Re-cyclized octreotide analogues: A structure-activity relationship study

Abstract

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Objectives: The β turn conformation of the somatostatin (Sst) pharmacophore sequence, Phe-Trp-Lys-Thr, is critical in order to achieve high affinity receptor binding for Sst receptor imaging and radiotherapy agents. Therefore, we determined the three dimensional solution phase structures of several Re-cyclized octreotide analogues by high resolution NMR spectroscopy and correlated our findings with Sst receptor binding affinities.

Methods: We previously reported the synthesis of four Re(V)-cyclized peptides from reduced linear peptides (octreotide, Ac-octreotide, Tyr³-octreotate, and Ac-Tyr³-octreoate) via transchelation reactions with [ReOCl₃(OPPh₃)(SMe₂)] in aqueous methanol solutions (Bigott, et al. J. Labelled Cpd. Radiopharm. 48:S235;2005). For each reaction, the predominant isomer with the expected molecular weight was isolated in pure form by preparative RP-HPLC and confirmed by LCMS. The disulfide-cyclized parent peptides (Re-free) were also synthesized, purified, and confirmed by LCMS. Two dimensional NMR experiments were performed to assess the Re(V) coordination spheres and three dimensional structures of the purified Re(V)-peptides. The parent disulfide peptides were also studied by NMR for comparison with the Re(V)-peptide data. Sst receptor IC₅₀ values were obtained for the Re-peptides and correlated to the NMR structural findings.

Results: In each of the peptides, the ReO metal center was coordinated to the two cysteine sulfurs and the amide nitrogen of the third amino acid in the sequence. The remaining coordination site on the Re metal was filled either by the N-terminal amine nitrogen or, in the case of the N-acetylated peptides, the amide nitrogen of the sixth amino acid. Sst receptor binding affinities were in the 10^-7 to 10^-8 M range, in a binding order of Ac-octreotide < octreotide < Ac-Tyr³-octrotate < Tyr³-octreotate.

Conclusions: Re-cyclization of these octreotide analogues resulted in low Sst receptor binding affinities, likely due to the perturbation of the β turn relative to the parent disulfide-cyclized peptide structure. Altering the N-terminus of these peptides had a significant effect on the Re(V) coordination sphere, overall peptide structure, and Sst receptor binding affinity. The proximity of the metal center to the β turn in the N-acetylated peptides disrupted the binding to a greater degree than when the N-terminus was involved in metal binding. Further structural modifications to distance the Re binding sphere from the Sst receptor pharmacophore may lead to new radiopharmaceutical agents with enhanced receptor binding.