Oxyresveratrol: Structural modification and evaluation of biological activities

Article abstract of DOI:10.3390/molecules21040489

Oxyresveratrol (2,4,3' ,5' -tetrahydroxystilbene, 1), a phytoalexin present in large amounts in the heartwood of Artocarpus lacucha Buch.-Ham., has been reported to possess a wide variety of biological activities. As part of our continuing studies on the structural modification of oxyresveratrol, a library of twenty-six compounds was prepared via O-alkylation, aromatic halogenation, and electrophilic aromatic substitution. The two aromatic rings of the stilbene system of 1 can be chemically modulated by exploiting different protecting groups. Such a strategy allows for selective and exclusive modifications on either ring A or ring B. All compounds were evaluated in vitro for a panel of biological activities, including free radical scavenging activity, DNA protective properties, antiherpetic activity, inhibition of α-glucosidase and neuraminidase, and cytotoxicity against some cancer cell lines. Several derivatives were comparably active or even more potent than the parent oxyresveratrol and/or the appropriate positive controls. The partially etherified analogs 5' -hydroxy-2,3' ,4-trimethoxystilbene and 3' ,5' -dihydroxy-2,4-dimethoxystilbene demonstrated promising anti-herpetic and DNA protective activities, offering new leads for neuropreventive agent research, whereas 5' -hydroxy-2,3' ,4,-triisopropoxystilbene displayed anti-α-glucosidase effects, providing a new lead molecule for anti-diabetic drug development. 3' ,5' -Diacetoxy-2,4-diisopropoxystilbene showed potent and selective cytotoxicity against HeLa cancer cells, but the compound still needs further in vivo investigation to verify its anticancer potential.

Full text of DOI:10.3390/molecules21040489

molecules
Article
Oxyresveratrol: Structural Modification and
Evaluation of Biological Activities
Nutputsorn Chatsumpun 1, Taksina Chuanasa 1, Boonchoo Sritularak 1, Vimolmas Lipipun 2,
Vichien Jongbunprasert 1, Somsak Ruchirawat 3, Poonsakdi Ploypradith 3,* and
Kittisak Likhitwitayawuid 1,
*
1
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand; manee_many@hotmail.com (N.C.);
s_taksina@yahoo.com (T.C.); Boonchoo.Sr@chula.ac.th (B.S.); Vichien.J@chula.ac.th (V.J.)
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand; Vimolmas.L@chula.ac.th
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, and Program in Chemical Biology,
Chulabhorn Graduate Institute, 54 Kampaeng Phet 6 Road, Bangkok 10210, Thailand; somsak@cri.or.th
Correspondence: poonsakdi@cri.or.th (P.P.); Kittisak.L@chula.ac.th (K.L.);
2
3
*
Tel.: +66-2-553-8555 (P.P.); +66-2-218-8360 (K.L.)
Academic Editors: Dominique Vervandier-Fasseur, Norbert Latruffe and Ole Vang
Received: 29 February 2016; Accepted: 11 April 2016; Published: 19 April 2016
Abstract: Oxyresveratrol (2,4,31,51-tetrahydroxystilbene,
1), a phytoalexin present in large amounts
in the heartwood of Artocarpus lacucha Buch.-Ham., has been reported to possess a wide variety
of biological activities. As part of our continuing studies on the structural modification of
oxyresveratrol, a library of twenty-six compounds was prepared via O-alkylation, aromatic
halogenation, and electrophilic aromatic substitution. The two aromatic rings of the stilbene system
of
1 can be chemically modulated by exploiting different protecting groups. Such a strategy
allows for selective and exclusive modifications on either ring A or ring B. All compounds were
evaluated in vitro for a panel of biological activities, including free radical scavenging activity, DNA
protective properties, antiherpetic activity, inhibition of
α-glucosidase and neuraminidase, and
cytotoxicity against some cancer cell lines. Several derivatives were comparably active or even
more potent than the parent oxyresveratrol and/or the appropriate positive controls. The partially
etherified analogs 51-hydroxy-2,31,4-trimethoxystilbene and 31,51-dihydroxy-2,4-dimethoxystilbene
demonstrated promising anti-herpetic and DNA protective activities, offering new leads
for neuropreventive agent research, whereas 51-hydroxy-2,31,4,-triisopropoxystilbene displayed
anti-α-glucosidase effects, providing a new lead molecule for anti-diabetic drug development.
31,51-Diacetoxy-2,4-diisopropoxystilbene showed potent and selective cytotoxicity against HeLa
cancer cells, but the compound still needs further in vivo investigation to verify its anticancer potential.
Keywords: oxyresveratrol; stilbene; free radicals;
Artocarpus lacucha; Artocarpus lakoocha
α-glucosidase; herpes simplex; cytotoxicity;
1. Introduction
Stilbenes are a group of plant polyphenolic compounds derived from the mixed shikimate-acetate
pathway. They are produced as phytoalexins, with limited but heterogeneous distribution in the plant
kingdom [
significant research attention due to their elaborate and diverse structures, and wide-ranging biological
activities [ ]. Historically, these compounds have been studied for antioxidant, antibacterial, antiviral
and anticancer activities [ ]. However, recent efforts have been made to explore other potential uses
1]. Over the years, these C6-C2-C6 compounds, as well as their oligomers, have attracted
2
3,4
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Molecules 2016, 21, 489
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such as therapeutic or preventive agents against non-communicable diseases (NCDs), for example,
diabetes mellitus (DM), cardiovascular illnesses, and Parkinson’s disease (PD) [5–7].
Oxyresveratrol (2,4,31,51-tetrahydroxystilbene,
1
, Scheme 1) is present in large amounts in the
heartwood of Artocarpus lacucha Buch.-Ham. (also known as Artocarpus lakoocha Roxb.) [ ], and thus
offers opportunities for chemical and biological studies. Our previous in vitro and in vivo experiments
have shown that is a potent tyrosinase inhibitor, and subsequent studies in human volunteers have
confirmed it as an effective skin-whitening agent [ 10]. Despite its moderate in vitro inhibitory activity
against herpes simplex virus type I (HSV-1) [11], compound , in the form of topical cream, exhibited
8
1
9,
1
high therapeutic efficacy in mice cutaneously infected with HSV-1, with a potency comparable to that
of the antiviral drug acyclovir [12]. Currently, mounting evidence has indicated HSV-1 infection in the
brain as one of the key causative factors in the pathogenesis of Alzheimer’s disease (AD) [13], and
this has suggested another medicinal importance for oxyresveratrol. Regarding the AD preventive
potential,
1
has been reported to inhibit
β-amyloid-induced neurotoxicity in cultured cortical neurons
and in vitro activity of
β
-secretase, an enzyme vital for the production of the A
β
protein [14 15].
,
In addition, the compound has been studied for protective activity against Parkinson’s disease
(PD) in cultured neuronal cells [16], and demonstrated to prevent neurotoxicity and DNA damage
through its antioxidant properties [17
H5N1 neuraminidase (an enzyme found in avian influenza virus) [19], but strong inhibition against
-glucosidase, an enzyme responsible for the breaking down of starch and polysaccharides into glucose
in the intestine [20]. In spite of all the potential uses mentioned above, seems to have rather limited
,18]. Compound 1 showed modest inhibitory activity against
α
1
clinical application. The compound has been reported to undergo extensive hepatic metabolism and
rapid urinary elimination, resulting in a short half-life time, and thereby restricting its clinical use [21].
Moreover, its postulated function as a preventive agent for neurodegenerative disorders, such as AD
or PD, appears to be hindered by its low ability to cross the blood-brain barrier (BBB) [22].
In our earlier report, we described a successful structural modification of
analog with enhanced tyrosinase inhibitory activity [ ]. In this investigation, we broadened our
chemical studies and expanded the biological evaluations with the intention of improving the following
biological activities of : antioxidant and DNA protective properties, inhibitory activities against the
1, which gave an
9
1
enzymes α-glucosidase and neuraminidase, and ability to inhibit the growth of herpes simplex virus
and some cancer cells.
2. Results and Discussion
2.1. Chemistry
2.1.1. O-Alkylation/O-Acylation
When
tetra-O-methylated products (
(Scheme 1). The alkylation of
and tetra-O-isopropyl compounds (
It should be noted that it was difficult to avoid the formation of
1
was allowed to react with MeI in acetone at room temperature, di-, tri- and
and ) were obtained in 9%, 25%, and 45% yields, respectively
with 2-bromopropane/K2CO3 in DMF gave the corresponding di-, tri-
and ) in 24%, 30%, and 28% yields, respectively (Scheme 1).
, perhaps due to the greater solubility
2,
3
4
1
5
,
6
7
7
of the more alkylated ethers in DMF under the reaction conditions. In both O-alkylation reactions,
the di-O-alkylation occurred on the two hydroxy groups of ring A, and tri-O-alkylation occupied
both hydroxy groups of ring A and a hydroxy group of ring B. These results suggested that the
phenolic groups on ring A were more reactive than their counterparts on ring B, most likely due to the
conjugation of the hydroxy groups of ring A with the olefinic bond, which is not possible for those on
ring B. This difference in the reactivity of the hydroxy groups was exploited in the subsequent studies
on the aromatic electrophilic substitution reactions, in which the isopropyl group was found to be a
better O-protecting group than the methyl after some experimentation (see below). Acetylation of
with AcCl gave 8 in 70% yield.
1

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O-Alkylation of the tri-O-isopropyl compound
6
with ethyl bromoacetate under basic conditions
went smoothly, to furnish in 84% yield the ethyl ester 9, which then underwent BCl3-mediated
deprotection of the isopropyl groups to give the trihydroxy compound 10 in 61% yield. Subsequent
saponification of the ethyl ester using 5% KOH in ethanol provided the corresponding acid 11 in a
moderate 40% yield (Scheme 1).
OR3
R6
2
1
(a)
OR1
4
2
3
4
+ (45%)
(9%)
+
(25%)
B
2
OR4
(b)
(c)
1
A
5 (24%) + 6 (30%) + 7 (28%)
8 (70%)
R2O
1
4
R5
(g)
36%
(d)
84%
OH
O iPr
Cl
OH
O iPr
OH
OCH2COOEt
HO
iPr O
12
9
(e)
61%
OH
36%
(h)
OMe
Cl
OH
OMe
OCH2COOEt
OMe
HO
MeO
10
13
R1 R2 R3 R4
40%
(f)
R5 R6
H
H
H
H
H
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
Me Me H
Me Me Me H
OH
Me Me Me Me
iPr iPr H
H
H
OCH2COOH
iPr iPr iPr
HO
11
iPr iPr iPr iPr
Ac Ac Ac Ac
Scheme 1. O-Alkylation, acylation, and aromatic chlorination of
) 2-bromopropane, K2CO3, DMF, 55 ˝C; (
K2CO3, acetone, rt; ( ) AcCl, Et3N, DMAP, CH2Cl2, rt; (
rt, Ar; ( ) 5% KOH, EtOH, rt; (g
1. Reagents and conditions: (a) MeI,
b
c
d
)
ethyl bromoacetate, K2CO3, DMF, rt; (
NCS, AcOH (glacial), rt; (h) MeI, K2CO3, acetone, 55 ˝C.
e
) BCl3, CH2Cl2,
´
78 ˝C
Ñ
f
)
2.1.2. Halogenation
It was expected that aromatic halogenation on
1 would be facile due to the high electron density
contributed by the hydroxy groups. Due to the conjugation between the hydroxy groups on ring A
1
1
and the olefin, the aromatic B ring should be more nucleophilic, particularly at the positions 2 /6 , and
this difference should facilitate regioselective electrophilic aromatic halogenation under appropriate
a1nd mild conditions. Using N-chlorosuccinimide (NCS) and glacial acetic acid, the corresponding
1 in 36% yield (Scheme 1). Chlorination on 7 and 20
2 -monochlorinated product 12 was obtained from
(prepared from
full methylation to give the tetramethylether 13 in 36% yield. Several attempts to carry out aromatic
bromination on or with N-bromosuccinimide under various conditions were performed, but failed
to give the brominated product. Iodination of 1 with N-iodosuccinimide was also not successful.
5) did not give the desired chlorinated product. Compound 12 smoothly underwent
1
4

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2.1.3. Aromatic Electrophilic Substitution
When the tetra-O-isopropyl compound
7
reacted under the Vilsmeier-Haack formylation
conditions, the corresponding ring B-formylated product 14 was obtained in good yield (80%)
(Scheme 2).
Scheme 2. Selective aromatic formylation of ring B of
of the aldehyde. Reagents and conditions: (
) POCl3, DMF, 0 ˝C, Ar; (
2-methyl-2-butene, acetone, rt; ( ) BCl3, CH2Cl2, rt, Ar; ( ) p-TsOH
78 ˝C
7
to 14 and the subsequent derivatizations
) NaClO2, NaH2PO4 2H2O,
H2O, H2O2, 0 ˝C, Ar; (
a
b
¨
c
´
Ñ
d
¨
e)
BBr3, CH2Cl2, ´78 ˝C Ñ rt, Ar; (f) EtOOCCH=PPh3, CH2Cl2, 0 ˝C.
The aldehyde functional group could be further oxidized to the corresponding carboxylic acid 15
in 79% yield using sodium chlorite. The aldehyde 14 smoothly underwent BCl3-mediated deprotection
1
of the isopropyl groups to provide the 2 -formyloxyresveratrol 16 in moderate 48% yield. In addition,
the aldehyde 14 could also undergo the acid-mediated Baeyer-Villiger-type Dakin reaction, which
converted the aldehyde into the corresponding formate. Subsequent in situ cleavage of the formate
1
under the reaction conditions furnished 2 -hydroxy-tetra-O-isopropyl oxyresveratrol 17 in good yield
(66%). Unfortunately, all attempts to deprotect the isopropyl groups of 17 failed, and only a mixture of
decomposed products was obtained. The deisopropylation of 15 with BBr3 under similar conditions
unexpectedly gave the isochromanone 18 in 20% yield, a result of the Lewis acid-mediated removal
of the O-isopropyl groups followed by C-O bond formation on the olefinic carbon of the stilbene
system. In addition, the aldehyde 14 could undergo a smooth Wittig olefination to furnish the stilbenyl
cinnamate 19 in 68% yield.
In order to effect the reactions on ring A of oxyresveratrol, the di-O-isopropyl compound 5 was
acetylated under standard conditiond to provide ring A-di-O-isopropyl ring B-di-O-acetyl compound
20 in 74% yield (Scheme 3).

Molecules 2016, 21, 489
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Scheme 3. Protecting group-directed selective aromatic formylation of ring A of 20 to 21 and the
subsequent derivatizations of the aldehyde. Reagents and conditions: ( ) Ac2O, Et3N, CH2Cl2, rt; (
2H2O, 2-methyl-2-butene, acetone, rt; ( ) BCl3, CH2Cl2,
a
b)
POCl3, DMF, 0 ˝C, Ar; (
c
) NaClO2, NaH2PO4
¨
d
´78 ˝C Ñ rt, Ar; (e) 5% KOH, EtOH, rt; (f) p-TsOH¨ H2O, H2O2, 0 ˝C.
After some experimentation, ring A was formylated on the 5-position to give 21 exclusively,
˝
in 60% yield, under the best Vilsmeier-Haack conditions (10 eq of POCl3 at 0 C
Ñ
rt overnight
under argon). Subsequent oxidation of the aldehyde 21 to the corresponding acid 22 using sodium
chlorite gave the latter in 73% yield. BCl3-mediated cleavage of all the O-isopropyl groups as well
as the O-acetyl groups of 21 and 22 finally gave the desired 5-formyl- and 5-carboxyoxyresveratrols
23 and 24 in 78% and 63% yields, respectively. Saponification of 21 and 22 using 5% KOH in ethanol
to cleave the acetate groups gave 5-formyl- and 5-carboxy-di-O-isopropyl oxyresveratrol 25 and 26
in 93% and 40% yields, respectively. The aldehyde 21 was subjected to the Dakin reaction to give
5-hydroxy-di-O-acetyl-di-O-isopropyl oxyresveratrol 27 in 47% yield.
It is worth noting that the differences between protecting groups could be used to manipulate
the selectivity of the aromatic electrophilic reactions. In the Vilsmeier-Haack reaction of the
tetra-O-isopropyl compound 7, all the protecting groups were electron-donating groups, which made
the 21 and 61 positions of ring B the most reactive ones, but in the Vilsmeier-Haack reaction of the
di-O-acetyl-di-O-isopropyl compound 20, the acetyl groups were electron-withdrawing groups, and
thus reduced the electron density of ring B. This was virtually equivalent to the deactivation of ring B,
and therefore directed the formylation to occur on ring A. This synthetic strategy could be a useful tool
for the synthesis of other similar analogs derivable from exploiting the difference in electron density of
each aromatic ring.
2.2. Biological Activity
Since
antioxidant activity and inhibition of the
(vide supra), all structurally modified analogs of
1
was previously studied and reported to exhibit an array of biological activities including
-glucosidase and neuraminidase enzymes and HSV-1
were subjected to the related assays to study the
α
1
effects of such modification on each biological activity. In addition, these compounds were also
evaluated for cytotoxic potential against four cancer cell lines of three different cancer types (breast,
cervical and lung).

Molecules 2016, 21, 489
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2.2.1. Free Radical Scavenging Activity
First, examinations were conducted on the relationships between the phenolic and olefinic
functionalities of the core stilbene structure and the free radical scavenging and DNA protective
activities. Then, investigations were carried out to study how the nature and position of substituents
with electron-withdrawing (i.e., Cl, CHO and COOH) or electron-donating (i.e., OH) properties on the
aromatic rings affected these biological activities. Compounds were first evaluated at 100
those that showed >80% inhibition were further analyzed to determine their IC50 values.
Oxyresveratrol ( ) showed weaker DPPH free radical scavenging activity than Trolox (Table 1).
The phenolic groups were essential since the activity was reduced or lost when some or all of the
phenolic groups of were esterified/etherified, as observed in compounds and 20. The only
exception was compound , which showed higher activity
, the 2,4,31-tri-O-methylated version of
µg/mL, and
1
1
2–8
3
1
against the superoxide anion radical than the parent compound. Interestingly, this compound also
showed higher DNA protective properties.
Table 1. IC50 values (µM) for free radical scavenging activities and DNA protective properties of 1
and derivatives.
DNA Protective
Activity
Free Radical Scavenging Activity
Compound
DPPH
Superoxide
1
2
3
11.7 ˘ 0.4
77.0 ˘ 6.4 *
nd
147.7˘ 9.8 *
9.7 ˘ 0.2 *
19.4 ˘ 3.1 *
14.7 ˘ 0.6 *
16.5 ˘ 2.8 *
11.7 ˘ 0.3
nd
303.1 ˘ 7.9
43.3 ˘ 6.7
39.4 ˘ 1.2
6.3 ˘ 0.8 *
59.1 ˘ 9.9
19.9 ˘ 2.8 *
28.6 ˘ 3.8 *
32.0 ˘ 1.4 *
32.2 ˘ 4.8
nd
28.3 ˘ 4.3 *
81.4 ˘ 4.2 *
54.7 ˘ 8.3
79.4 ˘ 14.9 *
111.8 ˘ 7.3 *
104.7 ˘ 8.5 *
18.6 ˘ 3.7 *
113.1 ˘ 4.6 *
nd #
120.1 ˘ 14.9 *
nd
5
10
11
12
16
17
18
22
23
24
25
26
27
Trolox
154.9 ˘ 14.7 *
81.9 ˘ 12.3 *
98.4 ˘ 7.0 *
43.4 ˘ 4.4 *
nd
107.3 ˘ 8.7 *
17.7 ˘ 3.5 *
88.3 ˘ 9.7 *
38.6 ˘ 1.4 *
nd
nd
nd
48.3 ˘ 5.8 *
nd
nd
nd
nd
7.0 ˘ 0.2 *
8.7 ˘ 1.1 *
293.5 ˘ 19.3
#: nd = not determined due to <80% inhibition at 100 µg/mL.; *: Significantly different from that of 1 (p < 0.05).
The olefinic bridge, which provided conjugation (and thus electron delocalization) between the
two aromatic rings, was also required for DPPH radical scavenging ability, as demonstrated from the
marked decrease in activity of 18. However, when evaluated against the superoxide anion, 18 displayed
higher activity than 1. This enhanced activity was most likely due to the electron-withdrawing effect
of the C=O of the dihydropyrone ring which helped stabilize the negatively-charged stilbene radical
formed from 18 after interaction with the superoxide anion.
Introducing an electron-donating group such as OH to the aromatic rings could restore the lost
DPPH scavenging activity caused by phenolic esterification/etherification. This was inferred by
comparing the activity of the following pairs of structures:
7 (no activity) vs. 17, and 20 (no activity) vs.
27. However, these sets of compounds did not give similar results in the superoxide assay.
Placing an electron-withdrawing substituent, such as Cl, CHO and COOH, onto the aromatic ring
of
1 yielded compounds 12, 16, 23 and 24 which showed weaker activity against the DPPH radical,
but enhanced activity against the superoxide anion. The improved activity was likely attributable to
the electron-withdrawing nature of these groups that helped to distribute the negative charge of the
radicals generated from the reactions of these stilbenoids with the superoxide anion.

Molecules 2016, 21, 489
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2.2.2. DNA Protective Property
Riboflavin (RF), when exposed to light, generates several kinds of free radicals, including
), neutral oxidized (RF( H) ) and cation (RF )
triplet (3RF +) riboflavin radical. The (3RF
‚
´
‚
‚
‚
radical then transfers energy to oxygen to form reactive oxygen species (ROS) such as singlet
oxygen (1O2) and superoxide (O2‚´) radicals [23
,24]. These free radicals, with their high energy,
can each damage DNA by attacking the purine base guanine to form 8-hydroxyguanine and
2,6-diamino-4-hydroxy-5-formamidopyrimidine [23
,25–27]. Any compound that can efficiently protect
DNA from the damage induced by photosensitized riboflavin must be capable of scavenging or
neutralizing these free radicals. In this study, compounds were first evaluated at 100
with >80% inhibition were further analyzed for their IC50 values (Table 1).
µg/mL, and those
Compound
the DPPH assay (Table 1). Total O-alkylation or O-acylation of
the lost or diminished activity for 14 15 17 19 and 20. The lost activity of 20 could be rescued
by placing an OH substituent on ring B (27), an outcome similar to the case of the DPPH radical. The
partially O-alkylated products of , including 10, and 11 showed enhanced DNA protective activity,
1
showed higher DNA protective activity than Trolox, despite its inferior activity in
1
destroyed the activity, as observed in
4,
7
,
8,
9,
,
,
,
1
3,
but the tri-O-isopropylated product 6 displayed markedly reduced activity.
Compound 18 did not lose the DNA protective activity despite the removal of the C=C bond.
With the aid of the C=O of the dihydro-
than . The placement of the electron-donating group OH on ring A of 20 could restore the lost activity,
giving 27, which is a better DNA protectant than . The introduction of an electron-withdrawing
substituent to the aromatic rings of gave unclear results. Placing Cl on ring B (i.e., compound 12
α-pyrone ring, this compound showed even higher activity
1
1
1
)
slightly increased the activity, but introducing COOH onto ring A reduced the activity (compound 24).
2.2.3. α-Glucosidase Inhibitory Activity
Almost all of the stilbenoids 2´27 prepared from oxyresveratrol exhibited reduced or lost
inhibitory activity against
α
-glucosidase (the positive control acarbose: IC50 745.9
˘
88.4
(IC50 19.5
has been suggested to be a promising structure for anti-diabetic drug
]. However, the compound, after oral absorption, was found to undergo extensive
hepatic metabolism and rapid urinary elimination, resulting in a short half-life time (~0.96 h) [21].
Compound , with three isopropoxy groups, is predicted to have much slower metabolism and may
µM). The
only exception was
1.7 M). In a previous study,
development [
6, which showed activity (IC50 19.6
˘
3.6
µM) comparable to that of
1
˘
µ
1
7
6
be considered as a better candidate for the preclinical and clinical studies for anti-DM drugs.
2.2.4. Neuraminidase Inhibitory Activity
In this study, oxyresveratol and all of its derivatives
2
–27 exhibited no activity against the enzyme
neuraminidase (positive control oseltamivir, IC50 0.5 nM), although
1
has been earlier described as
having modest activity [19]. It should be mentioned that resveratrol, a closely related stilbene, and
several of its synthetic and semisynthetic analogs have been recently reported to possess significant
inhibitory activity against influenza neuraminidase [28].
2.2.5. Antiherpetic Activity
Compounds
>50% inhibition at this concentration were further studied to determine their IC50 values (Table 2).
Three analogs, including compounds , and , showed IC50 values significantly lower than that
. Stilbenes and (di- and tri-O-methylated products of ) displayed 3- to 4-fold higher antiherpetic
1–27 were first evaluated for anti-HSV-1 activity at 100 µg/mL. Those that showed
2,
3
6
of
1
2
3
1
activity than the parent compound. Recently, compelling evidence has suggested that HSV-1 infection
of neuronal and glial cells plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD), and the
use of antiviral drugs in the treatment of AD has been proposed [13]. As mentioned earlier,
1, despite
its moderate anti-herpetic activity in vitro, exhibited potent activity in vivo in mice cutaneously infected

Molecules 2016, 21, 489
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with HSV-1 [12].However, due to its poor BBB permeability [22], oxyresveratrol (
1) seems to have
limited clinical application. In this regard, stilbenes
2 and 3, with their higher lipophilicity, may be
better candidates for further investigation on HSV infection of the CNS, as well as neurodegenerative
diseases such as AD or PD.
Table 2. Anti-herpetic activity of 1 and derivatives.
Compounds
IC50 (µM)
1
2
3
147.1 ˘ 16.8
40.2 ˘ 6.8 *
32.8 ˘ 5.9 *
107.0 ˘ 9.8 *
182.3 ˘ 28.9
130.3 ˘ 14.9
123.5 ˘ 19.3
289.3 ˘ 40.8 *
188.3 ˘ 25.1
207.4 ˘ 16.9 *
344.7 ˘ 32.5 *
226.8 ˘ 35.7 *
1.6 ˘ 0.0 *
6
10
14
15
18
21
22
23
26
ACV
* Significantly different from that of 1 (p < 0.05).
2.2.6. Cytotoxicity against Cancer Cells
All compounds ( 27) were initially evaluated at 50
inhibition were subjected to IC50 determination (Table 3).
1–
µg/mL, and those demonstrating >50%
Table 3. Cytotoxicity against cancer cells of 1 and some derivatives.
IC50 (µM)
Compounds
T47-D
HeLa
A549
H69AR
1
2
3
4
5
6
8
10
12
13
14
15
16
20
21
23
152.7 ˘ 4.5
114.2 ˘ 2.0 *
87.1 ˘ 9.5 *
nd
117.4 ˘ 11.9 *
37.7 ˘ 12.9 *
103.0 ˘ 4.3 *
88.6 ˘ 2.9 *
156.6 ˘ 6.0
nd
126.2 ˘ 0.3
28.4 ˘ 7.0 *
13.1 ˘ 2.1 *
12.1 ˘ 1.5 *
11.0 ˘ 2.7 *
25.7 ˘ 2.1 *
nd
159.8 ˘ 0.0
117.0 ˘ 11.1 *
52.4 ˘ 0.9 *
nd
55.5 ˘ 3.5 *
28.8 ˘ 5.1 *
nd
137.7 ˘ 8.4 *
120.7 ˘ 2.9 *
nd
nd #
180.7 ˘ 3.4
36.7 ˘ 3.3
26.8 ˘ 7.6
39.2 ˘ 6.5
54.5 ˘3.0
nd
100.9 ˘ 4.7 *
33.4 ˘ 2.9 *
18.7 ˘ 4.2 *
69.1 ˘ 3.3 *
83.2 ˘ 9.1 *
74.4 ˘ 4.8 *
5.7 ˘ 0.6 *
18.9 ˘ 0.7 *
77.9 ˘ 2.1 *
22.4 ˘ 0.4 *
79.8 ˘ 4.7 *
0.7 ˘ 0.2 *
nd
111.2 ˘ 3.1
65.7 ˘ 3.2
nd
nd
nd
nd
101.4 ˘ 9.1 *
99.3 ˘ 3.1 *
81.3 ˘ 0.6 *
54.5 ˘ 2.0 *
82.9 ˘ 2.2 *
44.7 ˘ 2.6 *
83.2 ˘ 10.6 *
0.5 ˘ 0.00 *
nd
nd
138.0 ˘ 3.1 *
51.7 ˘ 1.7 *
71.3 ˘ 3.2 *
143.2 ˘ 3.3 *
75.1 ˘ 0.7 *
104.4 ˘ 5.3 *
0.4 ˘ 0.1 *
38.0 ˘ 2.2
nd
nd
25
27
111.0 ˘ 2.3
nd
Doxorubicin
21.7 ˘ 0.9
#: nd = not determined due to <50 % inhibition at 50
µg/mL.; *: Significantly different from that of 1
(p < 0.05). Cell lines: T47D = human hormone-dependent breast cancer, HeLa = human cervical adenocarcinoma,
A549 = human non-small-cell lung carcinoma, H69AR = human multidrug-resistant small-cell lung carcinoma.
Seventeen derivatives demonstrated higher cytotoxicity than the parent compound
1, which was
virtually non-cytotoxic against all cancer cell lines. The partially (di- or tri-) O-alkylated products (
2, 3,

Molecules 2016, 21, 489
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5
4
and
showed enhanced selective cytotoxicity against HeLa and H69AR cells, but no activity against T47-D
against the multidrug-resistant small lung cancer cells
(H69AR) was in the same range as that of doxorubicin (p > 0.05). The fully O-isopropylated products,
including 17, and 19, did not show cytotoxicity in the assays, probably a result of their poor
aqueous solubility. With regard to the esterified analogs, the fully O-acetylated compound showed
slightly improved activity selectively against T47-D cells, but less activity against HeLa and A549 cells
when compared with . Compound 20, a di-O-isopropylated-di-O-acetylated product of , showed
better cytotoxicity than 1 in all cancer cell lines, with selective potency against HeLa cells.
Different impacts on the cytotoxicity were observed when an electron-withdrawing substituent
was attached to the aromatic rings of . Chlorination of
at C-21 gave 12, a product with increased
6) showed increased cytotoxicity against all types of cancer cells. The fully O-methylated product
and A549 cells. Interestingly, the IC50 value of
4
7, 9,
8
1
1
1
1
cytotoxicity against almost all cancer cell lines, except T47D cells. Similar cytotoxicity profiles were
observed for compounds 16 and 23, the 5- and the 21-formylated analogs, both of which displayed
increased activity against T47-D, HeLa and A549, but no activity against H69AR cells. Further
structural modification of 23 by converting the CHO into a COOH group (compound 24) destroyed
the cytotoxicity in all cancer cell lines. When
7, the fully O-isopropylated analog, was formylated or
carboxylated at C-21, partial restoration of cytotoxicity was observed (compounds 14 and 15). For 20
,
placing a CHO group at C-5 gave a product 21 with mixed results since the cytotoxicity against TD47-D
cells was enhanced, but the activity against HeLa A549 and H69AR cells was weakened (p < 0.05).
Transforming the CHO at this position of 21 into a COOH group abolished the activity (compound 22).
An electron-donating group such as hydroxy group, when added to the aromatic rings, could not
induce cytotoxicity, as reflected from the non-cytotoxicity of both
7 and 17, but instead seemed to
weaken the cytotoxicity, as evident when comparing the strong activity of 20 with that of 27 which
showed less cytotoxicity in HeLa and A549 cancer cells (p < 0.05).
3. Materials and Methods
3.1. General Information
All chemical reagents were purchased from commercial suppliers. Melting points (uncorrected)
were determined on a Fisher-Johns hot stage melting point apparatus. IR spectra were performed
using Universal Attenuated Total Reflectance (UATR) on a 2000 FT-IR system (Perkin Elmer, Boston,
MA, USA) or an A-30 spectrophotometer (Jasco, Easton, MD, USA). High resolution mass spectra were
taken on a MicroTOF instrument (Bruker, Billerica, MA, USA) using atmospheric pressure chemical
1
ionization (APCI) or electrospray ionization (ESI) in positive or negative mode. H- and 13C-NMR
were recorded on an Avance III 300 MHz spectrometer or 400 MHz spectrometer (Bruker, Billerica,
MA, USA). All chemical shift values were reported as δ (ppm) and coupling constant values J were
measured in Hz. Oxyresveratrol (1) was isolated and purified from the heartwood of Artocarpus lacucha
Buch.-Ham. (A. lakoocha Roxb.) as previously described [11,29,30].
3.2. Synthesis
3.2.1. Preparation of Compounds 2–4
K2CO3 (680 mg, 4.92 mmol) and MeI (0.11 mL, 1.8 mmol) were added to the solution of
0.82 mmol) in acetone (4 mL) at room temperature. The reaction mixture was stirred overnight. Then,
the reaction mixture was diluted with water (5 mL) and extracted with EtOAc (3 5 mL). The EtOAc
1 (200 mg,
ˆ
layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give a crude product which, after purification by preparative TLC (50% EtOAc/hexanes),
furnished 2 (19 mg, 9%), 3 (60 mg, 25%) and 4 (112 mg, 45%).
1
1
3 ,5 -Dihydroxy-2,4-dimethoxystilbene (
(300 MHz, CDCl3), (ppm): 7.43 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 16.5 Hz, 1H), 6.82 (d, J = 16.2 Hz, 1H),
2
): viscous brown oil; IR (UATR, cm´1): 3374, 2931, 1601; 1H-NMR
δ

Molecules 2016, 21, 489
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6.55 (d, J = 2.1 Hz, 2H), 6.48 (dd, J = 8.7, 2.1 Hz, 1H), 6.44 (d, J = 2.1 Hz, 1H), 6.24 (t, J = 2.1 Hz, 1H),
3.82 (s, 3H), 3.80 (s, 3H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 160.6, 158.1, 156.9, 141.0, 127.3, 126.2,
124.1, 119.2, 105.9, 105.1, 101.7, 98.5, 55.5, 55.4; TOF-HRMS m/z [M + H]+, calcd for C16H17O4: 273.1121;
found: 273.1118.
1
1
5 -Hydroxy-2,3 ,4-trimethoxystilbene (
(300 MHz, CDCl3), (ppm): 7.46 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 16.5 Hz, 1H), 6.88 (d, J = 16.5 Hz, 1H),
6.62 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.49 (dd, J = 8.4, 2.4 Hz, 1H), 6.45 (d, J = 2.4 Hz, 1H),
6.30 (t, J = 2.1 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H); 13C-NMR (75 MHz, CDCl3),
(ppm):
3
): viscous brown oil; IR (UATR, cm´1): 3408, 2938, 1590; 1H-NMR
δ
δ
160.9, 160.5, 158.0, 156.9, 140.6, 127.3, 126.6, 123.9, 119.3, 105.8, 105.0, 104.6, 100.4, 98.5, 55.5, 55.4, 55.3;
TOF-HRMS m/z [M + H]+, calcd for C17H19O4: 287.1278; found: 287.1285.
1
1
˝
2,3 ,4,5 -Tetramethoxystilbene (4 δ (ppm): 7.46
): white solid, mp 65–68 C; 1H-NMR (300 MHz, CDCl3),
(d, J = 8.4 Hz, 1H), 7.36 (d, J = 16.2 Hz, 1H), 6.92 (d, J = 16.5 Hz, 1H), 6.66 (d, J = 2.4 Hz, 2H), 6.47 (dd,
J = 8.4, 2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.34 (t, J = 2.1 Hz, 1H), 3.81 (s, 3H), 3.79 (s, 6H), 3.77 (s, 3H);
13C-NMR (75 MHz, CDCl3),
δ (ppm): 160.8, 160.5, 158.0, 140.3, 127.2, 126.8, 123.7, 119.1, 104.9, 104.2,
99.2, 98.3, 55.3, 55.2, 55.1; TOF-HRMS m/z [M + H]+, calcd for C18H21O4: 301.1434; found: 301.1440.
The spectroscopic data were in agreement with previously reported values [9].
3.2.2. Preparation of Compounds 5–7
To a solution of
1 (100 mg, 0.41 mmol) in DMF (2 mL), K2CO3 (283 mg, 2.05 mmol) and
2-bromopropane (2.30 mL, 24.6 mmol) were added. Then 2-bromopropane (1.15 mL, 12.3 mmol)
˝
was added every 24 h. The reaction mixture was stirred at 55 C for 3 days and monitored by
TLC. After completion, water (5 mL) was added, and the reaction was extracted with EtOAc
(3
ˆ
5 mL). The organic phase was washed with water (7
ˆ
5 mL) and brine, dried over Na2SO4,
filtered and concentrated. Column chromatographic purification with gradient EtOAc/hexanes (10%
EtOAc/hexanes Ñ 80% EtOAc/hexanes) gave 5 (32 mg, 24%), 6 (46 mg, 30%) and 7 (47 mg, 28%).
1
31,51-Dihydroxy-2,4-diisopropoxystilbene (
5
): brown oil; IR (UATR, cm´1): 3379, 2976, 1598; H-NMR
(400 MHz, CDCl3),
6.56 (d, J = 2.1 Hz, 2H), 6.48 (dd, J = 8.5, 2.3 Hz, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.25 (br s, 1H), 4.52 (m,
2H), 1.35 (d, J = 6 Hz, 6H), 1.33 (d, J = 6 Hz, 6H); 13C-NMR (100 MHz, CDCl3),
(ppm): 158.6, 157.0,
δ (ppm): 7.43 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 16.4 Hz, 1H), 6.84 (d, J = 16.4 Hz, 1H),
δ
156.5, 14´1.0, 127.4, 126.1, 124.3, 120.3, 107.5, 105.8, 103.2, 101.7, 71.2, 70.1, 22.1, 22.0; TOF-HRMS m/z
[M ´ H] , calcd for C20H23O4: 327.1602; found: 327.1594.
1
1
5 -Hydroxy-2,3 ,4,-triisopropoxystilbene (
MHz, CDCl3), (ppm): 7.45 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 16.4 Hz, 1H), 6.88 (d, J = 16.4 Hz, 1H), 6.61
6
): brown oil; IR (UATR, cm´1): 3393, 2976, 1588; 1H-NMR (400
δ
(t, J = 1.5 Hz, 1H), 6.56 (t, J = 1.6 Hz, 1H), 6.49 (dd, J = 8.5, 2.3 Hz, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.28 (t,
J = 2.1 Hz, 1H), 4.534 (m, 2H), 4.527 (m, 1H), 1.37 (d, J = 6 Hz, 6H), 1.343 (d, J = 6 Hz, 6H), 1.336 (d,
J = 6 Hz, 6H); 13C-NMR (100 MHz, CDCl3),
120.2, 107.4, 107.0, 105.4, 103.0, 101.8, 71.0, 70.0, 69.9, 22.2, 22.0; TOF-HRMS m/z [M
C23H29O4: 369.2071; found: 369.2066.
δ (ppm): 159.2, 158.6, 156.7, 156.5, 140.8, 127.4, 126.2, 124.4,
´
H]´, calcd for
2,31,4,51-Tetraisopropoxystilbene (
7
): white solid, mp 58–60 ˝C; IR (UATR, cm´1): 2976, 1585; 1H-NMR
(300 MHz, CDCl3), (ppm): 7.46 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 16.4 Hz, 1H), 6.91 (d, J = 16.4 Hz, 1H),
δ
6.62 (d, J = 2.2 Hz, 2H), 6.49 (dd, J = 8.5, 2.3 Hz, 1H), 6.45 (d, J = 3.9 Hz, 1H), 6.34 (t, J = 2.1 Hz, 1H),
4.55 (m, 4H), 1.37 (d, J = 6 Hz, 6H), 1.35 (d, J = 6 Hz, 12H), 1.34 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz,
CDCl3),
δ (ppm): 159.1, 158.6, 156.6, 140.5, 127.4, 126.7, 124.2, 120.4, 107.3, 106.4, 103.0, 102.5, 71.0, 69.9,
69.8, 22.2, 22.1; TOF-HRMS m/z [M + H]+, calcd for C26H37O4: 413.2686; found: 413.2679.
1
1
3.2.3. Preparation of 2,3 ,4,5 -Tetraacetoxystilbene (8)
Compound was prepared as previously described [31]. To a well-stirred solution of
0.21 mmol) in CH2Cl2 (2 mL), Et3N (0.17 mL, 1.23 mmol), DMAP (63 mg, 0.51 mmol) and acetyl
8
1 (50 mg,

Molecules 2016, 21, 489
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chloride (0.04 mL, 0.61 mmol) were added at room temperature, and the reaction mixture was stirred
for 3 h. After completion of the reaction, it was extracted with EtOAc (3 5 mL). The EtOAc layer was
washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a
crude product. Purification with column chromatography eluting with 40% EtOAc/hexanes gave
ˆ
8
˝
(60 mg, 70%). White solid, mp 129–131 C; IR (UATR, cm´1): 1762, 1605; 1H-NMR (300 MHz, CDCl3),
(ppm): 7.61 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 2.1 Hz, 2H), 7.05 (d, J = 15.9 Hz, 1H), 7.03 (dd, J = 8.4,
2.4 Hz, 1H), 6.97 (d, J = 15.9 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.85 (t, J = 2.1 Hz, 1H), 2.36 (s, 3H), 2.31 (s,
δ
6H), 2.29 (s, 3H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 168.93, 168.89, 168.8, 151.3, 150.5, 148.5, 139.4,
129.5, 127.2, 127.1, 123.5, 119.5, 117.0, 116.4, 114.8, 21.10, 21.09, 20.99; TOF-HRMS m/z [M + Na]+, calcd
for C22H20NaO8: 435.1056; found: 435.1050. The spectroscopic data were in agreement with earlier
reported values [32].
1
1
3.2.4. Preparation of 3 -O-Carbethoxymethyl-2,4,5 -triisopropoxystilbene (9)
To a solution of (100 mg, 0.27 mmol) in DMF (4 mL), K2CO3 (56 mg, 0.40 mmol) and ethyl
6
bromoacetate (0.04 mL, 0.4 mmol) were added at room temperature, and the reaction mixture was
stirred overnight. After completion of the reaction, water (5 mL) was added and the mixture was
extracted with EtOAc (3
ˆ
5 mL). The organic layer was washed with water (7
ˆ
5 mL) and brine,
dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude product, which
was purified by column chromatography on silica (15% EtOAc/hexanes) to furnish
Light brown oil; IR (UATR, cm´1): 2977, 1760, 1588; 1H-NMR (300 MHz, CDCl3),
9
(103 mg, 84%).
δ
(ppm): 7.45 (d,
J = 8.4 Hz, 1H), 7.33 (d, J = 16.5 Hz, 1H), 6.90 (d, J = 16.5 Hz, 1H), 6.68 (d, J = 1.5 Hz, 1H), 6.62 (d,
J = 1.8 Hz, 1H), 6.48 (dd, J = 8.7, 2.4 Hz, 1H), 6.45 (d, J = 2.1 Hz, 1H), 6.36 (t, J = 2.2 Hz, 1H), 4.62 (s, 2H),
4.54 (m, 3H), 4.26 (q, J = 7.1 Hz, 2H), 1.38 (d, J = 6 Hz, 6H), 1.344 (d, J = 6 Hz, 6H), 1.339 (d, J = 6 Hz,
6H), 1.30 (t, J = 6.8 Hz, 3H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 168.9, 159.0, 158.7, 156.6, 140.7, 127.4,
126.3, 124.6, 120.1, 107.6, 107.3, 104.7, 102.9, 101.4, 70.9, 69.9, 65.5, 61.8, 61.3, 22.1, 22.0, 14.1; TOF-HRMS
m/z [M + Na]+, calcd for C27H36NaO6: 479.2404; found: 479.2388.
1
1
3.2.5. Preparation of 3 -O-Carbethoxymethyl-2,4,5 -trihydroxystilbene (10)
A solution of BCl3 (3.07 mL, 3. 07 mmol) was added to a solution of
9 (233 mg, 0.51 mmol)
in CH2Cl2 (8 mL) at
stirred overnight. The reaction mixture was quenched with water (10 mL) and then extracted with
EtOAc (3 10 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and
´
78 ˝C under argon. Then it was allowed to warm to room temperature and
ˆ
concentrated under reduced pressure to give a crude product which was purified by preparative TLC
(50% EtOAc/hexanes) to give 10 (102 mg, 61%). Pale yellow solid, mp 198–200 ˝C; IR (UATR, cm´1):
3369, 1731, 1591; 1H-NMR (300 MHz, acetone-d6),
δ (ppm): 7.43 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 16.5 Hz,
1H), 6.97 (d, J = 16.5 Hz, 1H), 6.70 (br s,1H), 6.63 (br s,1H), 6.49 (d, J = 2.1 Hz, 1H), 6.49 (br s, 1H), 6.43
(dd, J = 8.4, 2.1 Hz, 1H), 6.34 (t, J = 2.1 Hz, 1H), 4.70 (s, 2H), 4.21 (q, J = 6.9 Hz, 2H), 1.25 (t, J = 6.9 Hz,
3H); 13C-NMR (75 MHz, acetone-d6),
δ (ppm): 169.4, 160.4, 159.4, 159.1, 156.9, 141.7, 128.4, 126.0, 124.9,
117.1, 108.4, 107.1, 104.4, 103.5, 101.6, 65.8, 61.3, 14.4; TOF-HRMS m/z [M + Na]+, calcd for C18H18NaO6:
353.0996; found: 353.0989.
1
1
3.2.6. Preparation of 3 -O-Carboxymethyl-2,4,5 -trihydroxystilbene (11)
To 10 (13 mg, 0.03 mmol), KOH (5% in EtOH, 1 mL) was added at room temperature, and the
reaction mixture was stirred for 10 min. The reaction was acidified with 2 N HCl to pH 5. The material
was extracted with EtOAc (3
filtered, and concentrated under reduced pressure to provide 11 (3 mg, 40%). Brown solid; IR (UATR,
cm´1): 3322, 2921, 2851, 1713; 1H-NMR (300 MHz, methanol-d4),
(ppm): 7.33 (d, J = 8.9 Hz, 1H), 7.30
(d, J = 16.4 Hz, 1H), 6.86 (d, J = 16.4 Hz, 1H), 6.58 (d, J = 1.6 Hz, 1H), 6.56 (d, J = 1.8 Hz, 1H), 6.31 (dd,
J = 5.9, 2.3 Hz, 1H), 6.30 (s, 1H), 6.25 (t, J = 2.1 Hz, 1H), 4.63(s, 2H); 13C-NMR (75 MHz, methanol-d4),
ˆ
5 mL), and the combined organic layers were dried over Na2SO4,
δ
δ

Molecules 2016, 21, 489
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(ppm):172.9, 160.8, 159.7, 159.4, 157.4, 142.4, 128.5, 126.2, 125.4, 117.7, 108.4, 107.3, 104.8, 103.6, 101.7,
´
65.9; TOF-HRMS m/z [M ´ H] , calcd for C16H13O6: 301.0718; found: 301.0708.
1
1
1
3.2.7. Preparation of 2 -Chloro-2,3 ,4,5 -tetrahydroxystilbene (12)
A mixture of (100 mg, 0.41 mmol) and N-chlorosuccinimide (54.8 mg, 0.41 mmol) in glacial
1
acetic acid (4 mL) was stirred at room temperature under argon for 3 h. After completion, solvent was
removed under reduced pressure. Purification by column chromatography (10% MeOH/CH2Cl2) gave
of 12 (42 mg, 36% yield). Light brown solid, mp 185–187 ˝C; IR (UATR, cm´1): 3196, 1599; 1H-NMR
(300 MHz, acetone-d6),
1H), 6.78 (d, J = 2.7 Hz, 1H), 6.45 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.7 Hz, 1H), 6.41 (dd, J = 8.4, 2.4 Hz, 1H);
13C-NMR (75 MHz, acetone-d6),
(ppm): 158.6, 156.5, 156.3, 153.9, 137.9, 127.9, 126.3, 121.3, 116.2, 110.4,
107.6, 103.8, 102.7, 102.3; TOF-HRMS m/z [M + H]+, calcd for C14H12ClO4: 279.0419; found: 279.0408.
δ (ppm): 7.44 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 16.4 Hz, 1H), 7.32 (d, J = 16.4 Hz,
δ
1
1
1
3.2.8. Preparation of 2 -Chloro-2,3 ,4,5 -tetramethoxystilbene (13)
To a well-stirred solution of 12 (88 mg, 0.3 mmol) in acetone (15 mL), K2CO3 (265 mg, 1.92 mmol)
and MeI (0.11 mL, 1.8 mmol) were added. The reaction mixture was stirred at 55 ˝C overnight and
monitored by TLC. After completion of the reaction, the mixture was diluted with water (10 mL), and
extracted with EtOAc (3
dried over Na2SO4, filtered and evaporated in vacuo. The residue obtained was purified over silica gel
using 20% EtOAc/hexanes to furnish 13 (40 mg, 36%). White solid; 1H-NMR (300 MHz, CDCl3),
ˆ
10 mL). The organic phase was washed with brine. The organic extract was
δ
(ppm): 7.56 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 16.5 Hz, 1H), 7.33 (d, J = 16.5 Hz, 1H), 6.83 (d, J = 2.7 Hz, 1H),
6.52 (dd, J = 8.7, 2.1 Hz, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.42 (t, J = 2.7 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 3H),
3.85 (s, 3H), 3.83 (s, 3H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 160.9, 158.6, 158.2, 155.9, 137.7, 127.7,
126.0, 123.2, 119.2, 113.8, 105.0, 101.9, 98.6, 98.4, 56.2, 55.6, 55.5, 55.4; TOF-HRMS m/z [M]+, calcd for
C18H20ClO4: 335.1045; found: 335.1035.
1
1
1
3.2.9. Preparation of 2 -Formyl-2,3 ,4,5 -tetraisopropoxystilbene (14)
POCl3 (0.16 mL, 1.69 mmol) was stirred with dry DMF (4 mL) at room temperature for 2 h under
argon. A solution of
mixture was further stirred overnight. After completion of the reaction, cool water (10 mL) was added,
and the mixture was extracted with EtOAc (3 10 mL). The organic layer was washed with water
(7 10 mL) and brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give
7
(200 mg, 0.48 mmol) in dry DMF (4 mL) was added at 0 ˝C and the reaction
ˆ
ˆ
a crude product, which was purified by column chromatography on silica (15% EtOAc/hexanes) to
furnish 14 (171 mg, 80%). Viscous yellow oil; IR (UATR, cm´1): 2976, 1671, 1586; 1H-NMR (300 MHz,
CDCl3),
6.75 (d, J = 2.1 Hz, 1H), 6.50 (dd, J = 8.6, 2.3 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 6.35 (d, J = 2.1 Hz, 1H),
4.69 (sept, J = 6 Hz, 1H), 4.48–4.63 (m, 3H), 1.33–1.41 (m, 24H); 13C-NMR (75 MHz, CDCl3),
(ppm):
δ (ppm): 10.53 (s, 1H), 8.08 (d, J = 16.4 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 16.4 Hz, 1H),
δ
191.1, 163.6, 162.7, 158.9, 156.7, 143.9, 128.1, 127.3, 125.4, 120.4, 116.8, 107.4, 105.0, 102.9, 100.1, 71.4, 70.9,
70.1, 69.9, 22.2, 22.04, 21.98; TOF-HRMS m/z [M + H]+, calcd for C27H37O5: 441.2635; found: 441.2639.
1
1
1
3.2.10. Preparation of 2 -Carboxy-2,3 ,4,5 -tetraisopropoxystilbene (15)
A solution of NaClO2 (260 mg, 2.88 mmol) and NaH2PO4 2H2O (447 mg, 2.86 mmol) in water
¨
(1.5 mL) was added to the solution of 14 (157 mg, 0.36 mmol) and 2-methyl-2-butene (0.13 mL, 1.5 mmol)
in acetone (1.5 mL) at room temperature. The reaction mixture was stirred for 1 h. After completion,
water (5 mL) was added, and the reaction mixture was extracted with EtOAc (3
ˆ
5 mL). The EtOAc
phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure
to give crude product, which was purified by column chromatography on silica (40% EtOAc/hexanes)
to furnish 15 (129 mg, 79%). Pale yellow solid, mp 154–156 ˝C; IR (UATR, cm´1): 2976, 1730, 1697,
1592; 1H-NMR (300 MHz, CDCl3),
δ
(ppm): 7.85 (d, J = 16.2 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.19 (d,
J = 16.2 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.49 (dd, J = 8.4, 2.1 Hz, 1H), 6.44 (d, J = 2.1 Hz, 1H), 6.42

Molecules 2016, 21, 489
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(d, J = 2.1 Hz, 1H), 4.68 (m, 2H), 4.54 (m, 2H), 1.44 (d, J = 6.3 Hz, 6H), 1.40 (d, J = 6.3 Hz, 6H), 1.37
(d, J = 6.3 Hz, 6H), 1.34 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz, CDCl3),
(ppm): 166.0, 160.9, 158.8,
δ
158.0, 156.6, 145.6, 128.1, 126.6, 126.5, 120.4, 107.4, 103.0, 101.5, 74.1, 71.0, 70.2, 70.0, 22.2, 22.1, 22.0, 21.9;
TOF-HRMS m/z [M + H]+, calcd for C27H37O6: 457.2585; found: 457.2587.
1
1
1
3.2.11. Preparation of 2 -Formyl-2,3 ,4,5 -tetrahydroxystilbene (16)
A solution of BCl3 (2.17 mL, 2.17 mmol) was added to a solution of 14 (119 mg, 0.27 mmol) in
CH2Cl2 (4 mL) at
and stirred overnight. Then, water (5 mL) was added, and the reaction was extracted with EtOAc
(3 5 mL). The EtOAc layer was washed with brine, dried over Na2SO4, filtered and concentrated
´
78 ˝C under argon. The reaction was allowed to warm up to room temperature
ˆ
under reduced pressure to give a crude product which was purified by u˝sing preparative TLC (40%
EtOAc/hexanes) to give 16 (35 mg, 48%). Yellow solid, decomposed >218 C; IR (UATR, cm´1): 3337,
1
1602; H-NMR (300 MHz, methanol-d4),
δ (ppm): 10.17 (s, 1H), 7.55 (d, J = 15.9 Hz, 1H), 7.38 (d,
J = 9 Hz, 1H), 7.23 (d, J = 15.9 Hz, 1H), 6.56 (d, J = 2 Hz, 1H), 6.33 (dd, J = 8.6, 2.3 Hz, 1H), 6.32 (br s,
1H), 6.14 (d, J = 2 Hz, 1H); 13C-NMR (75 MHz, methanol-d4), δ (ppm): 194.3, 167.3, 167.2, 160.1, 158.0,
148.0, 131.8, 129.6, 120.4, 117.2, 112.8, 108.5, 107.4, 103.6, 102.0; TOF-HRMS m/z [M
C15H11O5: 271.0612; found: 271.0607.
´
H]´, calcd for
1
1
1
3.2.12. Preparation of 2 -Hydroxy-2,3 ,4,5 -tetraisopropoxystilbene (17)
Compound 14 (121 mg, 0.27 mmol) and p-TsOH H2O (15 mg, 0.09 mmol) were dissolved in
¨
˝
methanol (1 mL). Then 30% hydrogen peroxide (0.07 mL, 0.54 mmol) was added at 0 C, and the
reaction was then warmed to room temperature and stirred for 1 h. Half-saturated sodium sulfite
solution (1 mL) was added, and the mixture was extracted with CH2Cl2 (3
was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to
ˆ
5 mL). The organic phase
give a crude product. Purification with preparative TLC (10% EtOAc/hexanes) gave 17 (78 mg, 66%).
Pale yellow solid; IR (UATR, cm´1): 3536, 2975, 1600; 1H-NMR (300 MHz, CDCl3),
δ (ppm): 7.53 (d,
J = 8.4 Hz, 1H), 7.38 (d, J = 16.8 Hz, 1H), 7.32 (d, J = 16.5 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.48 (dd,
J = 10.8, 2.4 Hz, 1H), 6.46 (br s, 1H), 6.40 (d, J = 2.4 Hz, 1H), 4.49 (m, 4H), 1.31–1.40 (m, 24H); 13C-NMR
(75 MHz, CDCl3),
δ (ppm): 158.4, 156.5, 150.7, 145.0, 138.8, 127.4, 124.6, 124.1, 121.1, 120.8, 107.5, 104.9,
103.3, 102.6, 71.8, 71.1, 71.0, 69.9, 22.2, 22.14, 22.13, 22.10; TOF-HRMS m/z [M + H]+, calcd for C26H37O5:
429.2635; found: 429.2643.
3.2.13. Preparation of 3-(2,4-Dihydroxyphenyl)-6,8-dihydroxyisochroman-1-one (18)
To a solution of 15 (104 mg, 0.23 mmol) in dry C˝H2Cl2 (2 mL), a solution of BBr3 (0.96 mL,
0.96 mmol) in CH2Cl2 was added under argon at
´
78 C, and the mixture was stirred for 20 min.
After completion of the reaction, water (5 mL) was added, and the mixture was extracted with EtOAc
(3 5 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated
ˆ
under reduced pressure to give a crude product. Purification with column chromatography eluting
with 5% MeOH/CH2Cl2 gave 18 (13 mg, 20%). Light brown solid, mp 210–212 ˝C; IR (UATR, cm´1):
3340, 3190, 1611; 1H-NMR (300 MHz, acetone-d6),
δ
(ppm): 7.28 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 2.1 Hz,
1H), 6.43 (dd, J = 8.4, 2.4 Hz, 1H), 6.35 (br s, 1H), 6.30 (d, J = 2.1 Hz, 1H), 5.83 (dd, J = 12, 3 Hz, 1H),
3.29 (dd, J = 16.5, 12.3 Hz, 1H), 3.04 (dd, J = 16.5, 3.3 Hz, 1H); 13C-NMR (75 MHz, acetone-d6),
δ
(ppm): 171.1, 165.13, 165.07, 159.5, 156.3, 143.8, 128.9, 117.2, 107.9, 107.3, 103.4, 101.95, 101.88, 76.5, 34.4;
TOF-HRMS m/z [M + Na]+, calcd for C15H12NaO6: 311.0526; found: 311.0529.
1
1
1
3.2.14. Preparation of 2 -(E)-Carbethoxyethenyl-2,3 ,4,5 -tetraisopropoxystilbene (19)
A solution of 14 in dry CH2Cl2 (4 mL) was added to EtO2CCH=PPh3 (176 mg, 0.51mmol)
˝
at 0 C under argon. Then the reaction mixture was allowed to warm to room temperature and
stirred overnight. After completion of the reaction, water (5 mL) was added, and the mixture was
extracted with EtOAc (3
ˆ
5 mL). The organic layer was washed with brine, dried over Na2SO4,

Molecules 2016, 21, 489
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filtered and concentrated under reduced pressure to give a crude product. Purification with column
chromatography eluting with 50% CH2Cl2/hexanes gave 19 (134 mg, 68%). Pale yellow oil; IR (UATR,
cm´1): 2977, 2932, 1706, 1589; 1H-NMR (300 MHz, CDCl3),
δ (ppm): 8.03 (d, J = 16.2 Hz, 1H), 7.45
(d, J = 8.4 Hz, 1H), 7.37 (d, J = 16.2 Hz, 1H), 7.15 (d, J = 16.2 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H), 6.50 (d,
J = 16.2 Hz, 1H), 6.49 (dd, J = 8.4, 2.4 Hz, 1H), 6.48 (br s, 1H), 6.39 (d, J = 2.1 Hz, 1H), 4.58 (m, 4H), 4.24
(q, J = 7.1 Hz, 2H), 1.28–1.40 (m, 27H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 168.2, 159.4, 158.7, 156.7,
142.3, 139.0, 128.4, 127.4, 125.6, 120.3, 120.1, 115.8, 107.2, 105.6, 102.8, 100.8, 71.0, 70.7, 69.9, 69.8, 59.9,
22.09, 22.04, 22.00, 14.3; TOF-HRMS m/z [M + H]+, calcd for C31H43O6: 511.3054; found: 511.3077.
1
1
3.2.15. Preparation of 3 ,5 -Diacetoxy-2,4-diisopropoxystilbene (20)
Et3N (0.05 mL, 0.33 mmol) and acetic anhydride (0.03 mL, 0.33 mmol) were added to a solution of
5
(50 mg, 0.15 mmol) in CH2Cl2 (2 mL) at room temperature. The reaction mixture was stirred for 2 h.
Water (5 mL) was added, and the reaction mixture was extracted with EtOAc (3 5 mL). The organic
ˆ
phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure
to give a crude product. Purification with column chromatography eluting with 20% EtOAc/hexanes
˝
1
gave 20 (46 mg, 74%). White solid, mp 65–67 C; IR (UATR, cm´1): 2978, 1768, 1599; H-NMR
(300 MHz, CDCl3),
6.94 (d, J = 16.2 Hz, 1H), 6.78 (t, J = 2.1 Hz, 1H), 6.46 (dd, J = 8.1, 2.4 Hz, 1H), 6.45 (br s, 1H), 4.51 (m,
2H), 2.25 (s, 6H), 1.36 (d, J = 6 Hz, 6H), 1.31 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz, CDCl3),
(ppm):
δ (ppm): 7.41 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 16.5 Hz, 1H), 7.08 (d, J = 2.1 Hz, 2H),
δ
168.7, 158.8, 156.6, 151.0, 140.7, 127.6, 125.8, 124.6, 119.4, 116.3, 113.3, 106.9, 102.5, 70.6, 69.7, 21.9, 21.8,
20.8; TOF-HRMS m/z [M + Na]+, calcd for C24H28NaO6: 435.1778; found: 435.1796.
1
1
3.2.16. Preparation of 3 ,5 -Diacetoxy-5-formyl-2,4-diisopropoxystilbene (21)
POCl3 (0.16 mL, 1.77 mmol) was stirred with dry DMF (1 mL) at room temperature for 2 h under
˝
argon. The solution of 20 (73 mg, 0.18 mmol) in dry DMF (1 mL) was added at 0 C. Then the reaction
mixture was allowed to warm to room temperature and stirred overnight. After completion of the
reaction, cool water (5 mL) was added, and the reaction mixture was extracted with EtOAc (3
ˆ
5 mL).
The organic layer was washed with water (7 5 mL) and brine, dried over Na2SO4, filtered and
ˆ
concentrated under reduced pressure to give a crude product, which was purified by preparative TLC
(20% EtOAc/hexanes) to furnish 21 (46 mg, 60%). Viscous yellow oil; IR (UATR, cm´1): 2979, 1768,
1671, 1593; 1H-NMR (300 MHz, CDCl3),
δ
(ppm): 10.32 (s, 1H), 8.03 (s, 1H), 7.27 (d, J = 16.5 Hz, 1H),
7.08 (d, J = 1.8 Hz, 2H), 7.03 (d, J = 16.5 Hz, 1H), 6.81 (t, J = 2.1 Hz, 1H), 6.44 (s, 1H), 4.66 (m, 2H), 2.29
(s, 6H), 1.43 (d, J = 6 Hz, 6H), 1.40 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz, CDCl3),
(ppm): 188.1, 168.7,
δ
161.8, 161.4, 151.0, 140.1, 126.6, 126.3, 124.2, 119.6, 118.8, 116.4, 113.8, 98.1, 71.2, 70.9, 21.67, 21.66, 20.8;
TOF-HRMS m/z [M + Na]+, calcd for C25H28NaO7: 463.1727; found: 463.1743.
1
1
3.2.17. Preparation of 3 ,5 -Diacetoxy-5-carboxy-2,4-diisopropoxystilbene (22)
A solution of NaClO2 (85 mg, 0.93 mmol) and NaH2PO4 2H2O (145 mg, 0.93 mmol) in water
¨
(0.5 mL) was added to a solution of 21 (51 mg, 0.12 mmol) and 2-methyl-2-butene (0.04 mL, 0.49 mmol)
in acetone (0.5 mL) at room temperature. The reaction mixture was stirred for 1 h. After completion,
water (5 mL) was added, and the reaction mixture was extracted with EtOAc (3
ˆ
5 mL). The organic
layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to
give a crude product, which was purified by column chromatography on silica (40% EtOAc/hexanes)
˝
to furnish 22 (39 mg, 73%). White solid, mp 106–108 C; IR (UATR, cm´1): 3267, 2980, 1769, 1732, 1601;
1H-NMR (300 MHz, CDCl3),
(d, J = 16.5 Hz, 1H), 6.82 (t, J = 2.1 Hz, 1H), 6.50 (s, 1H), 4.83 (sept, J = 6 Hz, 1H), 4.65 (sept, J = 6 Hz,
1H), 2.31 (s, 6H), 1.51 (d, J = 6 Hz, 6H), 1.45 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz, CDCl3),
(ppm):
δ (ppm): 8.35 (s, 1H), 7.26 (d, J = 16.5 Hz, 1H), 7.09 (d, J = 2.1 Hz, 2H), 7.08
δ
168.9, 165.3, 160.2, 157.2, 151.2, 140.1, 132.1, 127.5, 123.9, 121.4, 116.8, 114.2, 110.9, 99.0, 74.2, 71.4, 21.92,
21.88, 21.0; TOF-HRMS m/z [M + Na]+, calcd for C25H28NaO8: 479.1676; found: 479.1695.

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1
1
3.2.18. Preparation of 5-Formyl-2,3 ,4,5 -tetrahydroxystilbene (23)
A solution of BCl3 (0.42 mL, 0.42 mmol) was added to a solution of 21 (30 mg, 0.07 mmol)
in CH2Cl2 (2 mL) at
78 ˝C under argon. Then it was allowed to warm to room temperature and
stirred overnight. Water (5 mL) was then added, and the reaction mixture was extracted with EtOAc
(3 5 mL). The EtOAc was washed with brine, dried over Na2SO4, filtered and concentrated under
´
ˆ
reduced pressure to give a crude product which was purified by preparative TLC (40% EtOAc/hexanes)
to give 23 (15 mg, 78%). Yellow solid, decomposed >205 ˝C; IR (UATR, cm´1): 3208, 1626; 1H-NMR
(300 MHz, methanol-d4),
δ (ppm): 9.69 (s, 1H), 7.76 (s, 1H), 7.27 (d, J = 16.5 Hz, 1H), 6.95 (d, J = 16.5 Hz,
1H), 6.47 (d, J = 2.4 Hz, 2H), 6.25 (s, 1H), 6.16 (t, J = 2.1 Hz, 1H); 13C-NMR (75 MHz, methanol-d4),
δ
(ppm): 194.9´, 167.8, 164.8, 159.6, 141.6, 133.2, 128.4, 123.8, 121.0, 115.5, 105.9, 103.7, 102.7; TOF-HRMS
m/z [M ´ H] , calcd for C15H11O5: 271.0612; found: 271.0603.
1
1
3.2.19. Preparation of 5-Carboxy-2,3 ,4,5 -tetrahydroxystilbene (24)
A solution of BCl3 (0.88 mL, 0.88 mmol) was added to a solution of 22 (68 mg, 0.15 mmol) in
CH2Cl2 (2 mL) at
´
78 ˝C under argon. Then the reaction was allowed to warm to room temperature
and stirred overnight. Water (5 mL) was added, and the mixture was extracted with EtOAc (3
ˆ
5 mL).
The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under
reduced pressure to give a crude product which was purified by Sephadex LH20 (methanol) to
give 24 (27 mg, 63%). Yellow solid, decomposed >230 ˝C; IR (UATR, cm´1): 3337, 1616; H-NMR
1
(300 MHz, methanol-d4),
δ (ppm): 8.01 (s, 1H), 7.24 (d, J = 16.5 Hz, 1H), 6.92 (d, J = 16.5 Hz, 1H), 6.49 (d,
J = 2.1 Hz, 2H), 6.36 (s, 1H), 6.19 (t, J = 2.1 Hz, 1H); 13C-NMR (75 MHz, methanol-d4),
δ
(ppm): 173.5,
´
164.2, 162.8, 159.6, 141.6, 130.0, 128.5, 123.8, 118.9, 106.3, 105.8, 103.3, 102.7; TOF-HRMS m/z [M
calcd for C15H11O6: 287.0561; found: 287.0549.
´
H] ,
1
1
3.2.20. Preparation of 5-Formyl-3 ,5 -dihydroxy-2,4-diisopropoxystilbene (25)
To 21 (59 mg, 0.12 mmol), KOH (5% in EtOH, 1 mL) was added at room temperature, and
the reaction mixture was stirred for 10 min. After completion of the reaction, the reaction was
extracted with EtOAc (3
filtered and concentrated under reduced pressure to give a crude product. Purification with column
chromatography eluting with 50% EtOAc in hexanes gave 25 (41 mg, 93%). Yellow solid, mp 186–188 C;
IR (UATR, cm´1): 3355, 2979, 1588; 1H-NMR (300 MHz, acetone-d6),
(ppm): 10.32 (s, 1H), 7.99 (s, 1H),
7.30 (d, J = 16.5 Hz, 1H), 7.05 (d, J = 16.5 Hz, 1H), 6.80 (s, 1H), 6.59 (d, J = 2 Hz, 2H), 6.29 (t, J = 2 Hz,
1H), 4.91 (m, 2H), 1.43 (d, J = 6.3 Hz, 6H), 1.41 (d, J = 6.4 Hz, 6H); 13C-NMR (75 MHz, acetone-d6),
ˆ
5 mL). The EtOAc phase was washed with brine, dried over Na2SO4,
˝
δ
δ
(ppm): 188.0, 162.7, 162.3, 159.6, 140.9, 129.2, 126.6, 122.9, 121.1, 120.1, 105.8, 102.9, 100.0, 72.1, 71.8,
22.2, 22.1; TOF-HRMS m/z [M + H]+, calcd for C21H25O5: 357.1696; found: 357.1712.
1
1
3.2.21. Preparation of 5-Carboxy-3 ,5 -dihydroxy-2,4-diisopropoxystilbene (26)
To 22 (30 mg, 0.07 mmol), KOH (5% in EtOH, 1 mL) was added at room temperature, and the
reaction mixture was stirred for 10 min. The reaction was acidified with 2 N HCl to pH 5. The material
was extracted with EtOAc (3
filtered, and concentrated under reduced pressure to provide 26 (10 mg, 40%). Light brown solid, mp
112–114 ˝C; 1H-NMR (300 MHz, acetone-d6),
(ppm): 8.23 (s, 1H), 7.31 (d, J = 16 Hz, 1H), 7.07 (d,
J = 16 Hz, 1H), 6.88 (s, 1H), 6.59 (d, J = 2 Hz, 2H), 6.30 (t, J = 2 Hz, 1H), 5.04 (sept, J = 6 Hz, 1H), 4.89
(sept, J = 6 Hz, 1H), 1.46 (d, J = 6.3 Hz, 6H), 1.43 (d, J = 6 Hz, 6H); 13C-NMR (75 MHz, acetone-d6),
ˆ
5 mL), and the combined organic layers were dried over Na2SO4,
δ
δ
(ppm): 165.7, 160.8, 159.6, 158.5, 140.8, 131.5, 129.6, 122.9, 122.0, 112.7, 108.0, 103.0, 101.1, 74.4, 71.9,
´
22.2, 22.1; TOF-HRMS m/z [M ´ H] , calcd for C21H23O6: 357.1500; found: 371.1502.

Molecules 2016, 21, 489
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1
1
3.2.22. Preparation of 3 ,5 -Diacetoxy-5-hydroxy-2,4-diisopropoxystilbene (27)
A solution of 30% hydrogen peroxide (0.03 mL, 0.192 mmol) was added to a methanolic mixture of
˝
21 (44 mg, 0.10 mmol) and p-TsOH
¨
H2O (5 mg, 0.03 mmol) at 0 C. The reaction mixture was allowed
to warm to room temperature and stirred for 1 h. Half-saturated sodium sulfite solution (1 mL) was
added, and the mixture was extracted with CH2Cl2 (3 5 mL). The CH2Cl2 layer was washed with
brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude product.
ˆ
Purification with preparative TLC (30% EtOAc/hexanes) gave 27 (19 mg, 47%). Pale yellow solid,
1
mp 138–140 ˝C; IR (UATR, cm´1): 3525, 2977, 1767; H-NMR (300 MHz, CDCl3),
δ (ppm): 7.34 (d,
J = 16.2 Hz, 1H), 7.13 (s, 1H), 7.07 (d, J = 2.1 Hz, 2H), 6.87 (d, J = 16.2 Hz, 1H), 6.79 (t, J = 1.8 Hz, 1H),
6.50 (s, 1H), 4.54 (sept, J = 6 Hz, 1H), 4.33 (sept, J = 6 Hz, 1H), 2.30 (s, 6H), 1.36 (d, J = 6.3 Hz, 6H), 1.34
(d, J = 6.3 Hz, 6H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 169.0, 151.1, 149.4, 145.0, 141.4, 140.6, 125.3,
125.2, 120.9, 116.5, 113.7, 111.3, 103.7, 73.3, 71.8, 22.2, 22.0, 21.0; TOF-HRMS m/z [M + Na]+, calcd for
C24H28NaO7: 451.1727; found: 451.1723.
3.3. Biological Activities
In this study, statistical analysis of the biological data was performed using the Student's t-test.
All results were expressed as arithmetic mean ˘ standard deviation (SD).
3.3.1. DPPH Radical Assay
The experiment was performed according to an established method [33]. The experiment was
performed in a 96-well plate. The reduction of the DPPH was measured by reading the absorbance
at 510 nm with a Perkin Elmer Victor3 multilabel counter. Oxyresveratrol and Trolox were used as
positive controls.
3.3.2. Superoxide Radical Assay
The assay was based on the capacity of the sample to inhibit the reduction of nitroblue
tetrazolium (NBT) in the riboflavin-light-NBT system [18
positive controls.
,34]. Oxyresveratrol and Trolox were used as
3.3.3. Inhibitory Effect on Supercoiled DNA Breakage
The assay was performed as previously described [18]. This assay measured the ability of the test
sample to protect DNA against damage induced by the photochemical reaction of riboflavin, with
oxyresveratrol and Trolox used as positive controls.
3.3.4. Determination of Anti-Herpes Simplex Virus Activity
Anti-HSV activities of the compounds were determined using a modified plaque reduction assay,
with acyclovir as a positive control [35].
3.3.5. Neuraminidase (NA) Inhibition Assay
This assay was performed using the method previously described by Potier and co-workers [36].
The fluorescence was measured using SpectraMax M5 multi-detection microplate reader (Molecular
Devices, Sunnyvale, CA, USA) with excitation and emission wave lengths of 365 and 450 nm,
respectively. Oseltamivir was used as a positive control.
3.3.6. Determination of α-Glucosidase Inhibitory Activity
The assay was performed in a 96-well plate and based on the capacity of the sample to inhibit the
hydrolysis of p-nitrophenyl-α-D-glucoside (PNPG) by α-glucosidase to release p-nitrophenol (PNP), a
yellow color agent that can be monitored at 405 nm [20]. Acarbose was used as a positive control.

Molecules 2016, 21, 489
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3.3.7. Determination of Cytotoxic Activity
The assays for cytotoxicity were carried out according to a previous report [37]. Cell viability was
determined using MTT assay. Doxorubicin was used as the positive control.
4. Conclusions
A total of 26 derivatives were prepared from oxyresveratrol (1) through several types of reactions.
Selective di-O-alkylation is possible because the two hydroxy groups at C-2 and C-4 of ring A of
1
are
obviously more reactive than those on ring B. Electrophilic substitution on the B ring is more facile
than on the A ring. However, the relative reactivity can be reversed by manipulating the type and
the number of protecting groups. Through this strategy, electrophilic substitution reactions can be
selectively directed to ring A or B. This finding has provided a new and useful approach for the future
chemical modification of similarly oxygenated aromatic structures.
Compound
1
and analogs
2
–
27 were evaluated for a number of biological activities, including free
-glucosidase
radical scavenging activity, DNA protective properties, antiherpetic activity, inhibition of
α
and neuraminidase, and cytotoxicity against some cancer cell lines. Some of the derivatives were
equally active or more potent than the parent compound. It is interesting to note that the partially
1
1
1
1
etherified products 3 ,5 -dihydroxy-2,4-dimethoxystilbene (2) and 5 -hydroxy-2,3 ,4-trimethoxystilbene
) demonstrated encouraging antiherpetic and DNA protective activities. Compared with the parent
compound, and should have higher lipophilicity, and thus should have greater potential as
preventive agents for neurodegenerative diseases. 51-Hydroxy-2,31,4,-triisopropoxystilbene (
), an
etherified analog with retained anti- -glucosidase activity but with a potentially slower rate of
metabolism than that of
(3
2
3
6
α
1
, should be a better lead for oral anti-diabetic drug development. In addition,
the mixed esterified/etherified derivative 31,51-diacetoxy-2,4-diisopropoxystilbene (20) displayed
pronounced in vitro cytotoxicity against HeLa cells, and warrants further investigation in animals.
Acknowledgments: We are grateful to the Thailand Research Fund for a research grant (BRG 5580004). Thanks
are also due to the Research Instrument Center of the Faculty of Pharmaceutical Sciences, Chulalongkorn
University for providing research facilities. For cytotoxicity assays, Pakamas Intachote (Laboratory of Immunology,
Chulabhorn Research Institute) and Suchada Sengsai (Integrated Research Unit, Chulabhorn Research Institute)
are greatly appreciated.
Author Contributions: Nutputsorn Chatsumpun, Poonsakdi Ploypradith and Kittisak Likhitwitayawuid
conceived and designed the research, and prepared the manuscript. Nutputsorn Chatsumpun, Taksina
Chuanasa, Boonchoo Sritularak, Vimolmas Lipipun and Vichien Jongbunprasert performed the experiments.
Somsak Ruchirawat contributed chemical reagents and analysis tools. Poonsakdi Ploypradith and Kittisak
Likhitwitayawuid co-supervised the project.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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