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Radiolabeled gold nanoparticles (AuNPs) have been widely used for cancer diagnosis and therapy over recent decades. In this study, we focused on the development and in vitro evaluation of four new Au nanoconjugates radiolabeled with technetium-99m ( 99m Tc) via thiol-bearing ligands attached to the NP surface. More specifically, AuNPs of two different sizes (2 nm and 20 nm, referred to as Au (2) and Au (20) , respectively) were functionalized with two bifunctional thiol ligands (referred to as L 1 H and L 2 H). The shape, size, and morphology of both bare and ligand-bearing AuNPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. In vitro cytotoxicity was assessed in 4T1 murine mammary cancer cells. The AuNPs were successfully radiolabeled with 99m Tc-carbonyls at high radiochemical purity (>95%) and showed excellent in vitro stability in competition studies with cysteine and histidine. Moreover, lipophilicity studies were performed in order to determine the lipophilicity of the radiolabeled conjugates, while a hemolysis assay was performed to investigate the biocompatibility of the bare and functionalized AuNPs. We have shown that the functionalized AuNPs developed in this study lead to stable radiolabeled nanoconstructs with the potential to be applied in multimodality imaging or for in vivo tracking of drug-carrying AuNPs.

The synthesis of M(I)(CO)(3)(NNO) (M = Re, (99m)Tc) complexes conjugated to the antitumor agent 2-(4'-aminophenyl)benzothiazole and to its 6-methyl derivative, as well as their in vitro and in vivo biological evaluation as breast cancer radiopharmaceuticals, is reported. The Re complexes displayed under the fluorescence microscope clear uptake by the sensitive to the 2-(4'-aminophenyl)benzothiazole pharmacophore breast cancer cell lines MCF-7 and T47D, while uptake by less sensitive lines and by normal fibroblasts was much weaker. In accordance, uptake of the corresponding radioactive (99m)Tc complexes was clearly higher in the breast cancer cell lines MCF-7 and MDA-231 compared to normal fibroblasts. Biodistribution of the (99m)Tc complexes in SCID mice bearing MCF-7 xenografts showed appreciable tumor uptake. A tumor/muscle ratio of 2.2 was measured for the complex conjugated to 2-(4'-aminophenyl)benzothiazole that led to successful tumor imaging. The results render the 2-(4'-aminophenyl)benzothiazole complexes potential candidates for imaging ((99m)Tc) and targeted radiotherapy ((188)Re) of breast cancer.

The synthesis and biological evaluation of new M(I)(CO)3(NNO) (M = Re, 99mTc) complexes attached to the antitumor agent 2-(4-aminophenyl)benzothiazole are reported. The fluorescent rhenium complex enters MCF-7 breast cancer cells but does not enter normal HFFF-2 and MRC-5 cells. The analogous radioactive 99mTc complex produces fast blood and soft tissue clearance when administered to healthy mice. These complexes are promising candidates for developing radiopharmaceuticals for imaging (99mTc) and targeted radiotherapy (186Re, 188Re) of breast cancer.

The reaction of 2-(2'-pyridyl)benzothiazole, [NN], with the ReO(V)(3+) and TcO(V)(3+) cores in the presence of thiophenols, [S] (RC(6)H(4)SH, R = H, 4-CH(3), 4-OCH(3)), as coligands led to the isolation of hexacoordinated complexes of the MO[NN][S](3) type (M = Re, Tc). In all cases, two geometric mer isomers were formed, as evidenced by NMR spectroscopy and confirmed by X-ray crystallography. In both isomers, the coordination geometry about the metal ion is a distorted octahedral defined by the two nitrogen atoms of the bidentate ligand, the three sulfur atoms of the monodentate thiols, and the oxygen atom of the oxo group. The apical positions of the octahedron are occupied by the oxygen of the oxo group and, in one of the isomers, the nitrogen of the pyridyl moiety of 2-(2'-pyridyl)benzothiazole, while, in the second isomer, the imine nitrogen of 2-(2'-pyridyl)benzothiazole. The complexes are stable, neutral, and lipophilic. Complete (1)H and (13)C NMR assignments are reported for all complexes. The synthetic reaction was also successfully transferred at the technetium-99m tracer level by ligand exchange reaction using (99m)Tc-glucoheptonate as precursor in the presence of 2-(2'-pyridyl)benzothiazole and 4-CH(3)C(6)H(4)SH. The structure of the technetium-99m complex was established by high-performance liquid chromatographic comparison with the analogous oxotechnetium and oxorhenium complexes. The 2-(2'-pyridyl)benzothiazole ligand serves as a preliminary model for 2-(4-aminophenyl)benzothiazole, which possesses interesting properties for the development of technetium and rhenium radiopharmaceuticals for tumor imaging and/or radiotherapy as well as in vivo diagnosis of Alzheimer's disease.

The synthesis and characterization of oxotechnetium and oxorhenium mixed-ligand complexes of the general formula MO[NN][S](3) (M = (99)Tc and Re), where NN represents the bidentate ligand 2,2'-bipyridine and S represents a monodentate thiophenol, is reported. The complexes were prepared by ligand exchange reactions using (99)Tc-gluconate and ReOCl(3)(PPh(3))(2) as precursors for the oxotechnetium and oxorhenium complexes, respectively. Compound 1 (M = (99)Tc, S = 4-methylthiophenol) crystallizes in the monoclinic space group P2(1)/a, a = 23.12(1) A, b = 14.349(6) A, c = 8.801(4) A, beta = 94.81(2) degrees, V = 2918(2) A(3), Z = 4. Compound 3 (M = Re, S = 4-methylthiophenol) crystallizes in the monoclinic space group P2(1)/a, a = 23.018(9) A, b = 14.421(5) A, c = 8.775(3) A, beta = 94.78(1) degrees, V = 2903(2) A(3), Z = 4. Compound 4 (M = Re, S = 4-methoxythiophenol) crystallizes in the orthorhombic space group Pbca, a = 16.32(1) A, b = 24.55(2) A, c = 16.94(1) A, V = 6788(9) A(3), Z = 8. In all cases, the coordination geometry around the metal is distorted octahedral with the equatorial plane being defined by the three sulfur atoms of the thiophenols and one nitrogen atom of 2,2'-bipyridine, while the apical positions are occupied by the second nitrogen atom of 2,2'-bipyridine and the oxygen of the M=O core. The complexes are stable, neutral, and lipophilic. Complete (1)H and (13)C NMR assignments are reported for all complexes. The analogous oxotechnetium complexes have been also synthesized at tracer level ((99m)Tc) by mixing the 2,2'-bipyridine and the corresponding thiol with Na(99m)TcO(4) generator eluate using NaBH(4) as reducing agent. Their structure was established by chromatographic comparison with authentic oxotechnetium and oxorhenium complexes using high performance liquid chromatography techniques.

Synthesis of the 2,9-dimethyl-4,7-diaza-4-alkyl-2,9-decanedithiol (1, alkyl = morpholinylethyl in a, and alkyl = pyrrolidinylethyl in b), following a widely used synthetic scheme for diaminedithiol (DADT) ligands, led to the isolation of 1-alkyl-2-(1'-methyl-1'-sulfanylethyl)-3-(2' '-methyl-2' '-sulfanylpropyl)diazolidine (3) as the major product. Both ligands 1 and 2 gave complexes with the oxorhenium ReO(V) core. Ligand 1 gave the expected ReO[SNNS] complex (2) with the side chain on nitrogen in the syn configuration. Ligand 3 gave, in the presence of a monodentate aromatic thiol, complexes of the ReO[SNN][S][S] (4) and ReO[SNN][S] type (5), respectively, in which the diazolidine ring has rearranged to a thiazolidine ring. Crystallographic analysis showed that in 4 the coordination geometry about the metal is distorted octahedral where the equatorial plane is defined by the sulfur and one of the nitrogen atoms of the ligand and the two sulfurs of the aromatic thiols, while the axial positions are occupied by the oxygen of the ReO core and the second nitrogen of the ligand. Specifically, complex 4a crystallizes in space group P2(1)/c, a = 15.63(1) A, b = 15.28(2) A, c = 16.07(1) A, beta = 113.78(2) degrees, V = 3512(5) A(3), Z = 4. Complex 4b crystallizes in space group P2(1)/n, a = 14.560(9) A, b = 14.804(9) A, c = 19.85(1) A, beta = 90.94(2) degrees, V = 4278(1) A(3), Z = 4. In 5b, the coordination geometry is distorted square pyramidal with the SNN donor atom of the ligand and the aromatic thiol defining the equatorial plane and the doubly bonded oxygen occupying the apex of the pyramid. Complex 5b crystallizes in space group P(-)1, a = 9.387(5) A, b = 11.306(5) A, c = 14.040(6) A, alpha = 84.51(1) degrees, beta = 84.45(2) degrees, gamma = 87.17(1) degrees, V = 1475(1) A(3), Z = 2. All isolated complexes are neutral and lipophilic. Complete assignments of (1)H and (13)C NMR resonances are reported.

Two novel [99mTc](SNS/S) mixed ligand complexes carrying a pendant ester function on the monothiolate coligand were synthesized. The corresponding oxorhenium and [99gTc]oxotechnetium complexes prepared at the macroscopic level and chemically characterized were used for structure assignment of [99mTc](SNS/S) complexes prepared at the nanomolar level. Enzymatic hydrolysis of the pendant ester group of [99mTc](SNS/S) mixed ligand complexes by esterase was investigated in vitro and compared with that of the ethyl cysteinate dimer, [99mTc]ECD. Preliminary biodistribution data in mice shows that the complexes are lipophilic and exhibit significant initial uptake in rodent brain.

Two series of [99mTc](SNS/S) mixed ligand complexes each carrying the N-diethylaminoethyl or the N-ethyl-substituted bis(2-mercaptoethyl)amine ligand (SNS) are produced at tracer level using tin chloride as reductant and glucoheptonate as transfer ligand. The identity of [99mTc](SNS/S) complexes is established by high-performance liquid chromatographic (HPLC) comparison with authentic rhenium samples. The para substituent R on the phenylthiolate coligand (S) ranges from electron-donating (-NH2) to electron-withdrawing (-NO2) groups, to study complex stability against nucleophiles as a result of N- and R-substitution. The relative resistance of [99mTc](SNS/S) complexes against nucleophilic attack of glutathione (GSH), a native nucleophilic thiol of 2 mM intracerebral concentration, is investigated in vitro by HPLC. The reaction of [99mTc](SNS/S) complexes with GSH is reversible and advances via substitution of the monothiolate ligand by GS- and concomitant formation of the hydrophilic [99mTc](SNS/GS) daughter compound. The N-diethylaminoethyl complexes are found to be more reactive against GSH as compared to the N-ethyl ones. Complex reactivity as a result of R-substitution follows the sequence -NO2 >> -H > -NH2. These in vitro findings correlate well with in vivo distribution data in mice. Thus, brain retention parallels complex susceptibility to GSH attack. Furthermore, isolation of the hydrophilic [99mTc](SNS/GS) metabolite from biological fluids and brain homogenates provides additional evidence that the brain retention mechanism of [99mTc](SNS/S) complexes is GSH-mediated.

A series of neutral, lipophilic 99mTc mixed-ligand complexes of the general formula 99mTcOL1L2, where L1H2 is an N-substituted bis-(2-mercaptoethyl)amine, [X-CH2CH2N(CH2CH2SH)2], [SNS], and L2H is a monodentate thiol (RSH), [S], has been synthesized and evaluated in rodents for potential use in brain blood flow imaging. The complexes were prepared by ligand exchange reaction using 99mTc(V)O-glucoheptonate as precursor and equimolar quantities of the two ligands. In all cases the syn isomer was formed in a high yield, whereas the anti isomer was not always present. The formation of two isomeric complexes-syn and anti-was expected, since the N-substituent (X-CH2CH2N) can assume syn or anti configuration with respect to the 99mTcO3+ core during complexation. One anti and all syn isomers were isolated by HPLC. Their identity was confirmed by comparative HPLC studies with the analogous 99Tc complexes of established structure. In vivo distribution, in particular brain uptake and retention, greatly depended on the type of either tridentate (L1H2) or monodentate (L2H) ligand. All 99mTc complexes showed significant brain uptake in mice (0.78-4.35% injected dose per organ at 5 min postinjection). This initial uptake remained nearly constant for at least 30 min for most of the complexes. Structure-activity relationships of novel 99mTc(V)O SNS/S complexes in mice are reported and discussed. Selected complexes were further studied in rats. High brain uptake, comparable to that of 99mTc-d,l-HMPAO, and sufficient retention 60 min postinjection were provided with complex 18 [X = (C2H5)2N and R = p-CH3OC6H4CH2].

The simultaneous action of the tridentate ligand (C(2)H(5))(2)NCH(2)CH(2)N(CH(2)CH(2)SH)(2) and the monodentate coligand HSC(6)H(4)OCH(3) on a suitable ReO(3+) precursor results in a mixture of syn- and anti-oxorhenium complexes, ReO[(C(2)H(5))(2)NCH(2)CH(2)N(CH(2)CH(2)S)(2)] [SC(6)H(4)OCH(3)], in a ratio of 25/1. The complexes are prepared by a ligand exchange reaction using ReO(eg)(2) (eg = ethylene glycol), ReOCl(3)(PPh(3))(2), or Re(V)-citrate as precursor. Both complexes have been characterized by elemental analysis, FT-IR, UV-vis, X-ray crystallography, and NMR spectroscopy. The syn isomer C(17)H(29)N(2)O(2)S(3)Re crystallizes in the monoclinic space group P2(1)/n, a = 14.109(4) Å, b = 7.518(2) Å, c = 20.900(5) Å, beta = 103.07(1) degrees, V = 2159.4(9) Å(3), Z = 4. The anti isomer C(17)H(29)N(2)O(2)S(3)Re crystallizes in P2(1)/n, a = 9.3850(7) Å, b = 27.979(2) Å, c = 8.3648(6) Å, beta = 99.86(1) degrees, V = 2163.9(3) Å(3), Z = 4. Complete NMR studies show that the orientation of the N substituent chain with respect to the Re=O core greatly influences the observed chemical shifts. Complexes were also prepared at the tracer ((186)Re) level by using (186)Re-citrate as precursor. Corroboration of the structure at tracer level was achieved by comparative HPLC studies.

A new approach to the "3 + 1" mixed ligand oxotechnetium complexes of the general formula TcOL1L2, with ligands (L1H(2)) containing the SNN donor set and various monodentate thiols as coligands (L2H) is reported. The ligands L1H(2) (1-3, general formula R(1)CH(2)CH(2)NHCH(2)C(R(2))(2)SH where R(1) = N(CH(3))(2) and R(2) = H in 1, R(1) = pyrrolidin-1-yl and R(2) = H in 2, and R(1) = piperidin-1-yl and R(2) = CH(3) in 3) act as tridentate SNN chelates to the TcO(3+) core, leaving open one coordination site cis to the oxo group. In the presence of a monodentate thiol (L2H) and using (99)Tc(V)-gluconate as precursor, the vacancy is filled by the thiol which acts as the coligand. With this approach four neutral oxotechnetium complexes (4-7, general formula TcO[R(1)CH(2)CH(2)NCH(2)C(R(2))(2)S][SR] where RSH = p-methoxybenzenethiol, or p-methylbenzenethiol or benzyl mercaptan) were prepared in high yield by reacting L1H(2) and L2H with Tc(V)-gluconate in a ratio 1:1:1. The complexes were characterized by elemental analysis and spectroscopic methods. Complete assignments of (1)H and (13)C NMR resonances were made for all complexes. X-ray crystallographic studies of 5 (R(1) = pyrrolidin-1-yl, R(2) = H, RSH = p-methylbenzenethiol) and 7 (R(1) = piperidin-1-yl, R(2) = CH(3), RSH = benzyl mercaptan) showed that the complexes crystallize in the monoclinic space group P2(1)/n (a = 10.223(1) Å, b = 9.283(1) Å, c = 18.337(2) Å, beta = 97.262(2) degrees, V = 1726.3(4) Å(3), Z = 4; a = 11.876(2) Å, b = 10.470(2) Å, c = 17.098(3) Å, beta = 105.990(4) degrees, V = 2043.8(6) Å(3), Z = 4, for 5 and 7, respectively). Complexes5 and 7 have distorted square pyramidal coordination geometry with the oxo ligand in the axial position. The steric requirements of the oxo group cause the Tc atom to be displaced 0.68 Å out of the mean equatorial plane of the NNSS donor atoms in both complexes.

A series of 22 mixed-ligand complexes of the general formula TcOL(1)L(2), where L(1)H(2) are N-substituted bis(2-mercaptoethyl)amine ligands, [SN(R)S], and L(2)H are monodentate thiols as coligand, is reported. The complexes were prepared by the ligand exchange method using Tc-gluconate as precursor and equimolar quantities of the two ligands. In all cases the syn stereoisomer was formed in high yield and isolated as a crystalline product. In four cases HPLC analysis demonstrated the presence of the anti stereoisomer in the reaction mixture. Although the yield was less than 1%, one anti isomer, 4a, was successfully isolated as brown crystals. The isolated complexes were characterized by spectroscopic methods and elemental analysis. The formation of the two diastereomers, syn and anti, was expected due to the configuration of the nitrogen substituent (R) with respect to the central TcO core. The X-ray crystallography showed that the coordination geometry of the syn isomers 9, 11, and 18 is trigonal bipyramidal while for the anti isomer 4a it is distorted square pyramidal. This is the first documentation of syn/anti isomerism in N-substituted TcO[SN(R)S][S] mixed-ligand complexes.

In developing 99mTc complexes as potential brain-imaging agents, we investigated the coordination chemistry of ligands containing sulfur and nitrogen donor atoms with the general formula R-CH2CH2N(CH2CH2SH)2 (R = C2H5S, (C2H5)2N). These ligands act as tridentate SNS chelates to the TcO3+ core, leaving open one coordination site cis to the oxo group. In reactions with the highly reactive [99TcOCl4]- precursor, this vacancy was occupied by a chlorine atom. When the ligands reacted in the presence of 4-methoxythiophenol, using 99Tc(V)-gluconate as precursor, the vacancy was filled with 4-methoxythiophenol, which acted as coligand. Thus neutral mixed ligand complexes of the general formula [TcO((SCH2CH2)2NCH2CH2R)X], where X = Cl or 4-methoxythiophenol, were synthesized. The complexes were characterized by UV-vis, IR, 1H NMR, crystallographic, and elemental analyses. The crystal structures of 3a (R = C2H5S, X = Cl) and 4b (R = (C2H5)2N, X = 4-methoxythiophenol) demonstrated that the coordination geometry is trigonal bipyramidal with the N1 and Cl or S3 occupying the apical positions and the basal plane defined by the S1 and S2 of the tridentate ligand and the oxo group. The complexes 4a(99mTc) (R = C2H5S, X = 4-methoxythiophenol) and 4b(99mTc) were prepared using 99mTc-glucoheptonate as precursor and were purified by HPLC. Biodistribution in mice showed high initial brain uptake (3.68% and 3.56% dose/organ for 4a(99mTc) and 4b(99m-Tc), respectively). Complex 4b(99mTc) displayed significantly higher brain/blood values and prolonged retention in brain as well. The results suggest that structural modifications based on configurations 4a,b may provide novel 99mTc brain-imaging agents with improved biological characteristics.