First example of well-characterized Re and Tc tricarbonyl complexes of ciprofloxacin and norfloxacin in the development of infection-specific imaging agents

Aiming at the development of ^99mTc-based infection-specific imaging agents, the synthesis and characterization of rhenium and technetium-99m tricarbonyl complexes with derivatized ciprofloxacin and norfloxacin is hereby reported. The ligands were prepared by coupling the tridentate chelator picolylamino-N,N-diacetic acid (PADA) with the piperazinyl (NH) nitrogen of ciprofloxacin or norfloxacin, through the employment of the PADA anhydride. The corresponding rhenium complexes were synthesized using the fac-[NEt₄]₂[ReBr₃(CO)₃] precursor and were fully characterized by elemental analysis and NMR spectroscopy. X-ray crystallography of the ciprofloxacin complex showed that the geometry about rhenium is distorted octahedral defined by the NNO donor atom set of the tridentate chelator and the three carbonyl groups. The analogous technetium-99m complexes were prepared quantitatively through the use of the fac-[^99mTc(H₂O)₃(CO)₃]⁺ precursor and their structure was established by comparative HPLC studies using the well-characterized rhenium complexes as reference. Preliminary studies with the technetium-99m complexes showed high bacterial uptake in vitro.     

The synthesis and toxicity of tripodal tricarbonyl rhenium complexes as radiopharmaceutical models

We report the synthesis and toxicity of a series of rhenium(I) tricarbonyl complexes incorporating the trisaminomethylethane (TAME) ligand. Compounds with the (TAME)Re(CO)₃⁺ cation were synthesized via several routes, including by use of Re(CO)₅X precursors as well as the aqueous cation Re(CO)₃(H₂O)₃⁺. Salts of the formula [(TAME)Re(CO)₃]X where X = Br⁻, Cl⁻, NO₃⁻, PF₆⁻ and ClO₄⁻ were evaluated using two cell lines: the monoclonal S3 HeLa line and a vascular smooth muscle cell line harvested from mice. All compounds have isostructural cations and differ only in the identity of the non-coordinating anion. None of the complexes exhibited any appreciable toxicity in the HeLa line up to the solubility limit. In the vascular smooth muscle cell line, the bromide salt exhibited some cytotoxicity, but this observation most likely results from the presence of bromide anion, which has been shown to have limited toxicity.     

Biological evaluation of glucose and deoxyglucose derivatives radiolabeled with [99mTc(CO)3(H2O)3]+ core as potential melanoma imaging agents

Glucose 9 and 2-deoxyglucose 10 were successfully synthesized and radiolabeled with [(99m)Tc(CO)(3)(H(2)0)(3)](+) intermediate in high yield. The complexes were characterized by HPLC and its stability with histidine over time was challenged. Cell uptake and biodistribution studies in melanoma-bearing C57BL/6 mice were performed. Both compounds showed accumulation in tumor tissue with high tumor-to-muscle ratios. Thus, D-glucose- and D-2-deoxyglucose-(99m)Tc complex could be considered as agents for melanoma diagnosis.     

Design of bifunctional radiopharmaceutical for the development of 99mTc complexes for myocardial imaging agents

Several bifunctional radiopharmaceuticals (BR), molecules containing a neutral ^99mTc-dithiosemicarbazone (DTS) structure as a chelating site, along with a functional amino group (primary tertiary and quaternary amino group) are tested for their chemical or biological functionalities as myocardial agents. Investigation of these amino compounds is carried out in vitro and in vivo. Mice (ddY) distribution studies show a high radioactivity distribution in heart with every tested derivative, but the highest heart to blood ratio of 2.92 (1h) is achieved with the quaternary amino DTS. Also, this derivative displays low lung uptake inducing a proper target/non-target ratio for myocardial agent. The role conveyed by the various amino containing side chain in the biodistribution and validity of DTS derivative as BR are discussed.     

Development of 99mTc radiolabeled A85380 derivatives targeting cerebral nicotinic acetylcholine receptor: Novel radiopharmaceutical ligand 99mTc-A-YN-IDA-C4

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been implicated in higher brain functions. To elucidate the functional mechanisms underlying nAChRs and contribute significantly to development of drugs targeting neurological and neuropsychiatric diseases, non-invasive nuclear medical imaging can be used for evaluation. In addition, technetium-99m (^99mTc) is a versatile radionuclide used clinically as a tracer in single-photon emission computed tomography. Because A85380 is known as a potent α4β2-nAChR agonist, we prepared A85380 derivatives labeled with ^99mTc using a bifunctional chelate system. A computational scientific approach was used to design the probe efficiently. We used non-radioactive rhenium (Re) for a ^99mTc analog and found that one of the derivatives, Re-A-YN-IDA-C4, exhibited high binding affinity at α4β2-nAChR in both the docking simulation (?19.3 kcal/mol) and binding assay (Ki = 0.4 ± 0.04 nM). Further, ^99mTc-A-YN-IDA-C4 was synthesized using microwaves, and its properties were examined. Consequently, we found that ^99mTc-A-YN-IDA-C4, with a structure optimized by using computational chemistry techniques, maintained affinity and selectivity for nAChR in vitro and possessed efficient characteristics as a nuclear medicine molecular imaging probe, demonstrated usefulness of computational scientific approach for molecular improvement strategy.     

A click procedure with heterogeneous copper to tether technetium-99m chelating agents and rhenium complexes. Evaluation of the chelating properties and biodistribution of the new radiolabelled glucose conjugates

An efficient protocol was developed to tether chelating agents and rhenium complexes onto a glucoside scaffold with a heterogeneous copper catalyst via click chemistry. The supported catalyst avoids the formation of unwanted copper complexes during the cyclisation step. The possibility to graft a pre-chelated M(CO)₃ core by click chemistry onto a biomolecule was highlighted for the first time. ^99mTc(CO)₃-glucoconjugates displayed excellent in vitro stability, a fast in vivo blood clearance and a low specific organ uptake or long-term retention in spleen and stomach.     

A novel [99mTc[triple bond]N]2+ complex of metronidazole xanthate as a potential agent for targeting hypoxia

A xanthate derivative (L) at the pendant hydroxy group of metronidazole, a nitroimidazole known to possess affinity for hypoxic tumors, has been used as the carrier molecule for targeted delivery of the gamma-emitting radioisotope 99mTc to tumors. The xanthate residues (S2(-)) from two molecules of this ligand (L) were used for chelation with the [99mTcN]2+ intermediate to form a square pyramidal and neutral [99mTcN/L2] complex in >95% yield using a low ligand concentration of 1 mg/mL (approximately 3 x 10(-3) M). Biodistribution studies carried out in Swiss mice bearing fibrosarcoma tumor showed selective accumulation of the injected activity in the tumor (1.44 +/- 0.26% per gram 1 h pi) with major clearance through hepatobiliary route. The complex showed high tumor/muscle ratio (2.15 and 3.35 at 1 and 3 h post-injection, respectively) and tumor/blood ratio, which were comparable to hypoxia targeting agents 99mTc-BMS181321 and 99mTc-BRU59-21 reported earlier.     

A study of the concept of non-radioactive unit-dosed reagent kits [cold unit doses (CUDs)] as an efficient and cost-saving method for 99mTc radiopharmaceutical preparation

Traditionally, when preparing ^99mTc-labeled radiopharmaceuticals, [^99mTc]pertechnetate is added to the entire contents of a vial of reagent kit, and patient doses are subsequently withdrawn from the vial. This technique of compounding can be potentially wasteful for two reasons: (1) once reconstituted with ^99mTc, most reagent kits have a relatively short shelf-life, and thus the entire contents may not be used before expiration and (2) due to a need to conserve radioactivity in many hospitals, enough [^99mTc]pertechnetate is added to the reagent kit in order to retrieve only 1–2 patient doses, even though adequate chemicals (ligand, reducing agent, etc.) are present in the reagent kit to supply as many as 5–10 doses. Hence, a method for optimizing the efficient use of reagent kits would be desirable. The purpose of this study was to determine the feasibility of unit-dosing non-radioactive reagent kits and storing these cold unit doses (CUDs) for eventual labeling with ^99mTc. To evaluate this concept, unit doses were prepared from reagent kits of medronate (MDP) and pentetate (DTPA). The specific variables studied in this research were the effects of storage time, storage temperature and reconstitution volume (dilution) on the unit doses. These effects were monitored by measuring the radiochemical and biodistribution properties of the unit doses following their final reconstitution with [^99mTc]pertechnetate. The labeling efficiency was determined using instant thin layer chromatography (ITLC), and the biodistribution patterns of these radiolabeled CUDs were studied in mice. The results showed that MDP- and DTPA-CUDs stored at −18 °C retained the properties which resulted in acceptable radiochemical purity and biodistribution in mice for as long as 30 days. On the other hand, the radiochemical purity of MDP and DTPA unit doses stored at 25 °C deteriorated rapidly. Mean radiochemical purities as low as 0.58–19.4% were observed on day 30. Altered biodistributions were observed in a manner consistent with the decreased labeling efficiencies. The CUDs of lower dilution (3 mL) appeared to be more stable than the CUDs of higher dilution (10 mL). However, the effect of reconstitution volume was much less significant than the temperature effect on the CUDs. In conclusion, the concept of unit-dosing non-radioactive reagent kits appears to provide an efficient and cost-saving method for preparing infrequent and emergency radiopharmaceutical doses. The study also showed that the storage temperature of these unit doses is critical to the success of the procedure. The volume of reconstitution has a minimal impact on the stability of CUDs if stored at the appropriate temperature.     

Ligand substitution reactions of the [ReX6] (X = Cl, Br) anions. Synthesis and crystal structure of novel oxalato complexes of rhenium(IV)

The reaction of K₂[ReX₆] (X = Cl, Br) with oxalic acid and triethylamine in dimethylformamide solution yields the substituted complexes [ReX₄(ox)]²⁻ and cis-[ReX₂(ox)₂]²⁻, which can be obtained separately depending on the amount of added amine. The crystal structures of (PPh₄)₂[ReBr₄(ox)], cis-(PPh₄)₂[ReBr₂(ox)₂] and cis-(AsPh₄)₂[ReCl₂(ox)₂] have been determined by single-crystal X-ray diffraction. The anionic complexes are octahedral with only slight distortions. The direct isolation of the pure complexes as well as the formation of only the cis isomers - without the presence of trans isomers and/or [Re(ox)₃]²⁻ - is probably due to the kinetic inertness of Re(IV)-X bonds, which increases with the number of oxalato ligands bound to the metal ion.     

Syntheses and properties of organocyanamide, cyanoguanidine and dinitrogen complexes of rhenium. Crystal structure of mer-[ReCl2(NCNEt2) (PMePh2)3]

Treatment of a THF solution of trans-[ReCl(N₂)L₄] (L = PMePh₂) with a cyanamide, NCNR₂ (R = Me, Et or H) or with cyanoguanidine, NCNC(NH₂)₂ , yields mer-[ReCl(N₂)(NCNR₂)L₃] (1) or mer-[Re(N₂)[Re(N₂){NCNC(NH₂)₂}₂L₃]Cl (2), respectively, which, to our knowledge, are the first mixed dinitrogen-cyanamide-type complexes to be reported. The former products (1, R=Me or Et) can also be obtained from reaction of the benzoyldiazenido complex [ReCl₂(NNCOPh)L₃] with NCNR₂ in refluxing methanol; the Re(II) complex mer-[ReCl₂(NCNEt₂)L₃] (3) is also formed (conceivably via an unusual homolysis of the C---N bond of the benzoyldiazenido ligand) and its crystal structure is reported. It shows an unusual pyramidal conformation at the amine N atom of the diethylcyanamide ligand which also exhibits a significant structural trans influence on the phosphine, behaving as a stronger net electron donor than the latter ligand.     

Syntheses, structures, and properties of tricarbonyl rhenium(I) heteronuclear complexes with the multidentate bridging ligand containing bis(2-pyridine) and carboxylic acid

By deprotonation reaction of the rhenium(I) tricarbonyl complex, ClRe(CO)₃(H₂bpydt) (2, H₂dpydt = 2-(di(2-pyridyl)methylene)-1,3-dithiole-4,5-dicarboxylic acid, our previous work in J. Organomet. Chem. 694 (2009) 763), complex 3, [Bu₄N][ClRe(CO)₃(Hbpydt)], is synthesized and characterized. Using 3 as the starting material, two trinuclear heterometallic complexes M(MeOH)₄[ClRe(CO)₃(Hbpydt)]₂·2MeOH (M = Cu, 4; M = Mn, 5) are obtained. The crystal structures of 2-5 have been determined by X-ray crystallography. Complexes 4 and 5 are isostructural. Their absorption and emission properties are studied. The magnetic properties of complexes 4 and 5 have also been investigated.     

Carbamoylphosphate serves as the source of CN(-), but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha

Within the catalytic centre of [NiFe]-hydrogenases one carbonyl and two cyanide ligands are covalently attached to the iron. To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of Ralstonia eutropha H16 were heterologously overproduced in E. coli grown in the presence of L-[ureido-(13)C] citrulline and NaH(13)CO(3). Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO(2) and carbamoylphosphate. Incorporation of label from (13)CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.     

Five-coordinate rhenium(III)-thiolato complexes: the structure of [Re(SCH2PhOCH3)3(PPh3)2]

A novel five-coordinate rhenium(III)-thiolato complex, Re(SCH₂C₆H₄OCH₃-p)₃(PPh₃)₂ has been isolated during the reaction of trans-ReOCl₃(PPh₃)₂ with p-methoxybenzyl mercaptan. In the unexpected structure that was acquired, the central metal has undergone a reduction from Re(V) to Re(III). The five-coordinate Re(III) complex has been characterized by spectroscopic methods, elemental analysis and X-ray crystallography. X-ray crystallographic studies showed the coordination geometry around rhenium to be that of a trigonal bipyramid. The basal plane is defined by three sulfur atoms of the monodentate ligand, while the two apical positions are occupied by two phosphines of the precursor.     

Influence of the molecular charge on the biodistribution of bombesin analogues labeled with the [99mTc(CO)3]-core

The overexpression of Bombesin (BBS) receptors on a variety of human cancers make them interesting targets for tumor imaging and therapy. Analogues of the neuropeptide BBS have been functionalized with the (NalphaHis)- chelator for labeling with the 99mTc-tricarbonyl core. The introduction of a betaAla-betaAla linker between the stabilized BBS binding sequence and the chelator led to increased tumor uptake but still rather unfavorable in ViVo properties. Novel polar linkers, with different charge, have been introduced in the molecule and tested for their influence on the biodistribution. The new analogues showed a shift in hydrophilicity from a Log D=0.9 to Log D values between 0.4 and -2.2. All compounds kept the increased stability in both human plasma (t(1/2)>16 h) and in tumor cells (t(1/2)=30-40 min). The compounds with Log D values between +1 and -1 showed the highest binding affinities with Kd values of <0.5 nM, as well as the highest cellular uptake. However, higher hydrophilicity (Log D < -1.8) led to lower affinity and a substantial decrease of internalization. The introduction of a positive charge (beta3hLys) resulted in unfavorable biodistribution, with increased kidney uptake. The introduction of an uncharged hydroxyl group (beta3hSer) improved the biodistribution, resulting in significantly better tumor-to-tissue ratios. The compound with one single negative charge (beta3hGlu) showed a significant increase in the tumor uptake (2.1+/-0.6% vs 0.80+/-0.35% ID/g in comparison to the betaAla-betaAla analogue) and also significantly higher tumor-to-tissue ratios. The specificity of the in ViVo uptake was confirmed by coinjection with natural BBS. Moreover, the analogue provided a much clearer image of the tumor xenografts in the SPECT/CT studies. The introduction of a single negative charge may be useful in the development of new BBS analogues to obtain an improved biodistribution profile, with increased tumor uptake and better imaging.     

99TcmO4 as an indicator for specialized cell function in organ cultures of the human submaxillary gland

Summary Salivary gland tissue was cultured in vitro and viable cells were present during 14 days. The specific glandular cell function was estimated from the capacity to accumulate ⁹⁹Tc^mO₄ from the culture medium. The morphology of the cultured specimens was examined by light and electron microscopy. Structural changes and loss of the ⁹⁹Tc^mO₄ accumulation capacity increased with increasing culture time in vitro. A correlation was observed between the loss of ⁹⁹Tc^mO₄ accumulation capacity and the disappearance of zymogen granules from the cultured cells. However, the causative link between these two phenomena requires further analysis.Supported by grants from Karolinska Institutet     

A new hexanuclear rhenium cluster complex with six terminal acetate ligands: Synthesis, structure, and properties of K4[Re6S8(CH3COO)6]·8H2O

A room-temperature reaction between [Re₆S₈(OH)₆]⁴⁻ and acetic acid in an aqueous solution resulted in the substitution of all terminal hydroxo groups by acetate ligands, affording a new hexanuclear anionic rhenium cluster complex [Re₆S₈(CH₃COO)₆]⁴⁻. The complex was isolated as a potassium salt with the composition of K₄[Re₆S₈(CH₃COO)₆]·8H₂O (1) and characterized by X-ray single-crystal diffraction and elemental analyses, IR, ¹H NMR, UV-Vis, and luminescence spectroscopies.     

Octahedral rhenium cluster complexes with organic ligands: Synthesis, structure and properties of [Re6Q8(3,5-Me2PzH)6]Br2 · 2(3,5-Me2PzH) (Q = S, Se)

Two new octahedral cluster complexes - [Re₆S₈(3,5-Me₂PzH)₆]Br₂ · 2(3,5-Me₂PzH) (1) and [Re₆Se₈(3,5-Me₂PzH)₆]Br₂ · 2(3,5-Me₂PzH) (2), where 3,5-Me₂PzH is 3,5-dimethylpyrazole, have been synthesized using reaction of rhenium chalcobromide complexes Cs₄[Re₆S₈Br₆] · 2H₂O and Cs₃[Re₆Se₈Br₆] · H₂O, respectively, with molten 3,5-dimethylpyrazole. Both compounds synthesized were characterized by X-ray single-crystal diffraction and chemical analysis, IR and luminescent spectra.     

New copper(II), nickel(II) and zinc(II) complexes with 1,2,4-triazolo[1,5-a]pyrimidines and the chelating ligand 1,3-propanediamine: An unexpected coordination behavior for the 7-amine-derivative

Five new metal complexes with the metal ions Cu(II), Ni(II) and Zn(II) and containing 1,2,4-triazolo[1,5-a]pyrimidine derivatives and 1,3-propanediamine (tn) are described. The structural morphology of these coordination compounds depends on the triazolopyrimidine derivative used, being mononuclear for 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) and 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO), and 1D-polymeric for 7-amine-1,2,4-triazolo[1,5-a]pyrimidine (7atp). In the 7atp case, this ligand does not coordinate through N3 atom, as expected, but through N1, N4 and N71 in a bridging fashion. This unexpected coordination mode seems to be induced by the stability of the polynuclear metal complex in presence of tn ligand. All isolated metal complexes have been characterized by single-crystal X-ray diffraction, IR and UV-Vis spectroscopies, and EPR measurements. Moreover, luminescence measurements have been carried out for 7atp ligand and its polynuclear complex with Zn(II).     

Preparation and biodistribution of a 99mTc tricarbonyl complex with deoxyglucose dithiocarbamate as a tumor imaging agent for SPECT

The deoxyglucose dithiocarbamate (DGDTC) was successfully labeled with the ^99mTc(CO)₃ core to provide the corresponding ^99mTc(CO)₃–DGDTC complex in good yields. The radiochemical purity of the ^99mTc(CO)₃–DGDTC complex was over 90%, as measured by high performance liquid chromatography (HPLC). The complex possessed good stability in saline at room temperature and in mouse plasma at 37 °C. Its partition coefficient result indicated that it was a hydrophilic complex. The electrophoresis results showed the complex was neutral. The biodistribution of ^99mTc(CO)₃–DGDTC in mice bearing S 180 tumor showed that the complex clearly accumulated in tumor, exhibiting high tumor/blood and tumor/muscle ratios and good tumor retention. Single photon emission computed tomography (SPECT) image studies showed there was a visible uptake in tumor sites, suggesting ^99mTc(CO)₃–DGDTC could be considered as a potential tumor imaging agent.     

Synthesis and biological evaluation of tricarbonyl Re(I) and Tc(I) complexes anchored by poly(azolyl)borates: application on the design of radiopharmaceuticals for the targeting of 5-HT<Subscript>1A</Subscript> receptors

The building blocks fac-[^99mTc{κ³-HB(tim^Me)₃}(CO)₃] and fac-[^99mTc{κ³-R(μ-H)B(tim^Me)₂}(CO)₃] [R is H (4a), Ph (5a); tim^Me is 2-mercapto-1-methylimidazolyl] were obtained almost quantitatively by reacting fac-[^99mTc(CO)₃(H₂O)₃]⁺ with the corresponding scorpionate. These compounds cross the intact blood–brain barrier in mice, with significant retention in the case of 4a and 5a. Using 4a as the lead structure, we have synthesized the functionalized complexes fac-[M{κ³-H(μ-H)B(tim^Bu-pip)₂}(CO)₃] [M is Re (8), ^99mTc (8a); tim^Bu-pip is methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-mercapto-1-methylimidazol-5-yl)methanamide] and fac-[M{κ ³-H(μ-H)B(tim^Me)(tim^Bu-pip)}(CO)₃] [M is Re (9), ^99mTc (9a)] and evaluated their potential as radioactive probes for the targeting of brain 5-HT_1A serotonergic receptors. The Re complexes exhibit excellent affinity [IC₅₀=0.172 ± 0.003 nM (8); IC₅₀=0.65 ± 0.01 nM (9)] for the 5-HT_1A receptor. The radioactive congeners (^99mTc) have shown an initial brain uptake of 1.38 ± 0.46%ID g⁻¹ (8a) and 0.43 ± 0.12%ID g⁻¹ (9a), but suffer from a relatively fast washout.