There is evidence to suggest that increasing the level of saturation (that is, the number of sp-hybridized carbon atoms) of small molecules can increase their likelihood of success in the drug discovery pipeline. Owing to their favourable physical properties, alkylamines have become ubiquitous among pharmaceutical agents, small-molecule biological probes and pre-clinical candidates. Despite their importance, the synthesis of amines is still dominated by two methods: N-alkylation and carbonyl reductive amination. Therefore, the increasing demand for saturated polar molecules in drug discovery has continued to drive the development of practical catalytic methods for the synthesis of complex alkylamines. In particular, processes that transform accessible feedstocks into sp-rich architectures provide a strategic advantage in the synthesis of complex alkylamines. Here we report a multicomponent, reductive photocatalytic technology that combines readily available dialkylamines, carbonyls and alkenes to build architecturally complex and functionally diverse tertiary alkylamines in a single step. This olefin-hydroaminoalkylation process involves a visible-light-mediated reduction of in-situ-generated iminium ions to selectively furnish previously inaccessible alkyl-substituted α-amino radicals, which subsequently react with alkenes to form C(sp)-C(sp) bonds. The operationally straightforward reaction exhibits broad functional-group tolerance, facilitates the synthesis of drug-like amines that are not readily accessible by other methods and is amenable to late-stage functionalization applications, making it of interest in areas such as pharmaceutical and agrochemical research.

Due to its 3 carbonic acid groups being available for bioconjugation, the TRAP chelator (1,4,7-triazacyclononane-1,4,7-tris(methylene(2-carboxyethylphosphinic acid))) is chosen for the synthesis of trimeric bioconjugates for radiolabelling. We optimized a protocol for bio-orthogonal TRAP conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), including a detailed investigation of kinetic properties of Cu(II)-TRAP complexes. TRAP building blocks for CuAAC, TRAP(alkyne)3 and TRAP(azide)3 were obtained by amide coupling of propargylamine/3-azidopropyl-1-amine, respectively. For Cu(II) complexes of neat and triply amide-functionalized TRAP, the equilibrium properties as well as pseudo-first-order Cu(II)-transchelation, using 10 to 30 eq. of NOTA and EDTA, were studied by UV-spectrophotometry. Dissociation of any Cu(II)-TRAP species was found to be independent on the nature or excess of a competing chelator, confirming a proton-driven two-step mechanism. The respective thermodynamic stability constants (log K(ML): 19.1 and 17.6) and dissociation rates (k: 38 × 10(-6) and 7 × 10(-6) s(-1), 298 K, pH 4) show that the Cu(II) complex of the TRAP-conjugate possesses lower thermodynamic stability but higher kinetic inertness. At pH 2-3, its demetallation with NOTA was complete within several hours/days at room temperature, respectively, enabling facile Cu(II) removal after click coupling by direct addition of NOTA trihydrochloride to the CuAAC reaction mixture. Notwithstanding this, an extrapolated dissociation half life of >100 h at 37 °C and pH 7 confirms the suitability of TRAP-bioconjugates for application in Cu-64 PET (cf. t(1/2)(Cu-64) = 12.7 h). To showcase advantages of the method, TRAP(DUPA-Pep)3, a trimer of the PSMA inhibitor DUPA-Pep, was synthesized using 1 eq. TRAP(alkyne)3, 3.3 eq. DUPA-Pep-azide, 10 eq. Na ascorbate, and 1.2 eq. Cu(II)-acetate. Its PSMA affinity (IC50), determined by the competition assay on LNCaP cells, was 18-times higher than that of the corresponding DOTAGA monomer (IC50: 2 ± 0.1 vs. 36 ± 4 nM), resulting in markedly improved contrast in Ga-68-PET imaging. In conclusion, the kinetic inertness profile of Cu(II)-TRAP conjugates allows for simple Cu(II) removal after click functionalisation by means of transchelation, but also confirms their suitability for Cu-64-PET as demonstrated previously (Dalton Trans., 2012, 41, 13803).

Expression of the cellular transmembrane receptor avb6 integrin is essentially restricted to malignant epithelial cells in carcinomas of a broad variety of lineages, whereas it is virtually absent in normal adult tissues. Thus, it is a highly attractive target for tumor imaging and therapy. Furthermore, avb6 integrin plays an important role for the epithelial-mesenchymal interaction and the development of fibrosis. Methods: On the basis of the 68 Ga chelators TRAP (triazacyclononane-triphosphinate) and NODAGA, we synthesized mono-, di-, and trimeric conjugates of the avb6 integrin-selective peptide cyclo(FRGDLAFp(NMe)K) via click chemistry. These were labeled with 68 Ga and screened regarding their suitability for in vivo imaging of avb6 integrin expression by PET and ex vivo biodistribution in severe combined immunodeficiency mice bearing H2009 tumor (human lung adenocarcinoma) xenografts. For these, avb6 integrin expression in tumor and other tissues was determined by b6 immunohistochemistry. Results: Despite the multimers showing higher avb6 integrin affinities (23-120 pM) than the monomers (260 pM), the best results-that is, low background uptake and excellent tumor delineation-were obtained with the TRAP-based monomer 68 Ga-avebehexin. This compound showed the most favorable pharmacokinetics because of its high polarity (log D 5 -3.7) and presence of additional negative charges (carboxylates) on the chelator, promoting renal clearance. Although tumor uptake was low (0.65% 6 0.04% injected dose per gram tissue [%ID/g]), it was still higher than in all other organs except the kidneys, ranging from a maximum for the stomach (0.52 6 0.04 %ID/g) to almost negligible for the pancreas (0.07 6 0.01 %ID/g). A low but significant target expression in tumor, lung, and stomach was confirmed by immunohistochemistry. Conclusion: Because of highly sensitive PET imaging even of tissues with low avb6 integrin expression density, we anticipate clinical applicability of 68 Ga-avebehexin for imaging of avb6 tumors and fibrosis by PET.

Despite in vivo mapping of integrin α v β 3 expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α 5 β 1 appears to be a promising target, for which purpose we designed the PET radiopharmaceutical 68 Ga-aquibeprin. Methods: 68 Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α 5 β 1 integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated 68 Ga labeling. Integrin α 5 β 1 and α v β 3 affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α 5 and β 3 subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. Results: 68 Ga-aquibeprin shows high selectivity for integrin α 5 β 1 (50% inhibition concentration [IC 50 ] 5 0.088 nM) over α v β 3 (IC 50 5 620 nM) and a pronounced hydrophilicity (log D 5 -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the β 3 integrin subunit but also an intense expression of the α 5 integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. 68 Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for 68 Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer 68 Ga-avebetrin, which shows an inverse selectivity for integrin α v β 3 (IC 50 5 0.22 nM) over α 5 β 1 (IC 50 5 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (∼5 mg/kg) of the respective other compound. Conclusion: 68 Ga-aquibeprin and 68 Ga-avebetrin are recommendable for complementary mapping of integrins α 5 β 1 and α v β 3 by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.