Synthetic neovibsanes and their ability to induce neuronal differentiation in PC12 cells

Article abstract of DOI:10.1016/j.tet.2010.06.056

A series of neovibsanin A and B derivatives and lower homologues were synthesized to study their neurotrophic ability with PC12 cells. 4,5-Bis-epi-neovibsanin A displayed prominent ability to induce neurite outgrowth compared to control cultures. Herein w

Full text of DOI:10.1016/j.tet.2010.06.056

Tetrahedron 66 (2010) 6842e6850
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthetic neovibsanes and their ability to induce neuronal differentiation
in PC12 cells
a
b
b
a,
*
Annette P.-J. Chen , C. Catharina Müller , Helen M. Cooper , Craig M. Williams
a
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, St. Lucia 4072, Queensland, Australia
Queensland Brain Institute, University of Queensland, Brisbane, 4072, Queensland, Australia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 April 2010
Received in revised form 4 June 2010
Accepted 21 June 2010
Available online 25 June 2010
A series of neovibsanin A and B derivatives and lower homologues were synthesized to study their
neurotrophic ability with PC12 cells. 4,5-Bis-epi-neovibsanin A displayed prominent ability to induce
neurite outgrowth compared to control cultures. Herein we describe the total synthesis of 4,5-bis-
epi-neovibsanin A and B as well as comparing the biological activity of several neovibsane derivatives.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Neovibsanin A
Neovibsanin B
Natural product
Neurotrophic activity
1
. Introduction
reservoir of natural products, which are continuously being
screened for compounds displaying neurotrophic activity. Two
natural products isolated by Fukuyama, neovibsanin A (1) and B
(2), have been shown to induce neurite outgrowth in primary rat
11
Adult neurogenesis and the regenerative potential of neural stem
cells offer a tantalizing possibility for restoring neurons and lost
neural circuitry in neurodegenerative disorders such as Alzheimer’s
and Parkinson’s disease. Neurotrophins, such as nerve growth factor
cortical neurons at 0.01
icity [KB cells (IC50 30 and 33
mM, whilst displaying very weak cytotox-
12
mM, respectively) Fig. 1].
(
NGF), neurotrophin 3 (NT3), neurotrophin 4/5 (NT4/5) and brain-
derived neurotrophic factor (BDNF), are a family of polypeptides,
which are essential for the differentiation, growth, development,
survival and functional maintenance of neurons and several other
1
e4
neuroectoderm-derived cellular populations.
Deregulation of
neurotrophins or their receptors have been implicated in neuro-
degeneration, neuropathies, pain and cancer, thus providing a ra-
tionale for the synthetic development of neurotrophic factors for
5
,6
possible therapeutic applications in disease. So far, peptidyl neu-
rotrophic factors have been tested in neurodegenerative disorders
but none, however, have been effective due to the drawbacks gen-
7,8
erally associated with using large polypeptides as drugs. These
drawbacks include short half-lives in vivo, poor pharmacokinetic
profiles, proteolytic degradation and undesired pleiotropic effects.
The problems associated with protein-based therapies have
encouraged the development of small molecule agents, which can
mimic or enhance the neurotrophic activities of endogenous neu-
rotrophins.5
e10
These compounds are often sourced from the
Figure 1. Structures of neovibsanin A (1) and B (2) and the natural product epimers
4,5-bis-epi-neovibsanin A (3) and B (4).
*
Corresponding author. E-mail address: c.williams3@uq.edu.au (C.M. Williams).
0
040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.056

A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
6843
Neovibsanin A (1) and B (2) belong to a family of rare vibsane
compounds, many of which are of biological and synthetic interest.
one-carbon unit integration using lithium dithiazide, providing
compound 12 in 57% yield, followed by a mercury mediated
deprotection to reveal the aldehyde unit 13 (73% yield). Alde-
hyde 13 was subjected to prolonged reflux in chloroform with
ylide 1-(triphenylphosphoranylidene)propanone in an E selective
olefination to obtain enone 14 in 94% yield.
13
Recently, Imagawa, Fukuyama and Nishizawa reported the first total
synthesis of (ꢀ)-neovibsanin B (2). Tested for its neurotrophic ac-
tivity using PC12 cells (derived from rat pheochromocytoma),
a widely used model for studying NGF-induced neuronal differen-
tiation,14 (ꢀ)-neovibsanin B displayed comparable neurotrophic
Treatment of enone 14 with an excess of concentrated sulfuric
15
ꢁ
activities to the natural product (þ)-neovibsanin B. This suggests
that both enantiomers are able to induce neuronal differentiation.
Our research efforts towards synthesizing neovibsanin A (1) and
B (2) resulted in the total synthesis of (ꢀ)-4,5-bis-epi-neovibsanin
A (3) and B (4). This paper describes the syntheses of these natural
product diastereomers and an extensive evaluation of their activity
compared to other structural analogues by quantifying the differ-
entiation of PC12 cells.
acid in anhydrous methanol at 4 C promoted a five-step cascade
reaction to afford the tricyclic methyl esters 19 and 20 in 73%
overall yield in a 5:1 ratio, respectively (Scheme 2). The cascading
steps involve an initial deprotection of the TBS ether, leading to
a Michael addition of the primary alcohol to give 16, which un-
derwent a lactone ring opening with the solvent to form ester 17,
followed by hemiacetal formation and ketalization with methanol
to provide 19 and 20. In parallel, the two epimeric esters were re-
duced using LiAlH
4
then oxidized using pyridine-buffered
DesseMartin periodinane in an overall yield of 54%. Finally the enol
ester side chain was installed using microwave irradiation to give
2
2
. Results and discussion
.1. Chemistry
4,5-bis-epi-neovibsanin A (3) and B (4) (30%, E/Z ratio 5:1; 14% E/Z
ratio 3:2, respectively).
The H and 13C NMR spectroscopic data of the natural products
and the diastereomers are quite similar (Table 1). Notable differ-
ences occur at position 5 where the stereoconfiguration differs,
especially between neovibsanin B (2) and its diastereomer (4) with
1
(ꢀ)-4,5-Bis-epi-neovibsanin A (3) and B (4) were synthesized
in 12 steps via a modified biomimetic approach involving an
acid-catalyzed, one-pot, five-step cascade reaction to access the
1
6e24
tricyclic core. Based on previous work
and on the synthesis
of (ꢀ)-2-O-methylneovibsanin H, enone 14 was prepared from
-methylcyclohexanone 5 (Scheme 1). Ketone 6 was prepared
via a copper mediated 1,4-addition to cyclohexanone 5 in 83%
1
13
2
5
a difference of 0.54 ppm and 3.6 ppm in the H and C data, re-
spectively. Position 9 is also worth noting with a proton chemical
shift difference of about 0.7 ppm for the neovibsanin A comparison
and 0.8 ppm for the neovibsanin B comparison.
3
2
6,27
yield, followed by dehydrogenation with IBX$NMO
the -unsaturated ketone 7 in 78% yield. The allylic alcohol 8
was obtained using a modified BayliseHillman reaction in wa-
to afford
a,b
2
8,29
ter,
in 54% yield over two steps. Alkylation using lithium diiso-
propylamide with iodoacetate gave 3:2 mixture of di-
which was protected as the tert-butyldimethylsilyl ether
2.2. Biological evaluation
9
a
In view of the prominent biological activity of natural neo-
vibsanin A (1) and B (2), as well as the activity of the racemic
mixture synthesized by Fukuyama, compounds 19e22 and the two
natural product diastereomers 3 and 4 were examined for their
ability to stimulate neurite outgrowth in PC12 cells.
astereomers with the major isomer 10 having the correct
configuration (61% yield, 80% based on recovered starting ma-
terial). Compound 11 could be epimerized with LDA to furnish
further supplies of 10. To obtain enone 14, required an initial
ꢁ
Scheme 1. Reagents and conditions: (a) 5-bromo-2-methyl-2-pentene, Mg, CuI, THF, 83%; (b) IBX$NMO, DMSO, 45 C, 78%; (c) DMAP, CH
2
O, SDS, H
2
O, 56%; (d) Imidazole, TBSCl,
3
PCHCOCH , CHCl ,
Et, THF, 50 C, 61%; (f) LDA, 85%; (g) N-methyl dithiazine, n-BuLi, THF, ꢂ78 ꢁC, 57%; (h) Hg(ClO
ꢁ
CH
2
Cl
2
, 97%; (e) LDA, ICH
2
CO
2
4
)
2 3
, CaCO , H O/THF, 73%; (i) Ph
2
3
3
reflux, 94%.

6844
A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
ꢁ
ꢁ
Scheme 2. Reagents and conditions: (a) H
SO
2 4
, MeOH, 4 C, 73%; (b) LiAlH
4
2
, Et O, DMP, CH
2
Cl
2
/pyr, 55%; (c) [(CH
3
)
2
CCHCO]
2
O, DMAP, Toluene, MW, 110 C, 30% and 14% for 3 and 4,
respectively.
Table 1
Comparison of selected 1H and 13C NMR spectroscopic data between compounds 1e4
Position
Neovibsanin A (1)
4,5-bis-epi-neovibsanin A (3)
Neovibsanin B (2)
4,5-bis-epi-neovibsanin B (4)
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
3
4
5
8
9
d
137.8
90.7
87.7
137.4
113.1
50
d
139.0
90.9
89.2
137.1
111.5
48.7
d
137.9
91.6
86.4
137.8
113.0
48.8
d
139.2
90.6
90.0
136.8
112.2
49.5
d
d
d
d
4.56
7.50
5.00
3.32
4.31
7.36
5.7
4.62
7.5
5.22
3.32
4.08
7.42
6.02
3.28
OCH
3
3.14
Cells were exposed to compounds at a concentration of 40
m
M in
majority of cells dying, which may be due to the higher dose of
DMSO. No compounds, however, were capable of inducing neurite
outgrowth in the absence of NGF, suggesting a synergistic effect
between the compounds and NGF (data not shown).
combination with NGF (20 ng/mL) for 48 to 72 h. As shown in
Figure 2 and 3, a significant increase of neuronal differentiation
determined by the outgrowth of neurites could be observed after
7
2 h for all compounds (Fig. 2a/b).
In particular, compound 3, the diastereomer of natural product
In terms of total outgrowth, all compounds significantly pro-
moted neurite outgrowth compared to the control (þNGFþDMSO)
(Fig. 2b). Compounds 19, 22, 3 and 4 appeared to promote a higher
neovibsanin A, showed remarkable neuritogenic activity with an
1.5-fold increase in the percentages of neurite bearing cells com-
1
activity (*** p<0.001) than compounds 20 and 21 (
p<0.05). In-
*
pared to the control cultures containing NGF and DMSO (1.33%), the
compound solvent. A 5-fold increase, however, was observed when
compound 3 was compared to experiments controlling for the ef-
fect of DMSO (þNGFꢂDMSO). This suggests that 1.33% of DMSO is
slightly cytotoxic to PC12 cells. A comparison between the 48 h and
terestingly, all compounds induced less overall outgrowth com-
pared to control cultures without DMSO (þNGFꢂDMSO). However,
the reverse is observed for individual primary process lengths
(Fig. 5a). This apparent contradiction in behaviour may be due to
DMSO, restricting the ability of the cells to branch out (as observed
in the highly branched nature of cells cultured without DMSO
(Fig. 5b)).
72 h incubation time (Fig. 4), indicated that diastereomer 3 appears
to reach its maximum effect after 48 h as there is no significant
increase in neuronal differentiation after a further 24 h (timepoint
Comparing the biological activity with the structures of in-
dividual compounds, it is hard to determine a definite structural
feature that can help to explain the different neurogenic activities.
With regards to the three different side chains, both compounds
with an enol ester unit (i.e., 3 and 4) appear to promote greater
overall outgrowth (Fig. 2b). In comparison, only one of two com-
pounds with the ester (i.e., 19) or aldehyde (i.e., 22) side chain
showed comparable levels of activity (based on p values) to those of
72 h). The other compounds, however, appear to show a continuous
increase in outgrowth from 48 h to 72 h. In addition, compound 3
promotes a biological response faster as the number of cells with
neurites is already higher after 48 h compared to the values of other
compounds at 72 h.
Although higher concentrations (60
mM) induced neurite out-
growth, they also appeared to have cytotoxic effects with the

A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
6845
Figure 2. Quantitative analysis of neuronal differentiation of PC12 cells with compounds 19e22, 3, and 4 at 40
mM concentrations in the presence of NGF (20 ng/mL) for 72 h. (a)
The percentages of cells with longer processes than one and a half cell body lengths and (b) the total neurite outgrowth were plotted as an index of neuronal differentiation. Data are
presented as meanꢀSE (n¼3). Statistical significance was tested against controlþNGFþDMSO using a one-way ANOVA followed by Bonferroni post hoc means comparison with
*
* p<0.01 and *** p<0.001.
Figure 3. Morphological appearance of PC12 cells after treatment with compounds for 72 h. Neurite outgrowth was assessed by the presence, length and number of processes on
cells and compared to controls (þNGFþDMSO, þNGFꢂDMSO). Cells were stained for
b
III-tubulin with Tuj1 (red) and the cell nuclei (DAPI; blue) (Bar¼50
mm).
3
and 4. Of the compounds 19, 22, 3 and 4, both stereo-
configurations of the methoxy group are present in equal numbers.
In terms of the percentages of cells with neurites (Fig. 2a),
compound 3 with the enol ester side chain showed higher levels of
activity than compounds with the same stereoconfiguration of the
methoxy group but with the ester (19) or aldehyde (21) unit. For the
other methoxy group stereoconfiguration, however, it is the alde-
hyde (22), which induced higher biological activity then com-
pounds with ester (20) or enol ester (4) side chains. Therefore,
minor differences in the side chain functional groups, perhaps in
combination with the different stereoconfiguration of the methoxy
group appear to influence these cellular responses.
A stability check on all compounds after an extended (6 months)
stay in DMSO revealed that all compounds were stable except in the
case of compound 21 and 3 in which the methoxy group at position
7
was eventually replaced by a hydroxyl group to give 23 and 24,
respectively, most likely due to moisture in the DMSO. Compound
3 underwent further rearrangement to compound 26 as outlined
2
in Scheme 3.
Interestingly, a comparison between the three experiments per-
formed for compound 21 indicated a significant drop in biological
activity for experiment 3, suggesting that the rearrangement
Figure 4. Neuronal differentiation of PC12 cells at 48 h and 72 h. Data are presented as
meanꢀSE (n¼3). Statistical analysis was performed between 48 h and 72 h using a t-
test with ** p<0.01 and *** p<0.001.

6846
A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
Figure 5. (a) Quantitative analysis of primary process length and (b) number of primary branched processes per cell at 72 h. Data are presented as meanꢀSE (n¼3). Statistical
significance was tested against controlþNGFþDMSO using a one-way ANOVA followed by Bonferroni post hoc means comparison with ** p<0.01 and *** p<0.001.
Evaluation of these derivatives suggests that there is no adverse
effect on the biological activity with the replacement of the
methoxy group, but a considerable decrease in activity was ob-
served when the tricyclic core is replaced with a lactol ketone
structure. Therefore, an oxygen at position 7 and the tricyclic core
may be of importance in conferring biological activity.
3. Conclusion
Various compounds containing the tricyclic core of neovibsane
natural products were synthesized and evaluated for neurotrophic
activity. The diastereomer (3) of neovibsanin A (1) markedly pro-
moted neuritogenesis in a synergistic manner with NGF. Further
synthetic exploration to increase solubility, stability and drug-
likeness will be reported in due course.
4
4
4
. Experimental
Scheme 3. Hydroxyl derivatives 23 and 24 derived from compounds 21 and 3, re-
spectively. Hemiacetal 23 further rearranges to lactol ketone 26.
.1. Chemistry
.1.1. General. 1H and 13C NMR spectra were recorded on Bruker
described in Scheme 3 occurred between experiment 2 and 3 (Fig. 6).
All statistical analyses were therefore calculated using only experi-
ment 1 and 2 for compound 21. Compound 24, however, did not
rearrange further and the data obtained for compound 3 showed no
decrease in activity and all three data points were included.
AV300
00.62 MHz), DRX500 (500.13 MHz; 125.76 MHz) and AV900
900.13 MHz; 226.36 MHz) instruments in deuteriochloroform
CDCl ) or hexadeuteriobenzene (C ) unless otherwise stated.
(300.13 MHz;
75.47 MHz),
AV400
(400.13 MHz;
1
(
(
3
6 6
D
Coupling constants are given in hertz and chemical shifts are
expressed as values in parts per million. IR spectra were measured
d
on a Perkin Elmer FTIR spectrometer (Spectrum 2000) with
a Smiths detection (DuraSamplerIR II). GC/MS data were recorded
on a Shimadzu GC-17A Ver.3, mass-spectrometer: MS QP5050A,
ionisation at 70 eV, column: DB-5ms 30 m 0.25 mm, carrier-gas:
He, total flow 32.2 mL/min, column flow 1.3 mL/min, injector
ꢁ
ꢁ
temperature: 250 C, standard program: 2 min at 100 C, followed
ꢁ
ꢁ
by a temperature increase of 16 C/min and held at 250 C for
0 min. Low resolution electrospray ionization mass spectrometry
1
measurements (LRESIMS) were recorded in positive ionization
mode on a Bruker Esquire HCT (High Cpacity 3D ion trap) in-
strument with a Bruker ESI source. High resolution electrospray
ionization (HRESIMS) accurate mass measurements were recorded
in positive mode on a Bruker MicrOTOF-Q (quadrupole-Time of
Flight) instrument with a Bruker ESI source. Accurate mass mea-
surements were carried out with external calibration using sodium
formate as a reference calibrant. High and low resolution EI mass
spectral data were obtained on a Finnigan MAT900. Column chro-
matography was undertaken on silica gel (Flash Silica gel
Figure 6. Experiments conducted for compound 21 on the percentages of cells bearing
neurites. Data are presented as meanꢀSE (n¼1).

A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
6847
2
30e400 mesh), with distilled solvents. Moisture sensitive exper-
4.1.1.3. 2-Hydroxymethyl-5-methyl-5-(4-methylpent-3-enyl)cy-
clohex-2-enone (8). Based on the procedure reported by Porzelle,
2
9
iments were conducted in oven or flame-dried glassware under an
atmosphere of argon. Anhydrous solvents used in moisture sensi-
tive reactions were dried according to Perin and Armarego, ‘Puri-
fication of laboratory solvents’, third Ed. THF was freshly distilled
from sodium/benzophenone. Melting points were determined on
a Fisher Johns melting point apparatus and are uncorrected. Fine
chemicals were purchased from the Aldrich Chem. Co.
sodium dodecyl sulfate (772 mg, 2.68 mmol) and 4-(dimethyl-
amino)pyridine (3.76 g, 30.7 mmol) were added to a mixture of
distilled water (25.8 mL) and compound 7 (5.15 g, 26.8 mmol).
After stirring for 15 min, formalin (36.0 mL) was added and stir-
red for 16 h at rt. The mixture was then quenched with brine
(50 mL) and extracted with EtOAc (2ꢃ70 mL). The combined or-
4
ganic layers were dried over MgSO and the solvent was removed
4
.1.1.1. 3-Methyl-3-(4-methylpent-3-enyl) cyclohexanone (6).
in vacuo. The residue was purified by column chromatography
(ethyl acetate/petroleum ether, 1:2) to give the titled compound
2
3
Following the procedure developed by Heim,
magnesium
turnings (4.62 g, 190 mmol) were stirred under high vacuum for
min before an argon atmosphere was introduced. Iodine crys-
tals (15 mg) were added followed by freshly distilled THF
15 mL). 5-Bromo-2-methyl-2-pentene (14.1 g, 127 mmol),
a portion (2 mL) of which was added directly to the THF sus-
pension to initiate the reaction, was dissolved in anhydrous THF
(3.32 g, 56%) as a light yellow oil. IR
n
max(ATR) 3600e3100 (br),
ꢂ1
5
2963, 2923, 2873, 1666, 1380, 1027 cm
;
d
H
(500 MHz, CDCl
CH ), 1.57 (3H, s,]
CMe), 1.65 (3H, s,]CMe), 1.88e1.97 (2H, m, CCH CH ), 2.21 (1H,
dddd, J 19.0, 4.7, 2.6, 1.3 Hz, CCH CH]), 2.27 (1H, dd, J 16.1,
1.1 Hz, COCH C), 2.32e2.38 (1H, m, CCH CH]), 2.35 (1H, d, J
16.1 Hz, COCH C), 2.41 (1H, t, J 6.5 Hz, OH), 4.25 (2H, ddd, J 6.5,
2.6, 1.1 Hz, CH OH), 5.01e5.06 (1H, m, CH]CMe ), 6.76 (1H, ddt, J
4.7, 3.8, 1.1 Hz, COCR]CH); (125 MHz, CDCl ) 17.6, 22.4, 24.7,
25.6, 36.7, 38.0, 41.3, 50.3, 60.6, 124.1, 131.7, 137.5, 144.2, 200.4;
3
)
1.01 (3H, s, Me), 1.34 (2H, dd, J 9.5, 7.5 Hz, CCH
2
2
(
2
2
a b
H
a
H
b
a b
H
(
40 mL) and the solution added dropwise to the above suspen-
a b
H
sion over 20 min with heating. The mixture was stirred at rt until
completion of the reaction (monitored by TLC). In a second flask,
CuI (602 mg, 3.17 mmol) was suspended in anhydrous THF
2
2
d
C
3
ꢁ
þ
ꢄ
(
25 mL). The solution was then cooled to ꢂ20 C and the Grignard
GC/MS: m/z (%): 222 [M ] (9), 204 (14), 189 (10), 164 (8), 161(15),
139 (22), 121 (86), 109 (53), 97 (22), 83 (18), 69 (49), 55 (39), 41
(100).
ꢁ
solution added via syringe over 10 min. After 40 min at ꢂ20 C 3-
methyl-2-cyclohexenone (5) (6.5 mL, 57.1 mmol, 0.9 equiv) in
ꢁ
THF (20 mL) was added dropwise at ꢂ20 C. The reaction was
ꢁ
stirred at ꢂ20 C for 30 min, then at rt for 1 h. The reaction was
4.1.1.4. 2-[(tert-Butyldimethylsilyloxy)methyl]-5-methyl-5-(4-meth-
ylpent-3-enyl)cyclohex-2-enone (9). Imidazole (1.33 g, 19.5 mmol)
was added to a solution of compound 8 (2.17 g, 9.80 mmol) in anhy-
4
quenched by the dropwise addition of saturated NH Cl solution
(
50 mL) and the phases separated. The aqueous solution was
extracted with Et
were washed with brine (50 mL) and dried over MgSO
removal of the solvent in vacuo the residue was subjected to
vacuum distillation (66 C/0.02 mmHg) yielding the title com-
2
O (4ꢃ100 mL) and the combined organic layers
2 2
drous CH Cl (40 mL) followed by the addition of tert-butyldime-
thylsilyl chloride (2.21 g, 14.7 mmol) under an argon atmosphere. The
reaction mixture was stirred for 30 min before being diluted with
4
. After
ꢁ
CH
saturated NH
layer was dried over MgSO
was then purified by column chromatography (Et
2
2
Cl
2
(30 mL) and washed with saturated NaHCO
Cl solution (70 mL) and then brine (70 mL). The organic
and concentrated in vacuo. The residue
O/petroleum ether,
3
solution (70 mL),
pound as a yellow oil (9.20 g, 83%). IR
n
max(ATR) 2961, 2917, 2876,
(300 MHz, CDCl ) 0.92 (3H,
), 1.51e1.67 (2H, m, CH ), 1.58
3H, br s,]CMe), 1.66 (3H, br d, J 1.0 Hz,]CMe), 1.81e1.96 (4H, m,
CH ), 2.09 (1H, d, J 13.5 Hz, COCH C), 2.19 (1H, d, J 13.5 Hz,
COCH C), 2.25 (2H, t, J 6.8 Hz, CH ), 5.02e5.09 (1H, m, CH]
CMe ); (75 MHz, CDCl ) 17.6, 22.1, 22.2, 24.9, 25.7, 35.9, 38.6,
1.0, 41.7, 53.7, 124.3, 131.6, 212.1; GC/MS: m/z (%): 194 [M ] (14),
79 (11), 161 (11), 151 (55), 133 (6), 112 (44), 111 (100), 109 (75),
7 (34), 83 (31), 69 (76), 55 (81), 41 (78).
4
ꢂ1
1712, 1681, 1453, 1380, 1227 cm
;
d
H
3
4
s, Me), 1.23e1.29 (2H, m, CCH
2
CH
2
2
(
1:10) to give the titled compound (3.18 mg, 97%) as a light yellow oil. IR
ꢂ1
2
2
a
H
b
n
max(ATR) 2956, 2930, 2858, 1673, 1254, 1059, 835, 776 cm
(400 MHz, CDCl ) 0.05 (6H, s, SiMe ), 0.90 (9H, s, SiCMe
Me), 1.33 (2H, dd, J 9.6, 7.5 Hz, CCH CH ), 1.57 (3H, s, ]CMe), 1.65 (3H,
br s,]CMe), 1.86e1.99 (2H, m, CCH CH ), 2.17e2.25 (1H, m,
CCH CH]), 2.23 (1H, dd, J 15.8, 1.1 Hz, COCH C), 2.32 (1H, d,
J¼15.8 Hz, COCH C), 2.35 (1H, ddd, J 18.7, 6.4, 3.1 Hz, CCH CH]),
.34 (2H, ddd, J 4.8, 2.4, 2.4 m, CH OSi), 5.00e5.09 (1H, m, CH]CMe ),
6.78e6.85 (1H, m, COCR]CH); (100 MHz, CDCl
) ꢂ5.4 (2C), 17.6,
18.4, 22.4, 24.8, 25.6, 25.9 (3C), 36.7, 38.0, 41.5, 50.5, 59.9, 124.2, 131.7,
; d
H
a
H
b
2
3
2
3
), 0.99 (3H, s,
2
d
C
3
2
2
þ
ꢄ
4
1
9
2
2
H
a b
a b
H
a
H
b
a b
H
4
2
2
4.1.1.2. 5-Methyl-5-(4-methylpent-3-enyl)-2-cyclohexenone (7).
d
C
3
2
3
Based on the procedure reported by Heim , a mixture of o-
iodoxybenzoic acid30 (23.2 g, 82.9 mmol) and N-methyl morpho-
line N-oxide (11.6 g, 86.3 mmol) was dissolved in DMSO (65 mL)
and heated to 45 C. 3-Methyl-3-(4-methylpent-3-enyl)cyclohexa-
none (6) (6.70 g, 34.5 mmol) was added at 45 C and the mixture
137.5, 141.5, 199.2; HRMS (ESIþ) m/z found 359.2377; C20
2
H36NaO Si
requires 359.2382.
ꢁ
ꢁ
4.1.1.5. Ethyl 2-{3-[(tert-butyldimethylsilyloxy) methyl]-6-methyl-
heated at that temperature for 18 h. On cooling the mixture was
extracted with petroleum ether (6ꢃ80 mL) and the combined ex-
tracts washed with brine (200 mL). The combined organic phase
6-[4-methylpent-3-enyl]-2-oxocyclohex-3-enyl}acetate (10) & (11).
To
a stirred solution of diisopropylamine (331 mg, 450 mL,
3.27 mmol) in anhydrous THF (30 mL) was added n-butyl lithium
ꢁ
was dried over MgSO
4
and the solvent was removed in vacuo to
(1.47 M in hexane, 2.23 mL, 3.27 mmol) at 0 C under an argon at-
give the title compound (5.17 g, 78%) as a pale yellow oil, which was
mosphere. The reaction mixture was stirred at this temperature for
ꢁ
carried to the next step without further purification. IR
n
max(ATR)
15 min. The solution was then cooled to ꢂ50 C, to which was
ꢂ1
2
964, 2916, 2874, 1678, 1384, 1247, 734 cm
;
d
H
(300 MHz, CDCl
3
)
added the protected alcohol (1.00 g, 2.98 mmol) dissolved in an-
ꢁ
1
.01 (3H, s, Me), 1.35 (2H, dd, J 9.8, 7.3 Hz, CCH
2
CH
2
), 1.57 (3H, br s,]
hydrous THF (2 mL) and the reaction mixture stirred at ꢂ50 C for
CMe), 1.65 (3H, br d, J 1.1 Hz,]CMe), 1.93 (2H, dt, J 15.0, 7.3 Hz,
CCH CH ), 2.16 (1H, dddd, J 18.8, 4.5, 1.9, 1.0 Hz, CCH CH]), 2.23
1H, d, J 15.9 Hz, COCH C), 2.31 (1H, ddd, J 18.8, 3.6, 2.4 Hz, m,
CH]), 2.33 (1H, d, J 15.9 Hz, COCH C), 5.01e5.08 (1H, m,
), 6.00 (1H, dt, J 10.0, 1.9 Hz, COCH]CH), 6.83 (1H, ddd, J
0.0, 4.5, 3.6, COCH]CH); (75 MHz, CDCl ) 17.6, 22.4, 24.8, 25.7,
6.6, 38.2, 41.6, 50.2, 124.0, 129.1, 131.8, 148.2, 199.9; GC/MS: m/z
30 min. A solution of ethyl iodoacetate (764 mg, 3.57 mmol) in
anhydrous THF (2 mL) was added and the mixture was stirred at
2
2
a b
H
ꢁ
(
a
H
b
ꢂ50 C for 20 min before allowing the mixture to warm to rt and
ꢁ
CCH
a
H
b
a
H
b
then heated at 45 C for 17 h. The reaction was quenched by
CH]CMe
2
pouring the reaction mixture into a saturated NH
(20 mL) containing ice. The mixture was extracted with Et
(3ꢃ30 mL) and washed with saturated NaHCO solution (40 mL),
distilled water (40 mL) and brine (40 mL). The combined organic
phase was dried over MgSO and the solvent removed in vacuo. The
4
Cl solution
1
3
d
C
3
2
O
3
þ
ꢄ
(%): 192 [M ] (16), 149 (25), 121 (35), 109 (100), 95 (22), 81 (41), 69
51), 55 (36), 41 (72).
(
4

6848
A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
residue was then purified by silica gel column chromatography
4.1.1.7. 7-[(tert-Butyldimethylsilyloxy)methyl]-4-methyl-4-(4-
methylpent-3-enyl)-2-oxo-2,3,3a,4,5,7a-hexahydrobenzofuran-7a-
(
Et
2
O/petroleum ether, 1:15) to give the titled compounds 10
ꢁ
ꢁ
(
468 mg, 37%, mp 54e55 C) and 11 (304 mg, 24%, mp 50e51 C) as
carbaldehyde (13). A mixture of 12 (104 mg, 0.203 mmol), CaCO
3
crystalline solids. Compound 10: IR
n
max(ATR) 2957, 2930, 2858,
(203 mg, 2.03 mmol) and mercury(II) perchlorate hydrate (203 mg,
0.448 mmol) in THF (4 mL) and dimineralized water (1 mL) was
stirred at rt for 5 min and quenched by adding saturated NaHCO
3
solution (4 mL). The mixture was filtered through Celite and
washed with ether (10 mL). The filtrant was washed with saturated
ꢂ1
1
0
737, 1661, 1169, 1119, 1099, 837, 775 cm
.04 (6H, s, SiMe
CH
.9 Hz,]CMe), 1.66 (3H, d, J 0.9 Hz,]CMe), 1.89e2.05 (2H, m,
CH ), 2.11e2.22 (1H, m, CCH CH]), 2.22 (1H, dd, J 16.1,
.2 Hz, COCH CH), 2.54e2.65 (1H, m, CCH CH]), 2.57 (1H, dd,
J 16.1, 9.7 Hz, COCH CH), 3.03 (1H dd, J 9.7, 3.2 Hz, COCH CHCO),
.14 (2H, qd, J 7.1, 1.4 Hz, OCH CH ), 4.20e4.45 (2H, m, CH OSi),
.00e5.11 (1H, m, CH]CMe ), 6.80 (1H, ddd, J 6.3, 4.4, 2.1 Hz,
(75 MHz, CDCl
) ꢂ5.5 (2C), 14.2, 17.6, 18.4, 20.1, 22.1,
5.7, 25.9 (3C), 28.9, 37.6, 39.5, 41.2, 52.1, 59.9, 60.5, 123.8, 132.0,
;
d
H
(300 MHz, CDCl
), 1.25 (3H, t, J
), 1.58 (3H, d, J
3
)
2
), 0.81 (3H, s, Me) 0.89 (9H, s, SiCMe
), 1.30e1.42 (2H, m, CCH CH
3
7.1 Hz, OCH
2
3
2
2
0
CCH
3
2
2
a
H
b
NaHCO
solvent removed in vacuo. The residue was then purified by silica
gel column chromatography (Et O/petroleum ether, 1:1) affording
3 4
solution (5 mL), brine (5 mL), dried over MgSO and the
a
H
b
a b
H
a
H
b
2
2
4
5
2
3
2
the aldehyde as a low melting off-white solid (60 mg, 73%). IR
ꢂ1
2
n
max(ATR) 2955, 2930, 2858, 1776, 1724, 1254, 1115, 835, 776 cm
(300 MHz, CDCl
s, Me), 0.85 (9H, s, SiCMe
1.29e1.45 (1H, m, CCH CH
CMe),1.90 (2H, q, J 7.9 Hz, CCH
CCH CH]), 2.11 (1H, dt, J 17.5, 2.5 Hz, CCH
(3H, m, COCH CH, COCH CH), 4.08 (1H, dd, J 12.2, 0.7 Hz, CH
OSi), 4.32 (1H, d, J 12.2 Hz, CH OSi), 5.02 (1H, m, CH]CMe
6.02e6.16 (1H, m, COCR]CH), 9.38 (1H, s, CHO); (100 MHz,
CDCl
) ꢂ5.7, ꢂ5.6, 17.6, 18.3, 19.6, 22.0, 25.6, 25.8 (3C), 29.4, 34.0,
35.0, 40.5, 43.4, 63.9, 87.3, 123.6, 129.5, 132.0, 132.2, 175.3, 195.5;
;
COCR]CH);
2
d
C
3
d
H
3
) 0.02 (3H, s, SiMe), 0.03 (3H, s, SiMe), 0.84 (3H,
), 1.13e1.28 (1H, m, CCH CH ),
), 1.57 (3H, s,]CMe), 1.65 (3H, s,]
CH ), 2.01 (1H, ddd, J 17.5, 5.5,1.3 Hz,
CH]), 2.33e2.58
H-
),
3
a
H
b
2
1
36.9, 140.1, 173.2, 199.5; HRMS (ESIþ) m/z found 445.2760;
a
H
b
2
C
2
24
H
42NaO
4
Si requires 445.2750. Compound 11: IR
n
max(ATR) 2960,
2
2
ꢂ1
930, 2887, 2857, 1736, 1673, 1174, 1116, 1102, 855, 835, 774 cm
(400 MHz, CDCl ) 0.03 (6H, s, SiMe
CH
), 1.24e1.33 (1H, m, CCH
;
a
H
b
a b
H
d
H
3
2
), 0.88 (9 Hs, SiCMe
3
),
2
2
a
1.00e1.09 (1H, m, CCH
a
H
b
2
), 1.05 (3H, s, Me), 1.23 (3H, t, J 7.1 Hz,
CH ), 1.52 (3H, s,]CMe), 1.61
CH ), 2.25 (1H, dd, J 16.0,
CH), 2.26 (1H, ddd, J 18.8, 6.2, 3.2 Hz, CCH CH]),
.44 (1H, dd, J 18.8, 5.7 Hz, CCH CH]), 2.63 (1H, dd, J 16.0, 9.3 Hz,
CH), 2.95 (1H, dd, J 9.3, 3.8 Hz, COCH CHCO), 4.12 (2H, qd, J
.1, 2.1 Hz, OCH CH ), 4.22e4.39 (2H, m, CH OSi), 4.91e4.99 (1H, m,
CH]CMe ), 6.71e6.78 (1H, m, COCR]CH); (100 MHz, CDCl
ꢂ5.5 (2C), 14.1, 17.5, 18.3, 22.7, 25.6, 25.8, 25.9 (3C), 29.0, 33.0, 36.7,
9.7, 55.3, 59.9, 60.5, 123.9, 131.7, 137.6, 139.6, 173.3, 199.2; HRMS
ESIþ) m/z found 445.2745; C24 42NaO Si requires 445.2750.
b
a
H
b
2
OCH
2
CH
3
a
H
b
2
d
C
(
3H, s,]CMe), 1.69e1.90 (2H, m, CCH
2
2
3
3
.8 Hz, COCH
a
H
b
a b
H
2
a
H
b
HRMS (ESIþ) m/z found 429.2423;
23 4
C H38NaO Si requires
COCH
7
a
H
b
2
429.2432.
2
3
2
2
d
C
3
)
4.1.1.8. (E)-7-[(tert-Butyldimethylsilyloxy)methyl]-4-methyl-4-(4-
methylpent-3-enyl)-7a-(3-oxobut-1-enyl)-3,3a,4,5-tetrahy-
drobenzofuran-2(7aH)-one (14). A mixture of the aldehyde (300 mg,
3
(
H
4
0.737 mmol)
500 mg,1.57 mmol) in anhydrous CHCl
The solvent was removed from the reaction mixture, and the
remaining material was purified by column chromatography (Et O/
and
1-(triphenylphosphoranylidene)
acetone
(
3
was heated at reflux for 5 d.
4
.1.1.6. 7-[(tert-Butyldimethylsilyloxy)methyl]-4-methyl-7a-(5-
methyl-1,3,5-dithiazinan-2-yl)-4-(4-methylpent-3-enyl)-3,3a,4,5-tet-
2
rahydrobenzofuran-2(7aH)-one (12). n-BuLi (1.49 M in hexane,
petroleum ether,1:1). The titled compound 14 was isolated (300 mg,
ꢁ
2
.50 mL, 3.73 mmol) was added dropwise to a solution of 5-
94%) as a slightly yellow liquid, which solidified (mp 80e82 C);
d
H
methyl-1,3,5-dithiazine (521 mg, 3.86 mmol) in anhydrous THF
(300 MHz, CDCl
0.89 (3H, s, Me), 1.18e1.31 (1H, m, CCH
CCH CH ), 1.56 (3H, s,]CMe), 1.66 (3H, d, J 0.9 Hz,]CMe),
1.83e1.98 (2H, m, CCH CH ), 2.02e2.11 (2H, m, CCH CH]), 2.24 (3H,
s, COMe), 2.31e2.61 (3H, m, COCH CH, COCH CH), 4.06 (1H, ddd, J
14.4, 2.0, 1.8, CH OSi), 4.17 (1H, ddd, J 14.4, 2.0, 1.8, CH OSi),
4.92e5.15 (1H, m, CH]CMe ), 5.98e6.19 (1H, m, COCR]CH), 6.35
(1H, d, J 15.8 Hz, COCH]CH), 6.64 (1H, d, J 15.8 Hz, COCH]CH);
(100 MHz, CDCl
3
) 0.02 (6H, d, J 0.7 Hz, SiMe
2
), 0.87 (9H, s, SiCMe
3
),
ꢁ
(
10 mL) at ꢂ78 C and warmed to rt over 1 h and cooled again to
a
H
b
CH
2
), 1.36e1.49 (1H, m,
ꢁ
ꢂ78 C. A solution of compound 10 (1.05 g, 2.49 mmol) in an-
a
H
b
2
ꢁ
hydrous THF (3 mL) was added at ꢂ78 C and stirred for 120 min
2
2
2
ꢁ
at ꢂ78 C. The reaction was quenched by pouring the reaction
2
2
mixture into a saturated NH
4
Cl solution (50 mL) containing ice.
a
H
b
a b
H
The mixture was extracted with Et
layer was washed with distilled water (25 mL) and brine (25 mL).
The combined organic phase was dried over MgSO and the
2
O (3ꢃ50 mL) and the organic
2
d
C
4
3
) ꢂ5.5, ꢂ5.4, 17.7, 18.3, 21.9, 22.0, 25.6, 25.9 (3C),
solvent removed in vacuo. Petroleum spirit was added and the
28.5, 30.5, 34.2, 34.5, 40.3, 48.4, 61.4, 84.7, 123.6, 124.8, 128.4, 132.2,
flask cooled in the freezer and a white precipitate was formed,
133.2, 146.3, 175.2, 197.0; HRMS (ESIþ) m/z found 469.2736;
which was isolated by filtration to give compound 12 (730 mg,
26 4
C H42NaO Si requires 469.2745.
ꢁ
5
7%) as a white solid (mp 160e162 C). [Found: C, 61.12; H, 9.17;
N, 2.74; S 12.53. C26
H
45NO
max(ATR) 2926, 2882, 2856, 1765, 1096, 836, 780,
(500 MHz, CDCl ) 0.07 (3H, s, SiMe), 0.08 (3H, s,
), 1.23 (1H, ddd, J 13.7,
), 1.36 (1H, ddd, J 13.7, 10.3, 6.2 Hz,
), 1.60 (3H, s, ]CMe), 1.67 (3H, s,]CMe), 1.88e2.05
4H, m, CCH CH , CCH CH]), 2.46 (1H, dd, J 18.9, 1.8 Hz,
COCH CH), 2.52 (3H, s, NMe), 2.83 (1H, dd, J 10.2, 1.8 Hz,
COCH CH), 3.04 (1H, dd, J 18.9, 10.2 Hz, COCH CH), 4.05 (1H,
dd, J 13.3, 1.4 Hz, CH OSi), 4.11 (1H, dd, J 13.3, 1.2 Hz, CH
OSi), 4.28 (1H, dq, J 14.0, 1.7HZ, S CH N) 4.41 (1H, dq, J 14.0,
.0HZ, S CH N), 4.59 (1H, d, J 13.5 Hz, S CH N), 4.62 (1H, d, J
CH N), 4.80 (1H, s, SCHS), 5.05e5.11 (1H, m, CH]
), 6.19e6.24 (1H, m, COCR]CH); (100 MHz, CDCl
) ꢂ5.3,
3
S
2
Si requires C, 61.01; H, 8.86; N, 2.74;
4.1.1.9. Methyl 2-[2-methoxy-2,8-dimethyl-8-(4-methylpent-3-
en-1-yl)-3,3a,5,7,8,9-hexahydro-2H-furo[3,2-c]isobenzofuran-9-yl]
S 12.53%]. IR
n
ꢂ1
5
58 cm
;
d
H
3
acetate 19 & 20. Compound 14 (100 mg, 0.224 mmol) was dis-
ꢁ
SiMe), 0.76 (3H, s, Me), 0.91 (9H, s, SiCMe
1.1, 6.4 Hz, CCH CH
CCH CH
3
solved in anhydrous MeOH (20 mL) and cooled to 0 C before
1
a
H
b
2
2 4
freshly opened concentrated (98%) H SO (59 mL) was slowly added
dropwise. The solution was sealed under argon and placed in a re-
frigerator overnight for 17 h. The reaction was quenched by pouring
a
H
b
2
(
2
2
2
H
a b
the reaction mixture into an ice cold saturated NaHCO
(50 mL). The mixture was extracted with CH Cl
(3ꢃ50 mL) and the
organic layer was washed with brine (50 mL), dried over MgSO
and the solvent removed in vacuo. The residue was then purified by
silica gel column chromatography (Et O/petroleum spirit, 1:5) to
give compounds 19 (47 mg, 55%) and 20 (15 mg, 18%) as colourless
3
solution
2
a
H
b
2
2
a
H
b
a
H
b
4
1
a b
H
2
2
a
H
b
1
a
H
b
2
13.5 Hz, S
2
a b
H
CMe
2
d
C
3
oils. Compound 19: IR
n
max(ATR) 2958, 2934, 2883, 1730, 1163, 1095,
ꢂ1
ꢂ5.2, 17.6, 18.5, 18.9, 22.0, 25.7, 26.0 (3C), 31.6, 35.1, 35.3, 37.4,
1.7, 45.7, 60.3, 60.9 (2C), 62.1, 87.4, 124.2, 127.9, 131.8, 133.4,
76.1; HRMS (ESIþ) m/z found 512.2695; C26 45NNaO Si re-
quires 512.2683.
1063, 1028, 969, 875, 841 cm
Me), 1.19e1.33 (2H, m, CCH
CMe), 1.65 (3H, d, J 0.7 Hz,]CMe), 1.81 (1H, dd, J¼14.5, 5.9 Hz,
OCHCH C), 1.87e1.94 (3H, m, CCH CH , CCH CH]), 2.20e2.26
;
d
H
3
(500 MHz, CDCl ) 0.95 (3H, s,
4
1
2
CH
2
), 1.45 (3H, s, OCMe), 1.56 (3H, s,]
H
3 2
S
a
H
b
2
2
a b
H

A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
6849
(
1H, m, CCH
dd, J 7.4, 3.3 Hz, COCH
.43 (1H, dd, J 17.1, 3.3 Hz, COCH
s, ester OMe), 4.10 (1H, dd, J 10.1, 0.9 Hz,]CCH
a
H
b
CH]), 2.23 (1H, d, J 14.5 Hz, OCHCH
CH), 2.36 (1H, dd, J 17.1, 7.4 Hz, COCH
CH), 3.19 (3H, s, OMe), 3.65 (3H,
O), 4.43 (1H, d, J
C), 4.59 (1H, ddt, J 10.1, 3.1, 3.1 Hz,]CCH O),
.01e5.06 (1H, m, CH]CMe ), 5.62e5.66 (1H, m, OCH C]CH);
) 17.6, 21.5, 22.1, 22.5, 25.7, 29.9, 35.1, 37.8, 41.6,
3.2, 47.3, 48.9, 51.8, 70.3, 87.8, 90.3, 108.8, 121.4, 124.4, 131.4, 138.0,
34NaO requires
max(ATR) 2953, 2924, 2856, 1737, 1375,
a
H
b
C), 2.25 (1H,
J 20.1, 2.1 Hz, COCH
(1H, dd, J 7.7, 2.1 Hz, COCH
dd, J 20.1, 7.7 Hz, COCH H
a b
a
H
b
CH), 2.28 (1H, d, J 14.7 Hz, OCHCH
a
H
b
C), 2.40
2
a
H
b
CH),
2
CH), 3.18 (3H, d, J 0.6 Hz, OMe), 3.28 (1H,
CH), 4.13 (1H, dd, J 10.7. 1.0 Hz,]
O), 4.49 (1H, d, J
), 5.19e5.23 (1H, m,
(101 MHz, C ) 17.6, 22.3,
2
a
H
b
a
H
b
CCH
4.7 Hz, OCHCH
OCH C]CH), 9.51e9.55 (1H, m, CHO);
a
H
b
O), 4.43 (1H, dt, J 10.7, 2.2 Hz,]CCH
a b
H
5
5
(
4
1
.9 Hz, OCHCH
2
a
H
b
2
C), 5.08e5.14 (1H, m, CH]CMe
2
2
2
d
C
2
d
C
6 6
D
101 MHz, CDCl
3
22.4, 23.9, 25.8, 35.0, 37.7 (2C), 40.1, 41.8, 47.4, 49.4, 69.9, 89.0, 91.5,
110.9, 120.2, 125.0, 131.2, 139.3, 201.1; HRMS (ESIþ) m/z found
74.4; HRMS (ESIþ) m/z found 401.2291; C22
H
5
4
371.2193; C21H32NaO requires 371.2193.
4
01.2298. Compound 20: IR
n
ꢂ1
1
1
1
1
173, 1116, 1090, 1028 cm
.17e1.37 (2H, m, CCH
.65 (3H, s,]CMe), 1.77e1.87 (2H, m, CCH
8.7, 4.4, 3.1, 1.7 Hz, CCH CH]), 1.93 (1H, dd, J 14.7, 4.8 Hz,
OCHCH C), 2.19e2.25 (1H, m, CCH CH]), 2.22 (1H, dd, J 7.3,
.5 Hz, COCH CH), 2.26 (1H, d, J 14.7 Hz, OCHCH C), 2.41 (1H, dd,
J 18.7, 2.5 Hz, COCH CH), 3.07 (1H, dd, J 18.7, 7.3 Hz, COCH CH),
.31 (3H, s, OMe), 3.66 (3H, s, ester OMe), 4.22 (1H, dd, J 11.0,
.7 Hz,]CCH O), 4.42 (1H, d, J 4.8 Hz, OCHCH C), 4.40e4.45 (1H,
O), 5.01e5.06 (1H, m, CH]CMe ), 5.60e5.63 (1H, m,
C]CH); (126 MHz, CDCl ) 17.5, 21.8, 22.1, 23.9, 25.7, 28.8,
5.3, 37.7, 41.1, 41.4, 47.3, 49.6, 51.8, 69.8, 88.7, 91.2, 110.6, 120.5,
24.4, 131.5, 138.5, 175.1; HRMS (ESIþ) m/z found 401.2304;
;
d
H
(500 MHz, CDCl
3
) 0.97 (3H, s, Me),
4.1.1.11. 4,5-bis-epi-Neovibsanin A (3). To aldehyde 21 (10.0 mg,
28.7 mol) dissolved in anhydrous toluene (0.6 mL) under an argon
atmosphere was added 4-(N,N-dimethylamino)pyridine (4.21 mg,
2
CH
2
), 1.50 (3H, s, OCMe), 1.55 (3H, s,]CMe),
CH ), 1.88 (1H, dddd, J
m
2
2
a
H
b
34.5
34.5
m
mol) and 3-methylbut-2-enoic anhydride (6.29 mg,
a
H
b
a
H
b
mmol). The mixture was heated under microwave irradiation
ꢁ
2
2
a
H
b
for 24 h (maximum temperature 110 C, 250 W). On cooling, Et
2
O
a
H
b
a
H
b
(5 mL) was added and the organic layer washed with saturated
NaHCO solution (5 mL). The aqueous phase was extracted with
Et O (5 mL), dried over Na SO and concentrated in vacuo. Column
chromatography (Et O/petroleum spirit, 1:4, with 1% triethyl-
3
0
3
a
H
b
2
2
2
4
m,]CCH
OCH
3
1
C
a
H
b
2
2
2
d
C
3
amine) provided 4,5-bis-epi-neovibsanin A (3) (3.7 mg, 30%) as
a mixture of unresolvable E/Z (5:1) diastereomers (colourless oil);
d
H
(900 MHz, C
6
D
6
) 1.22 (3H, s, Me), 1.17e1.37 (2H, m, CCH
2
CH
2
),
0
22
H34NaO
5
requires 401.2298.
1.32 (3H, s, OCMe), 1.35 (3H, d, J 1.0 Hz,]CMe ), 1.63 (3H, s,]CMe),
1
.67 (3H, s,]CMe), 1.77e1.80 (1H, m, CCH
10.9 Hz, CHCH]CHO), 1.84 (1H, dd, J 14.0, 5.9 Hz, OCHCH
1.86e1.90 (1H, m, CCH CH]), 1.90e1.98 (2H, m, CCH CH ), 2.03
(3H, d, J 1.0 Hz,]CMe ), 2.44 (1H, d, J 14.0 Hz, OCHCH C), 3.14
(3H, s, OMe), 4.23 (1H, d, J 10.0 Hz,]CCH O), 4.31 (1H, d, J 5.8 Hz,
OCHCH C), 4.89 (1H, ddd, J 10.0, 4.9, 3.0 Hz,]CCH O), 5.17e5.20
(1H, m, CH]CMe ), 5.33e5.34 (1H, m, OCH C]CH), 5.64 (1H, dt, J
2.6, 1.3 Hz, OCOCH]CMe ), 5.70 (1H, dd, J 12.7, 10.9 Hz, CHCH]
CHO), 7.36 (1H, d, J 12.7 Hz, CHCH]CHO); (226 MHz, C ) 17.7,
a
H
b
CH]), 1.82 (1H, d, J
4.1.1.10. 2-[2-Methoxy-2,8-dimethyl-8-(4-methylpent-3-en-1-yl)-
a b
H
C),
3
,3a,5,7,8,9-hexahydro-2H-furo[3,2-c]isobenzofuran-9-yl]acetalde-
a
0
H
b
2
2
hyde 21 & 22. To a solution of compounds 19 and 20 (118 mg,
.312 mmol) in anhydrous Et O (10 mL) at rt was added LiAlH
35.5 mg, 0.936 mmol) in one portion and stirred under an argon
a b
H
0
2
4
a b
H
(
2
a b
H
atmosphere for 10 min. Sodium sulfate dodecahydrate (300 mg)
was added to the reaction until the suspension turned white in
2
2
2
colour. The slurry was then filtered and washed with Et
The filtrate was then concentrated in vacuo to give a pale yellow oil
104 mg, 95%). The crude was considered sufficiently pure to be
used in the next step without further purification. The above crude
was stirred at rt in a solution of anhydrous CH Cl and pyridine (1:1,
0 mL) under an argon atmosphere and DesseMartin periodinane
212 mg, 0.50 mmol), dissolved in CH Cl and pyridine (1:1, 10 mL),
was added in one portion. The reaction was stirred until completion
TLC, w4 h), then filtered through a plug of cotton wool and silica,
and washed with CH Cl (20 mL). The filtrate was then concen-
trated in vacuo and then diluted with Et O (20 mL) and washed
with brine (20 mL), dried over MgSO and the solvent removed in
vacuo. The residue was then purified by silica gel column chro-
matography (Et O/petroleum spirit, 1:5) to give compounds 21
46 mg, 42%) and 22 (13 mg, 12%) as colourless oils. Compound 21:
2
O (30 mL).
d
C
6 6
D
20.2, 21.4, 22.0, 22.6, 25.8, 27.0, 34.9, 38.2, 42.8, 47.6, 48.3, 48.7, 71.0,
89.2, 90.9, 109.4, 111.5, 115.2, 120.7, 125.5, 130.8, 137.1, 139.0, 159.6,
(
163.2; HRMS (ESIþ) m/z found 453.2620; C26
5
H38NaO requires
2
2
453.2611.
1
(
2
2
4.1.1.12. 4,5-bis-epi-neovibsanin B (4). Aldehyde 22 was sub-
jected to the same conditions as above to provide 4,5-bis-epi-
neovibsanin B (4) in 14% yield as a mixture of E/Z (3:2) di-
astereomers (colourless oil). Further careful chromatography gave
(
2
2
2
a 10:1 E/Z mixture that was used for characterisation. IR
n
max(ATR)
ꢂ1
4
1731, 1643, 1378, 1132, 1078, 955, 839 cm
H 6 6
; d (900 MHz, C D )
1.23 (1H, ddd, J 13.6, 11.8, 4.7 Hz, CCH
(3H, d, J 1.2 Hz,]CMe ), 1.41 (1H, ddd, J 13.6, 11.8, 4.7 Hz,
a
H
b
CH
2
), 1.24 (3H, s, Me), 1.33
0
2
(
CCH
a
H
b
CH
2
), 1.49 (3H, s, OCMe), 1.65 (3H, s,]CMe), 1.67 (3H, s,]
CH]), 1.81 (1H, d, J 11.2 Hz, CHCH]
CH]), 1.95e2.01 (2H, m, CCH CH ),
C),
C), 3.28 (3H, s, OMe), 4.08 (1H,
C), 4.20 (1H, dd, J 11.0, 1.1 Hz,]CCH O), 4.53
(1H, ddd, J 11.0, 5.5, 3.4 Hz, ]CCH O), 5.20e5.23 (1H, m, CH]
C]CH), 5.57e5.58 (1H, m, OCOCH]
), 6.02 (1H, dd, J 12.5, 11.2 Hz, CHCH]CHO), 7.42 (1H, d, J
(226 MHz, C ) 17.7, 20.2, 20.9, 22.6,
IR
n
max(ATR) 2934, 1722, 1379, 1222, 1139, 1096, 1065, 1025, 982,
CMe), 1.74e1.78 (1H, m, CCH H
a b
ꢂ1
8
78, 845 cm
;
d
H
(500 MHz, C
), 1.24 (3H, s, OCMe), 1.56 (1H, dd, J 14.4, 6.3 Hz,
C), 1.57 (3H, s,]CMe), 1.64e1.70 (1H, m, CCH CH]),
.69 (3H, s,]CMe), 1.73e1.87 (2H, m, CCH CH ), 1.87e1.93 (1H, m,
CCH CH]), 2.14e2.19 (3H, m, COCH CH, COCH CH), 2.37 (1H, d, J
4.4 Hz, OCHCH C), 3.09 (3H, s, OMe), 4.15 (1H, d, J 10.0 Hz,]
CCH O), 4.40 (1H, d, J 6.3 Hz, OCHCH C), 4.84 (1H, J 10.0, 2.9,
O), 5.09e5.14 (1H, m, CH]CMe ), 5.28e5.32 (1H,
C]CH), 9.35e9.36 (1H, m, CHO); (101 MHz, C ) 17.7,
6
D
6
) 1.01 (3H, s, Me), 1.11 (2H, dd, J
CHO),1.83e1.87 (1H, m, CCH
2.03 (3H, d, J 1.1 Hz,]CMe ), 2.30 (1H, d, J 14.0 Hz, OCHCH
a
H
b
2
2
0
9
.1, 7.9 Hz, CCH
2
CH
2
a
H
b
OCHCH
1
a
H
b
a
H
b
2.37 (1H, dd, J 14.0, 4.6 Hz, OCHCH
d, J 4.6 Hz, OCHCH
a b
H
2
2
2
a b
H
a
H
b
2
2
a b
H
1
a
H
b
CMe
CMe
2
), 5.23e5.25 (1H, m, OCH
2
a
H
b
2
2
1.9 Hz,]CCH
a
H
b
2
12.5 Hz, CHCH]CHO);
d
C
6 6
D
m, OCH
2
d
C
6
D
6
24.1, 25.8, 26.9, 35.1, 38.2, 42.8, 47.3, 47.6, 49.5, 70.5, 90.0, 90.6,
110.9, 112.2, 115.6, 119.9, 125.6, 130.8, 136.8, 139.2, 158.9, 163.3;
2
9
1.5, 22.5, 22.7, 25.8, 35.1, 38.0, 40.8, 40.9, 42.1, 47.6, 48.6, 70.6, 88.3,
0.7, 108.9, 121.0, 125.0, 131.2, 138.9, 200.6; HRMS (ESIþ) m/z found
5
HRMS (ESIþ) m/z found 453.2619; C26H38NaO requires 453.2611.
371.2192; C21
H32NaO
4
requires 371.2193. Compound 22: IR
n
max(-
ꢂ1
ATR) 2960, 2922,1719,1463,1380,1261,1226,1091,1024, 800 cm
(400 MHz, C
CCH CH ),1.18 (1H, ddd, J 13.8,11.9, 5.5 Hz, CCH
s, OCMe), 1.55 (3H, s,]CMe), 1.56e1.62 (1H, m, CCH
3H, s,]CMe), 1.73e1.88 (2H, m, CCH CH ), 1.88e1.94 (1H, m,
CCH CH]), 1.91 (1H, dd, J 14.7, 4.7 Hz, OCHCH C), 2.22 (1H, dd,
;
4.2. Neurite outgrowth screening method
d
H
6
D
6
) 1.02 (3H, s, Me),1.11 (1H, ddd, J 13.8,11.9, 5.3 Hz,
CH ),1.44 (3H,
CH]), 1.69
a
H
b
2
a
H
b
2
PC12 cells were plated (2 coverslips per condition) at a density
5
a
H
b
of 0.5ꢃ10 cells/well on 0.0015% poly-
L-ornithine (Sigma, USA)
(
2
2
coated glass coverslips in 24 well plates and cultured in growth
medium [DMEM; 10% normal horse serum, 5% foetal calf serum,
a
H
b
a b
H

6850
A.P.-J. Chen et al. / Tetrahedron 66 (2010) 6842e6850
penicillin (50 U/mL), streptomycin sulfate (50 mg/mL)]. Twenty-
four hours after plating, growth medium was replaced with dif-
ferentiation medium [DMEM; 1.5% normal horse serum, penicillin
Supplementary data
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2010.06.056
(
50 U/mL), streptomycin sulfate (50 mg/mL)] containing test com-
pounds (40 M) and NGF (20 ng/mL) (Millipore, USA). Compounds
were dissolved in DMSO (Sigma) to a stock concentration of 3 mM
and diluted to a final concentration of 40 M (1.33% DMSO) in
m
References and notes
m
culture medium. After a further 48 h, medium was replaced with
fresh differentiation medium containing test compounds and NGF
at the above concentrations. At indicated times, cells were fixed in
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Acknowledgements
We thank the University of Queensland and the Australian Re-
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