2,2′-Dihydroxy-3,3′-dimethoxy-5,5′-dimethyl-6,6′-dibromo-1,1′-biphenyl: preparation, resolution, structure and biological activity

Article abstract of DOI:10.1016/j.tetasy.2007.01.017

The preparation and resolution of the titled conformationally stable biphenyl 1 has been performed in high chemical yield starting from creosol 2. Enantiopure biphenyls (aR)-(+)-1 and (aS)-(-)-1 were obtained by the corresponding menthylcarbonate diastere

Full text of DOI:10.1016/j.tetasy.2007.01.017

Tetrahedron: Asymmetry 18 (2007) 414–423
2,20-Dihydroxy-3,30-dimethoxy-5,50-dimethyl-6,60-dibromo-1,10-
biphenyl: preparation, resolution, structure and biological activity
Davide Fabbri,a Maria Antonietta Dettori,a Giovanna Delogu,a,* Alessandra Forni,b
Gianluigi Casalone,b Giuseppe Palmieri,a Marina Pisanoa and Carla Rozzoa
aIstituto CNR di Chimica Biomolecolare, Traversa la Crucca 3, I-07040 Sassari, Italy
bIstituto CNR di Scienze e Tecnologie Molecolari, Via Golgi 19, I-20133 Milan, Italy
Received 22 December 2006; accepted 8 January 2007
Available online 21 February 2007
Abstract—The preparation and resolution of the titled conformationally stable biphenyl 1 has been performed in high chemical yield
starting from creosol 2. Enantiopure biphenyls (aR)-(+)-1 and (aS)-(ꢀ)-1 were obtained by the corresponding menthylcarbonate diaste-
reomer and successive reduction. The absolute configuration and specific rotation were correlated by X-ray analysis of the crystal struc-
ture of diastereopure menthylcarbonate (aS,1R,10R,2S,20S,5R,50R)-(+)-16. Preliminary biological evaluation of both racemic
enantiomers of 1 has been carried out on melanoma cell lines and significant and selective anticancer activity has been observed for
the enantiomer (aS)-(ꢀ)-1.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
hydroxylated biphenyls, simple synthetic strategies and
often straightforward resolution methodologies are
required in the preparation of this class of compounds.7
It is generally assumed that the stereogenic axis is involved
in pharmacological activities4–6 of hydroxylated biphenyls
as well as in the chirality transfer in catalytic and
stoichiometric processes.8
The rich structural diversity and complexity of natural
products has attracted large interest of synthetic chemists
due to the interesting stereochemical features and biologi-
cal activities.1 Due to the importance of chirality, great
efforts in synthetic strategy have also been devoted to the
preparation of enantiopure analogues of natural occurring
compounds.2
Given the importance of the biphenyl hydroxylated unit,
increasing attention has been paid to this class of com-
pounds from a synthetic, stereochemical and biological
point of view.
The field of synthetic methods is highly advanced at present
but, at the same time, is still lacking in many aspects such as
economy and easy feasibility.3 This aspect is often a restric-
tive factor in the preparation of naturally occurring com-
pounds of high stereochemical and functional complexity.
In a previous article, we reported the synthesis of the 6,60-
dibromo-dehydrodieugenol 3 in its enantiomerically pure
form starting from 2-methoxy-4-allylphenol (eugenol) 4.9
Although biphenyl 3 retains the natural configurationally
flexible biphenyl dehydrodieugenol 5, two atropisomers
of compound 3 were isolated due to the hindered rotation
around the stereogenic biaryl axis. We have observed that
both racemic and the enantiomers produce significant
antinociceptive effects as revealed by the reduction of the
number of acetic acid-induced writhing responses.10
Hydroxylated biphenyl units are present in a large number
of naturally occurring compounds, such as vancomycin,
biphenomycin, ellagitannins; the latter occurs in Nature
in more than 500 derivatives.4–6 Generally, hydroxylated
biphenyl derivatives have less complex structures compared
to naturally occurring compounds lacking in this unit. In
virtue of the axial chirality shown in rotationally hindered
Hydroxylated bromo biphenyls play an important role in
therapy as antibacterial, anti-HIV-1 agents11 and more
recently have displayed significant cytotoxicity against
*
Corresponding author. Tel.: +39 079 3961033; fax: +39 079 3961036;
e-mail: giovanna.delogu@icb.cnr.it
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.01.017

D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
415
human solid tumour cell lines in bromotyrosine dimer
derivatives.12 It is known that the presence of either a bro-
mo or chloro functionality in hydroxylated biaryls makes
them effective chiral ligands or chiral activators in asym-
metric catalytic processes since halides improve Lewis acid-
ity.13 In particular, the presence of a bromo group at the
ortho–ortho0 positions significantly influences the value of
the dihedral angle of biphenol and thus provides an impor-
tant change in the efficiency of asymmetric catalysis.13,14
would be expected in brominated biphenyl 1, whose struc-
ture is similar to dehydrodicreosol 6.
2. Results and discussion
According to the literature, commercial creosol 2 was trea-
ted at 0 ꢁC with a solution of methyltributylammonium
permanganate (MTBAP) in dichloromethane to give
dehydrodicreosol 6 in 85% yield.17b Biphenyl 6 was
obtained as a colourless solid.
As part of our continuing search among hydroxylated
biphenyls15 as building blocks for the preparation of prom-
ising pharmaceutics as well as new ligands, we have
selected 2,20-dihydroxy-3,30-dimethoxy-5,50-dimethyl-1,10-
biphenyl 6, namely dehydrodicreosol, as a scaffold for pre-
paring ortho–ortho0-dibromo-dehydrodicreosol derivatives.
We have explored different bromination conditions in
order to address selective bromination. Our aim was to
brominate the reactive 6,60-positions of the aromatic rings
maintaining the methyl groups or to transform the methyl
into a bromomethyl group, excluding further ring-bromin-
ation. Direct bromination of dehydrodicreosol 6 with
2.2 equiv of Br2 in ether at rt gave biphenyl 1 in 90% yield.
Ether appeared to be the most convenient solvent com-
pared to the other solvents generally used in the bromina-
tion reaction in the presence of Br2 (Scheme 1).23
5,50-Disubstituted biphenyl structures (C–C linkages) occur
frequently in softwood lignins because they come from the
symmetric coupling of the corresponding monomers.16
Dehydrodicreosol 6, a conformationally flexible biphenyl,
is the symmetrical dimer of 2-methoxy-4-methyl phenol
(creosol) 2.17 Compound 6 has been intensively studied as
a model compound in the coupling of lignin monomers18
and displays higher cytotoxic activity and antiradical effi-
ciency than that observed in creosol 2 (Fig. 1).19 High cyto-
toxic activity was observed when a human submandibular
gland carcinoma cell line was treated with dehydrodicreo-
sol 6, which, on the contrary, manifests sevenfold less cyto-
toxic activity against human gingival fibroblasts.20 In
virtue of this selectivity, 6 is a promising antitumoural
agent. The cytotoxicity of dehydrodicreosol 6 against
tumourþcells is likely associated with the high production
of CH3 intermediates instead of quinone methide radicals
as observed for monomer 2 and generally for O-methoxy-
4-alkylphenols.21 In fact, the presence of two methyl
groups at the 5,50-positions and four hydroxylated groups
seems to be a chemostructural requirement for the high
cytotoxic activity.22
With a slight adaptation of the method reported in the
literature for related structures,24 the hydroxyl groups were
protected with tetraethylene glycol to obtain biphenyl 7
starting from the reaction of biphenyl 1 with tetraethylene
glycol ditosylate in the presence of K2CO3 in DMF at
60 ꢁC (Scheme 1). Biphenyl 7, obtained in 65% overall
yield starting from 6 (Scheme 1, paths a and b), is also
achievable with comparable overall yield, by direct protec-
tion of the hydroxyl groups of dehydrodicreosol 6 with
tetraethylene glycol to give 8 and selective bromination at
the 6,60 positions of the aromatic rings with 2.2 equiv of
NBS, benzyl peroxide (BPO) in CCl4 at rt (Scheme 1,
path b–c).
Both biphenyls 7 and 8 have a tetraethylene glycol ring that
should be a suitable ligand to complex a Na+ ion.24,25
Biphenyl 7 is conformationally stable and, thus, two enan-
tiomers can be evidenced at rt. In order to have a multi-
functional ligand, we thought to transform the methyl
groups of 7 into suitable functional groups that could be
subjected to further transformations.26 According to the
general trend of the bromination reaction carried out in
the presence of NBS and BPO in CCl4 on methyl-substi-
tuted anisoles, side-chain brominations would be expected
Our starting point was to insert bromine at the 6,60-posi-
tion of dehydrodicreosol 6 in order to achieve configura-
tional stability, as well as a more decisive influence on
the torsional angle and, therefore, on the reactivity and ste-
reoselectivity of biphenyl 1. According to the anticancer
activity observed in hydroxylated biphenyls containing
bromo groups, an increase in the antitumoural activity
OCH
3
OCH
R
OCH
3
3
R
OCH
OH
3
Br
Br
OH
OH
Br
Br
OH
OH
OH
OH
OCH
OCH
3
R
OCH
3
3
(aR)-(+)-3
(aS)-(-)-3
2 R = CH3
4 R = allyl
5 R = allyl
6 R = CH3
Figure 1.

416
D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
OCH
H C
3
OCH
3
H C
3
OCH
3
3
Br
d
O
O
Br
Br
O
R
O
Br
Br
O
O
c
or
Br
Br
OH
OH
b
O
Br
OCH
H C
3
OCH
H C
3
OCH
3
3
3
7
9
1
R = -[(CH ) -O-(CH ) ] -O
2 2
2 2 2
a
c
H C
3
OCH
OCH
3
3
Br
O
O
O
O
O
O
O
d
b
O
O
6
O
Br
H C
3
OCH
OCH
3
3
10
8
Scheme 1. Synthesis of hydroxylated biphenyls containing bromo group. Reagents and conditions: (a) 2.2 equiv Br2, Et2O, rt, 2 h (90%);
(b) TsO[(CH2)2O(CH2)2]2OTs, K2CO3, DMF, 60 ꢁC (7: 12 h, 40%; 8: 14 h, 50%); (c) 2.2 equiv NBS, BPO, CCl4 (7: rt, 12 h, 64%; 9: rt to 70 ꢁC, 4 h,
60%); (d) 2.2 equiv NBS, hm (200 W), CCl4, reflux, 1 h (30%).
for biphenyl 7.23d,e Biphenyl 9 was obtained in 60% yield
after purification by flash chromatography (Scheme 1, path
c). Repeated attempts at changing the reaction conditions
(stoichiometry of NBS, temperature, time) did not improve
the yield of biphenyl 9.
Our aim was to produce hydroxylated biphenyl bromo
containing in enantiopure form. Thus, we had planned to
resolve them by transformation into diastereomer deriva-
tives, which would allow us to assign also the absolute con-
figuration for each enantiomer.
The light-induced reaction of side-chain bromination of
anisole derivatives with NBS in CCl4 has been studied by
Bickelhaupt et al.23d According to their report, the side-
chain bromination of compound 7 would be preferred to
aromatic bromination since radical generation at the
methyl group would be more favoured. We subjected
biphenyl 7 to light-induced bromination in the presence
of NBS in CCl4 in such a way that a gentle reflux was
maintained (Scheme 1, path d). Under these conditions,
biphenyl 7 was scarcely reactive, giving only 20% yield of
dibromomethyl biphenyl 9. At first, we attributed this poor
reactivity of biphenyl 9 to the bulkiness of the two bromo
groups at the ortho–ortho0 positions that would exert a neg-
ative effect ion in the formation of the methide radical,
which would be the key intermediate of the side-chain bro-
mination under light irradiation.23c,d Nevertheless, only a
slight increase in the yield of methyl bromination was ob-
served when we performed the same reaction on biphenyl
8, which is lacking in bromo atoms at the ortho–ortho0 posi-
tions (Scheme 1, path d). Biphenyl 10 was isolated in 30%
yield by flash chromatography and no nuclear aromatic
bromination was observed. Other steric and electronic fac-
tors should be taken into account in order to justify the
unfavourable generation of the methide radical and its res-
onance stabilization. According to what was observed such
as in biphenyl analogues, in biphenyl 10, the relative orien-
tation of the methoxyl groups with respect to the aromatic
ring should also be considered.27
We prepared phosphorothioamidate 11 treating racemic 1
with (S)-(ꢀ)-Cl2P(S)NHCH(CH3)Ph 12 in the presence of
refluxing pyridine.28 In this case we chose an inexpensive
chiral source, (S)-(ꢀ)-a-methylbenzylamine, which was
used in an equimolar ratio and was expected to be recov-
ered, under the reduction conditions, without a loss of
enantiomeric purity. The two phosphorothioamidates
(aR,S)-11 and (aS,S)-11 were obtained in 80% yield after
flash chromatography. All attempts to isolate one single
diastereomer proved unsuccessful (Scheme 2, path a). A
sharp difference in the solubility of two diastereomers 11
was reached after N-methylation. When an equimolar mix-
ture of phosphorothioamidates (aR,S)-11 and (aS,S)-11
was treated with CH3I under phase-transfer catalysis, N-
methyl phosphorothioamidates (aR,S)-13 and (aS,S)-13
were achieved in 74% yield in a 1:1 ratio (Scheme 2, path
b). Each diastereomer 13 was separated after flash chroma-
H C
3
OCH
O
3
H C
3
OCH
O
3
CH
S
3
CH
3
S
c
Br
Br
a
3
1
P
P
N
R
Ph
O
N
Ph
O
CH
OCH
3
H C
3
OCH
3
H C
3
14
11 R = H
13 R = CH
b
3
All compounds prepared were solids, air stable and easily
separated and purified by flash chromatography using
appropriate solvent mixtures.
Scheme 2. Synthesis of phosphorothioamidate diastereomers. Reagents
and conditions: (a) compound 12, py, reflux, 12 h (11: 80%); (b) CH3I,
TBAOH, CH2Cl2, H2O (13: 74%); (c) LiAlH4, THF (94%).

D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
417
tography in 15% yield with 99% de. Most diastereomers 13
were collected as a mixture of both. Recrystallization of
one diastereomer 13 from n-hexane allowed us to collect
crystals that, unfortunately, were not suitable for X-ray
analysis. The cleavage of N-methyl phosphothioamidate
biphenyl derivatives requires stoichiometric quantities of
LiAlH4 in THF under heating at 60 ꢁC. All attempts to
deprotect the phenolic hydroxyls of diastereomers 13 under
these conditions failed. The reaction afforded, even at room
temperature, only debromination products as expected for
aromatic rings containing bromo groups when treated with
hydrides (Scheme 2, path c).29 Biphenyl 14 was recovered
from the reaction in virtually quantitative yield. The failure
to recover pure diastereomers 13 in high yield as well as to
collect suitable crystals for diffraction analysis prompted us
to investigate another resolving agent.
After the reduction of the carbonate group in the presence
of a 1 M solution of LiAlH4 in THF at rt, each diastereo-
mer 16 gave the two atropo-enantiomers (+)-1 and (ꢀ)-1 in
90% yield (Scheme 3). In this case, although these condi-
tions are favourable for the debromination reaction,29 the
high reactivity of the carbonate group allowed us to obtain
biphenol 1 in 90% yield with traces of debrominated
product.
The enantiomeric purity of each biphenol 1 was >99%, and
related to the diastereomeric excess of the corresponding
menthylcarbonate 16.
Atropo-enantiomer 1 has a quite high interconversion
barrier in solution in most solvents. Interconversion of
biphenyl (aS)-1 was monitored by polarimetric measure-
ments, whereas interconversion of diastereomer (aR,1R,
10R,2S,20S,5R,50R)-(ꢀ)-16 was detected by 1H NMR, both
at different temperatures and times. Enantiopure biphenol
1 does not racemize in xylene even when heated to
100 ꢁC for 12 h. Bromobiphenol 1 and diastereomer
(aR,1R,10R,2S,20S,5R,50R)-(ꢀ)-16 are thermally and chemi-
cally stable.
In a previous article, we have successfully applied the
bromination reaction in a C2 symmetry flexible biphenol
bearing two menthylcarbonate groups at the ortho–ortho0
positions in order to achieve bromination and configura-
tional stability at the stereogenic axis.30
We decided to transform dehydrodicreosol 6 in the corre-
sponding bis-menthylcarbonate (10R,2S,20S,5R,50R)-15 by
treatment with 2.2 equiv of (ꢀ)-menthylchloroformate
and triethylamine at rt using toluene as a solvent. Bis-men-
thylcarbonate (10R,2S,20S,5R,50R)-15 was achieved in 90%
yield (Scheme 3, path a).
2.1. X-ray analysis and computational studies
A perspective view of (aS,1R,10R,2S,20S,5R,50R)-(+)-16 is
shown in Figure 2. This compound crystallizes in the rather
unusual (for organic compounds) hexagonal P3121 space
group with half a molecule in the asymmetric unit. The
molecule is completed by the (ꢀx, ꢀx + y, 1/3 ꢀ z) symme-
try operation, denoted here and in Figure 2 by a super-
script. The methoxy carbon atom C8 is located
significantly far from the plane through the atoms of the
When the bromination reaction was carried out on dicar-
bonate 15 using 4 equiv of [BTEAÆBr3] and 5 equiv of
ZnCl2 in CH3COOH, after 2 h at 60 ꢁC, a mixture of the
two diastereomers (aR,1R,10R,2S,20S,5R,50R)-16 and
(aS,1R,10R,2S,20S,5R,50R)-16 was obtained in a 1:1 ratio
and 80% yield (Scheme 3, path b). Complete regioselectiv-
ity as well as conformational stability of dibromo derivate
16 was achieved in only one reaction step. Both diastereo-
mers of 16 were readily separated by flash chromatography
with 99% de. Despite the complex structure of the diaste-
reomers of 16, the presence of a C2 symmetry axis allowed
us to have simplified NMR spectra and thus calculate the
diastereomeric ratio.
˚
aromatic ring (0.44(1) A). The dihedral angle s between
the least-squares planes through the biphenyl rings,
81.3(2)ꢁ, is indicative of high configurational stability. This
value is in the range (76–88ꢁ) observed for not constrained
ortho–ortho0 dibromo-substituted biphenyls, as found
through a survey on the Cambridge Structural Database
(CSD version Nov. 2005, no refcode restrictions applied).32
Among the retrieved structures, the biphenyl derivative
previously reported by us,30 differing from 16 for the ab-
sence of the methyl groups at the 5,50 positions, showed
dihedral angles s slightly larger with respect to 16, that is
86.1(2)ꢁ and 87.6(3)ꢁ for the two molecules of the asymmet-
ric unit. No difference, within the experimental error, was
otherwise observed between the biphenyl moieties of the
Suitable crystals were recovered after recrystallization from
n-hexane, which, when subjected to X-ray analysis, allowed
us to unequivocally assign both the structure and absolute
configuration of (aS,1R,10R,2S,20S,5R,50R)-(+)-16.
H C
3
OCH
3
H C
3
OCH
3
O
menthyl
R
R
O
O
O
O
a
OH
OH
(aS)-(-)-1
(aR)-(+)-1
c, d
menthyl
O
H C
3
OCH
3
H C
3
OCH
3
6
15 R = H
16 R = Br
b
Scheme 3. Resolution of 1 via menthylcarbonate diastereomers. Reagents and conditions: (a) (1R,2S,5R)-(ꢀ)-menthyl chloroformate, Et3N, toluene (15:
90%); (b) BTEAÆBr3, ZnCl2, AcOH, 60 ꢁC, 2 h (16: 80%); (c) separation of diastereomers by flash chromatography; (d) LiAlH4, THF, 0 ꢁC to rt, 90%.

418
D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
Figure 3. Electrostatic potential surfaces for model compounds 16A (left)
and 16B (right). Absolute isovalue = 0.05 a.u.; violet: positive isosurface,
brown: negative isosurface.
the molecule, with respect to 16B. In view of this observa-
tion, the proximity of two oxygen atoms (at 2,20 and 3,30
positions) in such biphenyl derivatives appears to be an
important structural factor. No relevant variations of the
EP surfaces are observed on the side of the bromine atoms.
Figure 2. ORTEP plot31 of diastereomer (aS,1R,10R,2S,20S,5R,50R)-(+)-
16. Displacement ellipsoids are drawn at the 20% probability level.
two structures. Owing to the recognized importance of the
methyl groups at the 5,50-positions in determining the cyto-
toxic activity of the parent dehydrodicreosol 6, we decided
to undertake a computational study on the two model com-
pounds, aimed at investigating the effect of these groups on
the structural and electronic properties of the biphenyl
unit.
2.2. Cell growth inhibition
To investigate the potential anticancer activity of biphenyl
( )-1, we performed in vitro cell proliferation assays on
three human melanoma cell lines. Cells were grown in the
presence of scalar doses of ( )-1, (aR)-(+)-1 and (aS)-
(ꢀ)-1, as described in the experimental section, to test the
capability of these compounds to inhibit cancer cells
growth (Fig. 4). The graphic reports results obtained with
the CN Mel-A cell line, which are representative for all
the melanoma cell lines tested. Results are expressed as a
mean percentage of cell growth in the presence of the spe-
cific molecule compared to the control (cells not treated).
Each bar is representative of three experiments standard
deviation. The three compounds showed growth inhibition
activity, particularly the enantiopure form (aS)-(ꢀ)-1.
After 5 days of cell culture in the presence of 1, we calcu-
lated the IC50 to be around 25–30 lM for the (aS)-(ꢀ)-1
form, IC50 above 40 lM for the (aR)-(+)-1 form and
IC50 higher than 60 lM for the racemic mixture. These re-
sults indicate that 1 has a potential anticancer activity in its
(aS)-(ꢀ)-1 enantiomeric form, since it consistently inhibits
melanoma cells proliferation. More accurate investigations
are needed to clarify the dose/time of action of (aS)-(ꢀ)-1
on different tumours types and its specificity for tumour
cells.
DFT geometry optimizations were performed on two mod-
el compounds obtained from 16 by substituting the men-
thyl with a methyl group (16A) and then removing the
methyl groups at the 5,50 positions (16B). Calculations
were performed at the B3LYP/6-31G** level with the
Gaussian03 package,33 starting from the X-ray experimen-
tal geometry of 16.
Analysis of the optimized bond distances in 16A and 16B
indicates that the corresponding experimental structures
are well reproduced by calculations (for example, the C–
˚
Br distances are reproduced within 0.02 A). The computed
dihedral angle between the least-squares planes through the
biphenyl rings is 86.6ꢁ in both model compounds, suggest-
ing that the experimentally observed difference is probably
attributable to crystal packing effects. Calculations then
confirm that no structural differences can be observed
between the two compounds. To investigate the changes
in the electron distribution arising from the presence of
the methyl groups at the 5,50-biphenyl positions, we have
computed the electrostatic potential (EP) of both 16A
and 16B compounds. Such a property is in fact highly
suitable to help understand the initial steps of recognition
processes (e.g., drug–receptor interactions), which are in
fact electrostatic in nature.34 In Figure 3, two illustrative
plots of EP surfaces of 16A (left) and 16B (right) are
reported. It can be easily seen that the negative regions
associated with the lone pairs of all the oxygen atoms are
more extended in 16A, indicating that an approaching
electrophile ‘sees’ a greater negative area on this side of
3. Conclusions
A straightforward procedure for preparing 2,20-dihydroxy-
3,30-dimethoxy-5,50-dimethyl-6,60-dibromo-1,10-biphenyl 1,
a C2-configurationally stable creosol-related dimer, has
been set up. The synthetic strategy entailed the preparation
of dehydrodicreosol 6 from creosol 2, then, aromatic bro-
mination to give racemic 1 in 80% overall yield. Both enan-
tiopure bromobiphenols 1 were achieved by a reaction

D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
419
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
( )-1
(aR)-(+)-1
(aS)-(-)-1
100 μM
80 μM
60 μM
40 μM
20 μM
10 μM
Figure 4. Anti-proliferative effect of ( )-1, (aR)-(+)-1 and (aS)-(ꢀ)-1 on melanoma cell lines.
path, which involved the transformation of dehydrodicreo-
sol 6 in menthylcarbonate 15, aromatic bromination, sepa-
ration of the two homochiral atropo-diastereomers
(aR,1R,10R,2S,20S,5R,50R)-(ꢀ)-16 and (aS,1R,10R,2S,20S,
5R,50R)-(+)-16, and final reduction of the menthyl
carbonate groups to give enantiopure 1 in 65% of overall
yield.
purified by flash chromatography using CH2Cl2 as an elu-
ent, to give 1 (1.41 g, 90%): mp 246 ꢁC. 1H NMR d 2.46 (s,
6H), 3.95 (s, 6H), 5.58 (br s, 2H), 6.88 (s, Ar, 2H); 13C
NMR d 23.56, 56.03, 112.57, 117.46, 124.81, 129.45,
141.85, 145.54; Anal. Calcd for C16H16Br2O4: C, 44.47;
H, 3.73. Found: C, 44.73; H, 3.60.
4.2. 3,30-Dimethoxy-5,50-dimethyl-[1,10-biphenyl]-2,20-diyl-
O,O0-bis[5-methyl-2-(1-methylethyl)-cyclohexyl]-carbonic
ester 15
We were able to correlate, from the crystal structure of dia-
stereomer (aS,1R,10R,2S,20S,5R,50R)-(ꢀ)-16, the absolute
configuration with the specific rotation of 1 and to calcu-
late the quite high racemization barrier. The high configu-
rational stability has also been confirmed by the high
value of dihedral angle calculated by the crystal structure
of 16.
A solution of 1 (7 g, 25.50 mmol) and Et3N (5 mL) in dry
toluene (30 mL) was added, dropwise, to a solution of
(ꢀ)-(1R,2S,5R)-menthyl chloroformate (12.2 g, 56 mmol)
in toluene (30 mL) at rt under N2. The solution was stirred
at rt for 1 h, washed with 10% HCl and water and the
organic phase extracted with CH2Cl2. The crude was dried
over Na2SO4 to give 15 as a colourless oil that was used in
the following reaction without purification (14.6 g, 90%):
1H NMR d 0.79 (d, J = 6.9 Hz, 6H), 0.95 (d, J = 6.9 Hz,
6H), 1.05 (d, J = 6.9 Hz, 6H), 0.80–1.95 (series of m,
18H), 2.40 (s, 6H), 3.87 (s, 6H), 4.54 (m, 2H), 6.75 (s, Ar,
2H), 6.80 (s, Ar, 2H); 13C NMR d 16.24, 20.71, 21.48,
22.03, 23.33, 25.93, 31.35, 34.10, 40.49, 46.87, 55.94,
78.91, 112.70, 122.77, 130.65, 135.70, 135.78, 151.23,
152.86; Anal. Calcd for C36H50O8: C, 70.79; H, 8.25.
Found: C, 70.83; H, 8.10.
Preliminary investigations on the potential anticancer
activity of 1 in racemic as well as in enantiopure form have
been carried out. Cell proliferation assays showed a strong
cell growth inhibition due to treatments of melanoma cells
with the (aS)-(ꢀ)-1 form, confirming selectivity of atropoi-
somers (aS)-(ꢀ)-1 and (aR)-(+)-1 in the anticancer activity.
The biologic activity related to the stereoaxis of hydroxyl-
ated biphenyl bromo-containing suggests the influence of
this geometrical element in the search of new anticancer
therapeutics containing biphenyls.
4. Experimental
4.3. 3,30-Dimethoxy-5,50-dimethyl-6,60-dibromo-[1,10-bi-
phenyl]-2,20-diyl-O,O0-bis[5-methyl-2-(1-methylethyl)-cyclo-
hexyl]-carbonic ester 16
4.1. 2,20-Dihydroxy-3,30-dimethoxy-5,50-dimethyl-6,60-di-
bromo-1,10-biphenyl 1
To a solution of 15 (1.5 g, 2.35 mmol) in acetic acid
(20 mL), BTEAÆBr3 (4 g, 9.4 mmol) and ZnCl2 (1.6 g,
11.7 mmol) were added in one pot. The reaction mixture
was stirred at 60 ꢁC for 2 h until the initial orange colour
faded. Aqueous Na2S2O5 was added to the mixture and
the organic phase extracted with CH2Cl2. The organic layer
was dried over Na2SO4 to obtain a 1:1 mixture of the two
diastereomers (aR,1R,10R,2S,20S,5R,50R)-16 and (aS,1R,
To a stirred solution of 6 (1 g, 3.64 mmol) in dry diethyl
ether (25 mL) at 0 ꢁC and under N2, Br2 (1.15 g,
7.29 mmol) was added. The cold bath was removed and
the mixture stirred at rt for 2 h. Aqueous Na2S2O5 was
added and the organic phase was extracted with diethyl
ether. The organic extract was dried over Na2SO4 and con-
centrated to afford a brown solid. The crude material was

420
D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
10R,2S,20S,5R,50R)-16 as a brown solid. The two diastereo-
mers were purified and separated by flash chromatography
using a 4:6 mixture of CH2Cl2–petroleum as an eluent
(1.49 g, 80%): (aR,1R,10R,2S,20S,5R,50R)-16: first diaste-
diastereomers) 76.25, 77.43; Anal. Calcd for C24H26Br2-
NO4PS: C, 46.84; H, 4.26. Found: C, 46.91; H, 4.34.
4.7. Dibenzo-(d,f)(1,3,2)-dioxaphosphepin-6-amine-1,11-di-
bromo-2,10-dimethyl-4,8-dimethoxy-N,N0-methyl-(1-phenyl-
ethyl)-6-sulfide 13
1
reomer eluted, 99% de: mp 199 ꢁC. H NMR d 0.74 (d,
J = 6.4 Hz, 6H), 0.85 (d, J = 6.4 Hz, 6H), 0.92 (d,
J = 6.6 Hz, 6H), 0.85–2.10 (series of m, 18H), 2.47 (s,
6H), 3.81 (s, 6H), 4.50 (m, 2H), 6.94 (s, 2H); 13C NMR d
16.47, 20.67, 22.07, 23.46, 24.04, 25.99, 31.54, 34.13,
40.35, 47.08, 56.03, 79.20, 114.22, 116.75, 131.87, 136.32,
To a solution of 11 (1 g, 1.62 mmol) in CH2Cl2 (20 mL)
and H2O (15 mL), tetrabutylammonium hydroxide
(TBAOH) (4.86 mmol, 40% aqueous solution) and CH3I
(0.7 g, 4.86 mmol) were added. The reaction mixture was
stirred at rt under N2. After 12 h, water and 10% HCl were
cautiously added. The organic phase was extracted with
CH2Cl2. The organic layer was dried over Na2SO4 to
obtain a colourless solid that was purified by flash chromato-
graphy using a 2:8 mixture of ethyl acetate–petroleum, as
an eluent, to give 13 (0.75 g, 74%): compound (ꢀ)-13, first
diastereomer eluted: 99% de, mp 98–99 ꢁC. 1H NMR d 1.53
136.93, 150.37, 151.94; Anal. Calcd for C38H52Br2O8: C,
20
57.29; H, 6.58. Found: C, 57.22; H, 6.50; ½aꢁD ¼ ꢀ72:9 (c
1, CHCl3). (aS,1R,10R,2S,20S,5R,50R)-16: second diaste-
1
reomer eluted, 99% de: mp 102 ꢁC. H NMR d 0.72 (d,
J = 6.8 Hz, 6H), 0.86 (d, J = 6.8 Hz, 6H), 0.92 (d, J =
6.8 Hz, 6H), 0.82–2.01 (series of m, 18H), 2.48 (s, 6H),
3.87 (s, 6H), 4.50 (m, 2H), 6.94 (s, Ar, 2H); 13C NMR d
16.18, 20.74, 22.05, 23.22, 24.02, 25.83, 31.28, 34.06,
40.23, 46.81, 55.90, 79.13, 114.08, 116.48, 131.82, 136.34,
3
3
(d, JHP = 7.2 Hz, 3H, CH–CH3), 2.26 (d, JHP = 10.4 Hz,
3H, N–CH3), 2.46 (s, 3H), 2.51 (s, 3H), 3.64 (s, 3H), 3.99 (s,
3H), 5.59 (m, 1H), 6.81 (s, Ar, 1H), 7.03 (s, Ar, 1H), 7.33
(d, J = 8.0 Hz, Ar, 1H), 7.41 (t, J = 8.0 Hz, Ar, 2H), 7.55
(d, J = 8 Hz, Ar, 2H); 13C NMR d (aliphatic only) 16.40
136.78, 150.28, 151.57; Anal. Calcd for C38H52 Br2O8: C,
20
57.29; H, 6.58. Found: C, 57.33; H, 6.67; ½aꢁD ¼ þ38:7
(c 1, CHCl3).
4.4. (aR)-(+)-2,20-Dihydroxy-3,30-dimethoxy-5,50-dimethyl-
2
2
(d, JCP = 2.8 Hz, N–CH3), 24.33 (d, JCP = 5.6 Hz, CH–
6,60-dibromo-1,10-biphenyl 1
2
CH3), 28.30, 29.74, 55.65, 56.13 (d, JCP = 10.5 Hz,
20
CH–CH3), 56.70; 31P NMR d 80.42; ½aꢁD ¼ ꢀ136:4 (c
A solution of (aR)-16 (99% de) (0.5 g, 0.62 mmol) in dry
THF (30 mL) was cooled at 0 ꢁC under N2. LiAlH4 (1 M
in THF) (6 mL, 6 mmol) was added with vigorous mag-
netic stirring. After 12 h, water and 10% HCl were cau-
tiously added. The organic phase was extracted with
ether, dried over Na2SO4 and evaporated to afford a col-
ourless solid. After purification by flash chromatography
using CH2Cl2 as an eluent, enantiomerically pure (aR)-1
0.5, CHCl3) Anal. Calcd for C25H28Br2NO4PS: C, 47.71;
H, 4.48. Found: C, 47.63; H, 4.44. Compound (+)-13, sec-
ond diastereomer eluted: 99% de, mp 244–245 ꢁC. 1H
NMR d 1.68 (d, J = 6.8 Hz, 3H, CH–CH3), 2.16 (d,
3JHP = 9.6 Hz, 3H, N–CH3), 2.51 (s, 6H), 3.89 (s, 3H),
4.00 (s, 3H), 5.76 (m, 1H), 6.91 (s, Ar, 1H), 7.05 (s, Ar,
1H), 7.20–7.40 (series of m, Ar, 5H); 13C NMR d (aliphatic
2
only) 16.19 (d, JCP = 2.66 Hz, N–CH3), 24.29 (d
2JCP = 5.6 Hz, CH–CH3), 28.37, 28.38, 55.33 (d, JCP
=
2
(0.24 g, 90%) and enantiomerically pure (ꢀ)-menthol
20
(0.82 g, 85%) were obtained. (aR)-(+)-1: ½aꢁD ¼ þ13:1 (c
20
7.9 Hz, CH–CH3), 55.85, 56.80; 31P NMR d 80.96;
20
0.5, CHCl3); ½aꢁ365 ¼ þ103:0 (c 0.5, CHCl3).
½aꢁD ¼ þ209:9 (c 0.5, CHCl3); Anal. Calcd for
C25H28Br2NO4PS: C, 47.71; H, 4.48. Found: C, 47.88; H,
4.41.
4.5. (aS)-(ꢀ)-2,20-Dihydroxy-3,30-dimethoxy-5,50-dimethyl-
6,60-dibromo-1,10-biphenyl 1
4.8. Dibenzo-(d,f)(1,3,2)-dioxaphosphepin-6-amine-2,10-
dimethyl-4,8-dimethoxy–N,N0-methyl-(1-phenylethyl)-6-
sulfide 14
Using the above procedure, diastereomer (aS)-16 (99% de)
20
gave (aS)-(ꢀ)-1: (0.24 g, 90%): ½aꢁD ¼ ꢀ13:1 (c 0.8, CHCl3);
20
½aꢁ365 ¼ ꢀ102:9 (c 0.8, CHCl3). Enantiomerically pure (ꢀ)-
menthol (0.79 g, 82%) was recovered.
A solution of (ꢀ)-13 (99% de) (0.3 g, 0.47 mmol) in dry
THF (30 mL) was cooled at 0 ꢁC under N2. LiAlH4 (1 M
in THF) (1 mL, 1 mmol) was added with vigorous mag-
netic stirring. After 12 h, water and 10% HCl were cau-
tiously added. The organic phase was extracted with
ether, dried over Na2SO4 and evaporated to afford a col-
ourless solid. After purification by flash chromatography
by using CH2Cl2 as an eluent, 14 (0.2 g, 90%) was obtained:
4.6. Dibenzo-(d,f)(1,3,2)-dioxaphosphepin-6-amine-1,11-di-
bromo-2,10-dimethyl-4,8-dimethoxy-N-(1-phenylethyl)-6-
sulfide 11
N-((S)-a-Methylbenzyl)-dichlorothiophosphoroamidate 12
(1.41 g, 5.5 mmol) was added dropwise to a solution of 1
(2 g, 4.62 mmol) in dry pyridine (50 mL) at rt under N2.
After 12 h at reflux, the reaction mixture was cooled and
made acid with 10% H2SO4. Water was added and the
organic phase extracted with CH2Cl2, dried over Na2SO4
and evaporated to dryness to obtain a colourless solid.
The crude was purified by flash chromatography using a
1:1 mixture of CH2Cl2–petroleum, as an eluent, to give
11 (2.7 g, 80%) as a colourless oil (1:1 inseparable
diastereomeric mixture). 31P NMR d (mixture of two
1
14: mp 99–100 ꢁC. H NMR d 1.68 (d, J = 6.8 Hz, 3H,
CH–CH3), 2.38 (d, J = 10.4 Hz, 3H, N–CH3), 2.41 (s,
3H), 2.42 (s, 3H), 3.85 (s, 3H), 3.91 (s, 3H), 5.75 (m, 1H),
6.80 (s, Ar, 1H), 6.83 (s, Ar, 1H), 6.89 (s, Ar, 1H), 6.90
(s, Ar, 1H), 7.27 (d, J = 7.2 Hz, Ar, 1H), 7.40 (t,
J = 7.2 Hz, Ar, 2H), 7.53 (d, J = 7.2 Hz, Ar, 2H); 13C
2
NMR d (aliphatic only) 16.39 (d, JCP = 3.1 Hz, N–CH3),
21.56 (d J = 2.9 Hz, CH–CH3), 28.24, 55.70 (d,
2JCP = 7.7 Hz, CH–CH3), 56.13, 56.16; 31P NMR d 84.07;

D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
421
Anal. Calcd for C25H30NO4PS: C, 63.67; H, 6.41. Found:
C, 63.56; H, 6.66.
7.06 (s, Ar, 2H); 13C NMR d 35.05, 56.00, 69.80, 70.13,
70.84, 71.86, 114.14, 116.93, 132.21, 134.43, 147.03,
151.93; Anal. Calcd for C24H28Br4O7: C, 38.53; H, 3.77.
Found: C, 38.26; H, 3.99.
4.9. 2,20-(17-crown-5)-3,30-Dimethoxy-5,50-dimethyl-1,10-
biphenyl 8
4.12. 2,20-(17-crown-5)-3,30-Dimethoxy-5,50-dibromomethyl-
To a stirred solution of K2CO3 (6 g, 44 mmol) in dry DMF
(200 mL) were added under N2 a solution of 6 (1.22 g,
4.45 mmol) in dry DMF (200 mL) and a solution of tetra-
ethyleneglycol ditosylate (2.23 g, 4.45 mmol) in dry DMF
(200 mL) at rt. The mixture was stirred at 60 ꢁC for 12 h.
Water (1000 mL) was added, and the organic phase
extracted with diethyl ether. The organic extract was dried
over Na2SO4 and concentrated to afford a brown solid. The
crude material was purified by flash chromatography using
a 1:1 mixture of ethyl acetate–petroleum, as eluent, to give
1,10-biphenyl 10
To a stirred solution of 8 (0.32 g, 0.7 mmol) in dry CCl4
(15 mL) was added NBS (0.3 g, 1.63 mmol) at rt under
N2. The solution was irradiated with a 200 W lamp for
1 h at reflux and then filtered. The solvent was evapo-
rated to obtain a brown solid. The crude material was
purified by flash chromatography using a 2:1 mixture
of acetone–petroleum, as eluent, to give 10 (0.21 g,
30%). Compound 10: mp 64–65 ꢁC. 1H NMR 3.94 (s,
6H), 3.35–4.20 (series of m, 16H), 4.51 (s, 4H), 6.96 (d,
J = 2.4 Hz, Ar, 2H), 7.01 (d, Ar, J = 2.4 Hz, Ar, 2H);
13C NMR d 34.14, 55.97, 70.15, 70.27, 70.48, 71.96,
112.16, 124.26, 129.88, 131.57, 132.50, 153.06; Anal.
Calcd C24H30Br2O7: C, 48.83; H, 5.12. Found: C,
48.99; H, 5.08.
1
8 (0.96 g, 50%): 8: mp 82–83 ꢁC. H NMR d 2.35 (s, 6H),
3.91 (s, 6H), 3.50–4.20 (series of m, 16H), 6.73 (s, Ar,
4H); 13C NMR d 21.32, 55.93, 70.11, 70.32, 70.70, 71.62,
112.41, 123.82, 132.21, 132.62, 143.77, 152.49; Anal. Calcd
for C24H32O7: C, 66.63; H, 7.46. Found: C, 66.55; H, 7.49.
4.10. 2,20-(17-crown-5)-3,30-Dimethoxy-5,50-dimethyl-6,60-
dibromo-1,10-biphenyl 7
4.13. Interconversion measurements of (aR)-(+)-1
From 1: To a stirred solution of K2CO3 (21 g, 151 mmol)
in dry DMF (400 mL) were added a solution of 1 (6.56 g,
15.18 mmol) in dry DMF (200 mL) and a solution of
tetraethyleneglycol ditosylate (7.6 g, 15.18 mmol) in dry
DMF (200 mL) at rt under N2. The mixture was stirred
at 60 ꢁC for 16 h. Water (1000 mL) was added, and the
solution extracted with diethyl ether. The organic extract
was dried (Na2SO4) and concentrated to afford a brown
solid. The crude material was purified by flash chromatog-
raphy using a 4:6 mixture of ethyl acetate–petroleum, as
eluent, to give 7 (3.58 g, 40%). From 8: To a stirred solu-
tion of 8 (3.2 g, 7 mmol) in CCl4 (100 mL) were added
NBS (3 g, 16.3 mmol) and BPO (0.17 g, 0.7 mmol) at rt
and under N2. The solution was stirred at rt for 12 h
and then filtered. The solvent was evaporated to obtain a
brown solid. The crude material was purified by flash chro-
matography using a 4:6 mixture of ethyl acetate–petro-
leum, as eluent, to give 7 (2.64 g, 64%). Compound 7:
mp 124 ꢁC. 1H NMR d 2.43 (s, 6H), 3.90 (s, 6H),
3.40–4.30 (series of m, 16H), 6.90 (s, Ar, 2H); 13C
NMR d 23.84, 55.91, 69.75, 70.13, 70.99, 71.52, 113.98,
116.81, 133.13, 134.19, 144.62, 151.45; Anal. Calcd for
C24H30Br2O7: C, 48.83; H, 5.12. Found: C, 48.85; H,
5.32.
Racemization measurements were performed by detection
of the optical rotatory activity of (aR)-(+)-1 in xylene after
heating the solution at 100 and 130 ꢁC at 0, 15, 30, 60, 120,
240, and 720 min, respectively. Optical rotatory activity
was unaffected at 100 ꢁC after 720 min at least, whereas lit-
tle but significant decrease of [a]D value was observed with-
in 15 min when (aR)-(+)-1 was heated at 130 ꢁC.
4.14. Interconversion measurements of
(aR,1R,10R,2S,20S,5R,50R)-(ꢀ)-16
Racemization measurements were performed by
detection of the presence of the other diastereomer
(aS,1R,10R,2S,20S,5R,50R)-(+)-16 when
a solution of
(aR,1R,10R,2S,20S,5R,50R)-(ꢀ)-16 in DMSO-d6 was
recorded by 1H NMR after heating at 100, 120 and 130 ꢁC.
4.15. X-ray structure determination of
(aS,1R,10R,2S,20S,5R,50R)-(+)-16
Crystal description: Colourless prism 0.32 · 0.26 ·
0.23 mm. Mr = 796.62, hexagonal, space group P3121,
3
˚
˚
a = b = 12.3105(17), c = 23.674(5) A, V = 3107.0(9) A ,
Z = 3, T = 293(2) K, l = 2.002 mmꢀ1. X-ray data were
collected on a Bruker Smart Apex CCD area detector using
4.11. 2,20-(17-crown-5)-3,30-Dimethoxy-5,50-dibromomethyl-
graphite-monochromated
Mo Ka
radiation
(k =
6,60-dibromo-1,10-biphenyl 9
0.71073 A). Data reduction was made using SAINT pro-
grams; absorption corrections based on multiscan were ob-
tained by SADABS.35 71,155 measured reflections, 7664
independent reflections, 3665 reflections with I > 2r(I),
3.82 < 2h < 66.40ꢁ, Rint = 0.052. The structure was solved
˚
To a stirred solution of 7 (1 g, 1.69 mmol) in dry CCl4
(15 mL) were added NBS (0.66 g, 3,72 mmol) and BPO
(0.04 g, 0.17 mmol) at rt under N2. The solution was stirred
at 70 ꢁC for 4 h and then filtered. The solvent was evapo-
rated to obtain a brown solid. The crude material was puri-
fied by flash chromatography using a 1:1 mixture of ethyl
acetate–petroleum, as eluent, to give 9 (0.76 g, 60%). Com-
by SIR-92 and refined on F2 by full-matrix least-squares
36
using SHELXL-97.37 Refinement on 7664 reflections, 217
parameters. Flack parameter38 for determination of the
absolute configuration = 0.005(9). Final R = 0.0407,
wR = 0.1077 for data with F2 > 2r(F2), (D/r)max = 0.001,
1
pound 9: mp 144–145 ꢁC. H NMR d 3.89 (s, 6H), 3.40–
Dqmax = 0.51, Dqmin = ꢀ0.27 Aꢀ3. The detailed structural
˚
4.40 (series of m, 16H), 4.63 (AB system, J = 10 Hz, 4H),

422
D. Fabbri et al. / Tetrahedron: Asymmetry 18 (2007) 414–423
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Am. Chem. Soc. 2003, 125, 15433–15442; (d) Solladie, G.
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parameters have been deposited with Cambridge Crystallo-
graphic Data Centre under the number CCDC 610996.
4.16. Cell lines and proliferation assays of ( )-1, (aR)-(+)-1
and (aS)-(ꢀ)-1
Three human malignant melanoma cell lines (LCM–Mel,
GR-Mel, CN Mel–A), obtained from Istituto Dermopatico
dell’Immacolata, Department of Molecular and Cellular
Biology, Rome, were used to evaluate potential anti-prolif-
erative activity of biphenyl 1, as racemic mixture ( )-1 and
in enantiopure forms (aR)-(+)-1 and (aS)-(ꢀ)-1. Before
testing, cells were grown to confluence in tissue culture
flasks using RPMI medium (Invitrogen, Carlsbad, CA,
USA) supplemented with 10% FBS and penicillin/strepto-
mycin [100 IU (50 lg)/mL] in a humidified 5% CO2 atmo-
sphere at 37 ꢁC. For proliferation assays cells were then
plated in 96-well plates (3 · 103/well) in their complete
medium, in quadruplicate. After 24 h, the medium was re-
moved and replaced on days 1, 3, and 5, by the same med-
ium (control) or supplemented with various doses of 1 at
final concentrations comprising between 10 and 100 lM.
The cells were observed with an inverted microscope every
24 h to check on morphological changes, suffering or cell
death. The percentage of cell proliferation was estimated
on day 6 by the colorimetric assay of Kueng et al.39 mod-
ified as follows: cells were fixed for 20 min at room temper-
ature with 4% paraformaldehyde (PFA), stained with 0.1%
crystal violet in 20% methanol for 20 min, washed in PBS,
solubilized with 10% acetic acid and read at 595 nm in a
microplate reader (VersamaxTM, Molecular Devices, USA).
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Yamashita, M.; Komori, T.; Umehara, K.; Imanaka, H.
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Acknowledgements
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This work was supported by the C.N.R.—Rome. We wish
to thank Dr. Daniele Castiglia from Istituto Dermopatico
dell’Immacolata, Department of Molecular and Cellular
Biology, Rome, Italy, for giving melanoma cell lines.
´
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