Gallium Complexation, Stability, and Bioconjugation of 1,4,7-Triazacyclononane Derived Chelators with Azaheterocyclic Arms

Abstract

We have recently introduced a 1,4,7-triazacyclononane (TACN) based chelating system with additional five-membered azaheterocyclic substituents for complexation of radioactive Cu ions. In this work, we investigated the complexation properties of these novel chelators with Ga. In labeling experiments, we could show that the penta- and hexadentate imidazole derivatives NODIA-Me 4 and NOTI-Me 1 can be labeled with Ga in specific activities up to ∼30 MBq nmol, while the corresponding thiazole derivative NOTThia 2 did not label satisfactorily under identical conditions. NMR studies on the Ga complexes of 1 and the model compound NODIA-Me-NH-Me 5 revealed formation of rigid 1:1 chelates with a slow macrocyclic interconversion and inert Ga-N bonds to the methylimidazole residues on the NMR time scale. The TACN-derived bifunctional chelator NODIA-Me was furthermore conjugated to a prostate-specific membrane antigen (PSMA) targeting moiety to give the corresponding bioconjugate NODIA-Me-PSMA 7. Serum stability and ligand challenge experiments of Ga-7 confirmed formation of a stable complex for up to 4 h. The remaining coordination site of five-coordinate Ga complexes was found to be occupied by monodentate ligands including hydroxide and chloride anions depending on the conditions. According to density functional theory calculations, coordination of monodentate ligands as well as of the amide group for the bioconjugated ligand are energetically plausible. Finally, the labeled bioconjugateGa-7 exhibited rapid renal clearance in biodistribution studies performed by small animal PET imaging with no indication of transchelation/demetalation in vivo. Altogether, our results provide strong evidence for a stable Ga complexation of our novel TACN-based chelators bearing imidazole arms. Despite the formation of two complexes incorporating different monodentate ligands in vitro, the imidazole type ligands show promise as chelating agents for the future development of gallium based radiopharmaceuticals.

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector

“…Fmoc-3-(2-naphthyl)-L-alanine was obtained from IRIS Biotech (Marktredwitz, Germany). The bifunctional chelator NODIA-Me (2-(4,7-bis((1-methyl-1Himidazol-2-yl)methyl)-1,4,7-triazonan-1-yl)acetic acid) 1 was prepared as previously described [58]. The radiotracer [ 68 Ga]Ga-PSMA-HBED-CC was prepared as previously described [59].…”

“…LR-ESI-MS calcd m/z for C 43 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 Page 11 (20.6 mg, 0.021 mmol, 91%). 1 68 Ga radiolabelling was accomplished by using the Modular-Lab PharmTracer automated synthesis module (Eckert&Ziegler, Berlin, Germany) as previously described [58]. The reaction vial contained 20 µg of 4 in a mixture of 400 µL sodium acetate buffer (pH 4.5) and 2 mL water.…”

“…Our previously described triply substituted ligands lack a functionality for the covalent conjugation of biologically active targeting vectors [57]. For this reason, we developed the BFC NODIA-Me 1, in which we replaced one methylimidazole arm with an acetic acid group (Scheme 2) [58]. This acetic acid group serves as site for the attachment of appropriate targeting vectors via peptide bond formation.…”

“…Corresponding data are given in the Supporting Information. We recently showed that the remaining coordination site of the gallium NODIA-Me complex can be occupied by monodentate ligands such as hydroxide or chloride [58]. The NODIA-Me complex charge can thus be estimated to +2 for [ 68…”

“…Experiments showed that corresponding methylimidazole-type ligands (e.g. NODIA-Me in Scheme 1) that incorporate borderline azaheterocyclic N donors according to the HSAB (hard and soft acids and bases) principle form rigid and stable Ga 3+ complexes allowing their application also with the PET-nuclide gallium-68 [58]. This study reports the in vitro and in vivo evaluation of the BFC NODIA-Me…”