Introduction The brain serotonin-7 receptor (5-HT7) is the most recently discovered serotonin receptor. It is targeted by several drug-candidates in psychopharmacology and neuropharmacology. In these fields, positron emission tomography (PET) is a molecular imaging modality offering great promise for accelerating the development process from preclinical discovery to clinical phases. We recently described fluorinated 5-HT7 radioligands, inspired by the structure of SB269970, the prototypical 5-HT7 antagonist. Although these results were promising, it appeared that the radiotracer-candidates suffered, among other drawbacks, from too low a 5-HT7 receptor affinity. Methods In the present study, seven structural analogs of SB269970 were synthesized using design strategies aiming to improve their radiopharmacological properties. Their 5-HT7 binding properties were investigated by cellular functional assay. The nitro-precursors of the analogs were radiolabeled by [18 F-]nucleophilic substitution, and in vitro autoradiography was performed in rat brain, followed by in vivo microPET. Result The chemical and radiochemical purity of the fluorine radiotracers was > 99% with specific activity in the 40-129 GBq/μmol range. The seven derivatives presented heterogeneous binding affinities toward 5-HT7 and 5-HT1A receptors. While [18 F]2F3P3 had promising characteristics in vitro, it showed poor brain penetration in vivo, partially reversed after pharmacological inhibition of P-glycoprotein. Conclusions These results indicated that, while chemical modification of these series improved several radiotracer-candidates in terms of 5-HT7 receptor affinity and specificity toward 5-HT1A receptors, other physicochemical modulations would be required in order to increase brain penetration.