Synthesis of?stilbene derivatives with inhibition of?SARS coronavirus replication

Article abstract of DOI:10.1016/j.ejmech.2006.03.024

Stilbene derivatives have wide range of activities. In an effort to find other potential activities of this kind of compounds, 17 derivatives, including resveratrol, were synthesized. Twelve of them were evaluated for their antiviral potential against severe acute respiratory syndrome (SARS)-CoV-induced cytopathicity in Vero E6 cell culture. The result showed that SARS virus was totally inhibited by compounds 17 and 19 ( ≤ 0.5?mg?ml^-1) and no significant cytotoxic effects were observed in vitro.

Full text of DOI:10.1016/j.ejmech.2006.03.024

European Journal of Medicinal Chemistry 41 (2006) 1084–1089
Short Communication
http://france.elsevier.com/direct/ejmech
Synthesis of stilbene derivatives
with inhibition of SARS coronavirus replication
Yue-Qing Li a, Ze-Lin Li b, Wei-Jie Zhao a,*, Rui-Xing Wen b, Qing-Wei Meng a, Yi Zeng b
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, 158 Zhongshan Road, BOX 90, Dalian, Liaoning Province 116012, China
b College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, China
Received 30 June 2005; received in revised form 3 March 2006; accepted 9 March 2006
Available online 27 July 2006
Abstract
Stilbene derivatives have wide range of activities. In an effort to find other potential activities of this kind of compounds, 17 derivatives,
including resveratrol, were synthesized. Twelve of them were evaluated for their antiviral potential against severe acute respiratory syndrome
(SARS)-CoV-induced cytopathicity in Vero E6 cell culture. The result showed that SARS virus was totally inhibited by compounds 17 and 19
( ≤ 0.5 mg ml–1) and no significant cytotoxic effects were observed in vitro.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Severe acute respiratory syndrome; Coronavirus; Stilbene derivatives
1. Introduction
pounds to the urgent antiviral filtration in vitro. The result was
exciting that the change of hydroxyl group’s site and the intro-
duction of nitrogen atom were beneficial to the anti-SARS ac-
tivity. Especially the substituted sites of hydroxyl groups in
compounds 17 and 19 have been demonstrated to be a key
structure element of anti-SARS virus.
No one knows the likelihood of evolution of SARS-CoV in
human and animals. Moreover the complete understanding of
pathogenesis of SARS remains tentative. In this study, we
evaluated such stilbene derivatives for their potential to inhibit
SARS-CoV replication for the first time and thought this dis-
covery would be beneficial to the anti-SARS-CoV develop-
ment.
Stilbene derivatives are widely distributed in nature, which
are thought to be phytoalexins. There is a growing interest in
stilbene derivatives because many activities have been ob-
served in some of the naturally occurring as well as some of
the synthetic stilbenes. Activities include antimicrobial [1–3],
antioxidant [4,5], antileukemic [6], anti-platelet aggregative [7,
8], protein tyrosine kinase inhibitory [9], anti-inflammatory
[10,11], anticarcinogenic activity [12,13], anti-HIV [14,15]
and anti-herpes simplex virus [16]. In the course of our re-
search for potential activities of stilbene derivatives, we de-
signed different hydroxyl substituted sites against resveratrol
and kept the trans structure to simulate this kind of phytoalex-
ins.
2. Chemistry
In early 2003, severe acute respiratory syndrome (SARS)
broke out in China and other countries. Many scholars and
researchers were engaged in the search of anti-SARS agents
and vaccines. At the same time, we also used this kind of com-
In order to prepare a variety of stilbene derivatives with hy-
droxyl groups, we used methoxyl materials as starting materi-
als to avoid the oxidation of hydroxyl compounds. Also we
introduced one pyridine ring in place of one of the benzene
rings for the purpose of evaluation the activity change. Pre-
paration of aim compounds was accomplished as given in
Scheme 1. According to this scheme, 3,5-dimethoxybenzyl
bromide, 2-chloromethyl-3, 4-dimethoxy pyridine or 2-chloro-
Abbreviations: CPE, cytopathic effect; SARS, severe acute respiratory
syndrome.
* Corresponding author.
E-mail address: zyzhao@chem.dlut.edu.cn (W.-J. Zhao).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.024

Y.-Q. Li et al. / European Journal of Medicinal Chemistry 41 (2006) 1084–1089
1085
Scheme 1. Synthesis of compounds 5–21. (a) P(OCH2CH3)3, (n-Bu)4NI, 100–120 °C; (b) methoxy benzaldehyde, NaH, THF, room temperature; (c) BBr3, CH2Cl2,
20–40 °C.
methyl-3,5-dimethyl-4-methoxypyridine were heated with
triethyl phosphite in the presence of (n-Bu)4NI to give the
phosphonates (2–4) (Michaelis–Arbuzov reaction [17]). In
turn, the reaction of aryl aldehyde with the anion of phosphates
formed in situ with sodium hydride gave (E)-stilbene deriva-
tives (5–14) (with almost none of (Z)-isomer) together with
water soluble diethyl phosphate (Wittig–Horner reaction [18,
19]). But the yield of pyridine containing derivatives is poor.
Further methoxyl derivatives were demethylated with BBr3 in
dichloromethane.
reveal the relationship between the substituent and activity (Ta-
bles 1 and 2).
4. Conclusions
We have synthesized (E)-stilbene derivatives bearing hydro-
xyl groups and in some of them used pyridine ring in place of
one benzene ring. Two of these compounds possessed antiviral
activity against SARS in vitro and till now this has never been
reported.
3. Biological results and discussion
5. Experimental protocols
In this study the antiviral potential of twelve compounds (7,
8, 10, 11, 14–21) was evaluated in vitro for their inhibitory
effect of SARS virus. Cytotoxicity in Vero E6 cells was mea-
sured before the antiviral activity. The compounds that showed
cytotoxity to Vero E6 were weeded out first. The remainder
was studied their inhibitory activity. Only compounds 17 and
19 could inhibit the replication of SARS virus in Vero E6 cells
in concentration ≤ 0.5 mg ml–1 (2.05 mM). There was no cyto-
toxicity to Vero E6 in concentration ≥ 2 mg ml–1 (8.20 mM)
(Table 3). It showed that both of them could inhibit SARS
virus in vitro. Compounds 17 and 19 clearly inhibited the cyto-
pathic effect (CPE) induced by infection with SARS-CoV
(Fig. 1). As compared with cinanserin which inhibits SARS
virus at 66 μM (IC90) [20], the result in our research seems
to be not so good. But the concentration 0.5 mg ml–1 is not
the terminal of the inhibition, because our research could not
undergo in present situation in our country.
The methoxy stilbene derivatives (7, 8, 10, 11, 14) showed
no cytotoxity. Compounds 7 and 10, which were methyl ethers
of compounds 17 and 19, respectively, could inhibit 50% re-
plication of SARS virus in Vero E6 cells in concentration
1 mg ml–1. Compounds 8, 11 and 14 had no inhibition in the
evaluation. Although compounds 17 and 19 had one same part,
the compound 21 that had the same part showed no activity. It
seemed that the whole molecular structure was necessary to the
inhibitory activity and hydroxy group was prior to methoxy
group. However, the structure–activity relationship was still
unclear in our present study. Maybe the further research will
5.1. Synthesis
Melting points were determined in capillary tubes on a Bu-
chi oil bath apparatus and uncorrected. Spectra were obtained
as follows: LC/Q-Tof MS, 1H NMR spectra on Varian INOVA
400 MHz spectrometer with TMS as the internal standard, IR
spectra on FT-IR Nicolet 20 spectrometer. Thin layer chroma-
tography (TLC) was carried out on Si gel plates (60 F254
Merck).
,
All reagents were commercially available and used as re-
ceived.
5.1.1. Diethyl [3,5-dimethoxybenzyl]phosphonate (2) [21]
Triethyl phosphate (2.7 ml, 16 mmol) was added to the 3,5-
dimethoxybenzyl bromide (2.3 g, 10 mmol) containing a cata-
lytic amount of tetrabutyl-ammonium iodine, and the mixture
was heated at 110–130 °C for 5–6 h. Excess triethyl phosphite
was removed by heating at 80–90 °C under vacuum (< 5 kPa)
1
to yield 2 as a light yellow oil. H NMR (CDCl3) δ 1.27 (t,
3JHH = 7.2 Hz, 6H, OCH2CH3), 3.09 (d, JHP = 21.6 Hz, 2H,
2
PCH2), 3.78 (s, 6H, OCH3), 4.04 (quint, JHH=3JHP = 7.2 Hz,
3
4H, OCH2CH3), 6.35 (s, 1H, Hpara of C6H4), 6.46 (s, 2H, Hortho
of C6H4) ppm; IR (KBr) 2939, 2839, 1685, 1598, 1512, 1461,
1257 (vs, P=O), 1206, 1160 (w, P–O–C), 1029 (vs, P–O), 969,
835 cm−1.

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Y.-Q. Li et al. / European Journal of Medicinal Chemistry 41 (2006) 1084–1089
Fig. 1. Cytopathic effect (CPE) of compounds 17 and 19 on replication of SARS-CoV in Vero E6 cells. (a) The normal Vero E6 cells were cultivated with Eagle’s
medium containing 10% fetal calf serum. (b) The Vero E6 cells infected with 100TCID50 SARS virus. (c) The infected cells were treated with compound 17 for 72h.
(d) The infected cells were treated with compound 19 for 72h.
Table 1
Substituent for compounds 2–14
Compounds
2
3
4
5
6
7
8
X
C
N
N
C
C
C
C
N
N
N
N
N
N
R1
OCH3
H
R2
H
H
CH3
H
H
H
H
H
H
R3
R4
H
OCH3
CH3
H
H
H
H
OCH3
OCH3
OCH3
CH3
CH3
CH3
R5
R6
R7
R8
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
OCH3
H
H
OCH3
H
H
OCH3
H
OCH3
H
H
OCH3
H
H
OCH3
H
H
H
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
OCH3
H
9
10
11
12
13
14
H
CH3
CH3
CH3
OCH3
OCH3
H
H
OCH3
Table 3
Inhibitory effect of compounds 17 and 19 on SARS virus in vitro
Com-
pounds
Experiment
number
Chemical
control
Final concentration Viral
Table 2
Substituent for compounds 15–21
of compound
control
(mg ml–1
)
(100TCI-
D50)
+++
+++
+++
+++
+++
+++
Compounds
X
C
C
C
N
N
N
N
R1′
OH
OH
OH
H
H
H
H
R2′
H
H
H
H
R3′
R4′
H
H
R5′
H
H
OH
H
R6′
H
OH
H
OH
H
R7′
OH
H
H
H
H
H
H
R8′
H
2
–
–
–
–
–
–
1
–
–
–
–
–
–
0.5
–
–
–
–
15
16
17
18
19
20
21
OH
OH
OH
17
19
1
2
3
1
2
3
–
–
–
–
–
–
OH
OH
OH
OH
OH
OH
H
OH OH
OH OH OH
H
–
–
CH3 OH CH3
CH3 OH CH3 OH
H
OH
H
– = no cytotoxicity, no CPE; +++ = CPE > 75% cells with CPE.

Y.-Q. Li et al. / European Journal of Medicinal Chemistry 41 (2006) 1084–1089
1087
5.1.2. Diethyl [3,4-dimethoxypyridine-2-methylene]
5.1.6. (E)-2′,3,5,5′-Tetramethoxystilbene (7)
phosphonate (3)
The compound was synthesized in the same manner as for 5
in 72.6% yield as a milk white amorphous solid, m.p.
54–55 °C dec. H NMR (CDCl3) δ 3.81(s, 3H, OCH3), 3.83
The compound was synthesized in the same manner as for 2
1
as a viscous orange liquid and used directly in the next step. 1H
3
(s, 6H, OCH3), 3.84 (s, 3H, OCH3), 6.39 (t, J = 1.6 Hz, 1H, H-
NMR (CDCl3) δ 1.28 (t, JHH= 7.2 Hz, 6H, OCH2CH3), 2.23
3
4
2
4), 6.69 (d, J = 1.6 Hz, 2H, H-2, 6), 6.79 (dd, J = 8.4 Hz, J
= 2.8 Hz, 1H, H-4′), 6.83(d, J = 8.4 Hz, 1H, H-3′), 7.13 (d,
J = 2.8 Hz, 1H, H-6′), 7.02, 7.42 each 1H (d, J = 16.4 Hz, H-
α, β) ppm; IR (KBr) 3005, 2941, 2833, 1593, 1498, 1463,
1240, 1206, 1154, 1063, 1052, 966, 862, 804 cm−1.
(d, J = 1.2 Hz, 3H, CH3), 2.32 (s, 3H, CH3), 3.42 (d, JHP
3
= 22.0 Hz, 2H, PCH2), 3.75 (s, 3H, OCH3), 4.09 (quint, JHH
= 3JHP = 7.2 Hz, 4H, OCH2CH3), 8.19 (s, 1H, H of C5NH)
ppm; IR (KBr) 2982, 2944, 1583, 1489, 1447, 1425, 1302,
1253 (vs, P=O), 1163 (w, P–O–C), 1053 (vs, P–O), 1027 (vs,
P–O), 965, 827, 781 cm−1.
5.1.7. (E)-2′,3,5,4′-Tetramethoxystilbene (8)
The compound was synthesized in the same manner as for 5
in 83.0% yield as a pale yellow crystal, m.p. 81–82 °C dec. 1H
NMR (CDCl3) δ 3.82 (s, 9H, OCH3), 3.86 (s, 3H, OCH3), 6.36
5.1.3. Diethyl [3,5-dimethyl-4-methoxypyridine-2-methylene]
phosphonate (4)
The compound was synthesized in the same manner as for 2
4
4
(t, J = 2.0 Hz, 1H, H-4), 6.46 (d, J = 2.4 Hz, 1H, H-3′), 6.51
1
as a viscous liquid and used directly in the next step. H NMR
4
3
4
(dd, J = 2.4 Hz, J = 8.4 Hz, 1H, H-5′), 6.67 (d, J = 2.0 Hz,
(CDCl3) δ 1.30 (t, 3JHH = 7.2 Hz, 6H, OCH2CH3), 3.48 (d, 2JHP
3
2H, H-2, 6), 7.49 (d, J = 8.4 Hz, 1H, H- 6′), 7.36, 6.94 each
1H (d, J = 16.4 Hz, 2H, H-α, β) ppm; IR (KBr) 3001, 2944,
= 22.4 Hz, 2H, PCH2), 3.90 (s, 3H, OCH3), 3.92 (s, 3H,
3
3
OCH3), 4.13 (quint, JHH = 3JHP= 7.2 Hz, 4H, OCH2CH3),
2840, 1629, 1590, 1504, 1461, 1428, 1295, 1196, 1155, 1062,
3
6
6.76 (dd, JHH=5.6 Hz, JHP = 1.6 Hz, 1H, Hpara of C5NH2),
1029, 968, 837, 818 cm−1.
3
8.19(d, JHH = 5.6 Hz, 1H, Hmeta of C5NH2) ppm; IR (KBr)
2983, 2933, 1590, 1566, 1475, 1396, 1253 (vs, P=O), 1164
(w, P–O–C), 1054 (vs, P–O), 1029 (vs, P–O), 965, 873, 850,
807, 777, 753 cm−1.
5.1.8. (E)-3′,5,5′, 6-Tetramethoxystilbene-2-nitrogen (9)
The compound was synthesized in the same manner as for 5
in 23.2% yield as a pale yellow solid, m.p. 88–90 °C dec. H
1
NMR (CDCl3) δ 3.83(s, 6H, OCH3), 3.88(s, 3H, OCH3), 3.93
(s, 3H, OCH3), 6.43(t, J = 2.0 Hz, 1H, H-4′), 6.78(d,
J = 2.0 Hz, 2H, H-2′, 6′), 6.75 (d, J = 5.2 Hz, 1H, H-4), 8.27
(d, J = 5.2 Hz, 1H, H-3), 7.47, 7.71 each 1H (d, J = 16.0 Hz,
2H, H-α, β) ppm; IR (KBr) 2927, 2836, 1597, 1476, 1458,
1423, 1280, 1270, 1203, 1158, 1148, 980, 818 cm−1.
5.1.4. (E)-3,4′,5-Trimethoxystilbene (5)
The product of first step was dissolved in dry THF (20 ml)
and stirred at 0–5 °C. Sodium hydride (0.6 g, 25 mmol) was
added to the well-stirred phosphonate ester solution. After
30 min, the aldehyde (10 mmol) in THF (30 ml) was added
dropwise, and the mixture was allowed to stir at room tempera-
ture for 8–16 h. The mixture was then cooled to 0 °C, and the
excess sodium hydride was quenched with water (10 ml). The
reaction mixture was then poured on ice, followed by addition
of 1 M HCl to pH 6, and the product was extracted with ethyl
acetate(4 × 50 ml). The organic layers were combined and
washed with saturated solution of sodium chloride (2 ×
30 ml). The ethyl acetate layer was dried over anhydrous so-
dium sulfate and evaporated. The residue was purified by re-
crystallization and gave 1.7 g (65.9%, total yield of the first
two steps) of 5 as a slightly yellow crystal, m.p. 56–57 °C dec.
1H NMR (CDCl3) δ 3.83 (s, 9H, OCH3), 6.38 (s, 1H, H-4),
6.65 (s, 2H, H-2,6), 6.90 (d, J = 8.8 Hz, 2H, H-3′,5′), 6.91 (d,
J = 16.4 Hz, 1H, H-α), 7.04 (d, J = 16.4 Hz, 1H, H-β), 7.44 (d,
J = 8.8 Hz, 2H, H-2′,6′) ppm; IR (KBr) 2936, 2836, 1598,
1512, 1459, 1426, 1253, 1154, 962, 832 cm−1.
5.1.9. (E)-2′,5,5′, 6-Tetramethoxystilbene-2-nitrogen (10)
The compound was synthesized in the same manner as for 5
in 20.7% yield as a pale yellow crystal, m.p. 100–101 °C dec.
1H NMR (CDCl3) δ 3.82(s, 3H, OCH3), 3.85(s, 3H, OCH3),
3
3.87(s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.73 (d, J = 5.6 Hz,
1H, H-4), 7.23 (d, 4J = 2.4 Hz, 1H, H-6′), 6.83 (dd, 3J
4
3
= 8.8 Hz, J = 2.4 Hz, 1H, H-4′), 6.85 (d, J = 8.8 Hz, 1H,
3
H-3′), 8.28 (d, J= 5.6 Hz, 1H, H-3), 8.08, 7.52 each 1H (d,
3J = 16.4 Hz, 2H, H-α, β) ppm; IR (KBr) 2995, 2944, 2833,
1629, 1573, 1495, 1286, 1241, 1208, 1069, 994, 978, 854,
817, 802 cm−1.
5.1.10. (E)-2′,5,4′, 6-Tetramethoxystilbene-2-nitrogen (11)
The compound was synthesized in the same manner as for 5
in 27.5% yield as a white amorphous solid, m.p. 131–133 °C
1
dec. H NMR (CDCl3) δ 3.84 (s, 3H, OCH3), 3.86 (s, 3H,
4
5.1.5. (E)-3,3′,5,5′-Tetramethoxystilbene (6)
OCH3), 3.87 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.47 (d, J
3
4
The compound was synthesized in the same manner as for 5
in 83.0% yield as a white crystal, m.p. 135–136 °C dec. H
NMR (CDCl3) δ 3.84 (s, 12H, OCH3), 6.40 (t, J = 2.0 Hz,
2H, H-4, 4′), 6.67(d, J = 2.0 Hz, 4H, H-2, 2′,6, 6′), 7.01(s,
2H, H-α, β) ppm; IR (KBr) 2999, 2939, 2839, 1594, 1462,
1428, 1207, 1194, 1151, 1065, 943, 865, 837, 825 cm−1.
= 2.0 Hz, 1H, H-3′), 6.52 (dd, J = 8.4, J = 2.0 Hz, 1H, H-5′),
6.70 (d, 3J = 5.2 Hz, 1H, H-4), 7.61 (d, 3J = 8.4 Hz, 1H, H-6′),
8.27 (d, 3J = 5.2 Hz, 1H, H-3), 7.43, 8.03 each 1H (d, 3J
= 16.4 Hz, 2H, H-α, β) ppm; IR (KBr) 2961, 2940, 2837,
1624, 1602, 1573, 1504, 1478, 1290, 1278, 1207, 1063,
1031, 998, 939, 821 cm−1.
1

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Y.-Q. Li et al. / European Journal of Medicinal Chemistry 41 (2006) 1084–1089
5.1.11. (E)-4,6-Dimethyl-3′,5,5′-trimethoxystilbene-2-nitrogen
(12)
= 1.6 Hz, 4H, H-2,2′,6, 6′), 6.79(s, 2H, H-α, β) ppm; IR
(KBr) 3538, 3449, 3240, 1602, 1516, 1462, 1387, 1346,
1312, 1259, 1158, 1005, 966, 956, 836 cm−1.
The compound was synthesized in the same manner as for 5
in 34.7% yield as a white needle crystal, m.p. 94–95 °C dec. 1H
NMR (CDCl3): δ 2.27 (s, 3H, CH3), 2.35 (s, 3H, CH3), 3.77
5.1.16. (E)-2′,3,5′,5-Tetrthydroxystilbene (17)
4
(s, 3H, OCH3), 3.83 (s, 6H, OCH3), 6.42 (t, J = 2.0 Hz, 1H,
The compound was synthesized in the same manner as for
4
H-4′), 6.75 (d, J = 2.0 Hz, 2H, H-2′, 6′), 8.26 (s, 1H, H-3),
15 in 84.5% yield as pale pink amorphous solid, m.p.
3
7.29, 7.63 each 1H (d, J = 15.6 Hz, 2H, H-α, β) ppm; IR
1
212–214 °C dec. H NMR (DMSO-d6) δ 6.29 (s, 1H, H-4),
(KBr) 2959, 2936, 2838, 1632, 1592, 1465, 1266, 1207,
4
6.53 (d, J = 2.0 Hz, 2H, H-2, 6), 6.60 (m, 1H, H-4), 6.71 (d,
1159, 1071, 994, 964, 819 cm−1.
4
3J = 8.4 Hz, 1H, H-3), 6.99 (d, J = 2.8 Hz, 1H, H-6), 7.34,
3
6.87 each 1H (d, J = 16.4 Hz, 2H, H-α, β) ppm; IR (KBr)
5.1.12. (E)-4,6-Dimethyl-2′,5,5′-trimethoxystilbene-2-nitrogen
(13)
3589, 3359, 1621, 1599, 1507, 1493, 1476, 1351, 1340,
1202, 1163, 1148, 989, 966, 862, 831, 810 cm−1; API-ES:
243 (M – H+), 279 (M + Cl–), 487 (2M – H+), 523 (2M +
Cl–). Q-TOFMS m/z [M + 1]+ 245.0811 (calculated for
C14H13O4, 245.0814).
The compound was synthesized in the same manner as for 5
as a golden oil; 1H NMR (CDCl3) δ 2.24 (s, 3H, CH3), 2.33 (s,
3H, CH3), 3.74 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 6.79 (dd, 3J = 9.0 Hz, 4J = 2.8 Hz, 1H, H-4′), 6.83
4
(d, 3J = 9.0 Hz, 1H, H-3′), 7.16(d, J = 2.8 Hz, 1H, H-6′), 8.25
5.1.17. (E)-3′,5,5′,6-Tetrhydroxystilbene-2-nitrogen (18)
(s, 1H, H-3), 7.37, 7.95 each 1H (d, J = 16.0 Hz, 2H, H-α, β)
ppm; IR (KBr) 2939, 2834, 1629, 1581, 1551, 1496, 1469,
1277, 1220, 1076, 1045, 1027, 978, 880, 853, 803 cm−1.
The compound was synthesized in the same manner as for
15 in 98.9% yield as pale yellow amorphous solid, m.p. >
1
4
300 °C; H NMR (DMSO-d6) δ 6.17(d, J = 2.2 Hz, 2H, H-
4
3
2′, 6′), 6.22 (t, J = 2.2 Hz, 1H, H-4′), 7.51 (d, J = 6.5 Hz,
1H, H-3), 6.18 (d, J = 6.5 Hz, 1H, H-4), 6.99, 7.54 each 1H
5.1.13. (E)-4,6-Dimethyl-2′,5,4′-trimethoxystilbene-2-nitrogen
(14)
3
(d, 3J = 16.5 Hz, 2H, H-α, β) ppm; IR (KBr) 3414, 3330, 1620,
1609, 1597, 1555, 1507, 1446, 1356, 1144, 1107, 1063, 1008,
991, 962, 825, 794, 734, 680 cm−1. API-ES: 244 (M – H+),
280 (M + Cl–), 489 (2M – H+), 525 (2M + Cl–). Q-TOFMS
m/z [M + 1]+ 246.0767 (calculated for C13H12NO4, 246.0766).
The compound was synthesized in the same manner as for 5
in 34.7% yield as a yellow needle crystal, m.p. 108–110.5 °C
dec. 1H NMR (CDCl3): δ 2.25 (s, 3H, CH3), 2.33(s, 3H, CH3),
3.75 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
6.51 (dd, 3J = 8.4 Hz, 4J = 2.0 Hz, 1H, H-5′), 6.47 (d, 4J
3
= 2.0 Hz, 1H, H-3′), 7.53 (d, J = 8.4 Hz, 1H, H-6′), 8.25 (s,
3
1H, H-3), 7.30, 7.90 each 1H (d, J = 15.6 Hz, 2H, H-α, β)
5.1.18. (E)-2′,5,5′,6-Tetrahyroxystilbene-2-nitrogen (19)
ppm; IR (KBr) 2993, 2938, 2837, 1620, 1604, 1580, 1506,
1469, 1296, 1279, 1209, 1104, 1074, 1033, 1004, 986, 943,
818 cm−1.
The compound was synthesized in the same manner as for
15 in 99.7% yield as pale yellow amorphous solid, m.p. >
1
3
300 °C dec. H NMR (DMSO-d6)δ 6.90(d, J = 8.8 Hz, 1H,
4
4
3
H-3′), 6.73(d, J = 3.0 Hz, 1H, H-6′), 6.58(dd, J = 3.0 Hz, J
5.1.14. (E)-3,4′,5-Trihydroxystilbene (15)
= 8.8 Hz, 1H, H-4′), 7.47(d, 3J = 6.7 Hz, 1H, H-3), 6.18(d, 3J
Boron tribromide (3.4 ml, 36 mmol) in CH2Cl2 (20 ml) was
added to a stirred solution of 5 (4 mmol) in CH2Cl2 (50 ml) at
–4 to 0 °C. The mixture was allowed to warm to 25–35 °C,
stirred for 3 h, then poured into ice-water, and extracted with
ethyl acetate (80 ml × 3). The organic layers were combined
and washed with saturated solution of sodium chloride
(1 × 30 ml). The ethyl acetate layer was dried over anhydrous
sodium sulfate and evaporated. The residue was purified by
recrystallization and gave 0.7–0.9 g (75.0–90.2%) of 15 as
amorphous solid, m.p. 251–252 °C dec [2]. 1H NMR (DMSO-
d6) δ 6.23 (s, 1H, H-4), 6.44 (s, 2H, H-2,6), 6.74 (d, 3J
3
= 6.7 Hz, 1H, H-4), 7.16, 7.54 each 1H (d, J = 16.9 Hz, 2H,
H-α, β) ppm; IR (KBr) 3246, 3145, 2690, 1639, 1603, 1503,
1414, 1373, 1243, 1211, 1004, 980, 969, 857, 842, 816 cm−1;
API-ES: 244 (M – H+), 280 (M + Cl–), 489 (2M – H+), 525
(2M + Cl–). Q-TOFMS m/z [M + 1]+ 246.0777 (calculated for
C13H12NO4, 246.0766).
5.1.19. (E)-4,6-Dimethyl-3′,5,5′-trihydroxystilbene-2-nitrogen
(20)
The compound was synthesized in the same manner as for
3
= 16.4 Hz, 1H, H-α), 6.90 (d, J = 16.4 Hz, 1H, H-β), 6.80 (d,
15 in 94.0% yield as pale yellow amorphous solid, m.p. 260 °C
3J = 8.2 Hz, 2H, H-3′,5′), 7.31(d, 3J = 8.2 Hz, 2H, H-2′,6′)
ppm; IR (KBr) 3298, 1606, 1589, 1514, 1445, 1327, 1249,
1154, 1010 ,988, 966, 832 cm−1.
1
(oxy.) dec. H NMR (DMSO-d6) δ 1.91 (s, 3H, CH3), 2.06 (s,
3H, CH3), 6.47 (d, 4J = 2.2 Hz, 2H, H-2′, 6′), 6.23 (t, 4J
= 2.2 Hz, 1H, H-4′), 7.56 (s, 1H, H-3), 7.05, 7.11 each 1H (d,
3J = 16.6 Hz, 2H, H-α, β) ppm; IR (KBr) 3239, 1624, 1594,
1478, 1442, 1377, 1343, 1310, 1281, 1157, 1091, 1013, 997,
961, 834 cm−1; API-ES: 292(M + Cl–), 513 (2M – H+), 549
(2M + Cl–). Q-TOFMS m/z [M + 1]+ 258.1136 (calculated for
C15H16NO3, 258.1130).
5.1.15. (E)-3,3′,5,5′-Tetrahydroxystilbene (16)
The compound was synthesized in the same manner as for
15 in 96.0% yield as pale yellow crystal, m.p. > 300 °C dec
1
4
[2]. H NMR (DMSO-d6) δ 6.11(s, 2H, H-4,4′), 6.36(d, J

Y.-Q. Li et al. / European Journal of Medicinal Chemistry 41 (2006) 1084–1089
1089
[2] M.A. Ali, K. Kondo, Y. Tsuda, Chem. Pharm. Bull. (Tokyo) 40 (1992)
5.1.20. (E)-4,6-Dimethyl-2′,5,5′-trihydroxystilbene-2-nitrogen
(21)
1130–1136.
[3] C. Boonlaksiri, W. Oonanant, P. Kongsaeree, P. Kittakoop, M. Tanti-
The compound was synthesized in the same manner as for
15 in 62.8% yield as pale yellow amorphous solid, m.p. 240 °C
charoen, Y. Thebtaranonth, Phytochem. 54 (2000) 415–417.
[4] Y.J. Cai, J.G. Fang, L.P. Ma, L. Yang, Z.L. Liu, Biochim. Biophys. Acta
1
(oxy.) dec. H NMR (DMSO-d6) δ 1.89(s, 3H, CH3), 2.03(s,
1637 (2003) 31–38.
3H, CH3), 6.72 (d, 3J = 8.5 Hz, 1H, H-3′), 6.95 (d, 4J = 3.0 Hz,
[5] H. Matsuda, T. Morikawa, I. Toguchida, J.Y. Park, S. Harima, M. Yoshi-
4
3
1H, H-6′), 6.61 (dd, J = 3.0 Hz, J = 8.5 Hz, 1H, H-4′), 7.47
kawa, Bioorg. Med. Chem. 9 (2001) 41–50.
3
(s, 1H, H-3), 7.14, 7.39 each 1H (d, J = 16.6 Hz, 2H, H-α, β)
[6] J.B. Zheng, V.D. Ramirez, Biochem. Biophys. Res. Commun. 261
(1999) 499–503.
ppm; IR (KBr) 3313, 1641, 1606, 1589, 1537, 1500, 1446,
1379, 1279, 1238, 1173, 1092, 970, 823 cm−1; MS: 258(M +
H); Q-TOFMS m/z [M + 1]+ 258.1141 (calculated for
C15H16NO3, 258.1130).
[7] F. Orsini, F. Pelizzoni, L. Verotta, T. Aburjai, J. Nat. Prod. 60 (1997)
1082–1087.
[8] T.A. Aburjai, Phytochem. 55 (2000) 407–410.
[9] K. Thakkar, R.L. Geahlen, M. Cushman, J. Med. Chem. 36 (1993)
2950–2955.
5.2. Assay method
[10] G.H. Chen, W. Liu, J.X. Li, W.M. John, P.C.T. Application, 2001 WO
0142231.
Vero E6 cells were cultured in our laboratory, College of
Life Science and Bioengineering, Beijing University of Tech-
nology. BJ 9-2b SARS virus was provided by Institute of Mi-
crobiology and Epidemiology, Academy of Military Medical
Sciences. Vero E6 cells (1.2 × 104 cells per well) were inocu-
lated in triplicate in 200 μl of Eagle’s medium containing 10%
fetal calf serum in a 96 wells plate. After 2 days cultivation, the
cells grew to a thin layer, then the cells were infected with 100
TCID50 SARS virus in 0.1 ml medium and incubated at 37 °C,
5% CO2, humidified incubator for 2 hours. After a 2 h adsorp-
tion, the supernatant was discarded to remove the free virus.
Chemicals in a series of 2 × dilation in 0.2 ml medium were
added into the cell wells, the chemical control without virus
and the viral control without chemical in 0.2 ml medium were
also added into the cell wells, they were then cultivated in the
same incubator for 3 days and the CPE of cells were recorded.
[11] G.H. Chen, W. Liu, J.X. Li, W.M. John, P.C.T. Application, 2002 WO
02057219.
[12] M.S. Jiang, L.N. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas,
C.W.W. Beecher, H.H.S. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G.
Mehta, R.C. Moon, J.M. Pezzuto, Science, 275 (1997) 218–220.
[13] L.M. Yang, S.J. Lin, F.L. Hsu, T.H. Yang, Bioorg. Med. Chem. Lett. 12
(2002) 1013–1015.
[14] X. Wang, A. Heredia, H. Song, Z. Zhang, B. Yu, C. Davis, R. Redfield,
J. Pharm. Sci. 93 (2004) 2448–2457.
[15] K. Likhitwitayawuid, B. Sritularak, K. Benchanak, V. Lipipun,
J. Mathew, R.F. Schinazi, Nat. Prod. Res. 19 (2005) 177–182.
[16] J.J. Docherty, M.M.H. Fu, B.S. Stiffler, R.J. Limperos, C.M. Pokabla,
A.L. DeLucia, Antivir. Res. 43 (1999) 135–145.
[17] A.K. Bhatacharya, G. Thyagarajan, Chem. Rev. 81 (1981) 415–430.
[18] C. Piechucki, Synthesis 3 (1976) 187–188.
[19] R. Baker, R.J. Sims, Synthesis 2 (1981) 117.
[20] L. Chen, C. Gui, X. Luo, Q. Yang, S. Günther, E. Scandella, C. Drosten,
D. Bai, X. He, B. Ludewig, J. Chen, H. Luo, Y. Yang, Y. Yang, J. Zou,
V. Thiel, K. Chen, J. Shen, X. Shen, H. Jiang, J. Virol. 11 (2005) 7095–
7103.
References
[21] M. Murias, N. Handler, T. Erker, K. Pleban, G. Ecker, P. Saiko, T. Sze-
[1] P. Wieslaw, K. Bogdan, IL Farmaco 54 (1999) 584–587.
keres, W. Jäger, Bioorg. Med. Chem. 12 (2004) 5571–5578.