We describe an easy synthesis of original C-functionalized cyclam derivatives based on the efficient bisaminal template method. In the perspective of developing bifunctional chelating agents (BCAs), this new synthetic strategy offers the possibility of introducing various coupling functions on one carbon atom in the β-N position of the macrocycle, leaving the four nitrogen atoms available for the introduction of pendant coordinating arms. The methodology is based on a keystone C-functionalized oxo-cyclam bisaminal intermediate that is obtained by cyclization of a preorganized tetraamine using various methyl acrylate analogues. These compounds constitute valuable precursors for selective preparation of mono- and di-N-protected C-functionalized cyclams and C-functionalized cyclams, cross-bridged cyclams, and oxo-cyclam derivatives. This approach was successfully adapted to the synthesis of three BCAs with great interest especially for biomedical applications: TETA, TE2A, and CB-TE2A. The structures of different intermediates and Cu(II) complexes of C-functionalized cyclam derivatives were confirmed using single-crystal X-ray diffraction, while reactivity of the key intermediates was rationalized by the analysis of the electrostatic potentials calculated at the TPSSh/6-311G(d,p) level.

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In view of the excellent copper(ii) and 64-copper(ii) complexation of a TE1PA ligand, a monopicolinate cyclam, in both aqueous medium and in vivo, we looked for a way to make it bifunctional, while maintaining its chelating properties. Overcoming the already known drawback of grafting via its carboxyl group, which is essential to the overall properties of the ligand, a TE1PA bifunctional derivative bearing an additional isothiocyanate coupling function on a carbon atom of the macrocyclic ring was synthesized. This led to an architecture that is comparable to that of other commercially available bifunctional copper(ii) chelators such as p-SCN-Bn-DOTA already used in clinical trials for 64Cu-immuno-PET imaging. The C-functionalization of TE1PA on one carbon atom in the β-N position of the cyclam backbone was successfully achieved by adapting our patented methodology to the huge challenge, allowing the regiospecific mono-N-functionalization of the unsymmetrical ligand. The obtained ligand p-SCN-Bn-TE1PA was coupled to a 9E7.4 murine antibody (mAb), an IgG2a anti CD-138 for multiple myeloma (MM) targeting. The conjugation efficiency was assessed by looking at the 64Cu radiolabeling and the radiopharmaceutical 64Cu-9E7.4-p-SCN-Bn-TE1PA immunoreactivity, and in particular by comparing with 9E7.4-p-SCN-Bn-NOTA and 9E7.4-p-SCN-Bn-DOTA obtained from commercial and presumably highly efficient chelators NOTA and DOTA, respectively. The results are quite clear, showing that p-SCN-Bn-TE1PA has a coupling rate 5 times higher and an immunoreactivity 1.5 to 2 times greater than those of its two competitors. p-SCN-Bn-TE1PA also outperforms TE1PA conjugated via its carboxylic function on the same antibody. The first 64Cu-immuno-PET preclinical study in a syngeneic model of MM was performed, confirming the good in vivo properties of 64Cu-9E7.4-p-SCN-Bn-TE1PA for PET imaging, considering the high clearance even after 24 h and the particularly important tumor-to-liver ratio that was increasing at 48 h.