Synthesis and structure-immunosuppressive activity relationships of bakuchiol and its derivatives

Article abstract of DOI:10.1016/j.bmc.2007.11.054

A series of derivatives of bakuchiol were synthesized and tested in vitro for their cytotoxicity, and inhibition of T cell proliferation and B cell proliferation. The data obtained provided preliminary structure-activity relationships of the compounds as immunosuppressive activity.

Full text of DOI:10.1016/j.bmc.2007.11.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2403–2411
Synthesis and structure–immunosuppressive activity
relationships of bakuchiol and its derivatives
Hongli Chen, Xiaolong Du, Wei Tang, Yu Zhou, Jianping Zuo,
*
Huijin Feng and Yuanchao Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Road Zu Chong Zhi,
Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
Received 25 October 2007; revised 20 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—A series of derivatives of bakuchiol were synthesized and tested in vitro for their cytotoxicity, and inhibition of T cell
proliferation and B cell proliferation. The data obtained provided preliminary structure–activity relationships of the compounds
as immunosuppressive activity.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Bakuchiol is a prenylated phenolic monoterpene iso-
lated from the seeds of Psoralea corylifolia L. (Legumi-
noesae) which has been used to treat a wide variety of
diseases and conditions in both Chinese and Indian
folkloric medicine.1 Bakuchiol is one of the active com-
pounds reported to exert anti-oxidative, anti-microbacteri-
al activity, inhibit iNO expression, and inhibit
mitochondrial lipid peroxidation.2 Also, bakuchiol has
been demonstrated to possess anti-inflammatory activity
since it is able to control leukocytic functions such as
eicosanoid production, migration, and degranulation
in the inflammatory site and it is a weak inhibitor of
secretory and intracellular phospholipase A2 (PLA2)
but dose-dependently reduces the formation of LTB4
and TXB2 by human neutrophils and platelet micro-
somes, respectively.3–5 In addition, it can inhibit COX-
1 (cyclooxygenase), COX-2, and 5-lipoxygenase.6 It also
inhibits the expression of the inducible nitric oxide syn-
thase gene via the inactivation of nuclear transcription
factor-kappaB in RAW 264.7 macrophages.7 On the
other hand, it has been reported that cyclobakuchiols
exhibited higher anti-inflammatory potency than bak-
uchiol.4,5 These findings prompted us to synthesize the
Figure 1. Bakuchiol and cyclobakuchiols.
derivatives of bakuchiol and investigate the structure–
activity relationships of bakuchiol and its analogues as
immunosuppressive activity (Fig. 1).
A series of bakuchiol derivatives modified at C-12, 13, a
second series modified at C-9, and a third series with
modified aryl groups were prepared and tested in vitro
for their cytotoxicity on murine splenocytes, and inhibi-
tion activity on concanavalin A (ConA) induced T cell
proliferation and on lipopolysaccharide (LPS) induced
B cell proliferation.8–11 Through the examination of
the T/B lymphocyte proliferative reaction, we could
understand the in vitro immunosuppression function
of the compounds.
2. Results and discussion
2.1. Chemistry
Keywords: Bakuchiol; Synthesis; Cytotoxicity; Immunosuppressive
activity.
In prior studies, the diastereomeric mixture of cyclo-
bakuchiols A and B, extracted from Psoralea glandulosa,
could increase the anti-inflammatory activity in compar-
*
Corresponding author. Tel.: +86 21 50806600 3502; fax: +86 21
50807288; e-mail: ycli@mail.shcnc.ac.cn
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.054

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H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
Next, we began the modification of aromatic ring
while keeping the side chain fixed. In the first ap-
proach, compound 16 was prepared by methylation
of bakuchiol (1). In the second approach, we intro-
duced a variety of groups at ortho-position of the phe-
nol. The selective ortho-formylation product 17 was
obtained by the reported procedures starting from
bakuchiol (1)15 which reacted with C6H5CH2P+Ph3Brꢀ
to afford 18 and with p-CH3OC6H5CH2P+Ph3Brꢀ to
afford 20. Alternatively, compound 17 was treated
with NaBH4 in methanol to give 19. On the other
hand, nitration of bakuchiol (1) with nitric acid gave
21 which was reacted with Zn–Fe to yield 22. Treat-
ment of 22 with acetic anhydride furnished 23, with
urea gave 24, and with carbon disulfide gave 25.16
Synthetic methods are shown in Scheme 3.
Scheme 1. Reagents and conditions: (a) m-CPBA, CH2Cl2, rt, 3 h,
78%; (b) 70% H2SO4, dioxane: H2O = 1:1, rt, 1 min, 99%; c. p-TsOH,
CH2Cl2, rt, 0.5 h, 64% (5), 20% (4).
ison with its biogenetic precursor, bakuchiol. Therefore,
we initially focused our attention on the modification of
the side chain. First, D12,13 double bond is investigated
and the synthesis of the derivatives was illustrated in
Scheme 1. Key intermediate 2 was prepared by epoxida-
tion of bakuchiol (1) with m-chloroperbenzoic acid12
followed by ring opening with 70% H2SO4 in dioxane
and water (1:1) to give 3. Alternatively, the epoxy ring
of 2 could undergo rearrangement in the presence of
p-TsOH to give compounds 4 and 5.13
2.2. Biological activity
All of the compounds were tested in MTT assay for their
cytotoxicity, T cell and B cell functional assays for eval-
uating their immunosuppressive activity in vitro. The
pharmacological results of these compounds are sum-
marized in Tables 1–3.
Then, in order to probe the effect of chiral quaternary
center (C-9), D7,8 double bond, and D17,18 double bond,
we designed the following synthetic approach to get
( )bakuchiol and its derivatives (Scheme 2). Key inter-
mediate 27 was prepared by 1,4-addition procedure with
citral (26) and vinylmagnesium bromide. Treatment of
27 with p-methoxyphenylmagnesium bromide furnished
the alcohol 6, with (3-fluoro-4-methoxyphenyl)magne-
sium bromide furnished the alcohol 7, and with (3,4-
dimethoxyphenyl)magnesium bromide followed by
dehydration furnished 11. Compounds 9 and 10 were
obtained by dehydration with 6 and 7, respectively.
Compounds 9, 10, and 11 were demethylated with
MeMgI to produce 8, 12, and 13, respectively.14 Com-
pound 15 was obtained by 1,4-addition procedure with
citral (26) and p-methoxybenzylmagnesium chloride fol-
lowed by dehydration and demethylation. Citral (26)
could convert to compound 14 in one step by an 1,2-
addition procedure, which probably underwent a rear-
rangement progress.
The effect of D12,13 modifications showed that all the C-
12,13 derivatives (2, 3, 4, 5) exhibited higher bioactivity
than bakuchiol on the Con A induced T cell prolifera-
tion. In the inhibition of LPS induced B cell prolifera-
tion, 2, 3 exhibited lower bioactivity and 4, 5 exhibited
slightly higher bioactivity at the concentration of 1 lM,
however, all the compounds (2, 3, 4, 5) exhibited high-
er bioactivity at the concentration of 10 lM compared
to bakuchiol (1). The entire D12,13 series also showed
an increase in cytotoxicity compared to bakuchiol (1),
especially for compound 3 which exhibited cytotoxicity
at a lower concentration (10 lM). In this series, com-
pound 2 was the most active analogue.
To our disappointment, the C-9, D7,8 double bond, and
D17,18 double bond were not good sites to modify since
the derivatives did not show obviously improved activ-
ity while exhibiting higher cytotoxicity. ( )bakuchiol
(8) exhibited slightly improved inhibitory activity both
on ConA-induced T cell proliferation and on LPS-in-
duced B cell proliferation meanwhile increased the
cytotoxicity compared to bakuchiol (1). The C-7,8
modifications (6, 7) and C-17,18 modifications (14,
15) showed lower inhibitory activity on ConA-induced
T cell proliferation and almost the same or slightly im-
proved inhibitory activity on LPS-induced B cell prolif-
eration compared to ( )bakuchiol (8). For the
extraordinary cytotoxicity of compound 10, we pre-
sumed that it was because the compound itself at the
higher concentration (10 and 100 lM) would absorb
the photons which influenced the OD value. Compound
9 showed no significant inhibitory activity on ConA-in-
duced T cell proliferation and there was no dose-depen-
dent relationship.
Scheme 2. Reagents and conditions: (a) CuI, R1MgBr, THF, ꢀ40 °C,
2–4 h, 40–60%; (b) ArMgBr THF, 0 °C, 91%; (c) POCl3, Py., 140 °C,
1 h, 95%; (d) MeMgI, 180 °C, 10 min, 91%; (e) p-CH3OC6H4MgBr,
THF, 0 °C–rt, 4 h, 95%.
On the basis of the pharmacological results of aryl sub-
stitutions, several structure–activity relationship conclu-
sions were summarized as follows:

H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
2405
Scheme 3. Reagents and conditions: (a) CH3I, K2CO3, acetone, 5 h, 90%; (b) (CH2O)n, MgCl2, Et3N, THF, reflux, 62%; (c) 50% NaOH,
C6H5CH2P+Ph3Brꢀ, CH2Cl2, rt, 85% (recovered starting material); (d) NaBH4, CH3OH, rt, 96%; (e) 50% NaOH, p-CH3OC6H5CH2P+Ph3Brꢀ,
CH2Cl2, rt, 85% (recovered starting material); (f) HNO3, CH3COOH: cyclohexane = 1:3, 40–50 °C, 70%; (g) Zn–Fe, CH2Cl2, 5% HCl, 40 °C, 93%;
(h) (CH3CO)2O, Py., rt, 99%; (i) NH2CONH2, Py., reflux, 99%; (j) KOH, CS2, CH3OH, H2O, 97%.
Table 1. Effect of Compounds 1–5 on murine lymphocyte proliferation induced by Concanavalin A (ConA) (5 mg/mL) or lipopolysaccharide (LPS)
(10 mg/mL)
Compound
Concentration (M)
Cytotoxicity
Inhibitory rate (%)
ConA-induced T cell proliferationa
LPS-induced B cell proliferation
1
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ14
ꢀ27
ꢀ18
ꢀ30
ꢀ93
ꢀ100
ꢀ30
ꢀ89
ꢀ100
ꢀ15
ꢀ47
ꢀ100
ꢀ21
ꢀ39
ꢀ32
ꢀ14
ꢀ89
ꢀ99
ꢀ16
ꢀ83
ꢀ99
ꢀ25
ꢀ45
ꢀ100
2
3
4
5
10ꢀ6
10ꢀ5
10ꢀ4
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
+
+
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
+
+
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ22
ꢀ75
ꢀ100
ꢀ22
ꢀ63
ꢀ99
a Student’s t-test was used to determine significance. Once the OD value was reduced and P < 0.05 was considered significant.
1. Protecting the phenol with methyl (16) could slightly
improve the inhibitory activity.
3. Introducing electron-donating groups (19, 22) did not
cause obvious change of the inhibitory effect but
improved the cytotoxicity. If held in the phenol and
the amino (23, 24), it could decrease the cytotoxicity
but the thiazole compound 25 exhibited considerable
cytotoxicity.
2. Introducing conjugated groups to the ortho-position
of the phenol (17, 18) led to higher immunosup-
pressive activity, except the nitro compound (21),
which relatively increased the cytotoxicity and
their (17, 18) inhibitory activity on LPS-induced B
cell proliferation was more potent than on
ConA-induced T cell proliferation. It was notewor-
thy that 20 slightly strengthened the T lymphocyte
proliferation.
3. Conclusions
In summary, a series of derivatives of bakuchiol were syn-
thesized and assessed in vitro for their cytotoxicity of lym-

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H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
Table 2. Effect of compounds 6–15 on murine lymphocyte proliferation induced by Concanavalin A (ConA) (5 mg/mL) or lipopolysaccharide (LPS)
(10 mg/mL)
Compound R1
R2ðR02Þ
R3ðR03Þ
Concentration (M) Cytotoxicitya
Inhibitory rate (%)
ConA-induced LPS-induced B
T cell proliferation cell proliferation
6
7
CH2CH
OCH3
H
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ9
ꢀ21
ꢀ38
ꢀ99
ꢀ8
ꢀ93
CH2CH
CH2CH
CH2CH
CH2CH
CH2CH
CH2CH
CH2CH
OH
OCH3
OH
F
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
ꢀ
ꢀ20
ꢀ11
ꢀ100
ꢀ36
ꢀ40
ꢀ100
8
H
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ19
ꢀ45
ꢀ100
ꢀ26
ꢀ42
ꢀ99
9
OCH3
OCH3
OCH3
OH
H
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
ꢀ
ꢀ13
ꢀ10
+2
ꢀ29
ꢀ35
ꢀ26
10
11
12
13
14
15
F
10ꢀ6
10ꢀ5
10ꢀ4
+
ꢀ17
ꢀ20
1
ꢀ33
ꢀ38
ꢀ9
ꢀ
ꢀ
R3=R2
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ26
ꢀ22
ꢀ100
ꢀ29
ꢀ51
ꢀ99
F
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ8
ꢀ30
ꢀ48
ꢀ100
ꢀ46
ꢀ100
OH (OCH3) OCH3 (OH) 10ꢀ6
ꢀ
+
+
ꢀ10
ꢀ33
ꢀ100
ꢀ99
10ꢀ5
10ꢀ4
ꢀ100
ꢀ100
OCH3
H
H
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ24
ꢀ24
ꢀ97
ꢀ31
ꢀ45
ꢀ98
CH2C6H4OCH3-p OCH3
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ2
ꢀ18
ꢀ32
ꢀ100
ꢀ2
ꢀ100
a Student’s t-test was used to determine significance. Once the OD value was reduced and P < 0.05 was considered significant.
phocyte, inhibitory activity on Con A-induced T cell pro-
liferation and LPS-induced B cell proliferation in com-
parison with bakuchiol. In the current study, it was
found that C-12,13 was a potential site to be modified that
could improve the inhibitory activity and the introduction
of conjugated groups to the ortho-position of the phenol
was probably a good way to improve the immunosuppres-
sive activity. The chiral quaternary center (C-9) was essen-
tial since ( )bakuchiol could increase the cytotoxicity.
ical, China). TLC: GF 254 silica gel plates (Yantai Mar-
ine Chemical Co. Ltd, China); detection by spraying
with 5% H2SO4 soln. followed by heating. HPLC: Agi-
lent 1100 series. Solvents were distilled from the appro-
priate drying agents before use.
4.1.1. Preparation of 12,13-epoxybakuchiol (2). Bakuch-
iol (5.0 mmol) was dissolved in 30 ml CH2Cl2 at 0 °C
and m-CPBA (m-chloroperbenzoic acid, 5.5 mmol) in
60 ml CH2Cl2 was added slowly. After being stirred
for 3 h at 25 °C, aqueous saturated NaHSO3 solution
was added and stirred for an additional 0.5 h. The reac-
tion mixture was extracted with CH2Cl2 and the organic
layer was washed with water, dried, and evaporated.
The product was purified by column chromatography
using hexane–EtOAc as eluent to give 2 (78%).
4. Experimental
4.1. Chemistry
NMR: Bruker 400 MHz instrument; TMS as internal
standard; d in ppm, J in Hz. EI-MS, HR-EI-MS: Finn-
igan LCQ-DECA instrument. Column chromatography
(CC): silica gel (200–300 mesh; Qingdao Marine Chem-
Compound 2: 1H NMR (400 MHz, CD3COCD3) d 1.19
(6H, s), 1.22 (3H, s), 1.58 (4H, m), 2.64 (1H, t,

H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
2407
Table 3. Effect of compounds 16–25 on murine lymphocyte proliferation induced by Concanavalin A (ConA) (5 mg/mL) or lipopolysaccharide (LPS)
(10 mg/mL)
Compound
Concentration (M)
Cytotoxicitya
Inhibitory rate (%)
ConA-induced T cell proliferation LPS-induced B cell proliferation
16
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ16
0
ꢀ33
ꢀ22
ꢀ69
ꢀ41
ꢀ90
ꢀ100
ꢀ24
ꢀ57
ꢀ100
ꢀ25
ꢀ34
ꢀ100
ꢀ74
ꢀ21
ꢀ79
ꢀ100
ꢀ13
ꢀ34
ꢀ100
ꢀ22
ꢀ23
ꢀ100
17
18
19
20
21
22
23
24
25
10ꢀ6
10ꢀ5
10ꢀ4
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
+
+
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
16
24
3
ꢀ8
ꢀ100
ꢀ10
ꢀ16
ꢀ9
ꢀ23
ꢀ40
ꢀ100
ꢀ14
ꢀ46
ꢀ100
ꢀ16
ꢀ14
ꢀ19
ꢀ19
ꢀ33
ꢀ99
ꢀ100
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
+
+
ꢀ22
ꢀ27
ꢀ9
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
+
+
ꢀ36
48
ꢀ100
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
+
ꢀ21
ꢀ52
ꢀ100
ꢀ21
ꢀ16
ꢀ16
ꢀ30
ꢀ43
ꢀ100
10ꢀ6
10ꢀ5
10ꢀ4
ꢀ
ꢀ
ꢀ
ꢀ
+
+
10ꢀ6
10ꢀ5
10ꢀ4
a Student’s t-test was used to determine significance. Once the OD value was reduced and P < 0.05 was considered significant.
J = 6.0 Hz), 5.01 (1H, m), 5.05 (1H, m), 5.92 (1H, m), 6.09
(1H, d, J = 16.2 Hz), 6.30 (1H, d, J = 16.2 Hz), 6.78 (2H,
d, J = 8.5 Hz), 7.27 (2H, d, J = 8.5 Hz); 13C NMR
(100 MHz, CD3COCD3) d 18.6, 23.3, 23.4, 24.7, 38.0,
42.5, 57.9, 64.2, 112.0, 115.8, 127.7, 127.8, 129.9, 134.7,
146.5, 157.3; EI-MS m/z 272 (M+); HR-EI-MS m/z calcd
C18H24O2: 272.1776, found: 272.1779.
d, J = 15.9 Hz), 6.72 (2H, d, J = 8.5 Hz), 7.20 (2H, d,
J = 8.5 Hz); 13C NMR (100 MHz, CDCl3) d 23.6, 24.8,
25.6, 26.9, 39.2, 42.7; 72.5, 79.4, 111.6, 115.8, 127.3,
127.7, 130.1, 135.3, 147.0, 157.2; EI-MS m/z 290 (M+);
HR-EI-MS m/z calcd C18H26O3: 290.1882. Found:
290.1880.
4.1.3. Preparation of 12-oxobakuchiol (4) and D,14 12-
hydroxybakuchiol (5). p-TsOH (0.1 mmol) was added to
a solution of compound 2 (1 mmol) in CH2Cl2 at room
temperature and the resulting solution was stirred for
0.5 h. The reaction mixture was quenched by aqueous
saturated NaHCO3 solution and extracted with CH2Cl2.
The organic layer was washed with brine, dried, and
evaporated. The product was purified by column chro-
matography using hexane–EtOAc as eluent to give 4
(20%) and 5 (64%).
4.1.2. Preparation of 12,13-diolbakuchiol (3). Compound
2 (1.0 mmol) was dissolved in the mixture of dioxane
and water (1:1, 15 ml) at 25 °C and 2–3 drops of 70%
H2SO4 were added. After 1 min, the reaction mixture
was quenched by aqueous saturated NaHCO3 solution
and extracted with CH2Cl2. The organic layer was
washed with brine, dried, and evaporated. The product
was purified by column chromatography using hex-
ane–EtOAc as eluent to give 3 (99%).
1
1
Compound 3: H NMR (400 MHz, CDCl3) d 1.05 (6H,
Compound 4: H NMR (400 MHz, CDCl3) d 1.06 (3H,
s), 1.13 (3H, s), 1.23 (1H, m), 1.40 (1H, m), 1.54 (1H, m),
1.86 (1H, m), 3.18 (1H, dd, J = 5.0, 12.0 Hz), 4.95 (1H,
d, J = 10.8 Hz), 4.96 (1H, d, J = 17.6 Hz), 5.86 (1H, dd,
J = 10.8, 17.6 Hz), 6.05 (1H, d, J = 15.9 Hz), 6.23 (1H,
s), 1.08 (3H, s), 1.17 (3H, s), 1.79 (2H, t, J = 7.8 Hz),
2.45 (2H, t, J = 7.8 Hz), 2.60 (1H, m), 5.02 (1H, dd,
J = 1.2, 17.5 Hz), 5.06 (1H, dd, J = 1.2, 10.8 Hz), 5.83
(1H, dd, J = 10.8, 17.5 Hz), 5.98 (1H, d, J = 16.2 Hz),

2408
H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
6.25 (1H, d, J = 16.2 Hz), 6.79 (2H, d, J = 8.6 Hz), 7.23
(2H, d, J = 8.6 Hz); 13C NMR (100 MHz, CDCl3) d
18.3, 23.4, 34.1, 35.8, 41.0, 42.0, 112.5, 11.4, 127.1,
127.3, 130.2, 134.6, 145.1, 155.1, 215.8; IRmmax (KBr):
3382.6, 2968.0, 1695.1, 972.0, 813.8 cmꢀ1; EI-MS m/z
272 (M+); HR-EI-MS m/z calcd C18H24O2: 272.1777.
Found: 272.1767.
orated. The product was purified by column chromatog-
raphy using hexane–EtOAc as eluent.
Compound 6: (2 steps, overall yield: 54%) 1H NMR
(400 MHz, CDCl3) d 1.10 (3H, s), 1.38 (2H, m), 1.59
(3H, m), 1.67 (3H, m), 1.84 (4H, m), 3.78 (3H, s), 4.76
(1H, m), 5.08 (3H, m), 5.89 (1H, m), 6.87 (2H, m),
7.25 (2H, m). EI-MS m/z 288 (M+); HR-EI-MS m/z
calcd C19H28O2: 288.2089. Found: 288.2077.
1
Compound 5: H NMR (400 MHz, CDCl3) d 1.18 (3H,
s), 1.56 (2H, m), 1.70 (3H, s), 1.77 (2H, m), 4.06 (1H, m),
4.95 (4H, m), 5.73 (1H, br s), 5.85 (1H, dd, J = 10.8,
17.6 Hz), 6.01 (1H, d, J = 16.2 Hz), 6.23 (1H, d,
J = 16.2 Hz), 6.76 (2H, d, J = 8.4 Hz), 7.21 (2H, d,
J = 8.4 Hz); 13C NMR (100 MHz, CDCl3) d 17.4, 23.4,
26.9, 29.5, 36.7, 42.1, 111.6, 112.1, 115.4, 126.7, 127.3,
130.4, 135.3, 145.7, 147.0, 154.9; EI-MS m/z 272 (M+);
HPLC 98.2% (the ratio of diastereoisomeric mixture is
almost 1:1).
Compound 7: (2 steps, overall yield: 51%) 1H NMR
(400 MHz, CDCl3) d 1.11 (3H, m), 1.34 (2H, m), 1.58
(3H, m), 1.67 (3H, m), 1.85 (4H, m), 3.88 (3H, m),
4.75 (1H, m), 5.08 (3H, m), 5.89 (1H, m), 6.90 (1H,
m), 7.01 (1H, m), 7.07(1H, m). EI-MS m/z 306 (M+);
HR-EI-MS m/z calcd C19H27FO2: 306.1995, Found:
306.1981.
Compound 8: (Yield: 88–92%) 1H NMR (400 MHz,
CDCl3) d 1.20 (3H, s), 1.50 (2H, m), 1.59 (3H, s), 1.68
(3H, s), 1.96 (2H, m), 5.02 (1H, d, J = 17.5 Hz), 5.04
(1H, d, J = 10.8 Hz), 5.12 (1H, t, J = 7.5 Hz), 5.89
(1H, dd, J = 10.8, 17.5 Hz), 6.06 (1H, d, J = 16.2 Hz),
6.26 (1H, d, J = 16.2 Hz), 6.78 (2H, d, J = 8.5 Hz),
7.25 (2H, d, J = 8.5 Hz). EI-MS: 256 (M+); HR-EI-MS
m/z calcd C18H24O: 256.1827; found: 256.1829.
4.1.4. The procedures for preparation of compounds 6–15.
A solution of Grignard reagent (2 equiv) in an inert ar-
gon atmosphere was added to a suspension of cuprous
iodide (0.5 equiv) in THF and cooled to ꢀ40 °C. After
stirring for 15 min at ꢀ40 °C, a solution of citral
(1 equiv) in THF was added to the reaction mixture
and the mixture was stirred for an additional 2 h at
ꢀ40 °C. After quenching with an aqueous saturated
NH4Cl solution, the mixture was diluted with Et2O
and successively washed twice with water and once with
brine. The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The prod-
uct was purified by column chromatography using hex-
ane–EtOAc as eluent.
Compound 9: (Yield: 90–96%) 1H NMR (400 MHz,
CDCl3) d 1.19 (3H, s), 1.50 (2H, m), 1.58 (3H, s), 1.67
(3H, s), 1.93 (2H, m), 3.80 (3H, s), 5.01 (1H, d,
J = 17.8 Hz), 5.03 (1H, d, J = 10.5 Hz), 5.10 (1H, t,
J = 7.4 Hz), 5.88 (1H, dd, J = 10.4, 17.8 Hz), 6.06 (1H,
d, J = 16.6 Hz), 6.26 (1H, d, J = 16.6 Hz), 6.84 (2H, d,
J = 8.6 Hz), 7.29 (2H, d, J = 8.6 Hz). EI-MS m/z 270
(M+); HR-EI-MS m/z calcd C19H26O: 270.1984. Found:
270.1985.
Subsequently, another Grignard reagent was added to
the aldehyde obtained above in dry THF with cooling
(0 °C) and stirring. After stirring for another hour, the
reaction mixture was allowed to stand overnight at
room temperature. The contents were cooled to 0 °C,
decomposed with cooled solution of ammonium chlo-
ride, and worked up in the usual manner. The product
was purified by column chromatography using hex-
ane–EtOAc as eluent to give the alcohol as diastereoiso-
meric mixtures.
1
Compound 10: (Yield: 90–96%) H NMR (400 MHz,
CDCl3) d 1.19 (3H, s), 1.49 (2H, m), 1.58 (3H, s), 1.67
(3H, s), 1.93 (2H, m), 3.88 (3H, s), 5.03 (2H, m), 5.10
(1H, t, J = 7.1 Hz), 5.87 (1H, dd, J = 10.8, 17.5 Hz),
6.06 (1H, d, J = 16.2 Hz), 6.21 (1H, d, J = 16.2 Hz),
6.88 (1H, t, J = 8.5 Hz), 7.02 (1H, d, J = 9.4 Hz), 7.12
(1H, dd, J = 2.1, 12.5 Hz); EI-MS m/z 288 (M+); HR-
EI-MS m/z calcd C19H25FO: 288.1890. Found:
288.1881.
The mixture of alcohols was dissolved in dry pyridine
and phosphoryl chloride was added dropwise with stir-
ring. The mixture was heated under reflux at 140 °C
(bath) for 1 h, cooled, diluted with light petroleum,
and washed with dilute hydrochloric acid, saturated so-
dium hydrogen carbonate, and water. The light petro-
leum layer was dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The product was purified
by column chromatography using light petroleum as
eluent.
1
Compound 11: (2 steps, overall yield: 86%) H NMR
(400 MHz, CDCl3) d 1.19 (3H, s), 1.49 (2H, m), 1.58
(3H, s), 1.67 (3H, s), 1.94 (2H, m), 3.88 (3H, s), 3.91
(3H, s), 5.04 (2H, m), 5.11 (1H, t, J = 7.1 Hz), 5.87
(1H, dd, J = 10.8, 17.3 Hz), 6.06 (1H, d, J = 16.2 Hz),
6.27 (1H, d, J = 16.2 Hz), 6.81 (1H, d, J = 8.7 Hz),
6.88 (1H, d, J = 8.7 Hz), 6.92 (1H, s); EI-MS m/z 300
(M+); HR-EI-MS m/z calcd C20H28O2: 300.2089.
Found: 300.2097.
The product obtained above was added to a solution of
methylmagnesium iodide in anhydrous ether. All the
solvent was evaporated off under vacuum, and the resi-
due, under argon, was heated at 180 °C for 10–20 min.
After cooling the mixture to rt, ether and dilute hydro-
chloric acid were added. The mixture was shaken and
the ether layer was washed with water, dried, and evap-
1
Compound 12: (Yield: 88–92%) H NMR (400 MHz,
CDCl3) d 1.19 (3H, s), 1.48 (2H, m), 1.58 (3H, s), 1.67
(3H, s), 1.93 (2H, m), 5.05 (3H, m), 5.86 (1H, dd,
J = 10.7, 17.5 Hz), 6.06 (1H, d, J = 16.1 Hz), 6.21 (1H,
d, J = 16.1 Hz), 6.92 (1H, t, J = 8.6 Hz), 7.02 (1H, d,
J = 9.4 Hz), 7.12 (1H, dd, J = 1.9, 12.0 Hz). EI-MS

H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
2409
m/z 274 (M+); HR-EI-MS m/z calcd C18H23FO:
274.1733. Found: 274.1732.
284 (M+); HR-EI-MS m/z calcd C19H24O2: 284.1777.
Found: 284.1776.
1
Compound 13: (Yield: 88–92%) H NMR (400 MHz,
4.1.7. Preparation of 3-(E)-styrylbakuchiol (18) and 3-
methoxystyrylbakuchiol (20). Compound 17 (1 mmol)
and the Wittig salt (1.2 mmol) were dissolved in CH2Cl2
(10 ml) followed by adding 50% NaOH (7 mmol) drop-
wise. After 3 h, the reaction mixture was quenched by
aqueous saturated NH4Cl solution and extracted with
CH2Cl2. The organic layer was washed with brine, dried,
and evaporated. The product was purified by column
chromatography using hexane–EtOAc as eluent.
CDCl3) d 1.19 (1.5H, s), 1.20 (1.5H, s), 1.49 (2H, m),
1.58 (3H, s), 1.67 (3H, s), 1.94 (2H, m), 3.86 (1.5H, s),
3.89 (1.5H, s), 5.02 (2H, m), 5.10 (1H, m), 5.87 (1H,
m), 6.04 (1H, m), 6.23 (1H, m), 6.79 (1H, m), 6.86
(1H, m), 7.01 (1H, s). EI-MS m/z 286 (M+); HR-EI-
MS m/z calcd C19H26O2: 286.1933. Found: 286.1928.
Compound 14: (Yield: 95%) 1H NMR (400 MHz,
CDCl3) d 1.37(3H, s), 1.59 (3H, s), 1.65 (2H, m), 1.68
(3H, s), 2.07 (2H, m), 3.81 (3H, s), 5.14 (1H, m), 6.14
(1H, d, J = 16.2 Hz), 6.53 (1H, d, J = 16.2 Hz), 6.86
(1H, d, J = 8.7 Hz), 7.32 (1H, d, J = 8.7 Hz); 13C
NMR (100 MHz, CDCl3) d 17.7, 23.0, 25.7, 28.3, 42.6,
55.3, 73.4, 113.9, 124.3, 126.5, 127.5, 129.8, 132.0,
134.5, 159.0; EI-MS m/z 260 (M+); HR-EI-MS m/z calcd
C17H24O2: 260.1776. Found: 260.1774.
Compound 18: (Yield: 85%) 1H NMR (400 MHz,
CD3COCD3) d 1.15 (3H, s), 1.45 (2H, m), 1.51 (3H, s),
1.59 (3H, s), 1.92 (2H, m), 4.98 (2H, m), 5.07 (1H, m),
5.88 (1H, dd, J = 10.2, 18.0 Hz), 6.12 (1H, d,
J = 16.5 Hz), 6.26 (1H, d, J = 16.5 Hz), 6.84 (1H, d,
J = 8.2 Hz), 7.14 (1H, dd, J = 2.0, 8.2 Hz), 7.18 (1H, m),
7.23 (1H, d, J = 16.4 Hz), 7.30 (2H, t, J = 7.6 Hz), 7.45
(1H, d, J = 16.4 Hz), 7.52 (2H, d, J = 7.1 Hz), 7.62 (1H,
d, J = 2.0 Hz); IRmmax (KBr): 3253.4, 2969.9, 2927.5,
1498.4, 966.2, 507.2 cmꢀ1; EI-MS m/z 358 (M+); HR-
EI-MS m/z calcd C26H30O: 358.2297. Found: 358.2292.
1
Compound 15: (3 steps, overall yield: 36%) H NMR
(400 MHz, CDCl3) d 1.05 (3H, s), 1.41 (2H, m),1.57
(3H, s), 1.66 (3H, s), 1.94 (2H, m), 2.62 (1H, s), 2.63
(1H, s), 3.78 (3H, s), 3.81 (3H, s), 5.09 (1H, m), 6.00
(1H, d, J = 16.2 Hz), 6.11 (1H, d, J = 16.2 Hz), 6.78
(1H, d, J = 8.8 Hz), 6.85 (1H, d, J = 8.7 Hz) 7.03 (1H,
d, J = 8.8 Hz),7.28 (1H, d, J = 8.7 Hz); EI-MS m/z 364
(M+); HR-EI-MS m/z calcd C25H32O2: 364.2402.
Found: 364.2406.
Compound 20: (Yield: 85%) 1H NMR (400 MHz,
CDCl3) d 1.21 (3H, s), 1.51 (2H, m), 1.59 (3H, s), 1.68
(3H, s), 1.97 (2H, m), 3.84 (3H, s), 5.04 (2H, m), 5.12
(1H, m), 5.89 (1H, dd, J = 10.6, 17.3 Hz), 6.10 (1H, d,
J = 16.2 Hz), 6.28 (1H, d, J = 16.2 Hz), 6.75 (1H, d,
J = 8.2 Hz), 6.90 (2H, d, J = 2.0, 8.6 Hz), 7.07 (1H, d,
J = 16.2 Hz), 7.15 (1H, dd, J = 2.2, 8.2 Hz), 7.20 (1H,
d, J = 16.2 Hz), 7.48 (3H, m); EI-MS m/z 388 (M+);
HR-EI-MS m/z calcd C27H32O2: 388.2403. Found:
388.2404.
4.1.5. Preparation of methyl ether of bakuchiol (16). Bak-
uchiol (2 mmol) was dissolved in acetone (5 ml) and
anhydrous K2CO3 (3 mmol) was added followed by
2 mmol of MeI. The mixture was allowed to reflux for
8 h. The reaction mixture was cooled, filtered, and evap-
orated. The product was purified by column chromatog-
raphy using hexane–EtOAc as eluent to give 16 (90%).
4.1.8. Preparation of 3-hydroxymethylbakuchiol (19).
Compound 17 (1 mmol) was dissolved in CH3OH
(10 ml) and NaBH4 was added. After 0.5 h, the reaction
mixture was poured into 20 ml water and extracted with
Et2O. The organic layer was washed with brine, dried,
and evaporated. The product was purified by column
chromatography using hexane–EtOAc as eluent to give
19 (96%).
The NMR spectra data of compound16 are the same as
those with that of compound 9.
4.1.6. Preparation of 3-formylbakuchiol (17). A mixture
of bakuchiol (10 mmol), MgCl2 (20 mmol), Et3N
(20 mmol) and paraformaldehyde (30 mmol) under ar-
gon and in THF (15 ml) was heated under reflux for
3 h. The reaction mixture was cooled to room tempera-
ture and ether was added. The resulting organic phase
was washed successively with 1 N HCl and water, dried
over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The product was purified by column chromatog-
raphy using hexane–EtOAc as eluent to give 17 (62%).
Compound 19: 1H NMR (400 MHz, CDCl3) d 1.19 (3H,
s), 1.48 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.95 (2H, m),
2.27 (1H, t, J = 4.5), 4.86 (2H, d, J = 4.5 Hz), 5.02 (2H,
m), 5.09 (1H, m), 5.86 (1H, dd, J = 10.8, 17.2 Hz), 6.04
(1H, d, J = 16.1 Hz), 6.22 (1H, d, J = 16.1 Hz), 6.83 (1H,
d, J = 8.1 Hz), 7.06 (1H, s), 7.21 (1H, dd, J = 8.1 Hz);
EI-MS m/z 286 (M+); HR-EI-MS m/z calcd C19H26O2:
286.1933. Found: 286.1941.
Compound 17: 1H NMR (400 MHz, CDCl3) d 1.21 (3H,
s), 1.51 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.95 (2H, m),
5.06 (3H, m), 5.88 (1H, dd, J = 10.6, 17.5 Hz), 6.12 (1H,
d, J = 16.2 Hz), 6.28 (1H, d, J = 16.2 Hz), 6.94 (1H, d,
J = 8.6 Hz), 7.50 (1H, d, J = 2.0 Hz), 7.56 (1H, dd,
J = 2.0, 8.6 Hz), 9.89 (1H, s), 10.93 (1H, s); 13C NMR
(100 MHz, CDCl3) d 17.6, 23.2, 25.7, 41.2, 42.6, 112.2,
117.7, 120.4, 124.6, 125.2, 130.2, 131.0, 131.4, 134.5,
137.4, 145.5, 160.6, 196.6; IRmmax (KBr) 2966.0,
2921.7, 1660.4, 1484.9, 970.0, 914.1 cmꢀ1; EI-MS m/z
4.1.9. Preparation of 3-nitrobakuchiol (21). The mixture
of HNO3 and HOAc (12:24 mmol) was added to the
solution of bakuchiol (10 mmol) in HOAc and cyclohex-
ane (1:3, 50 ml) with heating (48 °C) and stirring. 10 min
later, the reaction mixture was poured into water and
extracted with Et2O. The organic layer was washed with
brine, dried, and evaporated. The product was purified
by column chromatography using hexane–EtOAc as
eluent to give 21 (70%).

2410
H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
Compound 21: 1H NMR (400 MHz, CDCl3) d 1.21 (3H,
s), 1.51 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.92 (2H, m),
5.05 (3H, m), 5.86 (1H, dd, J = 10.6, 17.5 Hz), 6.17 (1H,
d, J = 16.2 Hz), 6.26 (1H, d, J = 16.2 Hz), 7.10 (1H, d,
J = 8.9 Hz), 7.62 (1H, dd, J = 2.2, 8.9 Hz), 8.03 (1H, d,
J = 2.2 Hz), 10.54 (1H, s); 13C NMR (100 MHz, CDCl3)
d 17.4, 22.9, 23.0, 25.5, 40.9, 42.6, 112.6, 120.1, 121.9,
124.6, 131.0, 131.7, 133.6, 135.1, 139.4, 145.3, 154.2;
EI-MS m/z 301 (M+); HR-EI-MS m/z calcd
C18H23NO3: 301.1677, Found: 301.1685.
d, J = 16.4 Hz), 6.30 (1H, d, J = 16.4 Hz), 7.08 (1H, dd,
J = 1.7, 8.3 Hz), 7.12 (2H, m), 9.00 (1H, br s); IRmmax
(KBr): 3216.7, 2923.6, 1778.1, 1463.7, 968.1, 709.7
cmꢀ1; EI-MS m/z 297 (M+); HR-EI-MS m/z calcd
C19H23NO2: 297.1729. Found: 297.1733.
4.1.13. Preparation of 3,4-benzo[d]oxazole-20-thion-
ebakuchiol (25). A solution of compound 22 (0.5 mmol),
KOH (0.5 mmol), and CS2 (0.5 mmol) in methanol
(1 ml) and water (0.15 ml) was refluxed for 3 h. The
reaction mixture was poured into water, extracted with
EtOAc. The organic layer was washed with brine, dried,
and evaporated. The product was purified by column
chromatography using hexane–EtOAc as eluent to give
19 (97%).
4.1.10. Preparation of 3-aminobakuchiol (22). Five per-
centage of HCl (1 ml) and Fe–Zn (3:3 mmol) were
added to the solution of compound 21 in CH2Cl2
(10 ml) with heating (40 °C) and stirring. 1 h later,
the reaction mixture was cooled, filtered. The filtrate
was extracted with CH2Cl2 and washed with aqueous
saturated NaHCO3 solution. The organic layer was
dried and evaporated. The product was purified by col-
umn chromatography using hexane–EtOAc as eluent to
give 22 (93%).
Compound 25: 1H NMR (400 MHz, CDCl3) d 1.21 (3H,
s), 1.51 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.95 (2H, m),
5.06 (3H, m), 5.87 (1H, dd, J = 10.8, 17.5 Hz), 6.18 (1H,
d, J = 16.3 Hz), 6.32 (1H, d, J = 16.3 Hz), 7.18 (1H, d,
J = 1.5 Hz), 7.22(1H, dd, J = 1.5, 8.5 Hz), 7.26 (1H, d,
J = 8.5 Hz), 10.6 (1H, br s); IR mmax (KBr): 3178.2,
2966.0, 1459.9, 933.4, 628.7 cmꢀ1; EI-MS m/z 313
(M+); HR-EI-MS m/z calcd C19H23NOS: 313.1501.
Found: 313.1518.
Compound 22: 1H NMR (400 MHz, CDCl3+D2O) d
1.17 (3H, s), 1.47 (2H, m), 1.57 (3H, s), 1.67 (3H, s),
1.93 (2H, m), 4.78 (1H, br s), 5.01 (2H, m), 5.10 (1H,
m), 5.86 (1H, dd, J = 10.8, 17.5 Hz), 6.00 (1H, d,
J = 16.2 Hz), 6.17 (1H, d, J = 16.2 Hz), 6.64 (1H, d,
J = 8.0 Hz), 6.68 (1H, dd, J = 1.8, 8.0 Hz), 6.81 (1H, d,
J = 1.8 Hz); 13C NMR (100 MHz, CDCl3) d 17.5, 23.0,
23.1, 25.5, 41.1, 42.3, 111.9, 114.4, 115.3, 118.1, 124.9,
126.9, 131.4, 131.6, 134.3, 135.7, 143.6, 146.2; EI-MS
m/z 271 (M+); HR-EI-MS m/z calcd C18H25NO:
271.1937. Found: 271.1950.
4.2. Biological assays
4.2.1. Reagents. Concanavalin A (Con A), lipopolysac-
charide (LPS, Escherichia coli 055:B5), 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT),
and 3,30,5,50-tetramethylbenzidine (TMB) were Sigma
(St. Louis, MO, USA) products. RPMI (Roswell Park
Memorial Institute) 1640 medium was purchased from
GibcoBRL, Life Technologies (USA). Fetal bovine ser-
um (FBS) was purchased from HyClone Laboratories
(Utah, USA). [3H]thymidine (1 mCi/ml) was purchased
from the Shanghai Institute of Atomic Energy.
4.1.11. Preparation of 3-acetamido-4-acetylbakuchiol
(23). Compound 22 was acetylated by the conventional
manner, using the Ac2O/pyridine, to give the product
23 (99%).
Compound 23: 1H NMR (400 MHz, CDCl3) d 1.19 (3H,
s), 1.48 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.93 (2H, m),
2.19 (3H, s), 2.36 (3H, s), 5.01 (2H, m), 5.09 (1H, t,
J = 7.2 Hz), 5.86 (1H, dd, J = 10.8, 17.4 Hz), 6.17 (1H,
d, J = 16.4 Hz), 6.29 (1H, d, J = 16.4 Hz), 7.05 (1H, d,
J = 8.3 Hz),7.13 (1H, d, J = 8.3 Hz), 8.16 (1H, s); 13C
NMR (100 MHz, CDCl3) d 17.6, 21.1, 23.2, 24.7, 25.7,
41.1, 42.7, 112.2, 120.5, 121.9, 122.2, 124.6, 126.3,
129.6, 131.4, 136.5, 138.8, 139.3, 145.5, 168.1, 168.7;
EI-MS m/z 355 (M+); HR-EI-MS m/z calcd
C22H29NO3: 355.2148. Found: 355.2159.
4.2.2. Animal. BALB/c mice, used at 6 to 8 weeks of age,
were purchased from Shanghai Experimental Animal
Center and were housed in a controlled environment
(12-h light/12-h dark photoperiod, 22
1
°C,
55% 5% relative humidity). All husbandry and exper-
imental contact made with the mice maintained specific
pathogen-free conditions. All mice were allowed to
acclimatize in our facility for 1 week before any experi-
ments were started. All experiments were carried out
according to the NIH Guide for Care and Use of
Laboratory Animals and were approved by the Bioeth-
ics Committee of the Shanghai Institute of Materia
Medica.
4.1.12. Preparation of 3,4-benzo[d]oxazole-20-onebakuch-
iol (24). A solution of compound 22 (0.5 mmol) and urea
(0.5 mmol) in dry pyridine (0.5 ml) was refluxed for 10–
12 h and poured into ice-cold water. The resulting mix-
ture was extracted with EtOAc and washed with brine.
The organic layer was dried and evaporated. The prod-
uct was purified by column chromatography using hex-
ane–EtOAc as eluent to give 24 (99%).
4.2.3. Preparation of spleen cell suspensions. Mice were
sacrificed and their spleens were removed aseptically.
A single spleen cell suspension was prepared and cell
debris and clumps were removed. Erythrocytes were
lysed with Tris-buffered ammonium chloride (0.155 M
NH4Cl, 16.5 mM Tris, pH 7.2) as previously de-
scribed,8–11 with slight modifications. Mononuclear cells
were washed and resuspended in RPMI-1640 medium
supplemented with 10% FBS, penicillin (100 U/ml),
and streptomycin (100 mg/ml).
Compound 24: 1H NMR (400 MHz, CDCl3) d 1.21 (3H,
s), 1.54 (2H, m), 1.58 (3H, s), 1.67 (3H, s), 1.95 (2H, m),
5.06 (3H, m), 5.87 (1H, dd, J = 10.8, 17.4 Hz), 6.14 (1H,

H. Chen et al. / Bioorg. Med. Chem. 16 (2008) 2403–2411
2411
2. Park, E. J.; Zhao, Y. Z.; Kim, Y. C.; Sohn, D. H. Planta
Med. 2005, 71, 508, and references cited therein.
3. Backhouse, C. N.; Delporte, C. L.; Negrete, R. E., et al.
J. Ethnopharmacol. 2001, 78, 27.
4. Ferrandiz, M.; Gil, B.; Sanz, M. J., et al. J. Pharm.
Pharmacol. 1996, 48, 975.
5. Backhouse, C. N.; Delporte, C. L.; Negrete, R. E., et al.
Phytochemistry 1995, 40, 325.
6. Jia, Q.; Hong, M. F. U.S. patent 0,251,749, 2006.
7. Pae, H. O.; Cho, H.; Oh, G. S., et al. Int. Immunophar-
macol. 2001, 1, 1849.
4.2.4. MTT assay. Cytotoxicity was assessed by the
MTT assay as previously described,8–11 with slight mod-
ifications. Briefly, spleen cells were cultured in triplicate
for 48 h with compounds. The cells with media alone
were used as controls. MTT (5 mg/ml) reagent was
added 4 h before the end of culture, and then cells were
lysed with 10% sodium dodecyl sulfate (SDS), 50% N,N-
dimethyl formamide, pH 7.2. OD values were read at
570 nm 6 h later and the cell death percent was calcu-
lated. Five mice were analyzed for each data point.
8. Yang, Z. S.; Zhou, W. L.; Sui, Y., et al. J. Med. Chem.
2005, 48, 4608.
9. Yao, G. M.; Ding, Y.; Zuo, J. P., et al. J. Nat. Prod. 2005,
68, 392.
10. Yang, Z. S.; Wang, J. X.; Zhou, Y., et al. Bioorg. Med.
Chem. 2006, 14, 8043.
11. Zhang, Y. N.; Zhong, X. G.; Zheng, Z. P., et al. Bioorg.
Med. Chem. 2007, 15, 988.
12. Ryu, S. Y.; Choi, S. U.; Lee, C. O.; Zee, O. P. Arch.
Pharm. Res. 1992, 15, 356.
13. Kita, Y.; Furukawa, A.; Futamura, J.; Ueda, K.; Sawama,
Y.; Hamamoto, H.; Fujioka, H. J. Org. Chem. 2001, 66,
8779.
4.2.5. Mitogen-induced proliferation assay. Spleen cells
were cultured in triplicate for 48 h with 5 mg/ml of Con-
canavalin A (Con A) or 10 mg/ml of lipopolysaccharide
(LPS) plus compounds. Cells were pulsed with 0.5 lCi/
well of [3H]thymidine for 8 h and harvested onto glass
fiber filters. The incorporated radioactivity was then
counted using a Beta Scintillation Counter (MicroBeta
Trilux, PerkinElmer Life Sciences). Five mice were ana-
lyzed for each data point.
14. This work will be detailed in a recent report.
15. Hansen, T. V. Org. Synth. 2005, 82, 64; Hansen, T. V.;
Skattebol, L. Tetrahedron Lett. 2005, 46, 3357.
16. Varma, R. S.; Verma, K. K. Indian J. Chem. 1988, 27B,
698.
References and notes
1. Sun, N. J.; Woo, S. H.; Cassady, J. M.; Snapka, R. M.
J. Nat. Prod. 1998, 61, 362, and references cited therein.