Synthesis and Evaluation of Nuclear and Optical Tetrazine-6-Hydrazinopyridine-3-Carboxylic Acid Probes for Molecular Imaging using Pretargeting and Bioorthogonal Chemistry

The aim of this thesis was to develop a new platform for the creation of targeted nuclear and optical molecular imaging probes. Current probes suffer from low target-to- background ratios, which can hinder diagnosis and monitoring of disease treatments due to poor image acquisition. To overcome this limitation, the approach taken involved coupling 6-hydrazinopyridine-3-carboxylic acid (HYNIC) to a tetrazine, where the product could be used to create targeted 99mTc and near-infrared dye probes. Targeting could then be accomplished using bioorthogonal chemistry with trans-cyclooctene (TCO) functionalized biomolecules, through an inverse electron demand Diels-Alder cycloaddition. The assessment for the clinical translation of 99mTc-HYNIC based nuclear probes was evaluated to determine the feasibility of using a ‘kit-like’ formulation system. A limitation associated with the development of HYNIC-based radiopharmaceuticals is the need for HPLC purification. The investigation into the need for HPLC purification was performed, where it was determined that there was no significant difference in target uptake between the HPLC-purified and non-purified 99mTc-HYNIC probe 6 when used with bone-seeking TCO-derived bisphosphonate (4.73 ± 0.47 versus 4.15 ± 0.27%ID/g in shoulder, 6.32 ± 0.21 versus 5.94 ± 0.59%ID/g in knee, respectively). This suggests that a kit-like system would be practical; thus streamlining the chemistry required for radiopharmaceutical development. Efforts to reduce non-target organ uptake observed with using 6 was accomplished through the synthesis of a more polar construct 12. While the compound rapidly cleared from non-target organs, it also displayed lower target uptake compared to 6 (1.62 ± 0.22 versus 4.15 ± 0.27%ID/g in shoulder, 2.39 ± 0.22 versus 5.94 ± 0.59%ID/g in knee, respectively), demonstrating the need for further pharmacokinetic optimization. The synthesis and evaluation of optical imaging probes was also completed, where the HYNIC-tetrazine core was linked to a commercially-available near-infrared dye through a hydrazone linkage. The bacterial binding capabilities of fluorescent compounds 22 and 24 (analogous to the 99mTc-labelled 6 and 12) were investigated in vitro using Gram-positive Staphylococcus aureus with TCO-modified vancomycin. The targeted samples exhibited a 3.8-fold (22) and 8.6-fold (24) increase in fluorescent intensity when compared to the blocking control used to account for binding specificity. Fluorescent microscopy images were also obtained, however only bacterial aggregates were observed, indicating the need for optimization of microscopy procedures. As such, a HYNIC-tetrazine platform was successfully developed as a means synthesize various targeting probes for molecular imaging applications using pretargeted, bioorthogonal chemistry.