Synthesis of novel trans-stilbene derivatives and evaluation of their potent antioxidant and neuroprotective effects

Article abstract of DOI:10.1016/j.ejmech.2009.03.011

A convenient synthesis and the biological properties of new amides, esters and other derivatives of trans-stilbene are described. The key synthetic strategies involve the Wittig-Horner reaction of a phosphonium salt 9 and an aldehyde 10 to generate (E)- or (Z)-olefins and a coupling reaction of an acid 12 and various amines 13a-n to give trans-stilbene derivatives 15a-n in high yields. A amide derivative 15g showed three times more in vitro free radical-scavenging activity than resveratrol, while another 15d exhibited strong inhibitory activity against lipopolysaccharide (LPS)^a-induced NO generation. Allylamide analogue 15a showed the most potent neuroprotective activity in glutamate-induced primary cortical neuron cells.

Full text of DOI:10.1016/j.ejmech.2009.03.011

European Journal of Medicinal Chemistry 44 (2009) 3166–3174
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel trans-stilbene derivatives and evaluation of their potent
antioxidant and neuroprotective effects
Jae-Chul Jung a, Eunyoung Lim b, Yongnam Lee b, Jun-Mo Kang a, Heejeong Kim a, Soyong Jang a,
**
,
b,
*
Seikwan Oh a , Mankil Jung
a Department of Neuroscience and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea
b Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient synthesis and the biological properties of new amides, esters and other derivatives of trans-
stilbene are described. The key synthetic strategies involve the Wittig–Horner reaction of a phosphonium
salt 9 and an aldehyde 10 to generate (E)- or (Z)-olefins and a coupling reaction of an acid 12 and various
amines 13a–n to give trans-stilbene derivatives 15a–n in high yields. A amide derivative 15g showed
three times more in vitro free radical-scavenging activity than resveratrol, while another 15d exhibited
strong inhibitory activity against lipopolysaccharide (LPS)a-induced NO generation. Allylamide analogue
15a showed the most potent neuroprotective activity in glutamate-induced primary cortical neuron cells.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 8 October 2008
Received in revised form
7 March 2009
Accepted 12 March 2009
Available online 24 March 2009
Keywords:
trans-Stilbene derivatives
Wittig–Horner reaction
Free radical-scavenging effects
LPS-induced NO generation
Neuroprotective activity
1. Introduction
[19–22]. They exhibit biological activities that inhibit lipopolysac-
charide (LPS)-induced production of PGE2 and NO in the mouse
E-Resveratrol (3,40,5-trihydroxy-trans-stilbene) is a phytoalexin
found in grapes, berries, peanuts, and red wine, and its derivatives
have been recognized to exhibit a wide range of biological and
pharmacological properties [1–3]. They were shown to promote
nitric oxide production [4,5], to inhibit platelet aggregation [6–8],
and to increase high-density lipoprotein cholesterol [4,9,10] as well
as to function as antitumor agents [11–14] and antioxidants [15,16].
The structural features of natural and synthetic resveratrol
analogues consist of diaryl groups on either end of an active double
bond to generate the stilbene skeleton. Although the presence of
the double bond in resveratrol gives rise to trans- and cis-isomeric
forms of resveratrol [(E)- and (Z)-diastereomers, respectively], the
trans-form is the thermodynamically more stable form (Fig. 1)
[17,18].
macrophage cell line RAW 264.7 [23,24]. As part of its anti-
inflammatory action, resveratrol attenuates expression of the
NF-kappaB-dependent inflammatory markers inducible nitric
oxide synthase and IL-6 [25]. trans-Resveratrol has been shown to
exert potent neuroprotective actions, which are attributed to its
antioxidant properties [26]. Chronic administration of resveratrol
to animal models of neurodegenerative injury and excitotoxic brain
damage resulted in partial neuroprotective action [27]. Resveratrol
also attenuated oxidized low density lipoprotein (oxLDL)-induced
cytotoxicity in PC12 cells with neuroprotection [28]. These studies
suggest that trans-resveratrol derivatives are the bioactive
components that suppress NO generation in microglia cells, and
that are responsible for the neuroprotective action in primary
cortical neuron cells. In spite of extensive efforts, the mechanism of
action related to the free radical-scavenging effects of trans-stil-
bene derivatives has not yet been established.
In a preliminary communication [29], we reported the design
and synthesis of resveratrol derivatives based on lithospermic
acid B as inhibitors of protein tyrosine phosphatase 1B. As part of
our ongoing studies on the development of antioxidative and
neuroprotective stilbene-derived drug candidates, we report in
this paper the convenient synthesis of nineteen novel trans-stil-
bene derivatives and their in vitro free radical-scavenging effects,
Recently, a great deal of interest has been generated by resver-
atrol and its analogues due to their antioxidative effects against
reactive oxygen species (ROS), which cause oxidative damage to
biological substances and are involved in aging and inflammation
* Corresponding author. Tel.: þ82 2 2123 2648; fax: þ82 2 364 7050.
** Corresponding author. Tel.: þ82 2 2650 5749; fax: þ82 2 2653 8891.
E-mail addresses: skoh@ewha.ac.kr (M. Oh), mjung@yonsei.ac.kr (M. Jung).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.011

J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
3167
R8
R9
R4
OH
R5
R6
HO
HO
O
R1
R2
O
R7
OH
R3
1-4
5-8
1 R1=R2=R3=R4=R5=R6=H, Stilbene
5 R7=R8=OH, R9=H, Piceid
2 R1=R3=R5=OH, R2=R4=R6=H, Resveratrol
3 R1=R3=R4=R5=OH, R2=R6=H, Piceatannol
6 R7=R9=OH, R8=OMe, Rhaponticin
7 R7=R8=OH, R9=OMe, Isorhaponticin
4 R1=R3=OH, R2=R4=R6=H, R5=OMe, 3,5-Dihydroxy-4'-methoxystilbene 8 R7=OH, R8=OMe, R9=H, Desoxyrhaponticin
Fig. 1. Chemical structures of resveratrol and its analogues.
inhibition of LPS-induced NO generation, and neuroprotective
effects.
separated by column chromatography. In the proton NMR anal-
yses, we found that the vinyl protons for the (E)-form of
compound 11 showed at 7.13 ppm and 6.95 ppm as a doublet
(J ¼ 16.0 Hz), while the vinyl protons for the (Z)-form of
compound 11 exhibited at 6.61 ppm and 6.51 ppm as a doublet
(J ¼ 12.2 Hz). According to a published procedure [29], E-olefin
compound 11 was converted to the protected acid 12 in three
steps in a 66% overall yield. The protected acid 12 was coupled
with several amines 13a–n [allyl amine, n-decyl amine,
tetrahydrofurfuryl amine, furfuryl amine, cyclohexyl amine,
(30-aminopropyl)-2-pyrrolidinone, 2-fluorobenzylamine, aniline,
2. Results and discussion
2.1. Chemistry
A series of trans-stilbene analogues 15a–n, 17–18, 22, 24, and
26 were prepared from commercially available 4-(chlor-
omethyl)benzoic acid and 3,4-dihydroxybenzaldehyde as starting
materials. The phosphonium salt 9 was prepared from commer-
cially available 4-(chloromethyl)-benzoic acid 19 in three steps in
an overall 79% yield according to a previously described proce-
dure [29]. A Wittig–Horner reaction of 9 with freshly protected
tert-butyl dimethylsilanyloxy (TBS)-aldehyde 10 [30,31] gave ester
11 (3:2 ratio of E/Z, 86% combined yield), which was cleanly
ethyl-3-aminoethylbenzoate,
morpholine,
1-allylpiperazine,
1-benzoylpiperazin, 4-methylpiperidine, 4-benzylpiperidine] to
give amides 14a–n, which were subjected to removal of a TBS
group from the dihydroxy groups to yield new trans-stilbene
derivatives 15a–n in good yields (Scheme 1).
O
H
O
N
O
R1O
R1O
O
OCH3
d or e
-IPh3+P
R
16 R1 = TBS
18 R1 = H
9
b
R1O
a
+
R1O
O
11 R = CO2Me, R' = TBS
R
TBSO
TBSO
CHO
ref.29
R1O
R1O
12 R = CO2H, R' = TBS
17 R = CO2Me, R' = H
b
13a-n
c
14a-n R1 = TBS
15a-n R1 = H
10
b
15a-n; R
H
N
O
N
N
O
N
H
H
N
H
N
H
C9H19
N
H
N
H
F
O
a
c
e
g
b
d
f
N
N
N
N
OEt
N
N
N
H
N
O
N
H
O
O
m
n
j
h
i
k
l
Scheme 1. Synthesis of new trans-stilbene derivatives 15a–n. Reagents and conditions: (a) NaH (60% dispersion in paraffin liquid, 1.3 equiv), CH2Cl2, 0 ꢀC, 16 h; (b) TBAF (2.5 equiv),
THF, 0 ꢀC, 30 min to 1 h. (c) 13a–n, EDCI (3.0 equiv), HOBt (3.0 equiv), DMF, 1 h; (d) 11, DIBAL-H (2.5 equiv), CH2Cl2, ꢁ78 ꢀC, 1 h, and then EDCI (3.0 equiv), HOBt (3.0 equiv), hippuric
acid (1.5 equiv), CH2Cl2, 40 ꢀC, 36 h; (e) 17, LAH (1.5 equiv), THF, ꢁ10 ꢀC, 1 h, and then EDCI (3.0 equiv), HOBt (3.0 equiv), hippuric acid (1.5 equiv), CH2Cl2, 40 ꢀC, 16 h.

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J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
R
O
CO2H
a
R1O
R1O
5 steps
ref. 29
OMe
Cl
(EtO)2OP
21 R = CO2Me, R1 = TBS
22 R = CO2Me, R1 = H
23 R = CH2OH, R1 = TBS
24 R = CH2OH, R1 = H
25 R = CHO, R1 = TBS
26 R = CHO, R1 = H
19
20
b
b
b
c
d
Scheme 2. Synthetic route for the preparation of compounds 22, 24 and 26. Reagents and conditions: (a) 10, NaH (1.3 equiv), CH2Cl2, 0 ꢀC, 16 h; (b) TBAF (2.5 equiv), THF, 0 ꢀC,
30 min; (c) DIBAL-H (2.5 equiv), CH2Cl2, ꢁ78 ꢀC, 1 h; (d) TEMPO (0.3 equiv), PhI(OAc)2 (3.0 equiv), CH2Cl2, rt, 7 h.
Treatment of ester 11 with 2.5 equiv of diisobutylalumi-
niumhydride (DIBAL-H) in dry CH2Cl2 led to the reduction of the
ester group, and the resulting primary alcohol underwent
coupling with 1.5 equiv of hippuric acid using ethyl (dimethyla-
minopropyl)carbodiimide (EDCI) and 1-hydroxy-benzotriazole
(HOBt) to afford 16 in a 55% yield in two steps [32,33]. At this
stage, coupling of the primary alcohol with hippuric acid was also
accomplished by dicyclohexylcarbodiimide (DCC)/CH2Cl2 [34,35],
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluraniumhexa-
fluorophosphate (HATU)/CH2Cl2 [36,37], and bis(2-oxo-3-oxazoli-
dinyl)phosphinic chloride (BOPCl) in CH2Cl2 [38,39]. Although
these latter conditions were more convenient for handling, the
HOBt/EDCI method afforded a superior yield. Removal of the TBS
group of 16 was achieved using trifluoroacetic acid (TFA) or tet-
rabutylammonium fluoride (TBAF) conditions to give 18 in 87% or
90% yields, respectively. In addition, the deprotection of ester 11
was accomplished by a TBAF condition with 72% yield. Reduction
of 17 using lithium aluminum hydride (LAH) in THF afforded the
terminal alcohol, which was coupled with hippuric acid in the
presence of HOBt and EDCI to give 18 in two steps with a 57%
overall yield (Scheme 1).
compound 20 with 10 in the presence of sodium hydride (NaH, 60%
dispersion in paraffin liquid) in CH2Cl2 afforded 21 in 76% yield.
Treatment of 21 with TBAF afforded 22 in 37% yield. Reduction of 21
with DIBAL-H in CH2Cl2 gave primary alcohol 23, which was
subsequently treated with TBAF to afford 24 in 70% yield. Treatment
of 23 with PhI(OAc)2 in the presence of 2,2,6,6-tetramethyl-1-
piperidinyloxy, free radical (TEMPO) gave aldehyde 25 in 71% yield,
and 25 was readily subjected to deprotection of the TBS group for
the desired product 26 in 70% yield (Scheme 2).
2.2. Chemical evaluation
2.2.1. Radical-scavenging activity
DPPH (diphenyl-1-picrylhydrazyl) radicals can be used in
preliminary screening of compounds capable of scavenging reac-
tive oxygen species, since these nitrogen radicals are much more
stable and easier to handle than oxygen free radicals. The radical-
scavenging activities of the new trans-stilbene analogues were
evaluated by the published test method [40,41] over the
concentration range of 10–200 mM (Table 1). Favorable scavenging
ratios were found for almost all of compounds 15a–n, 17–18, 22,
24, and 26 ranging from 30 to 75% at the highest concentration,
Methyl-(E)-3-{4-[(diethoxyphosphinyl)methyl]phenyl}acrylate
(20) was also prepared from commercially available 4-(chlor-
omethyl)benzoic acid (19) in five steps in an overall 27% yield
according to a previously published procedure [29]. Coupling of
200 mM (Fig. 2). To our surprise, most of the trans-stilbene
derivatives showed good DPPH radical-scavenging activity
compared with resveratrol, although compounds 15c, 15j, 15n, 17,
22, and 24 were less effective than the reference material. Among
these analogues, amide derivative 15g exhibited tentatively potent
radical-scavenging activity. Furthermore we found that acyclic
amine moieties (15a–15g) showed better radical-scavenging
activity than the cyclic amine moieties (15h–15n) as shown in
Table 1.
Table 1
Rate (IC50 mM) of scavenging DPPH radical of resveratrol and trans-stilbene
analogues 15a–n, 17–18, 22, 24, and 26 and resveratrol.
Compound
DPPH radical-scavenging activity (%)
10 50 100
IC50 (mM)
m
M
m
M
m
M
200 mM
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
17
15.00
18.48
16.73
21.95
17.19
12.88
29.82
10.22
21.27
5.70
24.76
17.19
15.07
4.75
2.31
21.22
5.91
29.01
41.95
17.99
35.06
22.46
22.16
42.23
17.58
29.28
10.19
31.18
24.58
21.34
10.12
8.95
41.70
75.17
74.13
79.08
31.74
79.17
68.68
44.03
87.98
59.81
62.79
29.40
62.37
59.07
48.63
25.80
29.28
46.91
5.29
98.06
47.87
>200
69.0
127.93
>200
43.59
>200
126.63
>200
140.96
184.55
>200
>200
>200
>200
>200
>200
>200
>200
26.46
47.99
40.22
28.45
52.32
27.15
44.13
19.14
40.05
36.73
31.49
17.41
16.50
33.17
5.73
18
22
24
26
28.15
5.31
8.64
21.25
8.44
6.82
13.15
9.45
11.48
28.77
28.39
17.28
54.38
33.79
Resveratrola
Fig. 2. DPPH bleaching kinetics in the presence of different concentrations (10, 50, 100,
and 200 mM) of prepared novel trans-stilbene derivatives 15a–n, 17–18, 22, 24, and 26.
a
Resveratrol is a reference material.

J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
3169
120
110
100
90
80
70
60
50
40
30
20
10
0
control
1 µM
10 µM
50 µM
sham MeOH LPS
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
120
110
100
90
80
70
60
50
40
30
20
10
0
control
1 µM
10 µM
50 µM
sham MeOH
LPS
15k
15l
15m
15n
17
18
22
24
26
Fig. 3. Effect of trans-resveratrol derivatives 15a–n, 17–18, 22, 24, and 26 on nitrite production in LPS-stimulated BV-2 microglia cells. Cells were treated with 100 ng/mL LPS, then
various concentrations of these compounds (1 M, 10 M, and 50
M) were added for 24 h at 37 ꢀC. Values indicate nitrite production from culture supernatants of LPS-treated cells
m
m
m
with or without compounds. Data represent the mean ꢂ standard deviation of three observations.
Fig. 4. Phase-contrast micrographs showing glutamate-induced neurotoxicity and neuroprotection of compound 26 in cultured cortical neurons. Neurons without compound
treatment showed healthy shapes (A) and compound 26 alone did not induce neurotoxicity (B). Compound 26 (50
treatment showed neuroprotection (C), while glutamate alone (50
M) induced neurotoxicity (D) (original magnification, 200). Bar ¼ 1 mm.
mM) was given for 24 h with glutamate (50 m
M) at 37 ꢀC and co-
m

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J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
80
70
60
50
40
30
20
10
0
control
1 µM
10 µM
50 µM
sham
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
Glu
80
70
60
50
40
30
20
10
0
control
1 µM
10 µM
50 µM
sham
15k
15l
15m
15n
17
18
22
24
26
Glu
Fig. 5. Protection of glutamate-induced neurotoxicity in cultured cortical neurons for compounds 15a–n, 17–18, 22, 24, and 26. Compounds were co-administered with glutamate
(50
observations.
m
M) for 24 h at 37 ꢀC. Lactate dehydrogenase (LDH) was measured at 340 nm using a microplate spectrophotometer. Data represent the mean ꢂ standard deviation of three
2.3. Biological assays in cultured cortical neuron and BV-2
microglia cells
(50 mM) induced neurotoxicity (Fig. 4). Allylamide derivative 15a
showed the most potent neuroprotective activity in glutamate-
induced primary cortical neuron cells (Fig. 5). Among the prepared
trans-stilbene analogues, compounds 18 and 26 exhibited good
2.3.1. Nitric oxide assay
Nitrite was used as a measure of NO production. The in vitro
suppression of LPS-induced NO generation of the prepared trans-
stilbenes was evaluated by the published test method [21] and the
results are summarized in Fig. 3. Most of the trans-stilbenes
inhibited nitrite accumulation in LPS-stimulated microglia BV-2
cells, with amide derivative 15d exhibiting the greatest inhibitory
activity for LPS-induced NO generation. Interestingly, compound
15n and propenylaldehyde analogue 26 effectively inhibited NO
anti-neurotoxicity activities at the lowest dose (1
compounds 15a, 15d, 15n, and 26 showed favorable anti-neuro-
toxicity activity at higher doses (10–50 M, Fig. 5). Excitotoxic
neuronal death was prevented by inclusion of 1–50 mM of
compounds 15a, 15d, and 26 in the culture media.
mM), while
m
3. Conclusion
production at the lowest concentration of 1
15a and 15d exhibited considerable inhibition of NO production at
10–50 M concentrations in LPS-stimulated BV-2 cells (Fig. 3).
These results also showed that compounds 15a, 15d, 15n, and 26
possessed good anti-inflammatory activity.
m
M, while compounds
An efficient method for synthesis of new trans-stilbene
analogues 15a–n, 17–18, 22, 24, and 26 has been described.
Furthermore, all analogues were evaluated for free radical-scav-
enging activity, suppression of LPS-induced NO generation, and
anti-excitotoxicity in vitro. Most of the trans-stilbene derivatives
showed 2–3 times more radical-scavenging activity than resvera-
trol. They were also potent inhibitors of nitrite accumulation in
LPS-stimulated microglia BV-2 cells.
m
2.3.2. Lactate dehydrogenase (LDH) assay
Lactate dehydrogenases are valuable in vitro markers for cellular
toxicity. In these studies, LDH activity was used as a measure of the
neuroprotective effects of the novel trans-stilbene compounds.
Compounds were added to the culture medium with glutamate,
and neuroprotection was observed via microscopic images. Phase-
contrast micrographs showed glutamate-induced neurotoxicity
and neuroprotection in cultured cortical neurons for the tested
compounds. Cultured neurons with neither glutamate nor
compound showed healthy cellular shapes, while glutamate
Amide derivatives 15a, 15d, and 15n (ED50; 1.95, 0.82, 1.12
respectively) and propenylaldehyde analogue 26 (ED50, 1.51
m
M,
mM)
were especially effective in inhibiting nitrite production. In addi-
tion, excitotoxic neuronal death was prevented by inclusion of 1–
50 mM of compounds 15a, 15d, and 26 (ED50, 29.6, 58.3, 42.3 mM,
respectively). Of these analogues, allylamide derivatives 15a
showed the most potent protective activity against glutamate-
induced neurotoxicity and was a potent anti-inflammatory agent.

J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
3171
4. Experimental
1511, 1471, 1422, 1297, 1254, 1228, 1123, 1007, 988, 838, 781 cmꢁ1
1H NMR (250 MHz, CDCl3)
;
d
¼ 7.91 (d, 2H, J ¼ 8.3 Hz, aromatic H),
4.1. Chemistry
7.33 (d, 2H, J ¼ 8.3 Hz, aromatic H), 6.70–6.67 (m, 3H, aromatic H),
6.60 (d, 1H, J ¼ 12.0 Hz, vinyl CH), 6.50 (d, 1H, J ¼ 12.0 Hz, vinyl CH),
3.90 (s, 3H, OCH3), 0.98–0.89 (m, 18H, tert-butyl H), 0.20–0.04 (m,
All commercial reagents and solvents were used as received
without further purification unless specified. Reaction solvents
were distilled from calcium hydride for dichloromethane and from
sodium metal and benzophenone for tetrahydrofuran. The reac-
tions were monitored and the Rf values determined using analytical
thin layer chromatography (TLC) with Merck silica gel 60 and F-254
precoated plates (0.25-mm thickness). Spots on the TLC plates were
visualized using ultraviolet light (254 nm) and a basic potassium
permanganate solution or cerium sulfate/ammonium dimolybdate/
sulfuric acid solution followed by heating on a hot plate. Flash
column chromatography was performed with Merck silica gel 60
(230–400 mesh). 1H NMR spectra were recorded on Bruker DPX-
250 or Varian Unity-Inova 500 spectrometers. Proton chemical
12H, dimethyl H); 13C NMR (63 MHz, CDCl3)
d
¼ 167.0, 146.7, 142.8,
132.1, 130.2, 129.8, 128.9, 128.4, 127.9, 122.5, 121.4, 120.9, 52.1, 26.0,
26.0, 18.6, 18.4, ꢁ3.9, ꢁ4.1; m/z 499.2 (Mþ þ 1).
4.2.2. trans-3,4-Di-(tert-butyldimethylsilanyloxy)-40-
(carboxy)stilbene (12)
Yield: 96%. Rf ¼ 0.26 (hexanes/ethyl acetate, 2:1, v/v); IR nmax
(CHCl3) 2928, 2855, 1681, 1605, 1595, 1564, 1513, 1421, 1286, 1251,
1177, 1126, 916, 837 cmꢁ1 1H NMR (250 MHz, DMSO-d6)
; d 12.8
(s, 1H, COOH), 7.98 (d, 2H, J ¼ 8.0 Hz, aromatic H), 7.75 (d, 2H,
J ¼ 8.1 Hz, aromatic H), 7.41 (d, 1H, J ¼ 16.2 Hz, vinyl CH), 7.23
(3H, m, aromatic H, vinyl CH), 6.94 (d, 1H, aromatic H), 1.02–1.01
(m, 18H, tert-butyl H), 0.25–0.24 (m, 12H, dimethyl H); 13C NMR
shifts are reported in ppm (
(TMS, 0.00) or with the solvent reference relative to TMS
employed as the internal standard (CDCl3, 7.26 ppm; d4-CD3OD,
3.31 ppm, d6-DMSO, 2.50 ppm). Data are reported as follows:
d) relative to internal tetramethylsilane
d
(63 MHz, DMSO-d6) d 167.6, 147.1, 146.8, 142.2, 131.3, 131.1, 130.2,
d
129.6, 126.7, 126.0, 121.5, 120.9, 119.9, 26.2, 18.7, ꢁ3.7; m/z 485.4
d
d
(Mþ þ 1).
chemical shift {multiplicity [singlet (s), doublet (d), triplet (t),
quartet (q), and multiplet (m)], coupling constants [Hz], integra-
tion}. 13C NMR spectra were recorded on Bruker DPX-250 (63 MHz)
or Varian Unity-Inova 500 (125 MHz) spectrometers with complete
4.3. General procedure for the preparation of deprotected
dihydroxy stilbenes (15a–n, 17–18, 22, 24, and 26)
proton decoupling. Carbon chemical shifts are reported in ppm (
relative to TMS with the respective solvent resonance as the
internal standard (CDCl3, 77.0 ppm; d4-CD3OD, 49.0 ppm,
d6-DMSO, 39.5 ppm). Infrared (IR) spectra were recorded on
a Nicolet Model Impact FT-IR 400 spectrometer. Data are reported
in wave numbers (cmꢁ1). Mass spectra were recorded on a MALDI-
TOF Voyager-DE STR (Applied Biosystems 4700 proteomics
d)
TBAF (0.25 mmol, 1 M solution in THF) was added dropwise to
a stirred solution of 11, 14a–n, 16, 21, 23, and 25 (0.10 mmol) in dry
THF (5 mL) at 0 ꢀC under nitrogen atmosphere, and the mixture was
stirred at room temperature for 30 min. The reaction mixture was
then quenched with water and acidified with 10% HCl, followed by
dilution with ethyl acetate (10 mL) and a brine wash (7 mL). The
organic phase was separated, and the aqueous phase was extracted
with ethyl acetate (2 ꢃ10 mL). The combined organic phases were
washed with brine (15 mL), dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure to yield dihydroxy stil-
benes, which were purified by flash column chromatography (silica
gel, CH2Cl2/methanol, 15/1–10/1, v/v) to give 15a–n, 17–18, 22, 24
and 26.
d
d
d
analyzer spectrometer, Palo Alto, CA) with an
ycinnamic acid ( -CHCA) matrix.
a-cyano-4-hydrox-
a
4.2. General procedure and spectral data of compounds 11 and 12
4.2.1. trans-3,4-Di-(tert-butyldimethylsilanyloxy)-40-
(methoxycarbonyl)stilbene/cis-3,4-Di-(tert-
butyldimethylsilanyloxy)-40-(methoxycarbonyl)stilbene (11)
To a solution of phosphonium salt 9 (1.0 g, 3.6 mmol) in dry
CH2Cl2 (10 mL) was added portionwise a suspension of NaH (0.16 g,
60% dispersion in paraffin liquid) in dry CH2Cl2 (10 mL) at 0 ꢀC and
the mixture was stirred at 0 ꢀC for 30 min. The protected aldehyde
10 (1.0 g, 2.7 mmol) in dry CH2Cl2 (10 mL) was added dropwise to
the reaction mixture and the resulting mixture was stirred at 0 ꢀC
for 16 h. The reaction mixture was quenched by slow addition of
water (20 mL) and extracted with ethyl acetate (2 ꢃ 30 mL). The
combined organic phases were washed with NaHCO3 (50 mL) and
brine (50 mL). The organic layer was separated, dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (silica gel,
hexanes/ethyl acetate, 20/1, v/v) to yield coupling products 11
(1.15 g, 86%, combined yield, E/Z ¼ 3:2) as a white solid and
a colorless syrup. (E)-form: Rf ¼ 0.41 (hexanes/ethyl acetate, 10:1,
v/v), IR nmax (CHCl3) 2941, 2854, 1729, 1672, 1601, 1515, 1464, 1429,
4.3.1. trans-3,4-Dihydroxy-40-(N-allylaminocarbonyl)stilbene
(15a)
Yield: 64%. Rf ¼ 0.06 (CH2Cl2/methanol, 20:1, v/v); IR nmax
(CHCl3) 3326, 1728, 1634, 1601, 1539, 1524, 1441, 1373, 1288, 1189,
1112, 1041 cmꢁ1
; d 7.82 (d, 2H,
1H NMR (250 MHz, CD3OD)
J ¼ 8.3 Hz, aromatic H), 7.57 (d, 2H, J ¼ 8.2 Hz, aromatic H), 7.20–
6.89 (m, 4H, aromatic H, vinyl CH), 6.63 (d, 1H, J ¼ 8.2 Hz, aromatic
H), 6.01–5.86 (m, 1H, vinyl CH), 5.27–5.11 (m, 2H, vinyl CH), 4.00–
3.98 (m, 2H, N–CH2); 13C NMR (63 MHz, CD3OD)
d 169.7,147.1,146.6,
142.8, 135.6, 133.4, 132.1, 130.6, 128.7, 127.1, 125.5, 120.7, 116.4, 116.2,
114.1, 43.2; m/z 296.1 (Mþ þ 1).
4.3.2. trans-3,4-Dihydroxy-40-(N-decylaminocarbonyl)stilbene
(15b)
Yield: 60%. Rf ¼ 0.47 (CH2Cl2/methanol, 10:1, v/v); IR nmax
(CHCl3) 3317, 2951, 2923, 2851, 1734, 1630, 1602, 1541, 1524, 1466,
1281, 1179, 1113 cmꢁ1
;
1H NMR (250 MHz, CDCl3)
d
¼ 8.02 (d, 2H,
1439, 1269, 963, 859 cmꢁ1 1H NMR (250 MHz, CD3OD)
; d 7.79 (d,
J ¼ 8.2 Hz, aromatic H), 7.55 (d, 2H, J ¼ 8.2 Hz, aromatic H), 7.13 (d,
1H, J ¼ 16.0 Hz, vinyl CH), 7.00 (s, 2H, aromatic H), 6.94 (d, 1H,
J ¼ 16.0 Hz, vinyl CH), 6.84 (d, 1H, J ¼ 8.7 Hz, aromatic H), 3.91
(s, 3H, OCH3), 1.01–0.99 (m, 18H, tert-butyl H), 0.23–0.22 (m, 12H,
2H, J ¼ 8.3 Hz, aromatic H), 7.58 (d, 2H, J ¼ 8.3 Hz, aromatic H), 7.17
(d, 1H, J ¼ 16.3 Hz, vinyl CH), 7.05–6.89 (m, 3H, aromatic H, vinyl
CH), 6.77 (d, 1H, J ¼ 8.2 Hz, aromatic H), 3.39–3.36 (m, 2H, N–CH2),
1.62–1.59 (m, 2H, alkyl chain), 1.34–1.29 (m, 12H, alkyl chain),
dimethyl H); 13C NMR (63 MHz, CDCl3)
d
¼ 167.1, 147.7, 147.2, 142.3,
1.00–0.87 (m, 5H, alkyl chain); 13C NMR (63 MHz, CD3OD)
d 169.7,
131.1, 130.6, 130.1, 128.6, 126.2, 125.7, 121.4, 119.6, 119.4, 52.4, 25.9,
25.4, 18.6, ꢁ3.8, ꢁ4.1; m/z 499.2 (Mþ þ 1). (Z)-form: Rf ¼ 0.48
(hexanes/ethyl acetate, 10:1, v/v), IR nmax (CHCl3) 2955, 2929, 2858,
147.10, 146.59, 142.7, 133.8, 132.1, 130.2, 128.7, 127.0, 125.5, 120.7,
116.4, 114.1, 41.1, 33.1, 30.2, 30.5, 30.5, 28.1, 23.7, 14.5: m/z 396.3
(Mþ þ 1).

3172
J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
4.3.3. trans-3,4-Dihydroxy-40-[N-(tetrahydrofuran-2-yl-
methyl)aminocarbonyl]stilbene (15c)
4.3.8. trans-3,4-Dihydroxy-40-(N-phenylaminocarbonyl)stilbene
(15h)
Yield: 60%. Rf ¼ 0.31 (CHCl3/methanol, 9:1, v/v); IR nmax (CHCl3)
Yield: 62%. Rf ¼ 0.26 (CH2Cl2/methanol, 9:1, v/v), IR nmax (CHCl3)
3444, 2079, 1633, 1556, 1287 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
3442, 1646, 1600, 1525, 1441 cmꢁ1 1H NMR (250 MHz, CD3OD);
;
d
¼ 7.80 (d, 2H, J ¼ 8.3 Hz, aromatic H), 7.58 (d, 2H, J ¼ 8.4 Hz,
d
¼ 7.93–7.89 (d, 2H, J ¼ 8.4 Hz, aromatic H), 7.71–7.60 (m, 4H,
aromatic H), 7.17 (d, 1H, J ¼ 16.3 Hz, vinyl CH), 7.05–6.90 (m, 3H,
aromatic H, vinyl CH), 6.77 (d, 1H, J ¼ 8.2 Hz, aromatic H), 4.10–4.09
(m, 1H, tetrahydrofuran H), 3.91–3.88 (m, 1H, tetrahydrofuran H),
3.82–3.75 (m, 1H, tetrahydrofuran H), 3.50–3.39 (m, 2H, CH2–
C4H7O), 2.00–1.91 (m, 3H, tetrahydrofuran H), 1.71–1.63 (m, 1H,
aromatic H), 7.41–7.33 (m, 3H, aromatic H), 7.20–7.17 (m, 2H,
aromatic H, vinyl CH), 7.07–6.91 (m, 2H, aromatic H, vinyl CH),
6.78–6.75 (d, 1H, J ¼ 8.2 Hz, aromatic H). 13C NMR (63 MHz,
CD3OD)
d
¼ 168.6, 147.2, 146.6, 143.1, 139.9, 134.1, 132.4, 130.6,
129.8, 129.1, 127.1, 125.6, 125.5, 122.4, 120.7, 116.4, 114.1; m/z 322.3
(Mþ þ 1).
tetrahydrofuran H); 13C NMR (63 MHz, CD3OD)
d
¼ 170.2, 144.3,
143.8, 140.0, 129.4, 127.8, 126.7, 126.0, 124.2, 122.7, 117.9, 113.7, 111.3,
76.3, 66.3, 42.1, 27.1, 23.8; m/z 340.4 (Mþ þ 1).
4.3.9. trans-3,4-Dihydroxy-40-[N-(3-
ethoxycarbonylphenyl)aminocarbonyl]stilbene (15i)
Yield: 74%. Rf ¼ 0.26 (CH2Cl2/methanol, 10:1, v/v); IR nmax
(CHCl3) 3345, 2924, 2853, 1716, 1651, 1598, 1544, 1437, 1290, 1259,
4.3.4. trans-3,4-Dihydroxy-40-[N-(furan-2-yl-
methyl)aminocarbonyl]stilbene (15d)
1108, 1112, 1025, 1002, 957, 756 cmꢁ1 1H NMR (250 MHz, CD3OD)
;
Yield: 73%. Rf ¼ 0.1 (CH2Cl2/methanol, 20:1, v/v); IR nmax (CHCl3)
3431, 2924, 2958, 2853, 1726, 1634, 1601, 1523, 1442, 1289, 1189,
d
8.40–8.39 (m, 1H, aromatic H), 7.99–7.90 (m, 3H, aromatic H),
1112, 1043, 1101 cmꢁ1 1H NMR (250 MHz, CD3OD)
; d 7.81 (d, 2H,
7.80 (d, 1H, J ¼ 8.3 Hz, aromatic H), 7.63 (d, 2H, J ¼ 8.3 Hz, aromatic
H), 7.49 (t, 1H, J ¼ 7.9 Hz, aromatic H), 7.20 (d, 1H, J ¼ 16.3 Hz, vinyl
CH), 7.06–6.90 (m, 3H, aromatic H, vinyl CH), 6.78 (d, 1H,
J ¼ 8.2 Hz, aromatic H), 4.41 (q, 2H, J ¼ 7.1 Hz, O–CH2), 1.42 (t, 3H,
J ¼ 8.4 Hz, aromatic H), 7.55 (d, 2H, J ¼ 8.4 Hz, aromatic H), 7.42 (m,
1H, furfuran H), 7.15 (d, 1H, J ¼ 16.3 Hz, vinyl CH), 7.05–6.88 (m, 3H,
aromatic H, vinyl CH), 6.78 (d, 1H, J ¼ 8.2 Hz, aromatic H), 6.35–
6.28 (m, 2H, furfuran H), 4.55 (m, 2H, NCH2); 13C NMR (63 MHz,
J ¼ 7.1 Hz, CH3); 13C NMR (63 MHz, CD3OD)
d 168.6, 167.8, 147.2,
CD3OD)
d 169.7, 153.2, 147.1, 146.6, 143.2, 142.8, 133.2, 130.6, 128.8,
146.6, 143.2, 140.4, 133.7, 130.5, 132.6, 132.2, 129.9, 129.1, 127.1,
126.5, 126.2, 125.4, 123.0, 120.8, 116.5, 114.1, 62.3, 14.6: m/z 404.7
(Mþ þ 1).
127.0, 125.4, 120.7, 116.4, 114.1, 111.4, 108.1, 37.6: m/z 336.3
(Mþ þ 1).
4.3.5. trans-3,4-Dihydroxy-40-(N-
cyclohexylaminocarbonyl)stilbene (15e)
4.3.10. trans-3,4-Dihydroxy-40-(N-morpholinecarbonyl)stilbene
(15j)
Yield: 63%. Rf ¼ 0.28 (CH2Cl2/methanol, 10:1, v/v); IR nmax
Yield: 66%. Rf ¼ 0.36 (CH2Cl2/methanol,15:1, v/v), IR nmax (CHCl3)
(CHCl3) 3393, 2930, 2854, 1724, 1628, 1603, 1526, 1507, 1447, 1374,
3375, 2964, 2925 2854, 1733, 1627, 1540, 1506, 1449, 1374, 1257,
1257 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
7.78 (d, 2H, J ¼ 8.3,
1188, 1045, 857 cmꢁ1
;
1H NMR (250 MHz, CD3OD);
d
¼ 7.54 (d, 2H,
aromatic H), 7.55 (d, 2H, J ¼ 8.3, aromatic H), 7.15 (d, 1H, J ¼ 16.3 Hz,
vinyl CH), 7.04–6.89 (m, 3H, aromatic H, vinyl CH), 6.78 (d, 1H,
J ¼ 8.2 Hz, aromatic H), 3.87–3.85 (m, 1H, NH–CH), 2.02–1.93 (m,
2H, cyclohexane), 1.79–1.66 (m, 4H, cyclohexane), 1.42–1.35 (m, 4H,
J ¼ 8.3 Hz, aromatic H), 7.32 (d, 2H, J ¼ 8.3 Hz, aromatic H), 7.12 (d,
1H, J ¼ 16.3 Hz, vinyl CH), 7.12–6.81 (m, 3H, aromatic H, vinyl CH),
6.69 (d, 1H, J ¼ 8.1 Hz, aromatic H), 3.52–3.43 (m, 8H, morpholine
H); 13C NMR (63 MHz, DMSO-d6)
d
¼ 168.6,145.5,145.0,138.5,133.1,
cyclohexane); 13C NMR (63 MHz, CD3OD)
d 169.2, 147.1, 146.6, 142.6,
129.8, 127.9, 127.2, 125.4, 123.6, 118.5, 115.3, 113.0, 65.7, 27.9; m/z
326.4 (Mþ þ 1).
134.0, 132.0, 129.9, 128.7, 127.0, 125.5, 120.7, 116.5, 114.1, 50.5, 33.8,
26.6, 26.4; m/z 338.2 (Mþ þ 1).
4.3.11. trans-3,4-Dihydroxy-40-[(4-
allylpiperazinyl)carbonyl]stilbene (15k)
4.3.6. trans-3,4-Dihydroxy-40-{N-[3-(2-oxopyrrolidin-1-
yl)propyl]aminocarbonyl}stilbene (15f)
Yield: 65%. Rf ¼ 0.33 (CH2Cl2/methanol, 10:1, v/v); IR nmax
Yield: 66%. Rf ¼ 0.41 (CH2Cl2/methanol, 10:1, v/v); IR nmax
(CHCl3) 3445, 2925, 2854, 1738, 1603, 1463, 1456, 1441, 1374, 1245,
(CHCl3) 3444, 2924, 2958, 2853, 1732, 1646, 1455, 1383, 1242, 1123,
1115, 1024 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
¼ 7.60 (d, 2H,
1086, 1025 cmꢁ1
; d 7.82 (d, 2H,
1H NMR (250 MHz, CD3OD)
J ¼ 8.0 Hz, aromatic H), 7.40 (d, 2H, J ¼ 8.0 Hz, aromatic H), 7.15 (d,
1H, J ¼ 16.3 Hz, vinyl CH), 7.03–6.88 (m, 3H, aromatic H, vinyl CH),
6.77 (d, 1H, J ¼ 8.2 Hz, aromatic H), 5.87–5.80 (m, 1H, vinyl CH),
5.24–5.20 (m, 2H, vinyl CH), 3.75–3.53 (m, 4H, piperazine H), 3.10
(d, 2H, J ¼ 6.0 Hz, N–CH2), 2.53–2.03 (m, 4H, piperazine H); 13C
J ¼ 8.4 Hz, aromatic H), 7.59 (d, 2H, J ¼ 8.4 Hz, aromatic H), 7.17 (d,
1H, J ¼ 16.3 Hz, vinyl CH), 7.05–6.89 (m, 3H, aromatic H, vinyl CH),
6.77 (d, 1H, J ¼ 8.2 Hz, aromatic H), 3.51–3.46 (m, 2H, NH–CH2),
3.40–3.29 (m, 4H, CH2N–CH2), 2.40–2.36 (m, 2H, pyrrolidone H),
2.11–2.02 (m, 2H, pyrrolidone H), 1.87–1.80 (m, 2H, CH2); 13C NMR
NMR (63 MHz, CD3OD)
d
¼ 172.4, 147.1, 146.6, 141.41, 135.1, 134.5,
(63 MHz, CD3OD)
d 178.1, 169.8, 147.1, 146.6, 142.8, 133.5, 132.2,
131.8, 130.6, 128.7, 127.2, 125.5, 120.6, 119.5, 116.5, 114.1, 62.3, 54.1,
130.6, 128.7, 127.1, 125.5, 120.7, 116.5, 114.1, 41.2, 38.1, 32.0, 30.8,
43.1; m/z 365.3 (Mþ þ 1).
27.9, 18.8: m/z 381.6 (Mþ þ 1).
4.3.7. trans-3,4-Dihydroxy-40-[N-(2-
4.3.12. trans-3,4-Dihydroxy-40-[(4-
fluorobenzyl)aminocarbonyl]stilbene (15g)
benzoylpiperazinyl)carbonyl]stilbene (15l)
Yield: 64%. Rf ¼ 0.28 (CH2Cl2/methanol, 10:1, v/v); IR nmax
Yield: 64%. Rf ¼ 0.46 (CH2Cl2/methanol, 10:1, v/v); IR nmax
(CHCl3) 3349, 2924, 2853, 1726, 1636, 1601, 1541, 1505, 1456, 1374,
(CHCl3) 3403, 2924, 2853, 1734, 1560, 1508, 1460, 1431, 1372, 1256,
1270, 1190, 1108, 962, 757 cmꢁ1; 1H NMR (250 MHz, CD3OD)
d
7.83
1002 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
7.60 (d, 2H, J ¼ 8.1 Hz,
(d, 2H, J ¼ 8.0, aromatic H), 7.57 (d, 2H, J ¼ 8.6 Hz, aromatic H), 7.42–
7.23 (m, 3H, aromatic H), 7.15–6.89 (m, 4H, aromatic H, vinyl CH),
6.78 (d, 2H, J ¼ 8.1 Hz, aromatic H), 4.63 (s, 2H, CH2); 13C NMR
aromatic H), 7.46–7.39 (m, 7H, aromatic H), 7.15 (d, 1H, J ¼ 16.3 Hz,
vinyl CH), 7.03–6.88 (m, 3H, aromatic H, vinyl CH), 6.77 (d, 1H,
J ¼ 8.1 Hz, aromatic H), 3.91–3.61 (m, 8H, piperazine H); 13C NMR
(63 MHz, CD3OD)
d
170.0, 164.1 (C–F), 147.1, 146.6, 142.9, 133.3,
(63 MHz, CD3OD) d 172.8, 147.1, 146.6, 141.6, 136.4, 134.2, 131.9,
132.2, 130.6, 130.2, 130.0, 128.8, 127.1, 125.5, 125.3, 120.7, 116.5,
131.4, 130.6, 129.8, 128.8, 128.2, 127.2, 125.4, 120.6, 116.4, 114.1, 30.8:
116.3, 115.9, 114.1, 38.3: m/z 364.3 (Mþ þ 1).
m/z 329.2 (Mþ þ 1).

J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
3173
4.3.13. trans-3,4-Dihydroxy-40-[N-(4-
methylpiperidine)carbonyl]stilbene (15m)
1112, 959, 853 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
¼ 7.49 (d, 2H,
J ¼ 8.2 Hz, aromatic H), 7.39 (d, 2H, J ¼ 8.3 Hz, aromatic H), 7.08 (d,
1H, J ¼ 16.4 Hz, vinyl CH), 6.99–6.84 (m, 3H, vinyl CH, aromatic H),
6.74 (d, 1H, J ¼ 8.11 Hz, aromatic H), 6.56 (d, 1H, J ¼ 16.1 Hz, vinyl
CH), 6.41–6.37 (m, 1H, vinyl CH), 4.13–4.09 (m, 2H, CH2O); 13C NMR
Yield: 65%. Rf ¼ 0.49 (CH2Cl2/methanol, 10:1, v/v), IR nmax (CHCl3)
3408, 2957, 2924, 2854,1736,1601,1508,1446,1373,1273,1251,1197,
1114, 1045, 969 cmꢁ1 1H NMR (250 MHz, CD3OD);
; d 7.56 (d, 2H,
J ¼ 8.0 Hz, aromatic H), 7.35 (d, 2H, J ¼ 8.0 Hz, aromatic H), 7.13–6.88
(m, 4H, aromatic H, vinyl CH), 6.77 (d, 1H, J ¼ 8.2 Hz, aromatic H),
4.60–4.54 (m, 1H, piperidine H), 3.71–3.50 (m, 1H, piperidine H),
3.05–2.80 (m, 2H, piperidine H), 1.78–1.57 (m, 4H, piperidine H),1.26
(m, 1H, piperidine H), 1.05 (d, 3H, J ¼ 6.6 Hz, CH3); 13C NMR (63 MHz,
(63 MHz, DMSO-d6)
d
¼ 146.1, 145.9, 136.9, 135.9, 130.8, 129.1, 129.0,
128.7, 126.9, 126.7, 125.1, 119.1, 116.2, 113.7, 62.0; m/z 291.2 (Mþ þ 1).
4.3.19. trans-3,4-Dihydroxy-40-(2-formyl-1-ethenyl)stilbene (26)
Yield: 70%. Rf ¼ 0.08 (CH2Cl2/methanol, 30:1, v/v); IR nmax
(CHCl3) 3395, 2923, 2958, 2855, 1673, 1620, 1594, 1382, 1129, 1019,
CD3OD)d172.4,147.0, 146.6, 141.1, 135.3,130.7, 131.7, 128.4, 127.1, 125.6,
120.6, 116.5, 114.0. 43.8, 35.8, 34.9, 32.2, 22.0: m/z 338.1 (Mþ þ 1).
965 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
¼ 10.07 (d, 1H, J ¼ 7.8 Hz,
–COH), 8.22–8.01 (m, 5H, vinyl H, aromatic H), 7.66 (d, 1H,
4.3.14. trans-3,4-Dihydroxy-40-[N-(4-
benzylpiperidine)carbonyl]stilbene (15n)
J ¼ 16.3 Hz, vinyl H), 7.43 (m, 4H, vinyl H, aromatic H), 7.16 (d, 1H,
J ¼ 8.1 Hz, aromatic H); 13C NMR (63 MHz, CD3OD)
¼ 193.8, 152.4,
d
Yield: 68%. Rf ¼ 0.46 (CH2Cl2/methanol,10:1, v/v); IR nmax (CHCl3)
145.6,144.8,140.1,132.0,130.4,128.8,127.9,127.2,126.1,123.6,118.6,
3395, 2923, 2851,1593,1558,1445,1838,1237,1086,1021, 963 cmꢁ1
;
115.2, 113.1; m/z 267.4 (Mþ þ 1).
1H NMR (250 MHz, CD3OD)
d
7.59 (d, 2H, J ¼ 8.2 Hz, aromatic H), 7.37
(d, 2H, J ¼ 8.2 Hz, aromatic H), 7.32–7.27 (m, 2H, aromatic H),
7.23–7.14 (m, 3H, aromatic H), 7.29–6.88 (m, 4H, aromatic H), 6.77
(d, 1H, J ¼ 8.2 Hz, aromatic H), 4.37 (m, 1H, piperidine H), 3.57 (m,
1H, piperidine H), 2.71–2.60 (2H, piperidine H), 2.60 (d, 2H,
J ¼ 6.6 Hz, CH2-Ph),1.74–1.55 (m, 4H, piperidine H),1.21–1.18 (m,1H,
4.4. Chemical evaluation
4.4.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging
effects
We used DPPH to assess free radical-scavenging activity [42,43].
DPPH is one of the few stable and commercially available organic
nitrogen radicals and has a UV-vis absorption maximum at 515 nm.
Upon reduction, the solution color fades; the reaction progress is
conveniently monitored by a spectrophotometer [44]. To test free
radical-scavenging effects using DPPH, compounds 15a–n, 17–18,
22, 24, and 26 were adjusted with methanol solution to final
piperidine H); 13C NMR (63 MHz, CD3OD)
d 168.8,145.9,145.5,140.1,
138.7, 134.5, 130.1, 129.0, 128.4, 128.2, 127.3, 125.9, 124.1, 118.9, 115.7,
113.5, 42.1, 41.5, 37.6, 31.8–31.6: m/z 414.8 (Mþ þ 1).
4.3.15. trans-3,4-Dihydroxy-40-(methoxycarbonyl)stilbene (17)
Yield: 32%. Rf ¼ 0.10 (hexanes/ethyl acetate, 2/1, v/v). IR nmax
(CHCl3) 3423, 1678, 1629, 1595, 1525, 1438, 1296, 1231, 1115,
concentrations of 10–200 mM. Tris-base buffer (0.1 mM) was added,
956 cmꢁ1
;
1H NMR (250 MHz, CD3OD);
d
¼ 7.92 (d, 2H, J ¼ 8.3 Hz,
and after 5 min, a DPPH radical-ethanol solution (1 mL, 0.5 mM)
was added. The mixture was warmed in a water bath for 25 min at
37 ꢀC. After 20 min, absorbance was measured with a spectropho-
tometer (515 nm). The DPPH radical-scavenging rate of each sample
and the 50% scavenging concentration based on the DPPH radical-
scavenging rate were calculated using the following rate (%):
aromatic H), 7.67 (d, 2H, J ¼ 8.3 Hz, aromatic H), 7.27 (d, 1H,
J ¼ 16.4 Hz, vinyl H), 7.04–6.90 (m, 3H, aromatic H), 6.77 (d, 1H,
J ¼ 8.1 Hz, aromatic H), 3.83 (s, 3H, OCH3); 13C NMR (63 MHz,
CD3OD)
d
¼ 167.2, 147.1, 146.4, 143.6, 132.9, 130.7, 129.3, 128.6, 127.2,
124.9, 120.4, 116.8, 114.6, 53.1; m/z 271.1 (Mþ þ 1).
ꢀ
ꢁ
4.3.16. trans-3,4-Dihydroxy-40-[2-
(benzamido)acetoxymethyl]stilbene (18)
A ꢁ C
DPPH radical-scavenging rate ð%Þ ¼ 1 ꢁ
ꢃ 100
B
Yield: 90%. Rf ¼ 0.30 (CHCl3/methanol, 9:1, v/v), IR nmax (CHCl3)
where A is the absorbance of the sample (DPPH þ compounds)
when a blank was substituted for the tris-base buffer, B is the
absorbance of the DPPH radical-ethanol solution when a blank was
substituted for the tris-base buffer, and C is the absorbance of the
sample (compounds) alone.
3343, 2955, 2929, 2895, 2857, 1749, 1652, 1601, 1515, 1422, 1298,
1253, 1194, 982, 839, 781 cmꢁ1
;
1H NMR (250 MHz, CD3OD)
d
¼ 7.86–7.83 (m, 2H, aromatic H), 7.55–7.44 (m, 5H, aromatic H),
7.36 (d, 2H, J ¼ 8.2 Hz, aromatic H), 7.01–6.86 (m, 4H, aromatic H,
vinyl CH), 6.76 (d,1H, J ¼ 8.2 Hz, aromatic H), 5.18 (s, 2H, CH2O), 4.16
(s, 2H, CH2N); 13C NMR (63 MHz, CDCl3)
d
¼ 171.3, 170.7, 146.7,
The IC50 values were calculated by linear regression of plots
where x-axis represented the various concentrations (10–200 mM)
146.5, 139.5, 135.7, 135.0, 133.0, 130.9, 130.5, 129.7, 129.6, 128.4,
127.2, 126.1, 120.3, 116.4, 113.9, 67.8, 42.6; m/z 404.1 (Mþ þ 1).
of test trans-stilbenes while the y-axis represented the percentage
of free radical-scavenging activities. The IC50 values of samples
were compared against the standards, resveratrol, and the lower
the IC50 of synthesized trans-stilbenes, the better it is as an anti-
oxidant [45].
4.3.17. trans-3,4-Dihydroxy-40-(methoxycarbonyl-1-
ethenyl)stilbene (22)
Yield: 37%. Rf ¼ 0.47 (hexanes/ethyl acetate, 1/1, v/v); IR nmax
(CHCl3) 3413, 3020, 1634, 1439, 1251 cmꢁ1 1H NMR (500 MHz,
;
CD3OD)
d
¼ 7.72 (d, 1H, J ¼ 16.0 Hz, vinyl H), 7.60–7.55 (m, 4H,
4.5. Biological evaluation
aromatic H), 7.20 (d, 1H, J ¼ 16.0 Hz, vinyl H), 7.07 (s, 1H, aromatic
H), 6.98 (d, 1H, J ¼ 16.5 Hz, vinyl H), 6.94 (d, 1H, J ¼ 9.0 Hz, aromatic
H), 6.80 (d, 1H, J ¼ 8.5 Hz, aromatic H), 6.55 (d, 1H, J ¼ 16.5 Hz, vinyl
4.5.1. Cortical neuron and BV-2 microglia culture
Cortical cell cultures were prepared from embryos of ICR mice at
a gestational age of 15 days. The cortex was dissected and kept in an
ice-cold solution. The cortical tissues were dissociated to single cells
by gentle suspension. The cell suspension was centrifuged at
1000 rpm for 5 min, and the resulting pellets were resuspended in
minimal essential media (MEM), supplemented with 5% heat-
inactivated fetal calf serum, mouse serum, glutamine, and glucose.
H), 3.82 (s, 3H, OCH3); 13C NMR (125 MHz, CD3OD)
d
¼ 169.4, 154.7,
147.1, 146.0, 141.8, 134.2, 131.6, 130.8, 129.7, 127.6, 125.8, 120.7, 117.6,
116.5, 114.0, 52.2; m/z 297.3 (Mþ þ 1).
4.3.18. trans-3,4-Dihydroxy-40-(3-hydroxyprop-1-enyl)stilbene
(24)
Yield: 70%. Rf ¼ 0.18 (CH2Cl2/methanol,15:1, v/v); IR nmax (CHCl3)
The cells were plated on plates coated with poly-D-lysine and lam-
3326, 2924, 2853, 1728, 1634, 1601, 1538, 1525, 1441, 1374, 1289,
inin at a density of 4.8 ꢃ 105 cells/well in 24-well cultured plates.

3174
J.-C. Jung et al. / European Journal of Medicinal Chemistry 44 (2009) 3166–3174
The cells were cultured in a CO2 incubator (5% [v/v], 37 ꢀC). Seven
days after plating, cells were treated with 10 M cytosine arabino-
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m
furanoside (Ara C) to reduce the growth of contaminating non-
neuronal cells. After treatment for 48 h, cells were fed with fresh
media (without fetal calf serum). The murine BV-2 microglia cell line
was maintained in DMEM supplemented with 10% FBS and peni-
cillin/streptomycin at 37 ꢀC in a humidified incubator under 5% CO2.
For all experiments, cells were plated at a density of 1 ꢃ105 cells/mL
in 24-well plates and then treated with 100 ng/mL LPS alone or with
various concentrations of compounds for 24 h at 37 ꢀC.
4.5.2. Nitric oxide assay
The Griess reaction was used to perform nitrite assays [46–48].
Cells were incubated with LPS (lipopolysaccharide, 100 ng/mL) and
various concentrations of trans-stilbene derivatives for 24 h at
37 ꢀC. The culture media were then mixed with an equal volume of
reagent (1 part 0.1% N-1-naphthylethylenediamine dihydro-
chloride, 1 part 1% sulfanilamide in 5% phosphoric acid) in 96-well
plates. The absorbance was determined at 540 nm using a micro-
plate reader. Data are reported as the mean ꢂ the standard devia-
tion of three observations.
4.5.3. Lactate dehydrogenase (LDH) assay
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This work was supported by a KOSEF Brain Neurobiology Grant
(2007), Ewha Global Challenge (BK21) grant, grant (20070301-034-
026-007-04-00) from the BioGreen 21 Program, Rural Development
Administration, and Korean Research Foundation Grant funded by
the Korean Government (MOEHRD) (KRF-2006-312-C00267), the
Republic of Korea. E.L. and Y.L. received fellowships from the BK 21
program of the Ministry of Education and Human Resources
Development, the Republic of Korea and the Seoul Science Fellow-
ship Program.
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Appendix. Supplementary data
General procedure and spectral data of compounds,14a–14n,16,
20, 21, 23, and 25. This material is available online at www.
sciencedirect.com. Supplementary data associated with this
article can be found in the online version, at doi:10.1016/j.ejmech.
2009.03.011.
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LPS: lipopolysaccharide
DPPH: diphenyl-1-picrylhydrazyl
LDH: lactate dehydrogenase
EDCI: ethyl(dimethylaminopropyl)carbodiimide
MEM: minimal essential media