Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among these analogues, FHBG (18F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission tomography) probe for monitoring reporter gene expression. To evaluate whether or not imposing rigidity into the flexible side chain of FHBG 4 could also impact its interaction, with amino acid residues within the binding site of HSV1-TK (Herpes Simplex Virus-1 Thymidine Kinase), thus influencing its cytotoxic activity. Herein, the synthesis of a new fluorinated nucleoside analogue 6 (conceived via ligand-docking studies) is reported. Agent 6 demonstrates selective activity against HeLa cells stably transfected with mutant HSV1-sr39TK and is also 47-fold more potent than FHBG.