The enantiomers of epiboxidine and of two related analogs: Synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1002/chir.22052

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (-)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.

Full text of DOI:10.1002/chir.22052

CHIRALITY (2012)
The Enantiomers of Epiboxidine and of Two Related Analogs:
Synthesis and Estimation of their Binding Affinity at a4b2 and a7
Neuronal Nicotinic Acetylcholine Receptors
1
1
1
1
2
2
*
CLELIA DALLANOCE, CARLO MATERA, MARCO DE AMICI, LUCA RIZZI, LUCA PUCCI, CECILIA GOTTI,
FRANCESCO CLEMENTI,2 AND CARLO DE MICHELI1
1Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Milano, Italy
2CNR, Istituto di Neuroscienze, Milano, Italy
ABSTRACT
Epiboxidine hydrochlorides (+)-2 and (ꢀ)-2, which are the structural analogs of
the antipodes of epibatidine (ꢁ)-1, as well as the enantiomeric pairs (+)-3/(ꢀ)-3 and (+)-4/(ꢀ)-4
were synthesized and tested for binding affinity at a4b2 and a7 nicotinic acetylcholine receptor
(nAChR) subtypes. Final derivatives were prepared through the condensation of racemic
N-Boc-7-azabicyclo[2.2.1]heptane-2-one (ꢁ)-5 with the resolving agent (R)-(+)-2-methyl-2-
propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric
pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a
result similar to that reported for both natural and unnatural epibatidine enantiomers at the
same investigated receptor subtypes. Chirality 00:000–000, 2012. © 2012 Wiley Periodicals, Inc.
KEY WORDS: epibatidine; epiboxidine and analogs; neuronal nicotinic acetylcholine receptors;
chiral resolution; enantiopure nicotinic ligands; binding affinity
INTRODUCTION
nAChR subtypes. Overall, epibatidine was characterized as
an exceptionally potent, unselective nicotinic agonist with
a marginal enantioselectivity at all the tested nAChRs,
among them the heteromeric a4b2 and homomeric a7
receptors, which are the subtypes with the highest expression
levels in the mammalian CNS.11–14
Nicotinic acetylcholine receptors (nAChRs) are members of
the Cys-loop family of ligand-gated pentameric ion channels,
which includes also GABAA, glycine, and 5-HT3 receptors.1,2
If the clinical application of selective nicotinic antagonists at
the muscular nAChRs has now well settled, research lines
from private companies as well as public institutions are
focused on the full physiological and pharmacological charac-
terization of each of the subtypes expressed in the central
nervous system (CNS), aiming at the identification of specific
and innovative therapeutic targets.3–5 One major issue of
the research on compounds acting at neuronal nAChRs is
the development of functionally selective ligands, mainly
agonists or partial agonists, which could be of potential
therapeutic significance for application to CNS pathologic
conditions. Among them are Alzheimer’s and Parkinson’s
diseases, epilepsy, schizophrenia, Tourette’s syndrome, atten-
tion deficit hyperactivity disorder, pain, depression, and
tobacco addiction.5–7
In addition to subtype selectivity, a further major aspect
of the ligand/receptor interactions is the degree of stereose-
lectivity shown by chiral derivatives in their molecular
recognition of receptor proteins. Within the superfamily of
cholinergic receptors, the structure/activity relationships of
chiral nicotinic ligands suggest an overall lower impact
of stereoselectivity and enantioselectivity when compared
with chiral muscarinic ligands. In this respect, (ꢀ)-epibatidine
1 (Figure 1), the alkaloid isolated in trace amounts from the
Ecuadorian poison frog Epipedobates tricolor,8 is a meaning-
ful example. This natural toxin was initially tested for its
antinociceptive activity, and epibatidine-induced analgesia,
markedly higher than that of morphine, was found to be
mediated by nAChRs.8,9 Next, several synthetic approaches
to (ꢁ)-epibatidine and its enantiomers10 allowed an accurate
analysis of their pharmacological profile at the various
Among the various epibatidine-related compounds reported
in the literature,13 epiboxidine (ꢁ)-215 (Fig. 1), in which
the 3-methylisoxazole ring bioisosterically replaced the 2-
chloropyridine moiety of the reference compound, behaved
as a potent a4b2 nicotinic agonist, retaining almost entirely
the antinociceptive potency of epibatidine but with a lower
toxicity.15 Recently, we have expanded the study of epiboxi-
dine structure/activity relationships through the elaboration
of an alternative synthetic approach to (ꢁ)-216 as well as the
preparation and biological screening of new epiboxidine-
related compounds.17,18 Among them, the close analog of
epiboxidine (ꢁ)-316 and the permanently charged derivative
(ꢁ)-418 emerged as novel interesting nicotinic ligands. As
part of a research line focused on the design, synthesis, and
pharmacological investigation of ligands acting at neuronal
nAChR subtypes,19,20 we aimed at extending our study
to the enantiomers of (ꢁ)-2, (ꢁ)-3, and (ꢁ)-4. In this
article, the preparation of the three target enantiomeric
pairs (+)-2/(ꢀ)-2, (+)-3/(ꢀ)-3, and (+)-4/(ꢀ)-4, depicted in
Contract grant sponsor: MIUR (FIRB); Contract grant number: (RBNE03FH5Y).
Contract grant sponsor: MIUR (PRIN); Contract grant number: (2009-R7WCZS).
Contract grant sponsor: EU “NeuroCypres” project; Contract grant number:
(202088).
*Correspondence to: C. Dallanoce, Dipartimento di Scienze Farmaceutiche
“Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133
Milano, Italy. E-mail: clelia.dallanoce@unimi.it
Received for publication 17 January 2012; Accepted 19 March 2012
DOI: 10.1002/chir.22052
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2012 Wiley Periodicals, Inc.

DALLANOCE ET AL.
Fig. 1. Structure of natural epibatidine (ꢀ)-1, epiboxidine (ꢁ)-2, its analogs (ꢁ)-3 and (ꢁ)-4, and the three couples of enantiomers (+)-2/(ꢀ)-2, (+)-3/(ꢀ)-3, and
(+)-4/(ꢀ)-4 examined in this study.
Figure 1, and the results of their binding affinity at neuronal
a4b2 and a7 nAChR subtypes will be presented and discussed.
(2 ꢃ 40 ml), then dried over anhydrous Na2SO4, filtered, and the
solvent was evaporated at reduced pressure. The residue underwent
two sequential silica gel flash chromatographies (petroleum ether/ethyl
acetate 9:1 ! 3:1), which allowed complete separation of the two
diastereomers (ꢀ)-6 (1.68 g, 41% yield) and (ꢀ)-7 (2.19 g, 53% yield).
(ꢀ)-(1R,4S,9R)-6: Colorless prisms (from diisopropyl ether), mp
63–64 ꢂC. Rf = 0.25 (petroleum ether/ethyl acetate 4:1). HPLC analysis,
MERCK LiChrospher Si 60 column (15 cm ꢃ 4.6 mm, 5 mm), mobile
phase: n-hexane/ethyl acetate 3:2; flow rate: 0.5 ml/min; l 254 nm;
retention time: 7.04 min. [a]2D5 = ꢀ261.2 (c 1.01, CHCl3). 1H NMR: 1.18
(s, 9H), 1.39 (s, 9H), 1.42–1.49 (m, 1H), 1.56–1.64 (m, 1H), 1.84–1.91
(m, 1H), 1.94–2.03 (m, 1H), 2.57–2.68 (m, 1H), 2.93–3.00 (m, 1H), 4.35–4.37
(m, 1H), 4.47–4.49 (m, 1H). 13C NMR: 22.61, 27.75, 28.35, 41.54, 44.50,
57.09, 57.34, 65.00, 80.77, 155.41, 183.86. MS (ESI) m/z [M + H]+ Calcd
for C15H26N2O3S: 314.4. Found: 315.5. Anal. Calcd for C15H26N2O3S
(314.44): C, 57.30; H, 8.33; N, 8.91. Found: C, 57.63; H, 8.47; N, 9.11.
(ꢀ)-(1S,4R,9R)-7: Colorless prisms (from diisopropyl ether), mp
96–97 ꢂC. Rf = 0.18 (petroleum ether/ethyl acetate 4:1). HPLC analysis,
MERCK LiChrospher Si 60 column (15cmꢃ 4.6 mm, 5 mm), mobile phase:
n-hexane/ethyl acetate 3:2; flow rate: 0.5 ml/min; l 254 nm; retention time:
8.45min. [a]2D5 = ꢀ269.1 (c 1.01, CHCl3). 1H NMR: 1.22 (s, 9H), 1.43 (s, 9H),
1.47–1.65 (m, 2H), 1.85–1.94 (m, 1H), 1.98–2.07 (m, 1H), 2.57–2.69 (m, 1H),
2.88 (d, J = 18.2 Hz, 1H), 4.44–4.47 (m, 1H), 4.55–4.57 (m, 1H). 13C NMR:
22.46, 27.90, 28.41, 40.27, 45.18, 56.94, 57.33, 64.69, 80.85, 155.17, 184.68.
MS (ESI) m/z [M + H]+ Calcd for C15H26N2O3S: 314.4. Found: 315.2. Anal.
Calcd for C15H26N2O3S (314.44): C, 57.30; H, 8.33; N, 8.91. Found:
C, 57.42; H, 8.55; N, 8.72.
EXPERIMENTAL
Materials and Methods
Racemic N-Boc-7-azabicyclo[2.2.1]heptan-2-one (ꢁ)-5 was prepared
according to a literature method.21,16 (R)-(+)-2-Methyl-2-propanesulfinamide
was purchased from Aldrich. 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 20ꢂC. Chemical shifts (d)
are expressed in ppm and coupling constants (J) in Hz. Thin layer chroma-
tography (TLC) analyses were performed on commercial silica gel 60 F254
aluminum sheets; spots were further evidenced by spraying with a dilute
alkaline potassium permanganate solution or a phosphomolybdic acid
solution and, for amines, with the Dragendorff reagent. Rotary power deter-
minations (sodium D line, 589nm) were carried out with a Jasco P-1010
Polarimeter coupled with a Huber thermostat. High performance liquid
chromatography (HPLC) analyses were performed with a Jasco PU-980
pump equipped with a UV–vis detector Jasco UV-975. Melting points were
determined on a model B-540 Büchi apparatus and are uncorrected. Electro-
spray ionization (ESI) mass spectra were obtained on a Varian 320 LC-MS/
MS instrument. Data are reported as mass-to-charge ratio (m/z) of the
corresponding positively charged molecular ions. Microanalyses (C, H,
and N) of new compounds agreed with the theoretical value within ꢁ0.4%.
Synthetic Chemistry
(ꢀ)-(1R,4S)-tert-Butyl-2-{(R)-tert-butylsulfinylimino}-7-azabicyclo
[2.2.1]heptane-7-carboxylate 6 and (ꢀ)-(1S,4R)-tert-butyl-2-{(R)-
tert-butylsulfinylimino}-7-azabicyclo[2.2.1]heptane-7-carboxylate
7.
(R)-(+)-2-Methyl-2-propanesulfinamide (1.58 g, 13.06 mmol) and
(ꢀ)-(1R,4S)-tert-Butyl-2-oxo-7-azabicyclo[2.2.1]heptane-7-
Ti(OEt)4 (6.07 ml, 24.2 mmol) were added to a stirred solution of (ꢁ)-5
(2.76 g, 13.06 mmol) in dry THF (40 ml), and the reaction was heated
at reflux under nitrogen, monitoring the disappearance of the starting
material by TLC (petroleum ether/ethyl acetate 4:1). After 2 h, the mix-
ture was cooled at room temperature, concentrated in vacuo, and the
residue was dissolved in ethyl acetate (150 ml) and treated with
brine (40 ml). After stirring for 15 min followed by filtration over a
CeliteW pad, the organic phase was separated and treated with water
Chirality DOI 10.1002/chir
carboxylate 5.
4N AcOH (50 ml) was added portionwise to a solution
of (ꢀ)-6 (775 mg; 2.46mmol) in MeOH (50ml), and the reaction mixture
was stirred and heated at 40ꢂC per 18h. The solution was concentrated at
reduced pressure, and the aqueous acidic phase was extracted with
Et2O (3ꢃ 20 ml). The pooled organic phases were sequentially treated with
H2O (20ml), saturated aqueous NaHCO3 solution (2 ꢃ 20 ml), brine
(20 ml), and H2O (20ml). After standard workup, the crude solid (510mg,
98% yield) was purified by crystallization.

THE ENANTIOMERS OF EPIBOXIDINE AND OF TWO RELATED ANALOGS
(1R,4S)-(ꢀ)-5: Colorless powder (from diisopropyl ether), mp 44–45ꢂC.
(5 ml) and anhydrous ZnCl2 (107 mg, 0.79 mmol). The mixture was quickly
warmed at room temperature and stirred overnight under argon until
disappearance of the starting material (TLC, petroleum ether/ethyl acetate
9:1). After addition of brine (20 ml), the crude reaction was repeatedly
extracted with ethyl acetate (4 ꢃ 20 ml). The combined organic extracts
were dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate 9:1) to afford (ꢀ)-9
(201 mg, 92%).
(ꢀ)-(1R,4S)-9: Colorless powder (from diisopropyl ether/ethyl acetate
1:1), mp 147–148 ꢂC. Rf = 0.46 (petroleum ether/ethyl acetate 4:1). Chiral
HPLC analysis, DAICEL Chiralcel OD-H column (25 cm ꢃ 4.6 mm, 5 mm),
mobile phase: n-hexane/2-propanol 99:1; flow rate: 0.8 ml/min; l 254 nm;
retention time: 12.13 min. E.e. 99%. [a]2D5 = ꢀ16.9 (c 1.05, CHCl3). 1H
NMR: 1.24 (m, 2H), 1.41 (s, 9H), 2.00 (m, 2H), 2.30 (s, 3H), 4.82
(bs, 1H), 4.94 (m, 1H), 6.10 (s, 1H), 6.68 (bs, 1H). 13C NMR: 11.62,
24.53, 25.49, 28.40, 60.89, 61.14, 102.22, 133.35, 135.17, 155.19, 160.24,
163.95. MS (ESI) m/z [M + H]+ Calcd for C15H20N2O3: 276.3. Found:
277.1. Anal. Calcd for C15H20N2O3 (276.33): C, 65.20; H, 7.30; N, 10.14.
Found: C, 65.49; H, 7.14; N, 10.39.
Rf = 0.20 (petroleum ether/ethyl acetate 95:5). Chiral HPLC analysis,
DAICEL Chiralpak AD column (25 cmꢃ 4.6 mm, 10mm), mobile phase:
n-hexane/2-propanol 98:2; flow rate: 1.0 ml/min; l 254nm; retention
time: 13.08 min. E.e. 98%. [a]2D5 = ꢀ73.1 (c 1.0, CHCl3) {lit.22: [a]D22 = ꢀ73.6
(c 1.10, CHCl3)}. 1H NMR: 1.40 (s, 9H), 1.50–1.65 (m, 2H), 1.88–2.07
(m, 3H), 2.43 (dd, J = 17.3 and 5.2 Hz, 1H), 4.21 (m, 1H), 4.53 (m, 1H). 13
C
NMR: 24.61, 27.74, 28.39, 45.42, 56.24, 64.13, 81.02, 155.26, 209.76. MS
(ESI) m/z [M+ H]+ Calcd for C11H17NO3: 211.3. Found: 212.1. Anal. Calcd
for C11H17NO3 (211.26): C, 62.54; H, 8.11; N, 6.63. Found: C, 62.63; H,
7.98; N, 6.72.
(+)-(1S,4R)-tert-Butyl-2-oxo-7-azabicyclo[2.2.1]heptane-7-
carboxylate 5.
The title compound was prepared from diastereomer
(ꢀ)-7 (820 mg, 2.61 mmol) following the aforementioned procedure
described for (ꢀ)-5. The crude ketone (518 mg, 94% yield) was purified
by crystallization.
(1S,4R)-(+)-5: Colorless powder (from diisopropyl ether), mp 43–44 ꢂC.
Chiral HPLC analysis was performed in the same conditions as those
reported for (ꢀ)-5. Retention time: 12.06 min. E.e. 98%. [a]2D5 = +75.7
(c 1.0, CHCl3) {lit.22: [a]D22 = +73.5 (c 1.0, CHCl3)}. The NMR spectroscopic
data matched those of (ꢀ)-5. MS (ESI) m/z [M + H]+ Calcd for
(+)-(1S,4R)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]
C
11H17NO3: 211.3. Found: 212.1. Anal. Calcd for C11H17NO3 (211.26): C,
62.54; H, 8.11; N, 6.63. Found: C, 62.72; H, 8.03; N, 6.84.
hept-2-ene-7-carboxylate 9.
The 7-azabicyclo[2.2.1]hept-2-ene
derivative (+)-9 was prepared from enoltriflate (+)-8 (508 mg,
1.48 mmol) following the aforementioned procedure described for
(ꢀ)-9. The crude reaction mixture was purified by silica gel column
chromatography (petroleum ether/ethyl acetate 9:1) to afford (+)-9
(364 mg, 89% yield).
(+)-(1S,4R)-9: Colorless powder (from diisopropyl ether/ethyl
acetate 1:1), mp 147–148 ꢂC. Chiral HPLC analysis was performed in
the same conditions as those reported for (ꢀ)-9. Retention time:
13.11 min. E.e. 99%. [a]2D5 = +16.1 (c 0.88, CHCl3). The NMR spectro-
scopic data matched those of (ꢀ)-9. MS (ESI) m/z [M + H]+
Calcd for C15H20N2O3: 276.3. Found: 277.4. Anal. Calcd for
(ꢀ)-(1R,4S)-tert-Butyl-2-(trifluoromethylsulfonyloxy)-7-azabicyclo
[2.2.1]hept-2-ene-7 carboxylate 8.
A solution of ketone (ꢀ)-5
(467 mg, 2.21 mmol) in anhydrous THF (5ml) was slowly added dropwise
to a stirred solution of KHMDS (5.52 ml of a 0.5 M KHMDS solution
in toluene) in anhydrous 1,2-dimethoxyethane (DME, 15 ml) cooled
at ꢀ78ꢂC under argon. After 40min, a solution of N-(5-chloro-2-pyridyl)bis
(trifluoromethanesulfonimide) (1.04g, 2.65mmol) in anhydrous DME
(5 ml) was dropped, and the reaction was kept under stirring for further
2 h at ꢀ78ꢂC, then for 3 days at room temperature, monitoring the
disappearance of the starting material (TLC, petroleum ether/ethyl
acetate 9:1). The solvent was then evaporated in vacuo, and the brown oily
residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate 9:1) to provide 387 mg (51% yield) of the desired
methanesulfonate.
C
15H20N2O3 (276.33): C, 65.20; H, 7.30; N, 10.14. Found: C, 65.48;
H, 7.22; N, 10.41.
(ꢀ)-(1R,2R,4S)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo
[2.2.1]heptane-7-carboxylate 10.
A suspension of (ꢀ)-9 (260 mg,
(ꢀ)-(1R,4S)-8: Colorless oil. Rf = 0.62 (petroleum ether/ethyl acetate 9:1).
[a]2D5 = ꢀ21.1 (c 1.02, CHCl3). 1H NMR: 1.13–1.50 (m, 2H), 1.40 (s, 9H),
1.95–2.10 (m, 2H), 4.65 (m, 1H), 4.73 (m, 1H), 5.92 (bs, 1H). 13C NMR:
25.40, 28.23, 29.91, 60.51, 81.21, 116.60, 120.86, 155.02. MS (ESI) m/z
[M + H]+ Calcd for C12H16F3NO5S: 343.3. Found: 344.1. Anal. Calcd for
0.94 mmol) and 10% Pd/C (35 mg) in MeOH (25 ml) was stirred at room
temperature for 5 h under hydrogen at atmospheric pressure, monitoring
the disappearance of the starting material (TLC, petroleum ether/
ethyl acetate 85:15). The reaction mixture was filtered over a CeliteW
pad, and the filtrate was concentrated to dryness at reduced pressure
affording (ꢀ)-10 (241 mg, 92%).
C
12H16F3NO5S (343.32): C, 41.98; H, 4.70; N, 4.08. Found: C, 42.10; H,
4.52; N, 4.22.
(ꢀ)-(1R,2R,4S)-10: Colorless powder (from diisopropyl ether/ethyl ac-
etate 1:1), mp 86–87 ꢂC. Rf = 0.51 (petroleum ether/ethyl acetate 7:3).
Chiral HPLC analysis, DAICEL Chiralpak AD column (25 cm ꢃ 4.6 mm,
10 mm), mobile phase: n-hexane/2-propanol 98:2; flow rate: 1 ml/min; l
254 nm; retention time: 12.72 min. E.e. 99%. [a]2D5 = ꢀ4.0 (c 1.0, CHCl3).
1H NMR: 1.30–1.40 (m, 2H), 1.42 (s, 9H), 1.50–1.65 (m, 2H), 1.67–1.80
(m, 1H), 2.13–2.28 (m, 1H), 2.27 (s, 3H), 3.35 (m, 1H), 4.20 (m, 1H),
4.35 (m, 1H), 5.82 (s, 1H). 13C NMR: 11.57, 24.62, 28.43, 29.84, 34.71,
39.31, 57.03, 59.61, 80.13, 102.93, 155.59, 159.90, 172.61. MS (ESI) m/z
[M + H]+ Calcd for C15H22N2O3: 278.4. Found: 279.2. Anal. Calcd for
(+)-(1S,4R)-tert-Butyl-2-(trifluoromethylsulfonyloxy)-7-azabicyclo
[2.2.1]hept-2-ene-7-carboxylate 8.
The title compound was
prepared from ketone (+)-5 (639mg, 3.02 mmol) following the aforemen-
tioned procedure described for (ꢀ)-8. The crude reaction mixture was
purified by silica gel column chromatography (petroleum ether/ethyl
acetate 9:1) to afford (+)-8 (592 mg, 57% yield).
(+)-(1S,4R)-8: Colorless oil. [a]2D5 = +21.5 (c 0.88, CHCl3). The NMR
spectroscopic data matched those of (ꢀ)-8. MS (ESI) m/z [M + H]+ Calcd
for C12H16F3NO5S: 343.3. Found: 344.2. Anal. Calcd for C12H16F3NO5S
(343.32): C, 41.98; H, 4.70; N, 4.08. Found: C, 41.85; H, 4.63; N, 4.15.
C15H22N2O3 (278.35): C, 64.73; H, 7.97; N, 10.06. Found: C, 64.85; H,
8.14; N, 10.15.
(ꢀ)-(1R,4S)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]
hept-2-ene-7-carboxylate 9.
To a magnetically stirred solution of
(+)-(1S,2S,4R)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo
enoltriflate (ꢀ)-8 (270 mg, 0.79 mmol) in anhydrous THF (15 ml), kept at
ꢀ78 ꢂC under argon, were sequentially added [Pd2(dba)3]ꢄCHCl3 (70 mg,
0.06 mmol), triphenylphosphine (15 mg, 0.02 mmol), then a solution of
3-methyl-5-tributylstannylisoxazole16 (880 mg, 2.40 mmol) in anhydrous THF
[2.2.1]heptane-7-carboxylate 10.
Alkene (+)-9 (215 mg,
0.78 mmol), which was reacted following the aforementioned described
procedure, gave 206 mg (95% yield) of the 7-azabicyclo[2.2.1]heptane
derivative (+)-10.
Chirality DOI 10.1002/chir

DALLANOCE ET AL.
(+)-(1S,2S,4R)-10: Colorless powder (from diisopropyl ether/ethyl
(ꢀ)-(1S,2R,4R)-2 ꢃ HCl: Colorless prisms (from 2-propanol), mp
186–190 ꢂC, dec. [a]2D5 = ꢀ18.8 (c 0.50, MeOH). The NMR spectroscopic
data matched those of (+)-2 hydrochloride. MS (ESI) m/z [M+ H]+ Calcd
for C10H15ClN2O: 214.7. Found: 179.2. Anal. Calcd for C10H15ClN2O
(214.69): C, 55.94; H, 7.04; N, 13.05. Found: C, 55.79; H, 7.11; N, 13.17.
acetate 1:1), mp 82–84 ꢂC. Chiral HPLC analysis was performed in the
same conditions as those reported for (ꢀ)-10. Retention time:
11.80 min. E.e. 99%. [a]2D5 = +4.4 (c 1.0, CHCl3). The NMR spectroscopic
data matched those of (ꢀ)-10. MS (ESI) m/z [M + H]+ Calcd for
C
15H22N2O3: 278.4. Found: 279.2. Anal. Calcd for C15H22N2O3 (278.35):
(+)-5-{(1R,4S)-7-Azabicyclo[2.2.1]hept-2-en-2-yl}-3-methylisoxazole
C, 64.73; H, 7.97; N, 10.06. Found: C, 64.96; H, 7.63; N, 10.35.
3.
The N-Boc protected derivative (ꢀ)-9 (122 mg, 0.44 mmol) was
dissolved in 4N HCl in anhydrous dioxane (2 ml) at a 0 ꢂC. The reaction
mixture was allowed to warm at room temperature, and then it was stirred
for about 1 h monitoring the disappearance of the starting material (TLC,
petroleum ether/ethyl acetate 85:15). The reaction mixture was concentrated
under reduced pressure, then dissolved in water (5 ml), and treated with
diethyl ether (3 ꢃ 5 ml). The residual aqueous phase was made basic by
addition of solid Na2CO3 and extracted with dichloromethane (3 ꢃ 5ml). After
standard workup, the pooled organic phases provided the free secondary
base (+)-3 (57 mg, 73% yield).
(ꢀ)-(1R,2S,4S)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo
[2.2.1]heptane-7- carboxylate 11.
Potassium tert-butoxide
(438 mg, 3.90 mmol) was added to a stirred solution of the endo-epimer
(ꢀ)-10 (182 mg, 0.65 mmol) in tert-butanol (50 ml). The reaction was
heated at reflux for 30 h until a maximum conversion of about 60%
(TLC monitoring, petroleum ether/ethyl acetate 4:1). The crude
mixture was concentrated in vacuo, and the two endo/exo-epimers
were separated by silica gel column chromatography (petroleum
ether/ethyl acetate 95:5 ! 3:1), which provided 106 mg (58% yield) of
pure (ꢀ)-11.
(+)-(1R,4S)-3: Pale yellow viscous oil. Rf = 0.57 (dichloromethane/
methanol 9:1). [a]2D5 = +63.8 (c 0.85, CHCl3). 1H NMR: 1.16–1.27
(m, 2H), 1.93–2.10 (m, 3H), 2.30 (s, 3H), 4.34 (bs, 1H), 4.47 (bs, 1H),
6.05 (s, 1H), 6.72 (bs, 1H). 13C NMR: 11.67, 23.79, 24.74, 61.17, 61.38,
102.20, 135.41, 136.48, 160.29, 164.02. MS (ESI) m/z [M + H]+ Calcd for
(ꢀ)-(1R,2S,4S)-11: Pale yellow viscous oil. Rf = 0.25 (petroleum ether/
ethyl acetate 85:15). Chiral HPLC analysis, DAICEL Chiralpak AD
column (25 cm ꢃ 4.6 mm, 10 mm), mobile phase: n-hexane/2-propanol
98:2; flow rate: 1 ml/min; l 254nm; retention time: 13.62min. E.e. 99%.
[a]2D5 = ꢀ13.9 (c 1.0, CHCl3). 1H NMR: 1.33 (s, 9H), 1.40–1.57 (m, 2H),
1.70–1.85 (m, 2H), 1.90 (d, J = 6.6 Hz, 2H), 2.20 (s, 3H), 2.99
(t, J = 6.8 Hz, 1H), 4.32 (bs, 1H), 4.34 (bs, 1H), 5.82 (s, 1H). 13C NMR:
11.64, 28.35, 28.99, 29.10, 36.90, 41.23, 55.52, 60.36, 79.91, 101.40, 155.02,
159.80, 175.36. MS (ESI) m/z [M+ H]+ Calcd for C15H22N2O3: 278.4. Found:
279.5. Anal. Calcd for C15H22N2O3 (278.35): C, 64.73; H, 7.97; N, 10.06.
Found: C, 64.92; H, 7.77; N, 10.34.
C
10H12N2O: 176.2. Found: 177.1. Anal. Calcd for C10H12N2O (176.22): C,
68.16; H, 6.86; N, 15.90. Found: C, 68.45; H, 6.73; N, 16.12.
(ꢀ)-5-{(1S,4R)-7-Azabicyclo[2.2.1]hept-2-en-2-yl}-3-methylisoxazole
3.
Reaction of (+)-9 (225 mg, 0.81 mmol), according to the aforemen-
tioned described procedure, gave (ꢀ)-3 (113 mg, 79% yield).
(1S,4R)-(ꢀ)-3: Pale yellow viscous oil. [a]2D5 = ꢀ65.9 (c 1.0, CHCl3). The
NMR spectroscopic data matched those of the enantiomer (+)-3. MS
(ESI) m/z [M + H]+ Calcd for C10H12N2O: 176.2. Found: 177.0. Anal. Calcd
for C10H12N2O (176.22): C, 68.16; H, 6.86; N, 15.90. Found: C, 68.53; H,
6.51; N, 15.68.
(+)-(1S,2R,4R)-tert-Butyl-2-(3-methylisoxazol-5-yl)-7-azabicyclo
[2.2.1]heptane-7-carboxylate 11.
The endo-derivative (+)-10
(145 mg, 0.52mmol), which was reacted following the aforementioned
described procedure, gave 81mg (56% yield) of the exo-epimer (+)-11.
(+)-(1S,2R,4R)-11: Pale yellow viscous oil. Chiral HPLC analysis
was performed in the same conditions as those reported for (ꢀ)-11.
Retention time: 12.70 min. E.e. 99%. [a]2D5 = +13.4 (c 1.0, CHCl3). The
NMR spectroscopic data matched those of (ꢀ)-11. MS (ESI) m/z
[M + H]+ Calcd for C15H22N2O3: 278.4. Found: 279.3. Anal. Calcd for
(+)-5-{(1R,4S)-7-Azabicyclo[2.2.1]hept-2-en-2-yl}-3-methylisoxazole
fumarate 3 ꢃ C4H4O4 and (ꢀ)-5-{(1S,4R)-7-azabicyclo[2.2.1]hept-2-
en-2-yl}-3-methylisoxazole fumarate 3 ꢃ C4H4O4.
A solution of
fumaric acid (33 mg, 0.28 mmol) in MeOH was added to a solution of the
free base (+)-3 or (ꢀ)-3 (50 mg, 0.28 mmol) in MeOH (2 ml). After stirring
for 3 h at room temperature, the solvent was removed at reduced pressure,
and the crude salt was obtained quantitatively.
C
15H22N2O3 (278.35): C, 64.73; H, 7.97; N, 10.06. Found: C, 64.65; H,
8.22; N, 10.19.
(+)-(1R,4S)-3 ꢃ C4H4O4: Colorless prisms (from 2-propanol/diisopropyl
ether 2:1), mp 172–174 ꢂC, dec. [a]2D5 = +26.6 (c 0.50, MeOH). 1H NMR
(CD3OD): 1.49–1.67 (m, 2H), 2.19–2.25 (m, 2H), 2.30 (s, 3H), 4.86 (bs,
1H), 5.09 (bs, 1H), 6.57 (s, 1H), 6.67 (s, 2H), 6.83 (bs, 1H). 13C NMR
(CD3OD): 9.94, 21.46, 22.32, 60.53, 61.22, 103.84, 129.77, 132.66, 134.97,
160.86, 161.75, 170.04. MS (ESI) m/z [M+ H]+ Calcd for C14H16N2O5:
292.3. Found: 177.1. Anal. Calcd for C14H16N2O5 (292.29): C, 57.53; H,
5.52; N, 9.58. Found: C, 57.78; H, 5.46; N, 9.71.
(ꢀ)-(1S,4R)-3 ꢃ C4H4O4: Colorless prisms (from 2-propanol/diisopropyl
ether 2:1), mp 174–175 ꢂC, dec. [a]2D5 = ꢀ29.5 (c 0.50, MeOH). The NMR
spectroscopic data matched those of the enantiomer (+)-3 fumarate. MS
(ESI) m/z [M + H]+ Calcd for C14H16N2O5: 292.3. Found: 177.2. Anal. Calcd
for C14H16N2O5 (292.29): C, 57.53; H, 5.52; N, 9.58. Found: C, 57.64; H,
5.78; N, 9.61.
(+)-5-{(1R,2S,4S)-7-Azabicyclo[2.2.1]heptan-2-yl}-3-methylisoxazole
hydrochloride 2 ꢃ HCl.
The N-Boc protected derivative (ꢀ)-11
(120 mg, 0.43 mmol) was dissolved in 4N HCl in anhydrous dioxane
(2 ml) at 0 ꢂC. The reaction mixture was allowed to warm at room temper-
ature, and then it was stirred for about 1 h monitoring the disappearance
of the starting material (TLC, petroleum ether/ethyl acetate 85:15). The
reaction mixture was then concentrated to dryness under reduced pressure,
and the residue was purified by crystallization to afford 63mg (73% yield)
of (+)-2 hydrochloride.
(+)-(1R,2S,4S)-2 ꢃ HCl: Colorless prisms (from 2-propanol), mp
1
184–192 ꢂC, dec. [a]D25 = +19.5 (c 0.50, MeOH). H NMR (D2O): 1.65–1.95
(m, 4H), 2.01–2.08 (m, 1H), 2.13 (s, 3H), 2.24–2.29 (m, 1H), 3.42
(dd, J = 5.5 and 9.1 Hz, 1H), 4.26 (bs, 1H), 4.38 (bs, 1H), 6.10 (s, 1H). 13C
NMR (D2O): 11.64, 28.35, 28.99, 29.10, 36.90, 41.23, 55.52, 60.36, 79.91,
101.40, 155.02, 159.80, 175.36. MS (ESI) m/z [M + H]+ Calcd for
(+)-3-Methyl-5-{(1R,4S)-7-methyl-7-azabicyclo[2.2.1]hept-2-en-2-
yl}isoxazole 12.
Sodium cyanoborohydride (137 mg, 2.2 mmol) was
C
10H15ClN2O: 214.7. Found: 179.1. Anal. Calcd for C10H15ClN2O (214.69):
added in portions to a mixture of (+)-3 (180mg, 1.02mmol), a 37% aqueous
solution of formaldehyde (0.4 ml) and acetonitrile (10 ml) at 0ꢂC. The reac-
tion mixture was stirred at room temperature for 1 h, then after concentra-
tion in vacuo, ethyl acetate (5 ml) and water (5 ml) were added. The two
layers were separated and the residual aqueous phase was extracted with
ethyl acetate (2 ꢃ 5 ml). After standard workup, the crude reaction mixture
C, 55.94; H, 7.04; N, 13.05. Found: C, 56.14; H, 6.95; N, 13.36.
(ꢀ)-5-{(1S,2R,4R)-7-Azabicyclo[2.2.1]heptan-2-yl}-3-methylisoxazole
hydrochloride 2 ꢃ HCl.
Reaction of (+)-11 (75mg, 0.27mmol), accord-
ing to the aforementioned described procedure, gave (ꢀ)-2 hydro-
chloride (35 mg, 61% yield), which was purified by crystallization.
Chirality DOI 10.1002/chir

THE ENANTIOMERS OF EPIBOXIDINE AND OF TWO RELATED ANALOGS
underwent a silica gel column chromatography (dichloromethane/metha-
a-bungarotoxin binding receptors, the membrane homogenates were
preincubated with 2 mM a-bungarotoxin and then with [3H]epibatidine.
Saturation experiments were performed by incubating aliquots of
cortex membrane homogenates with 0.01–2.5 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. After incubation, 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.
nol 9:1), which afforded the N-methylated compound (+)-12 (130mg,
67% yield).
(+)-(1R,4S)-12: Yellow oil. Rf = 0.56 (dichloromethane/methanol 9:1).
[a]2D5 = +29.7 (c 1.0, CHCl3). 1H NMR: 1.08–1.25 (m, 2H), 1.93–2.03
(m, 2H), 2.15 (s, 3H), 2.30 (s, 3H), 3.91 (m, 1H), 4.02 (m, 1H), 6.04
(s, 1H), 6.51 (bs, 1H). 13C NMR: 11.59, 24.90, 25.74, 34.52, 67.73,
101.94, 131.17, 160.18, 165.15, 175.78. MS (ESI) m/z [M + H]+ Calcd for
C11H14N2O: 190.2. Found: 191.1. Anal. Calcd for C11H14N2O (190.24): C,
69.45; H, 7.42; N, 14.73. Found: C, 69.87; H, 7.12; N, 14.92.
125I]a-Bungarotoxin binding.
Saturation binding experiments
(ꢀ)-3-Methyl-5-{(1S,4R)-7-methyl-7-azabicyclo[2.2.1]hept-2-en-
[
2-yl}isoxazole 12.
Reaction of (ꢀ)-3 (115mg, 0.65mmol), according
were performed using aliquots of cortex membrane homogenates incu-
bated overnight with 0.1–10 nM concentrations of [125I]a-bungarotoxin
(specific activity 200–213 Ci/mmol, 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 earlier, and the bound radioactivity was directly
counted in a g counter.
to the aforementioned described procedure, gave (ꢀ)-12 (68 mg, 55% yield).
(ꢀ)-(1R,4S)-12: Light yellow oil. [a]2D5 = ꢀ31.8 (c 1.0, CHCl3). The
NMR spectroscopic data matched those of the enantiomer (+)-12. MS
(ESI) m/z [M + H]+ Calcd for C11H14N2O: 190.2. Found: 191.1. Anal. Calcd
for C11H14N2O (190.24): C, 69.45; H, 7.42; N, 14.73. Found: C, 69.79; H,
7.24; N, 14.55.
(+)-3-Methyl-5-{(1R,4S)-7-methyl-7-azabicyclo[2.2.1]hept-2-en-
2-yl}isoxazole methyl iodide 4 and (ꢀ)-3-methyl-5-{(1S,4R)-7-
methyl-7-azabicyclo[2.2.1]hept-2-en-2-yl}isoxazole methyl iodide
Affinity of derivatives (+)-2/(ꢀ)-2, (+)-3/(ꢀ)-3, and (+)-4/(ꢀ)-4
for nAChRs.
Inhibition of radioligand binding by epibatidine and
the test compounds was measured by preincubating cortex homogenates
with increasing doses (10 pM–10 mM) of the reference nicotinic agonist
epibatidine or nicotine, or the drugs to be tested, for 30 min at room
temperature. This was followed by overnight incubation with a final
concentration of 0.075 nM [3H]epibatidine (for the a4b2 subtype) or
1 nM [125I]a-bungarotoxin (for the a7 subtype) at the same temperatures
as those used for the saturation experiments. These ligand concentra-
tions were used for the competition binding experiments because they
are within the range of the ligand KD values of the ligands for the two
different classes of nAChRs. For each compound, experimental data
obtained from three saturation and three competition binding experi-
ments were analyzed by means of a nonlinear least square procedure,
using the LIGAND program as described by Munson and Rodbard.23
The binding parameters were calculated by simultaneously fitting three
independent saturation experiments, and the Ki values were determined
by fitting the data of three independent competition experiments. 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).
4.
Iodomethane (156 ml, 2.52 mmol) was added to a solution of the
tertiary base (+)-12 or (ꢀ)-12 (65 mg, 0.34 mmol) in methanol (2 ml),
and the resulting mixture was stirred for 3 h at room temperature. The
solvent was removed under reduced pressure to quantitatively yield
the crude quaternary salt as a yellow solid residue, which was washed
three times with small amounts of diethyl ether.
(+)-(1R,4S)-4: Colorless prisms (from abs. ethanol), mp 150–
155 ꢂC, dec. [a]D25 = +62.3 (c 0.50, MeOH). 1H NMR (D2O): 1.48–1.64
(m, 2H), 2.12 (s, 3H), 2.28–2.41 (m, 2H), 2.93 (s, 3H), 2.96 (s, 3H),
4.62 (bs, 1H), 4.83 (bs, 1H), 6.37 (s, 1H), 6.73 (bs, 1H). 13C NMR
(D2O): 10.52, 21.09, 21.79, 44.99, 45.36, 74.54, 75.77, 104.85, 129.73,
131.04, 161.31, 161.95. MS (ESI) m/z [M]+ Calcd for C12H17IN2O:
332.2. Found: 205.3. Anal. Calcd for C12H17IN2O (332.18): C, 43.39; H,
5.16; N, 8.43. Found: C, 43.52; H, 5.37; N, 8.34.
(ꢀ)-(1S,4R)-4: Colorless prisms (from abs. ethanol), mp 150–155 ꢂC,
dec. [a]2D5 = ꢀ64.5 (c 0.50, MeOH). The NMR spectroscopic data matched
those of the enantiomer (+)-4. MS (ESI) m/z [M]+ Calcd for C12H17IN2O:
332.2. Found: 205.2. Anal. Calcd for C12H17IN2O (332.18): C, 43.39; H,
5.16; N, 8.43. Found: C, 43.48; H, 5.01; N, 8.56.
RESULTS AND DISCUSSION
Receptor Binding Assays
As illustrated in Scheme 1, the synthetic approach to the
enantiomerically pure final derivatives started with the resolu-
tion of 7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptan-2-one
(ꢁ)-5,21,16 an advanced key intermediate in a few syntheses
of (ꢁ)-epibatidine.24 Moreover, the two enantiomers of (ꢁ)-5,
that is, (1R,4S)-(ꢀ)-5 and (1S,4R)-(+)-5, were obtained by
enantiospecific synthesis,22 through enantiotopic discrimination
of a suitable meso-intermediate25 or exploiting the formation of
diastereomeric aminales with (R,R)-1,2-diphenylehylenedia-
mine.24 In this study, we made use of commercial (R)-(+)-2-
methyl-2-propanesulfinamide26–28 which, in the presence of the
Lewis acid Ti(OEt)4,29 efficiently condensed with (ꢁ)-5 provid-
ing a mixture of the two epimeric tert-butanesulfinyl ketimines
(ꢀ)-(1R,4S,9R)-6 and (1S,4R,9R)-(ꢀ)-7 in comparable amounts
[(ꢀ)-6/(ꢀ)-7 about 43:57] (Scheme 1). After separation by
silica gel flash chromatography, the two isomers were individu-
ally treated with 4N acetic acid in methanol, which allowed
to isolate the enantiomers (1R,4S)-(ꢀ)-5 (from (ꢀ)-6) and
(1S,4R)-(+)-5 (from (ꢀ)-7) and, accordingly, to assign the
absolute configuration to the precursor ketimines. Both
Chirality DOI 10.1002/chir
Tissue preparation.
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 ethylenediaminetetraacetic
acid [EDTA], 2 mM ethylene glycol tetraacetic acid [EGTA], and 2 mM
phenylmethylsulfonyl fluoride [PMSF], pH 7.4) using a potter homoge-
nizer; the homogenates were then diluted and centrifuged at 60,000 g
for 1.5 h. Total membrane homogenization, dilution, and centrifugation
procedures were performed twice, then the pellets were collected, rapidly
rinsed with a buffer solution (50 mM Tris–HCl, 120 mM NaCl, 5 mM KCl,
1 mM MgCl2, 2.5 mM CaCl2, and 2 mM PMSF, pH 7), and resuspended in
the same buffer containing a mixture of 20 mg/ml of each of the following
protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin.
[3H]Epibatidine binding.
(ꢁ)-[3H]Epibatidine with a specific
activity of 56–60 Ci/mmol was purchased from Perkin Elmer (Boston
MA); the nonradioactive a-bungarotoxin, epibatidine, and nicotine were
purchased from Sigma Aldrich (Italy). It has been previously reported
that [3H]epibatidine also binds to a-bungarotoxin binding receptors
with nM affinity.11 To prevent the binding of [3H]epibatidine to the

DALLANOCE ET AL.
and (1S,2R,4R)-(ꢀ)-2, respectively. The same protocol was
applied to the two intermediate olefins (1R,4S)-(ꢀ)-9 and
(1S,4R)-(+)-9, but owing to the inherent hygroscopicity of the
two hydrochlorides, we isolated the free bases (1R,4S)-(+)-3
and (1S,4R)-(ꢀ)-3, which were then transformed into their
crystalline 1:1 fumarates (Scheme 2). Alternatively, (1R,4S)-
(+)-3 and (1S,4R)-(ꢀ)-3 were reacted with aqueous formalde-
hyde, followed by reduction of the intermediate aldimines with
sodium cyanoborohydride, which provided the corresponding
tertiary amines (1R,4S)-(+)-12 and (1S,4R)-(ꢀ)-12. These
enantiomers were treated with excess iodomethane to give
the dimethylammonium salts (1R,4S)-(+)-4 and (1S,4R)-(ꢀ)-4
(Scheme 2).
The three couples of derivatives under study (+)-2/(ꢀ)-2,
(+)-3/(ꢀ)-3, and (+)-4/(ꢀ)-4 were assayed for binding affin-
ity at rat a4b2 and a7 nAChRs, using [3H]epibatidine and
[
125I]a-bungarotoxin as radioligands, respectively. The Ki
values, calculated from the competition curves of three separate
experiments by means of the LIGAND program,23 are reported
in Table 1 and compared with the known Ki values of (ꢁ)-1 and
its enantiomers and those previously obtained by us for (ꢁ)-2,
(ꢁ)-3, and (ꢁ)-4.
Inspection of the binding data at the explored nAChRs
evidences the identity of the absolute configuration at the
common stereogenic centers for the antipodes with the
highest affinity. Indeed, within each enantiomeric pair,
hydrochloride (1R,2S,4S)-(+)-2, fumarate (1R,4S)-(+)-3, and
iodomethylate (1R,4S)-(+)-4 were found to be the eutomers
at both a4b2 and a7 nAChRs. Worth noting, the free base
natural epibatidine (1R,2R,4S)-(ꢀ)-1 and hydrochloride
(1R,2S,4S)-(+)-2 share the same spatial arrangement around
the stereogenic centers and have opposite polarimetric rotation
angles. The two enantiomeric epibatidines (1R,2R,4S)-(ꢀ)-1
and (1S,2S,4R)-(+)-1 displayed very high affinity at the a4b2
nAChRs (Ki values equal to 0.020 and 0.019 nM,12 respectively),
but their molecular interaction was characterized by the
absence of enantioselectivity. Similarly, our data obtained at
the same subtype for the enantiomeric epiboxidine hydrochlor-
ides (1R,2S,4S)-(+)-2 and (1S,2R,4R)-(ꢀ)-2 (Ki values equal to
0.21 and 0.45nM, respectively) and their structural analogs
(1R,4S)-(+)-3 and (1S,4R)-(ꢀ)-3 (Ki values equal to 29 and
73 nM, respectively) revealed a lack of enantioselectivity (E.R.
values of 2 and 2.5 for the two pairs) in addition to a progressive
reduction in affinity. A lower affinity was detected at the a7
subtype for the epiboxidine enantiomers (Ki values of 5.2 and
15.5nM), an outcome comparable with that reported for the
epibatidine enantiomers12 (Ki values of 7.0 and 4.9 nM), both
pairs showing a negligible enantioselectivity (E.R. values of 3
and 0.7, respectively). On the other hand, the two antipodes
of the “unsaturated” epiboxidine analog (ꢁ)-3 evidenced a
marked reduction in affinity (Ki values of 0.17 and 2.8 mM) at
the a7 subtype together with a gain of the enantioselectivity
(E.R. value of 16.5).
Scheme 1. Reagents and conditions: (a) Ti(OEt)4, THF, 65 ꢂC, 2 h and (b)
4N CH3COOH, MeOH, 40 ꢂC, 18 h.
enantiomeric ketones were characterized by high enantiomeric
purity (e.e. = 98%) and spectroscopic/polarimetric features in
line with those reported in the literature.22 Moreover, the lack
of diastereoselection associated to the initial addition provided
ketones (ꢀ)-5 and (+)-5 in comparable yields, and we could
carry out the overall synthetic sequence in a parallel fashion.
The N-Boc protected enantiomeric ketones were then con-
verted into their corresponding trifluoromethanesulfonates
(1R,4S)-(ꢀ)-8 and (1S,4R)-(+)-8, respectively, by sequential
treatment with potassium bis(trimethylsilyl)amide, to form
the enolate, and N-(5-chloro-2-pyridyl)bis(trifluoromethane-
sulfonimide).30,31 Next, an efficient Stille–Miyaura cross-
coupling protocol was carried out by reacting the enantiomers
(1R,4S)-(ꢀ)-8 and (1S,4R)-(+)-8 with 3-methyl-5-tributylstanny-
lisoxazole in the presence of the tris(dibenzylideneacetone)
dipalladium(0)-chloroform adduct, triphenylphosphine, and
anhydrous ZnCl2.16 The two enantiomeric isoxazole derivatives
(1R,4S)-(ꢀ)-9 and (1S,4R)-(+)-9 (Scheme 2), obtained in 92%
and 89% yield, were then submitted to a standard catalytic
hydrogenation over 10% Pd/C, which gave the two N-Boc-
endo-epiboxidines (1R,2R,4S)-(ꢀ)-10 and (1S,2S,4R)-(+)-10 as
the sole isomers, as already found for the reduction of (ꢁ)-916
and similarly to what observed in a parallel preparation of the
corresponding intermediate in the synthesis of (ꢁ)-epibati-
dine.32 The subsequent epimerization reaction to the desired
exo-isomers was achieved, with a conversion of about 60%, by
prolonged heating of the two endo-isomers with potassium
tert-butoxide in refluxing tert-butanol,21 followed by a column
chromatography purification. The two enantiomeric N-Boc-
exo-epiboxidines (1R,2S,4S)-(ꢀ)-11 and (1S,2R,4R)-(+)-11 un-
derwent removal of the N-Boc protection with a 4N solution of
HCl in dioxane, which produced the corresponding epiboxi-
dine antipodes as crystalline hydrochlorides (1R,2S,4S)-(+)-2
In terms of preferred recognition of ligands by the assayed
nAChRs, the exceptional affinity shown by racemic epibatidine
for the a4b2 subtype determines the high degree of a4b2
versus a7 subtype selectivity (Table 1), which is drastically
reduced on passing from (ꢁ)-1 (291)33 to (ꢁ)-2 (30)18 and
(ꢁ)-3 (32).16 Such selectivity profiles are retained when the
affinity data obtained on the enantiomeric pairs (ꢀ)-1/(+)-1
(350/258), (+)-2/(ꢀ)-2 (25/34), and (+)-3/(ꢀ)-3 (5.5/38) are
taken into account, none of the tested individual isomers is able
to substantially alter the nAChR subtype discrimination
Chirality DOI 10.1002/chir

THE ENANTIOMERS OF EPIBOXIDINE AND OF TWO RELATED ANALOGS
Scheme 2. Reagents and conditions: (a) KHMDS, 0.5 M in toluene, N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonimide), DME, ꢀ78 ꢂC to room
temperature (rt), 3 days; (b) [Pd2(dba)3] ꢄ CHCl3, PPh3, anhydrous ZnCl2, THF, ꢀ78 ꢂC to rt, 3 days; (c) H2, 10% Pd/C, MeOH, rt, 5 h; (d) tert-BuOK/tert-BuOH,
reflux, 30 h (about 60% conversion), flash chromatography; (e) 4N HCl, dioxane, rt, 1 h; (f) aq. Na2CO3, solvent extraction; (g) HCHO (37% aq.), NaBH3CN, CH3CN,
rt, 1 h; (h) C4H4O4,MeOH, rt, 3 h; (i) CH3I, MeOH, rt, 3 h.
TABLE 1. Affinity of enantiomeric pairs (+)-2/(ꢀ)-2, (+)-3/(ꢀ)-3, and (+)-4/(ꢀ)-4 for native a4b2 and a7 nAChR subtypes present
in rat cortical membranes labeled, respectively, by [3H]epibatidine and [125I]a-bungarotoxin
Entry
a4b2 [3H]Epi Kia (nM)
a7 [125I]a-BgTx Kia (nM)
Selectivity a4b2/a7
(ꢁ)-1
0.035b
0.020c
0.019c
1
10.2b
291
350
258
(ꢀ)-(1R,2R,4S)-1
(+)-(1S,2S,4R)-1
E.R.d
7.0c
4.9c
0.7
(ꢁ)-2
0.24e
7.3e
30
25
34
(+)-(1R,2S,4S)-2 ꢃ HCl
(ꢀ)-(1S,2R,4R)-2 ꢃ HCl
E.R.d
0.21 (17)
0.45 (19)
2
5.2 (22)
15.5 (18)
3
(ꢁ)-3
50f
1600f
32
5.5
38
(+)-(1R,4S)-3 ꢃ C4H4O4
(ꢀ)-(1S,4R)-3 ꢃ C4H4O4
E.R.d
29 (22)
73 (17)
2.5
170 (34)
2800 (38)
16.5
(ꢁ)-4
13.3e
1.6e
0.12
0.11
0.34
(+)-(1R,4S)-4
(ꢀ)-(1S,4R)-4
E.R.d
10.2 (28)
67 (24)
6.6
1.1 (30)
23 (29)
21
aKi values were derived from three competition binding experiments. Numbers in brackets refer to the percent coefficients of variation. Literature data available for
(ꢁ)-1, (+)-1, (ꢀ)-1, (ꢁ)-2, (ꢁ)-3, and (ꢁ)-4 have been added for comparison.
bRef. 33.
cRef. 12.
dE.R., eudismic ratio; ratio between the Ki value of distomer and the Ki value of eutomer.
eRef. 18.
fRef. 16.
Chirality DOI 10.1002/chir

DALLANOCE ET AL.
9. Badio B, Garraffo HM, Spande TF, Daly JW. Epibatidine: discovery and
detected for the corresponding racemate. Worth pointing out,
the permanently charged ammonium salt (ꢁ)-4 showed an
opposite affinity trend compared with that of the other investi-
gated ligands because it preferentially bound the a7 subtype.18
Both individual enantiomers of (ꢁ)-4 preserved the affinity
profile of the racemate, and the eutomer (1R,4S)-(+)-4 [Ki = 10.2
nM (a4b2) and 1.1 nM (a7)] should be included in the number
of high affinity unselective nicotinic ligands at a4b2 and a7
receptors. In addition, the (+)-4/(ꢀ)-4 couple exhibited an
increase of enantioselectivity at both receptor subtypes [E.
R. = 6.6 (a4b2) and 21 (a7)] when compared with the other
investigated enantiomeric pairs.
definition as a potent analgesic and nicotinic agonist. Med Chem Res
1994;4:440–448.
10. Broka C. Synthetic approaches to epibatidine. Med Chem Res
1994;4:449–460.
11. Gerzanich V, Peng X, Wang F, Wells G, Anand R, Fletcher S, Lindstrom J.
Comparative pharmacology of epibatidine: a potent agonist for neuronal
nicotinic acetylcholine receptors. Mol Pharmacol 1995;48:774–782, and
references cited therein.
12. Cox CD, Malpass JR, Gordon J, Rosen A. Synthesis of epibatidine isomers:
endo-5- and 6-(6’-chloro-3’-pyridyl)-2-azabicyclo[2.2.1]heptanes. J Chem Soc
Perkin Trans 1 2001;2372–2379.
13. Carroll FI. Epibatidine structure-activity relationships. Bioorg Med Chem
Lett 2004;14:1889–1996, and references cited therein.
14. White R, Malpass JR, Handa S, Baker SR, Broad LM, Folly L, Mogg A.
Epibatidine isomers and analogues: structure-activity relationships.
Bioorg Med Chem Lett 2006;16:5493–5497.
CONCLUSIONS
15. Badio B, Garraffo HM, Plummer CV, Padgett WL, Daly JW. Synthesis and
nicotinic activity of epiboxidine: an isoxazole analogue of epibatidine. Eur
J Pharmacol 1997;321:189–194.
16. Rizzi L, Dallanoce C, Matera C, Magrone P, Pucci L, Gotti C,
Clementi F, De Amici M. Epiboxidine and novel-related analogues: a
convenient synthetic approach and estimation of their affinity at
neuronal nicotinic acetylcholine receptor subtypes. Bioorg Med Chem
Lett 2008;18:4651–4654,and references cited therein.
17. Dallanoce C, Magrone P, Bazza P, Grazioso G, Rizzi L, Riganti L, Gotti C,
Clementi F, Frydenvang K, De Amici M. New analogues of epiboxidine
incorporating the 4,5-dihydroisoxazole nucleus: synthesis, binding affinity
at neuronal nicotinic acetylcholine receptors, and molecular modeling
investigations. Chem Biodivers 2009;6:244–259.
18. Dallanoce C, Matera C, Pucci L, Gotti C, Clementi F, De Amici M, De
Micheli C. Synthesis and binding affinity at a4b2 and a7 nicotinic acetyl-
choline receptors of new analogues of epibatidine and epiboxidine con-
taining the 7-azabicyclo[2.2.1]hept-2-ene ring system. Bioorg Med Chem
Lett, 2012;22: 829–832.
In this study, we prepared hydrochlorides (ꢀ)-2 and (+)-2,
the two enantiomers of epiboxidine, whose racemate is one
of the most interesting synthetic analogs of natural (ꢀ)-epiba-
tidine. Resolution of the racemic N-Boc-7-azabicyclo[2.2.1]
heptane-2-one coupled with a Suzuki cross-coupling reaction
was the key step of the applied synthetic sequence. With
the same approach, we prepared two further epiboxidine-
related nicotinic ligands, that is, the “unsaturated” fumarates
of the secondary bases (ꢀ)-3 and (+)-3 as well as the
dimethylammonium iodides (ꢀ)-4 and (+)-4. The three
enantiomeric pairs were assayed in binding experiments to
evaluate their affinity for the neuronal a4b2 and a7 nAChR
subtypes. On the whole, the degree of the observed enantios-
electivity was poor for the three pairs of enantiomers at both
investigated receptors, a result which to a large extent
matches that known for the antipodes of epibatidine. Interest-
ingly, both antipodes of (ꢁ)-4, at variance with those of (ꢁ)-2
and (ꢁ)-3, preferentially recognized the a7 receptor, although
with a low subtype selectivity.
19. Dallanoce C, Magrone P, Matera C, Frigerio F, Grazioso G, De Amici M,
Fucile S, Piccari V, Frydenvang K, Pucci L, Gotti C, Clementi F, De
Micheli C. Design, synthesis, and pharmacological characterization of
novel spirocyclic quinuclidinyl-2-isoxazoline derivatives as potent and
selective agonists of a7 nicotinic acetylcholine receptors. ChemMedChem
2011;6:889–903.
20. Pucci L, Grazioso G, Dallanoce C, Rizzi L, De Micheli C, Clementi F,
Bertrand S, Bertrand D, Longhi R, De Amici M, Gotti C. Engineering of
a-conotoxin MII-derived peptides with increased selectivity for native
a6b2* nicotinic acetylcholine receptors. FASEB J 2011;25:3775–3789.
ACKNOWLEDGMENTS
Carlo Matera wishes to thank “Dote ricerca”: FSE, Regione
Lombardia for co-financing his postdoctoral position.
21. Zhang C, Trudell ML. A short and efficient total synthesis of (ꢁ)-epibati-
dine. J Org Chem 1996;61:7189–7191.
22. Hernández A, Marcos M, Rapoport H. Synthesis of (+) and (ꢀ)-N-BOC-7-
azabicyclo[2.2.1]heptan-2-ones, versatile intermediates for the enantiospe-
cific synthesis of (+)- and (ꢀ)-epibatidine and analogues. J Org Chem
1995;60:2683–2691.
LITERATURE CITED
1. Sine SM, Engel AG. Recent advances in Cys-loop receptor structure and
function. Nature 2006;440:448–455.
23. Munson PJ, Rodbard D. LIGAND: a versatile computerized approach
for characterization of ligand-binding systems. Anal Biochem
1980;107:220–239.
24. Moreno-Vargas AJ, Vogel P. Efficient resolution of N-Boc-7-azabicyclo
[2.2.1]hept-5-en-2-one: formal syntheses of natural epibatidine and its
enantiomer. Tetrahedron: Asymmetry 2003;14:3173–3176, and references
cited therein.
25. Pandey G, Tiwari SK, Singh RS, Mali RS. A short and efficient synthesis of
enantiopure (+)-N-Boc-7-azabicyclo[2.2.1]heptan-2-one utilizing an asym-
metric desymmetrization protocol: formal total synthesis of (ꢀ)-epibatidine.
Tetrahedron Lett 2001;42:3947–3949.
26. Ellman JA, Owens TD, Tang TP. N-tert-butanesulfinyl imines: versatile
intermediates for the asymmetric synthesis of amines. Acc Chem Res
2002;35:984–995.
2. Tsetlin V, Kuzmin D, Kasheverov I. Assembly of nicotinic and other
Cys-loop receptors. J Neurochem 2011;116:734–741.
3. Albuquerque EX, Pereira EFR, Alkondon M, Rogers, SW. Mammalian
nicotinic acetylcholine receptors: from structure to function. Physiol Rev
2009;89:73–120.
4. Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic
cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol
Toxicol 2007;47:699–729.
5. Jensen AA, Frølund B, Liljefors T, Krogsgaard-Larsen P. Neuronal nicotinic
acetylcholine receptors: structural revelations, target identifications, and
therapeutic inspirations. J Med Chem 2005;48:4705–4745.
6. Cincotta SL, Yorek MS, Moschak TM, Lewis SR, Rodefer JS. Selective nicotinic
acetylcholine receptor agonists: potential therapies for neuropsychiatric
disorders with cognitive dysfunction. Curr Opin Investig Drugs 2008;9:47–56.
27. McMahon JP, Ellman JA. High stereoselective addition of organometallic
reagents to N-tert-butanesulfinyl imines derived from 3- and 4-substituted
cyclohexanones. Org Lett 2004;6:1645–1647.
7. Taly A, Corringer PJ, Guedin D, Lestage P, Changeux JP. Nicotinic receptors:
allosteric transitions and therapeutic targets in the nervous system. Nat Rev
Drug Discov 2009;8:733–750.
28. Morton D, Stockman RA. Chiral non-racemic sulfinimines: versatile
reagents for asymmetric synthesis. Tetrahedron 2006;62:8869–8905.
8. Spande TF, Garraffo HM, Edwards MW, Yeh JC, Pannell L, Daly JW.
Epibatidine:
a novel (chloropyridyl)azabicyloheptane with potent
29. Chelucci G, Baldino S, Chessa S, Pinna GA, Soccolini F. An easy route to
optically active 1-substituted-1-pyridyl-methylamines by diastereoselective
analgesic activity from an Ecuadorian poison frog. J Am Chem Soc
1992;114:3475–3478.
Chirality DOI 10.1002/chir

THE ENANTIOMERS OF EPIBOXIDINE AND OF TWO RELATED ANALOGS
reduction of enantipoure N-tert-butanesulfinyl ketimines. Tetraedron:
Asymmetry 2006;17:3163–3169.
and 3-azatetracyclo[6.1.1.0.2,705,19
] decanes. J Org Chem 2004;69:
3758–3764.
30. Carroll FI, Liang F, Navarro HA, Brieaddy LE, Abraham P, Damaj MI,
Martin BR. Synthesis, nicotinic acetylcholine receptor binding, and anti-
nociceptive properties of 2-exo-2-(2’-substituted-5’-pyridinyl)-7-azabicyclo
[2.2.1]heptanes, epibatidine analogues. J Med Chem 2001;44:2229–2237.
31. Choi Y, White JD. Intramolecular photocycloaddition of unsaturated
isoquinuclidines. Synthesis of 2-azatetracyclo[4.0.0.4,907,10]decanes
32. Fletcher SR, Baker R, Chambers MS, Herbert RH, Hobbs SC, Thomas SR,
Verrier HM, Watt AP, Ball RG. Total synthesis and determination of the
absolute configuration of epibatidine. J Org Chem 1994;59:1771–1778.
33. Zhang N, Tomizawa M, Casida JE. 5-Azidoepibatidine: an exceptionally
potent photoaffinity ligand for neuronal a4b2 and a7 nicotinic acetylcho-
line receptors. Bioorg Med Chem Lett 2003;13:525–527.
Chirality DOI 10.1002/chir