RU2663929C2 - Use of thiochromeno[2,3-c]quinoline-12-one compound for treating non-small cells lung cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to oncology, and can be used for treating non-small cells lung cancer (NSCLC) with resistance to tyrosine kinase inhibitors of epidermal growth factor receptors. According to the invention, methods include administering a thiochromeno[2,3-c]quinoline-12-one – derivative N19.

EFFECT: use of the inventions allows treating resistant NSCLC by suppressing HSP90, epidermal growth factor receptors, and cMET without retinal cytotoxicity.

13 cl, 4 ex, 14 dwg

Description

Technical field

The present invention relates to the use of a derivative of a thiochromeno [2,3-c] quinolin-12-one compound for the treatment of non-small cell lung cancer (NSCLC) with resistance to epidermal growth factor receptor tyrosine kinase inhibitors.

State of the art

Mutations in the epidermal growth factor receptor (EGFR) are considered promising biomarkers for therapy using tyrosine kinase inhibitors (ITCs) as a treatment for non-small cell lung cancer (NSCLC) (See refs. 1, 2, 3).

The mechanisms of primary TTC resistance are not yet fully understood, but overexpression of paxillin (PXN) provides primary TTC resistance in NSCLC by modulating the resistance of the Msd-1 and BIM proteins due to activation of the extracellularly regulated ERK kinase (See ref. 6). The combination of TKI with an ERK inhibitor, selumetinib, improves TKI sensitivity and results in cell and animal models (See ref. 7, 8). Unfortunately, no benefits have yet been established from the combination of an ERK inhibitor and ITC in the treatment of patients with NSCLC.

Resistance to ITC is often found in patients with REFR-mutated NSCLC who underwent treatment with ITC, and this resistance is considered acquired (secondary) (See reference 4, 5). The most common mutation of acquired resistance in EGFR is T790M in exon 20 (See ref. 9, 10). The REFR-T790M mutation and the cMET amplification are 50-60% and 5-20%, respectively, of the observed ITR REFR resistance in patients with NSCLC (See ref. 9, 10). Protein expression and phosphorylation of REFR-T790M and cMET are associated with primary and acquired resistance to TTI-targeted therapy in such patients.

Thus, the development of a new generation of inhibitors of ITR REFR and cMET is the most important strategy for overcoming ITC REFR resistance in NSCLC (See ref. 11-19). Unfortunately, REFR-independent mechanisms of acquired resistance to AZD9291, a third-generation TEC, have already been detected in REFR-E790M-positive patients with NSCLC (See Ref. 20).

In mouse models with lung cancer, with marked DEL19-T790M or L858R-T790M REFR mutations, with simultaneous overexpression of cMET, there was no significant regression of the tumor in response to monotherapy targeting only REFR or cMET (See ref. 21). On the contrary, combination therapy, which was simultaneously aimed at both REFR and cMET, was extremely effective against NTC REFR-resistant tumors caused jointly by Del19-T790M or L858R-T790M and cMET. Despite such a promising result, the same combined approach with REFR + cMET ITC inhibitors was not successful in clinical trials in humans with NSCLC with REFR mutations (See Ref. 22).

Thus, in order to develop a direction for the treatment of NSCLC with REFR mutations, the inventor is trying to find a double REFR and cMET inhibitor in order to get a better treatment effect than with the combination of a REFR inhibitor and cMET inhibitor. This is an important issue that this invention is directed to.

Previous patents TW 1488843, US 8927717 and journal articles by the inventor (Ref. 23) have shown that derivatives of thiochromeno [2,3-c] quinolin-12-one inhibit the proliferation of DU-145 and PC-3 NSCLC. In the present invention, the inventor attempts to further investigate the existence of a thiochromeno [2,3-c] quinolin-12-one derivative that can be used to suppress EGFR and cMET in order to cure NSCLC cells with resistance to ITR EGFR.

Summary of the invention

On the one hand, the present invention provides a method for the treatment of non-small cell lung cancer with resistance to ITR REFR, which consists in administering a therapeutically effective amount of an active compound selected from the groups comprising derivatives of the compound thiochromeno [2,3-c] quinolin-12-one, their pharmaceutically acceptable salts

stereoisomers and enantiomers, where the derivative compound thiochromeno [2,3-c] quinolin-12-one has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, where n1 = 3 and n2 = 2.

According to the invention, TTI resistance includes primary TTI resistance or acquired TTI resistance.

According to the invention, primary resistance to TTI is caused by overexpression of paxillin.

According to the invention, acquired resistance to ITC is a mutation of REFR T790M in exon 20.

According to the invention, the treatment of non-small cell lung cancer is provided by apoptosis.

According to the invention, the active compound is administered with at least one pharmaceutically acceptable base, diluent or excipient.

On the other hand, the invention provides a method of suppressing HSP90 for the treatment of non-small cell lung cancer with resistance to epidermal growth factor receptor tyrosine kinase inhibitors, which consists in administering a therapeutically effective amount of an active compound selected from groups including derivatives of the compound

thiochromeno [2,3-c] quinolin-12-one, their pharmaceutically acceptable salts, stereoisomers and enantiomers, where the derivative of thiochromeno [2,3-c] quinolin-12-one has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, n1 = 3, and n2 = 2.

According to the invention, a derivative compound

thiochromeno [2,3-c] quinolin-12-one reduces the expression of p-EGFR, p-Src, pY118-PXN, p-AKT, p-ERK, or p-cMET or increases the expression of BIM.

According to the invention, wherein the active compound is administered with at least one pharmaceutically acceptable base, diluent or excipient.

On the other hand, the invention provides a method for suppressing EGFR and cMET for the treatment of non-small cell lung cancer with resistance to epidermal growth factor receptor tyrosine kinase inhibitors, which consists in administering a therapeutically effective amount of an active compound selected from the groups comprising thiochromeo derivatives [2,3-c ] quinolin-12-one, their pharmaceutically acceptable salts, stereoisomers and enantiomers, where the derivative compound thiochromeno [2,3-c] quinolin-12-one has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, where n1 = 3 and n2 = 2.

According to the invention, the suppression of EGFR and cMET is a decrease in the expression of EGFR and cMET.

According to the invention, the suppression of EGFR and cMET is the destruction of EGFR and cMET via ubiquitinylation.

According to the invention, the active compound is administered with at least one pharmaceutically acceptable base, diluent or excipient.

A brief description of the graphic materials

In FIG. Figure 1 shows the effect on NSCLC cells with an REFR mutation by gefitinib. PXN expression and viability of gefitinib cells for 48 hours among NSCLC cells with REFR mutation.

In FIG. Figure 2 shows the effect of N19 on NSCLC cells with an REFR mutation. Cell viability with N19 for 48 hours.

In FIG. Figure 3 shows the effects of gefitinib and N19 on the colony formation of NSCLC cells.

In FIG. Figure 4 shows the effects of gefitinib and N19 on apoptosis of NSCLC cells.

In FIG. Figure 5 shows the effects of gefitinib and N19 on the expression of EGFR protein, cMET, and their molecules.

In FIG. Figure 6 shows the effect of N19 on EGFR and cMET via the proteasome.

In FIG. Figure 7 shows the effect of N19 on endogenous EGFR and cMET.

In FIG. Figure 8 shows the effect of N19 on the polyubiquitinylation of EEGF and cMET.

In FIG. Figure 9 shows the effects of N19 on EGFR and cMET and apoptosis.

In FIG. Figure 10 shows the effect of N19 on the degradation of EGFR and cMET protein.

In FIG. Figure 11 shows the effect of N19 on degradation of EGFR and cMET protein.

In FIG. Figure 12 shows the effect of N19 on the interaction between HSP90 and REFR or cMET.

In FIG. 13 shows the effect of N19 on tumor growth.

In FIG. Figure 14 shows the cytotoxic effect of N19 on ARPE-19 retinal cells.

Detailed Description of a Preferred Embodiment

Unless otherwise specified, the technical and scientific terms in this manual refer to common meanings that are known to the ordinary technician in this field.

The terms “treatment”, “on treatment” and similar terms refer to methods that can slow down, improve, reduce or reverse the disease or related symptoms that the patient is currently suffering from, as well as the prevention of the disease or any symptoms that arise.

The term “medically acceptable” means that the material or compound should be compatible with the other ingredients of the composition and should not harm the patient.

The present invention is demonstrated by the following examples, but is not limited to. Medicines and biomaterials are marketed and readily available. Below is just a channel through which they can be obtained.

Chemicals and antibodies: Gefitinib, SU11274, 17-DMAG and 17-AAG were obtained from Selleckchem (Houston, Texas, USA). All other chemicals were purchased from Sigma Chemical (St. Louis, Missouri, USA), unless otherwise indicated. Anti-REFR antibodies, anti-ERK (total), anti-phosphorylated (p) -ERK, anti-AKT (total), and anti-p-AKT were obtained from Cell Signaling (Danvers, Mass.) , USA). Anti-HSP70 and anti-H8P90 antibodies were obtained from Genetex (Irvine, California, USA). All other antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

The thiochromeno [2,3-c] quinolin-12-one derivative used in the examples has the following structure:

R group: -NH (CH ₂ ) ₃ NH (CH ₂ ) ₂ OH, name according to the International Union of Theoretical and Applied Chemistry: 10-Chloro-6 - ((3 - ((2-hydroxyethyl) amino) propyl) amino) - 12H-thiochromeno [2,3-c] quinolin-12-one, hereinafter TC19 (N19, NSC777201).

Animal model experiments: Tumor cells were injected subcutaneously into the back of 4-5 week old female nude mice. The xenograft tumor size was measured every 3 days, and the tumor volume was determined as (length × width ² ) / 2. When the tumors grew to 50 mm, the mice were divided into the following groups in random order: base (DMSO), gefitinib (5 mg / kg) or N19 (5 mg / kg). Drugs were administered by intraperitoneal injection every 3 days.

Plasmid Construction and Transfection: The PXN overexpression plasmid was kindly provided by Dr. Salgia (University of Chicago, Chicago, Illinois, USA). REFR (TRCN0000121068) and CMET (TRCN0000009850) were purchased from the RINA National Profile Center, Sinik Academy (Taipei, Taiwan). These plasmids were temporarily transfected into lung cancer cells (1 × 10 ⁶ ) using Turbofect reagent (Formentas, Glen Burnie, Maryland, USA). After 48 hours, cells were collected for analysis in subsequent experiments.

MTT cytotoxicity analysis: Before treatment, cells in the exponential growth phase were pretreated with overexpression and knockdown plasmids for 24 hours. The in vitro cytotoxic effects of this treatment were determined by MTT analysis.

(3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide) (550 nm), and cell viability was expressed as a percentage of control (untreated) cells (% control).

Annexin V-PI staining assay: Cells were harvested by trypsinization and centrifugation at 1000 × g for 5 min. After resuspension in the fixing buffer solution (10 mM HEPES-NaOH, 140 mM NaCl, 2.5 mM CaCl ₂ ) with a final cell density of 1-2 × 10 ⁶ cells per ml, 100 μl of a single cell suspension (1-2 × 10 ⁵ cells) was kept together with 5 μl of annexin V-FITC and 5 μl of protease inhibitor for 15 minutes at room temperature in the dark. After adding 400 μl of fixative buffer solution, the samples were analyzed using a BD FACS Calibur flow pitometer (BD Biosciences, San Jose, California, USA) for 1 hour. For each sample, 10,000 events were counted.

Colony Formation Analysis: Cell culture was performed in 6-well plates in complement medium overnight. After incubation, the culture media were replaced with fresh medium containing gefitinib, N19 and DMSO as a solvent control for 48 hours. The treated cells were seeded in new medium supplemented with 10% fetal bovine serum for another 10 days. Before the pictures of the colonies, the cells were stained with 0.01% crystal violet for 1 h at room temperature.

Western blotting and immunoprecipitation: Total cell lysates were prepared in immunoprecipitated lysis buffer (20 mM Tris, pH 7.5, 100 mM sodium chloride, 1% IGEPAL CA-630, 100 μM Na ₃ VO ₄ , 50 mM NaF, 30 mM sodium pyrophosphate) containing a complete mixture of protease inhibitors with or without EDTA (Roche Diagnostics, Basel, Switzerland). Protein concentrations were measured using a Bio-Rad protein assay (Bio-Rad (Bio-Rad), Richmond, California, USA). Proteins were immunoprecipitated with 1 μg of primary antibodies and Protein A pellets (Sigma, Danvers, Mass., USA) for 4 hours and then washed three times with lysis buffer. Protein samples were separated by 10% gel in the presence of sodium dodecyl sulfate, transferred to a polyvinylidene difluoride membrane (Millipore, Bill Erica, Massachusetts, USA), and finally, immunoblotting with primary antibodies was performed. Primary antibodies for immunoblotting were used in solution from 1: 500 to 1: 1000. Secondary mouse, goat and rabbit antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, Texas, USA) were used in a 1: 5000 solution. Protein signals were detected using chemiluminescent reagents (Amersham Pharmacia, Piscataway, New Jersey, USA).

The present invention is demonstrated by the following examples, but is not limited to.

Example 1. N19 is more effective than gefitinib for apoptotic suppression of cell viability and colony formation in NSCLC cells with an REFR mutation

PXN confers primary resistance to TTC in NSCLC cells with an REFR mutation. The value of IC ₅₀ for gefitinib in six cell lines of NSCLC with a mutation of REFR was estimated using MTT analysis. The IC ₅₀ value for gefitinib in H1975, H1650, CL97, and PC9GR cells (PC9 cells resistant to gefitinib) ranged from 13.2 to 13.8 μM. The lowest IC ₅₀ value was observed in PC9 cells, but the IC ₅₀ value significantly increased (up to 14.6 μM) due to ectopic expression of PXN in these cells (Fig. 1).

Interestingly, the IC ₅₀ value for N19 in these cell lines ranged from 5.5 to 7.0 μM, but a relatively low IC ₅₀ value (4.9 μM) for N19 was observed in PC9 cells. According to an MTT assay, N19 did not show cytotoxicity in normal lung cell lines WI38 and Beas-2B (Fig. 2).

In FIG. Figure 3 shows representative growth of agar plate colonies. N19 is more effective than gefitinib in suppressing colony formation in all types of cells except for PC9 cells (Fig. 3, bottom panel). Representative apoptotic profiles of these cells after treatment with gefitinib or N19 and analysis with V-PI annexin staining are shown in FIG. 4. The percentage of apoptotic cells was higher after treatment with N19 than after treatment with gefitinib in all types of cells except for PC9 cells (Fig. 4, bottom panel).

These results clearly showed that with N19, a stronger suppression of cell viability and colony formation is obtained than with gefitinib in NSCLC cells with an REFR mutation, and that such suppression occurs due to apoptosis.

Example 2. N19 kills NSCLC cells with an REFR mutation by apoptosis caused by the destruction of the REFR and cMET proteins

We examined the possibility that the death of NSCLC cells with an REFR mutation in response to N19 can occur due to the destruction of the REFR and cMET proteins. Forty-eight hours after treatment with gefitinib (10 μM) and N19 (10 μM), the cell lysate was collected and analyzed by western blotting. Western blotting showed a decrease in the expression levels of p-REFR and p-AKT after gefitinib treatment of PC9, PC9 cells with overexpression of PXN (PC9-PXN) and HI650, but not after gefitinib treatment of PC9GR and H1975 cells (Fig. 5). The expression levels of REFR, AKT, ERK, and cMET did not change after treatment with gefitinib in all types of cells. Interestingly, the expression of p-REFR, REFR, p-cMET, cMET, p-AKT, AKT and p-ERK was significantly suppressed by treatment with N19. Similar changes in protein expression were observed in response to treatment with gefitinib or N19 of CL97 cells with high PXN expression.

However, a decrease in the expression of EGFR and cMET from N19 in these cell types was reversed in the presence of MG132, compared with cells without N19 treatment (Fig. 6).

PC9-PXN, H1650, and PC9GR cells were treated with 100 μg / ml cycloheximide for the indicated number of hours with or without 5 μM N19. Levels of EGFR and cMET proteins were evaluated using Western blotting. The pulse chase experience with PC9-PXN, H1650, and PC9GR cells showed that the expression of REFR and cMET proteins gradually decreased from N19 during the indicated time intervals; however, the levels of both proteins remained relatively unchanged after treatment with a control solvent (Fig. 7).

PC9-PXN, H1650, and PC9GR cells were treated with 5 μM N19 for 5 hours. The polyubiquitination of EGFR and cMET was evaluated by Western blotting in immunoprecipitation. The ubiquitin ladder of the REFR and cMET proteins was evident in these cell types after treatment with N19, but was absent in the control solvent (Fig. 8). Thus, the destruction of EGFR and cMET proteins from N19 occurs due to the post-translational mechanism mediated by the ubiquitin proteasome, and not due to the modulation of the translation apparatus.

Then we checked whether cell apoptosis caused by N19 actually occurred due to the destruction of the EGFR and cMET proteins. Three types of cells (PC9-PXN, PC9GR, and HI650) were transfected with short hairpin RNA (kshRNA) REFR (kshREFR) and ksh-c-MET, individually or in combination, and then treated with 5 μM N19 for 24 hours. Western blotting showed that the levels of expression of REFR and cMET were significantly reduced after treatment with N19 in these types of cells transfected with non-specific kshRNA (NC, Fig. 9). Then, the expression of the REFR protein and cMET was reduced in cells transfected with r-REFR, r-cc-MET, or r-REFR + r-cc-MET and subjected to N19 treatment (Fig. 9). Cell apoptosis was measured using annexin V-PI staining using flow cytometry. The percentage of apoptotic cells varied with different treatment and depended on the levels of expression of EGFR and cMET in these cell types. The results showed that N19 kills NSCLC cells with an REFR mutation by apoptosis, mainly due to the destruction of the EGFR proteins and the cMET ubiquitin proteasomes.

Example 3. N19 probably acts as an inhibitor of HSP90 and kills NSCLC cells with the REFR mutation due to the simultaneous destruction of the REFR and cMET ubiquitin proteasome proteins.

REFR and CMET are HSP90 client proteins. Therefore, we hypothesized that N19 could act as an inhibitor of HSP90 and kill NSCLC cells with the REFR mutation due to the destruction of the REFR and cMET ubiquitin proteasome proteins. Forty-eight hours after treatment with 17-AAG (5 μM) or N19 (5 μM), cell lysates were collected and analyzed for signal changes using Western blotting with the indicated antibodies. Western blotting showed that expression of EGFR and cMET in PC9, PC9-PXN, H1650 and H1975 cells was almost completely suppressed by the HSP90 17-AAG or N19 inhibitor. The levels of p-AKT and p-ERK expression in these cell types were also significantly reduced after treatment with 17-AAG or N19 (Fig. 10), while the same treatment significantly increased the level of HSP70 expression in these cell types. The growth of Mc1-1 and a decrease in BIM in these cell types was reversed by treatment with 17-AAG or N19. Changes in the levels of the Mc1-1 and BIM proteins corresponded to the percentage of apoptotic cells observed after treatment with 17-AAGmraN19.

PC9-PXN and HI 650 cells were treated with gefitinib, a cET MET inhibitor SU11274, a combination of gefitinib + SU11274, 17-AAG or N19 to determine whether PXN mediated resistance to ITC can be overcome with gefitinib + SU11274, as well as 17-AAG or N19. Cells were treated with gefitinib, SU11274, gefitinib + SU11274, 17-AAG or N19 for 48 hours. Cell apoptosis was measured using annexin V-PI staining using flow cytometry. Cell lysates were collected and analyzed for signal changes using Western blotting with the indicated antibodies. Western blotting showed that the expression levels of p-REFR, p-Src, pY118-PXN, p-ACT, p-ERK and p-cMET were significantly reduced from gefitinib + SU11274, 17-AAG or N19 in both types of cells, but only partially reduced from one gefitinib or SU11274 (Fig. 11). The appearance of HSP70 expression was observed in both types of cells, but only after treatment with 17-AAG or N19, and not other types of treatment (Fig. 11). The expression of Mc1-1 and BIM was reversed by gefitinib + SU 11274, and such reversal was also observed after treatment with 17-AAG or N19. A similarly high percentage of apoptotic cells was observed in both types of cells after treatment with gefitinib + SU11274, 17-AAG or N19, but not after treatment with gefitinib or SU11274 alone (Fig. 11). H1650 cells were treated with N19 (10 μM) for 43 hours, and then MG132 for another 5 hours. Cell lysates were immunoprecipitated with anti-HSP90-conjugated granules. Immunoprecipitates were evaluated to determine the expression of EGFR and cMET using Western blotting. β-actin was used as a control for application. Immunoprecipitation analysis showed that the interaction of HSP90 with EGFR or cMET in HI 650 cells decreased with N19 treatment in the presence or absence of MG132 (Fig. 12). These results show that N19 probably acts as an inhibitor of HSP90 and kills NSCLC cells with the REFR mutation by apoptosis due to the destruction of the REFR and cMET proteins.

Then we compared the inhibition of subcutaneous tumor growth in athymic mice caused by the PC9-PX # stable clone due to treatment with N19 and gefitinib. Representative tumor mass and subcutaneous tumor mice are shown in FIG. 13. The photographs depict representative tumor masses in mice after administration of the drug for 27 days via intraperitoneal injection. Representative tumor samples were excised on day 27. The effect of N19 on the body weight of mice. The mean ± standard deviation was calculated by tumor volume and body weight of five nude mice in each group. The tumor mass in athymic mice caused by the PC9-VC or PC9-PXN-stable clone was almost completely suppressed by N19. The inhibitory effect of gefitinib was also observed in the tumor mass of athymic mice caused by vector control cells. However, tumor mass in athymic mice caused by PC9-PXN-stable clone or PC9-VC cells gradually decreased over 27 days while the mice were treated with gefitinib or a control solvent. The body weight of the mice did not change during treatment with N19 or gefitinib during the 27-day study. A similar inhibitory effect was observed in N19 for tumor mass caused by PC9GR in cell and animal models. These results clearly showed that N19 completely suppresses tumor growth caused by the PC9-PXN-stable clone and PC9GR cells. Therefore, we believe that N19 effectively suppresses tumor xenograft formation with primary and acquired resistance to TTI.

Example 4. Analysis of retinal cytotoxicity

It is known that all existing HSP90 inhibitors have retinal cytotoxicity, and clinical trials are impossible, so we use the MTT assay to find out if N19 has cytotoxicity for ARPE-19 retinal cells. MTT analysis and annexin V-PI staining analysis were performed on ARPE cells treated with N19, 17-AAG or 17-DMAG. All data were collected from three independent experiments. The mean and standard deviation were indicated as a column with error bars. As shown in FIG. 14, N19 at a concentration of 10 μM (this concentration can effectively suppress HSP90) does not have cytotoxicity for ARPE-19 cells, but other 17-AAG or 17-DMAG HSP90 inhibitors show cytotoxicity for ARPE-19 cells.

This invention solves the problem of the previous treatment of NSCLC, an EGFR inhibitor and a cMET inhibitor are not yet effective for patients with NSCLC with a mutation of ITR REFR, and all HSP90 inhibitors (in the opposite direction from REFR, cMET) have a deficiency in the form of cytotoxicity for retinal cells. The invention has the following effects: 1) N19 has a therapeutic effect in NSCLC with primary resistance to TEC due to overexpression of PXN, 2) N19 has a therapeutic effect in NSCLC with acquired resistance to TEC due to T790M mutation, 3) N19 destroys REFR and cMET due to ubiquitinylation and leads to apoptosis of NSCLC with the REFR mutation, 4) N19 is a double inhibitor of EGF and cMET, and 5) N19 is an inhibitor of HSP90 and does not have cytotoxicity for retinal cells. As an inhibitor of HSP90, N19 can overcome the resistance of EGFR to ITC and kill NSCLC with the mutation of EGFR due to the destruction of EGFR and cMET ubiquitin proteasomes. Compared to other HSP90 inhibitors, N19 is not cytotoxic to retinal cells.

The above data are individual for practical use of the present invention. Practical cases limit the scope of the patented invention. All equal effective applications and changes consistent with the spirit of the invention should be included within the scope of the patented invention.

The multiple effect described above is fully consistent with the requirements of novelty and progressive legal monopoly. Therefore, in accordance with the law, we ask that the present invention be approved for promotion.

Bibliography

1. Mock TS. Personalized medicine in lung cancer: what we need to know. Nat Rev Clin Oncol 2011; 8: 661-668.

2. Hang Z, Stiegler AL, Boggon TJ, Kobayashi S, Halmos B. EGFR-mutated lung cancer: a paradigm of molecular oncology. Oncotarget 2010; 1: 497-514.

3. Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009; 361: 958-967.

4. Gazdar AF. Activating and resistance mutations of EGFR in non-small-cell lung cancer: role in clinical response to EGFR tyrosine kinase inhibitors. Oncogene 2009; 28 (Suppl 1): S24-S31.

5. Tan CS, Gilligan D, Pacey S. Treatment approaches for EGFR-inhibitor-resistant patients with non-small-cell lung cancer. Lancet Oncol 2015; 16: e447-e459.

6. Wu DW, Chen CY, Chu CL, Lee H. Paxillin confers resistance to tyrosine kinase inhibitors in EGFR-mutant lung cancers via modulating BIM and Mcl-1 protein stability. Oncogene 2015; 35: 621-630.

7. Huang MH, Lee J, Chang YJ, Tsai HH, Lin YL, Lin AM et al. MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib. Mol Oncol 2013; 7: 112-120.

8. Eberlein CA, Stetson D, Markovets AA, Al-Kadhimi KJ, Lai Z, Fisher PR et al. Acquired resistance to the mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models. Cancer Res 2015; 75: 2489-2500.

9. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007; 316: 1039-1043.

10. Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA 2007; 104: 20932-20937.

11. Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov 2014; 4: 1046-1061.

12. Nanjo S, Yamada T, Nishihara H, Takeuchi S, Sano T, Nakagawa T et al. Ability of the Met kinase inhibitor crizotinib and new generation EGFR inhibitors to overcome resistance to EGFR inhibitors. PLoS One 2013; 8: e84700.

13. Janne PA, Yang JC, Kim DW, Planchard D, Ohe Y, Ramalingam SS et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N Engl J Med 2015; 372: 1689-1699.

P,

I, Baska F,

C, Greff Z et al. Discovery and biological evaluation of novel dual EGFR/c-Met inhibitors. ACS Med Chem Lett 2014; 5: 298-303.14. Szokol B,

P

I, Baska F,

C, Greff Z et al. Discovery and biological evaluation of novel dual EGFR / c-Met inhibitors. ACS Med Chem Lett 2014; 5: 298-303.

15. Takeuchi S, Wang W, Li Q, Yamada T, Kita K, Donev IS et al. Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer. Am J Pathol 2012; 181: 1034-1043.

16. Rho JK, Choi YJ, Kim SY, Kim TW, Choi EK, Yoon SJ et al. MET and AXL inhibitor NPS-1034 exerts efficacy against lung cancer cells resistant to EGFR kinase inhibitors because of MET or AXL activation. Cancer Res 2014; 74: 253-262.

17. Nakade J, Takeuchi S, Nakagawa T, Ishikawa D, Sano T, Nanjo S et al. Triple inhibition of EGFR, Met, and VEGF suppresses regrowth of HGF-triggered, erlotinib-resistant lung cancer harboring an EGFR mutation. J Thorac Oncol 2014; 9: 775-783.

18. Sano Y, Hashimoto E, Nakatani N, Abe M, Satoh Y, Sakata K et al. Combining onartuzumab with erlotinib inhibits growth of non-small cell lung cancer with activating EGFR mutations and HGF overexpression. Mol Cancer Ther 2015; 14: 533-541.

19. Sequist LV, Rolfe L, Allen AR. Rociletinib in EGFR-mutated non-small-cell lung cancer. N Engl J Med 2015; 373: 578-579.

F, Gobert A, Auger N, Lacroix L et al. EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients. Ann Oncol 2015; 26: 2073-2078.20. Planchard D, Loriot Y,

F, Gobert A, Auger N, Lacroix L et al. EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients. Ann Oncol 2015; 26: 2073-2078.

21. Xu L, Kikuchi E, Xu C, Ebi H, Ercan D, Cheng KA et al. Combined EGFR / MET or EGFR / HSP90 inhibition is effective in the treatment of lung cancers codriven by mutant EGFR containing T790M and MET. Cancer Res 2012; 72: 3302-3311.

22. Sequist LV, von Pawel J, Garmey EG, Akerley WL, Brugger W, Ferrari D et al. Randomized phase II study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non-small-cell lung cancer. J Clin Oncol 2011; 29: 3307-3315.

23. Chen TC, Wu CL, Lee CC, Chen CL, Yu DS, Huang HS. Structure-based hybridization, synthesis and biological evaluation of novel tetracyclic heterocyclic azathioxanthone analogues as potential antitumor agents. Eur J Med Chem 2015; 103: 615-627.

Claims (19)

1. A method for the treatment of non-small cell lung cancer with resistance to epidermal growth factor receptor tyrosine kinase inhibitors, which consists in administering a therapeutically effective amount of an active compound selected from the groups comprising thiochromeno [2,3-c] quinolin-12-one derivatives thereof, pharmaceutically acceptable salts, stereoisomers and enantiomers, wherein the thiochromeno [2,3-c] quinolin-12-one derivative has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, where n1 = 3 and n2 = 2. 2. The method of claim 1, wherein the resistance to tyrosine kinase inhibitors comprises primary resistance to tyrosine kinase inhibitors or acquired resistance to tyrosine kinase inhibitors. 3. The method of claim 2, wherein the primary resistance to tyrosine kinase inhibitors is caused by overexpression of paxillin. 4. The method of claim 2, wherein the acquired resistance to tyrosine kinase inhibitors is a mutation of the T790M epidermal growth factor receptor in exon 20. 5. The method according to claim 1, where the treatment of non-small cell lung cancer is provided by apoptosis. 6. The method of claim 1, wherein the active compound is administered with at least one pharmaceutically acceptable base, diluent or excipient. 7. A method of suppressing HSP90 for the treatment of non-small cell lung cancer with resistance to epidermal growth factor receptor tyrosine kinase inhibitors, which comprises administering a therapeutically effective amount of an active compound selected from the groups consisting of thiochromeno [2,3-c] quinolin-12-one derivatives , their pharmaceutically acceptable salts, stereoisomers and enantiomers, wherein the thiochromeno [2,3-c] quinolin-12-one derivative has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, where n1 = 3 and n2 = 2. 8. The method of claim 7, wherein the derivative compound thiochromeno [2,3-c] quinolin-12-one reduces the expression of p-REFR, p-Src, pY118-PXN, p-AKT, p-ERK, or p- cMET or increases the expression of BIM. 9. The method of claim 7, wherein the active compound is administered with at least one pharmaceutically acceptable base, diluent or excipient. 10. A method of suppressing epidermal growth factor receptors and cMET for the treatment of non-small cell lung cancer with resistance to epidermal growth factor receptor tyrosine kinase inhibitors, which consists in administering a therapeutically effective amount of an active compound selected from the groups including thiochromeo derivatives [2,3-c] quinolin-12-one, their pharmaceutically acceptable salts, stereoisomers and enantiomers, where the derivative compound thiochromeno [2,3-c] quinolin-12-one has the following structure:

in which the R group is —NH (CH ₂ ) _n1 NH (CH ₂ ) _n2 OH, where n1 = 3 and n2 = 2. 11. The method according to p. 10, where the suppression of epidermal growth factor receptors and cMET is a decrease in the expression of epidermal growth factor receptors and cMET. 12. The method according to p. 10, where the suppression of epidermal growth factor receptors and cMET is the destruction of epidermal growth factor receptors and cMET through ubiquitinylation. 13. The method of claim 10, wherein the active compound is administered with at least one pharmaceutically acceptable base, diluent, or excipient.

Images