Technetium(I) Complexes Bearing <scp>l</scp> - or <scp>d</scp> -Amino Acids as Tumor-Seeking Agents

Metabolic alterations in cancer cells present an opportunity for the development of new targeted radiopharmaceuticals for cancer imaging and therapy. Radiotracers targeting transporters of amino acids (Aa) hold great potential as agents for cancer theranostics but very few Aa-based radiotracers have been explored, even less based on metallic complexes. To address this challenge, we describe herein the (radio)-synthesis and biological characterization of novel [^99mTc]-Tc-(I) complexes bearing pendant l- or d-amino acids for SPECT imaging. The complexes of the type <i>fac</i>-[M-(CO)₃(k³-<b>L</b>)]⁺ (<b>ML1</b>-<b>ML8</b>, M = Re, ^99mTc) were prepared by reaction of <i>fac</i>-[M-(CO)₃(H₂O)₃]⁺ with conjugates combining a pyrazolyl-diamine (Pz) chelating unit and both enantiomeric forms (l/d) of Arg (<b>L1</b>/<b>L2</b>), Lys (<b>L3</b>/<b>L4</b>), His (<b>L5</b>/<b>L6</b>) and Trp (<b>L7</b>/<b>L8</b>). The NMR spectral data is compatible with tridentate coordination of the Pz unit in <b>ReL1-ReL4</b> and <b>ReL6</b>/<b>ReL7</b>. Reaction of <b>L5</b>/<b>L6</b> with the Re precursor under acidic conditions yielded a major species in which His is directly involved in metal coordination, the so named β-form of <b>ReL5</b>/<b>ReL6</b>. Under basic reaction conditions, an increase of the so named α-species, where the metal center is stabilized by the Pz unit, is observed. All [^99mTc]-Tc-(I) complexes, obtained at neutral pH, displayed significantly higher uptake and internalization in a panel of cancer cell lines than the amino acid-free control complex, <b>[</b> ^<b>99m</b> <b>Tc]-TcL0</b>, with <b>[</b> ^<b>99m</b> <b>Tc]-TcL5</b> and <b>[</b> ^<b>99m</b> <b>Tc]-TcL7</b> emerging as the best performing molecules. Aimed at assessing the influence of the <b>α</b>/<b>β</b>-<b>[</b> ^<b>99m</b> <b>Tc]-TcL5</b> ratio in the cellular uptake, we reacted <b>L5</b> with the <b>[</b> ^<b>99m</b> <b>Tc]-Tc</b> precursor at low or high pH (favoring <b>β-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> or <b>α-[</b> ^<b>99m</b> <b>Tc]-TcL5</b>, respectively) and evaluated the cell uptake properties of the resulting species. Interestingly, the putative <b>β-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> species presented an approximately 3× higher uptake than the putative <b>α-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> radioactive species, surpassing all the other Aa-containing complexes. This result suggests that the pendant Pz unit may act as an uptake vector. Mechanistic studies aimed to understand the biological properties of <b>β-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> were also performed but we were unable to correlate the uptake with the levels of Aa transporters expression. Considering that <b>β-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> presented similar uptake in the MCF7 breast and A549 lung cancer cell lines, we assessed its tumor-targeting properties in mouse models bearing these 2 types of cancer xenografts. Low tumor accumulation in the breast cancer model was observed, with overall poor tumor-to-nonorgan ratio. In contrast, the biodistribution results of <b>β-[</b> ^<b>99m</b> <b>Tc]-TcL5</b> in mice-bearing A549 xenografts were encouraging as moderate tumor accumulation was observed at all time points with promising tumor-to-nontarget-organ ratios. These results lead to the conclusion that the Pz moiety bears unknown/unexplored biological properties that should be further investigated.