6,6-Spiroimine analogs of (-)-gymnodimine A: Synthesis and biological evaluation on nicotinic acetylcholine receptors

Article abstract of DOI:10.1039/c1ob06257c

Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric al

Full text of DOI:10.1039/c1ob06257c

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 8112
www.rsc.org/obc
PAPER
6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological
evaluation on nicotinic acetylcholine receptors†
Leslie Duroure,a Thierry Jousseaume,a Ro´mulo Ara´oz,b Elvina Barre´,a Pascal Retailleau,a Laurent Chabaud,*a
Jordi Molgo´*b and Catherine Guillou*a
Received 26th July 2011, Accepted 5th September 2011
DOI: 10.1039/c1ob06257c
Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and
optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael
addition of a b-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same b-ketoester
was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched
mixtures were tested for their biological activities on Xenopus oocytes either expressing (human a4b2)
or having incorporated (Torpedo a12bgd) nicotinic acetylcholine receptors (nAChRs). These spiroimine
analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active
than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of
nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins.
subset of muscle and neuronal nicotinic acetylcholine receptors
(nAChRs).7 The biological target was then confirmed by the co-
Introduction
(-)-Gymnodimine A (1), first isolated from oysters collected
off the coast of New Zealand,1 is a macrocyclic imine phyco-
toxin produced by the dinoflagelate Karenia selliformis (Fig. 1).2
Shellfish can concentrate this phycotoxin through filter-feeding
on toxic dinoflagellates, acting thereafter as primary vectors for
transferring this toxic chemical compound to crabs, fish, birds,
marine mammals and ultimately to humans.3
crystallization of two representative members of the spiroimine
family, (-)-gymnodimine A (1) and 13-desmethyl spirolide C, with
the acetylcholine binding protein (AChBP), a soluble surrogate
for the ligand binding domain of a7 nAChR subtype.7 This
study allowed the identification of the structural determinants
in both (-)-gymnodimine A (1) and AChBP, which are important
for toxin–receptor interaction. Among them, it was shown that
the protonated imine nitrogen of the toxin plays a key role in
its binding to the acetylcholine binding site of AChBP. These
results corroborate the hypothesis that the spiroimine is one of the
pharmacophores of (-)-gymnodimine A (1) and other members
of the family.8,9
Given the importance of the spiroimine core as a key structural
feature responsible for the toxicity of the phycotoxin (1), we report
herein racemic and enantioselective syntheses of 6,6-spiroimine
analogs of (-)-gymnodimine A (1) and their biological activities
on nAChRs.
Fig. 1 Structure of (-)-gymnodimine A (1) and simple models studied.
(-)-Gymnodimine A (1) exhibits acute toxicity in a mouse
bioassay4 by intraperitoneal injection (LD50 = 80 mg kg-1)5 or
by oral administration (LD50 = 755 mg kg-1).6 Recently, (-)-
gymnodimine A (1) was shown to bind and block different
Results
aCentre de Recherche de Gif, Institut de Chimie des Substances Naturelles,
CNRS, 1, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. E-mail:
guillou@icsn.cnrs-gif.fr, chabaud@icsn.cnrs-gif.fr; Fax: +33 (0)1 69 07 72
47; Tel: +33 (0)1 69 82 30 75
Synthesis of racemic and optically active spiroimines 9 and 11
The synthesis of racemic spiroimines ( )-9 and ( )-11 were
achieved starting from unsaturated b-ketoester 2 (Scheme 1).10
Conjugate addition of b-ketoester 2 with acrolein was catalyzed
by triethylamine and afforded the aldehyde ( )-3 in 61% yield.
Double reduction of the keto-aldehyde ( )-3 was carried out in
a sequential one-pot operation. Treatment of ( )-3 with NaBH4
was followed, upon completion of the reduction of the aldehyde,
by addition of CeCl3·7H2O and additional NaBH4. This one-pot
bCentre de Recherche de Gif, CNRS, Institut Fe´de´ratif de Neurobiologie
Alfred Fessard FRC2118, Laboratoire de Neurobiologie et De´veloppement,
UPR 3294, 1 Avenue de la Terrasse, baˆtiments 32-33, 91198 Gif sur Yvette
cedex, France. E-mail: Jordi.Molgo@inaf.cnrs-gif.fr; Fax: +33 (0)1 69 82
41 41; Tel: +33 (0)1 69 82 36 42
† Electronic supplementary information (ESI) available. CCDC reference
numbers 828699. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c1ob06257c
8112 | Org. Biomol. Chem., 2011, 9, 8112–8118
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
derived from Cinchona alkaloids in the asymmetric Michael
addition of 2 with acrolein resulted in low e.e. The asymmetric
syntheses of spiroimines 9 and 11 were thus achieved by using
an asymmetric allylic alkylation.13 To the best of our knowledge,
there has been no example of asymmetric allylic alkylation with
unsaturated b-ketoesters such as 2. We thus tested a range of chiral
ligands (Table 1) and found that (S,S)-DACH phenyl Trost ligand
gave◦the best results in terms of yield and enantiomeric ratio at
-20 C (entry 9).14
Functionalization of the allyl group was achieved by olefin
cross-metathesis of (+)-12 with vinyl pinacol boronate, catalyzed
by Hoveyda–Grubbs second generation catalyst 13,15 to give
vinyl boronate 14 in 90% yield as a mixture of isomers (E/Z =
80/20) (Scheme 2).16 This intermediate underwent oxidation upon
treatment with sodium perborate to afford aldehyde 3, which was
directly reduced under previously optimized conditions to give diol
(+)-4 in 51% yield from 14.17 The optically active spiroimines (+)-
9 and (+)-11 (e.r. = 91 : 9) were obtained as described in Scheme
1. Both spiroimines were converted into the hydrochloride salts
(+)-15 and (-)-10 to evaluate their biological activities.
Scheme 1 Racemic syntheses of spiroimines ( )-9 and ( )-11.
Table 1 Ligand screening results
Entry Ligand
T/◦C Yield (%) e.r.
1
2
3
4
5
6
7
8
(R)-BINAP
25
25
25
25
25
25
35
26
27
41
—
59
30
40
87
50
40 : 50
52 : 48
52 : 48
62 : 38
—
14 : 86
43 : 57
88 : 12
91 : 9
(R,R)-DUPHOS
(S,S)-DIOP
(S)-tBuPHOX
(R,R)-DACH pyridyl Trost ligand
(R,R)-DACH naphtyl Trost ligand
(R,R)-ANDEN phenyl Trost ligand 25
(S,S)-DACH phenyl Trost ligand
(S,S)-DACH phenyl Trost ligand
(S,S)-DACH phenyl Trost ligand
25
-20
-78
9
10
82 : 18
procedure allowed us to obtain diol ( )-4 in 74% isolated yield
(d.r. = 90/10). The major diastereomer was then separated by
chromatography and treated with DBU in acetonitrile to afford the
key lactone ( )-5 in 75% yield, with relative configuration secured
by X-ray analysis.11 The allylic alcohol ( )-5 was protected with a
TBS group to give lactone ( )-6 in 87% yield. Lactone ring opening
with methyl magnesium bromide selectively afforded the methyl
ketone ( )-7 in good yield (71%). The primary alcohol of ( )-7
was then converted into the azide ( )-8, which was reduced with
supported triphenylphosphine to give spiroimine ( )-9. The silyl
group was removed under acidic conditions, which proved to be
compatible with the imine functionality.12 The resulting iminium
salt ( )-10 was then purified under basic conditions to afford the
imine ( )-11. It is worth mentioning that ( )-9 and ( )-10 exist
in solution as their imine and iminium forms, respectively. By
contrast, compound ( )-11 is in equilibrium with its enamine form
in CDCl3 (imine/enamine: 93/7).
Scheme 2 Enantioselective syntheses of spiroimines (+)-9 and (+)-11 and
their corresponding iminiums (+)-15, (-)-10.
Biological evaluation of spiroimines 9, 11 and their hydrochloride
salts 10, 15 on nAChRs
To determine whether the 6,6-spiroimine group plays a key role
in the interaction of (-)-gymnodimine A (1) with nAChRs,
we first evaluated the biological activity of ( )-9 on Xenopus
oocytes expressing the human a4b2 nAChR using the voltage-
clamp technique7 (Fig. 2A). Maximal peak amplitude of the
acetylcholine (ACh)-induced current (IACh) was reached follow-
ing 4 s superfusion with 150 mM ACh, after which nAChRs
became slowly desensitized (Fig. 2A, a, black superimposed trace).
Superfusion of 2 mM ( )-9 by itself did not elicit nicotinic current
responses, but in the presence of the ACh agonist (150 mM), it
reduced the peak amplitude and modified the kinetics of the IACh
(Fig. 2A, b, red superimposed trace). IACh peak current blockade
With a reliable route to racemic spiroimines, we then focused on
an enantioselective synthesis of 9 and 11. The use of chiral amines
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8112–8118 | 8113
©

View Article Online
Fig.
3
Inhibition of ACh-induced nicotinic current by synthetic
spiroimines in oocytes expressing the human neuronal a4b2 nAChR
(white colums), or having incorporated to their membrane the muscle-type
Torpedo a12bgd nAChR (black columns). Oocytes were voltage-clamped
at -60 mV and first perfused with 150 mM ACh for 15 s, followed by 150 s
washout of ACh with the Ringer solution in order to obtain the control
current values. Thereafter, the same oocyte was perfused with 2 mM of one
of the synthetic spiroimines for 60 s followed by 15 s perfusion with 150 mM
ACh + 2 mM of one of the synthetic spiroimines. Each column represents
mean values SEM; with n = 5–8 oocytes (from at least 3 different donors
per experimental condition). The ACh peak currents in the presence of the
spiroimine compounds were normalized to control currents recorded from
the same oocyte, to yield fractional (%) response data.
The biological activity of racemic ( )-11 and the enantiomer
(+)-11 (e.r. = 91/9) compounds, lacking the TBS group, were
examined on the human a4b2 nAChR in the same experimental
conditions as described above (2 mM ( )-11 and 2 mM (+)-11 in
the presence of 150 mM ACh). Both spiroimines showed a weak
antagonistic activity towards the a4b2 nAChR and did not affect
current kinetics, as shown in the representative recordings (Fig.
2B and D). Furthermore, as shown in Fig. 2 E and F, the iminium
salts (+)-15 and (-)-10 exhibited similar activities regarding both
current kinetics and percent blockade of the IACh peak amplitude
(84 4.76 and 16.34 8.73, respectively, n = 4) as (+)-9 and (+)-11
(Fig. 2, C, D).
In addition, spiroimines (+)-9, ( )-11, (+)-11, (+)-15 and
(-)-10 were tested on oocytes having the Torpedo muscle-type
a12bgd nAChR incorporated.7 As shown in Fig. 3, (+)-9 and
(+)-15 exhibited the highest blocking potency, as compared to
equimolecular concentrations of ( )-11, (+)-11 and (-)-10, not
only towards the muscle-type a12bgd nAChR, but also towards
the a4b2 nAChR. It should be noted that blockade of nicotinic
currents induced by ACh on both Torpedo a12bgd and human
a4b2 nAChRs by (+)-9 and (+)-15 was obtained with 40–45
times lower (-)-gymnodimine A concentrations,7 indicating that
the spiroimine analogs are less active than the native toxin.
Fig. 2 Examples of the effects of racemic (A,B), optically active (C,D) and
hydrochloride salts (E,F) of synthetic spiroimines on human a4b2 nAChRs
expressed in Xenopus oocytes. (A,B) ACh-evoked current recorded at a
holding potential of -60 mV, before (black trace, a), and during the action
of 2 mM ( )-9 with 150 mM ACh (red trace b, in A), or 2 mM ( )-11 with 150
mM ACh (red trace b in B), followed by 150 s washout of the spiroimine
compounds from the medium (blue traces c in A and B). The lines above
the current traces indicate the time ACh was perfused, before during or
150 s after the spiroimine compounds. (C,D) show ACh-evoked current
recorded before (black trace, a), during the action of 2 mM (+)-9 with 150
mM ACh (C) and 2 m M (+)-11 with 150 mM ACh (D) (red trace b in C and
D), and after 150 s washout of the spiroimine molecules from the medium
(blue trace c in C and D). (E,F) Data obtained with the hydrochloride salts
(+)-15 and (-)-10 (2 mM) under the same experimental conditions as in
A–D.
by ( )-9 was not immediately reversible after 150 s wash out of the
compound from the medium (Fig. 2A, c, blue superimposed trace).
Interestingly, upon 150 s wash out of ( )-9, the kinetics of the IACh
were similar to the control despite the incomplete recovery of the
peak amplitude. The optically active spiroimine (+)-9 (e.r. = 91/9)
showed a similar, but stronger, blocking potency on the human
a4b2 nAChR than did the racemic ( )-9 (Fig. 2C). Thus, (+)-9,
when tested at a concentration of 2 mM in the presence of 150 mM
ACh, blocked 89 4.76% (n = 6) of the IACh peak amplitude (Fig.
2C, b, and Fig. 3) compared to the 31.2 3.02% blockade by ( )-9
at the same concentration (n = 6) (Fig. 2A, b). Furthermore, the
current kinetics was 3.4 0.15 times faster with the enantiomeric
(+)-9 (1.87 0.3 s) than with the racemic compound ( )-9 (6.37
0.39 s), as evaluated by determining the half decay time of the
currents.
Discussions
The present work shows for the first time that synthetic spiroimines
possess an antagonist activity towards both neuronal- (a4b2)
and muscle-type (a12bgd) nAChRs. Such activity not only differs
in potency between ( )-9 and ( )-11 in both a4b2 and a12bgd
nAChRs as compared to (+)-9 and (+)-11 (the racemic compounds
8114 | Org. Biomol. Chem., 2011, 9, 8112–8118
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
being in general less potent), but also compounds 9 affected the
kinetics of the ACh-induced current while compounds 11 did
not. A possible explanation for the blocking activity revealed by
compounds 9 may arise from the presence of the TBS groups used
to protect the allylic alcohol 5. Since both compounds 9 and 11
were unable, by themselves, to exert an agonist action on nAChRs
and to induce nicotinic currents through the nAChR, it is likely
that the changes in nicotinic current kinetics induced by 9 may
reflecta nAChRchannelblock triggered once the nAChRchannels
are being activated by ACh. Alternatively, changes in current
kinetics may reflect changes in the desensitization rate of nAChRs.
Further work will be needed to clarify these two possibilities. The
imines (+)-9 and (+)-11 and their corresponding iminium salts
(+)-15 and (-)-10 showed similar activities. Considering the pKa
of imines (+)-9 and (+)-11 (pKa = 8.8 0.4),18 they might be
protonated in the physiological medium buffered at pH = 7.4. This
could explain why both spiroimines (+)-9, (+)-11 and iminiums
(+)-15, (-)-10 have a similar activity on nAChRs. Whatever
the molecular mechanism involved in the action of compounds
9 and 11, our results show that the spiroimine constituent of
gymnodimines A, B and C is one of the essential groups for
interacting with the nAChRs. In contrast, the tetrahydrofuran part
of (-)-gymnodimine A did not inhibit the binding of biotinylated
a-bungarotoxin to muscle-type (a12bgd) nAChRs, as recently
reported using a non-radioactive ligand binding assay.19 These
results support the hypothesis that the 6,6-spiroimine core is the,
or one of, the main pharmacophore(s) for the gymnodimine family
of phycotoxins.
MHz, CDCl3) d (ppm): 9.7 (t, 1H, J = 1.5 Hz), 6,88–6.79 (m, 1H),
5.97 (dt, 1H, J = 10.0, 2.0 Hz), 4.11 (q, 2H, J = 7.1 Hz), 2.70–2.20
(m, 5H), 2.20–1.94 (m, 2H), 1.93–1.80 (m, 1H), 1.17 (t, 3H, J =
7.1 Hz). 13C NMR (75 MHz, CDCl3) d (ppm): 201.2 (Cq), 196.1
(Cq), 171.4 (Cq), 149.2 (CH), 129.2 (CH), 61.5 (CH2), 55.9 (Cq),
39.5 (CH2), 31.0 (CH2), 25.8 (CH2), 23.6 (CH2), 14.1 (CH3). IR
n (neat): 2929, 2359, 2339, 1723, 1681, 1446, 1388, 1240, 1192,
1094, 1021 (cm-1). MS (ESI) m/z: 247.1 [M + Na]+, 279.1 [M +
Na + MeOH]+. HRMS calcd for C12H16O4Na+: 247.0946, found:
247.0941.
( )-Ethyl 2-hydroxy-1-(3-hydroxypropyl)cyclohex-3-enecarbo-
xylate 4. To a solution of ketoaldehyde 3 (70 mg, 0.312 mmol,
1 eq.) in MeOH (3.1 mL) was added NaBH4 (5.9 mg, 0.156 mmol,
0.5 eq.) at -78 ◦C. The conversion was monitored by TLC. After
completion of the reaction, CeCl3·7H2O (128 mg, 0.343 mmol,
1.1 eq.) was added at -78 ◦C. The suspension was warmed to -50
◦C until all CeCl3·7H2O was dissolved. The solution was cooled
back to -78 ◦C, then NaBH4 (13 mg, 0.343 mmol, 1 eq.) was
◦
added. The solution was stirred for 1 h at -78 C then saturated
NH4Cl was added dropwise at this temperature. The mixture was
warmed to RT and then water was added. The aqueous layer
was extracted twice with EtOAc. The combined organic layers
were washed with water then brine, and dried over Na2SO4. The
solvent was removed in vacuo, then the crude mixture purified
through silica gel (heptane to heptane/EtOAc 5/5) to afford the
1
major isomer as a colorless oil (m = 52.9 mg, 74%). H NMR:
(500 MHz, CDCl3) d 5.75–5.65 (m, 2H), 4.35 (m, 1H), 4.06 (q, J =
7.1 Hz, 2H), 3.59–3.41 (m, 4H), 2.02–1.92 (brm, 2H), 1.83–1.65
(m, 3H), 1.53–1.43 (m, 2H), 1.37 (m, 1H), 1.17 (t, J = 7.0 Hz, 3H).
13C NMR: (75 MHz, CDCl3) d 176.0 (Cq), 129.6 (CH), 128.1
(CH), 67.6 (CH), 63.0 (CH2), 60.6 (CH2), 48.9 (Cq), 28.3 (CH2),
27.3 (CH2), 25.8 (CH2), 22.8 (CH2), 14.2 (CH3). IR n (neat):
3343, 2933, 1719, 1018 cm-1. MS: (ESI) m/z: 251 (100) [M + Na].
HRMS: calcd for C12H20O4Na: 251.1259. found: 251.1265.
Conclusions
In conclusion, we have developed new synthetic routes to the
6,6-spiroimine skeleton of (-)-gymnodimine A 1. The quaternary
carbon of racemic spiroimines has been created by means of
a Michael addition of a cyclic b-ketoester to acrolein. In turn,
optically active spiroimines have been synthesized by using for the
first time an asymmetric allylic alkylation. Biological evaluations
revealed that racemic and optically active spiroimines 9 and 11,
and their corresponding hydrochloride salts 10 and 15, blocked
both neuronal- (a4b2) and muscle-type (a12bgd) nAChRs. These
results indicate that the 6,6-spiroimine moiety is important for the
blockade of nAChRs. A detailed structure–activity relationship
study on the 6,6-spiroimine skeleton is currently under investiga-
tion, and will be reported in due time.
(+)-(6R,7S)-7-Hydroxy-2-oxaspiro[5.5]undec-8-en-1-one
5.
The diol 4 (52.9 mg, 0.232 mmol, 1 eq.) was dissolved in
acetonitrile (3 mL) and DBU (41 mL, 0.278 mmol, 1.2 eq.) was
added. The solution was stirred at room temperature for 7 h and
saturated NH4Cl was added. The aqueous layer was extracted
with EtOAc (¥3). The combined organic layers were washed
with brine, dried over Na2SO4 and the solvent removed in vacuo.
The crude mixture was purified through silica gel (heptane to
heptane/EtOAc: 6/4) to afford a white solid (m = 31.6 mg, 75%).
1H NMR (500 MHz, CDCl3) d (ppm): 5.69 (dd, 1H, J = 9.8,
2.2 Hz), 5.55 (dd, 1H, J = 9.8, 1.6 Hz), 4.92 (s, 1H), 4.36 (m,
2H), 2.15–2.04 (m, 4H), 1.96–1.86 (m, 2H), 1.71–1.66 (m, 2H).
13C NMR (75 MHz, CDCl3) d (ppm): 176.8 (Cq), 129.6 (CH),
127.0 (CH), 71.2 (CH), 70.2 (CH2), 46.8 (Cq), 30.0 (CH2), 21.4
(¥2) (CH2), 21.1 (CH2). IR n (neat): 3419, 1693, 1260, 1164, 1061
(cm-1). MS (ESI, m/z): 205.1 [M + Na]+. HMRS (ESI, m/z):
Calcd for C10H14O3Na+: 205.0841, found: 205.0834. m.p. = 97 ◦C.
Experimental
Procedures and characterizations of new compounds
( )-Ethyl 2-oxo-1-(3-oxopropyl)cyclohex-3-enecarboxylate 3.
To a solution of b-ketoester 2 (200 mg, 1.19 mmol, 1eq.) and Et3N
(0.017 mL, 0.120 mmol, 0.1 eq.) in dry DMF (2.4 mL) was added
acrolein (0.120 mL, 1.78 mmol, 1.5 eq.). The mixture was stirred
overnight at room temperature and then water was added. The
aqueous layer was extracted twice with EtOAc and the combined
organic layers washed with water, then brine. The organic layer was
dried over Na2SO4 and the solvent removed in vacuo. The crude
mixture was purified over silica gel (heptane to heptane/EtOAc
(+)-(6R,7S)-7-((tert-Butyldimethylsilyl)oxy)-2-oxaspiro[5.5]-
undec-8-en-1-one 6. To a solution of allylic alcohol 5 (29.1 mg,
0.160 mmol, 1 eq.) in dry dichloromethane (0.53 mL) was added
2,6-lutidine (27.9 mL, 0.240 mmol, 1.5 eq.). The reaction mixture
◦
was cooled to 0 C and TBSOTf (40.3 mL, 0.176 mmol, 1.1 eq.)
1
8/2) to afford a yellow oil (m = 160.7 mg, 61%). H NMR (300
was added dropwise. The solution was stirred for 1 h at room
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8112–8118 | 8115
©

View Article Online
temperature and then quenched with a saturated solution of
NH4Cl. The aqueous layer was extracted with dichloromethane
(¥3). The combined organic extracts were washed with water,
brine and dried over MgSO4. The yellow residue was purified
by flash chromatography (heptane to heptane/EtOAc 8/2) to give
the protected alcohol as a yellow oil (m = 41.2 mg, 87%). 1H NMR
(500 MHz, CDCl3) d (ppm): 5.63 (d, 1H, J = 10.5 Hz), 5.44 (d,
1H, J = 10.5 Hz), 4.83 (s, 1H), 4.32 (m, 2H), 2.17 (td, 1H, J =
11.8, 3.9 Hz) 2.04–1.83 (m, 5H), 1.61 (m, 2H), 0.88 (s, 9H), 0.08
(s, 3H), 0.06 (s, 3H). 13C NMR (75 MHz, CDCl3) d (ppm): 176.3
(Cq), 130.5 (CH), 126.2 (CH), 72.7 (CH), 70.2 (CH2), 47.1 (Cq),
30.4 (CH2), 25.8 (CH3), 22.0 (CH2), 21.4 (¥2) (CH2), 17.9 (Cq),
-4.6 (CH3), -5.1 (CH3). IR n (neat): 1705, 1255, 833, 774 (cm-1).
MS (ESI, m/z): 319.2 [M + Na]+. HMRS (ESI, m/z): Calcd for
C16H28O3SiNa+: 319.1705, found: 319.1700. [a]2D5 +114.3 (c 0.222,
CHCl3, e.r. 91/09).
(Cq), 129.2 (CH), 128.9 (CH), 66.6 (CH), 55.5 (Cq), 51.9 (CH2),
30.1 (CH2), 26.0 (CH3), 25.9 (CH2), 25.8 (CH3), 23.7 (CH2), 23.1
(CH2), 18.0 (Cq), -0.35 (CH3), -4.8 (CH3). IR n (neat): 2093,
1698; 1250, 1054, 834; 774 (cm-1). MS: (ESI, m/z): 360.2 [M +
Na]+. HMRS: (ESI, m/z) Calcd for C17H31N3O2SiNa+: 360.2083,
found: 360.2072. [a]2D5 +103.8 (c 0.34, CHCl3, e.r. 91/09).
(+)-(6S,7S)-7-((tert-Butyldimethylsilyl)oxy)-1-methyl-2-azas-
piro[5.5]undeca-1,8-diene 9. To a solution of azide 8 (38.7 mg,
0.115 mmol, 1 eq.) in a mixture of THF/H2O (v/v = 9/1) (1 mL)
was added supported triphenylphosphine (1.6 mmol g-1) (107.5
mg, 0.172 mmol, 1.5 eq). The mixture was stirred at RT overnight
then filtered. The polymer was washed with CH2Cl2 (¥3) and the
solvent removed in vacuo. The crude mixture was purified over
silica gel (DCM(1%NH3)/MeOH: 100/0 to 95/5) to afford a
1
colorless oil (m = 27 mg, 80%). H NMR (500 MHz, CDCl3)
d (ppm): 5.65 (dd, 1H, J = 10.5, 1.9 Hz), 5.48 (dq, 1H, J = 10.5,
1.9 Hz), 4.64 (s, 1H), 3.55 (m, 2H), 2.09 (s, 3H), 2.07–2.01 (m,
3H), 1.85 (m, 2H), 1.56 (m, 2H), 1.47 (m, 1H), 0.87 (s, 9H), 0.08
(s, 3H), 0.06 (s, 3H). 13C NMR (75 Mhz, CDCl3) d (ppm): 171.9
(Cq), 130.7 (CH), 126.8 (CH), 71.4 (CH), 49.8 (CH2), 43.3 (Cq),
29.7 (CH2), 25.7 (CH3), 22.5 (CH3), 22.2 (CH2), 21.6 (CH2), 19.9
(CH2), 18.0 (Cq), -3.9 (CH3), -4.8 (CH3). IR n (neat): 2927, 1649,
1253, 1093, 836 (cm-1). MS: (ESI, m/z): 294.2 [M + H]+. HMRS:
(ESI, m/z) Calcd for C17H32NOSi+: 294.2253, found: 294.2255.
[a]2D5 +81.0 (c 0.21, CHCl3, e.r. 91/09).
(+)-1-((1R,2S)-2-((tert-Butyldimethylsilyl)oxy)-1-(3-hydroxy-
propyl)cyclohex-3-en-1-yl)ethanone 7. To a solution of lactone 6
◦
(31.8 mg, 0.107 mmol, 1 eq.) in dry THF (0.36 mL) at 0 C was
added MeMgBr (229 mL, 0.321 mmol, 3 eq.) and the reaction
mixture was allowed to warm to room temperature. After 2 h,
a saturated solution of NH4Cl was added. The aqueous layer
was extracted with dichloromethane (¥3). The combined organic
extracts were washed with water and brine, and dried over MgSO4.
The yellow residue was purified by flash chromatography (heptane
to heptane/EtOAc 7/3) to give the methyl ketone 6 as a colorless
1
(+)-((6S,7S)-7-((tert-Butyldimethylsilyl)oxy)-1-methyl-2-azas-
piro[5.5]undeca-1,8-dien-2-ium chloride 15. To a solution of imine
9 (6 mg, 0.020 mmol) in DCM/MeOH (v/v = 9/1, 2 mL) was
added HCl 6 N (0.04 mL) and the mixture was stirred at room
temperature for 30 min. The solvent was evaporated in vacuo to
afford 15 as a colorless oil (m = 6.5 mg, quant.).
oil (m = 23.7 mg, 71%). H NMR (500 MHz, acetone) d (ppm):
5.77 (m, 1H), 5.66 (m, 1H), 4.45 (d, 1H, J = 4.1 Hz), 3.53 (t,
1H, J = 6.4 Hz), 3.47 (m, 2H), 2.12 (s, 3H), 2.02–1.95 (m, 2H),
1.90–1.78 (m, 3H), 1.60 (td, 1H, J = 12.3, 3.4 Hz), 1.47–1.40 (m,
1H), 1.28–1.20 (m, 1H) 0.90 (s, 9H), 0.11 (s, 6H). 13C NMR (75
MHz, acetone) d (ppm): 210.9 (Cq), 130.4 (CH), 129.6 (CH), 67.6
(CH), 62.9 (CH2), 56.2 (Cq), 28.3 (CH3), 26.7 (CH2), 26.3 (¥3)
(CH3), 26.3 (CH2), 26.1 (CH2), 23.9 (CH2), 18.7 (Cq), -3.3 (CH3),
-4.5 (CH3). IR n (neat): 3388, 1697, 1250, 1052, 832, 772 (cm-1).
MS (ESI, m/z): 335.2 [M + Na]+. HMRS (ESI, m/z) Calcd for
C17H32O3SiNa+: 335.2018, found: 335.2021. [a]2D5 +118.2 (c 0.29,
CHCl3, e.r. 91/09).
1H NMR (500 MHz, CD3OD) d (ppm): 5.80 (m, 1H), 5.59 (m,
1H), 4.90 (s, 1H), 3.66 (m, 2H), 2.51 (s, 3H), 2.23–2.13 (m, 4H),
2.08 (m, 1H), 1.88 (m, 1H), 1.79 (m, 1H), 1.73 (m, 1H), 0.90 (s, 9H),
0.17 (s, 3H), 0.11 (s, 3H). 13C NMR (75 MHz, CD3OD) d (ppm):
197.9 (Cq), 129.8 (CH), 128.8 (CH), 73.1 (CH), 47.4 (Cq), 46.8
(CH2), 30.7 (Cq), 30.0 (CH2), 26.2 (CH3), 22.0 (CH2), 21.9 (CH2),
18.6 (CH2), -3.8 (CH3), -4.7 (CH3). IR n (neat): 3376, 3032–2856,
1681, 1090, 836 (cm-1). MS: (ESI, m/z): 294.2 [M]+. HMRS: (ESI,
m/z) Calcd for C17H32NOSi+: 294.2253, found: 294.2253. [a]2D5 +50
(c 0.26, MeOH, e.r. 91/09).
(+)-1-((1R,2S)-1-(3-Azidopropyl)-2-((tert-butyldimethylsilyl)-
oxy)cyclohex-3-en-1-yl)ethanone 8. To a solution of alcohol 7
(63.3 mg, 0.202 mmol, 1 eq.) in dry dichloromethane (2 mL) was
added Et3N (113 mL, 0.808 mmol, 4 eq.), t◦hen methanesulfonyl
chloride (62.5 mL, 0.808 mmol, 4 eq.) at 0 C. The mixture was
stirred for 3 h and then a saturated solution of NH4Cl was added.
The aqueous layer was extracted with dichloromethane (¥3). The
combined organic extracts were washed with water, brine and
dried over MgSO4. The crude mixture was dissolved in DMSO
(2 mL) and NaN3 (39.4 mg, 0.606 mmol, 3 eq.) was added at RT.
The mixture was stirred overnight at RT then brine was added.
The aqueous layer was extracted with dichloromethane (¥3). The
combined organic extracts were washed with water, brine and dried
over Na2SO4. The residue was purified by flash chromatography
(heptane to heptane/EtOAc 9/1) to give the desired alkyl azide as
a colorless oil (m = 38.7 mg, 57%). 1H NMR (500 MHz, CDCl3) d
(ppm): 5.78–5.70 (m, 2H), 4.43 (d, 1H, J = 3.8 Hz), 3.32 (m, 1H),
3.20 (m, 1H), 2.14 (s, 3H), 2.08–1.94 (m, 2H), 1.92–1.87 (m, 1H),
1.87–1.84 (m, 2H), 1.59–1.54 (m, 2H), 1.38–1.30 (m, 1H), 0.88 (s,
9H), 0.07 (s, 6H). 13C NMR (75 MHz, CDCl3) d (ppm): 211.5
(+)-(6S,7S)-1-Methyl-2-azaspiro[5.5]undeca-1,8-dien-7-ol
11. To a solution of imine 9 (30.3 mg, 0.103 mmol, 1 eq.) in
MeOH (1 mL) was added concentrated HCl (0.1 mL) and the
mixture was stirred at 50 ◦C for 3 h. The crude mixture was purified
over silica gel (DCM(1%NH3)/MeOH: 100/0 to 90/10) to afford
11 as a white solid (m = 17 mg, 92%). 1H NMR (500 MHz, CDCl3)
d (ppm): imine 5.68 (m, 1H), 5.54 (dd, 1H, J = 10.1, 1.8 Hz),
4.66 (s, 1H), 3.53 (m, 2H), 2.94 (brs, 1H), 2.05 (m, 2H), 2.01
(brs, 3H), 1.90–1.78 (m, 2H), 1.72 (m, 1H), 1.62–1.54 (m, 2H),
1.51 (m, 1H). Enamine 5.85 (m, 1H), 5.75 (m, 1H), 4.05 (d, 1H,
J = 4.5 Hz), 3.68 (m, 1H), 2.28 (m, 1H), other signal are masked
by the imine form. 13C NMR (75 Mhz, CDCl3) d (ppm): 172.4
(Cq), 129.7 (CH), 127.7 (CH), 70.6 (CH), 49.6 (CH2), 43.1 (Cq),
29.3 (CH2), 22.3 (CH3), 21.6 (CH2), 21.6 (CH2), 19.7 (CH2). IR n
(neat): 3112, 3022–2742, 1645, 1068 (cm-1). MS: (ESI, m/z): 180.1
[M + H]+. HMRS: (ESI, m/z) Calcd for C11H18NO: 180.1388,
8116 | Org. Biomol. Chem., 2011, 9, 8112–8118
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
found: 180.1380. mp = 110–112 ◦C. [a]D25 +5.0 (c 0.42, CHCl3, e.r.
91/09).
mL min-1: e.r. 91/09. [a]D25 +106.4 (c 0.232, CHCl3). Reported for
S enantiomer e.r. 93/07: [a]2D5 -120.7 (c 1.0, CHCl3).
(-)-(6S,7S)-7-Hydroxy-1-methyl-2-azaspiro[5.5]undeca-1,8-dien-
2-ium chloride 10. To a solution of imine 11 (5 mg, 0.028 mmol)
in MeOH (0.17 mL) was added concentrated HCl (0.02 mL), and
the mixture was stirred at room temperature for 3 h. The solvent
was evaporated in vacuo to afford 10 as a colorless oil (m = 6 mg,
(R)-Ethyl 2-oxo-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)allyl)cyclohex-3-enecarboxylate 14. To a stirred and degassed
solution of allyl ketone 12 (300.0 mg, 1.44 mmol, 1.0 eq.) and vinyl
boronic acid pinacol ester (1.2 mL, 7.2 mmol, 5.0 equiv.) in toluene
(14.5 mL) was added Hoveyda–Grubbs’ catalyst II 13 (90.2 mg,
0.14 mmol, 10 mol%). The mixture was heated at 80 ◦C for 20 h
then cooled to ambient temperature. Solvent was removed in vacuo
and the residue was purified by flash column chromatography
(heptane to heptane/EtOAc 90/10). The product 14 was obtained
as a slightly pink oil (418.4 mg, 90%) as a mixture of (E)- and (Z)-
1
quant.). H NMR (500 MHz, CD3OD) d (ppm): 5.78 (m, 1H),
5.59 (m, 1H), 4.74 (s, 1H), 3.67 (m, 2H), 2.51 (s, 3H), 2.19–2.13
(m, 2H), 2.12–1.98 (m, 3H), 1.91 (m, 1H), 1.83–1.71 (m, 2H). 13
C
NMR (75 MHz, CD3OD) d (ppm): 196.0 (Cq), 130.2 (CH), 128.3
(CH), 70.7 (CH), 46.9 (CH2), 46.7 (Cq), 30.2 (CH2), 22.0 (CH2),
21.1 (CH3), 21.0 (CH2), 18.4 (CH2). IR n (neat): 3331, 2996–2652,
1679, 1066 (cm-1). MS: (ESI, m/z): 180.1 [M]+. HMRS: (ESI,
m/z) Calcd for C11H18NO: 180.1388, found: 180.1383. [a]2D5 -8.3
(c 0.12, MeOH, e.r. 91/09).
1
boronate (80/20). H NMR (300 MHz, CDCl3) d (ppm): major
6.92 (td, 1H, J = 10.0, 3.7 Hz), 6.53 (td, 1H, J = 17.8, 7.1 Hz), 6.06
(dt, 1H, J = 10.1, 2.0 Hz), 5.54 (d, J = 17.7 Hz), 4.19 (q, 2H, J =
7.1 Hz), 2.78 (dd, 1H, J = 13.9, 7.1 Hz), 2.67 (dd, 1H, J = 13.9, 7.1
Hz), 2.58–2.48 (m, 1H), 2.45 (dt, 1H, J = 13.6, 4.3 Hz), 2.39–2.30
(m, 1H), 1.97 (ddd, 1H, J = 13.7, 9.0, 4.7 Hz), 1.32–1.22 (m, 15H).
13C NMR (75 MHz, CDCl3) d (ppm): 195.4 (Cq), 171.0 (Cq),
149.7 (CH), 148.1 (CH), 129.0 (CH), 83.4 (Cq), 83.2 (Cq), 61.4
(CH2), 56.6 (Cq), 40.2 (CH2), 29.8 (CH2), 24.8 (2 ¥ CH3), 24.7 (2 ¥
CH3), 23.6 (CH2), 14.1 (CH3). IR n (neat): 2978, 1729, 1686, 1637,
1444, 1359, 1323, 1143, 970, 848 (cm-1). MS (ESI+) m/z: 357.2
[M + Na]+. HRMS calcd for C18H27O5Na11B: 357.1849, found:
357.1852.
(+)-(R)-Ethyl 1-allyl-2-oxocyclohex-3-enecarboxylate 12. To a
degassed solution of allylpalladium chloride dimer (3.26 mg,
0.0089 mmol, 0.5 mol%) and (S,S)-DACH-phenyl Trost ligand
(14.8 mg, 0.0214 mmol, 1.2 mol%) in toluene (3.7 mL) was
added degassed allyl acetate (289 mL, 2.675 mmol, 1.5 eq.). The
initial clear orange solution faded and became cloudy. Degassed
1,1,3,3-tetramethylguanidine (269 mL, 2.14 mmol, 1.1 eq.) was
added and the mixture returned to a clear orange solution. A
solution of ethyl-2-oxocyclohex-3-ene-1-carboxylate 2 (293.3 mg,
1.74 mmol, 1 eq.) in toluene (0.7 mL) was added slowly and the
reaction stirred overnight at -20 ◦C under argon atmosphere. The
reaction mixture was quenched with saturated aqueous NH4Cl
and the layers were separated. The aqueous layer was extracted
with Et2O. The combined organic extracts were washed with brine
and water, dried over MgSO4, filtered and concentrated in vacuo.
The resulting oil was purified by flash chromatography (heptane
to heptane/EtOAc 90/10) to give the allyl ketone 12 as a colorless
(+)-Ethyl 2-hydroxy-1-(3-hydroxypropyl)cyclohex-3-enecarbo-
xylate 4. To a stirred solution of boronate ester 14 (728.6 mg,
2.18 mmol, 1.0 equiv.) in THF/H2O (v/v: 1/1) (44 mL) was added
NaBO3 (368.9 mg, 2.40 mmol, 1.1 equiv.) and the resulting mixture
was stirred at RT for 3 h 30 min. The reaction mixture was diluted
with methyl tert-butyl ether (MTBE) and water was added. The
aqueous layer was extracted with MTBE (¥3) and the combined
organic layers washed with brine then dried over Na2SO4. The
solvent was removed in vacuo. The crude mixture was dissolved
in MeOH (22 mL) and NaBH4 (41.2 mg, 1.09 mmol, 0.5 eq.)
at -78 ◦C was added. The conversion was monitored by TLC.
After completion of the reaction, CeCl3·7H2O (893 mg, 2.40 mmol,
1.1 eq.) was added at -78 ◦C. The suspension was warmed to -50
◦C until all CeCl3·7H2O was dissolved. The solution was cooled
back to -78 ◦C, then NaBH4 (82.4 mg, 2.18 mmol, 1 eq.) was
added. The solution was stirred for 1 h at -78 ◦C, then saturated
NH4Cl was added dropwise at this temperature. The mixture was
warmed to RT, then water was added. The aqueous layer was
extracted twice with EtOAc. The combined organic layers were
washed with water then brine, and dried over Na2SO4. The solvent
was removed in vacuo, then the crude mixture purified through
silica gel to afford the major isomer as a colorless oil (heptane to
heptane/EtOAc 5/5) (m = 253.8 mg, 51%). [a]2D5 +130.9 (c 0.202,
CHCl3, e.r. 91/09).
1
oil (317.6 mg, e.r. 91/09. 82%). H NMR (300 MHz, CDCl3) d
(ppm): 6.91 (dt, 1H, J = 10.1, 4.2 Hz), 6.05 (dt, 1H, J = 10.0, 2.0
Hz), 5.85–5.70 (m, 1H), 5.13 (m, 1H), 5.08 (m, 1H), 4.17 (q, 2H,
J = 7.2 Hz), 2.67 (dt, 1H, J = 13.9, 7.3 Hz), 2.54 (dt, 1H, J = 13.9,
7.3 Hz), 2.55–2.22 (m, 3H), 2.01–1.91 (m, 1H), 1.24 (t, 3H, J =
7.2 Hz). 13C NMR (75 MHz, CDCl3) d (ppm): 195.7 (Cq), 171.1
(Cq), 149.6 (CH), 133.3 (CH), 129.0 (CH), 118.8 (CH2), 61.3 (Cq),
56.6 (CH2), 38.3 (CH2), 29.9 (CH2), 23.6 (CH2), 14.1 (CH3). IR n
(neat): 1739, 1726, 1678, 1622, 1190, 919, 670 (cm-1). MS (ESI+)
m/z: 231.1 [M + Na]+. HRMS calcd for C12H16O3Na: 231.0997,
found: 231.0994. HPLC: Heptane/iPrOH (90/10), column IC 5
mm (4.6 ¥ 250mm), injection volume 10 mL, flow: 0.8 mL min-1:
e.r. 91/09; [a]2D5 +54.3 (c 0.2, CHCl3, e.r. 91/09).
Absolute configuration determination: To a solution of (R)-ethyl
1-allyl-2-oxocyclohex-3-enecarboxylate (100.0 mg, 0.48 mmol, 1.0
eq.; e.r. 91/9) in THF (960 mL) at -78 ◦C, was added L-selectride
(480 mL, 0.48 mmol, 1.0 eq.). After 1 h at this temperature,
the mixture was quenched with water and extracted with ethyl
acetate. The combined organic extracts were washed with brine
and water, dried over MgSO4, filtered and concentrated in vacuo.
The resulting oil was purified by flash chromatography (heptane
to heptane/EtOAc 90/10) to give the allyl ketone as a colorless
oil (72.0 mg, 71%). All spectroscopic data are in agreement with
those reported by Trost et al.13e HPLC: heptane/iPrOH (95/5),
column IC 5 mm (4,6 ¥ 250mm), injection volume 10 mL, flow: 0.8
Experimental of biological evaluation
Animals and biological materials. Adult female Xenopus laevis
frogs were obtained from the Centre de Ressources Biologiques
Xe´nopes – CNRS (Universite´ de Rennes 1, France). Live Torpedo
marmorata fishes were purchased from the Service Mode`les
Biologiques of the Station Biologique de Roscoff (France). Live
animals were maintained in the Animal Campus facility of Gif sur
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8112–8118 | 8117
©

View Article Online
Yvette. Experiments were performed in accordance with European
Community guidelines for laboratory animal handling and with
the official edict presented by the French Ministry of Agriculture
and the recommendations of the Helsinki Declaration. The cDNA
coding for human a4b2 nAChR was kindly provided by Pr. J.-P.
Changeux and Dr P. J. Corringer (Pasteur Institute, Paris, France)
Acknowledgements
We thank the CNRS and ICSN for financial support. L. Duroure
and T. Jousseaume were supported by fellowships from the
Institut de Chimie des Substances Naturelles (ICSN-CNRS).
This work was also supported in part by the Agence Nationale
de la Recherche (grant PCV07-194417-NEUROSPIROIMINE),
and by grants from EU 7th Frame Program STC-CP2008-1-
555612 (ATLANTOX) and 2009-1/117 PHARMATLANTIC.
We also thank Pr. Yannick Landais, Dr Vale´rie Desvergnes (ISM,
University of Bordeaux) and Dr Bogdan Iorga (ICSN, Gif-sur-
Yvette) for helpful discussions.
Torpedo membrane preparation and microtransplantation to
oocytes. Torpedo marmorata specimens kept in conventional ar-
tificial seawater were anaesthetized with tricaine (Sigma-Aldrich.)
at a concentration of 0.03% in seawater, before surgical excision
of electric organs. The a12bgd nAChR-rich membranes were
prepared at 4 ◦C from freshly dissected and sliced Torpedo electric
organs using procedures previously described,20 resuspended in
5 mM glycine, and stored aliquotted at -80 ◦C until use.
Microtransplantation of Torpedo nAChR used microinjection into
the oocyte cytoplasm of a membrane suspension (50 nL at 3.5 mg
mL-1 protein) from a Nanoliter2000 Micro4 Controller (World
Precision Instruments, Inc., UK) mounted on a microscope.
Notes and References
1 T. Seki, M. Satake, L. Mackenzie, H. F. Kaspar and T. Yasumoto,
Tetrahedron Lett., 1995, 36, 7093–7096.
2 A. J. Haywood, K. A. Steidinger, E. W. Truby, P. R. Bergquist, P. L.
Bergquist, J. Adamson and L. J. Mackenzie, J. Phycol., 2004, 40, 165–
179.
3 F. M. Van Dolah, Environ. Health Perspect., 2000, 108, 133–141.
4 C. O. Miles, A. L. Wilkins, D. J. Stirling and A. L. MacKenzie, J. Agric.
Food Chem., 2000, 48, 1373–1376.
5 R. Kharrat, D. Servent, E. Girard, G. Ouanounou, M. Amar, R.
Marrouchi, E. Benoit and J. Molgo´, J. Neurochem., 2008, 107, 952–
963.
6 R. Munday, N. R. Towers, L. Mackenzie, V. Beuzenberg, P. T. Holland
and C. O. Miles, Toxicon, 2004, 44, 173–178.
7 Y. Bourne, Z. Radic, R. Ara`oz, T. Talley, E. Benoit, D. Servent, P.
Expression of human a4b2 nAChR in Xenopus oocytes. Oocytes
were surgically removed from mature female Xenopus laevis frogs
under anesthesia using ethyl-3-amino benzoate methanesulfonate
salt (Sigma-Aldrich, Saint Quentin Fallavier, France) solution
(1.5 g l-1), were treated for 10 min with 1 mg ml-1 collagenase
type I (Sigma-Aldrich) in calcium-free medium containing (in
mM): NaCl 88, KCl 2.5, MgCl2 1, HEPES 5 (pH 7.6), and were
manually defolliculated. Following an extensive washing with this
solution, oocytes were transferred to Barth’s solution containing
(in mM): NaCl 88, KCl 1, MgSO4 0.33, CaCl2 0.41, MgSO4 0.82,
Ca(NO3)2 0.33, NaHCO3 2.4, HEPES 10 (pH 7.2) supplemented
with streptomycin sulphate and 0.01 mg ml-1 penicillin-G. Oocytes
were maintained at 19 ◦C for up to 4 days in this solution. Stage
V–VI oocytes were selected and microinjected with 50 nL human
a4b2 cDNA (1 mg ml-1), as reported previously.7 Recordings were
performed 3 to 4 days after cDNA injection.
Taylor, J. Molgo and P. Marchot, Proc. Natl. Acad. Sci. U. S. A., 2010,
´
107, 6076–6081.
8 M. Stewart, J. W. Blunt, M. H. Munro, W. T. Robinson and D. J.
Hannah, Tetrahedron Lett., 1997, 38, 4889–4890.
9 T. Hu, J. M. Curtis, J. A. Walter and J. L. C. Wright, Tetrahedron Lett.,
1996, 37, 7671–7674.
10 For the synthesis of b-ketoester 2 see: T. B. Poulsen, L. Bernardi, J.
Aleman, J. Overgaard and K. A. Jorgensen, J. Am. Chem. Soc., 2006,
129, 441–449.
11 X-Ray data for ( )-5 has been deposited with the Cambridge Crystal-
lographic Data Centre, deposition number 828699†.
12 Y. Ahn, G. I. Cardenas, J. Yang and D. Romo, Org. Lett., 2001, 3,
751–754.
Voltage-clamp recording on oocytes. ACh-evoked currents
were recorded with a standard two-microelectrode voltage-clamp
amplifier (OC-725B, Warner Instrument Corp., Hamden, CT,
USA) at a holding potential of -60 mV. Voltage and current
micro-electrodes were pulled from borosilicate glass to reach 0.5–
1.5 MX tip resistance when filled with 3 M KCl. Data were
acquired with a pCLAMP-9/Digidata-1322A system (Molecular
Devices, Union City, CA, USA). The recordin◦g chamber (capacity
300 mL) was superfused (8–12 mL min-1; 20 C) with a modified
Ringer’s solution containing (in mM): NaCl 100, KCl 2.8, BaCl2
0.3, HEPES 5 (pH 7.4), where BaCl2 substitution to CaCl2 prevents
secondary activation of a Ca2+-dependent Cl- current.21 Oocytes
were initially incubated for 3 min with spiroimines, and then ACh
was applied for 15 s in oocytes expressing the human a4b2 as well
as for the Torpedo (a12bgd) nAChRs incorporated to the oocyte
membrane, using a computer-controlled solution exchange system
(VC-6; Warner Instruments). Between successive ACh applica-
tions, 4 min perfusion intervals with modified Ringer’s solution
were maintained to ensure receptor recovery from desensitization.
13 (a) B. M. Trost and D. L. Van Vranken, Chem. Rev., 1996, 96, 395–422;
(b) B. M. Trost, Acc. Chem. Res., 1996, 29, 355–364; (c) B. M. Trost
and M. L. Crawley, Chem. Rev., 2003, 103, 2921–2943; (d) Z. Lu and
S. Ma, Angew. Chem., Int. Ed., 2008, 47, 258–297; (e) B. M. Trost,
R. Radinov and E. M. Grenzer, J. Am. Chem. Soc., 1997, 119, 7879–
7880.
14 The absolute configuration was established on ethyl 1-allyl-2-
oxocyclohexanecarboxylate, obtained by 1,4-reduction of 12. See the
Experimental section.
15 S. B. Garber, J. S. Kingsbury, B. L. Gray and A. H. Hoveyda, J. Am.
Chem. Soc., 2000, 122, 8168–8179.
16 C. Morrill and R. H. Grubbs, J. Org. Chem., 2003, 68, 6031–6034.
17 G. W. Kabalka and H. C. Hedgecock Jr., J. Org. Chem., 1975, 40,
1776–1779.
18 The pKa value has been estimated using ACD/I-Lab 2.0. In the buffer
solution, pH = 7.4, about 96% of the imine should be protonated.
19 S. Toumieux, R. Beniazza, V. Desvergnes, R. Ara´oz, J. Molgo´ and
Yannick Landais, Org. Biomol. Chem., 2011, 9, 3726–3732.
20 F. Krieger, A. Mourot, R. Ara`oz, F. Kotzyba-Hibert, J. Molgo´,
E. Bamberg and M. Goeldner, ChemBioChem, 2008, 9, 1146–
1153.
21 S. B. Sands, A. C. S. Costa and J. W. Patrick, Biophys. J., 1993, 65,
2614–2621.
8118 | Org. Biomol. Chem., 2011, 9, 8112–8118
This journal is
The Royal Society of Chemistry 2011
©