Development and fluorine-18-radiolabeling of benzodioxolylpyrimidine EphB4 receptor inhibitors

Objectives: Ephrins and Eph receptors were found to be disregulated in several human tumor entities like breast, colon, liver, prostate or melanoma [1]. Concerning Eph/ephrin signalling, the EphB4/ephrinB2 system seems to play a major role in tumor angiogenesis [2]. Several inhibitors were reported based either on peptides which block the extracellular ephrin binding domain [3] or small organic molecules which block the intracellular kinase domain of Eph receptors. For this purpose, promising EphB4 kinase inhibitors based on the dioxolylpyrimidine scaffold were used for the development of a novel radiofluorinated PET tracer candidate [4].
Methods:
Compound 1 (figure 1) represents an advantageous molecule for the development of a fluorine-18 containing radiotracer. It shows an adequate affinity to EphB4 (90 nM) and consists of two structural parts. For the synthesis of the new radiotracer [¹⁸F]2, the methylsulfonylphenyl residue in 1 was replaced by a 3-fluoropropylsulfonylphenyl moiety (part B). This part of the molecule was prepared starting from sodium benzenesulfinate and 1-bromopropanol in 5 synthesis steps. Part A of the radiotracer as well as the lead compound containing the benzodioxole moiety was synthesized as described previously [4]. Finally, the non-radioactive reference 2 was obtained after 6 steps, whereas the respective tosylate precursor was prepared within 7 steps. Cellular binding and internalization of [¹⁸F]2 was investigated in human A375 melanoma cells as well as A375 cells stably transfected with human EphB4 receptor or mock vector. Furthermore, specific binding and potential transport mechanisms were analyzed by application of different specific inhibitors.
Results:
Starting from a 2 GBq [¹⁸F]fluoride batch, 65±5 MBq of [¹⁸F]2 were obtained with >95% radiochemical purity within 60 min synthesis time (including HPLC purification tR = 3.9 min, acetonitrile:water, 1:1 + 0.1% TFA). The best conditions for radiolabeling of the tosylate precursor were found to be acetonitrile as solvent at 90 °C for 15 min. Afterwards, water was added and the mixture was filtered prior to the HPLC purification. Cell association studies in melanoma cells showed time dependent increase in binding and internalization of [¹⁸F]2. Both inhibition with lead compound 1 and [¹⁹F]2 significantly decreased binding and internalization of [¹⁸F]2
indicating specific EphB4 binding. However, involvement of other kinases for binding can not be excluded. Moreover, participation of ATP-binding cassette transporters for cellular uptake is indicated.
Conclusions:
A novel potential EphB4 inhibitor based on the dibenzodioxolylpyrimidine scaffold has been synthesized and successfully radiolabeled with fluorine-18. Further biological studies including dynamic small animal PET are currently in progress.
References:
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Davis, R. Abagyan, H. Widmer, E. B. Pasquale, P. Kuhn, (2007), J. Chem. Biol. 282, 36505, [4] C. Bardelle, T.
Coleman, D. Cross, S. Davenport, J. G. Kettle, E. J. Ko, A. G. Leach, A. Mortlock, J. Read, N. J. Roberts, P.
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