Neuronal growth promoting sesquiterpene-neolignans; Syntheses and biological studies

Article abstract of DOI:10.1039/c1ob06363d

The use of small molecules that can promote neuronal growth represents a promising approach to regenerative science. Along these lines we have developed separate short or modular syntheses of the natural products caryolanemagnolol and clovanemagnolol, sma

Full text of DOI:10.1039/c1ob06363d

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PAPER
Neuronal growth promoting sesquiterpene–neolignans; syntheses and
biological studies
Xu Cheng,a Nicole Harzdorf,a Zin Khaing,b Danby Kang,a Andrew M. Camelio,a Travis Shaw,a
Christine E. Schmidtb and Dionicio Siegel*a
Received 10th August 2011, Accepted 26th September 2011
DOI: 10.1039/c1ob06363d
The use of small molecules that can promote neuronal growth represents a promising approach to
regenerative science. Along these lines we have developed separate short or modular syntheses of the
natural products caryolanemagnolol and clovanemagnolol, small molecules previously shown to
promote neuronal growth and induce choline acetyltransferase activity. The postulated biosynthetic
pathways, potentially leading to the assembly of these molecules in nature, have guided the laboratory
syntheses, allowing the preparation of both natural products in as few as two steps. With synthetic
access to the compounds as single enantiomers we have examined clovanemagnolol’s ability to promote
the growth of embryonic hippocampal and cortical neurons. Clovanemagnolol has been shown to be a
potent neurotrophic agent, promoting neuronal growth at concentrations of 10 nM.
The lack of regeneration in the central nervous system (CNS) has
been attributed to two factors; the inhibitory extrinsic environment
formed after injury and the absence of intrinsic growth signals.
In the 1980’s Aguayo and coworkers showed that some axons,
unable to regrow in their native CNS environment, have the ability
to grow and extend into transplanted peripheral nervous system
(PNS) nerve graphs.1 This finding led to 30 years of research to
uncover the basis of the growth inhibitory environment of the
CNS.2 Unfortunately, efforts to promote regeneration through the
removal of these inhibitory signals have produced only limited
results. However, regeneration in the CNS is possible, passing
through the inhibitory glial environment, if appropriate signaling
pathways are first activated. This was discovered in dorsal root
ganglion (DRG) neurons, which have projections into both the
PNS and CNS. If the peripheral branches of DRG neurons are
cleaved, providing a “conditioned lesion”, followed by severing
the CNS branch, regeneration can occur in the CNS portion
through a series of transcriptional events that enhance the intrinsic
growth potential.3 The identification of these and other growth
promoting pathways, particularly ones that can be controlled
by small molecules, could provide significant advancements to
regenerative medicine. The recent development of a variety of
hydrogels and other systems for continuous delivery, if coupled
with a validated small molecule, would provide a unique and
promising platform for therapeutic development.4
pharmacologically appealing strategy of using small molecules,
however, has received considerably less attention.5 This follows
from a poor understanding of how small molecules, traditionally
used as inhibitors, can result in a gain of biological function.
In an effort to identify small molecules with neurotrophic activity
the sesquiterpene–neolignan caryolanemagnolol was isolated from
the bark of Magnolia obovata in yields ranging from 0.0035% to
0.00022% (Fig. 1).6 The highly lipophilic molecule was identified
with seven other structurally related compounds in a screen for
compounds with neurotrophic activity. Treatment of primary
neuronal cell cultures with caryolanemagnolol leads to increased
neurite outgrowth relative to controls, with pronounced growth
at 0.1 mM, the lowest concentration reported.6a Treatment of
cultured neurons at the same concentration resulted in choline
acetyltransferase (ChAT) activity being increased by 163% relative
to control cultures.6a Similarly, an isomeric natural product
The majority of approaches toward inducing CNS regeneration
focus on the use of gene therapy, growth factors, and stem cells. The
aChemistry and Biochemistry, The University of Texas at Austin, 1 University
Station, A5300 Austin, Texas, 78712, United States
bBiomedical Engineering, The University of Texas at Austin, Austin, Texas,
United States
Fig. 1 Caryolanemagnolol and clovanemagnolol.
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Fig. 2 The proposed biosynthesis of clovanemagnolol.
clovanemagnolol was shown to have related activity, promoting
neurite growth at 0.1 mM and ChAT induction at 1 mM.6a
formation followed by base had failed, instead generating allylic
halides through an ene-type process. This unexpected reaction is
likely due to the lack of an appropriate pathway for the attack of
water, opening the bromonium intermediate, from the inside of the
nine-membered ring. Of the reagents examined, the Shi catalyst
10 proved successful in overriding the inherent bias of the system,
providing epoxide 4 as the major product in a 2.2 : 1 ratio of a
to b.9
In a single reaction we were able to convert epoxides 4 and
5 to caryolanemagnolol and clovanemagnolol, respectively, using
diphenyl phosphate and magnolol (8) in dichloromethane at 23 ◦C
(Fig. 4). Significant optimization of the acid component was
necessary as a number of other Brønsted and Lewis acids failed
to induce the desired reaction. Acid mediated rearrangement of
epoxide 4 with diphenyl phosphate and trapping with magnolol
led to the formation of caryolanemagnolol (1) in 15% yield. The
major by-product in this reaction was competitive trapping of the
cation with diphenyl phosphate. Analogously, the rearrangement
of epoxide 5 in the presence of diphenyl phosphate generated
clovanemagnolol in 10% yield. Elimination proceeding the trap-
ping of the cation proved to be the major competitive pathway,
generating clovene as the major by-product. While low yielding
we have found these single-step transformations starting from the
corresponding epoxides have proven to scale well providing the
natural products on the multigram scale. In addition, the reaction
can be run on the diastereomeric mixture of epoxides, generating
both natural products.
To examine the initially reported growth promoting activities of
caryolanemagnolol and clovanemagnolol and to prepare biologi-
cally optimized analogs we have developed two syntheses for each
of the natural products, allowing either the large scale synthesis
or the preparation of analogs.7 The proposed biosyntheses have
provided insight into the development of short, flexible laboratory
syntheses of both compounds commencing from the readily
accessible starting material, (-)-caryophyllene, a plant-derived
commodity chemical used in the flavor and fragrance industry.
The proposed biosynthetic pathway is initiated by the oxida-
tion of (-)-caryophyllene, providing diastereomeric epoxides, a-
caryophyllene oxide (4) or b-caryophyllene oxide (5) (Fig. 2).
Brønsted activation of the epoxides 4 or 5 leads to an intramolec-
ular attack of the exocyclic alkene, generating diastereomeric
tricyclic intermediates 6 and 7 bearing bridgehead carbocations.
In the caryolanemagnolol sequence the cation 6 is trapped
by magnolol (8), directly forming the natural product. In the
clovanemagnolol sequence the cation 7 has correctly aligned
orbitals to enable a ring expansion of the adjacent cyclobutane
ring, thus releasing ring strain, forming the cationic tricycle
9. This secondary cation is subsequently trapped by magnolol
(8), forming clovanemagnolol (2). In their work elucidating the
structure of caryophyllene, Barton and coworkers were the first to
describe these rearrangements as well as the generation of clovane
and caryolane based structures from the parent caryophyllene
compounds.8
In addition to the single step reactions, multi-step syntheses to
both molecules were developed to access analogs. In an optimized
procedure the rearrangement of a-epoxide 4 was achieved using
an in situ aluminum phenoxide reagent generated through the
combination of 4-bromophenol and trimethylaluminum in a 3 : 1
ratio in dichloromethane at 0 ◦C.10 This reagent, when combined
with 4, provided the carbocyclic core of caryolanemagnolol in a
single operation, yielding bromide 11 in 69% yield (Scheme 1). In
In the laboratory setting, the syntheses of caryolanemagnolol
and clovanemagnolol were initiated with diastereoselective epox-
idation reactions. The system proved to have a relatively strong
preference for the formation of the b-epoxide 5 as epoxidation of
(-)-caryophyllene with mCPBA generated epoxides 4 and 5 in a
1 : 5 ratio, favoring the b-epoxide 5 (Fig. 3). Attempts to change the
diastereoselectivity of the epoxidation reaction using halohydrin
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Fig. 3 The diastereoselective epoxidation of (-)-caryophyllene 3.
Fig. 4 Single step syntheses of caryolanemagnolol and clovanemagnolol following from the proposed biosynthetic pathway.
addition to NMR, the structure of bromide 11 was confirmed
by X-ray diffraction of the 3,5-dinitrobenzoyl derivative (Fig.
5). Due to the inability of 2,4-dibromophenol or any ortho-
substituted phenols to participate in the reaction the bromination
of 11 was required. Preliminary attempts at bromination were
competitive with oxidation of the secondary alcohol, forming the
corresponding ketone of 11, therefore, protection of the secondary
alcohol was required. An acetate was appended under standard
conditions followed by the installation of a second bromide using
bromine and sodium acetate at elevated temperature (60 ◦C) in
acetic acid, generating dibromide 12 in 77% yield over the two steps
(Scheme 1). With the bromine atoms installed, Suzuki reactions
were used to sequentially form the allyl appendage and the biaryl
bond.11 In the majority of allylation reactions examined, Suzuki
cross-coupling led to rapid bisallylation of 14. However, using
pinacol allylboronate 13, tetrakis(triphenylphosphine)palladium
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Scheme 1 The multistep synthesis of caryolanemagnolol (1).
steric and electronic factors. A second Suzuki reaction between
monobromide 14 and pinacol ester 15 using conditions devel-
oped by Fu and coworkers formed the hindered biaryl bond,
generating 16 in 71% yield (Scheme 1).12 The removal of the
acetate and carbamoyl groups was simultaneously achieved with
lithium aluminum hydride in THF heated to reflux, providing
caryolanemagnolol (1) in 71% yield. While the synthetic material
matched the spectra for isolated caryolanemagnolol, we found that
the original isolation report incorrectly stated the chemical shifts
for caryolanemagnolol.6a,7
Fig. 5 The X-ray crystal structure of the 3,5-dinitrobenzoyl derivative of
bromide 11.
The synthesis of clovanemagnolol was achieved over six-steps
using similar transformations to those used for the synthesis
of caryolanemagnolol (Scheme 2). Unfortunately, the aluminum
phenoxide reagent used in the first step of the caryolanemagnolol
catalysis, and close monitoring of the reaction by TLC compound
14 could be isolated in 72% yield. Selective cross-coupling of
the para-bromide over the ortho can be attributed to both
Scheme 2 The multistep synthesis of clovanemagnolol (2).
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Table 1 Neurite outgrowth of embryonic hippocampal neurons after
treatment with magnolol and clovanemagnolol
Table 2 Neurite outgrowth of embryonic cortical neurons after treatment
with magnolol and clovanemagnolol
Average Neurite Length
per Neuron (mm)
Percent Change
from Control
Average Neurite Length
per Neuron (mm)
Percent Change
from Control
Treatment
Treatment
Control
DMSO
176.6 27.0
168.7 21.5
265.1 67.6
218.8 40.7
54.7 17.3
287.6 82.8
223.4 49.9
28.5 10.9
—
Control
DMSO
352.7 54.4
347.2 82.8
424.1 24.2
548.8 124.0
57.8 27.4
508.5 87.0
432.9 64.5
68.2 22.2
—
-4.5
50.1
23.9
-69.0*
62.8
26.5
-83.9
-1.6
20.2
55.6
-74.6*
44.2
22.7
-80.7*
Mag (0.01 mM)
Mag (0.1 mM)
Mag (1.0 mM)
CM (0.01 mM)
CM (0.1 mM)
CM (1.0 mM)
Mag. (0.01 mM)
Mag. (0.1 mM)
Mag. (1.0 mM)
CM (0.01 mM)
CM (0.1 mM)
CM (1.0 mM)
sequence failed to convert epoxide 5 to the carbocyclic clovane
core. Therefore, starting from the recrystallized caryophyllene b-
oxide (5) the rearrangement was achieved using diphenyl phos-
phate and 4-bromophenol, providing the clovane core structure
17 in 35% yield with clovene as a major by-product. Conversion
of bromide 17 to clovanemagnolol was achieved in 28% yield
over the five steps using the same set of transformations as the
caryolanemagnolol approach. The spectral data of isolated and
synthetic clovanemagnolol fully matched.
With access to clovanemagnolol we have been able to test
and quantify the small molecule’s effects on different neuronal
types. In preliminary studies we found that while clovanemagnolol
consistently promoted outgrowth, caryolanemagnolol appeared
to induce neuronal pruning in embryonic hippocampal neurons.
This effect will be reported in due course. We therefore studied
the small molecule-mediated outgrowth using clovanemagnolol.13
For comparison, we measured the effects of clovanemagnolol
versus the effects to the related and more extensively studied
compound magnolol (8).14 In both hippocampal and cortical
neurons we found clovanemagnolol to be more effective at
promoting neuronal growth (Fig. 6 and 7.). Interestingly, both
compounds demonstrated biphasic dose profiles.
clovanemagnolol’s effects on growth were shown to be greater
than magnolol. After 2 days in vitro we found that treatment with
magnolol resulted in enhanced neurite outgrowth at both 0.01 and
0.1 mM concentrations, although only treatment at 0.1 mM reached
statistical significance (p < 0.03). Treatment with clovanemagnolol
resulted in a significant increase in neurite outgrowth at 0.01 mM
(p < 0.01), whereas treatment at 0.1 mM resulted in a slight
increase in average neurite length (p > 0.05) (Table 2). At higher
concentration (1 mM), the addition of both compounds resulted
in significant reduction for both magnolol and clovanemagnolol
treated cells (p < 0.001) compared to neurons in the control
culture. Therefore, similar to hippocampal neurons, we found that
treatment with either magnolol or clovanemagnolol has neurite
outgrowth enhancing properties for embryonic cortical neurons,
and this effect is concentration dependent.
Cell culture and isolation of primary cells
Primary neuronal cells were obtained from E16–E18 mouse em-
bryos. Pregnant mice were sacrificed using isoflurane asphyxiation
and embryos were collected in 1x Hank’s buffered salt solution
(HBSS) with gentamycin. After removing the meninges, cortical
tissue or hippocampi were dissected away from the surrounding
tissue. Samples were treated with 0.25% Trypsin/EDTA (Sigma–
Aldrich) for 20 min and were mechanically dissociated using fire
polished Pasteur pipettes. Cells were plated on 12 mm round
glass coverslips coated with Poly-D-Lysine (20 mg ml-1) at a
density of 30 000 cells per coverslip in a DMEM/F12 media
supplemented with N2 in the absence of serum. All cultures
were grown at standard condition with 37 ◦C in 5% CO2. Cells
were fed every 3 days by replacing half of the media with fresh
medium.
Effects of clovanemagnolol and magnolol on hippocampal neurons
in vitro is concentration dependent
Dissociated embryonic hippocampal neurons were cultured in
vitro in the presence of control, DMSO, or varying amounts
of magnolol or clovanemagnolol (Fig. 6). After 2 days in vitro,
we found that treatment with either magnolol (0.01 mM; p <
0.02) or clovanemagnolol (0.01 mM; p < 0.03) was able to
significantly enhance the neurite outgrowth of neurons compared
to control cultures. At 0.1 mM concentration, the addition of either
molecule resulted in a modest enhancement in neurite outgrowth.
At micromolar concentrations, treatment with either compound
resulted in a significant decrease in the average neurite outgrowth
(69.45 and 84.08% reduction for magnolol and clovanemagnolol,
respectively). Therefore, we found that both magnolol and clo-
vanemagnolol have neurite outgrowth enhancing properties in
embryonic hippocampal neurons in a concentration dependent
manner (Table 1).12
Immunostaining
After 2–4 days in culture, cells were fixed with 2% paraformalde-
hyde at room temperature for 20 min, and incubated in blocking
buffer (3% normal goat serum + 0.3% Triton X-100 in PBS)
for 1 h at room temperature. Primary antibody staining against
betaIII tubulin (1 : 1000, Abcam) was used to visualize neurons.
Samples were incubated with primary antibody in blocking
buffer overnight at 4 ◦C. Samples were washed with PBS and
incubated with goat anti-mouse IgG conjugated to AlexaFluor 488
(Invitrogen). Samples were counter stained with DAPI (1 : 1000,
Sigma–Aldrich) to visualize all nuclei, and mounted onto glass
slides using Fluoromount G (SouthernBiotech, Birmingham,
Effects of clovanemagnolol and magnolol on cortical neurons in
vitro is concentration dependent
Dissociated embryonic cortical neurons were treated with either
magnolol or clovanemagnolol (Fig. 7). At lower concentrations
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Fig. 6 Embryonic hippocampal neurons showed enhanced neurite outgrowth after treatment with magnolol (Mag) and clovanemagnolol (CM).
Dissociated E18 mouse hippocampal neurons were cultured in control (CTR, medium only), DMSO (medium + DMSO), magnolol (0.01–1.0 mM), or
clovanemagnolol (0.01–1.0 mM). After 2 days in vitro, 0.01 mM concentration magnolol can significantly increase the neurite length (48%, p < 0.02).
Treatment with clovanemagnolol also resulted in significant increase in neurite outgrowth at 0.01 mM (63%, p < 0.03). However, treatment with higher
concentration of either compound resulted in fewer numbers of neurons, bearing significantly shorter neurites for both.12
Fig. 7 Embryonic cortical neurons showed enhanced neurite outgrowth after treatment with magnolol (Mag) and clovanemagnolol (CM). Dissociated
E18 mouse hippocampal neurons were cultured in control (CTR, medium only), DMSO (medium + DMSO), magnolol (0.01–1.0 mM), or clovanemagnolol
(0.01–1.0 mM). After 2 days in vitro, 0.1 mM concentration magnolol can significantly increase the neurite length (56%, p < 0.03). Treatment with
clovanemagnolol also resulted in significant increase in neurite outgrowth at 0.01 mM (44%, p < 0.01). However, treatment with higher concentration of
either compound resulted in fewer numbers of neurons, bearing significantly shorter neurites.12
Alabama). Three to five random areas (covering 30% of the total
surface area of the 12 mm glass coverslip) were recorded using
an Olympus IX70 (Olympus America, Melville, NY) inverted
microscope and the images were captured with an Optronics
MagnaFire (Goleta, CA) digital color camera. Acquired images
were analyzed using ImageJ, and composite figures were produced
using Adobe Photoshop CS4.
Analysis of neurite outgrowth
To determine the average neurite length, samples were imaged
at 3–5 random areas (covering over 30% of the glass coverslip).
Using the ImageJ Plug-in “Neurite Tracer” program automated
quantification of neurite growth was determined as previously
described.15
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aqueous solution) and K2CO3 aqueous solution (9.3 g, 67.3 mmol,
6.73 equiv. in water (65 mL) in separate syringes over 1.5 h using
a syringe pump. After complete addition, the reaction mixture
was extracted with hexane (100 mL ¥ 3). The combined organic
extracts were washed with brine (50 mL), dried over Na2SO4,
filtered and concentrated. The crude oil was purified by silica
gel chromatography (hexane/EtOAc = 95 : 5 as eluent) to give a
light yellow oil, 1.64 g, yield 74%. TLC Rf 0.24, Hex/EA = 95 : 5.
1H NMR showed a 2.2 : 1 diastereomeric ratio of epoxides 4 and
5, respectively, with epoxide 4 as the major isomer. NMR data
matches that reported.16
To a solution of (b)-carophyllene (5.6 g (90% purity), 24.7 mmol,
1 equiv.) in CH2Cl2 (30 mL) under argon at 0 ◦C was added
m-CPBA (4.51 g, 25.9 mmol, 1.05 equiv.) in CH2Cl2 (30 mL)
over 30 min via syringe pump, the reaction mixture became
heterogeneous after 15 min. After stirring for an additional hour
at 0 ◦C the mixture was filtered through cotton. The filtrate
was concentrated, diluted with hexane (40 mL), and washed
with 1 N NaOH (30 mL ¥ 2). The organic extract was dried
over Na2SO4, filtered, and concentrated. Purification by silica gel
chromatography (hexane/EtOAc = 95 : 5 as eluent) provided a
colorless oil (4.5 g, 83%). TLC Rf 0.24, Hex/EA = 95 : 5. 1H NMR
showed a 1 : 5 diastereomeric ratio of epoxides 4 and 5, respectively.
NMR data matches that reported.16
Conclusion
The previous data for clovanemagnolol in primary neuronal
cultures had reported the growth effects at 100 nM.6 In this
study we have shown that clovanemagnolol, in primary neuronal
culture, demonstrates a biphasic dose response an shows activity
at the lower concentration than previously reported, 10 nM.
This effect was seen in both hippocampal and cortical neurons.
Magnolol showed a similar effect in cortical but not hippocampal
neurons. These interesting biological effects warrant further study
to determine the mechanism of action. This biphasic activity leads
us to hypothesize that clovanemagnolol is functioning by inducing
the dimerization of cell surface receptors similar to proteinaceous
growth factors. These studies will be reported in due course.
Experimental
General
All reactions were performed in flame dried round bottom or
modified Schlenk (Kjeldahl shape) flasks fitted with rubber septa
under a positive pressure of argon, unless otherwise indicated.
Air- and moisture-sensitive liquids and solutions were transferred
by syringe or canula. Where necessary (so noted), solutions were
deoxygentated by alternative freeze (liquid N2)/evacuation/thaw
cycles (≥three iterations). Organic solutions were concentrated
by rotary evaporation at ~20 Torr. Analytical thin-layer chro-
matography (TLC) was carried out using 0.2 mm commercial
silica gel plates (silica gel 60, F254, EMD chemical). Thin layer
chromatography plates were visualized by exposure to ultraviolet
light and/or exposure to an acidic solution of p-anisaldehyde or
ceric ammonium molybdate solution followed by heating on a hot
plate. Dichloromethane, tetrahydrofuran, toluene, and benzene
were purified using a Pure-Solv MD-5 Solvent Purification System
(Innovative Technology). All commercial reagents were used di-
rectly, without further purification unless otherwise notes. Nuclear
magnetic resonance spectra (1H NMR and 13C NMR) were
recorded with a Varian Gemini (400 MHz) or Varian (300 MHz)
spectrometer. Chemical shifts are reported as parts per million
(ppm) downfield of tetramethylsilane and referenced relative to
residual protium in NMR solvents (CHCl3: d 7.26 ppm, C6D5H: d
7.15 ppm: D2HCOD: d 3.31 ppm). Chemical shifts for carbon are
reported in ppm downfield of tetramethylsilane and are referenced
to the carbon resonances of the solvent (CDCl3 d 77.0, C6D6
d 128.0, D3COD: d 44.9). Infrared spectra (IR) were recorded
on a Nicolet 380 FTIR using thin film (neat). Optical rotations
were measured on an ATAGO AP-300 automatic polarimeter
with a path length of 100 mm. Melting points were measured on
a MEL-TEMP device without correction. High-resolution mass
spectra (HRMS) were obtained on a Karatos MS9 at the UT Mass
Spectrometry Facility.
Caryolanemagnolol (1)
To a solution of (b)-caryophyllene a-oxide 4 (40 mg, 2.2 : 1 ratio of
a-oxide 4 to b-oxide 5, 0.125 mmol of 4, 1 equiv. of 4) and magnolol
(8) (49.3 mg, 0.185 mmol, 1.5 equiv.) in dichloromethane (2 mL)
at 23 ◦C was added a solution of diphenylphosphate (15.4 mg,
0.062 mmol, 0.5 equiv.) in dichloromethane (1 mL) over 3 min.
Then the orange solution was stirred at 38 ◦C for 2.5 h the
solvent was removed and the residue was diluted with hexane
(50 mL). The hexane solution was washed with phosphate buffer
(pH 7.4, 2 ¥ 10 mL) and 1 N NaOH (3 ¥ 10 mL (to recover the
magnolol)). The organic layer was washed with brine (10 mL)
and dried over Na2SO4. The solution was concentrated and the
residue purified by silica gel chromatography (hexane/EtOAc =
85 : 15 as eluent) to yield a mixture of caryolanemagnolol 1 and
clovanemagnolol 2. The mixture was purified by a second round
of silica gel chromatography (100% dichloromethane as eluent) to
provide pure caryolanemagnolol 1 as a colorless oil (9.1 mg, 15%
yield). TLC Rf 0.25, hexane/EtOAc = 80 : 20; [a]2D6 = 61.6, (c 2.1,
CHCl3); 1HNMR (400 MHz, C6D6): d 0.60 (s, 3H), 0.80 (dt, J =
4.2, 14.7 Hz, 1H), 0.88 (s, 3H), 1.08 (s, 3H), 1.12–1.41 (m, 6H),
1.44–1.59 (m, 2H), 1.64–1.82 (m, 2H), 1.91–2.03 (m, 2H), 2.16
(ddd, J = 1.9, 10.2, 20.1 Hz, 1H), 2.90 (t, J = 3.0 Hz, 1H), 3.16 (d,
J = 6.8 Hz, 2H), 3.24 (d, J = 6.6 Hz, 2H), 4.94–5.04 (m, 4H), 5.82
(ddt, J = 6.8, 10.1, 16.8 Hz, 1H), 5.94 (ddt, J = 6.6, 10.2, 20.1 Hz,
1H), 6.92 (d, J = 8.5 Hz, 1H, Ar-H), 6.97 (dd, J = 2.2, 8.3 Hz, 1H),
7.03 (dd, J = 2.2, 8.2 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.22 (d,
J = 2.2 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 7.58 (s, 1H); 13C NMR
(100 MHz, C6D6): d 20.2, 20.5, 26.3, 28.4, 29.2, 30.0, 34.6, 35.3,
36.1, 38.7, 39.2, 39.41, 39.44, 39.5, 43.8, 71.0, 84.1, 115.0, 118.4,
123.7, 128.2, 128.7, 129.4, 131.3, 132.0, 133.5, 135.6, 137.2, 138.1,
149.4, 153.0; FTIR: 3361, 1638, 1491, 1209, 1045, 938, 822 cm-1.
MS (ESI): M+Na+ 509. HRMS (ESI+), calculated for C33H41O3
(M-(H-)) 485.3060, found 485.3061.
Epoxides 4 & 5
To a solution of (b)-caryophyllene (2.27 g (90% purity), 10 mmol,
1 equiv.) in acetonitrile (50 mL) and dimethoxymethane (100 mL)
was added a buffer solution (100 mL, 0.05 M Na2B4O7 10 H2O
in 4 ¥ 10-4 M Na2(EDTA)), n-Bu4HSO4 (140 mg, 0.4 mmol,
∑
0.04 equiv.), L-Shi catalyst 10 (2.58 g, 10 mmol, 1 equiv.), and
◦
R
cooled to 0 C. Addition of Oxoneꢀ (8.5 g, 13.8 mmol, 1.38 equiv.)
in buffer aqueous solution (65 mL, 4 ¥ 10-4 M Na2EDTA
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Note: reported optical rotations [a]2D3.5 = 11.2, synthetic material
[a]2D6 = 61.6, see ref. 6.
(100 MHz, CDCl3): d 20.8, 21.1, 26.4, 28.3, 30.3, 30.5, 34.7, 36.0,
37.5, 39.2, 40.3, 41.9, 44.8, 71.6, 80.7, 114.0, 123.2, 131.5, 154.7;
FTIR: 3394, 1638, 1493, 1232 cm-1; HRMS (ESI+) calculated for
C21H29BrO2Na 415.1249, found 415.1243.
Clovanemagnolol (2)
To a solution of recrystallized b-caryophyllene b-oxide 5 (60 mg,
0.259 mmol, 1 equiv.) and magnolol (8) (103 mg, 0.388 mmol,
1.5 equiv.) in dichloromethane (2 mL) was added a solution
of diphenylphosphate (32.4 mg, 0.129 mmol, 0.5 equiv.) in
dichloromethane (1 mL) over 3 min. The orange solution was
stirred at 38 ◦C for 2.5 h then the solvent was removed. The residue
was diluted with hexane (50 mL) and washed with phosphate
buffer (pH 7.4, 2 ¥ 10 mL) and 1 N NaOH (3 ¥ 10 mL (to recover
the magnolol)). The organic layer was washed with brine (10 mL)
and dried over Na2SO4 and then concentrated. The residue was
purified by silica gel chromatography (hexane/EtOAc = 85 : 15 as
eluent) to yield clovanemagnolol (2) as a colorless oil (12 mg, 10%
yield). TLC Rf 0.22, hexane/EtOAc = 80 : 20; [a]2D6 = 96.0, (c 1.5,
Dibromide 12
To a solution of bromide 11 (1.23 g, 3.13 mmol, 1 equiv.),
triethylamine (0.87 mL, 6.25 mmol,
2 equiv.), and 4-
dimethylaminopyridine (0.382 g, 3.13 mmol, 1 equiv.) in CH2Cl2
(40 mL) at 0 ◦C was added acetic anhydride (0.443 mL, 4.69 mmol,
1.5 equiv.). The solution was stirred at 23 ◦C for 20 min then
the mixture was diluted with CH2Cl2 (30 mL) and washed with
2 N HCl (30 mL), 1 N NaOH (20 mL), and brine (10 mL).
The organic extract was dried over MgSO4, filtered, and con-
centrated. The residue was purified by silica gel chromatography
(hexane/EtOAc = 95 : 5 as eluent) to yield the acetate of 11 as a
colorless oil (1.14 g, 84%). TLC Rf 0.28, hexane/EtOAc = 95 : 5;
1HNMR (400 MHz, CDCl3): d 0.87 (s, 3H), 0.99 (s, 3H), 1.01 (s,
3H), 1.20–1.28 (m, 1H), 1.39–2.06 (m, 9H), 1.91–2.06 (m, 3H),
2.01 (s, 3H), 2.36 (ddd, J = 1.3, 10.2, 11.7 Hz, 1H), 4.71 (t, J =
3.7 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 8.9 Hz, 2H);
13C NMR (100 MHz, CDCl3): d 20.8, 21.1, 21.2, 25.7, 26.6, 30.3,
34.7, 35.9, 37.8, 38.3, 40.2, 43.1, 44.9, 73.6, 80.4, 113.8, 122.7,
131.6, 154.7, 170.6. FTIR: 1734, 1486, 1245 cm-1; HRMS (ESI+),
calculated for C23H31BrO3Na 457.1349, found 457.1349.
To a reaction flask containing the acetate of 11 (0.832 g,
1.91 mmol, 1 equiv.) and sodium acetate (0.314 g, 3.82 mmol,
2 equiv.) in acetic acid (10 mL) was added bromine (0.404 mL,
7.64 mmol, 4 equiv.) dissolved in acetic acid (5 mL) in a single
portion. The reaction was heated to 60 ◦C for 4 h. The reaction was
diluted with hexane (100 mL) and washed with water (100 mL ¥
3), 1 N NaOH (20 mL ¥ 3), and then brine (30 mL). The
organic extract was dried over Na2SO4, filtered, and concentrated.
Purification by silica gel chromatography (hexane/EtOAc = 95 : 5)
provided dibromide 12 as a colorless oil (0.902 g, 92%). TLC Rf
0.28, hexane/EtOAc = 95 : 5; 1HNMR (400 MHz, CDCl3): d 0.89
(s, 3H), 1.01 (s, 3H), 1.02 (s, 3H), 1.23–1.29 (m, 1H), 1.39–2.01 (m,
11H), 2.03 (s, 3H), 2.13 (m, 1H), 2.29–2.43 (m, 1H), 4.71 (t, J =
3.2 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 2.4, 8.8 Hz, 1H),
7.65 (d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 20.8,
21.0, 21.2, 25.7, 26.7, 30.3, 30.9, 34.8, 35.8, 37.7, 38.4, 40.3, 42.4,
44.7, 73.6, 83.0, 114.0, 117.9, 122.5, 130.2, 135.4, 152.1, 170.6.
FTIR: 1738, 1467, 1040, 1017 cm-1; HRMS (ESI+), calculated for
C23H30Br2O3Na 535.0455, found 535.0454.
1
CHCl3); HNMR (400 MHz, C6D6): d 0.64 (s, 3H), 0.70 (d, J =
12.8 Hz, 1H), 0.83 (s, 3H), 0.83 (m, 1H), 0.92 (s, 3H), 0.98–1.18
(m, 4H), 1.26 (dd, J = 5.5, 11.4 Hz, 1H), 1.35 (m, 1H), 1.50–171
(m, 4H), 1.79 (dt, J = 3.7, 13.3 Hz, 1H), 3.04 (s, 1H), 3.18 (d,
J = 6.8 Hz, 2H), 3.23 (d, J = 6.6 Hz, 2H), 4.10 (dd, J = 5.7,
8.5 Hz, 1H), 4.96–5.01 (m, 3H), 5.03 (dd, J = 1.7, 3.4 Hz, 1H),
5.81–5.98 (m, 2H), 6.58 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 7.02
(dd, J = 2.3, 8.2 Hz, 1H), 7.04 (dd, J = 2.3, 8.4 Hz, 1H), 7.16 (d,
J = 2.3 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.19 (d, J = 8.3 Hz,
1H); 13C NMR (100 MHz, C6D6): d 20.6, 25.1, 26.5, 26.7, 28.3,
30.9, 32.8, 34.4, 35.4, 37.4, 39.3, 39.4, 44.2, 45.0, 49.9, 74.3, 89.4,
115.0, 115.4, 115.9, 117.4, 127.1, 128.9, 129.2, 129.4, 131.2, 131.8,
132.5, 133.8, 137.4, 138.0, 152.8, 154.2; FTIR: 3394, 1638,1232,
822 cm-1; HRMS (CI+), calculated for C33H42O3 486.3131, found
486.3134.
Note: reported optical rotations [a]2D5 = 21.0, synthetic material
[a]2D6 = 96.0, see ref. 6.
Bromide 11
To a solution of p-bromophenol (1.93 g, 11.2 mmol, 1.5 equiv.)
in CH2Cl2 (20 mL) at 0 ◦C was added a solution of AlMe3 in
hexane (1 M, 3.72 mL, 3.72 mmol, 0.5 equiv.) over 5 min, followed
by stirring at 0 ◦C for 30 min, warmed to 23 ◦C, and stirred
for 1 h. Then the solution was concentrated and p-bromophenol
(1.93 g, 11.2 mmol, 1.5 equiv.) in CH2Cl2 (30 mL) was added at
0 ◦C dropwise. After stirring for 10 min a solution of caryophyllene
oxide (1.64 g (2.2 : 1 ratio of epoxides), 5.1 mmol of 4, 1 equiv. of 4)
in CH2Cl2 (20 mL) was added over 30 min. The mixture was stirred
at 0 ◦C for an additional 2 h then the solvent was removed and the
residue was suspended with hexane (200 mL). The organic layer
was washed with 2 N HCl (50 mL), 1 N NaOH (50 mL), and brine
(30 mL). The organic extract was dried over MgSO4, filtered, and
concentrated to provide a viscous residue. The residue was purified
by silica gel chromatography (hexane/EtOAc = 9 : 1 as eluent) to
give bromide 11 as a clear oil (1.37 g, yield 69% from 4). TLC
Acetate 14
To a mixture of dibromide 12 (640 mg, 1.24 mmol, 1 equiv.),
Pd(PPh3)4 (288 mg, 0.249 mmol, 0.2 equiv.), and NaOH (0.995 g,
24.9 mmol, 20 equiv.) was added toluene (18 mL), water (9 mL),
and pinacol allylboronate 13 (4.0 mL, 16.0 mmol, 13 equiv.)
followed by vigorous stirring (1200 rpm) at 90 ◦C for 55 min. The
organic layer was separated and the aqueous layer was extracted
with EtOAc (30 mL). The combined organic extracts were
combined with silica gel (1 g) and the sample was concentrated
to dryness. The sample was purified by silica gel chromatography
(hexane/EtOAc = 95 : 5 as eluent) to provide 14 as a colorless oil
Rf 0.24, hexane/EtOAc = 85 : 15. [a]26 = 90.3◦, (c 1.5, CHCl3);
D
1HNMR (400 MHz, CDCl3): d 0.92 (s, 3H), 0.99 (s, 3H), 1.00 (s,
3H), 1.14–1.21 (m, 1H), 1.37–1.87 (m, 10H), 1.96–2.06 (m, 2H),
2.34 (ddd, J = 2.1, 3.4, 18.2 Hz, 1H), 3.49 (dd, J = 3.0, 4.6 Hz,
1H), 6.73 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 9.0 Hz, 2H); 13C NMR
1
(430 mg, 72%). TLC Rf 0.30, hexane/EtOAc = 95 : 5; HNMR
390 | Org. Biomol. Chem., 2012, 10, 383–393
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(400 MHz, CDCl3): d 0.88 (s, 3H), 1.01 (s, 3H), 1.02 (s, 3H),
1.21–1.27 (m, 1H), 1.39–1.65 (m, 4H), 1.73 (dd, J = 8.0, 9.6 Hz,
1H), 1.88–1.91 (m, 5H), 1.95–2.00 (m, 1H), 2.02 (s, 3H), 2.16 (m,
1H), 2.37 (ddd, J = 1.7, 3.6, 18.2 Hz, 1H), 3.28 (d, J = 6.7 Hz,
2H), 4.70 (t, J = 3.0 Hz, 1H), 5.03–5.06 (m, 1H), 5.08–5.09 (m,
1H), 5.86–5.96 (m, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 2.2,
8.4 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 20.8, 21.0, 21.2, 25.7, 26.7, 30.3, 30.8, 34.8, 35.8, 37.5, 38.4, 39.0,
40.3, 42.4, 44.6, 73.8, 82.2, 116.1, 117.1, 121.7, 127.4, 133.1, 134.8,
136.9, 150.9, 170.6; FTIR: 1737, 1486, 1247 cm-1; HRMS (ESI+),
calculated for C26H35BrO3Na 497.1665, found 497.1662.
64.7 mmol, 3 equiv.) in CH2Cl2 (80 ml) at 0 ◦C was added a solution
of diphenylphosphate (2.70 g, 10.8 mmol, 0.5 equiv.) in CH2Cl2
(20 mL) over 30 min. The gray solution was stirred at 0 ◦C for 4
h and then the solvent was removed under vacuum. The residue
was diluted with hexane (300 mL) and washed with 1 N NaOH
(50 mL ¥ 3). The organic layer was washed with brine (10 mL) and
dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel chromatography (hexane/EtOAc = 90 : 10 as
eluent) to provide bromide 17 as a white solid (3.1 g, ◦35% yield).
26
TLC Rf: 0.34 hexane/EtOAc = 85 : 15; Mp 114–116 C; [a]d
=
93.1◦, (c 1.7, CHCl3); 1HNMR (400 MHz, CDCl3): d 0.94 (s, 3H),
0.95 (s, 3H), 0.96–1.04 (m, 2H), 1.06 (s, 3H), 1.09–1.21 (m, 1H),
1.32–1.43 (m, 3H), 1.48–1.68 (m, 6H), 1.79 (dt, J = 4.7, 13.6 Hz,
1H), 1.85 (dd, J = 5.4, 12.7 Hz, 1H), 1.98–2.07 (m, 1H), 3.34
(bs, 1H), 4.19 (dd, J = 5.4, 7.9 Hz, 1H), 6.76 (d, J = 9.0 Hz,
2H), 7.33 (d, J = 9.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): d
20.9, 25.7, 26.6, 27.2, 28.3, 31.6, 33.1, 34.7, 36.0, 38.3, 43.8, 45.3,
50.0, 74.9, 86.5, 112.3, 117.5, 132.1, 158.1; FTIR: 3416, 1486,
1243 cm-1; HRMS (ESI+), calculated for C21H28BrO (M-H2O)
375.1323, found 375.1318.
Carbamate 16
To a flask containing acetate 14 (330 mg, 0.694 mmol, 1 equiv.),
borate ester 15 (997 mg, 2.78 mmol, 4 equiv.), and NaOH
(333 mg, 8.33 mmol, 12 equiv.) followed by addition of toluene
(7 mL) and water (5 mL, sparged with argon for 5 min). A
solution of Pd2(dba)3 (39.9 mg, 0.069 mmol, 0.1 equiv.) and
tBu3P (51 mL, 0.208 mmol, 0.3 equiv.) in toluene (3 mL) was
then added. The mixture was immediately placed in a 90 ◦C
oil bath and stirred at 1200 rpm for 30 min (color transition
of red to yellow to brown to dark brown). After cooling the
mixture was diluted with EtOAc (50 mL) and water (10 mL).
The layers were separated and the aqueous phase was extracted
with EtOAc (20 mL). The combined organic extracts were dried
over MgSO4, filtered, and concentrated. Purification by silica gel
chromatography (hexane/EtOAc = 90 : 10 as eluent) provided 16
as colorless oil (308 mg, 71%). TLC Rf 0.42, hexane/EtOAc =
Dibromide 18
To a solution of bromide 17 (2.6 g, 6.38 mmol, 1 equiv.) and
4-dimethylam◦inoyridine (0.78 g, 6.38 mmo, 1 equiv.) in CH2Cl2
(40 mL) at 0 C was added triethylamine (1.78 mL, 12.8 mmol,
2 equiv.). Subsequently, acetic anhydride (1.21 mL, 12.8 mmol,
2 equiv.) was added dropwise over 10 min. The solution was
stirred at 0 ◦C for 1 h then diluted with CH2Cl2 (100 mL) washed
with 1 N NaOH (30 mL), 2 N HCl (30 mL) and brine (20 mL).
The organic layer was dried over Na2SO4, filtered, and concen-
trated. The residue was purified by silica gel chromatography
(hexane/EtOAc = 95 : 5) to provide the acetate of 17 as a colorless
oil (2.50 g yield 87%). TLC Rf 0.32, hexane/EtOAc = 95 : 5;
1HNMR (400 MHz, CDCl3): d 0.85 (s, 3H), 0.94 (s, 3H), 1.05
(s, 3H), 1.09 (d, J = 12.6 Hz, 1H), 1.21–1.28 (m, 1H), 1.33–1.74
(m, 9H), 1.85 (d, J = 5.4, 12.8 Hz, 1H), 1.93–1.99 (m, 1H), 2.02 (s,
3H), 4.20 (dd, J = 1.9, 7.4 Hz, 1H), 4.57 (b, 1H), 6.77 (d, J = 9.0 Hz,
2H), 7.34 (d, J = 9.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): d
20.8, 21.3, 24.1, 25.8, 27.8, 28.1, 31.7, 32.8, 33.6, 36.9, 38.5, 43.7,
45.3, 50.0, 86.5, 112.4, 117.6, 132.1, 157.9, 170.8; FTIR: 1733,
1486 cm-1; HRMS (ESI+), calculated for C23H31BrO3Na 457.1356,
found 457.1349.
1
80 : 20; HNMR (300 MHz, C6D6): d 0.72 (s, 3H), 0.77 (t, J =
7.2 Hz, 3H), 0.82–0.87 (m, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.03–
1.60 (m, 10H), 1.66 (s, 3H), 1.68–1.82 (m, 2H), 1.88 (t, J = 9.7 Hz,
1H), 2.09 (dd, J = 8.8, 20.8 Hz, 1H), 2.90–3.12 (m, 4H), 3.21 (t, J =
8.0 Hz, 4H), 4.23 (bs, 1H), 4.91–5.03 (m, 4H), 5.82–5.96 (m, 2H),
6.87 (d, J = 8.3 Hz, 1H), 6.97 (dd, J = 1.9, 8.5 Hz, 2H), 7.20 (d, J =
1.9 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H); 13C NMR (75 MHz, C6D6):
d 13.4, 14.0, 20.6, 20.9, 24.8, 25.9, 26.7, 30.3, 30.7, 34.6, 35.8, 36.7,
38.3, 39.7, 39.9, 40.3, 41.5, 41.9, 42.0, 44.2, 73.5, 80.8, 115.5, 115.7,
122.5, 123.3, 131.9, 132.4, 132.8, 133.1, 133.3, 135.8, 136.7, 137.6,
137.7, 137.8, 148.1, 151.7, 153.8, 169.5. FTIR: 1737, 1486, 1247
cm-1; HRMS (CI+), calculated for C40H53NO5 627.3924, found
627.3921.
Caryolanemagnolol (1)
To a solution of the acetate of 17 (2.00 g, 4.45 mmol, 1 equiv.)
and sodium acetate (0.73 g, 8.9 mmol, 2 equiv.) in acetic acid
(30 mL) was added a solution of bromine (0.918 mL, 17.8 mmol,
4 equiv.) in acetic acid (10 mL) over 1 min. The flask was
stirred at 60 ◦C for 4 h. The mixture was diluted with hexane
(200 mL) and washed with water (3 ¥ 100 mL), 1 N NaOH
(3 ¥ 100 mL), and brine (50 mL). The organic layer was dried
over Na2SO4, filtered, and concentrated. Purification by silica gel
chromatography (hexane/EtOAc = 95 : 5 as eluent) gave dibromide
18 as a colorless oil (2.01 g, 85%). TLC Rf 0.32, hexane/EtOAc =
To the solution of carbamate 16 (80 mg, 0.127 mmol, 1 equiv.) in
tetrahydrofuran (2 mL) was added LiAlH4 solution in tetrahydro-
furan (1 M, 2.55 mL, 2.55 mmol, 20 equiv.) dropwise at 23 ◦C.
The mixture was heated to reflux for 2 h and then cooled to
◦
23 C and cautiously quenched with water (5 mL). The mixture
was diluted with diethyl ether (10 mL) and 2 N HCl (5 mL).
The organic layer was collected and washed with brine (20 mL).
The organic phase was concentrated and the residue purified by
silica gel chromatography (hexane/EtOAc = 85 : 15) to provide
caryolanemagnolol (1) as a colorless pasty oil (44 mg, 71%).
1
95 : 5; HNMR (400 MHz, CDCl3): d 0.87 (s, 3H), 0.96 (s, 3H),
1.08 (s, 3H), 1.10 (d, J = 13.2 Hz, 1H), 1.24–1.31 (m, 1H), 1.35–1.48
(m, 1H), 1.53–1.85 (m, 7H), 1.88 (dd, J = 5.3, 13.0 Hz, 1H), 1.96–
2.01 (m, 1H), 2.03 (s, 3H), 4.21 (dd, J = 0.9, 6.5 Hz, 1H), 4.58 (bs,
1H), 6.73 (d, J = 8.9 Hz, 1H), 7.32 (dd, J = 2.4, 8.8 Hz, 1H), 7.64
(d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 20.8, 21.3,
Bromide 17
To a solution of b-caryophyllene b-oxide (recrystallized from
benzene)3 (5 g, 21.6 mmol, 1 equiv.) and p-bromophenol (11.9 g,
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24.3, 25.9, 27.9, 28.1, 31.8, 32.8, 33.6, 36.8, 39.0, 43.7, 45.9, 49.9,
87.9, 112.3, 114.0, 115.4, 130.9, 135.5, 154.4, 170.8; FTIR: 1735,
1246, 1042 cm-1; HRMS (ESI+), calculated for C23H30Br2O3Na
535.0455, found 535.0454.
122.9, 129.1, 131.7, 131.8, 132.1, 135.7, 137.4, 137.8, 148.0, 153.5,
154.7, 169.4; FTIR: 1720, 1246, cm-1; HRMS (CI+), calculated
for C40H54O5N 628.4002, found 628.3986.
Clovanemagnolol (2)
Acetate 19
To a solution of carbamate 20 (100 mg, 0.159 mmol, 1 equiv.) in
tetrahydrofuran (4 mL) was added LiAlH4 solution in tetrahydro-
furan (1 M, 3.19 mL, 3.19 mmol, 20 equiv.) dropwise at 23 ◦C.
The mixture was heated to reflux for 2 h and then cooled to
To a flask charged with substrate 18 (0.488 g, 0.948 mmol,
1
equiv.), NaOH (0.758 g, 17.0 mmol, 18 equiv.), and
tetrakis(triphenylphosphine)palladium (0.219 g, 0.190 mmol,
0.2 equiv.) under argon was added toluene (10 mL), water (5 mL,
sparged with argon for 5 min), and allyl pinacol borate 13
(3.05 mL, 15.17 mmol, 16 equiv.). The biphasic mixture was stirred
at 1200 rpm at 90 ◦C for 50 min whereupon TLC (hexane/EtOAc =
97 : 3) showed full conversion. The mixture was cooled to 23 ◦C and
extracted with EtOAc (50 mL). The organic layer was washed with
brine (50 mL), dried over Na2SO4, and concentrated. The residue
was purified by silica gel chromatography (hexane/EtOAc = 97 : 3
as eluent) to provide acetate 19 as a colorless oil (0.316 g, yield
70%). TLC Rf 0.33, hexane/EtOAc = 95 : 5; 1HNMR (400 MHz,
CDCl3): d 0.87 (s, 3H), 0.95 (s, 3H), 1.09 (s, 3H), 1.10 (d, J = 7.2 Hz,
1H), 1.25–1.48 (m, 4H), 1.53–1.78 (m, 5H), 1.88 (dd, J = 5.3,
12.9 Hz, 2H), 1.96–2.01 (m, 1H), 2.03 (s, 3H), 3.29 (d, J = 6.7 Hz,
2H), 4.22 (dd, J = 5.4, 7.0 Hz, 1H), 4.58 (b, 1H), 5.04–5.07 (m, 1H),
5.07–5.09 (m, 1H), 5.86–5.96 (m, 1H), 6.79 (d, J = 8.4 Hz, 1H),
7.03 (dd, J = 2.2, 8.3 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 20.8, 21.3, 24.2, 25.8, 27.9, 28.1, 31.8, 32.8,
33.6, 36.8, 38.8, 38.9, 43.9, 45.7, 49.9, 87.7, 113.0, 114.4, 116.0,
128.2, 133.2, 133.3, 137.1, 153.5, 170.9; FTIR: 1735, 1248 cm-1;
HRMS (ESI+), calculated for C26H35BrO3Na 497.1665, found
497.1662.
◦
23 C and the excess LiAlH4 was carefully quenched with water
(10 mL). The mixture was diluted with Et2O (40 mL) and 2 N HCl
(20 mL). The organic layer was collected and washed with brine
(10 mL). The concentrated organic phase was purified by silica
gel chromatography with hexane/EtOAc = 85 : 15 to provide the
clovanemagnolol 2 as a colorless oil (57 mg, 73%).
Pinacol ester 15
To a solution of 4-allylphenol (2.0 g, 14.9 mmol, 1 equiv.) and
4-dimethylamino pyridine (3.64 g, 29.8 mmol, 2 equiv.) in CH2Cl2
(40 mL) was added a solution of diethyl chlorocarbamate (3.77
ml, 29.8 mmol, 2 equiv.) in CH2Cl2 (20 mL) at 0 ◦C. The solution
was stirred at 0 ◦C for 2 h, 23 ◦C for 4 h, then diluted with CH2Cl2
(200 mL), washed with 1 N NaOH (100 mL), 2 N HCl (100 mL),
and brine (50 mL). The organic extract was dried over Na2SO4,
filtered, and concentrated. Purification of the residue by silica
gel chromatography (hexane/EtOAc = 85 : 15 as eluent) provided
the carbamate as a yellow oil (2.7 g, 78% yield). TLC Rf 0.45,
hexane/EtOAc = 80 : 20.
To THF (40 mL) under argon at -78 ◦C was added s-BuLi
(1.21 M, 12.75 mL, 15.4 mmol, 1.2 equiv.) followed by TMEDA
(2.33 mL, 15.4 mmol, 1.2 equiv.) providing a light yellow solution.
After 30 min, a solution of the carbamate prepared above (3.0 g,
12.9 mmol) in THF (10 mL) was added over 10 min. The yellow
solution was stirred for 2 h at -78 ◦C then trimethyl borate
(2.87 mL, 25.7 mmol, 2 equiv.) was added followed by warming
to 23 ◦C over 1 h (color fade upon addition of trimethyl borate).
A solution of 0.1 N HCl (100 mL) was added and the aqueous
phase was extracted with ether (20 mL ¥ 3). The combined organic
extracts were dried over Na2SO4, filtered, and concentrated. The
oily residue was dissolved in methanol (40 mL) and pinacol (4.56 g,
38.6 mmol, 3 equiv.) was added as a solid. The solution was stirred
Carbamate 20
To a flask charged with acetate 19 (200 mg, 0.421 mmol, 1 equiv.),
the pinacol ester 15 (604 mg, 1.683 mmol, 4 equiv.), and NaOH
(202 mg, 5.05 mmol, 12 equiv.) under argon was added toluene
(6 mL), water (4 mL, sparged with argon for 5 min) and a solution
of Pd(dba)2 (24.2 mg, 0.042 mmol, 0.1 equiv.), and tBu3P (31 mL,
0.126 mmol, 0.3 equiv.) solution in toluene (2 mL, stirring for 10
min before addition). The mixture was immediately placed in a
90 ◦C oil bath and stirred at 1200 rpm for 30 min (red to yellow to
brown to dark brown color sequence) then the reaction was diluted
with EtOAc (30 mL) and water (10 mL). The phases were separated
and the aqueous phase was extracted with EtOAc (20 mL). The
combined organic extracts were dried over MgSO4, filtered, and
concentrated. Silica gel chromatography (hexane/EtOAc = 90 : 10
as eluent) provided carbamate 20 as a colorless pasty oil (196 mg,
74%). TLC Rf 0.40, hexane/EtOAc = 80 : 20; 1HNMR (400 MHz,
C6D6): d 0.70 (s, 3H), 0.81 (s, 3H), 0.86 (s, 3H), 0.71–0.90 (m, 8H),
0.95–1.24 (m, 5H), 1.44 (d, J = 12.7 Hz, 1H), 1.50–1.70 (m, 5H),
1.80 (s, 3H), 2.91 (d, J = 5.4 Hz, 2H), 3.00 (d, J = 5.6 Hz, 2H), 3.18
(t, J = 7.5 Hz, 4 H), 4.01 (dd, J = 5.8, 15.6 Hz, 1H), 4.68 (b, 1H),
4.93–5.02 (m, 4H), 5.80–6.00 (m, 2H), 6.85 (d, J = 8.4 Hz, 1H),
6.92 (dd, J = 2.3, 8.3 Hz, 1H), 7.01 (dd, J = 2.3, 8.3 Hz, 1H), 7.16
(d, J = 2.1 Hz, 1H, Ar-H), 7.20 (d, J = 2.3 Hz, 1H, Ar-H), 7.31 (d,
J = 8.3 Hz, 1H, Ar-H); 13C NMR (100 MHz, C6D6): d 13.1, 13.7,
18.1, 20.6, 24.2, 25.5, 27.8, 28.0, 31.4, 32.7, 33.3, 36.6, 38.2, 39.4,
39.6, 41.3, 41.7, 43.7, 45.4, 49.7, 76.3, 87.2, 115.0, 115.1, 115.4,
◦
at 23 C for 1 h and the mixture was concentrated. The viscous
residue was dissolved in EtOAc (30 mL) and washed with water
(20 mL ¥ 3). The organic phase was dried over Na2SO4, filtered,
and concentrated. Silica gel chromatography (hexane/EtOAc =
90 : 10 as eluent) yielded the pinacol ester 15 as a colorless oil
(3.8 g, 82% yield). TLC Rf 0.37, hexane/EtOAc = 80 : 20; 1HNMR
(300 MHz, CDCl3): d 1.19 (t, J = 7.0 Hz, 3H), 1.28 (t, J =
7.0 Hz, 3H), 1.30 (s, 12H), 3.37 (q, J = 7.2 Hz, 2H), 3.37 (d,
J = 8.0 Hz, 2H), 3.50 (q, J = 7.2 Hz, 2H), 5.03–5.09 (m, 2H),
5.95 (ddt, J = 6.7, 10.1, 16.8 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H,
7.24 (d, J = 2.3, 8.3 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d 13.4, 14.1, 24.9, 39.5, 41.6, 41.9, 83.4, 115.7,
122.2, 132.3, 136.2, 136.3, 137.5, 154.5, 154.9; FTIR: 1720, 1510
cm-1; HRMS (CI+), calculated for C20H31O4NB 360.2348, found
360.2346.
392 | Org. Biomol. Chem., 2012, 10, 383–393
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