Structural modification of honokiol, a biphenyl occurring in magnolia officinalis: The evaluation of honokiol analogues as inhibitors of angiogenesis and for their cytotoxicity and structure-activity relationship

Article abstract of DOI:10.1021/jm200830u

Honokiol, widely known as an antitumor agent, has been used as an antiangiogenesis drug lead. In this paper, 47 honokiol analogues and derivatives were investigated for their antiangiogenic activity by application of the transgenic zebrafish screening model, antiproliferative and cytotoxic activity against HUVECs, and three tumor cell lines by MTT assay. 3′,5-Diallyl-2, 4′-dihydroxy-[1,1′-biphen-yl]-3,5′-dicarbaldehyde (8c) was found to suppress the newly grown segmental vessels from the dorsal aorta of zebrafish and prevent inappropriate vascularization as well as exhibit more potent inhibitory effects on the proliferation of HUVECs, A549, HepG2, and LL/2 cells (IC₅₀ = 15.1, 30.2, 10.7, and 21.7 μM, respectively) than honokiol (IC₅₀ = 52.6, 35.0, 16.5, and 65.4 μM, respectively). Analogue 8c also effectively inhibited the migration and capillary-like tube formation of HUVECs in vitro. The antiangiogenic effect and antiproliferative activity of these structurally modified honokiol analogues and derivatives have led to the establishment of a structure-activity relationship.

Full text of DOI:10.1021/jm200830u

ARTICLE
pubs.acs.org/jmc
Structural Modification of Honokiol, a Biphenyl Occurring in Magnolia
officinalis: the Evaluation of Honokiol Analogues as Inhibitors of
Angiogenesis and for Their Cytotoxicity and StructureꢀActivity
Relationship
Liang Ma,† Jinying Chen,† Xuewei Wang,† Xiaolin Liang, Youfu Luo, Wei Zhu, Tianen Wang, Ming Peng,
Shucai Li, Shi Jie, Aihua Peng, Yuquan Wei, and Lijuan Chen*
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4,
Gaopeng Street, Chengdu 610041, China
S Supporting Information
b
ABSTRACT: Honokiol, widely known as an antitumor agent, has
been used as an antiangiogenesis drug lead. In this paper, 47
honokiol analogues and derivatives were investigated for their
antiangiogenic activity by application of the transgenic zebrafish
screening model, antiproliferative and cytotoxic activity against
HUVECs, and three tumor cell lines by MTT assay. 30,5-Diallyl-2,
40-dihydroxy-[1,10-biphen-yl]-3,50-dicarbaldehyde (8c) was found
to suppress the newly grown segmental vessels from the dorsal
aorta of zebrafish and prevent inappropriate vascularization as well
as exhibit more potent inhibitory effects on the proliferation of HUVECs, A549, HepG2, and LL/2 cells (IC50 = 15.1, 30.2, 10.7, and
21.7 μM, respectively) than honokiol (IC50 = 52.6, 35.0, 16.5, and 65.4 μM, respectively). Analogue 8c also effectively inhibited the
migration and capillary-like tube formation of HUVECs in vitro. The antiangiogenic effect and antiproliferative activity of these
structurally modified honokiol analogues and derivatives have led to the establishment of a structureꢀactivity relationship.
’ INTRODUCTION
leukemia integration-1 (fli-1:) enhanced GFP transgenics express
GFP in the entire ECs, facilitating imaging of the vascular anatomy.7
ECs play pivotal roles in a range of physiological processes such
as angiogenesis and as the selective blood barrier. They are also
involved in many pathophysiological events including arterial
disease and cancer development. One of the principal targets of
antiangiogenic therapy is genetically stable, nontransformed ECs
which are less prone to acquire drug resistance.8 When tumor cells
secrete pro-angiogenic growth factors that bind to receptors on
dormant ECs, leading to ECs activation, stimulation, vasodilata-
tion, and an up-regulation of vessel permeability, these activated
ECs could rapidly detach from the extracellular matrix and base-
ment membrane. They further migrate, proliferate to sprout and
self-assemble into new branches, followed by the formation of a
new basement membrane.9,10 Therefore, inhibition of the prolif-
eration, migration, and tubeformationof ECs might be aneffective
therapy for suppressing tumor progression and metastasis. Up to
now, more than 300 angiogenesis inhibitors have been developed.
There are 80 antiangiogenic drugs currently in clinical trials.
Notably many inhibitors of angiogenesis have been discovered in
natural resources, such as fungi, mushrooms, shark muscle and
cartilage, sea coral, green tea, ginseng, and garlic by screening of
ECs cultures.11
Angiogenesis is a critical step for tumor cell proliferation, inva-
sion, and metastasis in several solid tumors and hematological
malignancies.1 Tumor growth depends on angiogenesis, i.e., the
recruitment of new blood vessels from pre-existing vasculature.2
Without the development and progression of new blood vessels,
tumors cannot deteriorate beyond a critical size or metastasize
to other organs.3 Angiogenesis also is a complicated multistep
process involving endothelial cell (EC) activation, invasion, mi-
gration, proliferation, tube formation, and finally capillary network
formation.4
The zebrafish (Danio rerio) screening model for antiangiogen-
esis has first emerged as an important vertebrate model organism
in the 1970s and has since been increasingly applied to the study
of human diseases.5 The advantages of zebrafish as a valid tumor
model system for antiangiogenesis screening are well documen-
ted, including its low cost, easy maintenance, rapid generation
time (about three months), large clutch size (number of offspring),
the presence of transparent embryos, and ex utero embryonic
development.6 By the application of transgenesis, particular ECs
of zebrafish could be easily observed for gene expression or
cellular morphology before and after drug exposure. In the
transgenic zebrafish model, fetal liver kinase-1 (flk-1:) enhanced
green fluorescent proteins (GFP)-nuclear localization signal
(NLS) transgenics express GFP in the nucleus of ECs and friend
Received: January 7, 2011
Published: August 19, 2011
r
2011 American Chemical Society
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Figure 1. Optimization of building blocks for the synthesis of the biphenolic scaffold.
Scheme 1. Synthesis of Compounds 1, 2aꢀba
a Reagents and conditions: (a) py HBr3, glacial acetic acid; (b) Zn dust, glacial acetic acid; (c) Pd(OAc)2, PPh3, K2CO3(2 M), DMF, N2 atmosphere,
3
90 °C; (d) BBr3, ꢀ15 °C to room temperature.
Honokiol, a biphenolic neolignan with inappreciable toxicity
isolated from Magnolia officinalis, has been reported to possess
antiangiogenic and antitumor property in several tumor cell lines
and tumor xenograft models.12,13 However, only a few studies
have focused on the structural modification and structureꢀactivity
relationship (SAR) ofhonokiol analoguesand derivatives targeting
angiogenesis or cancer. Disconnections and chemical synthesis
have been undertaken to develop novel honokiol analogues and
derivatives to improve biological activity or clarify the SAR.14
Recent studies in our laboratory were devoted to the structurally
modified honokiol witha continuingeffort todevelopmorepotent
antitumor molecules.15,16
In this study, a series of honokiol analogues and derivatives were
designed (Figure 1), synthesized, and subsequently screened. Among
them, 8c effectively suppressed the formation of new blood vessels in
zebrafish-based assay and exhibited a medium inhibitory effect on
HUVECs (human umbilical vein endothelial cells; IC50 = 15.1 μM)
in contrast to honokiol (IC50 = 52.6 μM). Analogue 8c moderately
dust (Scheme 1). Suzuki coupling of 4-allyl-2-bromo-anisole with
appropriate arylboronic acids led to the corresponding 2-O-methyl
biphenyl derivatives (1aꢀb), which then was demethylated under
boron tribromide (BBr3) to yield 2-phenolic hydroxyl biphenyl
derivatives (2aꢀb).
In Scheme 2, the intermediate 3 was prepared through Suzuki
cross-coupling reaction. Two cross-coupling methods were devel-
oped for condensation of appropriate arylboronic acids with
1-(allyloxy)-4-bromobenzene under palladium catalyst. The O-
allylation of 3 with 3-bromoprop-1-ene or 1-bromo-3-methyl-
but-2-ene was performed in the presence of potassium carbonate
as base and acetone as solvent, and then derivatives (4aꢀl, and
5aꢀb) were synthesized through Claisen rearrangement in N,N-
diethylaniline as a valid solvent. Moreover, compounds 6aꢀf and
7aꢀb were obtainedby following similar synthetic methods
(Scheme 3).
Williamson alkylation of alkyl halide with phenolic hydroxyl
groups of honokiol was accomplished by employing potassium
carbonate as base, and DMF as solvent. ReimerꢀTiemann
reaction was performed for the introduction of aldehyde group
to the honokiol scaffold using chloroform and sodium hydroxide
at 50 °C. In theory, three formylated honokiol analogues should
be synthesized, and in fact, only two formylated analogues (i.e.,
3-formylated honokiol, 8a, and 3,50-diformylated honokiol, 8c)
were obtained because of low ortho-regioselectivity in the Reimerꢀ
Tiemann reaction. Because the chemical environment of two
phenolic hydroxyl groups of honokiol occurring was similar,
highly regioselective ReimerꢀTiemannreactionandchemoselective
Williamson alkylation were not facilely achieved (Scheme 4). There-
fore, the crude synthetic samples of analogues were separated by
blocked the proliferation of A549 (human lung carcinoma; IC50
30.2 μM), HepG2 (human hepatocellular liver carcinoma; IC50
=
=
10.7 μM), and LL/2 cells (Lewis lung carcinoma; IC50 = 21.7 μM)
superior to honokiol. Importantly, 8c exerted more potent inhibitory
potencies against the migration and tube formation of HUVECs than
those of honokiol. The results of in vivo antiangiogenic effect and in
vitro antiproliferative activity of honokiol analogues and derivatives
have led to the establishment of a SAR.
Chemistry. 4-Allyl-2-bromoanisole was synthesized from com-
mercially available 4-allylanisole according to the previously reported
method17 and employed 2.8 equiv of pyridinium hydrobromide
perbromide (py HBr3) followed by further debromination with zinc
3
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Scheme 2. Synthesis of Compounds 4aꢀl and 5aꢀba
a Reagents and conditions: (a) BrCH2CHdCH2 or BrCH2CHdC(CH3)2, K2CO3, acetone, reflux, 5 h; (b) method A: Pd(OAc)2, PPh3, K2CO3 (2 M),
isopropyl alcohol, N2 atmosphere, 90 °C, overnight; method B: Pd(PPh3)4, DMF, K3PO4 3H2O (2 M), N2 atmosphere, 100 °C, overnight; (c) N,N-
3
diethylaniline, reflux, overnight.
Scheme 3. Synthesis of Compounds 6aꢀf, and 7aꢀba
a Reagents and conditions: (a) CH3I or BrCH2CHdCH2, K2CO3, acetone, reflux, overnight; (b) N,N-diethylaniline, reflux, overnight.
reversed-phase high-performance liquid chromatography (prepara-
tive HPLC) and high-performance counter-current chromatogra-
phy (HPCCC).18 At this stage, the products were fully analyzed and
characterized by NMR, MS, and HPLC before being submitted to
the biological screening.
the exception of 8a, 8c, and honokiol. The remaining analogues
and derivatives (not listed) were inactive even at concentrations
exceeding 40 μM. However, honokiol and 8a were toxic or lethal
to zebrafish embryos at a concentration of 40 μM. The antiangio-
genic effects of 9aand10a weremoderate at20μM and not greatly
improved when the concentration reached 40 μM (Figure 2).
Importantly, analogue 8c inhibited intersomitic vessel sprouts
arising from the dorsal aorta and exhibited the higher antiangio-
genic activity in a concentration-dependent manner (Figure 3).
Inspection of their structural features and antiangiogenic
activities highlighted that all pharmacologically active analogues
and derivatives based on a biphenyl scaffold should possess the
2- and/or 40-OH group. Introduction of electron-withdrawing
groups (EWG; e.g., nitrated in 4l, 5b, and 7a; trifluoromethylated
in 4iꢀk; monofluorinated in 4g; and difluorinated in 4h) and
electron-donating groups (EDG; e.g., hydroxylated in 2b; mono-
methoxylated in 4bꢀc; dimethoxylated in 4eꢀf; methylthiolated
in 4d; and allylated in 7b) to the biphenyl moiety failed to im-
prove their antiangiogenic potencies in contrast to honokiol.
’ RESULTS AND DISCUSSION
Antiangiogenic Effects in the Transgenic Zebrafish Model.
Recently, zebrafish has been extensively used as an in vivo
drug screening model for investigating the formation of new
blood vessels during the stages of angiogenesis.19 Zebrafish embryos
are transparent and inhibitors or drugs dissolved in dimethylsulf-
oxide (DMSO) are readily permeable through the chorion. In the
present study, all honokiol analogues and derivatives were incubated
with the transgenic fli-1: enhanced GFP zebrafish embryos that
carried a 15-kb fli-1 promoter which could drive the expression of
GFP in the entire endothelium.7c
At a concentration of 10 μM, the antiangiogenic rates of the
majority of compounds listed in Table 1 were less than 25%, with
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Scheme 4. Synthesis of Various Analogues from Honokiol Scaffolda
a Reagents and conditions: (a) CH3I; C2H5I; CH3CH2Br; BrCH2COOC2H5; (CH3)2CHI; DMF, K2CO3, 30 °C, 6 h; (b) CHCl3, 35% NaOH (aq),
50 °C, 1 h; (c) methanol, NaBH(OAc)3, glacial acetic acid, ice bath to room temperature, overnight.
Table 1. Antiangiogenic Effects in Zebrafish Embryos
antiangiogenic effectsa
compds
5 μM
10 μM
20 μM
40 μM
honokiol
2a
+
O
O
O
+
++
+
+++
+
deadb
ndc
nd
+
2b
+
+
4i
+
+
4h
+
+
+
4k
O
O
O
O
+
+
+
+
5b
O
+
+
+
7a
+
+
7b
+
+
+
8a
++
++
+
++
+++
++
+
dead
++++
++
nd
+
8c
+
9a
+
9d
O
O
+
+
9g
+
+
9h
+
+
+
Figure 2. Effects on neovascularization in zebrafish embryos. The trans-
genic fli-1: enhanced GFP zebrafish embryos in embryo water (0.2 g/L of
instant ocean salt in distilled water) incubated with the indicated concen-
trations of compounds at 6 h postfertilization (hpf) for 24 h. Zebrafish
embryos were imaged and the number of angiogenic vessels in the trunk was
quantified. The left boxed area (red arrowhead) were magnified and shown
in the right section (magnification: left, 50ꢁ; right, 100ꢁ).
10a
10b
10c
+
+
++
+
++
+
+
+
O
+
+
+
a The semiquantitative scale for angiogenic inhibitory rates: O, inactive;
++++, > 75% angiogenic inhibition; +++, 50ꢀ75%; ++, 25ꢀ50%; +,
< 25% suppression of angiogenesis as compared to the vehicle treated
zebrafish embryos. b Zebrafish embryos treated with the indicated
concentration of compound were dead. c Not determined.
and 3,50-diformylated in 8c) seemed to be crucial for the anti-
angiogenic effects.
The alkylation of the phenolic hydroxyl group of 6, 9, and 10 also
lowered the inhibitory activity. With respect to these modified
analogues based on honokiol, we found that the presence of an
aldehyde group at the 3- or/and 50-position (3-formylated in 8a,
Antiproliferative Activity and SAR Study. An important
strategy for antiangiogenic therapy is effectively inhibiting the
proliferation of ECs. Thus, the IC50 value against HUVECs was
selected as the main index. Because few analogues and derivatives
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Table 2. IC50 Values of Derivative (1ꢀ2 and 4ꢀ7) against
HUVECs, A549, HepG2, and LL/2 Cell Lines
Figure 3. Effects of different concentrations of 8c on neovascularization
in zebrafish embryos. Analogue 8c concentration-dependently (10, 20,
and 40 μM) suppressed the intersomitic vessel sprouts and dorsal
longitudinal anastomotic vessels at 6 hpf for 48 h.
IC50 (μM)
compd
HUVECs
A549
HepG2
LL/2
were found to possess antiangiogenic potency, we further
evaluated their antiproliferative effects on HUVECs and cyto-
toxic activities against tumor cell lines (A549, HepG2, and LL/2)
by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay.
As shown in Table 2, 2-O-methyl derivatives (1aꢀb) exhibited
no inhibitory activity against the proliferation of HUVECs compared
to the corresponding 2-O-demethyl derivatives (2aꢀb). Although2b
bearing a 40-OH group manifested the higher potency against
HUVECs than 30-nitro derivative 2a, the antiproliferative
activities of 2a and 2b were both inferior to that of honokiol.
From these results, we could predict that the 2- and 40-OH
group of the biphenyl moiety might be essential for inhibiting
the proliferation of HUVECs. Likewise, the number of allyl
group potentially affected the cytotoxicity.
honokiol
1a
1b
2a
2b
4a
4b
4c
52.6
>100.0
>100.0
79.1
35.0 a
>100.0
95.4
16.5 b
>100.0
>100.0
62.3
65.4
89.4
71.1
>100.0
76.2
61.4
64.3
89.0
>100
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
78.2
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
80.0
>100.0
>100.0
>100.0
66.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
52.4
4d
4e
4f
>100.0
>100.0
>100.0
>100.0
59.7
4g
4h
4i
82.0
60.3
Subsequently, we investigated the antiproliferative and cytotoxic
activities of the 3-allylbiphenyl-4-ols (4aꢀl, and 5aꢀb) and 3-allyl-
4-alkyloxybiphenyls (6aꢀf). Additionally, 3,5-diallylbiphenyl-4-ols
(7aꢀb) were tested for a better understanding of the relationship
between the activity and the position and number of the allyl group.
In detail, 11 of these derivatives (4aꢀc, 4eꢀg, 4l, 6aꢀb, 6d, and 6e)
were completely inactive against the tested cell lines and all pharma-
cological profiles were summarized in Table 2. Trifluoromethyl deriva-
tives (20-CF3 in 4i, 30-CF3 in 4j, or 40-CF3 in 4k) exerted less
potent antiproliferative activities against HUVECs than hono-
kiol. Although O-alkylated derivatives (O-methyl in 6c and O-
allyl in 6f) showed weak cytotoxic activities against HepG2 and
LL/2 cells, they were unable to suppress the growth of HUVECs.
Inspection of their structural features and the activities indicated
that lack of the 2-OH and 4-allyl group of the biphenyl scaffold
approved to be unfavorable for the inhibition of HUVECs. Further-
more, 3,5-diallylbiphenyl-4-ols (7aꢀb) also did not achieve the
improvement in inhibitory potency in comparison to honokiol
which contained 30,5-diallyl groups. The results suggested that
the orientation of two allyl groups of the biphenyl scaffold was
responsible for the antiproliferative activity. In accordance with
in vivo antiangiogenic data from our synthetic derivatives, the
introduction of any EDGs or EWGs to the biphenyl scaffold was
dispensable for the activity.
The above analysis based on the position of phenolic hydroxyl
group and allyl group of the biphenyl scaffold was summarized.
Unexpectedly, all aforementioned derivatives showed less potent
antiproliferative activities against HUVECs than honokiol. The
retention of any reactive functional groups (phenolic hydroxyl
grouporallylgroup) of thebiphenyl scaffold has been proven to be
required for the inhibitory potency. Consequently, our synthetic
strategy was further devoted to keep the honokiol moiety intact.
As shown in Table 3, if the 2-phenolic hydroxyl group of
honokiol was alkylated, the antiproliferative effect on HUVECs
decreased with the increase of alkyl chain length (methyl in 9a;
ethyl in 9b; propyl in 9c; and isopropyl in 9d) compared to that
4j
76.0
73.0
58.3
65.9
4k
4l
74.0
76.0
60.3
74.8
>100.0
>100.0
77.0
>100.0
>100.0
64.0
>100.0
91.0
>100.0
>100.0
45.1
5a
5b
6a
6b
6c
56.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
80.0
>100.0
>100.0
89.0
>100.0
>100.0
78.8
6d
6e
6f
>100.0
>100.0
60.1
>100.0
>100.0
65.1
7a
7b
80.5
56.5
75.6
69.2
60.3
a Data was cited from ref 13. b Data was cited from ref 14e.
of honokiol, suggesting that the free 2-phenolic hydroxyl group
potentially affected the antiproliferative activity and that the alkyla-
tion of honokiol was detrimental to the activity. This consistent
tendency also occurred in 40-O-alkyl analogues (9eꢀf), and yet their
inhibitory potencies were less strong than those of analogues 9aꢀd,
stating that the contribution of 2-OH group was slightly superior to
the 40-OH of honokiol. When both the phenolic hydroxyl groups of
honokiol were alkylated, the corresponding analogues (9iꢀm) were
almost inactive against the proliferation of HUVECs, indicating that
the number of the bare phenolic hydroxyl group contributed to
antiproliferation.
To our surprise, the introduction of the aldehyde group at 3- and/
or 50-position of honokiol exhibited a remarkable enhancement of
antiproliferative effect on HUVECs and cytotoxicity against tumor
cells (Table 3). Analogue 8c (IC50 = 15.1 μM) achieved 3.5- and
3.0-fold improvements in the inhibition of HUVECs compared to
honokiol (IC50 = 52.6 μM) and 8a (IC50 = 46.2 μM). Meanwhile,
8c moderately suppressed the growth of A549, HepG2, and LL/2
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Table 3. IC50 Values of Honokiol Analogues (8ꢀ11) against
HUVECs, A549, HepG2, and LL/2
Figure 4. Structureꢀactivity relationships of honokiol analogues and
derivatives.
IC50 (μM)
change. If the position and number were modified, the corre-
sponding compounds become inactive (e.g., 7b vs. honokiol).
Honokiol is also a potent antiviral,14a,b neurotrophic,14c and
antioxidative agent.14d The antiproliferative SAR of honokiol analo-
gues and derivatives was similar to ones of the previous reports with
the difference occurring in the structural modification of the aldehyde
group at the 3- and/or 50-position (Figure 4).14 The introduction of
two aldehyde groups to honokiol achieved improvements in the
in vivo antiangiogenic potency and in vitro antiproliferative activity
against the four cell lines. The aforementioned results validated that
honokiol as a drug-like lead was extremely promising.
compd
HUVECs
A549
HepG2
LL/2
8aa
8c
46.2
15.1
43.6
30.2
ndb
32.9
10.7
65.3
21.7
9a
55.8
55.3
68.8
9b
9c
67.1
51.0
73.8
61.6
89.3
82.4
>129.9
48.4
64.8
9d
9e
70.5
42.7
49.1
61.8
nd
57.5
108.3
67.6
9f
68.2
81.6
71.5
Effects on the HUVECs Migration. The migration of ECs is
an important process of chemotaxis and an indispensable step to
generate new blood vessels. Inhibition on this process will block
the formation of new blood vessels. Therefore, wound-healing
migration assay was applied to assess the HUVECs migration.
With comparable or superior to honokiol in antiangiogenic and
antiproliferative activity, analogues 8a, 8c, 9a, and 10a were next
selected for biological evaluation. As illustrated in Figure 5, the
HUVECs actively migrated into the wound area (between the
two white lines) under the compound-free condition (control).
At a concentration of 20 μM, analogues 8a and 8c manifested
nearly equal potency to that of honokiol, and stronger inhibition
than those of 9a and 10a. While the tested compounds’
concentration reached 40 μM, the HUVECs migratory rates of
8a, 8c, 9a, 10a, and honokiol were 34.2, 21.2, 54.2, 63.2, and
38.5%, respectively. Analogue 8c statistically exerted the higher
potent inhibitory effect on the migration of HUVECs, reaching a
1.5-fold improvement over honokiol.
9g
73.3
53.8
49.9
45.8
9h
9i
65.5
53.2
63.5
48.4
>100.0
>100.0
>100.0
>100.0
>100.0
32.5
nd
>135.98
>124.2
>114.2
>114.2
68.9
>136.00
>124.2
>114.2
>114.2
>91.3
20.4
9j
>124.2
>114.2
>114.2
>91.3
28. 6
42.6
9k
9l
9m
10a
10b
10c
10d
11a
11b
20.9
39.1
53.9
40.8
37.9
39.0
28.5
31.9
86.9
>85.8
>135.0
>122.6
>85.2
>135.0
>122.6
26.1
>100.0
>135.0
>122.6
>100.0
a Data was cited from ref 16. b Not determined.
tumor cells, with the IC50 values of 30.2, 10.7, and 21.7 μM,
respectively. The O-alkylation of 8a led to four correspond-
ing analogues (10aꢀd), and their antiproliferative potencies
against HUVECs in comparison to those of 8c were not notably
improved. As a consequence, we could surmise that the phenolic
hydroxyl groups of honokiol play critical roles in the inhibitory
activity. Chemical reduction of the aldehyde group of 8a and 8c
rendered their counterparts (11a and 11b) inactive, highlighting
that the reactive aldehyde group was profitable to the activity.
Overall, a SAR of honokiol analogues and derivatives (Figure 4)
has been summarized as follows: (a) All the biologically active
honokiol analogues and derivatives possessed the 2- and/or 40-OH
of the biphenyl scaffold. As for the position of phenolic hydroxyl
groups of honokiol, the C-2 site was slightly superior to the C-40
site (e.g., 9a vs 9e). The O-alkylation inevitably reduced the
activity (e.g., 9i vs honokiol). (b) The introduction of the aldehyde
group at the 3- and/or 50-position of honokiol has been approved
to be critical for antiangiogenic and antiproliferative activity (e.g.,
8c vs. honokiol). A similar study has been reported that the
formylated compound exerted a favorable inhibitory effect on the
migration and invasion of HT1080 cells.20 (c) The introduction of
any EWGs or EDGs did not achieve major improvements in the
inhibitory potency (e.g., 4i vs. honokiol). (d) The position and
number of the allyl groups of honokiol should be forbidden to
Effects on the HUVECs Tube Formation. In the later stages
of angiogenesis, ECs will assemble into an interconnected tubular
network which is nearly identical to in vivo capillary vascular beds.
Inhibition on the formation of capillary-like tube networks will ter-
minate the development of new blood vessels. Thus, a tube for-
mationassaybyplatingHUVECsonMatrigel was performed. In the
control, cells showed the high mobility on Matrigel and formed an
intact tubular network in 12 h (Figure 6I). In comparison with the
control group, the inhibitory rates of tube formation treated with 8a,
8c, 9a, 10a, and honokiol at a concentration of 20 μM were 41.2,
21.3, 34.9, 82.5, and 51.4%, respectively (Figure 6II). Our observa-
tion indicated that 8c approximately achieved a 2.5-fold improve-
ment in the inhibition of tube formation compared to that of
honokiol at the same concentration and the intact tubular structures
disrupted by 20 μM 8c were sparse and incomplete.
’ CONCLUSION
The strategy of antiangiogenic therapy provides an alternative
that uses the evolving vessels, which nourishes the growth and
metastasis of tumor, as the attractive and prime target.21 The ECs
activation, proliferation, invasion, migration, and tube formation
are the fundamental steps for angiogenesis. However, acquired
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Figure 5. Effects on the HUVECs migration. (I) HUVECs suspended in serum-free Dulbecco’s Modified Eagle Medium (DMEM) containing
compounds (20 or 40 μM) for 24 h were photographed under a phase contrast microscopy (magnification: 50ꢁ). Control was treated with serum-free
DMEM. (II) Inhibitory rates of compounds on the HUVECs migration. Data represented the mean ( standard error (SE) from three independent
experiments. *P < 0.05; **P < 0.01.
Figure 6. Effects on the HUVECs tube formation. (I) HUVECs (1 ꢁ 104 cells) suspended in DMEM containing each compound (20 μM) were
added to the Matrigel. Control was treated with DMEM alone. After incubation for 12 h at 37 °C, capillary networks were photographed
and quantified (magnification: 100ꢁ). (II) Inhibitory rates of compounds on the HUVECs tube formation. The number of intact tubes was
counted in five randomly chosen regions and expressed as the percentage of the control. The results were expressed as mean ( SE *P < 0.05;
**P < 0.01; ***P < 0.005.
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drug resistance remains a major obstacle of tumor targeting
therapy.22 In comparison with genetically unstable tumor cells,
the ECs recruited by new tumor vasculatures are genetically more
stable and less susceptible to acquired drug resistance.23 There-
fore, blocking angiogenesis, specifically targeting ECs, has been
proven to be a feasible strategy for preventing angiogenesis-
dependent tumor growth and metastasis.
angiogenesis. Zebrafish embryos were generated by natural pairwise mating
and raised at 28.5 °C in embryo water (0.2 g/L of Instant Ocean Salt in
distilled water). At about 6 hpf, the embryos were sorted in the 6-well plate
(6 embryos/well), removing dead and unhealthy embryos. Then the
embryos were treated with the indicated concentrations of compounds
which were added into embryo water. After incubation for 24 or 48 h, the
embryos were anesthetized using 0.05% 2-phenoxyethanol in embryo water,
photographed, and quantified.
Herein, 47 honokiol analogues and derivatives represented an
allylated biphenolic scaffold and possessed a unique mode of
action in antiangiogenic and antitumor activity. In detail, 8c
moderately blocked the newly grown segmental vessels from the
dorsal aorta in the transgenic zebrafish-based assay, exhibited the
strongest inhibitory effects on the proliferation, migration, and
tube formation of HUVECs featuring antiangiogenic property in
this study, and also exerted its medium cytotoxicity against the
selected tumor cell lines (A549, HepG2, and LL/2). Addition-
ally, the further mechanism of antiangiogenesis (e.g., targeting
vascular endothelial growth factor (VEGF) or fibroblast growth
factor (FGF)), in vivo antitumor evaluation, and structural
modification of 8c are in progress.
Chemistry. NMR spectra were recorded at 400 MHz on a Varian
spectrometer (Varian, Palo Alto, CA, USA) model Gemini 400 and
reported in parts per million. Chemical shifts (δ) are quoted in ppm relative
to tetramethylsilane (TMS) as an internal standard, where (δ) TMS = 0.00
ppm. The multiplicity of the signal is indicated as s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet, defined as all multipeak signals where overlap
or complex coupling of signals makes definitive descriptions of peaks difficult.
MS spectra were measured by quadrupole-time-of-flight (Q-TOF) Premier
mass spectrometer utilizing electrospray ionization (ESI) (Micromass,
Manchester, UK). Room temperature is within 20ꢀ25 °C. The purity of
compound was determined to be g97% by HPLC analysis (Supporting
Information). The synthetic methods of 1aꢀb and 2aꢀb were referred from
the reported literature.17 The structural elucidations of 8a, 8c, 10a, and 10b
have been reported in our previous literatures.18
5-Allyl-2-methoxy-30-nitro-1,10-biphenyl (1a). Yield 87.0%. 1H NMR
(400 MHz, CDCl3): δ 8.58 (t, 1H, J = 2.0 Hz, 20-H), 8.22ꢀ8.18 (m, 1H,
40-H), 7.92ꢀ7.78 (m, 2H, 50- and 60-H), 7.68ꢀ7.53 (m, 2H, 4- and 6-H),
6.93ꢀ6.91 (m, 1H, 3-H), 6.19ꢀ6.11 (m, 1H, ꢀCHdCH2), 5.34ꢀ5.30
(m, 2H, ꢀCHdCH2), 3.83 (s, 3H, ꢀOCH3), 3.49 (d, 2H, J = 5.4 Hz,
ꢀCH2CH). HRMS [M + H]+ calcd 270.1130; found 270.1122.
5-Allyl-2,40-dimethoxy-1,10-biphenyl (1b). Yield 65.7%. 1H NMR
(400 MHz, CDCl3): δ 7.69ꢀ7.61 (m, 2H, 20- and 60-H), 7.23ꢀ7.18 (m,
2H, 30- and 40-H), 7.07ꢀ7.01 (m, 1H, 6-H), 6.98ꢀ6.96 (m, 2H, 3- and
4-H), 6.12ꢀ6.05 (m, 1H, ꢀCHdCH2), 5.31ꢀ5.29 (m, 2H, ꢀCHdCH2),
3.89 (s, 3H, 40-OCH3), 3.85 (s, 3H, 2-OCH3), 3.49 (d, 2H, J = 5.2 Hz,
ꢀCH2CH). HRMS [M + H]+ calcd 255.1385; found 255.1401.
’ EXPERIMENTAL SECTION
Cell Culture. HUVECs were isolated from human umbilical cord
with collagens. After dissociation, the cells were collected and cultured
on gelatin-coated culture flasks in M-199 medium with 20% fetal bovine
serum (FBS), 10 ng/mL basic FGF, 2 ng/mL VEGF, 100 IU/mL
penicillin, and 100 μg/mL streptomycin. Subcultures were performed
with trypsinꢀethylene diamine tetraacetic acid (EDTA). Media were
refreshed every two days. HUVECs were confirmed by their cobblestone
morphology and strong positive immunoreactivity to von Willebrand
factor. All cells were incubated in an atmosphere containing 5% CO2
at 37 °C.
MTT Assay. MTT assay was performed to evaluate the cytotoxic and
antiproliferative acclivities of all compounds. Cells were treated with
various concentrations of compound in 96-well culture plates for 24 h in
final volumes of 200 μL (5 ꢁ 103 cells/well). Then 20 μL of MTT
solution (5 mg/mL) was added to each well, and cells were incubated for
an additional 3 h. Then the medium was carefully removed, and
precipitates were dissolved in 150 μL of DMSO, shaken mechanically
for 30 min, and then absorbance values at a wavelength of 570 nm were
taken on a spectrophotometer (Molecular Devices, Sunnyvale, USA).
IC50 values were calculated using percentage of growth versus untreated
control.
5-Allyl-30-nitro-[1,10-biphenyl]-2-ol (2a). Yield 71.3%. 1H NMR
(400 MHz, CDCl3): δ 8.60 (s, 1H, 20-H), 8.20ꢀ8.18 (m, 1H, 40-H),
7.91ꢀ7.89 (m, 1H, 60-H), 7.65ꢀ7.57 (m, 2H, 50- and 6-H), 6.96ꢀ6.91
(m, 2H, 3- and 4-H), 6.22ꢀ6.18 (m, 1H, ꢀCHdCH2), 5.58 (s, 1H,
ꢀOH), 5.32ꢀ5.28 (m, 2H, ꢀCHdCH2), 3.49 (d, 2H, J = 5.4 Hz,
ꢀCH2CH). HRMS [M ꢀ H]ꢀ calcd 254.0817; found 254.0814.
1
5-Allyl-[1,10-biphenyl]-2,40-diol (2b). Yield 54.9%. H NMR (400
MHz, CDCl3): δ 7.62ꢀ7.58 (m, 3H, Ar-H), 7.33ꢀ7.30 (m, 1H, Ar-H),
6.96ꢀ6.94 (m, 3H, Ar-H), 6.10ꢀ6.03 (m, 1H, ꢀCHdCH2), 5.31ꢀ5.29
(m, 2H, ꢀCHdCH2), 3.48 (d, 2H, J = 5.4 Hz, ꢀCH2CH). HRMS
[M ꢀ H]ꢀ calcd 225.0916; found 225.0899.
Wound-Healing Assay. HUVECs were seeded in 6-well plates
precoated with 0.1% gelatin and grown overnight to confluence. The
monolayer cells were wounded by scratching with 10 μL pipet tips and
washed twice with serum-free DMEM to remove the nonadherent cells
and then replaced by serum-free DMEM with the indicated concentra-
tions of compounds for 24 h. Images were taken at 0 h and 24 h inde-
pendently after incubation at 37 °C, 5% CO2. The migrated HUVECs
were manually counted. The values were observed from three randomly
selected fields. Similar patterns of the inhibition were observed in three
independent experiments.
Tube Formation Assay. Matrigel was dissolved at 4 °C overnight.
Each well of prechilled 96-well plates was coated with 50 μL of Matrigel,
incubated and solidified at 37 °C for 45 min. After removing the unsolid
fluid, HUVECs at the density of 1 ꢁ 104 were cultured in DMEM
containing the indicated concentrations of compounds for 24 h. Con-
trols are treated with the DMEM alone. Images were digitally captured
and quantitatively analyzed (Olympus).
Typical Procedure for the Synthesis of 4aꢀl, and 5aꢀb. Step I:
Synthesis of 1-Allyloxy-4-bromobenzene. To a mixture of 4-bromophenol
(3.5 g, 20.0 mmol) and anhydrous potassium carbonate (3.6 g, 26.0 mmol,
1.3 equiv) in acetone (25 mL), allyl bromide (1.9 mL, 22.0 mmol, 1.1 equiv)
was added dropwise. The mixture was heated at 60 °C for 5 h. After
cooling, the mixture was filtered and the solution was evaporated to
dryness. Then the filtrate was extracted with diethyl ether (20 mL ꢁ 3)
and 10% NaOH (20 mL ꢁ 1) and the organic layer was washed by brine
(20 mL ꢁ 2), dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure to afford a colorless oil product (4.1 g, 19.0 mmol,
95.0%).
Step II: Synthesis of Intermediates 3 by Suzuki-Coupling Reaction.
Method A (for 4aꢀf). 1-Allyloxy-4-bromobenzene (1.0 mmol) and
arylboronic acid (1.2 mmol) were dissolved in isopropyl alcohol (4 mL)
at room temperature. After becoming a clear solution, Pd(OAc)2 (0.01
mmol), PPh3 (0.03 mmol), and a solution of anhydrous potassium
carbonate (2.0 mmol) in water (1 mL) were added at N2 atmosphere,
and the resulting mixture was stirred for further 18 h at 90 °C. The
mixture was filtered and extracted with ethyl acetate (3 ꢁ 10 mL).
Zebrafish Embryos Assay. Using transgenic fli-1: enhanced GFP
zebrafish embryos, we examined the effects of all compounds on embryonic
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The extract was washed by brine (2 ꢁ 10 mL), dried over anhydrous sodium
sulfate, and concentrated. The residue was purified by gel chromato-
graphy (ethyl acetate:petroleum ether = 1:10) to give our target
compound with a satisfactory yield.
Method B (for 4gꢀl). 1-Allyloxy-4-bromobenzene (1.0 mmol) and
arylboronic acid (1.2 mmol) were dissolved in DMF (4 mL) at room
temperature. After becomig a clear solution, Pd(PPh3)4 (0.01 mmol)
(d, 1H, J = 8.0 Hz, 5-H), 6.56ꢀ6.53 (m, 2H, 30- and 50-H), 6.08ꢀ6.00
(m, 1H, ꢀCHdCH2), 5.23ꢀ5.14 (m, 2H, ꢀCHdCH2), 5.11 (s, 1H,
ꢀOH), 3.84 (s, 3H, 4-OCH3), 3.78 (s, 3H, 3-OCH3), 3.44 (d, 2H, J =
6.4 Hz, ꢀCH2CH). HRMS [M + Na]+ calcd 293.1154; found 293.1150.
1
3-Allyl-40-fluoro-[1,10-biphenyl]-4-ol (4g). Yield 23.9%. H NMR
(400 MHz, CDCl3): δ 7.50ꢀ7.46 (m, 2H, 2- and 6-H), 7.32ꢀ7.30 (m,
2H, 20- and 60-H), 7.11ꢀ7.07 (m, 2H, 30- and 50-H), 6.88 (d, 1H, J =
8.4 Hz, 5-H), 6.11ꢀ6.00 (m, 1H, ꢀCHdCH2), 5.23ꢀ5.18 (m, 2H,
ꢀCHdCH2), 5.05 (s, 1H, ꢀOH), 3.47 (d, 2H, J = 4.8 Hz, ꢀCH2CH).
HRMS [M + H]+ calcd 229.1029; found 229.0982.
and a solution of K3PO4 3H2O (2.0 mmol) in water (1 mL) were added
3
at N2 atmosphere, and the mixture was stirred for further 18 h at 100 °C
(TLC monitoring). The mixture was filtered and extracted with ethyl
acetate (3 ꢁ 10 mL). The extract was washed by brine (2 ꢁ 10 mL),
dried over anhydrous sodium sulfate, and concentrated. The residue was
further purified by gel chromatography (ethyl acetate:petroleum ether =
1:10) to give our target compound.
3-Allyl-30,50-difluoro-[1,10-biphenyl]-4-ol (4h). Yield 34.8%. 1H NMR
(400 MHz, CDCl3): δ 7.35ꢀ7.31 (m, 2H, 2- and 6-H), 7.08ꢀ7.03 (m, 2H,
20- and 60-H), 6.89 (d, 1H, J = 8.0 Hz, 5-H), 6.76ꢀ6.69 (m, 1H, 40-H),
6.10ꢀ5.97 (m, 1H, ꢀCHdCH2), 5.24ꢀ5.18 (m, 2H, ꢀCHdCH2), 5.07
(s, 1H,ꢀOH), 3.48 (d, 2H, J = 6.0 Hz, ꢀCH2CH). HRMS [M + H]+ calcd
269.0754; found 269.0752.
Step III: Synthesis of 4aꢀl and 5aꢀb. Allyl bromide or 1-bromo-
3-methylbut-2-ene (1.3 mmol) was added to the solution of 3 (1.0
mmol) and anhydrous potassium carbonate (2.0 mmol) and refluxed for
5 h (TLC monitoring). After cooling, the mixture was filtered and the
solution was evaporated to dryness. Then the crude was extracted by
diethyl ether (2 ꢁ 10 mL), and the organic layer was washed by brine
(2 ꢁ 10 mL), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to afford the product. And then the product was
dissolved in N,N-diethylaniline and refluxed overnight, monitored by
thin-layer chromatography (TLC). After reaction was completed, the
solution was adjusted to pH = 4 by 3N HCl and extracted with ethyl
acetate (2 ꢁ 10 mL) and the organic layer was washed by water and
brine, dried over anhydrous magnesium sulfate, and concentrated under
reduced pressure to afford the crude product. The residue was further
purified by silica gel column chromatography (ethyl acetate:petroleum
ether = 1:10) to give our target compounds. The yield was depicted as
the total yield of four steps.
3-Allyl-20-(trifluoromethyl)-[1,10-biphenyl]-4-ol (4i). Yield 43.0%.
1H NMR (400 MHz, CDCl3): δ 7.72 (d, 1H, J = 7.2 Hz, 60-H), 7.53 (t,
1H, J = 7.2 Hz, 30-H), 7.43 (t, 1H, J = 7.2 Hz, 50-H), 7.32 (d, 1H, J =
7.2 Hz, 40-H), 7.10ꢀ7.08 (m, 2H, 2- and 6-H), 6.84 (d, 1H, J = 8.0 Hz,
5-H), 6.09ꢀ5.99 (m, 1H, ꢀCHdCH2), 5.19ꢀ5.15 (m, 2H, ꢀCHdCH2),
3.44 (d, 2H, J = 6.0 Hz, ꢀCH2CH). HRMS [M ꢀ H]ꢀ calcd 277.0840;
found 277.0840.
3-Allyl-30-(trifluoromethyl)-[1,10-biphenyl]-4-ol (4j). Yield 59.8%.
1H NMR (400 MHz, CDCl3): δ 7.77 (s, 1H, 20-H), 7.72ꢀ7.70 (m, 1H,
60-H), 7.56ꢀ7.49 (m, 2H, 2- and 6-H), 7.39ꢀ7.35 (m, 2H, 30- and 40-H),
6.91 (d, 1H, J = 8.0 Hz, 5-H), 6.11ꢀ6.01 (m, 1H, ꢀCHdCH2),
5.24ꢀ5.19 (m, 2H, ꢀCHdCH2), 3.49 (d, 2H, J = 6.4 Hz, ꢀCH2CH).
HRMS [M + H]+ calcd 301.0816; found 301.0820.
3-Allyl-40-(trifluoromethyl)-[1,10-biphenyl]-4-ol (4k). Yield 51.8%.
1H NMR (400 MHz, CDCl3): δ 7.67ꢀ7.62 (m, 4H, Ar0-H), 7.40ꢀ7.36
(m, 2H, 2- and 6-H), 6.92 (d, 1H, J = 8.0 Hz, 5-H), 6.10ꢀ6.01 (m, 1H,
ꢀCHdCH2), 5.24ꢀ5.19 (m, 2H, ꢀCHdCH2), 3.49 (d, 2H, J = 6.0 Hz,
ꢀCH2CH). HRMS [M + H]+ calcd 279.0997; found 279.0897.
3-Allyl-30-nitro-[1,10-biphenyl]-4-ol (4l). Yield 81.0%. 1H NMR
(400 MHz, CDCl3): δ 8.41 (t, 1H, J = 2.0 Hz, 20-H), 8.16ꢀ8.14 (m,
1H, 40-H), 7.88ꢀ7.86 (m, 1H, 60-H), 7.57 (t, 2H, J = 8.4 Hz, 50-H),
7.43ꢀ7.39 (m, 2H, 2- and 6-H), 6.94 (d, 1H, J = 8.0 Hz, 5-H), 6.12ꢀ6.02
(m, 1H, ꢀCHdCH2), 5.25ꢀ5.20 (m, 2H, ꢀCHdCH2), 3.51 (d, 2H,
J = 5.4 Hz, ꢀCH2CH). HRMS [M + H]+ calcd 278.0793; found 278.0785.
40-Methoxy-3-(2-methylbut-3-en-2-yl)-[1,10-biphenyl]-4-ol (5a).
3-Allyl-[1,10-biphenyl]-4-ol (4a). Yield 32.8%. 1H NMR (400 MHz,
CDCl3): δ 7.55ꢀ7.53 (m, 2H, 2- and 6-H), 7.43ꢀ7.36 (m, 4H, Ar0-H),
7.32ꢀ7.28 (m, 1H, 40-H), 6.89 (d, 1H, J = 8.4 Hz, 5-H), 6.11ꢀ6.01 (m,
1H, ꢀCHdCH2), 5.24ꢀ5.18 (m, 2H, ꢀCHdCH2), 4.98 (br s, 1H,
ꢀOH), 3.48 (d, 2H, J = 5.4 Hz, ꢀCH2CH). HRMS [M + Na]+ calcd
233.0942; found 233.0939.
3-Allyl-40-methoxy-[1,10-biphenyl]-4-ol (4b). Yield 58.9%. 1H
NMR (400 MHz, CDCl3): δ 7.48ꢀ7.46 (m, 2H, 20- and 60-H),
7.33ꢀ7.30 (m, 2H, 2- and 6-H), 6.96ꢀ6.94 (m, 2H, 30- and 40-H),
6.86 (d, 1H, J = 8.4 Hz, 5-H), 6.11ꢀ6.01 (m, 1H, ꢀCHdCH2),
5.23ꢀ5.17 (m, 2H, ꢀCHdCH2), 3.84 (s, 3H, 4-OCH3), 3.47 (d, 2H,
J = 5.4 Hz, ꢀCH2CH). RMS [M + Na]+ calcd 263.1048; found
263.1056.
1
Yield 22.0%. H NMR (400 MHz, CDCl3): δ 7.48 (m, 2H, J = 8.4
Hz, 20- and 60-H), 7.32ꢀ7.28 (m, 2H, 2- and 6-H), 6.96 (d, 2H, J = 8.8
Hz, 30- and 50-H), 6.85 (d, 1H, J = 8.8 Hz, 5-H), 5.50 (s, 1H, ꢀOH), 5.12
(s,1H, ꢀCHdCH2), 5.06 (s, 1H, ꢀCHdCH2), 3.84 (s, 3H, 4-OCH3),
3.62 (d, 1H, J = 7.2 Hz, ꢀCH2CH), 1.70 (s, 3H, ꢀCH3), 1.17 (d, 3H, J =
6.8 Hz, ꢀCH3). HRMS [M + H]+ calcd 269.1542; found 269.1539.
3-(2-Methylbut-3-en-2-yl)-30-nitro-[1,10-biphenyl]-4-ol (5b). Yield
58.0%. 1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H, 20-H), 8.15ꢀ8.13
(m, 1H, 40-H), 7.87 (d, 1H, J = 7.6 Hz, 60-H), 7.57 (t, 1H, J = 8.0 Hz,
50-H), 7.41ꢀ7.38 (m, 2H, 2- and 6-H), 6.94 (d, 1H, J = 8.0 Hz, 5-H),
5.13 (s, 1H, ꢀCHdCH2), 5.09 (s, 1H, ꢀCHdCH2), 3.71 (q, 1H, J =
7.2 Hz, ꢀCH2CH), 1.73 (s, 3H, ꢀCH3), 1.49 (d, 3H, J = 6.8 Hz,
ꢀCH3). HRMS [M + H]+ calcd 284.1287; found 284.1289.
3-Allyl-30-methoxy-[1,10-biphenyl]-4-ol (4c). Yield 51.9%. 1H NMR
(400 MHz, CDCl3): δ 7.39ꢀ7.32 (m, 3H, Ar-H), 7.15 (d, 1H, J = 7.6
Hz, 6-H), 7.09 (s, 1H, 20-H), 6.90ꢀ6.85 (m, 2H, 40- and 5-H),
6.08ꢀ6.04 (m, 1H, ꢀCHdCH2), 5.24ꢀ5.18 (m, 2H, ꢀCHdCH2),
3.87 (s, 3H, 3-OCH3), 3.49 (d, 2H, J = 6.4 Hz, ꢀCH2CH). HRMS
[M + H]+ calcd 241.1229; found 241.1225.
1
3-Allyl-30-(methylthio)-[1,10-biphenyl]-4-ol (4d). Yield 76.9%. H
NMR (400 MHz, CDCl3): δ 7.42 (s, 1H, 2-H), 7.37ꢀ7.29 (m, 4H,
Ar-H), 7.21ꢀ7.18 (m, 1H, 20-H), 6.89 (d, 1H, J = 8.4 Hz, 5-H),
6.11ꢀ6.01 (m, 1H, ꢀCHdCH2), 5.24ꢀ5.18 (m, 2H, ꢀCHdCH2),
3.48 (d, 2H, J=6.4Hz,ꢀCH2CH), 2.53 (s, 3H, 3-SCH3).HRMS[M+H]+
calcd 257.1000; found 257.1002.
Synthesis of 6aꢀf: Following Step I of Typical Procedure
for the Synthesis of 4aꢀl. 3-Allyl-4,40-dimethoxy-1,10-biphenyl
(6a). Yield 89.5%. 1H NMR (400 MHz, CDCl3): δ 7.48 (d, 2H, J = 8.4
Hz, 20- and 60-H), 7.38ꢀ7.34 (m, 2H, 2- and 6-H), 6.95 (d, 2H, J = 8.4
Hz, 30- and 50-H), 6.91 (d, 1H, J = 8.4 Hz, 5-H), 6.07ꢀ6.00 (m, 1H,
ꢀCHdCH2), 5.12ꢀ5.05 (m, 2H, ꢀCHdCH2), 3.86 (s, 3H, 4-OCH3),
3.84 (s, 3H, 40-OCH3), 3.44 (d, 2H, J = 6.4 Hz, ꢀCH2CH). HRMS [M +
Na]+ calcd 277.1204; found 277.1191.
1
3-Allyl-30,40-dimethoxy-[1,10-biphenyl]-4-ol (4e). Yield 65.9%. H
NMR (400 MHz, CDCl3): δ 7.34ꢀ7.30 (m, 2H, 2- and 6-H), 7.09ꢀ7.05
(m, 2H, 20- and 60-H), 6.93ꢀ6.88 (m, 2H, 5- and 50-H), 6.12ꢀ6.02 (m,
1H, ꢀCHdCH2), 5.23ꢀ5.17 (m, 2H, ꢀCHdCH2), 3.95 (s, 3H,
4-OCH3), 3.91 (s, 3H, 3-OCH3), 3.48 (d, 2H, J = 5.4 Hz, ꢀCH2CH).
HRMS [M + H]+ calcd 271.1334; found 271.1343.
3-Allyl-4-(allyloxy)-40-methoxy-1,10-biphenyl (6b). Yield 95.9%. 1H
NMR (400 MHz, CDCl3): δ 7.47 (d, 2H, J = 8.4 Hz, 20- and 60-H),
1
3-Allyl-20,40-dimethoxy-[1,10-biphenyl]-4-ol (4f). Yield 55.9%. H
NMR (400 MHz, CDCl3): δ 7.28ꢀ7.19 (m, 3H, 2-, 6-, and 60-H), 6.82
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7.03ꢀ7.40 (m, 2H, 2- and 6-H), 6.95 (d, 2H, J = 8.4 Hz, 30- and 50-H),
6.89 (d, 1H, J = 9.2 Hz, 5-H), 6.12ꢀ5.09 (m, 2H, ꢀOCH2CHdCH2
and ꢀCHdCH2), 5.47ꢀ5.27 (m, 2H, ꢀOCH2CHdCH2), 5.13ꢀ5.05
(m, 2H, ꢀCHdCH2), 4.58 (d, 2H, J = 5.2 Hz, ꢀOCH2CHdCH2),
3.84 (s, 3H, 4-OCH3), 3.47 (d, 2H, J = 6.8 Hz, ꢀCH2CH). HRMS
[M + H]+ calcd 281.1542; found 281.1524.
The mixture was distilled in oil bath under reduced pressure, and the
residue was takeninto water and extractedwith ethyl acetate (3 ꢁ 10 mL).
The organic layer was dried by anhydrous sodium sulfate. The solvent was
evaporated to get crude product, which was further purified by column
chromatography using petroleum ether/ethyl acetate (30/1) as an eluent.
1
30,5-Diallyl-40-methoxy-[1,10-biphenyl]-2-ol (9a). Yield 40.0%. H
3-Allyl-4-methoxy-40-(trifluoromethyl)-1,10-biphenyl (6c). Yield 91.8%.
1H NMR (400 MHz, CDCl3): δ 7.56ꢀ7.45 (m, 4H, Ar0-H), 7.33ꢀ7.28
(m, 2H, 2- and 6-H), 6.82 (d, 1H, J = 8.0 Hz, 5-H), 6.00ꢀ5.95 (m, 1H,
ꢀCHdCH2), 5.20ꢀ5.10 (m, 2H, ꢀCHdCH2), 3.89 (s, 3H, 40-OCH3),
3.42 (d, 2H, J = 6.0 Hz, ꢀCH2CH). HRMS [M + H]+ calcd 293.1153;
found 293.1158.
NMR (400 MHz, CDCl3): δ 7.22ꢀ7.29 (m, 2H, Ar-H), 7.06 (d, 1H, J =
2.0 Hz, 60-H), 7.03 (d, 1H, J = 2.0 Hz, 4-H), 6.95 (d, 1H, J = 8.0 Hz, 3-H),
6.90 (d, 1H, J = 8.0 Hz, 50-H), 5.92ꢀ6.04 (m, 2H, 5-CHdCH2 and
30- CHdCH2), 5.04ꢀ5.15 (m, 4H, 5-CHdCH2 and 30-CHdCH2),
3.88 (s, 3H, 40-OCH3), 3.43 (d, 2H, J = 6.4 Hz, 5-CH2CH), 3.34 (d, 2H,
J = 6.8 Hz, 30-CH2CH). 13C NMR (400 MHz, CDCl3): δ 157.2, 150.8,
137.8, 136.7, 132.3, 130.7, 130.5, 130.2, 129.3, 129.0, 128.7, 127.9, 116.1,
115.8, 115.7, 110.9, 55.8, 39.6, 34.4. HRMS [M ꢀ H]ꢀ calcd 279.1385;
found 279.1379.
3-Allyl-4-methoxy-30-nitro-1,10-biphenyl (6d). Yield 87.0%. 1H
NMR (400 MHz, CDCl3): δ 8.40 (s, 1H, 20-H), 8.13 (d, 1H, J = 8.0
Hz, 40-H), 7.87 (d, 1H, J = 8.0 Hz, 60-H), 7.56 (t, 1H, J = 8.0 Hz, 50-H),
7.47 (d, 1H, J = 8.0 Hz, 6-H), 7.41 (d, 1H, J = 2.4 Hz, 2-H), 6.96 (d, 1H,
J = 8.4 Hz, 5-H), 6.08ꢀ5.98 (m, 1H, ꢀCHdCH2), 5.13ꢀ5.08 (m, 2H,
ꢀCHdCH2), 3.89 (s, 3H, 40-OCH3), 3.46 (d, 2H, J = 6.8 Hz,
ꢀCH2CH). HRMS [M ꢀ H]ꢀ calcd 268.0974; found 268.0965.
30,5-Diallyl-40-ethoxybiphenyl-2-ol (9b). Yield 40.2%. 1H NMR (400
MHz, CDCl3): δ 7.28 (d, 1H, J = 2.4 Hz, 6-H), 7.25 (d, 1H, J = 8.4 Hz,
20-H), 7.07 (d, 1H, J = 2.0 Hz, 60-H), 7.04 (s, 1H, 4-H), 6.95 (d, 1H, J =
8.0 Hz, 3-H), 6.92 (d, 1H, J = 8.0 Hz, 50-H), 5.96ꢀ6.06 (m, 2H,
4-CHdCH2 and 40-CHdCH2), 5.05ꢀ5.18 (m, 4H, 5-CHdCH2 and
30-CHdCH2), 4.10 (q, 2H, J = 20.8 Hz, ꢀCH2CH3), 3.46 (d, 2H, J = 6.8
Hz, 5-CH2CH), 3.37 (d, 2H, J = 6.8 Hz, 30-CH2CH), 1.47 (t, 3H, J =
14.0 Hz, ꢀCH2CH3). 13C NMR (400 MHz, CDCl3): δ 156.5, 151.0,
138.0, 136.8, 132.2, 130.6, 130.3, 129.9, 129.1, 128.8, 128.1, 128.0, 115.9,
115.7, 115.6, 111.9, 63.8, 39.5, 34.6, 15.0. HRMS [M ꢀ H]ꢀ calcd
293.1542; found 293.1439.
1
3-Allyl-4-(allyloxy)-30-nitro-1,10-biphenyl (6e). Yield 97.8%. H NMR
(400 MHz, CDCl3): δ 8.41 (s, 1H, 20-H), 8.15 (d, 1H, J = 8.4 Hz, 40-
H), 7.88 (d, 1H, J = 8.0 Hz, 60-H), 7.58 (t, 1H, J = 8.4 Hz, 50-H),
7.42ꢀ7.44 (m, 2H, 2- and 6-H), 6.96 (d, 1H, J = 8.4 Hz, 5-H), 6.13ꢀ
6.02 (m, 2H, ꢀOCH2CHdCH2 and ꢀCHdCH2), 5.49ꢀ5.44 (m,
1H, ꢀOCH2CHdCH2), 5.34ꢀ5.31 (m, 1H, ꢀOCH2CHdCH2),
5.16ꢀ5.09 (m, 2H, ꢀCHdCH2), 4.63 (d, 2H, J = 4.8 Hz,
ꢀOCH2CHdCH2), 3.51 (d, 2H, J = 6.4 Hz, ꢀCH2CH). HRMS
[M ꢀ H]ꢀ calcd 294.1130; found 294.1121.
30,5-Diallyl-40-propoxybiphenyl-2-ol (9c). Yield 21.0%. 1H NMR
(400 MHz, CDCl3): δ 7.26 (d, 1H, J = 2.0 Hz, 6-H), 7.23 (d, 1H, J =
2.0 Hz, 20-H), 7.05 (d, 1H, J = 2.0 Hz, 60-H), 7.02 (d, 1H, J = 2.4 Hz,
4-H), 6.92 (d, 1H, J = 2.0 Hz, 3-H), 6.88 (q, 1H, J = 14 Hz, 50-H),
5.92ꢀ6.03 (m, 2H, 5-CHdCH2 and 30-CHdCH2), 5.03ꢀ5.12 (m, 4H,
5-CHdCH2 and 30-CHdCH2), 3.97 (t, 2H, J = 12.8 Hz, ꢀCH2CH2),
3.43 (d, 2H, J = 6.8 Hz, 5-CH2CH), 3.34 (d, 2H, J = 6.4 Hz, 30-CH2CH),
1.84 (q, 2H, J = 20.8 Hz, ꢀCH2CH2), 1.07 (t, 3H, J = 14.8 Hz,
ꢀCH2CH3). 13C NMR (400 MHz, CDCl3): δ 156.6, 151.0, 138.0,
136.8, 132.2, 130.6, 130.4, 129.9, 129.1, 128.8, 128.1, 128.0, 115.9, 115.7,
115.6, 111.8, 69.7, 39.6, 34.7, 22.8, 10.9. HRMS [M ꢀ H]ꢀ calcd
307.1698; found 307.1687.
N-(30-Allyl-40-methoxy-[1,10-biphenyl]-3-yl)acetamide (6f). Yield
93.8%. 1H NMR (400 MHz, DMSO- d6): δ 9.97 (s, 1H, ꢀNH), 9.54
(s, 1H, 20-H), 7.74 (s, 1H, 2-H), 7.52 (d, 1H, J = 8.4 Hz, 50-H),
7.33ꢀ7.28 (m, 2H, 6- and 60-H), 7.20 (d, 1H, J = 8.0 Hz, 40-H),
6.89ꢀ6.87 (m, 1H, 4-H), 6.05ꢀ5.95 (m, 1H, ꢀCHdCH2), 5.11ꢀ5.02
(m, 2H, ꢀCHdCH2), 3.86 (s, 3H, 40-OCH3), 3.36ꢀ3.34 (m, 2H,
ꢀCH2CH), 2.05 (s, 3H, ꢀCOCH3). HRMS [M + Na]+ calcd 304.1313;
found 304.1298.
Synthesis of 7aꢀb: Followed by Step III of Typical Proce-
dure for the Synthesis of 4aꢀl. 3,5-Diallyl-30-nitro-[1,10-biphenyl]-
30,5-Diallyl-40-isopropoxybiphenyl-2-ol (9d). Yield 35.3%. 1H NMR
(400 MHz, CDCl3): δ 7.25 (d, 1H, J = 1.2 Hz, 6-H), 7.22 (s, 1H, 20-H),
7.05 (d, 1H, J = 2.0 Hz, 60-H), 7.03 (d, 1H, J = 2.0 Hz, 4-H), 6.94 (d, 1H,
J = 8.0 Hz, 3-H), 6.90 (d, 1H, J = 8.4 Hz, 50-H), 5.91ꢀ6.04 (m, 2H,
5-CHdCH2 and 30-CHdCH2), 5.02ꢀ5.16 (m, 4H, 5-CHdCH2 and
30-CHdCH2), 4.56ꢀ4.62 (m, 1H, ꢀCH(CH3)2), 3.41 (d, 2H, J = 6.8
Hz, 5-CH2CH), 3.34 (d, 2H, J = 6.8 Hz, 30-CH2CH), 1.38 (s, 3H,
ꢀCH3), 1.36 (s, 3H, ꢀCH3). 13C NMR (400 MHz, CDCl3): δ 155.4,
151.0, 137.9, 136.9, 132.2, 130.8, 130.7, 130.3, 128.9, 128.8, 128.1, 127.9,
115.8, 115.7, 115.6, 113.4, 70.2, 39.5, 34.8, 22.3. HRMS [M ꢀ H]ꢀ calcd
307.1698; found 307.1627.
1
4-ol (7a). Yield 31.0%. H NMR (400 MHz, CDCl3): δ 7.99ꢀ7.86
(m, 2H, 20- and 40-H), 7.21 (m, 2H, 50- and 60-H), 7.77ꢀ7.64 (m, 2H,
2- and 6 -H), 6.11ꢀ6.05 (m, 2H, ꢀCHdCH2), 5.34ꢀ5.26 (m, 4H,
ꢀCHdCH2), 3.86 (s, 1H,ꢀOH), 3.51 (d, 4H, J = 6.2 Hz, ꢀCH2CH).
HRMS [M + H]+ calcd 296.1287; found 296.1291.
3,5-Diallyl-40-methoxy-[1,10-biphenyl]-4-ol (7b). Yield 23.0%. 1H
NMR (400 MHz, CDCl3): δ 7.49ꢀ7.46 (m, 2H, 20- and 60-H), 7.21
(m, 2H, 2- and 6-H), 7.07ꢀ6.94 (m, 2H, 30- and 50-H), 6.09ꢀ6.01 (m,
2H, ꢀCHdCH2), 5.23ꢀ5.16 (m, 4H, ꢀCHdCH2), 3.87 (s, 3H,
4-OCH3), 3.76 (s, 1H,ꢀOH), 3.47 (d, 4H, J = 6.4 Hz, ꢀCH2CH).
HRMS [M + H]+ calcd 281.1542; found 281.1539.
3,50-Diallyl-20-methoxy-[1,10-biphenyl]-4-ol (9e). Yield 30.4%. 1H
NMR (400 MHz, CDCl3): δ 7.27ꢀ7.31 (m, 2H, Ar-H), 7.1 (d, 1H, J =
2.4 Hz, 60-H), 7.08 (d, 1H, J = 2.4 Hz, 4-H), 6.90 (d, 1H, J = 8.0 Hz,
50-H), 6.85 (d, 1H, J = 8.4 Hz, 3-H), 6.10ꢀ5.92 (m, 2H, 5-CHdCH2
and 30-CHdCH2), 5.03ꢀ5.24 (m, 4H, 5-CHdCH2 and 30-CHdCH2),
3.77 (s, 3H, 2-OCH3), 3.44 (d, 2H, J = 6.8 Hz, 5-CH2CH), 3.34 (d, 2H,
J = 6.8 Hz, 30-CH2CH). 13C NMR (400 MHz, CDCl3): δ 154.8, 153.2,
137.8, 136.5, 132.3, 131.5, 131.2, 130.9, 130.3, 129.0, 127.9, 124.8, 116.5,
115.5, 115.4, 111.4, 55.7, 39.4, 35.3. HRMS [M ꢀ H]ꢀ calcd 279.1385;
found 279.1360.
Typical Procedure for ReimerꢀTiemann Reaction. Com-
pounds 8 were prepared as follows. The solution of honokiol (5.0 mmol,
2.7 g), CHCl3 (30 mL), and 10 mL of 35% NaOH was stirred at 50 °C
for 1 h. The crude product was buffered to a pH of 6ꢀ7 with 5%
hydrochloric acid. The mixture was initially extracted with ethyl acetate
(3 ꢁ 50 mL). The combined organic layers were washed by brine
(3 ꢁ 50 mL) and evaporated under reduced pressure. The sample was
separated by preparative HPLC using methanol/water (30/70) as an
eluent.
Typical Procedure for the Synthesis of the Alkylation of
Honokiol Analogues 9aꢀm and 10aꢀd. Compound 9a was
prepared as follows. The mixture of honokiol (2.7 g, 5.0 mmol), DMF
(30 mL), and anhydrous potassium carbonate (10 mmol, 2.8 g) was
stirred at room temperature, and then iodomethane (10.0 mmol) diluted
with 5 mL of DMF was added dropwise and stirred at 30 °C for 6 h.
3,50-Diallyl-20-ethoxybiphenyl-4-ol (9f). Yield 20.4%. 1H NMR (400
MHz, CDCl3): δ 7.39 (d, 1H, J = 2.4 Hz, 6-H), 7.37 (d, 1H, J = 2.4 Hz,
20-H), 7.34 (d, 1H, J = 2.8 Hz, 60-H), 7.12 (d, 1H, J = 2.0 Hz, 4-H), 7.06
(d, 1H, J = 4.2 Hz, 50-H), 7.03 (d, 1H, J = 2.4 Hz, 3-H), 5.92ꢀ6.08 (m,
2H, 5-CHdCH2 and 30-CHdCH2), 5.01ꢀ5.12 (m, 4H, 5-CHdCH2
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Journal of Medicinal Chemistry
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1
and 30-CHdCH2), 4.10 (dd, 2H, J = 21.2 Hz, 5-CH2CH), 3.42 (dd, 2H,
J = 20.0 Hz, 30-CH2CH), 3.40 (d, 2H, J = 6.8 Hz, ꢀCH2CH3), 1.42 (t,
3H, J = 14.0 Hz, ꢀCH2CH3). 13C NMR (400 MHz, CDCl3): δ 154.2,
153.1, 137.9, 136.6, 132.4, 131.7, 131.3, 130.9, 130.6, 128.9, 128.0, 124.8,
116.4, 115.6, 113.4, 113.1, 64.3, 39.5, 35.1, 14.9. HRMS [M ꢀ H]ꢀ calcd
293.1542; found 293.1580.
30,5-Diallyl-2,40-diisopropoxybiphenyl (9l). Yield 19.3%. H NMR
(400 MHz, CDCl3): δ 7.39 (d, 1H, J = 2.0 Hz, 6-H), 7.34 (d, 1H, J =
2.0 Hz, 20-H), 7.13 (d, 1H, J = 2.4 Hz, 60-H,), 7.02 (d, 1H, J = 2.4 Hz,
4-H), 6.86ꢀ6.70 (m, 2H, Ar-H), 5.93ꢀ6.07 (m, 2H, 5-CHdCH2 and
30-CHdCH2), 5.01ꢀ5.11 (m, 4H, 5-CHdCH2 and 30-CHdCH2),
4.53ꢀ4.62 (m, 1H, 2-OCH), 4.31ꢀ4.40 (m, 1H, 40-OCH), 3.40 (d, 2H,
J = 6.8 Hz, 5-CH2CH), 3.35 (d, 2H, J = 6.8 Hz, 30-CH2CH), 1.35 (d, 6H,
J = 6.0 Hz, 2-OCH(CH)3), 1.22 (d, 6H, J = 6.0 Hz, 40-OCH(CH)3). 13C
NMR (400 MHz, CDCl3): δ 154.5, 153.2, 137.9, 137.3, 132.6, 131.9,131.3,
131.1, 130.8, 128.8, 128.0, 127.6, 115.8, 115.5, 115.3, 112.4, 71.0, 70.0, 39.5,
34.7, 22.3, 22.1. HRMS [M + H]+ calcd 350.2246; found 350.2313.
Diethyl 2,20-((30,5-diallyl-[1,10-biphenyl]-2,40-diyl)bis(oxy))diacetate
(9m). Yield 89.3%. 1H NMR (400 MHz, CDCl3): δ 7.55 (s, 1H, 6-H),
7.45 (d, 1H, J = 2.0 Hz, 20-H), 7.43 (d, 1H, J = 2.0 Hz, 60-H), 7.14 (d, 1H,
J = 2.0 Hz, 4-H), 7.07 (d,1H, J = 2.4 Hz, 5-H), 6.79 (d, 1H, J = 2.0 Hz,
50-H), 5.91ꢀ6.11 (m, 2H, 5-CHdCH2 and 30-CHdCH2), 5.03ꢀ5.13
(m, 4H, 5-CHdCH2 and 30-CHdCH2), 4.66 (s, 2H, 2-OCH2), 4.53 (s,
2H, 40-OCH2), 4.20ꢀ4.30 (m, 4H, 2- and 40-COOCH2), 3.50 (d, 2H, J =
6.8 Hz, 5-CH2CH), 3.36 (d, 2H, J = 6.8 Hz, 30-CH2CH), 1.32 (t, 3H, J =
14.4 Hz, 2-COOCH2CH3), 1.27 (t, 3H, J = 14.4 Hz, 40-COOCH2CH3).
13C NMR (400 MHz, CDCl3): δ 169.1, 169.0, 154.9, 153.3, 137.6, 136.9,
133.7, 131.5, 131.4, 131.3, 130.9, 128.8, 128.5, 128.0, 115.7, 115.6, 113.2,
111.1, 66.2, 65.9, 61.3, 61.2, 39.4, 34.5, 14.2. HRMS [M + Na]+ calcd
461.1935; found 461.2194.
3,50-Diallyl-20-propoxybiphenyl-4-ol (9g). Yield 32.2%. 1H NMR
(400 MHz, CDCl3): δ 7.32 (d, 1H, J = 2.0 Hz, 6-H), 7.30 (d, 1H, J =
2.0 Hz, 20-H), 7.12 (d, 1H, J = 2.0 Hz, 60-H), 7.06 (d, 1H, J = 2.4 Hz,
4-H), 7.04 (d, 1H, J = 2.0 Hz, 50-H), 6.86 (q, 1H, J = 14 Hz, 3-H),
5.93ꢀ6.10 (m, 2H, 5-CHdCH2 and 30-CHdCH2), 5.03ꢀ5.22 (m, 4H,
5-CHdCH2 and 30-CHdCH2), 3.89 (d, 2H, J = 12.8 Hz, ꢀCH2CH2),
3.44 (d, 2H, J = 6.4 Hz, 5-CH2CH), 3.36 (d, 2H, J = 6.8 Hz, 30-CH2CH),
1.82 (q, 2H, J = 20.4 Hz, ꢀCH2CH2), 0.96 (t, 3H, J = 14.8 Hz,
ꢀCH2CH3). 13C NMR (400 MHz, CDCl3): δ 154.4, 154.3, 137.9,
137.8, 132.2, 131.7, 131.3, 130.9, 130.5, 129.9, 128.9, 127.9, 115.6, 115.5,
115.3, 112.7, 70.2, 39.5, 35.20, 22.7, 10.8. HRMS [M ꢀ H]ꢀ calcd
307.1698; found 307.1627.
3,50-Diallyl-20-isopropoxybiphenyl-4-ol (9h). Yield 20.4%. 1H NMR
(400 MHz, CDCl3): δ 7.35 (d, 1H, J = 1.2 Hz, 6-H), 7.32 (s, 1H, 20-H),
7.30 (d, 1H, J = 2.0 Hz, 60-H), 7.12 (d, 1H, J = 2.0 Hz, 4-H), 7.06 (d, 1H,
J = 8.0 Hz, 50-H), 6.90 (d, 1H, J = 8.4 Hz, 3-H), 5.91ꢀ6.04 (m, 2H,
5-CHdCH2 and 30-CHdCH2), 5.02ꢀ5.16 (m, 4H, 5-CHdCH2 and
30-CHdCH2), 4.56ꢀ4.62 (m, 1H, ꢀCH(CH3)2), 3.41 (d, 2H, J = 6.8
Hz, 5-CH2CH), 3.34 (d, 2H, J = 6.8 Hz, 30-CH2CH), 1.38(s, 3H,
ꢀCH3), 1.36 (s, 3H, ꢀCH3). 13C NMR (400 MHz, CDCl3): δ 153.2,
153.0, 137.9, 136.7, 132.8, 132.0, 131.7, 131.6, 131.1, 128.8, 127.9, 124.8
116.4, 116.1, 115.6, 115.3, 71.4, 39.6, 35.1, 22.2. HRMS [M ꢀ H]ꢀ calcd
307.1698; found 307.1627.
30,5-Diallyl-2-hydroxy-40-methoxybiphenyl-3-carbaldehyde (10a).
Yield 21.4%.
30,5-Diallyl-2,40-dimethoxybiphenyl-3-carbaldehyde (10b). Yield 43.2%.
30,5-Diallyl-2,40-diethoxybiphenyl-3-carbaldehyde (10c). Yield 65.3%.
1H NMR (400 MHz, CDCl3): δ 10.46 (s, 1H, ꢀCHO), 7.61 (d, 1H, J =
2.4 Hz, 6-H), 7.36ꢀ7.40 (m, 3H, Ar-H), 6.90 (d, 1H, J = 8.4 Hz, 50-H),
5.91ꢀ6.06 (m, 2H, 5-CHdCH2 and 30-CHdCH2), 5.04ꢀ5.13 (m, 4H,
5-CHdCH2 and 30-CHdCH2), 4.09 (q, 2H, J = 21.2 Hz, 2-OCH2), 3.62
(q, 2H, J = 21.2 Hz, 20-OCH2), 3.43 (m, 4H, 5-CH2CH and 30-CH2CH),
1.45 (t, 3H, J= 14.4 Hz, 2-CH2CH3), 1.14 (t, 3H, J= 14.0 Hz, 20-CH2CH3).
13C NMR (400 MHz, CDCl3): δ 190.7, 158.3, 156.4, 137.4, 136.9, 136.7,
136.1, 136.0, 130.4, 129.9, 129.3, 128.8, 127.7, 126.2, 116.5, 115.5, 111.2, 70.8,
63.7, 39.5, 34.6, 15.2, 14.9. HRMS [M + H]+ calcd 351.1955; found 351.1913.
Diethyl 2,20-((30,5-Diallyl-3-formyl-[1,10-biphenyl]-2,40-diyl)bis(oxy))
30,5-Diallyl-2,40-dimethoxy-1,10-biphenyl (9i). Yield 33.1%. 1H NMR
(400 MHz, CDCl3): δ 7.34ꢀ7.42 (m, 2H, Ar-H), 7.15 (d, 1H, J = 2.4
Hz, 60-H), 7.12 (d, 1H, J = 2.4 Hz, 4-H), 6.96 (d, 1H, J = 8.0 Hz, 3-H),
6.92 (d, 1H, J = 8.0 Hz, 50-H), 5.97ꢀ6.12 (m, 2H, 5-CHdCH2 and 30-
CHdCH2), 5.06ꢀ5.15 (m, 4H, 5-CHdCH2 and 30-CHdCH2), 3.89
(s, 3H, 2-OCH3), 3.81 (s, 3H, 40-OCH3), 3.46 (d, 2H, J = 6.8 Hz,
5-CH2CH), 3.40 (d, 2H, J = 6.8 Hz, 30-CH2CH). 13C NMR (400 MHz,
CDCl3): δ 156.4, 155.0, 137.8, 137.1, 132.3, 131.0, 130.7, 130.5, 128.4,
128.1, 127.9, 120.9, 115.5, 115.4, 111.4, 110.0, 55.7, 55.5, 39.4, 34.4.
HRMS [M ꢀ H]ꢀ calcd 293.1542; found 293.1622.
1
diacetate (10d). Yield 71.2%. H NMR (400 MHz, CDCl3): δ 10.67-
30,5-Diallyl-2,40-diethoxybiphenyl (9j). Yield 21.2%. 1H NMR (400 MHz,
CDCl3): δ 7.39 (d, 1H, J = 2.4 Hz, 6-H), 7.37 (d, 1H, J = 2.4 Hz, 20-H), 7.34
(d, 1H, J = 2.8 Hz, 60-H), 7.12 (d, 1H, J = 2.0 Hz, 4-H), 7.06 (d, 1H, J = 4.2
Hz, 3-H), 7.03 (d, 1H, J = 2.4 Hz, 40-H), 5.92ꢀ6.08 (m, 2H, 5-CHdCH2
and 30-CHdCH2), 5.01ꢀ5.12 (m, 4H, 5-CHdCH2 and 30-CHdCH2),
4.10 (q, 2H, J = 21.2 Hz, 2-OCH2CH3), 3.42 (q, 2H, J = 20 Hz,
40-OCH2CH3), 3.40 (d, 2H, J = 6.8 Hz, 5-CH2CH), 3.35(d, 2H, J =
6.4 Hz, 30-CH2CH), 1.42 (t, 3H, J = 14.0 Hz, 2-OCH2CH3), 1.32 (t, 3H, J =
14 Hz, 40-OCH2CH3). 13C NMR(400 MHz, CDCl3): δ 155.8, 154.4, 138.0,
137.3, 132.4, 131.3, 131.0, 130.8, 128.3, 128.1, 127.9, 115.6, 115.4, 113.0,
111.0, 64.3, 63.7, 39.6, 34.6, 15.1, 15.0. HRMS [M + H]+ calcd 322.2006;
found 323.1999.
(s, 1H, ꢀCHO), 7.66 (d, 1H, J = 2.4 Hz, 6-H), 7.40 (d, 1H, J = 2.0 Hz,
20-H), 7.26ꢀ7.38 (m, 2H, Ar-H), 6.81 (d, 1H, J = 8.4 Hz, 50-H),
5.90ꢀ6.11 (m, 2H, 5-CHdCH2 and 30-CHdCH2), 5.07ꢀ5.14 (m,
4H, 5-CHdCH2 and 30-CHdCH2), 4.70 (s, 2H, 2-OCH2), 4.29 (q,
2H, J = 21.6 Hz, 2-COOCH2), 4.14 (q, 2H, J = 21.2 Hz, 20-COOCH2),
4.12 (s, 2H, 20-OCH2), 3.51 (d, 2H, J = 6.8 Hz, 4-CH2CH) 3.41 (d, 2H,
J = 6.8 Hz, 40-CH2CH), 1.33 (t, 3H, J = 7.2 Hz, 2-COOCH2CH3), 1.21 (t,
3H, J = 14.4 Hz, 20-COOCH2CH3). 13C NMR (400 MHz, CDCl3):
δ 191.0, 168.8, 168.8, 156.9, 155.6, 137.4, 136.7, 136.5, 136.5, 134.2, 130.7,
130.1, 129.9, 129.8, 127.7, 126.6, 116.7, 115.9, 111.7, 69.9, 65.7, 61.4, 61.2,
39.4, 34.4, 14.2, 14.1. HRMS [M + H]+ calcd 467.2064; found 467.2027.
Typical Procedure for the Synthesis of 11. Compound 11a
was prepared as follows. Sodium triacetoxyborohydride (317.8 mg, 1.5
mmol) was added to the solution of 8a (294.3 mg, 1.0 mmol), glacial
acetic acid (86 μL, 1.5 mmol), and methanol (10 mL) in an ice bath. The
mixture was stirred and allowed to warm up to room temperature. The
reaction was monitored by TLC. The mixture was filtered, evaporated,
and extracted with ethyl acetate (3 ꢁ 10 mL) and brine (2 ꢁ 10 mL) and
dried by anhydrous sodium sulfate. Finally, the solvent was evaporated
under reduced pressure and the product was obtained.
30,5-Diallyl-2,40-dipropoxybiphenyl (9k). Yield 13.4%. 1H NMR (400
MHz, CDCl3): δ 7.38 (d, 1H, J = 2.0 Hz, 6-H), 7.35 (d, 1H, J = 2.0 Hz, 20-
H), 7.33 (d, 1H, J = 2.0 Hz, 60-H), 7.12 (d, 1H, J = 2.4 Hz, 4-H), 7.04 (d,
1H, J = 2.0 Hz, 3-H), 6.86 (q, 1H, J = 14 Hz, 40-H), 5.93ꢀ6.05 (m, 2H,
5-CHdCH2 and 30-CHdCH2), 5.01ꢀ5.10 (m, 4H, 5-CHdCH2 and 30-
CHdCH2), 3.96 (t, 2H, J = 12.4 Hz, 2-OCH2), 3.86 (t, 2H, J = 13.2 Hz,
40-OCH2), 3.42 (d, 2H, J = 6.8 Hz, 5-CH2CH), 3.35 (d, 2H, J = 6.8 Hz, 30-
CH2CH), 1.82 (q, 2H, J = 20.8 Hz, 2-OCH2CH2), 1.80 (q, 2H, J = 20.8
Hz, 40-OCH2CH2), 1.07 (t, 3H, J = 14.8 Hz, 2-OCH2CH2CH3), 0.96 (t,
3H, J = 14.8 Hz, 40-OCH2CH2CH3). 13C NMR (400 MHz, CDCl3): δ
155.8, 154.5, 138.0, 137.3, 132.2, 131.3, 131.0, 130.7, 130.6, 128.3, 128.0,
127.8, 115.5, 115.3, 112.7, 110.7, 70.2, 69.6, 39.5, 34.7, 22.9, 22.7, 10.9,
10.8. HRMS [M ꢀ H]ꢀ calcd 349.2168; found 349.2227.
30,5-Diallyl-3-(hydroxymethyl)biphenyl-2,40-diol (11a). Yield 81.3%.
1H NMR (400 MHz, CDCl3): δ 7.27 (d, 1H, J = 2.4 Hz, 60-H), 7.25 (d,
1H, J = 3.2 Hz, 20-H), 7.02(d, 1H, J = 2.4 Hz, 6-H), 6.94 (d, 1H, J = 2.0 Hz,
4-H), 6.89 (d, 1H, J = 7.6 Hz, 30-H), 5.90ꢀ6.09 (m, 2H, 5-CHdCH2 and
30-CHdCH2), 5.04ꢀ5.23 (m, 4H, 5-CHdCH2 and 30-CHdCH2), 4.81
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(s, 1H, 3-CH2OH), 3.46 (d, 2H, J = 6.4 Hz, 5-CH2CH), 3.33 (d, 2H, J =
6.8 Hz, 30-CH2CH), 2.37(s, 1H, 3-CH2OH). 13C NMR (400 MHz,
CDCl3): δ 153.9, 150.1, 137.7, 136.2, 131.8, 131.2, 130.2, 129.5, 128.6,
128.5, 127.7, 126.2, 125.9, 116.7, 116.2, 115.7, 63.6, 39.4, 35.1. HRMS
[M + Na]+ calcd 319.1310; found 319.1317.
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1
70.4%. H NMR (400 MHz, CDCl3): δ 7.20 (d, 1H, J = 7.20 Hz,
20-H), 7.03 (d, 1H, J = 7.0 Hz, 60-H), 6.97 (d, 1H, J = 2.0 Hz, 6-H), 6.89
(d, 1H, J = 2.0 Hz, 4-H), 5.88ꢀ6.08 (m, 2H, 5-CHdCH2 and
30-CHdCH2), 5.03ꢀ5.16 (m, 4H, 5-CHdCH2 and 30-CHdCH2),
4.82 (s, 1H, 3-CH2OH), 4.75 (s, 1H, 50-CH2OH), 3.44 (d, 2H, J = 6.4
Hz, 5-CH2CH), 3.31 (d, 2H, J = 6.8 Hz, 30-CH2CH), 2.80 (s, 1H,
3-CH2OH), 2.60 (s, 1H, 50-CH2OH). 13C NMR (400 MHz, CDCl3):
δ 153.5, 150.0, 137.6, 136.5, 131.8, 130.7, 130.2, 128.8, 128.6, 127.7,
127.5, 126.8, 125.8, 124.9, 116.1, 115.7, 64.1, 63.5, 39.3, 34.2. HRMS
[M + Na]+ calcd 349.1410; found 349.1420.
’ ASSOCIATED CONTENT
S
Supporting Information. HPLC purity and chromatograms
b
of honokiol analogues and derivatives. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 86-28-85164063. Fax: 86-28-85164060. E-mail: lijuan17@
hotmail.com.
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J. Y.; Huang, W. C.; Xu, G. B.; Peng, M.; Han, J.; Li, R.; Yang, S. Y.; Wei,
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Author Contributions
†These authors contributed equally.
’ ACKNOWLEDGMENT
We are grateful to the National Key Programs of China during
the 11th Five-Year Plan Period (2009ZX09501-015).
’ ABBREVIATIONS LIST
EC, endothelial cell; flk-1, fetal liver kinase-1; GFP, green fluor-
escent protein; NLS, nuclear localization signal; fli-1, friend leukemia
integration-1; SAR, structureꢀactivity relationship; DMSO, di-
methylsulfoxide; IC50, half-maximum inhibitory concentration;
EDG, electron-donating group; EWG, electron-withdrawing group;
HUVEC, human umbilical vein endothelial cell; A549, human
lung carcinoma; HepG2, human hepatocellular liver carcinoma;
LL/2, Lewis lung carcinoma; HPCCC, high performance coun-
ter-current chromatography; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide; DMEM, Dulbecco’s modified
eagle medium; VEGF, vascular endothelial growth factor;
FGF, fibroblast growth factor
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honokiol. Tetrahedron Lett. 2009, 50, 1151–1152.
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He, S. C.; Li, S. C.; Luo, Y. F.; Wei, Y. Q.; Chen, L. J. Purification of
honokiol derivatives from one-pot synthesis by high-performance coun-
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Novel Tool for Cardiovascular Drug Discovery. Recent Pat. Cardiovasc.
Drug Discovery 2009, 4, 1–5.
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