Development of a new 18F-labeled radioligand for imaging of sigma2 receptors by positron emission tomography

Objectives: Sigma2 receptors (S2R) have been found in CNS, liver, kidney, as well as endocrine glands and are suggested to play important roles in the regulation of cell differentiation. Besides their overexpression in various tumor cell lines, derived from e.g. breast, brain, colon, lung, pancreas, and prostate, they show a 10-fold higher expression in the proliferating vs quiescent status and thus are possible markers of solid tumor’s proliferative status. To quantify the S2R availability in living subjects, we aim for the development of a new class of S2R ligands that could be labeled by fluorine-18.
Methods: Starting from structural motifs known for S2R ligands [1, 2], we modified the indole ring system in A and synthesized a novel series of fluorine containing indole and aza-indole derivatives (1a-d and 2-6 in Fig. 1). Their binding affinities towards sigma2 and sigma1 receptors were determined by radioligand-binding assays, and 2 was selected for synthesis of a boronic acid pinacol ester precursor for radiolabeling. Synthesis of [18F]2 was optimized starting from 100-500 MBq of 18F-fluoride, using Kryptofix (K2.2.2.)/ K2CO3 (0.18-1.8 µmol/ 0.04-0.35 µmol) as well as TBAHCO3 (2.3 and 7.5 µmol) and 2-4 mg of precursor 7, in the presence of Cu(OTf)2py4 (0.4-6.8 eq.) in various solvent systems at 80-135 °C, monitored for 5-20 min. For monitoring, several analytical methods (radio-UHPLC, -HPLC, and -TLC) have been established, e.g. on the basis of RP18 und RP8 stationary phases for LC systems. Besides, different techniques for purification and isolation were investigated, including a substitution of semi-preparative HPLC by time-saving cartridge systems.
Results: By altering the heterocyclic system of A, a small series of fluorinated aza-indoles was synthesized (Fig. 1), of which 2 showed most promising binding affinity and selectivity (Ki(S2R) = 1.6 nM; Ki(S1R) = 691 nM). Radiosynthesis of [18F]2 was achieved with RCYs in a range of 20-45% (n = 2, all non-isolated, radio-UHPLC) by use of 2.0 mg of precursor 7 (4.1 µmol) and 3.6 eq. of Cu(OTf)2py4 at 115 °C within 10 min. The reaction was accompanied by the formation of a by-product (bp) which increased over time. Using the K2.2.2./ K2CO3 system resulted in RCYs of 21.5% (bp 5.5%) and 27.7 % (bp 4.6%), in DMF and DMA/ n-BuOH, respectively. Application of TBAHCO3 showed further increased conversions, represented by a RCY of 44.8% (bp 9.1%) in DMA/ n-BuOH. For subsequent semi-preparative HPLC, separation conditions were optimized, but still lack from low recoveries. As an alternative, SPE procedures using cartridge systems (SiO2, RP18) are being established and could be used as a time saving technique for the isolation of [18F]2.
Conclusions: A novel S2R-affine aza-indole derivative 2 was synthesized and radiofluorination of the appropriate boronic acid pinacol ester precursor afforded [18F]2 in RCYs of up to 45% (non-isolated). The optimal parameters for the radiosynthesis, conducted in a synthesis automat or module, have to be determined to setup a procedure for the production of [18F]2, which enables detailed preclinical in vitro and in vivo studies of this promising radioligand.
Acknowledgement: The authors would like to thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (BR 1360/13-1).
References:
[1] Georgiadis, M.-O. et al. Molecules 2017, 22, 1408;
[2] Wang, L. et al. Bioorg Med. Chem. 2017, 25, 3792-3802