A small-molecule compound D6 overcomes EGFR-T790M-mediated resistance in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a deadly and highly prevalent malignancy. Targeting activated-EGFR mutations in NSCLC via EGFR tyrosine kinase inhibitor (EGFR-TKI) initially achieves a profound therapeutic response, but resistance frequently evolves, reducing treatment options. Here, we present a small-molecule compound D6 which selectively inhibits tumor cell growth and migration in NSCLC cells with EGFR-TKI-resistant T790M-EGFR-activated mutations (T790M-EGFR-AM), e.g., L858R/T790M, 19Del/T790M and L858R/T790M/C797S. D6 mimics a natural product isolated from the roots of Codonopsis pilosula and selectively competes with T790M-EGFR-AM to bind to HSP90, thus facilitating the ubiquitination dependent proteasomal degradation of T790M-EGFR-AM. By contrast, D6 has little impact on typical HSP90 chaperone activity, suggesting low systemic toxicity. Promisingly, D6 combined with erlotinib or osimertinib shows efficacy in overcoming the EGFR-TKIs-resistance in NSCLCs. Our study raises an alternative strategy to overcome T790M-mediated EGFR-TKI resistance in NSCLC via targeting the protein–protein interaction of HSP90 and T790M-EGFR by intervention with D6.


Extraction, isolation and structure identification of natural D6-1
The roots of Codonopsis pilosula (16.0 kg) were powdered and extracted under reflux with 80% EtOH (3 × 40 L × 1 h) to give a crude extract, which was suspended in water followed by successive partition with petroleum ether and EtOAc to afford an EtOAc soluble extract (220 g). This extract was divided into nine parts (

Preparation of D6
The target compound D6 was accomplished by a four-step reaction with a 10% yield.
The detailed reaction processes are as follows:
The pH was adjusted to 5.0 by adding 10% K 2 CO 3 aqueous solution over 24 h. The filtrated solids were collected and washed in a small volume of EA before vacuum drying to afford compound 1 (9.08 g, 70% yield).
SOCl 2 (5 mL) at room temperature was dropped slowly into a solution of compound 1 (9.0 g, 42 mmol) in 200 mL of dry methanol and stirred for 4 h. Then a reflux reaction was conducted for 4 h before thin layer chromatography (TLC) to complete the reaction. The reaction mixture was concentrated under reduced pressure and dried before proceeding directly to the next reaction.
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (7.0 g, 46 mmol) was added to a solution of the methyl ester of compound 1 (7.6 g, 33 mmol) in 50 mL of DMSO. After stirring for 48 h, the solution was poured into 400 mL ice-cold water and the reaction mixture was successively extracted with EA and dichloromethane (DCM). After drying with anhydrous sodium sulfate, the extraction solution was rotated and dried by silica gel column chromatography to obtain compound 2 (6.02 g, 80% yield).
BuLi (65 mL, 1.6 M, 105 mmol) at -78°C was added to a solution of dimethyl methylphosphonate (13.0 g, 105 mmol) in 150 mL of dry tetrahydrofuran (THF), and stirred for 0.5 h. Then, compound 2 (6.0 g, 26.3 mmol) was dissolved in 50 mL dry THF and added to the above reaction solution, and stirred for 1 h at -78°C . The reaction system was added to 100 mL saturated ammonium chloride solution and extracted three times with EA. The extracted phase was combined, dried by anhydrous sodium sulfate, concentrated, and then residue silica gel column chromatography was performed to obtain compound 3 (4.18 g, 50% yield).