Alpha-Synuclein PET Tracer Development—An Overview About Current Efforts

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Review Alpha-Synuclein PET Tracer Development—An Overview about Current Efforts

Špela Korat 1, Natasha Shalina Rajani Bidesi 2, Federica Bonanno 3, Adriana Di Nanni 3, Anh Nguyên Nhât Hoàng 4,5, Kristina Herfert 3, Andreas Maurer 3 , Umberto Maria Battisti 2, Gregory David Bowden 3 , David Thonon 4, Daniëlle Vugts 1, Albert Dirk Windhorst 1 and Matthias Manfred Herth 2,6,*

1 Department of Radiology and Nuclear Medicine, Amsterdam UMC, Vrije Universeit Amsterdam, De Boelelaan 1085c, 1081 HV Amsterdam, The Netherlands; [email protected] (Š.K.); [email protected] (D.V.); [email protected] (A.D.W.) 2 Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 160, 2100 Copenhagen, Denmark; [email protected] (N.S.R.B.); [email protected] (U.M.B.) 3 Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University Tübingen, Röntgenweg 15, 72076 Tübingen, Germany; [email protected] (F.B.); [email protected] (A.D.N.); [email protected] (K.H.); [email protected] (A.M.); [email protected] (G.D.B.) 4 Elysia Raytest, Rue du Sart-Tilman 375, 4031 Liège, Belgium; [email protected] (A.N.N.H.); [email protected] (D.T.) 5 GIGA Cyclotron Research Centre In Vivo Imaging, Department of Chemistry, University of Liège, Allée du 6 Août 8, 4000 Liège, Belgium 6 Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark Citation: Korat, Š.; Bidesi, N.S.R.; * Correspondence: [email protected] Bonanno, F.; Di Nanni, A.; Hoàng, A.N.N.; Herfert, K.; Maurer, A.; Abstract: Neurodegenerative diseases such as Parkinson’s disease (PD) are manifested by inclusion Battisti, U.M.; Bowden, G.D.; Thonon, bodies of alpha-synuclein (α-syn) also called α-synucleinopathies. Detection of these inclusions is D.; et al. Alpha-Synuclein PET Tracer thus far only possible by histological examination of postmortem brain tissue. The possibility of Development—An Overview about α Current Efforts. Pharmaceuticals 2021, non-invasively detecting -syn will therefore provide valuable insights into the disease progression 14, 847. https://doi.org/10.3390/ of α-synucleinopathies. In particular, α-syn imaging can quantify changes in monomeric, oligomeric, ph14090847 and fibrillic α-syn over time and improve early diagnosis of various α-synucleinopathies or monitor treatment progress. Positron emission tomography (PET) is a non-invasive in vivo imaging technique Academic Editor: Irina Velikyan that can quantify target expression and drug occupancies when a suitable tracer exists. As such, novel α-syn PET tracers are highly sought after. The development of an α-syn PET tracer faces Received: 6 July 2021 several challenges. For example, the low abundance of α-syn within the brain necessitates the Accepted: 17 August 2021 development of a high-affinity ligand. Moreover, α-syn depositions are, in contrast to amyloid Published: 26 August 2021 proteins, predominantly localized intracellularly, limiting their accessibility. Furthermore, another challenge is the ligand selectivity over structurally similar amyloids such as amyloid-beta or tau, Publisher’s Note: MDPI stays neutral which are often co-localized with α-syn pathology. The lack of a defined crystal structure of α-syn with regard to jurisdictional claims in has also hindered rational drug and tracer design efforts. Our objective for this review is to provide a published maps and institutional affil- comprehensive overview of current efforts in the development of selective α-syn PET tracers. iations.

Keywords: alpha-synuclein; imaging; synucleinopathies; positron emission tomography

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Abnormal alpha-synuclein (α-syn) depositions (α-synucleinopathies) in neurons, conditions of the Creative Commons nerve ﬁbers, or glia cells are the hallmark for many neurodegenerative diseases such as Attribution (CC BY) license (https:// Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy creativecommons.org/licenses/by/ (MSA) [1]. PD, for example, is the most common progressive neurodegenerative movement 4.0/). disorder. Today, approximately 7–10 million patients worldwide are affected. In Europe,

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the proportion of the people suffering from PD is 1.6% for those aged 65+ and up to 3% for those aged 80+. As the average age of the population in the EU continues to rise, the overall number of PD patients also increases, placing a significant burden on society due to associated healthcare costs [2–4]. Currently, the combined direct and indirect cost of PD, including treatment, social security payments, and lost income from an inability to work, is estimated to be nearly EUR 42 billion in the United States of America (USA) alone. Although symptoms can, to a certain extent, be medically remediated, there is currently no cure for PD. Medication costs for an individual with PD average approximately EUR 2100 per year [5]. The underlying biochemical mechanism of PD is not fully understood, and it is difficult to ascertain possible causes of the disease from its effects. More importantly, the clinical diagnosis of PD is not straightforward in the early phases of the illness. This seriously complicates the identification of targets for novel therapies and the interpretation of treatment outcomes. It is imperative to correctly diagnose the illness as early as possible since early intervention is key to limiting the neurodegenerative process. The pathological hallmark of PD is deposition of α-syn protein in the brain followed by dopaminergic neuronal loss [6–8]. Currently, α-syn deposition cannot be assessed accurately in vivo. The inability to quantify these abnormal aggregates limits the accurate clinical diagnosis of PD as well as DLB and MSA, and also hinders research into the understanding of these diseases; thus, impeding the development of new treatments. Positron emission tomography (PET) molecular imaging has the potential to reveal the pathogenesis of brain disorders when suitable tracers are available. PET can localize and quantify drug targets, monitor treatment effect, or image disease pathophysiology on a molecular basis. Various studies have indicated toward the importance of imaging with respect to patient inclusion in clinical trials, especially for PD [9]. Currently, imaging of dopaminergic neurons has been used to monitor the effects of investigational disease-modifying drugs in PD, but dopamine cell loss is a downstream effect of α-syn deposition and thus, there is great interest in imaging this specific protein in PD therapies for monitoring progression as well as for early diagnosis. Cerebrospinal fluid biomarkers, skin tissue biopsies, and other imaging techniques will likely be developed in the future as well. However, a PET tracer in the central nervous system (CNS) has the potential to provide evidence for central target engagement, following disease progression and drug treatment efficacy. By developing a tracer that selectively identifies α-syn deposition, the correct cohort of patients may be included in clinical trials and the readout of these trials may be correlated with the underlying disease pathology. Thus, the ability to image α-syn deposition will be a game-changing achievement [7] and will facilitate the development of effective treatments. Presently, there are no tracers available for the detection of α-syn deposition in patients. The main obstacles are difficulties in identifying selective α-syn deposition binding ligands (to be used as tracers) that are suitable for PET measurements. Considering this, the Michael J. Fox Foundation for Parkinson’s Research (MJFF) announced a challenge to develop such a tracer sponsoring a USD 2 million prize to the first successful team. In this review, we aim to give a detailed summary of the potential radiotracers targeting α-syn developed thus far. For this reason, a bibliographic search was performed from various sources, including PubChem, ChEMBL, Reaxys, World Intellectual Property Organization (WIPO), PubMed and other online resources (in Table S1, Supporting Infor- mation). We describe the rationality in their design, highlight lessons to be learned from these efforts, and give suggestions on how to move forward.

2. Alpha-Synuclein and PET Imaging 2.1. Alpha-Synuclein within the Central Nervous System α-Syn is an abundant protein throughout the CNS. It represents approximately 1% of cytosolic proteins in the brain and is predominantly found in the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum [10]. Although α-syn has been localized within the nucleus of mammalian neurons [11,12], the vast majority of α-syn has been found in presynaptic termini in a free or membrane-bound form [13]. The normal func- Pharmaceuticals 2021, 14, 847 tional role of α-syn is still not completely understood, nor is its involvement in 3CNS of 45 diseases [14]. However, α-syn has been suggested to play a specific role in synaptic plasticity. For example, a sustained reduction in α-syn expression levels was detected in specific zebra finch brain regions, which were implicated during song acquisition [15]. α-Syn has presynaptic termini in a free or membrane-bound form [13]. The normal functional role alsoof beenα-syn shown is still to not be completely an inhibitor understood, of phospholipase nor is its involvement D2 (PLD2), in which CNS diseases indicates [14 that]. it hasHowever, a specific αfunction-syn has in been membrane suggested trafficking to play a [14,16]. specific Furthermore, role in synaptic there plasticity. is evidence For that α-synexample, is involved a sustained in neurotransmitter reduction in α-syn release expression [14] and levels more was importantly detected in specific in the zebraformation of toxicfinch radical brain regions, species which [17]. Finally, were implicated α-syn has during also songbeen acquisitionsuggested [to15 ].beα involved-Syn has also in DNA repairbeen processes, shown to including be an inhibitor the repair of phospholipase of double-strand D2 (PLD2), breaks which [18]. indicates that it has a specific function in membrane trafficking [14,16]. Furthermore, there is evidence that α-syn Alpha-Synucleinopathiesis involved in neurotransmitter in PD, release DLB and [14] MSA and more importantly in the formation of toxic radical species [17]. Finally, α-syn has also been suggested to be involved in DNA repair processes,PD patients including show the abnormal repair of intraneuronal double-strand breaks inclusions [18]. named Lewy bodies (LB) and Lewy neurites (LN), which are predominantly present in the substantia nigra. Both fila- mentAlpha-Synucleinopathies types contain α-syn as in PD,a major DLB andcomponent. MSA DLB patients also possess LB and LN filaments.PD In patients contrast show to abnormalPD, these intraneuronalfilaments are inclusions mainly distributed named Lewy in bodies the cerebral (LB) and cortex [19].Lewy The neuritesmajor filamentous (LN), which are component predominantly of glial present and in neuronal the substantia cytoplasmic nigra. Both inclusions filament in MSAtypes patients contain isα also-syn asα-syn a major [20]. component. Contrary DLB to inclusions patients also found possess in LBPD and and LN DLB, filaments. in MSA, filamentousIn contrast components to PD, these are filaments not only are found mainly in distributed neurons inbut the also cerebral within cortex oligodendrocytes [19]. The major filamentous component of glial and neuronal cytoplasmic inclusions in MSA patients and in the cytoplasm of nerve cells [19,21–23] (Figure 1). Patient-derived α-syn fibrils from is also α-syn [20]. Contrary to inclusions found in PD and DLB, in MSA, filamentous differentcomponents α-synucleinopathies, are not only found namely in neurons PD, DLB, but also and within MSA, oligodendrocytes possess structural and differences. in the Specifically,cytoplasm PD of nerve and cellsMSA [19 fibrils,21–23 exhibit] (Figure flat1). Patient-derived and twisted polymorphs,α-syn fibrils from while, different in contrast,α- DLBsynucleinopathies, fibrils are cylindrical namely with PD, DLB,no twist and MSA,[24]. The possess precise structural role differences.of α-syn in Specifically, PD, DLB, and MSAPD is and still MSA not fibrilsunderstood exhibit flat[25]. and However, twisted polymorphs, early α-syn while, aggregates in contrast, (protofibrils) DLB fibrils and are oli- gomercylindrical species with that no are twist formed [24]. Theduring precise the role aggregation of α-syn in process PD, DLB, are and believed MSA is stillto mediate not neurotoxicityunderstood through [25]. However, the disruption early α-syn of aggregates cellular homeostasis (protofibrils) and oligomerother effects species on that various intracellularare formed targets during leading the aggregation to mitochondria process arel believedtoxicity, to enhancement mediate neurotoxicity of inflammatory through re- the disruption of cellular homeostasis and other effects on various intracellular targets sponses, and synaptic and endoplasmic dysfunction [26–31]. Furthermore, α-syn leads to leading to mitochondrial toxicity, enhancement of inflammatory responses, and synaptic the andrelease endoplasmic of radical dysfunction species, which [26–31 ].is Furthermore,suggested toα -syntrigger leads neurodegeneration. to the release of radical Finally, DNAspecies, repair which function is suggested also appears to trigger to be neurodegeneration. compromised in Finally, LB inclusion DNA repair bearing function neurons [17].also Several appears overview to be compromised reviews about in LB the inclusion toxic effect bearing of neurons α-syn have [17]. Severalbeen published overview and summarizereviews aboutthe current the toxic state-of-the-art effect of α-syn knowledge have been published [28,32,33]. and summarize the current state-of-the-art knowledge [28,32,33].

Figure 1. Simplified illustration of the differences of α-syn deposition and structure in PD, DLB, and MSA neurodegenerative Figure 1. Simplified illustration of the differences of α-syn deposition and structure in PD, DLB, and MSA neurodegener- diseases. In particular, LB-rich brain regions deposited in the cingulate cortex are thought to be specific for PD and DLB, ative diseases. In particular, LB-rich brain regions deposited in the cingulate cortex are thought to be specific for PD and while depositions within the cerebellum are specific for MSA. Additionally, inclusions of α-syn are found in neuronal cells DLB, while depositions within the cerebellum are specific for MSA. Additionally, inclusions of α-syn are found in neuronal in PD and DLB. Conversely, in MSA these inclusions are found in oligodendrocytes ((left), visualized with BioRender). Structural differences of α-syn have been reported in patient-derived α-syn fibrils (right). (Adapted from [24] and visualized with BioRender). 2.2. The Structure of α-Syn The native form: Considerable effort has been made to reveal the native state of α-syn. However, it is still debated if native α-syn exists as an unfolded monomer [34], Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 47

cells in PD and DLB. Conversely, in MSA these inclusions are found in oligodendrocytes ((left), visualized with BioRen- der). Structural differences of α-syn have been reported in patient-derived α-syn fibrils (right). (Adapted from [24] and visualized with BioRender).

2.2. The Structure of α-Syn Pharmaceuticals 2021, 14, 847 4 of 45 The native form: Considerable effort has been made to reveal the native state of α- syn. However, it is still debated if native α-syn exists as an unfolded monomer [34], as a helically folded tetramer [35], as multimer conformations [36], or as a mixture of all these formsas a helically[36]. In contrast, folded tetramer the primary [35], asstructure multimer of α conformations-syn is well described. [36], or as It a is mixture encoded of by theall SNCA these formsgene and [36]. encompasses In contrast, the140 primaryamino acids structure (Figure of 2A).α-syn The is wellprotein described. is divided It is into threeencoded domains: by the the SNCA N-terminal gene and amphipathic encompasses region 140 amino (1–60 acids residues), (Figure 2theA). non-amyloid- The protein β componentis divided (NAC) into three hydrophobic domains: theregion N-terminal (61–95 re amphipathicsidues), and region the C-terminal (1–60 residues), acidic region the (96–140non-amyloid- residues)β component [37]. Several (NAC) isoforms hydrophobic of α-syn region exist (61–95 and are residues), formed and through the C-terminal alternative α splicingacidic region [38,39]. (96–140 The synuclein residues) [protein37]. Several family isoforms is reported of -syn to existundergo and areextensive formed post-trans- through alternative splicing [38,39]. The synuclein protein family is reported to undergo extensive lational modifications (PTMs) in vivo including phosphorylation, truncation, acetylation, post-translational modiﬁcations (PTMs) in vivo including phosphorylation, truncation, or nitration [40]. acetylation, or nitration [40].

Figure 2. (A) Primary structure of α-syn with highlighted regions: N-terminal (in blue), NAC (in grey), and C-terminal

(in red). (B) Conformational states of α-syn (adapted from [41] and visualized with BioRender). (C) Solid-state NMR Figureatomic-resolution 2. (A) Primary (4.8 structure Å) of α of-syn α-syn fibrils with (PDB highlighted ID: 2n0a, regions: adapted N-terminal from Twohig (in etblue), al. [ 42NAC]) with (in grey), putative and binding C-terminal sites: (in red). (B) Conformational states of α-syn (adapted from [41] and visualized with BioRender). (C) Solid-state NMR atomic- binding site 2 (BS2; in blue), binding site 9 (BS9; in pink), binding site 3/13 (BS3/13; in orange) on α-syn fibrils (reprinted resolution (4.8 Å) of α-syn fibrils (PDB ID: 2n0a, adapted from Twohig et al. [42]) with putative binding sites: binding site with permission from [43]. Copyright 2018 American Chemical Society. Structure of α-syn was imported from PDB and 2 (BS2; in blue), binding site 9 (BS9; in pink), binding site 3/13 (BS3/13; in orange) on α-syn fibrils (reprinted with permis- visualized with MOE 2018). (D) The α-syn pathology of PD with two pigmented nerve cells containing LB (red arrows) and sion from [43]. Copyright 2018 American Chemical Society. Structure of α-syn was imported from PDB and visualized LN (black arrows). (Adapted from [19]). with MOE 2018). (D) The α-syn pathology of PD with two pigmented nerve cells containing LB (red arrows) and LN (black arrows). (Adapted from [19]). α-Syn fibril formation: Under certain physiological conditions, native α-syn can ag- gregate. Thus far, it is unclear if a specific conformation of α-syn or other processes trigger aggregationα-Syn fibril and formation: ultimately, convertUnder certain soluble physiological native α-syn into conditions,β-sheet rich, native tightly α-syn stacked can ag- gregate.fibrils. However,Thus far, aggregationit is unclear isif thoughta specific to occurconformation via a highly of α dynamic-syn or other equilibrium processes between trigger aggregationnumerous conformations, and ultimately, various convert oligomeric soluble native states andα-syn (proto)fibrils into β-sheet(Figure rich, 2tightlyB) [36] stacked. Un- fibrils.derstanding However, the aggregation processis thought is difficult to occur since via ita highly is highly dynamic complex equilibrium and can be be- tweenaffected numerous by a broad conformations, set of parameters. various For instance,oligomeric various states environmental and (proto)fibrils factors (Figure appear 2B) [36].to have Understanding an influence the on anaggregation increased risk process for PD, is difficult such as exposure since it is to highly pesticides, complex herbicides, and can beexposure affected toby heavy a broad metals, set of organic parameters. solvents, For and instance, various various sources environmental of oxidative stress factors [44]. ap- pearConversely, to have an it has influence also been on shown an increased that certain risk mutationsfor PD, such within as exposure the α-syn to encoding pesticides, SNCA herbicides,gene enhanceexposure or to reduce heavy the metals, formation organic of protofibrils solvents, and [45]. various Changes sources within of the oxidativeα-syn NAC stress domain appear pivotal for α-syn fibrillation, especially within the amino acid sequences 71VTGVTAVAQKTV82 or 66VGGAVVTGV74. Removal of either of these sequences abro- gated the fibril formation completely [46,47]. PTMs have also been shown to influence the aggregation process. For example, phosphorylation on residue S129 leads to extensive aggregation (Figure2D) [ 48]. This PTM appears to be pathologically relevant since it is Pharmaceuticals 2021, 14, 847 5 of 45

present in >90% of all α-syn derived from PD patients, but only seen in 4% of native soluble α-syn [49]. Three-dimensional (3D) structure and possible binding sites for small molecules: To date, no high-resolution and detailed 3D structure of α-syn fibrils has been reported. The highly repetitive nature of the respective secondary structure and the residue-type degeneracy pose significant challenges to this endeavor [50]. The lack of knowledge of the function of α-syn, its binding partners, their binding mechanism, and the broad set of possible PTMs have been found to be additional complications [37]. Nevertheless, certain structural secrets of α-syn fibrils have been revealed [50–52]. For example, Tuttle et al. could have elucidated the structure of a single filament fibril of α-syn using solid-state nuclear magnetic resonance (ssNMR) (Figure2C) [50]. Years later, Li and colleagues were the first to distinguish between two structurally different forms of fibril species, termed protofilaments, using cryogenic electron microscopy [53]. In 2018, Hsieh et al. conducted a molecular blind docking study using the structural data obtained from the ssNMR study by Tuttle et al. This study aimed to identify possible binding regions of small molecules toward α-syn fibrils [43]. Three putative binding sites, namely binding site 2 (Y39-S42-T44; BS2), 9 (G86-F94-K96; BS9), and 3/13 (K45-V48-H50 and K43-K45-V48-H50; BS3/13) were identified (Figure2C). To test these virtual hits, chemically diverse putative binders were synthesized, and their binding profile was examined using photoaffinity labeling and radioligand binding studies. Results strongly indicated that there are several binding sites [43]. How this knowledge can be used to design selective and high-affinity α-syn ligands has to be further investigated.

2.3. Challenges—Why Is It Difficult to Develop an α-Syn PET Tracer? Challenge 1. The target The development of an α-syn PET tracer is more challenging in comparison to the development of tracers for other amyloids such as Aβ or tau. A key reason for this is that the absolute concentration of α-syn aggregates within the brain is thought to be 10- to 50-fold lower than that of Aβ or tau (Figure3A) [ 7,54,55]. Consequently, high affinity of a putative tracer, most likely in the subnanomolar range, is required. Next, co-existence and co-localization of α-syn with Aβ and tau fibrils requires a PET tracer with high selectivity over Aβ and tau in order to be able to image α-syn at all (Figure3C) [ 56]. For instance, Aβ pathology can be found in more than 80% of all synucleinopathies. In DLB, Aβ deposits are detected in approximately 85% of all cases and tau pathology is observed in approximately 30% [7,54,57,58]. Unfortunately, Aβ or tau fibrils form structurally similar β-pleated sheets (Figure3B), complicating the development of selective α-syn binding ligands. Furthermore, and in contrast to Aβ, the highest concentration of α-syn inclusions can be found intracellularly (Figure3D). Therefore, any successful α-syn PET tracer has to be able to cross cell membranes, in addition to the blood-brain-barrier (BBB) [54,56]. Usually, this is not considered the biggest hurdle, but it may lead to issues, e.g., the D2/3 PET tracer [11C]raclopride can pass the BBB, but it cannot pass cell membranes [59]. It might also be that extensive in vivo PTMs possibly result in a variety of substructures that might not be recognized by the tracer developed for the unmodified structure (Figure3E) [54,56] . Challenge 2. The design Another aspect that in general challenges the development of tracers for amyloids is the standard development strategies that have been established over the last decades for more traditional targets such as receptors and enzymes that cannot be applied to these protein-based structures without precisely defined binding pockets. The underling binding mechanism is poorly understood and studies identifying for example binding sites, or to reveal kinetic and thermodynamic ligand-binding parameter using computational or other strategies are in its infancy [43,60,61]. Conversely, structure-based drug design of these ligands is hampered by the limited availability of high-resolution X-ray crystal structures of α-syn fibrils. Moreover, it is questionable if these structures are able to mimic the aggregates and conditions of the fibrils in vivo. For this reason, the only option available Pharmaceuticals 2021, 14, 847 6 of 45

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to develop an α-syn PET tracer is a ligand-based approach. However, this strategy has several limitations mainly related to the small number of ligands and data available for limitedthe binding. and application Furthermore, of high-throughput the extensive literature screening and methods structures to available this material on A isβ currentlyor tau nottracers available. cannot This be exploited obviously to slow designs down new compoundsthe development in order process. to avoid selectivity issues.

Figure 3. Challenges and characteristics of an ideal α-syn tracer. (Reprinted with permission from [56]. Copyright 2014 the Figure 3. Challenges and characteristics of an ideal α-syn tracer. (Reprinted with permission from [56]. Copyright 2014 the Society of Nuclear Medicine and Molecular Imaging, Inc.) (A) Low density and concentration of α-syn in comparison with Society of Nuclear Medicine and Molecular Imaging, Inc.) (A) Low density and concentration of α-syn in comparison with other targets. (B) Structural similarity with other proteins such as Aβ and tau. (C) Coexistence and colocalization with other targets. (B) Structural similarity with other proteins such as Aβ and tau. (C) Coexistence and colocalization with structural similar amyloids such as Aβ and tau. (D) Intracellular localization. (E) Multiple post-translational modifications structural similar amyloids such as Aβ and tau. (D) Intracellular localization. (E) Multiple post-translational modifications such as phosphorylation (P), ubiquitination (U), and nitration (N). (F) Various structural forms such as fibrils, oligomers, such as phosphorylation (P), ubiquitination (U), and nitration (N). (F) Various structural forms such as fibrils, oligomers, andand misfolded misfolded proteins. proteins. (Visualized (Visualized with with BioRender). BioRender). Challenge 3. The evaluation 2.4. SuccessThe reliability Criteria for and a predictabilitySmall Molecule of αin-Syn vitro PETbinding Tracer assays is crucial in any PET tracer developmentA successful and PET is a bigtracer challenge for neuroimaging for α-syn tracer is dependent development. on Itseveral has been key postulated parameters (Figurethat structural 4) [73]. First, variation the ligand of α-syn must between be labeled patient in brain-derivedsufficient amountsin vivo (activityaggregates yield and > 500 MBq).in vitro Usually,α-syn fibrilscarbon-11 can explain(11C) or thefluorine-18 difficulties (18F) in are translating the nuclides new ofα-syn choice PET for tracers small tomol- ecule-basedclinical research PET neuroimaging [62]. Structural tracers. heterogeneity These nuclides between displayin vivo excellentbrain-derived decay material character- isticsfrom for PD PET and imaging MSA patients (short havehalf-life, been good reported branching and support ratio, and this short hypothesis positron [62 ,range)63]. Do [74] andthese radiotracers conformational labeled differences using these have radionuclides an influence oncan the also binding be produced pattern in of good potential radio- chemicalligands thatyields have (RCY) still toin bemost elucidated, well-equippe i.e., ared bindingradiopharmacies motifs in different(activity conformational yield > 500 MBq) α-syn fibrils changed in such a way that prevent ligands to bind universally to all forms [75]. Additionally, potential ligands can be radiolabeled with these isotopes without in- (Figure3F)? troducing bulk and charge, which often prevents sufficient blood-brain barrier (BBB) penetrance. BBB permeability should be high with an early standard uptake value (SUV) > 1.0 [54] (within several minutes, for example 5-10 min). Brain uptake for amyloid tracers is recommended to be ≥ 0.4% ID/g in rat brain or ≥ 4.0%ID/g in mouse brain [54]. Addition- ally, the plasma clearance half-time should be fast (less than 30 min) [54] to reduce back-

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Similar considerations also apply to α-syn depositions formed in animal models [7]. Are these forms structurally related to those formed in humans and can they be used for screening purposes to identify selective ligands? In this context the selection of the animal model plays a crucial role in the outcome of these experiments. In general, in vivo animal testing for tracers targeting brain aggregates is currently performed in wild-type healthy animals such as mice, rats, and non-human primates (NHP). However, recent studies are moving toward the use of disease models since they are able to mimic intracellular inclusion bodies that are as similar to those found in humans. Currently, several genetically modified rodent models are available. These are usually robust, but the long time required for the development of the pathology (several months) and the high costs limit the use of these animals. For these reasons, the most employed rodent models are the seeding model with pre-formed fibrils (PFF), viral vector model, and the combination of viral vectors and PFF [64–66]. The combination of a viral vector and PFFs have shown all the important hallmarks of α-synucleinopathies and was reported to accelerate the induction of α-syn aggregates as rapid as within 10 days after PFF injection [65] Another quick, robust, and easy to implement model was developed by Verdurand et al. by injecting fibrils into striatum of rats and conducting in vivo imaging 7 days after inoculation [67]. Rodents may not provide all the data (safety, P-gP efflux, metabolites, etc.) for the tracer, and they need to be tested in larger animals. NHP models with α-syn inclusion have been created with inoculation of fibrils, LB, and viral vectors but all the models require months to form the pathology, making them expensive [68–70]. The optimum would be to develop a rodent model of α-synucleinopathy that is suitable for in vivo PET imaging of α-syn pathological forms. The model must fulfil the criteria of rapid, abundant, and progressive pathology development over time. Another animal that has gained popularity in brain imaging is the domestic pig [71,72]. Creating an α-syn pig model that is appropriate, fast, and inexpensive will drastically reduce the cost of developing such tracers. It is doubtful if in vitro assays or in vivo models based on fibrils are predictive and clinically relevant; models to study specific uptake in vivo are lacking. For this reason, exclusively patient-derived material should be used for screening new α-syn tracers. How- ever, access to human tissue that is well characterized in respect to its pathology is limited and application of high-throughput screening methods to this material is currently not available. This obviously slows down the development process.

2.4. Success Criteria for a Small Molecule α-Syn PET Tracer A successful PET tracer for neuroimaging is dependent on several key parameters (Figure4)[ 73]. First, the ligand must be labeled in sufficient amounts (activity yield >500 MBq). Usually, carbon-11 (11C) or fluorine-18 (18F) are the nuclides of choice for small molecule-based PET neuroimaging tracers. These nuclides display excellent decay characteristics for PET imaging (short half-life, good branching ratio, and short positron range) [74] and radiotracers labeled using these radionuclides can also be produced in good radiochemical yields (RCY) in most well-equipped radiopharmacies (activity yield >500 MBq) [75]. Additionally, potential ligands can be radiolabeled with these isotopes without introducing bulk and charge, which often prevents sufficient blood-brain barrier (BBB) penetrance. BBB permeability should be high with an early standard uptake value (SUV) >1.0 [54] (within several minutes, for example 5-10 min). Brain uptake for amyloid tracers is recommended to be ≥ 0.4% ID/g in rat brain or ≥ 4.0%ID/g in mouse brain [54]. Additionally, the plasma clearance half-time should be fast (less than 30 min) [54] to reduce background signal from blood. Ideally, the tracer binds reversibly to its target and no metabolites exist within the brain. These characteristics greatly simplify kinetic modeling and allow for appropriate quantification and reproducibility [56]. The tracer should show a low non-displaceable binding component to increase signal-to-noise ratio within the brain [54] and has to be accessible in high molar activity (Am) (>50 GBq/µmol, we believe) to avoid target saturation or non-negligible self-blocking [76,77]. An additional key parameter for any successful neuro PET tracer is its selectivity toward off-targets Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 47

ground signal from blood. Ideally, the tracer binds reversibly to its target and no metabolites exist within the brain. These characteristics greatly simplify kinetic modeling and allow for appropriate quantification and reproducibility [56]. The tracer should show a low non-displaceable binding component to increase signal-to-noise ratio within the brain [54] and has to be accessible in high molar activity (Am) (> 50 GBq/µmol, we believe) to avoid target saturation or non-negligible self-blocking [76,77]. An additional key parameter for any successful neuro PET tracer is its selectivity toward off-targets expressed or deposited in same brain regions. A recommended selectivity over these off-targets is thought to be > 30-100 [78]. For a successful α-syn PET tracer, the MJFF has published a criteria list, which has to be fulfilled to receive the MJFF Alpha-synuclein Imaging Prize [79]. We have adapted this list and will use it to assess the ability of published structures to image α-syn via PET (Figure 4).

Suggested Success Criteria for a α-Syn PET Ligand in the preclinic setting • Radiolabeling with 11C or 18F should be achievable in a RCY of > 10% (activity yield > 500 MBq) and in a high molar activity (> 50 GBq/µmol); • Affinity toward α-syn around 1 nM or lower; • The radioligand enables quantification of lower density α-syn targets in the presence of higher density Aβ or tau proteins; • The binding affinity or density selectivity for α-syn over Aβ or tau should be at least 30-50 fold (~1 nM vs. 50 nM); • Off-target binding > 500 nM; • Selective binding to α-synuclein-rich brain homogenates from PD patients (versus Pharmaceuticals 2021, 14, 847 8 of 45 Aβ, tau rich homogenates); • Binds to LBs/LNs in human tissue; • BBB permeability, with early peak uptake of SUV > 1.5; •expressed ≥ 0.4% or ID/g deposited in rat brain in same or ≥ brain 4.0% regions. ID/g in Amouse recommended brain; selectivity over these off-targets is thought to be > 30–100 [78]. For a successful α-syn PET tracer, the MJFF • No or minimal radiometabolites within the brain; •has published a criteria list, which has to be fulﬁlled to receive the MJFF Alpha-synuclein Imaging Low Prize non-displaceable [79]. We have adapted binding this component list and will (rule use it of to assessthumb: the logD ability7.4 of< 3 published [80]; • structures Reversible to image bindingα-syn kinetics via PET (Figurewith rapid4). plasma clearance (< 30 min).

FigureFigure 4. The approachapproach for for the the development development of α of-syn α-syn selective selective PET tracers.PET tracers. The initial The searchinitial was search was conductedconducted fromfrom various various sources sources (in Table(in Table S1, Supporting S1, Supporting Information) Information) in order toin create order an to overview create an over- of compounds that have been designed and developed to image α-syn. The identiﬁed structures should be screened using criteria similar to the structures proposed by the MJFF for a successful α-syn PET tracer and are based on in vitro assays and PET imaging technique.

Suggested Success Criteria for a α-Syn PET Ligand in the Preclinic Setting • Radiolabeling with 11C or 18F should be achievable in a RCY of >10% (activity yield >500 MBq) and in a high molar activity (>50 GBq/µmol); • Affinity toward α-syn around 1 nM or lower; • The radioligand enables quantification of lower density α-syn targets in the presence of higher density Aβ or tau proteins; • The binding affinity or density selectivity for α-syn over Aβ or tau should be at least 30–50 fold (~1 nM vs. 50 nM); • Off-target binding >500 nM; • Selective binding to α-synuclein-rich brain homogenates from PD patients (versus Aβ, tau rich homogenates); • Binds to LBs/LNs in human tissue; • BBB permeability, with early peak uptake of SUV >1.5; •≥ 0.4% ID/g in rat brain or ≥4.0% ID/g in mouse brain; • No or minimal radiometabolites within the brain; • Low non-displaceable binding component (rule of thumb: logD7.4 < 3 [80]; • Reversible binding kinetics with rapid plasma clearance (<30 min). Pharmaceuticals 2021, 14, 847 9 of 45

3. Alpha-Synuclein PET Tracer Development—From a Molecular Development Point of View Over the past decades, a set of chemically diverse compounds have been developed that target α-syn. The development was based on various strategies including rational drug design and high-throughput screening approaches. In this section, we will present a selection of these structures and highlight their potential to be used as α-syn selective PET tracers. Additionally, we will critically reflect their in vitro and in vivo biological evaluation results. Historically, the first investigated α-syn binders were based on fluorescent dyes such as Thioflavin-T (ThT) or Thioflavin-S (ThS) (Figure5A,B) [ 81]. These structures, however, were not selective and consequently, numerous attempts to develop fluorescent dyes with an improved binding affinity have been made over the years. For example, Celej and colleagues studied the effect of N-arylaminonaphthalene sulfonate derivatives upon interaction with conjugated α-syn fibrils. 2,6-ANS and 2,6-TN (Figure5A) showed little fluorescence in the presence of non-aggregated α-syn, but much more fluorescence, occasionally with spectral shifts, when applied to fibrils [82]. Affinities were, however, only in the low micromolar range. In the same year, Volkova et al. conducted a similar study with monomethine cyanines. T-284 and SH-516 were shown to associate with fibrillar α-syn with affinities of 560 nM and 650 nM, respectively (Figure5A) [ 81,83]. To better understand the interaction of these structures, especially SH-516 was studied in a model proposed by Krebs et al. that describes the binding of dyes to amyloids [84]. It was suggested that monomethine cyanines bind in a long channel that run parallel to the length of the fibril axis (Figure5C) [ 85]. This information was used to develop second generation dyes. The resulting tri- and pentamethine cyanines displayed selectivity toward fibrillar or oligomeric α-syn over monomeric α-syn [83]. Interestingly, the pentamethine cyanine dye SL-631 (Figure5A) also bound better to less dense and structured oligomers compared to beta-sheet containing fibrils. In contrast, the trimethine cyanine SH-299 (Figure5A) was able to bind to β-sheets. This distinct binding pattern was attributed to the distinct steric characteristics of both compounds. The more sterically hindered structure SL-631 is supposed to be incapable of entering and binding in the groove formed by β-sheets, whereas the less bulky structure SH-299 is thought to be capable. SH-299 may serve as an imaging agent for both oligomeric and fibrillar α-syn species whereas SL-631 is better suited for oligomeric forms [86]. In 2013, Neal et al. developed three heterogeneous structures to image α-syn selectively. LDS-798 and the phenoxazine dyes Nile Red and Nile Blue (Figure5A) were able to selectively bind to LB in postmortem human tissue of DLB or PD patients. Selectivity over amyloid or tau pathology in human AD tissue was detected. Surprisingly, to date no further attempts concerning radiolabeling and binding affinity measurements have been conducted [87]. From a broader point of view, the studies mentioned above show that it is possible to selectively bind and image α-syn in postmortem human tissue. However, most probes displayed low affinity and poor selectivity toward α-syn and are, as such, not suitable for development as PET tracers. Conversely, these ligands were considered excellent lead structures and were consequently employed as a starting point to develop other α-syn selective ligands in many of the studies discussed below. Pharmaceuticals 2021, 14, x 847 FOR PEER REVIEW 1010 of of 47 45

Figure 5. ((AA)) Chemical Chemical structures structures of of fluore fluorescentscent probes and cyanine dyes. ( (BB)) ThS ThS positive neurons confirming confirming α-syn pathology (adapted from Patterson et al.) [88]. [88]. (C)) Hypothetical presentation presentation of of how mono- and trimethine cyanine dyes interact with α-syn fibrils (reprinted with permission from [83]. Copyright 2008 Elsevier). interact with α-syn fibrils (reprinted with permission from [83]. Copyright 2008 Elsevier).

3.1.3.1. Tricyclic Tricyclic Analogues Analogues (TCAs) 3.1.1.3.1.1. Phenothiazine Phenothiazine Analogues Analogues PhenothiazinePhenothiazine analogues analogues (SIL (SILs)s) have have widely widely been been studied studied as aspotentially potentially selective selective α- synα-syn PET PET ligands. ligands. They They were were developed developed based based on rational on rational drug drug design design considerations. considerations. For example,For example, structurally structurally related related bisarylimines bisarylimines have have been been proven proven to show to show neuroprotective neuroprotective ef- fectseffects in inPD PD related related models, models, most most likely likely due due to their to their antioxidant antioxidant properties properties [89]. [ Within89]. Within this structuralthis structural class, class, several several phenothiazines phenothiazines were were found found to inhibit to inhibit insoluble insoluble α-synα-syn filament filament ag- gregationaggregation at atlow low micromolar micromolar IC IC50 50concentrationsconcentrations [89–91]. [89–91 ].Encouraged Encouraged by by these these findings, findings, severalseveral phenothiazines analoguesanalogues were were synthesized synthesized and and their their binding binding properties properties evaluated evalu- atedbased based on a fluorescenton a fluorescent ThT competitionThT competition assay. assay. Several Several ligands ligands with affinities with affinities between between 30 and 3080 and nM were80 nM identified were identified (Figure6 (Figure). The first 6). promisingThe first promising structure identifiedstructure withinidentified this within study thiswas study a dimethoxy was a dimethoxy substituted substituted phenothiazine phenothiazine analogue withanalogue an affinity with an of approximatelyaffinity of ap- proximately120 nM. Replacement 120 nM. Replacement of one of theof one methoxy of the methoxy group with group a cyano-group with a cyano-group or an amine or an aminedecreased decreased the affinity the affinity more thanmore 3 than to 5-fold. 3 to 5- Infold. contrast, In contrast, replacement replacement with awith nitro-group a nitro- SIL5 groupincreased increased affinity affinity approximately approximately 4-fold (4-fold, Figure (SIL56, )[Figure92]. Further6) [92]. attemptsFurther attempts to improve to the affinity by altering this position with other substituents failed in a follow-up study [93]. improve the affinity by altering this position with other substituents failed in a follow-up Structure activity relationship (SAR) studies around the methoxy group of SIL5 (K between study [93]. Structure activity relationship (SAR) studies around the methoxy igroup of

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32.1 nM and 83.3 nM, Figure6) revealed that the binding affinity toward α-syn decreased SIL5 (Ki between 32.1 nM and 83.3 nM, Figure 6) revealed that the binding affinity toward in the following order: –OCH > –Br > –I > –H. However, the fluoroethoxy (SIL26) and the α-syn decreased in the following3 order: –OCH3 > –Br > –I > –H. However, the fluoroethoxy (3-iodoallyl)oxy(SIL26) and the (3-iodoallyl)oxy (SIL23) derivatives (SIL23 showed) derivatives only slightly showed reducedonly slightly affinity reduced in the affinity order of 50in nM the [order92]. Furthermore, of 50 nM [92].N Furthermore,-substitution N of-substitution phenothiazines of phenothiazines greatly reduced greatly affinity reduced against αaffinity-syn fibrils against [92, 94α-syn]. In fibrils 2018, [92,94]. binding In of 201 SILs8, binding toward ofα -synSILs fibrilstoward was α-syn studied. fibrils Inwas particular, stud- SIL23ied. Inand particular,SIL26 preferentially SIL23 and SIL26 bind preferentially to BS 3/13 of bindα-syn to BS fibrils 3/13 [of43 α].-syn fibrils [43].

General structure 1

Ki Ki Ki [nM] a [nM] b [nM] c # R1 R2 R3 clogP d* cLogD7.4 ** Fibrils Human PD brain homogenates α-syn α-syn Aβ1-42 tau

1 OCH3 OCH3 H 122 ± 5.1 n.d. n.d. n.d. n.d. 4.01 4.7

2 OCH3 CN H 346 ± 37 n.d. n.d. n.d. n.d. 4.07 4.1

3 OCH3 NH2 H >500 n.d. n.d. n.d. n.d. 2.79 3.4

SIL5 OCH3 NO2 H 32.1 ± 1.3 66.2 110 136 83.1 4.32 4.2

SIL22 Br NO2 H 75.3 ± 8.4 31.9 102 173 57.1 5.31 4.5

SIL23 OCH2CHCHI NO2 H 57.9 ± 2.7 148 * 635 * 230 * 119–168 * 5.87 5.3

SIL26 OCH2CH2F NO2 H 49.0 ± 4.9 15.5 103 125 33.5 4.57 4.3

4 OCH3 NO2 CH3 >500 n.d. n.d. n.d. n.d. 4.44 4.3

SIL3B H NO2 H n.d. 19.9 71.5 52.3 49.4 4.02 4.3

a FigureFigure 6. Phenothiazine6. Phenothiazine analogues analogues with with binding binding propertiesproperties determined from from two two different different assays. assays. Affinitya Affinity toward toward α-synα-syn fibrils determined by ThT fluorescence assay. [92] b Affinity toward recombinant α-syn and tau and synthetic Aβ1-42 fibrils fibrils determined by ThT fluorescence assay [92]. b Affinity toward recombinant α-syn and tau and synthetic Aβ fibrils determined by [125I]SIL23 competition assay [55]. c Affinity toward human PD brain homogenate determined by [1251–42I]SIL23 125 c 125 determinedcompetition by [assayI]SIL23 [55]. * competition Kd value determined assay [55 ].withAffinity the radioligand toward human [125I]SIL23 PD brainon fibrils homogenate or human determined PD brain homogenate. by [ I]SIL23 125 competition* clogP computed assay [55 from]. * KBioByted value software. determined ** clogD with7.4 the computed radioligand from [ACD/Percepta.I]SIL23 on fibrils n.d.: not or humandetermined. PD brain homogenate. * clogP computed from BioByte software. ** clogD7.4 computed from ACD/Percepta. n.d.: not determined. Inspired by these findings, Bagchi et al. radiolabeled SIL23 with iodine-125 and eval- uatedInspired its selectivity by these profile findings, against Bagchi Aβ1-42 et and al. tau radiolabeled fibrils as wellSIL23 as itswith binding iodine-125 properties and at eval- uatedLBs and its selectivityLNs present profile in human against PD Abrains.β1–42 andIn this tau study, fibrils SIL23 as well showed as its a binding Kd value properties of 148 atnM LBs on and α-syn LNs fibrils present and incomparable human PD affinity brains. in human In this study,tissue. TheseSIL23 valuesshowed are, a however, Kd value of 148a 2-fold nM on lowerα-syn than fibrils the and affinity comparable determined affinity by in the human fluorescent tissue. TheseThT competition values are, however,assay, ahighlighting 2-fold lower the than variations the affinity between determined both assays. by the Its fluorescentselectivity toward ThT competition Aβ1-42 and tau assay, fibrils high- lightingis low and the as variations such SIL23 between is not suitable both assays. for the Its development selectivity towardof a PET A αβ-syn1–42 tracer.and tau How- fibrils isever, low and[125I]SIL23 as such is,SIL23 nonetheless,is not suitablean excellent for thetool development to evaluate the of binding a PET α profile-syn tracer. of other How- ever,phenothiazine[125I]SIL23 analogues.is, nonetheless, SIL26 showed an excellent the best tool selectivity to evaluate profile the bindingin this assay. profile A 6-fold of other phenothiazineand 8-fold selectivity analogues. for αSIL26-syn overshowed Aβ1-42 the and best tau selectivity fibrils was profile detected, in this respectively. assay. A 6-fold In andgeneral, 8-fold Ki selectivity values obtained for α -synfromover PD brain Aβ1–42 homogeand taunate fibrils assay waswere detected,comparable respectively. with those In obtained from α-syn fibrils. This indicated that not only can the [125I]SIL23 competition general, Ki values obtained from PD brain homogenate assay were comparable with those obtainedassay accurately from α-syn predict fibrils. binding This in indicated human tissue, that notbut onlyalso that can the fibrillar[125I]SIL23 α-syncompetition binding assaysite is accurately conserved predictbetween binding recombinant in human α-syn tissue, fibrils but and also the thatin vivo the formed fibrillar speciesα-syn in binding PD sitepatients is conserved [55]. Although between SIL5 recombinant and SIL26α did-syn not fibrils show and particularly the in vivo highformed binding species affinity in PD patientsand selectivity, [55]. Although they showedSIL5 andmostSIL26 promisingdid not results show among particularly the tested high phenothiazines binding affinity and and selectivity, they showed most promising results among the tested phenothiazines and were therefore radiolabeled for in vivo PET studies (Figure7A). [11C]SIL5 was isolated in a RCY

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were therefore radiolabeled for in vivo PET studies (Figure 7A). [11C]SIL5 was isolated in a RCY of 35–45% (decay-corrected to EOB) and an Am of >363 GBq/µmol and [18F]SIL26 in 18 ofa RCY 35–45% of 55–65% (decay-corrected (decay-corrected to EOB) to andEOB) an and Am anof A >363m of > GBq/200 GBq/µmolµmol and [95].[ F]SIL26 Both tracersin a RCYwere of able 55–65% to pass (decay-corrected the BBB and tohad EOB) homogeneous and an Am ofdistribution > 200 GBq/ throughoutµmol [95]. Boththe brain tracers in weremale ableSprague-Dawley to pass the BBB rats. and Total had homogeneousbrain uptake for distribution [11C]SIL5 throughoutwas 0.953%ID/g the brain at 5 inmin male p.i. 11 Sprague-Dawleyand 0.287%ID/g at rats. 30 min Total p.i. brain In contrast, uptake for [18F]SIL26[ C]SIL5 showedwas 0.953%ID/g a lower total at brain 5 min uptake p.i. and of 18 0.287%ID/g0.758%ID/g at at 5 30 min min p.i. p.i. but In a contrast, higher uptake[ F]SIL26 than showed[11C]SIL5 a lower(0.359%ID/g) total brain at 120 uptake min p.i. of 11 0.758%ID/gThe decreased at washout 5 min p.i. may but be a a higher result uptakeof the increased than [ C]SIL5 lipophilicity(0.359%ID/g) of [18F]SIL26 at 120 in com- min 18 p.i.parison The decreasedwith [11C]SIL5 washout. Faster may washout be a result kinetics of the are increased generally lipophilicity preferred in of PET[ F]SIL26 studies, 11 in[96] comparison and consequently, with [ C]SIL5[11C]SIL5. Faster was selected washout for kinetics in vivo arePET generally imaging preferredstudies in inhealthy PET 11 studies,NHP (Figure [96] and 7B). consequently, Expected homogenous[ C]SIL5 wasuptake selected within for thein brain vivo PET was imagingobserved studies (since inno healthytarget should NHP (Figurebe present7B). in Expected healthy homogenoussubjects) [95]. uptakeStandardized within uptake the brain value was (SUV) observed peak (sinceuptake no was target > 3 at should approximately be present 7 min in healthy and slowly subjects) decreased [95]. Standardizedand flattened over uptake 70 min value to (SUV)values peak around uptake 1 SUV. was This > 3value at approximately is a good indicator 7 min for and the slowly non-displaceable decreased and binding flattened com- overponent 70 minof the to valuestracer and around may 1 SUV.be too This high value, taking is a goodinto account indicator the for low the non-displaceableamount of α-syn bindingfibrils that component should be of present the tracer in diseased and may animals. be too high, As such, taking a intofurther account study the should low amount be per- α offormed-syn in fibrils α-syn that positive should animals, be present e.g. in transgenic diseased animals. mice expressing As such, a α further-syn fibrils, study to should see if α α be[11C]SIL5 performed can selectively in -syn positive image α animals,-syn. It has e.g. been transgenic shown mice that, expressingat least for SIL23,-syn fibrils,the bind- to [11C]SIL5 α SIL23, seeing ifsite in micecan expressing selectively the image human-syn. α-syn It has transgene been shown containing that, at the least A53T for mutationthe binding site in mice expressing the human α-syn transgene containing the A53T mutation (M83 line) is present in this transgenic mouse model for PD, whereas mice expressing the (M83 line) is present in this transgenic mouse model for PD, whereas mice expressing WT human α-syn transgene (M7 line) did not show any specific SIL23 in brain tissue ho- the WT human α-syn transgene (M7 line) did not show any specific SIL23 in brain tissue mogenates. Since SIL5 is a close analogue of SIL23 and can therefore be displaced by homogenates. Since SIL5 is a close analogue of SIL23 and can therefore be displaced by SIL23, it is likely that both compounds bind at the same binding site. As such, the A53T SIL23, it is likely that both compounds bind at the same binding site. As such, the A53T transgenic mouse model appears to be a suitable in vivo model to test the in vivo binding transgenic mouse model appears to be a suitable in vivo model to test the in vivo binding characteristics of [11C]SIL5 or related compounds [95]. characteristics of [11C]SIL5 or related compounds [95].

FigureFigure 7.7. RadiolabeledRadiolabeled phenothiazinephenothiazine analogues. ( (AA)) Chemical Chemical structures structures of of [[1818F]SIL26F]SIL26 andand [11[11C]SIL5C]SIL5. (.(B)B TACs) TACs from from in invivo vivo evaluation evaluation of of [11[C]SIL511C]SIL5 in inhealthy healthy NHP NHP (adapted (adapted from) from) [95]. [95 ].

3.1.2.3.1.2. PhenoxazinePhenoxazine andand PhenazinePhenazine AnaloguesAnalogues InIn parallelparallel to to the the phenothiazines, phenothiazines, their their isosteres, isosteres, the the phenoxazines phenoxazines and theand phenazines, the phena- werezines, also were evaluated also evaluated as potential as potentialα-syn PET α-syn tracers. PET Tu tracers. et al. synthesized Tu et al. synthesized a small compound a small librarycompound consisting library of consisting 8 compounds of 8 compounds and determined and theirdetermined binding their affinity binding toward affinity recom- to- binantward recombinantα-syn fibrils byα-syn a ThT-based fibrils by fluorescence a ThT-based assay. fluorescence Several ligands assay. showedSeveral affinityligands inshowed the nanomolar affinity in range the nanomolar (Figure8A) range and among(Figure them 8A) andTZ-2-39 amongwith them the TZ-2-39 lowest Kwithi value the of 9.5 nM [97]. In comparison with the phenothiazines, the phenoxazines showed better lowest Ki value of 9.5 nM [97]. In comparison with the phenothiazines, the phenoxazines affinity toward α-syn fibrils. For example, SIL5 showed a K of 32.1 nM while TZ-2-33 a showed better affinity toward α-syn fibrils. For example, SIL5i showed a Ki of 32.1 nM Ki of 25.7 nM. Values were determined using the same binding assay set-up. No further while TZ-2-33 a Ki of 25.7 nM. Values were determined using the same binding assay set- attempts have been made thus far to improve the binding profile of this structural class. up. No further attempts have been made thus far to improve the binding profile of this TZ-2-48 showed displaceable binding and was shown to bind preferentially to the BS 3/13 structural class. TZ-2-48 showed displaceable binding and was shown to bind preferen- position of α-syn fibrils [43]. Interestingly, the phenazines (Figure8B) did not show any tially to the BS 3/13 position of α-syn fibrils [43]. Interestingly, the phenazines (Figure 8B) significant affinity toward α-syn, highlighting the importance of oxygen or sulfur in the did not show any significant affinity toward α-syn, highlighting the importance of oxygen bridge between the aromatic rings [97]. or sulfur in the bridge between the aromatic rings [97].

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General structure 2

Ki # R1 R2 R3 X [nM]

TZ-2-33 OCH3 H H CH 25.7

TZ-2-39 H OCH3 H CH 9.5

TZ-2-48 H OCH3 H N 26.8

Figure 8. GeneralFigure structure8. General of structure phenoxazine of phenoxazine and phenazine and analogues. phenazine ( Aanalogues.) Chemical (A structure) Chemical of phenoxazinestructure of analogues phenoxazine analogues with binding properties utilized from ThT fluorescence assay using α-syn with binding properties utilized from ThT fluorescence assay using α-syn fibrils [97]. (B) Chemical structure of phenazine fibrils [97]. (B) Chemical structure of phenazine analogues TZ16-133-2 with binding affinity Ki > analogues TZ16-133-2 with binding affinity K > 1000 nM [97]. 1000 nM [97]. i 3.2. Benzoxazole Derivatives 3.2. Benzoxazole3.2.1. Derivatives BF-227 3.2.1. BF-227 BF-227 was initially developed as a PET ligand to image amyloid plaques in AD pa- BF-227 wastients initially (Figure developed9A) [ 98]. as However, a PET ligand during to itsimage biological amyloid evaluation plaques BF-227in AD pa-was found to bind tients (Figure 9A)also [98]. to α -synHowever, fibrils withduring an its affinity biological of Kd 9.63evaluation nM, 10-fold BF-227 lower was than found its affinityto to Aβ1–42 bind also to α-synfibrils fibrils (Kd 1.31with nM). an affinity Inspired of byKd these9.63 nM, results, 10-foldBF-227 lowerwas than evaluated its affinity in human to brain tissue. Aβ1-42 fibrils (KdContradictory 1.31 nM). Inspired results by with these respect results, to BF-227 the ability was ofevaluatedBF-227 to in detect humanα -synbrain in human brain tissue. Contradictorytissue haveresults been with reported respect [to99 –the102 ability]. For example,of BF-227 Fodero-Tavoletti to detect α-syn in and human colleagues reported brain tissue havethat beenBF-227 reportedbinds [99–102]. to α-syn inFor PD, example, but not Fodero-Tavoletti in DLB patients [ 99and]. Acolleagues follow-up study analyz- reported that BF-227ing the binds binding to α properties-syn in PD, of butBF-227 not inin DLB human patients postmortem [99]. A follow-up tissue using study brain specimens analyzing the bindingfrom PD properties and DLB of patients BF-227 in showed human that postmortemBF-227 can tissue detect usingα-syn brain fibrils spec- in all cases [99]. imens from PDA and PET DLB study patients in MSA showed patients that confirmedBF-227 can that detect[11 C]BF-227α-syn fibrils(Figure in all9 A,B)cases is suitable for [99]. A PET studyimaging in MSAα-syn patients deposition confirmed in these that [ patients11C]BF-227 [100 (Figure], but a 9A,B) recent is publicationsuitable for by Verdurand imagingα-syn depositionet al. questioned in these the patients binding [100], of BF-227 but a recentto pathological publication forms by Verdurand of α-syn. Autoradiography et al. questioned (ARG)the binding on MSA of BF-227 and control to pathological brain sections forms without of α-syn.α-syn Autoradiography pathology were investigated. (ARG) on MSAIn and contrast control to brain previous sections studies, without Verdurand α-syn pathology and colleagues were investigated. selected the In medulla oblon- contrast to previousgata asstudies, their targetVerdurand region and to studycolleagues the binding selected of theBF-227 medulla. The oblongata reason for as this is that the their target regionmedulla to study oblongata the binding is systematically of BF-227. The affected reason during for this disease is that progression the medulla in MSA without 18 11 oblongata is systematicallydeveloping Aaffectedβ co-pathology. during disease[ F]BF-227 progression(which in isMSA chemically without identical develop- to [ C]BF-227) ing Aβ co-pathology.could not [18F]BF-227 detect any (whichα-syn is inclusions chemically when identical compared to [11 toC]BF-227 healthy) controlscould not [101 ]. This study detect any α-synconcluded inclusions that whenBF-227 comparedcannot beto usedhealthy as ancontrolsα-syn PET[101]. ligand This study [99,100 con-]. The discrepancy cluded that BF-227between cannot results be used was explainedas an α-syn with PET concentration ligand [99,100]. differences The discrepancy of BF-227 be-used in ARG and tween results wasfluorescent explained measurements. with concentration In contrast differences to fluorescence of BF-227 measurements, used in ARG and where micromolar fluorescent measurements.concentrations In arecontrast required, to fluorescence only nanomolar measurements, concentrations where are micromolar needed in ARG. The con- concentrations centrationare required, differences only nanomolar stem from concentrations the sensitivity are needed of fluorescence in ARG. comparedThe con- radioactivity centration differencesdetection. stem Authors from the exemplified sensitivity the of latter fluorescence by conducting compared fluorescence radioactivity study and exposing MSA patient at 100 µM of unlabeled BF-227 without corresponding ARG detection at detection. Authors exemplified the latter by conducting fluorescence study and exposing 1 nM (Figure9C) [ 101]. However, the given explanation cannot explain the positive results MSA patient at 100 µM of unlabeled BF-227 without corresponding ARG detection at 1 gained within the PET study of [11C]BF-227 [100]. Verdurand and colleagues suggest that nM (Figure 9C) [101]. However, the given explanation cannot explain the positive results PET differences in this study are unlikely related to α-syn binding. More importantly, they gained within the PET study of [11C]BF-227 [100]. Verdurand and colleagues suggest that showed that BF-227 can selectively bind to Aβ and as such suggesting that mixed Aβ or PET differences in this study are unlikely related to α-syn binding. More importantly, they α-syn pathologies, which often coexist, could have been imaged in the aforementioned showed that BF-227 can selectively bind to Aβ and as such suggesting that mixed Aβ or PET study [101].

Pharmaceuticals 2021, 14, x FOR PEER REVIEW 14 of 47

α-syn pathologies, which often coexist, could have been imaged in the aforementioned PET study [101]. These findings highlight the difficulties of validating the binding properties of a given chemical structure for its suitability for imaging α-syn fibrils. Control experiments are of crucial importance to rule out binding to other fibrillar structures such as Aβ1-42, especially in human tissue. Evaluation assays should be carefully validated with respect to their ability to predict whether a chemical structure is able to image α-syn fibrils in human tissue. Different conformational forms and densities of α-syn in MSA, DLB, and PD tissue can also influence the outcome of a study and lead to false conclusions. In summary, further experiments are required to validate the usefulness of BF-227 as an α-syn Pharmaceuticals 2021, 14, 847 14 of 45 PET ligand. Current results suggest that BF-227 is not capable of imaging α-syn fibrils in vivo.

N N N N 18 F O F N F N O O O O O S 11CH N S N 3 S N

BF-227 [11C]BF-227 [18F]BF-227

B C a b

11 18 11 Figure 9. (A) Chemical structures ofof BF-227BF-227,, [[11C]BF-227 and [18F]BF-227F]BF-227..( (BB)) TACs TACs of [11C]BF-227C]BF-227 inin putamen putamen in in normal normal controlscontrols and MSA patients (Reprinted with with permission permission from from [100]. [100]. Copyright Copyright 2010 2010 Ox Oxfordford University University Press). Press). ( (CC)) Fluores- Fluores- cence detection of BF-227 (at 100 µM) using MSA brain sections. (a) Tissue autofluorescence (in green) that is imaged prior cence detection of BF-227 (at 100 µM) using MSA brain sections. (a) Tissue autofluorescence (in green) that is imaged prior to BF-227. (b) BF-227 labeling (in blue) of few inclusions. Microscopic examination under DAPI (blue, exposure 500 ms) to BF-227. (b) BF-227 labeling (in blue) of few inclusions. Microscopic examination under DAPI (blue, exposure 500 ms) and and FITC (green, exposure 100 ms) filters (adapted from [101]). FITC (green, exposure 100 ms) filters (adapted from [101]). 3.2.2. SAR Studies of BF-227 These findings highlight the difficulties of validating the binding properties of a given chemicalA series structure of BF-277-like for its suitability compounds for imaging were desiα-syngned fibrils. in order Control to increase experiments the affinity are of andcrucial selectivity importance profile to rule of BF-227 out binding (Figure to other10A) toward fibrillar α structures-syn. Various such oxyethylene as Aβ1–42, especially groups (in5-7 human) as well tissue. as hydrogen, Evaluation iodine assays (8) and should fluorine be carefully (9) were introduced validated with to study respect the tosubsti- their tutionability pattern to predict of whetherthe 6-position a chemical of the structure benzo[d is]oxazole. able to imageIntroductionα-syn fibrils of these in human oxyethylene tissue. groupsDifferent did conformational not alter the affinity forms or and the densities selectivity of profileα-syn inof MSA,the compound DLB, and at PD all. tissue Substitu- can tionalso of influence the fluoroethoxy the outcome group of awith study an andiodi leadne increased to false conclusions.the selectivity In approximately summary, further 10- fold,experiments whereas aresubstitution required towith validate fluorine the or usefulness hydrogen ofloweredBF-227 theas affinity an α-syn toward PET ligand.α-syn fibrilsCurrent from results 53 nM suggest to 286 that nMBF-227 (Figureis 10) not [103. capable Interestingly, of imaging thisα-syn research fibrils insuggested vivo. that the binding affinity of BF-227 toward α-syn fibrils was significantly different when com- pared3.2.2. SARto the Studies affinity of initially BF-227 reported by Fodero-Tavoletti et al. [99] (Figure 10). A possible explanationA series for of this BF-277-like discrepancy compounds may be were that designeddifferent in ordervitro assays to increase as well the as affinity different and fibrilselectivity preparations profile ofwereBF-227 used(Figure in these 10 studies.A) toward α-syn. Various oxyethylene groups (5–7) as well as hydrogen, iodine (8) and fluorine (9) were introduced to study the substitution 3.2.3.pattern Rational of the 6-positionDrug Design of thearound benzo[ BF-227d]oxazole. Introduction of these oxyethylene groups did notIn 2018, alter theVerdurand affinityor et theal. applied selectivity a rational profile of drug the compounddesign approach at all. Substitutionto two other of lead the structurefluoroethoxy scaffolds group (b withenzoimidazoles an iodine increased and TCAs) the selectivity based on approximatelybenzoxazoles. 10-fold,Computational whereas substitution with fluorine or hydrogen lowered the affinity toward α-syn fibrils from 53 nM to 286 nM (Figure 10)[103]. Interestingly, this research suggested that the binding affinity of BF-227 toward α-syn fibrils was significantly different when compared to the affinity initially reported by Fodero-Tavoletti et al. [99] (Figure 10). A possible explanation for this discrepancy may be that different in vitro assays as well as different fibril preparations were used in these studies. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 15 of 47

modeling was applied, and various combinations of lead structure motifs were docked into an α-syn fibril model [67]. Among the 10 virtually designed compounds, two ligands (2FBox and 4FBox) were considered worthwhile to be synthesized and in vitro tested. 18F- Radiolabeling of these molecules succeeded with a RCY of 10–19% and an Am = 68–543 GBq/µmol. Affinity and selectivity were determined with an in vitro saturation assay using synthetic α-syn and Aβ1-42 fibrils. [18F]2FBox (Figure 10B) showed the best binding characteristics with an affinity of Kd = 3.3 ± 2.8 nM against α-syn fibrils and a roughly 50- fold selectivity over Aβ1-42 fibrils. In vitro ARG of rat brains showed that [18F]2FBox was able to detect α-syn fibrils previously injected. Interestingly, preclinical imaging using the same animal model showed that [18F]2FBox was not able to image these α-syn fibrils in vivo, although [18F]2FBox showed sufficient brain uptake and an acceptable, but slow clearance profile. Peak brain uptake was 0.47% ID/g at 12 min p.i. (SUV = 1.6), and the tracer was washed out slowly, lowering its peak uptake only 20% within 50 min. The authors suggested that the discrepancy between the in vitro to in vivo results might be due to the low spatial resolution of PET imaging in comparison to in vitro ARG. The high non- displaceable binding component of [18F]2FBox could also possibly prevent selective imaging. However, [18F]2FBox also failed to detect selective binding on postmortem brain tissue (medulla oblongata) from PD and MSA patients using ARG [67]. The discrepancy between results obtained from computational modeling, postmortem human tissue, and recombinant human fibrils are concerning, but support the hypothesis that fibrils formed in vitro (and virtual models thereof), may not sufficiently represent in vivo forms in all cases. Further validation of this hypothesis is urgently needed. Pharmaceuticals 2021, 14, 847 Since 2FBox did not show selective binding to α-syn in postmortem tissue, we believe that15 of 45 2FBox is not a promising starting point for the development of suitable α-syn PET radioligands.

# R1 R2 R3 [nM] clogP * cLogD7.4 **

α-syn Aβ1-42

BF-227 H OCH2CH2F N(CH3)2 9.63 a; 53 b; 14.03 ± 43.52 c 1.31 and 80 a; 12 b; 4.3 ± 1.5 d; - -

[18F]2FBox - - - 3.3 ± 2.8 c 145 ± 114.5 c 4.76 4.0

General structure 3

5 H OCH2CH2OCH2F N(CH3)2 83 11 - -

6 H O(CH2CH2O)2CH2F N(CH3)2 52 13 - -

7 H O(CH2CH2O)4CH2F N(CH3)2 75 18 - -

8 H I N(CH3)2 74 223 - -

9 H F H 286 1446 - -

FigureFigure 10. 10.(A )(A General) General structure structure of of BF-227-likeBF-227-like compounds compounds with with bindin bindingg properties properties specified specified in table. in table.Affinities Affinities were de- were termined for α-syn and Aβ1-42 fibrils. Binding affinities were determined by several techniques: a Kd values identified with determined for α-syn and Aβ fibrils. Binding affinities were determined by several techniques: a K values identified Scatchard analysis [99,103].1–42 b In vitro saturation assay using synthetic α-syn and Aβ1-42 fibrils [67]. c In vitrod saturation b c with Scatchard analysis [99,103]. In vitro saturation assay using synthetic α-syn and Aβ1–42 fibrils [67]. In vitro saturation d 125 binding assay [67]. In vitro binding assays (Ki) determined for Aβ1–42 fibril in competitive binding assay using [ I]BF- 180 [98]. * clogP values were calculated by BioByte. ** clogD values calculated by ACD/Percepta. (B) The chemical 7.4 structure of [18F]2FBox with the TAC from small-animal PET imaging of fibril-injected rats (controls were taken from the striata regions) (reprinted with permission from [67]. Copyright 2018 American Chemical Society).

3.2.3. Rational Drug Design around BF-227 In 2018, Verdurand et al. applied a rational drug design approach to two other lead structure scaffolds (benzoimidazoles and TCAs) based on benzoxazoles. Compu- tational modeling was applied, and various combinations of lead structure motifs were docked into an α-syn fibril model [67]. Among the 10 virtually designed compounds, two ligands (2FBox and 4FBox) were considered worthwhile to be synthesized and in vitro tested. 18F-Radiolabeling of these molecules succeeded with a RCY of 10–19% and an Am = 68–543 GBq/µmol. Affinity and selectivity were determined with an in vitro satura- 18 tion assay using synthetic α-syn and Aβ1–42 fibrils. [ F]2FBox (Figure 10B) showed the best binding characteristics with an affinity of Kd = 3.3 ± 2.8 nM against α-syn fibrils and a roughly 50-fold selectivity over Aβ1–42 fibrils. In vitro ARG of rat brains showed that [18F]2FBox was able to detect α-syn fibrils previously injected. Interestingly, preclinical imaging using the same animal model showed that [18F]2FBox was not able to image these α-syn fibrils in vivo, although [18F]2FBox showed sufficient brain uptake and an acceptable, but slow clearance profile. Peak brain uptake was 0.47% ID/g at 12 min p.i. (SUV = 1.6), and the tracer was washed out slowly, lowering its peak uptake only 20% within 50 min. The authors suggested that the discrepancy between the in vitro to in vivo results might be due to the low spatial resolution of PET imaging in comparison to in vitro ARG. The high non-displaceable binding component of [18F]2FBox could also possibly prevent selective imaging. However, [18F]2FBox also failed to detect selective binding on postmortem brain tissue (medulla oblongata) from PD and MSA patients using ARG [67]. Pharmaceuticals 2021, 14, 847 16 of 45

The discrepancy between results obtained from computational modeling, postmortem human tissue, and recombinant human fibrils are concerning, but support the hypothesis that fibrils formed in vitro (and virtual models thereof), may not sufficiently represent in vivo forms in all cases. Further validation of this hypothesis is urgently needed. Since 2FBox did not show selective binding to α-syn in postmortem tissue, we believe that 2FBox is not a promising starting point for the development of suitable α-syn PET radioligands.

3.3. Indolinones and Indolinone-Dienes In 2007, Honson et al. reported that indolinone structures confer moderate affinity, but poor selectivity toward α-syn over Aβ1–42 and tau fibrils [104]. Neal et al. later demonstrated that the two fluorescent dyes LDS 798 and LDS 730 (Figure5A) were capable of detecting LB in postmortem PD tissue [87]. This inspired Chu et al. to combine the key features, namely the double bond fragment of the dyes and the indolinone structure, into one new lead. With these modifications, they hoped to increase the selectivity toward α- syn [105]. A series of over 40 compounds was synthesized, and the novel indolinone-diene derivatives displayed higher selectivity toward α-syn. Single double bonds only showed moderate affinity and no selectivity while diene analogues showed improved affinity and selectivity. We speculate that the increased length of these diene analogues is responsible for this and allow for a stronger interaction between the ligand and the binding site. The Z, E configuration was shown to be more active (Figure 11A,B). Five indolinone-diene analogues showed an affinity of Ki < 25 nM toward α-syn fibrils and > 5-fold selectivity over Aβ1–42 and tau fibrils. Compound 15a appeared to be most promising with the highest observed affinity for α-syn (Ki of 2.08 nM) and an approximately 68-fold and 40-fold lower affinity for Aβ and tau fibrils, respectively. Affinities were measured using a ThT based competitive binding assay. These promising data prompted Chu et al. to 18F-radiolabel 15a (Figure 11B) resulting in an isolated activity of 0.71 ± 0.23 GBq (decay corrected) and 18 an Am ranging from 29.6 to 185 GBq/µmol. Kd measurements of [ F]15a confirmed the affinity and selectivity profile measured by aforementioned competition binding assay, i.e., 15a can selectively be displaced from its binding site [43]. Binding affinities toward α-syn, Aβ and tau fibrils were determined to be 8.9, 271 and 50 nM, respectively and 15a was identified to preferential bind to BS 3/13 of α-syn fibrils [43]. Despite these encouraging results, [18F]15a has not been tested on patient-derived tissues, and the compound has not been thoroughly evaluated in preclinical PD animal models. The authors reasoned that 15a possesses a logP of 4.18, which is outside of the standard range for CNS tracers [80] and furthermore, they were concerned that the nitro- group of 15a could be reduced in vivo [105]. A limited in vivo evaluation in NHP was nevertheless conducted and [18F]15a showed slow clearance [54]. Since 15a is one of the most selective α-syn ligands ever designed, we believe this compound should have been tested in preclinical PD animal models and in vitro on patient-derived tissues. Why another compound with lower affinity and selectivity, namely compound 10, was instead evaluated on brain tissue from PD/DLB and PD patients with fluorescence microscopy (Figure 12) remains unclear. Unfortunately, E,E and E,Z stereoisomers were not isolated, and the compound was applied as a mixture with a 1:1 ratio. 10 was clearly able to stain LB. Aβ plaques from AD patients may also be stained, bringing the α-syn selectivity of 10 into question [105]. Nevertheless, this study showed that radiolabeled derivatives from this structural class are able to detect α-syn fibrils only when the latter are present in high concentrations. Future studies based on 15a are warranted and should focus first on the in vitro binding profile of patient-derived α-syn tissue at low concentration. If promising, extensive SAR studies should be conducted to develop better compounds with improved pharmacodynamics and α-syn selectivity. Pharmaceuticals 2021, 14, 847 17 of 45 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 17 of 47

General structure 4

Ki # cLogD7.4 ** R1 R2 E,E or Z,E [nM] clogP a

α-syn Aβ1-42 tau 10 - - mixture 40.7 ± 8.7 27.6 ± 4.8 53.7 ± 9.7 3.85 3.6 11a H H Z,E 25.0 ± 12.7 214.2 ± 52.1 121.5 ± 18.8 5.4 4.6

12a H OCH3 Z,E 12.9 ± 4.9 130.8 ± 64.4 72.4 ± 24.1 5.3 4.3

13a OCH3 H Z,E 3.8 ± 0.6 109.7 ± 0.4 228.6 ± 113.7 5.3 4.3

14a OCH3 OCH3 Z,E 3.5 ± 0.2 73.6 ± 27.3 151.7 ± 64.7 4.8 4.4

15a H OCH2CH2F Z,E 2.08 ± 0.3 142.4 ± 36.9 80.1 ± 12.0 5.6 4.5

15b H OCH2CH2F E,E 76.1 ± 41.2 125.3 ± 40.1 455.7 ± 66.1 5.6 4.5

Figure 11. Chemical structures of indolidone-diene analogues with key properties. (A) General structure of indolidone- Figure 11. Chemical structures of indolidone-diene analogues with key properties. (A) General structure of indolidone- dienes and their binding affinity toward recombinant α-syn, tau, and synthetic Aβ1-42 fibrils determined by ThT competi- dienes and their binding affinitya toward recombinant α-syn, tau, and synthetic Aβ fibrils determined by ThT competitive Pharmaceuticalstive 2021 binding, 14, x assays FOR PEER [105]. REVIEW clogP were calculated from BioByte software. ** clogD1–427.4 values calculated by ACD/Percepta. 18 of 47 a 18 binding(B) assaysChemical [105 structures]. clogP of [ wereF]15a calculated (Z,E configuration) from BioByte and 15b software. (E,E configuration) ** clogD [105].7.4 values calculated by ACD/Percepta. (B) Chemical structures of [18F]15a (Z,E configuration) and 15b (E,E configuration) [105]. Despite these encouraging results, [18F]15a has not been tested on patient-derived tissues, and the compound has not been thoroughly evaluated in preclinical PD animal models. The authors reasoned that 15a possesses a logP of 4.18, which is outside of the standard range for CNS tracers [80] and furthermore, they were concerned that the nitro-group of 15a could be reduced in vivo [105]. A limited in vivo evaluation in NHP was nevertheless conducted and [18F]15a showed slow clearance [54]. Since 15a is one of the most selective α-syn ligands ever designed, we believe this compound should have been tested in preclinical PD animal models and in vitro on patient-derived tissues. Why another compound with lower affinity and selectivity, namely compound 10, was instead evaluated on brain tissue from PD/DLB and PD patients with fluorescence microscopy (Figure 12) remains unclear. Unfortunately, E,E and E,Z stereoisomers were not isolated, and the compound was applied as a mixture with a 1:1 ratio. 10 was clearly able to stain LB. Aβ FigureFigure 12. Chemical structure ofof 1010 and fluorescentfluorescent microscopymicroscopy studies studies on on brain brain tissue tissue from from PD/DLB PD/DLB plaques from AD patients may also be stained, bringing the α-syn selectivity of 10 into andand PDPD patients (reprinted(reprinted withwith permissionpermission fromfrom [[105]105] CopyrightCopyright 20152015 AmericanAmerican ChemicalChemical Society).Society). question [105]. Nevertheless, this study showed that radiolabeled derivatives from this 3.4.3.4.structural Thiazole Derivativesclass are able to detect α-syn fibrils only when the latter are present in high concentrations. Future studies based on 15a are warranted and should focus first on the in TheThevitro thiazolethiazole binding profile moietymoiety of isis patient-derived aa widely applied α-syn scaffoldscaffold tissue at toto low enhanceenhance concentration. affinityaffinity If andand promising, selectivityselectivity ofof potentialpotentialextensive αα-syn -synSAR targetingstudiestargeting should compounds.compounds. be conducted For to instance,instance, develop better thethe benzothiazolebenzothiazole compounds with ringring improved isis presentpresent inin cyaninecyaninepharmacodynamics dyesdyes ((T-284T-284 and andand SH-516SH-516α-syn selectivity.,, FigureFigure5 5)) and and fluorescent fluorescent dyes dyes ( (ThTThT,, FigureFigure5 5)) that that have have previouslypreviously beenbeen shownshown toto bindbind toto αα-syn-syn fibrils.fibrils. InIn previousprevious sections,sections, thethe thiazolethiazole moietymoiety alreadyalready appearedappeared forfor exampleexample withinwithin thethe ligandligand BF-227BF-227.. InIn thisthis section,section, wewe willwill furtherfurther discussdiscuss itsits pastpast rolerole inin thethe designdesign ofof aa selectiveselective αα-syn-syn ligand.ligand.

3.4.1.3.4.1. Diphenylthiazole Derivatives Several diphenylthiazole, diphenyloxazoles and diphenylthiophenes compounds were Several diphenylthiazole, diphenyloxazoles and diphenylthiophenes compounds developed as potential α-syn imaging agents [106]. However, only one diphenylthiazole, were developed as potential α-syn imaging agents [106]. However, only one diphenylthiazole, compound 17 (Figure 13) has thus far shown a reasonable affinity (Ki = 89 nM) toward α-syn aggregates in affinity measurements conducted using a competition binding assay. Compound 17 also did not show any significant binding for Aβ in AD tissue.

Figure 13. Chemical structure of 17 with binding affinity Ki = 89 nM [106].

3.4.2. Bithiazole Derivatives Bithiazole derivatives were reported as promising new ligands for the detection of neuropathological aggregates, including α-syn. Unfortunately, the only available literature source, to date, is patented and no additional articles are available on the subject. Researchers from the Technical University of Munich developed these bithiazoles and among them compounds 18, 19 and 24 (Figure 14) showed high affinity for α-syn and good selectivity over Aβ1-42 and tau fibrils (as determined by saturation binding assays). Several bithiazoles from the same family were identified as potential ligands for targeting Aβ and tau. Potential ligands for α-syn are summarized in Figure 14 with limited binding affinity data. Compound 18 demonstrated the highest affinity (Ki = 3 nM) and a selectivity of 127-fold and >300-fold over Aβ1-42 and tau, respectively. Additionally, compounds 20, 21, 22 and 23 (Figure 14) were also reported to be selective for α-syn, although no binding data was published [107]. Just recently, [18F]S3-1 (Figure 14) a novel structurally related α-syn PET tracer was evaluated in rats, NHPs and humans following a conference abstract [108]. [18F]S3-1 showed binding affinity of 3 nM for α-syn and high selectivity of 120-fold for Aβ and tau. Brain kinetics were investigated in E46K rats demonstrating statistically significant difference between E46K and health controls. Furthermore, in NHPs initial brain update was up to 1.4% ID and the SUV was 1.3 at 30 min with rapid clearance (SUV

Pharmaceuticals 2021, 14, x FOR PEER REVIEW 18 of 47

3.4. Thiazole Derivatives The thiazole moiety is a widely applied scaffold to enhance affinity and selectivity of potential α-syn targeting compounds. For instance, the benzothiazole ring is present in cyanine dyes (T-284 and SH-516, Figure 5) and fluorescent dyes (ThT, Figure 5) that have previously been shown to bind to α-syn fibrils. In previous sections, the thiazole moiety already appeared for example within the ligand BF-227. In this section, we will further discuss its past role in the design of a selective α-syn ligand.

3.4.1. Diphenylthiazole Derivatives Pharmaceuticals 2021, 14, 847 18 of 45 Several diphenylthiazole, diphenyloxazoles and diphenylthiophenes compounds were developed as potential α-syn imaging agents [106]. However, only one diphenylthiazole, compound 17 (Figure 13) has thus far shown a reasonable affinity (Ki = 89 nM) to- compound 17 (Figure 13) has thus far shown a reasonable affinity (Ki = 89 nM) toward αward-syn aggregatesα-syn aggregates in affinity measurementsin affinity measurements conducted using conducted a competition using binding a competition assay. binding Compoundassay. Compound17 also did 17 not also show did any not significant show any binding significant for Aβ in binding AD tissue. for Aβ in AD tissue.

Figure 13. Chemical structure of 17 with binding affinity K = 89 nM [106]. Figure 13. Chemical structure of 17 with bindingi affinity Ki = 89 nM [106]. 3.4.2. Bithiazole Derivatives 3.4.2.Bithiazole Bithiazole derivatives Derivatives were reported as promising new ligands for the detection of neuropathologicalBithiazole derivatives aggregates, including were reportedα-syn. Unfortunately, as promising the new only ligands available for liter- the detection of ature source, to date, is patented and no additional articles are available on the subject. neuropathological aggregates, including α-syn. Unfortunately, the only available litera- Researchers from the Technical University of Munich developed these bithiazoles and amongture source, them compounds to date, 18is, 19patentedand 24 (Figure and 14no) showedadditional high affinityarticles for areα-syn available and good on the subject. selectivityResearchers over Afromβ1–42 theand tauTechnical fibrils (as University determined by of saturation Munich binding developed assays). these Several bithiazoles and bithiazolesamong them from thecompounds same family 18, were 19 identified and 24 as(Figure potential 14) ligands showed for targeting high affinity Aβ and for α-syn and tau. Potential ligands for α-syn are summarized in Figure 14 with limited binding affinity good selectivity over Aβ1-42 and tau fibrils (as determined by saturation binding assays). data. Compound 18 demonstrated the highest affinity (K = 3 nM) and a selectivity of Several bithiazoles from the same family were identifiedi as potential ligands for targeting 127-fold and >300-fold over Aβ1–42 and tau, respectively. Additionally, compounds 20, 21, 22Aβand and23 tau.(Figure Potential 14) were ligands also reported for toα-syn be selective are summarized for α-syn, although in Figure no binding 14 with data limited binding 18 wasaffinity published data. [ 107Compound]. Just recently, 18 demonstrated[ F]S3-1 (Figure the 14) highest a novel structurally affinity (K relatedi = 3 nM)α-syn and a selectivity PET tracer was evaluated in rats, NHPs and humans following a conference abstract [108]. of 127-fold and >300-fold over Aβ1-42 and tau, respectively. Additionally, compounds 20, [18F]S3-1 showed binding affinity of 3 nM for α-syn and high selectivity of 120-fold for Aβ and21, 22 tau. and Brain 23 kinetics (Figure were 14) investigated were also in reported E46K rats demonstratingto be selective statistically for α-syn, significant although no binding 18 differencedata was between published E46K [107]. and health Just controls.recently, Furthermore, [ F]S3-1 (Figure in NHPs initial14) a brainnovel update structurally related wasα-syn up toPET 1.4% tracer ID and was the evaluated SUV was 1.3 in at rats, 30 min NHPs with rapid and clearancehumans (SUV following ~ 0.1) but a conference with abstract marginally[108]. [18F]S3-1 increases showed in the later binding timepoints affinity (SUV of = 3 0.4 nM at 120formin). α-syn Dynamic and high PET selectivity studies of 120-fold 18 α showedfor Aβ specificand tau. binding Brain of [kineticsF]S3-1 in were patients investigated with underlying in E46K-synucleinopathies rats demonstrating [108]. statistically In summary, bithiazole derivatives present a class of compounds with excellent affinity andsignificant selectivity, difference however more between detailed E46K scientific and literature health iscontrols. needed in Furthermore, order to provide in NHPs initial morebrain thorough update conclusions.was up to 1.4% ID and the SUV was 1.3 at 30 min with rapid clearance (SUV

3.4.3. Benzothiazole Derivatives Benzothiazole analogues have recently attracted attention as potentially selective α-syn PET ligands. Among this class, [11C]PBB-3 was initially developed to image namely tau aggregates [109]. Its binding affinity toward tau was 1.8 nM (neocortex/hippocampus transgenic mice) [110]. During preclinical evaluation, binding toward α-syn was detected. Koga et al. were the first to examine the ability of [11C]PBB-3 (Figure 15A) to bind to brain-derived LB, LN, and glial cytoplasmic inclusions (GCI) from patients with α-synucleinopathies (MSA and DLB). Fluorescence imaging showed that PBB3 can bind to α-synucleinopathies only at high concentrations (32.3 µM). For example, immunofluorescence double-staining clearly demonstrated this binding (Figure 15B). In ARG and 11 at a low concentration (10 nM, Am = 133 GBq/µmol), [ C]PBB-3 could only bind to the high density GCIs from MSA patients. No specific binding was detected in DLB patients (Figure 15C) [111]. Recently, an additional compound based on the benzothiazole scaffold was identified. PP-BTA-4, a push-pull (PP) benzothiazole derivative, was shown to bind to α-syn. PP-BTA-4 (Figure 15A) strongly increased its fluorescence intensity upon binding to the protein aggregates in solution. A Kd of 48.0 ± 0.6 nM was determined by an in vitro saturation binding assay on α-syn aggregates from human PD brains. PP-BTA-4 also bond Pharmaceuticals 2021, 14, 847 19 of 45

to Aβ1–42 (derived from AD patients) with an affinity in the same order of magnitude [112]. Other benzothiazole derivatives of interest are compounds 25, 26, and 27 (Figure 15A). In a fluorescence-based assay using amygdala brain sections derived from fully developed PD and AD patients (Braak stage V -VI) selective binding of these compounds to α-syn over Aβ plaques was detected. The compounds were incubated on the brain sections at a high concentration of 100 µM. Further binding affinity measurements revealed Kd values for Pharmaceuticals 2021, 14, x FOR PEER REVIEW 19 of 47 compounds 25 and 26 of 50 nM and 14 nM, respectively [113]. Affinities were determined by backscattering interferometry on human PD brain homogenates (cortex). Affinity data for 27 is not reported [113]. Just recently, a novel analogue of PBB3, C05-01 (Figure 15A), ~ 0.1) but with marginally increases in the later timepoints (SUV = 0.4 at 120 min). Dynamic has been discovered showing a Ki of 3.5 nM for brain homogenates and a Ki of 25 nM for PET studies showed specific binding of [18F]S3-1 in patients with underlying α-synucle- α-syn fibrils. C05-01 was derived from a library of 44 compounds that were structurally inopathies [108]. In summary, bithiazole derivatives present a class of compounds with similar to PBB3 and was selected based on quantitative evaluation using fluorescence excellent affinity and selectivity, however more detailed scientific literature is needed in microscopyorder to provide and a semiquantitativemore thorough conclusions. approach using a tissue microarray (TMA).

General structure 5

Ki # % inhibition of [3H]PIB b Lipophilicity a R1 R2 [nM]

α-syn Aβ1-42 tau Aβ1-42 clogP c logP d clogP * cLogD7.4 **

18 H F 3 382 >1000 45 ± 2 5.47 n.d. 5.47 4.5

19 CH3 H 10 386 558 45 ± 2 5.95 n.d. 5.98 4.8

20 CH2F H 3 *** >360 *** >360 *** n.d. n.d. 2.6 5.57 4.7 ([18F]S3-1) (CH2[18F])

21 CH2O(CH2)2F H n.d. n.d. n.d. n.d. n.d. 2.5 6.06 4.6 22 F H n.d. n.d. n.d. n.d. n.d. 2.6 5.62 4.4

23 CH2(OCH2CH2)2F H n.d. n.d. n.d. n.d. n.d. 2.3 5.28 4.5 24 - - 6 492 >1000 35 ± 2 n.d. 3.2 6.00 4.7

Figure 14. Chemical structures of bithiazole derivatives with key properties. Chemical structure of potential α-syn ligands Figure 14. Chemical structures of bithiazole derivatives with key properties. Chemical structure of potential α-syn ligands with binding properties toward recombinant α-syn, Aβ1-42, and tau fibrils. a Affinity toward α-syn fibrils, synthetic amyloid α β a α with bindingpeptides properties (Aβ1-42) and toward human recombinant recombinant tau-441-syn, A determined1–42, and by tau saturation fibrils. bindingAffinity assay toward [107].-syn b % inhibition fibrils, synthetic of [3H]PIB amyloid b 3 peptidesdetermined (Aβ1–42) by and competition human recombinant binding assay tau-441 [107]. cdetermined calculated clogP by saturationaccording to binding the reference assay [[107].107]. d Measured% inhibition logP offrom[ H]PIB determinedoctanol/PBS by competition study according binding to the assay refere [nce107 ].[107].c calculated n.d.: not determin clogP accordinged. * clogP tovalues the referencewere calculated [107]. byd BioByte.Measured ** logP from octanol/PBSclogD7.4 values study calculated according by ACD/Perc to the referenceepta. *** Literature [107]. n.d.: source not [108]. determined. * clogP values were calculated by BioByte. ** clogD values calculated by ACD/Percepta. *** Literature source [108]. 7.4 3.4.3. Benzothiazole Derivatives TMABenzothiazole is an effective analogues approach have forrecently high-throughput attracted attention molecular as potentially analysis selective of pathological α- 11 tissues,syn PET as itligands. makes Among use of paraffinthis class, blocks, [ C]PBB-3 which was allow initially one developed to measure to severalimage namely tissue cores withouttau aggregates consuming [109]. the Its tissuebinding block. affinity A to furtherward tau advantage was 1.8 nM of (neocortex/hippocampus TMA is that it can be used transgenic mice) [110]. During preclinical evaluation, binding toward α-syn was detected. to examine tissue from multiple patients. TMA also allows multiplexing of histological Koga et al. were the first to examine the ability of [11C]PBB-3 (Figure 15A) to bind to brain- analysisderived including LB, LN, and immunohistochemistry, glial cytoplasmic inclusions immunofluorescence, (GCI) from patients histological with α-synucleinop- staining. The bindingathies profile(MSA and of C05-01DLB). Fluorescencewas determined imaging on showed recombinant that PBB3α-syn can fibrilsbind to using α-synucle- saturation 3 andinopathies competition only at binding high concentrations assay with [(32.3H]C05-01 µM). Forand example,C05-01 immunofluorescenceand an affinity in dou- the range 25–30ble-staining nM was clearly determined. demonstrated Further this studies binding using (Figure brain 15B). homogenates In ARG and from at a low DLB concen- (amygdala) 11 revealedtration different(10 nM, Am affinities = 133 GBq/µmol), to those [ previously11C]PBB-3 could reported only bind by Kogato the ethigh al. density [111]. [GCIsC]PBB3 showedfrom MSA a moderate patients. affinity No specific (Kd bindingof 58 nM) was while detected non-labeled in DLB patientsC05-01 (Figuredemonstrated 15C) [111]. a high affinityRecently, (Ki ofan 3.5additional nM). These compound data based once againon the benzothiazole showed that scaffold the tertiary was identified. structure PP- of fibrils andBTA-4 brain, a homogenates push-pull (PP) can benzothiazole be different. de Anotherrivative, was reason shown for theto bind observed to α-syn. discrepancy PP-BTA- may 4 (Figure 15A) strongly increased its fluorescence intensity upon binding to the protein aggregates in solution. A Kd of 48.0 ± 0.6 nM was determined by an in vitro saturation binding assay on α-syn aggregates from human PD brains. PP-BTA-4 also bond to Aβ1-42 (derived from AD patients) with an affinity in the same order of magnitude [112]. Other

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be the variance of experiments. Further characterization of selectivity using PD, MSA, AD and Pick’s disease TMA sections and fresh frozen tissue confirmed the binding of C05-01 to α-syn, however, binding to other amyloids was also shown [114]. Recently, new ThT analogues, namely RB1 and RB2 (Figure 15A), were developed in order to achieve stronger selectivity for α-syn over amyloid fibrils. Key structural features include introduction of a double bond and cyclic nitrogen donors (either piperidine or piperazine). Binding affinities were tested using α-syn fibrils (concentration < 30 µM) and revealed Kd values of 30 nM and ~4.4 µM for RB1 and RB2, respectively. The authors hypothesize that the piperidine moiety fits better in the hydrophobic pocket of α-syn fibrils and therefore RB1 shows higher affinity for α-syn than the lead compound. No selectivity data over Aβ or tau have been reported. Encouraged by these results the authors studied RB1 and its applicability for live-cell imaging using the SH-SY5Y human neuroblastoma cell line. Cultured cells were incubated with short α-syn fibrils. Then, these cells were incubated with RB1 at concentration 500 nM and demonstrated selective staining of α-syn fibrils in the cytosol. This study showed an approach to implement the design and development Pharmaceuticals 2021, 14, x FOR PEER REVIEW 21 of 47 of new compounds. However, binding affinity and selectivity profile need to be further improved and investigated [115].

Figure 15. Chemical structures and examples of fluorescence and ARG. (A) Chemical structures of [11C]PBB-3, PP- Figure 15. Chemical structures and examples of fluorescence and ARG. (A) Chemical structures of [11C]PBB-3, PP-BTA-4, compoundsBTA-4, compounds 25, 26, 2725,, and 26, RB1 27, and, RB2RB1. (B,) RB2PBB-3.(B fluorescence) PBB-3 fluorescence labeling and labeling immu andnofluorescence immunofluorescence double staining double for staining α-syn (NACP)for α-syn and (NACP) phosphor-tau and phosphor-tau (CP13) in DLB (CP13) from inbrainstem DLB from type brainstem LB (reprinted type with LB (reprinted permission with from permission [111]. Copyright from [2017111]. InternationalCopyright 2017 Parkinson International and Movement Parkinsonand Disorder Movement Society). Disorder (C) Autoradiographic Society). (C) Autoradiographic labeling using labeling [11C]PBB-3 using in[ 11amygdalaC]PBB-3 ofin DLB amygdala patients of DLB (reprinted patients with (reprinted permission with from permission [111]. fromCopyright [111]. 2017 Copyright Internat 2017ional International Parkinson Parkinsonand Movement and Movement Disorder Society).Disorder Society).

3.4.4. InThienopyridine, conclusion, benzothiazole Thiazolo Pyridine derivatives Derivatives appear to be structural leads which may be helpRecently, in the development a new heterogeneou of an α-syns class selective of compounds compound. was Extensive discovered, SAR which studies may are stain still αneeded-syn aggregates to learn more as well about as howAβ plaques. binding andFibrils selectivity were derived can be from improved. fully developed PD and AD patients (Braak stage V-VI) and a library of 130 compounds were incubated at 100 µM and their neuropathological staining was confirmed by staining with primary antibodies. Of the 130 tested compounds, only several (28-34) showed selectivity for α-syn aggregates over Aβ plaques (Figure 16, determine with fluorescence staining). The only compound that was tested for affinity was compound 34, although it only shows weak staining of α-syn aggregates. This decision was most likely taken since 34 did not stain Aβ plaques. An affinity (Kd) of 9 nM was determined by backscattering interferometry on human derived tissue (PD brain homogenates) [113]. To further evaluate this class of compounds, a comprehensive in vivo evaluation, in vitro ARG at lower concentrations and radiolabeling studies should be performed.

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3.4.4. Thienopyridine, Thiazolo Pyridine Derivatives Recently, a new heterogeneous class of compounds was discovered, which may stain α-syn aggregates as well as Aβ plaques. Fibrils were derived from fully developed PD and AD patients (Braak stage V-VI) and a library of 130 compounds were incubated at 100 µM and their neuropathological staining was confirmed by staining with primary antibodies. Of the 130 tested compounds, only several (28–34) showed selectivity for α-syn aggregates over Aβ plaques (Figure 16, determine with fluorescence staining). The only compound that was tested for affinity was compound 34, although it only shows weak staining of α-syn aggregates. This decision was most likely taken since 34 did not stain Aβ plaques. An affinity (Kd) of 9 nM was determined by backscattering interferometry on human derived tissue (PD brain homogenates) [113]. To further evaluate this class of Pharmaceuticals 2021, 14, x FOR PEER REVIEW 22 of 47 compounds, a comprehensive in vivo evaluation, in vitro ARG at lower concentrations and radiolabeling studies should be performed.

Kd Staining Staining Aβ Staining Aβ # R1 R2 R3 R4 [nM] a α-Syn aggregates b plaques c plaques d α-Syn General structure 6

28 N(CH3)2 NHCH3 - - n.d. Strong Weak n.d. 29 H 4-morpholine - - n.d. Strong Weak n.d. General structure 7 30 H H H 3-fluoropiperidin-1-yl n.d. Strong n.d. No 31 H H H pyrrolidin-1-yl n.d. Strong n.d. No

32 H CH3 CH3 pyrrolidin-1-yl n.d. Strong n.d. No

33 CH3 H H pyrrolidin-1-yl n.d. Strong n.d. No 34 - - - - 9 Weak No n.d.

Figure 16. Chemical structures of thienopyridine, thiazolo pyridine derivatives with key properties. Chemical structures Figure 16. Chemical structures of thienopyridine, thiazolo pyridine derivatives with key properties. Chemical structures of of compounds 28-34 and general structures with key binding properties. a Affinity toward α-syn fibrils aggregates deter- compounds 28–34 and general structures with key binding properties. a Affinity toward α-syn fibrils aggregates determined mined by backscattering interferometry on PD brain homogenates. b Staining of α-syn aggregates on PD sections. c Staining b α c β byof A backscatteringβ plaques on interferometryAD sections. d Staining on PD brain of A homogenates.β plaques on PDStaining sections of with-syn mixed aggregates pathology. on PD n.d.: sections. not determined.Staining ofLiter- A d plaquesature source on AD [113]. sections. Staining of Aβ plaques on PD sections with mixed pathology. n.d.: not determined. Literature source [113]. 3.5. Benzoimidazole Derivatives 3.5. Benzoimidazole Derivatives 3.5.1. Styryl Benzodiazole Derivatives Styryl benzazole derivatives have beenbeen shownshown toto interactinteract withwith differentdifferent amyloidamyloid fib- fi- brilsrils [ 116[116].]. This This inspired inspired Watanabe Watanabe et et al. al. to to test test if thisif this structural structural class class of compoundsof compounds can can be beused used to developto develop an anα-syn α-syn selective selective compound compound [117 [117,118],118]. Three. Three novel novel probes probes were were synthe- syn- thesizedsized and and subsequently subsequently labeled labeled with with iodine-125 iodine-125 (Figure (Figure 17). 17). Watanabe Watana etbe al. et speculated al. speculated that thatincorporation incorporation of dienes of dienes in the in linker the lin wouldker would enhance enhance the affinitythe affinity toward towardα-syn α-syn aggregates aggre- gatesand that and bulky that bulky substituents substituents like benzyl like benzyl groups groups might increase might increase selectivity selectivity toward α toward-syn over α- Asynβ aggregates.over Aβ aggregates. These considerations These considerations followed thefollowed theory the developed theory developed by Chu et abyl. duringChu et al.the during development the development of compound of 15acompound[105]. Saturation 15a [105]. binding Saturation assays binding based assays on fluorescence based on fluorescenceintensity measurements intensity measurements on recombinant on recombinantα-syn and Aα-synβ1–42 andaggregates Aβ1-42 aggregates showed thatshowed the that the developed probes possessed a Kd in the range of 99.5–874 nM. [125I]BI-2 showed the best affinity and selectivity profile (Kd 99.5 ± 20.8 nM and 7-fold selective over Aβ1-42). These results suggest that bulky substituents enhance affinity as well as selectivity. Con- sequently, BI-2 was studied further using fluorescent staining on PD and AD brain sections (at 200 µM). Stained sections were compared with immunohistochemistry and colocalization was observed in the case of α-syn positive tissue, but not for Aβ positive tissue. These results encouraged the research to label two additional derivatives of BI-2 and evaluate the in vivo biological behavior of all three compounds. Only [125I]BI-3 showed a moderate brain uptake of 2.0% ID/g at 10 min post injection and moderate clearance within 60 min resulting in 0.5% ID/g (Figure 17) [117]. Further improvements regarding affinity, selectivity and lipophilicity are needed to develop an α-syn selective ligand based on this scaffold.

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developed probes possessed a Kd in the range of 99.5–874 nM. [125I]BI-2 showed the best affinity and selectivity profile (Kd 99.5 ± 20.8 nM and 7-fold selective over Aβ1–42). These results suggest that bulky substituents enhance affinity as well as selectivity. Consequently, BI-2 was studied further using fluorescent staining on PD and AD brain sections (at 200 µM). Stained sections were compared with immunohistochemistry and co-localization was observed in the case of α-syn positive tissue, but not for Aβ positive tissue. These results encouraged the research to label two additional derivatives of BI-2 and evaluate the in vivo biological behavior of all three compounds. Only [125I]BI-3 showed a moderate Pharmaceuticals 2021, 14, x FOR PEER REVIEW brain uptake of 2.0% ID/g at 10 min post injection and moderate clearance23 of 47 within 60 min resulting in 0.5% ID/g (Figure 17)[117]. Further improvements regarding affinity, selectivity and lipophilicity are needed to develop an α-syn selective ligand based on this scaffold.

General structure 8

# R1 [nM] a clogP * cLogD7.4 **

α-Syn Aβ1-42

[125I]BI-1 CH2C6H5 485 ± 160 179 ± 59.1 6.28 7.12

[125I]BI-2 p-CH2C6H4OCH3 99.5 ± 20.8 727 ± 227 6.23 7.04

[125I]BI-3 CH3 874 ± 169 271 ± 67.3 4.14 5.6

Figure 17. General structure of radio-iodinated benzoimidazole derivatives and their brain uptake Figure 17. General structure of radio-iodinated benzoimidazole derivatives and their brain uptake after intravenous injection. after intravenous injection. Binding affinities were determined on recombinant α-syn and Aβ1-42 ag- Binding affinities were determined on recombinant α-syn and Aβ aggregates. (Reprinted with permission from [117]. gregates. (Reprinted with permission from [117]. Copyright1–42 2017 Elsevier). * clogP values were cal- Copyright 2017 Elsevier). * clogP values were calculated by BioByte. ** clogD values calculated by ACD/Percepta. culated by BioByte. ** clogD7.4 values calculated by ACD/Percepta.7.4 3.5.2. Azaindole Derivatives 3.5.2. Azaindole Derivatives A recently published study by Routier et al. reported on a new set of compounds A recentlyfeaturing published an study azaindole by Routier moiety et and al. reported a phenyl on ring a linkednew set via of acompounds triple bond. The process featuring an azaindoleleading tomoiety the discovery and a phenyl of these ring structures linked via was a not triple discussed. bond. The Their process binding affinity and leading to the discovery of these structures was not discussed. Their binding affinity and selectivity were measured by ThT fluorescence assays using synthetic α-syn, Aβ1–42, and selectivity weretau measured fibrils. Of by the ThT nine fluorescence tested compounds, assays using compound synthetic39 αwas-syn, the Aβ most1-42, and promising ligand tau fibrils. Of the nine tested compounds, compound 39 was the most promising ligand (Figure 18A), with a > 22-fold selectivity over Aβ1–42 fibrils but only limited selectivity (Figure 18A), withover a tau> 22-fold fibrils. selectivity SAR studies over conductedAβ1-42 fibrils on but the only heteroaromatic limited selectivity substituents over at the 5-, 6- tau fibrils. SARand studies 7-position conducted (35 vs.on the36 vs.heteroaromatic37, Figure 18 substituents) of the indole at the scaffold 5-, 6- and revealed 7- that the 7- position (35 vs.azaindole 36 vs. 37,structure Figure 18) (Figure of the 18 indoleA) resulted scaffold in revealed the best selectivitythat the 7-azaindole profile. The influence of structure (Figure2-thienyl, 18A) resulted 3-thienyl, in the p-F-pyridyl, best selectivity fluoride profile. substitution The influence on the of 6-position 2-thienyl, were 3- also studied thienyl, p-F-pyridyl,(38 vs. fluoride39 vs. 40substitutionvs. 41, Figure on the 18 6-position). In particular, were also substitution studied ( with38 vs. 2-thienyl 39 gave the vs. 40 vs. 41, Figurebest affinity18). In particular, but also resulted substitution in limited with selectivity.2-thienyl gave The the only best compound affinity but radiolabeled and also resulted inevaluated limited selectivity.in vivo was The[ 18onlyF]42 compound(Figure 18 C).radiolabeled The in vivo andstudy evaluated confirmed in vivo it to can cross the was [18F]42 (FigureBBB despite18C). The its in relatively vivo study high confir clogDmed [119 it]. to Notwithstanding can cross the BBB the despite limited its amount of peer relatively high reviewedclogD [119]. literature Notwithstanding on this class the of compounds,limited amount its structural of peer reviewed similarities litera- with C05-01 make ture on this classit aof lead compounds, compound its structural for α-syn PETsimilarities tracer developmentwith C05-01 make [114]. it Althougha lead com- it is challenging pound for α-syn PET tracer development [114]. Although it is challenging to draw solid conclusions from two different classes of compounds and without any knowledge on their binding profile, we are speculating that the triple bond linker and its induced planarity can enhance the binding of ligands to β-sheets. A similar hypothesis was drawn by Kaide and colleagues [120]. However, due to the structural similarity between different amyloids and their frequent colocalization, high planarity compounds will not be able to discriminate between abnormal aggregates; thus, leading to a moderate to low selectivity profile which is the case for azaindole derivatives. Interestingly, this compound class showed moderate selectivity over Aβ but no selectivity over tau. As such, further SAR

Pharmaceuticals 2021, 14, 847 23 of 45

to draw solid conclusions from two different classes of compounds and without any knowledge on their binding proﬁle, we are speculating that the triple bond linker and its induced planarity can enhance the binding of ligands to β-sheets. A similar hypothesis was drawn by Kaide and colleagues [120]. However, due to the structural similarity between different amyloids and their frequent colocalization, high planarity compounds will not Pharmaceuticals 2021, 14, x FOR PEER REVIEW 24 of 47 be able to discriminate between abnormal aggregates; thus, leading to a moderate to low selectivity proﬁle which is the case for azaindole derivatives. Interestingly, this compound class showed moderate selectivity over Aβ but no selectivity over tau. As such, further SARstudies, studies, in vivoin vivoevaluationevaluation and ex and vivo ex AR vivoG on ARG postmortem on postmortem tissue are tissue needed are to neededconclu- to conclusivelysively determine determine if azaindole if azaindole derivatives derivatives are promising are promising scaffolds for scaffolds the future for develop- the future developmentment of α-syn of selectiveα-syn selective ligands. ligands.

General structure 9

Ki [nM] a # X1 X2 X3 R1 R2 R3 cLogP * cLogD7.4 ** α-Syn Aβ1-42 tau 35 N C C H H H 0.53 ± 0.2 11.8 ± 3 0.44 ± 0.1 3.98 4.1 36 C N C H H H 1.4 ± 1 5.2 ± 3 0.49 ± 0.2 3.98 3.5 37 C C N H H H 1.0 ± 0.5 3.6 ± 2 2.4 ± 0.9 3.98 3.3 38 N C C F H H 0.79 ± 0.4 16.8 ± 2 n.d. 4.61 4.0 39 N C C 2-thienyl H H 0.49 ± 0.2 4.9 ± 0.6 1.67 ± 0.5 6.37 5.1 40 N C C 3-thienyl H H 0.73 ± 0.3 4.27 ± 0.6 n.d. 6.16 5.0 41 N C C p-F-pyridyl H H 1.27 ± 0.4 3.1 ± 1 n.d. 5.28 4.4 42 ------2.4 ± 1 1.2 ± 1 0.97 ± 0.4 4.63 4.1

Figure 18. Chemical structures of azaindole derivatives with key properties. (A) Chemical structure of compound 39. (B) Figure 18. Chemical structures of azaindole derivatives with key properties. (A) Chemical structure of compound 39. General structure of azaindole derivatives with identified in vitro Ki and Kd binding properties [119]. (C) Chemical struc- (B) General structure of azaindole derivatives with identified in vitro K and K binding properties [119]. (C) Chemical ture of [18F]42 that was used for in vivo study. * clogP values were calculatedi by dBioByte. ** clogD7.4 values calculated by 18 structureACD/Percepta. of [ F]42 that was used for in vivo study. * clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. 3.6. Diphenylpyrazole Derivatives 3.6. Diphenylpyrazole Derivatives High-throughput screening of more than 20.000 compounds identified 3,5-diphe- nylpyrazoleHigh-throughput (DPP) as screening a promising of more small than molecule 20,000 compoundsscaffold to detect identified aggregated 3,5-diphenylpyrazole α-syn in- (DPP)clusions. as a DDP promising based smallcompounds molecule inhibi scaffoldted pathological to detect aggregated aggregationα of-syn α-syn inclusions. in vivo by DDP basedbinding compounds to α-syn fibrils inhibited as demonstrated pathological by aggregation fluorescence of αspectroscopy.-syn in vivo Theby binding most promis- to α-syn fibrilsing drug as demonstrated candidate, anle138b by fluorescence (Figure 19A) spectroscopy. was identified The mostto be promisingan oligomer drug modulator candidate, anle138b[121]. Interestingly,(Figure 19 A)Wagner was identifiedet al. demonstrated to be an that oligomer anle138b modulator binds only [121 to]. pathological Interestingly, Wagneraggregates et al. of demonstrated prion protein and that αanle138b-syn in vivobinds and not only to to natural pathological monomers aggregates of α-syn of[121]. prion proteinIn addition, and α DPP-syn derivativesin vivo and were not toshown natural to directly monomers bind of toα α-syn-syn [ 121fibrils]. In in addition, vitro [122]. DPP derivativesNotably, anle138b were shown may inhibit to directly 77% of bind α-syn to α oligomer-syn fibrils formation,in vitro which[122]. can Notably, be shownanle138b in mayseveral inhibit PD animal 77% of models.α-syn oligomerIt also slowed formation, down disease which progression. can be shown Anle138b in several can be PD ad- ani- malministered models. orally, It also crosses slowed the down BBB and disease does progression.not show any Anle138bsignificant cantoxicity be administeredafter long- orally,term therapeutic crosses the application. BBB and does The notintrinsic show fluorescence any significant of anle138b toxicity was after used long-term to study thera-its peuticbinding application. properties Theagainst intrinsic α-syn fluorescence[122]. The fluorescence of anle138b intensitywas used of anle138b to study increased its binding propertiesmore than against 30-fold αafter-syn the [122 addition]. The fluorescenceof aggregated intensityα-syn, but of didanle138b not changeincreased after the more thanaddition 30-fold of monomeric after the addition α-syn. ofThis aggregated change suggestedα-syn, but a st didrong not and change specific after structure-de- the addition pendent binding of anle138b. A Kd of 190 ± 120 nM to aggregated α-syn was determined [122]. However, anle138b also inhibited the aggregation of other common protein aggregates involved in neurodegenerative diseases [121,123–127]. This selectivity challenge prompted the group to conduct a comprehensive study to increase affinity and selectivity. This led to the identification of anle253b [121]. In a competition assay with [3H]anle138b using recombinant human α-syn fibrils, anle253b (Figure 19A) showed a high affinity for

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of monomeric α-syn. This change suggested a strong and specific structure-dependent binding of anle138b.AKd of 190 ± 120 nM to aggregated α-syn was determined [122]. However, anle138b also inhibited the aggregation of other common protein aggregates involved in neurodegenerative diseases [121,123–127]. This selectivity challenge prompted the group to conduct a comprehensive study to increase affinity and selectivity. This led to the identification of anle253b [121]. In a competition assay with [3H]anle138b using recombinant human α-syn fibrils, anle253b (Figure 19A) showed a high affinity for α-syn aggregates with an IC50 value of 1.6 nM. It also showed preferential binding to α-syn fibrils compared to other α-syn states such as monomers or oligomers. These promising results motivated the carbon-11 labeling of the structure [128]. [11C]anle253b was synthesized with a high RCY (47%), but with a low Am of 15.1 ± 3.4 GBq/µmol at EOS. Further improvement of the molar activity is most likely needed to optimally image low-density targets such as α-syn aggregates. [11C]anle253b was evaluated in healthy rats and showed complete excretion after 75 min from the whole animal and low brain uptake with atypical brain kinetics (Figure 19B). For the latter, the authors hypothesized that the high logP (clogP = 5.61 calculated by BioByte) could be the reason. No radiometabolites were detected using radio-HPLC [128]. Presently, no DPP derivatives have been evaluated on human pathological tissues for more accurate understanding of their affinity and selectivity. Recently, a new derivative within this compound class has been reported which has a lower lipophilicity, called MODAG-001 (clogP = 3.85). MODAG-001 displayed strong binding to α-syn fibrils (Kd = 0.6 ± 0.1 nM), and a moderate affinity to tau (Kd = 19 ± 6.4 nM) and Aβ fibrils (Kd = 20 ± 10 nM). MODAG-001 could be isolated in 266 ± 113 MBq and with 11 an Am of 98.6 ± 24.7 GBq/µmol. [ C]MODAG-001 (Figure 19C) showed excellent BBB permeability in mice and was able to visualize fibril-inoculations in rat striata using PET imaging. However, [11C]MODAG-001 was not able to detect aggregated α-syn in human brain sections from DLB patients. Future studies should be designed to reveal the reason for this behavior Furthermore, no evidence for binding toward tau in AD and PSP brain tissues have been detected by ARG. Authors also questioned binding toward Aβ studied in AD brain tissue, however, additional studies should be conducted [129]. Overall, this compound class is considered promising and may represent a lead compound for future SAR studies.

Furan-2-yl-1H-pyrazoles Inspired by anle138b and in particular by the pyrazole ring interactions with the peptide backbone of α-syn (Figure 20), Ryan et al. proposed a novel class of compounds, namely furan-2-yl-1H-pyrazoles. ThT florescence and mass spectrometry (MS) binding assays were performed in order to characterize the inhibitory activity and to understand the binding interactions of this compound class with the protein. As a result, several new compounds were identified. Compound 43 (Figure 20) was identified as a lead pyrazole and compounds 44 and 45 (Figure 20) as lead pyrazolines for further development. These compounds showed the highest ability to inhibit α-syn aggregation. Further in vivo studies are needed to fully characterize them. Pharmaceuticals 20212021,, 1414,, 847x FOR PEER REVIEW 2526 of 4547

11 Figure 19. ((AA)) Chemical Chemical structures structures of of the the 3, 3,5-diphenylpyrazole5-diphenylpyrazole derivatives derivatives anle138banle138b andand [ C]anle253b[11C]anle253b. (B.() TACsB) TACs of the of Pharmaceuticals 2021[,11 14C]anle253b, x FOR PEER obtained REVIEW from the in vivo study in healthy rats (Adapted from [128]). (C) Chemical27 structure of 47 of the [11C]anle253b obtained from the in vivo study in healthy rats (Adapted from [128]). (C) Chemical structure of [11C]MODAG-001 with TACs and PET images in mouse (Adapted from [129]). [11C]MODAG-001 with TACs and PET images in mouse (Adapted from [129]). Furan-2-yl-1H-pyrazoles Inspired by anle138b and in particular by the pyrazole ring interactions with the peptide backbone of α-syn (Figure 20), Ryan et al. proposed a novel class of compounds, namely furan-2-yl-1H-pyrazoles. ThT florescence and mass spectrometry (MS) binding assays were performed in order to characterize the inhibitory activity and to understand the binding interactions of this compound class with the protein. As a result, several new compounds were identified. Compound 43 (Figure 20) was identified as a lead pyrazole and compounds 44 and 45 (Figure 20) as lead pyrazolines for further development. These compounds showed the highest ability to inhibit α-syn aggregation. Further in vivo studies are needed to fully characterize them.

Figure 20. ChemicalFigure structures 20. Chemical of furan-2-yl-1H-pyrazoles structures of furan-2-yl-1H-pyrazoles derivatives 43 derivatives, 44, and 45 43and, 44 proposed, and 45 and binding proposed scheme of a peptide backbonebinding with scheme a furan-2-yl-1H-pyrazole of a peptide backbone with with highlighted a furan-2-yl-1H-pyrazole HBD and HBA features with highlighted (reprinted withHBD permissionand from [130]. CopyrightHBA features 2020 American (reprinted Chemical with permission Society). from [130]. Copyright 2020 American Chemical Society).

3.7. Chalcone Derivatives 3.7.1. Radioiodinated Diphenyl Derivatives Chalcone derivatives were originally developed to inhibit the formation of Aβ aggregates [131]. Four chalcone derivatives (Figure 21) were radiolabeled with iodine-125 (Am = 81.4 GBq/µmol, RCY range = 19–60%) and subsequently evaluated for their selectivity profile toward α-syn fibrils [132]. Affinities on recombinant α-syn fibrils in the range of 5.4–217 nM were reported, as well as moderate selectivity over recombinant Aβ fibrils (0.2–3.0-fold). [125I]IDP-4 showed the most promising affinity and selectivity profile with a Kd of 5.4 ± 1.5 nM for α-syn and 3-fold selectivity over Aβ. Fluorescent and immunohistochemical staining on human brain slices showed that IDP-4 (at 20 µM) was able to visualize α-syn and LBs. IDP-3 and IDP-4 displayed a similar binding pattern. In contrast, IDP-1 and IDP-2 did not show specific α-syn binding on post-mortem brain samples from PD patients. All compounds were evaluated in vivo. [125I]IDP-1 had an initially high brain uptake (2.43% ID/g), while all other derivatives displayed low brain uptake (range 0.45– 0.81% ID/g) at 2 min p.i. as exemplified in Figure 21. The reason for the relatively low brain uptake of [125I]IDP-4 is unknown but may be attributed to its high molecular weight [132]. Further improvement with respect to selectivity and affinity are most likely needed to selectively image α-syn depositions.

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3.7. Chalcone Derivatives 3.7.1. Radioiodinated Diphenyl Derivatives Chalcone derivatives were originally developed to inhibit the formation of Aβ aggregates [131]. Four chalcone derivatives (Figure 21) were radiolabeled with iodine-125 (Am = 81.4 GBq/µmol, RCY range = 19–60%) and subsequently evaluated for their selectivity profile toward α-syn fibrils [132]. Affinities on recombinant α-syn fibrils in the range of 5.4–217 nM were reported, as well as moderate selectivity over recombinant Aβ fibrils (0.2–3.0-fold). [125I]IDP-4 showed the most promising affinity and selectivity profile with a Kd of 5.4 ± 1.5 nM for α-syn and 3-fold selectivity over Aβ. Fluorescent and immunohistochemical staining on human brain slices showed that IDP-4 (at 20 µM) was able to visualize α-syn and LBs. IDP-3 and IDP-4 displayed a similar binding pattern. In contrast, IDP-1 and IDP-2 did not show specific α-syn binding on post-mortem brain samples from PD patients. All compounds were evaluated in vivo. [125I]IDP-1 had an initially high brain uptake (2.43% ID/g), while all other derivatives displayed low brain uptake (range 0.45–0.81% ID/g) at 2 min p.i. as exemplified in Figure 21. The reason for the relatively low brain uptake of [125I]IDP-4 is unknown but may be attributed to its high molecular weight [132]. Further improvement with respect to selectivity and affinity are most likely needed to selectively image α-syn depositions.

3.7.2. Chalcone and Five-Membered Heterocyclic Isosteres Hsieh et al. attempted to diversify the structure of the chalcone core by substituting the phenyl group of the “benzoyl moiety” with a 2-benzothiazolyl and a 2-thiazolyl group (compounds 46, 47, Figure 22) in the development of an α-syn PET tracer. Surprisingly, the five-membered heterocyclic isosteres conferred much higher α-syn selectivity than the benz-fused aromatic constituent. Substitution of the “styrene moiety” at 4-position had a major influence on the affinity and selectivity profile of the compound [133]. Methoxy substitution resulted in the highest affinity and selectivity for α-syn, and further SAR studies were centered around this key structure. For example, replacement of the enone moiety with an isoxazole (48, Figure 22) or a pyrazole structure (50, Figure 22) was investigated. Both of these derivatives were further modified via extension with a styrene moiety which was substituted with a methoxy group at 4-position. A ThT competition binding assay performed with these compounds revealed that the isoxazole and pyrazole derivatives with a double bond (compounds 49, 51 in Figure 22) bind with higher affinity to α-syn fibrils compared to their analogues without the double bond linker. They also displayed a higher affinity for Aβ fibrils, whereas the affinity toward tau fibrils were not altered [133]. Subsequently, a molecular-modeling approach was conducted to study which properties influenced the binding of these structure toward α-syn, Aβ or tau fibrils. 3D geometric characteristics such as the shape of the molecule (linearity and flatness), topological diameters or intramolecular distance between hydrogen bond receptors were calculated for various structures. Interestingly, the order of binding affinity (49b > 47 > 48) correlated to the order of distance between the isoxazole oxygen and the methoxy group (9.98 > 8.44 > 7.72 Å). Moreover, it appeared that compounds with higher degrees of flatness and linearity such as isoxazole and pyrazole analogues showed increased affinity for α-syn [133]. Finally, compounds 49b and 51 displayed a preference for BS2 as indicated by molecular docking [43]. In general, the presented structures display good affinity and selectivity over amyloid fibrils. For example, the two isoxazole derivatives 49a and 49b showed a ~ 50 fold selectivity over tau fibrils and moderate affinity (18.5 ± 9.2 nM) toward α-syn fibrils. Presently, none of these compounds have been radiolabeled and evaluated on human brain tissue or in in vivo animal models. More comprehensive studies on this family of compounds should be conducted. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 28 of 47 Pharmaceuticals 2021, 14, 847 27 of 45

General structure 10

# n [nM] a clogP * cLogD7.4 ** α-Syn Aβ [125I]IDP-1 1 n.d. 12.88 ± 2.02 4.45 4.8 [125I]IDP-2 2 217. 87 ± 73.87 37.14 ± 10.80 4.9 4.5 [125I]IDP-3 3 23.28 ± 2.78 13.35 ± 5.67 5.51 5.0 [125I]IDP-4 4 5.40 ± 1.50 16.24 ± 4.78 5.51 5.5

FigureFigure 21. Chemical21. Chemical structure structure of radioiodinated of radioiodinated diphenyl derivatives diphenyl and derivatives brain uptake and of brain radioactivity uptake after of intravenousradioac- injection.tivity after Binding intravenous afﬁnity values injection. are determined Binding for affinity recombinant valuesα-syn are anddetermined Aβ ﬁbrils. for Reprinted recombinant with permission α-syn and from

OnoAβ et fibrils. al. [132 ].Reprinted Copyright 2016with Royal permission Society offrom Chemistry. Ono et * clogPal. [132]. values Copyright were calculated 2016 byRoyal BioByte. Society ** clogD of Chem-7.4 values calculatedistry. * byclogP ACD/Percepta. values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. 3.8. Quinolinyl Analogues 3.7.2. Chalcone and Five-Membered Heterocyclic Isosteres Several compounds containing a quinolinyl moiety and six-membered aromatic rings Hsieh et al. attemptedlinked either to diversify by a double the bond struct or anure oxadiazole of the chalcone bridge were core designed by substituting by Yue et al. for the phenyl group ofα the-syn “benzoyl imaging [134 moiety”]. A rational with designa 2-benzothiazolyl approach was applied and a based2-thiazolyl on previous group findings (compounds 46, 47,combining Figure 22) key in features the development of the compounds of anLDS α-syn 798 and PET2,6-ANS tracer.(Figure Surprisingly,5). In particular, the incorporation of double bonds into the side chain, the addition of heteroatoms on the five-membered bothheterocyclic aromatic rings,isosteres the use conferred of secondary much amines higher or oxadiazoleα-syn selectivity fragments than to bridge the both benz-fused aromaticaromatic constituent. rings were Substitution explored (Figure of the 23 “styreneA). As a result,moiety” 25 newat 4-position quinolinyl analogshad a were major influence on synthesizedthe affinity and and tested selectivityin vitro. profile Only three of the derivatives compound displayed [133]. K i Methoxyvalues < 52 nM. substitution resultedAffinity in the measurements highest affinity were conducted and selectivity using a ThT for competitive α-syn, and binding further fluorescence SAR assay α studies were centeredwith around recombinant this key-syn struct fibrils.ure. Despite For these example, moderate replacement affinities, all of three the compounds enone (52, 53, 54) were radiolabeled with carbon-11 or fluorine-18 (Figure 23B). Using recombinant moiety with an isoxazoleα-syn fibrils(48, Figure and brain 22) ADor a tissue pyrazole homogenates, structure the (50, affinity Figure of 22) these was compounds inves- was tigated. Both of thesedetermined derivatives to be were 21, 79, furt 18her nM formodified[11C]52, via[18F]53 extension, [18F]54, with respectively. a styrene Affinity moi- toward ety which was substitutedAβ fibrils with was a determinedmethoxy group to be inat the4-position. same order A ofThT magnitude competition [134]. binding These binding assay performed withcharacteristics these compounds discouraged revealed the authors that from the isoxazole further evaluating and pyrazole the compounds deriva-in vivo. 125 tives with a double Recently,bond (compounds another quinolinyl 49, 51 analogue in Figure[ 22)I]TZ6184 bind (Figurewith higher 23C) has affinity been reported to α- with limited information. In vitro characterization of this radiotracer on recombinant α-syn syn fibrils compared to their analogues without the double bond linker. They also dis- fibrils showed that it possesses an extremely good Kd of 0.39 nM [135]. However, no played a higher affinityselectivity for profileAβ fibrils, has been whereas reported the and affinity thereby notoward thorough tau conclusions fibrils were can benot made. altered [133]. Subsequently, a molecular-modeling approach was conducted to study which properties influenced the binding of these structure toward α-syn, Aβ or tau fibrils. 3D geometric characteristics such as the shape of the molecule (linearity and flatness), topological diameters or intramolecular distance between hydrogen bond receptors were calculated for various structures. Interestingly, the order of binding affinity (49b > 47 > 48) correlated to the order of distance between the isoxazole oxygen and the methoxy group (9.98 > 8.44 > 7.72 Å). Moreover, it appeared that compounds with higher degrees of flatness and linearity such as isoxazole and pyrazole analogues showed increased affinity for α-syn [133]. Finally, compounds 49b and 51 displayed a preference for BS2 as indicated by molecular docking [43]. In general, the presented structures display good affinity and selectivity over amyloid fibrils. For example, the two isoxazole derivatives 49a and 49b showed a ~ 50 fold selectivity over tau fibrils and moderate affinity (18.5 ± 9.2 nM) toward

Pharmaceuticals 2021, 14, x FOR PEER REVIEW 29 of 47

Pharmaceuticals 2021, 14, 847 28 of 45 α-syn fibrils. Presently, none of these compounds have been radiolabeled and evaluated on human brain tissue or in in vivo animal models. More comprehensive studies on this family of compounds should be conducted.

# [nM] clogP * cLogD7.4 **

α-Syn Aβ1-42 Tau 46 530.5 ± 64.3 353.0 ± 29.7 716.5 ± 58.7 3.72 3.7 47 53.0 ± 19.8 > 500 >1000 2.13 2.7 48 133.5 ± 78.5 > 1000 >1000 3.03 2.5 49a,b 18.5 ± 9.2 91.5 ± 58.7 >1000 3.4 2.9 50 162.5 ± 41.7 >1000 >1000 3.16 2.6 51 59.0 ± 11.3 327.0 ± 76.4 >1000 3.54 3.0

Figure 22. Chemical structures of chalcone derivatives and their affinity values expressed as Ki values toward α-syn, Aβ Figure 22.Pharmaceuticals 2021, 14, x FOR PEER REVIEW 30 ofα 47 β Chemicaland tau fibrils structures [133]. * clogP ofchalcone values were derivatives calculated byand BioByte. their ** clogD afﬁnity7.4 values values calculated expressed by ACD/Percepta. as Ki values toward -syn, A and tau ﬁbrils [133]. * clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. 3.8. Quinolinyl Analogues Several compounds containing a quinolinyl moiety and six-membered aromatic rings linked either by a double bond or an oxadiazole bridge were designed by Yue et al. for α-syn imaging [134]. A rational design approach was applied based on previous findings combining key features of the compounds LDS 798 and 2,6-ANS (Figure 5). In particular, the incorporation of double bonds into the side chain, the addition of heteroatoms on both aromatic rings, the use of secondary amines or oxadiazole fragments to bridge both aromatic rings were explored (Figure 23A). As a result, 25 new quinolinyl analogs were synthesized and tested in vitro. Only three derivatives displayed Ki values < 52 nM. Affinity measurements were conducted using a ThT competitive binding fluorescence assay with recombinant α-syn fibrils. Despite these moderate affinities, all three compounds (52, 53, 54) were radiolabeled with carbon-11 or fluorine-18 (Figure 23B). Using recombinant α-syn fibrils and brain AD tissue homogenates, the affinity of these compounds was

determined to be 21, 79, 18 nM for [11C]52, [18F]53, [18F]54, respectively. Affinity toward Ki [nM] Aβ fibrils was determinedKd [nM]to be in the same order of magnitude [134]. These binding char- # acteristics discouraged the authors from further evaluatingclogP the * compounds cLogD in vivo.7.4 ** Re- α-syn a α-syn b Human AD tissue homogenates c cently, another quinolinyl analogue [125I]TZ6184 (Figure 23C) has been reported with lim- 11 [ C]52 52 ± 21 ited information. 21 In vitro characterization 13 of this radiotracer 4.20 on recombinant α 3.4-syn fibrils 18 [ F]53 18 ± 1 showed that 79 it possesses an extremely 55 good Kd of 0.39 nM [135]. 4.45 However, no 3.5 selectivity [18F]54 15 ± 3 profile has 18 been reported and thereby 25 no thorough conclusions 3.78 can be made. 3.8

Figure 23. Chemical structures of new quinolinyl analogs. (A) General structures 11 and 12 of quinolinyl analogs. (B) Figure 23. Chemical structures of new quinolinyl analogs. (A) General structures 11 and 12 of quinolinyl analogs. Chemical structures of new radiolabeled quinolinyl analogs [11C]52, [18F]53 and [18F]54. a Binding affinities expressed as Ki (B) Chemicalvalues structures determined of new by ThT radiolabeled indirect competitive quinolinyl binding analogs using recombinant[11C]52, [ 18α-synF]53 fibrils.and b[ Binding18F]54. affinitiesa Binding expressed affinities as expressed Kd values and determined by direct radioligand competitive binding using recombinant α-syn fibrils.b c Binding affinities as Ki values determined by ThT indirect competitive binding using recombinant α-syn fibrils. Binding affinities expressed expressed as Kd values and determined by direct radioligand competitive binding assay on tissue homogenates from AD c as Kd valuespatients and determined [134]. * clogP by values direct were radioligand calculated by competitive BioByte. ** clogD binding7.4 values using calculated recombinant by ACD/Percepta.α-syn fibrils. (C) ChemicalBinding affinities expressed asstructure K values of [125 andI]TZ6184 determined with binding by affinity direct K radioligandd = 0.39 nM [135]. competitive binding assay on tissue homogenates from AD d patients [134]. * clogP values were calculated by BioByte. ** clogD values calculated by ACD/Percepta. (C) Chemical 3.9. N-Substituted Phenyl Amides 7.4 structure of [125I]TZ6184 with binding affinity K = 0.39 nM [135]. 3.9.1. N-Phenylbenzamided Analogues 3.9. N-SubstitutedIn a seriesPhenyl of over Amides170 compounds, a new class of α-syn binders was identified by Borroni and colleagues [136]. The initial idea about the molecular design of these N-phe- N 3.9.1. nylbenzamide-Phenylbenzamide derivatives Analoguesis not disclosed in the patent where these molecules are pre- Insented. a series The binding of over profile 170 compounds,of [3H]55 (Figure a 24A) new was class studied of α by-syn ARG binders on A30P was trans- identified by Borronigenic and mouse colleagues and human [ 136PD brain]. The sections, initial and idea the results about encouraged the molecular the authors design to use of these N- this compound for displacement studies for a set of other N-phenylbenzamides. Results phenylbenzamide derivatives is not disclosed in the patent where these molecules are are expressed as % displacement. Six compounds (56-61) were identified as high affinity 3 presented.ligands Thewith a binding displacement profile of >87% of [ (inH]55 A30P(Figure mice, at 241 µM),A) waswhich studied were tritiated by ARG to on A30P transgenicevaluate mouse their binding and human profile in PD ex vivo brain ARG sections, in A30P andmice. the High results brain accumulation encouraged as the authors to usewell this as compoundgood target engagement for displacement in the midbrain, studies pons forand subthalamic a set of other brainN regions-phenylbenzamides. was detected. No significant off-target binding was reported. The selectivity of all tritiated compounds was assessed with an in vitro displacement assay using Aβ AD human brain tissue. The experiments showed that all compounds also bind to Aβ (Figure 24B). As such, further structure activity studies are needed to increase the selectivity of these compounds to α-syn [136]. Finally, their binding interactions with α-syn was studied using molecular docking techniques, suggesting that the compounds bind preferentially within BS 9 [43].

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Results are expressed as % displacement. Six compounds (56–61) were identified as high affinity ligands with a displacement of >87% (in A30P mice, at 1 µM), which were tritiated to evaluate their binding profile in ex vivo ARG in A30P mice. High brain accumulation as well as good target engagement in the midbrain, pons and subthalamic brain regions was detected. No significant off-target binding was reported. The selectivity of all tritiated compounds was assessed with an in vitro displacement assay using Aβ AD human brain tissue. The experiments showed that all compounds also bind to Aβ (Figure 24B). As such, further structure activity studies are needed to increase the selectivity of these compounds Pharmaceuticals 2021, 14, x FOR PEER REVIEWα α 31 of 47 to -syn [136]. Finally, their binding interactions with -syn was studied using molecular docking techniques, suggesting that the compounds bind preferentially within BS 9 [43].

General structure 13

# X1 X2 R1 R2 R3 cLogP * clogD7.4 **

56 N C OCH2CH2OCH2CH2F H H 3.44 3.6

57 N C OCH2CH2OCH2CH2F CN CN 2.88 2.9

58 C C OCH2CH2OCH2CH2F CN CN 4.03 3.5

59 C N OCH2F H H 3.61 2.9

60 N C OCH2F H H 3.61 3.3

61 N C OCH2F CN CN 3.06 2.6

62 N C OCH3 F H - -

FigureFigure 24. Chemical24. Chemical structures structures of ofN N--phenylbenzamidephenylbenzamide analogues.analogues. ( (AA) )General General structure structure of ofN-Nphenylbenzamide-phenylbenzamide analogues analogues (general(general structure structure 13) 13) that that showed showed toto be high high affinity affinity displacers displacers and and chemical chemical structure structure of [3H]55 of .[ 3KeyH]55 properties. Key properties are sum- are 3 summarizedmarized in in table table below below [136]. [136]. (B (B) Chemical) Chemical structure structure of of[ H]59[3H]59 andand its itsbinding binding to human to human AD ADbrain brain tissue tissue (cortical (cortical tissue) tissue) and comparison with staining of anti-Aβ mAb (adapted from Borroni et al. [136]). * clogP values were calculated by Bi- and comparison with staining of anti-Aβ mAb (adapted from Borroni et al. [136]). * clogP values were calculated by BioByte. oByte. ** clogD7.4 values calculated by ACD/Percepta. ** clogD7.4 values calculated by ACD/Percepta. 3.9.2. N-Substituted Phenyl Amides Analogues 3.9.2. N-Substituted Phenyl Amides Analogues Several N-substituted phenyl amide derivatives bearing phenothiazine, phenoxa- Several N-substituted phenyl amide derivatives bearing phenothiazine, phenoxazines, zines, quinolinyl, and styryl pyridinyl substituents were also reported to be potential α- quinolinyl, and styryl pyridinyl substituents were also reported to be potential α-syn syn binders [94]. Moderate binding affinity with Ki values < 100 nM were determined by bindersa ThT competitive [94]. Moderate assay binding on α-syn affinity fibrils. with N-substituted Ki values phenyl < 100 nMamides were appeared determined to be by a ThTmost competitive promising assay(Figure on 25)α -synand were fibrils. studiedN-substituted further. Substitution phenyl amides at the appeared 4-position to of bethe most promisingphenyl moiety (Figure showed 25) and that were the studiedbinding further.affinity is Substitution reduced in the at the sequence: 4-position Br > of OMe the phenyl> I moiety> OEtF. showed Compound that the63 (Figure binding 25) affinity showed is reducedthe highest in theaffinity sequence: (ca. 10Br nM). > OMe No further > I > OEtF . Compoundstudies concerning63 (Figure selectivity 25) showed or in vivo the highestevaluation affinity have been (ca. reported 10 nM). up No till further now [94]. studies concerningIn a different selectivity study, Xu or etin al. vivo usedevaluation automated have MS as been a technique reported to up quickly till now detect [94 the]. In a direct interaction between molecules and proteins. Using this method 2500 compounds were screened for α-binding and resulted in one hit: compound 64 (Figure 25) Further- more, the increase of fluorescence during aggregation process was evaluated during the ThT assay, showing that compound 64 was able to strongly inhibit α-syn aggregation by 91% [137]. Currently, there are no known PET tracers developed from compound 64 and no binding affinity or selectivity profile are known. Thus, additional studies are needed.

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different study, Xu et al. used automated MS as a technique to quickly detect the direct interaction between molecules and proteins. Using this method 2500 compounds were screened for α-binding and resulted in one hit: compound 64 (Figure 25) Furthermore, the increase of fluorescence during aggregation process was evaluated during the ThT assay, showing that compound 64 was able to strongly inhibit α-syn aggregation by 91% [137]. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 32 of 47 Currently, there are no known PET tracers developed from compound 64 and no binding affinity or selectivity profile are known. Thus, additional studies are needed.

Figure 25. ChemicalChemical structures structures of of NN-substituted-substituted phenyl phenyl amide amide analogues. analogues. From From left left to toright: right: general general structure structure 14 of 14 N of- substituted phenyl amide analogues. Chemical structure of compounds 63 and 64. N-substituted phenyl amide analogues. Chemical structure of compounds 63 and 64.

3.9.3. 2-Phenoxy- 2-Phenoxy-NN-(3-phenylisoxazol-5-yl)acetamide-(3-phenylisoxazol-5-yl)acetamide Analogues Analogues An exemplar-based in in silico screening campaign was was used used to to identify potential potential lig- lig- ands for α-syn.-syn. An An exemplar exemplar is is defined defined as as an an ideal ideal pseudoligand pseudoligand that would optimally fit fit inin the protein surface pocket; therefore, it is exploited as a templatetemplate forfor pharmacophorepharmacophore screening [138]. [138]. An An in in silico silico assay assay was was design designeded to highlight to highlight compounds compounds that thatbind bind to two to previouslytwo previously identified identified binding binding sites—BS2 sites—BS2 and BS9—within and BS9—within the α-syn the fibrilsα-syn (2n0a, fibrils Figure (2n0a, 2C).Figure Approximately2C) . Approximately 10 million 10 million molecules molecules from the from ZINC15 the ZINC15 database database were werescreened screened and ledand to led the to identification the identification of 17 of chemically 17 chemically diverse diverse potential potential α-synα-syn binders. binders. Compounds Compounds 65 3 and65 and 66 66(Figure(Figure 26A,B) 26A,B) were were of ofspecial special interest interest since since they they were were able able to to displace displace [3H]Tg-H]Tg- 190b—a selective ligand for binding site BS2 (Figure 2626A)—fromA)—from α-syn fibrils.fibrils. Inhibitory concentrations, IC IC5050 values,values, were were 490 490 nM nM and and 9.49 9.49 nM nM for for 6565 andand 6666,, respectively. respectively. Com- Com- pound 66 was used as a lead and the molecule was deconstructed to identify the essential featuresfeatures needed needed for for binding binding to to αα-syn.-syn. The The new new resulting resulting scaffold scaffold was was compound compound 67—a67—a 2- phenoxy-2-phenoxy-N-(3-phenylisoxazol-5-yl)acetamide.N-(3-phenylisoxazol-5-yl)acetamide. This This template template was was then then used used to to find find struc- struc- turallyturally similar similar compounds from the Mcule compoundcompound library. 39 promising structures were discovered using this approach. All All compounds compounds were were tested tested using using the the aforemen- aforemen- 3 3 tionedtioned competition binding binding assay with [3H]Tg-190bH]Tg-190b and [3H]BF2846H]BF2846 (Figure 26B).26B). More importantimportant SAR SAR were were obtained obtained when when different different heterocycles were introduced to the core structurestructure within rings A, B or C (Figure 26B).26B). For example, replacement of the A ring with a pyridine pyridine ( 68),), furan furan ( (69)) or or thiophene thiophene ( (70)) group group did did not not improve improve binding. binding. However, However, we were not able toto drawdraw thesethese conclusionsconclusions since since the the identical identical compounds compounds with with phenyl phenyl moiety moi- as the A ring, for compounds 69 and 70, were not presented in the publication. Conversely, ety as the A ring, for compounds 69 and 70, were not presented in the publication. Con- para substitution on the A ring improved binding toward α-syn fibrils in many cases (see versely, para substitution on the A ring improved binding toward α-syn fibrils in many for example 76 vs. 66). Also, the electronic nature of ring A showed to be essential. In cases (see for example 76 vs. 66). Also, the electronic nature of ring A showed to be essen- particular, a bromo substituted version (73) showed to have a higher affinity than 71, when tial. In particular, a bromo substituted version (73) showed to have a higher affinity than a methoxy group is present on ring C. Also, comparing compounds 72 and 66 revealed 71, when a methoxy group is present on ring C. Also, comparing compounds 72 and 66 that a bromo substituent has a significant effect on binding affinity. Moreover, the authors revealed that a bromo substituent has a significant effect on binding affinity. Moreover, studied the influence of additional fluoro substituents (75) which showed to have a positive the authors studied the influence of additional fluoro substituents (75) which showed to effect on affinity (75 vs. 76). have a positive effect on affinity (75 vs. 76). Replacement of the B ring with an oxadiazole heterocycle lowered the affinity. All compounds possessing an oxadiazole (for example 77) showed no displacement of [3H]Tg-190b (Figure 26C), no matter the substituents on rings A or C. In contrast, replacement of B with a pyrazole only increased the affinity moderately (compounds 78 and 79 (Figure 26C). In addition, from the comparison of heterocyclic differentiations in the B ring we cannot conclude which of the heterocyclic rings had the best influence on affinity. Substitution on the C ring revealed the following: ortho substituents decrease binding, while meta and para substituents improve binding. In addition, the electronic nature of the C ring for para substituents is essential. Since para substituents and in particular fluoro substituents show decreased binding, while moderate to strong binding is demonstrated by derivatives bearing electron donating groups such as methyl and methoxy groups. As

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a general trend, non-planar compounds showed weaker binding than the planar compounds exemplified in Figure 26D (80 and 81) [121,139]. Similar observations were previously drawn for other structural classes. The most promising compound identified in this study was 82 (Figure 26E). It displayed an IC50 of 3.32 nM and of 12.6 nM for BS2 and BS9, respectively. The iodinated derivative 83 (Figure 26E) was selected for labeling with iodine-125. Binding experiments with [125I]83 (RCY = 57%, Am = 81 GBq/µmol) on α-syn fibrils demonstrated excellent binding (Kd = 1.06 nM), but only moderate selectivity over Aβ1-42 fibrils (4-fold, Kd = 4.56 nM). Binding to other proteins in mouse brain lysate was found to be high. In vitro ARG was performed using brain tissue from A53T PD mice (15 Pharmaceuticals 2021, 14, 847 months old). Images confirmed binding of [125I]83 to α-syn rich brain regions [139]. Fur-31 of 45 ther efforts are needed to decrease unspecific protein binding and increase selectivity. Ad- ditional data for human tissue are highly needed to further develop this class of molecules.

General structure 15

# R1 R2 R3 R4 Site 2 IC50 [nM] Site 9 IC50 [nM] cLogP * clogD7.4 **

66 H F CH3 CH3 9.49 109 4.67 3.5

71 H H H OCH3 95.1 557 3.65 2.8

72 H Br CH3 CH3 3.32 12.6 5.39 4.4

73 H Br H OCH3 68.7 37.0 4.53 3.5

74 F F H OCH3 95.7 >1000 3.88 2.4

75 F F CH3 CH3 19.6 >1000 4.74 3.5

76 H H CH3 CH3 30.7 >1000 4.51 3.7

Figure 26. Cont.

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FigureFigure 26. 26. ChemicalChemical structures structures of of 2-phenoxy- 2-phenoxy-NN-(3-phenylisoxazol-5-yl)acetamide-(3-phenylisoxazol-5-yl)acetamide analogues. analogues. (A (A) )Chemical Chemical structure structure of of 3 3 compoundscompounds 6565, ,[[H]Tg-190b3H]Tg-190b andand [ [H]BF28463H]BF2846. (.(BB) Chemical) Chemical structure structure of of compounds compounds 6666, 67, 67 andand general general structure structure of of most most 3 3 potent α-syn binders. Table represents IC50 values screened for BS2 and BS9 using [ H]Tg-190b3 and [ H]BF28463 [139]. * potent α-syn binders. Table represents IC50 values screened for BS2 and BS9 using [ H]Tg-190b and [ H]BF2846 [139]. clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. (C) Chemical structures of com- * clogP values were calculated by BioByte. ** clogD values calculated by ACD/Percepta. (C) Chemical structures of pounds 68-70, 77-79 (D) Models of likely 3D structures7.4 of compounds 80 and 81 (visualized by Molinspiration Galaxys 3D Structurecompounds Generator68–70, 77 v2018.01–79 (D) Modelsbeta). Methyl of likely substituent 3D structures of compound of compounds 81 generates80 and 81 (visualizednon-planar bystructure Molinspiration in comparison Galaxys with3D Structurecompound Generator 80. (E) Chemical v2018.01 structure beta). Methyl of compound substituent 82 ofand compound [125I]83. 81 generates non-planar structure in comparison with compound 80.(E) Chemical structure of compound 82 and [125I]83. 3.10. Flavonoids Replacement of the B ring with an oxadiazole heterocycle lowered the affinity. All compoundsFlavonoids possessing act as scavenger an oxadiazole for free (for radicals, example lowering77) showed as such no displacementoxidative stress, of [which3H]Tg- has190b been(Figure shown 26C), to nobe matterrelevant the in substituents PD pathogenesis on rings due A orto C.generation In contrast, of replacementspecies such ofas B freewith radicals a pyrazole and onlysuperoxide. increased For the this affinity reason moderately, several flavonoids (compounds were78 and assessed79 (Figure for their 26C). abilityIn addition, to inhibit from α-syn the comparison fibrillation in of heterocyclicvitro [140]. The differentiations assay, based in on the ThT B ring fluorescence, we cannot showedconclude that which several of theof these heterocyclic compounds rings can had inhibit the best α-syn influence fibrillation on affinity. (Figure Substitution 27) [140]. Completeon the C ringα-syn revealed fibrillation the inhibition following: wasortho obtainedsubstituents using decrease 50 µM of binding, compounds while 6-HPmeta andand Baicaleinpara substituents (Figure 27). improve Furthermore, binding. significant In addition, inhibition the electronic was also nature detected of the for C 7 ring addi- for tionalpara substituents flavonoids: isEriodictoyl, essential. Since22-324,para myricetin,substituents EGCG, and inT-415, particular 22-340/tricetin fluoro substituents, and 22- 341show (Figure decreased 27). The binding, flavonoids while have moderate not yet to strongbeen tested binding in vivo is demonstrated for their ability by derivatives to image αbearing-syn fibrils. electron Hence, donating more comprehensive groups such as methylstudies andshould methoxy be conducted groups. As[141]. a general trend, non-planar compounds showed weaker binding than the planar compounds exemplified in Figure 26D(80 and 81)[121,139]. Similar observations were previously drawn for other structural classes. The most promising compound identified in this study was 82 (Figure 26E). It displayed an IC50 of 3.32 nM and of 12.6 nM for BS2 and BS9, respectively. The iodinated derivative 83 (Figure 26E) was selected for labeling with iodine-125. Binding 125 experiments with [ I]83 (RCY = 57%, Am = 81 GBq/µmol) on α-syn fibrils demonstrated excellent binding (Kd = 1.06 nM), but only moderate selectivity over Aβ1–42 fibrils (4-fold, Kd = 4.56 nM). Binding to other proteins in mouse brain lysate was found to be high. In vitro ARG was performed using brain tissue from A53T PD mice (15 months old). Images confirmed binding of [125I]83 to α-syn rich brain regions [139]. Further efforts are

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needed to decrease unspeciﬁc protein binding and increase selectivity. Additional data for human tissue are highly needed to further develop this class of molecules.

3.10. Flavonoids Flavonoids act as scavenger for free radicals, lowering as such oxidative stress, which has been shown to be relevant in PD pathogenesis due to generation of species such as free radicals and superoxide. For this reason, several flavonoids were assessed for their ability to inhibit α-syn fibrillation in vitro [140]. The assay, based on ThT fluorescence, showed that several of these compounds can inhibit α-syn fibrillation (Figure 27)[140]. Complete α-syn fibrillation inhibition was obtained using 50 µM of compounds 6-HP and Baicalein (Figure 27). Furthermore, significant inhibition was also detected for 7 addi- Pharmaceuticals 2021, 14, x FOR PEER tionalREVIEW flavonoids: Eriodictoyl, 22-324, myricetin, EGCG, T-415, 22-340/tricetin, and 22-34135 of 47 (Figure 27). The flavonoids have not yet been tested in vivo for their ability to image α-syn fibrils. Hence, more comprehensive studies should be conducted [141].

FigureFigure 27. KineticKinetic profiles profiles of of the the fibril fibril formation formation (ada (adaptedpted from from Meng Meng et et al. [140]) [140]) and chemical structuresstructures of of αα-syn-syn fibrillation fibrillation inhibiting inhibiting flavonoids. flavonoids.

3.11.3.11. Bicyclic Bicyclic Sulfur-Contai Sulfur-Containingning 2-Pyridone 2-Pyridone Analogues Analogues FN075FN075 waswas initially initially studied studied as as an an antibiotic antibiotic (Figure (Figure 28)28) as as it it has has been been shown shown to to inhibit inhibit thethe formation formation of of bacterial bacterial amyloid amyloid fibers. fibers. Interestingly, Interestingly, and and contrary contrary to to its its behavior in bacteria,bacteria, FN075FN075 waswas also also identified identified as as a potent α-syn-syn fibril fibril promoter [142]. [142]. Prompted Prompted by by thesethese results, results, a a limited limited SAR SAR study study on FN075 was conducted. Derivatives altered the lag timetime of of α-syn-syn aggregation in in several animal models. Unfortunately, Unfortunately, no no affinity affinity studies werewere conducted making making it it difficult difficult to to ration rationalizealize the the data data obtained [143–145]. [143–145]. However, However, thethe carboxylic carboxylic acid acid moiety moiety appeared appeared to to be be an an important feature feature for for the the activity since the α correspondingcorresponding esters esters lost lost their their ability ability to toinfluence influence the theα-syn-syn fibril fibril formation formation [142]. [ 142In 2018,]. In Cairns2018, Cairns et al. developed et al. developed a first aPET first tracer PET tracerbased basedon FN075 on FN075. In order. In orderto increase to increase the brain the accumulation,brain accumulation, they exploited they exploited a prodrug a prodrug approach, approach, masking masking the carboxylic the carboxylic acid as an acid ace- as an acetoxymethyl ester, which was designed to be unstable in vivo and release FN075 toxymethyl ester, which was designed to be unstable in vivo and release FN075 within within the brain [143,146]. The authors reasoned that the carboxylic acid moiety disabled the brain [143,146]. The authors reasoned that the carboxylic acid moiety disabled the the compound’s ability to enter the brain as it becomes deprotonated at physiological compound’s ability to enter the brain as it becomes deprotonated at physiological pH. A pH. A methoxy derivative was also synthesized and evaluated as possible candidate for methoxy derivative was also synthesized and evaluated as possible candidate for radio- radiolabeling. In vitro α-syn fibrillization was also triggered by this methoxy analogue 84, labeling. In vitro α-syn fibrillization was also triggered by this methoxy analogue 84, while the acetoxymethyl analogue was inactive. This promising result prompted the group while the acetoxymethyl analogue was inactive. This promising result prompted the to radiolabel the compound. They reported an Am = > 37 GBq/µmol, an RCP of >97%, group to radiolabel the compound. They reported an Am = > 37 GBq/µmol, an RCP of but no RCY. [11C]84 (Figure 28A) as well as its protected derivative [11C]85 (Figure 28A) >97%, but no RCY. [11C]84 (Figure 28A) as well as its protected derivative [11C]85 (Figure were evaluated in vivo in healthy NHP by dynamic PET imaging (Figure 28B). [11C]85 was 28A) were evaluated in vivo in healthy NHP by dynamic PET imaging (Figure 28B). able to cross the BBB, showing a slow washed out whereas [11C]84 did not enter the brain [11C]85 was able to cross the BBB, showing a slow washed out whereas [11C]84 did not enter the brain (Figure 28B). The authors speculated that [11C]85 was hydrolyzed within the brain however this needs to be proved conclusively [146]. Further studies are needed to determine the potential of FN075 analogues as possible agents for the imaging of α-syn fibrillization. In particular, affinity and selectivity measurements should be conducted.

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(Figure 28B). The authors speculated that [11C]85 was hydrolyzed within the brain however Pharmaceuticals 2021, 14, x FOR PEERthis REVIEW needs to be proved conclusively [146]. Further studies are needed to determine36 the of 47 potential of FN075 analogues as possible agents for the imaging of α-syn ﬁbrillization. In particular, afﬁnity and selectivity measurements should be conducted.

Figure 28. The chemical structures of FN075 and its analogues. (A) The chemical structures of FN075 and radiolabeled acetoxymethyl ester [11C]84 and [11C]85. (B) TACs in the whole brain derived from healthy NHP following the IV injection of [11C]84 and [11C]85 (reprinted with permission from [146]. Copyright 2018 American Chemical Society). FigureFigure 28. 28.The The chemical chemical structures structures of ofFN075 FN075and and its its analogues. analogues. ( A(A) The) The chemical chemical structures structures of ofFN075 FN075and and radiolabeled radiolabeled 11 11 acetoxymethyl ester [ 11C]84 and [ 11C]85.(B) TACs in the whole brain derived from healthy NHP following the IV injection acetoxymethylNext, ester Singh [ C]84 et andal. synthesized [ C]85. (B) TACs several in the wholestruct brainural derivedanalogues from around healthy NHP a tricyclic following skeleton the IV injection ofof[11 [11C]84C]84and and[ 11[11C]85C]85(reprinted (reprinted withwithpermission permission from from [ 146[146].].Copyright Copyright 20182018 AmericanAmerican ChemicalChemical Society).Society). (general structure 16, Figure 29) [147]. ThT displacement assays (expressed as relative fluorescence %) on α-synNext, andNext, A Singh Singhβ1-40 fibrils et et al. al. synthesized synthesizedindicated that several several p-nitrophenyl structural structural analogues analogues derivatives around around (86b a a tricyclic ,tricyclic f, o, skeleton skeleton p, 87a, i, k-o) displayed(general(general structureaffinity structure 16,toward 16, Figure Figure both 29 29))[ 147 fibril[147].]. ThT ThTtypes. displacement displacement Other electron-withdrawing assays assays (expressed (expressed as as relative relative fluo- flu- rescence %) on α-syn and Aβ fibrils indicated that p-nitrophenyl derivatives (86b, f, o, p, groups such as 4-trifluoromethylorescence %) on andα-syn 4-cyanophenyl and 1–40Aβ1-40 fibrils did indicated not achieve that p-nitrophenyl comparable derivativesbinding (86b, f, o, results. 87ap, ,87ai, k,– oi,) k-o displayed) displayed affinity affinity toward toward both fibril both types. fibril Othertypes. electron-withdrawing Other electron-withdrawing groups suchgroups as 4-trifluoromethyl such as 4-trifluoromethyl and 4-cyanophenyl and 4-cyanophenyl did not achieve did not comparable achieve comparable binding results. binding results.

General structure 16 General structure 16 # R R1 # R R1 86b cyclopropyl Ph 86b cyclopropyl Ph 86f Ph cyclopropyl 86f Ph cyclopropyl 86o H Ph 86o H Ph 86p cyclopropyl 3-trifluoromethylphenyl 86p cyclopropyl 3-trifluoromethylphenyl 87a 87acyclopropyl cyclopropyl H H 87i 87i Ph Ph H H 87k 87k H H H H 87l 3-hydroxyphenyl87l 3-hydroxyphenyl H H 87m 3-methylphenyl87m 3-methylphenyl H H 87n 3-acetylphenyl87n 3-acetylphenyl H H 87o 3-thienyl H 87o 3-thienyl H Figure 29. Chemical structures of novel pyridine-fused 2-pyridones (86b, f, o, p, 87a, i, k-o). Figure 29.Figure Chemical 29. Chemical structures structures of novel of novel pyridine-fused pyridine-fused 2-pyridones 2-pyridones (86b (86b, ,f,f ,oo, ,pp,, 87a87a, ii,, kk-o–o).). A non-pyridine-based fused-peptidomimetic scaffold was also synthesized (88, Fig- A non-pyridine-basedure 30), but fused-peptidomimet did not show any significantic scaffold binding was alsotoward synthesized α-syn fibrils. (88, This Fig- indicates that ure 30), but did nota showpyridine-fused any significant tricyclic binding backbone toward in this α class-syn offibrils. compounds This indicates is critical that for α-syn fibril a pyridine-fused tricyclicbinding backbone[147]. in this class of compounds is critical for α-syn fibril binding [147].

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A non-pyridine-based fused-peptidomimetic scaffold was also synthesized (88, Figure 30), PharmaceuticalsPharmaceuticals 2021 2021, 14, 14, x, FORx FOR PEER PEER REVIEW REVIEW α 37 of37 47 of 47 but did not show any significant binding toward -syn fibrils. This indicates that a pyridine- fused tricyclic backbone in this class of compounds is critical for α-syn fibril binding [147].

Figure 30. Chemical structure of compound 88. FigureFigure 30.30. ChemicalChemical structurestructure ofof compoundcompound 8888.. 3.12. N,N-Dibenzylcinnamamide Derivatives 3.12.3.12. N,N-DibenzylcinnamamideN,N-Dibenzylcinnamamide DerivativesDerivatives A recently published study by Chen et al. utilized Surface Plasmon Resonance (SPR) A recently published study by Chen et al. utilized Surface Plasmon Resonance (SPR) screeningA recently to identify published N,N-dibenzylcinnamamide study by Chen et al. utilized (DBC) deri Surfacevatives Plasmon as high Resonanceaffinity α-syn (SPR) screening to identify N,N-dibenzylcinnamamide (DBC) derivatives as high affinity α-syn screeningbinders [148]. to identify Among N,N- 39 compoudibenzylcinnamamidends, one hit compound (DBC) was deri identifiedvatives as with high an affinity adequate α-syn binders [148]. Among 39 compounds, one hit compound was identified with an adequate bindersKd of 8.21 [148]. nM Among (89, Figure 39 compou 31). Basednds, on one these hit compoundresults, eight was novel identified DBC analogues with an adequate were K of 8.21 nM (89, Figure 31). Based on these results, eight novel DBC analogues were Kdesigned,dd of 8.21 nMsynthesized, (89, Figure and 31). evaluated Based againston these α -synresults, fibrils. eight Two novel compounds DBC analogues (91 and 93 were) designed, synthesized, and evaluated against α-syn fibrils. Two compounds (91 and 93) designed,showed good synthesized, binding affinitiesand evaluated with K againstd values α of-syn 1.03 fibrils. nM and Two 10.90 compounds nM, respectively. (91 and 93) showed good binding affinities with Kd values of 1.03 nM and 10.90 nM, respectively. showedThese affinity good bindingdata lead affinities to three hypotheses.with Kd values Firstly, of fluorine1.03 nM as and R1 substituent10.90 nM, respectively.appeared These affinity data lead to three hypotheses. Firstly, fluorine as R substituent appeared to Theseto have affinity an essential data lead role tofor three α-syn hypotheses. binding. For Firstly, example, fluorine compound as1 R1 substituent91 showed signifi-appeared have an essential role for α-syn binding. For example, compound 91 showed significantly tocantly have betteran essential affinity role than for compound α-syn binding. 97. Secondly, For example, furan-2-yl compound as the R 291 substituent showed signifi- in- better affinity than compound 97. Secondly, furan-2-yl as the R substituent increased cantlycreased better the affinity affinity substantially than compound in comparison 97. Secondly, to either furan-2-yl phenyl or as2 2-CH the 3RO-phenyl2 substituent sub- in- the affinity substantially in comparison to either phenyl or 2-CH O-phenyl substituents. creasedstituents. the Thirdly, affinity fluorine substantially as the Rin3 substituentcomparison drastically to either phenylincreased3 or binding2-CH3O-phenyl affinity in sub- Thirdly, fluorine as the R3 substituent drastically increased binding affinity in comparison stituents.comparison Thirdly, to a cyano fluorine group as [148]. the RNo3 substituent further evaluation drastically studies increased have been binding reported affinity thus in to a cyano group [148]. No further evaluation studies have been reported thus far for this comparisonfar for this compound to a cyano class,group including [148]. No select furtherivity evaluation and affinity studies data from have post-mortem been reported PD thus compound class, including selectivity and affinity data from post-mortem PD brain tissue farbrain for tissuethis compound on PD. However, class, including this compound selectsivity class and nonetheless affinity datawarrants from further post-mortem investi- PD on PD. However, this compounds class nonetheless warrants further investigation. braingation. tissue on PD. However, this compounds class nonetheless warrants further investigation.

General structure 17 Kd # R1 R2 GeneralR3 structure 17 cLogP * clogD7.4 ** [nM] Kd # 90 R1F RPh2 CNR3 484.6 4.16cLogP * clogD 3.9 7.4 ** [nM] 91 F furan-2-yl F 1.03 4.04 3.5 90 F Ph CN 484.6 4.16 3.9 92 F furan-2-yl CN n.d. 3.33 3.2 91 F furan-2-yl F 1.03 4.04 3.5 93 F Ph F 10.90 4.87 4.1 92 F furan-2-yl CN n.d. 3.33 3.2 94 H Ph F 47.34 4.72 4.2 93 F Ph F 10.90 4.87 4.1 95 F 2-CH3O-Ph CN n.d. 4.07 3.7 94 H Ph F 47.34 4.72 4.2 96 F 2-CH3O-Ph F 331.7 4.78 4.2 95 97 F H 2-CHfuran-2-yl3O-Ph CN F n.d.n.d. 3.9 4.07 3.6 3.7

96 F 2-CH3O-Ph F 331.7 4.78 4.2 FigureFigure 31. 31.The The DBC DBC derivatives derivatives tested tested in in SPR SPR assayassay andand structure of DBC DBC compound compound with with binding binding affinities afﬁnities [148]. [148 *]. clogP * clogP 97values were calculatedH by BioByte.furan-2-yl ** clogD 7.4 values calculated F by ACD/Percepta.n.d. 3.9 3.6 values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. Figure 31. The DBC derivatives tested in SPR assay and structure of DBC compound with binding affinities [148]. * clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta.

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3.13. Benzofuranone3.13. Benzofuranone Due their structuralDue their similarity structural with similarityknown α-syn with ligands known suchα-syn as ligands15a (Figure such 11), as 15aa (Figure 11), small set of benzofuranonea small set of derivatives benzofuranone (Figure derivatives 32) was synthesized (Figure 32 )and was tested synthesized for its abil- and tested for its ity to bind to αability-syn fibrils to bind [149]. to α -synCompound fibrils [149 98 ].was Compound identified98 towas be identifiedthe most promising to be the most promising structure with structurea Ki of 2.32 with nM a for Ki theof 2.32 α-syn nM fibril for theBS9α and-syn an fibril IC50 BS9 value and of an 1.18 IC 50nMvalue for BS2. of 1.18 nM for BS2. 3 These affinity dataThese were affinity determined data were by determined a competition by abinding competition assay bindingusing [3H]Tg-190b assay using [ H]Tg-190b 3 and [3H]BF-2846and (Figure[ H]BF-2846 26A). Encouraged(Figure 26A). by Encouragedthese results, by the these binding results, properties the binding of 98 properties of were evaluated98 (atwere 10 µM) evaluated on human (at 10 post-mortemµM) on human PD, post-mortemAD, and MSA PD, brain AD, sections. and MSA In- brain sections. terestingly, 98 Interestingly,was able to distinguish98 was able between to distinguish LB, LN, between and GCI LB, pathology. LN, and GCI LB from pathology. PD LB from PD brain sections brain(cortex) sections and A (cortex)β plaques and from Aβ ADplaques brain from tissue AD demonstrated brain tissue demonstratedthe highest the highest binding. Conversely,binding. a weak Conversely, binding a for weak of GCI binding from for MSA of GCI brain from section MSA (cerebellum) brain section and (cerebellum) and no binding to LNno bindingfrom PD tobrain LN sections from PD was brain obtained sections (Figure was obtained 32) [149]. (Figure Presently, 32)[ no149 in]. Presently, no vivo study hasin been vivo conducted study has with been these conducted compounds. with these compounds.

General structure 18 Site 2 Site 9 # X R cLogP * clogD7.4 ** IC50 [nM] Ki [nM]

98 CH OCH3 1.18 2.32 5.3 4.0

99 N OCH3 25.8 18.3 4.09 3.5 100 CH H 38.6 33.8 5.92 4.6 101 - - 222 35.1 3.26 2.6

Figure 32. Chemical structure of 99-101 with binding IC50 and Ki values. Fluorescence microscopy Figure 32. Chemical structure of 99–101 with binding IC50 and Ki values. Fluorescence microscopy studies of 98 in studies of 98 in postmortem PD tissue. Image shows LB immunostained with syn303 antibody and postmortem PD tissue. Image shows LB immunostained with syn303 antibody and 98.Reprinted with permission from [149]. 98.Reprinted with permission from [149]. Copyright 2020 The Royal Society of Chemistry. * clogP Copyright 2020 The Royal Society of Chemistry. * clogP values were calculated by BioByte. ** clogD7.4 values calculated by values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. ACD/Percepta.

3.14. Bisquinolines3.14. Bisquinolines Bisquinolines areBisquinolines a new class are of acompou new classnds ofthat compounds were identified that were as potential identiﬁed α-syn as potential α-syn ligands just recentlyligands [120]. just A recently library of [120 44.000]. A compounds library of 44.000 was screened compounds for structures was screened with for structures high planarity with(to recognize high planarity β-sheet (to structures recognize of βamyloids-sheet structures), moderate of amyloids),molecular weight moderate molecular

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weight(in order (in to order pass to the pass BBB), the and BBB), the and ability the ability to introduce to introduce a radionuclide a radionuclide into the into lead the struc- lead structure.ture. Based Based on these on these criteria, criteria, 150 compounds 150 compounds were wereselected selected and fu andrther further evaluated evaluated using usingan inhibition an inhibition ThT ThTbinding binding assay. assay. KPTJ10017KPTJ10017 (Figure(Figure 33) 33 )showed showed the the highesthighest inhibitioninhibition (62.1%).(62.1%). Next,Next, Kaide Kaide and and colleagues colleagues modified modified the the chemical chemical structure structure of of the the lead lead with with the the aimaim ofof introducing introducing aa fluorine fluorine atom atom into into the the molecule molecule while while only only minimally minimally changing changing its its physicochemicalphysicochemical properties.properties. ThisThis ledled toto thethe identificationidentification ofof BQ1BQ1 andand BQ2BQ2 (Figure(Figure 3333),), whichwhich showedshowed KKi i valuesvalues ofof 17.017.0 andand 11.611.6 nMnMrespectively, respectively, in in a a ThT ThT competition competition assays assays usingusing recombinant recombinantα α-syn-syn aggregates. aggregates. Selectivity Selectivity of of both both compounds compounds for forα α-syn-syn aggregates aggregates overover A Aββwas was notnot optimaloptimal andand furtherfurther SARSARstudies studiesare are neededneeded toto reducereduce the the ligandsligands affinity affinity toto off-targetoff-target proteinsproteins (Figure(Figure 3333).). However, However, BQ1BQ1 andand BQ2BQ2 cancan detect detect αα-syn-syn aggregates aggregates in inhuman human PD PD brain brain sections sections using using a fluo a fluorescentrescent staining-based staining-based experiments. experiments. BQ1BQ1 and andBQ2 BQ2werewere also alsoable ableto detect to detect Aβ fibrils Aβ fibrils of AD of patients AD patients in brain in brain tissue tissue whereas whereas no binding no binding to tau tofibrils tau fibrilswas observed. was observed. Finally, Finally, compound compound [18F]BQ2[18 F]BQ2(Figure(Figure 33) was 33 radiolabeled) was radiolabeled (RCY = (RCY1.2%, = A 1.2%,m = 8.9A mGBq/µmol)= 8.9 GBq/ andµmol evaluated) and evaluated in vivo in vivonaïvein mice. naïve A mice. moderate A moderate brain uptake brain uptake(1.59% (1.59%ID/g at ID/g2 min) at and 2 min) slow and clearance slow clearance (1.35% ID/g (1.35% at 60 ID/g min) atwas 60 detected. min) was This detected. reten- Thistion retentioncould be couldcaused be by caused binding by bindingto myelin to myelinsheaths, sheaths, monoamine monoamine oxidases, oxidases, or neuro- or neuromelanins.melanins. Finally, Finally, ARG ARGstudies studies were conducted were conducted on PD on brain PD sections. brain sections. A high A non-specific high non- specificbinding binding component component in the whole in the wholeregion regionwas detected, was detected, suggesting suggesting that [18 thatF]BQ2[18F]BQ2 as suchas is suchnot suitable is not suitable for α-syn for α PET-syn imaging PET imaging [120]. [120 Further]. Further SAR SAR studies studies are are therefore therefore needed needed to toimprove improve the the affinity affinity and and selectivity selectivity profile profile of of BQ BQ analogues. analogues.

# [nM] clogP * cLogD7.4 ** α-Syn Aβ KPTJ10017 3.4 ± 0.1 1.1 ± 0.2 4.08 2.6 BQ1 17.0 ± 3.5 8.5 ± 3.6 4.58 3.5 BQ2 11.6 ± 2.6 7.3 ± 0.7 4.83 4.4

Figure 33. Chemical structure of KPTJ10017, BQ1, BQ2, and18 [18F]BQ2 with binding affinities (Ki values) for recombinant Figure 33. Chemical structure of KPTJ10017, BQ1, BQ2, and [ F]BQ2 with binding affinities (Ki values) for recombinant α-syn and Aβ aggregates [120].* clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. α-syn and Aβ aggregates [120].* clogP values were calculated by BioByte. ** clogD7.4 values calculated by ACD/Percepta. 4.4. ConclusionsConclusions ConsiderableConsiderable research research has has been been devoted, devoted, over over the the past past decades, decades, to theto the identification identification of αof-syn α-syn ligands ligands and aand variety a variety of chemically of chemically diverse diverse compounds compounds have been have evaluated. been evaluated. Approxi- matelyApproximately 37 different 37 chemical different classes chemical have beenclasses developed have been and amongdeveloped them and 23 compounds among them were 23 furthercompounds radiolabeled were further and evaluated radiolabeled in preclinical and evaluated studies in ( Tablepreclinical S2, Supporting studies (Table Information) S2, Sup-. Onlyporting two information). compounds (Only[11C]MODAG-001 two compoundsand ([11[125C]MODAG-001I]TZ6184) showed and high[125I]TZ6184 affinity) (<1 showed nM) whilehigh theaffinity rest showed(<1 nM) moderatewhile the biding rest showed toward moderateα-syn fibrils. biding Four toward compounds α-syn ( [fibrils.18F]2FBox Four, [18compoundsF]15a, [11C]MODAG-001 ([18F]2FBox, [18F]15a,and [18 [F]S3-111C]MODAG-001) showed high and selectivity [18F]S3-1) (30–50-fold) showed high over selectiv- struc- turallyity (30–50-fold) related amyloid over structurally proteins asrelated Aβ and amyloid tau fibrils. proteins Most as A importantly,β and tau fibrils. only Most 12 were im- evaluatedportantly, usingonly 12 human were derivedevaluated brain using material human and derived among brain them material six showed and among binding them to αsix-syn. showed However, binding these to α compounds-syn. However, were these either compounds unable to were selectively either bindunable to toα-syn selectively or its bindingbind to wasα-syn tested or its only binding by fluorescent was tested staining only by and fluorescent not ARG whichstaining is consideredand not ARG to bewhich the “goldis considered standard”. to Inbeour the opinion,“gold standard”. the absence In ofour a validopinion, candidate the absence for α-syn of a PETvalid imaging candidate is for α-syn PET imaging is mainly related to the lack of reliable and reproducible assays.

Pharmaceuticals 2021, 14, 847 38 of 45

mainly related to the lack of reliable and reproducible assays. As a matter of fact, the most challenging aspect over the years has been the evaluation process of these compounds. Thus far, a variety of in vitro approaches for the determination of binding affinity on fibrils have been described. However, it is unclear if the specific assays used for compound screening are able to accurately mimic the pathophysiology present in human derived tissue. Recently, Schweighauser et al. [150] found structural discrepancies in MSA α-syn filaments in comparison to in vitro “synthetic” filaments (from recombinant wild-type and mutant α-syn). In particular, the size and the packaging of protofilaments seems to differ in human derived tissue compared to “synthetic” fibrils [150]. Moreover, the authors suggested that different strains of α-syn might exist in DLB and MSA patients. Similar hypothesis has been put forward by several other scientists [63,151]. By contrast, Shah- nawaz et al., employing tissues derived from MSA and PD patients, demonstrated with cryo-electron tomography that the structure of α-syn fibrils amplified from human material is consistent with the structure of “synthetic” α-syn fibrils prepared in vitro [63]. The discrepancy in results between these two publications is concerning and questions current approaches to design, develop and evaluate α-syn PET tracers. A possible alternative to synthetic fibrils may be sarkosyl-insoluble α-syn material which has been used in more recent studies [150]. Up-to-date, ThT competition assays are often used as the only method to evaluate binding, although it has been proved that α-syn fibrils contain multiple binding sites [43]. It might be possible that ThT based assays cannot distinguish between compounds that do not bind to the same binding site. Furthermore, many scientists have questioned the reproducibility of these assays, especially given the discrepancies in the reported binding affinities of the studied compounds [67,98,99,114]. The observed discrepancies may be related to several factors, such as, the difference in fibril preparation or practicality of the assay, for example. More reliable and reproducible binding assays are needed in the future to address this challenge. High throughput screening (HTS) may represent an alternative approach to identify α-syn ligands. Until now, only two HTS studies using human derived tissues have been reported [113,114]. Rational drug design is as a matter of fact another alternative development approach. However, this strategy, and in particular structure-based drug design, is hindered as a high resolution crystal structure of α-syn fibril remains elusive. Despite this, several groups have based their research on explorative computational modeling and have identified putative binding sites on α-syn fibrils [43]. We speculate that cryo-electron microscopy (cryo-EM) studies of α-syn from PD, MSA or DLB patients will be a valuable tool in the future that will help to guide the design and development of new imaging probes providing a clearer picture of available binding sites. Finally, a completely different solution to develop α-syn imaging agents is to engineer specific antibodies. A similar approach has been already successfully employed in PET imaging of Aβ [152–154]. In conclusion, an α-syn selective PET tracer is still missing and no clear pathway is foreseen to develop such a tracer in the near future. Recent findings studying PD revealed that Lewy pathology is complex and consists of a variety of fragmented membranes, organelles and vesicles [151]. This may explain why the development of α-syn PET tracer has faced several challenges. However, the first promising results have been published and hope is on the rise, as outlined in this review.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ph14090847/s1, Table S1: Summary of search strategy based on PRISM guidelines, Table S2: Summary table of all radiolabeled compounds with reported results from in vitro assays and preclinical studies. Author Contributions: All the authors contributed to the drafting and other preparation of this manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant, agreement no 813528. Pharmaceuticals 2021, 14, 847 39 of 45

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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