Derivatives of dibenzothiophene for positron emission tomography imaging of α7-nicotinic acetylcholine receptors

Article abstract of DOI:10.1021/jm401184f

A new series of derivatives of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl) dibenzo[b,d]thiophene 5,5-dioxide with high binding affinities and selectivity for α7-nicotinic acetylcholine receptors (α7-nAChRs) (K_i = 0.4-20 nM) has been synthesized for positron emission tomography (PET) imaging of α7-nAChRs. Two radiolabeled members of the series [¹⁸F]7a (K_i = 0.4 nM) and [¹⁸F]7c (K_i = 1.3 nM) were synthesized. [¹⁸F]7a and [¹⁸F]7c readily entered the mouse brain and specifically labeled α7-nAChRs. The α7-nAChR selective ligand 1 (SSR180711) blocked the binding of [¹⁸F]7a in the mouse brain in a dose-dependent manner. The mouse blocking studies with non-α7-nAChR central nervous system drugs demonstrated that [ ¹⁸F]7a is highly α7-nAChR selective. In agreement with its binding affinity the binding potential of [¹⁸F]7a (BP_ND = 5.3-8.0) in control mice is superior to previous α7-nAChR PET radioligands. Thus, [¹⁸F]7a displays excellent imaging properties in mice and has been chosen for further evaluation as a potential PET radioligand for imaging of α7-nAChR in non-human primates.

Full text of DOI:10.1021/jm401184f

Article
pubs.acs.org/jmc
Derivatives of Dibenzothiophene for Positron Emission Tomography
Imaging of α7-Nicotinic Acetylcholine Receptors
Yongjun Gao,† Kenneth J. Kellar,‡ Robert P. Yasuda,‡ Thao Tran,‡ Yingxian Xiao,‡ Robert F. Dannals,†
and Andrew G. Horti*,†
†Russell H. Morgan Department of Radiology and Radiological Sciences, Division of Nuclear Medicine, The Johns Hopkins
University School of Medicine, 600 North Wolfe Street, Baltimore, Maryland 21287-0816, United States
‡Georgetown University, 3900 Reservoir Road, Washington, D.C. 20007, United States
ABSTRACT: A new series of derivatives of 3-(1,4-
diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-di-
oxide with high binding affinities and selectivity for α7-
nicotinic acetylcholine receptors (α7-nAChRs) (Ki = 0.4−20
nM) has been synthesized for positron emission tomography
(PET) imaging of α7-nAChRs. Two radiolabeled members of
the series [18F]7a (Ki = 0.4 nM) and [18F]7c (Ki = 1.3 nM)
were synthesized. [18F]7a and [18F]7c readily entered the
mouse brain and specifically labeled α7-nAChRs. The α7-
nAChR selective ligand 1 (SSR180711) blocked the binding of
[18F]7a in the mouse brain in a dose-dependent manner. The mouse blocking studies with non-α7-nAChR central nervous
system drugs demonstrated that [18F]7a is highly α7-nAChR selective. In agreement with its binding affinity the binding
potential of [18F]7a (BPND = 5.3−8.0) in control mice is superior to previous α7-nAChR PET radioligands. Thus, [18F]7a
displays excellent imaging properties in mice and has been chosen for further evaluation as a potential PET radioligand for
imaging of α7-nAChR in non-human primates.
standing of α7-nAChR-related CNS disorders and could also
facilitate novel α7-nAChR drug development. Positron
emission tomography (PET) is the most advanced technique
to quantify neuronal receptors and their occupancy in vivo, and
the development of a suitable PET radiotracer for α7-nAChRs
would be of particular interest.
Many lead structures of α7-nAChR ligands have been
identified within various structural classes. A number of these
ligands have been radiolabeled for PET ([18F], [11C]) and
single-photon emission computed tomography (SPECT)
([123I]) (Table 1) and studied in mice, pigs, and non-human
primates as potential α7-nAChR probes.18−29 Most of these
radioligands entered the animal brain but manifested relatively
low specific binding (for review, see refs 30−32) and
insufficient BPND values (BPND < 1) (Table 1). [11C]CHIBA-
1001 is the only α7-nAChR PET radioligand so far that has
been studied in human subjects,25 but it also exhibits low
specific binding (Table 1).
INTRODUCTION
■
Cerebral neuronal nicotinic cholinergic receptors (nAChRs)
are ligand-gated ion channels composed of α (i.e., α2−α10)
and β (i.e., β2−β4) subunits that can assemble in multiple
combinations of pentameric structures. Among the many
nAChRs subtypes in the human CNS, heteropentameric
α4β2-nAChRs and homopentameric α7-nAChRs are predom-
inant.1,2 α7-nAChRs are composed of five identical α7 subunits,
and each subunit provides an orthosteric binding site for its
neurotransmitter acetylcholine.3 Many lines of evidence
associate α7-nAChRs with the pathophysiology of a variety of
disorders such as schizophrenia and AD, anxiety, depression,
traumatic brain injury, multiple sclerosis, inflammation, and
drug addiction.4−12
Clinical experiments with α7-nAChR agonists have demon-
strated that selective activation of the receptor is a viable
approach toward improving cognitive performance in patients
with schizophrenia.13,14
Because of the importance of the α7-nAChR in human
neurophysiology and as a potential drug target, synthesis and
preclinical examination of α7-nAChR subtype selective
compounds receive substantial interest in industry and
academia.9,14 A number of α7-nAChR drugs are currently in
various stages of the development for treatment of a variety of
disorders including schizophrenia, AD, multiple sclerosis,
depression, asthma, and type 2 diabetes.15−17
Because of the exceptionally low concentration (Bmax) of
cerebral α7-nAChR binding sites in the human (5−15 fmol/mg
protein)33 and animal brain (1.5−12 fmol/mg tissue),34,35
a
PET radioligand with high specific brain uptake for this
receptor subtype must exhibit very high binding affinity and
selectivity, along with other important properties (e.g.,
lipophilicity, polar surface area, suitability for radiolabeling) in
In vivo imaging and quantification of α7-nAChR binding in
Received: May 3, 2013
humans would provide a significant advance in the under-
© XXXX American Chemical Society
A
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Table 1. In Vitro Properties and Binding Potential in Cortex (BPND) of the Previously Published PET/SPECT Radioligands for
Imaging of α7-nAChR
B
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Table 1. continued
a
The BPND values in the cortex were taken directly from the corresponding references or estimated as VT/VND − 1 or (cortex uptake/cerebellum
uptake) − 1.41,42
an appropriate range (for details, see refs 30, 32, 36, 37). The
general aptness of a PET radioligand for quantitative imaging
studies is defined by a conventional criterion Bmax/KD ≥ 10.37
This equation predicts that a picomolar range of the binding
affinity is required for a good α7-nAChR PET radioligand (KD
≤ 0.15−1.2 nM), whereas the most previously published α7-
nAChR radioligands exhibited nanomolar binding affinities
(Table 1). It is noteworthy, however, that the inhibition
binding assays of the published compounds have been
performed under a variety of assay conditions, and thus, the
values of Ki listed in Table 1 may not be directly comparable to
one another (see Results and Discussion for details).
envisioned the synthesis of fluoro derivatives of 5 producing a
new set of compounds with similar or even better binding
affinities with the potential for radiolabeling with [18F] for PET
imaging. The rationale for the design of fluorine-bearing
analogues of compound 5 has been strengthened by reports
that fluorine substituents can increase the metabolic stability
and the rate and extent of blood−brain barrier penetration of
radiotracers.44
In this study, we describe the design, synthesis and in vitro
and in vivo characterization in mice of a series of high α7-
nAChR binding affinity derivatives of 5 as potential probes for
PET imaging of α7-nAChR receptor.
Recently Abbott Laboratories has reported 3-(1,4-
diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-diox-
ide 5 (Figure 1) as an α7-nAChR selective antagonist with
extraordinarily high binding affinity, Ki = 0.023 nM.43 We
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of α7-nAChR Ligands. The fluoro
derivatives 7a−e of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-
dibenzo[b,d]thiophene 5,5-dioxide 5 were synthesized via the
Buchwald−Hartwig cross-coupling reaction between the
respective fluorobromo compounds 6a−e with 1,4-
diazabicyclo[3.2.2]nonane (Scheme 1).
The nitro derivatives of (1,4-diazabicyclo[3.2.2]nonan-4-
yl)dibenzo[b,d]thiophene 5,5-dioxide 10 and 11 were synthe-
sized similarly starting with respective nitrobromodibenzothio-
phene derivatives 8 and 9 (Scheme 2). Reduction of nitro
groups in 10 and 11 with iron powder gave corresponding
Figure 1. 3-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]-
thiophene 5,5-dioxide 5, an α7-nAChR antagonist with very high
binding affinity43 that was used as the lead compound in this report.
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a
Scheme 1
a
Reagents and conditions: (a) Pd2(dba)3, rac-BINAP, toluene, 1,4-diazabicyclo[3.2.2]nonane, Cs2CO3, 85 °C, 24 h.
a
Scheme 2
a
Reagents and conditions: (a) Pd2(dba)3, rac-BINAP, toluene, 1,4-diazabicyclo[3.2.2]nonane, Cs2CO3, 85 °C, 24 h; (b) iron powder, NH4Cl, THF,
MeOH, water, 80 °C, 3 h; (c) (i) 4N H2SO4, CH3CN, NaNO2, −5 °C, 30 min; (ii) NaI, CuI, water 70 °C, 30 min.
a
Scheme 3
a
Reagents and conditions: (a) Cs2CO3, DMF, 5 h; (b) iron powder, NH4Cl, THF, MeOH, water, 80 °C, 3 h; (c) (i) NaNO2, 37% HCl, 0−5 °C, 30
min; (ii) NaBF4, 0−5 °C, 30 min; (iii) Cu2O, 0.1 N H2SO4, 35−40 °C 30 min; (d) 30% H2O2, acetic acid, 60 °C, 24 h.
a
Scheme 4
a
Reagents and conditions: (a) H2SO4, HNO3, 0 °C; (b) 30% H2O2, acetic acid, 60 °C, 24 h; (c) NBS, conc H2SO4 24 h; (d) SnCl2·2H2O, 37% HCl,
HOAc, 100 °C, 60 min or iron powder, NH4Cl, THF, MeOH, water, 80 °C, 3 h; (f) (i) 48% HBF4, 0−5 °C 10 min; (ii) NaNO2, 0−5 °C, 1 h; (iii)
xylene, 135 °C, 30 min.
D
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a
anilines 12 and 13 in high yield. Diazotization−iodination of 12
and 13 yielded corresponding iodides 14 and 15 (Scheme 2).
Synthesis of Intermediate Compounds. The synthesis of
intermediate fluorobromide 6a was performed in four steps
(Scheme 3). Coupling of commercially available 4-bromo-2-
fluoronitrobenzene 16 and 2-fluorothiophenol 17 gave nitro-
diaryl thioether 18 that was reduced to aniline 19. Aniline 19
was treated with sodium nitrite at 0 °C in the presence of
hydrochloric acid and sodium tetrafluoroborate to yield a
corresponding diazonium tetrafluoroborate derivative (not
shown). The intramolecular deazotization/cyclization of the
diazonium salt in the presence of copper(I) oxide and 0.1 N
sulfuric acid afforded fluorobromodibenzothiophene derivative
20, which in turn was oxidized with hydrogen peroxide to 6a in
high yield (Scheme 3).
The fluorobromo isomers 6b and 6c were synthesized in four
steps via the commercially available dibenzo[b,d]thiophene 5,5-
dioxide 21 and 2-nitrodibenzo[b,d]thiophene 22, respectively
(Scheme 4). In brief, nitration of compound 21 and oxidation
of compound 22 gave compounds 23 and 24, respectively.
Bromination of compounds 23 and 24 provided monobromo
derivatives 25 and 9 that sequentially were reduced to anilines
26 and 27, respectively, in high yields. The anilines 26 and 27
were converted to fluorides 6b and 6c in moderate yields by the
Schiemann reaction via the corresponding intermediate
diazonium fluoroborates (structures not shown). The diazo-
nium salts precipitated in the reaction mixture and were
isolated by filtration in high yields.
Scheme 6
a
Reagents and conditions: (a) 30% H2O2, acetic acid, 60 °C, 24 h; (b)
NBS, H2SO4, 24 h.
shares 30% homology with the 5-HT3 receptor and first
generation α7-nAChR radioligands exhibited low α7-nAChR/
5-HT3 selectivity,27 the in vitro binding affinity at the 5-HT3
receptor was also determined for selected compounds of our
series (Table 2).
α7-nAChR Assays. The α7-nAChR assays for 7a−e, 10, 11,
14, and 15 were performed using a commercial assay consisting
of rat cortical membranes (rich in α7-nAChR) in competition
against 0.1 nM [125I]α-bungarotoxin, an α7-nAChR antagonist
with a KD of 0.7 nM. These assays were performed
independently in duplicate, each twice (Table 2). Assays for
two reference compounds, methyllycaconitine (MLA), a
conventional reference α7-nAChR antagonist, and compound
5,43 a lead of our series, were also performed (Table 3).
The new series of fluoro isomers 7a−d exhibited high
binding affinity at α7-nAChRs with Ki values in the range 0.3−
2.5 nM, whereas the binding affinity of isomer 7e was lower
(Table 2). The Ki values of the fluoro derivatives 7a−d (Table
2) were better than that of the conventional reference α7-
nAChR ligand MLA (Table 3). Among all fluoro isomers
compound 7a manifested the best α7-nAChR binding affinity
that was an order of magnitude better than MLA and at least
comparable to the nonfluorinated lead 5 (Tables 2 and 3).
Within the series 7a−e, two fluoro derivatives 7a and 7c were
selected for further evaluation. This selection was based on the
high α7-nAChR binding affinity and selectivity of 7a and 7c
(see Table 2) and the suitability of these compounds for
radiolabeling with [18F]. The radiolabeling of [18F]7a and [18F]
7c was anticipated to be accomplished by a direct nucleophilic
substitution (SNAr) with [18F]fluoride via the nitro 10 and 11
or iodo derivatives 14 and 15, respectively. The leaving nitro
groups in 10 and 11 or iodo groups in 14 and 15 are activated
for SNAr fluorination by the powerful electron-withdrawing
SO2Ar on the ortho and para positions, respectively.47−50 We
did not find in the literature an example of fluorination of nitro-
or iododibenzothiophene 5,5-dioxides, but the structural
analogue of 11, 4,4′-sulfonylbis(p-nitrobenzene), has been
converted to the corresponding fluoro derivative with good
yield.51
The brominated isomers 6d and 6e were prepared by
bromination of 4-fluorodibenzo[b,d]thiophene 5,5-dioxide 29
starting with 4-fluorodibenzo[b,d]thiophene 28 (Scheme 5).45
a
Scheme 5
a
Reagents and conditions: (a) 30% H2O2, acetic acid, 60 °C, 24 h; (b)
NBS, H2SO4, 24 h.
Oxidation of 28 with hydrogen peroxide gave dioxide 29 in
nearly quantitative yield. Bromination of 29 with 1 equiv of
NBS in H2SO4 afforded two isomeric bromides: 6d as the main
product in 24% yield and 6e as a minor product in 13% yield. A
substantial amount of compound 29 (about 50%) was
recovered from the reaction mixture. Isomers 6d and 6e were
readily separated by silica gel chromatography.
3-Bromo-6-nitrodibenzo[b,d]thiophene 5,5-dioxide 8 was
synthesized in two steps: (1) oxidation of 4-nitrodibenzo[b,d]-
thiophene 3046 gave 4-nitrodibenzo[b,d]thiophene 5,5-dioxide
31 in 90% yield; (2) bromination of compound 31 provided
compound 8 as the only product in 77% yield (Scheme 6).
In Vitro Inhibition Binding Assay. The results of the α7-
nAChR in vitro inhibition binding assays for compounds 7a−e,
10, 11, 14, and 15 are shown in Table 2. In order to determine
α7-nAChR selectivity of new compounds vs other nAChR
subtypes, binding assays for the main cerebral heteromeric
nAChR subtypes (α2β2, α2β4, α3β2, α3β4, α4β2, and α4β4)
were also performed (Table 2). In addition, because α7-nAChR
The fluoro derivative 7b that also exhibited high α7-nAChR
binding affinity was not selected for further studies because the
activating SO2Ar was located on the meta position to the
leaving group and direct radiolabeling of [18F]7b via its nitro or
iodo derivative was less likely. A detailed search of the literature
for SNAr reactions for 3-nitrodibenzo[b,d]thiophene 5,5-
dioxide, a potential precursor for [18F]7b, or its structural
analogues (1-nitro-3-(phenylsulfonyl)benzene, etc.) did not
reveal any previous publications.
The potential precursors 10, 11, 14, and 15 for 18F-
fluorination of [18F]7a and [18F]7c were studied in the same
α7-nAChR inhibition binding assay. The nitro compounds 10
and 11 exhibited α7-nAChR binding affinities comparable to
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Table 2. Inhibition in Vitro Binding Affinities (Ki, nM) of the New Series 7a−e, 10, 11, 14, and 15 toward α7-nAChR,
Heteromeric nAChR Subtypes, and 5-HT3
b
heteromeric nAChR subtype
selectivity
a
c
compd
α7-nAChR
α2β2
α2β4
α3β2
α3β4
α4β2
α4β4
5-HT3
α7/α4β2
α7/5HT3
7a
7b
7c
7d
7e
10
11
14
15
0.37, 0.45
1.02, 1.37
1.32, 1.35
1.83, 2.45
17.8, 20.3
0.34, 0.35
3.41, 6.21
0.93, 1.93
6.46, 8.77
>10000
4000
1000
709
562
1000
230
1370
561
d
d
d
d
d
d
d
nt
nt
nt
nt
nt
nt
nt
1000
292
8000
838
2000
678
5000
3000
261
885
3000
1000
251
505
663
66
378
d
141
nt
d
>10000
562
2000
4000
nt
210
d
d
d
d
d
d
d
nt
nt
nt
nt
nt
nt
nt
d
d
d
d
d
d
d
nt
nt
nt
nt
nt
nt
nt
d
d
d
d
d
d
d
nt
nt
nt
nt
nt
nt
nt
d
784
6000
1000
9000
477
5000
nt
63
a
b
Rat cortical membranes, radiotracer [125I]α-bungarotoxin (0.1 nM). KD = 0.7 nM. Inhibition in vitro binding assay of all heteromeric nAChR
subtypes was performed with stably transfected HEK293 cells and [3H]epibatidine (0.5 nM). KD = 0.021 nM (α2β2-nAChR). KD = 0.084 nM
(α2β4-nAChR). KD = 0.034 nM (α3β2-nAChR). KD = 0.29 nM (α3β4-nAChR). KD = 0.046 nM (α4β2-nAChR). KD = 0.094 nM (α4β4-nAChR).55
c
d
Human 5-HT3 recombinant/HEK293 cells, radiotracer [3H]GR65630 (0.35 nM). KD = 0.5 nM nt = not tested.
versus heteromeric nAChR including the main cerebral subtype
α4β2-nAChR (Table 2). Interestingly, the α7/α4β2 selectivity
of iodo derivative 15 is 10 times lower than the corresponding
fluoro derivative 7c.
5-HT3 Assay. The in vitro binding affinity of the most
promising members of the series, compounds 7a and 7c, at the
5-HT3 receptor was determined commercially using membrane
preparations from HEK293 cells expressing transfected human
5-HT3R in competition with 0.35 nM [3H]GR65630, a 5-HT3R
antagonist with a KD of 0.5 nM. The assay demonstrated that
fluoro compounds 7a and 7c manifest relatively low 5-HT3
binding affinities and they are highly α7-nAChR/5HT3
selective (Table 2).
Lipophilicity of 7a and 7c. Lipophilicity (log D7.4) is
considered an important property of CNS radioligand because
it has been linked to the blood−brain barrier permeability and
nonspecific binding.35,37,38 The lipophilicity values for 7a and
7c (log D7.4 = 2.0) were calculated with ACD Labs Structure
Designer Suite (ACD Labs, Toronto, Canada) and fall within
the conventional range for CNS PET radioligands.
Radiochemistry. We radiolabeled the fluoro isomers 7a
and 7c that exhibited the highest binding affinity within the
series with fluorine-18. The radiosyntheses were performed
remotely in one step by 1,10-diaza-4,7,13,16,21,24-
hexaoxabicyclo[8.8.8]hexacosane (Kryptofix-222) assisted ra-
diofluorination of the respective nitro precursors 10 and 11
(Scheme 7) or iodo precursors 14 and 15 using a radio-
Table 3. Inhibition in Vitro Binding Affinities (Ki, nM) of
Reference Compounds toward α7-nAChR
a
compd
α7-nAChR
MLA
2.91 0.76 (n = 9)
2
3
4
5
20.4
38.0
3.3
0.30, 0.50
a
The binding assay conditions are the same as those in Table 2.
those of the corresponding fluorides 7a and 7c, whereas the
binding affinities of iodo derivatives 14 and 15 were lower.
Currently, there is no conventional in vitro competition
binding assay for α7-nAChR. Different research groups use
different radioligands ([125I]α-bungarotoxin, [3H]α-bungaro-
toxin, [3H]MLA, [125I]iodo-MLA, [3H]A-585539, etc.) and
different sources of receptor tissue (cell lines, brain, adrenal
glands) under different conditions for this assay.26,29,52−54 It is
not surprising that the difference in the Ki values for the same
compound under different assay conditions can exceed an order
of magnitude.53,54 Therefore, a direct comparison of Ki values
of the previously published α7-nAChR ligands with compounds
of our new series is not practical.
For the purpose of comparison, we determined the Ki values
of the three most recently published α7-nAChR PET
radioligands [11C]2,26 [18F]3,29 and [18F]427 (Table 3) under
the same assay conditions as those of our series (Table 2). It
was noteworthy that the α7-nAChR binding affinities of the
best compounds of our series 7a and 7c were substantially
better than those of the previous radioligands.
Scheme 7. Radiosynthesis of [18F]7a and [18F]7c
Heteromeric nAChR Subtypes Assays. The heteromeric
nAChR subtypes assays (α2β2-, α2β4-, α3β2-, α3β4-, α4β2-,
α4β4-nAChR) were performed in our laboratories using
membrane preparations from HEK293 cells expressing the
transfected nAChR under test in competition with 0.5 nM
[3H]epibatidine to investigate the specificity of the ligand for
each receptor (Table 2).
The heteromeric nAChR Ki values of the tested compounds
7a, 7c−e, and 15 were substantially greater than the
corresponding α7-nAChR Ki values, indicating a high α7-/
heteromeric-nAChR subtype selectivity of all studied com-
pounds (Table 2). Thus, the fluoro isomer 7a with the best α7-
nAChR binding affinity also manifested an excellent selectivity
chemistry synthesis module (Microlab, GE) followed by the
semipreparative HPLC separation and formulation of [18F]7a
and [18F]7c as sterile apyrogenic solutions in 7% ethanolic
saline.
It is noteworthy that the radiotracer product yields from iodo
precursors 14 and 15 were substantially lower than those of the
nitro precursors 10 and 11. The conventional Kryptofix-222/
potassium carbonate assisted radiofluorination of both iodo
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Figure 2. Regional distribution of [18F]7a (left) and [18F]7c (right) in CD-1 mice. Data: mean % injected dose/g tissue
SD (n = 3).
Abbreviations: Coll, superior and inferior colliculus; Hipp, hippocampus; FrCtx, frontal cortex; Rest, rest of brain; Th, thalamus; Str, striatum; CB,
cerebellum.
Figure 3. Self-blockade study of [18F]7a and [18F]7c in CD-1 mice. Left: Inhibition of [18F]7a (0.07 mCi, specific radioactivity of 9200 mCi/μmol,
iv) accumulation by intravenous co-injection with 7a (0 mg/kg (white) and 0.3 mg/kg (black)) in the mouse brain regions 90 min after the
injection: (∗) P < 0.01, significantly different from controls; (∗∗) P = 0.04, insignificantly different from controls (ANOVA). Right: Inhibition of
[18F]7c (0.07 mCi, specific radioactivity of 12 000 mCi/μmol, iv) accumulation by intravenous co-injection with 7c (0 mg/kg (white) and 0.2 mg/kg
(black)) in the mouse brain regions 90 min after the injection: (∗) P ≤ 0.01, (∗∗) P = 0.015, significantly different from controls; (∗∗∗) P = 0.5,
insignificantly different from controls (ANOVA). Data are the mean % injected dose/g tissue SD (n = 3). Abbreviations: Coll, superior and
inferior colliculus; Hipp, hippocampus; FrCtx, frontal cortex; Str, striatum; Rest, rest of brain, CB, cerebellum.
derivatives 14 and 15 in DMSO at 130−180 °C produced [18F]
7a and [18F]7c with radiochemical yields below 0.5%, and this
radiosynthesis pathway was not optimized further (not shown).
The radiofluorination of nitro derivative 10 or 11 (Scheme
7) in the presence of Kryptofix-222/potassium carbonate at 160
°C produced [18F]7a or [18F]7c in a slightly better yield (2−
3%). Further optimization of this radiosynthesis suggested that
both final products [18F]7a and [18F]7c rapidly decomposed in
the DMSO reaction solution in the presence of highly basic
K2CO3, but the radiochemical yield was improved if the less
basic potassium oxalate was used. In the presence of potassium
oxalate, the final products [18F]7a and [18F]7c were prepared
under similar reaction conditions with comparable radio-
preparative HPLC and were not detected by analytical HPLC
in the final products [18F]7a and [18F]7c (see Table 6 for the
HPLC details).
Biodistribution Studies of [18F]7a and [18F]7c in Mice.
Baseline Studies in Mice. Radioligands [18F]7a and [18F]7c
were evaluated in mice as potential PET tracers for imaging α7-
nAChRs. After intravenous injection, [18F]7a and [18F]7c
exhibited robust initial brain uptake followed by washout. The
highest accumulation of radioactivity of both radioligands
occurred in the superior/inferior colliculus, hippocampus, and
frontal cortex. Moderate uptake was observed in thalamus and
striatum, and the lowest radioactivity was seen in cerebellum
(Figures 2−4). This distribution of radioactivity was similar to
the previously published in vitro data on the distribution of α7-
nAChRs in rodents.56,57 The clearance rate of [18F]7a and [18F]
7c from cerebellum was higher than that from any other region
studied. The ratios of tissues to cerebellum increased steadily
over the 90 min, reaching values of 10 for [18F]7a and 4.5 for
chemical yields of 16
6% (n = 14) (nondecay-corrected),
with specific radioactivities in the range 330−1260 GBq/μmol
(9−34 Ci/μmol) and a radiochemical purity greater than 99%.
The nitro precursors 10 and 11 that exhibited substantial α7-
nAChR binding affinity (Table 2) were fully separated by
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Figure 4. Blocking of [18F]7a and [18F]7c with α7-nAChR-selective ligands in CD-1 mice. Left: Dose dependent blockade of [18F]7a (0.07 mCi,
specific radioactivity of 7900 mCi/μmol, iv) accumulation by intravenous coinjection with 1 (doses 0.02, 0.2, 1, 3 mg/kg) in the mouse brain regions
90 min after the injection: (∗) P ≤ 0.01, significantly different from controls (ANOVA). Right: Dose dependent blockade of [18F]7c (0.07 mCi,
specific radioactivity of 11 000 mCi/μmol, iv) accumulation by intravenous co-injection with 5 (doses 0.001, 0.0045, 0.014 mg/kg) in the mouse
brain regions 90 min after the injection: (∗) P < 0.01, significantly different from controls; (∗∗) P = 0.06, insignificantly different from control
(ANOVA). Data are the mean % injected dose/g tissue SD (n = 3). Abbreviations: Coll, superior and inferior colliculus; Hipp, hippocampus; Ctx,
cortex; Str, striatum; Th, thalamus; Rest, rest of brain; CB, cerebellum.
[18F]7c. The better ratios for [18F]7a vs [18F]7c are in
agreement with in vitro α7-nAChR binding affinity of these
compounds (Table 2).
the blockade was significant only with the highest dose of 1 (3
mg/kg) (Figure 4, left). A similar dose−response study was
performed with [18F]7c using compound 5, a selective α7-
nAChR antagonist, as a blocker (Figure 4, right). These studies
confirmed that the in vivo binding of [18F]7a and [18F]7c was
specific and mediated by α7-nAChR. The dose-escalation
response demonstrated that both radioligands are suitable tools
for evaluation of new α7-nAChR drug candidates.
Specificity and Selectivity of [18F]7a and [18F]7c Binding in
the Mouse Brain. A conventional in vivo blockade method-
ology with CNS drugs is used here for demonstration of
specificity and selectivity at the α7-nAChR receptor in the
mouse brain. A self-blockade study with a nonradioactive form
of a radioligand estimates whether or not the binding is specific.
A blockade study with a drug that is highly selective at the
target binding site is expected to show the selectivity and
specificity of the radioligand binding. A dose-escalation
blockade with such a target selective drug provides further
evidence of the radioligand specificity and selectivity, and it is
useful for demonstration of the radioligand suitability for
evaluation of conventional drug candidates. In addition,
blockade with CNS drugs that do not bind at the target site
provides more evidence of the radioligand selectivity versus
other cerebral binding sites.
It is noteworthy that the doses of 1 that significantly blocked
the [18F]7a binding in CD1 mice were comparable to the doses
of 1 that significantly improved cognitive deficit in the various
rodent models of schizophrenia.59,60 This finding suggests that
[18F]7a is a suitable radioprobe for in vivo studies in mice with
pharmacologically relevant doses of α7-nAChR drugs.
Because the lowest regional uptake of [18F]7a and [18F]7c
was seen in the cerebellum, the regional BPND values in mice
were approximated for a single time point measurement (90
min) as BPND = (regional uptake/cerebellum uptake) − 1 42
(Table 4). The substantially higher BPND values for [18F]7a are
Self-Blockade Studies. Self-blockade studies of [18F]7a with
7a (Figure 3, left) and of [18F]7c with 7c (Figure 3, right)
demonstrated a reduction of the radioligand uptake in most
brain regions except the cerebellum, a region with low density
of α7-nAChRs. The studies showed that accumulation of [18F]
7a and [18F]7c radioactivity in the mouse brain was specific.
When the specific binding of the radioligands in the
hippocampus and colliculus was estimated by using the
radioactivity concentration in the blocked cerebellum as
nonspecific binding, the specific binding value amounted to
94% and 80% and the baseline-to-blockade ratio in the α7-
nAChR-rich regions was 13 and 5 for [18F]7a and [18F]7c,
respectively. This result also demonstrated that [18F]7a
exhibited a higher level of specificity and greater uptake in
the mouse brain versus [18F]7c.
Neither behavioral nor locomotor activity changes were
observed in the mice in the blockade studies with 7a (0.3 mg/
kg, iv) or 7c (0.2 mg/kg, iv).
Blocking with Selective α7-nAChR Ligands. A blockade
study of [18F]7a with 1, a selective α7-nAChR partial agonist
with a Ki of 22 nM,58 showed a dose dependent blockade in all
regions studied. However, in the α7-nAChR-poor cerebellum,
Table 4. Approximate BPND Values (Unitless) of [18F]7a and
a
[18F]7c in the Mouse Brain Regions
region
compd
Coll
Hipp
Ctx
[18F]7a
[18F]7c
8.0 1.6
2.0 0.5
5.5 1.7
3.1 0.7
5.3 1.2
2.0 0.3
a
Data: mean SD (n = 6). Abbreviations: Coll, superior and inferior
colliculus; Hipp, hippocampus; Ctx, cortex.
in agreement with greater binding affinity of this compound
versus [18F]7c (Table 2; also see Figure 7). The BPND values
for both radioligands [18F]7a and [18F]7c were superior to all
previously published α7-nAChR PET radioligands (Table 1).
Blocking with Nicotine and α4β2-nAChR Selective
Cytisine. The blockade of [18F]7a in CD1 mouse brain with
cytisine, a partial nicotinic agonist selective for α4β2-nAChR
and other β2/β4-containing heteromeric nAChR subtypes
while exhibiting low α7-nAChR binding affinity,52,55,61 showed
insignificant reduction of radioactivity accumulation in all
regions studied (Figure 5). This result demonstrated that [18F]
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7a manifested insignificant binding at α4β2-nAChRs in the
mouse brain.
Figure 6. Effect of various CNS drugs (Table 5) on accumulation of
[18F]7a in CD-1 mouse brain regions 90 min after injection of tracer
expressed as % ID/g tissue. Abbreviations: Coll, superior and inferior
colliculus; Hipp, hippocampus; Ctx, cortex; CB, cerebellum; REST,
rest of brain. Data are the mean
SD (n = 3): (∗) P < 0.01,
significantly different from controls. Columns that do not include the
asterisk are insignificantly different from controls (P > 0.05) (ANOVA,
single-factor analysis). The graph demonstrates that unlike the positive
control (1) all non-α7-nAChR CNS drugs do not have an effect on the
cerebral uptake of [18F]7a and the radiotracer is α7-nAChR selective
in vivo.
Figure 5. Blockade of [18F]7a accumulation in CD-1 mouse brain
regions by injection of cytisine (1 mg/kg, sc) and nicotine (5 mg/kg,
sc) (both 90 min after the injection). Data are the mean % injected
dose/g tissue SD (n = 3). Abbreviations: Coll, superior and inferior
colliculus; Hipp, hippocampus; Ctx, cortex; CB, cerebellum; Rest, rest
of brain. The effect of cytisine was insignificant in all regions studied
(P > 0.05, asterisk is not shown). The difference between control and
nicotine was significant ((∗) P ≤ 0.01) in all regions except CB ((∗∗)
P = 0.9) (ANOVA). The study demonstrates that [18F]7a does not
bind in vivo at the main cerebral α4β2-nAChR subtype and it is
suitable for nicotine blockade studies.
Table 5. CNS Drugs (2 mg/kg, sc) for α7-nAChR Selectivity
a
Studies in Mice
dose
time of administration
drug
target receptor
(mg/kg) before radiotracer, min
1
selective α7-nAChR
2
2
2
10
10
10
partial agonist
ondansetron selective 5-HT3
antagonist
The blockade study of [18F]7a with nicotine that binds at all
nAChR subtypes including α7-nAChR52 showed significant
blockade in all regions except the nAChR-poor cerebellum.
This study suggests that [18F]7a can be used for nicotine
addiction or smoking studies in mice. The lesser blockade of
[18F]7a with nicotine (Figure 5) in comparison with 1 (Figure
4) is due to the rather modest binding affinity of nicotine at α7-
nAChR (Ki = 610 nM).52
Blocking with Non-a7-nAChR CNS Ligands. For determi-
nation of in vivo selectivity of [18F]7a for α7-nAChRs vs several
major CNS receptor systems, we compared the regional
distribution (Figure 6) of the radiotracer in control CD-1 mice
vs mice preinjected with various CNS active drugs or the
positive control 1 (see Table 5 for the drug list). None of the
drugs except 1 reduced accumulation of radioactivity when
compared with controls (Figure 6). The absence of blockade
with the 5-HT3-selective drug ondansetron was especially
remarkable because α7-nAChR ligands often bind to this
receptor subtype. This finding suggests that in the mouse brain
the radioligand [18F]7a was selective for α7-nAChRs versus
several major cerebral binding sites.
SCH23390
D1- and D5-antagonist
and 5-HT1C/2C
agonist
spiperone
ketanserin
naltrindole
D2-like and 5-HT2A
receptor antagonist
2
2
2
10
10
10
5-HT2/5-HT2C
antagonist
selective δ-opioid
antagonist
a
5-HT = 5-hydroxytryptoamine (serotonin).
and BPND (R2 = 0.05, not shown). It was likely that the lack of
correlation was due to the wide variability in binding assay
conditions for these compounds when performed by various
research groups (see Results and Discussion).
When the α7-nAChR binding assay for the radioligands is
performed under the same assay conditions (Tables 2 and 3),
the binding affinities 1/Ki correlate linearly (Figure 7) with the
cortical BPND values of [18F]7a and [18F]7c (Table 4) and
[11C]2, [18F]3, and [18F]4 (Table 1). This finding may explain
why the specific binding of the very high affinity radioligands
[18F]7a and [18F]7c is superior to the previous α7-nAChR
radioligands with lower binding affinities. This result
emphasizes further the importance of high binding affinity for
the imaging properties of α7-nAChR radioligands.
Comparison of Imaging Properties of [18F]7a and [18F]
7c with Previous α7-nAChR PET Radioligands. Binding
potential (BPND), a measure of in vivo specific binding and one
of the most important imaging characteristics of a PET
radioligand, is defined as the ratio of Bmax (receptor density) to
KD (radioligand equilibrium dissociation constant) or the
product of Bmax and binding affinity.41,62 Therefore, the binding
affinities (1/Ki) of α7-nAChR radioligands should correlate
linearly with their BPND values.
CONCLUSION
■
A series of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]-
thiophene 5,5-dioxide derivatives with high binding affinities for
α7-nAChRs (Ki = 0.4−20 nM) has been synthesized with
potential application for PET imaging of α7-nAChRs. Two
members of the series, 7a and 7c, with the best α7-nAChR
The comparison of all previously published α7-nAChR
radioligands (Table 1) revealed little correlation between 1/Ki
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C-18 10 μm columns (analytical 4.6 mm × 250 mm and preparative 10
mm × 250 mm).
A dose calibrator (Capintec 15R) was used for all radioactivity
measurements. Radiofluorination was performed with a modified GE
MicroLab radiochemistry box.
Chemistry. Typical Procedure for Reduction of Nitro
Derivatives to Anilines 12, 13, 19, 26, 27. A mixture of nitro
compound (1 mmol), iron powder (4 mmol), ammonium chloride
(1.2 mmol) in methanol (6 mL), THF (6 mL), and water (2 mL) was
heated to reflux (80 °C) for 3 h. The resulting mixture was diluted
with ethanol and concentrated and dried under vacuum. The residue
was purified by silica gel column chromatography (CHCl3/i-PrOH/
Et3N 10:1:0.1 to 10:30:4) to give the corresponding aniline derivative.
Typical Procedure for Bromination. N-Bromosuccinimide
(NBS) (1 mmol) was added to a solution of the starting 1,4-
dibenzothiophene derivative (1 mmol) in concentrated H2SO4 (3.6
mL) at room temperature. After 24 h, the solution was carefully
poured into ice/water. The solids were filtered and washed with water
and methanol. The obtained solids were recrystallized from 95%
EtOH to afford the bromo compounds.
cortex
Figure 7. Correlation of the BPND
(unitless) vs 1/Ki (nM−1) of
α7-nAChR PET radioligands [11C]2, [18F]3, [18F]4, [18F]7a, and [18F]
7c (y = 1.91x + 0.52; R2 = 0.98). The BPND values are shown in Tables
1 and 3. The SD values are available for [18F]7a and [18F]7c only. All
Ki values were obtained in this study under the same binding assay
conditions (Tables 2 and 3).
Typical Procedure for Oxidation of 1,4-Dibenzothiophene
Derivatives. 1,4-Dibenzothiophene derivative (1 mmol) was
dissolved in glacial acetic acid (2.8 mL) at room temperature.
Aqueous hydrogen peroxide (30%, 1.4 mL) was added in small
portions to the stirred solution. The addition of H2O2 resulted initially
in some precipitation. The mixture was stirred at 60 °C for 24 h, then
cooled to room temperature. The solid was filtered off, sequentially
washed with 70% aqueous acetic acid, then 30% aqueous acetic acid,
then water, and dried to afford the title compound.
binding affinities (Ki of 0.4 and 1.3 nM, respectively) and high
selectivity vs other nicotinic subtypes and 5-HT3, were
radiolabeled with 18F.
[18F]7a and [18F]7c readily entered the mouse brain and
specifically and selectively labeled cerebral α7-nAChR
receptors. The binding potential (BPND) values in mouse
cortex of [18F]7a, [18F]7c, and previously published α7-nAChR
radioligands correlated linearly with their binding affinities (1/
Ki) when the binding affinity values were determined under the
same assay conditions. In agreement with the binding affinity of
[18F]7a its BPND value in mice was substantially better than
those of the previous α7-nAChR radioligands.
3-Bromo-6-fluorodibenzo[b,d]thiophene 5,5-Dioxide (6a).
The typical procedure for oxidation of 1,4-dibenzothiophene was
followed, starting with 20 (600 mg, 2.13 mmol). The title compound
1
6a (648 mg, 97%) was obtained as white crystals. H NMR (CDCl3,
400 MHz) δ 7.97 (s, 1H), 7.81 (dd, J = 12.0, 1.8 Hz, 1H), 7.68 (d, J =
8.0 Hz, 1H), 7.66 (dd, J = 8.0, 4.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H),
7.24 (t, J = 8.0 Hz, 1H).
The best PET radioligand of this new series [18F]7a exhibits
excellent α7-nAChR imaging properties in the mouse brain.
Therefore, [18F]7a holds promise as a highly specific PET
radioligand for quantification of α7-nAChR receptors, and
further evaluation of this radioligand in baboon PET studies is
underway.
3-Bromo-7-fluorodibenzo[b,d]thiophene 5,5-Dioxide (6b). A
mixture of 26 (620 mg, 2 mmol) and 48% tetrafluoroboric acid
(HBF4) (4 mL) was stirred at 0−5 °C for 10 min. A cold solution of
sodium nitrite (204 mg in 0.8 mL of water, 3 mmol) was added
dropwise with stirring. After the mixture was stirred for 1 h at 0−5 °C
the precipitated intermediate diazonium tetrafluoroborate was
collected by filtration, washed with cold tetrafluoroboric acid (5%)
and water and Et2O, and dried under vacuum. The diazonium
tetrafluoroborate was boiled in xylene (135 °C) for 120 min. The
solvent was evaporated under reduced pressure. The residue was
extracted with a mixture of chloroform and water. The chloroform
layer was separated and concentrated. The residue was chromato-
graphed on silica gel using hexanes−EtOAc (4:1) as eluent to give 6b
as a pale yellow solid (330 mg, 53%). 1H NMR (CDCl3, 400 MHz) δ
7.96 (d, J = 2.0 Hz, 1H), 7.81−7.77 (m, 2H), 7.64 (d, J = 8.0 Hz, 1H),
7.54 (dd, J = 8.0, 4.0 Hz, 1H), 7.40−7.35 (m, 1H)
EXPERIMENTAL SECTION
■
All reagents were used directly as obtained commercially unless
otherwise noted. Reaction progress was monitored by TLC using silica
gel 60 F254 (0.040−0.063 mm) with detection by UV. All moisture-
sensitive reactions were performed under an argon atmosphere using
oven-dried glassware and anhydrous solvents. Column flash
chromatography was carried out using E. Merck silica gel 60F
(230−400 mesh). Analytical thin-layer chromatography (TLC) was
performed on aluminum sheets coated with silica gel 60 F254 (0.25 mm
thickness, E. Merck, Darmstadt, Germany). Melting points were
1
determined with a Fisher-Johns apparatus and were not corrected. H
7-Bromo-2-fluorodibenzo[b,d]thiophene 5,5-Dioxide (6c). A
mixture of 27 (310 mg, 1 mmol) and 48% tetrafluoroboric acid
(HBF4) (2 mL) was stirred at 0−5 °C for 10 min. A cold solution of
sodium nitrite (102 mg, 1.5 mmol) in 0.4 mL of water was added
dropwise with stirring. After the mixture was stirred for 1 h at 0 −5 °C,
the precipitated diazonium tetrafluoroborate was collected by
filtration, washed with cold tetrafluoroboric acid (5%) and water and
Et2O, and dried under vacuum. The diazonium tetrafluoroborate was
boiled in xylene (135 °C) for 30 min. The solvent was evaporated
under reduced pressure. The residue was treated with chloroform and
water. The chloroform layer was separated and concentrated. The
residue was chromatographed on silica gel using hexanes−EtOAc
NMR spectra were recorded with a Bruker-400 NMR spectrometer at
nominal resonance frequencies of 400 MHz in CDCl3 or DMSO-d6
(referenced to internal Me4Si at δH 0 ppm). The chemical shifts (δ)
were expressed in parts per million (ppm). First order J values were
given in hertz. Splitting patterns are described as singlet (s), doublet
(d), triplet (t), quartet (q), and broad (br). High resolution mass
spectra were recorded utilizing electrospray ionization (ESI) at the
University of Notre Dame Mass Spectrometry facility. All compounds
that were tested in the biological assays were analyzed by combustion
analysis (CHN) to confirm the purity of >95%. Elemental analyses
were determined by Galbraith Laboratories, Inc. (Knoxville, TN). The
HPLC system consisted of two Waters model 600 pumps, two
Rheodyne model 7126 injectors, an in-line Waters model 441 UV
detector (254 nm), and a single sodium iodide crystal flow
radioactivity detector. All HPLC chromatograms were recorded with
Varian Galaxy software (version 1.8). The analytical and semi-
preparative chromatographies were performed using Waters XBridge
1
(4:1) as eluent to give 6c as a pale yellow solid (156 mg, 50%). H
NMR (DMSO-d6, 400 MHz) δ 8.38 (d, J = 4.0 Hz, 1H), 8.23−8.18
(m, 2H), 8.13−8.07 (m, 2H), 7.54 (t, J = 8.0 Hz, 1H).
3-Bromo-4-fluorodibenzo[b,d]thiophene 5,5-Dioxide (6d)
and 1-Bromo-4-fluorodibenzo[b,d]thiophene 5,5-Dioxide
(6e). The typical procedure for bromination was followed, starting
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with 29 (905 mg, 3.86 mmol). Separation of the crude reaction
product by silica gel chromatography using hexanes/ethyl acetate
(5:2) yielded two isomers 6d (285 mg, 0.91 mmol, 23.6%) and 6e
(160 mg, 0.51 mmol, 13%). The isomer 6e was in the first
chromatography fraction, whereas 6d was in the second fraction.
6d: Rf = 0.31 (hexanes/EtOAc 2:1); 1H NMR (CDCl3, 400 MHz) δ
7.86−7.80 (m, 3H), 7.70 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H),
7.49 (d, J = 8.0 Hz, 1H).
3-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-4-fluorodibenzo[b,d]-
thiophene 5,5-Dioxide (7d). The typical procedure for Buchwald−
Hartwig cross-coupling reaction was followed, starting with 6d (0.246
g, 0.78 mmol), and the title compound 7d was obtained as a yellow
1
solid (170 mg, 0.47 mmol, 60% yield). Free base: H NMR (DMSO-
d6, 400 MHz) δ 8.06 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H),
7.36 (t, J = 8.0 Hz, 1H), 3.82 (s, 1H), 3.43−3.40 (m, 2H), 3.03−3.00
(m, 2H), 2.93−2.89 (m, 4H), 2.00−1.97 (m, 2H), 1.74−1.66 (m, 2H);
HRMS calculated for C19H20FN2O2S ([M + H]) 359.1224; found,
359.1246.
6e: Rf = 0.5 (hexanes/EtOAc 2:1); 1H NMR (CDCl3, 400 MHz) δ
8.94 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.83−7.79 (m, 1H),
7.75 (dd, J = 8.0, 4.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 8.0
Hz, 1H).
TSA salt: 1H NMR (DMSO-d6, 400 MHz) δ 10.16 (s, 1H), 8.12 (br
s, 1H), 7.94−7.90 (m, 2H), 7.78 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0
Hz, 1H), 7.50−7.40 (m, 3H), 7.12 (m, 2H), 4.01 (s, 1H), 3.54−3.38
(m, 6H), 2.30 (s, 3H), 2.19 (s, 2H), 2.07 (s, 2H), 1.09−1.03 (m, 2H).
Elemental analysis for C26H27FN2O5S2·0.5H2O, calcd: C, 57.87; H,
5.23; N, 5.19. Found: C, 58.21; H, 5.56; N, 4.88.
Typical Procedure for Buchwald−Hartwig Cross-Coupling
Reaction. 3-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-6-
fluorodibenzo[b,d]thiophene 5,5-Dioxide (7a). A catalyst sol-
ution was prepared by mixing tris(dibenzylideneacetone)dipalladium
(Pd2(dba)3, 58 mg, 0.063 mmol; Aldrich) and racemic BINAP (39 mg,
0.125 mmol; Strem) in toluene (4 mL) and heating the mixture to 90
°C for 15 min. The solution was cooled and then added to a mixture
of 1,4-diazabicyclo[3.2.2]nonane (200 mg, 1.58 mmol) and 6a (0.492
g, 1.58 mmol), in toluene (12 mL). Cs2CO3 (766 mg, 2.4 mmol;
Aldrich) was then added, and the reaction mixture was flushed with
nitrogen and heated overnight at 80−85 °C. After cooling to room
temperature, the mixture was concentrated and purified by silica gel
flash chromatography (CHCl3/i-PrOH/Et3N 10:1:0.2). The title
compound 7a (227 mg, 40% yield) was obtained as a yellow solid.
1H NMR (DMSO-d6, 400 MHz) δ 7.89 (d, J = 8.0 Hz, 1H), 7.77 (d, J
= 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.29−7.24 (m, 2H), 7.12 (d, J
= 8.0 Hz, 1H), 4.19 (s, 1H), 3.70−3.67 (m, 2H), 2.98−2.91 (m, 4H),
2.88−2.82 (m, 2H), 1.99 (m, 2H), 1.72−1.66 (m, 2H); HRMS
calculated for C19H20FN2O2S ([M + H]) 359.1224; found, 359.1240.
Preparation of 7a·p-TSA Salt. A mixture of 7a (30 mg, 0.084
mmol) and p-toluenesulfonic acid monohydrate (19 mg, 0.099 mmol)
was stirred in EtOAc−EtOH (2 mL, 10:1) at room temperature for 2
h. The resulting solid was collected, washed with EtOAc−EtOH (2
mL, 10:1) and EtOAc (3 mL), and dried under vacuum to afford the
title compound as a yellow solid (32 mg, 72% yield). 1H NMR
(DMSO-d6, 400 MHz) δ 10.10 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.85
(d, J = 8.0 Hz, 1H), 7.79−7.73 (m, 1H), 7.49−7.45 (m, 3H), 7.32 (t, J
= 8.0 Hz, 1H), 7.25 (dd, J = 8.0, 4.0 Hz, 1H), 7.12 (br s, 1H), 7.10 (br
s, 1H), 4.47 (s, 1H), 3.95−3.93 (m, 2H), 3.49−3.39 (m, 6H), 2.29 (s,
3H), 2.19 (m, 2H), 2.05 (m, 2H). Elemental analysis for
C26H27FN2O5S2, calcd: C, 58.85; H, 5.13; N, 5.28. Found: C, 58.57;
H, 5.04; N, 5.18.
1-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-4-fluorodibenzo[b,d]-
thiophene 5,5-Dioxide (7e). The typical procedure for Buchwald−
Hartwig cross-coupling reaction was followed, starting with 6e (0.112
g, 0.36 mmol). The title compound 7e was obtained as a yellow solid
1
(52 mg, 0.15 mmol, 40% yield). H NMR (CDCl3, 400 MHz) δ 8.50
(d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H),
7.55 (t, J = 8.0 Hz, 1H), 7.43 (dd, J = 8.0, 4.0 Hz, 1H), 7.14 (t, J = 8.0
Hz, 1H), 3.66−3.63 (m, 1H), 3.29−3.21 (m, 5H), 3.14−3.09 (m, 2H),
2.16−2.10 (m, 2H), 1.87−1.71 (m, 3H); HRMS calculated for
C19H20FN2O2S ([M + H]) 359.1224; found, 359.1215. Elemental
analysis for C19H19FN2O2S·1.5H2O, calcd: C, 59.20; H, 5.75; N, 7.27.
Found: C, 58.90; H, 5.76; N, 7.10.
3-Bromo-6-nitrodibenzo[b,d]thiophene 5,5-Dioxide (8). The
typical procedure for bromination was followed, starting with 31 (1.96
g, 7.5 mmol), and compound 8 was obtained as a pale brown solid
(1.73 g, 77%). 1H NMR (DMSO-d6, 400 MHz) δ 8.70 (d, J = 8.0 Hz,
1H), 8.45−8.43 (m, 2H), 8.28 (d, J = 8.0 Hz, 1H), 8.14−8.09 (m,
2H). HRMS calculated for C12H6BrNNaO4S ([M + Na]+) 361.9093;
found, 361.9080.
7-Bromo-2-nitrodibenzo[b,d]thiophene 5,5-Dioxide (9). The
typical procedure for bromination was followed, starting with 24 (1.82
g, 6.95 mmol), and compound 9 (2.1 g, 89%) was obtained as a pale
1
yellow solid. H NMR (DMSO-d6, 400 MHz) δ 9.10 (s, 1H), 8.44−
8.47 (m, 3H), 8.33 (d, J = 8.0 Hz, 1H), 8.11 (dd, J = 8.0, 4.0 Hz, 1H).
3-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-6-nitrodibenzo[b,d]-
thiophene 5,5-Dioxide (10). The typical procedure for Buchwald−
Hartwig cross-coupling reaction was followed starting with 8 (0.129 g,
0.38 mmol). Note that the reaction mixture was heated at 105 °C for
48 h. The title compound 10 was obtained as a reddish solid (80 mg,
4-(7-Fluorodibenzo[b,d]thiophen-3-yl)-1,4-diazabicyclo-
[3.2.2]nonane 5,5-Dioxide (7b). The typical procedure for
Buchwald−Hartwig cross-coupling reaction was followed, starting
with 6b (0.2 g, 0.64 mmol). The title compound 7b was obtained as a
1
55% yield). H NMR (DMSO-d6, 400 MHz) δ 8.40 (d, J = 4.0 Hz,
1H), 8.15 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0
Hz, 1H), 7.26 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H), 4.21 (s,
1H), 3.71 (m, 2H), 3.00−2.85 (m, 6H), 2.00 (s, 2H), 1.71 (m, 2H);
HRMS calculated for C19H20N3O4S ([M + H]) 386.1169; found,
386.1150. Elemental analysis for C19H19N3O4S·H2O, calcd: C, 56.56;
H, 5.25; N, 10.42. Found: C, 56.65; H, 4.99; N, 10.50.
7-(1, 4-Diazabicyclo[3.2.2]nonan-4-yl)-2-nitrodibenzo[b,d]-
thiophene 5,5-Dioxide (11). The typical procedure for Buch-
wald−Hartwig cross-coupling reaction was followed, starting with 9
(1.83 g, 5.38 mmol). The title compound 11 was obtained as a reddish
solid (0.836 g, 61% yield). 1H NMR (DMSO-d6, 400 MHz) δ 8.77 (s,
1H), 8.20−8.12 (m, 3H), 7.32 (d, J = 4.0 Hz, 1H), 7.16 (dd, J = 8.0,
4.0 Hz, 1H), 4.23 (s, 1H), 3.72 (m, 2H), 3.00−2.88 (m, 6H), 2.00 (br
s, 2H), 1.74−1.69 (m, 2H); HRMS calculated for C19H20N3O4S ([M +
H]) 386.1169; found, 386.1152. Elemental analysis for C19H19N3O4S·
1.25H2O, calcd: C, 55.94; H, 5.31; N, 10.30. Found: C, 55.98; H, 5.17;
N, 10.15.
1
yellow solid (104 mg, 0.29 mmol, 45% yield). H NMR (DMSO-d6,
400 MHz) δ 7.99 (dd, J = 8.0, 4.0 Hz, 1H), 7.89 (dd, J = 8.0, 3.0 Hz,
1H), 7.86 (d, J = 8.0 Hz, 1H), 7.55 (m, 1H), 7.25 (d, J = 4.0 Hz, 1H),
7.11 (dd, J = 8.0, 4.0 Hz, 1H), 4.17 (s, 1H), 3.66 (m, 2H), 2.99−2.91
(m, 3H), 2.87−2.82 (m, 3H), 2.00−1.97 (m, 2H), 1.71−1.65 (m, 2H).
Elemental analysis for C19H19FN2O2S·0.1H2O, calcd: C, 62.37; H,
5.17; N, 7.58. Found: C, 62.25; H, 5.44; N, 7.19.
7-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo[b,d]-
thiophene 5,5-Dioxide (7c). The typical procedure for Buchwald−
Hartwig cross-coupling reaction was followed, starting with 6c (0.226
g, 0.72 mmol), and the title compound 7c (140 mg, 54% yield) was
1
obtained as a yellow solid. H NMR (DMSO-d6, 400 MHz) δ 7.93−
7.87 (m, 3H), 7.26−7.21 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 4.20 (s,
1H), 3.71−3.68 (m, 2H), 3.01−2.83 (m, 6H), 2.00 (br s, 2H), 1.73−
1.67 (m, 2H); HRMS calculated for C19H20FN2O2S ([M + H])
359.1224; found, 359.1241.
1
TSA salt: H NMR (DMSO-d6, 400 MHz) δ 10.08 (s, 1H), 8.00−
6-Amino-3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-dibenzo[b,d]-
thiophene 5,5-Dioxide (12). The typical procedure for reduction of
nitro derivatives was followed starting with 10 (0.34 g, 0.88 mmol),
7.94 (m, 3H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (m, 2H), 7.32−7.25 (m,
2H), 7.12 (br, 1H), 7.10 (br, 1H), 4.48 (s, 1H), 3.94 (m, 2H), 3.47−
3.38 (m, 6H), 2.29 (s, 3H), 2.19 (m, 2H), 2.06−1.99 (m, 2H).
Elemental analysis for C26H27FN2O5S2·0.75H2O, calcd: C, 57.39; H,
5.28; N, 5.15. Found: C, 57.22; H, 5.11; N, 5.12.
1
and compound 12 (146 mg, 46%) was obtained as a yellow solid. H
NMR (DMSO-d6, 400 MHz) δ 7.73 (d, J = 12.0 Hz, 1H), 7.25 (t, J =
8.0 Hz, 1H), 7.11 (br s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 4.0
K
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Journal of Medicinal Chemistry
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Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 5.87 (br s, 2 H), 4.17 (s, 1H), 3.66
(m, 2H), 2.98−2.91 (m, 6H), 2.01 (s, 2H), 1.72 (m, 2H).
H), 7.10−7.00 (m, 2H), 6.92−6.87 (m, 1H), 6.69 (d, J = 8.0 Hz, 1H),
4.37 (br s, 2H).
2-Amino-7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]-
thiophene 5,5-Dioxide (13). The typical procedure for reduction of
nitro derivatives was followed starting with 11 (0.68 g, 1.76 mmol),
3-Bromo-6-fluorodibenzo[b,d]thiophene (20). Compound 19
(1.18 g, 3.96 mmol) was dissolved in 37% HCl (11 mL), and the
solution was cooled below 5 °C. To this reaction mixture, sodium
nitrite (408 mg, 5.93 mmol) was added slowly at a temperature below
5 °C. After addition, the mixture was stirred for 30 min below 5 °C.
Then sodium tetrafluoroborate (865 mg, 7.92 mmol) was added, and
the reaction mixture was stirred for another 30 min at a temperature
below 5 °C. This reaction solution was then added to the stirred
solution of copper(I) oxide (1.14 mg, 7.92 mmol) in 0.1 N sulfuric
acid (390 mL) at 35−40 °C. The reaction mixture was stirred for 15−
30 min. Ethyl acetate was added to the reaction mixture, and the
mixture was filtered to remove inorganic compound. The filtrate was
then extracted with ethyl acetate (3 × 120 mL). The organic extract
was washed with water followed by brine and then concentrated under
vacuum. The residue was purified by silica gel chromatography
1
and compound 13 was obtained as a yellow solid (585 mg, 93%). H
NMR (DMSO-d6, 400 MHz) δ 7.67 (d, J = 12 Hz, 1H), 7.43 (d, J = 12
Hz, 1H), 7.27 (s, 1H), 7.14 (d, J = 8.0, 4.0 Hz, 1H), 6.91 (s, 1H), 6.53
(d, J = 8.0, 4.0 Hz, 1H), 6.17 (s, 2H), 4.40 (s, 1H), 3.87 (br s, 3H),
3.07 (m, 1H), 2.15 (br s, 3H), 2.02 (br s, 3H), 1.20 (m, 2H).
3-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-6-iododibenzo[b,d]-
thiophene 5,5-Dioxide (14). Compound 12 (143 mg, 0.4 mmol)
was dissolved in a mixture of 4 N H2SO4 (0.8 mL) and CH3CN (1
mL), and the solution was cooled to −5 °C. Sodium nitrite (55 mg,
0.8 mmol) dissolved in H2O (0.5 mL) was added dropwise at the same
temperature. After the mixture was stirred for 60 min a solution of
diazonium salt was formed. To a mixture of CuI (268 mg, 1.4 mmol)
and saturated KI solution (2.5 mL) at 70 °C was added the above-
prepared solution of diazonium salt dropwise over 10 min, and the
mixture was further stirred at 70 °C for 30 min. The reaction mixture
was cooled, and 28% ammonia solution was added (2 mL). The
aqueous suspension was repeatedly extracted with CHCl3 and the
combined organic layers were washed with brine (10 mL), dried
(Na2SO4), and concentrated in vacuo. The crude product was purified
by flash chromatography on silica gel (CHCl3/i-PrOH/Et3N 10:1:0.1
to 3:1:0.2) to give 14 (28 mg, 15%). 1H NMR (DMSO-d6, 400 MHz)
δ 7.96 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 4.0 Hz, 1H), 7.81 (d, J = 8.0
Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 4.0 Hz, 1H), 7.18 (d, J =
8.0 Hz, 1H), 4.34 (s, 1H), 3.82 (m, 2H), 3.22−3.18 (m, 6H), 2.09 (m,
2H), 1.87 (m, 2H). Elemental analysis for C19H19IN2O2S·2.5H2O,
calcd: C, 44.63; H, 4.73; N, 5.48. Found: C, 44.88; H, 4.41; N, 5.48.
7-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-2-iododibenzo[b,d]-
thiophene 5,5-Dioxide (15). Compound 13 (285 mg, 0.8 mmol)
was dissolved in a mixture of 4 N H2SO4 (1.5 mL) and CH3CN (2
mL), and the solution was cooled to −5 °C. NaNO2 (110 mg, 1.6
mmol, 2 equiv) dissolved in H2O (1 mL) was added dropwise at the
same temperature. After the mixture was stirred for 60 min a solution
of diazonium salt was formed. To a mixture of CuI (536 mg, 2.8 mmol,
3.5 equiv) and saturated KI solution (2.5 mL) at 70 °C was added
above-prepared solution of diazonium salt dropwise over 10 min and
further stirred at 70 °C for 30 min. The reaction mixture was cooled,
and saturated NH4OH was added (4 mL). The aqueous suspension
was repeatedly extracted with CHCl3, and the combined organic layers
were washed with brine (10 mL), dried (Na2SO4), and concentrated in
vacuo. The crude product was purified by flash chromatography on
silica gel (CHCl3/i-PrOH/Et3N 10:1:0.1 to 3:1:0.2) to give 15 (75
mg, 20%). 1H NMR (DMSO-d6, 400 MHz) δ 8.40 (d, J = 4.0 Hz, 1H),
7.93 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.0, 1.8 Hz, 1H), 7.60 (d, J =
12.0 Hz, 1H), 7.25 (d, J = 1.8 Hz, 1H), 7.11 (dd, J = 8.0, 4.0 Hz, 1H),
4.20 (s, 1H), 3.70 (m, 2H), 2.98−2.88 (m, 6H), 2.00 (s, 2H), 1.72 (m,
2H); HRMS calculated for C19H20IN2O2S ([M + H]) 467.0285;
found, 467.0306;. Elemental analysis for C19H21IN2O3S, calcd: C,
47.12; H, 4.37; N, 5.78. Found: C, 47.24; H, 4.53; N, 5.87.
1
(hexanes) to give 20 (600 mg, 54%). H NMR (CDCl3, 400 MHz) δ
8.04 (d, J = 4.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz,
1H), 7.62 (dd, J = 8.0, 4.0 Hz, 1H), 7.47 (ddd, J = 12.0, 8.0, 4.0 Hz,
1H), 7.22 (t, J = 8.0 Hz, 1H).
3-Nitrodibenzo[b,d]thiophene 5,5-Dioxide (23). Dibenzo-
[b,d]thiophene 5,5-dioxide 21 (10 g, 46 mmol, Aldrich) was slowly
added to a stirred mixture of glacial acetic acid (34 mL) and sulfuric
acid (96%, 34 mL). The slurry was stirred, and red fuming nitric acid
(36 mL) was added dropwise over a period of 90 min at temperature
−5 °C to 5 °C. The slurry was stirred for another 30 min and poured
over ice. The precipitate was filtered, rinsed with water, and dried at
room temperature. The crude product was recrystallized with
acetonitrile to give 23 as yellow crystals (8.7 g, 72%). 1H NMR
(DMSO-d6, 400 MHz) δ 8.84 (d, J = 8.0 Hz, 1H), 8.65 (dd, J = 8.0, 2.0
Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.13 (d, J
= 8.0 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H).
2-Nitrodibenzo[b,d]thiophene 5,5-Dioxide (24). The typical
procedure for oxidation of 1,4-dibenzothiophene was followed starting
with 22 (489 mg, 2.13 mmol, Oakwood Chemical), and the title
compound 24 (510 mg, 90%) was obtained as white crystals. 1H NMR
(CDCl3, 400 MHz) δ 8.66 (d, J = 4 Hz, 1H), 8.43 (dd, J = 8, 4 Hz,
1H), 8.04 (d, J = 8 Hz, 1H), 7.96 (d, J = 8 Hz, 1H), 7.92 (d, J = 8 Hz,
1H), 7.79 (t, J = 8 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H).
3-Bromo-7-nitrodibenzo[b,d]thiophene 5,5-Dioxide (25).
The typical procedure for bromination was followed starting with 23
(2.59 g, 9.9 mmol), and brown solid was obtained and recrystallized
1
with benzene to yield 25 as a yellow solid (1.73 g, 51%). H NMR
(DMSO-d6, 400 MHz) δ 8.89 (d, J = 4.0 Hz, 1H), 8.67 (dd, J = 8.0, 3.0
Hz, 1H), 8.52−8.49 (m, 2H), 8.35 (d, J = 8.0 Hz, 1H), 8.15 (dd, J =
8.0, 2.0 Hz, 1H).
7-Bromodibenzo[b,d]thiophen-3-amine 5,5-Dioxide (26). A
solution of stannous chloride dihydrate (12.4 g, 56 mmol) in 37%
hydrochloric acid (21 mL) was added to a mixture of 25 (1.7 g, 5
mmol) in glacial acetic acid (50 mL). The reaction mixture was stirred
at 100 °C for 60 min and cooled to 5 °C. The precipitate was filtered
off, rinsed with water on the filter, and dispersed in water. The
dispersion was made basic (pH 10) by addition of an excess of 1 M
sodium hydroxide and stirred for 3 h. The precipitate was filtered off,
rinsed with water, and dried overnight on the filter to yield 26 (0.7 g,
45%) as a pale white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.12 (s,
1H), 7.87−7.77 (m, 3H), 6.95 (s, 1H), 6.87 (dd, J = 8, 4 Hz, 1H), 6.20
(br s, 2H).
2-Amino-7-bromodibenzo[b,d]thiophene 5,5-Dioxide (27).
The typical procedure for reduction of nitro derivatives was followed
starting with 9 (0.60 g, 1.76 mmol). The title compound 27 (496 mg,
91%) was obtained as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ
8.16 (d, J = 4 Hz, 1H), 7.93(d, J = 8.0 Hz, 1H), 7.87 (d, J = 12 Hz,
1H), 7.56 (d, J = 8 Hz, 1H), 7.08 (s, 1H), 6.71 (d, J = 8 Hz, 1H), 6.36
(br s, 2H).
(5-Bromo-2-nitrophenyl)(2-fluorophenyl)sulfane (18). Cesi-
um carbonate (4.3 g, 13.2 mmol) was added to a solution of 4-bromo-
2-fluoronitrobenzene 16 (2.42, 11 mmol, Aldrich) and 2-fluoroben-
zene thiol 17 (1.4 g, 11 mmol, Aldrich) in DMF (60 mL), and the
mixture was stirred for 5 h at room temperature. Water (200 mL) and
ethyl acetate (100 mL) were added. The organic layer was separated
and washed sequentially with water (100 mL) and then brine (100
mL). The organic phase was separated, dried, and concentrated to
yield a yellow solid that was purified by silica gel chromatography
(hexanes/EtOAc 8:1 to 3:1) to give 18 (2.88 g, 80%). 1H NMR
(CDCl3, 400 MHz) δ 8.17 (d, J = 8.0 Hz, 1H), 7.68−7.60 (m, 2H),
7.41−7.29 (m, 3H), 6.95 (s, 1H).
4-Bromo-2-((2-fluorophenyl)thio)aniline (19). The typical
procedure for reduction of nitro derivatives was followed, starting
with 18 (3.2 g, 9.75 mmol), and the title compound 19 was obtained
4-Fluorodibenzo[b,d]thiophene 5,5-Dioxide (29). The typical
procedure for oxidation of 1,4-dibenzothiophene was followed starting
with 4-fluorodibenzo[b,d]thiophene 2845 (1.62 g, 8 mmol). The title
1
as a brown solid (2.46 g, 85%). H NMR (CDCl3, 400 MHz) δ 7.59
1
(d, J = 4.0 Hz, 1H), 7.33 (dd, J = 8.0, 4.0 Hz, 1H), 7.21−7.15 (m, 1
compound 29 (1.8 g, 96%) was obtained as white crystals. H NMR
L
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Table 6. HPLC Conditions for [18F]7a and [18F]7c
flow rate,
mL/min
product retention
time, min
nitro precursor
retention time, min
column
mobile phase
[18F]7a, preparative
[18F]7a, analytical
[18F]7c, preparative
[18F]7c, analytical
XBridge C18 column,
CH3OH/CH3CN/H2O/Me3N
260:120:620:2
12
32
21
10 μm (250 mm × 10 mm)
XBridge C18 column,
5 μm (250 mm × 4.6 mm)
XBridge C18 column,
10 μm (150 mm × 10 mm)
XBridge C18 column,
3.5 μm (100 mm × 4.6 mm)
CH3CN/H2O/Et3N 390:610:1
CH3CN/H2O/NH3 280:720:1
CH3CN/H2O/NH3 380:620:1
2
7.4
20
5.5
27
10
2
3.4
5.2
(CDCl3, 400 MHz) δ 7.85 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz,
1H), 7.71−7.57 (m, 4H), 7.20 (t, J = 8.0 Hz, 1H).
Membrane Homogenate Preparation (Heteromeric nAChR).
Membrane homogenates for ligand binding assays were made as
described previously.52,55 Briefly, cultured cells at >90% confluency
were removed from the culture flask (80 cm2) with a disposable cell
scraper and placed in 10 mL of 50 mM Tris-HCl buffer (pH 7.4, 4
°C). The cell suspension was centrifuged at 1000g for 5 min, and the
pellet was collected. The cell pellet was then homogenized in 10 mL of
buffer with a Polytron homogenizer for 20 s and centrifuged at 35000g
for 10 min at 4 °C. Membrane pellets were resuspended in fresh
buffer.
4-Nitrodibenzo[b,d]thiophene 5,5-Dioxide (31). The typical
procedure for oxidation of 1,4-dibenzothiophene was followed starting
with 3046 (1.08 g, 4.71 mmol). The final compound 31 (1.1g, 90%)
1
was obtained as pale yellow crystals. H NMR (DMSO-d6, 400 MHz)
δ 8.69 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.33 (d, J = 8.0
Hz, 1H), 8.11 (t, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.88 (t, J =
8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H). HRMS calculated for
C12H7NNaO4S ([M + Na]) 283.9988; found, 283.9994.
Radiosynthesis of [18F]7a and [18F]7c. The same radiolabeling
method was used for both radioligands [18F]7a and [18F]7c. A solution
of the [18F]fluoride, 15−20 mg of Kryptofix 222, and 1−2 mg of
K2C2O4 in 1 mL of 50% aqueous acetonitrile was added to a reaction
vessel of a GE MicroLab box. The mixture was heated at 120−135 °C
under a stream of argon, while water was evaporated azeotropically
after the addition of 2 mL of CH3CN. A solution of the corresponding
nitro precursor (10 or 11) (2 mg) in anhydrous DMSO (0.8 mL) was
added to the reaction vessel and heated at 160 °C for 12 min. The
reaction mixture was cooled, diluted with 0.7 mL of water, and injected
onto the reverse-phase semipreparative HPLC column (Table 6). The
radioactive peak was collected in 50 mL of HPLC water. The water
solution was transferred through an activated Waters C-18 Oasis HLB
light solid-phase extraction (SPE) cartridge. After the SPE was washed
with 10 mL of saline, the product was eluted with a mixture of 1 mL of
ethanol and 0.04 mL of 1 N HCl through a 0.2 μm sterile filter into a
sterile, pyrogen-free multidose vial and 10 mL of 0.9% saline and 0.05
mL of sterile 8.4% solution sodium bicarbonate were added through
the same filter. The final products [18F]7a and [18F]7c were then
analyzed by analytical HPLC (Table 6) using a UV detector at 340 nm
to determine the radiochemical purity and specific radioactivity at the
time synthesis ended. The total synthesis time including QC was 70−
80 min.
In Vitro Binding Assay. α7-nAChR Assay with Rat Brain
Membranes. The assay was done commercially by Caliper
PerkinElmer (Hanover, MD). In brief, rat cortical membranes were
incubated with [125I]α-bungarotoxin (KD = 0.7 nM) at 0.1 nM in a
buffer consisting of 50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 0.003 mM atropine sulfate at pH 7.4 for 150 min
at 0 °C.63 The binding was terminated by rapid vacuum filtration of
the assay contents onto GF/C filters presoaked in PEI. Radioactivity
trapped onto the filters was assessed using a γ-counter. Nonspecific
binding was defined as that remaining in the presence of 1 μM α-
bungarotoxin. The assays were done two times independently, each in
duplicate, at multiple concentrations of the test compounds. Binding
assay results were analyzed using a one-site competition model, and
IC50 curves were generated based on a sigmoidal dose response with
variable slope. The Ki values were calculated using the Cheng−Prusoff
equation. Methyllycaconitine (MLA) was used as a reference
compound in all assays.
Binding to Heteromeric nAChR. Binding to heteromeric nAChR
subunit combinations, which represent possible heteromeric nAChRs,
was measured with 0.5 nM [3H]epibatidine in HEK cells expressing
these subunits (KD = 0.021 nM (α2β2-nAChR), KD = 0.084 nM
(α2β4-nAChR), KD = 0.034 nM (α3β2-nAChR), KD = 0.29 nM
(α3β4-nAChR), KD = 0.046 nM (α4β2-nAChR), KD = 0.094 nM
(α4β4-nAChR)).55 Aliquots of the membrane homogenates contain-
ing 30−200 μg of protein were used for the binding assays, which were
carried out in a final volume of 100 μL in borosilicate glass tubes. After
incubation at 24 °C for 2 h, the samples were collected with a cell
harvester (Brandel M-48) onto Whatman GF/C filters prewet with
0.5% polyethylenimine. After the samples were harvested, the filters
were washed three times with 5 mL of 50 mM Tris-HCl buffer and
then counted in a liquid scintillation counter. Nonspecific binding was
measured in samples incubated in parallel containing 300 μM nicotine
for [3H]epibatidine binding. Specific binding was defined as the
difference between total binding and nonspecific binding. Data from
these competition binding assays were analyzed using Prism 5
(GraphPad Software, San Diego, CA).
5-HT3(h) Binding Assay. The assay was done commercially by
Caliper PerkinElmer (Hanover, MD) using recombinant HEK293 cells
and 0.35 nM [3H]GR65630 (KD = 0.5 nM).
Biodistribution Studies in CD-1 Mice. Baseline Study. Male
CD-1 mice weighing 25−30 g from Charles River Laboratories
(Wilmington, MA) were used for biodistribution studies. The animals
were sacrificed by cervical dislocation at various times following
injection of [18F]7a or [18F]7c (70 μCi, specific radioactivity 8000−
12000 mCi/μmol, in 0.2 mL of saline) into a lateral tail vein, three
animals per time point. The brains were rapidly removed and dissected
on ice. The brain regions of interest were weighed, and their
radioactivity content was determined in an automated γ-counter with a
counting error below 3%. Aliquots of the injectate were prepared as
standards, and their radioactivity content was counted along with the
tissue samples. The percent of injected dose per gram of tissue (%ID/g
tissue) was calculated. All experimental protocols were approved by
the Animal Care and Use Committee of the Johns Hopkins Medical
Institutions.
Self-Blockade of [18F]7a Binding with 7a. In vivo saturation
blockade studies were done by iv coadministration of the radiotracer
[18F]7a (70 μCi, SA = 9200 mCi/μmol, 0.2 mL) with various doses of
“cold” 7a per animal (0 μg (vehicle), 0.0048 μg, 7.2 μg). Compound
7a was dissolved in saline at pH 5.5. At 90 min after administration of
the tracer and blocker, brain tissues were harvested, and their regional
radioactivity content was determined. The self-blockade of [18F]7c
with 7c was done similarly.
HEK 293 Cell Culture and Stable Transfections (Heteromeric
nAChR). HEK 293 cells (ATCC CRL 1573) were maintained at 37
°C with 5% CO2 in a humidified incubator. Growth medium for the
HEK 293 cells was the minimum essential medium supplemented with
10% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL
streptomycin. Transfections of these cells and selection and establish-
ment of stable cell lines were carried out as described previously.52,55
Blockade of [18F]7a Binding with 1. In vivo α7-nAChR receptor
blocking studies were done by intravenous coadministration of the
M
dx.doi.org/10.1021/jm401184f | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
radiotracer [18F]7a (70 μCi, SA = 7900 mCi/μmol, 0.2 mL) with
various doses of 1 (0 μg (vehicle), 0.02 mg/kg, 0.2 mg/kg, 1 mg/kg,
and 3 mg/kg). Three animals per dose were used. 1 was dissolved in a
vehicle (saline/alcohol (9:1) at pH 5.5). At 90 min after
administration of the tracer, brain tissues were harvested, and their
regional radioactivity content was determined. The dose-dependent
blockade study of [18F]7c with 5 was done the same way.
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Blockade of [18F]7a with Nicotine and Cytisine. In vivo CB1
receptor blocking studies were carried out by subcutaneous (sc)
administration of (−)-nicotine tartrate (5 mg/kg) or cytisine (1 mg/
kg) followed by iv injection of the radiotracer [18F]7a (70 μCi, specific
radioactivity of ∼14 000 mCi/μmol, 0.2 mL) 5 min thereafter. The
drugs were dissolved in saline and administered in a volume of 0.1 mL.
Control animals were injected with 0.1 mL of saline. At 90 min after
administration of the tracer, brain tissues were harvested, and their
radioactivity content was determined.
Blockade of [18F]7a with Non-α7-nAChR Drugs. In vivo
receptor blocking studies were performed by administration of six
drugs (Table 5), followed by iv injection of the radiotracer [18F]7a (70
μCi, specific radioactivity of ∼14 000 mCi/μmol, 0.2 mL). The drugs
(2 mg/kg, sc) were dissolved in a vehicle (saline/DMSO 5:1) and
administered in a volume of 0.1 mL. Control animals were injected
with 0.1 mL of the vehicle solution. At 90 min after administration of
the tracer, brain tissues were harvested, and their radioactivity content
was determined.
AUTHOR INFORMATION
Corresponding Author
*Phone: 410-614-5130. Fax: 410-614-0111. E-mail: ahorti1@
jhmi.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Richard Wahl for fruitful discussions. We thank
Dr. Hiroto Kuwabara for reading the manuscript and for his
useful comments. We thank Dr. Yuchuan Wang for useful
comments and references. We are grateful to Paige Finley and
Gilbert Green for their valuable help with animal experiments.
We are thankful to Judy W. Buchanan for editorial help. We
very much thank Drs. Peter Brust (Helmholtz-Zentrum
Dresden-Rossendorf Institute of Radiopharmacy) and Dan
Peters (DanPET AB) for the samples of compounds 2 and 3
and Dr. Martin Pomper for the sample of 4 for our inhibition
binding assay studies. This research was supported by NIH
Grants MH079017 and AG037298 (A.G.H.) and in part by the
Division of Nuclear Medicine of The Johns Hopkins University
School of Medicine.
(17) Wallace, T. L.; Bertrand, D. Alpha7 neuronal nicotinic receptors
as a drug target in schizophrenia. Expert Opin. Ther. Targets 2013, 17,
139−155.
ABBREVIATIONS USED
(18) Pomper, M. G.; Phillips, E.; Fan, H.; McCarthy, D. J.; Keith, R.
A.; Gordon, J. C.; Scheffel, U.; Dannals, R. F.; Musachio, J. L.
Synthesis and biodistribution of radiolabeled alpha 7 nicotinic
acetylcholine receptor ligands. J. Nucl. Med. 2005, 46, 326−334.
(19) Hashimoto, K.; Nishiyama, S.; Ohba, H.; Matsuo, M.; Kobashi,
T.; Takahagi, M.; Iyo, M.; Kitashoji, T.; Tsukada, H. [11C]CHIBA-
1001 as a novel PET ligand for alpha7 nicotinic receptors in the brain:
a PET study in conscious monkeys. PLoS One 2008, 3, e3231.
(20) Ogawa, M.; Nishiyama, S.; Tsukada, H.; Hatano, K.; Fuchigami,
T.; Yamaguchi, H.; Matsushima, Y.; Ito, K.; Magata, Y. Synthesis and
evaluation of new imaging agent for central nicotinic acetylcholine
receptor alpha7 subtype. Nucl. Med. Biol. 2010, 37, 347−355.
(21) Dolle, F.; Valette, H.; Hinnen, F.; Vaufrey, F.; Demphel, S.;
Coulon, C.; Ottaviani, M.; Bottlaender, M.; Crouzel, C. Synthesis and
preliminary evaluation of a carbon-11-labelled agonist of the α7
nicotinic acetylcholine receptor. J. Labelled Compd. Radiopharm. 2001,
44, 785−795.
■
BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; Bmax, re-
ceptor density in tissue; BPND, binding potential; EtOAc, ethyl
acetate; EtOH, ethanol; Et2O, diethyl ether; Et3N, triethyl-
amine; KD, dissociation constant of the radioligand−receptor
complex; nAChR, nicotinic acetylcholine receptor; i-PrOH,
isopropyl alcohol
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