EGFR (epidermal growth factor receptor) targeting small molecular compound and application thereof

Abstract

The invention belongs to the technical field of tumor targeted therapy, and particularly relates to a targeted EGFR small molecule compound and application thereof. The present invention provides a small molecule compound targeting EGFR or a pharmaceutically acceptable salt thereof. The spiro alkaloid compound provided by the invention contains a heterocyclic framework with an arylated quaternary stereo center, comprises a helical heterocyclic ring, a distributed heterocyclic ring, a fused heterocyclic ring and a bridged heterocyclic ring, and can be applied to preparation of EGFR (epidermal growth factor receptor) targeted inhibition drugs # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeted therapy, and specifically relates to small molecule compounds targeting EGFR and applications thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, often accompanied by metastasis, recurrence, treatment failure, and poor prognosis. Clinically, most HCCs are already in the advanced stage, and when the initial diagnosis is made, treatment options are limited. In recent years, a large number of research advances in molecular targeted therapy and immunotherapy have brought new hope to patients with advanced HCC. Among them, sorafenib, lenvatinib, and azotocin / bevacizumab combination 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, so new treatment strategies are urgently needed to overcome drug resistance.

[0003] EGFR, also known as ERBB1 or HER1, belongs to the subfamily of RTKs. The cognate ligands of EGFR include EGF and TGFα, which induce EGFR homodimerization or heterodimerization, leading to the phosphorylation of intracellular tyrosine residues to activate downstream pathways (such as RAS-RAF-MEK-ERK, JAK-STAT and PI3K-AKT pathways) and regulate various biological processes such as cell proliferation, anti-apoptosis, metastasis and metabolism. Disorders of EGFR promote the occurrence and development of a variety of tumors. EGFR overexpression occurs in 40-70% of human hepatocellular carcinomas and is associated with the occurrence and development of tumors. In clinical trials, erlotinib showed moderate effects in phase II treatment, while gefitinib and cetuximab provided only poor results in patients with advanced HCC. In addition, the phase III study of erlotinib did not show therapeutic effects in patients with advanced HCC. Some studies have reported that the use of EGFR inhibitors can lead to different degrees of drug resistance, such as secondary mutations of EGFR, activation of NF-kB and JAK-STAT pathways, etc. Therefore, a better understanding of the mechanisms by which EGFR signaling 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. The kinase is activated upon ligand-induced conformational changes in the receptor, thereby regulating the expression of downstream signaling molecules. Many studies have shown that the JAK / STAT pathway is frequently deregulated in cancer, including HCC. In fact, it has been reported that JAK-STAT is abnormally activated in up to 60% of HCC cases. Since JAK functions upstream of STATs in STATs, it can serve as a viable target for inhibiting the downstream effects of the JAK / STAT pathway. Activation of the JAK / STAT signaling pathway is a well-reported phenomenon in many cancers, including HCC. The diverse cellular effects of the JAK / STAT signaling pathway and its relationship with other signaling pathways have been shown to contribute to many key hallmarks of cancer development and progression. Therefore, inhibition of the activated JAK / STAT pathway is a novel therapeutic strategy. In fact, a large number of clinical trials and studies on JAK / STAT inhibitors have demonstrated the potential and effectiveness of these compounds in alleviating various cancers. However, in the case of HCC, clinical studies on these compounds are still in the early stages, and limited benefits have been observed. Summary of the invention

[0005] The invention provides a spiro alkaloid compound.

[0006] The present invention provides the use of the above compound.

[0007] According to a specific embodiment of the present invention, the spiroalkaloid compound is selected from Formula I or a pharmaceutically acceptable salt thereof.

[0008]

[0009] The compound of the present invention is a heterocyclic framework containing an aromatic quaternary stereocenter, including a spiral heterocycle, a distributed heterocycle, a fused heterocycle and a bridged heterocycle.

[0010] The present invention provides the use of the above compound in preparing a drug for treating cancer. Preferably, the cancer is liver cancer.

[0011] Preferably, the present invention provides the use of the above-mentioned compound in the preparation of a drug for treating liver cancer by targeted inhibition of EGFR.

[0012] The present invention also provides a pharmaceutical composition, which comprises the above compound and a pharmaceutically acceptable carrier.

[0013] The present invention also provides a pharmaceutical composition for treating cancer, comprising the above compound and a pharmaceutically acceptable carrier. The cancer includes 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 RTK. After binding to a ligand, EGFR is activated and leads to subsequent excitement of intracellular signaling pathways, such as EGFR-PI3K-Akt, EGFR-MAPK and EGFR-JAK-STAT. They are involved in the proliferation, differentiation, migration and apoptosis of certain cells. High expression of EGFR and overactivation of 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, liver cancer and other tumors. In order to reduce the impact of the EGFR pathway on cancer, the present invention provides an EGFR inhibitor that specifically binds to the EGFR tyrosine kinase domain and inhibits its activity.

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

[0016] The EGFR signaling pathway is also an important molecule in the initiation and clinical outcome of many respiratory diseases. Dysregulation of the EGFR pathway leads to abnormal EGFR signaling, which is associated with the early pathogenesis of pulmonary fibrosis, cancer, and a variety of airway hypersecretion diseases (including chronic obstructive pulmonary disease, asthma, and cystic fibrosis).

[0017] Mucopolysaccharidosis (MPS) is a group of inherited metabolic diseases that belong to lysosomal storage diseases and are caused by the deficiency of lysosomal enzymes required for the metabolism of glycosaminoglycans (GAGs). The specific activation of EGFR and the increased phosphorylation level of the downstream extracellular signal-regulated kinase ERK lead to lysosomal distortion and hypertrophy.

[0018] Chronic kidney disease (CKD) is characterized by persistent inflammation and progressive fibrosis, ultimately leading to end-stage renal disease. Although many studies have investigated the factors involved in the progressive deterioration of renal function, current treatment strategies can only delay disease progression, without meeting the need for effective therapeutic interventions targeting the causes behind the inflammatory process that could slow or reverse the development and progression of CKD. EGFR is a membrane tyrosine kinase receptor expressed in the kidney that is activated following renal injury, and preclinical studies have demonstrated its potential as a therapeutic target for CKD.

[0019] EGFR activity is essential for normal cardiac development, and in vasculature, endothelial and smooth muscle cells are both sources and targets of EGF-like ligands. Activation of EGFR has been implicated in blood pressure regulation, endothelial dysfunction, neointimal hyperplasia, atherosclerosis, and cardiac remodeling. Furthermore, increases in circulating EGF-like ligands may mediate accelerated vascular disease associated with chronic inflammation.

[0020] Viruses are intracellular pathogens that exploit host cell machinery for their propagation. Extensive studies on virus-host interactions have revealed an alternative antiviral strategy targeting host cellular factors. The epidermal growth factor receptor (EGFR) is a multifunctional signal transducer involved in a range of cellular processes. Numerous studies have revealed how viruses exploit the functions of EGFR at different stages of the viral life cycle. In general, viruses attach to the host cell surface and interact with EGFR to facilitate viral entry, viral replication and spread, and evasion of host immune surveillance. In addition, virus-induced activation of EGFR signaling has been associated with mucin expression, tissue damage, and carcinogenesis, leading to serious complications.

[0021] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that primarily affects articular dialysis joints and is characterized by synovitis, synovial tissue hyperplasia, cartilage and bone destruction, and eventual physical disability. In inflammatory RA, activated EGFR / JAK / STAT confers resistance to apoptosis of synovial cells, leading to a significant increase in the number of synovial cells, and EGFR / JAK / STAT has also been shown to play an anti-apoptotic role through translational regulation. Binding of ligands to their receptors induces JAK phosphorylation, which in turn promotes STAT phosphorylation, regulates gene transcription of proinflammatory cytokines and chemokines, and directly leads to tissue damage in RA. Therefore, the key 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 in the central nervous system (brain, spinal cord, and optic nerves). The EGFR / JAK / STAT pathway is important in the pathogenesis of MS / EAE (experimental autoimmune encephalomyelitis, an animal model of MS). Preclinical studies in animal models have shown that inhibition of the EGFR / JAK / STAT pathway disrupts neuroinflammatory and neurodegenerative processes. The EGFR / JAK / STAT signaling pathway plays a crucial role in the pathogenesis of MS.

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

[0024] Sepsis is a systemic inflammatory response syndrome (SIRS) caused by severe, life-threatening infection 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 (MPNs), cutaneous T-cell lymphoma (CTCL), lung cancer, gastric cancer, prostate cancer, colon cancer, etc. The JAK / STAT signaling pathway has been reported to be activated in different solid tumors and is generally considered to be a new therapeutic target for various cancer types. The JAK / STAT signaling pathway is widely present in a variety of tissues and cells, and its overactivation is closely related to the occurrence, progression, invasion and metastasis of a variety of 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 powder for injection, water injection, sodium chloride or glucose intravenous infusion.

[0027] In the above-mentioned medicine, the oral preparation includes additives, and the additives are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, 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, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0029] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0030] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

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

[0032] The surfactant includes Tween-80, sodium lauryl sulfate, sodium stearate sulfonate and the like.

[0033] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

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

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

[0036] The method of using the above-mentioned drug includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection or transdermal permeation, and the drug is applied to a patient in need of treatment.

[0037] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, and a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the composition of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

[0038] The present invention also provides a method for preparing the above compound:

[0039]

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

[0041] (1) With 2-methoxy-p-benzoquinone and a 3-substituted isatin derivative as reactants and chiral β-ICD as a catalyst, an asymmetric Michael addition reaction takes place in an organic solvent to obtain an intermediate.

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

[0043] Beneficial effects of the present invention:

[0044] (1) The present invention provides a method for preparing a spiroalkaloid compound having anti-tumor activity.

[0045] (2) The spiroalkaloid compound I provided by the present invention can be applied to EGFR targeted inhibitors against liver cancer, as shown in the following:

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

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

[0048] c. The anti-liver cancer effect of compound I of the present invention is dependent on EGFR. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 Compound I inhibits the proliferation of various liver cancer cells in vitro. (Left and middle) Proliferation curves of liver cancer cells after the action of compound I (Huh7 and HepG2 cells, 72h as an example); (right) Proliferation curves of normal cells after the action of compound I.

[0051] Figure 2 Shows the CETSA binding analysis of compound I and cellular EGFR protein molecules. (Left) Western-blot analysis of EGFR protein expression; (Right) Grayscale analysis of EGFR protein relative expression, with the grayscale value of β-tubulin protein as the internal reference.

[0052] Figure 3 Shows SPR binding analysis of compound I and EGFR protein molecules.

[0053] Figure 4 The results show that the liver cancer inhibitory activity of compound I is dependent on the expression of EGFR. (Top) SRB analysis of the cytotoxicity of compound I on Huh7 cells after EGFR overexpression for 72 hours. (Bottom) SRB analysis of the cytotoxicity of compound I on Huh7 cells after EGFR knockout for 72 hours.

[0054] Figure 5 The results show that compound I plays a role in the treatment of 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, ns No significant difference compared with the control group. DETAILED DESCRIPTION

[0055] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0056] The experimental materials involved in the embodiments of the present invention are:

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

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

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

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

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

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

[0063] Balb / c nude mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

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

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

[0066] Example 1 Synthesis of Active Spiroalkaloid Compound I

[0067]

[0068] The 4-methoxy-substituted isatin derivative B (114.4 mg, 0.3 mmol), β-ICD (1 mg, 0.003 mmol), and dichloromethane (2 mL) were added to the reaction flask in sequence, and 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. The raw material B disappeared as detected by TCL, and the mixture was transferred to room temperature and concentrated under reduced pressure to remove dichloromethane to obtain a crude product C. C was redissolved in methanol (2 mL), p-toluenesulfonic acid (5.1 mg, 0.03 mmol) was added, and stirred at room temperature for 3 hours. The intermediate C disappeared as detected by TCL, and concentrated under reduced pressure, and then a white solid D was obtained by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1), i.e., 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] + Calculate 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. SRB method to detect the proliferation rate of liver cancer cells

[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 plated in a 96-well plate with a single-well volume of 90 μL and a cell density of 4×103 / well, placed in a 37°C, 5% CO2 incubator, and cultured until completely adhered.

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

[0078] (4) SRB method for OD detection 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 for more than 5 times and dry naturally 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 discard the SRB.

[0082] d. Wash with 1% glacial acetic acid for more than 5 times and dry naturally 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 on an ELISA reader and measure the OD value at 515 nm.

[0085] The cell proliferation rate was calculated according to the following formula:

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

[0087] (6) GraphPad was used to draw a graph and calculate the half inhibitory concentration (IC) 50 .

[0088] The results are as follows Figure 1 The results showed that compound I could inhibit the proliferation of Huh7 and HepG2 cells in a time- and dose-dependent manner. 50The inhibition rates of 20 μM in normal cells were 49.83% for Huvec, 34.74% for Beas-2b, and 21.08% for L02. It can be seen that compound I has a stronger inhibitory ability against liver cancer cells and a weaker inhibitory ability against normal cells, indicating that compound I not only has an anti-cancer effect, but also has a certain safety.

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

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

[0091] Dosing: a solvent control group and a drug-dosing group were set up separately, wherein DMSO was added to the solvent control group, and compound I was added to the drug-dosing group to a final concentration of 4 μM, and the cells were cultured in an incubator for 3 h.

[0092] Collect cells: discard the culture medium after aspirating with the pipette tip, then wash the cells twice with PBS, digest the cells with trypsin, add culture medium to terminate digestion, centrifuge at 1200rpm for 5min. After centrifugation, resuspend with PBS, wash the cells twice, resuspend with PBS, and divide each group equally into 4 PCR tubes.

[0093] Gradient heating: Place the PCR tube in a PCR instrument for gradient heating, and set the temperatures to 38.4, 40.6, 47.6, and 52.1°C.

[0094] Cell lysis: After the gradient heating is completed, transfer the cell suspension to a 1.5 ml EP tube and place it in liquid nitrogen for lysis. Do this 3 to 4 times, each time for 3 minutes.

[0095] Collect samples: Place the lysed cells in a 4°C refrigerated centrifuge at a centrifugal speed of 20,000 × g for 20 min. Collect the supernatant and add Loading Buffer to resuspend, boil in boiling water for 15 min, and store at -20°C for later use.

[0096] The interaction between compound I and protein can help EGFR have stronger stability in high temperature environment. By comparing the degradation of EGFR protein in the DMSO group and the compound treatment 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 increase, indicating that compound I can bind to the EGFR protein of the cell.

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

[0099] Surface plasma resonance (SPR) technology was used to analyze the binding of compound I to EGFR protein.

[0100] The SpotBot3 microarray control software in the SpotBot3 needle platform coupled compound I to the surface of the biochip implant, and UV light was used to crosslink the compound on the chip surface for 15 minutes. EGFR protein was diluted to 50, 100 nM, 200 nM and 400 nM with PBS and injected for detection.

[0101] The obtained data were analyzed and fitted using PLEXERASPR Data Analysis Module (DAM) analysis 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 and a moderate binding strength.

[0103] 4. Detection of the effect of compound D on Huh7 cell proliferation by overexpression or knockout of EGFR

[0104] (1) When the confluency of Huh7 cells reaches about 70%, transfection is performed.

[0105] (2) Prepare two EP tubes for each group. In one tube, add 3.75 μL of transfection reagent to 125 μL of serum-free and dual antibody-free culture medium. In the other tube, add 10 μL of EGFR interference fragment (EGFR interference fragment sequence 5'-GAGGCAAAGUGCCUAUCAATT-3'; NC sequence 5'-UUCUCCGAACGUGUCACGUTT-3') or 6 ug of plasmid (the plasmid is a plasmid containing EGFR fragment, purchased from Addgene, catalog number 11011) to 125 μL of serum-free and dual antibody-free culture medium. Gently mix and let stand.

[0106] (3) Add the culture medium containing the interference 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 culture medium without serum and dual antibody.

[0108] (5) After 6 hours, each well was replaced with 2 mL of complete medium. After 48 hours of transfection, a portion of the cells were sampled to detect the transfection efficiency, and the remaining portion was re-plated and counted. The cells were seeded in a 96-well plate, and after the cells adhered to the wall, compound I was added to detect the effect on Huh7 proliferation.

[0109] The results are as follows Figure 4 , high expression of EGFR enhanced the proliferation ability of liver cancer cells of compound I. On the contrary, after knocking down EGFR, the cytotoxicity of compound I to liver cancer was weakened. This shows that EGFR plays a key role in the anti-cancer activity of compound I. Based on the above studies, it can be concluded that compound I exerts its anti-liver cancer function by directly targeting EGFR.

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

[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 Each) and monitor tumor growth. When the tumor volume reaches 100 mm 3 Start treatment when

[0113] (2) The treatment group was given Compound I (two doses, once a day), and the control group was given normal saline (solvent), and the medication was given for 12 days.

[0114] (3) Analysis of treatment effects:

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

[0116] Tumor volume = long diameter × short diameter 2 ×1 / 2

[0117] b. At the end time, the mice were killed and dissected to obtain the 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 was calculated according to the following formula:

[0119]

[0120] Changes in animal weight and organ indexes can be used to determine whether there are obvious toxic side effects and show its safety.

[0121] The results are as follows Figure 5 As shown, compound I significantly inhibited the growth of Huh7 xenograft tumors in vivo, with the tumor inhibition rate reaching 64% at a dose of 50 mg / kg and 47.7% at a dose of 25 mg / kg, and the tumor weight was significantly reduced. At the same time, there was no difference in the body weight of mice in the treatment group and the control group, indicating that the treatment at this dose was relatively safe.

[0122] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A compound selected from Formula I or a pharmaceutically acceptable salt thereof, 2. Use of the compound according to claim 1 in the preparation of a drug for treating cancer.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

5. Use of the compound according to claim 1 in the preparation of EGFR targeted inhibitory drugs.

6. An EGFR targeted inhibitory drug, characterized in that: The targeted inhibition drug comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

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