**1. Introduction**

Receptor-specific targeting ligands were recently harnessed to transport and selectively deliver cytotoxic payloads to cancer tissues in vivo. Payloads may vary from chemotherapeutics to radionuclides and are usually strongly tethered to a chemically modified endogenous ligand or a synthetic agent that expresses a high affinity for the selected receptors [1,2]. Focusing on radioactive cargos, the use of metal radionuclides allows for the achievement of both diagnostic and therapeutic purposes since the same targeting vector can be labelled with metals exhibiting different emissions but similar or equal chemical features. A stable bond between a radiometal and ligand is usually provided by using

**Citation:** Verona, M.; Rubagotti, S.; Croci, S.; Sarpaki, S.; Borgna, F.; Tosato, M.; Vettorato, E.; Marzaro, G.; Mastrotto, F.; Asti, M. Preliminary Study of a 1,5-Benzodiazepine-Derivative

Labelled with Indium-111 for CCK-2 Receptor Targeting. *Molecules* **2021**, *26*, 918. https://doi.org/10.3390/ molecules26040918

Academic Editors: Alessandra Boschi and Petra Martini Received: 30 December 2020 Accepted: 3 February 2021 Published: 9 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

proper bifunctional chelators, i.e., chemical compounds covalently bind to the backbone of the targeting vector and are able to form thermodynamically and kinetical stable complexes with the metal [3,4].

Among target receptors, cholecystokinin-2 receptor (CCK-2R) is a G-protein-coupled receptor that is normally expressed in the central nervous system and gastric mucosa, where it conveys a regulatory function. Endogenous ligands for CCK-2R are low molecularweight peptides mainly synthesized in the central nervous system and the gastrointestinal tract such as gastrin and cholecystokinin. CCK-2R is also overexpressed in various human cancers (e.g., lung, medullary thyroid, pancreatic, colon, and gastrointestinal stromal tumors), where it stimulates cell growth, migration, and tumor metastasis, but it displays limited expression in other normal tissues [5–8]. For this reason, CCK-2R is a suitable target for functional imaging and therapy with radiopharmaceuticals, and its potential has been largely explored in the last few years by using various radiolabeled derivatives of its endogenous activators [9]. However, the use of peptide-based ligands still raises some concerns because derivatives exhibiting the highest tumor uptake are also characterized by high retention in the kidneys. Conversely, radiolabeled peptides that display low accumulation in kidneys show little retention in tumor tissues as well. Secondly, being the peptide chains prone to degradation by endogenous peptidases and physiological oxidation on methionine residue, the affinity for receptors can be thwarted during circulation, thus resulting in a suboptimal distribution with a consequent accumulation of high radioactive doses in healthy tissues. Finally, the use of agonist such as CCK- or gastrin-derivatives may activate the receptor signal stimulating the growth, proliferation, and survival of cancer cells [10,11]. As a consequence, the development of a peptide-based CCK-2R-targeting molecule for radiotherapeutic applications has been hampered so far, although many improvements have been obtained over the years through several interesting approaches [12–14].

An alternative path to outperform these issues could be achieved by labelling antagonist ligands based on small organic molecules rather than amino acid sequences. As opposed to agonists, antagonists do not activate the signaling cascade when bound to the receptor, and a non-peptide-based structure should provide a higher resistance to enzymatic degradation. Nastorazepide (Z-360) is a selective 1,5-benzodiazepine-derivative CCK-2 receptor antagonist with potential antineoplastic activity. Z-360 binds to CCK-2R, leading to the avoidance of its activation with sub-nanomolar affinity and high selectivity, in contrast to CCK-1R (K<sup>d</sup> = 0.47 nmol/L; selectivity relative to CCK-1R = 672) [15].

The usefulness of Z-360 as directing moiety for the preparation of radiopharmaceutical has already been demonstrated in the literature. For example, a series of nastorazepidebased derivatives have been synthesized and tethered to a N3S- or N4-system bifunctional chelator through different spacers [16,17]. The introduction of these chelators provided a suitable moiety for technetium-99m (t1/2 = 6 h; E<sup>γ</sup> = 140 keV) complexation, while appropriate spacers were introduced to optimize affinity and water solubility for CCK-2R. As a result, it has been demonstrated that Z360-based radiopharmaceuticals are valuable tools that are able to yield high-resolution images of CCK-2R-expressing tumors, especially at longer acquisition times.

Based on these groundbreaking results, in the present study, a 1,5-benzodiazepinebased ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator was synthesized in order to yield a precursor with theragnostic potential. DOTA is an almost universal chelator that is able to complex (with high stability) several metal radionuclides currently applied in clinical practice such as gallium-68 (t1/2 = 68 m; Eβ<sup>+</sup> = 0.89 MeV), indium-111 (t1/2 = 2.8 d; E<sup>γ</sup> = 173 and 247 keV), lutetium-177 (t1/2 = 6.7 d; Eβ(max) = 497, 384, and 176 keV; E<sup>γ</sup> = 113 and 208 keV), just to mention a few [18]. Herein, the labelling of the DOTA-functionalized nastorazepide derivative was carried out with indium-111. Indium-111 was preferred among the other diagnostic radionuclides for its established use in pre-clinical and clinical studies, as well as its half-life that is suitable for long-term evaluations. In fact, in our explorative experiment, indium-111 was particularly

adapted to shed light on the uptake and pharmacokinetic of the radiotracer for several hours, giving better insights into the structure–metabolism relationship.
