Do spiroindolines have the potential to replace vesamicol as lead compound for the development of radioligands targeting the vesicular acetylcholine transporter?

Article abstract of DOI:10.1016/j.bmc.2017.03.028

The vesicular acetylcholine transporter (VAChT) is an important target for in vivo imaging of neurodegenerative processes using positron emission tomography (PET). So far the development of VAChT PET radioligands is based on the single known lead compound

Full text of DOI:10.1016/j.bmc.2017.03.028

Accepted Manuscript
Do spiroindolines have the potential to replace vesamicol as lead compound for
the development of radioligands targeting the vesicular acetylcholine transport-
er?
Marcel Lindemann, Winnie Deuther-Conrad, Rares Moldovan, Kondapalli
Venkata Gowri Chandra Sekhar, Peter Brust, Barbara Wenzel
PII:
S0968-0896(17)30529-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.028
BMC 13626
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 January 2017
13 March 2017
15 March 2017
Please cite this article as: Lindemann, M., Deuther-Conrad, W., Moldovan, R., Sekhar, K.V.G., Brust, P., Wenzel,
B., Do spiroindolines have the potential to replace vesamicol as lead compound for the development of radioligands
targeting the vesicular acetylcholine transporter?, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.03.028
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Do spiroindolines have the potential to replace vesamicol as lead compound for the development
of radioligands targeting the vesicular acetylcholine transporter?
Marcel Lindemann1, Winnie Deuther-Conrad1, Rares Moldovan1, Kondapalli Venkata Gowri Chandra
Sekhar2, Peter Brust1, Barbara Wenzel1*
1
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Research Site
Leipzig, Germany;
2
Department of Chemistry, Birla Institute of Technology & Science, Pilani, Hyderabad Campus, Jawahar Nagar,
Hyderabad, Telangana, India;
* corresponding author
Abstract
The vesicular acetylcholine transporter (VAChT) is an important target for in vivo imaging of
neurodegenerative processes using positron-emission-tomography (PET). So far the development of
VAChT PET radioligands is based on the single known lead compound vesamicol. In this study we
investigated a recently published spiroindoline based compound class (Sluder et al. 2012), which was
suggested to have potential in the development of VAChT ligands. Therefore, we synthesized a small
series of N,N-substituted spiro[indoline-3,4’-piperidine] derivatives and determined their in vitro
binding affinities toward the VAChT. In order to investigate the selectivity, the off-target binding
toward σ1 and σ2 receptors were determined. The compounds possessed VAChT affinities with Ki
values in the range of 39 to 376 nM. Binding affinities toward the σ1 and σ2 receptors are in a similar
range indicating that the strong structural difference between the spiroindolines and vesamicol did
not improve the selectivity. The observed potential to additionally bind to σ receptors let us assume
that the herein investigated spiroindolines are not suitable to replace vesamicol as lead compound
for the development of VAChT ligands.
Keywords: VAChT, Vesamicol, Spiroindolines, PET, sigma receptors
1. Introduction
The vesicular acetylcholine transporter (VAChT) is a transmembrane protein located on presynaptic
vesicles of cholinergic neurons. It transports the neurotransmitter acetylcholine into the vesicles for
2
storage and controlled release and thus is an essential part of cholinergic neurotransmission.1,
Exclusively expressed in the presynaptic part of cholinergic neurons, the VAChT is considered as
valuable target for the investigation of cholinergic degeneration as a key feature related to the
cognitive decline in Alzheimer's Disease (AD).3-6 Noninvasive in vivo imaging based on radiolabeled
VAChT ligands using PET (positron emission tomography) or SPECT (single-photon emission
1

computed tomography) is a promising option for visualization of such cholinergic neuronal processes
and thus might support early diagnostics of AD.7, 8
So far, the development of VAChT radiotracers is based on vesamicol (trans-2-(4-
phenylpiperidino)cyclohexanol, Figure 1), the single known chemical lead compound, that binds with
high affinity to an allosteric site of the transporter.9, 10 However, radiolabeled vesamicol itself is not
suitable for specific VAChT imaging due to its insufficient selectivity caused by the additional binding
to the σ1 and σ2 receptors11, 12, which are both as well expressed in cholinergic brain regions. Because
so far only vesamicol-like structures are known to be tolerated by this allosteric binding site at
VAChT, the development of selective VAChT ligands has focused mainly on structural modifications of
vesamicol with the aim to keep or improve the affinity for VAChT and to reduce the affinity for sigma
receptors. Several classes of vesamicol analogs have been studied such as benzovesamicols,13-18
trozamicols,19, 20 morpholino vesamicols,21, 22 and azaspirovesamicols23. For some of these compounds
moderate or high binding affinities to VAChT were determined in vitro and in vivo. In particular
benzovesamicol derivatives labelled with fluorine-18 or iodine-123 have been used to investigate
cholinergic pathways in humans by measuring VAChT expression via PET imaging.14, 24, 25 However,
despite promising results none of the ligands has been proven to be optimal for clinical application so
far.
When studying the literature in detail, it turned out that the investigation of target selectivity of
vesamicol analogs has been often neglected. To get a deeper insight into the structure-affinity and
-selectivity relationship of vesamicol analogs, we recently synthesized a library of systematically
modified derivatives and investigated them regarding their in vitro binding toward the VAChT and the
off-target binding toward σ1 and σ2 receptors.26 A main result of this study was the observation that
an increase of VAChT affinity was often accompanied by an increase in σ1 and/or σ2 receptor affinity.
Finally, our results demonstrated the challenges in the development of VAChT PET imaging ligands
based on vesamicol as lead compound.
In 2012, Sluder et al. reported on N,N-substituted spiro[indoline-3,4’-piperidine] derivatives27 and the
linkage of their toxic action to the VAChT in insects.28 The authors demonstrated that the binding of
the selected spiroindoline derivative [3H]SYN876 (Figure 1) in insect tissue is related to a single
binding site in rat PC12 cells transfected with the VAChT gene of Drosophila melanogaster.
Accordingly, they proposed this compound class as alternative to vesamicol for the development of
VAChT ligands for PET imaging of neurodegenerative diseases.
In order to examine the potential of these spiroindolines as a new lead compound class for the
development of VAChT PET ligands, we i) synthesized selected derivatives of the reported series and
developed new derivatives with slight structural modifications, ii) determined their in vitro binding
2

affinity toward VAChT as well as toward σ1 and σ2 receptors and iii) investigated the binding
mechanism of SYN876 and vesamicol using rat PC12 cells transfected with rat VAChT.
Figure 1. Known VAChT ligands vesamicol, benzovesamicol and spiroindoline [3H]SYN876 as potential lead
compounds in the development of VAChT PET radiotracers
2. Results and discussion
In order to investigate the in vitro binding affinities of the N,N-substituted spiro[indoline-3,4’-
piperidine] derivatives for the VAChT, we synthesized the already published28 derivatives 2 (SYN876)
and 3 (Scheme 1). With respect to the development of an 18F-labelled PET radiotracer, we
additionally designed four new compounds (4-7) bearing fluorine containing groups substituted at
the indoline nitrogen. The in vitro binding affinities (Ki values) of all derivatives toward the VAChT and
the σ1 and σ2 receptors were determined by radioligand displacement studies. Since vesamicol binds
to an allosteric site at the vesicular acetylcholine transporter, we also investigated whether the
binding site of compound 2 is identical to the vesamicol binding site.
2.1 Chemistry
The synthesis of the derivatives 2-7 was performed on the basis of the synthetic route reported by
Sluder at al.28 In a first step, compound 1 was synthesized according to the already in detail described
procedure28 (Scheme is given in the supplementary material). Briefly, 4-chlorocinnamyl chloride was
coupled with 1,4-dioxa-8-azaspiro[4,5]decane and deprotected to the corresponding 4-piperidinone
derivative. Subsequent Wittig reaction with methoxymethyltriphenylphosphonium chloride gave the
corresponding methoxymethylenepiperidine which was allowed to react with 4-
fluorophenylhydrazine and trifluoroacetic acid in a Fischer-Indole like reaction to give the
spiroindoline derivative 1. Compound 1 served as starting material to substitute different aroyl (2-6)
groups at the secondary nitrogen of the indoline core by using triethylamine (TEA) and (benzotriazol-
3

1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (Scheme 1). For the synthesis
of compound 7 we selected a simple and mild method for introducing the 2-fluoroethylester group.
Thus, 2-fluoroethyl methanesulfonate and potassium carbonate as source for the carbonyl
functionality reacted with the amine 1 to give the carbamate 7 in moderate yield. The formation of
the corresponding N-alkylated product was not observed. Similar carbamation reactions of amines
have been already described in literature and are beneficial in terms of handling when comparing to
the common method of using alkyl chloroformates.29, 30
Scheme 1. Reagents and conditions: a) BOP, TEA, 2-chloroisonicotinic acid for 2, 2-fluoroisonicotinic acid for 3,
6-bromonicotinic acid for 4, 6-fluoronicotinic acid for 5 and 4-fluorobenzoic acid for 6, CH2Cl2, rt, 24 h; b) K2CO3,
DMF, 2-fluoroethyl methanesulfonate, 90 °C, 19 h.
2.2 In vitro binding affinities
The compounds 2-7 were assessed regarding their affinities toward rat VAChT, rat σ1 receptor and
rat σ2 receptor. The Ki values and experimental conditions are presented in Table 1.
Table 1. In vitro binding affinities of spiroindolines 2-7 and reference compounds (±)-vesamicol and
(±)-benzovesamicol
Ki (σ1)b
Ki (σ2)c
Compd
Ki (VAChT)a
( )-vesamicold
47.4 ± 6.3
20.4 ± 4.7
112 ± 36
4

( )-benzovesamicold
1.32 ± 0.25
153 ± 49
110 ± 38
39.2 ± 8.4
376 ± 95
243 ± 78
328 ± 42
26.1 ± 4.5
137 ± 32
55.4 ± 14.8
179 ± 14
282 ± 85
293 ± 47
44.1 ± 7.6
96.4 ± 22.3
1185 ± 484
748 ± 229
60.1 ± 17.8
> 10000
2
3
4
5
6
7
1205 ± 128
1520 ± 169
Ki values in nM (mean ± S.D., n≥2) were derived from IC50 values according to the Cheng-Prusoff equation: Ki =
IC50/(1+C/KD), where C is the concentration of the radioligand and KD the dissociation constant of the
b
corresponding radioligand; a (-)-[3H]vesamicol: KD = 25.6 nM to VAChT on rVAChT-PC12 cells;
(+)-
c
[3H]pentazocine: KD = 6.9 nM to σ1 receptors on rat cortex homogenates; [3H]DTG with blocking of σ1
receptors using 1 µM dextrallorphan: KD = 29.0 nM to σ2 receptors on rat liver homogenates; d Ki values were
taken from Barthel et al.26
The bromonicotinyl substituted derivative 4 shows the highest affinity for the VAChT within the
investigated spiroindoline series with a Ki value close to that of vesamicol (39.2 nM vs. 47.4 nM).
Interestingly, the substitution of the bromine of 4 by a fluorine atom to achieve compound 5 caused
an almost 10-fold reduction of the VAChT binding affinity. According to our data, the two published 28
derivatives 2 and 3 show only moderate binding to the rat VAChT with Ki values of 153 and 110 nM,
respectively. In particular compound 2 was proposed by Sluder et al.28 as alternative for the
development of VAChT PET radioligands. However, when comparing the binding affinity of 2 with (±)-
benzovesamicol (Ki(VAChT) = 1.32 nM), compound 2 is much less suitable because of a more than
100-fold lower VAChT affinity. The introduction of a fluorobenzoyl (6) and a fluoroethyl carboxylate
(7) function at the indoline nitrogen did not improve the binding affinity for VAChT compared to 2
and 3.
Surprisingly, the investigated N,N-substituted spiro[indoline-3,4’-piperidine] derivatives
demonstrated certain affinity for the σ1 receptor. The Ki values of compounds 2-7 for this target are
in the range of about 45-300 nM. Moreover, derivatives 3 and 7 even show higher binding affinity for
the σ1 receptor than for the VAChT. This observed off-target binding has to be interpreted as a
serious drawback of the compound class. Beside an improvement of the VAChT binding affinity
additionally a lowering of the σ1 receptor binding would have to be achieved when developing new
VAChT ligands based on this spiroindoline scaffold.
.
5

Generally, the binding affinities of the investigated derivatives 2-7 for the σ2 receptor are
considerably lower. Only compound 4 (Ki = 60.1 nM) shows a higher affinity for this target which
impairs its selectivity for the VAChT.
2.3 Binding sites of compound 2 and vesamicol
Besides the orthosteric binding site of VAChT for the natural ligand acetylcholine, the transporter
protein possess an allosteric binding site for the inhibitor vesamicol. To assess the potential of
compound 2 and derivatives thereof for molecular imaging of VAChT, it is crucial to analyse in detail
the mechanisms of interaction between the protein and the ligands. In a first attempt, we performed
radioligand displacement studies to investigate, if compound 2 competes with (±)-vesamicol for the
same binding site at VAChT.31
As shown in Figure 2, increasing concentrations of compound 2 (0 nM, 100 nM, 1000 nM) result in a
rightward shift of the binding curves reflecting an increase in the apparent value of Ki of (±)-
vesamicol. By linearization of this set of sigmoid curves according to Hanes-Woolf32, 33 we obtained a
pattern of almost parallel lines (Figure 3). Because such pattern can be taken as indicative for a
competition mechanism34, we assume that compound 2 and vesamicol bind to the same allosteric
binding site of VAChT. This finding is quite interesting, because so far only vesamicol like compounds
have been described to bind with comparatively high affinity to the VAChT. Obviously, the binding
pocket is more flexible than expected, since the molecular structure of compound 2 is much different
compared to vesamicol.
Figure 2. Representation of binding data as non-linearized inhibition curves (one out of three experiments; n=3
per data point).
6

Figure 3. Representation of binding data after Hanes-Woolf linearization.
4. Conclusion
In order to investigate the potential of the recently published N,N-substituted spiro[indoline-3,4’-
piperidine] derivatives28 as new lead compounds for the development of PET radioligands for imaging
of the vesicular acetylcholine transporter, we synthesized a small series of derivatives (2-7) and
determined their in vitro binding affinities for the VAChT and the main off-target binding to σ1 and σ2
receptors. According to our data, all derivatives bind with moderate to low affinities toward the
VAChT and show in addition considerable affinities for σ1 and/or σ2 receptors. Because of this
general potential to bind to σ receptors, we conclude that the herein investigated spiroindolines are
not suitable to replace vesamicol analogues as future lead compounds for the development of VAChT
PET radioligands.
5. Material and methods
5.1. Chemistry
Analysis of all compounds was performed by HRMS, HPLC, TLC, and NMR spectroscopy. The spectra
and chromatograms are given in the Supplementary material.
High resolution mass spectra were recorded on a ESI-qTOF Impact II (Bruker Daltonik GmbH) and on
a ESI-TOF micrOTOF (Bruker Daltonik GmbH) using ElectroSpray Ionization (ESI). NMR spectra (1H, 13C,
7

13C-APT, 19F, H,H-COSY, HSQC, HMBC) were recorded on spectrometers from Varian. Splitting
patterns have been designated as follows: s: singlet, d: doublet, bs: broad singlet, m: multiplet, t:
triplet, dd: doublet of doublet, td: triplet of doublet; dt: doublet of triplet.
Analytical thin-layer chromatography (TLC) was performed on silica gel coated plates (Machery-
Nagel, ALUGRAM SIL G/UV254). The spots were identified using a UV lamp or by spraying a solution of
0.1% ninhydrine in EtOH/H2O 1:10 or dipping into a KMnO4-solution (3.0 g KMnO4, 20 g K2CO3, 0.25
mL glacial acid, 300 mL water).
For purification of final products flash column chromatography was used with silica gel ZEOsorb 60 /
40-63 microns from Apollo Scientific Ltd.
The chemical purity of all compounds is ≥ 97% and was controlled by HPLC using a 150 × 3 mm
Reprosil-Pur Basic HD - 3 µm column (Dr. Maisch GmbH, Germany). These analytical chromatographic
separations were performed on a Dionex Ultimate 3000 system, incorporating a LPG-3400SD pump,
an autosampler WPS-3000 TSL, a column compartment TCC-3000SD and a diode array detector
DAD3000 (monitoring from 254-720 nm). Mixtures of acetonitrile (ACN) and aqueous 20 mM NH4OAc
were used as eluents in a linear gradient system (5 min at 25% ACN, 5-30 min to 95% ACN, 30-35 min
at 95% and 35-45 min at 25% ACN)
Chemical names of compounds were generated by ChemDraw Professional 16.0.
(E)-1'-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4'-piperidine] (1): The already published
derivative 1 was synthesized in quantities of 1.6 g according to the procedure as described.28 Rf =
1
0.54 (ethyl acetate/n-hexane/NH3 3:1:0.1); H NMR (CDCl3, 300 MHz): δ = 1.75 (d; J = 11.1 Hz; 2H),
1.92 (td; J = 12.6 Hz; J = 4.0 Hz; 2H), 2.11 (td; J = 11.9 Hz; J = 2.6 Hz; 2H), 2.94 (d; J = 11.9 Hz; 2H), 3.17
(dd; J = 6.7 Hz; J = 1.3 Hz; 2H), 3.44 (s; 2H), 3.56 (bs; -NH), 6.28 (dt; J = 15.9 Hz; J = 6.7 Hz; 1H), 6.46 (s;
1H), 6.52 (dd; J = 4.4 Hz; J = 4.2 Hz; 1H), 6.72 (ddd; J = 9.1 Hz; J = 8.4 Hz; J = 2.6 Hz; 1H), 6.79 (dd;
J = 8.6 Hz; J = 2.6 Hz; 1H), 7.24-7.32 (m; 4H) ppm; 13C NMR (CDCl3, 75 MHz): δ = 35.9, 44.7 (J = 1.8 Hz),
50.9, 61.3, 77.29, 110.0 (J = 2.1 Hz), 110.1 (J = 30.3 Hz), 113.7 (J = 23.5 Hz), 127.4, 127.5, 128.7, 131.8,
133.1, 135.4, -, 138.8 (J = 6.9 Hz), 146.4, 157.1 (J = 235.5 Hz) ppm; 19F NMR (CDCl3, 282 MHz): δ = -
125.73 ppm; MS (ESI-(+)): m/z calcd. for C21H23ClFN2 [M+H]+ 357.1; found 357.2.
(E)-(1'-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4'-piperidin]-1-yl)(2-chloropyridin-4-
yl)methanone (2, SYN876): The synthesis of the already published derivative 2 was slightly modified
by using 2-chloroisonicotinic acid instead of the corresponding acid chloride as described.28 2-
Chloroisonicotinic acid (0.043 g; 0.308 mmol; 1.9 eq.), 0.057 g (0.159 mmol; 1.0 eq.) 1, 0.161 g (0.364
mmol; 2.3 eq.) BOP and 0.12 mL (0.085 g; 0.840 mmol; 5.3 eq.) triethylamine (TEA) were dissolved in
8 mL dichloromethane under argon atmosphere. The solution was stirred for 24 h. Then 50 mL water
were added and the aqueous layer was extracted with CHCl3 (4 x 25 mL). The combined organic
8

layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was
purified by column chromatography (SiO2; ethyl acetate/n-hexane/NH3 1:1:0.1) to afford the titled
1
compound 2 (0.059 g; 0.123 mmol; 77%). Rf = 0.48 (ethyl acetate/n-hexane/NH3 1:1:0.1); H NMR
(DMSO-d6, 400 MHz): δ = 1.66 (m; 2H), 1.87 (m, 4H), 2.80 (s; 2H), 3.04 (m, 2H), 3.87 (s; 2H), 6.29 (dt; J
= 6.2 Hz; J = 13.2 Hz; 1H), 6.49 (d; J = 15.9 Hz; 1H), 7.09 (t; J = 7.9 Hz; 1H), 7.26 (m; 1H), 7.35 (d;
J = 8.4 Hz; 2H), 7.44 (d; J = 8.3 Hz; 2H), 7.66 (d; J = 4.4 Hz; 1H), 7.78 (s; 1H), 8.12 (dd; J = 4.7 Hz;
J = 8.3 Hz; 1H), 8.59 (d; J = 5.0 Hz; 1H) ppm; 13C NMR (DMSO-d6, 100 MHz): δ = 36.3, 43.5, 50.4, 59.6,
60.6, 110.9 (J = 24.4 Hz), 114.5 (J = 23.1 Hz), 118.3 (J = 7.6 Hz), 121.0, 122.3, 128.3, 128.8, 129.0,
131.1, 132.2, 136.1, 138.0, 143.5 (J = 7.4 Hz), 147.7, 151.2 (J = 5.9 Hz), 159.9 (J = 240.5 Hz), 164.7
ppm; 19F NMR (DMSO-d6, 376 MHz): δ = -117,0 ppm; HRMS (ESI-(+)): m/z calcd. for C27H25Cl2FN3O
[M+H]+ 496.1246; found 496.1351 [M+H]+.
(E)-(1'-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4'-piperidin]-1-yl)(2-fluoropyridin-4-
yl)methanone (3): The synthesis of the already published derivative 3 was slightly modified by using
fluoroisonicotinic acid instead of the corresponding acid chloride as described.28 2-Fluoroisonicotinic
acid (0.043 g; 0.308 mmol; 1.9 eq.), 0.057 g (0.159 mmol; 1.0 eq.) 1, 0.161 g (0.364 mmol; 2.3 eq.)
BOP and 0.12 mL (0.085 g; 0.840 mmol; 5.3 eq.) triethylamine were dissolved in 8 mL
dichloromethane under argon atmosphere. The solution was stirred for 24 h. Then 50 mL water were
added and the aqueous layer was extracted with CHCl3 (4 x 25 mL). The combined organic layers
were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by
column chromatography (SiO2; ethyl acetate/n-hexane/NH3 1:1:0.1) to afford the titled compound 3
(0.059 g; 0.123 mmol; 77%). Rf = 0.42 (ethyl acetate/n-hexane/NH3 1:1:0,1); 1H NMR
(CDCl3, 400 MHz): δ = 1.72 (m; 2H), 1.99 (m; 4H), 3.01 (m; 2H), 3.16 (s; 2H), 3.80 (s; 2H), 6.24 (m; 1H),
6.48 (d; J = 15.6 Hz; 1H), 6.95 (dd; J = 2.4 Hz; J = 8.1 Hz; 1H), 7.00 (m; 1H); 7.09 (s; 1H), 7.29-7.32 (m;
5H), 8.20 (bs; 1H), 8.41 (d; J = 4.6 Hz; 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ = 36.4, 43.5, 50.6, 60.5,
61.1, 107.6 (J = 38.6 Hz), 110.1 (J = 24.1 Hz), 114.8 (J = 24.5 Hz), 118.9 (J = 14 Hz), 119.0, 127.0, 127.5,
128.8, 132.1, 133.3, 135.2, 137.0, 142.0, 149.0 (J = 14.9 Hz), 149.3, 160.4 (J = 243.7 Hz), 163.9
(J = 241.3 Hz), 164.5 ppm; 19F NMR (CDCl3, 376 MHz): δ = -65.1, -117.6 ppm; HRMS (ESI-(+)): m/z
calcd. for C27H25ClF2N3O [M+H]+ 480.1649; found 480.1647 [M+H]+.
(E)-(6-bromopyridin-3-yl)(1’-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4’-piperidin]-1-
yl)methanone (4): 6-Bromopyridine-3-carboxylic acid (0.13 g, 0.672 mmol, 1.2 eq.), 0.20 g (0.560
mmol; 1.0 eq.) 1, 0.370 g (0.840 mmol; 1.5 eq.) BOP and 0.27 mL (0.20 g; 1.960 mmol; 3.5 eq.)
triethylamine were dissolved in 15 mL dichloromethane under argon atmosphere. The solution was
stirred for 24 h. Additional 0.056 g (0.280 mmol; 0.5 eq.) 6-bromopyridine-3-carboxylic acid, 0.124 g
9

(0.280 mmol; 0.5 eq.) BOP and 76.7 µl (0.056 g; 0.560 mmol; 1.0 eq.) triethylamine were added and
stirred for further 48 h. After addition of 50 mL water, the aqueous layer was extracted with CHCl3 (4
x 25 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by column chromatography (SiO2; ethyl acetate/n-hexane/NH3
1:1:0.1) to afford the titled compound 4 (0.102 g; 0.188 mmol; 34%). Rf = 0.30 (ethyl acetate/n-
1
hexane/NH3 1:1:0.1); H NMR (DMSO-d6, 300 MHz): δ = 1.64 (m; 2 H), 1.87 (m; 4 H), 2.8 (m; 2 H),
3.05 (m; 2 H), 3.93 (s; 2 H), 6.28 (dt; J = 15.7 Hz; J = 6.4 Hz; 1 H), 6.48 (d; J = 15.8 Hz; 1 H), 7.03 (m; 1
H), 7.23 (dd; J = 8.7 Hz; J = 2.7 Hz; 1 H), 7.33 (d; J = 8.6 Hz; 2 H), 7.42 (d; J = 8.5 Hz; 2 H), 7.79 (d; J =
8.1 Hz; 1 H), 8.02 (m; 3 H), 8.65 (m; 1 H) ppm; 13C NMR (DMSO-d6, 75 MHz): δ = 36.2, 43.3, 50.4, 59.9,
60.6, 110.9 (J = 22.9 Hz), 114.4 (J = 23.2 Hz), 118.2 (J = 8.4 Hz), 128.3, 128.5, 128.7, 129.0, 131.2,
132.2, 132.6, 136.0, 138.2, 138.7, 143.2, 143.5, 149.3, 159.7 (J = 240.5 Hz), 165.2 ppm; 19F NMR
(DMSO-d6, 282 MHz): δ = -117.3 ppm; HRMS (ESI-(+)): m/z calcd. for C27H25BrClFN3O [M+H]+
540.0854; found 540.0846.
(E)-
(1’-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4’-piperidin]-1-yl)(6-fluoropyridin-3-
yl)methanone (5): 6-Fluoropyridine-3-carboxylic acid (0.095 g, 0.672 mmol, 1.2 eq.), 0.20 g (0.560
mmol; 1.0 eq.) 1, 0.370 g (0.840 mmol; 1.5 eq.) BOP and 0.27 mL (0.20 g; 1.960 mmol; 3.5 eq.)
triethylamine were dissolved in 15 mL dichloromethane under argon atmosphere. The solution was
stirred for 48 h. Then additional 0.039 g (0.280 mmol; 0.5 eq.) 6-fluoropyridine-3-carboxylic acid,
0.124 g (0.280 mmol; 0.5 eq.) BOP and 76.7 µL (0.056 g; 0.560 mmol; 1.0 eq.) TEA were added and
stirred for further 48 h. After addition of 50 mL water, the aqueous layer was extracted with CHCl3 (4
x 25 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by column chromatography (SiO2; ethyl acetate/n-hexane/NH3
1:1:0.1) to afford the titled compound 5 (0.118 g; 0.246 mmol; 44%). Rf = 0.16 (ethyl acetate/n-
1
hexane/NH3 1:1:0.1); H NMR (DMSO-d6, 400 MHz): δ = 1.64 (m; 2H), 1.85 (m; 4 H), 2.80 (m; 2H),
3.04 (d; J = 6.4 Hz; 2H), 3.94 (s; 2H), 6.27 (dt; J = 15.9 Hz; J = 6.4 Hz; 1H), 6.47 (d; J = 15.9 Hz; 1H), 7.03
(bs; 1H), 7.22 (dd; J = 8.7 Hz; J = 2.7 Hz; 1H), 7.32 (m; 3H), 7.41 (m; 2H), 8.09 (bs; 1H), 8.27 (m, 1H),
8.54 (bs; 1H) ppm; 13C NMR (DMSO-d6, 100 MHz): δ = 36.2, 43.3, 50.4, 60.0, 60.6, 110.2 (J = 37.5 Hz),
110.8 (J = 23.8 Hz), 114.3 (J = 23.1 Hz), 115.7 (J = 22.5 Hz), 118.2, 128.3, 128.7, 129.0, 131.1, 131.6,
132.2, 136.0, 138.2, 141.8 (J = 8.7 Hz), 143.5 (J = 7.8 Hz), 147.2 (J = 15.8 Hz), 159.6 (J = 240.6 Hz),
163.8 (J = 239.0 Hz), 165.3 ppm; 19F NMR (DMSO-d6, 282 MHz): δ = -65.9, -117.4 ppm; HRMS (ESI-
(+)): m/z calcd. for C27H25ClF2N3O [M+H]+ 480.1654; found 480.1647.
(E)-(1'-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4'-piperidin]-1-yl)(4-
fluorophenyl)methanone (6): In a round bottom flask 0.072 g (0.202 mmol; 1.0 eq.) of 1, 0.034 g
10

(0.242 mmol; 1.2 eq.) of 4-fluorobenzoic acid, 0.134 g (0.303 mmol; 1.5 eq.) BOP and 0.10 mL
(0.071 g; 0.707 mmol; 3.5 eq.) of triethylamine were dissolved in 8 mL dichloromethane under argon
atmosphere. The solution was stirred for 1 day at room temperature. Then 50 mL of water were
added and the aqueous layer was extracted with chloroform (4 × 25 mL). The combined organic
layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was
purified by column chromatography (SiO2; ethyl acetate/n-hexane/NH3 1:1:0.1) to afford the titled
1
compound 6 (0.029 g; 0.060 mmol; 30%). Rf = 0.56 (ethyl acetate/n-hexane/NH3 1:1:0,1); H NMR
(DMSO-d6, 300 MHz): δ = 1.64 (m; 2H), 1.87 (m; 4H), 2.82 (m; 2H), 3.05 (m; 2H), 3.89 (s; 2H), 6.29
(dt; J = 6.4 Hz; J = 15.7 Hz; 1H), 6.49 (d; J = 15.9 Hz; 1H), 7.02 (br s; 1H), 7.22 (dd; J = 2.6 Hz; J = 8.6 Hz;
1H), 7.33 (m; 4H), 7.43 (m; 2H), 7.67 (m; 2H), 8.02 (br s; 1H) ppm; 13C NMR (DMSO-d6, 75 MHz): δ =
36.1, 43.1, 50.5, 60.2, 60.7, 110.8 (J = 24.1 Hz); 114.3 (J = 23.1 Hz), 116.0 (J = 21.7 Hz), 118.1, 128.3,
128.7, 129.0, 130.2 (J = 8.6 Hz), 131.2, 132.2, 133.5, 136.0, 138.4, 143.4 (J = 7.1 Hz), 159.5
(J = 240.3 Hz), 163.4 (J = 247.8 Hz), 167.4 ppm; 19F NMR (DMSO-d6, 282 MHz): δ = -110.0, -117.9
ppm; HRMS (ESI-(+)): m/z calcd. for C28H26ClF2N2O [M+H]+ 479.1701; found 479.1693.
2-Fluoroethyl (E)-1'-(3-(4-chlorophenyl)allyl)-5-fluorospiro[indoline-3,4'-piperidine]-1-carboxylate
(7): In a round bottom flask 0.044 g (0.123 mmol; 1.0 eq.) of 1, 0.026 g (0.185 mmol; 1.5 eq.) of 2-
fluoroethyl methanesulfonate and 0.061 g (0.369 mmol; 3.0 eq.) potassium carbonate were dissolved
in 2 mL DMF under argon atmosphere and stirred at 90 °C for 19 h. Then additional 2-fluoroethyl
methanesulfonate (8.7 mg, 0.061 mmol, 0.5 eq.) was added and the mixture heated for further 10 h.
After addition of 50 mL of water, the aqueous layer was extracted with chloroform (4 × 25 mL) and
the combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo.
The residue was purified by column chromatography (SiO2; ethyl acetate/n-hexane/NH3 1:2:0.1) to
afford the titled compound 7 (0.029 g; 0.065 mmol; 54%). Rf = 0.27 (ethyl acetate/n-hexane/NH3
1:2:0.1); 1H NMR (CDCl3, 300 MHz): δ = 1.70 (d; J = 12.7 Hz; 2H), 1.95 (td; J = 3.3 Hz; J = 12.9 Hz; 2H),
2.12 (t; J = 12.0 Hz; 2H), 3.00 (d; J = 11.6 Hz; 2H), 3.20 (d; J = 6.7 Hz; 2H), 3.91 (s; 2H), 4.47 (d;
J = 28.3 Hz; 2H), 4.70 (d; J = 47.6 Hz; 2H), 6.28 (dt; J = 6.67 Hz; J = 14.3 Hz; 1H), 6.50 (d; J = 15.9 Hz;
1H), 6.86 (s; 1H), 6.88 (s; H), 7.27-7.33 (m; 4H), 7.79 (s; 1H) ppm; 13C NMR (CDCl3, 75 MHz): δ = 37.1,
42.4, 50.5, 57.7, 61.3, 64.4 (J = 20.8 Hz), 81.7 (J = 170.5 Hz), 110.1 (J = 23.1 Hz), 114.5 (J = 23.1 Hz),
115.6, 127.1, 127.5, 128.7, 132.0, 133.2; 135.3; 137.4; 140.6; 152.4; 159.2 (J = 241.3 Hz) ppm; 19F
NMR (CDCl3, 282 MHz): δ = 5.3, -119.9 ppm; HRMS (ESI-(+)): m/z calcd. for C24H26ClF2N2O2 [M+H]+
447.1645; found 447.1656.
5.2. In vitro binding experiments
11

5.2.1 Preparation of homogenates of rVAChT-PC12 cells
rVAChT-PC12 cells (by courtesy of Prof. Ali Roghani, Texas Tech University, Lubbock, USA) were
grown in DMEM (high glucose, 5% cosmic calf serum, 10% equine serum, 100 U/mL penicillin-
streptomycin, 0.125 g/L G-418) to confluence (37 °C, 5% CO2). Cells were harvested by scraping into
the medium and pelleted by centrifugation (800 rpm, 3 min). The cell pellet was resuspended in
chilled 50 mM TRIS-HCl, pH 7.4 and triturated to effect hypotonic lysis. The suspension was
centrifuged at 15.000 rpm at 4 °C for 15 min. The obtained crude membrane fraction was maintained
at -25 °C until assay. For the binding experiments aliquots were thawed and resuspended in a desired
volume of assay buffer.
5.2.2 Preparation of rat brain and liver tissue homogenates
The studies involving experiments with animal tissues were performed in accordance with the
national law on the care and use of laboratory animals and approved by the local Ethics Committee
(Landesdirektion Sachsen, T 48/14).
Female Sprague Dawley rats (150–200 g) were anaesthetized and decapitated. Their brains and livers
were rapidly removed from the skull. Brain tissue without cerebellum as well as liver samples were
homogenized in 10 volumes (w/v) of chilled 50 mM TRIS-HCl, pH 7.4, using a Teflon-glass
homogenizer (10 strokes, 1500 rpm). The respective homogenates were centrifuged (14.000 rpm,
4°C, 15 min), the resulting supernatants were decanted and the pellets resuspended with the same
buffer. The homogenization-centrifugation was performed three times, and the finally obtained
washed pellets were aliquoted in the same buffer and stored at -25 °C until assay.
5.2.3 Binding experiments
For VAChT binding assays, the crude membrane fraction of VAChT-PC12 cells was incubated with (-)-
[3H]vesamicol ([3H]2-(4-phenylpiperidinyl)cyclohexanol, molar activity 1258 GBq/mmol, PerkinElmer
Life Sciences, Boston, MA, USA; 1.5 nM) and up to seven concentrations of the test compound (0.01
nM – 10 µM) in a volume of 1 mL (50 mM TRIS-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1
mM MgCl2) in duplicate for 120 min at room temperature under agitation (200 min-1). Nonspecific
binding of the radioligand was determined by co-incubation with 10 µM (±)-vesamicol.
For comparison of the bindings sites of compound 2 and vesamicol at VAChT, competition assays
were performed as described above by using (±)-vesamicol as test compound alone or in the
presence of 100 nM or 1000 nM compound 2 in the same assay, i.e., with absolute identical test
conditions.
For σ1 and σ2 receptor binding assays, the crude membrane fractions of rat brain and liver were
incubated with (+)-[3H]pentazocine (specific activity: 1070 GBq/mmol, PerkinElmer Life Sciences,
12

Boston, USA) and [3H]DTG (specific activity: 1110 GBq/mmol, PerkinElmer Life Sciences, Boston, USA)
in the presence of 1 µM of the σ1 receptor masking dextrallorphan (by courtesy of Roche AG, Basel,
Switzerland), respectively, in a volume of 1 mL (50 mM TRIS-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2
mM CaCl2, 1 mM MgCl2) in duplicate for 120 min at room temperature under agitation (200 min-1).
Nonspecific binding of both radioligands was determined in the presence of 10 µM haloperidol.
The incubations were terminated by rapid filtration through Whatman GF/B glass-fiber filters
presoaked in 0.3% polyethylenimine for 60 min at room temperature using a rapid vacuum filtration
system (cell harvester Brandel Gaithersburg, MD, USA), and the filters were washed three times with
4 mL chilled 50 mM TRIS-HCl, pH 7.4. Filterbound radioactivity was measured by liquid scintillation
counting (Tri-Carb2900TR, PerkinElmer Life Sciences Boston, MA, USA).
IC50 values were calculated from competitive binding curves by nonlinear curve fitting (GraphPad
Prism, version 3; GraphPad Software, Inc., San Diego, USA). The apparent inhibition constant (Ki) was
derived from IC50 values according to the CHENG–PRUSOFF equation: Ki = IC50/(1+C/KD) where C is the
concentration of the radioligand and KD is the dissociation constant of the radioligand (in-house
determined values: KD (-)-[3H]vesamicol = 25.6 nM to VAChT on rVAChT-PC12 cells, KD (+)-
[3H]pentazocine = 6.9 nM to σ1 receptors on rat cortical membranes, KD [3H]DTG = 29 nM for σ2
receptors on rat liver membranes with masking of σ1 receptors using dextrallorphan).
Linear transformation according to Hanes-Woolf was performed using the protocols implemented in
GraphPad Prism (Version 3; GraphPad Software, Inc., San Diego, USA).
Acknowledgment
The authors thank Dr. L. Hennig from the University of Leipzig for measuring the NMR spectra, Dr.
Susan Billig and Ramona Oehme from the University of Leipzig for measuring the high resolution
mass spectra and Tina Spalholz for performing the in vitro binding studies. We also thank Dr. Markus
Leutzsch from the University of Cambridge for his support in the analysis of NMR-data. We are very
grateful to Prof. Ali Roghani from the Texas Tech University, Lubbock, USA, for providing the rVAChT-
PC12 cell line .
Supplementary material
Supplementary material associated with this article can be found in the online version at:
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Figure captions:
Figure 1. Known VAChT ligands vesamicol, benzovesamicol and spiroindoline [3H]SYN876 as potential
lead compounds in the development of VAChT PET radiotracers
Figure 2. Representation of binding data as non-linearized inhibition curves (one out of three
experiments; n=3 per data point).
Figure 3. Representation of binding data after Hanes-Woolf linearization.
Scheme 1. Reagents and conditions: a) BOP, TEA, 2-chloroisonicotinic acid for 2, 2-fluoroisonicotinic
acid for 3, 6-bromonicotinic acid for 4, 6-fluoronicotinic acid for 5 and 4-fluorobenzoic acid for 6,
CH2Cl2, rt, 24 h; b) K2CO3, DMF, 2-fluoroethyl methanesulfonate, 90 °C, 19 h.
16