H(2)azapa: a versatile acyclic multifunctional chelator for (67)Ga, (64)Cu, (111)In, and (177)Lu

Abstract

Preliminary experiments with the novel acyclic triazole-containing bifunctional chelator H2azapa and the radiometals (64)Cu, (67)Ga, (111)In, and (177)Lu have established its significant versatile potential as an alternative to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for metal-based radiopharmaceuticals. Unlike DOTA, H2azapa radiolabels quantitatively with (64)Cu, (67)Ga, (111)In, and (177)Lu in 10 min at room temperature. In vitro competition experiments with human blood serum show that (64)Cu remained predominantly chelate-bound, with only 2% transchelated to serum proteins after 20 h. Biodistribution experiments with [(64)Cu(azapa)] in mice reveal uptake in various organs, particularly in the liver, lungs, heart, intestines, and kidneys. When compared to [(64)Cu(DOTA)](2-), the lipophilic neutral [(64)Cu(azapa)] was cleared through the gastrointestinal tract and accumulated in the liver, which is common for lipophilic compounds or free (64)Cu. The chelator H2azapa is a model complex for a click-based bifunctional chelating agent, and the lipophilic benzyl "place-holders" will be replaced by hydrophilic peptides to modulate the pharmacokinetics and direct activity away from the liver and gut. The solid-state molecular structure of [In(azapa)(H2O)][ClO4] reveals a very rare eight-coordinate distorted square antiprismatic geometry with one triazole arm bound, and the structure of [(64)Cu(azapa)] shows a distorted octahedral geometry. The present study demonstrates significant potential for bioconjugates of H2azapa as alternatives to DOTA in copper-based radiopharmaceuticals, with the highly modular and "clickable" molecular scaffold of H2azapa easily modified into a variety of bioconjugates. H2azapa is a versatile addition to the "pa" family, joining the previously published H2dedpa ((67/68)Ga and (64)Cu), H4octapa ((111)In, (177)Lu, and (90)Y), and H5decapa ((225)Ac) to cover a wide range of important nuclides.

Figure 1

¹H NMR spectra (DMSO-d₆, 25 °C) of (top) H₂azapa, 300 MHz; (middle) [Ga(azapa)]⁺, 600 MHz; (middle) [In(azapa)]⁺, 400 MHz; and (bottom, 300 MHz, D₂O) [Lu(azapa)]⁺, showing simple diastereotopic splittings for a single isomer of [Ga(azapa)]⁺ and major peak broadening for solution fluxional interconversion between multiple isomers of [In(azapa)]⁺ and, to a lesser extent, [Lu(azapa)]⁺.

Figure 2

Overlay of ¹H NMR spectra of [In(azapa)]⁺ (400 MHz, DMSO-d₆) at temperatures ranging from 25 to 135 °C, showing a gradual averaging effect from increasingly rapid interconversion between multiple coordination isomers at higher temperatures.

Figure 3

ORTEP drawing of the solid-state molecular structure of [In(azapa)(H₂O)][ClO₄] obtained by X-ray diffraction. Hydrogen atoms are omitted for clarity.

Figure 4

ORTEP drawing of the solid-state molecular structure of [Cu(azapa)]. Hydrogen atoms are omitted for clarity.

Figure 5

Biodistribution of [⁶⁴Cu(azapa)] and [⁶⁴Cu(DOTA)]²⁻ showing organ and tissue uptake as percent injected dose per gram (% ID/g) obtained over 24 h in healthy athymic nude mice; Y-axis is normalized at 25% ID/g for clarity.

Figure 6

PET images of two (A and B) healthy female nude athymic mice injected with [⁶⁴Cu(azapa)] and imaged at 1, 4, and 24 h post injection, showing rapid clearance through the gut, with ~8%ID/g remaining in the liver after 24 h.

Scheme 1

Synthesis of H₂azapa: Reagents and Conditionsa ^a(i) THF, RT, 3 h; (ii) Propargyl bromide, NaO^tBu, TBAI, MeCN, 0 °C–RT, 48 h; (iii) 2:1 DCM/TFA, RT, 1 h; (iv) Methyl 6-bromomethyl picolinate, Na₂CO₃, MeCN, RT, 48 h; (v) BnN₃, sodium ascorbate (0.2 equiv), Cu(OAc)₂ (2.1 equiv), 1:1 t-BuOH/H₂O, RT, 16 h; (vi) Na₂S, 1:1 THF/H₂O, 24 h.

Chart 1

Structures of Selected State-of-the-Art “Gold Standard” Chelators, and Chelators Studied or Discussed in This Work