Novel analogs of antitumor agent calixarene 0118: Synthesis, cytotoxicity, click labeling with 2-[18F]fluoroethylazide, and in vivo evaluation

• Calixarene 0118 is a promising anti-angiogenic drug currently in phase I clinical trial.
• 8 new analogs were synthesized, some proved more cytotoxic in vitro than parent 0118.
• 18F-labeled analogs were prepared using Cu-catalyzed ‘click chemistry’.
• No tumor uptake was observed in dynamic PET-scans until 3 h.
• Radiolabeled 0118 analogs allow pharmacokinetic profiling and lead optimization.

Calixarene 0118 is a potent anti-angiogenic agent that effectively inhibited tumor growth in preclinical studies, and is currently being evaluated in a phase I clinical trial. We have designed two close mimetics of calixarene 0118 containing a terminal alkynyl-functional group, and developed an optimized semi-automated procedure for radiolabeling with 2-[18F]fluoroethylazide using click chemistry. Following semi-preparative HPLC purification and formulation, the lower-rim modified analog [18F]6 and the equatorially labeled [18F]13 were obtained in >97% radiochemical purity and overall decay-corrected isolated radiochemical yields of 18.7 ± 2.7% (n = 4) and 10.2 ± 5.0% (n = 4), respectively, in a total synthesis time of about 2 h. Preliminary in vivo studies in nude mice bearing human tumor xenografts revealed highest accumulation of both tracers in the liver, followed by spleen, kidney, lung and bone, with no substantial uptake in the tumor. Still, these first-in-class radiotracers are a valuable tool for pharmacokinetic profiling and improvement of calixarene-based anti-angiogenic therapeutics in the future, as similar radiolabeling strategies may be applied to other compounds in the calixarene series. The cold reference compounds of the radiotracers were characterized in terms of cytotoxicity and anti-proliferative effects on HUVEC cells and on MA148 human ovarian carcinoma cells, along with the respective precursors, a small series of 0118 analogs modified with short-chain linear alkyl substituents, and a PEG3-spaced calixarene dimer. While all of the new analogs proved at least equipotent to parent 0118, some of them inhibited HUVEC and MA148 cell growth almost 4- and 10-fold more effectively, rendering these analogs promising candidates for further evaluation in anti-angiogenic cancer therapy.

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