Synthesis and evaluation of bakuchiol derivatives as potential anticancer agents

Article abstract of DOI:10.3390/molecules23030515

A series of bakuchiol derivatives were synthesized and evaluated for their anti-proliferative and the inhibitory activities on SMMC7721 cell line migration using PX-478 as a positive control. The results showed (S,E)-4-(7-methoxy-3,7-dimethyl-3-vinyloct-1

Full text of DOI:10.3390/molecules23030515

molecules
Article
Synthesis and Evaluation of Bakuchiol
Derivatives as Potential Anticancer Agents
Cheng-Zhu Wu †, Da-Chuan Liu †, Xing Guo, Yiqun Dai, Tao Ma, Hong-Mei Li * and Qiang Huo *
School of Pharmacy, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, China;
wuchengzhu0611@foxmail.com(C.-Z.W.); dcliu1226@outlook.com (D.-C.L.); guoxing7010@163.com (X.G.);
daiyiqun25@126.com (Y.D.); matao1992@foxmail.com (T.M.)
* Correspondence: athongmei@foxmail.com (H.-M.L.); huoqiang3@sohu.com (Q.H.);
Tel.: +86-552-3171262(H.-M.L.); +86-552-3173343 (Q.H.)
† These authors contributed equally to the work.
Received: 13 January 2018; Accepted: 14 February 2018; Published: 26 February 2018
Abstract: A series of bakuchiol derivatives were synthesized and evaluated for their anti-proliferative
and the inhibitory activities on SMMC7721 cell line migration using PX-478 as a positive control.
The results showed (S,E)-4-(7-methoxy-3,7-dimethyl-3-vinyloct-1-en-1-yl)phenol (10) to have the best
activity among the tested compounds, which included PX-478. In addition, compound 10 showed
greater inhibitory activity than that of bakuchiol in the transwell migration and invasion assays at
every dose. In western blotting tests, compound 10 showed a promising ability to downregulate
the expression of HIF-1α and its associated downstream proteins MMP-2 and MMP-9. Moreover,
this effect was dose-dependent and could represent a possible mechanism of action for the anticancer
activity of compound 10.
Keywords: bakuchiol derivatives; semi-synthesis; cytotoxicity; migration; invasion
1. Introduction
As sources of new drugs natural products of plant origin play one of the most important
roles in the drug research and development field. Bakuchiol (
from the seeds of Psoralea corylifolia L. (Fabaceae). Since its discovery, bakuchiol has exhibited
various biological properties, including anticancer [ ], antioxidant [ ], estrogenic-like [ ],
antimicrobial [ ], antifungal [ ], liver protective [10], human carboxylesterase 2 (hCE2) inhibitory [11],
1) is an isoprene terpenoid isolated
1
–
4
5
6,7
8
9
and immune-suppressive activities [12]. This evidence suggests that bakuchiol has potential for further
development and application.
A few studies have reported the semi-synthesis of bakuchiol and its various activities [
In our previous studies, we demonstrated the inhibitory effect of bakuchiol on hypoxia-inducible factor
(HIF-1 ), with an IC50 value of 6.1 M, in a screening experiment [15]. HIF-1 is an important
1,12–14].
1
α
α
µ
α
transcription factor that induces hypoxic conditions. It plays a dominant role in the expressions
of correlative genes and is involved in the proliferation, migration, invasion, vascularization,
and glycometabolism of tumor cells. Therefore, HIF-1
α
inhibitors are considered potential therapeutic
agents for cancer [16,17].
In this study, the benzene ring, hydroxyl group, and side-chain of bakuchiol (
1) were modified
to obtain a series of bakuchiol derivatives, and their pharmacological activities inhibiting migration
and invasion of SMMC7721 cells was evaluated in transwell assays under CoCl2-induced hypoxic
conditions. Their ability to inhibit the expression of relevant proteins, including HIF-1
α and its
associated downstream proteins, was evaluated by western blotting.
Molecules 2018, 23, 515; doi:10.3390/molecules23030515
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2. Results and Discussion
2.1. Chemistry
Bakuchiol (
gel column chromatography. The other target compounds
synthesized from in one- or two-step reactions according to the general pathway outlined in Scheme 1.
Compound was synthesized from compound by an alkylation reaction using bromopropane,
with anhydrous sodium carbonate in acetone as the catalyst. Compounds were obtained by
esterification of compound using acetic anhydride (compound ), propionic anhydride (compound ),
decyl chloride (compound ), and benzoyl chloride (compound ), respectively, with DMAP as the
catalyst. Because the acyl chlorides are unstable, decyl chloride and benzoyl chloride should be used
immediately after the compounds are obtained. Compound was obtained by nitration of compound
with nickel nitrate in acetone, using p-TSA as catalyst. Further, compound was reacted with propionic
anhydride to obtain compound . Bakuchiol was reacted with H2O and methanol to obtain compounds
and 10, respectively, and with 3-chloro- peroxybenzoic acid to form compound 11. The detailed
synthetic processes are described in the Materials and Methods section. The compounds 11 were
1) was extracted and isolated from the seeds of P. corylifolia L. and purified by silica
2
–
11 mentioned in this research were
1
2
1
3–6
1
3
4
5
6
7
1
7
8
9
2–
purified by silica gel column chromatography and their chemical structures were characterized using
1H-NMR, 13C-NMR, ESI-MS and (HR)-ESI-MS.
Scheme 1. Synthetic routes for bakuchiol derivatives. Reagents and conditions: (
K2CO3, acetone, reflux, 8 h. ( ) acetic anhydride, propionic anhydride, decyl chloride or benzoyl
chloride, catalytic amount of DMAP, methylene chloride, rt. ( ) nickel nitrate, catalytic amount of
a) bromopropane,
b
c
p-TSA, acetone, reflux. (
sulphuric acid, H2O or methanol, rt. (
0.5 h.
d
) propionic anhydride, catalytic amount of◦DMAP, acetone, rt. (
e) dilute
f) m-CPBA, methylene chloride, 0 C to rt, 0.5 h. rt, 3 h. NaHSO3,
2.2. Anti-proliferative Activity
All compounds were evaluated for their cytotoxicity in vitro against four human cancer
cell lines using MTT assays, with ADM (adriamycin) and PX-478 (S-2-amino-3-[40-N,N,-bis-(2-

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chloroethyl)amino]phenylpropionic acid N-oxide dihydrochloride) as positive control. PX-478 is
an inhibitor of constitutive hypoxia-induced HIF-1 and thus HIF-1 transcription factor activity in
human tumor xenografts and has marked antitumor activity against larger tumor xenografts, which has
a positive correlation with HIF-1 levels [18]. The MTT assays indicated cancer cell viability in response
α
α
α
to the different compounds, and the data reflected the inhibition effect of the bakuchiol derivatives on
cancer cell proliferation. Based on the results of the MTT assays in Table 1, most of compounds showed
cytotoxic activity against various cancer cell lines. Compound
against four cancer cell lines, however, the inhibition activity almost disappeared when the 4-hydroxy
group was alkylated by propane (compound ). Among the 4-acylates, compound showed the best
exhibited reduced activities as the carbon chain length
, replaced by benzoyl chloride, only showed little inhibitory
) also exhibited good cytotoxic
activity, which was better than its propionyl derivative (compound ), which had an IC50 of more
than 100 M for inhibition of HepG2. Compounds and 11 also showed commendable activities in
MTT assays. Among all the compounds assayed, compound 10 exhibited the best inhibition activity
with a low IC50. It is worth mentioning that eight compounds, compounds 10, and 11,
1 exhibited significant cytotoxic activity
2
3
cytotoxic activity, while compounds
gradually increased, and compound
4 and 5
6
effect on SW480. The nitration product of bakuchiol (compound
7
8
µ
9
1
,
3,
4,
7,
8,
9
,
exhibited better activities when compared with the PX-478, on human liver carcinoma SMMC7721 cells.
Table 1. Anti-proliferative activity of compounds 1–11 (IC50, µM, 48 h).
HepG2 b
SW480 d
Compound
SMMC7721 a
MDA-MB-231 c
1
2
15.44 ± 3.91 e
21.18 ± 1.16
12.97 ± 1.22
>100
9.96 ± 0.15
>100 f
>100
>100
3
4
5
6
7
8
9
10
11
10.65 ± 0.56
19.84 ± 0.66
46.80 ± 5.20
>100
9.97 ± 0.11
12.95 ± 0.60
9.62 ± 0.23
8.53 ± 0.88
23.26 ± 0.48
0.77 ± 0.08
27.57 ± 0.45
16.17 ± 0.06
27.95 ± 1.06
>100
9.28 ± 1.02
20.75 ± 0.91
>100
14.66 ± 0.19
27.66 ± 0.16
>100
>100
>100
59.88 ± 1.62
30.63 ± 1.43
39.94 ± 1.57
15.35 ± 0.34
5.01 ± 0.42
32.78 ± 0.54
0.26 ± 0.06
35.42 ± 0.39
19.06 ± 0.55
25.20 ± 1.44
15.58 ± 0.72
26.66 ± 1.14
8.79 ± 0.53
14.93 ± 0.64
2.23 ± 0.05
38.63 ± 0.53
>100
17.42 ± 1.32
4.60 ± 0.13
7.22 ± 0.70
3.05 ± 0.32
28.67 ± 0.68
ADM g
PX-478
a: SMMC7721: human liver carcinoma cells; b: HepG2: human liver hepatocellular carcinoma cells; c: MDA-MB-231:
human breast cancer cells; d: SW480: human colorectal carcinoma cells; e: The half maximal inhibitory concentration
± standard deviation (SD); f: No inhibitory activity at 100 µM; g: Adriamycin.
2.3. Preliminary Screening of the Inhibitory Effect on SMMC7721 Migration
Compound
1 and its derivatives 2–11 were screened, with PX-478 as the positive control,
using transwell assays, which enables us to identify the compounds that inhibit cancer cell migration
in vitro. The following observations were obtained and are listed in Figure 1. The preliminary
pharmacological screening revealed that three compounds (1, 8 and 10) exhibited the best inhibitory
activities on the migration of SMMC7721. Compounds 1 and 8 were almost equivalent to the PX-478;
it is also worth mentioning that compound 10 had the best anti-migration activity in the series of
derivatives, and was found to be better than PX-478. Because compound
8 could not inhibit all kinds
of cancer cells in the MTT assay, before, we selected only compounds and 10 for further experiments.
1

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Figure 1. Preliminary screening of the effects on migration of SMMC7721 cells under hypoxia conditions
of compounds 11 (20 M) compared with positive control (PX-478, 25 M) by transwell assays.
Hypoxia culture condition was established by 150 M CoCl2 induction for 24 h. 1
105 cells were
1
–
µ
µ
µ
×
seeded into the transwell migration chamber and exposed to compounds for 24 h. (A) The migrated
cells at the lower side of the membrane were then stained using Crystal Violet. (B) The quantification
analysis. Column, mean; bar, standard deviation; n = 3.
2.4. Ability to Inhibit Cell Migration and Invasion at Various Concentrations of Compounds 1 and 10
On the basis of the preliminary screening results, the cells were incubated with compounds
1 and
10 in various concentrations to investigate the effect of both inhibiting cell migration (Figure 2) and
invasion (Figure 3). The results showed that the migration and invasion abilities of the SMMC7721
cell line were suppressed by compounds
concentration was decreased, the anti-migratory and anti-invasive effects of the compounds was
reduced too. Both compounds and 10 showed strong anti-migratory activity and anti- invasive
activity at 10 and 20 M, which were better than that exhibited by the positive control, PX-478 (at a dose
of 25 M). The compounds also showed commendable activities when the dose was reduced to 5 M.
Compound 10, in particular, showed better anti-migratory and anti-invasive activities when compared
1 and 10 in a dose-dependent manner. In other words, as the
1
µ
µ
µ

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with that of compound
1
at every dose of the transwell assays, and continued to show some activity
even at a dose to 2.5 µM.
Figure 2. Effects of selected compounds
1 and 10 under the lower concentration gradient with positive
control (PX-478) on migration of SMMC7721 cells under hypoxia conditions by transwell assays.
1 × 105 cells were seeded onto the transwell migration chamber and exposed to compounds for 24 h.
(
(
A) The migrated cells at the lower side of the membrane were then stained using Crystal Violet.
B
) The quantification analysis. Column: mean; bar: standard deviation; n = 3. *p < 0.05 compared with
the control.
Figure 3. Cont.

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Figure 3. Effects of compounds
(PX-478) on invasion of SMMC7721 cell under hypoxia conditions by transwell assays. 1.5
were seeded into the matrigel coated (matrigel:DMEM = 1:8) transwell invasion chamber and exposed
to compounds for 36 h. ( ) The invaded cells at the lower side of the membrane were then stained
using Crystal Violet. ( ) The quantification analysis. Column: mean; bar: standard deviation; n = 3.
1
and 10 under the lower concentration gradient with positive control
×
105 cells
A
B
*p < 0.05 compared with the control.
2.5. Compound 10 Inhibits the Expression of Relevant Proteins
Since HIF-1α was reported, its status has grown dramatically, especially in cancer biology [19,20].
Hypoxic conditions are considered as one of the common characteristics of solid tumors [21], which can
facilitate tumor metastasis, migration, invasion, and many other important and fatal physiological
activities. Therefore, pharmacological inhibition of HIF-1α could be a valuable strategy to modulate
tumor apoptotic processes. In this manuscript, we discuss the anticancer activity of compound 10 by
testing its ability to inhibit HIF-1α expression with western blotting (Figure 4).
Figure 4. Effects of compound 10 on the expression level of HIF-1α and its associated down- stream
proteins: MMP-2 and MMP-9.
The western blot results clearly showed that expression of HIF-1
α protein was reduced by
compound 10 in a concentration-dependent manner under CoCl2-induced hypoxic conditions with
PX-478 as the positive control. Compared to PX-478, compound 10 showed significant inhibition,
with compound 10 showing slightly better inhibition than that shown by compound
Therefore, we continued to study the effect of compound 10 on the expression of MMP-2 and MMP-9
proteins, which are the associated downstream proteins of HIF-1 . We found compound 10 could
1 (Figure S22).
α

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also inhibit the expression of the two downstream proteins significantly, with PX-478 as the positive
control, and its suppression effect was concentration-dependent as well.
3. Materials and Methods
3.1. General Information
All reagents used for chemical synthesis were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Solvents were distilled before use. All the reactions were monitored by TLC on silica gel
F254 plates (Qingdao Haiyang Inc., Qingdao, China) for detection of the spot. Silica-gel column
chromatography was performed for purification of the products. All NMR spectra were recorded
on a DPX 600 instrument (Bruker, Fallanden, Switzerland) using CDCl3 as the solvent with TMS
as the internal standard. Chemical shifts are expressed in delta (δ) units and coupling constants in
Hz. The chemical shifts are reported in parts per million (ppm) and the following abbreviations
are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and doublet of doublets (dd).
Mass spectra were recorded on an ESI-Esquire 3000 instrument (Bruker Daltonics Inc., Billerica,
MA, USA). The purity of all compounds (bakuchiol and its derivatives) was determined by HPLC
(Shimadzu Co., Tokyo, Japan) and found to be in the 95–99% range.
3.2. Isolation of Bakuchiol (1)
The crude extract of bakuchiol was isolated from the seeds of P. corylifolia L. Then the bakuchiol
(S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1- yl)phenol, 1, purity > 95%), was purified by silica gel
column chromatography and characterized by spectroscopic and chemical methods [15]. Rf = 0.45
(n-hexane: ethyl acetate = 10:1).
3.3. Synthesis of (S,E)-1-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)-4-propoxybenzene (2)
A mixture of bakuchiol (1, 256 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) in acetone
(5 mL) was stirred at reflux temperature for 1 h. Then bromopropane (185 mg, 1.5 mmol) was added,
and the mixture was refluxed for another 5 h approximately. After cooling to the room temperature
and removing the solution, the reaction was poured in some water and stirred for 0.5 h to wash
away the remaining potassium carbonate. Then the solid was obtained by filtered and purified by
silica gel column chromatography with 50:1 n-hexane:ethyl acetate mixture to obtain compound
2
.
1
The yield was 52.0%. Rf = 0.85 (n-hexane: ethyl acetate = 10:1). H-NMR (CDCl3)
δ 7.27 (d, J = 8.0 Hz,
2H, Ar-H), 6.82 (d, J = 8.3 Hz, 2H, Ar-H), 6.25 (d, J = 15.9 Hz, 1H, 1-ene-H), 6.04 (d, J = 16.3 Hz, 1H,
2-ene-H), 5.87 (dd, J = 17.4, 10.7 Hz, 1H, 3-ethenyl-H), 5.10 (t, J = 6.4 Hz, 1H, 6-ene-H), 5.05–4.95 (m,
2H, 3-ethenyl-2H), 3.90 (t, J = 6.4 Hz, 2H, -OCH2-), 2.00–1.88 (m, 2H, -CH2-), 1.79 (dd, J = 13.9, 6.9 Hz,
2H, -CH2-), 1.66 (s, 3H, -CH3), 1.57 (s, 3H, -CH3), 1.52–1.43 (m, 2H, -CH2-), 1.18 (s, 3H, 3-CH3), 1.02 (t,
J = 7.2 Hz, 3H, -CH3). 13C-NMR (CDCl3)
δ 158.3, 146.1, 135.7, 131.3, 130.5, 127.1 (2C), 126.6, 124.9,
114.6 (2C), 111.8, 69.6, 42.5, 41.3, 25.7, 23.4, 23.3, 22.6, 17.6, 10.5. ESI-MS: m/z, [M + H]+ 299.2.
3.4. General Procedure for the Synthesis of (S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl) Phenyl Substituted
Carboxylic Esters 3–6
Compounds 3–6 were synthesized from compound 1 with an appropriate alkanoic anhydride
or acyl chloride in the presence of a catalytic amount of N,N-dimethylaminopyridine with 5 mL
dichloromethane (DCM) as solvent. Reactions were monitored by TLC. After the reaction was
complete, the mixture was poured in ice water and the resulting oily material, which was water
insoluble, was separated and dissolved in DCM. Then the organic layer was washed with water for
twice, then dried by anhydrous calcium chloride and concentrated under reduced pressure. The acyl
derivatives were purified by silica gel column chromatography with a 40:1 n-hexane:ethyl acetate
mixture as eluent to give compounds
3–6. The yields, Rf (n-hexane:ethyl acetate = 10:1) and the spectral
data of compounds 3–6 are given below.

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1
(S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)phenyl acetate (3). The yield was 90.6%. Rf = 0.70. H-NMR
(CDCl3)
δ 7.38–7.32 (m, 2H, Ar-H), 7.04–6.99 (m, 2H, Ar-H), 6.30 (d, J = 16.2 Hz, 1H, 1-ene-H), 6.16 (d,
J = 16.2 Hz, 1H, 2-ene-H), 5.87 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.10 (dd, J = 7.8, 6.4 Hz, 1H,
6-ene-H), 5.03 (ddd, J = 18.7, 14.1, 1.2 Hz, 2H, 3-ethenyl-2H), 2.29 (s, 3H, -COCH3), 1.95 (dd, J = 16.2,
7.7 Hz, 2H, -CH2-), 1.67 (s, 3H, -CH3), 1.58 (s, 3H, -CH3), 1.53–1.46 (m, 2H, -CH2-), 1.20 (s, 3H, 3-CH3).
13C-NMR (CDCl3)
42.7, 41.2, 25.7, 23.3, 23.2, 21.1, 17.7. ESI-MS: m/z, [M + H]+ 299.2.
δ 169.5, 149.6, 145.7, 138.2, 135.8, 131.4, 127.0 (2C), 126.3, 124.7, 121.6 (2C), 112.1,
(S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)phenyl propionate (
4). The yield was 61.5%. Rf = 0.77.
1H-NMR (CDCl3)
δ
7.35 (d, J = 8.6 Hz, 2H, Ar-H), 7.05–6.97 (m, 2H, Ar-H), 6.30 (d, J = 16.3 Hz,
1H, 1-ene-H), 6.15 (d, J = 16.3 Hz, 1H, 2-ene-H), 5.88 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.10 (t,
J = 7.1 Hz, 1H, 6-ene-H), 5.03 (ddd, J = 18.7, 14.1, 1.2 Hz, 2H, 3-ethenyl-2H), 2.58 (q, J = 7.5 Hz, 2H,
-CH2-), 1.95 (d, J = 8.3 Hz, 2H, -CH2-), 1.67 (s, 3H, -CH3), 1.58 (s, 3H, -CH3), 1.52–1.47 (m, 2H, -CH2-),
1.26 (t, J = 7.6 Hz, 3H, -CH3), 1.20 (s, 3H, 3-CH3). ESI-MS: m/z, [M + H]+ 313.2.
(S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)phenyl decanoate (
5). The yield was 11.7%. Rf = 0.80.
1H-NMR (CDCl3)
δ
7.41–7.37 (m, 2H, Ar-H), 7.06–7.02 (m, 2H, Ar-H), 6.34 (d, J = 16.2 Hz, 1H, 1-ene-H),
6.19 (d, J = 16.2 Hz, 1H, 2-ene-H), 5.92 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.15 (td, J = 7.1, 3.6 Hz,
1H, 6-ene-H), 5.07 (ddd, J = 18.8, 14.1, 1.3 Hz, 2H, 3-ethenyl-2H), 2.58–2.55 (m, 2H, -CH2-), 1.99 (dd,
J = 16.2, 7.6 Hz, 2H, -CH2-), 1.79–1.76 (m, 2H, -CH2-), 1.71 (s, 3H, -CH3), 1.62 (s, 3H, -CH3), 1.46–1.40
(m, 4H, -(CH2)2-), 1.35–1.31 (m, 10H, -(CH2)5-), 1.24 (s, 3H, 3-CH3), 0.93–0.92 (m, 3H, -CH3). 13C-NMR
(CDCl3)
δ 172.2, 149.9, 145.7, 138.2, 135.7, 131.2, 127.0 (2C), 126.4, 124.9, 121.7 (2C), 112.2, 42.7, 41.4,
34.4, 32.0, 29.9, 29.7, 29.6, 29.5, 29.3, 25.7, 25.1, 23.3, 22.8, 17.6, 14.2. ESI-MS: m/z, [M + H]+ 411.3.
(S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)phenyl benzoate (
). The yield was 13.9%. Rf = 0.70. 1H-NMR
(CDCl3) 8.20 (dd, J = 8.3, 1.2 Hz, 2H, ph-H), 7.65–7.61 (m, 1H, ph-H), 7.51 (t, J = 7.8 Hz, 2H, ph-H),
6
δ
7.41 (d, J = 8.5 Hz, 2H, Ar-H), 7.15 (d, J = 8.6 Hz, 2H, Ar-H), 6.34 (d, J = 16.3 Hz, 1H, 1-ene-H), 6.19 (d,
J = 16.3 Hz, 1H, 2-ene-H), 5.89 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.12 (t, J = 7.2 Hz, 1H, 6-ene-H),
5.04 (ddd, J = 18.8, 14.1, 1.3 Hz, 2H, 3-ethenyl-2H), 1.97 (dd, J = 16.2, 7.4 Hz, 2H, -CH2-), 1.68 (s, 3H,
-CH3), 1.59 (s, 3H, -CH3), 1.52 (dd, J = 9.6, 7.8 Hz, 2H, -CH2-), 1.22 (s, 3H, 3-CH3). 13C-NMR (CDCl3)
δ
165.2, 149.8, 145.7, 138.3, 135.8, 133.6, 131.4, 130.2 (2C), 129.6, 128.6(2C), 127.1 (2C), 126.4, 124.7,
121.7 (2C), 112.1, 42.7, 41.3, 25.7, 23.3, 23.3, 17.7. Its HR-ESI-MS exhibited a pseudo molecular ion peak
at m/z 361.2162 [M + H]+ consistent with the molecular formula C25H28O2.
3.5. Synthesis of (S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)-2-nitrophenol (7)
Compound
a catalytic amount of p-TSA (0.01 mmol) were added. The mixture was refluxed until compound
was fully consumed, and then the acetone was removed under vacuum to get the crude product.
After extraction with water and DCM, the organic layer was dried with anhydrous calcium chloride
overnight and concentrated under reduced pressure. The pure compound was obtained by silica gel
1 (0.5 mM) was dissolved in acetone (10 mL), then nickel (II) nitrate (1 mmol) and
1
7
column chromatography with n-hexane:ethyl acetate mixture (40:1) as eluent. The yield of compound
1
7
was 51.8%. Rf = 0.85 (n-hexane:ethyl acetate = 10:1). H-NMR (CDCl3)
δ
10.52 (s, 1H, Ar-OH), 8.04 (s,
1H, Ar-H), 7.61 (dd, J = 8.3, 1.7 Hz, 1H, Ar-H), 7.10 (d, J = 8.7 Hz, 1H, Ar-H), 6.26 (d, J = 15.8 Hz, 1H,
1-ene-H), 6.17 (d, J = 16.3 Hz, 1H, 2-ene-H), 5.87 (dd, J = 17.4, 10.6 Hz, 1H, 3-ethenyl-H), 5.15–5.09 (m,
1H, 6-ene-H), 5.10–4.98 (m, 2H, 3-ethenyl-2H), 1.95 (dd, J = 16.4, 8.7 Hz, 2H, -CH2-), 1.68 (s, 3H, -CH3),
1.58 (s, 3H, -CH3), 1.53 - 1.47 (m, 2H, -CH2-), 1.21 (s, 3H, -CH3). ESI-MS: m/z, [M + H]+ 302.3.
3.6. Synthesis of (S,E)-4-(3,7-dimethyl-3-vinylocta-1,6-dien-1-yl)-2-nitrophenyl Propionate (8)
Using the preparation and purification described above for compound 4, and using 7, propionic
anhydride and catalytic amount of N,N-dimethylaminopyridine in 5 mL DCM as solvent, compound
1
8
was prepared in 67.2% yield. Rf = 0.50 (n-hexane:ethyl acetate = 20:1). H-NMR (CDCl3)
δ
8.04 (d,
J = 2.1 Hz, 1H, Ar-H), 7.59 (dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 7.15 (d, J = 8.4 Hz, 1H, Ar-H), 6.30 (q,

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J = 16.2 Hz, 2H, 1-ene-2H), 5.87 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.09 (dt, J = 6.0, 4.8 Hz, 2H,
3-ethenyl-H and 6-ene-H), 5.03 (dd, J = 17.5, 1.1 Hz, 1H, 3-ethenyl-H), 2.67 (q, J = 7.5 Hz, 2H, -CH2-),
1.98 - 1.92 (m, 2H, -CH2-), 1.68 (s, 3H, -CH3), 1.58 (s, 3H, -CH3), 1.52 (ddd, J = 8.3, 5.2, 1.7 Hz, 2H, -CH2-),
1.29 (t, J = 7.5 Hz, 3H, -CH3), 1.22 (s, 3H, -CH3). 13C-NMR (CDCl3)
δ 172.2, 144.9, 142.6, 141.8, 141.5,
137.1, 131.8, 131.6, 125.2, 124.5, 122.8, 112.8, 42.9, 41.1, 27.5, 25.7, 23.2, 23.1, 17.8, 8.7. Its HR-ESI-MS
exhibited a pseudo molecular ion peak at m/z 358.2013 [M + H]+ consistent with the molecular formula
C21H27NO4.
3.7. Synthesis of (S,E)-4-(7-hydroxy-3,7-dimethyl-3-vinyloct-1-en-1-yl)phenol (
dimethyl-3-vinyloct-1-en-1-yl)phenol (10)
9) and (S,E)-4-(7-methoxy-3,7-
Compound
1 (0.5 mM, Rf = 0.70, DCM:methanol = 50:1) was dissolved in 5 mL methanol,
and dilute sulphuric acid (1 mL, 3 M) was slowly added with ice bath cooling. Then the temperature
was raised up to 45 ◦C and the mixture was stirred for 8 h until the reaction was complete (monitored
by TLC). After the methanol was removed under reduced pressure, the solid residue was extracted
with 250 mL water and the same volume of DCM. Then the organic layer was washed with water,
dried over anhydrous sodium sulfate overnight, and concentrated under reduced pressure. The residue
was then purified by column chromatography on silica gel, using DCM and methanol mixture (80:1)
as eluent, to give compounds 9 and 10.
(S,E)-4-(7-hydroxy-3,7-dimethyl-3-vinyloct-1-en-1-yl)phenol (
9). The yield was 16.4%. Rf = 0.20 (DCM:
methanol = 50:1). 1H-NMR (CDCl3)
δ
7.26–7.24 (m, 1H, Ar-H), 7.24–7.23 (m, 1H, Ar-H), 7.10 (s, 1H),
6.77–6.76 (m, 1H, Ar-H), 6.75–6.74 (m, 1H, Ar-H), 6.24 (d, J = 16.2 Hz, 1H, 1-ene-H), 6.05 (d, J = 16.2 Hz,
1H, 2-ene-H), 5.87 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.02 (ddd, J = 18.9, 14.1, 1.3 Hz, 2H,
3-ethenyl-H), 1.48–1.44 (m, 4H, -CH2-), 1.37–1.33 (m, 2H, -CH2-), 1.26 (s, 1H, Ar-OH), 1.20 (s, 6H,
7-CH3 and 8-CH3), 1.19 (s, 3H, 3-CH3). 13C-NMR (CDCl3)
δ 154.9, 146.0, 135.8, 127.4 (2C), 126.6, 116.0,
115.4 (2C), 111.9, 71.4, 44.6, 42.6, 41.9, 29.3 (2C), 23.5, 19.3. ESI-MS: m/z, [M − H]+ 273.2.
(S,E)-4-(7-methoxy-3,7-dimethyl-3-vinyloct-1-en-1-yl)phenol (10). The yield was 20.0%. Rf = 0.40 (DCM:
1
methanol = 50:1). H-NMR (CDCl3)
δ 7.25–7.24 (m, 1H, Ar-H), 7.24–7.23 (m, 1H, Ar-H), 6.78–6.76
(m, 1H, Ar-H), 6.76–6.75 (m, 1H, Ar-H), 6.24 (d, J = 16.2 Hz, 1H, 1-ene-H), 6.05 (d, J = 16.2 Hz, 1H,
2-ene-H), 5.87 (dd, J = 17.5, 10.7 Hz, 1H, 3-ethenyl-H), 5.04–4.98 (m, 2H, 3-ethenyl-2H), 3.16 (s, 3H,
-OCH3), 1.47–1.41 (m, 4H, -CH2-), 1.34–1.28 (m, 2H, -CH2-), 1.18 (s, 3H, 3-CH3), 1.13 (s, 6H, -CH3).
13C-NMR (CDCl3)
δ 155.0, 146.1, 135.8, 130.6, 127.4 (2C), 126.6, 115.4 (2C), 111.9, 75.0, 49.1, 42.6, 42.0,
40.5, 25.0 (2C), 23.5, 18.8. Its HR-ESI-MS exhibited a pseudo molecular ion peak at m/z 289.2139
[M + H]+ consistent with the molecular formula C19H28O2.
3.8. Synthesis of 4-((3S,E)-3-(2-(3,3-dimethyloxiran-2-yl)ethyl)-3-methylpenta-1,4-dien-1-yl) phenol (11)
Compound 1 (1.5 mmol) was dissolved in DCM (6 mL). 3-Chloroperoxybenzoic acid (m-CPBA,
3 mmol) dissolved in 5 mL DCM was added dropwise to the solution under ice bath cooling, and then
the mixture was stirred at room temperature for 4.5 h, until the reaction was complete as monitored by
TLC. Saturated sodium bicarbonate was added and stirred for 1 h to neutralize the excess m-CPBA.
After pouring into 250 mL water, the aqueous layer was extracted thrice with 90 mL dichloromethane
(30 mL
×
3). The combined dichloromethane layers were dried over anhydrous sodium sulfate
overnight. Evaporation of the solvent gave a crude product, which was purified by silica gel column
chromatography with n-hexane:ethyl acetate mixture (10:1) as eluent to give the title compound in
1
27.4% yield. Rf = 0.30 (n-hexane:ethyl acetate mixture = 10:1). H-NMR (CDCl3)
δ
7.25–7.24 (m, 1H,
Ar-H), 7.24–7.23 (m, 1H, Ar-H), 6.78–6.77 (m, 1H, Ar-H), 6.76–6.75 (m, 1H, Ar-H), 6.28–6.24 (m, 1H,
1-ene-H), 6.07–6.01 (m, 1H, 2-ene-H), 5.89–5.83 (m, 1H, 3-ethenyl-H), 5.07–5.02 (m, 2H, 3-ethenyl-2H),
2.71 (t, J = 5.7 Hz, 1H, 30-CH-), 1.58–1.54 (m, 6H, -CH3), 1.31–1.28 (m, 2H, -CH2-), 1.26–1.24 (m, 2H,
-CH2-), 1.20 (s, 3H, -CH3). 13C-NMR (CDCl3)
112.2, 64.7, 58.5, 42.2, 37.5, 24.9, 24.2, 23.4, 18.7. ESI-MS: m/z, [M + H]+ 273.2.
δ 154.8, 145.5, 135.2, 129.8, 127.4 (2C), 126.9, 115.4 (2C),

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3.9. Cell Culture, Growth Conditions, and Treatments
SMMC7721 (human liver carcinoma cells), HepG2 (human liver hepatocellular carcinoma cells)
and SW480 (human colorectal carcinoma cells) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM), which was obtained from Gibco (Grand Island, NY, USA). MDA-MB-231 cells (human breast
cancer cells) were cultured in minimum essential medium (MEM, Gibco, Grand Island, NY, USA)
supplemented with 10% fetal bovine serum (FBS, Millipore, Billerica, MA, USA) and maintained
at 37 ◦C in a humidified incubator with 5% CO2. All four types of cancer cells were obtained from
the Shanghai Cell Bank of the Chinese Academy of Sciences (CAS). To establish hypoxic conditions,
the culture medium was supplemented with 150
dimethyl sulfoxide (DMSO, BIOSHARP, Hefei, China). PX-478 (Selleck Chemicals, Houston, TX, USA),
a selective HIF-1 inhibitor, was dissolved in ddH2O. The control group was treated with DMSO only
µM CoCl2 for 24 h. The compounds were dissolved in
α
under identical conditions.
3.10. MTT Assay
Cells were seeded in 96-well microtiter plates at a density of 5
×
103 cells per well and treated with
different concentrations of the compounds and adriamycin for 48 h. At the end of the incubation period,
15 L of MTT (purchased from Sigma, St. Louis, MO, USA), at a dose of 5 mg/mL in phosphate-buffered
saline (PBS), was added to each well and incubated at 37 ◦C for 4 h. After 4 h, the MTT solution was
replaced with 150 L of DMSO to dissolve the formazan crystals. The plates were further incubated
µ
µ
for 30 min, and the cell viability was determined by measuring absorbance at a test wavelength of 490
nm with the microplate reader (Synergy HT, BioTek, Winooski, VT, USA).
3.11. Cell Migration Assay
The migration assay, which was used to screen the active compounds from the target products,
was performed using a 24-well cell culture plate with 8.0 µm pore size membrane inserts without
matrigel. First, 5
×
104◦ SMMC7721 cells were added to the upper wells, and the chambers were
incubated for 24 h at 37 C. The lower chamber was filled with 800 µL of 10% FBS as a chemoattractant.
After 24 h under normoxic conditions, the cells that had migrated were stained (Crystal Violet) and
photographed under a light microscope at 200 magnification. The number of migratory cells was then
×
counted and analyzed to determine statistically significant differences. Each condition was assayed in
triplicate, the experiments were performed independently at least three times, and the results were
expressed as the number of cells/field. One-way analysis of variance was used to determine significant
differences. The assay described here was also used to determine the influence of the compounds
1
and 10 at different concentrations on the migration of SMMC7721 cells.
3.12. Cell Invasion Assay
The invasion assay was performed using a 24-well cell culture plate with 8.0
µm pore size
membrane inserts. The membrane undersurface was coated with 50
µL of matrigel with serum-free
medium for 30 min at 37 ◦C. The rest of the operational steps and the conditions of the invasion assay
were the same as those described for the cell migration assay.
3.13. Western Blotting
The SMMC7721 cells were plated in a 6-well culture plate at a density of 5
After incubation with drugs, the cells were lysed with SDS buffer containing 200 mM Tris (pH 6.8),
40% glycerol, and 10% mercaptoethanol. The protein (30 g) obtained from cell extracts was separated
×
105 cells per well.
µ
using 10–15% SDS/polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene
difluoride (PVDF) membrane. Membranes were blocked with 10% skimmed milk in Tris-buffered
saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight at
◦
4 C with respective primary antibodies [anti-HIF-1
α
(Epitomics, Burlingame, CA, USA), anti-MMP-2

Molecules 2018, 23, 515
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(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-MMP-9 (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA), and anti- -actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA)]
β
diluted to 1:1000–2000 in TBST containing 5% skimmed milk. After washing three times with TBST,
the membranes were incubated with horseradish peroxidase- conjugated anti-rabbit and anti-mouse
serum, which were used as secondary antibodies (diluted 1:10,000 in TBST containing 5% skimmed
milk) for 1 h at room temperature. After incubation, the antigen–antibody complexes were visualized
using the ECL kit and β-actin was analyzed as a loading control.
3.14. Statistical Analysis
All experiments were repeated three times. Results are expressed as the mean
±
standard
deviation. Differences were evaluated using one-way analysis of variance with the least significant
difference post hoc test for multiple comparisons via SPSS (version 17.0, SPSS Inc., Chicago, IL, USA).
p < 0.05 was considered to indicate a statistically significant difference.
4. Conclusions
In summary, a series of bakuchiol derivatives 2–11 were synthesized and evaluated in vitro for
their anti-proliferative, anti-migratory, and anti-invasive activities. Our data revealed that compound
10 showed the best activity among all the assayed compounds. We would like to mention that this was
the first time the compound 10 was synthesized, evaluated, and explored to study its mechanisms,
and a more in-depth and comprehensive study to confirm these results would be worthwhile. Further
study and evaluation of bakuchiol derivatives has broad prospects and is important in the search for
new anticancer agents.
Supplementary Materials: The following are available online, Figures S1–S22.
Acknowledgments: This research was financially supported by the Foundation for Young Talents in College
of Anhui Province under Grant (2013SQRL048ZD); Education Department of Anhui Natural Science Research
Project China under Grant (KJ2016A465, KJ2017A233).
Author Contributions: Qiang Huo and Hong-Mei Li designed the research; Cheng- Zhu Wu, Da-Chuan Liu,
and Xing Guo performed experiments; Yiqun Dai and Tao Ma analyzed the data; Cheng-Zhu Wu contributed to
the writing of the manuscript.
Conflicts of Interest: There are no conflicts of interest.
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Sample Availability: Samples of the compounds 1–11 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).