Evaluation of Rhenium and Technetium-99m Complexes Bearing Quinazoline Derivatives as Potential EGFR Agents.

Τhe Epidermal Growth Factor Receptor tyrosine kinase inhibitor (EGFR-TKI) 6-amino-4-[(3-bromophenyl) amino]quinazoline was derivatized with 6-bromohexanoyl-chloride and coupled with the tridentate chelating agents N-(2-pyridylmethyl) aminoethyl acetic acid (PAMA) and L(+)-cysteine bearing the donor atom set NNO and SNO, respectively. The rhenium precursors ReBr(CO)5 and fac-[NEt4]2[ReBr3(CO)3] were used for the preparation of the Re complexes fac-[Re(NNO)(CO)3] (5a) and fac-[Re(SNO)(CO)3] (7a) which were characterized by NMR and IR spectroscopies. Subsequently, the new potential EGFR inhibitors were labeled with the fac-[99mTc(CO)3]+ core in high yield and radiochemical purity (>90%) by ligand exchange reaction using the fac-[99mTc][Tc(OH2)3(CO)3]+ precursor. The radiolabeled complexes were characterized by comparative HPLC analysis with the analogous rhenium (Re) complexes as references. In vitro studies in the A431 cell lines showed that both ligands and Re complexes inhibit A431 cell growth. Complex 5a demonstrated the highest potency (IC50 = 8.85 ± 2.62 µM) and was further assessed for its capacity to inhibit EGFR autophosphorylation, presenting an IC50 value of 26.11 nM. Biodistribution studies of the 99mTc complexes in healthy mice showed high in vivo stability for both complexes and fast blood and soft tissue clearance with excretion occurring via the hepatobiliary system.

Synthesis and preclinical evaluation of rhenium and technetium-99m "4 + 1" mixed-ligand complexes bearing quinazoline derivatives as potential EGFR imaging agents.

Epidermal growth factor receptors (EGFR) of tyrosine kinase (TK) have shown high expression levels in most cancers and are considered a promising target for cancer diagnosis and therapy. Expanding the investigation for novel targeted radiopharmaceuticals, an EGFR inhibitor such as 4-aminoquinazoline derivatives along with a radionuclide such as technetium-99m (99mTc) could be ideal. Thus, we report herein the synthesis, characterization, and biological evaluation of new "4 + 1" mixed-ligand ReIII- and 99mTcIII-complexes of the general formula [99mTc][Tc(NS3)(CN-R)] bearing tris(2-mercaptoethyl)-amine (NS3) as the tetradentate tripodal ligand and a series of isocyanide derivatives (CN-R) of tyrosine kinase inhibitor (3-bromophenyl)quinazoline-4,6-diamine as the monodentate ligand. The quinazoline isocyanide derivatives 4a-d were prepared in two steps and reacted with the [Re(NS3)PMe2Ph] precursor leading to the final complexes 5a-d in high yield. All compounds were characterized by elemental analysis, IR, and NMR spectroscopies. In vitro studies, for their potency to inhibit the cell growth, using intact A431 cells indicate that the quinazoline derivatives 4a-d and the Re complexes 5a-d significantly inhibit the A431 cell growth. In addition, the EGFR autophosphorylation study of complex 5b shows an IC50 value in the nanomolar range. The corresponding "4 + 1" 99mTc-complexes 6a-d were prepared by employing the [99mTc]TcEDTA intermediate and the appropriate monodentate 4a-d in a two-step synthetic procedure with a radiochemical yield (RCY) from 63 to 77 % and a radiochemical purity (RCP) > 99 % after HPLC purification. Their structures have been established by HPLC comparative studies using the well-characterized Re-complexes 5a-d as reference. All 99mTc-complexes remain stable for at least 6 h, and their logD7.4 values confirmed their anticipated lipophilic character. Biodistribution studies in healthy Swiss albino mice of 99mTc-complexes showed hepatobiliary excretion and initial fast blood clearance. Complex 6b was also tested in Albino SCID mice bearing A431 tumors and showed rapid tumor uptake at 5 min (2.80 % ID/g) with a moderate tumor/muscle ratio (2.06) at 4 h p.i. The results encourage further investigation for this type of 99mTc-complexes as single-photon emission computed tomography (SPECT) radio agents for imaging tumors overexpressing EGFR.

Synthesis and Characterization of Novel [2 + 1] Tricarbonyl Rhenium Complexes with the Hydrophilic Phosphine Ligands PTA and CAP.

In the pursuit of hydrophilic model fac-[Re(CO)3]+ complexes for (radio) pharmaceutical applications, six novel [2 + 1] mixed-ligand complexes of the general type fac-[Re(CO)3(bid)P] were synthesized and characterized, where bid is a bidentate ligand bearing either (N, O) or (S, S') donor atom sets and P is the hydrophilic phosphine 1,3,5-triaza-7-phosphoadamantane (PTA) or its macrocyclic homologue 1,4,7-triaza-9-phosphatricyclo[5.3.2.1]tridecane (CAP). The (N, O) ligands used in this study were picolinic and quinaldic acid, while the (S, S') ligand was diethyldithiocarbamate. The complexes were synthesized in generally high yields and purity and the characterization was performed by spectroscopic methods, IR, NMR, and elemental analysis. Detailed X-ray crystallographic study of molecular packing by using Hirshfeld analysis tools revealed a plethora of intermolecular interactions such as hydrogen bond, π⋯π, C-H⋯π, and carbonyl-carbonyl interactions. To our knowledge, the CAP complexes reported herein are the first example of [2 + 1] mixed-ligand fac-[Re(CO)3]+ complexes with CAP. The new complexes might have the potential to serve as platforms for the design of target-specific complexes with favorable pharmacokinetics.

Design, synthesis, structural optimization, SAR, in silico prediction of physicochemical properties and pharmacological evaluation of novel & potent thiazolo[4,5-d]pyrimidine corticotropin releasing factor (CRF) receptor antagonists.

Corticotropin-releasing factor (CRF) is a 41-amino-acid neuropeptide secreted from the hypothalamus and is the main regulator of the hypothalamus-pituitary-adrenocortical (HPA) axis. CRF is the master hormone which modulates physiological and behavioral responses to stress. Many disorders including anxiety, depression, addictive disorders and others are related to over activation of the CRF system. This suggests that new molecules which can interfere with CRF binding to its receptors may be potential candidates for neuropsychiatric drugs to treat stress-related disorders. Previously, three series of pyrimidine and fused pyrimidine CRF1 receptor antagonists were synthesized by our group and specific binding assays, competitive binding studies and determination of the ability to antagonize the agonist-stimulated accumulation of cAMP (the second messenger for CRF receptors) were reported. In continuation of our efforts in this direction, in the current manuscript, we report the synthesis & biological evaluation of a new series of CRF1 receptor antagonists. Seven compounds showed promising binding affinity with the best two compounds (compounds 6 & 43) displaying a superior binding affinity to all of our previous compounds. Compounds 6 & 43 have only 4 times and 2 times less binding affinity than the standard CRF antagonist antalarmin, respectively. Thus, our two best lead compounds (compound 6 & 43) can be considered potent CRF receptor antagonists with binding affinity of 41.0 & 19.2 nM versus 9.7 nM for antalarmin.

Effective Labeling of Amine Pharmacophores through the Employment of 2,3-Pyrazinedicarboxylic Anhydride and the Generation of fac-[M(CO)3(PyA)P] and cis-trans-[M(CO)2(PyA)P2] Complexes (PyA = Pyrazine-2-carboxylate, P = Phosphine, M = Re, 99mTc).

The fac-[M(CO)3(PyA)(P)] and cis-trans-[M(CO)2(PyA)(P)2] neutral complexes (M is Re or 99mTc), based on the mixed ligand strategy with pyrazine-2-carboxylic acid (PyAH) as the bidentate N,O and triphenylphosphine as the monodentate P ligand, are presented. Through the employment of the anhydride of pyrazine-2,3-dicarboxylic acid (PyDA), the PyAH scaffold was conveniently derivatized with the model bioactive amine 1-(2-methoxyphenyl)piperazine, the active part of the 5-HT1A antagonist WAY100635. Reaction of either PyAH or the pharmacophore-bearing PyAH ligand (L1H) with fac-[M(CO)3]+ core in water yielded the intermediate fac-[M(CO)3(PyA)(H2O)] complexes. The labile aqua ligand was easily replaced by PPh3 to yield the fac-[Re(CO)3(PyA)(PPh3)] complexes, while in toluene under reflux, the cis-trans-[Re(CO)2(PyA)(PPh3)2] complexes were obtained. The latter complexes were alternatively obtained from mer-[Re(CO)3(PPh3)2Cl] by refluxing with the PyA ligand in toluene. The analogous 99mTc complexes were synthesized quantitatively, showing excellent stability in competition studies. The methodology described herein represents a practical procedure for the effective integration of the fac-[M(CO)3]+ core with amine-bearing biologically active compounds for diagnosis/therapy.

Synthesis and In Vitro Evaluation of Gold Nanoparticles Functionalized with Thiol Ligands for Robust Radiolabeling with 99mTc.

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 (99mTc) 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 L1H and L2H). 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 99mTc-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.

Synthesis and evaluation of new mixed "2 + 1" Re, 99mTc and 186Re tricarbonyl dithiocarbamate complexes with different monodentate ligands.

A series of "2 + 1" mixed ligand tricarbonyl complexes of the general formula fac-[Re/99mTc/186Re(CO)3(DDTC)(L)] containing diethyldithiocarbamate (DDTC) as a monoanionic bidentate ligand and a series of monodentate ligands L was synthesized, characterized and evaluated. The impact of ligand L on the radiochemical yield (RCY) and biodistribution of the final compounds was also investigated. DDTC and the appropriate L ligand [cyclohexyl isocyanide (cisc), tert-butyl isocyanide (tbi), triphenylphosphine (PPh3), methyldiphenylphosphine (PPh2Me), triphenylarsine (AsPh3), imidazole (im), and 4-aminopyridine (4AP)] readily reacted in equimolar amounts with the [Et4N]2[Re(CO)3Br3] precursor to afford fac-[Re(CO)3(DDTC)(cisc)], Re1, fac-[Re(CO)3(DDTC)(tbi)], Re2, fac-[Re(CO)3(DDTC)(PPh3)], Re3, fac-[Re(CO)3(DDTC)(PPh2Me)], Re4, fac-[Re(CO)3(DDTC)(AsPh3)], Re5, fac-[Re(CO)3(DDTC)(im)], Re6 and fac-[Re(CO)3(DDTC)(4AP)], Re7, complexes in high yields (>80%). All Re complexes were fully characterized by IR, NMR, and in addition Re4, Re5, and Re7 with X-ray crystallography. Analogous reactions as performed with Re were subsequently explored on the 99mTc and 186Re-tracer levels using the corresponding fac-[99mTc/186Re(CO)3(H2O)3]+ precursor. Complexes 99mTc1 - 99mTc5, 186Re1 and 186Re3 were obtained in high radiochemical yield (>91%), while the complexes 99mTc6, 99mTc7 and 186Re7 formed with radiochemical yields of 55%, 28%, and 75%, respectively. The 99mTc and 186Re-complexes were characterized by comparative HPLC analysis using the analogous Re complexes. During histidine and cysteine challenge experiments at 37 °C through 6 h, complexes 99mTc1 - 99mTc5 remained > 92% stable, while complexes 99mTc6 and 99mTc7 remained only 8% stable through 3 h. Similar studies for 186Re-complexes showed that 186Re1 and 186Re3 remained > 95% stable for up to 48 h, while 186Re7 had decreased to 7% after 3 h. LogD7.4 data of 99mTc1 - 99mTc5, 186Re1, and 186Re3 complexes, which ranged from 2.59 to 3.39, suggested high lipophilicity. Biodistribution studies in healthy Swiss albino mice showed hepatobiliary excretion for 99mTc1, 99mTc2, and 99mTc4, fast blood clearance for 99mTc4, while high liver uptake and retention for 99mTc3 and 99mTc5 were measured. Moreover, 99mTc2 showed high accumulation in the lungs with sustained retention (52.80% ID/g at 4 h p.i.) and significant brain uptake at 2 min p.i. (1.89% ID/g). The study showed the great influence of monodentate ligand in the synthesis and biodistribution of the mixed ligand complexes.

Comparative Study of a Series of 99mTc(CO)3 Mannosylated Dextran Derivatives for Sentinel Lymph Node Detection.

Sentinel lymph node detection (SLND) is rapidly entering common practice in the management of patients with tumors. The introduction of mannose molecules to 99mTc-labeled dextrans, so far, showed that the sentinel node could trap these agents due to their recognition by the mannose receptors of lymph node macrophages. The current study aimed to synthesize, characterize, and biologically evaluate a series of mannosylated dextran derivatives labeled with 99mTc for potential use in SLND. The compounds were designed to have a dextran with a molecular weight of 10-500 kDa as a backbone, S-derivatized cysteines, efficient SNO chelators, and mannose moieties for binding to mannose receptors. They were successfully synthesized, thoroughly characterized using NMR techniques, and labeled with the fac-[99mTc(CO)3]+ synthon. Labeling with high yields and radiochemical purities was achieved with all derivatives. In vivo biodistribution and imaging studies demonstrated high uptake in the first lymph node and low uptakes in the following node and confirmed the ability to visualize the SLN. Among the compounds studied, 99mTc-D75CM demonstrated the most attractive biological features, and in combination with the high radiochemical yield and stability of the compound, its further evaluation as a new radiopharmaceutical for sentinel lymph node detection was justified.

Synthesis, characterization and evaluation of 68Ga labelled monomeric and dimeric quinazoline derivatives of the HBED-CC chelator targeting the epidermal growth factor receptor.

Tyrosine kinase (TK) receptors including epidermal growth factor receptors (EGFRs) are known to be overexpressed in a wide variety of solid tumors associated with poor prognosis. The HBED-CC chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid 1 was coupled via one or both its propionic acid moieties with the quinazoline EGFR-TK inhibiting pharmacophore 4-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)butanamide 3 resulting in either a monomeric 4 or a dimeric 5 species. Ligands 4 and 5 reacted with Ga3+ generating the corresponding complexes Ga4 and Ga5. Both ligands and complexes were characterized with mass spectrometry and NMR spectroscopy and evaluated in vitro with MTT assays in A431 cells, where they showed IC50 values in the range 51.6 to 68.8 µM. Labeling of ligands 4 and 5 with the PET radionuclide 68Ga was quantitative and resulted in tracers [68Ga]Ga4 and [68Ga]Ga5 with radiochemical purities greater than 98%, which were also characterised by comparative RP-HPLC studies with Ga4 and Ga5 respectively. Radiotracers [68Ga]Ga4 and [68Ga]Ga5 were stable (intact tracer over 98%) in the reaction mixture (120 min) and in human serum (30 min). Both tracers were evaluated in vivo with biodistribution experiments in SCID mice bearing A431 tumors presenting tumor uptake of 1.34 for [68Ga]Ga4 and 1.01 %ID/g for [68Ga]Ga5 at 5 min, which was slightly decreased at 60 min p.i. and then remained stable until 120 min p.i. To the best of our knowledge, this is the first report of monomeric and dimeric quinazoline conjugates with the chelator HBED-CC, which can serve as a basis for further development of EGFR-TKI targeting tracers.

Mitochondrial D-loop Sequence Variability in Three Native Insular Griffon Vulture (Gyps fulvus) Populations from the Mediterranean Basin.

The islands of Sardinia, Crete, and Cyprus are hosting the last native insular griffon populations in the Mediterranean basin. Their states have been evaluated from "vulnerable" to "critically endangered". The sequence analysis of molecular markers, particularly the mtDNA D-loop region, provides useful information in studying the evolution of closely related taxa and the conservation of endangered species. Therefore, a study of D-loop region sequence was carried out to estimate the genetic diversity and phylogenetic relationship within and among these three populations. Among 84 griffon specimens (44 Sardinian, 33 Cretan, and 7 Cypriot), we detected four haplotypes including a novel haplotype (HPT-D) that was exclusively found in the Cretan population with a frequency of 6.1%. When considered as a unique population, haplotype diversity (Hd) and nucleotide diversity (π) were high at 0.474 and 0.00176, respectively. A similar level of Hd and π was found in Sardinian and Cretan populations, both showing three haplotypes. The different haplotype frequencies and exclusivity detected were in accordance with the limited matrilineal gene flow (FST = 0.07097), probably related to the species reluctance to fly over sea masses. The genetic variability we observe today would therefore be the result of an evolutionary process strongly influenced by isolation leading to the appearance of island variants which deserve to be protected. Furthermore, since nesting sites and food availability are essential elements for colony settlement, we may infer that the island's colonization began when the first domestic animals were transferred by humans during the Neolithic. In conclusion, our research presents a first contribution to the genetic characterization of the griffon vulture populations in the Mediterranean islands of Sardinia, Crete and Cyprus and lays the foundation for conservation and restocking programs.

Crystal structure of fac-aqua-[(E)-4-(benzo[d]thia-zol-2-yl)-N-(pyridin-2-yl-methyl-idene)aniline-κ2 N,N']tricarbonylrhenium(I) hexa-fluorido-phosphate methanol monosolvate.

In the title compound, fac-[Re(C19H13N3S)(CO)3(H2O)]PF6·CH3OH, the coordination environment of the ReI atom is octa-hedral with a C3N2O coordination set. In this mol-ecule, the N,N' bidentate ligand, (E)-4-(benzo[d]thia-zol-2-yl)-N-(pyridin-2-yl-methyl-idene)aniline, and the monodentate aqua ligand occupy the three available coordination sites of the [Re(CO)3]+ core, generating a '2 + 1' mixed-ligand complex. In this complex, the Re-C bonds of the carbonyl ligands trans to the coordinating N,N' atoms of the bidentate ligand are longer than the Re-C bond of the carbonyl group trans to the aqua ligand, in accordance with the intensity of their trans effects. The complex is positively charged with PF6 - as the counter-ion. In the structure, the complexes form dimers through π-π inter-molecular inter-actions. O-H⋯O and O-H⋯N hydrogen bonds lead to the formation of stacks parallel to the a axis, which further extend into layers parallel to (01). Through O-H⋯F hydrogen bonds between the complexes and the PF6 -counter-anions, a three-dimensional network is established.

Remarkable Brain Penetration of Cyclopentadienyl M(CO)3+ (M = 99mTc, Re) Derivatives of Benzothiazole and Benzimidazole Paves the Way for Their Application as Diagnostic, with Single-Photon-Emission Computed Tomography (SPECT), and Therapeutic Agents for Alzheimer's Disease.

The synthesis and evaluation of three novel 99mTc complexes (99mTc-1-3) and their corresponding Re complexes (Re-1-3), in which the phenyl ring of 2-phenylbenzothiazole or 2-phenylbenzimidazole is replaced by the cyclopentadienyl tricarbonyl [Cp99mTc(CO)3] core, are reported. Both 99mTc and Re complexes were prepared from the corresponding ferrocenyl derivatives, and the Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography. The complexes exhibit effective in vitro binding to ß-amyloid (Aß) plaques and fibrils, inhibit Aß fibril formation, and significantly reduce Aß-induced cytotoxicity and reactive oxygen species production in neuronal cell cultures. The brain uptake of the 99mTc complexes ranges between 7.94 and 3.99% ID/g at 2 min p.i., being the highest recorded for potential 99mTc Aß plaque imaging probes in mice. Powered by their high brain uptake, the complexes represent strong theranostic candidates against Alzheimer's disease combining single-photon-emission computed tomography diagnostic (99mTc complexes) and antiamyloid therapeutic (Re complexes) potential.

Simultaneous determination of paraquat and atrazine in water samples with a white light reflectance spectroscopy biosensor.

An optical immunosensor based on White Light Reflectance Spectroscopy for the simultaneous determination of the herbicides atrazine and paraquat in drinking water samples is demonstrated. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum due to reaction to effective biomolecular layer thickness. Dual-analyte determination is accomplished by functionalizing spatially distinct areas of the chip with protein conjugates of the two herbicides and scanning the surface with an optical reflection probe. A competitive immunoassay format was adopted, followed by reaction with secondary antibodies for signal enhancement. The sensor was highly sensitive with detection limits of 40 and 50 pg/mL for paraquat and atrazine, respectively, and the assay duration was 12 min. Recovery values ranging from 90.0 to 110% were determined for the two pesticides in spiked bottled and tap water samples, demonstrating the sensor accuracy. In addition, the sensor could be regenerated and re-used at least 20 times without significant effect on the assay characteristics. Its excellent analytical performance and short analysis time combined with the small sensor size should be helpful for fast on-site determinations of these analytes.

Dicarbonyl cis-[M(CO)2(N,O)(C)(P)] (M = Re, 99mTc) Complexes with a New [2 + 1 + 1] Donor Atom Combination.

The synthesis and characterization of the dicarbonyl mixed ligand cis-[Re(CO)2(quin)(cisc)(PPh3)] complex, 4, where quin is the deprotonated quinaldic acid, cisc is cyclohexyl isocyanide, and PPh3 is triphenylphosphine, is presented. The synthesis of 4 proceeds in three steps. In the first, the intermediate fac-[Re(CO)3(quin)(H2O)] aqua complex 2 is generated from the fac-[NEt4]2[Re(CO)3Br3] precursor, together with the brominated products fac-[Re(CO)3(quinH)(Br)] 1a and fac-[NEt4][Re(CO)3(quin)(Br)] 1b, in low yield. In the following step, replacement of the aqua ligand of complex 2 by the monodentate isocyanide ligand leads to the formation of fac-[Re(CO)3(quin)(cisc)], 3. In the third step replacement of the species trans to the isocyanide carbonyl group of 3 by a phosphine generates complex 4. The Re complexes 2-4 were prepared in high yield and fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography. At the technetium-99m (99mTc) tracer level, the analogous complexes 3' and 4' were produced in high radiochemical purity, characterized by comparative reverse phase high-performance liquid chromatography and showed high resistance to transchelation by histidine or cysteine. This new [N,O][C][P] donor atom combination with the cis-[M(CO)2]+ core (M = Re, 99mTc) is a promising scaffold for the development of novel diagnostic and therapeutic targeted radiopharmaceuticals.

Electrospray ionization study of tricarbonyl fac-[Re(CO)3 (PO)(X)]-type complexes: influence of ancillary co-ligands in the release of carbon monoxide.

RATIONALE: fac-[Re(CO)3 (PO)(X)]-type complexes (PO = chelated bidentate tertiary phosphine (1-), X = various neutral, mono-dentate ligands) represent a class of compounds that meets the synthetic criteria for the preparation of potential carbon monoxide (CO) release molecules (CORMs) for medicinal application. The aim of our investigation was to achieve qualitative information whether the nature of the ancillary X ligand might influence the release of CO. METHODS: The release of CO has been investigated by means of product ion spectrometry of electrospray ionization (ESI)-generated [M + H]+ species, produced by multiple collisional experiments, using an ion trap mass spectrometer. RESULTS: Tandem mass spectrometry applied to the protonated species [Re(CO)3 (PO)(X) + H]+ of seven complexes (those including X = OH2 (1), isonitrile (2, 3), imidazole (4), pyridine (5) and phosphine (6, 7)) shows initial loss of coordinated water (1) or pyridine (5), whereas the majority of investigated entries display initial, sequential release of CO groups. The energetics of CO release have been investigated by breakdown curves for selected collisionally activated decomposition processes involving CO, and compared with those involving X groups. CONCLUSIONS: The nature of the co-ligand X drives the primary loss in the MSn processes of [Re(CO)3 (PO)(X) + H]+ compounds. When X = solvent, the energetics of these decompositions follow the trend H2 O < MeOH < CO. In each case, loss of CO is a favored fragmentation route with associated energies following the trend: N-py ≤ P-phosphine < C-isonitrile. Overall, MSn pathways indicate that [Re(PO)] (Re with chelated PO phosphine) constitutes the residual moiety. This behavior indicates that the presence of a functionalized phosphine is essential for a sequential, controlled release of CO.

Synthesis and evaluation of 99mTc/Re-tricarbonyl complexes of the triphenylphosphonium cation for mitochondrial targeting.

INTRODUCTION: Lipophilic delocalized cations accumulate in tumor cell mitochondria due to their higher transmembrane potential. In this work, this strategy was adopted for the development of 99mTc tumor-targeted imaging agents. METHODS: Two tridentate ligands containing the triphenylphosphonium cation, L1 (S-cysteinyl) and L2 (N-iminodiacetate) as well as the respective 99mTc/ReL1 and 99mTc/ReL2 tricarbonyl complexes were synthesized. The effect of the ligands and the Re complexes on cell growth in U-87 MG glioblastoma cells was assessed. In vitro stability studies and measurement of logP of the 99mTc tracers was performed. The cellular and mitochondrial uptake of the 99mTc tracers in U-87 MG cells was evaluated. Biodistribution of 99mTcL1 and 99mTcL2 were performed on SCID mice bearing U-87 MG tumors. RESULTS: The ligands L1, L2 and the Re1 and ReL2 complexes were characterized spectroscopically. Single products 99mTcL1 and 99mTcL2, >90% stable in rat serum, were obtained. LogP was 0.40±0.14 for 99mTcL1 and -0.02±0.07 for 99mTcL2. L1, ReL1 and ReL2 caused no notable cytotoxicity and L2 was found to infer 40% inhibition of cellular growth at 10-5M as well as 80% cell death in culture at 10-4M. The cell uptake of 99mTcL1 and 99mTcL2 over 4h was 1.26±0.08% and 0.06±0.01% respectively, of which 13.41±3.63% and 18.61±6.19% was distributed in the mitochondria respectively. The initial tumor uptake in mice was found to be >1% ID/g for both 99mTc tracers. CONCLUSIONS: In vitro mitochondrial and in vivo tumor targeting was observed, better in 99mTcL1, however these properties should be optimized in future studies. Advances in Knowledge and Implications for Patient Care: Continuous efforts in this direction may lead to a suitable mitochondrial-targeted 99mTc imaging agent for tumor detection.

2-(4'-Aminophenyl)benzothiazole Labeled with 99mTc-Cyclopentadienyl for Imaging ß-Amyloid Plaques.

The development of a 99mTc-radiotracer for imaging of ß-amyloid (Aß) plaques with single photon emission computed tomography (SPECT) is strongly anticipated to provide a low cost and broadly accessible diagnostic tool for Alzheimer's disease (AD). Within this framework, 2-(4'-aminophenyl)benzothiazole, known to display affinity and specificity for Aß plaques, has been joined to the tricarbonyl fac-[M(CO)3]+ (M = Re(I), 99mTc(I)) core through the cyclopentadienyl moiety to yield stable, neutral, and lipophilic complexes (Re-1 and 99mTc-1, respectively). The Re-1 complex was completely characterized with spectroscopic methods and was shown to selectively stain Aß plaques on sections of human AD brain tissue. The 99mTc-1 complex displayed satisfactory initial brain uptake (0.53% ID/g at 2 min) and in vivo stability in healthy mice, while in transgenic 5xFAD mice, models for AD, a notable retention in the brain was noted (1.94% ID/g at 90 min). The results are encouraging and contribute to the effort of developing a SPECT amyloid imaging agent.

Rhenium(I) Tricarbonyl Complexes with (2-Hydroxyphenyl)diphenylphosphine as PO Bidentate Ligand.

In the present work, we investigated potential means to obtain neutral tricarbonyl mixed-ligand fac-[M(CO)3L1L2] complexes (M = Re, 99mTc) containing the (2-hydroxyphenyl)diphenylphosphine (POH) bidentate ligand (L1H) and a series of monodentate ligands (L2). First, fac-[Re(CO)3(PO)(H2O)], 1, was synthesized by reaction of POH and [Et4N]2[Re(CO)3Br3] in equimolar amounts in MeOH at room temperature. Interestingly, with excess of POH this reaction afforded fac-[Re(CO)3(PO)(POH)], 2, with POH operating both as a bidentate and as a monodentate ligand. Owing to the presence of the labile aqua ligand, which can be readily replaced by various monodentate ligands, 1 was further used as a precursor to generate a small library of the desired fac-[M(CO)3L1L2] complexes. Specifically, by reaction of triphenylphosphine (PPh3), imidazole (im), pyridine (py), cyclohexyl isocyanide (cisc), and tert-butyl isocyanide (tbi), the following products were readily obtained in excellent yields (92%-95%): fac-[Re(CO)3(PO)(PPh3)], 3, fac-[Re(CO)3(PO)(im)], 4, fac-[Re(CO)3(PO)(py)], 5, fac-[Re(CO)3(PO)(cisc)], 6, and fac-[Re(CO)3(PO)(tbi)], 7. All compounds were fully characterized by elemental analysis, IR and NMR spectroscopies, and electrospray ionization(+) mass spectrometry. Their solid-state structure was elucidated by X-ray crystallography. Of considerable interest is the fact that the corresponding 2'-7' were easily accessible at the 99mTc-tracer level in quantitative yields after reaction of POH and the respective monodentate ligand L2 with fac-[99mTc(CO)3(H2O)3]+ in aqueous MeOH, as verified by comparative chromatographic methods adopting dual photo- and radiometric detection modes. The high stability displayed by all 99mTc complexes during histidine and cysteine challenge assays underscored the suitability of the fac-[M(CO)3(PO)L2] system for radiopharmaceutical development purposes.

Prothymosin Alpha: An Alarmin and More...

BACKGROUND/OBJECTIVE: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as "alarmins". Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα. RESULTS: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger. CONCLUSION: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis.