Improving the Stability and Kinetic Inertness of Mn(II) Complexes by Increasing the Bridge Length in Bicyclic CDTA-Like Ligands.

Kinetic inertness of Mn(II)-based MRI contrast agents can be improved by increasing the rigidity of the polydentate ligand that tightly coordinate the metal ion. Taking inspiration from the remarkable increase in kinetic inertness of [Mn(CDTA)]2- compared to [Mn(EDTA)]2- due to the cyclohexyl backbone rigidity, we devised that bicyclic ligands would further improve the kinetic inertness of the Mn(II) complexes. The length of the alkyl bridge on the cyclohexane ring was varied from methylene (BCH-DTA), ethylene (BCO-DTA) to propylene (BCN-DTA) to evaluate the influence of the different trans-diaminotetraacetate ligands on relaxometric, thermodynamic and kinetic properties of the Mn(II) complexes. 1H and 17O NMR relaxometric studies showed a slight increase in relaxivity and a faster water exchange rate in these Mn(II)-complexes with respect to [Mn(CDTA)]2-. Solution studies revealed that the conditional stability (pMn) and dissociation half-life (t1/2) at pH 7.4 follow the order [Mn(BCH-DTA)]2-<[Mn(BCO-DTA)]2-<[Mn(BCN-DTA)]2- highlighting the effect of the bridge length on the overall stability of the Mn(II) complexes. Remarkably, [Mn(BCN-DTA)]2- shows an improved pMn value and a 7-times higher kinetic inertness than [Mn(CDTA)]2-. NMR studies on the Zn(II) analogues confirm the rigidity of the bicyclic complexes with an isomerization process at >313 K for the smaller bridged complex [Zn(BCH-DTA)]2-.

Thermodynamic and Kinetic Stabilities of Al(III) Complexes with N2O3 Pentadentate Ligands.

Al(III) complexes have been recently investigated for their potential use in imaging with positron emission tomography (PET) by formation of ternary complexes with the radioisotope fluorine-18 (18F). Although the derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) are the most applied chelators for [Al18F]2+ labelling and (pre)clinical PET imaging, non-macrocyclic, semi-rigid pentadentate chelators having two N- and three O-donor atoms such as RESCA1 and AMPDA-HB have been proposed with the aim to allow room temperature labelling of temperature-sensitive biomolecules. The paucity of stability data on Al(III) complexes used for PET imaging instigated a complete thermodynamic and kinetic solution study on Al(III) complexes with aminomethylpiperidine (AMP) derivatives AMPTA and AMPDA-HB and the comparison with a RESCA1-like chelator CD3A-Bn (trans-1,2-diaminocyclohexane-N-benzyl-N,N',N'-triacetic acid). The stability constant of [Al(AMPDA-HB)] is about four orders of magnitude higher than that of [Al(AMPTA)] and [Al(CD3A-Bn)], highlighting the greater affinity of phenolates with respect to acetate O-donors. On the other hand, the kinetic inertness of the complexes, determined by following the Cu2+-mediated transmetallation reactions in the 7.5-10.5 pH range, resulted in a spontaneous and hydroxide-assisted dissociation slightly faster for [Al(AMPTA)] than for the other two complexes (t1/2 = 4.5 h for [Al(AMPTA)], 12.4 h for [Al(AMPDA-HB)], and 24.1 h for [Al(CD3A-Bn)] at pH 7.4 and 25 °C). Finally, the [AlF]2+ ternary complexes were prepared and their stability in reconstituted human serum was determined by 19F NMR experiments.

AAZTA-Like Ligands Bearing Phenolate Arms as Efficient Chelators for 68 Ga Labelling in†vitro and in†vivo.

The introduction of a phenolate pendant arm in place of an acetate on AAZTA- and DATA-like ligands resulted in hepta- and hexadentate chelators able to form Ga(III) complexes with thermodynamic stability and kinetic inertness higher than that of other Ga(III) complexes based on the parent 6-amino-6-methylperhydro-1,4-diazepine scaffold. In particular, the heptadentate AAZ3A-endoHB with a phenolate arm on an endocyclic N-atom shows a logKGaL of 27.35 and a remarkable resistance to hydroxide coordination up to basic pH (pH>9). This behaviour allows to also improve the kinetic inertness of the complex showing a dissociation half-life (t1/2 ) at pH 7.4 of 76 h. Although also the hexadentate AAZ2A-exoHB chelator forms a stable (logKGaL =24.69) and inert (t1/2 =33 h at pH 7.4) Ga(III) complex, the 68 Ga labelling showed a better radiochemical yield with AAZ3A-endoHB, especially at room temperature. Thus, a bifunctional chelator of AAZ3A-endoHB was synthesized bearing an isothiocyanate group that was conjugated to the N-terminus of a c(RGD) peptide for integrin receptor targeting. Finally, the conjugate was successfully labelled with 68 Ga isotope, and the resulting radiotracer tested for its stability in human serum and then in vivo for targeting B16-F10 tumours with miniPET imaging.

Semi-Rigid (Aminomethyl) Piperidine-Based Pentadentate Ligands for Mn(II) Complexation.

Two pentadentate ligands built on the 2-aminomethylpiperidine structure and bearing two tertiary amino and three oxygen donors (three carboxylates in the case of AMPTA and two carboxylates and one phenolate for AMPDA-HB) were developed for Mn(II) complexation. Equilibrium studies on the ligands and the Mn(II) complexes were carried out using pH potentiometry, 1H-NMR spectroscopy and UV-vis spectrophotometry. The Mn complexes that were formed by the two ligands were more stable than the Mn complexes of other pentadentate ligands but with a lower pMn than Mn(EDTA) and Mn(CDTA) (pMn for Mn(AMPTA) = 7.89 and for Mn(AMPDA-HB) = 7.07). 1H and 17O-NMR relaxometric studies showed that the two Mn-complexes were q = 1 with a relaxivity value of 3.3 mM-1 s-1 for Mn(AMPTA) and 3.4 mM-1 s-1 for Mn(AMPDA-HB) at 20 MHz and 298 K. Finally, the geometries of the two complexes were optimized at the DFT level, finding an octahedral coordination environment around the Mn2+ ion, and MD simulations were performed to monitor the distance between the Mn2+ ion and the oxygen of the coordinated water molecule to estimate its residence time, which was in good agreement with that determined using the 17O NMR data.

Polymerizable Gd(iii) building blocks for the synthesis of high relaxivity macromolecular MRI contrast agents.

A new synthetic strategy for the preparation of macromolecular MRI contrast agents (CAs) is reported. Four gadolinium(iii) complexes bearing either one or two polymerizable methacrylamide groups were synthesized, serving as monomers or crosslinkers for the preparation of water-soluble, polymeric CAs using Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. Using this approach, macromolecular CAs were synthesized with different architectures, including linear, hyperbranched polymers and gels. The relaxivities of the polymeric CAs were determined by NMR relaxometry, revealing an up to 5-fold increase in relaxivity (60 MHz, 310 K) for the linear polymers compared with the clinically used CA, Gd-DOTA. Moreover, hyperbranched polymers obtained from Gd(iii) crosslinkers, displayed even higher relaxivities up to 22.8 mM-1 s-1, approximately 8 times higher than that of Gd-DOTA (60 MHz, 310 K). A detailed NMRD study revealed that the enhanced relaxivities of the hyperbranched polymers were obtained by limiting the local motion of the crosslinked Gd(iii) chelate. The versatility of RAFT polymerization of Gd(iii) monomers and crosslinkers opens the doors to more advanced polymeric CAs capable of multimodal, bioresponsive or targeting properties.

Rigid and Compact Binuclear Bis-hydrated Gd-complexes as High Relaxivity MRI Agents.

The first binuclear Gd-complex of the 12-membered pyridine-based polyaminocarboxylate macrocyclic ligand PCTA was synthesized by C-C connection of the pyridine units through two different synthetic procedures. A dimeric AAZTA-ligand was also synthesized with the aim to compare the relaxometric results or the two ditopic Gd-complexes. Thus, the 1 H relaxometric study on [Gd2 PCTA2 (H2 O)4 ] and on [Gd2 AAZTA2 (H2 O)4 ]2- highlighted the remarkable rigidity and compactness of the two binuclear complexes, which results in molar relaxivities (per Gd), at 1.5 T and 298 K of ca. 12-12.6 mM-1 s-1 with an increase of ca. 80 % at 1.5 T and 298 K (+70 % at 310 K) with respect to the corresponding mononuclear complexes.

Towards Enhanced MRI Performance of Tumor-Specific Dimeric Phenylboronic Contrast Agents.

It is known that phenylboronic acid (PBA) can target tumor tissues by binding to sialic acid, a substrate overexpressed by cancer cells. This capability has previously been explored in the design of targeting diagnostic probes such as Gd- and 68Ga-DOTA-EN-PBA, two contrast agents for magnetic resonance imaging (MRI) and positron emission tomography (PET), respectively, whose potential has already been demonstrated through in vivo experiments. In addition to its high resolution, the intrinsic low sensitivity of MRI stimulates the search for more effective contrast agents, which, in the case of small-molecular probes, basically narrows down to either increased tumbling time of the entire molecule or elevated local concentration of the paramagnetic ions, both strategies resulting in enhanced relaxivity, and consequently, a higher MRI contrast. The latter strategy can be achieved by the design of multimeric GdIII complexes. Based on the monomeric PBA-containing probes described recently, herein, we report the synthesis and characterization of the dimeric analogues (GdIII-DOTA-EN)2-PBA and (GdIII-DOTA-EN)2F2PBA. The presence of two Gd ions in one molecule clearly contributes to the improved biological performance, as demonstrated by the relaxometric study and cell-binding investigations.

Solid-phase synthesis and evaluation of tumour-targeting phenylboronate-based MRI contrast agents.

Paramagnetic macrocycles functionalized with phenylboronic moieties have proven to be interesting for MRI applications based on their ability to recognize cancer cells and generate local contrast. However, full use of the potential of this class of compounds is hampered by laborious and inefficient synthetic and, especially, purification procedures. The amphiphilic character of water-soluble phenylboronates renders them difficult compounds to be prepared through conventional solution synthesis due to the tendency to aggregate and form adducts with other nucleophiles. The new strategy described herein exploits the advantage of solid-phase synthesis with the application of DEAM-PS resin for anchorage and the subsequent simplified derivatization of boronates. GdDOTA-EN-PBA and its fluorinated analogue GdDOTA-EN-F2PBA were synthesized in a much easier, faster and economically convenient way to achieve good yields and purity. Furthermore, the effect of electron-withdrawing fluorine atoms on the aromatic ring of the latter compound was investigated by comparing the physico-chemical properties of both compounds as well as their binding affinity towards melanoma cancer cells.

Selective functionalization of 6-amino-6-methyl-1,4-perhydrodiazepine for the synthesis of a library of polydentate chelators.

Polydentate chelators are an important part of an imaging probe, which consists of an agent that usually produces signals for imaging purposes connected to a targeting moiety. The goal of this study was to set up a generic protocol to prepare a library of polydentate ligands having a 6-amino-6-methyl-1,4-perhydrodiazepine (AMPED) core and able to chelate metal ions of interest for various diagnostic imaging techniques, including Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT). These ions, among which we can include Mn(ii), Cu(ii), Al(iii) or Ga(iii), require penta- or hexa-dentate chelators for this purpose, and the AMPED scaffold has considerable potential to support various pendant arms for coordination of such ions. AMPED already has three amino nitrogen donors; thus, only two or three additional arms should be introduced to obtain penta- or hexa-dentate systems. This condition implies that symmetrical or asymmetrical structures have to be developed, depending on the functionalization of cyclic and exocyclic amines. Starting from easily available materials, we have designed a convenient protocol for the preparation of multiple AMPED-based ligands endowed with different characteristics, several of which were synthesized as examples.

Room Temperature Al18 F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging.

Positron emission tomography (PET) is a non-invasive molecular imaging technology that is constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i. e. Fab, svFab, nanobodies) is increasing and has led us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which were investigated as such for {Al18 F}2+ -core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed under various conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240 min in human serum (HS), EDTA 5 mM, PBS and 0.9 % NaCl solutions. The in vivo stability of [Al18 F(2-AMPDA-HB)]- was assessed in healthy nude mice (n=6). Radiochemical yields between 55 % and 81 % were obtained at pH 5 and room temperature. High stability in HS was measured for [Al18 F(2-AMPDA-HB)]- , with 90 % of F-18 complexed after 120 min. High stability in vivo, rapid hepatobiliary and renal excretion, with low accumulation of free F-18 in bones were measured. Thus, this new Al18 F-chelator may have a great impact on immuno-PET radiopharmacy, by facilitating the development of new fluorine-18-labeled heat-sensitive biomolecules.

Preclinical Evaluation of the Hsp70 Peptide Tracer TPP-PEG24-DFO[89Zr] for Tumor-Specific PET/CT Imaging.

High precision in vivo PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG24-DFO[89Zr] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG24 moiety supported tracer stability and improved biodistribution characteristics in vivo The K d of the tracer ranged in the low nanomolar range (18.9 ± 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG24-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1+, a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG24-DFO[89Zr] in vivo enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1+ (6.2 ± 1.1%ID/g), 4T1 (4.3 ± 0.7%ID/g), and CT26 (2.6 ± 0.6%ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 ± 0.03%ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG24-DFO[89Zr] to detect mHsp70 in vivo suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70.Significance: A novel peptide-based PET tracer against the oligomerization domain of Hsp70 has potential for universal tumor-specific imaging in vivo across many tumor type. Cancer Res; 78(21); 6268-81. ©2018 AACR.

Mesoscopic FRET Antenna Materials by Self-Assembling Iridium(III) Complexes and BODIPY Dyes.

This study presents a new design of light-harvesting antenna materials using two dyes organised into mesoporous silica: an iridium(III) complex and a BODIPY-derived surfactant that undergo Förster resonance energy transfer (FRET), acting, respectively, as donor and acceptor. The chemical structure of each dye determines the position taken within the micellar templates used for the synthesis of the silica host, which maintains mesopore order as shown by TEM imaging. Steady-state and time-resolved UV-visible spectroscopy revealed that incorporation of the iridium complex into the silica shields it from oxygen-induced quenching and allows a degree of control over the donor-acceptor distance, yielding FRET efficiencies from 24 to 76 % and tuneable emission ranges. Such silica-based antennae show promising properties for the realisation of polychromatic sensitisers for photovoltaics and photocatalysis.

Surface PEG Grafting Density Determines Magnetic Relaxation Properties of Gd-Loaded Porous Nanoparticles for MR Imaging Applications.

Surface PEGylation of nanoparticles designed for biomedical applications is a common and straightforward way to stabilize the materials for in vivo administration and to increase their circulation time. This strategy becomes less trivial when MRI active porous nanomaterials are concerned as their function relies on water/proton-exchange between the pores and bulk water. Here we present a comprehensive study on the effects of PEGylation on the relaxometric properties of nanozeolite LTL (dimensions of 20 × 40 nm) ion-exchanged with paramagnetic GdIII ions. We evidence that as long as the surface grafting density of the PEG chains does not exceed the "mushroom" regime (conjugation of up to 6.2 wt % of PEG), Gd-LTL retains a remarkable longitudinal relaxivity (38 s-1 mM-1 at 7 T and 25 °C) as well as the pH-dependence of the longitudinal and transverse relaxation times. At higher PEG content, the more compact PEG layer (brush regime) limits proton/water diffusion and exchange between the interior of LTL and the bulk, with detrimental consequences on relaxivity. Furthermore, PEGylation of Gd-LTL dramatically decreases the leakage of toxic GdIII ions in biological media and in the presence of competing anions, which together with minimal cytotoxicity renders these materials promising probes for MRI applications.

Fate of Organic Functionalities Conjugated to Theranostic Nanoparticles upon Their Activation.

Neutron activation is widely applied for the preparation of radioactive isotopes to be used in imaging and/or therapy. The type of diagnostic/therapeutic agents varies from small chelates coordinating radioactive metal ions to complex nanoparticulate systems. Design of these agents often relies on conjugation of certain organic functionalities that determine their pharmacokinetics, biodistribution, targeting, and cell-penetrating abilities, or simply on tagging them with an optical label. The conjugation chemistry at the surface of nanoparticles and their final purification often require laborious procedures that become even more troublesome when radioactive materials are involved. This study represents a thorough investigation on the effects of neutron activation on the organic moieties of functionalized nanoparticles, with special focus on (166)Ho2O3 particles conjugated with PEG-fluorescein and PEG-polyarginine motives. Spectroscopic and thermogravimetric analyses demonstrate only a limited degradation of PEG-fluorescein upon irradiation of the particles up to 10 h using a thermal neutron flux of 5 × 10(16) m(-2) s(-1). Cell experiments show that the polyarginine-based mechanisms of membrane penetration remain unaltered after exposure of the functionalized particles to the mixed field of neutrons and gammas present during activation. This confirms that radiation damage on the PEG-polyarginines is minimal. Intrinsic radiations from (166)Ho do not seem to affect the integrity of conjugated organic material. These findings open up a new perspective to simplify the procedures for the preparation of functionalized metal-based nanosystems that need to be activated by neutron irradiation in order to be applied for diagnostic and/or therapeutic purposes.

Dendrimeric ß-cyclodextrin/Gd(III) chelate supramolecular host-guest adducts as high-relaxivity MRI probes.

We have synthesized a new macromolecular architecture, (PAMAM)-CD8 , which consists of eight ß-cyclodextrin units (ß-CD) attached to a generation 1 poly(amidoamine) (PAMAM) dendrimer through a disulfide bond, which can be cleaved under reducing conditions. This system shows a pronounced hosting capability towards Gd(III) chelates functionalized with hydrophobic groups, thus leading to well-defined supramolecular adducts. (1)H NMR relaxometric investigations were carried out to follow the formation of adducts with three Gd(III) chelates based on the ligand architectures of 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid (AAZTA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) suitably functionalized with benzyl or adamantyl (Ad) pendant groups. In particular, the ditopic complex composed of two AAZTA chelating units connected to a central aromatic ring that bears an adamantyl group showed a strong affinity (ca. 10(6) M(-1)) for the CD units of the dendrimer, which is two orders of magnitude higher than toward human serum albumin (HSA). Remarkable relaxivity enhancements (i.e., up to 71% at 1 T and 25 °C) were observed upon the formation of the macromolecular host-guest adducts due to a decrease in the molecular tumbling rate and fast water-exchange. Reduction experiments and competition studies between the paramagnetic dendrimer and HSA were carried out by relaxometric techniques. The results show that the metal complexes are not displaced by the protein, thus suggesting that this novel macromolecular probe is potentially suitable for applications in vivo.

Dendrimersomes: a new vesicular nano-platform for MR-molecular imaging applications.

A new class of nanovesicles formed by the self-assembly of amphiphilic Janus dendrimers, dendrimersomes, loaded with hydrophilic or amphiphilic magnetic resonance imaging chelates shows promising properties as a novel, efficient and versatile nanoplatform for biomedical imaging.

Synthesis of 6-substituted 6-nitroperhydro-1,4-diazepines via novel tandem retro-Henry and Mannich/Michael reactions.

N,N'-Dibenzyl-6-hydroxymethyl-6-nitroperhydro-1,4-diazepine was converted into a nitronate via retro-Henry reaction, followed by either Michael reaction with several acrylic derivatives or Mannich reaction with different amines, thus leading to 6-substituted 6-nitroperhydro-1,4-diazepines. The tandem retro-Henry/Mannich reaction was also carried out using benzylamine as base, solvent, and reagent at the same time. Selective hydrogenation of the nitro group and complete hydrogenolysis were also successfully achieved.

Cleavable ß-cyclodextrin nanocapsules incorporating Gd(III)-chelates as bioresponsive MRI probes.

Perthiolated ß-cyclodextrin-based nanocapsules incorporating diaquo Gd(III)-complexes represent a promising new type of bioresponsive MRI contrast agent, showing a pronounced relaxivity change upon degradation in a reducing environment.

Coordination chemistry of amide-functionalised tetraazamacrocycles: structural, relaxometric and cytotoxicity studies.

Three different tetraazamacrocyclic ligands containing four amide substituents that feature groups (namely allyl, styryl and propargyl groups) suitable for polymerisation have been synthesised. Gadolinium(III) complexes of these three ligands have been prepared as potential monomers for the synthesis of polymeric MRI contrast agents. To assess the potential of these monomers as MRI contrast agents, their relaxation enhancement properties and cytotoxicity have been determined. A europium(III) complex of one of these ligands (with propargyl substituents) is also presented together with its PARACEST properties. In addition, to gain further insight into the coordination chemistry of the tetra-propargyl substituted ligand, the corresponding zinc(II) and cadmium(II) complexes have been prepared. The X-ray crystal structures of the tetra-propargyl ligand and its corresponding gadolinium(III), zinc(II) and cadmium(II) complexes are also presented.