A Bone-Seeking trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using ^99mTc- and ¹⁷⁷Lu-Labeled Tetrazines

Abstract

A high yield synthesis of a novel, small molecule, bisphosphonate-modified trans-cyclooctene (TCO-BP, 2) that binds to regions of active bone metabolism and captures functionalized tetrazines in vivo, via the bioorthogonal inverse electron demand Diels-Alder (IEDDA) cycloaddition, was developed. A Tc-labeled derivative of 2 demonstrated selective localization to shoulder and knee joints in a biodistribution study in normal mice. Compound 2 reacted rapidly with aLu-labeled tetrazine in vitro, and pretargeting experiments in mice, using 2 and the Lu-labeled tetrazine, yielded high activity concentrations in shoulder and knee joints, with minimal uptake in other tissues. Pretargeting experiments with 2 and a novelTc-labeled tetrazine also produced high activity concentrations in the knees and shoulders. Critically, both radiolabeled tetrazines showed negligible uptake in the skeleton and joints when administered in the absence of 2. Compound 2 can be utilized to target functionalized tetrazines to bone and represents a convenient reagent to test novel tetrazines for use with in vivo bioorthogonal pretargeting strategies.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…11 These IEDDA reactions have second-order rate constants ranging from k 2  = 10 3 –10 6 M −1 s −1 , which are primarily driven by expulsion of N 2 and TCO strain relief following the cycloaddition 12,13 Furthermore, electron withdrawing substituents attached to the tetrazine confer increased reaction rates between the dienophile and diene. 14,15 Successful pretargeted in vivo nuclear imaging has been demonstrated in preclinical models using PET isotopes, including 11 C (t 1/2 20.3 minutes), 16,17 18 F (t 1/2 110 minutes) 18–20 and 64 Cu (t 1/2 12.7 hours), 4,21–24 as well as SPECT isotopes including 99m Tc (t 1/2 6 hours), 25–27 111 In (t 1/2 2.8 days) 28–30 and 177 Lu (t 1/2 6.7 days). 27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups.…”

“…14,15 Successful pretargeted in vivo nuclear imaging has been demonstrated in preclinical models using PET isotopes, including 11 C (t 1/2 20.3 minutes), 16,17 18 F (t 1/2 110 minutes) 18–20 and 64 Cu (t 1/2 12.7 hours), 4,21–24 as well as SPECT isotopes including 99m Tc (t 1/2 6 hours), 25–27 111 In (t 1/2 2.8 days) 28–30 and 177 Lu (t 1/2 6.7 days). 27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups. 12,34–37 Most examples incorporate TCO on the slower clearing molecule followed by a fast clearing radiolabeled tetrazine tracer, but some groups have explored reversing the chemistry.…”

Technetium‐99m radiochemistry for pharmaceutical applications

“…The conjugation of the pharmacophore unit to the chelator usually is achieved via methods that are fast and selective under mild conditions, like amidation. Furthermore, the regioselective alkyne‐azide cycloaddition as well as other “click” reactions have been proposed . In the case where the pharmacophore is a bioactive peptide, the bifunctional chelator may be an amino acid itself, added at the end of the peptide sequence.…”

“…Furthermore, the regioselective alkyne-azide cycloaddition as well as other "click" reactions have been proposed. 117,118 In the case where the pharmacophore is a bioactive peptide, the bifunctional chelator may be an amino acid itself, added at the end of the peptide sequence. For example, suitable tripeptide or tetrapeptide chelators may be incorporated at the end of the peptide sequence to label with the oxotechnetium (V) core.…”

Synthesis and evaluation of a novel 99mTc nitrido radiopharmaceutical with alendronate dithiocarbamate as a potential bone‐imaging agent

“…In this complex, the L,L-ethylene dicysteine group acted as a bifunctional chelator to link to the AMDP and coordinate with 99m Tc. [21] Due to the similar coordination chemistry of rhenium and technetium, theranostic application of 99m Tc/ 186/188 Re complexes is possible. Ogawa et al chose mercaptoacetylglycylglycylglycine (MAG3) and 6-hydrazinopyridine-3-carbox ylic acid (HYNIC) as chelating sites to develop 99m Tc-MAG3-HBP (HBP = alendronate) and 99m Tc-HYNIC-HBP.…”