Synthesis of C- and O-prenylated tetrahydroxystilbenes and O-prenylated cinnamates and their action towards cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.02.017

Synthesis of the naturally occurred C- and O-prenylated tetrahydroxystilbenes and O-prenylated cinnamates was carried out by decarbonylative Heck reaction and selenium dioxide catalysed oxidation, respectively. In the decarbonylative Heck synthetic route, fusion of benzoyl chloride and styrene derivatives was catalysed by an N-heterocyclic carbene system generated in situ by palladium acetate and 1,3-bis(2,6-diisopropylphenyl) imidazolinium chloride to form a E-tetrahydroxystilbene derivative. Formation of allyl ether was subsequently carried out by reaction of the deprotected OH in the A phenyl ring of the stilbene with 3,3-dimethylallyl bromide and a base (sodium hydride) to form O-prenylated tetrahydroxystilbene derivatives. [1,5]-Rearrangement of the isoprenyl unit from O- to C-position in the A ring was carried out at elevated temperature in the presence of magnesium silicate (Florisil) to form the corresponding C-prenylated tetrahydroxystilbene. Formation of O-prenylated cinnamate was first carried out by base catalysed allyl ether formation between 3,3-dimethylallyl bromide and hydroxycinnamic acid methyl ester. The methyl group of the isoprenyl unit was subsequently oxidized using selenium dioxide to form a terminal hydroxyl group. The prenylated tetrahydroxystilbenes and cinnamate synthesized in this study were novel derivatives of piceatannol and methyl 4-(3′-methylbut-2′-enyloxy) cinnamate isolated from propolis in Kangaroo Island, South Australia. The synthetic compounds were tested against K562 cancer cells and potent growth inhibitory activity was observed for E-1-[5-hydroxy-3-methoxy-2-(3-methyl-2- butenyl)phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene, IC₅₀ = 0.10 μM.

Full text of DOI:10.1016/j.ejmech.2013.02.017

European Journal of Medicinal Chemistry 63 (2013) 415e422
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of C- and O-prenylated tetrahydroxystilbenes and
O-prenylated cinnamates and their action towards cancer cells
,
Nooshin Koolaji a, Abdallah Abu-Mellal a b, Van H. Tran a, Rujee K. Duke c,
,
Colin C. Duke a
*
a Faculty of Pharmacy, University of Sydney, NSW 2006, Australia
b Faculty of Pharmacy, Al Ain University of Science and Technology, Abu Dhabi, United Arab Emirates
c Department of Pharmacology, Faculty of Medicine, University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of the naturally occurred C- and O-prenylated tetrahydroxystilbenes and O-prenylated cin-
namates was carried out by decarbonylative Heck reaction and selenium dioxide catalysed oxidation,
respectively. In the decarbonylative Heck synthetic route, fusion of benzoyl chloride and styrene de-
rivatives was catalysed by an N-heterocyclic carbene system generated in situ by palladium acetate and
1,3-bis(2,6-diisopropylphenyl)imidazolinium chloride to form a E-tetrahydroxystilbene derivative. For-
mation of allyl ether was subsequently carried out by reaction of the deprotected OH in the A phenyl ring
of the stilbene with 3,3-dimethylallyl bromide and a base (sodium hydride) to form O-prenylated tet-
rahydroxystilbene derivatives. [1,5]-Rearrangement of the isoprenyl unit from O- to C-position in the
A ring was carried out at elevated temperature in the presence of magnesium silicate (Florisil) to form the
corresponding C-prenylated tetrahydroxystilbene. Formation of O-prenylated cinnamate was first carried
out by base catalysed allyl ether formation between 3,3-dimethylallyl bromide and hydroxycinnamic acid
methyl ester. The methyl group of the isoprenyl unit was subsequently oxidized using selenium dioxide
to form a terminal hydroxyl group. The prenylated tetrahydroxystilbenes and cinnamate synthesized in
this study were novel derivatives of piceatannol and methyl 4-(30-methylbut-20-enyloxy)cinnamate
isolated from propolis in Kangaroo Island, South Australia. The synthetic compounds were tested against
K562 cancer cells and potent growth inhibitory activity was observed for E-1-[5-hydroxy-3-methoxy-2-
Received 12 October 2012
Received in revised form
8 February 2013
Accepted 14 February 2013
Available online 27 February 2013
Keywords:
C- and O-prenylated tetrahydroxystilbenes
O-prenylated cinnamates
Decarbonylative Heck reaction
Selenium dioxide catalysed oxidation
Anticancer activity
(3-methyl-2-butenyl)phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene, IC50 ¼ 0.10
mM.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
cholesterol in blood, stimulate development of bones and carti-
lages, increase blood clotting by blocking of blood clotting in-
hibitors, widen blood vessels, improve circulation and increase
penetration through blood vessel walls [3e5]. Research has
revealed that the E form exhibits more potent activity than the
corresponding Z form across the biological screens including anti-
cancer and anti-oxidant activities [6].
The most well-known and well-characterized stilbene is
resveratrol, (E)-3,40,5-trihydroxystilbene, a natural phytoalexin
found in grapes, red wine, mulberries, peanuts and other food
products, including a rare report of its occurrence in propolis [1].
Resveratrol is synthesized by plants using stilbene synthase
enzyme [1]. Resveratrol has been reported to play a role in the
prevention of heart diseases and diabetes, alteration of eicosanoid
synthesis, modulation of lipid, lipoprotein metabolism and to be
the main protagonist for the so-called “French paradox” [7]. Sci-
entists have attributed this phenomenon to moderate consumption
of anti-inflammatory and anti-oxidant polyphenolic compounds,
Stilbenes, C6eC2eC6, and cinnamates, C6eC3, have been found in
a wide range of plant sources, aromatherapy products and dietary
supplements. The naturally occurring stilbenes and cinnamates
exist in the E form and normally occur as hydroxylated compounds
commonly found in coniferous plants such as fir tree Abies species,
spruce Picea species, pine Pinus species, juniper Juniperus species,
also, in rhubarb Rheum species and mulberry Morus species [1].
Over the years, stilbene compounds have attracted the attention
of many researchers due to their wide range of biological activities
including stimulation of protein synthesis process, increase reten-
tion of nitrogen and accelerate cellular growth and development
[2]. Moreover, the stilbenes have shown to increase the concen-
tration of
a-lipoprotein and decrease the concentration of
* Corresponding author. Tel.: þ612 93512321; fax: þ612 93514391.
E-mail address: colin.duke@sydney.edu.au (C.C. Duke).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.017

416
N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
such as piceatannol and resveratrol, in red wine [8,9]. Piceatannol,
(E)-3,30,40,5-tetrahydroxystilbene, is another naturally occurring
hydroxystilbene found in grapes, sugar cane, berries and peanuts
[10e12]. Piceatannol, a 3-hydroxylated resveratrol, is synthesized
in plants in response to fungal attack, ultraviolet exposure, and
microbial infection [13]. Induction of piceatannol synthesis is also
evident during the ripening of grapes and increases during the
cross-coupling of a benzoyl chloride with a styrene as shown in
Scheme 2. The stilbene was then reacted with 3,3-dimethylallyl
bromide to form O-prenylated tetrahydroxystilbene. The allyl
group subsequently underwent a [1,5]-rearrangement to form the
corresponding C-prenylated tetrahydroxystilbene as shown in
Scheme 3. It is interesting to note that the [1,5]-rearrangement of the
allyl group was rather unexpected. It is known that under Claisen
rearrangement the allyl group migrates primarily to adjacent
unsubstituted ortho position and to para position when both ortho
positions are blocked. However, there are cases that the allyl group
migrates to para position despite the availability of an unsubstituted
ortho position. This behaviour is commonly seen in poly-
hydroxybenzenes or in the presence of a catalyst. In these circum-
stances the allyl group migrates to the most stable position. In
current study, in the presence of magnesium silicate, the allyl group
migrated from O-3 to C-6 position to form the C-prenylated tetra-
hydroxystilbene as shown in Scheme 3. Spectroscopic data of com-
pound 17 was identical with one of the compounds isolated from
Kangaroo Island propolis [21].
In this study, inexpensive and readily accessible starting mate-
rial resorcylic acid (3,5-dihydroxybenzoic acid) 1 was used to
prepare benzoyl chlorides (Scheme 1) as precursors for decarbon-
ylative Heck reaction. It provides the option of introduction of
various ether substituents or different protecting groups for se-
lective introduction of isoprenyl units. As a result, three protected
benzoyl chlorides were successfully formed. The main drawback in
this synthetic route was undesirably low yield of mono-benzyl or
mono-methyl ethers obtained due to formation of di-substituted
compounds despite only one equivalent molar of benzyl bromide
used in the reaction. This resulted in incomplete benzylation of
resorcylic acid.
process of wine production due to b-glucosidase activity of bacteria
[14]. Piceatannol has been shown to exert various pharmacological
effects such as anticancer, anti-inflammatory, anti-oxidant, anti-
aging, anti-diabetic and cardio-protective activities [15e17].
Furthermore, the potency of piceatannol usually surpasses the
resveratrols [15,17e19].
In past decade, natural products containing prenyl groups have
been recognized as valuable biologically active phytochemicals.
Prenylation is a chemical or enzymatic addition of a hydrophobic
isoprenoid side chain to an accepting molecule such as “flavonoid
or polyhydroxy aromatic compound. From a pharmacological point
of view, the prenylated compounds appear to exert more potent or
higher efficacy than the parent compound. These natural products
represent new leads for the development of novel drugs [20].
Furthermore, prenylated aromatic secondary metabolites play a
critical role in the biosynthesis of a wide range of molecules
exerting valuable pharmacological effects across phylogenetically
different classes of living organisms, from bacteria to mammals and
plants. The most commonly reported of these groups are preny-
lated cinnamic acids and their derivatives, while prenylated flavo-
noids are less common, and prenylated benzophenones are rare
natural occurrence. Prenylated piceatannol derivatives are also
examples of rare natural products.
Recently, our research group has discovered a series of novel C-
and O-prenylated piceatannols from propolis in Kangaroo Island of
South Australia [21]. We report here the total synthesis of these
novel prenylated piceatannols. An E-tetrahydroxystilbene was first
formed via decarbonylative Heck reaction of a benzoyl chloride and
a styrene catalysed by an N-heterocyclic carbene generated in situ
by palladium acetate and 1,3-bis(2,6-diisopropylphenyl)imidazoli-
nium chloride. An allyl ether was formed at the O-3 position of the
stilbene using 3,3-dimethylallyl bromide and NaH to form O-pre-
nylated tetrahydroxystilbene. Rearrangement of the isoprenyl unit
from O-3 to C-6 was carried out at elevated temperature (110 ꢀC) in
the presence of magnesium silicate (Florisil) to form the corre-
sponding C-prenylated tetrahydroxystilbene. Synthesis of hydroxyl
derivative of O-prenylated cinnamate was carried out first by base
catalysed allyl ether formation between 3,3-dimethylallyl bromide
and para-hydroxycinnamic acid methyl ester. The methyl group of
the isoprenyl unit was subsequently oxidized using selenium di-
oxide to form a terminal hydroxyl group.
Preparation of the styrene intermediates 10a and 10b was
accomplished by Wittig reaction. Treatments of protected vanillin
or isovanillin with methyltriphenylphosphonium bromide in the
presence of potassium tert-butoxide afforded styrenes 10a or 10b,
respectively, at reasonably high yield (Scheme 1).
Decarbonylative coupling reactions are useful synthetic routes to
form CeC or Ceheteroatom bonds. The main advantage of decar-
bonylative Heck reaction is its outstanding E selectivity, whilst
others generally give a mixture of E/Z-isomers [22]. Overall, mod-
erate to high yields were achieved in all steps except for the removal
of benzyl protecting group in O-prenylated hydroxystilbene. It ap-
pears that the E-conjugated system stabilizes the benzyl protecting
group against removal. This led to hydrogenation of the double bond
seen from the formation of compounds 15a, 15b and 18 (Schemes 2
and 3, Fig. 1). Different protecting groups other than benzyl group
such as silyl ether protective groups could be used to avoid hydro-
genation, thereby improving the yield of both O- and C-prenylated
tetrahydroxystilbenes. Rearrangement of isoprenyl group from O- to
C-position can also be improved by variation of reaction conditions
and catalysts such as silica or alumina particles.
2. Chemistry
In this study we report total synthesis of the novel prenylated E-
tetrahydroxystilbenes including E-1-[5-hydroxy-3-(3-methylbut-2-
enyloxy)phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene (14a), E-
1-[5-hydroxy-3-(3-methylbut-2-enyloxy)phenyl]-2-[3-hydroxy-4-
methoxyphenyl]ethene (14b) and E-1-[5-hydroxy-3-methoxy-2-(3-
methylbut-2-enyloxy)phenyl]-2-[4-hydroxy-3-methoxyphenyl]eth-
ene (17) and their dihydro analogues including 15a, 15b and 18 as
shown in Fig. 1. The prenylated stilbenes 14a and 17 are naturally
occurring piceatannol derivatives isolated from propolis in Kangaroo
Island [21]. Synthesis of the C-prenylated hydroxystilbene was suc-
cessively achieved first by decarbonylative Heck reaction to form an
E-hydroxystilbene. A reaction employing an N-heterocyclic carbene
system generated in situ from 1,3-bis(2,6-diisopropylphenyl)imida-
zolinium chloride with palladium acetate to catalyse a carbonylative
Esters of cinnamic acid and esters of substituted cinnamic acid
are well-known compounds in propolis such as benzyl caffeate,
cinnamyl caffeate, CAPE, isopentenyl cinnamates, isopentyl ferulate
and benzyl ferulate which are isolated from poplar type propolis
[23,24]. In Brazilian propolis, cinnamic acid derivatives are found to
be C-prenylated cinnamic acid derivatives. Examples of these
compounds are artepillin C, baccharin, drupanin, 3-prenyl-4-
hydroxycinnamic acid (PHCA), 2,2-dimethyl-6-carboxyethenyl-8-
prenyl-2H-1-benzopyran (DPB) and 2,2-dimethyl-6-carboxyeth
enyl-2H-1-benzopyran (DCBEN) [25].
The prenylated hydroxylated cinnamic acid derivative (com-
pound 21) synthesized in this study is a novel compound first
isolated from Kangaroo Island propolis. It is an O-prenylated cin-
namic methyl ester with a terminal hydroxyl group attached to one

N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
417
Fig. 1. Structures of prenylated tetrahydroxystilbenes and their dihydro analogues and prenylated cinnamates.
of the vinyl methyl of the isoprenyl unit (compound 21, Fig. 1) and
found in abundance in the propolis. This type of hydroxylation is
not common in plants and could involve very specific enzymatic
activity.
compound 17 was a remarkably potent inhibitor with an IC50
value of 0.10
value of 21.0
m
m
M, followed by 18 (IC50 value of 10
M), 15b (IC50 value of 31.6 M), 14b (IC50 value of
M).
mM), 14a (IC50
m
46.8
mM) and 15a (IC50 value of 63.0
m
The parent compound (compound 20, Fig. 1) has also been
isolated in very small amount from the same propolis. This com-
pound was first reported in 1968 from Australian native plant
Cotula australis (Asteraceae).
It is clear that the conjugated double bond, which bridges the
two aromatic rings, is an important structural feature in the inhi-
bition of cancer cell proliferation. Reduction of this bond of 17 to
give 18 has dramatically reduced (about 100-fold) the potency of
the compound. The role of the double bond is to keep the resorcinol
and catechol rings in a co-planar orientation. It is well documented
that a number of beneficial effects of stilbenes such as anti-oxidant
properties, relate to the resonance effects [26e28]. In fact, the
unpaired electrons are mainly distributed to the O-atom in para
position, double bond, and B-benzene ring. Without the double
bond the resonance effects between the two benzene rings is
totally blocked and co-planarity is lost resulting in major changes in
both electrostatic and steric properties. In addition, C-linkage of
isoprenyl to aromatic moiety appears to enhance significantly the
inhibitory potency of the compound in comparison with O-linkage
3. Pharmacology
The determination of drug safety and cell toxicity measure is a
critical step in pre-clinical drug discovery process. To test the
toxicity of the final compounds towards the K562 leukemic cancer
cell line, MTT cytotoxicity assay was performed.
The MTT antiproliferative assay against leukemia K562 cell line
revealed that these prenylated tetrahydroxystilbenes inhibited cell
growth in a concentration-dependent manner. The inhibitory po-
tency is dependent on their structures. As shown in Table 1
Scheme 1. Preparation of compounds 10a and 10b: (i) anhyd. MeOH, CH3COCl, reflux under N2, 2 h, 2: 90%; (ii) DMF, NaH, N2 atm, 1 equiv. PhCH2Br, rt, 2 h, 3: 42%; (iii) DMF, NaH, N2
atm, MeI, rt, 2 h, 4: 41%; (iv) MeOH, 1M NaOH, N2 atm, rt, 2 h, 5a and 5b: 94%; (v) Ac2O, pyridine, rt, 2 h, 6a and 6b: 94%; (vi) SOCl2, reflux, 4 h, 7a and 7b: quantitative yield; (vii)
DMF, NaH, 1.2 equiv. PhCH2Br, rt, 3 h, 9a and 9b: 92%; (viii) THF, CH3P(Ph)3Br, N2 atm, (CH3)3COK, rt, 2 h, 10a and 10b: 85%.

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N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
Scheme 2. Preparation of compound 15a: (i) xylene, 5% Pd(AcO)2, 1,3-bis-(2,6-diisopropylphenyl)imidazolinium chloride, N-ethylmorpholine, reflux under N2 atm, 18e22 h, 11a:
34%; (ii) MeOHeTHF, 1 M NaOH, N2 atm, rt, 3 h, 12a: 44%; (iii) DMF, NaH, N2 atm, C5H9Br, rt, 3 h, 13a: 43%; (iv) 1,4-cyclohexadieneeEtOH, 10% PdeC, reflux under N2 atm, 14a: 20%,
15a: 15%.
of isoprenyl. Consequently, 14a was considerably less active in
comparison to 17 and 18.
4. Conclusion
It is also noticeable that vanilloid moiety (3-methoxyl-4-
hydroxyphenyl) appears to contribute to inhibitory activities of
the stilbenes in this study. As a result, exchanging the methoxy and
hydroxyl groups from vanilloid to isovanilloid moiety as shown in
14b reduces the inhibition of 14a by approximately three fold. The
cell proliferation assay suggests the variation of the hydroxyl group
pattern of the B phenyl ring, concerning number and position,
resulted in the differences in activity shown. It is of further interest
that hydroxyl group at the 40-position contributes more to cytotoxic
activity than the other hydroxyl group. Therefore, the 40-position in
stilbene provides the most acidic hydrogen and its removal from
the 40-position is highly preferred [26,29]. It is well accepted that
phenol compounds are not active as anti-oxidants unless substi-
tution at either the ortho or para position has increased the electron
density at the hydroxy group and lowered the oxygenehydrogen
bond energy, in effect increasing the reactivity towards the lipid
free radicals. Substitution in phenolic compounds at the meta po-
sition has a rather limited effect, and compounds like resorcinol
with a hydroxyl group in the meta position are thus poor scaven-
gers of free radicals compared to similar compounds with substi-
tution at the ortho or para position. The presence of additional
hydroxy groups, either at the ortho or para position, further in-
creases the antioxidative activity of the compound as intra-
molecular hydrogen bonds stabilize the phenoxyl radicals [30].
The MTT antiproliferative assay against leukemia K562 cell line
revealed significant variation in activity between the tested com-
pounds. Among all the cinnamic acid derivatives tested, the alde-
With careful selection of starting precursors of interest, a
number of derivatives of prenylated mono-, di-, tri-, or poly-
hydroxystilbenes can be prepared for structureeactivity relation-
ship analysis. As described in the Introduction, natural products
that contain prenyl groups are of particular interest in drug dis-
covery and development. From a pharmacological point of view, the
prenylated compounds appear to exert more potent or higher ef-
ficacy than their parent compounds. These natural products thus
represent new leads for the development of novel drugs [20].
5. Experimental
5.1. General procedures
Commercially available reagents were used without purification
unless otherwise stated. Anhydrous tetrahydrofuran (THF) was
freshly distilled from sodium hydride under a nitrogen atmosphere.
Anhydrous dimethylformamide (DMF) was distilled from calcium
hydride under a nitrogen atmosphere. All air- and moisture-
sensitive reactions were carried out under a dry nitrogen atmo-
sphere in oven-dried glass-ware. Analytical thin-layer chromatog-
raphy was carried out on aluminum backed plates coated with silica
gel (Merck, 60, F254) and visualized under UV light at 254 nm.
Chromatographic purification was carried out by short column
vacuum chromatography using Merck silica gel (60H). Melting
points were taken on a hot stage microscope and are uncorrected.
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
in CDCl3 on a Varian-Gemini 400MR (Palo Alto, CA, USA) with
VNMRj 2.2C Varian software. TMS signal was used as reference.
Low- and high-resolution mass spectra were measured by direct
hyde derivative 22 showed the highest activity (IC50 ¼ 57.5
then the hydroxylated compound 21 (IC50 ¼ 70.8 M) with the
parent compound 20 showing lowest activity (IC50 ¼ 97.7 M).
mM)
m
m
Scheme 3. Preparation of compounds 17 and 18: (i) Toluene, 10ꢁ equiv. Florisil, reflux under N2 atm, 4 h, 16: 59%; (ii) 1,4-cyclohexadieneeEtOH, 10% PdeC, reflux under N2 atm, 17:
65%, 18: 25%.

N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
419
phase to give 11a as yellowish oil (0.43 g, 34%). 1H NMR (400 MHz,
Table 1
Cytotoxicity activities of compounds 14aeb, 15aeb,
17, 18 and 20e22. Antiproliferative activity was
determined by MTT assay as shown in Experimental
part 5.2. All data are presented as mean values of two
independent experiments. Coefficients of variation
were <10%. IC50: concentration of the tested com-
pound that inhibits 50% of cell growth.
CDCl3)
d
(ppm) 7.45e7.28 (10 H, m, 2 ꢁ benzyl Ph), 7.06 (1H, d,
J ¼ 1.8 Hz, H-20), 7.02 (1H, d, J ¼ 16.4 Hz, H-
a
), 6.97 (1H, dd, J ¼ 6.4,
b), 6.87e6.85 (3H, m, H-
1.8 Hz, H-60), 6.89 (1H, d, J ¼ 16.0 Hz, H-
2,50,6), 6.62 (1H, t, J ¼ 2.2 Hz, H-4), 5.18 (2H, s, benzyl CH2), 5.07 (2H,
s, benzyl CH2), 3.94 (3H, s, OCH3), 2.30 (3H, s, COCH3); 13C NMR
(100 MHz, CDCl3)
d (ppm) 169.39 (CO), 159.78, 151.81, 149.79,
Compound
IC50 (mM)
148.32, 139.82, 137.00, 136.57, 130.48, 129.78, 128.63 (2C), 128.58
(2C), 128.10, 127.88, 127.55 (2C), 127.24 (2C), 126.02, 119.98, 113.95,
112.02, 110.50, 109.48, 107.28, 71.00 (benzyl CH2), 70.27 (benzyl
CH2), 56.02 (30-OCH3), 21.19 (CH3CO); MS (ESI) m/z (%): 503
([M þ Na]þ, 98), 481 ([M þ H]þ, 100), 412 (19), 408 (45), 241 (17);
HRMS (ESI) (m/z): calcd for C31H28O5 [M þ Na]þ 503.1829, found
503.1828.
14a
14b
15a
15b
17
18
20
21
22
21.0
46.8
63.0
31.6
0.10
10.0
97.7
70.8
57.5
5.3. Preparation of E-1-[3-acetoxy-5-benzyloxyphenyl]-2-[3-
benzyloxy-4-methoxyphenyl]ethene (11b)
infusion electrospray ionization tandem mass spectrometry and
reported as mass to charge ratio (m/z) and related intensity (%).
Low-resolution ESI-MS was recorded on a Thermo-Finnigan TSQ
7000 (LC-MS/MS system). High resolution ESI-MS was measured on
a Bruker Daltonics Apex Ultra Fourier Transform Ion Cyclotron
Resonance 7 T Mass Spectrometer.
Preparation of intermediates such as 3-acetoxy-5-
benzyloxybenzoyl chloride (7a) and 3-acetoxy-5-methoxybenzoyl
chloride (7b), 4-benzyloxy-3-methoxy-1-ethenylbenzene (10a)
and 3-benzyloxy-4-methoxy-1-ethenylbenzene (10b) for the syn-
thetic routes as described in Scheme 1, and methyl (E)-4-(30-
methylbut-2-enyloxy)cinnamate (20) for Scheme 4 were carried
out by methods as described in literature. For details of these
syntheses refer to Supplementary data.
The title compound was prepared similar to that described for
11a with the condensation of 7a and 10b to give 11b. The product
was recrystallised from a mixture of hexane/toluene (2:1) to afford
yellowish needle crystals (0.4 g, 32%), mp 133e134 ꢀC. 1H NMR
(400 MHz, CDCl3)
d
(ppm) 7.49e7.29 (10H, m, 2 ꢁ benzyl Ph), 7.07
(1H, d, J ¼ 2.0 Hz, H-20), 7.05 (1H, dd, J ¼ 8.4, 2.0 Hz, H-60), 6.97 (1H,
d, J ¼ 16.2 Hz, H- ), 6.95 (1H, t, J ¼ 1.8 Hz, H-6), 6.89 (1H, d,
a
J ¼ 8.4 Hz, H-50), 6.84 (1H, t, J ¼ 1.8 Hz, H-2), 6.82 (1H, d, J ¼ 16.2 Hz,
H-b
), 6.61 (1H, t, J ¼ 2.2 Hz, H-4), 5.19 (2H, s, benzyl CH2), 5.07 (2H,
s, benzyl CH2), 3.90 (3H, s, OCH3), 2.30 (3H, s, COCH3); 13C NMR
(100 MHz, CDCl3) (ppm) 169.39 (CO), 159.76, 151.80, 149.90,
d
148.34, 139.83, 137.05, 136.58, 129.94, 129.78, 128.64 (2C), 128.60
(2C), 128.11, 127.94, 127.58 (2C), 127.38 (2C), 125.88, 120.59, 112.00,
111.91, 111.81, 110.52, 107.22, 71.16, 70.27, 56.06 (40-OCH3), 21.18
(CH3CO); MS (ESI) m/z (%): 503 ([M þ Na]þ, 98), 481 ([M þ H]þ, 32),
393 (58), 323 (100); HRMS (ESI) (m/z): calcd for C31H28O5
[M þ Na]þ 503.1829, found 503.1832.
Refer to Supplementary data for plotted 1H NMR and 13C NMR
spectra of the key compounds including E-1-[5-hydroxy-3-(3-
methylbut-2-enyloxy)-phenyl]-2-[3-hydroxy-4-methoxyphenyl]
ethene (14b), 1-[5-hydroxy-3-(3-methylbut-2-enyloxy)-phenyl]-2-
[4-hydroxy-3-methoxyphenyl]ethane (15a), 1-[5-hydroxy-3-(3-
methylbut-2-enyloxy)-phenyl]-2-[3-hydroxy-4-methoxyphenyl]
ethane (15b), 1-[5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)
phenyl]-2-[4-hydroxy-3-methoxyphenyl]-ethane (18) and methyl
(E)-4-(30-carbonylbut-(E)-20-enyloxy)cinnamate (22).
5.4. Preparation of E-1-[3-acetoxy-5-methoxyphenyl]-2-[4-
benzyloxy-3-methoxyphenyl]-ethene (11c)
The title compound was prepared similar to that described for
11a with the condensation of 7b and 10a to give 11c. The product
was purified on silica gel using hexane/ethyl acetate (3:1) as mo-
bile phase to give 11c as off-white solid (0.45 g, 43%), mp 104e
5.2. Preparation of E-1-[3-acetoxy-5-benzyloxyphenyl]-2-[4-
benzyloxy-3-methoxyphenyl]-ethene (11a)
106 ꢀC. 1H NMR (400 MHz, CDCl3)
d 7.45e7.28 (5H, m, phenyl
5.2.1. General method
Ph), 7.07 (1H, d, J ¼ 2.4 Hz, H-20), 7.03 (1H, d, J ¼ 16.2 Hz, H-
a
), 6.98
To a solution of 1,3-bis(2,6-diisopropylphenyl)imidazolinium
chloride (0.093 g, 10%, 0.217 mmol) in xylene (3 mL) under nitrogen
was added palladium acetate (0.048 g, 10%, 0.217 mmol). The
mixture was stirred for 30 min at room temperature followed by
the addition of the solution of 7a (0.7 g, 2.17 mmol) in xylene
(2 mL), N-ethylmorpholine (0.04 mL, 0.316 mmol) and the solution
of styrene 10a (0.627 g, 2.61 mmol) in xylene (3 mL). The resulting
mixture was refluxed at 130 ꢀC for 18e22 h and monitored by TLC.
The solvent was evaporated under reduced pressure. The residue
was purified on silica gel using hexane/ethyl acetate (3:1) as mobile
(1H, dd, J ¼ 8.4, 2.0 Hz, H-60), 6.90 (1H, d, J ¼ 16.8 Hz, H-
b), 6.88e
6.84 (3H, m, H-2,50,6), 6.53 (1H, d, J ¼ 2.0, H-4), 5.18 (2H, s, benzyl
CH2), 3.95 (3H, s, OCH3), 3.83 (3H, s, OCH3), 2.31 (3H, s, COCH3); 13
C
NMR (100 MHz, CDCl3) d 169.41 (CO), 160.58, 151.83, 149.78, 148.31,
139.76, 137.00, 130.49, 129.72, 128.58 (2C), 127.88, 127.24 (2C),
126.07, 119.97, 113.94, 111.73, 109.67, 109.46, 106.54, 71.01, 56.02
(30-OCH3), 55.50 (5-OCH3), 21.18 (CH3CO); MS (ESI) m/z (%): 405
([M þ H]þ, 57), 363 (93), 315 (62), 273 (100), 239 (54), 91 (30);
HRMS (ESI) (m/z): calcd for C25H24O5 [M þ H]þ 405.1696, found
405.1695.
Scheme 4. Preparation of compounds 20e22: (i) anhyd. MeOH, CH3COCl, N2 atm, rt, 24 h; (ii) anhyd. MeOH, NaH, N2 atm, C5H9Br, rt, 3 h, 20: 26%; (iii). DCM, SeO2, TBHP, rt, 48 h, 21:
38%, 22: 12%.

420
N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
5.5. Preparation of E-1-[5-benzyloxy-3-hydroxyphenyl]-2-[4-
benzyloxy-3-methoxyphenyl]-ethene (12a)
100), 363 ([M þ H]þ, 18); 323 (40); HRMS (ESI) (m/z): calcd for
C23H22O4 [M þ H]þ 363.1591, found 363.1592.
5.5.1. General method
5.8. Preparation of E-1-[5-benzyloxy-3-(3-methylbut-2-enyloxy)
To the solution of 11a (0.04 g, 0.083 mmol) in a mixture of
equal part of methanol, tetrahydrofuran and water (9 mL) at 0 ꢀC
under nitrogen was added sodium hydroxide (0.02 g, 0.35 mmol).
The reaction mixture was warmed up to room temperature and
stirred for further 3 h. The product was acidified with 0.1 M hy-
drochloric acid (5 mL) then extracted with ethyl acetate
(3 ꢁ 30 mL). The combined organic layers were washed with 20%
aqueous sodium chloride (10 mL), dried over sodium sulfate and
evaporated under reduced pressure. The residue was purified on
silica gel using hexane/ethyl acetate (2:1), followed by recrystal-
lisation from a mixture of hexaneeethyl acetate (2:1) to afford
12a as an off-white powder (0.016 g, 44%), mp 114e115 ꢀC. 1H
phenyl]-2-[4-benzyloxy-3-methoxyphenyl]ethene (13a)
5.8.1. General method
To the suspension of sodium hydride (60% dispersion in mineral
oil, 0.005 g, 0.125 mmol) in anhydrous N,N-dimethylformamide
(5 mL) at 0 ꢀC under nitrogen was added dropwise the solution of
12a (0.04 g, 0.091 mmol) in N,N-dimethylformamide (3 mL) fol-
lowed by the addition of 3,3-dimethylallyl bromide (0.011 mL,
0.091 mmol) dropwise [31]. The resulting mixture was stirred at
room temperature for 2e3 h, quenched with cold distilled water
(5 mL), acidified with cold 0.1 M hydrochloric acid (5 mL) and
extracted with diethyl ether (3 ꢁ 10 mL). The combined organic
layers were washed with distilled water (2 ꢁ 10 mL) and dried over
sodium sulfate. The solvent was evaporated under reduced pres-
sure. The residue was purified on silica gel using hexane/ethyl ac-
etate (2:1) as mobile phase to afford 13a as yellowish oil (0.02 g,
NMR (400 MHz, Methanol-d4)
d (ppm) 7.45e7.28 (10H, m,
2 ꢁ benzyl Ph), 7.06 (1H, d, J ¼ 2.0 Hz, H-20), 7.00 (1H, d,
J ¼ 16.2 Hz, H-
a
), 6.98 (1H, dd, J ¼ 8.4, 2.0 Hz, H-60), 6.87 (1H, d,
J ¼ 8.4 Hz, H-50), 6.86 (1H, d, J ¼ 16.2 Hz, H-
b), 6.71 (1H, t,
J ¼ 2.0 Hz, H-6), 6.59 (1H, t, J ¼ 2.0 Hz, H-2), 6.38 (1H, t, J ¼ 2.2 Hz,
H-4), 5.18 (2H, s, benzyl CH2), 5.07 (2H, s, benzyl CH2), 4.82 (1H, s
br, OH), 3.95 (3H, s, OCH3); 13C NMR (100 MHz, Methanol-d4)
yield 43%). 1H NMR (400 MHz, CDCl3)
d (ppm) 7.46e7.28 (10H, m,
2 ꢁ benzyl Ph), 7.07 (1 H, d, J ¼ 2.0 Hz, H-20), 7.01 (1H, d, J ¼ 16.4 Hz,
H-
a
), 6.96 (1H, d, J ¼ 8.0, 2.0 Hz, H-60), 6.90 (1H, d, J ¼ 16.4 Hz, H-
b),
d
(ppm) 160.29, 156.78, 149.76, 148.19, 139.92, 136.98, 136.84,
6.87 (1H, d, J ¼ 8.4 Hz, H-50), 6.73 (1H, t, J ¼ 2.0 Hz, H-6), 6.68 (1H, t,
J ¼ 2.0 Hz, H-2), 6.48 (1H, t, J ¼ 2.2 Hz, H-4), 5.52e5.49 (1H, m, H-
200), 5.17 (2H, s, benzyl CH2), 5.07 (2H, s, benzyl CH2), 4.52 (2H, d,
J ¼ 6.8 Hz, H-100), 3.95 (3H, s, OCH3), 1.80 (3H, d, J ¼ 0.8 Hz, H-400),
130.65, 129.20, 128.61 (2C), 128.57 (2C), 128.02, 127.88, 127.49
(2C), 127.26 (2C), 126.54, 119.94, 113.96, 109.45, 105.97, 105.69,
101.54, 71.02 (benzyl CH2), 70.01 (benzyl CH2), 56.03 (30-OCH3);
MS (ESI) m/z (%): 461 ([M þ Na]þ, 100), 439 ([M þ H]þ, 18); HRMS
1.76 (3H, d, J ¼ 0.8 Hz, H-500); 13C NMR (100 MHz, CDCl3)
d (ppm)
(ESI) (m/z): calcd for C29H26O4 [M
439.1908.
þ
H]þ 439.1904, found
160.21, 160.12, 149.78, 148.14, 139.49, 138.35, 137.03, 136.94, 130.76
(C-b), 128.94, 128.59 (2C), 128.56 (2C), 127.94, 127.86, 127.54 (2C),
127.24 (2C), 126.97 (C-a), 119.87, 119.53, 113.98, 109.43, 105.38,
5.6. Preparation of E-1-[5-benzyloxy-3-hydroxyphenyl]-2-[3-
benzyloxy-4-methoxyphenyl]ethene (12b)
105.32, 101.14, 71.01 (benzyl CH2), 70.09 (benzyl CH2), 64.84 (C-100),
56.03 (30-OCH3), 25.85 (C-400), 18.22 (C-500); MS (ESI) m/z (%): 529
([M þ Na]þ, 100), 507 ([M þ H]þ, 30); HRMS (ESI) (m/z): calcd for
C34H34O4 [M þ H]þ 507.2530, found 507.2528.
The title compound was prepared similar to that described for
12a above with the use of 11b to give 12b as colorless solid (0.017 g,
yield 46%), mp 117e119 ꢀC. 1H NMR (400 MHz, Methanol-d4)
5.9. Preparation of E-1-[5-benzyloxy-3-(3-methylbut-2-enyloxy)
d
(ppm) 7.49e7.30 (10H, m, 2 ꢁ benzyl Ph), 7.07 (1H, d, J ¼ 2.0 Hz, H-
phenyl]-2-[3-benzyloxy-4-methoxyphenyl]ethene (13b)
20), 7.06 (1H, dd, J ¼ 8.2, 2.0 Hz, H-60), 6.96 (1H, d, J ¼ 16.2 Hz, H-
a),
6.87 (1H, d, J ¼ 8.2 Hz, H-50), 6.79 (1H, d, J ¼ 16.2 Hz, H-
b
), 6.99 (1H,
The title compound was prepared similar to that described for
t, J ¼ 1.8 Hz, H-6), 6.57 (1H, t, J ¼ 1.8 Hz, H-2), 6.38 (1H, t, J ¼ 2.2 Hz,
13a with the use of 12b to give 13b as off-white solid (0.022 g, yield
H-4), 5.19 (2H, s, benzyl CH2), 5.07 (2H, s, benzyl CH2), 3.90 (3H, s,
47%), mp 88e90 ꢀC. 1H NMR (400 MHz, CDCl3)
d (ppm) 7.49e7.32
OCH3). 13C NMR (ppm) (100 MHz, Methanol-d4)
d
160.20, 158.30,
(10H, m, 2 ꢁ benzyl Ph), 7.08 (1H, d, J ¼ 2.0 Hz, H-20), 7.06 (1H,
149.68, 148.25, 139.70, 137.41, 137.31, 130.62, 128.16, 128.07 (2C),
128.05 (2C), 127.52, 127.41 (3C), 127.15 (2C), 126.62, 120.35, 111.95
(2C), 105.67, 104.09, 101.11, 70.88 (benzyl CH2), 69.56 (benzyl CH2),
55.10 (40-OCH3); MS (ESI) m/z (%): 461 ([M þ Na]þ, 100), 439
([M þ H]þ, 30); HRMS (ESI) (m/z): calcd for C29H26O4 [M þ H]þ
439.1904, found 439.1907.
dd, J ¼ 8.4, 2.0 Hz, H-60), 6.97 (1H, d, J ¼ 16.4 Hz, H-
a), 6.89 (1H, d,
J ¼ 8.4 Hz, H-50), 6.82 (1H, d, J ¼ 16.4 Hz, H-
), 6.71 (1H, t, J ¼ 1.6 Hz,
b
H-6), 6.66 (1H, t, J ¼ 1.6 Hz, H-2), 6.47 (1H, t, J ¼ 2.0 Hz, H-4), 5.53e
5.49 (1H, m, H-200), 5.20 (2H, s, benzyl CH2), 5.07 (2H, s, benzyl CH2),
4.52 (2H, d, J ¼ 6.4 Hz, H-100), 3.91 (3H, s, OCH3), 1.80 (3H, d,
J ¼ 0.8 Hz, H-400), 1.75 (3H, d, J ¼ 0.8 Hz, H-500); 13C NMR (100 MHz,
CDCl3)
137.67, 130.91 (C-
128.05 (2C), 127.50 (C-
d (ppm) 161.04, 160.95, 150.51, 149.11, 140.24, 139.03, 137.81,
5.7. Preparation of E-1-[3-hydroxy-5-methoxyphenyl]-2-[4-
benzyloxy-3-methoxyphenyl]ethene (12c)
b
), 129.57, 129.27 (4C), 128.66, 128.58, 128.22 (2C),
), 121.10, 120.20, 112.48, 112.40, 105.95,
a
105.86, 101.60, 71.55 (benzyl CH2), 70.47 (benzyl CH2), 65.18 (C-100),
56.36 (40-OCH3), 26.00 (C-400), 18.33 (C-500); MS (ESI) m/z (%): 529
([M þ Na]þ, 88), 507 ([M þ H]þ, 18), 323 (100); HRMS (ESI) (m/z):
calcd for C34H34O4 [M þ H]þ 507.2530, found 507.2533.
The title compound was prepared similar to that described for
12a with the use of 11c to prepare 12c as yellowish solid (0.019 g,
yield 53%), mp 110e112 ꢀC. 1H NMR (400 MHz, CDCl3)
d (ppm)
7.45e7.28 (5H, m, benzyl Ph), 7.07 (1H, d, J ¼ 2.0 Hz, H-20), 7.00 (1H,
d, J ¼ 16.6 Hz, H-
a
), 6.98 (1H, dd, J ¼ 8.0, 2.0 Hz, H-60), 6.87 (1H, d,
5.10. Preparation of E-1-[3-(3-methylbut-2-enyloxy)-5-
methoxyphenyl]-2-[4-benzyloxy-3-methoxyphenyl]ethene (13c)
J ¼ 16.8 Hz, H-
b
), 6.85 (1H, d, J ¼ 8.4 Hz, H-50), 6.62 (1H, t, J ¼ 1.8 Hz,
H-6), 6.57 (1H, t, J ¼ 1.8 Hz, H-2), 6.31 (1H, t, J ¼ 2.2 Hz, H-4), 5.17
(2H, s, benzyl CH2), 4.94 (1H, s, OH), 3.94 (3H, s, OCH3), 3.81 (3H, s,
The title compound was prepared similar to that described for
OCH3); 13C NMR (100 MHz, CDCl3)
d
(ppm) 161.08, 156.86, 149.73,
13a with the use of 12c to give 13c as off-white solid (0.011 g, yield
148.16, 139.85, 136.96, 130.69, 129.11, 128.57 (2C), 127.90, 127.29
(2C), 126.61, 119.95, 113.97, 109.47, 105.72, 104.73, 100.72, 71.0þ3,
56.03 (30-OCH3), 55.36 (5-OCH3); MS (ESI) m/z (%): 385 ([M þ Na] ,
46%), mp 66e69 ꢀC. 1H NMR (400 MHz, CDCl3)
d 7.45e7.28 (5H, m,
benzyl Ph), 7.08 (1H, d, J ¼ 2.0 Hz, H-20), 7.02 (1H, d, J ¼ 16.0 Hz, H-
a
), 6.98 (1H, dd, J ¼ 8.0, 2.0 Hz, H-60), 6.90 (1H, d, J ¼ 16.0 Hz, H-
b),

N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
421
6.87 (1H, d, J ¼ 8.4 Hz, H-50), 6.67 (1H, t, J ¼ 1.8 Hz, H-6), 6.64 (1H, t,
J ¼ 1.8 Hz, H-2), 6.40 (1H, t, J ¼ 2.2 Hz, H-4), 5.53e5.49 (1H, m, H-
200), 5.17 (2H, s, benzyl CH2), 4.53 (2H, d, J ¼ 6.8 Hz, H-100), 3.96 (3H,
s, OCH3), 3.81 (3H, s, OCH3), 1.81 (3H, d, J ¼ 0.8 Hz, H-400), 1.76 (3H, d,
6.34 (1H, t, J ¼ 1.9 Hz, H-6), 6.26 (1H, t, J ¼ 1.9 Hz, H-2), 6.24 (1H, t,
J ¼ 1.8 Hz, H-4), 5.46e5.70 (1 H, m, H-200), 5.46 (1H, s, OH), 4.68 (1H,
s, OH), 4.46 (2H, d, J ¼ 6.8 Hz, H-100), 3.84 (3H, s, OCH3), 2.85e2.76
(4H, m, CH2CH2),1.80 (3H, d, J ¼ 0.4 Hz, H-400), 1.73 (3H, d, J ¼ 0.4 Hz,
J ¼ 0.8 Hz, H-500); 13C NMR (100 MHz, CDCl3)
d
160.91, 160.23,
H-500); 13C NMR (100 MHz, CDCl3)
d (ppm) 160.11, 156.44, 146.22,
149.76, 148.14, 139.46, 138.35, 137.04, 130.78 (C-
b), 128.90, 128.56
144.46, 143.73, 138.23, 133.61, 120.95, 119.55, 114.15, 111.10, 107.89,
107.58, 99.62, 64.72 (C-100), 55.84 (30-OCH3), 38.29 (CH2CH2), 37.23
(CH2CH2), 25.84 (C-400), 18.18 (C-500); MS (ESI) m/z (%): 351
([M þ Na]þ, 100); HRMS (ESI) (m/z): calcd for C20H24O4 [M þ Na]þ
351.1566, found 351.1566.
(2C), 127.87, 127.25 (2C), 127.02 (C-a), 119.86, 119.56, 113.98, 109.44,
104.96, 104.49, 100.37, 71.02 (benzyl CH2), 64.82 (C-100), 56.04 (30-
OCH3), 55.36 (5-OCH3), 25.86 (C-400), 18.22 (C-500); MS (ESI) m/z (%):
453 ([M þ Na]þ, 85); HRMS (ESI) (m/z): calcd for C28H30O4
[M þ Na]þ 453.2036, found 453.2036.
5.14. 1-[5-hydroxy-3-(3-methylbut-2-enyloxy)phenyl]-2-[3-
5.11. Preparation of E-1-[5-hydroxy-3-(3-methylbut-2-enyloxy)
hydroxy-4-methoxyphenyl]ethane (15b)
phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene (14a)
The title compound was obtained as a yellowish oil (0.0027 g,
5.11.1. General method
18%). 1H NMR (400 MHz, CDCl3)
d
(ppm) 6.79 (1H, d, J ¼ 2.0 Hz, H-
To the solution of 13a (0.02 g, 0.035 mmol) in absolute ethanol
(8 mL), was added 1,4-cyclohexadiene (3 mL, 0.030 mmol) and
palladium on charcoal (10%, 0.002 g). The reaction mixture was
refluxed with stirring under nitrogen, for 1e2 h and monitored by
TLC. The product was filtered and evaporated under reduced
pressure to give a yellowish residue which was purified by pre-
parative HPLC on a silica gel column, eluted with hexane/ethyl
acetate (2:1) to afford 14a as yellowish oil (0.0025 g, yield 20%). 1H
20), 6.77 (1H, d, J ¼ 8.0 Hz, H-50), 6.66 (1H, dd, J ¼ 8.0, 2.0 Hz, H-60),
6.35 (1H, t, J ¼ 2.0 Hz, H-6), 6.27e6.25 (2H, m, H-2, 4), 5.56 (1H, s,
OH), 5.50e5.46 (1H, m, H-200), 4.74 (1H, s, OH), 4.46 (2H, d,
J ¼ 6.8 Hz, H-100), 3.88 (3H, s, OCH3), 2.80e2.78 (4H, m, CH2CH2),
1.80 (3H, d, J ¼ 1.2 Hz, H-400), 1.74 (3H, d, J ¼ 1.2 Hz, H-500); 13C NMR
(100 MHz, CDCl3)
d (ppm) 160.11, 156.42, 145.41, 144.80, 144.51,
138.18, 135.07, 119.73, 119.59, 114.59, 110.55, 107.81, 107.49, 99.64,
64.73 (C-100), 55.00 (40-OCH3), 38.05 (CH2CH2), 36.89 (CH2CH2),
25.83 (C-400), 18.18 (C-500); MS (ESI) m/z (%): 351 ([M þ Na]þ, 100);
HRMS (ESI) (m/z) calcd for C20H24O4 [M þ Na]þ 351.1566, found
351.1566.
NMR (400 MHz, CDCl3)
d
(ppm) 7.02 (1H, d, J ¼ 2.0 Hz, H-20), 7.00
(1H, d, J ¼ 16.4 Hz, H-
a
), 6.99 (1H, d, J ¼ 8.0 Hz, H-50), 6.91 (1H, dd,
J ¼ 8.0, 2.0 Hz, H-60), 6.85 (1H, d, J ¼ 16.4 Hz, H-
b), 6.64 (1H, t,
J ¼ 1.6 Hz, H-2), 6.56 (1H, t, J ¼ 1.6 Hz, H-6), 6.33 (1H, t, J ¼ 2.0 Hz, H-
4), 5.53e5.48 (1H, m, H-200), 4.52 (2H, d, J ¼ 6.8 Hz, H-100), 3.95 (3H,
s, OCH3), 1.82 (3H, d, J ¼ 1.0 Hz, H-400), 1.76 (3H, d, J ¼ 1.0 Hz, H-500);
5.15. Preparation of E-1-[5-hydroxy-3-methoxy-2-(3-methyl-2-
butenyl)phenyl]-2-[4-benzyloxy-3-methoxyphenyl]ethene (16)
13C NMR (100 MHz, CDCl3)
d (ppm) 160.38, 156.70, 146.67, 145.69,
139.87, 138.37, 129.73, 129.29 (C-
b
), 126.15 (C-
a
), 120.63, 119.49,
To a solution of 13c (0.024 g, 0.061 mmol) in toluene (30 mL)
was added 60e100 mesh Florisil (0.24 g, 10ꢁ). The reaction mixture
was refluxed at 110 ꢀC under nitrogen, for 4 h, then filtered and
evaporated under reduced pressure to give a brownish residue
which was purified on silica gel using hexaneeethyl acetate (2:1) to
afford a yellowish solid. The product was recrystallised from a
mixture of hexane/ethyl acetate (3:1) affording 16 as an off-white
powder (0.012 g, 59%), mp 161e162 ꢀC. 1H NMR (400 MHz, CDCl3)
114.54, 108.24, 105.58, 105.38, 101.29, 64.85 (C-100), 55.92 (30-OCH3),
25.85 (C-400), 18.22 (C-500); MS (ESI) m/z (%): 325 ([M ꢂ H], 100);
HRMS (ESI) (m/z): calcd for C20H22O4 [M þ H]þ 327.1590, found
327.1588.
5.12. Preparation of E-1-[5-hydroxy-3-(3-methylbut-2-enyloxy)
phenyl]-2-[3-hydroxy-4-methoxyphenyl]ethene (14b)
d
(ppm) 7.46e7.28 (5H, m, benzyl Ph), 7.19 (1H, d, J ¼ 16.0 Hz, H-
a),
The title compound was prepared similar to that described for
14a above with the use of 13b to give 14b as yellowish oil (0.003 g,
7.06 (1H, d, J ¼ 2.0 Hz, H-20), 6.97 (1H, dd, J ¼ 8.4, 2.0 Hz, H-60), 6.88
(1H, d, J ¼ 16.0 Hz, H-
b
), 6.87 (1H, d, J ¼ 8.4 Hz, H-50), 6.66 (1H, t,
yield 21%). 1H NMR (400 MHz, CDCl3)
d
(ppm) 7.13 (1H, d, J ¼ 2.0 Hz,
J ¼ 2.4 Hz, H-6), 6.40 (1H, t, J ¼ 2.4 Hz, H-4), 5.18 (2H, s, benzyl CH2),
5.13e5.09 (1H, m, H-200), 4.64 (1H, s, OH), 3.94 (3H, s, OCH3), 3.80
(3H, s, OCH3), 3.42 (2H, d, J ¼ 6.4 Hz, H-100), 1.80 (3H, d, J ¼ 1.2 Hz, H-
H-20), 6.98 (1H, d, J ¼ 16.0 Hz, H-
), 6.97 (1H, dd, J ¼ 8.0, 2.2 Hz, H-
a
60), 6.86 (1H, d, J ¼ 8.0 Hz, H-50), 6.86 (1H, d, J ¼ 16.0 Hz, H-
b), 6.64
(1H, t, J ¼ 2.0 Hz, H-2), 6.57 (1H, t, J ¼ 2.0 Hz, H-6), 6.33 (1H, t,
J ¼ 2.0 Hz, H-4), 5.60 (1H, s, OH), 5.52e5.48 (1H, m, H-200), 4.77 (1H,
s, OH), 4.52 (2H, d, J ¼ 6.8 Hz, H-100), 3.91 (3H, s, OCH3), 1.82 (3H, d,
J ¼ 0.8 Hz, H-400), 1.76 (3H, d, J ¼ 0.8 Hz, H-500); 13C NMR (100 MHz,
400),1.68 (3H, d, J ¼ 1.2 Hz, H-500); 13C NMR (100 MHz, CDCl3)
d
(ppm)
),
a), 123.61, 120.78,
158.56, 154.35, 149.76, 148.07, 138.11, 137.04, 131.21, 130.63 (C-
b
130.24, 128.56 (2C), 127.86, 127.22 (2C), 124.73 (C-
119.84, 113.99, 109.42, 103.88, 98.21, 71.03 (benzyl CH2), 55.97 (30-
OCH3), 55.68 (3-OCH3), 25.77 (C-100), 24.46 (C-400), 17.98 (C-500); MS
(ESI) m/z (%): 453 ([M þ Na]þ, 100), 431 ([M þ H]þ, 75); HRMS (ESI)
(m/z): calcd for C28H30O4 [M þ H]þ 431.2217, found 431.2217.
CDCl3)
d (ppm) 160.37, 156.69, 146.51, 145.75, 139.83, 138.36, 130.87,
128.92 (C-
b
), 126.78 (C-a), 119.51, 119.40, 111.84, 110.62, 105.66,
105.48, 101.33, 65.18 (C-100), 56.36 (40-OCH3), 26.00 (C-400), 18.33 (C-
500); MS (ESI) m/z (%): 349 ([M þ Na]þ, 24); HRMS (ESI) (m/z) calcd
for C20H22O4 [M þ Na]þ 349.1410, found 349.1408.
5.16. Preparation of E-1-[5-hydroxy-3-methoxy-2-(3-methyl-2-
butenyl)phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene (17)
Prenylated tetrahydroxystilbenes 15a and 15b were formed as
side products as the result of saturation of the double bond of 13a
and 13b, respectively. These compounds were purified and char-
acterised as follows.
The title compound was prepared similar to that described for
14a with the use of 16 to give 17 as yellowish oil (0.013 g, 65%) after
purification by silica gel column chromatography using hexanee
ethyl acetate (2:1) and HPLC using hexaneeisopropanol (2:1). 1H
5.13. 1-[5-hydroxy-3-(3-methylbut-2-enyloxy)phenyl]-2-[4-
hydroxy-3-methoxyphenyl]ethane (15a)
NMR (400 MHz, CDCl3)
d
(ppm) 7.17 (1H, d, J ¼ 16.0 Hz, H-
a), 7.01
(1H, dd, J ¼ 2.0 Hz, H-20), 7.0 (1H, d, J ¼ 8.2, 2.0 Hz, H-60), 6.91 (1H, d,
The title compound was obtained as a yellowish oil (0.002 g,
J ¼ 8.0 Hz, H-50), 6.87 (1H, d, J ¼ 16.0 Hz, H-
), 6.66 (1H, t, J ¼ 2.4 Hz,
b
15%). 1H NMR (400 MHz, CDCl3)
d
(ppm) 6.84 (1H, d, J ¼ 2.0 Hz, H-
H-6), 6.36 (1H, t, J ¼ 2.4 Hz, H-4), 5.66 (1H, s, OH), 5.15e5.09 (1H, m,
20), 6.69 (1H, dd, J ¼ 8.0, 2.0 Hz, H-60), 6.62 (1H, d, J ¼ 8.0 Hz, H-50),
H-200), 4.64 (1H, s, OH), 3.94 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.42

422
N. Koolaji et al. / European Journal of Medicinal Chemistry 63 (2013) 415e422
(2H, d, J ¼ 6.8 Hz, H-100), 1.81 (3H, d, J ¼ 1.0 Hz, H-400), 1.68 (3H, d,
72 h at 37 ꢀC in a humidified incubator with 5% of carbon dioxide.
Data is the average of two independent assays each performed in
duplicate. Two concentrations were used for each compound in
order to estimate the IC50 value. DMSO was used as vehicle at the
final concentration of 0.5% and at this concentration showed no
effect on cell growth (data not shown).
J ¼ 1.0 Hz, H-500); 13C NMR (100 MHz, CDCl3)
d (ppm) 158.57, 154.37,
146.68, 145.60, 138.18, 130.61, 130.44, 130.27, 124.28, 123.67, 120.73,
120.58, 114.55, 108.26, 103.88, 98.18, 55.88 (30-OCH3), 55.71 (3-
OCH3), 25.79 (C-100), 24.48 (C-400), 17.98 (C-500); MS (ESI) m/z (%):
339 ([M ꢂ H], 100); HRMS (ESI) (m/z): calcd for C21H24O4 [M þ Na]þ
363.1566, found 363.1566.
Acknowledgements
5.17. 1-[5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)phenyl]-2-
[4-hydroxy-3-methoxyphenyl]ethane (18)
Financial support was provided by the Australian National
Health and Medical Research Council. The authors wish to thank Mr
Bruce Tattam for his expertise and assistance with mass spectral
analyses.
The title compound was formed as side product as the result of
saturation of the double bond of 17. The product was obtained as
yellowish oil (0.005 g, 25%). 1H NMR (400 MHz, CDCl3)
d (ppm) 6.86
(1H, d, J ¼ 8.0 Hz, H-50), 6.70 (1H, dd, J ¼ 2.0 Hz, H-20), 6.64 (1H, d,
J ¼ 8.0, 2.0 Hz, H-60), 6.30 (1H, d, J ¼ 2.4 Hz, H-6), 6.24 (1H, d,
J ¼ 2.4 Hz, H-4), 5.48 (1H, s, OH), 5.07e5.03 (1H, m, H-200), 4.67 (1H,
s, OH), 3.85 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.28 (2H, d, J ¼ 6.4 Hz,
H-100), 2.82e2.73 (4H, m, CH2CH2), 1.74 (3H, d, J ¼ 1.2 Hz, H-400), 1.66
Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.02.
017. These data include MOL files and InChiKeys of the most
important compounds described in this article.
(3H, d, J ¼ 1.2 Hz, H-500); 13C NMR (100 MHz, CDCl3)
d (ppm) 158.63,
154.23, 146.23, 143.73, 142.01, 133.93, 130.59, 123.87, 120.92, 120.67,
114.21, 111.02, 107.96, 96.90, 55.84 (30-OCH3), 55.60 (3-OCH3), 37.19,
35.49, 25.76 (C-100), 24.38 (C-400), 17.95 (C-500); MS (ESI) m/z (%):381
([M þ K]þ26), 365 ([M þ Na]þ, 100), 286 (20), 137 (28); HRMS (ESI)
(m/z): calcd for C21H26O4 [M þ Na]þ 365.1723, found 365.1723.
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CDCl3)
d
(ppm) 7.64 (1H, dd, J ¼ 15.9 Hz), 7.46 (2H, dd, J ¼ 8.8 Hz),
6.90 (2H, dd, J ¼ 8.8 Hz), 6.30 (1H, dd, J ¼ 15.9 Hz), 5.76 (1H, bt,
J ¼ 6.5 Hz), 4.62 (2H, dd, J ¼ 6.5 Hz), 4.08 (2H, s), 3.79 (3H, s), 1.77
(3H, s); 13C NMR (100 MHz, CDCl3)
d (ppm) 167.9,160.5,144.6,140.5,
129.7, 127.1, 119.3, 115.2, 115.0, 67.6, 64.4, 51.6, 14.0; HRMS(ESI)
(m/z): calcd for C15H18O4 [M þ Na]þ 285.1097, found 285.1096.
In addition to the production of the title compound 21, there
was formation of minor aldehyde derivative as white solid (0.017 g,
yield 12%), mp 110e112 ꢀC determined as methyl (E)-4-(30-car-
bonylbut-(E)-20-enyloxy)cinnamate (22). 1H NMR (400 MHz, CDCl3)
d
(ppm) 9.49 (1H, s), 7.66 (1H, dd, J ¼ 16 Hz), 7.50 (2H, dd, J ¼ 8.8 Hz),
6.92 (2H, dd, J ¼ 8.8 Hz), 6.67 (1H, tm, J ¼ 5.6 Hz), 6.33 (1H, dd,
J ¼ 16 Hz), 4.91 (2H, dd, J ¼ 5.6 Hz), 3.80 (3H, s), 1.85 (3H, dd,
J ¼ 0.8 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm) 193.9, 167.6, 159.7,
146.8, 144.2, 140.1, 129.8, 127.9, 115.8, 114.9, 64.8, 51.6, 9.7; HRMS
(ESI) (m/z): calcd for C15H16O4 [M þ Na]þ 283.0941, found 283.0941.
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5.19. Cytotoxicity activity
K562 cell line was maintained and incubated. Cells were treated
with the compounds at a range of concentrations (0.01e100
mM) for
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