N,N-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(NNC₅H₄NH_x)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.