Use of a novel epidermal growth factor receptor degradation proteolytic targeting chimera DC1-206 in the preparation of a medicament for treating EGFR mutant non-small cell lung cancer

Abstract

The application relates to the technical field of non-small cell lung cancer treatment, and in particular to application of a novel epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 in preparation of a drug for treating EGFR mutant non-small cell lung cancer, wherein the targeting chimera DC1-206 structure comprises an E3 ubiquitin ligase ligand, a target protein ligand EGFR-TKI and a special structure Linker connecting the two active ligands, and finally forms a triplet EGFR-PROTAC active drug molecule; in the patient's body, the target protein ligand of the PROTAC is combined with the target protein, the E3 ubiquitin ligase ligand is combined with the substrate binding area of the intracellular E3 ubiquitin ligase, the target protein is pulled close to the side of the E3 ubiquitin ligase through the Linker, the UPS system degrades the target protein, the proliferation of lung cancer cells is effectively inhibited, the lung cancer cell apoptosis is promoted, and the high-efficiency tumor-targeting 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] .

[0009] Furthermore, the EGFR mutant non-small cell lung cancer cells are HCC827 cells.

[0010] Furthermore, the prepared pharmaceutical dosage form is any one of tablets, capsules, granules, and injections.

[0011] Furthermore, the method for preparing the targeted chimera DC1-206 comprises the following steps:

[0012] Step 1. (1s,4s)-4-aminocyclohexanecarboxylic acid methyl ester hydrochloride is subjected to a two-step nucleophilic substitution reaction to generate intermediate A;

[0013] 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.

[0014] 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;

[0015] 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.

[0016] 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.

[0017] Furthermore, the preparation reaction equation of the targeted chimera DC1-206 is as follows:

[0018] .

[0019] 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.

[0020] 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;

[0021] 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;

[0022] Wherein, the hydrolysis reagents in the second step nucleophilic substitution reaction are lithium hydroxide monohydrate, methanol and water.

[0023] 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.

[0024] Further, the condensing agent used in the condensation reaction in step 3 is HBTU, the base used is DIPEA, the reaction temperature is room temperature, and the reaction time is > 12h; the auxiliary agent used in the cyclization reaction is HOAc, the reaction temperature is 70℃, and the time is 1h.

[0025] Further, the reagent used for the removal of Boc and tert-butyl in step 4 is trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, the reaction temperature is 0℃, and the reaction time is 1h; the base used for the substitution reaction to generate intermediate D-2 and the substitution reaction to generate intermediate D-3 in step 4 is potassium carbonate.

[0026] Further, the solvent used in the condensation in step 5 is DMF, the condensing agent used is HATU, and the base used is DIPEA.

[0027] The advantages of the present application are:

[0028] The novel epidermal growth factor receptor degradation proteolytic targeting chimera DC1-206 provided by the present application can be used for preparing a drug for treating EGFR mutant non-small cell lung cancer. The structure thereof comprises an E3 ubiquitin ligase ligand, a target protein ligand EGFR-TKI, and a special structure Linker connecting the two active ligands, and finally forms a triple PROTAC active form. In the patient's body, the target protein ligand and the target protein of the PROTAC are combined, the E3 ubiquitin ligase ligand and the substrate binding region of the intracellular E3 ubiquitin ligase are combined, so as to pull the target protein close to the E3 ubiquitin ligase through the Linker, realize the degradation of the target protein by the UPS system, effectively target the epidermal growth factor receptor mutant lung cancer cells, especially the epidermal growth factor receptor exon 19 deletion of HCC827 cells, without affecting the epidermal growth factor receptor T790M / L858R of H1975 cells and the epidermal growth factor receptor WT of H1299 cells, effectively inhibiting the proliferation of lung cancer cells without showing obvious in vivo toxicity and drug resistance, and significantly promoting the apoptosis of lung cancer cells, realizing high-efficiency targeted inhibition of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The cell activity curve diagram of H1975, HCC827 and H1299 cells after being treated by DC1-206 for 48 hours;

[0030] Figure 2 The cell activity curve diagram of H1975, HCC827 and H1299 cells after being treated by gefitinib for 48 hours;

[0031] Figure 3 The influence of EGFR protein level after different concentrations of DC1-206 treating different cells for 12h Figure 3A is H1975 cells treated with Figure 3 B is HCC827 cells treated with Figure 3 C is H1299 cells treated with

[0032] Figure 4 Standardized results of EGFR protein levels after treating three types of cells with different concentrations of DC1-206 for 12 hours;

[0033] Figure 5 Results of time-dependent degradation of EGFR in HCC827 cells treated with DC1-206 at a concentration of 20 μM;

[0034] Figure 6 Standardized results of EGFR protein levels in HCC827 cells treated with DC1-206 at a concentration of 20 μM;

[0035] Figure 7 Results of treating HCC827 cells with DC1-206 at a concentration of 20 μM for 12 hours; Homoligand Changes in EGFR protein levels after pretreating cells with the indicated inhibitors for 8 hours and then treating HCC827 cells with DC1-206 for 12 hours;

[0036] Figure 8 Changes in EGFR protein levels after pretreating cells with different inhibitors at the indicated concentrations for 8 hours and then treating HCC827 cells with DC1-206 for 12 hours;

[0037] Figure 9 Effects of EGFR and its related downstream signaling pathways after treating HCC827 cells with different concentrations of DC1-206 for 12 hours;

[0038] Figure 10 Changes in cell cycle after treating HCC827 cells with different concentrations of DC1-206 for 12 hours;

[0039] Figure 11 Changes in apoptosis rate after treating HCC827 cells with different concentrations of DC1-206 for 12 hours;

[0040] Figure 12 Flow chart of the study of the anti-tumor effect of epidermal growth factor receptor degrading proteolysis targeting chimera DC1-206 in tumor-bearing mice;

[0041] Figure 13 Mouse tumors obtained after dissecting tumor-bearing mice treated with epidermal growth factor receptor degrading proteolysis targeting chimera DC1-206;

[0042] Figure 14 Changes in tumor volume after treating tumor-bearing mice with epidermal growth factor receptor degrading proteolysis targeting chimera DC1-206;

[0043] Figure 15 Figure 4 is a graph showing the change in tumor weight in tumor-bearing mice treated with the proteolysis targeting chimera DC1-206 for degradation of epidermal growth factor receptor;

[0044] Figure 16 Figure 5 is a graph showing the change in body weight in tumor-bearing mice treated with the proteolysis targeting chimera DC1-206 for degradation of epidermal growth factor receptor;

[0045] Figure 17 Figure 6 is a nuclear magnetic resonance hydrogen spectrum of the targeting chimera DC1-206 of the present application;

[0046] Figure 18 Figure 7 is a structural formula of DC1-35 in Example 3. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0048] It should be noted that each installation method and each technical term mentioned in the present application are technical terms well known in the art, and therefore will not be explained in more detail. In addition, the same reference numerals are used for the same components, but this does not affect or should not affect the accurate understanding of the technical solutions by those skilled in the art.

[0049] Example 1 This example provides a new proteolysis targeting chimera DC1-206 for preparing a drug for treating EGFR mutant non-small cell lung cancer, which is designed and synthesized based on E3 ligase GID4, and the structural formula is:

[0050] .

[0051] The reaction equation for preparing the targeting chimera DC1-206 is as follows:

[0052] .

[0053] The specific preparation steps of the targeting chimera DC1-206 are as follows (the overnight reaction time involved in this example is > 12 h):

[0054] Step 1. Preparation of intermediate A:

[0055] 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;

[0056] 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.

[0057] The reaction equation is:

[0058] .

[0059] The H NMR spectrum data of the intermediate A obtained are:

[0060] 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).

[0061] Step 2. Preparation of intermediate B:

[0062] To 5.70 mmol of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, 11.40 mmol of triethylamine and 2.85 mmol of DMAP in dichloromethane was added 11.40 mmol of TsCl (p-toluenesulfonyl chloride) dropwise at 0°C, and stirred at room temperature overnight. The light yellow oil was obtained after washing and drying; 3.13 mmol of the light yellow oil, 6.26 mmol of 4-amino-3-nitrophenol and 6.26 mmol of potassium carbonate were dissolved in acetonitrile, and refluxed for 12 h to obtain the intermediate B-1;

[0063] The intermediate B-1 was dissolved in methanol, and 10% Pd / C catalyst was added. After vacuum replacement with air, hydrogen was introduced, and stirred at room temperature overnight to obtain the intermediate B by hydrogen gas reduction;

[0064] The reaction equation is as follows:

[0065] .

[0066] Step 3. Preparation of the intermediate C:

[0067] The intermediate A obtained in step 1, the intermediate B (crude product was directly used) obtained in step 2 and 0.28 mmol of HBTU were dissolved in acetonitrile at 0°C, and 0.55 mmol of DIPEA was slowly added. After condensation reaction, the obtained product was added with HOAc, and reacted at 70°C for 1 h to obtain the intermediate C;

[0068] The reaction equation is as follows:

[0069] .

[0070] The obtained intermediate C has the following nuclear magnetic resonance hydrogen spectrum data:

[0071] 1HNMR (400 MHz, Chloroform-d) δ 7.70 (d, J = 7.3 Hz, 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).

[0072] Step 4. Preparation of intermediate D:

[0073] At 0 °C, 5.70 mmol of 4-(3-hydroxypropyl)piperazine-1-carboxylic acid tert-butyl ester, 11.40 mmol of triethylamine and 2.85 mmol of DMAP were dissolved in dichloromethane, 11.40 mmol of TsCl was added dropwise after stirring uniformly, and the temperature was slowly increased to room temperature. After stirring overnight, the product was washed and dried, and then purified using petroleum ether / ethyl acetate to obtain intermediate D-1;

[0074] Then 3.09 mmol of intermediate D-1 and 6.18 mmol of K2CO3 were dissolved in acetonitrile, 4.65 mmol of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol was slowly added, and the reaction was carried out at reflux for 12 h to obtain the substitution product. The substitution product was added to a mixture of trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, and the reaction was carried out at 0 °C for 1 h to remove the Boc group and obtain 0.32 g of intermediate D-2;

[0075] Then 0.72 mmol of intermediate D-2 and 1.43 mmol of K2CO3 were dissolved in acetonitrile, 1.08 mmol of tert-butyl 2-bromoacetate was slowly added, and the reaction was carried out at reflux for 12 h to obtain intermediate D-3;

[0076] Finally, intermediate D-3 was added to a mixture of trifluoroacetic acid and dichloromethane in a volume ratio of 1:1, and the reaction was carried out at 0 °C for 1 h to remove the tert-butyl group and obtain intermediate D;

[0077] The reaction equation is as follows:

[0078] .

[0079] The obtained intermediate D has the following nuclear magnetic resonance hydrogen spectrum data:

[0080] 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).

[0081] Step 5. Preparation of targeting chimera DC1-206:

[0082] 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.

[0083] The reaction equation is:

[0084] .

[0085] The NMR spectrum of the obtained targeted chimera DC1-206 is as follows Figure 17 As shown, the corresponding H NMR spectrum data is:

[0086] 1HNMR (400 MHz, Methanol-d4) δ 8.38 (s, 1H), 7.95 (dd, J = 6.7, 2.7 Hz, 1H), 7.63 - 7.56 (m, 2H), 7.32 (dd, J = 8.2, 5.9 Hz, 2H), 7.19 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.06 (s, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.94 - 6.89 (m, 1H), 6.87 - 6.79 (m, 2H), 6.22 (s, 1H), 4.10 (t, J = 5.7, 3.6 Hz, 2H), 4.05 (t, J = 6.2 Hz, 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, 3.0 Hz, 2H), 3.60 (dd, J = 5.8, 3.0 Hz, 2H), 3.52 (t, J = 5.2 Hz, 2H), 3.37 (t, J = 5.2 Hz, 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).

[0087] Experimental Example 1 This experimental example verifies the effect of the new epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 designed and synthesized in Example 1 based on E3 ligase GID4 on the proliferation of human lung adenocarcinoma cell lines, and the experimental process is as follows:

[0088] (1) Select H1975 (epidermal growth factor receptor T790M / L858R mutant), HCC827 (epidermal growth factor receptor exon 19 deletion), and H1299 (epidermal growth factor receptor wild type) three human lung adenocarcinoma cell lines, respectively, and use DMEM medium containing 10% FBS to culture, place the cells in a 10 cm cell culture dish, and place it in a 37°C, 5% CO2 saturated humidity incubator, until the cells are in the logarithmic growth phase, use 0.25% trypsin-EDTA digestion solution for cell passage; at the same time, dissolve the targeting chimera DC1-206 of Example 1 in DMSO solution as the experimental group, and select the first generation of EGFR-TKI gefitinib as the control group.

[0089] (2) Take the logarithmic phase growth phase cells, 5x103 cells per well are plated in 96-well plates, and the 96-well plates are placed in the cell incubator overnight. After the cells adhere, the cells are treated with DC1-206 of the experimental group and gefitinib of the control group, respectively. The drug concentration gradient of each group is 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, respectively, with 4 replicate wells in each group. The culture medium liquid is 100 μL per well. After 48 h of treatment, the 96-well plates are taken out, 10 μL of CCK-8 solution is added to each well, and the incubation is continued at 37°C in the dark for 1 h. The OD 450 nm absorbance value of each well is measured by an enzyme-labeled instrument.

[0090] (3) Each group of experiments is repeated 3 times, and finally, the average value of each replicate well is taken. The drug inhibition rate calculation formula is: (absorbance value of the unadded drug group-absorbance value of the experimental group) / absorbance value of the unadded drug group x 100%. The IC50 concentration of DC1-206 and gefitinib (as shown in Table 1) and the cell activity curve (as shown in FIG. 2) are finally obtained. Figure 1 and Figure 2

[0091] Figure 1 It can be seen that the IC50 of DC1-206 in H1975 is 66.72 μmol / L, the IC50 of HCC827 is 11.57 μmol / L, and the IC50 of H1299 is 144.6 μmol / L. It can be seen from Figure 2 that the IC50 of gefitinib in H1975 is 4.875 μmol / L, the IC50 of HCC827 is 1.698 μmol / L, and the IC50 of H1299 is 8.458 μmol / L. The above results show that, compared with the control group of gefitinib, the novel epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 designed and synthesized in Example 1 has a significant anti-proliferation effect on human lung adenocarcinoma cell lines with mutant EGFR.

[0092] Table 1: Results of the half-inhibitory concentration IC50 of DC1-206 and gefitinib

[0093]

[0094] ​​Experimental Example 2 This experimental example further verifies the degradation effect of the new epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 designed and synthesized in Example 1 on mutant EGFR in human lung adenocarcinoma cell lines. In order to further study whether DC1-206 can degrade epidermal growth factor receptor protein, the targeting chimera DC1-206 is used to treat three human lung adenocarcinoma cell lines in this experimental example, and the carrier (0.1% dimethyl sulfoxide) is used as a control group. The experimental process is as follows:

[0095] Cell seeding was performed one day before drug treatment to ensure that the confluence of the cells reached approximately 60% when the drugs were 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 plates containing the target cells. The target cells were treated according to the concentration gradient: 0 μmol / L, 1 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L and the time gradient: 0 h, 3 h, 6 h, 9 h, 12 h, 15 h. Subsequently, the protein samples were prepared according to the Western Blot experimental procedure to evaluate the effect of PROTACs on target proteins in cells. Finally, the protein was extracted and the change in epidermal growth factor receptor protein level was detected by Western blot analysis.

[0096] The results are shown in Figure 3 , and Figure 3 A and 3B can be seen that the targeting chimera DC1-206 of the experimental group induces the degradation of mutant epidermal growth factor receptor in lung cancer cell line HCC827 in a concentration-dependent manner. The effect of DC1-206 in inducing WTEGFR degradation in H1299 is shown in Figure 3 C, Figure 3 C can be seen that DC1-206 does not show significant WT epidermal growth factor receptor degradation. In addition, the band intensities of epidermal growth factor receptor and GAPDH in Western blot were quantified using ImageJ software, and their ratios were calculated as the normalization results. Nonlinear regression curve fitting was performed on the data using GraphPad Prism 8.0, and the degradation concentration when EGFR reached half-maximal degradation rate (DC50) was calculated. As shown in Figure 4 , it is calculated that the DC50 of DC1-206 for mutant EGFR in HCC827 cells is 2.330 μM, and the maximum degradation rate of mutant EGFR in HCC827 cells at a concentration of 20 μM is 88.85%.

[0097] The above results further confirm the targeting selectivity of the PROTACs for the epidermal growth factor receptor 19 exon 19del, and subsequently, the effect of target protein degradation was verified in the HCC827 cell line with the addition of the degrader DC1-206 at the same starting time and different time gradients. The results are shown in Figure 5 and Figure 6 DC1-206 induced the degradation of mutant epidermal growth factor receptors in a time-dependent manner, and when the concentration was 20 μM, the protein levels of the two mutant epidermal growth factor receptors decreased over time, and the time required for DC1-206 to degrade 50% of the protein (t1 / 2) was 6.81 h. Further illustrate that the novel epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 of the application can effectively target epidermal growth factor receptor mutant lung cancer cells, especially the epidermal growth factor receptor 19 exon deletion of HCC827 cells, without affecting the epidermal growth factor receptor T790M / L858R of H1975 cells and the epidermal growth factor receptor WT of H1299 cells.

[0098] Experimental Example 3 This experimental example further verifies the mechanism of the novel epidermal growth factor receptor degradation proteolysis targeting chimera DC1-206 designed and synthesized in Example 1 to degrade mutant EGFR. In order to study whether the degradation of mutant EGFR by DC1-206 is dependent on the binding of the ligand to the target, the cells were inoculated one day before drug treatment to ensure that the confluence of the cells reached about 60% when the drug was added. The experimental process is as follows:

[0099] (1) In order to determine whether the degradation of the target protein by EGFR-PROTAC is dependent on the binding of EGFR ligand and GID4 E3 ubiquitin ligand to EGFR and GID4 E3 ubiquitin ligand, respectively, DC1-206 was dissolved in an appropriate amount of DMSO, and then the cells were treated with DC1-206 as the experimental group, and EGFR ligand gefitinib, no EGFR ligand gefitinib, and E3 ligase ligand DC1-35 (structure as shown in Figure 18 ) were used as group control groups, respectively. Subsequently, protein samples were prepared according to the Western Blot experimental procedure to evaluate the effect of PROTACs on target proteins in cells, and then the proteins were extracted and analyzed by Western blot to detect the changes in epidermal growth factor receptor protein levels. The results are shown in Figure 7 It can be concluded that EGFR ligand gefitinib and E3 ligase ligand DC1-35 can competitively bind with DC1-206, and weaken the effect of PROTACs on degrading target proteins.

[0100] (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.

[0101] 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:

[0102] 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.

[0103] 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:

[0104] 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 and 11 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.

[0105] 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:

[0106] 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.

[0107] The present application is not limited to the details of the foregoing exemplary embodiments and can be practiced with modification and alteration within the scope of the present application, which is not limited to the details of the foregoing exemplary embodiments. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense. It is intended that the present application be construed as including all such modifications and alterations insofar as they come within the scope of the claims appended hereto along with the equivalents thereof. No limitation is intended to any of the drawings' figures, which are to be considered as illustrative only and non-limiting.

[0108] The above description is merely illustrative of the application and not restrictive.

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: ； The EGFR mutant non-small cell lung cancer cells are HCC827 cells.

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

3. 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.

4. The use according to claim 3, 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.

5. The use according to claim 4, 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.

6. The use according to claim 3, 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.

7. The use according to claim 3, 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.

8. The use according to claim 3, 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.

9. The use according to claim 3, 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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