Synthesis of novel chiral Δ2-isoxazoline derivatives related to ABT-418 and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes

Article abstract of DOI:10.1016/j.ejmech.2010.09.009

The enantiopure diastereomeric Δ²-isoxazoline derivatives (2S,5′R)-5a-10a and (2S,5′S)-5b, (2S,5′S)-9b, (2S,5′S)-11b, which are structural analogues of both ABT-418 2 and oxyimino ethers (S)-3 and (Z)-(S)-4, were synthesized through cycloaddition reactions involving nitrile oxides as 1,3-dipoles and (S)-N-Boc-2- vinylpyrrolidine-13 as the dipolarophile. The absolute configuration was unequivocally assigned to target compounds by means of an X-ray analysis. The derivatives under study were assayed at neuronal acetylcholine nicotinic receptors (nAChRs), where they showed a meaningful reduction in affinity at the heteromeric α4β2 subtype when compared to the reference molecules. Conversely, anti (2S,5′S)-5b and syn (2S,5′R)-10a isomers showed an affinity for the α7 nAChRs comparable to that observed for the model compound ABT-418.

Full text of DOI:10.1016/j.ejmech.2010.09.009

European Journal of Medicinal Chemistry 45 (2010) 5594e5601
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel chiral
D
2-isoxazoline derivatives related to ABT-418
and estimation of their affinity at neuronal nicotinic acetylcholine receptor
subtypes
,
a
a
b
a
Clelia Dallanoce a , Pietro Magrone , Carlo Matera , Leonardo Lo Presti , Marco De Amici ,
*
Loredana Riganti c, Francesco Clementi c, Cecilia Gotti c, Carlo De Micheli a
a Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b Dipartimento di Chimica Fisica ed Elettrochimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy
c 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
a r t i c l e i n f o
a b s t r a c t
The enantiopure diastereomeric D2-isoxazoline derivatives (2S,50R)-5ae10a and (2S,50S)-5b, (2S,50S)-9b,
(2S,50S)-11b, which are structural analogues of both ABT-418 2 and oxyimino ethers (S)-3 and (Z)-(S)-4,
were synthesized through cycloaddition reactions involving nitrile oxides as 1,3-dipoles and (S)-N-Boc-2-
vinylpyrrolidine-13 as the dipolarophile. The absolute configuration was unequivocally assigned to target
compounds by means of an X-ray analysis. The derivatives under study were assayed at neuronal
acetylcholine nicotinic receptors (nAChRs), where they showed a meaningful reduction in affinity at the
Article history:
Received 22 June 2010
Received in revised form
1 September 2010
Accepted 2 September 2010
Available online 17 September 2010
heteromeric a4b
2 subtype when compared to the reference molecules. Conversely, anti (2S,50S)-5b and
Keywords:
syn (2S,50R)-10a isomers showed an affinity for the
a
7 nAChRs comparable to that observed for the model
Neuronal nicotinic acetylcholine receptors
ABT-418
1,3-Dipolar cycloaddition
Nicotinic ligands
compound ABT-418.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Binding affinity
1. Introduction
compounds selective for a specific nAChR subtype is to target its
allosteric binding sites, which are usually less conserved than the
acetylcholine recognition site (orthosteric site) [9,10].
The potential therapeutic impact of novel drug candidates tar-
geting nAChRs is strictly reliant on their selectivity for a given
subtype. Among the 12 different neuronal nAChRs characterized so
far in the vertebrate brain [11,12], the most expressed are the het-
Neuronal nicotinic acetylcholine receptors (nAChRs), which
belong to the family of Cys-loop ligand-gated ion channels, are
responsible for rapid excitatory cholinergic transmission. The
various physiological functions regulated by nAChRs are due either
to their synaptic activation or to their involvement in the modu-
lation of neurotransmitter systems other than the cholinergic one
[1,2]. Accordingly, nAChRs have been identified as major thera-
peutic targets mainly for pathological conditions of the central
nervous system, such as Alzheimer’s and Parkinson’s diseases,
Tourette’s syndrome, schizophrenia, attention deficit/hyperactivity
disorder (ADHD), pain, substance abuse, anxiety and depression
[3e6]. In addition, some subtype selective radioligands with
improved brain penetration are under development for mapping
and recording the expression of central nAChRs [7,8]. Worth
mentioning, an alternative approach to the design and synthesis of
eromeric a4b2 channels and the homomeric a7 channels, which
represent preferred biological targets for the design of new selec-
tive nicotinic agonists/partial agonists [1,5,11]. Among the various
approaches involving the chemical manipulation of naturally
occurring nicotinic ligands [1,11,13], structure activity studies on
(S)-nicotine 1, the active ingredient of tobacco, afforded ABT-418 2,
a potent and rather selective
a4b2 nAChR agonist, in which
a 3-methylisoxazol-5-yl moiety bioisosterically replaced the pyri-
dine ring of the parent compound (Fig. 1) [14]. Notably, ABT-418 has
demonstrated signals of efficacy in adults with ADHD [15]. More
recently, a group of ABT-418 analogues, in which an oxyimino ether
moiety replaced the 3-methyl-5-isoxazolyl residue, was synthe-
sized and tested [16]. In particular, the two-enantiopure oxime
* Corresponding author. Tel.: þ39 02 50319327; fax: þ39 02 50319326.
E-mail address: clelia.dallanoce@unimi.it (C. Dallanoce).
ethers (S)-3 and (Z)-(S)-4 (Fig. 1) displayed
a moderate
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.009

C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
5595
N
O
O
N
N
N
N
N
H
H
H
R
ABT-418 2
(S)-Nicotine 1
(S)-3: R = CH
3
(Z)-(S)-4: R = H
O
O
2' N
N
1'
2
5'
3'
4'
N
HH
R2
N
R2
H
H
R1
R1
(2S,5'R)-(−)-5a: R = CH , R = H
(2S,5'S)-(+)-5b: R = CH , R = H
1
3
2
1
3
2
(2S,5'R)-(−)-6a: R = CH , R = CH
(2S,5'S)-(+)-9b: R = OCH , R = H
1 3 2
1
3
2
3
(2S,5'R)-(−)-7a: R = Br, R = H
(2S,5'S)-(+)-11b: R = Ot-Bu, R = H
1 2
1
2
(2S,5'R)-(−)-8a: R = Br, R = CH
3
1
2
(2S,5'R)-(−)-9a: R = OCH , R = H
1
3
2
(2S,5'R)-(−)-10a: R = OCH , R = CH
3
1
3
2
Fig. 1. Structures of the heterocyclic derivatives investigated in this study.
submicromolar affinity for the
a
4
b
2 subtype coupled with an
separated (2S,50R)-(ꢀ)-19a from (2S,50S)-(þ)-19b as well as
(2S,50R)-(ꢀ)-22a from (2S,50S)-(þ)-22b. Due to the lability of the
trityl-protecting group on silica gel, the flash chromatography was
performed on neutral alumina. Both pericyclic reactions produced
a mixture of cycloadducts in about 3:2 syn/anti ratio. Worth noting,
in the case of 17a and 17b, the above reported conversions and the
related chromatographic purification gave the expected 3-bromo-
substituted isomer syn (2S,50R)-(ꢀ)-20a along with the unexpected
3-tert-butoxy-substituted counterpart anti (2S,50S)-(ꢀ)-21b.
Evidently, the 3-bromo group of 17b was prone to the nucleophilic
displacement by tert-butanol, most likely during the N-Boc
cleavage step.
interesting 2 vs 7 selectivity profile [16].
a
4
b
a
Based on the above discussed results, as an extension of our
studies on novel selective heterocyclic ligands targeting neuronal
nAChR subtypes [17e19] we report in this paper the synthesis of
D
2-isoxazolines (2S,50R)-5ae10a, (2S,50S)-5b, (2S,50S)-9b, and
(2S,50S)-11b, which may be regarded as hybrid structural analogues
of isoxazole 2 and oxime ethers 3 and 4 (Fig. 1). The 3-Br or 3-OMe
substituted derivatives were prepared on the basis of recent results
obtained by our research group with a series of spirocyclic D
2-iso-
xazolinyl nicotinic ligands [17]. The target compounds were evalu-
ated for their binding affinity at both rat neuronal nAChR subtypes
(
a
4
b
2 and
2. Results and discussion
As first step of the synthetic sequence, we converted
a
7) and human cloned muscarinic receptors (hm1-5).
The relative stereochemistry to the syn/anti diastereomers, and
hence the absolute configuration to the 50-stereogenic center, was
preliminarily assigned by comparing the values of the chemical
shifts and coupling constants of protons H-2 and H-50 in the two
couples of N-trityl intermediates (ꢀ)-19a/(þ)-19b and (ꢀ)-22a/
a
commercially available (S)-(ꢀ)-proline 12 into (S)-(ꢀ)-tert-butyl-2-
vinylpyrrolidine-1-carboxylate 13, according to a published proce-
dure [20] (Scheme 1). The latter alkene was utilized as dipolarophile
in 1,3-dipolar cycloaddition reactions involving acetonitrile oxide
and bromonitrile oxide, generated in situ in a basic medium from
their stable halogenoxime precursors, i.e. cloroacetaldoxime 14 [21]
and dibromoformaldoxime 16 [22], respectively (Scheme 1). The
(þ)-22b. As previously observed with structurally related
D
2-iso-
xazolines [23,24], the H-50 of syn isomers (ꢀ)-19a and (ꢀ)-22a
resonated at lower field strength (5.16 and 5.22 d, respectively) than
the H-50 of the corresponding anti isomers (þ)-19b and (þ)-22b
0
(4.89 and 4.97
d
, respectively). Additionally, J2e5 for the syn isomers
(ꢀ)-19a and (ꢀ)-22a (J ¼ 0.5 and 1.8 Hz) was found to be smaller
0
than J2e5 for the anti isomers (þ)-19b and (þ)-22b (J ¼ 5.7 and
syn/anti diastereomeric mixtures of
D
2-isoxazolines 15a/15b and
5.7 Hz) [23,24]. The attribution was subsequently confirmed by an
X-ray analysis carried out on derivative 7a (see below).
17a/17b were obtained in high yield but the cycloadducts in each
couple turned out to be inseparable by means of a silica gel column
chromatography. Replacement of the 3-bromo group of diastereo-
mers 17a/b with the 3-methoxy moiety to yield derivatives 18a/
The six N-trityl derivatives (ꢀ)-19a, (þ)-19b, (ꢀ)-20a, (ꢀ)-21b,
(ꢀ)-22a, and (þ)-22b, depicted in Scheme 2, were then converted
in a mild acidic medium into the corresponding final secondary
amines (ꢀ)-5a, (þ)-5b, (ꢀ)-7a, (þ)-11b, (ꢀ)-9a, and (þ)-9b. The
most abundant syn isomers (ꢀ)-5a, (ꢀ)-7a, and (ꢀ)-9a were
submitted to a reductive amination, performed with aqueous
formaldehyde and sodium cyanoborohydride, to provide the cor-
responding tertiary amines (ꢀ)-6a, (ꢀ)-8a, and (ꢀ)-10a,
respectively.
b
was smoothly accomplished by refluxing the mixture of
compounds 17a/b with a methanol suspension of potassium
carbonate. Once again, the mixture of diastereomers 18a/b could not
be separated by a preparative column chromatography (Scheme 1).
In a further attempt to split the three diastereomeric mixtures
15a/b, 17a/b and 18a/b, we removed the N-Boc protection by
a standard treatment with trifluoracetic acid in dichloromethane.
As illustrated in Scheme 2, the goal was finally achieved via func-
tionalization of the pyrrolidine nitrogen with the trityl group. We
To confirm the syn/anti attribution, we decided to perform
a single-crystal X-ray diffraction experiment on the fumarate of
(ꢀ)-7a, since the presence of a bromine atom in this derivative

5596
C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
O
O
4 steps
ref. 20
a
N
Cl
H C
N H OH
H
N
H
Boc
N
HH
Boc
CH3
NOH
3
14
(S)-(−)-12
(S)-(−)-13
(2S,5'R)-15a
(2S,5'S)-15b
O
O
N
b
c
N
(S)-(−)-13
Br
Br
N
Boc
N
H
Boc
H
H
H
Br
OCH3
NOH
16
(2S,5'R)-17a
(2S,5'S)-17b
(2S,5'R)-18a
(2S,5'S)-18b
Scheme 1. Reagents and conditions: (a) TEA, CH2Cl2, r.t., 1 week; (b) NaHCO3, EtOAc, r.t., 48 h; (c) K2CO3, MeOH, r.t., 12 h.
would allow the resolution of the absolute configuration by
anomalous dispersion. After several fruitless attempts to grow
single-crystals of adequate dimensions of (ꢀ)-7a fumarate, we
finally succeeded to obtain a sample of racemic (ꢁ)-7a fumarate of
suitable quality for the structure solution. We therefore determined
the relative configuration of the two stereocenters in the racemic
crystal since the molecules present in the crystallographic unit cell
were found to have (2S,50R) or (2R,50S)-configuration (Fig. 2) [25].
The target derivatives (ꢀ)-5a-(ꢀ)-10a, (þ)-5b, (þ)-9b, and
(þ)-11b were assayed for binding affinity at rat 2 and
nAChRs, using [3H]epibatidine and [125I]
-bungarotoxin as radi-
oligands, respectively. The Ki values, calculated from the compe-
tition curves of three separate experiments by means of the
LIGAND program [26], are reported in Table 1 and compared with
those previously obtained by some of us for reference compounds
1e4.
a
4
b
a7
a
O
(2S,5'R)-(−)-19a: R = Tr
(2S,5'R)-(−)-5a: R = H
N
c
d
N
HH
CH3
(2S,5'R)-(−)-6a: R = CH
3
R
O
a
b
N
(2S,5'R)-15a
(2S,5'S)-15b
N
H
HH
CH3
O
O
N
N
(2S,5'S)-(+)-19b: R = Tr
(2S,5'S)-(+)-5b: R = H
c
(2S,5'R)-(−)-5a
(2S,5'S)-(+)-5b
N
R
HH
HH
CH3
(2S,5'R)-(−)-20a: R = Tr
(2S,5'R)-(−)-7a: R = H
(2S,5'R)-(−)-8a: R = CH
c
d
N
R
Br
3
O
a
b
(2S,5'R)-17a
(2S,5'S)-17b
N
N
H
HH
X
O
O
N
N
(2S,5'S)-(−)-21b: R = Tr
(2S,5'S)-(+)-11b: R = H
c
(2S,5'R)-(−)-7a: X = Br
(2S,5'S)-(+)-11b: X = Ot-Bu
N
R
HH
HH
Ot-Bu
(2S,5'R)-(−)-22a: R = Tr
(2S,5'R)-(−)-9a: R = H
OCH3 (2S,5'R)-(−)-10a: R = CH
c
d
N
R
3
O
N
a
b
(2S,5'R)-18a
(2S,5'S)-18b
N
H
H
H
OCH3
O
N
(2S,5'S)-(+)-22b: R = Tr
(2S,5'S)-(+)-9b: R = H
c
(2S,5'R)-(−)-9a
(2S,5'S)-(+)-9b
N
R
HH
OCH3
Scheme 2. Reagents and conditions: (a) 30% CF3COOH in CH2Cl2, 0 ꢂC to r.t., 1 h; (b) (C6H5)3CCl, TEA, CH2Cl2, r.t., 24 h; (c) CF3COOH-H2O (2:1), CH2Cl2, 0 ꢂC, 1 h; (d) 37% aq. HCHO,
NaBH3CN, CH3CN, r.t., 1 h.

C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
5597
subtypes (hm1e5) in transfected Chinese Hamster Ovary (CHO)
cells labeled with [3H]quinuclidinyl benzylate. The compounds
(10 mM) were preliminarily tested at hm2 and hm5, representative
of the two subgroups (M2, M4 and M1, M3, M5, respectively) of
mAChRs. None of the investigated derivatives displayed a percent
inhibition of the radioligand binding higher than 50%. As a conse-
quence, they were not further assayed at all five mAChRs, and their
Ki values may be considered higher than 10 mM at all the subtypes.
3. Conclusion
We prepared a set of novel enantiopure chiral pyrrolidine-iso-
xazolinyl stereoisomers using as a key step the 1,3-dipolar cyclo-
addition of nitrile oxides to (S)-(ꢀ)-N-Boc-2-vinylpyrrolidine. The
target derivatives were assayed for their binding affinity at nicotinic
as well as muscarinic acetylcholine receptors. All the compounds
displayed a drastic reduction of affinity at their key biological
Fig. 2. Room temperature experimental X-ray geometry of enantiomer (2S,50R)-7a in
the 7a fumarate racemic crystal, with the atom numbering scheme. Thermal ellipsoids
are drawn at 25% probability level.
Inspection of the data listed in Table 1 clearly shows that
insertion of the
at the 2 nAChR subtype. Indeed, the binding affinity of the syn
isomer (ꢀ)-6a (Ki ¼ 12 M) is reduced by two orders of magnitude
when compared to that of ABT-418 (Ki ¼ 0.020 M), its closest
analogue. A similar trend can be noticed if the comparison is made
with oxyimino ethers 3 and 4 (Ki ¼ 0.52 and 0.33 M, respectively).
Worth noting, the binding affinity at the
7 nAChRs of the novel D2
D
2-isoxazoline moiety sharply reduces the affinity
target, i. e. the a4b2 nAChR, which is rather selectively recognized
a4b
by the model ligands ABT-418 and related oxyimino ethers. Among
the investigated derivatives, the anti isomer (2S,50S)-(þ)-5b and the
syn isomer (2S,50R)-(ꢀ)-10a retained the affinity of ABT-418 at the
m
m
a
7 nAChR subtype. On the whole, replacement of the isoxazole ring
m
with a D
2-isoxazoline moiety in the molecular skeleton of ABT-418
a
-
led to a group of minor nicotinic ligands, both in terms of affinity
and subtype selectivity. This result further emphasizes the impor-
tance for high-affinity/selectivity nicotinic ligands of appropriate
isoxazoline derivatives is similar or even better than that reported
for oxyimino ethers 3 and 4. In particular, derivative (þ)-5b has a Ki
value two orders of magnitude lower than that of (S)-3 and roughly
three fold lower than that reported for ABT-418 (Ki ¼ 0.44 vs
planar heteroaromatic rings acting as hydrogen bond acceptor-
p
moieties. Moreover, the derivatives under study did not show any
appreciable affinity at the different muscarinic receptor subtypes.
1.20
mM) [16]. Furthermore, at the homomeric a7 receptor,
a pronounced stereochemical effect among couples of diastereo-
mers can be appreciated. In fact, the affinity of the anti (þ)-5b
derivative is two orders of magnitude higher than that of its related
4. Experimental protocols
syn (ꢀ)-5a isomer (Ki ¼ 0.44 vs 40
characterizes the anti (þ)-9b/syn (ꢀ)-9a couple (Ki ¼ 11.30 vs
277
mM), and a comparable trend
4.1. Chemistry
m
M). Finally, by comparing the data of (þ)-9b with those of
The spectroscopic and polarimetric data for alkene (S)-(ꢀ)-13,
prepared following a known procedure [20], matched those known
from the literature. Chloroacetaldoxime [21] and dibromo-
formaldoxime [22] were obtained according to known experi-
mental protocols. 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
(þ)-11b, we can deduce that an increase in the bulkiness of the
3-substituent is detrimental to the binding at both nAChRs,
particularly at the
a
7 subtype (Ki ¼ 11.30 vs > 500
mM).
The synthesized compounds were also assayed for binding
affinity at human muscarinic acetylcholine receptor (mAChR)
shifts (d) are expressed in ppm and coupling constants (J) in Hz. TLC
Table 1
analyses were performed on commercial silica gel 60 F254
aluminium sheets; spots were further evidenced by spraying with
a dilute alkaline potassium permanganate solution or the Dra-
gendorff reagent. 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 UVevis detector Jasco
UV-975 using a KROMASIL 5-AmyCoat column (0.46 cm ꢃ 25 cm).
Melting points were determined on a model B 540 Büchi apparatus
and are uncorrected. Microanalyses (C, H, N) of new compounds
agreed with the theoretical value within ꢁ0.4%.
Affinity of (S)-nicotine 1, ABT-418 2, (S)-3, (Z)-(S)-4, and derivatives (ꢀ)-5ae10a,
(þ)-5b, (þ)-9b, and (þ)-11b for native
a4b2 and a7 nAChR subtypes present in rat
cortical membranes and labeled by [3H]epibatidine and [125I]
a-bungarotoxin. The Ki
values were derived from three competitionebinding experiments. The numbers in
brackets refer to the % CV.
Entry
a
4
b2
a7
[3H]Epi
Ki ( M)
[3H]
a
-BgTx
m
Ki (m
M)
(2S,50R)-(ꢀ)-5a
(2S,50S)-(þ)-5b
(2S,50R)-(ꢀ)-6a
(2S,50R)-(ꢀ)-7a
(2S,50R)-(ꢀ)-8a
(2S,50R)-(ꢀ)-9a
(2S,50S)-(þ)-9b
(2S,50R)-(ꢀ)-10a
(2S,50S)-(þ)-11b
(S)-Nicotine 1
ABT-418 2
18 (34)
5.10 (21)
12 (17)
4.20 (19)
3.80 (15)
28 (20)
50 (24)
10.30 (23)
184 (32)
0.002a
40 (37)
0.44 (28)
325 (24)
35 (22)
43 (18)
277 (42)
11.30 (14)
2.90 (12)
>500
4.1.1. Mixture of tert-butyl-2-(3-methyl-4,5-dihydroisoxazol-5-yl)
pyrrolidine-1-carboxylates syn (2S,50R)-15a and anti (2S,50S)-15b
To a magnetically stirred solution of (S)-tert-butyl-2-vinyl-
pyrrolidine-1-carboxylate (ꢀ)-13 (1.50 g, 7.60 mmol) in
dichloromethane (60 mL) were added chloroacetaldoxime 14
(1.07 g, 11.41 mmol) and triethylamine (1.60 mL, 11.41 mmol). The
reaction mixture was stirred at r.t. for one week with addition of
further amounts of 14 (1.07 g ꢃ 10) and TEA (1.60 mL ꢃ 10). After
completion of the cycloaddition, the crude mixture was filtered,
evaporated and the residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate 4:1) to afford the
0.469a
0.020a
1.20a
(S)-3
(Z)e(S)-4
0.52a
49a
0.33a
>50a
(ꢁ)-Epibatidine
0.021 nMb
n.d.
0.71 nM
a
-BgTx
0.90 nMb
a
Ref. [16].
Kd values.
b

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C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
mixture of syn/anti cycloadducts 15a and 15b (1.82 g, 94% overall
yield) as a light yellow viscous oil [Rf ¼ 0.26 (petroleum ether/
ethyl acetate 4:1)]. 1H NMR (toluene d8, 100 ꢂC): 1.61 (s, 9H),
1.67e1.95 (m, 4H), 2.07 (s, 3H), 2.57e2.73 (m, 1H), 2.77e2.90 (m,
1H), 3.35e3.63 (m, 2H), 3.92e4.17 (m, 1H), 4.64e4.80 (m, 1H).
aluminium oxide flash chromatography (petroleum ether/ethyl
acetate 99:1), which gave the syn (ꢀ)-19a (630 mg) and anti
(þ)-19b (314 mg) isomers, and an intermediate (1.52 g) fraction of
their mixture (76% overall yield).
(2S,50R)-(ꢀ)-19a: Crystallized from n-hexane/ethyl acetate as
colorless prisms, mp 147.5e149.5 ꢂC. Rf ¼ 0.43 (petroleum ether/
20
4.1.2. Mixture of tert-butyl-2-(3-bromo-4,5-dihydroisoxazol-5-yl)
pyrrolidine-1-carboxylates syn (2S,50R)-17a and anti (2S,50S)-17b
ethyl acetate 85:15); [
a]
¼ ꢀ125.4 (c ¼ 1.0, CHCl3). 1H NMR: 0.46
D
(m, 1H), 0.80 (m, 1H), 1.25 (m, 1H), 1.40 (m, 1H), 1.92 (s, 3H), 2.39
(dd, 1H, J ¼ 9.1 and 17.2), 2.85 (dd, 1H, J ¼ 11.0 and 17.2), 3.02 (m,
1H), 3.42 (m, 2H), 5.16 (ddd, 1H, J ¼ 0.5, 11.0 and 11.0), 7.12e7.30
(m, 9H), 7.60 (d, 6H, J ¼ 7.3). 13C NMR: 13.6, 25.0, 25.4, 43.0, 51.4,
64.6, 78.4, 86.2, 126.2, 127.7, 129.8, 145.6, 155.0. Anal. Calcd for
C27H28N2O (396.52): C, 81.78; H, 7.12; N, 7.06. Found: C, 82.09; H,
6.91; N, 7.31.
To
a magnetically stirred suspension of (S)-tert-butyl-2-
vinylpyrrolidine-1-carboxylate (ꢀ)-13 (1.85 g, 9.34 mmol) and
sodium hydrogen carbonate (10.24 g, 0.122 mol) in ethyl acetate
(25 mL) was added dibromoformaldoxime 16 (1.90 g, 9.34 mmol).
The reaction mixture was stirred at r.t. for 2 days with addition of
a further amount (1 ꢃ 1.90 g) of 16. After completion of the
cycloaddition, Celite was added and the resulting slurry was
filtered under vacuum and washed with ethyl acetate. The
solvent was evaporated and the residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate 95:5) to
give the mixture of syn/anti cycloadducts 17a and 17b (2.27 g,
76% overall yield) as a yellow viscous oil [Rf ¼ 0.31 (petroleum
ether/ethyl acetate 9:1)]. 1H NMR (toluene d8, 100 ꢂC): 1.47
(s, 9H), 1.55e1.90 (m, 4H), 2.62e2.78 (m, 1H), 2.92e3.04 (m, 1H),
3.14e3.23 (m, 1H), 3.25e3.38 (m, 1H), 3.68e4.03 (m, 1H),
4.48e4.64 (m, 1H).
(2S,50S)-(þ)-19b: Yellow viscous oil, Rf ¼ 0.33 (petroleum ether/
20
ethyl acetate 85:15); [
a
]
¼ þ11.7 (c ¼ 1.0, CHCl3). 1H NMR: 0.35
D
(m, 1H), 1.13e1.30 (m, 3H), 1.99 (s, 3H), 2.82 (dd, 1H, J ¼ 9.7 and
17.6), 3.02e3.20 (m, 3H), 3.75 (m, 1H), 4.89 (ddd, 1H, J ¼ 5.7, 10.5
and 10.5), 7.14e7.34 (m, 9H), 7.58 (d, 6H, J ¼ 7.3). 13C NMR: 13.6,
24.6, 26.6, 41.0, 51.8, 62.0, 78.3, 83.1, 126.4, 127.8, 129.9, 145.2, 155.0.
Anal. Calcd for C27H28N2O (396.52): C, 81.78; H, 7.12; N, 7.06. Found:
C, 81.95; H, 6.80; N, 6.87.
To a solution of (ꢀ)-19a (395 mg, 1.0 mmol) in dichloro-
methane (3.7 mL) at 0 ꢂC were added water (38
mL) and tri-
fluoroacetic acid (77
mL, 0.99 mmol). The reaction mixture was
4.1.3. Mixture of tert-butyl-2-(3-methoxy-4,5-dihydroisoxazol-5-
yl)pyrrolidine-1-carboxylates syn (2S,50R)-18a and anti (2S,50S)-
18b
stirred at r.t. for 1 h and evaporated under vacuum. The residue
was added to 0.1 N HCl (15 mL) and extracted with diethyl ether
(3 ꢃ 10 mL). The residual aqueous phase, made basic by portion-
wise addition of K2CO3, was extracted with dichloromethane
(8 ꢃ 10 mL). The pooled organic extracts were dried over anhy-
drous sodium sulfate, the solvent was evaporated giving 98 mg of
To a solution of the isomeric mixture of 17a and 17b (3.04 g,
9.52 mmol) in methanol (80 mL) was added potassium carbonate
(13.15 g, 95 mmol), and the reaction mixture was magnetically
stirred at r.t. for 12 h. After completion of the reaction, Celite was
added and the resulting slurry was filtered under vacuum and
washed with dichloromethane. The concentrated residue was
diluted with water (50 mL) and extracted with dichloromethane
(5 ꢃ 30 mL). The residue of the pooled organic phases was purified
by silica gel column chromatography (petroleum ether/ethyl
acetate 4:1), and afforded the mixture of isomers 18a and 18b
(2.26 g, 88% overall yield) as a colorless viscous oil [Rf ¼ 0.34
(petroleum ether/ethyl acetate 4:1)]. 1H NMR: 1.45 (s, 9H), 1.85 (m,
2H), 1.99 (m, 2H), 2.80e3.03 (m, 2H), 3.27e3.48 (m, 2H), 3.82 (s,
3H), 3.84e4.12 (m, 1H), 4.52e4.87 (m, 1H).
(ꢀ)-5a (64% yield).
20
(2S,50R)-(ꢀ)-5a: Light yellow oil, [
a
]
D
¼ ꢀ118.7 (c ¼ 1.0, CHCl3).
1H NMR: 1.51 (m, 1H), 1.78 (m, 2H), 1.88 (m, 1H), 1.98 (s, 3H), 2.16
(bs, 1H), 2.84 (dd, 1H, J ¼ 16.9 and 8.1), 2.88e3.02 (m, 3H), 3.24
(m, 1H), 4.54 (ddd, 1H, J ¼ 5.5, 7.7 and 8.1). 13C NMR: 13.6, 25.8,
28.0, 41.4, 47.1, 61.2, 82.7, 155.5. Anal. Calcd for C8H14N2O (154.21):
C, 62.31; H, 9.15; N, 18.17. Found: C, 62.58; H, 9.07; N, 17.95.
The same procedure, performed on (þ)-19b (270 mg,
0.68 mmol), afforded 55 mg of (þ)-5b (52% yield).
20
(2S,50S)-(þ)-5b: Colorless oil, [
a
]
¼ þ104.1 (c ¼ 1.05, CHCl3). 1H
D
NMR: 1.44 (m, 1H), 1.81 (m, 3H), 1.99 (s, 3H), 2.20 (bs, 1H), 2.75 (dd,
1H, J ¼ 7.7 and 17.2), 2.87 (m, 1H), 3.00 (dd, 1H, J ¼ 10.3 and 17.2),
3.11 (m, 2H), 4.48 (ddd, 1H, J ¼ 2.9, 7.7 and 10.3). 13C NMR: 13.5,
25.3, 27.9, 42.0, 46.7, 62.3, 83.0, 155.7. Anal. Calcd for C8H14N2O
(154.21): C, 62.31; H, 9.15; N, 18.17. Found: C, 62.09; H, 9.38; N,
17.92.
4.1.4. 3-Methyl-5-pyrrolidin-2-yl-4,5-dihydroisoxazoles syn
(2S,50R)-(ꢀ)-5a and anti (2S,50S)-(þ)-5b
To the mixture of cycloadducts 15a and 15b (1.82 g, 7.16 mmol)
dissolved in dichloromethane (15 mL) at 0 ꢂC was added tri-
fluoroacetic acid (2.76 mL, 35.8 mmol) dropwise. After 30 min the
reaction mixture was further stirred at r.t. for 1 h. After evaporation
under vacuum, the residue was diluted with 0.1 N HCl (50 mL,
pH ¼ 2e3) and extracted with diethyl ether (3 ꢃ 30 mL). The
residual aqueous phase, made basic by portionwise addition of
NaHCO3, was extracted with dichloromethane (6 ꢃ 30 mL). The
organic layers were dried over anhydrous sodium sulfate and the
solvent was evaporated at reduced pressure. The crude residue was
purified by silica gel column chromatography (dichloromethane/
methanol 9:1) to obtain the syn/anti mixture of isomers (ꢀ)-5a and
(þ)-5b (904 mg, 82% overall yield) as a dark yellow oil [Rf ¼ 0.38
(dichloromethane/methanol 9:1)].
4.1.5. 3-Methyl-5-(1-methylpyrrolidin-2-yl)-4,5-dihydroisoxazole
syn (2S,50R)-(ꢀ)-6a
To a solution of (ꢀ)-5a (140 mg, 0.91 mmol) in acetonitrile
(15 mL), under N2, were added formaldehyde (0.34 mL, 4.54 mmol)
and NaBH3CN (114 mg, 1.82 mmol). The reaction mixture was
stirred for 1 h at r.t., then 2 N HCl (8 mL) was added. After stirring
for 2 h, the mixture was extracted with diethyl ether (3 ꢃ 5 mL). The
residual aqueous phase, made basic by portionwise addition of
NaHCO3, was extracted with dichloromethane (6 ꢃ 3 mL). After the
usual work-up, the residue was purified by silica gel column
chromatography (dichloromethane/methanol 95:5), providing
(ꢀ)-6a (100 mg, 65% yield).
To a solution of 5a and 5b (1.26 g, 8.18 mmol) in dichloro-
methane (80 mL), under N2, were added triphenylchloromethane
(2.28 g, 8.18 mmol) and triethylamine (3.41 mL, 24.54 mmol), and
the reaction mixture was stirred at r.t. for 24 h. After evaporation of
the solvent the residue was diluted with petroleum ether and
filtered under vacuum. The crude filtrate was submitted to a neutral
(2S,50R)-(ꢀ)-6a: Colorless oil, Rf ¼ 0.41 (dichloromethane/
20
methanol 9:1); [
a
]
D
¼ ꢀ102.8 (c ¼ 0.95, CHCl3); 1H NMR: 1.53 (m,
1H),1.67 (m, 2H), 1.83 (m, 1H), 1.96 (s, 3H), 2.24 (m, 1H), 2.36 (s, 3H),
2.46 (m, 1H), 2.85 (m, 2H), 3.06 (m, 1H), 4.60 (ddd, 1H, J ¼ 3.6, 9.9
and 9.9). 13C NMR: 13.5, 23.0, 27.1, 40.5, 42.1, 58.2, 67.6, 81.7, 155.6.

C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
5599
Anal. Calcd for C9H16N2O (168.24): C, 64.25; H, 9.59; N, 16.65.
Found: C, 64.52; H, 9.37; N, 16.48.
propanol 4:1; flow rate: 0.8 mL/min;
l
220 nm; tR 7.13 min.
E.e. > 98%. 1H NMR (D2O): 1.61 (m, 1H), 1.77e1.96 (m, 2H), 2.01
(m, 1H), 3.07 (dd, 1H, J ¼ 7.6 and 18.2), 3.19 (m, 2H), 3.49 (dd, 1H,
J ¼ 11.1 and 18.2), 3.72 (m, 1H), 5.02 (ddd, 1H, J ¼ 3.2, 7.6 and 11.1),
6.53 (s, 1H). 13C NMR (D2O): 23.4, 23.5, 44.2, 46.2, 61.5, 79.1, 134.8,
140.3, 171.8. Anal. Calcd for C11H15BrN2O5 (335.15) C, 39.42; H, 4.51;
N, 8.36. Found: C, 39.60; H, 4.32; N, 8.12.
4.1.6. 3-Bromo-5-pyrrolidin-2-yl-4,5-dihydroisoxazole syn (2S,50R)-
(ꢀ)-7a and 3-tert-butoxy-5-pyrrolidin-2-yl-4,5-dihydroisoxazole
anti (2S,50S)-(þ)-11b
The mixture of syn/anti cycloadducts 17a and 17b (2.27 g,
7.11 mmol) was reacted with trifluoroacetic acid following the
procedure described for 15a and 15b. The crude residue was puri-
fied by silica gel column chromatography (dichloromethane/
methanol 9:1) producing 1.04 g of yellow oil as a mixture of (ꢀ)-7a
and (þ)-11b [Rf ¼ 0.42 (dichloromethane/methanol 9:1)].
A mixture of 7a and 11b (350 mg) was reacted with triphe-
nylchloromethane according to the procedure applied to 5a and
5b. The crude reaction mixture underwent a neutral aluminium
oxide flash chromatography (petroleum ether/ethyl acetate 99:1),
which gave the trityl derivatives (ꢀ)-20a (513 mg) and (ꢀ)-21b
(106 mg).
The corresponding (ꢁ)-7a fumarate was obtained, with
a
comparable overall yield, through the reaction sequence
described for the synthesis of the syn laevorotatory enantiomer.
The intermediate derivatives and the final salt had the same
spectroscopic features described above for the pure antipode. The
resulting salt afforded crystals suitable to perform X-ray analysis
(see belo0w).
(2S*,5 R*)-(ꢁ)-7a ꢃ C4H4O4: Crystallized from 2-propanol as
colorless prisms, mp 128.5e130 ꢂC. Anal. Calcd for C11H15BrN2O5
(335.15) C, 39.42; H, 4.51; N, 8.36. Found: C, 39.57; H, 4.67; N, 8.48.
(2S,50R)-(ꢀ)-20a: Crystallized from n-hexane as colorless
4.1.8. 3-Bromo-5-(1-methylpyrrolidin-2-yl)-4,5-dihydroisoxazole
syn (2S,50R)-(ꢀ)-8a
prisms, mp 78.5e80.5 ꢂC. Rf ¼ 0.64 (petroleum ether/ethyl acetate
9:1); [
a
]
¼ ꢀ83.2 (c ¼ 0.36, CHCl3). 1H NMR: 0.46 (m, 1H), 0.83
The free base (ꢀ)-7a (200 mg, 0.91 mmol) was reacted following
the protocol applied to the synthesis of (ꢀ)-6a. After the usual
work-up, the residue was purified by silica gel column chroma-
tography (dichloromethane/methanol 9:1), providing the tertiary
amine (ꢀ)-8a (75 mg, 35% yield).
20
D
(m, 1H), 1.25 (m, 1H), 1.40 (m, 1H), 2.70 (dd, 1H, J ¼ 9.9 and 17.2),
3.03 (m, 1H), 3.12 (dd, 1H, J ¼ 11.0 and 17.2), 3.44 (m, 2H), 5.27
(ddd, 1H, J ¼ 2.2, 11.0 and 11.0), 7.14e7.33 (m, 9H), 7.58 (d, 6H,
J ¼ 7.5). 13C NMR: 25.1, 29.9, 45.4, 54.5, 64.1, 78.3, 87.8, 126.4, 127.9,
129.8, 136.9, 146.4. Anal. Calcd for C26H25BrN2O (461.39): C, 67.68;
H, 5.46; N, 6.07. Found: C, 67.87; H, 5.23; N, 6.29.
(2S,50R)-(ꢀ)-8a: Light yellow oil, Rf ¼ 0.68 (dichloromethane/
20
methanol 9:1); [
a
]
¼ ꢀ131.4 (c ¼ 0.51, CHCl3). 1H NMR: 1.52
D
(2S,50S)-(ꢀ)-21b: Crystallized from n-hexane/ethyl acetate as
(m, 1H), 1.75 (m, 2H), 1.93 (m, 1H), 2.31 (m, 1H), 2.39 (s, 3H), 2.60
(m, 1H), 3.09 (m, 1H), 3.15 (dd, 1H, J ¼ 10.8 and 17.0), 3.26 (dd, 1H,
J ¼ 9.4 and 17.0), 4.77 (ddd, 1H, J ¼ 3.2, 9.7 and 9.7). 13C NMR: 23.3,
27.5, 42.4, 42.9, 58.3, 66.7, 84.0, 137.6. Anal. Calcd for C8H13BrN2O
(233.11): C, 41.22; H, 5.62; N, 12.02. Found: C, 40.87; H, 5.88; N,
12.35.
colorless prisms, mp 161.5e164.5 ꢂC. Rf ¼ 0.52 (petroleum ether/
20
ethyl acetate 9:1); [
a]
¼ ꢀ46.6 (c ¼ 1.0, CHCl3). 1H NMR: 0.56 (m,
D
1H), 0.81 (m, 1H), 1.24e1.41 (m, 2H), 1.46 (s, 9H), 2.42e2.58 (m, 2H),
3.07 (m,1H), 3.27 (m,1H), 3.46 (d,1H, J ¼ 8.8), 4.90 (ddd,1H, J ¼ 1.8,
7.5 and 7.5), 7.14e7.28 (m, 9H), 7.58 (m, 6H). 13C NMR: 25.2, 25.4,
27.8, 39.5, 51.4, 59.3, 62.8, 78.3, 82.3, 126.4, 127.8, 129.7, 145.4, 169.7.
Anal. Calcd for C30H34N2O2 (454.60): C, 79.29; H, 7.54; N, 6.16.
Found: C, 78.91; H, 7.80; N, 5.85.
4.1.9. 3-Methoxy-5-pyrrolidin-2-yl-4,5-dihydroisoxazoles syn
(2S,50R)-(ꢀ)-9a and anti (2S,50S)-(þ)-9b
A solution of (ꢀ)-20a (490 mg, 1.06 mmol) was reacted as
The mixture of syn/anti cycloadducts 18a and 18b (2.15 g,
7.95 mmol) was reacted with trifluoroacetic acid following the
procedure described in Section 4.1.4 for 15a and 15b. The crude
residue was purified by silica gel column chromatography
(dichloromethane/methanol 9:1) affording 1.26 g of yellow oil (93%
overall yield) as a mixture of (ꢀ)-9a and (þ)-9b [Rf ¼ 0.44
(dichloromethane/methanol 9:1)].
described for (ꢀ)-19a, and 104 mg of the free base (ꢀ)-7a were
obtained (45% yield).
20
(2S,50R)-(ꢀ)-7a: Light yellow viscous oil, [
a]
¼ ꢀ43.3 (c ¼ 1.0,
D
CHCl3). 1H NMR: 1.49 (m, 1H), 1.82 (m, 2H), 1.92 (m, 1H), 2.28
(bs, 1H), 2.98 (m, 2H), 3.20 (m, 2H), 3.38 (m, 1H), 4.67 (ddd, 1H,
J ¼ 5.5, 9.9 and 9.9). 13C NMR: 25.7, 27.8, 44.0, 47.0, 60.5, 84.5, 137.6.
Anal. Calcd for C7H11BrN2O (219.08): C, 38.38; H, 5.06; N, 12.79.
Found: C, 38.70; H, 4.87; N, 12.56.
Derivatives (ꢀ)-9a and (þ)-9b (1.25 g, 7.34 mmol) were reacted
with triphenylchloromethane as described in Section 4.1.4 for 5a
and 5b. The crude reaction mixture was submitted to a neutral
aluminium oxide flash chromatography (petroleum ether/ethyl
acetate 99:1), which afforded the syn (ꢀ)-22a (605 mg) and anti
(þ)-22b (170 mg) trityl isomers, and an intermediate (1.82 g)
fraction of their mixture (86% overall yield).
Similarly, the N-trityl derivative (þ)-21b (250 mg, 0.55 mmol)
produced 70 mg of the free base (þ)-11b (60% yield).
20
(2S,50S)-(þ)-11b: Light yellow viscous oil, [
a
]
D
¼ þ17.4 (c ¼ 1.12,
CHCl3). 1H NMR: 1.34 (m, 1H), 1.40 (s, 9H), 1.65e1.88 (m, 3H), 2.22
(bs, 1H), 2.49 (dd, 1H, J ¼ 8.5 and 16.2), 2.71 (dd, 1H, J ¼ 7.7 and
16.2), 2.95 (m, 2H), 3.24 (dd,1H, J ¼ 7.4 and 7.4), 4.12 (dd,1H, J ¼ 8.5
and 8.5). 13C NMR: 25.3, 27.4, 27.5, 38.9, 46.7, 59.2, 60.2, 80.6, 169.2.
Anal. Calcd for C11H20N2O2 (212.29) C, 62.23; H, 9.50; N, 13.20.
Found: C, 62.47; H, 9.58; N, 12.97.
(2S,50R)-(ꢀ)-22a: Crystallized from ethyl acetate as colorless
prisms, mp 165.5e166.5 ꢂC. Rf ¼ 0.47 (petroleum ether/ethyl
20
acetate 85:15); [
a]
¼ ꢀ81.7 (c ¼ 1.19, CHCl3). 1H NMR: 0.44 (m,
D
1H), 0.86 (m, 1H), 1.45 (m, 2H), 2.53 (dd, 1H, J ¼ 10.3 and 16.7), 2.81
(dd, 1H, J ¼ 10.3 and 16.7), 3.04 (m, 1H), 3.42 (m, 2H), 3.82 (s, 3H),
5.22 (ddd, 1H, J ¼ 1.8, 9.4 and 9.4), 7.13e7.27 (m, 9H), 7.58
(d, 6H, J ¼ 7.3). 13C NMR: 25.0, 25.2, 36.5, 51.4, 57.3, 63.9, 78.3, 87.3,
126.3, 127.8, 129.8, 145.6, 167.5. Anal. Calcd for C27H28N2O2 (412.52):
C, 78.61; H, 6.84; N, 6.79. Found: C, 78.49; H, 6.88; N, 6.97.
4.1.7. Fumarates of 3-bromo-5-pyrrolidin-2-yl-4,5-dihydroisoxazole
syn (2S,50R)-(ꢀ)-7a and syn (2S*,50R*)-(ꢁ)-7a for X-ray analysis
To a solution (ꢀ)-7a (180 mg, 0.82 mmol) in methanol (1.5 mL)
was added a solution of fumaric acid (96 mg, 0.82 mmol) in
methanol (1.5 mL), and the mixture was stirred at r.t. for 16 h. The
corresponding salt was obtained quantitatively after removal of the
solvent.
(2S,50S)-(þ)-22b: Colorless viscous oil, Rf ¼ 0.40 (petroleum
20
ether/ethyl acetate 85:15); [
a]
¼ þ3.8 (c ¼ 0.64, CHCl3). 1H NMR:
D
0.36 (m, 1H), 1.10e1.43 (m, 3H), 2.87 (dd, 1H, J ¼ 10.1 and 16.3),
2.98e3.16 (m, 3H), 3.74 (m, 1H), 3.83 (s, 3H), 4.97 (ddd, 1H, J ¼ 5.7,
10.1 and 10.1), 7.10e7.36 (m, 9H), 7.58 (d, 6H, J ¼ 7.3). 13C NMR:
24.6, 26.5, 34.9, 51.6, 57.4, 61.9, 78.0, 84.3, 126.4, 127.8, 129.9, 145.2,
(2S,50R)-(ꢀ)-7a ꢃ C4H4O4: Crystallized from 2-propanol/ethyl
20
acetate as a pale yellow powder, mp 120e122.5 ꢂC [
a
]
D
¼ ꢀ160.4
(c ¼ 1.0, MeOH). Chiral HPLC analysis, mobile phase: n-hexane/2-

5600
C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
168.0. Anal. Calcd for C27H28N2O2 (412.52): C, 78.61; H, 6.84; N,
6.79. Found: C, 78.92; H, 7.12; N, 6.50.
4.3. Receptor binding assays
A solution of (ꢀ)-22a (595 mg, 1.44 mmol) was reacted as
4.3.1. Membranes binding of [3H]epibatidine and [125I]
bungarotoxin
a-
described for (ꢀ)-19a, giving the free base (ꢀ)-9a (101 mg, 41%
yield).
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
20
(2S,50R)-(ꢀ)-9a: Pale yellow oil, [
a]
¼ ꢀ52.3 (c ¼ 1.28, CHCl3).
D
1H NMR: 1.43 (m, 1H), 1.69 (m, 2H), 1.81 (m, 1H), 1.93 (bs, 1H),
2.79e2.93 (m, 4H), 3.21 (dd, 1H, J ¼ 7.1 and 7.1), 3.75 (s, 3H), 4.47
(ddd, 1H, J ¼ 5.8, 9.1 and 9.1). 13C NMR: 25.7, 27.9, 35.0, 47.1, 57.4,
60.5, 84.5, 167.8. Anal. Calcd for C8H14N2O2 (170.21): C, 56.45; H,
8.29; N, 16.46. Found: C, 56.77; H, 8.01; N, 16.70.
Similarly, (þ)-22b (320 mg, 0.78 mmol) provided 90 mg the free
base (þ)-9b (68% yield).
20
(2S,50S)-(þ)-9b: Pale yellow oil, [
a
]
¼ þ42.8 (c ¼ 1.33, CHCl3).
D
1H NMR: 1.40 (m, 1H), 1.67e1.88 (m, 3H), 2.07 (bs, 1H), 2.75
(dd, J ¼ 8.2 and 16.2, 1H), 2.90 (m, 1H), 2.98 (m, 2H), 3.19 (m, 1H),
3.86 (s, 3H), 4.49 (ddd, 1H, J ¼ 1.4, 7.1 and 8.2). 13C NMR: 25.3, 27.7,
35.9, 46.7, 57.4, 61.8, 84.3, 167.8. Anal. Calcd for C8H14N2O2
(170.21): C, 56.45; H, 8.29; N, 16.46. Found: C, 56.17; H, 8.52; N,
16.27.
buffer containing a mixture of 20 mg/mL of each of the following
protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin.
4.3.2. [3H]Epibatidine binding
(ꢁ)-[3H]Epibatidine with a specific activity of 56e60 Ci/mmol
4.1.10. 3-Methoxy-5-(1-methylpyrrolidin-2-yl)-4,5-dihydroisoxazole
syn (2S,50R)-(ꢀ)-10a
was purchased from PerkineElmer (Boston MA); the nonradioac-
tive
from SigmaeAldrich (Italy). It has been previously reported that
[3H]epibatidine also binds to
-bungarotoxin binding receptors
with nM affinity [27]. In order to prevent the binding of [3H]epi-
batidine to the -bungarotoxin binding receptors, the membrane
homogenates were pre-incubated with 2 -bungarotoxin and
a-bungarotoxin, epibatidine, and nicotine were purchased
The free base (ꢀ)-9a (500 mg, 2.94 mmol) was reacted following
the protocol applied to the synthesis (ꢀ)-6a. After the usual work-
up, the residue was purified by silica gel column chromatography
(dichloromethane/methanol 95:5), providing the tertiary amine
(ꢀ)-10a (260 mg, 48% yield).
a
a
mM a
(2S,50R)-(ꢀ)-10a: Light yellow oil, Rf ¼ 0.40 (dichloromethane/
then with [3H]epibatidine. The saturation experiments were per-
formed by incubating aliquots of cortex membrane homogenates
with 0.01e2.5 nm concentrations of (ꢁ)-[3H]epibatidine overnight
at 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
20
methanol 9:1); [
a]
¼ ꢀ73.4 (c ¼ 1.16, CHCl3). 1H NMR: 1.62
D
(m,1H),1.76 (m, 2H),1.91 (m, 1H), 2.26 (m,1H), 2.41 (s, 3H), 2.53 (m,
1H), 2.88 (dd, 1H, J ¼ 10.1 and 16.3), 2.99 (dd, 1H, J ¼ 9.7 and 16.3),
3.09 (m, 1H), 3.85 (s, 3H), 4.68 (ddd, 1H, J ¼ 4.0, 10.1 and 10.1). 13C
NMR: 23.3, 27.4, 34.4, 42.4, 57.3, 58.3, 67.3, 83.4, 167.9. Anal. Calcd
for C9H16N2O2 (184.24): C, 58.67; H, 8.75; N, 15.21. Found: C, 58.95;
H, 8.51; N, 14.97.
counted in a
b
counter.
4.2. Single-crystal X-ray diffraction analysis of (ꢁ)-7a fumarate
4.3.3. [125I]
a-Bungarotoxin binding
The saturation binding experiments were performed using
aliquots of cortex membrane homogenates incubated overnight
A batch consisting of about 5 mg of (ꢁ)-7a fumarate was dis-
solved in methanol and then crystallized by slow evaporation of the
solvent at T ¼ 4 ꢂC. After 5 days, a solid precipitate appeared, which
was made up by some white, very thin needles together with
a small quantity of colorless, larger prisms. After testing some
with 0.1e10 nm concentrations of [125I]
a-bungarotoxin (specific
activity 200e213 Ci/mmol, Amersham) at r.t. Nonspecific binding
was determined in parallel by means of incubation in the presence
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
useless
specimens,
one
of
the
latter
(dimensions:
0.100 ꢃ 0.050 ꢃ 0.025 mm3) was selected for the X-ray analysis.
Crystal data: title compound: C7H11N2OBr/C4H4O4, Mr 335.15
a.m.u.; monoclinic; space group P21/c (No 14), centrosymmetric;
unit cell (Å, Å3): a ¼ 5.6092 (12), b ¼ 25.018 (5), c ¼ 9.7897 (19),
g
4.3.4. nACh Receptor affinity of compounds (ꢀ)-5a, (þ)-5b, (ꢀ)-6a,
(ꢀ)-7a, (ꢀ)-8a, (ꢀ)-9a, (þ)-9b, (ꢀ)-10a, and (þ)-11b
V ¼ 1333.3 (3); Z: 4; Dc: 1.670 g/cm3; F(000): 680;
m
: 3.101 mmꢀ1
.
The inhibition of radioligand binding by epibatidine and the test
compounds was measured by pre-incubating cortex homogenates
with increasing doses (10 pMe10 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
The sample was twinned, with relative weight of the minor twin
fraction being as large as 16.7(6) %. A full sphere of 20,099
diffraction data (both components) was collected within sinw/
l
¼ 0.55 Åꢀ1 (Mo source); the data were subsequently corrected for
X-ray absorption by an empirical procedure and merged, giving
a final set of 1734 independent, observed reflections. It was
impossible to obtain reliable data at greater resolution due to the
very low sample dimensions. In the final least-square model, riding
motion constraints were applied to the hydrogen atoms, whose
isotropic thermal parameters Uiso were estimated as 1.2 Uiso of the
heavy atom to which they were bonded. Moreover, the anisotropic
thermal motion of the C, N and O atoms was constrained to be equal
among the atoms belonging to the same ring. More information can
be found on the deposited data [25].
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
[26]. The binding parameters were calculated by simultaneously

C. Dallanoce et al. / European Journal of Medicinal Chemistry 45 (2010) 5594e5601
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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).
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When final compound concentrations up to 500 mM did not inhibit
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based on the Cheng and Prusoff’s equation [28].
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Acknowledgments
This research was financially supported by the Italian FIRB grant
# RBNE03FH5Y and PRIN grant # 20072BTSR2. We are indebted to
Prof. P. Ernsberger, Case Western Reserve University, Cleveland, OH,
USA, for the affinity data of the studied compounds at the human
cloned muscarinic receptor subtypes.
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