Cross-Isotopic Bioorthogonal Tools as Molecular Twins for Radiotheranostic Applications

Abstract

Radiotheranostics are designed by labeling targeting (bio)molecules with radionuclides for diagnostic or therapeutic application. Because the pharmacokinetics of therapeutic compounds play a pivotal role, chemically closely related imaging agents are used to evaluate the overall feasibility of the therapeutic approach. "Theranostic relatives" that utilize different elements are frequently used in clinical practice. However, variations in pharmacokinetics, biodistribution, and target affinity due to different chemical properties of the radioisotopes remain as hurdles to the design of optimized clinical tools. Herein, the design and synthesis of structurally identical compounds, either for diagnostic (¹⁸ F and a stable metal isotope) or therapeutic application (radiometal and stable ¹⁹ F), are reported. Such "molecular twins" have been prepared by applying a modular strategy based on click chemistry that enables efficient radiolabeling of compounds containing a metal complex and a tetrazine moiety. This additional bioorthogonal functionality can be used for subsequent radiolabeling of (bio)molecules or pretargeting approaches, which is demonstrated in vitro.

Keywords: bioorthogonal chemistry; click chemistry; isotopes; radiochemistry; radiopharmaceuticals.

Figure 1

Concept of “cross‐isotopic” theranostic tools (CITs). A trifunctional precursor can be used to introduce a stable metal isotope and subsequently be radiofluorinated through copper‐catalyzed click chemistry. An additional bioorthogonal moiety can be applied for A) radiolabeling of (bio)molecules or B) pretargeting approaches through in vivo click chemistry.

Figure 2

A) Chemical synthesis of tetrazine‐modified metal complexes by using stable metal isotopes of holmium, lutetium, and yttrium. B) Preparation of precursor compounds (containing stable fluorine‐19) for subsequent labeling with therapeutic radiometals. C) Click‐radiofluorination (top) and radiometalation (bottom) to afford diagnostic (diaCITs) and therapeutic CITs (theraCITs), respectively, in good to excellent radiochemical yields. Boc: tert‐butyloxycarbonyl, HBTU: 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate, DIPEA: N,N‐diisopropylethylamine, TFA: trifluoroacetic acid.

Figure 3

A) Verification of click radiofluorination and radiometalation by HPLC for holmium CITs. B) Neutron activation analysis of radiofluorinated diaCITs, showing sufficient stability of the metal–DOTA complexes during radiolabeling through copper‐catalyzed click chemistry.

Figure 4

A) Synthesis of an ¹⁸F‐labeled and yttrium‐DOTA modified ACUPA conjugate.

Figure 5

A) The application of [¹⁸F]F‐Y‐ACUPA to study uptake in PSMA‐positive LNCaP cells. B) Treatment of LNCaP cells first with ACUPA‐TCO (11), followed by [¹⁸F]F‐Y‐10, to investigate the application of CITs towards pretargeting approaches. C) The PSMA inhibitor 2‐PMPA was used in control experiments (blocking).

Figure 6

A) Results of in vitro targeting and B) in vitro pretargeting by applying different concentrations of 11 (500, 1000 nm) for labeling and pretargeting (n=3).