applied sciences

Review A Picture of Modern Tc-99m Radiopharmaceuticals: Production, Chemistry, and Applications in Molecular Imaging

Alessandra Boschi 1 , Licia Uccelli 2,3 and Petra Martini 2,4,*

1 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari, 46, 44121 Ferrara, Italy; [email protected] 2 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via Luigi Borsari, 46, 44121 Ferrara, Italy; [email protected] 3 Nuclear Medicine Unit, University Hospital, Via Aldo Moro, 8, 44124 Ferrara, Italy 4 Legnaro National Laboratories, Italian National Institute for Nuclear Physics (LNL-INFN), Viale dell’Università, 2, 35020 Legnaro (PD), Italy * Correspondence: [email protected]; Tel.: +39-0532-455354



Received: 15 May 2019; Accepted: 19 June 2019; Published: 21 June 2019


Abstract: Even today, techentium-99m represents the radionuclide of choice for diagnostic radio-imaging applications. Its peculiar physical and chemical properties make it particularly suitable for medical imaging. By the use of molecular probes and perfusion radiotracers, it provides rapid and non-invasive evaluation of the function, physiology, and/or pathology of organs. The versatile chemistry of technetium-99m, due to its multi-oxidation states, and, consequently, the ability to produce a variety of complexes with particular desired characteristics, are the major advantages of this medical radionuclide. The advances in technetium coordination chemistry over the last 20 years, in combination with recent advances in detector technologies and reconstruction algorithms, make SPECT’s spatial resolution comparable to that of PET, allowing 99mTc radiopharmaceuticals to have an important role in nuclear medicine and to be particularly suitable for molecular imaging. In this review the most efficient chemical methods, based on the modern concept of the 99mTc-metal fragment approach, applied to the development of technetium-99m radiopharmaceuticals for molecular imaging, are described. A specific paragraph is dedicated to the development of new 99mTc-based radiopharmaceuticals for prostate cancer.

Keywords: technetium-99m; chemistry; radiopharmaceuticals

1. Introduction Molecular Imaging (MI) is a type of medical procedure that provides the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in living systems [1]. Nuclear medicine is a branch of MI that, through the use of radiopharmaceuticals, allows physicians to see and to measure functional, metabolic, chemical, and biological processes within the body and/or to treat malignant tumours, cancer, and other diseases [2–5]. In general, a radiopharmaceutical is a medicinal product that is, usually, administered intravenously to the patient. The radiopharmaceutical is the result of the linkage of two elements, a carrier and at least one radioactive atom that, with its nuclear properties, defines the diagnostic and/or therapeutic nature of the radioactive compound. The carrier plays an important role in the selective transport of the radionuclide to a specific biological target (Figure1).

Appl. Sci. 2019, 9, 2526; doi:10.3390/app9122526 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, x FOR PEER REVIEW 2 of 16

Radioactive atoms decay through a nuclear process that leads to the production of γ-rays used in the diagnostic imaging field. In this case, the radioactivity coming from the radiopharmaceutical, carrying the diagnostic information outward from the body, is detected and transformed in a very precise picture of the radiopharmaceutical’s distribution in the body by means of so-called “gamma cameras” working with sophisticated computerized algorithms. On the contrary, radionuclides decaying through the emission of massive particles, such as electrons or alphas, can be used in nuclearAppl. Sci. medicine2019, 9, 2526 for radionuclide therapy to treat certain types of cancer and other diseases [6–8].2 of 16

Figure 1. Schematic representation of linkage of the carrier and the radioactive atom to form the Figure 1. Schematic representation of linkage of the carrier and the radioactive atom to form the radiopharmaceutical that interacts with a specific biological target. radiopharmaceutical that interacts with a specific biological target. Radioactive atoms decay through a nuclear process that leads to the production of γ-rays used in theIdeally, diagnostic for diagnostic imaging field.nuclear In thismedicine case, theimaging radioactivity applications, coming each from nuclear the radiopharmaceutical, decay should yield acarrying monochromatic the diagnostic gamma-ray information in the energy outward range from 100 the–600 body, keV, is and detected the half-life and transformed of the radionuclide in a very shouldprecise picturebe on ofthe the radiopharmaceutical’sorder of a few hours distribution to avoid in theunnecessary body by means exposure of so-called to radiation. “gamma Radiopharmaceuticalscameras” working with for sophisticatedsingle-photon computerizedcomputed tomography algorithms. (SPECT) On the are contrary,generally radionuclidescomposed of singledecaying small through molecules the emission (the size ofcan massive range from particles, 10−12 such to 10 as−6 m) electrons radiolabelled or alphas, wi canth a begamma-emitting used in nuclear isotope,medicine such for as radionuclide indium-111, therapy iodine-123, to treat gallium-67, certain types or technetium-99m. of cancer and other diseases [6–8]. AdvancedIdeally, for methods diagnostic to nuclearspecifically medicine and selectively imaging applications, incorporate each medical nuclear radionuclides, decay should such yield as a radiometals,monochromatic into gamma-ray targeting invectors, the energy based range on 100–600an improved keV,and understanding the half-life of theof radionuclidetheir coordination should chemistrybe on the orderand ofthe a fewdevelopment hours to avoid of unnecessarycreative labelling exposure strategies, to radiation. are Radiopharmaceuticals the core of modern for radiopharmaceuticalsingle-photon computed research tomography [9–11]. (SPECT) are generally composed of single small molecules (the Among the SPECT12 isotopes6 currently in use, technetium-99m has become the workhorse of size can range from 10− to 10− m) radiolabelled with a gamma-emitting isotope, such as indium-111, diagnosticiodine-123, nuclear gallium-67, medicine or technetium-99m. and technetium-99m-based radiopharmaceuticals are still the most used 99m radioactiveAdvanced chemical methods compounds to specifically in hospitals’ and selectively clinical practice incorporate [12–15]. medical The main radionuclides, reasons for suchTc’s as continuingradiometals, usage into targeting are its ideal vectors, nuclear based onproperti an improvedes and understandingits convenient of supply their coordination method through chemistry a commercialand the development generator system. of creative Techentium-99m labelling strategies, decays arethrough the core the emission of modern of radiopharmaceutical140 keV γ-rays (89% abundance),research [9– 11which]. is ideal for imaging with medical gamma cameras, and can be administered to 99m patientsAmong in low-radiation the SPECT isotopesdoses. Moreover, currently its in 6-h use, half-life technetium-99m is sufficient has for become the preparation the workhorse of Tc of radiopharmaceuticalsdiagnostic nuclear medicine (in hospitals and technetium-99m-basedor centralized radiopharmacies), radiopharmaceuticals their possible aredistribution, still the most the performanceused radioactive of quality chemical controls, compounds administration in hospitals’ to the clinicalpatient, practice accumulation [12–15 in]. Thethe target main organ, reasons and for image99mTc’s acquisition. continuing usageEven areif itsthe ideal use nuclear of radiopha propertiesrmaceuticals and its convenient labelled supplywith positron method throughemitters a radionuclidescommercial generator challenged system. the field Techentium-99m of SPECT tracers, decays over through 70% of the diagnostic emission investigations of 140 keV γ-rays are (89%still performedabundance), with which this issingle ideal isotope for imaging for imaging with medical of bone, gamma renal, cameras,hepatic, andhepatobiliary, can be administered cardiac, and to oncologicalpatients in diseases low-radiation or other doses. pathologies. Moreover, Until its some 6-h half-life years ago, is su theffi cientinferior for sensitivity, the preparation temporal of 99m andTc spatialradiopharmaceuticals resolution of SPECT (in hospitals cameras or compared centralized to radiopharmacies), positron emission their tomography possible distribution,(PET) cameras, the togetherperformance with ofthe quality complex controls, and contrived administration inorganic to the chemistry patient, accumulation of this metal, in were thetarget considered organ, key and image acquisition. Even if the use of radiopharmaceuticals labelled with positron emitters radionuclides challenged the field of SPECT tracers, over 70% of diagnostic investigations are still performed with this single isotope for imaging of bone, renal, hepatic, hepatobiliary, cardiac, and oncological diseases or other pathologies. Until some years ago, the inferior sensitivity, temporal and spatial resolution of SPECT cameras compared to positron emission tomography (PET) cameras, together with the complex and contrived inorganic chemistry of this metal, were considered key problems in the development of new technetium-99m compounds for the imaging of more specific molecular targets [16]. Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 16 Appl. Sci. 2019, 9, 2526 3 of 16 problems in the development of new technetium-99m compounds for the imaging of more specific molecular targets [16]. Advances inin technetiumtechnetium chemistry chemistry over over the the last last 20 20 years years have have facilitated facilitated the developmentthe development of new of technetiumnew technetium radiopharmaceuticals radiopharmaceuticals with great with potential great pote inntial clinical in practice.clinical practice. In addition, In addition, recent advances recent inadvances detector in technologies detector technologies and reconstruction and reconstruc algorithmstion clearlyalgorithms showed clearly that showed the spatial that resolution the spatial od SPECTresolution is approaching od SPECT is that approaching of PET without that of a concomitantPET without decrease a concomitant in sensitivity. decrease In particular,in sensitivity. novel In multi-pinholeparticular, novel collimators, multi-pinhole coupled collimators, with cadmium coupled zinc with telluride cadmium (CZT) zinc solid-state telluride photon (CZT) detectors solid-state to furtherphoton improvedetectors image to further quality improve and reduce image scannerquality and size, reduce reduce scanner the imaging size, reduce time and the radiation imaging dose.time Theand newradiation SPECT dose. scanners The new have SPECT demonstrated scanners up have to a 7-folddemonstrated increase inup photon to a 7-fold sensitivity increase and in an photon up to 2-foldsensitivity improvement and an up in to image 2-fold resolution. improvement Furthermore, in image theresolution. adoption Furthermore, of CZT involves the aadoption superior of energy CZT resolutioninvolves a comparedsuperior energy to a conventional resolution comp gammaared camera to a conventional [17]. gamma camera [17]. The aboveabove considerationsconsiderations lead lead us us to to believe believe that that99m Tc-radiopharmaceuticals99mTc-radiopharmaceuticals will will continue continue to play to anplay important an important role in role nuclear in nuclear medicine medicine and MI. and The MI. versatile The versatile chemistry chemistry of technetium-99m of technetium-99m is a major is a advantagemajor advantage in radiopharmaceutical in radiopharmaceutical development. development. The aim of this review is to describe and report the most eefficientfficient labelling methods to produce modern technetium-99mtechnetium-99m radiopharmaceuticals radiopharmaceuticals for for nuclear nuclear medicine. medicine Highly. Highly specific specific activities activities needed forneeded targeting for targeting low-concentration low-concentration targets are targets easily achievableare easily achievable through advanced through chemical advanced procedures chemical involvingprocedures the involving99mTc-metal the 99m fragmentTc-metal approach.fragment approach. A section A is section dedicated is dedicated to the development to the development of new 99mof newTc-based 99mTc-based radiopharmaceuticals radiopharmaceutica for prostatels for prostate cancer. cancer.

2. Techentium-99m Production RoutesRoutes Techentium-99m is easily produced by the decaydecay ofof thethe parentparent molybdenum-99molybdenum-99 by elutingeluting 99 99m compact and transportabletransportable 99Mo/Mo/99mTcTc generator generator systems systems that that are are almost always available in nuclear medicine departments. The generator system is a simplesimple apparatus composed of an alumina column 99 99 2 99 on which the 99 Mo is absorbed in the chemical form of molybdate99 [ Mo]MoO2− . The99 Mo decay on which the Mo is absorbed in the chemical form of molybdate [ Mo]MoO4 .4 The− Mo decay into 99m 99m into99m Tc leads to the formation of pertechnetate99m [ Tc]TcO− , which is less tightly bound to the Tc leads to the formation of pertechnetate [ Tc]TcO4 , which4− is less tightly bound to the alumina aluminacolumn because column of because its single of negative its single charge negative compared charge comparedto the double to thenegative double charge negative of molybdate. charge of 99m molybdate.99m [ Tc]NaTcO can be easily eluted from the column with a saline solution thanks to the [ Tc]NaTcO4 can be easily4 eluted from the column with a saline solution thanks to the depression depressioncaused by an caused under-vacuum by an under-vacuum vial appositely vial insert appositelyed in insertedthe pertechnetate in the pertechnetate collecting area collecting (Figure area 2). 99m (FigureThe obtained2). The sodium obtained 99m sodiumTc-pertechnetateTc-pertechnetate is ready for is ready injection for injectionor for the or forpreparation the preparation of other of otherradiopharmaceuticals radiopharmaceuticals [18,19]. [18 ,19].

Figure 2. Schematic representation of the 99Mo/99mTc generator system. Figure 2. Schematic representation of the 99Mo/99mTc generator system. The worldwide demand for molydbenum-99 is satisfied by nuclear reactors via the 235U(n,f)99Mo fissionThe route worldwide on Highly demand Enriched for Uraniummolydbenum-99 targets (HEU,is satisfied enrichment by nuclear over reactors 80% in via235U). the Alternatively,235U(n,f)99Mo molibdenum-99fission route on Highly can be Enriched produced Uranium by bombarding targets (HEU, molybdenum-98 enrichment withover high-intensity80% in 235U). Alternatively, neutrons to generatemolibdenum-99 sufficient can amounts be produced of molybdenum-99 by bombarding [20 ].molybdenum-98 with high-intensity neutrons to generateNuclear sufficient reactors amounts involved of molybdenum-99 in the supply of [20]. this isotope are listed in Table1. In the last decade, theyNuclear have su ffreactorsered planned involved and in unplannedthe supply shutdownsof this isotope for are maintenance listed in Table or breakdowns, 1. In the last causing,decade, firstthey inhave 2009 suffered and then planned in 2012 and and unplanned 2013, a shortageshutdown ins Mo-99for maintenance production or andbreakdowns, therefore causing, in Tc-99m’s first availabilityin 2009 and for then clinical in 2012 studies. and Recently,2013, a shortage significant in interruptionsMo-99 production in the and supply therefore of technetium-99m in Tc-99m’s Appl. Sci. 2019, 9, 2526 4 of 16 occurred in 2016 and 2018, the years when the French OSIRIS and Canadian NRU reactors, respectively, stopped operating.

Table 1. Nuclear reactors involved in 99Mo supply for generator medical devices.

Reactors Countries Six-Day GBq/Mo-99 GBq/year Start of Service End of Service LVR-15 Czech Republic 3330000 1957 2028 MARIA Poland 3515000 1974 2030 OPAL Australia 3420650 2007 2055 HFR The Netherlands 8946600 1961 2024 BR2 Belgium 6060600 1961 >2026 RA-3 Argentina 680800 1967 2027 SAFARI-1 South Africa 4835900 1965 2030

In addition to the reactors reported in Table1, smaller amounts of molybdenum-99 are produced in the RBT-6 and RBT-10 reactors in Russia: irradiating HEU targets have a production capacity of 37,000 GBq/week, and WWR-c has an available production capacity of 12,950 GBq/week. All Mo-99 suppliers except those in Russia produce molybdenum-99 using LEU targets or are in the final stages of converting production from HEU to LEU (Low Enriched Uranium, enrichment below 20% in 235U) targets [21]. The supply of molybdenum-99 and technetium-99m is particularly sensitive to reactor shutdowns because, after production, they cannot be stored for future needs due to their short half-lives. Several alternative production routes have been evaluated and some of them have become reliable thanks to the worldwide research efforts of the last 10 years [22–25]. Alternative strategies for the production of molybdenum-99 include the use of linear accelerators, where a molybdenum-100 source is irradiated with gamma rays, as the 100Mo(γ,n)99Mo reaction [26]. The direct production of 99mTc on cyclotrons, involving the proton bombardment of a solid 100Mo source in the 100Mo(p,2n)99mTc reaction, is now a reality [23,27,28]. A list of various cyclotron and non-cyclotron production routes is given in Table2.

Table 2. Tc-99m alternative production routes.

Reactor Based Accelerator Based LEU-235U(n,f)99Mo 238U(γ,f)99Mo nat,98Mo(n,γ)99Mo 96Zr(α,n)99Mo 100Mo(γ,n)99Mo 100Mo(p,pn)99Mo 100Mo(p,2n)99mTc

3. Chemical Approaches to Bioactive Molecule Labelling with Technetium-99m In the last decay, the labelling of targeting vectors has become the main object of modern radiopharmaceutical research. Technetium is a transition metal and, as such, presents a major disadvantage with respect to other radionuclides in combining with biologically active molecules. For example, 99mTc cannot be a substitute for a carbon or a hydrogen atom in a targeting molecule, as happens for the labelling with carbon-11, or fluorine-18 and iodine-123 respectively. The development of technetium imaging agents requires both a familiarity with the coordination chemistry of the group 7 metals and an understanding of the design of suitable ligands that provide robust molecular imaging probes. Deep knowledge of inorganic chemistry allows for developing convenient approaches to introduce stable technetium-99m into a bioactive molecule with the aim of not affecting its bioactivity. A number of inorganic technetium functional groups, also called “cores” or “metal fragments,” have been identified so far (Figure3). These groups are chemical motifs comprising a characteristic arrangement of atoms bonded to the metallic centre and determining the formation of a variety of coordination complexes and molecular geometries. They can be prepared in physiological solution, have technetium Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 16 Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 16 For example, 99mTc cannot be a substitute for a carbon or a hydrogen atom in a targeting molecule, as Forhappens example, for 99mtheTc cannotlabelling be awith substitute carbon-11, for a carbon or fluorine-18 or a hydrogen and atom iodine-123 in a targeting respectively. molecule, The as happensdevelopment for theof technetiumlabelling withimaging carbon-11, agents requiror fluorine-18es both a andfamiliarity iodine-123 with respectively.the coordination The developmentchemistry of theof grouptechnetium 7 metals imaging and an agentsunderstanding requires of both the designa familiarity of suitable with ligands the coordination that provide chemistryrobust molecular of the group imaging 7 metals probes. and Deep an understanding knowledge of of inorganic the design chemistry of suitable allows ligands for that developing provide robustconvenient molecular approaches imaging to introduce probes. Deep stable knowledg technetium-99me of inorganic into a bioactive chemistry molecule allows forwith developing the aim of convenientnot affecting approaches its bioactivity. to introduce A number stable of inorganic technetium-99m technetium into functional a bioactive groups, molecule also with called the “cores” aim of notor “metalaffecting fragments,” its bioactivity. have A beennumber identified of inorganic so fa technetiumr (Figure 3). functional These groups groups, are also chemical called “cores” motifs orcomprising “metal fragments,” a characteristic have arrangement been identified of atoms so fa bondedr (Figure to the3). metallicThese groups centre areand chemicaldetermining motifs the Appl.comprisingformation Sci. 2019 of ,a9 ,acharacteristic 2526 variety of coordination arrangement complexes of atoms and bonded molecular to the geometries.metallic centre They and can determining be prepared5 ofthe 16in formationphysiological of a solution,variety of have coordination technetium complexes in a reduc anded molecular oxidation geometries. state, and haveThey labilecan be coordination prepared in physiologicalpositions, which solution, can be have conveniently technetium exploited in a reduc toed introduce oxidation the state, selected and have bioactive labile molecule.coordination As in a reduced oxidation state, and have labile coordination positions, which can be conveniently positions,previously which described, can be technetium-99m, conveniently exploited whether to produced introduce bythe a selected 99Mo/99m bioactiveTc generator molecule. or by As a exploited to introduce the selected bioactive molecule. As previously described, technetium-99m, previouslycyclotron, is described, obtained as technetium-99m, [99mTc]NaTcO4 in whethera physiological produced solution. by aConsequently, 99Mo/99mTc generator in order to or link by the a 99 99m 99m whether produced by a Mo/ Tc generator or by a cyclotron, is obtained as [ Tc]NaTcO4 in a cyclotron,radionuclide is obtained to a bioactive as [99m molecule,Tc]NaTcO the4 in Tc(VII)a physiological metal must solution. be reduced Consequently, to a suitable in order oxidation to link state the physiological solution. Consequently, in order to link the radionuclide to a bioactive molecule, the radionuclidein the presence to ofa bioactive appropriate molecule, ligands. the Tc(VII) metal must be reduced to a suitable oxidation state Tc(VII)in the presence metal must of appropriate be reduced ligands. to a suitable oxidation state in the presence of appropriate ligands. R N O N R N O N OC Tc N Tc N OC Tc Tc Tc Tc Tc Tc OC O OC CO O CO Tc-Oxo Tc-Nitrido Tc-HYNIC fac-Tc-tris-Carbonyl Tc-Oxo Tc-Nitrido Tc-HYNIC fac-Tc-tris-Carbonyl Figure 3. Inorganic technetium functional groups useful for labelling bioactive molecules. HYNIC =6- Figurehydrazinonicotinamide. 3. 3. InorganicInorganic technetium technetium functional functional groups groups useful for useful labelling for bioactive labelling molecules. bioactive HYNIC molecules.=6- HYNIChydrazinonicotinamide.=6-hydrazinonicotinamide. The metal fragments reported in Figure 3, in combination with an appropriate set of coordinatingThe metalmetal atoms, fragments fragments provide reported reported an effective in Figurein Figurestrategy3, in combination 3, for in tethering combination with a anbiologically appropriatewith an activeappropriate set of moiety coordinating withset of a atoms,coordinatingtechnetium-99m provide atoms, an complex. eff ectiveprovide The strategy an metal effective for fragment tethering strategy strategy a biologicallyfor tethering mainly active ainvolves biologically moiety a two-component with active a technetium-99m moiety systemwith a complex.technetium-99mcomposed The of (1) metal complex.the fragmentradioactive The strategy metal metal fragment mainly fragment involves strategy and (2) a two-componentmainlyan appropriate involves chelating system a two-component composed group eventually of system (1) the radioactivecomposedbound to the of metal (1)selected the fragment radioactive bioactive and molecule (2)metal an appropriatefragment through an a chelatingd spacer (2) an group. appropriate group The eventually high chelating affinity bound group of to the the eventually precursor selected bioactiveboundmetal fragment to moleculethe selected for throughthe bioactive specific a spacer bindingmolecule group. sites through The on the high a bioactivespacer affinity group. ofligand the The precursor allows high for affinity metal obtaining fragmentof the a precursorconjugate for the specificmetalcomplex fragment binding resulting for sites from the on specific the fitting bioactive binding of the ligand sites two allowson carefully the bioactive for selected obtaining ligand molecular a conjugate allows building for complex obtaining blocks resulting a (Figureconjugate from 4). thecomplex fitting resulting of the two from carefully the fitting selected of the molecular two carefully building selected blocks molecular (Figure4 ).building blocks (Figure 4).

Figure 4. Schematic representation of the metal fragment strategy for bioactivebioactive moleculemolecule labelling.labelling . Figure 4. Schematic representation of the metal fragment strategy for bioactive molecule labelling. InIn thethe followingfollowing section,section, descriptionsdescriptions ofof complexescomplexes containingcontaining thethe mostmost importantimportant technetiumtechnetium 99m functionalfunctionalIn the groups followinggroups are given.aresection, given. Some descriptions ofSome these complexes of complexesthese arecomplexes also containing examples are the of mostalsoTc-radiopharmaceuticals importantexamples technetiumof 99mTc- thatfunctionalradiopharmaceuticals have been groups employed are that in given.have clinical been studiesSome employed orof are these currentlyin clinical complexes used studies as diagnostic orare are currentlyalso agents examples used in clinical as diagnosticof practice.99mTc- radiopharmaceuticalsagents in clinical practice. that have been employed in clinical studies or are currently used as diagnostic 3.1. The 99mTc(V)-Oxo Core agents in clinical practice. 3.1. TheThe 99m99mTc(V)-oxoTc(V)-oxo core core represents the most extensively studied metal fragment. Complexes based 3.1. The 99mTc(V)-oxo core on thisThe core 99mTc(V)-oxo are generally core pentacoordinated, represents the most adopting extensiv squareely studied pyramidal metal geometry fragment. with Complexes the π-bonding based oxo-groupon thisThe core99m inTc(V)-oxo theare apicalgenerally core position. represents pentacoordinated, Due the to themost strong extensiv adoptingtranselyinfluence squarestudied pyramidalmetal of the fragment. oxo-group, geometry Complexes six coordinatedwith thebased π- technetium-oxoonbonding this core oxo-group are compounds generally in the apical arepentacoordinated, relatively position. uncommon. Due adopting to the The strong square oxo-core trans pyramidal is influence stabilized geometry of by theσ- andoxo-group, withπ-donating the sixπ- atomsbonding coming oxo-group from in amino, the apical amido, position. and thiolate Due to ligands, the strong as well trans as influence the tetradentate of the oxo-group, ligands of thesix N4-xSx class [29–32]. These ligands are extremely efficient at forming more stable 99mTc-oxo complexes compared with those derived from bidentate ligands. These compounds can be prepared through the direct reduction of the 99mTc-pertechnetate anion in the presence of tin chloride, which is the ubiquitous preliminary step in the preparation of conventional 99mTc-radiopharmaceuticals, or via a ligand exchange reaction with [99mTc]Tc-glucoheptonate (Figure5). Appl. Sci. 2019, 9, x FOR PEER REVIEW 6 of 16

coordinated technetium‒oxo compounds are relatively uncommon. The oxo-core is stabilized by σ- and π-donating atoms coming from amino, amido, and thiolate ligands, as well as the tetradentate ligands of the N4-xSx class [29–32]. These ligands are extremely efficient at forming more stable 99mTc-oxo complexes compared with those derived from bidentate ligands. These compounds can be prepared through the direct reduction of the 99mTc-pertechnetate anion in the presence of tin chloride, which is the ubiquitous 99m Appl.preliminary Sci. 2019, 9, 2526 step in the preparation of conventional Tc-radiopharmaceuticals, or via a6 ofligand 16 exchange reaction with [99mTc]Tc-glucoheptonate (Figure 5).

O O + Tc +3H O Tc O OH O OH OH

+Sn2+ l,l-ECD

O OH O O NH N O O + Tc Sn S S HO OH

l,l-[99mTc]Tc-ECD

Figure 5. Reaction scheme of the preparation of the technetium-99m N,N-1,2-ethylene diylbis-L-cysteine diethylFigure ester 5. dihydrochlorideReaction scheme from of the99m Tc-pertechnetate.preparation of the technetium-99m N,N-1,2-ethylene diylbis-L- cysteine diethyl ester dihydrochloride from 99mTc-pertechnetate. A representative example is provided by the complex technetium-99m N,N-1,2-ethylene diylbis-L-cysteineA representative diethyl example ester dihydrochloride is provided by (thel,l-[ 99mcomplexTc]Tc-ECD), technetium-99m a brain perfusion N,N-1,2-ethylene imaging agent diylbis- onL-cysteine the market diethyl with the ester name dihydrochloride Neurolite®. ( Thel,l-[99m complexTc]Tc-ECD), is neutral a brain and perfusion formed byimaging multiple agent Tc onO the ≡ bondsmarket coordinated with the byname a tetradentate Neurolite® ligand. The complex with two is nitrogen neutral andand twoformed sulphur by multiple donor atoms Tc≡O inbonds a squarecoordinated/pyramidal by arrangement. a tetradentate The ligand high symmetry with two makes nitrogen the complexand two hydrophobic. sulphur donor Because atoms of itsin a lipophilicity,square/pyramidal it can cross arrangement. the blood-brain The barrierhigh symmetry (BBB). The makes mechanism the complex of brain hydrophobic. localization isBecause based onof its thelipophilicity, transformation it can by cross esterase the enzymesblood‒brain of one barrier of the (BBB). lateral The ester mechanism groups (–COOEt)of brain localiza in a carboxyliction is based groupon the (–COOH), transformation with the by subsequent esterase enzymes formation of ofone a negativelyof the lateral charged ester groups more hydrophilic (⎯COOEt) in compound a carboxylic thatgroup is unable (⎯COOH), to cross thewith BBB the and subsequent thus is trapped. formation It is important of a negatively to note that charged only 99m moreTc-ECD hydrophilic having a l,lcompoundconfiguration that undergoes is unable to ester cross hydrolysis, the BBB and making thus thisis trapped. compound It is important a true metabolic to note marker that only of the99mTc- brain’sECD esterase having enzymes.a l,l configuration undergoes ester hydrolysis, making this compound a true metabolic markerMixed of aminothiol-based the brain’s esterase chelators, enzymes. most notably N2S2 [e.g., bis(aminoethanethiol) (BAT)] and N3S [e.g.,Mixed mercaptoacetylglycylglycylglycine aminothiol-based chelators, most (MAG notably3)], have N2S been2 [e.g., used bis(aminoethanethiol) to label a selected bioactive(BAT)] and moleculeN3S [e.g., with mercaptoacetylglycylglycylglycine technetium-99m [33–37]. These tetradentate(MAG3)], have chelating been used systems to label allow a selected us to produce bioactive moremolecule stable Tc(V)–oxo with technetium-99m complexes in comparison[33–37]. These with tetradentate those produced chelating with systems similar bidentate allow us chelators. to produce moreRepresentative stable Tc(V)–oxo examples complexes from this in class comparison of compounds with arethose99m producedTc-TRODAT-1, with asimilar probe forbidentate the diagnosischelators. of Parkinson’s disease, and 99mTc-Depreotide (P829), a Tc(V)-oxo somatostatin analogue complexRepresentative [38–42]. TRODAT-l examples (Figure from6) isthis currently class of considered compounds a usefulare 99mTc-TRODAT-1, imaging agent fora probe the early for the detectiondiagnosis of Parkinson’s of Parkinson’s disease, disease, where and a phenyltropanepharmacophore99mTc-Depreotide (P829), a Tc(V)-oxo is used assomatostatin a targeting vectoranalogue forcomplex specific monoamine[38–42]. TRODAT-l transporter (Figure proteins, 6) is currently and the N considered2S2 chelate system,a useful connectedimaging agent to the for tropane the early molecule,detection has of been Parkinson’s introduced disease, to coordinate where a thephenyltrop technetium-oxoanepharmacophore core without is a usedffecting as a the targeting affinity vector for thefor biological specific target. monoamine transporter proteins, and the N2S2 chelate system, connected to the tropane Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 of 16 molecule, has been introduced to coordinate the technetium‒oxo core without affecting the affinity H N for the biological target.O NH 2 S S 2 Tc Phe(Me-N) Tyr D-Trp N N N O O H2N O O N N N Hcy H Tc Val Lys O H Cl S N N H2

99m 99m Tc-TRODAT-1 Tc-P829 Figure 6. ChemicalChemical structure structure of the of 99m theTc-TRODAT-199mTc-TRODAT-1 and 99m andTc-Depreotide99mTc-Depreotide (P829) (P829)radiopharmaceuticals. radiopharmaceuticals.

3+ Likewise, a a tripeptide tripeptide chain chain provides provides a N a3S N 3bindingS binding group group for (Tc for≡O) (Tc3+ O)core incore the in99m theTc- 99m ≡ DepreotideTc-Depreotide radiopharmaceutical radiopharmaceutical (Figure (Figure6) and 6the) and bioactive the bioactive part of the part original of the octreotide original octreotide structure structurehas been modified has been modifiedto eliminate to eliminatethe disulphide the disulphide bridge, which bridge, would which be reduced would be during reduced the during formation the 3+ 99m formation of the3+ (Tc O) core from the generator-eluted99m Tc-pertechnetate. of the (Tc≡O) core ≡from the generator-eluted Tc-pertechnetate.

3.2. The 99mTc(V)- Hydrazido Metal Fragment Metalorganohydrazine chemistry is an alternative approach to the design of radiopharmaceuticals based on a stable and substitution inert 99mTc-metal fragment [43]. 99mTc- hydrazido metal fragment is formed by a technetium‒HYNIC (HYNIC = 6-hydrazinonicotinamide) core composed of the metal in the V oxidation state and occupying the ligand one or two coordination sites. HYNIC ligands are particularly convenient for radiolabelling biomolecules, with 99mTc being the active ester most readily combined with small molecules, proteins, or different targeting vectors. However, even if the metal‒organohydrazino unit can be easily prepared from the metal‒oxo core by a simple condensation reaction, the intimate details of the chemistry are more complex and dependent upon reaction conditions and the presence of co-ligands, which can influence the stability, hydrophilicity, and pharmacokinetics of the resulting 99mTc-compounds [44]. A large number of co-ligands have been tested, including ethylenediaminediacetic acid (EDDA), tricine, glucoheptonate, mannitol, thiolates, glucamine, and phosphines forming different binary and ternary systems [45]. The EDDA co-ligand is in general preferred to tricine when preparing 99mTc‒ HYNIC complexes with higher stability in vivo and fewer isomeric forms. Unfortunately, while many efforts have been made to completely establish the identities of these complexes, the chemistry remains unclear. The dependence of the coordination chemistry on the reaction condition of this technology has led to a certain difficulty in the development of 99mTc-HYNIC radiopharmaceuticals; moreover, the increasing regulators’ requirements of providing fully characterized products does not seem to have promoted HYNIC inclusion in clinical practice. Nevertheless, a wide range of bioactive molecules have been labelled with the HYNIC strategy, among which the most investigated are undoubtedly the somatostatin derivatives for neuroendocrine tumour (NETs) imaging. Most published data concern the commercially available 99mTc‒[HYNIC, Tyr(3)]octreotide (99mTc‒HYNIC‒ TOC, Figure 7) prepared through a two-vial kit formulation containing EDDA as a co-ligand [46–48]. Appl. Sci. 2019, 9, 2526 7 of 16

3.2. The 99mTc(V)- Hydrazido Metal Fragment Metalorganohydrazine chemistry is an alternative approach to the design of radiopharmaceuticals based on a stable and substitution inert 99mTc-metal fragment [43]. 99mTc- hydrazido metal fragment is formed by a technetium-HYNIC (HYNIC = 6-hydrazinonicotinamide) core composed of the metal in the V oxidation state and occupying the ligand one or two coordination sites. HYNIC ligands are particularly convenient for radiolabelling biomolecules, with 99mTc being the active ester most readily combined with small molecules, proteins, or different targeting vectors. However, even if the metal-organohydrazino unit can be easily prepared from the metal-oxo core by a simple condensation reaction, the intimate details of the chemistry are more complex and dependent upon reaction conditions and the presence of co-ligands, which can influence the stability, hydrophilicity, and pharmacokinetics of the resulting 99mTc-compounds [44]. A large number of co-ligands have been tested, including ethylenediaminediacetic acid (EDDA), tricine, glucoheptonate, mannitol, thiolates, glucamine, and phosphines forming different binary and ternary systems [45]. The EDDA co-ligand is in general preferred to tricine when preparing 99mTc-HYNIC complexes with higher stability in vivo and fewer isomeric forms. Unfortunately, while many efforts have been made to completely establish the identities of these complexes, the chemistry remains unclear. The dependence of the coordination chemistry on the reaction condition of this technology has led to a certain difficulty in the development of 99mTc-HYNIC radiopharmaceuticals; moreover, the increasing regulators’ requirements of providing fully characterized products does not seem to have promoted HYNIC inclusion in clinical practice. Nevertheless, a wide range of bioactive molecules have been labelled with the HYNIC strategy, among which the most investigated are undoubtedly the somatostatin derivatives for neuroendocrine tumour (NETs) imaging. Most published data concern the commercially available 99mTc-[HYNIC, Tyr(3)]octreotide (99mTc-HYNIC-TOC, Figure7) preparedAppl. Sci. 2019 through, 9, x FOR a PEER two-vial REVIEW kit formulation containing EDDA as a co-ligand [46–48]. 8 of 16

Figure 7. The figure illustrates the chemical structure of the complex 99mTc-HYNIC-TOC, an imaging Figure 7. The figure illustrates the chemical structure of the complex 99mTc‒HYNIC‒TOC, an imaging agent for neuroendocrine tumours. agent for neuroendocrine tumours. The complex is formed by a technetium atom bound to a ligand composed of two functional The complex is formed by a technetium atom bound to a ligand composed of two functional parts, and a HYNIC group linked to an octapeptide. The EDDA ancillary ligand is required to parts, and a HYNIC group linked to an octapeptide. The EDDA ancillary ligand is required to stabilize the coordination geometry. 99mTc-HYNIC-TOC represents a selective receptor imaging agent stabilize the coordination geometry. 99mTc‒HYNIC‒TOC represents a selective receptor imaging for neuroendocrine tumours and, even if the true molecular structure of 99mTc-HYNIC-TOC is not agent for neuroendocrine tumours and, even if the true molecular structure of 99mTc‒HYNIC‒TOC is exactly determined, its biological properties can be understood by considering the bioactive group not exactly determined, its biological properties can be understood by considering the bioactive group octreotide and selectively targeting the somatostatin receptors (SSR) overexpressed on the membranes of neuroendocrine tumours. This tracer showed greater sensitivity compared to 111In-octreotide, such as specific and high receptor affinity, good biodistribution, faster renal excretion, lower radiation exposure, and high imaging quality as well as the superior capability of 99mTc‒TOC to visualize extrahepatic lesions [49].

3.3. The [99mTc(N)PNP]2+ metal fragment

The nitride technetium mixed ligand complexes [99mTc][Tc(N)(PNP)Cl2] provide a further example of the metal fragment approach, where the Tc(N) group is coordinated to a chelating diphosphine ligand of the PNP type and the pentacoordinated geometry is saturated by two chloride atoms. These atoms can be easy replaced by bidentate ligands (Y~Z) having in their molecular structure electron-rich coordinating atoms (Y, Z) highly reactive towards the electrophilic [99mTc(N)(PNP)]2+ metallic block to afford the asymmetrical complexes [99mTc(N)(PNP)(Y~Z)]0/+ [50,51]. From this class of asymmetrical complexes, a novel class of myocardial tracers exhibiting superior qualities was obtained, among which 99mTc-N-DBODC and 99mTc-N-MPO are the most representative (Figure 8) [52,53].

O O O

N O P S Tc N N P S P S Tc O P O N N O O O N O O O 99m TcN-DBODC 99mTcN-MPO Figure 8. Chemical structure of two not-commercial imaging agents for the myocardial perfusion, 99mTc‒N‒DBODC and 99mTcN‒MPO . Appl. Sci. 2019, 9, x FOR PEER REVIEW 8 of 16

Figure 7. The figure illustrates the chemical structure of the complex 99mTc‒HYNIC‒TOC, an imaging agent for neuroendocrine tumours.

The complex is formed by a technetium atom bound to a ligand composed of two functional parts, and a HYNIC group linked to an octapeptide. The EDDA ancillary ligand is required to stabilize the coordination geometry. 99mTc‒HYNIC‒TOC represents a selective receptor imaging 99m Appl.agent Sci. for2019 neuroendocrine, 9, 2526 tumours and, even if the true molecular structure of Tc‒HYNIC‒TOC8 of 16is not exactly determined, its biological properties can be understood by considering the bioactive group octreotide and selectively targeting the somatostatin receptors (SSR) overexpressed on the octreotidemembranes and of selectivelyneuroendocrine targeting tumours. the somatostatin receptors (SSR) overexpressed on the membranes of neuroendocrineThis tracer showed tumours. greater sensitivity compared to 111In-octreotide, such as specific and high 111 receptorThis affinity, tracer showed good biodistribution, greater sensitivity faster compared renal excretion, to In-octreotide, lower radiation such asexposure, specific and high receptorimaging quality affinity, as good well biodistribution,as the superior capability faster renal of 99m excretion,Tc‒TOC lowerto visualize radiation extrahepatic exposure, lesions and high[49]. imaging quality as well as the superior capability of 99mTc-TOC to visualize extrahepatic lesions [49]. 3.3. The [99mTc(N)PNP]2+ metal fragment 3.3. The [99mTc(N)PNP]2+ Metal Fragment The nitride technetium mixed ligand complexes [99mTc][Tc(N)(PNP)Cl2] provide a further 99m exampleThe nitrideof the technetiummetal fragment mixed approach, ligand complexes where [the Tc][Tc(N)(PNP)ClTc(N) group is 2coordinated] provide a further to a chelating example ofdiphosphine the metal fragmentligand of the approach, PNP type where and the pentacoordinated Tc(N) group is coordinated geometry is to saturated a chelating by diphosphinetwo chloride ligandatoms. ofThese the PNP atoms type can and be the easy pentacoordinated replaced by bidentate geometry ligands is saturated (Y~Z) by having two chloride in their atoms. molecular These atomsstructure can beelectron-rich easy replaced coordinating by bidentate ligandsatoms (Y~Z)(Y, Z) having highly in theirreactive molecular towards structure the electron-richelectrophilic 99m 2+ coordinating[99mTc(N)(PNP)] atoms2+ metallic (Y, Z) highly block reactive to afford towards the theasymmetrical electrophilic complexes [ Tc(N)(PNP)] [99mTc(N)(PNP)(Y~Z)]metallic block to0/+ 99m 0/+ a[50,51].fford the From asymmetrical this class complexesof asymmetrical [ Tc(N)(PNP)(Y~Z)] complexes, a novel[ 50class,51]. of From myocardial this class tracers of asymmetrical exhibiting complexes,superior qualities a novel was class obtained, of myocardial among tracers which exhibiting 99mTc-N-DBODC superior and qualities 99mTc-N-MPO was obtained, are the among most 99m 99m whichrepresentativeTc-N-DBODC (Figure 8) and [52,53].Tc-N-MPO are the most representative (Figure8)[52,53].

O O O

N O P S Tc N N P S P S Tc O P O N N O O O N O O O 99m TcN-DBODC 99mTcN-MPO Figure 8. Chemical structure of two not-commercial imag imaginging agents for the myocardial perfusion, 99mTcTc-N-DBODC‒N‒DBODC andand 99m99mTcNTcN-MPO.‒MPO .

The first compound is prepared through a two-vial kit formulation following a two-step preparation 99m procedure. To the first vial, the generator eluted Tc-pertechnetate is added in the presence of SnCl2 and succinic dihydrazide (SDH), required to form the Tc N group. The second vial contains the ≡ sodium salt of DBODC (bis-N-ethoxyethyl-dithiocarbamato), the bis(dimethoxypropylphosphinoethyl)- ethoxyethylamine [PNP5; (CH3OC3H6)2P(CH2)2-N(C2H4-OCH2CH3)-(CH2)2P(C3H6OCH3)2], and γ-cyclodextrin, used as a solubilizing agent for the diphosphine ligand. After the reconstitution of the contents of the second lyophilized vial with saline, a part of the resulting solution is added to the 99m first vial and heated at 100 ◦C for 15 min to obtain the final Tc-N-DBODC compound with yield >95%. A similar procedure is applied to the preparation of the cationic nitride complex Tc-N-MPO ([(99mTc-N(mpo)(PNP5)]+: mpo = 2-mercaptopyridine oxide). 99mTc-N-MPO showed favourable biodistribution properties and myocardial uptake with rapid liver clearance in Sprague-Dawley rats [52,53]. The [99mTc(N)PNP]2+ metal fragment has been efficiently employed for connecting a bioactive molecule with technetium-99m, providing that the biomolecule includes the appropriate set of coordinating atoms. In particular, it was found that the amino acid cysteine shows excellent coordinating 99m 2+ properties toward the electrophilic [ Tc(N)PNP] metal fragment, either through the (NH2,S−) pair or the (O−,S−) pair alternatively, giving rise to highly specific and quantitative reactions. Therefore, a selected bioactive molecule, such as a peptide or a small protein, can be conveniently combined with a cysteine residue through the terminal carboxylic group to form the corresponding COO-functionalized N,S-cysteine ligand or through the terminal amino group to give an N- functionalized O,S-cysteine ligand. The resulting bioactive cysteine derivative is then reacted with [99mTc(N)PNP]2+ to produce the mixed [99mTc(N)PNP-cysteine-bioactive]0/+ complex. This approach has been used for the preparation Appl. Sci. 2019, 9, 2526 9 of 16 of receptor-specific complexes for imaging benzodiazepine receptors and receptor-specific tracers for 5HT1A receptors [54,55].

99m + 3.4. The [ Tc][Tc(CO)3] Metal Fragment 99m + The core [ Tc][Tc(CO)3] is a chemically robust organometallic moiety formed by technetium in 99m + the I oxidation state coordinated to three carbonyl groups. The precursor [ Tc][Tc(CO)3(H2O)3] can be readily prepared from the generator-eluted pertechnetate under the reducing conditions developed by Alberto et al. [56–58]. A large number of different Tc(I) complexes can be prepared starting from this stable water-soluble synthon, because the water ligands can be easily replaced by a variety of donor groups [59,60]. 99m + High yields of the [ Tc][Tc(CO)3(H2O)3] metal fragment are obtained in a single-step 99m procedure in the presence of the appropriate buffers, via the reduction of [ Tc]TcO4− by potassium boranocarbonate K2[BH3CO2], which also acts as a source of carbonyl ligands. With the aim of making this labelling chemistry readily accessible, a kit-based formulation for producing 99m + [ Tc][Tc(CO)3(H2O)3] synthon is available under the name IsoLink. 6 99m + When the metal has a d low spin configuration, the complexes containing the [ Tc][Tc(I)(CO)3] core are typically inert and maintain their integrity under different reaction conditions. While the robustness of complexes is purely kinetic, essentially all types of donor atoms have been used and numerous examples of bidentate and tridentate chelators have been reported, including derivatives that can be linked to targeting molecules [61]. As opposed to the traditional 99mTc(V)-oxo approach, 99m + where the choice of ligand is quite limited, for [ Tc][Tc(I)(CO)3] almost any monodentate, bidentate, ot tridentate donor ligands can be used. Among them, anionic bidentate ligands are more efficient than neutral ones, and tridentate ligands react much faster than bidentate ones, providing the additional advantage of shielding the metal centre from cross-reactions, as often occurs for the 99m + 99m + [ Tc(CO)3(H2O)(B)] and [ Tc(CO)3Cl(B)] complexes (B, bidentate ligand). The most studied tridentate ligands are aliphatic triamines, trithiacyclonane, bis-benzimidazole amine, pyrazolyl borates, and histidine derivatives, which can be conveniently modified to provide complexes with an anionic, cationic, or neutral overall charge and specific pharmacokinetic profiles to achieve the desired 99m + biodistribution when the [ Tc][Tc(I)(CO)3] is incorporated into targeting vectors. Of these ligands, the histidine (his) derivative can be easily connected to biomolecules through the introduction of an acetic acid group at Nε in histidine to yield a Nα carboxylate histidine ready to be condensed to a selected biomolecule. The use of histidine has the additional advantage of not being sensitive 99m to reduction and the resulting [ Tc][Tc(CO)3(his)] can be prepared in a single reaction step from 99mTc-pertechnentate.

4. The Metal Fragments Approach in the Development of New 99mTc-Based Radiopharmaceuticals for Prostate Cancer Prostate-specific membrane antigen (PSMA), a glutamate carboxypeptidase enzyme overexpressed in 95% of advanced prostate cancer (PCa) cases, is a molecular target for the imaging and radionuclide therapy of PCa using specific radiopharmaceuticals. In particular, the inhibition of its activity has been attributed to the urea-based Lys(b-naphthyl alanine)-NH-CO-NH-Glu(Lys(Nal)-Urea-Glu fragment, which, interacting electrostatically with the active peptide sites of the enzyme, has been shown to image advantageously PSMA-expressing in PCa [62]. Furthermore, when the expression levels in healthy tissue are low, PSMA has the potential for high-dose radiotherapeutic treatment, with minimized radioactivity-related side effects. Recent studies on 177Lu-PSMA-617 have demonstrated a high tumour to normal tissue uptake, with prolonged retention of radionuclides in tumour-bearing areas in men with metastatic prostate cancer [63–66]. Before therapeutic treatment, the imaging of radiopharmaceutical uptake in tumours or metastases is essential and can be performed by positron emitter or single-photon emitter compounds. For these two categories of radiopharmaceuticals, β+ particle emitter [68Ga]Ga-DOTA derivatives for Appl. Sci. 2019, 9, x FOR PEER REVIEW 10 of 16

through the introduction of an acetic acid group at Nε in histidine to yield a Nα carboxylate histidine ready to be condensed to a selected biomolecule. The use of histidine has the additional advantage of not being sensitive to reduction and the resulting [99mTc][Tc(CO)3(his)] can be prepared in a single reaction step from 99mTc-pertechnentate.

4. The metal fragments approach in the development of new 99mTc-based radiopharmaceuticals for prostate cancer Prostate-specific membrane antigen (PSMA), a glutamate carboxypeptidase enzyme overexpressed in 95% of advanced prostate cancer (PCa) cases, is a molecular target for the imaging and radionuclide therapy of PCa using specific radiopharmaceuticals. In particular, the inhibition of its activity has been attributed to the urea-based Lys(b-naphthyl alanine)-NH-CO-NH-Glu(Lys(Nal)- Urea-Glu fragment, which, interacting electrostatically with the active peptide sites of the enzyme, has been shown to image advantageously PSMA-expressing in PCa [62]. Furthermore, when the expression levels in healthy tissue are low, PSMA has the potential for high-dose radiotherapeutic treatment, with minimized radioactivity-related side effects. Recent studies on 177Lu-PSMA-617 have demonstrated a high tumour to normal tissue uptake, with prolonged retention of radionuclides in tumour-bearing areas in men with metastatic prostate cancer [63–66]. Appl. Sci.Before2019, 9therapeutic, 2526 treatment, the imaging of radiopharmaceutical uptake in tumours10 of 16or metastases is essential and can be performed by positron emitter or single-photon emitter compounds. For these two categories of radiopharmaceuticals, β+ particle emitter [68Ga]Ga-DOTA γ PETderivatives imaging for have PET beenimaging developed have been [67 developed,68], while [67,68], in the while case ofin the-emitter case of compounds, γ-emitter compounds, different 99m differentTc-radiopharmaceuticals 99mTc-radiopharmaceuticals have been have reported been reported for SPECT for SPECT PCa imaging. PCa imaging. In this In lastthis case,last case, the possibilitythe possibility of choosing of choosing from from different different synthesis synthesis strategies strategies to ensure to ensure robust robust radiolabelling radiolabelling has beenhas been the drivingthe driving force force in the in developmentthe development of compounds of compounds with with improved improved biodistribution biodistribution properties. properties. Based Based on the chemistry of the organometallic fragment [99mTc][Tc(CO) (H O) ]+, two promising compounds, on the chemistry of the organometallic fragment [99mTc][Tc(CO)3 3(H2 2O)3 3]+, two promising compounds, MIP-1404MIP-1404 andand MIP-1405,MIP-1405, bothboth basedbased onon anan imidazoleimidazole modificationmodification ofof thethe PSMAPSMA inhibitor,inhibitor, werewere developed (Figure9). developed (Figure 9).

99mTc-MIP-1405 OH O O HO O H O N H N N HO OH O O H H O N N HO O N HO OH O O N COOH H N HO O O NH OC N N O HO N Tc O CO N OC N NH CO N 2 OH Tc N O O CO N O CO N OH OH O 99m Tc-MIP-1404 99m FigureFigure 9. The 9. chemicalThe chemical structure structure of the ofcomplexes the complexes [99mTc][Tc(CO) [ Tc][Tc(CO)3-MIP-1404,3-MIP-1404, MIP-1405]. MIP-1405].

The preparation of the metal complexes [99mTc][Tc(CO) -MIP-1404, MIP-1405] was accomplished The preparation of the metal complexes [99mTc][Tc(CO)33-MIP-1404, MIP-1405] was accomplished usingusing aa standardstandard methodologymethodology andand commerciallycommercially availableavailable IsoLinkIsoLink kitskits (Covidien,(Covidien, Dublin,Dublin, Ireland)Ireland) andand the imidazole imidazole chelator, chelator, which which contains contains three three nitrogen nitrogen atomatom suitable suitable for binding for binding to the 99m toTc(I) the- 99m tricarbonyl-core.Tc(I)-tricarbonyl-core. The radiochemical The radiochemical yield of the yield final of radiopharmaceuticals the final radiopharmaceuticals is only 70% is onlyand post- 70% andlabelling post-labelling purification purification is needed is [69] needed. Both [ 69compounds]. Both compounds were used were to visualize used to visualizetumours tumoursin PCa and in PCametastases and metastases in lymph in lymphnodes an nodesd bones. and bones. In particular, In particular, the MIP-1404 the MIP-1404 compound, compound, also also known known as 99m as[99m [ Tc]Tc-trofolastat,Tc]Tc-trofolastat, has has demonstrated demonstrated superior superior biod biodistributionistribution properties properties with with low low uptake in thethe kidneykidney andand moremore lesionslesions thanthan MIP-1405MIP-1405 inin aa phasephase IIII clinicalclinical trialtrial [[58].58]. The different different pharmacokinetics hashas beenbeen attributed attributed to theto carboxymethylthe carboxymethyl group group attached attached to the imidazole.to the imidazole. In the MIP-1405 In the compound MIP-1405 there is only a terminal carboxymethyl group on the imidazole moiety, while MIP-1404 has a biscarboxymethyl amino-2-oxoethyl group linked to each imidazole (see Figure9). The technology of 99mTc-hydrazinonicotinyl was also applied in the development of a PSMA inhibitor for 99mTc-based SPECT. Ferro-Flores et al. [70] reported in vitro and in vivo studies of 99mTc–EDDA–HYNIC–iPSMA demonstrating that this radiopharmaceutical could detect tumour and metastases of PCa as well as 68Ga–PSMA–617. 99mTc–EDDA–HYNIC–iPSMA was quickly prepared from a kit with radiochemical yield >95%. Recently, a new 99mTc radiopharmaceutical, known as 99mTc–PSMA–I&S (I&S = investigation and surgery), is available for SPECT imaging and radio-guided surgery (RGS) for clinical application in urology [71]. This compound can be produced through a robust and reliable freeze-dried kit, facilitating the on-site production. The radiopharmaceutical is based on the molecule DOTAGA-(3-iodo-y)-f-k-Sub(KuE), in which the DOTA chelator is present to coordinate indium-111. Due to the limitations associated with the high cost, poor availability, and non-ideal nuclear properties of this radionuclide, a technetium-99m analogue has been proposed. The DOTA chelator, specifically used for indium-111 labelling, has been replaced with the hydrophilic MAG3- analogue MAS3 (mercaptoacetyl triserina), with the aim of stably binding the technetium-99m in the V oxidation state (Figure 10). At first, the use of the ease and efficient MAG3 labelling procedure allowed the 99m formation of the Tc–MAS3–y–nal–k(Sub–KuE) complex, where the 3–iodo–D–Tyr–D–Phe–sequence in the linker moiety was replaced by a D–Tyr–D–2–Nal–sequence to enhance its interaction with a Appl. Sci. 2019, 9, 2526 11 of 16

99m remote arene binding site. Then, a second, more stable compound, Tc–mas3–y–nal–k(Sub–KuE), was developed, where all the L–serines in the chelator MAS3 (2–mercaptoacetyl–L–Ser–L–Ser–l–Ser–), more susceptibleAppl. Sci. 2019, to 9, x proteolytic FOR PEER REVIEW degradation, have been replaced with the corresponding D–serine–(mas3)11 of 16 (Figure 10). The high in vivo stability of the resulting 99mTc–PSMA–I&S compound represents a major compound there is only a terminal carboxymethyl group on the imidazole moiety, while MIP-1404 prerequisite for RGS clinical application performed on the day after the tracer injection for practical has a biscarboxymethyl amino-2-oxoethyl group linked to each imidazole (see Figure 9). reasons [71]. 99mTc–PSMA–I&S is a promising gamma probe that could potentially be used for in vivo The technology of 99mTc-hydrazinonicotinyl was also applied in the development of a PSMA intraoperative measurements to facilitate localizing metastases and removing recurrent PCa lesions. A inhibitor for 99mTc-based SPECT. Ferro-Flores at al. [70] reported in vitro and in vivo studies of 99mTc– very recent study also demonstrated the feasibility of using a 99mTc–PSMA–I&S radiopharmaceutical EDDA–HYNIC–iPSMA demonstrating that this radiopharmaceutical could detect tumour and to guide intraoperative identification and surgical removal of metastatic lymph nodes in PCa patients metastases of PCa as well as 68Ga–PSMA–617. 99mTc–EDDA–HYNIC–iPSMA was quickly prepared scheduled for salvage surgery [72]. The PSMA radio–guided surgery essentially involves, first, the from a kit with radiochemical yield >95%. selection of patients based on 68Ga–PSMA–PET results and clinical history, then the injection of Recently, a new 99mTc radiopharmaceutical, known as 99mTc–PSMA–I&S (I&S = investigation and 99mTc–PSMA–I&S and subsequent SPECT/CT imaging aiming to confirm the technetium uptake in surgery), is available for SPECT imaging and radio-guided surgery (RGS) for clinical application in the same lesions of the preoperative gallium-68 compound. Finally, PSMA radio-guided surgery is urology [71]. This compound can be produced through a robust and reliable freeze-dried kit, performed with in vivo and ex vivo gamma probes to reliably identify the metastatic prostate cancer facilitating the on-site production. The radiopharmaceutical is based on the molecule DOTAGA-(3- lesions. Figure 11 is a graphical representation of the steps of PSMA radio-guided surgery. iodo-y)-f-k-Sub(KuE), in which the DOTA chelator is present to coordinate indium-111.

. Figure 10. The chemical structure of the complexes 111In–PSMA–I&T and 99m FigureTc–MAS3 10. The/mas3–y–nal–k–Sub–KuE, chemical structure of the and complexes a graphical 111In–PSMA–I&T representation and of the 99m interactionTc–MAS3/mas3–y–nal– between the radiotracerk–Sub–KuE, and and PSMA. a graphical representation of the interaction between the radiotracer and PSMA.

Due to the limitations associated with the high cost, poor availability, and non-ideal nuclear properties of this radionuclide, a technetium-99m analogue has been proposed. The DOTA chelator, specifically used for indium-111 labelling, has been replaced with the hydrophilic MAG3- analogue MAS3 (mercaptoacetyl triserina), with the aim of stably binding the technetium-99m in the V oxidation state (Figure 10). At first, the use of the ease and efficient MAG3 labelling procedure allowed the formation of the 99mTc–MAS3–y–nal–k(Sub–KuE) complex, where the 3–iodo–D–Tyr–D–Phe– sequence in the linker moiety was replaced by a D–Tyr–D–2–Nal–sequence to enhance its interaction with a remote arene binding site. Then, a second, more stable compound, 99mTc–mas3–y–nal–k(Sub– KuE), was developed, where all the L–serines in the chelator MAS3 (2–mercaptoacetyl–L–Ser–L–Ser– l–Ser–), more susceptible to proteolytic degradation, have been replaced with the corresponding D– serine–(mas3) (Figure 10). The high in vivo stability of the resulting 99mTc–PSMA–I&S compound represents a major prerequisite for RGS clinical application performed on the day after the tracer injection for practical reasons [71]. 99mTc–PSMA–I&S is a promising gamma probe that could potentially be used for in vivo intraoperative measurements to facilitate localizing metastases and removing recurrent PCa lesions. A very recent study also demonstrated the feasibility of using a Appl. Sci. 2019, 9, x FOR PEER REVIEW 12 of 16

99mTc–PSMA–I&S radiopharmaceutical to guide intraoperative identification and surgical removal of metastatic lymph nodes in PCa patients scheduled for salvage surgery [72]. The PSMA radio–guided surgery essentially involves, first, the selection of patients based on 68Ga–PSMA–PET results and clinical history, then the injection of 99mTc–PSMA–I&S and subsequent SPECT/CT imaging aiming to confirm the technetium uptake in the same lesions of the preoperative gallium-68 compound. Finally, PSMA radio-guided surgery is performed with in vivo and ex vivo gamma probes to reliably identify

Appl.the metastatic Sci. 2019, 9, 2526 prostate cancer lesions. Figure 11 is a graphical representation of the steps of PSMA12 of 16 radio-guided surgery.

Figure 11. The steps of PSMA radio-guided surgery. Figure 11. The steps of PSMA radio-guided surgery. 5. Conclusions 5. Conclusions The scientific advances of the last 20 years in the development of chemical approaches dedicated to selectivelyThe scientific introducing advances and of stabilizingthe last 20 years the metal in the technetium-99m development of intochemical a bioactive approaches construct, dedicated such asto theselectively “99mTc-metal introducing fragment and approach,”stabilizing the have metal allowed technetium-99m the development into a bioactive of new technetium-99m construct, such 99m radiopharmaceuticalsas the “ Tc-metal fragment for tumour approach,” imaging, have previously allowed thought the development to be a prerogative of new oftechnetium-99m PET and other radionuclides.radiopharmaceuticals The recent for tumour impressive imaging, results previously achieved thought with di tofferent be a prerogative PSMA target of derivatives,PET and other in combinationradionuclides. with The improvement recent impressive of the SPECT results technology, achieved with indicate different that 99m PSMATc radiopharmaceuticals target derivatives, are in 99m farcombination from obsolete with and improvement will continue of tothe play SPECT an important technology, and indicate renewed that role inTc nuclear radiopharmaceuticals medicine. are far from obsolete and will continue to play an important and renewed role in nuclear medicine. Author Contributions: Conceptualization, A.B. and L.U.; writing—original draft preparation, P.M.; writing—reviewAuthor Contributions: and editing, Conceptualization, P.M. A.B. and L.U.; writing—original draft preparation, P.M.; writing— review and editing, P.M. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References References 1. Mankoff, D.A. A definition of molecular imaging. J. Nucl. Med. 2007, 48, 18N–21N. [PubMed] 2.1. Pysz,Mankoff, M.A.; D.A. Gambhir, A definition S.S.; Willmann,of molecular J.K. imaging. Molecular J. Nucl. Imaging: Med. 2007 Current, 48, 18N–21N. Status and Emerging Strategies. 2. Clin.Pysz, Radiol. M.A.; Gambhir,2010, 65, 500–516.S.S.; Willmann, [CrossRef J.K.][ MolecularPubMed] Imaging: Current Status and Emerging Strategies. Clin. 3. Qaim,Radiol. S.M.; 2010 Spahn,, 65, 500–516. I. Development of novel radionuclides for medical applications. J. Label. Comp. Radiopharm. 3. 2018Qaim,, 61 S.M.;, 126–140. Spahn, [CrossRef I. Development][PubMed ]of novel radionuclides for medical applications. J. Label. Comp. 4. Boschi,Radiopharm A.;. Martini,2018, 61, 126–140. P.; Uccelli, L. 188Re(V) Nitrido Radiopharmaceuticals for Radionuclide Therapy. 4. PharmaceuticalsBoschi, A.; Martini,2017, 10P.;, 12.Uccelli, [CrossRef L. 188][Re(V)PubMed Nitrido] Radiopharmaceuticals for Radionuclide Therapy. 5. Mottaghy,Pharmaceuticals F.M. 2017 Current, 10, 12. and future aspects of molecular imaging. Methods 2009, 48, 81–82. [CrossRef] [PubMed] 6. Uccelli, L.; Martini, M.; Cittanti, C.; Carnevale, A.; Missiroli, L.; Giganti, M.; Bartolomei, M.; Boschi, A. Therapeutic Radiometals: Worldwide Scientific Literature Trend Analysis (2008–2018). Molecules 2019, 24, 640. [CrossRef][PubMed] 7. Qaim, S.M. Nuclear data for medical applications: An overview. Radiochim. Acta 2001, 89, 189–196. [CrossRef] 8. Boschi, A.; Martini, P.; Janevik-Ivanovska, E.; Duatti, A. The emerging role of copper-64 radiopharmaceuticals as cancer theranostics. Drug Discov. Today 2018, 23, 1489–1501. [CrossRef] Appl. Sci. 2019, 9, 2526 13 of 16

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