Synthesis and preliminary pet imaging of ¹¹C- and ¹⁸F-isotopologues of the ROS1/ALK inhibitor PF-06463922 in nonhuman primates

Objectives A next-generation small molecule inhibitor of the orphan receptor tyrosine kinase c-ros oncogene 1 (ROS1) which has a kinase domain that is physiologically related to anaplastic lymphoma kinase (ALK), PF-06463922¹ (1) is undergoing Phase I/II clinical trial investigations in ROS1 and ALK fusion-positive non-small cell lung cancers. We set out to investigate the potential of PF-06463922 (1) as a novel PET tracer for oncology and to determine if it crosses the blood-brain-barrier, by preparing the ¹¹C- and ¹⁸F- isotopologues, and confirming brain penetration in non-human primates.

Methods [¹¹C] and [¹⁸F] PF-06463922 (1) were synthesized, by a unique 2-step automated carbon-11 methylation of the carboxamide functionality and the use of an iodonium ylide² precursor for reaction with [¹⁸F]fluoride, respectively (Scheme 1). Attempted fluorodenitration with [¹⁸F]TEAF was carried out using the nitro precursor (3-4 mg) in DMSO and heating at 215 °C for 15 minutes, however, [¹⁸F] PF-06463922 could not be identified in the reaction mixture. The radiosynthesis was then attempted using the Discovery mode of the Advion NanoTek microfluidic system and the optimal conditions were determined to be ~14 mg / ml (3 mg) of the nitro precursor in DMSO (200 µL), a 100 µm x 2 m reactor at a temperature of 220°C, with a flow rate of 40 µL/min. To ensure complete deprotection of all labeled materials, the product was hydrolyzed using 1 ml of ethanolic HCl, at 100°C for 3 minutes, however, complete HPLC separation of the nitro precursor from the final product proved to be problematic. Employing the iodonium ylide precursor and [¹⁸F]TEAF, heating at 80 °C for 10 min, followed by hydrolysis and purification by HLB SepPak, the desired product, [¹⁸F]PF-06463922, was prepared in high chemical and radiochemical purity. The C-11 isotopologue, was prepared using a 2-step labeling of the protected des-methyl precursor employing the “Loop Method”³, followed by purification and hydrolysis on a modified TracerLab FxFn synthesizer.

Results The iodonium ylide method proved to be effective for preparation of the ¹⁸F isotopologue in a 14% non-decay corrected (NDC) radiochemical yield (RCY), relative to starting [¹⁸F]fluoride as compared to the 1% yield by microfluidic fluorodenitration, and in a radiochemical purity of >97%. By using the two-step methylation procedure, the labeled intermediate could be easily separated from the precursor by HPLC and then was rapidly hydrolyzed and purified by HLB SepPak during the reformulation. The final product was obtained in an isolated yield of 3% (NDC), with a S.A. of > 3 Ci/ μmol and with > 95% radiochemical purity. Studies were performed on two adult male rhesus macaques (8-10 kg), and all experiments were performed in compliance with procedures authorized by the Institutional Animal Care and Use Committee. Whole body dynamic PET imaging revealed radioactivity primarily in liver and kidneys, the latter exhibiting signal primarily at early time points, which progressively shifted to the urinary bladder over time. High initial uptake was also seen in the brain, consistent with previous suggestions of the brain penetration properties of the non-radioactive molecule. The peak measured brain concentrations regionally exceeded 2 SUV at approximately 9-10 min post-injection. Whole brain time activity curves exhibit rapid uptake followed by fast washout of the radiotracer in normal non-human primates.

Conclusions Carbon-11 and Fluorine-18 isotopologues of PF-06463922 (1) could be prepared in good radiochemical yield and purity via a unique 2-step ¹¹C-labeling strategy or a robust iodonium ylide radiofluorination strategies, respectively. [¹¹C]PF-06463922 was routinely produced and used to perform preliminary PET imaging studies in non-human primates, confirming that this radiotracer is indeed capable of penetrating the blood brain barrier and may be suitable for oncology imaging applications in the future.

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