Effects of flavonoids on cell proliferation and caspase activation in a human colonic cell line HT29: An SAR study

Article abstract of DOI:10.1021/jm040770b

A library of 42 natural and synthetic flavonoids has been screened for their effect on cell proliferation and apoptosis in a human colonic cell line (HT-29). Examples of different classes of flavonoids have been screened, and the effects of hydroxylation, methoxylation and/or C-alkylation at various positions in the A- and B-rings have been assessed. Flavones and flavonols possess greater antiproliferative activity than chalcones and flavanones. With respect to structural modification of flavonoids, C-isoprenylation was by far the most effective, with substitution at the 8-position and longer chains, such as geranyl giving the best results. Finally, most compounds that significantly reduced cell survival also increased caspase activity, suggesting that at least part of their antiproliferative activity might be attributable to an apoptotic response.

Full text of DOI:10.1021/jm040770b

2790
J. Med. Chem. 2005, 48, 2790-2804
Effects of Flavonoids on Cell Proliferation and Caspase Activation in a Human
Colonic Cell Line HT29: An SAR Study
Jean-Baptiste Daskiewicz,† Flore Depeint,‡ Lionel Viornery,† Christine Bayet,† Geraldine Comte-Sarrazin,†
Gilles Comte,† Jennifer M. Gee,‡ Ian T. Johnson,‡ Karine Ndjoko,§, Kurt Hostettmann,§, and Denis Barron*,|
Laboratoire des Produits Naturels, CNRS-UMR 5013, UFR de Chimie-Biochimie, Universite´ Claude Bernard Lyon 1,
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France, Institute of Food Research, Norwich Research Park,
Colney, Norwich NR4 7UA, United Kingdom, Institut de Pharmacognosie et Phytochimie (IPP), Baˆtiment de l’Ecole de
Pharmacie (BEP), Universite´ de Lausanne, CH-1015 Lausanne-Dorigny, Suisse, and Department of Food Sciences,
Nestle´ Research Center, PO Box 44, CH-1000 Lausanne 26, Switzerland
Received January 13, 2004
A library of 42 natural and synthetic flavonoids has been screened for their effect on cell
proliferation and apoptosis in a human colonic cell line (HT-29). Examples of different classes
of flavonoids have been screened, and the effects of hydroxylation, methoxylation and/or
C-alkylation at various positions in the A- and B-rings have been assessed. Flavones and
flavonols possess greater antiproliferative activity than chalcones and flavanones. With respect
to structural modification of flavonoids, C-isoprenylation was by far the most effective, with
substitution at the 8-position and longer chains, such as geranyl giving the best results. Finally,
most compounds that significantly reduced cell survival also increased caspase activity,
suggesting that at least part of their antiproliferative activity might be attributable to an
apoptotic response.
Introduction
The citrus flavone tangeretin was first reported in 1995
to inhibit leukaemic HL-60 cell growth through the
induction of apoptosis,25 and more recently metabolites
of the lemon fruit flavanone eriocitrin were shown to
induce apoptosis in the same cell line.26 However,
studies on ways in which flavonoids interact with the
apoptotic sequence of programmed cell death have
increased dramatically in the past 4 years. Flavone has
been shown to be a potent apoptosis inducer in human
colon carcinoma cells, and this effect appears to be
restricted to cancer cells.27 Similarly, selective induction
of apoptosis in human prostate cancer cells by api-
genin28,29 and in human myeloid leukaemia HL-60 cells
by luteolin has been recently reported.30 Induction of
apoptosis by wogonin and fisetin has been demonstrated
to involve activation of the caspase 3 cascade in hepato-
cellular carcinoma SK-HEP-1 cells31 and human leu-
kemia HL-60 cells.32 In contrast, wogonin inhibited
apoptosis of activated rat glial cells, but this effect was
shown to be related to an inhibition of NO production.33
There are also numerous reports of the activation of
apoptosis by baicalin and baicalein.34-36 Polymethoxy-
flavonoids,37 the polyprenylated flavanone sophor-
anone,38 lavandulyl flavanones and flavanonols,39 and
various naturally occurring isoprenoid flavones40 are
also inducers of apoptosis. However, epicatechin and its
metabolite 3′-O-methylepicatechin protect human fibro-
blasts41 and neurons42 from oxidative-stress-induced cell
death. Apoptosis inhibition by galangin was shown to
involve blockage of aryl hydrocarbon receptor activa-
tion.43 Naringin prevented cell death in normal liver
cells, but not in cancer cells.44
Polyphenolic compounds are plant products which
represent common constituents of fruits and vegetables.1
The major group of plant polyphenols is represented by
flavonoids, and a recent review has estimated their
number as 6500 in the plant kingdom.2 A large number
of biological effects have been attributed to these
compounds.3 More precisely, the interaction of dietary
flavonoids with the gut has numerous implications for
human health,4 and flavonoid compounds in the diet
may act as chemopreventive agents against the devel-
opment of cancers in the alimentary tract.5,6 In fact, a
number of studies have demonstrated antiproliferative
activity associated with flavonoids,3 for example green
tea7 and wine8 polyphenols, flavanone and flavanonol
derivatives such as pinostrobin,9 liquiritigenin,10 and
silibinin,11 chalcones12 such as isoliquiritigenin and
butein,13 flavone and flavonol derivatives such as
chrysin,14 wogonin,10 baicalein,10,15 7-O-methylgalan-
gin,14 luteolin, and quercetin,16 polymethoxyflavones17,18
including nobiletin10 and ternatin,19 prenylated fla-
vonoids from hops,20 and isoflavones such as genistein.10
Potentially protective substances may exert their anti-
proliferative effects through a number of mechanisms.
For example, it has been demonstrated that antiprolif-
erative isoflavones, such as biochanin A and genistein,
act through induction of apoptosis.21 Similar effects have
been described for two synthetic chalcones,22 and the
role of dietary compounds in the modulation of apoptosis
in the colorectal mucosa has been recently reviewed.23,24
* To whom correspondence should be addressed. Tel: 41 21 785 94
97. Fax: 41 21 785 85 54. E-mail: Denis.Barron@rdls.nestle.com.
† Universite´ Claude Bernard Lyon 1.
A study on a wide range of 30 flavonoid compounds
was carried out by Kuntz et al.,45 but the authors could
not draw clear structure-activity relationships (SAR)
regarding their apoptotic or antiproliferative effects. In
fact, and perhaps surprisingly, very few studies have
‡ Institute of Food Research.
§ Universite´ de Lausanne.
| Nestle´ Research Center.
Present address: Universite´ de Gene`ve, Sciences II, Section des
Sciences Pharmaceutiques, Quai Ernest-Ansermet 30, CH-1211 Gene`ve
4, Switzerland.
10.1021/jm040770b CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/30/2005

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2791
allyl)chrysin 16,52 8-(1,1-dimethylallyl)-3-O-methyl-
galangin 34,52 6-prenylnorwogonin 24,53 8-(1,1-dimethyl-
allyl)galangin 27,54 and 8-(1,1-dimethylallyl)kaempfer-
ide 3154 were prepared according to published proce-
dures. Naringenin chalcone 1 is a well-known natural
product55 and was obtained here by alkaline treatment
of narigenin. 4-Methoxy-2′,4′-â-trihydroxychalcone 2
was prepared by LiHMDS-mediated condensation of
resacetophenone 43 with anisoyl chloride 44 (Scheme
Scheme 1. Synthesis of
4-Methoxy-2′,4′,â-trihydroxychalcone 2
1
1), after adaptation of a reported procedure.56 The H
NMR of 2 in acetone-d6 was in accordance with pub-
lished data.57 The 13C NMR of 2 was similar to previous
data,58 except for the carbonyl, the C4, and the C4′
signals that we have reassigned after analysis of the
proton-carbon long distance correlation (HMBC) spec-
trum of 2. In the synthesis of dimethylallylchalcone 3,
O-prenylation of resacetophenone 43 with 1 equivalent
of prenyl bromide 45 yielded 2′-hydroxy-4′-prenyloxy-
acetophenone 4659 in 44% yield (Scheme 2). Aldol
condensation of the latter compound with benzaldehyde
47 produced cordoin 4860 (54%). An attempt to re-
arrange 48 by conventional boiling acetic anhydride/
sodium acetate conditions52 was unsuccessful since
cordoin remained unchanged. Thus we applied more
drastic conditions to provoke the sigmatropic rearrange-
ment. Indeed, microwave irradiation of cordoin in a
mixture of N,N-diethylaniline and benzoic anhydride
followed by saponification produced 2′,4′-dihydroxy-5′-
(1,1-dimethylallyl)chalcone 3, albeit in small amounts
(15% yield). The second product of the reaction, 2′,4′-
dihydroxychalcone 49,61 resulted from the elimination
of the O-prenyl chain of cordoin with no rearrangement.
The original synthesis of 5-O-Methylchrysin 6 was first
reported 42 years ago,62 but it has been recently identi-
fied as a natural constituent of Urugayan propolis.63 In
this study, it has been synthesized by methylation of
7-O-benzoylchrysin 50, followed by saponification of the
7-benzoyl group (Scheme 3). The complete spectral
characterization of 6 is given here for the first time. 5-O-
prenylchrysin 12 resulted from the saponification of 7-O-
benzoyl-5-O-prenylchrysin 52 (Scheme 3). O-Prenylation
of chrysin 5 with geranyl bromide or farnesyl bromide
yielded 7-O-geranylchrysin 17 (91%) and 7-O-farnesyl-
chrysin 21 (89%), respectively (Scheme 4). Sigmatropic
rearrangements of 17 and 21, using boiling acetic
anhydride/sodium acetate, gave rise, after saponifica-
tion, to the 8-linalyl product 20 (49%) and the 8-
nerolidyl product 22 (41%), respectively (Scheme 4). The
attempted to draw structure-activity relationships in
this respect, especially as many studies agree on the
necessary presence of the 4-carbonyl46,47 and of the 2,3-
double bond in the C ring.47,48 The effect of 3-hydroxyl-
ation is unclear, being either considered as unpredict-
able46 or without effect.48 A- and B-ring substitutions
were much more difficult to interpret with respect to
antiproliferative activity. A broader study on 21 fla-
vonoid compounds has recently been carried out by
Nagao et al. using three tumor cell lines.49 Although
they found it difficult to draw clear conclusions, sub-
stitution profiles such as 5,7-dihydroxy, 5,7-dihydroxy-
6-methoxy, or 5,6,7-trihydroxy in ring A, and 3′,4′-
dihydroxy or 3′,4′-dihydroxy-5′-methoxy in ring B, were
considered activating. According to another study, the
presence of at least three adjacent methoxyl groups in
the molecule can confer a more potent antiproliferative
effect.48 As part of our investigations of the impact of
flavonoids on the prevention of colon cancer, we have
screened a library of 42 natural and synthetic flavonoids
for effects on cell proliferation and apoptosis in a human
colon carcinoma cell line (HT-29). Examples of different
classes of flavonoids have been screened, and the effects
of hydoxylation, methoxylation and/or C-alkylation at
various positions of the A- and B-rings have been
assessed.
Results and Discussion
Chemistry. Tables 1-5 display the structures of the
42 studied flavonoids, of which compounds 3, 12, 17,
20-22, 33, 36, 38, and 40-42 are novel. Naringenin 4,
chrysin 5, polymethoxyflavones 25 and 26, kaempferol
28, kaempferide 30, and quercetin 32 were from com-
mercial sources. Chrysin derivatives 7-11, 13, 15, 18,
19,50 6-(1,1-dimethylallyl)chrysin 14,51 8-(1,1-dimethyl-
Scheme 2. Synthesis of 2′,4′-Dihydroxy-5′-(1,1-dimethylallyl)chalcone 3a
a Reagents and conditions: (I) K2CO3, tetrabutylammonium bromide, DMF, 44%; (II) Aq. KOH, MeOH, 54%; (III) Bz2O, N,N-
diethylaniline, MWI, 15.5% of 3 and 14% of 49.

2792 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
Scheme 3. Preparation of 5-Alkylated Chrysins 6 and
pound 33 have been published. In the present paper,
we describe the first efficient regioselective syntheses
of compounds 23, 29, and 33. The selective syntheses
of 8-prenylapigenin 23 and 8-prenylkaempferol 29 fol-
lowed the strategy that we previously developed for the
preparation of 8-prenylchrysin.69 This involved O-
prenylation of the 5-hydroxyl after protection of all other
hydroxyls by methoxymethyl (MOM) groups, followed
by a sigmatropic rearrangement in N,N-diethylaniline
at 217 °C and by acidic removal of the protective groups
(Scheme 5). The 13C NMR data for 8-prenylapigenin 23
(licoflavone C) was in accordance with previous data,64
except for the C8 and C10 NMR signals that we have
12a
a Reagents and conditions: (I) ICH3, tetrabutylammonium
hydroxide, CH2Cl2/toluene; (II) 5% KOH in MeOH, ultrasound,
21% of 6 from 50; (III) prenyl bromide, tetrabutylammonium
hydroxide, CH2Cl2/toluene; (IV) 5% KOH in MeOH, ultrasound,
50% of 12 from 50.
1
reversed, based on the analysis of the 2D H-13C long
distance correlation spectrum of compound 23. In fact,
the signal at 108.39 ppm was unequivocally assigned
to C8 since it displayed a strong 2J correlation with H1′′
of the 3,3-dimethylallyl chain. The signal at 106.36 ppm
was correlated with both H3 and H6, in accordance with
its attribution to C10. Complete NMR data in acetone-
d6 for 8-prenyl kaempferol 29 (noranhydroicaritin) is
reported here for the first time. The synthesis of
8-prenylquercetin 33 caused specific problems. Indeed,
reaction of quercetin 32 with chloromethyl methyl ether
using N,N-diisopropylamine as a base only lead to
protection of the most acidic 7-, 4′-, and 3-hydroxyls.
Besides the fact that the 3′-hydroxyl could not be
protected, even the reaction of the most acidic hydroxyl
was incomplete and a mixture of products was obtained.
Only the use of sodium hydride as a base would have
allowed complete protection but obviously such strong
alkaline conditions would lead to extensive degradation
of quercetin. We solved this problem by adopting the
following strategy (Scheme 6). The preparation of
3,7,3′,4′-tetrakis-O-methoxymethylquercetin 56 was ac-
complished in three steps with an overall yield of 57%
from quercetin. None of the intermediate products was
isolated, and the three steps were performed on line.
Thus quercetin 32 was first reacted with 6 equivalents
of chloromethyl methyl ether and 6 equivalents of N,N-
diisopropylethylamine in DMF. The mixture of partially
protected products, now stable in strong alkaline condi-
Scheme 4. Syntheses of 8-Linalylchrysin 20 and of
8-Nerolidylchrysin 22a
a Reagents and conditions: (I) Geranyl bromide or farnesyl
bromide, anhydrous K2CO3, Me2CO; (II) Ac2O/NaOAc, reflux; (III)
5% KOH in MeOH, ultrasound.
8-(3,3-dimethylallyl) derivatives of apigenin 2364 and
kaempferol 2965 have been previously described as plant
constituents. 8-Prenylkaempferol 29 has been prepared
by an established chemical synthesis involving direct
prenylation of kaempferol with 2-methyl-3-buten-2-ol in
the presence of BF3 etherate.66 However, this method
gives quite a low yield from a mixture of analogues.
Finally, although there have been several reports of
synthesis of 8-prenylquercetin 33,67,68 neither a clear
synthetic procedure nor the characterization of com-
Scheme 5. Syntheses of the 8-Prenyl Derivatives of Apigenin 23, Kaempferol 29, and Quercetin 33a
a Reagents and conditions: (I) 54 and 55: MOM chloride, iPr2Net, DMF; 56: see Scheme 6; (II) prenyl bromide, tetrabutylammonium
hydroxide, CH2Cl2/toluene; (III) N,N-diethylaniline, 217 °C; (IV) dilute HCl, MeOH, reflux.

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2793
Scheme 6. Strategy for the Preparation of 3,3′,4′,7-Tetrakis-O-methoxymethylquercetin 56a
a Reagents and conditions: (I) MOM chloride; iPr2Net, DMF; (II) MOM chloride, NaH, DMF; (III) dilute HCl, EtOH, room temperature,
57% of 56 from 32.
Scheme 7. Preparation of 3-Methoxyflavones 35-41, and Their 8-(1,1-Dimethylallyl) Derivatives 36 and 38a
a Reagents and conditions: (I) Et3N, reflux; (II) KOH/MeOH, reflux; (III) prenyl bromide, K2CO3/Me2CO; (IV) NaOAc/Ac2O, reflux; (V)
OH-; VI) H+.
Scheme 8. Synthesis of 3-O-Methylplatanetin 42a
well plate methodology to detect changes in viability by
neutral red uptake. All compounds were tested at a
concentration of 50 µM in the culture medium. Cell
survival data after 6 and 24 h incubation are listed in
Tables 1-5. The conclusions summarized in the follow-
ing paragraph are based on the significance (no more
than 90% cell survival and P < 0.05) or not of differences
in antiproliferative effects after 24 h incubation. Among
the different classes of flavonoid compounds, chalcones
1 and 2 as well as the flavanone naringenin 4 failed to
reduce proliferation. Only flavones such as chrysin 5,
flavonols such as kaempferol 28 and quercetin 32
displayed some effect. This is in agreement with previ-
ous studies that have pointed out the importance of the
2,3-double bond on the C-ring. In addition, methylation
of one or two positions greatly reduced the activity of
chrysin (6, 7, 8) but increased the activity of the
kaempferol derivative (30) after 24 h incubation. Thus
elements other than presence or absence of a methyl
moiety are involved in SAR affecting the antiprolif-
erative potential of flavonoids. Polymethoxylated struc-
tures were, however, mainly inactive (25, 26, 39-41)
with the exception of 5,7-dihydroxy-3,3′,4′trimethoxy-
flavone (37: 74.2 ( 6.6% at 24 h). This is in contrast to
previous findings for polymethoxyflavones;10,18,19,48 how-
ever, these were carried out on human leukemic HL-60
cells,10 CEM leukemia, CDD 922 skin fibroblasts and
SW 1573 lung tumor cells,19 or melanoma cell lines.48
Only in one case18 have HT-29 human colon cancer cells
been used as models, and the authors pointed out the
fact that, among the various cell lines employed, colon
cancer cell lines were less sensitive to the effects of
polymethoxyflavones. Thus our observations that ex-
a Reagents and conditions: (I) K2S2O8, aq. tetramethylammo-
nium hydroxide; (II) prenyl bromide, aq. tetramethylammonium
hydroxide, 39% of 42 from 72.
tions, was further alkylated with 6 equivalents of
chloromethyl methyl ether and 9 equivalents of sodium
hydride, yielding 3,5,7,3′,4′-pentakis-O-methoxymethyl-
quercetin 63. Exposure of compound 63 to dilute etha-
nolic HCl at room-temperature resulted in the selective
cleavage of the 5-MOM group, giving rise to the desired
3,7,3′,4′-tetrakis-O-methoxymethylquercetin 56. The
preparation of a series of 3-methoxyflavones 35-41,
with various methoxylation patterns at positions 2′, 3′,
and 5′, involved Allan-Robinson condensations of 2′,4′,6′-
trihydroxy-2-methoxyacetophenone 64 (Scheme 7) with
the properly substituted benzoic anhydrides 65-69.
Flavones 35 and 37 were further O-prenylated at
position 7 and rearranged in boiling acetic anhydride/
sodium acetate, to give the 8-(1,1-dimethylallyl) flavones
36 and 38 (Scheme 7). 3-O-Methylplatanetin 42 has
been synthesized by an adaptation (Scheme 8) of our
previous method53 based on the online 8-oxidation and
6-prenylation of flavones in aqueous tetramethyl-
ammonium hydroxide.
Antiproliferative Activity on Human Colonic
Cell Line (HT-29) in Vitro. Fourty-two compounds
have been tested for antiproliferative activity using 96-

2794 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
Table 1. Stuctures and Antiproliferative Activity of Chalcones 1-3 and Flavanone 4a
compound substitution
antiproliferative
antiproliferative
effect (24 h)
caspase
induction (5 h)
R1
R2
R3
R4
effect (6 h)
1
2
3
4
H
H
OH
H
H
H
OH
H
OH
OMe
H
102.0 ( 9.6
96.2 ( 12.2
35.4 ( 15.6
102.2 ( 11.7
94.1 ( 11.8
96.6 ( 14.0
15.6 ( 8.8
94.8 ( 8.2
1,1-DMA
7.3
a Antiproliferative effects are expressed as a percentage of the response of the control cells (mean ( SD); n > 18 replicates per experiment,
experiment repeated at least four times. The lower the value the higher the antiproliferative activity. Caspase 3/7 activity is expressed
as a ratio of caspase activation in treated cells to that of untreated control cells. Data presented here are the mean of duplicate wells on
the same plate. The higher the value the greater the caspase activity.
Table 2. Structures and Antiproliferative Activity of Chrysin Derivatives 5-22a
compound substitution
antiproliferative
effect (6 h)
antiproliferative
effect (24 h)
caspase
induction (5 h)
R1
R2
R3
R4
5
OH
OMe
OH
OH
OH
OH
OH
H
OH
H
H
H
H
H
H
Bn
H
H
H
86.6 ( 10.0
104.7 ( 5.2
104.0 ( 4.1
92.2 ( 4.7
96.6 ( 5.0
65.3 ( 7.5
103.2 ( 4.6
17.0 ( 5.1
87.9 ( 8.9
90.4 ( 8.3
86.3 ( 8.3
76.7 ( 5.5
90.4 ( 4.2
58.2 ( 12.7
18.0 ( 7.4
28.4 ( 10.0
87.8 ( 4.4
43.3 ( 9.5
62.8 ( 6.2
107.6 ( 8.1
100.8 ( 4.9
83.8 ( 7.2
87.1 ( 8.4
57.6 ( 7.0
97.4 ( 5.7
19.4 ( 3.2
87.6 ( 6.8
79.1 ( 5.5
75.5 ( 7.8
57.7 ( 5.0
84.3 ( 3.4
53.9 ( 6.5
7.6 ( 4.3
0.8
6
H
OH
7
Me
Me
H
Bn
H
H
OH
8
OMe
O-Bn
OH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
OH
O-(3,3-DMA)
OH
OH
1.4
3,3-DMA
OH
OH
1,1-DMA
OH
OH
H
OH
3,3-DMA
0.9
1.1
1.2
1.9
9.0
8.9
OH
H
OH
1,1-DMA
H
H
OH
H
O-Ger
OH
OH
Ger
H
OH
OH
OH
O-Far
OH
Ger
Lin
H
OH
H
28.1 ( 5.4
83.3 ( 7.6
24.5 ( 4.5
OH
H
OH
H
Ner
6.9
a Antiproliferative effects are expressed as a percentage of the response of the control cells (mean ( SD); n > 18 replicates per experiment,
experiment repeated at least four times. The lower the value the higher the antiproliferative activity. Caspase 3/7 activity is expressed
as a ratio of caspase activation in treated cells to that of untreated control cells. Data presented here are the mean of duplicate wells on
the same plate. The higher the value the greater the caspase activity.
tensive methoxylation renders such compounds inactive
against human colon cancer cells are not entirely
inconsistent. Aromatic substitution of the chrysin mol-
ecule increases antiproliferative activity at position 6
(10) but not at position 7 (9) or 8 (11). In addition,
aliphatic C-substitutions were more activating at posi-
tion 8 than 6 as evidenced by data for prenyl- (13, 15),
geranyl- (18, 19), and 1,1-dimethylallylchrysin (14, 16).
In term of O-substitutions, an aliphatic chain in position
5 (12) greatly increased the activity, while any substitu-
tions at position 7 (5 < 9 ∼ 17 ∼ 21) showed decreased
activity. The order of potency of the different aliphatic
chains depended on the position of substitution (at C6:
geranyl 18 ∼ benzyl 10 < 5 (aglycone) < 1,1-dimethyl-
allyl 14 < prenyl 13 while at C8: geranyl 19 < nerolidyl
22 < linalyl 20 < 1,1-dimethylallyl 16 < 5 (aglycone) <
prenyl 15 < benzyl 11), but indications of a pattern
emerged. A geranyl group at position 6 or 8 (18, 19)
increased potency of chrysin while a prenyl group at
these two positions (13, 15) decreased its effect. Longer
aliphatic chains were also consistently more activating
than shorter ones (19 < 15, and 22 < 20 < 16). Finally,
1,1-dimethylallyl substitutions at position 8 increased
the activity of the aglycone form in all cases (16 < 5,
31 < 30, 36 < 35, 38 < 37). However the order of
potency of substituted compounds did not strictly follow
that of the corresponding aglycones. Substitution at
position 8 with a prenyl group increased the activity of

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2795
Table 3. Structures and Antiproliferative Activity of Miscellaneous Flavones 23-26a
compound substitution
antiproliferative
antiproliferative
effect (24 h)
caspase
induction (5 h)
R1
R2
R3
R4
R5
R6
R7
effect (6 h)
23
24
25
26
OH
H
OH
3,3-DMA
H
OH
H
OMe
OMe
H
76.9 ( 12.0
67.4 ( 6.1
95.4 ( 9.7
94.2 ( 10.6
56.2 ( 5.0
71.7 ( 5.2
102.1 ( 7.5
100.7 ( 5.0
OH
3,3-DMA
OMe
OH
OH
H
H
H
H
1.6
OMe
OMe
OMe
OMe
OH
OMe
H
OMe
OMe
a Antiproliferative effects are expressed as a percentage of the response of the control cells (mean ( SD); n > 18 replicates per experiment,
experiment repeated at least four times. The lower the value the higher the antiproliferative activity. Caspase 3/7 activity is expressed
as a ratio of caspase activation in treated cells to that of untreated control cells. Data presented here are the mean of duplicate wells on
the same plate. The higher the value the greater the caspase activity.
Table 4. Structures and Antiproliferative Activity of Flavonols 27-33a
compound substitution
antiproliferative
effect (6 h)
antiproliferative
effect (24 h)
caspase
induction (5 h)
R1
R2
R3
27
28
29
30
31
32
33
1,1-DMA
H
3,3-DMA
H
1,1-DMA
H
3,3-DMA
H
H
76.7 ( 5.5
92.0 ( 5.9
78.8 ( 12.5
99.7 ( 7.7
56.4 ( 7.2
61.4 ( 9.6
68.0 ( 8.5
63.0 ( 6.3
89.6 ( 7.3
71.0 ( 7.1
84.7 ( 4.1
42.1 ( 4.8
51.3 ( 5.2
67.0 ( 4.1
1.1
1.8
2.7
1.0
H
OH
H
OH
H
OMe
OMe
OH
H
OH
OH
1.0
1.9
OH
a Antiproliferative effects are expressed as a percentage of the response of the control cells (mean ( SD); n > 18 replicates per experiment,
experiment repeated at least four times. The lower the value the higher the antiproliferative activity. Caspase 3/7 activity is expressed
as a ratio of caspase activation in treated cells to that of untreated control cells. Data presented here are the mean of duplicate wells on
the same plate. The higher the value the greater the caspase activity.
Table 5. Structures and Antiproliferative Activity of 3-Methoxyflavones 34-42a
compound substitution
antiproliferative
effect (6 h)
antiproliferative
effect (24 h)
caspase
induction (5 h)
R1
R2
R3
R4
R5
R6
34
35
36
37
38
39
40
41
42
H
H
H
H
H
H
H
H
1,1-DMA
H
H
H
H
79.3 ( 5.0
92.0 ( 8.4
34.5 ( 15.4
87.9 ( 8.2
8.5 ( 2.2
101.9 ( 6.4
89.7 ( 7.6
90.0 ( 8.3
59.8 ( 7.2
79.4 ( 8.5
77.1 ( 5.5
20.1 ( 9.3
74.2 ( 6.6
4.7 ( 0.6
101.8 ( 6.5
91.5 ( 6.2
92.5 ( 7.8
71.8 ( 3.5
1.3
1.2
8.1
1.1
5.2
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
1,1-DMA
H
H
H
H
H
OMe
OMe
OMe
OMe
H
H
1,1-DMA
H
H
H
H
OMe
H
H
OMe
OMe
H
H
OMe
H
3,3-DMA
OH
H
3.5
a Antiproliferative effects are expressed as a percentage of the response of the control cells (mean ( SD); n > 18 replicates per experiment,
experiment repeated at least four times. The lower the value the higher the antiproliferative activity. Caspase 3/7 activity is expressed
as a ratio of caspase activation in treated cells to that of untreated control cells. Data presented here are the mean of duplicate wells on
the same plate. The higher the value the greater the caspase activity.
apigenin and kaempferol (23, 29) but decreased that of
chrysin and quercetin (15, 33). Similarly to the effect
observed for methylation patterns, this type of substitu-
tion appears to have variable and unpredicatable effect
on antiproliferative potential. The distribution of iso-
prenoid flavonoids in the plant kingdom is restricted to
a handful of families.70 Hops is one of the rare isoprenoid
flavonoid-rich plants that can participate in the elabora-

2796 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
caspase response (1.8 to 2 times the control value), and
12 did not increase caspase activity significantly (less
than 1.5 times the control value). The remaining five
compounds were not tested due to limited availability.
Caspase activity at 5 h was inversely correlated to cell
survival at 6 h (R2 ) 0.56; P < 0.01) and 24 h (R2 )
0.60; P < 0.01) (Figure 1), with most compounds that
reduced cell survival to less than 60% being capable of
inducing an apoptotic response, as detected by caspase
assay. One noticeable exception was 5-O-prenylchrysin
12 which failed to induce a caspase response. These data
suggest that a free hydroxyl group at position 5 may be
a contributory factor in caspase activation. However this
observation is based on only one compound not fitting
the model and requires substantiation, bearing in mind
that factors such as the size of the substituent at
position 5 and its hydrophobicity may also be relevant.
Both quercetin 32 and chrysin (5) failed to induce
apoptosis, as measured by increased caspase activity.
The data for quercetin is in contrast to published
literature, some of it based on HT-29 cells.71 In the latter
study, however, quercetin was used at a higher concen-
tration (100 µM) as compared to the present work (50
µM). There were two further compounds with unex-
pected caspase activity in relation to their antiprolif-
erative potential at 6 h. These were the 8-prenyl
conjugates of quercetin 33 and kaempferol 29, the
former only a moderate activator of caspase. Again this
suggests that a complex combination of structural
factors is involved in antiproliferative activity and
caspase induction.
Conclusion
Despite the atypical behavior of a limited number of
compounds, clear structure-activity relationships are
apparent from the present study. Flavones and flavonols
possess greater antiproliferative activity than chalcones
and flavanones. With respect to substitutions, C-iso-
prenylation was by far the most effective, with the
localization at position 8 and substitution with longer
chains such as geranyl producing the best increase in
antiproliferative potential. Among the flavonoids tested
that are dietary components, only quercetin, kaempfer-
ol, and less methoxylated flavones such as kaempferide
showed antiproliferative activity at a concentration of
50 µM. In contrast, at this concentration, we were
unable to obtain evidence of antiproliferative activity
for highly methoxylated flavones. Most compounds
reducing cell survival to less than 60% after 6 h
incubation also provoked a caspase response, suggesting
that at least part of their antiproliferative activity might
be attributable to an apoptotic response. It is noteworthy
that the structural requirements for flavonoids demon-
strated by this study, to give the best antiproliferative
activity and caspase activation, are similar to those we
previously established for the inhibition of P-glyco-
protein-mediated efflux of drugs,50 especially concerning
the beneficial effects of C-isoprenylation. P-glycoprotein
(Pgp) is one of the transporter involved in multidrug
resistance (MDR), and it has been recently established72
that some MDR modulators may also induce apoptosis
by elevation of the level in ceramide, now recognonized
as an intracellular signal for apoptosis.73,74 Lucci et al.75
have reported a synergestic action of different MDR
modulators on resistant human breast carcinoma MCF-
Figure 1. Correlation between the viability of HT29 cells
treated with compounds 3, 5, 12, 15,16,17,18,19, 20, 22, 24,
27, 28, 29, 30, 32,33, 34, 35, 36, 37, 38, and 42 for (a) 6 h or (b)
24 h and caspase activation by these compounds. Point marked
with /* is 5-O-prenylchrysin; n ) 23 and P < 0.01 for both
comparisons.
tion of a food product. Miranda et al.20 have recently
reported antiproliferative activity for hops isoprenoid
chalcones and suggested chemopreventive potential.
Their study demonstrated once again that HT-29 colon
cancer cells were more resistant than human breast
cancer cells to the antiproliferative effects of these
flavonoids. Since most of the studied isoprenoid chal-
cones from hops carry an average of three to four
hydroxyl groups, our observations, that a reduced
antiproliferative effect on HT-29 cells is associated with
increasing hydroxylation pattern of isoprenoid fla-
vonoids, are consistent with this previous study.
Effects of Flavonoids on Caspase Activation on
Human Colonic Cell Line HT29. Of the compounds
studied, 15 failed to reduce viability below the effective
threshold of 90% of the untreated cells. These com-
pounds were therefore not investigated any further, and
only compounds causing a greater reduction in the
uptake of neutral red were assessed for induction of
caspase 3 and 7 activity. Caspase-induction data, where
available, is included in Tables 1-5. Of the molecules
tested, eight increased caspase levels to more than twice
the control value; another three caused a moderate

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2797
bromide 45, dropwise at room temperature. After addition of
all of the reagents, the reaction mixture was incubated for 2.5
h at room temperature with stirring. The resulting solution
was diluted with 200 mL of H2O plus 100 mL of 20% aq.
Na2CO3 and extracted with 250 mL of EtOAc. The EtOAc
extract was rinsed by 3 × 100 mL of 20% aq. Na2CO3 and by
2 × 200 mL of H2O. Using this procedure, all the unreacted
resacetophenone remained in solution in the aqueous layers,
leaving the prenylresacetophenone dissolved in the EtOAc
extract. The latter was purified by VLC on silica gel using a
gradient of EtOAc in hexane as solvent, leading to the isolation
of 4.87 g (22 mmol, 44%) of compound 46.59 1H NMR (200 MHz,
CDCl3) δ 1.75 (brs, 1 H, H5′′), 1.80 (brs, 1 H, H4′′), 2.55 (s, 3
H, H2), 4.55 (d, 2 H, J ) 6.7 Hz, H1′′), 5.47 (brt, 1 H, J ) 6.7
Hz, H2′′), 6.45 (m, 2 H, H3′ + H5′), 7.62 (d, 1 H, J ) 9.2 Hz,
H6′), 12.73 (s, 1 H, 2′OH): in accordance with previously
published partial 1H NMR data;76 13C NMR (50 MHz, CDCl3):
δ 18.11 (C5′′), 25.67 (C4′′), 26.01 (C2), 65.07 (C1′′), 101.43 (C3′),
108.03 (C5′), 113.71 (C1′), 118.65 (C2′′), 132.15 (C6′), 138.91
(C3′′), 165.11 (C2′), 165.37 (C4′), 202.38 (C1).
7-AdrR cells, leading to increased ceramide levels and
associated apoptosis. Thus combined MDR modulators,
by synergistic effects on drug efflux and induction of
apoptosis, may offer new strategies in the prevention
and treatment of cancers. As Pgp modulators, the action
of isoprenoid flavonoids on the events such as ceramide
formation in apoptotic signaling warrant further atten-
tion in the future.
Experimental Section
General Chemistry Methods. Resacetophenone was from
Acros. Chrysin, naringenin, and prenyl, geranyl, and farnesyl
bromides were from Aldrich. Apigenin, kaempferol, and
kaempferide were purchased from Extrasynthe`se (Genay,
France). Quercetin monohydrate was from Sigma. 3′-Hydroxy-
4′,5,6,7-tetramethoxy-, and 3′,4′,5,5′,6,7-hexamethoxyflavones
were obtained from Lancaster. Vacuum liquid chromatography
(VLC) was performed using Lichroprep Si 60 40-63 µm as
stationary phase, and a gradient of EtOAc in hexane as
solvent. Medium-pressure liquid chromatography (MPLC) was
carried out using a Buchi B688 pump equipped with a Buchi
B687 gradient maker, a Lichroprep Diol 40-63 µm or a
Lichroprep Si 60 40-63 µm as stationnary phases, and a
gradient of EtOAc in hexane as solvent. High Performance
Liquid Chromatography (HPLC) for cordoin quantification was
carried out on a Lichrospher RP18 column (250 × 4.6 mm,
particle size 5 µm) using a gradient of MeOH in H2O as solvent
and UV detection at 254 nm. Analyses were performed using
a standard compound of pure cordoin from our laboratory
collection. Microwave irradiation (rearrangement of cordoin)
was carried out using a domestic Bluewind 1797 oven. Gel
filtration (purification of the products of the rearrangement
of cordoin) was performed on Sephadex LH-20 in a 400 × 20
mm open column. 1H NMR spectra at 300 and 500 MHz were
recorded on a Bruker DPX 300 and a Varian Unity Inova NMR
spectrometer, respectively. The signals were assigned with the
help of direct and long distance heteronuclear correlation
spectra. Chemical ionization mass spectra were recorded in
the positive mode, on a Finnigan MAT95XL apparatus, using
isobutane as gas reagent. High-resolution mass spectra were
obtained on a TOF mass spectrometer (Micromass, Manches-
ter, UK), using the following conditions: loop injections in H2O
(0.1% TFA)-MeCN (0.1% TFA); capillary voltage: 3200 V;
extraction cone voltage: 3 V; desolvatation temperature: 150
°C; sample cone voltage: 32; source temperature: 120 °C;
nebulization gas (N2): 515 L/h; calibration with PEG in
positive mode: 250-610 mau; reference: leucine enkephalin
(1 ng/mL, injection of 5 µL/min).
Cordoin (48). An amount of 4.87 g (22 mmol) of 2′-hydroxy-
4′-prenyloxyacetophenone 46 and 2.2 mL (22 mmol) of benz-
aldehyde 47 were dissolved in 10 mL of MeOH in a round-
bottom flask. To this solution, 40 mL of H2O and 50 mL of
50% aq. KOH were added successively, and the mixture was
refuxed for 30 min. After cooling, the solution was poured on
crushed ice and acidified (6 N HCl). This led to the formation
of a yellow precipitate which was isolated by filtration. Yield
in crude solid: 5.78 g. The amount of pure cordoin 4860 present
in this crude solid was estimated as 3.66 g (11.87 mmol, 54%)
by HPLC analysis. A sample of pure 48 was obtained by
dissolving the crude solid in 200 mL of MeOH:Me2CO (6:4) at
40 °C. The solution was left to concentrate in a beaker at room
temperature in the dark for 72 h. The yellow crystals of cordoin
that have appeared were isolated by filtration and rinsed with
cold MeOH. Yield in pure cordoin 48: 2.1 g. More crystals can
be obtained by allowing the filtrate to further concentrate at
1
room temperature. H NMR (200 MHz, CDCl3) δ 1.74 (brs, 1
H, H5′′), 1.79 (brs, 1 H, H4′′), 4.54 (d, 2 H, J ) 6.7 Hz, H1′′),
5.46 (brt, 1 H, J ) 6.7 Hz, H2′′), 6.48 (m, 2 H, H3′ + H5′), 7.40
(m, 3 H, H3 + H4 + H5), 7.55 (d, 1 H, J ) 15.5 Hz, HR), 7.62
(m, 2 H, H2 + H6), 7.80 (d, 1 H, J ) 9.6 Hz, H6′), 7.86 (d, 1 H,
J ) 15.5 Hz, Hâ), 13.41 (s, 1 H, 2′OH); 13C NMR (50 MHz,
CDCl3): δ 18.20 (C5′′), 25.75 (C4′′), 65.16 (C1′′), 101.72 (C3′),
108.25 (C5′), 113.96 (C1′), 118.67 (C2′′), 120.35 (CR), 128.46
(C2 + C6), 128.93 (C3 + C5), 130.57 (C4), 131.17 (C6′), 134.80
(C1), 139.06 (C3′′), 144.25 (Câ), 165.59 (C4′), 166.83 (C2′),
191.73 (CdO).
2′,4′-Dihydroxy-5′-(1,1-dimethylallyl)chalcone (3). A
homogenized mixture of 0.4 g (1.3 mmol) of cordoin 48 and of
1.46 g (6.45 mmol, 5 equiv) of benzoic anhydride were
introduced into a 10 mL Teflon bottle, and 3 mL of freshly
distilled N,N-diethylaniline was added. The bottle was well
stoppered and submitted to 15 min of microwave irradiation
at 750 W (CAUTION, overpressure may develop inside the
bottle). After complete cooling, the content of the bottle was
taken up into a solution of 5% KOH in MeOH (250 mL), and
the solution was subjected to ultrasound for 25 min (saponi-
fication of the benzoate esters). The medium was diluted with
H2O, acidified (pH 1, 6 N HCl), and extracted with EtOAc,
and the EtOAc extract was rinsed with H2O. The first
purification of the EtOAc extract was carried out by gel
filtration on Sephadex LH-20 using hexane:EtOAc:Me2CO (5:
1:1) as solvent. This resulted in the separation of two products.
The first fraction to be eluted from the gel filtration column
contained crude 2′,4′-dihydroxy-5′-(1,1-dimethylallyl)chalcone
3, which was purified by MPLC on diol-bonded silica to give
62 mg (0.2 mmol, 15.5%) of pure 3. 1H NMR (300 MHz,
acetone-d6) δ 1.55 (s, 6 H, H4′′ + H5′′), 5.00 (d, 1 H, J ) 10.5
Hz, H3′′), 5.02 (d, 1 H, J ) 17.5 Hz, H3′′), 6.32 (dd, 1 H, J )
10.5 and 17.5 Hz, H2′′), 6.44 (brs, 1 H, H3′), 7.48 (m, 3 H, H3
+ H4 + H5), 7.83 (m, 2 H, H2 + H6), 7.87 (s, 2 H, HR + Hâ),
7.96 (s, 1 H, H6′), 13.33 (s, 1 H, 2′OH); 13C NMR (75 MHz,
acetone-d6): δ 27.04 (C4′′ + C5′′), 40.35 (C1′′), 104.31 (C3′),
4-Methoxy-2′,4′-â,trihydroxychalcone 2. Forty milliliters
of 1 M LiHMDS in THF (40 mmol) was added to a mixture of
1.52 g of resacetophenone 43 (10 mmol) in 40 mL of dry THF,
under argon at -78 °C. The reaction mixture was stirred at
-78 °C for 1 h and at -10 °C for 2 h. After cooling again at
-78 °C, 1.7 g of anisoyl chloride 44 (10 mmol) in 40 mL of dry
THF was added. The mixture was allowed to warm to room
temperature and stirred overnight. The solution was acidified
and extracted by EtOAc, the solvent was removed, and the
residue was purified by MPLC on DIOL-bonded silica (gradient
of ethyl acetate in hexane), to give 1.46 g of compound 2 (5.1
mmol; yield 51%). UV, MS, and 1H NMR (acetone-d6) data:
see reference.57 13C NMR (acetone-d6, 75 MHz): δ: 50.77 (C2
diketone), 57.00 (OMe enol), 92.56 (CR enol), 104.52 (C3′
diketone), 105.05 (C3′ enol), 110.14 (C5′ enol), 113.83 (C1′
enol), 115.99 (C3+C5 enol), 127.65 (C1 enol), 130.68 (C2+C6
enol), 133.42 (C6′ enol), 165.19 (C4 enol), 166.39* (C2′ enol),
167.07* (C4′ enol), 178.09 (Câ enol), 196.24 (CdO enol); MS
(CI+) m/z 287 (M+H); HRCIMS (C16H14O5) calcd 287.0919,
found 287.0914. Anal. (C16H14O5 0.30MeOH) C, H.
2′-Hydroxy-4′-prenyloxyacetophenone (46). To a stirred
solution of 7.6 g (50 mmol) of resacetophenone 43 in 20 mL of
dry DMF were added successively 7.6 g (50 mmol, 1 equiv) of
anhydrous K2CO3, 1.61 g (5 mmol, 0.1 equiv) of tetrabutyl-
ammonium bromide, and 5.2 mL (45 mmol, 0.9 equiv) of prenyl

2798 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
110.36 (C3′′), 113.10 (C1′), 121.27 (CR), 128.00 (C5′), 129.06
(C2 + C6), 129.37 (C3 + C5), 129.69 (C6′), 130.89 (C4), 135.46
(C1), 143.96 (Câ), 148.03 (C2′′), 164.52 (C4′), 165.49 (C2′),
192.21 (CdO); UV (λ nm (log ꢀ)) 314 (4.12); MS (TOF ES+)
m/z 309 [M + H]+; HRMS (C20H21O3) calcd 309.1491, found
309.1494. Anal. (C20H20O3) C, H. The second fraction to be
eluted from the gel filtration column contained crude 2′,4′-
dihydroxychalcone 49,61 which was again purified by MPLC
on diol-bonded silica, yielding 43.9 mg (0.18 mmol, 15%) of pure
49.
5-O-Methylchrysin (6). To a stirred solution of 102.8 mg
(0.29 mmol) of 7-O-benzoylchrysin51 50 in 5 mL of CH2Cl2 and
5 mL of toluene were added, successively 30 µL (0.48 mmol,
1.6 equiv) of methyl iodide and 225 mg (0.28 mmol, 1 equiv)
of tetrabutylammonium hydroxide 30 hydrate. After 2 h
reaction at room temperature under stirring, the medium was
diluted with crushed ice, acidified (1 N HCl) and extracted with
EtOAc. The EtOAc extract was rinsed with H2O and evapo-
rated under reduced pressure. The residue of crude 51 was
taken with 20 mL of 5% KOH in MeOH. The solution was
ultrasounded for 5 min, diluted with H2O, acidified (1 N HCl),
and extracted with EtOAc, and the EtOAc extract was rinsed
with H2O. Evaporation of the solvent yielded 68.6 mg of a
residue which was purified by VLC on silica gel, leading to
the isolation of 14 mg (0.06 mmol, 21%) of pure 6.62
mixture was refluxed for 3 days. After cooling, the medium
was diluted with crushed ice, allowed to warm to room
temperature (hydrolysis of acetic anhydride), and extracted
with EtOAc, and the EtOAc extract was rinsed with H2O.
Evaporation of the extract under reduced pressure produced
an oily residue which was taken up in a solution of 5% KOH
in MeOH. After 1 h at room temperature (cleavage of acetates),
the medium was diluted with crushed ice, acidified (1 N HCl),
and extracted with EtOAc, and the EtOAc extract was rinsed
with H2O. The EtOAc extract was purified by MPLC on silica
gel, to give 0.49 g (1.25 mmol, 49%) of compound 20. 1H NMR
(300 MHz, CDCl3) δ 1.40 (s, 3 H, H10′′), 1.55 (s, 3 H, H9′′),
1.74 (s, 3 H, H4′′), ca. 1.87-2.04 (m, 2 H, H5′′ + H6′′), ca. 2.18-
2.25 (m, 1 H, H5′′), 4.93 (m, 1 H, H7′′), 5.45 (d, 1 H, J ) 10.6
Hz, H3′′), 5.48 (d, 1 H, J ) 17.9 Hz, H3′′), 6.35 (s, 1 H, H6),
6.55 (dd, 1 H, J ) 10.6 and 17.9 Hz, H2′′), 6.65 (s, 1 H, H3),
7.36 (brs, 1 H, 7OH), 7.57 (m, 3 H, H3′ + H4′ + H5′), 7.84 (m,
2 H, H2′ + H6′), 13.17 (s, 1 H, 5OH); 13C NMR (75 MHz,
CDCl3): δ 17.86 (C10′′), 24.12 (C6′′), 25.99 (C9′′), 26.84 (C4′′),
40.26 (C5′′), 44.75 (C1′′), 102.15 (C6), 106.61 (C10), 107.30 (C3),
109.28 (C8), 114.90 (C3′′), 124.13 (C7′′), 127.35 (C2′ + C6′),
129.53 (C3′ + C5′), 131.99 (C8′′), 132.20 (C4′), 132.45 (C1′),
148.14 (C2′′), 157.07 (C9), 161.14 (C5), 162.63 (C7), 165.43 (C2),
183.29 (C4); MS (TOF ES+) m/z 391 [M + H]+; HRMS
(C25H27O4) calcd 391.1909, found 391.1924. Anal. (C25H26O4)
C, H.
5-O-Prenylchrysin (12). To a stirred mixture of 1 g (2.8
mmol) of 7-O-benzoylchrysin51 50 in 50 mL of CH2Cl2 and 50
mL of toluene were added 4.5 g (5.6 mmol, 2 equiv) of
tetrabutylammonium hydroxide 30 hydrate and 0.5 mL (4.3
mmol, 1.5 equiv) of prenyl bromide, successively. After 3 h
stirring at room temperature, H2O was added and the medium
was extracted with EtOAc. After evaporation of the solvent,
the residue of 52 was taken up in 20 mL of 5% KOH in MeOH,
and ultrasounded for 8 min. The medium was diluted with
crushed ice, acidified (1 N HCl), and extracted by EtOAc.
Purification of the EtOAc extract by MPLC on diol-bonded
silica yielded 0.45 g (1.4 mmol, 50%) of compound 12. 1H NMR
(500 MHz, acetone-d6) δ 1.78 (s, 3 H, H5′′), 1.80 (s, 3H, H4′′),
4.66 (d, 2 H, J ) 6.0 Hz, H1′′), 5.54 (brt, 1 H, J ) 6.0 Hz,
H2′′), 6.49 (d, 1 H, J ) 2.1 Hz, H6), 6.58 (s, 1 H, H3), 6.65 (d,
1 H, J ) 2.1 Hz, H8), 7.58 (m, 3 H, H3′ + H4′ + H5′), 8.02 (m,
2H, H2′ + H6′), 9.63 (brs, 1 H, 7OH)); 13C NMR (125 MHz,
acetone-d6): δ 18.29 (C5′′), 25.79 (C4′′), 66.86 (C1′′), 96.32 (C8),
98.86 (C6), 109.27 (C3), 109.54 (C10), 120.90 (C2′′), 121.71 (C2′
+ C6′), 129.83 (C3′ + C5′), 131.87 (C4′), 132.68 (C1′), 137.48
(C3′′), 160.62 (C9*), 160.65 (C2*), 161.39 (C5), 163.08 (C7),
176.32 (C4); UV (λ nm (log ꢀ)) 264 (3.77), 305 sh. Anal.
(C20H18O4 0.90EtOAc) C, H.
7-O-Geranylchrysin (17). To a solution of 2 g (7.86 mmol)
of chrysin 5 in 300 mL of Me2CO (gentle warming) were added
2.4 g (17.38 mmol, 2.2 equiv) of anhydrous K2CO3, and 3.4 mL
(17.07 mmol, 2.2 equiv) of geranyl bromide. The mixture was
refluxed for 3 h. After cooling, the solid K2CO3 was eliminated
by filtration. The filtrate was evaporated under reduced
pressure, to give a residue which was crystallized from 150
mL of MeOH, to give 2.8 g (7.2 mmol, 91%) of pure 7-O-
geranylchrysin 17. 1H NMR (500 MHz, CDCl3) δ 1.61 (s, 3 H,
H10′′), 1.68 (s, 3 H, H9′′), 1.76 (s, 3H, H8′′), ca. 2.08-2.20 (m,
4 H, H4′′ + H5′′), 4.62 (d, 2 H, J ) 6.2 Hz, H1′′), 5.10 (m, 1 H,
H6′′), 5.49 (m, 1 H, H2′′), 6.38 (d, 1 H, J ) 2.4 Hz, H6), 6.51
(d, 1 H, J ) 2.4 Hz, H8), 6.66 (s, 1 H, H3), 7.54 (m, 3 H, H3′
+ H4′ + H5′), 7.88 (m, 2H, H2′ + H6′), 12.66 (brs, 1 H, 5OH));
13C NMR (125 MHz, CDCl3): δ 16.73 (C8′′), 17.68 (C10′′), 25.64
(C9′′), 26.23 (C5′′), 39.49 (C4′′), 65.52 (C1′′), 93.34 (C8), 98.80
(C6), 105.60 (C10), 105.81 (C3), 118.36 (C2′′), 123.61 (C6′′),
126.27 (C2′ + C6′), 129.05 (C3′ + C5′), 131.34 (C1′), 131.78
(C4′), 131.94 (C7′′), 142.29 (C3′′), 157.75 (C9), 162.08 (C5),
163.94 (C2), 164.97 (C7), 182.45 (C4); UV (λ nm (log ꢀ)) 268
(4.40), 310 sh; MS (TOF ES+) m/z 391 [M + H]+; HRMS
(C25H27O4) calcd 391.1909, found 391.1912. Anal. (C25H26O4
0.40EtOAc) C, H.
7-O-Farnesylchrysin (21). Same procedure as the prepa-
ration of 17 was followed, except that geranyl bromide was
replaced by 4.6 mL of farnesyl bromide (17.07 mmol, 2.2 equiv).
After evaporation of the Me2CO filtrate, the residue was
purified by MPLC on diol-bonded silica, to give 3.2 g (7 mmol,
89%) of pure 7-O-farnesylchrysin 21. 1H NMR (500 MHz,
CDCl3) δ 1.59 (s, 3 H, H15′′), 1.61 (s, 3 H, H13′′), 1.67 (s, 3 H,
H14′′), 1.77 (s, 3H, H12′′), ca. 1.94-2.00 (m, 2 H, H8′′), ca.
2.02-2.08 (m, 2 H, H9′′), ca. 2.10-2.18 (m, 4 H, H4′′ + H5′′),
4.61 (d, 2 H, J ) 6.3 Hz, H1′′), 5.08 (m, 1 H, H10′′), 5.11 (m,
1 H, H6′′), 5.49 (m, 1 H, H2′′), 6.38 (d, 1 H, J ) 2.2 Hz, H6),
6.50 (d, 1 H, J ) 2.2 Hz, H8), 6.65 (s, 1 H, H3), 7.53 (m, 3 H,
H3′ + H4′ + H5′), 7.87 (m, 2H, H2′ + H6′), 12.69 (s, 1 H, 5OH));
13C NMR (125 MHz, CDCl3): δ 16.01 (C13′′), 16.73 (C12′′),
17.64 (C15′′), 25.65 (C14′′), 26.12 (C5′′), 26.67 (C9′′), 39.49
(C4′′), 39.64 (C8′′), 65.49 (C1′′), 93.30 (C8), 98.77 (C6), 105.58
(C10), 105.72 (C3), 118.38 (C2′′), 123.47 (C6′′), 124.26 (C10′′),
126.25 (C2′ + C6′), 129.03 (C3′ + C5′), 131.28 (C11′′), 131.33
(C1′), 131.76 (C4′), 135.56 (C7′′), 142.28 (C3′′), 157.73 (C9),
162.08 (C5), 163.91 (C2), 164.96 (C7), 182.43 (C4); UV (λ nm
(log ꢀ)) 268 (4.42), 311 sh.
8-Nerolidylchrysin (22). One gram (2.18 mmol) of com-
pound 21 was rearranged following the same procedure,
workup, and purification of the product as used in the
preparation of 20. This resulted in the isolation of 0.41 g (0.89
mmol, 41%) of compound 22. 1H NMR (300 MHz, CDCl3) δ
1.40 (s, 3 H, H10′′), 1.56 (s, 3 H, H15′′), 1.66 (s, 3 H, H14′′),
1.74 (s, 3 H, H4′′), ca. 1.75-1.85 (m, 2 H, H9′′), ca. 1.85-1.95
(m, 6 H, H5′′ + H6′′ + H11′′), 4.95 (m, 1 H, H7′′), 5.02 (m, 1
H, H12′′), 5.44 (d, 1 H, J ) 10.7 Hz, H3′′), 5.48 (d, 1 H, J )
17.8 Hz, H3′′), 6.35 (s, 1 H, H6), 6.56 (dd, 1 H, J ) 10.7 and
17.8 Hz, H2′′), 6.65 (s, 1 H, H3), 7.38 (brs, 1 H, 7OH), 7.54 (m,
3 H, H3′ + H4′ + H5′), 7.84 (m, 2 H, H2′ + H6′), 13.16 (s, 1 H,
5OH); 13C NMR (75 MHz, CDCl3): δ 16.22 (C10′′), 18.06 (C15′′),
24.22* (C11′′), 26.07 (C14′′), 26.86 (C4′′), 26.97* (C6′′), 39.97
(C9′′), 40.33 (C5′′), 45.74 (C1′′), 102.14 (C6), 106.65 (C10),
107.30 (C3), 109.28 (C8), 114.85 (C3′′), 123.90 (C7′′), 124.57
(C12′′), 127.34 (C2′ + C6′), 129.52 (C3′ + C5′), 131.73 (C13′′),
132.00 (C4′), 132.44 (C1′), 135.89 (C8′′), 148.16 (C2′′), 157.06
(C9), 161.13 (C5), 162.64 (C7), 165.42 (C2), 183.28 (C4); UV (λ
nm (log ꢀ)) 273 (4.40), 314 sh; MS (TOF ES+) m/z 459 [M +
H]+; HRMS (C30H35O4) calcd 459.2535, found 459.2539. Anal.
(C30H34O4) C, H.
7,4′-Bis-O-methoxymethylapigenin (54). A stirred solu-
tion of 0.25 g (0.92 mmol) of apigenin 53 in 10 mL of dry DMF
was cooled to 4 °C in an ice bath, and 0.64 mL (3.68 mmol; 4
equiv) of N,N-diisopropylethylamine, followed by 0.3 mL (3.68
mmol; 4 equiv) of bromomethyl methyl ether, were added
8-Linalylchrysin (20). One gram (2.56 mmol) of compound
17 was dissolved in 20 mL of acetic anhydride, 0.6 g (7.31
mmol, 2.8 equiv) of freshly fused NaOAc was added, and the

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2799
dropwise. Reaction was allowed to take place for 1 h under
stirring at room temperature. The medium was diluted with
H2O, adjusted to pH 1 (1 N HCl), and extracted with EtOAc.
The EtOAc extract was rinsed with H2O, concentrated under
reduced pressure, and purified by MPLC on diol-bonded silica,
to give 0.26 g (0.72 mmol; 78%) of compound 54. 1H NMR (300
MHz, acetone-d6) δ 3.47 (s, 3 H, 4′MOMb), 3.49 (s, 3 H,
7MOMb), 5.32 (s, 2 H, 4′MOMa), 5.33 (s, 2 H, 7MOMa), 6.42
(d, 1H, J ) 2.1 Hz, H6), 6.74 (s, 1 H, H3), 6.77 (d, 1H, J ) 2.1
Hz, H8), 7.22 (d, 2H, J ) 9.0 Hz, H3′ + H5′), 8.05 (d, 2H,
J ) 9.0 Hz, H2′ + H6′), 12.92 (s, 1 H, 5OH); 13C NMR (75
MHz, acetone-d6): δ 57.41 (4′MOMb), 57.59 (7MOMb), 96.11
(4′MOMa), 96.25 (7MOMa), 96.33 (C8), 101.45 (C6), 106.09
(C3), 107.71 (C10), 118.50 (C3′ + C5′), 126.35 (C1′), 130.14 (C2′
+ C6′), 159.55 (C9), 162.46 (C4′), 164.09 (C5), 165.17 (C7),
166.11 (C2), 184.32 (C4); UV (λ nm (log ꢀ)) 269 (4.33), 319;
MS (TOF ES+) m/z 359 [M + H]+; HRMS (C19H19O7) calcd
359.1131, found 359.1149. Anal. (C19H18O7‚0.10hexane) C, H.
3,4′,7-Tris-O-methoxymethylkaempferol (55). Similar
reaction of 0.5 g (1.75 mmol) of kaempferol 28 in 20 mL of dry
DMF with 0.86 mL (10.2 mmol, 6 equiv) of bromomethyl
methyl ether in the presence of 1.8 mL (10.2 mmol, 6 equiv)
of N,N-diisopropylethylamine yielded, after similar purification
of the EtOAc extract by MPLC on diol-bonded silica, 0.38 g
(0.91 mmol, 52%) of compound 55. 1H NMR (300 MHz, acetone-
d6) δ 3.20 (s, 3H, 3MOMb), 3.47 (s, 3H, 4′MOMb), 3.48 (s, 3H,
7MOMb), 5.21 (s, 2H, 3MOMa), 5.32 (s, 2H, 4′MOMa), 5.33
(s, 2H, 7MOMa), 6.42 (d, 1H, J ) 2.1 Hz, H6), 6.73 (d, 1H, J
) 2.1 Hz, H8), 7.22 (d, 2H, J ) 9.0 Hz, H3′ + H5′), 8.11 (d,
2H, J ) 9.0 Hz, H2′ + H6′), 12.63 (s, 1H, 5OH); 13C NMR (75
MHz, acetone-d6): δ 57.32 (4′MOMb), 57.55 (7MOMb), 58.74
(3MOMb), 95.95 (4′MOMa), 96.00 (C8), 96.14 (7MOMa), 99.37
(3MOMa), 101.07 (C6), 108.03 (C10), 117.79 (C3′ + C5′), 125.64
(C1′), 132.47 (C2′ + C6′), 137.26 (C3), 158.60* (C9), 158.64*
(C2), 161.39 (C4′), 163.79 (C5), 165.11 (C7),180.49 (C4); UV (λ
nm (log ꢀ)) 266 (4.35), 340; MS (TOF ES+) m/z 419 [M + H]+;
HRMS (C21H23O9) calcd 419.1342, found 419.1350. Anal.
(C21H22O9 0.79hexane) C, H.
(4′MOMb), 56.02 (7MOMb), 57.01 (3MOMb), 93.86 (7MOMa),
94.30 (4′MOMa), 94.34 (C8), 95.03 (3′MOMa), 97.22 (3MOMa),
99.16 (C6), 105.61 (C10), 115.82 (C5′), 117.66 (C2′), 123.33
(C1′), 123.55 (C6′), 134.97 (C3), 146.12 (C3′), 149.42 (C4′),
155.92 (C9), 156.24 (C2), 160.83 (C5), 162.50 (C7), 177.90 (C4);
UV (λ nm (log ꢀ)) 250, 266 (4.28), 340. Anal. (C23H26O11) C, H.
7,4′-Bis-O-methoxymethyl-5-O-prenylapigenin (57). An
amount of 0.24 g (0.67 mmol) of compound 54, 1.08 g (1.35
mmol, 2 equiv) of tetrabutylammonium hydroxide 30 hydrate,
10 mL of CH2Cl2, and 10 mL of toluene were stirred at room
temperature until complete disappearance of solid and forma-
tion of a biphase. To this medium was added 0.12 mL (1 mmol,
1.5 equiv) of prenyl bromide, and the reaction was allowed to
proceed for 3 h at room temperature and gentle stirring.
Dilution of the medium with H2O, extraction with EtOAc, and
purification of the extract by MPLC on diol-bonded silica
afforded 0.2 g (0.47 mmol, 70%) of compound 57. 1H NMR (300
MHz, acetone-d6) δ 1.77 (s, 3 H, H5′′), 1.78 (s, 3 H, H4′′), 3.46
(s, 3 H, 4′MOMb), 3.49 (s, 3 H, 7MOMb), 4.66 (d, 2 H, J ) 6.4
Hz, H1′′), 5.30 (s, 2 H, 4′MOMa), 5.34 (s, 2 H, 7MOMa), 5.52
(brt, 1 H, J ) 6.4 Hz, H2′′), 6.51 (s, 1 H, H3), 6.58 (d, 1H, J )
2.1 Hz, H6), 6.84 (d, 1H, J ) 2.1 Hz, H8), 7.19 (d, 2H, J ) 9.0
Hz, H3′ + H5′), 7.97 (d, 2H, J ) 9.0 Hz, H2′ + H6′); 13C NMR
(75 MHz, acetone-d6): δ 19.32 (C5′′), 26.82 (C4′′), 57.28
(4′MOMb), 57.54 (7MOMb), 68.02 (C1′′), 95.95 (4′MOMa),
96.14 (7MOMa), 97.56 (C8), 100.46 (C6), 109.20 (C3), 111.80
(C10), 118.29 (C3′ + C5′), 121.89 (C2′′), 126.71 (C1′), 129.41
(C2′ + C6′), 138.61 (C3′′), 161.27 (C9), 161.67 (C4′), 161.82 (C2),
161.98 (C5), 163.23 (C7), 177.27 (C4); MS (TOF ES+) m/z 427
[M + H]+; HRMS (C24H27O7) calcd 427.1757, found 427.1776.
Anal. (C24H26O7 0.30CH2Cl2) C, H.
5-O-Prenyl-3,4′,7-tris-O-methoxymethylkaempferol (58).
Similar reaction of 0.38 g (0.91 mmol) of compound 55 in 12
mL of CH2Cl2 and 12 mL of toluene, with 1.46 g (1.8 mmol, 2
equiv) of tetrabutylammonium hydroxide 30 hydrate and 0.16
mL (1.37 mmol, 1.5 equiv) of prenyl bromide, gave, after
purification of the EtOAc extract by MPLC on diol-bonded
silica, 0.33 g (0.68 mmol, 75%) of compound 58. 1H NMR (300
MHz, acetone-d6) δ 1.78 (s, 3H, H5′′), 1.79 (s, 3H, H4′′), 3.18
(s, 3H, 3MOMb), 3.46 (s, 3H, 4′MOMb), 3.48 (s, 3H, 7MOMb),
4.68 (d, 2H, J ) 6.0 Hz, H1′′), 5.17 (s, 2H, 3MOMa), 5.30 (s,
2H, 4′MOMa), 5.33 (s, 2H, 7MOMa), 5.55 (brt, 1H, J ) 6 Hz,
H2′′), 6.57 (d, 1H, J ) 2.3 Hz, H6), 6.78 (d, 1H, J ) 2.3 Hz,
H8), 7.19 (d, 2H, J ) 8.9 Hz, H3′ + H5′), 8.07 (d, 2H, J ) 8.9
Hz, H2′ + H6′); 13C NMR (75 MHz, acetone-d6): δ 19.34 (C5′′),
26.84 (C4′′), 57.26 (4′MOMb), 57.54 (7MOMb), 58.57 3MOMb),
68.03 (C1′′), 95.96 (4′MOMa), 96.18 (7MOMa), 97.15 (C8),
98.93 (3MOMa), 99.96 (C6), 111.84 (C10), 117.68 (C3′ + C5′),
121.79 (C2′′), 126.23 (C1′), 132.01 (C2′ + C6′), 138.76 (C3′′),
139.40 (C3), 154.51 (C2), 160.38 (C9), 160.78 (C4′), 162.13 (C5),
163.35 (C7), 174.12 (C4); UV (λ nm (log ꢀ)) 263 (4.32), 331.
5-O-Prenyl-3,3′,4′,7-tetrakis-O-methoxymethylquerce-
tin (59). Similar reaction of 1 g (2.1 mmol) of compound 56 in
40 mL of CH2Cl2 and 40 mL of toluene, with 6.22 g (4.2 mmol,
2 equiv) of tetrabutylammonium hydroxide 30 hydrate and
0.68 mL (3.15 mmol, 1.5 equiv) of prenyl bromide, gave, after
purification of the EtOAc extract by MPLC on diol-bonded
silica, 0.65 g (1.19 mmol, 57%) of compound 59. 1H NMR (300
MHz, acetone-d6) δ 1.78 (s, 3 H, H5′′), 1.79 (s, 3 H, H4′′), 3.20
(s, 3 H, 3MOMb), 3.48 (s, 3 H, 7MOMb), 3.49 (s, 3 H, 4′MOMb),
3.51 (s, 3 H, 3′MOMb), 4.68 (d, 2 H, J ) 6.4 Hz, H1′′), 5.19 (s,
2 H, 3MOMa), 5.28 (s, 2 H, 3′MOMa), 5.31 (s, 2 H, 4′MOMa),
5.33 (s, 2 H, 7MOMa), 5.54 (brt, 1 H, J ) 6.4 Hz, H2′′), 6.57
(d, 1H, J ) 2.1 Hz, H6), 6.77 (d, 1H, J ) 2.1 Hz, H8), 7.29 (d,
1 H, J ) 8.7 Hz, H5′), 7.75 (dd, 1 H, J ) 2.1 and 8.7 Hz, H6′),
7.95 (d, 1 H, J ) 2.1 Hz, H2′); 13C NMR (75 MHz, acetone-d6):
δ 19.33 (C5′′), 26.83 (C4′′), 57.40 (3′MOMb), 57.44 (4′MOMb),
57.55 (7MOMb), 58.63 (3MOMb), 68.02 (C1′′), 96.15 (7MOMa),
96.89 (4′MOMa), 97.15 (C8), 97.53 (3′MOMa), 98.93 (3MOMa),
99.92 (C6), 111.80 (C10), 118.19 (C5′), 120.24 (C2′), 121.77
(C2′′), 125.19 (C6′), 126.78 (C1′), 138.76 (C3′′), 139.50 (C3),
148.82 (C3′), 151.45 (C4′), 154.18 (C2), 160.34 (C9), 162.12 (C5),
163.35 (C7), 174.11 (C4); UV (λ nm (log ꢀ)) 264 (3.82), 333.
3,3′,4′,7-Tetrakis-O-methoxymethylquercetin (56). To
a cooled solution (4 °C, ice bath) of 2.22 g (6.93 mmol) of
quercetin monohydrate 32 in 50 mL of dry DMF was slowly
added dropwise under stirring 5.1 mL (44.1 mmol, 6.4 equiv)
of N,N-diisopropylethylamine, followed by 3 mL (44.1 mmol,
6.4 equiv) of bromomethyl methyl ether. After complete
addition of the reagents, the ice bath was removed and reaction
was allowed to take place for 1 h at room temperature under
stirring. The medium was diluted with H2O, acidified (pH 1,
1 N HCl), and extracted with EtOAc. The EtOAc extract was
rinsed with H2O, evaporated under reduced pressure, and
dried under vaccuum. The residue was taken up in 50 mL of
dry DMF, and 1.75 g of NaH (75% in mineral oil, 66.1 mmol)
was added under stirring. After 5 min, 2.8 mL (44.1 mmol) of
bromomethyl methyl ether was added dropwise, and reaction
was allowed to take place under stirring for 1 h at room
temperature. Dilution of the medium with water, acidification
(pH 1, 1 N HCl), extraction with EtOAc, rinsing of the EtOAc
extract with H2O, and evaporation of the organic layer to
dryness yielded a residue of crude 3,5,7,3′,4′-pentakis-O-
methoxymethylquercetin 63 which was not isolated. The
residue was taken up in 40 mL of ethanolic HCl (3% concen-
trated HCl in EtOH) and stirred at room temperature for 30
min (selective cleavage of the 5-MOM protective group). After
dilution with H2O, the solution was extracted with EtOAc, and
the EtOAc extract was rinsed with H2O, concentrated under
reduced pressure, and purified by VLC on silica gel, to give
1.89 g (3.95 mmol, 57% from 32) of 3,3′,4′,7-tetrakis-O-
1
methoxymethylquercetin 56. H NMR (300 MHz, DMSO-d6)
δ 3.16 (s, 3 H, 3MOMb), 3.41 (s, 3 H, 7MOMb), 3.42 (s, 3 H,
4′MOMb), 3.44 (s, 3 H, 3′MOMb), 5.14 (s, 2 H, 3MOMa), 5.26
(s, 2 H, 3′MOMa), 5.32 (s, 2 H, 7MOMa), 5.33 (s, 2 H, 4′MOMa),
6.46 (d, 1H, J ) 2.1 Hz, H6), 6.79 (d, 1H, J ) 2.1 Hz, H8),
7.29 (d, 1 H, J ) 8.7 Hz, H5′), 7.73 (dd, 1 H, J ) 2.1 and 8.7
Hz, H6′), 7.86 (d, 1 H, J ) 2.1 Hz, H2′), 12.51 (s, 1 H, 5OH);
13C NMR (75 MHz, DMSO-d6): δ 55.78 (3′MOMb), 55.89

2800 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
7,4′-Bis-O-methoxymethyl-8-prenylapigenin (60). An
amount of 100 mg (0.23 mmol) of compound 57 and 10 mL of
freshly distilled N,N-diethylaniline were heated at 217 °C for
3 h under stirring. After cooling, the medium was diluted with
H2O, acidified (pH 1, 1 N HCl), and extracted with EtOAc.
The EtOAc extract was rinsed with H2O and evaporated under
reduced pressure, and the residue was purified by MPLC on
diol-bonded silica. This afforded 56.2 mg (0.13 mmol, 56.5%)
130.24 (C2′ + C6′), 133.04 (C3′′), 157.01 (C9), 162.00 (C5),
162.90 (C4′), 163.25 (C7), 166.03 (C2), 184.43 (C4); MS (CI+)
m/z 339 [M + H]+; HRMS (C20H19O5) calcd 339.1232, found
339.1238.
8-Prenylkaempferol (29). Similar deprotection of 28.1 mg
(0.06 mmol) of compound 61 and similar purification of the
hydrolysis product by MPLC on diol-bonded silica yielded 15.5
mg (0.044 mmol, 73%) of compound 29.65,66 1H NMR (300 MHz,
acetone-d6) δ 1.66 (brs, 3 H, H4′′), 1.81 (brs, 3 H, H5′′), 3.56
(d, 2 H, J ) 6.8 Hz, H1′′), 5.28 (brt, 1 H, J ) 6.8 Hz, H2′′),
6.36 (s, 1H, H6), 7.03 (d, 2H, J ) 8.9 Hz, H3′ + H5′), 8.18 (d,
2H, J ) 8.9 Hz, H2′ + H6′), 12.10 (s, 1 H, 5OH); 13C NMR (75
MHz, acetone-d6): δ 19.16 (C5′′), 23.28 (C1′′), 26.83 (C4′′), 99.93
(C6), 105.29 (C10), 108.30 (C8), 117.44 (C3′ + C5′), 124.64
(C2′′), 124.77 (C1′), 131.51 (C2′ + C6′), 133.14 (C3′′), 137.54
(C3), 148.07 (C2), 156.20 (C9), 161.01 (C5), 161.21 (C4′), 163.25
(C7), 163.18 (C7), 177.91 (C4); MS (CI+) m/z 355 [M + H]+;
HRMS (C20H19O6) calcd 355.1182, found 355.1181.
8-Prenylquercetin (33). Similar deprotection of 40 mg
(0.07 mmol) of compound 62, followed by similar purification
of the hydrolysis product by MPLC on diol-bonded silica,
yielded 18.5 mg (0.05 mmol, 71%) of compound 33.67,68 1H NMR
(300 MHz, acetone-d6) δ 1.66 (brs, 3 H, H4′′), 1.82 (brs, 3 H,
H5′′), 3.56 (d, 2 H, J ) 7.0 Hz, H1′′), 5.28 (brt, 1 H, J ) 7.0
Hz, H2′′), 6.35 (s, 1H, H6), 7.01 (d, 1 H, J ) 8.5 Hz, H5′), 7.75
(dd, 1 H, J ) 2.3 and 8.5 Hz, H6′), 7.88 (d, 1 H, J ) 2.1 Hz,
H2′), 12.10 (s, 1 H, 5OH); 13C NMR (75 MHz, acetone-d6): δ
19.13 (C5′′), 23.23 (C1′′), 26.85 (C4′′), 99.76 (C6), 105.15 (C10),
108.20 (C8), 116.82 (C2′), 117.21 (C5′), 122.41 (C6′), 124.44
(C2′′), 125.15 (C1′), 133.20 (C3′′), 137.58 (C3), 146.87 (C3′),
147.89 (C2), 149.27 (C4′), 156.04 (C9), 160.91 (C5), 163.06 (C7),
177.78 (C4); MS (CI+) m/z 371 [M + H]+; HRMS (C20H19O7)
calcd 371.1131, found 371.1123.
1
of compound 60. H NMR (300 MHz, acetone-d6) δ 1.67 (s, 3
H, H4′′), 1.84 (s, 3 H, H5′′), 3.47* (s, 3 H, 4′MOMb), 3.48* (s,
3 H, 7MOMb), 3.59 (d, 2 H, J ) 7.0 Hz, H1′′), 5.26 (brt, 1 H,
J ) 7.0 Hz, H2′′), 5.33# (s, 2 H, 4′MOMa), 5.38# (s, 2 H,
7MOMa), 6.57 (s, 1H, H6), 6.72 (s, 1 H, H3), 7.24 (d, 2H, J )
9.0 Hz, H3′ + H5′), 8.04 (d, 2H, J ) 9.0 Hz, H2′ + H6′), 12.93
(s, 1 H, 5OH); 13C NMR (75 MHz, acetone-d6): δ 19.16 (C5′′),
23.65 (C1′′), 26.85 (C4′′), 57.42* (4′MOMb), 57.62* (7MOMb),
96.13# (4′MOMa), 96.36# (7MOMa), 99.57 (C6), 105.83 (C3),
107.36 (C10), 110.58 (C8), 118.57 (C3′ + C5′), 124.40 (C2′′),
126.75 (C1′), 130.12 (C2′ + C6′), 133.36 (C3′′), 156.40 (C9),
162.26 (C5), 162.44 (C4′ + C7), 166.03 (C2), 184.69 (C4); MS
(TOF ES+) m/z 427 [M + H]+; HRMS (C24H27O7) calcd
427.1557, found 427.1776. Anal. (C24H26O7 0.60CH2Cl2) C, H.
7,4′-Bis-O-methoxymethyl-8-prenylkaempferol (61).
Similar rearrangement of 0.1 g (0.20 mmol) of compound 58
in 10 mL of N,N-diethylaniline at 217 °C, yielded, after usual
purification of the product by MPLC on diol-bonded silica,
0.063 g (0.14 mmol, 70%) of compound 61. The formation of
compound 61 involved the loss of the 3-MOM group during
the course of the reaction, which did not affect the rearrange-
ment of the 5-prenyl chain. 1H NMR (300 MHz, CDCl3) δ 1.69
(s, 3 H, H4′′), 1.83 (s, 3 H, H5′′), 3.49* (s, 3 H, 4′MOMb), 3.51*
(s, 3 H, 7MOMb), 3.57 (d, 2H, J ) 6.6 Hz, H1′′), 5.22 (brt, 1H,
J ) 6.6 Hz, H2′′), 5.26 (s, 2H, 4′MOMa), 5.28 (s, 2H, 7MOMa),
6.62 (s, 1H, H6), 7.18 (d, 2H, J ) 8.9 Hz, H3′ + H5′), 8.18 (d,
2H, J ) 8.9 Hz, H2′ + H6′), 11.71 (s, 1H, 5OH); 13C NMR (75
MHz, CDCl3): δ 17.97 (C5′′), 21.86 (C1′′), 25.73 (C4′′), 56.19
(4′MOMb), 56.35 (7MOMb), 94.18 (4′MOMa), 94.30 (7MOMa),
97.23 (C6), 104.17 (C10), 108.87 (C8), 116.19 (C3′ + C5′),
122.45 (C2′′), 124.64 (C1′), 129.39 (C2′ + C6′), 132.04 (C3′′),
135.60 (C3), 145.58 (C2), 153.58 (C9), 158.68 (C4′), 158.96 (C5),
160.42 (C7), 175.67 (C4). UV (λ nm (log ꢀ)) 270 (4.30), 319,
372.
General Procedure for the Synthesis of Benzoic An-
hydrides. This was done according to the method described
by Nangia and Chandrasekaran.77 A solution of 16 mL of
triethylamine in 14 mL of THF was added dropwise to a THF
solution (100 mL) containing 2 equiv of benzoic acid and 1
equiv of methanesulfonyl chloride at 0 °C. The mixture was
stirred for 1 h and concentrated under vacuum. A 50 mL
volume of 10% aq NaHCO3 was then added, and the medium
was extracted by EtOAc. Evaporation of the EtOAc extract
yielded the pure anhydride.
8-Prenyl-3,3′,4′,7-tetrakis-O-methoxymethylquerce-
tin (62). Similar rearrangement of 0.1 g (0.18 mmol) of
compound 59 in 10 mL of N,N-diethylaniline at 217 °C, yielded,
after usual purification of the product by MPLC on diol-bonded
silica, 0.05 g (0.09 mmol, 50%) of compound 62. 1H NMR (300
MHz, acetone-d6) δ 1.65 (s, 3 H, H4′′), 1.79 (s, 3 H, H5′′), 3.23
(s, 3 H, 3MOMb), 3.48 (s, 3 H, 7MOMb), 3.50 (s, 3 H, 4′MOMb),
3.54 (s, 3 H, 3′MOMb), 3.55 (d, 2 H, J ) 7.0 Hz, H1′′), 5.22 (s,
2 H, 3MOMa), 5.26 (brt, 1 H, J ) 7.0 Hz, H2′′), 5.29 (s, 2 H,
3′MOMa), 5.34 (s, 2 H, 4′MOMa), 5.38 (s, 2 H, 7MOMa), 6.58
(s, 1 H, H6), 7.34 (d, 1 H, J ) 8.7 Hz, H5′), 7.82 (dd, 1 H, J )
2.3 and 8.7 Hz, H6′), 8.00 (d, 1 H, J ) 2.3 Hz, H2′), 12.62 (s,
1 H, 5OH); 13C NMR (75 MHz, acetone-d6): δ 19.09 (C5′′), 23.40
(C1′′), 26.80 (C4′′), 57.38 (3′MOMb), 57.51 (4′MOMb), 57.54
(7MOMb), 58.91 (3MOMb), 96.19 (7MOMa), 96.85 (4′MOMa),
97.56 (3′MOMa), 99.16 (C6), 99.38 (3MOMa), 107.67 (C10),
110.30 (C8), 118.13 (C5′), 120.44 (C2′), 124.10 (C2′′), 125.72
(C6′), 126.42 (C1′), 133.41 (C3′′), 137.11 (C3), 148.94 (C3′),
152.05 (C4′), 155.40 (C9), 158.12 (C2), 161.92 (C5), 162.28 (C7),
180.73 (C4); UV (λ nm (log ꢀ)) 272 (4.43), 321, 350 sh.
8-Prenylapigenin (23). An amount of 44.9 mg (0.105
mmol) of compound 60 were dissolved in 40 mL of MeOH. After
addition of 8 mL of 1 N HCl, the solution was refluxed for 1.5
h. After cooling, the medium was diluted with H2O and
extracted with EtOAc, and the EtOAc extract was rinsed with
H2O, concentrated under reduced pressure, and purified by
MPLC on diol-bonded silica, to give 16.4 mg (0.05 mmol, 48%)
of compound 23.64 UV in MeOH, and 1H NMR data in acetone-
d6 were in accordance with that previously reported for
licoflavone C;64 13C NMR (75 MHz, acetone-d6): δ 19.12 (C5′′),
23.37 (C1′′), 26.83 (C4′′), 100.34 (C6), 104.89 (C3), 106.36 (C10),
108.39 (C8), 117.89 (C3′ + C5′), 124.58 (C2′′), 124.64 (C1′),
Anisic Anhydride (65). Reaction of 10 g of anisic acid (65.7
mmol) and 2.6 mL of methanesulfonyl chloride (33.6 mmol)
1
yielded 6.13 g of 6577 (21.4 mmol; yield 65%). H NMR (200
MHz, CDCl3) δ 3.90 (s, 6H, OCH3), 6.98 (d, 4H, J ) 9.0 Hz,
H3 + H5), 8.10 (d, 4H, J ) 9.0 Hz, H2 + H-6); 13C NMR (50
MHz, CDCl3) δ 55.48 (OCH3), 114.04 (C3 + C5), 121.21 (C1),
132.71 (C2 + C6), 162.18 (CdO), 164.47 (C4).
3,4-Dimethoxybenzoic Anhydride (66). Similar reaction
of 10 g (55 mmol) of 3,4-dimethoxybenzoic acid and 2.2 mL of
methanesulfonyl chloride (28.4 mmol) yielded 8.47 g of 6678
(24.5 mmol; yield 89%). 1H NMR (300 MHz, CDCl3) δ 3.91 (s,
6 H, 4OMe), 3.94 (s, 6H, 3OMe), 6.91 (d, 2H, J ) 8.5 Hz, H5),
7.58 (d, 2H, J ) 2.0 Hz, H2), 7.75 (dd, 2H, J ) 8.4 and 2.0 Hz,
H6); 13C NMR (75 MHz, CDCl3) δ 56.00 (3OMe + 4OMe),
110.40 (C5), 112.52 (C2), 121.18 (C1), 124.93 (C6), 148.99 (C3),
154.31 (C4), 162.31 (CdO).
3,4,5-Trimethoxybenzoic Anhydride (67). Similar reac-
tion of 10 g (47.1 mmol) of 3,4,5-trimethoxybenzoic acid and
1.8 mL of methanesulfonyl chloride (23.3 mmol) yielded 8.6 g
of 3,4,5-trimethoxybenzoic anhydride 6779,80 (21.2 mmol; yield
90%). 1H NMR (300 MHz, CDCl3) δ 3.92 (s, 12H, 3OMe +
5OMe), 3.96 (s, 6H, 4-OMe), 7.38 (s, 4H, H2 + H6); 13C NMR
(75 MHz, CDCl3) δ 58.37 (3OMe + 5OMe), 61.01 (4-OMe),
108.00 (C2 + C6), 123.52 (C1), 143.91 (C4), 153.24 (C3 + C5),
162.22 (CdO).
2,3,4-Trimethoxybenzoic Anhydride (68). Reaction of 10
g (47.3 mmol) of 2,3,4-trimethoxybenzoic acid and 1.5 mL (18.8
mmol) of methanesulfonyl chloride in 100 mL of THF with 9.2
mL of triethylamine in 11 mL of THF yielded 6.51 g of 2,3,4-
trimethoxybenzoic anhydride 6881 (16.0 mmol; yield 85%). 1H
NMR (acetone-d6, 300 MHz) δ 3.83 (s, 6H, 3OMe), 3.89 (s, 6H,

Effects of Flavonoids on Cell Proliferation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2801
2OMe), 3.97 (s, 6H, 4OMe), 6.98 (s, 2H, J ) 8.9 Hz, H5), 7.75
(d, 2H, J ) 8.9 Hz, H6); 13C NMR (acetone-d6, 75 MHz) δ 57.64
(4OMe), 62.05 (3OMe), 63.15 (2OMe), 109.59 (C5), 117.98 (C1),
129.87 (C6), 145.03 (C3), 157.35 (C2), 160.83 (C4), 163.03
(CdO).
2,4,5-Trimethoxybenzoic Anhydride (69). Similar reac-
tion of 10 g of 2,4,5-trimethoxybenzoic acid (47.1 mmol) and
1.9 mL of methanesulfonyl chloride (23.6 mmol) yielded 9.06
g of 2,4,5-trimethoxybenzoic anhydride 6978 (22.3 mmol; yield
95%). 1H NMR (300 MHz, acetone-d6) δ 3.83 (s, 6H, OMe), 3.85
(s, 6H, OMe), 3.96 (s, 6H, OMe), 6.81 (s, 2H, H3), 7.50 (s, 2H,
H6); 13C NMR (75 MHz, acetone-d6) δ 57.42 (OMe), 57.79
(OMe), 58.00 (OMe), 99.80 (C3), 110.65 (C1), 117.22 (C6),
144.88 (C5), 157.62 (C4), 158.78 (C2), 163.43 (CdO).
General Procedure for the Synthesis of 3-Methoxy-
flavones. To a well homogenized mixture (Mortar) of 1 equiv
of 2′,4′,6′-trihydroxy-2-methoxyacetophenone 6482 and of 3
equiv of the properly substituted benzoic anhydride was added
1.5 mL of triethylamine. The solution was refluxed for 5 min.
The residue was taken up in 150 mL of 5% KOH in MeOH
and sonicated for 20 min. The medium was diluted with 300
mL of water and neutralized with 6 N HCl. After storage at 4
°C for 24 h, the precipitate formed was isolated by filtration
and crystallized in hexane:acetone (10:1).
10.82 (s, 1H, 7OH), 12.61 (s, 1H, 5OH); In the 13C NMR
spectrum (75 MHz, DMSO-d6), the chemical shifts of C5, C9,
and C5′ have been reassigned as compared to published
values,85 on the basis of direct and long distance carbon-
proton correlation experiments: δ 55.57 (3′OMe + 4′OMe),
59.70 (3OMe), 93.80 (C8), 98.55 (C6), 104.19 (C10), 111.22
(C2′), 111.52 (C5′), 121.83 (C6′), 122.12 (C1′), 137.98 (C3),
148.38 (C3′), 151.15 (C4′), 155.05 (C2), 156.31 (C9), 161.15 (C5),
164.15 (C7), 177.83 (C4). Anal. (C18H16O7‚0.20CH2Cl2) C,H.
5-Hydroxy-7-prenyloxy-3,3′,4′-trimethoxyflavone (71).
A solution of 0.34 g of compound 37 (0.98 mmol), 0.25 mL of
prenyl bromide (1.97 mmol) and 0.4 g of anhydrous K2CO3 (2.0
mmol) in 50 mL of acetone was refluxed for 5 h. After filtration
and evaporation of the filtrate, the residue was crystallized
in methanol to give 0.3 g of compound 71 (0.73 mmol; yield
74%). 1H NMR (200 MHz, DMSO-d6): δ 1.72 (s, 3H, H5′′), 1.75
(s, 3H, H4′′), 3.81 (s, 3H, 3OMe), 3.85 (s, 6H, 3′OMe + 4′OMe),
4.63 (d, 2H, J ) 6.7 Hz, H1′′), 5.44 (m, 1H, H2′′), 6.34 (d, 1H,
J ) 2.2 Hz, H6), 6.76 (d, 1H, J ) 2.2 Hz, H8), 7.14 (d, 1H, J
) 8.6 Hz, H5′), 7.63 (d, 1H, J ) 2.0 Hz, H2′), 7.70 (dd, 1 H, J
) 8.5 and 2.0 Hz, H6′), 12.58 (s, 1H, 5OH); 13C NMR (50 MHz,
DMSO-d6): δ 17.98 (C5′′), 25.34 (C4′′), 55.60 (3′OMe + 4′OMe),
59.71 (3OMe), 65.28 (C1′′), 92.99 (C8), 98.20 (C6), 105.10 (C10),
111.25 (C2′), 111.54 (C5′), 118.86 (C2′′), 121.96 (C1′ + C6′),
138.19 (C3 + C3′′), 148.41 (C3′), 151.27 (C4′), 155.33 (C2),
156.18 (C9), 160.79 (C5), 164.32 (C7), 177.94 (C4); MS (CI) m/e
413 [M + H]+; HRMS (C23H24O8) calcd 413.1600, found
413.1600. Anal. (C23H24O7. 0.2 toluene + 0.1 hexane) C, H.
Ermanine (35). Reaction of 0.6 g of 64 (3 mmol) and 2.6 g.
1
of 65 (9 mmol) yielded 0.763 g of 3582 (2.43 mmol; 81%). H
NMR and 13C NMR spectra (DMSO-d6) were in accordance
with published values.82,83 Anal. (C17H14O6‚0.10MeOH) C,H.
5,7-Dihydroxy-8-(1,1-dimethylallyl)-3,3′,4′-trimethoxy-
flavone (38). An amount of 0.3 g of compound 71 (0.73 mmol)
and 0.12 g of anhydrous sodium acetate in 20 mL of acetic
anhydride were refluxed for 48 h. After dilution and extraction
by chloroform, the residue was stirred with 70 mL of 5%
methanolic KOH for 4 h at room temperature. The solution
was then acidified and extracted with ethyl acetate and
crystallized in hexane:Me2CO (50:5) to yield 0.183 g of pure
compound 38 (0.44 mmol; yield 60%). 1H NMR (300 MHz,
CDCl3): δ 1.68 (s, 6H, H4′′ + H5′′), 3.82 (s, 3H, 3OMe), 3.94*
(s, 3H, 3′OMe), 3.97* (s, 3H, 4′OMe), 5.39 (d, 1H, J ) 10.7 Hz,
H3′′), 5.46 (d, 1H, J ) 17.7 Hz, H3′′), 6.29 (s, 1H, H6), 6.46
(dd, 1H, J ) 17.7 and 10.7 Hz, H2′′), 7.00 (d, 1H, J ) 8.7 Hz,
H5′), 7.25 (s, 1H, 7OH), 7.53 (d, 1H, J ) 2.1 Hz, H2′), 7.63
(dd, 1H, J ) 8.7 and 2.1 Hz, H6′), 13.03 (s, 1H, 5OH); 13C NMR
(75 MHz, CDCl3): δ 28.06 (C4′′ + C5′′), 40.64 (C1′′), 55.97*
(3′OMe), 55.98* (4′OMe), 60.29 (3-OMe), 101.17 (C6), 106.95
(C10), 109.70 (C8), 110.84 (C5′), 111.54 (C2′), 113.68 (C3′′),
122.53 (C6′), 122.74 (C1′), 138.65 (C3), 148.69 (C3′), 148.96
(C2′′), 151.08 (C4′), 155.27 (C9), 156.54 (C2), 160.54 (C5),
161.48 (C7), 179.07 (C4); MS (CI) m/e 413 [M + H]+; HRMS
(C23H24O8) calcd 413.1600, found 413.1600. Anal. (C23H24O7)
C, H.
5-Hydroxy-7-prenyloxy-3,4′-trimethoxyflavone (70). A
solution of 0.30 g of compound 35 (0.95 mmol), 0.2 mL of prenyl
bromide (1.73 mmol), and 0.4 g of anhydrous K2CO3 (2.0 mmol)
in 50 mL of acetone was refluxed for 5 h. After filtration and
evaporation of the filtrate, the residue was crystallized in
methanol to give 0.33 g of compound 70 (0.86 mmol; yield 91%).
1H NMR (200 MHz, DMSO-d6): δ 1.71 (s, 3H, H4′′), 1.74 (s,
3H, H5′′), 3.79 (s, 3H, 3OMe), 3.84 (s, 3H, 4′OMe), 4.61 (d,
2H, J ) 6.6 Hz, H1′′), 5.43 (brt, 1H, J ) 6.6 Hz, H2′′), 6.32 (d,
1H, J ) 1.8 Hz, H6), 6.70 (d, 1H, J ) 1.8 Hz, H8), 7.11 (d, 2H,
J ) 8.8 Hz, H3′ + H5′), 8.02 (d, 2H, J ) 8.8 Hz, H2′ + H6′),
12.58 (s, 1H, 5OH); 13C NMR (50 MHz, DMSO-d6): δ 17.96
(C4′′), 25.32 (C5′′), 55.37 (4′OMe), 59.66 (3OMe), 65.27 (C1′′),
92.86 (C8), 98.16 (C6), 105.10 (C10), 114.15 (C3′ + C5′), 118.90
(C2′′), 122.03 (C1′), 129.92 (C2′ + C6′), 138.07 (C3 + C3′′),
155.37 (C2), 156.20 (C9), 160.83 (C5), 161.35 (C4′), 164.30 (C7),
177.98 (C4); MS (CI) m/e 383 [M + H]+; HRMS (C22H22O7) calcd
383.1495, found 383.1497. Anal. (C22H22O6) C, H.
5,7-Dihydroxy-8-(1,1-dimethylallyl)-3,4′-trimethoxy-
flavone (36). An amount of 0.3 g of compound 70 (0.78 mmol)
and 0.12 g of anhydrous sodium acetate in 20 mL of acetic
anhydride, were refluxed for 48 h. After dilution and extraction
by chloroform, the residue was stirred with 70 mL of 5%
methanolic KOH for 4 h at room temperature. The solution
was then acidified and extracted by ethyl acetate, to give 0.21
g of pure compound 36 (0.55 mmol; yield 71%). 1H NMR (200
MHz, DMSO-d6): δ 1.55 (s, 6H, 1′′Me), 3.75 (s, 3H, 3OMe),
3.84 (s, 3H, 4′OMe), 4.77 (dd, 1H, J ) 10.5 and 1.1 Hz, H3′′),
4.80 (dd, 1H, J ) 17.5 and 1.1 Hz, H3′′), 6.27 (dd, 1H, J )
17.5 and 10.5 Hz, H2′′), 6.30 (s, 1H, H6), 7.10 (d, 2H, J ) 9.0
Hz, H3′ + H5′), 7.91 (d, 2H, J ) 9.0 Hz, H2′ + H6′), 10.52
(brs, 1H, 7OH), 13.00 (s, 1H, 5OH); 13C NMR (50 MHz, DMSO-
d6): δ 29.54 (1′′Me), 40.34 (C1′′), 55.25 (4′OMe), 59.66 (3OMe),
99.60 (C6), 105.02 (C10), 108.36 (C3′′), 110.75 (C8), 113.80 (C3′
+ C5′), 121.95 (C1′), 130.33 (C2′ + C6′), 137.42 (C3), 149.66
(C2′′), 154.75 (C2), 156.07 (C9), 158.98 (C5), 161.03 (C4′),
162.89 (C7), 178.13 (C4); MS (CI) m/e 383 [M + H]+; HRMS
(C22H22O7) calcd 383.1495, found 383.1500. Anal. (C22H22O6)
C, H.
5,7-Dihydroxy-3,3′,4′,5′-tetramethoxyflavone (39). Re-
action of 1.37 g of 64 (6.9 mmol) and 8.6 g of 67 (21 mmol)
yielded 1.6 g of 39 (4.3 mmol; yield 62%) which was crystallized
in hexane:Me2CO to give 0.67 g of pure 39.86 Anal. (C19H18O8‚
0.70MeOH) C, H.
5,7-Dihydroxy-2′,3,3′,4′-tetramethoxyflavone (40). Re-
action of 1.0 g of 64 (5.0 mmol) and 6.2 g of 68 (15 mmol)
1
yielded 1.6 g of 40 (4.3 mmol; yield 86%). H NMR (acetone-
d6, 300 MHz) δ 3.76 (s, 3H, 3OMe), 3.86 (s, 3H, 3′OMe), 3.91
(s, 3H, 2′OMe), 3.94 (s, 3H, 4′OMe), 6.27 (d, 1H, J ) 2.1 Hz,
H6), 6.39 (d, 1H, J ) 2.1 Hz, H8), 6.94 (d, 1H, J ) 8.7 Hz,
H5′), 7.24 (d, 1H, J ) 8.7 Hz, H6′), 12.75 (s, 1H, 5OH); 13C
NMR (acetone-d6, 75 MHz) δ 57.51 (4′OMe), 61.72 (3OMe),
61.95 (3′OMe), 62.85 (2′OMe), 95.50 (C8), 100.44 (C6), 107.39
(C10), 109.36 (C5′), 119.39 (C1′), 127.23 (C6′), 141.54 (C3),
144.36 (C3′), 154.18 (C2′), 158.02 (C4′), 158.50 (C2), 159.33
(C9), 164.40 (C5), 165.89 (C7), 180.58 (C4); MS (CI) m/e 375
[M + H]+; HRMS (C19H18O9) calcd 375.1078, found 375.1078.
Anal. (C19H18O8‚0.1 MeOH + 0.2 H2O) C, H.
5,7-Dihydroxy-3,3′,4′-trimethoxyflavone (37). Reaction
of 1.6 g of 64 (8.1 mmol) and 8.4 g of 66 (21.0 mmol) yielded
2.2 g of 37 (5.9 mmol; yield 74%) which was crystallized in
hexane:Me2CO to give 0.85 g of pure 37.84 1H NMR (200 MHz,
DMSO-d6): δ 3.80 (s, 3H, 3OMe), 3.84 (brs, 6H, 3′OMe +
4′OMe), 6.19 (d, 1H, J ) 1.6 Hz, H6), 6.46 (d, 1H, J ) 1.6 Hz,
H8), 7.13 (d, 2H, J ) 8.5 Hz, H5′), 7.64 (m, 2H, H2′ + H6′),
5,7-Dihydroxy-2′,3,4′,5′-tetramethoxyflavone (41). Re-
action of 1.3 g of 64 (6.6 mmol) and 8.0 g of 69 (19.7 mmol)
yielded 1.2 g of 41 (3.2 mmol; yield 48%). 1H NMR (300 MHz,
DMSO-d6): δ 3.70 (s, 3H, 3OMe), 3.73 (s, 3H, 5′OMe), 3.81*

2802 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Daskiewicz et al.
(3) Middleton, E., Jr; Kandaswami, C.; Theoharides, T. C. The
effects of plant flavonoids on mammalian cells: implication for
inflammation, heart disease and cancer. Pharmacol. Rev. 2000,
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(4) Gee, J. M.; Johnson, I. T. Polyphenolic compounds: interaction
with the gut and implications for human health. Curr. Med.
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(s, 3H, 2′OMe), 3.88* (s, 3H, 4′OMe), 6.21 (d, 1H, J ) 1.9 Hz,
H6), 6.34 (d, 1H, J ) 1.9 Hz, H8), 6.84 (s, 1H, H3′), 7.07 (s,
1H, H6′); 13C NMR (75 MHz, DMSO-d6): δ 55.79* (4′OMe),
177.86 (C4), 56.27 (5′OMe), 56.37* (2′OMe), 59.88 (3OMe),
93.65 (C8), 98.02 (C3′), 98.53 (C6), 104.52 (C10), 109.74 (C1′),
113.66 (C6′), 139.00 (C3), 142.29 (C5′), 151.76‡ (C4′), 151.97‡
(C2′), 156.03 (C2), 156.89 (C9), 161.33 (C5), 164.08 (C7); MS
(CI) m/e 375 [M + H]+; HRMS (C19H18O9) calcd 375.1078, found
375.1077. Anal. (C19H18O8) C, H.
3-O-Methylplatanetin (42). Potassium persulfate (9.5 g;
35.1 mmol) in 250 mL of water was added to 5 g of 7282,83 (17.6
mmol) in 50 mL of 20% aq. tetraethylammonium hydroxide.
The solution was stirred for 2 h in the dark under nitrogen.
After neutralization with a saturated aq solution of KH2PO4,
the mixture was extracted with Et2O, followed by butanol.
After evaporation of the butanolic extract, the residue was
dissolved in 60 mL of 10% aq. tetramethylammonium hydrox-
ide and, after addition of 6 mL of prenyl bromide (52 mmol),
stirred for 1 h. The reaction was stopped by acidification with
6 N HCl. After 4 h at room temperature, the solution was
extracted with EtOAc. 2.5 g of 42 (6.8 mmol; yield 39% from
72) were obtained after purification of the extract by MPLC
on polyamide SC6 (toluene). 1H NMR (200 MHz, DMSO-d6) δ
1.60 (brs, 3H, H4′′), 1.70 (brs, 3H, H5′′), 3.24 (d, 2H, J ) 6.8
Hz, H1′′), 3.77 (s, 3H, 3OMe), 5.15 (brt, 1H, J ) 6.8 Hz, H2′′),
7.57 (m, 3H, H3′ + H4′ + H5′), 8.12 (m, 2H, H2′ + H6′), 9.00
(6) Birt, D. F.; Hendrich, S.; Wang, W. Dietary agents in cancer
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T.; Reuhl, K. R.; Yang, C. S.; Conney, A. H. Inhibitory effect of
green tea on the growth of established skin papillomas in mice.
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gou, E.; Gemetzi, C.; Kouroumalis, E.; Martin, P. M.; Castanas,
E. Wine antioxidant polyphenols inhibit the proliferation of
human prostate cancer cell lines. Nutr. Cancer 2000, 37, 223-
233.
(9) Le Bail, J. C.; Aubourg, L.; Habrioux, G. Effects of pinostrobin
on estrogen metabolism and estrogen receptor transactivation.
Cancer Lett. 2000, 156, 37-44.
(10) Hirano, T.; Gotoh, M.; Oka, K. Natural flavonoids and lignans
are potent cytostatic agents against human leukemic HL-60
cells. Life Sci. 1994, 55, 1061-1069.
(11) Singh, R. P.; Dhanalakshmi, S.; Tyagi, A. K.; Chan, D. C. F.;
Agarwal, C.; Agarwal, R. Dietary feeding of silibinin inhibits
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(brs, 1H, 8OH), 10.00 (brs, 1H, 7OH), 12.33 (s, 1H, 5OH); 13
C
NMR (50 MHz, DMSO-d6) δ 17.86 (C5′′), 21.52 (C1′′), 25.62
(C4′′), 60.16 (3OMe), 103.73 (C10), 111.16 (C6), 122.32 (C2′′),
123.99 (C8), 128.59 (C2′ + C6′), 128.78 (C3′ + C5′), 130.38
(C1′), 130.95 (C3′′), 131.14 (C4′), 138.65 (C3), 143.87 (C9),
150.80 (C5), 152.69 (C7), 155.00 (C2), 178.55 (C4); MS (CI) m/e
369 [M + H]+; HRMS (C21H21O6) calcd 369.1338, found
369.1341. Anal. (C21H21O6) C, H.
Cell Viability. Cells were cultured into 96-well plates
(15 000 cells per well in 200 µL medium; DMEM, 5% fetal calf
serum) for 24 h prior to treatment. The medium was subse-
quently removed and replaced by fresh medium containing the
test compound (50 µM) in the presence of sodium ascorbate
(200 µM). At the end of the incubation time (6 and 24 h) the
plates were treated according to Fautz et al.87 with a minor
modifications. In brief, cells were incubated in neutral red dye
in medium (50 µg/mL) for 2 h, fixed in aqueous formaldehyde:
CaCl2 (0.5: 0.1) for 30 s and washed twice in phosphate
buffered saline (PBS). After air-drying, 200 µL of aqueous
ethanol:acetic acid (50: 1) was added to each well to release
the intracellular dye from lysed cells. The optical density of
solutions in each well was assessed using an automatic
spectrophotometric plate reader at 550 nm.
Apoptosis. The protocol followed the manufacturer’s in-
structions (ApopOne kit, Promega). Briefly, cells were cultured
in 96-well plates (15 000 cells per well in 100 µL medium).
After 5 h incubation with the test molecule, 100 µL of apoptosis
reagent (including caspase substrate and lysis buffer) was
added to the well and plates were incubated for 45 min at 37
°C. Finally plates were assessed in an automatic fluorescence
plate reader (excitation 485 nm, emission 538 nm).
Acknowledgment. This work was supported by
grants from the European Community (Polybind project
QLK1-1999-00505). Jean-Baptiste Daskiewicz was the
recipient of a fellowship from the French Ministry of
Research and Education.
Supporting Information Available: Elemental analyses
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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