Synthesis of (±)-5′-methoxyhydnocarpin-D, an inhibitor of the Staphylococcus aureus multidrug resistance pump

Article abstract of DOI:10.1016/j.tet.2005.02.024

The total synthesis of regioisomerically pure (±)-5′- methoxyhydnocarpin-D (6) from commercially available vanillin (7), methyl gallate (9) and 2′,4′,6′-trihydroxyaceophenone (10) is achieved.

Full text of DOI:10.1016/j.tet.2005.02.024

Tetrahedron 61 (2005) 4149–4156
Synthesis of (G)-50-methoxyhydnocarpin-D, an inhibitor of the
Staphylococcus aureus multidrug resistance pump
*
Kin-Fai Chan, Yunzhe Zhao, Larry M. C. Chow and Tak Hang Chan
Department of Applied Biology and Chemical Technology and the Open Laboratory for Chiral Technology, the Institute of Molecular
Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR,
The State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Schenzhen, China
Received 9 November 2004; revised 24 January 2005; accepted 1 February 2005
Abstract—The total synthes0is of regioisomerically pure (G)-50-methoxyhydnocarpin-D (6) from commercially available vanillin (7),
methyl gallate (9) and 20,40,6 -trihydroxyaceophenone (10) is achieved.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitor.6 In 2003, Pettit and Musumeci also reported the
isolation of 6 from the plant Hymeneae palustris and
Berberis aetnensis.7 The naturally occurring 6 was optically
inactive and has no antimicrobial activity by itself. It
potentiates the growth inhibitory activity of the natural
antibacterial alkaloid berberine and the commonly used
fluoroquinolone antibiotics norfloxacin. Further structure
activity relationships (SARs) studies also provided detailed
structural requirements to the success of NorA efflux pump
inhibition.8 Recently, Guz reported a synthesis of regio-
isomeric mixture of racemic 6 by employing the silver ion
Multidrug resistance (MDR) remains a significant impedi-
ment to the successful chemotherapeutic treatment of
bacterial infections and cancer.1 This resistance is mainly
attributed to the ubiquitous transmembrane efflux proteins
that actively export a wide variety of structurally unrelated
therapeutics from the cell, resulting in a low intracellular
concentration of the drug. To date, many such efflux pump
proteins have been identified and classified.2 One of these
well-characterized membrane proteins is the NorA efflux
pump, which is found in bacteria Staphylococcus aureus3
and employs the transmembrane electrochemical gradient
of protons to drive the extrusion of drugs from the cell. The
NorA pump in S. aureus also extrudes a broad spectrum of
substrates from amphiphilic ethidium bromide to basic
puromycin and neutral fluoroquinolone antibiotics. As
MDR has already emerged in some bacteria in clinical
settings, the identification and development of effective
bacterial efflux pump inhibitors is needed. There are only a
few known inhibitors of NorA MDR pump (Fig. 1). The first
identified NorA MDR pump inhibitor was the alkaloid
reserpine (1), which is toxic at high dosages.4 Other
modulators of NorA MDR pump have since been dis-
covered including synthetic amide 2, amino alcohol 3,
amide 4, and naturally occurring phenylpiperidine 5.5
Recently, Stermitz reported that a flavonolignan, namely
(G)-50-methoxyhydnocarpin-D (6), isolated from the plant
Berberis fremontii, was a potent NorA MDR pump
Keywords: Staphylococcus aureus; Multidrug resistance; NorA; 50-Meth-
oxyhydnocarpin-D; Mitsunobu reaction.
*
Corresponding author. Tel.: C852 2766 5605; fax: C852 2364 9932;
e-mail: bcchanth@polyu.edu.hk
Figure 1.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.02.024

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K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
mediated coupling of selgin and coniferyl alcohol.9 To the
best of our knowledge, no report on the synthesis of
regioisomerically pure 6 has appeared up to now in the
literature. Although the exact binding site of NorA inhibitor
remains unclear, it is believed that such inhibitors bind
directly to the hydrophobic region of NorA efflux pump thus
preventing the drug efflux.10 Information from the binding
studies of synthetic analogs will be useful in delineating the
pharmacophore for the inhibition of NorA.11 Herein we
describe the regioselective total synthesis of 6.
phenyl) in refluxing toluene furnished ester 11 in good yield
(78%). The presence of electron-donating p-methoxy
functionality in the acetal probably activated such
transacetalization process.
Of the various methods available for the ether formation, the
Mitsunobu reaction14 is undoubtedly the reaction of choice.
Initial attempts to synthesize the alkene 12 by reaction of
ester 11 and allyl alcohol 8 under the standard protocol of
using triphenylphosphine (PPh3) and diethyl azodicarboxyl-
ate (DEAD) in THF were accomplished in low yield and
low regioselectivity (Table 1, Entry 1). From proton NMR
of the product mixture, the desired branched isomer 12
derived from SN2 reaction showed a multiplet signal at
6.1 ppm and the linear isomer 13 derived from SN20 showed
a characteristic doublet of triplet at 6.3 ppm. Integrations of
both proton signals revealed that the ratio of 12/13 was
3.8:1. Similar results were obtained when using toluene as
solvent (Table 1, Entry 3). In both cases, removal of the
highly soluble phosphine oxide from the reaction mixture
was a tedious process. Improved regioselectivity was
obtained by using dioxane as solvent with a ratio of 12/13
at 7.8:1, but the yield was still unacceptable at 38%
(Table 1, Entry 2). The success of replacing PPh3 by 1,2-
bis(diphenylphosphino)ethane (dppe) for ease of products
separation prompted us to use the diphosphine ligands for
the Mitsunobu reaction.15 Initial attempts to use bis(diphe-
nylphosphino)methane (dppm) as phosphine source failed
to improve the amount of alkene 12 (Table 1, Entry 4).
Interestingly, high regioselectivity and reasonably good
yield were finally achieved by using dppe (Table 1, Entry 5).
Increasing the chain length by using 1,3-bis(diphenyl-
phosphino)propane (dppp) had a similar result as dppe
(Table 1, Entry 6). We also observed that those diphosphine
oxides were insoluble in dioxane, therefore they could be
removed by simple filtration through a short pad of silica
gel.
2. Results and discussion
Our retrosynthetic approach to the total synthesis of 6 is to
assemble the skeleton with synthons 8, 9 and 10 as depicted
in Scheme 1. The allyl alcohol 8 can in turn be derived from
the commercially available vanillin (7) according to
literature.12 Then, stepwise assembly of the allyl alcohol 8
with methyl gallate (9) at the meta-position followed by
cyclization at para-phenoxy moiety should furnish the 1,4-
benzodioxane moiety of 6 with control of regiochemistry.
This necessitates the selective protection of the hydroxyl
groups0 in 9. Final construction of the flavone core with
20,40,6 -trihydroxyacetophenone (10) should be easily
achieved.
Table 1. Mitsunobu reaction of compounds 8 and 11
Scheme 1.
Entry Conditions
12:13a
Yield (%)
2.1. Regioselective connection of 8 and 9
1
2
3
4
5
6
DEAD, PPh3, THF, 0 8C to rt
3.8:1
7.8:1
4.3:1
2.6:1
17.5:1
13.7:1
36
38
44
52
48
55
DEAD, PPh3, Dioxane, 0 8C to rt
DEAD, PPh3, PhMe, 0 8C to rt
DEAD, dppm, Dioxane, 0 8C to rt
DEAD, dppe, Dioxane, 0 8C to rt
DEAD, dppp, Dioxane, 0 8C to rt
With the requisite alcohol 8 in hand,12 we embarked on the
regioselective connection of 8 and 9 as shown in Scheme 2.
Selective protection of the 3,4-dihydroxy function of methyl
gallate (9) with either dichlorotoluene or acetone gave poor
yield (!40%).13 Instead, acid-catalyzed reaction of ester 9
with p-ansialdehyde dimethyl acetal (PMP: p-methoxy-
a Determined from the purified product by NMR spectroscopy.
2.2. Construction of the 1,4-benzodioxane moiety
The construction of 1,4-benzodioxane moiety in a trans
manner with control of regiochemistry presented a more
difficult challenge. An extensive search of chemical
literature yielded three examples: the first, described by
Pan,16 reported an intramolecular ring opening of a chiral
oxirane. A second route, described by Buchwald,17 reported
a palladium-catalyzed intramolecular etherification of
hydroxyl aryl halides. A third route, described by Valoti,18
reported an intramolecular substitution of ditosylates. Since
the Buchwald route requires an aryl halide,19 we did not
attempt that approach. For the Pan approach,18 suffice it to
say that we have prepared the epoxide 14, but numerous
Scheme 2.

K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
4151
attempts to selectively remove the PMP protecting group
invariably led to the opening of the epoxide first. We
therefore turned our attention on the Valoti approach20
proceeding from synthon 8.
the para-phenoxy moiety under basic conditions gave 1,4-
benzodioxane 20. The trans disposition of the two
substituents on the dioxane ring in 20 was evident from
the coupling constants of the hydrogens on the dioxane ring.
Methylation of 20 with dimethyl sulfate under basic
conditions followed by sequential deprotection of benzyl
group of 21 and pivaloyl group of 22 furnished ester 23 in
high yield. Interestingly, successful intramolecular nucleo-
philic substitution under basic conditions only proceeded
with tosylate 19, but not tosylate 18. We attributed the
difference to the fact that two bulky groups (OPiv and
substituted benzene ring) were sufficiently far away from
each other in tosylate 19 and the intramolecular substitution
was not hindered (Fig. 2). An unidentified alkene byproduct
was observed in the tosylate 18 cyclization reaction,
presumably due to the tosylate group elimination. Finally,
efforts to convert 18 to an iodide with the same
stereochemistry as 19 have thus far not been successful.
As outlined in Scheme 3, the successful reaction sequence
for the construction of 1,4-benzodioxane moiety consisted
in the following steps: (a) dihydroxylation of terminal
alkene 12 using catalytic amount of Osmium tetroxide
(OsO4) and 4-methylmorpholine N-oxide (MNO) gave diol
15 in reasonably good yield as a mixture of diastereomers;
(b) selective protection of the primary OH of 15 with
pivaloyl chloride (PivCl) in pyridine at 0 8C furnished
alcohol 16 in good yield; (c) tosylation of the secondary OH
in 16 with tosyl chloride in pyridine gave the tosylate 17 in
high yield; (d) acid-promoted cleavage of the acetal
functionality of 17 without destroying the tosyl and pivaloyl
moiety gave isomeric tosylates 18 and 19 in high yield in an
isolated ratio of 1:1. The two tosylates were separable by
chromatography and they showed the same number of
proton and carbon signals in the NMR spectroscopy; (e)
intramolecular nucleophilic substitution of tosylate 19 by
Figure 2.
2.3. Completionof synthesis of 50-methoxyhydnocarpin-D
A number of synthetic routes are known for the preparation
of flavones.19 The most commonly used precursors were 20-
hydroxychalcones, which could be oxidatively cyclized to
give flavones in high yield. As shown in Scheme 4, further
protection of ester 23 with tert-butyldimethylsilyl chloride
(TBSCl) gave disilyl ether 24. The ester functionality of 24
was successfully converted to the corresponding aldehyde
26 in high yield by sequential diisobutylaluminium hydride
(DIBAL-H) reduction in THF followed by pyridinium
dichromate (PDC) oxidation. Attempts to synthesize
Scheme 3.
Scheme 4.

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K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
Table 2. Antibiotic activities of selected compounds against Staphylococcus aureus in the absence and presence of synthetic 6
Drug
EC50
without addition
of 6 (mg/mL)
EC50
with addition
of 10 mg/mL 6 (mg/mL)
Activity
potentiated
Norfloxacin
0.32
1.57
4.53
O64
0.16
0.27
2.14
5.3
2-fold
6-fold
2-fold
O12-fold
Ethidium bromide
Tetraphenyl-phosphonium
Pentamidine
aldehyde 26 by one step DIBAL-H reduction of ester 24 at
lower temperature were unsuccessful. The dimethoxy-
methyl-protected acetophenone was prepared from
20,40,60-trihydroxyacetophenone (10) according to the
literature.20 Then condensation of aldehyde 32 with
acetophenone was accomplished in basic medium to give
chalcone 27, together with the cleavage of both silyl groups.
It should be mentioned that cleavage of mono-silyl ether
was observed when reaction time was insufficient. Selenium
dioxide mediated cyclization of chalcone 27 in the presence
of catalytic amount of dimethylsulfoxide (DMSO) followed
by acetic acid deprotection of methoxymethyl (MOM)
group of 28 furnished 6. Initial attempts of deprotection of
28 using 6 M HCl resulted in polymerization of 6. Its
spectroscopic data were similar to those reported in the
literature for 50-methoxyhydnocarpin-D.9
4.2. Materials
All reagents and solvents were reagent grade. Reagents
were purchased from commercial suppliers and were used
without further purification unless otherwise stated.
Solvents were purified and dried using standard procedures
when necessary. All organic solvents were evaporated under
reduced pressure with a rotary evaporator. The plates used
for thin-layer chromatography (TLC) were E. Merck Silica
Gel 60F254 (0.25-mm thickness) precoated on an aluminum
plate and they were visualized under short (254-nm) UV
light. Compounds on TLC plate were visualized with a
spray of 5% w/v dodecamolybdo-phosphoric acid in
ethanol, or with acidified potassium permanganate solution,
and with subsequent heating. Chromatographic purifications
were carried out using MN silica gel 60 (230–400 mesh).
4.2.1. Methyl 7-hydroxy-2-(4-methoxyphenyl)-1,3-
benzo-dioxole-5-carboxylate (11). To a round-bottom
flask equipped with a Dean-Stark trap and water condenser
was charged with methyl gallate (9) (3.7 g, 20 mmol),
p-ansi-aldehyde dimethyl acetal (4.6 g, 25 mmol),
p-toluene-sulfonic acid monohydrate (10 mg) and toluene
(80 mL). The reaction mixture was then heated to reflux for
2 h. The solvent collected at the side arm of the Dean-Stark
trap was continuously removed. When TLC indicated
complete consumption of ester 9, the reaction mixture was
then cooled and diluted with CH2Cl2 (80 mL). The mixture
was poured into a separating funnel containing saturated
Na2CO3 solution (150 mL) and extracted with CH2Cl2
(20 mL!3). The combined organic layers were washed
with brine, dried over MgSO4, filtered and evaporated to
30 mL. After addition of n-hexane (100 mL), precipitation
and filtration, the ester 11 (4.7 g, 78%) was obtained as an
off-white solid: mp 142–144 8C; 1H NMR (CDCl3) d 3.82 (s,
3H), 3.86 (s, 3H), 6.93 (d, JZ8.4 Hz, 2H), 6.96 (s, 1H), 7.13
(s, 1H), 7.38 (s, 1H), 7.47 (d, JZ8.4 Hz, 2H); 13C NMR
(CDCl3) d 52.3, 55.4, 102.9, 111.9, 114.1, 114.1, 124.1,
127.3, 128.0, 138.7, 138.9, 148.9, 161.3, 166.9; LRMS m/z
303 (MCCH, 100); HRMS Calcd for C16H15O6 (MCCH)
303.0869, found 303.0843.
2.4. Biological assays
We have tested the potencies of 6 in terms of its ability to
potentiate a wide range of NorA substrates (norfloxacin,
ethidium bromide, tetraphenylphosphonium and pentami-
dine) against Staphylococcus aureus using the Lewis’s
method.6a The results obtained are summarized in Table 2.
In all cases, the synthetic 6 showed MDR modulating
activity.
3. Conclusion
We have described the first regioselective total synthesis of
(G)-50-methoxyhydnocarpin-D (6) from commercially
available vanillin (7), methyl gallate (9) and 2,4,6-
trihydroxy-acetophenone (10). The key steps included the
Mitsunobu reaction of 11 with 8 and the intramolecular
substitution of tosylate 19 to give 1,4-benzodioxane moiety.
The enantioselective synthesis of 6 is now in process.
4. Experimental
4.2.2. Methyl 7-[3-(3-methoxy-4-benzyloxyphenyl)prop-
1-enyloxy]-2-(4-methoxyphenyl)-1,3-benzodioxole-5-
carboxylate (12). To a well-stirred solution of ester 11
(0.27 g, 0.89 mmol), allyl alcohol 8 (0.30 g, 1.1 mmol) and
1,2-bis(diphenylphosphino)ethane (0.31 g, 0.78 mmol) in
dioxane (10 mL) at 0 8C was added diethyl azodicarboxlate
(0.3 mL, 1.9 mmol). The mixture was allowed to stir
vigorously and warm slowly to room temperature over
16 h. The reaction mixture was filtered through a short pad
of silica gel and the filtrate was then poured into a separating
funnel containing 0.5 M HCl (100 mL) and extracted with
4.1. General
All NMR spectra were recorded on a Bruker MHz DPX400
1
spectrometer at 400.13 MHz for H and 100.62 MHz for
13C. Low-resolution and high-resolution mass spectra were
obtained on a HP 5989B Mass Spectro-meter by electron
spray ionization mode at an ionizing voltage of 30 eV.
Melting points were measured using Electrothermal IA9100
digital melting point apparatus and were uncorrected.

K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
4153
1
CH2Cl2 (10 mL!3). The combined organic layers were
washed with saturated NaHCO3 (100 mL), dried over
MgSO4, filtered and evaporated to afford a brown oil,
which was subjected to flash column chromatography (20%
EtOAc in n-hexane) on silica gel (25 g) to afford a mixture
of terminal alkene 12 (0.24 g, 48%) and internal alkene 13.
The pure alkene 12 for characterizations was obtained by
afford alcohol 16 (1.23 g, 84%) as white foam: H NMR
(CDCl3) d 1.20 (s, 9H), 2.49 (br, 1H), 3.67–3.85 (m, 10H),
4.16–4.19 (m, 1H), 4.31 (dd, JZ4.8, 10.2 Hz, 1H), 5.09 (s,
2H), 5.27–5.32 (m, 1H), 6.90–6.94 (m, 6H), 7.14 (s, 1H),
7.26–7.42 (m, 8H); 13C NMR (CDCl3) d 26.9, 27.0, 38.7,
52.0, 55.2, 55.7, 55.8, 64.7, 70.8, 72.6, 72.6, 73.3, 81.9,
104.0, 104.1, 111.7, 111.8, 113.4, 113.5, 113.9, 115.1,
120.0, 124.1, 127.2, 127.3, 127.8, 128.0, 128.0, 128.4,
129.3, 129.4, 136.8, 136.8, 140.1, 140.8, 149.0, 149.1,
149.6, 149.7, 161.3, 166.0, 178.6; LRMS m/z 695 (MCC
Na, 20); HRMS Calcd for C38H40O11Na (MCCNa)
695.2468, found 695.2520.
1
further flash column chromatography as white foam: H
NMR (CDCl3) d 3.81–3.90 (m, 9H), 5.12 (s, 2H), 5.24 (d,
JZ10.4 Hz, 1H), 5.33 (dd, JZ8.8, 16.8 Hz, 1H), 5.75 (d,
JZ6.0 Hz, 1H), 6.06–6.10 (m, 1H), 6.82–6.96 (m, 6H), 7.18
(s, 1H), 7.29–7.47 (m, 8H); 13C NMR (CDCl3) d 52.0, 55.3,
55.9, 55.9, 70.9, 82.3, 82.4, 103.9, 103.9, 110.4, 110.5,
111.7, 113.6, 113.7, 114.0, 115.2, 115.3, 119.4, 119.4,
124.0, 127.2, 127.5, 127.8, 128.1, 128.1, 128.5, 132.4,
137.0, 137.2, 137.3, 140.5, 140.5, 148.0, 149.1, 149.7,
161.3, 166.3; LRMS m/z 557 (MCCNa, 12); HRMS Calcd
for C33H30O8Na (MCCNa) 577.1838, found 577.1822.
4.2.5. Methyl 7-[3-(3-methoxy-4-benzyloxyphenyl)-1-
pivaloyloxy-2-tosyloxypropyloxy]-2-(4-methoxyphenyl)-
1,3-benzodioxole-5-carboxylate (17). p-Toluenesulfonyl
chloride (0.90 g, 4.7 mmol) was added to a stirred solution
of alcohol 16 (1.23 g, 1.8 mmol) in anhydrous pyridine
(6 mL) at room temperature. The mixture was stirred for
further 16 h. The reaction was quenched by addition of 1 M
HCl (100 mL) and extracted with CH2Cl2 (10 mL!3). The
combined organic layers were washed with saturated
NaHCO3 (100 mL). After drying over MgSO4, filtration
and evaporation, a pale yellow oil was obtained, which was
subjected to flash column chromatography on silica gel
(20 g) with gradient elution (10–30% EtOAc in n-hexane) to
4.2.3. Methyl 7-[3-(3-methoxy-4-benzyloxyphenyl)-1,2-
dihydroxypropyloxy]-2-(4-methoxyphenyl)-1,3-benzo-
dioxole-5-carboxylate (15). To a round-bottomed flask was
charged with t-BuOH (10 mL), water (10 mL) 4-methyl-
morpholine N-oxide monohydrate (0.54 g, 4 mmol) and
alkene 12 (0.54 g, 1 mmol). Then catalytic amount of OsO4
(0.1 mL, 2.5 wt% solution in t-BuOH) was added and the
resulting mixture was stirred vigorously for 40 h. The
mixture was then cooled to 0 8C and Na2S2O3 (3 g) was
added. The resulting mixture was allowed to warm to room
temperature and was stirred for a further 1 h after which
time the layers were separated. The aqueous layer was
further extracted with CH2Cl2 (10 mL!3) and the com-
bined organic layers were washed with water (100 mL).
After drying over MgSO4, filtration, and evaporation, a pale
yellow oil was obtained, which was subjected to flash
column chromatography on silica gel (10 g) with gradient
elution (20–60% EtOAc in n-hexane) to afford diol 15
(0.28 g, 48%) as white foam: 1H NMR (CDCl3) d 2.52–2.65
(m, 1H), 3.48–3.56 (m, 1H), 3.72–3.79 (m, 1H), 3.80 (s,
9H), 3.96–3.98 (m, 1H), 5.08 (s, 2H), 5.26–5.32 (m, 1H),
6.80–6.93 (m, 6H), 7.13 (s, 1H), 7.25–7.42 (m, 8H); 13C
NMR (CDCl3) d 52.0, 55.3, 55.8, 55.9, 62.2, 62.8, 70.8,
74.3, 75.4, 76.7, 81.9, 82.2, 104.0, 104.1, 110.6, 111.8,
113.4, 114.0, 114.5, 114.8, 119.8, 119.9, 124.0, 127.2,
127.3, 127.8, 128.0, 128.0, 128.5, 129.6, 129.9, 136.8,
140.2, 140.3, 140.4, 140.4, 140.7, 148.3, 148.9, 148.9,
149.0, 149.6, 149.7, 161.3, 166.1; LRMS m/z 611 (MCC
Na, 37); HRMS Calcd for C33H32O10Na (MCCNa)
611.1893, found 611.1909.
1
afford tosylate 17 (1.06 g, 72%) as white foam: H NMR
(CDCl3) d 1.16 (s, 9H), 2.34 (s, 3H), 3.64–3.85 (m, 9H),
3.69–3.97 (m, 1H), 4.31–4.52 (m, 1H), 5.00–5.08 (m, 3H),
5.41–5.49 (m, 1H), 6.96–7.53 (m, 19H); 13C NMR (CDCl3)
d 21.4, 26.9, 38.6, 38.6, 51.9, 55.2, 55.3, 55.6, 70.6, 70.6,
80.5, 80.6, 95.5, 111.8, 113.9, 113.9, 124.0, 127.0, 127.1,
127.1, 127.5, 127.5, 127.6, 127.7, 127.8, 127.8, 127.9,
128.0, 128.4, 128.4, 129.3, 129.4, 136.6, 136.7, 140.5,
144.5, 149.0, 161.2, 165.8, 165.8; LRMS m/z 849 (MCC
Na, 13); HRMS Calcd for C45H46O13NaS (MCCNa)
849.2557, found 849.2606.
4.2.6. syn-methyl 3,4-dihydroxy-5-[3-(3-methoxy-4-ben-
zyloxy-phenyl)-1-pivaloyloxy-2-tosyloxypropyloxy]-ben-
zoate (18) and anti-methyl 3,4-dihydroxy-5-[3-(3-
methoxy-4-benzyloxyphenyl)-1-pivaloyloxy-2-tosyloxy-
propyloxy]-benzoate (19). p-Toluenesulfonic acid mono-
hydrate (0.70 g, 3.7 mmol) was added to a stirred solution of
tosylate 17 (1.06 g, 1.3 mmol) in CH2Cl2 (20 mL) at room
temperature. The mixture was stirred for further 1 h. The
reaction was quenched by addition of saturated NaHCO3
(100 mL) and extracted with CH2Cl2 (10 mL!3). The
combined organic layers were dried over MgSO4, filtered
and evaporated to furnish pale brown oil, which was
subjected to flash column chromatography on silica gel
(25 g) with gradient elution (20–50% EtOAc in n-hexane) to
afford the diastereomeric tosylates. Less polar tosylate syn-
18 (0.38 g, 42%) as white foam: 1H NMR (CDCl3) d 1.15 (s,
9H), 2.42 (s, 3H), 3.64 (dd, JZ3.2, 12.8 Hz, 1H), 3.76 (s,
3H), 3.87 (s, 3H), 4.35 (dd, JZ2.0, 12.8 Hz, 1H), 5.12–5.18
(m, 4H), 6.91 (m, 4H), 7.27–7.80 (m, 10H); 13C NMR
(CDCl3) d 21.6, 27.0, 38.8, 51.9, 56.1, 61.6, 70.9, 80.1, 80.5,
107.6, 109.7, 111.1, 114.0, 119.5, 121.2, 127.3, 127.7,
127.8, 128.0, 128.6, 129.8, 132.8, 136.6, 137.9, 144.2,
144.8, 145.5, 149.2, 150.3, 166.6, 177.7; LRMS m/z 731
(MCCNa, 9); HRMS Calcd for C37H40O12NaS (MCCNa)
731.2138, found 731.2183. More polar tosylate anti-19
4.2.4. Methyl 7-[3-(3-methoxy-4-benzyloxyphenyl)-1-
pivaloyloxy-2-hydroxypropyloxy]-2-(4-methoxyphenyl)-
1,3-benzodioxole-5-carboxylate (16). Pivaloyl chloride
(0.5 mL, 4.1 mmol) was added slowly to a stirred solution
of diol 15 (1.21 g, 2.2 mmol) in anhydrous pyridine (15 mL)
at 0 8C under N2. The mixture was stirred for further 0.5 h.
The reaction was quenched by addition of 1 M HCl
(100 mL) and extracted with CH2Cl2 (10 mL!3). The
combined organic layers were washed with saturated
NaHCO3 (100 mL). After drying over MgSO4, filtration
and evaporation, a pale yellow oil was obtained, which was
subjected to flash column chromatography on silica gel
(20 g) with gradient elution (10–30% EtOAc in n-hexane) to

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K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
(0.42 g, 46%) was isolated as white foam: 1H NMR (CDCl3)
d 1.15 (s, 9H), 2.41 (s, 3H), 3.75 (s, 3H), 3.78 (s, 3H), 3.90
(dd, JZ7.6, 12.4 Hz, 1H), 4.23 (dd, JZ4.0, 12.0 Hz, 1H),
5.07 (s, 2H), 5.31 (d, JZ3.6 Hz, 1H), 5.42–5.46 (m, 1H),
6.79–6.96 (m, 4H), 7.26–7.41 (m, 8H), 7.74 (d, JZ8.4 Hz,
2H); 13C NMR (CDCl3) d 21.6, 27.0, 38.7, 51.9, 55.7, 62.1,
70.8, 78.2, 80.2, 107.7, 110.5, 111.2, 113.2, 120.1, 121.1,
126.3, 127.2, 127.4, 127.9, 128.5, 129.8, 133.4, 136.7,
138.3, 143.6, 144.2, 145.3, 148.8, 149.7, 166.6, 178.0;
LRMS m/z 731 (MCCNa, 28); HRMS Calcd for
C37H40O12NaS (MCCNa) 731.2138, found 731.2114.
charged with ester 21 (0.23 g, 0.42 mmol), 5% palladium
on activated carbon (15 mg) and EtOAc (20 mL). The
reaction mixture was then subjected to a H2 atmosphere at
balloon pressure and stirred for 14 h. When TLC indicated
complete consumption of ester 21, the reaction mixture was
filtered through and the solvent was evaporated. The
resulted residue was subjected to flash column chromato-
graphy (15 g) with gradient elution (10–30% EtOAc in
n-hexane) to afford ester 22 (0.19 g, 99%) as white foam: 1H
NMR (CDCl3) d 1.17 (s, 9H), 3.84 (s, 6H), 3.88 (s, 3H), 3.99
(dd, JZ3.6, 12.4 Hz, 1H), 4.28 (dd, JZ3.6, 7.0 Hz, 1H),
4.36 (dd, JZ2.4, 12.2 Hz, 1H), 4.62 (d, JZ8.0 Hz, 1H),
6.03 (s, 1H), 6.83–6.92 (m, 3H), 7.21 (s, 1H), 7.33 (s, 1H);
13C NMR (CDCl3) d 27.0, 38.7, 52.0, 55.8, 56.2, 62.5, 75.9,
76.2, 105.8, 109.4, 111.9, 114.7, 120.6, 122.2, 127.0, 137.0,
143.6, 146.5, 146.9, 148.5, 166.5, 177.7; LRMS m/z 483
(MCCNa, 100); HRMS Calcd for C24H28O9Na (MCCNa)
483.1631, found 483.1632.
4.2.7. trans-methyl 2-pivaloyloxymethyl-8-hydroxy-3-(3-
methoxy-4-benzyloxyphenyl)-2,3-dihydrobenzo[1,4]-
dioxane-6-carboxylate (20). To a round-bottom flask was
charged with tosylate 19 (0.42 g, 0.59 mmol), K2CO3
(0.16 g, 1.2 mmol) and acetone (50 mL). The reaction
mixture was then heated to reflux for 2 h. When TLC
indicated complete consumption of tosylate 19, the reaction
mixture was cooled and filtered through a short pad of silica
gel. The solvent was evaporated and the resulting residue
was subjected to flash column chromatography on silica gel
(20 g) with gradient elution (10–30% EtOAc in n-hexane) to
afford ester 20 (0.25 g, 79%) as white foam: 1H NMR
(CDCl3) d 1.20 (s, 9H), 3.84 (s, 3H), 3.87–3.91 (m, 1H),
3.89 (s, 3H), 4.27–4.30 (m, 1H), 4.52 (dd, JZ2.4, 12.4 Hz,
1H), 4.91 (d, JZ8.4 Hz, 1H), 5.16 (s, 2H), 6.25 (br, 1H),
6.89–6.92 (m, 3H), 7.26–7.30 (m, 3H), 7.34–7.38 (m, 2H),
7.42–7.44 (m, 2H); 13C NMR (CDCl3) d 27.0, 38.8, 52.0,
56.0, 62.0, 70.8, 76.3, 76.6, 109.7, 110.4, 110.6, 113.7,
119.9, 122.9, 127.1, 127.8, 128.5, 135.0, 136.6, 143.5,
144.9, 149.0, 149.9, 166.5; LRMS m/z 559 (MCCNa, 49);
HRMS Calcd for C30H32O9Na (MCCNa) 559.1944, found
559.1970.
4.2.10. trans-methyl 2-hydroxymethyl-8-methoxy-3-(3-
methoxy-4-hydroxyphenyl)-2,3-dihydrobenzo[1,4]-diox-
ane-6-carboxylate (23). To a round-bottom flask was
charged with ester 22 (0.14 g, 0.30 mmol), K2CO3 (0.12 g,
0.87 mmol) and methanol (20 mL). The reaction mixture
was then heated to reflux for 2 h. When TLC indicated
complete consumption of ester 22, the reaction mixture was
then cooled and neutralized with 1 M HCl solution and
extracted with CH2Cl2 (10 mL!3). The combined organic
layers were dried over MgSO4, filtered and evaporate to
furnish a colorless oil, which was subjected to flash column
chromatography on silica gel (15 g) with gradient elution
(20–40% EtOAc in n-hexane) to afford ester 23 (0.11 g,
1
96%) as white solid: mp 138–140 8C; H NMR (CDCl3) d
3.02 (br, 1H), 3.57 (br, 1H), 3.86 (s, 6H), 3.87–3.89 (m, 2H),
3.91 (s, 3H), 4.02–4.05 (m, 1H), 4.94 (d, JZ8.0 Hz, 1H),
6.03 (br, 1H), 6.90–6.92 (m, 3H), 7.22 (d, JZ1.2 Hz, 1H),
7.35 (d, JZ1.2 Hz, 1H); 13C NMR (CDCl3) d 52.1, 55.9,
56.1, 61.2, 75.9, 78.9, 105.3, 109.6, 112.2, 114.7, 120.6,
122.1, 127.5, 137.1, 143.8, 146.3, 146.9, 148.3, 166.6;
LRMS m/z 399 (MCCNa, 49); HRMS Calcd for
C19H20O8Na (MCCNa) 399.1056, found 399.1054.
4.2.8. trans-methyl 2-pivaloyloxymethyl-8-methoxy-3-(3-
methoxy-4-benzyloxyphenyl)-2,3-dihydrobenzo[1,4]-
dioxane-6-carboxylate (21). To a round-bottom flask was
charged with ester 20 (0.25 g, 0.47 mmol), K2CO3 (0.12 g,
0.87 mmol), dimethyl sulfate (0.15 g, 5.8 mmol) and
acetone (50 mL). The reaction mixture was then heated to
reflux for 2 h. When TLC indicated complete consumption
of ester 20, the reaction mixture was cooled and filtered
through a short pad of silica gel. The solvent was washed
with 0.5 M NaOH solution and extracted with CH2Cl2
(10 mL!3). The combined organic layers were evaporated
and the residue was subjected to flash column chromato-
graphy (15 g) with gradient elution (10–20% EtOAc in
n-hexane) to afford product 21 (0.23 g, 90%) as colorless
4.2.11. trans-methyl 2-tert-butyldimethylsilyloxymethyl-
8-methoxy-3-(3-methoxy-4-tert-butyldimethylsilyloxy-
phenyl)-2,3-dihydrobenzo[1,4]dioxane-6-carboxylate
(24). To a round-bottom flask was charged with ester 23
(0.14 g, 0.37 mmol), imidazole (76 mg, 1.12 mmol) and
anhydrous THF (20 mL). tert-butyldimethylsilyl chloride
(TBSCl) (0.11 g, 0.73 mmol) was added in one portion and
the reaction mixture immediately became milky. When
TLC indicated complete consumption of ester 23 (typically
2 h), the reaction mixture was quenched by washing with
saturated NaHCO3. The aqueous layer was extracted with
CH2Cl2 (10 mL!3). The combined organic layers were
dried over MgSO4, evaporated. The resulting residue was
subjected to flash column chromatography on silica gel
(15 g) with elution (5% EtOAc in n-hexane) to afford ester
1
oil: H NMR (CDCl3) d 1.19 (s, 9H), 3.86 (s, 3H), 3.89 (s,
3H), 3.91 (s, 3H), 4.01 (dd, JZ4.0, 12.0 Hz, 1H), 4.29–4.31
(m, 1H), 4.38 (dd, JZ3.2, 12.4 Hz, 1H), 4.90 (d, JZ8.0 Hz,
1H), 5.16 (s, 2H), 6.87–6.90 (m, 3H), 7.24–7.44 (m, 7H);
13C NMR (CDCl3) d 27.0, 38.7, 52.0, 55.9, 56.2, 62.4, 70.7,
75.8, 76.1, 105.7, 110.3, 111.9, 113.6, 119.8, 122.2, 127.1,
127.8, 128.1, 128.4, 136.6, 136.9, 143.6, 148.5, 148.8,
149.8, 166.4, 177.6; LRMS m/z 573 (MCCNa, 54); HRMS
Calcd for C31H34O9Na (MCCNa) 573.2101, found
573.2054.
1
24 (0.21 g, 93%) as colorless oil: H NMR (CDCl3) d 0.06
(s, 6H), 0.15 (s, 6H), 0.88 (s, 9H), 0.99 (s, 9H), 3.58 (dd, JZ
2.4, 11.8 Hz, 1H), 3.80 (s, 3H), 3.85 (s, 3H), 3.91 (s, 3H),
3.94 (dd, JZ2.4, 12.0 Hz, 1H), 4.03–4.05 (m, 1H), 4.99 (d,
JZ8.0 Hz, 1H), 6.85–6.93 (m, 3H), 7.23 (d, JZ1.6 Hz, 1H),
7.36 (d, JZ1.6 Hz, 1H); 13C NMR (CDCl3) d K5.7, K5.4,
4.2.9. trans-methyl 2-pivaloyloxymethyl-8-methoxy-3-(3-
methoxy-4-hydroxyphenyl)-2,3-dihydrobenzo[1,4]-diox-
ane-6-carboxylate (22). To a round-bottom flask was

K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
4155
K4.7, K4.7, 18.2, 18.3, 25.6, 25.6, 25.7, 51.9, 55.3, 56.2,
62.0, 75.7, 78.8, 105.7, 111.1, 112.1, 119.9, 120.9, 121.7,
129.5, 137.7, 143.7, 145.4, 148.6, 151.0, 166.7; LRMS m/z
627 (MCCNa, 100); HRMS Calcd for C31H48O8NaSi2
(MCCNa) 627.2785, found 627.2796.
added at once and the reaction mixture turned yellow
immediately and was stirred for 24 h at room temperature.
When TLC indicated complete consumption of aldehyde 26,
water (100 mL) was added. The resulting mixture was
extracted with EtOAc (10 mL!3). The combined organic
layers were dried over MgSO4, evaporated and subjected to
flash column chromatography on silica gel (15 g) with
elution (20% EtOAc in n-hexane) to afford chalcone 27
(84 mg, 75%) as yellow foam: 1H NMR (CDCl3) d 2.77 (br,
1H), 3.47 (s, 3H), 3.51 (s, 3H), 3.52–3.53 (m, 1H), 3.87–
3.90 (m, 1H), 3.90 (s, 3H), 3.92 (s, 3H), 4.04–4.08 (m, 1H),
4.96 (d, JZ8.4 Hz, 1H), 5.17 (s, 2H), 5.26 (s, 2H), 5.97 (s,
1H), 6.21 (d, JZ2.0 Hz, 1H), 6.30 (d, JZ2.0 Hz, 1H), 6.78
(d, JZ1.6 Hz, 1H), 6.91–6.95 (m, 4H), 7.67 (d, JZ15.6 Hz,
1H), 7.79 (d, JZ15.2 Hz, 1H), 13.90 (s, 1H); 13C NMR
(CDCl3) d 56.0, 56.4, 56.9, 61.3, 76.1, 78.8, 94.0, 94.7, 95.1,
97.4, 104.4, 107.4, 109.6, 110.4, 114.7, 120.7, 125.8, 127.6,
128.0, 135.1, 142.7, 144.5, 146.4, 146.9, 148.8, 159.7,
163.3, 167.2, 192.6; LRMS m/z 585 (MCCH, 35), HRMS
Calcd for C30H33O12 (MCCH) 585.5093, found 585.5085.
4.2.12. trans-2-tert-butyldimethylsilyloxymethyl-8-meth-
oxy-3-(3-methoxy-4-tert-butyldimethylsilyloxy-phenyl)-
2,3-dihydrobenzo[1,4]dioxane-6-methanol (25). To a
well-stirred solution of ester 24 (0.20 g, 0.33 mmol) in
anhydrous THF (10 mL) at 0 8C under N2 atmosphere was
added diisobutylaluminum hydride (DIBAL-H) (1 M in
hexane, 0.8 mL, 0.8 mmol). The mixture was stirred for
further 15 minutes at 0 8C and at room temperature for 2 h.
The reaction was quenched by addition of Et2O (50 mL),
H2O (1 mL) and 2 M NaOH (2 mL) until a heavily white
precipitate was formed. The precipitate was removed by
filtration and the filtrate was washed with saturated NH4Cl
solution, separated and dried over MgSO4. Removal of the
solvent under reduced pressure gave alcohol 25 (0.18 g,
94%) as colorless oil: 1H NMR (CDCl3) d 0.06 (s, 6H), 0.16
(s, 6H), 0.88 (s, 9H), 0.98 (s, 9H), 2.15 (br, 1H), 3.56 (dd,
JZ2.8, 11.6 Hz, 1H), 3.80 (s, 3H), 3.86 (s, 3H), 3.92 (dd,
JZ2.4, 11.8 Hz, 1H), 3.96–3.99 (m, 1H), 4.53 (s, 2H), 4.98
(d, JZ7.6 Hz, 1H), 6.56 (s, 1H), 6.60 (s, 1H), 6.85–6.93 (m,
3H); 13C NMR (CDCl3) d K5.6, K5.3, K4.7, 18.2, 18.4,
25.6, 25.8, 55.3, 56.2, 62.2, 65.2, 75.8, 78.3, 103.6, 108.5,
111.2, 120.0, 120.8, 130.0, 132.8, 132.9, 144.1, 145.3,
148.9, 150.9; LRMS m/z 599 (MCCNa, 100); HRMS Calcd
for C30H48O7NaSi2 (MCCNa) 599.2836, found 599.2814.
4.2.15. trans-5-hydroxy-2-(2-hydroxymethyl-8-methoxy-
3-(3-methoxy-4-hydroxyphenyl)-2,3-dihydrobenzo[1,4]-
dioxin-6-yl)-7-methoxymethoxychromen-4-one (28). To
a round-bottom flask was charged with chalcone 27 (70 mg,
0.12 mmol), selenium dioxide (53 mg, 0.48 mmol), three
drops of DMSO and t-butanol (10 mL). The reaction
mixture was heated to reflux for 48 h until 1H NMR
indicated complete consumption of chalcone 27. If the
reaction did not complete, additional portions of selenium
dioxide and DMSO were added. The reaction mixture was
then quenched by adding water (100 mL) and the aqueous
layer was then extracted with EtOAc (10 mL!3). The
combined organic layers were dried over MgSO4, evapor-
ated to afford chromenone 28 (46 mg, 75%) as yellow foam;
1H NMR (CDCl3) d 3.48 (s, 3H), 3.56–3.59 (m, 1H), 3.90 (s,
3H), 3.94 (s, 3H), 4.06–4.11 (m, 1H), 4.98 (d, JZ8.4 Hz,
1H), 5.22 (s, 2H), 6.54 (s, 1H), 6.60 (s, 1H), 6.63 (s, 1H),
6.87–6.95 (m, 3H), 7.21 (s, 1H), 12.60 (br, 1H); LRMS m/z
539 (MCCH, 100); HRMS Calcd for C28H27O11 (MCCH)
539.1553, found 539.1547.
4.2.13. trans-2-tert-butyldimethylsilyloxymethyl-8-meth-
oxy-3-(3-methoxy-4-tert-butyldimethylsilyloxy-phenyl)-
2,3-dihydrobenzo[1,4]dioxane-6-carbaldehyde (26). To a
well-stirred mixture of pyridinium dichromate (PDC)
˚
(0.16 g, 0.43 mmol) and 4 A powdered molecular sieves
(1 g) in anhydrous CH2Cl2 (20 mL) at room temperature,
was added alcohol 25 (0.12 g, 0.21 mmol) dissolved in
CH2Cl2 (10 mL). The resulting mixture darkened immedi-
ately and was stirred under N2 atmosphere for 12 h. The
resulting dark brown mixture was filtered through a short
pad of silica gel and the filter cake was washed with EtOAc
(50 mL). The pale brown filtrate was evaporated and the
residue was chromatographed on silica gel (20 g, 15%
EtOAc in n-hexane) to afford aldehyde 26 (0.1 g, 84%) as
colorless oil: 1H NMR (CDCl3) d 0.06 (s, 6H), 0.16 (s, 6H),
0.88 (s, 9H), 0.98 (s, 9H), 3.59 (dd, JZ2.4, 12.0 Hz, 1H),
3.81 (s, 3H), 3.93 (s, 3H), 3.96 (dd, JZ2.4, 12.2 Hz, 1H),
4.06–4.08 (m, 1H), 5.00 (d, JZ7.6 Hz, 1H), 6.87–6.93 (m,
3H), 7.10 (d, JZ1.6 Hz, 1H), 7.13 (d, JZ1.6 Hz, 1H), 9.77
(s, 1H); 13C NMR (CDCl3) d K5.6, K5.4, K4.7, 18.2, 18.4,
25.6, 25.8, 55.4, 56.2, 61.9, 75.8, 79.0, 103.3, 111.1, 114.5,
120.0, 121.0, 128.9, 129.2, 139.3, 144.3, 145.6, 149.6,
151.1, 190.8; LRMS m/z 597 (MCCNa, 100); HRMS Calcd
for C30H46O7NaSi2 (MCCNa) 597.2680, found 597.2674.
4.2.16. (G)-50-Methoxyhydnocarpin-D (6). To a 50 mL
round-bottom flask was charged with chromenone 28
(40 mg, 0.07 mmol) and 20% AcOH in H2O. The reaction
mixture was heated to reflux for 1 h. The reaction mixture
was then quenched by neutralizing with saturated NaHCO3
solution and the aqueous layer was then extracted with
EtOAc (10 mL!3). The obtained organic layer was dried
over MgSO4, evaporated to afford 6 (19 mg, 52%) as yellow
solid. The pure sample was obtained by by washing the
yellow solid with boiling chloroform: mp 210 8C (decomp.);
1H NMR (d6-DMSO) d 3.51–5.44 (m, 1H), 3.82–3.86 (m,
1H), 3.95 (s, 3H), 3.97 (s, 3H), 4.17–4.19 (m, 1H), 5.04 (d,
JZ8.0 Hz, 1H), 6.23 (d, JZ1.6 Hz, 1H), 6.55 (s, 1H), 6.71
(s, 1H), 6.89 (d, JZ8.0 Hz, 1H), 6.98–7.00 (m, 1H), 7.14 (d,
JZ4.4 Hz, 1H), 7.25 (s, 2H), 9.77 (br, 1H), 12.90 (s, 1H);
13C NMR (d6-DMSO) d 56.1, 56.5, 60.3, 76.2, 78.7, 94.6,
99.3, 103.2, 104.2, 104.5, 108.5, 112.2, 115.7, 121.0, 122.7,
127.5, 137.0, 144.6, 147.5, 148.0, 149.5, 157.8, 161.8,
163.4, 164.6, 182.2; LRMS m/z 495 (MCCH, 100); HRMS
Calcd for C26H23O10 (MCCH) 495.1291, found 495.1327.
4.2.14. trans-3-[2-hydroxymethyl-8-methoxy-3-(3-meth-
oxy-4-hydroxyphenyl)-2,3-dihydrobenzo[1,4]-dioxin-6-
yl]-1-[2-hydroxy-4,6-bis(methoxymethoxy)phenyl]pro-
penone (27). To a round-bottom flask was charged with
aldehyde 26 (0.11 g, 0.19 mmol), 2-hydroxy-4,6-bis-
(methoxymethoxy)-acetophenone (86 mg, 0.34 mmol) and
ethanol (5 mL). 2 M KOH solution in ethanol (10 mL) was

4156
K.-F. Chan et al. / Tetrahedron 61 (2005) 4149–4156
Rapisarda, A.; Pappalardo, M. S.; Iauk, L.; Int J. Antimicrob.
Acknowledgements
Agents 2003, 22, 48–53.
We thank the Areas of Excellence Scheme established under
the University Grants Committee of the Hong Kong
Administrative Region, China (Project Nos.: 5296/01P and
AoE/P- 10/01) and The Hong Kong Polytechnic University
for financial support.
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