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The translocator protein 18kDa (TSPO) is today a validated target for a number of therapeutic applications, but also a well-recognized diagnostic/imaging biomarker for the evaluation of inflammatory related-disease state and progression, prompting the development of specific and dedicated TSPO ligands worldwide. For this purpose, pyrazolo[1,5-a]pyrimidine acetamides constitute a unique class of high affinity and selectivity TSPO ligands; it includes DPA-714, a fluorine-containing derivative that has also been labelled with the positron-emitter fluorine-18, and is nowadays widely used as a Positron Emission Tomography imaging probe. Recently, to prevent defluorination issues encountered invivo with this tracer, a first series of analogues was reported where the oxygen atom bridging the phenyl ring of the core structure and the fluorinated moiety was replaced with a more robust linkage. Among this new series, CfO-DPA-714 was discovered as a highly promising TSPO ligand. Herein, a novel series of fluorinated analogues of the latter molecule were synthesized and invitro characterized, where the pharmacomodulation at the amide position of the molecule was explored. Thirteen compounds were thus prepared from a common key-ester intermediate (synthesized in 7 steps from 4-iodobenzoate - 11% overall yield) and a set of commercially available amines and obtained with moderate to good yields (23-81%) and high purities (>95%). With one exception, all derivatives displayed nanomolar to subnanomolar affinity for the TSPO and also high selectivity versus the CBR (Ki (CBR)/Ki (TSPO)>103). Within this series, three compounds showed better Ki values (0.25, 0.26 and 0.30nM) than that of DPA-714 (0.91nM) and CfO-DPA-714 (0.37nM), and favorable lipophilicity for brain penetration (3.67.4&nbsp

DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to thephenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (Ki: 0.35 nM) and selectiveligand targeting the translocator protein 18 kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [18F]fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, andformulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2GBq of [18F]DPA-C5yne,ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to110GBq/μmol within 50–60 min, starting from a 30GBq [18F]fluoride batch (14–17%). LogP and LogD of [18F]DPA-C5yne weremeasured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studiesperformed on slices of ((R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a hightarget-to-background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstratedusing DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore,DPA-C5yne proved to be stable in plasma at 37°C for at least 90min.