Synthesis and biological evaluation of novel radioiodinated imidazopyridine derivatives for amyloid-β imaging in Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2014.05.043

Non-invasive detection for amyloid-β peptide (Aβ) deposition has important significance for the early diagnosis and medical intervention for Alzheimer's disease (AD). In this study, we developed a series of imidazopyridine derivatives as potential imaging agents for single-photon emission computed tomography (SPECT). Two of them, compounds DRK092 and DRM106, showed higher affinity for synthetic human Aβ 1-40 fibrils than did the well-known amyloid-imaging agent IMPY. A metabolite analysis revealed brain-permeable radioactive metabolites of ¹²⁵I-labeled DRK092 and IMPY; no radioactive metabolites from ¹²⁵I-labeled DRM106 ([ ¹²⁵I]DRM106) were detected. In addition, in vitro autoradiography clearly demonstrated specific binding of [¹²⁵I]DRM106 in the hippocampal region of AD enriched with Aβ plaques. Thus, our results strongly suggested that compound DRM106 can be used as an imaging agent for SPECT to detect Aβ deposition in AD brain.

Full text of DOI:10.1016/j.bmc.2014.05.043

Accepted Manuscript
Synthesis and biological evaluation of novel radioiodinated imidazopyridine
derivatives for amyloid-β imaging in Alzheimer’s disease
Chun-Jen Chen, Kazunori Bando, Hiroki Ashino, Kazumi Taguchi, Hideaki
Shiraishi, Osuke Fujimoto, Chiemi Kitamura, Satoshi Matsushima, Masayuki
Fujinaga, Ming-Rong Zhang, Hiroyuki Kasahara, Takao Minamizawa, Cheng
Jiang, Maiko Ono, Makoto Higuchi, Tetsuya Suhara, Kazutaka Yamada, Bin Ji
PII:
S0968-0896(14)00401-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.043
BMC 11604
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 March 2014
20 May 2014
21 May 2014
Please cite this article as: Chen, C-J., Bando, K., Ashino, H., Taguchi, K., Shiraishi, H., Fujimoto, O., Kitamura,
C., Matsushima, S., Fujinaga, M., Zhang, M-R., Kasahara, H., Minamizawa, T., Jiang, C., Ono, M., Higuchi, M.,
Suhara, T., Yamada, K., Ji, B., Synthesis and biological evaluation of novel radioiodinated imidazopyridine
derivatives for amyloid-β imaging in Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2014), doi: http://
dx.doi.org/10.1016/j.bmc.2014.05.043
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Synthesis and biological evaluation of novel radioiodinated imidazopyridine
derivatives for amyloid- imaging in Alzheimer’s disease
Chun-Jen Chena, b, c, Kazunori Bandoa, Hiroki Ashinoa, Kazumi Taguchia, Hideaki
Shiraishia, Osuke Fujimotoa, Chiemi Kitamuraa, Satoshi Matsushimaa, Masayuki
Fujinagab, Ming-Rong Zhangb, Hiroyuki Kasaharaa, Takao Minamizawaa, Cheng Jiangd,
Maiko Onob, Makoto Higuchib, Tetsuya Suharab, Kazutaka Yamadac, # and Bin Jib, *
a Fujifilm RI Pharma Co. LTD, Chiba, Japan
b Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
c The United Graduate School of Veterinary Science, Gifu University, Gifu, Japan
d School of Pharmacy, Fudan University, Shanghai, China
* Corresponding author: Bin Ji, Ph.D.
Molecular Imaging Center, National Institute of Radiological Sciences
4-9-1, Anagawa, Inage-ku, Chiba, Chiba 263-8555, Japan
Phone: +81-43-206-3251. Fax: +81-43-253-0396.
E-mail: kihin@nirs.go.jp
1

# Co-corresponding author: Kazutaka Yamada, Ph.D.
The United Graduate School of Veterinary Science, Gifu University
1-1 Yanagido, Gifu City 501-1193, Japan
Phone: +81-155-49-5395. Fax : +81-155-49-5398
E-mail: kyamada@obihiro.ac.jp
2

Abstract
Non-invasive detection for amyloid- peptide (A) deposition has important
significance for the early diagnosis and medical intervention for Alzheimer’s disease
(AD). In this study, we developed a series of imidazopyridine derivatives as potential
imaging agents for single-photon emission computed tomography (SPECT). Two of
them, compounds DRK092 and DRM106, showed higher affinity for synthetic human
A1-40 fibrils than did the well-known amyloid-imaging agent IMPY. A metabolite
analysis revealed brain-permeable radioactive metabolites of 125I-labeled DRK092 and
IMPY; no radioactive metabolites from 125I-labeled DRM106 ([125I]DRM106) were
detected. In addition, In vitro autoradiography clearly demonstrated specific binding of
[125I]DRM106 in the hippocampal region of AD enriched with A plaques. Thus, our
results strongly suggested that compound DRM106 can be used as an imaging agent for
SPECT to detect A deposition in AD brain.
3

Keywords: Alzheimer’s disease (AD); amyloid- peptide (A); autoradiography;
positron emission tomography (PET); single-photon emission computed tomography
(SPECT)
4

1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by
the appearance of amyloid-β peptide (Aβ) plaques and neurofibrillary tangles (NFTs).
Since fibrillary Aβ can accumulate in the brain for decades before the appearance of
AD,1 in-vivo non-invasive detection of Aβ deposition is crucial for the early diagnosis
and treatment of AD at the prodromal stage. Nuclear imaging by positron emission
tomography (PET) and single-photon emission computed tomography (SPECT) are
clinically applicable for functional examination, and are widely used in translational
research. Amyloid imaging with PET tracers has been successfully employed in
preclinical research and clinical trials.2-5 11C-labeled Pittsburgh compound B ([11C]PiB)
is a widely used PET ligand that has been employed to quantitatively detect amyloid
deposition in AD model animals and patients.2,3,6 It has also been used as an imaging
biomarker for AD diagnosis as well as a prognostic index for the conversion from mild
cognitive impairment (MCI) to AD.7-9 To overcome the disadvantage of the short
half-life (approximately 20 min) of 11C-labeled tracers, ligands labeled with 18F
(half-life: approximately 110 min) have also been developed for routine medical
needs.5,10-12 Two such radioligands, [18F]florbetapir and [18F]flutemetamol, have already
5

been approved by the US Food and Drug Administration (FDA) as radioactive
diagnostic agents to detect amyloid deposition in adult patients with cognitive
impairment.
In comparison with PET, radioisotopes used in SPECT, such as 123I (half-life: 13.22
hours), have a longer half-life and can therefore achieve a longer distance delivery.
More SPECT scanners have also been installed for routine clinical examination.
Although inferior to PET in terms of sensitivity and quantitative performance, SPECT is
more suitable for the primary screening of prodromal AD patients, owing to lower
operating and installation costs. In the past decade, several SPECT ligands with
different scaffolds have been developed that can be used to successfully visualize
amyloid deposition in brain sections of AD models and patients with AD in vitro. To
date, however, there has been lack of SPECT ligands available for amyloid imaging to
distinguish between normal subjects and patients with AD. In the present study, we
developed a series of novel imidazopyridine derivatives to be employed as SPECT
agents in the in-vivo detection of A pathology.
6

2. Results and discussion
To date, a number of iodinated ligands, such as dipheny-1,3,4-oxadiazole (1,3,4-DPOD)
derivatives,13 aurone derivatives,14 1,4-diphenyltriazole derivatives,15 pyridyl
benzofuran derivatives,16 and imidazo[1,2-a]pyridine derivatives,17 have been employed
for amyloid imaging with SPECT (Fig.1). IMPY, an imidazo[1,2-a]pyridine derivative,
is the only ligand for SPECT that has been tested in human subjects18. Although this
ligand has shown excellent properties as an imaging probe in preclinical studies,19,20
including good brain permeability and rapid off-target washout, the preliminary clinical
data failed to demonstrate the ability of this ligand to distinguish between AD and a
cognitively normal brain. This may due to an insufficient affinity for A fibrils or a
metabolic instability21. In this study, novel compounds for SPECT amyloid imaging
using the core structure of IMPY were developed.
The novel imidazo[1,2-a]pyridine derivatives were synthesized from bromoketone 1 as
shown in Scheme 1. Synthesis of 2 bearing imidazo[1,2-a]pyridine skeleton was
achieved by reaction of bromoketone 1 with 2-aminopyridines under mild basic
conditions. The cyano derivative 2 was used as a common intermediate in the synthesis
of DRM105, DRK092, DRM101, and DRM102. The tetrazol derivative DRM105 was
7

synthesized by reaction of azidotrimethylsilane with trimethylaluminum used as a
catalyst. Transformation of cyano derivative 2 into 3 with DAIBAL-H, followed by
treatment with p-toluenesulfonylmethylisocyanide, gave DRK092. The thioamidation of
2 with thioacetamide under acidic conditions led to the formation of 4, which was
treated with chloroacetaldehyde in the presence of Et3N to produce the thiazole
derivative DRM101. In addition, reaction of 2 with hydroxylamine produced amidine
derivatives 5, followed by cyclization of amidine 5 with trimethylorthoformate leading
to the oxadiazole derivative DRM102. Additional reaction of aldehyde 3 with
methylmagnesium bromide produced the alcohol derivative 6, which was treated with
manganese dioxide to give the acetyl derivatives 7. Condensation of the acetyl
derivative 7 with dimethylformamide dimethylacetal (DMFDMA) led to the synthesis
of -dimethylaminovinylketone derivatives 8, and subsequent cyclization of the
-dimethylaminovinylketone group with hydrazine produced DRM106.
The precursors for radioiodination, tributyltin derivatives DRM015 and DRN059, were
synthesized by stannylation of iodo-derivatives DRK092 or DRM106 with
bis(tributyltin) in the presence of tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4]
and subsequent treatment of 9 with Boc2O. Synthesis of [125I]DRK092 and
[125I]DRM106 was carried out as shown in Scheme 1. The [125I]iodination of the
8

tributylstannyl precursors DRM015 or DRN059 with [125I]NaI was performed by
iododestannylation with chloramine T as a catalyst. When DRN059 was used as
precursor, deprotection of the Boc group in the pyrazole ring was performed by
treatment with a 12 N solution of NaOH at 70 °C for 35 min. After these reactions were
completed, a semi-preparative HPLC system with a radioactive detector was used and
the radioactive fraction corresponding to [125I]DRK092 or [125I]DRM106 collected. The
eluent was diluted with water, and the solution was loaded onto a pre-conditioned C18
Sep-Pak light cartridge. Finally, the desired compound, [125I]DRK092 or [125I]DRM106,
was eluted from the cartridge with EtOH to produce the final product. The successful
synthesis of [125I]DRK092 and [125I]DRM106 was confirmed by comparison with
non-radiolabeled DRK092 or DRM106 using HPLC. As for [125I]DRM106 (Fig. 2A),
the radiochemical purity of [125I]DRK092 and [125I]DRM106 was analyzed by using
thin-layer chromatography (TLC). In the final products, no significant chemical
impurities were found; the radiochemical purity of radioligands remained larger than
95% after keeping the products at room temperature for 24 h. Additionally, the
experimental condition of radiolabeling of DRM106 was also examined by cold run as
described in Supplementary information. We confirmed that the resulting product
derived from NaI under the condition of cold run was DRM106 by comparing the
9

retention time with that of synthesized standard DRM106 characterized with 1H and 13C
NMR (Supplementary information).
The affinity of the imidazo[1,2-a]pyridine derivatives was evaluated based on the
binding inhibition of [125I]IMPY to synthetic human A(1-40) fibrils. As shown in Table
1, the half-maximal inhibitory concentration (IC50) values ranged from approximately 1
to 740 nM. Compounds DRK092 and DRM106 showed a higher affinity (approximately
1.1 and 1.9 nM respectively) than IMPY (approximately 2.9 nM). In addition,
acetaminophen, allopurinol, antipyrine, bifonazole, caffeine, chloramphenicol,
chlorpheniramine, cimetidine, clonidine, clozapine, estradiol, haloperidol,
hydrocortisone, naphthalene, prednisolone, risperidone, testosterone, triphenylene, and
warfarin with published log D7.4 values were used to construct the standard curve to
estimate the lipophilicity parameter.22,23 The logarithms of the capacity factors (log kw)
of these compounds measured in this work showed a strong correlation with the log D7.4
values reported in previous studies (Fig. 2B). As shown in Table 1, the log D7.4 values of
DRK092 and DRM106 (3.6 and 3.2 respectively) calculated from their log kw suggest
that these compounds may have suitable brain permeability properties. Further analysis
of these two promising compounds was thus performed.
As expected, [125I]IMPY, [125I]DRK092, and [125I]DRM106 showed excellent brain
10

uptake. In particular, the initial peaks (ranging approximately between 0.8% and 1.3%
of ID/g brain) were rapidly eliminated from normal rat brains (Fig. 3A). The analysis of
metabolites by TLC clearly demonstrated that no detectable metabolites from
[125I]DRM106 in the brain over the observation period were present. In contrast, at least
two metabolites from [125I]DRK092 and [125I]IMPY, respectively, were detected in the
brain samples in the initial phase after the radiolabeled ligands were injected into
normal rats (Fig. 3B-D). Within 30 min of intravenous injection in the AD mouse
model20, the percentage of intact IMPY in plasma and brain decreased to approximately
20% and 40%, respectively. This finding confirmed the poor metabolic stability of
IMPY, which in turn may be one of the reasons why AD cannot be distinguished from
cognitively normal brains when IMPY is used. Our data also showed that DRK092 has a
similar metabolic instability to IMPY (Fig. 3B and C). IMPY and DRK092 metabolites
were not fully identified in the present study; however, considering that a metabolic
enzyme-catalyzed N-demethylation is likely to occur, we speculate that the observed
radioactive IMPY metabolites may include N-demethyl derivatives. Given the
difference in chemical structure between DRK092 and DRM106, the oxazole ring of
DRK092 may be the reason for its metabolic instability. Taken into account all these
findings, DRM106 was selected for further investigation. The biodistribution analysis
11

demonstrated that [125I]DRM106 was mainly accumulated in kidney and liver among
peripheral organs in the initial phase. Notably, no relevant abnormal accumulation in
special organs was measured during the observation period after the bolus injection,
which confirmed that the use of DRM106 should not raise safety concerns regarding
abnormal distribution. Thyroid uptake hardly increased with time within 1h, suggesting
less concern about deiodination of [125I]DRM106 in further SPECT imaging. After the
intravenous administration, brain-to-blood ratios were measured to be approximately
1.29, 1.21, 1.18, 0.66, and 0.25 at 2, 5, 15, 30, and 60 min, respectively; these values are
higher than those measured for [125I]IMPY (approximately 0.15, 0.04 and 0.02 at 2, 30,
60 min post-injection, respectively) as reported in previous studies.20
In addition, in-vitro autoradiography showed that the binding of [125I]DRM106 is higher
in the hippocampal region of AD brains enriched with A plaques than in the
corresponding region of healthy-control (HC) brain. Furthermore, the addition of
non-radiolabeled DRM106 significantly decreased these bindings, demonstrating that a
specific binding of [125I]DRM106 occurs (Fig. 4A). The spatial consistency of plaque
counterstaining with the fluorescent dye FSB and the accumulation of [125I]DRM106
clearly confirmed the specific binding of [125I]DRM106 to A plaques (red arrowheads
in Fig. 4B-D).
12

3. Conclusion
In the present study, we synthesized a series of novel imidazopyridine derivatives with
a chemical structure similar to that of IMPY. We obtained a promising compound
(DRM106) with excellent brain permeability and a rapid off-target washout, a sufficient
affinity to A aggregate, and an ideal metabolic stability. These data strongly suggest
that DRM106, labeled with iodine-123, can be efficiently employed as SPECT agent for
the detection of the amyloid pathology in the brains of patients with AD.
4. Experiments
Melting points (mp) were measured using a micromelting point apparatus (MP-500P,
1
Yanaco, Tokyo, Japan). H and 13C NMR spectra were recorded on a JNM-AL-400
spectrometer (JEOL; Tokyo, Japan) using tetramethylsilane as an internal standard. All
chemical shifts () were reported in parts per million (ppm) downfield from the standard.
High-resolution (HR) fast atom bombardment-mass spectrometry (FAB–MS) was
performed using a JEOL NMS-SX102 spectrometer (JEOL). Column chromatography
was performed on a Wakogel C-200 (Wako Pure Chemical Industries, Ltd.; Osaka,
Japan). HPLC was performed using a JASCO HPLC system (JASCO; Tokyo, Japan),
13

and effluent radioactivity was monitored using a NaI (Tl) scintillation detector system.
All chemical reagents were purchased of the highest grade commercially available from
Sigma-Aldrich Corporation (Milwaukee, WI), Wako Pure Chemical Industries, Ltd., or
Tokyo Chemical Industries, Ltd. (Tokyo, Japan).
4.1. Chemical synthesis
4.1.1. 4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)benzonitrile (2)
NaHCO3 (382 mg, 4.55 mmol) was added to a solution of 2-amino-5-iodopyridine (1.0
g, 4.55 mmol) and 1 (1.02 g, 4.55 mmol) in EtOH (30 mL) and heated at reflux for 16 h.
After having quenched the reaction mixture with water (10 mL), the resulting
precipitate was collected by filtration to give 2 (1.27 g, 81%) as a pale yellow powder;
1H NMR (400 MHz, DMSO-d6) δ 7.54 (1H, d, J = 9.5 Hz), 7.62 (1H, dd, J = 1.2, 9.3
Hz), 7.94 (2H, d, J = 8.3 Hz), 8.14 (2H, d, J = 8.5 Hz), 8.56 (1H, s), 9.02 (1H, s).
EI–MS m/z: 345 (M+).
4.1.2.
6-Iodo-2-[4-(1H-1,2,3,4-tetrazol-5-yl)phenyl]imidazo[1,2-a]pyridine
(DRM105)
Under a nitrogen atmosphere, trimethylaluminum (43.3 mg, 0.30 mmol in 150 μL
14

toluene) and azidotrimethylsilane (42 μL, 0.32 mmol) were added to a solution of 2
(104 mg, 0.30 mmol) in dry toluene (1 mL). The reaction mixture was stirred at 80 °C
for 2 h. After cooling the reaction mixture to room temperature, 1 mL of 6 M HCl was
added to the solution. The resulting precipitate was collected by filtration to give
DRM105 (17 mg, 15%) as a colorless powder; mp: >300 °C. 1H NMR (400 MHz,
DMSO-d6) δ 7.55–7.68 (2H, m), 8.18 (4H, q, J = 8.3 Hz), 8.54 (1H, s), 9.05 (1H, s).
HRMS (FAB) calcd for C14H10IN6 (M+), 389.0012; found: 389.0022.
4.1.3. 4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)benzaldehyde (3)
Diisobutylaluminum hydride in hexane (7.8 mL, 1.0 M; 7.8 mmol) was added to a
solution of 2 (1.27 g, 3.68 mmol) in THF (15 mL) and CH2Cl2 (15 mL) at 0 °C and
stirred for 15 min and then at room temperature for 4 h under a nitrogen atmosphere.
After the reaction mixture was quenched with saturated aqueous NH4Cl (2 mL), the
solution was stirred at room temperature for 1 h. MgSO4 and diethyl ether were added
to the reaction mixture and stirred for 1 h. Removal of solvent under reduced pressure
gave a residue, which was then purified by chromatography on a silica gel column using
CH2Cl2/MeOH (95:5, v/v) to give 3 (1.03 g, 81%) as a colorless powder; 1H NMR (400
MHz, DMSO-d6) δ 7.47 (2H, d, J = 1.2 Hz), 7.98 (2H, d, J = 8.3 Hz), 8.18 (2H, d, J =
15

8.3 Hz), 8.49 (1H, s), 8.95 (1H, t, J = 1.2 Hz), 10.02 (1H, s).
4.1.4. 5-[4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)phenyl]-1,3-oxazole (DRK092)
A mixture of 3 (647 mg, 1.86 mmol), K2CO3 (321 mg, 2.32 mmol) and
p-toluenesulfonylmethylisocyanide (454 mg, 2.33 mmol) in MeOH (10 mL) was heated
at reflux for 13 h. The precipitate was collected by filtration to give DRK092 (330 mg,
1
46%) as a colorless powder; mp: 284-286 °C. H NMR (400 MHz, DMSO-d6) δ7.45
(2H, d, J = 1.2 Hz), 7.74 (1H, s), 7.81 (2H, d, J = 8.5 Hz), 8.06 (2H, d, J = 8.5 Hz), 8.39
(1H, s), 8.47 (1H, s), 8.92 (1H, t, J = 1.2 Hz). 13C NMR (100 MHz, DMSO-d6) δ 76.14,
109.37, 117.92, 122.12, 124.46, 126.22, 126.73, 131.46, 132.60, 133.51, 143.48,
143.87, 150.34, 151.83. HRMS (FAB) calcd for C16H11IN3O (M+), 387.9947; found:
387.9905.
4.1.5. 4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)-1-benzenecarbothioamide (4)
Thioacetamide (150 mg, 2.0 mmol) and 2 (345 mg, 1.0 mmol) were added to a solution
of saturated hydrogen chloride in DMF (5 mL) and the reaction mixture was stirred at
80 °C for 4 h. After solvent removal under reduced pressure, aqueous saturated
NaHCO3 was added to the residue. The resulting precipitate was collected by filtration
16

1
to give 4 (269 mg, 71%) as a colorless powder; H NMR (400 MHz, DMSO-d6) δ 7.45
(2H, d, J = 1.0 Hz), 7.98 (4H, s), 8.41 (1H, s), 8.92 (1H, s), 9.52 (1H, s), 9.86 (1H, s).
FAB–MS m/z: 380 (M+ + H).
4.1.6. 2-[4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)phenyl]-1,3-thiazole (DRM101)
40% (w/w) chloroacetaldehyde in water (155 μL, 0.95 mmol) and Et3N (91 μL, 0.65
mmol) were added to a solution of 4 (249 mg, 0.66 mmol) in EtOH (10 mL) and heated
at reflux for 18 h. The reaction mixture was quenched with water and extracted with
CH2Cl2/MeOH. The organic layer was washed with water, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH, 100/5, v/v) to give DRM101 (103 mg, 39%) as a
colorless powder; mp: 240–241°C. 1H NMR (400 MHz, DMSO-d6) δ 7.45 (2H, s), 7.80
(1H, dd, J = 0.7, 3.2 Hz), 7.94 (1H, dd, J = 0.7, 3.2 Hz), 8.02 (2H, d, J = 8.1 Hz), 8.08
(2H, d, J = 8.3 Hz), 8.41 (1H, s), 8.93 (1H, d, J = 1.0 Hz). HRMS (FAB) calcd for
C16H11IN3S (M+), 403.9718; found: 403.9739.
4.1.7. N-Hydroxy-4-(6-iodoimidazo[1,2-a]pyridin-2-yl)benzamidine (5)
Hydroxylamine hydrochloride (208 mg, 3.0 mmol) and potassium carbonate (415 mg,
17

3.0 mmol) were added to a solution of 2 (345 mg, 1.0 mmol) in MeOH (10 mL). The
reaction mixture was heated at reflux for 14 h. After the reaction mixture was cooled to
room temperature, the resulting precipitate was collected by filtration and washed with
1
water to give 5 (279 mg, 74%) as a pale yellow powder; H NMR (400 MHz,
DMSO-d6) δ 5.82 (2H, s), 7.43 (2H, s), 7.74 (2H, d, J = 8.3 Hz), 7.94 (2H, d, J = 8.3
Hz), 8.34 (1H, s), 8.91 (1H, s), 9.65 (1H, s). EI–MS m/z: 378 (M+).
4.1.8. 3-[4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)]-1,2,4-oxadiazole (DRM102)
A mixture of 5 (265 mg, 0.70 mmol) and trimethylorthoformate (2.3 mL, 21.0 mmol)
was heated at reflux for 18 h. After the reaction mixture was cooled at room
temperature, the solvent was removed under reduced pressure and then CH2Cl2/MeOH
was added. The resulting precipitate was collected by filtration to give DRM102 (217
mg, 80%) as a yellow powder; mp: 230-232 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.46
(2H, s), 8.11 (2H, d, J = 8.3 Hz), 8.16 (2H, d, J = 8.3 Hz), 8.44 (1H, s), 8.94 (1H, d, J =
1.0 Hz), 9.71 (1H, d, J = 0.5 Hz). HRMS (FAB) calcd for C15H10IN4O (M+), 388.9899;
found: 388.9901.
4.1.9. 1-[4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)phenyl]-1-ethanol (6)
18

Methyl magnesium bromide in diethyl ether (787 μL, 3.0 M, 2.36 mmol) was added
dropwise to a solution of 3 (820 mg, 2.36 mmol) in THF (25 mL) at 0 °C under a
nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 15 min and then at
room temperature for 5 h. After being quenched with aqueous saturated ammonium
chloride (25 mL) and stirred at room temperature for 1 h, the reaction mixture was
extracted with CH2Cl2/MeOH. The organic layer was washed with water, dried over
Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 97/3, v/v) to give 6 (579 mg, 63%) as yellow
1
crystals; H NMR (400 MHz, CDCl3) δ 1.53 (3H, d, J = 6.3 Hz), 4.93 (1H, q, J = 6.5
Hz), 7.32 (1H, dd, J = 1.7, 9.4 Hz), 7.41 (2H, d, J = 7.6 Hz), 7.44 (1H, s), 7.78 (1H, s),
7.89 (2H, d, J = 8.1 Hz), 8.37 (1H, s).
4.1.10. 1-[4-(6-Iodoimidazo[1,2-a]pyridin-2-yl)phenyl]-2-ethanone (7)
Manganese dioxide (691 mg, 7.95 mmol) was added to a solution of 6 (579 mg, 1.59
mmol) in CHCl3 (50 mL); the reaction mixture was heated at reflux for 8 h. After the
suspension was filtered through Celite, the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 95/5, v/v) to give 7 (371 mg, 64%) as pale yellow crystals; 1H NMR
19

(400 MHz, DMSO-d6) δ 2.60 (3H, s), 7.46 (2H, s), 8.03 (2H, d, J = 8.3 Hz), 8.09 (2H, d,
J = 8.3 Hz), 8.46 (1H, s), 8.93 (1H, s). EI–MS m/z: 362 (M+).
4.1.11. (E)-3-(Dimethylamino)-1-[4-(6-iodoimidazo[1,2-a]pyridin-2-yl)phenyl]-2-
propen-1-one (8)
Under an argon atmosphere, N,N-dimethylformamide dimethyl acetal (363 μL, 2.73
mmol) was added dropwise to a solution of 7 (495 mg, 1.37 mmol) in DMF (50 mL);
the reaction mixture was heated at 150 °C for 16 h. After the reaction mixture was
concentrated under reduced pressure and the residue was purified by silica gel column
chromatography (CH2Cl2/MeOH, 97/3, v/v) to give 8 (274 mg, 48%) as yellow crystals;
1H NMR (400 MHz, CDCl3) δ 2.97 (3H, br), 3.16 (3H, br), 5.77 (1H, d, J = 12.4 Hz),
7.35 (1H, dd, J = 1.5, 10.9 Hz), 7.44 (1H, d, J = 9.3 Hz), 7.83 (1H, d, J = 12.4 Hz), 7.86
(1H, s), 7.98 (4H, d, J = 0.5 Hz), 8.40 (1H, s). EI–MS m/z: 386 (M+).
4.1.12. 6-Iodo-2-[4-(1H-3-pyrazolyl)phenyl]imidazo[1,2-a]pyridine (DRM106)
Hydrazine monohydrate (100 μL, 2.06 mmol) was added dropwise to a solution of 8
(344 mg, 0.82 mmol) in EtOH (30 mL) and heated at reflux for 3 h. After the reaction
mixture was cooled at room temperature, the resulting precipitate was collected by
20

filtration to give DRM106 (290 mg, 92%) as a colorless powder; mp: >300 °C. 1H NMR
(400 MHz, DMSO-d6) δ 6.75 (1H, d, J = 1.7 Hz), 7.44 (2H, t, J = 9.8 Hz), 7.79 (1H, br),
7.87 (2H, d, J = 8.0 Hz), 7.98 (2H, d, J = 8.0 Hz), 8.34 (1H, s), 8.91 (1H, s), 12.90 (1H,
13
br). C NMR (100 MHz, DMSO-d6) δ 75.8, 101.8, 108.8, 117.8, 125.4, 125.9, 129.8,
131.3, 132.3, 133.4, 143.4, 144.5, 149.7. HRMS (FAB) calcd for C16H12IN4 (M+),
387.0107; found: 387.0117.
4.1.13. 5-{4-[6-(Tributylstannyl)imidazo[1,2-a]pyridin-2-yl]phenyl}-1,3-
oxazole (DRM015)
A mixture of DRK092 (580 mg, 1.50 mmol), bis(tributyltin) (1.74 g, 3.0 mmol), and
Pd(PPh3)4 (173 mg, 0.15 mmol) in 1,4-dioxane (30 mL) was heated at reflux for 14 h
under an argon atmosphere. After the solvent was removed under reduced pressure, the
residue was purified by silica gel column chromatography (CH2Cl2/EtOAc, 7/3, v/v) to
1
give DRM015 (400 mg, 49%) as brown oil; H NMR (400 MHz, DMSO-d6) δ
0.89–0.93 (9H, m), 1.07-1.15 (6H, m), 1.31–1.39 (6H, m), 1.53–1.61 (6H, m), 7.18
(1H, d, J = 8.8 Hz), 7.39 (1H, s), 7.60 (1H, d, J = 8.8 Hz), 7.73 (2H, d, J = 8.3 Hz), 7.88
(1H, s), 7.93 (1H, s), 8.00 (1H, s), 8.03 (2H, d, J = 8.3 Hz). ESI–MS m/z: 552 (M+).
21

4.1.14. 2-[4-(1H-3-Pyrazolyl)phenyl]-6-(tributylstannyl)imidazo[1,2-a]pyridine (9)
A mixture of DRM106 (500 mg, 1.30 mmol), bis(tributyltin) (1.51 g, 2.6 mmol), and
Pd(PPh3)4 (150 mg, 0.13 mmol) in 1,4-dioxane (30 mL) was heated at reflux for 16 h
under an argon atmosphere. After the solvent was removed under reduced pressure, the
residue was purified by silica gel column chromatography (CH2Cl2/MeOH, 95/5, v/v) to
1
give 9 (300 mg, 42%) as a yellow oil; H NMR (400 MHz, CDCl3) δ 0.89–0.92 (9H,
m), 1.06-1.25 (6H, m), 1.31–1.40 (6H, m), 1.53–1.63 (6H, m), 6.67 (1H, d, J = 2.4 Hz),
7.17 (1H, d, J = 8.8 Hz), 7.61 (1H, d, J = 8.8 Hz), 7.64 (1H, d, J = 2.4 Hz), 7.83 (2H, d,
J = 8.3 Hz), 7.88 (1H, s), 8.00 (1H, s), 8.03 (2H, d, J = 8.3 Hz).
4.1.15. 2-{4-[1-(tert-Butoxycarbonyl)-3-pyrazoyl]phenyl}-6-(tributylstannyl)-
imidazo[1,2-a]pyridine (DRN059)
Di-tert-butyl dicarbonate (1.1 mL, 4.79 mmol) was added to a solution of 9 (113 mg,
0.21 mmol) and dimethylaminopyridine (26 mg, 0.21 mmol) in CH2Cl2 (3 mL) at 0 °C.
The reaction mixture was then stirred at room temperature for 8 h. This mixture was
quenched with water and extracted with CH2Cl2. The organic layer was washed with
water and saturated saline, dried over Na2SO4 and evaporated under reduced pressure.
The residue was purified by silica gel chromatography (CH2Cl2/MeOH, 97/3, v/v) and
22

produced DRN059 (127 mg, 93%) as colorless crystals; 1H NMR (400 MHz, CDCl3) δ
0.91 (9H, t, J = 7.3 Hz), 1.06–1.26 (6H, m), 1.31–1.40 (6H, m), 1.47–1.61 (6H, m), 1.68
(9H, s), 6.76 (1H, d, J = 2.9 Hz), 7.16 (1H, d, J = 8.8 Hz), 7.61 (1H, d, J = 8.5 Hz), 7.89
(1H, s), 7.98–8.04 (5H, m), 8.11 (1H, d, J = 3.0 Hz). FAB–MS m/z: 651 (M+ + H).
4.2. Radiosynthesis
4.2.1. 6-[125I]Iodo-2-[4-(1H-3-pyrazolyl)phenyl]imidazo[1,2-a]pyridine (DRM106)
Chloramine T (20 μL, 0.25 mg/mL) was added to a mixture of DRN059 (20 μL, 1
mg/mL in 2-propanol), sodium phosphate buffer (70 μL, 0.3 M, pH 5.5), and Na[125I]
(300 MBq, Nordion International, Ltd., Ottawa, ON, Canada) in a glass vial. The
reaction was allowed to proceed for 5 min at room temperature; after this time, it was
quenched by addition of 10 μL of a solution of NaOH (12 N). Subsequently, 100 μL of
ethanol were added into the reaction vial and the reaction mixture was heated at 75 °C
for 35 min, followed by cooling for 5 min. The reaction mixture was purified by HPLC,
using a Capcellpak C18 UG-120 column (6.0150 mm; Shiseido, Tokyo, Japan) with a
mobile phase (EtOH/H2O, 45/55, v/v) at a flow rate of 1.0 mL/min. Eluent volumes
were diluted with a 4-fold volume of 20% EtOH, then loaded onto and passed through a
pre-conditioned C18 Sep-Pak light cartridge (Waters Corporation; Milford, MA). After
23

the cartridge was washed with distilled water (5 mL) and absolute EtOH (150 μL),
[125I]DRK092 was eluted by additional absolute EtOH (1 mL); ascorbic acid (final
concentration: 5 mM) was added as a stabilizer to prevent oxidation and deiodination.
The final product [125I]DRM106 was obtained by filtration with a 0.20-µm filter. Its
radiochemical purity was analyzed by TLC. [125I]DRM106 was applied to TLC plates
(Whatman KC-18F) and eluted with an elution buffer, i.e., MeOH/H2O/NH3=80/19/1.
Radioactivity of separated spots was quantified by autoradiography. The specific
activity and the radiochemical purity were determined to be 81.4 GBq/μmol and >95%,
respectively. The solution was stored at –20 °C until used.
4.2.2. 5-[4-(6-[125I]Iodoimidazo[1,2-a]pyridine-2-yl)phenyl]-1,3-oxazole (DRK092)
Chloramine T (20 μL, 0.10 mg/mL) was added to a mixture of DRM015 (20 μL,
1mg/mL in EtOH), sodium phosphate buffer (70 μL, 0.3 M, pH 5.5), and Na[125I] (300
MBq) in a glass vial. After the reaction was allowed to proceed for 2 min at room
temperature, the reaction was quenched with aqueous sodium metabisulfite (100 μL, 2.0
mg/mL). The reaction mixture was purified following a protocol similar to that
employed for [125I]DRM106; however, a modified mobile phase (MeOH/H2O, 60/40,
v/v) for HPLC was used. The specific activity and the radiochemical purity were
24

determined to be 81.4 GBq/μmol and >95%, respectively. The solution was stored at
–20 °C until used.
4.3. Binding assay for IC50 estimation
Lyophilized synthetic Aβ1-40 was purchased from Peptide Institute Inc. (Osaka, Japan).
Aβ fibrils were generated by spontaneous assembly. Aβ1-40 peptides (final
concentration: 100 nM) were dissolved in 10 mM potassium phosphate buffered saline
(PBS, pH7.4) containing 1 mM EDTA and incubated at 37 °C for 4 days with gentle and
constant shaking. Aggregated Aβ fibrils were stored at 4 °C until used.
The 100-µL aliquots of synthetic human A fibrils were combined with 900 µL
of PBS containing [125I]IMPY (0.1 nM), 1% EtOH, and 20 mM ascorbic acid in the
presence of compounds with a concentration ranging from 0.04 to 1000 nM.The
reaction mixture was incubated at 37°C for 3 h, followed by vacuum filtration through
Whatman GF/B filters using a Brandel cell harvester (Model M-24R, Biomedical
Research. Lab., Gaithesburg, MD). The GF/B filters were counted by gamma counter
after three rapid washes with 2 mL of ice-cold PBS containing 0.1mM ascorbic acid. No
and full inhibitory effects were measured with the addition of vehicle and
nonradiolabeled IMPY (500 nM), respectively. The IC50 was generated by Scatchard
25

analysis using GraphPad Prism (GraphPad Software, version 4.0, San Diego, CA).
4.4. LogD7.4 measurements. The experimental determination of the partition
coefficients was performed according to a method previously published, which employs
HPLC.22,23 The compounds for the standard curve were analyzed by HPLC with the
following conditions: symmetry C18 column (3.9×150 mm; Waters Corporation);
mobile phase, e.g., 20 mM 3-morpholinopropanesulfonic acid (pH 7.4) containing
various methanol concentration (from 60% to 85%), flow rate (1.0 ml/min), UV
wavelength (256 or 300 nm. The capacity factor at various methanol concentrations
(k%) was calculated according to a method previously published24, using the following
equation: k%=(t%-t0)/t0, where t% and t0 are the retention times of the ligand and the
non-retained peak, respectively. The logarithms of the capacity factors (log k%) were
plotted versus methanol concentrations; log kw was obtained by linear extrapolation to
0% methanol concentration. The log D7.4 values of standard compounds were obtained
from previously published data22,23; those of novel compounds were calculated in this
work from the standard curve based on their log kw values.
4.5. Biodistribution and metabolite analysis in normal rats
26

Normal Sprague-Dawley (SD) rats (8 weeks old) for biodistribution and metabolite
analyses were purchased from Charles River Laboratories Japan Inc. (Yokohama,
Japan). The rats were administered with radioligands (1.85 MBq/kg body weight for
each ligand) via the tail vein, and euthanized by cervical dislocation under isoflurane
anesthesia at designed time points. Radioactivity in the organs of interest was measured
by gamma counter (Wallac Wizard 1480; PerkinElmer Wallac Inc, Turku, Finland). The
uptake of radiolabeled ligands was expressed as a percentage of the injected dose per
gram of tissue (% of ID/g tissue), except for the thyroid uptake, which was expressed as
percentage of the injected dose (% of ID).
In order to carry out the metabolite analysis, the brain samples were collected at specific
times in additional experiments. In particular, the collected brain tissues were
homogenized in 2-fold volume of saline, containing 0.1% tween 80 and 40 mM sodium
L-ascorbate. 50-µL aliquots of samples were then added to the same volumes of
acetonitrile, and the mixtures were applied to a Mini-UniPrep (UN203NPUORG
0.45um, GE Healthcare Bio-Sciences, Piscataway Township, NJ), followed by filtration
to remove the denatured proteins. The filtrates were applied to TLC plates and eluted by
80% ethanol in water. Radioactivity of separated spots was quantified by
autoradiography.
27

4.6. In-vitro autoradiographic analysis
Postmortem human brains for in vitro autoradiographic analysis were obtained from the
Center for Neurodegenerative Disease Research at the University of Pennsylvania
(Perelman School of Medicine), from patients with AD. Deparaffinized brain sections
(thickness of 10 μm) were preincubated with a blocking solution (1% BSA, 4% ethanol,
40 mM sodium L-ascorbate in PBS) for 30 min, and then incubated with [125I]DRM106
(0.5 nM) at room temperature for 3 h, with/without non-radiolabeled DRM106 (10 μM).
The brain sections were then dipped in PBS for 10 s and rinsed with 20% ethanol for 1
min, followed by rinsing with PBS six times, for 5 min each, and finally dipped into
distilled water for 10 s. The brain sections were blow-dried with warm air and attached
to imaging plates (BAS-MS2025; Fujifilm, Tokyo, Japan) overnight. Radiolabeling was
then detected by scanning the imaging plate, using the BAS5000 system (Fujifilm) or
Typhoon FLA 7000 scanner system (GE Healthcare). The same brain sections were used
for
counterstaining
of
plaques
with
0.01%
(E,E)-1-fluoro-2,5-bis(3-hydroxycarbony-4-hydroxy)styrylbenzene (FSB; Dojindo
Laboratories, Kumamoto, Japan) as described in our previous study.2
28

Acknowledgments
The authors thank Prof. John Q. Trojanowski and Prof. Virginia M.-Y. Lee (Center for
Neurodegenerative Disease Research, University of Pennsylvania) for kindly providing
human tissue. This work was supported in part by Grants-in-Aid for Japan Advanced
Molecular Imaging Program and Core Research for Evolutional Science and
Technology (T. S.), and Scientific Research on Innovative Areas (“Brain Environment”)
23111009 (M. H.) from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
29