A somatostatin receptor-targeting peptide labeled with a Re(I) tricarbonyl core via a clickable tridentate bifunctional chelator

Objectives We previously reported on the formation of Re- and 99mTc-tricarbonyl complexes using a tridentate chelator that coordinates the fac-[Re/99mTcI(CO)3]+ cores via imidazole N, thioether S, and carboxylate O donor atoms to yield stable neutral complexes. [1] Here, we report the synthetic modification of this ligand to generate a novel bifunctional chelator designed for both facile peptide conjugation and tridentate [NSO] coordination of Re/ 99mTc tricarbonyl cores. The synthesis, characterization and in vitro receptor affinity evaluation of a new Re(I)-labeled somatostatin receptor (SSTR)-targeting antagonist peptide are also presented.

Methods The novel tridentate chelating ligand, 2-(but-3-yn-1-ylthio)-3-(1H-imidazol-4-yl)propanoic acid (L), was synthesized by reacting 2-chloro-3-(1H-imidazol-4-yl)propanoic acid with S-(but-3-yn-1-yl) ethanethioate in a NaOH solution. The peptide sst2-ANT [2,3] [4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr- Cys)-DTyr-NH2] was first functionalized with an N-terminal azide via reaction with N3CH2COOH and then conjugated to the alkyne of L via a Cu(I)-assisted click reaction. The resulting L-sst2-ANT bioconjugate was reacted with the [Re(CO)3(H2O)3]+ intermediate [4] to yield Re(CO)3(L-sst2-ANT). The IC50 value of Re(CO)3(L-sst2-ANT) was determined via a challenge experiment in SSTR-expressing AR42J cells with 125I-Tyr11-somatostatin-14 as the competing radioligand.

Results The bifunctional chelating ligand L was synthesized in relatively low yield (29 %) and characterized by NMR and LCMS (m/z = 225.0 for [M+H]+, calc. 225.1). Click conjugation of L and resin-attached N3-sst2-ANT proceeded cleanly to yield L-sst2-ANT (m/z = 1462.5 for [M+H]+, calc. 1462.5). The synthesis of Re(CO)3(L-sst2-ANT) yielded two isomeric products in a 0.8:1 ratio with an 84% yield on average (by HPLC). HRMS analysis of the HPLC-purified Re products confirmed the expected m/z of 1732.6 (calc. 1732.4) for [M+H]+ and 866.8 (calc. 866.7) for [M+2H]2+, as well as the predicted isotopic pattern for Re. An IC50 value of 28 nM ± 13 nM was obtained for Re(CO)3(L-sst2-ANT), showing that acceptable SSTR affinity was retained after the addition of the metal complex.

Conclusions A novel bifunctional chelator for the fac-[Re/99mTcI(CO)3]+ cores was synthesized and successfully conjugated to sst2-ANT via an azide-alkyne cycloaddition reaction. Re(CO)3(L-sst2-ANT) was prepared in high yield and demonstrated nanomolar in vitro SSTR affinity, warranting further study of this new potential neuroendocrine cancer targeting agent. Ongoing tracer level experiments with 99mTc are promising.

• Download figure
• Open in new tab
• Download powerpoint