Design, synthesis, and anti-proliferative evaluation of [1,1′-biphenyl]-4-ols as inhibitor of HUVEC migration and tube formation

Article abstract of DOI:10.3390/molecules17078091

Allylated biphenol neolignans contain a variety of chemopreventive entities that have been used as anti-tumor drug leads. Herein, 37 allylated biphenols were evaluated for anti-proliferative activity by the MTT assay and inhibitory effect on the migration and tube formation of HUVECs featuring anti-angiogenic properties. 3-(2-Methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1, 1′-biphenyl]-4-ol (5c) exerted an inhibitory effect on HUVECs compared to honokiol (IC₅₀ = 47.0 vs. 52.6 μM) and showed significant blocking effects on the proliferation of C26, Hela, K562, A549, and HepG2 (IC ₅₀ = 15.0, 25.0, 21.2, 29.5, and 13.0 μM, respectively), superior to those of honokiol (IC₅₀ = 65.1, 62.0, 42.0, 75.0, and 55.4 μM, respectively). Importantly, compound 5c inhibited the migration and capillary-like tube formation of HUVECs in vitro.

Full text of DOI:10.3390/molecules17078091

Molecules 2012, 17, 8091-8104; doi:10.3390/molecules17078091
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis, and Anti-Proliferative Evaluation of
[1,1′-biphenyl]-4-ols as Inhibitor of HUVEC Migration and
Tube Formation
Yan Ran 1,†, Liang Ma 2,†, Xuewei Wang 2, Jinying Chen 2, Guangcheng Wang 2, Aihua Peng 2
and Lijuan Chen 1,2,
*
1
Pharmacy College of West China of Sichuan University, Chengdu, Sichuan 610065, China
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School,
Sichuan University, Chengdu, Sichuan 610041, China
2
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: lijuan17@hotmail.com;
Tel.: +86-28-8516-4063; Fax: +86-28-8516-4060.
Received: 10 May 2012; in revised form: 11 June 2012 / Accepted: 20 June 2012 /
Published: 5 July 2012
Abstract: Allylated biphenol neolignans contain a variety of chemopreventive entities that
have been used as anti-tumor drug leads. Herein, 37 allylated biphenols were evaluated for
anti-proliferative activity by the MTT assay and inhibitory effect on the migration and tube
formation of HUVECs featuring anti-angiogenic properties. 3-(2-Methylbut-3-en-2-yl)-
3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c) exerted an inhibitory effect on HUVECs
compared to honokiol (IC50 = 47.0 vs. 52.6 μM) and showed significant blocking effects
on the proliferation of C26, Hela, K562, A549, and HepG2 (IC50 = 15.0, 25.0, 21.2, 29.5,
and 13.0 μM, respectively), superior to those of honokiol (IC50 = 65.1, 62.0, 42.0, 75.0,
and 55.4 μM, respectively). Importantly, compound 5c inhibited the migration and
capillary-like tube formation of HUVECs in vitro.
Keywords: biphenol; endothelial cell; anti-proliferative; migration; tube formation

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1. Introduction
Angiogenesis is a complicated multistep process involving endothelial cell (EC) activation, invasion,
migration, proliferation, tube formation, and finally capillary network formation in several solid tumors
and haematological malignancies [1]. Tumor growth depends on the recruitment of new blood vessels
from pre-existing vasculature. Without the development and progression of new blood vessels, tumors
cannot deteriorate beyond a critical size or metastasize to other organs [2–4].
Endothelial cells play crucial roles in a series of physiological processes (e.g., wound healing,
reproduction, and embryonic development) and pathophysiological events (e.g., solid tumor growth,
psoriasis, and diabetic retinopathy). The well-established target of anti-angiogenesis is genetically stable,
non-transformed ECs, which are less prone to acquire drug resistance [5,6]. When tumor cells secrete
pro-angiogenic growth factors that bind to receptors on dormant ECs, leading to ECs activation,
stimulation, vasodilatation and an up-regulation of vessel permeability, the activated ECs rapidly detach
from the extracellular matrix and basement membrane by secreting proteases (matrix metalloproteinases).
Subsequently, they migrate and proliferate to sprout and self-assemble into new branches from the
pre-existing vasculatures [7–9]. Therefore, suppression of EC activation, invasion, migration,
proliferation, tube formation, and finally capillary network formation seem to be an effective
and relevant strategy for blocking tumor progression and cancer development. Up to now, more than
80 anti-angiogenic drugs are currently in clinical trials. Notably, many angiogenesis inhibitors have been
discovered in natural resources, such as fungi, mushrooms, shark muscle and cartilage, sea corals, green
tea, ginseng, and garlic by screening of EC cultures [10].
Biphenols are widely found in naturally products, including lignans, flavonoids, tannins, together with
coumarins, peptides, glycopeptides, etc. [11]. Natural biphenol products contained a range of chemo-
therapeutic and chemo-preventive entities that have the capacity of preventing and inhibiting the
development of malignancies [12–14]. However, only a few studies have focused on the structural
modification and structure-activity relationship (SAR) of biphenols targeting angiogenesis or
cancer [15,16]. Disconnections and chemical synthesis have been undertaken to develop potent allylated
biphenols to improve biological activity or clarify the SAR in our previous study [17].
In this study, 37 allylated biphenols were designed, synthesized, and subsequently screened. Among
our synthetic compounds, 3-(2-methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol
(5c) exhibited a medium inhibitory effect on human umbilical vein endothelial cells (HUVECs;
IC50 = 40.0 μM) in contrast to honokiol (IC50 = 57.0 μM) and blocked the proliferation of C26
(murine colon adenocarcinoma, IC50 = 15.0 μM), Hela (human cervical carcinoma, IC50 = 25.0 μM),
K562 (human erythromyeloblastoid leukemia; IC50 = 21.2 μM), A549 (human lung carcinoma;
IC50 = 29.5 μM), and HepG2 (human hepatocellular liver carcinoma; IC50 = 13.0 μM) showing superior
activity to honokiol (IC50 = 65.1, 62.0, 42.0, 75.0, and 55.4 μM, respectively). Importantly, 5c showed
much more potent inhibitory potency against the migration and tube formation of HUVECs than those
of honokiol. The anti-proliferative activity of allylated biphenols led to the establishment of a
structure-activity relationship. In contrast to our previous report, compound 5c exhibited comparable
inhibitory activity to honokiol, but less potent in vitro inhibitory potency than 3,5′-diformalhonokiol
(compound 8c in reference [17]) against HUVECs, A549, and, HepG2. Although compound 5c could
significantly suppress HUVEC migration and tube formation, 5c was not found to prevent the

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newly-grown segmental vessels from the dorsal aorta of zebrafish, and inappropriate vascularisation in
the transgenic zebrafish screening model.
2. Results and Discussion
2.1. Chemistry
As depicted in Scheme 1, 1-allyloxy-4-bromobenzene (2) was prepared in a satisfactory yield (95%)
by a convenient procedure starting from commercially available p-bromophenol and employing 1.1
equiv of allyl bromide in the presence of anhydrous K2CO3 (1.3 equiv) as base and acetone as solvent.
The intermediates 3a–t were obtained through Suzuki-Miyaura reactions [17,18]. Two cross-coupling
methods were developed for the condensation of appropriate arylboronic acids with 2 under palladium
catalysis under a N2 atmosphere. Method A involved the use of Pd(OAc)2 as a cross-coupling catalyst,
PPh3 as a reducing agent and K2CO3 (2.0 M) as a base in isopropanol as solvent. The intermediates
3a–i was obtained by Method A in 55–80% yields. Another coupling method (used for 3j–t) was
carried out by using Pd(PPh3)4 as a catalyst, 2.0 M K3PO4·3H2O as a base and DMF as solvent. The
intermediates 3j–t were obtained by Method B in much better yields (75–90%).
Scheme 1. Synthesis of compounds 4a–t and 5a–c.
Reagents and conditions: (a) Br-CH2CH=CH2, acetone, reflux, 5 h; (b) Method A: Pd(OAc)2, PPh3,
.
K2CO3, isopropanol, H2O, N2 atmosphere, 90 °C; Method B: Pd(PPh3)4, DMF, H2O, K3PO4 3H2O,
N2 atmosphere, 100 °C; (c) N,N-diethylaniline, reflux, overnight.
The O-allylation of 3 with 3-bromoprop-1-ene or 1-bromo-3-methylbut-2-ene was performed in a
good yield in the presence of anhydrous K2CO3. Next, the corresponding compounds 4a–t and 5a–c
were synthesized through the Claisen rearrangement, which belongs to the [3,3]-sigmatropic concerted
rearrangement category, in N,N-diethylaniline (boiling point = 216 °C) as solvent [19,20]. Moreover, the
other allylated biphenols 6a–j and 7a–d were prepared following similar synthetic methods (Scheme 2).

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Scheme 2. Synthesis of compounds 6a–j, and 7a–d.
Reagents and conditions: (a) CH3-I or Br-CH2CH=CH2, acetone, reflux, overnight;
(b) N,N-diethylaniline, reflux, overnight.
2.2. Anti-Proliferative Activity and SAR Study
In order to find new potential angiogenic inhibitors, 37 allylated biphenols were primarily
investigated for anti-proliferative activity on C26 and Hela tumor cells by the MTT assay and these
results are shown in Table 1. In addition, predicted octanol/water log P (Clog P, miLog P, and Xlog P)
were calculated using the ChemDraw software, Molinspiration online service and XlogP3 online service,
respectively, to provide a measure of lipophilicity. Honokiol, a potent anti-angiogenic and anti-tumor
drug lead, was selected as a positive control.
As for the twenty 3-allyl-[1,1′-biphenyl]-4-ols 4a–t, the preliminary IC50 values indicated that only six
substituted derivatives (i.e., 3′-SCH3 in 4c, 2′-CF3 in 4j, 2′,4′-diCF3 in 4m, 3′,5′-diCF3 in 4n, 2′-Cl,4′-CF3
in 4o, and 3′-NO2 in 4t) exhibited comparable or superior activity to honokiol (65.1 μM for C26, and
62.0 μM for Hela) on anti-proliferative activity, with 4n being the most efficient (34.5 μM for C26, and
36.0 μM for Hela). Importantly, 4n achieved an approximately 2.0-fold improvement in inhibitory
activities against C26 and Hela compared to those of honokiol. With respect to all the O-alkylated
biphenyls (compounds 6a–j), the introduction of a methyl or allyl group did not provide any advantage
for the anti-proliferative effects. Although O-methylation of 4g (3′,4′,5′-triOCH3) led to the
corresponding derivative 6c, which showed weak inhibitory activity (76.0 μM for C26), it still was
inactive in blocking the proliferation of Hela cells (>100.0 μM). Inspection of the structural features of
4a–t, and 6a–j and activity indicated that the phenolic hydroxyl group at the C-4 position of the biphenyl
motif was very essential for anti-proliferation activity because the O-alkylated compounds were inactive
(e.g., 4n vs. 6f; 4t vs. 6i) and the number of the trifluoromethyl substituents potentially affected the
inhibitory potency (e.g., one CF3 in 4j vs. two CF3 in 4n).

Molecules 2012, 17
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Table 1. IC50 values against C26 and Hela cells and calculated properties of allylated biphenols.
IC50 (μM)
Compd MW a cLogP b miLogP c xLogP d
C26
65.1
69.0
>100.0
63.0
67.0
96.0
90.0
74.0
80.0
>100.0
53.1
54.0
61.7
45.1
34.5
59.5
57.1
88.1
67.2
>100.0
48.5
74.0
58.0
15.0
Hela
62.0
96.0
82.0
62.0
81.5
58.0
>100.0
95.5
89.5
76.5
52.3
70.1
68.5
48.7
36.0
50.0
94.5
72.5
>100.0
80.0
56.5
95.0
55.0
25.0
K562
42.0
NI e
NI
71.2
NI
90.0
NI
110.5
NI
Honokiol 266.33
5.03
4.37
4.25
4.81
4.21
4.12
4.12
3.99
4.15
3.88
5.29
5.29
5.29
6.21
6.21
5.85
4.53
4.69
3.28
2.59
3.95
5.05
4.96
7.01
4.51
5.20
4.26
4.41
5.56
7.17
7.17
3.54
4.47
5.03
5.04
5.30
7.27
8.07
5.01
4.47
4.51
4.88
4.53
4.51
4.12
4.10
4.36
3.98
5.32
5.34
5.37
6.19
6.19
5.97
4.64
4.73
3.67
3.68
4.41
5.50
5.38
7.16
4.60
5.24
4.17
4.43
5.44
6.90
7.94
3.74
4.48
5.12
4.95
5.07
6.74
7.71
4.98
4.26
4.23
4.78
4.23
4.21
4.21
4.18
4.08
3.98
5.15
5.15
5.15
6.03
6.03
5.78
4.36
4.46
3.44
3.23
4.09
5.11
4.97
6.91
4.56
5.20
4.50
4.40
5.47
7.00
7.86
3.77
4.42
5.06
5.16
5.31
7.10
7.98
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
210.10
240.12
256.36
240.30
270.03
270.13
300.35
254.09
268.31
278.27
278.09
278.09
346.08
346.08
312.05
228.26
246.25
267.13
303.09
255.09
268.35
283.12
374.32
254.32
280.15
314.15
268.11
292.11
386.11
414.14
281.35
269.11
295.12
295.12
280.15
386.33
NI
41.6
61.5
55.8
65.8
56.3
62.4
82.0
78.0
NI
NI
4t
71.0
80.0
53.0
21.2
NI
NI
NI
NI
NI
NI
NI
58.0
NI
NI
55.2
73.4
37.3
42.1
5a
5b
5c
6a
6b
6c
6d
6e
6f
>100.0 >100.0
>100.0 >100.0
76.0
>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 >100.0
44.7
65.0
25.5
6g
6h
6i
6j
7a
7b
7c
7d
51.3
72.0
36.0
40.0
414.38
29.0
a
b
c
Molecular weight; Calculated by ChemDraw Ultra, version 10.0; Calculated by Molinspiration
online service; d Calculated by XLOGP3 online service; e no inhibition.
In order to explore the SAR information based on the aforementioned analysis, we further synthesized
compounds 5a–c, in which the allyl group at the C-3 site was replaced by a 2-methylbut-3-en-2-yl

Molecules 2012, 17
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substituent, and 7a–d with the introduction of one more allyl (compounds 7a–c) or a 2-methylbut-3-en-2-yl
(7d) group at the C-5 position of the biphenol scaffold. Interestingly, 3-(2-methylbut-3-en-2-yl)-3′,5′-
bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c) was found to exhibit more potent inhibitory activity against
C26 and Hela tumor cells (IC50 = 15.0, and 25.0 μM, respectively) than those of honokiol and the
chemical entity also contained the two trifluoromethyl (CF3) substituents. However, the further
modification of 5c that led to 7c and 7d didn’t achieve an effective improvement in inhibitory potency
compared to honokiol, indicating that the best number of allyl or 2-methylbut-3-en-2-yl moieties in the
same phenyl ring seemed to be less than two, as well as highlighting that the introduction of allyl or
2-methylbut-3-en-2-yl groups at the C-5 position of the biphenol was forbidden.
A relevant strategy for anti-angiogenesis is effectively inhibition of the proliferation of ECs. Thus, the
result of anti-proliferative activity against HUVECs was selected as the main index and the IC50 values
against A549 and HepG2 cells were also outlined in Table 2. With comparable or superior inhibitory
potency against C26 and Hela cells compared to honokiol, nine allylated biphenols were next selected for
biological evaluation, with 5c being the most efficient. Compound 5c exhibited a moderate inhibitory
activity against the proliferation of HUVECs (IC50 = 57.0 μM) in contrast to honokiol (IC50 = 40.0 μM)
and exerted remarkable cytotoxic activities against A549 and HepG2 cells (IC50 = 29.5 and 13.0 μM,
respectively). At the same time, 7c presented comparable inhibitory potencies. The anti-proliferative
effects on HUVECs and the inhibitory activities against A549 and HepG2 of tested compounds
showed consistent tendencies. In addition, there was a potential positive correlation between the
lipophilicity (Log P and cLogP) and inhibitory potency.
Table 2. IC50 values against HUVECs and A549, HepG2 cells of the selected allylated biphenols.
IC50 (μM)
Compd.
HUVEC
40.0
A549
75.0
59.7
73.0
76.0
50.0
72.0
64.0
29.5
30.0
32.0
HepG2
55.4
52.4
58.3
60.3
37.5
68.0
56.0
13.0
26.5
33.0
Honokiol
4j
4k
4l
4n
4o
5b
5c
7c
7d
82.0
76.0
74.0
>100.0
>100.0
77.0
47.0
48.0
70.0
2.3. Effects on HUVEC Migration
EC migration is a relevant process in chemotaxis and an indispensable step to form new blood
vessels. Inhibition on the process could block the formation of new blood vessels and further suppress
the development of cancer. Therefore, to characterize the effects of allylated biphenols 5c and 7c on
HUVEC migration, an in vitro migration assay was performed by the application of a slightly modified
Boyden chamber. As depicted in Figure 1I, the HUVECs actively migrated to the serum-containing
lower chamber within 6 h under the untreated conditions (control). Compared to the control, the mean

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number of invaded HUVECs (% of control group) treated with 5c and 7c at the concentration of 40 μM
were 7.1 and 14.2%, respectively, and the cell migration was not likely to occur [Figure 1(II)]. After
treatment with 5c and 7c, even at 20 μM, the inhibitory effects on cell migration were also effective
and the mean invasion rates remained below 50.0%. However, at 10 μM, the two biphenols hardly
inhibited HUVEC migration. As a consequence, 5c and 7c exerted the potent inhibitory activity on the
migration of HUVECs in a concentration-dependent manner and 5c was more potent than 7c.
Figure 1. Effects of 5c and 7c on the HUVECs migration. (I) HUVECs seeded in 24-well
Chambers were incubated for 6 h with medium alone (control) or contained the indicated
concentration (10, 20, and 40 μM) of 5c and 7c. The photographs exhibited the migrated
cells on the lower surface of the filter stained with 1% crystal violet under a phase contrast
microscopy (magnification: 200×). (II) Mean invaded HUVECs (% of control) were
quantitatively analyzed by the inhibitory effects of 5c and 7c. Data represented the
mean ± standard error (SE) from three independent experiments.
** P < 0.01; *** P < 0.005.

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In the later stages of angiogenesis, ECs will assemble into an interconnected tubular network which is
similar to in vivo capillary vascular beds. Inhibition on this formation of capillary-like tube networks will
block the formation of new blood vessels. A tube formation assay was performed by plating HUVECs on
Matrigel. In the blank control, the cells exhibited high mobility on Matrigel and constructed an intact
tube network in 24 hours [Figure 2(I)]. Compared to the control, the mean number of tube formation
(% of control) treated with 5c and 7c at the concentration of 40 μM were 3.7 and 4.7%, respectively,
and the disrupted tubular structures were sparse and incomplete [Figure 2(II)]. After the treatment with
5c and 7c, even at 20 μM, the inhibitory effects were also effective and the mean tube formation
remained at 23.4 and 40.6%, respectively. However, served at the low concentration of 10 μM, the two
compounds were totally unable to inhibit tube formation of HUVECs. Our observation indicated
that allylated biphenols (5c and 7c) could effectively terminate the formation of capillary-like tube
networks and the effect of 5c was superior to that of 7c at the same concentration.
Figure 2. Effects of 5c and 7c on the HUVECs Tube Formation. (I) HUVECs (1 × 104 cells)
suspended in DMEM containing the tested compound (10, 20, and 40 μM) were added
to the Matrigel. Control was treated with DMEM alone. After incubation for 24 hours at
37 °C, capillary networks were photographed and quantified (magnification: 200×).
(II) The number of mean tube formation 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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3. Experimental
3.1. Chemistry
Chemical reagents of analytical grade were purchased from Chengdu Changzheng Chemical Factory
1
(Sichuan, China). H-NMR spectra were recorded at 400 MHz on a Varian Gemini 400 spectrometer
(Varian, Palo Alto, CA, USA) and are reported in parts per million. Chemical shifts (δ) are quoted in
ppm relative to the internal standard tetramethylsilane (TMS), where (δ) TMS = 0.00 ppm. The
multiplicity of the signals is indicated as s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet
defined as all multipeak signals where overlap or complex coupling of signals makes definitive
descriptions of peaks difficult. Mass spectra were measured by a Premier quadrupole-time of flight
(Q-TOF) mass spectrometer (Micromass, Manchester, UK) utilizing electrospray ionization (ESI). The
purity of compounds was determined to be ≥97% by HPLC analysis using a photodiode array detector
(Waters, Milford, MA, USA) and the chromatographic column was an Atlantis C18 (150 mm × 4.6 mm,
i.d. 5 μm) (Waters, Milford, Ireland). All compounds were dissolved as 0.1 mg/mL solutions in HPLC
quality methanol with 10 μL injected on a partial loop fill at a flow rate of 1 mL/min and the column
chamber was kept at 20 °C for the analysis. The moble phases were 70% methanol and 30% water (0.1%
formic acid).
3.1.1. General Procedure Step I for the Preparation of 1-Allyloxy-4-Bromobenzene (2)
Allyl bromide (1.9 mL, 22 mmol) was slowly added into a solution of p-bromophenol (3.46 g,
20 mmol) and anhydrous K2CO3 (3.6 g, 26 mmol) in acetone (25 mL), and the mixture was refluxed
for 5 hours (TLC monitoring). After completing and cooling, the mixture was filtered to remove the
solid and the filtrate was evaporated to dryness. The residue was extracted with diethyl ether (20 mL × 3)
and 10% NaOH (20 mL × 2) and then the organic layer was combined and washed by brine (20 mL × 2),
dried over anhydrous MgSO4, and concentrated under reduced pressure to afford a colorless oil
(4.05 g, 19.01 mmol, 95.0%).
3.1.2. Step II for the Preparation of Intermediates 3 by Suzuki-Coupling Reaction
Method A (for 3a-i): Compound 2 (1.0 mmol) and arylboronic acids (1.2 mmol) were dissolved in
isopropanol (4 mL) at room temperature and stirred for 10 min. After a clear solution was formed,
Pd(OAc)2 (0.01 mmol), PPh3 (0.03 mmol) and anhydrous K2CO3 (2.0 mol/L, 1 mL) were quickly added
under a N2 atmosphere, and the resulting mixture was stirred for a further 18 hours at 90 °C (TLC
monitoring). After completion of the reaction, the mixture was filtered, and extracted with ethyl acetate
(10 mL × 3). The extracts were combined and washed with brine (10 mL × 2), dried over anhydrous
MgSO4, and concentrated. The residue was purified by silica gel column chromatography (ethyl
acetate/petroleum ether = 1:10) to give the intermediates 3a–i in satisfactory yields (55–80%).
Method B (for 3j–t): Compound 2 (1.0 mmol) and arylboronic acids (1.2 mmol) were dissolved in
DMF (4 mL) at room temperature and stirred for 5 min. After a clear solution was formed, Pd(PPh3)4
(0.01 mmol), and a solution of K3PO4·3H2O (2.0 mmol) in water (1 mL) were quickly added under a N2
atmosphere, and the mixture was heated for 18 h at 100 °C (TLC monitoring). After completion, the

Molecules 2012, 17
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reaction mixture was filtered, and extracted with ethyl acetate (10 mL × 3). The extract was combined
and washed with brine (10 mL × 2), dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified by gel chromatography (ethyl acetate/petroleum ether = 1:10) to give
the intermediates 3j–t in satisfactory yields (75–90%).
3.1.3. Step III for the Preparation of 4a–t
Allyl bromide or 1-bromo-3-methylbut-2-ene (1.3 mmol) was added to the solution of 3 (1.0 mmol)
and anhydrous K2CO3 (2.0 mmol) and refluxed for 5 hours. After completion (TLC monitoring) and
cooling, the mixture was filtered, and the solution was evaporated to dryness. Next, the crude product
was dissolved in N,N-diethylaniline and refluxed for 10 hours under a N2 atmosphere (TLC monitoring).
After completion and cooling, the solution was adjusted to pH = 4 with 2.5 N HCl and extracted with
ethyl acetate (10 mL × 2) and the organic layer was combined and washed with water (10 mL × 2) and
brine (10 mL × 1), dried over anhydrous MgSO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1:10) to
give the targeted 4a–t. The yield represents the total yield of the above four steps. The chemical and
structural elucidation of eighteen compounds (4a–g, 4j, 4t, 5a–b, 6a–b, 6d–e, 6i–j, and 7a–b) have
been reported in our previous publication [17].
1
2-Allyl-4-(benzo[d][1,3]dioxol-5-yl)phenol (4h). Yield 40.0%; HPLC: 99.3%. H-NMR (CDCl3)
δ 7.29–7.26 (m, 2H, 2- and 6′-H), 7.02–6.98 (m, 2H, 2′- and 6-H), 6.86–6.84 (m, 2H, 5- and
5′-H), 6.09–6.02 (m, 1H, -CH=CH2), 5.98 (s, 2H, -OCH2O-), 5.23–5.17 (m, 2H, -CH=CH2), 3.46 (d, 2H,
J = 6.8 Hz, -CH2CH); HRMS [M + Na]+ calcd. 255.1021; found 255.1028.
1
2-Allyl-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenol (4i). Yield 68.0%; HPLC: 97.8%. H-NMR
(CDCl3) δ 7.31–7.26 (m, 2H, 2- and 6-H), 7.06–7.01 (m, 2H, 2′- and 6′-H), 6.90 (d, 1H, J = 8.4 Hz, 5-H),
6.85 (d, 1H, J = 8.0 Hz, 5′-H), 6.10–6.00 (m, 1H, -CH=CH2), 5.23–5.17 (m, 2H, -CH2CH), 4.97 (s, 1H,
-OH), 4.29 (s, 4H, -OCH2CH2O-), 3.46 (d, 2H, J = 6.4 Hz, -CH2CH); HRMS [M + H]+ calcd. 269.1178;
found 269.1172.
3-Allyl-2′-(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4l). Yield 43.0%; HPLC: 97.8%. 1H-NMR (CDCl3): δ
7.72 (d, 1H, J = 7.2 Hz, 6′-H), 7.53 (t, 1H, J = 7.2 Hz, 3′-H), 7.43 (t, 1H, J = 7.2 Hz, 5′-H), 7.32 (d,
1H, J = 7.2 Hz, 4′-H), 7.10–7.08 (m, 2H, 2- and 6-H), 6.84 (d, 1H, J = 8.0 Hz, 5-H), 6.09–5.99 (m, 1H,
-CH=CH2), 5.19–5.15 (m, 2H, -CH=CH2), 3.44 (d, 2H, J = 6.0 Hz, -CH2CH); HRMS [M − H]− calcd.
277.0840; found 277.0840.
1
3-Allyl-3′-(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4k). Yield 59.8%; HPLC: 99.5%. H-NMR (CDCl3): δ
7.77 (s, 1H, 2′-H), 7.72–7.70 (m, 1H, 6′-H), 7.56–7.49 (m, 2H, 2- and 6-H), 7.39–7.35 (m, 2H, 3′- and
4′-H), 6.91 (d, 1H, J = 8.0 Hz, 5-H), 6.11–6.01 (m, 1H, -CH=CH2), 5.24–5.19 (m, 2H, -CH=CH2), 3.49
(d, 2H, J = 6.4 Hz, -CH2CH); HRMS [M + H]+ calcd. 301.0816; found 301.0820.
1
3-Allyl-4′-(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4l). Yield 51.8%; HPLC: 98.9%. H-NMR (CDCl3): δ
7.67–7.62 (m, 4H, Ar′-H), 7.40–7.36 (m, 2H, 2- and 6-H), 6.92 (d, 1H, J = 8.0 Hz, 5-H), 6.10–6.01

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(m, 1H, -CH=CH2), 5.24–5.19 (m, 2H, -CH=CH2), 3.49 (d, 2H, J = 6.0 Hz, -CH2CH); HRMS [M + H]+
calcd. 279.0997; found 279.0993.
1
3-Allyl-2′,4′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4m). Yield 21.2%; HPLC: 98.7%. H-NMR
(CDCl3): δ 7.90 (s, 1H, 3′-H), 7.38–7.35 (m, 2H, 2′- and 6′-H), 7.15–7.11 (m, 2H, 2- and 6-H), 6.95–6.81
(m, 1H, 5-H), 6.10–5.97 (m, 1H, -CH=CH2), 5.20–5.10 (m, 2H, -CH2CH), 3.48 (d, 2H, J = 6.4 Hz,
-CH=CH2). HRMS [M + H]+ calcd. 347.0871; found 247.0869.
3-Allyl-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4n). Yield 41.2%; HPLC: 98.0%. 1H-NMR
(CDCl3): δ 7.95 (s, 1H, 4′-H), 7.40–7.37 (m, 2H, 2′- and 6′-H), 7.15–7.11 (m, 1H, 2-H), 6.95–6.81
(m, 2H, 5- and 6-H), 6.12–6.00 (m, 1H, -CH=CH2), 5.23–5.14 (m, 2H, -CH2CH), 3.50 (d, 2H, J = 6.0 Hz,
-CH=CH2). HRMS [M + H]+ calcd. 347.0871; found 347.0870.
1
3-Allyl-4′-chloro-3′-(trifluoromethyl)-[1,1′-biphenyl]-4-ol (4o). Yield 18.9%; HPLC: 97.5%. H-NMR
(CDCl3): δ 7.93 (s, 1H, 3′-H), 7.41–7.36 (m, 2H, 2′- and 6′-H), 7.18–7.14 (m, 2H, 2- and 6-H), 7.05–7.01
(m, 1H, 5-H), 6.16–5.99 (m, 1H, -CH=CH2), 5.25–5.16 (m, 2H, -CH2CH), 3.54 (d, 2H, J = 6.4 Hz,
-CH=CH2). HRMS [M + Na]+ calcd. 335.0426; found 335.0422.
3-Allyl-4′-fluoro-[1,1′-biphenyl]-4-ol (4p). Yield 23.9%; HPLC: 99.0%. 1H-NMR (CDCl3): δ 7.50–7.46
(m, 2H, 2- and 6-H), 7.32–7.30 (m, 2H, 2′- and 6′-H), 7.11–7.07 (m, 2H, 3′- and 5′-H), 6.88 (d, 1H,
J = 8.4 Hz, 5-H), 6.11–6.00 (m, 1H, -CH=CH2), 5.23–5.18 (m, 2H, -CH=CH2), 5.05 (s, 1H, -OH), 3.47
(d, 2H, J = 4.8 Hz, -CH2CH); HRMS [M + H]+ calcd. 229.1029; found 229.1022.
1
3-Allyl-3′,5′-difluoro-[1,1′-biphenyl]-4-ol (4q). Yield 34.8%; HPLC: 98.6%. H-NMR (CDCl3): δ
7.35–7.31 (m, 2H, 2- and 6-H), 7.08–7.03 (m, 2H, 2′- and 6′-H), 6.89 (d, 1H, J = 8.0 Hz, 5-H), 6.76–6.69
(m, 1H, 4′-H), 6.10-5.97 (m, 1H, -CH=CH2), 5.24-5.18 (m, 2H, -CH=CH2), 5.07 (s, 1H,-OH), 3.48
(d, 2H, J = 6.0 Hz, -CH2CH); HRMS [M + H]+ calcd. 269.0754; found 269.0749.
1
N-(3′-Allyl-4′-hydroxy-[1,1′-biphenyl]-3-yl)acetamide (4r). Yield 56.9%; HPLC: 99.2%. H-NMR
(DMSO-d6) δ 9.97 (s, 1H, -NH), 9.54 (s, 1H, -OH), 7.74 (s, 1H, 2′-H), 7.52 (d, 1H, J = 8.4 Hz, 4′-H),
7.32–7.28 (m, 2H, 2- and 6-H), 7.20 (d, 1H, J = 8.0 Hz, 5′-H), 6.89–6.87 (m, 1H, 5-H), 6.05–5.95 (m,
1H, -CH=CH2), 5.11–5.02 (m, 2H, -CH2CH), 3.36–3.34 (m, 2H, -CH=CH2), 2.05 (s, 3H, -CH3); HRMS
[M + H]+ calcd. 268.1338; found 268.1347.
1
N-(3′-Allyl-4′-hydroxy-[1,1′-biphenyl]-3-yl)methanesulfonamide (4s). Yield 20.9%; HPLC: 99.7%. H-
NMR (CDCl3): δ 7.41–7.33 (m, 5H, Ar-H), 7.18–7.16 (m, 1H, 2′-H), 6.89 (d, 1H, J = 8.0 Hz, 5-H), 6.74
(s, 1H, -NH), 6.05 (m, 1H, -CH=CH2), 5.22–5.17 (m, 2H, -CH2CH), 3.47 (d, 2H, J = 6.4 Hz, -CH=CH2),
3.04 (s, 3H, -CH3); HRMS [M + Na]+ calcd. 326.0827; found 326.0829.
3.1.4. Syntheses of 5a–c followed the general procedure
3-(2-Methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c). Yield 19.5%; HPLC:
98.9%. 1H-NMR (CDCl3): δ 7.95 (s, 1H, 4′-H), 7.41–7.37 (m, 1H, Ar-H), 6.95–6.91 (m, 3H, Ar-H), 6.73

Molecules 2012, 17
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(d, 1H, J = 8.0 Hz, 5-H), 6.11–5.97 (m, 1H, -CH=CH2), 5.07 (s, 1H, -CH=CH2), 4.83 (s, 1H, -CH=CH2),
1.74 (s, 3H, -CH3), 1.71 (s, 3H, -CH3). HRMS [M + H]+ calcd. 378.1184; found 378.1180.
3.1.5. Syntheses of 6a–j Following Step III of the General Procedure
1
3′-Allyl-3,4,4,5′-tetramethoxy-1,1′-biphenyl (6c). Yield 97.5%; HPLC: 98.4%. H-NMR (CDCl3):
δ 7.39–7.37 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz, 2-H), 7.32 (d, 1H, J = 2.4 Hz, 6-H), 6.91 (d, 1H, J = 8.4 Hz,
5-H), 6.72 (s, 2H, 2′- and 6′-H), 6.08–5.98 (m, 1H, -CH=CH2), 5.12–5.05 (m, 2H, -CH=CH2), 3.93 (s,
6H, -CH3), 3.88 (s, 3H, -CH3), 3.87 (s, 3H, -CH3), 3.44 (d, 2H, J = 6.4 Hz, -CH2CH). HRMS [M + Na]+
calcd. 337.1416; found 337.1323.
1
3-Allyl-4-(allyloxy)-3′,5′-bis(trifluoromethyl)-1,1′-biphenyl (6f). Yield 95.8%; HPLC: 97.9%. H-NMR
(CDCl3): δ 8.10 (s, 1H, 4′-H), 7.55–7.50 (m, 2H, 2′- and 6′-H), 7.35–7.31 (m, 1H, 2-H), 7.05–7.00
(m, 2H, 5- and 6-H), 6.15–6.00 (m, 2H, -CH=CH2 and -OCH2CH=CH2), 5.53–5.48 (m, 1H,
-OCH2CH=CH2), 5.41–5.36 (m, 1H, -OCH2CH=CH2), 5.26–5.19 (m, 2H, -CH=CH2), 4.65 (d, 2H,
J = 4.8 Hz, -OCH2CH=CH2), 3.54 (d, 2H, J = 6.4 Hz, -CH2CH); HRMS [M + H]+ calcd. 387.1184;
found 387.1180.
3-Allyl-4-((3-methylbut-2-en-1-yl)oxy)-3′,5′-bis(trifluoromethyl)-1,1′-biphenyl (6g). Yield 89.5%; HPLC:
98.5%. 1H-NMR (CDCl3): δ 8.01 (s, 1H, 4′-H), 7.45–7.40 (m, 2H, 2′- and 6′-H), 7.35–7.30 (m, 1H, 2-H),
7.08–7.02 (m, 2H, 5- and 6-H), 6.05–5.97 (m, 2H, -CH=CH2 and -OCH2CH=CH2), 5.50–5.45 (m, 1H,
-OCH2CH=CH2), 5.40–5.35 (m, 1H, -OCH2CH=CH2), 5.27–5.21 (m, 2H, -CH=CH2), 4.55 (d, 2H,
J = 4.8 Hz, -OCH2CH=CH2), 3.59 (d, 2H, J = 6.4 Hz, -CH2CH), 1.56 (s, 3H, -CH3), 1.53 (s, 3H, -CH3);
HRMS [M + H]+ calcd. 415.1497; found 415.1495.
1
N-(3′-Allyl-4′-methoxy-[1,1′-biphenyl]-3-yl)acetamide (6h). Yield 93.8%; HPLC: 99.5%. H-NMR
(DMSO-d6): δ 9.97 (s, 1H, -NH), 9.54 (s, 1H, 2′-H), 7.74 (s, 1H, 2-H), 7.52 (d, 1H, J = 8.4 Hz, 5′-H),
7.33–7.28 (m, 2H, 6- and 6′-H), 7.20 (d, 1H, J = 8.0 Hz, 4′-H), 6.89–6.87 (m, 1H, 4-H), 6.05–5.95 (m,
1H, -CH=CH2), 5.11–5.02 (m, 2H, -CH=CH2), 3.86 (s, 3H, 4′-OCH3), 3.36–3.34 (m, 2H, -CH2CH), 2.05
(s, 3H, -COCH3); HRMS [M+Na]+ calcd. 304.1313; found 304.1298.
3.1.6. Syntheses of 7a–d Following Step III of the General Procedure
1
3,5-Diallyl-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (7c). Yield 65.4%; HPLC: 98.4%. H-NMR
(CDCl3): δ 8.12 (s, 1H, 4′-H), 7.51-7.46 (m, 2H, 2′- and 6′-H), 7.30-7.24 (m, 1H, 2-H), 7.05–7.01 (m,
2H, 5- and 6-H), 6.16–6.03 (m, 2H, -CH=CH2), 5.29–5.15 (m, 4H, -CH=CH2), 4.65–4.43 (m, 4H,
-CH2CH); HRMS [M + H]+ calcd. 409.1003; found 409.0998.
3-Allyl-5-(2-methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (7d). Yield 65.4%;
1
HPLC: 97.4%. H-NMR (CDCl3): δ 8.09 (s, 1H, 4′-H), 7.48–7.44 (m, 2H, 2′- and 6′-H), 7.21–7.18 (m,
1H, 2-H), 7.10–7.06 (m, 2H, 5- and 6-H), 6.13–6.04 (m, 2H, -CH=CH2), 5.25–5.13 (m, 4H, -CH=CH2),
4.55–4.36 (m, 4H, -CH2CH), 1.66 (s, 3H, -CH3), 1.63 (s, 3H, -CH3); HRMS [M + H]+ calcd. 437.1316;
found 437.1315.

Molecules 2012, 17
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4. Conclusions
To date, suppression of angiogenesis-dependent tumor growth has been a widely accepted strategy
for cancer therapy. Although acquired drug resistance remains an insurmountable obstacle of tumor-
targeting therapy, it is unlikely to occur or at least at a low rate if the genetically stable ECs are
targeted. Thus, ECs have been proven to be an attractive and potent target for angiogenesis
therapy [21]. In the present study, 37 allylated biphenols represented a novel biphenyl structural motif
and possessed a unique mode of action in anti-angiogenic and anti-tumor activity. In detail,
3-(2-methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c) showed the strongest
inhibitory effects on the proliferation, migration and tube formation of HUVECs featuring
anti-angiogenic properties, and its average anti-proliferative activities against four tumor cell lines
(C26, Hela, A549, and HepG2) demonstrated that the biologically active allylated biphenol molecule
has dual functions and the inhibitory effect of 5c was specific on HUVEC migration and tube
formation, rather than resulting from its cytotoxicity.
Acknowledgment
We are grateful to the National Key Programs of China during the 11th Five-Year Plan Period
(2009ZX09102-045 and 2009ZX09501-015).
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Sample Availability: Samples of the compounds 4a–7d are available from the authors.
© 2012 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 license
(http://creativecommons.org/licenses/by/3.0/).