A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid ( 6), has been synthesized and its complexation behavior toward Cu2+ ions investigated. The ligand 6 has been characterized by spectroscopic methods, and a molecular structure of a corresponding Cu(II) complex has been elucidated by single-crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), beta Ala-beta Ala-[Cha13, Nle14]BN(7-14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with 64Cu2+, and the resulting complexes, 64Cu subset6 and 64Cu subset8 , were found to be stable in the presence of a large excess of a competing ligand (cyclam) or copper-seeking superoxide dismutase (SOD), as well as in rat plasma. Biodistribution studies of 64Cu subset8 in Wistar rats showed a high activity uptake into the pancreas (5.76 +/- 0.25 SUV, 5 min p.i.; 3.93 +/- 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is overexpressed in a large number of breast and prostate carcinomas. The novel 64Cu subset6 complex had a dominating influence on the nonspecific activity biodistribution of its BN conjugate, since the distribution data of 64Cu subset6 are similar to those of 64Cu subset8 . The 64Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of 64CuCl 2, suggesting that 64Cu subset6 does not undergo significant demetalation, but rather exhibits high in vivo stability.

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2''-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.

The quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and (3-bromophenyl)quinazoline-4,6-diamine (3) were labelled with (99m)Tc using the "4 + 1" mixed-ligand system [Tc(NS3)(CN-R)] and the tricarbonyl moiety fac-[Tc(CO)3]+. In the "4 + 1" approach the technetium(iii) is stabilized by a monodentate isocyanide bearing a quinazoline fragment (L1,L2 ) and by the tetradentate tripodal ligand tris(2-mercaptoethyl)-amine (NS3). In the "4 + 1" approach, 99mTc-labelling was performed in a two-step procedure, the complexes [Tc(NS3)(L1)] (7a) and [Tc(NS3)(L2)] (8a) being obtained in about 50-70% yield. In the tricarbonyl approach, the fac-[Tc(CO)3]+ unit is anchored by two different monoanionic chelators bearing the quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and (3-bromophenyl)quinazoline-4,6-diamine (3). Both chelators have a N2O donor atom set, but one contains a pyrazolyl ring (L5H) and the other contains a pyridine unit (L6H). In both cases the conjugation of the quinazoline to the chelator was done through the secondary amine of the potentially tridentate and monoanionic chelators, the corresponding 99mTc-complexes (10a, 11a) being obtained in quantitative yield. The identities of the 99mTc-labelled quinazolines (7a, 8a, 10a, 11a) were confirmed by comparison with the HPLC profiles of the analogous Re compounds (7, 8, 10, 11). All these Re complexes were characterized by NMR and IR spectroscopy, elemental analysis and in some cases by MS and X-ray diffraction analysis. In vitro studies indicate that the quinazoline fragments, after conjugation to the cyano group (L1, L2) or to the pyrazolyl containing chelator (L5H), as well as the corresponding Re complexes (7, 8, 10) inhibit significantly the EGFR autophosphorylation and also inhibit A431 cell growth. These two effects were also found for the pyridine-containing chelator (L6H) and corresponding Re complex (11), although to a lesser extent.

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EXAFS analyses were successfully used to determine the structure of Re and Tc carbonyl thioether complexes in solid and liquid samples. In connection with chromatographic and mass spectrometric methods the behaviour of Tc carbonyl dithioether complexes in aqueous solution was studied. It was found that complexes containing a bidentate thioether ligand are able to react with water by exchange of the chloride ion, which results in the formation of a cationic complex. As expected, the exchange proceeds faster at the n.c.a. level because of concentration differences.