preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Preparation and Evaluation of <sup>99m</sup>Tc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

McMaster University

The aim of this work was to investigate the relative radiolabelling kinetics and affinity of a series of ligands for the [(99m)Tc(CO)(3)](+) core, both in the absence and in the presence of competing donors. This information was used to select a suitable ligand for radiolabelling complex peptide-based targeting vectors in high yield under mild conditions.

Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor.

Ultrasound (US) contrast agents based on microbubbles (MBs) are being investigated as platforms for drug and gene delivery. A methodology for determining the distribution and fate of modified MBs quantitatively in vivo can be achieved by tagging MBs directly with (99m)Tc. This creates the opportunity to employ dual-modality imaging using both US and small animal SPECT along with quantitative ex vivo tissue counting to evaluate novel MB constructs.

The synthesis, magnetic purification and evaluation of 99mTc-labeled microbubbles.

A new prosthetic group referred to as the triazole appending agent (TAAG) was developed as a means to prepare targeted radioiodine-based molecular imaging and therapy agents. Tributyltin-TAAG and the fluorous analogue were synthesized in high yield using simple click chemistry and the products labeled in greater than 95% RCY with (123)I. A TAAG derivative of an inhibitor of prostate-specific membrane antigen was prepared and radiolabeled with (123)I in 85% yield where biodistribution studies in LNCap prostate cancer tumor models showed rapid clearance of the agent from nontarget tissues and tumor accumulation of 20% injected dose g(-1) at 1 h. The results presented demonstrate that the TAAG group promotes minimal nonspecific binding and that labeled conjugates can achieve high tumor uptake and exquisite target-to-nontarget ratios.

Triazole Appending Agent (TAAG): A New Synthon for Preparing Iodine-Based Molecular Imaging and Radiotherapy Agents.

Dipeptidyl (acyloxy)methyl ketones (AOMKs) were functionalized with different iodine-containing prosthetic groups to generate a library of candidate cathepsin B probes. Compound 23a, (S)-20-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-1-(4-iodophenyl)-1,14,21-trioxo-5,8,11-trioxa-2,15-diazadocosan-22-yl 2,4,6-trimethylbenzoate, was identified as a potential lead through in vitro screening, having a Ki = 181 ± 9 nM and demonstrating the ability to effectively label active cathepsin B in vitro. Its less potent analogue 11a, (S)-3-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-7-[6-(4-iodobenzamido)hexanamido]-2-oxoheptyl 2,4,6-trimethylbenzoate, was also tested as a comparison. Biodistribution studies of the iodine-125-labeled compounds in MDA-MB-231 mouse xenografts exhibited tumor uptake of 0.58% ± 0.06% injected dose per gram (ID/g) for [(125)I]11a and 1.12% ± 0.08% ID/g for [(125)I]23a at 30 min. The tumor-to-blood ratios reached 1.2 for [(125)I]23a and 1.6 for [(125)I]11a after 23 h. The more hydrophilic [(125)I]23a showed an improved clearance profile with a superior tumor-to-muscle ratio of 7.0 compared to 3.4 for [(125)I]11a at 23 h. Iodinated AOMK ligands are suitable in vitro probes for cathepsin B and hold promise as a platform to develop molecular imaging probes.

Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B.

The synthesis, stability, and photophysical properties of [2 + 1] Re(I)/Tc(I) complexes derived from bipyridine and a series of imidazole derivatives were investigated as a means of identifying complexes suitable for creating targeted isostructural optical/nuclear molecular imaging probes. To prepare the desired complexes, [Re(CO)3(H2O)3]Br was combined with 2,2'-bipyridine (bipy) to give [Re(CO)3(bipy)Br], which in turn was converted to the desired complexes by treatment with functionalized imidazoles, yielding crystal structures of two new Re complexes. The corresponding (99m)Tc complexes [(99m)Tc(CO)3(bipy)(L)](+) (L = imidazole derivatives) were prepared by combining [(99m)Tc(CO)3(bipy)(H2O)]Cl with the same series of ligands and heating at 40 or 60 °C for 30 min. Quantitative transformation to the final products was confirmed in all cases by HPLC, and the nature of the complexes was verified by comparison to the authentic Re standards. Incubation in saline and plasma, and amino acid challenge experiments showed that N-substituted imidazole derivatives, bearing electron donating groups, exhibited superior stability to analogous metal complexes derived from less basic ligands. Imaging studies in mice revealed that with the appropriate choice of monodentate ligand, it is possible to prepare robust [2 + 1] Tc complexes that can be used as the basis for preparing targeted isostructural optical and nuclear probes.

Imidazole-based [2 + 1] Re(I)/99mTc(I) complexes as isostructural nuclear and optical probes.

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 (99m)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 a (177)Lu-labeled tetrazine in vitro, and pretargeting experiments in mice, using 2 and the (177)Lu-labeled tetrazine, yielded high activity concentrations in shoulder and knee joints, with minimal uptake in other tissues. Pretargeting experiments with 2 and a novel (99m)Tc-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.

A Bone-Seeking trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using (99m)Tc- and (177)Lu-Labeled Tetrazines.

Hydrocyanine dyes are sensitive "turn-on" type optical probes that can detect reactive oxygen species (ROS). We have developed a method to prepare an (18) F-labeled hydrocyanine dye as a multi-modal PET and optical "turn-on" probe. A commercially available near infrared (NIR) dye was modified with a fluorinated prosthetic group that did not alter its ROS sensing properties in the presence of superoxide and hydroxyl radicals. The (18) F-labeled analogue was produced using a single-step terminal fluorination procedure. Positron emission tomography (PET) imaging and quantitative in vivo biodistribution studies indicated this novel probe had remarkably different pharmacokinetics compared to the oxidized cyanine analogue. The chemistry reported enables the use of quantitative and dynamic PET imaging for the in vivo study of hydrocyanine dyes as ROS probes.

Preparation of an (18) F-Labeled Hydrocyanine Dye as a Multimodal Probe for Reactive Oxygen Species.

A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with 99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The 99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability in vitro and in vivo. A trans-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with 99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with 99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control 99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting Staphylococcus aureus infection in vitro and in vivo. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the 99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.

Article	Year
Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor. Nuclear medicine and biology\| PubMed ID: 19875047	2009
The synthesis, magnetic purification and evaluation of 99mTc-labeled microbubbles. Nuclear medicine and biology\| PubMed ID: 21741260	2011
Triazole Appending Agent (TAAG): A New Synthon for Preparing Iodine-Based Molecular Imaging and Radiotherapy Agents. ACS medicinal chemistry letters\| PubMed ID: 24900470	2012
Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B. Journal of medicinal chemistry\| PubMed ID: 25360988	2014
Imidazole-based [2 + 1] Re(I)/99mTc(I) complexes as isostructural nuclear and optical probes. Inorganic chemistry\| PubMed ID: 25634699	2015
A Bone-Seeking trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using (99m)Tc- and (177)Lu-Labeled Tetrazines. Journal of medicinal chemistry\| PubMed ID: 27676258	2016
Preparation of an (18) F-Labeled Hydrocyanine Dye as a Multimodal Probe for Reactive Oxygen Species. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 27768812	2017
A 99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule trans-Cyclooctene Derivatives. PloS one\| PubMed ID: 27936007	2016

Shannon Czorny

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