Click Chemistry and Radiochemistry: An Update.

The term "click chemistry" describes a class of organic transformations that were developed to make chemical synthesis simpler and easier, in essence allowing chemists to combine molecular subunits as if they were puzzle pieces. Over the last 25 years, the click chemistry toolbox has swelled from the canonical copper-catalyzed azide-alkyne cycloaddition to encompass an array of ligations, including bioorthogonal variants, such as the strain-promoted azide-alkyne cycloaddition and the inverse electron-demand Diels-Alder reaction. Without question, the rise of click chemistry has impacted all areas of chemical and biological science. Yet the unique traits of radiopharmaceutical chemistry have made it particularly fertile ground for this technology. In this update, we seek to provide a comprehensive guide to recent developments at the intersection of click chemistry and radiopharmaceutical chemistry and to illuminate several exciting trends in the field, including the use of emergent click transformations in radiosynthesis, the clinical translation of novel probes synthesized using click chemistry, and the advent of click-based in vivo pretargeting.

Site-selective radiolabeling using mushroom tyrosinase and the strain-promoted oxidation-controlled 1,2-quinone cycloaddition.

We report the in vitro characterization and in vivo evaluation of a novel 89Zr-labeled radioimmunoconjugate synthesized using a site-selective bioconjugation strategy based on the oxidation of tyrosinase residues exposed by the deglycosylation of the IgG and the subsequent strain-promoted oxidation-controlled 1,2-quinone cycloaddition between these amino acids and trans-cyclooctene-bearing cargoes. More specifically, we site-selectively modified a variant of the A33 antigen-targeting antibody huA33 with the chelator desferrioxamine (DFO), thereby producing an immunoconjugate (DFO-SPOCQhuA33) with equivalent antigen binding affinity to its parent immunoglobulin but attenuated affinity for the FcγRI receptor. This construct was subsequently radiolabeled with [89Zr]Zr4+ to create a radioimmunoconjugate - [89Zr]Zr-DFO-SPOCQhuA33 - in high yield and specific activity that exhibited excellent in vivo behavior in two murine models of human colorectal carcinoma.

Site-Specific Radiohalogenation of a HER2-Targeted Single-Domain Antibody Fragment Using a Novel Residualizing Prosthetic Agent.

Because of their rapid tumor accumulation and normal tissue clearance, single-domain antibody fragments (sdAbs) are an attractive vehicle for developing radiotherapeutics labeled with the α-emitter 211At. Herein, we have evaluated iso-[211At]AGMB-PODS, a prosthetic agent that combines a functionality for residualizing radiohalogens with a phenyloxadiazolyl methylsulfone (PODS) moiety for site-specific sdAb conjugation. Iso-[211At]AGMB-PODS and its radioiodinated analogue were evaluated for thiol-selective conjugation to anti-HER2 5F7 sdAb bearing a C-terminus GGC tail. Both radiohalogenated PODS-5F7GGC conjugates were synthesized in good radiochemical yields and retained high binding affinity on HER2-positive BT474 breast carcinoma cells. Iso-[211At]AGMB-PODS-5F7GGC was considerably more stable in vitro than its maleimide analogue in the presence of cysteine and human serum albumin (HSA) and exhibited excellent tumor uptake and high in vivo stability. Superior tumor-to-kidney activity ratios were seen for both radiohalogenated PODS-5F7GGC conjugates compared with [177Lu]Lu-DOTA-PODS-5F7GGC. These results suggest that iso-[211At]AGMB-PODS-5F7GGC warrants further evaluation for the treatment of HER2-expressing malignancies.

Design and preclinical evaluation of nanostars for the passive pretargeting of tumor tissue.

INTRODUCTION: Pretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. METHODS: Nanostars were synthesized, consisting of 7-8 center-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2'-(7-(2-(tetrazine-poly(ethyleneglycol)11-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-PEG11-NODA) or 2,2'-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). RESULTS: Highest tumor accumulation of [18F]F-Tz-PEG11-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4%ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG12 (P < 0.05) or TCO-PEG106 (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([18F]F-Tz-NODA). CONCLUSIONS: A pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.

Preparation of Titanocene-Gold Compounds Based on Highly Active Gold(I)-N-Heterocyclic Carbene Anticancer Agents: Preliminary in†vitro Studies in Renal and Prostate Cancer Cell Lines.

Heterometallic titanocene-based compounds containing gold(I)-phosphane fragments have been extremely successful against renal cancer in vitro and in vivo. The exchange of phosphane by N-heterocyclic carbene ligands to improve or modulate their pharmacological profile afforded bimetallic complexes effective against prostate cancer, but less effective against renal cancer in vitro. Herein we report the synthesis of new bimetallic Ti-Au compounds by the incorporation of two previously reported highly active gold(I)-N-heterocyclic carbene fragments derived from 4,5-diarylimidazoles. The two new compounds [(η5 -C5 H5 )2 TiMe(µ-mba)Au(NHC)] (where NHC=1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene, NHC-Bn 2 a; or 1,3-diethyl-4,5-diphenylimidazol-2-ylidene, NHC-Et 2 b) with the dual linker (-OC(O)-p-C6 H4 -S-) containing both a carboxylate and a thiolate group were evaluated in vitro against renal and prostate cancer cell lines. The compounds were found to be more cytotoxic than previously described Ti-Au compounds containing non-optimized gold(I)-N-heterocyclic fragments. We present studies to evaluate their effects on cell death pathways, migration, inhibition of thioredoxin reductase (TrxR) and vascular endothelial growth factor (VEGF) in the PC3 prostate cancer cell line. The results show that the incorporation of a second metallic fragment such as titanocene into biologically active gold(I) compounds improves their pharmacological profile.

Customizing Morphology, Size, and Response Kinetics of Matrix Metalloproteinase-Responsive Nanostructures by Systematic Peptide Design.

Overexpression and activation of matrix metalloproteinase-9 (MMP-9) is associated with multiple diseases and can serve as a stimulus to activate nanomaterials for sensing and controlled release. In order to achieve autonomous therapeutics with improved space-time targeting capabilities, several features need to be considered beyond the introduction of an enzyme-cleavable linker into a nanostructure. We introduce guiding principles for a customizable platform using supramolecular peptide nanostructures with three modular components to achieve tunable kinetics and morphology changes upon MMP-9 exposure. This approach enables (1) fine-tuning of kinetics through introduction of ordered/disordered structures, (2) a 12-fold variation in hydrolysis rates achieved by electrostatic (mis)matching of particle and enzyme charge, and (3) selection of enzymatic reaction products that are either cell-killing nanofibers or disintegrate. These guiding principles, which can be rationalized and involve exchange of just a few amino acids, enable systematic customization of enzyme-responsive peptide nanostructures for general use in performance optimization of enzyme-responsive materials.

Bimetallic titanocene-gold phosphane complexes inhibit invasion, metastasis, and angiogenesis-associated signaling molecules in renal cancer.

Following promising recent in vitro and in vivo studies of the anticancer efficacies of heterometallic titanocene-gold chemotherapeutic candidates against renal cancer, we report here on the synthesis, characterization, stability studies and biological evaluation of a new titanocene complex containing a gold-triethylphosphane fragment [(η-C5H5)2TiMe(µ-mba)Au(PEt3)] (4) Titanofin. The compound is more stable in physiological fluid than those previously reported, and it is highly cytotoxic against a line of human clear cell renal carcinoma. We describe here preliminary mechanistic data for this compound and previously reported [(η-C5H5)2TiMe(µ-mba)Au(PPh3)] (2) Titanocref which displayed remarkable activity in an in vivo mouse model. Mechanistic studies were carried out in the human clear cell renal carcinoma Caki-1 line for the bimetallic compounds [(η-C5H5)2TiMe(µ-mba)Au(PR3)] (PR3 = PPh32 Titanocref and PEt34 Titanofin), the two monometallic gold derivatives [Au(Hmba)(PR3)] (PR3 = PPh31 cref; PEt33 fin), titanocene dichloride and Auranofin as controls. These studies indicate that bimetallic compounds Titanocref (2) and Titanofin (4) are more cytotoxic than gold monometallic derivatives (1 and 3) and significantly more cytotoxic than titanocene dichloride while being quite selective. Titanocref (2) and Titanofin (4) inhibit migration, invasion, and angiogenic assembly along with molecular markers associated with these processes such as prometastatic IL(s), MMP(s), TNF-α, and proangiogenic VEGF, FGF-basic. The bimetallic compounds also strongly inhibit the mitochondrial protein TrxR often overexpressed in cancer cells evading apoptosis and also inhibit FOXC2, PECAM-1, and HIF-1α whose overexpression is linked to resistance to genotoxic chemotherapy. In summary, bimetallic titanocene-gold phosphane complexes (Titanocref 2 and Titanofin 4) are very promising candidates for further preclinical evaluations for the treatment of renal cancer.