Design of α7 nicotinic acetylcholine receptor ligands in quinuclidine, tropane and quinazoline series. Chemistry, molecular modeling, radiochemistry, in vitro and in rats evaluations of a [18F] quinuclidine derivative

Article abstract of DOI:10.1016/j.ejmech.2014.04.057

In this report, we describe the synthesis of a novel library of α7 nAChR ligands based on the modulation of the quinuclidine, quinazoline and tropane moieties. Spirane derivatives were newly synthesized under stereo specific 1,3 dipolar cylcoadditions. On

Full text of DOI:10.1016/j.ejmech.2014.04.057

Accepted Manuscript
Design of α7 Nicotinic Acetylcholine Receptor Ligands in Quinuclidine, Tropane and
Quinazoline Series. Chemistry, Molecular Modeling, Radiochemistry, in vitro and in
18
Rats Evaluations of a [ F] Quinuclidine Derivative
Frédéric Pin, Johnny Vercouillie, Aziz Ouach, Sylvie Mavel, Zuhal Gulhan, Gabrielle
Chicheri, Christian Jarry, Stephane Massip, Jean-Bernard Deloye, Denis Guilloteau,
Franck Suzenet, Sylvie Chalon, Sylvain Routier
PII:
S0223-5234(14)00378-X
10.1016/j.ejmech.2014.04.057
EJMECH 6928
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2014
Revised Date: 17 April 2014
Accepted Date: 19 April 2014
Please cite this article as: F. Pin, J. Vercouillie, A. Ouach, S. Mavel, Z. Gulhan, G. Chicheri, C. Jarry,
S. Massip, J.-B. Deloye, D. Guilloteau, F. Suzenet, S. Chalon, S. Routier, Design of α7 Nicotinic
Acetylcholine Receptor Ligands in Quinuclidine, Tropane and Quinazoline Series. Chemistry, Molecular
18
Modeling, Radiochemistry, in vitro and in Rats Evaluations of a [ F] Quinuclidine Derivative, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.04.057.
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Graphical abstract

ACCEPTED MANUSCRIPT
Design of α7 Nicotinic Acetylcholine Receptor Ligands
in Quinuclidine, Tropane and Quinazoline Series.
Chemistry, Molecular Modeling, Radiochemistry, in vitro
1
8
and in Rats Evaluations of a [ F] Quinuclidine Derivative.
a
b
a
b
b
Frédéric Pin, Johnny Vercouillie, Aziz Ouach, Sylvie Mavel, Zuhal Gulhan,
b
c
c
d
Gabrielle Chicheri, Christian Jarry, Stephane Massip, Jean-Bernard Deloye, Denis
b
a
b,*
a,*
Guilloteau, Franck Suzenet, Sylvie Chalon and Sylvain Routier
a
Université d’Orléans, CNRS, ICOA, UMR 7311, F-45067 Orleans, France.
b
UMR Inserm U930, Université François Rabelais de Tours, CHRU de Tours, Tours, France.
Université de Bordeaux, Pharmacochimie, FRE CNRS 3396, 136 Rue Léo Saignat, 33076 Bordeaux Cedex, France
Laboratoires Cyclopharma, Biopôle Clermont-Limagne, 63360 Saint-Beauzire, France.
c
d
Keywords: alpha 7nicotinic acetylcholine receptors, quinuclidine, tropane, synthesis,
SAR, radiochemistry, molecular modeling, in vivo evaluation.
*
Corresponding authors
Email address: sylvie.chalon@univ-tours.fr and sylvain.routier@univ-orleans.fr

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Abstract
In this report, we describe the synthesis of a novel library of α7 nAChR ligands based
on the modulation of the quinuclidine, quinazoline and tropane moieties. Spirane derivatives
were newly synthesized under stereospecific 1,3 dipolar cylcoadditions. Only amide
derivatives bonded efficiently to the receptor with Ki measured between 14 and 133 nM. The
1
8
best fluorinated candidate was selected and radiolabeled. The potent [ F]4 PET tracer was
evaluated in rats and its brain accumulation quantified.

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1
. Introduction
The neurotransmitter acetylcholine (ACh) exerts its effects on the central nervous
system (CNS) through two distinct muscarinic mAChRs and nicotinic nAChRs receptor
types. nAChRs belong to the superfamily of ligand-gated ion channels possessing a
pentameric structure [1]. These receptors are composed of multiple subunits, which have been
divided into muscle-type (α1, β1, δ, γ and ε) and neuronal (α2-α10 and β2-β4) subunits [2].
In the brain, one of two major nAchRs is the α4β2 subtype, distinguished by its high affinity
binding to acetylcholine and nicotine.
Homomeric α7-subtype receptors are highly permeable to calcium and are distinguished
by their tight binding to α-bungarotoxin. Previous studies indicated that neuronal nicotinic
receptors play important roles in modulating neurotransmission, cognition, sensory gating and
anxiety [3]. Thus, there has been renewed interest in the use of nicotinic agonists to treat CNS
diseases [4], such as Parkinson’s disease, depression, schizophrenia, epilepsy and Alzheimer’s
disease (AD).
Because of their distribution and abundance in the CNS (hippocampus, cortex,
cerebellum, olfactory bulb, striatum, thalamus, and spinal cord) [2], the α7 subtypes are
potential therapeutic targets for these disorders. Their early reduction fully correlates with
cognitive functions, favours the internalization of Aβ peptide and increases neurotoxicity. In
addition, they act on signaling pathways which prevent inflammatory processes that are fully
involved in AD [5]. Subsequently, α7nAChR agonists were identified and allowed the design
of novel therapeutic agents for AD. It is indubitably crucial to have in hand a human
1
8
compatible [ F] positron emission tomography (PET) tracer to make an early diagnosis or to
validate the efficiency of drugs in clinical trials for the treatment of AD.
Several α7 receptor ligands have been reported in the literature and among them, many
quinuclidine derivatives have been synthesized (Figure 1). These aliphatic hindered tertiary
amines, considered by organic chemists as a super base, have punctuated the history of
medicinal chemistry and have been used as pharmacophoric elements in antibiotic structures
as well as in the design of potent cannabinoid receptors, muscarinic, serotoninergic and
squalene synthase inhibitors [6]. In α7 ligand discovery, this skeleton is mostly functionalized
at the C-3 position by ethers [7], amides [8], carbamates [9] or ureas [10] and other
representatives are also found with a C-2,-3 quinuclidinic disubstitution [11].

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1
8
Figure 1. Representative α7 nAchR ligands and reported F PET imaging agents.
In addition, following a restrictive conformation strategy, several spiro derivatives have
been synthesized (I-II) [12]. Lastly, the quinuclidine scaffold has been successfully engaged
in a bio-isosteric strategy and replaced by other polycyclic tertiary amines (III) in order to
modulate the pharmacologic properties and explore SAR [12,13].
Based on our expertise in heterocyclic bio-mimetic development [14], we envisioned
synthesizing and evaluating the potency of novel α7 nAChR ligands and their transformation
18
into PET tracers following an approach close to one we previously used to develop a [ F]-
11
labeled tracer of the dopamine transporter. In this emerging area, only a small number of [ C]
1
8
short half-life tracers have been developed and even fewer [ F]-labeled PET tracers have
1
8
18
18
18
been described, such as [ F]NS10743, [ F]NS14490, [ F]AZ11637326 and [ F]IV (Fig 1).
18
Most of them display low or modest brain uptake and/or a lack of specificity except [ F]IV
for which early evaluations in rodents seem promising [15]. Based on available SAR studies,
we synthesized a library of potent α7 nAChR ligands containing a quinuclidine, a tropane or a
8
H-quinolizine moiety. Some amides of type V were synthesized and evaluated in our
biological system in order to complete unachieved SAR in those series. Original
spiroisoxazoline derivatives of type VI and VII were additionally prepared using 1,3-dipolar
cylcoadditions (Figure 2).
Figure 2. Main synthetic objectives.
We report herein methods to access spirane derivatives whose structure was formally
established by X-ray crystal analysis. Affinity of each final compound was determined and
molecular modeling docking studies confirmed the binding mode of the best two ligands. A
1
8
18
fluorinated derivative was selected and used to design a novel [ F] α7 nAChR tracer [ F]4
which was characterized ex vivo in the rat.
2
. Chemistry
2
.1. Synthesis of amides V.
Synthesis of amides was performed starting from quinuclidinone and tropanone
derivatives to form the primary amine, stored as chlorhydrate salts 1 and 2 (Scheme 3) [16].
Freshly prepared compounds 1 and 2 were required to ensure further cycloaddition success
(see section 2.2.).

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Scheme1.
Peptide bond formations were achieved from 1 and 2 using various (het)aryl carboxylic
acids. Several experimental conditions were used in order to isolate the expected amides 3-11
in satisfactory yields. Standard molecules 3-6 were prepared for binding test validation
according to reference procedures, while the other amides 7-11 were obtained using DCC and
DMAP as cross coupling reagents; the use of acylchlorides in basic media gave only poor
yields.
Table 1. Amides of type IV starting from 1 or 2.
Additionally, bromo derivative 9 (Table 1, entry 7) was successfully used in a Suzuki-
Miyaura reaction, using 2-thiophenyl and 2-furanyl boronic acids in a mixture of
toluene/EtOH at 150°C in presence of potassium carbonate (Scheme 2) [17]. Under
microwave irradiation, cross coupling occurred very quickly and after only 20 minutes, final
tropanamides 12 and 13 were isolated in 84 % and 90 % yields, respectively.
Scheme 2.
2.2. Synthesis of spiro derivatives VI and VII.
The next objective was to link the tropane and the 8H-quinazoline scaffolds with a spiro
isoxazoline skeleton. Many synthetic approaches are available to achieve the isoxazoline ring
formation. Among them, we identified 1,3-dipolar cycloaddition as an interesting key step to
reach the synthetic objectives in a straightforward pathway. For this purpose, the tropane or
8
H-quinazoline exomethylenes were prepared following literature procedure and a few
(het)aryl chlorooximes were prepared in situ just before the cycloaddition reaction.
8
H-Quinolizin-1-one 15 was prepared following the Barker procedure [18]. A Wittig
condensation starting from tropinone and 15 involving MePPh Br in the presence of t-BuOK
3
furnished exomethylene derivatives 14 [19] and 16 in good yields. The strong instability of
this alkene 16 prompted us to prevent its degradation by direct preparation of its borane
complex. 17 was isolated as a white solid and appeared sufficiently stable to be stored without
any particular precautions.
Scheme 3.
We also freshly prepared six different (het)arylchlorooximes 24-29 (Table 2) via the
condensation of the chosen aldehydes with hydroxylamine hydrochloride at room temperature
in presence of Et N (a syn/anti isomer mixture) and subsequent chlorination of the oxime
3
intermediates 18-23 using NCS at 0°C.

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Table 2. Preparation of chlorooximes 24-29.
The exomethylene and chlorooxime reagents were engaged in a 1,3-dipolar
cycloaddition [19]. A period of 24 h was necessary to complete the conversion and offer the
spiro-isoxazole units. Under these optimized experimental conditions, compounds 30-34 were
isolated in moderate yields. For each run, we were unable to prevent nitrile oxide dimerization
and early degradation due to instability of chlorooximes 24-29. Unfortunately, derivative 35
was not pure enough to engage it in cross coupling reactions and increase diversity.
Table 3. Synthesis of spiranic tropane derivatives VI.
All 1,3-dipolar cycloadditions were fully regio and stereo specific. Only one
diastereoisomer of compound 30-34 was isolated and characterized by NMR. Isolation of a
single crystal of 30 gave access to its X-ray analysis (Figure 3). The ORTEP representation
clearly shows the spiranic carbon configuration and the formation of the oxazole ring fused to
the tropane moiety.
Figure 3.
Starting from the non-basic 8H-quinolizine boron complex 17, the experimental
procedure was simplified. All reagents were mixed without any special precautions and spiro
derivatives 36-41 were isolated in 49-64% yield (Table 4). BH protection was removed under
3
the reaction conditions.
Table 4. Synthesis of spiranic 8H-quinazoline derivatives VII.
Regio and stereo selectivity of the cycloaddition in quinazoline series was fully
established. An X-ray analysis of 41 completed our spectroscopic data and the ORTEP
representation (Figure 4) clearly shows the quinazolin/oxazolin spiranic ligation enchainment.
The N,O oxazolin heteroatoms direct towards the same side as the C-16 quinuclidine
hydrogen atom. Oxazoline and bromothiophene moieties constitute a single plane.
Figure 4.
3
. In vitro Biological assays.
All synthesized compounds were evaluated for their inhibitory effect on the binding of
125
[
I]α-bungarotoxin as reference ligand. Measured affinities are reported in Table 5. In our
hands, Ki for derivatives 3 and 4 were found in the nanomolar range whereas compounds 5
and 6 appeared less efficient at binding to α7 nAChR (Table 5, entries 1-4). These results are
in agreement with the reported EC50 and selectivity (vs 5-HT receptors) for 4 and its
3

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chlorinated derivative 3 (PNU-282987), which was the most potent derivative of the series
[
8]. Product 4 appeared therefore as a good candidate for in vivo exploration of the α7 nAChR
18
by PET after labeling with [ F].
More derivatives were synthesized in the tropane amide series (entries 5-11).
Surprisingly, 4-chlorophenyl and thiophenyl derivatives 7 and 8 were inactive, indicating a
radical change in the binding mode to receptor. Fortunately, α7 nAChR binding can be
restored by increasing the size and lipophilicity of the amide substituent thanks to a bromine
at the C-5 position of the thiophene ring. Compound 9 exhibited an encouraging Ki of 78 nM
(entry 7). Increasing the size of this aromatic substituent was detrimental for affinity.
Benzofurane and benzothiophene derivatives 10 and 11 (entries 8, 9) which contain planar
and aromatic fused systems showed lower affinities (a reduction of 30% was observed).
Table 5. Results of in vitro assays.
This tropane amide SAR study prompted us to introduce a bisthiophene and a
furanylthiophene substituent. Resulting derivatives 12 and 13 exhibited Ki of 12 and 14 nM,
respectively (Table 5, entries 10, 11). The latter two derivatives are indubitably lead
1
8
compounds for novel SAR optimizations and for designing original F tracers, whereas spiro
derivatives of type V and VI were inactive (Ki > 1 µM).
From these in vitro results, a QSAR study (Discovery Studio® 2.5, Accelrys) was
done to improve our knowledge of new ligands design for α7 nAChR, in order to point out
molecular requirements in the tropaneamide bis(het)aryl series [20-22]. The QSAR equation
with the regression statistics is first given (see supplementary data, Fig.S1). As the coefficient
in the equation is negative for the conformational descriptor mentioned as Apol (“Sum of
atomic polarizabilities”), the less polar the structure, the higher the pKi. Concerning the
structural properties, HBD_Count indicates the importance of “number of hydrogen bond
donating groups” (one or more H-bond donating groups is a plus). BIC is a “graph-theoretical
info-content descriptor” concerning “Bonding Information Content”. As the equation
coefficient is negative, the less compact the structure is, the higher the pKi will be. Also as the
2
D topological descriptors, IAC_Mean (“Mean Information of Atomic Composition”) and IC
(“Information Content”, depending on the size, degree of branching and flexibility of the
structure), have a positive coefficient, the higher the values, the better. The equation showed
occurrences of 3D Jurs descriptors, suggesting the importance of charge distribution and
surface areas (Jurs_WNSA_3 depends on total molecular solvent-accessible surface area), and
should be a high value, indicative of non-compact structures.

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Furthermore, a 3D-QSAR model (Discovery Studio® 2.5, Accelrys) was developed
Figure 5). Several key features are predicted to increase α7 nAChR affinity: i) reinforcement
(
of the negative environment in a zone 10Å from the N-1 tertiary nitrogen atom (toward the
second (het)aryl group); ii) decrease of the negative environment in a zone 7Å from the N-1
nitrogen atom and minimization of the electro negativity of the first aromatic ring and iii)
minimization of Van der Waals interactions around this first aromatic group. Chemical assays
are also in progress to optimize our series using these preliminary 3D data.
Figure 5.
5
. Radiolabeling
Among the compounds developed, 4 demonstrated a good affinity and selectivity to
α
7 nAchR and presented a fluorine atom on the aromatic ring, allowing preparation of its
radiofluorinated analogue.
1
8
F labeling of compound 4 was envisaged by aromatic nucleophilic substitution
starting from the nitro derivative 6. As the electron withdrawal effect of the amide function is
weak, the radionuclide incorporation by conventional thermal heating was very low, around 2
%
(decay corrected from EOB) after 20 min reaction. We set up the reaction in a microwave
designed for radionuclide applications (PET wave, CEM). Under microwave irradiation at
1
8
1
00 W for 10 min, yield was improved to 10 % in a shorter time and afforded [ F]4 with a
specific activity of 26 ±10 GBq/µmole. Moreover, the use of microwave implies the use of an
additional device compared to the thermal approach which was entirely accomplished in an
FX-FN automaton (GE healthcare). It thus generates high dead volumes and considerable
losses during transfer of crude product between the two devices.
Scheme 4.
6
. In vivo studies
To date it can be argued that an ideal α7 nAChR tracer requires i) a passage through
the blood-brain barrier followed by a preferential accumulation in brain areas known to
contain α7 nAChR, ii) a decreased accumulation after the MLA α7 nAChR antagonist pre-
treatment as indicative for in vivo specific binding [23]. To evaluate these parameters, we
performed preliminary brain biodistribution experiments in rats.
1
8
As can be seen in Figure 6, [ F]4 entered the rat brain but after one hour post i.v.
injection, the residual amount of compound was low and rather homogeneously distributed

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between the studied regions (0.028 – 0.037% ID/g tissue in the control group). This result
may indicate the penetration of our compound into the brain but it is possible that a very high
release occurs. Although there is undoubtedly a differential blood-brain barrier crossing level
1
8
depending on the animal species, it is clear that [ F]4 had a lower brain uptake in rats than
1
8
the recently developed tracer [ F]IV in mice, and that its distribution among brain regions
18
was rather homogenous, in contrast to results obtained with [ F]IV [15]. However, the
1
8
residual amount of [ F]4 in rats pre-injected with the α7 nAChR antagonist MLA showed a
slight decrease in accumulation (5 – 25%) in almost all studied brain regions, which could
indicate a partial in vivo binding of our tracer to α7 nAChR. The much better affinity of
1
8
18
[
F]IV than [ F]4 for α7 nAChR (0.4 vs 18 nM) is probably correlated with the results
1
8
obtained in vivo and we are pursuing efforts to develop F tropanamide and other
quinuclidinic triazole series with improved affinity [24].
Figure 6.
7
. Conclusions
In this paper, we have reported the synthesis of novel amide and spiro derivatives as
potent ligands for 7 nicotinic receptors. Structures of novel spiro derivatives were formally
α
established by X-Ray analysis. Our compound library was evaluated for its affinity to α7
nAChR. Tropane and quinuclidine amides remain the best ligands but their binding mode
differs considerably. Among the quinuclidine amides, we selected the fluorinated benzamide 4
which had a rather good affinity for α7 nAChR of 18 nM. Radiolabeling of 4 led to the PET
1
8
ligand [ F]4 dedicated to α7 nAchR imaging. Even if the brain biodistribution studies
1
8
revealed that [ F]4 is not fully suitable as a specific in vivo PET tracer, this report indicates
the potential of this quinuclidinic series. Other efforts are in progress in other series to
increase the brain penetration and modulate the brain distribution. In tropanamide series, a
novel SAR potency emerged, offering us the possibility to develop other original tracer series.
8
. Experimental section.
8.1. Chemistry.
1
13
H NMR and C NMR spectra were recorded on a Bruker DPX 250 MHz or 400 MHz
instrument using CDCl or DMSO-d . The chemical shifts are reported in parts per million (δ
3
6
scale) and all coupling constant (J) values are in Hertz (Hz). The following abbreviations were
used to explain the multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet)
and dd (doublet doublet). Melting points are uncorrected. IR absorption spectra were obtained

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-1
on a Perkin Elmer PARAGON 1000 PC and values are reported in cm . HRMS were
recorded on a Bruker maXis mass spectrometer by the "Fédération de Recherche"
ICOA/CBM (FR2708) platform. Monitoring of the reactions was performed using silica gel
TLC plates (silica Merck 60 F254). Spots were visualized by UV light at 254 nm and 356 nm.
Column chromatographies were performed using silica gel 60 (0.063-0.200 mm, Merck).
Microwave irradiation was carried out in sealed 2-5 mL vessels placed in a Biotage Initiator
system using a standard absorbance level (300 W maximum power). The temperatures were
measured externally by an IR probe that determined the temperature on the surface of the vial
and could be read directly from the instrument screen. The reaction time was measured from
when the reaction mixture reached the stated temperature for temperature-controlled
experiments. Pressure was measured by a non-invasive sensor integrated into the cavity.
8
.1.2. General procedure for the synthesis of amide derivatives 7-11.
To a solution of 3-endo-tropanamine hydrochloride salt 2 (213 mg, 1.00 mmol) in 10 mL of
CH Cl were added successively the corresponding carboxylic acid (1.5 mmol), the N,N’-
2
2
dicyclohexylcarbodiimide (310 mg, 1.5 mmol), the 4-dimethylaminopyridine (12.0 mg, 0.10
mmol) and the triethylamine (420 µl, 3.0 mmol). The reaction mixture was refluxed for 6 h.
after cooling, the mixture was diluted with CH Cl (10 mL) and the organic layer was washed
2
2
with a saturated solution of NaHCO (10 mL), dried over MgSO , filtered and evaporated
3
4
under reduced pressure. The resulting product was purified by chromatography on a silica gel
column eluting with CH Cl /MeOH/28% aq. NH OH (9/1/0.1) to give the expected products
2
2
4
in appreciable yields.
.1.3. 4-Chloro-N-[(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] benzamide 7.
8
Compound 7 was prepared from the 4 chlorobenzoic acid and was isolated after purification
as a white solid (205 mg, 74%). mp 170-172 °C. Rf (CH Cl /MeOH 9/1) 0.11. IR (ATR
2
2
-1
diamond, cm ) ν 760, 1016, 1046, 1088, 1141, 1291, 1340, 1486, 1519, 1593, 1630, 2922,
1
3
351. H NMR (250 MHz, CDCl )
δ
1.70-1.90 (m, 4H), 2.20-2.25 (m, 2H), 2.30-2.40 (m,
3
5
H), 3.26 (br s, 2H), 4.25 (q, 1H, J = 6.5 Hz), 6.49 (br s, 1H), 7.40 (d, 2H , J = 8.5 Hz), 7.66
Ar
1
3
(
d, 2H , J = 8.5 Hz). C NMR (63 MHz, CDCl ) δ 26.1 (2 x CH ), 36.4 (2 x CH ), 40.3
Ar 3 2 2
(
CH ), 42.2 (CH), 60.3 (2 x CH), 128.3 (2 x CH ), 129.2 (2 x CH ), 133.5 (C ), 137.9 (C ),
3 Ar Ar q q
+
1
65.6 (C=O). HRMS (ESI): m/z calculated for C H ClN O (MH ): 279.1264; Found:
15 20 2
2
79.1259.

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8.1.4.
N-[(1R,5S)-8-Methyl-8-azabicyclo[3.2.1]octan-3-yl]thiophene-2-
carboxamide 8.
Compound 8 was prepared from the 2-thiophenecarboxylic acid and was isolated after
purification as a white solid (140 mg, 56%). mp 138-140 °C. Rf (CH Cl /MeOH 9/1) 0.13. IR
2
2
-1
(
ATR diamond, cm ) ν 1037, 1077, 1113, 1298, 1341, 1421, 1452, 1510, 1541, 1615, 2944,
1
3
286. H NMR (250 MHz, CDCl )
δ
1.73-1.88 (m, 4H), 2.15-2.40 (m, 4H), 2.32 (s, 3H), 3.21
3
(
br s, 2H), 4.25 (q, 1H, J = 6.7 Hz), 6.35 (br s, 1H), 7.08 (dd, 1H , J = 3.9 Hz, 0.9 Hz), 7.44-
Ar
1
3
7
4
.48 (m, 2H ). C NMR (63 MHz, CDCl ) δ 26.1 (2 x CH ), 36.5 (2 x CH ), 40.3 (CH ),
Ar 3 2 2 3
2.1 (CH), 60.2 (2 x CH), 127.9 (CH ), 128.1 (CH ), 129.8 (CH ), 139.5 (C ), 161.1
Ar
Ar
Ar
q
+
(
C=O). HRMS (ESI): m/z calculated for C H N OS (MH ): 251.1218; Found: 251.1213
13 19 2
8
.1.5. 5-Bromo-N-[(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] thiophene-2-
carboxamide 9.
Compound 9 was prepared from the 5-bromothiophene-2-carboxylic acid and was isolated
after purification as a white solid (170 mg, 52%). mp 197-199 °C. R (CH Cl /MeOH 9/1)
f
2
2
-1
0
.18. IR (ATR diamond, cm ) ν 747, 1039, 1104, 1290, 1420, 1435, 1540, 1622, 2905, 3326.
1
H NMR (250 MHz, CDCl3)
δ 1.65 – 1.8 (m, 4H), 2.10 – 2.30 (m, 4H), 2.31 (s, 3H), 3.20 (br
s, 2H), 4.21 (q, 1H, J = 6.9 Hz), 6.19 (br s, 1H), 7.04 (d, 1H , J = 3.9 Hz), 7.18 (d, 1H , J =
Ar
Ar
1
3
3
.9 Hz). C NMR (63 MHz, CDCl ) δ 26.1 (2 x CH ), 36.4 (2 x CH ), 40.3 (CH ), 42.2 (CH),
3 2 2 3
6
0.2 (2 x CH), 117.7 (C ), 128.0 (CH ), 130.9 (CH ), 140.8 (C ), 160.0 (C=O). HRMS
q
Ar
Ar
q
+
(
ESI): m/z calculated for C H BrN OS (MH ): 329.0323; Found: 329.0335.
13 18 2
8
.1.6. N-[(1R,5S)-8-Methyl-8-azabicyclo[3.2.1]octan-3-yl] benzothio- phene-5-
carboxamide 10.
Compound 10 was prepared from the 1-benzothiophene-5-carboxylic acid and was isolated
after purification as a white solid (240 mg, 80%). mp 154-156 °C. R (CH Cl /MeOH 9/1)
f
2
2
-1
0
.15. IR (ATR diamond, cm ) ν 757, 1044, 1102, 1147, 1253, 1339, 1436, 1532, 1625, 2911,
1
3
309. H NMR (400 MHz, CDCl )
δ 1.79 (d, 2H, J = 14.4 Hz), 1.84-1.95 (m, 2H), 2.18-2.25
3
(m, 2H), 2.28-2.35 (m, 5H), 3.21 (br s, 2H), 4.30 (q, 1H, J = 6.8 Hz), 6.56 (d, 1H, J = 6.3 Hz),
7
.40 (d, 1H , J = 5.4 Hz), 7.52 (d, 1H , J = 5.4 Hz), 7.67 (dd, 1H , J = 8.4 Hz, J = 1.6 Hz),
Ar Ar Ar
13
7
.92 (d, 1H , J = 8.4 Hz), 8.20 (d, 1H , J = 1.6 Hz). C NMR (100 MHz, CDCl ) δ 26.2 (2 x
Ar
Ar
3
CH ), 36.6 (2 x CH ), 40.4 (CH ), 42.2 (CH), 60.2 (2 x CH), 122.3 (CH ), 122.5 (CH ),
2
2
3
Ar
Ar
1
22.9 (CH ), 124.4 (CH ), 128.2 (CH ), 131.6 (C ), 139.7 (C ), 142.8 (C ), 166.8 (C=O).
Ar Ar Ar q q q
+
HRMS (ESI): m/z calculated for C H N OS (MH ): 301.1375; Found: 301.1373.
1
7
21
2

ACCEPTED MANUSCRIPT
8
.1.7. N-[(1R,5S)-8-Methyl-8-azabicyclo[3.2.1]octan-3-yl]benzo
furan-5-
carboxamide 11.
Compound 11 was prepared from the 1-benzofuran-5-carboxylic acid and was isolated after
purification as a white solid (218 mg, 77%). mp 135-137 °C. R (CH Cl /MeOH 9/1) 0.16. IR
f
2
2
-1
(
ATR diamond, cm ) ν 731, 1025, 1110, 1130, 1299, 1345, 1434, 1459, 1536, 1631, 2933,
1
3
319. H NMR (400 MHz, CDCl )
δ
1.80 (d, 2H, J = 14.6 Hz), 1.86-1.92 (m, 2H), 2.14-2.26
3
(m, 2H), 2.30-2.40 (m, 5H), 3.24 (br s, 2H), 4.30 (q, 1H, J = 6.8 Hz), 6.50 (d, 1H, J = 5.9 Hz),
6
.84 (d, 1H , J = 2.0 Hz), 7.54 (d, 1H , J = 8.6 Hz), 7.63-7.71 (m, 2H ), 8.00 (d, 1H , J =
Ar Ar Ar Ar
13
1
.6 Hz). C NMR (100 MHz, CDCl )
δ 26.2 (2 x CH ), 36.4 (2 x CH ), 40.3 (CH ), 42.1
2 2 3
3
(
CH), 60.3 (2 x CH), 107.2 (CH ), 111.7 (CH ), 120.5 (CH ), 123.2 (CH ), 127.9 (C ),
Ar Ar Ar Ar q
1
30.4 (C ), 146.6 (CH ), 156.7 (C ), 166.9 (C=O). HRMS (ESI): m/z calculated for
q Ar q
+
C H N O (MH ): 285.1603; Found: 285.1606.
17
21
2
2
8
.1.8. N-[(1R,5S)-8-Methyl-8-azabicyclo[3.2.1]octan-3-yl]-5-(2-thienyl)thiophene-
2
-carboxamide 12.
Under argon, in a sealed vial, compound 9 (100 mg, 0.30 mmol) was dissolved in a mixture of
toluene/EtOH (3/1, 4 mL), and 2-thiophene boronic acid (47 mg, 0.36 mmol), K CO (85 mg,
2
3
0
.60 mmol) and Pd(PPh ) (18 mg, 15.6 ꢀmol) were successively added. The reaction mixture
3 4
was heated under microwave irradiation at 150 °C for 15 min. After cooling, water (15 mL)
was added, and extractions were performed with CH Cl (2 x 15 mL). The combined organic
2
2
layers were dried with MgSO , filtered and the volatiles were removed under reduced
4
pressure. The crude residue was purified by flash chromatography using silica gel eluting with
CH Cl /MeOH/NH OH (9/1/0.1) to afford compound 12 (85 mg, 84%) as a white solid. mp
2
2
4
-1
>
250 °C. R (CH Cl /MeOH 9/1) 0.15. IR (ATR diamond, cm ) ν 714, 1036, 1076, 1116,
f 2 2
1
1
1
3
5
δ
291, 1339, 1434, 1452, 1526, 1611, 1629, 2931, 3317. H NMR (400 MHz, DMSO-d ) δ
6
.82-2.24 (m, 8H), 2.32 (s, 3H), 3.27 (br s, 2H), 3.92 (q, 1H, J = 6.9 Hz), 7.15 (dd, 1H , J =
Ar
.5 Hz, J = 5.0 Hz), 7.36 (d, 1H , J = 3.9 Hz), 7.45 (d, 1H , J = 3.5 Hz), 7.62 (d, 1H , J =
Ar
Ar
Ar
13
.0 Hz), 7.77 (d, 1H , J = 3.9 Hz), 8.04 (d, 1H, J = 3.6 Hz). C NMR (100 MHz, DMSO-d )
Ar
6
24.8 (2 x CH ), 34.2 (2 x CH ), 39.2 (CH ), 41.5 (CH), 59.6 (2 x CH), 124.2 (CH ), 125.1
2
2
3
Ar
(CH ), 126.5 (CH ), 128.5 (CH ), 129.4 (CH ), 135.8 (C ), 138.3 (C ), 140.4 (C ), 160.9
Ar Ar Ar Ar q q q
+
(
C=O). HRMS (ESI): m/z calculated for C H N OS (MH ): 333.1095; Found: 333.1091.
1
7
21
2
2
8
.1.9. 5-(2-Furyl)-N-[(1R,5S)-8-methyl-8-azabicyclo[3.2.1] octan-3-yl ] thiophene-
2
-carboxamide 13.

ACCEPTED MANUSCRIPT
Compound 13 was synthesized as described for 12, starting from furan-2-boronic acid (41 mg,
0
.36 mmol), and was purified by flash chromatography using silica gel eluting with
CH Cl /MeOH/NH OH (9/1/0.1) as a white solid (85 mg, 90%). mp >250 °C. R
f
2
2
4
-1
(
CH Cl /MeOH 9/1) 0.16. IR (ATR diamond, cm ) ν 747, 1013, 1086, 1112, 1289, 1341,
2 2
1
1
439, 1514, 1536, 1616, 1630, 2925, 3326. H NMR (400 MHz, DMSO-d )
δ
1.89-2.30 (m,
6
8
H), 2.38 (s, 3H), 3.37 (br s, 2H), 3.93 (q, 1H, J = 6.9 Hz), 6.66 (dd, 1H, J = 3.4 Hz, J = 1.8
Hz), 6.94 (d, 1H , J = 3.4 Hz), 7.41 (d, 1H , J = 3.9 Hz), 7.77-7.87 (m, 2H ), 8.11 (d, 1H, J
Ar
Ar
Ar
1
3
=
3.6 Hz). C NMR (100 MHz, DMSO-d )
δ
24.5 (2 x CH ), 33.7 (2 x CH ), 39.1 (CH ), 41.3
6
2
2
3
(
CH), 59.9 (2 x CH), 107.2 (CH ), 112.4 (CH ), 123.0 (CH ), 129.3 (CH ), 136.5 (C ),
Ar Ar Ar Ar q
1
38.1 (C ), 143.3 (CH ), 148.0 (C ), 161.1 (C=O). HRMS (ESI): m/z calculated for
q Ar q
+
C H N O S (MH ): 317.1324; Found: 317.1312.
17
21
2
2
8
.1.12.
Borane-9-methylene-8H-quinolizine complex 17.
Under argon at -10°C, a solution of B H •THF (1M in THF) (9.0 mL, 9.0 mmol) was added
2
6
dropwise to a solution of the olefin 16 (1.36 g, 9.0 mmol) in 75 mL of dry THF. The mixture
was stirred at the same temperature for 1 h. A brine solution (30 mL) was added and the layers
were separated. The aqueous layer was extracted with ethyl acetate (2x50 mL) and the organic
layers were combined, dried over MgSO and evaporated under reduced pressure. The
4
resulting white precipitate was filtered and washed with petroleum ether (20 mL) to give the
borane tertiary derivative 17 (1.44 g, 97%) as a white solid. R (petroleum ether/EtOAc 9/1)
f
-1
1
0
.89. mp 97-99 °C. IR (ATR diamond, cm ) ν 916, 1033, 1137, 1295, 1397, 3277. H NMR
(
250 MHz, CDCl3) 1.25-1.73 (m, 7H), 1.85-2.02 (m, 1H), 2.07-2.29 (m, 2H), 2.39-2.66 (m,
δ
1
3
5
H), 2.82 (d, 1H, J = 11.8 Hz), 3.14 (d, 2H, J = 9.5 Hz), 4.81 (s, 1H), 4.97 (s, 1H). C NMR
21.1 (CH ), 21.8 (CH ), 23.6 (CH ), 24.4 (CH ), 33.9 (CH ), 65.0 (CH ),
(63 MHz, CDCl3)
δ
2
2
2
2
2
2
+
6
5.8 (CH ), 69.6 (CH), 111.4 (CH ), 142.9 (C ). MS (ESI): m/z 152.5 (M+H ).
2
2
q
8
.1.13.
General procedure for the synthesis of oximes 18-23.
The chosen aldehyde (5.0 mmol) was dissolved in dry CH Cl (10 mL). Then, triethylamine
2
2
(1.05 mL, 7.5 mmol) and hydroxylamine hydrochloride (420 mg, 6.0 mmol) were added to
the above solution and the mixture was stirred for 12 at room temperature. The reaction was
diluted with CH Cl (10 mL), washed with a saturated solution of NaHCO (10 mL), dried
2
2
3
over MgSO and evaporated under reduced pressure. Oximes were obtained as a mixture of
4
syn and anti stereoisomers. A simple chromatography on a silica gel led to enough pure
1
compound (verified by H NMR) to convert them into chlorooxime derivative.

ACCEPTED MANUSCRIPT
8
.1.14.
General procedure for the synthesis of chlorooximes 24-29.
At 0°C, the syn and anti oxime 18-23 (7.0 mmol) was dissolved in dry DMF (30 mL) and the
N-chlorosuccinimide (1.03 g, 7.7 mmol) was slowly added. The reaction was stirred for 12 h
and the solvent was carefully evaporated under reduced pressure (without heating above
4
0°C). The residue was solubilized into ethyl acetate (30 mL) and washed five times with
water (50 mL). The organic layer was dried over anhydrous MgSO and carefully
4
concentrated under vacuum to afford the crude chlorooxime 24-29. Their purities were
1
verified by H NMR prior to use the chlorooximes in the next cyclo-addition step.
8
.1.15.
General procedure A for the synthesis of spiranic derivatives 30, 31,
3
2 and 34.
A solution of chlorooximes 24-28 (2.0 mmol) in CH Cl (10 mL) in a gas-tight syringe was
2
2
added via a syringe pump over 48 h to a solution of exomethylene (14) (137 mg, 1.0 mmol)
and NaHCO (420 mg, 5.0 mmol) in dry CH Cl (20 mL). The reaction was then stirred at
3
2
2
ambient temperature for a further 24 h period which was achieved by addition of water (15
mL). The layers were separated, and the aqueous phase was extracted with CH Cl (2 x 10
2
2
mL). The organic extracts were combined and washed with brine (10 mL), dried over
anhydrous sodium sulfate, filtered and concentrated under reduced pressure. A flash column
chromatography on silica gel, eluting with CH Cl /MeOH/ NH OH (9/1/0.1) afforded the title
2
2
4
compound 30-34.
.1.16.
8
(1'R,5'S)-3-(4-Fluorophenyl)-8'-methyl-spiro[4H-isoxazole-5,3'-8-
azabicyclo[3.2.1]octane] 30.
Compound 30 was obtained as described in general procedure A from chlorooxime 24 as a
white solid (123 mg, 45%). mp 122-124 °C. R (CH Cl /MeOH 9/1) 0.22. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 837, 917, 1067, 1089, 1159, 1219, 1351, 1412, 1427, 1513, 1605, 1933. H NMR (400
MHz, CDCl3) 1.73-1.84 (m, 4H), 2.03-2.10 (m, 2H), 2.32 (dd, 2H, J = 13.8 Hz, 3.1 Hz),
.39 (s, 3H), 3.29 (s, 2H), 3.31 (s, 2H), 7.07 (t, 2H , J = 8.6 Hz), 7.62 (dd, 2H , J = 8.6 Hz, J
δ
2
Ar
Ar
1
3
=
3.2 Hz). C NMR (100 MHz, CDCl ) δ 26.1 (2 x CH ), 38.2 (CH ), 40.6 (2 x CH ), 48.8
3 2 3 2
(
CH ), 60.3 (2 x CH), 85.1 (C ), 116.0 (d, 2 x CH , J = 21.9 Hz), 126.6 (d, C , J = 3.4 Hz),
2 q Ar q
1
28.5 (d, 2 x CH , J = 8.3 Hz), 155.2 (C ), 169.8 (d, C , J = 250.3 Hz). HRMS (ESI): m/z
Ar q q
+
calculated for C H FN O (MH ): 275.1560; Found: 275.1566.
1
6
20
2
8
.1.17.
(1'R,5'S)-3-(4-Chlorophenyl)-8'-methyl-spiro[4H-isoxazole-5,3'-8-
azabicyclo[3.2.1]octane] 31.

ACCEPTED MANUSCRIPT
Compound 31 was obtained as described in general procedure A from chlorooxime 25 as a
white solid (127 mg, 44%). mp 155-157 °C. R (CH Cl /MeOH 9/1) 0.30. IR (ATR diamond,
f
2
2
-1
cm ) ν 914, 975, 1011, 1068, 1087, 1135, 1216, 1231, 1346, 1402, 1425, 1452, 1491, 1552,
1
1
593, 2937, 2962. H NMR (400 MHz, CDCl )
δ
1.74-1.84 (m, 4H), 2.05-2.12 (m, 2H), 2.32
3
(
dd, 2H, J = 13.9 Hz, J = 3.3 Hz), 2.40 (s, 3H), 3.31 (br s, 4H), 7.35 (d, 2H , J = 8.6 Hz),
Ar
1
3
7
.58 (d, 2H , J = 8.6 Hz). C NMR (100 MHz, CDCl ) δ 26.1 (2 x CH ), 38.1 (CH ), 40.6 (2
Ar 3 2 3
x CH ), 48.6 (CH ), 60.2 (2 x CH), 85.3 (C ), 127.8 (2 x CH ), 128.9 (C ), 129.1 (2 x CH ),
2
2
q
Ar
q
Ar
+
1
35.9 (C ), 155.3 (C ). HRMS (ESI): m/z calculated for C H ClN O (MH ): 291.1264;
q q 16 20 2
Found: 291.1249.
8
.1.18.
(1'R,5'S)-3-(1,3-Benzodioxol-5-yl)-8'-methyl-spiro[4H-isoxa-zole-
5
,3'-8-azabicyclo[3.2.1]octane] 32.
Compound 32 was obtained as described in general procedure A from chlorooxime 26 as a
-
white solid (141 mg, 47%). mp >250 °C. R (CH Cl /MeOH 9/1) 0.14. IR (ATR diamond, cm
f
2
2
1
1
)
ν 918, 976, 1031, 1101, 1236, 1259, 1379, 1434, 1453, 1504, 2502. H NMR (400 MHz,
CDCl3) 1.70-1.82 (m, 4H), 2.00-2.20 (m, 2H), 2.32 (dd, 2H, J = 14.1 Hz, J = 3.3 Hz), 2.42
δ
(
s, 3H), 3.29 (s, 2H), 3.32 (br s, 2H), 6.00 (s, 2H), 6.81 (d, 1H , J = 8.1 Hz), 7.00 (dd, 1H , J
Ar
Ar
1
3
=
8.1 Hz, J = 1.5 Hz), 7.26 (d, 1H , J = 1.5 Hz). C NMR (100 MHz, CDCl )
δ
26.1 (2 x
Ar
3
CH ), 37.9 (CH ), 40.4 (2 x CH ), 49.0 (CH ), 60.3 (2 x CH), 84.5 (C ), 101.6 (CH ), 106.5
2
3
2
2
q
2
(CH ), 108.3 (CH ), 121.2 (CH ), 124.5 (C ), 148.3 (C ), 149.3 (C ), 155.8 (C ). HRMS
Ar Ar Ar q q q q
+
(
ESI): m/z calculated for C H N O (MH ): 301.1552; Found: 301.1537.
17
21
2
3
8
.1.19.
(1'R,5'S)-3-(Benzothiophen-5-yl)-8'-methyl-spiro[4H-isoxazole-5,3'-
8
-azabicyclo[3.2.1]octane] 33.
Compound 33 was obtained as described in general procedure A from chlorooxime 27 as a
white solid (121 mg, 39%). mp 162-164 °C. R (CH Cl /MeOH 9/1) 0.31. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 931, 1049, 1105, 1148, 1236, 1309, 1363, 2923. H NMR (400 MHz, CDCl )
δ
1.79-
3
1
.97 (m, 4H), 2.08-2.27 (m, 2H), 2.40 (dd, 2H, J = 14.1 Hz, J = 3.0 Hz), 2.49 (s, 3H), 3.40 (br
s, 2H), 3.44 (s, 2H), 7.36 (d, 1H , J = 5.4 Hz), 7.49 (d, 1H , J = 5.4 Hz), 7.76 (d, 1H , J =
Ar
Ar
Ar
1
3
8
.5 Hz), 7.88 (d, 1H , J = 8.5 Hz), 7.97 (s, 1H ). C NMR (100 MHz, CDCl )
δ
26.0 (2 x
Ar
Ar
3
CH ), 37.7 (CH ), 40.2 (2 x CH ), 48.9 (CH ), 60.4 (2 x CH), 84.4 (C ), 122.0 (CH ), 122.4
2
3
2
2
q
Ar
(CH ), 123.0 (CH ), 124.2 (CH ), 126.6 (C ), 127.8 (CH ), 139.8 (C ), 141.3 (C ), 156.4
Ar Ar Ar q Ar q q
+
(
C ). HRMS (ESI): m/z calculated for C H N OS (MH ): 313.1375; Found: 313.1375.
q
18 21
2

ACCEPTED MANUSCRIPT
8
.1.20.
(1'R,5'S)-3-(Benzofuran-5-yl)-8'-methyl-spiro[4H-isoxazole-5,3'-8-
azabicyclo[3.2.1]octane] 34.
Compound 34 was obtained as described in general procedure A from chlorooxime 28 as a
-
yellow solid (98 mg, 33%). mp >250 °C. R (CH Cl /MeOH 9/1) 0.17. IR (ATR diamond, cm
f
2
2
1
1
)
ν 929, 1026, 1106, 1131, 1173, 1231, 1264, 1346, 1367, 1437, 1477, 1615, 2550, 3405. H
NMR (400 MHz, DMSO-d6) 2.05-2.22 (m, 6H), 2.33 (d, 2H, J = 12.7 Hz), 2.58 (s, 3H),
.31 (br s, 2H), 3.65 (s, 2H), 7.06 (d, 1H , J = 2.1 Hz), 7.68-7.76 (m, 2H ), 8.01 (s, 1H ),
δ
3
8
3
Ar
Ar
Ar
1
3
.10 (d, 1H , J = 2.1 Hz). C NMR (100 MHz, DMSO-d )
δ 23.4 (2 x CH ), 39.6 (CH ),
2 3
Ar
6
9.8 (2 x CH ), 47.2 (CH ), 61.4 (2 x CH), 82.5 (C ), 106.9 (CH ), 111.6 (CH ), 120.2
2
2
q
Ar
Ar
(CH ), 122.8 (CH ), 124.8 (C ), 127.4 (C ), 147.1 (CH ), 154.9 (C ), 156.7 (C ). HRMS
Ar Ar q q Ar q q
+
(
ESI): m/z calculated for C H N O (MH ): 297.1603; Found: 297.1606.
18
21
2
2
8
.1.21.
General procedure B for the synthesis of spiranic derivatives 36-41.
To a suspension of borane-9-methylene-8H-quinolizine complex (200 mg, 1.21 mmol) and
potassium carbonate (508 mg, 6.05 mmol) in ethyl acetate (15 mL) were added the
corresponding chlorooximes (2.42 mmol). The reaction mixture was stirred at room
temperature for 48 h. after completion of the cycloaddition, the solvent was evaporated and
the residue was purified by silica gel column chromatography eluting with
CH Cl /MeOH/NH OH (9/1/0.1) to afford the corresponding spiranic adduct.
2
2
4
8
.1.22.
(9S,9aS)-3'-(4-Fluorophenyl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 36.
Compound 36 was obtained as described in general procedure B from chlorooxime 24 as a
-1
white solid (188 mg, 54%). mp 102-104 °C. R 0.15. IR (ATR diamond, cm ) ν 915, 1103,
f
1
1
118, 1130, 1166, 1221, 1290, 1355, 1443, 1514, 1603, 2940. H NMR (400 MHz, CDCl ) :
δ
3
0
3
.89-1.06 (m, 1H), 1.14-1.31 (m, 1H), 1.40-1.87 (m, 7H), 1.90-2.12 (m, 4H), 2.75-2.96 (m,
1
3
H), 3.48 (d, 1H, J = 17.2 Hz), 7.02-7.13 (m, 2H ), 7.61-7.70 (m, 2H ). C NMR (100
Ar
Ar
MHz, CDCl ) :
δ 23.1 (CH ), 24.0 (CH ), 24.8 (CH ), 25.9 (CH ), 37.2 (CH ), 40.6 (CH ),
2 2 2 2 2 2
3
5
6.5 (CH ), 56.9 (CH ), 68.2 (CH), 88.2 (C ), 116.0 (d, 2 x CH , J = 21.9 Hz), 126.3 (d, C , J
2 2 q Ar q
=
3.3 Hz), 128.6 (d, 2 x CH , J = 8.4 Hz), 155.3 (C ), 163.8 (d, C , J = 250.3 Hz). HRMS
Ar q q
+
(
ESI): m/z calculated for C H FN O (MH ): 289.1716; Found: 289.1707.
17 22 2
8
.1.23.
(9S,9aS)-3'-(4-Chlorophenyl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 37.

ACCEPTED MANUSCRIPT
Compound 37 was obtained as described in general procedure B from chlorooxime 25 as a
white solid (184 mg, 50%). mp 133-135 °C. R (CH Cl /MeOH 9/1) 0.19. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 1014, 1027, 1090, 1107, 1119, 1289, 1352, 1405, 1442, 1495, 1596, 2934. H NMR
400 MHz, CDCl3) 0.91-1.02 (m, 1H), 1.19-1.30 (m, 1H), 1.48 (q, 1H, J = 12.8 Hz), 1.55-
.80 (m, 6H), 1.92-2.10 (m, 4H), 2.75-2.94 (m, 2H), 2.92 (d, 1H, J = 17.2 Hz), 3.47 (s, 1H, J
(
δ
1
1
3
=
17.2 Hz), 7.37 (d, 2H , J = 8.5 Hz), 7.60 (d, 2H , J = 8.5 Hz). C NMR (100 MHz,
Ar Ar
CDCl3)
δ 23.1 (CH ), 24.0 (CH ), 24.8 (CH ), 25.8 (CH ), 37.2 (CH ), 40.4 (CH ), 56.5
2 2 2 2 2 2
(
CH ), 56.8 (CH ), 68.2 (CH), 88.5 (C ), 127.9 (2 x CH ), 128.6 (C ), 129.2 (2 x CH ),
2 2 q Ar q Ar
+
1
36.0 (C ), 155.4 (C ). HRMS (ESI): m/z calculated for C H ClN O (MH ): 305.1421;
q q 17 22 2
Found: 305.1432.
8
.1.24.
(9S,9aS)-3'-(1,3-benzodioxol-5-yl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 38.
Compound 38 was obtained as described in general procedure B from chlorooxime 26 as a
white solid (198 mg, 52%). mp 218-220 °C. R (CH Cl /MeOH 9/1) 0.25. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 920, 1035, 1114, 1222, 1250, 1348, 1453, 1496, 2930. H NMR (400 MHz, CDCl )
δ
3
0
3
.85-1.05 (m, 1H), 1.12-1.31 (m, 1H), 1.39-1.83 (m, 7H), 1.87-2.10 (m, 4H), 2.75-2.89 (m,
H), 3.44 (d, 1H, J = 17.2 Hz), 5.97 (s, 2H), 6.79 (d, 1H , J = 8.1 Hz), 7.02 (dd, 1H , J = 8.1
Ar
Ar
1
3
Hz, J = 1.7 Hz), 7.26 (d, 1H , J = 1.7 Hz). C NMR (100 MHz, CDCl )
δ
23.1 (CH ), 24.0
Ar
3
2
(
CH ), 24.7 (CH ), 25.8 (CH ), 37.2 (CH ), 40.7 (CH ), 56.5 (CH ), 56.8 (CH ), 68.3 (CH),
2 2 2 2 2 2 2
8
1
7.8 (C ), 101.6 (CH ), 106.5 (CH ), 108.3 (CH ), 121.4 (CH ), 124.2 (C ), 148.2 (C ),
q 2 Ar Ar Ar q q
+
49.3 (C ), 155.8 (C ). HRMS (ESI): m/z calculated for C H N O (MH ): 315.1709;
q
q
18 23
2
3
Found: 315.1693.
8.1.25.
(9S,9aS)-3'-(benzothiophen-5-yl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 39.
Compound 39 was obtained as described in general procedure B from chlorooxime 27 as a
white solid (220 mg, 56%). mp 113-115 °C. R (CH Cl /MeOH 9/1) 0.22. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 933, 1025, 1055, 1119, 1183, 1288, 1366, 1442, 2749, 2795, 2866, 2934. H NMR
400 MHz, CDCl3) 0.95-1.10 (m, 1H), 1.14-1.34 (m, 1H), 1.38-1.88 (m, 7H), 1.93-2.15 (m,
H), 2.76-2.98 (m, 2H), 3.04 (d, 1H, J = 17.2 Hz), 3.60 (d, 1H, J = 17.2 Hz), 7.35 (d, 1H , J
(
δ
4
Ar
=
5.4 Hz), 7.49 (d, 1H , J = 5.4 Hz), 7.77 (dd, 1H , J = 8.5 Hz, 1.4 Hz), 7.88 (d, 1H , J =
Ar
Ar
Ar
1
3
8
.5 Hz), 8.01 (d, 1H , J = 1.4 Hz). C NMR (100 MHz, CDCl )
δ
23.2 (CH ), 24.1 (CH ),
Ar
3
2
2
2
4.8 (CH ), 25.9 (CH ), 37.2 (CH ), 40.8 (CH ), 56.6 (CH ), 56.9 (CH ), 68.4 (CH), 88.0
2
2
2
2
2
2

ACCEPTED MANUSCRIPT
(
C ), 122.2 (CH ), 122.4 (CH ), 123.0 (CH ), 124.3 (CH ), 126.4 (C ), 127.7 (CH ),
q
Ar
Ar
Ar
Ar
q
Ar
+
1
39.8 (C ), 141.2 (C ), 156.5 (C ). HRMS (ESI): m/z calculated for C H N OS (MH ):
q
q
q
19 23
2
3
27.1531; Found: 327.1534.
8.1.26.
(9S,9aS)-3'-(benzofuran-5-yl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 40.
Compound 40 was obtained as described in general procedure B from chlorooxime 28 as a
white solid (184 mg, 49%). mp 92-94 °C. R (CH Cl /MeOH 9/1) 0.21. IR (ATR diamond,
f
2
2
-1
1
cm ) ν 883, 907, 932, 1031, 1105, 1129, 1228, 1266, 1366, 1439, 1470, 2931. H NMR (400
MHz, CDCl3) 0.92-1.10 (m, 1H), 1.13-1.37 (m, 1H), 1.41-1.86 (m, 7H), 1.94-2.13 (m, 4H),
.80-2.95 (m, 2H), 3.00 (d, 1H, J = 17.2 Hz), 3.59 (d, 1H, J = 17.2 Hz), 6.78 (dd, 1H , J =
δ
2
2
Ar
.2 Hz, J = 1.0 Hz), 7.50 (dt, 1H , J = 8.7 Hz, J = 1.0 Hz), 7.65 (d, 1H , J = 2.2 Hz), 7.72
Ar
Ar
1
3
(
dd, 1H , J = 8.7 Hz, J = 1.7 Hz), 7.86 (d, 1H , J = 1.7 Hz). C NMR (100 MHz, CDCl ) δ
Ar Ar 3
2
3.2 (CH ), 24.1 (CH ), 24.8 (CH ), 25.9 (CH ), 37.2 (CH ), 41.0 (CH ), 56.6 (CH ), 56.9
2 2 2 2 2 2 2
(CH ), 68.4 (CH), 87.9 (C ), 107.0 (CH ), 112.0 (CH ), 120.0 (CH ), 123.2 (CH ), 125.2
2 q Ar Ar Ar Ar
(
C ), 127.9 (C ), 146.1 (CH ), 155.9 (C ), 156.4 (C ). HRMS (ESI): m/z calculated for
q
q
Ar
q
q
+
C H N O (MH ): 311.1760; Found: 311.1770.
19
23
2
2
8
.1.27.
(9S,9aS)-3'-(5-bromo-2-thienyl)-spiro[8H-quinolizine-9,5'-4H-
isoxazole] 41.
Compound 41 was obtained as described in general procedure B from chlorooxime 29 as a
white solid (274 mg, 64%). mp 123-125 °C. R (CH Cl /MeOH 9/1) 0.19. IR (ATR diamond,
f
2
2
-1
cm ) ν 914, 952, 984, 1108, 1126, 1160, 1179, 1230, 1297, 1350, 1445, 2755, 2799, 2852,
1
2
7
3
2
928. H NMR (400 MHz, CDCl )
δ
0.87-1.05 (m, 1H), 1.13-1.33 (m, 1H), 1.40-1.83 (m,
3
H), 1.88-2.12 (m, 4H), 2.75-2.90 (m, 3H), 3.45 (d, 1H, J = 17.0 Hz), 6.89 (d, 1H, H , J =
Ar
1
3
.9 Hz), 7.00 (d, 1H, H , J = 3.9 Hz). C NMR (100 MHz, CDCl ) δ 23.1 (CH ), 24.0 (CH ),
2 2
Ar
3
4.7 (CH ), 25.8 (CH ), 37.0 (CH ), 40.7 (CH ), 56.5 (CH ), 56.8 (CH ), 68.1 (CH), 88.8
2
2
2
2
2
2
(
C ), 115.7 (C ), 128.2 (CH ), 130.3 (CH ), 134.2 (C ), 151.6 (C ). HRMS (ESI): m/z
q q Ar Ar q q
+
calculated for C H BrN OS (MH ): 355.0480; Found: 355.0475.
1
5
20
2
8
.2. Molecular modeling
Computational results were obtained using software programs from Accelrys Software Inc.
The molecules were built and minimized in a molecular package (Discovery Studio® 2.5.5,
Accelrys, San Diego, CA) by CHARMm with the CFF partial charge estimation method. The
3
D structures were generated and optimized using a CHARMm forcefield with a root mean

ACCEPTED MANUSCRIPT
squared (RMS) difference of energy gradient of 0.1 kcal/mol. The GFA model in the QSAR
protocol was used with a population size of 100 and 5000 maximum generations, in linear
model form. The descriptors correlation matrix and the results are given in the Supplementary
data.
For the 3D-QSAR model, all the structures were superimposed by choosing N-1 and C-4
(spiranic carbon) on the piperidine scaffold and C-1’ on the first aromatic cycle as tethers to
superimpose. The grid spacing was 1.5 Å. For docking studies, the crystal structure of the
pentamer AChBP obtained from Aplysia californica (Ac-AChBP) with 2-methoxy-4-OH-
benzylidene anabaseine (pdb code 2WN9) was used as template [22]. Interaction sites were
calculated within a radius of 11.0 Å. The fit was achieved with a maximum rms deviation of
0
.7 Å from the interaction sites for each structure, at physiological pH, using the DeNovo
protocol.
8
.3. Biological studies.
Experiments were performed on male Wistar rats weighing 250-300 g (Centre d’Elevage R.
Janvier, Le Genest St Isle, France). All animal use procedures were conducted in accordance
with the requirements of the European Community Council Directive 2010/63/EU for the care
of laboratory animals and with the authorization of the Regional Ethical Committee. Rats
were kept in a temperature (23 ± 0.5°C) and humidity (43 ± 8%) controlled environment
under a 12 h light-dark cycle with food and water available ad libitum. All efforts were made
to minimize animal suffering and discomfort.
Stable α-bungarotoxin and methyllycaconitine were obtained from Tocris Bioscience (R&D
1
25
Systems, Lille, France) and [ I]α-bungarotoxin (specific activity 81.4 TBq/mmol) from
Perkin-Elmer (Courtaboeuf, France).
8
.4.1. In vitro binding assays.
Animals were killed by decapitation on the day of the assay and both frontal cortices of each
animal were removed on ice and weighed (2 rats were used for each experiment). The tissue
was homogenized in 10 vol of a pH 7.4 HEPES 15 mM buffer containing 120 mM NaCl, 5.4
mM KCl, 0.8 mM MgCl and 1.8 mM CaCl using an Ultraturrax T25. After 45,000×g
2
2
centrifugation at 4°C for 10 min (J2-21M/E, Beckman), the supernatant was eliminated and
the pellet was suspended in 2 mL of the same buffer. The protein concentration was measured
according to Bradford using bovine serum albumin as standard.

ACCEPTED MANUSCRIPT
125
For competition studies, [ I]α-bungarotoxin (2 nM) was incubated in the presence or
-6
-10
absence of each tested compound (10 to 10 M) with 25 µg protein in a total volume of 1
mL in a pH 7.4 Tris-HCl buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2
and 2.5 mM CaCl ) for 3 h at 22°C. Nonspecific binding was determined in the presence of 1
2
µM α-bungarotoxin. After incubation, samples were diluted in 3 mL of Tris-HCl buffer at 4°C
and then rapidly filtered through Whatman GF/C fiber filters soaked with 0.05%
polyethylenimine (Sigma, St Quentin-Fallavier, France). The filters were washed twice with 3
mL of 4°C buffer, and the residual radioactivity was measured in a γcounter (2480 Gamma
counter Wizard, Perkin Elmer).The IC50 values were determined graphically for each
compound and the Ki calculated according to Cheng & Prussof [25].
8
.4.2. Cerebral biodistribution studies.
1
8
Rats were injected i.v. under isofurane gas anesthesia in the tail vein with 0.3 mL of [ F]4
5±2 MBq) in the control group (n = 5). In the MLA group (n = 5), injection of the tracer was
(
preceded (15 min) by i.v. injection of methyllycaconitine (3 mg/kg). Rats were killed by
decapitation at 1 h post injection of the tracer. The whole brain was quickly removed and
dissected into segments consisting of the cortex, striatum, hippocampus, hypothalamus,
midbrain and cerebellum. Samples were weighed and radioactivity was measured in the same
1
8
γ counter as above (counting efficiency of 48% for F), and the percent injected dose per
gram of tissue (%ID/g) was calculated by comparison with samples to standard dilutions of
the injected solution.
8
.5. Radiochemistry.
.5.1. N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-[ F]fluorobenzamide [ F]4.
18
18
8
1
8
No-carrier-added aqueous F-fluoride ion was produced on a cyclotron (PET trace, GE
1
8
18
18
Healthcare) by irradiation of enriched O H O with protons via the O(p,n) F nuclear
2
1
8
reaction. F-Fluoride was transferred to a GE TRACERlab FX-FN synthesizer and passed
through an anion-exchange resin (Waters Sep-Pak Accell Light QMA cartridge in the
1
8
carbonate form). Trapped F-fluoride was isolated by elution with a solution of aqueous
eluent solution containing K CO (7 mg in 300 µL of pure water), acetonitrile (300 µL), and
2
3
2
2 mg of Kryptofix-222 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8] hexacosane).
Azeotropic drying by addition of ACN (1 mL) was performed. The evaporation was
performed at 90°C under helium flow and vacuum, and the operation was repeated twice.
1
8
ACN (300 µl) was added to the reactor to solubilize the F fluoride which was transferred to

ACCEPTED MANUSCRIPT
the 2 mL reactor of the microwave (PET wave, CEM) containing 2 mg of nitrobenzamide 6
dissolved in DMSO (1 mL). The solution was gently heated to 95 °C for 2 min under helium
flow to remove the volatiles. Then, the solution was heated under microwave irradiation at
1
00 W for 10 min. The solution was cooled and transferred back to the FX-FN synthesizer.
The crude product was injected onto a Gemini C18 column (10x250 mm, 10ꢀm) and
purification occurred at 5 mL/min using NH OAc 0.1M (0.2% Et N)/ACN (82:18) as mobile
4
3
1
8
phase. The radioactive peak corresponding to [ F]4 was collected and diluted with water (30
mL). The radioactive product was trapped on a tC18 cartridge (Waters Sep-Pak Accell Light
1
8
tC18 cartridge. The cartridge was rinsed with 5 mL of water and [ F]4 was eluted from the
tC18 with EtOH (0.5 mL). Formulation was completed by addition of 0.9 % NaCl solution
(4.5 mL).
8
.6. Cristallography.
X-ray analyses were carried out on a Bruker Enraf Nonius CAD4 diffractometer with a Cu
sealed tube. The structures were solved by direct methods and refined using the Shelx 97 suite
of programs [26] in the integrated WinGX system [27]. The positions of the H atoms were
deduced from coordinates of the non-H atoms and confirmed by Fourier synthesis. The non-H
atoms were refined with anisotropic temperature parameters. H atoms were included for
structure factor calculations but not refined. The program PLATON [28] was used for analysis
and drawing figures with thermal ellipsoids at a 30% probability level. Crystallographic data
have been deposited in the Cambridge Crystallographic Data Centre (CCDC numbers 952168
and 952169 for compounds 30 and 26 respectively). Copies of these data can be obtained free
of charge from the Director CCDC 12 Union Road, Cambridge, CB2 1EZ, UK.
Fax:
+44
1223
336
033;
e-mail:
deposit@ccdc.cam.ac.uk
or
http://www.ccdc.cam.ac.uk/data_request/cif.
9. Supporting information
1
13
H and C spectra copy for compounds 23, 25-27, 31-54, QSAR study and correlation matrix
of molecular property vs properties as a heat map and additional cristallographic data are
furnished.
10. Acknowledgements
This research was supported by grants from the Région Centre the ANR Malz program (ANR-
0-MALZ-0004) and France Alzheimer. The two teams (ICOA, U930) involved in the project
are members of Labex IRON (ANR-11-LABX-0018-01). The authors thank Cyclopharma
1

ACCEPTED MANUSCRIPT
1
8
Laboratories for kindly supplying F and for use of their laboratory (Laboratoires
Cyclopharma, Biopôle Clermont-Limagne, 63360 Saint-Beauzire, France).
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ACCEPTED MANUSCRIPT
List of figure captions :
1
8
Figure 1. Representative α7 nAchR ligands and reported F PET imaging agents.
Figure 2. Main synthetic objectives.
Figure 3. ORTEP representation of X-Ray analysis of 30.
Figure 4. ORTEP representation of X-Ray crystal structures of 41.
Figure 5. Isosurface of the 3D-QSAR model coefficients in a single picture.
1
8
Figure 6. Cerebral biodistribution of [ F]4 quantified after 60 min in rat.

ACCEPTED MANUSCRIPT
N
N
N
N
NH
O
NH
NH
NH
O
O
O
N
O
N
S
F
O
F
Amid
PHA-543613)
Urea
Ether
Carbamate
JN403)
(
(
O
R
N
O
O
Ia R = H (AR-R17779)
N
Me
N
Ib
N
I
Ic
S
O
N
NH
O
N
N
N
O
N
N
IIIb
N
IIIa (PHA-709829)
18F
N
N
18F
O
N
O
N
[18
F]NS10743
[18
F]AZ11637326
N
1
8
F
18F
O
S
(
)
2
O
N
N
N
N
O
N
N
[18
F]IV
[18
F]NS14490
N
1
8
Figure 1. Representative α7 nAchR ligands and reported F PET imaging agents.
Figure 2. Main synthetic objectives.