Synthesis and cytotoxicity studies of newly designed benzyl-hydroquinone derivatives

Article abstract of DOI:10.1007/s00044-017-2127-z

Abstract: A series of new benzyl-substituted hydroquinones has been prepared by short and flexible routes. Such molecules are simplified analogs of bioactive natural terpenes. Preliminary studies have established, for some of these derivatives, promising cytotoxicity data against human carcinoma cancer cell lines.

Full text of DOI:10.1007/s00044-017-2127-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2127-z
ORIGINAL RESEARCH
Synthesis and cytotoxicity studies of newly designed benzyl-
hydroquinone derivatives
Olivier Tasseau1 Paul Mosset1 Sophie Barillé-Nion2 Fabien Gautier2,3,4
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Philippe Juin2,3 Nicolas Levoin5 Niharika Amireddy6 Shasi Kalivendi6
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1
René Grée
Received: 21 September 2017 / Accepted: 9 December 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Graphical abstract
Abstract A series of new benzyl-substituted hydro-
quinones has been prepared by short and flexible routes.
Such molecules are simplified analogs of bioactive natural
terpenes. Preliminary studies have established, for some of
these derivatives, promising cytotoxicity data against
human carcinoma cancer cell lines.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2127-z) contains supplementary
material, which is available to authorized users.
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Keywords Hydroquinones Terpenes Cancer HCT cells
* Sophie Barillé-Nion
●
MDAMB231 cells MDAMB468 cells
sophie.barille@univ-nantes.fr
* René Grée
rene.gree@univ-rennes1.fr
1
Université de Rennes 1, Institut des Sciences Chimiques de
Rennes, CNRS UMR 6226, Avenue du Général Leclerc, 35042
Rennes Cedex, France
Introduction
2
UMR 892 INSERM/6299 CNRS/Université de Nantes, Team
Hydroquinones are common components of bioactive nat-
ural products, as well as privileged scaffolds in bioorganic
and medicinal chemistry. Representative examples are the
terpene-hydroquinones and sesquiterpene-hydroquinones
(Bertanha et al. 2014), generally isolated from marine
sponges (Gordaliza 2012), such as Avarol 1, Isozonarol 2,
Neoavarol 3 (Perez-Garcia et al. 2005) and Yahazunol 4 or
Zonarol 5 (Laube et al. 2005). Other, more complex,
bioactive meroterpene-hydroquinones have also been
described recently with linear chains (Menna et al. 2013) or
bicyclic systems, such as the Fascioquinols (for instance 6)
(Zhang et al. 2011) or the Strongylophorines such as 7
(Noda et al. 2015; Lee et al. 2015; Abdjul et al. 2016) as
8 “Cell Survival and Tumor Escape in Breast Cancer”, Institut de
Recherche Thérapeutique de l’Université de Nantes, 8 quai
Moncousu, BP 70721, 44007 Nantes Cedex 1, France
3
Institut de Cancérologie de l’Ouest, Centre de Lutte contre le
Cancer René Gauducheau, Boulevard Jacques Monod, 44805
Saint Herblain-Nantes Cedex, France
4
Plateforme IMPACT®, Biogenouest Institut de Recherche
Thérapeutique de l’Université de Nantes, 8, quai Moncousu, BP
70721 44007 Nantes Cedex 1, France
5
Bioprojet-Biotech, 4 rue du Chesnay Beauregard, BP 96205,
35762 Saint Grégoire, France
6
CSIR-Indian Institute of Chemical Technology, Uppal Road,
Hyderabad 500007 Telangana, India

Med Chem Res
HO
HO
HO
HO
OH
OH
OH
OH
OH
1
3
4
2
Avarol
HO
Isozonarol
Neoavarol
Yahazunol
OH
HO
HO
OH
OH
H
OH
7
H
5
6
H
H
O
O
Strongylophorine-8
Zonarol
Fascioquinol B
Fig. 1 Representative examples of bioactive sesquiterpene and meroterpenes isolated from marine sponges
Therefore, it appears of interest to design new, and more
Table 1 Calculated properties of terpene-hydroquinones
simple, molecules inspired from such structures and to
study their biological properties in particular for their
cytotoxic and antiproliferative activities (Simon-Levert
et al. 2010). In continuation with our research dealing with
the anticancer properties of hydroquinones and quinones
designed from embelin, a known XIAP inhibitor (Viault
et al. 2011, 2013), we became interested in molecules such
as 8 and 11 having an hydroquinone connected to various
types of benzylic or polyaromatic entities (Fig. 2). Such
species could be considered as very simplified analogs of
previous bioactive terpenes and compound 11b for instance
has a SAscore = 0.31, together with a QED = 0.67. In a few
cases, to extend the biological structure–activity relation-
ships studies in these series, we have considered the cor-
responding dimethylethers 9 as well as the quinones 10. Up
to now, few type-8 molecules have been prepared (Ozaki
et al. 1997; Sampath Kumar et al. 1999) and only one group
reported some anticancer properties for four of them,
through inhibition of the sarco/endoplasmic reticulum cal-
cium ATPase (Paula et al. 2009; Deye et al. 2009).
QED
SAscore
Avarol 1
0.54
0.52
0.55
0.69
0.54
0.43
0.47
0.67
0.05
Isozonarol 2
Neoavarol 3
Yahazunol 4
Zonarol 5
0.05
0.06
0.04
0.06
Fascioquinol B
Strongylophorin-8
11b
−0.01
−0.08
0.31
QED scores drug-likeness, with mean QE = 0.67 for favorable
molecules. SAscore quantizes the chemical accessibility, normalized
to [−1, +1] with higher values indicating easier to synthesize
described in Fig. 1. This family of terpenes-hydroquinones,
and corresponding quinones, has already demonstrated an
impressive range of bioactivities including cytotoxic
(Müller et al. 1985; Crispino et al. 1989; De Guilio et al.
1990; Pereira et al. 2015), anti-inflammatory (Ferrandiz
et al. 1994), and anti-HIV properties (Sarin et al. 1987).
They also act as inhibitors for receptors of protein kinases
(Yoo et al. 2003), and interleukin-8 (Patil et al. 1997).
Although these molecules are associated with many
promising biological properties, their drug-likeness is rather
low, as judged from their QED score (Bickerton et al. 2012)
(Table 1). This is unfortunately not rare for natural products
whose chemical complexity, further, makes their supply
difficult. These terpenes-hydroquinones are usually isolated
in very small amounts and their total synthesis represents
significant challenges (Bernet et al. 2003), as confirmed by
their calculated synthetic accessibility (SAscore, Table 1)
(Ertl and Schuffenhauer 2009).
The goal of this publication is first to describe simple and
flexible strategies to prepare the selected molecules 8–11.
The second goal is to report preliminary biological studies
performed with these molecules and we focused mainly on
anticancer properties.
Results and discussion
The synthesis of the first generation of molecules followed
literature procedures (Scheme 1). Condensation of 1,4

Med Chem Res
HO
Fig. 2 Design of the target
molecules 8–11
OH
Terpene part
HO
analogues
generation 1
MeO
O
OMe
9
OH
8
O
R1
R1
R1
10
analogues
generation 2
HO
OH
11
R2
HO
Scheme 1 Synthesis of the first
generation of target molecules
OHC
13
O
LiCl
+
R1
OH
a: R1= H
Py, Δ
b: R1= 2-Br
c: R1= 3-Br
d: R1= 4-Br
O
R1
90%
8
12
MeO
O
MeI, K2CO3
DMF
MnO2
Toluene
OMe
8
O
90%
90%
R1
R1
9
10
a: R1= H
a: R1= H
b: R1= 2-Br
c: R1= 3-Br
d: R1= 4-Br
b: R1= 2-Br
c: R1= 3-Br
d: R1= 4-Br
cyclohexanedione 12 with various aromatic aldehydes 13a–
d were carried out in the presence of LiCl, in pyridine at
reflux, affording the desired 2-benzyl hydroquinones targets
8a–d in good yields (Ozaki et al. 1997; Sampath Kumar
et al. 1999; de Abreu Lima et al. 2016). In order to explore
more systematically the biological properties of these
molecules, we prepared also the bis-methylethers and the
corresponding quinones. Therefore for the first series of
molecules, bismethylation was performed by a standard
method using MeI and K2CO3 in N,N-dimethylformamide
(DMF) to give the desired bisethers 9a–d in good yields
(Wessig and Möllnitz 2008; Paduraru et al. 2008). For the
second series, oxidation with MnO2 in presence of a
desiccant in toluene has been found to be the method of
choice. It gave the quinones 10a–d in a cleaner form than
oxidation by ceric ammonium nitrate (Passerieux et al.
1989) or DDQ (Singh and Turner 1972). All these deriva-
tives 8, 9, and 10 have been fully characterized by their
spectral and analytical data.
These compounds were first evaluated for their anti-
proliferative activity against the human colorectal carci-
noma cell line HCT116, using the cell viability assay based
on tetrazolium reduction, and the results are given in Fig. 3.
Among these compounds, two molecules, one hydro-
quinone 8b and one bis-ether 9a displayed significant effect
on this cancer cell line since both compound decreased cell
proliferation (35%) after 48 h treatment at 10 μM. On the
other hand, the quinones 10 were inactive in this test in the
same settings.

Med Chem Res
Therefore, we selected the bromo hydroquinone deriva-
tives 8b–8d as starting points to design and prepare a sec-
ond generation of compounds by Suzuki-type reactions
(Scheme 2), in line with our previous work on phenolic
derivatives (Viault et al. 2011). So, coupling reactions of
brominated 2-benzyl hydroquinones with boronic acids
were carried out in a dioxane/water mixture with Na2CO3 as
the base and in the presence of Pd(PPh3)4 at 5% (Gan et al.
2009; Song et al. 2005). Starting from the ortho-8b, the
meta-8c and the para-derivative 8d, the desired products
11b–11d were obtained in fair to good yields. The structure
of all these new molecules 11 were confirmed by their
spectral and analytical data.
These second-generation compounds were also evaluated
for their antiproliferative activity against the human color-
ectal carcinoma cell line HCT116, as previously described.
Among them, three molecules 11b, 11c-1, and 11c-2 dis-
played significant antiproliferative effect at a concentration
of 10 µM on HCT116 cell line, the molecule 11b showing
the strongest effect (Fig. 4).
1.0
Since the ortho derivative 11b showed a very promising
cytotoxic activity, we chose to prepare and screen a com-
plete series of close analogs (12b) with various substituents
(Cl, Me, OMe, OPh) in the three positions around the
second aromatic ring. Their syntheses, using again Suzuki-
type reactions, are reported in Scheme 3. All these deriva-
tives have spectral and analytical data consistent with their
structures.
Among this series of compounds, only molecules 12b-3,
12b-11, and 12b-12 significantly decreased cell viability in
HCT116 cells when used at 10 μM (Fig. 5). However, these
compounds were not more active than 11b on this cell line.
**
***
0.5
0.0
8a 8b 8c 8d 9a 9b 9c 9d 10a 10b 10c 10d
Fig. 3 Effects of the first series of compounds on cancer cell’s via-
bility. All compounds were tested on HCT116 cancer cells using 10
μM for 48 h. Cell viability was then assessed using the MTT assay.
DMSO 0.1% was used as negative control and all data were normal-
ized using this control (DO ratio = 1). Experiments were performed
three times. **p < 0.01; ***p < 0.001
Scheme 2 Synthesis of the
second generation of target
molecules
HO
HO
PhB(OH)2 (2 equiv)
Na2CO3 (2 equiv)
OH
OH
Pd(PPh3)4 (5 mol%)
Br
dioxane/water 5/1
11b
80°C, ca. 15 h
8b
62%
HO
HO
R2
B(OH)2
OH
OH
11c-1; R2=H
Na2CO3 / Pd(PPh3)4 cat.
dioxane/water 5/1
11c-2; R2=3-OH
80°C
8c
Br
11c-3; R2=3-CHO
R2
HO
HO
R2
B(OH)2
OH
OH
Na2CO3 / Pd(PPh3)4 cat.
dioxane/water 5/1
80°C
8d
11d-1; R2=H
Br
11d-2; R2=3-OH
11d-3; R2=3-CHO
R2

Med Chem Res
Embelin and analogs are well known as potent inhibitors
of the XIAP protein (Nikolovska–Coleska et al. 2004; Chen
et al. 2006; Viault et al. 2011, 2013). Therefore, we
attempted to define whether the antiproliferative effect
exerted by some of the compounds above was dependent on
their putative molecular target XIAP. Thus, we used the
XIAP KO isogenic cell line HCT116 (obtained from B.
Vogelstein, Baltimore, MD USA), in parallel to its parental
cell line HCT116 in MTT experiments. However, we could
not detect any difference between these cell lines, arguing
against a role of XIAP in the antiproliferative effects
observed (data not shown).
We completed the analysis in using two breast cancer
cell lines MDAMB231 and MDAMB468 in proliferation
and cell death assays. Our data indicate that the molecule 8b
efficiently decreased proliferation as observed in HCT116
cells (Figs. 3, 6). Interestingly, treating these breast cancer
cells with 11d-1 and 11d-2 also led to significant decrease
of proliferation, as compared to HCT116 cells for which no
effect could be detected. In contrast, the molecule 9a had no
effect on breast cancer cells as compared to HCT116 cells.
These results suggest that the biological effect triggered by
some of these molecules may depend on cancer cells
background such as oncogenic pathways that sustain their
proliferation.
performed cell death assays using Annexin V staining
procedure (Fig. 7). We pointed out that these three mole-
cules were able to trigger apoptosis in breast cancer cells.
This proapoptotic activity turned out to be particularly
effective in MDAMB468.
Finally, we considered also another possible option for
the mechanism of action of such molecules. It has been
demonstrated that the strongylophorines function as protea-
some inhibitors (Noda et al. 2015). Therefore, it appeared of
interest to check if compounds 12b-1 to 12b-12, as well as
11b the most active in previous experiments, could have a
similar action. Proteasome activity assay demonstrated that
the compounds 12b-1, 12b-3 to 12b-6 exhibited a small, but
significant, inhibition against the chymotrypsin-like activity
of proteasome (Fig. 8). Though 12b-2 also inhibited the
activity to similar extent, the values were not significant.
However, none of the above compounds were found to
inhibit the trypsin-like activity of the proteasome.
Conclusion
New benzylic analogs of sesquiterpene/meroterpene-
hydroquinones have been prepared by short and efficient
routes. A few of them have demonstrated promising cyto-
toxic properties, in particular, 11b against the colon cancer
cell line HCT116, while 11d-1 and 11d-2 exhibited good
antiproliferative and proapoptotic effects against the MDA-
MB468 and the MDA-MB231 breast cancer cell lines. This
type of benzylic hydroquinones and quinones constitues a
new pattern for further development but more studies are
required in order to identify the target(s) at the molecular
level and the mechanism(s) of action of these derivatives.
To further characterize the effects of molecules 8b, 11d-
1, and 11d-2 selected from previous experiments, we
1.0
*
*
0.5
0.0
****
Experimental part
11b
11c-1
11c-2
11c-3
11d-1
11d-2
11d-3
Chemical synthesis
Fig. 4 Effects of the second series of compounds on cancer cell’s
viability. All compounds were tested on HCT116 cancer cells using
10 μM for 48 h. Cell viability was then assessed using the MTT assay.
DMSO 0.1% was used as negative control and all data were normal-
ized using this control (DO ratio = 1). Experiments were performed
three times. *p < 0.05; ****p < 0.0001
General remarks for chemistry section
All reagents and solvents were purchased from Acros
Fisher, Sigma-Aldrich, Alfa Aesar, and Fluka France and
HO
R2
HO
12b-7; R2= 2-OMe
12b-8; R2= 3-OMe
12b-9; R2= 4-OMe
12b-10; R2= 2-OPh
12b-11; R2= 3-OPh
12b-12; R2= 4-OPh
12b-1; R2= 2-Cl
12b-2; R2= 3-Cl
12b-3; R2= 4-Cl
12b-4; R2= 2-Me
12b-5; R2= 3-Me
12b-6; R2= 4-Me
B(OH)2
R2
OH
OH
Br
Na2CO3 / Pd(PPh3)4 cat.
dioxane/water 5/1
130°C
8b
Scheme 3 Synthesis of a focused library of 12 analogs of 11b

Med Chem Res
Fig. 5 Effects of the third series
of compounds on cancer cell’s
viability. All compounds were
tested on HCT116 cancer cells
using 10 μM for 48 h. Cell via-
bility was then assessed using
the MTT assay. DMSO 0.1%
was used as negative control and
all data were normalized using
this control (DO ratio = 1).
Experiments were performed
three times
1.0
0.5
0.0
12b-1 12b-2 12b-3 12b-4 12b-5 12b-6 12b-7 12b-8 12b-9 12b-10 12b-11 12b-12
MDAMB468
MDAMB231
100
100
*
**
***
****
***
50
50
****
0
0
8b
9a
11b
11c-1 11c-2 11d-1 11d-2
8b
9a
11b
11c-1 11c-2 11d-1 11d-2
using DMSO 0.1% as negative control. All data were normalized using
this control. Experiments were performed three times. **p < 0.01;
***p < 0.001; ****p < 0.0001
Fig. 6 Effects of selected compounds on cancer cell’s viability using
MDAMB468 and MDAMB231 breast cancer cell lines after a 48 h
treatment with 10 μM. Cell viability was assessed using the MTT assay
MDAMB468
MDAMB231
Fig. 7 Proapoptotic effect of
100
80
60
40
20
0
selected compounds on the
breast cancer cell lines
100
80
60
40
20
0
****
***
**
MDAMB468 and MDAMB231.
The Annexin V cell death assay
was carried out after 48h-
treatment with 10 μM of com-
pounds. DMSO 0.1% was used
as a negative control. Included
data indicate % of dying and
died cancer cells after treatment.
Experiments were performed
three times and means and
standard errors are shown. *p <
0.05; **p < 0.01; ***p < 0.001;
****p < 0.0001
*
DMSO
8b
11d-1 11d-2
DMSO
8b
11d-1 11d-2
were used without further purification. All the reactions
requiring anhydrous conditions were carried out under inert
atmosphere (argon or nitrogen) using glassware previously
dried in an oven. The solvents used were freshly distilled
under nitrogen according to the standard procedures
described in the literature such as diethyl ether (Et2O) was
distilled over sodium/benzophenone; dichloromethane
(CH2Cl2) and toluene were distilled over calcium hydride.
The reaction mixtures were magnetically stirred with Teflon
stirring bars, and the temperatures were measured
externally. Reactions were monitored by thin-layer chro-
matography (TLC) performed on 0.2-mm precoated plates
of silica gel 60 F-254 (Merck) with appropriate eluent.
Detection was carried out with ultraviolet light (254 and
365 nM) and/or p-anisaldehyde staining solution. Merck
silica gel (60 Å, 40–63 μM, 230–400 Mesh particule sizes)
was mainly used for the column chromatography. Solvents
were evaporated with a Büchi rotary evaporator.
Nuclear magnetic resonance spectra were recorded on
Bruker Avance 300, 400, and 500 spectrometers, with multi-

Med Chem Res
Fig. 8 The effect of compounds
12 and 11b on proteasome
activity was measured using the
AMC-conjugated peptide sub-
strates
for
trypsin
and
chymotrypsin-like activities of
the 20s proteasome in the pre-
sence or absence of test com-
pounds (10 μM) or the positive
control, bortezomib (Bort, 1 μM)
as described in the “Materials
and methods” section. Data are
the mean SD of three samples
(n = 3) and the experiment has
been performed two times. *p <
0.01 as compared to controls
core probes (BBO-type double and TBI-type triple probes).
For 1H NMR spectra, chemical shifts δ (H) are expressed in
parts per million downfield from tetramethylsilane (TMS),
and deuterated solvent peaks using [δ (CDCl3) = 7.26 ppm,
δ (DMSO-d6) = 2.50 ppm, δ (Acetone-d6) = 2.05 ppm, δ
(CD3OD) = 3.31 ppm] as internal references. For 13C NMR
spectra (75 MHz), δ (C) also, are given in ppm relative to
TMS, using [δ (CDCl3) = 77.16 ppm, δ (DMSO-d6) =
39.52 ppm, δ (Acetone-d6) = 29.84 ppm, δ (CD3OD) =
49.00 ppm] as internal references (Gottlieb et al. 1997). So,
data are given in the following order: δ value; then multi-
plicities were designated as following: singlet (s), doublet
(d), triplet (t), quadruplet (q), multiplet (m), or br (broad),
then the number of atoms (protons), and the coupling con-
stants J are given in Hertz. Mass spectral analyses have been
performed at the Centre Régional de Mesures Physiques de
l’Ouest (CRMPO, SFS, ScanMAT) in Rennes (France). The
high-resolution mass spectra were recorded in positive mode
using direct electrospray ionization, respectively, on a
Waters Q-Tof 2 (ESI-MS/ASAP-MS) and on a Thermo
Fisher Scientific Q-Exactive spectrometers. The thermal
behavior of compounds was studied using Kofler bench
apparatus and melting points were determined. Measures
were performed in triplicate, after calibration with a standard
(control) substance to determine an average value without
other correction.
monitored by TLC. After completion of the reaction, the
mixture was cooled to room temperature and poured in to an
ice cold water (100 mL). Reaction crude was extracted three
times with Et2O (3 × 150–200 mL). The organic layers
were combined, washed with 0.5 N hydrochloric acid
solution and with brine. The resulting organic phase was
dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure. The crude products
were purified by flash chromatography on a silica-gel
(Fluka, 60 Å, 40–60 μm, 230–400 Mesh) column with
gradient elution with 10–30% EtOAc in Pentane (EtOAc:
ethyl acetate) and recrystallization in chloroform (CHCl3) to
afford compound 8a–d as solid powder.
2-Benzylbenzene-1,4-diol (8a) Compound 8a was pre-
pared from cyclohexane-1,4-dione 12 (3 g, 26.76 mmol)
with benzaldehyde 13a (2.72 mL, 26.76 mmol) in presence
of lithium chloride (1.13 g, 26.76 mmol) in pyridine (11 mL),
according to the standard procedure. The obtained brown
ocher oil was purified by chromatography column and
recrystallization from CHCl3. The compound was obtained
as a white solid which takes a pink tint if exposed to the
ambient air (3.80 g, 71%); mp 105 °C; Rf: 0.40 (Pentane/
EtOAc: 9/1). The spectral data of 8a are in agreement with
literature (Ozaki et al. 1997; Sampath Kumar et al. 1999).
2-(2-Bromobenzyl)benzene-1,4-diol (8b) Compound 8b
was prepared from cyclohexane-1,4-dione 12 (4 g, 35.67
mmol) with 2-bromobenzaldehyde 13b (4.16 mL, 35.67
mmol) in presence of lithium chloride (1.51 g, 35.67 mmol)
in pyridine (15 mL). The crude compound 8b was obtained
as wine red viscous liquid, which was purified by silica-gel
column chromatography (60–120 Mesh) as described
above. After recrystallization from chloroform, the ortho-
substituted compound was obtained as a brown solid (6.04 g,
61%); mp 126 °C; Rf: 0.35 (Pentane/EtOAc: 9/1). The
Standard procedure for the preparation of 2-
benzylhydroquinones, compounds 8a–d
In a glass three-neck round-bottom flask, cyclohexane-1,4-
dione 12 (1 equiv), benzaldehyde derivatives (1 equiv) 13a–
d, and lithium chloride (1 equiv) were dissolved in pyridine.
The reaction medium was placed under an inert atmosphere,
with magnetic stirring. The reaction temperature was slowly
raised to reflux and stirred for 2 h. Reaction mixture was

Med Chem Res
spectral data of 8b are in agreement with literature (de
Abreu Lima et al. 2016).
washed with water and saturated NaCl solution, several
times. The resulting organic phase was dried over anhy-
drous magnesium sulfate, filtered, and concentrated under
reduced pressure. The crude product was purified by chro-
matography on a silica-gel column with gradient elution
from EtOAc: 5–20% in Pentane as described in general
remarks and the desired compound 9a–d was obtained as
colorless or brown oil.
2-(3-Bromobenzyl)benzene-1,4-diol (8c) Compound 8c
was prepared from cyclohexane-1,4-dione 12 (4 g, 35.67 mmol)
with 3-bromobenzaldehyde 13c (4.16 mL, 35.67 mmol)
in presence of lithium chloride (1.51 g, 35.67 mmol) in
pyridine (15 mL), according to the standard procedure. The
obtained brown ocher oil is purified on silica gel column
chromatography (60–120 Mesh) as described above. After
recrystallization from chloroform, the meta-substituted
compound was obtained as a brown solid (6.44 g, 65%); mp
2-Benzyl-1,4-dimethoxybenzene (9a) Compound 9a was
prepared from 2-benzylbenzene-1,4-diol 8a (0.8 g, 4 mmol)
and K2CO3 (3.31 g, 24 mmol) dissolved in DMF (7 mL) and
methyl iodide was added (3.6 mL, 16 mmol), according to
the standard procedure. After purification on silica gel
column, the product 9a was obtained as colorless oil (0.65
1
120 °C; Rf: 0.5 (Pentane/AcOEt: 9/1); H NMR (300 MHz,
DMSO-d6) δ: 8.71 (s, 1H, OH), 8.59 (s, 1H, OH), 7.36 (m,
2H, H-12, H-13), 7.23 (broad s, 1H, H-11), 7.21 (broad s,
1H, H-15), 6.61 (dm, J = 8.1 Hz, 1H, H-1), 6.46 (s, 1H, H-
2), 6.43 (d with a roof effect, J = 2.9 Hz, 1H, H-4), 3.79 (s,
2H, CH2); 13C NMR (75 MHz, DMSO-d6) δ: 149.77 (Car-
OH, C-6), 147.29 (Car-OH, C-3), 144.31 (Cquat, C-10),
131.21 (Car-H, C-13), 130.33 (Car-H, C-15), 128.50 (Car-H,
C-12), 127.77 (Car-H, C-11), 127.23 (Car-H, C-5), 121.47
(Car-Br, C-13), 116.82 and 115.72 (2Car-H, C-1, C-2),
113.71 (Car-H, C-4), 34.93 (CH2, C-9); HRESIMS (ASAP)
m/z (pos): [M]+ 277.9942 C13H11O279Br (calcd. 277.9942),
[M + H]+ 279.0021 C13H12O279Br (calcd. 279.0020).
1
g, 72%); Rf: 0.81 (Pentane/EtOAc: 8/2); H NMR (300
MHz, DMSO-d6) δ: 7.29–7.12 (m, 5H, H-11, H-12, H-13,
H-14, H-15), 6.89 (d, J = 8.7 Hz, 1H, H-1), 6.74 (dd with a
roof effect, J = 3.1, 8.7 Hz, 1H, H-2), 6.70 (broad d (small
coupling with CH2), J = 3.1 Hz, 1H, H-4), 3.86 (broad s,
2H, CH2), 3.71 (s, 3H, CH3), 3.65 (s, 3H, CH3); 13C NMR
(75 MHz, DMSO-d6) δ: 153.03 (Car–OCH3, C-6), 151.04
(Car–OCH3, C-3), 140.69 (Cquat, C-10), 130.17 (Car-H, C-
13), 128.55 (2Car-H, C-12, C-14), 128.15 (2Car-H, C-11, C-
15), 125.70 (Cquat, C-5), 116.51 (Car-H,C-4), 111.74 and
111.32 (2Car-H, C-1, C-2), 55.78 (OCH3, C-16), 55.22
(OCH3, C-17), 35.26 (CH2, C-9); HRESIMS (ASAP) m/z
(pos): 228.1161 [M]+ C15H16O2 (calcd. 228.1150),
229.1230 [M + H]+ C15H17O2 (calcd. 229.1228).
2-(4-Bromobenzyl)benzene-1,4-diol (8d) Compound 8d
was prepared from cyclohexane-1,4-dione 12 (3 g, 26.76 mmol)
with 4-bromobenzaldehyde 13d (4.95 g, 26.76 mmol) in
presence of lithium chloride (1.13 g, 26.76 mmol) in pyr-
idine (11 mL), according to the standard procedure. The
obtained beige oil is purified on silica gel column as
described above. After recrystallization from chloroform,
the para-substituted compound was obtained as a white
solid powder (5.2 g, 70%); mp 117 °C; Rf: 0.45 (Pentane/
EtOAc: 9/1). The spectral data of 8d are in agreement with
literature (de Abreu Lima et al. 2016).
2-(2-Bromobenzyl)-1,4-dimethoxybenzene
(9b) Com-
pound 9b was prepared from 2-(2-bromobenzyl)benzene-
1,4-diol 8b (4 g, 14.4 mmol) and K2CO3 (11.9 g, 86.4
mmol) dissolved in DMF (36 mL) and methyl iodide was
added (3.6 mL, 57.6 mmol), according to the standard pro-
cedure. After purification on silica gel column, the product
9b was obtained as a brown red oil (4.2 g, 95%); Rf: 0.80
1
(Pentane/EtOAc: 8/2); H NMR (300 MHz, DMSO-d6) δ:
7.61 (dd, J = 7.9, 1.3 Hz, 1H, H-14), 7.29 (ddd, J = 7.6,
7.4, 1.3 Hz, 1H, H-12), 7.16 (ddd, J = 7.8, 7.4, 1.7 Hz, 1H,
H-13), 7.09 (dd, 1H, H-11, J = 7.6, 1.7 Hz), 6.92 (d with
roof effect, J = 8.9 Hz, 1H, H-1), 6.79 (dd, J = 8.8, 3.1 Hz,
1H, H-2), 6.49 (d, J = 3.1 Hz, 1H, H-4), 3.95 (broad s, 2H,
CH2), 3.72 (s, 3H, CH3), 3.63 (s, 3H, CH3); 13C NMR (75
MHz, DMSO-d6) δ: 153.01 (Car–OCH3, C-6), 151.18
(Car–OCH3, C-3), 139.27 (Cquat, C-10), 132.38 (Car-H, C-
14), 130.79 (Car-H, C-13), 128.15 (2Car-H, C-11, C-12),
127.69 (Cquat, C-5), 124.15 (Car–Br, C-15), 116.36 (Car-H,
C-4), 111.60 and 111.38 (2Car-H, C-1, C-2), 55.75 (OCH3,
C-16), 55.18 (OCH3, C-17), 35.40 (CH2, C-9); HRESIMS
(ASAP) m/z (pos): 306.0254 C15H15O279Br [M]+ (calcd.
306.0255), 307.0323 [M + H]+ C15H16O279Br (calcd.
307.0333).
Standard procedure for the preparation of 2-benzyl-1,4-
dimethoxybenzene derivatives, compounds 9a–d
In a two-neck round-bottom flask, the corresponding 2-
benzylhydroquinone (or 2-benzylbenzene-1,4-diol) deriva-
tive 8a–d was dissolved in DMF. The anhydrous potassium
carbonate in large excess (6 equiv) was added with small
amount of DMF, and the reaction medium was placed under
nitrogen atmosphere. Then, the methyl iodide (4 equiv) was
poured dropwise in reaction mixture under vigorous mag-
netic stirring. The reaction was controlled by TLC and left
overnight at room temperature. Then, the crude mixture was
washed with water and extracted three times with CH2Cl2
(3 × 50 to 100 mL). The combined organic layers were

Med Chem Res
2-(3-Bromobenzyl)-1,4-dimethoxybenzene
(9c) Com-
distillated toluene was added to this reaction mixture which
was stirred for 15–20 min at room temperature until no
evolution was observed through TLC monitoring. Then, the
suspension was filtered through celite (R-545, Acros
Organics), washed with dichloromethane, and the filtrate
was concentrated under reduced pressure. The desired
compound 10a–d was obtained as red oil which was placed
in small volume of Et2O at −4 °C to 0 °C in order to give a
crystallized solid.
pound 9c was prepared from 2-(3-bromobenzyl)benzene-
1,4-diol 8c (3 g, 10.8 mmol) and K2CO3 (8.9 g, 64.8 mmol)
dissolved in DMF (27 mL) and methyl iodide was added
(2.7 mL, 43.2 mmol), according to the standard procedure.
After purification on silica gel column, the product 9c was
obtained as viscous brown oil (3.1 g, 94%); Rf: 0.85 (Pen-
tane/EtOAc: 8/2); 1H NMR (300 MHz, CDCl3) δ:
7.40–7.37 (m, 1H, H-15), 7.35–7.30 (m, 1H, H-13),
7.18–7.10 (m, 2H, H-11, H-12), 6.82 (d with a roof effect, J
= 8.8 Hz, 1H, H-1), 6.75 (dd, J = 8.8, 3.0 Hz, 1H, H-2),
6.68 (broad d, J = 3.0 Hz, 1H, H-4), 3.93 (broad s, 2H,
CH2), 3.78 (s, 3H, CH3), 3.75 (s, 3H, CH3); 13C NMR (75
MHz, DMSO-d6) δ: 153.08 (Car–OCH3, C-6), 151.01
(Car–OCH3, C-3), 143.71 (Cquat, C-10), 131.16 (Car-H, C-
13), 130.39 (Car-H, C-15), 129.37 (Car-H, C-12), 128.69
(Car-H, C-11), 127.69 (Cquat, C-5), 121.51 (Car–Br, C-14),
116.65 (Car-H, C-4), 111.86 and 111.65 (2Car-H, C-1, C-2),
55.81 (OCH3, C-16), 55.29 (OCH3, C-17), 34.95 (CH2, C-
9); HRESIMS (ASAP) m/z (pos): 306.0266 [M]+
C15H15O279Br (calcd. 306.0255), 307.0332 [M + H]+
C15H16O279Br (calcd. 307.0333).
2-Benzyl-1,4-benzoquinone (10a) Compound 10a was
prepared from 2-benzylbenzene-1,4-diol 8a (0.4 g, 2 mmol)
with manganese dioxide (0.14 g, 16 mmol) in distillated
toluene (10 mL), according to the standard procedure. The
desired compound 10a was obtained as a yellow-orange
crystallized solid (0.387 g, 97%); mp 44 °C; Rf: 0.50
(Pentane/EtOAc: 9/1). The spectral data of 10a are in
agreement with literature (Murahashi et al. 1996).
2-(2-Bromobenzyl)-1,4-benzoquinone (10b) Compound
10b was prepared from 2-(2-bromobenzyl)-benzene-1,4-
diol 8b (0.2 g, 0.72 mmol) with manganese dioxide (0.5 g,
5.7 mmol) in distillated toluene (5 mL), according to the
standard procedure. The ortho-substituted desired product
10b was obtained as a brown orange oil (0.177 g, 89%); Rf:
0.40 (Pentane/EtOAc: 9/1); 1H NMR (300 MHz, DMSO-d6)
δ: 7.65 (dm, J = 7.7 Hz, 1H, H-14), 7.41–7.32 (m, 2H, H-
11, H-13), 7.24 (ddd, J = 7.9, 6.3, 2.7 Hz, 1H, H-12), 6.94
(d with a roof effect, J = 10.1 Hz, 1H, H-1), 6.86 (dd with a
roof effect, J = 10.1, 2.5 Hz, 1H, H-2), 6.13 (dt, J = 2.5,
1.7 Hz, 1H, H-4), 3.84 (d, J = 1.7 Hz, 2H, CH2); 13C NMR
(75 MHz, DMSO-d6) δ: 187.38 (C=O, C-6), 186.72 (C=O,
C-3), 146.64 (Cquat, C-5), 136.75 and 136.37 (2Car-H,C-1,
C-2), 136.32 (Cquat, C-10), 132.77 (Car-H, C-14), 132.72
(Car-H, C-4), 131.67 (Car-H, C-13), 129.06 (Car-H, C-11),
128.05 (Car-H, C-12), 124.07 (Car-Br, C-15), 34.53 (CH2,
C-9); HRESIMS (ASAP) m/z (pos): 276.9864 [M + H]+
C13H10O279Br (calcd. 276.9864).
2-(4-Bromobenzyl)-1,4-dimethoxybenzene
(9d) Com-
pound 9d was prepared from 2-(3-bromobenzyl)benzene-
1,4-diol 8d (5 g, 18 mmol) and K2CO3 (14.9 g, 108 mmol)
dissolved in DMF (45 mL) and methyl iodide was added
(4.4 mL, 72 mmol), according to the standard procedure.
After purification on silica gel column, the product 9d was
obtained as a purple oil (5.05 g, 92%); Rf: 0.83 (Pentane/
1
EtOAc: 8/2); H NMR (300 MHz, CDCl3) δ: 7.37 (half of
an A2X2 system, 2H, H-12, H-14), 7.08 (half of an A2X2
system, 2H, H-11, H-15), 6.79 (d with a roof effect, J = 8.8
Hz, 1H, H-1), 6.72 (dd, J = 8.8, 3.0 Hz, 1H, H-2), 6.64
(broad d, J = 3.0 Hz, 1H, H-4), 3.89 (broad s, 2H, CH2),
3.76 (s, 3H, CH3), 3.73 (s, 3H, CH3); 13C NMR (75 MHz,
CDCl3) δ: 153.50 (Car–OCH3, C-6), 151.58 (Car–OCH3, C-
3), 139.85 (Cquat, C-10), 131.31 (2Car-H, C-12, C-14),
130.66 (2Car-H, C-11, C-15), 130.20 (Cquat, C-5), 119.65
(Car–Br, C-13), 116.84 (Car-H, C-4), 111.45 and 111.41
(2Car-H, C-1, C-2), 55.99 (OCH3, C-16), 55.67 (OCH3, C-
17), 35.56 (CH2, C-9); HRESIMS (ASAP) m/z (pos):
306.0254 [M]+ C15H15O279Br (calcd. 306.0255), 307.0324
[M + H]+ C15H16O279Br (calcd. 307.0333).
2-(3-Bromobenzyl)-1,4-benzoquinone (10c) Compound
10c was prepared from 2-(3-bromobenzyl)-benzene-1,4-diol
8c (0.57 g, 2 mmol) with manganese dioxide (1.39 g, 16
mmol) in distillated toluene (10 mL), according to the
standard procedure. The meta-substituted desired product
10c was obtained as a brown solid (0.33 g, 60%); mp 101 °
C; Rf: 0.50 (Pentane/EtOAc: 9/1); 1H NMR (300 MHz,
DMSO-d6) δ: 7.70–7.67 (m, 1H, H-13), 7.60 (dt, J = 7.0,
2.0 Hz, 1H, H-15), 7.47–7.32 (m, 2H, H-11, H-12), 6.99 (d
with a roof effect, J = 10.1 Hz, 1H, H-1), 6.93 (dd with a
roof effect, J = 10.1, 2.4 Hz, 1H, H-2), 6.71 (dt, J = 2.4,
1.4 Hz, 1H, H-4), 3.95 (d, J = 1.3 Hz, 2H, CH2); 13C NMR
(75 MHz, DMSO-d6) δ: 187.72 (C=O, C-6), 186.98 (C=O,
C-3), 147.26 (Cquat, C-5), 140.20 (Cquat, C-10), 136.66 and
Standard procedure for the preparation of 2-benzyl-
benzoquinone derivatives, compounds 10a–d
In a two-neck round-bottom flask, powdered manganese
dioxide (8 equiv) was added to previously dried under
vacuum 2-benzylhydroquinone derivative 8a–d with a few
molecular sieve beads. Under nitrogen atmosphere,

Med Chem Res
136.40 (2Car-H, C-1, C-2), 133.24 (Car-H, C-4), 131.74
(Car-H, C-13), 130.60 (Car-H, C-15), 129.46 (Car-H, C-12),
128.22 (Car-H, C-11), 121.71 (Car-Br, C-14), 33.90 (CH2,
C-9); HRESIMS (ASAP) m/z (pos): 276.9864 [M + H]+
C13H10O279Br (calcd. 276.9864).
7.2 mmol), and sodium carbonate (0.76 g, 7.2 mmol) in 14 mL
of a dioxane/water (5/1) mixture, according to the standard
procedure. Dried under vacuum, the desired product 11b
was obtained as brown oil (0.61 g, 62%); Rf: 0.60 (Pentane/
EtOAc: 6/4); 1H NMR (300 MHz, CDCl3) δ: 7.42–7.15 (m,
9H, H-11, H-12, H-13, H-14, H-17, H-18, H-19, H-20, H-
21), 6.58 (d with a roof effect, J = 8.5 Hz, 1H, H-1), 6.51
(dd, J = 8.5, 2.9 Hz, 1H, H-2), 6.33 (d, J = 2.9 Hz, 1H, H-
4), 3.86 (s, 2H, CH2); 13C NMR (75 MHz, CDCl3) δ:
149.37 (Car–OH, C-6), 147.36 (Car–OH, C-3), 142.21
(Cquat, C-16), 141.40 (Cquat, C-15), 136.78 (Cquat, C-10),
130.23 (Car-H, C-13), 129.80 (Car-H, C-19), 129.21 (2Car-
H, C-18, C-20), 128.80 (Car-H, C-12), 128.21 (2Car-H, C-
17, C-21), 127.71 (Car-H, C-14), 127.07 (Car-H, C-11),
126.48 (Cquat, C-5), 117.44 and 116.50 (2Car-H, C-1, C-2),
113.84 (Car-H, C-4), 33.54 (CH2, C-9); HRESIMS (ASAP)
m/z (pos): 299.1047 [M + Na]+ C19H16O2Na (calcd.
299.1048).
2-(4-Bromobenzyl)-1,4-benzoquinone (10d) Compound
10d was prepared from 2-(4-bromobenzyl)-benzene-1,4-
diol 8d (0.57 g, 2 mmol) with manganese dioxide (1.39 g,
16 mmol) in distillated toluene (10 mL), according to the
standard procedure. The para-substituted desired product
10d was obtained as oil which crystallizer as a orange foam
(0.620 g, 75%); mp 104 °C; Rf: 0.45 (Pentane/EtOAc: 9/1);
1H NMR (300 MHz, DMSO-d6) δ: 7.50 (half of an A2X2
system, 2H, H-11, H-15), 7.22 (half of an A2X2 system, 2H,
H 12, H 14), 6.87 (d with a roof effect, J = 10.1 Hz, 1H, H-
1), 6.82 (dd with a roof effect, J = 10.1, 2.4 Hz, 1H, H-2),
6.55 (dt, J = 2.4, 1.4 Hz, 1H, H-4), 3.69 (d, J = 0.8 Hz, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) δ: 187.73 (C=O, C-
2-(3-Phenylbenzyl)benzene 1,4-diol (11c-1) Compound
11c-1 was prepared from 2-(3-bromobenzyl)benzene-1,4-
diol 8c (1 g, 3.6 mmol), tetrakis(triphenylphosphine)palla-
dium(0) (0.21 g, 0.18 mmol, 5 mol%), phenylboronic acid
(0.878 g, 7.2 mmol), and sodium carbonate (0.76 g,
7.2 mmol) in 14 mL of a dioxane/water (5/1) mixture,
according to the standard procedure. Dried under vacuum,
the desired product 11c-1 was obtained oil which crystallize
to brown solid (0.76 g, 70%) in Et2O; mp 94 °C; Rf: 0.70
(Pentane/EtOAc: 6/4); 1H NMR (300 MHz, CDCl3) δ:
7.58–7.52 (m centered at 7.55 ppm, 2H, H-17, H-21), 7.47
(t, J = 2.0 Hz, 1H, H-15), 7.42–7.24 (m, 5H, H-11, H-13,
H-18, H-20, H-19 with H 19(para) at 7.28 ppm, ddt, J = 8.2,
6.3, 1.2 Hz), 7.19 (dt, J = 7.5, 1.4 Hz, 1H, H-14), 6.52 (d
with a strong roof effect, J = 2.5 Hz, 1H, H-4); 6.64–6.62
(symmetrical m, 1H, H-1), 6.50 (dd, J = 8.1, 3.0 Hz, 1H, H-
2), 3.95 (s, 2H, CH2); 13C NMR (75 MHz, CDCl3) δ:
151.09 (Car–OH, C-6), 149.12 (Car–OH, C-3), 143.18
(Cquat, C-16), 142.70 (Cquat, C-12), 142.42 (Cquat, C-10),
130.00 (Car-H, C-15), 129.72 (2Car-H, C-18, C-20), 129.69
(Car-H, C-19); 128.98 (Car-H, C-14), 128.68 (Car-H, C-11),
128.12 (Car-H, C-13), 127.97 (2Car-H, C-17, C-21), 125.46
(Cquat, C-5), 116.80 and 118.07 (2Car-H, C-1, C-2), 114.57
(Car-H, C-4), 36.77 (CH2, C-9); HRESIMS (ASAP) m/z
(pos): 299.1047 [M + Na]+ C19H16O2Na (calcd. 299.1048).
6), 187.00 (C=O, C-3), 147.45 (Cquat, C-5), 136.82 (Cquat
,
C-10), 136.69 and 136.38 (2Car-H, C-1, C-2), 133.10 (Car-
H, C-4), 131.36 (2Car-H, C-11, C-15), 131.35 (2Car-H, C-
12, C-14), 119.73 (Car-Br, C-13), 33.75 (CH2, C-9);
HRESIMS (ASAP) m/z (pos): 276.9863 [M + H]+
C13H10O279Br (calcd. 276.9864).
Standard procedure for the preparation of 2-(phenylbenzyl)
benzene-1,4-diol derivatives, compounds 11b, 11c1-3,
11d1-3
In a glass three-neck round-bottom flask, the 2-(bromo-
benzyl)benzene-1,4-diol (8b–d) (1 equiv), the correspond-
ing boronic acid derivative (2 equiv), tetrakis
(triphenylphosphine)palladium(0) (5 mol%), and sodium
carbonate (2 equiv) as inorganic base were diluted in a
dioxane/water (5/1) mixture. The reaction was stirred at 80 °C
under an inert atmosphere overnight (ca. 15 h). After cool-
ing to room temperature, the reaction mixture was extracted
with CH2Cl2, three times. The combined organic layers
were washed with water and brine, several times. The
resulting organic phase was dried over anhydrous magne-
sium sulfate, filtered, and concentrated under reduced
pressure. The crude product was purified by flash chroma-
tography on silica-gel column column with gradient elution
with 5–20% EtOAc in Pentane. The desired compound 11b,
or 11c(1-3), or 11d(1-3) was obtained as brown oil which
was crystallized, for some, in Et2O.
2-[3-Hydroxy-(3-phenylbenzyl)]benzene 1,4-diol (11c-2)
Compound 11c-2 was prepared from 2-(3-bromobenzyl)
benzene-1,4-diol 8c (0.5 g, 1.8 mmol), tetrakis(triphenyl-
phosphine)palladium(0) (0.105 g, 0.09 mmol, 5 mol%), 3-
hydroxyphenylboronic acid (0.5 g, 3.6 mmol), and sodium
carbonate (0.38 g, 3.6 mmol) in 10 mL of a dioxane/water
(5/1) mixture, according to the standard procedure. Dried
under vacuum, the desired product 11c-2 was obtained as
2-(2-Phenylbenzyl)benzene 1,4-diol (11b) Compound 11b
was prepared from 2-(2-bromobenzyl)benzene-1,4-diol 8b
(1 g, 3.6 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.21 g, 0.18 mmol, 5 mol%), phenylboronic acid (0.878 g,

Med Chem Res
oil which crystallized to red-brown solid (0.24 g, 45%) in
Et2O; mp 103 °C; Rf: 0.42 (Pentane/EtOAc: 6/4); H NMR
standard procedure. Dried under vacuum, the desired pro-
duct was obtained as dark red solid powder (0.76 g, 70%);
mp 134 °C; Rf: 0.65 (Pentane/EtOAc: 6/4). The spectral
data of 11d-1 are in agreement with literature (de Abreu
Lima et al. 2016).
1
(300 MHz, Acetone-d6) δ: 8.41 (envelope from 8.7 to 8.2
ppm which topped at 8.41 ppm, 1H, OH), 7.75 (broad s, 1H,
OH), 7.62 (envelope from 7.7 to 7.55 ppm which topped at
7.62 ppm, 1H, OH), 7.51 (half of an A2X2 system, 2H),
7.33 (half of an A2X2 system with further weak coupling
with CH2, 2H), 7.25 (t, J = 8.1 Hz, 1H), 7.13–7.07 (m, 2H),
7.09 (broad s, 2H), 6.81 (ddd, J = 8.0, 2.4, 1.1 Hz, 1H),
6.72 (d with a roof effect, J = 8.5 Hz, 1H), 6.60 (broad d, J
= 2.8 Hz, 1H), 6.54 (dd with a roof effect, J = 8.5, 3.0 Hz,
1H), 3.95 (s, 2H, CH2); 13C NMR (75 MHz, Acetone-d6) δ:
158.67 (Cquat, Car-OH), 151.24 (Cquat, Car-OH), 148.66
(Cquat, Car-OH), 143.31 (Cquat), 141.60 (Cquat), 139.33
(Cquat), 130.66 (Car-H), 130.18 (2Car-H), 129.30 (Cquat),
127.47 (2Car-H), 118.83 (Car-H), 117.93 (Car-H), 116.56
(Car-H), 114.89 (Car-H), 114.41 (Car-H), 114.36 (Car-H),
36.02 (CH2); HRESIMS m/z (pos): 315.0999 [M + Na]+
C19H16O3Na (calcd. 315.0997).
2-((3′-Hydroxy-[1,1′-biphenyl]-4-yl)methyl)benzene-1,4-
diol (11d-2) Compound 11d-2 was prepared from 2-(4-
bromobenzyl)benzene-1,4-diol 8d (0.5 g, 1.8 mmol), tetra-
kis(triphenylphosphine)palladium(0) (0.105 g, 0.09 mmol,
5 mol%), 3-hydroxyphenylboronic acid (0.5 g, 3.6 mmol),
and sodium carbonate (0.38 g, 3.6 mmol) in 10 mL of a
dioxane/water (5/1) mixture, according to the standard
procedure. Dried under vacuum, the desired product 11d-2
was obtained as brown oil which crystallized in beige solid
(0.26 g, 50%) in Et2O; mp 189 °C; Rf: 0.55 (Pentane/
EtOAc: 6/4); 1H NMR (300 MHz, Acetone-d6) δ: 8.41
(envelope from 8.6 to 8.2 ppm which topped at 8.41 ppm,
1H, OH, H-22), 7.75 (envelope from 7.82 to 7.69 ppm
which topped at 7.75 ppm, 1H, OH, H-8), 7.62 (envelope
from 7.67 to 7.57 ppm which topped at 7.62 ppm, 1H, OH,
H-7), 7.51 (half of A2X2, 2H, H-12, H-14), 7.33 (half of
A2X2 with further weak coupling with CH2, 2H, H-11, H-
15), 7.25 (dd, J = 8.1, 8.1 Hz, 1H, H-17), 7.13–7.06 (m, 2H,
H-18, H-21), 6.81 (ddd, J = 8.0, 2.4, 1.1 Hz, 1H, H-19),
6.72 (d, J = 8.5 Hz, 1H, H-1), 6.60 (d, J = 2.8 Hz, 1H, H-4),
6.54 (dd, J = 8.5, 3.0 Hz, 1H, H-2), 3.95 (s, 2H, CH2, H-
9);13C NMR (75 MHz, Acetone-d6) δ: 158.68 (Car–OH, C-
20), 151.24 (Car–OH, C-6), 148.66 (Car–OH, C-3), 143.31
(Cquat, C-13), 141.60 (Cquat, C-16), 139.32 (Cquat, C-10),
130.66 (Car-H, C-18), 130.18 (2Car-H, C-11, C-15), 129.30
(Cquat, C-5), 127.47 (2Car-H, C-12, C-14), 118.83 (Car-H,
C-2), 117.93 (Car-H, C-1), 116.56 (Car-H, C-17), 114.89
(Car-H, C-18), 114.41 (Car-H, C-4), 114.36 (Car-H, C-21),
36.02 (CH2, C-9). HRESIMS m/z (pos): 315.0998 [M +
Na]+ C19H16O3Na (calcd. 315.0997).
3′-(2,5-Dihydroxybenzyl)-[1,1′-biphenyl]-3-carbaldehyde
(11c-3) Compound 11c-3 was prepared from 2-(3-bromo-
benzyl)benzene-1,4-diol 8c (0.5 g, 1.8 mmol), tetrakis(tri-
phenylphosphine)palladium(0) (0.105 g, 0.09 mmol, 5 mol
%), 3-formylphenylboronic acid (0.54 g, 3.6 mmol), and
sodium carbonate (0.38 g, 3.6 mmol) in 10 mL of a dioxane/
water (5/1) mixture, according to the standard procedure.
Dried under vacuum, the desired product 11c-3 was obtained
as orange oil which crystallized to beige solid (0.25 g, 46%)
1
in Et2O; mp 109.5 °C; Rf: 0.40 (Pentane/EtOAc: 6/4); H
NMR (300 MHz, Acetone-d6) δ: 10.13 (s, 1H, CHO), 8.18
(ddd, J = 2.0, 1.6, 0.6 Hz, 1H), 7.96 (ddd, 1H, J = 7.7, 2.0,
1.2 Hz, 1H), 7.91 (ddd, 1H, J = 7.6, 1.4, 1.3 Hz, 1H),
7.72–7.65 (m, 2H), 7.55 (ddd, 1H, J = 7.6, 1.9, 1.3 Hz, 1H),
7.40 (td, J = 7.6, 0.6 Hz, 1H), 7.32 (dddt, J = 7.6, 1.7, 1.3,
0.6 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 3.0 Hz,
1H), 6.53 (dd, J = 8.5, 3.0 Hz, 1H), 4.01 (s, 2H, CH2); 13C
NMR (75 MHz, Acetone-d6) δ: 193.09 (CHO, C-22), 151.15
(Cquat, Car-OH, C-6), 148.54 (Cquat, Car-OH, C-3), 143.32
(Cquat, C-14), 142.91 (Cquat, C-16), 140.35 (Cquat), 138.21
(Cquat), 133.50 (Car-H), 130.51 (Car-H), 129.80 (Car-H, C-
12), 129.51 (Car-H, C-11), 129.19 (Cquat), 128.89 (Car-H, C-
15), 128.67 (Car-H, C-18), 128.48 (Car-H, C-13), 125.27
(Car-H), 117.82 (Car-H, C-2), 116.46 (Car-H, C-1), 114.34
(Car-H, C-4), 36.46 (CH2, C-9); HRESIMS m/z (pos):
327.0998 [M + Na]+ C20H16O3Na (calcd: 327.0997).
4′-(2,5-Dihydroxybenzyl)-[1,1′-biphenyl]-3-carbaldehyde
(11d-3) Compound 11d-3 was prepared from 2-(4-bro-
mobenzyl)benzene-1,4-diol 8d (0.28 g, 1 mmol), tetrakis
(triphenylphosphine)palladium(0) (0.058 g, 0.05 mmol,
5 mol%), 3-formylphenylboronic acid (0.3 g, 2 mmol), and
sodium carbonate (0.22 g, 2 mmol) in 7 mL of a dioxane/
water (5/1) mixture, according to the standard procedure.
Dried under vacuum, the desired product 11d-3 was
obtained as pale-yellow solid (0.15 g, 50%); mp 135 °C; Rf:
1
0.36 (Pentane/EtOAc: 6/4); H NMR (300 MHz, Acetone-
2-(4-Phenylbenzyl)benzene 1,4-diol (11d-1) Compound
11d was prepared from 2-(4-bromobenzyl)benzene-1,4-diol
8d (1 g, 3.6 mmol), tetrakis(triphenylphosphine)palladium
(0) (0.21 g, 0.18 mmol, 5 mol%), phenylboronic acid (0.878
g, 7.2 mmol), and sodium carbonate (0.76 g, 7.2 mmol) in
14 mL of a dioxane/water (5/1) mixture, according to the
d6) δ: 10.11 (s, 1H, CHO), 8.17 (s, 1H, OH), 7.94 (dd, 2H,
J = 7.8 Hz, H-21, H-17), 7.81 (s, 1H, OH), 7.70–7.59 (m,
4H, H-12, H-14 and H-11, H-15), 7.40 (d with roof effect,
2H, J = 8.1 Hz, H-18, H-19), 6.74 (d, 1H, J = 8.5 Hz, H-1),
6.63 (d, 1H, J = 2.0 Hz, H-4), 6.56 (dd, 1H, J = 8.5, 2.8 Hz,
H-2), 3.98 (s, 2H, CH2); 13C NMR (75 MHz, Acetone-d6) δ:

Med Chem Res
193.08 (CHO, C-23), 151.25 (Car–OH, C-6), 148.67
(Car–OH, C-3), 142.65 (Cquat, C-13), 142.43 (Cquat, C-16),
138.22 (Cquat, C-10), 137.86 (Cquat, C-20), 133.30 (Car-H,
C-19), 130.49 (Car-H, C-21), 130.45 (2Car-H, C-11, C-15),
129.16 (Cquat, C-5), 128.60 (2Car-H, C-12, C-14), 127.62
(2Car-H, C-17, C-18), 117.97 (Car-H,C-2), 116.59 (Car-H,
C-1), 114.45 (Car-H, C-4), 36.08 (CH2, C-9). HRESIMS m/
CDCl3) δ: 7.34–7.25 (m, 5H), 7.25–7.18 (m, 2H),
7.17–7.13 (m, 1H), 6.60 (dd, J = 8.5, 0.5 Hz Hz, 1H), 6.54
(dd, J = 8.5, 2.9 Hz, 1H), 6.33 (dq, J = 2.9, 0.7 Hz, 1H),
5.2–3.8 (two partly superimposed envelopes, one forming
an inflexion point at 4.51 ppm and the other topping at 4.32
ppm, 2H, OH), 3.85 (s, 2H, CH2); 13C NMR (75 MHz,
CDCl3) δ: 149.36 (Cquat), 147.29 (Cquat), 143.21 (Cquat),
140.83 (Cquat), 136.87 (Cquat), 133.96 (Cquat), 130.05 (CH),
129.95 (CH), 129.38 (CH), 129.33 (CH), 128.55 (Cquat),
128.12 (CH), 127.39 (CH), 127.16 (CH), 126.57 (CH),
117.43 (CH), 116.41 (CH), 113.92 (CH), 33.51 (CH2);
z
(pos): 327.0997 [M + Na]+ C20H16O3Na (calcd:
327.0995).
HRESIMS
m/z
(pos):
333.0655
[M + Na]+
Standard procedure for the preparation of 2-
(phenylbenzyl)-benzene-1,4-diol derivatives, compounds
12b-1 to 12b-12
C19H15O235ClNa (calcd. 333.0653).
2-((4′-Chlorobiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
3) Very dark red-brown oil (yield: 48%); H NMR (300
1
To a stirred solution of 2-(2-bromobenzyl)benzene-1,4-diol
8b (100 mg, 0.358 mmol) in a mixture of 1,4-dioxane: H2O
(3 mL, 5:1) was added tetrakis(triphenylphosphine)palla-
dium(0) (0.2 equiv), substituted phenylboronic acids (2.0
equiv), and Na2CO3 (2.0 equiv) at room temperature under
N2 atmosphere in 10 mL microwave vial. The whole reac-
tion mixture was degassed with N2 for 20 min. Then, the
vial was placed in a (Anton Paar, Monowave 300) micro-
wave reactor and irradiated at 130 °C for 30 min. Reaction
was monitored by TLC, after completion of the reaction
volatiles were concentrated under reduced pressure. Diluted
the reaction mixture with water (10 mL) and EtOAc (10 mL).
Aqueous layer was re-extracted with EtOAc (2 × 10 mL).
The combined organic layers were washed with brine (10
mL), concentrated to dryness and the obtained crude pro-
duct was purified by silica-gel (60–120 mesh) column
chromatography [gradient elution with 10–30% EtOAc in
Hexanes] to afford compounds 12b-1 to 12b-12.
MHz, CDCl3) δ: 7.36–7.26 (m, 4H), 7.24–7.17 (m, 4H),
6.59 (d, J = 8.5 Hz, 1H), 6.53 (dd, J = 8.5, 2.9 Hz, 1H),
6.32 (dq, J = 2.9, 0.6 Hz, 1H), 4.65–4.43 (broad envelope
which topped at 4.52 ppm, 1H, OH), 4.41–4.25 (broad
envelope which topped at 4.33 ppm, 1H, OH), 3.84 (s, 2H,
CH2); 13C NMR (75 MHz, CDCl3) δ: 149.39 (Cquat), 147.29
(Cquat), 141.04 (Cquat), 139.84 (Cquat), 136.79 (Cquat),
133.10 (Cquat), 130.51 (2CH), 130.14 (CH), 130.02 (CH),
128.65 (Cquat), 128.33 (2CH), 127.99 (CH), 126.58 (CH),
117.35 (CH), 116.33 (CH), 113.84 (CH), 33.47 (CH2);
HRESIMS
m/z
(pos):
333.0651
[M + Na]+
C19H15O235ClNa (calcd. 333.0653).
2-((2′-Methylbiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
4) Light brown glassy solid which melted near room
1
temperature (yield: 55%); H NMR (300 MHz, CDCl3) δ:
7.36–7.16 (m, 6H), 7.16–7.06 (m, 2H), 6.57 (dd, J = 8.5,
0.6 Hz, 1H), 6.51 (dd, J = 8.5, 2.9 Hz, 1H), 6.23 (dq, J =
2.9, 0.6 Hz, 1H), 4.42 (envelope from 5.1 ppm to higher
field, partly superimposed with the neighboring one, topped
and centered at 4.42 ppm, 1H, OH), 4.04 (envelope which is
partly superimposed with the neighboring one, centered at
4.04 ppm, 1H, OH), 3.72 (d of AB system, J = 15.6 Hz, 1H,
CH2), 3.61 (d of AB system, J = 15.6 Hz, 1H, CH2), 1.95
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ: 149.30
(Cquat), 147.53 (Cquat), 141.57 (Cquat), 140.85 (Cquat),
137.40 (Cquat), 136.36 (Cquat), 129.99 (CH), 129.91 (CH),
129.45 (2CH), 128.26 (Cquat), 127.62 (CH), 127.48 (CH),
126.44 (CH), 125.60 (CH), 117.61 (CH), 116.82 (CH),
113.91 (CH), 33.84 (CH2), 19.80 (CH3); HRESIMS m/z
(pos): 313.1200 [M + Na]+ C20H18O2Na (calcd. 313.1199).
2-((2′-Chlorobiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
1
1) Brown oil (yield: 72%); H NMR (300 MHz, CDCl3)
δ: 7.48–7.43 (m, 1H), 7.34–7.23 (m, 4H), 7.21–7.16 (m,
3H), 6.60 (dd, J = 8.6, 0.4 Hz, 1H), 6.54 (dd, J = 8.6, 2.9
Hz, 1H), 6.33 (dq, J = 2.9, 0.6 Hz, 1H), 4.8–4.4 (broad
envelope which topped at 4.55 ppm, 1H, OH), 4.4–4.1
(broad envelope which topped at 4.31 ppm, 1H, OH), 3.77
(d of AB system, J = 16.2 Hz, 1H, CH2), 3.66 (d of AB
system, J = 16.2 Hz, 1H, CH2); 13C NMR (75 MHz,
CDCl3) δ = 149.27 (Cquat), 147.63 (Cquat), 139.93 (Cquat),
139.39 (Cquat), 137.48 (Cquat), 133.42 (Cquat), 131.22 (CH),
129.97 (CH), 129.45 (CH), 129.11 (CH), 128.83 (CH),
128.38 (CH), 127.70 (Cquat), 126.67 (CH), 126.45 (CH),
117.70 (CH), 116.56 (CH), 114.09 (CH), 33.82 (CH2);
HRESIMS
m/z
(pos):
333.0653
[M + Na]+
2-((3′-Methylbiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
C19H15O235ClNa (calcd. 333.0653).
1
5) Brown oil (yield: 64%); H NMR (400 MHz, CDCl3)
δ: 7.33–7.17 (m, 5H), 7.15 (broad d, J ≈ 7.5 Hz, 1H),
7.11–7.06 (m, 2H), 6.60 (d, J = 8.5 Hz, 1H), 6.53 (dd, J =
8.5, 3.0 Hz, 1H), 6.36 (d, J = 3.0 Hz, 1H), 4.48 (broad s,
2-((3′-Chlorobiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
2) Colorless wax (yield: 45%); 1H NMR (300 MHz,

Med Chem Res
1H, OH), 4.19 (broad s, 1H, OH), 3.86 (s, 2H, CH2), 2.35
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ: 149.39
(Cquat), 147.39 (Cquat), 142.33 (Cquat), 141.31 (Cquat),
137.83 (Cquat), 136.75 (Cquat), 130.18 (CH), 130.01 (CH),
129.72 (CH), 128.82 (Cquat), 128.09 (CH), 127.82 (CH),
127.62 (CH), 126.45 (CH), 126.23 (CH), 117.46 (CH),
116.58 (CH), 113.86 (CH), 33.61 (CH2), 21.44 (CH3);
HRESIMS m/z (pos): 313.1199 [M + Na]+ C20H18O2Na
(calcd. 313.1199).
(CH), 128.87 (Cquat), 127.77 (CH), 126.50 (CH), 121.63
(CH), 117.42 (CH), 116.50 (CH), 114.64 (CH), 113.83
(CH), 113.02 (CH), 55.20 (CH3), 33.60 (CH2); HRESIMS
m/z (pos): 329.1146 [M + Na]+ C20H18O3Na (calcd.
329.1148).
2-((4′-Methoxybiphenyl-2-yl)methyl)benzene-1,4-diol
(12b-9) Brown oil (yield: 45%); 1H NMR (300 MHz,
CDCl3) δ: 7.29–7.18 (m with an half part of an A2X2 sys-
tem at 7.21 ppm, 6H), 6.91 (half part of an A2X2 system,
2H), 6.60 (dd, J = 8.5, 0.4 Hz, 1H), 6.53 (dd, J = 8.5, 3.0
Hz, 1H), 6.35 (dq, J = 3.0, 0.6 Hz, 1H), 4.80–4.48 (broad
envelope which topped at 4.62 ppm, 1H, OH), 4.42–4.23
(broad envelope which topped at 4.33 ppm, 1H, OH), 3.87
(broad s [q with improving the resolution, J = 0.6 Hz], 2H,
CH2), 3.82 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3) δ:
158.69 (Cquat), 149.38 (Cquat), 147.38 (Cquat), 141.83
(Cquat), 136.87 (Cquat), 133.78 (Cquat), 130.41 (CH), 130.30
(2CH), 129.82 (CH), 128.87 (Cquat), 127.48 (CH), 126.49
(CH), 117.38 (CH), 116.46 (CH), 113.82 (CH), 113.68
(2CH), 55.33 (CH3), 33.60 (CH2); HRESIMS m/z (pos):
329.1149 [M + Na]+ C20H18O3Na (calcd. 329.1148).
2-((4′-Methylbiphenyl-2-yl)methyl)benzene-1,4-diol (12b-
1
6) Very deep red oil (yield: 48%); H NMR (300 MHz,
CDCl3) δ: 7.29–7.23 (m, 3H), 7.23–7.15 (m, 5H), 6.61 (d, J
= 8.5 Hz, 1H), 6.54 (dd, J = 8.5, 2.9 Hz, 1H), 6.38 (d, J =
2.9 Hz, 1H), 4.55 (broad s, 1H, OH), 4.25 (broad s, 1H,
OH), 3.87 (s, 2H, CH2), 2.38 (s, 3H, CH3); 13C NMR (75
MHz, CDCl3) δ: 149.39 (Cquat), 147.39 (Cquat), 142.13
(Cquat), 138.40 (Cquat), 136.79 (Cquat), 136.74 (Cquat),
130.30 (CH), 129.73 (CH), 129.10 (2CH), 128.94 (2CH),
128.85 (Cquat), 127.56 (CH), 126.48 (CH), 117.42 (CH),
116.52 (CH), 113.84 (CH), 33.53 (CH2), 21.17 (CH3);
HRESIMS m/z (pos): 313.1201 [M + Na]+ C20H18O2Na
(calcd. 313.1199).
2-((2′-Phenoxybiphenyl-2-yl)methyl)benzene-1,4-diol
1
2-((2′-Methoxybiphenyl-2-yl)methyl)benzene-1,4-diol
(12b-7) Brown oil (yield: 54%); 1H NMR (400 MHz,
CDCl3) δ: 7.37 (ddd, J = 8.2, 7.5, 1.8 Hz, 1H), 7.30–7.21
(m, 2H), 7.20–7.15 (m, 2H), 7.06 (td, J = 7.4, 1.0 Hz, 1H),
7.08–7.04 (m, 1H), 7.00 (dd, J = 8.3, 1.0 Hz, 1H), 6.68 (d,
J = 8.6 Hz, 1H), 6.60 (dd, J = 8.6, 3.0 Hz, 1H), 6.52 (d, J =
3.0 Hz, 1H), 5.24 (s, 1H, OH), 4.51 (s, 1H, OH), 3.81 (d of
AB system, J = 16.6 Hz, 1H, CH2), 3.77 (s, 3H, OCH3),
3.53 (d of AB system, J = 16.6 Hz, 1H, CH2); 13C NMR
(75 MHz, CDCl3) δ: 155.66 (Cquat), 148.84 (Cquat), 148.31
(Cquat), 138.33 (Cquat), 137.43 (Cquat), 131.22 (CH), 130.43
(Cquat), 130.24 (CH), 129.03 (CH), 128.10 (CH), 127.99
(CH), 127.00 (Cquat), 126.65 (CH), 121.38 (CH), 118.17
(CH), 116.71 (CH), 114.48 (CH), 111.11 (CH), 55.85
(CH3), 34.30 (CH2); HRESIMS m/z (pos): 329.1151 [M +
Na]+ C20H18O3Na (calcd. 329.1148).
(12b-10) Reddish oil (yield: 56%); H NMR (300 MHz,
CDCl3) δ: 7.32 (ddd, J = 8.1, 7.2, 1.9 Hz, 1H), 7.29–7.15
(m, 7H), 7.10–7.04 (m, 1H), 7.02 (ddt, J = 7.9, 7.0, 1.1 Hz,
1H, Hpara), 6.98 (dd, J = 8.0, 1.1 Hz, 1H), 6.91–6.85 (m,
2H), 6.67 (d, J = 8.6 Hz, 1H), 6.58 (dd, J = 8.6, 3.0 Hz,
1H), 6.45 (dq, J = 3.0, 0.6 Hz, 1H), 4.81 (s, 1H, OH), 4.47
(s, 1H, OH), 3.92 (broad d of AB system, J = 16.4 Hz, 1H,
CH2), 3.73 (broad d of AB system, J = 16.4 Hz, 1H, CH2);
13C NMR (100 MHz, CDCl3) δ: 157.10 (Cquat), 153.93
(Cquat), 149.07 (Cquat), 148.02 (Cquat), 137.73 (Cquat),
137.56 (Cquat), 133.08 (Cquat), 131.62 (CH), 130.22 (CH),
129.59 (2CH), 129.03 (CH), 128.47 (CH), 128.05 (CH),
127.38 (Cquat), 126.35 (CH), 123.82 (CH), 123.18 (CH),
119.10 (CH), 118.65 (2CH), 117.98 (CH), 116.71 (CH),
114.31 (CH), 34.00 (CH2); HRESIMS m/z (pos): 391.1303
[M + Na]+ (calcd. for C25H20O3Na 391.1305).
2-((3′-Methoxybiphenyl-2-yl)methyl)benzene-1,4-diol
(12b-8) Colorless oil (yield: 27%); H NMR (300 MHz,
2-((3′-Phenoxybiphenyl-2-yl)methyl)benzene-1,4-diol
(12b-11) Brown oil (yield: 50%); 1H NMR (300 MHz,
CDCl3) δ: 7.36–7.23 (m, 6H), 7.22–7.16 (m, 1H), 7.09 (ddt,
J = 7.8, 7.0, 1.1 Hz, 1H, Hpara), 7.05–6.96 (m, 4H), 6.92
(ddd, J = 2.5, 1.6, 0.4 Hz, 1H), 6.59 (dd, J = 8.5, 0.5 Hz,
1H), 6.53 (ddt, J = 8.5, 2.9, 0.5 Hz, 1H), 6.31 (dq, J = 2.9,
0.6 Hz, 1H), 4.65–4.35 (broad envelope which topped at
4.49 ppm, 1H, OH), 4.37–4.17 (broad envelope which
topped at 4.27 ppm, 1H, OH), 3.87 (s [q with improving the
resolution, J = 0.6 Hz], 2H, CH2); 13C NMR (75 MHz,
CDCl3) δ: 157.15 (Cquat), 156.92 (Cquat), 149.33 (Cquat),
147.28 (Cquat), 143.13 (Cquat), 141.51 (Cquat), 136.75
1
CDCl3) δ: 7.34–7.26 (m, 4H), 7.25–7.19 (m, 1H),
6.92–6.86 (m, 2H), 6.82–6.79 (m, 1H), 6.61 (dd, J = 8.5,
0.4 Hz, 1H), 6.54 (ddt, J = 8.5, 2.9, 0.6 Hz, 1H), 6.36 (dq, J
= 2.9, 0.6 Hz, 1H), 4.53–4.43 (broad envelope which top-
ped at 4.48 ppm, 1H, OH), 4.31–4.21 (broad envelope
which topped at 4.26 ppm, 1H, OH), 3.87 (broad s [q with
improving the resolution, J = 0.6 Hz], 2H, CH2), 3.73 (s,
3H, OCH3); 13C NMR (100 MHz, CDCl3) δ: 159.33 (Cquat),
149.42 (Cquat), 147.39 (Cquat), 142.79 (Cquat), 142.13
(Cquat), 136.71 (Cquat), 130.09 (CH), 129.90 (CH), 129.23

Med Chem Res
(Cquat), 130.05 (CH), 129.89 (CH), 129.78 (2CH), 129.48
(CH), 128.60 (Cquat), 127.86 (CH), 126.49 (CH), 124.00
(CH), 123.40 (CH), 119.45 (CH), 119.09 (2CH), 117.37
(CH), 117.35 (CH), 116.41 (CH), 113.83 (CH), 33.46
(CH2); HRESIMS m/z (pos): 391.1307 [M + Na]+
C25H20O3Na (calcd. 391.1305).
and data were analyzed using GraphPad Prism 5.0 Software
and Student’s t-test was used for statistical analysis. Errors
bars represent standard errors of mean. The symbols cor-
respond to a P-value inferior to *0.05, **0.01, ***0.001,
and ****0.0001.
Cell death assay (Annexin V binding assay)
2-((4′-Phenoxybiphenyl-2-yl)methyl)benzene-1,4-diol
(12b-12) Light pink foam (yield: 46%); H NMR (300
1
After 48 h of incubation with selected molecules used at
10 μM, breast cancer cells were collected and re-suspended
in Annexin V binding buffer containing 1 µL Annexin
V-FITC (Miltenyi Biotec) for 15 min on ice in the dark.
Subsequently, cells were analyzed by flow cytometry, using
a FACS Calibur operated by CellQuest software. Apoptotic
cells were considered as Annexin V-positive cells and data
are represented as % of positive cells among total cell
populations. Experiments were performed three times and
means and corresponding standard errors are shown. Stu-
dent’s t-test was used for statistical analysis.
MHz, CDCl3) δ: 7.39–7.31 (almost symmetrical m, 2H),
7.31–7.17 (m, 6H), 7.12 (ddt, J = 7.8, 7.0, 1.1 Hz, 1H,
H
para), 7.08–7.03 (m, 2H), 7.00 (half part of an A2X2 sys-
tem, 2H), 6.60 (d, J = 8.5 Hz, 1H), 6.53 (dd, J = 8.5, 2.9
Hz, 1H), 6.35 (dq, J = 2.9, 0.6 Hz, 1H), 4.63–4.48 (broad
envelope which topped at 4.55 ppm, 1H, OH), 4.39–4.25
(broad envelope which topped at 4.32 ppm, 1H, OH), 3.89
(s, 2H, CH2); 13C NMR (75 MHz, CDCl3) δ: 157.05 (Cquat),
156.51 (Cquat), 149.38 (Cquat), 147.39 (Cquat), 141.62
(Cquat), 136.88 (Cquat), 136.27 (Cquat), 130.52 (2CH),
130.36 (CH), 129.91 (CH), 129.80 (2CH), 128.75 (Cquat),
127.69 (CH), 126.56 (CH), 123.43 (CH), 119.11 (2CH),
118.39 (2CH), 117.42 (CH), 116.45 (CH), 113.85 (CH),
33.66 (CH2); HRESIMS m/z (pos): 391.1309 [M + Na]+
C25H20O3Na (calcd. 391.1305).
Proteasome inhibition
Measurement of proteasome activities HeLa cells were
washed with buffer I (50 mM Tris-HCl, pH 7.4, 2 mM
dithiothreitol, 5 mM MgCl2, 2 mM ATP) and then homo-
genized with buffer I containing 250 mM sucrose. The
lysate was centrifuged at 10,000 x g and the clarified
supernatant was used for enzyme activity determination.
Briefly, the peptidase activities of proteasome were mea-
sured by the addition of 50 μM fluorogenic peptide sub-
strates, Suc–Leu–Leu–Val–Tyr–AMC (for chymotrypsin-
like), Z–Ala–Arg–Arg–AMC (for trypsin-like), and
Z–Leu–Leu–Glu–AMC (for caspase-like activity) in assay
buffer (50 mM Tris-HCl, pH 7.8, 20 mM KCl, 5 mM
MgCl2, and 1 mM dithiothreitol). The reaction mixture was
incubated for 30 min at 37 °C (Vangala et al. 2014). The
released fluorescence was measured at 360/460 nm of
excitation and emission wavelengths in EnSpire multimode
plate reader (Perkin Elmer). Enzymatic activities are
expressed as percentage of activities relative to DMSO
control.
Biological studies
Material and methods
Cell viability assay (MTT)
Cells from the human colorectal carcinoma cell line
HCT116, and breast cancer cell lines (MDAMB231 and
MDAM468), were grown in DMEM medium supplemented
with L-glutamine (2 mM), streptomycin (50 units/mL),
penicillin (550 ng/mL), and 5% FBS to confluence in a
humidified atmosphere (37 °C, 5% CO2), seeded (6000/
well, 50 μL) in 96-well plates, and allowed to attach for 24
h. Compounds were diluted in DMSO and added to cells at
10 μM for 48 h. 50 μL of MTT solution ((3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium)
prepared at the concentration of 2.5 mg/mL in PBS was then
applied to each well and incubated for 3.5 h. Medium was
subsequently removed from wells and resulting formazan
crystals solubilized in 100 μL DMSO. Culture plates were
rocked gently for 30 min to solubilize before optical density
was measured using a microplate reader (BioRad) at 570
nm. Cells that incubated in 0.1% DMSO were used as 100%
proliferation control and the relative growth for each com-
pound was obtained by the normalization using the control.
Three separate experiments were performed in triplicates
Acknowledgements Financial support of this program by the “Ligue
contre le Cancer, Conseil Interrégional Grand Ouest” is gratefully
acknowledged. This research has also been performed as part of the
Indo–French Joint Laboratory “Natural Products and Synthesis toward
Affordable Health”. We thank University of Rennes1, University of
Nantes, INSERM, CNRS, and CSIR for financial support. We thank
CRMPO (Rennes) for the mass spectral studies. We thank Mr. Q.
Raynal, Mrs. C. Tesniere, and M. Sturtz for their work on this topic.
We thank Chemveda Ltd. for the preparation of compounds 12b.

Med Chem Res
Compliance with ethical standards
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Conflict of interest The authors declare that they have no conflict of
interest.
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