18F-Labeled 1,4-Dioxa-8-azaspiro[4.5]decane Derivative: Synthesis and Biological Evaluation of a σ1 Receptor Radioligand with Low Lipophilicity as Potent Tumor Imaging Agent

Article abstract of DOI:10.1021/acs.jmedchem.5b00593

We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ₁ receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K_i = 5.4 ± 0.4 nM) for σ₁ receptors and selectivity for σ₂ receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [¹⁸F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/μmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [¹⁸F]5a to σ₁ receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ₁ receptors could be used for tumor imaging.

Full text of DOI:10.1021/acs.jmedchem.5b00593

Subscriber access provided by UNIV OF MISSISSIPPI
Article
18F-labeled 1,4-Dioxa-8-azaspiro[4.5]decane Derivative:
Synthesis and Biological Evaluation of a Sigma-1 Receptor
Radioligand with Low Lipophilicity as Potent Tumor Imaging Agent
Fang Xie, Ralf Bergmann, Torsten Kniess, Winnie Deuther-Conrad, Constantin Mamat,
Christin Neuber, Boli Liu, Jörg Steinbach, Peter Brust, Jens Pietzsch, and Hongmei Jia
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00593 • Publication Date (Web): 19 Jun 2015
Downloaded from http://pubs.acs.org on June 23, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
8
Fꢀlabeled 1,4ꢀDioxaꢀ8ꢀazaspiro[4.5]decane Derivative: Synthesis and
Biological Evaluation of a Sigmaꢀ1 Receptor Radioligand with Low
Lipophilicity as Potent Tumor Imaging Agent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1,2
1
1
1
1,3
Fang Xie , Ralf Bergmann , Torsten Kniess , Winnie DeutherꢀConrad , Constantin Mamat ,
1
2
1,3
1
1,3
2*
Christin Neuber , Boli Liu , Jörg Steinbach , Peter Brust , Jens Pietzsch , Hongmei Jia
1) HelmholtzꢀZentrum DresdenꢀRossendorf, Institute of Radiopharmaceutical Cancer
Research, POB 510119, Dꢀ01314 Dresden, Germany
2
) Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of
Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
3) Technische Universität Dresden, Department of Chemistry and Food Chemistry, Dꢀ01062
Dresden, Germany
Corresponding author: Tel./fax: +86 10 58808891. Eꢀmail address: hmjia@bnu.edu.cn
(
Hongmei Jia).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
We report the syntheses and evaluation of series of novel piperidine compounds with low
lipophilicity as receptor ligands.
ꢀ(4ꢀ(2ꢀFluoroethoxy)benzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (5a) possessed high affinity
(K = 5.4 ± 0.4 nM) for σ receptors and selectivity for σ receptors (30ꢀfold) and the
σ
1
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
1
2
18
vesicular acetylcholine transporter (1404ꢀfold). [ F]5a was prepared using a oneꢀpot,
twoꢀstep labeling procedure in an automated synthesis module, with a radiochemical purity
of > 95%, and a specific activity of 25–45 GBq/µmol. Cellular association, biodistribution
1
8
and autoradiography with blocking experiments indicated specific binding of [ F]5a to σ
1
receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging
using mouse tumor xenograft models demonstrated a high accumulation both in human
carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation
of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity
and appropriate affinity for σ receptors could be used for tumor imaging.
1
Keywords: Carcinoma, melanoma, sigmaꢀ1 receptors, piperidine compounds
ACS Paragon Plus Environment

Page 3 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
1
The sigma (σ) receptor was originally postulated as a class of opioid receptor subtype.
2,3
Currently, two subtypes of σ receptors were confirmed, namely σ
1
and σ
2
.
1
The σ receptor
4
ꢀ6
is a unique 25.3 kDa protein containing 223 amino acids and two transmembrane regions.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
It represents a “receptor chaperone” and has been shown to be involved in the regulation of a
6
ꢀ9
variety of cellular functions. They are linked to CNS diseases, such as depression, drug
8ꢀ15
addiction, Parkinson’s disease (PD), Alzheimer’s disease (AD), tumors and heart failure.
On the other hand, the structure of the σ
2
receptor was not identified so far. However, 21.5
2
kDa was estimated as its molecular weight. Of note, progesterone receptor membrane
16
2
component 1 (PGRMC1) was considered as σ binding site recently.
Both of the σ receptors were reported to be highly expressed not only in various brain
tumors, like glioma and neuroblastoma, but also in a variety of peripheral neoplasia, like
breast cancer, malignant melanoma, colon carcinomas, prostate cancer, renal carcinomas, and
12,13,17ꢀ22
small cell lung carcinoma.
Higher level of σ receptor expression in cancer cells was
1
2
0
found than that in the corresponding nonꢀcancerous cells. Furthermore, the σ
2
receptors are
Thus, the σ
21,22
also highly expressed in rapidly proliferative cells than in quiescent cells.
receptors represent valuable targets for tumor imaging.
Currently,
Nꢀ(4ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)butyl)ꢀ2ꢀ(2ꢀ
18
18
[ F]fluoroethoxy)ꢀ5ꢀmethylbenzamide ([ F]ISOꢀ1, Figure 1) is in clinical trials for in vivo
23,24
imaging of σ
imaging,
2
receptors in tumors.
1
Among the investigated σ receptor radiotracer for PET
11
1ꢀ(3ꢀmethoxyꢀ4ꢀ[ C]methoxyphenethyl)ꢀ4ꢀ(3ꢀphenylpropyl)piperazine
11
18
18
(
[ C]SA4503) and 1ꢀ3ꢀ[ F]fluoropropylꢀ4ꢀ((4ꢀcyanophenoxy)ꢀmethyl)piperidine ([ F]FPS)
2
5,26 11
have been used in human brain study.
[ C]SA4503 was also found to have a high tumor
uptake in C6 tumor bearing rats with tumorꢀtoꢀplasma and tumorꢀtoꢀmuscle ratios of 13.4 and
27
5
.0, respectively at 1 h postinjection. However, this radiotracer seemed to be not suitable as
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a diagnostic tool for visualization of non small cell lung cancer (NSCLC) and its brain
metastases in cancer patients because of the relative high background uptake in lung and
28
18
brain. [ F]FPS exhibited high B16 melanoma tumor uptake (11.14% ID/g) with
tumorꢀtoꢀblood ratio of 123.8 at 4 h postinjection, but the high uptake and tumorꢀtoꢀblood
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ratio were not a result of the σ receptor binding which restricted its application as tumor
2
9
imaging. Until now, there are no successful tumor probes for imaging of σ
1
receptors in the
clinical trial.
In the past decade, series of spirocyclic piperidine compounds have been reported as σ
1
3
0ꢀ39
receptor
ligands.
Among
these
ligands,
1
'ꢀbenzylꢀ3ꢀmethoxyꢀ3Hꢀspiro(2ꢀbenzofuranꢀ1,4'ꢀpiperidine) (1a) possessed nanomolar
affinity for σ receptors and high selectivity against the σ receptors and more than 60 other
receptors, transporters and ion channels. Introduction of the fluorine atom into the carbon
side chain of the spirocyclic benzofuran system with twoꢀcarbon length led to the discovery
1
2
3
0ꢀ32
18
of [ F]fluspidine with the potential for neuroimaging and quantitation of σ receptors in vivo
1
33
(
Figure 1). In our previous work, we made structural modifications on the Nꢀbenzyl moiety
while eliminating the substituent on the spirocyclic benzofuran section (Figure 2). We
introduced an iodine at the paraꢀposition of the benzyl moiety (compound 2) and found that
1
25
radioligand [ I]2 exhibited high initial brain uptake and specific binding to σ receptors in
1
34
vivo. Later, we replaced the iodine atom at the pꢀposition of the Nꢀbenzyl moiety with a
fluoroꢀethoxylated
chain
(compound
3).
The
radioligand
1
8
18
1′ꢀ(4ꢀ(2ꢀ[ F]fluoroethoxy)benzyl)ꢀ3Hꢀspiro(2ꢀbenzofuranꢀ1,4′ꢀpiperidine) ([ F]3) was found
2
to possess subnanomolar affinity and excellent selectivity towards σ receptors and the
35
vesicular acetylcholine transporter (VAChT). It also exhibited high initial brain uptake and
35
specific
binding
to
σ
1
receptors
in
vivo.
Moreover,
1'ꢀbenzylꢀ3ꢀmethoxyꢀ3,4ꢀdihydrospiro(2ꢀbenzopyranꢀ1,4'ꢀpiperidine) (1b) possessed high
ACS Paragon Plus Environment

Page 5 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
0
affinity for σ
1
receptors and high subtype selectivity. Replacement of the benzene moiety
of spirocyclic compound 1a and 1b by pyrazole (4a, 4b) or thiophene residue (4c) preserved
3
6ꢀ39
the affinity and selectivity.
1
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this study, we used [ F]3 as a lead compound to develop a novel radiotracer for imaging
of σ receptors in the tumor. Our design concept is shown in Figure 2. First, we eliminated
1
the benzene ring of the spirocyclic benzofuran moiety and introduced a cyclic acetalic
function (5a–5e). The resulting 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane residue (5a) possessed
much lower lipophilicity than the former spirocyclic benzofuran piperidine moiety and was
thus expected to exhibit fast background washout. We also replaced the 2ꢀfluoroethoxy group
of 5a with halogen (5b–5d) or a methoxy group (5e) to assess better affinity and selectivity.
Moreover,
we
replaced
the
Nꢀ(2ꢀfluoroethoxy)benzyl
group
with
Nꢀ1ꢀ(4ꢀfluorophenyl)propanꢀ1ꢀone (6a) or Nꢀ1ꢀ(4ꢀfluorophenyl)butanꢀ1ꢀone moiety (6b) to
examine effects of distance between the basic nitrogen atom and the benzene ring. Finally, by
keeping the Nꢀ1ꢀ(4ꢀfluorophenyl)propanꢀ1ꢀone or Nꢀ1ꢀ(4ꢀfluorophenyl)butanꢀ1ꢀone moiety
constant, we substituted the 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane moiety with simple piperidine
residue without or with different substituents such as methyl, hydroxyl and hydroxymethyl
(
7a–7h) to verify the importance of the Oꢀheterocyclic residue. Herein we report the
synthesis and evaluation of these compounds as potent σ receptor ligands and their potential
1
applications for imaging σ receptors in the tumor.
1
RESULTS
Chemistry. The synthetic routes of compounds 5a–5e, 6a, 6b and 7a–7h are shown in
Scheme 1 and Scheme 2, respectively. Compound 8 was obtained from the reaction of
pꢀcresol and 1ꢀbromoꢀ2ꢀfluoroethane followed by radical bromination with a total yield of
35
39% according to the reported method in the literature.
Nꢀalkylation of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (9) gave compounds 5a–5e, 6a and 6b with yields of
39–87%. Reactions between compounds 10a–10d with the corresponding bromides provided
compounds 7a–7h with yields of 42–69%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vitro Radioligand Competition Studies. The affinities of the novel ligands 5a–5e, 6a, 6b
and 7a–7h for the σ
1
and σ
2
receptors and for the VAChT were determined with radioligand
35,40
3
competition
assays
as
reported
previously.
(+)ꢀ[ H]pentazocine,
3
3
[
H]ꢀ1,3ꢀdiꢀoꢀtolylꢀguanidine ([ H]DTG) (in the presence of 1 µM dextrallorphan to mask the
3
σ
1
receptors) and (ꢀ)ꢀ[ H]ꢀvesamicol were used for the σ
1
and σ
receptors and VAChT,
2
3
5,40
respectively.
In general, the novel compounds 5a–5e, 6a and 6b with 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane
moiety and compounds 7a–7h with simple piperidine moiety still preferred to bind to σ
receptors. Replacement of 3Hꢀspiro(2ꢀbenzofuranꢀ1,4’ꢀpiperidine) moiety with
The results are provided in Table 1.
1
1
,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane moiety slightly decreased the affinity and selectivity (3 vs
a). But this series of compounds (5a–5e, 6a and 6b) still displayed nanomolar affinity for σ
values of 3.3–11.2 nM and high subtype selectivity towards σ receptors
(σ ) = 16–71). Similarly to the findings for the previously reported spirocyclic
5
1
receptors with K
(K (σ )/K
piperidine derivatives, a small group at the paraꢀposition of the benzyl group with F, Br, I,
OCH , and OCH CH F, still maintained the high affinity and selectivity for the σ receptors
5a–5e). Replacement of 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane moiety with 4ꢀmethylpiperidine
moiety also preserved nanomolar affinity and subtype selectivity (6b vs 7a). The piperidine
group decreased the σ receptor affinity slightly (6b vs 7c). However, less lipophilic groups
i
2
i
2
i
1
3
2
2
1
3
4,35
(
1
such as OH, CH OH at the paraꢀposition of the piperidine group decreased the affinity and
2
selectivity considerably (6b vs 7e, 7g). If the number of carbon atoms between the carbonyl
group attached to 4ꢀfluorophenyl and piperidine moiety was increased from 3 to 4, increased
ACS Paragon Plus Environment

Page 7 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
affinity both for σ
1
and σ
2
receptors was observed (7a vs 7b, 7c vs 7d, 7e vs 7f, 7g vs 7h),
suggesting that the carbon linker length affected the affinity and selectivity of the compounds
for σ receptors.
Considering the high affinity and subtype selectivity of compound 5a for σ
1
receptors as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
8
well as convenient radiosynthesis of the corresponding [ F]5a, compound 5a was further
investigated for its affinity for VAChT. Compound 5a displayed extremely low affinity for
VAChT (K
i 1
= 7.58 ± 0.98 ꢁM) and was thus characterized by a higher selectivity for σ
3
5
receptors (K
i
(VAChT)/K
i
(σ
1
i i 1
) = 1404) compared to compound 5 (K (VAChT)/K (σ ) = 799).
18
Radiochemistry. The synthesis of [ F]5a is presented in Scheme 3. Reductive amination of
compound 9 in the presence of NaBH(OAc)
3 with a yield of 46%. The formation of radiotracer [ F]5a was achieved through a oneꢀpot,
twoꢀstep radiolabeling procedure, which was developed for the radiosynthesis of
3
with 4ꢀhydroxybenzaldehyde led to precursor
18
1
1
8
42
18
Fꢀradiolabeled COXꢀ2 inhibitors. In the first step, 2ꢀ[ F]fluoroethylꢀ1ꢀtosylate was
18
prepared from ethylene 1,2ꢀditosylate by nucleophilic fluorination with [ F]fluoride. Then
1
8
precursor 13 in DMF was added to the reaction vial. Fꢀfluroethylation of compound 13
provided the desired radiotracer. After purification via semiꢀpreparative high performance
1
8
liquid chromatography (HPLC), [ F]5a was obtained in about 15% radiochemical yield (n =
1
2, decayꢀcorrected) with a radiochemical purity (RCP) of > 95% and a specific activity (A
S
)
of 25–45 GBq/µmol. The total synthesis time was 100 min.
Evaluation of the Radiolabeled Compound.
18
Lipophilicity. The apparent distribution coefficient of the Fꢀlabeled radiotracer was
35,40
determined using a shakeꢀflask method as previously reported.
The log D7.4 value of
18
[
1
F]5a was 0.81 ± 0.13, indicating a low lipophilicity of this series of σ receptor ligands
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane moiety.
18
In Vitro Stability. The in vitro stability of [ F]5a in ethanol, saline, or human plasma was
evaluated by measuring the RCP at different time points. After 2 h of incubation with ethanol,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
saline, or human plasma, the RCP of [ F]5a was more than 92% as shown in the Supporting
Information.
Cell Uptake Experiments. Four different human tumor cell lines, comprising carcinoma
(PC3, DU145, MCF7) and melanoma (A375), which show different level of expression of σ1
1
8
2
or/and σ receptors were used to study the cellular association of compound [ F]5a in vitro
(Figure 3). An increase in the radiotracer association was observed with longer incubation
1
9
time. Especially in DU145 cells with high density of σ
1
receptors, substantial cellular
1
8
association of [ F]5a (18.55% ID/mg protein) was found at 120 min. The cellular association
18
of [ F]5a was about 9.73% and 8.08% ID/mg protein in A375 and PC3 cell lines,
respectively. The lowest cellular association (5.48% ID/mg protein at 120 min) was observed
17
in MCF7 cell lines, which was in good agreement with their low expression of σ receptors.
1
Blocking studies were performed with haloperidol, SA4503 and fluspidine as blocking agents.
After incubation with 10 ꢁM of haloperidol, SA4503 or fluspidine, the cellular association
was significantly reduced in the above four tumor cell lines. Preꢀincubation with haloperidol
and SA4503 led to marked reduction of 85% and 88% in DU145 cell lines (2.85 and 2.26%
vs 18.55% ID/mg protein) at 120 min. Preꢀincubation with haloperidol, SA4503 and
fluspidine also resulted in a significant blocking of cellular association in A375 and PC3 cell
lines (2.81–4.99% ID/mg protein). Moreover, a further reduction was observed when
preꢀincubated with haloperidol and SA4503 (3.28 and 2.85% ID/mg protein, respectively) in
18
MCF7 cell lines. These data demonstrated specific binding of [ F]5a to σ receptors in
1
ACS Paragon Plus Environment

Page 9 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
carcinoma (PC3, DU145, MCF7) and melanoma (A375) cell lines.
18
Biodistribution Studies in Rats. To evaluate the kinetics of [ F]5a, biodistribution studies
were performed in male Wistar rats without (control) and with simultaneous injection of
haloperidol (1 mg/kg body weight) as blocking agent. The results are summarized in Figure 4
and in the Supporting Information. The standardized uptake value (SUV) and percentage of
injected dose (%ID) are applied to evaluate the accumulation of radiotracer in rat organs and
tissues.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
In controls, [ F]5a displayed high initial brain uptake (3.67 %ID and SUV, 2.89) with high
brainꢀtoꢀblood ratio of 7.81 at 5 min postinjection. Among peripheral organs, the pancreas
exhibited the highest uptake (SUV, 6.33 ± 1.15), followed by the liver, adrenals, kidneys,
spleen, and lung, which is in accordance with the σ
1
receptor densities in these organs. In
1
receptors displayed a relatively fast
addition, the above organs with high expression of σ
clearance rate of 31–63% after 60 min.
1
8
To detect the specific binding of [ F]5a to σ receptors in vivo, the effects of simultaneous
injection of haloperidol on the biodistribution of the radiotracer in various organs in male
Wistar rats were examined. A remarkable reduction of the radiotracer accumulation (55%)
was observed in the brain at 5 min postinjection. Moreover, radiotracer accumulation in the
1
pancreas, lungs and spleen, which are known to possess high expression of σ receptors, was
significantly reduced by 48%, 51% and 52%, respectively. At 60 min, the uptake in brain,
pancreas, spleen and liver was decreased by 25–64%. These results indicated the specific
1
8
binding of [ F]5a to σ receptors in vivo.
18
Imaging of Rat Brains. To further confirm the specific binding of [ F]5a to σ receptors in
vivo, microꢀPET/MRI and autoradiography studies were performed in adult male Wistar rats.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The binding specificity was evaluated in a blocking study with simultaneous injection of
haloperidol (1 mg/kg body weight). Representative brain images (projections and sections) of
the radiotracer at 5 and 60 min postinjection and muscle sections of identical animals for
18
comparison are presented in Figure 5. [ F]5a crossed the bloodꢀbrain barrier (BBB) rapidly
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and showed high accumulation in the brain regions such as cortex, cerebellum, and thalamus
1
known to express high density of σ receptors at 5 min postinjection. When animals were
simultaneously injected with haloperidol, accumulation of the radiotracer decreased in the
above brain regions, consistent with the results of the biodistribution studies.
Pꢀglycoprotein (Pꢀgp) is highly expressed in the blood−brain barrier and many tumors. To
18
test whether [ F]5a is a substrate for Pꢀgp in vivo, dynamic PET brain imaging with
cyclosporine A, an inhibitor of Pꢀgp, was carried out in Wistar rats (n = 2). Either saline or
cyclosporine A (5 mg/kg body weight) was injected 10 min prior to the radiotracer injection.
The results are summarized in Figure 6. Administration of cyclosporine A did not change the
bioavailability of the radiotracer as represented by the area under the curve values (AUCs).
The small difference between AUCs in control and that in blocked animals was not
18
significant, suggesting that [ F]5a is not a substrate for Pꢀgp.
Small Animal PET Imaging in Mouse Tumor Xenograft Models. To further evaluate the
18
potential applications of [ F]5a for imaging of σ receptors in tumors, small animal dynamic
1
PET studies were performed in four different xenograft models. The results are presented in
Figure 7. Among the investigated models, the highest accumulation of the radiotracer was
observed in the PC3 tumors (human prostate cancer, n = 3), followed by A431 (human
squamous cell carcinoma, n = 5), A375 (human melanoma, n = 2), and DU145 (human
prostate cancer, n = 4) tumors. Administration of haloperidol (1 mg/kg body weight)
significantly reduced the accumulation of the radiotracer in PC3 (n = 2) or A431 (n = 5)
ACS Paragon Plus Environment

Page 11 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
8
tumors (p < 0.05), indicating specific binding of [ F]5a to σ receptors in these tumors in
vivo.
18
Representative PET imaging with [ F]5a in NMRI nu/nu mice bearing four different
tumors is presented in Figure 8. Perfusion background and radiotracer accumulation in an
A431 tumor bearing NMRI nu/nu mouse are depicted in images A (midframe time 3 min
postinjection) and B (midframe time 50 min postinjection), respectively. The signal intensity
in image A subtracted that in image B resulted in difference image C clearly illustrating
tumor accumulation with high tumorꢀtoꢀbackground ratio. Substantial tumor accumulation of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
[ F]5a and high tumorꢀtoꢀbackground ratio were also observed in (D) A375, (E) PC3, and (F)
DU145 tumor bearing NMRI nu/nu mice. Additionally, PET imaging in mice showed
substantial accumulation of radiotracer in intestine and bladder indicating the major
elimination routes (Figure 8). A partial contribution of radiotracer accumulation in pancreas
to the overall radiotracer accumulation in the abdominal region should be considered and
would be consistent with biodistribution data obtained in rats. However, this cannot be
differentiated in detail using the imaging approach used in this study.
1
8
In Vivo Metabolic Stability. Metabolic stability of [ F]5a was investigated in arterial blood
plasma, urine, and brain samples from Wistar rats. The HPLC chromatograms of the
radioactive compounds in arterial blood plasma samples at 0, 5, and 60 min postinjection and
in a urine sample at 60 min postinjection are presented in the Supporting Information.
1
8
Metabolite fraction of [ F]5a in arterial blood plasma is shown in the Supporting
Information. The HPLC chromatograms of the radiotracer in brain and blood plasma samples
at 5 min postinjection without and with haloperidol blocking is provided in the Supporting
Information. In arterial blood plasma samples, three less lipophilic metabolites, M1, M2, and
M3 were observed at retention times of 4.3, 10.3 and 10.9 min, respectively. At 60 min, the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
polar metabolite M1 was found to be the main metabolite. The biological halfꢀlife of [ F]5a
in arterial blood plasma was 4.1 min. In control brain samples, the intact parent tracer
accounted for about 95% of the total activity, and only very small amounts of the metabolite
M1 was observed. Treatment of haloperidol resulted in an increment of metabolite M1 in the
brain.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DISCUSSION
Experimental and clinical evidence suggest that both σ
1
and σ
2
receptors could play a
tumor
1
8
significant role in cancer biology. Until now, [ F]ISOꢀ1 is the most successful σ
2
23,24
probe.
However, there is no successful tumor probe for imaging of σ
1
receptors in clinical
1
1
18
trial. The high background uptake of [ C]SA4503 and the nonꢀspecific binding of [ F]FPS
12,28,29
in B16 tumor restricted their applications in tumor detection.
If a σ receptor probe is
1
more hydrophilic, its background uptake is expected to be lower and thus advantageously for
tumor imaging. Based on these prerequisites, our aim was to develop a tumor radiotracer with
suitable lipophilicity and appropriate affinity for σ receptors.
1
In our previous work, we found that compound 3 possessed subnanomolar affinity and
18
high selectivity for σ
1
receptors. Moreover, [ F]3 showed very high brain uptake and specific
35
binding to σ
1
receptors in vivo. For this series of spirocyclic piperidine derivatives, Wünsch
considered the benzene ring of the Oꢀheterocycle as the “primary hydrophobic region” and
the phenyl group of the Nꢀsubstituent as the “secondary hydrophobic region” of the σ ligands
1
3
9
by Glennon’s pharmacophore model. Recently, several σ
receptor residues have been
1
identified to play a central role in binding of different series of structurally unrelated
43
compounds. In order to design compounds with lower liphophilicity and nanomolar affinity,
we replaced the spirocyclic piperidine moiety in compound 3 with a more hydrophilic group
1
,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane and simple piperidine. In vitro binding assays showed that
ACS Paragon Plus Environment

Page 13 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5a–5e, 6a and 6b with 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane and 7a, 7b with simple piperidine
still exhibited nanomolar affinity and subtype selectivity for σ receptors. Moreover,
1
compound 5a also exhibits 1404ꢀfold less affinity for VAChT. That is to say, the lack of the
benzene moiety connected to the spirocyclic piperidine residue could preserve the nanomolar
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
affinity for σ receptors while it could decrease the affinity for VAChT. Some other less
1
lipophilic and smaller moieties, like methyl may serve as “primary hydrophobic region” in
the piperidine ligands. And thus the “primary hydrophobic region” in the Glennon’s
pharmacophore model appears more flexible.
11
Considering nanomolar affinity and comparable selectivity to that of [ C]SA4503 as well
18
as the convenient synthesis of the corresponding radiotracer, [ F]5a was prepared to detect
the kinetic profiles in vivo. For brain imaging probes, moderate lipophilicity (log D = 1–3) of
4
4
the tracers is considered to be favorable for penetrating the BBB. But for tumor imaging
agent, suitable lipophilicity needs to be optimized in order to find the potential radiotracer
with high tumor uptake and low background accumulation. Consistent with our prediction,
1
8
11
the lipophilicity of [ F]5a (log D = 0.81) is much lower than that of [ C]SA4503 (log D
7.4
7
.4
4
5
18
46 18
33
18
= 2.52), [ F]FPS (log D7.5 = 2.80), [ F]fluspidine (log D7.2 = 2.57), and [ F]3 (log D7.4
3
5
18
=
2.41). Moreover, [ F]5a displayed high in vitro stability in ethanol, saline and human
plasma at room temperature (r. t.) for 2 h. These in vitro properties encouraged us to perform
18
the in vivo evaluation of [ F]5a as tumor imaging agent.
18
In biodistribution studies in rats, [ F]5a (SUV, 2.89) showed higher brain uptake than that
11
of [ C]SA4503 (SUV, 1.50 in cortex and SUV, 1.66 in cerebellum at 60 min postinjection,
47ꢀ49
SUV, 1.55 in cortex and SUV, 1.21 in cerebellum at 90 min postinjection) in rats.
In
addition, this radiotracer exhibited low initial blood uptake and thus high brainꢀtoꢀblood ratio.
Simultaneous administration of haloperidol resulted in a significantly decreased accumulation
1
in organs and tissues with high expression of σ receptors, including brain, liver, lung, kidney,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
and pancreas, indicating specific binding of [ F]5a to σ receptors in vivo.
In autoradiography studies in rats, high accumulation of the radiotracer was observed in
brain areas with high expression of σ
1
receptors, such as cortex, cerebellum, and thalamus at
1
8
5
min postinjection. Treatment with haloperidol also reduced the accumulation of [ F]5a in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
these regions. The results of rat brain PET images in control and blocking animals were in
good agreement with those of the autoradiography. Cerebellum, thalamus, and cortex
containing high density of σ receptors showed high accumulation. Blocking with haloperidol
1
and SA4503 significantly decreased the uptake, especially in these regions. Considering the
low affinity of 5a for VAChT and the high selectivity of SA4503 towards 36 other receptors,
50
ion channels and components of second messenger systems, we conclude that the results of
1
8
1
the blocking study suggest specific binding of [ F]5a to σ receptors in the rat brain in vivo.
It is well known that Pꢀgp is highly expressed at the bloodꢀbrain barrier and in tumors. The
identification of a radiotracer as a substrate of Pꢀgp is very important in the development of
5
1
18
brain or tumor imaging agent. Coꢀinjection of cyclosporine A with [ F]5a did not increase
the brain uptake significantly (p > 0.05) in the PET studies indicating that the radiotracer is
18
not a Pꢀgp substrate. This finding may explain the high initial brain uptake of [ F]5a despite
low lipophilicity and encourage us to further evaluate this radiotracer as tumor imaging
agent.
1
8
In order to confirm the specific binding [ F]5a to σ receptors in tumor cell lines, the
1
18
cellular association of [ F]5a was measured in four kinds of cell lines with different levels of
σ
1
receptor expression. Among the four cell lines, DU145 cells were reported to possess high
expression of σ and σ receptors with Bmax values of 1,800 and 1,930 fmol/mg protein,
respectively. High σ receptor expression in PC3 cell line investigated with real time PCR
was also reported. A375 cells exhibited relatively low expression of σ receptors but high
expression of σ receptors (with Bmax values 34 and 3,403 fmol/mg protein toward σ and σ
1
2
1
9
1
1
2
1
2
ACS Paragon Plus Environment

Page 15 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
7,20
receptors, respectively).
1
Even lower σ receptor expression was reported in MCF7
17,20
cells.
Consistent with the expression levels of σ receptors in these cells, highest cellular
1
18
association of [ F]5a was observed in DU145 cells, followed by A375, PC3 and MCF7 cells.
4
1,48,52
Treatment with SA4503 (σ
1
agonist with nanomolar affinity and high selectivity),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
3,54
33
haloperidol (a potent σ and σ
1
2
antagonist)
1
or fluspidine (a potent σ probe) reduced the
18
cellular association of [ F]5a significantly. These results demonstrated the specific binding
18
of [ F]5a to σ receptors in these cell lines in vitro.
1
To further determine the specific binding in tumors in vivo, small animal dynamic PET
studies were investigated in four different xenograft models in mice. Significant radiotracer
accumulation in the tumors of the radiotracer was observed in all models. Different from
what was found in the cellular association studies, the highest tumor uptake was obtained in
the PC3 tumor, followed by in the A431, A375 and DU145 tumor. Such discrepancy may be,
in part, due to differences between the used carcinomas and melanoma in tumor growth rate,
vascularization, perfusion and cellular microenvironment. However, the uptake in these
11
tumor bearing mice was higher than those of [ C]SA4503 (SUV, 0.65 ± 0.15) and
18
27
[
F]FEꢀSA5845 (SUV, 0.92 ± 0.39) in the C6 tumor. Treatment with haloperidol
significantly reduced the accumulation in the PC3 and A431 tumors, which is similar to the
11
27
uptake of [ C]SA4503 in the C6 tumor (SUV, 0.43 ± 0.15). These data indicate specific
18
binding of [ F]5a to σ receptors in the tumors in vivo. Moreover, PET revealed considerable
1
tumor uptake and a significant blocking effect of haloperidol in A431 tumor bearing NMRI
18
nu/nu mice. These results further demonstrate the promising features of [ F]5a as tumor
imaging agent.
CONCLUSION
Series of compounds with more hydrophilic group 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
simple piperidine moieties were synthesized and found to possess nanomolar affinity,
subtype selectivity for σ
primary hydrophobic region” in the Glennon’s pharmacophore model provides the chance to
develop σ receptor radiotracers with low lipophilicity for tumor imaging. The novel
1
receptors and high selectivity for VAChT. The more flexible
“
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
8
radiotracer [ F]5a not only possessed the nanomolar affinity and selectivity for σ
but also showed specific binding to σ receptors in the normal organs known to express high
level of σ receptors, tumor cell lines, and tumors in vivo. The clear visualization of the tumor
in PET images with this radiotracer indicates a progress in the development of σ receptor
radiotracer with appropriate affinity and suitable lipophilicity as tumor imaging agent.
1
receptors,
1
1
1
18
1
Further evaluation of [ F]5a and the development of improved σ receptor tumor radiotracers
are planned.
EXPERIMENTAL SECTION
General Methods and Materials. All the chemicals or reagents were purchased from
1
13
19
commercial suppliers and used without further purification. H NMR, C NMR, and
F
NMR spectra were recorded on a Varian Inovaꢀ400 spectrometer at 400, 101, and 376 MHz,
1
13
respectively. Chemical shifts are reported in ppm with tetramethylsilane ( H, C) and
19
trichlorofluoromethane ( F) as internal standards, respectively and coupling constants (J) are
reported in Hertz (Hz). MS spectra were obtained by Quattro micro API ESI/MS (Waters,
USA) and Quattro micro WATERS Xevo TQꢀS. High resolution mass spectrometry (HRMS)
was performed on a JEOL NMSꢀSX102 spectrometer (JEOL). HPLC analyses were
performed using a Luna C18 column (250 × 4 mm, 5 µm) (Phenomenex) using an isocratic
eluent of acetonitrile/Na
2
HPO
4
(0.05 M) = 50/50 by a gradient pump L2500 (Merck, Hitachi)
CN/NH OAc (0.01 M) by
and Agela Venusil MP C18 (5 ꢁm, 4.6 × 250 mm) using CH
3
4
Shimadzu SCLꢀ20 AVPHPLC system with a flow rate of 1 mL/min. The products were
ACS Paragon Plus Environment

Page 17 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
monitored by a UV detector L4500 (Merck, Hitachi) and SPDꢀM20AUVꢀVIS detector at 254
nm and by γꢀdetection with a scintillation detector Gabi (Raytest). The purities of the final
products were determined by the HPLC method, all the final compounds were higher than
95% and the purities are shown in the Supporting Information. Noꢀcarrierꢀadded aqueous
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
[ F]fluoride was produced using a CYCLONE 18/9 cyclotron (IBA) by irradiation of
18
18
18
18
[
2
O]H O via the O(p,n) F nuclear reaction. Synthesis of [ F]5a was performed in an
automated nucleophilic synthesizer TracerLABFXN (GE). Semiꢀpreparative purification was
performed with a Nucleosilꢀ100 C18 column (250 × 21 mm, 7 µm) (MachereyꢀNagel) using
2 4
an isocratic eluent of acetonitrile/Na HPO (0.05 M) = 60/40 by a S1122 HPLC pump
(Sykam) with a flow rate of 5 mL/min. The product was monitored by a Kꢀ2001 filer
photometer (Knauer) at 254 nm and by a γꢀdetector integrated in the synthesis module.
Animal experiments were carried out according to the guidelines of the German
Regulations for Animal Welfare. The protocol was approved by the local Ethical Committee
for Animal Experiments.
Chemistry.
General Procedure for the Syntheses of 5a–5e, 6a, 6b and 7a–7h. All of these products
were synthesized under the similar conditions. 1.0 Equiv of the corresponding bromide
compound, 1.2 equiv of 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane or corresponding piperidine, K CO ,
2
3
3
and NaI in catalytic amounts were added into the CH CN, the mixture was refluxed for 4 h.
After cooling and filtration, the solvent was removed under reduced pressure. Finally, the
crude product was purified by column chromatography (silica gel, petroleum ether:ethyl
acetate = 1:2 or 1:1 (v/v)) to afford the final products.
8ꢀ(4ꢀ(2ꢀFluoroethoxy)benzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (5a). Compound 8 (306 mg,
1.31 mmol), compound 9 (225 mg, 1.57 mmol), K CO (217 mg, 1.57 mmol), and NaI in
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
catalytic amounts were added into CH
3
CN (30 mL) and afforded 5a (208 mg, 54%) as
1
lightꢀyellow oil. H NMR (400 MHz, CDCl ) δ 7.20 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz,
3
2H), 4.72 (dt, J = 47.4, 4.2 Hz, 2H), 4.16 (dt, J = 27.8, 4.2 Hz, 2H), 3.91 (s, 4H), 3.43 (s, 2H),
1
3
2
3
.47 (s, 4H), 1.70 (t, J = 5.6 Hz, 4H). C NMR (101 MHz, CDCl ) δ 157.7, 131.5, 130.5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
114.5, 107.5, 82.2, 67.3, 64.4, 62.2, 51.4, 35.0. F NMR (376 MHz, CDCl
3
) δ –224.3. MS
+
+
(
ESI+) m/z: 295.92 [M + H] . HRMS (EI) m/z: calcd for C16
H22FNO
3
[M + H] 296.1662,
found 296.1653.
8ꢀ(4ꢀFluorobenzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane
(5b).
1ꢀ(Bromomethyl)ꢀ4ꢀfluorobenzene (436 mg, 2.31 mmol), compound 9 (395 mg, 2.76 mmol),
K
2
CO
3
3
(319 mg, 2.31 mmol), and NaI in catalytic amounts were added into CH CN (25 mL)
1
and afforded 5b (344 mg, 59%) as yellow oil. H NMR(400 MHz, CDCl
3
) δ 7.33–7.22 (m,
13
2H), 6.93–7.00 (m, 2H), 3.92 (s, 4H), 3.46 (s, 2H), 2.49 (s, 4H), 1.71 (t, J = 5.4 Hz, 4H). C
19
NMR (101 MHz, CDCl
3
) δ 161.9, 134.3, 130.4, 114.9, 107.2, 64.2, 61.8, 51.2, 34.8.
F
+
NMR (376 MHz, CDCl ) δ –224.4. MS (ESI+) m/z: 251.95 [M + H] . HRMS (EI) m/z: calcd
3
+
for C14
H
18FNO
2
[M + H] 252.1400, found 252.1409.
8
ꢀ(4ꢀIodobenzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (5c). 1ꢀ(Bromomethyl)ꢀ4ꢀiodobenzene
520 mg, 1.76 mmol), compound 9 (301 mg, 2.10 mmol), K CO (150 mg, 2.10 mmol), and
CN (35 mL) and afforded 5c (392 mg, 62%) as
(
2
3
NaI in catalytic amounts were added into CH
3
1
lightꢀyellow oil. H NMR (400 MHz, CDCl ) δ 7.60 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.0 Hz,
3
13
2H), 3.91 (s, 4H), 3.43 (s, 2H), 2.48 (s, 4H), 1.71 (t, J = 5.5 Hz, 4H). C NMR(101 MHz,
CDCl
3
) δ 138.4, 137.2, 130.9, 107.1, 92.3, 64.2, 62.0, 51.2, 34.8. MS (ESI+) m/z: 359.77 [M
+
+
+
H] . HRMS (EI)m/z: calcd for C14
H18INO
2
[M + H] 360.0461, found 360.0455.
8ꢀ(4ꢀBromobenzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane
(5d).
1ꢀBromoꢀ4ꢀ(bromomethyl)benzene (400 mg, 1.60 mmol), compound 9 (275 mg, 1.92 mmol),
K
2
CO
3
3
(265 mg, 1.92 mmol), and NaI in catalytic amounts were added into CH CN (25 mL)
ACS Paragon Plus Environment

Page 19 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
and afforded 5d (437 mg, 87%) as lightꢀyellow oil. H NMR (400 MHz, CDCl
3
) δ 7.37 (d, J
=
8.3 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 3.88 (s, 4H), 3.41 (s, 2H), 2.45 (s, 4H), 1.68 (t, J =
13
5
3
.6 Hz, 4H). C NMR(101 MHz, CDCl ) δ 132.8, 126.5, 125.9, 116.0, 102.4, 59.4, 57.1,
81
79
+
4
6.4, 30.0. MS (ESI+) m/z: 314.08 [M + H, Br], 312.02 [M + H, Br] . HRMS (EI) m/z:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
calcd for C14
H
18BrNO
2
[M + H] 312.0599, found 312.0614.
8
ꢀ(4ꢀMethoxybenzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane
(5e).
1
ꢀ(Bromomethyl)ꢀ4ꢀmethoxybenzene (760 mg, 3.78 mmol), compound 9 (649 mg, 4.54
mmol), K
2
CO
3
3
(626 mg, 4.54 mmol), and NaI in catalytic amounts were added into CH CN
1
(
45 mL) and afforded 5e (708 mg, 71%) as yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.20 (d,
J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 3.91 (s, 4H), 3.77 (s, 3H), 3.44 (s, 2H), 2.48 (s, 4H),
1
3
1
.71 (t, J = 5.6 Hz, 4H). C NMR(101 MHz, CDCl
3
) δ 158.7, 130.2, 113.5, 110.0, 107.3,
+
6
4.2, 62.0, 55.2, 51.1, 34.7. MS (ESI+) m/z: 264.09 [M + H] . HRMS (EI) m/z: calcd for
+
C
15
H
21NO
3
[M + H] 264.1600, found 264.1607.
1ꢀ(4ꢀFluorophenyl)ꢀ3ꢀ(1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decanꢀ8ꢀyl)propanꢀ1ꢀone
(6a).
3
ꢀBromoꢀ1ꢀ(4ꢀfluorophenyl)propanꢀ1ꢀone (620 mg, 2.68 mmol), compound 9 (460 mg, 3.22
mmol), K
2
CO
3
(444 mg, 3.22 mmol), and NaI in catalytic amounts were added into CH
3
CN
1
(
25 mL) and afforded 6a (303 mg, 39%) as lightꢀyellow oil. H NMR (400 MHz, CDCl
3
) δ
7.96–7.86 (m, 2H), 7.11–7.01 (m, 2H), 3.88 (s, 4H), 3.09 (t, J = 7.4 Hz, 2H), 2.79 (t, J = 7.4
1
3
Hz, 2H), 2.53 (t, J = 5.0 Hz, 4H), 1.68 (t, J = 5.6 Hz, 4H). C NMR(101 MHz, CDCl ) δ
3
19
1
97.6, 165.7, 133.4, 130.6, 115.6, 107.0, 64.2, 52.7, 51.4, 36.5, 34.8. F NMR (376 MHz,
+
CDCl
3
) δ –106.4. MS (ESI+) m/z: 293.95 [M + H] . HRMS (EI)m/z:calcd for C16
H
20FNO
3
+
[
M + H] 294.1505, found 294.1510.
1ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ(1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decanꢀ8ꢀyl)butanꢀ1ꢀone
(6b).
4
ꢀBromoꢀ1ꢀ(4ꢀfluorophenyl)butanꢀ1ꢀone (300 mg, 1.22 mmol), compound 9 (210 mg, 1.47
mmol), K
2
CO
3
3
(202 mg, 1.47 mmol) , and NaI in catalytic amounts were added into CH CN
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
(35 mL) and afforded 6b (168 mg, 45%) as colorless solid. H NMR (400 MHz, CDCl
3
) δ
7
.98–7.90 (m 2H), 7.12–7.00 (m, 2H), 3.87 (s, 4H), 2.92 (t, J = 7.1 Hz, 2H), 2.49 (s, 4H),
13
2
.40 (t, J = 7.1 Hz, 2H), 1.95–1.84 (m, 2H), 1.65 (t, J = 5.6 Hz, 4H). C NMR(101 MHz,
1
9
CDCl
3
) δ 193.6, 160.8, 128.8, 125.9, 110.8, 102.3, 59.4, 52.4, 46.4, 31.4, 29.8, 17.1.
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
NMR (376 MHz, CDCl
3
) δ –106.2. MS (ESI+) m/z: 308.21 [M + H] . HRMS (EI) m/z: calcd
+
for C17
H
22FNO
3
[M + H] 308.1662, found 308.1654.
1
4
0
ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ(4ꢀmethylpiperidinꢀ1ꢀyl)butanꢀ1ꢀone
(7a).
Compound
11,
ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)butanꢀ1ꢀone (160 mg, 0.65 mmol), 4ꢀmethylpiperidine (83 mg,
.84 mmol), K
2
CO
3
(116 mg, 0.84 mmol) and NaI in catalytic amounts were added into
1
CH
3
CN (25 mL) afforded 7a (118 mg, 69%). H NMR (400 MHz, CDCl
3
) δ 8.04–7.93 (m,
2
2
3
H), 7.11 (t, J = 8.5 Hz, 2H), 3.05–2.98 (m, 2H), 2.54 (t, J = 7.4 Hz, 4H), 2.11 (t, J = 11.1 Hz,
H), 2.07–1.94 (m, 2H), 1.64 (d, J = 11.0 Hz, 2H), 1.46–1.26 (m, 3H), 0.92 (d, J = 5.7 Hz,
1
3
3
H). C NMR (101 MHz, CDCl ) δ 198.1, 165.6, 133.3, 130.6, 115.6, 57.6, 53.5, 36.1, 33.2,
19
+
+
30.3, 21.5, 20.9. F NMR (376 MHz, CDCl ) δ –106.52. MS (ESI ): m/z 264.06 [M+H] .
3
+
HRMS (EI)m/z:calcd for C16
H
23FNO [M + H] 264.1764, found 264.1773.
1
ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ(4ꢀmethylpiperidinꢀ1ꢀyl)pentanꢀ1ꢀone
(7b).
Compound
12,
5
ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)pentanꢀ1ꢀone (300 mg, 1.15 mmol), 4ꢀmethylpiperidine (146 mg,
1.47 mmol), K
2
CO
3
3
(202 mg, 1.47 mmol) and catalytic amount NaI were added into CH CN
1
(40 mL) and afforded 7b (278 mg, 63%). H NMR (400 MHz, CDCl ) δ 7.99–7.94 (m, 2H),
3
7
.10 (t, J = 8.4 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H), 2.88 (d, J = 11.5 Hz, 2H), 2.35 (t, J = 11.0
Hz, 2H), 1.90 (t, J = 11.2 Hz, 2H), 1.78–1.68 (m, 2H), 1.63–1.54 (m, 4H), 1.39–1.18 (m, 3H),
13
0
.89 (d, J = 6.2 Hz, 3H). C NMR (101 MHz, CDCl
3
) δ 198.5, 165.6, 133.3, 130.6, 115.5,
19
58.6, 54.0, 38.3, 34.2, 30.7, 26.5, 22.4, 21.8. F NMR (376 MHz, CDCl ) δ –105.72. MS
3
+
+
+
(
ESI ): m/z 278.21 [M+H] . HRMS (EI)m/z:calcd for C17
H25FNO [M + H] 278.1920, found
278.1928.
ACS Paragon Plus Environment

Page 21 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ(piperidinꢀ1ꢀyl)butanꢀ1ꢀone (7c). Compound 11 (426 mg, 1.73
mmol), piperidine (188 mg, 2.21 mmol), K CO (305 mg, 2.21 mmol) and NaI in catalytic
2
3
1
amounts were added into CH
MHz, CDCl ) δ 7.96–7.83 (m, 2H), 6.99 (t, J = 8.5 Hz, 2H), 2.86 (t, J = 7.0 Hz, 2H), 2.32 (t,
J = 6.9 Hz, 6H), 1.94–1.78 (m, 2H), 1.53–1.41 (m, 4H), 1.37–1.23 (m, 2H). C NMR (101
3
CN (40 mL) and afforded 7c (296 mg, 69%). H NMR (400
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
19
3
MHz, CDCl ) δ 193.4, 160.7, 128.6, 125.8, 110.7, 53.3, 49.4, 31.4, 20.6, 19.3, 16.4. F NMR
+
+
(
376 MHz, CDCl ) δ –106.81. MS (ESI ): m/z 249.88 [M+H] . HRMS (EI)m/z:calcd for
3
+
C
15
H21FNO [M + H] 250.1607, found 250.1606.
1ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ(piperidinꢀ1ꢀyl)pentanꢀ1ꢀone (7d). Compound 12 (200 mg, 0.77
mmol)，piperidine (140 mg, 0.98 mmol), K CO (135 mg, 0.95 mmol) and NaI in catalytic
2
3
1
amounts were added into CH
MHz, CDCl ) δ 7.98–7.89 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H),
2.38–2.23 (m, 6H), 1.78–1.64 (m, 2H), 1.60–1.48 (m, 6H), 1.40–1.34 (m, J = 2.2 Hz, 2H).
3
CN (20 mL) and afforded 7d (85 mg, 42%). H NMR (400
3
1
3
3
C NMR (101 MHz, CDCl ) δ 193.8, 160.8, 128.6, 125.9, 110.9, 54.3, 49.8, 33.6, 21.7, 21.2,
19
+
+
1
9.7, 17.7. F NMR (376 MHz, CDCl ) δ –106.74. MS (ESI ): m/z 265.05 [M+H] . HRMS
3
+
(EI)m/z:calcd for C16
H
23FNO [M + H] 264.1764, found 264.1766.
1ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ(4ꢀhydroxypiperidinꢀ1ꢀyl)butanꢀ1ꢀone (7e). Compound 11 (384
mg, 1.57 mmol), 4ꢀhydroxypiperidine (190 mg, 1.88 mmol), K CO (259 mg, 0.74 mmol)
CN (50 mL) and afforded 7e (275 mg,
2
3
and NaI in catalytic amounts were added into CH
3
1
6
6%). H NMR (400 MHz, CDCl
3
) δ 8.04–7.95 (m, 2H), 7.13 (t, J = 8.7 Hz, 2H), 3.85 (s,
1H), 3.05 (t, J = 6.9 Hz, 2H), 2.99–2.85 (m, 2H), 2.63 (t, J = 7.3 Hz, 2H), 2.52 (s, 2H),
1
3
2.17–2.01 (m, 4H), 1.76 – 1.56 (m, 2H). C NMR (101 MHz, CDCl ) δ 197.9, 165.7, 133.2,
3
19
1
30.7, 115.7, 66.8, 57.2, 50.1, 36.0, 32.9, 20.6. F NMR (376 MHz, CDCl
3
) δ –106.21. MS
+
+
(
ESI ): m/z 266.16 [M+H] .
1ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ(4ꢀhydroxypiperidinꢀ1ꢀyl)pentanꢀ1ꢀone (7f). Compound 12 (195
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mg, 0.75 mmol), 4ꢀhydroxypiperidine (91 mg, 0.90 mmol), K
2
CO
3
(124 mg, 0.90 mmol) and
1
NaI in catalytic amounts were added into CH CN (25 mL) and afforded 7f (107 mg, 52%). H
3
3
NMR (400 MHz, CDCl ) δ 8.01–7.94 (m, 2H), 7.16–7.07 (m, 2H), 3.80–3.71 (m, 1H), 2.97 (t,
J = 7.1 Hz, 2H), 2.89–2.81 (m, 2H), 2.46 (t, J = 7.6 Hz, 2H), 2.33–2.24 (m, 2H), 2.02–1.93
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
(m, 2H), 1.79–1.57 (m, 6H). C NMR (101 MHz, CDCl
3
) δ 198.5, 165.6, 133.3, 130.61,
19
1
15.6, 67.0, 58.0, 50.7, 38.1, 33.7, 26.2, 22.1. F NMR (376 MHz, CDCl ) δ –106.48. MS
3
+
+
(
ESI ): m/z 280.15 [M+H] .
1
ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ(4ꢀ(hydroxymethyl)piperidinꢀ1ꢀyl)butanꢀ1ꢀone (7g). Compound 11
CO (208 mg, 1.51
CN (35 mL) and afforded 7g (217
(309 mg, 1.26 mmol), 4ꢀpiperidinemethanol (174 mg, 1.51 mmol), K
2
3
mmol) and NaI in catalytic amounts were added into CH
3
1
mg, 62%). H NMR (400 MHz, CDCl
3
) δ 8.07–7.91 (m, 2H), 7.16–7.04 (m, 2H), 3.47 (d, J =
6.4 Hz, 2H), 3.09–2.92 (m, 4H), 2.47 (t, J = 7.3 Hz, 2H), 2.11–1.94 (m, 4H), 1.73 (d, J = 13.0
13
Hz, 2H), 1.59–1.46 (m, 1H), 1.37–1.21 (m, 2H). C NMR (101 MHz, CDCl
3
) δ 198.3, 165.6,
1
9
133.4, 130.6, 115.6, 67.6, 57.8, 53.3, 38.3, 36.2, 28.3, 21.3. F NMR (376 MHz, CDCl ) δ
3
+
+
–
106.61. MS (ESI ): m/z 280.18 [M+H] .
1ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ(4ꢀ(hydroxymethyl)piperidinꢀ1ꢀyl)pentanꢀ1ꢀone (7h). Compound 12
(160 mg, 0.62 mmol), 4ꢀpiperidinemethanol (85 mg, 0.74 mmol), K
2
CO
3
(102 mg, 0.74 mmol)
and NaI in catalytic amounts were added into CH
3
CN (25 mL) and afforded 7h (98 mg, 54%).
1
H NMR (400 MHz, CDCl ) δ 8.03–7.91 (m, 2H), 7.12 (t, J = 8.6 Hz, 2H), 3.52 (d, J = 5.7
3
Hz, 2H), 3.25 (d, J = 11.9 Hz, 2H), 3.01 (t, J = 6.5 Hz, 2H), 2.69 (t, J = 7.3 Hz, 2H), 2.37 (t, J
1
3
=
11.6 Hz, 2H), 1.94–1.53 (m, 9H). C NMR (101 MHz, CDCl
3
) δ 198.3, 165.7, 133.2,
1
9
1
30.6, 115.7, 66.9, 58.0, 53.1, 37.9, 37.5, 27.5, 25.3, 21.8. F NMR (376 MHz, CDCl ) δ
3
+
+
–106.25. MS (ESI ): m/z 294.11 [M+H] .
4ꢀ(1,4ꢀDioxaꢀ8ꢀazaspiro[4.5]decanꢀ8ꢀylmethyl)phenol (13). The precursor of 5a was
35
synthesized by a similar reported method. Compound 9 (670 mg, 4.68 mmol) and
ACS Paragon Plus Environment

Page 23 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4ꢀhydroxybenzaldehyde (629 mg, 5.15 mmol) were dissolved in anhydrous dichloromethane
(
40 mL) and stirring at room temperature for 2 h. Then NaBH(OAc) (1.48 g, 7.02 mmol)
3
was added and the mixture was stirred at room temperature over night. After washing with
saturated NaHCO solution and extraction with dichloromethane, the combined organic
layers were dried by Na SO . The solvent was removed under reduced pressure, and the
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4
residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:2 (v/v))
1
to afford compound 13 (530 mg, 46%) as a colorless solid. H NMR (400 MHz, CDCl ) δ
3
7.08 (d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.5 Hz, 2H), 3.94 (s, 4H), 3.48 (s, 2H), 2.59 (s, 4H),
1
3
1
3
.78 (t, J = 5.7 Hz, 4H). C NMR(101 MHz, CDCl ) δ 155.6, 131.0, 128.2, 115.3, 107.2,
+
64.2, 62.0, 50.9, 34.3. MS (ESI+) m/z: 250.14 [M + H] .
1
Radioligand Competition Studies. The σ assay was performed with the radioligand
3
(
+)ꢀ[ H]pentazocine (1288 GBq/µmol; PerkinElmer) using membrane homogenates obtained
from rat cortex (SPRD, female, 10–12 weeks). The σ assay was performed with the
2
3
radioligand [ H]DTG (1761 GBq/µmol; PerkinElmer) using membrane homogenates
obtained from rat liver (SPRD, female, 10–12 weeks). The VAChT assay was performed with
3
radioligand ( )ꢀ[ H]ꢀvesamicol (1 nM) using membrane homogenates from PC12 cells
−
transfected with rat VAChT.
The procedures for the radioligand competition assays of 5a–5e, 6a, 6b and 7a–7h were
35,40
previously described.
Detailed procedures are presented in the Supporting Information.
Radiochemistry. The TRACERLabFXꢀFN synthesis module (GE) was used for the
18
production of [ F]5a. A cleaning program was carried out prior to the start of the
radiosynthesis. The corresponding starting materials were stocked in the storage vessels and
18
in the mixing flask. All reactions were performed with the starting [ F]fluoride activity of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
5–15 GBq and the [ F]fluoride aqueous solution was trapped on the anion exchange
cartridge (QMA, Waters) and eluted into the reaction vessel with a solution of Kryptofix 222
(
2 3 2 3
15 mg, 40 µmol), K CO (2.7 mg, 20 µmol) and Cs CO (30 mg, 92.1 µmol) in 1.5 mL of
18
acetonitrile/water = 1:1 (v/v). The [ F]fluoride eluate was dried under vacuum, a nitrogen
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
stream at 95 °C and addition of 3 × 1 mL of acetonitrile. After the completed drying, ethylene
1
,2ꢀditosylate (6.0 mg, 16.2 mmol) in 0.64 mL of acetonitrile was added into the reaction
vessel. The mixture was heated at 90 °C for 15 min. After cooling, hydroxyl precursor 13 (9.0
mg, 36.1 mmol) in 0.44 mL of DMF was added in the reaction vessel in succession. After
heating at 110 °C for 10 min, the mixture was diluted with 1 mL of HPLC eluent and
transferred to the semiꢀpreparative HPLC with a flow of 5 mL/min. The fraction with a
18
retention time about 20 min containing the radiotracer [ F]5a was transferred into the mixing
flask containing 30 mL of water where the product was trapped by a reversed phase C18
18
cartridge, After washing with water, [ F]5a was eluted off with 1 mL of ethanol and diluted
with sterile saline for in vitro and in vivo experiments.
Determination of log D Value. Flask shake method with 1ꢀoctanol/phosphateꢀbuffered
18
saline (PBS, 0.05 mol/L, pH = 7.4) was applied to determine the log D value of [ F]5a
3
5,40
according to literature.
The detailed procedure is shown in the Supporting Information.
18
In Vitro Stability. The in vitro stability of [ F]5a was evaluated by monitoring the
radiochemical purity at 2 hour. The procedures to determine the in vitro stability in ethanol,
35,40
saline and human plasma were similar to those in the literature.
in the Supporting Information.
The details are provided
18
Cell Uptake. Cellular uptake experiments for evaluation of [ F]5a in vitro were performed
ACS Paragon Plus Environment

Page 25 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in the following tumor cell models using a protocol published elsewhere with some
5
5
modifications.
A375 (human malignant melanoma line; ATCC CRLꢀ1619), PC3
(androgenꢀindependent human malignant prostate adenocarcinoma line, ATCC CRLꢀ1435),
DU145 (androgenꢀindependent human metastatic prostate adenocarcinoma line; ATCC
HTBꢀ81), and MCF7 (human metastatic mammary adenocarcinoma line; ATCC HTBꢀ22)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cells were used in this experiment. Substantial expression of σꢀreceptor subtypes σ
1 2
and σ as
well as binding of specific σꢀreceptor ligands has been demonstrated in the literature for all
19,56,57
selected cell lines.
5
5
Cells were routinely cultivated as the reported method. Cells were seeded in 24ꢀwell
5
5
plates at a density of 1.3 × 10 cells/mL (A375, PC3, MCF7) and 1.5 × 10 cells/mL (DU145)
18
and grown to confluence, The cell tracer uptake experiments using compound [ F]5a
(0.4–0.7 MBq/mL; specific activity at application time: 30 GBq/µmol) were performed in
quadruplicate in PBS at 37°C for 1, 10, 30, 60, and 120 min (2ꢀ3 independent experiments
without blocking; 1ꢀ2 independent experiments with blocking). For blocking experiments, the
cells were preꢀincubated for 10 min with 10 µM of the following σꢀreceptor ligands:
haloperidol, fluspidine or SA4503. The details of cell culture and cell uptake experiments are
shown in the Supporting Information.
Generation of Mouse Tumor Xenografts. For the generation of subcutaneous tumors PC3,
DU145, A375 and A431 (human epidermoid (squamous) cell carcinoma, ATCC CRLꢀ1555)
cells were used. The latter were cultivated following the same protocol reported above for
cellular uptake experiments. Nine weeks old male and female NMRI (nu/nu) mice were
subcutaneously xenotransplanted into the legs with these cells according to published
58,59
protocol.
The procedures are shown in the Supporting Information.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biodistribution. The male Wistar rats (n = 32, Wistar Unilever, HsdCpb:WU, Harlan
Winkelmann GmbH, Borchen, Germany) were between 7 and 9 weeks of age and body
weight of 125 ± 14 g were used in the biodistribution study. The procedures are based on the
58ꢀ61
reported literature and shown in the Supporting Information.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Metabolite Analysis. Metabolic stability studies were performed on male Wistar rats.
Arterial blood samples were collected at 1, 3, 5, 10, 20, 30, and 60 min after injections of
18
[
F]5a. Blood cells were separated by centrifugation (5 °C, 5 min, 8000 rpm), and plasma
proteins were precipitated using 60% acetonitrile and subsequent centrifugation (5 °C, 5 min,
8000 rpm). The deproteinated supernatant was analyzed by radioꢀHPLC. The radioꢀHPLC
system (Agilent 1100 series) applied for metabolite analysis was equipped with UV detection
(254 nm) and an external radiochemical detector (Ramona, Raytest GmbH, Straubenhardt,
Germany). Analysis was performed on a Zorbax C18 300SB (250 × 9.4 mm; 4 ꢁm) column
with an eluent system C (water + 0.1% TFA) and D (acetonitrile + 0.1% TFA) in a gradient 5
min 95% C, 10 min to 95% D, and 5 min at 95% D at a flow rate of 3 mL/min. HPLC were
performed on arterial blood samples from 1 to 60 min after injections and on urine sample
from 60 min after injection. The metabolite fraction in the blood plasma was calculated. The
extracts from other tissues were prepared by ultrasound homogenization as 10% solution in
PBS, centrifugation (5 °C, 70 000 g × min) and subsequent precipitation by 60% acetonitrile.
The supernatant was analyzed by the described radioꢀHPLC.
Small Animal PET. Evaluation of tumor metabolism was performed by dynamic small
18
animal positron emission tomography using the radiotracer [ F]5a. Transmission scans, CT
55,58,61
and PET acquisition followed the protocol given by us in detail elsewhere.
procedures are shown in the Supporting Information.
The
ACS Paragon Plus Environment

Page 27 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Ex Vivo Autoradiography. Four adult male Wistar rats weighing 119−132 g were allowed
18
free access of food and water. [ F]5a was injected in physiological saline of 0.5 mL of
1
8
[
F]5a containing 40 MBq into two rats (control) and 1 mg/kg body weight
haloperidol (blocked) was administered intravenously under anesthesia with a gas mixture
of 10% desfluran and 30% oxygen/air. After injection, anesthesia was discontinued for the
h incubation animals and for the animals with 5 min incubation time was the anesthesia
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
,
1
continued. At 5 or 60 minutes after injection, the animals were sacrificed and the brains were
quickly removed and frozen in powdered dry ice/isopentane. Coronal brain slices (10 µm)
were cut with a microtomeꢀcryostat (CM 1850, Leica Instruments, Germany), thawꢀmounted
onto glass slides (SuperFrost, Menzel, Germany), airꢀdried for 60 min and exposed to
o
imaging plates (BASꢀSR; FujiꢀPhoto Film Co., Ltd., Tokyo) or stored at ꢀ20 C for 12 h in
standard xꢀray cassettes. The autoradiograms were scanned by the bioimaging analyzer
system (BAS 5000, FujiPhoto Film Co., Ltd.).
Statistical Analysis. Values are expressed as mean ± SD or SE. Values were compared using
an unpaired Student’s tꢀtest with Welch’s correction and an Fꢀtest to compare the variances
(GraphPad Prism 5.02 for Windows, GraphPad Software, San Diego, CA). The
nonparametric Wilcoxon signed rank test and the D’AgostinoꢀPearson normality test were
used for statistical evaluation for some of the data. p < 0.05 was considered significant and is
indicated by an asterisk.
ASSOCIATED CONTENT
Supporting Information
18
General information and some parts of evaluation of [ F]5a in the Experimental Section,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
radiochemical purity and biodistribution of [ F]5a, rat brain PET imaging and metabolic
18
1
studies of [ F]5a, purities and the H NMR of the final compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
AUTHOR INFORMATION
Corresponding Author
*
Phone: +86ꢀ10ꢀ58808891; Fax: +86ꢀ10ꢀ58808891; Eꢀmail: hmjia@bnu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The excellent technical assistance of Aline Morgenegg, Catharina Heinig, Tina Spalholz,
Peggy Wecke, Martin Ullrich and Sebastian Meister is greatly acknowledged. This work was
supported by the National Natural Science Foundation of China (Grant No. 21471019) and
by Deutsche Forschungsgemeinschaft Germany (Grant No. STE 601/11ꢀ1). This work was
further supported in part by the Helmholtz Association within HelmholtzꢀPortfolio Topic
"Technologie und Medizin–Multimodale Bildgebung zur Aufklaerung des InꢀvivoꢀVerhaltens
von polymeren Biomaterialien". Fang Xie is the recipient of an academic exchange
scholarship funded by the China Scholarship Council (CSC).
ABBREVIATIONS USED
S
AD, Alzheimer’s disease; A , specific activity; AUCs, area under the curve values; BAT,
11
brown
adipose
tissue;
BBB,
bloodꢀbrain
barrier;
[ C]SA4503,
11
18
1
ꢀ(3ꢀmethoxyꢀ4ꢀ[ C]methoxyphenethyl)ꢀ4ꢀ(3ꢀphenylpropyl)piperazine;
[ F]FPS,
ACS Paragon Plus Environment

Page 29 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
8
18
1ꢀ3ꢀ[ F]fluoropropylꢀ4ꢀ((4ꢀcyanophenoxy)ꢀmethyl)
piperidine;
[ F]ISOꢀ1,
1
8
Nꢀ(4ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)butyl)ꢀ2ꢀ(2ꢀ[ F]fluoroethoxy)ꢀ5ꢀmeth
3
3
ylbenzamide; [ H]DTG, [ H]ꢀ1,3ꢀdiꢀoꢀtolylꢀguanidine; HPLC, high performance liquid
chromatography; HRMS, high resolution mass spectrometry; Hz, Hertz; ID, injected
dose; %ID, percentage of injected dose; NSCLC, non small cell lung cancer; PBS,
phosphateꢀbuffered saline; PD, Parkinson’s disease; PET, positron emission tomography;
Pꢀgp, Pꢀglycoprotein; PGRMC1, progesterone receptor membrane component 1; RCP,
radiochemical purity; r. t., room temperature; SUV, standardized uptake value; VAChT,
vesicular acetylcholine transporter; WAT, white adipose tissue.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
1
.
Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. The effects of
morphineꢀand nalorphineꢀlike drugs in the nondependent and morphineꢀdependent
chronic spinal dog. J. Pharmacol. Exp. Ther. 1976, 197, 517−532.
2
.
Hellewell, S. B.; Bowen, W. D. A sigmaꢀlike binding site in rat pheochromocytoma
(
PC12) cells: decreased affinity for (+)ꢀbenzomorphans and lower molecular weight
suggest a different sigma receptor form from that of guinea pig brain. Brain Res. 1990,
527, 244ꢀ253.
3
4
5
.
.
.
Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien. J. L.; Musacchio, J. M.; Rothman, R. B.;
Su, T.ꢀP.; Tam, S. W.; Taylor, D. P. A proposal for the classification of sigma binding
sites. Trends Pharmacol. Sci. 1992, 13, 85ꢀ86.
Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H.ꢀG.; Striessnig, J.; Kempner, E.;
Glossmann, H. Purification, molecular cloning, and expression of the mammalian
1
sigma ꢀbinding site. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 8072−8077.
Kekuda, R.; Prasad, P. D.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V. Cloning and
ACS Paragon Plus Environment