Synthesis and Evaluation of Fluorine-18 Labeled 2-Phenylquinoxaline Derivatives as Potential Tau Imaging Agents

Article abstract of DOI:10.1021/acs.molpharmaceut.0c01078

In this study, three pairs of optically pure 18F-labeled 2-phenylquinoxaline derivatives were evaluated as Tau imaging agents for the diagnosis of Alzheimer's disease (AD). The chiral 2-fluoromethyl-1,2-ethylenediol side chain was attached to the 2-phenyl

Full text of DOI:10.1021/acs.molpharmaceut.0c01078

pubs.acs.org/molecularpharmaceutics
Article
Synthesis and Evaluation of Fluorine-18 Labeled
2
‑Phenylquinoxaline Derivatives as Potential Tau Imaging Agents
Kaixiang Zhou, Fan Yang, Yuying Li, Yimin Chen, Xiaojun Zhang, Jinming Zhang, Junfeng Wang,
Jiapei Dai, Lisheng Cai,* and Mengchao Cui*
Cite This: Mol. Pharmaceutics 2021, 18, 1176−1195
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sı Supporting Information
ABSTRACT: In this study, three pairs of optically pure 18F-
labeled 2-phenylquinoxaline derivatives were evaluated as Tau
imaging agents for the diagnosis of Alzheimer’s disease (AD). The
chiral 2-fluoromethyl-1,2-ethylenediol side chain was attached to
the 2-phenylquinoxaline backbone to increase hydrophilicity,
thereby improving the binding affinity of the probe to tangles
and their selectivity toward Tau tangles over β-amyloid plaques
(
Aβ). These probes displayed excellent fluorescent properties and
high selectivity for tangles on brain sections from transgenic mice
rTg4510) and AD patients. Quantitative binding assays with AD
homogenates showed that the probes (R)-5 and (S)-16 have a high affinity (K = 4.1 and 10.3 nM, respectively) and high selectivity
(
i
18
18
(
30.5-fold and 34.6-fold, respectively) for tangles over Aβ. The high affinity and selectivity of (R)-[ F]5 and (S)-[ F]16 for tangles
were further confirmed with autoradiography on AD brain tissue in vitro. In addition, they displayed sufficient blood-brain barrier
penetration (7.06% and 10.95% ID/g, respectively) and suitable brain kinetics (brain2 min/brain60 min = 10.1, 6.5 respectively) in
normal mice. Ex vivo metabolism studies and micro-positron emission computed tomography (PET) revealed high brain biostability,
18
18
good brain kinetic properties, and low nonspecific binding for (S)-[ F]16. Together, these results demonstrate that (R)-[ F]5 and
1
8
(S)-[ F]16 are promising PET probes for Tau tangles imaging.
KEYWORDS: Alzheimer’s disease, Tau tangles, PET imaging, quinoxaline
INTRODUCTION
with an Aβ-positive PET scan displayed normal cognitive
7,8
■
functions. As such, other hallmarks of the disease, down-
Alzheimer’s disease (AD) is a type of neurodegenerative
disorder and the main cause of dementia, resulting in serious
threats to physical and mental health of the aged. The
number of AD patients worldwide is predicted to exceed 106
million by the year 2050. However, the definitive etiopatho-
genesis of AD is still unclear. Post-mortem assessment of brain
tissue from AD patients reveals two major pathological
biomarkers: extracellular senile plaques consisting of β-amyloid
Aβ) peptides and intracellular neurofibrillary tangles (NFTs)
consisting of hyperphosphorylated Tau proteins, as the gold
stream after the aggregation of Aβ in brain, may be more
9
1
,2
appropriate to achieve diagnosis or staging of AD. For
instance, the levels of misfolded Tau proteins in the brain
3
correlate with the degree of neuronal degeneration and
10−12
cognitive decline in AD.
The Tau protein is a micro-
tubule-associated protein naturally existing in axons that
stabilize microtubules, regulate the plasticity of the cytoskele-
ton, and promote neurite outgrowth. The metabolic pathway
of the Tau protein is controlled by hyperphosphorylation and
dephosphorylation. In abnormal cases, the hyperphosphory-
lated Tau proteins accumulate as paired helical filaments,
(
4
standard biomarkers for the diagnosis of AD. Besides its
complicated pathogenesis, the development of AD therapeutics
is also impeded by the lack of diagnostic tools that may be used
further leading to cytoskeletal dissociation and neurodeteriora-
13,14
tion.
In addition, abnormal Tau aggregation is also
5
to stage and monitor the progression of the disease. It is
associated with varieties of neurodegenerative disorder such
as progressive supranuclear palsy, sporadic corticobasal
therefore imperative to develop positron emission computed
tomography (PET) imaging probes targeting the hallmarks of
6
AD.
Received: November 1, 2020
Revised: December 27, 2020
Accepted: December 28, 2020
Published: January 21, 2021
Aβ may not be a suitable biomarker for the monitoring of
AD disease progression, especially in the later stage of the
disease. Multiple PET probes have been developed and studied
in a clinical setting to monitor AD progression. However, the
correlation between the burden of Aβ plaques and cognitive
impairment in AD is not well-established, as some patients
©
2021 American Chemical Society
https://dx.doi.org/10.1021/acs.molpharmaceut.0c01078
1
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Figure 1. Chemical scaffolds evaluated as PET probes for Tau.
degeneration, Pick’s disease, frontotemporal dementia, and
human PET studies. However, the E/Z isomerization of the
probe during radiochemical synthesis, even in the presence of
room light, may be problematic and should not be
15
Parkinsonism linked to chromosome 17. The quantitative
evaluation of Tau level in the cerebrospinal fluid would
advance the understanding of tauopathies, providing informa-
tion on curative effects and, ultimately, accelerating drug
32−34
ignored.
The first-generation ligands have been deeply
studied in human clinical trials but demonstrated varying
degrees of off-target binding.
9
,16,17
discovery in antitau therapeutic trials.
However, lumbar
puncture is invasive, and the Tau level in the cerebrospinal
Subsequent to the optimization of binding properties and
pharmacokinetics, second-generation Tau tracers are now
under investigation and are being processed in clinical trials.
fluid does not reflect the regional tissue distribution of Tau and
1
8
35
its aggregates. Therefore, PET probes with specific binding
with Tau aggregates would allow for the noninvasive
visualization and subsequent quantification of Tau burdens
in AD brains.
With the aid of suitable screening, structure activity relation-
18
ship (SAR) analysis, and structural optimization, [ F]MK-
6240 was identified as the most potent Tau tracer, exhibiting
18
36 18
The first generation of PET Tau tracers, namely, [ F]T807
high specificity and selectivity to NFTs. [ F]MK-6240 is
1
8
37
and [ F]T808, showed a high selectivity for Tau aggregates
over Aβ plaques (25- and 27-fold, respectively, Figure 1). The
currently under phase I clinical trials. More recently, other
18
scaffolds, including 1,5-naphthyridine derivative [ F]JNJ-
1
8
38
18
39,40
probe [ F]T808 showed metabolic defluorination, while
311, isoquinoline derivative [ F]JNJ-067
, imidazo[1,2-
1
8
11
[
F]T-807 was approved by the Food & Drug Administration
FDA) to image Tau pathology in patients being evaluated for
a]pyridine derivative [ C]RO963, imidazo[1,2-a]-
1
1
(
pyrimidinederivative [ C]RO643, pyrrolo[2,3-b:4,5-c′]-
18
41,42
AD. This significant progress gave a sign that these Tau tracers
dipyridine derivative [ F]RO948
, naphthylethylidene
44,45
19−24
18
43
18
hold great promise for AD evaluation and diagnosis.
THK
malononitrile derivative [ F]1f, and [ F]PI-2620
and
18
46
18
18
derivatives with a 2-arylquinoline scaffold are another class of
[ F]GTP1 as the derivatives of [ F]T807 and [ F]T808,
were all evaluated as novel PET imaging probes for Tau.
We recently reported that the 2-phenylquinoxaline scaffold
can recognize the β-sheet structure, including those of Aβ
2
5,26
PET probe of Tau aggregates.
The first in-human PET
1
8
study of [ F]THK-5117 demonstrated that the PET images
reflect the known NFTs distribution in an AD brain. However,
this probe suffered from binding to subcortical white matter
plaques and tangles. We developed Aβ imaging agents based
27−29
47−49
and unsuitable pharmacokinetics.
A structural analogue,
on the scaffold.
During those investigations, we found that
1
8
[
F]THK-5351, featuring a single (S)-enantiomer and
some of these quinoxaline derivatives were capable of binding
with NFTs on brain sections of AD patients in vitro. This
greatly inspired us to develop Tau PET probes based on the
optimization of the 2-phenylquinoxaline scaffold. We followed
the successful optimization strategy of THK derivatives, in
order to achieve an appropriate Tau targeting probe with high
pyridine ring, displayed improved pharmacokinetics and
showed a high signal-to-noise ratio in PET images, when
30,31
applied in AD patients.
[
Another promising Tau probe,
C]PBB3, with two trans carbon−carbon double bonds,
showed selective affinity for Tau aggregates in both in vitro and
1
1
1
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Article
Figure 2. Design strategy for the 2-phenylquinoxaline scaffolds as PET probes of Tau.
binding affinity and selectivity over Aβ plaques with low off-
target binding and suitable in vivo pharmacokinetics. When a
unique fluoropropanol side chain was introduced to the THK
isopropyl alcohol, while phase B was hexane) with the flow rate
at 0.5 mL/min. The fluorescence imaging was performed using
an EVOS FL fluorescence microscope imaging system (Life
Technologies) equipped with 4′,6-diamidino-2-phenylindole
(DAPI), green fluorescent protein (GFP), red fluorescent
protein (RFP), and Cy5.0 filter sets. Autoradiography images
were scanned using a Cyclone Plus storage phosphor system
(PerkinElmer). In vivo micro-PET/CT imaging was performed
on micro-PET/CT (Super Nova, PINGSENG Healthcare).
Male ICR mice (5 weeks, 18−22 g, male), SD rats (8−9 weeks,
300−350 g, male), and WT mice (C57BL6, 12 months old,
male) used for biodistribution and in vitro fluorescent staining
were purchased from Vital River Laboratories. Post-mortem
brain tissues of Tg-tau mice (C57BL6, rTg4510, 7 months old,
female) were kindly provided by Xuanwu Hospital, Capital
Medical University. Brain tissue samples from an autopsy
confirmed the AD patients (91 years old, male, temporal lobe;
85 years old, male, entorhinal cortex) were obtained from the
Chinese Brain Bank Center. All protocols involving the use of
animals were granted and supervised by the animal care
committee of Beijing Normal University.
18
27
18
30,31
scaffold, to give [ F]THK-5117 and [ F]THK-5351,
they showed higher selectivity for Tau and more favorable in
vivo kinetics. In addition, during the development of
1
1
[
C]PBB3, the investigators showed that lower lipophilicity
in their ligands synthesized improved the binding selectivity of
3
2
Tau versus Aβ. In this study, we introduced the chiral
fluoropropanol side chain at the phenolate position and N,N-
dimethylamino/N-monomethylamino group at position 6 or 7
of the 2-phenylquinoxaline scaffold, to find compounds with
increased binding affinity and selectivity toward NFTs (Figure
2
). Thus, three pairs of 18F-labeled chiral 2-phenylquinoxaline
derivatives were designed, synthesized, and evaluated, as PET
imaging probes for tangles.
EXPERIMENTAL SECTION
■
General Information. All reagents used for chemical
synthesis and biological evaluation were purchased from
commercial available suppliers; there is no further purification
unless otherwise stated. Chemical reaction processes were
monitored by Merck thin layer chromatography plates
Synthesis. 4-(6-Nitroquinoxalin-2-yl)phenol (1). 4-Nitro-
o-phenylenediamine (3.1 g, 20.0 mmol) was diluted in
dimethyl sulfoxide (DMSO) (8 mL) and placed at 0 °C, and
then 2-bromo-4′-hydroxyacetophenone (4.3 g, 20.0 mmol)
diluted in DMSO (5 mL) was added dropwise into the
solution. The reaction mixture was placed at r.t. with stirring
for 10 min. After collection, the precipitate was washed with
(
aluminum sheets covered with silica gel 60 F254 plates), and
products were purified using flash column chromatography
1
13
with silica gel (45−75 μm) matrix. H NMR and C NMR
spectra were conducted on a Bruker Avance 400 MHz, JEOL
JNM-ECZ 400 or 600 MHz spectrometer in CDCl3 or
1
1
00 mL of EtOH and 200 mL of deionized water. Compound
was gained as a yellow crystalline solid (3.1 g, 58%). H
deuterated dimethyl sulfoxide (DMSO-d ) solutions at r.t., and
6
1
the deuterated solvent resonance was employed as an internal
standard. Chemical shifts (δ) are reported in ppm, coupling
constants (J) are reported in hertz (Hz), and the multiplicity is
defined by s (singlet), d (doublet), t (triplet) or m (multiplet).
Low-resolution mass spectrometry (MS) spectra were acquired
using a Thermo Surveyor MSQ Plus (ESI) instrument, and
high-resolution mass spectra (HRMS) were acquired using a
Thermo Scientific Q-Exactive (ESI) mass spectrometer. The
fluorescence spectra were obtained using RF-5301PC spectro-
fluorophotometer (Shimadzu). The absolute fluorescence
quantum yields were determined by Absolute PL Quantum
Yield Spectrometer C11347 (Hamamatsu). The radioactivity
was determined using an automatic γ-counter (WALLAC/
Wizard 2480). The radioligands were purified using an SCL-20
AVP high-performance liquid chromatography (HPLC)
system (Shimadzu) equipped with an SPD-20A UV detector
NMR (400 MHz, DMSO-d ) δ 10.27 (s, 1H), 9.68 (d, J = 4.5
Hz, 1H), 8.81 (dd, J = 13.2, 2.5 Hz, 1H), 8.50 (dd, J = 9.2, 2.6
Hz, 1H), 8.29 (d, J = 8.8 Hz, 2H), 8.23 (t, J = 9.0 Hz, 1H),
6
6
.98 (d, J = 8.7 Hz, 1H). Electrospray ionization mass
spectrometry (ESIMS): m/z calcd for C H N O 268.1;
found 267.7, [M + H] .
-(6-Aminoquinoxalin-2-yl)phenol (2). N H ·H O (5.1 g,
14 10
3
3
+
4
2
4
2
4
(
8.0 mmol) was added slowly into the solution of compound 1
4.3 g, 16.1 mmol) and 10% Pd/C (0.8 g, 16.1 mmol) in 150
mL MeOH. The mixture was refluxed at 90 °C with stirring for
h. After the removal of Pd/C by filtration, the filtrate was
2
concentrated under vacuum to yield crude compound 2, which
was recrystallized in MeOH to acquire a yellow crystalline solid
1
(3.4 g, 89%). H NMR (400 MHz, DMSO-d
) δ 9.86 (s, 1H),
6
9.21 (s, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 9.0 Hz,
1H), 7.28 (dd, J = 9.0, 2.4 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H),
(λ = 254 nm) and γ-radiation scintillation detector (Bioscan
Flow Count 3200 NaI/PMT). The HPLC conditions were: a
reverse-phase semipreparative Venusil MP C18 column
6.98 (s, 1H), 6.95 (s, 1H), 6.03 (s, 2H). ESIMS: m/z calcd for
+
C
H
12
N
3
O 238.1; found 237.7, [M + H] .
14
(
Bonna-Agela, 5 μm, 10 mm × 250 mm), isocratic solvent
4-(6-(Methylamino)quinoxalin-2-yl)phenol (3). To a sol-
ution of intermediate 2 (1.2 g, 5.0 mmol) and paraformalde-
hyde (0.6 g, 20.0 mmol) in MeOH (100 mL) was added
system (phase B was acetonitrile, while phase A was water)
with the flow rate at 4.0 mL/min. The optical purity was
determined on a Primaide HPLC System (Hitach) equipped
with a Chiralpak AS-RH column (Daicel, 5 μm, 4.6 × 150
mm) eluted with an isocratic solvent system (phase A was
CH ONa (5 M in MeOH) (2.0 mL, 10.0 mmol). The mixture
3
was refluxed at 90 °C with stirring for 2 h. After this mixture
was allowed to cool to r.t., NaBH (0.8 g, 20.0 mmol) was
4
1
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1
added slowly, and then the reaction mixture was refluxed for an
additional 3 h. Water (200 mL) was added after the solvent
was removed under reduced vacuum and the mixture was
neutralized by HCl (1 M). After filtration, the crude product
was collected, suspended in 100 mL of deionized water, and
treated with ultrasound for 15 min. Compound 3 was collected
68%). [α]22 −7.2 (C 1.0, MeOH). H NMR (400 MHz,
D
CDCl ) δ 9.07 (s, 1H), 8.08 (d, J = 8.5 Hz, 2H), 7.89 (d, J =
3
8.9 Hz, 1H), 7.16 (d, J = 9.3 Hz, 1H), 7.10−6.97 (m, 3H),
4.63 (dt, J = 47.1, 5.2 Hz, 2H), 4.31 (d, J = 17.9 Hz, 1H), 4.17
1
3
(d, J = 4.9 Hz, 2H), 3.02 (s, 3H). C NMR (100 MHz,
DMSO-d ) δ 159.85, 151.06, 145.82, 144.18, 142.88, 136.22,
6
1
by filtration as a yellow solid (1.2 g, 95%). H NMR (400
130.10, 129.80, 128.30, 123.11, 115.44, 101.56, 84.93 (d, J =
166.7 Hz), 68.78 (d, J = 7.7 Hz), 68.16 (d, J = 19.1 Hz), 30.06.
HRESIMS: m/z calcd for C H FN O 328.14502; found
MHz, DMSO-d ) δ 9.78 (s, 1H), 9.16 (s, 1H), 8.03 (d, J = 8.2
6
Hz, 2H), 7.71 (d, J = 9.1 Hz, 1H), 7.23 (d, J = 9.1 Hz, 1H),
1
8
19
3
2
+
6
1
.89 (d, J = 8.3 Hz, 2H), 6.71 (s, 1H), 6.59 (d, J = 4.4 Hz,
328.14558, [M + H] .
H), 2.81 (d, J = 4.4 Hz, 3H). ESIMS: m/z calcd for
(R)-2-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
phenyl)-N-methylquinoxalin-6-amine [(R)-6]. A mixture of
compound 3 (756.4 mg, 3.0 mmol), (R)-(−)-2,2-dimethyl-4-
(hydroxymethyl)-1,3-dioxolane-p-toluenesulfonate (1030.8
mg, 3.6 mmol), and CsF (1.4 g, 9.0 mmol) in 30 mL of
DMF was placed at 65 °C for 2 h. After it cooled to r.t., the
reaction mixture was diluted with 150 mL of deionized water,
and the formed precipitate was washed by deionized water
three times after filtration and collection. The crude product
was purified by flash column chromatography (petroleum
+
C H N O 252.1; found 252.2, [M + H] .
1
5
14
3
(
R)-N-Methyl-2-(4-(oxiran-2-ylmethoxy)phenyl)-
quinoxalin-6-amine [(R)-4]. A mixture of compound 3 (0.5 g,
.0 mmol), (R)-glycidyl 3-nitrobenzenesulfonate (0.6 g, 2.5
2
mmol), and CsF (0.9 g, 6.0 mmol) in 25 mL of
dimethylformamide (DMF) was heated to 65 °C with stirring
for 2 h. After it cooled to r.t., the reaction mixture was diluted
with 150 mL of deionized water. After filtration, the precipitate
was washed by deionized water three times. The crude product
was purified by flash column chromatography (petroleum
ether/ethyl acetate = 2:1, v/v) to give the final product as a
yellow solid (1043.2 mg, 95%). H NMR (400 MHz, CDCl )
1
ether/ethyl acetate = 2:1, v/v) to generate the final product as
3
1
a yellow solid (365.1 mg, 59%). H NMR (400 MHz, CDCl )
δ 9.08 (s, 1H), 8.06 (d, J = 7.6 Hz, 2H), 7.84 (d, J = 9.0 Hz,
1H), 7.12 (d, J = 9.2 Hz, 1H), 7.09−7.02 (m, 2H), 6.98 (s,
1H), 4.58−4.46 (m, 1H), 4.18 (dd, J = 15.2, 8.6 Hz, 1H), 4.14
(dd, J = 9.5, 5.5 Hz, 1H), 4.02 (dd, J = 9.4, 5.9 Hz, 1H), 3.94
(dd, J = 8.4, 5.9 Hz, 1H), 2.99 (s, 3H), 1.48 (s, 3H), 1.42 (s,
3H). ESIMS: m/z calcd for C H N O 366.2; found 366.4,
3
δ 9.05 (s, 1H), 8.13−7.98 (m, 2H), 7.89 (d, J = 9.1 Hz, 1H),
7
.17 (dd, J = 9.1, 2.4 Hz, 1H), 7.12−7.01 (m, 3H), 4.32 (dd, J
=
11.0, 3.1 Hz, 1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.40 (ddd,
J = 7.0, 5.7, 2.9 Hz, 1H), 3.02 (s, 3H), 2.98−2.85 (m, 1H),
.80 (dd, J = 4.9, 2.6 Hz, 1H). ESIMS: m/z calcd for
2
2
1
24
3
3
+
+
C H N O 308.1; found 308.3, [M + H] .
[M + H] .
1
8
18
3
2
(
S)-N-Methyl-2-(4-(oxiran-2-ylmethoxy)phenyl)-
(S)-2-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
phenyl)-N-methylquinoxalin-6-amine [(S)-6]. The proce-
dures were similar to those used for the preparation of
compound (R)-6; the final product was gained as a yellow
solid (1035.9 mg, 94%). H NMR (400 MHz, CDCl ) δ 9.06
(s, 1H), 8.19−7.95 (m, 2H), 7.82 (d, J = 9.0 Hz, 1H), 7.15 (d,
J = 9.2 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.98 (s, 1H), 4.67−
4.43 (m, 1H), 4.19 (dd, J = 8.5, 6.4 Hz, 1H), 4.11 (d, J = 5.4
Hz, 1H), 4.07−3.97 (m, 1H), 3.93 (dd, J = 8.5, 5.9 Hz, 1H),
2.98 (s, 3H), 1.48 (s, 3H), 1.42 (s, 3H). ESIMS: m/z calcd for
quinoxalin-6-amine [(S)-4]. The procedures were similar to
those used for the preparation of compound (R)-4, and the
1
final product was gained as a yellow solid (378.4 mg, 62%). H
1
NMR (400 MHz, CDCl ) δ 9.07 (s, 1H), 8.14−7.99 (m, 2H),
3
3
7
.88 (d, J = 9.1 Hz, 1H), 7.15 (dd, J = 9.1, 2.5 Hz, 1H), 7.07
(
1
=
(
d, J = 8.9 Hz, 2H), 7.01 (d, J = 2.2 Hz, 1H), 4.31 (dd, J =
1.0, 3.1 Hz, 1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.40 (ddd, J
7.0, 5.8, 2.9 Hz, 1H), 3.01 (s, 3H), 2.98−2.91 (m, 1H), 2.80
dd, J = 4.9, 2.6 Hz, 1H). ESIMS: m/z calcd for C H N O
2
1
8
18
3
+
+
3
08.1; found 308.3, [M + H] .
S)-1-Fluoro-3-(4-(6-(methylamino)quinoxalin-2-yl)-
phenoxy)propan-2-ol [(S)-5]. A solution of compound (R)-4
C H N O 366.2; found 366.3, [M + H] .
2
1
24
3
3
(
(R)-3-(4-(6-(Methylamino)quinoxalin-2-yl)phenoxy)-
propane-1,2-diol [(R)-7]. A solution of compound (R)-6
(1030.3 mg, 2.8 mmol) in 10 mL of THF and 30 mL of HCl
(1 M) was placed at 90 °C with stirring and reflux for 30 min.
After the completion of the reaction, the THF was evaporated
under reduced vacuum, and the mixture was neutralized by
100 mL of saturated NaHCO3 solution; the crystalline
(
(
283.4 mg, 0.9 mmol) and tetrabutylammonium fluoride
TBAF) (1 M in tetrahydrofuran (THF), 10.0 mL, 10.0
mmol) in anhydrous THF was placed at 90 °C with stirring
and reflux for 6 h. After the completion of the reaction, the
mixture was evaporated under vacuum and then purified by
flash column chromatography (petroleum ether/ethyl acetate
precipitate was filtrated to yield compound (R)-7 as a yellow
solid (910.2 mg, 99%). H NMR (400 MHz, DMSO-d ) δ 9.21
1
=
2:1, v/v). The final product was acquired as a yellow
6
22
crystalline solid (230.6 mg, 76%). [α] +7.2 (C 1.0, MeOH).
(s, 1H), 8.13 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 9.1 Hz, 1H),
7.24 (dd, J = 9.1, 1.7 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 6.72
(s, 1H), 6.62 (d, J = 4.8 Hz, 1H), 4.96 (d, J = 4.9 Hz, 1H),
4.67 (t, J = 5.4 Hz, 1H), 4.07 (dd, J = 9.8, 4.0 Hz, 1H), 3.93
(dd, J = 9.7, 6.2 Hz, 1H), 3.87−3.66 (m, 1H), 3.46 (t, J = 5.3
Hz, 2H), 2.81 (d, J = 4.8 Hz, 3H). ESIMS: m/z calcd for
D
1
H NMR (400 MHz, CDCl ) δ 9.08 (s, 1H), 8.08 (d, J = 8.0
3
Hz, 2H), 7.88 (d, J = 9.1 Hz, 1H), 7.15 (dd, J = 9.1, 2.6 Hz,
1
H), 7.07 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 2.1 Hz, 1H), 4.64
(
dt, J = 47.0, 4.6 Hz, 2H), 4.38−4.26 (m, 1H), 4.21−4.12 (m,
13
2
1
1
7
H), 3.01 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 159.85,
6
+
51.06, 145.82, 144.18, 142.88, 136.21, 130.09, 129.79, 128.30,
23.12, 115.45, 101.54, 84.92 (d, J = 167.7 Hz), 68.78 (d, J =
.7 Hz), 68.15 (d, J = 19.2 Hz), 30.06. HRESIMS: m/z calcd
C H N O 326.1; found 326.4, [M + H] .
1
8
20
3
3
(S)-3-(4-(6-(Methylamino)quinoxalin-2-yl)phenoxy)-
propane-1,2-diol [(S)-7]. The procedures were similar to those
used for the preparation of compound (R)-7, and the final
+
for C H FN O 328.14558; found 328.14502, [M + H] .
1
8
19
3
2
1
(
R)-1-Fluoro-3-(4-(6-(methylamino)quinoxalin-2-yl)-
product was gained as a yellow solid (1064.3 mg, 99%). H
phenoxy)propan-2-ol [(R)-5]. The procedures were similar to
those used for the preparation of compound (S)-5; the final
product was gained as a yellow crystalline solid (183.4 mg,
NMR (400 MHz, DMSO-d ) δ 9.21 (s, 1H), 8.13 (d, J = 8.8
Hz, 2H), 7.73 (d, J = 9.1 Hz, 1H), 7.24 (dd, J = 9.1, 2.5 Hz,
1H), 7.07 (d, J = 8.9 Hz, 2H), 6.72 (d, J = 2.4 Hz, 1H), 6.62
6
1
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(
q, J = 4.7 Hz, 1H), 4.96 (d, J = 5.2 Hz, 1H), 4.66 (t, J = 5.7
4.5 mL, 4.5 mmol) in anhydrous THF was placed at 90 °C
with stirring and reflux for 3 h. The solvent was evaporated
under vacuum, and the product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 1:4, v/v).
Hz, 1H), 4.07 (dd, J = 9.9, 4.1 Hz, 1H), 3.93 (dd, J = 9.9, 6.2
Hz, 1H), 3.81 (td, J = 12.2, 5.6 Hz, 1H), 3.45 (t, J = 5.7 Hz,
2
H), 2.81 (d, J = 4.9 Hz, 3H). ESIMS: m/z calcd for
+
1
C H N O 326.1; found 326.3, [M + H] .
The final product was gained as a yellow solid (0.5 g, 74%). H
1
8
20
3
3
(
S)-2-(4-(2,3-Bis((tert-butyldimethylsilyl)oxy)propoxy)-
NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.17 (d, J = 7.6 Hz,
3
phenyl)-N-methylquinoxalin-6-amine [(S)-8]. To a solution
of compound (R)-7 (0.6 g, 2.0 mmol) and tert-butyldime-
thylsilyl chloride (TBDMSCl) (1.8 g, 12.0 mmol) in
acetonitrile (100 mL) was added imidazole (1.1 g, 16.0
mmol), and the mixture was placed at r.t. with stirring for 12 h.
After evaporation to remove the solvent, the crude product was
purified by flash column chromatography (petroleum ether/
ethyl acetate = 3:1, v/v). The final product was gained as a
yellow solid (1.0 g, 94%). H NMR (400 MHz, CDCl ) δ 9.07
s, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 9.0 Hz, 1H),
2H), 8.09 (dd, J = 9.0, 3.8 Hz, 1H), 7.91 (s, 2H), 7.10 (d, J =
8.1 Hz, 2H), 4.21−4.10 (m, 3H), 3.89 (dd, J = 11.4, 3.5 Hz,
1H), 3.80 (dd, J = 11.4, 5.0 Hz, 1H), 3.44 (s, 3H), 1.51 (s,
9H). ESIMS: m/z calcd for C H N O 426.2; found 426.3,
2
3
28
3
5
+
[M + H] .
(S)-tert-Butyl (2-(4-(2,3-dihydroxypropoxy)phenyl)-
quinoxalin-6-yl)(methyl)carbamate [(S)-10]. The procedures
were similar to those used for the preparation of compound
1
(R)-10; the final product was gained as a canary solid (0.6 g,
3
1
(
91%). H NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.17 (d, J
3
7
3
1
.15 (d, J = 9.1 Hz, 1H), 7.09−6.89 (m, 3H), 4.18 (dd, J = 9.5,
.3 Hz, 1H), 4.14−4.02 (m, 1H), 3.95 (dd, J = 9.4, 6.8 Hz,
H), 3.67−3.65 (dd, J = 5.6, 2.9 Hz, 2H), 3.01 (s, 3H), 0.91
= 8.9 Hz, 2H), 8.11 (d, J = 9.0 Hz, 1H), 7.99−7.84 (m, 2H),
7.10 (d, J = 8.9 Hz, 2H), 4.16 (d, J = 2.8 Hz, 3H), 3.89 (dd, J =
11.4, 3.6 Hz, 1H), 3.80 (dd, J = 11.4, 5.0 Hz, 1H), 3.45 (s,
3H), 1.51 (s, 9H). ESIMS: m/z calcd for C H N O 426.2;
found 426.4, [M + H] .
(S)-3-(4-(6-((tert-Butoxycarbonyl)(methyl)amino)-
quinoxalin-2-yl)phenoxy)-2-hydroxypropyl 4-methylbenze-
(
d, J = 1.5 Hz, 18H), 0.12 (d, J = 2.6 Hz, 6H), 0.08 (s, 6H).
2
3
28
3
5
+
ESIMS: m/z calcd for C H N O Si 554.3; found 554.7, [M
3
0
48
3
3
2
+
+
H] .
R)-2-(4-(2,3-Bis((tert-butyldimethylsilyl)oxy)propoxy)-
(
phenyl)-N-methylquinoxalin-6-amine [(R)-8]. The proce-
dures were similar to those used for the preparation of
nesulfonate [(S)-11]. Et N (2 mL) was added into the
solution of compound (R)-10 (0.4 g, 1.0 mmol) and TsCl (0.2
3
g, 1.0 mmol) in anhydrous CH Cl ; the mixture was placed at
2
2
1
r.t. with stirring for 12 h. The solvent was evaporated under
vacuum, and the product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 1:1, v/v).
1
The product was gained as a yellow oil (160.1 mg, 28%). H
NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.16 (d, J = 8.7 Hz,
3
=
2.3 Hz, 18H), 0.12 (d, J = 3.0 Hz, 6H), 0.08 (d, J = 0.7 Hz,
2H), 8.09 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 15.8 Hz, 2H), 7.81
(d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.7
Hz, 2H), 4.40−4.18 (m, 3H), 4.10 (d, J = 4.5 Hz, 2H), 3.44 (s,
3H), 2.43 (s, 3H), 1.51 (s, 9H). ESIMS: m/z calcd for
6
H). ESIMS: m/z calcd for C H N O Si 554.3; found 554.6,
30
48
3
3
2
+
[M + H] .
(
S)-tert-Butyl (2-(4-(2,3-bis((tert-butyldimethylsilyl)oxy)-
+
propoxy)phenyl)quinoxalin-6-yl)(methyl)carbamate [(S)-9].
C H N O S 580.2; found 580.4, [M + H] .
3
0
34
3
7
A solution of compound (S)-8 (1.0 g, 2.0 mmol) and
(R)-3-(4-(6-((tert-Butoxycarbonyl)(methyl)amino)-
(
Boc) O (4.4 g, 20.0 mmol) in anhydrous THF was placed at
0 °C with stirring and reflux for 12 h. The solvent was
quinoxalin-2-yl)phenoxy)-2-hydroxypropyl 4-methylbenze-
nesulfonate [(R)-11]. The procedures were similar to those
used for the preparation of compound (S)-11; the final
2
9
evaporated under vacuum and purified by flash column
1
chromatography (petroleum ether/ethyl acetate = 5:1, v/v).
product was gained as a yellow oil (210.5 mg, 33%). H NMR
1
The final product was gained as a yellow solid (1.0 g, 81%). H
(400 MHz, CDCl ) δ 9.25 (s, 1H), 8.16 (d, J = 7.6 Hz, 2H),
3
NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.16 (d, J = 8.5 Hz,
8.10 (s, 1H), 7.91 (s, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.34 (d, J
= 8.4 Hz, 2H), 7.02 (d, J = 7.6 Hz, 2H), 4.48−4.20 (m, 3H),
4.10 (d, J = 4.4 Hz, 2H), 3.45 (s, 3H), 2.43 (s, 3H), 1.51 (s,
9H). ESIMS: m/z calcd for C H N O S 580.2; found 580.6,
3
2
8
H), 8.10 (d, J = 9.1 Hz, 1H), 7.99−7.64 (m, 2H), 7.09 (d, J =
.5 Hz, 2H), 4.21 (dd, J = 9.4, 3.1 Hz, 1H), 4.10 (s, 1H), 3.97
(
1
6
dd, J = 8.9, 7.2 Hz, 1H), 3.76−3.46 (m, 2H), 3.44 (s, 3H),
3
0
34
3
7
+
.51 (s, 9H), 0.91 (d, J = 1.5 Hz, 18H), 0.12 (d, J = 3.1 Hz,
H), 0.08 (s, 6H). ESIMS: m/z calcd for C H N O Si 654.4;
[M + H] .
(2S)-3-(4-(6-((tert-Butoxycarbonyl)(methyl)amino)-
quinoxalin-2-yl)phenoxy)-2-((tetrahydro-2H-pyran-2-yl)-
oxy)propyl 4-methylbenzenesulfonate [(S)-12]. A solution of
compound (S)-11 (60.3 mg, 0.1 mmol), 3,4-dihydropyran
(80.4 mg, 1.0 mmol), and pyridinium toluene-4-sulfonate
(50.4 mg, 0.2 mmol) in anhydrous CH Cl was placed at r.t.
35
56
3
5
2
+
found 654.5, [M + H] .
R)-tert-Butyl (2-(4-(2,3-bis((tert-butyldimethylsilyl)oxy)-
(
propoxy)phenyl)quinoxalin-6-yl)(methyl)carbamate [(R)-9].
The procedures were similar to those used for the preparation
of compound (S)-9; the final product was gained as a yellow
2
2
1
solid (1.2 g, 90%). H NMR (400 MHz, CDCl ) δ 9.25 (s,
with stirring for 10 h. After the solvent was removed by
evaporation under vacuum, the crude product was purified by
flash column chromatography (petroleum ether/ethyl acetate
3
1
H), 8.17−8.10 (m, 3H), 7.92−7.87 (m, 2H), 7.09 (d, J = 8.7
Hz, 2H), 4.21 (dd, J = 9.6, 3.4 Hz, 1H), 4.15−4.05 (m, 1H),
3
3
.97 (dd, J = 9.5, 6.7 Hz, 1H), 3.67 (t, J = 5.6 Hz, 2H), 3.44 (s,
H), 1.51 (s, 9H), 0.91 (d, J = 2.5 Hz, 18H), 0.12 (d, J = 3.4
= 2:1, v/v). The final product was gained as a yellow oil (60.6
1
mg, 90%). H NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.16−
3
Hz, 6H), 0.08 (d, J = 0.7 Hz, 6H). ESIMS: m/z calcd for
8.10 (m, 3H), 7.90 (d, J = 12.6 Hz, 2H), 7.79 (dd, J = 8.2, 5.8
Hz, 2H), 7.30 (d, J = 7.1 Hz, 2H), 7.08−6.93 (m, 2H), 4.36−
4.17 (m, 3H), 4.13−4.06 (m, 1H), 3.92−3.80 (m, 2H), 3.51−
3.49 (m, 2H) 3.45 (d, J = 1.3 Hz, 3H), 2.41 (s, 3H), 1.82−1.67
(m, 2H), 1.53−1.51 (m, 4H), 1.51 (d, J = 0.8 Hz, 9H).
+
C H N O Si 654.4; found 654.6, [M + H] .
3
5
56
3
5
2
(
R)-tert-Butyl (2-(4-(2,3-dihydroxypropoxy)phenyl)-
quinoxalin-6-yl)(methyl)carbamate [(R)-10]. A solution of
compound (S)-9 (1.0 g, 1.5 mmol) and TBAF (1 M in THF,
1
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ESIMS: m/z calcd for C H N O S 664.3; found 664.6, [M +
NMR (400 MHz, CDCl ) δ 9.09 (s, 1H), 8.21−8.01 (m, 2H),
35
42
3
8
3
+
H] .
2R)-3-(4-(6-((tert-Butoxycarbonyl)(methyl)amino)-
7.94 (d, J = 9.3 Hz, 1H), 7.39 (dd, J = 9.4, 2.8 Hz, 1H), 7.09
(d, J = 2.8 Hz, 1H), 7.06 (d, J = 1.9 Hz, 2H), 4.31 (dd, J =
11.0, 3.1 Hz, 1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.42−3.38
(m, 1H), 3.15 (s, 6H), 3.00−2.89 (m, 1H), 2.80 (dd, J = 4.9,
2.6 Hz, 1H). ESIMS: m/z calcd for C H N O 322.1; found
(
quinoxalin-2-yl)phenoxy)-2-((tetrahydro-2H-pyran-2-yl)-
oxy)propyl 4-methylbenzenesulfonate [(R)-12]. The proce-
dures were similar to those used for the preparation of
1
9
20
3
2
+
compound (S)-12, and the final product was gained as a yellow
322.3, [M + H] .
1
oil (100.7 mg, 72%). H NMR (400 MHz, CDCl ) δ 9.25 (s,
(S)-N,N-Dimethyl-2-(4-(oxiran-2-ylmethoxy)phenyl)-
quinoxalin-6-amine [(S)-15]. The procedures were similar to
those used for the preparation of compound (R)-4; the final
3
1
H), 8.35−8.07 (m, 3H), 7.93 (d, J = 11.6 Hz,2H), 7.80 (dd, J
11.5, 8.0 Hz, 2H), 7.32 (dd, J = 14.0, 8.1 Hz, 2H), 7.08−6.86
=
1
(
m, 2H), 4.38−3.99 (m, 5H), 3.90−3.70 (m, 2H), 3.49 (s,
product was gained as a yellow solid (532.5 mg, 85%). H
1
H), 3.45 (s, 3H), 2.41 (s, 3H), 1.78−1.70 (m, 4H), 1.52 (s,
NMR (400 MHz, CDCl ) δ 9.07 (s, 1H), 8.08 (d, J = 8.9 Hz,
3
2
H), 1.52 (s, 9H). ESIMS: m/z calcd for C H N O S 664.3;
2H), 7.96 (d, J = 9.4 Hz, 1H), 7.41 (dd, J = 9.4, 2.7 Hz, 1H),
7.13 (d, J = 2.5 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 4.32 (dd, J =
11.0, 3.1 Hz, 1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.40 (dt, J =
8.6, 3.0 Hz, 1H), 3.17 (s, 6H), 2.94 (t, J = 4.5 Hz, 1H), 2.80
(dd, J = 4.9, 2.6 Hz, 1H). ESIMS: m/z calcd for C H N O
35
42
3
8
+
found 664.6, [M + H] .
1
1
N ,N -Dimethyl-4-nitrobenzene-1,3-diamine (13). A sol-
ution of 5-chloro-2-nitroaniline (12.5 g, 72.5 mmol) and
K CO (36.0 g, 26.1 mmol) in 150 mL of DMF was placed at
2
3
19 20
3
2
+
1
50 °C with vigorous stirring overnight. After the reaction, 500
322.1; found 322.3, [M + H] .
mL of deionized water was added into the mixture, and the
formed precipitate was gathered by filtration and washed by
deionized water three times to receive the desired product as a
(S)-1-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-3-
fluoropropan-2-ol [(S)-16]. The procedures were similar to
those used for the preparation of compound (S)-5; the final
product was yielded as a yellow crystalline solid (187.2 mg,
1
yellow solid without further purification (12.4 g, 94%). H
2
2
1
NMR (400 MHz, CDCl ) δ 8.02 (d, J = 9.7 Hz, 1H), 6.15 (dd,
85%). [α]
+7.7 (C 1.0, MeOH). H NMR (400 MHz,
3
D
J = 9.7, 2.6 Hz, 1H), 5.78 (d, J = 2.6 Hz, 1H), 3.05 (s, 6H).
ESIMS: m/z calcd for C H N O 182.1; found 182.2, [M +
CDCl ) δ 9.08 (s, 1H), 8.08 (d, J = 8.8 Hz, 2H), 7.95 (d, J =
3
9.3 Hz, 1H), 7.41 (dd, J = 9.4, 2.7 Hz, 1H), 7.12 (s, 1H), 7.07
(d, J = 8.8 Hz, 2H), 4.75−4.64 (m, 1H), 4.62−4.50 (m, 1H),
8
12
3
2
+
H] .
-(6-(Dimethylamino)quinoxalin-2-yl)phenol (14) and 4-
7-(dimethylamino)quinoxalin-2-yl)phenol (21). In a solution
1
3
4
4.41−4.23 (m, 1H), 4.16 (d, J = 4.8 Hz, 2H), 3.16 (s, 6H). C
(
NMR (400 MHz, DMSO-d ) δ 160.01, 151.00, 146.53, 143.44,
6
of compound 13 (3.6 g, 20.0 mmol) in 150 mL of MeOH was
added with 10% Pd/C (1.0 g, 20.0 mmol) at r.t.; the mixture
143.23, 135.56, 129.96, 129.84, 128.46, 120.50, 115.47, 105.36,
84.92 (d, J = 167.7 Hz), 68.80 (d, J = 7.7 Hz), 68.14 (d, J =
19.1 Hz), 40.57. HRESIMS: m/z calcd for C H FN O
was placed under H atmosphere (1 atm). The mixture was
2
19 21
3
2
+
placed at r.t. for 6 h with stirring. After the solvent was
342.16123; found 342.16089, [M + H] .
4
4
removed under vacuum, N ,N -dimethylbenzene-1,2,4-triami-
newas obtained as a pure white solid. Then a solution of 2-
bromo-4′-hydroxyacetophenone (4.3 g, 20.0 mmol) diluted
with 10 mL of DMSO was added. The reaction mixture was
stirred at r.t. for 10 h. After the reaction, 150 mL of deionized
water was added, and the product was extracted with ethyl
acetate (10 × 50 mL). The organic layer was combined and
(R)-1-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-3-
fluoropropan-2-ol [(R)-16]. The procedures were similar to
those used for the preparation of compound (S)-5; the product
was gained as a yellow crystalline solid (168.2 mg, 82%).
2
2
1
[α] −7.7 (C 1.0, MeOH). H NMR (400 MHz, CDCl ) δ
D
3
9.09 (s, 1H), 8.08 (dd, J = 9.0, 2.4 Hz, 2H), 7.94 (d, J = 9.3
Hz, 1H), 7.39 (dd, J = 9.4, 2.6 Hz, 1H), 7.07 (dt, J = 9.1, 2.6
Hz, 3H), 4.76−4.63 (m, 1H), 4.62−4.51 (m, 1H), 4.38−4.25
dried over anhydrous MgSO , then filtered and concentrated in
4
1
3
vacuo. The crude product was purified by flash column
chromatography (dichloromethane/ethyl acetate = 5:1, v/v) to
give a mixture of regioisomers of 4-(6-(dimethylamino)-
quinoxalin-2-yl)phenol (14) and 4-(7-(dimethylamino)-
quinoxalin-2-yl)phenol (21). The regioisomers were separated
by recrystallization with H C O ·2H O in hot EtOH. After the
(m, 1H), 4.16 (dd, J = 4.0, 1.7 Hz, 2H), 3.15 (s, 6H).
C
NMR (400 MHz, DMSO-d ) δ 160.00, 150.98, 146.52, 143.42,
6
143.22, 135.56, 129.96, 129.83, 128.45, 120.47, 115.46, 105.36,
84.92 (d, J = 167.7 Hz), 68.79 (d, J = 7.7 Hz), 68.15 (d, J =
19.1 Hz), 40.56. HRESIMS: m/z calcd for C H FN O
1
9
21
3
2
+
342.16123; found 342.16052, [M + H] .
2
2
4
2
mixture cooled to r.t., the oxalate of compound 14 precipitated
first and was collected by filtration and washed by cold EtOH
and neutralized with NH ·H O to give pure compound 14 as a
(R)-2-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
phenyl)-N,N-dimethylquinoxalin-6-amine [(R)-17]. The pro-
cedures were similar to those used for the preparation of
3
2
1
yellow solid (0.6 g, 10%). H NMR (400 MHz, DMSO-d ) δ
compound (R)-6; the final product was gained as a yellow
6
1
9
1
.21 (s, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 9.3 Hz,
H), 7.48 (dd, J = 9.2, 1.9 Hz, 1H), 6.95−6.86 (m, 3H), 3.06
solid (673.1 mg, 94%). H NMR (400 MHz, CDCl ) δ 9.09 (s,
3
1H), 8.12−8.04 (m, 2H), 7.94 (d, J = 9.3 Hz, 1H), 7.39 (d, J =
9.3 Hz, 1H), 7.10−7.05 (m, 3H), 4.52 (d, J = 5.8 Hz, 1H),
4.20 (dd, J = 8.5, 6.4 Hz, 1H), 4.14 (dd, J = 9.5, 5.5 Hz, 1H),
4.03 (dd, J = 9.5, 5.8 Hz, 1H), 3.94 (dd, J = 8.5, 5.9 Hz, 1H),
3.16 (d, J = 3.0 Hz, 6H), 1.49 (s, 3H), 1.42 (s, 3H). ESIMS:
(
[
s, 6H). ESIMS: m/z calcd for C H N O 266.1; found 266.3,
M + H] . The filtrate was neutralized by NH ·H O to gain
pure compound 21 as a yellow solid (2.0 g, 30%). H NMR
400 MHz, DMSO-d ) δ 9.05 (s, 1H), 8.12 (d, J = 8.4 Hz,
16 16 3
+
3
2
1
(
6
+
2
H), 7.80 (d, J = 9.3 Hz, 1H), 7.41 (dd, J = 9.2, 1.9 Hz, 1H),
m/z calcd for C H N O 380.2; found 380.4, [M + H] .
2
2
26
3
3
6
.93 (dd, J = 12.6, 5.1 Hz, 3H), 3.08 (s, 6H). ESIMS: m/z
(S)-2-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
phenyl)-N,N-dimethylquinoxalin-6-amine [(S)-17]. The pro-
cedures were similar to those used for the preparation of
+
calcd for C H N O 266.1; found 266.2, [M + H] .
1
6
16
3
(
R)-N,N-Dimethyl-2-(4-(oxiran-2-ylmethoxy)phenyl)-
quinoxalin-6-amine [(R)-15]. The procedures were similar to
those used for the preparation of compound (R)-4; the final
product was obtained as a yellow solid (547.3 mg, 85%). H
compound (R)-6; the final product was gained as a yellow
1
solid (660.1 mg, 91%). H NMR (400 MHz, CDCl ) δ 9.09 (s,
3
1
1H), 8.07 (d, J = 8.9 Hz, 2H), 7.95 (d, J = 9.3 Hz, 1H), 7.48−
1
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Article
7
.30 (m, 1H), 7.11 (s, 1H), 7.07 (t, J = 5.9 Hz, 2H), 4.59−4.40
sulfonate [(R)-20]. The procedures were similar to those
used for the preparation of compound (S)-12, and the final
product was gained as a yellow oil (100.7 mg, 90%). H NMR
(
m, 1H), 4.20 (dd, J = 8.5, 6.4 Hz, 1H), 4.14 (dd, J = 9.5, 5.5
1
Hz, 1H), 4.03 (dd, J = 9.5, 5.8 Hz, 1H), 3.94 (dd, J = 8.5, 5.9
Hz, 1H), 3.16 (s, 6H), 1.49 (s, 3H), 1.42 (s, 3H). ESIMS: m/z
(400 MHz, CDCl ) δ 9.05 (s, 1H), 8.04 (d, J = 8.3 Hz, 2H),
3
+
calcd for C H N O 380.2; found 380.4, [M + H] .
7.98 (d, J = 9.1 Hz, 1H), 7.84−7.74 (m, 2H), 7.45 (d, J = 7.6
Hz, 1H), 7.29 (d, J = 7.9 Hz, 2H), 7.18 (s, 1H), 6.95 (dd, J =
8.8, 5.6 Hz, 2H), 4.36−4.14 (m, 4H), 4.13−4.01 (m, 2H), 3.49
(s, 2H), 3.19 (s, 6H), 2.41 (s, 3H), 1.92−1.64 (m, 2H), 1.53
(s, 4H). ESIMS: m/z calcd for C H N O S 578.2; found
2
2
26
3
3
(
R)-3-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-
propane-1,2-diol [(R)-18]. The procedures were similar to
those used for the preparation of compound (R)-7; the final
product was gained as a yellow solid (594.2 mg, 96%). H
1
3
1
36
3
6
+
NMR (400 MHz, DMSO-d ) δ 9.27 (s, 1H), 8.17 (d, J = 8.9
578.4, [M + H] .
6
Hz, 2H), 7.85 (d, J = 9.3 Hz, 1H), 7.50 (dd, J = 9.4, 2.8 Hz,
(R)-N,N-Dimethyl-3-(4-(oxiran-2-ylmethoxy)phenyl)-
quinoxalin-6-amine [(R)-22]. The procedures were similar to
those used for the preparation of compound (R)-4; the final
1
H), 7.08 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 2.8 Hz, 1H), 4.98
(
d, J = 4.8 Hz, 1H), 4.68 (s, 1H), 4.07 (dd, J = 9.9, 4.1 Hz,
1
1
3
H), 3.93 (dd, J = 9.9, 6.2 Hz, 1H), 3.81 (d, J = 5.0 Hz, 1H),
product was gained as a yellow solid (536.1 mg, 83%). H
.45 (t, J = 4.8 Hz, 2H), 3.09 (s, 6H). ESIMS: m/z calcd for
NMR (400 MHz, CDCl ) δ 8.94 (s, 1H), 8.12 (d, J = 8.8 Hz,
3
+
C H N O 340.2; found 340.3, [M + H] .
2H), 7.90 (d, J = 9.3 Hz, 1H), 7.33 (dd, J = 9.3, 2.8 Hz, 1H),
7.15 (s, 1H), 7.08 (dd, J = 6.9, 4.9 Hz, 2H), 4.32 (dd, J = 11.0,
3.1 Hz, 1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.44−3.36 (m,
1H), 3.15 (s, 6H), 2.97−2.91 (m, 1H), 2.80 (dd, J = 4.9, 2.6
Hz, 1H). ESIMS: m/z calcd for C H N O 322.1; found
1
9
22
3
3
(
S)-3-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-
propane-1,2-diol [(S)-18]. The procedures were similar to
those used for the preparation of compound (R)-7; the final
1
product was gained as a yellow solid (578.4 g, 94%). H NMR
1
9
20
3
2
+
(
400 MHz, DMSO-d ) δ 9.27 (s, 1H), 8.17 (d, J = 8.9 Hz,
322.2, [M + H] .
6
2
7
1
9
3
3
H), 7.85 (d, J = 9.3 Hz, 1H), 7.50 (dd, J = 9.4, 2.8 Hz, 1H),
.08 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 2.8 Hz, 1H), 4.97 (s,
H), 4.68 (s, 1H), 4.07 (dd, J = 9.9, 4.1 Hz, 1H), 3.93 (dd, J =
.9, 6.2 Hz, 1H), 3.83−3.80 (m, 1H), 3.45 (d, J = 5.7 Hz, 2H),
.09 (s, 6H). ESIMS: m/z calcd for C H N O 340.2; found
(S)-N,N-Dimethyl-3-(4-(oxiran-2-ylmethoxy)phenyl)-
quinoxalin-6-amine [(S)-22]. The procedures were similar to
those used for the preparation of compound (R)-4; the final
1
product was gained as a yellow solid (581.2 mg, 91%). H
NMR (400 MHz, CDCl ) δ 8.94 (s, 1H), 8.12 (d, J = 8.8 Hz,
19
22
3
3
3
+
40.4, [M + H] .
S)-3-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-2-
2H), 7.89 (d, J = 9.3 Hz, 1H), 7.33 (dd, J = 9.3, 2.8 Hz, 1H),
7.13 (s, 1H), 7.10−7.03 (m, 2H), 4.32 (dd, J = 11.0, 3.1 Hz,
1H), 4.04 (dd, J = 11.0, 5.7 Hz, 1H), 3.44−3.36 (m, 1H), 3.15
(s, 6H), 2.95−2.88 (m, 1H), 2.80 (dd, J = 4.9, 2.6 Hz, 1H).
ESIMS: m/z calcd for C H N O 322.1; found 322.3, [M +
(
hydroxypropyl 4-methylbenzenesulfonate [(S)-19]. The
procedures were similar to those used for the preparation of
compound (S)-11; the desired product was gained as a yellow
oil (265.2 mg, 38%). H NMR (400 MHz, CDCl ) δ 9.08 (s,
1
9
20
3
2
1
+
H] .
3
1
(
H), 8.06 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 9.3 Hz, 1H), 7.80
d, J = 8.3 Hz, 2H), 7.41 (d, J = 9.4 Hz, 1H), 7.32 (d, J = 8.0
Hz, 2H), 7.11 (s, 1H), 6.96 (d, J = 8.6 Hz, 2H), 4.39−4.12 (m,
H), 4.07 (d, J = 4.5 Hz, 2H), 3.16 (s, 6H), 2.42 (s, 3H).
ESIMS: m/z calcd for C H N O S 494.2; found 494.4, [M +
(S)-1-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-3-
fluoropropan-2-ol [(S)-23]. The procedures were similar to
those used for the preparation of compound (S)-5; the final
product was gained as a yellow crystalline solid (196.5 mg,
3
2
2
1
85%). [α]
CDCl ) δ 8.94 (s, 1H), 8.14 (d, J = 8.8 Hz, 2H), 7.92 (d, J =
+7.5 (C 1.0, MeOH). H NMR (400 MHz,
26
28
3
5
D
+
H] .
R)-3-(4-(6-(Dimethylamino)quinoxalin-2-yl)phenoxy)-2-
hydroxypropyl 4-methylbenzenesulfonate [(R)-19]. The
procedures were similar to those used for the preparation of
compound (S)-11; the desired product was gained as a yellow
3
(
9.3 Hz, 1H), 7.35 (dd, J = 9.3, 2.8 Hz, 1H), 7.20 (s, 1H), 7.08
(d, J = 8.9 Hz, 2H), 4.63 (dt, J = 47.0, 4.7 Hz, 2H), 4.33−4.28
(m, 1H), 4.18−4.16 (dd, J = 4.6, 2.6 Hz, 2H), 3.17 (s, 6H).
1
3
C NMR (100 MHz, DMSO-d ) δ 160.54, 151.65, 150.87,
6
1
oil (193.6 mg, 36%). H NMR (400 MHz, CDCl ) δ 9.08 (s,
143.97, 138.46, 135.17, 129.83, 129.58, 129.17, 119.39, 115.45,
105.33, 84.91 (d, J = 167.7 Hz), 68.82 (d, J = 7.7 Hz), 68.13
(d, J = 19.1 Hz), 40.54. HRESIMS: m/z calcd for
3
1
H), 8.06 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 9.4 Hz, 1H), 7.81
d, J = 8.3 Hz, 2H), 7.41 (dd, J = 9.4, 2.7 Hz, 1H), 7.33 (d, J =
.3 Hz, 2H), 7.12 (s, 1H), 6.98 (t, J = 7.0 Hz, 2H), 4.28−4.22
m, 3H), 4.07 (d, J = 4.5 Hz, 2H), 3.17 (s, 6H), 2.42 (s, 3H).
ESIMS: m/z calcd for C H N O S 494.2; found 494.4, [M +
(
8
(
+
C H FN O 342.16123; found 342.16092, [M + H] .
1
9
21
3
2
(R)-1-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-3-
fluoropropan-2-ol [(R)-23]. The procedures were similar to
those used for the preparation of compound (S)-5; the final
product was gained as a yellow crystalline solid (158.3 mg,
26
28
3
5
+
H] .
2S)-3-(4-(6-(Dimethylamino)naphthalen-2-yl)phenoxy)-
-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzene-
(
2
2
1
2
87%). [α]
−7.5 (C 1.0, MeOH). H NMR (400 MHz,
D
sulfonate [(S)-20]. The procedures were similar to those
used for the preparation of compound (S)-12; the final
CDCl ) δ 8.94 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 8.08 (d, J =
3
8.8 Hz, 1H), 7.94 (d, J = 9.4 Hz, 1H), 7.37 (dd, J = 9.4, 2.4 Hz,
1H), 7.10 (d, J = 8.9 Hz, 2H), 4.63 (dt, J = 47.1, 5.0 Hz, 2H),
1
product was gained as a yellow oil (127.4 mg, 87%). H NMR
1
3
(
400 MHz, CDCl ) δ 9.06 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H),
4.34−4.29 (m, 1H), 4.18−4.17 (m, 2H), 3.19 (s, 6H).
C
3
7
.97 (d, J = 9.3 Hz, 1H), 7.79 (dd, J = 8.2, 5.7 Hz, 2H), 7.43
NMR (100 MHz, DMSO-d ) δ 160.53, 151.64, 150.87, 143.96,
6
(
dd, J = 9.4, 2.6 Hz, 1H), 7.29 (d, J = 7.3 Hz, 2H), 7.15 (s,
138.46, 135.17, 129.83, 129.57, 129.16, 119.37, 115.44, 105.33,
84.91 (d, J = 167.7 Hz), 68.82 (d, J = 7.7 Hz), 68.14 (d, J =
19.1 Hz), 40.53. HRESIMS: m/z calcd for C H FN O
1
3
1
H), 6.95 (dd, J = 8.8, 5.7 Hz, 2H), 4.42−4.13 (m, 4H), 4.12−
.99 (m, 2H), 3.52−3.46 (m, 2H), 3.18 (s, 6H), 2.40 (s, 3H),
1
9
21
3
2
+
.82−1.59 (m, 2H), 1.53 (s, 4H). ESIMS: m/z calcd for
342.16123; found 342.16064, [M + H] .
+
C H N O S 578.2; found 578.5, [M + H] .
(R)-3-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
phenyl)-N,N-dimethylquinoxalin-6-amine [(R)-24]. The pro-
cedures were similar to those used for the preparation of
3
1
36
3
6
(
2R)-3-(4-(6-(Dimethylamino)naphthalen-2-yl)phenoxy)-
2
-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzene-
1
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Article
compound (R)-6; the final product was gained as a yellow
(2S)-3-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-2-
((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzenesul-
fonate [(S)-27]. The procedures were similar to those used for
the preparation of compound (S)-12; the final product was
1
solid (756.0 mg, 99%). H NMR (400 MHz, CDCl ) δ 8.93 (s,
3
1
H), 8.13 (d, J = 3.2 Hz, 2H), 7.92 (s, 1H), 7.35 (s, 1H), 7.18
s, 1H), 7.08 (s, 2H), 4.69−4.40 (m, 1H), 4.20 (dd, J = 8.5, 6.4
Hz, 1H), 4.14 (dd, J = 9.5, 5.5 Hz, 1H), 4.05 (d, J = 5.8 Hz,
(
1
gained as a yellow oil (123.5 mg, 77%). H NMR (400 MHz,
1
1
3
H), 3.94 (dd, J = 8.5, 5.9 Hz, 1H), 3.17 (s, 6H), 1.48 (s, 3H),
CDCl ) δ 8.94 (s, 1H), 8.11 (d, J = 8.7 Hz, 2H), 7.91 (d, J =
3
.32 (s, 3H). ESIMS: m/z calcd for C H N O 380.2; found
9.3 Hz, 1H), 7.79 (dd, J = 8.2, 5.6 Hz, 2H), 7.35 (dd, J = 9.3,
2.7 Hz, 1H), 7.32−7.27 (m, 2H), 7.22 (s, 1H), 7.08−7.06 (m,
1H), 6.96 (dd, J = 8.8, 5.8 Hz, 1H), 4.36−4.05 (m, 6H), 3.57−
2
2
26
3
3
+
80.4, [M + H] .
S)-3-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)-
(
3
1
5
.47 (m, 2H), 3.17 (s, 6H), 2.40 (s, 3H), 1.82−1.67 (m, 2H),
phenyl)-N,N-dimethylquinoxalin-6-amine [(S)-24]. The pro-
cedures were similar to those used for the preparation of
.56−1.50 (m, 4H). ESIMS: m/z calcd for C H N O S
31 36
3
6
+
78.2; found 578.6, [M + H] .
2R)-3-(4-(7-(Dimethylamino)naphthalen-2-yl)phenoxy)-
-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzene-
compound (R)-6; the final product was gained as a canary
1
(
yellow solid (630.2 mg, 83%). H NMR (400 MHz, CDCl ) δ
3
2
8.93 (s, 1H), 8.13 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 9.3 Hz,
1H), 7.35 (dd, J = 9.4, 2.4 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H),
4.51 (dd, J = 11.8, 5.9 Hz, 1H), 4.20 (dd, J = 8.5, 6.5 Hz, 1H),
4.14 (dd, J = 9.6, 5.5 Hz, 1H), 4.04 (dd, J = 9.5, 5.8 Hz, 1H),
3.93 (dd, J = 8.5, 5.9 Hz, 1H), 3.18 (s, 6H), 1.48 (s, 3H), 1.42
sulfonate [(R)-27]. The procedures were similar to those
used for the preparation of compound (S)-12; the final
1
product was gained as a yellow oil (64.7 mg, 74%). H NMR
(
8
400 MHz, CDCl ) δ 8.93 (s, 1H), 8.21 (s, 2H), 8.04 (d, J =
.0 Hz, 1H), 7.97 (d, J = 10.3 Hz, 1H), 7.79 (dd, J = 8.2, 5.8
3
(
3
s, 3H). ESIMS: m/z calcd for C H N O 380.2; found
22 26 3 3
+
Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 7.0 Hz, 2H),
80.4, [M + H] .
R)-3-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-
7
3
2
.06−6.88 (m, 2H), 4.40−4.14 (m, 4H), 4.12−4.06 (m, 2H),
.50 (s, 2H), 3.23 (s, 6H), 2.42 (s, 3H), 1.53 (s, 4H), 1.26 (s,
H). ESIMS: m/z calcd for C H N O S 578.2; found 578.6,
(
propane-1,2-diol [(R)-25]. The procedures were similar to
those used for the preparation of compound (R)-7; the final
3
1
36
3
6
+
1
[M + H] .
product was gained as a yellow solid (676.3 mg, 98%). H
1
8 −
Radiochemistry. [ F ] Fluoride trapped on a QMA
NMR (400 MHz, DMSO-d ) δ 9.10 (s, 1H), 8.22 (d, J = 8.5
6
cartridge was eluted with 1 mL (acetonitrile/H O, v/v = 4/1)
2
Hz, 2H), 7.82 (d, J = 9.3 Hz, 1H), 7.43 (dd, J = 9.3, 2.0 Hz,
of Kryptofix-222/K CO solution (13 mg of Kryptofix-222 and
2
3
1
H), 7.09 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 1.9 Hz, 1H), 4.98
1
.1 mg of K CO ) into a 10 mL reaction vial. The vial was
2 3
(
d, J = 5.0 Hz, 1H), 4.69 (t, J = 5.6 Hz, 1H), 4.08 (dd, J = 9.8,
heated to 120 °C with nitrogen gas flow to remove solvent for
3
4
5
.0 Hz, 1H), 3.94 (dd, J = 9.8, 6.2 Hz, 1H), 3.82 (dd, J = 10.1,
min, and then the residue was dried azeotropically with three
.2 Hz, 1H), 3.46 (t, J = 5.6 Hz, 2H), 3.09 (s, 6H). ESIMS: m/
separate additions of anhydrous acetonitrile (1 mL). After the
drying, the appropriate solution of tosylated precursors (S)/
+
z calcd for C H N O 340.2; found 340.4, [M + H] .
1
9
22
3
3
(
S)-3-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-
(
R)-12, (S)/(R)-20, or (S)/(R)-27 (3.0 mg) in anhydrous
propane-1,2-diol [(S)-25]. The procedures were similar to
those used for the preparation of compound (R)-7; the final
acetonitrile (600 μL) was transferred into the vial and heated
to 100 °C for 7 min. Then 400 μL of aqueous HCl (1 M) was
added and heated at 100 °C for an additional 5 min. After it
cooled to r.t., the solution was neutralized by the addition of an
1
product was gained as a yellow solid (500.4 mg, 88%). H
NMR (400 MHz, DMSO-d ) δ 9.28 (s, 1H), 8.19 (d, J = 8.5
6
Hz, 2H), 7.84 (d, J = 9.3 Hz, 1H), 7.47 (d, J = 9.2 Hz, 1H),
aqueous solution of NaHCO (1 M). The reaction mixture was
3
7
1
3
3
.09 (t, J = 7.7 Hz, 2H), 6.97 (s, 1H), 4.98 (s, 1H), 4.68 (s,
H), 4.09−4.05 (m, 1H), 4.02−3.88 (m, 1H), 3.81 (s, 1H),
.46 (s, 2H), 3.09 (s, 6H). ESIMS: m/z calcd for C H N O
3
diluted with 10 mL of deionized water and then passed
through a preconditioned Sep-Pak Plus-C18 cartridge
1
9
22
3
(
Cleanert IC-C18, Bonna-Agela Technologies). The cartridge
+
40.2; found 340.5, [M + H] .
was washed with 10 mL of deionized water, and then the
trapped product was eluted with acetonitrile (2 × 1 mL) and
subjected to HPLC purification on a Venusil MP C18 reverse-
phase column (5 μm, 10 × 250 mm; Green Technologies).
The HPLC fraction containing the product was collected and
concentrated under vacuum, and it was diluted with saline
containing 10% ethanol for biological evaluation.
In Vitro Fluorescent Staining. Paraffin-embedded brain
slices (8-μm) from Tg-tau (C57BL6, rTg4510, 7 months old,
female, temporal lobe), WT mouse (C57BL6, 12 months old,
male), and AD patients (91 years old, male, temporal lobe; 85
years old, male, entorhinal cortex) were used for multi-
fluorescent staining studies. Brain slices were deparaffinized by
fresh xylene for 5 min and washed with EtOH and water for 1
min. Then the brain slice was incubated with prepared
solutions of fluorinated compounds (1 μM, 10% ethanol),
DANIR 3b (1 μM, 10% ethanol), and DAPI (1 μg/mL, in
phosphate-buffered saline (PBS)), respectively. The staining
was performed at r.t. for 15 min and washed with 40% EtOH
solution. Fluorescent images were acquired on an EVOS FL
fluorescence microscope imaging system (Life Technologies)
equipped with GFP, RFP and DAPI filter sets.
(
S)-3-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-2-
hydroxypropyl 4-methylbenzenesulfonate [(S)-26]. The
procedures were similar to those used for the preparation of
compound (S)-11; the final product was gained as a yellow oil
1
(
260.5 mg, 38%). H NMR (400 MHz, CDCl ) δ 8.91 (s, 1H),
3
8
.08−8.06 (m, 3H), 7.90 (d, J = 9.3 Hz, 1H), 7.79 (d, J = 8.3
Hz, 1H), 7.34−7.29 (m, 3H), 7.10 (d, J = 2.6 Hz, 1H), 7.07−
7
3
.02 (m, 1H), 6.94 (dd, J = 8.8, 3.9 Hz, 1H), 4.33−4.18 (m,
H), 4.12 (d, J = 7.2 Hz, 1H), 4.05 (d, J = 4.5 Hz, 1H), 3.15
(
4
s, 6H), 2.40 (s, 3H). ESIMS: m/z calcd for C H N O S
26 28 3 5
+
94.2; found 494.5, [M + H] .
R)-3-(4-(7-(Dimethylamino)quinoxalin-2-yl)phenoxy)-2-
(
hydroxypropyl 4-methylbenzenesulfonate [(R)-26]. The
procedures were similar to those used for the preparation of
compound (S)-11; the final product was gained as a yellow oil
1
(
173.3 mg, 34%). H NMR (400 MHz, CDCl ) δ 8.92 (s, 1H),
3
8
.21−7.99 (m, 2H), 7.90 (d, J = 9.3 Hz, 1H), 7.80 (d, J = 8.3
Hz, 2H), 7.35−7.30 (m, 3H), 7.11 (d, J = 2.5 Hz, 1H), 6.98−
6
.89 (m, 2H), 4.28−4.21 (m, 3H), 4.13−3.97 (m, 2H), 3.16
(
s, 6H), 2.41 (s, 3H). ESIMS: m/z calcd for C H N O S
2
6
28
3
5
+
4
94.2; found 494.7, [M + H] .
1
183
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Article
Immunofluorescent Staining. Immunofluorescent stain-
ing was performed on the brain slices from Tg-tau (C57BL6,
rTg4510, 7 months old, female) and AD patients (91 years old,
male, temporal lobe; 85 years old, male, entorhinal cortex).
Deparaffinization was performed according to the same
methods described above. All steps that involved staining
were completed in the moist cassette at r.t. Antigen retrieval
was implemented with immersion of the brain slices into 0.01
M citric acid/sodium citrate acid buffer (pH = 6.0) at 95 °C
for 10 min. After they cooled to r.t., the brain slices were
washed with PBS (0.1 μM, pH = 7.4, 3 × 5 min). The antigens
were blocked by incubation of the brain slices with 3% bovine
serum albumin for 2 h, and the blocking buffer was removed by
PBS (0.1 μM, pH = 7.4, 3 × 5 min). Subsequently, the brain
slices were treated with antiphospho-tau monoclonal antibody
into a solution containing 3.0 mL of n-octanol and 3.0 mL of
PBS (pH 7.4). The mixture was vortexed for 5 min and then
centrifuged for 5 min at 3000 rpm/min. Samples from n-
octanol (100 μL) and PBS (500 μL) were carefully taken in
triplicate and tested by an automatic γ counter (WALLAC/
Wizard 1470, PerkinElmer). Samples from the n-octanol layer
(2.0 mL) were added into a solution containing 1.0 mL of n-
octanol and 3.0 mL of PBS (pH 7.4) to repartition until
consistent distribution coefficient values were obtained.
Ex Vivo Biodistribution Studies. The ex vivo biodis-
tribution was performed on normal ICR mice (5 weeks, 18−22
1
8
g, male). F-labeled ligand (6 MBq/mL, 100 μL) in 10%
EtOH saline solution was intravenously (i.v.) injected to each
mouse through the tail vein. The mice (n = 5) were sacrificed
by decapitation at 2, 10, 30, and 60 min after i.v. injection,
respectively. The organs of interest were removed and
weighed; the radioactive counts of the organs were recorded
by an automatic γ counter (WALLAC/Wizard 1470,
PerkinElmer). The percent dose per gram of wet tissue (%
ID/g) was calculated by the ratio of the tissue counts to
diluted aliquots of the injected dose. The mean ± standard
deviation (SD) was expressed as the uptake value for five
parallel tests.
(
AT8) (5 μg/mL, Thermo scientific) at 4 °C for 20 h. After
they were washed by PBS (0.1 μM, pH = 7.4, 3 × 5 min) at r.t.,
the brain slices were incubated with a secondary antibody
(
donkey antimouse IgG) (4 μg/mL, Alexa Fluor594, Abcam
plc) for 2 h. After the brain slices were washed by PBS (0.1
μM, pH = 7.4, 3 × 5 min), the staining results were acquired
on an EVOS FL fluorescence microscope imaging system (Life
Technologies) equipped with a Cy5.0 filter set.
In Vitro Autoradiography. The deparaffinized brain slices
In Vivo Metabolism Studies. In vivo metabolism studies
were performed on normal ICR mice rats (8−9 weeks, 300−
from an AD patient (85 years old, male, entorhinal cortex)
18
18
were incubated with 7 MBq/mL of F-labeled tracers (10%
ethanol) alone or 18F-labeled tracers with 200 nM blocking
compound ((S)-16, THK-5317, or T807) at r.t. for 40 min and
then washed briefly with a 25% EtOH aqueous solution. After
they were dried, the slices were exposed to a medium
phosphorus plate (PerkinElmer) for 30 min, and the in vitro
autoradiography images were recorded by a Cyclone Plus
storage phosphor system (PerkinElmer).
350 g, male). The radiolabeled ligand (S)-[ F]16 (18.5−74
MBq, 200 μL) in 10% EtOH saline solution was injected via
tail vein, and the mice were sacrificed at 10 and 60 min
postinjection. The blood was collected as much as possible and
centrifuged at 3000 rpm/min for 5 min to obtain plasma.
Brain, liver, feces, and urine were also harvested and
homogenized in 1−2 mL of acetonitrile. The obtained
homogenates were passed through a disposable polypropylene
3
In Vitro Inhibition Binding Assay. [ H]THK523,
H]T807, and [ H]PIB were used as radioligands to
(
PP) syringe microporous filter (0.22 μm) and added with 300
[3
3
μL of acetonitrile to further precipitate protein. The final
samples were filtered and analyzed with a radio-HPLC
quantitatively determine the binding affinity. The AD brain
tissue was homogenized in an assay medium (0.1% Tween-20
in PBS) and diluted 500 times in volume. Aliquots (100 μL) of
this brain homogenates were transferred to 48 tubes. Then 100
(
2
Shimadzu) equipped with an SPD-20A UV detector (λ =
54 nm) and γ-radiation scintillation detector (Bioscan Flow
Count 3200 NaI/PMT). HPLC conditions: reverse-phase
semipreparative Venusil MP C18 column (Bonna-Agela, 5 μm,
3
3
μL of solution of radioligand ([ H]THK523, [ H]T807, or
3
[
H]PIB, 10 Bq/μL) in an assay medium was added into the
1
0 mm × 250 mm), 4 mL/min, acetonitrile/H O = 60%/40%.
2
−
5
−10
tube; 10 μL of nonradiolabeled probes (10 to 10 M in
DMSO) was added into each tube and diluted with an assay
medium to the final volume of 1 mL. Then the mixture was
vortexed and incubated at 37 °C for 2 h. After separation of the
mixture with a cell harvester, the filter paper (Whatman, GF/B;
preprocessed by 0.5% polyethylenimine solution) was washed
with 3 mL of PBS three times and then placed in 7 mL plastic
vials. Four milliliters of scintillation fluid was added to each
plastic vial and vortexed until the filter was floating. After
incubation overnight, the radioactivity of scintillation vials was
counted. The data were analyzed by GraphPad Prism version
In Vivo Micro-PET/CT Studies. In vivo micro-PET/CT
imaging was performed in SD rats (n = 2−3, 300−350 g). The
rat was anesthetized with 2.5% isoflurane gas in an oxygen flow
during the imaging process and positioned on the bed of a
micro-PET/CT scanner (Super Nova, PINGSENG Health-
care). An attenuation correlation was performed with an X-ray
transmission scan, and then the radiolabeled ligands
18
18
[
F]THK-5351 or (S)-[ F]16 (18.5 MBq, 2 mL) in 10%
EtOH saline solution were i.v. bolus injected into the rat.
Subsequently, dynamic brain PET scans (0 to 60 min) were
started, and whole-body scans were also obtained at 60 min.
We employed a three-dimensional ordered subset expectation
maximization (3D OSEM) algorithm for image reconstruction.
The time frames for brain PET imaging were 30 × 120 s.
Region of interest (ROI) outlines were drawn manually on the
whole brain. The resulting ROI was then transferred to
coregistered PET images. Standardized uptake values (SUVs)
were calculated for all ROIs by a normalization of the injected
dose and body weight. Time−Activity curves (TACs) were
calculated from this.
4
.03 (GraphPad Software). The inhibition constant (K ) was
i
59
calculated using the Cheng-Prusoff equation
K = IC /(1 + [L]/K )
i
50
d
where [L] is the concentration (0.4 nM). K is the equilibrium
d
dissociation constant of the control radioligand, and IC is the
5
0
half-maximal inhibitory concentration. K was calculated by a
d
“Scatchard analysis of homologous displacement” from multi-
ple runs with self-displacement from an AD brain.
Distribution Coefficient (Log D) Determination.
HPLC-purified tracer (120 MBq/mL, 100 μL) was added
Mao-B Binding Assays in Liver Homogenates. The
fresh liver tissue of an SD rat was washed in 0.2 M cold PBS
1
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Article
a
Scheme 1. Reagents and Conditions
a
(
a) DMSO, r.t., 10 min, 58%; (b) N H ·H O, Pd/C, MeOH, 90 °C, 2 h, 89%; (c) (1) (CH O) , CH ONa, MeOH, 90 °C, 5 h, (2) NaBH , 90
2 4 2 2 n 3 4
°
C, 2 h, 95%; (d) CsF, DMF, 65 °C, 2h, (R)/(S)-4 = 59%/62%, (R)/(S)-6 = 95%/94%; (e) TBAF (1 M in THF), anhydrous THF, reflux for 6 h,
(
S)/(R)-5 = 76%/68%; (f) HCl (1 M), THF, 90 °C, reflux for 30 min, NaHCO , (R)/(S)-7 = 99%/99%; (g) TBDMSCl, imidazole, acetonitrile,
3
r.t., 12 h, (S)/(R)-8 = 94%/87%; (h) (Boc) O, anhydrous THF, 90 °C, 12 h, (S)/(R)-9 = 81%/90%; (i) TBAF (1 M in THF), anhydrous THF, 90
2
°C, 3 h, (R)/(S)-10 = 74%/91%; (j) TsCl, TEA, CH Cl , r.t., 12 h, (S)/(R)-11 = 28%/33%; (k) 3,4-dihydropyran, PPTS, CH Cl , r.t., 10 h, (S)/
2
2
2
2
(R)-12 = 90%/72%.
(
−4 °C, pH = 7.6), followed by homogenization in 50 mL of
pellet after the removal of supernatants. The pellet was diluted
with 4 mL of sucrose (0.3 M, −4 °C) and stored at −80 °C
until required. The preparation of the enzyme−substrate
solution was performed by dissolving 10 μL of 4-
(trifluoromethyl)benzylamine in 9990 μL of PBS (0.2 M, pH
7.6). The chromogenic solution prepared for inclusion in the
assay mixture contained vanillic acid (2 mM), 4-amino-
antipyrine (1 mM), and peroxidase (250 IU/ml) in PBS (0.2
sucrose (0.3 M, −4 °C) under an ice−water bath.
Subsequently, the liver homogenates were centrifuged at
1
000g for 10 min; the supernatants were separated and stored
at −4 °C. The residues were further centrifuged at 1200g for
5 min with 10 mL of sucrose (0.3 M, −4 °C). All
supernatants were gathered together and centrifuged at
0 000g for 15 min to obtain the deposition of mitochondrial
1
1
1
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a
Scheme 2. Reagents and Conditions
a
(
a) DMF, K CO , 150 °C, overnight, 94%; (b) H , Pd/C, MeOH, r.t., 6 h; (c) DMSO, r.t., 10 h, 14/21 = 10%/30%; (d) CsF, DMF, 65 °C, 2 h,
2
3
2
(
R)/(S)-15 = 85%/85%, (R)/(S)-17 = 94%/91%, (R)/(S)-22 = 83%/91%, (R)/(S)-24 = 99%/83%; (e) TBAF (1 M in THF), anhydrous THF,
reflux for 6 h, (S)/(R)-16 = 85%/82%, (S)/(R)-23 = 85%/87%; (f) HCl (1M), THF, 90 °C, reflux for 30 min, (R)/(S)-18 = 96%/94%, (R)/(S)-
5 = 98%/88%; (g) TsCl, TEA, CH Cl , r.t., 12 h, (S)/(R)-19 = 38%/36%, (S)/(R)-26 = 38%/34%; (h) 3,4-dihydropyran, PPTS, CH Cl , r.t., 10
2
2
2
2
2
h, (S)/(R)-20 = 87%/90%, (S)/(R)-27 = 77%/74%.
M, pH 7.6). Clorgyline solutions were prepared on a daily basis
RESULTS AND DISCUSSION
■
(
(
1 μM). The assay was performed on a black 96-well plate
Costar), incubated at 37 °C for 30 min with 50 μL of tissue
Chemistry. The reference compounds for radioligands (S)-
5 and (R)-5 were synthesized in five steps, with total overall
yields of 22% and 21%, respectively. The 6-NO substituted 2-
homogenates and 25 μL of clorgyline solution per well. After
that, 50 μL of probe (concentration gradient manner) was
added and incubated for another 30 min, with continuous
incubation for 90 min with a successive addition of enzyme−
substrate solution (120 μL) and chromogenic solutions (40
μL). The absorbance of each well was measured on
InfiniteM200 pro microplate readers (Tecan) at 490 nm.
The data were analyzed by GraphPad Prism version 4.03
2
phenylquinoxaline backbone (1) was synthesized as the major
product by rapid cyclization of 4-nitro-o-phenylenediamine
and 2-bromo-4′-hydroxyacetophenone in DMSO. Here,
DMSO serves as both solvent for the reaction mixture and
oxidant for the cyclized intermediate. After filtration, the
compound was washed with EtOH (100 mL), which left a
yellow crystal with high purity (Scheme 1). After an efficient
catalytic reduction of the nitro group into an amino group to
obtain 2, the N-monomethylation was performed through a
reductive methylation under basic conditions, to obtain
(
GraphPad Software).
1
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Table 1. Fluorescent Properties and Binding Affinity of the 2-Phenylquinoxaline-Based Tau Probes
a
b
c
Φ,
DCM
%
λem/λex (nm)
Ki (nM) at
d
e
probes
S)-5
R)-5
S)-16
R)-16
S)-23
R)-23
PBS
DCM
PBS
THK site T807 site PIB site selectivity fold
IC50 (μM) for Mao-B
clog P
(
(
(
(
(
(
58.6
58.0
70.7
68.8
22.4
22.9
12.3
16.2
4.6
7.3
4.2
493/411
493/410
500/426
499/424
502/422
499/424
554/418
553/419
578/441
571/437
584/425
579/426
36
>1000
630
611
385
>1000
>1000
87
2.4
1.21
0.33
>10
>10
>10
>10
0.49
2.98
f
f
4.1
10.3
23
13
30
125
356.3
66
94
188
30.5
34.6
2.9
7.2
6.3
2.98/2.36
3.01/2.61
3.01
3.02
3.02
5.8
g
f
THK-5351
2.9
0.7
3.02/2.71
a
b
c
Absolute quantum yield measured in CH Cl (100 nM) and PBS (100 nM). Emission maxima determined in CH Cl and PBS. K values
2
2
2
2
i
d
e
determined at the THK site, T807 site, and PIB site using AD brain homogenates. Selectivity fold of K values for Tau over Aβ. Calculated using
the online ALOGPS 2.1 program. Measured log D value by testing the distribution coefficient between n-octanol and PBS. K values adapted from
i
f
g
d
ref 30.
a
Scheme 3. Reagents and Conditions
a
a) (1) 18F , K CO , Kryptofix-222, anhydrous acetonitrile, 100 °C; (2) (i) HCl (1 M), (ii) NaHCO .
−
(
2
3
3
compound 3. We selected (R)/(S)-glycidyl-3-nitrobenzenesul-
fonate with the highly efficient leaving group as the reagents to
synthesize the chiral epoxides (R)/(S)-4 in moderate yields of
(S)-7 in high yields of 99%. To eliminate the influence of
secondary aryl amino group on the radiofluorination reaction,
we protected the amino group. First, the free dihydroxyl
groups were protected by TBDMSCl, to give (R)/(S)-8.
Second, the aryl methylamino group was protected using tert-
butyloxycarbonyl (Boc) group, to give (R)/(S)-9. The silyl
protecting group was removed selectively by TBAF to give
(R)/(S)-10 in yields of 74−91%. The terminal less-hindered
hydroxyl group in (R)/(S)-10 was selectively tosylated by p-
tosyl chloride (TsCl) in CH Cl . To prevent the influence of
the residual tertiary hydroxyl group in the radiofluorination, it
was protected by a tetrahydropyranyl (THP) group, to give the
desired precursors (S)/(R)-12.
The reference compounds for radioligand (S)/(R)-16 and
(S)/(R)-23 were synthesized in four steps, with total overall
yields of 7%, 7%, 20%, and 22%, respectively (Scheme 2). The
N,N-dimethylamino group in (S)/(R)-16 and (S)/(R)-23
could not be synthesized from normal methylation reactions,
5
9−62%. The final fluorinated 2-phenylquinoxaline derivatives
were obtained by a regioselective epoxy ring-opening reaction
using tetra-n-butylammonium fluoride (TBAF) in yields of
6
8−76%, with an optical purity of the final products (S)/(R)-5
greater than 99% (Figure S1 and Table S1).
The precursors (R)/(S)-12 for radioligands (S)-5 and (R)-5
were synthesized in seven steps from compound 3, with total
overall yields of 13% and 16%, respectively (Scheme 1). We
used (S)-(+)-2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane-p-
toluenesulfonate and (R)-(−)-2,2-dimethyl-4-(hydroxymeth-
yl)-1,3-dioxolane-p-toluenesulfonate, instead of (S)-(+)- or
2
2
(
R)-(−)-3-chloropropane-1,2-diol building block, to generate
the chiral dioxolane derivatives (R)/(S)-6 in high yields of 94−
5%, selectively, at the phenolate position instead of secondary
9
aryl amino group. The isopropylidene group was removed by
acidic hydrolysis to give the free propanediol derivatives (R)/
including reductive amination and direct CH I methylation.
3
1
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Figure 3. In vitro fluorescent staining on brain slices from Tg-tau mouse (C57BL6, rTg4510, 7 months old, female) by (R)-5 (A, GFP filter set,
0×) and (S)-16 (E, GFP filter set, 20×). The location of NFTs was confirmed by immunofluorescent staining using AT8 antibody(B, F, Cy5.0
2
filter set, 20×). Merged images of fluorescent staining and immunofluorescent staining results (C, G, and K). Fluorescent staining on brain slices
from WT mouse (C57BL6, 12 months old, male) by (R)-5 (D, GFP filter set, 20×) and (S)-16 (H, GFP filter set, 20×). Red squares mark areas
for magnification as presented in bottom panels (I, J).
Figure 4. In vitro multifluorescent staining on brain slices from two AD patients. Staining results of (R)-5 (A, F) (1 μM, 10% ethanol) and (S)-16
(
K, P) (1 μM, 10% ethanol) on GFP channel. The same brain slices were costained by AT8 antibody on Cy5.0 channel (B, G, L, Q); Aβ probe
DANIR 3b (1 μM, 10% ethanol) on RFP channel (C, H, M, R); cell nucleus dye DAPI (1 μg/mL, in PBS) on DAPI channel (D, I, N, S). Merged
images for the corresponding staining results (E, J, O, T). Rows 1 and 3: (85 years old, male, entorhinal cortex; 20×); Rows 2 and 4: (91 years old,
male, temporal lobe; 40×).
Thus, we designed a new synthetic route to introduce a
dimethylamino group from the reaction of the commercially
available 5-chloro-2-nitroaniline in DMF, which serves as both
solvent and dimethylamine precursor, to afford 13. After a
1
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catalytic reduction of the nitro group, the 2-phenylquinoxaline
backbone was synthesized analogously as described in Scheme
able fluorescent properties of high quantum yield and emission
maxima around 550 nm in PBS, suitable for fluorescence
staining. As shown in Figure 3, Figure 4, and Figures S3−S6,
the hyperphosphorylated Tau deposits shown in the circled
shape in the Tg-tau mouse and in the flame shape in the AD
patients were illuminated clearly by (R)-5 and (S)-16 (GFP
filter set). When the same tissue region was costained by DAPI
to stain the cell nucleus (em: 461 nm, DAPI filter set) and
DANIR 3b to specifically stain Aβ (RFP filter set), no
overlapped spots were observed in the merged images of the
three staining images described above. This shows that our
probes bind with protein targets other than Aβ. When the
same tissue region was stained using immunofluorescence, with
the first antibody, antiphospho-tau monoclonal antibody
1
, to afford compounds 14 and 21, depending on the position
of the dimethylamino group (Scheme 2). Flash column
chromatography was not effective to separate these two
regioisomers, due to their similar structure and solubility in
organic solvents. We separated these two regioisomers by a
recrystallization of their oxalates in ethanol, due to their
solubility difference. After it was neutralized with ammonium
hydroxide, we obtained pure 14 and 21 with 10% and 30%
yields, respectively. The remaining two steps for the synthesis
of (S)/(R)-16 and (S)/(R)-23 are in accordance with those
described in Scheme 1 (detailed in Scheme 2). Similarly, the
precursors for radioligands (S)/(R)-16 and (S)/(R)-23,
namely, (S)/(R)-20 and (S)/(R)-27, were synthesized in
four steps from compounds 14 and 21, with total overall yields
of 30%, 28%, 28%, and 18%, respectively (Scheme 2). The
optical purity of the final products (S)/(R)-5, (S)/(R)-16, and
50
(
(
AT8), and the secondary antibody, donkey antimouse IgG
Cy5.0 filter set), extensive overlap was observed in the
merged images of the probes, (R)-5 and (S)-16, and of the
antibodies, confirming that our probes stain the hyper-
phosphorylated Tau deposits specifically. The AT8 antibody
recognizes a phosphatase-sensitive epitope containing the
phosphorylated Ser202 residue (numbering of human
Tau40). Different from immunofluorescent staining, our
probe recognizes the β-sheet structure of Tau tangles, which
could explain that our probe performed more pronounced
staining for Tau tangles than immunofluorescent staining. No
specific signals were observed on brain slices from wild-type
(
S)/(R)-23 was determined to be more than 99% by chiral
1
13
HPLC analysis. All original H NMR, C NMR, MS, and
HRMS spectra are described in the Supporting Information.
Fluorescent Properties. Compared with the 2-phenyl-
26
quinoline derivatives, 2-phenylquinoxaline derivatives dis-
played a stronger fluorescence with a red-shift (Table 1). The
quantum yield of these compounds depends on both chemical
structures and solvent used. The stronger electron-donating
ability of the N,N-dimethylamino group gave both a higher
quantum yield and a longer emission maxima than that of the
N-monomethylamino group, (S/R)-16 versus (S/R)-5. How-
ever, if the N,N-dimethylamino group is not in direct
(
WT) mouse. In addition, our probes stain NFTs consistently
and rapidly, within 30 s even without washing. Compared with
other time-consuming and expensive immunofluorescent
staining, we believe that the fluorescent probes described
here would also be useful for an in vitro detection of NFTs.
Under the same experimental conditions, when we used
2
conjugation with the electron-poorer sp hybridized nitrogen,
the quantum yield is lower, (S/R)-23 versus (S/R)-16. In
addition, these ligands exhibited moderate solvatochromism,
with a moderate red-shift of fluorescence emission maxima
from CH Cl to PBS. These ligands gave the highest quantum
32
PBB3 as a probe for fluorescent staining, it displayed
considerable staining of Aβ plaques (Figure S7).
2
2
Determining Binding Constants. We determined the
binding affinity of our probes using AD brain homogenates
competing with three reference radioligands, namely,
yield in CH Cl , with ∼70% quantum yield for (S/R)-16.
2
2
However, the fluorescence was quenched significantly in PBS.
These 2-phenylquinoxaline derivatives may serve as useful
fluorescent probes for an in vitro detection of NFTs, due to
their favorable fluorescent properties.
3
3
3
[
H]THK-523, [ H]T807, and [ H]PIB. The first two
radioligands are for the two different binding sites on Tau
aggregates, respectively, and the last is for the binding site on
Aβ plaques. In some studies, the heparin-induced recombinant
Tau fragments (K18Δ280 or K18) were usually used for in
18
18
Radiochemistry. The F-labeled tracers, (S/R)-[ F]5, 16
and 23, were prepared from the nucleophilic substitution of
the tosylate in precursors, (S)/(R)-12, 20 and 27, by
51
1
8
vitro affinity measurements. However, the inner conforma-
tion of a chemically induced Tau fibril may not be a reliable
model for native NFTs, with differences including size,
[
(
F]fluoride in the presence of K CO and Kryptofix-222
2 3
Scheme 3). After radiofluorination, removal of the N-boc or
THP protecting groups by HCl (1 M) in the same reaction
vessel was achieved in one pot yielding the crude tracers. The
radiotracers were purified by semipreparative HPLC, and their
identities were confirmed by HPLC coinjection analysis, with
radiochemical yields greater than 20% and radiochemical
purity greater than 98% (Figure S2 and Table S2). The molar
activity of these tracers was calculated to be 90−123.5 GBq/
μmol.
52
morphology, and hyperphosphorylation. The K values of
i
our probes on different binding sites are shown in Table 1, and
the probes exhibited a high affinity (<40 nM) on the THK site
of NFTs, a moderate affinity (87−356.3 nM) on the PIB site
of Aβ plaques, and a poor affinity (>1000 nM) on the T807
site of NFTs. These results indicate that the 2-phenylquinoxa-
line scaffold shares the same THK binding site but not the
5
3
T807 site of NFTs. Since the concentration of Tau
aggregates is ∼5−20 times lower than that of Aβ aggregates,
and since the two aggregates usually coexist in the brain of AD,
a high affinity and selectivity for Tau aggregates are required
In Vitro Histological Staining. In this study, multi-
fluorescent staining of these 2-phenylquinoxaline derivatives
was performed on brain slices from transgenic (Tg) mouse
(C57BL6, rTg4510, 7 months old, female) and two autopsy-
5
4,55
confirmed AD patients to demonstrate their selective binding
with Tau deposits instead of Aβ plaques. In vitro fluorescent
staining is one of the rapid and efficient methods to screen
fluorescent probes that have a high binding affinity and
selectivity for specific targets at the tissue level. As mentioned
before, these 2-phenylquinoxaline derivatives exhibited favor-
for the PET imaging agents of Tau aggregates.
Our probes,
= 4.1
(R)-5 and (S)-16, displayed a high affinity to NFTs (K
i
and 10.3 nM) and outstanding selectivity over Aβ aggregates
(31- and 35-fold selectivity). They showed a similar affinity
30
and selectivity with both THK-5351 (2.9 nM) and T807
53
(2.6 nM).
1
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Figure 5. In vitro autoradiography of 18F-radiolabeled ligands (R)-[ F]5 (A) and (S)-[ F]16 (D) on brain slices from an AD patient (85 years
18
18
old, male; entorhinal cortex). The presence and location of NFTs were confirmed by fluorescent staining with the nonradiolabeled probes (R)-5
(
B) and (S)-16 (E) or immunofluorescent staining using antiphospho-tau monoclonal antibody (AT8) (C). Red squares mark areas for
18
magnification as presented in the middle panels (F, G, H, I, J). Totaling binding of (S)-[ F]16 (K); self-blocking with 200 nM (S)-16 (L);
blocking with 200 nM THK-5317 (M); blocking with 200 nM T807 (N).
Distribution Coefficient (Log D) Determination. To
validate the effectiveness of introducing a fluoropropanol side
chain to reduce lipophilicity, we determined the distribution
coefficient between n-octanol and PBS (log D) and calculated
the log P value using the online ALOGPS 2.1 program (clog P)
for these 2-phenylquinoxaline derivatives. The lipophilicity
Ex Vivo Biodistribution Studies. An ex vivo biodis-
tribution study is a simple and efficient way to quantitatively
determine the distribution of radiotracers in different organs,
and it may also give information on metabolites. Three pairs of
1
8
18
F-labeled tracers [(S)/(R)-[ F]5, 16, and 23] were
investigated in normal ICR mice to evaluate their pharmaco-
1
8
1
8
kinetic properties, as well as [ F]THK-5351 (leading
compound of THK series). Similar to the requirements of
PET Aβ imaging agents, a desirable PET Tau imaging probe
should possess a high initial uptake and fast washout in a
normal brain. As shown in Figure 6A and Table 2, S3−S9,
these radiotracers penetrated the blood-brain barrier (BBB)
with greater than 6% ID/g in 2 min postinjection. The
values for (S)-[ F]16 with a dimethylamino group (log D =
1
8
2
.61 ± 0.05, clog P = 3.01) and (R)-[ F]5 with a
monomethylamino group (log D = 2.36 ± 0.14, clog P =
18
2
.98) compared to dimethylamino derivatives [ F]4a with a
49
fluoropegylated chain (log D = 3.14 ± 0.23, clog P = 3.56)
showed a significant decrease in lipophilicity by adding a
fluoropropanol side chain, which may be the key factor that
these probes possess high binding affinity and selectivity
toward Tau over Aβ. In addition, these quinoxaline derivatives
displayed similar lipophilicity values compared with those of
18
dimethylamino derivatives (S)/(R)-[ F]16 showed the high-
est initial brain uptake (10.95% and 10.97% ID/g,
18
respectively), which is pretty higher than [ F]THK-5351
(
6.46 ± 0.43% ID/g). Although the (R)/(S)-enantiomers
1
8
[
F]THK-5351 (log D = 2.71 ± 0.05, clog P = 3.02).
showed a similar ability to cross the BBB, they differ in brain
washout rate. The S-enantiomers (S)-[ F]5, 16, and 23 with
In Vitro Autoradiography. We used autoradiography
18
studies of our radioligands on Tau-rich brain tissue from AD
patient (entorhinal cortex), to confirm the specific binding
ability of radiolabeled tracers with NFTs. As shown in Figure 5,
a high density of specific radioactive signals in the entorhinal
cortex was observed with high resolution to be NFTs, with
their locations confirmed by fluorescent staining with the
nonradiolabeled probes or immunofluorescent staining using
antiphospho-tau monoclonal antibody (AT8). Moreover, (S)-
brain2 min/brain60 min = 15.9, 6.5, and 19.2, respectively, showed
a faster brain washout rate than the R-enantiomers (R)-[ F]5,
18
1
6, and 23, with brain2 min/brain60 min = 10.1, 3.8, and 10.7,
respectively. The same pattern was also observed for the PET
1
8
imaging of F-labeled Aβ imaging agents, as well as for the
1
8
6,30,56
F-labeled THK derivatives.
As expected, the faster brain
washout of the S-enantiomers also showed a faster excretion
from intestine (Figure 6B). The brain washout rate also
depends on lipophilicity and the rate of metabolism. The
1
8
[
F]16 showed a strong signal with a high amount of
displaceable binding in the region of high NFT load (Figure
K) by self-blocking (Figure 5L) and THK-5317 blocking
Figure 5M) or partial blocking by T807 (Figure 5N).
18
tracers, ((S)/(R)-[ F]16), with dimethylamino group at the 6
position, showed a much slower brain washout than ((S)/(R)-
5
(
18
[ F]5), with a monomethylamino group. However, (R/S)-
1
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1
8
18
18
Figure 6. Ex vivo kinetic curves of brain (A), intestine (B), and bone (C) uptake for (S)/(R)-[ F]5, (S)/(R)-[ F]16, (S)/(R)-[ F]23, and
[
18
F]THK-5351 in normal ICR mice at different time points postinjection. Error bars represent standard deviations for 5−6 mice at each time
point. (D) HPLC profiles of the parent compound and metabolites extracted from the brain and liver of normal ICR mice after i.v. injection of (S)-
18
[
F]16.
Table 2. Brain and Intestine Kinetics of 18F-Labeled Tracers in Normal ICR Mice
a
a
brain uptake (% ID/g)
intestine uptake (% ID)
ligands
2 min
60 min
clearance rate
2 min
60 min
1
8
(
(
(
(
(
(
[
S)-[ F]5
7.79 ± 0.77
7.06 ± 0.63
10.95 ± 1.29
10.97 ± 1.37
9.00 ± 0.64
10.05 ± 0.43
6.46 ± 0.43
0.49 ± 0.08
0.7 ± 0.08
1.69 ± 0.17
2.85 ± 0.55
0.47 ± 0.06
0.94 ± 0.10
0.47 ± 0.08
15.9
10.1
6.5
10.22 ± 1.32
11.43 ± 0.64
13.79 ± 1.09
9.67 ± 1.21
9.89 ± 0.67
12.03 ± 1.08
12.70 ± 1.85
55.99 ± 3.6
48.74 ± 3.45
43.65 ± 4.88
35.63 ± 5.28
57.35 ± 2.49
43.71 ± 2.48
60.12 ± 2.90
1
8
R)-[ F]5
1
8
S)-[ F]16
1
8
R)-[ F]16
3.8
1
8
S)-[ F]23
19.2
10.7
13.7
1
8
R)-[ F]23
1
8F]THK-5351
a
Each value was given as the mean ± SD for 5−6 mice at each time interval.
[18
F]23 with a dimethylamino group at the 7 position showed
shown in Figure 6D and Table S10, three radioactive
metabolites were observed, and they presented less lip-
ophilicity than the parent compound with a shorter retention
time in the HPLC profile. Liver is the main metabolic organ;
the percent of metabolites increased from 10 to 60 min
a much faster brain washout rate than these with a
dimethylamino group at the 6 position. The former may
have a faster metabolic breakdown than the latter, because of
the electronic conjugation difference discussed above. In
1
8
18
addition, the brain clearance rate of [ F]THK-5351
brain2 min/brain60 min = 13.7) is equivalent to that of the S-
postinjection. It is worth mentioning that (S)-[ F]16
(
exhibited excellent biostability in the brain, and the percentage
of the parent compound is more than 90% at 60 min
postinjection. This also means that the polar metabolites
generated in the liver cannot penetrate the BBB by
bloodstream transportation. Furthermore, to make a direct
enantiomers of quinoxaline derivatives. Low levels of bone
uptake (<4% ID/g) during the whole investigation indicate
that our tracers and [ F]THK-5351 were stable against in
18
vivo defluorination (Figure 6C).
18
In Vivo Metabolism and Micro-PET/CT Studies. To
explore the in vivo biostability and metabolic mechanics, (S)-
comparison of brain kinetic properties between (S)-[ F]16
18
and [ F]THK-5351, dynamic micro-PET/CT studies were
performed on SD rats. Although the peak brain uptake of (S)-
1
8
[
F]16 was i.v. injected into normal ICR mice, the mice were
1
8
18
sacrificed at 10 and 60 min postinjection, and samples of the
brain, liver, plasma, urine, and feces were collected and
extracted with acetonitrile and analyzed with radio-HPLC. As
[ F]16 (6 min) was slightly slower than that of [ F]THK-
18
5351, (S)-[ F]16 exhibited a much higher BBB penetration
than [ F]THK-5351 (SUV, 2.2 vs 1.3); the brain region
18
1
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18
18
Figure 7. Dynamic micro-PET/CT study of (S)-[ F]16 and [ F]THK-5351 in SD rats (n = 2−3). (A) Summed PET images from rats overlaid
on CT images in a transverse, coronal, and sagittal plane at time points of 2, 10, and 60 min. (B) Whole brain region time−activity curves after i.v.
injection of tracers. Radioactivity concentration is expressed as SUV that is normalized to the injected dose and body weight.
1
8
18
became clearly visible after i.v. injection (Figure 7). However, a
very high nonspecific retention in the submandibular gland of
detected by (R)-[ F]5 and (S)-[ F]16, and the results were
further confirmed by fluorescent and immunofluorescent
staining. Ex vivo biodistribution in mice and micro-PET/CT
studies in rats demonstrated that these probes could penetrate
BBB sufficiently and actualize the brain region visible, and they
showed fast washout from the normal brain quickly.
1
8
[
1
F]THK-5351 was observed from a scanning video at 2 and
0 min (see Supporting Information Video), which greatly
suppressed the signals from the brain region. Although the
1
8
18
washout rate of (S)-[ F]16 is slower than that of [ F]THK-
5
18
18
351 (3.1 vs 8.2), the radioactivity for (S)-[ F]16 in the brain
Furthermore, (S)-[ F]16 displayed good stability in the
brain and low inhibition to MAO-B. The parallel comparison
still could be cleared at 60 min and concentrated in the
intestine and stomach for these two tracers (see Figure 7 and
Supporting Information Video). In addition, the results of in
vivo PET studies are very consistent with ex vivo
18
18
between (S)-[ F]16 and [ F]THK-5351 revealed our tracers
have many advantages such as high binding affinity, selectivity,
initial brain uptake, and low off-target binding. On the basis of
the results of these preclinical evaluations, (S)-16 satisfied the
criteria to be promising PET probes for Tau imaging, and
clinical validation studies of (S)-[ F]16 in AD patients and
healthy subjects are undergoing; the results will be published in
due course.
18
biodistribution and indicated (S)-[ F]16 possessed good
pharmacokinetic properties.
18
In Vitro MAO-B Inhibition Binding Measurements.
Monoamine oxidase B (MAO-B) is a flavoenzyme in the
mitochondrial outer membrane and plays a major role in the
metabolism of neuroactive and vasoactive amines. In the
previous studies, the MAO-B binding was also considered as
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c01078.
■
57
sı
*
important off-target binding of THK probes. Thus, the off-
target effects of our Tau probes to MAO-B were assessed by an
inhibition of the enzyme−substrate with the spectro-photo-
metric assay, which was the similar method used for the test of
39
MAO inhibition with JNJ-067. As shown in Table 1 and
Figure S8, (R/S)-5 and THK-5351 exhibited a moderate to
poor inhibition ability (IC , 0.33−10 μM) to MAO-B. To
Additional figures, tables, NMR and MS spectra (PDF)
Micro-PET/CT brain scanning video of [ F]THK-
5351 at 2 min after i.v. injection (AVI)
Micro-PET/CT brain scanning video of [ F]THK-
5351 at 10 min after i.v. injection (AVI)
Micro-PET/CT whole-body scanning video of (S)-
[ F]16 at 60 min after i.v. injection (AVI)
Micro-PET/CT whole-body scanning video of
[ F]THK-5351 at 2 min after i.v. injection (AVI)
18
50
18
make a comparison, selegiline (IC50 = 22.17 ± 8.42 nM), an
irreversible MAO-B inhibitor, was also assayed under the same
condition. Selegiline could efficiently inhibit the enzyme−
substrate, and the results were consistent with the previously
18
58
reported value (IC50 = 19.60 ± 0.86 nM). Compared with
THK-5351 (IC50 = 491.25 nM), some of the quinoxaline
derivatives displayed poor MAO-B affinity, for example, (R/S)-
18
1
6 and (R/S)-23. Thus, the MAO-B off-target binding of these
AUTHOR INFORMATION
Corresponding Authors
■
quinoxaline derivatives is expected to be very low.
In the present study, we designed, synthesized, and
1
8
Mengchao Cui − Key Laboratory of Radiopharmaceuticals,
Ministry of Education, Beijing Normal University, Beijing
evaluated three pairs of F-labeled 2-phenylquinoxaline
derivatives as novel Tau probes. Excellent fluorescent proper-
ties and in vitro fluorescent staining results validate that these
probes could selectively bind to NFTs on brain sections from
Tg-tau mouse and AD patients. Meanwhile, high quantum
yield, selective binding, and fast labeling promoted them as
100875, China; orcid.org/0000-0002-3488-7864;
Email: cmc@bnu.edu.cn; Fax: +86-13811995064
Lisheng Cai − Molecular Imaging Branch, National Institute
of Mental Health, National Institutes of Health, Bethesda
2
0892, Maryland, United States; Email: lishengcai@
fluorescence dyes to detect NFTs with high sensitivity in vitro.
K values determined using competitive ligands ([ H]THK-
mail.nih.gov; Fax: +1-301-480-5112
3
i
3
3
523, [ H]T807, and [ H]PIB) on AD brain homogenates
Authors
revealed that (R)-5 and (S)-16 possessed a high affinity to Tau
tangles and selectivity over Aβ plaques. Autoradiography
studies on AD brain tissue demonstrated that NFTs could be
Kaixiang Zhou − Key Laboratory of Radiopharmaceuticals,
Ministry of Education, Beijing Normal University, Beijing
100875, China
1
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Article
Fan Yang − Key Laboratory of Radiopharmaceuticals,
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00875, China
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Yuying Li − Key Laboratory of Radiopharmaceuticals,
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aggregation in Alzheimer’s disease: An attractive target for the
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00875, China
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PLA General Hospital, Beijing 100853, China
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Tau Positron Emission Tomography (PET) Imaging: Past, Present,
and Future. J. Med. Chem. 2015, 58, 4365−4382.
Jinming Zhang − Department of Nuclear Medicine, Chinese
PLA General Hospital, Beijing 100853, China
Junfeng Wang − Gordon Center for Medical Imaging,
Department of Radiology, Massachusetts General Hospital/
Harvard Medical School, Charlestown 02129, Massachusetts,
United States
Jiapei Dai − Wuhan Institute for Neuroscience and
Neuroengineering, South-Central University for Nationalities,
Wuhan 430074, China; orcid.org/0000-0002-1474-
(7) Shaw, L. M.; Vanderstichele, H.; Knapik-Czajka, M.; Clark, C.
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1
(
509−1517.
9) Jack, C. R.; Knopman, D. S.; Jagust, W. J.; Shaw, L. M.; Aisen, P.
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Okamura, N. The challenges of tau imaging. Future Neurol. 2012, 7,
M.C. and K.Z. conceived and designed the experiments. K.Z.,
M.C., L.C., F.Y., Y.L., and Y.C. performed the experiments.
K.Z., M.C., and L.C. analyzed the data. M.C., J.Z., X.Z., J.D.,
and L.C. contributed reagents, materials, and analysis tools.
K.Z., M.C., J.W., and L.C. wrote the paper.
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This work was funded by the Beijing Municipal Natural
Science Foundation (No. 7182089) and the National Natural
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Notes
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ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, β-amyloid; NFTs, neurofibrillary
tangles; PET, positron emission computed tomography; FDA,
Food & Drug Administration; DMSO, dimethyl sulfoxide; r.t.,
room temperature; MeOH, methanol; DMF, N,N-dimethyl-
formamide; TBAF, tetrabutylammonium fluoride; THF,
tetrahydrofuran; HCl, hydrochloric acid; TBDMSCl, tert-
butyldimethylsilyl chloride; Boc, tert-butyloxycarbonyl; TsCl,
p-tosyl chloride; TEA, trimethylamine; PPTS, 4-toluenesul-
fonic acid pyridine salt; THP, tetrahydropyranyl; PBS,
phosphate buffer saline; Tg, transgenic; WT, wild-type; SD,
standard deviation; ID, injected dose; i.v., intravenous; BBB,
blood-brain barrier; NMR, nuclear magnetic resonance; J,
coupling constant (in NMR spectrometry); MS, mass
spectrometry; ESI, electron spray ionization; HRMS, high-
resolution mass spectrometry; HPLC, high-performance liquid
chromatography; DAPI, 4′,6-diamidino-2-phenylindole; GFP,
green fluorescent protein; ROI, region of interest; SUVs,
standardized uptake values
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Pani, L. Qualification opinion of novel methodologies in the
predementia stage of Alzheimer’s disease: cerebro-spinal fluid related
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