Development of Pyridothiophene Compounds for PET Imaging of α-Synuclein

: Aggregated α-synuclein (α-syn) protein is a pathological hallmark of Parkinson’s disease (PD) and Lewy body dementia (LBD). Development of positron emission tomography (PET) radiotracers to image α-syn aggregates has been a longstanding goal. This work explores the suitability of a pyridothiophene scaffold for α-syn PET radiotracers, where 47 derivatives of a potent pyridothiophene (asyn-44; K d = 1.85 nM) were synthesized and screened against [ 3 H]asyn-44 in competitive binding assays using post-mortem PD brain homogenates. Equilibrium inhibition constant (K i ) values of the most potent compounds were determined, of which three had K i 's in the lower nanomolar range (12-15 nM). An autoradiography study confirmed that [ 3 H]asyn-44 is promising for imaging brain sections from multiple system atrophy and PD donors. Fluorine-18 labelled asyn-44 was synthesized in 6±2% radiochemical yield (decay-corrected, n=5) with a molar activity of 263±121 GBq/µmol. Preliminary PET imaging of [ 18 F]asyn-44 in rats showed high initial brain uptake (>1.5 standardized uptake value (SUV)), moderate washout (~0.4 SUV at 60 min), and low variability. Radiometabolite analysis showed 60-80% parent tracer in the brain after 30 and 60 mins. While [ 18 F]asyn-44 displayed good in vitro properties and acceptable brain uptake, troublesome radiometabolites precluded further PET imaging studies. The synthesis and in vitro evaluation of additional pyridothiophene derivatives are underway, with the goal of attaining improved affinity and metabolic stability.


Introduction
Parkinson's disease (PD), Lewy body dementia (LBD) and multiple system atrophy (MSA) are diseases characterized by the presence of pathological α-synuclein (α-syn) aggregates and are therefore termed α-synucleinopathies. [1,2] α-Syn is a small presynaptic protein of 140 amino acids.In its physiological state, α-syn is unfolded, soluble, and can be found abundantly throughout the central nervous system (CNS).While its exact function is unknown it is purported that α-syn plays a role in synaptic plasticity. [3,4]In α-synucleinopathies, α-syn forms insoluble aggregates which are stabilized by a ß-sheet structure. [5,6]In PD and LBD, these aggregates are found in neuronal cell bodies (Lewy bodies) as well as axons and dendrites (Lewy neurites), with their distribution depending on the disease type and state.In MSA, the α-syn aggregates present as filamentous inclusions in oligodendrocytes (glia cell inclusions). [7,8]Typically, α-syn aggregation starts at the brain stem regions and spreads throughout the brain as the disease progresses.11] Since disease progression correlates with the accumulation of aggregated α-syn, there is a tremendous interest in developing positron emission tomography (PET) radiotracers targeting α-syn as a biomarker for PD, LBD and MSA. [5]Such a radiotracer would be valuable for target engagement studies, and to serve in the early diagnosis of α-synucleinopathies as well as in patient stratification for α-syn-targeted therapy. [12,13]Consequently, numerous PET tracers targeting α-syn have been developed. [4]owever, to date, none of these tracers have proven ideal for PET imaging of PD patients. [14]During the preparation of this manuscript, the development of an α-syn PET radiopharmaceutical was reported, [ 18 F]ACI-12589, which bound to brain regions affected by -synuclein pathology in patients with MSA; however, there was limited binding in PD. [15] The development of α-syn PET tracers for PD remains to be amongst the greatest challenges in PET neuroimaging and is hampered by several factors: First, the density of aggregated αsyn is low; about 10-50 times lower than the density of other protein aggregates such as amyloid beta (Aß).Therefore, a successful tracer needs to have a very high binding affinity (likely <1 nM) to increase the specific binding signal.Furthermore, the characteristic ß-sheet structure of aggregated α-syn is shared with other protein aggregates such as Aß and tau.Since α-syn accumulation is often accompanied by Aß and tau aggregation, it is imperative that the α-syn radiotracer has a high selectivity over these targets (at least 30-50 fold) in addition to other CNS targets.Another challenge is the intracellular location of the α-syn aggregates which requires the radiotracer to cross both the bloodbrain barrier as well as cell membranes. [13,14,16]A recent review by Korat et.al. discusses these and other challenges in detail and gives a comprehensive list of suggested success criteria for α-syn PET tracers. [4]he goal of the present work is to address the need for an α-syn PET radiotracer by further exploring a lead compound based on a pyridothiophene scaffold.This scaffold was disclosed in a patent by AC Immune from a heterogenous class of bicyclic compounds that were assayed for binding to α-syn and Aß plaques. [17](R)-5-(6-(3-Fluoropyrrolidin-1-yl)pyridin-3-yl)-2-(pyridin-3-yl)thieno [3,2b]pyridine (asyn-44; Figure 1) was shown to be potent (α-syn Kd = 9 nM; consistent with our assays) and had negligible staining of Aß plaques. [17]In the present study, we synthesized 47 derivatives of asyn-44 and screened them against [ 3 H]asyn-44 in competitive binding assays using post-mortem PD brain homogenates.Ki values of the most potent compounds were determined.We also synthesized [ 18 F]asyn-44 and evaluated this lead compound in healthy rats as a potential PET radiotracer for α-syn.

Results and Discussion
Chemistry A library of pyridothiophenes was synthesized by varying R and R´ substituents of the scaffold, resulting in four subclasses (I-IV) of compounds (Figure 2).In order to access the compounds of subclass I (Scheme 1), the commercially available pyridothiophene core 1 was cross-coupled with commercially available (6-fluoropyridin-3-yl)boronic acid in a Suzuki-Miyaura reaction to obtain 2 with a yield of 58.8%.After radical bromination with N-bromosuccinimide (87.5% yield), the resulting compound 3 was subjected to Suzuki-Miyaura coupling with aryl boronic esters to obtain compounds 4-7 in yields ranging from 4 to 15%.To access compounds of subclass II, 2 was reacted with 4fluoropiperidine hydrochloride, followed by bromination with Nbromosuccinimide to obtain 9 in 30% isolated yield over two steps (Scheme 2).Compound 9 was further derivatized into a variety of compounds.These were mostly obtained via a palladiumcatalysed cross-coupling reaction with aryl boronic acids (compounds 10-25).Buchwald-Hartwig amination of 9 with three primary amines yielded compounds 26-28.Pseudo-halogen exchange of 9 with CuCN in DMF yielded 23% of cyanocompound 29.Further expansion of the library was achieved by reacting 9 with carbon monoxide to form the ethyl ester 30 with a yield of 81%, which served as an intermediate in the synthesis of five additional derivatives, 31-35 (Scheme 3).To access 31 and 32, 30 was hydrolysed to 31 (92%) and subsequently reacted with dimethylamine hydrochloride to form the amide 32 (38%).Reaction of 30 with dimethylamine directly led to the formation of 33 (52%).34 was synthesized by reduction of 30 with lithium aluminium hydride (22%), which was then methylated with methyl iodide in presence of sodium hydride, yielding 48% of 35.Derivatives of subclass III were obtained via compound 37. 37 was synthesized in two steps from 1, which was previously obtained during the synthesis of subclass I derivatives: 1 was brominated by use of elemental bromine and n-butyllithium, followed by palladium catalyzed cross-coupling with 3-methyl-5-Bpin-pyridazine (Scheme 4).Compound 37 was then further coupled with boronic acid pinacol esters under palladium catalysis to form compounds 38-41.Boronic acid pinacol esters were obtained by Buchwald-Hartwig amination of the dihalogenated arenes with corresponding amines and subsequent Miyaura borylation.Buchwald-Hartwig amination of 37 yielded compounds 42 and 43.

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This article is protected by copyright.All rights reserved.Derivatives of subclass IV were synthesized via two different routes.Derivatives 45-48 were synthesized in two steps from 36: First, 36 was subjected to a Suzuki-Miyaura coupling with pyridin-3-ylboronic acid to form compound 44, which was subsequently reacted with a variety of amines (free base or hydrogen chloride salt) to yield the respective derivative (Scheme 5).To access derivatives 50-55, compound 49 was synthesized in two steps from 1 and subsequently coupled to the respective nitrogen nucleophile to yield 50-55 (Scheme 6).

In vitro evaluation of asyn-44
[ 3 H]Asyn-44 was characterized in vitro to determine its suitability as a lead for α-syn PET tracer development.First, the equilibrium dissociation constant (Kd), and maximal number of binding sites (Bmax) were determined in PD and Alzheimer's disease (AD) tissue.It was found that asyn-44 bound with high affinity in PD tissues (Kd=1.85±0.38 nM, Bmax=55±9 nM, n=3) and was much less potent in AD tissues (Kd=170±60 nM, Bmax=420±200 nM, n=3).

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In vitro evaluation of the pyridothiophene derivatives
The synthesized derivatives were evaluated in vitro to determine the most promising compounds for use as α-syn PET radiotracers.Most of the compounds were initially screened in a two-point assay, similarly to our previous report. [21]The objective of this screen was to determine which structural derivatives displaced [ 3 H]aysn-44 in a dose-dependent manner and to select the leads with the most promise for full competition binding assays.The compounds were screened against [ 3 H]asyn-44 in human PD tissue homogenates at two concentrations (30 and 300 nM).
Figure 5 shows a heat map of the screening results, with exact values in the SI (Table S1).Even though many compounds showed good inhibition (<50% remaining bound [ 3 H]asyn-44) at 300 nM concentration, only a few showed good inhibition at 30 nM.
For compounds with very good inhibition (<30% bound [ 3 H]asyn-44) at 300 nM and moderate to good inhibition (<70% bound [ 3 H]asyn-44) at 30 nM, as well as for compounds that had not been screened in the two-point assay, we determined the full competition binding curve to calculate their equilibrium inhibition constant (Ki).The results of the compounds with a Ki < 50 nM are shown in Table 2.The only compounds in the lower nanomolar range were 13 (Ki 12.1 nM), 55 (Ki 12.2 nM) and 51 (Ki 14.6 nM).These compounds are structurally very similar to the lead asyn-44: They had minor modifications at the fluoropyrrolidine ring and, in case of 13, a small modification at the pyridine ring.However, none of these three compounds showed higher binding affinity than the lead compound, and we proceeded with asyn-44 for further in vivo characterization.Overall, we found that only limited modifications of the lead structure retained high affinity to the target.The ring size of the 3fluoropyrrolidinyl group could be increased or decreased; the fluorine atom was, however, important to retain high affinity.This is probably due to the "fluorine effect" where the electrochemical effect of fluorine influences physicochemical properties of a compound.[22,23]   The terminal pyridyl group was modified, however, nitrogen-bearing heterocyles generally retained the highest affinities.An exception is replacement of the pyridyl group by thiophene, bromide or cyanide, which also resulted in moderate-high affinity.
The limited success to synthesize compounds for α-syn with sub-nM affinity can be attributed to α-syn being an intrinsically disordered protein which can adopt a range of structural conformations.The availability of high-resolution X-ray crystal structures of α-syn aggregates is limited, which further hampers a structure-based drug design approach.Therefore, it is difficult to predict which structural modifications on the ligands will improve binding.

In vivo evaluation of [ 18 F]asyn-44
To investigate if asyn-44 is a suitable candidate for the imaging of α-syn in vivo, we conducted preliminary PET imaging studies with [ 18 F]asyn-44 in male and female wild-type rats.After intravenous (i.v.) administration of the tracer, the radioactivity in the entire brain peaked at an initial maximum standardized uptake value (SUV; semiquantitative measure of tracer uptake, defined as the activity concentration in a region of interest normalized to the total injected amount of radioactivity per body weight) of >1.5 within 5 minutes of injection, gradually decreasing to 0.4 SUV over the course of the 120-minute PET scan (Figure 6).These findings revealed that [ 18 F]asyn-44 has good brain permeability and moderate brain clearance, which are suitable characteristics for a PET tracer targeting α-asyn.No difference between sexes was observed in our preliminary study.A radiometabolite studies of [ 18 F]asyn-44 was carried out to assess potential brain-penetrant metabolites which could confound PET imaging.Blood plasma as well as brain homogenates were analysed 30 and 60 minutes post injection of the radiotracer (Table 3) which revealed a low plasma stability of the tracer for both sexes with less than 50% intact tracer after 30 minutes.While this is not necessarily a problem for brain tracers, radiometabolites were identified in the brain homogenates at a higher rate than what would be expected in a non-perfused brain.In females 30-40% radiometabolites (polar and lipophilic) were present after 30-60 minutes, and males showed even higher average percentage of radiometabolites at both timepoints.While the polar metabolites were likely formed in the brain, the lipophilic metabolites may also cross the blood brain barrier from the plasma into the brain (SI, Figures S7 and S8).To avoid the formation of brain-penetrant radiolabelled metabolites in future derivatives, alternative labelling sites could be considered and/or structural modifications of the scaffold need to be explored. [24,25]Based on common metabolic reactions, the lipophilic metabolites might result from oxidation of the N-heterocycles, either at the carbon or nitrogen atoms.A potential strategy for the future development of asyn-44 derivatives could be to reduce likelihood of oxidation by decreasing the electron density in the N-heterocycles (e.g. through introduction of more nitrogen atoms), to explore radiolabelling at the central or terminal rings, or by attempting to block metabolic positions by introducing suitable substituents. [26,27]

Conclusion
This work demonstrates that asyn-44 is a potent (Kd = 1.85 nM) lead compound and the pyridothiophene scaffold is promising for the development of an α-syn PET tracer in both PD and MSA.While [ 18 F]asyn-44 exhibited favourable in vitro and in vivo characteristics for neuroimaging, the presence of radiometabolites in rat brain precluded further PET imaging studies.Our future efforts are focused on the synthesis and in vitro evaluation of additional derivatives of asyn-44 with the goal of attaining improved affinity and metabolic stability.

Experimental General
Ligands 50-55 were provided by MedChem Imaging, Inc. (Boston, USA) with >95% purity.All other ligands and labelling precursors were provided by Sai Life Sciences Ltd. (Telangana, India) with >90% purity.[ 3 H]Asyn-44 (1.5 GBq/μmol, 36 MBq/mL; Figure 3) was labelled with tritium gas from the respective dibromo precursor at Novandi Chemistry AB (Södertälje, Sweden).All other chemicals and reagents were obtained from commercial vendors and were used as received without further purification.[ 18 F]Fluoride was produced with a MC-17 cyclotron (Scanditronix, Sweden) from an oxygen-18 enriched water target.Analytical HPLC was performed on a 1260 Infinity II HPLC system (Agilent, CA) with a M177 γ-radiation detector (Ludlum Instruments, TX) connected in series after the UV detector.Radiochemistry was performed in lead-shielded hot cells in a laboratory designed to handle radioactive material.The work was carried out in accordance with the radiation protection guidelines and regulations from the Canadian Nuclear Safety Commission and internal safety policies and procedures.Animal studies were carried out in accordance with the guidelines put forth by the institutional animal care and use committees at CAMH (Protocol 891).

2-Bromo-5-(6-fluoro-3-pyridyl)thieno[3,2-b]pyridine 3:
To a solution of 5-(6-fluoro-3-pyridyl)thieno[3,2-b]pyridine 2 (600 mg, 2.60 mmol, 1 eq) in CCl4 (10 mL) was added NBS (2.32 g, 13.04 mmol, 5 eq) and AIBN (90 mg, 0.52 mmol, 0.2 eq).The resulting reaction mixture was heated at 100-110 °C for 18 h.The progress of the reaction was monitored by TLC and LCMS.After completion, the reaction mixture was concentrated in vacuo and purified by combi flash chromatography (15% EtOAc/Heptane) to afford 2-bromo-5-(6-fluoro-3-pyridyl)thieno[3,2-b]pyridine 3 (700 mg, 87.5 %) as an off white solid.General procedure 4-7: To a stirred solution of 2-bromo-5-(6fluoropyridin-3-yl)thieno[3,2-b]pyridine 3 (1 eq) and the respective aryl boronic ester (1.5 eq) in 1,4-dioxane/water (4:1) (5-10 mL) was added Cs2CO3 or t-BuONa (2.5 eq).The reaction mixture was degassed with nitrogen gas for 10-15 min, then Pd(dppf).CH2Cl2 (0.1 eq) was added, and the mixture was again degassed with nitrogen gas for 10-15 min.Then, the mixture was heated at 110 °C for 1 h in a microwave.The progress of the reaction was monitored by TLC and LCMS.After completion, the reaction mixture was cooled to RT, filtered through celite and washed with EtOAc.The organic layer was dried over Na2SO4 and concentrated in vacuo.The crude was purified by preparative HPLC to afford the product (4-15%).) and the respective boronic acid precursor (1.3-7 eq) in 1,4-dioxane/water (4:1) was added Cs2CO3 (2.5 eq).The reaction mixture was degassed with nitrogen gas for 15-30 mins, then Pd(dppf).CH2Cl2 (0.1 eq) was added, and the mixture was again degassed with nitrogen gas for 15-30 mins.The reaction was allowed to stir at 70-110 °C for 5-16 h.The progress of the reaction was monitored by TLC.After completion, the reaction mixture was cooled to RT, filtered through celite and washed with EtOAc.The organic layer was dried over Na2SO4, concentrated in vacuo and purified by combi flash column chromatography or preparative HPLC to afford the product.General procedure 26-28: To the stirred solution of 2-bromo-5-(6-(4-fluoropiperidin-1-yl)pyridin-3-yl)thieno[3,2-b]pyridine 9 (1 eq) and R-NH2 (1.2 eq) in toluene (6 mL) was added sodium tertbutoxide (3 eq).After stirring the reaction mixture for 10 min under argon gas purging, BINAP (0.15 eq) and Pd2(dba)3 (0.1 eq) were added at room temperature and the reaction mixture was stirred at 100 °C for 50 min under microwave irradiation.The progress of the reaction was monitored by TLC and LCMS.The reaction mixture was filtered through celite and washed with DCM.The filtrate was concentrated under reduced pressure to get the crude compound which was purified by Combi-flash column chromatography.Pure fractions were concentrated under reduced pressure to get the desired product.MeNMe2.HCl (42 mg, 0.52 mmol, 2.5 eq) in DMF (5 mL) was added DIPEA (0.109 mL, 0.62 mmol, 3 eq) and HATU (159 mg, 0.41 mmol, 2 eq) at 0 °C.The resulting reaction mixture was allowed to stir at RT for 16 h.The progress of the reaction was monitored by TLC.After completion, the reaction mixture was  .17mmol, 1 eq) in DMF (5 mL) was added NaH (13 mg, 0.34 mmol, 2 eq) and MeI (0.016 mL, 0.26 mmol, 1.5 eq) at 0 °C.The resulting reaction mixture was allowed to stir at RT for 3 h.The progress of the reaction was monitored by TLC.After the completion of the reaction, ice cold water was added, and the mixture was extracted with EtOAc (2 x 10 mL).The organic layer was washed with water, dried over sodium sulfate and concentrated in vacuo.The crude was purified by combi-flash using 10% EtOAc/heptane to afford 5-( 6 (1.5 eq) were added to a stirred solution of the respective boronic acid (1-1.2eq) in 1,4-dioxane (3 mL) and water (0.1 mL).The reaction mixture was purged for 10 min with argon gas.Pd(dppf)Cl2 (0.1 eq) was added at room temperature and the reaction mixture was stirred at 90-100 °C for 40-60 min under microwave irradiation.The progress of reaction was monitored by TLC and LCMS.The reaction mixture was filtered through celite and washed with CH2Cl2.The filtrate was concentrated under reduced pressure and purified by Combi-flash column chromatography.Pure fractions were concentrated under reduced pressure to obtain the desired compound (9.5-30%).General procedure 42-43: To the stirred solution of 5-chloro-2-(6-methylpyridazin-4-yl)thieno[3,2-b]pyridine 37 (1 eq) and the respective amine (1.2 eq) in toluene (3 mL) was added sodium tert-butoxide (3 eq).The reaction mixture was purged for 10 min with argon gas.BINAP (0.15 eq) and Pd2(dba)3 (0.1 eq) were added at room temperature and the reaction mixture was stirred at 100 °C for 40 min under microwave irradiation.The progress of reaction was monitored by TLC and LCMS.The reaction mixture was filtered through celite and washed with CH2Cl2.The filtrate was concentrated under reduced pressure and purified by Combiflash column chromatography.Pure fractions were concentrated under reduced pressure to obtain the desired compound (8.5-33%).General procedure 45-48: To a stirred solution of 5-chloro-2-(pyridin-3-yl)thieno[3,2-b]pyridine 44 (100 mg, 1 eq) and the respective amine (hydrochloride) (7 eq) in DMSO (0.1-3 mL) was added K2CO3 (10 eq).The resulting reaction mixture was heated at 140 °C for 18 h to 4 days.The progress of the reaction was monitored by TLC and LCMS.After completion, the reaction mixture was cooled to RT, quenched with water (20 mL), extracted with 5% MeOH in DCM (2 x 30 mL) and washed with water.The organic layer was dried over Na2SO4, concentrated in vacuo and purified by preparative HPLC to afford the product (6-29%).General procedure 50-55, Asyn-44: The synthesis was performed according to a literature procedure. [17]In brief, to a microwave tube were added compound 49 (1 eq), the respective

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This article is protected by copyright.All rights reserved.nitrogen nucleophile (5 eq), n-BuOH, followed by N,N'diisopropylethylamine (7 eq).The tube was sealed and heated at 200 °C for 1 hour using a microwave reactor.The solvent was removed under reduced pressure and the residue was taken up in dichloromethane, washed with ammonium chloride saturated solution and water, dried over Na2SO4, and concentrated under reduced pressure.The residue was purified by flash column chromatography to afford the desired product.

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Radiosynthesis of [ 18 F]asyn-44
The synthesis was carried out on a TRACERlab FX2 N radiosynthesis module.[ 18 F]Fluoride (5-20 GBq) was trapped on an anion exchange cartridge (HCO3; Synthra, Germany) and was eluted with a solution of 1.35 mg K2CO3 and 14 mg Kryptofix® 222 in 0.9 mL MeOH and 0.1 mL water.It was azeotropically dried at 85-110 °C under addition of 1 mL MeCN with a N2 flow and vacuum.The reaction vessel was then cooled to 60°C.The precursor (1 mg in 0.7 mL DMSO) was added, and the reaction mixture was stirred and heated at 100 °C.After 10 minutes, the reactor was cooled to 40 °C and the mixture was diluted with 4 mL mobile phase (MeCN/0.1Mammonium formate 45:55).The product was purified by semi-preparative HPLC (Luna C18 column (5 µm, 250 x 10 mm), MeCN/0.1Mammonium formate 45:55, 6 mL/min) and the fraction eluting at ~25 min was collected in a flask containing 25 mL water and passed over a C18 solidphase extraction cartridge.The product was eluted with 1 mL ethanol, followed by 9 mL saline.[ 18 F]Asyn-44 was obtained in a total synthesis time of 1 hour with a radiochemical purity of >95%, a radiochemical yield of 6±2% (decay-corrected, n=5) and a molar activity of 263±121 GBq/µmol.

LogD7.4 determination
The logD7.4 of [ 18 F]asyn-44 was determined via the shake-flask method: [19] Briefly, 50 mL 1-octanol was added to a separatory funnel and washed three times with potassium phosphate buffer (50 mL, 0.025M, pH 7.4).Then, 100 µL of the formulated [ 18 F]asyn-44 solution was added to the 1-octanol phase and was washed twice more with the potassium phosphate buffer.Eight 3-mL aliquots were taken of the 1-octanol layer and added to test tubes containing 3 mL buffer solution.The tubes were shaken for 10 min and centrifuged (~3500-4000 rpm) for 5 min.Aliquots of the 1-octanol layers (0.5 mL each) and buffer layers (1.5 mL each) were taken, weighed and measured in a gamma counter (Perkin Elmer; Waltham, Massachusetts, US).The logD7.4 was calculated by taking the natural logarithm of the ratio of counts per mL of the 1-octanol and corresponding buffer aliquots and was 4.16±0.04(n=8).

Postmortem tissues
The binding assays utilized fresh frozen blocks (1 cm 3 ) of autopsy-confirmed human tissues at the University of Pittsburgh under the approval of the Committee for Oversight of Research and Clinical Training Involving Decedents (CORID).PD anterior cingulate cortex brain tissue was obtained from Dr. Thomas Beach at Banner/Sun Health Arizona, USA and contained frequent α-synuclein aggregates and no other detectable aggregated amyloid or TDP-43 species.AD and normal control brain tissues were obtained from the Neurodegenerative Disease Brain Bank at the University of California, San Francisco, USA and contained only frequent tau neurofibrillary tangles and A plaque aggregates (AD tissue, middle frontal gyrus) and no other detectable aggregated amyloid or TDP-43 species (control tissue, middle frontal gyrus).The frozen tissue blocks were prepared for the binding assays as previously described. [21]he autoradiography and IHC studies utilized post-mortem paraffin embedded tissue blocks from different brain regions of control donors and donors with confirmed α-syn and tau pathology, which were acquired from the University of Pennsylvania Brain Bank.The presence or absence of pathological α-syn, tau and A aggregates in tissue sections from α-synucleinopathy, tauopathies and control donors was confirmed by immunostaining.

In Vitro Competition (Ki) Assays
The equilibrium inhibition constant (Ki) values of the unlabelled compounds (asyn-44 derivatives as well as tau ligands) were determined versus tritium-labelled asyn-44 according to our published methods. [21]Frozen aliquots (−80 °C) of homogenized human PD (asyn-44 derivatives) or AD (tau ligands) tissue (10 mg/mL in phosphate buffered saline) were thawed and diluted in 50 mM tris buffer (pH = 7.0) to 1 mg/mL.The unlabelled competitors were dissolved in DMSO at a concentration of 400 µM and diluted to 20 µM with tris buffer, resulting in a 5% DMSO solution in tris buffer.The subsequent serial dilution (6 µM to 4 nM) was made with 5% DMSO in tris buffer to maintain a constant DMSO concentration.50 µL of the respectively diluted solution was added to 50 µL of [ 3 H]asyn-44 and 800 µL of tris buffer/20% ethanol/0.1% bovine serum albumin (BSA), resulting in concentrations of 1 nM [ 3 H]asyn-44 and 0.2-1000 nM unlabelled  competitor.To this, 100 µL of the 1 mg/mL brain homogenate was added (100 µg/mL final tissue concentration) and the mixture was incubated for 60 min at room temperature, filtered through a Whatman GF/B glass filter via a Brandel M-24R cell harvester (Gaithersburg, MD, USA) and rapidly washed three times with 3 mL of tris buffer/20% ethanol/0.1%BSA.The filters were counted in CytoScint-ES after thorough vortexing.The concentration of bound compound was determined from the radioactivity retained on the filter (corrected for nonspecific binding (vide infra)) and the molar activity of the tritiated compound.
The equilibrium inhibition constant (Ki) values of the unlabelled asyn-44 was determined in AD brain homogenates versus tritiumlabelled Pittsburgh compound B according to our published methods. [28]Essentially, a similar procedure was carried out as outlined for [ 3 H]asyn-44 using PBS instead of tris buffer.

Two Point Screening Assays
Screening assays of test compounds were conducted at concentrations of 30 and 300 nM unlabelled competitor in PD brain homogenate versus tritium-labelled asyn-44.The assay was carried out similarly as described for the Ki assay (vide supra).No (0%) inhibition was defined by the number of counts without the addition of any unlabelled competitor and complete (100%) inhibition was defined as the number of counts at 1 μM of the unlabelled radioligand (nonspecific binding).The % inhibition of the radioligand by the test compound at 30 or 300 nM was defined by the number of counts specifically bound at 30 or 300 nM divided by the difference of counts at 0% inhibition minus the counts at 100% inhibition multiplied by 100.All assays were performed in triplicate at each concentration.

Equilibrium dissociation constant (Kd) and Bmax assays
Tritium-labelled asyn-44 homologous binding assays utilized PD, AD, or control brain homogenates to determine equilibrium dissociation constant (Kd) and Bmax values and were performed as previously described with minor modification, which included the use of a binding assay and filter wash solutions of tris buffer (50 mM, pH 7.0)/20% ethanol/0.1%BSA. [21]

Autoradiography
In vitro autoradiography was performed using 6-μm-thick deparaffinized sections derived from MSA, PD, AD, CBD, PSP and CT brains.Brain sections were first equilibrated for 20 min in PBS (Dulbecco's phosphate buffered saline) and then incubated with 4 nM [ 3 H]asyn-44 (specific activity 34 Ci/mmol) in assay binding buffer (PBS + 20% EtOH) for 90 min at RT.To determine the non-specific binding (NSB), adjacent brain sections were incubated with [ 3 H]asyn-44 mixed with 10 μM of unlabelled asyn-44.After incubation, slides were washed 3 x 5 min in cold washing buffer (PBS + 20% EtOH) to remove unbound tracer and then dipped briefly in distilled water.Slides were then allowed to air-dry before being exposed and scanned in a real-time autoradiography system (BeaQuant instrument, ai4R) for 5 h.ROI delimitation and quantification of signal was performed by using the image analysis software Beamage (ai4R).Specific binding was determined by subtracting the non-specific signal from the total signal and expressed as counts/min/mm 2 .Immunohistochemistry was performed on deparaffinized sections adjacent to those used for autoradiography.Sections were permeabilized with 0.1% Triton™ X-100 for 10 min followed by 3 x 5 min washes in PBS Tween20 buffer.Sections underwent hydrogen peroxide blocking for 15 min and PBS + 10% goat serum + 1% BSA + 0.1% Tween 20 blocking for 1 h at room temperature.Sections were immunostained using α-syn (phosphor S129) antibody (P-syn/81A) (Abcam), anti-phosphotau (Ser202, Thr205) antibody (ThermoFisher), used at 1:500 dilution overnight at 4 ºC.After a series of thorough washes with PBST buffer, the slides were incubated with the secondary antibodies Goat Anti-Mouse IgG H&L (HRP) (Abcam) at 1:10000 for 1 h at room temperature.The sections were washed again with PBST buffer for 3 x 5 min, treated with DAB substrate for 10 min and counterstained by Meyer's hematoxylin dye.Subsequently, the sections were mounted with Limonene mounting media and coverslips for microscopy.Images were captured with a Zeiss microscope at 10X magnification.

PET imaging
A total of 6 healthy adult rats (Sprague Dawley, 4-month old, 364±88 g; 3/3 M/F) underwent PET imaging following bolus injection of [ 18 F]asyn44.Rats were catheterized in the tail vein and positioned in a dedicated small animal PET/CT scanner (nanoScan™, Mediso Ltd., Budapest, Hungary).Anaesthesia was maintained throughout the scanning period while monitoring body temperature and respiration.A computed tomography (CT) was acquired before each PET scan and used for attenuation and scatter correction purposes as well as PET/CT co-registration and co-registration with a stereotactic MR atlas of rat brain [29] to define anatomical regions of interest (ROI).Dynamic PET scans were acquired for 120 minutes immediately after i.v.administration of [ 18 F]asyn-44 (18.3±1.8 (16.5-21.1)MBq, 0.19-1.05nmol/kg, 54-268 GBq/μmol).The acquired list mode data were sorted into 39 frames (3 × 5, 3 × 15, 3 × 20, 7 × 60, 17 × 180, and 6 × 600 s) 3D true sinograms (ring difference 84).The 3D sinograms were converted into 2D sinograms using Fourier-rebinning and reconstructed using a 2Dfiltered back projection (FBP) with a Hann filter at a cut-off of 0.50 cm -1 .Static images of the complete emission acquisition and different time frames of 0-5, 5-60 and 60-120 min were reconstructed with the manufacturer's proprietary iterative 3D algorithm (six subsets and four iterations).All image data were corrected for detector geometry and efficiencies, dead-time and decay-corrected to the start of acquisition, with corrections for attenuation and scatter using a CT-based material map.Image analyses and extraction of brain TACs from the dynamic FBP images were performed using VivoQuant® 2020 (2020patch1, Invicro, Needham, MA, USA).Standardized uptake values (SUV) were calculated by normalizing the regional radioactivity for injected radioactivity and body weight of the animal.

Figure 2 .
Figure 2. General structure and four subclasses of pyridothiophenes.

Table 1 .
Ki values (n =1) of [ 3 H]asyn-44 and various tau ligands in AD brain homogenates.were conducted in AD tissue homogenates to assess the affinity of asyn-44 in competition with [ 3 H]PiB binding to aggregated Aβ.The Ki of asyn-44 versus [ 3 H]PiB in AD brain homogenate was 9600±800 nM (n=3), indicating a relatively poor affinity of asyn-44 for the PiB binding site on aggregated Aβ.To assess the affinity of asyn-44 for different tau binding sites, [ 3 H]asyn-44 was screened against four tau-PET ligands, PI-2620, APN-1607 (a.k.a.PM-PBB3), OXD-2115 (a.k.a.CBD-2115) and MK-6240 ( [ 3 H]Asyn-44 gave a clear signal overlapping with α-syn neuropathology visualized using anti-pS129 α-syn immunohistochemistry (IHC).The specificity of the pathological α-syn signal was confirmed by the extent of co-localization with pS129 IHC as well as its displacement by an excess of unlabelled asyn-44.In contrast to the synucleinopathy samples, [ 3 H]asyn-44 showed only weak binding in AD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) pathologies and control case.Binding to the non-demented control case was due to the presence of a-syn inclusions.Selectivity over pathological tau was further assessed by IHC for misfolded tau and no clear co-localization between tau inclusions by IHC and [ 3 H]asyn-44 autoradiographic signal was observed.

Figure 6 .
Figure 6.In vivo brain PET study of [ 18 F]asyn-44 in rats; dynamic PET images were obtained for 120 min.Top: Representative example of summed images (0−5, 5-60, and 60-120 mins); Bottom: Time-activity curves of [ 18 F]asyn-44 in This article is protected by copyright.All rights reserved.
This article is protected by copyright.All rights reserved.
15213765, ja, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202303921 by University Of Toronto Librarie, Wiley Online Library on [15/02/2024].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License This article is protected by copyright.All rights reserved.
15213765, ja, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202303921 by University Of Toronto Librarie, Wiley Online Library on [15/02/2024].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Metabolites [ 18 F]Asyn-44 (19-48 MBq) was injected into the tail vein of healthy Sprague Dawley rats (5-8-month old, 409±96 g; 6/5 M/F) under isoflurane anaesthesia.The animals were recovered from anaesthesia after tracer injection and at 30 and 60 minutes post injection, they were sacrificed by decapitation and trunk blood samples were collected in heparinised tube (n=2-3 for each time point) and centrifuged at 2,500 RCF (Relative centrifugal force) for 5 min to collect plasma.Brains were excised quickly and homogenized with a BeadBug™ (Benchmark Scientific; Sayreville, NJ, USA) in 3.2 mL ice-cold acetonitrile together with 1 μg of unlabelled parent compound to facilitate tracer extraction, then 1.6 mL deionized water was added and mixed.The homogenate was centrifuged at 12,000 g for 5 min and the supernatant was collected.Plasma and brain extract were analysed by column-switching HPLC on a Luna C18(2) column (10 μm, 4.6 × 250 mm, Phenomenex) with mobile phase of 40:60 CH3CN/ 0.1 M ammonium formate in water at 2 mL/min.The HPLC data was analyzed with PowerChrom chromatography software (eDAQ Pty Ltd.; Colorado Springs, Colorado, US).