Characterization of Novel 18F-Labeled Phenoxymethylpyridine Derivatives as Amylin Imaging Probes

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00756

Deposition of islet amyloid consisting of amylin constitutes one of pathological hallmarks of type 2 diabetes mellitus (T2DM), and it may be involved in the development and progression of T2DM. However, the details about the relationship between the depos

Full text of DOI:10.1021/acs.molpharmaceut.8b00756

Article
pubs.acs.org/molecularpharmaceutics
Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX
18
Characterization of Novel F‑Labeled Phenoxymethylpyridine
Derivatives as Amylin Imaging Probes
Hiroyuki Watanabe,† Masashi Yoshimura,† Kohei Sano,‡ Yoichi Shimizu,‡ Sho Kaide,† Yuji Nakamoto,§
Kaori Togashi,§ Masahiro Ono,*,† and Hideo Saji†
†Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida
Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
‡Division of Clinical Radiology Service, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
§Department of Diagnostic Imaging and Nuclear Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
S
* Supporting Information
ABSTRACT: Deposition of islet amyloid consisting of amylin
constitutes one of pathological hallmarks of type 2 diabetes mellitus
(T2DM), and it may be involved in the development and progression
of T2DM. However, the details about the relationship between the
deposition of islet amyloid and the pathology of T2DM remain
unclear, since no useful imaging tracer enabling the visualization of
pancreatic amylin is available. In the present study, we synthesized
and evaluated six novel 18F-labeled phenoxymethylpyridine (PMP)
derivatives as amylin imaging probes. All 18F-labeled PMP derivatives
showed not only affinity for islet amyloid in the post-mortem T2DM
pancreatic sections but also excellent pharmacokinetics in normal
mice. Furthermore, ex vivo autoradiographic studies demonstrated
that [18F]FPMP-5 showed intense labeling of islet amyloids in the
diabetes model mouse pancreas in vivo. The preclinical studies suggested that [18F]FPMP-5 may have potential as an imaging
probe that targets amylin aggregates in the T2DM pancreas.
KEYWORDS: amylin, imaging, type 2 diabetes, fluorine-18
them, type 2 diabetes mellitus (T2DM) is the most common
form of diabetes, accounting for 90−95% of people with
diabetes mellitus.1 The current diagnosis of T2DM is based on
the blood glucose level and glucose tolerance.5−7 However,
this method is unable to detect T2DM in the early stage.8
Since T2DM is known to be a progressive disease, the
therapeutic intervention in the early phase is important.
Therefore, the development of a novel method for the early
diagnosis of T2DM is strongly expected.
The gradual and progressive loss of the β-cell mass (BCM)
is believed to be a characteristic of T2DM.9−11 Some reports
state that BCM in T2DM patients is reduced to less than 50%
of that of healthy controls.11 Therefore, a number of
radioactive agents for in vivo imaging of pancreatic β-cells
have been developed for the purpose of improving under-
standing of the pathophysiology of T2DM.12−15 In the future,
β-cell imaging may be used as a critical tool for understanding
the progression of T2DM. However, it is considered that the
INTRODUCTION
■
Diabetes mellitus is classified as metabolic disorders charac-
terized by hyperglycemia due to defects in insulin secretion,
insulin action, or both.1 The chronic hyperglycemia of diabetes
mellitus induces metabolic failure of major nutrients, such as
fat and protein.1 Moreover, diabetes mellitus is associated with
a number of complications, namely, diabetic nephropathy,
diabetic retinopathy, and diabetic neuropathy.1 These diabetic
complications not only greatly reduce the quality-of-life of
diabetes patients, but also shorten their lifespan. Recent
research revealed that adults with diabetes died 4.6 years earlier
than adults without diabetes.2 The World Health Organization
stated that the number of people with diabetes had risen
markedly and there were approximately 422 million diabetes
patients worldwide in 2014, which equates to 8.5% of the adult
population.3 Due to population aging, rapid urbanization, and
changes in dietary habits, the number of people suffering from
diabetes is still increasing throughout the world. The
International Diabetes Federation produced estimates of
diabetes prevalence in 2013, on the basis of which the number
is expected to rise to 592 million by 2035.4 According to its
cause, diabetes mellitus is classified into four categories, type 1,
type 2, other types, and gestational diabetes mellitus.1 Among
Received: July 16, 2018
Revised: October 9, 2018
Accepted: October 29, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.molpharmaceut.8b00756
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Figure 1. Chemical structures of [125I]IPBF and [99mTc]1.
Figure 2. Chemical structures of 18F-labeled PMP derivatives.
proliferation of β-cells and insulin secretion are promoted in
order to compensate for insulin resistance in the early phase of
T2DM,16,17 which means that the BCM may not decrease
monotonically. Previous research using rodent models
reported that BCM expansion was observed under insulin-
resistant conditions.18 From the above, we considered that it is
difficult to diagnose T2DM in the early phase solely based on
β-cell imaging.
low initial uptake by the pancreas of the normal mouse,
whereas it showed intensive accumulation in the liver.25 For in
vivo amylin imaging, a low initial uptake of an imaging probe
by the pancreas weakens the on-target signal; on the other
hand, a high accumulation in the liver leads to the noise signal.
Therefore, structural modification based on [99mTc]1 may be
essential in order to improve the pharmacokinetics. Herein, we
describe a series of phenoxymethylpyridine (PMP) derivatives,
with a structure simpler than PBF as a binding moiety.
Recently, benzyloxybenzene, whose chemical structure is
similar to PMP, was reported to be a promising scaffold for
imaging agents targeting β-amyloid (Aβ) plaques.26−28 In
addition, our previous research revealed the feasibility of
amylin imaging with Aβ imaging probes.22 Taken together, we
hypothesized that PMP derivatives have the potential to act as
amylin imaging probes. The iminodiacetic acid (IDA) core, the
99mTc-chelating moiety of [99mTc]1, is sometimes prone to
accumulation in the liver.29,30 Instead of 99mTc, we introduce
18F into PMP derivatives via variable hydrophilic linkers.
Considering the above, we designed, synthesized, and
evaluated six novel PMP derivatives as amylin imaging probes
(Figure 2). In our previous study, ex vivo experiments with
islet amyloid model mice established by the orthotopic
implantation method were carried out in order to determine
the in vivo binding affinity of ligands.25 These model mice
were suitable for the screening of amylin imaging probes in
vivo; however, they probably do not show symptoms of
diabetes. In this study, we conducted ex vivo experiments using
obese transgenic mice, which express human amylin peptides
and develop an islet pathology resembling that in humans with
T2DM.31,32 Therefore, the present study shows PMP
derivatives’ binding affinity in vivo for islet amyloids, which
was evaluated under conditions close to those of the living
T2DM pancreas.
Islet amyloid deposits are one of the characteristic
pathologic features of the T2DM pancreas.19 Islet amyloid
mainly forms by the aggregation of amylin, also known as islet
amyloid polypeptide (IAPP) consisting of a 37-amino acid
peptide.20 Amylin is a neuroendocrine peptide hormone that is
coproduced and cosecreted along with insulin from pancreatic
β-cells.20 The aggregation of amylin peptide has been
suggested to occur in a stepwise process. Monomeric amylin
peptide forms amyloid fibrils via soluble oligomers and
protofibrils, some of which are toxic to the pancreatic β-
cells.20 In addition, the amount of islet amyloid formation has
been reported to be correlated with a BCM reduction.21 Since
islet amyloid depositions are found in up to 90% of T2DM
patients at autopsy, it may be common to the T2DM
pathology.19 Taken together, islet amyloid deposition may be
involved in the pathogenesis of T2DM. Despite its potential
importance, the lack of a method for visualizing amyloid
depositions in the T2DM pancreas makes it difficult to
conduct research on islet amyloid in vivo. Therefore, the
development of a radiotracer for amylin imaging may facilitate
intensive research focusing on the pathogenesis of T2DM.
We previously found out that the Aβ imaging probe, 125I-
labeled pyridyl benzofuran (PBF), has the potential to serve as
an amylin imaging probe.22 More recently, other groups
reported that two Aβ imaging probes, [18F]FDDNP and
[18F]florbetapir, showed the feasibility for amylin imaging.23,24
However, the pharmacokinetics of these probes in pancreas
and its surrounding organs is not suitable for in vivo imaging of
amylin. We also reported that 99mTc-labeled PBF derivatives
meet the fundamental requirements for radioactive amylin
imaging probes (Figure 1).25 In particular, [99mTc]1 showed
specific binding to amylin aggregates transplanted into the
pancreas of a living mouse.25 However, [99mTc]1 displayed a
EXPERIMENTAL SECTION
■
General Remarks. All reagents were commercial products
and used without further purification unless indicated
otherwise. W-Prep 2XY (Yamazen Corporation, Osaka,
Japan) was used for silica gel column chromatography on a
B
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Hi Flash silica gel column (40 μm, 60 Å, Yamazen) or Hi Flash
aminopropyl silica gel column (40 μm, 60 Å, Yamazen). LC-
20AD (Shimadzu Corporation, Kyoto, Japan) equipped with
an SPD-20A UV detector (Shimadzu) was used for high-
performance liquid chromatography (HPLC). LC-9101 (Japan
Analytical Industry, Tokyo, Japan) with a JAIGEL-2H column
and CHCl3 as the mobile phase was used for recycling
0 °C for 2 h. Water (3 mL), 2 N NaOH (3 mL), and water (3
mL) were added successively at 0 °C, and the resulting mixture
was stirred vigorously. The mixture was then filtered through
Celite. The filtrate was extracted with AcOEt (70 mL × 2), and
the organic layers were combined and dried over Na2SO4.
Evaporation of the solvent afforded a residue, which was
purified by silica gel chromatography (CHCl3/MeOH = 10/1)
to give 355 mg of 4 (55.9%). 1H NMR (400 MHz, CDCl3, δ):
8.01 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.4, 2.4 Hz, 1H), 6.39
(d, J = 8.4 Hz, 1H), 4.59 (s, 1H), 4.53 (s, 2H), 2.91 (d, J = 5.2
Hz, 3H), 1.94 (s, 1H). MS (ESI) (m/z): 139 [MH+].
1
preparative HPLC. H NMR spectra were recorded using a
JEOL JNM-ECS400 or JEOL ECA-500 spectrometer, and
chemical shifts are reported in δ (ppm) relative to TMS as an
internal standard. Coupling constants are reported in Hertz.
Multiplicity was defined as singlet (s), doublet (d), triplet (t),
or multiplet (m). Low-resolution mass spectra (LRMS) were
obtained using a SHIMADZU LCMS-2020. High-resolution
mass spectrometry (HRMS) was conducted with a JEOL JMS-
700.
Chemistry. Methyl 6-(dimethylamino)nicotinate (1). To a
solution of methyl 6-aminonicotinate (1.52 g, 10 mmol) in
acetic acid (40 mL) was added paraformaldehyde (3.0 g, 100
mmol) and NaBH3CN (1.88 g, 30 mmol). The reaction
mixture was stirred at room temperature for 23 h and then
quenched with 2 N NaOH aq. The solution was extracted with
AcOEt (100 mL × 2). The organic layers were combined and
dried over Na2SO4. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
(AcOEt/hexane = 1/3) to give 1.54 g of 1 (85.5%). 1H NMR
(400 MHz, CDCl3, δ): 8.80 (d, J = 2.3 Hz, 1H), 8.00 (dd, J =
9.0, 2.0 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H), 3.86 (s, 3H), 3.17
(s, 6H). MS (ESI) (m/z): 181 [MH+].
2-(4-((6-(Dimethylamino)pyridin-3-yl)methoxy)phenoxy)-
ethyl 4-methylbenzenesulfonate (5). 2-(4-Hydroxyphenoxy)-
ethyl 4-methylbenzenesulfonate was prepared according to ref
28. To a solution of 2 (152 mg, 1 mmol) and 2-(4-
hydroxyphenoxy)ethyl 4-methylbenzenesulfonate (308.4 mg, 1
mmol) in THF (10 mL) was added triphenylphosphine (393.4
mg, 1.5 mmol), and then the mixture was cooled to 0 °C.
Diisopropyl azodicarboxylate (303.3 mg, 1.5 mmol) in THF (5
mL) was added dropwisely at 0 °C, and then the reaction
mixture was stirred at room temperature for 8.5 h. To the
solution, triphenylphosphine (78.7 mg, 0.3 mmol) and
diisopropyl azodicarboxylate (60.7 mg, 0.3 mmol) were
added, and then the solution was stirred at room temperature
for 18.5 h. Evaporation of the solvent afforded a residue, which
was purified by silica gel chromatography (AcOEt/hexane = 1/
1) to give 121 mg of 5 (27.3%). 1H NMR (400 MHz, CDCl3,
δ): 8.17 (d, J = 2.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.51 (dd,
J = 8.8, 2.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 9.2
Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 9.2 Hz, 1H),
4.84 (s, 2H), 4.34 (t, J = 4.8 Hz, 2H), 4.09 (t, J = 4.8 Hz, 2H),
3.09 (s, 6H), 2.45 (s, 3H). MS (ESI) (m/z): 443 [MH+].
2-(4-((6-(Methylamino)pyridin-3-yl)methoxy)phenoxy)-
ethyl 4-methylbenzenesulfonate (6). To a solution of 4
(138.2 mg, 1 mmol) and 2-(4-hydroxyphenoxy)ethyl 4-
methylbenzenesulfonate (277.5 mg, 0.9 mmol) in THF (6
mL) was added triphenylphosphine (524.6 mg, 2 mmol), and
then the mixture was cooled to 0 °C. Diisopropyl
azodicarboxylate (404.4 mg, 2 mmol) in THF (3 mL) was
added dropwisely at 0 °C, and then the reaction mixture was
stirred at room temperature for 5 h. Evaporation of the solvent
afforded a residue, which was purified by silica gel
chromatography (AcOEt/hexane = 3/1) and recycling
preparative HPLC with CHCl3 to give 83.5 mg of 6
(6-(Dimethylamino)pyridin-3-yl)methanol (2). A solution
of 1 (1.56 g, 8.63 mmol) in THF (20 mL) was added
dropwisely to a stirred suspension of lithium aluminum
hydride (655 mg, 17.3 mmol) in THF (20 mL) at −78 °C.
The reaction mixture was stirred at −78 °C for 4.5 h and then
at 0 °C for 1 h. Water (3 mL), 2 N NaOH (3 mL), and water
(3 mL) were added successively at 0 °C, and the resulting
mixture was stirred vigorously. The mixture was then filtered
through Celite. The filtrate was extracted with AcOEt (70 mL
× 2), and the organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which
was purified by silica gel chromatography (AcOEt/hexane = 5/
1
1) to give 1.02 g of 2 (77.4%). H NMR (500 MHz, CDCl3,
δ): 8.10 (d, J = 2.3 Hz, 1H), 7.50 (dd, J = 8.6, 2.3 Hz, 1H),
6.51 (d, J = 8.8 Hz, 1H), 4.53 (s, 2H), 3.09 (s, 6H), 1.70 (s,
1H). MS (ESI) (m/z): 153 [MH+].
1
(21.6%). H NMR (400 MHz, CDCl3, δ): 8.10 (d, J = 2.4
Methyl 6-(methylamino)nicotinate (3). To a solution of
methyl 6-chloronicotinate (2.0 g, 11.7 mmol) in DMF (20
mL) was added methylamine (30% in MeOH, 4 mL). The
reaction mixture was stirred at 50 °C for 19 h. The solution
was allowed to cool to room temperature and then extracted
with AcOEt (70 mL × 2). The organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
(AcOEt/hexane = 1/1) to give 764 mg of 3 (39.4%). 1H NMR
(400 MHz, CDCl3, δ): 8.75 (d, J = 2.0 Hz, 1H), 8.01 (dd, J =
8.8, 2.0 Hz, 1H), 6.36 (d, J = 9.2 Hz, 1H), 5.05 (s, 1H), 3.87
(s, 3H), 2.99 (d, J = 5.2 Hz, 3H). MS (ESI) (m/z): 167
[MH+].
Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.53 (dd, J = 8.4, 2.4 Hz,
1H), 7.34 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 9.2 Hz, 2H), 6.72
(d, J = 9.2 Hz, 2H), 6.43 (d, J = 8.4 Hz, 1H), 4.84 (s, 2H),
4.79 (s, 1H), 4.34 (t, J = 4.8 Hz, 2H), 4.10 (t, J = 4.8 Hz, 2H),
2.93 (d, J = 4.8 Hz, 3H), 2.45 (s, 3H). MS (ESI) (m/z): 429
[MH+].
2-(2-(2-(4-((6-(Dimethylamino)pyridin-3-yl)methoxy)-
phenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate
(7). 2-(2-(2-(4-Hydroxyphenoxy)ethoxy)ethoxy)ethyl 4-meth-
ylbenzenesulfonate was prepared according to ref 28. To a
solution of 2 (152.2 mg, 1 mmol) and 2-(2-(2-(4-
hydroxyphenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfo-
nate (396.5 mg, 1 mmol) in THF (6 mL) was added
triphenylphosphine (393.4 mg, 1.5 mmol), and then the
mixture was cooled to 0 °C. Diisopropyl azodicarboxylate
(303.3 mg, 1.5 mmol) in THF (2 mL) was added dropwisely
at 0 °C, and then the reaction mixture was stirred at room
temperature for 20 h. Evaporation of the solvent afforded a
(6-(Methylamino)pyridin-3-yl)methanol (4). A solution of
3 (764 mg, 4.60 mmol) in THF (15 mL) was added
dropwisely to a stirred suspension of lithium aluminum
hydride (349 mg, 9.20 mmol) in THF (15 mL) at −78 °C.
The reaction mixture was stirred at −78 °C for 3 h and then at
C
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residue, which was purified by silica gel chromatography
(AcOEt/hexane = 3/1) to give 279.1 mg of 7 (52.6%). H
organic layers were combined and dried over Na2SO4.
Evaporation of the solvent afforded a residue, which was
purified by silica gel chromatography (AcOEt/hexane = 3/1)
1
NMR (500 MHz, CDCl3, δ): 8.18 (d, J = 2.5 Hz, 1H), 7.80 (d,
J = 8.5 Hz, 2H), 7.53 (dd, J = 8.5, 2.5 Hz, 1H), 7.33 (d, J = 8.5
Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H),
6.53 (d, J = 8.5 Hz, 1H), 4.85 (s, 2H), 4.16 (t, J = 5.0 Hz, 2H),
4.06 (t, J = 4.5 Hz, 2H), 3.79 (t, J = 5.5 Hz, 2H), 3.70 (t, J =
5.0 Hz, 2H), 3.67−3.60 (m, 4H), 3.09 (s, 6H), 2.43 (s, 3H).
MS (ESI) (m/z): 531 [MH+].
1
to give 67.8 mg of 11 (59.7%). H NMR (500 MHz, CDCl3,
δ): 8.18 (d, J = 2.0 Hz, 1H), 7.52 (dd, J = 9.0, 2.5 Hz, 1H),
6.89−6.83 (m, 4H), 6.53 (d, J = 9.0 Hz, 1H), 4.85 (s, 2H),
4.62−4.51 (m, 2H), 4.09 (t, J = 4.5 Hz, 2H), 3.84 (t, J = 4.5
Hz, 2H), 3.80−3.71 (m, 6H), 3.09 (s, 6H). HRMS (FAB+)
(m/z): [MH+] calcd for C20H28FN2O4, 379.2033; found,
379.2043.
2-(2-(2-(4-((6-(Methylamino)pyridin-3-yl)methoxy)-
phenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate
(8). To a solution of 4 (138.2 mg, 1 mmol) and 2-(2-(2-(4-
hydroxyphenoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfo-
nate (396.5 mg, 1 mmol) in THF (6 mL) was added
triphenylphosphine (393.4 mg, 1.5 mmol), and then the
mixture was cooled to 0 °C. Diisopropyl azodicarboxylate
(303.3 mg, 1.5 mmol) in THF (2 mL) was added dropwisely
at 0 °C, and then the reaction mixture was stirred at room
temperature for 17 h. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
5-((4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)phenoxy)-
methyl)-N-methylpyridin-2-amine (12: FPMP-4). To a
solution of 8 (87.8 mg, 0.17 mmol) in THF (6 mL) was
added TBAF solution (1 M in THF, 0.68 mL). The reaction
mixture was refluxed for 2.5 h. The solution was allowed to
cool to room temperature and then extracted with AcOEt (60
mL × 2). The organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which
was purified by aminopropyl silica gel chromatography
1
(AcOEt/hexane = 3/1) to give 39.7 mg of 12 (64.1%). H
1
(AcOEt/hexane = 3/1) to give 115.1 mg of 8 (22.3%). H
NMR (400 MHz, CDCl3, δ): 8.12 (d, J = 2.4 Hz, 1H), 7.52
(dd, J = 8.4, 2.0 Hz, 1H), 6.89−6.84 (m, 4H), 6.41 (d, J = 8.8
Hz, 1H), 4.85 (s, 2H), 4.64−4.50 (m, 3H), 4.09 (t, J = 4.8 Hz,
2H), 3.84 (t, J = 5.2 Hz, 2H), 3.81−3.70 (m, 6H), 2.93 (d, J =
5.2 Hz, 3H). HRMS (FAB+) (m/z): [MH+] calcd for
C19H26FN2O4, 365.1877; found, 365.1884.
NMR (500 MHz, CDCl3, δ): 8.11 (d, J = 2.0 Hz, 1H), 7.80 (d,
J = 8.0 Hz, 2H), 7.53 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 8.0 Hz,
2H), 6.88 (d, J = 9.0 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 6.42
(d, J = 8.5 Hz, 1H), 4.85 (s, 2H), 4.68 (s, 1H), 4.16 (t, J = 4.5
Hz, 2H), 4.06 (t, J = 4.5 Hz, 2H), 3.80 (t, J = 5.0 Hz, 2H), 3.70
(t, J = 5.0 Hz, 2H), 3.66−3.60 (m, 4H), 2.93 (d, J = 5.5 Hz,
3H), 2.43 (s, 3H). MS (ESI) (m/z): 517 [MH+].
5-((4-(2-Fluoroethoxy)phenoxy)methyl)-N,N-dimethylpyr-
idin-2-amine (9: FPMP-1). To a solution of 5 (88.5 mg, 0.2
mmol) in THF (7 mL) was added TBAF solution (1 M in
THF, 0.64 mL). The reaction mixture was refluxed for 2 h.
The solution was allowed to cool to room temperature and
then extracted with AcOEt (70 mL × 2). The organic layers
were combined and dried over Na2SO4. Evaporation of the
solvent afforded a residue, which was purified by silica gel
chromatography (AcOEt/hexane = 1/1) to give 30.0 mg of 9
(51.6%). 1H NMR (500 MHz, CDCl3, δ): 8.18 (d, J = 2.5 Hz,
1H), 7.52 (dd, J = 9.0, 2.5 Hz, 1H), 6.91−6.85 (m, 4H), 6.53
(d, J = 9.0 Hz, 1H), 4.86 (s, 2H), 4.79−4.68 (m, 2H), 4.20−
4.13 (m, 2H), 3.09 (s, 6H). HRMS (FAB+) (m/z): [MH+]
calcd for C16H20FN2O2, 291.1509; found, 291.1506.
(5-(4-(Benzyloxy)phenoxy)-2,2-dimethyl-1,3-dioxolan-4-
yl)methyl 4-methylbenzenesulfonate (13). To a solution of
methyl 4-(benzyloxy)phenol (1.0 g, 5 mmol) in DMF (10 mL)
was added K2CO3 (1.38 g, 10 mmol) and (−)-1,4-di-O-tosyl-
2,3-O-isopropylidene-L-threitol (2.82 g, 6 mmol). The reaction
mixture was stirred at 70 °C for 21 h. The solution was allowed
to cool to room temperature and then extracted with AcOEt
(70 mL × 2). The organic layers were combined and dried
over Na2SO4. Evaporation of the solvent afforded a residue,
which was purified by silica gel chromatography (AcOEt/
1
hexane = 1/5) to give 1.64 g of 13 (66.0%). H NMR (400
MHz, CDCl3, δ): 7.80 (d, J = 8.0 Hz, 2H), 7.44−7.31 (m,
6H), 6.89 (d, J = 9.2 Hz, 2H), 6.77 (d, J = 9.2 Hz, 2H), 5.02
(s, 2H), 4.26 (dd, J = 9.6, 3.2 Hz, 1H), 4.20−4.05 (m, 5H),
3.93 (dd, J = 9.6, 4.8 Hz, 1H), 2.43 (s, 3H), 1.41 (s, 3H), 1.38
(s, 3H). MS (APCI) (m/z): 499 [MH+].
5-((4-(2-Fluoroethoxy)phenoxy)methyl)-N-methylpyridin-
2-amine (10: FPMP-2). To a solution of 6 (68.6 mg, 0.16
mmol) in THF (6 mL) was added TBAF solution (1 M in
THF, 0.64 mL). The reaction mixture was refluxed for 2 h.
The solution was allowed to cool to room temperature and
then extracted with AcOEt (60 mL × 2). The organic layers
were combined and dried over Na2SO4. Evaporation of the
solvent afforded a residue, which was purified by silica gel
chromatography (AcOEt/hexane = 3/1) to give 33.2 mg of 10
(75.1%). 1H NMR (400 MHz, CDCl3, δ): 8.12 (d, J = 2.4 Hz,
1H), 7.52 (dd, J = 8.4, 2.4 Hz, 1H), 6.91−6.85 (m, 4H), 6.41
(d, J = 8.8 Hz, 1H), 4.85 (s, 2H), 4.81−4.67 (m, 2H), 4.57 (s,
1H), 4.21−4.12 (m, 2H), 2.93 (d, J = 5.2 Hz, 3H). HRMS
(FAB+) (m/z): [MH+] calcd for C15H18FN2O2, 277.1352;
found, 277.1359.
5-((4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)phenoxy)-
methyl)-N,N-dimethylpyridin-2-amine (11: FPMP-3). To a
solution of 7 (159.2 mg, 0.3 mmol) in THF (6 mL) was added
TBAF solution (1 M in THF, 1.2 mL). The reaction mixture
was refluxed for 2 h. The solution was allowed to cool to room
temperature and then extracted with AcOEt (70 mL × 2). The
(5-(4-Hydroxyphenoxy)-2,2-dimethyl-1,3-dioxolan-4-yl)-
methyl 4-methylbenzenesulfonate (14). To a solution of 13
(1.64 g, 3.29 mmol) in a mixture of MeOH (25 mL) and THF
(10 mL) was added 10% Pd/C (150 mg). The reaction
mixture was stirred vigorously for 7 h at 50 °C under an H2
atmosphere. The reaction mixture was filtered through Celite,
and then the filtrate was evaporated. The residue was purified
by silica gel chromatography (AcOEt/hexane = 1/3) to give
1.01 g of 14 (75.2%). 1H NMR (400 MHz, CDCl3, δ): 7.80 (d,
J = 8.0 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 6.78−6.72 (m, 4H),
4.54 (s, 1H), 4.26 (dd, J = 9.6, 3.2 Hz, 1H), 4.20−4.05 (m,
4H), 3.93 (dd, J = 9.2, 4.0 Hz, 1H), 2.44 (s, 3H), 1.41 (s, 3H),
1.38 (s, 3H). MS (APCI) (m/z): 409 [MH+].
(5-(4-((6-(Dimethylamino)pyridin-3-yl)methoxy)-
phenoxy)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methyl-
benzenesulfonate (15). To a solution of 2 (152.2 mg, 1
mmol) and 14 (408.5 mg, 1 mmol) in THF (6 mL) was added
triphenylphosphine (393.4 mg, 1.5 mmol), and then the
mixture was cooled to 0 °C. Diisopropyl azodicarboxylate
(303.3 mg, 1.5 mmol) in THF (3 mL) was added dropwisely
at 0 °C, and then the reaction mixture was stirred at room
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temperature for 19 h. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
(AcOEt/hexane = 1/1) to give 339.5 mg of 15 (62.6%). H
NMR (400 MHz, CDCl3, δ): 8.18 (d, J = 2.0 Hz, 1H), 7.80 (d,
J = 8.0 Hz, 2H), 7.53 (dd, J = 8.8, 2.4 Hz, 1H), 7.34 (d, J = 8.4
Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H),
6.53 (d, J = 9.2 Hz, 1H), 4.86 (s, 2H), 4.26 (dd, J = 10.0, 3.2
Hz, 1H), 4.20−4.05 (m, 4H), 3.93 (dd, J = 9.2, 4.0 Hz, 1H),
3.10 (s, 6H), 2.43 (s, 3H), 1.41 (s, 3H), 1.38 (s, 3H). MS
(ESI) (m/z): 543 [MH+].
(2,2-Dimethyl-5-(4-((6-(methylamino)pyridin-3-yl)-
methoxy)phenoxy)-1,3-dioxolan-4-yl)methyl 4-methylben-
zenesulfonate (16). To a solution of 4 (138.2 mg, 1 mmol)
and 14 (408.5 mg, 1 mmol) in THF (6 mL) was added
triphenylphosphine (393.4 mg, 1.5 mmol), and then the
mixture was cooled to 0 °C. Diisopropyl azodicarboxylate
(303.3 mg, 1.5 mmol) in THF (3 mL) was added dropwisely
at 0 °C, and then the reaction mixture was stirred at room
temperature for 16 h. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
(AcOEt/hexane = 3/1) and aminopropyl silica gel chromatog-
raphy (AcOEt/hexane = 1/1) to give 231.2 mg of 16 (43.7%).
1H NMR (500 MHz, CDCl3, δ): 8.12 (d, J = 2.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 2H), 7.52 (dd, J = 8.5, 2.0 Hz, 1H), 7.33 (d, J =
8.5 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H),
6.41 (d, J = 8.5 Hz, 1H), 4.85 (s, 2H), 4.59 (s, 1H), 4.26 (dd, J
= 10.5, 3.5 Hz, 1H), 4.20−4.06 (m, 4H), 3.93 (dd, J = 9.5, 4.5
Hz, 1H), 2.93 (d, J = 5.0 Hz, 3H), 2.43 (s, 3H), 1.41 (s, 3H),
1.38 (s, 3H). MS (ESI) (m/z): 529 [MH+].
2.5 h at room temperature. The solution was quenched with
sat. NaHCO3 and 2 N NaOH at 0 °C and then extracted with
AcOEt (20 mL × 2). The organic layers were combined and
dried over Na2SO4. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
1
1
(AcOEt/hexane = 3/1) to give 38.5 mg of 19 (62.0%). H
NMR (400 MHz, CDCl3, δ): 8.18 (d, J = 2.4 Hz, 1H), 7.52
(dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 9.2 Hz, 2H), 6.84 (d, J =
9.2 Hz, 2H), 6.53 (d, J = 8.8 Hz, 1H), 4.86 (s, 2H), 4.66−4.47
(m, 2H), 4.08−4.03 (m, 4H), 3.10 (s, 6H), 2.63 (br, 2H).
HRMS (FAB+) (m/z): [MH+] calcd for C18H24FN2O4,
351.1720; found 351.1718.
1-Fluoro-4-(4-((6-(methylamino)pyridin-3-yl)methoxy)-
phenoxy)butane-2,3-diol (20: FPMP-6). To a solution of 18
(77.8 mg, 0.207 mmol) in MeOH (6 mL) was added 2 N
HCl−MeOH (2.5 mL). The reaction mixture was stirred for 3
h at room temperature. The solution was quenched with sat.
NaHCO3 and 2 N NaOH at 0 °C and then extracted with
AcOEt (30 mL × 2). The organic layers were combined and
dried over Na2SO4. Evaporation of the solvent afforded a
residue, which was purified by silica gel chromatography
1
(CHCl3/MeOH = 10/1) to give 53.3 mg of 20 (76.7%). H
NMR (400 MHz, DMSO-d6, δ): 8.02 (d, J = 2.4 Hz, 1H), 7.43
(dd, J = 8.4, 2.0 Hz, 1H), 6.91 (d, J = 9.2 Hz, 2H), 6.85 (d, J =
9.2 Hz, 2H), 6.55 (dd, J = 9.6, 4.4 Hz, 1H), 6.43 (d, J = 8.4 Hz,
1H), 5.13−5.05 (m, 2H), 4.09 (s 2H), 4.58−4.31 (m, 2H),
4.01−3.96 (m, 1H), 3.83−3.80 (m, 3H), 2.75 (d, J = 4.8 Hz,
3H). HRMS (FAB+) (m/z): [MH+] calcd for C17H22FN2O4,
337.1564; found 337.1563.
5-((4-((5-(Fluoromethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-
oxy)phenoxy)methyl)-N,N-dimethylpyridin-2-amine (17). To
a solution of 15 (271.3 mg, 0.5 mmol) in THF (7 mL) was
added TBAF solution (1 M in THF, 2.0 mL). The reaction
mixture was refluxed for 5.5 h. The solution was allowed to
cool to room temperature and then extracted with AcOEt (60
mL × 2). The organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which
was purified by silica gel chromatography (AcOEt/hexane = 1/
Radiolabeling with 18F. We produced [18F]fluoride
according to our method reported previously.33 Kryptofix222
(10 mg) was dissolved in the solution of [18F]fluoride in water.
The solvent was removed at 120 °C under a stream of argon
gas. The residue was azeotropically dried with 300 μL of
anhydrous acetonitrile three times at 120 °C under a stream of
nitrogen gas. To prepare [18F]FPMP-1−4, a solution of the
corresponding tosylate precursor (1.0 mg, purity: ≥ 95%) in
acetonitrile (200 μL) was added to the reaction vessel
containing the 18F activity. The mixture was heated at 100
°C for 10 min. The 18F-labeled compound was purified by
HPLC on a COSMOSIL 5C18-AR-II column with an isocratic
solvent of acetonitrile/20 mM phosphate buffer, pH 7.0, at a
flow rate of 1.0 mL/min ([18F]FPMP-1 and [18F]FPMP-3:
acetonitrile/20 mM phosphate buffer, pH 7.0 = 40/60.
[18F]FPMP-2 and [18F]FPMP-4: acetonitrile/20 mM phos-
phate buffer, pH 7.0 = 32/68.). To prepare [18F]FPMP-5−6, a
solution of the corresponding tosylate precursor (1.0 mg) in
acetonitrile (200 μL) was added to the reaction vessel
containing the 18F activity. The mixture was heated at 100
°C for 10 min and subsequently cooled down. Then, 1 N HCl
(100 μL) was added, and the mixture was heated at 100 °C
again for 5 min. The solution was allowed to cool to room
temperature, and then 300 μL of sat. NaHCO3 aq. was added
to adjust the pH. The mixture was extracted with ethyl acetate,
and the solvent was evaporated under a stream of nitrogen gas.
The residue was dissolved in the mobile phase (100 μL), and
the solution was passed through a filter. The radiofluorinated
ligand was purified by HPLC on a COSMOSIL 5C18-AR-II
column with an isocratic solvent of acetonitrile/20 mM
phosphate buffer, pH 7.0, at a flow rate of 1.0 mL/min
([18F]FPMP-5: acetonitrile/20 mM phosphate buffer, pH 7.0
= 28/72; [18F]FPMP-6: acetonitrile/20 mM phosphate buffer,
1
3) to give 69.2 mg of 17 (35.5%). H NMR (500 MHz,
CDCl3, δ): 8.18 (d, J = 2.5 Hz, 1H), 7.52 (dd, J = 8.5, 2.5 Hz,
1H), 6.89 (d, J = 9.0 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 6.53
(d, J = 8.5 Hz, 1H), 4.86 (s, 2H), 4.71−4.50 (m, 2H), 4.28−
4.13 (m, 3H), 4.01 (dd, J = 10.5, 3.5 Hz, 1H), 3.09 (s, 6H),
1.47 (s, 6H). MS (ESI) (m/z): 391 [MH+].
5-((4-((5-(Fluoromethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-
oxy)phenoxy)methyl)-N-methylpyridin-2-amine (18). To a
solution of 16 (157.6 mg, 0.3 mmol) in THF (5 mL) was
added TBAF solution (1 M in THF, 1.2 mL). The reaction
mixture was refluxed for 4 h. The solution was allowed to cool
to room temperature and then extracted with AcOEt (70 mL ×
2). The organic layers were combined and dried over Na2SO4.
Evaporation of the solvent afforded a residue, which was
purified by silica gel chromatography (AcOEt/hexane = 1/1)
1
to give 81.2 mg of 18 (72.4%). H NMR (400 MHz, CDCl3,
δ): 8.12 (d, J = 2.4 Hz, 1H), 7.52 (dd, J = 8.8, 2.4 Hz, 1H),
6.89 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H), 6.41 (d, J =
8.8 Hz, 1H), 4.85 (s, 2H), 4.71−4.48 (m, 2H), 4.30−4.13 (m,
3H), 4.01 (dd, J = 9.2, 5.2 Hz, 1H), 2.93 (d, J = 5.2 Hz, 3H),
1.47 (s, 6H). MS (ESI) (m/z): 377 [MH+].
1-(4-((6-(Dimethylamino)pyridin-3-yl)methoxy)phenoxy)-
4-fluorobutane-2,3-diol (19: FPMP-5). To a solution of 17
(69.2 mg, 0.177 mmol) in MeOH (6 mL) was added 2 N
HCl−MeOH (2.0 mL). The reaction mixture was stirred for
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Scheme 1. Synthetic Route of FPMP-1−4
pH 7.0 = 20/80). The purity of all F-18 labeled compounds
was determined by HPLC.
Ex Vivo Autoradiography Using Diabetes Model Mice. A
saline solution (150 μL) of [18F]FPMP-5 (74.0 MBq)
containing EtOH (15 μL) was injected through the tail vein
of hIAPPTg/o-Avy/A mice (21 or 23 months-old, male, n = 2)
and a WT-Avy/A mouse (21 months-old, male, n = 1). The
animals were sacrificed by decapitation at 60 min post-
injection. The pancreas was immediately removed, embedded
in super cryoembedding medium (SCEM) compound
(SECTION-LAB Co., Ltd., Hiroshima, Japan), and then
frozen in a dry ice/hexane bath. Frozen sections were prepared
at a 20-μm thickness. The sections were exposed to a BAS
imaging plate (Fuji Film) overnight. Autoradiographic images
were obtained using a BAS5000 scanner system (Fuji Film).
After autoradiographic examination, the adjacent sections were
stained with ThS and an anti-amylin antibody to confirm the
presence of islet amyloids.
Immunohistochemical Staining Using Pancreas Sections
from Diabetes Model Mice. The sections were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30
min at room temperature, followed by two 3 min washes in
PBS. They were then autoclaved for 20 min in target retrieval
solution (Agilent Technologies) for antigen retrieval, followed
by two 3 min incubations in PBS. The sections were incubated
with MOM mouse IgG blocking reagent (Mouse on Mouse
Elite Peroxidase kit, Vector Laboratories, Burlingame, CA,
USA) for 1 h at room temperature, followed by two 2 min
incubations in PBS. The sections were incubated with MOM
diluent (Mouse on Mouse Elite Peroxidase kit, Vector
Laboratories) for 5 min at room temperature, and then the
excess of MOM diluent was removed. The sections were
incubated with mouse monoclonal amylin primary antibody
(1:200; R10/99, GeneTex, Inc., San Antonio, TX, USA),
which is diluted with MOM diluent, for 30 min at room
temperature. After two 2 min washes in PBS, they were
incubated with biotinylated goat antimouse IgG (Mouse on
Mouse Elite Peroxidase kit, Vector Laboratories) at room
temperature for 10 min. After two 2 min washes in PBS, the
sections were incubated with the streptavidin−peroxidase
complex at room temperature for 5 min. After one 5 min
incubation in PBS and one 5 min incubation in TBS, they were
incubated with DAB as a chromogen for 5 min. After being
washed with water, the sections were observed under a
microscope (FSX100; Olympus).
In Vitro Autoradiography Using Human Pancreas
Sections. We performed in vitro autoradiography using
human pancreas sections according to our method reported
previously.25 In this assay, the sections were incubated with
18F-labeled PMP derivatives (370 kBq/1 μL) and were then
washed with 10%EtOH (two 1 min washes) and rinsed with
water (one 30-s wash).
ThS Staining of Islet Amyloids in Pancreas Sections. We
performed ThS staining of islet amyloids in pancreas sections
according to our method reported previously.25
Immunohistochemical Staining Using Human Pancreas
Sections. We performed immunohistochemical staining using
human pancreas sections according to our method reported
previously.25 In this study, the sections were autoclaved for 20
min in target retrieval solution (Agilent Technologies, Santa
Clara, CA, USA) for antigen retrieval.
Animals. Animal experiments were conducted in accord-
ance with our institutional guidelines and were approved by
the Kyoto University Animal Care Committee. Male ddY mice
were purchased from Japan SLC, Inc. (Shizuoka, Japan). ddY
mice were fed standard chow and had free access to water.
FVB/N-Tg(Ins2-IAPP)RHFSoel/J, wild-type FVB/NJ, and
B6.C3-Avy/J mice were purchased from The Jackson
Laboratory (Bar Harbor, ME, USA). Wild-type C57BL/6J
mice were purchased from Charles River (Yokohama, Japan).
FVB/N-Tg(Ins2-IAPP)RHFSoel/J hemizygotes were bred
with wild-type FVB/NJ mice to produce hemizygous hIAPP
transgenic mice (hIAPPTg/o). FVB/N-Tg(Ins2-IAPP)-
RHFSoel/J mice were crossed with B6.C3-Avy/J mice to
generate hIAPPTg/o mice and wild-type littermate controls with
Avy/A at the agouti locus. These mice were fed a high-fat diet
containing 45 kcal% fat (D12451, Research Diets, New
Brunswick, NJ, USA) and had free access to water.
In Vivo Biodistribution in Normal Mice. We performed in
vivo biodistribution in normal mice according to our method
reported previously.25 In this study, we used 18F-labeled PMP
derivatives (23.6−63.5 kBq in 100 μL).
Analysis of Radiometabolites in Blood and Pancreas. We
performed analysis of radiometabolites in blood and pancreas
according to our method reported previously.34,35 In this study,
we used [18F]FPMP-5 (22.2−29.6 MBq in 150 μL), and the
mice were sacrificed at 2, 30, and 60 min post-injection.
F
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Scheme 2. Synthetic Route of FPMP-5−6
Scheme 3. 18F Labeling of PMP Derivatives
precursor to generate [18F]FPMP-5 and [18F]FPMP-6 with
radiochemical yields of 21.1 and 13.3%, respectively. Each
radiolabeling was accomplished with a radiochemical purity
greater than 95% with specific activity grater than 2.39 GBq/
μmol. The radioactive PMP derivatives were identified by a
comparison of the retention time with that of the non-
radioactive compounds.
Autoradiography of Post-Mortem Pancreatic Sec-
tions with 18F-Labeled PMP Derivatives. First, we tried to
perform the competitive inhibition assay or the saturation
binding assay to evaluate the binding affinity of FPMP
derivatives. However, because FPMP derivatives weakly bind
to synthetic amylin aggregates, it was difficult to compare the
binding affinity of FPMP derivatives using these assays. A
similar phenomenon was frequently reported in some papers
describing the development of tau imaging probes. In these
papers, the binding affinity of some tau imaging probes was
evaluated with in vitro autoradiographic study using human
brain sections, not with the binding assay using recombinant
tau proteins.34,36,37 Therefore, to evaluate the binding affinity
for islet amyloids, in vitro autoradiographic studies with 18F-
labeled PMP derivatives were carried out using post-mortem
human pancreatic sections. As shown in Figure 3, the
autoradiographic images of each 18F-labeled PMP derivative
indicated their specific binding to islet amyloids. The pattern
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of nonradioactive FPMP-1−4
and FPMP-5−6 is shown in Schemes 1 and 2, respectively.
The phenoxymethylpyridine backbone (5, 6, 7, 8, 15, and 16)
was obtained by the Mitsunobu reaction between the
substituted phenols (2-(4-hydroxyphenoxy)ethyl 4-methylben-
zenesulfonate, 2-(2-(2-(4-hydroxyphenoxy)ethoxy)ethoxy)-
ethyl 4-methylbenzenesulfonate, and 14 and corresponding
benzyl alcohols (2 and 4) in 21.6−62.6% yields. The fluorine
derivatives (FPMP-1−4, 17, and 18) were obtained by
refluxing the corresponding tosylate precursors (5, 6, 7, 8,
15, and 16) with tetra-n-buthylammonium fluoride in
anhydrous THF. Compounds 19 and 20 were subsequently
deprotected of acetals to afford FPMP-5 and FPMP-6.
Radiolabeling. The desired radiofluorinated PMP deriva-
tives were prepared from their corresponding tosylate
precursors, as shown in Scheme 3. [18F]FPMP-1,
[18F]FPMP-2, [18F]FPMP-3, and [18F]FPMP-4 were obtained
via a nucleophilic displacement reaction with a [18F]fluoride
anion with radiochemical yields of 42.2, 34.3, 44.4, and 42.2%,
respectively. To prepare [18F]FPMP-5 and [18F]FPMP-6, the
corresponding precursors were reacted with [18F]fluoride and
then treated with aqueous HCl for the deprotection of acetals.
Radiolabeling with 18F was successfully performed on the
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images, and intensity (PSL/mm2) in each region was
calculated for each probe. Then, we calculated the T2DM/
healthy control ratio and compared the binding affinity for
amylin aggregates between FPMP derivatives (Figure S3). This
result showed that the dimethylamino derivatives ([18F]FPMP-
1, [18F]FPMP-3, and [18F]FPMP-5) have a higher binding
affinity than the monomethylamino derivatives ([18F]FPMP-2,
[18F]FPMP-4, and [18F]FPMP-6). Taken together,
[18F]FPMP-1, [18F]FPMP-3, and [18F]FPMP-5 showed a
sufficient potential to function as amylin imaging probes.
In Vivo Biodistribution in Normal Mice. Important
requirements for amylin imaging agents involve the in vivo
biodistribution characteristics, such as avoidance of accumu-
lation in organs located near the pancreas and high uptake by
the pancreas. Since the off-target accumulation in organs
located near the pancreas impairs the signal-noise ratio, it is a
serious problem for amylin imaging in vivo. Especially, the liver
is located very close to the pancreas. Moreover, many
radiotracers generally accumulate in the liver since it plays a
major role in drug metabolism. Therefore, we evaluated the
liver accumulation of 18F-labeled PMP derivatives using normal
ddY mice (Figure 4A). The biodistribution experiment in mice
revealed moderate accumulation in the liver (11.0−23.7%ID/g
at 2 min). Our previous study demonstrated that [99mTc]1,
which is a 99mTc-labeled PBF derivative developed as an amylin
imaging probe, displayed initial intensive accumulation in the
liver (62.1%ID/g at 2 min).23 18F-labeled PMP derivatives
showed much lower accumulation in the liver than [99mTc]1,
which is highly desirable for amylin imaging probes. Since
several imaging probes with a 99mTc-IDA core showed high
uptake by the liver despite their low-to-moderate lipophilicity
as shown in the previous reports,29,30 the high [99mTc]1 uptake
by the liver may also be due to the characteristics of its chelate.
Therefore, exchange into the 18F-labeled hydrophilic linker
could help reduce the accumulation in the liver.
Figure 3. In vitro autoradiography of [18F]FPMP-1 (A), [18F]FPMP-
2 (B), [18F]FPMP-3 (C), [18F]FPMP-4 (D), [18F]FPMP-5 (E), and
[18F]FPMP-6 (F) with pancreatic sections from a T2DM patient.
Adjacent sections were also stained with an antibody against amylin
(G) and ThS (H).
of radioactivity accumulation was correlated with amylin
immunostaining and thioflavin-S (ThS) staining in adjacent
sections (Figures 2G and 2H, respectively). In contrast, no
accumulation of radioactivity was observed on autoradiography
using normal adult pancreas sections (Figures S2A−F),
although a dense area was stained positively with amylin
immunostaining (Figure S2G). ThS is a fluorescent dye
commonly used for the detection of amyloid fibrils. Since there
is no ThS labeling in normal pancreas sections (Figure S2H),
they contained only natural amylin peptides stored in the
pancreatic β-cells. Therefore, 18F-labeled PMP derivatives
displayed specific binding to amylin aggregates in islet
amyloids, not to natural amylin peptides. The regions of
interest (ROIs) were set in the T2DM pancreas section and
healthy control pancreas section in the autoradiographic
The high initial uptake by the target organ, that is the
pancreas, is essential for amylin imaging in vivo, and moreover,
imaging probes also need to have fast washout kinetics from
normal pancreatic tissues. Therefore, we also evaluated
biodistribution kinetics in the pancreas of 18F-labeled PMP
derivatives using normal mice. As shown in Figure 4B and
Tables S1−6, each PMP derivative exhibited high initial uptake
by the pancreas (4.67−6.00%ID/g at 2 min) and fast washout
kinetics from the healthy pancreas (0.68−2.72%ID/g at 60
Figure 4. Comparison of liver (A) and pancreas (B) uptakes of radioactivity after injection of [18F]FPMP-1−6 and [99mTc]1 into normal ddY mice
(male, n = 5). The data on [99mTc]1 are reprinted from ref 25.
H
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Figure 5. HPLC analysis of radiometabolites in the blood (A) and the pancreas (B) after injection of [18F]FPMP-5 into normal ddY mice (male, n
= 3−5).
min). Especially, [18F]FPMP-5 and [18F]FPMP-6 showed
excellent initial uptake by the pancreas (6.00 and 5.92%ID/g at
2 min, respectively). According to our previous report, [99mTc]
1 displayed low initial uptake by the normal mouse pancreas
(0.74%ID/g). We successfully developed novel radiolabeled
tracers, which exhibited good in vivo kinetics, by reduction of
the intensive exposure to the liver. Additionally, no marked
uptake of 18F-labeled PMP derivatives in bone was observed
(1.23−4.89%ID/g at 60 min), indicating that they may show
little defluorination in vivo, and interference with the imaging
may be negligible (Tables S1−6). 18F-labeled PMP derivatives
exhibited low initial uptake by the stomach (2.62−3.91%ID at
2 min), but the accumulation of [18F]FPMP-2, [18F]FPMP-3,
and [18F]FPMP-5 in the stomach increased with time (11.3−
15.7%ID/g at 60 min). Because the stomach is also located
near the pancreas, this pharmacokinetics may be unfavorable
for amylin imaging. Therefore, further improvement of the
stomach uptake should be necessary to develop more useful
probes for amylin imaging. In general, the chirality may
influence the biological activities, including pharmacokinetics
and metabolism.38−41 Because there are two chiral carbons in
FPMP-5 and FPMP-6, separation of optical isomers may
improve the performance as the amylin imaging probe.
analyzed by HPLC, we identified a metabolic profile in the
pancreas similar to that in the blood, suggesting that
[18F]FPMP-5 is rarely metabolized in the pancreas (Figure
5B). The percent of the intact form sequentially decreased and
reached 93.7, 60.8, and 36.3% at 2, 30, and 60 min after
injection, respectively (Figure 5B).
Ex Vivo Autoradiography of [18F]FPMP-5 Using
Transgenic Mice Expressing Human Amylin. To assess
the binding ability of [18F]FPMP-5 for islet amyloid in vivo, ex
vivo autoradiography studies were carried out using an obese
transgenic mouse model of T2DM expressing human amylin
(hIAPP) under the rat insulin promoter in addition to the
dominant agouti viable yellow mutation (Avy), called the
hIAPPTg/o-Avy/A mouse. We also carried out the same
experiments using a control mouse with Avy/A, called the
WT-Avy/A mouse. In the male hIAPPTg/o-Avy/A mouse, the
progressive development of diabetes was observed by 15 weeks
of age, and the islet amyloid was clearly present by 4 months of
age and was more extensive by 10 months.31,32 We conducted
ex vivo autoradiography studies using 21-month-old male
hIAPPTg/o-Avy/A and WT-Avy/A mice (Figure 6). Ex vivo
autoradiograms after the injection of [18F]FPMP-5 into a
hIAPPTg/o-Avy/A mouse showed the dense accumulation of
islet amyloids in the pancreas (Figure 6A). The radioactivity in
the pancreas showed an association with the signals obtained
with ThS staining and amylin immunostaining using the
adjacent pancreatic sections (Figure 6E,F,G). Conversely, no
such [18F]FPMP-5 labeling was observed in the WT-Avy/A
mouse pancreas (Figure 6C). Since the WT-Avy/A mouse
pancreas section showed no ThS staining (Figure 6D),
[18F]FPMP-5 may not bind to the normal pancreatic tissue.
This was consistent with fast washout kinetics from the healthy
pancreas in the biodistribution experiments (Figure 4B) and
low nonspecific binding to the pancreas tissue from a healthy
adult (Figure S2E).
In Vivo Metabolic Stability of [18F]FPMP-5. Considering
the results of the binding to islet amyloid together with the
biodistribution studies, we selected [18F]FPMP-5 for further
biological evaluations. We determined the in vivo metabolic
stability of [18F]FPMP-5 in normal ddY mice (Figure 5). After
the injection of [18F]FPMP-5 into ddY mice, the radio-
metabolites in the pancreas and blood were analyzed by
HPLC. When radioactivity in mouse plasma was analyzed by
HPLC, we confirmed the conversion of [18F]FPMP-5 to
several different chemical forms (Figure 5A). The percent of
the intact form sequentially decreased and reached 93.1, 41.8,
and 27.5% at 2, 30, and 60 min after injection, respectively
(Figure 5A). When radioactivity in mouse pancreas was
I
DOI: 10.1021/acs.molpharmaceut.8b00756
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
Author Contributions
H.W. and M.Y. contributed to this work equally. H.W., M.Y.,
M.O., and H.S. designed the study. H.W. and M.Y. performed
the experiments. H.W., M.Y., S.K., M.O., and H.S. analyzed the
data. H.W., M.Y., and M.O. wrote the manuscript. All authors
contributed to discussions and manuscript review. All authors
have given approval for the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the Japan Society for
the Promotion of Science (JSPS) through the “Funding
Program for Next Generation World-Leading Researchers
(LS060)”, initiated by the Council for Science and Technology
Policy (CSTP), and JSPS Research Fellowships for Young
Scientists (14J05961).
ABBREVIATIONS
■
T2DM, type 2 diabetes mellitus; BCM, β-cell mass; IAPP, islet
amyloid polypeptide; Aβ, β-amyloid; PET, positron emission
tomography; IDA, iminodiacetic acid; ThS, thioflavin-S; PBS,
phosphate-buffered saline; TBS, tris-buffered saline; DAB, 3,3′-
diaminobenzidine; THF, tetrahydrofuran; DMF, N,N-dime-
thylformamide; (HCHO)n, paraformaldehyde; MeCN, aceto-
nitrile; DIAD, diisopropyl azodicarboxylate; TBAF, tetra-n-
butylammonium fluoride; AcOEt, ethyl acetate; NMR, nuclear
magnetic resonance; MS (ESI or APCI), mass spectrometry
(electrospray ionization or atmospheric pressure chemical
ionization); HRMS (FAB), high-resolution mass spectrometry
(fast atom bombardment); TMS, tetramethylsilane; J, coupling
constant (in NMR spectrometry); HPLC, high-performance
liquid chromatography.
Figure 6. Ex vivo autoradiography with hIAPPTg/o-Avy/A and WT-
Avy/A mice (male, 21 months old). Autoradiograms of [18F]FPMP-5
using hIAPPTg-Avy/A and WT-Avy/A mice (A and C, respectively).
ThS staining with the adjacent pancreatic sections (B and D,
respectively). High magnification images of A and B (E and F,
respectively). Amylin immunohistochemical staining with an adjacent
pancreatic section (G).
CONCLUSIONS
■
In the present research, we designed and synthesized six novel
18F-labeled PMP derivatives as amylin imaging probes in the
T2DM pancreas. Each derivative showed affinity for islet
amyloids in the T2DM pancreatic sections and good
pharmacokinetics in normal mice. Among them, [18F]FPMP-
5 displayed not only clear labeling of islet amyloids but also the
highest uptake by the mouse pancreas. In addition, ex vivo
autoradiographic studies with [18F]FPMP-5 revealed the
intense labeling of islet amyloids in the pancreas of the
diabetes model mouse in vivo. Taken together, [18F]FPMP-5
has the feasibility as the amylin imaging probe to understand
the T2DM pathology focusing on islet amyloid.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.8b00756.
■
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Masahiro Ono: 0000-0002-2497-039X
Hideo Saji: 0000-0002-3077-9321
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