Synthesis and structure–activity relationship (SAR) studies of 1,2,3-triazole, amide, and ester-based benzothiazole derivatives as potential molecular probes for tau protein

Abstract

The pyridinyl-butadienyl-benzothiazole (PBB3 15) scaffold was used to develop tau ligands with improved in vitro and in vivo properties for imaging applications to provide insights into the etiology and characteristics of Alzheimer's disease. The photoisomerisable trans-butadiene bridge of PBB3 was replaced with 1,2,3-triazole, amide, and ester moieties and in vitro fluorescence staining studies revealed that triazole derivatives showed good visualisation of Aβ plaques, but failed to detect the neurofibrillary tangles (NFTs) in human brain sections. However, NFTs could be observed using the amide 110 and ester 129. Furthermore, the ligands showed low to high affinities (K_i = >1.5 mM–0.46 nM) at the shared binding site(s) with PBB3.

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Introduction

Recent data indicate that Alzheimer's disease (AD) and other types of dementia are affecting approximately 35 million people worldwide.^1–3 AD is associated with cognitive decline, including spatial disorientation, memory loss,⁴ language impairment, mood swings, and behavioural issues.⁵ Although the underlying cause of AD is still unknown,⁶ accumulating evidence suggests multifactorial factors instead of a single cause.

Typical pathological hallmarks found in the brains of individuals with AD include the formation of extracellular amyloid plaques and phosphorylated tau fibrils. Overproduction and accumulation of amyloid in the brain has been considered as a major contributor to the pathogenic processes that can lead to early-onset AD. Alternatively, an imbalance between Aβ production and clearance may contribute to late-onset sporadic AD.⁷ Recent studies suggest that dysfunction in Aβ clearance in the central nervous system due to aging promotes the accumulation, aggregation, and deposition of Aβ1-40 and Aβ1-42, indicating that amyloid clearance may be clinically important in the development of AD.⁸

Hyperphosphorylated tau protein, or neurofibrillary tangle (NFT), is also a hallmark of AD pathology. The basic role of tau proteins is believed to be stabilisation of the microtubule cell cytoskeleton presenting as six different isoforms each comprising either 3-repeat units (3R) or 4-repeat units (4R) with different forms found in different tauopathies. In pathogenic conditions, amyloid plaques along with neuroinflammation lead to hyperphosphorylation of tau proteins, resulting in damage to the microtubules, affecting cell function and viability.⁹ It has been recognised that the density of amyloid plaques does not correlate with disease progression or cognitive impairment in AD. In contrast to the amyloid pathology, the density and neocortical spread of NFTs correlate well with progressive neuronal degeneration and cognitive decline in AD patients, thus making the non-invasive detection and measurement of NFTs using external imaging a desirable biomarker for AD.⁴ NFTs or paired helical filaments of tau protein are found within neurons whereas aggregates of amyloid-b protein (Ab) which are the constituents of plaques are distributed in the extracellular space.

During the last decade, several positron emission tomography (PET) probes have been developed for the in vivo detection of β-amyloid pathology, including [¹¹C]-Pittsburgh compound B ([¹¹C]PiB) 1, [¹¹C]SB13 2, [¹⁸F]BAY94-9172 (florbetaben or florpyramine) 3, [¹⁸F]AV-45 (florbetapir) 4, [¹⁸F]flutemetamol 5, [¹⁸F]AZD4694 6, and [¹¹C]AZD2184 7 (Fig. 1).¹⁰

Fig. 1 Structure of ¹¹C-labelled and ¹⁸F-labelled radiotracers for imaging Aβ-plaques in Alzheimer's disease patients.

The development of tau tracers with high specificity has lagged behind its amyloid counterparts. This is because tau is homologous in structure with β-amyloid, but is present in lower concentrations, thus requiring tau ligands to have selectivity (20–50 fold affinity) for tau over β-amyloid.^11–13 Consequently, discovery efforts have translated into the development of small molecule PET imaging radiotracers with high binding affinity to NFTs and selectivity over β-amyloid.¹² A number of PET tracers targeting aggregated forms of tau have been identified including first-generation tracers: [¹⁸F]FDDNP 8,¹⁴ [¹⁸F]THK5117 9, [¹⁸F]THK5351 10,¹⁵ [¹⁸F]-T807 (flortaucipir) (AV-1451) 11, [¹⁸F]-T808 (AV-680) 12 (Fig. 2)¹⁰ and PBB1–PBB5 13–17 (Fig. 3);¹⁶ and second-generation tracers: [¹⁸F]RO-948 18,¹⁷ [¹⁸F]MK6240 19,¹² and [¹⁸F]JNJ311 20,¹⁸ and [¹⁸F]PM-PBB3 (aka APN-1607) 21 (Fig. 4).¹⁹

Fig. 2 Structure of first-generation tau radiotracers.

Fig. 3 Structures and design of PBB1–PBB5 for labelling with carbon-11.

Fig. 4 Structure of second-generation tau radiotracers.

Recently a number of novel tau PET tracers based on different scaffolds have been reported.²⁰ Many of these have limitations, including off-target binding, poor specificity, and low stability which hinder their potential in the clinical settings.^21,22

[¹¹C]PBB3 15 is used clinically for the in vivo detection of tau deposits in AD and non-AD tauopathies.²³ Recent clinical studies showed that it differentiated tau in AD brains from healthy control brains, and specific binding was observed in the CA1 and subiculum areas in the hippocampus where a high accumulation of fibrillar tau protein exists.¹⁷

Despite the favourable in vivo properties, PBB3 15 incorporates a photoisomerisable trans-butadiene between the aminopyridine and the benzothiazole moieties. It was also found that only the (E,E)-isomers was active in binding to tau.²² Although, photoisomerisation can be entirely blocked using a UV-free LED light in the radiosynthesis and administration to subjects,²² the trans butadiene moiety of PBB3 15 provided an excellent structural platform for further tau ligand development. In this project, we focused on generating a new series of tau ligands by replacing the trans-butadiene bridge of PBB3 15 with 1,2,3-triazole, amide, and ester moieties (Fig. 5), with the aim of finding molecules with better stability whilst retaining its favourable in vivo properties. It was envisaged that a triazole would result in an inflexible linker whereas an amide and ester linker would result in a partially restricted linkage, thus generating a range of conformationally restricted examples. Furthermore, the latter amide and ester linkages attached to the benzothiazole could resemble the sulfoxide oxygen characteristic of the tau ligand [¹⁸F]N-methyllansoprazole.

Fig. 5 Strategy for the development of novel tau ligands through modification of the trans-butadiene bridge on PBB3 15.

We report the preliminary data on the synthesis, structure–activity relationship (SAR) studies and biological evaluation (binding properties and fluorescence study) of 1,2,3-triazole, amide, and ester-based benzothiazole derivatives as potential ligands for detection of tau protein.

Results and discussion

Chemistry

The core structure of PBB3 15 consists of two building blocks; a benzothiazole and a pyridine connected by a trans-butadiene bridge. Replacing this trans-butadiene bridge with other bridging moieties provided scope for structural modifications between the benzothiazole region, and pyridine while offering greater photostability, through a variety of substitution patterns. The key component in our synthetic strategy of the target molecules was the incorporation of 1,4-disubstituted 1,2,3-triazole derivatives prepared by a Cu(I)-catalyzed azide–alkyne cycloaddition (click reaction) using appropriately substituted substrates.

The synthesis of alkyne 25 for the first target was accomplished using a reported procedure²⁴ with minor modifications (Scheme 1). Diazotization of the commercially available 6-methoxybenzo[d]thiazol-2-amine 22, followed by Sandmeyer iodination gave the iodobenzothiazole 23 in 70% yield, which was subjected to a Sonogashira cross-coupling reaction in the presence of 2.0 equivalents of ethynyltrimethylsilane to generate the protected alkyne 24 in 66% yield. Deprotection of the protected alkyne 24 with KF in MeOH at rt for 4 h to give the free alkyne 25 in 98% yield.

Scheme 1 Synthesis of triazole derivatives 30 and 31. Reagents and conditions: (a) p-TsOH·H₂O, NaNO₂, KI, MeCN/H₂O (9 : 1), 0 °C – rt, 18 h, 70%. (b) PdCl₂(PPh₃)₂, CuI, ethynyltrimethylsilane, DMF/TEA (2 : 1), rt – 65 °C, 8 h, 66%. (c) KF, MeOH, rt, 4 h, 98%. (d) i. NaOMe, paraformaldehyde, MeOH, 65 °C, 6 h; ii. NaBH₄, 0–65 °C, 2 h, 70%. (e) Na ascorbate, N,N-dimethylethylenediamine, NaN₃, EtOH/H₂O (7 : 3), rt – 65 °C, 8 h, 23–45%. (f) Na ascorbate, CuSO₄·5H₂O, t-BuOH/H₂O (1 : 1), rt, 24 h, N₂, 59–69%. (g) 48% HBr in AcOH, 110 °C, 96 h, no conversion.

The aryl azide 28 was prepared by N-methylation of aryl bromide 26via reductive amination to give 27 in 70% yield. Azidation of 27 in the presence of NaN₃ and the stabilising ligand, N,N′-dimethylethylenediamine (DMEDA)²⁵ gave the azide 28 in 23% yield. The low yield was attributed to the incomplete conversion of 27, and therefore product azide 28 was obtained as a mixture along with 27 in approximately 1 : 1 ratio as indicated by analysis of the ¹H NMR spectrum. Attempts to separate the mixture using column chromatography and preparative TLC proved unsuccessful. Therefore, the azide 28 was used in the next reaction step as a mixture along with 27.

Aryl azide 29 was synthesised to that for azide 28. The reaction was optimised through careful choice of solvent mixture and temperature (Table 1) to give the aryl azide in 45% yield (entry 4), however, incomplete consumption of the starting bromide 26 was observed. Generally, a lower reaction temperature gave an incomplete conversion of aryl bromide 26 to azide 29 (entry 1–4), whereas at 70 °C the azide 29 eventually decomposed with no sign of the starting bromide 26 (entry 5). A compromise temperature of 65 °C allowed sufficient reactivity of the bromide 26 in the nucleophilic substitution to azide 29, while maintaining the stability of the in situ formed azide 29.

Table 1 Optimisation of the azidation reaction of aryl bromide 26 using DMEDA as a stabilising ligand for the generation of aryl azide 29

Entry	Solvent	Temp (°C)	Time (h)	Yield (%)	Unreacted aryl bromide^a (%)
a Unreacted aryl bromides were obtained as the percent recovery (%) after flash chromatography over SiO2 gel (CH2Cl2/MeOH – 90 : 10).
1	DMSO/H2O (1 : 1)	65	8	25	51
2	DMSO/H2O (7 : 3)	65	8	22	50
3	EtOH/H2O (1 : 1)	65	8	40	46
4	EtOH/H2O (7 : 3)	65	8	45	40
5	EtOH/H2O (7 : 3)	70	8	11	—

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The synthesis of triazoles 30 and 31 was achieved via CuAAC reactions of azide 28 (2.0 eq.) and 29 (2.0 eq.), and alkyne 25 (1.0 eq.) in the presence of Cu(II)/ascorbate in t-BuOH/H₂O at rt for 24 h. Attempts to synthesise triazole 32 using excess 48% HBr in AcOH were unsuccessful, likely due to the low solubility of the starting triazole 31 in AcOH; therefore a new synthetic pathway via alkyne 39 was developed (Scheme 2).

Scheme 2 Synthesis of triazole 32. Reagents and conditions: (a) HCl, EtOH, rt, 48 h, 72%. (b) TBDMS-Cl, imidazole, DMF, rt, 24 h, 41%. (c) p-TsOH·H₂O, NaNO₂, KI, MeCN/H₂O (28 : 1), 0 °C – rt, 18 h, 63%. (d) PdCl₂(PPh₃)₂, CuI, ethynyltrimethylsilane, DMF/TEA (4 : 1), rt – 65 °C, 6 h, 74%. (e) KF, MeOH, rt, 4 h, 86%. (f) Na ascorbate, CuSO₄·5H₂O, t-BuOH/H₂O (1 : 1), rt, 24 h, N₂, 53%.

The benzothiazole core 35 was synthesised in 72% yield by an acid-catalysed condensation reaction of benzoquinone 33 with thiourea 34 in EtOH at rt for 48 h, followed by protection of the phenol using t-butyldimethylsilyl (TBDMS) in the presence of imidazole in DMF to give 36 in 41% yield. Sandmeyer iodination of 36 in the presence of p-TsOH·H₂O, NaNO₂, and KI gave iodobenzothiazole 37 as a grey solid in 63% yield which was subjected to a Sonogashira cross-coupling reaction to afford the protected alkyne 38 in 74% yield. The synthesis of free alkyne 39 was achieved in 86% yield via fluoride desilylation using an excess of potassium fluoride (KF) and a solution of 38 in MeOH. Finally, the CuAAC reaction of free alkyne 39 with aryl azide 28 gave 32 in 53% yield (Scheme 2).

The triazole derivatives 30–32 were found to possess low solubility in most solvents, being only slightly soluble in DMSO and DMF. Therefore, in the search of analogues with improved solubility, additional derivatives were designed and synthesised incorporating different substitutions on the benzothiazole moiety (R) and phenyl ring (R₁ and R₂), and changes to the carbon linker (n) connecting the triazole unit and phenyl functionality (Fig. 6). To this end, free alkyne 43 was successfully synthesised using the same strategy as for alkyne 25 (Scheme 3).

Fig. 6 Rational design of triazole derivatives to target improved solubility.

Scheme 3 Synthesis of free alkyne 43. Reagents and conditions: (a) p-TsOH·H₂O, NaNO₂, KI, MeCN/H₂O (9 : 1), 0 °C – rt, 18 h, 71%. (b) PdCl₂(PPh₃)₂, CuI, ethynyltrimethylsilane, DMF/TEA (2 : 1), rt – 65 °C, 6 h, 76%. (c) KF, MeOH, rt, 2 h, 96%.

The synthesis of azides 54–63 was achieved in high to excellent yields (74–90%) by reacting the appropriate benzyl and phenylethyl bromides 44–53 with NaN₃ in DMF at 55 °C for 24 h (Table 2).

Table 2 Summary of the synthesis of azides 54–63

Azide	R1	R2	n	Yield (%)
54	F	H	1	83
55	H	F	1	75
56	CF3	H	1	87
57	H	H	1	90
58	NO2	H	1	77
59	CN	H	1	77
60	F	H	2	74
61	CF3	H	2	83
62	OH	H	2	78
63	NO2	H	2	78

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The target triazole derivatives were realised in moderate to high yields via CuAAC catalytic click reactions between the free alkyne 25 or 43 and the azides 54–63 (Table 3).

Table 3 Summary of the synthesis of triazole derivatives 64–78via CuAAC reactions

Compound	R	R1	R2	n	Yield (%)
64	OCH3	F	H	1	77
65	OCH3	F	H	2	69
66	OCH3	H	F	1	84
67	OCH3	CF3	H	1	74
68	OCH3	CF3	H	2	71
69	OCH3	H	H	1	86
70	OCH3	OH	H	2	70
71	OCH3	NO2	H	1	79
72	OCH3	NO2	H	2	75
73	OCH3	CN	H	1	73
74	CH3	F	H	1	78
75	CH3	F	H	2	69
76	CH3	CF3	H	1	73
77	CH3	OH	H	2	81
78	CH3	NO2	H	1	75

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Furthermore, to investigate the role of alkyl alcohol substituents for tau binding, triazole derivatives 89–93 were synthesised. The synthesis started with the azidation of bromo alkyl alcohols 79–83 using NaN₃ in DMF at 55 °C for 24 h, followed by CuAAC reactions with free alkyne 25 (Scheme 4).

Scheme 4 Synthesis of triazole derivatives 89–93. Reagents and conditions: (a) NaN₃, DMF, 55 °C, 24 h, 90–96%. (b) Na ascorbate, CuSO₄·5H₂O, t-BuOH/H₂O (1 : 1), rt, 24 h, N₂, 73–84%.

Standard amide coupling was used for the synthesis of amide derivatives from the benzothiazole carboxylic acid and commercially available amines. The synthesis of acid 95 was achieved through the addition of 1.0 mol L⁻¹ NaOH (1.1 eq.) to a solution of the commercially available benzothiazole nitrile 94 in EtOH and heating at reflux for 18 h. Acidification of reaction mixture to pH 3 with a solution of HCl (18%) gave the acid 95 in 92% yield (Scheme 5).

Scheme 5 Synthesis of benzothiazole carboxylic acid 95.

The synthesis of amide derivatives 109–121 was realised in high yields (70–77%) via amide coupling under EDCl and HOBT conditions in DMF at rt for 24 h (Table 4).

Table 4 Summary of the synthesis of amide derivatives 109–121via amide coupling

Compound		Yield (%)	Compound		Yield (%)
109		70	116		82
110		77	117		85
111		75	118		83
112		76	119		76
113		73	120		68
114		77	121		73
115		71

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As isosteres of the amide linker, a series of ester analogues were made by the conversion of acid 95 to the corresponding sodium salt 122 in 98% yield. Reaction of benzothiazole salt 122 with the commercially available alkyl bromides 123–126 in DMF at rt for 24 h gave the ester products 127–130 in high to excellent yields (75–88%) (Table 5).

Table 5 Summary of the synthesis of ester derivatives 127–130

Compound		Yield (%)
127		85
128		88
129		78
130		75

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Biological evaluation

Heterologous ligand binding results. A heterologous blocking assay was performed to quantitatively evaluate the binding properties of the selected tau ligands by obtaining the inhibitory constant (K_i) and % inhibition against [¹¹C]PBB3 15 in brain homogenates of a 58 year old male AD patient (middle frontal gyrus, Braak stage V/VI). The binding efficiency is categorised according to the % shared binding sites with [¹¹C]PBB3 and the affinity K_i at the shared binding site(s) with [¹¹C]PBB3 as outlined in Table 6. However, a technical limitation of the K_i of the test compound may not be accurately determined if the degree of the heterologous blockade of [¹¹C]PBB3 binding was low or very little.

Table 6 Binding efficiency of tau ligands in heterologous binding assay

Shared binding site with PBB3 (% inhibition)		Affinity at shared binding site(s) with PBB3 (Ki)
Full	95–100%	>PBB3 (high affinity, Ki < 4 nM)
Middle	50–94%
Low	25–49%	=PBB3 (similar to PBB3, 4 nM < Ki < 10 nM)
Very little	1–25%
None	0%, Ki not determinable	<PBB3 (low affinity, Ki > 10 nM)

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The results of the heterologous binding assays for selected tau ligands are shown in Table 7.

Table 7 Inhibitory constant (K_i) and inhibition values of selected ligands

Ligand	Inhibitory constant (Ki)	Inhibition
R ^2: R-square for the binding curve of each compound fitted with Prism 6J. ND: not determinable: the inability to obtain the IC50 (Ki) values with certainty when the nonlinear regression using values for each concentration of test compound added did not converge for either 1- or 2-site curve fitting.
30	=PBB3 (6.3 nM, R^2 = 0.8825)	Very little (19.9%)
31	<PBB3 (39.4 nM, R^2 = 0.9620)	Low (32.3%)
32	=PBB3 (8.1 nM, R^2 = 0.5741)	Very little (14.3%)
64	>PBB3 (3.8 nM, R^2 = 0.2613)	Very little (10.1%)
67	<PBB3 (84.8 nM, R^2 = 0.0659)	Very little (1.5%)
69	<PBB3 (341.4 nM, R^2 = 0.3130)	Very little (5.7%)
70	<PBB3 (171.3 nM, R^2 = 0.9215)	Low (27.2%)
71	<PBB3 (>1.5 mM, R^2 = 0.5021)	Very little (13.5%)
72	>PBB3 (28.1 nM, R^2 = 0.4872)	Very little (0.2%)
73	>PBB3 (0.46 nM, R^2 = 0.1674)	Very little (7.7%)
75	ND	ND
89	<PBB3 (59.4 nM, R^2 = 0.4957)	Very little (8.9%)
90	<PBB3 (28.5 nM, R^2 = 0.7500)	Very little (8.0%)
91	<PBB3 (45.5 nM, R^2 = 0.4785)	Very little (13.6%)
92	<PBB3 (241.7 nM, R^2 = 0.4666)	Very little (7.9%)
93	<PBB3 (13.6 nM, R^2 = 0.0798)	Very little (3.3%)
110	<PBB3 (761.0 nM, R^2 = 0.8849)	Low (36.8%)
111	<PBB3 (>1.5 mM, R^2 = 0.4912)	Very little (16.3%)
115	<PBB3 (215.0 nM, R^2 = 0.9685)	Low (34.7%)
127	ND	ND
128	<PBB3 (13.2 nM, R^2 = 0.3642)	Very little (7.9%)
129	>PBB3 (2.4 nM, R^2 = 0.2924)	ND

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Analysis of the binding affinity (K_i) revealed that ligands 30 and 31 exhibited nanomolar affinity at the shared binding site(s) with [¹¹C]PBB3 15 with K_i values of 6.3 and 39.4 nM, respectively. Substituting the methoxy unit on 31 with a hydroxy group (32) improved the affinity approximately 5-fold.

The replacement of the pyridine unit with fluorobenzene and the introduction of a one carbon linker between the triazole and phenyl moiety improved the affinity, as can be observed on ligand 64 (K_i = 3.8 nM). Furthermore, the substitution of the fluorine atom on 64 with a hydrogen atom (69) and a nitro group (71) decreased the binding affinity, suggesting that the high electronegative property of fluorine may be important for binding. The replacement of the fluorine atom with the polar nitrile group (73) led to improvement in affinity (K_i = 0.46 nM).

Ligands 89–93 showed nanomolar to submicromolar affinities at the shared binding site(s) with [¹¹C]PBB3 15 (K_i = 13.6–241.7 nM), and interestingly, ligand 93 showed the highest affinity (K_i = 13.6 nM) compared to other ligands in this series. The superior affinity of ligand 93 suggests that the additional oxygen on the molecule should favourably interact with the site of the protein.

Furthermore, amide derivatives 110, 111, and 115 exhibited low affinity at the shared binding site(s) with [¹¹C]PBB3 15 (K_i > 10 nM). By contrast, ester derivatives generally demonstrated better affinity than amide derivatives. Although ligand 129 seemed to display a high K_i value of 2.4 nM, this was associated with a high uncertainty and its shared binding status with PBB3 was not determinable.

Overall, the ligands showed no to low% inhibition (0.0–36.8%) at the shared binding site with [¹¹C]PBB3 15. The ligands with a pyridine moiety (30–32) demonstrated very little to low% inhibition. Among the examples of this subset, the ligand with the mono N-methyl group (31) exhibited better inhibition (32.3%) than the ligand with a free amine group (30) (19.9%), suggesting that methyl substituent at pyridine unit may play an important role for the binding. However, the substitution of the methoxy group on 31 with a hydroxy unit (32) led to a decrease in inhibition value (14.3%).

The substitution of the pyridine unit with a phenyl moiety resulted in lower inhibition of [¹¹C]PBB3 15 (0.2–13.5%). However, the hydroxy group attached to the phenyl ring in the para-position may be essential for achieving good inhibition, as exemplified by ligand 70 with an inhibition value of 27.2%.

Furthermore, the substitution of the pyridine unit with small alkyl alcohol (89–93) led to very little inhibitions of [¹¹C]PBB3 15 (3.3–13.6%). The ligand 91 with seven alkyl carbons was more effective among these derivatives, and lower inhibitions were observed by either increasing or decreasing the alkyl carbon length. Interestingly, the presence of an oxygen atom in the middle of the carbon chain led to a significant decrease in inhibition of [¹¹C]PBB3 15, as observed in ligand 93 (% inhibition = 3.3%).

In addition, three amide derivatives (110, 111, and 115) displaced 16.3–36.8% of [¹¹C]PBB3 15 binding in the AD tissue. Among these derivatives, generally the 1-pyridine (110) and 4-hydoxymethyl (115) units were associated with better activity than the 3-cyano unit (111). Testing of ester derivatives (127–129) indicated that these ligands minimally blocked the [¹¹C]PBB3 15.

Furthermore, the synthesised derivatives were subjected to calculated physiochemical properties to test the suitability of incorporation of the new linkers for future studies. The majority of the ligand derivatives showed good physicochemical properties according to Lipinski parameters, in relation to lipophilicity (clog P), number of hydrogen bond donor (HBD), number of hydrogen bond acceptor (HBA), polar surface area (PSA), and the ratio of the steady-state concentrations of a compound between the brain and the blood (log BB) (calculated by Molinspiration online services, ESI† S126–S128). In addition, the new derivatives are photo-stable, with some of them demonstrated moderate to high binding affinity against [¹¹C]-PBB3 15 in in vitro heterologous blocking assay. Hence, further studies, including in vivo imaging using their corresponding radiolabelled derivatives, especially for ligands 110 and 129, which showed the capability to detect NFTs, are needed.

Fluorescence staining. The in vitro fluorescence staining with the synthesised tau ligands was examined in the middle frontal gyrus sections of AD patient bearing β-amyloid and tau proteins. PBB3 15 was used as a positive control as it labels tau lesions and several types of senile plaques, particularly dense core plaques, at micromolar concentration.²⁶ Representative staining of dense core plaque, primitive plaque and NFT's with PBB3 is shown in Fig. 7.

Fig. 7 Representative staining of dense-core plaque, primitive plaque, and NFTs labeled with PBB3 (15), which are used as a positive control in these experiments.²⁶

The in vitro staining assay of selected triazole derivatives revealed that ligands with pyridine units (30–32) poorly stained Aβ plaques and NFTs. The observation that the Aβ plaques and NFTs were not detectable may be due to the poor solubility properties of these probes in 50% ethanol in water, which limits tissue penetration, leading to insufficient imaging. However, this assumption needs to be confirmed by improving the bioavailability of the molecules. The substitution of a pyridine unit with phenyl moieties containing different substituents, and the introduction of the carbon linker between triazole and phenyl moieties improved the histology results. For example, ligands 67 and 75 which contain fluorine atoms displayed good staining of both dense-core plaque and primitive plaque (Aβ plaques) in human brain sections. This also suggests that the substitution of a methoxy group with a methyl group in the benzothiazole moiety improved the imaging of Aβ plaques as can be observed on ligand 75. Furthermore, the introduction of either a nitro (72), nitrile (73), or hydroxy (77) substituent on the phenyl ring led to ligands that showed good visualisation of Aβ plaques. It was also found that unsubstituted phenyl (69) intensely stained Aβ plaques (Fig. 8). Unfortunately, all triazole derivatives failed to detect NFTs.

Fig. 8 Representative in vitro histochemical staining of postmortem AD brain tissues with selected ligands. White arrow: dense-core plaque, yellow arrow: primitive plaque, red arrow: NFT.

Unexpectedly, triazole ligands containing alkyl alcohols (89–92) and an alkoxy alcohol (93) failed to detect Aβ plaques and NFTs by fluorescence microscopy. In addition, most of the amide derivatives have failed to visualise Aβ plaques and NFTs. However, ligand 110 with a nitrogen atom at the 1-position of the phenyl ring selectively detected NFTs (Fig. 8). Furthermore, the ester derivative 129 bearing a trifluoro substituent on the para-position of the phenyl ring and one carbon linker between the ester group and the phenyl moiety exhibited specific staining to NFTs. However, ligands with a nitrile (127) and fluorine (128) substituent at the phenyl ring were unsuccessful in detecting Aβ and NFTs.

Experimental methods

Representative synthesis of triazole 32

2-Iodo-6-methoxybenzo[d]thiazole 23²⁴. To a solution of 6-methoxybenzo[d]thiazol-2-amine 22 (4.50 g, 25.00 mmol) and p-toluenesulfonic acid monohydrate (15.07 g, 87.50 mmol) in MeCN (150 mL) was added a solution of NaNO₂ (4.45 g, 50.00 mmol) and KI (10.79 g, 65.00 mmol) in H₂O (16 mL) at 0 °C. The reaction was allowed to reach rt, and was stirred for 18 h. The reaction mixture was quenched with H₂O (350 mL) and basified with 2 M NaHCO₃ to pH 8–9, followed by addition of Na₂S₂O₃ (20 mL), yielding a brown precipitate. The precipitate was collected by filtration, washed with H₂O (20 mL), and dried in vacuo to afford the iodobenzothiazole 23 (5.11 g, 70%) as a light brown solid. The product was used directly for the next step without further purification. The spectroscopic data was in agreement with that previously reported. TLC (EtOAc/hexanes – 60 : 40): R_f = 0.85. ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, J = 9.2 Hz, 1H), 7.28 (d, J = 2.8 Hz, 1H), 7.03 (dd, J = 9.2, 2.8 Hz, 1H), 3.86 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 158.1, 149.0, 140.4, 123.0, 115.6, 103.1, 101.5, 55.8. MS (ESI +ve) m/z 292 ([M + H]⁺, 100%).

6-Methoxy-2-((trimethylsilyl)ethynyl)benzo[d]thiazole 24²⁴. To a solution of iodobenzothiazole 23 (1.86 g, 6.39 mmol), PdCl₂(PPh₃)₂ (90 mg, 0.13 mmol), CuI (26 mg, 0.14 mmol), and triethylamine (4.0 mL, 28.70 mmol) in anhydrous DMF (8.0 mL) was added a solution of ethynyltrimethylsilane (1.26 g, 12.83 mmol). The resulting mixture was stirred at 65 °C for 8 h. After cooling to rt, the solution was filtered through a layer of celite, and concentrated. The residue was subjected to flash chromatography over SiO₂ gel (EtOAc/hexanes – 60 : 40) to afford the protected alkyne 24 (1.10 g, 66%) as a light yellow oil. The product was used in the next reaction step immediately or stored in the freezer for a limited time due to stability issues. The spectroscopic data was in agreement with that previously reported. TLC (EtOAc/hexanes – 60 : 40): R_f = 0.86. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J = 9.2 Hz, 1H), 7.25 (d, J = 2.8 Hz, 1H), 7.09 (dd, J = 9.2, 2.8 Hz, 1H), 3.85 (s, 3H), 0.30 (s, 9H). ¹³C NMR (400 MHz, CDCl₃) δ 158.9, 147.3, 145.6, 136.8, 124.3, 116.6, 103.4, 102.3, 97.0, 55.8, 0.5. MS (ESI +ve) m/z 262 ([M + H]⁺, 100%).

2-Ethynyl-6-methoxybenzo[d]thiazole 25²⁴. To a solution of the protected alkyne 24 (1.00 g, 3.83 mmol) in MeOH (30 mL) was added KF (1.00 g, 17.21 mmol), and the mixture was stirred at rt for 4 h. The solvent was removed, and the residue was subjected to flash chromatography over SiO₂ gel (EtOAc/hexanes – 60 : 40) to afford the free alkyne 25 (0.71 g, 98%) as a pale yellow solid. The spectroscopic data was in agreement with that previously reported. TLC (EtOAc/hexanes – 60 : 40): R_f = 0.84. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 9.2 Hz, 1H), 7.27 (d, J = 2.8 Hz, 1H), 7.12 (dd, J = 9.2, 2.8 Hz, 1H), 3.88 (s, 3H), 3.55 (s, 1H). ¹³C NMR (400 MHz, CDCl₃) δ 159.1, 147.3, 144.8, 136.9, 124.6, 116.8, 103.5, 83.4, 77.0, 55.9. MS (ESI +ve) m/z 190 ([M + H]⁺, 100%).

5-Bromo-N-methylpyridin-2-amine 27²⁷. To a stirred solution of 5-bromo-pyridin-2-amine 26 (2.50 g, 14.45 mmol) in anhydrous methanol (100 mL) was added NaOMe (3.90 g, 72.25 mmol), and followed by paraformaldehyde (2.17 g, 72.25 mmol). After the reaction mixture was stirred at 60 °C for 6 h, it was cooled to 0 °C with continued stirring. NaBH₄ (2.73 g, 72.25 mmol) was added partionwise. The mixture was heated at reflux for another 2 h, cooled in an ice bath (0 °C), H₂O (50 mL) was added, and the mixture was extracted with CH₂Cl₂ (3 × 150 mL). The combined organic layers were washed with H₂O (150 mL), brine (150 mL), dried (Na₂SO₄), filtered, and concentrated. The residue was subjected to flash chromatography over SiO₂ gel (CH₂Cl₂/MeOH – 90 : 10) to afford the mono-N-methyl 27 (1.90 g, 70%) as a pale yellow solid. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.49. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 2.0 Hz, 1H), 7.49 (dd, J = 8.8, 2.0 Hz, 1H) 6.30 (d, J = 8.8 Hz, 1H), 4.59 (br s, 1H), 2.89 (d, J = 4.8 Hz, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 158.2, 148.8, 139.9, 107.8, 107.0, 29.3. MS (ESI +ve) m/z 187 (⁷⁹Br [M + H]⁺, 100%), 189 (⁸¹Br [M + H]⁺, 100%).

5-Azido-N-methylpyridin-2-amine 28. A solution of the 5-bromo-N-methylpyridin-2-amine 27 (540 mg, 2.89 mmol), sodium ascorbate (30 mg, 0.15 mmol), CuI (58 mg, 0.31 mmol), and N,N′-dimethylethylenediamine (40 mg, 0.44 mmol) in EtOH/H₂O (2 : 1, 22.5 mL) was stirred at rt for 30 min. NaN₃ (0.38 g, 5.85 mmol) was added, and the reaction mixture was stirred at 65 °C for 8 h. The solution was cooled to rt, filtered through a layer of celite, concentrated, and extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with H₂O (150 mL), brine (150 mL), dried (Na₂SO₄), filtered, and concentrated. The residue was subjected to flash chromatography over SiO₂ gel (CH₂Cl₂/MeOH – 90 : 10) to afford the mixture of the azide 28 (100 mg, 23%) and starting material 5-bromo-N-methylpyridin-2-amine 27 (102 mg) as a grey solid which were unseparable. This mixture was used directly in the subsequent step. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.44. ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J = 2.8 Hz, 1H), 7.15 (dd, J = 8.8, 2.8 Hz, 1H) 6.29 (d, J = 8.8 Hz, 1H), 4.63 (br s, 1H), 2.89 (d, J = 4.8 Hz, 3H). MS (ESI +ve) m/z 150 ([M + H]⁺, 100%). HRMS (ESI +ve TOF) calcd for C₆H₈N₅⁺ 150.0774, found 150.0773 ([M + H]⁺).

5-Azidopyridin-2-amine 29. A solution of the 2-amino-5-bromopyridine 26 (1.00 g, 5.78 mmol), sodium ascorbate (60 mg, 0.30 mmol), CuI (116 mg, 0.61 mmol), and N,N′-dimethylethylenediamine (79 mg, 0.88 mmol) in EtOH : H₂O (2 : 1, 45 mL) was stirred at rt for 30 min. NaN₃ (0.76 g, 11.69 mmol) was added, and the reaction mixture was stirred at 65 °C for 8 h. The solution was cooled to rt, filtered through a layer of celite, concentrated, and extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with H₂O (150 mL), brine (150 mL), dried (Na₂SO₄), filtered, and concentrated. The residue was subjected to flash chromatography over SiO₂ gel (CH₂Cl₂/MeOH – 90 : 10) to afford the azide 29 as a grey solid (0.35 g, 45%) which was used directly in the subsequent step. The spectroscopic data was in agreement with that previously reported. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.41. ¹H NMR (400 MHz, CDCl₃) δ 7.84 (d, J = 2.8 Hz, 1H), 7.16 (dd, J = 8.8, 2.8 Hz, 1H) 6.53 (d, J = 8.8 Hz, 1H), 4.44 (br s, 2H). ¹³C NMR (400 MHz, CDCl₃) δ 156.0, 139.1, 128.9, 127.7, 109.5. MS (ESI +ve) m/z 136 ([M + H]⁺, 100%). HRMS (ESI +ve TOF) calcd for C₅H₆N₅⁺ 136.0618, found 136.0623 ([M + H]⁺).

5-(4-(6-Methoxybenzo[d]thiazol-2-yl)-1H-1,2,3-triazol-1-yl)pyridin-2-amine 30.
Following general procedure A₂ (see ESI†). Alkyne 25 (40 mg, 0.21 mmol), CuSO₄·5H₂O (5 mg, 0.02 mmol), sodium ascorbate (8 mg, 0.04 mmol), and azide 29 (57 mg, 0.42 mmol) were stirred in t-BuOH/H₂O (1 : 1, 4.2 mL) to afford the 1,4-disubtituted-1,2,3-triazole 30 (40 mg, 59%) as a pale yellow solid after sequential trituration with CH₂Cl₂, MeCN, and hot MeOH. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.03. Mp 182 °C (decomp). ¹H NMR (400 MHz, DMSO) δ 9.35 (s, 1H), 8.47 (d, J = 2.4 Hz, 1H), 7.93 (dd, J = 8.8, 2.4 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.16 (dd, J = 9.0, 2.4 Hz, 1H), 6.61 (d, J = 8.8 Hz, 1H), 6.50 (br s, 2H), 3.87 (s, 3H). ¹³C NMR (400 MHz, DMSO) δ 160.3, 157.6, 156.1, 147.6, 142.6, 140.8, 135.5, 130.7, 123.4, 123.1, 121.4, 116.0, 107.8, 105.0, 55.8. IR (neat) ν_max 3462 (m), 3331 (m), 3222 (m), 3051 (w), 1645 (s), 1608 (m), 1563 (s), 1516 (s), 1466 (m), 1430 (m), 1413 (m), 1324 (s), 1249 (m), 1225 (s), 1079 (m), 1018 (s), 942 (s), 887 (s) cm⁻¹. MS (ESI +ve) m/z 325 ([M + H]⁺, 100%). HRMS (ESI +ve TOF) calcd for C₁₅H₁₃N₆OS⁺ 325.0866, found 325.0872 ([M + H]⁺).

5-(4-(6-Methoxybenzo[d]thiazol-2-yl)-1H-1,2,3-triazol-1-yl)-N-methylpyridin-2-amine 31. The mixture of the azide 28 (100 mg, 0.67 mmol) and 5-bromo-N-methylpyridin-2-amine 27 (102 mg) was dissolved in t-BuOH/H₂O (1 : 1, 10 mL), and alkyne 25 (127 mg, 0.67 mmol), CuSO₄·5H₂O (18 mg, 0.07 mmol), and sodium ascorbate (67 mg, 0.34 mmol) were added, and the reaction was stirred at rt for 24 hours. The resulting precipitate was filtered, and triturated with hot MeCN (5 mL) to afford the 1,4-disubtituted-1,2,3-triazole 31 (156 mg, 69%) as a pale yellow solid. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.05. Mp 189 °C (decomp). ¹H NMR (400 MHz, DMSO) δ 9.35 (s, 1H), 8.55 (d, J = 2.4 Hz, 1H), 7.93 (dd, J = 8.8, 2.4 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.15 (dd, J = 9.0, 2.4 Hz, 1H), 7.07 (q, J = 4.8 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 3.86 (s, 3H), 2.84 (d, J = 4.8 Hz, 3H). ¹³C NMR (400 MHz, DMSO) δ 159.6, 157.6, 156.2, 147.7, 142.6, 140.6, 135.5, 130.2, 124.2, 123.1, 121.3, 116.0, 107.8, 105.0, 55.8, 28.0. IR (neat) ν_max 3317 (m), 3100 (m), 3051 (w), 1608 (s), 1537 (s), 1493 (m), 1453 (m), 1401 (m), 1313 (s), 1290 (m), 1255 (m), 1218 (s), 1182 (m), 1079 (m), 1058 (m), 945 (s), 871 (s) cm⁻¹. MS (ESI +ve) m/z 339 ([M + H]⁺, 100%). HRMS (ESI +ve TOF) calcd for C₁₆H₁₅N₆OS⁺ 339.1023, found 339.1028 ([M + H]⁺).

2-(1-(6-(Methylamino)pyridin-3-yl)-1H-1,2,3-triazol-4-yl)benzo[d]thiazol-6-ol 32. Method A:. A mixture of 1,4-disubtituted-1,2,3-triazole 31 (25 mg, 0.08 mmol) and 48% HBr in AcOH (10 ml) was stirred at 110 °C for 96 h. The mixture was allowed to cooling down at rt, quenched with H₂O (10 mL), and neutralised with aqueous 1 N NaOH solution. The precipitated was collected by filtration, washed with H₂O, and dried in vacuo to give 32 in 10% yield.
Method B: following general procedure A₂ (see ESI†). Alkyne 39 (37 mg, 0.21 mmol), CuSO₄·5H₂O (5 mg, 0.02 mmol), sodium ascorbate (8 mg, 0.04 mmol), and azide 28 (63 mg, 0.42 mmol) were stirred in t-BuOH/H₂O (1 : 1, 4.2 mL) to afford the 1,4-disubtituted-1,2,3-triazole 32 (36 mg, 53%) as a brown solid after sequential trituration with CH₂Cl₂, MeCN, and hot MeOH. TLC (CH₂Cl₂/MeOH – 90 : 10): R_f = 0.05. Mp 170 °C (decomp). ¹H NMR (500 MHz, DMSO, 75 °C) δ 9.99 (s, 1H), 9.36 (s, 1H), 8.58 (d, J = 2.4 Hz, 1H), 7.94 (dd, J = 8.8, 2.4 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.12 (br s, 1H), 7.01 (dd, J = 9.0, 2.4 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 2.83 (s, 3H). ¹³C NMR (500 MHz, DMSO) δ 159.6, 156.0, 155.1, 146.8, 142.8, 140.6, 135.5, 130.2, 123.3, 121.2, 116.2, 108.2, 107.0, 28.1. IR (neat) ν_max 3243 (m), 3096 (m), 1613 (s), 1555 (s), 1455 (m), 1403 (m), 1380 (m), 1260 (m), 1235 (m), 1168 (m), 1035 (m), 902 (s), 831 (m), 799 (m) cm⁻¹. MS (ESI +ve) m/z 325 ([M + H]⁺, 100%). HRMS (ESI +ve TOF) calcd for C₁₅H₁₃N₆OS⁺ 325.0866, found 325.0866 ([M + H]⁺).

[¹¹C]PBB3 15. The radiosynthesis was carried out according to the procedure reported previously.²² At the end of synthesis (EOS), [¹¹C]PBB3 of 1.6–3.1 GBq was obtained after 30–35 min proton bombardment at a beam current of 15 μA. The decay-corrected radiochemical yield of [¹¹C]PBB3 based on [¹¹C]CO₂ was 15.4 ± 2.8% (n = 10) at the end of bombardment (EOB), and the molar activity was 120 ± 38 GBq μmol⁻¹ (n = 10) at EOS. The averaged radiochemical purity of [¹¹C]PBB3 product in an umber vial was 98 ± 1.5% and remained >95% after staying the product for 60 min without fluorescence light.

Postmortem brain tissues. All brain autopsies were performed after consent of the legal next of kin or an individual with power of attorney. Post-mortem human brains were obtained from autopsies carried out at the Center for Neurodegenerative Disease Research of the University of Pennsylvania Perelman School of Medicine on patients with AD. Tissues for homogenate binding assays were frozen, and tissues for fluorescence staining were fixed in 10% neutral buffered formalin followed by embedding in paraffin blocks. Studies of autopsy samples were approved by the National Institutes for Quantum Science and Technology Certified Review Board (approval number: 14-015).

In vitro binding assay. Frozen tissues derived from the middle frontal gyrus of an AD patient were homogenized in 50 mM Tris-HCl buffer, pH 7.4, containing protease inhibitor cocktail (cOmplete™, EDTA-free; Roche), and stored at −80 °C until analyses. To assay radioligand binding with heterologous blockade, these homogenates (100 μg tissue) were incubated with 5 nM [¹¹C]PBB3 (molar activity: 76.8 ± 25 GBq μmol⁻¹) in the absence or presence of test compounds at varying concentrations ranging from 1 × 10⁻¹¹ to 1 × 10⁻⁶ M in Tris-HCl buffer containing 10% ethanol, pH 7.4, for 30 min at room temperature. Non-specific binding of [¹¹C]PBB3 was determined in the presence of 1 × 10⁻⁶ M PBB3. Inhibitory constant (K_i) was determined by using non-linear regression to fit a concentration–binding plot to one-site and two-site binding models derived from the Cheng–Prusoff equation with GraphPad Prism version 5.0 (GraphPad Software), followed by F-test for model selection.

Fluorescence staining. Six-μm thick deparaffinized postmortem human brain sections were incubated in 50% ethanol containing 0.001% (w/v) of ligand at room temperature for 30 min. The brain samples were rinsed with 50% ethanol for 5 min, dipped into distilled water twice for 3 min each, and mounted in VECTASHIELD. Fluorescence images were captured by a DM4000 microscope equipped with filter cube for DAPI.

Conclusions

A series of tau ligands containing 1,2,3-triazole, amide, and ester moieties were synthesised. The heterologous blocking assay revealed that several of these ligands exhibited low to high affinities (K_i = >1.5 mM–0.46 nM) at the shared binding site(s) with PBB3 15. Ligands 30 and 64 had a similar affinity with PBB3 15 with K_i values of 6.3 and 3.8 nM, respectively. Furthermore, the ligands showed none to low % inhibition (0.0–36.8%) against [¹¹C]PBB3 15. Results from in vitro fluorescence staining showed that some triazole derivatives showed good visualisation of Aβ plaques, but failed to detect the NFTs in human brain sections. However, NFTs were visualised with ligands 110 and 129. All except one of the novel tau ligands synthesised and tested thus far possess a methoxy or methyl substituent at the 6-position of the benzothiazole ring. PiB 1, PBB3 15 and many related amyloid and tau ligands are prepared with a free hydroxy group. It is not clear if biological efficacy can be improved following demethylation, although one analogue 32 bearing the hydroxy group did not show improved binding. Ligands 110 and 129 represent a good starting point for further tau ligand development.

Author contributions

Conceptualization: AK, PAK, MH. Formal analysis HW, MO, TY, KK, MH, M-RZ, MF, PAK, AK. Funding acquisition PAK, AK, MF, MH. Investigation HW, MO, TY, KK, MH, M-RZ. Methodology HW, AK, MO, MH, MF, PAK, AK. Project administration AK, PAK. Resources AK, PAK, MF, M-RZ, MH, MO. Supervision AK, PAK, MH, M-RZ. Validation HW, M-RZ, MO, PAK. Visualization HW, M-RZ, MO, PAK. Writing – original draft HW, AK, PAK. Writing – review & editing HW, AK, PAK, MO, M-RZ, MH, MF.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

HW thanks Ministry of Research and Technology/National Research and Innovation Agency (Indonesia) for PhD scholarship funding under Research and Innovation in Science and Technology Project (RISET-Pro) No. 8245-ID.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2md00358a

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