New spirocyclic Δ2-isoxazoline derivatives related to selective agonists of α7 neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.ejmech.2011.09.028

A set of structural analogues of spirocyclic quinuclidinyl- Δ²-isoxazolines, characterized as potent and selective α7 nicotinic agonists, was prepared and assayed for binding affinity at α7 and α4β2 neuronal nicotinic acetylcholine receptors (nAChRs). The investigated derivatives (3a-3c, 4a-4c, 5a-5c, 6a-6c, and 7a-7c), synthesized via the 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles, showed an overall reduced affinity at the α7 subtype when compared with their model compounds. Solely Δ²-isoxazolines 3a, 3b, and 6c maintained a binding affinity in the nanomolar range at the α7 nAChRs (K_i = 230, 420 and 700 nM, respectively). The quaternary ammonium salt 6c retained also a noteworthy α7 vs. α4β2 subtype selectivity, whereas 3a and 3b showed a sharp reduction in selectivity compared with 1a and 1b, their quinuclidinyl higher homologues.

Full text of DOI:10.1016/j.ejmech.2011.09.028

European Journal of Medicinal Chemistry 46 (2011) 5790e5799
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New spirocyclic
D
2-isoxazoline derivatives related to selective agonists of
a7
neuronal nicotinic acetylcholine receptors
,
a
a
a
a
Clelia Dallanocea , Fabio Frigerio , Giovanni Grazioso , Carlo Matera , Giacomo Luca Visconti ,
*
Marco De Amicia, Luca Puccib, Francesco Pistilloc, Sergio Fucilec d, Cecilia Gottib, Francesco Clementib,
,
Carlo De Michelia
a Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b CNR, Istituto di Neuroscienze, Farmacologia Cellulare e Molecolare e Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano,
Via Vanvitelli 32, 20129 Milano, Italy
c I.R.C.C.S. Neuromed, Istituto Neurologico Mediterraneo, Via Atinese 18, 86077 Pozzilli (Isernia), Italy
d Dipartimento di Fisiologia e Farmacologia, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of structural analogues of spirocyclic quinuclidinyl-
D
2-isoxazolines, characterized as potent and
7 and 2 neuronal
Received 28 February 2011
Received in revised form
12 July 2011
Accepted 19 September 2011
Available online 24 September 2011
selective
a
7 nicotinic agonists, was prepared and assayed for binding affinity at
a
a4b
nicotinic acetylcholine receptors (nAChRs). The investigated derivatives (3ae3c, 4ae4c, 5ae5c, 6ae6c,
and 7ae7c), synthesized via the 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles,
showed an overall reduced affinity at the
Solely
2-isoxazolines 3a, 3b, and 6c maintained a binding affinity in the nanomolar range at the
nAChRs (Ki ¼ 230, 420 and 700 nM, respectively). The quaternary ammonium salt 6c retained also
a noteworthy 7 vs. 2 subtype selectivity, whereas 3a and 3b showed a sharp reduction in selectivity
compared with 1a and 1b, their quinuclidinyl higher homologues.
Ó 2011 Elsevier Masson SAS. All rights reserved.
a7 subtype when compared with their model compounds.
D
a7
Keywords:
a
a4b
Neuronal nicotinic acetylcholine receptors
a
7 Selective nicotinic ligands
Cycloaddition reaction
Binding affinity
Electrophysiological assays
Molecular modeling
1. Introduction
prominent nAChRs in the CNS, especially in brain areas (cortex and
hippocampus) involved in the elaboration of cognitive processes
[3]. These two receptor subtypes also modulate the release of
Nicotinic acetylcholine receptors (nAChRs) are broadly charac-
terized transmembrane proteins which mediate the physiological
responses to the neurotransmitter acetylcholine. These receptors,
which are expressed in muscle, nerve and sensory cells, exist as
neurotransmitters such as acetylcholine, glutamate,
g-amino-
butyric acid, norepinephrine, histamine and dopamine, which are
essential for a wide range of CNS functions [3,5]. Functionally,
pentameric ligand-gated ion channels composed of
non- 1- 4, ε, , and ) subunits [1,2]. Homomeric channels are
composed of 7 or 9 subunits and the 10 subunit only gives rise
to functional receptors when it is co-expressed with the 9 subunit.
Among the subunits which form homomeric nAChRs, only the
one is widely distributed in the mammalian brain. The 2- 6 and
2- 4 subunits generate an array of heteromeric channels found in
a
(
a
1-
a
10) and
a
7 channels are distinguished from
lower affinity for acetylcholine, high affinity for the antagonist
a-bungarotoxin, fast desensitization, and higher permeability to
a4b2 receptors due to their
a
(b
b
g
d
a
a
a
a
calcium [3,4,6]. In the last two decades, an improved knowledge of
the role exerted by the two cited nAChR subtypes in cognitive
processes, mood, nociception, and neuroprotection has engendered
the development of subtype-selective compounds with therapeutic
potential for different CNS-related pathologies, including Alz-
heimer’s and Parkinson’s diseases, attention deficit hyperactivity
disorder, schizophrenia, epilepsy, Tourette’s syndrome, anxiety,
depression, and nicotine addiction [4,7]. Moreover, homopenta-
a
7
a
a
b
b
the central nervous system (CNS), and much effort is currently
devoted to completing the knowledge of their structure, distribu-
tion, and physiological role [3,4]. The heteromeric
a4b2 and the
homomeric 7 subtypes have been characterized as the most
a
meric
most of the anti-inflammatory effect of the vagal cholinergic
pathway [8]. Consequently, selective agonists have been
a7 channels were found to exert a pivotal role in mediating
* Corresponding author. Tel.: þ39 02 50319327; fax: þ 39 02 50319326.
E-mail address: clelia.dallanoce@unimi.it (C. Dallanoce).
a
7
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.028

C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
5791
investigated in different experimental models of inflammation,
CO2Et
O
X
3 steps
ref. 13
thus providing additional insight into the functional biology of this
nAChR subtype [8]. Furthermore, an anti-neuropathic effect has
been recently demonstrated, in terms of pain behavior and nervous
a
N
N
N
CO2Et
BH3
tissue protection, following acute and repeated treatment with
selective agonists in a model of peripheral neuropathy [9].
a7
8
9
10: X = O
The structural requirements of ligands selectively binding to
a7 or
b
a4b2 nAChR subtypes have been the subject of a number of investi-
gations, whose most relevant results were collected in comprehensive
literature reports [4,7a]. Interestingly, in a recent study, novel molec-
11: X = CH2
N
N
O
ularscaffoldswereidentified,capabletogeneratehighselectivityat
a7
O
R
R
or 2 nAChR subtype with simple changes in the substitution
a4b
c
f
11
pattern [10]. These structural variations on a common molecular
skeleton should contribute to better define differences in the ligand
binding domains of the two investigated nAChRs, particularly in
receptor areas outside the conserved cation binding crevice [10].
In the last years, our research group has been involved in the
study of the structureeactivity relationships of a number of
heterocyclic derivatives designed as selective ligands for neuronal
nAChR subtypes [11]. In this framework, we patented a series of
N
N
BH3
12a: R = Br
13a: R = Br
13b: R = OCH3
13c: R =C6H5
e
11
d
12b: R = OCH3
3a
g
compounds targeting the
spirocyclic quinuclidinyl-
emerged as the most selective
a
D
7 subtype [12a], and, among them, the
2-isoxazolines 1ae1c, 2a and 2b (Fig. 1)
7 vs. 2 ligands [12b]. In this
3b
3c
a
a4b
13a
13b
13c
paper, we discuss the preparation and pharmacological character-
ization of a group of structural analogues of the above cited qui-
nuclidinyl derivatives, i.e. their lower homologues containing the 1-
azabicyclo[2.2.1]heptane nucleus as well as simplified analogues
carrying the piperidine or the N-methylpiperidine moiety. Herein
4a
4b
4c
h
Scheme 1. Reagents and conditions: (a) 1.0 M BH3-THF complex (1 equiv), THF, r.t.,
10 min; (b) CH3P(C6H5)3Br (1.5 equiv), tert-BuOK (1.4 equiv), THF, reflux 45 min, then
r.t., 1h; (c) Br2C ¼ NOH (1.1 equiv), NaHCO3, AcOEt, r.t., 48 h; (d) K2CO3, MeOH, reflux,
4 h; (e) PhCH ¼ NOH (2.5 equiv), 3.5% aq NaClO (2.5 equiv), CH2Cl2, r.t., 13 h; (f)
CF3CO2H (7 equiv), acetone, r.t., 10 h; (g) C4H4O4 (1.1 equiv), MeOH, r.t., 16 h; (h) CH3I
(10 equiv), Et2O, r.t., 1 h.
we report the synthesis and the binding affinity data at
a7, a4b2
and 4 nAChRs of spirocyclic derivatives 3aec, 4aec, 5aec, 6aec,
a3b
and 7aec (Fig. 1). We chose to investigate even the quaternary
ammonium salts 4aec and 6aec in view of a potential interest in
selective ligands targeting the peripheral a7 nAChR population.
Treatment of 9 with a 1.0
M solution of borane-THF complex provided
2. Chemistry
the N-boranyl ketone 10, which underwent a smooth Wittig-type
methylenation to afford alkene 11. The latter was used as the dipo-
larophile in the pericyclic reactions with bromonitrile oxide and
benzonitrile oxide. In both cases, the 1,3-dipole was generated in situ
from a stable precursor, i.e. dibromoformoxime [14a] or benzald-
oxime [14b].
All spirocylic derivatives were synthesized as racemates taking
advantage of the1,3-dipolarcycloadditionofnitrileoxides toproperly
protected dipolarophiles [11c,12]. As illustrated in Scheme 1, diester 8
was initially converted into 3-oxo-1-azabicyclo[2.2.1]heptane 9
following an experimental protocol described in the literature [13].
N
N
O
O
R
R
N
H
N
CH3
I
C4H3O4
1a: R = Br
1b: R = OCH3
1c: R = C6H5
2a: R = Br
2b: R = OCH3
N
N
N
N
N
O
O
O
O
O
R
R
R
R
R
N
H H
N
H
N
CH3
N
N
I
I
C4H3O4
C4H3O4
C4H3O4
H3C
H
H3C CH3
3a: R = Br
3b: R = OCH3
3c: R = C6H5
4a: R = Br
4b: R = OCH3
4c: R = C6H5
5a: R = Br
5b: R = OCH3
5c: R = C6H5
6a: R = Br
6b: R = OCH3
6c: R = C6H5
7a: R = Br
7b: R = OCH3
7c: R = C6H5
Fig. 1. Molecular structure of model and target compounds.

5792
C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
N
O
N
O
X
R
OCH3
b or c
d
16a
N
N
N
Boc
Boc
Boc
15: X = CH2
16a: R = Br
16b
16c
H5
: R = C6
a
14
: X = O
N
N
O
O
R
R
16a
16b
16c
f
e
N
H
N
CH3
17a: R = Br
17b
18a
: R = Br
18b: R = OCH3
: R = OCH3
18c
: R = C6H5
17c
: R = C6H5
g
h
g
6a 6b 6c
,
7a 7b 7c
,
5a 5b 5c
,
,
,
,
Scheme 2. Reagents and conditions: (a) CH3P(C6H5)3Br (1.5 equiv), tert-BuOK (1.4 equiv), THF, reflux 45 min, then r.t., 1 h; (b) Br2C ¼ NOH (1.1 equiv), NaHCO3, AcOEt, r.t., 48 h; (c)
PhCH ¼ NOH (2.5 equiv), 3.5% aq NaClO (2.5 equiv), CH2Cl2, r.t., 13 h; (d) K2CO3, MeOH, reflux, 4 h; (e) 4N HCl (1.6 equiv), dioxane, r.t., 1 h (for 17a) or CF3CO2H (7 equiv), CH2Cl2, r.t.,
10 h (for 17b and 17c); (f) 37% aq HCHO (5 equiv), NaBH3CN (2.5 equiv), CH3CN, r.t., 20 min; (g) C4H4O4 (1.1 equiv), MeOH, r.t., 16 h; (h) CH3I (10 equiv), Et2O, r.t., 1 h.
Worth noting, while the first cycloaddition gave the expected 3-
bromo-N-boranyl derivative 12a in 88% yield, the second one did
not produce the corresponding 3-phenyl analogue but directly the
N-deprotected derivative 13c in less than 10% yield. The 3-bromo
intermediate 12a was submitted to the nucleophilic displacement
by methanol to provide the corresponding 3-methoxy derivative
12b in high yield. The N-boranyl group of compounds 12a and 12b
was then removed by treatment with a dichloromethane solution
of trifluoroacetic acid to yield the desired tertiary bases 13a and
13b, respectively. Amines 13aec were then treated with fumaric
acid to afford the related crystalline fumarates 3ae3c or, alterna-
tively, were transformed into the corresponding iodomethylates
4ae4c by reaction with excess iodomethane (Scheme 1).
The N-Boc-3-methylenepiperidine 15, obtained from commer-
cially available N-Boc-3-piperidone 14 through a Wittig reaction,
was submitted to the previously described cycloaddition reactions,
as reported in Scheme 2. In this instance, both bromo- and phenyl-
D
2-isoxazolines 16a and 16c were isolated in very high yield.
Likewise, the transformation of the 3-bromo derivative 16a into the
corresponding 3-methoxy analogue 16b occurred smoothly.
Table 1
Affinity of derivatives 3ae7a, 3be7b, and 3ce7c for native
epibatidine. Affinity of derivatives 3ae3c, 5b, 6c, 7b, and 7c for
a
7 and
4 nAChRs heterologously expressed in HEK 293 cells.
a4b a
2 nAChR subtypes present in rat cortical membranes, labeled by [125I] -bungarotoxin and [3H]
a3b
Compound
a
7 [125I]
Kia
), n ¼ 4
a
eBgTx
a4
b
2 [3H]Epibatidine
Ki>a
), n ¼ 4
a
3
b
4 [3H]Epibatidine
Ki>a
), n ¼ 4
(m
M
(
m
M
(mM
1a 3 0.5 C4H4O4
1b 3 1.5 C4H4O4
1c 3 0.5 C4H4O4
2a
0.0135 (29)b
0.0142 (32)b
0.025 (35)b
0.109 (23)b
0.0914 (36)b
0.23 (30)
0.42 (35)
3.60 (31)
6.20 (46)
2.90 (38)
5.10 (48)
6.60 (53)
5.50 (42)
30 (44)
3.70 (48)
13 (48)
0.70 (39)
4.30 (34)
3.70 (41)
4.50 (37)
0.64 (17)b
7.90 (16)b
22 (21)b
32.5 (18)b
175.4 (17)b
5 (14)
16 (22)
53 (32)
22 (20)
20 (28)
38 (31)
44 (38)
> 100
2b
3a 3 0.75 C4H4O4
3b 3 0.5 C4H4O4
3c 3 0.5 C4H4O4
4a
4b
4c
5a 3 0.75 C4H4O4
5b 3 0.75 C4H4O4
5c 3 0.75 C4H4O4
6a
6b
6c
13 (17)
46 (21)
16 (16)
Nt
Nt
Nt
Nt
91 (18)
Nt
Nt
Nt
> 100
50 (33)
94 (32)
> 100
58 (41)
> 100
6.60 (16)
Nt
75 (16)
43 (15)
7a 3 0.5 C4H4O4
7b 3 0.5 C4H4O4
7c 3 0.75 C4H4O4
> 100
a
The Ki values were derived from three competition-binding experiments. The numbers in brackets refer to the % coefficients of variation.
Ref. [12b]. Nt: not tested.
b

C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
5793
Removal of the N-Boc protective group from 16ae16c was carried
out under standard conditions to give secondary amines 17ae17c in
high yield (Scheme 2). The free bases were converted into the
related fumarates 7ae7c or were submitted to reductive amination
with aqueous formaldehyde and sodium cyanoborohydride, to
provide the corresponding tertiary amines 18ae18c in 90e97%
yield. The desired final salts were then obtained by treating these
tertiary bases either with fumaric acid to give fumarates 5ae5c or
with methyl iodide to produce iodomethylates 6ae6c.
transfected with human
doseeresponse relationship indicated an EC50 value of
84.1 ꢁ 0.8 M, with a mean maximal amplitude which was 76 ꢁ 5%
of the mean current amplitude elicited by ACh 1 mM (n ¼ 8, Fig. 3b).
By contrast, derivatives 5b and 6c were able to elicit 7-mediated
inward currents only when applied at the concentration of 1 mM,
with a mean amplitude reaching 7 ꢁ 4% and 26 ꢁ 6% of that evoked
by ACh 1 mM, respectively (n ¼ 8 and n ¼ 12, Fig. 3b). These results
indicate that the 3-bromo-derivative 3a retains the agonistic
pharmacological profile of its higher homologue 1a [12b] whereas
the 3-OMe-isoxazoline 5b and the 3-Ph-isoxazoline 6c behave as
partial agonists with different levels of potency.
a
7 cDNA, clamped at ꢀ70 mV (Fig. 3a). The
m
a
3. Pharmacology
Target compounds 3ae7a, 3be7b, and 3ce7c were tested for
binding affinity at rat
a
a7 and a4b
2 nAChR subtypes, using [125I]
4. Molecular modeling studies
-bungarotoxin and [3H]epibatidine as radioligands, respectively,
according to a previously described experimental protocol [11c].
The Ki values, which were calculated from the competition curves
of four separate experiments by means of the LIGAND program [15],
are gathered in Table 1 and compared with those of reference
analogues 1ae1c, 2a, and 2b [12b]. We also evaluated the binding
affinity of some of the derivatives under study, i.e. 3ae3c, 5b, 6c, 7b
With the aim of rationalizing the pharmacological results of this
study, we performed a conformational analysis on the group of
methoxy derivatives, which allowed to ascertain that the congeners
1e4 assume only one rigid boat conformation. Conversely, their
analogues 5e7 exist as two chair conformers, one of which is
and 7c, for heterologously expressed a3b4 nAChRs (Table 1). This
subtype is predominant in sensory and autonomic ganglia and in
the adrenal gland but it is also expressed in specific brain regions,
where it seems to be involved in addiction to nicotine and other
drugs of abuse [16a]. A relevant affinity for this “ganglionic nAChR
subtype” [16b] could potentially cause relevant adverse effects in
compounds designed as selective agonists at the a7 subtype.
The overall pharmacological data on the novel derivatives
confirm that the quinuclidine moiety is a crucial structural
requirement for the ligand recognition by the
a7 nAChRs [17].
Indeed, derivatives 3ae3c, which carry the 1-azabicyclo[2.2.1]
heptane nucleus and are the closest analogues of template agonists
1ae1c, show a significantly lower affinity than the reference
compounds. As a matter of fact, the binding affinity of compounds
3ae3c at the a7 subtype is 17-, 30- and 144-fold lower than that of
the corresponding one carbon higher homologues 1ae1c. Such
a reduction can reasonably be ascribed to a shape modification of
the basic portion of the ligands. Nevertheless, among the novel
derivatives, the 3-Br (3a) and the 3-OCH3 (3b) compounds dis-
played the highest affinity at
0.42 M, respectively), thus paralleling the biological trend of
leading compounds 1a and 1b.
The
2-isoxazolines bearing either the piperidine (7ae7c) or
the 1-methylpiperidine nucleus (5ae5c) behaved as low affinity
nicotinic ligands, with Ki values in the range 3.70e30
Furthermore, they exhibited one order of magnitude lower affinity
at the 2 subtype (Ki in the range 44 - > 100 ) compared with
that determined at the 7 subtype. Even the permanently charged
a7 nAChRs (Ki values of 0.23 mM and
m
D
a
7
m
M.
a
4b
mM
a
methiodides 4ae4c, 6a, and 6b can be classified as low affinity and
unselective nicotinic ligands but the 3-phenyl-substituted deriva-
tive 6c, which was able to discriminate the two receptor subtypes
under study. This derivative displayed a moderate affinity for the
a
7 subtype (Ki ¼ 0.70
nAChRs.
The binding data obtained on the
tested compounds are low affinity ligands at this subtype, with Ki
values in the range 6.60 M (6c) and 91 M (5b). As a consequence,
the best 7 nAChR ligands in the set of analogues, i.e. 3a, 3b and 6c,
display a certain degree of subtype selectivity vs. both investigated
heteromeric -containing nAChRs.
mM) and lacked any affinity for the a4b2
a3b4 nAChRs showed that the
m
m
a
a/b
Fig. 2. Binding modes of a) (R)-1b and b) (R)-3b in the active site of the a7 nAChR
Three of the studied compounds, i.e. 3a, 5b and 6c, were assayed
in electrophysiological experiments to assess their functional
subtype, after energy minimization of the complexes. Receptor model residues are
depicted as stick model and carbon atoms are colored in white; surfaces and carbon
atoms are colored in cyan for (R)-1b and in green for (R)-3b. Some receptor’s residues
have been omitted for clarity.
profile at the
a7 nAChR subtype. Bromo-isoxazoline 3a evoked
inward whole-cell currents when applied on GH4C1 cells

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C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
aromatic box which characterizes the
[12b], and, in particular, the cation- interaction with the side chain
of Trp54 was totally missing (Fig. 2b). Moreover, a steric clash
between the
2-isoxazoline moiety of (R)-3b and the backbone of
a7 subtype binding cleft
p
D
Trp148, proved by its relaxation during the energy minimization of
the complex, further worsened the overall quality of the binding
mode of this ligand. These findings may help explaining the parallel
reduction of the affinity showed by 3b and 3a in comparison with
1b and 1a, respectively.
5. Conclusion
In summary, the quinuclidine nucleus in the series of spi-
rocyclic derivatives under study maximizes their molecular inter-
action with the
relevant binding affinity and functional discrimination toward the
2 subtype. A progressive decrease in the 7 binding affinity has
a7 subtype, thus providing ligands with both
a4
b
a
been detected on passing from quinuclidines to 1-azabicyclo[2.2.1]
heptanes, and to N-methylpiperidines or piperidines. We found
that the inability of these simplified analogues to retain the
a7
affinity/selectivity profile of the model structures has to be
attributed to a decrease of the complementary fit within the
binding cleft of the
a7 receptor protein and, for the N-methyl-
piperidine or piperidine derivatives, to the loss of suitable
conformational requirements. In terms of functional profile, elec-
trophysiological assays suggest a comparable
a7-mediated acti-
vation pattern for 3a and 1a, i.e. the homologues having
a dimethylene or a monomethylene bridge. The lack of this
structural fragment causes a meaningful reduction of the func-
Fig. 3. a) Typical traces evoked by ACh (1 mM) or compound 3a (1 mM) on GH4C1
tional response at the
a7 subtype, as it has been observed for
cells stably transfected with human
a
7 nAChR subunit. b) Dose-normalized response
7 nAChRs.
derivatives 5b and 6c.
relationships of derivatives 3a, 5b, and 6c at human
a
6. Experimental protocols
dominating, i.e. 99% populated in the case of 7a. Thus, spirocyclic
derivatives 1e4 may be viewed as 3-substituted piperidines forced
in a boat conformation by insertion of a bridge formed by one or
two methylene groups. We superimposed the eutomer (R)-1a [12b]
with the two conformers of (S)-7a. We chose enantiomer (S)-7a
since it shares with (R)-1a the same spatial arrangement of the
substituents around the stereogenic center. The basic moiety of the
favored conformer of (S)-7a (represented in white in Fig. 1, Sup-
porting Information) overlapped perfectly with that of the (R)-1a
conformer, while the two spirocyclic rings assumed different
spatial orientations. On the contrary, the isoxazoline rings of (R)-1a
and the lowest populated conformer of (S)-7a (represented in green
in Fig. 1, Supporting Information) could be superimposed whereas
their basic nitrogen atoms did not fit. The poor overall superim-
position of both conformers of (S)-7a with (R)-1a may account for
the low affinity showed by ligands 5aec and 7aec when compared
to the corresponding quinuclidine-containing model compounds.
Another result which deserves further analysis is the drop in
affinity observed on passing from the two agonists 1a and 1b to
their congeners 3a and 3b. These derivatives share the boat
conformation of the piperidine-like basic moiety and the agonist
functional profile, as we proved for the couple 1a and 3a. To
rationalize the biological results we used the molecular model of
6.1. Chemistry
1H NMR and 13C NMR spectra were recorded with a Varian
Mercury 300 (1H, 300.063; 13C, 75.451 MHz) spectrometer in
CDCl3 solutions (unless otherwise indicated) at 20 ꢂC. Chemical
shifts (d) are expressed in ppm and coupling constants (J) in Hz.
TLC analyses were performed on commercial silica gel 60 F254
aluminum sheets; spots were further evidenced by spraying with
a dilute alkaline potassium permanganate solution or, for tertiary
amines, with the Dragendorff reagent. Melting points were
determined on a model B 540 Büchi apparatus and are uncor-
rected. ESI mass spectra of the final salts were obtained on a Var-
ian 320 LC-MS/MS instrument. Data are reported as mass-to-
charge ratio (m/z) of the corresponding positively charged
molecular ions. Microanalyses (C, H, Br, I, N) agreed with the
theoretical value within ꢁ0.4%.
6.1.1. 1-Boranyl-1-azabicyclo[2.2.1]heptan-3-one 10
A 1.0 M solution of borane-THF complex (10 mL) was added
under nitrogen to a stirred solution of 1-azabicyclo[2.2.1]heptan-3-
one 9 [13] (1.11 g, 10.0 mmol) in dry THF (20 mL) at 0 ꢂC. After
stirring for 10 min at r.t., the reaction mixture was concentrated in
vacuo and the residue was purified by silica gel column chroma-
tography (cyclohexane/ethyl acetate 7:3) to yield the N-boranyl
ketone 10 (612 mg, 49% yield).
10: Viscous colorless oil. Rf ¼ 0.55 (cyclohexane/ethyl acetate
1:1). 1H NMR: 1.91e2.01 (m, 1H), 2.32e2.43 (m, 1H), 3.01e3.39
(m, 5H), 3.27 (dd,1H, J ¼ 3.9 and 17.3), 3.46 (dd,1H, J ¼ 2.8 and 17.3).
13C NMR: 29.56, 50.49, 58.27, 59.98, 69.76, 210.25. Anal. Calcd for
C6H12BNO (124.98): C, 57.66; H, 9.68; N, 11.21. Found: C, 57.91; H,
9.45; N, 11.47.
the a7 nAChR subtype elaborated by our research group [18], and
performed docking calculations on (R)-1b [12b] and (R)-3b, the
putative eutomer. We found that the best pose obtained with (R)-
3b gave rise to the same polar contacts observed with (R)-1b, i.e.,
two hydrogen bonding with the side chains of Gln116 and Tyr92
(Fig. 2). However, at variance with what we could predict from the
2D structures of 1b and 3b, the basic moieties showed some
differences in their binding to the
a7 nAChR. In fact, we found that
the bicyclic ring of (R)-3b created weaker contacts with the

C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
5795
6.1.2. 1-Boranyl-3-methylene-1-azabicyclo[2.2.1]heptane 11
To an ice-bath cooled suspension of tert-BuOK (754 mg,
6.72 mmol) in anhydrous THF (25 mL), methyl triphenylphos-
phonium bromide (2.43 g, 7.20 mmol) was added. After 15 min,
the reaction mixture was heated at reflux for 45 min. After cooling,
a solution of 10 (600 mg, 4.80 mmol) in anhydrous THF (3 mL) was
added and the mixture was stirred at r.t. for 1 h. Then, the reaction
was quenched with acetone (8 mL) and the solid was filtered off.
The liquid phase was concentrated under reduced pressure and
the crude mixture was purified by silica gel column chromatog-
raphy (cyclohexane/EtOAc 95:5) to afford alkene 11 (573 mg, 97%
yield).
(dichloromethane/methanol 95:5) to afford the free amine 13a
(194 mg, 65% yield).
13a: Yellow oil. Rf ¼ 0.14 (dichloromethane/methanol 9:1). 1H
NMR: 1.19e1.28 (m, 1H), 1.53e1.65 (m, 1H), 2.33e2.42 (m, 2H),
2.50e2.57 (m, 2H), 2.69e2.80 (m, 1H), 2.84 (m, 1H), 3.00 (d, 1H,
J ¼ 17.6), 3.12 (dd, 1H, J ¼ 2.0 and 13.5), 3.32 (d, 1H, J ¼ 17.6). 13C
NMR: 24.15, 45.57, 46.91, 53.61, 59.84, 69.63, 94.69, 136.37. Anal.
Calcd for C8H11BrN2O (231.09): C, 41.58; H, 4.80; N, 12.12. Found: C,
41.26; H, 5.09; N, 12.37.
6.1.6. 3-Methoxy-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene
13b
11: Thick pale yellow oil. Rf ¼ 0.43 (cyclohexane/ethyl acetate
4:1). 1H NMR: 1.66e1.75 (m, 1H), 2.06e2.17 (m, 1H), 2.78e2.96 (m,
3H), 3.10e3.21 (m, 2H), 3.40 (dd, 1H, J ¼ 2.2 and 15.8), 3.63 (dd, 1H,
J ¼ 2.6 and 15.8), 4.75 (d, 1H, J ¼ 2.6), 4.99 (d, 1H, J ¼ 2.6). 13C NMR:
30.92, 45.12, 59.90, 66.30, 67.01, 104.35, 147.93; Anal. Calcd for
C7H14BN (123.00): C, 68.35; H, 11.47; N, 11.39. Found: C, 68.73; H,
11.21; N, 11.67.
Intermediate 12b (370 mg, 1.89 mmol) was reacted following
the protocol above described for the preparation of 13a, which
afforded the free amine 13b (179 mg, 52% yield).
13b: Light yellow oil. Rf ¼ 0.29 (dichloromethane/methanol 9:1).
1H NMR: 1.10e1.20 (m, 1H), 1.43e1.54 (m, 1H), 2.23 (dd, 1H, J ¼ 3.0
and 9.9), 2.26e2.33 (m, 1H), 2.42 (dd, 1H, J ¼ 3.0 and 13.2), 2.49 (d,
1H, J ¼ 4.7), 2.60e2.70 (m, 1H), 2.70 (d, 1H, J ¼ 16.8), 2.76 (dd, 1H,
J ¼ 1.7 and 9.9), 2.98 (dd, 1H, J ¼ 2.2 and 13.2), 2.99 (d, 1H, J ¼ 16.8),
3.67 (s, 3H). 13C NMR: 24.36, 36.71, 46.61, 53.58, 56.94, 59.43, 69.38,
93.41, 167.01. Anal. Calcd for C9H14N2O2 (182.22): C, 59.32; H, 7.74;
N, 15.37. Found: C, 59.56; H, 7.48; N, 15.11.
6.1.3. 3-Bromo-1-oxa-2,7-diaza-7-boranyl-7,9-methanospiro[4.5]
dec-2-ene 12a
To a suspension of 11 (500 mg, 4.07 mmol) and NaHCO3 (4.45 g,
52.91 mmol) in ethyl acetate (30 mL) was added dibromo-
formaldoxime (907 mg, 4.47 mmol). The reaction mixture was
stirred at room temperature for 2 days, then CeliteÒ was added, and
the resulting slurry was filtered under vacuum and washed with
ethyl acetate. The solvent was evaporated and the residue was
column chromatographed (cyclohexane/ethyl acetate 4:1) to afford
compound 12a (876 mg, 88% yield).
6.1.7. 3-Phenyl-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene 13c
A 3.5% aqueous solution of NaClO (9.7 mL, 4.56 mmol) was
added dropwise to a solution of dipolarophile 11 (561 mg,
4.56 mmol) in dichloromethane (15 mL) and benzaldoxime
(552 mg, 4.56 mmol). After stirring for 10 h at r.t., further 1.5
equiv of benzaldoxime and NaClO were added. The reaction
mixture was further stirred for 3 h, then poured into water
(10 mL) and the aqueous phase was extracted with dichloro-
methane (3 ꢃ 10 mL). The pooled organic extracts were dried
over anhydrous Na2SO4 and the residue was purified by silica gel
column chromatography providing cycloadduct 13c (94 mg, 9%
yield).
12: yellow oil: Rf ¼ 0.47 (cyclohexane/ethyl acetate 1:4). 1H
NMR: 1.59e1.69 (m, 1H), 2.02e2.14 (m, 1H), 2.80 (d, 1H, J ¼ 4.8),
2.86e2.94 (m, 2H), 3.09 (dd, 1H, J ¼ 2.6 and 13.6), 3.17 (d, 1H,
J ¼ 17.8), 3.15e3.32 (m, 2H), 3.33 (d, 1H, J ¼ 17.8), 3.47 (dd, 1H,
J ¼ 2.6 and 13.6). 13C NMR: 23.68, 45.68, 45.94, 58.39, 64.47, 71.61,
92.48, 137.26; Anal. Calcd for C8H14BBrN2O (244.92): C, 39.23; H,
5.76; N, 11.44. Found: C, 39.50; H, 5.47; N, 11.26.
13c: Yellow oil. Rf ¼ 0.25 (dichloromethane/methanol 9:1). 1H
NMR: 1.41e1.50 (m, 1H), 1.64e1.73 (m, 1H), 2.46e2.58 (m, 2H),
2.62e2.68 (m, 2H), 2.82e2.91 (m, 1H), 3.04 (m, 1H), 3.20 (d, 1H,
J ¼ 17.1), 3.27 (dd, 1H, J ¼ 2.2 and 11.6), 3.54 (d, 1H, J ¼ 17.1),
7.34e7.43 (m, 3H), 7.61e7.66 (m, 2H). 13C NMR: 24.19, 45.43, 46.67,
57.61, 61.87, 67.47, 94.35, 126.65, 128.59, 130.07, 156.35. Anal. Calcd
for C14H16N2O (228.29): C, 73.66; H, 7.06; N, 12.27. Found: C, 73.92;
H, 7.15; N, 12.03.
6.1.4. 3-Methoxy-1-oxa-2,7-diaza-7-boranyl-7,9-methanospiro
[4.5]dec-2-ene 12b
A stirred suspension of bromo-D
2-isoxazoline 12a (485 mg,
1.98 mmol) and K2CO3 (2.74 g, 19.80 mmol) in methanol (30 mL)
was stirred at reflux for 4 h. After addition of CeliteÒ and filtration
under vacuum, the crude filtrate was submitted to a silica gel
column chromatography (cyclohexane/ethyl acetate 1:1), which
gave the title compound 12b (373 mg, 96% yield).
12b: Colorless viscous oil. Rf ¼ 0.40 (cyclohexane/ethyl acetate
1:4). 1H NMR: 1.63e1.72 (m, 1H), 2.03e2.15 (m, 1H), 2.80e3.21 (m,
4H), 3.08 (dd, 1H, J ¼ 2.5 and 13.5), 2.97 (d, 1H, J ¼ 16.5), 3.11 (d, 1H,
J ¼ 16.5), 3.28 (m,1H), 3.46 (dd,1H, J ¼ 2.5 and 13.5), 3.85 (s, 3H). 13C
NMR: 23.83, 36.93, 45.65, 57.35, 58.35, 64.12, 71.67, 91.38, 166.97.
Anal. Calcd for C9H17BN2O2 (196.05): C, 55.14; H, 8.74; N, 14.29.
Found: C, 55.39; H, 8.87; N, 14.02.
6.1.8. tert-Butyl-3-bromo-1-oxa-2,7-diazaspiro[4.5]dec-2-ene-7-
carboxylate 16a
The pericyclic reaction was performed on dipolarophile 15
(3.0 g, 15.21 mmol) following the protocol above reported for olefin
12a. The crude reaction mixture was purified by column chroma-
tography (petroleum ether/ethyl acetate 9:1) to afford cycloadduct
16a (4.66 g, 96% yield).
16a: Crystallized from n-hexane as a colorless powder, mp
93e95 ꢂC. Rf ¼ 0.23 (petroleum ether/ethyl acetate 9:1). 1H NMR
(DMSO-d6): 1.38 (s, 9H), 1.41e1.48 (m, 1H), 1.59e1.71 (m, 1H),
1.79e1.84 (m, 2H), 3.00 (d, 1H, J ¼ 17.3), 3.11 (d, 1H, J ¼ 17.3),
3.19e3.28 (m, 1H), 3.30e3.36 (m, 1H), 3.31 (d, 1H, J ¼ 13.0), 3.40 (d,
1H, J ¼ 13.0). 13C NMR: 23.24, 28.56, 34.68, 43.17, 49.51, 50.96, 80.37,
85.65, 136.95, 154.86. Anal. Calcd for C12H19BrN2O3 (319.19): C,
45.15; H, 6.00; N, 8.78. Found: C, 45.27; H, 6.07; N, 8.64.
6.1.5. 3-Bromo-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene 13a
To an ice-bath cooled and stirred solution of 12a (316 mg,
1.29 mmol) in acetone (10 mL) at 0 ꢂC, trifluoroacetic acid (0.67 mL,
9.03 mmol) was added dropwise and the mixture was stirred at r.t.
for 10 h. After concentration at reduced pressure, the residue was
dissolved in water (10 mL) and treated with ether (3 ꢃ 5 mL). The
residual aqueous phase was made alkaline by portionwise addition
of solid K2CO3 (pH ¼ 10) and extracted with dichloromethane
(3 ꢃ 5 mL). The combined organic phases were dried over anhy-
drous Na2SO4, concentrated under reduced pressure, and the crude
residue was purified by silica gel column chromatography
6.1.9. tert-Butyl-3-methoxy-1-oxa-2,7-diazaspiro[4.5]dec-2-ene-7-
carboxylate 16b
Compound 16a (1.0 g, 3.13 mmol) was reacted following the
protocol previously described for 12b. The title methoxy derivative

5796
C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
16b was isolated (813 mg, 96% yield) by silica gel column
chromatography.
7.37e7.42 (m, 3 H), 7.63e7.68 (m, 2H). 13C NMR: 24.61, 35.16, 43.87,
44.00, 45.84, 54.77, 84.51, 126.64, 128.85, 130.15, 156.46. Anal. Calcd
for C13H16N2O (216.28): C, 72.19; H, 7.46; N, 12.95. Found: C, 72.42;
H, 7.21; N, 12.77.
16b: Yellow oil. Rf ¼ 0.32 (petroleum ether/ethyl acetate 4:1). 1H
NMR: 1.38 (s, 9H), 1.71e1.82 (m, 4H), 2.59e2.73 (m, 2H), 2.92e3.10
(m, 2H), 3.62e3.68 (m, 2H), 3.77 (s, 3H). 13C NMR: 23.39, 28.53,
35.03, 40.66, 43.17, 50.94, 57.15, 80.07, 84.42, 154.94, 167.04. Anal.
Calcd for C13H22N2O4 (270.32): C, 57.76; H, 8.20; N, 10.36. Found: C,
58.04; H, 8.47; N 10.08.
6.1.14. 3-Bromo-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 18a
A 37% aqueous solution of formaldehyde (0.628 mL, 8.4 mmol)
and NaBH3CN (211 mg, 3.36 mmol) were added portionwise to
a stirred solution of 17a (370 mg, 1.69 mmol) in CH3CN (10 mL).
After stirring at r.t. for 20 min, the solvent was removed, and the
residue was partitioned between dichloromethane and acidic
water (pH ¼ 3). The residual aqueous phase (about 10 mL), after
addition of solid NaHCO3 (pH ¼ 8), was extracted with dichloro-
methane (3 ꢃ 5 mL). The combined organic extracts were dried
over anhydrous Na2SO4, concentrated in vacuo, and column chro-
matographed to afford the tertiary amine 18a (360 mg, 92% yield).
18a: Yellow oil. Rf ¼ 0.58 (dichloromethane/methanol 9:1). 1H
NMR: 1.41e2.60 (m, 1H), 1.60e1.68 (m, 2H), 1.68e1.86 (m, 1H),
2.14e2.20 (m, 2H), 2.20e2.60 (m, 2H), 2.24 (s, 3H), 2.89 (d, 1H,
J ¼ 17.5), 3.06 (d, 1H, J ¼ 17.5). 13C NMR: 22.95, 34.01, 46.40, 47.78,
54.92, 63.23, 86.99, 148.47. Anal. Calcd for C8H13BrN2O (233.11): C,
41.22; H, 5.62; N, 12.02. Found: C, 41.47; H, 5.85; N, 11.79.
6.1.10. tert-Butyl-3-phenyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene-7-
carboxylate 16c
The title compound was prepared from dipolarophile 15
(300 mg, 1.52 mmol) following the procedure above described for
13c. The crude reaction mixture was purified by column chroma-
tography (petroleum ether/ethyl acetate 9:1) to afford cycloadduct
16c (467 mg, 97% yield).
16c: Crystallized from n-hexane/EtOAc (1:1) as a colorless
powder, mp 162e162.5 ꢂC. Rf ¼ 0.43 (petroleum ether/ethyl acetate
4:1). 1H NMR (DMSO-d6): 1.37 (s, 9H), 1.47e1.54 (m, 1H), 1.69e1.76
(m, 1H), 1.82e1.87 (m, 2H), 3.13 (m, 2H), 3.35 (m, 4H), 7.43e7.44 (m,
3H), 7.62e7.64 (m, 2H). 13C NMR: 23.55, 28.57, 28.64, 35.09, 43.12,
51.49, 80.18, 84.69, 126.73, 128.92, 130.04, 130.30, 155.05, 156.38.
Anal. Calcd for C18H24N2O3 (316.39): C, 68.33; H, 7.65; N, 8.85.
Found: C, 68.53; H, 7.70; N, 8.69.
6.1.15. 3-Methoxy-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 18b
The secondary amine 17b (236 mg, 1.39 mmol) was reacted
following the procedure above described for the preparation of 18a.
Tertiary amine 18b was isolated (243 mg, 95% yield) by silica gel
column chromatography.
18b: Light yellow oil. Rf ¼ 0.62 (dichloromethane/methanol 9:1).
1H NMR: 1.50e1.62 (m, 1H), 1.63e175 (m, 2H), 1.76e1.84 (m, 1H),
2.05e2.18 (m, 1H), 2.19e2.27 (m, 1H), 2.28 (s, 3H), 2.50e2.67 (m,
2H), 2.72 (d, 1H, J ¼ 16.5), 2.95 (d, 1H, J ¼ 16.5), 3.83 (s, 3H). 13C
NMR: 23.08, 34.04, 41.62, 46.27, 55.02, 56.64, 63.41, 84.79, 166.80.
Anal. Calcd for C9H16N2O2 (184.24): C, 58.67; H, 8.75; N, 15.21.
Found: C, 58.52, H, 9.03, N, 14.98.
6.1.11. 3-Bromo-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 17a
To a stirred ice cooled solution of cycloadduct 16a (400 mg,
1.25 mmol) in dioxane (3 mL) was added a 4N HCl solution in
dioxane (0.5 mL, 2 mmol). The reaction mixture was then stirred at
r.t. for about 1 h (TLC monitoring). The solvent was removed in
vacuo and a saturated NaHCO3 aqueous solution (about 8 mL) was
added (pH ¼ 8), which was extracted with ether (5 ꢃ 5 mL). The
pooled organic extracts were dried over anhydrous Na2SO4 and the
crude residue was purified by silica gel column chromatography
(dichloromethane/methanol 98:2) to provide the tertiary base 17a
(187 mg, 68% yield).
17a: Yellow oil. Rf ¼ 0.24 (dichloromethane/methanol 95:5). 1H
NMR: 1.45e1.57 (m, 1H), 1.73e1.83 (m, 1H), 1.92e1.98 (m, 2H), 2.46
(d, 1H, J ¼ 16.0), 2.56 (d,1H, J ¼ 16.0), 2.70e2.78 (m, 1H), 2.89 (d, 1H,
J ¼ 17.3), 2.94e2.96 (m, 1H), 3.02 (d, 1H, J ¼ 17.3). 13C NMR (DMSO-
d6): 23.85, 29.90, 34.67, 45.38, 50.45, 85.40, 137.14. Anal. Calcd for
C7H11BrN2O (219.08): C, 38.38; H, 5.06; N, 12.79. Found: C, 38.63; H,
4.88; N, 12.57.
6.1.16. 3-Phenyl-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 18c
The secondary amine 17c (200 mg, 0.92 mmol) was reacted
following the procedure above described for the preparation of 18a.
Tertiary amine 18c was isolated (192 mg, 90% yield) by silica gel
column chromatography.
18c: Light yellow oil. Rf ¼ 0.27 (dichloromethane/methanol 9:1).
1H NMR: 1.55e1.63 (m, 1H), 1.72e1.82 (m, 2H), 1.85e1.90 (m, 1H),
2.10e2.22 (m, 1H), 2.23e2.38 (m, 1H), 2.31 (s, 3H), 2.50e2.68 (m,
2H), 3.06 (d, 1H, J ¼ 16.7), 3.35 (d, 1H, J ¼ 16.7), 7.37e7.40 (m, 3H),
7.65e7.68 (m, 2H). 13C NMR: 23.52, 34.31, 44.34, 46.62, 55.32, 63.77,
85.41, 126.71, 128.85, 130.08, 130.25, 156.40. Anal. Calcd for
C14H18N2O (230.31): C, 73.01; H, 7.88; N, 12.16. Found: C, 73.40; H,
7.64; N, 11.91.
6.1.12. 3-Methoxy-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 17b
The protected amine 16b (400 mg, 1.48 mmol) was reacted
following the protocol previously described for the preparation of
the 13a. Secondary amine 17b was obtained (219 mg, 87% yield) by
silica gel column chromatography,
17b: Thick yellow oil. Rf ¼ 0.46 (dichloromethane/methanol
95:5). 1H NMR: 1.46e1.53 (m, 1H), 1.70e1.80 (m, 2H), 1.89e1.94 (m,
1H), 2.64e2.76 (m, 4H), 2.79e2.87 (m, 1H), 2.89e2.94 (m, 1H), 3.83
(s, 3H). 13C NMR: 24.19, 34.93, 41.56, 45.68, 54.41, 56.96, 83.93,
167.13; Anal. Calcd for C8H14N2O2 (170.21): C, 56.45; H, 8.29; N,
16.46. Found: C, 56.21; H, 8.52; N, 16.68.
6.1.17. General procedure for the preparation of fumarates
To a solution of the free base (0.8 mmol) in methanol (3 mL) was
added a solution of fumaric acid (102 mg, 0.88 mmol) in methanol
(2 mL). After stirring overnight at room temperature, the solvent
was removed at reduced pressure and the crude fumarate, which
was obtained quantitatively, was crystallized.
6.1.13. 3-Phenyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene 17c
3-Bromo-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene fuma-
rate 3a ꢃ 3/4 C4H4O4: Crystallized from 2-propanol as colorless
prisms, mp 169e170 ꢂC. 1H NMR (CD3OD): 1.74e1.84 (m, 1H),
2.06e2.18 (m, 1H), 2.97 (d, 1H, J ¼ 5.0), 3.10e3.19 (m, 1H), 3.24 (dd,
1H, J ¼ 2.5 and 9.5), 3.29e3.45 (m, 2H), 3.45 (d, 1H, J ¼ 18.2), 3.45
(dd, 1H, J ¼ 2.8 and 13.0), 3.62 (d, 1H, J ¼ 18.2), 3.65 (dd, 1H, J ¼ 2.8
and 13.0), 6.69 (s, 1.5H). 13C NMR (CD3OD): 21.31, 45.01, 51.67, 58.65,
64.93, 65.74, 91.25, 135.02, 138.06, 169.80. MS (ESI) m/z [M]þ Calcd
for C8H11BrN2O: 231.1. Found: 231.0. Anal. Calcd for C11H14BrN2O4
The protected amine 16c (430 mg, 1.36 mmol) was reacted
following the protocol previously described for the preparation of
the 13a. Secondary amine 17c (273 mg, 93% yield) was purified by
silica gel column chromatography.
17c: Thick light yellow oil. Rf ¼ 0.52 (dichloromethane/methanol
9:1). 1H NMR: 1.57e1.64 (m, 1H), 1.78e1.90 (m 2H), 1.96e2.02 (m,
1H), 2.73e2.82 (m, 1H), 2.80 (d, 1H, J ¼ 13.0), 2.86e2.92 (m, 1H),
2.96 (d, 1H, J ¼ 13.0), 3.00 (d, 1H, J ¼ 16.8), 3.16 (d, 1H, J ¼ 16.8),

C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
5797
(318.14): C, 41.53; H, 4.44; Br, 25.12; N, 8.81. Found: C, 41.77; H, 4.23;
Br, 24.86; N, 8.58.
(m, 2H), 2.89e3.06 (m, 4H), 3.18e3.24 (m, 1H), 3.31e3.37 (m, 1H),
3.83 (s, 3H), 6.63 (s, 1H). 13C NMR (CD3OD): 24.20, 37.68, 47.87,
49.08, 55.65, 64.87, 83.97, 134.49, 162.39, 168.27. MS (ESI) m/z
[M þ H]þ Calcd for C8H14N2O2: 170.2. Found: 171.1. Anal. Calcd for
C10H16N2O4 (228.25): C, 52.62; H, 7.07; N, 12.27. Found: C, 52.81; H,
7.25; N, 12.09.
3-Phenyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fumarate 7c ꢃ 3/4
C4H4O4: Crystallized from 2-propanol as colorless prisms, mp
199.5e201 ꢂC dec. 1H NMR (D2O): 1.82e1.90 (m, 3H), 1.97e2.02 (m,
1H), 2.90e2.97 (m, 1H), 3.07e3.12 (m, 1H), 3.30 (bs, 2H), 3.20e3.39
(m, 2H), 6.51 (s, 1.5H), 7.35e7.41 (m, 3H), 7.50e7.56 (m, 2H). 13C
NMR (CD3OD): 23.17, 32.15, 43.26, 59.38, 63.22, 81.97, 125.31,
127.90, 129.67, 131.02, 136.13, 155.27, 169.64. MS (ESI) m/z [M þ H]þ
Calcd for C14H18N2O: 216.3. Found: 217.0. Anal. Calcd for
C16H19N2O4 (303.33): C, 63.35; H, 6.31; N, 9.24. Found: C, 63.12; H,
6.47; N, 9.40.
3-Methoxy-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene
fumarate 3b ꢃ 1/2 C4H4O4: Crystallized from 2-propanol as colorless
prisms, mp 149e150 ꢂC. 1H NMR (CD3OD): 1.75e1.87 (m, 1H),
2.08e2.27 (m, 1H), 2.86e3.10 (m, 2H), 3.15e3.38 (m, 2H), 3.39e3.58
(m, 2H), 3.61e3.77 (m, 2H), 3.83 (s, 3H); 3.90e4.07 (m, 1H), 6.70 (s,
1H). 13C NMR (CD3OD): 21.40, 24.26, 44.80, 56.75, 58.43, 63.63,
64.92, 65.73, 134.63, 167.72, 168.58. MS (ESI) m/z [M þ H]þ Calcd for
C9H14N2O2: 182.2. Found: 183.1. Anal. calcd for C11H16N2O4
(240.26): C, 54.99; H, 6.71; N, 11.66. Found: C, 55.18; H, 6.53; N,
11.79.
3-Phenyl-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene fuma-
rate 3c ꢃ 1/2 C4H4O4: Crystallized from 2-propanol as colorless
prisms, mp 176.5e177.5 ꢂC. 1H NMR (CD3OD): 1.80e1.90 (m, 1H),
2.06e2.18 (m, 1H), 2.97 (d, 1H, J ¼ 4.7), 3.16e3.22 (m, 1H), 3.25 (dd,
1H, J ¼ 2.3 and 9.6), 3.30e3.44 (m, 1H), 3.44e3.58 (m, 2H), 3.54 (d,
1H, J ¼ 17.8), 3.68e3.77 (m, 1H), 3.73 (d, 1H, J ¼ 17.8), 6.53 (s, 1H),
7.34e7.41 (m, 3H), 7.54e7.57 (m, 2H). 13C NMR (CD3OD):0.21.34,
45.13, 51.55, 58.78, 65.06, 65.66, 91.65, 128.21, 128.86, 130.27,
131.03, 134.55, 156.22, 168.76. MS (ESI) m/z [M þ H]þ Calcd for
C14H16N2O: 228.3. Found: 229.1. Anal. Calcd for C16H18N2O3
(286.33): C, 67.12; H, 6.34; N, 9.78. Found: C, 67.35; H, 6.39; N,10.02.
3-Bromo-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fumarate
5a ꢃ 3/4 C4H4O4: Crystallized from 2-propanol as pale yellow
prisms, mp 121e122 ꢂC dec. 1H NMR (D2O): 1.65e1.80 (m, 1H),
1.80e1.90 (m, 2H), 1.90e2.21 (m, 1H), 2.75 (s, 3H), 2.79e2.90 (m,
1H), 3.00e3.30 (m, 3H), 3.32e3.45 (m, 1H), 3.48e3.65 (m, 1H), 6.54
(s, 1.5H). 13C NMR (CD3OD): 24.43, 31.35, 43.54, 53.28, 58.75, 63.57,
84.68, 135.32, 149.63, 170.40; MS (ESI) m/z [M]þ Calcd for
C8H13BrN2O: 233.1. Found: 233.1. Anal. Calcd for C11H16BrN2O4
(320.16): C, 41.27; H, 5.04; Br, 24.96; N, 8.75. Found: C, 41.38; H,
4.92; Br, 25.22; N, 8.89.
6.1.18. General procedure for the preparation of iodomethylates
To a solution of the free base (0.5 mmol) in methanol (3 mL) was
added iodomethane (310 mL, 5 mmol). The solution was left over-
night at room temperature, then the solvent was removed at
reduced pressure affording quantitatively the crude quaternary
salt, which was crystallized.
3-Bromo-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene
methyl iodide 4a: Crystallized from 2-propanol as colorless prisms,
mp 177e178 ꢂC. 1H NMR (D2O): 1.77e1.87 (m, 1H), 2.20e2.32 (m,
1H), 3.00 (m, 1H), 3.17 (s, 3H), 3.40e3.46 (m, 3H), 3.43 (d, 1H,
J ¼ 18.2), 3.58 (d, 1H, J ¼ 18.2), 3.58e3.61 (m, 1H), 3.71e3.82 (m,
2H). 13C NMR (D2O): 22.70, 45.21, 45.36, 62.02, 67.53, 73.81, 92.09,
100.24, 139.95. MS (ESI) m/z [M]þ Calcd for C9H14BrN2Oþ: 246.1.
Found: 245.0. Anal. Calcd for C9H14BrIN2O (373.03): C, 28.98; H,
3.78; Br, 21.42; I, 34.02; N, 7.51. Found: C, 28.75; H, 3.92; Br, 21.65; I,
33.85; N, 7.78.
3-Methoxy-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fuma-
rate 5b ꢃ 3/4 C4H4O4: Crystallized from 2-propanol as colorless
prisms, mp 136.5e137.5 ꢂC dec. 1H NMR (D2O): 1.62e1.78 (m, 1H),
1.78e1.95 (m, 2H), 1.95e2.05 (m, 1H), 2.74 (s, 3H), 2.80e2.90 (m,
2H), 2.90e3.02 (m, 2H), 3.30e3.38 (m, 1H), 3.49e3.56 (m, 1H), 3.70
(s, 3H), 6.54 (s, 1.5H). 13C NMR (CD3OD): 29.66, 31.00, 40.89, 53.42,
56.79, 58.87, 82.38, 92.82, 135.09, 167.82, 170.05. MS (ESI) m/z
[M þ H]þ Calcd for C9H16N2O2: 184.2. Found: 185.1. Anal. Calcd for
C12H19N2O5 (271.29): C, 53.13; H, 7.06; N, 10.33. Found: C, 53.27; H,
7.26; N, 10.15.
3-Methoxy-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene
methyl iodide 4b: Crystallized from 2-propanol as colorless prisms,
mp 167e167.5 ꢂC. 1H NMR (D2O): 1.75e1.91 (m, 1H), 2.18e2.30 (m,
1H), 2.98 (m, 1H), 3.15 (s, 3H), 3.16 (d, 1H, J ¼ 17.0), 3.27 (d, 1H,
J ¼ 17.0), 3.34e3.42 (m, 3H), 3.57 (m, 1H), 3.65 (dd, 1H, J ¼ 2.3 and
12.9), 3.71 (s, 3H), 3.76 (dd, 1H, J ¼ 2.4 and 12.9). 13C NMR (D2O):
23.30, 26.54, 45.34, 45.57, 51.02, 61.89, 67.46, 73.98, 92.25, 164.87.
MS (ESI) m/z [M]þ Calcd for C10H17N2Oþ2 : 197.3. Found: 197.1. Anal.
Calcd for C10H17IN2O2 (324.16): C, 37.05; H, 5.29; I, 39.15; N, 8.64.
Found: C, 36.86; H 5.43; I, 39.02; N, 8.59.
3-Phenyl-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fumarate
5c ꢃ 3/4 C4H4O4: Crystallized from 2-propanol as colorless prisms,
mp 171.5e172.5 ꢂC dec. 1H NMR (D2O): 1.78e2.02 (m, 4H), 2.75 (s,
3H), 2.84e2.94 (m, 1H), 3.06e3.10 (m, 1H), 3.29 (bs, 2H), 3.38e3.50
(m, 2H), 6.51 (s, 1.5H), 7.30e7.48 (m, 3H), 7.50e7.59 (m, 2H). 13C
NMR (CD3OD): 24.15, 31.14, 43.75, 53.52, 59.16, 63.60, 82.51, 126.71,
128.78, 129.17, 130.52, 134.73, 157.57, 169.01. MS (ESI) m/z [M þ H]þ
Calcd for C14H18N2O: 230.3. Found: 231.1. Anal. Calcd for
C17H21N2O4 (317.36): C, 64.34; H, 6.67; N, 8.83. Found: C, 64.57; H,
6.72; N, 8.65.
3-Phenyl-1-oxa-2,7-diaza-7,9-methanospiro[4.5]dec-2-ene
methyl iodide 4c: Crystallized from 2-propanol as a light yellow
powder, mp 173.5e174 ꢂC. 1H NMR (D2O): 1.91e1.98 (m, 1H),
2.23e2.33 (m, 1H), 2.97 (m, 1H), 3.18 (s, 3H), 3.39e3.50 (m, 3H),
3.55 (d, 1H, J ¼ 18.0), 3.62 (m, 1H), 3.71 (d, 1H, J ¼ 18.0), 3.71e3.83
(m, 2H), 7.34e7.42 (m, 3H), 7.52e7.56 (m, 2H). 13C NMR (CD3OD):
21.27, 45.11, 50.14, 51.64, 58.65, 64.54, 65.98, 91.11, 128.20, 128.83,
130.21, 131.06, 156.20. MS (ESI) m/z [M]þ Calcd for C15H19N2Oþ:
243.3. Found: 243.1. Anal. Calcd for C15H19IN2O (370.23): C, 48.66;
H, 5.17; I, 34.28; N, 7.57. Found: C, 48.83; H, 5.38; I, 34.47; N, 7.45.
3-Bromo-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fumarate 7a ꢃ 1/2
C4H4O4: Crystallized from 2-propanol/diethyl ether (1:1) as color-
less needles, mp 161.5e164 ꢂC. 1H NMR (D2O): 1.77e1.92 (m, 3H),
2.03e2.07 (m, 1H), 2.86e2.94 (m, 1H), 3.05e3.15 (m, 3H),
3.26e3.30 (m, 1H), 3.40e3.46 (m, 1H), 6.41 (s, 1H). 13C NMR
(CD3OD): 24.20, 31.72, 46.77, 48.77, 63.61, 83.63, 134.63, 149.83,
168.69. MS (ESI) m/z [M]þ Calcd for C7H11BrN2O: 219.1. Found:
218.9. Anal. Calcd for C9H13BrN2O3 (277.12): C, 39.01; H, 4.73; Br,
28.83; N, 10.11. Found: C, 39.19; H, 4.57; Br, 28.98; N, 10.02.
3-Methoxy-1-oxa-2,7-diazaspiro[4.5]dec-2-ene fumarate 7b ꢃ 1/2
C4H4O4: Crystallized from 2-propanol as colorless prisms, mp
161e163.5 ꢂC dec. 1H NMR (CD3OD): 1.75e1.89 (m, 2H), 1.94e2.08
3-Bromo-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene
methyl
iodide 6a: Crystallized from 2-propanol as colorless prisms, mp
198.5e199.5 ꢂC. 1H NMR (D2O): 1.65e1.75 (m, 1H), 1.79e1.94 (m,
1H), 2.00e2.19 (m, 2H), 3.08 (s, 3H), 3.13 (s, 3H), 3.12e3.29 (m, 3H),
3.37e3.49 (m, 2H), 3.70e3.75 (m, 1H). 13C NMR (CD3OD): 17.17,
22.15, 31.05, 33.46, 45.31, 54.54, 62.64, 86.65, 148.84. MS (ESI) m/z
[M þ H]þ Calcd for C9H16BrN2Oþ: 248.1. Found: 249.2. Anal. Calcd
for C9H16BrIN2O (375.04): C, 28.82; H, 4.30; Br, 21.31; I, 33.84; N,
7.47. Found: C, 29.09; H, 4.43; Br, 21.46; I, 33.69; N, 7.22.
3-Methoxy-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene methyl
iodide 6b: Crystallized from 2-propanol as yellow prisms, mp
187e188 ꢂC. 1H NMR (D2O): 1.60e1.75 (m, 1H), 1.76e1.92 (m, 1H),

5798
C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
1.98e2.16 (m, 2H), 2.85 (d, 1H, J ¼ 17.1), 2.97 (d, 1H, J ¼ 17.1), 3.06 (s,
3H), 3.13 (s, 3H), 3.18e3.24 (m, 1H), 3.30 (d, 1H, J ¼ 13.5), 3.39e3.48
(m, 1H), 3.68 (d, 1H, J ¼ 13.5), 3.72 (s, 3H). 13C NMR (CD3OD): 17.25,
30.78, 42.21, 50.54, 56.96, 61.88, 65.77, 83.11, 167.89. MS (ESI) m/z
[M]þ Calcd for C10H19N2O2þ: 199.3. Found: 199.1. Anal. Calcd for
C10H19IN2O2 (326.17): C, 36.82; H, 5.87; I, 38.91; N, 8.59. Found: C,
37.04; H, 5.71; I, 39.19; N, 8.44.
6.2.4. Binding to heterologously expressed
HEK 293 cells were grown in Dulbecco’s modified Eagle medium
supplemented with 10% fetal bovine serum, 1% -Glutamine, 100
units/ml penicillin G, and 100 g/streptomycin in a humidified
atmosphere containing 10% CO2. The cDNAs encoding 3 and
were transfected into the HEK 293 cells at 30% confluency. The cell
transfections were performed in 100 mm Petri dishes using 30 L of
JetPEIÔ (Polypus, France) (1 mg/ml, pH 7.2) and 10 g of cDNAs.
a3b4 receptors
L
m
a
b4
m
3-Phenyl-7-methyl-1-oxa-2,7-diazaspiro[4.5]dec-2-ene
methyl
m
iodide 6c: Crystallized from 2-propanol as colorless prisms, mp
197e198 ꢂC. 1H NMR (D2O): 1.71e1.92 (m, 2H), 2.08e2.20 (m, 2H),
3.09 (s, 3H), 3.18 (s, 3H), 3.22e3.40 (m, 3H), 3.40e3.55 (m, 2H),
3.60e3.72 (m, 1H), 7.31e7.49 (m, 3H), 7.51e7.60 (m, 2H). 13C NMR
(CD3OD): 17.60, 31.26, 45.44, 56.91, 61.86, 66.29, 83.11, 126.81,
128.86, 130.64, 157.39, 167.30. MS (ESI) m/z [M]þ Calcd for
C15H21N2Oþ: 245.3. Found: 245.1. Anal. Calcd for C15H21IN2O
(372.24): C, 48.40; H, 5.69; I, 34.09; N, 7.53. Found: C, 48.49; H, 5.82;
I, 33.91; N, 7.66.
After 48 h transfection, the cells were collected, washed with PBS
by centrifugation, and used for binding analysis.
6.2.5. Affinity of compounds 3aec, 4aec, 5aec, 6aec, and 7aec for
nAChRs
The inhibition of radioligand binding by epibatidine and the test
compounds was measured by pre-incubating cortex homogenates
with increasing doses (10 pM - 10 mM) of the reference nicotinic
agonists, epibatidine or nicotine, and the drug to be tested for
30 min at r.t., followed by overnight incubation with a final
6.2. Receptor binding assays
concentration of 0.075 nM [3H]epibatidine or 1 nM [125I]
a-bun-
garotoxin at the same temperatures as those used for the saturation
experiments. These ligand concentrations were used for the
competition-binding experiments because they are within the
range of the KD values of the ligands for the two different classes of
nAChRs. For each compound, the experimental data obtained from
the three saturation and three competition-binding experiments
were analyzed by means of a non-linear least square procedure,
using the LIGAND program as described by Munson and Rodbard
[15]. The binding parameters were calculated by simultaneously
fitting three independent saturation experiments and the Ki values
were determined by fitting the data of three independent compe-
tition experiments. The errors in the KD and Ki values of the
simultaneous fits were calculated using the LIGAND software, and
were expressed as percentage coefficients of variation (% CV).
6.2.1. Membranes binding of [3H]epibatidine and [125I]
bungarotoxin
a-
The cortex tissues were dissected, immediately frozen on dry ice
and stored at ꢀ80 ꢂC 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 ethylenediaminetetra-
acetic 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 60,000g for 1.5 h. The total membrane homogeni-
zation, dilution and centrifugation procedures were performed
twice, then the pellets were collected, rapidly rinsed with a buffer
solution (50 mM TriseHCl, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2,
2.5 mM CaCl2 and 2 mM PMSF, pH 7), and resuspended in the same
When final compound concentrations up to 100 mM did not inhibit
buffer containing a mixture of 20
protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin.
m
g/mL of each of the following
radioligand binding, the Ki value was defined as being > 100
based on the Cheng and Prusoff’s equation [20].
mM
Binding to HEK 293 transfected a3b4 receptors was performed by
6.2.2. [3H]Epibatidine binding
overnight incubation at 4 ꢂC with [3H]Epi at a concentration ranging
from 0.005 to 1 nM. All of the incubations were performed in a buffer
containing 50 mM TriseHCl, pH 7, 150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 2.5 mM CaCl2, 2 mg/ml BSA. Specific ligand binding was
defined as total binding minus the binding in the presence of 100 nM
cold Epi. The inhibition of [3H]Epi binding by compounds was
measured by incubating increasing concentration of the compounds
for 5 min followed by overnight incubation with 0.25 nM (in the case
(ꢁ)-[3H]Epibatidine with a specific activity of 56e60 Ci/mmol
was purchased from Perkin Elmer (Boston MA); the nonradioactive
a
-bungarotoxin, epibatidine, and nicotine were purchased from
Sigma Aldrich (Italy). It has been previously reported that [3H]
epibatidine also binds to -bungarotoxin binding receptors with
nM affinity [19]. In order to prevent the binding of [3H]epibatidine
to the -bungarotoxin binding receptors, the membrane homoge-
nates were pre-incubated with 2
a
a
m
M
a
-bungarotoxin and then with
of the
a3b4 subtype). After incubation, the membranes of HEK cells
[3H]epibatidine. The saturation experiments were performed by
incubating aliquots of cortex membrane homogenates with
0.01e2.5 nM concentrations of (ꢁ)-[3H]epibatidine overnight at
transfected with a3b4 receptors were washed seven times with ice-
cold PBS, and the bound [3H]Epi was then determined by means of
liquid scintillation counting in a beta counter.
4
ꢂC. 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
6.3. Electrophysiological recordings
The human
in the rat anterior pituitary GH4C1 cell line [21]. Transient trans-
fection was achieved by adding to each dish 1 g of human
subunit cDNA, along with 4 l of lipofectamine. All culture media
a7 nAChRs were expressed by transient transfection
counted in a
b
counter.
m
a7
m
6.2.3. [125I]
a-bungarotoxin binding
were purchased from Invitrogen (Italy). Whole-cell current
recordings were performed 2e3 days after plating. Recordings and
data analysis were performed by using borosilicate glass patch
The saturation binding experiments were performed using
aliquots of cortex membrane homogenates incubated overnight
with 0.1e10 nM concentrations of [125I]
a
-bungarotoxin (specific
pipette (3- to 6-MU tip resistance) connected to an Axopatch 200A
activity 200e213 Ci/mmol, Amersham) at r.t. Nonspecific binding
was determined in parallel by means of incubation in the presence
amplifier (Axon Instruments, Foster City, CA). Data were stored on
a PC computer by using PCLAMP10 software (Molecular Devices).
During the recording period, the cells were bathed in the following
solution (mM): 140 NaCl, 2 CaCl2, 2.8 KCl, 2 MgCl2, 10 Hepes/NaOH
and 10 glucose; pH 7.3. The patch pipettes were filled with
of 1
were filtered as described above and the bound radioactivity was
directly counted in a counter.
mM unlabelled a-bungarotoxin. After incubation, the samples
g

C. Dallanoce et al. / European Journal of Medicinal Chemistry 46 (2011) 5790e5799
5799
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(5e15 MU) were estimated from slow transient compensations. A
series resistance compensation of 85e90% was obtained in all
cases. The cells were voltage-clamped at a holding potential
of ꢀ70 mV and continuously perfused with a gravity-driven system
using independent external tubes for the control and agonist-
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n
ꢀ
ꢁo
I ¼ Imax ½CꢄnH
=
EC50nH þ ½CꢄnH
(1)
where I is the current amplitude induced by the agonist at
concentration [C], Imax is the maximum response of the cell, nH is
the Hill coefficient and EC50 is the concentration for which a half
maximum response is induced.
6.4. Molecular modeling
The structures of the target compounds were built by Gauss-
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considered in the ionized form to better simulate the physiological
conditions. The superimposition between both conformers of (S)-
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chains D and E of a published model [18] of a7 nAChRs were per-
formed by means of the program GOLD 4.0 [24]. The receptor
active-site radius was set equal to 11 Å from the indole nitrogen of
Trp148 responsible for the primary ligand anchoring point. The side
chain of Gln116 was not restrained during the docking calculation.
The goldscore fitness function and the distribution of torsion angles
were chosen as indicators of the quality of the docking results. Van
der Waals and hydrogen bonding radii were set respectively at 4.0
and 3.0 Å, while genetic algorithm parameters were kept at the
default value. The obtained complexes were further geometry
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J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers,
K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari,
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Acknowledgments
This research was financially supported by the Italian Ministry
of Education, University and Research (FIRB grant RBNE03H5Y to
CDM and PRIN grant 20072BTSR2 to FC and MDA). The EC Neuro-
cypres grant, the Terdismental grant nꢂ ID 16983 SAL-50 from
Regione Lombardia, the Compagnia San Paolo grant (2005-1964) to
CG, and a grant from Associazione Oasi Maria SS, Troina, Italy to FC
are also gratefully acknowledged.
References
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[24] GOLD v. 4.0, Cambridge Crystallographic data Centre: Cambridge, UK.
[25] Tripos Inc., 1699 South Hanley Rd., St. Louis, MO 63144.
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