Automated Radiosynthesis a Novel Mu Opioid Antagonist [18F]3FN and its Preclinical Evaluation

Introduction: Opioid receptors (mu, delta, kappa and nociceptin/orphanin FQ peptide) play a key role in the mechanism of action of both synthetic and natural analgesics. Since the 1970s, PET imaging of opioid receptors has provided a useful tool to understand the role of opioid receptors in pain research as well as in psychiatric and neurological disorders. Currently, the radioligand of choice for studies related to the mu opioid receptor is agonist [¹¹C]carfentanil. [¹¹C]Carfentanil is useful in its current role but presents safety issues because of its extremely high potency.¹ The development of a selective mu opioid receptor antagonist would both address safety issues and allow for studies using agonist/antagonist radioligand pairs. A series of opioid antagonists has been screeened (Fig 1A), revealing 3FN as a promising lead for labeling with ¹⁸F. The automated radiosynthesis of selective mu opioid antagonist [¹⁸F]3FN has been successfully carried out (Fig 1B), and the new radiotracer has been evaluated in vivo in non-human primates, NHP (Fig 1C).

Methods: Stannane precursor and ¹⁹F standard were produced from tert-butyl 4-oxopiperidine-1-carboxylate in 4 steps and were fully characterized by ¹H NMR, ¹³C NMR and ¹⁹F NMR. ¹⁸F-3FN radiosynthesis was automated in a TRACERLab FX_FN module. Briefly, cyclotron produced ¹⁸F was trapped on a QMA Sep-Pak and eluted into the reactor using KOTf (10 mg) and minimal K₂CO₃ (50 µg) in H₂O (0.5 mL). Azeotropic drying was then carried out using acetonitrile (1 mL). To the dried [¹⁸F]KF was added a solution of the stannane precursor (5.0 mg, 0.009 mmol) in DMA (847 µL) followed by addition of a solution of [Cu(OTf)₂(py)₄] (0.2 M stock solution in DMA, 90 µL, 0.02 mmol) and pyridine (1 M stock solution in DMA, 63 µL, 0.14 mmol). The reaction mixture was then heated to 100 °C and allowed to stir for 15 mins.²Upon cooling to 50 °C, 2 mL of buffer (55% acetonitrile, 10 mM NH₄HCO₃, pH 10) was added and after stirring for an additional 1 min was transferred to an HPLC loop for injection and purification by semi-preparative chromatography (Gemini 5 µm NXC18 110 Å, 250X10, 4 mL/min). The product peak (retention time ~ 18 min) was collected and diluted into 50 mL of MQ H₂O followed by trapping on a C18 extraction disk. The trapped product was washed with 10 mL of sterile water, eluted with 500 µL of EtOH and then rinsed with 4.0 mL of saline into the collection vial containing 5.5 mL of saline. The resulting 10 mL solution was then passed through a sterile filter into a sterile 10 mL dose vial. The identity and purity of [¹⁸F]3FN was then confirmed using HPLC (Luna C18(2), 150X4.6, 5µ, Buffer: 30% acetonitrile, 10 mM NH₄OAc, pH 5.0, 2 mL/min at 40 °C). Dynamic NHP PET imaging was conducted out to 120 min with and without naloxone blocking to show binding of [¹⁸F]-3FN to the mu receptor.

Results: The stannane precursor was successfully synthesized from tert-butyl 4-oxopiperidine-1-carboxylate in 24% total yield. The compound 3FN was selected based on results form an assay of a series of analogues to determine opioid selectivity and establish the agents as antagonists. [¹⁸F]3FN was synthesized in > 99% radiochemical purity with 6.6% activity yield (119 mCi; 66 min from EOB, Fig 1B). The reformulated dose was a clear, colorless solution with a pH of 5.0. ¹⁸F-3FN was found to be stable out to 4 h in 5% ethanol/saline. NHP imaging showed a pattern of uptake consistent with known mu opioid agent [¹¹C]carfentanil (Fig 1C).

Conclusions: An automated synthesis of [¹⁸F]3FN has been succesfully developed and preliminary in vivo evaluation in NHP revealed excellent brain uptake and kinetics. Further preclinical studies are planned to develop a PK model for 3FN in order to facilitate clinical translation.

Acknowledgements: Funding for this research was made possible in part by the Michigan Pioneer Fellows Program.

[1] Kaur, T. et al. ACS Chem. Neurosci. 2020;11;2906-2914.

[2] Makaravage, K. J. et al. Org. Lett. 2016;18,5440-5443.

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