Synthesis and pharmacological evaluation of methyl and fluoroethyl piperazine analogues as PET probes for sigma receptor imaging.

Both subtypes of sigma receptors, s1 and s2, are overexpressed in many cancers with s2 proposed as a biomarker of tumor proliferation. Sigma receptors are also abundant and are involved in various pathological processes in central nervous system. We are interested in developing a high affinity selective s2 radioligand, in particular for in vivo monitoring of proliferative status of solid tumors and response to anti‐cancer therapies. We report here the synthesis and evaluation of two piperazine analogues. 1‐cyclohexyl‐4‐[3‐(5‐methoxy‐1,2,3,4‐tetrahydronaphthalen‐1‐yl)propyl]piperazine (PB28) represents one of the lead candidates in the development of sigma receptor ligands for therapeutic and diagnostic applications. However, its utility is limited due to relatively high lipophilicity. We synthesized a more hydrophilic analogue 1 (Figure 1, logD7.4 = 2.38). Binding affinity (Ki) of its S‐enantiomer, (–)‐(S)‐1, to 2 was 5.92 ± 0.52 nM with ~15‐fold selectivity over 1 (Ki = 94.6 ± 12.6 nM). (–)‐(S)‐1 was radiolabeled with 11C via standard O‐alkylation with 11C‐methyliodide in an automated module. 11C‐(–)‐(S)‐1 was produced in 53 ± 7 % isolated decay‐corrected yield with radiochemical and chemical purity over 99% and specific activity greater than 100 GBq/micromol. Average synthesis time including semi‐preparative HPLC purification was 30 min. Fluoroethylation of desmethyl precursor 3 negatively affected binding to both sigma receptor subtypes (1 Ki = 316 ± 21 nM, 2 60% of 3H‐(+)‐DTG binding inhibition at 10‐11 M) and as a result the fluorinated compound was not pursued further. PET imaging was performed in mice bearing EMT6, C6 or PC3 tumors after i.v. injection of 11C‐(–)‐(S)‐ 1. For C6 xenografts low target‐to‐nontarget ratio did not allow clear tumor visualization. No accumulation was visible in EMT6 tumor, which is commonly used to evaluate s2 ligands, or PC3 tumor with SUV 0 suggesting poorly perfused tissue. Brain uptake was low and homogeneous (SUV 0.9‐1.3). High accumulation of radioactivity was observed in liver and intestine suggesting hepatobiliary clearance. In vitro autoradiography with 11C‐(–)‐(S)‐1 using rat brain slices resulted in a heterogeneous binding of the tracer with the highest accumulation in olfactory bulb, cerebellum, and cortex. This binding was successfully blocked by 10 μM of either, haloperidol, (+)‐(R)‐1, or PB28. In conclusion, despite excellent in vitro properties, 11C‐(–)‐(S)‐1 was not successful in imaging s2 receptors in vivo. Other analogues are currently being evaluated.