Highly Sensitive Near-Infrared Fluorophores for in Vivo Detection of Amyloid-β Plaques in Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.5b00861

Alzheimer's disease (AD) is pathologically characterized by the accumulation of β-amyloid (Aβ) deposits in the parenchymal and cortical brain. In this work, we designed, synthesized, and evaluated a series of near-infrared (NIR) probes with electron donor-acceptor end groups interacting through a π-conjugated system for the detection of Aβ deposits in the brain. Among these probes, 3b and 3c had excellent fluorescent properties (emission maxima > 650 nm and high quantum yields) and displayed high sensitivity and high affinities to Aβ aggregates (3b, K_d = 8.8 nM; 3c, K_d = 1.9 nM). Both 3b and 3c could readily penetrate the blood-brain barrier with high initial brain uptake and fast to moderate washout from the brain. In vivo NIR imaging revealed that 3b and 3c could efficiently differentiate transgenic and wild-type mice. In summary, our research provides new hints for developing smarter and more activatable NIR probes targeting Aβ.

Full text of DOI:10.1021/acs.jmedchem.5b00861

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Article
High Sensitive Near-Infrared Fluorophores for in Vivo
Detection of Amyloid-# Plaques in Alzheimer’s Disease
Hualong Fu, Mengchao Cui, Liu Zhao, Peiyu Tu, Kaixiang Zhou, Jiapei Dai, and Boli Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00861 • Publication Date (Web): 11 Aug 2015
Downloaded from http://pubs.acs.org on August 23, 2015
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High Sensitive Near-Infrared Fluorophores for in
Vivo Detection of Amyloid-β Plaques in
Alzheimer’s Disease
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†
*,†
†
†
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‡
Hualong Fu, Mengchao Cui, Liu Zhao, Peiyu Tu, Kaixiang Zhou, Jiapei Dai, and Boli Liu
†
†
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing
Normal University, Beijing 100875, P. R. China
‡
Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for
Nationalities, Wuhan 430074, P. R. China
ABSTRACT
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Alzheimer’s disease (AD) is pathologically characterized by the accumulation of β-amyloid (Aβ)
deposits in the parenchymal and cortical brain. In this article, we designed, synthesized and
evaluated a series of near-infrared (NIR) probes with electron donor-acceptor end groups
interacting through a π-conjugated system for the detection of Aβ deposits in the brain. Among
these probes, 3b and 3c had excellent fluorescent properties (emission maxima > 650 nm and
high quantum yields) and displayed high sensitivity and high affinities to Aβ aggregates (3b: Kd
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=
8.8 nM; 3c: K = 1.9 nM). Both 3b and 3c could readily penetrate the blood-brain barrier with
d
high initial brain uptake and fast to moderate washout from the brain. In vivo NIR imaging
revealed that 3b and 3c could efficiently differentiate transgenic and wild-type mice. In
summary, our research would provide new hints for developing smarter and more activatable
NIR probes targeting Aβ.
INSTRUCTION
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Alzheimer’s disease (AD) stands out among the etiologies of acquired dementia and is
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, 2
becoming an extensive health problem with the ever-increasing aging population. Generally, it
has been thought that the extracellular accumulation of β-amyloid (Aβ) peptides into plaques
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plays a vital role in AD onset. Therefore, research on the development of probes for the in vivo
detection of Aβ has emerged as a way to define the etiology of the dementia, deduce the
progression of AD and provide an accurate index of putative anti-Aβ agents.
In the past decades, substantial research work has been dedicated to developing probes capable
of crossing the blood-brain barrier (BBB) and selectively binding to Aβ plaques for use in
structural imaging techniques, such as magnetic resonance imaging (MRI), positron emission
tomography (PET) and optical imaging. MRI is a time-consuming tool in Aβ imaging due to the
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, 7
ambiguous signal contrast between amyloid plaques and surrounding tissue.
To date, most
radiolabeled small-molecule probes targeting Aβ have been developed for PET imaging, and
1
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three F-labeled agents have been approved by the FDA; however, the usage of short-lived
isotopes requires an on-site cyclotron and a team of radiochemistry staff, limiting its widespread
8, 9
application.
Optical imaging, including multiphoton microscopy and near-infrared (NIR)
imaging, permits high temporal and spatial resolution. Furthermore, with the aid of fluorescence
molecular tomographic (FMT) imaging, optical imaging has drawn more attention as an optimal
tool in the detection and quantification of biological processes, preclinical trials, and drug
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0-13
development.
Multiphoton microscopy has been used for in vivo imaging in small animals
14
and has served as a powerful approach to monitor the effects of anti-Aβ agents. However, such
microscopy is invasive and narrow-viewed, hence not applicable to the intact imaging of the
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brain compartment.
NIR imaging, particularly relying on rationally designed NIR imaging
agents (the NIR detection window is 650 - 900 nm), is a viable technique for non-invasive
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imaging due to several advantages, such as sufficient light penetration depth in living tissues,
avoidance of radiation, noninvasive operation, real-time imaging and inexpensive instruments.
Several NIR probes aimed at the early detection of AD have been reported to date (Figure. 1).
AOI-987 is a charged molecule with a small Stocks shift (the wavelength value derived from the
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difference between excitation and emission of a fluorophore, 20 nm) and moderate affinity (K =
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220 nM), which implies limited BBB permeability and obscured signal increase upon binding
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with Aβ plaques. Better yet, Ran et al. have exploited curcumin derivatives as NIR probes for
soluble or insoluble Aβ species. In 2009, CRANAD-2 was reported as a NIR probe for Aβ
plaques with advantages over other candidates such as its ideal binding constant (K = 38.7 nM)
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and optimal fluorescent properties (emission maximum at 805 nm). More recently, CRANAD-
58 has been designed and synthesized for detecting both insoluble and soluble Aβ species in
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vitro and in vivo. However, the slow brain washout rate caused by their high lipophilicity has
impeded their application in NIR imaging. Additionally, BAP-1 with high quantum yield (QY)
and high affinity to Aβ aggregates (K = 44.1 nM) was explored as another NIR probe targeting
d
Aβ plaques, but the small Stocks shift (34 nm) and high nonspecific uptake in the scalp hindered
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its practical use for in vivo imaging.
Figure 1. Structures of the reported NIR probes for detecting insoluble or soluble Aβ species.
RESULTS AND DISSCUSSION
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Design, synthesis and fluorescent properties. To design feasible imaging probes for the in
vivo detection of Aβ plaques in the brain, our group exploited the electron donor-acceptor (D-A)
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architecture as a preferable scaffold using the following rationale.
1) Due to the internal
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charge transfer (ICT) transition, these “D-A” molecules exhibit a striking discrepancy between
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dipole moments in their ground and lowest energy singlet excited states. Extended π-
conjugated systems could result in increased electronic delocalization, which induces a much
narrower band gap between the excited and ground states, leading to an emission wavelength in
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the deep-red or NIR spectral range.
Our probes displayed reasonable fluorescent properties
(moderate increase in fluorescence intensity upon binding to Aβ aggregates with a blue shift) and
moderate to high affinities to Aβ aggregates, among which DANIR 2c (Figure 2) had the highest
affinity (K = 26.9 nM). For NIR imaging, the spatial resolution is limited when probing through
d
the intact cranium and scalp. Thus, the quantification of the amyloid plaques in the brain
achieved by the spatial integration of fluorescence intensity mainly relies on the sufficient light
penetration depth and the high QY of the fluorophore. Thus the low QY of our D-A probes
impeded all their practical application in vivo. 2) Our previous work also suggested that the
conjugated double-bond bridge was crucial for Aβ binding and that the affinity increased with
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the extension of the π-bridge.
3) As reported, for aromatic hydrocarbons such as benzene,
naphthalene, anthracene and pyrene, a remarkable increase in QY was observed when the size
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increased from 1 to 4 rings. Therefore, it is reasonable to speculate that the QY could be
improved by replacing the benzene ring with a naphthalene ring. 4) Furthermore, 2-(1-(6-
(dimethylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP, Figure 2) with a hydrophobic
naphthalene ring was reported by Jacobson et al. as an environmentally sensitive, hydrophobic
and neural fluorophore with high dipole moment for fluorescence microscopy that was capable
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of readily passing cell membrane. In 2012, the analogs of DDNP were explored by Petric et al.
as probes for the neuroimaging of amyloids, and most of the probes displayed high affinities to
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Aβ aggregates. Additionally, 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene)
FDDNP, Figure 2), a fluorinated DDNP version with a high affinity for Aβ1-40 fibrils, has been
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reliably utilized for the histological staining of amyloid-like protein specimens and became the
first PET imaging probe for the location of Aβ and neurofibrillary tangles (NFTs) in the brains of
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AD patients.
However, the deficient emission wavelength has precluded their application as
NIR probes. 5) Finally, the N,N'-dimethylamino group is an ideal electron donating moiety and
an encouraging substituent for better Aβ binding affinity, and its planar geometry allows efficient
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conjugation with the naphthalene ring system.
Based on the above design rationale, we
report the synthesis, characterization, and biological evaluation of a more extensive series of D-
A molecules (DANIR 3a-e, Figure 2) as NIR imaging probes for Aβ plaques in AD. The binding
models of these probes to Aβ fibrils were explored by molecular docking simulation.
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Figure 2. Chemical structures of DANIR 2a-c, DDNP, FDDNP and newly designed DANIR
probes.
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The synthesis of the newly designed DANIR probes is shown in Scheme 1. The Wittig
reaction was employed to extend the polyenic chains in preparing naphthyl aldehydes. To obtain
the final probes, the naphthyl aldehydes were subjected to the Knoevenagel reaction with
malononitrile catalyzed by K CO , and most of the products (except 3e) could be crystallized
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from the reaction mixture in good yields (> 70%). The single crystal structures of 3b and 3c were
presented as ORTEPs (Figure 7), and relevant crystallographic data are listed in Table S5 in
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Supporting Information. In contrast to DDNP and its analogs,
each of the probes crystallized
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into one planar conformer. Single-crystal structures and H NMR data demonstrated that 3a-e
existed in the E isomer. The purity of 3a-e was determined to be higher than 98% by high-
performance liquid chromatography (HPLC).
As anticipated, with the lengthening of the polyenic chains, the probes showed significant
bathochromic shifts of absorption/emission maxima and solvent dependency in various solvents,
indicating a narrowing HOMO-LUMO gap and ongoing ICT transition (Figure S2 and S3, and
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Table S1 in Supporting Information). As shown in Table 1, the emission wavelengths of 3b-e
red-shifted to the NIR range (> 680 nm in PBS) with large Stokes shifts (> 120 nm). Due to the
longer polyenic chains, 3d and 3e exhibited emission wavelengths of 828 and 830 nm in PBS,
respectively, which were longer than the emission wavelength of CRANAD-2. Furthermore, 3b
and 3c exhibited high QYs of 29.9% and 9.0% in dichloromethane (DCM), respectively,
considerably higher than the QY of DANIR 2c (4%). As shown in Figure S1 in Supporting
Information, molecules presenting extended polyenic chains (3d and 3e) displayed an onset of
saturation of the excited state dipole (as well as the emission maxima). This result could be
related to both charge delocalization and structural effects, which counterbalance the length
effect. That is to say, the extension of the conjugated system results in increased photoinduced
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charge delocalization, leading to tapering-off of the excited state dipole. Concurrently, multiple
torsions along the single bonds of the polyenic chains could remarkably affect the global
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geometry of the molecules in solution, thus influencing the excited state dipole. These effects
also had a marked impact on the QYs of the probes. With increasing polyenic chain length, an
intense decrease in QY was observed from 3b to 3e (Table 1 and Figure S1 in Supporting
Information). As suggested by our results, lengthening the polyenic chains had a limited effect in
achieving compounds with longer emission maxima and might produce undesirable results,
including decreased QY. These results provide meaningful guidelines for the design of D-A
based NIR probes.
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a
Scheme 1. Synthesis of naphthyl aldehydes and DANIR probes.
a
Reagents and conditions: (a) Dimethylamine (33% in aqueous solution), Na S O , water, 140
2
2
5
°C, high pressure, 18 h. (b) (1) n-BuLi (2.5 M in hexane), anhydrous THF, -78 °C, 30 min, (2)
anhydrous DMF, r.t, 15 min, (3) HCl (1 M), r.t. (c) (1) ((1,3-Dioxolan-2-
yl)methyl)triphenylphosphonium bromide, anhydrous THF, NaH, 18-crown-6, r.t, 24 h; (2) HCl
(
1 M), r.t. (d) Malononitrile, K CO , methanol or THF, r.t, 5 min.
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Table 1. Fluorescent properties, binding data and calculated log P (clog P) values of 3a-e,
DANIR 2c, CRANAD-2 and BAP-1.
a
a
b
c
d
d
k
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Probe
λem1 (nm) λem2 (nm)
Φ (%)
Fold
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716
280
K (nM)
K (nM)
cLog P
3.46
4.05
4.56
5.10
5.62
3.37
6.16
-
d
i
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9
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0
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0
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0
3a
614
682
783
828
830
538
615
678
4.8/0.1
29.9/0.3
9.0/0.009
3.7/0
44.8 ± 15.3 156.4 ± 15.8
3b
8.8 ± 1.5
1.9 ± 1.1
n/o
96.7 ± 6.7
32.7 ± 3.4
197.4 ± 43.3
> 1000
3c
e
e
3d
716
n/o
625
n/o
5
3e
0.1/0
n/o
n/o
f
f
f
g
f
f
f
DANIR 2c
665
4.1 /0.2
12
26.9
36.9
g
h
j
g
g
7
49
05
70.3 /0.04
n/o
-
-
-
CRANAD-2
h
h,i
40 /0.6
h
h
h
8
715
70
38.69
j
g
g
j
BAP-1
648
648
13.5 /0
-
44.1
-
3.52
a
b
Determined in PBS (λ ) and upon binding with Aβ aggregates (λem2). Measured in
em1
c
DCM/PBS, respectively. Fold increase in fluorescence intensity upon binding with Aβ
aggregates. K and K values of 3a-e were measured in triplicate with results given as the mean
± SD. A solution of 3d in PBS (30% ethanol, 1 ꢀM) was used. Data from ref.[21]. Data were
measured in the same conditions with 3a-e. Data from ref.[17]. QY was measured after
d
d
i
e
f
g
h
i
j
k
CRANAD-2 bound to Aβ aggregates. Data from ref.[19]. The values were calculated using
40
the online ALOGPS 2.1 program. n/o: not observed.
In vitro characterizations. A valuable property of a fluorophore is its sensitivity to the
surrounding environment, so an ideal Aβ probe should have a “turn-on” mechanism after binding
to Aβ plaques. We first evaluated the fluorescence response of 3a-e toward the synthetic Aβ1-42
aggregates or bovine serum albumin (BSA) in PBS (pH = 7.4). As expected, in a solution
containing Aβ aggregates, most of the probes (except 3d and 3e) exhibited a remarkable increase
in fluorescence intensity (ranging from 280- to 716-fold) and significant blue shifts (ranging
from 35 to 79 nm) at a low final concentration of 50 nM, whereas 3d only showed a weak
response (5-fold) at a high concentration of 1 ꢀM (Table 1, Figure 3 and S4 in Supporting
Information). Moreover, no significant changes in the fluorescence intensity and emission
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wavelength were observed when 3e and CRANAD-2 were incubated with Aβ aggregates in the
experiment condition (Figure S4 in Supporting Information). The results also suggested that no
interaction occurred between our probes and BSA. The “turn-on” phenomenon of 3a-d toward
Aβ aggregates occurs mainly because these small molecules have intercalated into the
hydrophobic clefts of the aggregated amyloid fibrils with high affinities. This interpretation was
further confirmed by the significant changes of the QYs in DCM and PBS, solvents with
increasing polarity (Table 1). These results suggested that most of our probes have high
sensitivity to Aβ aggregates and can be selectively “turned on” upon binding with them.
Furthermore, the high sensitivity of 3a-c in detecting Aβ aggregates paves the way for their
potential application in fluorescence intensity distribution analysis during anti-Aβ drug
screening, which plays a significant role as a future readout technology for miniaturized high-
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throughput screening.
Various Aβ plaques ranging from diffuse to highly compacted (dense-core) have been proved
to be responsible for the neuropathology of AD. Another form of plaques distributed in the walls
of small arteries and arterioles within the leptomeninges and cortex play a central role in the
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onset of cerebral amyloid angiopathy (CAA). The selective binding of 3a-e to different forms
of amyloid plaques was determined by neuropathological fluorescence staining experiments
using 8-ꢀm brain sections from double transgenic (Tg) mice and four different AD/CAA
patients. As shown by the staining patterns of the brain slices from Tg mice (Figure 3A and B,
and S5 in Supporting Information), the presence and distribution of the fluorescent spots stained
by 3a-d were observed to colocalize with the Aβ plaques stained with thioflavin-S (Th-S, a
common dye for staining Aβ fibrils). After incubating the brain slices from AD patients with
solutions of 3a-e, different forms of Aβ plaques including diffuse plaques, dense-core plaques
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and plaques accumulating along the vessel walls were stained clearly by 3a-d with a high
contrast-to-background fluorescence ratio (Figure 3C and D, and S6 in Supporting Information).
However, 3e displayed no specific staining of the plaques in the brain slices from Tg mice or
AD/CAA patients. Based on the staining patterns, most of the new probes (3a-d) can bind
selectively to Aβ plaques in sections from both Tg mice and AD/CAA patients with high
sensitivity and clarity, indicating their superior affinities to Aβ aggregates.
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Figure 3. (A-D) Histological staining of brain slices from Tg mice (A and B, magnification: 10×)
and an AD patient (C and D, magnification: 10×). A) and C), B) and D) were stained by 3b and
3c, respectively. (E and F) Emission wavelengths of 3b (E) and 3c (F) upon binding with Aβ
aggregates or BSA.
The affinities of the probes for Aβ aggregates were quantitatively assessed through saturation
and inhibition binding assays. Table 1 summarizes the K and K values of 3a-d. In accordance
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with the Aβ aggregate binding and fluorescence staining results, 3a-d displayed moderate to high
affinities to Aβ1-42 aggregates, whereas 3e exhibited a poor affinity. It was found that 3a had
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comparable affinity (K = 44.8 nM) to CRANAD-2 (K = 38.7 nM) and DANIR 2c (K = 26.9
d
d
d
nM). More importantly, 3b and 3c exhibited very high affinities, with K values of 8.8 and 1.9
d
nM, respectively, higher than the values of all of the reported NIR Aβ probes. In the inhibition
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assays, most of the probes (except 3e) inhibited [ I]IMPY in a dose-dependent manner (Figure.
S7D in Supporting Information). In both assays, the binding affinities of 3a-c were improved
with the lengthening of the π-bridge. It was previously extrapolated that a conjugated and planar
architecture of a ligand is very important for Aβ binding, and small-size substitutions at the
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dicyanovinyl position could also improve the binding affinity.
Each of these probes has a
small-size hydrogen substitution at the dicyanovinyl position. Moreover, as proved by the crystal
and optimized structures (Figure 7 and S17 in Supporting Information), the N,N'-
dimethylnaphthalene and dicyanomethylene groups are approximately coplanar in the extended
π-conjugated system. In our case, 3d and 3e had the longest polyenic chains, but conversely
displayed decreased affinities to Aβ aggregates, mainly because the excessive torsions along the
single bonds induced by prolonging the polyenic chain length blocked their ready access to the
Aβ fibrils. Taken together, our probes exhibited high to poor affinities to Aβ aggregates.
Notably, 3b and 3c had very high affinities, highlighting their potential application as NIR
imaging probes in detecting and quantifying amyloid deposits within the tissue targets.
Biostability and BBB penetration. As the stability of the chemical entities in plasma is a
prerequisite for showing good in vivo efficacy, we examined the stability of 3b and 3c in mice
plasma. The results showed that the tested probes were stable with purities higher than 94% after
incubation with mice plasma for 30/60 min (Figure S8 in Supporting Information), indicating
that 3b and 3c had excellent biostability in plasma and could be developed for in vivo use.
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The lipophilicity (log P within 1-3.5 range) and molecular weight (< 600 Da) of the ligands are
two primary factors affecting the capability of BBB penetration. The molecular weights (M. W.)
and calculated log P (clog P) values of 3b (M. W. = 273.33, clog P = 4.05) and 3c (M. W. =
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99.37, clog P = 4.56) were lower than for CRANAD-2 (M. W. = 410.26, clog P = 6.16) and a
little higher than for DANIR 2c (M. W. = 249.31, clog P = 3.37). This result indicates that 3b
and 3c have great potential in BBB penetration. The brain uptake of 3b and 3c was intuitively
confirmed by dissecting brain samples at 2 min after intravenous (i.v.) injection of the probes
(Figure 4A). Under visible light, the brain dosed with 3b appeared in darker color than the
control brain. Due to the high QY and brain uptake of 3b, the corresponding brain appeared
shiny red under ultraviolet (UV) light (365 nm). The BBB penetrating ability of our probes was
quantified in ICR mice, and the data were expressed as the percent injected dose per gram of
tissue (% ID/g). As shown in Table 2, 3b displayed high initial brain uptake at 2 min (12.80 %
ID/g) and fast washout from the brain at 60 min post-injection (brain2 min/brain60 min = 21.3), vital
qualities in achieving a preferable signal-to-noise ratio. Due to higher lipophilicity and larger
molecular weight, 3c showed decreased brain uptake (4.44 % ID/g at 10 min post-injection) and
slower washout from the brain (brain10 min/brain60 min = 3.3), whereas 3d and 3e could not
penetrate the BBB. Determined in the same conditions, DANIR 2c could rapidly penetrate into
the brain with high initial uptake (16.88 % ID/g at 2 min post-injection), and wash out from the
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brain very fast (brain2 min/brain60 min = 42.2). In accordance with the NIR imaging results,
CRANAD-2 displayed slow washout from the brain (brain10 min/brain60 min = 2.4). BAP-1
displayed low brain uptake (2.37 % ID/g at 10 min post-injection), slow egress from the brain
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(brain10 min/brain60 min = 5.5), and significant instability in the living brain. As seen from the
HPLC profiles (Figure S10 in Supporting Information), 3b showed excellent biostability, while
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c was metabolized as of 10 min post-injection. The experimental log P values and kinetics of
b and 3c in the blood of mice has been evaluated. As shown in Table 3, the experimental log P
3
values were unanimous to the calculated ones (Table 1). Both 3b and 3c showed fast clearance
from the blood. Furthermore, 3c displayed considerably higher lipophilicity and initial uptake in
blood (log P = 4.48, blood2 min = 3.40 % ID/g) than for 3b (log P = 4.10, blood2 min = 1.90 %
ID/g), which may account for the lower brain uptake of 3c. In summary, the length of the
polyenic chains had a significant impact on the brain kinetics of the probes and the extension of
the polyenic chains clearly decreased the initial brain uptake and brain washout rate. Of note, 3b
displayed rapid penetration through the intact BBB, fast washout from the brain and excellent
biostability in the living brain, demonstrating that 3b is a promising Aβ probe for in vivo NIR
imaging.
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Figure 4. (A) Brains of ICR mice separated at 2 min after i.v. injection of 3b and 3c under
visible light (top row) and UV light (365 nm, bottom row). Brains were also dissected from mice
directly as a control. (B) Comparison of brain uptake of 3b, 3c, DANIR 2c, CRANAD-2 and
BAP-1 in ICR mice.
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Table 2. Brain kinetics of 3b, 3c, DANIR 2c, CRANAD-2 and BAP-1 in ICR mice.
Time after injection / % ID/g
Probe
Brain10 min/brain60 min
2 min
10 min
30 min
60 min
a
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b
12.80 ± 0.78 7.18 ± 0.50 2.88 ± 0.58 0.60 ± 0.15
3.65 ± 0.56 4.44 ± 0.20 2.05 ± 0.20 1.34 ± 0.22
21.3
3
c
3.3
a
DANIR 2c 16.88 ± 1.80 4.41 ± 0.92 0.70 ± 0.02 0.40 ± 0.05
42.2
CRANAD-2 4.06 ± 0.28 7.56 ± 1.22 5.82 ± 0.31 3.17 ± 0.42
2.4
b
BAP-1
2.23 ± 0.29 2.37 ± 0.42 1.00 ± 0.15 0.43 ± 0.07
5.5
a
b
The clearance rate of 3b and DANIR 2c are presented as brain2 min/brain60 min. Data from
ref.[20].
Table 3. The experimental log P values and blood clearance rate of 3b and 3c in ICR mice.
Time after injection / % ID/g
Probe
Log P
2 min
60 min
3b
4.10 ± 0.17
1.90 ± 0.12 0.25 ± 0.010
3c
4.48 ± 0.0045 3.40 ± 0.52 0.065 ± 0.0027
In vivo imaging and ex vivo fluorescence staining. To validate the feasibility of 3b and 3c
for in vivo NIR imaging, nude mice were i.v. injected with the probes to observe the brain
kinetics. As shown in Figure S11 and S12 in Supporting Information, nearly all of the
fluorescence signals were centralized in the brain compartments and could be captured
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efficiently, indicating that 3b and 3c can readily penetrate into the BBB and have potential
applications for in vivo NIR imaging of the brain compartment. In accordance with the brain
kinetics results, the brain egress of 3b was much faster than for 3c.
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Next, to confirm the great potential of 3b and 3c in non-intrusively detecting Aβ plaques in the
brain, we performed in vivo NIR imaging with Tg mice and age-matched wild-type (WT) mice.
Semi-quantitative information was obtained from the recorded images at different time points
before and after dye dosing following equation (1). As shown in Figure 5A, the fluorescence
signals of 3b displayed striking differences between the Tg and WT groups at the earliest time
point of 10 min after i.v. administration, earlier than for CRANAD-2 and AOI987 (t = 30 min).
Furthermore, as shown in Figure 5B, the fluorescence signals of 3b in the brains of Tg mice
decreased at a considerably slower rate, and the discrepancies of the fluorescence signals
between the Tg and WT mice [F(Tg)/F(WT) ranged from 1.0 to 2.1] became larger in the course
of the detection time (0 - 60 min), indicative of specific dye retention trapping by amyloid
plaques. However, due to its inadequate brain uptake, slow brain washout rate and instability in
living brains, 3c showed fluorescence signal differences between the Tg and WT groups at a
hysteretic time point of 30 min, and narrower discrepancies of the fluorescence signals
[
F(Tg)/F(WT) ranging from 1.0 to 1.5] were observed (Figure 5C and D). In consideration of the
blue-shift of the probes after binding to the Aβ plaques, the fluorescent light emitted at shorter
wavelengths (620 and 680 nm for 3b and 3c, respectively) from the mice brains was also
detected. As shown in Figure S13 and S14 in Supporting Information, the fluorescence signals of
3b at 620 nm displayed significant differences between the Tg and control groups from 10 min
post injection, with large discrepancies [F(Tg)/F(WT) ranging from 1.0 to 2.8]. However, for the
captured fluorescence behaviour of 3c, no noteworthy differences were obtained between the
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results under 780 and 680 nm. In conclusion, due to the high QY, excellent brain kinetics and
biostability, 3b presented advantages over 3c in in vivo NIR imaging and exhibited great
capacity to distinguish Tg and WT mice.
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Figure 5. In vivo imaging of Aβ deposits by 3b and 3c. (A and C) Representative images of Tg
mice (top row) and age-matched WT ones (bottom row) at selected time points before and after
i.v. injection of 3b (A) and 3c (C). (B and D) Relative fluorescence intensity in the brain
compartments of Tg mice (red line) and WT mice (black line) after i.v. injection of 3b (B) and 3c
(D). The differences of the fluorescence signals between Tg and WT mice were also illustrated
by the values of F(Tg)/F(WT) (right Y axis). The fluorescence signals of 3b and 3c were
recorded at 680 (A and B) and 780 nm (C and D), respectively.
The in vivo imaging results were further confirmed by ex vivo fluorescence staining of frozen
brain sections from Tg and WT mice after administration of 3b and 3c. Under fluorescence
microscopy, Aβ plaques were observed in the cortex (Figure 6) and cerebellum (Figure S15 in
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Supporting Information) regions after in vivo staining by 3b and 3c, respectively, whereas the
sections of WT mice showed no such fluorescent spots (Figure S16 in Supporting Information).
In addition, the distribution of the amyloid plaques was confirmed by staining the same sections
with Th-S (Figure S15 in Supporting Information). This result confirmed that 3b and 3c could
specifically label the amyloid plaques in vivo; hence, the discrepancies of the fluorescence
signals between the Tg and WT mice in NIR imaging were due to the specific retention of the
probes after binding to the Aβ plaques.
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Figure 6. Ex vivo histology results of brain slices of cortex regions of Tg mice after dosing with
3b (A and B) and 3c (E and F). (A) and (C) are partial enlarged views of homologous sections.
Magnification: 10×.
Computational studies. Computational approaches have been widely used in studying
protein-ligand interactions, including their applications in exploring the binding modes and
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interactions of probes such as thioflavin-T (Th-T), Congo Red (CR),
[Ru(bpy) dppz]
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and benzyloxybenzene derivatives toward Aβ. In this study, the binding modes of 3a-e to Aβ
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fibrils were examined by molecular docking simulations. The lowest-energy/top-ranked docking
poses are shown in Figure 7: 3a-e bound to the grooves on the KLVFFA fibril surface, an
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identical binding site to CR and IMPY , namely, a hydrophobic cleft oriented parallel to the
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main fibril axis formed by VAL18 and PHE20. Considering that the mature amyloid fibrils have
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lengths > 1 ꢀm, the long grooves on the amyloid fibril surface are ubiquitous. We could
extrapolate that this binding site is a general recognition mode of amyloid fibrils by planar
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molecules.
Analysis of the docking results showed that the molecules with larger conjugated
systems and dipole moments could better fit the binding channel with lower binding energies
(Table S6 in Supporting Information); concurrently, for 3a-c, a good correlation between the pKi
2
values and binding energies (R = 0.92) was observed. However, the K values (binding
i
affinities) of 3d and 3e did not coincide well with the computational binding energy. A more
detailed discussion about the docking results can be found in Supporting Information. Therefore,
our computational studies could partially reproduce our experimental trends and suggested that
3a-e likely filled in the grooves on the fibril surface formed by VAL18 and PHE20 with valid
shape complementarities.
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Figure 7. ORTEP structures of 3b (A) and 3c (B) and merged docking conformations of 3a-e (C
and D). The residues of Aβ fibrils are presented in the licorice style (C) and a molecule surface
representation (D).
CONCLUSIONS
In this report, we successfully designed and synthesized a new family of DANIR probes (3a-e)
with electron donor-acceptor architecture for the detection of Aβ plaques for NIR imaging. Most
of these probes (3a-c) could be “turned on” by Aβ aggregates with high sensitivity and
remarkable fluorescent property changes. The DANIR probes possess various affinities to Aβ
aggregates. Among them, 3b and 3c exhibit the highest affinities and meet all of the
17, 21
requirements of a NIR probe for detecting Aβ deposits noninvasively in vivo.
Our results
also demonstrated that polyenic chains (C=C bonds) have a significant impact on the
characteristics of the probe, such as the fluorescent properties, affinity and brain kinetics.
Increasingly, fluorescent imaging has been used for drug discovery because of the simple
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operation, short acquisition time and simultaneous measurement of several mice, compared with
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the much longer data acquisition time on a single animal by PET, SPECT or MRI. Thus, the
application of 3b and 3c could accelerate the throughput and development of anti-Aβ drugs. The
binding modes of 3a-e with Aβ fibrils were extensively studied with docking simulations and the
results suggested that our probes most likely bound to the clefts on the surface of the Aβ fibrils.
In summary, we designed and synthesized a series of NIR probes capable of sensing Aβ
aggregation/plaques in vitro or in vivo and our results could shed light on developing a new
generation of smarter and more activatable NIR probes targeting Aβ.
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EXPERIMENTAL SECTION
General Information. All chemicals were commercial products of analytical grade, and were
used without further purification unless otherwise indicated. Column chromatography
purification was performed on silica gel (Yantai Industry Research Institute, 80 - 100 Å) packed
into glass columns. The synthetic trifluoroacetic acid salt forms of amyloid-β (1-42) peptides
were obtained from Osaka PEPTIDE INSTITUTE, Inc. (Osaka, Japan), and aggregated for in
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vitro studies using the previously reported procedures. H and C NMR were obtained at 400
MHz and 100 MHz, respectively, on a Bruker spectrometer in CDCl solutions at room
3
temperature, and the chemical shifts were quoted in δ values (parts per million, ppm) downfield
relative to the internal TMS. Mass spectra and the high-resolution mass were acquired using a
Surveyor MSQ Plus (ESI) instrument and a GCT CA127 Micronass UK instrument.
Fluorescence and ultraviolet-visible spectra were measured using a spectrofluorophotometer
(RF-5301PC, Shimadzu, Japan) and a UV spectrophotometer (UV-2450, Shimadzu, Japan),
respectively. Fluorescence QYs were measured using an aqueous solution of Rhodamine 6G as a
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standard (Φ = 0.76). The purity and biostability in mice plasma of the key compounds were
determined by HPLC performed on a Shimadzu SCL-20 AVP system equipped with a SPD-20A
UV detector (λ = 254 nm). The HPLC analysis was performed on a Venusil MP C18 reverse
phase column (Agela Technologies, 5 µm, 4.6 mm × 250 mm). In the BBB penetration test, an
Agilent 1260 Infinity Quaternary LC (Agilent Technologies) system was used, and the HPLC
analyses were performed on a Cosmosil packed column (5C18-AR-II, 4.6 × 150 mm, Nacalai
Tesque). In the HPLC analyses, a binary gradient elution system was used, where the mobile
phases A and B were water and acetonitrile, respectively. Fluorescence observation was
performed on the Axio Observer Z1 (Zeiss, Germany) equipped with DAPI, AF488, AF546 and
Cy 5.0 filter sets. Human brain sections of AD/CAA patients were obtained from Chinese Brain
Bank Center.
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Normal ICR mice (5 weeks, male) were used for BBB penetration determination and normal
nude mice (BALB/c nude, 6 weeks, female) were used for in vivo imaging (purchased from Vital
River Laboratory Animal Technology Co. Ltd., Beijing, China). Tg mice (C57BL6,
APPswe/PSEN1, 14-19 months old, female), used as Alzheimer’s models, were purchased from
the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. All animal
experiments were approved by the animal care committee of Beijing Normal University.
Chemistry. 6-Bromo-N,N-dimethylnaphthalen-2-amine (1) Compound 1 was prepared by a
2
9
previously reported procedure and a white solid (4.43 g, 65.8%) was obtained.
-(Dimethylamino)-2-naphthaldehyde (2a) Compound 2a was prepared by a previously
6
2
9
reported procedure and a yellow solid (8.6 g, 93.5%) was obtained.
(
E)-3-(6-(Dimethylamino)naphthalen-2-yl)acrylaldehyde (2b) To a stirred solution of 2a
(
1.5 g, 7.5 mmol) in anhydrous THF (25 mL) was added (1,3-dioxolan-2-
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ylmethyl)triphenylphosphonium bromide (5.7 g, 13.2 mmol). NaH (60% dispersion in mineral
oil, 1.7 g) was then added to the suspension in small portions, after which 18-crown-6 was added
as catalyst. The stirring was continued for 24 h at room temperature, and then the reaction was
quenched with an aqueous solution of HCl (1 M), followed by neutralization with ammonia
water. The mixture was extracted with CH Cl (3×40 mL), and the organic phase was dried
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over anhydrous magnesium sulfate. After the solvent was removed in vacuum, the residue was
purified by flash column chromatography (petroleum ether/CH Cl / ethyl acetate = 6:6:1, v: v) to
2
2
1
give compound 3 as a yellow solid (770 mg, 45.4%). mp 145.3-146.6 ºC. H NMR (400 MHz,
CDCl ) δ: 9.71 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.67 (d, J = 8.6 Hz,
3
1H), 7.58 (d, J = 16.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H),
+
6
.75 (dd, J = 15.8 Hz, 7.8 Hz, 1H), 3.12 (s, 6H). MS: m/z calcd for [C H NO + H] 226.1,
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found 226.2.
(2E,4E)-5-(6-(Dimethylamino)naphthalen-2-yl)penta-2,4-dienal (2c) The same reaction
described above to prepare compound 2b was used, and the brown solid 2c was obtained (83.0
1
mg, 33.5%). mp 192.8-193.7 ºC. H NMR (400 MHz, CDCl ) δ: 9.61 (d, J = 8.0 Hz, 1H), 7.73
3
(s, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.30 (dd, J =
15.0 Hz, 10.6 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 15.3 Hz, 1H), 7.02 (dd, J = 15.3 Hz,
10.6 Hz, 1H), 6.89 (s, 1H), 6.26 (dd, J = 15.0 Hz, 8.0 Hz, 1H), 3.09 (s, 6H). MS: m/z calcd for
+
[
C H NO + H] 252.1, found 252.1.
1
7
17
(2E,4E,6E)-7-(6-(Dimethylamino)naphthalen-2-yl)hepta-2,4,6-trienal (2d) The same
reaction described above to prepare compound 2b was used, and the dark red solid 2d was
1
obtained (140.0 mg, 25.3%). mp 213.8-214.2 ºC. H NMR (400 MHz, CDCl ) δ: 9.58 (d, J = 8.0
3
Hz, 1H), 7.68 (d, J = 9.5 Hz,1H), 7.67 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H),
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.20 (dd, J = 15.1 Hz, 11.3 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.95-6.85 (m, 4H), 6.55 (dd, J =
13.9 Hz, 11.4 Hz, 1H), 6.18 (dd, J = 15.1 Hz, 8.0 Hz, 1H), 3.08 (s, 6H). MS: m/z calcd for
+
[
C H NO + H] 278.2, found 278.1.
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(2E,4E,6E,8E)-9-(6-(Dimethylamino)naphthalen-2-yl)nona-2,4,6,8-tetraenal (2e) The same
reaction described above to prepare compound 2b was used, and the black solid 2e was obtained
1
(30.0 mg, 36.7%). mp 172.1-173.2 ºC. H NMR (400 MHz, CDCl ) δ: 9.57 (d, J = 8.0 Hz, 1H),
3
7
.69 (d, J = 8.4 Hz,1H), 7.65 (s, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.16 (dd,
J = 15.1 Hz, 11.4 Hz, 1H), 6.97-6.67 (m, 5H), 6.47 (d, J =14.6 Hz, 1H), 6.48-6.42 (m, 1H), 6.16
+
(dd, J = 15.1 Hz, 8.0 Hz, 1H), 3.08 (s, 6H). MS: m/z calcd for [C H NO + H] 304.2, found
21
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3
04.0.
General procedure for Knoevenagel condensation: To a solution of naphthyl aldehyde in
methanol (2a-d) or THF (2e) was added an excess of malononitrile (2 equiv), and then a solution
of K CO in methanol (100 ꢀL) was added as catalyst. Precipitation of 3a-d was achieved after
2
3
stirring at r.t. for 5 min, and the precipitate was collected by filtration; 3e was obtained by flash a
column chromatography.
2-((6-(Dimethylamino)naphthalen-2-yl)methylene)malononitrile (3a) Compared with the
2
9
reported procedure, 3a was prepared in a much faster (5 min versus 2 h), environmentally
friendly (changed the reaction solvent pyridine with methanol) and straightforward (the product
crystallized from the reaction mixture versus isolated by chromatography and re-crystallization)
synthesis route in a higher yield (85.5% versus 76%) as a red solid. mp 203.0-204.3 ºC (literature
1
2
9: 211.0-214.5 ºC). H NMR (400 MHz, CDCl ) δ: 8.08 (d, J = 1.4 Hz, 1H), 7.96 (dd, J = 8.9
3
Hz, 1.9 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.70 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.17 (dd, J = 9.2
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+
Hz, 2.6 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 3.17 (s, 6H). MS: m/z calcd for [C H N + H]
1
6
13
3
248.1, found 248.0.
(
E)-2-(3-(6-(Dimethylamino)naphthalen-2-yl)allylidene)malononitrile (3b) A dark red solid
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was obtained, yield 75.0%. mp 191.7-192.9 ºC. H NMR (400 MHz, CDCl ) δ: 7.82 (s, 1H), 7.73
3
(d, J = 9.1 Hz, 1H), 7.55-7.64 (m, 3H), 7.34 (dd, J = 15.0 Hz, 2.6 Hz, 1H), 7.20-7.26 (m, 1H),
1
3
7
.16 (dd, J = 9.1 Hz, 2.4 Hz, 1H), 6.87 (s, 1H), 3.13 (s, 6H). C NMR (100 MHz, CDCl ) δ:
3
1
60.15, 151.34, 150.29, 137.29, 132.36, 130.36, 127.50, 127.19, 125.78, 124.21, 119.90, 116.35,
114.35, 112.46, 105.69, 79.26, 40.35. HRMS: m/z calcd for C H N 273.1266, found 273.1270.
1
8
15
3
2
-((2E,4E)-5-(6-(Dimethylamino)naphthalen-2-yl)penta-2,4-dien-1-ylidene)malononitrile
1
(
3c) A black solid was obtained, yield 93.3%. mp 240.0-240.9 ºC. H NMR (400 MHz, CDCl )
3
δ: 7.73 (s, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.46
(
d, J = 11.9 Hz, 1H), 7.15-7.07 (m, 3H), 7.00 (dd, J = 15.0 Hz, 11.0 Hz, 1H), 6.87 (s, 1H), 6.81-
1
3
6
1
1
.75 (m, 1H), 3.11 (s, 6H). C NMR (100 MHz, CDCl ) δ: 159.42, 151.19, 149.68, 146.00,
3
36.33, 130.08, 129.86, 129.71, 127.07, 124.80, 124.71, 123.94, 119.99, 116.41, 114.31, 112.32,
05.80, 79.78, 40.54. HRMS: m/z calcd for C H N 299.1422, found 299.1426.
2
0
17
3
2-((2E,4E,6E)-7-(6-(Dimethylamino)naphthalen-2-yl)hepta-2,4,6-trien-1-
1
ylidene)malononitrile (3d) A black solid was obtained, yield 74.0%. mp 226.4-227.2 ºC. H
NMR (400 MHz, CDCl ) δ: 7.71 (d, J = 8.2 Hz, 1H), 7.70 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.57
3
(d, J = 8.2 Hz, 1H), 7.44 (d, J = 11.9 Hz, 1H), 7.20-7.17 (m, 1H), 7.03-6.85 (m, 5H), 6.72 (dd, J
=
14.1 Hz, 12.0 Hz, 1H), 6.52 (dd, J = 13.5 Hz, 11.7 Hz, 1H), 3.11 (s, 6H). HRMS: m/z calcd for
+
[
C H N + H] 326.1652, found 326.1659.
22 19
3
2
-((2E,4E,6E,8E)-9-(6-(Dimethylamino)naphthalen-2-yl)nona-2,4,6,8-tetraen-1-
ylidene)malononitrile (3e) After the solvent was removed, the residue was purified by flash
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column chromatography (petroleum ether/CH Cl /ethyl acetate = 10: 10: 1, v: v) to give
2
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1
compound 3e as a black solid (5.0 mg, 29.0%). mp 234.3-235.2 ºC. H NMR (400 MHz, CDCl )
3
δ: 7.85-7.70 (m, 4H), 7.60 (s, 1H), 7.43 (d, J = 11.9 Hz, 1H), 7.00 (d, J = 14.1 Hz, 1H), 6.97 (d, J
0
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=
14.1 Hz, 1H), 6.90 (s, 1H), 6.86-6.68 (m, 4H), 6.55-6.44 (m, 2H), 3.13 (s, 6H). HRMS: m/z
+
calcd for [C H N + H] 352.1808, found 352.1821.
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X-ray Crystallography.10 mg of 3b and 3c were dissolved in 2 mL of DCM, respectively,
and then 0.5 mL petroleum ether was slowly added. Upon gentle evaporation at room
temperature, red (3b) or dark red (3c) crystals were yielded. The X-ray single-crystal structures
were determined on a Rigaku R-AXIS RAPID IP XRD system with a Saturn724+ charge-
coupled device (CCD) detector. Diffractometer at 153(2) K with graphite monochromated Mo
4
6
Kα radiation (λ = 0.71073 Å) was used. All structures were solved using SHELXL-97 program
2
and refined with full-matrix least-squares on F method. All the hydrogen atoms were
geometrically fixed using the riding model.
Fluorescence Spectral Assessment of 3a-e with Aβ1-42 and BSA. To the solutions of DANIR
probes (50 nM) in 3 mL PBS (pH = 7.4) was added 140 ꢀL suspension of the Aβ1-42 aggregate in
deionized water (for a final Aβ concentration of 10 ꢀg/mL) or 100 ꢀL solution of BSA in PBS
(0.3 mg/mL, for a final concentration of 10 ꢀg/mL). The mixture was incubated at 37 °C with
slight and constant shaking (100 r/min) for 1.5 h. Then, the mixture was transferred to a quartz
sampling cell and its fluorescence spectra were recorded by a RF-5301PC fluorescence
spectrophotometer. The spectra of 3a-e in PBS and of PBS buffer (as a blank control) were also
measured using the same parameters. The final spectra were corrected using the blank control.
For the characterization of 3d and 3e, a solution of 1 ꢀM in PBS (30% ethanol) was used.
CRANAD-2 (50 nM) was also measured in the same conditions.
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In Vitro Histological Staining of Brain Slices of Tg Mice and Human Brain. Paraffin-
embedded 8-ꢀm brain tissue sections from double Tg mice (C57BL6, APPswe/PSEN1, 22
months old, male), three AD patients [female, 64 years old (F64), male, 91 years old (M91) and
female, 71 years old (F71)] and a CAA patient [female, 68 years old (F68)] were used for in vitro
fluorescence staining. Before use, the slices were well deparaffinized by washing with ethanol
for 5 min after a 15-min immersion in xylene. The slices were incubated in aqueous solutions of
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3a-e (1 ꢀM, 2.5% DMSO, 10% ethanol) for 10 min at room temperature and then washed with
ethanol/water (v: v, 40%: 60%). Next, the brain sections were covered with cover glasses prior to
fluorescence observation using an Axio Observer Z1 (Zeiss, Germany) equipped with DAPI,
AF488, AF546, and Cy 5.0 filter sets. To confirm the substantial staining of the plaques, the
adjacent sections of Tg mice were stained with Th-S (0.125 % aqueous solution).
In Vitro Aβ Aggregates Binding Constant Determination. A suspension of 100 ꢀL Aβ1-42
aggregates (1.6 ꢀg per borosilicate glass tube) was added to a mixture containing 100 ꢀL 3a-e
(500 to 0.0051 nM in the final assay mixture) and 800 ꢀL of PBS (pH = 7.4) in a final volume of
1.0 mL. Nonspecific assays were defined without test probes. All assay mixtures were incubated
at 37 °C with slight and constant shaking (100 r/min) for 1.5 h. Next, the solutions were
transferred to black 96-well Costar assay plates, and the fluorescence intensity was measured
using a fully equipped POLARstar Omega microplate reader (BMG LABTECH, Germany).
Fluorescence intensity values of each probe were plotted versus their concentrations (Figure S7
in Supporting Information), and the binding constants were calculated using GraphPad Prism
5.0.
In Vitro Inhibition Binding Studies Using Aβ1−42 Aggregates. Inhibition experiments were
conducted in 12 × 75 mm borosilicate glass tubes according to the previously described
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procedures with certain modifications. Briefly, 100 ꢀL of Aβ aggregates (86 nM in the final
125
assay mixture) was added to a mixture containing 100 ꢀL of radioligand [ I]IMPY (140000
−
5
−10.5
cpm/100 ꢀL), 100 ꢀL inhibitors (3a-e, 10 to 10
M in ethanol), and 700 ꢀL 0.05% BSA
0
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aqueous solution in a total volume of 1.0 mL. The mixture was incubated for 2 h at 37°C, and
then the bound and free radioactive fractions were separated by vacuum filtration through
borosilicate glass fiber filters (Whatman GF/B) using a M-24 cell harvester (Brandel,
Gaithersburg, MD). The radioactivity from filters containing the bound 125I ligand was
determined in a γ-counter (Wallac/Wizard 1470) with 85% efficiency. The half maximal
inhibitory concentration (IC ) was determined using GraphPad Prism 5.0, and the inhibition
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constant (K ) was calculated using the Cheng-Prusoff equation: K = IC /(1 + [L]/K ), where
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d
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[L] is the concentration of [ I]IMPY used in the assay, and K is the dissociation constant of
d
IMPY. The K value of IMPY was assessed under the same conditions as a control.
i
The Stability Testing of 3b and 3c upon Incubated with Mice Plasma. To validate the
stability of the probes, the following procedures were used. Blood was drawn from normal ICR
mice (five weeks, male), and the plasma was separated by centrifugation and stored at -80 °C.
Step 1: To 500 ꢀL mice plasma was added 50 ꢀL 3b or 3c in ethanol (1 ꢀM) in a 5 mL glass
tube, and the mixture was incubated at 37 °C for 30/60 min. Step 2: After 30/60 min, the mixture
was treated with 300 ꢀL acetonitrile and filtered by flash nylon membrane (0.22 ꢀm); the filtrates
and the mice plasma (as blank control) were treated by the same method twice. Step 3: The
filtrates were subjected to HPLC analysis, and the spectra were recorded using acetonitrile: water
=
80%: 20% with a flow rate of 1 mL/min. The retention time and purity of 3b and 3c were
shown in Figure S8 in Supporting Information.
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BBB Penetration and Blood Clearance Rate Determination. To intuitively compare the
brain uptake of the probes, ICR mice (22-24 g, 5 weeks, male) were sacrificed at 2 min after i.v.
injection of the probes (2 mg/Kg, 45% DMSO and 55% propylene glycol, 50 ꢀL), and brain
samples were removed. Pictures of the brains were obtained under both visible light and UV
light (365 nm). The brains of ICR mice were also removed directly as controls (Figure S9 in
Supporting Information).
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The brain uptake rates of 3b (2 mg/Kg, 45% DMSO and 55% propylene glycol, 50 ꢀL) and 3c
(0.5 mg/Kg, 45% DMSO and 55% propylene glycol, 50 ꢀL) were analyzed by following our
20
previously established procedures. The brain uptake rates of DANIR 2c (2 mg/Kg) and
CRANAD-2 (0.5 mg/Kg) were also tested in the same condition as controls.
The blood clearance rate was implemented by following the same procedures in the test of
BBB penetration. In brief, ICR mice (22-24 g, 5 weeks, male, n = 3-4) were sacrificed at
different time points of 2 and 60 min after i.v. injection of the probes (3b, 2 mg/Kg; 3c, 0.5
mg/Kg). Blood samples were collected, anticoagulated with sodium heparin and homogenized
with 1.0 mL acetonitrile. Then, the remainder homogenate was extracted with 0.5 mL
acetonitrile. The extracting solution was filtered by flashing nylon membrane (0.22 ꢀm) and
dried over anhydrous sodium sulfate. After that, 100 ꢀL of the combined acetonitrile were
analyzed by HPLC. The uptake in blood was presented by % injected dose per gram (% ID/g).
Determination of the Partition Coefficient. A solution of 3b or 3c (5 mM) in octanol (3.0 g)
was subjected to partition with octanol-saturated PBS (pH = 7.4, 3.0 g) in a test tube. The
mixture was vortexed for 3 min, followed by a 5-min centrifugation at 3000 rpm. Two aliquots
from the octanol (10 ꢀL) and water (400 ꢀL) layers were analyzed by HPLC. The log P value
was calculated by the ratio of the peak area. The measurement was conducted in triplicate.
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