Metals in Imaging of Alzheimer's Disease

International Journal of Molecular Sciences

Review Metals in Imaging of Alzheimer’s Disease

Olga Krasnovskaya 1,2,* , Daniil Spector 1,2,* , Alexander Zlobin 1, Kirill Pavlov 1, Peter Gorelkin 1,2 , Alexander Erofeev 1,2, Elena Beloglazkina 1 and Alexander Majouga 1,2,3

1 Chemistry Department, Lomonosov Moscow State University, Leninskie Gory 1,3, 119991 Moscow, Russia; [email protected] (A.Z.); [email protected] (K.P.); [email protected] (P.G.); [email protected] (A.E.); [email protected] (E.B.); [email protected] (A.M.) 2 Department of Materials Science of Semiconductors and Dielectrics, National University of Science and Technology (MISIS), Leninskiy Prospect 4, 101000 Moscow, Russia 3 Mendeleev University of Chemical Technology of Russia, Miusskaya Ploshchad’ 9, 125047 Moscow, Russia * Correspondence: [email protected] (O.K.); [email protected] (D.S.)

Received: 10 November 2020; Accepted: 28 November 2020; Published: 2 December 2020

Abstract: One of the hallmarks of Alzheimer’s disease (AD) is the deposition of amyloid plaques in the brain parenchyma, which occurs 7–15 years before the onset of cognitive symptoms of the pathology. Timely diagnostics of amyloid formations allows identifying AD at an early stage and initiating inhibitor therapy, delaying the progression of the disease. However, clinically used radiopharmaceuticals based on 11C and 18F are synchrotron-dependent and short-lived. The design of new metal-containing radiopharmaceuticals for AD visualization is of interest. The development of coordination compounds capable of effectively crossing the blood-brain barrier (BBB) requires careful selection of a ligand moiety, a metal chelating scaffold, and a metal cation, defining the method of supposed Aβ visualization. In this review, we have summarized metal-containing drugs for positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) imaging of Alzheimer’s disease. The obtained data allow assessing the structure-ability to cross the BBB ratio.

Keywords: Alzheimer disease; amyloid; PET; SPECT; MRI

1. Introduction Alzheimer’s disease is the most common form of neurodegenerative disease. This pathology is characterized by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs) in the brain [1]. One of the hallmarks is the extracellular amyloid plaques in aggregated forms of a peptide called amyloid-β (Aβ), appearing years before the onset of symptoms [2–5]. Timely diagnostic imaging plays an important role in managing AD. Several positron emission tomography (PET) imaging agents have been developed that bind to different amyloids, such as 2-(1,1-dicyanopropen-2-yl)-6-(2-[18F]-fluoroethyl)-methylamino-naphthalene [18F]FDDNP, [11C]Pittsburgh Compound-B (PiB), [18F]Florbetapir, [18F]Florbetaben, and [18F]Flutemetamol, allow obtaining semiquantitative information about amyloid deposition in patients, which allows presaging the development of clinical symptoms of AD 7–15 years before their occurrence [6–10] (Figure1). But using these drugs requires an expensive laborious synthesis with confirmation of radio purity at each stage. The short half-lives of the currently used radionuclides 11C (20.4 min) and 18F (109.8 min) may also limit the widespread use of these imaging agents [11,12]. Although metal cations such as Cu(II), Zn(II), and Fe(III) proved to coordinate undesirably with histidine residues at the N-terminus of Aβ, promoting Aβ aggregation and stabilization of Aβ oligomers [13], an increased accumulation of these metals in Aβ-amyloids raises the possibility of

Int. J. Mol. Sci. 2020, 21, 9190; doi:10.3390/ijms21239190 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9190 2 of 35

Int. J. Mol. Sci. 2020, 21, x 2 of 37 designing Cu(II)-, Zn(II)-, and Fe(III)-based metal complexes for the diagnosis and theranostics of designing Cu(II)–, Zn(II)–, and Fe(III)–based metal complexes for the diagnosis and theranostics of 64 AD. ADAD. diagnosticAD diagnostic agents agents radiolabeled radiolabeled with with 64Cu are are attractive attractive not not only only due due to the to thesimple simple and fast and fast introductionintroduction of radionuclide of radionuclide at at the the last last stage stage ofof non-radioactivenon-radioactive synthesis, synthesis, but but also also due dueto the to 12.7 the h 12.7 h half-lifehalf-life of 64 ofCu 64Cu radionuclide, radionuclide, ideal ideal for for PETPET imagingimaging [14]. [14].

Figure 1. Pittsburgh compound B (PiB), [11C]4-N-Methylamino-4′-hydroxystilbene (SB–13), and Figure 1. Pittsburgh compound B (PiB), [11C]4-N-Methylamino-40-hydroxystilbene (SB-13), and FlorbetairFlorbetair (AV–45), (AV-45), AD AD PET PET imaging imaging agents. agents.

AnotherAnother promising promising PET PET radionuclide radionuclide isis 6868Ga. Positron–emitting Positron-emitting 68Ga68 Gacan canbe obtained be obtained from froma a 68 68 68Ge/68Ge/GaGa generator, generator, which which would would allowallow aa cyclotron-independentcyclotron-independent distribution distribution of PET. of PET. The Theparent parent nuclide, 68Ge, has a half-life of 271 days, and the generators can provide sufficient quantities of 68Ga nuclide, 68Ge, has a half-life of 271 days, and the generators can provide sufficient quantities for up to one year, resulting in a relatively inexpensive and reliable source of a positron-emitting of 68Ga for up to one year, resulting in a relatively inexpensive and reliable source of a radionuclide [15,16]. positron-emittingIn addition radionuclide to PET imaging [15,16 ].of amyloids, single-photon emission computed tomography In(SPECT) addition and tomagnetic PET imaging resonance of amyloids,imaging (MRI) single-photon are alternative emission diagnostic computed tools for tomographyAD visualization, (SPECT) and magneticable to overcome resonance the imaginglimitations (MRI) of PET are imaging alternative in terms diagnostic of cost and tools broad for ADaccessibility visualization, [17]. The able to overcometechnetium–99 the limitations m (99mTc) of PET radioisotope imaging for in termsSPECT of imaging cost and can broad be cyclotron-independently accessibility [17]. The technetium-99prepared m (99mbyTc) a 99 radioisotopeMo/99mTc generator for SPECT [18]. imagingThe MRI can imaging be cyclotron-independently allows nonradioactive diagnostics prepared byand a 99is Moalso/99m Tc generatorcheaper [18 and]. The faster MRI than imaging PET imaging. allows nonradioactiveThe Gd3+ PET imaging diagnostics agents andfor A isβ alsovisualization cheaper andare also faster of than PET imaging.interest [19]. The Gd3+ PET imaging agents for Aβ visualization are also of interest [19]. The development of effective diagnostic and therapeutic agents targeting amyloid is not a trivial The development of effective diagnostic and therapeutic agents targeting amyloid is not a task. The blood-brain barrier (BBB) is a highly selective, semipermeable barrier, consistent of trivial task. The blood-brain barrier (BBB) is a highly selective, semipermeable barrier, consistent of cerebrovascular endothelial cells, surrounded by extracellular matrix, astrocytes, and pericytes [20], cerebrovascularwhich prevents endothelial potential therapeutics cells, surrounded from reaching by extracellular the cerebral matrix, target, astrocytes,thus limiting and their pericytes efficacy [20], which[21]. prevents Various potential approaches therapeutics to effective from brain reaching delivery the are cerebraldeveloped, target, such thus as chemical limiting drug their delivery efficacy [21]. Varioussystems approaches [22], e.g., toa drug effective conjugation brain with delivery dihydropyridine, are developed, mannitol, such or aromatic as chemical substances drug [23], delivery systemsphysical [22], methods, e.g., a drug such conjugationas focused ultrasound with dihydropyridine, [24] or sonophoresis mannitol, [25], and or biological aromatic methods, substances e.g., [23], physicaldrug methods,conjugation such with aspolycationic focused ultrasoundproteins or amino [24] oracids sonophoresis [26]. [25], and biological methods, e.g., drugThe conjugation complexity with of the polycationic architecture of proteins the blood-brain or amino barrier, acids as [26 well]. as the significant difficulties Theaccompanying complexity the development of the architecture of drugs capable of the of blood-brain overcoming it, barrier, prompts as the well creation as the of in significant vitro models of the BBB, such as microfluidic models [27], brain organoids [28], and microvascular systems difficulties accompanying the development of drugs capable of overcoming it, prompts the [29]. creation ofThein BBB vitro permeabilitymodels of theof BBB,a compound such as microfluidicis related to modelsits lipophilicity, [27], brain expressed organoids by [28the], and microvascularwater/octanol systems partition [29]. coefficient, log Poct/water, molecular weight (MW), and plasma Thepharmacokinetics BBB permeability [30]. Low–MW of a compound amphiphilic is related molecules to its lipophilicity, with log Poct/water expressed ≈ 2 have by theoptimal water BBB/octanol partitionpenetration coeffi cient,[31]. Conjugating log Poct/water an A,β molecular-affinity moiety, weight a metal-chelating (MW), and plasmamoiety, and pharmacokinetics a metal cation in [30]. Low-MWone scaffold amphiphilic is often difficult, molecules and the with resu loglting Pdrugs are often2 unable have to optimal cross the BBB BBB.penetration [31]. oct/water ≈ ConjugatingSedgwick an A βet-a ffial.nity summarized moiety, ametal-based metal-chelating imaging moiety, agents and for aneurodegenerative metal cation in onedisease sca ffold is oftendiagnostics difficult, [32]. and Gomes the resulting et al. also drugs summarized are often an interaction unable to of cross metal the complexes BBB. with the Aβ peptide Sedgwick et al. summarized metal-based imaging agents for neurodegenerative disease diagnostics [32]. Gomes et al. also summarized an interaction of metal complexes with the Aβ peptide [33]. Liu et al. reported potential applications of metal-based agents in therapy, diagnosis, and theranosis of AD [34]. Int. J. Mol. Sci. 2020, 21, 9190 3 of 35

Int. J. Mol. Sci. 2020, 21, x 3 of 37 In this review, we summarize various solutions in the design of amyloid-affinity drugs capable of [33]. Liu et al. reported potential applications of metal-based agents in therapy, diagnosis, and β effectivelytheranosis crossing of ADthe [34]. BBB, and different approaches for designing A -affinity drugs for diagnosing AD. ThreeIn summarythis review, tables we summarize can be conveniently various solutions used in to the evaluate design of the amyloid-affinity structure of the drugs ligand capable and the resultof ofeffectively brain penetration crossing the by BBB, the coordinationand different compoundapproaches basedfor designing on it, notingAβ-affinity the successfuldrugs for and unsuccessfuldiagnosing attempts AD. Three to createsummary drugs tabl fores diagnosingcan be conveniently AD. This used review to evaluate will be usefulthe structure to researchers of the for developingligand and approaches the resultfor of designingbrain penetration Aβ-affi bynity the drugs coordination for both compound the therapy based and on diagnostics it, noting the of AD. successful and unsuccessful attempts to create drugs for diagnosing AD. This review will be useful 2. Copperto researchers Coordination for developing Compounds approaches for PET for designing Imaging ofAβ Alzheimer-affinity drugs Disease for both the therapy and diagnostics of AD. PET diagnostics is based on registering a pair of gamma quanta resulting from the annihilation of electrons2. Copper and Coordination positrons that Compounds arise during for thePET positron-beta Imaging of Alzheimer decay of Disease a radionuclide. Annihilation of the positron, which remained in the tissue, with one of the electrons of the medium, generates two PET diagnostics is based on registering a pair of gamma quanta resulting from the annihilation gamma quanta with the same energy, scattering in opposite directions along one straight line. A set of electrons and positrons that arise during the positron-beta decay of a radionuclide. Annihilation of detectorsof the positron, makes which it possible remained to obtain in the tissue, a three-dimensional with one of the electrons reconstruction of the medium, of the distribution generates two of the radionuclidegamma quanta in the with body the tissue same [energy,35]. scattering in opposite directions along one straight line. A set 64 + ofThe detectors radionuclide makes it possibleCu has to a obtain long half-life a three-dime (t1/2 nsional= 12.7 reconstruction h, β = 17%, ofβ− the= 39%,distribution e-capture of the decay EC =radionuclide43%, Emax in= 0.656the body MeV) tissue and [35]. can be considered an ideal PET tracer [36]. Copper-coordination compoundsThe radionuclide are promising 64Cu for has PET a long diagnostics half-life (t1/2 of =AD 12.7 becauseh, β+ = 17%, of notβ− = only39%, e–capture the emission decay properties EC = but also43%, theEmax increased= 0.656 MeV) affinity and ofcan amyloids be considered for copper an ideal cations, PET tracer which [36]. would Copper-coordination further increase the accumulationcompounds of are copper-containing promising for PET drugsdiagnostics in the of therapeutic AD because targetof not only [37]. the emission properties but alsoA standardthe increased approach affinity inof developingamyloids for AcoβpperPET cations, imaging which drugs would is further a conjugation increase the of an accumulation of copper-containing drugs in the therapeutic target [37]. Aβ-binding benzothiazole, benzofuran, or stilbene scaffold, with a metal-chelating moiety. A standard approach in developing Aβ PET imaging drugs is a conjugation of an Aβ-binding Thiosemicarbazone derivatives are often used as a metal-chelating agent, based on the diacetylbis(N(4)- benzothiazole, benzofuran, or stilbene scaffold, with a metal-chelating moiety. Thiosemicarbazone methylthiosemicarbazonatoderivatives are often used Cu-ATSM as a drugmetal-chelat [38]. ing agent, based on the diacetylbis(N(4)- methylthiosemicarbazonatoLim et al. [39] developed Cu-ATSM a bis(thiosemicarbazonato)copper(II) drug [38]. complex 1 (all numbers of coordinationLim et compounds al. [39] developed are bold a through bis(thiosemicarbazonato)copper(II) all the manuscript) conjugated complex with 1 a(all stilbene numbers functional of groupcoordination (Figure2). compounds A fluorescent are bold assay through with thioflavin-Tall the manuscript) (Th-T) conjugated showed with a drop a stilbene in the functional fluorescence (485group nm) after(Figure an 2). addition A fluorescent of coordination assay with thioflav compoundin–T (Th–T)1, meaning showed aa displacementdrop in the fluorescence of thioflavin. Also,(485 examination nm) after an by addition transmission of coordination electron compound microscopy 1, meaning (TEM) ofa displacement the structural of thioflavin. morphology Also, of the Aβ fibrilsexamination pre-treated by transmission with coordination electron microscopy compound (TEM)1 showed of the significantstructural morphology changes in of morphology. the Aβ Epi-fluorescencefibrils pre-treated microscopy with coordination of AD human compound brain sections1 showed with significant E18 antibody changes revealed in morphology. a co-localization Epi– fluorescence microscopy of AD human brain sections with E18 antibody revealed a co-localization of of the immunostained and epi-fluorescent images. Biodistribution of radiolabeled 64Cu-1 in wild-type the immunostained and epi-fluorescent images. Biodistribution of radiolabeled 64Cu–1 in wild–type mice and APP/PS1 transgenic mice (Tg-mice) after intravenous tail vein injection (85 MBq) showed a mice and APP/PS1 transgenic mice (Tg–mice) after intravenous tail vein injection (85 MBq) showed significantlya significantly higher higher brain brain uptake uptake in APPin APP/PS1/PS1 Tg-mice Tg–mice compared compared with with theirtheir wild type type (Table (Table 1).1).

Figure 2. 64Cu(II)-ATSM derivative 1 conjugated with stilbene functional group, designed for Aβ ﬁbrils visualization. Int. J. Mol. Sci. 2020, 21, 9190 4 of 35

Table 1. Cu(II)-based coordination compounds for positron emission tomography (PET) imagining of Alzheimer disease.

Coordination Brain Uptake, ID/g, 2 min Brain / 2min 60min Brain Tissue Experiments Aβ Binding Moiety Reference Compound Number Post-Injection, % (*Brain2min/30min) Ratio Cu(ATSM)-based coordination compounds 2.5 0.6 (APP/PS1 ± transgenic mice) Epi-fluorescence microscopy of 1 - Stilbene [39] 1.7 0.6 (Wild-type mice) AD human brain sections ± 7 min after injection Epi-fluorescence microscopy of 2–4 1.11 0.20 2.92 * 2-benzothiazole,3,4-styrylpyridine [40] ± AD human brain sections 1.39 0.06 ± 1.31 * Elemental composition of AD 1.06 0.43 5–8 ± 2.16 * human brain tissue using Benzofuran [41] 0.77 0.19 ± 1.05 * LA-ICP-MS 1.54 0.60 ± Epi-fluorescence microscopy of 9 -- Stilbene [42] AD human brain tissue (ligand) 2.2 0.6 6.47 Epi-fluorescence microscopy of 10–15 ± Styrylpyridine [43] 1.1 0.2 5 AD human brain sections ± 4.41 0.23 16–25 ± - PET imagine of BALB/c mice - [44] (23 h Post-injection similar) Other metal-chelating moieties 0.33 0.12 1.83 Fluorescent staining using brain 26, 27 ± Benzofuran [45] 0.36 0.10 2.11 sections from a Tg2576 mice ± 0.37 0.06 2.64 ± 0.17 0.02 1.30 ± Fluorescent imaging of amyloid 1.33 0.27 4.92 29–33 ± plaques in Tg2576 AD mice Benzothiazole [46,47] 0.49 0.01 2.22 ± brain sections 0.61 0.14 4.69 ± 0.75 0.16 2.88 ± Fluorescence imaging of 0.16 0.02 1.59 34–39 ± amyloid plaques in 5xFAD mice Benzothiazole [48] 0.99 0.04 4.95 ± brain sections

* Brain2min/30min ratio is indicated instead of Brain2min/60min ratio. Int. J. Mol. Sci. 2020, 21, x 5 of 37

Brain Uptake, Coordination Brain2min/60min ID/g, 2 Brain Tissue Aβ Binding Compound (*Brain2min/30min) Reference min Post– Experiments Moiety Number Ratio Injection, % from a Tg2576 mice Fluorescent 0.37 ± 0.06 2.64 imaging of 0.17 ± 0.02 1.30 amyloid 1.33 ± 0.27 4.92 29–33 plaques in Benzothiazole [46,47] 0.49 ± 0.01 2.22 Tg2576 AD 0.61 ± 0.14 4.69 mice brain 0.75 ± 0.16 2.88 sections Fluorescence imaging of 0.16 ± 0.02 1.59 amyloid 34–39 Benzothiazole [48] 0.99 ± 0.04 4.95 plaques in 5xFAD mice brain sections

* Brain2min/30min ratio is indicated instead of Brain2min/60min ratio. Int. J. Mol. Sci. 2020, 21, 9190 5 of 35

TheThe same same DonnellyDonnelly groupgroup reportedreported a a copper copper radiopharmaceutical radiopharmaceutical Cu Cu(II)-ATSM(II)–ATSM with with an an appendedappended styrylpyridine styrylpyridinefunctional functional group for A Aββ plaqueplaque imaging imaging [40] [40 (Figure] (Figure 3).3 ).Binding Binding of of3 and3 and 4 4 (coordination compound 2 was quite insoluble) to Aβ plaques was clearly evident, as demonstrated (coordination compound 2 was quite insoluble) to Aβ plaques was clearly evident, as demonstrated by by epi–fluorescence microscopy. The Aβ–specific 1E8 antibody was used as a control. The epi-ﬂuorescence microscopy. The Aβ-speciﬁc 1E8 antibody was used as a control. The biodistribution biodistribution of coordination compounds 3 and 4 radiolabeled with 64Cu in wild-type mice after of coordination compounds 3 and 4 radiolabeled with 64Cu in wild-type mice after intravenous tail intravenous tail injection (∼13 MBq) displayed good brain uptake of coordination compound 4 in injection ( 13 MBq) displayed good brain uptake of coordination compound 4 in 1.1%. 1.1%. ∼

FigureFigure 3. 3.64 64Cu(II)-ATSMCu(II)–ATSM derivativesderivatives conjugated 2–42–4 withwith benzothiazole/styrylpyrydine benzothiazole/styrylpyrydine functional functional group,group, designed designed for for A Aββﬁbrils fibrils visualization.visualization.

InIn 2019 2019 [ 41[41],], thethe DonnelyDonnely groupgroup reported a a sy synthesisnthesis of of four four hybrid hybrid thiosemicarbazonato- thiosemicarbazonato- benzofuran ligands and their copper complexes (Figure 4). Addition of either 6 or 8 to Aβ1−42 results benzofuran ligands and their copper complexes (Figure4). Addition of either 6 or 8 to Aβ1 42 results − ininInt. dramatic dramatic J. Mol. Sci. changes 2020changes, 21, x in in the the structural structural morphology,morphology, as as identified identified by by the the TEM TEM images. images. The The AD AD human human6 of 37 brainbrain tissue tissue samples samples treated treated withwith 88 werewere analyzed for for elemental elemental comp compositionosition using using the the laser laser ablation ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) assay by tracking the change in the inductively coupled plasma mass spectrometry (LA-ICP-MS) assay by tracking the change in the ratio 65Cu/63Cu. A sample of nonradioactive isotopically enriched 65Cu–8 was used to distinguish ratio 65Cu/63Cu. A sample of nonradioactive isotopically enriched 65Cu-8 was used to distinguish biologically present copper from the complex. Coordination compound 3 was used as a control. The biologically present copper from the complex. Coordination compound 3 was used as a control. benzofuran–containing complex 65Cu–8 appears to bind with improved differentiation compared The benzofuran-containing complex 65Cu-8 appears to bind with improved differentiation compared with the styryl-pyridine-containing complex 65Cu–3 and potentially offers better sensitivity for with the styryl-pyridine-containing complex 65Cu-3 and potentially offers better sensitivity for amyloid. amyloid. The complex preferentially binds to areas of the brain enriched with Aβ plaques, which was β Theconfirmed complex preferentiallyby immunohistochemistry binds to areas ofwith the brainan enrichedaged-match with control. A plaques, The whichbiodistribution was confirmed of bycoordination immunohistochemistry compoundswith 5–8 radiolabeled an aged-match with control. 64Cu in The wild-type biodistribution mice showed of coordination the best brain compounds uptake 64 5–results8 radiolabeled for coordination with Cu compound in wild-type 8 (1.54% mice of showed injected thedose best (ID)/g brain at uptake2 min after results injection, for coordination dropping compoundto 0.77% ID/g8 (1.54% at 30 ofmin). injected dose (ID)/g at 2 min after injection, dropping to 0.77% ID/g at 30 min).

FigureFigure 4. 4.64 64Cu(II)-ATSMCu(II)–ATSM derivatives derivatives5 5––88 conjugatedconjugated with benzofuran functional functional group, group, designed designed for for AβAﬁbrilsβ fibrils visualization. visualization.

McInne [42] incorporated a 4-vinylpyridine functional group to investigate whether the complex 9 binds to Aβ plaques with an additional pyridyl hydrogen bond acceptor at the expense of the electron-donating dimethlylamino and hydroxy groups (Figure 5). Comparing the fluorescence from the 9-treated AD human brain tissue with (1E8)-treated brain tissue revealed good co-localization.

Figure 5. Cu(II)–ATSM derivative conjugated with piridylstilbene functional group 9, designed for Aβ fibrils visualization.

This research group recently presented several structural analogues (10–5) of coordination compound 3, where the bis-(thiosemicarbazone) moiety is conjugated to stilbene functional groups [44] (Figure 6). All coordination compounds significantly alter the emission intensity of the ThT/Aβ conjugate. Compounds 11 and 15 were selected as lead compounds because of the ease of synthesis. The TEM of Aβ1−40 fibrils preincubated with 11 and 15 reveal a dramatic change in fibril morphology. Epi–fluorescence microscopy on human AD brain tissue proved an ability of 11 and 15 to bind amyloid-β plaques, which was also confirmed by Aβ-specific antibody (1E8) staining. Experiments

Int. J. Mol. Sci. 2020, 21, x 6 of 37 inductively coupled plasma mass spectrometry (LA–ICP–MS) assay by tracking the change in the ratio 65Cu/63Cu. A sample of nonradioactive isotopically enriched 65Cu–8 was used to distinguish biologically present copper from the complex. Coordination compound 3 was used as a control. The benzofuran–containing complex 65Cu–8 appears to bind with improved differentiation compared with the styryl-pyridine-containing complex 65Cu–3 and potentially offers better sensitivity for amyloid. The complex preferentially binds to areas of the brain enriched with Aβ plaques, which was confirmed by immunohistochemistry with an aged-match control. The biodistribution of coordination compounds 5–8 radiolabeled with 64Cu in wild-type mice showed the best brain uptake results for coordination compound 8 (1.54% of injected dose (ID)/g at 2 min after injection, dropping to 0.77% ID/g at 30 min).

Int. J. Mol.Figure Sci. 4.2020 64Cu(II)–ATSM, 21, 9190 derivatives 5–8 conjugated with benzofuran functional group, designed for6 of 35 Aβ fibrils visualization.

McInne [[42]42] incorporated a 4-vinylpyridine functional group to investigate whether the complex 9 binds to AAβ plaques with an additional pyridyl hydrogenhydrogen bond acceptor at the expense of the electron-donating dimethlylamino and hydroxy groups (Figure5 5).). ComparingComparing thethe ﬂuorescencefluorescence fromfrom the 9-treated ADAD humanhuman brainbrain tissuetissue withwith (1E8)-treated(1E8)-treated brainbrain tissuetissue revealedrevealed goodgood co-localization.co-localization.

Figure 5.5. Cu(II)-ATSMCu(II)–ATSM derivative derivative conjugated conjugated with with piridylstilbene piridylstilbene functional functional group group9, designed 9, designed for Aforβ ﬁbrilsAβ fibrils visualization. visualization.

This research groupgroup recentlyrecently presentedpresented severalseveral structuralstructural analoguesanalogues (10(10–5–15)) of coordinationcoordination compound 33,, wherewhere the the bis-(thiosemicarbazone) bis-(thiosemicarbazone) moiety moiety is conjugatedis conjugated to stilbeneto stilbene functional functional groups groups [44] (Figure[44] (Figure6). All 6). coordination All coordination compounds compounds significantly significantly alter alter the the emission emission intensity intensity of of the the ThT ThT/A/Aββ conjugate. Compounds 11 and 15 were selected as lead compounds because of the ease of synthesis. The TEM of Aβ1 40 fibrils preincubated with 11 and 15 reveal a dramatic change in fibril morphology. The TEM of Aβ1−−40 fibrils preincubated with 11 and 15 reveal a dramatic change in fibril morphology. Epi-fluorescenceInt.Epi–fluorescence J. Mol. Sci. 2020, 21 microscopy microscopy, x on on human human AD AD brain brain tissue tissue proved proved an ability an ofability11 and of15 11to and bind 15 amyloid- to 7bind of 37β plaques,amyloid- whichβ plaques, was alsowhich confirmed was also by confirmed Aβ-specific by antibody Aβ-specific (1E8) antibody staining. (1E8) Experiments staining. with Experiments wild-type micewith wild-type showed high mice brain showed uptake high forbrain both uptake11 and for 15bothat 11 2 minand after15 at 2 the min injection after the (2.2% injection and (2.2% 1.1%, respectively),and 1.1%, respectively), followed by followed rapid removal by rapid after removal 1 h. after 1 h.

Figure 6.6. Cu(II)-ATSMCu(II)–ATSM derivatives derivatives conjugated conjugated with with stilbene stilbene functional functional groups groups10– 1015–,15 designed, designed for Aforβ ﬁbrilsAβ fibrils visualization. visualization.

Observing the various design steps of the PET bindingbinding agentsagents developeddeveloped under Donnelly’s leadership, we note that they achieved signiﬁcantsignificant improvementsimprovements inin brainbrain uptakeuptake (Table(Table1 1,, lines lines 3–7). 3–7). Paterson et et al. al. [44] [44 developed] developed a series a seriesbis(thiosemicarbazones) bis(thiosemicarbazones) 16–25 with16– amine25 with and amine polyamine and polyaminefunctional groups functional in order groups to increase in order the to increaseBBB permeability the BBB permeability of the complexes of the (Figure complexes 7). Intracellular (Figure7). Intracellularuptake of the uptake complexes of the was complexes measured was by measured inductively by inductivelycoupled plasma coupled mass plasma spectrometry mass spectrometry (ICP–MS). (ICP-MS).Intracellular Intracellular accumulation accumulation decreased decreased in the order in the 17 order > [1917+2H]> [192+ >+ [2H]21+H]2+ +> >[ 21[23++H]H]+ >> [[2523+3H]+ H]3++. >Biodistribution[25 + 3H]3+. studies Biodistribution were performed studies using were small-animal performed using micro-PET small-animal imaging. micro-PETThe complexes imaging. with Thea secondary complexes amine, with 21, a secondary and a primary amine, amine21, and functional a primary group, amine 23, functional showed little group, to 23,no radioactivityshowed little in to nothe radioactivitybrain. The complex in the brain. with The a pendent complex secondary with a pendent amine, secondary 17, had a amine, relatively17, had high a relativelylevel of brain high leveluptake. of brain uptake. The authors designed these complexes not as PET imaging agents for amyloids, but as hypoxia- sensitive agents capable of accumulating in malignant tumors. But the impressive results of brain penetration shown by complex 17 (injected activity/per gramm IA/g at 23 h after injection was 2.43%) again convince us of the promising potential of copper-containing preparations as diagnostic agents for imaging brain pathologies. Ex vivo biodistribution analysis of 17–preinjected BALB/C mice bearing EMT6 tumors showed a 4.17% ± 1.03% injected activity per gram of tissue at 40 min post- injection, and 4.41% ± 0.23% injected activity per gram of tissue in the brain.

Int. J. Mol. Sci. 2020, 21, 9190 7 of 35

Int. J. Mol. Sci. 2020, 21, x 8 of 37

Figure 7. Cu(II)–ATSMCu(II)-ATSM derivatives derivatives conj conjugatedugated with with polyamines polyamines 16–2516, –designed25, designed for Aβ for fibrils Aβ ﬁbrilsvisualization. visualization.

TheTherefore, authors Сu–ATSM–based designed these agents complexes are interesting not as PET both imaging as redox–active agents for agents amyloids, sensitive but asto hypoxia-sensitivehypoxia, capable of agents accumulation capable of in accumulating solid tumors, in and malignant as highly tumors. penetrating But the agents impressive for therapy results and of braindiagnostics penetration of brain shown pathologies. by complex 17 (injected activity/per gramm IA/g at 23 h after injection was 2.43%)Conjugates again convince containing us of theAβ-binding promising and potential metal-chelating of copper-containing moieties were preparations found to modulate as diagnostic the agents for imaging brain pathologies. Ex vivo biodistribution analysis of 17-preinjected BALB/C aggregation of Aβ42 species [49,50]. Therefore, 64Cu coordination compounds based on them are mice bearing EMT6 tumors showed a 4.17% 1.03% injected activity per gram of tissue at 40 min expected to bind Aβ effectively. ± post-injection, and 4.41% 0.23% injected activity per gram of tissue in the brain. Watanabe at al. designed± and synthesized two novel 64Cu–labeled benzofuran derivatives 26 and 27 withTherefore, cyclen Cu-ATSM-based(1,4,7,10-tetraazacy agentsclododecane) are interesting or DOTA both as(1,4,7,10- redox-active tetraazacyclododecane-1,4,7,10- agents sensitive to hypoxia, capabletetraacetic of accumulationacid) as chelators in solid [45]tumors, (Figure and8). as highly penetrating agents for therapy and diagnostics of brainAn pathologies.in vitro binding assay with ([125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo [1,2- Conjugates containing Aβ-binding and metal-chelating moieties were found to modulate the a]pyridine) [125I] IMPY as the competitive ligand showed dose-dependent inhibition with Ki 33.7 ± 64 aggregation14.6, 243.5 ± 88.2. of A βFluorescent42 species [staining49,50]. Therefore, using Tg2576Cu mice coordination brain sections compounds proved the based amyloid-binding on them are expectedability of to26 bind to a Agreaterβ eﬀectively. extent than 27. Unfortunately, biodistribution studies revealed quite low brain uptake equal to 0.33% and 0.36%, respectively.

Int. J. Mol. Sci. 2020, 21, 9190 8 of 35

Watanabe et al. designed and synthesized two novel 64Cu-labeled benzofuran derivatives 26 and 27 with cyclen (1,4,7,10-tetraazacyclododecane) or DOTA (1,4,7,10- tetraazacyclododecane- 1,4,7,10-tetraacetic acid) as chelators [45] (Figure8). Int. J. Mol. Sci. 2020, 21, x 9 of 37

FigureFigure 8. Benzofuran8. Benzofuran moiety, moiety, conjugated conjugated with with metal-chelating metal-chelating cyclen cyclen26 or261,4,7,10- or 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetictetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) acid (DOTA)27, designed 27, designed for Aβ ﬁbrilsfor Aβ visualization. fibrils visualization.

An inSharma vitro binding et al. designed assay with a series ([125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo of copper-coordination compounds based on an A [1,2-a]β-binding 2- 125 pyridine)phenylbenzothiazole [ I] IMPY as the competitivemoiety, conjugated ligand showed with metal-chelating dose-dependent macrocyclic inhibition with 1,4,7-triazacyclononane Ki 33.7 14.6, ± 243.5 (tacn)88.2. Fluorescentand 2,11-diaza staining [3.3]- using(2,6)pyridinophane Tg2576 mice brain (N4H sections2) 29–33 proved [46,47]the (Figure amyloid-binding 9). The ThT abilityfluorescence ± of 26 tocompetition a greater extent assay than sugge27.sts Unfortunately, a good affinity biodistribution L29−L33 for studiesAβ40 fibrils. revealed Fluorescence quite low microscopy brain uptake studies equal toon 0.33% Tg2576 and APP 0.36%, Tg–mice respectively. brain sections, with amyloid–binding Congo Red as a control, showed a Sharmaspecific et binding al. designed for organic a series ligands of copper-coordination L29−L33. The ThT compoundscompetition basedassays on with an Acopperβ-binding complexes 2-phenylbenzothiazole29−33 also revealed moiety, a strong conjugated Aβ binding with affinity metal-chelating for 32. A specific macrocyclic binding 1,4,7-triazacyclononane of the 64Cu–labeled L29−L33 (tacn) andto A 2,11-diazaβ plagues was [3.3]-(2,6)pyridinophane proven using ex vivo (N autoradiogra4H2) 29–33 [phy46,47 studies] (Figure on9 ).brain The ThTsections fluorescence of Tg2576 mice competitionand wild-type assay suggests mice as a gooda control affinity in theL29 absence–L33 for and Aβ presence40 fibrils. of Fluorescence a known A microscopyβ-specific blocking studies agent on Tg2576(B1). APPCoordinationTg-mice compounds brain sections, 29−33 with showed amyloid-binding a significant A Congoβ binding: Red the asa autoradiography control, showed intensity a specificmarkedly binding fordecreased organic in ligands the presenceL29–L33 of. B1 The blocking ThT competition agent. Biodistribution assays with copperstudies complexesin normal CD–1 64 29–33 alsomice revealed showed a the strong highest Aβ binding brain uptake affinity of for 1.33%32. A specific± 0.27% binding ID/g at of 2 themin Cu-labeledpost-injectionL29 for–L33 29. The to Aβ plaguesPET/CT wasimaging proven of the using Tg2576 ex vivo mice autoradiography showed a radiotracer studies accumulation on brain sections in the of head Tg2576 and mice neck area and wild-typefor 29, 31 mice, and as 32 a. Coordination control in the compound absence and 29 presence shows the of highest a known brain Aβ-specific uptake of blocking 0.57% ± agent 0.05% ID/g (B1). Coordinationin post-PET biodistribution compounds 29– analysis.33 showed a significant Aβ binding: the autoradiography intensity markedly decreased in the presence of B1 blocking agent. Biodistribution studies in normal CD-1 mice showed the highest brain uptake of 1.33% 0.27% ID/g at 2 min post-injection for 29. The PET/CT ± imaging of the Tg2576 mice showed a radiotracer accumulation in the head and neck area for 29, 31, and 32. Coordination compound 29 shows the highest brain uptake of 0.57% 0.05% ID/g in post-PET ± biodistribution analysis. Huang et al. developed a series of compounds based on classical amyloid-binding moiety Pittsburg compound B and used fragments 1,4-dimethyl-1,4,7-triazacyclononane (tacn) as the metal-chelating group [48] (Figure 10). The ThT fluorescence competition assays showed nanomolar affinities for the Aβ1–40 for organic ligands L34 and L35. Staining with 5xFAD mice brain sections showed significant Aβ-binding affinity of the organic ligands L34–36 and L39. The Cu2+ complexes 35, 36, and 39 also showed significant Aβ binding. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) cell viability assays on mice neuroblastoma (N2a) cells showed that coordination compounds 35, 37, and 38 exhibit no appreciable cell toxicity. Unfortunately, determination of the octanol/phosphate-buffered saline (PBS) partition coefficient values revealed that 64Cu-labeled complexes 37 and 38 exhibit log Doct values of 0.6, suggesting that 2-pyridyl-benzothiazole derivatives may be too hydrophilic to cross the BBB.

Int. J. Mol. Sci. 2020, 21, x 9 of 37

Figure 8. Benzofuran moiety, conjugated with metal-chelating cyclen 26 or 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) 27, designed for Aβ fibrils visualization.

Sharma et al. designed a series of copper-coordination compounds based on an Aβ-binding 2- phenylbenzothiazole moiety, conjugated with metal-chelating macrocyclic 1,4,7-triazacyclononane (tacn) and 2,11-diaza [3.3]-(2,6)pyridinophane (N4H2) 29–33 [46,47] (Figure 9). The ThT fluorescence

competition assay suggests a good affinity L29−L33 for Aβ40 fibrils. Fluorescence microscopy studies on Tg2576 APP Tg–mice brain sections, with amyloid–binding Congo Red as a control, showed a specific binding for organic ligands L29−L33. The ThT competition assays with copper complexes 29−33 also revealed a strong Aβ binding affinity for 32. A specific binding of the 64Cu–labeled L29−L33 to Aβ plagues was proven using ex vivo autoradiography studies on brain sections of Tg2576 mice and wild-type mice as a control in the absence and presence of a known Aβ-specific blocking agent (B1). Coordination compounds 29−33 showed a significant Aβ binding: the autoradiography intensity markedly decreased in the presence of B1 blocking agent. Biodistribution studies in normal CD–1 mice showed the highest brain uptake of 1.33% ± 0.27% ID/g at 2 min post-injection for 29. The PET/CT imaging of the Tg2576 mice showed a radiotracer accumulation in the head and neck area Int. J.for Mol. 29 Sci., 312020, and, 21 32, 9190. Coordination compound 29 shows the highest brain uptake of 0.57% ± 0.05% ID/g9 of 35 in post-PET biodistribution analysis.

Int. J. Mol. Sci. 2020, 21, x 10 of 37

Figure 9. Benzothiazole moieties, conjugated with metal-chelating 1,4,7-triazacyclononane and 2,11- diaza[3.3]-(2,6)pyridinophane L29−L33, designed for Aβ fibrils binding, and model ligand L28 without benzotiazole moiety.

Huang et al. developed a series of compounds based on classical amyloid-binding moiety Pittsburg compound B and used fragments 1,4-dimethyl-1,4,7-triazacyclononane (tacn) as the metal- chelating group [48] (Figure 10). The ThT fluorescence competition assays showed nanomolar affinities for the Aβ1–40 for organic ligands L34 and L35. Staining with 5xFAD mice brain sections showed significant Aβ-binding affinity of the organic ligands L34–36 and L39. The Cu2+ complexes 35, 36, and 39 also showed significant Aβ binding. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide) cell viability assays on mice neuroblastoma (N2a) cells showed

that coordination compounds 35, 37, and 38 exhibit no appreciable cell toxicity. Unfortunately, determination Figure 9. Benzothiazole of the octanol/phosphate-buffered moieties, conjugated with saline metal-chelating (PBS) partition 1,4,7-triazacyclononane coefficient values revealed and that2,11-diaza[3.3]-(2,6)pyridinophane 64Cu–labeled complexes 37 andL29 38–L33 exhibit, designed log D foroct values Aβ ﬁbrils of 0.6, binding, suggesting and model that ligand2-pyridyl-L28 benzothiazolewithout benzotiazole derivatives moiety. may be too hydrophilic to cross the BBB.

FigureFigure 10. 10.Pittsburg Pittsburg compound compound B B derivatives, derivatives, conjugated with with metal-chelating metal-chelating 1,4-dimethyl-1,4,7- 1,4-dimethyl-1,4,7- triazacyclononanetriazacyclononaneL34 L34–L39–39,, designed designed for for A Aββ fibrilsﬁbrils binding. binding.

Ex vivo autoradiography studies using brain sections of 5xFAD Tg–mice confirmed an amyloid– binding specificity of radiolabeled coordination compounds 35, 36, and 39, but 64Cu–labeled 34 also exhibits nonspecific binding. The MW of 36 was found to be too large for efficient brain uptake. Biodistribution studies in normal CD–1 mice proved 39 to cross the BBB, while 35 showed low brain uptake.

Int. J. Mol. Sci. 2020, 21, 9190 10 of 35

Ex vivo autoradiography studies using brain sections of 5xFAD Tg-mice confirmed an amyloid-binding specificity of radiolabeled coordination compounds 35, 36, and 39, but 64Cu-labeled 34 also exhibits nonspecific binding. The MW of 36 was found to be too large for efficient brain uptake. Biodistribution studies in normal CD-1 mice proved 39 to cross the BBB, while 35 showed low brain uptake.

3. Gd3+ and Ga3+ Coordination Compounds for Aβ Visualization Another promising emerging radionuclide for PET is 68Ga. Positron-emitting 68Ga can be obtained from a 68Ge/68Ga generator, which would facilitate cyclotron-independent distribution of PET. The parent nuclide 68Ge has a half-life of 271 days, and the generators can provide sufficient quantities of 68Ga for up to one year, resulting in a relatively inexpensive and reliable source of a positron-emitting radionuclide [51]. Ga3+ is a hard acid metal that can make strong bonds with hard base ligands such as carboxylic acids, amino nitrogen hydroxamates, and phenolates [52], which leads to the tendency to use rigid oxygen-containing chelating structures in 68Ga-based drug candidates, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA. MRI is an imaging technique based on the physical phenomenon of nuclear magnetic resonance. Various structural and functional changes including atrophy, vascular dysfunction, or changes in the volume of the hippocampus can be quantified using anatomical MRI [53]. Gadolinium(III) is the constituent of most MRI contrast agents due to a large magnetic moment (spin only effective magnetic 1 moment µeff 4 7.94 BM, from seven half-filled f-orbitals) and a long electron-spin relaxation time (108 to 109 s, from the symmetric S electronic state) [54]. Table2 summarizes the coordination compounds for magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT) diagnostics of Alzheimer’s disease, based on amyloid-affinity ligands conjugated with various metal chelating moieties:

Table 2. Gd3+, Ga3+ coordination compounds for magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT) imaging of AD.

Diagnostic Metal- Chelating Aβ-binding № Brain Uptake, % Metal Reference Method Moiety Moiety Cellebrium 0.50 0.07 MRI 40–42 ± Gd3+, 111In3+ DO3A PiB [55] Cortex SPECT 0.36 0.03 ± - 43, 44 - MRI Gd3+ DO3A PiB [56] Benzothiazole DOTA 45–60 MRI Gd3+ Benzoxazole [57] PCTA Stilbene 61 - PET Ga3+ DOTA Benzofuran [58] 62–64 - PET Ga3+ DOTA PiB [59] 0.12 0.05 ± 0.17 0.05 ± 0.31 0.09 65–70 ± PET Ga3+ HBED-CC Styrylpyridine [60] 0.21 0.05 ± 0.22 0.03 ± 0.11 0.01 ± 71 1.24 0.31 PET Ga3+ Chalkone [61] ± 72 No brain uptake - Ga3+ Curcumin [62,63] 74 No biodistribution N O Schiﬀ- base 75 - Ga 2 2 Curcumin [64] experiment 3+ ligand NODAGA 76, 77 - Ga3+ Curcumin [65] AAZTA 0.21 0.07 (5 min PET Ga3+ 78–88 ± DOTA Tacrine [66] p.i.) SPECT 99mTc3+ Int. J. Mol. Sci. 2020, 21, x 12 of 37

78– 0.21 ± 0.07 (5 min PET Ga3+ DOTA Tacrine [66] 88 p.i.) SPECT 99mTc3+

Martins et al. have designed an amyloid-targeted ligand that can efficiently complex different metal ions for various imaging modalities, including Gd3+ for MRI and 111In3+ for SPECT imaging by a conjugation of a cyclen-based macrocycle DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) with a benzothiazole moiety [55]. Ligand L40–based complexes of Gd3+, Eu3+, and 111In3+ were obtained (Figure 11). Upon binding of 40 to Aβ plaques, higher relaxivity in nuclear magnetic relaxation dispersion (NMRD) profiles was observed due to the complex becoming immobilized during plaque binding. A binding affinity of 40 to Aβ1−40 was evaluated by surface plasmon resonance measurements and Int.yielded J. Mol. Sci.Kd =2020 (180, 21 ±, 9190 10) μM, and similar Kd values were also expected for the Eu3+ and In3+ analogues11 of 35 41 and 42. The binding affinity of 40 to HSA was assessed by proton relaxation enhancement measurements and yielded Kd = 110 ± 20 μM. A specific binding of 41 to Aβ deposits was proved on postmortemMartins ethuman al. have brain designed tissue of an AD amyloid-targeted patients using fluorescence ligand that staining can eﬃ cientlywith PiB complex and thioflavin- diﬀerent 3+ 111 3+ metalS as controls. ions for Unfortunat various imagingely, the modalities, log P oct/water including −0.15 value Gd forfor 40 MRI and and also theIn highfor MW SPECT = 842 imaging shows bythat a conjugationthe complex of is a not cyclen-based optimized macrocycleto cross the DO3A BBB. In (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic vivo biodistribution experiments with the 3+ 3+ 111 3+ acid)radiolabeled with a benzothiazole 111In–analogue moiety 42 in [55 adult]. Ligand maleL40 Swiss-based mice complexes showed of that Gd cortex, Eu ,and and cerebellumIn were obtainedpenetration (Figure ID/g 11 at). 2 min was 0.36% and 0.5%, respectively.

FigureFigure11. 11. SynthesisSynthesis ofof Aβ-specific-speciﬁc DO3A–benzothiazole DO3A-benzothiazole ligand ligand L40L40 andand coordination coordination compounds compounds 4040 3+ 3+ 3+ (Gd(Gd3+), 41 (Eu3+),), and and 4242 (In(In3)+ based) based on on it, it,designed designed for for MRI MRI and and SPECT SPECT Aβ A fibrilsβ ﬁbrils visualization. visualization.

UponMartins binding et al. of subsequently40 to Aβ plaques, presented higher two relaxivity novel inDO3A nuclear monoamide magnetic relaxation derivative dispersion ligands (NMRD)conjugated profiles to the was PiB observedmoiety, 43 due and to 44 the, via complex linkers becoming differing immobilizedin length and during chemical plaque structure binding. to improve the log P-value and to enhance BBB penetration of the complexes [56] (Figure 12). A binding affinity of 40 to Aβ1 40 was evaluated by surface plasmon resonance measurements − and yielded K = (180 10) µM, and similar K values were also expected for the Eu3+ and In3+ d ± d analogues 41 and 42. The binding affinity of 40 to HSA was assessed by proton relaxation enhancement measurements and yielded K = 110 20 µM. A specific binding of 41 to Aβ deposits was proved on d ± postmortem human brain tissue of AD patients using fluorescence staining with PiB and thioflavin-S as controls. Unfortunately, the log P oct/water 0.15 value for 40 and also the high MW = 842 shows − that the complex is not optimized to cross the BBB. In vivo biodistribution experiments with the radiolabeled 111In-analogue 42 in adult male Swiss mice showed that cortex and cerebellum penetration ID/g at 2 min was 0.36% and 0.5%, respectively. Martins et al. subsequently presented two novel DO3A monoamide derivative ligands conjugated to the PiB moiety, 43 and 44, via linkers differing in length and chemical structure to improve the log P-value and to enhance BBB penetration of the complexes [56] (Figure 12). The amphiphilic compounds 43 and 44 were found to form micelles in solution. Analysis of the rotational dynamics for micelles formed using the Lipari-Szabo approach indicated highly flexible large aggregates. The coordination compounds 43 and 44 were unable to cross the BBB, and the amount detected was found to be insufficient for MRI detection. Bort et al. reported amyloid-targeted hydroxybenzothiazole, hydroxybenzoxazole, and hydroxy- trans-stilbene moieties conjugated via neutral and positive-charged linkers with PCTA (3,6,9,15-tetraaza bicyclo[9.3.1]-pentadeca1(15),11,13-triene-3,6,9-triacetic acid) and DOTA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal-chelates, and Gd(III) complexes 45–60 based on them [57] (Figure 13). The affinity of the coordination compounds 45–60 for amyloid aggregates was determined in vitro using [125I]IMPY ([125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo [1,2-a]pyridine)-binding Int. J. Mol. Sci. 2020, 21, 9190 12 of 35

competition experiments on synthetic Aβ1–42 aggregates, with DOTA-(Lys)3-BTA being the most potent. To assess the BBB permeability of the coordination compounds, an in vitro model of BBB constituted of a co-culture of rat primary brain capillary endothelial cells and rat glial cells was used. Unfortunately, none of the designed complexes showed BBB penetration ability. Int.Int. J. J. Mol. Mol. Sci.Sci. 20202020,, 2121,, xx 1313 of of 37 37

FigureFigureFigure 12. 12.12.DO3A-PiB-based DO3A–PiB–basedDO3A–PiB–based Gd Gd3+3+3+coordination coordinationcoordination compounds compounds 4343 andand 4444,,, designeddesigned designed forfor for MRIMRI MRI visualizationvisualization visualization of Aofofβ AAplagues.ββ plagues.plagues.

WatanabeTheThe amphiphilicamphiphilic et al. designed compoundscompounds and 43 andand synthesized 44 were found68Ga-labeled to form micellesmicelles benzofuran inin solution.solution. derivative AnalysisAnalysis61 ofof thewiththe 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticrotationalrotational dynamicsdynamics forfor micellesmicelles formed using the acidLipari–Szabo (DOTA) approach as the metal-chelating indicatedindicated highlyhighly agent flexibleflexible [58 ] 125 (Figurelargelarge 14 aggregates.aggregates.). A competitive TheThe coordinationcoordination Aβ1–42 binding compoundscompounds experiment 43 ofand61 (with44 were [ unableunableI] (IMPY) toto ascrosscross the competitivethethe BBB,BBB, andand ligand) thethe showedamountamount a dose-dependent detecteddetected waswas foundfound inhibition to be insufficient and values for close MRI to thedetection. clinically applied IMPY. Neuropathological fluorescentBortBort stainingetet al.al. reportedreported of Tg2576 amyloid-targeted mice brain sections hydroxybenzothiazole, treated with coordination hydroxybenzoxazole,hydroxybenzoxazole, compound 61 andandwith Thioflavinhydroxy-trans-stilbenehydroxy-trans-stilbene S as a control moietiesmoieties proved aconjugated specific binding via neutral of the and coordination positive-charged compound linkerslinkers to withwith Aβ plaques.PCTAPCTA (3,6,9,15-tetraaza bicyclo[9.3.1]-pentadeca1(15),11,13-triene-3,6,9-triacetic acid) and DOTA (1,4,7,10- A biodistribution(3,6,9,15-tetraaza experiment bicyclo[9.3.1]-pentadeca1(15),11, in normal mice showed13-triene-3,6,9-triacetic brain uptake of the acid) coordination and DOTA compound (1,4,7,10-61 tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal-chelates, and Gd(III) complexes 45–60 based (0.45%tetraazacyclododecane-1,4,7,10- ID/g), which is too low fortetraacetic the compound acid) as to metal-chelates, serve as an MRI and agent. Gd(III) complexes 45–60 based onon themthem [57][57] (Figure(Figure 13).13).

Figure 13. Cont.

Int. J. Mol. Sci. 2020, 21, 9190 13 of 35

Int. J. Mol. Sci. 2020, 21, x 14 of 37

FigureFigure 13. 13.PCTA PCTA/DOTA–benzothiazole/benzoxazole/stilbene-based/DOTA-benzothiazole/benzoxazole/stilbene-based Gd Gd3+3 +coordinationcoordination compounds compounds Int. J. Mol.4545–60–60 Sci.designed 2020 designed, 21, x for for MRI MRI visualization visualization ofof AAββ plagues. 15 of 37

The affinity of the coordination compounds 45–60 for amyloid aggregates was determined in vitro using [125I]IMPY ([125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo [1,2-a]pyridine)-binding competition experiments on synthetic Aβ1–42 aggregates, with DOTA–(Lys)3–BTA being the most potent. To assess the BBB permeability of the coordination compounds, an in vitro model of BBB constituted of a co-culture of rat primary brain capillary endothelial cells and rat glial cells was used. Unfortunately, none of the designed complexes showed BBB penetration ability. Watanabe et al. designed and synthesized 68Ga–labeled benzofuran derivative 61 with 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as the metal-chelating agent [58] (Figure 14).

A competitive Aβ1–42 binding experiment of 61 (with [125I] (IMPY) as the competitive ligand) showed 33++ a dose-dependentFigure 14. DOTA–benzofuran–basedDOTA-benzofuran-based inhibition and values GdGd closecoordination to the compound clinically applied 61 designed IMPY. for MRI Neuropathological visualization β fluorescentof A β plagues. staining of Tg2576 mice brain sections treated with coordination compound 61 with Thioflavin S as a control proved68 a specific binding of the coordination compound to Aβ plaques. A Cressier et et al. al. reported reported 68Ga–labeledGa-labeled complexes complexes conjugated conjugated to Pittsburgh to Pittsburgh Compound Compound B, 2-(4 B,′- biodistribution11 experiment in normal mice showed brain uptake of the coordination compound 61 2-(411 -[ C]methylaminophenyl)-6-hydroxybenzothiazole (PIB) and DOTA via aromatic or alkyl pacers [ C]methylaminophenyl)-6-hydroxybenzothiazole(0.45%0 ID/g), which is too low for the compound to serve(PIB) asand an MRIDOTA agent. via aromatic or alkyl pacers L62–L64L62–L64 [59][59] (Figure (Figure 15).15). The The BBB BBB permeability permeability of of th thee complexes complexes was was insufficient, insuﬃcient, as as shown shown by by μµPET.PET. Moreover, the evaluation of the complexes 62–6462–64 through an autoradiograph autoradiographicic approach with human brain tissues failed to detect amyloid deposits.

Figure 15. DOTA–Pib–based ligands L62–L64.

Zha et al. reported 68Ga–labeled styrylpyridine derivatives 65–70 with high MW based on an N,N’-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N’-diacetic acid (HBED–CC) core for Ga3+ complexation derivatized with styrylpyridinyl groups [60] (Figure 16). An in vitro competitive binding assay was conducted to measure the inhibition of [125I]IMPY Aβ binding by coordination compounds 65–70. The monovalent conjugate 69 showed a low binding affinity. The in vitro autoradiography on AD brain sections showed a high binding affinity of 65–70 to Aβ plaques, but in vivo biodistribution studies in CD–1 mice showed low brain penetration. This may allow a selective labeling of Aβ plaques deposited on the walls of cerebral blood vessels, which could be a useful tool for diagnosing cerebral amyloid angiopathy (CAA), but not in the Aβ plaques in the parenchymal brain tissues.

Int. J. Mol. Sci. 2020, 21, x 15 of 37

Figure 14. DOTA–benzofuran–based Gd3+ coordination compound 61 designed for MRI visualization of Aβ plagues.

Cressier et al. reported 68Ga–labeled complexes conjugated to Pittsburgh Compound B, 2-(4′- [11C]methylaminophenyl)-6-hydroxybenzothiazole (PIB) and DOTA via aromatic or alkyl pacers L62–L64 [59] (Figure 15). The BBB permeability of the complexes was insufficient, as shown by μPET. Moreover,Int. J. Mol. Sci. the2020 evaluation, 21, 9190 of the complexes 62–64 through an autoradiographic approach with human14 of 35 brain tissues failed to detect amyloid deposits.

FigureFigure 15. 15. DOTA–Pib–basedDOTA-Pib-based ligandsligands L62L62–L64–L64..

68 Zha et al. reported 68Ga–labeledGa-labeled styrylpyridine styrylpyridine derivatives derivatives 65–7065–70 withwith high high MW MW based on an N,N’-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N’-diacetic acidacid (HBED-CC) (HBED–CC) core core for 3+ forGa Gacomplexation3+ complexation derivatized derivatized with styrylpyridinylwith styrylpyridi groupsnyl groups [60] (Figure [60] 16(Figure). An in 16). vitro Ancompetitive in vitro 125 competitivebinding assay binding was conducted assay was to conducted measure the to inhibitionmeasure the of [inhibitionI]IMPY of A β[125bindingI]IMPY byAβ coordination binding by coordinationcompounds 65compounds–70. The monovalent65–70. The monovalent conjugate conjugate69 showed 69 showed a low binding a low binding aﬃnity. affinity. The in The vitro in vitroautoradiography autoradiography on AD on AD brain brain sections sectio showedns showed a higha high binding binding a ﬃaffinitynity of of65 65–70–70 to to AAββ plaques, but in vivo biodistribution studies in CD–1 CD-1 micemice showedshowed low brain penetration. This may allow a selective labeling labeling of of A Aββ plaquesplaques deposited deposited on on the the walls walls of of cere cerebralbral blood blood vessels, vessels, which which could could be be a usefula useful tool tool for for diagnosing diagnosing cerebral cerebral amyloid amyloid angiopathy angiopathy (CAA), (CAA), but but not not in in the the A Aββ plaquesplaques in the parenchymal brain tissues. Int. J. Mol. Sci. 2020, 21, x 16 of 37

FigureFigure 16.16. HBED–CC–styrilpiridineHBED-CC-styrilpiridine coordination coordination compounds compounds 65–7065–,70 designed, designed for PET for PET imaging imaging of Aβ of Aplaques.β plaques.

CurcuminCurcumin (C21), (C21), (1E,6E)-1,7-bis(4-hydroxy-3-me (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione,thoxyphenyl)-1,6-heptadiene-3,5-dione, is a ispromising a promising organic organic motif for motif designing for designing biologically biologically active coordination active coordination compounds. compounds.Curcumin demonstrated high antiproliferative activity in vitro and in vivo [67] and is also known to accumulate in tumor cells, presumably due to the ability to bind the vitamin–D receptor [68]. Curcumin and its derivatives are widely studied as agents for diagnosis, prevention, and treatment of AD [69,70], and also proved to be an amyloid–specific dye [71,72]. It binds to soluble Aβ plagues [73] and is reported to have sufficient brain permeability and favorable amyloid-binding in APPsw Tg-mice [74]. Curcumin is currently regarded as a specific organic core for AD therapy and diagnostic drug development. Several curcumin-based fluorescent probes for Aβ imaging have been designed [75]. A number of research works are devoted to a curcumin-based metal-containing agent for MRI, SPECT, and PET diagnostics [76]. The affinity of curcumin for amyloid plaques has raised interest in chalcone derivatives as organic core for the development of Aβ-affinity diagnostic agents. In 2007, Ono et al. reported chalcone-based probes for in vivo imaging of Aβ plaques in Alzheimer’s brains [77]. Chauhan et al. reported a bis-chalcone Ga3+–based coordination compound 71 [61] (Figure 17). The stability of coordination compound 69 in HSA was proven using ITLC–SG. Also, the high Aβ-binding affinity of 69 to HAS was proven in a protein-binding assay. Aβ-binding studies on aggregated Aβ42 were performed, and Scatchard plots suggest one-site binding with a Kd of 3.46 ± 0.41 nM.

Figure 17. Chalchone-based ligand L71, designed for Aβ plaques binding.

Blood kinetics studies of coordination compound 71 in normal rabbits showed a fast clearance during the initial time period of 30 min. Biodistribution studies showed a high uptake level of 1.24%

Int. J. Mol. Sci. 2020, 21, x 16 of 37

Figure 16. HBED–CC–styrilpiridine coordination compounds 65–70, designed for PET imaging of Aβ plaques. Int. J. Mol. Sci. 2020, 21, 9190 15 of 35 Curcumin (C21), (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a promising organic motif for designing biologically active coordination compounds. Curcumin Curcumindemonstrated demonstrated high antiproliferative high antiproliferative activity in vitro activity and in vivoin vitro [67] andand is inalso vivo known[67] to and accumulate is also knownin tumor to cells, accumulate presumably in tumor due cells, to the presumably ability to bind due tothe the vitamin–D ability to receptor bind the [68]. vitamin-D receptor [68]. Curcumin andand its its derivatives derivatives are widelyare widely studied studied as agents as agents for diagnosis, for diagnosis, prevention, prevention, and treatment and oftreatment AD [69,70 of], AD and [69,70], also proved and also to be proved an amyloid-specific to be an amyloid–specific dye [71,72]. Itdye binds [71,72]. to soluble It binds Aβ toplagues soluble [ 73Aβ] andplagues is reported [73] and tois reported have suffi tocient have brainsufficient permeability brain permeability and favorable and favo amyloid-bindingrable amyloid-binding in APPsw in Tg-miceAPPsw Tg-mice [74]. Curcumin [74]. Curcumin is currently is currently regarded regarded as a specific as a organic specific core organic for AD core therapy for AD and therapy diagnostic and drugdiagnostic development. drug development. Several curcumin-based Several curcumin-based fluorescent probes fluorescent for Aβ probesimaging for have Aβ imaging been designed have been [75]. Adesigned number [75]. of research A number works of research are devoted works to aare curcumin-based devoted to a curcumin-based metal-containing metal-containing agent for MRI, SPECT, agent andfor MRI, PET diagnosticsSPECT, and[ 76PET]. diagnostics [76]. The aaffinityffinity of of curcumin curcumin for for amyloid amyloid plaques plaques has raisedhas raised interest interest in chalcone in chalcone derivatives derivatives as organic as coreorganic for thecore development for the development of Aβ-affinity of A diagnosticβ-affinity agents. diagnostic In 2007, agents. Ono etIn al.2007, reported Ono chalcone-basedet al. reported probeschalcone-based for in vivo probesimaging for in of vivo Aβ imagingplaques of in A Alzheimer’sβ plaques in brainsAlzheimer’s [77]. Chauhanbrains [77]. et Chauhan al. reported et al. a 3+ bis-chalconereported a bis-chalcone Ga -based Ga coordination3+–based coordination compound 71compound[61] (Figure 71 17[61]). The(Figure stability 17). ofThe coordination stability of compoundcoordination69 compoundin HSA was 69 provenin HSA usingwas proven ITLC-SG. using Also, ITLC–SG. the high Also, Aβ the-binding high A aβffi-bindingnity of 69 affinityto HAS of was proven in a protein-binding assay. Aβ-binding studies on aggregated Aβ were performed, 69 to HAS was proven in a protein-binding assay. Aβ-binding studies on aggregated42 Aβ42 were and Scatchard plots suggest one-site binding with a K of 3.46 0.41 nM. performed, and Scatchard plots suggest one-site bindingd with ±a Kd of 3.46 ± 0.41 nM.

FigureFigure 17.17. Chalchone-based ligand L71, designed for Aβ plaques binding.

Blood kinetics studies of coordinationcoordination compound 71 inin normalnormal rabbitsrabbits showedshowed aa fastfast clearanceclearance during the the initial initial time time period period of of30 30min. min. Biodistribution Biodistribution studies studies showed showed a high a highuptake uptake level of level 1.24% of 1.24% 0.31% with rapid excretion within an hour. Also, PET images in a normal adult male BALB/C ± mice during 2–30 m intravenous post-injection exhibited a significant activity in the brain at 2 min post-injection and rapid washout from the healthy brain. Thus, coordination compound 71 showed no specific binding or prolonged retention in the healthy brain, due to the absence of Aβ plagues. Asti et al. reported 68Ga-labeled complexes based on curcumin, diacetyl-curcumin (DAC), and bis(dehydroxy)curcumin (bDHC) 72–74 [62] (Figure 18). The affinity of nat/68Ga-Curcuminoid complexes 72–74 for Aβ1 40 amyloid synthetic fibrils was evaluated by measuring the radioactivity of − synthetic Aβ fibrils preincubated with complexes 72–74 and also using fluorescence microscopy with untreated fibrils as a negative control. A fluorescence microscopy study of drug-preincubated A-549 tumor cells confirmed an internalization of Ga3+-curcuminoid complexes in lung cancer cells. Continuing the study, Rubagotti et al. reported [63] an in vitro and in vivo investigation of the biological properties of coordination compounds 72–74. The in vivo brain uptake was assessed using a Tg2576 mice model. Although Aβ plagues were clearly visualized after brain section staining with coordination compounds, no brain uptake in vivo was observed. These results indicate a high Aβ-affinity of gallium complexes 72–74 along with an inability of the coordination compounds to cross the BBB in vivo. 3+ Lange et al. reported [64] a six-coordinate Ga complex 75 based on an N2O2 Schiff-base ligand and β-diketone curcumin, which is known to bind to Aβ plagues because of the structural similarity to Int. J. Mol. Sci. 2020, 21, x 17 of 37

± 0.31% with rapid excretion within an hour. Also, PET images in a normal adult male BALB/C mice during 2–30 m intravenous post-injection exhibited a significant activity in the brain at 2 min post- injection and rapid washout from the healthy brain. Thus, coordination compound 71 showed no Int.specific J. Mol. binding Sci. 2020, or21, prolonged 9190 retention in the healthy brain, due to the absence of Aβ plagues.16 of 35 Asti et al. reported 68Ga–labeled complexes based on curcumin, diacetyl-curcumin (DAC), and bis(dehydroxy)curcumin (bDHC) 72–74 [62] (Figure 18). The affinity of nat/68Ga–Curcuminoid Congo Red [78] (Figure 19). The ability of 75 to bind to Aβ plaques was assessed using epi-ﬂuorescence complexes 72–74 for Aβ1−40 amyloid synthetic fibrils was evaluated by measuring the radioactivity of λ λ microscopysynthetic Aβ ( fibrilsex = 359preincubated nm, em =with461 complexes nm) on AD 72–74 and and age-matched also using human fluorescence brain microscopy samples with with an 1E8-antibodyuntreated fibrils as as control. a negative The obtainedcontrol. A results fluoresc allowence suggestingmicroscopy some study degree of drug-preincubated of speciﬁcity of A–54973 for β Atumorplaques. cells confirmed an internalization of Ga3+–curcuminoid complexes in lung cancer cells.

Figure 18. Curcumin–basedCurcumin-based Ga Ga3+3 coordination+ coordination compounds compounds 72–7472–,74 designed, designed for forPET PET imaging imaging of A ofβ Int. J. Aplaques.Mol.β plaques. Sci. 2020 , 21, x 18 of 37

Continuing the study, Rubagotti et al. reported [63] an in vitro and in vivo investigation of the biological properties of coordination compounds 72–74. The in vivo brain uptake was assessed using a Tg2576 mice model. Although Aβ plagues were clearly visualized after brain section staining with coordination compounds, no brain uptake in vivo was observed. These results indicate a high Aβ- affinity of gallium complexes 72–74 along with an inability of the coordination compounds to cross the BBB in vivo. Lange et al. reported [64] a six-coordinate Ga3+ complex 75 based on an N2O2 Schiff-base ligand and β-diketone curcumin, which is known to bind to Aβ plagues because of the structural similarity to Congo Red [78] (Figure 19). The ability of 75 to bind to Aβ plaques was assessed using epi– fluorescence microscopy (λex = 359 nm, λem = 461 nm) on AD and age-matched human brain samples with an 1E8–antibody as control. The obtained results allow suggesting some degree of specificity of 33++ 73 forFigureFigure Aβ plaques. 19.19. Curcumin-basedCurcumin–based GaGa coordinationcoordination compound compound 7575 with a SchiSchiff-basedﬀ-based metal-chelatingmetal-chelating moiety,moiety, designeddesigned forfor PETPET imagingimaging ofof AAββ plaques.plaques.

OrtecaOrteca et etal. recently al. recently reported reportedcurcumin scaffolds curcumin conjugated scaﬀolds with conjugated 1,4,7-triazacyclononane,1- with 1,4,7- triazacyclononane,1-glutaric glutaric acid-4,7-acetic acid (NODAGA) acid-4,7-acetic and 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- acid (NODAGA) and 1,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepinemethylperhydro-1,4-diazepine (AAZTA) as metal chelators L76 (AAZTA) and L77 as[65] metal (Figure chelators 20). L76 and L77 [65] (Figure 20).

Figure 20. Curcumin–based Ga3+ coordination compounds 76 and 77 with NODAGA and AAZTA metal-chelating moieties, designed for PET imaging of Aβ plaques.

Gniazdowska et al. designed a series of tacrine analogues, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor [79], the enzymes responsible for the degeneration of the neurotransmitter acetylcholine and labeled with diagnostic radionuclides technetium–99m using bifunctional ligand Hynic [80] 78–85, and gallium–68, using macrocyclic ligand DOTA 84–86 [80] (Figure 21). The Log D values for the coordination compounds are presented in Table 3. Coordination compounds 82 and 86 with the highest Log D values were selected as lead compounds.

Int. J. Mol. Sci. 2020, 21, x 18 of 37

Figure 19. Curcumin–based Ga3+ coordination compound 75 with a Schiff-based metal-chelating moiety, designed for PET imaging of Aβ plaques.

Orteca et al. recently reported curcumin scaffolds conjugated with 1,4,7-triazacyclononane,1- glutaricInt. J. Mol. acid-4,7-acetic Sci. 2020, 21, 9190 acid (NODAGA) and 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-17 of 35 methylperhydro-1,4-diazepine (AAZTA) as metal chelators L76 and L77 [65] (Figure 20).

FigureFigure 20. 20. Curcumin–basedCurcumin-based Ga Ga3+3 +coordinationcoordination compounds compounds 7676 andand 7777 withwith NODAGA NODAGA and and AAZTA AAZTA metal-chelatingmetal-chelating moieties, moieties, designed designed for for PET PET imaging imaging of of A Aββ plaques.plaques.

GniazdowskaGniazdowska et et al. al. designed designed a aseries series of of tacrine tacrine analogues, analogues, acetylcholinesterase acetylcholinesterase (AChE) (AChE) and and butyrylcholinesterasebutyrylcholinesterase (BuChE) (BuChE) inhibitor inhibitor [79], [79], the the enzymes enzymes responsible responsible for for the the degeneration degeneration of of the the neurotransmitterneurotransmitter acetylcholine acetylcholine and and labeled labeled with with diagnostic diagnostic radionuclides radionuclides technetium–99m technetium-99m using using bifunctionalbifunctional ligand ligand Hynic Hynic [80] [80 ]78–8578–85, ,and and gallium–68, gallium-68, using using macrocyclic macrocyclic ligand ligand DOTA DOTA 84–8684–86 [80][80 ] (Figure(Figure 21). 21). The The Log Log D D values values for for the the coordination coordination compounds compounds are are presented presented in in Table Table 3.3. Coordination Coordination compounds 82 and 86 with the highest Log D values were selected as lead compounds. compoundsInt. J. Mol. Sci. 82 2020 and, 21 86, x with the highest Log D values were selected as lead compounds. 19 of 37

FigureFigure 21. 21.Tacrine-based Tacrine–based99m 99mTcTc33++ coordinationcoordination compounds compounds 78–8578–85 andand Ga Ga3+3 coordination+ coordination compounds compounds 8686–88–88 with with Hynic Hynic and and DOTA DOTA metal-chelatingmetal–chelating moieties,moieties, designed for for PET PET imaging imaging of of A Aβ βplaques.plaques.

An ability of coordinationTable 3. Log compounds D values for82 coordinationand 86 to inhibit compounds acetylcholinesterase78–88. (AChE) and butyrylcholinesterase (BuChE) was estimated using Ellman’s colorimetric assay. The half maximal (CH2)n Log D inhibitory concentration IC50 values for the tested derivatives are presented in Table 4. Tacrine was 99m 68 used as the reference inhibitor.[ Tc]Tc-Hynic-NH(CH 2)nTac [ Ga]Ga-DOTA-NH(CH2)nTac 78: n = 2 2.95 0.06 - − ± 79: n = 3 2.80 0.01 - Table 3.− Log ±D values for coordination compounds 78–88. 80: n = 4 2.53 0.02 - − ± 81: n = 5 2.41 0.01 - (CH2)n − Log± D 82: n = 6 2.08 0.01 - 99m − ± 68 83 : n = 7 [ Tc]Tc–Hynic–NH(CH1.86 0.02 2)nTac 86 : [ Ga]Ga–DOTA–NH(CH2.52 0.01 2)nTac − ± − ± 78:84 n: =n 2= 8 −2.951.50 ± 0.060.01 87 : 2.02– 0.01 − ± − ± 85: n = 9 1.38 0.01 88: 1.52 0.01 79: n = 3 −−2.80 ± 0.01 − – ± 80: n = 4 −2.53 ± 0.02 – 81: n = 5 −2.41 ± 0.01 – 82: n = 6 −2.08 ± 0.01 – 83: n = 7 −1.86 ± 0.02 86: –2.52 ± 0.01 84: n = 8 −1.50 ± 0.01 87: –2.02 ± 0.01 85: n = 9 −1.38 ± 0.01 88: –1.52 ± 0.01

Table 4. The activity of 82 and 86 against two cholinesterases.

IC50 ± SD ** (nM) Compound Selectivity for AChE a Selectivity for BuChE b AChE BuChE 82 0.10 ± 0.01 0.12 ± 0.02 1.2 0.83 86 290 ± 20 167 ± 9 0.57 1.75 Tacrine 107 ± 9 16 ± 1 0.15 6.67

a Selectivity for AChE is defined as IC50(BuChE)/IC50(AChE); b Selectivity for BuChE is defined as

IC50(AChE)/IC50(BuChE). ** half maximal inhibitory concentrations ± standard deviation

An in vivo pharmacodynamic study of coordination compound 86 allowed only a qualitative view because the brain penetration was low, 0.21%. The pharmacodynamic study of coordination compound 82 was incomplete due to the low activity of the compound, and the result was therefore omitted. But the ex vivo radioactivity measurement showed that both complexes can penetrate the BBB.

4. 99mTc3+–Based Coordination Compounds for SPECT Visualization of Aβ To overcome the limitations of PET imaging in terms of cost and broad accessibility, SPECT was proposed as alternative diagnostic tool [81]. Technetium–99m (99mTc) is a desirable radioisotope for

Int. J. Mol. Sci. 2020, 21, 9190 18 of 35

An ability of coordination compounds 82 and 86 to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was estimated using Ellman’s colorimetric assay. The half maximal inhibitory concentration IC50 values for the tested derivatives are presented in Table4. Tacrine was used as the reference inhibitor.

Table 4. The activity of 82 and 86 against two cholinesterases.

IC50 SD ** (nM) Compound ± Selectivity for Selectivity for AChE BuChE AChE a BuChE b 82 0.10 0.01 0.12 0.02 1.2 0.83 ± ± 86 290 20 167 9 0.57 1.75 ± ± Tacrine 107 9 16 1 0.15 6.67 ± ± a b Selectivity for AChE is deﬁned as IC50(BuChE)/IC50(AChE); Selectivity for BuChE is deﬁned as IC (AChE)/IC (BuChE). ** half maximal inhibitory concentrations standard deviation 50 50 ± An in vivo pharmacodynamic study of coordination compound 86 allowed only a qualitative view because the brain penetration was low, 0.21%. The pharmacodynamic study of coordination compound 82 was incomplete due to the low activity of the compound, and the result was therefore omitted. But the ex vivo radioactivity measurement showed that both complexes can penetrate the BBB.

4. 99mTc3+-Based Coordination Compounds for SPECT Visualization of Aβ To overcome the limitations of PET imaging in terms of cost and broad accessibility, SPECT was proposed as alternative diagnostic tool [81]. Technetium-99m (99mTc) is a desirable radioisotope for the preparation of SPECT radiopharmaceuticals because it has a rich chemistry, unique nuclear properties 99m (T1/2 = 6 h, E = 140 keV), and an easy cost-eﬀective availability. Tc can be readily prepared by a 99Mo/99mTc generator [82]. The development of a 99mTc-radiotracer for imaging Aβ plaques with SPECT is strongly expected to provide a low cost, broadly accessible diagnostic tool for AD. Table5 summarizes the coordination compounds for single-photon emission computed tomography (SPECT) diagnostics of Alzheimer’s disease:

Table 5. 99mTc coordination compounds for single-photon emission computed tomography (SPECT) visualization of AD.

Brain Uptake, ID/g, 2 Brain /Brain № 2 min 60 min Brain Tissue Experiments Ligand Reference Min Post-Injection % Ratio 4.10 0.38 ± /6.34 0.81 ± Fluorescent staining of Re 2.30 0.27 ± complexes on /3.68 0.07 ± 8.20 APPswe/PSEN1 mice and AD Chalcone-mimic moiety 89–91 4.18 patient brain sections [83] 1.11 0.34/ with [Cp99mTc(CO) ] ± 1.73 3 1.64 0.17 ± Autoradiography on a APPswe/PSEN1 model mice With/without PgP Blocked by Cyclosporin A Curcumin-like dibenzylideneacetone 0.49 0.08 6.13 ± In vitro ﬂuorescent staining conjugated with 0.47 0.11 3.92 92–95 ± of Re complexes of brain monoamineemonoamide [84] 0.48 0.06 5.33 ± tissue APPswe/PSEN1 mice dithiol (MAMA) and BAT 0.31 0.06 2.06 ± (bis(aminoethanethiol) as chelating moieties Benzotiasole/stilbene Autoradiography Tg2576 conjugated with 96–100 0.28 0.03 2.54 [85,86] ± and wild-type mice hydroxamamide (Ham) as chelating moiety Int. J. Mol. Sci. 2020, 21, 9190 19 of 35

Table 5. Cont.

Brain Uptake, ID/g, 2 Brain /Brain № 2 min 60 min Brain Tissue Experiments Ligand Reference Min Post-Injection % Ratio Styrilpyridyl conjugated 0.25 0.05 with ± SPECT images in APP/ PS1 101–104 0.24 0.02 1.26 pyridylamine-carboxylate [87] ± transgenic mice (wild type/APP mice) and dipyridylamine ligands as chelating moiety 1.10 0.08 3.54 ± In vitro autoradiography Arylbenzoxazole conjugated 0.96 0.13 6.40 105–107 ± Brain tissue from with bis (aminoethanethiol) [88] 1.55 0.51 3.87 ± APPswe/PSEN1 mice (BAT) as chelating moiety 1.24 0.17 8.64 ± Fluorescent staining of Re complexes with brain 0.80 0.17 26.66 sections of APPswe/PSEN1T ± Benzothiazole conjugated 0.61 0.08 3.38 mice and AD patients 109–116 ± with iminodiacetic acid [89] 0.88 0.14 6.68 In vitro autoradiography on ± (IDA) as chelating moiety 1.21 0.22 20.16 brain sections of ± APPswe/PSEN1T mice and AD patients Fluorescent staining of rhenium complexes on brain slices from APPswe/PSEN1 0.69 0.16 1.50 mice and AD patients. ± 0.46 0.09 1.15 ± 0.59 0.12 1.37 Autoradiography on brain Arylbenzoxazole conjugated ± 117–132 2.11 0.11 3.40 slices from APPswe/PSEN1 with bis (aminoethanethiol) [90] ± 0.92 0.09 1.46 mice (BAT) as chelating moiety ± 0.47 0.07 2.47 Ex vivo Autoradiography ± 0.60 0.05 2.07 APPswe/PSEN1 mice ± In vivo SPECT CT Imaging − in Rhesus Monkeys Styrilpyridyl conjugated Fluorescent staining or De with 2-aminoethyl-2- 131 -- complexes of AD human [91] hydroxybenzamide as brain tissue chelating moiety Brain /Brain 0.53 0.11 2 min 90 min Fluorescence staining of Re ± 2.0 Benzothiazole conjugated 132 0.52 0.08 complexes of AD patient [92] ± Brain /Brain with tricarbonyl [M(CO) ]+ (healthy/5xFAD mice) 2 min brain and 5x FAD mice 3 90min 2.1. Phenylquinoxaline Ex vivo autoradiography conjugated with bis 133–135 0.88 0.08 3.52 [93] ± using Tg2576 mice (aminoethanethiol) (BAT) as chelating moiety Styrilpyridyl/Benzofuran Fluorescence staining of Re conjugated with 136–140 _- complexes of AD patient [94] pyridylthiosemicarbazide as brain chelating moiety Arylimidazo[2,1-b] Autoradiography of AD rat benzothiazole conjugated 0.78 0.07 8.66 141, 142 ± model (vaccinated with Aβ with triazole-based N/N/O, [95] 0.86 0.07 7.16 ± solution) N/N/N, N/N/S ligands as chelating moieties 7.94 1.46 39.7 Benzothiazole with benzene ± Fluorescent staining of AD 143–145 3.99 0.60 99.75 ring replaced by the [96] ± patient brain 5.36 0.65 59.55 cyclopentadienyl tricarbonyl ± Planar scintigraphy, 0.38 0.03 ± autoradiography and 0.35 0.01 ± ﬂuorescent staining with (AD/normal rats) Brain2 min/brain30 Thioﬂavin S and Congo Red With/without blocked min 2.33 D-(FPLIAIMA)-NH 146 studies on prepared brain 2 [97] PgP (Cyclosporine A) Brain2 min/brain30 peptide slices of AD rats (vaccinated (10 min after injection) min 1.65 with Aβ1–42) and brain 0.27 0.01 ± sections of AD and 0.60 0.01 ± Schizophrenia patients.

Liu et al. designed and synthesized novel chalcone-mimic Re/99mTc Re-89–91/[99mTc]87–91 complexes [83] (Figure 22). Ferrocene complexes were synthesized as precursors for 99mTc coordination Int. J. Mol. Sci. 2020, 21, x 22 of 37

(10 min after rats (vaccinated with injection) Aβ1–42) and brain 0.27 ± 0.01 sections of AD and 0.60 ± 0.01 Schizophrenia Int. J. Mol. Sci. 2020, 21, 9190 patients. 20 of 35

Liu et al. designed and synthesized novel chalcone–mimic Re/99mTc Re–89–91/[99mTc]87 – 91 compounds.complexes Complexes[83] (FigureRe-90 22). andFerroceneRe-91 demonstratedcomplexes were a high synthesized affinity to as Aβ precursorsplaques in brainfor 99m tissueTc sectionscoordination from ADcompounds. patients andComplexes Tg-mice Re–90 (APPswe and/ PSEN1Re–91 ),demonstrated while demonstrating a high affinity no apparent to Aβ plaques labeling inin both brain normal tissue mice sections C57BL6 from and AD normal patients adult and brainTg–mice sections. (APPswe/PSEN1 The Ki value), while ranges demonstrating established usingno anapparent Aβ1 42 labelingbinding in assay both rangednormal frommice C57 899BL6 to 108 and nM. normal As anadult extension brain sections. of the conjugatedThe Ki valueπ rangessystem, − complexestablishedRe-91 usingdemonstrated an Aβ1−42 binding the highest assay a ffirangednity. Thefromin 899 vitro toautoradiography 108 nM. As an ofextension[99mTc]89–91 of theon Tg-miceconjugated brains π system, confirmed complex the ARe–91β affi demonstratednity of [99mTc]91 the highest(Ki = 108 affinity. nM). The In the in vitro biodistribution autoradiography studies, 99m 99m [99mof Tc]89[ Tc]89–91and [99m onTc]90 Tg–miceshowed brains excellent confirmed initial the uptakes Aβ affinity and fast of clearance[ Tc]91 (Ki (respectively = 108 nM). 4.10% In the and 99m 99m 2.30%)biodistribution in the brain, studies, while [[99mTc]89Tc]91 andshowed [ Tc]90 moderate showed brain excellent uptake initial (1.11%). uptakes and fast clearance (respectively 4.10% and 2.30%) in the brain, while [99mTc]91 showed moderate brain uptake (1.11%).

99m 99m FigureFigure 22. 22.[ 99m[ Tc]Tc] coordinationcoordination compounds [[99mTc]89–91Tc]89–91 basedbased on on chalchone–mimic chalchone-mimic scaffolds scaﬀolds and and theirtheir Re Re analogues analogues89–91 89–91..

AA biodistribution biodistribution in permeability-glycoprotein in permeability-glyco blockedprotein byblocked cyclosporin by A (ancyclosporin immunosuppressant A (an drug)immunosuppressant revealed an increase drug) of revealed BBB-penetrating an increase abilities of BBB–penetrating of the coordination abilities compounds of the coordination[99mTc]89–91 . 99m 99m Thiscompounds result may [ revealTc]89–91[99m. Tc]89–91This resultto bemay substrates reveal [ forTc]89–91 the rodent to be PgP substrates transporter. for the rodent PgP transporter.Yang et al. reported four 99mTc-labeled dibenzylideneacetone derivatives [99mTc]92–95 and Yang et al. reported four 99mTc–labeled dibenzylideneacetone derivatives [99mTc]92–95 and corresponding rhenium complexes 92–95 [84] (Figure 23). correspondingInt. J. Mol. Sci. 2020 rhenium, 21, x complexes 92–95 [84] (Figure 23). 23 of 37

O O O N N

M S N M S N N O O S N O O n S n

99m 99m 92: n = 2, M = Re; [ Tc]90: n = 2, M = Tc 94: n = 2, M = Re; [99mTc]92: n = 2, M = 99mTc 93: n = 4, M = Re; [99mTc]91: n = 4, M = 99mTc 99m 99m 95: n = 4, M = Re; [ Tc]93: n = 4, M = Tc FigureFigure 23. 23.Coordination Сoordination compounds compoundsRe Re//99m99mTc92Tc 92–95–95 based based on on dibenzylideneacetone dibenzylideneacetone sca scaffoldsﬀolds with with BAT (92,BAT 93 /([92,99m 93/[Tc]92,99mTc]92, [99mTc]93 [99mTc]93) and) MAMAand MAMA (94, ( 9594,/[ 95/[99mTc]94,99mTc]94, [99m [99mTc]95Tc]95) designed) designed for for SPECT SPECT imagingimaging of Aβofplaques. Aβ plaques.

The binding affinities of rhenium complexes 92–95 for Aβ1–42 aggregates were evaluated by competition binding assay using [125I]IMPY. Coordination compounds 92 and 93 with the BAT chelating moiety showed better Aβ1–42 affinity (Ki = 24.7 and 13.6 nM) compared with coordination compounds 94 and 95 with the MAMA chelating moiety (Ki = 120.9 and 59.1 nM). Increasing the length of the spacer was found to promote Aβ1–42 binding. All four rhenium complexes, 92–95, displayed excellent labeling of Aβ plaques in in vitro fluorescent staining on sections of brain tissue from a Tg–mice (C57BL6, APPswe/PSEN1) and age–matched control mice. Biodistribution experiments of 99mTc–labeled coordination compounds [99mTc]92–95 in normal ICR mice showed the highest initial uptake at 2 min post–injection (respectively 0.49%, 0.47%, 0.48%, and 0.31% ID/g), followed by rapid washout from the brain. Iikuni et al. designed five novel 99mTc–Ham complexes [99mTc]96–99 with a bivalent amyloid ligand based on stilbene/benzothiazole moieties and HAM as chelating agent [85] (Figure 24).

Figure 24. 99mTc–HAM complexes based on stilbene and benzothiazole moieties [99mTc]96–99, designed for SPECT imaging of Aβ plaques, and model coordination compound 100.

Coordination compounds [99mTc]96–99 displayed moderate affinity for amyloid aggregates (respectively 22.2%, 42.6%, 4.6%, 38.7%), while model compound [99mTc]100, which does not include any amyloid ligands, showed no affinity. In vitro autoradiography of Tg2576 mice brain section assay proved an ability of [99mTc]96, [99mTc]97, and [99mTc]99 to bind Aβ plaques. A biodistribution experiment of [99mTc]97 with the highest binding affinity in the inhibition assay in normal mice showed very low brain uptake (0.28% ID/g). Further, the authors of Reference [86] applied coordination compounds [99mTc]96–99 to CAA– specific imaging probes and evaluated their utility for CAA–specific imaging. An in vitro inhibition

Int. J. Mol. Sci. 2020, 21, x 23 of 37

O O O N N

M S N M S N N O O S N O O n S n

99m 99m 92: n = 2, M = Re; [ Tc]90: n = 2, M = Tc 94: n = 2, M = Re; [99mTc]92: n = 2, M = 99mTc 93: n = 4, M = Re; [99mTc]91: n = 4, M = 99mTc 99m 99m 95: n = 4, M = Re; [ Tc]93: n = 4, M = Tc Figure 23. Сoordination compounds Re/99mTc 92–95 based on dibenzylideneacetone scaffolds with Int. J. Mol.BAT Sci. (92,2020 93/[, 2199m,Tc]92, 9190 [99mTc]93) and MAMA (94, 95/[99mTc]94, [99mTc]95) designed for SPECT imaging21 of 35 of Aβ plaques.

The binding affinities of rhenium complexes 92–95 for Aβ1–42 aggregates were evaluated by The binding affinities of rhenium complexes 92–95 for Aβ1–42 aggregates were evaluated by competition binding assay using [125I]IMPY. Coordination compounds 92 and 93 with the BAT chelating competition binding assay using [125I]IMPY. Coordination compounds 92 and 93 with the BAT moiety showed better Aβ1–42 affinity (Ki = 24.7 and 13.6 nM) compared with coordination compounds chelating moiety showed better Aβ1–42 affinity (Ki = 24.7 and 13.6 nM) compared with coordination 94 and 95 with the MAMA chelating moiety (Ki = 120.9 and 59.1 nM). Increasing the length of the compounds 94 and 95 with the MAMA chelating moiety (Ki = 120.9 and 59.1 nM). Increasing the spacer was found to promote Aβ1–42 binding. All four rhenium complexes, 92–95, displayed excellent length of the spacer was found to promote Aβ1–42 binding. All four rhenium complexes, 92–95, labeling of Aβ plaques in in vitro fluorescent staining on sections of brain tissue from a Tg-mice displayed excellent labeling of Aβ plaques in in vitro fluorescent staining on sections of brain tissue (C57BL6, APPswe/PSEN1) and age-matched control mice. Biodistribution experiments of 99mTc-labeled from a Tg–mice (C57BL6, APPswe/PSEN1) and age–matched control mice. Biodistribution coordination compounds [99mTc]92–95 in normal ICR mice showed the highest initial uptake at 2 min experiments of 99mTc–labeled coordination compounds [99mTc]92–95 in normal ICR mice showed the post-injection (respectively 0.49%, 0.47%, 0.48%, and 0.31% ID/g), followed by rapid washout from highest initial uptake at 2 min post–injection (respectively 0.49%, 0.47%, 0.48%, and 0.31% ID/g), the brain. followed by rapid washout from the brain. Iikuni et al. designed five novel 99mTc-Ham complexes [99mTc]96–99 with a bivalent amyloid Iikuni et al. designed five novel 99mTc–Ham complexes [99mTc]96–99 with a bivalent amyloid ligand based on stilbene/benzothiazole moieties and HAM as chelating agent [85] (Figure 24). ligand based on stilbene/benzothiazole moieties and HAM as chelating agent [85] (Figure 24).

FigureFigure 24.24.99m 99mTc–HAMTc–HAM complexes complexes based based on stilbene on stilbene and benzothiazole and benzothiazole moieties [moieties99mTc]96 –[9999m,Tc]96–99 designed, fordesigned SPECT for imaging SPECT of imaging Aβ plaques, of Aβ and plaques, model and coordination model coordination compound compound100. 100.

CoordinationCoordination compoundscompounds [[99m99mTc]96–99Tc]96–99 displayed moderate aaffinityffinity forfor amyloidamyloid aggregatesaggregates (respectively(respectively 22.2%,22.2%, 42.6%,42.6%, 4.6%,4.6%,38.7%), 38.7%), while while model model compound compound[ 99m[99mTc]100,, whichwhich doesdoes notnot includeinclude anyany amyloid ligands, ligands, showed showed no no affinity. affinity. In Invitro vitro autoradiographyautoradiography of Tg2576 of Tg2576 mice mice brain brain section section assay assayproved proved an ability an ability of [ of99m[Tc]9699mTc]96, [99m, [Tc]9799mTc]97, and, and [99m[99mTc]99Tc]99 to tobind bind A Aββ plaques.plaques. A biodistributionbiodistribution experimentexperiment of of[99m [99mTc]97Tc]97with with the the highest highest binding binding affinity affinity in the in inhibition the inhibition assay inassay normal in micenormal showed mice veryshowed low very brain low uptake brain (0.28% uptake ID (0.28%/g). ID/g). Further,Further, the authors authors of of Reference Reference [86] [86 applied] applied coordination coordination compounds compounds [99mTc]96–99[99mTc]96 to– CAA–99 to CAA-specificspecific imaging imaging probes probes and evaluated and evaluated their utility their for utility CAA–specific for CAA-specific imaging. imaging.An in vitro An inhibitionin vitro inhibition assay using Aβ1–40 aggregates deposited mainly in CAA showed a high binding affinity of coordination compounds [99mTc]96–99. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in Tg-mice of coordination compounds [99mTc]97 and [99mTc]99. + Hayne et al. reported [87] tridentate ligands L101–L104 designed to bind to the [M(CO)3] core (M = Tc/Re) conjugated with a stilbene Aβ-binding moiety (Figure 25). The complexes 101 and 103 showed little to no plaque binding in brain tissue from AD-positive subjects. Epi-fluorescence Int.Int. J. J. Mol. Mol. Sci. Sci. 2020 2020, ,21 21, ,x x 2424 of of 37 37 assayassay usingusing AAββ1–401–40 aggregatesaggregates depositeddeposited mainlymainly inin CAACAA showedshowed aa highhigh bindingbinding affinityaffinity ofof coordinationcoordination compoundscompounds [[99m99mTc]96–99Tc]96–99. . InIn vitrovitro autoradiographyautoradiography ofof humanhuman CAACAA brainbrain sectionssections andand exex vivovivo autoradiographyautoradiography ofof Tg2576Tg2576 micemice displayeddisplayed excellentexcellent labelinglabeling ofof AAββ depositionsdepositions inin humanhuman CAACAA brain brain sections sections and and high high affinity affinity and and selectivity selectivity to to CAA CAA in in Tg–mice Tg–mice of of coordination coordination compounds compounds [[99m99mTc]97Tc]97 and and [ [99m99mTc]99Tc]99. . Int. J. Mol. Sci. 2020, 21, 9190 22+ of 35 HayneHayne etet al.al. reportedreported [87][87] tridentatetridentate ligandsligands L101–L104L101–L104 designeddesigned toto bindbind toto thethe [M(CO)[M(CO)33]]+core core (M(M == Tc/Re)Tc/Re) conjugatedconjugated withwith aa stilbenestilbene AAββ–binding–binding moietymoiety (Figure(Figure 25).25). TheThe complexescomplexes 101101 andand 103103 showed little to no plaque binding in brain tissue from AD–positive subjects. Epi–fluorescence microscopyshowed little of to tissue no plaque sections binding of the in frontal brain cortex tissue of from an AD-aAD–positiveffected brain subjects. treated Epi–fluorescence with 102 and microscopy of tissue sections of the frontal cortex of an AD–affected brain treated with 102 and 104 104microscopybearing anof tissue electron-donating sections of the dimethylamino frontal cortex functionalof an AD–affected group revealed brain treated good correlationwith 102 and of 104 the bearing an electron–donating dimethylamino functional group revealed good correlation of the complexesbearing an toelectron–donating Aβ plaques, and thedimethylamino E18 antibody functi was usedonal as group a control. revealed good correlation of the complexescomplexes to to A Aββ plaques, plaques, and and the the E18 E18 antibody antibody was was used used as as a a control. control.

FigureFigure 25. 25.25. Tridentate Tridentate ligands ligands L101–L104 L101L101–L104–L104 conjugated conjugatedconjugated with with a a stilbene stilbene A Aββ–binding–binding-binding moiety moietymoiety designed designeddesigned for for β AAAββ plaques plaques binding, binding, and and the the proposed proposedproposed structure structurestructure of ofof coordination coordinationcoordination compound compound 101 101.. .

99m The biodistribution of the radiolabeled coordination compound [99m Tc]103 was investigated in TheThe biodistributionbiodistribution ofof thethe radiolradiolabeledabeled coordinationcoordination compoundcompound [[99mTc]103Tc]103 waswas investigatedinvestigated inin both wild-type and APP/PS1 Tg-mice. Low brain uptake (~0.25%) was registered in both cases, and no bothboth wild–type wild–type and and APP/PS1 APP/PS1 Tg–mice. Tg–mice. Low Low brain brain uptake uptake (~0.25%) (~0.25%) was was registered registered in in both both cases, cases, and and nostatistically statistically signiﬁcant significant diﬀ differenceerence between between wild-type wild–type and and Tg-mice Tg–mice was was observed. observed. no statistically significant difference between99m wild–type and Tg–mice was observed. 99m Wang et al. reported four neutral Re99m/ Tc-labeled coordination compounds 105–10899m /[ Tc] WangWang et et al. al. reported reported four four neutral neutral Re/ Re/99mTc–labeledTc–labeled coordination coordination compounds compounds 105–108 105–108//[[99mTc]105–Tc]105– 105–108 based on arylbenzoxazole moieties conjugated with bis(aminoethanethiol) (BAT) as a chelating 108108 basedbased onon arylbenzoxazolearylbenzoxazole moietiesmoieties conjugatedconjugated withwith bis(aminoethanethiol)bis(aminoethanethiol) (BAT)(BAT) asas aa chelatingchelating moiety [88] (Figure 26). moietymoiety [88] [88] (Figure (Figure 26). 26).

3+ 99m 3+ 99m Figure 26. Re 3+( 105–108) 99mTc 3+ ([ 99mTc]105–108) complexes based on arylbenzoxazole with a BAT Figure 26.26. ReRe3+ ((105–108105–108)) 99mTcTc3 +([([99mTc]105–108)Tc]105–108 complexes) complexes based based on on arylbenzoxazole arylbenzoxazole with with a a BAT metal–chelating moiety, designed for SPECT imaging of Aβ plaques. metal-chelatingmetal–chelating moiety,moiety, designed designed for for SPECT SPECT imaging imaging of of A Aββplaques. plaques.

In vitro fluorescent staining with rhenium complexes 105–108 with Aβ plaques, neuropathological staining with the brain sections of a Tg-mice and an AD patient showed specific Aβ-binding of the complexes. An in vitro competition binding assay was performed using [125I] IMPY as the competing radioligand. A moderate Aβ-binding affinity of 105 and 106 (Ki = 128.21 and 393.18 nM) and a high affinity of complexes 107 and 108 (Ki = 15.86 and 37.19 nM) with N,N-dimethyl amino Int. J. Mol. Sci. 2020, 21, x 25 of 37

In vitro fluorescent staining with rhenium complexes 105–108 with Aβ plaques, neuropathological staining with the brain sections of a Tg–mice and an AD patient showed specific AInt.β–binding J. Mol. Sci. 2020of the, 21 ,complexes. 9190 An in vitro competition binding assay was performed using [125I] 23IMPY of 35 as the competing radioligand. A moderate Aβ–binding affinity of 105 and 106 (Ki = 128.21 and 393.18 nM) and a high affinity of complexes 107 and 108 (Ki = 15.86 and 37.19 nM) with N,N–dimethyl amino 99m group was was estimated. estimated. 99mTc–labeledTc-labeled complexes complexes were were prepared prepared by bya ligand a ligand exchange exchange reaction reaction from from the 99m intermediatethe intermediate 99mTc–glucoheptonate.Tc-glucoheptonate. In vitroIn vitro autoradiographyautoradiography in inTg–mice Tg-mice brain brain tissue tissue showed 99m labelinglabeling of cortex, hippocampus, and cerebellum regions byby [[99mTc]107Tc]107.. Biodistribution studies studies of coordination compounds compounds displayed displayed higher higher initial initial brain brain uptake uptake of N,N–dimethylated of N,N-dimethylated derivatives derivatives and and brain /brain ratio than the N-monomethylated analogs ([99mTc]105 vs [99mTc]107 and brain2min/brain2min 60min ratio60min than the N–monomethylated analogs ([99mTc]105 vs [99mTc]107 and [99mTc]106 99m 99m vs[ [99mTc]106Tc]108vs). [ Tc]108). JiaJia et al. reported reported a a design and biological evaluation evaluation of of a series of negatively charged imaging β probes with with limited limited BBB BBB penetration penetration for for thethe selective selective detection detection of vascular of vascular Aβ deposition A deposition [89]. Eight [89]. Eight 99mTc(CO) -labeled benzothiazole derivatives [99mTc]109–116 and their Re(III) analogues 109–116 99mTc(CO)3–labeled3 benzothiazole derivatives [99mTc]109–116 and their Re(III) analogues 109–116 β were designed as potential SPECT imaging probes for cerebrovascular A Aβ depositiondeposition (Figure (Figure 27).27). Rhenium surrogates 109–116 displayed high affinities to Aβ aggregates with K values ranging from Rhenium surrogates 109–116 displayed high affinities to Aβ aggregates with Ki values ranging from 42 to 106 106 nM, nM, rhenium rhenium complex 116 with the longest carbon linker length (n = 6)6) displayed displayed the the highest highest affinity to Aβ aggregates (K = 42.2 nM). Complex 115 also demonstrated unambiguous and affinity to Aβ1−142 aggregates42 (Ki = 42.2i nM). Complex 115 also demonstrated unambiguous and specific − β 99m labelingspecific labelingof Aβ plaques of A plaques in brain inbrain sections sections from from Tg–mice. Tg-mice. 99mTc–labeledTc-labeled coordination coordination compounds compounds [99mTc]109–116 were obtained by ligand exchange reactions with fac–[99mTc(CO) (H O) ]+. [99mTc]109–116 were obtained by ligand exchange reactions with fac–[99mTc(CO)3(H3 2O)2 3]3+.

Figure 27. Negatively charged imaging probes [99mTc]109–116 designed for the selective detection of Figure 27. Negatively charged imaging probes [99mTc]109–116 designed for the selective detection of vascular Aβ deposition, and their Re3+ analogues 109–116. vascular Aβ deposition, and their Re3+ analogues 109–116 .

Autoradiography studiesstudies in in AD AD human human brain brain tissue tissue proved proved the ability the of ability coordination of coordination compound compound[99mTc]116 to[99m bindTc]116 Aβ todeposits bind Aβ in deposits bloodvessels in blood but vessels not in but cerebral not in parenchymacerebral parenchyma on brain on sections brain sectionsof an AD of patient,an AD patient, while [ 125whileI]IMP [125 labeledI]IMP labeled both. both. Ex vivo Ex autoradiographyvivo autoradiography studies studies in Tg-mice in Tg–mice and andwild-type wild–type mice mice were were also also performed. performed. The The radioactive radioactive spots spots were were found found to to concentrate concentrate atat the site of the the blood blood vessels vessels in inthe the Tg–mice Tg-mice brain brain tissue, tissue, as identified as identified by in byvitroin vitrofluorescencefluorescence staining staining using thioflavin–S.using thioflavin-S. Biodistribution Biodistribution studies studies of [99mTc]116 of [99m Tc]116show ashow relatively a relatively low brain low uptake brain uptake equal to equal 1.21% to ±1.21% 0.22% ID/g0.22% at ID2 min/g at post–injection 2 min post-injection and rapid and blood rapid washout blood washout with an withapproximately an approximately 23–fold decline 23-fold ± indecline blood in radioactivity blood radioactivity at 60 min at 60post–injection. min post-injection. Other Othercomplexes complexes showed showed worse worse brain brainuptake. uptake. The authorsThe authors claimed claimed that coordination that coordination compounds compounds [99mTc]109–116 [99mTc]109–116 are prospectiveare prospective as cerebrovascular as cerebrovascular Aβ– visualizationAβ-visualization agents. agents. Zhang et al. designed designed a series of sixteen 99m99mTc–labeledTc-labeled imaging probes [99m99mTc]117–132Tc]117–132 for Aβ plaques basedbased on on 2-arylbenzothiazoles 2–arylbenzothiazoles conjugated conjugated with with a bis(aminoethanethiol) a bis(aminoethanethiol) (BAT) (BAT) chelating chelating moiety moietyand their and Re(III) their analogues Re(III) analogues117–132 [117–13290] (Figure [90] 28 (Figure). An in 28). vitro Anbinding in vitro a ffibindingnity of affinity rhenium of complexes rhenium complexes 117–132 to aggregated Aβ1−42 peptide was estimated by a competitive binding assay125 using 117–132 to aggregated Aβ1 42 peptide was estimated by a competitive binding assay using [ I]IMPY − [as125 aI]IMPY reference as ligand.a reference The results ligand. obtained The results proved obtain that bothed proved the introduction that both of athe dimethylamine introduction groupof a dimethylamineand an increase ingroup the lengthand an of increase the linker in betweenthe length the of amyloid the linker affinity between and the the metal-chelating amyloid affinity moiety and thepromotes metal–chelating Aβ binding moiety of the resultingpromotes coordination Aβ binding compounds. of the resulting Compounds coordination120 and 122compounds.showed a Compoundsbinding affinity 120 (respectivelyand 122 showed 8.4 and a binding 8.8 nM) affinity surpassing (respectively that of IMPY, 8.4 and a widely 8.8 nM) used surpassing imaging that agent. of Binding of the coordination compound to Aβ plaques in Tg-mice and AD brain tissue samples was also proven using in vitro fluorescent staining with thioflavin-S as a control. Int. J. Mol. Sci. 2020, 21, 9190 24 of 35

99mTc-labeled probes [99mTc]117–132 were obtained using a ligand exchange reaction with 99mTc glucoheptonate. The ability of the puriﬁed 99mTc-labeled probes [99mTc]118–134 to bind Aβ − plaques was tested in brain slices from Tg-mice. Biodistribution studies of 99mTc-labeled complexes were conducted. [99mTc]124 indicated its suitability as a diagnostic probe. 99mTc-labeled coordination Int. J. Mol. Sci. 2020, 21, x 26 of 37 compound [99mTc]124 showed relatively high initial brain uptake (2.11% ID/g at 2 min) and a reasonable IMPY,clearance a widely rate (0.62% usedID imaging/g at 60 min),agent. in Binding contrast of to the other coordination complexes, compound which exhibited to Aβ poor plaques brain in uptake Tg– mice(less and than AD 1% brain ID/ g tissue at 2 min) samples and slow was clearance,also proven presumably using in vitro because fluorescent of their staining higher lipophilicitywith thioflavin– and Snonspeciﬁc as a control. binding to plasma proteins.

FigureFigure 28. 28. Re(III)Re(III) coordination coordination compounds compounds 117–132117–132 basedbased on on 2–arylbenzothiazoles 2-arylbenzothiazoles conjugated conjugated with with a a BATBAT chelating chelating moiety. moiety.

SPECT images of coordination compound [99mTc]122 in rhesus monkeys were registered, and the 99mTc–labeled probes [99mTc]117–132 were obtained using a ligand exchange reaction with images revealed radioactivity accumulation in the brain, indicating permeation of [99mTc]121 through 99mTc−glucoheptonate. The ability of the purified 99mTc–labeled probes [99mTc]118–134 to bind Aβ the BBB (Table6). This is the first assessment of a 99mTc-labeled Aβ probe in nonhuman primates. plaques was tested in brain slices from Tg–mice. Biodistribution studies of 99mTc–labeled complexes 99m 99m were Tableconducted. 6. [ 99m[ Tc]122Tc]124brain indicated accumulation its suitability in rhesus as a diagnostic monkeys probe. (M04: 4-year-old,Tc–labeled male; coordination F27: 99m compound27-year-old, [ Tc]124 female). showed relatively high initial brain uptake (2.11% ID/g at 2 min) and a reasonable clearance rate (0.62% ID/g at 60 min), in contrast to other complexes, which exhibited poor brain uptake (less than0–10 1% Min ID/ g 10–20at 2 min) Min and 20–30 slow Min clearance, 30–40 presumably Min Clearance because Ratio of their higher lipophilicity M04and nonspecific 1.23 binding 1.13 to plasma proteins. 1.01 0.88 1.40 SPECT imagesF27 of coordination 0.78 0.70compound [99m 0.67Tc]122 in rhesus 0.64 monkeys were 1.22 registered, and the images revealed radioactivity accumulation in the brain, indicating permeation of [99mTc]121 throughHayne the etBBB al. reported(Table 6). oxotechentium(V) This is the first assessment and oxorhenium(V) of a 99mTc–labeled complexes [A99mβ probeTc]133 inand nonhuman133 based primates.on a styrylpyridyl functional group with 2-aminoethyl-2-hydroxybenzamide as a chelating moiety [91] (Figure 29). The affinity of 133 for Aβ1 42 fibrils was estimated to be Ki = 855 nM using a fluorescence − competitionTable 6. [ assay99mTc]122 against brain Thioflavin accumulation T. It in was rhesus also monkeys shown that (M04:133 4–year–old,binds to Amale;β plaques F27: 27–year–old, in human brain tissuefemale). using human AD brain sections. 99m 99m Kiritsis 0–10 et al. Min reported a10–20 2-(40-aminophenyl)benzothiazole-based Min 20–30 Min 30–40 MinTc-radioagent Clearance [ RatioTc]134 and its Re(III)M04 analogue1.23134 [92] (Figure1.13 30 ). A strong a1.01ffinity of 134 for0.88 Aβ plaques in brain sections1.40 from an ADF27 patient was0.78 proven using confocal0.70 microscopy.0.67 The binding affi0.64nity of 134 for Aβ421.22was measured in vitro by competition binding assay between the stable 134 and its radioactive 99mTc-labeled analogue 99m [ Tc]134Hayne, et and al. thereported obtained oxotec Ki washentium(V) 13.6 4.8 and nM. oxorhenium(V) complexes [99mTc]133 and 133 based 99m± on a styrylpyridylBiodistribution functional experiments group of with [ 2–aminoTc]134 inethyl–2–hydroxybenzamide Swiss albino mice revealed as a a chelating moderate moiety initial [91]brain (Figure uptake 29). of 0.53%The affinity ID/g atof 2 133 min for and Aβ slow1−42 fibrils clearance was ofestimated radioactivity to be fromKi = 855 the brainnM using with a a 99m fluorescencebrain2min/brain competition90min ratio assay of 2.1. against Administration Thioflavin of T. [ It wasTc]134 alsoin shown 5xFAD that Tg-mice 133 binds showed to A thatβ plaques 0.52% inID human/g of radioactivity brain tissue is using recorded human inthe AD brain brain at sections. 2 min, a result similar to that in healthy mice. But the significant increase of radioactivity in the brain of 5xFAD Tg-mice with time (1.94% ID/g at 90 min post-injection) is consistent with retention of [99mTc]134 through binding to Aβ plaques. Iikuni et al. reported three novel 99mTc complexes [99mTc]135–137 based on a phenylquinoxaline scaffold and their model Re(III) analogues 135–137 [93] (Figure 31).

Int. J. Mol. Sci. 2020, 21, x 27 of 37

Int. J. Mol. Sci. 2020, 21, 9190 25 of 35

Int. J. Mol. Sci. 2020, 21, x 27 of 37

Figure 29. Oxorhenium(V) complexes 133 based on a styrylpyridyl functional group with 2- aminoethyl-2-hydroxybenzamide as a chelating moiety, designed for SPECT imaging of Aβ plaques.

Kiritsis et al. reported a 2–(4′–aminophenyl)benzothiazole–based 99mTc–radioagent [99mTc]134 and its Re(III) analogue 134 [92] (Figure 30). A strong affinity of 134 for Aβ plaques in brain sections from an AD patient was proven using confocal microscopy. The binding affinity of 134 for Aβ42 was FigureFigure 29. 29. Oxorhenium(V)Oxorhenium(V) complexes complexes 133133 basedbased on ona styrylpyridyl a styrylpyridyl functional functional group group with with 2- 99m measuredaminoethyl-2-hydroxybenzamide2-aminoethyl-2-hydroxybenzamide in vitro by competition asbinding asa chelating a chelating assay moiety, moiety,between designed designed the sTable for for SPECT SPECT 134 imaging and imaging its ofradioactive of A Aββ plaques.plaques. Tc– labeled analogue [99mTc]134, and the obtained Ki was 13.6 ± 4.8 nM. Kiritsis et al. reported a 2–(4′–aminophenyl)benzothiazole–based 99mTc–radioagent [99mTc]134 and its Re(III) analogue 134 [92] (Figure 30). A strong affinity of 134 for Aβ plaques in brain sections from an AD patient was proven using confocal microscopy. The binding affinity of 134 for Aβ42 was measured in vitro by competition binding assay between the sTable 134 and its radioactive 99mTc– labeled analogue [99mTc]134, and the obtained Ki was 13.6 ± 4.8 nM.

Figure 30. 2-(4′-aminophenyl)benzothiazole-based99m 99mTc–radioagent99m [99mTc]134 and its Re(III) Figure 30. 2-(40-aminophenyl)benzothiazole-based Tc-radioagent[ Tc]134 and its Re(III) analogue analogue134, designed 134, fordesigned SPECT for imaging SPECT of imaging Aβ plaques. of Aβ plaques.

BiodistributionAn in vitro binding experiments experiment of [99m inTc]134 solution in showedSwiss albino promising mice revealed Aβ affinity a moderate for complex initial135 brainand uptakeaverage of binding 0.53% a ffiID/gnity forat complex2 min and136 .slow The afficlearancenity increased of radioactivity in the order from of the the N,N-dimethylated brain with a brainderivativeFigure2min/brain >30.N-monomethylated90min 2-(4 ratio′-aminophenyl)benzothiazole-based of 2.1. Administration derivative > primary of [99m 99mTc]134 aminoTc–radioagent in derivative. 5xFAD [99m Tg–miceTc]134 andshowed its Re(III)that 0.52% ID/ganalogue Theof radioactivity brain 134,uptake designed is recorded for for 99mSPECTTc-labeled in theimaging brain complexof at A 2β miplaques.n, [a99m result Tc]135 similarwas to found that in to healthy be 0.88%, mice. and But the brain2min/brain60min ratio was 3.52. An ex vivo autoradiographic examination was also performed usingBiodistribution a Tg2576 mice, experiments and [99mTc]135 of [99mshowedTc]134 intensivein Swiss albino radioactive mice revealed spots insections a moderate from initial the Tg2576 brain uptakemice but of not 0.53% from ID/g the age-matched at 2 min and mice. slow In addition, clearance these of spotsradioactivity corresponded from withthe Abrainβ depositions with a brainconfirmed2min/brain by90min fluorescent ratio ofstaining 2.1. Administration in the same sectionsof [99mTc]134 with thioflavin-S.in 5xFAD Tg–mice showed that 0.52% ID/g of radioactivity is recorded in the brain at 2 min, a result similar to that in healthy mice. But the

Int. J. Mol. Sci. 2020, 21, x 28 of 37 significant increase of radioactivity in the brain of 5xFAD Tg–mice with time (1.94% ID/g at 90 min post–injection) is consistent with retention of [99mTc]134 through binding to Aβ plaques. Int. J.Iikuni Mol. Sci. et2020 al. ,reported21, 9190 three novel 99mTc complexes [99mTc]135–137 based on a phenylquinoxaline26 of 35 scaffold and their model Re(III) analogues 135–137 [93] (Figure 31).

FigureFigure 31. 31. 99m99mTcTc complexes complexes [99m [99mTc]135–137Tc]135–137 basedbased on on a aphenylquinoxaline phenylquinoxaline scaffold scaﬀold and and their their model model Re(III)Re(III) analogues analogues 135–137,135–137, designeddesigned for for SPECT SPECT imaging imaging of of A Aββ plaques.plaques.

AnFletcher in vitro et binding al. reported experiment six Re(III) in solution complexes showed138–142 promisingbased onAβ styrilpyridylaffinity for complex and benzofuran 135 and averagemoieties binding [94] (Figure affinity 32 for). Ancomplex affinity 136 to. The Aβ affinityplagues increased was investigated in the order using of the a ThTN,N-dimethylated assay, and the derivativeobtained results> N-monomethylated suggested that derivative the complexes > primary either amino bind competitively derivative. with ThT to Aβ1–42 fibrils 99m 99m 99m or inhibitThe brain fibril uptake formation. for 99mTc–labeledTc-labeled coordinationcomplex [99m compoundsTc]135 was [foundTc]138 to andbe 0.88%, [ Tc]139 and werethe brainInt.also J. Mol. obtained.2min/brain Sci. 202060min, 21 , ratiox was 3.52. An ex vivo autoradiographic examination was also performed29 of 37 using a Tg2576 mice, and [99mTc]135 showed intensive radioactive spots in sections from the Tg2576 mice but not from the age–matched mice. In addition, these spots corresponded with Aβ depositions confirmed by fluorescent staining in the same sections with thioflavin–S. Fletcher et al. reported six Re(III) complexes 138–142 based on styrilpyridyl and benzofuran moieties [94] (Figure 32). An affinity to Aβ plagues was investigated using a ThT assay, and the obtained results suggested that the complexes either bind competitively with ThT to Aβ1–42 fibrils or inhibit fibril formation. 99mTc–labeled coordination compounds [99mTc]138 and [99mTc]139 were also obtained.

FigureFigure 32. Re(III)Re(III) complexescomplexes138 138–142–142 based based on styrilpyridylon styrilpyridyl and benzofuranand benzofuran moieties, moieties, and 99m andTc labeled 99mTc labeledcoordination coordination compounds compounds [99mTc]138 [99mandTc] [13899m Tc]and139, [99mdesignedTc]139, designed for SPECT for imaging SPECT of imaging Aβ plaques. of Aβ plaques.

Molavipordanjani et al. reported two novel radiolabeled 2–arylimidazo[2,1–b]benzothiazoles 143 and 144 [95] (Figure 33). The affinity of the coordination compounds for Aβ1–42 aggregates was evaluated, and both radiolabeled complexes showed a significant Aβ binding. Tissue staining and autoradiography with Congo Red as a control proved an ability of the obtained complexes 143 and 144 to bind to Aβ plaques in the brain sections of the rat AD model. Biodistribution studies in normal BALB/C mice showed an initial brain uptake of 0.78% and 0.86% ID/g respectively, for 143 and 144 in normal mice, followed by a nearly complete washout within an hour.

Figure 33. Radiolabeled 2–arylimidazo[2,1–b]benzothiazoles 143 and 144, designed for SPECT imaging of Aβ plaques.

Int. J. Mol. Sci. 2020, 21, x 29 of 37

Figure 32. Re(III) complexes 138–142 based on styrilpyridyl and benzofuran moieties, and 99mTc Int. J. Mol. Sci. 2020, 21, 9190 27 of 35 labeled coordination compounds [99mTc]138 and [99mTc]139, designed for SPECT imaging of Aβ plaques. Molavipordanjani et al. reported two novel radiolabeled 2-arylimidazo[2,1-b]benzothiazoles Molavipordanjani et al. reported two novel radiolabeled 2–arylimidazo[2,1–b]benzothiazoles 143 and 144 [95] (Figure 33). The aﬃnity of the coordination compounds for Aβ1–42 aggregates was 143 and 144 [95] (Figure 33). The affinity of the coordination compounds for Aβ1–42 aggregates was evaluated, and both radiolabeled complexes showed a signiﬁcant Aβ binding. Tissue staining and evaluated, and both radiolabeled complexes showed a significant Aβ binding. Tissue staining and 143 144 autoradiographyautoradiography with with Congo Congo Red Red as as a controla control proved proved an an ability ability of of the the obtained obtained complexes complexes 143and and β to144 bind to to bind A toplaques Aβ plaques in the in the brain brain sections sections of of the the rat rat AD AD model. model. Biodistribution studies studies inin normal normal BALBBALB/C/C mice mice showed showed an an initial initial brain brain uptake uptake of of 0.78% 0.78% and and 0.86% 0.86% IDID/g/g respectively,respectively, forfor 143143 andand 144 in normalin normal mice, mice, followed followed by a by nearly a nearly complete complete washout washout within within an an hour. hour.

FigureFigure 33. 33.Radiolabeled Radiolabeled 2-arylimidazo[2,1-b]benzothiazoles 2–arylimidazo[2,1–b]benzothiazoles143 143and 144,and designed144, designed for SPECT for SPECT imaging ofimaging Aβ plaques. of Aβ plaques. Int. J. Mol. Sci. 2020, 21, x 30 of 37 Sagnou et al. reported synthesis of three novel 99mTc complexes [99mTc]145–[99mTc]147 and Sagnou et al. reported synthesis of three novel 99mTc complexes [99mTc]145–[99mTc]147 and their 145 147 β theircorresponding corresponding Re analogues Re analogues 145–147, in– which, in the which phenyl the ring phenyl of the ringclassical of the Aβ–binding classical structures A -binding structures2–phenylbenzothiazole 2-phenylbenzothiazole or 2–phenylbenzimidazole or 2-phenylbenzimidazole is replaced is replaced by cyclopentadienyl by cyclopentadienyl tricarbonyl tricarbonyl 99m [Cp[Cp99mTc(CO)Tc(CO)3][3] 96[96]] (Figure (Figure 3434).).

FigureFigure 34. 34.99m 99mTcTc complexescomplexes [99m99mTc]145–Tc]145–[[99m99mTc]147Tc]147 andand their their corresponding corresponding Re analogues Re analogues 145 –145 147–147 designeddesigned for for SPECT SPECT imaging imaging of of A Aββplaques. plaques.

The affinity of complexes 145–147 for Aβ plaques was evaluated with confocal microscopy on human AD brain sections. All three complexes bind selectively to the Aβ plaques. Competition binding assays between the stable Re complexes 145–147 and their radioactive 99mTc counterparts [99mTc]145–[99mTc]147 showed Ki values of 65.8 ± 21.3, 7.0 ± 2.9, and 5.7 ± 2.9 nM. Biodistribution experiments showed brain uptake of [99mTc]145 (7.94 ± 1.46%) comparable to that of 18F–florbetapir (7.33% ID/g at 2 min), fast blood clearance, and lack of retention in brain tissue. Biodistribution of [99mTc]145 in 5xFAD Tg–mice showed AD brain accumulation of 3.90 ± 0.19 for Tg–mice and 2.68 ± 0.06 for wild–type mice (15 min post–injection). The Re complexes 145–147 also showed an anti–amyloid therapeutic potential. Jokar et al. designed a 99mTc agent 148 with a lipophilic peptide scaffold, 99mTc–Cp–GABA–D– (FPLIAIMA)–NH2 [97] (Figure 35).

Int. J. Mol. Sci. 2020, 21, x 30 of 37

Sagnou et al. reported synthesis of three novel 99mTc complexes [99mTc]145–[99mTc]147 and their corresponding Re analogues 145–147, in which the phenyl ring of the classical Aβ–binding structures 2–phenylbenzothiazole or 2–phenylbenzimidazole is replaced by cyclopentadienyl tricarbonyl [Cp99mTc(CO)3] [96] (Figure 34).

Int. J.Figure Mol. Sci. 34.2020 99m, 21Tc, 9190complexes [99mTc]145–[99mTc]147 and their corresponding Re analogues 145 – 14728 of 35 designed for SPECT imaging of Aβ plaques. The affinity of complexes 145–147 for Aβ plaques was evaluated with confocal microscopy on The affinity of complexes 145–147 for Aβ plaques was evaluated with confocal microscopy on human AD brain sections. All three complexes bind selectively to the Aβ plaques. Competition binding human AD brain sections. All three complexes bind selectively to the Aβ plaques.99m Competition assays between the stable Re complexes 145–147 and their radioactive 99mTc counterparts binding99m assays99m between the stable Re complexes 145–147 and their radioactive Tc counterparts [ Tc]145–[ Tc]147 showed Ki values of 65.8 21.3, 7.0 2.9, and 5.7 2.9 nM. Biodistribution [99mTc]145–[99mTc]147 showed Ki values of 65.8 ±± 21.3, 7.0 ±± 2.9, and 5.7 ±± 2.9 nM. Biodistribution experiments showed brain uptake of [99mTc]145 (7.94 1.46%) comparable to that of 18F-florbetapir experiments showed brain uptake of [99mTc]145 (7.94 ±± 1.46%) comparable to that of 18F–florbetapir (7.33% ID/g at 2 min), fast blood clearance, and lack of retention in brain tissue. (7.33% ID/g at 2 min), fast blood clearance, and lack of retention in brain tissue. Biodistribution of [99mTc]145 in 5xFAD Tg-mice showed AD brain accumulation of 3.90 0.19 for Biodistribution of [99mTc]145 in 5xFAD Tg–mice showed AD brain accumulation of 3.90± ± 0.19 Tg-mice and 2.68 0.06 for wild-type mice (15 min post-injection). The Re complexes 145–147 also for Tg–mice and 2.68± ± 0.06 for wild–type mice (15 min post–injection). The Re complexes 145–147 showed an anti-amyloid therapeutic potential. also showed an anti–amyloid therapeutic99m potential. Jokar et al. designed99m a Tc agent 148 with a lipophilic99m peptide scaffold, 99m Jokar et al. designed a Tc agent 148 with a lipophilic peptide scaffold, Tc–Cp–GABA–D– Tc-Cp-GABA-D-(FPLIAIMA)-NH2 [97] (Figure 35). (FPLIAIMA)–NH2 [97] (Figure 35).

99m Figure 35. Tc-Cp-GABA-D-(FPLIAIMA)-NH2 148 based on an Aβ-aﬃnitive peptide scaﬀold, designed for SPECT imaging of Aβ plaques.

Binding affinity studies were carried out on Aβ aggregation, and the respective observed values of K and Bmax were 20.22 7.26 µM and 201,700 8750.89 bound molecules/plaque. In vitro d ± ± autoradiography studies, scintigraphy, and fluorescence staining were performed on the brain sections of AD and normal rats and also on brain sections of AD, normal, and schizophrenia patients for better confirmation. The radiopeptide displayed a good binding affinity for the Aβ plaques on brain sections of AD rats and a significant binding affinity for Aβ plaques in human brain sections. Brain uptake in AD and normal rats was respectively 0.38% and 0.35%, and brain uptake of radiopeptide on AD brain increased 2 min post-injection and slowly dropped at 30 min, as compared with normal ones. Biodistribution studies in the presence of a p-glycoprotein (PgP) blocker and SPECT/CT imaging studies were also performed following intravenous administration of the probe. The analyzed images showed significant radioactivity uptake in the AD brains compared with uptake in normal rats.

5. Conclusions Among various strategies utilized to obtain copper-based AD imaging agents, compound 1 with a low molecular mass and ATSM chelating moiety demonstrated the highest level of brain uptake at 2 min post-injection. We note that modification of the ATSM moiety with polyamine led to a significant increase in brain uptake. Other Cu-chelating fragments such as DOTA lead to a decrease in brain uptake compared with Cu-ATSM-based complexes. Gd/Ga complexes designed for MRI and PET imaging of Aβ showed good in vitro activity, but when tested in vivo, those compounds showed little to no BBB penetration, which can result from the presence of rigid DOTA/DO3A, etc., scaffolds used to chelate Gd/Ga. The most potent compound Int. J. Mol. Sci. 2020, 21, 9190 29 of 35

71 demonstrated a brain uptake of 1.24% ID/g at 2 min post-injection despite a MW 1000, which is ≈ far beyond the optimal mass for BBB penetration. Some of the 99mTc-based coordination compounds demonstrated promising in vitro and in vivo activity. The most potent complexes for SPECT imaging were compounds 145–147 with piano stool moieties coupled with Aβ-binding benzothiazole scaffolds, with 145 showing a brain uptake of 7.94% at 2 min post-injection. When rigid chelating structures, long linkers, and heavy Aβ-binding fragments are used, the BBB penetrability of the resulting coordination compounds decreases dramatically, as shown for 92–95 and 107–132. Metal-based imaging agents for AD allow noninvasive imaging of Aβ plaques, a crucial procedure for successful AD diagnosis and therapy. There is a strong need for new efficient AD imaging probes, and this area of research is therefore thriving. The radioisotopes 64Cu, 68Ga, and 99mTc are promising and can be obtained either by cyclotrons or by radioisotope generators. They also have half-lives much longer than do 18F and 11C, which are currently used for imaging. Radioactive metal isotopes can be introduced at the last step of synthesizing an imaging agent, which reduces the potential activity loss. Among the vast variety of compounds considered in this review, the most promising results were shown by Cu2+-based coordination compounds 1 and 11 for PET imaging, Gd3+-based coordination compound 40 for MRI, and 99mTc-based coordination compound 145 for SPECT imaging, demonstrating the best Aβ-binding affinity and brain uptake at 2 min post-injection while being light-weight complexes with small Aβ-binding fragments.

Author Contributions: Conceptualization, O.K. and D.S.; Writing—Original Draft Preparation, O.K. and D.S.; Writing—Review & Editing, O.K., D.S., A.Z., K.P., P.G., A.E.; Visualization, D.S., A.Z., K.P.; Supervision, P.G., A.E.; Project Administration, E.B. A.M.; Funding Acquisition, P.G., A.E., A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Education and Science of the Russian Federation implemented by governmental decree No. 211 dated 16 March 2013, and by the Russian Science Foundation, Grant No. 20-14-00312. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

[125I]IMPY ([125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo [1,2-a]pyridine) ICP-MS inductively coupled plasma mass spectrometry [18F]FDDNP (1,1-dicyanopropen-2-yl)-6-(2-[18F]-ﬂuoroethyl)-methylamino-naphthalene SPECT single-photon emission computed tomography PET positron emission tomography MRI magnetic resonance imaging AD Alzheimer’s disease BBB Blood-brain barrier CAA cerebral amyloid angiopathy PgP P-glycoprotein TEM transmission electron microscopy Tg-mice transgenic mice ThT Thioﬂavin-T DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DO3A 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid PCTA 3,6,9,15-tetraaza bicyclo[9.3.1]-pentadeca1(15),11,13-triene-3,6,9-triacetic acid HBED-CC N,N’-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N’-diacetic acid NODAGA 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid AAZTA 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine BAT- Bis-amino bis-thiol MAMA Monoamine-monoamide dithiols IC50 The half maximal inhibitory concentration MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PIB 2-(40-[11C]methylaminophenyl)-6-hydroxybenzothiazole Int. J. Mol. Sci. 2020, 21, 9190 30 of 35

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