Efficient microwave-assisted prenylation of pinostrobin and biological evaluation of its derivatives as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2010.02.068

Pinostrobin (5-hydroxy-7-methoxyflavanone) obtained in relatively large amounts from fingerroot (Boesenbergia pandurata) was converted to its C-6 and C-8 prenylated derivatives. The Mitsunobu reaction, europium(III)-catalyzed Claisen-Cope rearrangement, and Claisen reaction coupled with cross-metathesis were used as the key steps. Using a sealed-vessel microwave reactor, the Mitsunobu and Claisen/Cope reactions occurred smoothly with short reaction times and in satisfactory yields. The target compounds and five new intermediary substances showed cytotoxic activity toward SK-BR-3, MCF-7, PC-3, and Colo-320DM human tumor cell lines, and all of them had significantly lower IC₅₀ (μM) values than pinostrobin. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2010.02.068

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2086–2089
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient microwave-assisted prenylation of pinostrobin and biological
evaluation of its derivatives as antitumor agents
Hadi Poerwono a, Shigeru Sasaki b, Yoshiyuki Hattori b, Kimio Higashiyama b,
*
a Faculty of Pharmacy, Airlangga University, Jalan Dharmawangsa Dalam, Surabaya 60286, Indonesia
b Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Pinostrobin (5-hydroxy-7-methoxyflavanone) obtained in relatively large amounts from fingerroot (Boe-
senbergia pandurata) was converted to its C-6 and C-8 prenylated derivatives. The Mitsunobu reaction,
europium(III)-catalyzed Claisen–Cope rearrangement, and Claisen reaction coupled with cross-metathe-
sis were used as the key steps. Using a sealed-vessel microwave reactor, the Mitsunobu and Claisen/Cope
reactions occurred smoothly with short reaction times and in satisfactory yields. The target compounds
and five new intermediary substances showed cytotoxic activity toward SK-BR-3, MCF-7, PC-3, and Colo-
Received 15 January 2010
Revised 13 February 2010
Accepted 17 February 2010
Available online 20 February 2010
Keywords:
5-Hydroxy-7-methoxyflavanone
Prenylation
320DM human tumor cell lines, and all of them had significantly lower IC50 (lM) values than pinostrobin.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Microwave-assisted reaction
Mitsunobu reaction
Claisen rearrangement
Cross-metathesis
Anticancer activity
Flavonoids, including flavones, flavanones, flavanols, flavonols,
and isoflavones, constitute the largest and most important group
of polyphenolic compounds in plants. Erdtman reported the pres-
ence of a new flavonoid named pinostrobin (1, Fig. 1) in the heart-
wood of pine (Pinus strobus) about 65 years ago.1 Since then, there
have been about 300 articles that have included this flavanone as
their subject. Besides its occurrence in plants belonging to Pina-
ceae,2 pinostrobin has also been found in the following families:
Fabaceae,3 Asteraceae,4 Salicaceae,5 Lauraceae,6 Zingiberaceae,7
Piperaceae,8 Chloranthaceae,9 Myrtaceae,10 Rosaceae,11 Pterida-
ceae,12 Annonaceae,13 Lamiaceae,14 Betulaceae,15 Juglandaceae,16
Polygonaceae,17 Santalaceae,18 Myricaceae,19 Muntingiaceae,20
Chenopodiaceae,21 Grossulariaceae,22 Notophagaceae,23 Melianth-
aceae,24 Acrobolbaceae (liverwort),25 and propolis.26
activity and decreased MCF-7 human breast cancer cell growth in-
duced by dehydroepiandrosterone sulfate and estradiol.36 In com-
parison with other prominent flavonoids found in honey,
pinostrobin has been shown to be the most potent inducer of
mammalian phase II chemoprotective and antioxidant enzymes
as a result of an examination conducted by Fahey and co-work-
ers.37 As we have done in this study, this group has also suggested
using the edible rhizomes of fingerroot (B. pandurata) as the most
promising source of pinostrobin.
Furthermore, it has been suggested that prenylated flavonoids
exhibit stronger bioactivities than those without prenyl substitu-
ents as assessed by the cAMP phosphodiesterase inhibition test, a
useful tool for screening biologically active compounds contained
in medicinal plants. The aforementioned test revealed that the
Pinostrobin has been the subject of a number of studies on its
bioactivities including cytotoxicity,27 quinone reductase induc-
tion,28 antioxidant activity,29 antimicrobial activity,30 virus prote-
ase inhibition,31 antinociceptive activity,32 anti-inflammatory
activity,33 and gastroprotective activity.34 Moreover, Sukardiman
et al. have suggested that the cytotoxicity of pinostrobin against
human mammary carcinoma emerged from its inhibition of DNA
Topoisomerase I activity.35 In line with the above result, Le Bail
et al. have confirmed that pinostrobin exhibited antiaromatase
* Corresponding author.
E-mail address: kimio@hoshi.ac.jp (K. Higashiyama).
Figure 1. Pinostrobin (1) and its prenylated derivatives (2 and 3).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.068

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2087
IC50 value of 8-prenylpinostrobin (3) was about one-sixth of that of
pinostrobin (1).38 More than a dozen years later, a group spanning
seven universities found that flavanones bearing a hydrophobic
prenyl, geranyl, or lavandulyl group at position 8 (in the A ring)
were shown to be the strongest multi-drug resistance protein 1
(MRP1)/ATP-binding cassette C1 (ABCC1) inhibitors in a study of
flavonoids as multi-drug resistance modifiers.39 These findings
have been ascertained by the very recent study conducted by Ka-
dota and co-workers reporting that an increase of prenyl groups
is one of the structural requirements for enhancing the cytotoxicity
of flavanone derivatives.40
The considerable and extensive interest in pinostrobin and its
prenylated derivatives has consequently led us to subject pinost-
robin to a prenylation procedure yielding 6-prenylpinostrobin (2,
Fig. 1) and 8-prenylpinostrobin (3, Fig. 1). Both the prenyl deriva-
tives are known as naturally occurring flavonoids. In 1980, three
different groups almost concurrently reported flavanone 3 as a
new compound isolated from Tephrosia sp. (Fabaceae).41 While
there are a number of articles referring to C-8 prenylflavanone
3,42 there is only one report on the occurrence of C-6 prenylated
congener 2 in a plant (Derris rariflora, Fabaceae).43 Claisen rear-
rangement, which is known as a useful method for regioselective
prenylation of phenolic natural products,44 was used to synthesize
the target compounds.
solvents, we treated flavanone 4 with catalytic amounts (10 mol %)
of Eu(fod)3 in dry chloroform48 at 60 °C for 12 h to give 6-(1,1-
dimethylallyl)pinostrobin (5) as the sole product. As reported by
other groups, however, similar reaction conditions of the Claisen
procedure have been applied to 7,40-diacetoxy-5-prenyloxyflava-
none, 7,40-diacetoxy-5-prenyloxyisoflavone, and 7,40-diacetoxy-5-
prenyloxyflavone, respectively, to give mainly the 8-prenylated
derivatives along with minor ortho-rearranged products.45 The pre-
dominance of para-rearranged products of the three 5-prenyloxy-
flavonoids suggests that a low temperature Claisen–Cope proce-
dure using Eu(fod)3 as the catalyst proceeds with high selectivity.
This was, of course, contrary to the results of our study. A plausible
source of the discrepancy is the electronic nature of the meta-sub-
stituents of phenolic substrates. It has been reported that electron-
withdrawing groups, including –OCOCH3, promote rearrangement
to the ortho position next to the group, while electron-donating
groups, including –OCH3, have the opposite effect.49
In order to convert the regioselectivity for C–O into that for C–C,
we attempted a microwave-assisted Claisen–Cope reaction cata-
lyzed by Eu(fod)3. To our knowledge, there is only one report to
date that has employed europium(III)-catalyzed sigmatropic rear-
rangement under microwave heating,50 and no report of such a
reaction applied to the construction of C-prenyl flavonoids. Table 1
lists the details of our novel and facile procedure for synthesizing
Hence, we embarked on synthesizing prenylflavanones 2 and 3
from flavanone 1, which was isolated from the rhizomes of temu
kunci (B. pandurata) grown in the East Java area of Indonesia. As
outlined in Scheme 1, the already existing 7-O-methyl functional-
ity of 1 serves favorably as a protective group. This sets the stage
for installing the requisite C-5 prenyl ether moiety, which will be
used as the substrate for the Claisen reaction. By using the Mitsun-
obu reaction, the condensation of flavanone 1 and 3-methyl-2-bu-
ten-1-ol in the presence of triphenylphosphine (PPh3) and diethyl
azodicarboxylate (DEAD) in dry tetrahydrofuran (THF) gave the de-
sired 5-O-prenylflavanone 4 in 81% yield after a reaction time of
18 h. The yield was in agreement with previous studies reporting
a similar O-prenylation of flavonoids using the classical Mitsunobu
procedure.45 The challenge of achieving both a shorter reaction
time and a higher yield could, however, be fulfilled by heating
the chemical reactions with microwave (MW) energy. Compared
with conventional heating methods, microwave heating has been
shown to dramatically decrease reaction times, increase product
yields, and enhance product purities by reducing unwanted side
reactions.46 A microwave-assisted Mitsunobu reaction is reliable,
as it has been employed by previous groups to synthesize target
molecules in satisfactory yields.47 Therefore, flavanone 1, and three
equivalents of prenyl alcohol, Ph3P, and diisoproyl azodicarboxyl-
ate (DIAD) were heated in dry THF in a sealed tube at 60 °C inside
a microwave reactor (Scheme 1). This resulted in the C-5 prenylat-
ed flavanone 4 in 96% yield after a reaction time of just 30 min—a
considerably higher percentage yield in a shorter reaction time
than previously achieved.
8-prenylflavanone 3 via a lanthanide-catalyzed Claisen–Cope
process of 5-O-prenylflavanone 4 under microwave irradiation.
Initially, reacting prenyl ether 4 with 10 mol % of the catalyst Eu(-
fod)3 in dry CHCl3 inside a sealed-vessel microwave reactor at
60 °C for 2 h afforded a 4:1 ratio of the major ortho-rearranged
product 5 and the minor para-rearranged target compound 3 (en-
try 1). Extending the reaction time to 4 h, however, produced no
significant changes in the composition and yield of the products
(entry 2). Given that microwave-accelerated procedure requires a
higher temperature to facilitate the tandem Claisen–Cope reac-
tion,44 we carried out europium(III)-catalyzed rearrangement of
5-O-prenyl substrate 4 under microwave irradiation at 100 °C for
30 min. This gave equal amounts of 6-(1,1-dimethylallyl)flavanone
5 and its cyclic derivative 6, in addition to the major product, 8-
prenylated flavanone 3 (entry 3). It is noteworthy that once the
6-rearranged product 5 is formed, the higher temperature enables
it to competitively undergo subsequent para-Cope rearrangement
to provide the 8-prenylflavanone 3, and to couple with the C-5 hy-
droxy group to give a diastereomeric mixture of coumaran 6. To
achieve complete para-rearrangement, the aforementioned micro-
wave-assisted reaction was allowed to occur for 30 min at 140 °C—
the maximum temperature to which the CHCl3 solvent could be
Table 1
Results of Claisen rearrangement of compound 4
Once the prenyl ether intermediate 4 was available, attention
was focused on C-prenylation at positions 6 or 8 of the flavanone
depending on the conditions of Claisen rearrangement. Instead of
using the conventional uncatalyzed condition in high boiling point
Entry
Microwave heating condition
Yield (%)
3
5
6
Total
1
2
3
4
60 °C, 2 h
60 °C, 4 h
100 °C, 30 min
140 °C, 30 min
18
20
39
51
68
63
20
0
Tracea
Tracea
27
86
83
86
85
34
Scheme 1. Preparation of prenyl ether 4. Reagents and conditions: (a) DEAD, PPh3,
THF, 0 °C to rt, 18 h, 81%; (b) DIAD, PPh3, THF, MW 60 °C, 30 min, 96%.
a
The compound was found in only trace amounts.

2088
H. Poerwono et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2086–2089
Table 2
raised inside the microwave reactor (entry 4). Surprisingly, the 6-
allylated product 5 was not detected. The predominant 8-prenyl
product 3 occurred in moderate yield along with the minor product
dihydrofurano-flavanone 6.
Cytotoxicity of compounds 1–8 against human tumor cell lines
Compounds
IC50
(l
M)
SK-BR-3
MCF-7
PC-3
Colo-320DM
Next, we sought to execute regioselective C-prenylation at the
C-6 position of the lead compound 1 to provide 6-prenylflavanone
2. The key steps of the synthetic strategy were C-6 allylation via
1
2
3
4
5
6
7
8
94.3
30.7
34.4
19.6
12.1
14.0
24.2
18.1
84.9
22.9
30.3
20.3
45.6
29.5
23.9
32.2
86.7
45.8
35.3
37.8
28.4
10.4
45.3
46.0
>100
52.2
24.2
37.5
36.5
18.6
37.5
68.5
europium(III)-catalyzed Claisen rearrangement, followed by
a
cross-metathesis (CM) similar to that in previous C-6 prenylations
of naringenin and genistein using conventional heating.51 Unlike
the O-prenylation of flavanone 1 described above, we discovered
that the classical Mitsunobu procedure using PPh3 failed to effect
the O-allylation of 1. A previously reported similar reaction on
7,40-diacetoxy-5-hydroxyflavanone, however, successfully gave
the appropriate 5-O-allyl derivative.51 It was assumed that the de-
crease of the phenolic –OH acidity of compound 1 caused by its 5-
methoxy group is responsible for the ineffectiveness of O-allylation
described above. We then examined the utilization of another re-
agent, triisopropylphosphite (TIP), instead of PPh3. Although the
use of TIP gave a moderate yield of allyl ether product 7, the isopro-
pyl moiety of TIP competitively reacted with the phenolic hydroxyl
group to afford isopropyl ether as a side product, which was not
easily separated from the major product 7. Building on the useful-
ness of the microwave-enhanced Mitsunobu reaction, Moody et al.
have realized a microwave-assisted combined Mitsunobu reac-
tion–Claisen rearrangement strategy that enables the rapid synthe-
sis of 1,4-benzoquinone derivatives.52 Therefore, we sought to
apply this combined protocol to our study via the reaction of 1
with allyl alcohol, Ph3P, and DIAD by heating in dry THF inside
the sealed tube of a microwave reactor at 180 °C. After a reaction
time of 60 min, however, the corresponding 6-allylpinostrobin 8
was obtained in only 33% yield together with an equal amount of
the recovered starting material. Consequently, we settled on a
step-by-step synthetic route (Scheme 2) starting with the reaction
of flavanone 1 and allyl alcohol under a microwave-assisted Mits-
unobu reaction at 60 °C for 30 min to give 5-O-allylpinostrobin (7)
in good yield. The allyl ether substrate 7 was then introduced to a
europium(III)-catalyzed Claisen rearrangement in a microwave
reactor at 120 °C for 2 h, to obtain C-6 allylated flavanone 8 in good
yield. To complete the construction of prenyl functionality, the al-
lyl moiety of 8 was subjected to the CM reaction with isobutylene
by using 3 mol % Grubbs catalyst in a sealed tube. The desired 6-
prenylflavanone 2 was obtained in high yield after 18 h of standard
heating at 45 °C.
was compared to that of the lead compound 1. All derivatives
exhibited significantly lower IC50 values than 1, and good cytotox-
icity against the four cell lines (IC50, 50 l
M or lower).54 The prenyl
moieties at the 6-C-, 8-C-, and 5-O-positions of pinostrobin re-
vealed no striking differences in the cytotoxic activity of prenylat-
ed derivatives 2–4, respectively, although compound 3 was two
times stronger than 2 against Colo-320DM. The O-prenyl function-
ality at C-5 of compound 4 and the O-allyl functional group at C-5
of compound 7 provided these compounds with equal cytotoxicity
against all cell lines used in this study. A similar phenomenon was
also observed when we evaluated the effect of the 6-C-prenyl sub-
stituent of 2 and the 6-C-allyl functional group of 8 on their cyto-
toxic activities. The 6-(1,1-dimethylallyl) substituent of 5 showed
higher cytotoxicity against SK-BR-3, PC-3 and Colo-320DM than
the 6-(3,3-dimethylallyl) substituent of 2. In contrast, compound
2 was two times more active than 5 against MCF-7. The introduc-
tion of the allyl or prenyl side chains on an appropriate position of
the flavanone structure was found to improve the cytotoxicity. Sur-
prisingly, furo-flavanone 6 was revealed in general to be the most
potent inhibitor of all evaluated cell lines due to its hydrophobic
dihydrofurano side ring.
In summary, we have developed an efficient microwave-as-
sisted procedure for the allylation and prenylation of pinostrobin
(1) via the Mitsunobu reaction, ortho-Claisen and para-Cope rear-
rangements, and cross-metathesis. Biological evaluations revealed
that the two target compounds (2 and 3) and five new intermedi-
ary substances (4–8) were more active than the lead compound 1
against a panel of cancer cell lines. The increase of the lipophilicity
of the derivatives to interact with biological targets provided by
the allyl or prenyl groups can be a basis to explain the improve-
ment of antitumor activity.
The biological activities of all resulting compounds were inves-
tigated against a panel of human tumor cell lines including SK-BR-
3 (breast), MCF-7 (breast), PC-3 (prostate), and Colo-320DM
(colon),53 as listed in Table 2. The cytotoxicity of the derivatives
Acknowledgments
This research was supported financially in part by a grant for
the Open Research Center Project. H.P. thanks Japan Student Ser-
vices Organization (JASSO) for the Follow-up Research Fellowship,
and Indonesian Directorate General of Higher Education (DGHE)
for the Competitive Grant Program B (PHK-B).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.068.
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