New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3? Nicotinic Receptors

Article abstract of DOI:10.1021/acs.jmedchem.8b01372

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [³H]-cytisine and [³H]-methyllycaconitine to measure their affinity for α4β2? and α7? nicotinic receptors. The new derivatives showed some preference for the α4β2? over the α7? subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest K_i values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4β2, α7, and α3β2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4β2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3β2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC₅₀ = 0.43 μM). The primary amines described here represent new chemotypes for the α7 and α3? receptor subtypes.

Full text of DOI:10.1021/acs.jmedchem.8b01372

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Article
New rigid nicotine analogs, carrying a norbornane moiety,
are potent agonists of #7 and #3* nicotinic receptors
Dina Manetti, Alexandra Garifulina, Gianluca Bartolucci, Carla Bazzicalupi, Cristina Bellucci, Niccolò
Chiaramonte, Silvia Dei, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Paola Gratteri, Ekaterina
Spirova, Irina Shelukhina, Elisabetta Teodori, Katia Varani, Victor Tsetlin, and Maria Novella Romanelli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01372 • Publication Date (Web): 25 Jan 2019
Downloaded from http://pubs.acs.org on January 25, 2019
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New rigid nicotine analogs, carrying a norbornane moiety, are potent agonists of α7 and α3*
nicotinic receptors
a,
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e
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Dina Manetti, * Alexandra Garifulina, Gianluca Bartolucci, Carla Bazzicalupi, Cristina Bellucci,
a
a
d
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Niccolò Chiaramonte, Silvia Dei, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Paola Gratteri,
b
b
a
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Ekaterina Spirova, Irina Shelukhina, Elisabetta Teodori, Katia Varani, Victor Tsetlin, Maria
Novella Romanellia
a
Department of Neuroscience, Psychology, Drug Research and Child’s Health (NEUROFARBA),
Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff 6,
5
0019 Sesto Fiorentino, Italy
b
Department of Molecular Basis of Neurosignaling, Shemyakin-Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str. 16/10, 117997
Moscow, Russia
c
Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia 3, 50019
Sesto Fiorentino.
d
Department of NEUROFARBA, Section of Pharmacology and Toxicology, University of
Florence, Viale G Pieraccini 6, 50139 Firenze, Italy
e
Department of NEUROFARBA, Laboratory of Molecular Modeling Cheminformatics & QSAR,
University of Firenze, via Ugo Schiff 6, I-50019 Sesto Fiorentino, Firenze, Italy.
f
Institute of Pharmacology, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara,
Italy
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Abstract
A 3D-database search has been applied to design a series of endo and exo
3‐(pyridin‐3‐yl)bicyclo[2.2.1]heptan‐2‐amines as nicotinic receptor ligands. The synthesized
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compounds were tested in radioligand binding assay on rat cortex against [ H]-cytisine and [ H]-
methyllycaconitine (MLA) to measure their affinity for α4β2* and α7* nicotinic receptors. The new
derivatives showed some preference for the α4β2* over the α7* subtype, their affinity being
dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo
primary amines displayed the lowest K values on both receptor subtypes. Selected compounds (1a,
i
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a, 3a and 6a) were tested on heterologously expressed α4β2, α7 and α3β2 receptors, and on
SHSY-5Y cells. Compounds 1a and 2a showed α4β2 antagonistic properties while behaved as full
agonists on recombinant α7 and on SHSY5Y cells. On the α3β2 subtype, only the chloro derivative
2
a showed full agonist activity and submicromolar potency (EC 0.43 μM). The primary amines
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described here represent new chemotypes for the α7 and α3* receptor subtypes.
Keywords
Nicotinic acetylcholine receptor; 42 antagonist; α7 agonist; α3 agonist; 3D-database search;
molecular modeling.
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Introduction
Nicotinic acetylcholine receptors (nAChRs) are considered as attractive targets for drug design
due to their involvement in many pathophysiological processes in the CNS and also in non-neuronal
systems.1-7 Several nicotinic ligands entered clinical trials, mainly for cognitive deficits associated
with neurological diseases, smoking cessation and pain. Several other therapeutic applications are
under study, and both agonists and antagonists may be useful depending on the targeted receptor
subtype and pathology.8
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Of the seventeen nAChR subunits cloned so far (α1-10, β1-4, γ, δ, ε) only sixteen have been
found in mammalian tissues (α8 has been found only in chicken). These subunits can assemble into
functional pentamers in many different ways, giving a large number of possible subtypes, the high
homology between them making the design of selective ligands difficult.8-9 The heteromeric α4β2*
(* indicates the possible presence of other subunits) and the homomeric α7 receptors, the most
abundant nAChRs in the CNS, are the most investigated subtypes. Some degree of selectivity for
ligands targeting these two subtypes has been achieved, since structural requirements are somehow
different. However, many nicotinic ligands, including compounds in clinical trials or approved for
therapy, have mixed pharmacological profile: as an example varenicline (Chart 1), approved for
smoking cessation, showed > 500-fold higher affinity for α4β2* over α7 receptors in binding
experiments, but only 8-fold higher potency in functional assays.10-11 In functional studies,
varenicline behaved as a partial agonist on α4β2* and full agonist on α7 receptors; it is possible that
both activities, as well as interaction with other subtypes, contribute to the pharmacological effects
of this compound.12-13 Other well characterized ligands have a mixed pharmacological profile, such
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as GTS-21, an α7 partial agonist and α4β2 antagonist, tested in clinical trials to treat cognitive
deficit associated with Alzheimer’s disease, Attention-Deficit Hyperactivity Disorder or
Schizophrenia (www.clinicaltrial.gov).
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Chart 1. Structure of nicotinic ligands
OMe
OMe
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Me
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NH
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GTS-21
varenicline
AT-1001: X = Br
AT-1012: X = I
N
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Other nAChR subtypes such as α3*, α5* and α6*, which are less abundant in the CNS,15-16 have
been studied to a lesser extent, also owing to the shortage of selective ligands. Indeed these
subtypes are also attractive targets, since they are involved in neurotransmitter release or in tobacco
dependence.17-18 Moreover, the α3β4* subtype is widely expressed in the peripheral nervous system
and also in non-neuronal tissues.19 In recent times, also compounds with selectivity for α3*
receptors have been discovered, such as the α3β4*partial agonists AT-1001 and AT-1012 (Chart 1);
AT-1001 gave promising results when tested in rat models of nicotine or cocaine addiction.20-21
Therefore, the design of new nicotinic modulators is still of interest.
Different strategies can be applied to search for new ligands endowed with improved activity or
selectivity. Some years ago, we used a 3D database searching approach to discover novel lead
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compounds. The requirements for the nicotinic pharmacophore, i.e. a basic nitrogen atom and an
H-bond acceptor group, their distance and orientation, were extracted from pyrido[3,4]homotropane
(
PHT), a fully rigid α4β2 ligand (Chart 2).23-24 These features were then transformed into a query to
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search within the Cambridge Structural Database (CSD), and resulted in several hits; optimization
of one of them resulted in quinoline analogues of nicotine.25-26
With the aim to find new chemotypes for the nicotinic receptors, later we repeated a similar
approach, and among the retrieved hits, the molecule LOYMOB (Chart 2) gave us the idea of using
a simple bicyclo[2.2.1]heptane moiety as a spacer between the pyridyl ring (H-bond acceptor) and
an aliphatic amino group (see compounds with general formula A). These molecules possess 4
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stereogenic centres, leading to 4 possible diasteromeric racemates; however, only the trans
derivatives shown in Chart 2 would comply with the criteria of nicotinic pharmacophore. In fact, in
these trans isomers, differing in endo/exo arrangement of the amino group, the distance between the
basic and the pyridyl nitrogen atoms is in the range of 4.9 - 5.6 Å, in accord with the well-known
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nicotinic pharmacophoric models. Therefore, a series of amines were designed (1a-b – 6a-b),
differing in the endo (a) or exo (b) arrangement of the amino group, the presence of a Cl atom on
the pyridyl ring, and the number of methyl groups on the amino moiety (0-2).
Chart 2. Design of compounds 1-6 from the lead PHT.
Cl
CH3
N
H C
3
N
HN
3D search
simplification
N
A
N
NR2
N
H C
N
3
O
NR R2
1
PHT
LOYMOB
N
X
1-6
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a, endo
R , R = H, CH
X = H, Cl
b, exo
NR R2
1
3
N
X
In this preliminary work we intended to assess the nicotinic potentiality of this new scaffold,
leaving to a future time the optimization and the study of enantioselectivity. In fact, the norbornane
ring is a structural feature also found in mecamylamine, a non competitive nicotinic antagonist, but
we reckoned that the presence on the molecule of a pyridyl ring could allow the interaction with the
orthosteric site, and possibly introduce agonistic properties. In addition, we were aware that the
extraction from PHT of only the essential pharmacophoric features for binding to the nicotinic
receptor could give new molecules which may not show subtype selectivity, since other important
properties such as shape and volume have not been taken into account in the initial design.
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Knowing these limitations, the designed compounds were synthesized and tested for their
affinity on 42* and 7* nicotinic receptors of rat brain. Since some of the new compounds
displayed interesting affinity, their functional properties were also assessed in vitro in SHSY5Y
cells and on the heterologously expressed individual α4β2, α7 and α3β2 subtypes.
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Methodology and results
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D search and design
The pharmacophoric search was performed on the Cambridge Structural Database (CSD). The
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queries, showed in Figure 1, contained the geometrical features expected for a nicotinic agonist, i.e.
a nitrogen atom, potentially cationic, and another heteroatom (as a pyridyl N, or a carbonyl O) as H-
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bond acceptor group. Some changes were introduced with respect to the first approach: 1) the H-
bond acceptor moiety could be also an oxygen atom; 2) the distance between the two
pharmacophoric heteroatoms has been defined without the addition of a “dummy” lone pair; 3) the
distance ranges were chosen larger than those previously reported, in order to explore either flexible
structures, or the so called “water-extension” concept, which suggests the bridging role of a water
molecule in the binding of ligands to the receptor.27, 29-30 R factor  0.05 was used as an additional
filter to reduce the number of structures for consideration.
From the resulted hit list we have chosen LOYMOB (Chart 2): this molecule shows a carbonyl
group as H-bond acceptor moiety, while the aromatic heterocycle could be replaced with
(cyclo)alkyl groups carrying or incorporating a basic nitrogen. However, we realized that there
could be other ways to modify this molecule: we replaced the aromatic ring with a pyridine moiety,
transformed the keto function into an amine, and removed the methyl groups on the norbornane
ring, leading to compounds with general formula A (Chart 2). By this way, the two pharmacophoric
moieties, a pyridine ring and a basic nitrogen, would be linked by a rigid spacer, the
bicyclo[2.2.1]heptane ring.
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Figure 1. Queries used in the CSD search: Qa = N,C; c subscript indicates atoms belonging to rings.
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Before the 3D structure of α4β2 nAChR X-ray structure was published in 2016 (XRD) and,
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more recently in 2018 (cryo-EM), comparative modeling was used by some of us to build the
three-dimensional model of the N-terminal extracellular portion (Ligand Binding Domain, LBD) of
the α4β2 nicotinic receptor.33 When our work started, this was the only chance to predict the
binding ability of the designed compounds. This model was used to test possible interactions of
compound 1a and 1b (see structure in Table 1) at the binding site formed by the α4 ((+) side) and
β2 ((-) side) subunits. Hydrogen bond, electrostatic and cation-π interactions with α4Y204 and
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α4W156 (residue numbers according to 5KXI ) stabilize the cationic head of compounds 1a and
b while the pyridine ring extends in a hydrophobic cavity lined by residues from both subunits
Figure S1, Supporting Information). The computational study predicted that 1a and 1b could have
1
(
high affinity for the α4β2 receptor, thus validating our design.
Chemistry
34
The key intermediates of the synthetic pathway were (E)-3-(2-nitrovinyl)pyridine 9 and (E)-2-
chloro-5-(2-nitrovinyl)pyridine 10;35 these compounds were synthesized by addition of
nitromethane to the commercially available aldehydes, followed by dehydration of the alcoholic
36
intermediates 7 and 8, according to Duursma (Scheme 1). Then, a Diels-Alder reaction of 9 and
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0 with cyclopenta-1,3-diene, obtained after thermal decomposition and distillation of
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commercially available dicyclopentadiene, gave compounds 11 and 12.
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Scheme 1. Synthesis of endo and exo 3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines 1a,b-6a,ba
OH
N
X
CHO
a
NO2
b
NO2
c
X
N
X
N
X
N
NO2
7
8
: X = H
: X = Cl
9: X = H
10: X = Cl
11a,b: X = H
12a,b: X = Cl
N
X
N
X
NH2
d
f
+
1b: X = H
2b: X = Cl
e for X = H
NO2
e for X = Cl
NH2
13a: X = H
13b: X = H
14a,b: X = Cl
N
1a: X = H
2a: X = Cl
X
g, h
X
g, h
i
N
i
NHMe
NMe2
N
X
3
4
a: X = H
a: X = Cl
NHMe
N
N
X
NMe2
3b
5a: X = H
6a: X = Cl
5b: X = H
6b: X = Cl
a
Reagents and conditions: (a) CH NO tBuOK, tBuOH; (b) (CF CO) O, Et N, -10°C; (c) cyclopenta-
3
2,
3
2
3
1
,3-diene, CH Cl ; (d) H /Pd/C; abs EtOH; (e) chromatographyc separation; (f) SnCl .2H O, abs EtOH,
2 2 2 2 2
D; (g) ClCOOEt, Et N, CH Cl ; (h) LiAlH THF; (i) HCOOH, HCHO, abs EtOH.
3
2
2
4,
The Diels-Alder reaction gave endo/exo mixtures, where the thermodynamically more stable
endo adduct was always the predominant isomer. The two isomers were clearly visible from the
NMR spectra: in fact, in the endo isomers, the proton geminal to the nitro group appears as a triplet
at 4.97 ppm for 11a and at 4.91 ppm for 12a. On the contrary, in the exo isomer the same proton
appears as a doublet at higher fields (4.52 ppm for 11b and 4.45 ppm for 12b). The isomeric ratios
and the overall yield were dependent on the solvent used: the Diels-Alder reaction performed on 9
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in toluene, 1,4-dioxane or dichloromethane gave endo/exo mixtures in 4.3:1 (68% yield), 7:1 (97%
yield) and 6.4:1 (99% yield), respectively. The substituent on the pyridine nucleus had also a small
effect: in dichloromethane the endo/exo ratios were 6.4:1 and 5.3:1 for 11 and 12, respectively.
Catalytic hydrogenation of the double bond of 11a,b and 12a,b gave compounds 13a,b and
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
1
4a,b, respectively; chromatographic separation was possible only on the former, giving 13a and
3
8
13b. Reduction of nitro group with SnCl in abs ethanol, and chromatographic separation gave the
2
desired primary amines 1a, 2a, and 1b, 2b. Reaction with ethyl chloroformate, followed by
reduction of the intermediate carbamates gave monomethyl derivatives 3a, 3b and 4a; these
reactions were not performed on 2b, due to its low amount. Reaction with formaldehyde and formic
acid on 1a, 1b, 2a and 2b gave tertiary amines 5a, 5b, 6a and 6b.
For biological tests, all compounds were transformed into the HCl salts.
Radioligand binding studies on rat brain.
To evaluate their affinity for the neuronal nAChRs, the synthesized compounds were tested in
2
6
vitro on rat brain in competition binding experiments according to a previously applied protocol ;
3
the results are reported in Table 1. [ H]-Cytisine was used to detect binding to the α4β2* receptor,
3
while [ H]-methyllycaconitine (MLA) allowed to measure the interaction with the α7* subtype.
Nicotine and MLA were taken as reference compounds.
As a general remark, amines 1-6 were more active on α4β2* than on α7* nicotinic receptors. The
K values of all compounds were in the nanomolar range for the α4β2* subtype, while for some of
i
them (1b, 3b, 5a, 5b, 6a and 6b) the affinity constants were not calculated on the α7* subtype, since
3
at 1 μM concentration the amount of displaced [ H]-MLA from rat midbrain was below 33%. Some
compounds displayed also a moderate selectivity: the endo secondary amines 3a and 4a showed,
respectively, a 34- and 37-fold higher affinity on α4β2* than on α7* nicotinic receptors.
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5
6
Endo-exo isomerism plays a crucial role in binding affinity: on both subtypes the endo
compounds 1a, 2a, 3a, 5a and 6a showed higher affinity than their exo isomers 1b, 2b, 3b, 5b and
6b; potency ratios ranged, on the α4β2* subtype, from 7 for 6a-6b, to 79 for 1a-1b; on α7*
nicotinic receptors the highest difference (22 times) was for 2a-2b. Endo-exo isomers differ in the
spatial orientation of the bicycloheptane moiety (shown in Figure 2), suggesting a limited space
available within the binding site for the bulky spacer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As far as methylation on the basic nitrogen atom is concerned, primary amines were more potent
than secondary and tertiary ones, in both endo and exo series; on the α7 subtype, tertiary amines
5
a,b and 6a,b showed poor interaction. Methylation affected also subtype selectivity: as mentioned
before, endo secondary amines 3a and 4a showed the highest selectivity ratios, while in the exo
series, the primary amine 2b showed the highest preference for 42* over 7* receptors (8 times).
This rank order of affinity is somehow unexpected, since removal of N-methyl groups of nicotinic
ligands often resulted in a lower activity.39 N-methylation should influence basicity and, as a
consequence, the extent of protonation of the amino group, but the prediction of pKa values (see
below) suggests for all amines a degree of protonation >99%.This detrimental effect of methylation
on the activity can be explained by an increase of steric hindrance, which is better tolerated on
+

42 rather than on 7 receptors. However, the presence of more than one NH moiety can give
additional interactions. In fact, while testing protonated secondary amines, Post et al found evidence
+
…
for a double involvement of the ammonium group, which established NH π interactions not only
40
with the tryptophan residue in loop B but also with a tyrosine residue in loop C. These authors
suggested that a smaller cation head could allow for stronger interactions with loop C in the binding
site, an hypothesis that could explain also the results of this study.
The insertion of a 6-Cl atom on the pyridine ring increased the affinity for both * and 7*
subtypes, even if the increment was small. The highest increase was found for the exo primary
amine 2b on both 42* (Ki /Ki = 6) and * (Ki /Ki >4). The small increase in affinity
1
b
2b
1b
2b
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slightly enhanced selectivity (compare 2b-1b, 4a-3a, 6a-5a and 6b-5b), with the exception of
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
primary amine 2a: the Ki /Ki
ratio for this compound was 8, while that of the unsubstituted
α4β2*
α7*
compound 1a was 22.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1: Binding affinity of compounds 1-6 on α4β2* and α7* receptors of rat brain, and functional
activity of compounds 1a, 2a, 3a and 6a.
1
2
N
X
NR R
b (exo)
a (endo)
NR R2
1
N
X
hα7c
rα7d hα3β2e SH-SY5Y
α4β2*
K (nM)
i
α7*Ki
K (α7)/
i
N
X
R1
R2
EC50
EC50
EC50
cellsf
a
(nM)b K (α4β2)
i
(μM)
(μM)
(μM)
EC (μM)
5
0
2
±
1
.11
0.18
66
± 14
.31 ±
.19
46.0
22
0.048
±0.013
5.98
±1.50
6.32
±1.07
1.92
±0.66
1a
H
H
H
H
H
H
H
H
H
H
H
± 4.0
>1000
[30%]
1
b
> 6
1
0
10.53
± 1.12
0.024
±0.006
2.71
±0.30
0.43
±0.10
0.22
±0.04
2
a
Cl
Cl
H
H
8
2
±
352
27
24
2
b
H
28 ± 2
10.22 ±
1
2
±
3
±0.27
5
±
5
± 49
43
±
2
±
2.31
8
34
2.29
±0.94
65.53
±5.74
3
a
CH₃
CH₃
CH₃
>10
N.d.
.09
06
18
± 32
>1000
[15%]
116
3b
H
> 5
37
.12
4
5
a
a
Cl
H
± 12
9
6
54
>1000
[8%]
>1000
[6%]
>1000
[33%]
>1000
[18%]
CH₃ CH₃
CH₃ CH₃
> 17
> 2
> 23
> 3
-
5
b
H
6.45
±0.41
2.86
±0.17
6
a
Cl CH₃ CH₃
Cl CH₃ CH₃
Nicotine
>10
N.d.
4
94
28
6
b
4.71
-
±1.44g
22.47±3.06
±
0.19
1
0
.42±
.17
Methyllycaconitine
-
-
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
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5
5
5
5
5
5
5
6
a
3
i
Displacement of [ H]-cytisine from rat cerebral cortex; K values are expressed as mean ±SEM (n = 4 indipendent
experiments). b Displacement of [ H]-MLA from rat midbrain; K
3
values are expressed as mean ±SEM (n = 4
i
indipendent experiment). Square brackets: % inhibition of [ H]-MLA binding at 1 µM. Increase in intracellular [Ca2+]
3
c
on Neuro2a cells expressing hα7 receptor, in the presence of PNU 120596 (10 μM). d Electrophysiology on Xenopus
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
e
f
oocytes, expressing rat α7 nAChR. Electrophysiology on Xenopus oocytes, expressing human α3β2 nAChR Increase
2
+
g
41
in intracellular [Ca ] on SH-SY5Y cells. From ref. . N.d: not determined.
Molecular modeling
The binding ability of the synthesized compounds was analyzed using the X-ray structure of the
3
1
human α4β2 nicotinic receptor, by means of docking simulations. Compounds 1a and 1b were
taken as representatives for all the derivatives reported in Table 1. Since these derivatives have been
tested only as racemates, both enantiomers of each compound, i.e. (1R,2R,3S,4S)-1a (1a1),
(
1S,2S,3R,4R)-1a (1a ), (1S,2R,3S,4R)-1b (1b ) and (1R,2S,3R,4S)-1b (1b , see structures in Figure
2 1 2
S2, Supporting Information) were submitted to docking simulations. Overall, the docked
compounds give rise to poses showing strong analogies with nicotine in the 5KXI crystal structure,
and possibly differing in the orientation of the pyridine ring which can be tilted by 180°. The
obtained poses and the selected contacts are shown in Figure 2 and Figure S3 (Supporting
Information), and distances/angles for each contact are listed in Table 2.
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2
2
2
2
2
2
2
2
2
3
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3
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0
1
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5
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9
0
1
2
3
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5
6
7
8
9
0
1
2
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8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Predicted poses for the enantiomers of 1b (A,B) and 1a (D-F) in comparison with nicotine (C)
(PDB 5KXI). The nitrogen atom of the pyridine ring is oriented towards α4Y204; poses with the pyridine
group tilted by 180° are reported in Figure S3 (Supporting Information). (A): 1b ;(B): 1b ; (C): cognate
1
2
nicotine in 5KXI; (D): 1a ; (E): 1a (pose I);( F): 1a (pose II). The molecular surface formed by residues
1
2
2
lining the hydrophobic pocket which accommodate the pyridine ring is shown in panel A and represented in
grey.
Interestingly, the two enantiomers of compound 1b (1b and 1b , exo configuration) gave a
1
2
3
1
+
fairly good overlap with nicotine in the crystal structure, with the position of the C - NH group
3
corresponding to the N – CH of the nicotine. The ammonium function in all poses gives rise to H-
3
bonds with both α4W156 and α4S155 carbonyl oxygens, as well as α4Y100 phenol oxygen. Indeed,
α4W156, which is involved in the most important contacts in the nicotine complex, still plays an
+
…
important role. As a matter of fact, the NH π contact formed by the ligand in the X-ray structure
…
with the indole ring of W156 (Figure 2C) is replaced in each pose of enantiomer 1b by a CH π
1
contact (Figure 2A). The 1b isomer points its CH bond towards the aromatic ring of Y204 (Figure
2
2
B).
As far as compound 1a (endo configuration) is concerned, different poses can be observed for
the two enantiomers. While 1a is always placed very similarly to 1b (Figure 2A and 2D, Table 2),
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for 1a two different poses are possible. In one of them (1a (I), Figure 2E), the ammonium group
2
2
still matches the position of the nicotine N-methyl group and gives the contacts previously
discussed for the exo enantiomers 1b and 1b , namely the α4W156 carbonyl oxygens, the α4Y100
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phenol oxygen, and the Y204 aromatic ring. On the contrary, in the other pose (1a (II), Figure 2F)
2
+
…
it does not match this position but it is involved in a very interesting NH π contact with the indole
ring of α4W156, thus restoring the pivotal π-cation interaction, and giving a possible explanation
for the difference in α4β2 affinity between 1a and 1b. Additional considerations could be made
regarding the pyridine group. Notably, in all poses found for 1a and 1b, the pyridine group occupies
a position mostly similar to that of the same group in the nicotine crystal structure. However, some
docking solutions orient the pyridine nitrogen in a similar fashion as the pyridine nitrogen in the
nicotine, and, as found in the crystal structure, apparently it doesn’t give any contact (Table 2,
Figure 2C and Figure S3C, Supporting Information). On the other hand, the poses of Figure 2 have
the pyridine group tilted by 180° and the nitrogen atom located about 3 Å apart from the phenol
oxygen of α4Y204.
Therefore, the outcome of computational studies can rationalize the difference in affinity for
exo/endo isomerism on the α4β2 subtype, and predicts a bifurcated H-bonds with several oxygen
atoms surrounding the cationic primary amine, possibly increasing binding strength.
Table 2. Selected contacts for the pose obtained from the MM calculations
N(py) O NH+ ….
…
.
O
NH+ ….O=C
NH+ ….π (centroid)
d(Å)
d(Å)/angle DH-A(°)
α4Y100
d(Å)/angle(°)
d(Å)
α4Y204
α4W156 α4S155
α4W156
-
2.2/122
2.3/137
2.4/131
2.3/145
2.2/147
2.9/128
2.9/128
3.1/131
3.1/132
-
-
-
-
1
1
a
1
3
-
.2
.2
2.1/130
2.2/155
b
1
3
2.2/153
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-
2.1/129
2.1/132
2.6/127
2.5/120
2.5/158
2.6/148
2.1/142
2.1/138
2.0/149
2.1/146
2.3/136
2.5/119
-
-
-
-
-
-
-
1b
2
2.8
-
-
-
1a
2
2
(pose I)
3
-
.2
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.6
2.6
1
a
(pose II)
3
.2
Calculated physicochemical properties
The calculated physicochemical properties of the compounds are reported in Table 3. Ligand
4
2
efficiency (LE) has been computed by the equation of Hopkins, considering the binding affinity
(
Ki) for α4β2* and α7*; it gives information on how efficiently a compound occupies the binding
site. As reported in Table 3, all the new derivatives possess LE values in the range 0.53-0.85
Kcal/mol for the α4β2* and 0.59-0.73 Kcal/mol for the α7* subtype On the latter, LE was
calculated only for compounds showing a K value. These values are higher than the mean value
i
(
0.45) calculated for oral drugs 43
A series of physicochemical properties involved in the CNS penetration were also calculated,
such as molecular weight (MW), polar surface area (PSA), basicity on the aliphatic (pKa1) and
aromatic (pKa2) nitrogen atoms, and lipophilicity (clogP). All derivatives have a MW<270 Da and
2
44
PSA< 40 Å . ClogP is in the range 1.7-3.2; however, according to Ghose only compounds 2a, 2b,
4a, 5a, 5b, 6a and 6b have lipophylicity values predictive of a good blood-brain barrier penetration
(
suggested range: 2.1-4-4). The logBB evaluation, as calculated by Clark45 combining the two
variables PSA and clogP, predicts that primary amines (logBB< 0) may have a medium distribution
to the brain, while compounds as 6a/6b with logBB >0.3 should readily cross the blood-brain
barrier.46
Table 3. Calculated physicochemical properties of compounds 1-6
1
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MW [Da] clogP PSA [Å2] logBB pKa1/pKa2
LE α4β2* LE α7*
[Kcal/mol] [kcal/mol]
N
1a 0.85
1b 0.66
2a 0.81
0.72
188.13
188.13
222.09
222.09
202.14
202.14
236.10
216.16
216.16
250.12
250.12
1.703 38.91
1.703 38.91
2.500 38.91
2.500 38.91
1.849 24.92
1.849 24.92
2.646 24.92
2.385 16.13
2.385 16.13
3.182 16.13
3.182 16.13
-0.178 10.03/3.74
-0.178 10.03/3.74
-0.056 10.03/-0.30
-0.056 10.03/-0.30
0.051 10.32/3.54
0.051 10.32/3.54
0.172 10.29/-0.50
0.262 9.81/4.17
0.262 9.81/4.17
0.383 9.77/0.13
0.383 9.77/0.13
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.73
2
3
3
4
5
5
6
6
b
a
b
a
a
b
a
b
0.69
0.73
0.61
0.73
0.62
0.53
0.59
0.53
0.60
0.59
-
0.59
-
-
-
-
LE: Ligand binding efficacy as reported by Hopkins].42 LE= ΔG/NHEA = -RTlnKi/NHEA= 1.372
[-log
*
Ki(mol)]/NHEA where NHEA is the number of non-hydrogen atoms. MW molecular weight and clogP were calculated
from Chembiodraw Ultra 14.0. PSA Polar Surface Area and pKa1/pKa2 were calculated at www.chemicalize.org.
45
logBB was calculated from Clark’s equation: logBB= -0.0148 PSA+0.152 clogP+ 0.139.
Functional studies on α4β2 receptor
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2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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5
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5
5
5
5
5
6
0
1
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6
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8
9
0
Figure 3. A) Representative nicotine (Nic)-evoked current traces mediated by human α4β2 nAChR in the
presence of 50ꢀµM of 1a, 2a, 3a or 6a. B) Bar graph for 1a, 2a, 3a and 6a (50 μM) inhibition of nicotine (20
μM)-evoked currents mediated by human α4β2 nAChR. Data are presented as mean ± SEM, n = 4 – 5. One-
way ANOVA with Tukey’s HSD test, black asterisks denote significant difference (p < 0.05) between
normalized nicotine-evoked current in the presence of 1a, 2a, 3a or 6a and normalized nicotine-evoked
current in the absence of compounds, p = 0.00000146
In order to measure the functional properties of the new ligands, some molecules (1a, 2a, 3a and
6
a) were chosen for further tests. Compound 2a was selected because in the binding tests it showed
the highest affinity on both subtypes, while 1a, 3a and 6a were chosen for their selectivity ratios
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(
Table 1). Due to low availability, no exo isomer was selected. These compounds were tested on
human α4β2 receptors expressed in Xenopus laevis oocytes. Two-electrode voltage clamp was used
to determine their mode of action. No direct activation of human α4β2 was detected upon
application of the compounds (data not shown). Application of 20 μM nicotine after 5 min
incubation with the tested compounds resulted in a decrease of agonist-evoked current, revealing
their antagonistic properties (Fig. 3). The most effective compounds were primary amines 1a and
2a, in accord with binding data (Table 1): a 50 μM concentration of these compounds was able to
block 91% and 81%, respectively, of nicotine-evoked currents. Compounds 3a and 6a demonstrated
a lower inhibitory activity on α4β2 nAChR: they were able to block 53% and 69% of nicotine-
evoked currents, respectively.
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Functional studies on α7 receptors
Figure 4 Functional activity of compounds 1a, 2a, 3a and 6a on α7 nAChR. A) Calcium rise of 1a
(open red squares), 2a (open black circles), 3a (open green triangles) or 6a (open blue triangles) on
human α7 nAChR expressed in Neuro2 cells. Peak amplitudes of compound-evoked currents were
normalized to current produced by acetylcholine (100 μM). The cells were preincubated with 10
μM PNU120596, a positive allosteric modulator of α7 nAChR, for 20 minutes before agonist
application. B) Agonist activity of 1a (open red squares), 2a (open black circles), 3a (open green
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triangles), and 6a (open blue triangles), (0.025, 0.25, 2.5, 25, 250 μM) on rat α7 nAChR expressed
in Xenopus oocytes. Peak amplitudes of compound-evoked currents were normalized to
acetylcholine (100 μM)-evoked peak current amplitude (grey dash line). Data are presented as mean
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±
SEM, n = 3. EC values are reported in Table 1.
50
The activity of compounds 1a, 2a, 3a and 6a was then examined on the human α7 nAChR
heterologously expressed in the neuroblastoma Neuro2a cell line, where the agonist-induced
2+
2+
increase in intracellular Ca concentration ([Ca ] ) is registered in the presence of PNU120596 (10
i
μM), a positive allosteric modulator (PAM).47-50 Nicotine was taken as positive control (Fig. S4,
Supporting Information). As shown in Fig. 4A, the tested compounds behaved as agonists: receptor
2+
activation, amplified by PAM co-application, produced a [Ca ] rise, with maximal activity similar
i
to that elicited by 100 μM ACh. Under these conditions the potency of the tested compounds was in
the nanomolar (1a and 2a) and micromolar (3a and 6a) range (Table 1).
The agonistic properties of the compounds were further confirmed in electrophysiological assay
on rat α7 receptors expressed in Xenopus laevis oocytes (Fig. 4B). In this test, performed in the
absence of PNU120596, compounds 1a and 2a were full agonists, showing potency in the
micromolar range (Table 1) and high efficacy: as a matter of fact, 1a and 2a were able to elicit a
current three-times higher than that induced by 100 μM ACh (Fig. 4B). Compounds 3a and 6a were
confirmed to be less potent also under these conditions.
Functional studies on α3β2 receptor
The agonist properties of the compounds were further tested in Xenopus oocytes, expressing
human 32 nAChR (Fig. 5). In two-electrode voltage clamp experiments, all compounds were
able to activate the receptor, although with different efficacy. Compound 2a was the most potent
(
EC 0.43 μM) and behaved as full agonist, with maximal activity about 160% with respect to
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51
nicotine at a rather high concentration, 50 μM. On the contrary, 1a was a partial agonist showing
maximal activity about 70% with respect to nicotine, while 3a and 6a activated the 32 nAChR
only marginally.
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Figure 5. Agonist activity of 1a (open red squares), 2a (open black circles), 3a (open green triangles), and
6
a (open blue triangles) on human 32 nAChR expressed in Xenopus oocytes. Peak amplitudes of
compound-evoked currents were normalized to nicotine (50 μM)-evoked peak current amplitude (grey dash
line). Data are presented as mean of three different oocytes ± SEM. EC values are reported in Table 1.
50
Functional studies on nicotinic receptors in SH-SY5Y cells
The pronounced agonistic activity of compounds 1a and 2a on heterologously expressed 7
and 32 nAChR encouraged us to check their ability to activate nicotinic receptors in human
neuroblastoma SH-SY5Y cell line. This cell line endogenously expresses the 3, 5, 7, 2 and 4
subunits; according to literature data, homopentameric α7 and heteropentameric α3β2, α3α5β2,
5
1
α3β2β4, α3α5β2β4, α3β4 and α3α5β4 can be present. In calcium imaging experiments compounds
2
+
1a and 2a behaved as full agonists (Fig. 6), being able to produce a [Ca ] rise, with EC
i
50
1
.92±0.66 μM and 0.22±0.04 μM, respectively, while nicotine EC50 was several times higher
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(22.47±3.06 μM). The chloro derivative 2a was about 8 times more potent than the unsubstituted
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analogue 1a. No increase of [Ca ] was observed after application of 3a and 6a.
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Figure 6. Agonist activity of 1a (open red squares), 2a (open black circles), and nicotine (open
yellow triangles) on nicotinic receptors endogenously expressed in SH-SY5Y cells. Peak
amplitudes of compound-evoked responses were normalized to response amplitude produced by
nicotine (100 μM). Data are presented as mean of three independent experiments ± SEM. Each
experiment includes 5-6 fluorescence measurements of one cell population (>10000 cells).
Discussion
In this work we report a new series of rigid nicotinic receptor ligands, in which the two
pharmacophoric groups, the pyridyl ring and the basic amine, are separated by a bulky bicyclic
spacer. The compounds represent rigid analogues of nicotine, the most potent being primary
amines. It can be noticed that some primary amines have been described as high affinity α4β2
ligands;52-56 only for few of them the interaction with the α7 subtype has been measured and found
negligible.53, 56 The endo primary amines reported here thus represent new chemotypes for the α7
and α3* subtypes.
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The tested compounds have a mixed pharmacological profile, being antagonists at the α4β2
receptor (Fig. 3) and agonists on human and rat α7 receptors. Agonist activity towards the α7
subtype was detected in calcium-imaging experiments on Neuro2a cell line (Fig. 4) in the presence
of the positive allosteric modulator PNU 120596, and confirmed also in two-electrode voltage
clamp studies on the rat α7 receptors expressed in Xenopus laevis oocytes. It must be highlighted
that the activity on this subtype was also revealed in competition binding experiments on rat brain,
as well as by calcium imaging on the human α7 receptor in the presence of the positive allosteric
modulator PNU 120596. This is important, because nicotinic receptors of the same subtype may
greatly differ depending on the receptor environment; for example, the natural product 6-
bromohypaphorin behaved as an agonist in the presence of PNU 120596 with the α7 receptor
heterologously expressed in the Neuro2a cell line, but did not reveal agonistic properties on the
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chicken α7/GlyR chimera expressed in Xenopus oocytes. Another factor may be the receptor
species specificity: for example, α-conotoxin RgIA was considered as potential analgesic but it was
found that its affinity for the human α9α10 receptor is about 100-lower than for the rat receptor.58
Of special interest is the activity of compounds 1a and 2a against α3β2 nAChR detected in
electrophysiology experiments (Fig. 5). The exact role of this subtype in the CNS is not completely
understood: in rodents this subtype has been found located in specific areas of CNS (habenula-
interpeduncular way, cerebellum, lateral geniculate nucleus and superior colliculus).15-16
Compounds which either potentiate or inhibit their activity are of undoubted value.
Since for a number of our compounds we detected the activities against three subtypes of
nAChRs, it was of interest to check their effects on the different cell lines either of normal or
malignant cells which contain the respective nAChRs in their “native “ environment and may be a
better approximation for assessing the suitability of these compounds as potential drugs or at least
hints to drugs. We took the human neuroblastoma SH-SY5Y cell line which is known to express
+
2
several nAChR subunits including α7, α3, β2. Fig. 6 shows that the increase in Ca concentration
can be induced by nicotine and, no less efficiently, by compounds 1a and 2a. Since without adding
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positive allosteric modulator PNU 120596 the α7 nAChR in this line cannot be activated,47, 59 the
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increase in Ca concentration can be ascribed to the action on the α3β2 receptor, especially in view
of Fig 5, demonstrating ion currents due to activation of this heterologously expressed receptor
subtype; however, the involvement of other α3* subtypes cannot be ruled out.
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Conclusions
In this work we reported a series of endo/exo 3-pyridyl-bicyclo[2.2.1]heptan-2-amines, designed
starting from the α4β2-selective ligand PHT. Their affinity was measured by means of binding
experiments on the α4β2* and α7* receptors of rat brain. The new compounds displayed nanomolar
affinity for the α4β2* subtype; on the α7* receptor the affinity was lower, but some compounds
(1a, 2a, 4a) were active in the low-medium nanomolar range. On both receptor subtypes the order
of potency was primary > secondary > tertiary amines, the endo series being more active than the
exo one.
In electrophysiological studies, several compounds (1a, 2a, 3a and 6a) displayed antagonistic
properties on hα4β2 receptors expressed in Xenopus laevis oocytes, whereas calcium imaging
experiments revealed agonist properties on the human and rat α7 subtypes. Interestingly,
electrophysiological experiments for several compounds showed an agonistic activity towards α3β2
+2
nAChR which agrees with their Ca increasing concentration revealed in the neuroblastoma SH-
SY5Y cells. Thus, although none of the novel synthesized primary amines possesses a strict
selectivity towards one distinct nAChR subtype, still they represent a novel chemotype for several
subtypes of neuronal nAChRs which also may be of value. Work is underway to probe in more
detail the structure-activity relationships of this class of compounds, including enantioselectivity, in
order to improve their potency and selectivity toward the α3* receptor.
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Experimental Section
Chemistry
Melting points were determined on a Büchi apparatus and are uncorrected. NMR spectra were
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recorded on a Brucker Avance 400 spectrometer (400 MHz for H NMR, 100 MHz for C). H and
1
3
1
13
C NMR spectra were measured at room temperature (25°C) in an appropriate solvent. H and C
chemical shifts are expressed in ppm () referenced to TMS. Spectral data are reported using the
following abbreviations: s = singlet, bs= broad singlet, d = doublet, dd = doublet of doublets, t =
triplet, app t= apparent triplet, m = multiplet, and coupling constants are reported in Hz, followed
by integration. Chromatographic separations were performed on a silica gel column by gravity
chromatography (Kieselgel 40, 0.063-0.2000 mm; Merck) or flash chromatography (Kieselgel 40,
0
.040-0.063 mm; Merck). Analytical TLC was performed on silica gel (200-300 mesh) GF/UV 254
plates, and the chromatograms were visualized under UV light at 254 nm. Yields are given after
purification, unless otherwise stated. The purity of the final compounds was determined by Agilent
1
200 liquid chromatography system composed by autosampler, binary pumps, column oven and
diode-array detector (LC-DAD) operating in UV range (210-400 nm). The analysis were carried out
using a Phenomenex Luna PFP column 100 mm length, 2 mm internal diameter and 3 m of
particle size. The analyte separation were ensured employing as mobile phase 10 mM ammonium
acetate solution (phase A) and methanol (phase B) in gradient elution. The time program elution
was as follows: initial 5% phase B for 1 min, then increase to 95% phase B in 18 min and kept for 6
-1
min. The analysis performed at constant flow of 0.35 mL min , temperature of 40°C and injecting
-1
1
0 L of a 10 g mL solution of each analyte. The obtained results displayed that all the studied
compound show a purity equal or major than 95%. The chromatographic profiles of LC-DAD
analysis and corresponding UV spectra were reported in Supporting Information (Figures S6-S8,
Supporting Information). High resolution mass spectrometry (HR-MS) analysis were performed
with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with an electrospray ionization
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source (ESI). The analysis were carried out introducing, via syringe pump at 10 L min , the
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sample solution (1.0 g mL in mQ water:acetonitrile 50:50), in positive ion mode. These
experimental conditions allow the monitoring of protonated molecules of the studied compounds
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(
[M+H] species), that they were measured with a proper dwell time to achieve 60,000 units of
resolution at Full Width at Half Maximum (FWHM). Elemental composition of compounds were
calculated on the basis of their measured accurate masses, accepting only results with an attribution
error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value, in order to
consider only the protonated species.51 When reactions were performed under anhydrous
conditions, the mixtures were maintained under nitrogen and the solvents were purified and dried
by standard methods. Compounds were named following IUPAC rules as applied by Reaxys
(
version 2.19790.2) software. For biological tests, amines were transformed into the corresponding
hydrochlorides, which were obtained as white solid, after crystallization from abs.
ethanol/anhydrous diethyl ether.
Procedure A: synthesis of nitrovinyl pyridine (9, 10)
Step 1 (7, 8). To a stirring solution of the proper aldehyde (1 equiv) in anhydrous THF (5 mL)
under nitrogen, tert-butyl alcohol (2.8 equiv) was added at rt. After cooling at 0°C, potassium tert-
butoxide (0.05 equiv) was added. The mixture was stirred at rt under nitrogen for 24 h; then it was
quenched with a saturated water solution of NaCl and extracted with CH Cl . The organic layer was
2
2
dried over Na SO and concentrated under reduced pressure to give alcohols 7-8, which were used
2
4
as such for the following step.
Step 2 (9, 10). To a solution of the alcohol (1 equiv) in anhydrous CH Cl (2,5 mL), cooled at -
2
2
10 °C and kept under nitrogen, trifluoroacetic anhydride (1 equiv) and triethylamine (2 equiv) were
added. The mixture was stirred at -10 °C for 30 min; then it was quenched with NaHCO (saturated
3
solution in water) and extracted with CH Cl . The organic layer was dried over Na SO and
2
2
2
4
concentrated under reduced pressure. The crude product was purified by column chromatography
using CH Cl /CH OH/NH 95/6/0.8 as eluting system, to give the desired nitrovinyl pyridine.
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Procedure B: Diels-Alder reaction, formation of bicyclic nucleus (11a, 11b, 12a, 12b)
To a stirred solution of (E)-3-(2-nitrovinyl)pyridine or (E)-2-chloro-5-(2-nitrovinyl)pyridine (9-
10, 1 equiv), in anhydrous CH Cl , cyclopenta-1,3-diene (1 equiv), obtained after thermal
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decomposition and distillation of commercial dicyclopentadiene dymer, was added. After 72 hours
stirring at rt, the solvent and the unreacted dymer were removed under reduced pressure to give the
desired compound as a mixture of endo/exo isomers.
Procedure C: hydrogenation of double bond (13a, 13b, 14a, 14b)
To a stirred solution of the suitable 3-nitrobicyclo[2.2.1]heptene derivative, in absolute EtOH (20
mL), 1.3 g Pd/C (10%) were added and the mixture was hydrogenated at 27 psi for 1 h. Filtration
and removal of the solvent gave a residue which was purified by chromatography or used as such
for the next step.
Procedure D: reduction of nitro group (1a, 1b, 2a, 2b)
To a stirring solution of nitro derivative (1 equiv) in abs EtOH (7 mL), SnCl 2H O (7 equiv) was
2
2
added and the mixture was heated under reflux at 80 °C for 3h. After cooling, the solution was
made alkaline with K CO (saturated solution in water), filtered over celite and extracted with
2
3
CH Cl The organic layer was dried over Na SO and concentrated under reduced pressure to give
2
2.
2
4
a residue that was purified by flash chromatography.
Procedure E: Amino Monomethylation (3a, 3b, 4a)
To a stirred solution of the suitable primary amine (1 equiv) in anhydrous CH Cl (5mL) cooled at
2
2
0°C, anhydrous triethylamine (1,1 equiv) and ethyl chloroformate (1,1 equiv) were added. The
mixture was stirred at rt for 24 h under nitrogen, then it was quenched with a saturated solution of
Na CO and extracted twice with CH Cl The organic layers were dried over Na SO and
2
3
2
2.
2
4
concentrated under reduced pressure to give a residue, which was used as such for the next step.
The crude mixture was cooled at 0°C and, under nitrogen, treated with anhydrous THF (5 mL) and
LiAlH (2 equiv). After heating under reflux for 4h, and stirring for 12 h at rt the reaction mixture
4
was quenched with H O, and NaOH (10% in H O) was added; the lithium salts were removed by
2
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filtration and the solution was extracted twice with CH Cl The organic layers were dried over
2
2.
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1
1
1
1
1
1
1
1
1
1
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5
5
5
5
5
6
Na SO and concentrated under reduced pressure. The crude product was purified by column
2
4
chromatography with the appropriate eluent.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
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6
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9
0
Procedure F: transformation of primary amines into N,N-dimethyl derivatives (5a, 5b, 6a, 6b)
To a stirring solution of the suitable primary amine (1 equiv) in EtOH 96% (4 mL), HCOOH (17
equiv) and CH O at 40% (5 equiv) were added and the solution was refluxed for 4h at 80°C. Then,
2
the reaction mixture was alkalinized with a saturated solution of NaHCO and was extracted with
3
CH Cl The organic layers were dried over Na SO and concentrated under reduced pressure to
2
2.
2
4
give the desired derivative, which usually did not require further purification.
Procedure G: general procedure for the synthesis of hydrochlorides
To obtain the hydrochloride salt, an excess of acetyl chloride (2 eq for each basic nitrogen atom
in the molecule) was added to anhydrous methanol (2-3 mL), then the amine (1 eq) was dissolved in
this solution. After stirring for 15 min, the solvent was removed under vacuum and the solid residue
was dried under vacuum and recrystallized from absolute ethanol/anhydrous diethyl ether.
endo 3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amine (1a): prepared from 13a (474.5 mg, 2.17
mmol) according to procedure D; eluent: CH Cl /CH OH/NH 90/10/1. Oil (315.0 mg, 77% yield).
2
2
3
3
1
H NMR (400 MHz, CDCl ) δ 1.24-1.43 (m, 3H, H5’ax+ H6’ax + H ); 1.44-1.51 (m, 2H, H5’eq+
3
7’
H6’eq); 1.53-1.65 (m, 1H, H ); 1.85 (d, J=5.2 Hz, 1H, H ); 2.02 (s, 1H, H ); 2.18 (d, J=3.2 Hz, 1H,
7’
2’
4’
H ); 3.00 (app t, J=8.4 Hz, J=4.4 Hz, 1H, H ); 7.00 (dd, J=8.0 Hz, J=5.0 Hz, 1H, H Ar); 7.38 (d,
1’
3’
5
1
3
J=8.0 Hz, 1H, H Ar); 8.20 (d, J=4.4 Hz, 1H, H Ar); 8.32 (d, J= 1.6 Hz, 1H, H Ar). C NMR (100
4
6
2
MHz, CDCl ) δ 19.73 (C ’); 30.97 (C ’); 36.88 (C ’); 43.30 (C ’); 43.31 (C ’); 55.21 (C ’); 62.61
3
7
5
6
1
4
2
(
C ’); 123.19 (C Ar); 133.81 (C Ar); 141.12 (C Ar); 146.99 (C Ar); 148.44 (C Ar). ESI-HRMS
3 5 4 3 6 2
+
(m/z) calculated for [M+H] ion species C H N = 189.1386, found 189.1383.
1
2
17
2
1
a.2HCl (prepared according to Procedure G): mp 145-146°C.
exo 3-(Pyridin-3-yl)bicyclo[2.2.1]heptan-2-amine (1b): prepared from 13b (210.0 mg, 0.96
mmol) according to procedure D; eluent CH Cl /CH OH/NH 90/10/1. Oil (54.3 mg, 30% yield).
2
2
3
3
2
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4
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5
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6
1
H NMR (400 MHz, CDCl ) δ 1.13-1.18 (m, 2H, H5’ax+ H ); 1.23-1.27 (m, 1H, H ); 1.39 (d,
3
7’
7’
J=10.0 Hz, 1H, H6’ax); 1.56-1.61 (m, 1H, H5’eq); 1.87 (d, J=9.6 Hz, 1H, H6’eq); 2.13 (d, J=4.4 Hz,
1H, H ); 2.44 (bs, 1H, H ); 2.76 (bs, 1H, H ); 3.06 (d, J= 3.6 Hz, 1H, H ); 3.30 (bs, 2H, NH); 7.20
2
’
4’
1’
3’
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(dd, J=8.0 Hz, J=4.8 Hz, 1H, H Ar); 7.50 (d, J=7.6 Hz, 1H, H Ar); 8.41 (d, J=4.8 Hz, 1H, H Ar);
5 4 6
1
3
8
4
.46 (s, 1H, H Ar). C NMR (100 MHz, CDCl ) δ 21.6 (C ’); 27.07 (C ’); 36.69 (C ’); 42.11 (C ’);
2 3 7 6 5 4
5.63 (C ’); 55.44 (C ’); 59.13 (C ’); 123.07 (C Ar); 135.41 (C Ar); 136.66 (C Ar); 147.31 (C Ar);
2
1
3
5
4
3
6
+
149.68 (C Ar). ESI-HRMS (m/z) calculated for [M+H] ion species C H N = 189.1386, found
2
12 17
2
1
89.1385.
b.2HCl (prepared according to Procedure G): mp >260°C.
1
endo 3-(6-chloropyridin-3-yl)bicyclo[2.2.1]heptan-2-amine (2a) and exo-3-(6-chloropyridin-3-
yl)bicyclo[2.2.1]heptan-2-amine (2b): prepared from 14a,b (822.6 mg, 3.26 mmol) according to
procedure D; after separation by column chromatography eluent: CH Cl /CH OH/NH 97/3/0.3.
2
2
3
3
Oils.
endo 2a (194.3 mg, 27.0% yield): H NMR (400 MHz, CDCl )  1.35-1.45 (m, 2H, CH ); 1.48-1.53
1
3
2
(m, 1H, CH); 1.63-1.71 (m, 4H, NH + 2CH); 1.76-1.83 (m, 1H, CH); 2.03 (app t, J=4.4 Hz, J=1.2
2
Hz, 1H, CH); 2.23 (s,1H, CH); 2.29 (d, J=3.6 Hz, 1H, CH); 3.16 (app t, J=4.4 Hz, J=4.0 Hz, 1H,
CH); 7.22 (d, J=8.0 Hz, 1H, H Ar); 7.54 (dd, J=8.0 Hz, J=2.4 Hz, 1H, H Ar); 8.26 (d, J=2.4 Hz,
4
5
1
3
1
H, H Ar). C NMR (100 MHz, CDCl )  19.73 (CH ); 31.26 (CH ); 36.87 (CH ); 43.33 (CH);
2
3
2
2
2
43.56 (CH); 54.63 (CH); 62.96 (CH); 123.89 (CH); 137.06 (CH); 140.19 (C); 148.20 (CH); 148.85
+
(C). ESI-HRMS (m/z) calculated for [M+H] ion species C H ClN = 223.0997, found 223.0995.
1
2
16
2
2
a.2HCl (prepared according to Procedure G): mp >260°C.
1
exo 2b (17.3 mg, 2.4% yield): H NMR (400 MHz, CDCl ) : 1.11-1.18 (m, 2H, CH ); 1.27-1.31
3
2
(m, 1H, CH); 1.40 (d, J=10.0 Hz, 1H, CH); 1.55-1.61 (m, 1H, CH); 1.62 (s, 2H, NH₂); 1.82 (d,
J=9.6 Hz, 1H, CH); 2.07 (s, 1H, CH); 2.44 (s, 1H, CH); 2.66 (s, 1H, CH); 2.96 (d, J=4.0 Hz, 1H,
1
3
CH); 7.24 (d, J=8.4 Hz, 1H, H Ar); 7.49 (d, J=8.4 Hz, 1H, H Ar); 8.24 (s, 1H, H Ar). C NMR
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5
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(
100 MHz, CDCl )  21.57 (CH ); 27.06 (CH ); 36.60 (CH ); 41.91 (CH); 45.89 (CH); 54.93 (CH);
3
2
2
2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
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2
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3
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3
3
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3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
9.71 (CH); 123.66 (CH); 136.03 (C); 137.98 (CH); 148.86 (C); 149.38 (CH). ESI-HRMS (m/z)
+
calculated for [M+H] ion species C H ClN = 223.0997, found 223.0998.
1
2
16
2
0
1
2
3
4
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3
4
5
6
7
8
9
0
2
b.2HCl (prepared according to Procedure G): mp >260°C.
endo N-methyl-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amine (3a): prepared from 1a (150.0 mg,
0.8 mmol) according to procedure E; eluent: CH Cl /CH OH/NH 95/6/0.8. Pale-yellow oil (80.8
2
2
3
3
1
mg. 50% yield). H NMR (400 MHz, CDCl ) δ 1.32-1.39 (m, 3H, H
+ H6’ax + H ); 1.61-1.69
7’
3
5’ax
(m, 3H, H5’eq + H6’eq + H ); 1.88 (bs, 1H, NH); 2.08 (dd, J= 5.3 Hz, J=1.5 Hz, 1H, H ); 2.21 (d,
7
’
2
’
J=3.8 Hz, 1H, H ); 2.27 (s, 3H, CH N); 2.41 (s, 1H, H ); 2.98-3.00 (m, 1H, H ); 7.16 (dd, J=7.9
1’
3
2’
3’
Hz, J=4.8 Hz, 1H, H Ar); 7.54 (d, J=7.9 Hz, 1H, H Ar); 8.36 (dd, J= 4.8 Hz, J=1.5 Hz, 1H, H Ar);
5
4
6
13
8
.48 (d, J=2.2 Hz, 1H, H Ar). C NMR (100 MHz, CDCl ) δ 20.01 (CH ); 31.26 (CH ); 35.28
2 3 2 2
(CH ); 36.23 (CH ); 39.37 (C ’); 43.52 (C ’); 53.73 (C ’); 70.20 (C ’); 123.29 (C Ar); 134.24
3 2 4 1 2 3 5
+
(C Ar); 141.45 (C Ar); 147.23 (C Ar); 148.90 (C Ar). ESI-HRMS (m/z) calculated for [M+H] ion
4
3
6
2
species C H N = 203.1543, found 203.1547.
1
3
19
2
3
a.2HCl (prepared according to Procedure G): low melting solid.
exo N-methyl-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amine (3b): prepared from 1b (54.0 mg,
0
.28 mmol) according to procedure E; eluent: CH Cl /CH OH/NH 90/10/1. Oil (11.6 mg, 20%
2
2
3
3
1
yield). H NMR (400 MHz, CDCl ) δ 1.12-1.31 (m, 3H, H
+ 2H ); 1.42-1.45 (m, 1H, CHH
3
6’ax
7’
Ax); 1.65-1.69 (m, 1H, CHH-Eq); 2.01 (d, J=8.8 Hz, 1H, CHH-Eq); 2.40 (s, 3H, CH N); 2.44 (s,
3
1H, H ); 2.48 (d, J=4.4 Hz, 1H, H ); 2.91 (d, J=4.4 Hz, 1H, H ); 3.00 (s, 1H, H ); 7.21-7.24 (m,
4
’
1’
3’
2’
13
1
H, H Ar); 7.50 (d, J=8.0 Hz, 1H, H Ar); 8.45 (d, J=4.8 Hz, 1H, H Ar); 8.47 (s, 1H, H Ar). C
5 4 6 2
NMR (100 MHz, CDCl ) δ 21.90 (C ’); 27.15 (CH ); 33.11 (CH N); 37.42 (CH ); 41.21 (CH);
3
7
2
3
2
42.85 (CH); 52.36 (C ’); 66.76 (C ’); 123.23 (C Ar); 135.31 (C Ar); 136.23 (C Ar); 147.74 (C Ar);
2
3
5
4
3
6
+
1
2
3
49.55 (C Ar). ESI-HRMS (m/z) calculated for [M+H] ion species C H N = 203.1543, found
2 13 19 2
03.1544.
b.2HCl (prepared according to Procedure G): low melting solid.
2
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