Technetium-99m Tricarbonyl Pyridylpiperazine Complexes as 5-HT₇ Receptor-Targeted SPECT Radiotracers for Glioblastoma Imaging

Abstract Technetium-99m radioligands targeting the 5-HT₇R may improve molecular imaging of glioblastoma by enabling visualization of receptor overexpression with SPECT. In this work, two new ligands ( 9 and 10 ) and their corresponding technetium tricarbonyl complexes ([ 99m Tc]Tc(CO) 3 - 9 and [ 99m Tc]Tc(CO) 3 - 10 ) were synthesized, radiolabeled in high radiochemical purity (> 96%), and showed good stability in saline and serum. Both complexes exhibited moderate lipophilicity (logD = 0.43 ± 0.07 and 0.72 ± 0.01, respectively), suitable for in vivo application. In vitro binding studies on U-87 MG, SKOV-3, HT-29, and A549 cells demonstrated that [ 99m Tc]Tc(CO) 3 - 10 exhibits specific, high-capacity 5-HT₇R binding on U-87 MG cells (Kᴅ = 5.88 ± 0.27 nM; Bmax = 7.83 ± 0.26×10⁴ fmol/10⁷ cells), while [ 99m Tc]Tc(CO) 3 - 10 shows only low, nonspecific binding and was not further pursued. In normal mice, [ 99m Tc]Tc(CO) 3 - 10 showed rapid blood clearance with predominant hepatobiliary and renal excretion and moderate but measurable brain uptake (0.70 ± 0.27%ID/g at 30 min). In U-87 MG xenografts, [ 99m Tc]Tc(CO) 3 - 10 accumulated in tumors with peak uptake of 3.19 ± 0.49%ID/g and a tumor-to-muscle ratio of 4.32 ± 0.97 at 30 min, while pimozide pre-blocking reduced tumor uptake and tumor-to-muscle ratio by up to 67%, confirming receptor-mediated binding. Planar scintigraphy at 60- and 120-min post-injection visualized U-87 MG tumors with imaging tumor-to-muscle ratios of 4.04 and 3.78, which decreased markedly in the blocked groups, indicating specific 5-HT₇R targeting. Overall, [ 99m Tc]Tc(CO) 3 - 10 emerges as a promising 5-HT₇R-targeted SPECT radiotracer for glioblastoma imaging and warrants further optimization and evaluation.