Novel O-[C]-methylated derivatives of the neprilysin inhibitor sacubitril: Radiosynthesis, autoradiography and plasma stability evaluation.

Introduction: The O-[C]methylated derivatives of the clinically used neprilysin inhibitor (NEPi) sacubitril ([C]SacOMe, (2R,4S)-ethyl 5-([biphenyl]-4-yl)-4-(4-[C]methoxy-4-oxobutanamido)-2-methylpentanoate) and LBQ657 ([C]MeOLBQ, (2R,4S)-5-(biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [C]methyl ester and [C]LBQOMe, (2R,4S)-5-(biphenyl-4-yl)-4-[(4-[C]methoxy-4-oxobutanamido)]-2-methylpentanoic acid) were evaluated to determine their potential as PET imaging tracers and investigate the effect of such labeling esterification on neprilysin (NEP) binding.

Methods: [C]MeOLBQ, [C]SacOMe and [C]LBQOMe were synthesized by O-[C]methylation using [C]methyl triflate. Binding of these radiolabeled derivatives (5 nM) were assessed by autoradiography on rat neprilysin rich kidney slices with or without 10 μM NEPi (thiorphan or sacubitril) for 20 min at 37 °C. [C]LBQOMe was further tested for binding selectivity in the presence of 10 μM of angiotensin-converting enzyme inhibitor (ACEi, captopril) or angiotensin II AT receptor blocker (AT1R, losartan). Radioligands were evaluated for their in vitro stability up to 20 min after incubation at 37 °C in rat and human plasma by reverse-phase column-switch HPLC. Non-radioactive SacOMe incubated in rat and human plasma was analyzed by HPLC-coupled with high resolution mass spectrometry (HRMS) to confirm the metabolites' identity. [C]SacOMe main labeled metabolite was further analyzed by HPLC after incubation in rat kidney slices at 37 °C.

Results: The novel [C]SacOMe and [C]LBQOMe were produced in 32 ± 3% RCY and 15 ± 6% at EOS (decay-corrected from [C]CO, n = 3), high molar activity (407 ± 92 GBq/μmol and 260 ± 92 GBq/μmol), and high chemical (≥90%) and radiochemical (≥99%) purities in a total synthesis time of 31 and 34 min, respectively. High accumulation of [C]SacOMe and [C]LBQOMe in kidneys was completely blocked (>99.9%) by pre-incubation with NEPi, whereas [C]MeOLBQ displayed negligible uptake in autoradiography studies. [C]LBQOMe binding was not affected by saturating doses of losartan or captopril indicating binding selectivity for NEP. While [C]SacOMe and [C]LBQOMe were stable in human plasma (>92%) even after 20 min incubation at 37 °C, rat plasma analyses exhibited >95% biotransformation of [C]SacOMe, 40% of [C]LBQOMe and >80% loss of the C-methyl group of [C]MeOLBQ after 5 min of incubation. Comparable results using the non-radioactive SacOMe were obtained by HPLC-HRMS. Radio-HPLC analysis of the extracted activity of rat kidney slices incubated with [C]SacOMe demonstrated that >95% of the radioactive signal corresponded to [C]LBQOMe as the main metabolite.

Conclusion: The desethyl active metabolite of [C]SacOMe, [C]LBQOMe, displayed stability in human plasma, binding selectivity for neprilysin over ACE or AT1R in rat kidney slices. Rapid plasmatic dealkylation at the 2-methylbutanoic acid position is in line with the necessity of incorporating the labeling group on oxobutanoic acid side in the strategy to develop a stable O-alkylated labeled derivative of sacubitril.