Application of novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 in preparation of medicine for treating EGFR mutant non-small cell lung cancer

Abstract

The invention relates to the technical field of non-small cell lung cancer treatment, in particular to application of a novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 in preparation of a medicine for treating EGFR mutant non-small cell lung cancer. The structure of the targeted chimera DC1-206 comprises an E3 ubiquitin ligase ligand, a target protein ligand EGFR-TKI and a Linker of a special structure for connecting two active ligands, a triad EGFR-PROTAC active drug molecule is finally formed, the target protein ligand of PROTAC is combined with a target protein in the body of a patient, and the targeted chimera DC1-206 can be used for preparing the targeted chimera DC1-206. According to the present invention, the E3 ubiquitin ligase ligand is combined with the substrate binding region of the E3 ubiquitin ligase in the cell, such that the target protein is pulled to the side of the E3 ubiquitin ligase through the Linker, the target protein is degraded by the UPS system, the lung cancer cell proliferation is effectively inhibited, the lung cancer cell apoptosis is promoted, and the efficient targeting tumor inhibition effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of non-small cell lung cancer treatment, and in particular to the use of a novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 in the preparation of drugs for treating EGFR mutant non-small cell lung cancer. Background Art

[0002] Lung cancer remains the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the most common type, accounting for approximately 85% of all lung cancer cases. In patients with NSCLC, epidermal growth factor receptor (EGFR) mutations are key driver mutations, closely associated with disease onset, progression, and treatment response, making them a focus of targeted therapy research.

[0003] Lung adenocarcinoma (LUAD) is the most common subtype of NSCLC. In East Asia, the epidermal growth factor receptor (EGFR) mutation rate in female LUAD patients is approximately 40%-50%. These mutations primarily occur in exons 18 to 21 of the EGFR, leading to constitutive activation of the receptor tyrosine kinase, thereby promoting sustained tumor cell proliferation, angiogenesis, and invasiveness, and promoting metastasis. Notably, the most common sensitizing mutations are exon 19 deletions and exon 21 L858R substitutions, which together account for approximately 85% of all EGFR mutations in NSCLC.

[0004] The discovery of EGFR mutations has opened the door to targeted therapy, with tyrosine kinase inhibitors (TKIs) becoming the first-line treatment for EGFR-positive NSCLC. EGFR TKIs specifically target and inhibit the abnormal signaling pathways caused by EGFR mutations, significantly slowing tumor growth and improving patient survival. This therapeutic effect is particularly pronounced in patients with EGFR exon 19 deletions or the L858R mutation in exon 21.

[0005] Although epidermal growth factor receptor inhibitors (EGFR-TKIs) have demonstrated significant efficacy, the development of drug resistance remains a major challenge. This resistance is often associated with secondary mutations (such as T790M), which reduce the drug's binding efficiency to the EGFR. Furthermore, during treatment, EGFR inhibitors may activate compensatory signaling pathways. For example, in HCC827 cells and NSCLC patients with pre-existing low-frequency MET amplification, the dominant MET-amplified clones survive and expand after EGFR-TKI treatment, thereby accelerating tumor progression and promoting the development of drug resistance. Therefore, there is an urgent need to develop new treatments to provide more treatment options for patients with EGFR mutations.

[0006] Currently, there is a lack of drugs on the market that are effective alone against EGFR mutations in non-small cell lung cancer and that inhibit the development of drug resistance. In this context, proteolysis-targeting chimera (PROTAC) technology has become a promising strategy. PROTAC is a multifunctional molecule composed of an E3 ligand, a target protein ligand, and a linker. It can recruit specific target proteins as ubiquitination substrates and hijack the ubiquitin-proteasome system (UPS) to promote their degradation. Unlike EGFR inhibitors, using PROTAC technology to degrade EGFR can more effectively inhibit the EGFR signaling pathway. In NSCLC cells, PROTAC can target and degrade mutant epidermal growth factor receptors, providing new therapeutic possibilities for inhibiting tumor cell growth and metastasis. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention provides a novel epidermal growth factor receptor degradation protein hydrolysis targeted chimera DC1-206 for use in the preparation of a drug for treating EGFR mutant non-small cell lung cancer. The structural formula of the targeted chimera DC1-206 is: .

[0008] Furthermore, the EGFR mutant non-small cell lung cancer cells are HCC827 cells.

[0009] Furthermore, the prepared pharmaceutical dosage form is any one of tablets, capsules, granules, and injections.

[0010] Furthermore, the method for preparing the targeted chimera DC1-206 comprises the following steps: Step 1. (1s,4s)-4-aminocyclohexanecarboxylic acid methyl ester hydrochloride is subjected to a two-step nucleophilic substitution reaction to generate intermediate A; Step 2. 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamic acid tert-butyl ester is subjected to sulfonylation and nucleophilic substitution to obtain intermediate B-1. Intermediate B-1 is then dissolved in methanol, 10% Pd / C catalyst is added, and the mixture is reduced under hydrogen atmosphere at room temperature to obtain intermediate B. Step 3. The intermediate A obtained in step 1 and the intermediate B obtained in step 2 are dissolved in acetonitrile, and the intermediate C is obtained through condensation and cyclization reaction; Step 4. 4-(3-hydroxypropyl)piperazine-1-carboxylic acid tert-butyl ester is reacted with TsCl and DMAP to obtain intermediate D-1. Intermediate D-1 is then dissolved in acetonitrile and added with 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol, followed by substitution reaction and de-Boc reaction to obtain intermediate D-2. Intermediate D-2 is then dissolved in acetonitrile and tert-butyl 2-bromoacetate for substitution to obtain intermediate D-3. Finally, intermediate D-3 is de-tert-butylated to obtain intermediate D. Step 5. Take the intermediate C obtained in step 3 and add trifluoroacetic acid and dichloromethane to remove Boc at 0°C. After reacting for 1 hour, warm to room temperature and add the intermediate D obtained in step 4 to cause condensation. Then, add piperidine to remove Fmoc to obtain the targeted chimera DC1-206.

[0011] Furthermore, the preparation reaction equation of the targeted chimera DC1-206 is as follows: .

[0012] Furthermore, the two-step nucleophilic substitution reaction in step 1 includes a first nucleophilic substitution reaction to generate intermediate A-1 and a second nucleophilic substitution reaction to introduce an Fmoc protecting group into intermediate A-1. The addition temperature of the two-step nucleophilic substitution is 0°C, and the reaction time is >12h.

[0013] Furthermore, the first step of the nucleophilic substitution reaction is to dissolve methyl 4-aminocyclohexanecarboxylate hydrochloride in dichloromethane, generate a substitution product under the action of chloroacetyl chloride and triethylamine, and then dissolve the obtained substitution product in acetonitrile, add 1-(1H-indol-2-yl)methylamine, and reflux under the action of sodium bicarbonate and potassium iodide to generate intermediate A-1; The second step of the nucleophilic substitution reaction is to hydrolyze the intermediate A-1 to obtain a hydrolysis product, dissolve the hydrolysis product in dioxane, and add Fmoc-OSu and Na2CO3 to introduce a protecting group to obtain intermediate A; Wherein, the hydrolysis reagents in the second step nucleophilic substitution reaction are lithium hydroxide monohydrate, methanol and water.

[0014] Furthermore, the reagents for the sulfonylation reaction in step 2 are TsCl, triethylamine and DMAP, the reaction temperature is room temperature, and the reaction time is greater than 12 hours; the reagents for the nucleophilic substitution reaction in step 2 are 4-amino-3-nitrophenol and potassium carbonate, the reaction temperature is reflux temperature, and the reaction time is 12 hours; the solvents for the sulfonylation reaction and the nucleophilic substitution reaction are both acetonitrile.

[0015] Furthermore, in the step 3, the condensation agent used in the condensation reaction is HBTU, the base used is DIPEA, the reaction temperature is room temperature, and the reaction time is greater than 12 h; the auxiliary agent used in the cyclization reaction is HOAc, the reaction temperature is 70° C., and the reaction time is 1 h.

[0016] Furthermore, the reagents used for de-Boc and de-tert-butylation in step 4 are trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, the reaction temperature is 0°C, and the reaction time is 1 h; the base used in the substitution reaction to generate intermediate D-2 and the substitution reaction to generate intermediate D-3 in step 4 is potassium carbonate.

[0017] Furthermore, the solvent for the condensation in step 5 is DMF, the condensing agent used is HATU, and the base used is DIPEA.

[0018] The advantages of the present invention are: The novel epidermal growth factor receptor degradation protein hydrolysis targeted chimera DC1-206 provided by the present invention can be used to prepare a drug for treating EGFR mutant non-small cell lung cancer. Its structure includes a linker with an E3 ubiquitin ligase ligand, a target protein ligand EGFR-TKI, and a special structure connecting the two active ligands, ultimately forming an active form of a triplet PROTAC. In the patient's body, the target protein ligand of PROTAC binds to the target protein, and the E3 ubiquitin ligase ligand binds to the substrate binding region of the E3 ubiquitin ligase in the cell, thereby Inker pulls the target protein close to the E3 ubiquitin ligase, enabling the UPS system to degrade the target protein. It can effectively target lung cancer cells with epidermal growth factor receptor mutations, especially HCC827 cells with epidermal growth factor receptor exon 19 deletion, without affecting H1975 cells with epidermal growth factor receptor T790M / L858R and H1299 cells with epidermal growth factor receptor WT. It effectively inhibits the proliferation of lung cancer cells without showing obvious in vivo toxicity and drug resistance, and can significantly promote lung cancer cell apoptosis, achieving efficient targeted tumor inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the cell viability of H1975, HCC827, and H1299 cells after treatment with DC1-206 for 48 hours; Figure 2 This is a cell viability curve of H1975, HCC827 and H1299 cells after treatment with gefitinib for 48 hours; Figure 3 Effects of different concentrations of DC1-206 on EGFR protein levels in different cells after 12 hours of treatment ( Figure 3 A is the treatment of H1975 cells, Figure 3 B is the treatment of HCC827, Figure 3 C is treated H1299 cells); Figure 4The results are the normalized results of EGFR protein levels after treatment of three types of cells with different concentrations of DC1-206 for 12 hours; Figure 5 The results show the time-dependent degradation of EGFR in HCC827 cells after administration of DC1-206 at a concentration of 20 μM; Figure 6 The results are normalized to the EGFR protein level in HCC827 cells after administration of DC1-206 at a concentration of 20 μM; Figure 7 For use Homoligand Changes in EGFR protein levels in HCC827 cells after pretreatment with the indicated concentrations for 8 h and then treatment with DC1-206 for 12 h; Figure 8 Figure 2 Changes in EGFR protein levels in HCC827 cells after pretreatment with different inhibitors at the indicated concentrations for 8 hours and then treatment with DC1-206 for 12 hours; Figure 9 The effects of different concentrations of DC1-206 on EGFR and its related downstream signaling pathways on HCC827 cells after 12 hours of treatment; Figure 10 The cell cycle changes of HCC827 cells after treatment with different concentrations of DC1-206 for 12 hours; Figure 11 The changes in apoptosis rate of HCC827 cells after treatment with different concentrations of DC1-206 for 12 hours; Figure 12 Flow chart for studying the anti-tumor effect of the proteolytic targeting chimera DC1-206 targeting epidermal growth factor receptor degradation in tumor-bearing mice; Figure 13 Figure 1 shows the mouse tumors obtained after the tumor-bearing mice were sacrificed and treated with the chimera DC1-206 targeting epidermal growth factor receptor degradation proteolysis. Figure 14 This is a graph showing the changes in tumor volume in tumor-bearing mice after treatment with the epidermal growth factor receptor proteolytic targeting chimera DC1-206; Figure 15 This is a graph showing the changes in tumor weight in tumor-bearing mice after treatment with the epidermal growth factor receptor proteolytic targeting chimera DC1-206; Figure 16 The graph shows the weight changes of tumor-bearing mice treated with the proteolytic targeting chimera DC1-206 for epidermal growth factor receptor degradation; Figure 17 This is the H NMR spectrum of the targeted chimera DC1-206 of the present invention; Figure 18 This is the structural diagram of DC1-35 in Experimental Example 3. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] It should be noted that the various installation methods and technical terms mentioned in the present invention are technical terms that have long been clearly known in the relevant technical field and therefore will not be further explained. In addition, the same reference numerals are used for the same components, but this does not affect nor constitute an accurate understanding of the technical solution by those skilled in the art.

[0022] Example 1 This example provides a novel epidermal growth factor receptor degradation protein hydrolysis targeted chimera DC1-206 for the preparation of a drug for treating EGFR mutant non-small cell lung cancer. The chimera DC1-206 is designed and synthesized based on the E3 ligase GID4 and has the structural formula: .

[0023] The reaction equation for preparing the targeting chimera DC1-206 is as follows: .

[0024] The specific preparation steps of the targeted chimera DC1-206 are as follows (the overnight reaction time involved in this example is all >12 h): Step 1. Preparation of intermediate A: 3.6 mmol of (1s,4s)-4-aminocyclohexane-1-carboxylic acid methyl ester hydrochloride and 10.8 mmol of triethylamine were dissolved in dichloromethane and slowly added dropwise to a dichloromethane solution of chloroacetyl chloride (a solution formed by adding 5.4 mmol of chloroacetyl chloride to dichloromethane at 0°C) at 0°C, and then stirred at room temperature overnight; the mixture was washed with 0.1 M HCl, NaHCO3, and brine in sequence, and the organic phase was dried over anhydrous Na2CO3 and dried under vacuum to obtain the substitution product, which was then dissolved in acetonitrile, and then 5.4 mmol of 1-(1H-indol-2-yl)methylamine, 10.8 mmol of NaHCO3, and 0.36 mmol of KI were added and refluxed for 12 h. After filtration, the solvent was removed under vacuum to purify the intermediate A-1; 3.49 mmol of intermediate A-1 was dissolved in methanol and water at 0°C, and 34.9 mmol of LiOH·H2O was slowly added. The mixture was stirred at room temperature overnight to allow hydrolysis to occur. The hydrolyzed product was dissolved in dioxane and water at 0°C, and then 5.2 mmol of Fmoc-OSu and Na2CO3 were added. The mixture was stirred at room temperature overnight. The reaction solution was extracted with ethyl acetate and purified to obtain intermediate A. The reaction equation is: .

[0025] The H NMR spectrum data of the intermediate A obtained are: 1 HNMR (400MHz, DMSO-d6) δ12.04 (s, 1H), 11.17 (s, 1H), 11.07 (s, 1H), 7.94-7.80 (m, 3H), 7.6 3(d, J=7.4Hz, 1H), 7.50-7.39(m, 3H), 7.39-7.27(m, 3H), 7.14-7.01(m, 2H), 7.01-6.91(m, 1H), 6.29(s, 1H), 6.04(s, 1H), 4.59(d, J=20.1Hz, 2H), 4.37-4.20(m, 3H), 3.91(s, 2H), 3.8 2-3.67 (m, 1H), 2.41-2.34 (m, 1H), 1.90-1.75 (m, 2H), 1.61-1.50 (m, 4H), 1.50-1.38 (m, 2H).

[0026] Step 2. Preparation of intermediate B: At 0°C, 5.70 mmol of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, 11.40 mmol of triethylamine, and 2.85 mmol of DMAP were added to dichloromethane, and then 11.40 mmol of TsCl (p-toluenesulfonyl chloride) was slowly added dropwise. The mixture was stirred at room temperature overnight, washed, and dried to obtain a light yellow oil. 3.13 mmol of the light yellow oil, 6.26 mmol of 4-amino-3-nitrophenol, and 6.26 mmol of potassium carbonate were then dissolved in acetonitrile and refluxed for 12 h to allow a substitution reaction to occur to obtain intermediate B-1. Then, the intermediate B-1 was dissolved in methanol, 10% Pd / C catalyst was added, hydrogen was introduced after vacuum ventilation, and the mixture was stirred at room temperature overnight, and reduced in hydrogen atmosphere to obtain the intermediate B; The reaction equation is: .

[0027] Step 3. Preparation of Intermediate C: At 0°C, 0.22 mmol of intermediate A obtained in step 1, 0.28 mmol of intermediate B obtained in step 2 (the crude product was used directly), and 0.28 mmol of HBTU were dissolved in acetonitrile. 0.55 mmol of DIPEA was slowly added. After condensation reaction, HOAc was added to the obtained product and the reaction was carried out at 70°C for 1 h to obtain intermediate C. The reaction equation is: .

[0028] The H NMR spectrum data of the obtained intermediate C are: 1 HNMR (400MHz, Chloroform-d) δ7.70 (d, J=7.3Hz, 2H), 7.59-7.42 (m, 3H), 7.42-7.27 (m, 4H), 7.24 -6.99(m, 5H), 6.96-6.86(m, 1H), 6.20(s, 1H), 5.11-4.94(m, 1H), 4.68-4.42(m, 3H), 4.38-4.28(m , 1H), 4.26-4.06 (m, 3H), 3.96-3.79 (m, 4H), 3.77-3.67 (m, 2H), 3.67-3.59 (m, 2H), 3.55 (t, J=5.1 Hz, 2H), 3.36-3.27(m, 2H), 2.98-2.86(m, 1H), 1.90-1.70(m, 4H), 1.69-1.52(m, 4H), 1.43(s, 9H).

[0029] Step 4. Preparation of Intermediate D: At 0°C, 5.70 mmol of tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate, 11.40 mmol of triethylamine, and 2.85 mmol of DMAP were dissolved in dichloromethane. After stirring evenly, 11.40 mmol of TsCl was slowly added dropwise. The temperature was slowly raised to room temperature and stirred overnight. After washing and drying, the mixture was purified using petroleum ether / ethyl acetate to obtain intermediate D-1. Then, 3.09 mmol of intermediate D-1 and 6.18 mmol of K2CO3 were dissolved in acetonitrile, and 4.65 mmol of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol was slowly added. After reflux for 12 h, the substitution product was obtained. The substitution product was added with trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, and the reaction was carried out at 0°C for 1 h. The Boc was removed to obtain 0.32 g of intermediate D-2. Then, 0.72 mmol of intermediate D-2 and 1.43 mmol of K2CO3 were dissolved in acetonitrile, and 1.08 mmol of tert-butyl 2-bromoacetate was slowly added. After reflux reaction for 12 h, intermediate D-3 was generated. Finally, intermediate D-3 was added with trifluoroacetic acid and dichloromethane in a volume ratio of 1:1 and reacted at 0°C for 1 h to remove the tert-butyl group to obtain intermediate D; The reaction equation is: .

[0030] The H NMR spectrum data of the obtained intermediate D are: 1 HNMR (400MHz, Methanol-d4) δ8.71 (s, 1H), 8.09 (s, 1H), 8.01 (dd, J=6.7, 2.6Hz, 1H), 7.77-7.69 (m, 1H), 7.36 (t, J=8.9H z, 1H), 7.30 (s, 1H), 4.42 (t, J=5.6Hz, 2H), 4.10 (s, 3H), 3.47 (s, 2H), 3.45-3.34 (m, 6H), 3.07 (s, 4H), 2.44-2.36 (m, 2H).

[0031] Step 5. Preparation of targeting chimera DC1-206: At 0°C, 0.07 mmol of intermediate C obtained in step 3 was dissolved in a trifluoroacetic acid and dichloromethane solution in a volume ratio of 1:1, and the reaction was carried out at 0°C for 1 h to remove Boc. The de-BOc product was then warmed to room temperature, dissolved in DMF, and 0.08 mmol of intermediate D obtained in step 4, 0.11 mmol of HATU, and 0.35 mmol of DIPEA were added. The reaction was stirred at room temperature overnight to obtain a condensation product. Finally, the condensation product was dissolved in dichloromethane and acetonitrile, piperidine was slowly added dropwise, and the product was stirred at room temperature for 2 h to remove Fmoc to obtain the targeted chimera DC1-206.

[0032] The reaction equation is: .

[0033] The NMR spectrum of the obtained targeted chimera DC1-206 is as follows Figure 17 As shown, the corresponding H NMR spectrum data is: 1HNMR (400MHz, Methanol-d4) δ8.38 (s, 1H), 7.95 (dd, J=6.7, 2.7Hz, 1H), 7.63-7.56 (m, 2H), 7.32 (dd, J=8.2, 5.9Hz, 2H), 7.19 (d, J=8.9Hz, 1H), 7.14 (d, J=8.2Hz, 1H), 7.06 (s, 1H), 6.98 (d, J=2.4Hz, 1H), 6.94-6.89 (m, 1H), 6.87- 6.79 (m, 2H), 6.22 (s, 1H), 4.10 (t, J=5.7, 3.6Hz, 2H), 4.05 (t, J=6.2Hz, 2H), 3.89(s, 3H), 3.87-3.84(m, 1H), 3.84(s, 2H), 3.83-3.80(m, 2H), 3.67(dd, J =5.7, 3.0Hz, 2H), 3.60(dd, J=5.8, 3.0Hz, 2H), 3.52(t, J=5.2Hz, 2H), 3.37( t, J=5.2Hz, 2H), 3.22(s, 2H), 2.93(s, 2H), 2.92-2.81(m, 1H), 2.53(t, 2H), 2.51-2.40 (m, 8H), 1.99-1.90 (m, 4H), 1.86-1.74 (m, 3H), 1.62-1.53 (m, 5H).

[0034] Experimental Example 1 This experimental example verifies the effect of the novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 designed and synthesized based on the E3 ligase GID4 in Example 1 on the proliferation of human lung adenocarcinoma cell lines. The experimental process is as follows: (1) Three human lung adenocarcinoma cell lines, H1975 (epidermal growth factor receptor T790M / L858R mutation), HCC827 (epidermal growth factor receptor exon 19 deletion), and H1299 (epidermal growth factor receptor wild type), were selected and cultured in DMEM medium containing 10% FBS. The cells were placed in a 10 cm cell culture dish and placed in an incubator at 37°C and 5% CO2 saturated humidity until the cells were in the logarithmic growth phase. The cells were then passaged using 0.25% trypsin-EDTA digestion solution. At the same time, the targeted chimera DC1-206 of Example 1 was dissolved in DMSO solution as the experimental group, and the first-generation EGFR-TKI gefitinib was selected as the control group.

[0035] (2) Cells in the logarithmic growth phase were plated on a 96-well plate at a density of 5×103 cells per well. The 96-well plate was placed in a cell incubator for overnight culture. After the cells adhered to the wall, the cells were treated with DC1-206 in the experimental group and gefitinib in the control group. The concentrations of the drugs in each group were increased in sequence according to the concentration gradient of 0 μmol / L, 0.3125 μmol / L, 0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L. Each group had 4 replicate wells, and the culture medium in each well was 100 μL. After 48 h of treatment, the 96-well plate was removed, 10 μL of CCK-8 solution was added to each well, and the cells were cultured at 37°C in the dark for 1 h. The absorbance of each well at OD 450 nm was measured using an enzyme marker.

[0036] (3) Each experiment was repeated three times. Finally, the average value of each replicate was taken and the drug inhibition rate was calculated according to the formula: (absorbance value of the untreated group - absorbance value of the experimental group) / absorbance value of the untreated group × 100%. Finally, the IC50 concentrations of DC1-206 and gefitinib (as shown in Table 1) and the cell activity curve (as shown in Table 1) were obtained. Figure 1 and Figure 2 shown).

[0037] Depend on Figure 1 It can be seen that the IC50 of DC1-206 in H1975 is 66.72μmol / L, the IC50 of HCC827 is 11.57μmol / L, and the IC50 of H1299 is 144.6μmol / L. Figure 2 It can be seen that the IC50 of gefitinib in H1975 is 4.875 μmol / L, the IC50 in HCC827 is 1.698 μmol / L, and the IC50 in H1299 is 8.458 μmol / L. The comparison of the above results shows that compared with gefitinib in the control group, the novel epidermal growth factor receptor degradation proteolysis-targeting chimera DC1-206 designed and synthesized in Example 1 of the present invention has a significant anti-proliferative effect on human lung adenocarcinoma cell lines carrying mutant EGFR.

[0038] Table 1 Results of half-inhibitory concentration IC50 of DC1-206 and gefitinib

[0039] Experimental Example 2 This experimental example further verified the degradation effect of the novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 designed and synthesized in Example 1 on mutant EGFR in human lung adenocarcinoma cell lines. To further study whether DC1-206 can degrade epidermal growth factor receptor protein, this experimental example used the targeted chimera DC1-206 as the experimental group to treat three human lung adenocarcinoma cell lines, and used a vehicle (0.1% dimethyl sulfoxide) as the control group. The experimental process is as follows: Cells were seeded the day before drug treatment to ensure that the cell confluency reached approximately 60% when the drug was added. According to the experimental protocol, DC1-206 was dissolved in an appropriate amount of DMSO, and then these drug solutions were added to the culture plate containing the target cells. The target cells were treated with a concentration gradient of 0 μmol / L, 1 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L and a time gradient of 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h. Subsequently, protein samples were prepared according to the Western Blot experimental steps to evaluate the effect of PROTACs on the target protein in the cells. Finally, the protein was extracted and the changes in the epidermal growth factor receptor protein level were detected by Western blot analysis.

[0040] The results are as follows Figure 3 As shown, Figure 3 Comparison between A and 3B shows that the targeted chimera DC1-206 in the experimental group induced the degradation of mutant epidermal growth factor receptor in lung cancer cell line HCC827 in a concentration-dependent manner. The effect of DC1-206 on inducing WTEGFR degradation in H1299 is shown in Figure 3. Figure 3 As shown in C, Figure 3 C shows that DC1-206 did not show significant degradation of WT EGFR. In addition, the band intensities of EGFR and GAPDH in Western blots were quantified using ImageJ software, and their ratio was calculated as the normalized result. GraphPad Prism 8.0 was used to fit the data with a nonlinear regression curve to calculate the degradation concentration at which EGFR reached half-maximal degradation rate (DC50). Figure 4 As shown, the calculated DC50 of DC1-206 for mutant EGFR in HCC827 cells was 2.330 μM, and the maximum degradation rate of mutant EGFR in HCC827 cells was 88.85% at a concentration of 20 μM.

[0041] The above results further confirmed the targeting selectivity of PROTACs for epidermal growth factor receptor 19 exon 19del. Subsequently, the degradation agent DC1-206 was added to the HCC827 cell line to verify the effect of target protein degradation at the same starting time and different time gradients. Figure 5 and Figure 6 As shown, DC1-206 induced the degradation of mutant EGFR in a time-dependent manner. At a concentration of 20 μM, the protein levels of both mutant EGFRs decreased over time, and the time required for DC1-206 to degrade 50% of the protein (t1 / 2) was 6.81 hours. This further demonstrates that the novel EGFR degradation proteolytic targeting chimera DC1-206 of the present invention can effectively target EGFR-mutated lung cancer cells, particularly HCC827 cells with EGFR exon 19 deletion, while having no effect on EGFR T790M / L858R in H1975 cells and EGFR WT in H1299 cells.

[0042] Experimental Example 3 This experimental example further verified the mechanism of degradation of mutant EGFR by the novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 designed and synthesized in Example 1. In order to study whether the degradation of mutant EGFR by DC1-206 depends on the binding of ligand to the target, cells were seeded one day before drug treatment to ensure that the cell confluency reached approximately 60% when the drug was added. The experimental process was as follows: (1) To determine whether EGFR-PROTAC degradation of target proteins depends on the binding of EGFR ligands and GID4 E3 ubiquitin ligands to EGFR and GID4 E3 ubiquitin ligands, DC1-206 was dissolved in an appropriate amount of DMSO, and then cells were treated with DC1-206 as experimental groups, with or without EGFR ligand gefitinib, and E3 ligase ligand DC1-35 (structure as shown in Figure 2). Figure 18 As shown in the figure, the control group and the protein samples were prepared according to the Western Blot experimental steps to evaluate the effect of PROTACs on the target protein in the cells. The protein was then extracted and the changes in the epidermal growth factor receptor protein level were detected by Western blot analysis. The results are shown in Figure 2. Figure 7 As shown, it can be concluded that the EGFR ligand gefitinib and the E3 ligase ligand DC1-35 can competitively bind to DC1-206, weakening the effect of PROTAC in degrading the target protein.

[0043] (2) In order to determine whether EGFR-PROTACs degradation of target proteins mediates the degradation of mutant epidermal growth factor receptors through the ubiquitin-proteasome pathway, DC1-206 was dissolved in an appropriate amount of DMSO, and then cells were treated with DC1-206 as the experimental group, and the cells were treated with or without the proteasome inhibitor MG132 and the ubiquitination inhibitor MLN4924 as the control group. Subsequently, protein samples were prepared according to the Western Blot experimental steps to evaluate the effect of PROTACs on the target protein in the cells. The proteins were then extracted and the changes in the epidermal growth factor receptor protein level were detected by Western blot analysis. The results are shown in Figure 2. Figure 8 As shown, it can be concluded that DC1-206 promotes the ubiquitination of EGFR, thereby leading to the degradation of EGFR through the proteasome pathway.

[0044] Experimental Example 4 In this experimental example, the novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 designed and synthesized in Example 1 was used to study the effects of DC1-206 on EGFR-related downstream pathways. To study the effects of DC1-206 on EGFR-related downstream pathways, cells were seeded one day before drug treatment to ensure that the cell confluency reached approximately 60% when the drug was added. The experimental process was as follows: DC1-206 was dissolved in an appropriate amount of DMSO, and then cells were treated with DC1-206. Subsequently, protein samples were prepared according to the Western Blot experimental steps, and then proteins were extracted and analyzed by Western blot analysis to detect changes in EGFR, phosphorylated EGFR, and EGFR downstream related AKT, phosphorylated AKT, ERK, and phosphorylated ERK in the cells to evaluate the effect of DC1-206 on EGFR related downstream pathways. The results are shown in FIG. Figure 9 As shown, it can be seen that DC1-206 can effectively inhibit the activation of phosphorylated EGFR, phosphorylated AKT, and phosphorylated ERK.

[0045] Experimental Example 5 This experimental example studies the effects of the novel epidermal growth factor receptor degradation protein hydrolysis-targeted chimera DC1-206 designed and synthesized in Example 1 on cell cycle and apoptosis. Cells were seeded one day before drug treatment to ensure that the cell confluency reached approximately 60% when the drug was added. The experimental process is as follows: To determine the effect of EGFR-PROTAC on cell cycle and apoptosis, DC1-206 was dissolved in an appropriate amount of DMSO, and then cells were treated with DC1-206. HCC827 cells were subjected to dual-parameter cell fluorescence assay of membrane-bound protein v and propidium iodide (PI). At the same time, whether DC1-206 could block the cell cycle of HCC-827 cells was detected. The results are as follows Figure 10 and11 As shown, it can be seen that DC1-206 can prevent HCC827 cells from entering the G1 phase in a concentration-dependent manner, and DC1-206 can induce HCC-827 cell apoptosis in a dose-dependent manner.

[0046] Experimental Example 6 This experimental example studies the anti-tumor effect of the EGFR-PROTAC targeting chimera DC1-206 designed and synthesized in Example 1 in tumor-bearing mice. The experimental process is as follows: The animals used were 6-week-old female nude mice BALB / cnu (Beijing BGI Biotechnology Co., Ltd.), which were cultured in a specific environment and 5×10 6 Cells were implanted subcutaneously in the right groin of mice, and when the tumor volume reached about 100 cubic millimeters (e.g. Figure 12 As shown in the figure, nude mice were randomly divided into three groups: ① control group: vehicle; ② experimental group: gefitinib; ③ experimental group: DC1-206 (n=6); and vehicle (5% DMSO + 5% PEG300 + 5% Tween80 + 85% H2O, ip / qd) served as a negative control group. The experimental groups received gefitinib and DC1-206 (20 mg / kg, ip / qd) treatment. Tumor volume and mouse body weight were measured every 3 days thereafter. The experiment was terminated after 20 days of medication. The mice were euthanized under deep anesthesia, and the subcutaneous tumors were dissected and removed, and images were collected (as shown in the figure). Figure 13 The tumor volume calculation formula used in this study is (length × width 2 ) / 2. The result is as follows Figure 14-16 As shown, DC1-206 can significantly inhibit tumor growth, and its efficacy is comparable to that of gefitinib. In addition, no obvious animal toxicity was observed throughout the treatment process, indicating that the novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 of the present application is both safe and effective as a degradation agent.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention may be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description. It is intended that all variations that fall within the meaning and range of equivalents of the claims be embraced within the present invention. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor modifications, equivalent replacements, and improvements made to the above embodiments based on the technical essence of the present invention shall be included in the scope of protection of the technical solution of the present invention.

Claims

1. Use of a novel epidermal growth factor receptor degradation protein hydrolysis targeting chimera DC1-206 in the preparation of a drug for treating EGFR mutant non-small cell lung cancer, characterized in that: The structural formula of the targeting chimera DC1-206 is: 。 2. The use according to claim 1, characterized in that The EGFR mutant non-small cell lung cancer cells are HCC827 cells.

3. The use according to claim 1, characterized in that The prepared pharmaceutical dosage form is any one of tablets, capsules, granules and injections.

4. The use according to claim 1, characterized in that The preparation method of the targeted chimera DC1-206 comprises the following steps: Step 1. (1S,4S)-4-aminocyclohexanecarboxylic acid methyl ester hydrochloride is subjected to a two-step nucleophilic substitution reaction to generate intermediate A; Step 2. 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamic acid tert-butyl ester is subjected to sulfonylation and nucleophilic substitution to obtain intermediate B-1. Intermediate B-1 is then dissolved in methanol, 10% Pd / C catalyst is added, and the mixture is reduced under hydrogen atmosphere at room temperature to obtain intermediate B. Step 3. The intermediate A obtained in step 1 and the intermediate B obtained in step 2 are dissolved in acetonitrile, and the intermediate C is obtained through condensation and cyclization reaction; Step 4. 4-(3-hydroxypropyl)piperazine-1-carboxylic acid tert-butyl ester is reacted with TsCl and DMAP to obtain intermediate D-1. Intermediate D-1 is then dissolved in acetonitrile and added with 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol, followed by substitution reaction and de-Boc reaction to obtain intermediate D-2. Intermediate D-2 is then dissolved in acetonitrile and tert-butyl 2-bromoacetate for substitution to obtain intermediate D-3. Finally, intermediate D-3 is de-tert-butylated to obtain intermediate D. Step 5. Take the intermediate C obtained in step 3 and add trifluoroacetic acid and dichloromethane to remove Boc at 0°C. After reacting for 1 hour, warm to room temperature and add the intermediate D obtained in step 4 to cause condensation. Then, add piperidine to remove Fmoc to obtain the targeted chimera DC1-206.

5. The use according to claim 4, characterized in that The two-step nucleophilic substitution reaction in step 1 includes a first nucleophilic substitution reaction to generate intermediate A-1 and a second nucleophilic substitution reaction to introduce an Fmoc protecting group into intermediate A-1. The addition temperature of the two nucleophilic substitution steps is 0° C., and the reaction time is >12 h.

6. The use according to claim 5, characterized in that The first step of the nucleophilic substitution reaction is to dissolve methyl 4-aminocyclohexanecarboxylate hydrochloride in dichloromethane, generate a substitution product under the action of chloroacetyl chloride and triethylamine, and then dissolve the obtained substitution product in acetonitrile, add 1-(1H-indol-2-yl)methylamine, and reflux under the action of sodium bicarbonate and potassium iodide to generate intermediate A-1; The second step of the nucleophilic substitution reaction is to hydrolyze the intermediate A-1 to obtain a hydrolysis product, dissolve the hydrolysis product in dioxane, and add Fmoc-OSu and Na2CO3 to introduce a protecting group to obtain intermediate A; Wherein, the hydrolysis reagents in the second step nucleophilic substitution reaction are lithium hydroxide monohydrate, methanol and water.

7. The use according to claim 4, characterized in that The reagents for the sulfonylation reaction in step 2 are TsCl, triethylamine and DMAP, the reaction temperature is room temperature, and the reaction time is greater than 12 hours; the reagents for the nucleophilic substitution reaction in step 2 are 4-amino-3-nitrophenol and potassium carbonate, the reaction temperature is reflux temperature, and the reaction time is 12 hours; the solvents for the sulfonylation reaction and the nucleophilic substitution reaction are both acetonitrile.

8. The use according to claim 4, characterized in that In the step 3, the condensation agent used in the condensation reaction is HBTU, the base used is DIPEA, the reaction temperature is room temperature, and the reaction time is greater than 12 hours; the auxiliary agent used in the cyclization reaction is HOAc, the reaction temperature is 70° C., and the reaction time is 1 hour.

9. The use according to claim 4, characterized in that The reagents used for de-Boc and de-tert-butylation in step 4 are trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, the reaction temperature is 0°C, and the reaction time is 1 h; the base used in the substitution reaction to generate intermediate D-2 and the substitution reaction to generate intermediate D-3 in step 4 is potassium carbonate.

10. The use according to claim 4, characterized in that The solvent for the condensation in step 5 is DMF, the condensation agent is HATU, and the base is DIPEA.

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