Synthesis and initial in vitro evaluation of olmutinib derivatives as prospective imaging probe for non-small cell lung cancer.

Introduction: Imaging a non-small cell lung cancer (NSCLC) using radiolabeled tyrosine kinase inhibitors (TKIs) has attracted attention due to their unique interaction with the target epidermal growth factor receptor (EGFR). Olmutinib (OTB) is one of the third-generation EGFR TKIs, which selectively inhibit EGFR L858R/T790M mutation. In this study, we aim to estimate the interaction of the iodinated OTB (I-OTB)-receptor complex by molecular docking. Furthermore, we will synthesize the I-OTB and evaluate its activity toward EGFR L858R/T790M by in vitro cytotoxicity assay. Methods: A molecular docking simulation was carried out using an AutoDock Vina program package to estimate the interaction of the ligand-receptor complex. The I-OTB, N-{3-iodo-5-[(2-{[4-(4-methylpiperazin-1-yl)phenyl]aminothieno{3,2-d}pyrimidin-4-yl)oxy]phenyl} acrylamide, was synthesized by introducing an iodine atom in the phenyl group in the 3-aryloxyanilide structure. The half inhibitory concentration (IC50) was determined by employing a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) assay to evaluate the activity of I-OTB. Results: The docking study exhibited that I-OTB could take an interaction similar to that of the parent compound. We successfully synthesized I-OTB and confirmed its structure by instrumental analysis. The binding energy of OTB and I-OTB in complex with EGFR T790M are -8.7 and -7.9 kcal/mol, respectively. The cytotoxicity assay showed that I-OTB also has an affinity towards the EGFR L858R/T790M mutation with the IC50 10.49 ± 5.64 𝜇M compared to the EGFR wild type with the IC50 over than 10 𝜇M. Conclusion: The cytotoxicity effect of I-OTB was comparable to that of OTB. This result indicates that the iodine substituent in OTB did not alter the parent compound selectivity toward double mutations EGFR L858R/T790M. Therefore, I-OTB is prominent for radioiodination, and [123/124I] I-OTB may be a promising candidate for EGFR L858R/T790M mutation imaging.

A Radiobrominated Tyrosine Kinase Inhibitor for EGFR with L858R/T790M Mutations in Lung Carcinoma.

Activating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as a positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the corresponding substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated [77Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N-chlorosuccinimide. Although we aimed to provide a novel PET imaging probe, 77Br was used as an alternative radionuclide for 76Br. We fundamentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975 (IC50 0.18 ± 0.06 µM) comparable to that of CO-1686 (IC50 0.14 ± 0.05 µM). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686. These results indicate that the binding site of the radiotracers should be identical to that of CO-1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 ± 0.17) was higher than that in H441 tumor (3.71 ± 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification.

Synthesis and Fundamental Evaluation of Radioiodinated Rociletinib (CO-1686) as a Probe to Lung Cancer with L858R/T790M Mutations of Epidermal Growth Factor Receptor (EGFR).

Rociletinib (CO-1686), a 2,4-diaminopyrimidine derivative, is a highly potent tyrosine kinase inhibitor (TKI) that acts on epidermal growth factor receptor (EGFR) with L858R/T790M mutations. We supposed radioiodinated CO-1686 would function as a useful tool for monitoring EGFR L858R/T790M mutations. To aid in patient selection before therapy with EGFR-TKIs, this study aimed to develop a 125I-labeled derivative of CO-1686, N-{3-[(2-{[4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl]amino}-5-(trifluoromethyl)pyrimidine-4-yl] amino}-5-([125I]iodophenyl)acrylamide ([125I]ICO1686) and evaluate its selectivity toward EGFR L858R/T790M. Radiosynthesis was performed by iododestannylation of the corresponding tributylstannyl precursor with [125I]NaI and N-chlorosuccinimide. The selectivity of the tracer for detecting EGFR L858R/T790M was evaluated using three relevant non-small cell lung cancer (NSCLC) cell lines-H1975, H3255 and H441 overexpressing the dual mutation EGFR L858R/T790M, active mutant EGFR L858R and wild-type EGFR, respectively. The nonradioactive ICO1686 and the precursor compound were successfully synthesized. A novel radiolabeled probe, [125I]ICO1686, was prepared with high radiochemical yield (77%) and purity (>99%). ICO1686 exhibited high cytotoxicity toward H1975 (IC50 0.20 ± 0.05 µM) and H3255 (IC50 0.50 ± 0.21 µM), which is comparable to that of CO-1686. In contrast, the cytotoxicity of ICO1686 toward H441 was 10-fold lower than that toward H1975. In the cell uptake study, the radioactivity uptake of [125I]ICO1686 in H1975 was 101.52% dose/mg, whereas the uptakes in H3255 and H441 were 33.52 and 8.95% dose/mg, respectively. The uptake of [125I]ICO1686 in H1975 was greatly reduced to 45.61% dose/mg protein by treatment with excess CO-1686. In vivo biodistribution study of the radiotracer found that its accumulation in H1975 tumor (1.77 ± 0.43% ID/g) was comparable to that in H3255 tumor (1.63 ± 0.23% ID/g) and the accumulation in H1975 tumor was not reduced by pretreatment with an excess dose of CO-1686. Although this radiotracer exhibited highly specific in vitro uptake in target cancer cells, structural modification is required to improve in vivo biodistribution.