Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-Isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1002/cmdc.201000514

A set of racemic spirocyclic quinuclidinyl-Δ²-isoxazoline derivatives was synthesized using a 1,3-dipolar cycloaddition-based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ²-Isoxazolines 3a (3-Br), 6a (3-OMe), 5a (3-Ph), 8a (3-OnPr), and 4a (3-Me) were the ligands with the highest affinity for the α7 subtype (K_i values equal to 13.5, 14.2, 25.0, 71.6, and 96.2nM, respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)-3-methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene sesquifumarate 6a were prepared using (+)-dibenzoyl-L- or (-)-dibenzoyl-D-tartaric acid as resolving agents. Enantiomer (R)-(-)-6a was found to be the eutomer, with K_i values of 4.6 and 48.7nM against rat and human α7 receptors, respectively. Spirocyclic is better! Significant binding selectivity and functional activity toward α7 nAChR subtypes were achieved with new chiral derivatives, characterized by a Δ²-isoxazoline ring linked to a quinuclidine moiety with a spirocylic junction. The eutomer of the most promising derivative in the series, the 3-O-methoxy analogue (R)-(-)-6, fit well to a model for the α7 nAChRs.

Full text of DOI:10.1002/cmdc.201000514

DOI: 10.1002/cmdc.201000514
Design, Synthesis, and Pharmacological Characterization
of Novel Spirocyclic Quinuclidinyl-D2-Isoxazoline
Derivatives as Potent and Selective Agonists of a7
Nicotinic Acetylcholine Receptors
Clelia Dallanoce,[a] Pietro Magrone,[a] Carlo Matera,[a] Fabio Frigerio,[a] Giovanni Grazioso,[a]
Marco De Amici,[a] Sergio Fucile,[b, c] Vanessa Piccari,[b] Karla Frydenvang,[d] Luca Pucci,[e]
Cecilia Gotti,[e] Francesco Clementi,[e] and Carlo De Micheli*[a]
A set of racemic spirocyclic quinuclidinyl-D2-isoxazoline deriva-
tives was synthesized using a 1,3-dipolar cycloaddition-based
approach. Target compounds were assayed for binding affinity
toward rat neuronal homomeric (a7) and heteromeric (a4b2)
nicotinic acetylcholine receptors. D2-Isoxazolines 3a (3-Br), 6a
(3-OMe), 5a (3-Ph), 8a (3-OnPr), and 4a (3-Me) were the li-
gands with the highest affinity for the a7 subtype (Ki values
equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nm, respectively), and
showed excellent a7 versus a4b2 subtype selectivity. These
compounds, tested in electrophysiological experiments against
human a7 and a4b2 receptors stably expressed in cell lines,
behaved as partial a7 agonists with varying levels of potency.
The two enantiomers of (ꢀ)-3-methoxy-1-oxa-2,7-diaza-7,10-
ethanospiro[4.5]dec-2-ene sesquifumarate 6a were prepared
using (+)-dibenzoyl-l- or (ꢁ)-dibenzoyl-d-tartaric acid as re-
solving agents. Enantiomer (R)-(ꢁ)-6a was found to be the eu-
tomer, with Ki values of 4.6 and 48.7 nm against rat and human
a7 receptors, respectively.
Introduction
Nicotinic acetylcholine receptors (nAChRs) are members of the
Cys-loop superfamily of ligand-gated ion channels, which, in
mammals, also encompasses the serotonin 5-HT3, g-aminobuty-
ric acid (GABAA and GABAC), and glycine receptors.[1] The
nAChRs, which are involved in physiological responses to the
neurotransmitter acetylcholine (ACh), are extensively distribut-
ed throughout the peripheral and central nervous system
(CNS).[2] These receptors are assemblies of five distinct trans-
membrane proteins (subunits), each characterized by a large
extracellular N-terminal domain, four membrane-spanning
alpha helices, and a short C-terminal domain.[3] Neuronal
nAChRs are localized on the postsynaptic neuronal terminals
or act as presynaptic receptors, thus indirectly modulating the
release of various key neurotransmitters including dopamine,
serotonin, GABA, glutamate, histamine, and norepinephrine.[4]
The nAChR subunits are encoded by seventeen genes, and
twelve subunits have been identified to date in the brain: nine
a (a2–a10) and three b (b2–b4) subunits, which assemble to
form either homopentamers (a7 or a9) or heteropentamers
(a9/a10 and a/b).[5] However, a relatively small subset of a/b
combinations generates functionally and physiologically rele-
vant channels. It has been postulated that a homomeric recep-
tors have five identical ACh binding sites per receptor com-
plex, each located at the subunit interface, while heteromeric
receptors have binding sites located solely at the interface be-
tween an a and a b subunit.[6] The subunit composition of
neuronal nAChRs affects both their response to exogenous li-
gands and their permeability to cations (Na+, K+ and Ca2+). In
general, receptors characterized by heteromeric combinations
of a4 and b2 subunits, with putative (a4)2(b2)3 stoichiometry,
show high affinity for the agonist (S)-nicotine. Conversely, ho-
mopentameric receptors, that is, the a7 subtype, strongly bind
antagonists such as a-bungarotoxin and methyllycaconitine. In
terms of intrinsic Ca2+ permeability, neuronal heteromeric a/b
nAChRs have a fractional Ca2+ current of 2–5%,[7] which
increases to 6–12% for homomeric a7 receptors.[7]
[a] Dr. C. Dallanoce, Dr. P. Magrone, Dr. C. Matera, Dr. F. Frigerio,
Dr. G. Grazioso, Prof. M. De Amici, Prof. C. De Micheli
Dipartimento di Scienze Farmaceutiche Pietro Pratesi
Universitꢀ degli Studi di Milano
Via Mangiagalli 25, 20133 Milan (Italy)
Fax: (+39)02-50319326
E-mail: carlo.demicheli@unimi.it
[b] Prof. S. Fucile, Dr. V. Piccari
Dipartimento di Fisiologia e Farmacologia
Universitꢀ di Roma La Sapienza
Piazzale A. Moro 5, 00185 Rome (Italy)
[c] Prof. S. Fucile
Istituto Neurologico Mediterraneo Neuromed
Via Atinese 18, 86077 Pozzilli (Italy)
[d] Prof. K. Frydenvang
Department of Medicinal Chemistry
The Faculty of Pharmaceutical Sciences, University of Copenhagen
Universitetsparken 2, 2100 Copenhagen (Denmark)
[e] Dr. L. Pucci, Dr. C. Gotti, Prof. F. Clementi
CNR, Istituto di Neuroscienze, Farmacologia Cellulare e Molecolare
Dipartimento Farmacologia, Chemioterapia e Tossicologia Medica
Universitꢀ degli Studi di Milano
Via Vanvitelli 32, 20129 Milan (Italy)
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The heteromeric a4b2 sub-
type is, by far, the most widely
distributed nAChR in the CNS,
while among the homomeric re-
ceptor population in the brain,
expression of the a7 subtype is
highest. The two subtypes are
differently localized throughout
the CNS; however, they are
coexpressed in the hippocampus
and the cortex,[4] which play a
major role in cognitive processes
such as learning and memory
consolidation. Ligands acting at
these two nAChR subtypes
could be promising drug candi-
dates for therapeutic interven-
tion in a range of pathologies,
including Alzheimer’s and Par-
kinson’s diseases, epilepsy, schiz-
ophrenia, Tourette’s syndrome,
attention deficit hyperactivity
disorder, pain, depression, and
tobacco addiction.[2,8] Initially,
a4b2 nAChRs were the privi-
Figure 1. Structures of selected reference a7 nAChR agonists/partial agonists and of the spirocyclic quinuclidinyl
derivatives investigated in this study.
leged molecular target of analogues of natural compounds
such as nicotine, epibatidine, and cytisine that were designed
by pharma companies and academia with the aim of discover-
ing selective agonists for development as drug candidates to
treat a number of neurodegenerative diseases.[9] Recently, a
growing number of reports has focused on derivatives de-
signed as selective a7 nAChR ligands.[3b,10] The interest in a7
selective agonists/partial agonists is mainly related to deficits
in auditory sensory processing, which lead to a state of sensory
overload and contribute to the attentional and cognitive dys-
functions that are the hallmark of various CNS diseases such as
schizophrenia.[11] Moreover, the administration of a7 selective
agonists, either alone or in association with antipsychotic
drugs, exhibited in vivo efficacy in preclinical models predictive
of cognitive-enhancing and antipsychotic-like activities.[12] Fur-
thermore, there is growing evidence that ligands selectively
targeting a7 nAChRs may modulate cellular functions beyond
synaptic transmission, making them potentially useful drugs
for innovative therapeutic indications other than CNS patholo-
gies. As the presence of a7 nAChRs in peripheral cholinergic
pathways has been demonstrated,[13] selective agonists of this
subtype are emerging as promising drug candidates for the
treatment of inflammation and pain.[14] Finally, a rapidly pro-
gressing research line is the allosteric modulation of nAChRs,
primarily focused on positive modulators of the a7 subtype,
that is, compounds which lack intrinsic efficacy for direct stim-
ulation of the receptor but enhance agonist-induced receptor
activation.[8c,15]
collected and are shown in Figure 1, including the quinuclidine
ligands AR-R17779,[10a,17] PNU-282987,[18] JN403,[19] and TC-
5619,[12b] and azabicyclic or diazabicyclic compounds
SSR180711[20] and PHA-709829.[21]
As part of an ongoing research program devoted to the
study of novel heterocyclic derivatives targeting neuronal
nAChRs,[22] we report herein the results obtained for a selected
group of nicotinic ligands which have been described in a
patent application.[22d] This paper deals with the synthesis of a
set of spirocyclic quinuclidinyl-D2-isoxazolines (1a–10a) and
two quinuclidinyl-isoxazolin-3-ones (11 a and 12a) whose
structure is reported in Figure 1. The corresponding quaternary
methyl iodides 1b–4b, 6b–10b, and 12b (Figure 1), ligands
potentially acting at peripheral nAChRs, were also prepared
and tested.
The target compounds were initially evaluated for their
binding affinity toward the a7 and a4b2 nAChR subtypes, fol-
lowed by a study of the electrophysiological profile of the
most relevant ligands. Furthermore, the two enantiomers of
the most promising a7 selective agonist, 6a, were prepared
and tested. The (R)-configuration was attributed to the euto-
mer by X-ray structural analysis, and its binding mode was in-
vestigated by molecular docking in a model of the a7 nAChR
subtype.
Results and Discussion
Our approach to target compounds is based on the versatile
1,3-dipolar cycloaddition of nitrile oxides to suitable dipolaro-
philes to yield D2-isoxazoline derivatives. We have used this
strategy extensively over the last decade to synthesize a large
number of biologically active compounds.[23] In the present
The rigid quinuclidine scaffold or similar bicyclic tertiary
bases represent first choice core fragments for molecular rec-
ognition of ligands by the a7 channel protein.[16] Structures of
some of the most promising full/partial a7 agonists have been
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Novel a7 Nicotinic Acetylcholine Receptor Agonists
to produce the corresponding alkoxy N-boranyl D2-
isoxazolines 18–22, which were readily transformed
into final tertiary bases 6–10. As illustrated in
Scheme 2, standard catalytic hydrogenation of 3-ben-
zyloxy-D2-isoxazoline 21 produced the cyclic carba-
mate 23,[28] which was then transformed into its N-
methylated analogue 24 by treatment with iodome-
thane in an acetone suspension of potassium carbon-
ate. Removal of the boranyl protecting group from
23 and 24 gave free bases 11 and 12 in high yield.
Tertiary amines 1–12 were treated with fumaric acid
to give the related crystalline salts 1a–12a. Alterna-
tively, tertiary amines 1–4, 6–10, and 12 were com-
bined with methyl iodide to afford the corresponding
iodomethylates 1b–4b, 6b–10b, and 12b
(Scheme 2).
Target compounds 1a–12a, 3b, 4b, and 6b–10b
were assessed for binding affinity to a7 and a4b2 rat
nAChR subtypes using
[
125I]a-bungarotoxin and
[3H]epibatidine as radioligands, respectively. Ki values,
calculated from competition curves using the
LIGAND program,[29] are presented in Table 1, with
values from a7 nAChR agonists in Figure 1 that were
used as reference compounds. These data show that
the simultaneous presence of the quinuclidinyl and
D2-isoxazolinyl moieties, coupled with the stereoelec-
tronic features of the substituent at position 3 of the
spirocyclic system, are crucial structural requirements
Scheme 1. Reagents and conditions: a) Et2OCC(Cl)=NOH (4 equiv), NaHCO3, dioxane,
reflux, 4 d, 13%; b) NaBH4, EtOH, RT, 5 h, 48%; c) 1.0 m BH3·THF complex, 08C!RT,
30 min, 84%; d) Br2C=NOH (3 equiv), K2CO3, EtOAc, RT, 5 d, 48%; e) CH3C(Cl)=NOH
(9 equiv), Et3N, CH2Cl2, RT, 48 h, 30%; f) C6H5CH=NOH (5 equiv), NaOCl(aq), CH2Cl2, RT, 5 d,
16%; g) CF3CO2H, acetone, RT, 12 h, 70%; h) MeOH (or EtOH), K2CO3, RT, 16 h, 81%;
i) NaH, THF, nPrOH (or C6H5CH2OH or HCꢂCCH2OH), 08C!RT, 16 h, 74%.
study, the racemic spirocylic D2-isoxazolines and isoxazolidin-3- for productive molecular recognition by the homomeric a7 re-
ones were synthesized by reacting 3-methylene-1-azabicyclo- ceptor subtype. Indeed, in the series of fumarates, D2-isoxazo-
[2.2.2]octane 13, or its borane complex 14, with various nitrile lines 3a (3-Br), 6a (3-OCH3), 5a (3-C6H5), 8a (3-OnPr), and 4a
oxides under a variety of experimental conditions (Scheme 1). (3-CH3) emerged as the ligands exhibiting the highest affinity
Initially, olefin 13,[24] in dioxane at reflux, was combined with for the a7 subtype (Ki values equal to 13.5, 14.2, 25.0, 71.6, and
excess ethoxycarbonylformonitrile oxide, generated in situ 96.2 nm, respectively). In addition, at heteromeric a4b2
with sodium bicarbonate from its stable precursor, ethyl 2- nAChRs, the same set of compounds displayed Ki values rang-
chloro-2-(hydroximino)-acetate.[25] This pericyclic process gave ing from 0.64 mm (3a) to 140 mm (7a).
the expected ester 1 in very low yield (13%); the cycloadduct
was converted into the related primary alcohol 2 by treatment 4a) possess remarkable a7 vs. a4b2 selectivity (47-, 554-, 880-,
with sodium borohydride. 922-, and 95-fold, respectively). A comparison of the data for
As shown, the above cited derivatives (3a, 6a, 5a, 8a, and
In order to improve the yield, dipolarophile 13 was trans- iodomethylates 1b–4b, 6b–10b, and 12b with data for their
formed into the more reactive N-boranyl alkene 14[26] using a corresponding fumarates demonstrated a general decrease in
1.0m solution of borane–THF complex (BH3·THF). As expected, binding affinity at both nAChR subtypes. Of these, 3-methoxy
reaction of 14 with bromonitrile oxide[27a] and acetonitrile derivative 6b emerged as the most selective ligand (about
oxide[27b] afforded spirocyclic cycloadducts 15 and 16 in higher 1900-fold), due to a six-fold reduction in affinity at the a7 sub-
yields (48% and 30%, respectively, Scheme 1). The correspond- type (Ki =91.4 nm), combined with a substantial inability to
ing 3-phenyl-D2-isoxazoline 17 was obtained in 16% yield fol- bind the a4b2 receptor (Ki =175.4 mm).
lowing the reaction of 14 with benzonitrile oxide, generated in
Based on the previously discussed binding and selectivity
situ from benzaldoxime and aqueous sodium hypochlorite.[27c] data, we wanted to fully characterize the cholinergic profile of
The N-boranyl moiety of intermediates 15, 16, and 17 was the most promising derivative in the series, the 3-methoxy-D2-
easily removed by treatment with an acetone solution of tri- isoxazoline 6a. To this end, we tested 6a with two additional
fluoroacetic acid, yielding the desired quinuclidinyl free bases nAChRs, the human a3b4 subtype, expressed in SH-SY5Y cells,
3, 4, and 5, respectively. Notably, compounds 4 and 5 were and the human a1b1gd receptor, expressed in TE671 cells. In
previously prepared and tested as potential acetylcholinester- addition, we evaluated the compound against five human
ase inhibitors.[24]
cloned muscarinic receptors (mAChRs, M1–M5). Derivative 6a
The intermediate D2-isoxazoline 15 underwent efficient nu- showed marginal binding affinity for both a3b4 (Ki =15 mm)
cleophilic displacement of bromine by various alcohols in basic and a1b1gd subtypes (Ki =78 mm), and behaved as a low affini-
medium (potassium carbonate or sodium hydride suspension) ty ligand for muscarinic receptors [Ki values between 1.95 mm
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Table 1. Affinity of derivatives 1a–12a, 3b, 4b, and 6b–10b for native
a4b2 and a7 nAChR subtypes present in rat cortical membranes, labeled
by [3H]epibatidine and [125I]a-bungarotoxin.
Compd
Ki[a] [nm], n=4
Selectivity
a7
125I]a-BgTx
a4b2
[3H]Epibatidine
a7/a4b2
[
1a
1b
2a
2b
132000 (63)
n.t.[b]
1450 (39)
n.t.[b]
34000 (14)
n.t.[b]
120000 (51)
n.t.[b]
0.3
83
3a
3b
4a
4b
5a
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11 a
13.5 (29)
109 (23)
96.2 (29)
756 (27)
25.0 (35)
14.2 (34)
91.4 (36)
194 (53)
439 (46)
71.6 (44)
833 (55)
326 (48)
3300 (43)
150 (24)
4250 (35)
5700 (43)
250 (55)
n.t.[b]
636 (17)
47
298
95
269
880
32500 (18)
9090 (13)
203000 (18)
22000 (22)
7860 (16) 554
175400 (17)
140000 (64)
204000 (61)
66000 (40)
173000 (53)
63000 (53)
106000 (53)
32100 (15)
148600 (19)
20000 (19)
2800 (18)
n.t.[b]
1919
721
465
922
208
193
32
214
35
3.5
12a
12b
11
(ꢀ)-Epibatidine
a-BgTx
AR-R17779
PNU-282987
SSR180711
TC-5619
0.71 (17)
0.90[c] (20)
92[e]
0.021[c] (15)
n.d.[d]
16000[e]
174
24[f]
14%[g]
22[h]
>5000[i]
1[l]
2100[l]
2100
[a] Ki values were derived from four competition binding experiments.
Numbers in brackets refer to the percent coefficients of variation. The
data on some known alpha 7 agonists have been added for comparison.
[b] n.t., not tested. [c] Kd values. [d] n.d., not determined. [e] Ref-
erence [17]. [f] Reference [30]. [g] Inhibition at 1 mm.[30] [h] Reference [20].
[i] IC50 value for the human nAChR subtype.[20] [l] Reference [12b].
Scheme 2. Reagents and conditions: a) H2, 10% Pd/C, MeOH, 1 atm, 5 h,
97%; b) CF3CO2H, acetone, RT, 12 h, 54%; c) CH3I, K2CO3, acetone, RT, 16 h,
88%; d) C4H4O4, MeOH, 12 h, 100%; e) CH3I, MeOH, 12 h, 100%; f) (R,R)-(ꢁ)-
O,O’-dibenzoyl-l-tartaric acid, EtOH (crystallizations from MeOH), 5 d, 18%;
g) 20% NaOH, CH2Cl2 extraction, 15 min, 100%; h) (S,S)-(+)-O,O’-dibenzoyl-d-
tartaric acid, EtOH (crystallizations from MeOH), 5 d, 18%.
(M3) and 6.55 mm (M4)]. As the biological data collected for 6a
appeared very promising, we tackled the preparation of its
single enantiomers. Racemic base (ꢀ)-6 was reacted with the
commercially available (R,R)-(ꢁ)-O,O’-dibenzoyl-l- or (S,S)-(+)-
O,O’-dibenzoyl-d-tartaric acid. Fractional crystallization of the
salts allowed us to obtain (R)-(ꢁ)-6 and (S)-(+)-6 in high enan-
tiomeric excess (Scheme 2). The absolute configuration of the
enantiomers was assigned by single crystal X-ray analysis of
the salt between (R)-(ꢁ)-6 and (R,R)-(ꢁ)-O,O’-dibenzoyl-l-tarta-
ric acid. The perspective drawing (ORTEP) of this salt is shown
in Figure 2.
Table 2. Comparison of the binding affinities of (R)-(ꢁ)-6a and (S)-(+)-6a
to membrane-bound a7 receptors in rat and human species.
Compd
Ki[a] [nm] ([125I]a-BgTx)
a7 (human)
a7 (rat)
(R)-(ꢁ)-6a
(S)-(+)-6a
E.R.[b]
4.6 (35)
110 (40)
24
48.7 (37)
672 (36)
14
[a] Ki values, determined for membranes obtained from rat cortex or
human IMR32 neuroblastoma cells, were derived from three competition
binding experiments. Numbers in brackets refer to the percent coeffi-
cients of variation. [b] E.R., eudismic ratio: (S)-(+)/(R)-(ꢁ).
Tertiary amines (R)-(ꢁ)-6 and (S)-(+)-6 were transformed into
sesquifumarates (R)-(ꢁ)-6a and (S)-(+)-6a, which were assayed
for binding affinity against membranes containing rat cortical
a7 receptors or a7 receptors obtained from human IMR32
neuroblastoma cells (Table 2). The (R)-(ꢁ)-6a enantiomer was
shown to be the eutomer, exhibiting a ten-fold higher binding
affinity for rat a7 (Ki =4.6 nm) than human a7 (Ki =48.7 nm) re-
ceptors. Moreover, the molecular recognition of 6a was char-
acterized by a higher degree of enantio-discrimination at rat
a7, as indicated by the eudismic ratios of 24 and 14 (rat a7
and human a7, respectively; see Table 2). Notably, the spatial
arrangement of the substituents around the stereogenic
center of (R)-6a is identical to that of the selective a7 agonist
(S)-AR-R17779 (Figure 1).
Compound (ꢀ)-6a, its eutomer (R)-(ꢁ)-6a, and the high af-
finity a7 nAChR ligands 3a, 5a, and 8a were submitted to
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Novel a7 Nicotinic Acetylcholine Receptor Agonists
binding affinity profile. Next, we tested eutomer (R)-(ꢁ)-6a in
the same cell lines, finding similar activation patterns (Fig-
ure 3b) with the remarkable difference of a higher response
toward human a7 nAChRs when compared to that of 200 mm
ACh (177ꢀ30%, n=6) and an EC50 value of 16ꢀ9 mm. As
(ꢀ)-6a and eutomer (R)-(ꢁ)-6a induced currents at human a7
nAChRs even in the absence of the exogenously added ago-
nist, it suggests that this nicotinic agonist recognizes the or-
thosteric site of the a7 receptor, although an additional effect
mediated by an allosteric site cannot be ruled out.
High concentrations of (ꢀ)-6a were able to activate human
5-HT3 receptors. In fact, inward currents were evoked by (ꢀ)-
6a (EC50 >100 mm, Figure 3c) when applied to GH4C1 cells
transfected with 5-HT3 or 5-HT3A and 5-HT3B subunit cDNAs ex-
pressing homomeric 5-HT3A and heteromeric 5-HT3AB receptors
(n=6 and n=5, respectively). The maximal current amplitudes
mediated by 5-HT3 receptors activated by 200 mm (ꢀ)-6a were
much smaller (12ꢀ5% and 5.7ꢀ0.9%) than those evoked by
200 mm serotonin (5-HT) in the same cells, indicating that the
compound is a very weak partial agonist of these receptors
(Figure 3d).
Similarly to (ꢀ)-6a, 3a was able to substantially activate
human a7 nAChRs (Figure 4a), with a maximal current ampli-
tude of 542ꢀ33 pA elicited by 200 mm 3a (130ꢀ8% of the re-
sponse induced by 200 mm ACh, n=6). However, analysis of
the dose–response relationship revealed an EC50 value higher
than 100 mm, indicating a much lower apparent affinity of 3a
compared to (ꢀ)-6a. In addition, 3a showed higher affinity in
binding experiments towards both the human a3b4 subtype
(Ki =1.1 mm) and the human a1b1gd receptor (Ki =26 mm) than
6a. Also, 3-bromo derivative 3a emerged as a selective agonist
for the a7 nAChRs, being unable to elicit significant inward
currents when applied to human a4b2 HEK cells (2.4ꢀ0.9% of
the response induced by 200 mm ACh, n=7, Figure 4b) and
human a3b4-transfected HEK cells (1.4ꢀ0.9% of ACh-induced
response, n=7, not shown). Likewise, derivative 3a weakly ac-
tivated human 5-HT3A and 5-HT3AB receptors, with maximal cur-
rent amplitudes representing 2.2ꢀ1.5% and 18ꢀ6%, respec-
tively (n=6, Figure 4c), of the current amplitudes evoked by
200 mm 5-HT in the same cell lines (380ꢀ30 and 1700ꢀ
700 pA, respectively).
Figure 2. Perspective drawing (ORTEP[41]) of the salt formed by (R)-6 and
(R,R)-(ꢁ)-O,O’-dibenzoyl-l-tartaric acid. Displacement ellipsoids of nonhydro-
gen atoms are shown at the 50% probability level. Hydrogen atoms are rep-
resented by spheres of arbitrary size. a) (R)-6; b) (R,R)-O,O’-dibenzoyl-l-tar-
trate.
electrophysiological experiments to assess their functional pro-
files. The compounds were evaluated against human a7
nAChRs expressed by stable transfection in the rat anterior pi-
tuitary GH4C1 cell line,[31a] as well as against human a4b2 and
a3b4 nAChRs heterologously expressed in the human HEK 293
cell line.[31b] In addition, we evaluated the ability of 6a and 3a
to activate or inhibit 5-HT3 serotonin channels using GH4C1
cells transiently transfected with the human 5-HT3A and 5-HT3B
cDNAs. In fact, some a7 selective agonists have been shown
to interact at 5HT3 receptors, behaving as antagonists or partial
agonists.[10b] Derivative (ꢀ)-6a was able to evoke inward
whole-cell currents when applied to human a7 GH4C1 cells
clamped at ꢁ70 mV (Figure 3a). The dose–response relation-
ship indicated an EC50 value of 7.7ꢀ0.6 mm, with a mean maxi-
mal amplitude of 334ꢀ21 pA, which was 80ꢀ5% of the mean
current amplitude elicited by 200 mm ACh (n=7, Figure 3d). In
contrast, (ꢀ)-6a evoked negligible currents in human a4b2
HEK cells, with a maximal mean amplitude of 7ꢀ2 pA (0.3ꢀ
0.1% of the ACh-induced response, n=6), and in human
a3b4-transfected HEK cells (3.3ꢀ0.7% of ACh-induced re-
sponse, n=9, data not shown). These data indicate that
(ꢀ)-6a behaves as a partial agonist at a7 nAChRs, exhibiting
noteworthy ability to discriminate between the homomeric a7
and the heteromeric nAChR subtypes and thus confirming the
The 3-phenyl and 3-n-propoxy analogues (5a and 8a, re-
spectively) were also tested for their ability to evoke whole-cell
currents mediated by human a7 and a4b2 nAChRs. These de-
rivatives were unable to activate the a4b2 nAChR subtype and
acted as weak partial agonists toward a7 nAChRs. Maximal cur-
rent amplitudes evoked by 5a and 8a (200 mm) represented
32ꢀ8% and 38ꢀ5% of the response induced by 200 mm ACh
(n=6, data not shown).
In summary, of the compounds evaluated, methoxy deriva-
tive 6a possesses the best structural requirements to act as an
agonist, as characterized by high affinity and selectivity for the
a7 nAChRs. The substituent at position 3 of the D2-isoxazoline
ring affected not only the affinities but also the functional fea-
tures of derivatives towards the a7 subtype. In fact, gradual
increase in the steric bulkiness of the substituent, from bromo
(3a) and methoxy (6a) to phenyl (5a) and n-propoxy (8a),
ChemMedChem 2011, 6, 889 – 903
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C. De Micheli et al.
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with an aromatic box delimited
by the four amino acid residues
Trp54, Trp148, Tyr187, and
Tyr194 (Figure 5).
In addition, we detected a hy-
drogen bond between the pro-
tonated nitrogen of the quinucli-
dine moiety and the side chain
of Tyr92. Moreover, the methoxy
group appended to the D2-isoxa-
zoline nucleus of (R)-6 projected
into a lipophilic pocket defined
by residues Cys189–Cys190 (SꢁS
bridge), Leu119, Leu108, Val107,
Gln116, and Tyr117. An addition-
al hydrogen bond between the
3-methoxy group of (R)-6a and
the side chain of Gln116 further
stabilized the proposed ligand
binding mode (Figure 5). The in-
teraction of distomer (S)-6a with
the binding site of the a7 recep-
tor subtype was similar to that
displayed by the eutomer. How-
ever, due to the opposite orien-
tation of the spirocyclic moiety,
the methoxy group of (S)-6a
was unable to form a hydrogen
bond with the side chain of
Gln116, which could account for
the moderate eudismic ratio
found for 6a at human a7
nAChRs.
Figure 3. Effects of 6a on different types of human ligand-gated receptors. a) (top) Typical traces evoked by ACh
(black bar, 200 mm) or (ꢀ)-6a (gray bar, 200 mm) on GH4C1 cells stably transfected with human a7 nAChR subunit
(*) or on HEK 293 cells stably transfected with human a4 and b2 nAChR subunits (*), as indicated. Cells were
held at ꢁ70 mV; (bottom) (ꢀ)-6a dose-normalized response relationship for human a7 nAChR (*, n=7) and for
human a4b2 nAChR (*, n=6). The Hill equation was best fitted to data obtained from a7 nAChRs, yielding
EC50 =7.7ꢀ0.6 mm and nH =3.5ꢀ0.7; b) (top) typical traces evoked by ACh or (R)-(ꢁ)-6a in a7 cells (*, n=6) or
in a4b2 cells (*, n=6), as indicated; (bottom) (R)-(ꢁ)-6a dose-normalized response relationship for human a7
nAChR (*) and for human a4b2 nAChR (*). Hill equation was best fitted to data obtained from a7 nAChRs,
yielding EC50 =17ꢀ10 mm and nH =1.2ꢀ0.9; c) (top) typical traces evoked by 200 mm 5-HT or 200 mm (ꢀ)-6a in
GH4C1 cells transiently transfected with the human 5-HT3A subunit (*) or with human 5-HT3A and 5-HT3B subunits
(*), as indicated; (bottom) (ꢀ)-6a dose-normalized response relationship for human 5-HT3A receptors (*, n=6)
and for human 5-HT3AB receptors (*, n=6); d) histograms showing the ratio between the mean current ampli-
tude evoked by 200 mm (ꢀ)-6a and 200 mm ACh on nicotinic receptors (left, as indicated) or 200 mm 5-HT on 5-
HT3 receptors (right, as indicated).
Conclusions
In this study, a set of spirocyclic
quinuclidinyl-D2-isoxazolines and
two quinuclidinyl-isoxazolidin-3-
caused a significant decrease in the a7-mediated electrophy-
siological response. This result is expected to be even more
severe for the remaining O-alkylated analogues 7a, 9a, and
10a, which are characterized by relatively moderate affinities
(Ki =194, 326, and 150 nm, respectively). The same gradual
drop in a7 affinity (Ki =91.4, 439, 833, 3300, and 4250 nm, re-
spectively) was observed for the set of O-alkylated methylam-
monium iodides 6b–10b which, presumably, is also associated
with a stepwise reduced ability to activate the receptors.
ones were designed and synthesized by exploiting 1,3-dipolar
cycloaddition chemistry. Two groups of target compounds—
the tertiary bases assayed as fumarates and their correspond-
ing methyl iodides—were prepared and tested for binding af-
finity at rat a7 and a4b2 nAChR subtypes. Among the deriva-
tives studied, (ꢀ)-3-methoxy-1-oxa-2,7-diaza-7,10-ethanospiro-
[4.5]dec-2-ene sesquifumarate 6a was characterized as a high
affinity a7 nAChR partial agonist with relevant a7 versus a4b2
selectivity in both binding and functional experiments. More-
over, derivative 6a did not bind significantly to a3b4 or
a1b1gd nAChRs and to the five mAChRs, and marginally acti-
vated human 5-HT3 receptors expressed in cell lines. We pre-
pared the two enantiomers of (ꢀ)-6a by a resolution protocol,
assigned absolute R configuration to the eutomer by X-ray
analysis, and determined the enantioselectivity ratio (R)-6a/(S)-
6a at rat as well as at human a7 nAChR subtypes. At present,
pharmacokinetic profiling of (ꢀ)-6a is under evaluation, as
well as the in vivo activity in animal models to assess applica-
The two enantiomers of 6a were docked into the binding
cleft of the human a7 homology model that we recently pub-
lished,[22b] with the aim of accounting for their differences in af-
finity. This model has been validated for nicotinic agonists
characterized by various structural skeletons.[22b] The binding
mode of (R)-6 and (S)-6 was refined by cluster analysis (CA)
and molecular mechanics geometry optimization. Similar to
other a7 agonists/partial agonists bearing a quinuclidine
group, the molecular fragment of (R)-6 efficiently interacted
894
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Novel a7 Nicotinic Acetylcholine Receptor Agonists
Figure 5. Binding modes of (R)-6 and (S)-6 in the active site of a7 nAChR.
Receptor model residues are depicted as a stick model with carbon atoms
colored in gray. The carbon atoms of (R)-6 and (S)-6 are green and cyan, re-
spectively. Some residues have been omitted for clarity.
bility of this compound to treat CNS cognitive diseases and
neuropathic pain.
Experimental Section
Chemistry
Materials and methods: Alkene 13,[24] ethyl 2-chloro-2-(hydrox-
imino)-acetate,[25] dibromoformaldoxime,[27a] and 1-chloroacetaldox-
1
ime[27b] were prepared according to literature procedures. H NMR
and 13C NMR spectra were recorded with a Varian Mercury 300 (1H:
300.063; 13C: 75.451 MHz) spectrometer at 208C. Chemical shifts
(d) are expressed in ppm, with coupling constants (J) in Hz. TLC
analyses were performed on commercial silica gel 60 F254 alumi-
num sheets; spots were further evidenced by spraying with a
dilute alkaline potassium permanganate solution or, for tertiary
amines, with Dragendorff reagent. Melting points were determined
on a model B 540 Bꢁchi apparatus and are uncorrected. Rotary
power determinations (sodium D line, 589 nm) were carried out
with a Jasco P-1010 polarimeter coupled with a Huber thermostat.
Chiral HPLC analyses were performed with a Jasco PU-980 pump
equipped with a UV-Vis detector (Jasco UV-975) using a Daicel Chir-
alpak AD column (0.46 cm
ꢂ 25 cm). Microanalyses (C, H, N)
agreed with theoretical values within ꢀ0.4%.
(ꢀ)-3-Ethyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene carb-
oxylate (1): A solution of ethyl 2-chloro-2-(hydroximino)-acetate
(2.46 g, 16.24 mmol) in dioxane (40 mL) was added to a magneti-
cally stirred suspension of 13 (1.0 g, 8.12 mmol) and NaHCO3
(6.82 g, 81.2 mmol) in dioxane (50 mL). The reaction mixture was
heated at reflux for 4 d with further addition of ethyl 2-chloro-2-
(hydroximino)-acetate (2.46 g, 16.24 mmol) in dioxane (40 mL).
After filtration and removal of the solvent at reduced pressure, the
residue was purified by silica gel column chromatography (CH2Cl2/
MeOH, 9:1) to afford cycloadduct 1 (251 mg, 13%) as a pale yellow
Figure 4. Effects of 3a on different types of human ligand-gated receptors.
a) (top) Typical traces evoked by ACh (black bar, 200 mm) or 3a (gray bar,
200 mm) in GH4C1 cells stably transfected with human a7 nAChR subunit
(*) or on HEK 293 cells stably transfected with human a4 and b2 nAChR
subunits (*), as indicated. Cells were held at ꢁ70 mV; (bottom) 3a dose-
normalized response relationship for human a7 nAChR (*, n=6) and for
human a4b2 nAChR (*, n=7); b) (top) typical traces evoked by 200 mm 5-
HT or 200 mm 3a in GH4C1 cells transiently transfected with human 5-HT3A
subunit (*) or with human 5-HT3A and 5-HT3B subunits (*), as indicated;
(bottom) 3a dose-normalized response relationship for human 5-HT3A recep-
tors (*, n=5) and for human 5-HT3AB receptors (*, n=5); c) histograms
showing the ratio between the mean current amplitude evoked by 200 mm
3a and 200 mm ACh on nicotinic receptors (left, as indicated) or 200 mm
5-HT on 5-HT3 receptors (right, as indicated).
1
viscous oil: Rf =0.67 (CH2Cl2/MeOH, 4:1); H NMR (300 MHz, CDCl3):
d=1.29 (t, J=7.0, 3H), 1.42 (m, 1H), 1.60 (m, 2H), 1.91 (br s, 1H),
2.07 (m, 1H), 2.75 (m, 2H), 2.87 (m, 3H), 2.91 (d, J=18.3, 1H), 3.22
(d, J=15.3, 1H), 3.27 (d, J=18.3, 1H), 4.27 ppm (q, J=7.0, 2H);
13C NMR (75 MHz, CDCl3): d=14.4, 21.3, 23.6, 31.3, 43.4, 46.6, 47.0,
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C. De Micheli et al.
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62.2, 63.2, 90.6, 150.7, 161.1 ppm; Anal. calcd for C12H18N2O3: C
60.49, H 7.61, N 11.76, found: C 60.80, H 7.28, N 12.03.
2.91–3.09 (m, 5H), 3.02 (d, J=17.2, 1H), 3.37 ppm (dd, J=2.5 and
14.6, 1H); 13C NMR (75 MHz, CDCl3): d=13.3, 20.5, 23.3, 30.7, 48.5,
51.2, 52.3, 63.3, 84.7, 154.2 ppm; Anal. calcd for C10H19BN2O: C
61.88, H 9.87, N 14.43, found: C 61.55, H 10.18, N 14.62.
(ꢀ)-3-Hydroxymethyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene (2): NaBH4 (480 mg, 12.69 mmol) was added portionwise to
a magnetically stirred solution of ester 1 (400 mg, 1.68 mmol) in
EtOH (10 mL) at RT. After 5 h, 5 mL of 2 n HCl were added (pHꢃ3),
the solvent was evaporated, and the acidic residue was extracted
with EtOAc (3ꢂ5 mL). The residual aqueous phase, basified by por-
tionwise addition of K2CO3, was then repeatedly extracted with
CH2Cl2 (15ꢂ5 mL). The pooled organic phases were dried over
anhyd Na2SO4, then filtered and concentrated in vacuo to provide
the primary alcohol 2 (158 mg, 48%) as a yellow viscous oil: Rf =
0.27 (CH2Cl2/MeOH, 4:1); 1H NMR (300 MHz, CD3OD): d=1.57 (m,
1H), 1.72 (m, 2H), 1.93 (m, 1H), 2.09 (m, 1H), 2.74–2.88 (m, 4H),
2.92 (d, J=17.9, 1H), 3.03 (d, J=15.0, 1H), 3.09 (d, J=15.0, 1H),
3.25 (d, J=17.9, 1H), 4.26 ppm (s, 2H); 13C NMR (75 MHz, CDCl3):
d=21.5, 23.8, 31.2, 45.0, 46.6, 46.9, 58.6, 63.5, 86.8, 157.9 ppm;
Anal. calcd for C10H16N2O2: C 61.20, H 8.22, N 14.27, found: C 60.92,
H 8.51, N 14.46.
(ꢀ)-3-Phenyl-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
dec-2-ene (17): NaClO (4 mL, 10% aq solution) was added drop-
wise to a solution of 14 (500 mg, 3.65 mmol) and benzaldoxime
(624 mg, 5.48 mmol) in CH2Cl2 (5 mL). The reaction mixture was
stirred at RT for 5 d with further addition of benzaldoxime (5ꢂ
500 mg) and NaClO (5ꢂ4 mL, 10% aqueous solution). The phases
were separated and the aqueous layer was extracted with CH2Cl2
(3ꢂ10 mL). After standard workup, the residue was purified by
silica gel column chromatography (PE/EtOAc, 7:3) to afford cyclo-
adduct 17 (150 mg, 16%), which crystallized as colorless prisms
from EtOAc (mp 181–1838C): Rf =0.23 (PE/EtOAc, 7:3); 1H NMR
(300 MHz, CDCl3): d=1.81 (m, 2H), 2.03 (m, 1H), 2.29 (m, 1H), 2.45
(m, 1H), 3.17–3.48 (m, 5H), 3.27 (d, J=17.2, 1H), 3.50 (d, J=17.2,
1H), 3.68 (dd, J=2.2 and 14.3, 1H), 7.43 (m, 3H), 7.65 ppm (m,
2H); 13C NMR (75 MHz, CDCl3): d=20.2, 22.0, 31.5, 45.2, 47.8, 49.5,
62.2, 84.6, 126.7, 129.0, 130.7, 156.0 ppm; Anal. calcd for
C15H21BN2O: C 70.33, H 8.26, N 10.94, found: C 70.59, H 8.38, N
10.71.
Borane-3-methylene-1-azabicyclo[2.2.2]octane complex (14): A
1.0 m solution of BH3·THF (18 mL) was added under nitrogen to a
stirred solution of 13 (2.20 g, 17.9 mmol) in dry THF (10 mL) at 08C.
After 30 min, the mixture was concentrated in vacuo and the resi-
due was purified by silica gel column chromatography (PE/EtOAc,
9:1) to yield 14 as a colorless solid (2.05 g, 84%), which was crystal-
lized from n-hexane/Et2O (mp 70–728C, literature:[26] 72–738C): Rf =
0.64 (PE/EtOAc, 4:1); 1H NMR (300 MHz, CDCl3): d=1.86 (m, 4H),
2.56 (m, 1H), 3.04 (m, 4H), 3.63 (br s, 2H), 4.74 (br s, 1H), 4.91 ppm
(br s, 1H); 13C NMR (75 MHz, CDCl3): d=26.4, 31.4, 54.1, 61.2, 106.7,
144.7 ppm.
(ꢀ)-3-Methoxy-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
dec-2-ene (18): A stirred suspension of bromo-D2-isoxazoline 15
(1.00 g, 3.86 mmol) and K2CO3 (5.34 g, 38.6 mmol) in MeOH (60 mL)
was stirred at RT for 16 h. After addition of Celiteꢃ and filtration
under vacuum, the crude filtrate was submitted to silica gel
column chromatography (PE/EtOAc, 1:4), affording desired me-
thoxy derivative 18 (654 mg, 81%) as colorless prisms from EtOAc/
1
Et2O; mp 108.5–1108C; Rf =0.34 (PE/EtOAc, 1:4); H NMR (300 MHz,
CDCl3): d=1.70 (m, 2H), 1.96 (m, 1H), 2.27 (br s, 1H), 2.34 (m, 1H),
2.86 (d, J=16.5, 1H), 2.95–3.14 (m, 6H), 3.37 (dd, J=2.2 and 14.3,
1H), 3.85 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=20.2, 22.3, 30.5,
42.5, 51.8, 52.5, 57.7, 65.8, 84.5, 166.6 ppm; Anal. calcd for
C10H19BN2O2: C 57.17, H 9.12, N 13.33, found: C 56.95, H 9.31, N
13.59.
(ꢀ)-3-Bromo-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]dec-
2-ene (15): Dibromoformaldoxime (6.66 g, 32.8 mmol) was added
to a suspension of 14 (4.50 g, 32.8 mmol) and K2CO3 (22.7 g,
164 mmol) in EtOAc (90 mL). The reaction mixture was stirred at RT
for five days with further addition of dibromoformaldoxime (5ꢂ
2.67 g). After completion of the cycloaddition, Celiteꢃ was added,
and the resulting slurry was filtered under vacuum and washed
with EtOAc. The solvent was evaporated, and the residue was puri-
fied by silica gel column chromatography (PE/EtOAc, 1:1) to afford
cycloadduct 15 (4.05 g, 48%), which crystallized as colorless prisms
from EtOAc (mp 127–1288C): Rf =0.18 (PE/EtOAc, 2:3); 1H NMR
(300 MHz, CDCl3): d=1.73 (m, 2H), 1.98 (m, 1H), 2.25 (br s, 1H),
2.33 (m, 1H), 2.98–3.18 (m, 5H), 3.13 (d, J=17.2, 1H), 3.32 (d, J=
17.2, 1H), 3.40 ppm (dd, J=1.8 and 14.6, 1H); 13C NMR (75 MHz,
CDCl3): d=20.1, 22.1, 30.7, 51.1, 51.7, 52.5, 65.5, 85.8, 136.4 ppm;
Anal. calcd for C9H16BBrN2O: C 41.74, H 6.23, N 10.82, found: C
41.60, H 6.47, N 11.07.
(ꢀ)-3-Ethoxy-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
dec-2-ene (19): The title compound, prepared by the procedure
described for analogue 18 using EtOH as a solvent, was obtained
as colorless prisms in 61% yield from EtOAc/n-hexane; mp 94–
1
958C; Rf =0.44 (PE/EtOAc, 1:4); H NMR (300 MHz, CDCl3): d=1.34
(t, J=7.0, 3H), 1.68 (m, 2H), 1.93 (m, 1H), 2.25 (br s, 1H), 2.33 (m,
1H), 2.85 (d, J=16.5, 1H), 2.95–3.08 (m, 5H), 3.10 (d, J=16.5, 1H),
3.36 (d, J=14.3, 1H), 4.16 ppm (q, J=7.0, 2H); 13C NMR (75 MHz,
CDCl3): d=14.8, 20.2, 22.3, 30.4, 42.8, 51.8, 52.4, 65.8, 66.6, 84.1,
165.9 ppm; Anal. calcd for C11H21BN2O2: C 58.95, H 9.44, N 12.50,
found: C 58.88, H 9.67, N 12.23.
(ꢀ)-3-Methyl-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro-
(ꢀ)-3-n-Propoxy-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
dec-2-ene (20): NaH (188 mg, 7.82 mmol) was added to a stirred
solution of n-propanol (0.54 mL, 7.11 mmol) in anhyd THF under ni-
trogen at 08C. After 30 min, a solution of 15 (920 mg, 3.55 mmol)
in anhyd THF was added dropwise and the mixture was stirred at
RT for 16 h. After addition of H2O (1 mL), the solvent was concen-
trated in vacuo, and the residue was diluted with H2O (5 mL) and
extracted with EtOAc (4ꢂ5 mL). After the usual workup, the crude
reaction mixture was purified by silica gel column chromatography
(PE/EtOAc, 2:3) to afford 20 (600 mg, 71%) as a colorless, viscous
[4.5]dec-2-ene (16): A solution of 1-chloroacetaldoxime (1.02 g,
10.96 mmol) in CH2Cl2 (5 mL) and Et3N (1.5 mL, 10.96 mmol) was
added to a solution of 14 (1.0 g, 7.30 mmol) in CH2Cl2 (50 mL). The
reaction mixture was stirred at RT for 2 d with further addition of
1-chloroacetaldoxime (5ꢂ1.02 g) and Et3N (5ꢂ1.5 mL). After com-
pletion of cycloaddition, the solvent was evaporated, and the resi-
due was diluted with H2O (30 mL) and extracted with CH2Cl2 (3ꢂ
30 mL). The combined organic layers were dried over anhyd
Na2SO4, then filtered and concentrated in vacuo. After silica gel
column chromatography (PE/EtOAc, 4:1), D2-isoxazoline 16
(430 mg, 30%) was obtained as a pale yellow oil: Rf =0.30 (PE/
EtOAc, 2:3); 1H NMR (300 MHz, CDCl3): d=1.68 (m, 2H), 1.86 (m,
1H), 1.99 (s, 3H), 2.05 (br s, 1H), 2.28 (m, 1H), 2.77 (d, J=17.2, 1H),
1
oil: Rf =0.53 (PE/EtOAc, 2:3); H NMR (300 MHz, CDCl3): d=0.96 (t,
J=7.3, 3H), 1.72 (m, 4H), 1.94 (m, 1H), 2.25 (br s, 1H), 2.33 (m,
1H), 2.86 (d, J=16.5, 1H), 2.95–3.12 (m, 5H), 3.04 (d, J=16.5, 1H),
3.35 (dd, J=1.8 and 14.3, 1H), 4.06 ppm (t, J=6.6, 2H); 13C NMR
896
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Novel a7 Nicotinic Acetylcholine Receptor Agonists
(75 MHz, CDCl3): d=10.2, 19.7, 21.9, 22.0, 30.0, 42.3, 51.4, 52.0,
65.4, 71.8, 83.6, 165.7 ppm; Anal. calcd for C12H23BN2O2: C 60.52, H
9.74, N 11.76, found: C 60.35, H 10.07, N 11.58.
addition of K2CO3 and extracted with CH2Cl2 (4ꢂ5 mL). After stan-
dard workup, the tertiary base 3 (459 mg, 76%) was obtained as a
colorless oil: Rf =0.57 (CH2Cl2/MeOH, 9:1); 1H NMR (300 MHz,
CDCl3): d=1.50 (m, 2H), 1.69 (1H, m), 2.01 (m, 1H), 2.12 (m, 1H),
2.68–2.92 (m, 4H), 2.97 (d, J=14.4, 1H), 3.00 (d, J=17.3,1H), 3.29
(d, J=14.4, 1H), 3.31 ppm (d, J=17.3, 1H); 13C NMR (75 MHz,
CDCl3): d=20.9, 23.3, 31.0, 46.4, 46.8, 51.2, 62.7, 88.1, 136.2 ppm;
Anal. calcd for C9H13BrN2O: C 44.10, H 5.35, N 11.43, found: C 44.38,
H 5.11, N 11.09.
(ꢀ)-3-Benzyloxy-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
dec-2-ene (21): The title compound, prepared by the procedure
described for analogue 20 using benzyl alcohol, was obtained in
77% yield as colorless prisms from EtOAc/n-hexane; mp 128–
1
1298C; Rf =0.44 (PE/EtOAc, 2:3); H NMR (300 MHz, CDCl3): d=1.70
(m, 2H), 1.94 (m, 1H), 2.28 (br s, 1H), 2.35 (m, 1H), 2.92 (d, J=16.5,
1H), 2.96–3.14 (m, 6H), 3.39 (dd, J=1.8 and 14.6, 1H), 5.13 (s, 2H),
7.37 ppm (m, 5H); 13C NMR (75 MHz, CDCl3): d=20.2, 22.3, 30.5,
42.7, 51.7, 52.4, 65.8, 72.4, 84.4, 128.7, 128.9, 129.0, 135.1,
165.8 ppm; Anal. calcd for C16H23BN2O2: C 67.15, H 8.10, N 9.79,
found: C 67.06, H 8.37, N 9.95.
(ꢀ)-3-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene (4):
The title compound was prepared starting from 16 (311 mg,
1.60 mmol) using the protocol described for 3. After standard
workup, tertiary amine 4 (193 mg, 67%) was obtained as a yellow
oil: Rf =0.38 (CH2Cl2/MeOH, 4:1); 1H NMR and 13C NMR data
matched those found in the literature.[24]
(ꢀ)-3-Propargyloxy-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro-
[4.5]dec-2-ene (22): The title compound, prepared by the proce-
dure described for analogue 20 using propargyl alcohol, was ob-
tained as colorless prisms in 74% yield from EtOAc/n-hexane; mp
(ꢀ)-3-Phenyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene (5):
The title compound was prepared starting from 17 (150 mg,
0.586 mmol) using the protocol described for 3. After standard
workup, tertiary amine 5 (50 mg, 35%) was obtained as a colorless,
viscous oil: Rf =0.30 (CH2Cl2/MeOH, 95:5); 1H NMR and 13C NMR
data matched those found in the literature.[24]
1
127–1298C; Rf =0.48 (PE/EtOAc, 3:7); H NMR (300 MHz, CDCl3): d=
1.68 (m, 2H), 1.95 (m, 1H), 2.28 (br s, 1H), 2.35 (m, 1H), 2.61 (t, J=
2.2, 1H), 2.91 (d, J=16.5, 1H), 2.95–3.11 (m, 5H), 3.09 (d, J=16.5,
1H), 3.39 (dd, J=1.8 and 14.3, 1H), 4.75 ppm (d, J=2.2, 2H);
13C NMR (75 MHz, CDCl3): d=19.1, 21.6, 30.2, 42.5, 51.3, 52.1, 58.1,
65.4, 80.5, 84.6, 165.7, 167.3 ppm; Anal. calcd for C12H19BN2O2: C
61.57, H 8.18, N 11.97, found: C 61.80, H 8.35, N 11.71.
(ꢀ)-3-Methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
(6): The title compound was prepared starting from 18 (640 mg,
3.05 mmol) using the protocol described for 3. After standard
workup, the desired reaction product 6 (403 mg, 67%) was ob-
tained as a colorless oil: Rf =0.30 (CH2Cl2/MeOH, 95:5); 1H NMR
(300 MHz, CDCl3): d=1.50 (m, 1H), 1.61 (m, 1H), 1.75 (m, 1H), 2.11
(br s, 1H), 2.23 (m, 1H), 2.80 (d, J=16.5, 1H), 2.87–3.05 (m, 5H),
3.07 (d, J=16.5, 1H), 3.33 (dd, J=1.8 and 14.3, 1H), 3.84 ppm (s,
3H); 13C NMR (75 MHz, CDCl3): d=21.0, 23.6, 30.9, 42.6, 46.7, 47.0,
57.3, 62.7, 86.5, 166.9 ppm; Anal. calcd for C10H16N2O2: C 61.16, H
8.22, N 14.27, found: C 61.45, H 8.49, N 13.92.
(ꢀ)-1-Oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]decan-3-one
(23): A suspension of benzyloxy derivative 21 (450 mg, 1.57 mmol)
and 10% Pd/C (45 mg) in MeOH (10 mL) was stirred under hydro-
gen at atmospheric pressure for 5 h. The reaction mixture was fil-
tered and the solvent was evaporated in vacuo to afford 23
(298 mg, 97%) as a colorless oil: Rf =0.23 (CH2Cl2/MeOH, 95:5);
1H NMR (300 MHz, CDCl3): d=1.72 (m, 2H), 1.96 (m, 2H), 2.20 (m,
1H), 2.38 (br s, 1H), 2.74 (d, J=16.5, 1H), 2.84 (d, J=16.5, 1H),
2.92–3.08 (m, 4H), 3.13 (d, J=14.3, 1H), 3.34 ppm (d, J=14.3, 1H);
13C NMR (75 MHz, CDCl3): d=19.4, 21.4, 29.1, 41.6, 51.5, 51.9, 63.5,
83.2, 172.6 ppm; Anal. calcd for C9H17BN2O2: C 55.14, H 8.74, N
14.29, found: C 55.48, H 8.48, N 13.97.
(ꢀ)-3-Ethoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene (7):
The title compound was prepared starting from 19 (450 mg,
2.01 mmol) using the protocol described for 3. After standard
workup, amine 7 (288 mg, 68%) was obtained as a colorless oil:
1
Rf =0.33 (CH2Cl2/MeOH, 95:5); H NMR (300 MHz, CDCl3): d=1.33 (t,
(ꢀ)-2-Methyl-1-oxa-2,7-diaza-7-boranyl-7,10-ethanospiro[4.5]
decan-3-one (24): Excess CH3I (1.5 mL) was added to a suspension
of isoxazolidin-3-one 23 (900 mg, 4.59 mmol) and K2CO3 (1.90 g,
13.75 mmol) in acetone (30 mL). After stirring at RT for 16 h, the
solvent was evaporated, and the residue was diluted with H2O
(10 mL) and extracted with CH2Cl2 (4ꢂ10 mL). After standard
workup, the crude reaction mixture was purified by silica gel
column chromatography (CH2Cl2/MeOH, 95:5) to afford N-methylat-
ed compound 24 (850 mg, 88%) as a colorless oil: Rf =0.51
(CH2Cl2/MeOH, 95:5); 1H NMR (300 MHz, CDCl3): d=1.71 (m, 2H),
1.97 (m, 1H), 2.22 (m, 1H), 2.33 (br s, 1H), 2.70 (d, J=16.5, 1H),
2.84 (d, J=16.5, 1H), 2.95–3.14 (m, 4H), 3.16 (s, 3H), 3.28 (dd, J=
2.2 and 15.0, 1H), 3.36 ppm (dd, J=2.2 and 15.0, 1H); 13C NMR
(75 MHz, CDCl3): d=20.0, 21.7, 29.6, 32.4, 42.5, 51.9, 52.4, 64.3,
80.3, 168.1 ppm; Anal. calcd for C10H19BN2O2: C 57.17, H 9.12, N
13.33, found: C 57.40, H 9.31, N 12.95.
J=7.0, 3H), 1.42 (m, 1H), 1.54 (m, 1H), 1.64 (m, 1H), 2.02 (m, 1H),
2.12 (m, 1H), 2.75 (d, J=16.5, 1H), 2.78–2.90 (m, 4H), 2.94 (dd, J=
2.2 and 14.7, 1H), 3.03 (d, J=16.5, 1H), 3.24 (dd, J=2.2 and 14.7,
1H), 4.15 ppm (q, J=7.0, 2H); 13C NMR (75 MHz, CDCl3): d=14.8,
21.3, 24.0, 31.0, 42.9, 46.8, 47.1, 63.1, 66.0, 86.4, 166.2 ppm; Anal.
calcd for C11H18N2O2: C 62.83, H 8.63, N 13.32, found: C 62.58, H
8.31, N 13.55.
(ꢀ)-3-n-Propoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
(8): The title compound was prepared starting from 20 (590 mg,
2.48 mmol) using the protocol described for 3. After standard
workup, the reaction product 8 (450 mg, 81%) was obtained as a
colorless oil: Rf =0.42 (CH2Cl2/MeOH, 95:5); 1H NMR (300 MHz,
CDCl3): d=0.95 (t, J=7.3, 3H), 1.37 (m, 1H), 1.48 (m, 1H), 1.64 (m,
1H), 1.70 (m, 2H), 2.03 (m, 1H), 2.08 (m, 1H), 2.75 (d, J=16.5, 1H),
2.74–2.88 (m, 4H), 2.95 (dd, J=1.8 and 14.6, 1H), 3.03 (d, J=16.5,
1H), 3.22 (dd, J=1.8 and 14.6, 1H), 4.05 ppm (t, J=6.6, 2H);
13C NMR (75 MHz, CDCl3): d=10.3, 20.9, 22.1, 23.6, 30.5, 42.4, 46.4,
46.6, 62.7, 71.2, 85.9, 166.0 ppm; Anal. calcd for C12H20N2O2: C
64.26, H 8.99, N 12.49, found: C 63.86, H 8.70, N 12.76.
(ꢀ)-3-Bromo-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene (3):
A solution of CF3CO2H (1 mL) in acetone (5 mL) was added drop-
wise to an ice-cooled, stirred solution of 15 (640 mg, 2.47 mmol) in
acetone (7 mL). After disappearance of the starting material, which
was monitored by TLC (PE/EtOAc, 1:4), toluene (5 mL) was added,
and the solvents and excess reagent were evaporated in vacuo.
The residue was diluted with H2O (5 mL) and extracted with Et2O
(3ꢂ5 mL). The residual aqueous phase was basified by portionwise
(ꢀ)-3-Benzyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
(9): The title compound was prepared starting from 21 (350 mg,
1.22 mmol) using the protocol described for 3. After standard
workup, the desired reaction product 9 (264 mg, 79%) was ob-
ChemMedChem 2011, 6, 889 – 903
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
897

C. De Micheli et al.
MED
1
tained as a pale yellow oil: Rf =0.25 (CH2Cl2/MeOH, 95:5); H NMR
(300 MHz, CDCl3): d=1.41 (m, 1H), 1.54 (m, 1H), 1.63 (m, 1H), 2.05
(br s, 1H), 2.14 (m, 1H), 2.72–2.91 (m, 4H), 2.78 (d, J=16.1, 1H),
2.96 (d, J=14.3, 1H), 3.09 (d, J=16.1, 1H), 3.26 (d, J=14.3, 1H),
5.12 (s, 2H), 7.38 ppm (m, 5H); 13C NMR (75 MHz, CDCl3): d=20.5,
23.6, 30.5, 42.1, 46.3, 47.1, 62.5, 71.5, 86.0, 128.5, 128.9, 129.1,
135.6, 165.8 ppm; Anal. calcd for C16H20N2O2: C 70.56, H 7.40, N
10.29, found: C 70.32, H 7.64, N 10.53.
(75 MHz, D2O): d=17.8, 19.4, 29.2, 44.1, 45.9, 46.4, 56.6, 58.9, 83.9,
134.8, 160.7, 171.5 ppm; Anal. calcd for C14H20N2O6: C 53.84, H 6.45,
N 8.97, found: C 53.97, H 6.12, N 8.64.
(ꢀ)-3-Bromo-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene fu-
marate 3ꢀ0.5 C4H4O4 (3a): Colorless prisms; mp: 145–1468C (2-
propanol/EtOAc); 1H NMR (300 MHz, D2O): d=1.73 (m, 2H), 1.88
(m, 1H), 2.11 (m, 1H), 2.22 (br s, 1H), 3.09 (m, 3H), 3.20 (m, 1H),
3.23 (d, J=18.2, 1H), 3.41 (dd, J=2.3 and 14.4, 1H), 3.44 (d, J=
18.2, 1H), 3.49 (dd, J=2.3 and 14.4, 1H), 6.40 ppm (s, 1H); 13C NMR
(75 MHz, D2O): d=17.5, 19.1, 29.1, 45.8, 46.4, 49.8, 58.7, 85.4, 135.1,
139.8, 172.7 ppm; Anal. calcd for C11H15BrN2O3: C 43.58, H 4.99, N
9.24, found: C 43.38, H 5.15, N 9.07.
(ꢀ)-3-n-Propargyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene (10): The title compound was prepared starting from 22
(278 mg, 1.19 mmol) using the protocol described for 3. After stan-
dard workup, the reaction product 10 (178 mg, 68%) was obtained
1
as a colorless oil: Rf =0.25 (CH2Cl2/MeOH, 95:5); H NMR (300 MHz,
(ꢀ)-3-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene fu-
marate 4ꢀ0.75 C4H4O4 (4a): Pale yellow powder; mp: 172–1738C
dec. (EtOAc/n-hexane/2-propanol); 1H NMR (300 MHz, D2O): d=
1.74 (m, 2H), 1.81 (s, 3H), 1.88 (m, 1H), 2.08 (m, 1H), 2.12 (m, 1H),
2.95 (d, J=17.9, 1H), 3.13 (m, 3H), 3.23 (m, 2H), 3.38 (dd, J=2.2
and 14.3, 1H), 3.43 (dd, J=2.2 and 14.3, 1H), 6.48 ppm (s, 1.5H);
13C NMR (75 MHz, D2O): d=12.3, 17.9, 19.5, 29.1, 45.9, 46.4, 47.6,
59.1, 83.2, 134.8, 159.6, 171.6 ppm; Anal. calcd for C13H19N2O4: C
58.41, H 7.16, N 10.48, found: C 58.65, H 6.88, N 10.18.
CDCl3): d=1.47 (m, 2H), 1.65 (m, 1H), 2.04 (br s, 1H), 2.11 (m, 1H),
2.58 (t, J=1.5, 1H), 2.70–2.90 (m, 5H), 2.95 (d, J=14.6, 1H), 3.09 (d,
J=16.5, 1H), 3.25 (d, J=14.6, 1H), 4.74 ppm (d, J=1.5, 2H);
13C NMR (75 MHz, CDCl3): d=19.1, 21.6, 30.2, 42.5, 46.4, 46.6, 58.1,
62.6, 85.8, 86.0, 167.3, 168.1 ppm; Anal. calcd for C12H16N2O2: C
65.43, H 7.32, N 12.72, found: C 65.12, H 7.45, N 12.47.
(ꢀ)-1-Oxa-2,7-diaza-7,10-ethanospiro[4.5]decan-3-one (11): The
title compound was prepared starting from 23 (400 mg,
2.04 mmol) using the protocol described for 3. After standard
workup, the desired tertiary amine 11 (202 mg, 54%) was obtained
as a yellow oil: Rf =0.18 (CH2Cl2/MeOH, 9:1); 1H NMR (300 MHz,
CDCl3): d=1.46 (m, 2H), 1.60 (m, 1H), 1.95 (m, 1H), 2.03 (br s, 1H),
2.54 (d, J=16.1, 1H), 2.58–2.95 (m, 7H), 3.08 ppm (d, J=14.6, 1H);
13C NMR (75 MHz, CDCl3): d=20.8, 23.5, 29.0, 43.0, 46.8, 48.2, 62.1,
82.9, 168.5 ppm; Anal. calcd for C9H14N2O2: C 59.32, H 7.74, N
15.37, found: C 59.66, H 7.38, N 15.02.
(ꢀ)-3-Phenyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene fu-
marate 5ꢀ0.5 C4H4O4 (5a): Colorless prisms; mp: 191–1928C (abs
1
EtOH); H NMR (300 MHz, D2O): d=1.75–1.95 (m, 3H), 2.18 (m, 2H),
3.15–3.33 (m, 4H), 3.35 (d, J=17.6, 1H), 3.47 (d, J=14.3, 1H), 3.53
(d, J=14.3, 1H), 3.63 (d, J=17.6, 1H), 6.48 (s, 1H), 7.31 (m, 3H),
7.50 ppm (m, 2H); 13C NMR (75 MHz, D2O): d=18.0, 19.6, 29.4, 44.3,
46.2, 46.6, 59.1, 84.3, 126.8, 128.0, 129.1, 131.2, 134.7, 159.0,
171.6 ppm; Anal. calcd for C17H20N2O3: C 67.98, H 6.71, N 9.33,
found: C 68.20, H 6.56, N 9.12.
(ꢀ)-2-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]decan-3-one
(12): The title compound was prepared starting from 24 (500 mg,
2.38 mmol) using the protocol described for 3. After standard
workup, the reaction product 12 (271 mg, 58%) was obtained as a
colorless oil: Rf =0.31 (CH2Cl2/MeOH, 9:1); 1H NMR (300 MHz,
CDCl3): d=1.47 (m, 1H), 1.54 (m, 1H), 1.69 (m, 1H), 2.01 (m, 1H),
2.08 (br s, 1H), 2.59 (d, J=16.1, 1H), 2.72–2.88 (m, 5H), 2.93 (d, J=
16.1, 1H), 3.14 (s, 3H), 3.15 ppm (dd, J=2.2 and 14.3, 1H); 13C NMR
(75 MHz, CDCl3): d=20.9, 23.4, 30.0, 32.1, 42.6, 46.7, 47.2, 61.8,
82.8, 168.9 ppm; Anal. calcd for C10H16N2O2: C 61.20, H 8.22, N
14.27, found: C 60.93, H 8.38, N 14.52.
(ꢀ)-3-Methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
fumarate 6ꢀ1.5 C4H4O4 (6a): Colorless prisms; mp: 139–1408C (2-
1
propanol); H NMR (300 MHz, D2O): d=1.76 (m, 2H), 1.93 (m, 1H),
2.14 (m, 1H), 2.30 (br s, 1H), 3.03 (d, J=16.9, 1H), 3.07–3.32 (m,
4H), 3.19 (d, J=16.9, 1H), 3.45 (d, J=14.7, 1H), 3.49 (d, J=14.7,
1H), 3.69 (s, 3H), 6.59 ppm (s, 3H); 13C NMR (75 MHz, D2O): d=
17.6, 19.4, 28.9, 41.3, 45.9, 46.5, 57.6, 58.7, 84.4, 134.5, 169.0,
170.5 ppm; Anal. calcd for C16H22N2O8: C 51.89, H 5.99, N 7.56,
found: C 51.67, H 6.03, N 7.30.
(ꢀ)-3-Ethoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene fu-
marate 7ꢀ0.75 C4H4O4 (7a): Colorless prisms; mp: 167–1688C
(abs EtOH); 1H NMR (300 MHz, D2O): d=1.12 (t, J=7.0, 3H), 1.71
(m, 2H), 1.86 (m, 1H), 2.07 (m, 1H), 2.23 (br s, 1H), 2.97 (d, J=16.8,
1H), 3.00–3.25 (m, 5H), 3.41 (m, 2H), 3.96 (q, J=7.0, 2H), 6.48 ppm
(s, 1.5H); 13C NMR (75 MHz, D2O): d=13.7, 17.6, 19.4, 28.9, 41.6,
45.9, 46.4, 58.6, 67.2, 83.9, 134.7, 168.2, 171.3 ppm; Anal. calcd for
C14H21N2O5: C 56.55, H 7.12, N 9.42, found: C 56.29, H 7.31, N 9.58.
General procedure for the preparation of fumarates: A solution
of fumaric acid (102 mg, 0.88 mmol) in MeOH (2 mL) was added to
a solution of the free base (0.8 mmol) in MeOH (3 mL). After stir-
ring overnight at RT, the solvent was removed at reduced pressure,
and the crude salt was obtained quantitatively.
(ꢀ)-3-Ethoxycarbonyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene fumarate 1ꢀ1.5 C4H4O4 (1a): Colorless prisms; mp: 164–
1668C (abs EtOH); 1H NMR (300 MHz, CD3OD): d=1.33 (t, J=7.0,
3H), 1.96 (m, 3H), 2.32 (m, 2H), 3.20–3.42 (m, 5H), 3.51 (d, J=18.0,
1H), 3.57 (dd, J=2.2 and 13.9, 1H), 3.62 (dd, J=2.2 and 13.9, 1H),
4.33 (q, J=7.0, 2H), 6.69 ppm (s, 3H); 13C NMR (75 MHz, D2O): d=
13.3, 17.6, 19.2, 29.4, 42.4, 45.9, 46.5, 58.8, 63.4, 87.6, 134.9, 152.8,
161.3, 171.6 ppm; Anal. calcd for C18H24N2O9: C 52.42, H 5.87, N
6.79, found: C 52.18, H 6.05, N 7.05.
(ꢀ)-3-n-Propoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
fumarate 8ꢀC4H4O4 (8a): Colorless prisms; mp: 116–1178C
(EtOAc/CH2Cl2/abs EtOH); 1H NMR (300 MHz, D2O): d=0.76 (t, J=
7.3, 3H), 1.56 (m, 2H), 1.74 (m, 2H), 1.91 (m, 1H), 2.12 (m, 1H), 2.26
(br s, 1H), 3.0 (dd, J=2.6 and 16.8, 1H), 3.02–3.27 (m, 5H), 3.44 (br
s, 2H), 3.91 (t, J=6.6, 2H), 6.57 ppm (s, 2H); 13C NMR (75 MHz,
D2O): d=9.7, 17.6, 19.4, 21.7, 28.9, 41.5, 46.0, 46.5, 58.6, 72.8, 84.0,
134.5, 168.4, 170.5 ppm; Anal. calcd for C16H24N2O6: C 56.46, H 7.11,
N 8.23, found: C 56.49, H 7.38, N 7.97.
(ꢀ)-3-Hydroxymethyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene fumarate 2ꢀC4H4O4 (2a): Colorless prisms; mp: 180–1828C,
1
dec. (abs EtOH); H NMR (300 MHz, D2O): d=1.75 (m, 2H), 1.89 (m,
(ꢀ)-3-Benzyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
fumarate 9ꢀ0.75 C4H4O4 (9a): Colorless prisms; mp: 122–1238C
1H), 2.08 (m, 1H), 2.14 (br s, 1H), 2.99 (d, J=17.9, 1H), 3.03–3.29
(m, 4H), 3.22 (d, J=17.9, 1H), 3.38 (dd, J=1.9 and 14.3, 1H), 3.45
(dd, J=1.9 and 14.3, 1H), 4.18 (s, 2H), 6.49 ppm (s, 2H); 13C NMR
1
(abs EtOH); H NMR (300 MHz, D2O): d=1.68 (m, 2H), 1.88 (m, 1H),
2.09 (m, 1H), 2.22 (br s, 1H), 2.99 (d, J=17.2, 1H), 3.10 (m, 4H),
898
www.chemmedchem.org
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ChemMedChem 2011, 6, 889 – 903

Novel a7 Nicotinic Acetylcholine Receptor Agonists
3.16 (d, J=17.2, 1H), 3.39 (d, J=14.3, 1H), 3.43 (d, J=14.3, 1H),
4.97 (s, 2H), 6.49 (s, 1.5H), 7.26 ppm (m, 5H); 13C NMR (75 MHz,
D2O): d=17.6, 19.4, 28.9, 41.5, 45.9, 46.5, 58.5, 72.4, 84.2, 128.3,
128.9, 129.0, 134.7, 135.0, 167.8, 171.5 ppm; Anal. calcd for
C19H23N2O5: C 63.50, H 6.45, N 7.79, found: C 63.77, H 6.22, N 7.54.
J=1.8 and 14.3, 1H); 13C NMR (75 MHz, [D6]DMSO): d=20.7, 24.4,
32.4, 44.8, 51.4, 55.0, 55.5, 65.9, 68.2, 143.5 ppm; Anal. calcd for
C10H16BrIN2O: C 31.03, H 4.17, N 7.24, found: C 31.22, H 3.98, N
7.38.
(ꢀ)-3-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (4b): Colorless prisms; mp: 201–2038C (2-propanol/
(ꢀ)-3-Propargyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-
ene fumarate 10ꢀ0.5 C4H4O4 (10a): Colorless prisms; mp: 193–
1968C (2-propanol/MeOH); 1H NMR (300 MHz, D2O): d=1.71 (m,
2H), 1.85 (m, 1H), 2.08 (m, 1H), 2.24 (m, 1H), 2.79 (t, J=2.2, 1H),
2.99 (d, J=16.8, 1H), 3.02–3.17 (m, 4H), 3.16 (d, J=16.8, 1H), 3.41
(m, 2H), 4.56 (d, J=2.2, 2H), 6.35 ppm (s, 1H); 13C NMR (75 MHz,
D2O): d=17.4, 19.3, 28.9, 41.3, 45.9, 46.4, 57.9, 58.6, 77.1, 77.5, 84.6,
135.2, 167.2, 173.5 ppm; Anal. calcd for C14H18N2O4: C 60.42, H 6.52,
N 10.07, found: C 60.11, H 6.85, N 10.38.
1
MeOH); H NMR (300 MHz, D2O): d=1.75–1.85 (m, 2H), 1.81 (s, 3H),
1.93 (m, 1H), 2.14 (m, 2H), 2.85 (s, 3H), 2.95 (d, J=17.9, 1H), 3.17
(d, J=17.9, 1H), 3.15–3.38 (m, 4H), 3.52 ppm (br s, 2H); 13C NMR
(75 MHz, D2O): d=12.4, 19.4, 20.9, 29.0, 47.6, 51.8, 56.2, 56.8, 68.6,
83.8, 159.5 ppm; Anal. calcd for C11H19IN2O: C 41.01, H 5.94, N 8.69,
found: C 41.11, H 5.75, N 8.87.
(ꢀ)-3-Methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (6b): Colorless prisms; mp: 221–2228C (MeOH);
1H NMR (300 MHz, D2O): d=1.79 (m, 2H), 1.95 (m, 1H), 2.15 (m,
1H), 2.29 (m, 1H), 2.85 (s, 3H), 2.99 (d, J=17.0, 1H), 3.15 (d, J=
17.0, 1H), 3.14–3.37 (m, 4H) 3.56 (m, 2H), 3.65 ppm (s, 3H);
13C NMR (75 MHz, D2O): d=19.1, 20.8, 28.9, 41.3, 51.8, 56.3, 56.8,
57.7, 68.1, 84.9, 168.9 ppm; Anal. calcd for C11H19IN2O2: C 39.07, H
5.66, N 8.28, found: C 39.35, H 5.81, N 7.97.
(ꢀ)-1-Oxa-2,7-diaza-7,10-ethanospiro[4.5]decan-3-one fumarate
11ꢀ0.75 C4H4O4 (11a): Colorless prisms; mp: 172–1738C (n-
hexane/EtOAc/MeOH); 1H NMR (300 MHz, D2O): d=1.76 (m, 2H),
1.94 (m, 1H), 2.17 (m, 1H), 2.37 (br s, 1H), 2.87 (d, J=16.7, 1H),
2.95 (d, J=16.7, 1H), 3.08–3.30 (m, 4H), 3.42 (dd, J=2.2 and 14.3,
1H), 3.51 (dd, J=2.2 and 14.3, 1H), 6.47 ppm (s, 1.5H); 13C NMR
(75 MHz, D2O): d=17.4, 18.9, 28.1, 41.9, 46.1, 46.5, 57.2, 80.5, 134.8,
166.7, 171.6 ppm; Anal. calcd for C12H17N2O5: C 53.52, H 6.36, N
10.40, found: C 53.23, H 6.58, N 10.72.
(ꢀ)-3-Ethoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (7b): Colorless prisms; mp: 180–1818C (abs EtOH);
1H NMR (300 MHz, D2O): d=1.14 (t, J=7.0, 3H), 1.79 (m, 2H), 1.95
(m, 1H), 2.15 (m, 1H), 2.28 (br s, 1H), 2.85 (s, 3H), 2.98 (d, J=17.0,
1H), 3.15 (d, J=17.0, 1H), 3.17–3.38 (m, 4H), 3.55 (m, 2H),
3.98 ppm (q, J=7.0, 2H); 13C NMR (75 MHz, D2O): d=13.8, 19.1,
20.8, 28.9, 41.6, 51.8, 56.3, 56.8, 67.3, 68.1, 84.5, 168.2 ppm; Anal.
calcd for C12H21IN2O2: C 40.92, H 6.01, N 7.95, found: C 50.16, H
5.87, N 8.12.
(ꢀ)-2-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]decan-3-one
fumarate 12ꢀ0.5 C4H4O4 (12a): Colorless prisms; mp: 162–1638C
(n-hexane/abs EtOH); 1H NMR (300 MHz, D2O): d=1.77 (m, 2H),
1.95 (m, 1H), 2.16 (m, 1H), 2.36 (br s, 1H), 2.84 (d, J=16.8, 1H),
2.89 (d, J=16.8, 1H), 3.03 (s, 3H), 3.08–3.32 (m, 4H), 3.40 (dd, J=2.
6 and 14.3, 1H), 3.56 (dd, J=2.6 and 14.3, 1H), 6.54 ppm (s, 1H);
13C NMR (75 MHz, D2O): d=17.4, 19.0, 28.4, 31.6, 41.6, 46.0, 46.4,
57.7, 80.7, 134.8, 168.4, 171.5 ppm; Anal. calcd for C12H18N2O4: C
56.68, H 7.13, N 11.02, found: C 56.90, H 7.19, N 10.76.
(ꢀ)-3-Propoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (8b): Colorless prisms; mp: 134–1358C (EtOAc/2-
1
propanol); H NMR (300 MHz, D2O): d=0.78 (t, J=7.3, 3H), 1.57 (m,
General procedure for the preparation of iodomethylates: CH3I
(450 mL, 7.23 mmol) was added to a solution of the free base
(0.5 mmol) in MeOH (3 mL). The solution was left overnight at RT,
then the solvent was removed under reduced pressure, quantita-
tively affording the crude quaternary salt.
2H), 1.83 (m, 2H), 1.99 (m, 1H), 2.18 (m, 1H), 2.32 (br s, 1H), 2.88
(s, 3H), 3.04 (d, J=17.2, 1H), 3.16–3.38 (m, 5H), 3.59 (m, 2H),
3.93 ppm (t, J=6.6, 2H); 13C NMR (75 MHz, D2O): d=9.7, 19.1, 20.9,
21.8, 28.9, 41.5, 51.8, 56.3, 56.8, 68.1, 72.9, 84.5, 168.3 ppm; Anal.
calcd for C13H23IN2O2: C 42.63, H 6.33, N 7.65, found: C 42.90, H
6.12, N 7.38.
(ꢀ)-3-Ethoxycarbonyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene methyl iodide (1b): Light yellow prisms; mp: 162–1638C
(abs EtOH); 1H NMR (300 MHz, D2O): d=1.23 (t, J=7.0, 3H), 1.93
(m, 2H), 2.07 (m, 1H), 2.27 (m, 1H), 2.34 (br s, 1H), 2.97 (s, 3H),
3.31 (d, J=18.4, 1H), 3.33–3.48 (m, 4H), 3.50 (d, J=18.4, 1H), 3.73
(br s, 2H), 4.26 ppm (q, J=7.0, 2H); 13C NMR (75 MHz, D2O): d=
13.5, 19.1, 20.6, 29.4, 42.6, 51.8, 56.3, 56.9, 63.5, 68.1, 88.0, 152.9,
161.2 ppm; Anal. calcd for C13H21IN2O3: C 41.07, H 5.57, N 7.37,
found: C 41.38, H 5.22, N 7.10.
(ꢀ)-3-Benzyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (9b): Yellow prisms; mp: 184–185.58C (abs EtOH);
1H NMR (300 MHz, D2O): d=1.77 (m, 2H), 1.94 (m, 1H), 2.14 (m,
1H), 2.27 (br s, 1H), 2.84 (s, 3H), 3.03 (d, J=17.1, 1H), 3.18 (d, J=
17.1, 1H), 3.12–3.38 (m, 4H), 3.54 (m, 2H), 4.98 (s, 2H), 7.27 ppm
(m, 5H); 13C NMR (75 MHz, D2O): d=19.1, 20.8, 28.9, 41.5, 51.8,
56.3, 56.8, 68.0, 72.5, 84.7, 128.4, 128.9, 129.1, 135.0, 167.7 ppm;
Anal. calcd for C17H23IN2O2: C 49.29, H 5.60, N 6.76, found: C 49.57,
H 5.87, N 6.58.
(ꢀ)-3-Hydroxymethyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-
2-ene methyl iodide (2b): Light yellow prisms; mp: 155–1708C,
dec. (2-propanol/abs EtOH); 1H NMR (300 MHz, CD3OD): d=2.01
(m, 2H), 2.14 (m, 1H), 2.29 (m, 1H), 2.39 (m, 1H), 3.04 (s, 3H), 3.17
(d, J=17.6, 1H), 3.36 (dd, J=17.6, 1H), 3.42–3.55 (m, 4H), 3.71 (dd,
J=2.5 and 13.5, 1H), 3.79 (dd, J=2.5 and 13.5, 1H), 4.30 ppm (s,
2H); 13C NMR (75 MHz, D2O): d=19.3, 20.8, 29.0, 44.2, 51.8, 56.2,
56.6, 56.8, 68.4, 84.5, 160.7 ppm; Anal. calcd for C11H19IN2O2: C
39.07, H 5.66, N 8.28, found: C 38.85, H 5.41, N 8.54.
(ꢀ)-3-Propargyloxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-
ene methyl iodide (10b): Colorless prisms; mp: 186–1878C (2-
propanol/MeOH); 1H NMR (300 MHz, D2O): d=1.74 (m, 2H), 1.92
(m, 1H), 2.12 (m, 1H), 2.26 (m, 1H), 2.78 (t, J=2.2, 1H), 2.81 (s, 3H),
2.99 (d, J=17.2, 1H), 3.12–3.37 (m, 5H), 3.54 (m, 2H), 4.56 ppm (d,
J=2.2, 2H); 13C NMR (75 MHz, D2O): d=18.9, 20.7, 28.8, 41.3, 51.8,
56.2, 56.7, 58.0, 68.0, 84.6, 85.1, 167.0, 169.0 ppm; Anal. calcd for
C13H19IN2O2: C 43.11, H 5.29, N 7.73, found: C 42.81, H 5.55, N 7.95.
(ꢀ)-3-Bromo-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene
methyl iodide (3b): Colorless prisms; mp: 112–1148C (MeOH);
1H NMR (300 MHz, CD3OD): d=2.01 (m, 2H), 2.13 (m, 1H), 2.37 (m,
2H), 3.05 (s, 3H), 3.34 (m, 2H), 3.40 (d, J=18.0, 1H), 3.51 (m, 2H),
3.60 (d, J=18.0, 1H), 3.78 (dd, J=1.8 and 14.3, 1H), 3.84 ppm (dd,
(ꢀ)-2-Methyl-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]decan-3-one
methyl iodide (12b): Colorless prisms; mp: 181–1828C (2-propa-
nol); H NMR (300 MHz, D2O): d=1.86 (m, 2H), 2.03 (m, 1H), 2.22
(m, 1H), 2.42 (br s, 1H), 2.88 (d, J=18.0, 1H), 2.92 (s, 3H), 3.03 (d,
J=18.0, 1H), 3.06 (s, 3H), 3.17–3.42 (m, 4H), 3.60 (dd, J=2.3 and
1
ChemMedChem 2011, 6, 889 – 903
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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899

C. De Micheli et al.
MED
14.3, 1H), 3.69 ppm (dd, J=2.3 and 14.3, 1H); 13C NMR (75 MHz,
D2O): d=18.9, 20.4, 28.4, 31.6, 41.5, 51.8, 56.3, 56.7, 67.1, 81.1,
168.3 ppm; Anal. calcd for C11H19IN2O2: C 39.07, H 5.66, N 8.28,
found: C 39.28, H 5.75, N 8.09.
10, (2.258<q<27.508). Data were reduced using the program
EvalCCD.[34] Absorption correction was applied using the program
NUMABS (Tmin =0.964; Tmax =0.992).[35,36] A total of 60896 reflec-
tions were averaged according to the point group symmetry 222,
2
resulting in 6138 unique reflections (Rint =0.045 on Fo ).
Preparation of (R)-(ꢁ)-6a and (S)-(+)-6a: A solution of (R,R)-(ꢁ)-
O,O’-dibenzoyl-l-tartaric acid (4.57 g, 12.75 mmol) in abs EtOH
(75 mL) was added to a solution of (ꢀ)-6 (2.50 g, 12.75 mmol) in
MeOH (75 mL). The solid which formed upon standing was filtered
(filtrate A) and, after three slow crystallizations from MeOH, solid
(ꢁ)-B, (mp 159–1608C, dec.), was submitted to X-ray analysis (see
below and Figure 2). [a]2D0 =ꢁ96.4 (c=0.70; CH3OH); 1H NMR
(300 MHz, [D6]DMSO): d=1.54–1.82 (m, 3H), 1.98 (m, 1H), 2.13 (br
s, 1H), 2.94–3.12 (m, 4H), 2.98 (d, J=16.8, 1H), 3.16 (d, J=16.8,
1H), 3.27 (dd, J=1.9 and 15.1, 1H), 3.34 (dd, J=1.9 and 15.1, 1H),
3.72 (s, 3H), 5.65 (s, 2H), 7.50 (m, 4H), 7.64 (m, 2H), 7.93 ppm (m,
4H); 13C NMR (75 MHz, [D6]DMSO): d=18.5, 20.4, 29.8, 41.4, 45.3,
45.7, 57.5, 59.1, 72.7, 84.5, 129.4, 129.9, 130.1, 134.2, 165.5, 167.5,
168.8 ppm; Anal. calcd for C28H30N2O10: C 60.64, H 5.45, N 5.05,
found: C 60.51, H 5.67, N 4.98. Solid (ꢁ)-B was dissolved in 20%
aqueous NaOH (50 mL), extracted with CH2Cl2 (3ꢂ50 mL), dried
over anhyd Na2SO4, and concentrated in vacuo. The residue was
dissolved in MeOH, and a solution of fumaric acid (1.5 equiv) in
MeOH was added to the mixture. After stirring at RT for 12 h, the
reaction mixture was concentrated under reduced pressure to
afford the crude fumarate quantitatively.
Structure solution and refinement: The structure was solved by
the direct method using the SHELXS97 program[37] and refined
using the program SHELXL97.[38] Full matrix least-squares refine-
ment was performed on F2, minimizing ꢀw(Fo ꢁFc2)2, with aniso-
2
tropic displacement parameters for non-hydrogen atoms. The tar-
taric acid and the tartrate ion are situated on a two-fold axis. The
two half moieties were located and refined. The positions of all hy-
drogen atoms were located on intermediate difference electron
density maps, and hydrogen atoms in aliphatic CH, as well as NH
and OH moieties, were included in the observed positions and re-
fined with fixed isotropic displacement parameters (Uiso =1.2 Ueq
for CH and NH, Uiso =1.5 Ueq for OH). The remaining hydrogen
atoms were included in the calculated positions with fixed isotrop-
ic displacement parameters (Uiso =1.2 Ueq for CH (aromatic), CH2,
Uiso =1.5 Ueq for CH3). The refinement (377 parameters, 6138 reflec-
tions) converged at RF =0.0288, wRF2 =0. 0739 for 5797 reflections
with Fo > 4s(Fo); w=1/[s2(Fo )+(0.0460P)2+0.4508P], where P=
2
2
(Fo +2Fc2)/3; S=1.062. In the final difference, Fourier map maxi-
mum and minimum electron densities were 0.213 and
ꢁ0.200 eꢄꢁ3, respectively. Absolute configuration could not be as-
signed based on structure determination [Flack parameter=
ꢁ0.3(5)];[39] however, the (R)-configuration of the basic moiety
(compound 6) could be appropriate, given its stereochemistry rela-
tive to the known configuration of the (R,R)-O,O’-dibenzoyl-l-tar-
trate ion. A very short hydrogen bond was observed between tar-
taric acid (donor) and the tartrate ion (acceptor) [OꢁH···Oꢁ1: OꢁH
1.04(2) ꢄ, H···Oꢁ1 1.44(2) ꢄ, O···Oꢁ1: 2.479(1) ꢄ, OꢁH···Oꢁ1: 173(2) ꢄ].
Complex atomic scattering factors for neutral atoms were incorpo-
rated as in SHELXL97.[38,40] Crystallographic data for the (R,R)-O,O’-
dibenzoyl-l-tartrate of (R)-6 have been deposited in the Cambridge
Crystallographic Data Centre (deposition number CCDC 761547).
Copies of the data can be obtained, free of charge, upon applica-
tion to the site: http://www.ccdc.cam.ac.uk/data_request/cif.
(R)-(ꢁ)-6ꢀ1.5 C4H4O4 [(R)-(ꢁ)-6a]: Colorless prisms; mp: 146.5–
147.58C (2-propanol); [a]D20 =ꢁ23.7 (c=0.68; CH3OH); Anal. calcd
for C16H22N2O8: C 51.89, H 5.99, N 7.56, found: C 52.15, H 6.17, N
7.29; Chiral HPLC analysis, eluent: n-hexane:2-propanol, 4:1; flow
rate=0.8 mLminꢁ1; l=220 nm; tR =7.80 min; ee=96.2%.
Filtrate A was converted into the free base by concentrating the
solution and dissolving the residue in 20% aqueous NaOH. Extrac-
tion with CH2Cl2 (3ꢂ50 mL), followed by drying over Na2SO4 and
concentration in vacuo, yielded a residue (740 mg) that was dis-
solved in MeOH (60 mL) and combined with a solution of (S,S)-(+)-
O,O’-dibenzoyl-d-tartaric acid (1.35 g) in MeOH (60 mL). The result-
ing salt was crystallized twice from MeOH [solid (+)-B; mp: 159–
1
1608C, dec.]; [a]2D0 =+97.5 (c=0.70; CH3OH); H NMR and 13C NMR
data matched those reported above for the enantiomer; Anal.
calcd for C28H30N2O10: C 60.64, H 5.45, N 5.05, found: C 60.72, H
5.50, N 4.94; Solid (+)-B was treated with fumaric acid and crystal-
lized as described above.
Receptor binding assays
Tissue preparation: Cortex tissues were dissected, immediately
frozen on dry ice, and stored at ꢁ808C for later use. For each ex-
periment, cortex tissues from two rats were homogenized in 10 mL
of a buffer solution (50 mm Na3PO4, 1 m NaCl, 2 mm ethylenediami-
netetraacetic acid (EDTA), 2 mm ethylene glycol tetraacetic acid
(EGTA), and 2 mm phenylmethylsulfonyl fluoride, pH 7.4) using a
potter homogenizer; the homogenates were then diluted and cen-
trifuged at 60000 g for 1.5 h. Total membrane homogenization, di-
lution, and centrifugation procedures were performed twice, then
the pellets were collected, rapidly rinsed with a buffer solution
(50 mm Tris-HCl, 120 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2.5 mm
CaCl2, and 2 mm phenylmethylsulfonyl fluoride, pH 7), and resus-
pended in the same buffer containing a mixture of 20 mgmLꢁ1 of
each of the following protease inhibitors: leupeptin, bestatin, pep-
statin A, and aprotinin.
(S)-(+)-6ꢀ1.5 C4H4O4 [(S)-(+)-6a]: Colorless prisms; mp: 146.5–
147.58C (2-propanol); [a]D20 =+23.1 (c=0.65; CH3OH); Anal. calcd
for C16H22N2O8: C 51.89, H 5.99, N 7.56, found: C 52.09, H 5.72, N
7.73; Chiral HPLC analysis (see above), tR =6.94 min; ee=95.3%.
X-Ray crystallography
X-Ray crystallographic analysis of the salt from (R)-6 and (R,R)-
(ꢁ)-O,O’-dibenzoyl-l-tartaric acid: Colorless single crystals were
obtained from a solution in MeOH. Crystal data: C10H17N2O2; 0.5
(C18H14O8); 0.5 (C18H12O8), Mr =554.55, orthorhombic, space group
P21212 (No. 18), a=22.9390(5) ꢄ, b=14.664(3) ꢄ, c=7.9210(16) ꢄ,
V=2664.4(8) ꢄ3, Z=4, 1cald =1.382Mgmꢁ3
, F(000)=1168, m(Mo
Ka)=0.106 mmꢁ1, T=122.0 (5) K, crystal dimensions=0.38ꢀ0.36ꢀ
[3H]Epibatidine binding: (ꢀ)-[3H]Epibatidine, with a specific activi-
ty of 56–60 Cimmolꢁ1, was purchased from Perkin–Elmer (Boston,
MA); the non radioactive a-bungarotoxin, nicotine, and epibatidine
were purchased from Sigma–Aldrich (Italy). It was previously re-
ported that [3H]epibatidine also binds to a-bungarotoxin binding
receptors with nanomolar affinity.[42] In order to prevent the bind-
0.09 mm.
Data collection and processing: Diffraction data were collected
on an Enraf–Nonius KappaCCD diffractometer using graphite mon-
ochromated Mo Ka radiation (l=0.71073 ꢄ).[32,33] Reflections were
measured within the range ꢁ29ꢄhꢄ29, ꢁ19ꢄkꢄ19, ꢁ10ꢄlꢄ
900
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ChemMedChem 2011, 6, 889 – 903

Novel a7 Nicotinic Acetylcholine Receptor Agonists
ing of [3H]epibatidine to a-bungarotoxin binding receptors, mem-
brane homogenates were pre-incubated with 2 mm a-bungarotoxin
and then with [3H]epibatidine. Saturation experiments were per-
formed by incubating aliquots of cortex membrane homogenates
with 0.01–2.5 nm concentrations of (ꢀ)-[3H]epibatidine overnight
at 48C. Nonspecific binding was determined in parallel by incuba-
tion in the presence of 100 nm unlabeled epibatidine. After incuba-
tion, the samples were filtered through a GFC filter soaked in 0.5%
polyethylenimine and washed with 15 mL of a buffer solution
(10 mm Na3PO4, 50 mm NaCl, pH 7.4), and the filters were counted
in a b-counter. (ꢀ)-[3H]Epibatidine binding to the a3b4 subtype
was determined as described above, using membranes obtained
from SH-SY5Y neuroblastoma cells preincubated with 2 mm a-bun-
garotoxin.[43]
Italy). Whole-cell current recordings were performed 2–3 days after
plating. Recordings and data analysis were carried out using boro-
silicate glass patch pipettes (3- to 6 MW tip resistance) connected
to an Axopatch 200 A amplifier (Axon Instruments, Foster City, CA).
Data were stored on a PC computer using the PCLAMP10 software
(Molecular Devices). During the recording period, cells were
bathed in the following solution: 140 mm NaCl, 2 mm CaCl2,
2.8 mm KCl, 2 mm MgCl2, 10 mm Hepes/NaOH, and 10 mm glucose;
pH 7.3. The patch pipettes were filled with a solution containing:
140 mm CsCl, 2 mm MgATP, 10 mm Hepes/CsOH, and 5 mm BAPTA;
pH 7.3. Whole-cell capacitance and patch series resistance (5–
15 MW) were estimated from slow transient compensations. A
series resistance compensation of 85–90% 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-con-
taining solutions. These tubes were positioned 50–100 mm from
the patched cell and connected to a fast exchanger system (RSC-
160, BioLogic, France). Dose–response relationships were con-
structed by sequentially applying different concentrations of ago-
nists and normalizing the resulting current amplitudes to the value
obtained by applying 200 mm ACh or 5-HT to the same cell, as ap-
propriate. For quantitative estimations of agonist actions, dose–
response relationships were fitted to the following Equation (1):
[
125I]a-Bungarotoxin binding: Saturation binding experiments
were performed using aliquots of cortex membrane homogenates
incubated overnight with 0.1–10 nm concentrations of [125I]a-bun-
garotoxin (specific activity=200–213 Cimmolꢁ1, Amersham) at
room temperature. Nonspecific binding was determined in parallel
by incubation in the presence of 1 mm unlabeled a-bungarotoxin.
After incubation, the samples were filtered as described above,
and the bound radioactivity was directly counted in a g-counter.-
[
125I]a-Bungarotoxin binding to the human a7 receptors and
muscle type a1b1gd receptors was determined as described above
using membrane obtained from IMR32 neuroblastoma cells in the
case of a7 human subtype,[44] and membranes obtained from
TE671 cell line in the case of a1b1gd muscle receptors.
nH
nH
I ¼ Imaxf½CꢅnH=ðEC50 þ½Cꢅ Þg
ð1Þ
where I is the current amplitude induced by the agonist at concen-
tration [C], Imax denotes the maximal response of the cell, nH is the
Hill coefficient, and EC50 is the concentration for which a half maxi-
mum response is induced.
Affinity of derivatives 1a–12a, 3b, 4b, and 6b–10b for nAChRs:
Inhibition of radioligand binding by epibatidine, nicotine, and the
test compounds was measured by pre-incubating cortex homoge-
nates with increasing doses (10 pm–10 mm) of the reference nico-
tinic agonists epibatidine or nicotine, or the drugs to be tested, for
30 min at room temperature. This was followed by overnight incu-
bation with a final [3H]epibatidine concentration of 0.075 nm (for
the a4b2 subtype), 0.15 nm (for the a3b4 subtype), or 1 nm [125I]a-
bungarotoxin (for both a7 and a1b1gd subtypes), at the same
temperatures as those used for the saturation experiments. These
ligand concentrations were used for the competition binding ex-
periments, because they are within the range of the ligand KD
values for the two different classes of nAChRs. For each com-
pound, experimental data obtained from three saturation and
three competition binding experiments were analyzed by means
of a non-linear least squares procedure, using the LIGAND program
as described by Munson and Rodbard.[29] The binding parameters
were calculated by simultaneously fitting three independent satu-
ration experiments, and the Ki values were determined by fitting
the data of three independent competition experiments. Errors in
the KD and Ki values of the simultaneous fits were calculated using
the LIGAND software and were expressed as percentage coeffi-
cients of variation.
Molecular modeling
Ligands docked into the receptor binding clefts were built using
Sybyl 8.0 (Tripos Inc., St. Louis, MO) and preliminarily minimized at
the DFT/b3Lyp/6–31 g* level as implemented in Gaussian09.[45] The
amino groups were considered in the ionized form to better simu-
late physiological conditions. Docking experiments of selected li-
gands in the binding site created by chains D and E of our pub-
lished model[22b] of a7 nAChRs were performed using the program
GOLD 4.0.[46] The receptor active site radius was set equal to 11 ꢄ
from the indole nitrogen of Trp148, responsible for the primary
ligand anchoring point. The side chain of Gln116 was not restrain-
ed 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 at 4.0 and 3.0 ꢄ, respectively, while genetic
algorithm parameters were kept at default values. Next, CA by hier-
archical-agglomerative script (AClAP)[47] was applied to select a so-
lution and, therefore, a statistically significant binding mode. The
resulting complexes were further optimized geometrically by
means of molecular mechanics methods (Tripos force field), imple-
memented in Sybyl 8.0. Figures were generated with PyMOL soft-
ware.[48]
Electrophysiological recordings: Human a4b2 nAChRs were ex-
pressed by stable transfection in the human HEK 293 cell line,[31b]
while the human a7 nAChRs were expressed by stable transfection
in the rat anterior pituitary GH4C1 cell line.[31a] The human 5-HT3A
and 5-HT3B cDNAs were kindly provided by Dr. E. F. Kirkness, Insti-
tute for Genomic Research (Rockville, MD, USA), and were used to
transiently transfect GH4C1 cells, plated at a density of 5–10ꢂ104
per 35 mm Petri dish and grown in Ham’s F10 nutrient mixture,
with 10% fetal bovine serum and 1% penicillin and streptomycin.
Transient transfection was achieved by adding 1 mg human 5-HT3
subunit cDNA to each dish, along with 4 mL lipofectamine. All cul-
ture media were purchased from Invitrogen (San Giuliano Milanese,
Acknowledgements
This research project was financially supported by the following
grants: FIRB RBNE03FH5Y (C.D.M.), PRIN 2005054943 and
20072BTSR2 (F.C., C.G., and M.D.A.), EC Neurocypres (C.G.), Fon-
dazione Cariplo 2006/0779/109251 (C.G.), Compagnia San Paolo
ChemMedChem 2011, 6, 889 – 903
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
901

C. De Micheli et al.
MED
Sun, R. Li, C. Li; J. Ye, Pharmacol. Biochem. Behav. 2010, 95, 192–197; J.
Ye, Pharmacol. Biochem. Behav. 2010, 95, 192–197.
grant 2005–1964 (C.G.), and a grant from the Associazione Oasi
Maria SS, Troina, Italy (F.C.). We are indebted to Prof. P. Ernsberg-
er (Case Western Reserve University; Cleveland, OH), for affinity
data for (ꢀ)-6a toward the human cloned muscarinic receptor
subtypes. The technical assistance of Mr. F. Hansen (University of
Copenhagen; Copenhagen, Denmark), for X-ray data collection is
gratefully acknowledged.
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Received: November 26, 2010
Revised: January 13, 2011
Published online on March 1, 2011
ChemMedChem 2011, 6, 889 – 903
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
903