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)

Abstract

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.

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

“…The log p value for [ 77 Br]9 was 1.72 ± 0.21, which indicates that [ 77 Br]9 has appropriate lipophilicity for passive membrane penetration. The log p value of [ 77 Br]9 was significantly lower than that of [ 125 I]ICO1686, which was 1.84 ± 0.01 (p = 0.0097) [9]. This result is consistent with the reverse-phase HPLC analysis, in which the retention time of [ 77 Br]9 was shorter than that of [ 125 I]ICO1686.…”

“…In our previous study, an in vitro kinase inhibition assay with ICO1686 and CO-1686 showed their high selectivity toward double mutations EGFR L858R/T790M compared with that toward wild-type EGFR [ 9 ]. Therefore, we conducted a further study by designing and synthesizing radiobrominated [ 77 Br] 9 as a surrogate of [ 76 Br] 9 , which can be used as a molecular imaging agent for EGFR L858R/T790M because bromine could be a bioisostere of iodine.…”

“…Intermediate compounds were synthesized according to the previous studies, with a slight modification [ 9 , 25 , 26 , 27 , 28 ]. For compounds 1 – 3 and tin precursor 10 , materials synthesized in the previous study [ 9 ] were used. NMR spectra are provided in the Supporting Information (Figures S3–S8) .…”

“…WST-8 assay was used to evaluate IC 50 of 9 toward NSCLC cell lines (H1975, H441 and H3255) as described previously [ 9 ]. The reference compounds; CO-1686, ICO-1686, and gefitinib, have been evaluated in our previous study using the same source of cell lines [ 9 ].…”

“…We recently synthesized and evaluated a radiotracer, [ 125 I]ICO1686, which is a derivative of one of the third-generation EGFR-TKIs, CO-1686, with a high affinity for EGFR with L858R/T790M double mutations. [ 125 I]ICO1686 could detect the EGFR with L858R/T790M double mutations in vitro; however, it showed insufficient tumor uptake and low tumor/blood ratio of radioactivity [ 9 ].…”