The preparation of novel histrionicotoxin analogues and their activity towards the α4β2 and α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.tet.2017.11.047

Four histrionicotoxin analogues were prepared in an efficient manner utilizing a nitrone dipolar cycloaddition reaction as the key step in forming tricyclic intermediate 13. The nitrile in intermediate 13 was reduced with DIBAL to an aldehyde which then underwent Z-selective Wittig reactions to produce intermediates containing the Z-alkene side-chain. Hydrogenation of the Z-alkenes produced saturated histrionicotoxin analogues whereas reduction with SmI₂ afforded the unsaturated histrionicotoxin analogues. The histrionicotoxin analogues were shown to be potent non-competitive antagonists of the α4β2 and α7 nAChR's with the most potent analogue 3 displaying IC₅₀'s of 0.10 μM and 0.45 μM against the α4β2 and α7 nAChR's, respectively. The unsaturated analogues 15 and 18 displayed Hill slope (n_H) of approximately 1 whilst the saturated analogues 16 and 3 had a n_H of approximately 0.5, which may indicate that the saturated analogues are binding to more than one binding site.

Full text of DOI:10.1016/j.tet.2017.11.047

Accepted Manuscript
The preparation of novel histrionicotoxin analogues and their activity towards the
α4β2 and α7 nicotinic acetylcholine receptors
Cecily Eldridge, Gracia Quek, Michael Sako, John H. Ryan, Simon Saubern, Mary
Chebib, James M. Macdonald
PII:
S0040-4020(17)31195-X
10.1016/j.tet.2017.11.047
TET 29127
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 September 2017
Revised Date: 14 November 2017
Accepted Date: 16 November 2017
Please cite this article as: Eldridge C, Quek G, Sako M, Ryan JH, Saubern S, Chebib M, Macdonald JM,
The preparation of novel histrionicotoxin analogues and their activity towards the α4β2 and α7 nicotinic
acetylcholine receptors, Tetrahedron (2017), doi: 10.1016/j.tet.2017.11.047.
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The preparation of novel histrionicotoxin
analogues and their activity towards the
α4β2 and α7 nicotinic acetylcholine receptors
Leave this area blank for abstract info.
a
b
b
a,
a
b,
Cecily Eldridge , Gracia Quek , Michael Sako , John H. Ryan *, Simon Saubern , Mary Chebib *, and James
a,
M. Macdonald *
a
CSIRO, Manufacturing, Bag 10, Clayton South, Victoria 3169, Australia
b
Faculty of Pharmacy, The University of Sydney, New South Wales, 2006, Australia

1
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Tetrahedron
journal homepage: www.elsevier.com
The preparation of novel histrionicotoxin analogues and their activity towards the
α4β2 and α7 nicotinic acetylcholine receptors
a†
b†
b
a
a
b,*
Cecily Eldridge , Gracia Quek , Michael Sako , John H. Ryan ,* Simon Saubern , Mary Chebib and
a,
James M. Macdonald ∗
a
CSIRO, Manufacturing, Bag 10, Clayton South, Victoria 3169, Australia
Faculty of Pharmacy, The University of Sydney, New South Wales, 2006, Australia
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Four histrionicotoxin analogues were prepared in an efficient manner utilizing a nitrone dipolar
cycloaddition reaction as the key step in forming tricyclic intermediate 13. The nitrile in
intermediate 13 was reduced with DIBAL to an aldehyde which then underwent Z-selective
Wittig reactions to produce intermediates containing the Z-alkene side-chain. Hydrogenation of
Available online
the Z-alkenes produced saturated histrionicotoxin analogues whereas reduction with SmI
2
afforded the unsaturated histrionicotoxin analogues. The histrionicotoxin analogues were shown
to be potent non-competitive antagonists of the α4β2 and α7 nAChR’s with the most potent
analogue 3 displaying IC50’s of 0.10 µM and 0.45µM against the α4β2 and α7 nAChR’s,
Keywords:
Histrionicotoxin
Poison arrow frog toxin
nicotinic acetylcholine receptor
antagonist
respectively. The unsaturated analogues 15 and 18 displayed Hill slope (n
whilst the saturated analogues 16 and 3 had a n of approximately 0.5, which may indicate that
the saturated analogues are binding to more than one binding site.
H
) of approximately 1
H
1
,3-dipolar cycloaddition
2
009 Elsevier Ltd. All rights reserved.
—
——
†
Equal first authors. ∗ Corresponding authors. Tel.: +61-3-9545-2538; +61-3-9545-2541; +61-2-9351-8584 e-mail:
james.macdonald@csiro.au; jack.ryan@csiro.au; mary.collins@sydney.edu.au

2
Tetrahedron
1
. Introduction
lithium acetylide of alkyne 5 efficiently opened lactone 4 to
ACCEPTED MANUSCRIPT
furnish the alkynone 6 in excellent yield. Next, catalytic
hydrogenation gave the saturated derivative 7 (Scheme 1.
Relative stereochemistry is depicted throughout).
The nicotinic acetylcholine receptor (nAChR) is a member of
the ligand-gated ion channel family that mediate fast synaptic
transmission of cations between cells both in the central
1
Scheme 1. Preparation of intermediate 7.
(neuronal type) and peripheral (muscle) nervous systems. These
receptors are a pentameric protein assembly that can be formed
from various subunit proteins which mix and match to form
either at neuromuscular junction or in mammal brain. In this
The next step was to activate the alcohol of compound 7 as a
good leaving group. H NMR spectra obtained for compound 7
1
1
revealed the four protons adjacent to the carbonyl significantly
under-integrated to what would be expected for the product 7 and
consistent with compound 7 being in equilibrium with its hemi-
ketal isomer 7a in an approximate ratio of (7/7a) 4:1. Indeed, an
attempted mesylation (0 °C) of this isomeric mixture gave the
desired, somewhat unstable mesylate in 74% yield with another
unstable product being isolated but not characterized (presumably
the mesylate product of isomer 7a). We reasoned that the reaction
rate of mesylation of a secondary alcohol relative to the hydroxyl
of a hemi-ketal should be faster. Gratifyingly, repeating this
mesylation procedure at −30 °C resulted in just a single product;
study, we focused on the neuronal α4β2 and α7 nAChR’s as
these subtypes are most widely expressed and are implicated in
processes as diverse as cognition, consciousness, mood, nicotine
2
addiction, and nociception among others.
3
Since their discovery, the histrionicotoxins such as 285A 1
isolated from the poison arrow frog Dendrobates histrionicus as
well as the unnatural perhydrohistrionicotoxin 2 have inspired a
4
wealth of synthetic approaches and total syntheses. In addition
to these challenging synthetic targets possessing an intriguing
molecular architecture, the histrionicotoxins are also potent
5
inhibitors of nAChR. Over the years, there have been some
biological and toxicity studies of histrionicotoxins but these
3
were limited to animal studies, although some pharmacological
6
studies were performed on nAChRs with frog nerve preparations
7
and on Torpedo electroplax membranes. To date, there have not
been any functional studies of histrionicotoxins on any
recombinant nAChRs. This paper seeks to prepare novel
unnatural histrionicotoxin variants such as compound 3 and test
their activity toward the neuronal α4β2 and α7 nAChR’s.
NH OH.HCl
2
NaHCO3
EtOH
OH
O
OEt
OEt
N
OEt
OEt
70 C
OMs
OMs
8
9
OEt
OEt
styrene
EtO
OEt
70
C
5
3%
N
O
N
(
2 steps)
Figure 1. Structures of histrionicotoxins.
O
Ph
i) aq HCl
10
11
2
. Results and discussion
THF
ii) nBuLi
toluene/
One synthetic strategy to generate histrionicotoxins involved a
(tBuO)Ph2SiCH2CN
8
EtOH (6:1)
nitrone dipolar cycloaddition reaction as the key step and was
9
THF
MW
CN
also utilized to successfully synthesize histrionicotoxins 1 and
8 o
180 o
C
7
C
9
,10
2
.
The efficiency and potential for late-stage divergency of
N
78%
these chemistries formed the inspiration to prepare novel
analogues such as 3.
O
i
Ph
i) Bu2AlH
12
toluene 78 o
C
Racemic lactone 4 was chosen as the starting material, being
readily available and already containing the 7-carbon alkyl chain
that would ultimately be reflected in the final product. The
n
+
ii) [Ph3P Pr] Br
nBuLi THF
7
8 o
C
N
O
CN
N
O
CN
78%
47%
13
SmI2
THF
N
38%
N
H
O
HO
14
15
H2 aq HCl
Pd(OH)2/C
THF
84%
N
H
HO
16

3
the desired mesylate isomer 8 in excellent yield
A
(S
C
ch
C
em
E
e
P
2
T
). ED MA4)
NUSCRIPT
.
Scheme 2. Preparation of intermediate 8.
Scheme 4. Preparation of histrionicotoxin analogues 18 and 3.
Treatment of mesylate 8 with hydroxylamine accomplished
the formation of intermediate oxime 9 and then N-nucleophilic
displacement of the mesylate to form the cyclic nitrone 10. The
nitrone was isolated and immediately treated with styrene which
underwent a 1,3-dipolar cycloaddition reaction to effectively
protect the nitrone functionality as its styrene adduct;
isoxazolidine 11. The next step involved elaboration of the
diethyl acetal of 11 to a Z-α,β-unsaturated nitrile functionality.
This was achieved by acid catalyzed hydrolysis of the diethyl
acetal to liberate the aldehyde followed by olefination employing
The effects of 16, 15, 3 and 18 were evaluated on rat α4β2
nAChRs recombinantly expressed in Xenopus oocytes using 2-
electrode voltage clamp electrophysiology. Neither 16, 15, 3 or
18 activated the α4β2 nAChRs on their own indicating that they
are neither agonists nor partial agonists (data not shown). We
subsequently evaluated the compounds for antagonist effect. In
order to evaluate antagonist effects, the compounds were pre-
incubated for 3 mins before being co-applied with ACh in order
to give them time to access their binding site. In the presence of
100 µM ACh, the ACh induced currents were reduced or
blocked, indicating that 16, 15, 3 and 18 were antagonists.
Inhibitory concentration response curves were constructed with
increasing concentrations of 16, 15, 3 and 18 in the presence of
100 µM ACh and the resulting currents normalised to 100 µM
11
the modified Peterson reagent as described by Kojima et. al. It
is noteworthy that a simple modification to the preparation of this
important reagent (using two equivalents of lithium diisopropyl
amide instead of one) resulted in a superior, and more
predictable, yield of tert-butoxydiphenylsilylacetonitrile (80% vs
previously reported 62%; see experimental section). Careful
chromatography separated the desired Z-isomer from trace
amounts of the E-isomer. In this manner, Z-α,β-unsaturated
nitrile 12 was prepared in 78% yield from diethylacetal 11. As
ACh alone (Figure 1). 15 was more potent than 16 (IC50
=
9.38 µM; 95% CI: 3.30 to 26.75 µM) while 3 (IC50 = 0.10 µM;
95% CI: 0.03 to 0.30 µM) was more potent than 18. The order of
potency is as follows: 3 > 15 > 18 > 16. Table 1 summarises the
data. Interestingly the Hill slope (n ) differed for saturated vs
H
8-10
described previously, simply heating compound 12 resulted in
unsaturated histrionicotoxins. Unsaturated 15 and 18 displayed a
nH of approximately 1 while the saturated analogues 16 and 3 had
a n of approximately 0.5 (Table 2). A shallow n i.e a slope < 1
the following reaction cascade; a 1,3-dipolar cycloreversion
(extrusion of styrene) followed by an intramolecular 1,3-dipolar
H
H
cycloaddition reaction between the liberated nitrone and α,β-
unsaturated nitrile functionalities (under thermodynamic control)
to generate tricycle 13. The nitrile functionality of tricycle 13 was
reduced to the aldehyde and then Wittig olefination gave
exclusively the Z-alkene 14. The isoxazolidine moiety of 14
may indicate that histrionicotoxins are binding to more than one
site. Indeed different histrionicotoxin analogues have been
reported to bind to more than one site.
7
We then individually evaluated the effect of a fixed
concentration of the histrionicotoxins in the presence of varying
concentrations of ACh. Histrionicotoxins 16 (30 µM), 15 (1 µM),
12
could be selectively reduced under Brandi (SmI ) conditions to
2
give rise to unnatural histrionicotoxin derivative 15.
Alternatively, catalytic hydrogenation gave the fully reduced
variant; histrionicotoxin analogue 16 (Scheme 3).
3
(1 µM) and 18 (1 µM) all significantly reduced the maximal
current elicited by a saturating concentration of ACh such that
the maximal current was reduced from 116% (95% CI: 106 to
Scheme 3. Preparation of histrionicotoxin analogues 15 and 16.
1
26) in the absence of histrionicotoxin to approximately 45% in
the presence of histrionicotoxin (F-test on n ; p<0.05) at the
H
α4β2 nAChR (Example shown in Figure 2 with 3). The reduction
in the maximal current indicates that high concentrations of ACh
cannot compensate for the antagonist effect. In addition, the EC50
values of ACh in the presence of 16, 15, 3 and 18 ranged from
Additionally, two further unnatural histrionicotoxin analogues
1
8 and 3 were prepared from tricycle 13. Relative to the majority
3
of histrionicotoxins found in Nature, these latter analogues
contain “arms” elongated with two carbon atoms each (Scheme
6
2.15 µM to 207.9 µM (Table 2), but this change was not
statistically different to the EC50 values in the absence of
compounds (136.2 µM, 95% CI: 99.5 to 186.4 µM; F-test on

4
Tetrahedron
TEno
logEC , p>0.05). This pattern is indica
A
tiv
C
e
C
o
E
f
P
a
D
n- MAth
N
e
U
ort
S
ho
C
st
RI
PTbinding site at α4β2 nAChRs. In summary,
eri
c
50
competitive antagonist that exerts its effects on saturating
concentrations of an agonist, inferring that binding of the
histrionicotoxins is to an allosteric site, rather than to the ACh or
histrionicotoxins 16, 15, 3 and 18 are potent non-competitive
antagonists of recombinant α4β2 nAChRs.
Figure 1: Inhibitory concentration response curves of increasing concentrations of A 16, B 15, C 3 and D 18 in the presence of 100 µM ACh at rat α4β2
nAChRs recombinantly expressed in Xenopus oocytes. Histrionicotoxins were pre-incubated for 3 mins before co-addition with 100µM ACh. Data are
normalised to 100 µM ACh and presented as mean ± SEM (n= 3-8 oocytes; >2 batches of oocytes).
Table 1: Pharmacological data of the inhibitory effects of histrionicotoxins on rat α4β2 nAChRs recombinantly expressed in
Xenopus oocytes.
1
6
15
3
18
a
IC50 (µM)
9.39
0.78
0.10
2.72
9
5% CI
3.30 to 26.75
0.51
0.13 to 4.75
1.02
0.03 to 0.30
0.49
0.87 to 8.52
0.96
n
H
9
5% CI
0.78 to 0.23
3.02 to 0.98
0.79 to 0.19
1.98 to 0.06
a
IC50 is the effective concentration that inhibits 50% of the current response produced by 100 µM ACh. n
confidence interval (CI).
H
is the Hill slope. Errors are expressed as 95%
Table 2: Pharmacological data of increasing concentrations of ACh alone and in the presence histrionicotoxis on α4β2 nAChRs
recombinantly expressed in Xenopus oocytes.
ACh
16
15
3
18
a
EC50 (µM)
136.2
182.6
207.9
94.94
62.15
9
5% CI
99.5 to 186.4
116
37.7 to 884.5
46
55.9 to 773.5
52
17.7 to 509.0
41
17.6 to 219.0
51
IMax (%)
9
5% CI
106 to 126
36 to 55
40 to 63
31 to 52
40 to 61
a
EC50 is the effective concentration that activates 50% of the receptors. IMax is the maximum normalised current produced by ACh alone (1 mM). Errors are
expressed as 95% confidence interval (CI).
concentration dependent manner. Histrionicotoxin 3 had an IC50
of 0.45 µM (95% CI 0.23-0.90 µM; Figure 3A) while 18 had an
IC50 of 0.62 µM (95% CI 0.10-3.7 µM; Figure 3B). Given that
both 3 and 18 had similar potencies on both α4β2 and α7
nAChRs indicates the histrionicotoxins are not very selective and
that there is a common binding at both receptor subtypes. In
summary, histrionicotoxins are potent antagonists of nAChRs
and may act at sites distinct to the ACh binding site.
Figure 2. Concentration response curves of increasing concentrations of
ACh alone (ꢀ) and in the presence of 1 µM 3(ꢀ) on rat α4β2 nAChRs
recombinantly expressed in Xenopus oocytes. Oocytes were pre-incubated for
3
mins with 1 µM 3 before the the co-application of ACh. Data are
normalised to 1mM ACh and presented as mean ± SEM (n= 3-6 oocytes; >2
batches of oocytes).
We then evaluated the most potent histrionicotoxin series, 3
and 18, on homomeric rat α7 nAChRs. Both 3 and 18 inhibited
the current exhibited by an EC50 concentration of ACh in a

5
ACCEPTED MAvo
N
lta
U
ge
S
o
C
f
R
50
I
V
P
a
T
nd a capillary voltage of 3.0kV. The sample
was introduced by direct infusion at a rate of 5ꢀl/min using
1
3
PEG400 as an internal calibrant. Flash chromatography was
carried out using Merck Kieselgel 60 (230-400 mesh; particle
size 0.04-0.63 mm) silica gel. Analytical thin layer
chromatography (TLC) was conducted on Sigma-Aldrich silica
gel coated aluminium sheets and visualised with UV and/or by
dipping in a phosphomolybdic S2 acid/EtOH solution and
heating at 400 °C. IR analysis was carried out using a Perkin
Elmer 2000 FT-IR spectrophotometer, in absorbance mode, using
NaCl disks as the background reference. Microwave reactions
were carried out in sealed reaction vessels using a Biotage
Initiator 2.0 (400W). Melting points were recorded on an
Electrothermal IA9300 digital melting point apparatus, and are
uncorrected.
4
.2. 4,4-Diethoxybut-1-yne (5)
Alkyne 5 was prepared from propargyl bromide (17.8 g, 0.150
14
1
mol) according to the literature procedure.
H NMR (CDCl3,
4
2
4
00 MHz) δ 1.18 (t, J = 7.1 Hz, 6H), 1.98 (t, J = 2.6 Hz, 1H),
.48 − 2.50 (m, 2H), 3.48 − 3.56 (m, 2H), 3.64 − 3.69 (m, 2H),
13
.63 (t, J = 5.6 Hz, 1H); C NMR (CDCl , 100 MHz)
3
δ 15.07, 24.69, 61.75, 69.80, 79.58, 100.57.
.3. 1,1-diethoxy-9-hydroxyhexadec-3-yn-5-one (6)
,4-Diethoxybut-1-yne 5 (6.66 g, 44.1 mmol) in dry THF (120
4
4
n
mL) was cooled to −78 °C. BuLi (27.7 mL of a 1.59 M solution
in hexanes, 44.0 mmol) was added dropwise (2 min), which gave
a pale yellow solution, that was stirred (10 min). A solution of δ-
dodecalactone 4 (6.99 g, 35.2 mmol) in dry THF (30 mL) was
added dropwise (5 min) and the solution was stirred (1 h). The
reaction was quenched with saturated aqueous NH Cl and
allowed to warm to room temperature. This mixture was diluted
4
Figure 3. Inhibitory concentration response curves of increasing
concentrations of A 3 and B 18 in the presence of 300 µM ACh at rat α7
nAChRs recombinantly expressed in Xenopus oocytes. Histrionicotoxins
were preincubated for 3 mins before co-addition with 300µM ACh. Data are
normalised to 300 µM ACh and presented as mean ± SEM (n= 3-8 oocytes;
with EtOAc and H O and the organic layer was separated. The
2
aqueous phase was re-extracted (2 times) with EtOAc. The
combined organics were washed sequentially with saturated
aqueous NaHCO , saturated aqueous NaCl, dried (MgSO ),
3
4
>
2 batches of oocytes).
filtered and concentrated. The residue was purified by flash
chromatography (EtOAc/hexanes, gradient; 10:90, 15:85; 25:75
then 30:70) to yield alkyne 6 (10.8 g 90%) as a clear yellow oil:
3
. Conclusion
R 0.34 (30:70 EtOAc/hexanes); IR (neat) 3448, 2937, 2858,
Four closely related histinicotoxin analogues were prepared in
f
−1 1
2
217, 1674, 1457, 1373, 1346, 1228, 1162, 1120, 1063 cm ; H
an efficient manner and shown to be potent non-competitive
antagonists of nAChR’s. While the unsaturated analogues appear
to have one binding site, the saturated analogues would appear to
have more than one binding site. Further work is required to
elucidate the binding sites and develop structure-activity
relationships.
NMR (CDCl , 400 MHz) δ 0.85 – 0.90 (m, 3H, CH3), 1.20 –
3
1
1
.25 (m, 6H, 2 X O-CH2-CH3), 1.25 – 1.53 (m, 15H), 1.61 (b s,
H, OH), 1.66 – 1.89 (m, 2H), 2.58 (t, J = 7.2 Hz, 2H,), 2.70 (d, J
=
=
5.6 Hz, 2H), 3.51 – 3.61 (m, 2H) 3.64 – 3.73 (m, 2H), 4.70 (t, J
13
5.6 Hz, 1H); C NMR (CDCl , 100 MHz) δ 14.02, 15.12,
3
2
4
0.00, 22.58, 25.39, 25.59, 29.22, 29.57, 31.76, 36.47, 37.45,
5.25, 62.08, 71.36, 81.73, 89.25, 100.96, 187.95; HRMS (EI)
4
. Chemistry Experimental Section
+•
m/z 339.2516. C H NO [M-1] requires 339.2530.
20
35
4
4
.1. General Experimental
4
.4. 1,1-Diethoxy-9-hydroxyhexadecan-5-one (7)
Hexanes refer to the fraction with a boiling point of 40-60 °C.
Dry tetrahydrofuran (THF), dichloromethane, and toluene were
obtained by passing these solvents through activated alumina
columns. Unless otherwise specified, all H and C NMR spectra
were recorded on a Bruker Av400 spectrometer at 400 MHz and
A mixture of the alkyne 6 (5.25 g, 16.0 mmol) and Pd(OH)
2
on carbon (520 mg of 20% Pd catalyst) in EtOAc (200 mL) was
shaken vigorously under an atmosphere of H (30 psi, 2 h). The
1
13
2
™
mixture was filtered through Celite , washing with EtOAc. The
combined filtrate and washings were concentrated. The residue
was purified by flash chromatography (EtOAc/hexanes, gradient;
15:85 then 40:60) to yield the alkane 7 (5.3 g, 99%) as white
1
5
00.6 MHz respectively, or, a Bruker DRX508 spectrometer at
00 MHz and 125.8 MHz respectively, using CDCl solutions.
3
Chemical shifts (δ) are measured in ppm. Positive ion EI mass
spectra were run on ThermoQuest MAT95XL mass
a
waxy solid: R
f
0.19 (30:70 EtOAc/hexanes); IR (neat) 3459,
spectrometer using an ionization energy of 70eV. Accurate mass
measurements were obtained with a resolution of 5000-10000
using perfluorokerosene as the reference compound. High
resolution positive ion electrospray mass spectra were acquired
with a Micromass Q-TOF II mass spectrometer using a cone
3010, 2930, 2872, 2858, 1710, 1458,1376, 1216, 1128, 1059,
757, 667 cm ; H NMR (CDCl , 400 MHz) Data for the major
3
isomer δ 0.83 – 0.88 (m, 3H), 1.18 (t, J = 7.1 Hz, 6H), 1.22 –
1.31 (m, 9H), 1.22 – 1.47 (m, 5H), 1.54 – 1.73 (m, 7H), 2.39 –
2.45 (m, 4H), 3.41 – 3.51 (m, 2H), 3.51 – 3.57 (m, 1H), 3.57 –
−1 1

6
Tetrahedron
13
3
.67 (m, 2H) 4.43 – 4.50 (m, 1H); C NMR (
A
CD
C
C
C
l ,
E
1
P
00
T
MH
E
D
z) MA67
N
.86,
U
S
77
C
.2
R
0,
I
102.66, 126.00, 127.00, 128.29, 142.03 ; HRMS
P
T
3
Data for the major isomer δ 14.03, 15.28, 19.03, 19.65, 22.60,
(EI) m/z 445.3552. C H NO requires 445.3550.
28
47
3
2
6
5.61, 29.23, 29.60, 31.77, 33.00, 36.79, 37.44, 42.31, 42.53,
t
11
4
.7. Butoxychlorodiphenylsilane Kojima et. al.
1.05, 71.40, 102.66, 210.99; HRMS (EI) m/z 326.2812.
+•
t
C H O [M−H O] requires 326.2815.
20
38
3
2
( BuO)Ph SiCl was prepared according to the literature
procedure. Ph SiCl (15.0 g, 59.2 mmol) was converted to
2
11
2
2
4
.5. 1,1-Diethoxy-9-methanesulfonylhexa-decan-5-one (8)
t
(
BuO)Ph SiCl and distilled to a clear colourless oil (12.2 g,
2
1
To a solution of alcohol 7 (5.00 g, 14.5 mmol) and Et N (8.10
76%). H NMR (CDCl , 400 MHz) δ 1.39 (s, 9H), 7.37 – 7.48
3
3
13
mL, 58.1 mmol) in dry CH Cl (150 mL) at −30 ºC, was added
(m, 6H), 7.70 – 7.74 (m, 4H); C NMR (CDCl , 100 MHz) δ
2
2
3
MsCl (2.25 mL, 29.3 mmol) in dry CH Cl (50 mL) dropwise (25
min) and stirred (30 min). The mixture was quenched with
31.70, 31.99, 76.37, 127.31, 127.87, 130.53, 134.30, 134.86,
135.17.
2
2
aqueous saturated NaHCO , the organic layer was separated,
t
11
3
4
.8. ( Butoxydiphenylsilyl)acetonitrile.
washed with saturated aqueous NaCl solution, dried (MgSO ,
4)
i
filtered, and concentrated. The residue was purified by flash
chromatography (EtOAc/hexanes, gradient; 10:90 then 30:70) to
To a solution of Pr NH (9.12 mL, 64.5 mmol) in dry THF
180 mL) at 0 °C, BuLi (38.7 mL of a 1.59 M solution in
hexanes, 61.5 mmol) was added dropwise (2 min) which gave a
clear yellow solution that was stirred (5 min). MeCN (1.70 mL,
32.3 mmol) was added dropwise (1 min) giving a pale purple
2
n
(
yield mesylate 8 (5.1 g, 91%) as an unstable, clear, colourless oil:
1
R 0.29 (30:70 EtOAc/hexanes); H NMR (CDCl , 400 MHz) δ
f
3
0
1
4
.83 – 0.93 (m, 3H, CH ), 1.19 (t, J =7.1 Hz, 6H, 2 x OCH CH ),
3
2
3
t
.23 – 1.43 (m, 10H), 1.55 – 1.72 (m, 10H), 1.98 – 2.46 (m,
H,CH COCH ), 3.00 (s, 3H, SO CH ), 3.41 – 3.53 (m, 2H, 2 x
solution that was stirred (30 min). ( BuO)Ph SiCl (8.94 g, 30.7
2
2
2
3
3
mmol) in THF (70 mL) was added dropwise (2 min), which gave
a bright yellow clear solution. The solution was stirred (7 min) at
0 °C and then allowed to warm to room temperature and stirred
(2.5 h). The reaction was then quenched with saturated aqueous
NH Cl. The organic layer was separated and the aqueous phase
was re-extracted with Et O. The combined organics were dried
OCHHCH ), 3.57 – 3.66 (m, 2H, 2 x OCHHCH ) 4.45 – 4.50
3
3
13
(
m, 1H, CH(OEt) ), 4.64 – 4.73 (m, 1H, CHSO ); C NMR
2
3
(
CDCl , 100 MHz) δ 14.03, 15.30, 18.83, 19.00, 22.57, 24.97,
3
2
6
9.06, 29.28, 31.70, 32.99, 33.74, 34.28, 38.66, 41.89, 42.41,
1.09, 83.50, 102.65, 210.10
4
2
(
MgSO ), filtered and concentrated. The residue was purified by
4
4
.6. (2S*,6R*,8R*)-2-(Heptyl)-6-(4',4'-diethoxy-1'-butyl)-8-
silica gel funnel chromatography (CH Cl /hexanes, gradient;
1
white solid: mp 54 °C (lit. 56°C). H NMR (CDCl , 400 MHz)
phenyl-1-aza-9-oxabicyclo[4,3,0]nonane (11)
2
2
t
0:90 then 40:60), to give ( BuO)Ph SiCH CN (7.3 g, 80%) as
2
2
11
1
To the mesylate 8 (5.92 g, 14.0 mmol) in EtOH (250 mL) was
3
added NaHCO (8.47 g, 100 mmol) and NH OH·HCl (3.58 g,
δ 1.30 (s, 9H), 2.15 (s, 2H), 7.36 – 7.51 (m, 6H), 7.51 – 7.52
3
2
13
9
8.1 mmol). This mixture was stirred at room temperature (15
(m,4H) ; C NMR (CDCl , 100 MHz) δ 5.66, 31.92, 75.12,
3
min) and then at 70 ºC (20 h). H O was added (250 mL); and
118.40, 128.11, 130.67, 133.65, 134.60.
2
then the mixture was concentrated to approximately 250 mL. The
mixture was diluted with CH Cl and the organic layer was
4
.9. (2S*,6S*,8R*)-2-(Heptyl)-6-(5'-cyanopent-4'-en-1'-yl)-8-
2
2
phenyl-1-aza-9-oxabicyclo[4,3,0]nonane (12)
separated. The aqueous phase was re-extracted (4 times) with
CH Cl . The combined organics were washed with aqueous
2
2
To diethylacetal 11 (1.71 mg, 3.84 mmol) in THF (50 mL)
was added aqueous HCl (5.00 mL of 2.00 M solution) and the
solution was stirred (1.5 h). The reaction was quenched with
saturated aqueous NaHCO and diluted with EtOAc. The organic
layer was separated, the aqueous phase was re-extracted (2 times)
with EtOAc. The combined organics were washed with saturated
saturated NaCl solution, dried (MgSO ), filtered and concentrated
4
to give, presumably, the somewhat unstable crude nitrone 10 (
crude 4.9 g) as an oil that was used immediately: [R 0.27 (8:92
f
3
1
EtOH/CH Cl ); H NMR (CDCl , 400 MHz) data from crude
2
2
3
nitrone δ 0.82 − 0.88 (m, 3H, CH ), 1.18 (t, J = 7.1 Hz, 6H), 1.21
3
−
(
1.35 (m, 10H), 1.53 −1.97 (m, 8H), 2.06 – 2.35 (m, 2H), 2.39
t, J = 6.3 Hz, 2H), 2.44 − 2.59 (m, 2H), 3.43 – 3.52 (m, 2H, 2 x
OCHHCH ), 3.57 − 3.65 (m, 2H, 2 x OCHHCH ), 3.65 – 3.73
aqueous NaCl, dried (MgSO ), filtered and concentrated. The
4
residue was purified by silica gel funnel chromatography
(EtOAc:hexanes, gradient; 25:75) to give a clear yellow oil,
presumably the aldehyde, t that was used immediately.
Meanwhile, to a solution of ( BuO)Ph SiCH CN (1.39 mg, 4.71
mmol) in dry THF (80 mL) at –78 °C was added BuLi (2.96 mL
of 1.59 M solution in hexanes, 4.71 mmol) dropwise (~1 min).
This mixture was allowed to warm to 0 °C and kept at this
temperature for 10 min, and then cooled to –78 °C. To this
mixture was added the crude aldehyde in dry THF (30 mL)
dropwise (8 min). The reaction was stirred (1 h) and quenched
with saturated aqueous NH Cl and allowed to warm to rt. The
organic layer was separated and the aqueous phase was re-
extracted with EtOAc (2 times). The combined organics were
washed with saturated aqueous NaCl, dried (MgSO ), filtered and
concentrated. The residue was purified by flash chromatography
3
3
+
13
(m, 1H, CHN ), 4.45 – 4.50 (m, 1H, CH(OEt) ); C NMR
2
(
CDCl , 100 MHz) δ 14.03, 15.30, 15.59, 19.78, 22.58, 26.11,
3
2
2
n
2
6
6.44, 28.85, 29.13, 29.41, 31.63, 31.75, 32.36, 33.62, 61.20,
1.34, 67.01, 102.66, 148.26. This oil (4.94 g) was taken up in
styrene (100 mL), and hydroquinone (~ 2 mg) was added. The
mixture was heated (70 °C) for 4 days and then concentrated. The
residue was purified by flash chromatography (EtOAc/hexanes,
gradient; 4:96, 5:95, 6:94 then 7:93) to give isoxazolidine 11 (2.9
g, 53% over 2 steps) as a clear pale yellow oil: R 0.21 (10:90
f
4
EtOAc/hexanes); IR (neat) 3081, 3061, 2928, 2864, 1603, 1494,
−1
1
1
453, 1374, 1127, 1063, 754, 699 cm ; H NMR (CDCl , 400
3
MHz) δ 0.83 − 0.95 (m, 3H, CH ), 1.10 – 1.19 (m, 6H, 2 x
3
4
OCH CH ), 1.20 – 1.32 (m, 10H), 1.34 – 1.72 (m, 11H), 1.78 –
2
3
1
.89 (m, 2H), 1.89 – 1.97 (m, 1H), 1.98 – 2.03 (dd, J = 12.4, 5.2
(EtOAc:hexanes, gradient; 1:99, 2:98, 3:97, 4:96 then 7:93) to
Hz, 1H), 2.58 – 2.67 (m, 1H), 2.68 – 2.76 (m, 1H), 3.32 – 3.47
yield nitrile 12 (1.2 g, 78%) as a clear yellow oil: R 0.21 (10:90
EtOAc/hexanes); IR (neat) 3063, 3029, 2927, 2857, 2219, 1685,
1494, 1456, 1375, 1074, 1028, 753, 700m cm ; H NMR (CDCl ,
400 MHz) δ ]0.84 − 0.90 (m, 3H, CH ), 1.06 − 1.45 (m, 14H),
1
1
f
(m, 2H, 2 x OCHHCH ), 3.49 – 3.61 (m, 2H, OCHHCH ), 4.39
3 3
-
1 1
(
t, J = 5.6, 1H, CH(OEt) ), 5.39 (dd, J = 10.0, 5.2 Hz, 1H,
2
3
13
CHPh), 7.20 – 7.40 (m, 5H, Ar); C NMR (CDCl , 100 MHz) δ
3
3
1
3
4.07, 15.32, 18.97, 19.99, 22.64, 25.81, 29.31, 29.48, 29.93,
1.28, 31.87, 33.96, 34.69, 41.58, 42.25, 59.37, 60.74, 61.04,
.52 − 1.70 (m, 5H), 1.77 − 1.85 (m, 2H), 1.96 (dd and m, J =
2.5, 5.0 Hz, 2H, CHHCHPh and CHH), 2.30 − 2.36 (m, 2H,
CH CH=CHCN), 2.65 – 2.75 (m, 2H, CHHCHPh and CHN),
2

7
5
5
7
1
3
1
.19 (d, J = 10.9 Hz, 1H, CH=CHCN), 5.37 −
A
5.4
C
3
C
(dd
E
,
P
J
T
=
E
10
D
.0, MA(C
N
D
U
Cl
S
,
C
10
R
0 MIP
Hz) δ 14.08, 14.39, 17.87, 19.65, 21.22, 22.65,
T
3
.1 Hz, 1H, CHPh), 6.31 (dt, J = 10.9, 7.6 Hz, 1H, CH=CHCN)
24.97, 26.02, 29.30, 29.81, 29.97, 31.86, 32.30, 34.14, 34.73,
42.56, 64.86, 75.56, 77.99, 123.32, 136.31; HRMS (EI) m/z
13
.22 − 7.39 (m, 5H, Ph); C NMR (CDCl , 100 MHz) δ 14.09,
3
+•
9.94, 22.65, 25.75, 29.32, 29.49, 29.93, 31.04, 31.87, 31.97,
4.61, 41.31, 42.08, 59.41, 67.58, 76.80, 99.43, 115.98, 125.90,
27.06, 128.35, 141.98, 155.03; HRMS (EI) m/z 394.2978.
319.2864 C H NO [M] requires 319.2870.
21 37
4
.12. (2S*,6R*,7S*,8S*)-(1'Z)-7-(But-1'-enyl)-2-( heptyl)-1-
+•
azaspiro[5.5]undecan-8-ol (15)
C H N O [M] requires 394.2979.
26
38
2
*
*
*
*
To alkene 14 (318 mg, 0.995 mmol) was added SmI in THF
(0.1 M, 14 mL, 1.4 mmol) in 1 mL portions over a week. Fresh
2
4
.10. (1R ,5R ,8S ,12R )-12-Cyano-5-(heptyl)-6-aza-7-
1,6
oxatricyclo-[6.3.1.0 ] dodecane (13)
SmI in THF was added whenever the solution turned yellow (~
2
A solution of the α,β-unsaturated nitrile 12 (2.38 g, 6.03
mmol) in toluene (72 mL) and EtOH (12 mL) was divided
equally into 6 vials. These vials were each sealed and irradiated
in a microwave reactor for 50 min at 180 °C (pressure was ~ 11
bar). The contents of the vials were combined, concentrated and
the residue was purified by flash chromatography
every morning and evening). MeOH (0.5 mL) and aqueous NH3
(14 M, 2 mL) were added and the mixture was stirred (30
min).The pH was adjusted to ~10 (2 mL 14 M NH ), then
3
saturated aqueous Na S O was added, and the mixture was
2
2
3
diluted with H O and Et O, and the organic layer was separated.
2
2
The aqueous layer was re-extracted (3 times) with Et O and then
2
(EtOAc/hexanes, gradient; 2:98, 3:97, 3.5:96.5 then 4:96) to
the combined organics were washed with saturated aqueous
yield the tricycle 13 (1.4 g, 78%) as a clear yellow oil: R 0.24
NaCl, dried (MgSO ), filtered and concentrated. The residue was
f
4
(
10:90 EtOAc/hexanes); IR (neat), 2926, 2855, 2240, 1457, 1376,
purified by flash chromatography (EtOAc/hexanes, gradient;
10:90, 20:80, 25:75, 30:70 then MeOH/CH Cl , 2:98; then 14 M
−1 1
1
0
1
5
360, 1115, 1084, 945, 931 cm ; H NMR (CDCl , 400 MHz) δ
3
2
2
.83 − 0.89 (m, 3H, CH ), 1.05 – 1.37 (m, 8H), 1.50 – 2.04 (m,
aq NH /MeOH/CH Cl , gradient; 1:2:87 and then 1:3:96) to give
3
3 2 2
5H), 2.12 – 2.20 (m, 1H) 2.33 – 2.42 (m, 1H, CHN), 3.43 (d, J =
bicycle 15 (124 mg, 38%, with 162 mg starting material
13
.8 Hz, 1H, CHCN), 4.66 – 4.74 (m, 1H, CHO); C NMR
recovered) as an oil: R 0.48 (1:5:94 16 M NH /MeOH/CH Cl );
f
3
2
2
(
2
6
CDCl , 100 MHz) δ 14.06, 17.52, 19.19, 22.62, 25.62, 27.14,
9.23, 29.59, 29.82, 31.81, 32.24, 34.23, 36.02, 38.11, 65.42,
IR (neat) 3257, 3011, 2929, 2856, 1457, 1375, 1088, 970m, 727w
−1 1
3
cm ; H NMR (CDCl , 400 MHz) δ 0.71 - 0.90 (m, 5H, CH ,
3 3
5.51, 75.55, 117.91; HRMS (EI) m/z 290.2351. C H N O
CH, CH), 0.98 (t, J = 7.5 Hz, 3H, CH ), 1.17 – 1.47 (m, 16H),
18
30
2
3
+•
[
M] requires 290.2353.
1.47 – 1.65 (m, 6H), 1.97 – 2.20 (m, 3H, CHCH=CHCH , CH ),
2
2
2
.88
CHCH=CHCH
2
–
2.90 (m, 1H, CHNH), 3.22
, 3.67 – 3.75 (m, 1H, CHOH), 5.16 – 5.25 (m,
H, CHCH=CHCH ), 5.39 – 5.47 (m, 1H, CHCH=CHCH ), 7.30
– 3.29 (m, 1H,
4
.11. (1R*,5R*,8S*,12S*)-1'-(Z)-12-(But-1'-enyl)-5-(heptyl)-6-
1,6
aza-7-oxatricyclo[6.3.1.0 ] dodecane (14)
1
–
1
3
1
2
2
13
8.20 (v br s, 1H, OH); C NMR (CDCl , 100 MHz) δ 14.05,
4.43, 15.29, 19.47, 20.82, 22.63, 25.93, 28.59, 29.17, 29.91,
1.81, 32.79, 36.55, 37.42, 37.74, 38.03, 50.32, 54.59, 72.43,
To a solution of the nitrile 13 (769 mg, 2.65 mmol) in toluene
3
i
(50 mL) at −78 °C was added Bu AlH in toluene (1.5 M, 2.20
2
mL, 3.30 mmol). After 2 h, MeOH (0.5 mL) was added and the
mixture was allowed to warm to room temperature. This mixture
was diluted with EtOAc (40 mL) and aqueous potassium sodium
tartrate (1.4 M, 70 mL) and stirred vigorously (16 h). The organic
layer was separated and the aqueous layer was re-extracted (2
times) with EtOAc and the combined organics were washed with
saturated aqueous NaCl, dried (MgSO4), filtered and
concentrated to give, presumably the crude aldehyde (550 mg,
+•
26.63, 133.86; HRMS (EI) m/z 321.3022 C H NO [M]
21 39
requires 321.3026.
4
.13. (2S*,6R*,7S*,8S*)-7-(Butyl)-2-(heptyl)-1-
azaspiro[5.5]undecan-8-ol (16)
Alkene 14 (132 mg, 0.413 mmol), was added to a mixture of
Pd(OH) on carbon (100 mg of 20% Pd catalyst) and aqueous
2
7
1% crude) as an unstable colourless oil: [R 0.48 (30:70
HCl (1.03 mL, 2 M solution, 2.06 mmol) in dry THF (50 mL).
This mixture was shaken vigorously under an atmosphere of H2
(25 psi, 16 h). The mixture was filtered through Celite washing
f
EtOAc/hexanes] that was used immediately. Meanwhile, to a
mixture of 1-propyltriphenylphosphonium bromide (1.53 g, 3.98
mmol) in dry THF (60 mL) at −78 °C was added BuLi (2.50 mL
n
with THF/H O (4:1, 100 mL). The mixture was then neutralised
2
of a 1.59 M solution in hexanes, 3.98 mmol) dropwise (3 min).
The bright yellow mixture was allowed to warm to 0 °C and
stirred for 30 min during which the mixture had turned orange.
The mixture was cooled (−78 °C) and the crude aldehyde (550
mg) in THF (30 mL) was added dropwise (10 min). This mixture
was allowed to slowly warm to room temperature (over ~ 4 h)
(NaHCO ) and concentrated until all the THF was removed and
3
then extracted (4 times) with CH Cl . The organic phase was
2
2
washed with aqueous saturated NaCl, dried (MgSO4), filtered
and concentrated. The residue was purified by flash
chromatography (EtOAc/hexanes, gradient; 10:90, 20:80, 25:75,
30:70; MeOH/CH Cl , gradient; 2:98 then 14
M
aq
2
2
and then stirred overnight. Saturated aqueous NH Cl was added,
NH /MeOH/CH Cl , gradient; 1:2:87 and then 1:3:96) to give
4
3
2
2
and the mixture was diluted with H O and EtOAc. The organic
bicycle 16 (130 mg, 84%) as waxy solid. R 0.38 (1:10:89 16 M
2
f
layer was separated, and the aqueous layer was re-extracted (2
times) with EtOAc. The combined organics were washed
NH /MeOH/CH Cl ); IR (thin film) 3253, 2929, 2857, 1715,
3
2
2
-
1 1
1537, 1377, 1067, 970 cm ; H NMR (CDCl , 400 MHz) δ 0.79
3
sequentially with saturated aqueous NaHCO , saturated aqueous
– 0.94 (m, 8H, 2 x CH , CH, CH), 1.00 – 1.15 (m, 2H), 1.18 –
3
3
NaCl, dried (MgSO ), filtered and concentrated. The residue was
purified by flash chromatography (EtOAc/hexanes, gradient;
1.44 (m, 18H), 1.46 – 1.85 (m, 8H), 1.95 – 2.90 (m, 1H, CH),
2.16 – 2.23 (m, 1H), 2.89 – 3.00 (m, 1H, CHNH), 3.90 – 3.98 (m,
4
13
2
:98, 2.5:97.5 then 3:97) to yield the alkene 14 (402 mg, 47%
1H, CHOH), 7.31 – 8.35 (v br s, 1H, OH); C NMR (CDCl , 100
3
over two steps) as a clear, yellow oil: Rf 0.33 (10:90
EtOAc/hexanes); IR (neat) 3024, 2928, 2858, 1650, 1460, 1375,
MHz) δ 14.01, 14.05, 15.18, 19.48, 22.63, 23.02, 25.89, 27.54,
27.64, 29.16, 29.88, 30.26, 31.81, 33.00, 36.78, 36.86, 37.68,
−1 1
1
0
2
2
1
113, 1075, 925, 721 cm ; H NMR (CDCl , 400 MHz) δ 0.83 –
38.05, 50.07, 55.34, 69.76; HRMS (EI) m/z 323.3174 C H NO
3
21 41
+•
.91 (m, 3H, CH ), 0.96 – 1.04 (m, 3H, CH ), 1.06 – 1.65 (m,
[M] requires 323.3183.
3
3
0H), 1.73 – 1.82 (m, 2H), 1.90 – 2.04 (m, 2H), 2.07 – 2.18 (m,
4
.14. (1R*,5R*,8S*,12S*)-1'-(Z)-5-(heptyl)-12-(hex-1'-enyl)-6-
H, CH=CHCH ), 2.60 – 2.70 (m, 1H, CHN), 3.39 – 3.48 (m,
1,6
2
aza-7-oxatricyclo[6.3.1.0 ] dodecane (17)
H, CHCH=CH), 4.35 – 4.43 (m, 1H, CHO), 5.34 – 5.48 (m, 1H,
13
CHCH=CH), 5.59 – 5.70 (m, 1H, CH=CHCH2); C NMR

8
Tetrahedron
To a solution of the nitrile 13 (699 mg, 2.41 mmol) in
4.16. (2S*,6R*,7S*,8S*)-(1'Z)-2-(Heptyl)-7-(hex-1'-enyl)-1-
ACCEPTED MANUSCRIPT
i
toluene (50 mL) at −78 °C was added Bu AlH in toluene (1.50
azaspiro[5.5]undecan-8-ol (18)
2
M, 1.93 mL, 2.89 mmol). After 2 h, MeOH (0.5 mL) was added
and the mixture was allowed to warm to room temperature. This
mixture was diluted with EtOAc (40 mL) and aqueous potassium
sodium tartrate (1.40 M, 70.0 mL) and stirred vigorously (16 h).
The organic layer was separated, the aqueous layer was re-
extracted (2 times) with EtOAc and the combined organics were
To alkene 17 (57 mg, 0.144 mmol) was added SmI in THF (0.1
M, 14.0 mL, 1.4 mmol) in 1 mL portions over a week. MeOH
2
(
0.5 mL) and aqueous NH (14 M, 2 mL) were added and the
3
mixture was stirred (30 min). The pH was adjusted to ~10 (2 mL
4 M NH ), then saturated aqueous Na S O was added, the
1
3
2
2
3
mixture was diluted with H O and Et O, and the organic layer
2
2
washed with saturated aqueous NaCl, dried (MgSO ), filtered and
4
was separated. The aqueous layer was re-extracted (3 times) with
concentrated to give, presumably the crude aldehyde as an
unstable colourless oil that was used immediately. Meanwhile, to
Et O. The combined organics were washed with saturated
2
aqueous NaCl, dried (MgSO ), filtered and concentrated. The
4
a mixture of 1-pentyltriphenylphosphonium bromide (1.49 g,
n
residue was purified by flash chromatography (EtOAc/hexanes,
gradient; 40:60, then MeOH/CH Cl , 5:95 and then 14 M aq
3
.61 mmol) in dry THF (60 mL) at −78 °C was added BuLi
2
2
(
(
2.27 mL of a 1.59 M solution in hexanes, 3.61 mmol) dropwise
3 min). The bright yellow mixture was allowed to warm to 0 °C
NH /MeOH/CH Cl , 1:10:89) to afford olefin 18 (30 mg, 52%,
3
2
2
1
5 mg starting material recovered) as an oil: R 0.46 (1:5:94 16 M
f
and stirred for 30 min after which the mixture had turned orange.
The mixture was cooled (−78 °C) and the crude aldehyde (751
mg) in THF (30 mL) was added dropwise (10 min). This mixture
was allowed to slowly warm to room temperature (over ~ 4 h)
NH /MeOH/CH Cl ); IR (neat) 3258, 3011, 2927, 2856, 1457,
3
2
2
−1 1
1
0
1
3
3
377, 1089, 971, 724 cm ; H NMR (CDCl , 400 MHz) δ 0.78 –
3
.97 (m, 8H, 2 x CH , CH, CH), 1.20 – 1.48 (m, 19H), 1.50 –
.77 (m, 7H), 1.98 – 2.09 (m, 1H), 2.10 – 2.19 (m, 2H), 2.92 –
.04 (m, 1H, CHNH), 3.26 (d, J = 10.2 Hz, 1H, CHCH=CH),
3
and then stirred overnight. Saturated aqueous NH Cl was added
4
and the mixture was diluted with H O and EtOAc. The organic
2
.70
– 3.80 (m, 1H, CHOH), 5.18 – 5.27 (m, 1H,
layer was separated and the aqueous layer was re-extracted (2
times) with EtOAc. The combined organics were washed
CHCH=CHCH ), 5.40 – 5.50 ( m, 1H, CHCH=CHCH ), 7.40 –
2
2
13
7
1
2
5
.80 (v br s, 1H, OH exchange); C NMR (CDCl , 100 MHz) δ
3
sequentially with saturated aqueous NaHCO , saturated aqueous
3
4.02, 14.05, 15.27, 19.41, 22.46, 22.63, 25.91, 27.30, 28.49,
9.17, 29.89, 31.81, 32.02, 32.41, 36.24, 37.11, 37.99, 50.55,
5.00, 72.33, 126.83, 132.57; HRMS (EI) m/z 349.3333
NaCl, dried (MgSO ), filtered and concentrated. The residue was
4
purified by flash chromatography (EtOAc/hexanes, gradient;
4
6
:96, 5:95, 6:94, 7:93 then 8:92) to yield the alkene 17 (179 mg,
+•
C H NO [M] requires 349.3339.
23
43
4%) as a clear yellow oil: R 0.27 (10:90 EtOAc/hexanes); IR
f
(
9
neat) 3024, 2928, 2858, 1651, 1460, 1377, 1109w, 1080w, 944,
5
. Pharmacology Experimental Section
−1
1
24, 699 cm ; H NMR (CDCl , 400 MHz) δ 0.82 − 0.95 (m,
3
6
H, 2 x CH ), 1.20 − 2.05 (m, 28H), 2.06 – 2.16 (m, 2H,
The cDNAs encoding rat nAChR for α4 subunit was
3
CH=CHCH ), 2.60 – 2.69 (m, 1H, CHN), 3.39 – 3.47 (m, 1H,
subcloned in pSP64, for α7 subunit subcloned in pBS SK (+),
and β2 subunit subcloned in the pSP65 vectors and were
generous gifts from Professor Jim Boulter (University of
California, Los Angeles, CA). Preparation of cRNAs for α4 and
β2 subunits and two-electrode voltage-clamp recordings were
undertaken as previously described. In brief, oocytes were
surgically removed from Xenopus laevis while under general
anaesthesia (tricaine, 850 mg/500 mL) in accordance with the
National Health and Medical Research Council of Australia’s
ethical guidelines and approved by the University of Sydney
Animal Ethics Committee. Harvested lobes were treated with
collagenase A (2 mg mL-1; Roche Diagnostics, Australia) in
oocyte releasing buffer 2 (OR-2; 82.5mM NaCl, 2 mM KCl, 1
mM MgCl2, 5 mM HEPES, pH 7.5) for 1.5 h. cRNAs (1-10 ng)
were mixed in a 1:1 ratio of α4:β2 in a total volume of 50.6 nL
and injected into stage V-VI oocytes. Injected oocytes were kept
2
CH=CHCH ), 4.34 – 4.40 (m, 1H, CHO), 5.42 – 5.50 (m,1H,
2
13
CHCH=CH), 5.59 – 5.68 (m, 1H, CH=CHCH2); C NMR
(
CDCl , 100 MHz) δ 13.98, 14.08, 17.89, 19.73, 22.35, 22.65,
3
2
3
5.00, 26.00, 27.62, 29.31, 29.83, 29.98, 31.86, 31.94, 32.34,
4.20, 34.78, 42.62, 64.78, 64.84, 77.98, 123.89, 134.74; HRMS
15
+•
(EI) m/z 347.3180. C H NO [M] requires 347.3183.
23 41
4
.15. (2S*,6R*,7S*,8S*)-2-(Heptyl)-7-(hexyl)-1-
azaspiro[5.5]undecan-8-ol (3)
Alkene 17 (167 mg, 0.48 mmol), was added to a mixture of
Pd(OH) on carbon (100 mg of 20% Pd catalyst) and aqueous
2
HCl (0.96 mL, 2 M solution, 1.92 mmol) in dry THF (50 mL).
This mixture was shaken vigorously under an atmosphere of H2
(25 psi, 16 h). The mixture was filtered through Celite washing
with THF/H O (4:1, 100 mL). The mixture was then neutralised
2
in frog Ringer solution (96mM NaCl, 2mM KCl, 1 mM MgCl2,
(
NaHCO ) and concentrated until all the THF was removed. It
3
1
.8 mM CaCl and 5 mM HEPES; pH 7.4) containing 2.5 mM
2
was then diluted with CH Cl2 and the organic layer was
2
sodium pyruvate, 0.5ꢀM theophylline, and 4ꢀg/mL of
kanamycin, and stored for 2-5 days at 18°C in an orbital shaker
before being used for recording. Oocytes expressing α4β2
nAChRs were clamped at -60 mV and continually perfused with
separated. The aqueous phase was re-extracted (3 times) with
CH Cl . The combined organics were washed with aqueous
2
2
saturated NaCl, dried (MgSO4), filtered and concentrated. The
residue was purified by flash chromatography (EtOAc/hexanes,
gradient; 40:60, then MeOH/CH Cl , 5:95 and then 14 M aq
2+
Ca -free solution (115 mM NaCl, 2.5 mM KCl, 1 mM MgCl , ,
2
2
2
1
.8 mM BaCl , 10 mM HEPES; pH 7.4). Glass electrodes used
NH /MeOH/CH Cl , 1:10:89) to give bicycle 3 (136 mg, 81%) as
2
3
2
2
for recording had a resistance of 0.2-2Mꢁ and were filled with 3
waxy crystals. R 0.39 (1:10:89 16 M NH /MeOH/CH Cl ); IR
f
3
2
2
M KCl. Compounds were stored at -20°C and made up to the
(
1
neat) 3172, 2932, 2860, 2649, 1777, 1678, 1556, 1459, 1154,
2+
−1 1
required concentrations in Ca -free solution before applying to
the oocyte by gravity flow. Whole-cell currents were measured
using a GeneClamp 500 amplifier (Axon Instruments, Foster
City, CA, USA), a MacLab 2e recorder (AD Instruments,
Sydney, NSW, Australia) and Chart Version 4.0.1 program.
061 cm ; H NMR (CDCl , 400 MHz) δ 0.68 – 0.94 (m, 8H, 2
3
x CH , CH, CH), 1.00- 1.05 (m, 2H), 1.16 – 1.44 (m, 23H), 1.44
3
–
2
1
1
2
3
3
1.67 (m, 5H), 1.67 – 1.84 (m, 2H), 1.95 – 2.10 (m, 1), 2.17 –
.25 (m, 1H) 2.83 – 2.97 (v br s, 1H, CHNH), 3.85 – 3.96 (br s,
13
H, CHOH) 7.30 – 7.90 (v br, OH exchange); C NMR (CDCl3,
00 MHz) δ 14.05, 14.08, 15.20, 19.56, 22.63, 25.91, 27.75,
7.94, 28.05, 29.16, 29.68, 29.91, 31.81, 31.81, 33.28, 37.02,
7.07, 37.90, 38.14, 49.98, 55.10, 69.81; HRMS (EI) m/z
Inhibitory concentration response curves were determined in
the presence of 100 µM ACh for α4β2 or 300 µM for α7
nAChRs by first incubating the histrionicotoxins 3 mins before
+•
51.3497. C H NO [M] requires 351.3496.
23
45

9
3
.
Daly, J.W.; Karle, I.; Myers, C.W.; Tokuyama, T.; Waters, J.A.;
Witkop, B. Proc. Nat. Acad. Sci. USA 1971, 68, 1870.
co-addition with 100 µM or 300 µM A
A
Ch
C
,
C
re
E
sp
P
ec
TED
ti
ve
ly. MANUSCRIPT
Concentration response curves were determined over an ACh
concentration range of 0.1-10000 µM either alone or in the
presence of 16 (30 µM), 15 (1 µM), 3 (1 µM) and 18 (1 µM)
Currents were normalised to the currents elicited by 100 µM ACh
and presented as mean ± SEM and fitted using sigmoidal fit
4
5
.
.
Sinclair, A.; Stockman, R. A. Nat. Prod. Rep. 2007, 24, 298−326.
(a) Spivak, C. E.; Maleque, M. A.; Oliveira, A. C.; Masukawa, L.
M.; Tokuyama, T.; Daly, J. W.; Albuquerque, E. X. Mol.
Pharmacol. 1981, 21, 351−361. (b) Daly, J.W.; Nishizawa, Y.;
Edwards, M.W.; Waters, J.A.; Aronstam, R.S.; Neurochem. Res.
1991, 16, 489−500.
(variable slope) equation from GraphPad Prism 4.0:
6
.
(a) Spivak, C.E.; Maleque, M. A.; Oliveira, A. C.; Masukawa, L.
M.; Tokuyama, T.; Daly, J. W.; Albuquerque, E.X. Mol
Pharmacol 1982, 21, 351. (b) Ogura, T.; Warashina, A. Comp
Biochem Physiol C 1987, 88, 249.
nH nH nH
I=IMAX([A] ꢂ([A]
+ EC
))
50
7
8
.
.
Aronstam, R.S.; King, C.T. Jr.; Albuquerque, E.X.; Daly, J.W.;
Feigl, D.M. Biochem Pharmacol 1985, 34, 3037.
Davison, E. C.; Fox, M. E.; Holmes, A. B.; Roughley, S. D.;
Smith, C. J.; Williams, G. M.; Davies, J. E.; Raithby, P. R.;
Adams, J. P.; Forbes, I. T.; Press, N. J.; Thompson, M. J. J. Chem.
Soc., Perkin Trans. 1 2002, 1494−1514.
where I is the current, IMAX is the current produced by ACh at
a given concentration (100 µM, 300 µM or 1 mM as specified),
[
A] is the ligand concentration and n is the Hill slope. From this
H
equation, the concentration of the agonist that activates 50% of
expressed receptors (EC ) or in the case of inhibitory
concentration response curves the concentration of the antagonist
that inhibits 50% of the evoked ACh current (IC ) were
calculated. Data are presented as mean (95% confidence
intervals) from a minimum of 3 oocytes over a minimum of 2
batches. Unless otherwise stated statistical differences between
groups were calculated using Student’s t-test.
5
0
9. Macdonald, J. M.; Horsley, H. T.; Ryan, J. H.; Saubern, S;
Holmes, A. B. Org. Lett. 2008, 10, 4227−4229.
5
0
1
1
0. Brasholz, M; Macdonald, J. M.; Saubern, S.; Ryan J. R.; Holmes,
A. B. Chem. Eur. J. 2010, 16, 11471–11480.
1. Kojima, S.; Fukuzaki, T.; Yamakawa, A.; Murai, Y. Org. Lett.
2
004, 6, 3917−3920.
12. Revuelta, J.; Cicchi, S.; Brandi, A. Tetrahedron Lett. 2004, 45,
375−8377.
13. Still, W.C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
4. Hénaff, N, Whiting, A. Tetrahedron 2000, 56, 5193-5204.
8
6
. Acknowledgments
1
We thank Professor Andrew Holmes for his support and
15. Quek, G. X. J.; Lin, D.; Halliday, J. I.; Absalom, N.; Ambrus, J. I.;
Thompson, A. J.; Lochner, M.; Lummis, S. C. R.; McLeod, M. D.;
Chebib, M. ACS Chem. Neurosci. 2013, 1, 796–809.
encouragement. Dr’s Roger Mulder, Jo Cosgriff and Carl
Braybrook are thanked for collection of NMR and MS data. CE
thanks Dr Bob Laslett of Swinburne University for his support
throughout her Honours’ year.
7
. References and notes
Supplementary Material
1
2
.
.
(a) Albuquerque, E.X.; Periera, E. F. R.; Alkondon, M.; Rogers, S.
W. Physiol. Rev. 2009, 89, 73. (b) Gotti, C.; Clementi, F. Prog.
Neurobiol. 2004, 74, 363. (c) Millar, N. S.; Gotti, C.
1
13
Copies of H and C NMR spectra for Compounds 6–8, 10–
7, 3 and 18.
1
Neuropharmacology 2009, 56, 237.
(a) Levin, E.D.; Hall, B. J.; Rezvani, A. H. Curr. Top. Behav.
Neurosci. 2015, 23, 87. (b) Corradi, J.; Bouzat, C. Mol.
Pharmacol. 2016, 90, 288. (c) Mazzaferro, S.; Bermudez, I.; Sine,
S. M. J. Biol. Chem. 2017, 292, 2729. (d) Beinat, C.; Banister, S.
D.; Herrera, M.; Kassiou, M., Curr. Pharma. Des. 2016, 22,
2
134.