Hypoxia-inducible factor-1 and nuclear factor-κB inhibitory meroterpene analogues of bakuchiol, a constituent of the seeds of Psoralea corylifolia

Article abstract of DOI:10.1016/j.bmcl.2008.03.028

Two new meroterpenoids, 12,13-dihydro-12,13-dihydroxybakuchiol (2) and (12′S)-bisbakuchiol C (3), were isolated from the seeds of Psoralea corylifolia L. (Fabaceae). The structures of 2 and 3 were elucidated by spectroscopic and chemical methods. Six mero

Full text of DOI:10.1016/j.bmcl.2008.03.028

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2619–2623
Hypoxia-inducible factor-1 and nuclear factor-jB inhibitory
meroterpene analogues of bakuchiol, a constituent
of the seeds of Psoralea corylifolia
Cheng-Zhu Wu,a Seong Su Hong,b Xing Fu Cai,a Nguyen Tien Dat,a Ji-Xing Nan,c
Bang Yeon Hwang,d Jung Joon Leea and Dongho Leeb,*
aMolecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
bDivision of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
cKey Laboratory of Organism Functional Factors of the Changbai Mountain, Ministry of Education and College of Pharmacy,
Yanbian University, Yanji 133002, China
dCollege of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
Received 31 January 2008; revised 4 March 2008; accepted 10 March 2008
Available online 14 March 2008
Abstract—Two new meroterpenoids, 12,13-dihydro-12,13-dihydroxybakuchiol (2) and (120S)-bisbakuchiol C (3), were isolated from
the seeds of Psoralea corylifolia L. (Fabaceae). The structures of 2 and 3 were elucidated by spectroscopic and chemical methods. Six
meroterpenoids isolated from P. corylifolia and three semi-synthetic analogues were evaluated for HIF-1 and NF-jB inhibition, and
O-methyl and O-ethylbakuchiols (6 and 7) inhibited HIF-1 and NF-jB activation without significantly decreasing the viability of the
AGS and HeLa cells, respectively.
Ó 2008 Elsevier Ltd. All rights reserved.
Bakuchiol (1), a meroterpenoid isolated from the seeds
of Psoralea corylifolia L. (Fabaceae), has been shown
to exhibit the various biological properties including
antidiabetic,1,2 antiinflammatory,3 antimicrobial,4 anti-
oxidant,5 cytotoxic,6 and liver protective activities.7,8
In our search for biologically active agents of natural
origin, a methanol extract of the seeds of P. corylifolia
potently inhibited hypoxia-inducible factor-1 (HIF-1)
activation induced by hypoxia (100% inhibition at
20 lg/mL) in a HIF-1-mediated reporter gene assay in
AGS human gastric cancer cells. HIF-1 controls a num-
ber of cellular events required for the adaptation of can-
cer cells to hypoxia. Therefore, HIF-1 inhibitors are
considered as potential therapeutic agents for cancer.9,10
In a preliminary communication, we reported the initial
phytochemical and biological investigation of this plant,
with the isolation of bakuchiol (1) as a HIF-1 inhibitory
constituent, as well as two novel dimeric meroterpe-
noids, bisbakuchiols A and B (8 and 9), which were
not active in the HIF-1 mediated reporter gene assay.11
Here, we report the isolation of two new meroterpenes
(2 and 3) and a known meroterpene, 12,13-dihydro-
12,13-epoxybakuchiol (4),12 and the preparation of
bakuchiol analogues (5–7) (Fig. 1), in addition to the
biological evaluation of these compounds obtained.
Compound 2 was obtained as yellow oil and shown to
possess a molecular formula of C18H26O3 by positive
1
HRFABMS (m/z [M+Na]+, 313.1776). The H and 13C
NMR spectra of 2 were comparable to those of bakuchiol
(1), suggesting that 2 is a modified bakuchiol and a mer-
oterpene (Table 1). Thus, AA’XX’-type proton signals at
dH 6.71 (2H, d, J = 8.8 Hz, H-3 and H-5) and 7.19 (2H, d,
J = 8.8 Hz, H-2 and H-6), trans double bond signals at dH
6.04 (1H, d, J = 16.4 Hz, H-8) and 6.25 (1H, d,
J = 16.4 Hz, H-7), vinyl group signals at dH 5.02 (overlap,
H-18) and 5.91 (1H, m, H-17), and three methyl signals at
dH 1.11 (3H, s, H-14), 1.15 (3H, s, H-15), and 1.20 (3H, s,
H-16) were apparent. However, the absence of the proton
signal of an olefinic methine (H-12) of bakuchiol (1) and
the presence of signals at dH 3.21 (1H, dd, J = 2.0 and
10.8 Hz, H-12), dC 74.0 (s, C-13), and dC 80.6 (d, C-12)
led to the inference that oxidative modification had oc-
curred at the C-12 and C-13 double bond of bakuchiol
Keywords: Psoralea corylifolia; Fabaceae; Meroterpenoids; Hypoxia-
inducible factor-1; Nuclear factor-jB.
*
Corresponding author. Tel.: +82 2 3290 3017; fax: +82 2 953 0737;
e-mail: dongholee@korea.ac.kr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.028

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C.-Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2619–2623
Figure 1. Structures of compounds 1–9.
1
(1).12 Based on these observations and by comparison of
its spectroscopic data with those of bakuchiol (1),12 com-
pound 2 was suggested to be a 12,13-dihydroxylated bak-
uchiol, confirmed by HMQC and HMBC NMR
experiments. Therefore, the structure of the new com-
pound 2 was determined to be 12,13-dihydro-12,13-
dihydroxybakuchiol.13 Mosher’s methodology was ap-
plied to determine the absolute stereochemistry of the
C-12 position according to established procedures.14–16
However, after several attempts, Mosher’s esters of 2
could not be prepared, so the absolute configuration of
the C-12 position remains unknown.
in the H NMR spectrum of 3. In the aliphatic region,
an oxygenated methine signal at dH 3.61 (1H, dd,
J = 1.6 and 9.2 Hz, H-120) and six methyl signals at dH
1.20 (3H, s, H-150), 1.21 (3H, s, H-140), 1.22 (6 H, s,
H-16 and H-160), 1.60 (3H, s, H-14), and 1.69 (3H, s,
H-15) were apparent. Therefore, it was inferred that
compound 3 is a dimeric meroterpene, in which bakuch-
iol (1) and compound 2 are connected by ether linkage,
which was supported by mass fragmentations (Fig. 2).
This inference was supported by 2D NMR experiments
and was consistent with the molecular formula obtained.
The connectivity between the two meroterpene units was
determined by the analysis of 13C NMR data. Thus, the
downfield shift of the C-130 (a carbon) signal of 3 and
the upfield shifts of the C-120, 140, and 150 (b carbons)
compared to the corresponding 13C NMR data of 2
clearly indicated the linkage of the two units (C-4–C-
130).17 The absolute configurations of the C-120 and C-
9 (C-90) positions were clarified by Mosher’s methodol-
ogy and biogenetic consideration, respectively. Mosher’s
esters (3s and 3r) indicated the S configuration at C-120,
because of the positive values (DdSꢀR) obtained for H-
140 and H-150 and the negative differences for H-100
and H-110 (Fig. 2).18 Based on biogenetic consideration
(given the occurrence of 9S-bakuchiol only from nat-
ure),12,19 the stereochemistry of the stereogenic centers
at C-9 and C-90 could be assumed. Accordingly, the
structure of the new dimeric meroterpene, (120S)-bis-
bakuchiol C (3), was elucidated.20 To the best of our
knowledge, (120S)-bisbakuchiol C (3) is the first example
Compound 3 was obtained as yellow oil and shown to
possess a molecular formula of C36H48O3 by positive
HRFABMS (m/z [M+Na]+, 551.3498). The 1H and
13C NMR spectra of 3 exhibited two characteristic sets
of signals corresponding to bakuchiol (1) and com-
pound 2, respectively (Table 1). Briefly, two sets of
AA’XX’-type proton signals at dH 6.91 (2H, d,
J = 8.4 Hz, H-3 and H-5) and 7.28 (2H, d, J = 8.4 Hz,
H-2 and H-6); 6.78 (2H, d, J = 8.4 Hz, H-30 and H-50)
and 7.24 (2H, d, J = 8.4 Hz, H-20 and H-60), proton sig-
nals of two trans double bonds at dH 6.13 (1H, d,
J = 16.0 Hz, H-8) and 6.30 (1H, d, J = 16.0 Hz, H-7);
6.06 (1H, d, J = 16.0 Hz, H-80) and 6.28 (1H, d,
J = 16.0 Hz, H-70), proton signals of two vinyl groups
at dH 5.90 (1H, dd, J = 10.4 and 17.2 Hz, H-17) and
5.04 (overlap, H-18); 5.89 (1H, dd, J = 10.4 and
17.2 Hz, H-170) and 5.04 (overlap, H-180) were observed

C.-Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2619–2623
Table 1. NMR spectroscopic data (CDCl3) for compounds 1–4a
2621
Position
dH (J in Hz)
dC
2b
3
1
2b
3
4
1
130.9 s
127.4 d
115.4 d
154.6 s
126.4 d
135.9 d
42.5 s
131.1 s
128.4 d
116.4 d
157.9 s
128.4 d
136.1 d
43.5 s
134.0 s
126.9 d
124.5 d
153.4 s
126.7 d
137.4 d
42.8 s
130.3 s
127.3 d
115.4 d
155.1 s
127.0 d
135.0 d
42.2 s
2, 6
3, 5
4
7.19 d (8.8)
6.71 d (8.8)
7.28 d (8.4)
6.91 d (8.4)
7
6.25 d (16.4)
6.04 brd (16.4)
6.30 d (16.0)
6.13 d (16.0)
8
9
10
11
12
13
14
15
16
17
18
10
1.45 m, 1.87 m
1.27 m, 1.63 m
3.21 dd (10.8, 2.0)
1.51c m
1.98c m
5.12 brt
41.3 t
23.2 t
40.2 t
27.4 t
41.5 t
23.4 t
37.5 t
24.1 t
124.8 d
131.3 s
17.6 q
25.7 q
23.3 q
145.9 d
111.9 t
80.6 d
74.0 s
125.0 d
131.6 s
17.9 q
25.9 q
23.5 q
146.0 d
112.4d
130.8 s
127.6 d
115.6 d
155.1 s
127.0 d
135.8 d
42.6 s
65.1 d
59.2 s
1.11 s
1.15 s
1.20 s
5.91 m
5.02c
1.60 s
1.69 s
25.0 q
26.0 q
24.2 q
147.8 d
112.7 t
18.6 q
24.8 q
23.3 q
145.4 d
112.4 t
1.22 s
5.90 dd (17.2, 10.4)
5.04b
t
20, 60
30, 50
40
7.24 d (8.4)
6.78 d (8.4)
70
6.28 d (16.0)
6.06 d (16.0)
80
90
100
110
120
130
140
150
160
170
180
1.50 m, 1.98c m
1.44 m, 1.50c m
3.61 dd (9.2, 1.6)
38.6 t
26.4 t
78.9 d
83.8 s
1.21 s
1.20 s
20.8 q
23.3 q
23.8 q
146.0 d
1.22 s
5.89 dd (17.2, 10.4)
5.04c
112.2d
t
a The assignments were based on the DEPT, HMQC, and HMBC experiments.
b Measured for CD3OD solution.
c Overlapping signals.
d Signals may be interchangeable.
Table 2. Biological activities of compounds 1–7 (IC50 values, lM)
AGSa HeLab
HIF-1
6.1
MTT
NF-jB
MTT
1
15.3
—
6.9
—
11.0
—
c
2
3
4
5
6
7
—
—
—
—
12.2
—
—
—
—
5.7
8.7
26.3
26.8
55.8
—
5.7
5.7
18.1
—
12.2
—
a AGS, human gastric cancer cell.
b HeLa, human cervical adenocarcinoma cell.
c IC50 value >50 lM; 17-desmethoxy-17-N,N-dimethylaminoethylami-
nogeldanamycin (HIF-1, IC50 0.0036 lM; MTT, IC50 16.0 lM) and
celastrol (NF-jB, IC50 0.15 lM; MTT, IC50 2.06 lM) were used as
positive controls.
Figure 2. Mass fragmentation of compound 3 and DdS-R values of
MTPA esters (3s and 3r) of compound 3 by Mosher’s methodology.
of a dimeric meroterpene in which two meroterpenes are
linked by ether linkage.
only HIF-1 inhibitory principle (IC50 value 6.1 lM) iso-
lated from P. corylifolia.11 Previously, bakuchiol (1) was
reported to suppress the activation of NF-jB,3 which
regulates a number of genes involved in inflammation
and cancer responses.24 This led us to evaluate the
meroterpenes obtained from P. corylifolia for NF-jB
inhibition. Bakuchiol (1) and bisbakuchiol C (3) inhib-
Compounds 2–4 were evaluated for their potential to in-
hibit the HIF-1 activation induced by hypoxia using a
HIF-1-mediated reporter gene assay in AGS human gas-
tric cancer cells,21–23 but were inactive (IC50 values
>20 lg/ml) in the assay (Table 2). Therefore, as reported
in our preliminary communication, bakuchiol (1) is the

2622
C.-Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2619–2623
ited the NF-jB activation induced by TNF-a in HeLa
human cervical adenocarcinoma cells with IC50 values
of 6.9 and 12.2 lM, respectively, while compounds 2
and 4 and bisbakuchiols A and B were inactive (IC50
values >50 lM) in the assay (Table 2).25–28
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
03.028.
However, bakuchiol (1) exhibited significant cytotoxic-
ity to the AGS and HeLa cells as measured by MTT as-
says (IC50 values 15.3 and 11.0 lM, respectively) (Table
2), suggesting that bakuchiol (1) may regulate the
expression of HIF-1 and NF-jB, and/or the stability
of the AGS and HeLa cells. These results prompted us
to prepare simple bakuchiol analogues in an attempt
to increase HIF-1 and NF-jB inhibitory efficacy, to re-
duce the cytotoxicity, and to obtain a preliminary no-
tion of structure–activity relationship of bakuchiol (1).
Acetylbakuchiol (5),29,30 and O-methyl5,31 and O-ethyl-
bakuchiols32 (6 and 7) were prepared as described in
the Supplementary data. These analogues were tested
in both assay systems and acetylbakuchiol (5) displayed
almost the same biological activities as those of bakuch-
iol (1). However, O-methyl and O-ethylbakuchiols (6
and 7) retained the inhibitory effects against HIF-1
(IC50 values, 8.7 and 26.3 lM, respectively) and NF-
jB (IC50 values, 5.7 and 12.2 lM, respectively) activa-
tion without significantly affecting the viability of AGS
and HeLa cells, respectively, at the concentration of
50 lM.
References and notes
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of bakuchiol (1) play important roles in the biological
profiles of bakuchiol (1) in HIF-1 and NF-jB inhibition.
Thus, a bulky substitution on the hydroxyl group of
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bakuchiol (1) is required for the HIF-1 and NF-jB
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1
25
13. Compound 2: yellow oil. ½aꢁD +7.0° (c 0.1, MeOH). H-
and 13C NMR data of 2, see Table 1. HMBC correlations
H-2/C-7; H-7/C-1, C-8, C-9; H-8/C-1, C-7, C-9, C-16, C-
17; H-12/C-11, C-13, C-14, C-15; H-14/C-12, C-13, C-15;
H-15/C-12, C-13, C-14; H-16/C-8, C-9, C-10, C-17; H-17/
C-C-8, C-9, C-10, C-16. UV kmax (MeOH) nm (loge): 262
(3.93), 206 (3.99). ESIMS m/z: [M+Na]+ 313.4, [2M+Na]+
603.6, [MꢀH]ꢀ 289.4, [2M-H]ꢀ 579.6. HRFABMS m/z
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logues for HIF-1 and NF-jB inhibition, and found
out that O-methyl and O-ethylbakuchiols (6 and 7)
inhibited HIF-1 and NF-jB activation without signifi-
cantly decreasing the viability of AGS and HeLa cells,
respectively. Further studies are needed to optimize the
biological profiles of the meroterpenoids and to eluci-
date how the meroterpenoids inhibit HIF-1 and NF-
jB activation.
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Acknowledgments
1
18. 3r: H NMR (400 MHz, CDCl3) d: 1.994 (H-100), 1.366
(H-110), 3.553 (H-120), 1.167 (H-140), 1.152 (H-150). 3s: 1H
NMR (400 MHz, CDCl3) d: 1.990 (H-100), 1.358 (H-110),
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This work was supported by the Korea Research Foun-
dation Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF-
2006-311-E00588 to D. Lee) and by a research Grant
(PF06204-00 to J.J. Lee) from Plant Diversity Research
Center of 21st Frontier Research Program funded by the
Korean Ministry of Science and Technology. We thank
the Korea Basic Science Institute for providing certain
instruments used in this study.
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25
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C.-Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2619–2623
2623
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