Synthesis and biological evaluation of 9-fluorenone derivatives for SPECT imaging of α7-nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmcl.2019.126724

The α7-nicotinic acetylcholine receptor (α7-nAChR) subtype, is found to have a connection with the pathogenesis of a variety of psychiatric and neurological disorders. Herein, we report the development of radioiodinated 9-fluorenone derivatives as single-photon emission computed tomography (SPECT) imaging tracers for α7-nAChRs. Among the derivatives, the best member of the series 10 (Ki = 2.23 nM) were radiolabeled with ¹²⁵I for in vitro and in vivo studies. The radiotracer [¹²⁵I]10 exhibited robust brain uptake and specifically labeled α7-nAChRs with a peak uptake value of 9.49 ± 0.87%ID/g in brain. Blocking studies demonstrated that the tracer was highly specific toward α7-nAChR. Furthermore, ex vivo autoradiography and micro-SPECT/CT dynamic imaging in mice confirmed the excellent imaging properties. In addition, molecular docking was also performed to rationalize the potency of the chosen compounds towards α7-nAChRs. To conclude, compound 10 could serve as a promising radiotracer for the α7-nAChRs.

Full text of DOI:10.1016/j.bmcl.2019.126724

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 9-fluorenone derivatives for SPECT
imaging of α7-nicotinic acetylcholine receptor
Hang Gao, Shuxia Wang, Yueheng Qi, Guoxue He, Bingchao Qiang, Sixuan Wang, Huabei Zhang⁎
Key Laboratory of Radiopharmaceuticals of Ministry of Education, College of Chemistry, Beijing Normal University, No. 19 Xinjiekouwai Street, Haidian District, Beijing
100875, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The α7-nicotinic acetylcholine receptor (α7-nAChR) subtype, is found to have a connection with the patho-
genesis of a variety of psychiatric and neurological disorders. Herein, we report the development of radio-
iodinated 9-fluorenone derivatives as single-photon emission computed tomography (SPECT) imaging tracers for
α7-Nicotinic acetylcholine receptors (α7-
nAChRs)
α7-nAChRs. Among the derivatives, the best member of the series 10 (Ki = 2.23 nM) were radiolabeled with 125
I
SPECT/CT
Radiotracers
Alzheimer’s disease
for in vitro and in vivo studies. The radiotracer [125I]10 exhibited robust brain uptake and specifically labeled α7-
nAChRs with a peak uptake value of 9.49
0.87%ID/g in brain. Blocking studies demonstrated that the tracer
was highly specific toward α7-nAChR. Furthermore, ex vivo autoradiography and micro-SPECT/CT dynamic
imaging in mice confirmed the excellent imaging properties. In addition, molecular docking was also performed
to rationalize the potency of the chosen compounds towards α7-nAChRs. To conclude, compound 10 could serve
as a promising radiotracer for the α7-nAChRs.
Introduction
inflammation and substance dependence.5
Due to the distribution and quantity of α7-nAChRs in the CNS, this
The neurotransmitter acetylcholine (ACh) exerts its work on the
cardiovascular, immune system and the central and peripheral nervous
systems. Acetylcholine receptors (AChRs) which comprised different
multimers, are divided into a neuronal type (α2-α10 and β2-β4) and a
muscle type (α1, β1, γ, δ and ε). Different subunits may assembled into
numerous receptor subtypes.1 Among subtypes, nicotinic acetylcholine
receptors (nAChRs) which belong to the superfamily of ligand-gated ion
channels, are composed of five transmembrane proteins in a pentameric
structure.2 In human brain, α7-nAChRs and α4β2 subtypes are pre-
dominant.3
receptor type has become a potential therapeutic target for mental
disorders. Clinical experiments of α7-nAChR agonists revealed that α7-
nAChRs partial agonists can help improve cognitive performance in
people with SCZ.6 Meanwhile, numerous postmortem studies have
shown a reduction of α7-nAChRs in the cerebral tissues of SCZ patients,
with similar findings appearing in AD patients.7 Although numerous
studies have tried to elucidate the mechanisms of α7-nAChRs, in the
brain these mechanisms are not fully understood. Nevertheless, a small
numbers of therapies aimed at α7-nAChRs has been applied to clinical
trials.8 However, invalid cases of the planned therapies occurred in
clinical trials, emphasized a strong demand for a biomarker to mon-
itoring the pathological processes in preclinical research. Therefore, it
is undoubtedly crucial to develop a human compatible α7-nAChR
radiotracer for early diagnosis and to evaluate the efficiency of drug
therapies in clinical trials for the treatment of SCZ or AD.
Research from biological studies revealed that the α4β2-nAChR
subtype in the central nervous system (CNS) has a high binding affinity
to nicotine, while homomeric α7-nAChRs possess strong binding to
methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) and have a high
permeability to calcium.4 While nAChRs coordinate synaptic delivery at
muscle end-plates or functioning autonomic ganglia in the peripheral
nervous system, different subtypes of nicotinic acetylcholine receptors
in the CNS play their respective roles on regulating physiology, such as
sensorimotor gating, memory, metabolism and inflammation. The α7-
nAChR subtype, is found to have a connection with the pathogenesis of
a variety of psychiatric and neurological disorders, including Alzhei-
mer’s disease (AD), schizophrenia (SCZ), anxiety, depressive disorder,
In vivo imaging and quantifying α7-nAChRs in the human brain
provide a solution to develop such a biomarker. Positron-emission to-
mography (PET) and single-photon emission computed tomography
(SPECT) offer an advanced and suitable way to quantify cerebral re-
ceptors and their occupancy in vivo.
Numerous α7-nAChR ligands have been labeled with 11C, 18F or
125I,9 such as [125I]1,10
[
125I]Iodo-ASEM,11
[
11C]CHIBA-1001,12
[
18F]
⁎ Corresponding author.
E-mail addresses: hgao@mail.bnu.edu.cn (H. Gao), hbzhang@bnu.edu.cn (H. Zhang).
https://doi.org/10.1016/j.bmcl.2019.126724
Received 2 August 2019; Received in revised form 22 September 2019; Accepted 28 September 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:HangGao,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126724

H. Gao, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Representative α7-nAChRs radioligands.
NS10743,13.
[
18F]ASEM,14
[
18F]DBT10 and [18F]2 (Fig. 1).15,16 Despite
iodide aqueous solution.
some of them showing low brain uptake or binding specificity, two
The synthesis routes of the 1,4-diazepan-1-yl substitution com-
pounds 11–17 are outline in Scheme 2. The compounds 11 and 12 were
prepared by Buchwald-Hartwig cross-coupling reaction with hetero-
cyclic ring and Boc-protected aniline 6. The final amino substituted 13
and 14 were further obtained by removal of the Boc-protecting group.
The fluorine substitutes compound 15 was obtained via the Schiemann
reaction, and the final fluorine substitutes compound 16, 17 and 18
were synthesized using the similar steps as 11, 12 and 14.
promising 18F labeled α7-nAChR ligands [18F]DBT10 (Ki = 1.3 nM) and
[
18F]ASEM (Ki = 0.4 nM) have been tracked in primates and non-
primates studies.15,17 Together with [18F]2 (Ki = 2.98 nM), these three
compounds are the most promising agents for human α7-nAChR PET
imaging. We envisioned the synthesis of iodo derivatives of 2 would
producing a new set of compounds with similar potency and the po-
tential for radiolabeling with 123I and 125I for SPECT imaging.
Here we report [125I]Iodo-10, the radio-iodinated analog of fluoren-
9-one designed for usage in cell-based assays of α7-nAChRs and as
potential probes for SPECT imaging in vivo studies. The studied ligand is
amenable to labeling with 123I and serve as human α7-nAChR radio-
tracers for SPECT studies. In addition, molecular docking confirmed the
binding modes of the promising ligand.
To measure the in vitro binding affinity and selectivity of the syn-
thesized α7-nAChRs ligands and precursors, in vitro competition binding
assays of α7-nAChRs and α4β2 subtypes were performed. Both of the α7
and α4β2-nAChR assays were performed using rat cortical membranes.
The binding assays of α7-nAChRs were tested in vitro competition with
0.4 nM [125I]α-bungarotoxin ([125I]α-Bgt), which is an α7-nAChR an-
tagonist (KD = 0.7 nM). Furthermore, MLA, a specific α7-nAChR an-
tagonist was tested as the reference compound to ensure the reliability
and comparison of the method (Table 1).
Results and discussions
Among the new series of compounds, 10 showed the highest
binding affinity for pentamer-α7-nAChRs with Ki values of 2.23 nM and
The synthetic routes for the ortho-iodine substitution compounds 10
are shown in Scheme 1. Fluoranthene (3) was oxidized to open the loop
by chromium (VI) oxide and efficiently brominated to carboxylic acid 5.
After Curtius rearrangement by a one pot reaction, a Boc-protected
aniline 6 was generated by replacing the carboxy group of 5. The Boc-
protecting group of 6 was removed by 1 N HCl and then diazotized
using sodium nitrite in hydrochloric acid solution. After the diazo
coupled with dibutyamine, a triazene compound 8 was generated. The
precursor 9 was synthesized by Buchwald-Hartwig cross-coupling re-
action between 1,4-diazabicyclo[3.2.2]nonane and compound 8. The
nonlabelled standard 10 was obtained by TFA pretreating 9 in a sodium
is almost equally matched to that previously reported
[
18F]2
(Ki = 2.98 nM) and are the same level as ASEM (Ki = 0.37 nM) and
iodo-ASEM (Ki = 0.93 nM).14,16 10 exhibited much the same as the
reference α7-nAChRs ligand MLA, whereas the binding affinity of het-
erocycle substitutes compounds were lower. The potential precursors 9
was studied under the same conditions but showed poor results. The
1,4-diazepan-1-yl and the 4-methyl-1,4-diazepan-1-yl substitutes
showed moderate binding affinities in the range of 70 to 100 nM. The
amino-substituted fluorenone exhibited a slight higher potency than
Scheme 1. Synthesis of iodinated ortho-substitution compound 10. Reagents and conditions: (a) CrO3, H2O, CH3COOH, 80–85 °C, 110–120 °C; (b) Br2, 80–85 °C; (c)
Et3N, DPPA, t-BuOH, toluene, 110 °C; (d) CH3CN, 1 N HCl, 80 °C, 1 N NaOH; (e) NaNO2, CH3CN, HCl, dibutylamine, K2CO3, −5 °C, RT; (f) Cs2CO3, Pd2(dba)3, BINAP,
toluene, 85 °C; (g) TFA, NaI, MeCN, H2O, 80 °C.
2

H. Gao, et al.
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Scheme 2. Synthesis of 11–17. Reagents and conditions: (h) Cs2CO3, Pd2(dba)3, BINAP, toluene, 85 °C; (i) CH3CN, 1 N HCl, 80 °C, 1 N NaOH; (j) Et3N, DPPA, t-BuOH,
toluene, 110 °C; (j) HBF4, 0–5 °C; NaNO2; H2O, 120 °C; (k) Cs2CO3, Pd2(dba)3, BINAP, toluene, 80 °C.
Table 1
in 0.1 N NaOH) in a mixed solution of acetonitrile and water as the
solvent. The obtained crude was purified by high-performance liquid
chromatography (HPLC) followed by extraction on an SPE cartridge.
The final product of [125I]10 was eluted at 41.61 min and obtained with
a radiochemical yield of 31.8% and a high radiochemical purity
In vitro binding affinities (Ki, nM) of MLA, nicotine, and the new series 9, 10,
13, 14, 15 for α7-nAChR and α4β2 subtypes.
Compd
Ki (nM)
Selectivity
α7a
α4β2b
α7/α4β2
(> 98%).The specific activity of
[
125I]10 was 62.9 GBq/mmol
MLA
Nicotine
10
2.68
ntc
0.85
ntc
(1700 Ci/mmol). [125I]10 was authenticated by comparing its retention
time with its standard substance 10 via coinjection in HPLC. The re-
tention times of [125I]10 and 10 were 7.26 min and 6.93 min respec-
tively which matched were within admissible error levels (Fig. 2).
To confirmed the stability of studied radiotracer, the in vitro stability
of [125I]10 in fetal bovine serum at 37 °C, and in saline at room tem-
perature was determined by assessing radiochemical purity via HPLC
after incubating respectively. The radiochemistry purities of the studied
compounds in serum and saline were all > 98% after 12 h (Fig. 3),
which demonstrated the high in vitro biostability of radioligands [125I]
10.
3.8
1.15
658
357
2.23
77.9
88.1
80.1
92.5
0.56
4.45
9.21
5.21
4.11
6517
9842
2922
126
13
14
> 10 μM
16
9631
514
120
18
> 10 μM
a
SD rat (female, 180–270 g) cerebral cortex; radiotracer,
[
125I]α-Bgt
(0.4 nM), KD = 0.7 nM; Ki values are the means
SD of assays performed in
triplicate.
b
SD rat (female, 180–270 g) cerebral cortex; radiotracer, [3H]cytisine
(1 nM), KD = 0.77 nM; Ki values are the means
SD of assays performed in
For the biodistribution studies of [125I]10, it showed a high brain
uptake after intravenous injection (0.2 μCi, in 0.1 mL saline, containing
triplicate.
c
nt: not tested.
10% ethanol). [125I]10 exhibited a robust brain uptake of 6.47
0.41
fluorine-substituted compounds, indicating an interaction with α7-
nAChRs may exist. For the heterocycle substitutes groups on the other
sides, a loss of potency occurred when changing the 1,4-diazabicyclo
[3.2.2]nonane to the diazepam substitutes.
%ID/g at 5 min postinjection, then quickly reached the peak uptake
value of 9.49
0.87 %ID/g in the brain at 15 min (Table 2). The
brain15min/brain120min value was 2.91 which was a reasonable
clearance rate for brain SPECT imaging. Among the remaining organs,
the thyroid uptake, which was crucial for 125I-radiolabeled ligands,
showed relatively low values within an hour. The highest uptake of
Heteromeric α4β2-nAChR is the main nicotinic acetylcholine re-
ceptor subtype, so we also performed the binding assays using [3H]
cytisine (1 nM) as the competing radioligand (Table 1). The α4β2 sub-
type affinities of the tested compounds were obviously larger than the
corresponding α7-nAChR affinities which indicate good α7-nAChR/
α4β2-nAChR selectivity. 10 showed excellent selectivity where the
diazepam substitutes 13 and 16 manifested lower selectivity.
We radiolabeled the 10 as shown in Scheme 3. [125I]10 was pre-
[
125I]10 in the thyroid was 3.61
0.21 %ID at 120 min, indicating a
good stability of the iodide group in vivo. The blood uptake was fairly
low, a high brain/blood ratio of 8.11 at 15 min postinjection was
measured. Lung uptake displayed an idiosyncratic value of
50.78
8.72 %ID/g at 5 min, but a fast clearance.
To better illustrate the distribution of the radiotracer in brain, [125I]
pared by the reaction of precursor 9 acidized by TFA and Na125I (Na125
I
Scheme 3. Radiosynthesis of [125I]10.
3

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Fig. 2. (A) HPLC chromatogram of preparation of [125I]10. (B) Coinjection of [125I]10 and 10.
Fig. 4. Biodistribution of [125I]10 in the cerebral regions of mice. Data are
Fig. 3. HPLC chromatograms of [125I]10 in fetal bovine serum and saline, for
expressed as the percentage of injected dose per gram (%ID/g), mean
SD,
n = 3. Abbreviations: FrCtx, frontal cortex; Hipp, hippocampus; Str, striatum;
Coll, superior and inferior colliculi; Th, thalamus; CB, cerebellum. Rest, rest of
the brain.
1–12 h.
10 were tested in Kunming mice as potential SPECT tracers for imaging
cerebral α7-nAChRs (Fig. 4). After tail vein injection, [125I]10 exhibited
rapid initial brain uptake and quickly reached peak uptake in the brain
at 15 min, followed by a moderate washout. The peak concentration of
radioactivity of iodine-radiotracer appeared in the frontal cortex,
striatum and the superior and inferior colliculi. The uptake values of
[
125I]10 at 30 min in frontal cortex, hippocampus or superior and in-
ferior colliculi were 7.22
0.62 %ID/g, 5.73
0.42 %ID/g and
6.02
0.35 %ID/g respectively. Moderate uptake of the ligand was
observed in the striatum and thalamus; while the cerebellum had the
lowest uptake. The distribution of radioiodo-labelled [125I]10 was in
Table 2
Biodistribution of [125I]10 in Kunming mice (female, 18–22 g).a
Organs
Time (min)
5 min
15 min
30 min
60 min
120 min
Blood
1.87
6.47
0.22
8.96
7.62
4.62
50.78
16.42
2.16
1.03
1.42
1.57
3.50
0.21
1.17
9.49
0.65
4.21
13.56
8.07
29.52
12.48
3.74
1.31
1.84
1.83
8.11
0.19
0.87
0.12
0.21
1.10
8.03
0.93
3.50
16.35
10.19
24.85
9.89
3.85
2.71
6.43
6.76
7.30
0.32
0.73
0.14
0.18
0.70
6.21
1.51
4.48
9.61
6.30
11.20
5.63
2.29
2.37
5.51
5.32
8.87
0.27
0.32
0.24
0.29
0.56
0.51
0.52
3.26
3.61
1.67
5.84
4.03
5.17
4.21
1.30
2.83
2.83
3.26
6.26
0.13
0.15
0.21
0.16
0.57
0.44
0.84
0.87
0.18
0.23
0.21
0.47
Brain
0.41
0.09
0.87
0.65
0.32
Thyroidb
Heart
Liver
0.86
0.41
Spleen
Lung
0.63
2.84
1.25
0.97
1.45
8.72
1.04
0.97
Kidney
Muscle
Bone
0.76
0.47
0.31
0.51
0.55
0.80
0.57
0.94
0.46
0.20
0.39
1.02
0.35
0.15
0.40
0.39
0.17
0.21
Intestine
Stomac
Brain/Blood
a
b
Data are expressed as the percentage of injected dose per gram (%ID/g), mean
SD, n = 3.
Data are expressed as the percentage of injected dose (%ID), mean
SD, n = 3.
4

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Fig. 5. Inhibition of [125I]10 in mice brain regions. Data are mean
SD
Fig. 6. Blocking studies of [125I]10 with selective α7-nAChR, α4β2-nAChR and
(n = 3). Abbreviations: FrCtx, frontal cortex; Hipp, hippocampus; Str, striatum;
Coll, superior and inferior colliculi; Th, thalamus; CB, cerebellum. Rest, rest of
the brain.
5-HT3 ligands in Kunming mice. Data are mean
SD (n = 3). Blocking studies
of [125I]10 with intravenous preinjection of SSR180711 (1 mg/kg), cytisine
(1 mg/kg) and ondansetron (2 mg/kg). Abbreviations: FrCtx, frontal cortex;
Hipp, hippocampus; Str, striatum; CB, cerebellum.
accordance with the biodistribution of α7-nAChRs in rodents.19 The
ratio of radiotracers uptake in the whole brain tissue to that in the
cerebellum was 7.38 at 15 min and reached 12.3 after 60 min. The
clearance rate of the studied tracer from the cerebellum was faster than
from other regions. The ratios of [125I]10 are highly consistent with the
results of the in vitro α7-nAChR binding assays.
highly expressed. To further determine whether the in vivo binding of
[
125I]10 in the brain could be inhibited by selective agonist, SSR180711
was used as a blocker. As shown in Fig. 7B the ligand was significantly
blocked in Kunming mice, which again confirmed its specificity and
Self-blockade studies of [125I]10 with its nonlabeled standards were
also conducted to ensure specificity (Fig. 5). The results showed an
evident reduction of uptake among all regions of the brain, which de-
monstrated the specificity of the studied radiotracers. For [125I]10, the
cortex, superior and inferior colliculi and hippocampus had a blockade
ratio of 76.2%, 69.8%.
selectivity.
[
125I]10 showed effective labeling of α7-nAChRs and
minimal background labeling proving that the tracer is a suitable
radioprobe of α7-nAChR for in vivo studies.
Dynamic SPECT/CT studies of [125I]10 was successively obtained
on Kunming mice (female, 18–22 g) after intravenous injection of
radioiodinated probes (0.1 mL, 80 μCi, 2.96 MBq). Mice were anesthe-
tized with isoflurane for imaging and conducted with the 60 min con-
secutive dynamics SPECT/CT study. Furthermore, two more static
scanning (90 min, 120 min postinjection) were also performed to study
the internal retention and metabolism of the radioligands in mice. As
shown in Fig. 8A, significant and robust brain uptake was observed
postinjection. In agreement with the biodistribution results discussed
and 75.1% respectively. The results indicated that the [125I]10 was
specific to regions rich in α7-nAChRs, except for the cerebellum which
still had a moderate concentration. The deviation may due to the time
point that brain tissues were harvested was corresponded to the peak
concentration of brain uptake, which leads to uncontrolled factors.
To further determine the specificities of [125I]10 to α7-nAChRs over
other nAChRs or similar subtypes, independent experiments of the
blocking studies were carried out by intravenous preinjection of three
blocking agents. We chose SSR180711, a selective α7-nAChR partial
agonist with good affinity, to study in vivo selectivity.20 For the non-α7-
nAChR, α4β2-nAChR and 5-HT3 receptors, we chose cytisine and on-
dansetron for the blocking agents.21 In this study, SSR180711, cytisine
and ondansetron were preinjected 15 min before the injection of the
radiotracers. Saline was preinjected either in the control group to avoid
issues of interference. The mice were sacrificed at 100 min postinjection
of the radiotracers, and brain tissues were harvested. The SSR180711
blocking group showed an obvious reduction of radioactivity, the in-
hibitor ratio was greater than 60% suggesting that [125I]10 was specific
to α7-nAChRs (Fig. 6).
above,
[
125I]10 reached maximum radioactive concentration at
15–20 min postinjection. Deiodination was hardly to be observed
during the study in 60 min, which was in accordance with the in vitro
stability study and further demonstrated its in vivo stability. [125I]10
had a good retention and moderate washout in the mouse brain. The
accumulation in brain was gradually decreased after 60 min and
reached the same radioactive level with thyroid at 120 min. The ima-
ging results are consistent with the experimental data performed above
and further demonstrate that [125I]10 could be a potential SPECT
radiotracer for in vivo imaging of human α7-nAChRs.
To further ensure the brain uptake or distribution of the radioactive
substance in α7-nAChR blockaded Kunming mice, blocking studies of
[
125I]10 compounds were conducted. As shown in Fig. 8B, following
Meanwhile in the group of cytisine and ondansetron blocking,
radioactivity accumulation in the frontal cortex, hippocampus and
striatum for [125I]10 was not reduced, with an inhibitor ratio less than
15% which indicated that [125I]10 was selective for α7-nAChRs versus
α4β2 subtypes and 5-HT3 receptors. Moreover, three blocking agents
had almost no effect on accumulation of radioactivity in the cere-
bellum, suggesting that [125I]10 in the cerebellum had nonspecific
binding.
injection of SSR180711 (1 mg/kg) 15 min before radiotracer injection,
a significant reduction of uptakes was observed at 30 min and 60 min
postinjection. Therefore, the SPECT/CT blocking imaging study, in
combination with the results of the dynamics studies and in vivo/vitro
experiments discussed above, indicated that radioiodinated tracer [125I]
10 was specific to rodent α7-nAChRs and that [123I]10 might be an
excellent selective human α7-nAChR imaging agent.
For the purpose of elucidating the binding mode and efficiency of
α7-nAChR and its high potency ligands, molecular docking was per-
formed. We chose 10 together with previously reported compounds
iodo-ASEM to reveal the binding mode and find the differences among
the three. The two ligands were successfully docked into the binding
pocket (see Fig. S1 in Supplementary Information). Docking results
The ex vivo autoradiography was performed and cross-referenced
with mouse brain atlases to characterize [125I]10 as possible imaging
probes.22 As shown in Fig. 7, the radiotracer [125I]10 exhibited in-
tensive labeling, showing a strong signal in the frontal cortex, thalamus,
hypothalamus, hippocampus and striatum, where α7-nAChRs were
5

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Fig. 7. Ex vivo autoradiography of
[
125I]10 in
Kunming mice. (A) [125I]10 with reference brain
sections. (B) Ex vivo autoradiography of [125I]10 in
Kunming mice brain sections and control sections
(coronal). The presence and distribution of α7-
nAChRs in the sections were confirmed by blocking
studies with selective α7-nAChR ligands.
revealed that the interactions of α7-nAChR with series ligands were
dominated by the substitution of the 1,4-Diazobicylco[3.2.2]nonane
group. The similar docking poses of the ligands indicate that the 3D
geometry of the diazabicyclo group may play a vital role in binding
affinity. A strong H-bond was observed between Trp145 and the pro-
tonated tertiary amine of the nitrogen heterocyclic ring. The protonated
tertiary amine laying at the center of the pocket formed three π-H in-
teractions with Trp145, Tyr184 and Tyr191. The predicted binding
models demonstrated that the diazobicylco group was important for the
studied ligands and may provide the structural basis for further mod-
ifications of α7-nAChR ligands.
derivatives derivatives as potent ligands for α7-nAChRs. Among the
series of compounds, 10 having the best α7 nicotinic receptor binding
affinity (Ki = 2.23
0.56 nM), and high selectivity versus α4β2 re-
ceptor subtypes, was radiolabeled with 125I.
[
125I]10 have good in vitro stability, and efficiently entered the
Kunming mouse brain, which was then specifically labeled with α7
nicotinic receptors postinjection. Furthermore, blocking studies, ex vivo
autoradiography and micro-SPECT/CT imaging in mice confirmed the
specificity and selectivity of the ligands with α7-nAChRs. Additionally,
docking studies were also performed to rationalize the different po-
tencies of 10 towards α7-nAChRs and may provide some ideas to design
new α7-nAChR ligands.
Taken together [125I]10 exhibits the excellent properties as a pro-
mising SPECT radiotracer for studying α7-nAChRs both in vivo and in
vitro. Further studies of 10 labeled with 123I and are underway.
Conclusion
In this paper, we have reported the synthesis, radiobiological eva-
luations and binding modes of a series of 9H-fluoren-9-one substitutes
Fig. 8. Dynamic micro-SPECT/CT studies of radio tracers in Kunming mouse (female, 18–22 g). (A) Dynamic micro-SPECT/CT images of [125I]10. (B) Micro-SPECT/
CT blocking studies of [125I]10.
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H. Gao, et al.
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Declaration of Competing Interest
References
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
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