In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for ⁸⁹Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the ⁸⁹Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate ⁸⁹Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for ⁸⁹Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new ⁸⁹Zr-based complex. High stability in vivo, with low accumulation of free ⁸⁹Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final ⁸⁹Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.

中文翻译：

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用于 89Zr PET 成像的半刚性新型异羟肟酸酯 AMPED 基配体

在这项工作中，我们设计、开发、表征和研究了一种用于⁸⁹ Zr 标记和 PET 成像的新型螯合剂及其双功能衍生物。在初步研究中，我们基于七元杂环 AMPED（6-amino-6-methylperhydro-1,4-diazepine）合成了两种名为 AAZTHAS 和 AAZTHAG 的六齿螯合剂，目的是增加⁸⁹ Zr 配合物的刚性通过使用连接到环状结构的N-甲基-N-（羟基）琥珀酰胺或N-甲基-N-（羟基）戊二酰胺悬垂臂。N-甲基异羟肟酸酯基团是选择的供体基团以有效协调⁸⁹锆 在体外稳定性测试后，我们选择了臂更长的螯合剂 AAZTHAG 作为⁸⁹ Zr的最佳络合剂，在人血清 (HS) 中至少 72 小时的稳定性为 86.4 ± 5.5%。然后在不同时间点（最多 24 小时）获得的小动物 PET/CT 静态扫描和离体器官分布研究在健康裸鼠（n = 3）中进行，以研究这种新的⁸⁹ Zr的稳定性和体内生物分布基于复杂。体内稳定性高，游离⁸⁹积累量低测量了骨骼和肾脏中的 Zr。此外，合成了 AAZTHAG 的活化酯功能化版本，以允许与生物分子（如抗体）结合。然后将双功能螯合剂与人抗 HER2 单克隆抗体曲妥珠单抗 (Tz) 缀合，作为与生物活性分子缀合的原理测试的证明。最终的⁸⁹ Zr 标记化合物通过放射性 HPLC 和 SDS-PAGE 进行表征，然后进行放射自显影，并评估其在不同溶液中的稳定性至少 4 天。