核医学显像可以从分子水平反映病变基因、分子、代谢及功能状态，是实现疾病精准诊断必不可少的重要工具，而放射性药物是进行核医学PET/SPECT显像的必要条件。^99mTc是SPECT显像中应用最广泛的核素，^99mTc标记放射性药物的标记通过金属核素与配体配位实现，因此研发性质优良的双功能连接剂是^99mTc标记放射性药物的研究热点。HBED-CC(L₁)常用于⁶⁸Ga标记正电子药物的制备，与Ga³⁺有极高的配位常数(logK_GaL = 38.5)，但其在^99mTc标记放射性药物中的应用尚未见报道。故本论文首次建立HBED-CC类配体的羰基锝标记方法，探讨^99mTc标记的HBED-CC衍生物在SPECT显像中的应用价值和潜力。另外，⁶⁸Ga核素可通过⁶⁸Ge/⁶⁸Ga发生器获得，使用成本较低，药物制备方法简单，神经内分泌肿瘤显像剂[⁶⁸Ga]Ga-DOTA-TATE和[⁶⁸Ga]Ga-DOTA-TOC已被美国FDA批准上市并可以结合¹⁷⁷Lu等治疗核素实现诊疗一体化。但是对于肾功能不全的肿瘤患者，在未知情况下进行肽受体放射性治疗可能会对肾脏造成严重损伤，因此本论文进行⁶⁸Ga标记的新型HBED-CC氨基酸衍生物的制备与生物评价，旨在研发具有临床应用价值的新型肾功能显像药物。最后通过配体交换法实现[⁶⁸Ga]Ga-N₂S₂类配合物的快速高效制备，解决了直接标记法标记效率低的问题，为[⁶⁸Ga]Ga-N₂S₂类靶向探针的开发应用奠定基础。研究内容具体如下：

一、 HBED-CC类配体的合成与^99mTc标记方法研究及应用

本研究设计合成了HBED-CC(L₁)的双硝基咪唑衍生物HBED-CC-NI(L₂)，使用[^99mTc][Tc(CO)₃(H₂O)₃]⁺ 对L₁和L₂进行了放射性标记并建立标记产物分析方法，优化得出最佳标记条件：pH = 5，95 - 100 °C，30分钟。制备了Re(CO)₃L₁(或L₂)配合物，进行谱图表征研究配合物结构，其中[^99mTc]Tc(CO)₃L₂和Re(CO)₃L₂的HPLC谱图表明配合物存在异构体。使用密度泛函理论进一步研究配合物结构，计算结果显示Tc(CO)₃L₁存在多种键合异构体。将L₁苯环上的羧基用硝基咪唑（一种肿瘤乏氧显像剂的靶向基团）取代得到L₂，Tc(CO)₃L₂仅存在一种稳定配位方式，但是存在两种能量相近的非对映异构体(?G_b1 = 29.9 kcal mol^-1，?G_b2 = 24.8 kcal mol^-1)，该结果与[^99mTc]Tc(CO)₃L₂和Re(CO)₃L₂的HPLC分析结果一致。此外，使用前线轨道理论与自然键轨道理论对配合物的各种构型进行稳定性、自然电荷总量等研究，计算结果与实验数据也保持了良好的一致性。最后，对[^99mTc]Tc(CO)₃L₂进行了作为肿瘤乏氧显像药物的生物评价。EMT-6肿瘤细胞摄取实验结果表明[^99mTc]Tc(CO)₃L₂有肿瘤细胞摄取的乏氧选择性，但在荷瘤小鼠体内的肿瘤摄取较低（0.29 ± 0.06 %ID/g，90分钟），肠道组织中摄取高（大肠：39.14 ± 2.09 %ID/g，90分钟），导致[^99mTc]Tc(CO)₃L₂在荷瘤小鼠体内的肿瘤显像质量不佳。

二、新型⁶⁸Ga标记的HBED-CC天冬氨酸衍生物的合成、标记与生物评价

本研究设计合成了HBED-CC的双天冬氨酸衍生物（HBED-CC-DiAsp）并在室温下、10分钟内完成[⁶⁸Ga]Ga-HBED-CC-DiAsp的快速高效制备(RCP > 98%)。脂水分配系数实验与体外稳定性实验证明标记物具有高亲水性和良好的体外稳定性。血红细胞结合实验与血浆蛋白结合实验结果表明：[⁶⁸Ga]Ga-HBED-CC-DiAsp与[⁶⁸Ga]Ga-EDTA具有相似且足够低的血红细胞结合率（1.3 ± 0.3% vs 1.6 ± 0.4%，10分钟）与血浆蛋白结合率（3.74 ± 0.45% vs 3.19 ± 0.27%，10分钟），这有助于标记物快速通过肾小球。健康小鼠生物分布研究结果显示标记物主要通过肾脏快速清除（12.51 ± 3.18 %ID/g，10分钟；5.51 ± 1.60 %ID/g，30分钟），肝脏、肠、胃等器官摄取很低，血液清除速率快（5.69 ± 1.53 %ID/g，10分钟；2.46 ± 0.49 %ID/g，30分钟）。通过大鼠动态PET/CT成像得到标记物在大鼠体内的肾脏摄取时间-活度曲线，标记物在注射后3.6分钟可以达到肾脏最大摄取，8.8分钟清除至最大摄取值的一半，50分钟后清除彻底。注射丙磺舒（肾小管排泄抑制剂）对标记物的肾脏摄取无显著影响，证明标记物无肾小管再摄取。以上评价实验结果表明[⁶⁸Ga]Ga-HBED-CC-DiAsp符合肾功能显像药物的条件参数要求。

Nuclear medicine imaging can detect changes in genes, molecules, metabolism and functional status at molecular level for different diseases. It is an essential tool to provide important information for accurate diagnosis of various diseases. Radiopharmaceutical (molecular probe) is one of the necessary tools for PET/SPECT imaging. ^99mTc is the most widely used nuclide in SPECT and the preparation of radiometal labelled imaging agent is based on metal coordination. Therefore, the development of bifunctional chelate with excellent properties is highly desirable for development of ^99mTc radiopharmaceuticals. HBED-CC(L₁) shows a very high coordination stability constant with ⁶⁸Ga³⁺ (logK_GaL = 38.5). However, its application in ^99mTc radiopharmaceuticals has not been investigated. For this purpose, a radiolabeling method for the preparation of [^99mTc]Tc(CO)₃-HBED-CC complexes was established in this thesis to explore potential application of ^99mTc-labeled HBED-CC derivatives in SPECT imaging. In addition, ⁶⁸Ga labelled radiopharmaceuticals have several advantages: 1) it could be obtained through the use of a ⁶⁸Ge/⁶⁸Ga generator; 2) the preparation of ⁶⁸Ga radiopharmaceuticals is simple and relatively inexpensive; 3) neuroendocrine tumor imaging agents [⁶⁸Ga]Ga-DOTA-TATE and [⁶⁸Ga]Ga-DOTA-TOC have been approved for clinical diagnosis by the US FDA. Combining with the ¹⁷⁷Lu agents this approach has establish an integrated approach, theragnostic approach, for diagnosis and treatment. Patients with renal insufficiency, peptide receptor radiotherapy under unknown conditions may cause severe damage to the kidneys. For this reason, a ⁶⁸Ga radiolabeled HBED-CC amino acid derivative was prepared and evaluated as a novel renal function PET imaging agent. In addition a fast and efficient preparation of [⁶⁸Ga]Ga-N₂S₂ complexes by ligand exchange reaction was studied. It shows higher labeling efficiency than that of direct labeling and is useful for the development and application of [⁶⁸Ga]Ga-N₂S₂ as alternative imaging agents. The specific research methods and results are as follows:

1.      Research and application of ^99mTc labeled HBED-CC complexes

A HBED-CC nitroimidazole derivative, HBED-CC-NI (L₂), was designed and synthesized. [^99mTc][Tc(CO)₃(H₂O)₃]⁺ was used to radiolabel L₁ (HBED-CC) and L₂, and the analytical method for products was established. The optimal radiolabeling parameters were pH = 5, 95 - 100 °C, 30 min. Re(CO)₃L₁/L₂ complexes were synthesized as surrogates to investigate the coordination of the complexes. The HPLC chromatograms of [^99mTc]Tc(CO)₃L₂ and Re(CO)₃L₂ showed that there are potential isomers in the complex. DFT (density functional theory) calculation results showed that [^99mTc]Tc(CO)₃L₁ has multiple bonding isomers in solution. However [^99mTc]Tc(CO)₃L₂ has only one coordination mode, but there are two possible isomers with similar energy (?G_b1 = 29.9 kcal mol^-1, ?G_b2 = 24.8 kcal mol^-1). Results are consistent with the HPLC chromatograms of [^99mTc]Tc(CO)₃L₂ and Re(CO)₃L₂, which showed two different peaks. In addition, frontier orbital theory and natural bond orbital theory were used to study the stability and sum of natural charges of complexes. Results of calculation and experimental data also maintain good consistency. Finally, the biological properties of [^99mTc]Tc(CO)₃L₂ were evaluated. The results show that [^99mTc]Tc(CO)₃L₂ is hypoxic selectivity in its in vitro uptake in EMT-6 tumor cells. But the tumor uptake in EMT-6 xenographed mice model is low (0.29 ± 0.06 %ID/g, 90 min) and high uptake in intestinal tissue (large intestine: 39.14 ± 2.09 %ID/g, 90 min) was found in the tumor model, resulting in poor imaging properties of tumor.

2.      Synthesis and evaluation of novel ⁶⁸Ga-labeled renal imaging agent

HBED-CC-DiAsp was synthesized and labeled with [⁶⁸Ga]GaCl₄^- at room temperature. Plasma protein and red blood cells (RBC) binding were also evaluated by in vitro assays. Biodistribution and dynamic PET imaging studies were performed in normal mice and rats, respectively.

[⁶⁸Ga]Ga-HBED-CC-DiAsp was radiolabeled at room temperature by a one-step kit formulation in high purity without any purification (radiochemical purity > 98%). Previous reports suggested that Ga-HBED-CC exhibited a higher stability constant and rapid chelating formation rate than that of Ga-EDTA (logK_GaL = 38.5 vs 22.0, respectively). In vitro stability studies indicated that it was stable in saline up to 120 min. The log D_OW value (partition coefficient) between n-octanol and water, was found to be -2.52 ± 0.08. Plasma protein and RBC binding were similar to that observed for [⁶⁸Ga]Ga-EDTA. Biodistribution and dynamic PET/CT imaging studies in rats revealed a rapid clearance primarily through the renal-urinary pathway. The PET-derived [⁶⁸Ga]Ga-HBED-CC-DiAsp renograms in rats showed an average time-to-peak of 3.6 ± 0.7 min which was similar to that observed for [⁶⁸Ga]Ga-EDTA (3.1 ± 0.5 min). The time-to-half-maximal activity was also comparable to that of [⁶⁸Ga]Ga-EDTA (8.8 vs 8.2 min, respectively). Pretreatment of probenecid, a renal tubular excretion inhibitor, showed no significant effect on renal excretion.

Results showed that, [⁶⁸Ga]Ga-HBED-CC-DiAsp could be prepared quickly at room temperature in high yield and purity. In vitro studies and in vivo biodistribution in mice and rats suggested that [⁶⁸Ga]Ga-HBED-CC-DiAsp might be useful as a PET imaging agent for measurement of glomerular filtration rate, a key factor in measuring renal function.

3.     Rapid preparation of [⁶⁸Ga]Ga-N₂S₂ complex by ligand exchange reaction

This work establishs a fast and efficient method to prepare [⁶⁸Ga]Ga-N₂S₂ complexes using a ligand exchange reaction. First, radiolabeled intermediate [⁶⁸Ga]Ga-Citrate was prepared to increase the stability of ⁶⁸Ga. Next, in neutral or alkaline conditions, [⁶⁸Ga]Ga-Citrate undergoes a ligand exchange reaction with BAT-TEM (a N₂S₂ ligand). Finally, [⁶⁸Ga]Ga-BAT-TEM was obtained. Results show that [⁶⁸Ga]Ga-BAT-TEM could be prepared over a wide pH range (pH = 5 - 10) through ligand exchange reaction and the ligand used is one-tenth (0.065 mM vs. 0.65 mM) compared with direct radiolabeling. Results provided critical information for future development of [⁶⁸Ga]Ga-N₂S₂ based PET molecular probes.