Here, we present the synthesis and characterization of a new potentially nonadentate chelator H4pypa and its bifunctional analogue tBu4pypa-C7-NHS conjugated to prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys). H4pypa is very functionally versatile and biologically stable. Compared to the conventional chelators (e.g., DOTA, DTPA), H4pypa has outstanding affinities for both 111In (EC, t 1/2 ≈ 2.8 days) and 177Lu (β–,γ, t 1/2 ≈ 6.64 days). Its radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 min, at a ligand concentration as low as 10–6 M, with excellent stability in human serum over at least 5–7 days (<1% transchelation). The thermodynamic stabilities of the [M­(pypa)]− complexes (M3+ = In3+, Lu3+, La3+) were dependent on the ionic radii, where the smaller In3+ has the highest pM value (30.5), followed by Lu3+ (22.6) and La3+ (19.9). All pM values are remarkably higher than those with DOTA, DTPA, H4octapa, H4octox, and H4neunpa. Moreover, the facile and versatile bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold (compound 14) allows incorporation of a variety of linkers for bioconjugation through easy nucleophilic substitution. In this work, an alkyl linker was selected to couple H4pypa to a PSMA-targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting nor the chelation properties. The biodistribution profiles of 111In- and 177Lu-labeled tracers are different, but promising, with the 177Lu analogue particularly outstanding.

Potentially nonadentate (NO) bifunctional chelator p-SCN-Bn-Hneunpa and its immunoconjugate Hneunpa-trastuzumab for In radiolabeling are synthesized. The ability of p-SCN-Bn-Hneunpa and Hneunpa-trastuzumab to quantitatively radiolabel InCl at an ambient temperature within 15 or 30 min, respectively, is presented. Thermodynamic stability determination with In, Bi, and La resulted in high conditional stability constant (pM) values. In vitro human serum stability assays have demonstrated both In complexes to have high stability over 5 days. Mouse biodistribution of [In][In(p-NO-Bn-neunpa)], compared to that of [In][In(p-NH-Bn-CHX-A″-diethylenetriamine pentaacetic acid (DTPA))], at 1, 4, and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates In-neunpa-trastuzumab andIn-CHX-A″-DTPA-trastuzumab demonstrate a similar distribution profile but with slightly lower tumor uptake of In-neunpa-trastuzumab compared to that ofIn-CHX-A″-DTPA-trastuzumab. These results were also confirmed by immuno-single photon emission computed tomography (immuno-SPECT) imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator p-SCN-Bn-Hneunpa to be a promising chelator for In (and other radiometals) with high in vitro stability and also show Hneunpa-trastuzumab to be an excellent In chelator with promising biodistribution in mice.

Three dipicolinic acid amine-derived compounds functionalized with a carboxylate (Hdpaa), phosphonate (Hdppa), and bisphosphonate (Hdpbpa), as well as their nonfunctionalized analogue (Hdpa), were successfully synthesized and characterized. The 1:1 lanthanum(III) complexes of Hdpa, Hdpaa, and Hdppa, the 1:2 lanthanum(III) complex of Hdpa, and the 1:1 gallium(III) complex of Hdpaa were characterized, including via X-ray crystallography for [La(dppa)(HO)] and [Ga(dpaa)(HO)]. Hdpa, Hdpaa, and Hdppa were evaluated for their thermodynamic stability with lanthanum(III) via potentiometric and either UV-vis spectrophotometric (Hdpaa) or NMR spectrometric (Hdpa and Hdppa) titrations, which showed that the carboxylate (Hdpaa) and phosphonate (Hdppa) containing ligands enhanced the lanthanum(III) complex stability by 3-4 orders of magnitude relative to the unfunctionalized ligand (comparing log β and pM values) at physiological pH. In addition, potentiometric titrations with Hdpaa and gallium(III) were performed, which gave significantly (8 orders of magnitude) higher thermodynamic stability constants than with lanthanum(III). This was predicted to be a consequence of better size matching between the dipicolinate cavity and gallium(III), which was also evident in the aforementioned crystal structures. Because of a potential link between lanthanum(III) and osteoporosis, the ligands were tested for their bone-directing properties via a hydroxyapatite (HAP) binding assay, which showed that either a phosphonate or bisphosphonate moiety was necessary in order to elicit a chemical binding interaction with HAP. The oral activity of the ligands and their metal complexes was also assessed by experimentally measuring log P values using the shake-flask method, and these were compared to a currently prescribed osteoporosis drug (alendronate). Because of the potential therapeutic applications of the radionuclides Ga, radiolabeling studies were performed withGa and Hdpaa. Quantitative radiolabeling was achieved at pH 6.5 in 10 min at room temperature with concentrations as low as 10 M, and human serum stability studies were undertaken.

An acyclic hexadentate oxine-derived chelating ligand, Hhox, was investigated as an alternative to current chelators for Ga. The straightforward preparation of Hhox, involving only one or two steps, obviates the synthetic challenges associated with many reported Ga chelators; it forms a Ga complex of great stability (log K = 34.4) with a remarkably high gallium scavenging ability (pGa = -log[Ga] = 28.3, ([Ga] = 1 μM; [L ] = 10 μM; pH 7.4, and 25 °C)). Moreover, Hhox coordinates Ga quantitatively in 5 min at room temperature in ligand concentrations as low as 1 × 10 M, achieving an unprecedented high molar activity of 11 ± 1 mCi/nmol (407 ± 3.7 MBq/nmol) without purification, suggesting prospective kit-based convenience. [Ga(hox)] showed no decomposition in a plasma challenge. Good in vivo stability and fast renal and hepatic clearance of the [Ga(hox)] complex were demonstrated using dynamic positron emission tomography/computed tomography imaging. The intrinsic fluorescence of [Ga(hox)] allowed for direct fluorescence imaging of cellular uptake and distribution, demonstrating the dual-channel detectability and intracellular stability of the metal complex.

Herein, we present the syntheses and characterization of a new undecadendate chelator, H4py4pa, and its bifunctional analog H4py4pa-phenyl-NCS, conjugated to the monoclonal antibody, Trastuzumab, which targets the HER2+ cancer. H4py4pa possesses excellent affinity for 225Ac (α, t 1/2 = 9.92 d) for targeted alpha therapy (TAT), where quantitative radiolabeling yield was achieved at ambient temperature, pH = 7, in 30 min at 10–6 M chelator concentration, leading to a complex highly stable in mouse serum for at least 9 d. To investigate the chelation of H4py4pa with large metal ions, lanthanum (La3+), which is the largest nonradioactive metal of the lanthanide series, was adopted as a surrogate for 225Ac to enable a series of nonradioactive chemical studies. In line with the 1H NMR spectrum, the DFT (density functional theory)-calculated structure of the [La­(py4pa)]− anion possessed a high degree of symmetry, and the La3+ ion was secured by two distinct pairs of picolinate arms. Furthermore, the [La­(py4pa)]− complex also demonstrated a superb thermodynamic stability (log K [La(py4pa)] – ∼ 20.33, pLa = 21.0) compared to those of DOTA (log K [La(DOTA)] – ∼ 24.25, pLa = 19.2) or H2macropa (log K [La(macropa)] – = 14.99, pLa ∼ 8.5). Moreover, the functional versatility offered by the bifunctional py4pa precursor permits facile incorporation of various linkers for bioconjugation through direct nucleophilic substitution. In this work, a short phenyl-NCS linker was incorporated to tether H4py4pa to Trastuzumab. Radiolabeling studies, in vitro serum stability, and animal studies were performed in parallel with the DOTA-benzyl-Trastuzumab. Both displayed excellent in vivo stability and tumor specificity.