heterodimerization, leading to phosphorylation of intracellular tyrosine residues, activating downstream pathways (such as RAS-RAF-MEK-ERK, JAK-STAT, and PI3K-AKT pathways), and regulating various biological processes, such as cell proliferation, anti-apoptosis, metastasis, and metabolism. EGFR dysregulation promotes the occurrence and development of various tumors. EGFR overexpression occurs in 40-70% of human hepatocellular carcinomas and is associated with tumor development and progression. In clinical trials, erlotinib showed moderate efficacy in phase II treatment, while gefitinib and cetuximab provided only poor results in patients with advanced HCC. Furthermore, a phase III study of erlotinib did not demonstrate therapeutic efficacy in patients with advanced HCC. Studies have reported that the use of EGFR inhibitors can lead to varying degrees of resistance, such as secondary mutations in EGFR, activation of the NF-κB and JAK-STAT pathways, etc. Therefore, a better understanding of the mechanisms by which the EGFR signaling pathway affects HCC progression is needed.

[0004] The JAK / STAT pathway can be activated by a variety of cytokines and growth factors, such as interleukins, interferons, and members of the EGF family, which bind to their respective transmembrane receptors. This kinase is activated upon ligand-induced conformational changes in the receptor, thereby regulating the expression of downstream signaling molecules. Numerous studies have shown that the JAK / STAT pathway is frequently deregulated in cancers, including hepatocellular carcinoma (HCC). In fact, aberrant JAK-STAT activation has been reported in up to 60% of HCC cases. Since JAK acts upstream of STATs, it can serve as a viable target for inhibiting downstream effects of the JAK / STAT pathway. Activation of the JAK / STAT signaling pathway is a widely reported phenomenon in many cancers, including HCC. The various cellular effects of the JAK / STAT signaling pathway and its relationships with other signaling pathways have been shown to contribute to many key markers of cancer development and progression. Therefore, inhibiting the activated JAK / STAT pathway is a novel therapeutic strategy. Indeed, numerous clinical trials and studies on JAK / STAT inhibitors have demonstrated the potential and efficacy of these compounds in alleviating various cancers. However, in the case of HCC, clinical research on these compounds is still in its early stages, with limited observed benefits. Summary of the Invention

[0005] This invention provides a spirocyclic alkaloid compound.

[0006] This invention provides applications of the above-mentioned compounds.

[0007] According to specific embodiments of the present invention, the spirocyclic alkaloid compound is selected from formula I or a pharmaceutically acceptable salt thereof.

[0008]

[0009] The compounds of the present invention are heterocyclic frameworks containing arylated quaternary stereocenters, including helical heterocycles, distributed heterocycles, fused heterocycles, and bridging heterocycles.

[0010] This invention provides the application of the above-mentioned compound in the preparation of a drug for treating cancer, preferably liver cancer.

[0011] Preferably, the present invention provides the use of the above-mentioned compound in the preparation of a medicament for treating liver cancer by targeting and inhibiting EGFR.

[0012] The present invention also provides a pharmaceutical composition comprising the above-described compounds and a pharmaceutically acceptable carrier.

[0013] The present invention also provides a pharmaceutical composition for treating cancer, comprising the above-described compound and a pharmaceutically acceptable carrier. The cancers include non-small cell lung cancer, breast cancer, head and neck cancer, colon cancer, ovarian cancer, and liver cancer. Preferably, the cancer is liver cancer.

[0014] EGFR is a transmembrane glycoprotein belonging to the ErbB family of RTKs. Upon binding to a ligand, EGFR is activated, leading to the excitation of subsequent intracellular signaling pathways, such as EGFR-PI3K-Akt, EGFR-MAPK, and EGFR-JAK-STAT. These pathways are involved in the proliferation, differentiation, migration, and apoptosis of certain cells. High expression of EGFR and overactivation of its signaling pathways have been detected in various malignant tumors, including non-small cell lung cancer, breast cancer, head and neck cancer, colon cancer, ovarian cancer, and liver cancer. To mitigate the impact of the EGFR pathway on cancer, this invention provides an EGFR inhibitor that specifically binds to and inhibits the activity of the EGFR tyrosine kinase domain.

[0015] An EGFR-targeting inhibitory drug according to a specific embodiment of the present invention comprises the above-described compound and a pharmaceutically acceptable carrier.

[0016] The EGFR signaling pathway is also a crucial molecule in initiating and determining clinical outcomes in many respiratory diseases. Dysregulation of the EGFR pathway leads to abnormal EGFR signaling and is associated with the early pathogenesis of pulmonary fibrosis, cancer, and various airway hypersecretion diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis.

[0017] Mucopolysaccharidosis (MPS) is a group of inherited metabolic disorders belonging to the lysosomal storage diseases, caused by a deficiency of lysosomal enzymes required for the metabolism of glycosaminoglycans (GAGs). Specific activation of EGFR and increased phosphorylation of downstream extracellular signal-regulated kinase ERK lead to lysosomal aberrations and hypertrophy.

[0018] Chronic kidney disease (CKD) is characterized by persistent inflammation and progressive fibrosis, ultimately leading to end-stage renal disease. Although numerous studies have investigated the factors involved in the progressive deterioration of kidney function, current treatment strategies only slow disease progression and do not meet the need for effective therapeutic interventions that target the underlying causes of the inflammatory process, interventions that could slow or reverse the development and progression of CKD. EGFR, a membrane tyrosine kinase receptor expressed in the kidneys and activated after kidney injury, has demonstrated its potential as a therapeutic target for CKD in preclinical studies.

[0019] EGFR activity is crucial for normal cardiac development. In blood vessels, endothelial cells and smooth muscle cells serve as both sources and targets of EGF-like ligands. EGFR activation is associated with blood pressure regulation, endothelial dysfunction, neointimal hyperplasia, atherosclerosis, and cardiac remodeling. Furthermore, increased circulating EGF-like ligands may mediate accelerated vascular disease associated with chronic inflammation.

[0020] Viruses are intracellular pathogens that utilize host cell mechanisms for reproduction. Extensive research into virus-host interactions has revealed an alternative antiviral strategy targeting host cytokines. Epidermal growth factor receptor (EGFR) is a multifunctional signal transducer involved in a range of cellular processes. Numerous studies have revealed how viruses utilize EGFR function at different stages of their life cycle. Generally, viruses attach to the surface of host cells and interact with EGFR to facilitate viral entry, replication, and spread, as well as evade host immune surveillance. Furthermore, virus-induced EGFR signaling activation is associated with mucin expression, tissue damage, and carcinogenicity, leading to serious complications.

[0021] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that primarily affects arthritic joints. It is characterized by synovitis, synovial tissue hyperplasia, cartilage and bone destruction, and ultimately, physical disability. In inflammatory RA, activated EGFR / JAK / STAT confers resistance to synovial cell apoptosis, leading to a significant increase in synovial cell numbers. Furthermore, EGFR / JAK / STAT has been shown to exert its anti-apoptotic effect through translational regulation. The binding of the ligand to its receptor induces JAK phosphorylation, which in turn promotes STAT phosphorylation, regulating the transcription of pro-inflammatory cytokines and chemokines, and directly contributing to tissue damage in RA. Therefore, the crucial role of JAK / STAT pathway activation in RA has been further established.

[0022] Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease characterized by demyelination, axonal destruction, and progressive neurological dysfunction of the central nervous system (brain, spinal cord, and optic nerves). The EGFR / JAK / STAT pathway plays a crucial role in the pathogenesis of MS / EAE (an experimental autoimmune encephalomyelitis, an animal model of MS). Preclinical studies in animal models have shown that inhibiting the EGFR / JAK / STAT pathway disrupts neuroinflammatory and neurodegenerative processes. The EGFR / JAK / STAT signaling pathway plays a vital role in the pathogenesis of MS.

[0023] Inflammatory bowel disease (IBD) is a chronic or relapsing inflammatory disease of the gastrointestinal tract characterized by dysregulation of intestinal homeostasis, marked by uncontrolled inflammation and abnormal activation of the mucosal immune system. Tofacitinib, a JAK inhibitor, was recently approved by the FDA for the treatment of adults with another chronic non-intestinal inflammatory disease, namely moderate to severe rheumatoid arthritis. It has shown promising results in the treatment of IBD, particularly ulcerative colitis, suggesting that inhibiting the EGFR / JAK / STAT pathway is an innovative therapeutic approach for IBD.

[0024] Sepsis is a systemic inflammatory response syndrome (SIRS) caused by severe, life-threatening infections or organ dysfunction. The JAK / STAT pathway is an important pathway for many key cytokines in the pathogenesis of sepsis.

[0025] The EGFR / JAK / STAT signaling pathway is associated with more than a dozen human tumors and cancers, including myeloproliferative neoplasms (MPN), cutaneous T-cell lymphoma (CTCL), lung cancer, gastric cancer, prostate cancer, and colon cancer. The JAK / STAT signaling pathway has been reported to be activated in various solid tumors and is often considered a novel therapeutic target for various cancer types. The JAK / STAT signaling pathway is widely present in various tissues and cells, and its overactivation is closely related to the occurrence, progression, invasion, and metastasis of various tumors.

[0026] The above-mentioned drugs can be prepared into oral preparations or injections; the oral preparations include, but are not limited to, capsules, tablets, granules, and oral liquids; the injections include, but are not limited to, sterile powders for injection, aqueous injection solutions, and intravenous infusions of sodium chloride or glucose.

[0027] In the aforementioned drugs, the oral formulation includes additives, which are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0028] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0029] The lubricant, flow aid, or anti-sticking agent includes stearic acid; metal stearate salts such as calcium stearate or magnesium stearate; talc; colloidal silica; micronized silica gel; hydrogenated vegetable oil; polyethylene glycol; lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicic anhydride or silicate hydrates, etc.

[0030] The adhesive includes distilled water, ethanol of different concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the excipients described above.

[0031] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or croscarmellose sodium; croscarmellose; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0032] The surfactants mentioned include Tween-80, sodium dodecyl sulfate, sodium stearate sulfonate, etc.

[0033] The antioxidants mentioned include sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0034] The preservatives or antibacterial agents mentioned include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens, benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethanol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil, etc.

[0035] The flavoring agents include sweeteners such as sodium saccharin, aspartame, syrup, steviol, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; acid flavoring agents such as citric acid, malic acid or tartaric acid; and flavoring agents such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and various flavorings.

[0036] The method of using the above-mentioned drugs includes administering an effective amount of the drug to the subject. The administration route may be oral, intravenous injection, or transdermal penetration, applied to the patient requiring treatment.

[0037] A pharmaceutically effective dose refers to the amount of medication sufficient to treat a disease with a reasonable benefit / risk ratio. The level of the effective dose can be determined based on several factors, including the patient's disease type and severity, the drug's activity, drug sensitivity, timing of administration, route of administration, excretion rate, treatment duration, concurrent medications, and other factors known in the pharmaceutical field. The drugs of this invention can be administered as standalone therapeutic agents or in combination with other therapeutic agents. Furthermore, the compositions of this invention can be added continuously or simultaneously to typical therapeutic agents, and the compositions can be administered single or multiple times. It is important to consider all the above factors and administer the minimum dose that produces the maximum effect without side effects, which can be determined by the physician based on the patient's condition, age, etc.

[0038] This invention also provides a method for preparing the above-mentioned compound:

[0039]

[0040] The preparation method of compound I according to a specific embodiment of the present invention includes the following steps:

[0041] (1) Using 2-methoxy-p-benzoquinone and 3-substituted indigo derivatives as reactants, and chiral β-ICD as catalyst, an asymmetric Michael addition reaction occurs in an organic solvent to obtain an intermediate.

[0042] (2) Using methanol as a solvent, under acidic conditions, the intermediate reacts with methanol to undergo an acetalization reaction to obtain compound I.

[0043] The beneficial effects of this invention are:

[0044] (1) This invention provides a method for preparing a spirocyclic alkaloid compound with antitumor activity.

[0045] (2) The spirocyclic alkaloid compound I provided by this invention can be applied as an EGFR-targeting inhibitor for treating liver cancer, as shown in the following specific manifestations:

[0046] a. Compound I of the present invention has a significant inhibitory effect on liver cancer cells in vitro and in vivo.

[0047] b. Compound I of the present invention can directly bind to EGFR and inhibit its activity.

[0048] c. The anti-hepatocellular carcinoma effect of compound I of the present invention is dependent on EGFR. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 Compound I inhibits the proliferation of various hepatocellular carcinoma cells in vitro. (Left, Middle) Proliferation curves of hepatocellular carcinoma cells after treatment with compound I (using Huh7 and HepG2 cells, 72h as examples); (Right) Proliferation curves of normal cells after treatment with compound I.

[0051] Figure 2 This shows the CETSA binding analysis of compound I with cellular EGFR protein molecules. (Left) Western blot analysis of EGFR protein expression; (Right) Gray-scale analysis of relative EGFR protein expression, with β-tubulin protein gray-scale value as an internal control.

[0052] Figure 3 This shows the SPR binding analysis of compound I with the EGFR protein molecule.

[0053] Figure 4 The hepatocellular carcinoma inhibitory activity of compound I is shown to be dependent on EGFR expression. (Top) SRB assay analysis of the cytotoxicity of compound I on Huh7 cells after 72 hours of EGFR overexpression. (Bottom) SRB assay analysis of the cytotoxicity of compound I on Huh7 cells after EGFR knockout after 72 hours of EGFR knockout.

[0054] Figure 5 This demonstrates the therapeutic effect of compound I on liver cancer in vivo. (A) Tumor tissue image; (B) Tumor growth curve; (C) Tumor weight; (D) Body weight curve. *p<0.05, **p<0.01, no significant difference in ns compared to the control group. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0056] Experimental materials involved in the embodiments of this invention:

[0057] Fetal bovine serum was purchased from Yisheng Biotechnology Co., Ltd.

[0058] MEM culture medium was purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0059] DMEM and RPMI-1640 media were purchased from Gino Biopharmaceutical Technology Co., Ltd.

[0060] HepG2 and Huh7 cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0061] Huvec, L02, and Beas-2b were purchased from the Shanghai Cell Bank;

[0062] BCA protein quantification kit (Beyotime), ECL chemiluminescence detection kit, protein-free rapid blocking solution (Yamei), and HRP-labeled secondary antibody (Yamei) were purchased from Qingdao Aifit Biotechnology Co., Ltd.

[0063] Balb / c nude mice; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0064] EGFR WT recombinant plasmid was purchased from Addgene; si-EGFR was purchased from Shanghai Gemma Gene.

[0065] Human EGFR monoclonal antibody, Nickel-Beads nickel magnetic agarose globin purification kit (Beaver), and PMSF were purchased from Shanghai Unimicron Biotechnology Co., Ltd.

[0066] Example 1: Synthesis of Active Spirocyclic Alkaloid Compound I

[0067]

[0068] In a reaction flask, 4-methoxy-substituted indigo derivative B (114.4 mg, 0.3 mmol), β-ICD (1 mg, 0.003 mmol), and dichloromethane (2 mL) were added sequentially. The mixture was stirred at -40 °C for 10 minutes, followed by the addition of 2-methoxy-p-benzoquinone A (41.4 mg, 0.3 mmol), and the reaction was continued at -40 °C for 5 hours. TCL detection showed that starting material B had disappeared. The mixture was then transferred to room temperature, and dichloromethane was removed by vacuum concentration to obtain crude product C. C was redissolved in methanol (2 mL), and p-toluenesulfonic acid (5.1 mg, 0.03 mmol) was added. The mixture was stirred at room temperature for 3 hours. TCL detection showed that intermediate C had disappeared. After vacuum concentration, the mixture was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1) to obtain a white solid D, namely compound I (93.7 mg, yield 59%, 97% ee, >20:1 dr).

[0069] 1H NMR (400MHz, CDCl3) δ7.92(d,J=8.2Hz,1H),7.71(d,J=7.5Hz,1H),7.53(d,J=8.6Hz,2H),7.31(t,J=7.9Hz,1H),7.15(t,J=7.6Hz,1H),6.91(d,J=8.6H z,2H),6.63(s,1H),6.13(s,1H),5.19(s,1H),3.87(s,3H),3.83(s,3H),3. 13(s,3H),2.71(d,J=14.3Hz,1H),2.43(d,J=14.3Hz,1H),1.61(s,9H)ppm.

[0070] 13 C NMR (100MHz, CDCl3) δ178.4,159.8,149.4,147.2,145.1,140.8,139.2,134.9,132.4,128.4,127.7,1 27.1,125.1,115.4,114.7,113.8,112.6,101.6,100.0,84.6,56.2,55.4,50.4,50.0,45.4,28.2ppm.

[0071] HRMS: [M+Na] + calcd for C 30 H 31 NNaO8 + 556.1942, found: 556.1943. [α] D 20 +15.92 (c=0.68in CHCl3).

[0072] HPLC analysis: DAICEL CHIRALPAK IA, n-hexane / isopropanol = 90 / 10, 1.0 mL / min, λ = 210 nm, t major =10.75min,t minor =11.95min, ee=97%.

[0073] Example 2

[0074] 1. Detection of liver cancer cell proliferation rate using the SRB method

[0075] (1) HepG2, Huh7, L02, Huvec and Beas-2b cells were digested with trypsin and resuspended in culture medium to form a single-cell suspension.

[0076] (2) After counting, the cells were seeded into 96-well plates with a single well volume of 90 μL and a cell density of 4 × 10⁻⁶ cells / well.3 / well, placed in a 37℃, 5% CO2 incubator, and incubated until completely adhered to the wall.

[0077] (3) Compound I at different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.31, 0.15 μmol / L) was added to the compound group, and DMSO diluted proportionally was added to the blank control group, 10 μL / well, and the reaction time was 72 h.

[0078] (4) OD detection by SRB method 515 value:

[0079] a. Discard the culture medium in the 96-well plate, add 10% pre-cooled TCA (100 μL per well) to fix the cells, and place in a 4°C refrigerator for more than 1 hour.

[0080] b. Wash with ddH2O more than 5 times and let it air dry at room temperature.

[0081] c. Add 100 μL of 4 mg / mL SRB solution to each well, stain for 15 min at room temperature in the dark, and then discard the SRB.

[0082] d. Wash with 1% glacial acetic acid more than 5 times, and let it air dry at room temperature.

[0083] e. Add 150 μL of 10 mmol / L Tris-base solution to each well until the stain is completely dissolved.

[0084] (5) Place the well plate at 515nm on the microplate reader to measure the OD value.

[0085] Calculate cell proliferation rate using the following formula:

[0086] Cell proliferation inhibition rate (%) = (OD) 对照组 -OD 给药组 ) / OD 对照组 ×100%.

[0087] (6) Plot the graph using GraphPad and calculate the half-maximal inhibitory concentration (IC50). 50 .

[0088] The results are as follows Figure 1 As shown, this result indicates that compound I can inhibit the proliferation of Huh7 and HepG2 cells in a time- and dose-dependent manner, with an IC50 value of 72 h. 50The concentrations were 0.3 μM and 1.5 μM, respectively. In normal cells, the inhibition rates at 20 μM were 49.83% for Huvec, 34.74% for Beas-2b, and 21.08% for L02. This indicates that compound I exhibits strong inhibitory activity against liver cancer cells but weaker inhibitory activity against normal cells, suggesting that compound I not only possesses anticancer effects but also exhibits a certain degree of safety.

[0089] 2. CETSA detection of the binding of compound I to EGFR molecules.

[0090] Seeding plates: Select Huh7 cells in good condition, digest the cells with trypsin and count them, at a density of 3 × 10⁻⁶. 5 10,000 cells per well were seeded in 6-well plates and placed in an incubator to allow the cells to adhere.

[0091] Drug addition: A solvent control group and a drug addition group were set up. The solvent control group was given DMSO, and the drug addition group was given compound I to a final concentration of 4 μM. The groups were incubated in an incubator for 3 h.

[0092] Cell collection: Discard the pipette tip after aspirating the culture medium. Next, wash the cells twice with PBS, digest the cells with trypsin, add culture medium to stop the digestion, centrifuge at 1200 rpm for 5 min. After centrifugation, resuspend the cells in PBS, repeat the washing process twice, and then resuspend the cells in PBS again. Divide each group into 4 PCR tubes.

[0093] Gradient heating: Place the PCR tubes in the PCR instrument for gradient heating, with temperatures set to 38.4, 40.6, 47.6 and 52.1℃ respectively.

[0094] Cell lysis: After the gradient heating is completed, the cell suspension is transferred to a 1.5 ml EP tube and placed in liquid nitrogen for lysis. The lysis is repeated 3 to 4 times, 3 min each time.

[0095] Sample collection: The lysed cells were placed in a refrigerated centrifuge at 4°C, centrifuged at 20000×g for 20 min, the supernatant was collected and resuspended in Loading Buffer, boiled in boiling water for 15 min, and stored at -20°C for later use.

[0096] The interaction between compound I and the protein can help EGFR have greater stability in high-temperature environments. By comparing the degradation of EGFR protein in the DMSO group and the compound-treated group as the temperature increases, it can be determined whether there is an interaction between compound I and EGFR.

[0097] The results are as follows Figure 2 As shown, the addition of compound I delayed the degradation of EGFR caused by temperature rise, indicating that compound I can bind to the EGFR protein in cells.

[0098] 3. SPR technology for detecting the binding of compound I to EGFR molecules

[0099] The binding of compound I to the EGFR protein was analyzed using ion surface resonance (SPR) technology.

[0100] Compound I was coupled to the surface of the biochip implant using the SpotBot3 microarray control software in the SpotBot3 needle platform, and the compound on the chip surface was cross-linked with ultraviolet light for 15 minutes. EGFR protein was diluted with PBS to 50, 100 nM, 200 nM, and 400 nM and then injected for detection.

[0101] The obtained data was analyzed and fitted using the PLEXERASPR Data Analysis Module (DAM) software to obtain the dissociation constant (KD).

[0102] The results are as follows Figure 3 As shown, compound I can bind to EGFR with a KD value of 0.133 μM, indicating moderate binding strength.

[0103] 4. Effects of EGFR overexpression or knockout on the proliferation of Huh7 cells by detecting the effects of compound D.

[0104] (1) Transfection was performed when the Huh7 cells reached about 70% confluence.

[0105] (2) Prepare two EP tubes for each group. Add 3.75 μL of transfection reagent to 125 μL of serum-free and antibiotic-free medium in one tube. Add 10 μL of EGFR interference fragment (EGFR interference fragment sequence 5'-GAGGCAAAGUGCCUAUCAATT-3'; NC sequence 5'-UUCUCCGAACGUGUCACGUTT-3') or 6 μg of plasmid (this plasmid contains the EGFR fragment; the plasmid was purchased from Addgene, catalog number 11011) to 125 μL of serum-free and antibiotic-free medium in the other tube. Mix gently and let stand.

[0106] (3) Add the culture medium containing the interfering fragment to the culture medium containing the transfection reagent, mix gently, and let stand for 15 minutes.

[0107] (4) Replace the culture medium in the six-well plate with 750 μL of serum-free and antibiotic-free culture medium.

[0108] (5) After 6 hours, each well was replaced with 2 mL of complete culture medium. 48 hours after transfection, a portion of the cells were collected to assess transfection efficiency, while the remaining cells were re-seeded and counted. The cells were seeded in 96-well plates, and after cell adhesion, compound I was added to assess its effect on Huh7 cell proliferation.

[0109] The results are as follows Figure 4 High expression of EGFR enhanced the proliferative capacity of compound I in liver cancer cells, while knockdown of EGFR weakened the cytotoxicity of compound I against liver cancer cells. This indicates that EGFR plays a crucial role in the anticancer activity of compound I. In conclusion, compound I exerts its anticancer function by directly targeting EGFR.

[0110] 5. Analysis of the in vivo therapeutic effect of compound I on liver cancer

[0111] 1. Nude mouse Huh7 cell xenograft tumor model

[0112] (1) Huh7 cells were subcutaneously inoculated into 6-week-old Balb / c-Nude mice (3×10⁻⁶ cells per cell line). 6 Each animal was monitored for tumor growth; when the tumor volume reached 100 mm... 3 Treatment should begin at that time.

[0113] (2) The treatment group received compound I (two doses, once daily), and the control group received physiological saline (solvent). The administration lasted for 12 days.

[0114] (3) Analysis of treatment effect:

[0115] a. Monitor changes in tumor volume and body weight. The volume is calculated using the following formula:

[0116] Tumor volume = major diameter × minor diameter 2 ×1 / 2

[0117] b. At the termination time, sacrifice the mice and dissect them to obtain tumor volume and weigh it.

[0118] c. Tumor volume and tumor weight directly reflect the in vivo therapeutic effect of compound I. The tumor inhibition rate is calculated using the following formula:

[0119]

[0120] Changes in animal weight and organ indices can indicate whether there are obvious toxic side effects, thus demonstrating its safety.

[0121] The results are as follows Figure 5 As shown, compound I significantly inhibited the growth of Huh7 xenograft tumors, with an inhibition rate of 64% at 50 mg / kg and 47.7% at 25 mg / kg, and a significant reduction in tumor weight. Meanwhile, the body weight of the treated mice was not different from that of the control group, indicating that the treatment at this dose was relatively safe.

[0122] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of a compound in the preparation of a medicament for treating liver cancer by targeting and inhibiting EGFR, said compound being selected from formula I or a pharmaceutically acceptable salt thereof. Formula I.

2. The application according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier.

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