Synthesis and evaluation of a C-6 alkylated pyrimidine derivative for the in vivo imaging of HSV1-TK gene expression

Abstract

Introduction: We report on the synthesis, radiolabeling, in vitro and in vivo characterization of N-Me-[(18)F]FHBT (6-(3-[(18)F]fluoro-2-(hydroxymethyl)propyl)-1,5-dimethylpyrimidin-2,4(1H,3H)-dione), a C-6-substituted N-1-methylated pyrimidine derivative as a reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-TK) expression.

Methods: N-Me-[(18)F]FHBT was synthesized via a standard nucleophilic substitution reaction followed by acidic cleavage of the methoxytrityl protecting group. Cell uptake was studied in vitro with control HEK293 (human embryonic kidney cells) and HEK293 cells stably transfected with nonmutant HSV1-tk (HEK293TK+ cells). Positron emission tomography (PET) imaging and biodistribution studies of N-Me-[(18)F]FHBT or [(18)F]FHBG were performed in nude mice bearing xenografts of HEK293 control and TK+ cells.

Results: N-Me-[(18)F]FHBT was obtained in a two-step reaction in an overall maximal radiochemical yield (decay-corrected) of 5% and a radiochemical purity >96%. The tracer uptake in HSV1-TK containing HEK293TK+ cells was 14.5 times (at 30 min) and 55.4 times (at 240 min) higher than in control HEK293 cells. In mice, N-Me-[(18)F]FHBT and [(18)F]FHBG accumulated significantly and exhibited similar radioactivity levels in the HEK293TK+ xenografts; however, standardized uptake values ratios between HEK293TK+ and HEK293 control xenografts were higher for [(18)F]FHBG than for N-Me-[(18)F]FHBT. Both tracers showed high gall bladder and abdominal activity.

Conclusion: The biological evaluations demonstrated the feasibility of using N-methylated C-6-substituted pyrimidine derivative N-Me-[(18)F]FHBT as a PET radiotracer for monitoring HSV1-TK expression in vivo.