Radiosynthesis and in vitro characterisation of a potent ¹⁸F-fluorinated N^ε-acryloyllysine as activity-based probe for transglutaminase 2

Transglutaminase 2 (TGase 2) represents an interesting target for the development of PET tracers for functional imaging of tumours. Among the TGase 2 inhibitors described in the literature, N^α-phenylacetyl-N^ε-acryloyl-L-lysine-4-(6-methylpyridin-2-yl)piperazide (1) [1] seems to be most suitable for radiotracer development as this compound exhibits both strong inhibitory potential and selectivity towards human (h) TGase 2. Extensive structure-activity relationship studies by our group revealed some potent fluorinated analogues of 1, of which compound 3 (methyl group is substituted by fluorine) is a potential candidate for PET tracer development due to its great inhibitory potency and promising pharmacokinetic properties.
For the radiosyntheses of [¹⁸F]3, 6-nitro (2a) and 6-trimethylammonio-pyridines (2b) were envisaged as precursors for direct ¹⁸F-fluorination. The fluorination reactions using [¹⁸F]fluoride were performed under various conditions. Labelling of 2a by [¹⁸F]fluoride resulted in only moderate radiochemical yields (≈20%) accompanied by the formation of two 18F-labelled side products. In contrast to this, ¹⁸F-fluorination of 2b yielded exclusively [¹⁸F]3 in high radiochemical yields (≈70%). Therefore, precursor 2b was chosen for further radiosyntheses. In vitro characterisation of [¹⁸F]3 with regards to its reactivity towards hTGase 2 as well as its selectivity and specificity was done by radio-TLC and radio-SDS-PAGE. Kinetic investigations by radio-TLC provided values for k_inact/K_I that are in good agreement with values obtained by fluorimetric activity assays. Incubation of whole cell lysates of different human tumour cell lines exhibiting a high expression of TGase 2 with [¹⁸F]3, followed by SDS-PAGE and measurement of the ¹⁸F activity revealed essentially a single band around the molecular mass of hTGase 2 (≈77 kDa). Accordingly, no band was observed for those tumour cells which do not express TGase 2. Further experiments with [¹⁸F]3 will include cell uptake studies in living tumour cells as well as stability, biodistribution and PET studies in mice.
[1] J. Wityak et al. ACS Med. Chem. Lett. 2012, 3, 1024-1028