Synthesis and in vitro evaluation of 5-substituted benzovesamicol analogs containing N-substituted amides as potential positron emission tomography tracers for the vesicular acetylcholine transporter

Article abstract of DOI:10.1016/j.bmc.2017.01.041

Herein, new ligands for the vesicular acetylcholine transporter (VAChT), based on a benzovesamicol scaffold, are presented. VAChT is acknowledged as a marker for cholinergic neurons and a positron emission tomography tracer for VAChT could serve as a tool for quantitative analysis of cholinergic neuronal density. With an easily accessible triflate precursor, aminocarbonylations were utilized to evaluate the chemical space around the C5 position on the tetrahydronaphthol ring. Synthesized ligands were evaluated for their affinity and selectivity for VAChT. Small, preferably aromatic, N-substituents proved to be more potent than larger substituents. Of the fifteen compounds synthesized, benzyl derivatives (±)-7i and (±)-7l had the highest affinities for VAChT. Compound (±)-7i was chosen to investigate the importance of stereochemistry for binding to VAChT and selectivity toward the σ₁ and σ₂ receptors. Enantiomeric resolution gave (+)-7i and (?)-7i, and the eutomer showed seven times better affinity. Although racemate (±)-7i was initially promising, the affinity of (?)-7i for VAChT was not better than 56.7 nM which precludes further preclinical evaluation. However, the nanomolar binding together with the ready synthesis of [¹¹C]-(±)-7i shows that (?)-7i can serve as a scaffold for future optimizations to provide improved ¹¹C-labelled VAChT PET tracers.

Full text of DOI:10.1016/j.bmc.2017.01.041

Accepted Manuscript
Synthesis and in vitro evaluation of 5-substituted benzovesamicol analogs con-
taining N-substituted amides as potential positron emission tomography tracers
for the Vesicular Acetylcholine Transporter
Sara Roslin, Maria De Rosa, Winnie Deuther-Conrad, Jonas Eriksson, Luke R.
Odell, Gunnar Antoni, Peter Brust, Mats Larhed
PII:
S0968-0896(17)30146-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.041
BMC 13520
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 November 2016
19 January 2017
23 January 2017
Please cite this article as: Roslin, S., Rosa, M.D., Deuther-Conrad, W., Eriksson, J., Odell, L.R., Antoni, G., Brust,
P., Larhed, M., Synthesis and in vitro evaluation of 5-substituted benzovesamicol analogs containing N-substituted
amides as potential positron emission tomography tracers for the Vesicular Acetylcholine Transporter, Bioorganic
&
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.041
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Synthesis and in vitro evaluation of 5-substituted benzovesamicol analogs
containing N-substituted amides as potential positron emission tomography
†

tracers for the Vesicular Acetylcholine Transporter
a
a
b
a
a
a
Sara Roslin , Maria De Rosa , Winnie Deuther-Conrad , Jonas Eriksson , Luke R. Odell , Gunnar Antoni , Peter
b
c
Brust and Mats Larhed *
a
Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, BMC Box 574 SE-751 23 Uppsala, Sweden
Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Research Site Leipzig, 04318 Leipzig,
b
Germany
c
Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC Box 574 SE-751 23 Uppsala, Sweden
*
Corresponding Author: Mats Larhed. Tel: +46 18 471 4667. Fax: +46 18 471 4474. E-mail: mats.larhed@orgfarm.uu.se.
The authors declare no competing interests.
†
Abstract
Herein, new ligands for the vesicular acetylcholine transporter (VAChT), based on a benzovesamicol
scaffold, are presented. VAChT is acknowledged as a marker for cholinergic neurons and a positron
emission tomography tracer for VAChT could serve as a tool for quantitative analysis of cholinergic
neuronal density. With an easily accessible triflate precursor, aminocarbonylations were utilized to
evaluate the chemical space around the C5 position on the tetrahydronaphthol ring. Synthesized
ligands were evaluated for their affinity and selectivity for VAChT. Small, preferably aromatic, N-
substituents proved to be more potent than larger substituents. Of the fifteen compounds
synthesized, benzyl derivatives (±)-7i and (±)-7l had the highest affinities for VAChT. Compound (±)-7i
was chosen to investigate the importance of stereochemistry for binding to VAChT and selectivity
towards the σ
showed seven times better affinity. Although racemate (±)-7i was initially promising, the affinity of (-
-7i for VAChT was not better than 56.7 nM which precludes further preclinical evaluation. However,
1
and σ
2
receptor. Enantiomeric resolution gave (+)-7i and (-)-7i, and the eutomer
)
1
1
the nanomolar binding together with the ready synthesis of [ C]-(±)-7i shows that (-)-7i can serve as
a scaffold for future optimizations to provide improved C-labelled VAChT PET tracers.
1
1
Keywords
1
1
Vesicular acetylcholine transporter, VAChT, PET tracer, aminocarbonylation, C-labelling
. Introduction
1
In the cholinergic nerve terminal, acetylcholine (ACh) is accumulated in synaptic vesicles for storage.
1
,2
This accumulation is accomplished by the vesicular acetylcholine transporter (VAChT). The
expression of VAChT has been shown to correlate well with the brain cholinergic innervation, which
3
–6
can be found in areas such as the striatum, cortex, hippocampus and amygdala. VAChT has been
acknowledged as a biological marker for the integrity of cholinergic neurons due to the presynaptic
location.

Abbreviations
Acetylcholine, ACh; vesicular acetylcholine transporter, VAChT; choline acetyltransferase ChAT; positron emission
tomography,
PET;
(±)-trans-2-(4-phenylpiperidino)cyclohexanol,
vesamicol;
3-(4-phenyl-piperidin-1-yl)-1,2,3,4-
3
3
tetrahydronaphthalen-2-ol, benzovesamicol; structure-activity relationship, SAR; [ H]-1,3-di-o-tolylguanidine, [ H]DTG;
trans-bisacetato)-bis[o-(di-o-tolyl-phosphino)benzyl]di-palladium(II), Herrmann’s palladacycle; 2-dicyclo-hexylphosphino-
2′,4′,6′-triisopropylbiphenyl, XPhos; (dimethylamino)pyridine (DMAP); Palladium(dibenzylidene acetone), Pd(dba)
2
; SFC,
Supercritical fluid chromatography.

The cholinergic system is involved in neurodegenerative diseases associated with cognitive deficits,
as the loss of cholinergic neurons has been correlated to the impairment of cognitive abilities and
7
,8
memory. In the case of Alzheimer’s disease (AD), this has been known as the cholinergic hypothesis
and it has stimulated significant research over the years although it has been demonstrated that not
9
only cholinergic neurons are degenerated in AD. The related research has shown a loss of cortical
cholinergic neurons in AD patients and a correlation of cortical synapse loss with cognitive
7
,10,11
impairment.
More recent studies have shown a reduced expression of VAChT in brains of AD
5
patients. Another neurodegenerative disease is Parkinson’s disease (PD), where the motor
symptoms are the most pronounced symptoms but non-motor co-morbidities are also likely to affect
PD patients. Dementia is such a co-morbidity and demented patients with PD have been shown to
have a loss of cholinergic neurons in the basal forebrain similar to that of AD patients. The study of
the cholinergic system via the study of VAChT would therefore offer insight into the disease
pathology and could be a complement in the diagnosis of neurodegenerative diseases where loss of
cholinergic neurons is part of the etiology.
VAChT has been studied with imaging techniques such as positron emission tomography (PET) and
single-photon emission computed tomography (SPECT). PET is a molecular imaging technique
1
2
13–15
1
6
where a tracer is used to investigate biochemical or physiological processes in vivo. The tracer is a
1
1
18
molecule that has been radiolabeled with a positron-emitting radionuclide, such as C and F.
Several attempts at synthesizing a PET tracer for VAChT have been made but only a limited number
1
7–24
have progressed to in vivo preclinical evaluations.
All VAChT ligands synthesized thus far are
structurally related to (±)-trans-2-(4-phenylpiperidino)-cyclohexanol (vesamicol, Figure 1). This single
2
5
lead compound has been invaluable in the discovery and subsequent characterization of VAChT.
Vesamicol acts as an allosteric inhibitor at a stereoselective binding site with (-)-vesamicol being the
2
,26,27
preferred enantiomer.
lack of a suitable position for labeling along with a high affinity to other receptors. Specifically, the
competitive binding to the σ and σ receptor causes a selectivity problem and for a successful PET
tracer, selectivity for the target is essential. This selectivity issue is, together with a low affinity for
Vesamicol itself is unsuitable as a PET tracer for VAChT, because of the
2
8
1
2
1
7,18
VAChT, the main reason for failure of new VAChT ligands.
The affinity criteria is set by the binding
potential (BP, BP = B m2 ax/K
D
) and for neuronal targets, the affinity should be in the low nanomolar to
9–32
subnanomolar range.
A
structurally related analog (3-(4-phenylpiperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol
(
benzovesamicol), Figure 1) of vesamicol, first synthesized by Rogers et al. [33], has also been used as
33,34
a scaffold for different VAChT ligands.
The benzovesamicol scaffold has shown a better selectivity
over the σ receptors, compared to vesamicol derivatives, and the tetrahydronaphthol ring allows for
3
0,35,36
18
introduction of different substituents and for different possibilities of labeling.
(-)-5-[ F]-
fluoroethoxybenzovesamicol is the most promising PET tracer candidate reported to date and has
1
8
37
been evaluated in healthy human volunteers ((-)-[ F]FEOBV, Figure 1). The tracer candidate
distributed to brain regions known to contain cholinergic innervation but long scan times were
needed to reach equilibrium in the binding to VAChT. The selectivity over the σ
1
receptor is however
a concern and has not been verified in in vivo studies. In vitro binding studies has measured the
3
8
K
i(VAChT) to 19.6 nM and the Ki(σ-1) to 210 nM for (-)-FEOBV.

Figure 1. Vesamicol and selected VAChT ligands.
In a previous paper, modifications of rings B and C were explored (see vesamicol, Figure 1) by
1
1
exchanging the piperidine ring with a piperazine ring to allow for [ C]-aminocarbonylations for
3
9
introduction of a carbonyl function between the B and C ring. The tolerance for these modifications
was however low, probably due to the conformation of the ligands imparted by the amide group. In
the continued effort to find a PET tracer for VAChT, we wanted to explore the chemical space around
the C5 position of a benzovesamicol scaffold and to evaluate the affinity and selectivity of potential
PET tracers for VAChT (Figure 1). VAChT ligands with substituents on the C5 position have previously
3
0,33,35,40
been shown to have a better binding to VAChT than their C8 regioisomers.
With PET and
radiochemistry in mind, an amide was placed in the C5 position to be able to label all potentially
interesting ligands. Inclusion of the amide functional group allowed the use of different amines to
explore a structure-activity relationship (SAR) around the above mentioned position. Furthermore,
1
1
the positron emitting radionuclide C can be readily introduced in the amide moiety through
1
1
incorporation of [ C]CO via an aminocarbonylation reaction.
2. Results and discussion
The implication of cholinergic dysfunction in neurodegenerative diseases makes a PET tracer for
VAChT a desirable tool in order to better understand the pathology of the neurodegenerative
diseases. The work presented herein reflects the struggle of finding such a PET tracer candidate, one
with high affinity and selectivity. The synthesis of fifteen ligands from a benzovesamicol scaffold via a
novel and facile synthetic route was followed by in vitro determination of the affinity for VAChT and
1
1
the σ
.1 Chemistry
The traditional route to 5-substitued benzovesamicol derivatives has started from 1-
1
and σ
2
receptors, as well as C-labeling of one ligand by aminocarbonylation.
2
3
3,35,40
aminonaphthalene and has led to 5-aminobenzovesamicol derivatives.
To synthesize
derivatives, the amino group has been transformed via diazotization reactions or protected with 4-
3
0,40,41
fluorobenzoyl chloride.
The ambition in this project was to explore the SAR around the 5
position by using aliphatic and aromatic substituents. We therefore sought a precursor that could
1
1
undergo an aminocarbonylation reaction, with both CO and [ C]CO. To avoid the use of highly
regulated and carcinogenic 1-aminonaphthalene, α-naphthol was identified as a viable alternative
from which a triflate precursor could be synthesized in four steps. Thus a shortened route and a
1
1
precursor for aminocarbonylation both with CO and [ C]CO was developed.

Scheme 1. a) Liquid ammonia, Li, absolute EtOH, 1 h, -78 °C, 73%; b) N-Phenyl-bis(trifluoromethane-
sulfonimide), K CO , THF, MW, 120 °C, 6 min, 62%; c) m-CPBA, DCM, 0 °C to rt, 16 h, 77%; d) LiClO , 4-
phenylpiperidine, MeCN, reflux, 16 h, 33% yield ((±)-5 ) and 49% yield ((±)-6).
2
3
4
Synthesis of (±)-5 started with a Birch reduction of commercially available α-naphthol (1) to 2
4
2
(
Scheme 1). The selectivity for reduction of the unsubstituted ring was >99%. Compound 2 was
then converted by a microwave-assisted triflation, using N-phenyl-bis(trifluoromethanesulfonimide)
4
3
as triflate source, to 3 in only 6 minutes. The isolated double bond in 3 was epoxidized, giving the
44
racemate (±)-4 after an overnight reaction. 4-Phenylpiperidine and (±)-4 were refluxed overnight in
4
5
acetonitrile (MeCN), affording (±)-5 and (±)-6. The C8 regioisomer (±)-6 was obtained in higher yield
than the desired C5 regioisomer (±)-5 (49% and 33%, respectively), possibly due to a steric clash
between the 4-phenylpiperidine on C3 and the triflate group on C5. Compound (±)-5 was next used
as starting material for the microwave-assisted aminocarbonylation to obtain (±)-7a-n (Scheme 2).
For the aminocarbonylation, molybdenum hexacarbonyl (Mo(CO)
6
) was used as a solid source of
carbon monoxide together with the appropriate amine and trans-bis(acetato)bis[o-(di-o-
4
6,47
tolylphosphino)-benzyl]-dipalladium(II) (Herrmann’s palladacycle) as the catalyst.
There are only
limited literature examples describing the use of aryl triflates as coupling partners in
aminocarbonylations, mainly because they are not as reactive as the corresponding iodides and are
46,48
generally more expensive than the corresponding bromide.
In our case, the triflate (±)-5 was
selected as the key intermediate due to its ease of synthesis as compared to the corresponding
iodide. The overall transformation was accomplished in only four reaction steps and provides a
straightforward route to the ligand precursor (±)-5. The aminocarbonylations were run at 160 °C for
3
-8 h with the ligand 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos), which recently
4
9,50
has been used in carbonylation reactions for synthesis of 1,4-diketones and ureas.
The long
reaction times are needed for full consumption of triflate (±)-5. The yields for compounds (±)-7a-n
were in the range of 5%-37%, after flash column chromatography followed by semi-preparative HPLC
1
9
and isolation as the TFA salts (see experimental section for F NMR of (±)-7g and (±)-7j). Part of the

starting material was reduced to the corresponding arene or formed the corresponding acid in the
aminocarbonylation.
The amines were chosen in order to explore the SAR around the C5 position. Both primary and
secondary amines with aliphatic and aromatic side chains were used. Previous studies has indicated
that a certain amount of bulk can be tolerated in this region, thus bulky aliphatic amines were
3
3,51,52
investigated.
With the possibility of finding a hydrogen bond interaction, amines with hydrogen
bond acceptors and donators were also used. As for the amines with aromatic rings in the side chain,
variation of the electron density in the ring was explored by different ring substituents and by using
heteroaromatic rings.

Scheme 2. a) Amine, Cs
60 °C, MW, 3-8 h, 5%-37% yield (isolated as TFA-salts, more information can be found in part 4.
Experimental section).
2
CO
3
, Mo(CO) , XPhos, Herrmann's palladacycle, DMAP, 1,4-dioxane or MeCN,
6
1
Scheme 3. a) Amine, Cs
2
CO
3
, Mo(CO) , XPhos, Herrmann's palladacycle, DMAP, 1,4-dioxane, 160 °C,
6
MW, 5 h, 22% yield (isolated as a TFA-salt).
Resolution of racemate (±)-7i into enantiomers (+)-7i and (-)-7i was performed by SFC on an YMC
Chiral Cellulose-SB column (10 mm x 250 mm) with 30% methanol and 0.2% diethylamine in CO as
2
eluent. Compound (+)-7i eluted after 2.2 min and compound (-)-7i eluted after 2.95 min, both
isolated in 99% enantiomeric excess.
In order to prove the difference in affinity for the C5 and C8 regioisomer, the C8 regioisomer (±)-8
was synthesized from (±)-6 (Scheme 3).
2.2 In vitro binding studies
Compounds (±)-7a-n, (+)-7i, and (-)-7i were evaluated for their affinity toward VAChT in an in vitro
binding study (Table 1). Compound (±)-8, the regioisomer of (±)-7i, was also investigated to confirm
the expected lower affinity for ligands with substituents in the C8 position. The binding study for
VAChT was performed on PC12 cells stably transfected with a human VAChT gene, using
3
53
[
H]vesamicol as a high affinity VAChT radioligand (K
D
= 2.39 nM). As affinity for σ
1
and σ receptors
2
is a known problem for VAChT ligands, the compounds were also evaluated for their affinity to the σ
and σ receptors and hence their selectivity for VAChT was determined (expressed as the selectivity
1
2
factor, Table 1). The binding studies for the σ receptors were performed on membrane homogenates
3
3
from rat cortex or rat liver using (+)-[ H]pentazocine (K
D
= 6.9 nM) and [ H]DTG (K
D
= 29 nM) as high
54
affinity radioligands, for σ
1
and σ
2
respectively. The ability of (±)-7a-n, (+)-7i, and (-)-7i to compete
value, which was determined according
3
with [ H]vesamicol for binding to VAChT is expressed by a K
i
5
5
to the Cheng-Prusoff equation.
For ligands (±)-7a-n, the affinity for VAChT seems to be more dependent on steric effects rather than
on electronic effects (see below), which is in line with previously reported data. Structures (±)-7a-f,
with aliphatic side chains on the nitrogen, generally showed a low affinity and low selectivity toward
VAChT. Here, (±)-7c and (±)-7f were exceptions with moderate affinities (Ki(VAChT) of 295 and 205 nM,
5
6
respectively, see Table 1). Ligand (±)-7c also showed a very good selectivity relative to the σ
1
and σ
2
receptors. Both (±)-7c and (±)-7f have small side chains, as compared to the other, larger aliphatic
derivatives with affinities close to or in the micromolar range. Most notable is nitrile (±)-7e with the

largest side chain and the lowest affinity towards VAChT. Another noteworthy point is the fact that
compounds (±)-7a, (±)-7b and (±)-7d have higher affinity for σ receptors than for VAChT. Again, a
common denominator is their large amide side chains.
Compounds (±)-7g-n, with an aromatic or heteroaromatic ring on the amide nitrogen, generally
showed a moderate to good affinity and selectivity towards VAChT with the benzyl derivatives (±)-7i-l
having the highest affinities for VAChT. As for the selectivity for VAChT, most of the compounds
showed a higher selectivity regarding the σ
and (±)-7l had a Ki(VAChT) of 67.0 nM and 63.5 nM, respectively, making them the best compounds in
the series. In addition, the selectivity of both compounds toward the σ and σ receptors is
1
than the σ
2
receptor. Benzyl substituted ligands (±)-7i
1
2
comparable. Methylated analog (±)-7j had an almost three times lower affinity than (±)-7i, an
indication that the N-H fragment might be engaged in a hydrogen bond to an amino acid residue in
VAChT. However, the loss in affinity for VAChT is not followed by the same loss in affinity for either σ
receptor. The selectivity factors for (±)-7i are 20 and 6 for the σ
whereas the selectivity factors are 10 and 3 for (±)-7j. This indicates that the affinities for VAChT and
the σ and σ receptors is not necessarily governed by the same factors. The impact of electron
1
and σ receptors, respectively,
2
1
2
density in the aromatic ring on affinity was investigated through compounds (±)-7g-h and (±)-7k-l and
found not to have any particular impact. This has previously been shown by Tu et al. [55], who also
5
6
modified the A-ring.
Comparing the effect of the position of the side chain on the fused benzo-cyclohexanol ring, the C5
position was more favorable than the C8 position. Compound (±)-7i had an affinity of 67 nM as
compared to the C8 regioisomer (±)-8 with an affinity of 795 nM. This regioisomeric difference is in
3
0,33,35
accordance with previously acquired results.
affinity for the σ receptor than for VAChT.
Interesting to note is that (±)-8 showed higher
1
As for ligands (±)-7a-n selectivity for VAChT over the σ receptors, the majority of the compounds had
a higher affinity for VAChT than for the σ receptors (Table 1). Racemic vesamicol had a higher affinity
for the σ receptor than for VAChT (Table 1). Although VAChT ligands with a benzovesamicol-based
1
scaffold often show a higher selectivity for VAChT than for σ receptors compared to vesamicol-based
ligands, the selectivity is still an issue and points to a close structural resemblance of VAChT ligands
and σ receptor ligands. A sterical effect seems to be the most important determinant for the
selectivity, and compounds (±)-7a, (±)-7b and (±)-7d with their large side chains even showed higher
affinities for either the σ
they had an aliphatic or aromatic side chain, showed a higher selectivity for VAChT over the σ
receptor than the σ receptor. This is in contrast to previously reported results for benzovesamicol-
based VAChT ligands, with modifications in the C-ring, which have shown higher selectivity for VAChT
1
or the σ
2
receptor than for VAChT. In general, most compounds, whether
1
2
1
9,20,30,35
over the σ
could have a negative effect on the affinity for the σ
designing future VAChT ligands.
Because of the aforementioned stereoselective binding properties of VAChT, resolution of racemate
2
receptor.
The results presented here indicate that modifications in the A-ring
2
receptor, a notion that can be of use when
(
(
(
±)-7i to enantiomers (+)-7i and (-)-7i was performed. In Table 1, the binding affinities are shown. The
-)-enantiomer (Ki(VAChT) = 56.7 nM) showed a seven times higher affinity than the (+)-enantiomer
K
i(VAChT) = 393 nM), a result in line with the enantioselective characteristics of VAChT reported in the
19,20,36
literature.
In addition, the eutomer (-)-7i shows higher selectivities over the σ receptors in
comparison to the racemate (±)-7i and the distomer (+)-7i.

Table 1. Inhibition constants (K
their binding to VAChT and the σ
i
) of (±)-vesamicol and compounds (±)-7a-n, (+)-7i, (-)-7i and (±)-8 for
and σ receptors.
1
2
a
b
Compound
K
i
(nM)
Selectivity factor
c
d
e
R
VAChT
47.7
σ
1
σ
2
K
i
(σ
1
/VAChT)
K
i
(σ2/VAChT)
f
(
±)-vesamicol
±)-7a
20.4
112
0.43
2.0
2.3
0.4
(
1 547;
4 687;
3 874
753; 863
2
666
(
±)-7b
953; 947
669;
359; 377
0.9
0.4
1
078
(
±)-7c
328; 262
3 500;
13 000;
900
15 000;
4 900
> 34
0.66
34
9
(
±)-7d
1 700;
2 100
> 10 000
> 3.4
2
300
(
±)-7e
> 10 000 > 100 000 > 30 000
-
-
(
±)-7f
197; 212
193; 219
665;
436; 513
6.0
9.2
2.3
7.8
1
785
(
±)-7g
1 844;
1 970;
1 245
1
961
(
(
(
±)-7h
256; 241
67.0 ± 25.4
393 ± 69.6
56.7 ± 20.8
614; 835
2 246;
2.9
20
18
60
6.9
5.7
2.7
12
1
190
g
±)-7i
1 326 ±
384 ± 36
142
g
+)-7i
6 214 ± 2 915 ± 81
44
1
g
(
-)-7i
2 974 ± 1
42
612 ±
270
2

(
±)-7j
177; 214
148; 145
1 909;
027
430; 566
10
2.5
1.9
2
(
±)-7k
707; 519 250;306
4.2
(
±)-7l
70.1; 53.5
1 495;
827
418; 518
26
7.4
1
(
±)-7m
694; 469
317; 364
8 358;
515
7 932;
6 447
12
12
5
(
±)-7n
> 10 000
1 302;
> 29
4.0
1
415
(
±)-8
834; 755
267; 215
2 397;
377
0.30
3.0
2
a
3
K
i
-values were determined in competitive binding studies using radiolabeled ligands ([ H]vesamicol for VAChT, (+)-
3
3
[
H]pentazocine for the σ
1
receptor and [ H]DTG for the σ
-values from the two experiments performed are shown.
-values.
Binding to membrane homogenates from PC12 cells stably transfected with human VAChT gene. The equilibrium
2
receptor) in the presence of the compounds in concentrations
ranging from 100 μM – 0.1 nM. Individual K
i
b
Calculated from the mean of the K
i
c
3
dissociation constant of [ H]vesamicol has been determined with K
D
= 2.39 nM in this assay. Nonspecific binding of
3
[
H]vesamicol was determined in the presence of 10 µM vesamicol.
d
3
Binding to membrane homogenates from rat cortex. The equilibrium dissociation constant of (+)-[ H]pentazocine has
3
been determined with K
of 100 µM haloperidol. σ
D
= 6.9 nM in this assay. Nonspecific binding of (+)-[ H]pentazocine was determined in the presence
1
receptors were masked with 1 µM dextrallorphan.
e
3
Binding to membrane homogenates obtained rat liver. The equilibrium dissociation constant of [ H]DTG has been
3
determined with K
D
= 29 nM in this assay. Nonspecific binding of [ H]DTG was determined in the presence of 100 µM
haloperidol.
f
3
Reported results were obtained with (-)-[ H]-vesamicol and vesicles from the electric organ of Torpedo californica.
g
i
The K -values are the mean ± SD (nM) of 5-6 experiments.
2.3 Radiochemistry
In order to investigate whether this C5 substituted benzovesamicol-based scaffold could be
evaluated using PET, radiolabelling of (±)-7i was pursued. Again using (±)-5 as a precursor together
1
1
11
with benzylamine and [ C]CO, [ C]-(±)-7i was labelled through an aminocarbonylation (Scheme 4).
1
1
The reaction was carried out in a disposable glass vial where [ C]CO was efficiently added in xenon
5
7,58
11
gas resulting in ambient pressure conditions.
Without much optimization of the method, [ C]-(±)-
7i could be isolated in 9 ± 0.3% decay-corrected radiochemical yield, >99% radiochemical purity and
with a molar activity of 55-78 GBq/μmol at the end of synthesis (n = 2). As an example, when starting
11
from 16.4 GBq of [ C]CO, the product was isolated with 411 MBq activity and a molar activity of 55
GBq/μmol. This amount of activity is sufficient to perform preclinical experiments. These results
show the usefulness of (±)-5 as a precursor for labeling of C5 substituted benzovesamicol-based
VAChT ligands in addition to its potential use as a precursor for synthesis of new ligands.

11
Scheme 4. a) Benzylamine, Pd(dba)
2
, XPhos, triethylamine, tetrabutylammonium iodide, [ C]CO,
THF, 150 °C, 5 min, RCY = 9 ± 0.3% (decay corrected, n = 2).
3. Conclusion
In summary, fifteen VAChT ligands with different amide N-substituents in the C5 position on the
tetrahydronaphthol ring were synthesized. With a novel synthetic route for VAChT ligands, a
benzovesamicol scaffold was synthesized in a facile manner for further modification by employing
aminocarbonylation reactions. When exploring both aliphatic and aromatic side chains on the amide
nitrogen, ligands with affinities extending from the micromolar to the nanomolar range were
obtained.
Benzyl substituted ligands had the highest affinities, with (±)-7i and (±)-7l showing the strongest
binding to VAChT and good selectivities towards both σ receptors. In order to prove the
stereoselectivity of VAChT’s binding site, the enantiomers of (±)-7i were evaluated for their affinity.
Eutomer (-)-7i showed the best affinity for VAChT. Although racemate (±)-7i initially looked
promising, the affinity of (-)-7i does not warrant further preclinical experiments but the nanomolar
affinity for VAChT and the high selectivity over the σ
1
receptor make (-)-7i an interesting lead
structure for future development. Compound (±)-5 could be used as a precursor for both synthesis of
1
1
11
ligands and as a precursor for C-labeling. As [ C]-(±)-7i could be isolated with radioactivity high
enough for preclinical PET experiments, the interest in (-)-7i as a scaffold for development of new
1
1
C-labelled VAChT PET tracers is further supported.
4
4
. Experimental Section
.1 General chemistry information
Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Fischer
Pittsburgh, PA, USA) and used without further purification. Thin layer chromatography (TLC) was
(
performed on aluminum sheets precoated with silica gel 60 F254 (0.2 mm, Merck KGaA, Darmstadt,
Germany). Column chromatography was performed using silica gel 60 (40–63 μm, Merck KGaA,
1
4
11
Darmstadt, Germany). Carbon-11 was prepared by the N(p,α) C nuclear reaction using 17 MeV
protons produced by a Scanditronix MC-17 Cyclotron at PET Centre, Uppsala University Hospital, and
1
1
obtained as [ C]carbon dioxide. The target gas used was nitrogen (AGA Nitrogen 6.0) containing
1
1
0
.05% oxygen (AGA Oxygen 4.8). The [ C]CO
2
was transferred to the low-pressure xenon-system in a
and [ C]CO were concentrated on anhydrous silica traps at -196 °C
and released from the same traps by removal of liquid nitrogen dewars and heating the traps with a
1
1
11
stream of helium gas. [ C]CO
2

1
1
11
coiled heater. [ C]CO
2
was reduced to [ C]CO over zinc heated to 400 °C and Ascarite was used to
1
1
11
remove residual [ C]CO
2
. Before transferring [ C]CO into the reaction vial through a transfer needle
placed in the capped reaction vial, the carrier gas was changed from helium to xenon ( >99.9%, 1.5
mL/min). For a schematic view of the process, see Eriksson et al. [57] and Chow et al. [58].
TM
TM
Microwave reactions were carried out in a Smith Synthesizer or in an Initiator single-mode
microwave cavity producing controlled irradiation at 2450 MHz. Analytical GC-MS were performed
on an Agilent Technologies system with a CP-SIL 8 CB low bleed (30 m × 0.25 mm) capillary column
using a 70−300 °C temperature gradient and an EI ionization at 70 eV. Analytical reversed phase
HPLC-MS was performed on a Dionex Ultimate 3000 system using MeCN/H O (0.05% HCOOH) as the
2
mobile phase with MS detection, equipped with a C18 (Phenomenex Kinetex SB-C18 (4.8 × 50 mm))
column using UV (214 or 254 nm) detection or on a Dionex Ultimate 3000 system using MeCN/0.05%
HCOOH in H O as the mobile phase with MS detection, equipped with a C18 (Phenomenex Kinetex
2
SB-C18 (4.8 × 50 mm)) column using a UV diode array detector. Semi-preparative reversed phase
HPLC was performed on a Gilson-Finnigan ThermoQuest AQA system equipped with a C8 (Zorbax SB-
C8 (5 μm, 150 × 21.2 mm)) column using MeCN/H
Gilson GX-271 system equipped with a C18 (Macherey-Nagel Nucleodur HTec (5 μm, 125 × 21 mm)).
Purity determinations were done on the Dionex Ultimate 3000 system with MeCN/H O (0.05%
2
O (0.1% TFA) as the mobile phase with UV or on a
2
HCOOH) as the mobile phase and the C18 (Phenomenex Kinetex SB-C18 (4.8 × 50 mm)) column with
UV detection at 254 nm. Resolution of enantiomers was performed with supercritical fluid
chromatography (SFC). Analytical measurements were performed on a SFC Waters Investigator
system connected to a Waters 2998 PDA detector. The column was an YMC Chiral Cellulose-SB (5
μm, 4.6 mm × 150 mm). The column temperature was set to 45 °C. An isocratic condition of 30%
methanol and 0.1% diethylamine in CO was applied at a flow rate of 5 mL/min. Preparative runs
2
were performed on a SFC Waters Investigator system connected to a Waters 2998 PDA detector. The
column was an YMC Chiral Cellulose-SB (5 μm, 10 mm × 250 mm). The column temperature was set
to 45 °C. An isocratic condition of 30% methanol and 0.2% diethylamine in CO was applied at a flow
2
1
1
rate of 15 mL/min. Preparative purification of C-labeled (±)-7i was done on a VWR La Prep Sigma
system with a LP1200 pump, 40D UV detector and a Bioscan flowcount radiodetector. The mobile
phase was MeCN/ammonium formate buffer (pH 3.5) and the column used was Phenomenex Kinetex
C18 (5 µm, 150x10.0 mm). The identities, concentration and radiochemical purities of the purified
1
1
labeled compound [ C]-(±)-7i were determined either with VWR Hitachi Elite LaChrom system,
consisting of L-2130 pump, L-2200 autosampler, L-2300 column oven, L-2450 diode array detector in
+
series with a Bioscan β -flowcount radiodetector or with Elite LaChrom VWR international, equipped
with a LaPrep P206 pump, an Elite LaChrom L-2400 UV detector in series with a Bioscan β+-flowcount
detector, and using (±)-7i as reference. Mobile phase consisted of MeCN/ammonium carbonate (8.1
mM) and column used was Reprosil-Pur basic C18 (5 µm, 100 x 4.6 mm) from Dr Maisch GmbH or
MeCN/ammonium formate buffer (50 mM) on a Merck Chromolith Performance RP-18 column (4.6 ×
100 mm). Optical rotation was measured on a Rudolph research analytical autopol II automatic
polarimeter at 23°C and wavelength 589 nm using a 100 mm cell. NMR spectra were recorded on a
1
13
19
Varian Mercury plus spectrometer ( H at 399.8 MHz, C at 100.5 MHz and F at 376.5 MHz) at
ambient temperature. Chemical shifts (δ) are reported in ppm, indirectly referenced to
1
tetrametylsilane (TMS) via the residual solvent signal ( H: CHCl
3
δ 7.26, CD
CN δ 1.32, (CD CO δ 29.84, 206.26.
2
HOD δ 3.31, CD HCN δ
2
1
3
1
.94, (CHD
2
)(CD
3
)CO; C: CDCl
3
δ 77.16, CD
3
OD δ 49.00, CD
3
)
3 2
1
1
4
.2 Synthetic procedures and characterization of compounds 2 - (±)-8 and [ C]-(±)-7i

4
2
4
.2.1 5,8-Dihydronaphthalen-1-ol (2)
A two-necked flask equipped with a cold finger was charged with α-naphthol (1, 2.0 g, 0.014 mol).
Whilst vigorously stirring, liquid ammonia (30 mL) was added. After the α-naphthol had gone into
solution (~30 min), lithium metal (0.41 g, 0.059 mol) was added in small pieces over 30 min. When
the addition of the metal was completed, the solution (blue in color) was stirred for additional 1 h
before absolute ethanol (4 mL) was added dropwise. The cold finger was removed, stirring was
continued and the excess ammonia was evaporated in a stream of air introduced through an inlet
tube. The residue was dissolved in water (20 mL) and washed with diethyl ether (5 mL). The aqueous
phase was carefully acidified (pH < 1) using conc. HCl. An off-white solid precipitated which was
extracted to diethyl ether (3 x 5 mL). The collected organic layers were washed with brine (2 x 5 mL),
dried over MgSO , filtered and the solvent was removed under reduced pressure, affording the crude
4
product which was purified by silica gel flash column chromatography, using ethyl acetate/petroleum
ether 1:19, affording 1.5 g of 2 as white solid (73% isolated yield), re-crystallized from petroleum
1
ether. H NMR (399.8 MHz, Chloroform-d
5
1
): δ 7.06-7.02 (m, 1H), 6.73-6.71 (m, 1H), 6.63-6.61 (m, 1H),
1
3
.91-5.90 (m, 2H), 4.62 (s, 1H, OH), 3.42-3.39 (m, 2H), 3.29-3.26 (m, 2H). C NMR (100.5 MHz,
Chloroform-d
+
1
): δ 153.1, 135.9, 126.7, 124.5, 123.8, 121.2, 121.1, 112.2, 29.6, 24.0. MS (EI): m/z [M] :
146.1 (100).
4.2.2 5,8-Dihydronaphthalen-1-yl trifluoromethanesulfonate (3)
A 2-5 ml processor vial was charged with 2 (256 mg, 1.75 mmol), N-phenyl-bis(trifluoromethane
sulfonamide) (614 mg, 1.74 mmol), K CO (734 mg, 5.31 mmol) and THF (3 mL). The vial was sealed
2
3
and irradiated in the reactor cavity at 120 °C for 6 min. After cooling to room temperature, the
reaction mixture was filtered through a plug of celite which was rinsed with ethyl acetate. The filtrate
was concentrated under reduced pressure affording the crude product, which was purified by silica
gel flash column chromatography using ethyl acetate/petroleum ether 1:19 as eluent, to get 383 mg
1
of 3 as yellow oil, in 79% isolated yield. H NMR (399.8 MHz, Chloroform-d
7
1
): δ 7.23 (t, J = 7.9 Hz, 1H),
): δ
), 118.5, 29.4, 24.4. MS (EI):
1
3
.16-7.11 (m, 2H), 5.92-5.91 (m, 2H), 3.46-3.39 (m, 4H). C NMR (100.5 MHz, Chloroform-d
47.9, 137.6, 128.4, 127.6, 127.1, 124.0, 123.1, 118.6 (d, J = 320 Hz, CF
1
1
3
+
m/z [M] : 278.0 (100).
4.2.3 1a,2,7,7a-Tetrahydronaphtho[2,3-b]oxiren-3-yl trifluoromethanesulfonate [(±)-4]
A solution of 3 (200 mg, 0.72 mmol) in dichloromethane (10 mL) was cooled to 0 °C and m-
chloroperbenzoic acid (186 mg, 1.08 mmol) was added. The reaction mixture was gradually warmed
to room temperature and stirring was continued over night (16 h). An aqueous solution of saturated
NaHCO
3
(5 mL) was added to quench the excess of acid and the mixture was stirred for 30 min.
Phases were separated and the organic layer was washed with brine (2 x 5 mL), dried over MgSO
4
,
filtered and the solvent was evaporated under reduced pressure affording the crude product, which
was purified by silica gel flash column chromatography using 15% ethyl acetate in petroleum ether,
1
to get 159 mg of (±)-4 as white solid, in 77% isolated yield. H NMR (399.8 MHz, Chloroform-d
1
): δ
7
.25-7.21 (m, 1H), 7.13-7.09 (m, 2H), 3.57-3.49 (m, 3H), 3.42-3.37 (m, 1H), 3.26-3.22 (m, 1H), 3.09-
1
3
3.04 (m, 1H). C NMR (100.5 MHz, Chloroform-d
1
): δ 148.3, 135.1, 129.5, 127.8, 125.3, 119.5, 118.7
+
(d, J = 318 Hz, CF
3
), 51.3, 50.7, 29.8, 24.1. HRMS for C11
H
9
F
3
O
4
S [M+H] : 295.0252, Found: 295.0251.

4.2.4 6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-1-yl trifluoromethane-
sulfonate [(±)-5] and 7-Hydroxy-6-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-1-yl
trifluoromethanesulfonate [(±)-6]
A solution of (±)-4 (60.0 mg, 0.20 mmol) in MeCN (5 mL) was treated with lithium perchlorate (32.0
mg, 0.30 mmol) under nitrogen flow and stirred at room temperature until complete dissolution of
the salt. Then 4-phenylpiperidine (138 mg, 0.86 mmol), was added and the reaction mixture was
heated and refluxed for 16 h. After cooling to room temperature, MeCN was concentrated under
reduced pressure affording the crude product, which was a mixture of the two regioisomers (±)-5
and (±)-6. The crude product was purified by silica gel flash column chromatography using 30% ethyl
acetate in petroleum ether, affording 30 mg of compound (±)-5 as white solid and 45 mg of
compound (±)-6 as off-white solid, in 33% and 49% isolated yields, respectively. (±)-5 was re-
1
crystallized from petroleum ether. H NMR (399.8 MHz, Chloroform-d
1
): δ 7.35-7.31 (m, 2H), 7.26-
7
1
.22 (m, 4H), 7.14 (dd, J = 7.5, 3.5 Hz, 2H), 4.35 (s, 1H, OH), 3.88 (td, J = 10.0, 6.0 Hz, 1H), 3.50 (dd, J =
6.8, 6.0 Hz, 1H), 3.04-2.75 (m, 6H), 2.70 (dd, J = 16.8, 10.0 Hz, 1H), 2.57 (tt, J = 11.6, 3.9 Hz, 1H), 2.39
1
3
(td, J = 11.6, 1.8 Hz, 1H), 1.96-1.72 (m, 4H). C NMR (100.5 MHz, Chloroform-d
1
): δ 148.2, 146.0,
1
4
>
7
3
38.7, 129.3, 128.6, 127.7, 127.5, 126.9, 126.5, 119.6 (d, J = 318 Hz, CF
3
), 119.1, 65.8, 65.3, 53.7, 45.3,
+
3.0, 34.3, 33.9, 32.4, 26.5. HRMS for C22
99%. (±)-6 was re-crystallized from petroleum ether. H NMR (399.8 MHz, Chloroform-d
H
24
F
3
NO
4
S [M+H] : 456.1456, Found: 456.1451. HPLC purity
1
1
): δ 7.65-
.61 (m, 2H), 7.57-7.50 (m, 4H), 7.47-7.41 (m, 2H), 4.67 (s, 1H, OH), 4.21 (td, J = 10.2, 5.7 Hz, 1H),
.68 (dd, J = 16.4, 5.7 Hz, 1H), 3.42 (dd, J = 15.9, 4.5 Hz, 1H), 3.31 (dt, J = 10.5, 3.4 Hz, 1H), 3.23-3.04
1
3
(m, 5H), 2.92 (tt, J = 11.9, 4.1 Hz, 1H), 2.75 (td, J = 11.4, 2.4 Hz, 1H), 2.29-2.07 (m, 4H). C NMR (100.5
MHz, Chloroform-d
J = 318 Hz, CF
1
): δ 148.4, 146.0, 137.8, 129.4, 128.6, 128.4, 127.8, 127.0, 126.5, 119.0, 118.8 (d,
+
3
), 66.0, 65.1, 53.7, 45.2, 42.8, 38.0, 34.2, 33.8, 20.8. HRMS for C22
H
24
F
3
NO
4
S [M+H] :
456.1456, Found: 456.1458. HPLC purity > 99%.
2D NMR experiments (COSY, HMBC) were also performed and confirmed the assignment of both
regioisomers, according ref. [33].
.2.4 General procedure for synthesis of compounds (±)-7a-n and (±)-8
To a 2-5 mL processor vial, (±)-5 or (±)-6 (1 equiv.), amine (3 equiv.), Cs CO
4
2
3
(3 equiv.), Mo(CO) (1-1.5
6
equiv.), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos, 7.5 mol%), trans-di(μ-
acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium(II) (Herrmann’s palladacycle, 2.5 mol%), 4-
(dimethylamino)pyridine (DMAP, 2 equiv.) and either 1,4-dioxane or MeCN was added. The reaction
was sealed and irradiated for 3-8 h at 160 °C. The reaction mixture was allowed to cool to room
temperature before filtration. The filtrate was washed with ethyl acetate and concentrated in vacuo.
The crude product was purified by silica gel flash column chromatography using either ethyl acetate
in i-hexane or methanol and triethylamine (NEt ) in dichloromethane, followed by a second
3
purification on semi-preparative reversed phase HPLC to isolate the compounds (±)-7a-n and (±)-8 as
their TFA-salts.
4.2.4.1
6-hydroxy-7-(4-phenylpiperidin-1-yl)-N,N-dipropyl-5,6,7,8-tetrahydro-naphthalene-1-
carboxamide [(±)-7a]
Synthesized according to the general procedure using (±)-5 (55.8 mg, 0.12 mmol), di-N-propylamine
(62.0 mg, 0.61 mmol), Cs
2
CO
3
(122 mg, 0.37 mmol), Mo(CO) (32.6 mg, 0.12 mmol), Xphos (4.3 mg, 9
6
μmol), Herrmann’s palladacycle (2.9 mg, 3 μmol) and 1,4-dioxane (1.5 mL) and irradiated for 8 h.

Crude product was purified first by silica gel flash column chromatography with 1:4 to 1:2 ethyl
acetate in i-hexane, followed by purification on semi-preparative HPLC (MeCN/H
mg of compound (±)-7a was isolated as the TFA-salt (16% yield). H NMR (399.8 MHz, Methanol-d
2
O (0.1% TFA)). 10.5
) δ
1
4
7
–
3
1
.36 – 7.23 (m, 7H), 7.13 (d, J = 7.2 Hz, 1H), 4.24 (s, 1H), 3.75 – 3.36 (m, 7H), 3.23 – 2.87 (m, 6H), 2.25
1.98 (m, 4H), 1.67 (d, J = 83.6 Hz, 4H), 1.04 (t, J = 7.4 Hz, 3H), 0.92 – 0.85 (m, 1H), 0.76 (t, J = 7.4 Hz,
1
3
H). C NMR (100.5 MHz, , Methanol-d ) δ 172.8, 144.9, 137.5, 136.1, 130.8, 129.8, 128.27, 128.0,
4
27.7, 125.5, 87.1, 85.5, 70.0, 68.2, 66.2, 52.1, 47.9, 41.1, 39.6, 32.0, 31.6, 22.8, 21.9, 11.9, 11.4 (One
+
aromatic carbon missing). HRMS for C30
H
39
F
3
N
2
O
4
[M+H] : 435.3012, Found: 435.3008. HPLC purity >
90%.
4.2.4.2
(6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydronaphthalen-1-yl)(4-isopropyl-
piperidin-1-yl)methanone [(±)-7b]
Synthesized according to the general procedure using compound (±)-5 (50.0 mg, 0.11 mmol), 4-
isopropylpiperidine (67.0 mg, 0.55 mmol), Cs
2
CO
3
(107 mg, 0.33 mmol), Mo(CO) (30.0 mg, 0.11
6
mmol), Xphos (3.9 mg, 8 µmol), Herrmanns palladacycle (2.6 mg, 3 µmol), DMAP (26.8 mg, 0.22 mol)
and 1,4-dioxane (1.5 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash
column chromatography with 1:1 ethyl acetate in i-hexane, followed by purification on semi-
preparative HPLC (MeCN/H
2
O (0.1% TFA)). 10.0 mg of compound (±)-7b was isolated as the TFA-salt
) δ 7.37 – 7.21 (m, 7H), 7.17 – 7.08 (m, 1H), 4.79 – 4.70
1
(16% yield). H NMR (399.8 MHz, Methanol-d
4
(
1
m, 1H), 4.33 – 4.18 (m, 1H), 3.80 – 3.39 (m, 6H), 3.28 – 2.76 (m, 7H), 2.11 (d, J = 32.6 Hz, 4H), 1.93 –
.85 (m, 1H), 1.76 – 1.61 (m, 1H), 1.55 – 1.11 (m, 4H), 0.97 – 0.89 (m, 6H). C NMR (100.5 MHz,
1
3
Chloroform-d
1
) δ 143.1, 129.9, 129.8, 129.0, 127.4, 127.4, 127.3, 126.9, 124.9, 124.5, 52.5, 52.32,
4
8.4, 48.0, 46.9, 46.4, 42.9, 42.6, 40.7, 38.4, 32.5, 30.7, 30.4, 29.8, 29.4, 19.8, 19.7 (The carbonyl
+
carbon was not detected). HRMS for C32
H
41
F
3
N
2
O
4
[M+H] : 461.3168, Found: 461.3166. HPLC purity >
93%.
4.2.4.3
N-ethyl-6-hydroxy-N-(2-hydroxyethyl)-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-
naphthalene-1-carboxamide [(±)-7c]
Synthesized according to the general procedure using compound (±)-5 (39.2 mg, 0.09 mmol), 2-
ethylaminoethanol (22.9 mg, 0.26 mmol), Cs
2
CO
3
(89.3 mg, 0.27 mmol), Mo(CO) (34.2 mg, 0.13
6
mmol), Xphos (4.0 mg, 8 µmol), Herrmanns palladacycle (2.3 mg, 2 µmol), DMAP (21.3 mg, 0.17 mol)
and MeCN (2.5 mL) and irradiated for 3 h. Crude product was purified first by silica gel flash column
chromatography with 1:19 methanol in dichloromethane with 0.1% NEt
3
, followed by two
O (0.1% TFA)). 8.1 mg of compound (±)-7c was
, a few drops of D O) δ 7.37 –
purifications on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (18%) yield. H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2
7
.17 (m, 7H), 7.13 – 7.04 (m, 1H), 4.25 – 4.15 (m, 1H), 3.82 – 3.75 (m, 1H), 3.68 – 3.20 (m, 10H), 2.95
1
3
–
2.83 (m, 3H), 2.17 – 2.00 (m, 4H), 1.23 (t, J = 7.1 Hz, 1H), 1.01 (t, J = 7.1 Hz, 2H). C NMR (100.5
, a few drops of D O) δ 172.2, 145.0, 130.3, 130.2, 129.7, 128.0, 127.8, 127.8,
27.7, 127.7, 124.7, 67.6, 65.8, 60.1, 59.1, 47.1, 45.0, 40.3, 40.2, 39.0, 31.4, 31.0, 14.0, 13.0. HRMS
MHz, Acetonitrile-d
1
3
2
+
for C28
H
35
F
3
N
2
O
5
[M+H] : 423.2648, Found: 423.2639. HPLC purity > 99%.
4.2.4.4
(6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydronaphthalen-1-yl)(4-(hydroxyl-
methyl)piperidin-1-yl)methanone [(±)-7d]
Synthesized according to the general procedure using compound (±)-5 (63.6 mg, 0.14 mmol), 4-
piperidinemethanol (49.5 mg, 0.43 mmol), Cs
2
CO
3
(150 mg, 0.46 mmol), Mo(CO) (39.4 mg, 0.15
6

mmol), Xphos (5.0 mg, 10 µmol), Herrmanns palladacycle (3.6 mg, 4 µmol), DMAP (36.6 mg, 0.30
mol) and 1,4-dioxane (3 mL) and MeCN (0.5 mL) and irradiated for 3 h. Crude product was purified
first by silica gel flash column chromatography with 1:19 methanol in dichloromethane with 1% NEt ,
3
followed by two purifications on semi-preparative HPLC (MeCN/H
compound (±)-7d was isolated as the TFA-salt (17% yield). H NMR (399.8 MHz, Acetonitrile-d
2
O (0.1% TFA)). 13.6 mg of
, a few
1
3
drops of D
2
O) δ 7.37 – 7.32 (m, 2H), 7.30 – 7.18 (m, 5H), 7.06 (dd, J = 22.5, 7.3 Hz, 1H), 4.60 (t, J =
1
1
3.1 Hz, 1H), 4.28 – 4.14 (m, 1H), 3.67 – 3.33 (m, 8H), 3.30 – 3.09 (m, 2H), 2.98 – 2.73 (m, 5H), 2.17 –
1
3
.98 (m, 5H), 1.87 – 1.56 (m, 3H), 1.24 – 1.18 (m, 1H). C NMR (100.5 MHz, Acetonitrile-d
O) δ 170.2, 145.0, 135.8, 130.4, 129.7, 129.4, 128.1, 128.0, 127.9, 127.7, 124.9, 85.4, 67.7,
7.0, 66.7, 65.7, 51.5, 47.6, 42.5, 40.3, 39.5, 39.4, 39.0, 31.4, 31.1, 9.1. HRMS for C30
3
, a few
drops of D
6
2
H
37
F
3
N
2
O
5
+
[
M+H] : 449.2804, Found: 449.2822. HPLC purity > 92%.
4.2.4.5
3-(4-(6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-naphthalene-1-carbonyl)-
piperazin-1-yl)propanenitrile [(±)-7e]
Synthesized according to the general procedure using compound (±)-5 (52.3 mg, 0.12 mmol), 3-(1-
piperazinyl)-propionitrile (33.6 mg, 0.24 mmol), Cs
2
CO
3
(119 mg, 0.37 mmol), Mo(CO) (38.7 mg, 0.15
6
mmol), Xphos (4.7 mg, 10 µmol), Herrmanns palladacycle (3.2 mg, 3 µmol), DMAP (30.2 mg, 0.25
mol) and 1,4-dioxane (2 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash
column chromatography with 1:4 methanol in dichloromethane with 1% NEt
3
, followed by
O (0.1% TFA)). 15.8 mg of compound (±)-7e was
) δ 7.69 (br. s, 1H), 7.39 –
purification on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (22% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
7
.34 (m, 2H), 7.32 – 7.23 (m, 5H), 7.16 – 7.13 (m, 1H), 4.24 (td, J = 10.3, 5.6 Hz, 2H), 3.68 – 3.40 (m,
1
3
11H), 3.30 (dd, J = 16.2, 5.6 Hz, 2H), 3.25 – 3.03 (m, 3H), 3.01 – 2.84 (m, 5H), 2.26 – 2.02 (m, 4H). C
) δ 169.6, 144.9, 135.8, 135.0, 131.0, 129.7, 128.1, 127.9, 127.8,
NMR (100.5 MHz, Acetonitrile-d
3
1
27.6, 125.4, 117.8, 67.9, 67.3, 65.7, 52.9, 52.7, 52.5, 51.5, 44.6, 40.2, 39.3, 39.0, 31.2, 14.2. HRMS
+
for C31
H
37
F
3
N
4
O
4
[M+H] : 473.2917, Found: 473.2904. HPLC purity > 99%.
4.2.4.6
N-(cyclopropylmethyl)-6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-
tetrahydronaphthalene-1-carboxamide [(±)-7f]
Synthesized according to the general procedure using compound (±)-5 (62.0 mg, 0.14 mmol),
aminomethylcyclopropane (29.1 mg, 0.41 mmol), Cs
0
0
2
CO
.22 mmol), Xphos (4.8 mg, 10 µmol), Herrmanns palladacycle (3.1 mg, 3 µmol), DMAP (33.5 mg,
.27 mol) and MeCN (2 mL) and irradiated for 5 h. Crude product was purified first by silica gel flash
, followed by
O (0.1% TFA)). 15.1 mg of compound (±)-7f was
, a few drops D O) δ 7.37 –
3
(133 mg, 0.41 mmol), Mo(CO)
6
(57.3 mg,
column chromatography with 1:19 methanol in dichloromethane with 0.5% NEt
3
purification on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (21% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2
7
3
2
1
3
.32 (m, 2H), 7.30 – 7.22 (m, 6H), 4.19 (td, J = 10.2, 5.6 Hz, 1H), 3.61 – 3.47 (m, 3H), 3.44 – 3.33 (m,
H), 3.28 – 3.14 (m, 4H), 2.96 – 2.85 (m, 2H), 2.22 – 1.99 (m, 4H), 1.12 – 1.01 (m, 1H), 0.53 – 0.47 (m,
1
3
H), 0.28 – 0.23 (m, 2H). C NMR (100.5 MHz, Acetonitrile-d
3
, a few drops D O) δ 170.0, 144.9,
2
37.8, 135.6, 131.4, 130.7, 129.7, 127.9, 127.9, 127.6, 126.4, 68.5, 65.9, 52.4, 49.3, 44.8, 40.3, 39.1,
+
1.4, 31.1, 26.3, 11.6, 3.9, 3.8. HRMS for C28
H
33
F
3
N
O
2 4
[M+H] : 405.2542, Found: 405.2550. HPLC
purity > 99%.
4.2.4.7 6-hydroxy-N-(4-methoxyphenyl)-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydronaphthalene-
1-carboxamide [(±)-7g]

Synthesized according to the general procedure using compound (±)-5 (50.0 mg, 0.11 mmol), 4-
methoxyaniline (29.1 mg, 0.54 mmol), Cs CO (108 mg, 0.33 mmol), Mo(CO) (29.0 mg, 0.22 mmol),
Xphos (3.9 mg, 8 µmol), Herrmanns palladacycle (2.7 mg, 3 µmol), DMAP (28.8 mg, 0.22 mol) and
,4-dioxane (2 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash column
chromatography with 1:1 ethyl acetate in i-hexane, followed by purification on semi-preparative
2
3
6
1
HPLC (MeCN/H
H NMR (399.8 MHz, Acetonitrile-d
2
O (0.1% TFA)). 11.0 mg of compound (±)-7g was isolated as the TFA-salt (18% yield).
) δ 8.57 (s, 1H), 7.61 – 7.56 (m, 2H), 7.44 (dd, J = 6.7, 2.3 Hz, 1H),
1
3
7
.37 – 7.21 (m, 7H), 6.97 – 6.92 (m, 2H), 4.20 (td, J = 9.9, 5.5 Hz, 1H), 3.79 (s, 3H), 3.64 – 3.55 (m, 2H),
.52 – 3.44 (m, 2H), 3.38 – 3.21 (m, 4H), 2.97 – 2.85 (m, 2H), 2.32 – 2.19 (m, 1H), 2.11 – 2.02 (m, 3H).
3
1
3
C NMR (100.5 MHz, Chloroform-d ) δ 167.9, 157.4, 142.9, 135.9, 135.2, 131.8, 130.6, 130.3, 129.0,
1
1
9
127.7, 127.4, 126.8, 125.6, 122.7, 114.6, 77.4, 68.0, 66.3, 55.7, 53.0, 46.8, 40.5, 38.4, 30.5, 25.4.
F
+
NMR (376.5 MHz, Acetonitrile-d
3
) δ -75.6. HRMS for C31
H
33
3
F N
2
O
5
[M+H] : 457.2491, Found:
457.2512. HPLC purity > 99%.
4.2.4.8 6-hydroxy-7-(4-phenylpiperidin-1-yl)-N-(3-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydro-
naphthalene-1-carboxamide [(±)-7h]
Synthesized according to the general procedure using compound (±)-5 (51.0 mg, 0.11 mmol), 3-
(trifluoromethyl)aniline (53.7 mg, 0.33 mmol), Cs
2
CO
3
(110 mg, 0.34 mmol), Mo(CO) (31.8 mg, 0.12
6
mmol), Xphos (5.2 mg, 11 µmol), Herrmanns palladacycle (2.6 mg, 3 µmol), DMAP (27.7 mg, 0.23
mol) and 1,4-dioxane (3 mL) and MeCN (0.5 mL) and irradiated for 8 h. Crude product was purified
first by silica gel flash column chromatography with 1:19 methanol in dichloromethane with 1% NEt ,
3
followed by purification on semi-preparative HPLC (MeCN/H
2
O (0.1% TFA)). 6.8 mg of compound (±)-
) δ 9.87 (s, NH), 8.34 (s,
1
7
1
7
5
1
1
4
h was isolated as the TFA-salt (10% yield). H NMR (399.8 MHz, Acetone-d
6
H), 8.02 (d, J = 8.3 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.36 – 7.30 (m, 6H), 7.25 –
.20 (m, 1H), 4.26 (td, J = 9.8, 5.4 Hz, 1H), 3.80 – 3.56 (m, 4H), 3.54 – 3.34 (m, 3H), 3.30 (dd, J = 16.0,
1
3
.4 Hz, 1H), 3.03 – 2.93 (m, 4H), 2.50 – 2.38 (m, 2H). C NMR (100.5 MHz, Acetonitrile-d ) δ 168.8,
3
45.1, 140.6, 136.8, 136.5, 136.4, 132.1, 131.6, 131.3, 131.2, 130.8, 129.7, 127.8, 127.8, 127.6, 126.9,
26.8, 124.5, 121.5, 68.3, 66.4, 52.6, 48.6, 40.3, 39.3, 31.3, 31.1, 26.3. HRMS for C29
95.2233, Found: 495.2259. HPLC purity > 99%.
+
H
29
F
3
N
2
O
2
[M+H] :
4.2.4.9
N-benzyl-6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-naphthalene-1-carbox-
amide [(±)-7i]
Synthesized according to the general procedure using compound (±)-5 (54.8 mg, 0.12 mmol),
benzylamine (36.7 mg, 0.36 mmol), Cs CO (139 mg, 0.43 mmol), Mo(CO) (63.4 mg, 0.24 mmol),
Xphos (8.7 mg, 18 µmol), Herrmanns palladacycle (5.6 mg, 6 µmol), DMAP (29.0 mg, 0.24 mol) and
,4-dioxane (1.5 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash column
2
3
6
1
chromatography with ethyl acetate with 1% NEt
MeCN/H
NMR (399.8 MHz, Acetonitrile-d
3
, followed by purification on semi-preparative HPLC
O (0.1% TFA)). 19.8 mg of compound (±)-7i was isolated as the TFA-salt (30% yield). H
1
(
2
3
) δ 9.15 (br. s, 1H), 7.44 – 7.22 (m, 13H), 5.23 (br. s, 1H), 4.54 (d, J =
.2 Hz, 2H), 4.17 (td, J = 10.1, 5.6 Hz, 1H), 3.61 – 3.50 (m, 2H), 3.47 – 3.39 (m, 1H), 3.29 (ddd, J = 28.3,
6
1
1
3
6.1, 5.5 Hz, 2H), 3.22 – 3.09 (m, 3H), 2.94 – 2.81 (m, 2H), 2.31 – 2.16 (m, 1H), 2.11 – 2.01 (m, 3H). C
) δ 170.0, 145.0, 140.4, 137.5, 136.0, 131.4, 131.0, 129.7, 129.6,
NMR (100.5 MHz, Acetonitrile-d
3
1
28.6, 128.1, 127.9, 127.8, 127.7, 126.3, 68.3, 66.3, 52.6, 48.6, 43.9, 40.4, 39.2, 31.3, 31.1, 26.2.
+
HRMS for C31
H
33
F
3
N
2
O
4
[M+H] : 441.2542, Found: 441.2527. HPLC purity > 99%.

Resolution of compound (±)-7i
Chromatographic resolution of compound (±)-7i was accomplished by SFC (30% MeOH and 0.2%
diethylamine in CO
retention time, 2.95 min). The specific rotation, [α]589, of (+)-7i was 34.1° and the optical rotation
2
) to give 33.0 mg of (+)-7i (retention time, 2.2 min) and 33.3 mg of (-)-7i
(
was 0.058° (c 0.17 g/100 mL in acetone, 23 °C). The specific rotation,[α]589, of (-)-7i was -26.4° and
the optical rotation was -0.058° (c 0.22 g/100 mL in acetone, 23 °C). The enantiomeric excess of (+)-7i
was 99% and the enantiomeric excess of (-)-7i was 99%.
4.2.4.10 N-benzyl-6-hydroxy-N-methyl-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydronaphthalene-1-
carboxamide [(±)-7j]
Synthesized according to the general procedure using compound (±)-5 (56.2 mg, 0.12 mmol), N-
methylbenzylamine (45.1 mg, 0.37 mmol), Cs
2
CO
3
(133 mg, 0.41 mmol), Mo(CO) (50.5 mg, 0.19
6
mmol), Xphos (5.8 mg, 12 µmol), Herrmanns palladacycle (3.4 mg, 4 µmol), DMAP (33.8 mg, 0.28
mol) and 1,4-dioxane (2 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash
column chromatography with 1:99 methanol in dichloromethane with 0.5% NEt
3
, followed by
O (0.1% TFA)). 24.7 mg of compound (±)-7j was
, a few drops of D O) δ 7.46 –
purification on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (34% yield). H NMR (399.8 MHz, Acetonitrile -d
2
1
3
2
7
.40 (m, 3H), 7.39 – 7.05 (m, 16H), 4.37 (s, 1H), 4.17 (td, J = 10.3, 5.6 Hz, 1H), 3.61 – 3.48 (m, 3H),
13
3
.26 – 3.16 (m, 2H), 3.08 (s, 2H), 2.94 – 2.83 (m, 3H), 2.74 (s, 2H), 2.20 – 1.97 (m, 6H). C NMR (100.5
, a few drops of D O) δ 172.5, 171.8, 145.0, 144.8, 138.3, 137.6, 135.7, 130.6,
30.5, 129.8, 129.8, 129.7, 129.4, 128.7, 128.6, 128.2, 128.2, 128.0, 127.9, 127.9, 127.7, 127.7, 125.1,
25.0, 67.3, 65.6, 51.9, 51.0, 40.4, 40.3, 38.9, 38.8, 37.0, 34.1, 31.3, 31.2, 31.0, 31.0. Because (±)-7j
MHz, Acetonitrile-d
1
1
3
2
1
13
exists as two conformers, the integrals in the H NMR and the number of carbons in the C NMR
does not add up. After running the NMR at 50°C, the integrals in the H NMR adds up. There are still
1
1
3
1
too many signals in the C NMR but fewer than when the sample was run at room temperature. H
NMR (399.8 MHz, Acetonitrile-d , a few drops of D O, 60°C) δ 7.48 – 7.40 (m, 2H), 7.40 – 7.18 (m,
H), 7.18 – 7.06 (m, 2H), 4.81 – 4.66 (m, 1H), 4.38 (s, 1H), 4.15 (s, 1H), 3.64 – 3.41 (m, 2H), 3.39 – 2.99
3
2
9
1
3
(
m, 5H), 2.98 – 2.83 (m, 3H), 2.76 (s, 2H), 2.29 – 2.15 (m, 1H), 2.15 – 2.02 (m, 4H). C NMR (100.5
MHz, Acetonitrile-d , a few drops of D O, 60°C) δ 171.8, 136.2, 130.7, 130.6, 129.9, 129.6, 129.4,
28.8, 128.8, 128.5, 128.4, 128.2, 128.1, 128.1, 127.9, 125.5, 125.3, 68.3, 66.4, 55.6, 51.3, 49.8, 49.2,
3
2
1
4
1
9
0.7, 39.2, 37.1, 34.1, 31.5, 31.3, 26.2. F NMR (376.5 MHz, Acetonitrile-d
3
) δ -75.6. HRMS for
+
C
32
H
35
F
3
N
2
O
4
[M+H] : 455.2699, Found: 455.2705. HPLC purity > 99%.
4.2.4.11
6-hydroxy-N-(4-methoxybenzyl)-7-(4-phenylpiperidin-1-yl)-5,6,7,8-
tetrahydronaphthalene-1-carboxamide [(±)-7k]
Synthesized according to the general procedure using compound (±)-5 (57.8 mg, 0.13 mmol), 4-
methoxybenzylamine (52.5 mg, 0.38 mmol), Cs
2
CO
3
(125 mg, 0.39 mmol), Mo(CO) (53.3 mg, 0.20
6
mmol), Xphos (5.2 mg, 11 µmol), Herrmanns palladacycle (3.3 mg, 4 µmol), DMAP (30.5 mg, 0.25
mol) and 1,4-dioxane (2 mL) and irradiated for 5 h. Crude product was purified first by silica gel flash
column chromatography with 1:99 methanol in dichloromethane with 0.5% NEt
3
, followed by
O (0.1% TFA)). 19.6 mg of compound (±)-7k was
, a few drops of D O) δ 7.38 –
purification on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (26% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2
7
.22 (m, 10H), 6.95 – 6.90 (m, 2H), 4.51 – 4.37 (m, 2H), 3.73 (s, 3H), 3.58 – 3.47 (m, 2H), 3.46 – 3.39
13
(m, 1H), 3.29 – 3.05 (m, 5H), 2.93 – 2.81 (m, 2H), 2.21 – 1.97 (m, 4H). C NMR (100.5 MHz, Acetone-

d
6
, a few drops of D O) δ 170.1, 159.6, 145.0, 137.1, 135.7, 132.2, 131.1, 130.9, 130.0, 129.4, 127.5,
2
1
C
27.5, 127.5, 126.2, 114.6, 67.9, 65.7, 55.5, 51.7, 48.9, 43.1, 40.4, 39.0, 31.3, 31.0, 26.4. HRMS for
+
32
H
35
F
3
N
2
O
5
[M+H] : 471.2648, Found: 471.2639. HPLC purity > 99%.
4.2.4.12 N-(4-fluorobenzyl)-6-hydroxy-7-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydronaphthalene-1-
carboxamide [(±)-7l]
Synthesized according to the general procedure using compound (±)-5 (56.2 mg, 0.12 mmol), 4-
fluorobenzylamine (46.0 mg, 0.37 mmol), Cs
2
CO
3
(119 mg, 0.37 mmol), Mo(CO) (52.1 mg, 0.20
6
mmol), Xphos (4.6 mg, 10 µmol), Herrmanns palladacycle (2.8 mg, 3 µmol), DMAP (29.5 mg, 0.24
mol) and 1,4-dioxane (2 mL) and irradiated for 5 h. Crude product was purified first by silica gel flash
column chromatography with 1:99 methanol in dichloromethane with 0.5% NEt
3
, followed by
O (0.1% TFA)). 26.5 mg of compound (±)-7l was
) δ 7.46 – 7.40 (m, 2H), 7.38 –
purification on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (37% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
7
–
–
1
.32 (m, 2H), 7.31 – 7.22 (m, 6H), 7.15 – 7.08 (m, 2H), 4.50 (s, 2H), 4.15 (td, J = 10.2, 5.6 Hz, 1H), 3.60
3.50 (m, 2H), 3.47 – 3.39 (m, 1H), 3.32 – 3.08 (m, 5H), 2.95 – 2.80 (m, 2H), 2.28 – 2.13 (m, 1H), 2.10
1
3
2.00 (m, 3H). C NMR (100.5 MHz, Acetonitrile-d
3
) δ 170.3, 162.9 (d, J = 242.8 Hz), 145.0, 137.2,
36.4 (d, J = 3.2 Hz), 136.0, 131.5, 130.9, 130.6 (d, J = 8.4 Hz), 129.7, 127.9, 127.8, 127.7, 126.3, 116.2
(
[
d, J = 21.5 Hz), 68.0, 66.0, 52.2, 48.7, 43.1, 40.4, 39.1, 31.2, 31.0, 26.3 .HRMS for C31
M+H] : 459.2448, Found: 459.2433. HPLC purity > 99%.
H
32
4
F N
2
O
4
+
4.2.4.13
6-hydroxy-7-(4-phenylpiperidin-1-yl)-N-(thiazol-2-ylmethyl)-5,6,7,8-tetrahydro-
naphthalene-1-carboxamide [(±)-7m]
Synthesized according to the general procedure using compound (±)-5 (56.2 mg, 0.12 mmol), 2-
aminomethylthiazole (60.4 mg, 0.53 mmol), Cs
2
CO
3
(130 mg, 0.40 mmol), Mo(CO) (54.6 mg, 0.21
6
mmol), Xphos (4.4 mg, 9 µmol), Herrmanns palladacycle (2.9 mg, 3 µmol), DMAP (31.5 mg, 0.26 mol)
and 1,4-dioxane (2.5 mL) and irradiated for 8 h. Crude product was purified first by silica gel flash
column chromatography with 1:99 methanol in dichloromethane with 0.5% NEt
3
, followed by two
O (0.1% TFA)). 3.1 mg of compound (±)-7m was
, a few drops of D O) δ 7.73
purifications on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (5% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2
(d, J = 3.3 Hz, 1H), 7.49 (d, J = 3.3 Hz, 1H), 7.38 – 7.33 (m, 3H), 7.31 – 7.26 (m, 4H), 7.26 – 7.23 (m,
1
H), 4.81 (s, 2H), 4.19 (td, J = 10.2, 5.6 Hz, 1H), 3.61 – 3.46 (m, 3H), 3.44 – 3.27 (m, 3H), 3.26 – 3.16
1
3
(m, 1H), 2.95 – 2.86 (m, 2H), 2.24 – 1.97 (m, 5H). C NMR (100.5 MHz, Acetonitrile-d , a few drops of
3
D
2
O) δ 170.5, 145.0, 143.3, 136.5, 135.9, 131.9, 131.0, 129.7, 127.9, 127.87, 127.7, 127.6, 126.5, 68.0,
+
65.7, 52.1, 49.0, 41.7, 40.3, 39.0, 31.3, 31.0, 26.4. HRMS for C28
48.2047. HPLC purity > 99%.
H
30
F
3
N
3
O
4
S [M+H] : 448.2059, Found:
4
4.2.4.14
6-hydroxy-7-(4-phenylpiperidin-1-yl)-N-(2-(pyridin-2-yl)ethyl)-5,6,7,8-tetrahydro-
naphthalene-1-carboxamide [(±)-7n]
Synthesized according to the general procedure using compound (±)-5 (61.7 mg, 0.14 mmol), 2-(2-
aminoethyl)pyridine (50.0 mg, 0.41 mmol), Cs
2
CO
3
(137 mg, 0.42 mmol), Mo(CO) (56.9 mg, 0.22
6
mmol), Xphos (5.2 mg, 11 µmol), Herrmanns palladacycle (3.2 mg, 3 µmol), DMAP (34.5 mg, 0.28
mol) and 1,4-dioxane (2 mL) and irradiated for 5 h. Crude product was purified first by silica gel flash
column chromatography with 1:19 methanol in dichloromethane with 0.5% NEt
3
, followed by two
O (0.1% TFA)). 4.8 mg of compound (±)-7n was
, a few drops of D O) δ 8.70 –
purifications on semi-preparative HPLC (MeCN/H
isolated as the TFA-salt (6% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2

8
2
3
2
1
5
4
.66 (m, 1H), 8.36 (td, J = 7.9, 1.6 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.80 – 7.75 (m, 1H), 7.37 – 7.32 (m,
H), 7.30 – 7.24 (m, 3H), 7.23 – 7.17 (m, 3H), 4.17 (td, J = 10.1, 5.6 Hz, 1H), 3.81 – 3.74 (m, 2H), 3.59 –
.46 (m, 3H), 3.44 – 3.28 (m, 5H), 3.23 (dd, J = 16.0, 5.6 Hz, 1H), 3.18 – 3.09 (m, 1H), 2.96 – 2.83 (m,
1
3
H), 2.07 (s, 4H). C NMR (100.5 MHz, Acetonitrile-d
3
, a few drops of D O) δ 170.7, 156.6, 146.3,
2
45.1, 143.1, 136.5, 136.0, 131.7, 131.2, 129.7, 128.4, 127.8, 127.7, 127.9, 126.6, 125.7, 68.1, 65.9,
+
1.5, 49.9, 40.4, 39.4, 39.1, 34.7, 31.4, 31.1, 26.7. HRMS for C31
56.2654. HPLC purity > 99%.
H
34
F
3
N
3
O
4
[M+H] : 456.2651, Found:
4.2.4.15 N-benzyl-7-hydroxy-6-(4-phenylpiperidin-1-yl)-5,6,7,8-tetrahydro-naphthalene-1-carbox-
amide [(±)-8]
Synthesized according to the general procedure using compound (±)-6 (67.0 mg, 0.15 mmol),
benzylamine (47.1 mg, 0.44 mmol), Cs
Xphos (5.1 mg, 11 µmol), Herrmanns palladacycle (3.9 mg, 4 µmol), DMAP (36.4 mg, 0.30 mol) and
,4-dioxane (2 mL) and irradiated for 5 h. Crude product was purified first by silica gel flash column
chromatography with 1:99 methanol in dichloromethane with 0.5% NEt , followed by purification on
O (0.1% TFA)). 18.2 mg of compound (±)-8 was isolated as the TFA-
, a few drops of D O) δ 7.39 – 7.32 (m, 6H), 7.32 –
2
CO
3
(160 mg, 0.49 mmol), Mo(CO)
6
(59.7 mg, 0.23 mmol),
1
3
semi-preparative HPLC (MeCN/H
salt (22% yield). H NMR (399.8 MHz, Acetonitrile-d
2
1
3
2
7
4
d
1
.22 (m, 7H), 4.56 – 4.45 (m, 2H), 4.14 (td, J = 10.2, 5.6 Hz, 1H), 3.64 – 3.45 (m, 3H), 3.44 – 3.26 (m,
13
H), 3.20 – 3.10 (m, 1H), 2.97 – 2.88 (m, 2H), 2.26 – 1.99 (m, 4H). C NMR (100.5 MHz, Acetonitrile-
, a few drops of D O) δ 170.1, 145.0, 140.1, 137.3, 134.2, 132.3, 131.6, 129.7, 129.5, 128.4, 128.1,
3
2
27.9, 127.7, 127.6, 126.7, 67.4, 66.2, 52.0, 48.9, 43.8, 40.4, 36.7, 31.3, 31.1, 28.8. HRMS for
+
C
31
H
33
F
3
N
2
O
4
[M+H] : 441.2542, Found: 441.2553. HPLC purity > 99%.
4.3 In vitro binding studies
3
The VAChT assay was performed with the radioligand [ H]vesamicol (1539 GBq/µmol; Perkin Elmer)
using membrane homogenates obtained from PC-12 cells stably transfected with human VAChT gene
(obtained from Ali Roghani, Texas Tech University, Lubbock, TX, USA). The thawed membrane
preparation (~100 µg protein) was incubated with seven concentrations of test compounds (100 µM
3
–
0.1 nM), 2 nM [ H]vesamicol, and buffer (50 mM TRIS, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM
CaCl
2
, 1 mM MgCl ) in a total volume of 1 mL for 60 min at room temperature. The incubation was
2
terminated by rapid filtration through GF-B glass fibre filters presoaked in PEI (0.3%, 60 min, room
3
temperature). The receptor-bound [ H]vesamicol trapped on the filters was measured by liquid
3
scintillation counting. Nonspecific binding of [ H]vesamicol was determined with 10 µM vesamicol.
3
The equilibrium dissociation constant of [ H]vesamicol has been determined with K
D
= 2.39 nM in this
assay.
3
The σ
1
assay was performed with the radioligand (+)-[ H]pentazocine (1288 GBq/µmol; Perkin Elmer)
using membrane homogenates obtained from rat cortex (SPRD, female, 10 – 12 weeks). The thawed
membrane preparation (~100 µg protein) was incubated with seven concentrations of test
3
compounds (100 µM – 0.1 nM), 2 nM (+)-[ H]pentazocine, and buffer (50 mM TRIS, pH 7.4, 120 mM
NaCl, 5 mM KCl, 2 mM CaCl
2
, 1 mM MgCl ) in a total volume of 1 mL for 60 min at room temperature.
2
The incubation was terminated by rapid filtration through GF-B glass fibre filters presoaked in
3
polyethyleneimine (PEI) (0.3%, 60 min, room temperature). The receptor-bound (+)-[ H]pentazocine
trapped on the filters was measured by liquid scintillation counting. Nonspecific binding of (+)-
3
[
(
H]pentazocine was determined with 100 µM haloperidol. The equilibrium dissociation constant of
3
+)-[ H]pentazocine has been determined with K
D
= 6.9 nM in this assay.

3
3
The σ
2
assay was performed with the radioligand [ H]-1,3-di-o-tolylguanidine ([ H]DTG, 1761
GBq/µmol; Perkin Elmer) using membrane homogenates obtained from rat liver (SPRD, female, 10 –
1
2 weeks). The thawed membrane preparation (~100 µg protein) was incubated with seven
3
concentrations of test compounds (100 µM – 0.1 nM), 2 nM [ H]DTG, 1 µM dextrallorphan (Roche)
and buffer (50 mM TRIS, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl
2
, 1 mM MgCl ) in a total volume
2
of 1 mL for 120 min at room temperature. The incubation was terminated by rapid filtration through
GF-B glass fibre filters presoaked in PEI (0.3%, 60 min, room temperature). The receptor-bound
3
[
[
H]DTG trapped on the filters was measured by liquid scintillation counting. Nonspecific binding of
H]DTG was determined with 100 µM haloperidol. The equilibrium dissociation constant of [ H]DTG
3
3
has been determined with K
D
= 29 nM in this assay.
4.4 Radiochemistry
A small ligand screen was performed in order to find conditions for labeling of 5-substituted
benzovesamicol ligands from triflate (±)-5 (see Table S1, Supporting information). Compound (±)-7i
was labeled by adding 1.10 mg of (±)-5, 0.75 mg Pd(dba) , 2.45 mg XPhos, 0.92 mg
2
tetrabutylammonium iodide, 5 µl benzylamine and 2.5 µl triethylamine to a 900 µl pear-shaped
reaction vial. 200 µl of freshly distilled THF was added and the vial was capped and flushed with
1
1
nitrogen gas. After collection of [ C]CO in the vial, the activity was measured before 5 min heating at
50 °C commenced. After the reaction was finished, the activity was measured again whereby the
1
1
1
vial was vented and flushed with nitrogen gas to remove unreacted [ C]CO or volatile compounds.
The reaction mixture was diluted with 200 µl of MeCN to improve chromatography. Preparative
purification was performed with 30% ammonium formate in MeCN as mobile phase (flow 5 mL/min)
1
1
and [ C]-(±)-7i eluted after 12.7 min. Analytical HPLC showed >99% radiochemical purity measured
using two methods with different columns and mobile phases (see Supporting information for
1
1
chromatograms). An aliquot of (±)-7i was added to the analytical sample of [ C]-(±)-7i and retention
times were compared to confirm the identity. The synthesis was repeated twice with decay-
1
1
corrected yields 8.9% and 9.3% (decay-corrected to collection of [ C]CO) and 61%-85% of the carbon
monoxide had been converted to non-volatile products at the end of the reaction (calculated by
dividing the decay-corrected activity after the reaction with the activity after the nitrogen purge). For
1
1
experiment 1, starting with 16.4 GBq of [ C]CO, the product was isolated with 411 MBq activity after
5 min from end of bombardment. The concentration of the product was 1.2 µg/mL (volume 2.7 mL,
4
concentration derived from HPLC analysis) and the molar activity was 55 GBq/µmol at the end of
synthesis (calculated as activity/amount of substance). For experiment 2, starting with 10.1 GBq of
1
1
[
C]CO, the product was isolated with 235 MBq activity after 47 min from end of bombardment. The
concentration of the product was 1.3 µg/mL (volume 2.0 mL) and the molar activity was 78
GBq/µmol at the end of synthesis.
Supporting information
Supplementary data associated with this article can be found in the online version.
Acknowledgments
We thank Hooman Hamdi for help with the chemistry and Dr Patrik Nordeman for help with the
radiochemistry. We gratefully acknowledge the financial support from the Disciplinary domain of
Medicine and Pharmacy, Uppsala University, and the Swedish Society for Medical Research (SSMF).
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Graphical abstract