Nuclear targeting with cell-specific multifunctional tricarbonyl M(I) (M is Re, 99mTc) complexes: synthesis, characterization, and cell studies

Abstract

Auger-emitting radionuclides such as (99m)Tc have been the focus of recent studies aiming at finding more selective therapeutic approaches. To explore the potential usefulness of (99m)Tc as an Auger emitter, we have synthesized and biologically evaluated novel multifunctional structures comprising (1) a pyrazolyl-diamine framework bearing a set of donor atoms to stabilize the [M(CO)(3)](+) (M is Re, (99m)Tc) core; (2) a DNA intercalating moiety of the acridine orange type to ensure close proximity of the radionuclide to DNA and to follow the internalization and subcellular trafficking of the compounds by confocal fluorescence microscopy; and (3) a bombesin (BBN) analogue of the type X-BBN[7-14] (where X is SGS, GGG) to provide specificity towards cells expressing the gastrin releasing peptide receptor (GRPr). Of the evaluated (99m)Tc complexes, Tc ( 3 ) containing the GGG-BBN[7-14] peptide showed the highest cellular internalization in GRPr-positive PC3 human prostate tumor cells, presenting a remarkably high nuclear uptake in the same cell line. Live-cell confocal imaging microscopy studies with the congener Re complex, Re ( 3 ), showed a considerable accumulation of fluorescence in the nucleus, with kinetics of uptake similar to that exhibited by Tc ( 3 ). Together, these data show that the acridine orange intercalator and the metal fragment are colocalized in the nucleus, which indicates that they remain connected despite the lysosomal degradation of Tc ( 3 )/Re ( 3 ). These compounds are the first examples of (99m)Tc bioconjugates that combine specific cell targeting with nuclear internalization, a crucial issue to explore use of (99m)Tc in Auger therapy.

Evaluation of novel 99mTc(I)-labeled homobivalent α-melanocyte-stimulating hormone analogs for melanocortin-1 receptor targeting

“…The Boc-protected chelator pz(Boc)(CO 2 H) 2 -B was synthesized by alkaline hydrolysis of the precursor pz(Boc) (CO 2 H)(CO 2 Et)-B, which was prepared as previously described [41]. The compounds were characterized by the usual analytical chemistry techniques, including RP-HPLC, multinuclear NMR spectroscopy, and ESI-MS, as described in ''Materials and methods.''…”

“…Fmoc-Lys(methyltrityl), the acid-labile Sieber resin, and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) were purchased from Novabiochem (Merck, Lisbon, Portugal). 4-((2-(tert-Butoxycarbonylamino)ethyl)(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl) amino)butanoic acid [pz(Boc)(CO 2 H)], tert-butyl 2-((3-aminopropyl)(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino) ethylcarbamate [pz(Boc)(NH 2 )], N-tert-butyloxycarbonyl-1,2-ethylenediamine (Boc-NHCH 2 CH 2 NH 2 ), and 2-(1-(2-((2-(tert-butoxycarbonylamino)ethyl)(4-ethoxy-4-oxobutyl) amino)ethyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid [pz(Boc)(CO 2 H)(CO 2 Et)-B] were prepared as described elsewhere [38][39][40][41]. 1 H and 13 C NMR spectra were recorded with a Varian Unity 300 MHz spectrometer at room temperature.…”

“…A solution of pz(Boc)(CO 2 H)(CO 2 Et)-B [41] (0.20 g, 0.44 mmol) and excess NaOH (0.75 g, 2.10 mmol) in H 2 O/ tetrahydrofuran was refluxed overnight. After neutralization with HCl 1 M at 0°C and evaporation of solvent, the obtained crude product was purified by solid phase extraction using Sep-Pack C 18 Synthesis of fac-[Re(CO) 3 (k 3 -pz(CO 2 H))] ?…”

A Bombesin-Shepherdin Radioconjugate Designed for Combined Extra- and Intracellular Targeting

“…With the same goal, the groups of Alberto and Santos have combined a 99m Tc-labeled bombesin (BBS) derivative with the DNA intercalator acridine orange, which simultaneously serves as a fluorescent probe for optical imaging. The in vitro results reported on the ability of the conjugates to target the cell nucleus are not consistent and data on the externalization of radioactivity from cells is not reported [11,12]. The Garrison group investigated the combination of a radiolabeled BBS derivative with 2-nitroimidazoles, a hypoxia-specific prodrug.…”

99mTc Pyrene Derivative Complex Causes Double-Strand Breaks in dsDNA Mainly through Cluster-Mediated Indirect Effect in Aqueous Solution

“…These electrons are highly effective at generating DSBs in nuclear DNA, and various conjugates are being investigated as potential candidates for cancer treatment [6], [7], [8], [9], [10], [11]. Several studies have attempted to characterize 99m Tc and its interactions with double-strand DNA (dsDNA).…”