Synthesis and biological evaluation of dihydrobenzofuran lignans and related compounds as potential antitumor agents that inhibit tubulin polymerization

Article abstract of DOI:10.1021/jm990251m

A series of 19 related dihydrobenzofuran lignans and benzofurans was obtained by a biomimetic reaction sequence involving oxidative dimerization of p-coumaric, caffeic, or ferulic acid methyl esters, followed by derivatization reactions. All compounds were evaluated for potential anticancer activity in an in vitro human disease-oriented tumor cell line screening panel that consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse histologies. Leukemia and breast cancer cell lines were relatively more sensitive to these agents than were the other cell lines. Compounds 2c and 2d, but especially 2b (methyl (E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-3- methoxycarbonyl-2,3-dihydro-1-benzofuran-5-yl]prop-2-enoate), the dimerization product of caffeic acid methyl ester, containing a 3',4'-dihydroxyphenyl moiety and a hydroxyl group in position 7 of the dihydrobenzofuran ring, showed promising activity. The average GI₅₀ value (the molar drug concentration required for 50% growth inhibition) of 2b was 0.3 μM. Against three breast cancer cell lines, 2b had a GI₅₀ value of < 10 nM. Methylation, reduction of the double bond of the C₃-side chain, reduction of the methoxycarbonyl functionalities to primary alcohols, or oxidation of the dihydrobenzofuran ring to a benzofuran system resulted in a decrease or loss of cytotoxic activity. Compound 2b inhibited mitosis at micromolar concentrations in cell culture through a relatively weak interaction at the colchicine binding site of tubulin. In vitro it inhibited tubulin polymerization by 50% at a concentration of 13 ± 1 μM. The 2R,3R-enantiomer of 2b was twice as active as the racemic mixture, while the 2S,3S- enantiomer had minimal activity as an inhibitor of tubulin polymerization. These dihydrobenzofuran lignans (2-phenyl- dihydrobenzofuran derivatives) constitute a new group of antimitotic and potential antitumor agents that inhibit tubulin polymerization.

Full text of DOI:10.1021/jm990251m

J . Med. Chem. 1999, 42, 5475-5481
5475
Brief Articles
Syn th esis a n d Biologica l Eva lu a tion of Dih yd r oben zofu r a n Lign a n s a n d Rela ted
Com p ou n d s a s P oten tia l An titu m or Agen ts th a t In h ibit Tu bu lin P olym er iza tion
Luc Pieters,*,† Stefaan Van Dyck,‡ Mei Gao,‡ Ruoli Bai,§ Ernest Hamel,| Arnold Vlietinck,† and Guy Lemie`re‡
Department of Pharmaceutical Sciences, University of Antwerp, B-2610 Antwerp, Belgium, Department of Chemistry,
University of Antwerp, B-2020 Antwerp, Belgium, Science Applications International CorporationsFrederick,
Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and Laboratory of Drug Discovery Research
and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702
Received May 20, 1999
A series of 19 related dihydrobenzofuran lignans and benzofurans was obtained by a biomimetic
reaction sequence involving oxidative dimerization of p-coumaric, caffeic, or ferulic acid methyl
esters, followed by derivatization reactions. All compounds were evaluated for potential
anticancer activity in an in vitro human disease-oriented tumor cell line screening panel that
consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse
histologies. Leukemia and breast cancer cell lines were relatively more sensitive to these agents
than were the other cell lines. Compounds 2c and 2d , but especially 2b (methyl (E)-3-[2-(3,4-
dihydroxyphenyl)-7-hydroxy-3-methoxycarbonyl-2,3-dihydro-1-benzofuran-5-yl]prop-2-eno-
ate), the dimerization product of caffeic acid methyl ester, containing a 3′,4′-dihydroxyphenyl
moiety and a hydroxyl group in position 7 of the dihydrobenzofuran ring, showed promising
activity. The average GI50 value (the molar drug concentration required for 50% growth
inhibition) of 2b was 0.3 µM. Against three breast cancer cell lines, 2b had a GI50 value of <10
nM. Methylation, reduction of the double bond of the C3-side chain, reduction of the
methoxycarbonyl functionalities to primary alcohols, or oxidation of the dihydrobenzofuran
ring to a benzofuran system resulted in a decrease or loss of cytotoxic activity. Compound 2b
inhibited mitosis at micromolar concentrations in cell culture through a relatively weak
interaction at the colchicine binding site of tubulin. In vitro it inhibited tubulin polymerization
by 50% at a concentration of 13 ( 1 µM. The 2R,3R-enantiomer of 2b was twice as active as
the racemic mixture, while the 2S,3S-enantiomer had minimal activity as an inhibitor of tubulin
polymerization. These dihydrobenzofuran lignans (2-phenyl-dihydrobenzofuran derivatives)
constitute a new group of antimitotic and potential antitumor agents that inhibit tubulin
polymerization.
In tr od u ction
chain, in neolignans the two C6C3 units are not linked
by a â-â bond.3 Lignans and neolignans display a wide
variety of chemical structures, are widespread in nature,
and exhibit a broad range of biological activities, includ-
ing for some classes antitumor activities, e.g., the
podophyllotoxin group of lignans.4
To explore the potential antiproliferative and antitu-
moral activity of 3′,4-di-o-methylcedrusin (4d ), obtained
in low yield from its natural source, a synthesis from
various C6C3 precursors was devised. In this way both
4d and a series of synthetic dihydrobenzofuran lignans
were obtained. Their cytotoxicity was determined in an
in vitro human disease-oriented tumor cell line screen-
ing panel. A structure-activity relationship could be
established, and the mechanism of action was deter-
mined.
3′,4-Di-O-methylcedrusin (4d ), 3-[2-(3,4-dimethox-
yphenyl)-3-hydroxymethyl-7-methoxy-2,3-dihydro-1-ben-
zofuran-5-yl]propan-1-ol, was identified as one of the
minor constituents of the red latex called ‘dragon’s blood’
(‘sangre de drago’) in traditional medicine. This latex
is obtained by slashing the bark of various South
American Croton species (Euphorbiaceae). Lignan 4d ,
belonging to the neolignans, was found to act as an
inhibitor of cell proliferation1 and has been assigned a
2R,3S-configuration.2 Lignans and neolignans are formed
in nature by the oxidative dimerization of various C6C3
phenols. While lignans are defined as those compounds
in which the two C6C3 units are linked by a bond
connecting the central (â) carbon atom of each side
* To whom correspondence should be addressed. E-mail: pieters@
uia.ua.ac.be.
Ch em istr y
† Department of Pharmaceutical Sciences, University of Antwerp.
‡ Department of Chemistry, University of Antwerp.
§ Science Applications International CorporationsFrederick.
| Laboratory of Drug Discovery Research and Development, NCI.
A series of 19 related dihydrobenzofuran lignans and
benzofurans was synthesized as shown in Scheme 1. A
biomimetic oxidative coupling of the p-coumaric (1a ),
10.1021/jm990251m CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/04/1999

5476 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Sch em e 1. Synthetic Routes
Brief Articles
F igu r e 1.
of 2b was assigned a 2R,3R-configuration and the
second eluting enantiomer a 2S,3S-configuration.
Resu lts a n d Discu ssion
(a) Evalu ation of th e Cytotoxicity of Dih ydr oben -
zofu r a n Lign a n s. All dihydrobenzofuran lignans syn-
thesized were evaluated in the in vitro human disease-
oriented tumor cell line screening panel developed at
the NCI. The average log GI50 values (GI50 being the
molar drug concentration required for half growth
inhibition) calculated from all cell lines tested, as well
as the log GI50 values for a series of representative cell
lines, are listed in Table 1 (see Supporting Information).
The complete set of data obtained in this screening
panel showed that the leukemia cell lines and the breast
cancer cell lines were relatively more sensitive to the
cytotoxic dihydrobenzofuran lignans than were other
cell lines. Three dihydrobenzofuran lignans (2b, 2c, and
2d (2R,3R)) showed an average log GI50 < -5 (corre-
sponding to average GI50 values of 0.3, 3.3, and 5.1 µM,
respectively) and were selected for further evaluation
as potential antitumor agents. Compound 2b was the
dimerization product of caffeic acid methyl ester, con-
taining a 3′,4′-dihydroxyphenyl moiety and a hydroxyl
group in position 7 of the dihydrobenzofuran ring
(Figure 1). Compound 2c was the dimerization product
of ferulic acid methyl ester, with only one free hydroxyl
group left in position 4′; compound 2d was the methyl-
ated derivative of 2c. All three compounds still con-
tained the methyl ester functionality and the double
bond in the side chain. Apparently methylation of the
hydroxyl groups reduced activity, since the caffeic acid
derivative 2b was 10 times more active than the ferulic
acid derivative 2c. Methylation of 2c led to a further
loss of activity. Of all the dihydrobenzofuran lignans
tested, compound 2b showed the greatest cytotoxicity
against every cancer cell line examined. Against three
breast cancer cell lines (MDA-MB-435, MDA-N, and BT-
549) the GI50 for 2b was <10 nM. Submicromolar GI50
values were observed against additional leukemia and
breast cancer cell lines evaluated in the NCI screen as
follows: 0.033 (CCRF-CEM), 0.081 (MOLT-4), 0.055
(RPMI-8226), 0.069 µM (SR) (leukemia cell lines); 0.029
(MCF7), 0.054 (MCF7/ADR-RES), 0.050 (MDA-MB-
231/ATCC), 0.045 µM (HS 578 T) (breast cancer cell
lines).
caffeic (1b), and ferulic acid (1c) methyl esters in the
presence of silver oxide is the crucial step in the reaction
sequence, generating the dihydrobenzofuran skeleton
with a 2,3-trans-configuration.5 The 4′-OH group of 2c
has been methylated with methyl iodide in the presence
of potassium carbonate. Hydrogenation of the double
bond of the propenoates 2a -d in the presence of Pd-C
yields the corresponding propanoates 3a -d . Prolonged
reaction times or large amounts of catalyst have to be
avoided since this results in opening of the dihydrofuran
ring as shown in the sequence 2d f 3d f 7d . LiAlH4
reduction of both ester functions of 3a -d gave the
primary alcohols 4a -d . Two benzofuran lignans were
prepared from 2d by dehydrogenation to 5d with DDQ,
followed by hydrogenation of the side chain in the
presence of Pd-C to 6d .
The racemic products 2b and 2d have been resolved
into their enantiomers by preparative-scale chiral HPLC.2
The enantiomers of 3d and 4d have been synthesized
from the corresponding enantiomers of 2d by the same
methodology as the racemic compounds (Scheme 1). The
absolute configuration of the enantiomers of 2d -4d was
determined from a comparison of their CD spectra with
the CD spectra of ephedradin A, a compound with a
similar chromophore and with a known absolute con-
figuration determined by anomalous dispersion X-ray
crystallography.6 In this way the naturally occurring
3′,4-di-O-methylcedrusin has been assigned a 2R,3S-
configuration. This absolute configuration has been
confirmed independently by chemical correlation with
a (1S,4R)-camphanoyl derivative of a dihydrobenzofuran
lignan whose absolute configuration was determined by
an X-ray crystallographic study.7 CD spectra of both
enantiomers of 2b have been recorded. Comparison with
the CD spectra of the enantiomers of 2d 2 allowed
assignment of the absolute configuration (see Support-
ing Information, Figure 2). The first eluting enantiomer
The series 2b f 3b f 4b suggests the following
structure-activity conclusions: reduction of the double
bond in the C3 side chain (2b f 3b) caused over a 10-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5477
fold decrease in activity (average log GI50 from -6.54
to -4.97), while additional reduction of the methyl ester
functionality to a primary alcohol (3b f 4b) led to an
additional 10-fold loss in activity (average log GI50 -4.01
for 4b). Most compounds tested were racemic mixtures,
except for 2d , 3d , and 4d . Compound 2d allows us to
conclude that activity was related to the stereochemistry
of the dihydrofuran ring, the 2R,3R-isomer being the
more active one. To our surprise the initial natural lead
compound 3′,4-di-O-methylcedrusin (4d ) showed no
activity in the 60 cell line panel up to the highest
concentration tested (100 µM).
For all compounds tested, including the most active
ones, no TGI (total growth inhibition) or LC50 (50% cell
kill) level was reached for most cell lines (log TGI and
log LC50 > -4.00, 100 µM being the highest concentra-
tion tested). This indicated that their mechanism of
action was cytostatic rather than cytotoxic within the
48 h time frame of the assay. The COMPARE algo-
rithm,8 a program that compares a complete set of cell
sensitivities to those of standard agents or other agents
present in the NCI database, showed a correlation
(correlation coefficient ) 0.735) between 2b and a
number of combretastatins,9 which are known to act as
antimitotic compounds by inhibiting tubulin polymer-
ization (Figure 1). Correlation coefficients >0.6 can be
considered significant. In addition, the flat configuration
of the dose-response curves, obtained when TGI or LC50
values are not reached in the screening assays, is
observed with many antitubulin agents.
potent: (14.0 ( 0.2)% inhibition occurred with the
compound at 5.0 µM and (53 ( 3)% at 50 µM 2b.
Compound 2b was also evaluated for its effects on the
growth of HL 60 human leukemia cells. An IC50 value
of 0.2 µM for 2b at 24 h on the growth of this cell line
(increase in cell number from time zero) was obtained.
Next the effect on the mitotic index at 10 µM was
examined. After 24 h of growth, 35% of the cells
displayed a mitotic configuration (condensed chromo-
somes), as compared with 2% in the control culture. The
enantiomers were also examined for inhibitory effects
on the growth of the HL 60 cells. The same relative
activities were observed as with inhibition of tubulin
polymerization. The IC50 value obtained with the 2R,3R-
enantiomer was 0.08 µM, versus 0.6 µM with the 2S,3S-
enantiomer. These values were obtained with freshly
dissolved compounds. There is probably racemization
of the enantiomers in solution. Preliminary experiments
have yielded smaller differences when older solutions
of the enantiomers were compared than when freshly
prepared solutions were evaluated. Finally, the 2R,3R-
enantiomer was similar to racemic 2b in causing a
marked increase in the mitotic index of HL 60 cells.
Structurally, compounds binding to the colchicine site
of tubulin are much simpler than those binding to the
vinca or taxoid domains. Many of them can be described
as biaryl systems connected by a hydrocarbon bridge of
variable length. These planar rings have to be tilted
with respect to each other.12 This may explain why the
benzofuran derivative 5d was not active in the 60 cell
line panel, in contrast to its 2,3-saturated analogue 2d .
In addition to colchicine and combretastatins, other
examples of naturally occurring or synthetic inhibitors
of tubulin polymerization acting through the colchicine
site of tubulin and possessing such a biaryl system
include 2-aryl-1,8-naphthyridin-4(1H)-ones,13 2-phenyl-
4-quinolones,10 both with a N-containing heterocyclic
unit, chalcones,14 and flavonoids such as centaureidin,
with a six-membered heterocyclic unit instead of a five-
membered one as in dihydrobenzofurans. A recent
screening of 79 flavones related to centaureidin (3,4′,6-
trimethoxy-3′,5,7-trihydroxyflavone) (Figure 1) showed
that maximum potencies for tubulin interaction were
observed only with compounds possessing hydroxyl
substituents at C-3′ and C-5 and methoxy groups at C-3
and C-4′.15 The 3-methoxy group may be related to the
loss of planarity, essential for binding to the colchicine
site. It has already been observed by Cheng in 1986 that
a tricyclic chemical structural pattern, consisting of a
phenyl ring attached in position 2 of a naphthalene
nucleus or composed of various heterocyclic units, was
present in a large number of antineoplasic compounds.16
In addition to the examples listed above, the dihy-
drobenzofuran lignans (2-phenyl-dihydrobenzofurans)
discussed here constitute a new group of antimitotic
agents that inhibit tubulin polymerization, fitting into
this general pattern.
(b) In ter a ction s w ith Tu bu lin . Microtubules are
an important target for anticancer chemotherapy. Chemi-
cals that interact with tubulin, the major structural
component of microtubules, can cause mitotic arrest by
inhibiting (e.g., vinca alkaloids, colchicine) or stimulat-
ing (e.g., taxoids) microtubule assembly. Interestingly,
all antimitotic agents currently in clinical use as
anticancer drugs are natural products or semisynthetic
derivatives.10,11 Six compounds were initially examined
for effects on the polymerization of purified bovine brain
tubulin: 2b, 2c, 2d (2S,3S), 2d (2R,3R), 3b, and 4b. All
of them, except for 2b, showed an IC50 > 40 µM (50%
inhibitory concentration). Compound 2b was compared
with the potent antimitotic drug combretatstatin A-4
and proved to be only about 1/10 as active: It inhibited
the extent of polymerization by 50% at a concentration
of 13 ( 1 (SD) µM (n ) 3), while the IC50 value for
combretastatin A-4, used as a positive control, was 1.2
( 0.03 (SD) µM. Subsequent chromatographic resolution
of 2b into its enantiomers allowed their evaluation for
antitubulin activity. The 2R,3R-enantiomer yielded an
IC50 value of 6.0 ( 0.4 µM, while the 2S,3S-enantiomer
was essentially inactive (IC50 > 40 µM).
Combretastatin A-4 as well as compound 2b inhibited
the binding of [3H]colchicine to tubulin, and their
relative activities were quantitatively consistent with
their relative effects on the polymerization reaction. In
reaction mixtures containing 1.0 µM tubulin and 5.0 µM
[3H]colchicine, binding of radiolabeled drug to the
protein was inhibited (88 ( 4)% by 1.0 µM combret-
astatin A-4, and total inhibition occurred with 5.0 µM
combretastatin A-4. Combretastatin A-4 is one of the
most potent competitive inhibitors of colchicine binding
to tubulin known to date. Compound 2b was less
(c) Hollow F iber Assa y for P r elim in a r y in Vivo
Testin g. Compounds 2b, 2c, and 2d (2R,3R) were
evaluated in the hollow fiber assay developed at the
NCI. This is a preliminary in vivo screening tool for
assessing the potential anticancer activity of compounds
selected in the in vitro cell screen. However, none of the

5478 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Brief Articles
1
1c: amorphous, mp 148 °C; H NMR (acetone-d6) δ 8.10 (s,
1 H, 4-OH), 7.59 (d, J ) 16.02 Hz, 1 H, H-7), 7.29 (d, J ) 1.89
Hz, 1 H, H-2), 7.12 (dd, J ) 8.24, 1.98 Hz, 1 H, H-6), 6.87 (d,
J ) 8.24 Hz, 1 H, H-5), 6.38 (d, J ) 15.94 Hz, 1 H, H-8), 3.91
(s, 3 H, 3-OCH3), 3.68 (s, 3 H, 9-OCH3); 13C NMR (acetone-d6)
δ 167.87 (C-9), 149.98 (C-4), 148.66 (C-3), 145.66 (C-7), 127.42
(C-1), 123.75 (C-6), 116.06 (C-5), 115.55 (C-8), 111.42 (C-2),
56.35 (C-11), 51.49 (C-10); DCI-MS (NH3) m/z 209 (MH+).
compounds tested was sufficiently active for further in
vivo testing in standard subcutaneous xenograft models.
Con clu sion
In a series of synthetic dihydrobenzofuran lignans (2-
phenyldihydrobenzofuran lignans), the dimerization
product of caffeic acid methyl ester (2b) showed the
highest activity in a screening panel consisting of 60
human tumor cell lines, with an average GI50 value of
0.3 µM and GI50 values <10 nM against some breast
cancer cell lines. It appeared to inhibit mitosis at
micromolar concentrations in cell culture through a
relatively weak interaction at the colchicine binding site
of tubulin, and these activities in the racemate probably
derive from activity limited to the 2R,3R-enantiomer.
These dihydrobenzofuran lignans (2-phenyldihydroben-
zofuran derivatives) constitute a new group of antimi-
totic agents that inhibit tubulin polymerization. We
think it likely that modification of substituents and/or
the core structure of the molecule will yield more active
agents, including compounds that will have antitumor
activity.
Meth yl (E)-3-[2-(4-h ydr oxyph en yl)-3-m eth oxycar bon yl-
2,3-dih ydr o-1-ben zofu r an -5-yl]pr op-2-en oate (2a) was pre-
pared according to the method of Lemie`re et al.2 using 4.86 g
(27.3 mmol) of methyl p-coumarate, 4.53 g (19.5 mmol) of
silver(I) oxide, 70 mL of anhydrous benzene, and 50 mL of
anhydrous acetone and a reaction time of 65 h. The product
was purified by column chromatography (3.8 × 30 cm, silica
gel 60, 0.040-0.063 mm) with ethyl acetate-n-heptane (1:3)
as the eluent. The product was obtained as an oil (23%). 1H
NMR (acetone-d6) δ 8.55 (s, 1 H, 4-OH), 7.68 (d, J ) 1.83 Hz,
1 H, H-6′), 7.55 (dd, J ) 8.39, 1.83 Hz, 1 H, H-2′), 7.27 (d, J )
8.24 Hz, 2 H, H-2, H-6), 6.89 (d, J ) 8.39 Hz, 1 H, H-3′), 6.87
(d, J ) 8.24 Hz, 2 H, H-3, H-5), 6.64 (d, J ) 7.33 Hz, 1 H,
H-7′), 6.38 (d, J ) 16.02 Hz, 1 H, H-8′), 6.02 (d, J ) 7.33 Hz,
1 H, H-7), 4.37 (d, J ) 7.33 Hz, 1 H, H-8), 3.80 (s, 3 H, 9-OCH3),
3.72 (s, 3 H, 9′-OCH3); 13C NMR (acetone-d6) δ 167.73 (C-9′),
162.10 (C-4), 158.61 (C-4′), 145.07 (C-7′), 131.84 (C-1), 131.54
(C-1′), 128.66 (C-2′), 128.38 (C-2, C-6), 116.34 (C-3, C-5), 126.69
(C-5′), 126.06 (C-6′), 116.08 (C-8′), 110.75 (C-3′), 87.72 (C-7),
55.60 (C-8), 52.97 (9-OCH3), 51.55 (9′-OCH3); DCI-MS (NH3)
m/z 355 (MH+). HPLC analysis: Column A methanol-water,
65:35, 5.14 min; Column B methanol-water, 50:20, 4.17 min.
Meth yl (E)-3-[2-(3,4-dih ydr oxyh en yl)-7-h ydr oxy-3-m eth -
o x y c a r b o n y l-2,3-d ih y d r o -1-b e n zo fu r a n -5-y l]p r o p -2-
en oa te (2b) was prepared according to the method of Lemie`re
et al.2 using 1.905 g (9.8 mmol) of methyl caffeate, 826 g (3.5
mmol) of silver(I) oxide, 40 mL of anhydrous benzene, and 20
mL of anhydrous acetone. The product was purified by column
chromatography (3.8 × 30 cm, silica gel 60, 0.040-0.063 mm)
with ethyl acetate-n-heptane (1:1) as the eluent. After evapo-
ration and lyophilization, a white foam was obtained (33%);
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Bu¨chi B-545 melting point apparatus. 1H and
13C NMR spectra were measured on a Varian Unity 400
spectrometer. Additional HETCOR and long-range HETCOR
measurements to verify the proposed assignments were per-
formed on the same spectrometer. Chemical shifts are reported
in δ (ppm). Numbering of the atoms is done in the same way
as Lemie`re et al.2 for ease of comparison.
DCI mass spectra were obtained on a Ribermag R-10-10B
mass spectrometer. Column chromatography was performed
on Merck silica gel 60, 0.040-0.063 mm, 230-400 mesh
ASTM. Precoated silica gel plates (kieselgel 60, F254, 0.2 mm)
were used for TLC analysis. All products and reagents were
puchased from Acros, Belgium.
Chiral separations were performed using a column (5 × 21
cm) packed with 0.25 kg of Chiralpack AD. Peak shaving and
closed loop recycling techniques were used during the process.
Ethanol was the solvent used for the chiral chromatography.
Analytical HPLC using two independent systems was per-
formed to check the purity of the products. Column A: Alltech
Econosil C8 (4.6 × 250 mm). Column B: BIO-RAD Bio-Sil C18
(4.6 × 150 mm). Experimental details of the HPLC analysis
are reported for all test compounds in the form column; mobile
phase; retention time. The flow rate was 1 mL/min.
P r ep a r a t ion of Dih yd r ob en zofu r a n Lign a n s. Methyl
cinnamates (1a -c) were prepared from a mixture of the
corresponding cinnamic acid (4 g) and Dowex 50 W × 8200-
400 (0.4 g) in 25 mL of absolute methanol. After the mixture
was heated under reflux for 1 night, the mixture was filtered
and evaporated under reduced pressure to afford the product
as a solid (100%), which was used without further purification.
1a : amorphous, mp 136 °C; 1H NMR (acetone-d6) δ 8.80 (s,
1 H, 4-OH), 7.60 (d, J ) 16.02 Hz, 1 H, H-7), 7.52 (d, J ) 8.65
Hz, 2 H, H-2, H-6), 6.89 (d, J ) 8.55 Hz, 2 H, H-3, H-5), 6.33
(d, J ) 16.02 Hz, 1 H, H-8), 3.70 (s, 3 H, 9-OCH3); 13C NMR
(acetone-d6) δ 167.85 (C-9), 160.47 (C-4), 145.33 (C-7), 130.85
(C-2, C-6), 126.99 (C-1), 116.65 (C-3, C-5), 115.32 (C-8), 51.47
(9-OCH3); DCI-MS (NH3) m/z 179 (MH+).
1
amorphous mp 159 °C; H NMR (acetone-d6) δ 8.05 (s, 2 H,
3-OH, 4-OH), 7.57 (d, J ) 16.02 Hz, 1 H, H-7′), 7.14 (s, 1 H,
H-6′), 6.90 (d, J ) 1.95 Hz, 1 H, H-2), 6.84 (d, J ) 8.24 Hz, 1
H, H-5), 6.79 (dd, J ) 8.24, 1.98 Hz, 1 H, H-6), 6.33 (d, J )
16.02 Hz, 1 H, H-8′), 5.97 (d, J ) 8.33 Hz, 1 H, H-7), 4.35 (d,
J ) 8.33 Hz, 1 H, H-8), 3.79 (s, 3 H, 9′-OCH3), 3.71 (s, 3 H,
9-OCH3), 3.38 (s, 1 H, 3′-OH); 13C NMR (acetone-d6) δ171.69
(C-9), 167.79 (C-9′), 150.07 (C-3′), 146.33 (C-4), 146.09 (C-3),
145.42 (C-7′), 142.53 (C-4′), 132.70 (C-1), 129.40 (C-1′), 127.33
(C-5′), 118.70 (C-6), 117.76 (C-2′), 117.33 (C-6′), 116.16 (C-5),
116.05 (C-8′), 113.93 (C-2), 87.88 (C-7), 56.29 (C-8), 52.95 (9-
OCH3), 51.55 (9′-OCH3); DCI-MS (NH3) m/z 387 (MH+). Chiral
HPLC analysis: Chiralpak AD, length 5 cm; n-hexane-
ethanol, 60:40. Anal. (C20H18O6) C, H.
Meth yl (E)-3-[2-(4-h yd r oxy-3-m eth oxyp h en yl)-7-m eth -
oxy-3-m eth oxyca r bon yl-2,3-d ih yd r o-1-ben zofu r a n -5-yl]-
p r op -2-en oa te (2c) was prepared according to the method of
Lemie`re et al.2 using an improved workup. After evaporation,
the residual brown oil was dissolved in methanol. The solution
was left to stand overnight. Crystals (white, mp 151 °C) were
formed, filtered, and washed with cold methanol (50%): 1H
NMR (acetone-d6) δ 7.68 (s, 1 H, 4-OH), 7.63 (d, J ) 16.02 Hz,
1 H, H-7′), 7.33 (s, 1 H, H-2′), 7.30 (s, 1 H, H-6′), 7.09 (d, J )
1.98 Hz, 1 H, H-2), 6.91 (dd, J ) 8.09, 1.98 Hz, 1 H, H-6), 6.85
(d, J ) 8.09 Hz, 1 H, H-5), 6.45 (d, J ) 16.02 Hz, 1 H, H-8′),
6.03 (d, J ) 8.02 Hz, 1 H, H-7), 4.48 (d, J ) 8.02 Hz, 1 H,
H-8), 3.93 (s, 3 H, 3′-OCH3), 3.83 (s, 3 H, 3-OCH3), 3.82 (s, 3
H, 9-OCH3), 3.74 (s, 3 H, 9′-OCH3); 13C NMR (acetone-d6) δ
171.73 (C-9), 167.86 (C-9′), 151.01 (C-4′), 148.75 (C-3), 148.21
(C-4), 145.80 (C-3′), 145.50 (C-7′), 131.81 (C-1), 129.42 (C-1′),
127.35 (C-5′), 120.18 (C-6), 119.02 (C-6′), 116.29 (C-8′), 116.02
(C-5), 113.46 (C-2′), 110.96 (C-2), 56.54 (3′-OCH3), 56.35 (3-
OCH3), 55.92 (C-8), 53.07 (9-OCH3), 51.67 (9′-OCH3); DCI-MS
(NH3) m/z 415 (MH+). HPLC analysis: Column A methanol-
1b: amorphous, mp 158 °C; 1H NMR (acetone-d6) δ 8.3 (s, 2
H, 3-OH, 4-OH), 7.53 (d, J ) 15.87 Hz, 1 H, H-7), 7.15 (d, J )
2.14 Hz, 1 H, H-2), 7.03 (dd, J ) 8.09, 2.14 Hz, H-6), 6.86 (d,
J ) 8.09 Hz, 1 H, H-5), 6.27 (d, J ) 16.02 Hz, 1 H, H-8), 3.70
(s, 3 H, 9-OCH3); 13C NMR (acetone-d6) δ 167.82 (C-9), 148.75
(C-4), 146.33 (C-3), 145.68 (C-7), 127.75 (C-1), 122.51 (C-6),
116.43 (C-5), 115.28 (C-2), 115.21 (C-8), 51.45 (9-OCH3); DCI-
MS (NH3) m/z 195 (MH+).

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5479
water, 65:35, 5.01 min; Column B methanol-water, 50:20, 4.93
Hz, 1 H, H-2), 6.92 (d, J ) 7.93 Hz, 1 H, H-6), 6.87 (dd, J )
7.93, 1.84 Hz, 1 H, H-5), 6.03 (d, J ) 8.54 Hz, 1 H, H-7), 5.72
(s, 1 H, 4-OH), 4.30 (d, J ) 8.54 Hz, 1 H, H-8), 3.98 (s, 3 H,
3′-OCH3), 3.87 (s, 3 H, 3-OCH3), 3.81 (s, 3 H, 9-OCH3), 3.68 (s,
3 H, 9′-OCH3), 2.91 (t, J ) 7.78 Hz, 2 H, H-7′), 2.62 (t, J )
7.78 Hz, 2 H, H-8′); 13C NMR (acetone-d6) δ 173.21 (C-9′),
171.73 (C-9), 146.68 (C-4′), 146.43 (C-3), 145.92 (C-4), 144.57
(C-3′), 136.12 (C-8′), 134.19 (C-1′), 131.97 (C-1), 130.85 (C-7′),
125.25 (C-5′), 119.40 (C-6), 116.50 (C-6′), 114.44 (C-5), 113.16
(C-2′), 108.86 (C-2), 86.75 (C-7), 77.32 (C-8), 77.00 (3-OCH3),
76.68 (3′-OCH3), 52.53 (9-OCH3), 51.50 (9′-OCH3); DCI-MS
(NH3) m/z 417 (MH+). HPLC analysis: Column A methanol-
water, 65:35, 3.88 min; Column B methanol-water, 50:20, 3.51
min.
min. Anal. (C22H22O8) C, H.
Met h yl (E)-3-[2-(3,4-d im et h oxyp h en yl)-7-m et h oxy-3-
m eth oxyca r bon yl-2,3-d ih yd r o-1-ben zofu r a n -5-yl]p r op -2-
en oa te (2d ) was prepared using the method of Lemie`re et
al.2 using an improved workup. After evaporation of the
reaction mixture, the resulting yellow oil was dissolved in
methanol. The solution was left to stand overnight. Crystals
(75%) were formed, filtered, and washed with cold methanol:
1
White crystals, mp 135 °C; H NMR (acetone-d6) δ 7.65 (d, J
) 15.91 Hz, 1 H, H-7′), 7.19 (s, 1 H, H-6′), 7.03 (s, 1 H, H-2′),
6.97 (dd, J ) 15.91, 1.99 Hz, 1 H, H-6), 6.92 (d, J ) 1.99 Hz,
1 H, H-2), 6.84 (d, J ) 8.35 Hz, 1 H, H-5), 6.32 (d, J ) 15.91
Hz, 1 H, H-8′), 6.13 (d, J ) 8.15 Hz, 1 H, H-7), 4.35 (d, J )
8.15 Hz, 1 H, H-8), 3.87 (s, 3 H, 4-OCH3), 3.86 (s, 3 H, 3-OCH3),
3.84 (s, 3 H, 9-OCH3), 3.80 (s, 3 H, 9′-OCH3), 3.92 (s, 3 H, 3′-
OCH3); 13C NMR (acetone-d6) δ 170.74 (C-9), 167.55 (C-9′),
150.14 (C-4′), 149.62 (C-4), 149.49 (C-3), 144.85 (C-3′), 144.72
(C-6′), 132.18 (C-1), 128.76 (C-1′), 125.88 (C-5′), 118.82 (C-6),-
117.99 (C-6′), 115.76 (C-8′), 112.53 (C-2′), 111.46 (C-5), 109.56
(C-2), 87.41 (C-7), 56.09 (3′-OCH3), 56.09 (4-OCH3), 56.05 (3-
OCH3), 52.83 (9-OCH3), 51.56 (9′-OCH3); DCI-MS (NH3) m/z
429 (MH+). HPLC analysis: Column A methanol-water, 65:
35, 5.06 min; Column B methanol-water, 50:20, 5.61 min.
Anal. (C23H24O8) C, H.
Meth yl 3-[2-(3,4-d im eth oxyp h en yl)-7-m eth oxy-3-m eth -
o x y c a r b o n y l -2 ,3 -d i h y d r o -1 -b e n z o fu r a n -5 -y l ]p r o -
p a n oa te (3d ) was prepared according to the method of
Lemie`re et al.2 The reaction was improved by using a Parr
apparatus at 60 psi hydrogen pressure for 20 min. Workup
was performed identically, resulting in a white powder (81%);
1
mp 97-99 °C; H NMR (CDCl3) δ 7.80 (s, 1 H, H-6′), 7.70 (s,
1 H, H-2′), 6.97 (dd, J ) 8.09, 1.99 Hz, 1 H, H-6), 6.95 (d, J )
1.84 Hz, 1 H, H-2), 6.83 (d, J ) 8.09 Hz, 1 H, H-6), 6.05 (d, J
) 8.39 Hz, 1 H, H-7), 5.70 (s, 1 H, 4-OH), 4.31 (d, J ) 8.39 Hz,
1 H, H-8), 3.98 (s, 3 H, 3′-OCH3), 3.86 (s, 3 H, 3-OCH3), 3.85
(s, 3 H, 9-OCH3), 3.68 (s, 3 H, 9′-OCH3), 2.91 (t, J ) 7.75 Hz,
2 H, H-7′), 2.62 (t, J ) 7.78 Hz, 2 H, H-8′); 13C NMR (CDCl3)
δ 173.16 (C-9′), 171.24 (C-9), 149.66 (C-4), 149.65 (C-3), 146.74
(C-4′), 144.50 (C-3′), 134.37 (C-1′), 133.04 (C-1), 125.51 (C-5′),
118.83 (C-6), 116.76 (C-6′), 113.74 (C-2′), 111.90 (C-5), 110.25
(C-2), 86.72 (C-7), 56.45 (3′-OCH3), 56.23 (C-8), 56.20 (4-OCH3),
56.15 (3-OCH3), 52.52 (9-OCH3), 51.46 (9′-OCH3), 36.17 (C-
7′), 30.96 (C-8′); DCI-MS (NH3) m/z 431 (MH+). HPLC analy-
sis: Column A methanol-water, 65:35, 4.50 min; Column B
methanol-water, 50:20, 4.26 min.
Meth yl (E)-3-[2-(4-h yd r oxyp h en yl)- 3-m eth oxyca r bo-
n yl-2,3-d ih yd r o-1-ben zofu r a n -5-yl]p r op a n oa te (3a ) was
prepared by dissolving 418 mg (1.18 mmol) of 2a in 30 mL of
acetone to which 220 mg of 5% Pd/C was added. The mixture
was put in a Parr apparatus under 60 psi hydrogen pressure
and was shaken for 20 min. After filtration and evaporation,
the crude oil was purified by column chromatography (3 × 20
cm, silica gel 60, 0.040-0.063 mm) with ethyl acetate-heptane
(3:1) as the eluent, resulting in a colorless oil (85%): 1H NMR
(CDCl3) δ 7.26 (d, J ) 8.70 Hz, 2 H, H-2, H-6), 7.17 (d, J )
1.83 Hz, 1 H, H-6′), 7.05 (dd, J ) 8.24, 1.83 Hz, 1 H, H-2′),
6.81 (d, J ) 8.70 Hz, 2 H, H-3, H-5), 6.74 (d, J ) 8.24 Hz, 1 H,
H-3′), 5.45 (s, 1 H, 4-OH), 4.24 (d, J ) 7.78 Hz, 1 H, H-7), 3.81
(s, 3 H, 9-OCH3), 3.68 (s, 3 H, 9′-OCH3), 2.91 (t, J ) 7.78 Hz,
2 H, H-8′), 2.61 (t, J ) 7.78, 2 H, H-7′); 13C NMR (CDCl3) δ
173.55 (C-9′), 171.46 (C-9), 157.89 (C-4), 155.97 (C-4′), 132.70
(C-1′), 129.55 (C-2′), 127.51 (C-2, C-6), 124.93 (C-6′), 124.24
(C-5′), 115.63 (C-3, C-5), 109.74 (C-3′), 85.78 (C-7), 55.74 (C-
8), 52.66 (C-9), 51.64 (C-9′), 36.22 (C-8′), 30.50 (C-7′); DCI-MS
(NH3) m/z 357 (MH+). HPLC analysis: Column A methanol-
water, 65:35, 4.02 min; Column B methanol-water, 60:40, 9.22
min.
Methyl(E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-3-methoxy-
ca r bon yl-2,3-d ih yd r o-1-ben zofu r a n -5-yl]p r op a n oa te (3b)
was prepared from 2b using the same method and concentra-
tions as for the synthesis of 3a . The crude oil was purified by
column chromatography (3 × 20 cm, silica gel 60, 0.040-0.063
mm) with ethyl acetate-n-heptane (1:2) as the eluent, result-
ing in a colorless oil (95%): 1H NMR (acetone-d6) δ 7.95 (s, 2
H, 3-OH, 4-OH), 6.88 (d, J ) 1.98 Hz, 1 H, H-2), 6.82 (d, J )
8.08 Hz, 1 H, H-5), 6.76 (dd, J ) 8.08, 1.98 Hz, 1 H, H-6), 6.71
(s, 1 H, H-2′), 6.68 (s, 1 H, H-6′), 5.88 (d, J ) 7.48 Hz, 1 H,
H-7), 4.24 (d, J ) 7.53 Hz, 1 H, H-8), 3.76 (s, 3 H, 9-OCH3),
3.60 (s, 3 H, 9′-OCH3), 2.80 (t, J ) 7.63 Hz, 2 H, H-7′), 2.55 (t,
J ) 7.63 Hz, 2 H, H-8′); 13C NMR (acetone-d6) δ 173.45 (C-9′),
172.06 (C-9), 146.18 (C-4′), 146.08 (C-3), 145.41 (C-4), 142.00
(C-3′), 135.26 (C-1′), 133.36 (C-1), 126.48 (C-5′), 118.60 (C-6),
117.78 (C-6′), 116.20 (C-2′), 116.13 (C-5′), 113.9 (C-2), 87.95
(C-7), 56.40 (C-8), 52.94 (9-OCH3), 51.53 (9′-OCH3), 36.54 (C-
8′), 31.20 (C-7′); DCI-MS (NH3) m/z 389 (MH+). HPLC analy-
sis: Column A methanol-water, 65:35, 6.25 min; Column B
methanol-water, 60:40, 3.45 min.
3-[2-(4-Hyd r oxyp h en yl)-3-h yd r oxym eth yl-2,3-d ih yd r o-
1-ben zofu r a n -5-yl]p r op a n -1-ol (4a ) was prepared from 3a
according to the method of Lemie`re et al.2 In the workup the
crude green oil was dissolved in a few drops of ethyl acetate
and left to stand overnight at 0 °C, resulting in the formation
of white crystals (40%); mp 122-125 °C; 1H NMR (acetone-
d6) δ 8.32 (s, 1 H, 4-OH), 7.68 (m, J ) 1.40 Hz, 1 H, H-6′), 7.24
(d, J ) 8.24 Hz, 2 H, H-2, H-6), 6.95 (dd, J ) 8.08, 1.40 Hz, 1
H, C-2′), 6.89 (d, J ) 8.08 Hz, 1 H, H-2′), 6.81 (d, J ) 8.24 Hz,
2 H, H-3, H-5), 6.69 (d, J ) 8.08 Hz, 1 H, H-3′), 5.50 (d, J )
6.10 Hz, 1 H, H-7), 4.37 (m, J ) 6.10 Hz, 1 H, H-8), 3.85 (m,
J ) 10.83, 5.34 Hz, 1 H, H-9a), 3.78 (m, J ) 10.83, 7.02 Hz, 1
H, H-9b), 2.89 (s, 2 H, 9-OH, 9′-OH), 2.61 (t, J ) 7.98 Hz, 2 H,
H-7′), 1.78 (m, J ) 7.94, 6.24 Hz, 2 H, H-8′); 13C NMR (acetone-
d6) δ 159.09 (C-4), 157.95 (C-4′), 135.49 (C-5′), 134.45 (C-1),
129.19 (C-2′), 128.87 (C-1′), 127.97 (C-2, C-6), 125.69 (C-6′),
116.06 (C-3, C-5), 109.35 (C-3′), 87.70 (C-7), 81.87 (C-9′), 65.09
(C-9), 54.79 (C-8), 35.99 (C-8′), 32.33 (C-7′); DCI-MS (NH3) m/z
301(MH+); DCI-MS (NH3) m/z 283 (MH+)-H2O. HPLC analy-
sis: Column A methanol-water, 65:35, 4.17 min; Column B
methanol-water, 60:40, 2.53 min.
3-[2-(3,4-Dih yd r oxyp h e n yl)-3-h yd r oxym e t h yl-7-h y-
dr oxy-2,3-dih ydr o-1-ben zofu r an -5-yl]pr opan -1-ol (4b) was
prepared from 3b according to the method of Lemie`re et al.2
In the workup the crude oil was dissolved in 3 mL of ethyl
acetate and left to stand overnight at 0 °C, resulting in the
formation of white crystals (20%); mp 123 °C; 1H NMR (CDCl3)
δ 6.84 (m, 1 H, H-2), 6.72 (m, 2 H, H-5, H-6), 6.60 (s, 1 H,
H-6′), 6.56 (s, 1 H, H-2′), 5.53 (d, J ) 5.95 Hz, 1 H, H-7), 3.81
(m, J ) 10.99, 5.49 Hz, 1 H, H-9a), 3.73 (m, J ) 10.99, 7.32
Hz, 1 H, H-9b), 3.56 (t, J ) 6.57 Hz, 2 H, H-9′), 2.56 (t, J )
7.47 Hz, 2 H, H-7′), 1.80 (m, J ) 7.48, 6.57 Hz, 2 H, H-8′); 13
C
Meth yl 3-[2-(4-h yd r oxy-3-m eth oxyp h en yl)-7-m eth oxy-
3-m et h oxyca r bon yl-2,3-d ih yd r o-1-ben zofu r a n -5-yl]p r o-
p a n oa te (3c) was prepared from 2c using the same method
and concentrations as for the synthesis of 3a . Workup was
performed identically, resulting in a white powder (95%); mp
85-87 °C; H NMR (acetone-d6) δ 7.79 (s, 1 H, H-2′), 7.69 (s,
1 H, H-6′), 6.93 (d, J ) 1.84 Hz, 1 H, H-2), 6.92 (d, J ) 1.84
NMR (CDCl3) δ 146.56 (C-4′), 146.37 (C-3), 146.14 (C-4), 141.76
(C-3′), 136.61 (C-1′), 135.34 (C-1), 129.88 (C-5′), 118.59 (C-6),
116.98 (C-2′), 116.71 (C-6′), 116.22 (C-5), 114.01 (C-2), 88.64
(C-7), 65.26 (C-9), 61.52 (C-9′), 55.68 (C-8), 35.73 (C-8′), 32.70
(C-7′); DCI-MS (NH3) m/z 333 (MH+); DCI-MS (NH3) m/z 315
(MH+)-H2O. HPLC analysis: Column A methanol-water, 65:
35, 3.01 min; Column B; methanol-water, 60:40, 2.41 min.
1

5480 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Brief Articles
3-[2-(4-Hyd r oxy-3-m eth oxyp h en yl)-3-h yd r oxym eth yl-
7-m eth oxy-2,3-dih ydr o-1-ben zofu r an -5-yl]pr opan -1-ol (4c)
was prepared from 3c according to the method of Lemie`re et
al.2 The crude yellow oil was purified by column chromatog-
raphy (3.8 × 30 cm, silica gel 60, 0.040-0.063 mm) with ethyl
acetate-n-heptane (2:1) as the eluent, resulting in a light
yellow oil (78%): 1H NMR (CDCl3) δ 6.93 (d, J ) 1.83 Hz, 1
H, H-2), 6.90 (dd, J ) 8.09, 1.83 Hz, 1 H, H-6), 6.86 (d, J )
8.09 Hz, 1 H, H-5), 6.67 (s, 1 H, H-2′), 6.65 (s, 1 H, H-6′), 5.54
(d, J ) 7.48 Hz, 1 H, H-7), 3.94 (dd, J ) 10.91, 4.42 Hz, 1 H,
H-9a), 3.88 (dd, J ) 10.91, 6.02 Hz, 1 H, H-9b), 3.87 (s, 1 H,
9-OH), 3.85 (s, 1 H, 9′-OH), 3.68 (t, J ) 6.41 Hz, 2 H, H-9′),
) 9.16 Hz, 1 H, H-5), 6.70 (d, J ) 1.38 Hz, 1 H, H-2′), 4.02 (s,
3 H, 3′-OCH3), 3.96 (s, 3 H, 3-OCH3), 3.94 (s, 3 H, 4-OCH3),
3.93 (s, 3 H, 9-OCH3), 3.70 (s, 3 H, 9′-OCH3), 3.06 (t, J ) 8.09
Hz, 2 H, H-7′), 2.70 (t, J ) 8.09 Hz, 2 H, H-8′); 13C NMR
(CDCl3) δ 173.33 (C-9′), 164.61 (C-9), 161.15 (C-7), 150.97 (C-
4), 148.52 (C-3), 144.82 (C-3′), 141.84 (C-4′), 137.46 (C-1′),
129.13 (C-5′), 123.13 (C-6), 122.21 (C-1), 113.81 (C-6′), 112.81
(C-2), 110.71 (C-5), 108.08 (C-2′), 77.24 (C-8), 56.20 (9′-OCH3),
56.15 (3-OCH3), 56.00 (9-OCH3), 51.62 (3′-OCH3), 51.55 (4-
OCH3), 36.40 (C-8′), 31.62 (C-7′); DCI-MS (NH3) m/z 425
(MH+). HPLC analysis: Column A methanol-water, 65:35,
8.61 min; Column B methanol-water, 50:10, 4.34 min.
3.59 (m, 1 H, H-8), 2.66 (t, 2 H, H-7′), 1.87 (m, 2 H, H-8′); 13
C
Meth yl 3-(2-[2-(3,4-d im eth oxyp h en yl)-1-m eth oxyca r -
bon yeth yl]-3-h yd r oxy-4-m eth oxyp h en yl)pr opa n oa te (7d )
is a side product of the hydrogenation of 2d at prolonged
reaction times without the use of a Parr apparatus. It is
obtained when a solution of 2d and Pd/C in ethyl acetate is
bubbled through with hydrogen gas for 5 min. The reaction
flask is then closed and stirred at room temperature. Reaction
times more than 4 h show the appearance of compound 7d .
This product can be isolated by column chromatography using
the same conditions as those for the separation of 2d . The
product is obtained as a colorless oil: 1H NMR δ 6.74 (s, 1 H,
H-2), 6.72 (s, 1 H, H-6′), 6.63 (m, 3 H, H-2, H-5, H-6), 5.92 (s,
1 H, 4′-OH), 4.24 (br t, J ) 8.40, 7.02 Hz, 1 H, H-8), 3.84 (s, 3
H, 3-OCH3), 3.82 (s, 3 H, 3′-OCH3), 3.78 (s, 3 H, 4-OCH3), 3.67
(s, 3 H, 9′-OCH3), 3.61 (s, 3 H, 9-OCH3), 3.28 (dd, J ) 13.60,
8.40 Hz, 1 H, H-7a), 2.96 (dd, J ) 13.60, 6.80 Hz, 1 H, H-7b),
2.84 (t, J ) 7.60 Hz, 2 H, H-7′), 2.55 (t, J ) 7.60 Hz, 2 H,
H-8′); DCI-MS (NH3) m/z 432 (MH+).
Cytotoxicity Assa ys. The cytotoxic activity of test com-
pounds was evaluated in the NCI’s in vitro human disease-
oriented antitumor screen. This screening panel consists of 60
human tumor cell lines, largely derived from solid tumors, plus
some leukemia cell lines. Nine subpanels represent diverse
histologies, i.e., nonsmall cell lung, colon, central nervous
system, renal, ovarian, prostate, and breast cancers, mela-
noma, and leukemia. Compounds were tested at a minimum
of five concentrations at 10-fold dilutions. Results are evalu-
ated in terms of specificity and potency. The cytotoxic effects
of each compound are expressed as the molar drug concentra-
tions required for 50% growth inhibition (GI50), total growth
inhibition (TGI), and 50% cell kill (LC50).17
In h ibition of Tu bu lin P olym er iza tion . Details of the
purification of tubulin and of the assays of tubulin polymer-
ization and colchicine binding have been previously pub-
lished.18 Briefly, effects of test compounds on tubulin poly-
merization were evaluated in a system consisting of pure
bovine brain tubulin (i.e., no microtubule-associated proteins)
at 10 µM, in which assembly is induced by 0.8 M glutamate
and requires GTP. Drug and tubulin are preincubated in the
glutamate for 15 min at 30 °C and chilled on ice, GTP is added,
and the assembly reaction started in a recording spectropho-
tometer with a 0-30 °C jump. After 20 min, the reaction
mixtures are chilled to 0 °C to verify the extent of ‘normal’
assembly (as opposed to paclitaxel-induced assembly or ag-
gregation reactions in which reversibility is generally affected).
An IC50 value is determined based on the inhibition of the
extent of assembly at a series of drug concentrations.
NMR (CDCl3) δ 146.70 (C-3), 149.07 (C-4), 146.62 (C-4′), 145.66
(C-4), 144.20 (C-3′), 135.37 (C-1′), 133.22 (C-1), 127.89 (C-5′),
119.36 (C-6), 116.07 (C-2′), 114.36 (C-5), 112.74 (C-6′), 108.92
(C-2), 87.84 (C-7), 64.04 (C-9), 62.26 (C-9′), 56.11 (3′-OCH3),
56.01 (3-OCH3), 53.83 (C-8), 34.57 (C-8′), 31.98 (C-7′); DCI-
MS (NH3) m/z 361(MH+); DCI-MS (NH3) m/z 343 (MH+)-H2O.
HPLC analysis: Column A methanol-water, 65:35, 2.78 min;
Column B methanol-water, 60:40, 1.35 min.
3-[2-(3,4-Dim eth oxyp h en yl)-3-h yd r oxym eth yl-7-m eth -
oxy-2,3-d ih yd r o-1-ben zofu r a n -5-yl]p r op a n -1-ol (4d ) was
prepared from 3d according to the method of Lemie`re et al.2
without further modification (56%). 1H NMR (CDCl3) δ 6.95
(m, 2 H, H-2, H-6), 6.82 (d, J ) 8.70 Hz, 1 H, H-5), 6.67 (s, 1
H, H-2′), 6.65 (s, 1 H, H-6′), 5.54 (d, J ) 7.17 Hz, 1 H, H-5),
3.93 (dd, J ) 10.82, 6.14 Hz, 1 H, H-9b), 3.89 (s, 3 H, 4-OCH3),
3.87 (dd, J ) 10.80, 4.91 Hz, 1 H, H-9a), 3.84 (s, 3 H, 4-OCH3),
3.67 (t, J ) 6.34 Hz, 2 H, H-9′), 3.59 (m, 1 H, H-8), 2.66 (t, 2
H, H-7′), 1.87 (m, 2 H, H-8′); 13C NMR (CDCl3) δ 149.26 (C-3),
149.07 (C-4), 146.62 (C-4′), 144.19 (C-3′), 135.37 (C-1′), 133.92
(C-1), 127.90 (C-5′), 118.64 (C-6), 116.11 (C-6′), 112.78 (C-2′),
111.30 (C-5), 109.63 (C-2), 87.70 (C-7), 64.04 (C-9), 62.22 (C-
9′), 56.12 (3′-OCH3), 55.97 (C-10), 55.99 (C-11), 53.81 (C-8),
34.55 (C-8′), 31.96 (C-7′); DCI-MS (NH3) m/z 375 (MH+); DCI-
MS (NH3) m/z 357 (MH+)-H2O. HPLC analysis: Column A
methanol-water, 65:35, 3.43 min; Column B methanol-water,
50:20, 2.01 min.
Met h yl (E)-3-[2-(3,4-d im et h oxyp h en yl)-7-m et h oxy-3-
m eth oxyca r bon yl-1-ben zofu r a n -5-yl]p r op -2-en oa te (5d )
was prepared from 210 mg (0.49 mmol) of 2d , 425 mg (1.87
mmol) of DDQ, and 25 mL of anhydrous dioxane. The resulting
yellow-green solution was refluxed for 48 h under nitrogen.
The mixture was cooled to room temperature, and the resulting
light brown precipitate was removed by filtration. The filtrate
was concentrated by evaporation and the residue purified by
column chromatography (3 × 20 cm, silica gel 60, 0.040-0.063
mm) with dichloromethane. Recrystallization in acetone yielded
white crystals (91%): mp 183-185 °C; 1H NMR (CDCl3) δ 7.80
(d, J ) 15.86 Hz, 1 H, H-7′), 7.78 (d, J ) 1.42 Hz, 1 H, H-6′),
7.74 (d, J ) 2.13 Hz, 1 H, H-2), 7.71 (dd, J ) 8.54, 2.13 Hz, 1
H, H-6), 7.00 (d, J ) 1.37 Hz, 1 H, H-2′), 6.93 (d, J ) 8.54 Hz,
1 H, H-5), 6.45 (d, J ) 15.86 Hz, 1 H, H-8′), 4.04 (s, 3 H, 3′-
OCH3), 3.98 (s, 3 H, 3-OCH3), 3.96 (s, 3 H, 4-OCH3), 3.95 (s, 3
H, 9-OCH3), 3.83 (s, 3 H, 9′-OCH3); 13C NMR (CDCl3) δ 167.37
(C-9′), 164.16 (C-9), 161.68 (C-7), 151.19 (C-4), 148.52 (C-3),
145.45 (C-7′), 145.25 (C-3′), 144.08 (C-4′),131.45 (C-1′), 129.37
(C-5′), 123.18 (C-6), 121.65 (C-1), 117.13 (H-8′), 116.06 (C-6′),
112.75 (C-12′), 110.68 (C-5), 108.08 (C-8), 106.06 (C-2′), 56.12
(3-OCH3), 56.14 (3′-OCH3), 55.94 (4-OCH3), 51.64 (9-OCH3),
51.61 (9′-OCH3); DCI-MS (NH3) m/z 427(MH+). HPLC analy-
sis: Column A methanol-water, 65:35, 8.15 min; Column B
methanol-water, 50:10, 6.89 min.
HL-60 human leukemia cells (a generous gift of Dr. T. R.
Breitman, National Cancer Institute) were grown and exam-
ined for mitotic arrest as described before.19 Combretastatin
A-4 was generously provided by Dr. G. R. Pettit, Arizona State
University.
Meth yl 3-[2-(3,4-d im eth oxyp h en yl)-7-m eth oxy-3-m eth -
oxyca r bon yl-1-ben zofu r a n -5-yl]p r op a n oa te (6d ) was pre-
pared from 190 mg (0.44 mmol) of 5d and 80 mg of 5% Pd/C
in 30 mL of THF. The mixture was shaken for 20 min in a
Parr apparatus under 60 psi. After filtration and evaporation,
an amorphous white powder was obtained which was purified
by column chromatography with ethyl acetate-hexane (3:1)
(2 × 10 cm, silica gel 60, 0.040-0.063 mm) (90%); 1H NMR
(CDCl3) δ 7.74 (d, J ) 1.98 Hz, 1 H, H-2), 7.71 (dd, J ) 8.54,
1.98 Hz, 1 H, H-6), 7.43 (d, J ) 1.42 Hz, 1 H, H-6′), 6.93 (d, J
Hollow F iber Assa y. The hollow fiber assay for assessing
the potential anticancer activity of test compounds has been
described by Hollingshead et al.20 Briefly, human tumor cells
were cultivated in polyvinylidene fluoride hollow fibers, and
a sample of each cell line was implanted into each of two
physiologic compartments (intraperitoneal and subcutaneous)
in mice. After treatment with test compounds [2b, 2c, and
2d (2R,3R)] at each of two test doses (100 and 150 mg/kg/dose,
intraperitoneal administration) using a QD × 4 schedule, fiber
cultures were collected and the viable cell mass was deter-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5481
(8) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A. P.; Scudiero,
D. A.; Rubinstein, L. V.; Plowman, J .; Boyd, M. R. Display and
Analysis of Patterns of Differential Activity of Drugs Against
Human Tumor Cell Lines: Development of Mean Graph and
COMPARE Algorithm. J . Natl. Cancer Inst. 1989, 81, 1088-
1092.
(9) Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmidt, J .
M. Isolation, Structure and Synthesis of Combretastatins A-1
and B-1, Potent New Inhibitors of Microtubule Assembly,
Derived from Combretum Caffrum. J . Nat. Prod. 1987, 50, 119-
131.
(10) Leping, L.; Wang, H.-K.; Kuo, S.-C.; Wu, T.-S.; Lednicer, D.; Lin,
C. M.;Hamel, E.;Lee, K.-H. Antitumor Agents. 150. 2′,3′,4′,5′,5,6,7-
Substituted 2-Phenyl-4-Quinolones and Related Compounds:
Their Synthesis, Cytotoxicity, and Inhibition of Tubulin Poly-
merization. J . Med. Chem. 1994, 37, 1126-1135.
(11) Hamel, E. Antimitotic Natural Products and Their Interactions
with Tubulin. Med. Res. Rev. 1996, 16, 207-231.
mined using a formazan dye conversion assay. A scoring
system was developed to simplify evaluation of the results.
The cell lines used were: NCI-H23 and NCI-H522 (lung
cancer), SF-295 and U251 (CNS cancer), LOX IMVI and
UACC-62 (melanoma), OVCAR-3 and OVCAR-5 (ovarian
cancer), COLO 205 and SW-620 (colon cancer), MDA-MB-231
and MDA-MB-435 (breast cancer).
Ack n ow led gm en t. This work was financially sup-
ported by the FWO (Fund for Scientific Research,
Flanders, Belgium) (Grant No. G.0119.96). Dr. A. Mauger
and Dr. M. Hollingshead (Developmental Therapeutics
Program, National Cancer Institute) are kindly acknowl-
edged for their cooperation. We are very indebted to
Prof. Dr. V. Buss (Gerhard-Mercator-Universita¨t-Gesa-
mthochschule Duisburg) for recording and interpreting
CD spectra.
(12) Woods, J . A.; Hadfield, J . A.; Pettit, G. R.; Fox, B. W.; McGown,
A. T. The Interaction With Tubulin of a Series of Stilbenes Based
on Combretastatin A-4. Br. J . Cancer 1995, 71, 705-711.
(13) Chen, K.; Kuo, S.-C.; Hsieh, M.-C.; Mauger, A.; Lin, C. M.;
Hamel, E.; Lee, K.-H. Antitumor Agents. 174. 2′,3′,4′,5,6,7-
Substituted 2-Phenyl-1,8-Naphthyridin-4-ones: Their Synthesis,
Cytotoxicity, and Inhibition of Tubulin Polymerization. J . Med.
Chem. 1997, 40, 2266-2275.
Su p p or tin g In for m a tion Ava ila ble: CD spectra of both
enantiomers of 2b (Figure 2) and/or table of inhibition of in
vitro tumor cell growth by synthetic dihydrobenzofuran lignans
(Table 1). This material is available free of charge via the
Internet at http://pubs.acs.org.
(14) Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S. Chalcones: A
New Class of Antimitotic Agents. J . Med. Chem. 1990, 33, 1948-
1954.
(15) Beutler, J . A.; Hamel, E.; Vlietinck, A. J .; Haemers, A.; Rajan,
P.; Roitman, J . N.; Cardellina, J . H. II; Boyd, M. R. Structure-
Activity Requirements for Flavone Cytotoxicity and Binding to
Tubulin. J . Med. Chem. 1998, 41, 2333-2338.
(16) Cheng, C. C. A Common Structural Pattern Among Many
Biologically Active Compounds of Natural and Synthetic Origin.
Med. Hypotheses 1986, 20, 157-172.
(17) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer
Drug Discovery Screen. Drug Dev. Res. 1995, 34, 91-109.
(18) Verdier-Pinard, P.; Lai, J .-L.; Yoo, H.-D.; Yu, J .; Marquez, D.
G.; Nagle, M.; Nambu, J . D.; White, J . R.; Falck, W. H.; Gerwick,
B. W.; Day, B. W.; Hamel, E. Structure-Activity Analysis of the
Interaction of Curacin A, the Potent Colchicine Site Antimitotic
Agent, with Tubulin and Effects of Analogues on the Growth of
MCF-7 Breast Cancer Cells. Mol. Pharmacol. 1998, 53, 62-76.
(19) Paull, K. D.; Lin, C. M.; Malspeis, L.; Hamel, E. Identification
of Novel Antimitotic Agents Acting at the Tubulin Level by
Computer-Assisted Evaluation of Differential Cytotoxicity Data.
Cancer Res. 1992, 52, 3892-3900.
Refer en ces
(1) Pieters, L.; De Bruyne, T.; Claeys, M.; Vlietinck, A.; Calomme,
M.; Vanden Berghe, D. Isolation of a Dihydrobenzofuran Lignan
from South American Dragon’s Blood (Croton spp.) as an
Inhibitor of Cell Proliferation. J . Nat. Prod. 1993, 56, 899-906.
(2) Lemie`re, G.; Gao, M.; De Groot, A.; Dommisse, R.; Lepoivre, J .;
Pieters, L.; Buss, V. 3′,4-Di-O-methylcedrusin: Synthesis, Reso-
lution and Absolute Configuration. J . Chem. Soc., Perkin Trans.
1 1995, 1775-1779.
(3) Ward, R. S. The Synthesis of Lignans and Neolignans. Chem.
Soc. Rev. 1982, 11, 75-125.
(4) MacRae, W. D.; Towers, G. H. N. Biological Activities of Lignans.
Phytochemistry 1984, 23, 1207-1220.
(5) Chioccara, F.; Poli, S.; Rindone, B.; Pilati, T.; Brunow, G.;
Pietika¨inen, P.; Seta¨la¨, H. Regio-Selective and Diastereo-Selec-
tive Synthesis of Dimeric Lignans Using Oxidative Coupling.
Acta Chem. Scand. 1993, 47, 610-616.
(6) Tamada, M.; Endo, K.; Hikino, H.; Kabuto, C. Structure of
Ephedradine A, a Hypotensive Principle of Ephedra Roots.
Tetrahedron Lett. 1979, 873-876.
(7) Yuen, M. S. M.; Xue, F.; Mak, T. C. W.; Wong, H. N. C. On the
Absolute Structure of Optically Active Neolignans Containing
a Dihydrobenzo[b]furan skeleton. Tetrahedron 1998, 54, 12429-
12444.
(20) Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott, B.
J .; Mayo, J . G.; Malspeis, L.; Grever, M. R. In Vivo Cultivation
of Tumor Cells in Hollow Fibers. Life Sci. 1995, 57, 131-141.
J M990251M