Bis(het)aryl-1,2,3-triazole quinuclidines as α7 nicotinic acetylcholine receptor ligands: Synthesis, structure affinity relationships, agonism activity, [18F]-radiolabeling and PET study in rats

Article abstract of DOI:10.1016/j.ejmech.2019.06.049

In this paper we describe the design and synthesis of bis(Het)Aryl-1,2,3-triazole quinuclidine α7R ligands using an efficient three-step sequence including a Suzuki-Miyaura cross coupling reaction with commercially available and home-made boron derivatives. The exploration of SAR required the preparation of uncommon boron derivatives. Forty final drugs were tested for their ability to bind the target and nine of them exhibited Ki values below nanomolar concentrations. The best scores were always obtained when the 5-phenyl-2-thiophenyl core was attached to the triazole. The selectivity of these compounds towards the nicotinic α4β2 and serotoninergic 5HT3 receptors was assessed and their brain penetration was quantified by the preparation and in vivo evaluation of two [¹⁸F] radiolabelled derivatives. It can be expected from our results that some of these compounds will be suitable for further developments and will have effects on cognitive disorders.

Full text of DOI:10.1016/j.ejmech.2019.06.049

Accepted Manuscript
Bis(het)aryl-1,2,3-triazole quinuclidines as α7 nicotinic acetylcholine receptor ligands:
18
Synthesis, structure affinity relationships, agonism activity, [ F]-radiolabeling and
PET study in rats
Aziz Ouach, Johnny Vercouillie, Emilie Bertrand, Nuno Rodrigues, Frederic Pin,
Sophie Serriere, Liliana Boiaryna, Agnes Chartier, Nathalie Percina, Pakorn
Tangpong, Zuhal Gulhan, Celine Mothes, Jean-Bernard Deloye, Denis Guilloteau,
Guylene Page, Franck Suzenet, Frederic Buron, Sylvie Chalon, Sylvain Routier
PII:
S0223-5234(19)30576-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.049
EJMECH 11452
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 April 2019
Revised Date: 16 June 2019
Accepted Date: 17 June 2019
Please cite this article as: A. Ouach, J. Vercouillie, E. Bertrand, N. Rodrigues, F. Pin, S. Serriere, L.
Boiaryna, A. Chartier, N. Percina, P. Tangpong, Z. Gulhan, C. Mothes, J.-B. Deloye, D. Guilloteau,
G. Page, F. Suzenet, F. Buron, S. Chalon, S. Routier, Bis(het)aryl-1,2,3-triazole quinuclidines as α7
nicotinic acetylcholine receptor ligands: Synthesis, structure affinity relationships, agonism activity,
18
[
F]-radiolabeling and PET study in rats, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.049.
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ACCEPTED MANUSCRIPT
Bis(het)aryl-1,2,3-triazole
quinuclidines
as
α7
nicotinic
acetylcholine receptor ligands: synthesis, structure affinity
1
8
relationships, agonism activity, [ F]-radiolabeling and PET study
in rats.
1
,5
2
1
1
1
Aziz Ouach, Johnny Vercouillie, Emilie Bertrand, Nuno Rodrigues, Frederic Pin, Sophie
2
1
1
1
2
Serriere, Liliana Boiaryna, Agnes Chartier, Nathalie Percina, Pakorn Tangpong, Zuhal
2
3
3
2
4
Gulhan, Celine Mothes, Jean-Bernard Deloye, Denis Guilloteau, Guylene Page, Franck
1
1
2,*
1,*
Suzenet, Frederic Buron, Sylvie Chalon and Sylvain Routier
1
Institut de Chimie Organique et Analytique, Université d’Orléans, UMR CNRS 7311, rue de
Chartres, BP 6759, 45067 Orleans Cedex 2, France.
2
UMR 1253, iBrain, Université de Tours, Inserm, Tours, France.
3
Zionexa, 42 avenue de la Grande Armée, 75017 Paris, France.
4
EA3808-NEUVACOD, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073
Poitiers, France Cedex 9.
5
Present address: CHU Nantes, délégation régionale à la recherche clinique et à l’innovation,
5
allée de l’île Gloriette, Nantes, 44000 France.
Corresponding authors: sylvain.routier@univ-orleans.fr and sylvie.chalon@univ-tours.fr
1
8
KEYWORDS. Triazole, Quinuclidine, SAR, Agonism, Radiolabeling, F PET, Alpha 7
nicotinic acetylcholine receptor.
1

ACCEPTED MANUSCRIPT
Abstract
In this paper we describe the design and synthesis of bis(Het)Aryl-1,2,3-triazole
quinuclidine α7R ligands using an efficient three-step sequence including a Suzuki-Miyaura
cross coupling reaction with commercially available and home-made boron derivatives. The
exploration of SAR required the preparation of uncommon boron derivatives. Forty final
drugs were tested for their ability to bind the target and nine of them exhibited Ki values
below nanomolar concentrations. The best scores were always obtained when the 5-phenyl-2-
thiophenyl core was attached to the triazole. The selectivity of these compounds towards the
nicotinic α4β2 and serotoninergic 5HT3 receptors was assessed and their brain penetration
1
8
was quantified by the preparation and in vivo evaluation of two [ F] radiolabelled derivatives.
It can be expected from our results that some of these compounds will be suitable for further
developments and will have effects on cognitive disorders.
2

ACCEPTED MANUSCRIPT
Introduction
The role of the brain cholinergic system is largely mediated through nicotinic receptors,
which are ligand-gated ion channels composed of 5 subunits belonging to α and/or β
subfamily. The rich diversity of expression and function of nicotinic receptors, with neuronal
but also non-neuronal localization, and several sub-types which can interact in the control of
different neurotransmission pathways, support their major and complex involvement in a
large variety of physiological and cognitive functions.[1] The predominant subtypes of
nicotinic receptors in human brain are the heteropentameric α4β2 and homopentameric α7
forms. The α7 nicotinic acetylcholine receptor (α7R) is of particular interest due to its
involvement in various brain disorders such as schizophrenia and Alzheimer’s disease,[2, 3]
associated to its significant role in neuroinflammation. [4, 5]
5
The crucial role of α7R in a number of specific functional systems and diseases has
recently been extensively reviewed. [6, 7]
The in vivo exploration of α7R by a molecular imaging method such as PET (positron
emission tomography) is a highly promising way to better understand the changes associated
with the appearance and progression of these brain diseases but also to monitor treatment
efficacy and to improve the development of new therapeutic strategies. The key step in this
exploration is the availability of a suitable radiotracer, and several recent papers have
reviewed the state of progress in this field (Figure 1). [8-10]
We recently designed a novel series of α7R ligands using a straightforward methodology
able to selectively furnish derivatives of type I having a 1,2,3- triazole group attached directly
to a (R) quinuclidine in C-3. [11, 12] As demonstrated by another research group, these
ligands appeared suitable for therapeutic use. [13-16]
3

ACCEPTED MANUSCRIPT
Figure 1. Some representative α7 nicotinic receptor ligands developed either for therapy or
PET imaging.
SAR studies in family I showed that meta and para (Het)Ar substitutions were tolerated but
that the best Ki values remained mainly up to 2 nM (compound 1). Following a disruptive
approach from two interesting benzothiofuran 2 and benzothiophen 3 derivatives, we
developed unfused bis(Het)Ar derivatives of type II (Figure 2).
Figure 2. Drug design strategies leading to the development of the bis(Het)Ar derivatives
II. Sat = Satellites.
In this paper, we present the SAR evolution leading to very efficient ligands exhibiting
improved inhibition constants in the (sub)nanomolar range. Each terminal (Het)Ar group was
2
additionally equipped with some satellite functions (Sat) in order to explore the SAR
4

ACCEPTED MANUSCRIPT
diversity, and modulate the binding and hydrophilic or lipophilic balance parameters.
Globally, to envision a further development of the available series, a set of 47 final derivatives
was evaluated and among them some were selected to determine their agonist potency as well
as their selectivity against α4β2 and 5-HT receptors. The most potent fluorinated compounds
3
1
8
were radiolabelled with F in order to evaluate their biological properties in vivo in rodents
and their CNS compatibility.
Results and discussion
Synthesis
The synthesis of the library of type II ligands was achieved from (R)-3-quinuclidine amine
bis-hydrochloride 4 after three steps (Scheme 1). The first step consists in the preparation of
the azide followed by direct Huisgen condensation using bromo (Het)Ar terminal alkynes,
1
leading to the regioselective synthesis of 1,2,3-triazole. The last step is the Suzuki cross
coupling reaction which was carried out between borylated building blocks and the previously
formed brominated intermediate.
Scheme 1. Synthetic pathway to generate α7R ligands II.
To achieve this synthetic pathway, it was first necessary to generate a library of
bromo(Het)Ar terminal alkynes (Table 1). All of them were prepared from commercially
available aldehydes in the presence of the Bestmann Ohira reagent and an excess of base.[17]
Acetylenes 6-9 were obtained in near stoichiometric yields and stored without any precaution.
Synthesis of the regioselective triazole was achieved from the azide of 4 (which was obtained
5

ACCEPTED MANUSCRIPT
using the highly unstable reagent 5) and the alkynes 6-9 under classical copper catalyzed
conditions to afford the brominated derivatives 10-13 in satisfying yields.[18]
Table 1. Synthesis of compounds 10-13.
Alkynyl
b
Entry
Aldehydes
Compounds
Brominated Compounds (Step 2 , Yields)
a
(
Step 1 , Yield)
[
19]
20]
c
c
1
6
7
(95%)
(92%)
10 (50%)
[
c
c
2
3
4
11 (54%)
[
21]
22]
c
c
c
8
9
(92%)
(90%)
12 (42%)
[
c
1
3 (50%)
a
b
Dimethyl (1-diazo-2-oxopropyl)phosphonate (1.2 eq.), K CO (2.0 eq.), MeOH, r.t., 12 h.
2
3
1
H-Imidazole-1-sulfonyl azide (1.0 eq.), CuSO .5H O 10 mol %, K CO (3.0 eq.), MeOH,
4 2 2 3
r.t., 6h and then alkyne (1.0 eq.), Na ascorbate (0.2 eq.), CuSO .5H O 10 mol %, MeOH, r.t.,
4
2
1
2h. Yields are indicated in isolated products.
The last step of the synthesis was carried out with compounds 10-13 and a set of boron
derivatives. In the first place, starting from 10 or 11, the cross coupling reactions were
achieved using Pd(PPh ) and K CO under microwave irradiation.[23] After 15 minutes at
3
4
2
3
1
50 °C, using commercially available boronic acids, the final derivatives 14-20 were obtained
in yields up to 81% (Table 2, entries 1-7).
6

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Table 2. Synthesis of the library of type II compounds by direct condensation of boronic
acids with derivatives 10-13.
a
a
Entry
SM
10
Compounds of type II (Yield)
Entry
SM
12
Compounds of type II (Yield)
1
4
24
(91%)
1
1
c
1
b
b
(
(
81%)
1
5
25
(73%)
2
3
10
10
12
13
12
c
92%)
2
6 b,d
1
6
b
12
12
(75%)
(
92%)
1
7
27
(72%)
4
5
6
7
8
11
11
11
11
12
b
b
14
15
16
17
18
b,d
c
(
(
(
80%)
1
8
2
8
1
2
81%)
(
77%)
1
9
29
(80%)
b
b
c
12
12
13
b,d
c
85%)
2
0
30
(85%)
(
86%)
2
1
31
(80%)
b,d
(
85%)
2
2
32
(79%)
9
12
12
b
19
20
13
13
c
(
83%)
2
3
33
(66%)
1
0
c
b,d
(
91%)
a
b
Yields are indicated in isolated products. Obtained with boronic derivative (1.2 eq.),
c
Pd(PPh ) 10 mol %, K CO (2.0 eq.), toluene / MeOH 2 / 1, µW, 150 °C, 15 min. Obtained
3
4
2
3
with boronic derivative (1.2 eq.), PdCl (dppf) 10 mol %, Na CO (2.0 eq.), toluene / MeOH 2
2
2
3
d
/
1, µW, 100 °C, 40 min. 20 minutes microwave irradiation reaction time.
Since some yields decreased dramatically and degradation appeared, the experimental
conditions were slightly modified. Several assays were carried out using only 20 minutes of
irradiation or by coupling PdCl (dppf) and Na CO and using microwave irradiation at 110 °C
2
2
3
to achieve satisfactory results. In the case of the bromo derivatives 12 or 13, the two catalyzed
conditions were suitable to achieve the cross coupling reactions and to furnish the derivatives
2
1-33 in good yields ranging from 66 to 92%. Next, we decided to extend the library of active
7

ACCEPTED MANUSCRIPT
molecules. The bromo derivatives 10-12 were reacted with (5-formylthiophen-2-yl)boronic
acid and crude material was directly involved in a formyl reduction to afford 34-36 in high
yields (Scheme 2). To improve diversity, and having in hand 26 and 27, we next decided to
substitute the alcohol by a fluorine. The chosen method consisted in the direct electrophilic
reaction which involved a slight excess of DAST and furnished the fluorinated derivatives 37
and 38 in moderate yields (Scheme 2).
Scheme 2. Reagents and conditions : i) formylated boronic acids (1.2 eq.), Pd(PPh ) 10 mol
3
4
%
, K CO (2.0 eq,), toluene / MeOH 2 / 1 , µW 150 °C, 15 minutes then NaBH (1.2 eq.),
2
3
4
CH Cl , 0 °C, 30 min. from 10 : 34 82%, from 11 : 35 85%, from 12 : 36 78%; ii) DAST (1.4
2
2
eq.), CH Cl , 0 °C, 1 h, from 26 : 37 39%, from 27 : 38 45%.
2
2
To pursue our SAR study, we performed the synthesis of the boronic ester library 45-50
which were easily prepared from bromoaryl derivatives 39-44 using PdCl dppf as catalyst and
2
KOAc as base after 16 h heating at 90 °C (Scheme 3).[24]
8

ACCEPTED MANUSCRIPT
Scheme 3. Reagents and conditions : i) Bis(pinacolato)diboron (1.0 eq.), PdCl dppf 10 mol
2
%
, KOAc (2.0 eq.), dioxane, 90 °C, 16 h, from 39 : 45 76%, from 40 : 46 65%, from 41 : 47
3%, from 42 : 48 74%, from 43 : 49 72%, from 44 : 50 75%, from 59 : 62 70%, from 60 : 63
5%, from 61 : 64 78%; ii) bromomethyl derivative, Et N (2.0 eq.), CH CN, r.t., 3 h,
6
6
3
3
secondary aliphatic amine (2.0 eq.), from 51 : 52 78%, 53 72%, 54 75%, 55 72%, 56 72%, 57
6
8%, from 58 : 59 75%, 60 80%, 61 78%.
In addition, several nucleophilic substitutions were performed on bromomethyl derivative 51
which led directly to the boron derivatives 52-57. However, this interesting strategy failed
using 1-bromo-3-(bromomethyl)benzene 58. To circumvent this problem, this starting
material was first used in a reaction with the nucleophiles in the presence of Et N in
3
acetonitrile at room temperature, to afford the resulting tertiary amine 59-61 which was next
subjected to the borylation step leading to 62-64 in yields ranging between 65 and 78% after
flash chromatography (Table 3). The use in palladium cross coupling reactions [25, 26] of the
newly prepared boron intermediates led as expected to 81-90 in satisfactory yields.[27]
9

ACCEPTED MANUSCRIPT
Table 3. Increase in the library size of type II .
a
a
Entry
Compounds of type II (Yield)
Entry
Compounds of type II (Yield)
76
SM
SM
1
0,
65
(75%)
12,
49
1
1
1
2
3
4
5
(63%)
1
1
1
0,
66
(65%)
12,
50
77
(63%)
2
3
5
2
0,
67
(62%)
12,
52
78
(75%)
14
15
5
3
0,
68
(78%)
12,
53
79
(83%)
4
5
4
1
1
1
0,
69
(68%)
12,
54
80
(75%)
5
6
7
16
17
18
6
2
0,
70
(71%)
12,
55
81
(68%)
6
3
0,
4
71
(73%)
12,
56
82
(61%)
6
1
4
2,
5
72
(75%)
12,
57
83
(67%)
8
9
19
1
4
2,
6
73
(75%)
12,
62
84
(60%)
20
21
1
4
2,
7
74
(79%)
12,
63
85
(63%)
1
1
0
1
1
4
2,
8
75
(62%)
12,
64
86
(83%)
22
a
SM: Starting Materials. Yields are indicated in isolated products. All derivatives were
obtained with bromo derivative (1.0 eq.), boronic derivative (1.2 eq.), Pd(PPh ) 10 mol%,
3
4
K CO (2.0 eq.), toluene / MeOH 2 / 1, µW, 150 °C, 15 min.
2
3
In vitro alpha 7 nicotinic acetylcholine receptor (α7R) binding assays
1
25
The binding competition assays against the radiolabeled α7R antagonist [ I]α-bungarotoxin
were performed for all final compounds on a rat brain membrane preparation. The large
family of derivatives led to clear SAR trends. Table 4 summarizes the inhibition constant (Ki)
obtained for the first set of type II derivatives. The nature of the (Het)Ar , (Het)Ar and
1
2
satellites Sat (see Scheme 1) was modified step by step and results were analyzed.
When (Het)Ar was a phenyl ring and the second aromatic fraction was attached in position
1
C-4, affinities were always up to 100 nM (17-20, 35). This loss of affinity reached a
1
0

ACCEPTED MANUSCRIPT
-6
maximum with a fluoropyridine core as 19 exhibited a Ki value up to 10 M. Conversely,
moving the link between the two (Het)Ar moieties to the meta position led to a significant
increase in affinity (nanomolar range), suggesting that a moderate curve in the aromatic
enchainment was necessary to fit with the α7R binding site (14-16, 34). Moreover, the
addition of 3- or 4- hydroxymethyl groups led to 14 and 15 with a preference for the meta
satellite position (Ki of 1.4 and 7 nM, respectively). Additionally, the use of a 5’-
hydroxymethyl-2’-thiophenyl system led to 34 which possessed a very high affinity (Ki of 0.2
nM) whereas a 2-fluoropyridine core in (Het)Ar significantly decreased this affinity (16, Ki
2
of 16 nM).
As a thiophenyl skeleton appeared as a very interesting partner to enhance the affinity, we
decided to link this heterocycle to the triazole, i.e. at the (Het)Ar position. The second
1
(
Het)Ar moiety was first attached in C-4 to furnish derivatives 31-33. The 2-fluoropyridine
2
compound 33 was still disappointing (Ki of 31 nM) whereas the affinity in the presence of 3-
or 4- hydroxymethyl phenyl groups was better (13 and 1.5 nM, for 31 and 32, respectively).
The C-4 position on the (Het)Ar seemed therefore to be favorable for binding, in agreement
2
with a probable adjustment of the ligand in the active site due to the (Het)Ar thiophen /
1
phenyl switch.
When the (Het)Ar) thiophenyl ring was linked by its C-5 position to a phenyl group, several
1
(Het)Ar2 were suitable to explore the corresponding SAR. With thiophene or
dihydrobenzofurane as well as benzodioxine, Ki remained in an interesting range (21-25). The
interactions with α7R increased strongly when hydroxy or amino methyl groups were used as
supplementary functions and subnanomolar affinities were obtained for the 26, 27, 28 and 36
derivatives (0.7, 0.6, 0.3 and 0.8 nM, respectively).
1
1

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Table 4. Structure affinity relationships on derivatives of type II (part 1).
a
Ki
Ki
Residues on
Type II
Residues on
Type II
Entry
Entry
(
nM)
(nM)
1
4
0.6±0.2
27
1
1.4±0.6
14
2
3
7±1
15
16
15
16
0
.3±0.2
28
29
16±4
13±2
4
5
100±21
175-100
17
18
17
18
90-80
13±3
30
31
6
>1000
19
19
1.5±0.6
32
7
8
9
110-160
43±4
20
21
22
23
24
25
26
20
21
22
23
24
25
31±4
0.2±0.2
200-120
0.8±0.2
42±2
33
34
35
36
37
38
10±1
1
1
1
1
0
1
2
3
60±5
140
20±2
5±2
0.7±0.3
a
Values are expressed as an average of 3 independent experiments ± SEM.
Removing the satellite hydrophilic function and using a fluorine atom, ester or fluoromethyl
groups decreased the binding efficiency overall (29, 30, 37, 38). A first analysis of the library
seemed to indicate that it was preferable to use a 3,4-biphenyl and a 2-thiophen-5-phenyl
1
2

ACCEPTED MANUSCRIPT
system to enhance activities. This motif induced a planar structure with a slight curve in the
plane. Some flexibility was provided with quinuclidine and (Het)Ar hydrophilic alcohols and
2
methylene amine groups which enhanced interaction more than the other satellite (Sat)
functions used.
In order to investigate the best Sat fractions, we focused on compounds possessing the best
1
,3-biphenyl and 5-phenyl-2-thiophenyl systems. Results are summarized in Table 5. First of
all, in the biphenyl series the incorporation of a fluorine atom led to a decrease in affinity as
can be seen by comparing 65 to its non-fluorinated analog 15. The same behavior was
observed with thiophenyl containing molecules 72 (Ki of 12 nM vs 1.5 nM for 32). The use of
piperidine, morpholine, and piperazine on the benzylic methylene group finely tuned the
activity, with the result that the para substitution remained the best in the 1,3 biphenyl series
(66-68 compared to 69-71 and 78-80 compared to 84-86).
Noteworthy, the introduction of a hydrophilic side chain in para afforded the novel
derivatives 78-80 with Ki values in the subnanomolar range. Progression in the design
prompted us finally to use functionalized piperidines. Hydroxylation led to a stabilization of
the activity with compound 81 at Ki = 0.3 nM.
In conclusion, the SAR studies, which helped us to define the general architecture of α7R
ligands, were very fruitful and our hypothetical pharmacophoric model was fully validated as
9
molecules exhibited Ki values below nanomolar concentrations. Binding assays, realized
with approximatively 40 tested derivatives, showed clearly the preference to define
thiophenyl as (Het)Ar and phenyl ring as (Het)Ar with the best thiophene bisubstitution in
1
2
C-2 and C-5.
1
3

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Table 5. Structure affinity relationships on type II derivatives (part 2).
a
Ki
Triazole substituents
on type II derivatives
Ki
Triazole substituents
on type II derivatives
Entry
Entry
(
nM)
(nM)
1
57±6
1
18±2
65
76
1
2
3
±0.7
66
67
13
14
340±88
0.3±0.1
77
78
2
3
12.5±2
4
5
6
7
1.9±0.2
122±8
49±11
39±4
68
69
70
71
15
16
17
18
0.9±0.2
0.3±0.1
0.3±0.1
2.8±0.7
79
80
81
82
8
9
12±1
72
73
19
20
21
1.4±0.2
17±2
83
84
85
1.5±0.3
1.7±0.2
7
4
1
1
0
1
16±1
140
75
22
16±2
86
a
Values are expressed as an average of 3 independent experiments ± SEM.
Additionally, substitutions could be supported by the (Het)Ar and by the phenyl ring in C-4
2
particularly. We proved that the choice of substitution modulates the efficacy of compound
fixation. The best results were obtained with a benzylic methylene equipped with a tertiary
cyclic amine with a preference for bearing an additional hydroxymethyl function in C-4.
Based on this robust study, some sub-nanomolar active drugs were chosen for in cellulo
1
4

ACCEPTED MANUSCRIPT
18
assays and some fluorinated molecules were transformed to their [ F]-radiolabeled
equivalents to evaluate their brain penetration and distribution in vivo.
In cellulo and selectivity assays
From the above SAR studies, it appeared that several compounds presented a sub-
nanomolar affinity for α7R but did not possess a fluorine atom in their structure, and were
1
8
thus inappropriate to be transformed into [ F]-radiolabeled PET tracers. This high affinity led
us to pursue the pharmacological characterization of two of these compounds (26, 78) with a
view to proposing them as pharmacological agents. Concurrently, we selected from our
library the fluorinated compounds 29 and 38 which had a suitable affinity to be evaluated as
18
potential [ F]-PET tracers.
In a first step, we evaluated the in vitro agonism potency of these compounds by measuring
the release of intracellular calcium from human SH-SY5Y cells as previously described.[28,
2
9] Our results showed that 26 had no effect on this release whereas compounds 29 and 38
induced a releasing effect similar to that obtained with the reference α7R agonist choline, this
effect being significantly reduced (by around 50%) in the presence of the reference α7R
antagonist α-bungarotoxin (Figure 3, panel A). We also observed that the compound 78
induced an 80% higher calcium release than choline, this effect being reduced by 57% in the
presence of α-bungarotoxin.
In a second step, we evaluated the in vitro cell toxicity of these three α7R agonists (29, 38
and 78). The results presented in Figure 3 (panel B) showed an absence of cellular toxicity for
all compounds. However, it must be emphasized that compound 29 decreased cell viability by
about 15% at the highest concentration (13 µM) and an acidification of the culture medium
was observed.
1
5

ACCEPTED MANUSCRIPT
It appeared therefore that our compound 78 possessed a high affinity for α7R (Ki of 0.3
nM), had high agonist properties and showed no cellular toxicity at the highest concentration
-6
tested (3 µM). In addition, this compound had a Ki above 10 M towards the nicotinic α4β2
-7
receptors and 5.6 10 M towards the serotonin 5HT3 receptors, using competition assays on
3
3
cells expressing human receptors with [ H]cytisine and [ H]BRL 43694 as reference tracers,
respectively. Regarding these promising properties, further investigations should be
performed in order to test the potency of 78 to improve cognitive functions as already
demonstrated for other closely related structure compounds.[13, 14]
A
B
8
6
4
2
0
0
0
0
0
**
*
*
1
1
50
00
*
*
5
0
0
t
t
t
t
t
g
g
g
g
g
B
B
B
B
B
α
α
α
α
α
+
e
+
+
+
+
6
9
8
8
n
2
2
3
7
i
l
o
h
C
Figure 3. Panel A: Intracellular calcium release in human SH-SY5Y cells expressing native
α7R. Cells were loaded with fluo-3 AM and preincubated with 100 nM α-bungarotoxin (α-
Bgt) for 20 min and PNU-120596 for 1 min before stimulation with 3 mM choline or
molecules 26 (7 nM), 29 (130 nM), 38 (50 nM) and 78 (3 nM), as described in the Materials
and Methods. In order to normalize fluo-3 AM signals (excitation λEx = 506 nm, emission
λEm = 526 nm), the maximum and minimum fluorescence from each well was determined by
addition of 0.3% Triton-X100 (Fmax) followed by 100 mM MnCl (Fmin). Drug-evoked
2
responses were expressed as a percentage of the corresponding (Fmax-Fmin) value.
Fluorescence was recorded in a Varioskan Flash spectral scanning multimode reader. Results
are the mean ± SEM of 5 independent experiments in duplicate. Panel B: Cell viability. SH-
SY5Y cells were incubated during 48 hours with increasing concentrations (10 Ki, 100 Ki and
1
6

ACCEPTED MANUSCRIPT
1
000 Ki) of molecules 29, 38 and 78. The MTS reagent was added and the reading of the
optical density of the formazan was carried out after 3 hours of incubation using the
Varioskan Flash spectral scanning multimode reader (490 nm). The results are expressed as
percent viability compared to control cells incubated with the vehicle and represented the
mean ± SEM of 4 independent experiments in triplicate.
Radiolabeling and stability studies
In order to obtain potential PET tracers labeled with fluor-18, chemistry efforts were
developed to prepare the precursors allowing a nucleophilic substitution (Scheme 4). The 2-
nitropyridine derivative 87 was obtained from 12 using the Suzuki-Miyaura conditions
depicted in Table 2 with some changes in temperature (110 °C) and reaction time (1h30) to
achieve complete conversion. After purification the final product was isolated in a 77% yield.
For nucleophilic aliphatic substitution, the chlorine was choosen as leaving group. Thus, the
corresponding precursors were prepared by direct chlorination of 27 and 81 using SOCl in
2
dichloromethane with a catalytic amount of DMF. While derivative 89 was obtained in
satisfactory yield, reaction with the benzylic alcohol 27 failed. To circumvent this problem,
quinuclidine was first protected on the nitrogen atom using BH .Me S complex and then
3
2
SOCl was used to obtain the borylated derivative 88 in 80% yield.
2
NO2
N
N
N
N
S
N
Br
i)
N
N
S
O
O2N
B
N
87
N
N
12
N
O
Cl
N
OH
N
S
ii)
N
N
N
N
S
then iii)
N
8
8
27
H3B
N
ii)
N
N
N
S
N
Cl
N
N
S
OH
N
8
9
N
HCl
N
81
Scheme 4. Reagents and conditions : i) 2-nitro-5-pyridineboronic acid pinacol ester (1.2 eq.),
Pd(PPh ) 10 mol %, K CO (2.0 eq.), toluene / MeOH 2 / 1, µW, 110 °C, 1h30, 77%, ii)
3
4
2
3
1
7

ACCEPTED MANUSCRIPT
from 81 : SOCl (2.0 eq.), DMF cat., CH Cl , r.t., 12 h, 89 80% and from 27: SOCl (2.0 eq.),
2
2
2
2
DMF cat., CH Cl , r.t., 12 h then BH Me S (1.0 eq.), CH Cl / THF (9/1), -10 °C, 1h, 88
2
2
3
2
2
2
8
0%.
1
8
The synthesis of the radiotracer [ F]29 was achieved through a two-step radiolabeling
procedure (Scheme 5). First nucleophilic substitution occurred on the pyridine ring and
followed by a reduction of the nitro group of the excess of precursor’s excess was performed
1
8
to allow a fast and efficient purification of [ F]29. After purification via semi preparative
1
8
high performance liquid chromatography (HPLC), [ F]29 was obtained in about 7.4%
radiochemical yield (n = 5, decay-corrected) with a radiochemical purity (RCP) greater than
18
9
5% and a molar activity (MA) of 41−73 GBq/µmol. [ F]38 was obtained in a one-pot
1
8 -
multistep reaction from the chlorinated analog 88. Chlorine was substituted by F followed
by a removal of the BH protective group on the quinuclidine under acidic conditions. As for
3
1
8
18
[
F]29, [ F]38 was purified by HPLC and was obtained in 24% radiochemical yield (n = 4,
decay-corrected). RCP was greater than 95% and MA was 39-86 GBq/µmol. The total
1
8
18
18
synthesis time was about 100 min for [ F]29 and 90 min for [ F]38. [ F]29 was formulated
classically in a sodium chloride 0.9%/ethanol solution suitable for in vivo injection to animals.
1
8
18
In this medium, a fast degradation of [ F]38 was observed but [ F]38 remained stable in
1
8
pure ethanol up to 4h. Thus, [ F]38 was extemporaneously prepared in an injectable form
prior to animal experiments. The stability of both compounds in rat plasma was checked up to
4
hours and the purity was greater than 95% at the latest time point.
1
8

ACCEPTED MANUSCRIPT
1
8
Scheme 5. Nucleophilic radiosynthesis of potent compounds with F starting from their
1
8
corresponding precursors. Reagents and conditions: i) [ F]KF / K2.2.2, DMSO, 150 °C, 10
18
min ; ii) SnCl /HCl, 15min ; iii) aq. NaOH 0.6N; iv) [ F]KF / K2.2.2, DMSO, 115 °C, 15
2
18
min ; v) HCl/acetone, 10 min ; vi) [ F]KF / K2.2.2, DMSO, µW 100 W, 5 min.
1
8
Surprisingly, we were not able to prepare [ F]82 by thermal heating but only under
microwave activation. Even with these harsh conditions, 100 W for 5 min, the amount of
1
8
[
F]82 was not sufficient to prepare it for in vivo evaluation.
In vivo brain biodistribution and imaging studies
After checking that they had low in vitro toxicity (Figure 3B), good stability and
1
8
18
radiochemical purity (>95%), we evaluated the in vivo properties of [ F]29 and [ F]38 in
rodents (Figures 4 and 5).
In a first step of experiments, we measured the uptake of each tracer in several rat brain
regions. These regions were chosen according to their known α7R density in the rodent brain,
i.e. high in the frontal cortex and hippocampus, intermediate in the hypothalamus and
striatum, and low in the cerebellum (Davies ARL et al. Neuropharmacology 1999, 38:679-
6
90; Whiteaker P et al. Eur J Neurosci 1999, 11:2689-2696). Each tracer was intravenously
i.v.) injected in a group of control rats (n=6) and in another group of rats (n=6) that received
5 min before the tracer injection, an i.v. injection of the high affinity α7R antagonist
(
1
1
9

ACCEPTED MANUSCRIPT
methyllycaconitine (MLA; Wonnacott S et al. Methods Neurosci 1993, 12:263-275) at the
dose of 1 mg/kg. It was previously showed that this experimental design allows blocking the
in vivo accumulation of α7R radiotracer in the rat brain (Maier DL et al. Neuropharmacology
2
011, 61:161-171). The radioactivity uptake was measured in each dissected brain region at 1
hour post-injection. Although we did not prove that this radioactivity was exclusively related
to the parent tracers, this can be expected as we previously checked that they were stable up to
4
hours in rat plasma.
1
0
0
0
0
0
0
0
0
0
0
.00%
.90%
.80%
.70%
.60%
.50%
.40%
.30%
.20%
.10%
.00%
[
[
[
[
18F]29 Control
18F]29 MLA
18F]38 Control
18F]38 MLA
FCx
HPC
HPT
STRI
CB
Figure 4. Brain biodistribution study in rats. Rats (n = 6 group) received an i.v. injection of
1
8
18
either [ F]29 or [ F]38 preceded (MLA) or not (Control) by an i.v. injection of
methyllycaconitine (1.0 mg/kg). They were sacrificed at 1 h post-injection of the tracer and
the radioactivity was measured in different dissected brain regions. The results are expressed
as mean percentage of injected dose per gram of tissue (%ID/g) ± SEM. FCx, frontal cortex;
HPC, hippocampus; HPT, hypothalamus; STRI, striatum; CB, cerebellum.
2
0

ACCEPTED MANUSCRIPT
1
8
The brain biodistribution studies (Figure 4) showed that in control rats, both [ F]29 and
1
8
[
F]38 were able to pass through the blood-brain barrier, with a range value of 0.4 to
0
.8%ID/g tissue, depending on the brain region. Indeed, it is known that suitable radiotracers
for central nervous system exploration show typically values from 0.5% ID/g tissue in the
rodent brain (Wong DF & Pomper MG Mol Imaging Biol 2003, 5:350-362.) However, a
1
8
18
significantly higher level of accumulation was observed for [ F]29 than for [ F]38 (p<0.05)
in all the brain regions explored (Cx: 0.85±0.06 vs 0.56±0.02 %ID/g; HPC: 0.78±0.05 vs
0
.43±0.02 %ID/g; HPT: 0.59±0.05 vs 0.42±0.02 %ID/g; STRI: 0.76±0.06 vs 0.44±0.03
ID/g; CB: 0.58±0.05 vs 0.44±0.02 %ID/g). The ratio of accumulation in the structures vs
cerebellum, considered to reflect non-specific binding, was slightly above 1 in the Cx
%
1
8
(
1.47±0.03), HPC (1.34±0.04) and STRI (1.31±0.02) for [ F]29 and only in the Cx
1
8
(
1.29±0.04) for [ F]38. However, pre-injection of the antagonist α7R MLA did not
significantly modify the brain accumulation of either tracer, whatever the region considered.
It can be pointed out that in contrast to this last result, the uptake of other described α7R
tracers have been obtained in rodent brain with MLA, the selective α7R ligand SSR180711,
or nicotine (Maier et al. 2011; Gao et al. 2013).
1
8
The imaging study (Figure 5) illustrated the overall higher brain uptake for [ F]29 than for
18
[
F]38, with a quite homogeneous distribution of both tracers between the different regions.
2
1

ACCEPTED MANUSCRIPT
18
18
Figure 5. Representative sagittal PET brain image of [ F]29 and [ F]38. The rats
1
8
18
received a bolus i.v. injection of 37 MBq of [ F]29 or [ F]38. The acquisition lasted 71 min
and the images were reconstructed using a 2-D OSEM algorithm. Images represent the sum of
the last frames corresponding to 49-71 min of acquisition after tracer injection. Brain regions
are represented on the template (PMOD Technologies, Zurich, Switzerland;
www.pmod.com). Cx, cortex; STRI, striatum; THAL, thalamus; HPC, hippocampus; Coll,
colliculus; CERV, cerebellum.
Our in vivo results thus indicated that although they passed through the BBB after i.v.
1
8
18
injection, neither [ F]29 nor [ F]38 specifically accumulated in brain regions rich in α7R
and were not significantly displaced by the α7R antagonist MLA. This disappointing result
did not appear to be due to a lack of specificity of our compounds, as we measured a Ki above
-6
-7
1
0 M towards the nicotinic α4β2 receptors for both 29 and 38, and a Ki of 4.4 10 M and
-6
above 10 M towards the serotonin 5HT3 receptors for 29 and 38, respectively. Although
18
many attempts have been performed to develop suitable [ F]-labeled PET tracers for brain
1
8
α7R imaging, to date only [ F]ASEM (Figure 1) has proved to be really useful.[30-33] This
is probably related to the very high affinity of this compound (Ki of 0.4 nM)[34] compared to
ours (13 and 5 nM) that allows ASEM to bind in vivo to α7R despite their low brain density
in rodent as well as in human brain.[35-37]
Conclusion
We have prepared a library of bis(Het)Aryl-1,2,3-triazole quinuclidine α7R ligands using
an efficient strategy involving a Suzuki-Miyaura cross coupling reaction. The exploration of
SAR required the preparation of uncommon boron derivatives. Forty final drugs were tested
for their ability to bind the target and nine of them exhibited Ki below nanomolar
concentrations. The best scores were always obtained when the 5-phenyl-2-thiophenyl core
was attached to the triazole. Furthermore, the introduction of phenyl substituents in para led to
2
2

ACCEPTED MANUSCRIPT
a set of very efficient derivatives, in particular compound 78 which has a very high affinity
for α7R (Ki of 0.3 nM), is selective towards the nicotinic α4β2 and serotoninergic 5HT3
receptors, and is a potent agonist of the target. It can be expected that this compound will have
a strong effect on cognitive disorders, as such effects have already been obtained with
agonists possessing much poorer affinity (around 100 nM) for α7R.[13]
1
8
Regarding the interest of our compounds as [ F]-labeled PET tracers, they have not the
18
requisite properties compared to [ F]ASEM, probably due to their lower affinity. This point
is indeed a key parameter for a good in vivo tracer of α7R, due to their low density in the
brain.[1, 15]
Experimental part
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
-1
were obtained on a Perkin Elmer PARAGON 1000 PC and values are reported in cm .
HRMS were recorded on a Bruker maXis mass spectrometer. Monitoring of the reactions was
performed using silica gel TLC plates (silica Merck 60 F ). Spots were visualized by UV
2
54
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.
2
3

ACCEPTED MANUSCRIPT
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 lid.
The chromatographic purity of the final compounds was determined using an Agilent
Technology 1260 Infinity HPLC system with a C-18 column (Agilent ZORBAX Eclipse plus
C18 3.5 µm, 4.6 mm × 100 mm) operating at 30 °C. Elution was carried out using acetonitrile
containing 0.1% formic acid as mobile phase A and water containing 0.1% formic acid as
mobile phase B. Elution conditions: at 0 min, phase A 10% + phase B 90%; at 5 min, phase A
5
0% + phase B 50%; at 7 min phase A 50% + phase B 50%, at 7.1 min, phase A 10% + phase
B 90%; at 20 min, phase A 10% + phase B 90%. The flow-rate of the mobile phase was 0.9
mL/min, and the injection volume of the sample was 2 µL. Peaks were detected at 254 nm and
3
00 nm. Purity of all the tested compounds was found to be >95% unless otherwise stated.
General procedure A to obtain ethynyl derivatives: To a solution of dimethyl 1-diazo-2-
oxopropylphosphonate (1.15 g, 6.00 mmol) in MeOH (60 mL) were added the corresponding
aldehydes (5.00 mmol) and K CO (1.38 g, 10.0 mmol). The reaction mixture was stirred at
2
3
room temperature for 12 h. After removing the volatiles without any heating, alkynes 6-9
were purified by flash chromatography using a mixture of petroleum ether and EtOAc 99/1 as
eluent
.
General procedure B for the synthesis of arylated quinuclinine triazoles: Under argon,
to a solution containing 3-aminoquinuclidine bishydrochloride salt 4 (212 mg, 1.00 mmol)
and 1H-imidazole-1-sulfonyle azide 5 (232 mg, 1.10 mmol) in MeOH (6 mL) was portion
wise added K CO (415 mg, 3.00 mmol) and next a catalytic amount of CuSO , 5H O (25 mg,
2
3
4
2
0
.10 mmol). The reaction mixture was stirred at room temperature for 6 h and then
concentrated under reduced pressure. The crude solid was solubilized in Et O (10 mL),
2
filtered and the precipitate washed with an additional amount of Et O (10 mL). The combined
2
2
4

ACCEPTED MANUSCRIPT
organic layers were reduced under reduced pressure and the intermediate used in the next
step. After addition of MeOH (6 mL), the desired terminal alkynes 6-9 (1.0 mmol) and next
CuSO , 5H O (25 mg, 0.10 mmol), sodium ascorbate (40 mg, 0.20 mmol) were successively
4
2
added. The reaction mixture was stirred for 12 h at room temperature. Volatiles were
evaporated under reduced pressure and the residue purified by flash chromatography. When
some traces of residual imidazole moiety were observed, EtOAc (20 mL) was added. After
extraction with water (2 x 10 mL), the organic layer was dried over MgSO , filtered and
4
evaporated under reduced pressure to afford the pure derivative of type IV (10-13).
(R)-3-[4-(3-Bromophenyl)-1H-1,2,3-triazol-1-yl]quinuclidine 10. Compound 10 was
obtained from 1-bromo-3-ethynylbenzene following the general procedure B and isolated as a
white solid in a 50% yield. R : 0.18 (CH Cl /MeOH : 97/3 + NH OH 10%) ; Mp : 188-190
f
2
2
4
-
1
°
C ; IR (ATR, Diamond)
ν
(cm ) : 970, 1020, 1041, 1060, 1069, 1233, 1341, 14237 1455,
470, 1603, 2868, 2938 ; H NMR (400 MHz, CDCl ) : (ppm) 1.44-1.50 (m, 1H), 1.61-1.65
m, 1H), 1.73-1.87 (m, 2H), 2.27 (q, J = 3.2 Hz, 1H), 2.86-3.00 (m, 3H), 3.09-3.17 (m, 1H),
1
1
δ
3
(
3
4
7
2
.49 (ddd, J = 2.2 Hz, J = 9.8 Hz, J = 14.4 Hz, 1H), 3.68 (dd, J = 14.4 Hz, J = 3.6 Hz, 1H),
.63-4.65 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H),
1
3
.83 (s, 1H), 7.98 (s, 1H) ; C NMR (100 MHz, CDCl ) :
δ (ppm) 20.0 (CH ), 26.0 (CH ),
2 2
3
8.1 (CH), 46.9 (CH ), 47.2 (CH ), 52.6 (CH ), 58.5 (CH), 119.3 (CH), 122.9 (C ), 124.1
2
2
2
q
(
CH), 128.6 (CH), 130.4 (CH), 131.0 (CH), 132.6 (C ), 146.1 (C ) ; HRMS (EI-MS): m/z
q q
+
calcd. for C H N Br [M+H] : 333.0715, found: 333.0709.
1
5
18
4
(R)-3-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)quinuclidine 11. Compound 11 was
obtained from 1-bromo-4-ethynylbenzene following the general procedure B and isolated as a
white solid in a 54% yield. R : 0.32 (CH Cl /MeOH 80/20 + NH OH 10%) ; Mp : 158-160
f
2
2
4
-1
°
C ; IR (ATR, Diamond)
ν
(cm ) : 972, 1009, 1043, 1060, 1070, 1186, 1218, 1315, 1424,
1
1
451, 1479, 2867, 2936 ; H NMR (250 MHz, CDCl ) : (ppm) 1.40-1.57 (m, 1H), 1.60-1.72
δ
3
2
5

ACCEPTED MANUSCRIPT
(m, 1H), 1.72-1.86 (m, 2H), 2.28 (q, J = 3.1 Hz, 1H), 2.86-3.00 (m, 3H), 3.07-3.22 (m, 1H),
3
4
.49 (ddd, J = 2.2 Hz, J = 9.8 Hz, J = 14.5 Hz, 1H), 3.69 (dd, J = 5.0 Hz, J = 14.4 Hz, 1H),
1
3
.58-4.71 (m, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H), 7.81 (s, 1H) ; C NMR
(ppm) 20.2 (CH ), 26.2 (CH ), 28.4 (CH), 47.1 (CH ), 47.5 (CH ), 52.9
(
(
(
100 MHz, CDCl ) :
δ
3
2
2
2
2
CH ), 58.7 (CH), 119.3 (CH), 122.2 (C ), 127.4 (2 CH), 129.8 (C ), 132.2 (2 CH), 146.8
2
q
q
+
C ) ; HRMS (EI-MS): m/z calcd. for C H N Br [M+H] : 333.0715, found: 333.0722.
q
15 18
4
(R)-3-(4-(5-Bromothiophen-2-yl)-1H-1,2,3-triazol-1-yl)quinuclidine 12. Compound 12
was obtained from 2-bromo-5-ethynylthiophene following the general procedure B and
isolated as a white solid in a 42% yield. R : 0.46 (CH Cl /MeOH 80/20 + NH OH 1 %) ;
f
2
2
4
-1
Mp : 146-148 °C ; IR (ATR, Diamond) ν (cm ) : 972, 1041, 1057, 1215, 1322, 1433, 1496,
1
1
645, 2867, 2934, 3356 ; H NMR (400 MHz, CDCl ) : δ (ppm) 1.44-1.51 (m, 1H), 1.59- 1.69
3
(m, 1H), 1.71 -1.87 (m, 2H), 2.25 (q, J = 3.0 Hz, 1H), 2.83-2.99 (m, 3H), 3.07-3.16 (m, 1H),
3
1
1
4
.47 (ddd, J = 2.2 Hz, J = 9.8 Hz, J = 14.4 Hz, 1H), 3.66 (ddd, J = 1.6 Hz, J = 5.1 Hz, J =
4.4 Hz, 1H), 4.57-4.63 (m, 1H), 7.00 (d, J = 3.8 Hz, 1H), 7.08 (d, J = 3.8 Hz, 1H), 7.69 (s,
13
H) ; C NMR (100 MHz, CDCl ) : δ (ppm) 20.1 (CH ), 26.1 (CH ), 28.3 (CH), 47.0 (CH ),
3
2
2
2
7.4 (CH ), 52.7 (CH ), 58.7 (CH), 112.1 (Cq), 118.7 (CH), 124.3 (CH), 130.6 (CH), 134.8
2
2
+
(
Cq), 142.1 (Cq) ; HRMS (EI-MS): m/z calcd. for C H N SBr [M+H] : 339.0279, found:
13 16 4
3
39.0283.
R)-3-(4-(4-Bromothiophen-2-yl)-1H-1,2,3-triazol-1yl)quinuclidine (13). Compound 13
(
was obtained from 4-bromo-2-ethynylthiophene following the general procedure B and
isolated as a white solid in a 50% yield. R : 0.24 (CH Cl /MeOH 98/2 + NH OH 1 %) ; Mp:
f
2
2
4
-
1
1
2
1
06-108 °C ; IR (ATR, Diamond)
ν
(cm ) : 786, 988, 1054, 1226, 1326, 1452, 1497, 2870,
1
938, 3107; H NMR (400 MHz, CDCl ) :
δ
(ppm) 1.45-1.52 (m, 1H), 1.61-1.69 (m, 1H),
3
.76-1.85 (m, 2H), 2.26 (q, J = 3.2 Hz, 1H), 2.85-3.00 (m, 3H), 3.09-3.16 (m, 1H), 3.49 (ddd,
J = 2.1 Hz, J = 10.0 Hz, J = 14.4 Hz, 1H), 3.66 (dd, J = 5.2 Hz, J = 14.4 Hz, 1H), 4.60-4.65
2
6

ACCEPTED MANUSCRIPT
1
3
(
m, 1H), 7.19 (s, 1H), 7.26 (s, 1H), 7.74 (s, 1H) ; C NMR (100 MHz, CDCl ) :
δ
(ppm) 19.9
3
(
CH ), 25.9 (CH ), 28.1 (CH), 46.9 (CH ), 47.2 (CH ), 52.6 (CH ), 58.6 (CH), 110.2 (C ),
2
2
2
2
2
q
1
18.7 (CH), 122.1 (CH), 126.4 (CH), 134.3 (C ), 141.5 (C ) ; HRMS (EI-MS): m/z calcd. for
q q
+
C H N SBr [M+H] : 339.0276, found: 339.0273.
13
16
4
General procedure C for the synthesis of derivatives of type II. : To a mixture of
derivative of type IV (50 mg, 0.147 mmol) in toluene (1.5 mL) and EtOH (0.75 mL) were
successively added the desired boronic ester or acid of type V (0.176 mmol, 1.2 eq.), K CO3
2
(
0.294 mmol, 2.0 eq.) and Pd(PPh ) (0.0147 mmol, 10 mol %). The reaction mixture was
3 4
degassed with Ar and irradiated under microwave for 20 minutes at 150 °C. Alternatively
PdCl (dppf) 10 mol %, could be used as catalyst. In this case the base was switched to
2
Na CO (2.0 eq.) and the irradiation performed for 40 minutes at 100 °C. In both cases, after
2
3
cooling, the volatiles were removed under reduced pressure and the crude material purified by
flash chromatography (CH Cl /MeOH 80/20 + NH OH 0.1 mL).
2
2
4
(R)-(3'-(1-(Quinuclidin-3-yl)-1H-1,2,3-triazol-4-yl)-[1,1'-biphenyl]-3-yl)methanol
14.
Compound 14 was obtained from compound 10 and (3-(hydroxymethyl)phenyl)boronic acid
following the general procedure C and isolated as a white solid in a 81% yield. R : 0.18
f
-1
(
CH Cl /MeOH 97/3 + NH OH 1 mL) ; Mp: 201-203 °C ; IR (ATR, Diamond) ν (cm ) : 793,
2 2 4
1
9
84, 1042, 1262, 1326, 1423, 1436, 1612, 1697, 1719, 2870, 2943, 3133 ; H NMR (400
(ppm) 1.34-1.48 (m, 2H), 1.69-1.75 (m, 2H), 2.20 (q, J = 2.8 Hz, 1H),
.75-2.79 (m, 3H), 2.94-3.01 (m, 1H), 3.35-3.48 (m, 2H), 4.58 (d, J = 5.6 Hz, 2H), 4.72-4.79
m, 1H), 5.25 (d, J = 5.6 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.51-7.63
MHz, DMSO-d6):
δ
2
(
(
1
3
m, 3H), 7.66 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H), 8.85 (s, 1H) ; C NMR (100
(ppm) 20.0 (CH ), 25.7 (CH ), 28.1 (CH), 46.8 (CH ), 47.0 (CH ), 52.2
MHz, DMSO-d ) :
δ
6
2
2
2
2
(
CH ), 57.8 (CH), 63.3 (CH ), 121.5 (CH), 123.8 (CH), 124.5 (CH), 125.2 (CH), 125.5 (CH),
2 2
2
7

ACCEPTED MANUSCRIPT
1
26.2 (CH), 126.5 (CH), 129.1 (CH), 129.9 (CH), 131.9 (C ), 140.1 (C ), 141.3 (C ), 143.7
q
q
q
+
(
C ), 146.5 (C ) ; HRMS (EI-MS): m/z calcd. for C H N O [M+H] : 361.2023, found:
q
q
22 25
4
3
61.2024.
(R)-(3'-(1-(Quinuclidin-3-yl)-1H-1,2,3-triazol-4-yl)-[1,1'-biphenyl]-4-yl)methanol
15.
Compound 15 was obtained from compound 10 and (4-(hydroxymethyl)phenyl)boronic acid
following the general procedure C and isolated as a white solid in a 92% yield. R : 0.18
f
-
1
(
CH Cl /MeOH9 7/3 + NH OH 10%) ; Mp : 214-216 °C ; IR (ATR, Diamond):
ν
(cm ): 793,
72, 1041, 1222, 1320, 1451, 1481, 1660, 2868, 2939, 3077 ; H NMR (400 MHz, DMSO-
(ppm) 1.3-1.49 (m, 2H), 1.70-1.74 (m, 2H), 2.20 (q, J = 2.8 Hz, 1H), 2.74-2.78 (m, 3H),
.94-3.01 (m, 1H), 3.38-3.46 (m, 2H), 4.55 (s, 2H), 4.74-4.78 (m, 1H), 5.23 (br s, 1H), 7.42
d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 7.6 Hz, 2H),
2
2
4
1
9
d6):
δ
2
(
13
7
.87 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H), 8.85 (s, 1H) ; C NMR (100 MHz, DMSO-d ):
δ
(ppm)
6
2
0.0 (CH ), 25.7 (CH ), 28.1 (CH), 46.8 (CH ), 47.0 (CH ), 52.3 (CH ), 57.8 (CH), 63.0
2
2
2
2
2
(
CH ), 121.5 (CH), 123.7 (CH), 124.3 (CH), 126.3 (CH), 126.8 (2 CH), 127.4 (2 CH), 129.9
2
(
CH), 131.9 (C ), 138.6 (C ), 141.0 (C ), 142.5 (C ), 146.5 (C ) ; HRMS (EI -MS): m/z calcd.
q
q
q
q
q
+
for C H N O [M+H] : 361.2023, found: 361.2023.
2
2
25
4
(R)-3-(4-(3-(6-Fluoropyridin-3-yl)phenyl)-1H-1,2,3-triazol-1-yl)quinuclidine
16.
Compound 16 was obtained from compound 10 and (6-fluoropyridin-3-yl)boronic acid
following the general procedure C and isolated as a white solid in a 82% yield. R : 0.23
f
-1
(
CH Cl /MeOH 97/3 + NH OH 10%) ; Mp : 164-166°C ; IR (ATR, Diamond)
ν
(cm ) : 789,
2
2
4
1
9
80, 1061, 1208, 1344, 1454, 1473, 1590, 2865, 2940, 3059 ; H NMR (400 MHz, CDCl ) :
δ
3
(ppm) 1.42-1.55 (m, 1H), 1.65-1.70 (m, 1H), 1.75-1.87 (m, 2H), 2.30 (q, J = 3.2 Hz, 1H),
2
.88-2.97 (m, 3H), 3.12-3.19 (m, 1H), 3.51 (ddd, J = 2.2 Hz, J = 9.8 Hz, J = 14.4 Hz, 1H),
.72 (dd, J = 4.0 Hz, J = 14.4 Hz, 1H), 4.66-4.68 (m, 1H), 7.01 (dd, J = 2.8 Hz, J = 8.4 Hz,
3
2
8

ACCEPTED MANUSCRIPT
1
2
H), 7.48-7.54 (m, 2H), 7.82 (d, J = 7.2 Hz, 1H), 7.89 (s, 1H), 8.01-8.06 (m, 2H), 8.46 (d, J =
13
.0 Hz, 1H) ; C NMR (100 MHz, CDCl ):
δ (ppm) 20.0 (CH ), 25.9 (CH ), 28.1 (CH), 46.9
2 2
3
(
CH ), 47.2 (CH ), 52.6 (CH ), 58.5 (CH), 109.4 (d, J = 37.0 Hz, CH), 119.2 (CH), 124.3
2 2 2
(
CH), 125.3 (CH), 126.7 (CH), 129.6 (CH), 131.6 (C ), 134.5 (d, J = 4.0 Hz, C ), 137.4 (C ),
q q q
1
39.8 (d, J = 8.0 Hz, CH), 145.8 (d, J = 15.0 Hz, CH), 147.0 (C ), 163.2 (d, J = 238.0 Hz,
q
+
C ) ; HRMS (EI-MS): m/z calcd. for C H N F [M+H] : 350.1775, found: 350.1777.
q
20 21
5
(R)-(4'-(1-(Quinuclidin-3-yl)-1H-1,2,3-triazol-4-yl)-[1,1'-biphenyl]-3-yl)methanol
17.
Compound 17 was obtained from compound 11 and (3-(hydroxymethyl)phenyl)boronic acid
following the general procedure C and isolated as a white solid in a 80% yield. R : 0.18
f
-
1
(
CH Cl /MeOH 97/3 + NH OH 10%) ; Mp: 207-209 °C ; IR (ATR, Diamond)
ν
(cm ) : 792,
82, 1038, 1225, 1324, 1437, 1455, 1603, 2874, 2941, 3367 ; H NMR (400 MHz, DMSO-
(ppm) 1.32-1.52 (m, 2H), 1.67-1.74 (m, 2H), 2.20 (q, J = 2.8 Hz, 1H), 2.73-2.80 (m,
2
2
4
1
9
d6) :
δ
3
7
H), 2.96-3.05 (m, 1H), 3.36-3.50 (m, 2H), 4.58 (s, 2H), 4.76-4.78 (m, 1H), 5.25 (br s, 1H),
.32 (d, J = 7.2 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.67 (s, 1H), 7.75
13
(
d, J = 8.2 Hz, 2H), 7.97 (d, J = 8.2 Hz, 2H), 8.79 (s, 1H) ; C NMR (100 MHz, DMSO-d ) :
6
δ
6
1
(ppm) 20.0 (CH ), 25.7 (CH ), 28.1 (CH), 46.8 (CH ), 47.0 (CH ), 52.2 (CH ), 57.8 (CH),
2 2 2 2 2
3.3 (CH ), 121.4 (CH), 124.9 (CH), 125.2 (CH), 126.1 (3 CH), 127.4 (2 CH), 129.1 (CH),
2
30.4 (C ), 139.8 (C ), 139.9 (C ), 143.7 (C ), 146.2 (C ) ; HRMS (EI-MS): m/z calcd. for
q
q
q
q
q
+
C H N O [M+H] : 361.2023, found: 361.2026.
22
25
4
(R)-(4'-(1-(Quinuclidin-3-yl)-1H-1,2,3-triazol-4-yl)-[1,1'-biphenyl]-4-yl)methanol
18.
Compound 18 was obtained from compound 11 and (4-(hydroxymethyl)phenyl)boronic acid
following the general procedure C and isolated as a white solid in a 81% yield. R : 0.18
f
-1
(
CH Cl /MeOH 97/3 + NH OH 10%) ; Mp: 250-252 °C ; IR (ATR, Diamond)
ν
(cm ) : 798,
2
2
4
1
9
73, 1042, 1223, 1344, 1429, 1451, 1661, 2869, 2938, 3119 ; H NMR (400 MHz, DMSO-
2
9