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

PAML's scheduling/ inspection solution. Interview by Carren Bersch.

The synthesis, radiolabelling and biodistribution of iodinated C-hydroxy-nido-carborane ligands is described. Microwave heating by using NaF in aqueous ethanol was used to prepare {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate], nido-carboranol} and {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate-8-carboxylic acid], nido-salborin} in 97 and 90 % yield, respectively. Radioiodination of these nido-carboranes was completed by using both (125)I and (123)I, and the products were obtained in high radiochemical purity (>99 %) and yield (72 to 87 %). The structures of the radiolabelled products were validated through comparison to authentic standards. Biodistribution studies in BALB/c mice showed low accumulation of the labelled compounds in the liver and intestines, which are sites where labelled carboranes typically localise. The labelled cluster bearing hydroxy and carboxylic acid groups on the two carbon vertices demonstrated preferential clearance through the kidneys and low thyroid uptake. This compound had substantially reduced non-specific binding than the deshydroxy analogue making it an attractive bifunctional ligand for preparing targeted molecular imaging and therapy agents.

Synthesis, characterisation, and biodistribution of radioiodinated C-hydroxy-carboranes.

A convenient method to prepare (99m)Tc analogues of a class of rhenium(I) luminophores was developed, creating isostructural pairs of nuclear and optical probes. A two-step procedure and a new one-pot procedure were used to produce a series of [2 + 1] complexes of the type [Tc(CO)3(bipy)L](+) in greater than 80% yield. The plasma stability of the reported compounds was evaluated, where the basicity of the monodentate pyridine type ligand (L) has a significant impact with half-lives ranging from 2 to 20 h. The ability to generate the radioactive complexes makes it possible to quantitate cell uptake of Re luminophores, which was demonstrated in MCF-7 breast cancer cells using (99m)Tc analogues of two Re(I)-based mitochondrial targeting dyes.

Isostructural nuclear and luminescent probes derived from stabilized [2 + 1] rhenium(I)/technetium(I) organometallic complexes.

A series of C-hydroxy carborane derivatives of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)-pentanedioic acid were prepared as a new class of boron rich inhibitors of prostate specific membrane antigen (PSMA), which is overexpressed on prostate cancer tumours and metastases. Closo-, nido- and iodo-carborane conjugates were prepared and screened in vitro where the water soluble iodinated cluster had the highest affinity with an IC50 value (73.2 nM) that was comparable to a known PSMA inhibitor 2-(phosphonomethyl)-pentanedioic acid (PMPA, 63.9 nM). The radiolabeled analogue was prepared using (123)I and the biodistribution determined in a prostate cancer model derived from a PSMA positive cell line (LNCaP) at 1, 2, 4, 6 and 24 h post injection (n = 4 per time point). The results showed good initial tumour uptake of 4.17% at 1 h, which remained at that level only decreasing somewhat at 6 h (3.59%). At the latter time point tumour-to-blood and tumour-to-muscle ratios peaked at 3.47 at 25.52 respectively. There was significant off-target binding particularly in the liver and gall bladder and a surprising amount of deiodination in vivo. Notwithstanding, this work demonstrates that carboranes can be used to prepare potent ligands for PSMA creating the opportunity to develop a new class of BNCT agents for prostate cancer.

Preparation and evaluation of carborane-derived inhibitors of prostate specific membrane antigen (PSMA).

Catching bubbles: targeting ultrasound microbubbles using bioorthogonal inverse-electron-demand Diels-Alder reactions.

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.

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 fifth generation aliphatic polyester dendrimer was functionalized with vinyl groups at the periphery and a dipicolylamine Tc(I) chelate at the core. This structure was PEGylated with three different molecular weight mPEGs (mPEG160, mPEG350, and mPEG750) using thiol-ene click chemistry. The size of the resulting macromolecules was evaluated using dynamic light scattering, and it was found that the dendrimer functionalized with mPEG750 was molecularly dispersed in water, exhibiting a hydrodynamic diameter of 9.2 ± 2.1 nm. This PEGylated dendrimer was subsequently radiolabeled using [(99m)Tc(CO)3(H2O)3](+) and purified to high (>99%) radiochemical purity. Imaging studies were initially performed on healthy rats to allow comparison to previous Tc-labeled dendrimers and then on xenograft murine tumor models, which collectively showed that the dendrimers circulated in the blood for an extended period of time (up to 24 h). Furthermore, the radiolabeled dendrimer accumulated in H520 xenograft tumors, which could be visualized by single-photon emission computed tomography (SPECT). The reported PEGylated aliphatic polyester dendrimers represent a new platform for developing tumor-targeted molecular imaging probes and therapeutics.

Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated High-Generation Polyester Dendrimers.

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels-Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with trans-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics.

(125)I-Tetrazines and Inverse-Electron-Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies.

The feasibility of developing a single agent that can deliver radioactive iodine and also direct cellular immune function by engaging endogenous antibodies as an antibody-recruiting small molecule (ARM) was determined. A library of new prostate-specific membrane antigen (PSMA)-binding ligands that contained antibody-recruiting 2,4-dinitrophenyl (DNP) groups and iodine were synthesized and screened in vitro and in vivo. A lead compound (9b) showed high affinity for PSMA and the ability to bind anti-DNP antibodies. Biodistribution studies of the iodine-125 analogue showed 3% ID/g in LNCaP xenograft tumors at 1 h postinjection with tumor-to-blood and tumor-to-muscle ratios of 10:1 and 44:1, respectively. The radiolabeled analogue was bound and internalized by LNCaP cells, with both functions blocked using a known PSMA inhibitor. A second candidate showed high tumor uptake (>10% ID/g) but had minimal binding to anti-DNP antibodies. The compounds reported represent the first examples of small molecules developed specifically for combination immunotherapy and radiotherapy for prostate cancer.

Preparation and Evaluation of Radiolabeled Antibody Recruiting Small Molecules That Target Prostate-Specific Membrane Antigen for Combined Radiotherapy and Immunotherapy.

Prostate-specific membrane antigen (PSMA) is frequently overexpressed and upregulated in prostate cancer. To date, various (18)F- and (68)Ga-labeled urea-based radiotracers for PET imaging of PSMA have been developed and entered clinical trials. Here, we describe an automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical models of prostate cancer.

Automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical prostate cancer models.

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
PAML's scheduling/ inspection solution. Interview by Carren Bersch. MLO: medical laboratory observer\| PubMed ID: 20690456	2010
Synthesis, characterisation, and biodistribution of radioiodinated C-hydroxy-carboranes. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 22807279	2012
Isostructural nuclear and luminescent probes derived from stabilized [2 + 1] rhenium(I)/technetium(I) organometallic complexes. Inorganic chemistry\| PubMed ID: 24228697	2013
Preparation and evaluation of carborane-derived inhibitors of prostate specific membrane antigen (PSMA). Dalton transactions (Cambridge, England : 2003)\| PubMed ID: 24481236	2014
Catching bubbles: targeting ultrasound microbubbles using bioorthogonal inverse-electron-demand Diels-Alder reactions. Angewandte Chemie (International ed. in English)\| PubMed ID: 24829138	2014
Triazole Appending Agent (TAAG): A New Synthon for Preparing Iodine-Based Molecular Imaging and Radiotherapy Agents. ACS medicinal chemistry letters\| PubMed ID: 24900470	2012
Imidazole-based [2 + 1] Re(I)/99mTc(I) complexes as isostructural nuclear and optical probes. Inorganic chemistry\| PubMed ID: 25634699	2015
Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated High-Generation Polyester Dendrimers. Biomacromolecules\| PubMed ID: 26295201	2015
(125)I-Tetrazines and Inverse-Electron-Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies. Bioconjugate chemistry\| PubMed ID: 26699913	2016
Preparation and Evaluation of Radiolabeled Antibody Recruiting Small Molecules That Target Prostate-Specific Membrane Antigen for Combined Radiotherapy and Immunotherapy. Journal of medicinal chemistry\| PubMed ID: 26894427	2016
Automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical prostate cancer models. EJNMMI research\| PubMed ID: 27142881	2016
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

Nancy Janzen

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