Abstract 10: Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis, radiolabeling and preliminary biological studies on crizotinib and alectinib analogues

Abstract

Abstract Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, has emerged as a therapeutic target in various cancers, including lung cancer. Although several ALK inhibitors have gained regulatory approval, some of them do not cross the blood-brain barrier;1 furthermore, non-invasive indicators of target engagement or biomarkers predicting response to these agents in vivo are lacking. To overcome these limitations, fluorinated analogues of the ALK inhibitors crizotinib and alectinib (fluoroethyl crizotinib [FECr] and fluoroethyl alectinib [FEAl]) were synthesised and radiolabeled. We report the radiosynthesis and preliminary biological results on these compounds, fluoroethyl crizotinib (19F/18F-FECr) and fluoroethyl alectinib (19F/18F-FEAl). Briefly, 19F/18F-FECr and 19F/18F-FEAl were synthesized as previously reported.2 19F/18F-fluoroethyl tosylate was prepared, purified and coupled with crizotinib or alectinib. The products were purified by flash column chromatography to yield 19F/18F-FECr and 19F/18F-FEAl. Alternatively, precursor compounds were synthesized, directly fluorinated with 19F/18F-fluoride, and purified to yield 19F/18F-FECr and 19F/18F-FEAl. In vitro cytotoxicity assays were performed in lung cancer ALK-positive H2228 and ALK-negative H441 cells using non-radioactive compounds. In vivo biodistribution and PET/CT imaging studies were performed on tumor-free nude mice using 18F-labeled compounds. Chemically, the first method produced 18F-FECr in 20-24% yield (n=8), molar activity of 37 GBq/μmol, and >99% purity. The second method produced 18F-FECr and 18F-FEAl in 40% (n=6) and 20% yields, respectively. In in vitro cytotoxicity assays with ALK-positive H2228 cells, FECr showed an IC50 = 7.5 μM and FEAl an IC50 = 40 nM, similar to their cognate parental compound, confirming that the modification was not detrimental to drug potency. Positron emission tomographic (PET) imaging in tumor-free mice showed significant brain uptake of the 18F-labeled analogues at 10 min post-injection (6.5 %/ID/g, 18F-FECr; 8.2 %ID/g, 18F-FEAl). In conclusion, novel fluoroethyl-modified crizotinib and alectinib and their 18F-labeled analogues were synthesized with good yields, high purity, and high specific activity. PET and biodistribution results suggest that these fluorinated analogues may penetrate the blood-brain barrier and be potential targeted drugs for treatment of lung cancer that has metastasised to brain.