<i>N</i>,<i>N</i>-Bis(2-mercaptoethyl)methylamine: A New Coligand for Tc-99m Labeling of Hydrazinonicotinamide Peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the {M(NNC5H4NHx)2} core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M{CH3N(CH2CH2S)2}(NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re{C5H3N-2,6-(CH2S)2}(NNC5H4N)(NHNC5H4N)]·CH2Cl2·0.5MeOH (7), [Re{SCH2CH2)2O} (NNC5H4N)(NHNC5H4N)] (8), and [Re{(SCH2CH2)2S}(NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3{NNC5H3NH(CO2R)} {NHNC5H3N(CO2R)}] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3{NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)} {NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)}]·2HCl (14·2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2{NNC5H3N(CO2Me)}{NHNC5H3N(CO2Me)}] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.烟酰肼（Hydrazinonicotinamide，HYNIC）可与氧代锝(V)物种（如锝-甘露醇或其他锝-多羟基配合物）反应，形成稳定的锝-99m（Tc-99m）配位配合物。然而，通过锝-多羟基配体标记的[Tc-For-MLFK-HYNIC]的放射性高效液相色谱（radio-HPLC）分析显示存在多种放射化学物种，各自具有独特的生物分布模式。这可能是因为锝既可以结合至肼基部分，也可结合至多羟基配体，且存在多种配位几何构型。 三齿配体（如双(巯基乙基)甲胺（bis(mercaptoethyl)methylamine，NS2））可限制可能的配位几何构型，提升整体稳定性。为探究这一点，我们合成了NS2，将[Tc-甘露醇-For-MLFK-HYNIC]转化为相应的含NS2配合物[Tc-NS2-For-MLFK-HYNIC]，并将其感染成像性能与生物分布特性与[Tc-甘露醇-For-MLFK-HYNIC]进行对比。通过高效液相色谱确认了NS2配合物的成功转化：色谱图显示出单一独特的疏水物种，其保留时间长于[Tc-甘露醇-For-MLFK-HYNIC]配合物。 我们对左侧大腿感染大肠杆菌（E. coli）的家兔开展了两种制剂的成像实验。组织放射性测量结果显示，相较于锝-甘露醇-肽，锝-NS2-肽在血液、心脏和正常肌肉中的摄取更低，而在脾脏、感染肌肉及脓液中的摄取更高（p < 0.01）。上述结果表明，锝-NS2-肽配合物在化学上更均一，且表现出更优的感染定位能力与生物分布特性。 为了模拟金属-HYNIC核心与NS2及相关配体的相互作用，我们研究了[{M(NNC5H4NHx)2}核心的有效结构类似物][ReCl3(NNC5H4NH)(NHNC5H4N)]与[99TcCl3(NNC5H4NH)(NHNC5H4N)]分别与NS2、C5H3N-2,6-(CH2SH)2、O(CH2CH2SH)2及S(CH2CH2SH)2的反应，并分离得到了配合物[M{CH3N(CH2CH2S)2}(NNC5H4N)(NHNC5H4N]（M = 99Tc（5a）、Re（5b））、[Re{C5H3N-2,6-(CH2S)2}(NNC5H4N)(NHNC5H4N)]·CH2Cl2·0.5MeOH（7）、[Re{(SCH2CH2)2O}(NNC5H4N)(NHNC5H4N)]（8）以及[Re{(SCH2CH2)2S}(NNC5H4NH)(NHNC5H4N)]Cl（9）。 类似地，[ReCl3(NNC5H4NH)(NHNC5H4N)]与双齿配体吡啶-2-甲硫醇及3-(三甲基硅基)吡啶-2-硫醇反应，分别分离得到了[ReCl(C5H4N-2-CH2S)(NNC5H4N)(NHNC5H4N)]（10）与[Re(2-SC5H3N-3-SiMe3)2(NNC5H4N)(NHNC5H4N)]（11）；而其与N-甲基咪唑-2-硫醇反应则得到双核配合物[Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2(NHNC5H4N)2]（12）。 我们还制备了类似的金属-(HYNIC-OH)前体[ReCl3{NNC5H3NH(CO2R)}{NHNC5H3N(CO2R)}]（R = H，13a；R = CH3，13b），并将其与赖氨酸偶联得到[RCl3{NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)}{NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)}]·2HCl（14·2HCl）；而甲酯前体13b与2-巯基吡啶反应则得到[Re(2-SC5H4N)2{NNC5H3N(CO2Me)}{NHNC5H3N(CO2Me)}]（15）。 尽管化学研究证实了M-HYNIC核心（M = Tc、Re）的稳定性，以及其在金属相邻配位位点发生配体取代反应时仍能保持完整，但寡聚结构的分离以及13、14、15的肽偶联物的不溶性，凸显了在宏观尺度下表征这类物质的难度——这一发现与示踪剂水平下试剂纯度与结构鉴定长期存在的顾虑息息相关。