Synthesis, characterization, and estrogen receptor binding affinity of flavone-, indole-, and furan-estradiol conjugates

Article abstract of DOI:10.1016/j.bmcl.2007.03.016

Different flavone-, indole-, and furan-17β-estradiol conjugates, linked via alkyl spacer chains extending from the 17α-position of the estradiol moiety, were synthesized by Pd-catalyzed cross-coupling reactions. Structures were assigned based on spectrosc

Full text of DOI:10.1016/j.bmcl.2007.03.016

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3212–3216
Synthesis, characterization, and estrogen receptor binding affinity
of flavone-, indole-, and furan-estradiol conjugates
Naseem Ahmed, Celena Dubuc, Jacques Rousseau,
*
´
Francois Benard and Johan E. van Lier
Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences,
Universite´ de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4
Received 22 January 2007; revised 6 March 2007; accepted 6 March 2007
Available online 12 March 2007
Abstract—Different flavone-, indole-, and furan-17b-estradiol conjugates, linked via alkyl spacer chains extending from the
17a-position of the estradiol moiety, were synthesized by Pd-catalyzed cross-coupling reactions. Structures were assigned based
on spectroscopic data. In vitro competitive binding assays for the estrogen receptor (a-ER), using [3H]estradiol (RBA = 100) as
a competitor, revealed that a two-carbon alkyl linker combined with a flavone conjugate provided the highest binding affinity
(RBA ꢀ 9), warranting further studies on their potential use as selective estrogen-receptor modulators (SERMs) for hormone-
replacement therapies.
Ó 2007 Elsevier Ltd. All rights reserved.
Breast cancer remains a major cause of death for women
in the western world (2–10 times higher than for oriental
women) with the global incidence estimated at 1.15 mil-
lion in 2002.1 The female sex hormones, estrogens, play
a central role in the development of breast tumors.
Many of these tumors are initially hormone-responsive
and circulating estrogens have a vital role in their
growth. Approximately 80% of breast cancer cases
occur in post-menopausal women whose ovarian estro-
gen production has ceased with remaining estrogens
originating in extra-glandular tissues.
organic structures, have found an increasing number
of applications in molecular and cell biology.
Conjugated groups provide biomolecules with novel
properties, such as fluorescence emission, catalytic activ-
ity, altered hydrophobicity or bio-affinity, resistance
toward biodegradation or the ability to carry metal
ions.4 Many naturally occurring steroid hormones5
and non-steroidal6 derivatives are recognized by steroid
hormone receptors (SHRs) either as agonists or antago-
nists depending on their interaction with the SHR. Both
agonists or antagonists are used for the treatment of
hormone-dependent breast cancers (HDBCs).5,6
Estrogen receptors (ER) are proteins present in target
cells in very low concentrations (1000–10,000 molecules
per cell).2 Unlike circulatory estrogen binding proteins
(with high capacity and low binding specificity) that
aid in transporting steroids, the ER have low capacity
but are highly specific in binding selected steroid hor-
mones. The mechanism by which steroids and their
receptors work together to effect their physiological
functions is well known.3 During the past decades
bioconjugates, that is, biomolecules bearing unnatural
Phytoestrogens are plant-derived compounds that struc-
turally or functionally mimic mammalian estrogens.
Similar to the numerous effects of estrogens on the
human body, these are known to exert broad beneficial
effects toward human health, especially against cancer,
osteoporosis, irregular menopause syndrome, cardiovas-
cular disease, and possibly neuro-degenerative diseases.
Recently, the prenylated flavanone, 8-prenylnaringenin
(8-PN) derived from hops (Humulus lupulus L.), has
been identified as a potent estrogen showing the highest
in vitro estrogenic activity among all phytoestrogens
known to date.7 Furthermore, some phytoestrogens,
indoles, and furans have been shown to function as selec-
tive estrogen-receptor modulators (SERMs), eliminating
Keywords: Estrogen receptor; Flavone-, indole-, furan-estradiol con-
jugates; Pd-catalyzed Sonogashira reaction; Receptor binding affinity.
*
Corresponding author. Tel.: +1 819 564 5409; fax: +1 819 564
5442; e-mail: johan.e.vanlier@USherbrooke.ca
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.016

N. Ahmed et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3212–3216
3213
unwanted side-effects often observed with synthetic estro-
gens used in hormone-replacement therapies.8 However,
the non-selectivity of phytoestrogens and their acute tox-
icity toward rapidly proliferating healthy tissues has de-
terred their use as antitumor agents.
The alkynone intermediates were prepared via Pd-cata-
lyzed Sonogashira cross-coupling reactions and subse-
quent iodine or monochloroiodine-induced cyclization
of the alkynone to give the iodoflavone (3) and iodofu-
ran (7) in high yields (Scheme 1).13 However, using a
similar approach to prepare the iodoindoles (4 and 5)
gave the latter in low yields (10–20%) likely due to the
presence of the free hydroxyl and amino groups on the
substrate. In the case of iodoflavone 6, iodination was
accomplished by heating equimolar amounts of flavone
(commercially available), I2, and Cu(OAc)2 at 60 °C in
acetic acid for 2–3 h. All products were characterized
on the basis of their spectroscopic data and comparison
with literature values.13 Based on the high electrophilic
nature of the C-8 of the A-ring, the iodine in compound
6 is assigned to C-8 position, although this is difficult to
One approach to overcome this non-selectivity is to cou-
ple the phytoestrogen to a carrier with strong affinity for
a particular tissue from which the tumor is derived.9a
This might enable the selective delivery of the cytotoxic
agent to the cells or tissues bearing a high concentration
of binding sites for the carrier.9b,c To target breast can-
cer, estrogens are of particular interest since the majority
of breast cancers are ER-positive and markedly concen-
trate estrogens within the cell nucleus.9d Several at-
tempts have been made to utilize this approach by
conjugating estradiol with alkylating moieties such as
nitrogen mustards,10a nitrosoureas,10b aziridines,10c
dichloroplatinum.10d Such conjugates exhibit antitumor
activity and also possess sufficient binding to the ER,
allowing selective accumulation of the conjugates in
ER-rich cells. Several conjugates of 17b-estradiol (E2)
linked directly or indirectly through an alkyne-type rigid
spacer at the 17a-position have been advanced as syn-
thetic ligands for therapeutic applications targeting the
ER.11
1
confirm by H NMR due to the clustering of the aro-
matic protons.
We previously showed that in the case of steroid-nucle-
oside conjugates increasing the length of the carbon
spacer chain at the 17a-position enhances ER-binding
affinities.12a Therefore, we synthesized 1,7-octadiynyl-
17a-estradiol (2) following the above procedure and
subsequent conjugation with 3, 6, and 7 using the Pd-
catalyzed reaction to yield the desired products 13–15
in good yield (Scheme 2). All conjugated products were
characterized on the basis of their spectroscopic data
(1H NMR, EIMS, and HR MS).14
In search for agents that would be useful for the man-
agement of estrogen-dependent pathologies, we previ-
ously synthesized steroid-nucleoside conjugates and
tested them for their ER-binding affinity and cytotoxic-
ity toward ER-positive animal tumors.12 Here we report
a similar approach to improve the binding affinity of
phytoestrogens for the ER by conjugating a series of fla-
vone, indole, and furan derivatives with estradiol
attached to the 17a-position via alkyne chains of varying
lengths.
The syntheses of E2 conjugates 8–12 were accomplished
by employing a palladium-catalyzed Sonogashira cross-
coupling reaction using equimolar amounts of 17a-eth-
ynylestradiol (1) and products 3, 4, 5, 6 or 7 (Scheme 3)
in the presence of CuI (0.04 mol) and Pd[PPh3]4
(0.02 mol) as catalysts and triethylamine base refluxed
at 70 °C for 3 h under an argon atmosphere. After cool-
O
O
O
H
Ph
(ii)
Cl
(i)
I
OMe
OMe
O
R
3
H
I
I
H
TMS
I
(i)
TBAF, THF, rt
NH2
(i), (ii)
N
NH2
R
H
R=OH (4)
R=OMe (5)
BnO
OBn
H
OBn
I
O
(ii)
I
(i)
TsO
OMe
TsO
OMe
OTs
7
Scheme 1. Reagents and conditions: (i) PdCl2(PPh3)2 (2 mol%), CuI (1 mol%), Et3N, THF, rt, 16 h; (ii) I2 or ICl, CH2Cl2, rt, 10–15 min.

3214
N. Ahmed et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3212–3216
O
OH
O
Bn
O
HO
OH
13
3
H
(i)
O
OH
(i)
7
OTs
HO
2
HO
I
15
O
(i)
O
O
O
6
OH
14
HO
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4 (2 mol%), CuI (4 mol%), Et3N, THF, 70 °C, 3 h.
OH
H
O
N
OH
OH
O
O
O
OH
HO
11
HO
8
(i)
HO
(i)
4
(i)
9
3
BnO
OH
OH
6
H
OMe
H
N
(i)
7
O
(i)
OH
5
HO
1
OTs
HO
HO
10
12
Scheme 3. Reagents and conditions: (i) Pd(PPh3)4 (2 mol%), CuI (4 mol%), Et3N, THF, 70 °C, 3 h.
ing, the solvent was evaporated and the residue was
purified by flash chromatography using 20–40%
EtOAc/hexane to give the corresponding desired prod-
ucts 8–12.
technology.15 The RBA values, taking estradiol as 100,
are summarized in Table 1.
In addition to the nature of the phytoestrogen and
length of the spacer chain, steric effects and changes in
the overall molecular conformation of the estradiol con-
jugates also modulate the binding affinity for the ER
(Table 1). Among the different heterocyclic moieties
the flavone conjugate 8, featuring a C-2 tether, exhibits
Relative binding affinity (RBA). Relative binding affini-
ties (RBA) were measured by a competitive assay
against [3H]estradiol (concentration: 0.6 pM/well) using
purified ERa (Panvera, Madison, WI) and FlashPlate

N. Ahmed et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3212–3216
3215
Table 1. Relative binding affinities (RBA) and Ki-values for ERa of flavone-, indole- and furan-estradiol conjugates
Heterocycle moiety
—
E2 conjugate
Estradiol
RBAa (C-2 tether)
RBAa (C-8 tether)
Ki (nM)
100
100
12
O
O
8
9.17
—
37
13
0.01
63,052
O
O
9
0.92
—
100
382
14
0.24
—
10
11
8.71
4.05
35
75
N
MeO
H
—
N
HO
O
H
12
15
0.80
—
332
OBn
TsO
0.01
17,879
a Relative binding affinities (RBA) of the estradiol conjugates for the ERa were determined by a competitive [3H]estradiol binding assay using Flash
Plate technology. Conjugates (0.1–10 lM) were incubated at 25 °C for 1–2 h to establish EC50 values from which the RBA were calculated, taking
estradiol as unity.
the highest binding affinity (RBA = 9.17), followed by
the indole conjugate 11 (RBA = 4.05). Increasing the
length of the spacer consistently diminishes the binding
affinity (i.e., 8 vs 13, 9 vs 14, and 12 vs 15). This con-
trasts our earlier findings with a series of nucleoside-
17b-estradiol conjugates, where an increase in the length
of the spacer augmented the binding affinities.12a Block-
ing the hydrogen bonding capacity of the indole moiety
(e.g., OMe in 10 vs an OH in 11) increases the binding
affinity twofold suggesting that the latter improves inter-
action with the receptor. Varying the position of attach-
ment in the estradiol–flavone conjugates (i.e., 8 vs 9 and
13 vs 14) also strongly affects RBA values likely reflect-
ing steric effects and changes in the molecular conforma-
tion. Overall our data indicate that the lengths of the
spacer chain combined with steric effects are important
factors affecting the ER binding capacity for these estra-
diol–phytoestrogen conjugates.
Acknowledgments
This work was supported by the Canadian Institutes of
Health Research (CIHR, Grant No. MOP-44065) and
the Canadian Breast Cancer Research Alliance
(CBCRA, Grant No. 015388).
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