Design, Synthesis, and Preliminary Evaluation of SPECT Probes for Imaging β-Amyloid in Alzheimer's Disease Affected Brain

Article abstract of DOI:10.1021/acschemneuro.8b00064

In this study, we synthesized of a series of 2-phenyl- and 2-pyridyl-imidazo[1,2-a]pyridine derivatives and examine their suitability as novel probes for single-photon emission computed tomography (SPECT)-based imaging of β-amyloid (Aβ). Among the 11 evaluated compounds, 10 showed moderate affinity to Aβ(1-42) aggregates, exhibiting half-maximal inhibitory concentrations (IC₅₀) of 14.7 ± 6.07-87.6 ± 39.8 nM. In vitro autoradiography indicated that ¹²³I-labeled triazole-substituted derivatives displayed highly selective binding to Aβ plaques in the hippocampal region of Alzheimer's disease (AD)-affected brain. Moreover, biodistribution studies performed on normal rats demonstrated that all ¹²³I-labeled probes featured high initial uptake into the brain followed by a rapid washout and were thus well suited for imaging Aβ plaques, with the highest selectivity observed for a 1H-1,2,3-triazole-substituted 2-pyridyl-imidazopyridine derivative, [¹²³I]ABC577. This compound showed good kinetics in rat brain as well as moderate in vivo stability in rats and is thus a promising SPECT imaging probe for AD in clinical settings.

Full text of DOI:10.1021/acschemneuro.8b00064

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Design, synthesis, and preliminary evaluation of SPECT probes
for imaging #-amyloid in Alzheimer’s disease-affected brain
Yuki Okumura, Yoshifumi Maya, Takako Onishi, Yoshinari Shoyama, Akihiro Izawa, Daisaku
Nakamura, Shigeyuki Tanifuji, Akihiro Tanaka, Yasushi Arano, and Hiroki Matsumoto
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00064 • Publication Date (Web): 27 Mar 2018
Downloaded from http://pubs.acs.org on March 31, 2018
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Design, synthesis, and preliminary evaluation of
SPECT probes for imaging β-amyloid in
Alzheimer’s disease-affected brain
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Yuki Okumura,*,†, ‡ Yoshifumi Maya,† Takako Onishi , Yoshinari Shoyama,† Akihiro Izawa,†
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Daisaku Nakamura,† Shigeyuki Tanifuji,† Akihiro Tanaka,† Yasushi Arano,‡ and Hiroki
Matsumoto†
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†Research Centre, Nihon Medi-Physics Co., Ltd., 3-1, Kitasode, Sodegaura City, Chiba 299-
0266, Japan
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‡Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical
Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
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ABSTRACT
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In this study, we synthesized of a series of 2-phenyl- and 2-pyridyl-imidazo[1,2-a]pyridine
derivatives and examine their suitability as novel probes for single-photon emission computed
tomography (SPECT)-based imaging of β-amyloid (Aβ). Among the 11 evaluated compounds,
10 showed moderate affinity to Aβ(1−42) aggregates, exhibiting half-maximal inhibitory
concentrations (IC50) of 14.7 ± 6.07–87.6 ± 39.8 nM. In vitro autoradiography indicated that 123I-
labeled triazole-substituted derivatives displayed highly selective binding to Aβ plaques in the
hippocampal region of Alzheimer’s disease (AD)-affected brain. Moreover, biodistribution
studies performed on normal rats demonstrated that all 123I-labeled probes featured high initial
uptake into the brain followed by a rapid washout and were thus well suited for imaging Aβ
plaques, with the highest selectivity observed for a 1H-1,2,3-triazole-substituted 2-pyridyl-
imidazopyridine derivative, [123I]ABC577. This compound showed good kinetics in rat brain as
well as moderate in vivo stability in rats and is thus a promising SPECT imaging probe for AD in
clinical settings.
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KEYWORDS
123I-ABC577, single-photon emission computed tomography, Alzheimer’s disease, β-amyloid,
imaging
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INTRODUCTION
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Alzheimer's disease (AD), the most common progressive neurodegenerative disorder
accounting for 60–70% of all dementia cases,1 is characterized by the presence of senile plaques
(containing β-amyloid (Aβ) peptides) and neurofibrillary tangles (NFTs; containing
hyperphosphorylated tau protein) in the brain. Among these pathological markers, Aβ plaques
are known as one of the earliest ones2-4 and are believed to play an important role in the
progression of AD.5–7 Therefore, non-invasive diagnostic imaging of these plaques using
methods such as positron emission tomography (PET) and single-photon emission computed
tomography (SPECT) could be useful for tracking amyloid pathology. Importantly, pre-
symptomatic changes in the brain of AD patients can be determined using certain promising PET
imaging agents such as [18F]florbetapir,8,9 [18F]flutemetamol,10–12 and [18F]florbetaben3,13,14
(Figure 1). Corresponding clinical studies show that PET can visualize Aβ present in the brain
with high sensitivity and specificity, thus being useful for discriminating non-AD-related
dementia. Based on these results, the aforementioned PET agents have been approved by the
United States Food and Drug Administration (FDA) and the European Medicines Agency
(EMA). In contrast, despite much effort devoted to their development, the corresponding SPECT
imaging agents are currently unavailable. Although the spatial resolution and quantitative
accuracy of SPECT are generally inferior to those of PET, the former modality is more available
and affordable than the latter, especially in developing countries, highlighting the need to
develop clinically useful SPECT imaging probes for Aβ plaques. Recently, we have developed
6-iodo-2-[2-(1H-1,2,3-triazole-1-yl)imidazo[1,2-a]pyridine (28, [123I]ABC577, Figure 1)15 as a
clinically useful SPECT imaging probe and reported the results from the initial clinical studies.
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Herein, we describe the design, synthesis, and preclinical evaluation of [123I]ABC577 and its
analogs.
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RESULTS AND DISCUSSION
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Design. Suitable Aβ imaging probes should exhibit high permeability through the blood-brain
barrier (BBB), high specific binding to Aβ, low non-specific binding, and rapid clearance from
normal brain regions. Based on these requirements, several Aβ imaging probes have been
designed from fluorescent dye compounds such as Congo Red (CR) and Thioflavin-T (ThT) and
evaluated for in vivo imaging of Aβ plaques in an AD brain. The first-generation [11C]PIB
amyloid probe (Figure 1B),16–18 frequently used as a benchmark for PET amyloid imaging
agents, was developed as a neutral ThT derivative with improved brain uptake, whereas [11C]SB-
13 (Figure 1B)19,20 was designed to reduce the molecular weight of CR. Since the use of these
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11C-labeled probes is limited by their short half-lives of 20.3 min, several F-labeled amyloid
PET probes have been developed, with three of them approved by the FDA as
radiopharmaceuticals. Since ThT has a lower molecular weight than CR, thus exhibiting better
BBB penetration, some research groups have worked on developing ThT-based SPECT imaging
agents (Figure 1A).21–25 Among these species, [123I]IMPY (Figure 1B)26–28 is the only SPECT
probe tested in humans. However, this compound exhibits unsatisfactory signal-to-noise ratios,
having certain undesirable characteristics such as high lipophilicity and in vivo instability.26,29
Since the increased lipophilicity of probes enhances their nonspecific uptake in the subcortical
white matter of the brain,30 18F-labeled amyloid imaging probes are commonly inferior to the less
lipophilic [11C]-PIB.31 Thus, since the lipophilicity of 123I-labeled SPECT imaging probes tends
to be much higher than that of 18F-labeled PET probes, the development of the former for
practical applications is correspondingly much more challenging.
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In the light of these findings, we attempted to develop novel radioiodinated imidazopyridine
derivatives as probes for SPECT amyloid imaging. The structures of 123I-labeled 2-phenyl- and
2-pyridyl-imidazo[1,2-a]pyridines that were designed and evaluated in this study are shown in
Figure 2. The IMPY-derived 2-phenyl-imidazo[1,2-a]pyridine scaffold was chosen as a core
structure for the developed probes due to its lower lipophilicity compared to these of 2-phenyl-
benzothiazole and stilbene scaffolds used in the approved agents (Figure 1). Most of the
previously reported probes feature electron-donating groups such as N-methylamino- or N,N-
dimethylaminophenyl on at least one end of their molecules. Therefore, we replaced these groups
with an alkoxy group or electron-rich five-membered heteroaromatic ring and compared the
lipophilicity of the resulting compounds with that of IMPY by calculating their cLogP (Table 1).
The replacement of a phenyl group by a pyridyl group is also reported to improve the
pharmacokinetics of Aβ imaging probes because it decreases their lipophilicity.32–34 Thus, based
on previously reported results, we synthesized and evaluated 2-pyridyl-imidazo[1,2-a]pyridine
derivatives in addition to their 2-phenyl analogs.
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Chemistry. The synthesis of 2-phenyl-imidazo[1,2-a]pyridine derivatives is outlined in Schemes
1 and 2. The core structure, 2-phenyl-imidazo[1,2-a]pyridine, was obtained by condensation of
the corresponding para-substituted phenacyl bromides with 2-amino-5-iodopyridine in
acetonitrile. The 2-phenyl-imidazo[1,2-a]pyridine derivatives bearing alkoxy groups were
synthesized from intermediate 2 (Scheme 1) prepared from the previously reported phenacyl
bromide 135 in 78% yield and subsequently converted to 3 (53% yield), 4 (95% yield), 7 (61%
yield), and 8 (78% yield) by O-alkylation with trimethylsilyldiazomethane in methanol/1,4-
dioxane or with 1-fluoro-2-(tosyloxy)ethane under basic conditions (K2CO3) in
dimethylformamide (DMF). The silyl group was cleaved by tetra-n-butylammonium fluoride
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(TBAF) to afford alcohols 9 and 10 in yields of 80 and 84%, respectively. Also, 1,2,3-triazole
derivative 14 (28% yield over three steps) was prepared by the reaction of 4-
azidophenacylbromide with 2-amino-5-iodopyridine followed by 1,3-dipolar addition to
trimethylsilylacetylene and trimethylsilyl group cleavage (Scheme 2).
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Next, 2-(3-pyridyl)imidazo[1,2-a]pyridine derivatives were prepared as shown in Scheme 3.
The methoxy derivative 18 was synthesized by cyclization of bromide 16 (prepared by
bromination of 5-acetyl-2-bromopyridine by CuBr2) with 2-amino-5-iodopyridine and a
subsequent reaction with sodium methoxide (20% yield over three steps). Five-membered
heteroaromatics (27, 28, 29, and 30) were also synthesized from 5-acetyl-2-bromopyridine. In
their syntheses, 5-acetyl-2-bromopyridine was reacted in DMF under basic conditions with the
required five-membered heteroaromatics to afford 5-acetylpyridine derivatives 19, 20, 21, and 22
in yields of 81, 30, 17, and 85%, respectively. These were subsequently converted to silyl enol
ethers during the reaction of trimethylsilyl bromide/triethylamine and brominated with N-
bromosuccinimide in CH2Cl2 to afford β-bromoketone derivatives 23, 24, 25, and 26 in yields of
72, 82, 45, and 97%, respectively. Then, the thus-obtained 23, 24, 25, and 26 were reacted with
2-amino-5-iodopyridine to obtain 27, 28, 29, and 30 in yields of 55, 46, 35, and 71%,
respectively.
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Tributylstannyl precursors 31–40 were prepared from the corresponding iodo compounds (3,
4, 9, 10, 14, 18, 27, 28, 29, and 30) through an iodo-to-tributylstannyl exchange reaction
catalyzed by Pd(Ph3P)4/triethylamine (Scheme 4) in yields of 41, 55, 73, 42, 61, 41, 36, 31, 22,
and 33%, respectively. Purities of key compounds were shown to be higher than 95%, as
determined by high-performance liquid chromatography (HPLC).
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Radiolabeling. Radioiodinated compounds [123I]3, [123I]4, [123I]9, [123I]10, [123I]14, [123I]18,
[123I]27, [123I]28, [123I]29, and [123I]30 were prepared from the corresponding tributylstannyl
precursors dissolved in CH3CN by an iodo-destannylation reaction using hydrogen
peroxide/hydrochloric acid as an oxidant under no-carrier-added conditions (Scheme 4). The
labeled compounds were purified by HPLC and solid-phase extraction, and their radiochemical
purities were determined to be above 95% by radio-thin layer chromatography (radio-TLC).
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In vitro Binding Assay to Aβ(1–42) Aggregates. The affinities of the imidazo[1,2-a]pyridine
derivatives to the abovementioned aggregates were evaluated based on the inhibition of
[123I]IMPY binding to synthetic human Aβ(1–42) fibrils and expressed as the half-maximal
inhibitory concentration (IC50) values (nM). The IC50 value of IMPY also measured using the
same system for comparison (Table 2). As for the compounds bearing an alkoxy group,
fluoroethoxy group-bearing 4 showed an affinity (20.0 ± 7.77 nM) comparable to that of IMPY
(24.8 ± 1.19 nM), and the other alkoxy-group-bearing compounds exhibited moderate affinities
(47.2–87.6 nM). The two compounds with a five-membered heterocyclic system registered
promising binding affinities with the highest affinity (IC50 = 14.7 ± 6.07 nM) observed for 28
featuring a 1H-1,2,3-triazole ring at the 2-position of the pyridine ring, followed by 27 bearing a
pyrazole ring (IC50 = 18.2 ± 1.11 nM). The increase in the A-binding affinities was also
observed with compounds bearing a similar ring system,36 suggesting that the incorporation of an
electron-donating group would be promising to the development of radiolabeled probes with
high binding affinities to Aβ instead of amino groups used in most of the previously reported
probes. On the other hand, the affinity of 2H-1,2,3-triazole derivative 29 was much lower than
that of its isomers, namely 1H-1,2,3-triazole derivative 28 and 1H-1,2,4-triazole derivative 30.
These findings indicated that the position of the heteroatoms in the five-membered ring
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significantly affects the affinity of the Aβ aggregates. The IC50 values of the imidazo[1,2-
a]pyridine derivatives (except for 29) were close to that of IMPY (24.8 ± 1.19 nM), which was
sufficient for the in vivo visualization of Aβ aggregates.
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Autoradiography of Postmortem AD Brain Sections. The binding of 123I-labeled probes to Aβ
plaques in the human brain was evaluated by in vitro autoradiography using tissue sections from
postmortem AD brains (Figure 3 and Table 3). The spots of radioactivity derived from 123I-
labeled probes in the cortex were confirmed by immunostaining the same brain sections with an
anti-Aβ antibody (Figure 4), and the white matter area in these sections was outlined by a
Klüver-Barrera staining (Figure 5). When AD brain sections were incubated with 123I-labeled
imidazo[1,2-a]pyridine derivatives, the obtained images revealed the visualization of the Aβ
plaques with minimal background noise. [123I]IMPY, which was used for comparison,
accumulated in the hippocampal region of the AD brain section, which was enriched with Aβ
plaques, in preference to the healthy control region (gray matter signal = 137 photostimulated
luminescence per square millimeter (PSL/mm2)). However, the non-specific accumulation of
[123I]IMPY in brain white matter was also high (Figure 3A), with the gray/white matter signal
ratio being as low as 1.2. Imidazo[1,2-a]pyridine derivatives bearing an alkoxy group exhibited a
lower amyloid affinity than [123I]IMPY (14.6–26.2 PSL/mm2), although their gray/white matter
signal ratio was slightly higher than that of [123I]IMPY (1.3–2.2). In contrast, imidazo[1,2-
a]pyridine derivatives with a five-membered heterocyclic ring system exhibited high affinities
(52.2–128 PSL/mm2), except for [123I]29 (9.82 PSL/mm2, IC50 = 451 ± 250 nM). All triazole
derivatives ([123I]14, [123I]28, [123I]29, and [123I]30) showed low non-specific binding to white
matter, with their notably high gray/white matter signal ratios (2.7–6.8) attributed to the lower
lipophilicity of these compounds due to the presence of a polar triazole moiety. Comparison of
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the 2-phenyl ([123I]3 and [123I]14) and 2-pyridyl ([123I]18 and [123I]28) derivatives showed that
the Aβ binding affinity was not affected by the replacement of a benzene ring with a pyridine
ring (26.2, 78.1, 23.0, and 128 PSL/mm2, respectively). However, the gray/white matter signal
ratio was notably improved by the abovementioned replacement (1.3, 2.7, 1.6, and 6.8,
respectively).
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In vivo Kinetics in Normal Rat Brain. The biodistribution of radiolabeled probes in normal
rats is widely used for selecting candidates appropriate from a biological point of view, i.e., those
featuring facile penetration through the intact BBB and rapid washout from the normal brain.
Table 4 summarizes the time−activity data in the brain of normal rats after intravenous
administration of 123I-labeled probes, showing that fluoroethoxy-group-bearing [123I]4,
hydroxyalkyl-group-bearing [123I]9 and [123I]10 showed low uptakes at 2 min (0.72, 0.97, and
1.05% injected dose per gram (ID/g), respectively) and thus exhibited low penetration ability
through the intact BBB. In contrast, the rest of the radiolabeled probes showed good uptake into
the brain (~1.09–2.09% ID/g brain). In general, optimal BBB penetration is observed for LogP =
1.5–2.7, with mean = 2.1.37 Since the LogPoctanol/water values of these ligands (Table 1) indicated
facile uptake into normal rat brains, the 123I-labeled probes that initially penetrated the rat brain
were rapidly washed out. The brain2 min/brain60 min ratio is commonly used as an important index
to select probes with appropriate in vivo kinetics. The corresponding values for the 123I-labeled
probes are shown in Table 5. It was found that all evaluated probes exhibited higher ratios than
[123I]IMPY (18.8–48.5 vs. 18.2). Moreover, the obtained results indicated that [123I]28 was best
suited for use as an Aβ imaging probe among the compounds tested in this study because it
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exhibited high initial uptake into the brain followed by quick washout (brain2 min/brain60 min
=
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41.0). The favorable in vivo kinetics of the abovementioned species may be attributable to their
reduced lipophilicity achieved by introducing a 1H-1,2,3-triazole moiety.
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In vivo Stability Studies in Normal Rats. Since [123I]14, [123I]28, and [123I]30 showed suitable
characteristics for Aβ imaging, we compared the in vivo stability of these probes in rat plasma
with that of [123I]IMPY. The results showed that the former exhibited higher stability than the
latter (Table 6). Thin layer chromatography analyses of 28 and 30 showed a few unidentified
metabolites with post-injection time. While [123I]IMPY also registered a few metabolites with
time, a metabolite in this case was generated as early as 2 min post-injection at an Rf value close
to that of [123I]IMPY, suggesting demethylation of the N-dimethyl group (Supplemental Figure
S1), which may account for its insufficient signal-to-noise ratios observed in the clinical
studies.26,29 These results also suggested that the compounds without N-alkyl groups such as
[123I]14, [123I]28, and [123I]30 would be more appropriate for in vivo imaging of Aβ.
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The autoradiography results revealed that 1H-1,2,3-triazole derivatives [123I]14 and [123I]28
and 1H-1,2,4-triazole derivative [123I]30 displayed excellent binding affinities to Aβ plaques in
the hippocampal region of an AD brain, exhibiting a higher selectivity than [123I]IMPY and
higher in vivo stability in normal rats. Furthermore, all these probes displayed good kinetics with
brain2 min/brain60 min ratios of 18.8–48.5. Thus, the obtained results suggested that the newly
prepared 2-phenyl- and 2-pyridyl-imidazo[1,2-a]pyridine derivatives containing an alkoxy group
or five-membered aromatic heteroring system are well suited for in vivo imaging of Aβ plaques.
In particular, 1H-1,2,3-triazole 2-pyridyl-imidazo[1,2-a]pyridine derivative [123I]28
([123I]ABC577) exhibited the highest gray/white matter selectivity according to the in vitro
autoradiography analysis, as well as good kinetics in rat brain, and moderate in vivo stability in
rats.
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CONCLUSION
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In this study, 2-phenyl- and 2-pyridyl-imidazo[1,2-a]pyridine derivatives containing an
alkoxy group or a five-membered aromatic heteroring system were successfully synthesized as
novel SPECT imaging probes for Aβ plaques. An in vitro binding assay to Aβ(1–42) aggregates
confirmed that these species possess high binding affinities (IC50) sufficient for in vivo
applications. A high performance was observed for some of the ligands containing a five-
membered heterocyclic system. The autoradiography results revealed that 1H-1,2,3-triazole
derivatives [123I]14 and [123I]28, and 1H-1,2,4-triazole derivative [123I]30 displayed excellent
binding affinities to Aβ plaques in the hippocampal region of an AD brain, exhibiting a higher
selectivity than [123I]IMPY and higher in vivo stability in normal rats. Furthermore, all these
probes displayed good kinetics, with brain2 min/brain60 min ratios of 18.8–48.5. These findings
suggested that the newly prepared 2-phenyl- and 2-pyridyl-imidazo[1,2-a]pyridine derivatives
containing an alkoxy group or five-membered aromatic heteroring system would be useful for
the in vivo imaging of Aβ plaques. In particular, 1H-1,2,3-triazole 2-pyridyl-imidazo[1,2-
a]pyridine derivative [123I]28 ([123I]ABC577) exhibited the highest gray/white matter selectivity
according to in vitro autoradiography analysis, good kinetics in rat brain, and moderate in vivo
stability in rats. In our previous report,15 [123I]ABC577 demonstrated high affinity to AD
homogenate, low off-target binding, and the possibility to be a useful SPECT imaging tool to
identify Aβ in the human brain in preliminary clinical studies．The current work could pave the
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way to the development of further SPECT imaging agents for AD brain imaging, with further
testing and clinical studies expected to be reported in due course.
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METHODS
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General Remarks. All utilized reagents were commercial products that were used without
further purification unless otherwise indicated. 1H NMR and 13C NMR spectra were recorded on
a JEOL 500-MHz or Bruker 500-MHz NMR spectrometer in CDCl3 or dimethyl sulfoxide
(DMSO)-d6, utilizing tetramethylsilane (TMS) as an internal standard. The coupling constants
were reported in Hertz (Hz) and the multiplicity was denoted as s (singlet), d (doublet), t (triplet),
q (quartet), quin (quintet), and m (multiplet). Mass spectra were acquired using a Waters Xevo
QTofMS (Waters Corporation, Milford, MA) instrument. Radiochemical purity was determined
by radio-TLC (Rita Star, Raytest, Straubenhardt, Germany) on silica gel 60 F254 plates using
EtOAc/CH3OH/Et2NH (100:4:1, v/v/v) as a mobile phase. HPLC was performed using an
Alliance 2695 system (Waters Corporation, Milford, MA) with Waters 2487 Detector (λ = 254
nm) using a phenyl column (Waters Corporation Phenyl, 4.6 mm × 150 mm, 5 μm) and
acetonitrile/H2O containing 0.1 vol% trifluoroacetic acid as the mobile phase at a flow rate of 1.0
mL/min. All key compounds showed >95% purity when tested with this method. Wistar rats
were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and were housed under conditions of
controlled temperature (18–28 °C) and lighting (12:12 h light–dark cycle) with free access to
food and water. The protocols for all animal experiments were approved by the committee on
animal welfare at Nihon Medi-Physics Co., Ltd. All reagents and solvents were obtained
commercially from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), and Sigma-Aldrich Co.
LLC. (St. Louis, MO).
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Chemistry. 2-(4-Hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (2). 2-Amino-5-iodopyridine
(449 mg, 2.00 mmol) was added to a solution of 1 (441 mg, 2.00 mmol) in CH3CN (15 mL), and
the reaction mixture was heated under reflux for 5 h. After the reaction was complete, the
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mixture was allowed to cool down to room temperature and filtered. The precipitate was washed
with CH3CN (10 mL), dried under reduced pressure, suspended in a mixture of water (10.0 mL)
and methanol (10.0 mL), and treated with saturated aqueous NaHCO3 (~10 mL), followed by a
5-min sonication. The resulting mixture was filtered, and the precipitate was washed with water
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(10 mL) and dried under reduced pressure to afford 2 (526 mg, 78.0%). H NMR (500 MHz,
DMSO-d6):  8.86–8.84 (m, 1H), 8.14 (s, 1H), 7.78–7.74 (m, 2H), 7.40–7.35 (m, 2H), 6.86–6.82
(m, 2H); 13C NMR (125 MHz, DMSO-d6):  158.1, 145.9, 143.9, 132.5, 131.7, 127.7, 125.0,
118.1, 116.1, 108.0, 75.9.
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2-(4-Methoxyphenyl)-6-iodoimidazo[1,2-a]pyridine
(3).
A
solution
of
trimethylsilyldiazomethane in hexane (2 M, 1.2 mL, 2.4 mmol) was added to a solution of 2 (400
mg, 1.20 mmol) in a mixture of methanol (30 mL) and 1,4-dioxane (50 mL). The reaction
mixture was stirred for 24.5 h at room temperature and subsequently concentrated under reduced
pressure. The residue was recrystallized from EtOAc to afford 3 (223 mg, 53.0%). 1H NMR (500
MHz, CDCl3):  8.37–8.34 (m, 1H), 7.88–7.84 (m, 2H), 7.72 (s, 1H), 7.40 (d, 1H, J = 9.4 Hz),
7.31 (dd, 1H, J = 9.4, 1.6 Hz), 6.99–6.95 (m, 2H), 3.86 (s, 3H); 13C NMR (125 MHz, DMSO-d6):
 159.9, 146.3, 144.2, 132.3, 130.3, 127.4, 125.9, 118.3, 114.2, 106.88, 74.8, 55.3; Accurate
mass (ES+) m/z 350.9998 [M+H]+ (calcd. for C14H12IN2O: 350.9994).
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2-[4-(2-Fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (4). K2CO3 (5.50 g, 41.4 mmol)
and 1-fluoro-2-(tosyloxy)ethane (4.52 g, 20.7 mmol) were added to a solution of 2 (4.65 g, 13.8
mmol) in DMF (70 mL) and the reaction mixture was heated to 90 °C for 1.5 h. The reaction was
quenched by the addition of saturated aqueous NaHCO3, and the mixture was extracted with
EtOAc (100 mL × 2). The extract was washed with water and brine, dried over anhydrous
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Na2SO4, and concentrated to dryness. The crude product was purified by flash silica gel column
1
chromatography (hexane:EtOAc = 1:2, v/v) to afford 4 (5.00 g, 94.9%). H NMR (500 MHz,
CDCl3):  8.36 (s, 1H), 7.86 (d, 2H, J = 8.2 Hz), 7.71 (s, 1H), 7.39 (d, 1H, J = 8.7 Hz), 7.32–7.30
(m, 1H), 6.99 (d, 2H, J = 8.25 Hz), 4.84–4.71 (dd, 2H, JHH = 4.1, 2JHF = 47.2 Hz), 4.30–4.22 (dd,
2H, JHH = 4.1, 3JHF = 27.5 Hz); 13C NMR (125 MHz, CDCl3):  158.6, 146.1, 144.2, 132.4, 130.3,
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127.4, 126.5, 118.3, 114.9, 107.0, 81.9 (d, JCF = 170.8 Hz), 74.8, 67.2 (d, JCF = 20.6 Hz); F
2
3
NMR (470 MHz, CDCl3):  –223.7 (dd, JHF = 47.4 Hz, JHF = 27.7 Hz); Accurate mass (ES+)
m/z 383.0042 [M+H]+ (calcd. for C15H13FIN2O: 383.0057).
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2-[4-(2-t-Butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (7). Compound 5
(51.8 g, 142 mmol) was added to a solution of 2 (40.0 g, 119 mmol) and K2CO3 (49.3 g, 357
mmol) in DMF (320 mL). The reaction mixture was stirred at 86 °C for 3 h, quenched with the
addition of brine, and extracted with EtOAc (500 mL × 2). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography (heptane:EtOAc = 1:1, v/v) to afford 7 (53.3 g,
1
60.7%). H NMR (500 MHz, CDCl3):  8.29 (s, 1H), 7.82 (d, 2H, J = 8.2 Hz), 7.72 (d, 4H, J =
6.0 Hz), 7.44–7.37 (m, 8H), 7.27 (dd, 1H, J = 9.4, 1.6 Hz), 6.93 (d, 2H, J = 6.9 Hz), 4.12 (t, 2H,
J = 5.1 Hz), 4.01 (t, 2H, J = 5.1 Hz), 1.07 (s, 9H).
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2-[4-(3-t-Butyldiphenysiloxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (8). Compound 6
(52.2 g, 138 mmol) was added to a solution of 2 (31.0 g, 92.2 mmol) and K2CO3 (38.2 g, 277
mmol) in DMF (260 mL). The reaction mixture was stirred at 88 °C for 3 h, quenched with the
addition of brine, and extracted with EtOAc (500 mL × 2). The combined organic layers were
dried over anhydrous MgSO4 and concentrated under reduced pressure. The crude product was
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purified by silica gel column chromatography (CHCl3:CH3OH = 50:1, v/v) to afford 8 (45.5 g,
78.0%). 1H NMR (500 MHz, CDCl3):  8.36 (d, 1H, J = 0.8 Hz), 7.85 (d, 2H, J = 9.2 Hz), 7.72
(s, 1H), 7.65 (dd, 4H, J = 6.4, 1.2 Hz), 7.42–7.29 (m, 8H), 6.95 (d, 2H, J = 9.2 Hz), 4.16 (t, 2H, J
= 6.0 Hz), 3.87 (t, 2H, J = 6.0 Hz), 2.04 (tt, 2H J = 6.0, 6.0 Hz), 1.05 (s, 9H).
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2-[4-(2-Hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (9). A 1.0 M TBAF solution in
tetrahydrofuran (THF, 101 mL) was added to a solution of 7 (52.0 g, 84.0 mmol) in THF (150
mL). The reaction mixture was stirred at room temperature for 2 h, followed by quenching with
saturated aqueous NH4Cl and the addition of CH3CN (50 mL). The obtained precipitate was
7
8
1
9
filtered and washed with water (200 mL) and CH3CN (200 mL) to afford 9 (25.5 g, 79.9%). H
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NMR (500 MHz, DMSO-d6):  8.86 (s, 1H), 8.19 (s, 1H), 7.86 (d, 2H, J = 11.2 Hz), 7.39 (s, 2H),
7.00 (d, 2H, J = 11.2 Hz), 4.81 (t, 1H, J = 5.5 Hz), 4.04 (t, 2H, J = 5.0 Hz), 3.74 (dt, 2H, J = 5.5,
5.0 Hz); 13C NMR (125 MHz, DMSO-d6):  158.9, 143.0, 142.4, 133.5, 131.5, 127.1, 124.4,
116.7, 114.8, 108.1, 76.7, 69.5, 59.4; Accurate mass (ES+) m/z 381.0094 [M+H]+ (calcd. for
C15H14IN2O: 381.0100); Anal. C, 47.29; H, 3.45; N, 7.21 (calcd. for C15H13IN2O: C, 47.39; H,
3.45; N, 7.37).
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2-[4-(3-Hydroxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (10). A 1.0 M TBAF solution
in THF (85.2 mL) was added to a solution of 8 (44.9 g, 71.0 mmol) in THF (142 mL). The
reaction mixture was stirred at room temperature for 1.5 h, quenched with saturated aqueous
NH4Cl, and diluted with water (142 mL) and CH3CN (142 mL). The precipitate was filtered and
washed with water (200 mL) and CH3CN (200 mL) to afford 10 (23.5 g, 83.9%). 1H NMR (500
MHz, DMSO-d6):  8.86 (s, 1H), 8.19 (s, 1H), 7.85 (d, 2H, J = 8.7 Hz), 7.39 (s, 2H), 6.99 (d, 2H,
J = 8.7 Hz), 4.49 (br s, 1H), 4.08 (t, 2H, J = 6.4 Hz), 3.57 (dt, 2H, J = 6.4 Hz), 1.88 (tt, 2H, J =
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6.4, 6.4 Hz); 13C NMR (125 MHz, DMSO-d6):  158.7, 144.6, 143.2, 132.1, 131.2, 127.0, 125.7,
117.6, 114.6, 107.8, 75.7, 64.6, 57.3, 32.1; Accurate mass (ES+) m/z 395.0252 [M+H]+ (calcd.
for C16H16IN2O2: 395.0257); Anal. C, 48.54; H, 3.83; N, 7.00 (calcd. for C16H15IN2O: C, 48.75;
H, 3.84; N, 7.11).
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2-(Azidophenyl)-6-imidazo[1,2-a]pyridine (12). A solution of 11 (218 mg, 0.909 mmol) and 2-
amino-5-iodopyridine (200 mg, 0.909 mmol) in CH3CN (1.0 mL) was heated to 80 °C for 3 h.
The mixture was allowed to cool down to room temperature and filtered, and the obtained
precipitate was washed with CH3CN (20 mL) and dried under reduced pressure. The crude
product was suspended in a mixture of water (3.0 mL) and methanol (3.0 mL), and treated with
saturated aqueous NaHCO3 (~4 mL), followed by a 5-min sonication. The obtained precipitate
was filtered, washed with water (10 mL), and dried under reduced pressure to afford 12 (214 mg,
65.2%). 1H NMR (500 MHz, CDCl3):  8.89 (s, 1H), 8.31 (s, 1H), 7.99 (d, 2H, J = 8.7 Hz), 7.42
(s, 1H), 7.42 (s, 1H), 7.19 (d, 2H, J = 8.7 Hz).
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6-Iodo-2-[4-(1H-1,2,3-triazol-1-yl)phenyl]imidazo[1,2-a]pyridine (14).
Trimethylsilylacetylene (0.164 mL, 1.18 mmol) and copper(II) sulfate pentahydrate (29.6 mg,
0.118 mmol) were added to a solution of 12 (214 mg, 0.593 mmol) in DMF (3.0 mL), and the
reaction mixture was stirred at 80 °C for 3 h. After the reaction was complete, the mixture was
allowed to cool down to room temperature and diluted with water (20 mL). The precipitate was
filtered, washed with water (10 mL), and dried under reduced pressure to obtain crude 13 (137
mg). A 1.0 M TBAF solution in THF (0.3 mL) was added to a suspension of crude 13 (137 mg)
in THF (3.0 mL), and the mixture was refluxed for 4 h. The solution was then allowed to cool
down to room temperature, and the obtained precipitate was filtered, successively washed with
THF (5.0 mL) and diethyl ether (5.0 mL), and dried under reduced pressure to afford 14 (97.8
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mg, 42.5%). H NMR (500 MHz, DMSO-d6):  8.92 (s, 1H), 8.84 (d, 1H, J = 0.9 Hz), 8.41 (s,
1H), 8.15 (d, 2H, J = 8.7 Hz), 7.98 (d, 2H, J = 8.7 Hz), 7.96 (d, 1H, J = 0.9 Hz), 7.45 (s, 1H); 13C
NMR (125 MHz, DMSO-d6):  144.2, 144.1, 136.7, 134.9, 134.3, 133.3, 132.1, 127.5, 123.6,
121.1, 118.6, 110.1, 76.6; Accurate mass (ES+) m/z 388.0060 [M+H]+ (calcd. for C15H11IN5:
388.0059). Anal. C, 46.46; H, 2.73; N, 17.83 (calcd for C15H10IN5: C, 46.53; H, 2.60; N, 18.09).
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2-(2-Bromopyridin-5-yl)-6-iodoimidazo[1,2-a]pyridine (17). CuBr2 (3.57 g, 16.0 mmol) was
added to a solution of 15 (1.50 g, 7.61 mmol) in EtOAc (30 mL), and the mixture was refluxed
for 5 h, allowed to cool down to room temperature, and concentrated under reduced pressure.
The obtained residue was purified by flash silica gel column chromatography (hexane:EtOAc =
7:1, v/v) to obtain crude 16 (2.23 g), which was used to prepare 17 following the protocol
described above for 2 (1.26 g, 52.6%). 1H NMR (500 MHz, DMSO-d6):  8.91–8.90 (d, 1H, J =
2.3 Hz), 8.41 (s, 1H), 8.20 (dd, 1H, J = 8.3, 2.3 Hz), 7.67 (d, 1H, J = 8.3 Hz), 7.42 (s, 2H).
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2-(2-Methoxypyridin-5-yl)-6-iodoimidazo[1,2-a]pyridine (18). Sodium methoxide (171 mg,
3.17 mmol) was added to a solution of 17 (200 mg, 0.634 mmol) in CH3OH (3.0 mL) and DMSO
(3.0 mL) at 0 °C. The reaction mixture was stirred for 3 h, quenched with saturated aqueous
NH4Cl, and extracted with EtOAc (30 mL × 2). The combined organic layers were washed with
water and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography (hexane:EtOAc = 1:1, v/v) to
afford 18 (86.0 mg, 38.6%). 1H NMR (500 MHz, DMSO-d6):  8.89 (s, 1H), 8.73 (s, 1H), 8.27 (s,
1H), 8.20 (d, 1H, J = 8.5 Hz), 7.42 (s, 2H), 6.89 (d, 1H, J = 8.5 Hz), 3.89 (s, 3H); 13C NMR (125
MHz, DMSO-d6):  163.9, 144.8, 144.1, 142.8, 137.1, 133.1, 123.7, 118.4, 111.2, 109.0, 76.4,
55.4, 53.9; Accurate mass (ES+) m/z 351.9938 [M+H]+ (calcd. for C13H11IN3O: 351.9947).
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1-(2-(Pyrazol-1-yl)pyridin-5-yl)ethane-1-one (19). Compound 15 (400 mg, 1.99 mmol) and
K2CO3 (828 mg, 5.99 mmol) were added to a solution of pyrazole (408 mg, 5.99 mmol) in DMF
(10 mL), and the reaction mixture was heated to 100 °C for 5 h. After the reaction was complete,
the reaction mixture was allowed to cool down to room temperature, quenched with saturated
aqueous NH4Cl, and extracted with CH2Cl2 (50 mL × 2). The combined organic fractions were
washed with water and brine, dried over anhydrous MgSO4, and concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography (CH2Cl2:EtOAc =
50:1, v/v) to afford 19 (304 mg, 81.4%). 1H NMR (500 MHz, CDCl3):  8.98 (d, 1H, J = 2.3 Hz),
8.63 (d, 1H, J = 2.7 Hz), 8.36 (dd, 1H, J = 8.5, 2.3 Hz), 8.08 (d, 1H, J = 8.5 Hz), 7.79 (d, 1H, J =
1.4 Hz), 6.51 (dd, 1H, J = 2.7, 1.4 Hz), 2.65 (s, 3H).
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1-(2-(1H-1,2,3-Triazol-1-yl)pyridin-5-yl)ethan-1-one (20). Compound 20 (57.2 mg, 30.4%)
was prepared as described above for 19. 1H NMR (500 MHz, CDCl3):  9.05 (d, 1H, J = 2.3 Hz),
8.65 (d, 1H, J = 1.1 Hz), 8.46 (dd, 1H, J = 8.7, 2.3 Hz), 8.34 (d, 1H, J = 8.7 Hz), 7.86 (d, 1H, J =
1.1 Hz), 2.68 (s, 3H).
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17
1-(2-(2H-1,2,3-Triazol-2-yl)pyridin-5-yl)ethan-1-one (21). Compound 21 (31.9 mg, 17.0%)
was prepared as described above for 19. 1H NMR (500 MHz, CDCl3):  9.13 (d, 1H, J = 2.3 Hz),
8.45 (dd, 1H, J = 8.6, 2.3 Hz), 8.21 (d, 1H, J = 8.6 Hz), 7.97 (s, 2H), 2.69 (s, 3H).
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19
20
1-(2-(1H-1,2,4-Triazol-1-yl)pyridin-5-yl)ethan-1-one (22). Compound 22 (480 mg, 85.0%)
was prepared as described above for 19. 1H NMR (500 MHz, CDCl3):  9.25 (s, 1H), 9.02 (d, 1H,
J = 2.0 Hz), 8.44 (dd, 1H, J = 8.5, 2.0 Hz), 8.14 (s, 1H), 8.02 (d, 1H, J = 8.5 Hz), 2.68 (s, 3H).
21
22
1-(2-(Pyrazol-1-yl)pyridin-5-yl)-2-bromoethan-1-one (23). Triethylamine (230 μL) and
bromotrimethylsilane (140 μL, 1.07 mmol) were added to a solution of 19 (100 mg, 0.534 mmol)
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in CH2Cl2 (4.0 mL) at 0 °C. The reaction mixture was stirred overnight at room temperature,
quenched with water, and extracted with CH2Cl2. (20 mL × 2). The combined organic layers
were washed with water and brine, dried over anhydrous MgSO4, and concentrated under
reduced pressure. The residue was dissolved in THF (4.0 mL) and treated with N-
bromosuccinimide (94.3 mg, 0.530 mmol) at 0 °C, and the obtained mixture was stirred at room
temperature for 1 h. After the reaction was complete, the solvent was evaporated, and the residue
was purified by silica gel column chromatography (CH2Cl2:EtOAc = 50:1, v/v) to afford 23 (100
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mg, 71.7%). H NMR (500 MHz, CDCl3):  9.02 (d, 1H, J = 2.3 Hz), 8.62 (d, 1H, J = 2.3 Hz),
9
8.39 (dd, 1H, J = 8.7, 1.9 Hz), 8.10 (d, 1H, J = 8.7 Hz), 7.79 (s, 1H), 6.52 (t, 1H, J = 1.9 Hz),
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4.41 (s, 2H).
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1-(2-(1H-1,2,3-Triazol-1-yl)pyridin-5-yl)-2-bromoethan-1-one (24). Compound 24 (66.4 mg,
81.9%) was prepared similarly to compound 23. 1H NMR (500 MHz, CDCl3):  9.12 (d, 1H, J =
2.2 Hz), 8.67 (d, 1H, J = 1.1 Hz), 8.51 (dd, 1H, J = 8.7, 2.2 Hz), 8.38 (d, 1H, J = 8.7 Hz), 7.87 (d,
1H, J = 1.1 Hz), 4.44 (s, 2H).
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1-(2-(2H-1,2,3-Triazol-2-yl)pyridin-5-yl)-2-bromoethan-1-one (25). Compound 25 (20.2 mg,
44.7%) was prepared similarly to compound 23. 1H NMR (500 MHz, CDCl3):  9.18 (d, 1H, J =
2.1 Hz), 8.49 (dd, 1H, J = 8.5, 2.1 Hz), 8.24 (d, 1H, J = 8.5 Hz), 7.98 (s, 2H), 4.45 (s, 2H).
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20
21
1-(2-(1H-1,2,4-Triazol-1-yl)pyridin-5-yl)2-bromoethan-1-one (26). Compound 26 (657 mg,
1
96.5%) was prepared similarly to compound 23. H NMR (500 MHz, CDCl3):  9.27 (br s, 1H),
9.07 (d, 1H, J = 2.2 Hz), 8.48 (dd, 1H, J = 8.5, 2.2 Hz), 8.15 (s, 1H), 8.06 (d, 1H, J = 8.5 Hz),
4.43 (s, 2H).
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6-Iodo-2-(2-pyrazol-1-yl)pyridin-5-yl])imidazo[1,2-a]pyridine (27). 2-Amino-5-iodopyridine
(83.6 mg, 0.380 mmol) was added to a solution of 23 (100 mg, 0.376 mmol) in CH3CN (3.8 mL),
and the reaction mixture was refluxed for 4 h. After the reaction was complete, the mixture was
allowed to cool down to room temperature and filtered. The precipitate was washed with CH3CN
(5.0 mL) and dried under reduced pressure. The crude crystalline solid was suspended in a
mixture of water (50 mL) and CH3OH (50 mL), treated with saturated aqueous NaHCO3 (~50
mL), and sonicated for 5 min. The obtained precipitate was filtered, washed with water (10 mL),
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and dried under reduced pressure to afford 27 (80.2 mg, 54.5%). H NMR (500 MHz, DMSO-
9
d6):  9.04 (d, 1H, J = 2.3 Hz), 8.97 (s, 1H), 8.66 (d, 1H, J = 2.3 Hz), 8.50 (dd, 1H, J = 8.3, 2.3
Hz), 8.46 (s, 1H), 8.01 (d, 1H, J = 8.3 Hz), 7.86–7.85 (m, 1H), 7.48 (s, 2H), 6.61–6.60 (m, 1H);
13C NMR (125 MHz, DMSO-d6):  149.7, 144.9, 143.1, 141.7, 140.7, 135.7, 132.3, 131.0, 127.0,
126.4, 117.4, 111.5, 109.0, 107.7, 75.7, Accurate mass (ES+) m/z 388.0064 [M+H]+ (calcd. for
C15H11IN5: 388.0059).
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6-Iodo-2-[2-(1H-1,2,3-triazol)-1-yl]pyridin-5-yl]imidazo[1,2-a]pyridine (28). Compound 28
1
(44.0 mg, 45.5%) was prepared similarly to compound 27. H NMR (500 MHz, DMSO-d6): 
9.11 (d, 1H, J = 2.3 Hz), 8.92 (br s, 1H), 8.82 (d, 1H, J = 0.9 Hz), 8.57 (dd, 1H, J = 8.5, 2.3 Hz),
13
8.47 (s, 1H), 8.17 (d, 1H, J = 8.5 Hz), 7.95 (d, 1H, J = 0.9 Hz), 7.45 (br s, 2H); C NMR (125
MHz, DMSO-d6):  148.3, 146.4, 144.3, 141.2, 137.1, 134.9, 133.7, 132.2, 130.3, 122.5, 118.6,
114.5, 110.7, 77.0, Accurate mass (ES+) m/z 389.0007 [M+H]+ (calcd. for C14H10IN6: 389.0012);
Anal. C, 43.32; H, 2.34; N, 21.44 (calcd. for C14H9IN6: C, 43.32; H, 2.34; N, 21.65).
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6-Iodo-2-[2-(2H-1,2,3-triazol-1-yl)pyridin-5-yl]imidazo[1,2-a]pyridine (29). Compound 29
1
(10.4 mg, 35.3%) was prepared similarly to compound 27. H NMR (500 MHz, DMSO-d6): 
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9.13 (d, 1H, J = 2.3 Hz), 8.96 (br s, 1H), 8.56 (dd, 1H, J = 8.7, 2.3 Hz), 8.50 (s, 1H), 8.17 (s, 2H),
8.10 (d, 1H, J = 0.9 Hz), 7.48 (br s, 2H); 13C NMR (125 MHz, DMSO-d6):  149.1, 145.3, 143.1,
140.3, 136.7, 135.7, 132.5, 131.1, 128.5, 117.4, 113.6, 109.5, 76.0, Accurate mass (ES+) m/z
389.0004 [M+H]+ (calcd. for C14H10IN6: 389.0012).
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6-Iodo-2-[2-(1H-1,2,4-triazol-1-yl)pyridin-5-yl]imidazo[1,2-a]pyridine (30). Compound 30
1
(675 mg, 70.7%) was prepared similarly to compound 27. H NMR (500 MHz, DMSO-d6): 
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9.38 (br s, 1H), 9.10 (d, 1H, J = 2.1 Hz), 8.96 (br s, 1H), 8.57 (dd, 1H, J = 8.5, 2.1 Hz), 8.49 (s,
13
8
1H), 8.31 (s, 1H), 7.96 (d, 1H, J = 8.5 Hz), 7.49 (br s, 2H); C NMR (125 MHz, DMSO-d6): 
9
153.5, 148.7, 146.2, 144.3, 142.4, 141.4, 137.2, 133.6, 132.2, 129.7, 118.5, 113.6, 110.5, 76.8,
10
Accurate mass (ES+) m/z 389.0008 [M+H]+ (calcd. for C14H10IN6: 389.0012).
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2-(4’-Methoxyphenyl)-6-tributylstannylimidazo[1,2-a]pyridine (31). Triethylamine (2.0 mL),
bis(tributyltin) (190 μL, 0.375 mmol), and tetrakis(triphenylphosphine)palladium (20.0 mg,
catalytic amount) were added to a solution of 3 (87.6 mg, 0.250 mmol) in dioxane (10.0 mL).
The reaction mixture was heated to 90 °C for 21.5 h and subsequently concentrated. The
obtained residue was purified by flash silica gel column chromatography (hexane:EtOAc = 3:1,
v/v) to afford 31 (53.0 mg, 41.2%). 1H NMR (500 MHz, CDCl3):  8.00–7.94 (m, 1H), 7.89 (d,
2H, J = 8.7 Hz), 7.74 (s, 1H), 7.58 (d, 1H, J = 8.7 Hz), 7.14 (d, 1H, J = 8.7 Hz), 6.97 (d, 2H, J =
8.7 Hz), 3.86 (s, 3H), 1.59–1.48 (m, 6H), 1.39–1.32 (m, 6H), 1.18–1.05 (m, 6H), 0.90 (t, 9H, J =
7.3 Hz); 13C NMR (125 MHz, CDCl3):  159.5, 145.6, 145.0, 131.0, 130.0, 127.2, 126.7, 121.7,
116.8, 114.1, 106.2, 55.2, 28.9, 27.2, 13.6, 9.7, Accurate mass (ES+) m/z 515.2076 [M+H]+
(calcd. for C26H39N2OSn: 515.2084).
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2-[4’-(2-Fluoroethoxy)phenyl]-6-tributylstannylimidazo[1,2-a]pyridine (32). Compound 32
(298 mg, 55.4%) was prepared similarly to compound 31. 1H NMR (500 MHz, CDCl3):  7.99–
7.93 (m, 1H), 7.89 (d, 2H, J = 8.5 Hz), 7.74 (s, 1H), 7.58 (d, 1H, J = 8.7 Hz), 7.14 (d, 1H, J = 8.7
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Hz), 6.99 (d, 2H, J = 8.7 Hz), 4.76 (dt, 2H, JHF = 47.2 Hz, JHH = 4.1 Hz), 4.25 (dt, 2H, JHF
=
28.0 Hz, JHH = 4.1 Hz), 1.64–1.47 (m, 6H), 1.38–1.31 (m, 6H), 1.18–1.04 (m, 6H), 0.90 (t, 9H, J
= 7.3 Hz); 13C NMR (125 MHz, CDCl3):  158.4, 145.7, 144.9, 131.3, 130.1, 127.4, 122.0, 117.0,
114.9, 106.5, 82.0 (d, 2JCF = 170.8 Hz), 67.2 (d, 3JCF = 21.1 Hz), 29.1, 27.4, 13.7, 9.9; Accurate
mass (ES+) m/z 547.2152 [M+H]+ (calcd. for C27H40N2OFSn: 547.2147).
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2-[4’-(2-Hydroxyethoxy)phenyl]-6-tributylstannylimidazo[1,2-a]pyridine (33). Compound
1
33 (73.0 mg, 73.1%) was prepared similarly to compound 31. H NMR (500 MHz, CDCl3): 
7.99–7.93 (m, 1H), 7.87 (d, 1H, J = 8.3 Hz), 7.74 (s, 1H), 7.57 (d, 1H, J = 8.7 Hz), 7.13 (d, 1H, J
= 8.7 Hz), 6.96 (d, 1H, J = 8.3 Hz), 4.11 (t, 2H, J = 4.6 Hz,), 3.97 (t, 2H, J = 4.6 Hz), 1.63–1.47
13
(m, 6H), 1.38–1.31 (m, 6H), 1.17–1.04 (m, 6H), 0.90 (t, 9H, J = 7.1 Hz); C NMR (125 MHz,
CDCl3):  158.6, 145.7, 145.1, 131.2, 130.0, 127.4, 127.3, 121.9, 116.9, 114.8, 106.4, 69.3, 61.5,
29.1, 27.4, 13.7, 9.9; Accurate mass (ES+) m/z 545.2192 [M+H]+ (calcd. for C27H41N2O2Sn:
545.2190); Anal. C, 59.48; H, 7.26; N, 4.98 (calcd. for C27H40N2O2Sn: C, 59.69; H, 7.42; N,
5.16).
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2-[4’-(2-Hydroxypropoxy)phenyl]-6-tributylstannylimidazo[1,2-a]pyridine (34). Compound
1
34 (68.0 mg, 42.2%) was prepared similarly to compound 31. H NMR (500 MHz, CDCl3): 
7.99–7.93 (m, 1H), 7.87 (d, 2H, J = 8.9 Hz), 7.74 (s, 1H), 7.57 (d, 1H, J = 8.9 Hz), 7.13 (d, 1H, J
= 8.9 Hz), 6.97 (d, 2H, J = 8.9 Hz), 4.17 (t, 2H, J = 6.0 Hz), 3.89 (t, 2H, J = 6.0 Hz), 2.07 (tt, 2H,
J = 6.0, 6.0 Hz), 1.60–1.52 (m, 6H), 1.38–1.31 (m, 6H), 1.17–1.04 (m, 6H), 0.90 (t, 9H, J = 7.3
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Hz); 13C NMR (125 MHz, CDCl3):  158.8, 145.7, 145.1, 131.2, 130.1, 127.4, 127.0, 121.9,
117.0, 114.8, 106.4, 65.9, 60.5, 32.1, 29.1, 27.4, 13.7, 9.9; Accurate mass (ES+) m/z 559.2358
[M+H]+ (calcd. for C28H43N2O2Sn: 559.2347); Anal. C, 60.08; H, 7.47; N, 4.87 (calcd. for
C28H42N2O2Sn: C, 60.34; H, 7.60; N, 5.03).
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2-[4-(1H-1,2,3-Triazol-1-yl)phenyl]-6-tributylstannylimidazo[1,2-a]pyridine (35).
1
Compound 35 (43.0 mg, 60.5%) was prepared similarly to compound 31. H NMR (500 MHz,
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CDCl3):  8.13 (d, 2H, J = 8.7 H z), 8.05 (d, 1H, J = 0.9 Hz), 8.01 (s, 1H), 7.90 (s, 1H), 7.87 (d,
1H, J = 0.9 Hz,), 7.82 (d, 2H, J = 8.7 Hz), 7.62 (d, 1H, J = 8.7 Hz), 7.20 (d, 1H, J = 8.7 Hz),
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13
9
1.65–1.49 (m, 6H), 1.40–1.32 (m, 6H), 1.20–1.06 (m, 6H), 0.91 (t, 9H, J = 7.3 Hz); C NMR
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(125 MHz, CDCl3):  145.9, 143.7, 136.3, 134.8, 134.5, 131.8, 131.2, 127.2, 122.6, 121.6, 120.9,
117.2, 107.7, 29.0, 27.3, 13.7, 9.9; Accurate mass (ES+) m/z 552.2153 [M+H]+ (calcd. for
C27H38N5Sn: 552.2149); Anal. C, 58.90; H, 6.82; N, 12.85 (calcd. for C27H37N5Sn: C, 58.93; H,
6.78; N, 12.73).
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2-(2-Methoxypyridin-5-yl)-6-tributylstannylimidazo[1,2-a]pyridine (36). Compound 36 (32.0
mg, 41.1%) was prepared similarly to compound 31. 1H NMR (500 MHz, CDCl3):  8.69 (d, 1H,
J = 2.3 Hz), 8.19 (dd, 1H, J = 8.7, 2.3 Hz), 7.99 (s, 1H), 7.77 (s, 1H), 7.59 (d, 1H, J = 8.7 Hz),
7.17 (d, 1H, J = 8.7 Hz), 6.83 (d, 1H, J = 8.7 Hz), 3.99 (s, 3H), 1.60–1.53 (m, 6H), 1.41–1.32 (m,
13
6H), 1.19–1.05 (m, 6H), 0.91 (t, 9H, J = 7.3 Hz); C NMR (125 MHz, CDCl3):  164.0, 145.9,
144.6, 142.6, 136.7, 131.5, 130.1, 123.8, 122.3, 117.0, 110.9, 106.5, 53.6, 29.1, 27.4, 13.7, 9.9,
Accurate mass (ES+) m/z 516.2042 [M+H]+ (calcd. for C25H38N3OSn: 516.2037).
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2-[2-(Pyrazol-1-yl)pyridin-5-yl]-6-tributylstannylimidazo[1,2-a]pyridine (37). Compound 37
1
(25.1 mg, 35.7%) was prepared similarly to compound 31. H NMR (500 MHz, CDCl3):  8.94
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(d, 1H, J = 2.3 Hz), 8.66 (d, 1H, J = 2.3 Hz), 8.40 (dd, 1H, J = 8.7, 2.3 Hz), 8.05 (d, 1H, J = 8.7
Hz), 8.01 (s, 1H), 7.89 (s, 1H), 7.75 (s, 1H), 7.61 (d, 1H, J = 8.7 Hz), 7.20 (d, 1H, J = 8.7 Hz),
6.48 (s, 1H), 1.59–1.53 (m, 6H), 1.39–1.32 (m, 6H), 1.14–1.09 (m, 6H), 0.90 (t, 9H, J = 7.4 Hz);
13C NMR (125 MHz, CDCl3):  150.8, 146.0, 145.6, 142.1, 141.7, 136.2, 131.9, 130.1, 128.2,
127.1, 122.7, 117.1, 112.4, 107.8, 107.4, 29.0, 27.3, 13.7, 9.9, Accurate mass (ES+) m/z
552.2150 [M+H]+ (calcd. for C27H38N5Sn: 552.2149).
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2-[2-(1H-1,2,3,-Triazol-1-yl)pyridin-5-yl]-6-tributylstannylimidazo[1,2-a]pyridine (38).
Compound 38 (8.7 mg, 31.1%) was prepared similarly to compound 31. 1H NMR (500 MHz,
CDCl3):  9.05 (d, 1H, J = 1.4 Hz), 8.63 (d, 1H, J = 0.9 Hz), 8.48 (dd, 1H, J = 8.4, 2.3 Hz), 8.27
(d, 1H, J = 8.4 Hz), 8.02 (s, 1H), 7.93 (s, 1H), 7.85 (d, 1H, J = 0.9 Hz), 7.61 (d, 1H, J = 9.2 Hz),
7.22 (d, 1H, J = 9.2 Hz), 1.65–1.48 (m, 6H), 1.39–1.32 (m, 6H), 1.20–1.06 (m, 6H), 0.91 (t, 9H,
J = 7.1 Hz); 13C NMR (125 MHz, CDCl3):  148.2, 146.2, 146.1, 141.2, 136.4, 134.2, 132.1,
130.5, 130.2, 123.1, 121.1, 117.3, 114.0, 107.9, 29.0, 27.3, 13.6, 10.0; MS (FAB+) m/z 553
[M+H]+; Accurate mass (ES+) m/z 553.2110 [M+H]+ (calcd. for C26H37N6Sn: 553.2102); Anal.
C, 56.65; H, 6.51; N, 15.25 (calcd. for C26H36N6Sn: C, 56.64; H, 6.58; N, 15.24).
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2-[2-(2H-1,2,3,-Triazol-1-yl)pyridin-5-yl]-6-tributylstannylimidazo[1,2-a]pyridine (39).
Compound 39 (2.3 mg, 22.2%) was prepared similarly to compound 31. 1H NMR (500 MHz,
CDCl3):  9.09 (s, 1H), 8.52 (d, 1H, J = 8.5 Hz), 8.16 (d, 1H, J = 8.5 Hz), 8.03 (s, 1H), 7.97 (s,
1H), 7.92 (s, 2H), 7.62 (d, 1H, J = 8.9 Hz), 7.23 (d, 1H, J = 8 .9 Hz), 1.60–1.50 (m, 6H), 1.40–
1.34 (m, 6H), 1.15–1.12 (m, 6H), 0.91 (t, 9H, J = 7.1 Hz); 13C NMR (125 MHz, CDCl3):  150.1,
146.3, 146.1, 141.1, 136.5, 136.2, 132.0, 130.2, 129.9, 122.9, 117.2, 113.9, 107.9, 29.0, 27.3,
13.7, 9.9, Accurate mass (ES+) m/z 553.2113 [M+H]+ (calcd. for C26H37N6Sn: 553.2102).
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2-[2-(1H-1,2,4,-Triazol-1-yl)pyridin-5-yl]-6-tributylstannylimidazo[1,2-a]pyridine (40).
Compound 40 (47.0 mg, 32.9%) was prepared similarly to compound 31. 1H NMR (500 MHz,
CDCl3):  9.21 (s, 1H), 8.99 (d, 1H, J = 1.8 Hz), 8.47 (dd, 1H, J = 8.4, 1.8 Hz), 8.12 (s, 1H), 8.03
(s, 1H), 7.98 (d, 1H, J = 8.4 Hz),7.93 (s, 1H), 7.62 (d, 1H, J = 8.7 Hz), 7.22 (d, 1H, J = 8.7 Hz),
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1.65–1.49 (m, 6H), 1.36–1.49 (m, 6H), 1.21–1.07 (m, 6H), 0.91 (t, J = 7.3 Hz, 9H); C NMR
(125 MHz, CDCl3):  152.8, 148.5, 146.1, 146.0, 141.5, 141.1, 136.5, 132.1, 130.2, 129.9, 123.0,
117.2, 113.0, 107.7, 29.0, 27.3, 13.7, 9.9, Accurate mass (ES+) m/z 553.2091 [M+H]+ (calcd. for
C26H37N6Sn: 553.2102).
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Radiolabeling. 123I-labeled probes, namely [123I]3, [123I]4, [123I]9, [123I]10, [123I]14, [123I]18,
[123I]27, [123I]28, [123I]29, [123I]30, and [123I]IMPY were prepared from the corresponding
tributyltin precursors via iododestannylation. Typically, a solution of the tributylstannyl
precursor in CH3CN (1 mg/mL, 15–90 μL) was treated with hydrochloric acid (1 or 2 M, 17–170
μL), carrier-free [123I]sodium iodide (30 μL, 66.6–1127 MBq), and 30 wt% hydrogen peroxide
(5–10 μL). After standing at 40 °C for 10 min, the reaction mixture was subjected to HPLC
(YMC PackPro C8, 4.6×150 mm, 5 μm), and the fractions containing the 123I-labeled probe were
isolated. The radiochemical yields were in the range of 7.3−49.2% (uncorrected for radioactive
decay), and the radiochemical purities of all probes exceeded 95%.
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LogPoctanol Measurement. Typically, the 123I-labeled probe was diluted to ~1 MBq/mL with 1-
octanol saturated with water, and a 10-μL aliquot of the thus-obtained solution was added to a
microtube containing water-saturated 1-octanol and 1-octanol-saturated water (100 L each).
The reaction mixture was stirred and subsequently shaken for 5 min (20 to 25 °C, 20 rpm). After
a 20-min centrifugation at 3000 rpm and 23 °C, the octanol and water layers were sampled (50
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μL each), and their radioactivities were determined using an autowell gamma counter. The
logPoctanol values were calculated as: log P = log[count1-octanol/countwater].
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In vitro Binding Assay. The binding of 123I-labeled probes to Aβ(1–42) was evaluated by
filtration techniques described elsewhere.38 A solid form of Aβ(1–42) was purchased from
Peptide Institute (Osaka, Japan). Aggregation was carried out by gently dissolving the peptide
(0.25 mg/mL) in a buffered solution (pH 7.4) containing 10 mM sodium phosphate and 1 mM
ethylenediaminetetraacetic acid (EDTA). The solution was incubated at 37 °C for 42 h upon
gentle and constant shaking. Subsequently, solutions of the tested compound (50 μL, 0.064–5
μM in 10% ethanol), [123I]IMPY (50 μL, 0.508–1.19 MBq/mL), Aβ aggregates (50 μL) and 10%
ethanol (100 μL) were mixed, and the resulting solution was incubated at 37 °C for 3 h.
Subsequently, the bound and free radioactivities were separated by filtration under reduced
pressure (MultiScreen HTS Vacuum Manifold; Merck Millipore), followed by three washes with
10% ethanol. The radioactivity retained on the filters was quantified by an automatic gamma
counter (ARC-7001; Hitachi Aloka Medical, Ltd.), and the IC50 values were determined using
GraphPad Prism (GraphPad Software Inc., La Jolla, USA).
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In vitro Autoradiography. Frozen brain samples from a control subject and AD patients (Figure
4A: 86-year-old female, Figure 4B: 79-year-old male) were cut into 5-μm sections using a
cryostat (Leica Microsystems, CM3050S, Nussloch, Germany). The obtained sections were
dipped into phosphate-buffered saline (PBS) for 15, 5, and 5 min each, and then dipped in PBS
containing 1% bovine serum albumin (BSA). The thus-treated sections were incubated with 123I-
labeled probes (10 kBq/mL) for 30 min at room temperature, washed with PBS containing 1%
BSA for 5 min, and subjected to two 5-min rinses with PBS. The non-specific binding was
determined in the presence of 5 μM non-labeled compounds using an identical procedure. After
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drying, the sections were exposed to imaging plates (Fujifilm) overnight. Autoradiographic
images were obtained using a BAS-2500 instrument (Fujifilm Co. Ltd., Tokyo, Japan). Adjacent
sections were immunostained using the anti-Aβ monoclonal antibody 82E1.
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In vivo Kinetics in Normal Rat Brain. Each 123I-labeled probe (4 MBq) was injected under
non-anesthesia into the iodine-pretreated tail veins of male Wistar rats (eight weeks old). The
rats were sacrificed at 2, 5, 15, 30, and 60 min after injection (three animals at each time). The
brains of the sacrificed rats were removed and weighed, and their radioactivity was measured
using a single-channel analyzer (Ohyo Koken Kogyo Co. Ltd., SP-20, Tokyo, Japan). Data were
expressed as the percent injected dose per gram tissue (%ID/g).
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In vivo Stability. Each 123I-labeled probe (4 MBq) was injected under non-anesthesia into the
iodine-pretreated tail veins of male Wistar rats (eight weeks old). The rats were sacrificed at 2, 5,
15, and 30 min after the injection (three animals at each time), and their blood samples were
centrifuged at 3,000 rpm for 10 min at 4 °C to separate the plasma. The plasma samples (400 μL)
were mixed with methanol (1 mL) and centrifuged at 4,400 rpm for 10 min at 4 °C to remove the
plasma proteins. The supernatant was analyzed by radio-TLC (Silica Gel 60F 254, mobile phase:
acetonitrile-water (10:1, v/v)) using a Raytest Rita 68000 linear analyzer (Raytest, Straubenhardt,
Germany).
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FIGURES
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Figure 1. Structures of SPECT and PET Aβ imaging agents evaluated in humans. (A) Agents
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approved by FDA and EMA; (B) first-generation imaging agents; (C) ABC577 (present study).
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Figure 2. Structures of the developed 123I-labeled 2-phenyl- and 2-pyridyl-imidazo[1,2-
a]pyridines.
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Figure 3. Comparison of in vitro autoradiography images of Alzheimer’s disease brain sections
obtained using [123I]IMPY (A), [123I]3 (B), [123I]4 (C), [123I]9 (D), [123I]10 (E), [123I]14 (F),
[123I]18 (G), [123I]27 (H), [123I]28 (I15 and J), [123I]29 (K), and [123I]30 (L).
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