Synthesis and preliminary in vitro cytotoxic activity of new triphenylethylene dimers

Article abstract of DOI:10.1006/bioo.1999.1141

We have synthesized a series of six nonsteroidal homo- and heterobifunctional estrogenic dimers designed for the treatment of breast cancer. They are made of two triphenylethylene moieties linked by an aliphatic chain. The synthesis used six steps, from the known alcohol 5, with an overall yield of more than 60%. This article describes the synthesis of these products and their in vitro biological activity on two human breast cancer cell lines: MCF-7 and MDA-MB-231. The dimers are generally less active than tamoxifen, which presents an IC₅₀ = 16 and 40 μm on MCF-7 and MDA-MB- 231 cell lines, respectively. However, the symmetrical dimer bearing six hydroxy functions possesses the best in vitro cytotoxic activity of the series, showing an IC₅₀ = 38 μm on both types of cells. It was observed that the cytotoxicity of the dimers increases with the number of hydroxy groups present on the aromatic rings.

Full text of DOI:10.1006/bioo.1999.1141

Bioorganic Chemistry 27, 383–394 (1999)
Article ID bioo.1999.1141, available online at http://www.idealibrary.com on
Synthesis and Preliminary in Vitro Cytotoxic Activity of
New Triphenylethylene Dimers
Serge Groleau, Jose´e Nault, Martine Lepage, Michelle Couture,
Normand Dallaire, Gervais Be´rube´,1 and Rene´ C.-Gaudreault*
De´partement de Chimie-Biologie, Universite´ du Que´bec a` Trois-Rivie`res, C.P. 500,
Trois-Rivie`res, Que´bec, Canada G9A 5H7; and *Unite´ de Biotechnologie, Institut des
Biomate´riaux du Que´bec, C.H.U.Q., Pavillon Saint-Francois d’Assise, 10 Rue de l’Espinay,
¸
Que´bec, Que´bec, Canada GIL 3L5
Received December 7, 1998
We have synthesized a series of six nonsteroidal homo- and heterobifunctional estrogenic
dimers designed for the treatment of breast cancer. They are made of two triphenylethylene
moieties linked by an aliphatic chain. The synthesis used six steps, from the known alcohol
5, with an overall yield of more than 60%. This article describes the synthesis of these products
and their in vitro biological activity on two human breast cancer cell lines: MCF-7 and MDA-
MB-231. The dimers are generally less active than tamoxifen, which presents an IC50 ϭ 16
and 40 ␮M on MCF-7 and MDA-MB-231 cell lines, respectively. However, the symmetrical
dimer bearing six hydroxy functions possesses the best in vitro cytotoxic activity of the series,
showing an IC50 ϭ 38 ␮M on both types of cells. It was observed that the cytotoxicity of the
dimers increases with the number of hydroxy groups present on the aromatic rings.
᭧ 1999
Academic Press
INTRODUCTION
The synthesis of new antiestrogenic molecules has attracted considerable interest
because of their great therapeutic value in treating a number of hormone-dependent
human cancers. For example, several research groups have reported various methodol-
ogies for the synthesis of steroidal and non-steroidal antiestrogens. The bulk of the
work led to the study of the estradiol pharmacophore and a recent proposal of a
model for the receptor binding site (1). Some new molecules have allowed a detailed
mechanistic study of antiestrogen action on human breast cancer cells (2–11).
The known antiestrogens are competitive inhibitors of estrogen binding to the
estrogen receptor (ER). Simply, they reduce the ability of estradiol to stimulate nuclear
transcription and ultimately cell growth. The exact mechanism(s) by which pure
antiestrogens achieve a complete ER blockade is still a matter of debate. Several
mechanisms of action were observed: pure antiestrogens (1) reduce DNA binding by
1 To whom correspondence should be addressed. Fax: (819) 376-5084. E-mail: gervais berube@uqtr.
uquebec.ca.
383
0045-2068/99 $30.00
Copyright ᭧ 1999 by Academic Press
All rights of reproduction in any form reserved.

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GROLEAU ET AL.
interfering with receptor dimerization (7); (2) induce conformational changes of the
receptor that allow binding to DNA but do not promote events needed for gene
transcription (inactivation of the two transcription activation functions AF1 and AF2)
(8); (3) cause a rapid disappearance of the ER from the target tissue (degradation of
the ER), resulting in an insufficient amount of ER to bind the native ligand (estradiol)
and elicit agonist responses (9,10); and (4) inhibit nucleocytoplasmic shuttling of the
ER (diffusion out of the nucleus and degradation) by blocking its nuclear uptake (11).
It should be pointed out that pure antiestrogens could block completely the ER function
by a combination of several of these processes.
DIMERS AS ANTIESTROGENS
The synthesis of an estrogenic bivalent ligand could, theoretically, interfere with
the step of receptor dimerization. This concept was first studied by the group of
Professor Katzenellenbogen (12). They made several dimeric molecules that were
analogs of hexestrol (see 2, Scheme 1). Some symmetrical dimers showed interesting
antiestrogenic activity (12). A bivalent ligand could act as an antiestrogen either by
preventing the ER dimerization or, if there is impaired dimerization, by preventing
its association to the estrogen response element located on DNA. These two modes
of action would inhibit cell growth.
In order to further investigate the effect of a bivalent ligand on the ER, we have
synthesized a series of nonsteroidal homo- and heterobifunctional dimers 1. The
chemical structure of these new dimers is based on a nonsteroidal triphenylethylene
system. It was previously demonstrated that this type of system bearing two or three
hydroxy groups can interact with the ER (13). The triphenylethylene portions are
joined together with an 11-carbon-atom aliphatic chain. The choice of the length of
the linking chain is important as it was reported that such length gives molecules a
good estrogen receptor binding affinity (RBA, relative binding affinity (estradiol,
RBA ϭ 100%)) (12). Hexestrol dimer 2 with a similar linking chain length possesses
a RBA value of 6.9% at 25ЊC. Therefore, the dimers are designed to interact with
the ER, particularly when the triphenylethylene portion is hydroxylated. The nonhy-
droxylated systems were made as reference compounds. The new dimers are analogs
of tamoxifen (3; TAM). Tamoxifen, a nonsteroidal antiestrogen, has been used for
the treatment and more recently for the prevention of breast cancer. This article
describes the synthesis of these new homo- and heterobifunctional dimers and reports
on their in vitro cytotoxic activity on two neoplastic human breast cancer cells: MCF-
7(ER+) and MDA-MB-231 (ERϪ).
EXPERIMENTAL
Materials and Methods
Anhydrous reactions were performed under an inert atmosphere, the setup assembled
and cooled under dry nitrogen. Unless otherwise noted, starting material, reagents,
and solvents were obtained commercially and were used as such or purified and dried
by standard means (14). Organic solutions were dried over anhydrous magnesium
sulfate and evaporated on a rotary evaporator and under reduced pressure. All reactions
were monitored by thin-layer chromatography (TLC). The plates were visualized by

NEW NONSTEROIDAL DIMERS
385
SCHEME 1
UV fluorescence at 254 nm. Commercial TLC plates were Sigma T 6145 (polyester
˚
silica gel 60 A, 0.25 mm). Flash chromatography was performed according to the
method of Still and co-workers on Merck Grade 60 silica gel, 230–400 mesh (15).
All solvents used in chromatography were distilled. Melting points (mp) were recorded
on an Electrothermal 9100 apparatus and are uncorrected. The infrared spectra (IR)
were taken on a Perkin–Elmer 1430 IR or on a Nicolet Impact 420 FT–IR spectropho-
tometer. Mass spectral assays (MS, m/z) were obtained using a VG Micromass 7070
HS instrument (Universite´ de Sherbrooke) using an ionization energy of 70 eV. Nuclear
magnetic resonance (NMR) spectra were obtained in CDCl3 solution on a Bruker
AMX2 (500 MHz) instrument. Chemical shifts were measured relative to internal
1
standards: tetramethylsilane (␦ 0.0 ppm) for H and CDCl3 (␦ 77.0 ppm) for 13C
NMR. The number of carbons assigned to a peak, on 13C NMR spectra, is indicated
in brackets when it is more than one carbon. Multiplicities are described by the
following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), 2d

386
GROLEAU ET AL.
(two doublets), dd (double doublet), dt (double triplet), m (multiplet), and so on. The
NMR assignments were assisted by heteronuclear multiple-quantum correlation and
by correlated spectroscopy 2-D spectra (16). Spectral data are presented completely
for only one member of every family of molecules with the exception of the final
dimers 1, for which spectral data are completely described.
Synthesis of Tosylates 6
Tosyl chloride (3.37 g, 17.68 mmol) dissolved in methylene chloride (15 ml) was
added to a mixture of alcohol 5 (1.83 g, 3.54 mmol) and triethylamine (1.79 g, 17.67
mmol) in methylene chloride (35 ml). The reaction mixture was stirred at room
temperature (22ЊC) for 18 h under a nitrogen atmosphere. Then, most of the solvent
was evaporated and the residue was transferred to an extraction flask with ether (20
ml) and water (5 ml). The organic phase was washed with water (5 ϫ 5 ml), dried,
filtered, and concentrated to a viscous oil. The crude tosylate 6 was purified by flash
column chromatography (hexane:acetone, 9:1 to 85:15). The yield was 95%.
12,13,13-Triphenyl-12-tridecenyl tosylate (6a). MS (m/z) 580 (M+), 408
(M+–C7H8O3S), 269 (M+–C17H27O3S): Exact mass calcd for C38H44O3S: 580.3011.
Found: 580.3016.
13,13-Bis(4Ј-methoxyphenyl)-12-phenyl-12-tridecenyl tosylate (6b). MS (m/z) 640
(M+), 468 (M+–C7H8O3S), 329 (M+–C17H27O3S): Exact mass calcd for C40H48O5S:
640.3222. Found: 640.3210.
12,13,13-Tris(4Ј-methoxyphenyl)-12-tridecenyl tosylate (6c): IR (thin film, ␷
,
max
cmϪ1) 1600 (CϭC), 1240 (CϪO); H NMR (␦ ppm) 7.78 and 7.31 (4H, 2ϫ d, J ϭ
7.8 Hz, CH3-Ar-SO2), 7.13 and 6.86, 7.01 and 6.70, 6.78 and 6.55 (12H, 3ϫ 2d,
J ϭ 8.1, 8.4, and 8.7 Hz, 3ϫ methoxyphenyl), 4.01 (2H, t, J ϭ 6.5 Hz, CH2O), 3.80,
3.74, and 3.67 (9H, 3ϫ s, 3ϫ OCH3), 2.42 (3H, s, Ar-CH3), 2.40 (2H, m, CϭCϪCH2),
1.65–1.58 (2H, m, CH2CH2O), 1.35–1.14 (16H, m, (CH2)8); 13C NMR (␦ ppm)
158.14, 157.67, 157.31, 144.58, 139.38, 137.52, 136.53, 136.11, 135.12, 133.32,
131.89 (2), 130.61 (4), 129.78 (2), 127.85 (2), 113.40 (2), 113.25 (2), 112.75 (2),
70.68, 55.17, 55.04, 54.97, 35.88, 29.71, 29.43, 29.40, 29.32, 29.26, 28.97, 28.89,
28.80, 25.31, 21.59; MS (m/z) 670 (M+), 498 (M+–C7H8O3S), 359 (M+–C17H27O3S):
Exact mass calcd for C41H50O6S: 670.3328. Found: 670.3336.
1
Synthesis of Iodides 7
A mixture of tosylate 6 (3.43 mmol) and sodium iodide (2.57 g, 17.15 mmol) in
anhydrous acetone (25 ml) was heated to reflux for 8 h. After evaporation, the residue
was diluted with ether (20 ml) and washed with water (5 ϫ 5 ml). The ethereal
phase was dried, filtered, and evaporated and the residue purified by flash column
chromatography (hexane:acetone, 95:5) to produce iodide 7 (99% yield) as a vis-
cous oil.
1-Iodo-12,13,13-triphenyl-12-tridecene (7a). MS (m/z) 536 (M+), 269
(M+–C10H20I). Exact mass calcd for C31H37I: 536.1940. Found: 536.1933.
1-Iodo-13,13-bis(4Ј-methoxyphenyl)-12-phenyl-12-tridecene (7b). MS (m/z) 596
(M+), 468 (M+–HI), 329 (M+–C10H20I). Exact mass calcd for C33H41O2I: 596.2151.
Found: 596.2145.
1-Iodo-12,13,13-tris(4Ј-methoxyphenyl)-12-tridecene (7c). IR (thin film, ␷
,
max

NEW NONSTEROIDAL DIMERS
1
387
cmϪ1) 1600 (CϭC), 1240 (CϪO); H NMR (␦ ppm) 7.13 and 6.86, 7.01 and 6.70,
6.78 and 6.55 (12H, 3ϫ 2d, J ϭ 8.8, 8.1, and 7.4 Hz, 3ϫ methoxyphenyl), 3.81,
3.75, and 3.68 (9H, 3ϫ s, 3ϫ OCH3), 3.17 (2H, t, J ϭ 7.3 Hz, CH2-I), 2.40 (2H, m,
CϭCϪCH2), 1.80 (2H, p, J ϭ 7.3 Hz, CH2CH2I), 1.40–1.10 (16H, m, (CH2)8); 13C
NMR (␦ ppm) 158.11, 157.64, 157.28, 139.38, 137.49, 136.54, 136.11, 135.12, 131.88
(2), 130.61 (4), 113.38 (2), 113.24 (2), 112.74 (2), 55.18, 55.06, 54.97, 35.87, 33.55,
30.48, 29.70, 29.45 (2), 29.36, 29.26, 28.97, 28.51, 7.25; MS (m/z) 626 (M+), 498
(M+–HI), 359 (M+–C10H20I): Exact mass calcd for C34H43O3I: 626.2257. Found:
626.2252.
Synthesis of Ketones 8
To a stirred suspension of sodium hydride (448 mg, 11.2 mmol, 60% dispersion
in mineral oil) in a mixture of tetrahydrofuran and dimethyl sulfoxide (THF:DMSO,
9:1, 150 ml) was added the appropriate ketone 4a, 4b, or 4c (10.2 mmol). The reaction
mixture was heated in a water bath (45ЊC) for 1 h under a nitrogen atmosphere. After
cooling, the required iodide 7a, 7b, or 7c (5.1 mmol) dissolved in THF (10 ml) was
added dropwise and the resulting mixture stirred overnight at room temperature (18
h, 22ЊC). Then, most of the solvent was evaporated and the residue was diluted with
ether (200 ml) and treated with an aqueous solution of sodium thiosulfate (5%, 50
ml). The ethereal phase was washed thoroughly with water (6 ϫ 50 ml), dried, filtered,
and evaporated to give an oil that was purified by flash column chromatography
(hexane:acetone, 98:2 to 9:1). A viscous oil was obtained. The yield was 85%.
NB: The following combinations 0-0, 0-1, 0-2, 0-3, 2-1, 2-2, 2-3, and 3-3, found
next to the products’ numbers, represent the number of methoxy or hydroxy groups
on the dimeric molecule.
1,2,14,15,15-Pentaphenyl-14-pentadecen-1-one (8, 0-0). MS (m/z) 604(M+), 269
(M+–C24H31O). Exact mass calcd for C45H48O: 604.3705. Found: 604.3701.
1-(4Ј-Methoxyphenyl)-2,14,15,15-tetraphenyl-14-pentadecen-1-one (8, 0-1). MS
(m/z) 634 (M+). Exact mass calcd for C46H50O2: 634.3811. Found: 634.3800.
1,2-Bis(4Ј-methoxyphenyl)-14,15,15-triphenyl-14-pentadecen-1-one (8, 0-2). MS
(m/z) 664 (M+), 529 (M+–C8H7O2), 269 (M+–C26H35O3): Exact mass calcd for
C47H52O3: 664.3916. Found: 664.3909.
1,15,15-Tris(4Ј-methoxyphenyl)-2,14-diphenyl-14-pentadecen-1-one (8, 2-1). MS
(m/z) 694 (M+), 329 (M+–C25H33O2): Exact mass calcd for C48H54O4: 694.4022.
Found: 694.4011.
1,14,15,15-Tetrakis(4Ј-methoxyphenyl)-2-phenyl-14-pentadecen-1-one (8, 2-2).
MS (m/z) 724 (M+), 359 (M+–C25H33O2): Exact mass calcd for C49H56O5: 724.4128.
Found: 724.4119.
1,2,14,15,15-Pentakis(4Ј-methoxyphenyl)-14-pentadecen-1-one (8, 3-2). IR (thin
1
film, ␷max, cmϪ1) 1670 (CϭO), 1600 (CϭC), 1245 (CϪO); H NMR (␦ ppm) 7.95
and 6.80, 7.22 and 6.84, 7.13 and 6.86, 7.02 and 6.69, 6.78 and 6.55 (20H, 5ϫ 2d,
J ϭ 8.7, 6.3, 8.1, 8.7, and 8.0 Hz, 5ϫ 4Ј-methoxyphenyl), 4.43 (1H, t, J ϭ 7.0 Hz,
CHϪCO), 3.78, 3.77, 3.72, 3.71, and 3.65 (15H, 5ϫ s, 5ϫ OCH3), 2.40 (2H, m,
CϭCϪCH2), 2.12 and 1.77 (2H, 2ϫ m, CH2CHϪCO), 1.30–1.14 (18H, m, (CH2)9);
13C NMR (␦ ppm) 198.83, 163.15, 158.43, 158.12, 157.65, 157.29, 139.42, 137.48,
136.52, 136.13, 135.12, 132.31, 131.89 (2), 130.86 (2), 130.60 (4), 129.99, 129.10

388
GROLEAU ET AL.
(2), 114.18 (2), 113.63 (2), 113.39 (2), 113.24 (2), 112.74 (2), 55.33, 55.14, 55.12,
55.01, 54.94, 52.32, 35.88, 34.07, 29.72, 29.64, 29.55 (2), 29.49, 29.46, 29.29, 28.98,
27.71; MS (m/z) 754 (M+), 359 (M+–C26H35O3): Exact mass calcd for C50H58O6:
754.4233. Found: 754.4227.
Procedure for the Preparation of p-Methoxyphenyllithium
4-Bromoanisole (145 ␮l, 1.16 mmol) was dissolved in a mixture of dry THF:Et2O
(6:4, 10 ml). The reaction mixture was cooled down to Ϫ110ЊC by means of a light
petroleum ether/acetone/isopropanol/liquid nitrogen slush bath and kept under nitrogen
(17). A solution of n-butyllithium in hexane (1.6 M, 725 ␮l, 1.16 mmol) was added
to the solution and the mixture stirred for 30 min. Stirring was continued for a further
90 min, as the temperature was allowed to rise to ca. Ϫ60ЊC. The freshly prepared
p-methoxyphenyllithium was used as such for the synthesis of the 1,14-pentadeca-
dienes 1 (R and RЈ ϭ H or OCH3). Phenyllithium is available commercially.
Synthesis of 1,14-Pentadecadienes 1 (R and RЈ ϭ H or OCH3)
The freshly prepared organolithium derivative was added to a solution of ketone
8 (0.29 mmol) in a mixture of dry THF:Et2O (6:4, 10 ml). The reaction mixture was
stirred at Ϫ110ЊC for 30 min under a nitrogen atmosphere. Afterward, the reaction
mixture was diluted with ether (40 ml) and a solution of ammonium chloride was
added (20 ml, 10% aqueous). The phases were separated and the organic phase was
washed with water (3 ϫ 10 ml), dried, and evaporated to give the tertiary alcohol
intermediate. This alcohol was dehydrated in 25 ml 95% ethanol in the presence of
p-toluenesulfonic acid (TsOH) (10 mg, 0.04 mmol) heated to reflux for 3 h. After
evaporation of the solvent, the residue was taken with ether (30 ml) and extracted
with water (3 ϫ 10 ml). The ethereal phase was dried, filtered, and evaporated to a
viscous oil. Flash column chromatography (hexane:acetone, 100:0 to 85:15) gave
compound 1 (0.26 mmol) (R and RЈ ϭ H or OCH3) in 90% yield either as a viscous
oil or as a solid.
1,1,2,14,15,15-Hexaphenyl-1,14-pentadecadiene (1, 0-0). mp 113–114ЊC; IR
1
(KBr, ␷max, cmϪ1) 1590 (CϭC); H NMR (␦ ppm) 7.35–6.80 (30H, m, Ar-H), 2.42,
(4H, m, 2ϫ CϭCϪCH2), 1.4–1.1 (18H, m,(CH2)9); 13C NMR (␦ ppm) 143.64 (2),
143.17 (2), 142.63 (2), 141.25 (2), 139.19 (2), 130.85 (4), 129.70 (4), 129.63 (4),
128.22 (4), 127.91 (4), 127.47 (4), 126.68 (2), 126.23 (2), 125.82 (2), 35.98 (2), 29.77
(2), 29.63, 29.58 (2), 29.37 (2), 28.94 (2); MS (m/z) 664 (M+), 269 (M+–C30H35).
Exact mass calcd for C51H52: 664.4069. Found: 664.4066.
15,15-Bis(4Ј-methoxyphenyl)-1,1,2,14-tetraphenyl-1,14-pentadecadiene (1, 0-2).
MS (m/z) 724 (M+), 329 (M+–C30H35). Exact mass calcd for C53H56O2: 724.4280.
Found: 724.4276.
14,15,15-Tris(4Ј-methoxyphenyl)-1,1,2-triphenyl-1,14-pentadecadiene (1, 0-3).
MS (m/z) 754 (M+), 359 (M+–C30H35). Exact mass calcd for C54H58O3: 754.4386.
Found: 754.4368.
1,1,15,15-Tetrakis(4Ј-methoxyphenyl)-2,14-diphenyl-1,14-pentadecadiene (1, 2-2).
MS (m/z) 784 (M+), 329 (M+–C32H39O2). Exact mass calcd for C55H60O4: 784.4491.
Found: 784.4484.
1,1,14,15,15-Pentakis(4Ј-methoxyphenyl)-2-phenyl-1,14-pentadecadiene (1, 2-3).

NEW NONSTEROIDAL DIMERS
389
MP 117ЊC; MS (m/z) 814 (M+), 359 (M+–C32H39O2). Exact mass calcd for C56H62O5:
814.4597. Found: 814.4610.
1,1,2,14,15,15-Hexakis(4Ј-methoxyphenyl)-1,14-pentadecadiene (1, 3-3). mp 101–
1
102ЊC; IR (KBr, ␷max, cmϪ1) 1600 (CϭC), 1240 (CϪO); H NMR (␦ ppm) 7.13 and
6.85, 7.01 and 6.69, 6.77 and 6.55 (24H, 3ϫ 2d, J ϭ 7.6, 8.1, and 8.2 Hz, 6ϫ 4Ј-
methoxyphenyl), 3.80, 3.74, and 3.68 (18H, 6ϫ s, 6ϫ OCH3), 2.38 (4H, m, 2ϫ
CϭCϪCH2), 1.3–1.0 (18H, m, (CH2)9); 13C NMR (␦ ppm) 158.17 (2), 157.70 (2),
157.33 (2), 139.47 (2), 137.52 (2), 136.59 (2), 136.18 (2), 135.19 (2), 131.94 (4),
130.66 (8), 113.43 (4), 113.29 (4), 112.79 (4), 55.22 (2), 55.10 (2), 55.03 (2), 35.94
(2), 35.79 (2), 29.58 (3), 29.37 (2), 29.04 (2); MS (m/z) 844 (M+), 359 (M+–C33H41O3).
Exact mass calcd for C57H64O6: 844.4703. Found: 844.4697.
Synthesis of Hydroxylated Dimers 1 (R or RЈ ϭ H or OH)
The 1,14-pentadecadiene 1 (R or RЈ ϭ H or OCH3) (0.54 mmol) was dissolved in
dichloromethane (15 ml) and the resulting solution was cooled down to Ϫ60ЊC, under
a nitrogen atmosphere. Then, a solution of boron tribromide (1.0 M in CH2Cl2, 1.89
mmol) was added and the mixture was stirred 15 min at Ϫ60ЊC. The mixture was
allowed to warm to room temperature (22ЊC) and stirred for 18 h. Afterward, the
mixture was refluxed for 2 h. The reaction was cooled down to 0ЊC, before 15 ml
methanol was added. The resulting solution was concentrated to 1–2 ml, treated with
saturated NaHCO3 (30 ml), and extracted with ethyl acetate (5 ϫ 5 ml). The organic
phase was dried, filtered, and evaporated to give a residue which was purified by
flash column choromatography (hexane:acetone, 9:1 to 7:3) to give the hydroxylated
dimer 1 (0.50 mmol) (R or RЈ ϭ H or OH) as a yellow or orange solid. The yield
of this reaction was 90%.
15,15-Bis(4Ј-hydroxyphenyl)-1,1,2,14-tetraphenyl-1,14-pentadecadiene (1, 0-2).
MP 97ЊC; IR (KBr, ␷max, cmϪ1) 3370 (OH), 1600 (CϭC); 1H NMR (␦ ppm) 7.35–6.75
(24H, m, Ar-H and 4Ј-hydroxyphenyl), 6.72 and 6.46 (4H, 2d, J ϭ 8.1 Hz, 4Ј-
hydroxyphenyl), 4.63 and 4.44 (2H, 2ϫ s, 2ϫ Ar-OH), 2.41 (4H, 2ϫ m, 2ϫ
CϭCϪCH2), 1.40–1.00 (18H, m, (CH2)9); 13C NMR (␦ ppm) 154.12, 153.31, 143.52,
143.06, 142.91, 142.52, 141.15, 140.21, 139.02, 137.80, 136.45, 136.09, 132.09 (2),
130.83 (2), 130.72 (2), 129.58 (4), 129.51 (2), 128.09 (2), 127.81 (2), 127.77 (2),
127.33 (2), 126.54, 126.08, 125.89, 125.67, 114.91 (2), 114.24 (2), 35.93, 35.84,
29.70, 29.64, 29.46 (3), 29.25 (2), 28.89, 28.80; MS (m/z) 696 (M+), 301 (M+–C30H35).
Exact mass calcd for C51H52O2: 696.3967. Found: 696.3961.
14,15,15-Tris(4Ј-hydroxyphenyl)-1,1,2-triphenyl-1,14-pentadecadiene (1, 0-3). mp
1
55–60ЊC; IR (KBr, ␷max, cmϪ1) 3389 (OH), 1608 (CϭC), 1254 (CϪO); H NMR
(DMSO-d6, ␦ ppm) 9.32, 9.16, 9.10 (3H, 3ϫ s, 3ϫ Ar-OH), 7.40–6.96 (15H, m, Ar-
H), 6.94 and 6.73, 6.86 and 6.58, 6.62 and 6.43 (12H, 3ϫ 2d, J ϭ 8.0, 8.1, and 8.2
Hz, 3ϫ 4Ј-hydroxyphenyl) 2.36 and 2.31 (4H, m, 2ϫ CϭCϪCH2), 1.3–1.0 (18H,
m, (CH2)9); 13C NMR (DMSO-d6, ␦ ppm) 153.75, 155.20, 154.87, 142.86, 142.59,
141.75, 140.43, 138.63, 138.11, 137.37, 134.44, 134.29, 132.93, 131.28 (2), 130.15
(2), 130.04 (2), 129.99 (2), 129.12 (2), 128.87 (2), 128.12 (2), 127.70 (2), 127.38
(2), 126.55, 126.09, 125.67, 114.72 (2), 114.62 (2), 114.14 (2), 35.20, 35.06, 29.00,
28.75, 28.73 (2), 28.62, 28.58, 28.40, 28.37, 27.91; MS (m/z) 712 (M+), 317
(M+–C30H35). Exact mass calcd for C51H52O3: 712.3916. Found: 712.3907.

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1,1,15,15-Tetrakis(4Ј-hydroxyphenyl)-2,14-diphenyl-1,14-pentadecadiene (1, 2-2).
1
MP 90–100ЊC; IR (KBr, ␷max, cmϪ1) 3396 (OH), 1609 (CϭC); H NMR (acetone-
d6, ␦ ppm) 8.27 and 8.03 (4H, 2ϫ s, 4ϫ Ar-OH), 7.20–6.40 (26H, m, 4ϫ 4Ј-
hydroxyphenyl and 2ϫ Ar-H), 2.45 (4H, m, 2ϫ CϭCϪCH2), 1.40–1.05 (18H, m,
(CH2)9); 13C NMR (acetone-d6, ␦ ppm) 156.38 (2), 155.56 (2), 143.53 (2), 139.37
(2), 139.14 (2), 135.44 (2), 135.10 (2), 132.02 (4), 130.73 (2), 130.85 (4), 129.85
(4), 127.97 (4), 125.98 (2), 115.09 (4), 115.40 (4), 35.92 (2), 29.56, 29.51 (2), 29.31
(2), 28.91 (2) (The secondary carbons (CH2) were determined using a distortionless
enhancement by polarization transfer experiment); MS (m/z) 728 (M+), 301
(M+–C30H35O2). Exact mass calcd for C51H52O4: 728.3865. Found: 728.3859.
1,1,14,15,15-Pentakis(4Ј-hydroxyphenyl)-2-phenyl-1,14-pentadecadiene (1, 2-3).
1
MP 98ЊC; IR (KBr, ␷max, cmϪ1) 3345 (OH), 1600 (CϭC); H NMR (acetone-d6, ␦
ppm) 8.32, 8.29, 8.14, 8.08, and 8.06 (5H, 5ϫ s, 5ϫ Ar-OH), 7.22–6.50 (25H, m,
5ϫ 4Ј-hydroxyphenyl and Ar-H), 2.50 and 2.42 (4H, m, 2ϫ CϭCϪCH2), 1.40–1.00
(18H, m, (CH2)9); 13C NMR (acetone-d6, ␦ ppm) 156.36, 156.22, 155.73, 155.54,
155.38, 143.52, 139.37, 139.13 (2), 138.22, 135.79, 135.51, 135.43, 135.10, 134.31,
132.02 (4), 130.87 (2), 130.74 (4), 129.84 (2), 127.96 (2), 125.97, 115.09 (2), 115.05
(2), 114.92 (2), 114.39 (4), 35.88 (2), 29.79, 29.66, 29.58 (2), 29.51, 28.89, 28.74 (2
carbons were hidden by acetone-d6); MS (m/z) 744 (M+), 317 (M+–C30H35O2). Exact
mass calcd for C51H52O5: 744.3814. Found: 744.3809.
1,1,2,14,15,15-Hexakis(4Ј-hydroxyphenyl)-1,14-pentadecadiene (1, 3-3). MP 90–
100ЊC; IR (KBr, ␷max, cmϪ1) 3340 (OH), 1616 (CϭC), 1239 (CϪO); 1H NMR (acetone-
d6, ␦ ppm) 8.28, 8.13, and 8.05 (6H, 3ϫ s, 6ϫ Ar-OH), 7.04 and 6.82, 6.96 and 6.64,
6.71 and 6.50 (24H, 3ϫ 2d, J ϭ 8.8, 7.8, and 8.4 Hz, 6ϫ 4Ј-hydroxyphenyl), 2.41
(4H, m, 2ϫ CϭCϪCH2), 1.4–1.1 (18H, m, (CH2)9); 13C NMR (acetone-d6, ␦ ppm)
156.81 (2), 156.32 (2), 155.98 (2), 139.74 (2), 138.83 (2), 136.38 (2), 136.11 (2),
134.91 (2), 132.62 (4), 131.48 (4), 131.35 (4), 115.66 (4), 115.53 (4), 114.99 (4),
35.88 (2) (9 carbons were hidden by acetone-d6); MS (m/z) 760 (M+), 317
(M+–C30H35O3). Exact mass calcd for C51H52O6: 760.3764. Found: 760.3765.
Cytotoxicity Assay
Chemosensitivity of cell lines to TAM and the dimers were determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (18). This
colorimetric assay uses the ability of viable cells to reduce a soluble tetrazolium salt,
MTT, into an insoluble formazan precipitate.
Materials. (a) Drugs: The dimers (1) were synthesized as described previously.
Tamoxifen was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI).
(b) Cell lines and culture: Human breast cancer cell lines MCF-7 and MDA-MB-231
were obtained from the American Type Culture Collection (Rockville, MD). Both
types of cells were propagated in RPMI 1640 supplemented with 2 mM glutamine,
10% bovine serum (Gibco, Burlington, Ontario, Canada), and 100 U gentamycin/ml
(Sigma Chemical Company, St. Louis, MO). (c) Phosphate-buffered saline (PBS; pH
7.4) was prepared from PBS tablets (Oxford, Unipath Ltd., England) dissolved in
water as per the manufacturer’s instructions. (d) Microtiter plates (96 wells) were
obtained from Flow Labs, Inc. (McClean, VA). (e) MTT and DMSO were obtained

NEW NONSTEROIDAL DIMERS
391
from Sigma Chemical Company. (f) Plate reader was the Behring Elisa Processor II
(Behring, Marburg, Germany).
Method. A stock solution of TAM or the dimers 1 (400 ␮M in fresh RPMI 1640,
1% DMSO) was prepared from an initial solution of 40 mM in DMSO. The cells
(2000 cells/well) were grown in sterile 96-well culture plates at 37ЊC in a humidified
incubator with 5% CO2 for 24 h and then incubated for 72 h with TAM or the dimers
1 diluted in fresh culture medium (final concentration 0.1 to 400 ␮M). The test was
performed in 8 wells for each dilution. After incubation, supernatant was discarded
and 50 ␮l MTT (2.5 mg/ml into PBS:RMPI 1640, 1:4 v/v) was added and reincubated
for 4 h at 37ЊC. The cells were washed with PBS, the formazan precipitate was
solubilized in DMSO (100 ␮l, containing 1% glycine:NaOH 0.1 M; pH 11-12), and
the plate was read spectrophotometrically at 570 nm. Absorbance was recorded and
percentage cell survival was determined by comparing TAM- and dimer-treated cells
with the untreated control.
RESULTS AND DISCUSSION
Synthesis of Homo- and Heterobifunctional Dimers 1
Scheme 2 presents the structure of three alcohols 5a (R ϭ RЈ ϭ H), 5b (R ϭ
OCH3, RЈ ϭ H), and 5c (R ϭ RЈ ϭ OCH3), available in our laboratory, which
were synthesized with the following starting materials: deoxybenzoin (4a), 1-(4Ј-
methoxyphenyl)-2-phenyl-1-ethanone (4b), and desoxyanisoin (4c). These products
were obtained with an overall yield of 65% (13). The alcohols were changed to the
corresponding iodide in two chemical steps. First, the alcohols 5 were transformed
into the corresponding tosylate intermediates 6 with a mixture of tosyl chloride and
triethylamine in methylene chloride. The yield of this reaction was 95%. Treatment
of the tosylate intermediates with sodium iodide in dry acetone gave the desired
iodides 7 quantitatively.
The alkylation of ketones 4 (Scheme 2) was done in a mixture of tetrahydrofuran
and dimethyl sulfoxide (9:1). Reaction of sodium hydride with the ketone 4 gave the
sodium enolate which was immediately treated with the appropriate iodide 7 to give
compound 8. The yield of this reaction was 90%. Then, the addition of n-butyllithium
to 4-bromoanisole in a mixture of dry tetrahydrofuran and ether (THF:Et2O, 6:4) at
Ϫ110ЊC gave the corresponding organolithium reagent (17). The organomagnesium
reagent derived from 4-bromoanisole could also be used; however, being less reactive
than the organolithium reagent it leads to lower yield of reaction. Phenyllithium is
available commercially.
Addition of an excess of p-methoxyphenyllithium (or phenyllithium) to the ketone
8 and subsequent treatment of the crude tertiary alcohol intermediate with TsOH in
ethanol heated to reflux gave the 1,14-pentadecadienes dimers 1 (R and RЈ ϭ H or
OCH3) with a yield of 90%. Finally, demethylation with an excess of boron tribromide
in dichloromethane gave the hydroxylated dimers 1 (R and RЈ ϭ H or OH). The
yield for this step was 93%. This highly efficient synthesis used six steps from the
known alcohol 5, with an overall yield of more than 60%. The global yield from
ketone 4, with nine chemical transformations, is 40%. All new compounds synthesized
1
were homogeneous on TLC and were fully characterized by their IR, H NMR, 13C
NMR, and mass spectra as well as high-resolution mass spectra.

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GROLEAU ET AL.
SCHEME 2
In vitro Cytotoxic Activity
The cytotoxic activity of our new dimers was evaluated in vitro on two human
breast cancer tumor cell lines: MCF-7 and MDA-MB-231. The MCF-7 cell line is
estrogen receptor positive (ER+) while the MDA-MB-231 cell line is estrogen receptor
negative (ERϪ) (19). The cytotoxicity of dimers was tested along with tamoxifen 3,
as the control antiestrogen, on both human mammary carcinoma cell lines in order
to assess the potential antineoplastic selectivity of these molecules. The cytotoxic
activity was evaluated with the colorimetric MTT assay (18). The results are indicated
in Table 1.

NEW NONSTEROIDAL DIMERS
TABLE 1
393
Inhibitory Concentration of TAM and the Dimers on both ER+ and ERϪ Breast Cancer Cell Lines
Number
OHa
MCF-7 (ER+)
MDA-MB-231 (ERϪ)
Dimers
IC50 (␮M)b
IC50 (␮M)b
TAM
0
6
5
4
3
2
0
16
38
74
40
38
50
1 (3-3)
1 (3-2)
1 (2-2)
1 (3-0)
1 (2-0)
1 (0-0)
N/A
90
Ͼ100
Insoluble
N/A
Ͼ100
Ͼ100
Insoluble
Note. N/A, data not available.
a Number of hydroxy functions on the molecule.
b Inhibitory concentration as obtained by the MTT assay.
As shown by the MTT assays, this type of molecule does not present selectivity
toward the ER+ breast cancer cells. The dimers 1 are generally less active than
tamoxifen, which presents an IC50 ϭ 16 and 40 ␮M on MCF-7 and MDA-MB-231
cell lines, respectively. However, the symmetrical dimer 1 (3-3) bearing six hydroxy
functions possesses the best in vitro cytotoxic activity of the series showing an
IC50 ϭ 38 ␮M on both type of cells. The cytotoxicity of the dimers increases with
the number of hydroxy functions on the aromatic rings. The dimer with no hydroxy
function (1 (0-0)) was insoluble and could not be tested. The biological activity seems
to be linked to the solubility of these molecules rather than the strategic location of
the hydroxy functions on the dimers. Comparative biological assays with dimers
bearing a polyethelene chain and those bearing aliphatic chain 1 could verify this
observation. Unfortunately, the present dimeric molecules provide little improvement
over the known hexestrol derivatives 2 (12). New dimers with enhanced cytotoxic
activity must be designed, using the strategy described herein, before a complete
biological evaluation of this type of dimeric molecule is done.
In conclusion, it was possible to synthesize and characterize six new nonsteroidal
triphenylethylene dimers containing an aliphatic linking chain. A polyvalent strategy
allowing the synthesis of symmetrical and asymmetrical molecules was used. This
synthetic path is highly efficient and used six chemical steps from alcohol 5 with an
overall yield of 60%. Also, it was found that cytotoxicity of the dimers 1 is correlated
to the number of hydroxy groups on the molecule.
Work is in progress to synthesize symmetrical hexahydroxylated dimers with poly-
ethylene side chains of varying length. This will allow us to perform comparative
biological tests with dimers bearing alkyl (less soluble) and polyethylene linking
chains (more soluble) in order to better understand the relation between the molecular
structure of the dimers and their antitumoral potential.
ACKNOWLEDGMENTS
This work was supported by the Universite´ du Que´bec a` Trois-Rivie`res. We are grateful to Mr. Jacques
Lacroix, C.H.U.Q., Pavillon Saint-Franc¸ois d’Assise, for biological evaluation studies. We also thank Dr.

394
GROLEAU ET AL.
Gilles Sauve´, Institut Armand-Frappier, for the 1H and 13C NMR spectra and Mr. Gaston Boulay, Universite´
de Sherbrooke, for the mass spectra.
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