A Small Library of 1,2,3-Triazole Analogs of CAP-55: Synthesis and Binding Affinity at Nicotinic Acetylcholine Receptors

Article abstract of DOI:10.1002/cbdv.201800210

Alpha7 nicotinic acetylcholine receptor is emerging as a central regulator in inflammatory processes, as documented by increasing studies reported in the literature. For instance, the activation of this nicotinic receptor subtype in resident macrophages inhibits the production of pro-inflammatory cytokines, thereby attenuating local inflammatory responses, and may open a new window in the treatment of chronic inflammatory disease, such as Crohn's disease, rheumatoid arthritis, psoriasis, and asthma. In continuation of our ongoing research for the development of new cholinergic drug candidates, we selected the nicotine derivative CAP55, which was previously shown to exert anti-inflammatory effects via nicotinic stimulation, as a suitable compound for lead optimization. Through the isosteric replacement of its 3,5-disubstituted 4,5-dihydroisoxazole core with a 1,4-disubstituted 1,2,3-triazole ring, we could rapidly generate a small library of CAP55-related analogs via a one-pot copper(I)-catalyzed azide-alkyne cycloaddition. Receptor binding assays at nAChRs led to the identification of two promising derivatives, compounds 4 and 10, worthy of further pharmacological studies.

Full text of DOI:10.1002/cbdv.201800210

Luca Rizzia, Cecilia Gottib, Marco De Amicia, Clelia Dallanocea, and Carlo Matera*,a,c
a Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica "Pietro Pratesi",
Università degli Studi di Milano, Via L. Mangiagalli 25, 20133 Milano, Italy.
b Consiglio Nazionale delle Ricerche (CNR), Istituto di Neuroscienze, Via Vanvitelli 32, 20129 Milano,
Italy; Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di
Milano, Via Vanvitelli 32, 20129 Milano, Italy.
c Current address: Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and
Technology (BIST), Parc Científic de Barcelona, Carrer Baldiri Reixac 15-21, 08028 Barcelona, Spain; *
email: cmatera@ibecbarcelona.eu
Alpha7 nicotinic acetylcholine receptor is emerging as a central regulator in inflammatory processes,
as documented by increasing studies reported in the literature. For instance, the activation of this
nicotinic receptor subtype in resident macrophages inhibits the production of pro-inflammatory
cytokines, thereby attenuating local inflammatory responses, and may open a new window in the
treatment of chronic inflammatory disease, such as Crohn’s disease, rheumatoid arthritis, psoriasis
and asthma. In continuation of our ongoing research for the development of new cholinergic drug
candidates, we selected the nicotine derivative CAP55, which was previously shown to exert anti-
inflammatory effects via nicotinic stimulation, as a suitable compound for lead optimization.
Through the isosteric replacement of its 3,5-disubstituted 4,5-dihydroisoxazole core with a 1,4-
disubstituted 1,2,3-triazole ring, we could rapidly generate a small library of CAP55-related
analogues via a one-pot copper(I)-catalyzed azide-alkyne cycloaddition. Receptor binding assays at
nAChRs led to the identification of two promising derivatives, compounds 4 and 10, worthy of
further pharmacological studies.
Keywords: click chemistry • nicotine • alpha7 • cholinergic • inflammation
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Introduction
Convergence from many unrelated research areas has identified a primary role in health and disease
for one of the most abundant subtype of the nicotinic acetylcholine receptor (nAChR), the 7
nAChR. The α7 nAChR belongs to the family of acetylcholine-gated ion channels and exhibits distinct
biophysical and pharmacological profiles relative to other nAChR subtypes. This receptor, expressed
in the CNS, in the autonomous nervous system, and in the vascular system, is emerging as a central
regulator in processes ranging from cognitive mechanisms through modulation of specific
neurotransmitters, neuroprotection following various insults (e.g., chemical toxicity and -amyloid-
induced cell death), normalization of sensory gating in schizophrenic patients, and inflammatory
processes. Resident macrophages express the 7 nAChR, and activation of this receptor inhibits the
production of pro-inflammatory cytokines (e.g., TNF, IL-1, IL-6, HMGB-1), but does not inhibit anti-
inflammatory cytokine production (e.g., IL-10 and TGF-), thereby attenuating local inflammatory
responses.1,2,3 Previous studies have demonstrated that targeting pro-inflammatory cytokines, such
as TNF, IL-1 and IL-6, represents a promising approach in the treatment of inflammatory disorders,
such as Crohn’s disease, psoriasis, rheumatoid arthritis and asthma.4,5,6 Therefore, α7 nAChRs may
represent a relevant target in view of new therapeutics for the treatment of such inflammatory
diseases.
Several studies supporting a role for α7 nAChR in inflammatory reflexes can be found in the
literature. For instance, GTS-21, an7 nAChR partial agonist, was shown to inhibit levels of serum
TNF-α, which was associated with improved survival in a murine endotoxemia and severe sepsis
model,7 and strongly decreased the severity of pancreatitis-associated pulmonary inflammation.8,9
Moreover, GTS-21 strongly inhibited LPS-induced TNF- release into the peritoneal cavity and the
circulation.10 In 2011, Li and co-workers described the α7-mediated anti-inflammatory effects of A-
833834, a new-generation α7 nAChR agonist endowed with exceptionally high binding affinity for
this receptor subtype.11 A-833834 inhibited LPS-induced TNF-α release in a methyllycaconitine
(MLA)-sensitive manner in macrophages. These effects were also confirmed using human whole
blood assay where A-833834 was active. The role for α7 nAChR in regulating TNF-α release was
further confirmed in two different in vivo mouse models, the i.v. LPS-induced TNF-α release from
whole blood and the i.p. zymosan-induced peritonitis models. Li and coll. also showed that A-833834
reduced LPS-induced release of IL-1β and IL-6 in human blood. This is consistent with recent reports
that acetylcholine and other α7 nAChR agonists including PNU-282987 and AR-R17779 reduced the
release of TNF-α and IL-6 in human fibroblast-like synoviocytes via α7 nAChR,12,13 even though the
precise mechanisms underlying α7 nAChR-mediated cytokine reduction remain to be elucidated.11
In 2005, Saeed and coll. developed a new synthetic derivative of the major tobacco alkaloid nicotine
(from Nicotiana tabacum), named CAP55, and investigated its anti-inflammatory effects, which were
shown to be mediated by selective activation of the 7 nAChR subtype (Figure 1).5,14 CAP55
significantly inhibited endothelial cell inflammatory responses in vitro and in vivo. Using the localized
Shwartzman reaction model, characterized by sustained E-selectin and VCAM-1 expression,15 CAP55
was found to be very effective in inhibiting endothelial cell activation in vivo. Moreover, CAP55
significantly blocked TNF-induced adhesion molecule expression and chemokine expression by
human microvascular endothelial cells (HuMVECs) in a dose-dependent manner. The inhibitory
effects of CAP55 on endothelial cell activation were reverted both in vitro and in vivo by
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mecamylamine, a noncompetitive nAChR blocker, suggesting that the observed anti-inflammatory
effects were mediated by nAChRs. In the carrageenan air pouch model, CAP55 was also found to
suppress leukocyte migration during inflammation in vivo, and again this effect was blocked by
administration of mecamylamine.5
"Click" Reaction
NaN3
Cu2SO4·5H2O
sodium ascorbate
B
N
N
+
A
N
N
L-Pro, Na2CO3
DMSO:H2O
65 °C
X
N
N
O
1,2,3-triazole analogues
CAP55
Figure 1. Chemical structure of the cholinergic agonist CAP55, design strategies of the 1,2,3-triazole analogues reported in this study and general scheme of
the “click” reaction applied. Blue rings represent aryl, pyridyl, benzyl and pyridylmethyl halides; red rings represent aryl and pyridyl alkynes.
The above discussed results demonstrate the suppressive effects of α7 nAChR agonists on
inflammation-related events and highlight the potential therapeutic usefulness of such agents in the
treatment of a variety of inflammatory disorders. In continuation of our ongoing research for the
development of selective nicotinic agents,16,17,18,19,20,21 we identified CAP55 as a suitable candidate
for lead optimization. In order to rapidly generate a small library of CAP55 analogues, we aimed at
applying a “click chemistry” approach.22 For its molecular structure, CAP55 is well-suited to this
purpose: in fact, we observed that the replacement of its 3,5-disubstituted 4,5-dihydroisoxazole core
with a 1,4-disubstituted 1,2,3-triazole ring would have allowed us to prepare a set of new derivatives
via one-pot copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (Figure 1).23 Herein, we report
the synthesis of a “click chemistry”-based small library of 1,2,3-triazole analogues of CAP55 and the
assessment of their binding affinity at α7 and α4β2 nAChR subtypes.
Results and Discussion
The desired 1,4-disubstituted 1,2,3-triazole derivatives 1-10 (Figure 2) were all prepared via a
copper(I)-promoted 1,3-dipolar cycloaddition between an in situ generated azide and a terminal
alkyne, following the one-pot two-step procedure described by Fokin and co-workers.24 The starting
halide and alkyne building blocks were all selected in such a way that the new derivatives differed
for the presence and/or the position of the two pyridine nitrogen atoms, which normally play an
important role in the orthosteric binding at nAChRs (Figure 2).25,26 Our hypothesis that a 1,2,3-
triazole could work as an effective isosteric replacing function for the 4,5-dihydroisoxazole ring was
supported by in silico conformational analyses (see Experimental section for details). In fact,
molecular overlay of the lowest energy conformers of a representative ligand (compound 4) with
CAP55 revealed a high degree of structural similarity, with an average root mean square deviation
(RMSD) of 1.785 Å, as calculated on the corresponding heavy atoms after best superimposition
(Figure 3).
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All the new derivatives were obtained by simply mixing and stirring the needed reagents in a sealed
vial at 65 °C and isolated in a pure form by filtration (see Experimental section for details). Through
this experimental protocol, we did not observe the formation of any undesired NH triazole
byproduct, which may result from the competing reaction between inorganic azide and an alkyne.
Moreover, yields ranged from fair to excellent and the expected regioselectivity was maintained.24 It
is indeed well known that copper(I)-catalyzed azide-alkyne cycloadditions lead exclusively to the 1,4-
regioisomer.27 On the other hand, the ruthenium-catalyzed version of the reaction affords mainly
the 1,5-regioisomer.28,29 Finally, we tested the binding affinity of CAP55 (so far not reported in the
literature) and the new analogues 1-10 towards α7 and α4β2 nAChR subtypes. Ki values obtained
from the competition assays are reported in Table 1.
a) aryl, pyridyl, benzyl and pyridylmethyl halides used in this work
N
N
N
Br
I
I
I
I
b) aryl and pyridyl alkynes used in this work
N
N
c) 1,2,3-triazole analogues of CAP55 synthesized in this work
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
1
2
3
4
5
(71%)
(59%)
(52%)
(63%)
(87%)
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
6
7
8
9
10
(65%)
(82%)
(70%)
(67%)
(72%)
Figure 2. Chemical structure of the CAP55 1,2,3-triazole analogues synthesized and their building blocks for the one-pot copper(I)-catalyzed azide-alkyne
cycloaddition. Reaction yields are given in brackets.
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Figure 3. Superimposition of CAP55 (lowest energy conformer, in purple) and a representative member of our set of 1,2,3-triazole analogues (compound 4,
five lowest energy conformers, in orange), showing a high degree of similarity (average RMSD = 1.785 Å; single RMSD values were calculated with UCSF
Chimera). Panels a and b are different views of the same superimposition. Oxygen and nitrogen atoms are coloured in red and blue, respectively; hydrogen
atoms are hidden for the sake of clarity.
Table 1. CAP55 and compounds 1–10: binding affinity for native receptor subtypes present in rat cortex and labelled by [125I]αBungarotoxin (α7 nAChRs) or
[3H]epibatidine (α4β2 nAChRs). Numbers in brackets represent the % coefficient of variation (CV).
α7
α4β2
Compound
Structure
[
125I]-Bungarotoxin (Ki, M)
[3H]Epibatidine (Ki, M)
CAP55
407 (83)
686 (88)
100 (65)
> 100
70 (28)
76 (12)
48 (46)
> 100
1
2
3
4
5
6
6.5 (48)
> 100
20 (75)
50 (53)
178 (14)
346 (125)
7
8
9
189 (94)
91 (85)
73 (64)
150 (15)
47 (55)
34 (58)
10
24 (63)
> 100
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The parent compound CAP55 showed a moderate affinity for both receptor subtypes, with a certain
preference for α4β2. Based on the effects reported for this compound as an α7 activator in in vitro
and in vivo models of inflammation,5 its binding profile may seem quite surprising. However, similar
nicotinic ligands, such as GTS-21, have been proven to selectively stimulate α7 nAChRs but also
inhibit some other nicotinic receptors, including those containing β2 subunits, and, in our opinion, it
is reasonable to hypothesize a similar scenario for CAP55.30,31 Most of the new derivatives showed a
similar binding profile, with moderate or poor affinity for both receptor subtypes. Indeed,
compounds 4, 8, 9 and 10 displayed an increased affinity for α7, with compounds 4 (60-fold
increase) and 10 (17-fold increase) —both characterized by the presence of a pyridine ring on the
two substituted positions of the triazole core— emerging as the most interesting analogues. In
addition, compound 10 also showed a moderate gain in selectivity versus the α4β2 subtype. Overall,
the replacement of the 4,5-dihydroisoxazole core of CAP55 with a triazole system turned out to be a
well-tolerated and in some cases a beneficial substitution. Interestingly, increasing the distance
between the two pyridine nitrogen atoms produced an inversion of the selectivity profile in
compound 10 compared to its lower homologue 5.
Conclusions
In conclusion, we generated a small library of CAP55 analogues via a click chemistry approach and
demonstrated that the 1,4-disubstituted 1,2,3-triazole system may serve as an effective bioisosteric
replacement of the 3,5-disubstituted 4,5-dihydroisoxazole nucleus. We have also reported for the
first time the binding affinity profile of the cholinergic agonist CAP55 at the two main nAChR
subtypes. Compounds 4 and 10, which both showed a relevant increase in affinity for the α7
subtype, emerged as the most promising derivatives. Since it has been demonstrated that the anti-
inflammatory effects of CAP55 are mediated by nAChRs, it is reasonable to expect that structural
analogues endowed with a better binding affinity could prove better drug candidates. Further
studies are ongoing to elucidate, on one hand, the functional and anti-inflammatory properties of
these two nicotinic ligands and, on the other, to deepen the structure-activity relationships in this
set of compounds through the rational design of novel analogues.
Experimental Section
Conformational analyses
Methods. The two-dimensional (2D) structures of CAP55 and compound 4 were drawn (.mol files),
converted to three-dimensional (3D) representations (.mol2 files), and energy-minimized using the
ChemAxon MarvinSketch software.32 Five low energy conformers were generated for compound 4
(DREIDING force field). Energy minimization and conformer generation were carried out using the
proper tools implemented in ChemAxon MarvinSketch. Each conformer of 4 was then superimposed
to CAP55 using the “Match” function in UCSF Chimera.33 The carbon-5, oxygen-1 and nitrogen-2
atoms of the 4,5-dihydroisoxazole ring in CAP55 and the corresponding nitrogen-1, nitrogen-2 and
nitrogen-3 of the 1,2,3-triazole ring in compound 4 were used for the superimposition. Root mean
square deviation (RMSD) values were calculated for each conformer of 4 over CAP55 to evaluate the
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goodness of the overall superimposition using the “RMSD” function in UCSF Chimera. Only heavy
atoms were considered for these calculations, namely all pyridine nitrogen atoms, oxygen-1 and
nitrogen-2 in CAP55, and nitrogen-2 and nitrogen-3 in compound 4.
Chemistry
Materials and methods. All chemicals were purchased from Sigma-Aldrich and used without any
1
further purification. H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 (1H,
300.063; 13C, 75.451 MHz) spectrometer at 20 °C. ESI Mass spectra were obtained on a Varian 320
LC-MS-MS instrument; data are reported as mass-to-charge ratio (m/z). TLC analyses were
performed on commercial Silica Gel 60 F254 aluminium sheets; the spots were further evidenced by
spraying with a dilute alkaline potassium permanganate solution or a phosphomolybdic acid solution
(10% in ethanol). Melting points were determined on a model B 540 Büchi apparatus and are
uncorrected.
Typical experimental procedure. The aryl- or arylmethyl halide (1 equiv) was mixed with
ethynylbenzene or ethynylpyridine (1 equiv) in a sealable round bottom vial. To the mixture were
added L-proline (0.2 equiv), Na2CO3 (0.2 equiv), NaN3 (1.2 equiv), sodium ascorbate (0.1 equiv), 9:1
DMSO/H2O and CuSO4·5H2O (0.05 equiv). The vial was sealed and the mixture was stirred overnight
at 65 °C. Upon completion (monitored by TLC), the crude mixture was poured into ice-cold water.
The off-white precipitate was isolated by filtration and washed with dilute NH4OH to remove any
traces of explosive copper azides before the final drying step.
1,4-Diphenyl-1H-1,2,3-triazole [1]. 71% yield, white powder, mp: 183-185 °C; Rf = 0.79 (DCM/MeOH
= 99:1); 1H NMR (DMSO-d6): δ (ppm) 9.29 (s, 1H), 7.95 (d, J = 8.1 Hz, 4H), 7.62 (t, J = 7.5 Hz, 2H), 7.52-
7.46 (m, 3H), 7.37 (t, J = 7.4 Hz, 1H); 13C NMR (DMSO-d6):δ (ppm) 148.0, 137.3, 131.0, 130.6, 129.7,
+
129.4, 128.9, 126.0, 120.7, 120.3; MS (m/z) calcd. for C14H12N3 [M+H+] 222.10, found 222.1.
3-(1-Phenyl-1H-1,2,3-triazol-4-yl)pyridine [2]. 59% yield, light yellow powder, mp: 170-173 °C; Rf =
0.20 (cyclohexane/AcOEt = 1:1); 1H NMR (CDCl3): δ (ppm) 9.09 (s, 1H), 8.62 (d, J = 4.7 Hz, 1H), 8.31 (t,
J = 1.9 Hz, 1H), 8.29 (s, 1H), 7.83-7.79 (m, 2H), 7.58 (t, J = 7.2 Hz, 2H), 7.50 (d, J = 7.4 Hz, 1H), 7.42
(dd, J = 8.0 and 5.0 Hz, 1H); 13C NMR (CDCl3):δ (ppm) 149.7, 147.3, 145.5, 133.4, 130.1, 129.8, 129.3,
+
126.7, 124.1, 120.9, 118.2; MS (m/z) calcd. for C13H11N4 [M+H+] 223.10, found 223.1.
3-(4-Phenyl-1H-1,2,3-triazol-1-yl)pyridine [3]. 52% yield, yellow powder, mp: 176-178 °C; Rf = 0.22
(cyclohexane/AcOEt = 1:1); 1H NMR (CDCl3): δ (ppm) 9.07 (s, 1H), 8.73 (d, J = 4.4 Hz, 1H), 8.25 (s, 1H),
8.23-8.19 (m, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.53 (dd, J = 8.3 and 5.0 Hz, 1H), 7.48 (t, J = 7.4 Hz, 2H),
7.41 (d, J = 7.7 Hz, 1H); 13C NMR (CDCl3): δ(ppm) 150.2, 149.2, 141.8, 134.0, 130.0, 129.2, 129.0,
+
128.3, 126.2, 124.5, 117.6; MS (m/z) calcd. for C13H11N4 [M+H+] 223.10, found 223.1.
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2-(1-(Pyridin-3-yl)-1H-1,2,3-triazol-4-yl)pyridine [4]. 63% yield, light brown powder, mp: 203-206 °C;
Rf = 0.50 (DCM/MeOH = 95:5); 1H NMR (CDCl3): δ (ppm) 9.20 (s, 1H), 8.81 (m, 1H), 8.69-8.60 (m, 2H),
8.24 (dd, J = 11.8 and 8.0 Hz, 2H), 7.84 (t, J = 7.7 Hz, 1H), 7.64-7-52 (m, 1H), 7.30 (dd, J = 7.2 and 5.0
Hz, 1H); 13C NMR (CDCl3): δ (ppm) 150.1, 149.5, 149.0, 137.5, 137.3, 134.0, 130.1, 129.2, 128.3,
+
126.2, 123.4, 117.6; MS (m/z) calcd. for C12H10N5 [M+H+] 224.09, found 224.0.
3,3'-(1H-1,2,3-Triazole-1,4-diyl)dipyridine [5]. 87% yield, white powder, mp: 218-220 °C; Rf = 0.45
1
(DCM/MeOH = 96:4); H NMR (CDCl3): δ (ppm) 9.10 (s, 1H), 9.08 (d, J = 2.7 Hz, 1H), 8.75 (dd, J = 4.7
and 1.1 Hz, 1H), 8.65-8.60 (m, 1H), 8.35 (s, 1H), 8.29 (dt, J = 8.0 and 1.7 Hz, 1H), 8.21 (dq, J = 8.3 and
1.7 Hz, 1H), 7.55 (dd, J = 8.3 and 4.7 Hz, 1H), 7.43 (dd, J = 8.0 and 5.0 Hz, 1H); 13C NMR (CDCl3): δ
(ppm) 150.5, 150.1, 147.4, 146.2, 141.9, 133.7, 133.5, 128.4, 126.3, 124.6, 124.1, 118.1; MS (m/z)
+
calcd. for C12H10N5 [M+H+] 224.09, found 224.1.
4-(4-Phenyl-1H-1,2,3-triazol-1-yl)pyridine [6]. 65% yield, light brown powder, mp: 170-172 °C; Rf =
1
0.30 (cyclohexane/AcOEt = 6:4); H NMR (CDCl3): δ (ppm) 8.83 (m, 2H), 8.30 (s, 1H), 7.92 (d, J = 7.2
Hz, 2H), 7.80 (d, J = 5.0 Hz, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.41 (d, J = 7.2 Hz, 1H); 13C NMR (CDCl3):
+
δ(ppm) 152.0, 149.4, 143.2, 129.8, 129.3, 129.1, 126.2, 116.8, 113.9; MS (m/z) calcd. for C13H11N4
[M+H+] 223.10, found 223.1.
1-Benzyl-4-phenyl-1H-1,2,3-triazole [7]. 82% yield, white powder, mp: 128-130 °C; Rf = 0.38
1
(cyclohexane/AcOEt = 7:3); H NMR (DMSO-d6): δ (ppm) 8.63 (s, 1H), 7.83 (d, J = 7.4 Hz, 2H), 7.45-
7.30 (m, 8H), 5.64 (s, 2H); 13C NMR (DMSO-d6): δ (ppm) 136.7, 131.4, 129.6, 129.5, 128.9, 128.6,
+
128.1, 125.8, 122.3, 120.7, 53.7; MS (m/z) calcd. for C15H14N3 [M+H+] 236.12, found 236.1.
3-(1-Benzyl-1H-1,2,3-triazol-4-yl)pyridine [8]. 70% yield, light brown powder, mp: 100-102 °C; Rf =
1
0.25 (cyclohexane/AcOEt = 3:7); H NMR (CDCl3): δ (ppm) 8.95 (bs, 1H), 8.56 (d, J = 3.4 Hz, 1H), 8.18
(ddd, J = 7.7, 3.9 and 2.2 Hz, 1H), 7.74 (s, 1H), 7.43-7.30 (m, 6H), 5.60 (s, 2H); 13C NMR (CDCl3): δ
(ppm) 149.4, 147.2, 145.4, 134.5, 133.1, 129.4, 129.1, 128.3, 126.9, 123.9, 120.0, 54.6; MS (m/z)
+
calcd. for C14H13N4 [M+H+] 237.11, found 237.1.
3-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)pyridine [9]. 67% yield, white powder, mp: 112-115 °C; Rf
= 0.40 (AcOEt); 1H NMR (CDCl3): δ (ppm) 8.67-8.62 (m, 2H), 7.79 (d, J = 6.9 Hz, 2H), 7.71 (s, 1H), 7.62
(d, J = 7.7 Hz, 1H), 7.41 (t, J = 6.9 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 5.60 (s, 2H); 13C NMR (CDCl3): δ
(ppm) 150.6, 149.4, 148.8, 135.9, 130.8, 130.5, 129.1, 128.6, 126.0, 124.2, 119.6, 51.8; MS (m/z)
+
calcd. for C14H13N4 [M+H+] 237.11, found 237.1.
3-((4-(Pyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)pyridine [10]. 72% yield, white powder, mp: 132-
1
134 °C; Rf = 0.33 (AcOEt); H NMR (CDCl3): δ (ppm) 8.97 (d, J = 1.9 Hz, 1H), 8.68 (d, J = 2.2 Hz, 1H),
8.65 (dd, J = 5.0 and 1.7 Hz, 1H), 8.58 (dd, J = 4.7 and 1.4 Hz, 1H), 8.19 (dt, J = 8.0 and 1.9 Hz, 1H),
7.80 (s, 1H), 7.66 (dt, J = 8.0 and 2.2 Hz, 1H), 7.39-7.33 (m, 2H), 5.64 (s, 2H); 13C NMR (CDCl3): δ (ppm)
150.7, 149.5, 148.9, 147.7, 147.4, 136.0, 134.6, 130.8, 130.6, 126.1, 124.2, 120.1, 51.8; MS (m/z)
+
calcd. for C13H12N5 [M+H+] 238.11, found 238.1.
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Receptor binding assays
Animal tissues. Adult male pathogen-free Sprague-Dawley rats (Harlan-Nossan, Milan, Italy) (at least
4-month-old) were used. Rats were reared on a 12 h light/dark cycle (animals were kept in a 12 h
light/12 h dark cycle) with free access to food and water. All experiments were conducted according
to Ministry of Health (the regulatory authority for controlling the use of laboratory animals and
ethics on animal experiments in Italy) guidelines (Legislative Decree n. 116/92) and in accordance
with the European Community guidelines European Directive 86/609/EEC). All the experiments were
performed according to protocols (n.2/2010) approved by the University of Milan Animal Care and
Use Committees and by the Ministry of Health.
Membranes binding of [125I]-Bungarotoxin and [3H]Epibatidine. The cortex tissues were dissected,
immediately frozen on dry ice and stored at –80 ºC for later use. In each experiment, the cortex
tissues from two rats were homogenized in 10 mL of a buffer solution (50 mM Na3PO4, 1 M NaCl, 2
mM ethylenediaminetetraacetic acid [EDTA], 2 mM ethylene glycol tetraacetic acid [EGTA] and 2
mM phenylmethylsulfonyl fluoride [PMSF], pH 7.4) using a potter homogenizer; the homogenates
were then diluted and centrifuged at 60,000 g for 1.5 h. The total membrane homogenization,
dilution and centrifugation procedures were performed twice, then the pellets were collected,
rapidly rinsed with a buffer solution (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM
CaCl2 and 2 mM PMSF, pH 7), and resuspended in the same buffer containing a mixture of 20 μg/mL
of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Ki values
were calculated from the competition curves with the LIGAND software.34
[125I]α-Bungarotoxin binding. The saturation binding experiments were performed using aliquots of
cortex membrane homogenates incubated overnight with 0.1-10 nM concentrations of [125I]α-
bungarotoxin (specific activity 200–213 Ci/mmol, GE Healthcare) at room temperature. Nonspecific
binding was determined in parallel by means of incubation in the presence of 1 μM unlabelled α-
bungarotoxin. After incubation, the samples were filtered as described above and the bound
radioactivity was directly counted in a γ counter.
[3H]Epibatidine binding. (±)-[3H]Epibatidine with a specific activity of 56–60 Ci/mmol was purchased
from Perkin–Elmer (Boston MA); the non radioactive α-bungarotoxin and epibatidine were
purchased from Sigma. It has been previously reported that [3H]epibatidine also binds to α-
bungarotoxin binding receptors with nM affinity.35 To prevent the binding of [3H]epibatidine to the
α-bungarotoxin binding receptors, the membrane homogenates were pre-incubated with 2 μM α-
bungarotoxin and then with [3H]epibatidine. The saturation experiments were performed by
incubating aliquots of cortex membrane homogenates with 0.01-2.5 nM concentrations of (±)-
[3H]epibatidine overnight at 4 °C. Nonspecific binding was determined in parallel by means of
incubation in the presence of 100 nM unlabelled epibatidine. At the end of the incubation, the
samples were filtered on a GFC filter soaked in 0.5% polyethylenimine and washed with 15 mL of a
buffer solution (10 mM Na3PO4, 50 mM NaCl, pH 7.4), and the filters were counted in a β counter.
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Supplementary Material
Supporting information for this article is available on the WWW under https://XXX.XXX.XXX
Acknowledgements
ChemAxon MarvinSketch was used for drawing chemical structures and generating low energy
conformers (MarvinSketch version 17.28.0, 2017, http://www.chemaxon.com). Molecular graphics
images and RMSD calculations were produced using the UCSF Chimera package from the Resource
for Biocomputing, Visualization, and Informatics at the University of California, San Francisco
(supported by NIH P41 RR-01081).
Author Contribution Statement
LR and CM designed and synthesized the new compounds; CG performed receptor binding assays;
MDA and CD supervised the project; CM and MDA wrote the manuscript with contributions from all
authors.
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