Summary

Bivalent ligands have been defined as “molecules having two pharmacophores covalently connected by a linker”.This category of compounds has been used to design new ligands for various G-protein coupled receptors (GPCR) including the opioid, cannabinoid, chemokine, or melanocortin receptor systems among others. One of the interests of heterobivalent ligand (HBL) resides is the fact that the combination of two ligands for two different receptors may lead to improved pharmacological properties. The improvement of the pharmacological properties of bivalent ligands can arise from an increased affinity of the ligand for tissues or cells coexpressing the receptors that the HBLs are aimed at. This could be due to the fact that the binding of a first ligand to its corresponding receptor might bring the second ligand close to its targeted receptor, thus increasing its local concentration and its possibility of binding to its corresponding receptor simultaneously or even after dissociation from the first receptor. In addition, since some biomarkers may have different expression profiles depending on the individual or within an individual (heterogeneity) or the stage of a disease (change in receptor expression level), HBLs allow to target a larger number of clinical cases with one single molecule.
HBLs find a particularly adequate application in nuclear imaging and therapy of tumors. This is due to the fact that tumors overexpress different receptors, thus making multireceptor targeting a way of gaining specificity in tumor targeting. HBL-based radiotracers are expected to display increased uptake in disease tissue and improved tumor/background ratios at later time points. A number of receptors such as regulatory peptide receptors and chemokine and integrin receptors, among others, have been identified at the surfaces of tumor cells and could be used for multireceptor targeting. An example for such multireceptor expression by tumor cells can be found in prostate cancer (PCa). Prostate-specific membrane antigen (PSMA) is overexpressed in the vast majority of malignant tumors with the PSMA expression level increasing with higher tumor stage and grade. The gastrin-releasing peptide receptor (GRPR) is also overexpressed by PCa cells, and much effort has been dedicated to the development of GRPR-seeking imaging agents. However, recent studies have shown that both receptors are differently overexpressed and that GRPR is strongly upregulated in the majority of PCa cases particularly on lower grade, whereas higher grade expresses abundantly the PSMA.
In the context of tumor targeting, where antibodies or peptides can be used for imaging and therapy, the generation of peptide-based HBLs is by far easier than the generation of antibody fragment-based HBL conjugates. Furthermore, peptide conjugates benefit from fast diffusion into the tumor as well as fast clearance from the body, and their use as dimers or multimers tends to increase their in vivo half-life. A downside of this class of compounds is the importance of the distance separating the two ligands. As a matter of fact, this distance is required to be optimized to maximize the benefits of ligand heterobivalency, implying the synthesis of libraries of compounds with varied spacer length and nature. With the increased interest of medicinal chemists and radiopharmacists in HBLs, it is of prime importance to provide easy and straightforward procedures to synthesize them. The efficient synthesis of unlabeled HBLs might be performed by linear, sequential, or convergent synthesis. However, the use of HBLs as imaging probes requires the addition of an imaging probe and adds another layer of complexity to the chemical synthesis process. The use of a third polyfunctional molecular entity like a probe (chelator for nuclear imaging or therapy, or an organic-based fluorophore for cellular microscopy or fluorescence imaging) can make classical linear peptide synthesis cumbersome due to the use of multiple orthogonal protective groups, tedious deprotection and coupling steps that require intermediate purifications, the need of manipulating fully protected peptides, which can lead to solubility problems, or the inherent instability of some reporter probes (such as organic fluorophores) in classical SPPS conditions.
Developing modular strategies to synthesize radiolabeled HBLs is particularly important, since these molecules often need to be optimized to maximize the interactions of the ligands with receptors. The use of a multifunctional molecular scaffold or template is a highly efficient strategy to put together elaborated building blocks. Moreover, when those building blocks are delicate or difficult to synthesize, it is highly desirable that they are incorporated onto the scaffold via orthogonal click reactions.
Some amino acids, like lysine, are natural trifunctional platforms that can be advantageously functionalized with orthogonally reactive bioconjugation moieties. Similarly, trifunctionalized benzene rings can also be used as a starting material to provide a useful scaffold. However, both options require multiple modifications of commercial compounds to decorate the chosen scaffold with orthogonally reactive moieties. Alternatively, trichloro-1,3,5-triazines have been noticed as a way to obtain clickable platforms from a readily available commercial substrate. However, the high reactivity of triazines makes them vulnerable to hydrolysis and a large range of nucleophiles (including amines) and therefore requires careful optimization and chemical expertise in their handling.
The authors have recently demonstrated that the use of disubstituted tetrazines bearing various chelating agents and fluorescent organic dyes was a promising way for the effective synthesis of antibody-based multimodal imaging probes. To demonstrate both utility and scope of this approach, they report herein the usefulness of commercial 3,6-dichloro-1,2,4,5-tetrazine (dichloro-s-tetrazine) as a trifunctional platform for the modular and straightforward synthesis of heterobivalent ligands functionalized with an imaging probe. To reach this objective, a series of tetrazines derivatized with different tumor targeting vectors of interest in nuclear medicine were synthesized and reacted with a fluorescent organic dye or a chelator to generate radiolabeled heterobivalent imaging and therapy agents (or precursors thereof). In an effort to make this methodology even more practical, the previously published stepwise procedure was further improved to be carried out in one pot.

Results from the nanoScan PET/CT

For the imaging studies, the tumor-bearing mice were injected intravenously (retro-orbital sinus) with 100 μL of saline solution containing 2 to 10 MBq of 68Ga-labeled ligands.
The animals were imaged under isoflurane in a NanoScan PET/CT (Mediso Medical Imaging Systems Ltd., Budapest, Hungary) during 20 min using multiple bed (3 mice simultaneously) or single bed (1 mouse). A CT scan was performed directly after the PET imaging for anatomical co-registration and attenuation correction.
Images were reconstructed using Nucline 2.03 and post-reconstruction analysis, including decay correction, was performed using InterviewFusion (Softwares from Mediso Medical Imaging Systems, Hungary). Images are presented as maximum intensity projections (MIP) or axial/transaxial slices. Tumor uptake was quantified by the following procedure: CT-aided regions of interest were drawn, and tissue concentration was expressed as percent injected dose per gram (%ID/g) ± standard deviation.
Images recorded 50–70 min post injection (p.i.) showed that both tracers were able to exclusively accumulate in the xenograft overexpressing their complementary receptor, which demonstrated uptake specificity (3.46% ID/g for [⁶⁸Ga]Ga-AMBA in PC3 tumor and 3.22% ID/g for [⁶⁸Ga]Ga-PSMA11 in 22Rv1 tumor, Figure 5.). Both radioconjugates show uptake in the kidneys, which is typical of peptide-based radiotracers that, with few exceptions, benefit from renal excretion. [⁶⁸Ga]Ga-AMBA shows uptake in the gastrointestinal tract which is due to the presence of GRPR in the pancreas, stomach and colon.
At only 30–50 min p.i., [⁶⁸Ga]Ga-18 (1.88 nmol, 4.1 MBq) already showed accumulation in targeted tumor xenografts with 1.76% ID/g for PC3; 1.66% ID/g for 22Rv1 (measured by quantitative PET imaging, see Supporting Information) and GRPR- or PSMA-positive tissues such as pancreas and kidneys (Figure 5., right). Quantitative PET measurements 20 min later did not show additional accumulation in both tumors (1.75% ID/g for PC3; 1.69% ID/g for 22Rv1). A significant decrease in the signal from the blood pool (1.02% ID/g for heart), showing circulation of the radiotracer and washout from the background, let us anticipate an improved contrast at later time points in comparison with the images at 30–50 min (Figure 5.). Most signal was observed in the kidneys and in the bladder, suggesting that [⁶⁸Ga]Ga-18 is eliminated mainly through renal/urinary excretion. A weak signal in the gallbladder and high background in the abdominal region suggested a small amount of hepatobiliary excretion. The uptake of [⁶⁸Ga]Ga-18 in each xenograft (1.75 and 1.69% ID/g in PC3 and 22Rv1 xenografts) at 50–70 min was lower than the reference compounds (3.46 and 3.22% ID/g respectively). The authors hypothesize that this difference might be due to the structure of 18, which has not been optimized like AMBA or PSMA11.

• In summary, a straightforward method based on the use of dichloro-s-tetrazine to quickly access labeled heterobivalent ligands has been successfully developed. 
• The authors have demonstrated that the method is suited to easily assemble thiol- and amino-functionalized peptide-based ligands and different labels useful for medical imaging.
• The strategy, based on the use of multiple chemoselective reactions, successfully provided complex molecular structures in a straightforward, time-saving, and modular manner. The efficiency of our procedure has been further increased by developing a one-pot approach giving 3 times higher yields in a shorter amount of time than the stepwise approach.
• In vitro and in vivo evaluation of the pharmacological properties of one of the conjugates demonstrated that the platform-based assembly method does not hamper the interaction of the ligands with their receptors.

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