A facile synthesis of C2-symmetric 17β-estradiol dimers

Article abstract of DOI:10.1016/S0960-894X(02)00987-3

A rapid and efficient synthesis of a series of C₂-symmetric 17β-estradiol dimers is described. The new molecules are linked at position 17α of the steroid nucleus with either an alkyl chain or a polyethylene glycol chain. They are made from estrone in five chemical steps with an overall yield exceeding 30%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER⁺ and ER^-) human breast tumor cell lines: MCF-7 and MDA-MB-231. Some of the dimers present selective cytotoxic activity against the ER⁺ cell line.

Full text of DOI:10.1016/S0960-894X(02)00987-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 557–560
A Facile Synthesis of C2-Symmetric 17ꢀ-Estradiol Dimers
Daniel Rabouin,a Valerie Perron,a Blaise N’Zemba,a Rene C.-Gaudreaultb and
Gervais Berubea,*
a
´
Departement de Chimie-Biologie, Universite du Quebec a Trois-Rivieres, C.P. 500, Trois-Rivieres, Quebec, Canada G9A 5H7
Unite de Biotechnologie, Institut des Biomateriaux de Quebec, C.H.U.Q., Hopital Saint-Franc¸ois d’Assise, 10 Rue de l’Espinay,
´
´
´
`
´
`
`
´
b
´
ˆ
´
Quebec, Canada G1L 3L5
Received 29May 2002; accepted 1 October 2002
Abstract—A rapid and efficient synthesis of a series of C2-symmetric 17b-estradiol dimers is described. The new molecules are
linked at position 17a of the steroid nucleus with either an alkyl chain or a polyethylene glycol chain. They are made from estrone
in five chemical steps with an overall yield exceeding 30%. The biological activity of these compounds was evaluated in vitro on
estrogen dependent and independent (ER+ and ERꢀ) human breast tumor cell lines: MCF-7 and MDA-MB-231. Some of the
dimers present selective cytotoxic activity against the ER+ cell line.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
estrogenic moieties analogues of hexestrol or triphenyl-
ethylene. It was demonstrated that some of the hexestrol
bivalent ligands possess antiestrogenic activity.9 In some
cases, a biphasic interaction with the estrogen receptor
was observed.9 On the other hand, it was demonstrated
that a symmetrical triphenylethylene dimer bearing six
hydroxy functions, possesses a cytotoxic activity similar
to that of tamoxifen. However, this type of molecule did
not present selectivity towards ER+ breast cancer cells.10
The design of C2-symmetric ligands (bivalent ligands) as
bioactive molecules have attracted considerable attention
over the years because of their promising therapeutic
value in treating a number of diseases. For example,
bivalent ligands were designed as antagonist of the mus-
carinic receptor and of the k opiod receptor.1,2 A variety
of dimeric enzyme inhibitors were also studied such as
HIV-1 protease inhibitors as well as the glycosidases
inhibitors.3,4 Some bivalent ligands were used as prodrugs
with androgenic and myotrophic activities and a dimer of
a naturally occurring monomeric naphthylisoquinoline
alkaloid was prepared yielding an analogue with high
antimalarial activity.5,6 Recently, we have reported the
synthesis of spermidine and norspermidine dimers as high
affinity polyamine transport inhibitors.7 It is generally
believed that a bivalent ligand would be expect to show
enhanced receptor affinity (or biological activity) relative
to the monovalent ligand.8 Hence, we can understand the
great interest in making such dimeric compounds.
Recently, we have reported the synthesis of estrone
dimers linked at position 16 of the steroid nucleus.11
These dimers were linked via two ester groups with an
alkyl chain or a polyethylene glycol (PEG) chain. It was
shown that the estrone dimers were non-toxic towards
ER+ (MCF-7) and ERꢀ (MDA-MB-231) human breast
cancer cell lines. In order to obtain dimers with a stron-
ger linkage than the ester bonds found in the previously
described molecules, we planned the synthesis of estra-
diol dimers which are linked together via ether bonds.
The ether connection should increase the stability of the
dimeric molecules while improving its solubility.
Furthermore, some attempts have been made to design
estrogenic bivalent ligands in order to interfere with the
process of estrogen receptor dimerization.9,10 These
dimeric molecules were constructed from non-steroidal
We believed that the used of the natural steroid nucleus
in the design of such dimeric molecule could increase
the chance of interactions with the estrogen receptor.
Therefore, by increasing its interaction with the ER, one
would theoretically enhance its biological activity as
compared to the non-steroidal dimers. It is also believed
that this type of molecules synthesized from the natural
*Corresponding author. Fax: +1-819-376-5084; e-mail: gervais_
berube@uqtr.ca
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00987-3

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D. Rabouin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 557–560
hormone would present essentially no toxicity towards
healthy or cancerous cells but could possess interesting
antiestrogenic activity. This manuscript describes the
synthesis of several members of this new family of C2-
symmetric 17b-estradiol dimers (see general structure 1).
It also reports the in vitro cytotoxic activity of the
dimers on two neoplastic human breast cancer cell lines:
MCF-7 and MDA-MB-231 (ER+ and ERꢀ).
shows that their solubility remain almost constant what-
ever the length the linking arms (see Table 1).
As shown in Scheme 2, the di-, tri- tetra-, penta- and
hexa-ethylene glycols were readily transformed into the
dibrominated PEGs in two chemical steps with high
yields. Initially, the PEGs were treated with methane-
sulfonyl chloride and triethylamine in ether to give the
dimesylate intermediate which was not isolated. The
precipitated triethylamine hydrochloride salt was fil-
tered off and the residue evaporated to an oil. After-
wards, the dimesylate was treated with lithium bromide
in acetone at reflux for 20 h to give the desired dibro-
mides 3 with an average overall yield of 70%.
As shown in Scheme 3, six estradiol dimers were obtained
using a straightforward reaction sequence. Initially,
estrone (4) was protected as a benzyl ether using phase
transfer catalysis (PTC) methodology. Thus, estrone was
treated with benzyl bromide and tetrabutylammonium
hydrogen sulfate in dichloromethane in the presence of a
10% aqueous sodium hydroxide solution.12 The yield of
the protection reaction is 98%. The derivative 5 was
transformed into the 17a-(prop-20-enyl) estradiol 6 upon
treatment with freshly prepared allylmagnesium bromide
in dry diethylether.13 Derivative 6 was obtained with
95% yield. The hydroboration-oxydation sequence per-
formed on compound 6 gave the diol 2 in 72% yield.13
Hydrogenolysis of this intermediate with 10% Pd/C in
tetrahydrofuran gave quantitatively the triol 7.12 Selec-
tive O-alkylation of diol 2 was performed upon treatment
with sodium hydride for 30 min in a mixture of dry tet-
rahydrofuran and dimethylformamide to which was
added the appropriate dihalogenated PEG chain 3 (or
a,o-dibromoalkane) at room temperature for 20 h. The
dimers were obtained with an average yield of 50%. The
final dimeric molecules were obtained quantitatively by
hydrogenolysis of the benzyl ether with 10% Pd/C in
tetrahydrofuran. The dimers were obtained with an
average overall yield varying from 27% to 36%. All new
compounds synthesized were characterized by their
respective IR, 1H NMR, 13C NMR and mass spectra.14
Chemistry
A retrosynthetic analysis of the dimers is presented in
Scheme 1. As one can observe, the 17a,170a-estradiol
dimer 1 possess a C2 symmetry. The dimers 1 can easily
be made from a suitable dihalogenated PEG 3 (or an a,o-
dibromoalkane) component and an adequately functio-
nalised estradiol derivative 2 by selective alkylation on
the primary alcohol. The latter can be prepared from
estrone 4 itself and allyl bromide via a Grignard reaction.
This analysis provides a simple path for the synthesis of a
large variety of new estradiol dimers. Moreover, the
dihalogenated PEG are ideal linkers because they are
inexpensive, water soluble, and available in a variety of
lengths. In addition, the calculated LogP of the dimers
In Vitro Antitumor Activity
The toxicity of the dimers was evaluated on two human
breast tumor cell lines using the Sulforhodamine B col-
Scheme 1. Retrosynthetic analysis for the 17-b-estradiol dimers.
Table 1. Inhibitory concentration of drug on both ER+ and ERꢀ breast cancer cell lines, chain length and CLogP
Compd
MCF-7 (ER+
)
MDA-MB-231 (ERꢀ)
Chain length
n or m
CLogPb
3.78
IC50 (mM)a
IC50 (mM)a
Estradiol
Tamoxifen
7
1a
1b
1c
1d
1e
1f
32
11
71
>100
19—
—
6.82
—
n=1
n=2
n=3
n=4
n=5
m=2
>100
>200
>100
>100
>100
>100
>100
3.37
8.22
8.28
8.35
8.41
8.47
9.32
>200
62
63
>100
>100
>100
aInhibitory concentration as obtained by the SRB assay. Experiments were performed in octuplicate, errors are within Æ10%.
bCalculated LogP as obtained with ChemDraw Ultra 6.0.

D. Rabouin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 557–560
559
16 and 19atoms long. The shorter linkage seen in deri-
vative 1a (13 atoms long) as well as the longer linkages
seen in compounds 1d and 1e (22 and 25 atoms long)
appear to be inadequate for selective biological activity
on ER+ cancer cells. The calculated LogP of the dimers
is relatively constant throughout the sequence varying
from 8.22 to 8.47. The dimer 1f bearing a carbon atom
linking chain is not toxic and possesses a CLogP of 9.32
and is therefore less soluble than the PEG dimeric ana-
logues. On the other hand, the triol 7 show some toxi-
city against the ER+ (MCF-7) breast cancer cells with a
IC50 of 71 mM.
In summary, this manuscript presents a facile synthesis
of C2-symmetric 17b-estradiol dimers. They are made
form estradiol in five chemical steps with an overall yield
exceeding 30%. The key steps for the synthesis of these
compounds is the selective O-alkylation of diol 2 with
the dibrominated PEG chains 3. The dimers are gen-
erally less toxic towards breast cancer cells as compare to
the cognate hormone 17b-estradiol and the antiestrogen
tamoxifen. Interestingly, dimers 1b and 1c show specific
toxicity towards MCF-7, an hormone-dependent breast
cancer cell line. Further investigation will be necessary to
assess the complete biological potential of these new C2-
symmetric 17b-estradiol dimers.
Scheme 2. Synthesis of dihalogenated PEG chains.
Acknowledgements
This work was supported by the Universite du Quebec a
Trois-Rivieres. We are grateful to Mr. Jacques Lacroix
for biological evaluation studies. We also thank Dr.
Gilles Sauve and Mr. Nicolas Le Berre, Pharmacor Inc.
for the 1H and 13C NMR spectra and Mr. Gaston
Boulay, Universite de Sherbrooke, for the mass spectra.
References and Notes
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Scheme 3. Synthesis of 17b-estradiol dimers.
orimetric assay.15,16 The cytotoxicity of the compounds
was tested along with controls (estradiol and tamoxifen)
on both estrogen-receptor positive (ER+, MCF-7) and
estrogen-receptor negative (ERꢀ, MDA-MB-231)
human mammary carcinomas.17
6. Bringmann, G.; Saeb, W.; Koppler, D. Tetrahedron 1996,
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As shown by the SRB assays on the two human breast
cancer cell lines, the dimers are not very toxic towards
breast cancer cells (Table 1). The reference products,
estradiol and tamoxifen are more toxic than the dimers.
Interestingly, there is a selective activity of dimers 1b
and 1c against the ER+ cell line. The lower homologue
1a and the higher homologues 1d and 1e present no
cytotoxicity showing an IC50 of more than 100 mM. The
total length of the chain linking compounds 1b and 1a is

560
D. Rabouin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 557–560
11. Rabouin D. M.Sc. Thesis, Universite du Quebec a Trois-
Rivieres, Trois Rivieres, August 2001.
(C-2), 83.1 (C-17), 63.5 (CH2OH), 50.5, 47.7, 44.8, 40.9, 34.8,
34.3, 32.6, 30.6 (hidden by acetone), 28.4, 28.1, 27.3, 27.2, 15.1
(18-CH3). MS (m/e): 330 (M+), 312 (M+ꢀH2O), 271
12. Greene, T. W.; Wuts, P. G. M. In Protective Groups in
Organic Synthesis, 3rd Ed., John Wiley & Sons: New York, 2000.
13. Dionne, P.; Tchedam Ngatcha, B.; Poirier, D. Steroids
1997, 62, 674.
(M+ꢀC3H7O).
Exact
mass:
calculated
for
C21H30O3=330.2195; found=330.2198.
Spectral data for 1,11-dibromo-3,6,9-trioxadecane (3c, n=3):
IR (NaCl, nmax, cmꢀ1): 1277 and 1116 (C–O). 1H NMR
(CDCl3, d ppm): 3.80 (4H, t, J=6.3 Hz, 2ꢁ BrCH2CH2O),
3.66 (8H, s, 4ꢁ CH2O), 3.46 (4H, t, J=6.3 Hz, 2ꢁ CH2Br).
13C NMR (CDCl3, d ppm): 71.2 (BrCH2CH2O), 70.6 and 70.5
(CH2O), 30.3 (CH2Br)
14. Spectral data for 3-O-benzyl-17a-(30-hydroxypropyl)-
1,3,5(10)-estratrien-17b-ol (2): IR (NaCl, nmax, cmꢀ1): 3360
1
(O–H), 1600 (C¼C), 1254 and 1017 (C–O). H NMR (CDCl3,
d ppm): 7.41 (2H, d, J=7.0 Hz, a-CH), 7.36 (2H, t, J=7.2 Hz,
b-CH), 7.30 (1H, d, J=6.7 Hz, c-CH), 7.18 (1H, d, J=8.5 Hz,
1-CH), 6.76 (1H, dd, J=7.9Hz and J=2.0 Hz, 2-CH), 6.71
(1H, br s, 4-CH), 5.00 (2H, s, CH2Ph), 3.67 (2H, m, 30-
CH2OH), 2.86 (2H, m, 6-CH2), 2.59(2H, br s, 2 ꢁ OH), 2.31–
1.20 (17H, #m, 3ꢁ CH and 7ꢁ CH2), 0.90 (3H, s, 18-CH3).
13C NMR (CDCl3, d ppm): 156.7 (C-3), 138.0 (CCH2O), 137.3
(C-5), 132.9(C-10), 128.5 (C-b), 127.8 (C-c), 127.4 (C-a), 126.2
(C-1), 114.8 (C-4), 112.2 (C-2), 83.2 (C-17), 69.9 (CH2Ph), 63.3
(C-30), 49.5, 46.7, 43.8, 39.6, 34.4, 33.4, 31.5, 29.8, 27.5, 26.9
(C-20), 26.3, 23.4, 14.3 (C-18). MS (m/e): 420 (M+), 402
(M+ꢀH2O), 362 (M+ꢀC3H6O). Exact mass: calculated for
C28H36O3=420.2664; found=420.2656.
Spectral data for dimer 1c (n=3): IR (NaCl, nmax, cmꢀ1): 3385
1
(O–H), 1610 (C¼C), 1287 and 1036 (C–O). H NMR (CDCl3,
d ppm): 7.05 (2H, d, J=8.5 Hz, 2ꢁ 1-CH), 6.59(2H, d, J=8.2
Hz, 2ꢁ 2-CH), 6,51 (2H, d, J=1,4 Hz, 2ꢁ 4-CH), 3.65–3.42
(20H, s and m, 10ꢁ CH2O), 3.06 (4H, br s, 3-OH and 17-OH),
2.76 (4H, m, 2ꢁ 6-CH2), 2.30–1.15 (34H, m, 14ꢁ CH2, 6ꢁ
CH), 0.84 (6H, s, 2ꢁ 18-CH3). 13C NMR (CDCl3, d ppm):
155.1 (C-3), 138.9(C-5), 132.7 (C-10), 127.1 (C-1), 116.2 (C-4),
113.6 (C-2), 84.0 (C-17), 73.0, 71.3, 70.8, 50.4, 47.7, 44.6, 40.6,
34.8, 34.2, 32.5, 30.6, 28.4, 27.2, 24.8, 24.3, 15.4 (C-18). MS
(m/e): 800 (M+ꢀH2O), 782 (M+ꢀ2H2O). Exact mass: calcu-
lated for C50H72O8 (M+ꢀH2O)=800.5227; found=800.5231.
NB: a-CH, b-CH and c-CH are ortho, meta and para protons
on the benzyl protecting group. Similarly, a-C, b-C and c-C
are the corresponding carbons on the benzyl group.
Spectral data for 3-hydroxy-17a-(30-hydroxypropyl)-1,3,5(10)-
estratrien-17b-ol (7): IR (NaCl, nmax, cmꢀ1): 3351 (O–H), 1156
and 1241 (C–O). 1H NMR (Acetone-d6, d ppm): 8.10–7.70
(1H, br s, 3-OH), 7.08 (1H, d, J=8.5 Hz, 1-CH), 6.76 (1H, dd,
J=1.9Hz and J=8.4 Hz, 2-CH), 6.59(1H, dd, J=2.4 Hz and
J=8.7 Hz, 2-CH), 6.52 (1H, d, J=1.6 Hz, 4-CH), 3.64– 3,50
(2H, m, CH2OH), 3.30 - 3.05 (2H, br s, 17-OH and CH2OH),
2.78 (2H, m, 6-CH2), 2.40–1.10 (17H, m, 7ꢁ CH2, 3ꢁ CH),
0.92 (3H, s, 18-CH3). 13C NMR (Acetone-d6, d ppm): 155.9
(C-3), 138.5 (C-5), 132.2 (C-10), 127.0 (C-1), 115.9(C-4), 113.6
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