6-Mercaptomethylpyridine-3-carboxylic acid (MEMNIC): A new reagent for peptide labeling with Tc-99m

As part of our ongoing research into the development of peptide based radiopharmaceuticals, we have explored 2-mercaptopyridines and 2-mercapto-pyrimidines (2MPs) as coligands to control the radiochemical speciation of ^99mTc-labeled hydrazinonicotinamide (HYNIC) derivatized chemotactic peptides. In an attempt to develop an aminothiol ligand with greater stability, we synthesized 6-mercaptomethylpyridine-3-carboxylic acid (MEMNIC), assuming a five-member chelate ring complex would be more stable than the four-membered ring possible with 2MPs. Initial experiments suggested that ^99mTc-MEMNIC was stable in vivo and led to our investigation of it as a bifunctional chelator for ^99mTc labeling of peptides. The utility of MEMNIC was tested in a rabbit model of infection. The N-epsilon MEMNIC derivative of For-MLFK was prepared using the N-hydroxysuccinimidyl ester of MEMNIC. For-MLFK-MEMNIC was labeled via ^99mTc-mannitol and its imaging and biodistribution properties compared to Tc-For-MLFK-HYNIC and Tc-For-MLFCys (labeled via the free thiol of cysteine). Sites of infection were produced in New Zealand white rabbits (n = 6/peptide) by injection of a suspension of Escherichia coli in a posterior thigh. Twenty-four hours after infection, the rabbits were injected intravenously with 0.5 mCi of HPLC purified ^99mTc-peptide and imaged at 2.5 and 18 h p.i. The animals were sacrificed at 18 h and tissue activity determined. At 2.5 and 18 h, the organ distribution of Tc-For-MLFK-HYNIC and Tc-For-MLFK-MEMNIC were qualitatively similar with the exception of higher renal accumulation of the latter, whereas Tc-For-MLFKCys showed rapid and prolonged accumulation in bowel. ROI analysis gave target/background ratios of 3.91 ± 0.32 and 11.89 ± 2.21 for Tc-For-MLFK-HYNIC, 5.09 ± 0.66 and 9.88 ± 1.12 for Tc-For-MLFK-MEMNIC and 2.59 ± 0.16 and 1.70 ± 0.37 for Tc-For-MLFCys, at 2.5 and 18 h. Tissue radioactivity measurements (18 h) demonstrated that compared to Tc-For-MLFK-HYNIC, the accumulation of Tc-For-MLFK-MEMNIC was less in all organs except in kidney which was greater. Direct labeling of the thiol group of cysteine led to lower accumulation in all organs compared to Tc-For-MLFK-MEMNIC, except in GI tract which was fivefold higher. While, Tc-For-MLFK-MEMNIC had half the accumulation of Tc-For-MLFK-HYNIC in infected muscle and pus, the lower accumulation in most organs and normal muscle resulted in good infection localization. These results indicate that MEMNIC can be successfully used to label peptides with ^99mTc while exhibiting good in vivo stability. The chemistry of MEMNIC with the {M(V)O}³⁺ core characteristic of technetiun and its Group V congener rhenium was modeled by investigating the reactions of 2-mercaptomethylpyridine with appropriate Re(V)-oxo precursors in the presence of a variety of coligands. With diolate type tridentate donors of the class {X(CH₂CH₂O)₂}^2- (X = -NR, O, S), the '3+2' compounds [ReO{η³-(OCH₂CH₂)₂S}{η ²-SCH₂C₅H₄N}] (2) and [ReO{η³-(OCH₂CH₂)₂NCH ₃}{η²-SCH₂C₅H₄N] (3) were isolated. In contrast, with the more sterically demanding dirmercapto class of tridentate ligands {S(CH₂CH₂S)₂}^2-, the '3+1' species [ReO{η³-(SCH₂CH₂)₂S}{η ¹-(SCH₂C₅H₄N)}] (1) was isolated.