Novel 7α-alkoxy-17α-(4′-halophenylethynyl)estradiols as potential SPECT/PET imaging agents for estrogen receptor expressing tumours: Synthesis and binding affinity evaluation

Article abstract of DOI:10.1016/j.steroids.2012.05.004

In order to develop potential radiolabelled probes for imaging estrogen receptor (ER) positive tumours, we have synthesized and characterized a series of novel 7α-alkoxy-17α-(4′-iodophenylethynyl)estra-1,3,5(10)- triene-3,17β-diols and 7α-alkoxy-17α-(4′- fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols. The fluoro-substituted compounds showed a higher ER binding affinity than the corresponding iodo-derivatives, where 7α-methoxy- and 17α-(4′- fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diol showed the highest ER binding affinities (RBA = 80.9% and 78.9%, respectively), among the halophenylethynyl compounds studied and should be further explored as potential PET biomarkers for imaging of ER expressing tumours.

Full text of DOI:10.1016/j.steroids.2012.05.004

Steroids 77 (2012) 1123–1132
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Novel 7a-alkoxy-17
a
-(40-halophenylethynyl)estradiols as potential SPECT/PET
imaging agents for estrogen receptor expressing tumours: Synthesis and
binding affinity evaluation
a
a
b
Carina Neto a, Maria Cristina Oliveira a, , Lurdes Gano , Fernanda Marques , Takumi Yasuda ,
⇑
Thies Thiemann b,c, Torsten Kniess d, Isabel Santos a
a Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10,
2686-953 Sacavém, Portugal
b Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1, Kasuga-koh-en, Kasuga-shi, Fukuoka 816-8580, Japan
c Department of Chemistry, Faculty of Science, United Arab Emirates University, United Arab Emirates
d Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf, e.V., POB 510119, D-01314 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop potential radiolabelled probes for imaging estrogen receptor (ER) positive tumours,
we have synthesized and characterized a series of novel 7
1,3,5(10)-triene-3,17b-diols and -alkoxy-17
-(40-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17b-
diols. The fluoro-substituted compounds showed a higher ER binding affinity than the corresponding
iodo-derivatives, where 7 -methoxy- and 17
-(40-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17b-diol
a-alkoxy-17a
-(40-iodophenylethynyl)estra-
Received 9 February 2012
Received in revised form 7 May 2012
Accepted 16 May 2012
7
a
a
Available online 24 May 2012
a
a
showed the highest ER binding affinities (RBA = 80.9% and 78.9%, respectively), among the halophenyle-
thynyl compounds studied and should be further explored as potential PET biomarkers for imaging of ER
expressing tumours.
Keywords:
Breast cancer
Estrogen receptor (ER)
Estradiol
Ó 2012 Elsevier Inc. All rights reserved.
Positron emission tomography (PET)
Single photon emission computerized
tomography (SPECT)
Imaging
1. Introduction
on ER-ligands are valuable tools for diagnosis and oriented cancer
therapy. Hence the search for novel ligands to specifically target ER
Malignant tumours overexpressing estrogen receptors (ER)
have a great impact on human health and welfare, regardless of
the progress in their early detection and treatment. The role of
the interaction of estrogens and ER in breast cancer has been inves-
tigated extensively, since many of these carcinoma are ER+,
although the exact processes remain far from clear, mechanisti-
cally. However, there is ample evidence for the involvement of
estrogens and their receptors in the development of gynaecological
and endocrine cancers [1–5]. Such cancers are often hormonally
regulated and may be associated with high levels of progesterone
and epidermal growth factor receptor (EGFR) expression. The bio-
logical effects of estrogens are exerted through two subtypes of ER
in tumours of different etiology continues to be a very important
task. Both gamma- and positron-emitting estradiol derivatives
have been developed to visualize ER in breast tumours through
radionuclide guided imaging techniques such as single-photon
emission computed tomography (SPECT) or positron emission
tomography (PET), but few of these agents have reached the clini-
cal stage [6–8].
During the last decades, several efforts have been made to syn-
thesize estradiol derivatives that could be labelled with gamma-
emitters. Several radioiodinated estradiol derivatives have been
studied [6,9,10]. Among them, both isomers of 11b-methoxy-
(17a
,20E/Z)-[123I]iodovinylestradiol have been clinically assessed,
(ER
a
and ERb), ligand-transcription factors whose activity as indu-
with the 20Z isomer giving the better images of ER-positive human
breast tumors. While both primary and metastatic tumours were
detected with good sensitivity and selectivity, extensive correla-
tions between imaging and clinical outcome have not been pro-
vided so far [11–15]. Efforts have also been directed to the
design of steroidal estrogen derivatives labelled with 99mTc, due
to its favourable nuclear decay properties, low cost and availability
cer or repressor of gene transcription depends on the nature of the
bound ligand. ER is a relevant tumour biomarker, and drugs based
⇑
Corresponding author. Tel.: +351 219946184; fax: +351 219550117.
E-mail address: cmelo@itn.pt (M.C. Oliveira).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.05.004

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C. Neto et al. / Steroids 77 (2012) 1123–1132
[16–18]. While most of the emphasis has been placed on the imag-
ing potential of these radioligands, the presence of Auger electrons
from the decay of 123/125I has initiated interest in their radiothera-
peutic applications, too. Studies have shown that estrogens la-
belled with Auger emitters can destroy ER+ cells, while sparing
ERꢀ cells [19–21]. Concerning PET-imaging, fluorine-18, a cyclo-
tron-produced radionuclide, has been the most explored radiohal-
ogen for in vivo imaging studies of estrogen receptors [22–26]. To
ERa, it was understood that subsequently only the most promising
compound(s) would be resynthesized in radiolabelled form.
2. Experimental
2.1. General methods and equipment
date, 16a
-[18F]-estradiol (18F-FES) has been the most successful
Unless stated otherwise, all commercial reagents and solvents
were of analytical grade and were used without further purifica-
tion. 1H NMR, 13C NMR and 19F NMR spectra were recorded at room
temperature in a Varian Unity 300 MHz spectrometer or a JEOL
270 MHz spectrometer. 1H NMR, 13C NMR and 19F NMR chemical
shifts are reported relative to residual solvent signals or TMS as ref-
erence. Mass spectra were obtained in a Bruker HCT mass spec-
trometer or a Finnigan MAT 312 mass spectrometer. Chemical
reactions were monitored by thin-layer chromatography (TLC) on
Merck plates pre-coated with silica gel F254. Column chromatogra-
phy was performed on silica gel 60 (Merck).
candidate for in vivo imaging of estrogen receptor-positive tu-
mours and is currently in phase II of a study to predict response
to first line hormone therapy in women with ER(+) metastatic
breast cancer [27–29]. 4-Fluoro-11b-methoxy-16a
-[18F]-fluoroest-
radiol (4FMFES) is a newly developed radiolabeled estradiol ana-
logue for ER imaging with PET [30]. This tracer shows favourable
biodistribution in small-animal experiments and achieves higher
target-to-nontarget uptake ratios than does FES [31,32]. Subse-
quent studies of human biodistribution and dosimetry using serial
whole-body PET/CT have highlighted the potential of 4FMFES for
ER imaging in patients with breast cancer [33].
However, as many of the radioligands developed till date still
exhibit low receptor binding affinities (RBA) and non ER-regulated
tissue uptake, our team is continuing to work on the development
of new radiohalogenated steroids as ER-based radiopharmaceuti-
cals. In line with known data on structure–activity relationships
between steroidal ligands and ER, based on the ER affinity of com-
pounds investigated to-date, their cell binding studies and biolog-
ical behaviour in animal models [34–36], the authors decided to
study the effect of a heteroatom with lone electron pairs directly
In the receptor-binding affinity assay a human estrogen recep-
tor-a (ERa, Pan Vera, Invitrogen Corporation, CA, USA) isolated
from recombinant baculovirus-infected insect cells was used and
maintained at ꢀ80 °C as indicated by the manufacturer. Tritiated
estradiol, [2,4,6,7-3H]E2 (specific activity of 84.0 Ci/mmol) was ob-
tained from Amersham, GE Healthcare, UK. Hydroxyapatite (HAP)
was obtained from Calbiochem (San Diego, CA, USA).
2.2. Chemical synthesis
connected to C7
a of the steroidal frame on the receptor binding
2.2.1. 3-O-Benzyl-estra-1,3,5(10)-trien-3-ol-17-one-17,17-(20-[50,50-
dimethyl-10,30-dioxane]) (2)
affinity and the efficacy of the compound. Estradiol derivatives
with a heteroatom or a heteroatom functionality linked to C7 in
close proximity to the steroidal skeleton have been investigated
previously in form of 7-cyano containing estradiols [37]. There,
the hetero-functionality exerts an electron-withdrawing effect on
its bonding partner. In the present study, alkoxy functional groups
are directly linked to C7 of the estradiol frame, which would allow
a (receptor) Hꢁ ꢁ ꢁO interaction and then would increase the avail-
ability of the protons at C6 for close contacts with the receptor
through the stabilisation of a partial positive charge at C7. Within
this context it must be noted that, apart from 4FMFES mentioned
above, a number of 11b-methoxy substituted estradiols have been
found to be good ligands for ER
shown that 11b- and 7 -substituted estradiols often exhibit simi-
lar trends in RBA within a series. It is known that C17 can accom-
modate larger substituents without an appreciable deterioration in
RBA. S. Top, G. Jaouen et al. have shown that in the 11b-substituted
estradiol series, larger substituents were reasonably tolerated by
A solution of 3-O-benzylestra-1,3,5(10)-trien-3-ol-17-one (1)
(4.1 g, 11.4 mmol), neopentylglycol (NPG) (1.9 g, 18.3 mmol) and
p-toluenesulfonic acid monohydrate (p-TsOH, 225 mg, 1.2 mmol)
in benzene (78 mL) was stirred for 90 min under reflux with con-
tinuous azeotropic removal of water (Dean–Stark condenser). After
cooling to room temperature, a saturated NaHCO3 aqueous solu-
tion was added and the mixture was extracted with diethyl ether
(3 ꢂ 50 mL). The organic phase was washed with water (50 mL),
dried over MgSO4 and evaporated. The residue was then purified
by column chromatography on silica gel (diethyl ether:n-hex-
ane:chloroform, 1:1:0.5) to give 3-O-benzyl-estra-1,3,5(10)-trien-
3-ol-17-one-17,17-(20-[50,50-dimethyl-1’,3’-dioxane]) (2) as a col-
ourless solid (2.8 g, 57%), mp. 78–81 °C.
a [14,38]. Previously, it has been
a
a
1H NMR (300 MHz, CDCl3) d: 0.71 (s, 3H, CH3), 0.82 (s, 3H, CH3),
1.15 (s, 3H, CH3), 1.3–1.94 (m, 11H), 2.27 (m, 2H), 2.82 (m, 2H),
3.37 (m, 2H), 3.47 (d, 1H, 2J = 11.1 Hz), 3.65 (d, 1H, 2J = 11.1 Hz),
5.01 (s, 2H), 6.69 (d, 1H, 4J = 2.4 Hz), 6.75 (dd, 1H, 4J = 2.4 Hz,
3J = 8.4 Hz), 7.20 (d, 1H, 3J = 8.4 Hz), 7.36 (m, 5H).
ER
17
a
a
and that 11b-methoxy substituted derivatives with larger
-substituents show better RBA than their at C11 non-substi-
tuted analogues [38]. It must be noted in this respect, though, that
in the 7a-series a potential synergy between the 7 substituents
and 17 -substituents in the binding of the ligand to the protein
can be expected for relatively small substituents, only. As 17 -eth-
ynylation often not only increases the RBA of the respective com-
pounds, but also renders them more resistant towards rapid
metabolisation, it was planned to attach the radioisotope on an
a
2.2.2. 3-O-Benzyl-6-oxoestra-1,3,5(10)-triene-3-ol-17-one-17,17-(20-
a
[50,50-dimethyl-10,30-dioxane]) (3)
a
A mixture of 3-O-benzyl-estra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-1’,3’-dioxane]) (2) (3.4 g, 7.5 mmol),
KMnO4 (4.9 g, 0.03 mol), Adogen 464 (0.6 g) and NaHCO3
(231 mg, 2.75 mmol) in benzene (30 mL) and water (30 mL) was
stirred for 3 h at reflux temperature. Thereafter, the reaction mix-
ture was cooled to room temperature and the organic phase was
extracted with diethyl ether (3 ꢂ 50 mL), washed with water
(100 mL), dried over anhydrous MgSO4, filtered and concentrated
to dryness. The resulting crude was purified by column chromatog-
raphy on silica gel (diethyl ether:chloroform:hexane, 1:1:7) to give
ethynylated substituent at C17
ethynyl-substituted compounds are less stable in vitro and in vivo,
a halophenylethynyl substituent was chosen [39]. Therefore, 7
alkoxy-17
-(40-halophenylethynyl)estra-1,3,5(10)-triene-3,17b-
a of the target compound. As halo-
a
-
a
diols of type 14/15 became the synthetic targets (Scheme 1). A
radiofluorinated analogue of 14c has already been successfully pre-
pared by Wüst et al. but no biological data was presented by the
authors [40]. After the evaluation of the relative binding affinities
of the individual compounds in non-radiolabelled form towards
3-O-benzyl-6-oxoestra-1,3,5(10)-triene-3-ol-17-one
17,17-(20-
[50,50-dimethyl-1’,3’-dioxane]) (3) as a colourless solid (770 mg,
22%), mp. 120–123 °C.

C. Neto et al. / Steroids 77 (2012) 1123–1132
1125
Scheme 1. Synthesis of 7a-methoxy-, 7a-propoxy- and 17a
-(40-halophenylethynyl)estra-1,3,5(10)-triene-3,17b-diol derivatives (14a–14c and 15a–15c).
1H NMR (300 MHz, CDCl3) d: 0.72 (s, 3H, CH3), 0.82 (s, 3H, CH3),
1.15 (s, 3H, CH3), 1.24–2.49 (m, 12H), 2.71 (dd, 1H, 3J = 3.3 Hz,
2J = 18.0 Hz), 3.37 (m, 2H), 3.46 (d, 1H, 2J = 11.1 Hz), 3.65 (d, 1H,
2J = 11.1 Hz), 5.08 (s, 2H), 7.16 (dd, 1H, 4J = 2.4 Hz, 3J = 8.4 Hz),
7.27–7.40 (m, 6H), 7.63 (d, 1H, 4J = 2.4 Hz).
2.2.3. 3-O-Benzyl-estra-1,3,5(10)-triene-3,6-diol-17-one-17,17-(20-
[50,50-dimethyl-10,30-dioxane]) (4)
A solution of 3-O-benzyl-6-oxoestra-1,3,5(10)-triene-3-ol-17-
one 17,17-(20-[50,50-dimethyl-1’,3’-dioxane]) (3) (1.7 g, 3.7 mmol)
in a mixture of methanol (24.7 mL) and diethyl ether (8.2 mL)

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C. Neto et al. / Steroids 77 (2012) 1123–1132
was cooled to 0 °C and NaBH4 (0.84 g, 22.1 mmol) was slowly
added. Next, the mixture was stirred for 1 h at room temperature.
The solvent was removed in vacuo and water (30 mL) was slowly
added to the residue. The resulting mixture was extracted with
diethyl ether (3 ꢂ 30 mL). The organic phase was dried over anhy-
drous MgSO4 and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (n-hexane:diethyl
ether:chloroform, 2:1:1) to give 3-O-benzyl-estra-1,3,5(10)-tri-
ene-3,6-diol-17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (4)
as a colourless solid (1.6 g, 94%), mp. 107–109 °C.
2.2.6. 3-O-Benzyl-estra-1,3,5(10)-trien-3,7a
-diol-17-one-17,17-(20-
[50,50-dimethyl-10,30-dioxane]) (7)
To a suspension of LiAlH4 (116 mg, 3.1 mmol) in dry THF (5 mL)
was added 3-O-benzyl-6 ,7 -epoxyestra-1,3,5(10)-trien-3-ol-17-
a
a
one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (6) (704 g, 1.53 mmol)
at 0 °C and under an argon atmosphere, and the solution was stir-
red for 3 h at room temperature. Ethyl acetate (2 mL) was slowly
added at 0 °C to quench the remaining LiAlH4. Thereafter, aqueous
NH4Cl was added until a pH of 3 was reached, and the mixture was
extracted with diethyl ether (3 ꢂ 20 mL). The organic phase was
washed with water (20 mL), dried over anhydrous MgSO4 and con-
centrated in vacuo to give 3-O-benzyl-estra-1,3,5(10)-trien-3,
1H NMR (300 MHz, CDCl3,) d: 0.71 (s, 3H, CH3), 0.81 (s, 3H, CH3),
1.15 (s, 3H, CH3), 1.24–1.73 (m, 9H), 1.89–1.95 (m, 1H), 2.22–2.33
(m, 4H), 3.37 (m, 2H), 3.46 (d, 1H, 2J = 11.1 Hz), 3.65 (d, 1H,
2J = 11.1 Hz), 4.81 (m, 1H), 5.04 (s, 2H), 6.84 (dd, 1H, 4J = 2.4 Hz,
3J = 8.4 Hz), 7.18–7.43 (m, 7H).
7a
-diol-17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (7) as a
colourless solid (578 mg, 82%), mp. 135–137 °C.
1H NMR (300 MHz, CDCl3) d: 0.71 (s, 3H, CH3), 0.82 (s, 3H, CH3),
1.15 (s, 3H, CH3), 1.37–1.97 (m, 9H), 2.26–2.38 (m, 3H), 2.58 (m,
1H), 2.85–2.94 (m, 1H), 3.07–3.13 (m, 1H), 3.35–3.48 (m, 3H),
3.63 (d, 1H, 2J = 11.1 Hz), 4.10 (m, 1H), 5.01 (s, 2H), 6.70 (d, 1H,
4J = 2.4 Hz), 6.78 (d, 1H, 3J = 8.4 Hz), 7.22–7.41 (m, 6H). 13C NMR
(67.8 MHz, CDCl3) d: 13.9, 22.0, 22.5, 26.1, 27.0, 29.3, 30.4, 35.5,
38.6, 42.6, 43.2, 47.4, 69.9, 70.6, 72.6, 108.4, 112.9, 115.5, 126.7,
127.4, 127.8, 128.5, 134.5, 156.9. HRMS: calculated for C30H39O4:
463.2848; found: 463.2849.
2.2.4. 3-O-Benzyl-estra-1,3,5(10),6(7)-tetraen-3-ol-17-one-17,17-(20-
[50,50-dimethyl-10,30- dioxane]) (5)
To a solution of 3-O-benzyl-estra-1,3,5(10)-triene-3,6-diol-17-
one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (4) (1.6 g, 3.4 mmol)
in benzene (39 mL) was added neopentylglycol (NPG, 1.55 g,
0.02 mol) and p-TsOH (87 mg, 0.46 mmol). The resulting mixture
was heated for 4 h under reflux with continuous azeotropic re-
moval of water (Dean–Stark condenser). The solution was cooled
to room temperature, poured into a 5% aqueous Na2CO3 solution
(30 mL), and the organic phase was separated. The organic phase
was washed with water (30 mL) as well as with a saturated NaCl
solution (30 mL), dried over anhydrous MgSO4, filtered and con-
centrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (n-hexane:ethyl acetate, 5:2) to afford 3-O-
benzyl-estra-1,3,5(10),6(7)-tetraen-3-ol-17-one-17,17-(20-[50,50-
dimethyl-10,30-dioxane]) (5) as a colourless solid (1.01 g, 66%), mp.
122–124 °C.
2.2.7. 3-O-Benzyl-7a-methoxyestra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-10,30-dioxane]) (8a)
To a solution of 3-O-benzyl-estra-1,3,5(10)-trien-3,7a-diol-17-
one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (7) (578 mg, 1.25 mmol)
in dry THF (20 mL) was added NaH (1.8 g, 75 mmol) and then
methyl iodide (1.78 g, 0.78 mL, 0.01 mol). The reaction mixture
was stirred at room temperature for 20 h. Thereafter, the mixture
was poured into cold water and extracted with diethyl ether
(3 ꢂ 30 mL). The organic phase was dried over anhydrous MgSO4
and the solvent removed. The residue was purified by column
chromatography on silica gel (hexane:diethyl ether:chloroform,
1H NMR (300 MHz, CDCl3) d: 0.79 (s, 3H, CH3), 0.91 (s, 3H, CH3),
1.25 (s, 3H, CH3), 1.27–2.43 (m, 11H), 3.44–3.53 (m, 3H), 3.73 (m,
1H), 5.08 (s, 2H), 6.04 (d, 1H, J = 9.6 Hz), 6.48 (d, 1H, J = 9.6 Hz),
6.79 (s, 1H), 6.85 (d, 1H, 3J = 8.4 Hz), 7.22 (d, 1H, 3J = 8.4 Hz),
7.36–7.50 (m, 5H).
5:1:1) to give 3-O-benzyl-7a-methoxyestra-1,3,5(10)-trien-3-
ol-17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (8a) as a col-
ourless solid (458 mg, 77%), mp. 124–126 °C.
1H NMR (300 MHz, CDCl3,) d: 0.71 (s, 3H), 0.80 (s, 3H), 1.15 (s,
3H), 1.25–2.05 (m, 6H), 2.35 (m, 2H), 2.65 (m, 1H), 2.83 (m, 1H),
3.09 (d, 1H, J = 18 Hz), 3.34 (s, 3H), 3.36–3.53 (m, 4H), 3.62–3.75
(m, 3H), 5.01 (s, 2H), 6.70 (d, 1H, 4J = 2.4 Hz), 6.77 (dd, 1H,
4J = 2.4 Hz, 3J = 8.4 Hz), 7.21–7.42 (m, 6H). HRMS: calculated for
2.2.5. 3-O-Benzyl-6a,7a-epoxyestra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-10,30-dioxane]) (6)
To a mixture of 3-O-benzyl-estra-1,3,5(10),6(7)-tetraen-3-ol-
C31H40O4: 477.3005; found: 477.3003.
17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane])
(5)
(1.0 g,
2.2 mmol) in a mixture of CH2Cl2 (44 mL) and phosphate buffer
(0.1 M, pH 8) (44 mL) was slowly added m-chloroperbenzoic acid
(380 mg, 2.2 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 5 h. Next, the organic layer was separated,
washed with saturated sodium thiosulfate (40 mL) and water
(40 mL), dried over anhydrous MgSO4 and the solvent was re-
moved. The residue was purified by column chromatography on
silica gel (n-hexane:ethyl acetate, 5:1) to afford 3-O-benzyl-
2.2.8. 3-O-Benzyl-7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-10,30-dioxane]) (8b)
To a solution of 3-O-benzyl-estra-1,3,5(10)-trien-3,7a-diol-17-
one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (7) (300 mg, 0.65 mmol)
in dry THF (10 mL) was added NaH (519 mg, 13.0 mmol) and n-
propyl iodide (1.10 g, 6.5 mmol) and the reaction mixture was stir-
red at reflux temperature for 20 h. Then, the mixture was poured
into cold water and extracted with diethyl ether (3 ꢂ 20 mL). The
organic phase was dried over anhydrous MgSO4 and the solvent re-
moved. The residue was purified by column chromatography on
silica gel (hexane:diethyl ether:chloroform, 8:1:1) to give 3-O-
6a,7a
-epoxyestra-1,3,5(10)-trien-3-ol-17-one 17,17-(20-[50,50-di-
methyl-10,30-dioxane]) (6) as a colourless solid (0.7 g, 68%), mp.
110–112 °C.
1H NMR (300 MHz, CDCl3) d:0.75 (s, 3H, CH3), 0.86 (s, 3H, CH3),
1.19 (s, 3H, CH3), 1.51–1.66 (m, 5H), 1.73 (t, 1H), 1.96–2.09 (m, 3H),
2.26–2.45 (m, 3H), 3.40–3.54 (m, 4H), 3.69 (d, 1H, 2J = 11.1 Hz),
3.82 (d, 1H, J = 4.2 Hz), 5.06 (s, 2H), 6.91 (dd, 1H, 4J = 2.4 Hz,
3J = 8.4 Hz), 7.05 (d, 1H, 4J = 2.4 Hz), 7.17 (d, 1H, 3J = 8.4 Hz),
7.32–7.46 (m, 5H). 13C NMR (67.8 MHz, CDCl3) d: 13.5, 22.0, 22.5,
23.3, 24.1, 27.1, 28.8, 30.4, 36.0, 37.6, 44.7, 47.9, 53.8, 56.1, 70.1,
70.6, 72.7, 108.2, 109.9, 114.1, 116.4, 125.2, 127.4, 127.9, 128.6,
benzyl-7a
-propoxyestra-1,3,5(10)-trien-3-ol-17-one-17,17-(20-[50,
50-dimethyl-10,30-dioxane]) (8b) as a colourless solid (263 mg,
80%), mp. 95–98 °C.
1H NMR (270 MHz, CDCl3) d: 0.71 (s, 3H, CH3), 0.79 (s, 3H, CH3),
0.83 (t, 3H, CH3), 1.15 (s, 3H, CH3), 1.31–1.72 (m, 9H), 1.89–2.13 (m,
2H), 2.23–2.38 (m, 2H), 2.65–2.89 (m, 2H), 3.03 (d, 1H, J = 17 Hz),
3.21–3.29 (m, 1H), 3.35–3.67 (m, 6H), 5.01 (s, 1H), 6.68 (d, 1H,
4J = 2.4 Hz), 6.76 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.20–7.43 (m,
6H). HRMS: calculated for C33H44O4: 504.3240; found: 504.3246.
132.7, 133.6, 137.0, 156.9. HRMS: calculated for
461.2692; found: 461.2699.
C30H37O4:

C. Neto et al. / Steroids 77 (2012) 1123–1132
1127
2.2.9. 7
a
-Methoxyestra-1,3,5(10)-trien-3-ol-17-one-17,17-(20-[50,50-
room temperature for 15 h. Then, the reaction mixture was poured
into an aqueous solution of Na2CO3 (50 mL). The mixture was ex-
tracted with diethyl ether (3 ꢂ 50 mL) and the organic phase was
washed with water (100 mL), dried over anhydrous MgSO4 and
the solvent was removed. The residue was purified by column
chromatography on silica gel (hexane:diethyl ether:chloroform,
dimethyl-10,30-dioxane]) (9a)
To a solution of 3-O-benzyl-7
a-methoxyestra-1,3,5(10)-trien-3-
ol-17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (8a) (458 mg,
0.96 mmol) in THF (20 mL) was added 10w% Pd/C (25 mg). Hydro-
gen was bubbled into the reaction mixture, and hydrogenolysis
was performed for 24 h at room temperature under 1 atmosphere
of hydrogen. Then, the solution was filtered over Celite, washed
with THF and the solvent removed. The residue was purified by
column chromatography on silica gel (hexane:diethyl ether:chlo-
4:1:1) to give 7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one (10b)
as a colourless solid (227 mg, quant.), mp. 210–212 °C.
1H NMR (270 MHz, CDCl3) d: 0.83–0.89 (s, t, 6H, 2 CH3), 1.51–
2.20 (m, 10H), 2.40–2.55 (m, 2H), 2.73–2.86 (m, 2H), 3.05–3.12
(m, 1H), 3.23–3.31(m, 1H), 3.57–3.65 (m, 1H), 3.76 (m, 1H), 4.73
(s, 1H), 6.57 (d, 1H, 3J = 2.4 Hz), 6.63–6.66 (dd, 1H, 4J = 8.4 Hz,
3J = 2.4 Hz), 7.16 (d, 1H, 4J = 8.4 Hz) 13C NMR (67.8 MHz, CDCl3) d:
10.7, 13.6, 21.4, 23.3, 25.9, 31.4, 33.6, 35.8, 36.2, 42.1, 45.8, 47.8,
70.7, 72.4, 113.0, 115.8, 126.6, 132.0, 135.4, 153.5, 221.0. HRMS:
calculated for C21H28O3: 328.2038; found: 328.2036.
roform, 4:1:1) to give 7a-methoxyestra-1,3,5(10)-trien-3-ol-17-
one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (9a) as a colourless
solid (105 mg, 28%), mp. 170–171 °C.
1H (300 MHz, CDCl3) d: 0.72 (s, 3H, CH3); 0.81 (s, 3H, CH3); 1.15
(s, 3H, CH3); 1.20–1.55 (m, 3H); 1.6–2.07 (m, 2H); 2.28–2.87 (m,
2H), 2.62–2.64 (m, 1H), 2.74–2.81 (m, 1H), 3.00–3.07 (m, 1H),
3.34 (s, 3H, OCH3), 3.37–3.77 (m, 8H), 5.26 (s, 1H), 6.51 (dd, 1H,
4J = 2.4 Hz), 6.59 (d, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.15 (d, 1H,
3J = 8.4 Hz). HRMS: calculated for C24H35O4: 387.2535; found:
387.2538.
2.2.13. 3-O-(tert-Butyldimethylsilyl)-7a-methoxyestra-1,3,5(10)-
trien-3-ol-17-one (11a)
A
solution of tert-butyldimethylsilyl chloride (146 mg,
0.97 mmol) solution in anhydrous DMF (1.7 mL) was added to a
solution of -methoxyestra-1,3,5(10)-trien-3-ol-17-one (10a)
2.2.10. 7
dimethyl-10,30-dioxane]) (9b)
To a solution of 3-O-benzyl-7
a
-Propoxyestra-1,3,5(10)-trien-3-ol-17-one-17,17-(20-[50,50-
7a
(242 mg, 0.80 mmol) and imidazole (137 mg, 2.02 mmol) in anhy-
drous DMF (3 mL) at 0 °C. The mixture was stirred for 16 h at room
temperature. Then, the solvent was removed and the residue was
purified by column chromatography on silica gel (hexane:diethyl
ether:chloroform, 4:1:1) to afford 3-O-(tert-butyldimethylsilyl)-
a-propoxyestra-1,3,5(10)-trien-3-
ol-17-one-17,17-(20-[50,50-dimethyl-10,30-dioxane]) (8b) (263 mg,
0.52 mmol) in THF (5 mL) was added 10w% Pd/C (27 mg). Hydro-
gen was bubbled into the reaction mixture, and hydrogenolysis
was performed for 24 h at room temperature under 1 atmosphere
of hydrogen. Then, the solution was filtered and the solvent re-
moved. The residue was purified by column chromatography on
7a-methoxyestra-1,3,5(10)-trien-3-ol-17-one (11a) as a slowly
crystallizing, colourless solid (305 mg, 91%).
1H NMR (300 MHz, CDCl3) d: 0.05 (s, 6H, 2 CH3), 0.87 (s, 3H,
CH3), 0.96 (s, 9H, But), 1.45–1.68 (m, 5H), 1.86–2.20 (m, 3H),
2.40–2.53 (m, 2H), 2.65 (m, 1H), 2.78–2.85 (m, 1H), 3.11 (d, 1H,
2J = 18 Hz), 3.36 (s, 3H), 3.66 (m, 1H), 6.56 (d, 1H, 4J = 2.4 Hz),
6.61 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.12 (d, 1H, 3J = 8.4 Hz). 13C
NMR (67.8 MHz, CDCl3) d: 4.4, 13.6, 18.2, 21.4, 25.7, 26.0, 31.5,
32.7, 35.8, 36.4, 42.0, 45.8, 47.8, 56.7, 74.3, 117.6, 120.5, 126.3,
132.0, 134.6, 153.6, 220.7. HRMS: calculated for C25H38O3Si:
414.2590; found: 414.2594.
silica gel (hexane:ether:chloroform, 4:1:1) to 33give 7a-propoxy-
estra-1,3,5(10)-trien-3-ol-17-one-17,17-(20-[50,50-dimethyl-10,30-
dioxane]) (9b) as a colourless solid (203 mg, 94%), mp. 167–169 °C.
1H NMR (270 MHz, CDCl3) d: 0.73 (s, 3H, CH3), 0.81 (s, 3H, CH3),
0.87 (t, 3H, CH3), 1.16 (s, 3H, CH3), 1.23–1.74 (m, 8H), 1.90–2.14 (m,
2H), 2.28–2.40 (m, 2H), 2.66–2.82 (m, 2H), 2.98–3.05 (m, 1H),
3.22–3.28 (m, 1H), 3.37–3.69 (m, 6H), 4.51 (s, 1H), 6.54 (d, 1H,
4J = 2.4 Hz), 6.62 (dd, 1H, 4J = 2.4 Hz, 3J = 8.4 Hz), 7.18 (d, 1H,
3J = 8.4 Hz). HRMS: calculated for C26H38O4: 414.2770; found:
414.2767.
2.2.14. 3-O-(tert-Butyldimethylsilyl)-7a-propoxyestra-1,3,5(10)-
trien-3-ol-17-one (11b)
2.2.11. 7
a
-Methoxyestra-1,3,5(10)-trien-3-ol-17-one (10a)
A
solution of tert-butyldimethylsilyl chloride (109 mg,
0.72 mmol) in anhydrous DMF (2 mL) was added to a solution of
7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one (10b) (197 mg,
A
solution of
7a-methoxyestra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-10,30-dioxane]) (9a) (410 mg, 1.1 mmol)
and p-TsOH (83 mg, 0.43 mmol) in acetone (83 mL) was stirred at
room temperature for 15 h. Then, the reaction mixture was poured
into an aqueous solution of Na2CO3 (80 mL). The mixture was ex-
tracted with diethyl ether (3 ꢂ 50 mL) and the organic phase was
washed with water (100 mL), dried over anhydrous MgSO4 and
the solvent was removed. The residue was purified by column
chromatography on silica gel (hexane:diethyl ether:chloroform,
0.60 mmol) and imidazole (102 mg, 1.5 mmol) in anhydrous DMF
(3 mL) at 0 °C. The mixture was stirred for 10 h at room tempera-
ture. Then, the solvent was evaporated and the residue was puri-
fied by column chromatography on silica gel to afford 3-O-(tert-
butyldimethylsilyl)-7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one
(11b) as a slowly crystallizing, colourless solid (262 mg, 99%).
1H NMR (270 MHz, CDCl3) d: 0.19 (s, 6H, 2CH3), 0.83–0.88 (s, t,
6H, 2 CH3) 0.98 (s, 9H, But), 1.46–1.68 (m, 8H), 1.89–2.20 (m, 4H),
2.41–2.55(m, 2H), 2.72–2.86 (m, 2H), 3.04–3.11 (m, 1H), 3.23–3.32
(m, 1H), 3.57–3.65 (m, 1H), 3.75 (m, 1H), 6.56 (d, 1H, 4J = 2.4 Hz),
6.61–6.65 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.20 (d, 1H, 3J = 8.4 Hz).
13C NMR (67.8 MHz, CDCl3) d: 4.4, 10.7, 13.6, 18.2, 23.3, 25.7,
25.9, 31.5, 33.6, 35.8, 36.2, 42.1, 45.8, 47.8, 70.6, 72.4, 117.5,
120.5, 126.2, 132.4, 135.0, 153.5, 220.9. HRMS: calculated for
4:1:1) to give 7a-methoxyestra-1,3,5(10)-trien-3-ol-17-one (10a)
as a colourless solid (242 mg, 78%), mp. 205–207 °C.
1H NMR (300 MHz, CDCl3) d: 0.87 (s, 3H, CH3), 1.24–1.68 (m,
6H), 1.83–2.24 (m, 3H), 2.39–2.54 (m, 2H), 2.63–2.69 (m, 1H),
2.79–2.86 (m, 1H), 3.11 (d, 1H), 3.38 (s, 3H, OCH3), 3.66 (m, 1H),
6.57 (d, 1H, 4J = 2.4 Hz), 6.63 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.13
(d, 1H, 3J = 8.4 Hz). 13C NMR (67.8 MHz, CDCl3) d: 13.6, 21.4, 26.0,
31.4, 32.74, 35.8, 36.3, 42.0, 45.8, 47.8, 56.8, 74.2, 113.2, 115.8,
126.7, 131.7, 134.9, 153.6, 220.9. HRMS: calculated for C19H24O3:
300.1725; found: 300.1725.
C
27H42O3Si: 422.2903; found: 442.2904.
2.2.15. 3-O-(tert-Butyldimethylsilyl)-7 -methoxy-17a-
a
trimethylsilylethynylestra-1,3,5(10)-trien-3,17b-diol (12a)
2.2.12. 7
a
-Propoxyestra-1,3,5(10)-trien-3-ol-17-one (10b)
To a solution of trimethylsilylacetylene (216 mg, 2.20 mmol) in
dry THF (8 mL) was added under nitrogen at ꢀ78 °C n-butyl lith-
ium (solution in n-hexane, 1.58 M, 1.1 mL), and the resulting mix-
ture was stirred at ꢀ78 °C for 30 min and for further 30 min at 0 °C.
A
solution of
7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one-
17,17-(20-[50,50-dimethyl-10,30-dioxane]) (9b) (283 mg, 0.68 mmol)
and p-TsOH (51 mg, 0.27 mmol) in acetone (51 mL) was stirred at

1128
C. Neto et al. / Steroids 77 (2012) 1123–1132
Then, the reaction mixture was re-cooled to ꢀ78 °C and a solution
of 3-O-(tert-butyldimethylsilyl)-7 -methoxyestra-1,3,5(10)-trien-
4J = 2.4 Hz), 7.17 (d, 1H, 3J = 8.4 Hz). 13C NMR (67.8 MHz, CDCl3)
d:12.4, 22.7, 26.6, 32.6, 33.2, 35.9, 38.9, 43.2, 44.8, 47.2, 56.1,
74.2, 74.9, 79.9, 87.5, 113.2, 115.9, 126.8, 132.2, 135.2, 153.4.
HRMS: calculated for C21H26O3: 326.1882; found: 326.1879.
a
3-ol-17-one (11a) (305 mg, 0.74 mmol) in dry THF (8 mL) was
added. The mixture was allowed to warm slowly to room temper-
ature and was stirred for 15 h. Then, a saturated NH4Cl solution
(2 M, 40 mL) was added and the mixture was extracted with
diethyl ether (3 ꢂ 50 mL). The organic phase was dried over anhy-
drous MgSO4 and the solvent was removed. The residue was puri-
fied by column chromatography on silica gel (hexane:diethyl
2.2.18. 7
To a solution of 3-O-(tert-butyldimethylsilyl)-7
trimethylsilylethynylestra-1,3,5(10)-trien-3,17b-diol
a
-Propoxy-17
a
-ethynylestra-1,3,5(10)-trien-3,17b-diol (13b)
-propoxy-17
(12b)
a
a-
(216 mg, 0.40 mmol) in THF (10 mL) was added at ꢀ10 °C tetrabu-
tylammonium fluoride (TBAF, 418 mg, 1.6 mmol). The reaction
mixture was stirred at room temperature for 2 h. Then, diethyl
ether (30 mL) was added, and the organic phase was extracted.
The organic phase was dried over anhydrous MgSO4 and the sol-
vent was evaporated. The residue was purified by column chroma-
tography on silica gel (hexane:diethyl ether:chloroform, 4:1:1) to
ether:chloroform, 4:1:1) to give 3-O-(tert-butyldimethylsilyl)-7
a-
methoxy-17 -trimethylsilylethynylestra-1,3,5(10)-trien-3,17b-
a
diol (12a) as a slowly crystallizing, colourless solid (280 mg, 74%).
1H NMR (300 MHz, CDCl3) d: 0.16 (2s, 15H, 5 CH3), 0.83 (s, 3H,
CH3), 0.95 (s, 9H, But), 1.40–2.81 (m, 13H), 3.20 (m, 1H), 3.36 (s, 3H,
OCH3), 3.42 (m, 1H), 6.53 (d, 1H, 4J = 2.4 Hz), 6.60 (dd, 1H,
3J = 8.4 Hz, 4J = 2.4 Hz), 7.14 (d, 1H, 3J = 8.4 Hz). 13C NMR
(67.8 MHz, CDCl3) d: ꢀ4.4, 0.0, 12.5, 18.2, 22.7, 25.7, 26.5, 32.7,
33.4, 36.2, 38.9, 43.1, 44.9, 47.3, 57.2, 75.1, 80.1, 90.3, 109.6,
117.6, 120.6, 126.5, 132.4, 134.8, 153.5. HRMS: calculated for
give
7a-propoxy-17a-ethynylestra-1,3,5(10)-trien-3,17b-diol
(13b) as a colourless solid (115 mg, 81%), mp. 196–198 °C.
1H NMR (270 MHz, CDCl3) d: 0.86 (s, 3H, CH3), 0.88 (t, 3H, CH3),
1.18–2.50 (m, 13H), 2.59 (s, 1H), 2.69–2.84 (m, 2H), 2.99–3.05 (m,
1H), 3.21–3.29 (m, 1H), 3.56–3.64 (m, 2H), 4.63 (s, 1H), 6.55 (d, 1H,
4J = 2.4 Hz), 6.61–6.64 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.17 (d, 1H,
3J = 8.4 Hz). 13C NMR (67.8 MHz, CDCl3) d: 10.7, 12.4, 22.6, 23.3,
26.4, 32.5, 34.0, 35.7, 38.9, 43.1, 44.6, 47.0, 70.9, 73.0, 74.0, 79.9,
87.2, 93.6, 112.9, 115.7, 126.6, 132.5, 135.6, 153.3. HRMS: calcu-
lated for C23H30O3: 354.2195; found: 354.2193.
C30H48O3Si2: 512.3142; found: 512.3143.
2.2.16. 3-O-(tert-Butyldimethylsilyl)-7 -propoxy-17a-
a
trimethylsilylethynylestra-1,3,5(10)-trien-3,17b-diol (12b)
To a solution of trimethylsilylacetylene (162 mg, 1.65 mmol) in
dry THF (6 mL) was added at ꢀ78 °C lithium diisopropylamide
(solution in THF/ethylbenzene/heptane, 2 M, 0.9 mL, 1.8 mmol),
and the resulting mixture was stirred for 30 min at ꢀ78 °C and
for an additional 30 min at 0 °C. Then, the reaction mixture was re-
cooled to ꢀ78 °C, and a solution of 3-O-(tert-butyldimethylsilyl)-
2.2.19. 7
triene-3,17b-diol (14a)
solution of -methoxy-17
3,17b-diol (13a) (80 mg, 0.24 mmol), 1,4-fluoro-iodobenzene
a-Methoxy-17a
-(40-fluorophenylethynyl)estra-1,3,5(10)-
A
7
a
a-ethynylestra-1,3,5(10)-trien-
7a-propoxyestra-1,3,5(10)-trien-3-ol-17-one
(11b)
(243 mg,
0.55 mmol) in dry THF (6 mL) was added. The mixture was allowed
to warm overnight (15 h). Then, NH4Cl (2 M, 30 mL) was added,
and the mixture was extracted with ether (3 ꢂ 30 mL). The organic
phase was dried over anhydrous MgSO4 and the solvent evapo-
rated. The residue was purified by column chromatography on sil-
ica gel (hexane:diethyl ether:chloroform, 4:1:1) to give 3-O-(tert-
(27
l
L,
0.24 mmol),
copper
(I)
iodide
(CuI,
1.8 mg,
9.6 ꢂ 10ꢀ3 mmol) and Pd[PPh3]4 (5.54 mg, 4.8 ꢂ 10ꢀ3 mmol) was
refluxed in a mixture of dry THF (5 mL) and dry triethylamine
(5 mL) for 3 h under a nitrogen atmosphere. After cooling to room
temperature, the solvent was evaporated, and the residue was
purified by column chromatography (ethyl acetate:n-hexane, 1:1)
butyldimethylsilyl)-7
a-propoxy-17a-trimethylsilylethynylestra-
to give 7a-methoxy-17a
-(40-fluorophenylethynyl)estra-1,3,5(10)-
1,3,5(10)-trien-3,17b-diol (12b) as a slowly crystallizing, colourless
triene-3,17b-diol (14a) as a light brown solid (82 mg, 80%), mp.
solid (220 mg, 74%).
109–110 °C.
1H NMR (270 MHz, CDCl3) d: 0.16–0.18 (2s, 15H, 5 CH3), 0.97 (s,
3H, CH3), 1.40–2.38 (m, 25H), 2.67–2.83 (m, 2H), 2.97–3.04 (m,
1H), 3.18–3.24 (m, 1H), 3.61–3.64 (m, 2H), 6.55 (d, 1H,
4J = 2.4 Hz), 6.61 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.16 (d, 1H,
3J = 8.4 Hz). 13C NMR (67.8 MHz, CDCl3) d: ꢀ4.3, 0.0, 11.0, 12.4,
22.6, 23.4, 25.8, 26.3, 32.6, 33.9, 35.9, 38.9, 43.1, 44.6, 47.1, 70.9,
73.3, 80.1, 90.0, 109.3, 117.4, 120.4, 120.5, 126.2, 132.8, 135.2,
1H NMR (300 MHz, CDCl3) d: 0.89 (s, 3H, CH3), 1.27–2.24 (m,
11H), 2.37–2.47 (m, 1H), 2.58–2.66 (m, 1H), 2.74–2.81 (m, 2H),
3.03 (d, 1H, 2J = 17.7 Hz), 3.34 (s, 3H, OCH3), 3.54 (m, 1H), 5.65 (s,
1H), 6.53 (d, 1H, 4J = 2.4 Hz), 6.60 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz),
6.98 (m, 2H), 7.13 (d, 1H, 3J = 8.4 Hz), 7.41 (m, 2H). 13C NMR
(75 MHz, CDCl3) d: 13.0, 23.0, 27.0, 33.2, 33.4, 36.3, 39.2, 43.4,
45.4, 48.0, 57.3, 75.3, 80.6, 85.3, 113.4, 115.7 (d, JCF = 22.0 Hz),
116.1, 119.3, 127.0, 131.9, 133.7, 133.8 (d, JCF = 9.3 Hz), 135.3,
154.0, 162.7 (d, JCF = 247.7 Hz). 19F-RMN (282 MHz, CDCl3) d:
ꢀ111.46 (quint.). ESI/MS: calculated for C27H29O3F: 419.2 [MꢀH]ꢀ;
found: 419.1 [MꢀH]ꢀ. C27H29O3F: calcd. C, 77.11; H, 6.95. Found. C,
77.20; H, 6.64.
153.3. HRMS: calculated for
540.3451.
C31H52O3Si2: 540.3455; found:
2.2.17. 7a-Methoxy-17a-ethynylestra-1,3,5(10)-trien-3,17b-diol
(13a)
To a solution of 3-O-(tert-butyldimethylsilyl)-7
trimethylsilylethynylestra-1,3,5(10)-trien-3,17b-diol
a
-methoxy-17
a-
(12a)
2.2.20. 7
triene-3,17b-diol (14b)
solution of
a
-Propoxy-17
a
-(40-fluorophenylethynyl)estra-1,3,5(10)-
(280 mg, 0.54 mmol) in THF (10 mL) was added at ꢀ10 °C tetrabu-
tylammonium fluoride (TBAF, 565 mg, 2.16 mmol). The reaction
mixture was stirred at room temperature for 2 h. Then, diethyl
ether (30 mL) was added, and the organic phase was extracted.
The organic phase was dried over anhydrous MgSO4 and the sol-
vent evaporated. The residue was purified by column chromatogra-
A
7
a
-propoxy-17 -ethynylestra-1,3,5(10)-trien-
a
3,17b-diol (13b) (40 mg, 0.11 mmol), 1,4-fluoro-iodobenzene
(12.7
l
L, 0.11 mmol), CuI (1.0 mg, 4.4 ꢂ 10ꢀ3 mmol) and Pd[PPh3]4
(5.54 mg, 4.8 ꢂ 10ꢀ3 mmol) was refluxed in a mixture of dry THF
(5 mL) and dry triethylamine (5 mL) for 3 h under nitrogen atmo-
sphere. After cooling to room temperature, the solvent was evapo-
rated, and the residue was purified by column chromatography
phy on silica gel (hexane:ether:chloroform, 4:1:1) to give 7
methoxy-17 -ethynylestra-1,3,5(10)-trien-3,17b-diol (13a) as
a-
a
a
colourless solid (133 mg, 75%), mp. 193–196 °C.
(ethyl acetate:n-hexane, 1:1) to give 7a-methoxy-17a
-(40-fluoro-
1H NMR (300 MHz, CDCl3) d: 0.86 (s, 3H, CH3), 1.27–2.53 (m,
10H), 2.61 (s, 1H), 2.59–2.70 (m, 1H), 2.76 (m, 1H); 2.85 (m, 1H),
3.07 (d, 1H, 2J = 17.5 Hz), 3.36 (s, 3H, OCH3), 3.53 (m, 1H), 4.62–
4.66 (m, 1H), 6.56 (d, 1H, 4J = 2.4 Hz), 6.64 (dd, 1H, 3J = 8.4 Hz,
phenylethynyl)estra-1,3,5(10)-triene-3,17b-diol (14b) as a light
brown solid (25 mg, 50%), mp. 164–166 °C.
1H NMR (300 MHz, CDCl3) d: 0.82 (s, 3H, CH3), 0.86 (t, 3H, CH3),
1.22–2.44 (m, 13H), 2.58 (m, 1H), 2.69–2.84 (m, 2H), 3.00 (m, 1H),

C. Neto et al. / Steroids 77 (2012) 1123–1132
1129
3.21–3.26 (m, 1H), 3.58–3.65 (m, 2H), 5.06 (s, 1H), 6.53 (d, 1H,
4J = 2.4 Hz), 6.61 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 6.97 (m, 2H),
7.15 (d, 1H, 3J = 8.4 Hz), 7.38 (m, 2H). 13C NMR (75 MHz, CDCl3)
d: 9.7, 11.4, 21.6, 22.3, 25.4, 31.8, 33.0, 34.8, 37.9, 42.2, 43.8,
46.5, 65.9, 72.2, 79.4, 84.0, 91.3, 111.9, 114.5 (d, JCF = 22 Hz),
114.8, 118.1, 125.6, 131.2, 132.4 (d, JCF = 8.3 Hz), 132.4, 134.5,
152.4, 161.4 (d, JCF = 247.7 Hz). 19F-RMN (282 MHz CDCl3) d:
(ethyl acetate:n-hexane, 1:1) to give 7
phenylethynyl)estra-1,3,5(10)-triene-3,17b-diol (15b) as a colour-
less solid (18.1 mg, 38%), 190–193 °C.
a
-propoxy-17
a
-(40-iodo-
1H NMR (CDCl3, 300 MHz) d: 0.82 (s, 3H, CH3), 0.86 (t, 3H, CH3),
1.21–2.43 (m, 13H), 2.58 (s, 1H), 2.66–2.82 (m, 2H), 2.99 (m, 1H),
3.22 (m, 1H), 3.55 (m, 2H), 4.72 (s, 1H), 6.55 (d, 1H, 4J = 2.4 Hz),
6.61 (dd, 1H, 3J = 8.4 Hz, 4J = 2.4 Hz), 7.14 (m, 3H), 7.61 (d, 2H,
3J = 8.4 Hz). 13C NMR (CDCl3, 75 MHz) d: 9.7, 11.4, 21.6, 22.4,
25.3, 31.5, 33.0, 34.6, 37.9, 42.2, 43.6, 46.0, 69.9, 72.0, 78.9, 84.1,
93.1, 111.9, 114.3, 121.5, 125.6, 130.3, 131.5, 134.6, 136.4, 152.3.
ESI/MS: calculated for C29H33O3I: 557.1 [M+H]+; found: 557.2
[M+H]+. C29H33O3I: calcd. C, 62.59; H, 5.98. Found. C, 62.36; H, 5.89.
ꢀ111.44 (quint.). ESI/MS: calculated for
C29H33O3F: 449.2
[M+H]+; found: 449.4 [M+H]+. C29H33O3F: calcd. C, 77.65; H, 7.42.
Found. C, 77.73; H, 7.71.
2.2.21. 17a
-(40Fluorophenylethynyl)estra-1,3,5(10)-triene-3,17b-diol
(14c)
Preparation of 14c followed the general literature procedure
[35]. solution of 17 -ethynylestradiol (13c) (100 mg,
L, 0.34 mmol), CuI
Pd[PPh3]4 (7.9 mg,
2.2.24. 17a
-(40-Iodophenylethynyl)estra-1,3,5(10)-triene-3,17b-diol
(15c)
A
a
0.34 mmol), 1,4-fluoro-iodobenzene (40
l
Preparation of 15c followed the general literature procedure
(2.7 mg,
1.4 ꢂ 10ꢀ2 mmol)
and
[40]. A solution of 17a-ethynylestradiol (13c) (50 mg, 0.17 mmol),
6.8 ꢂ 10ꢀ3 mmol) was refluxed in a mixture of dry THF (5 mL)
and dry triethylamine (5 mL) for 3 h under nitrogen atmosphere.
After cooling to room temperature, the solvent was evaporated,
and the residue was purified by column chromatography (ethyl
1,4-diiodobenzene
(56.1 mg, 0.17 mmol), CuI (1.3 mg,
6.8 ꢂ 10ꢀ3 mmol) and Pd[PPh3]4 (9.4 mg, 3.4 ꢂ 10ꢀ3 mmol) was re-
fluxed in a mixture of dry THF (5 mL) and dry triethylamine (5 mL)
for 3 h under nitrogen atmosphere. After cooling to room temper-
ature, the solvent was evaporated, and the residue was purified by
acetate:n-hexane, 1:1) to give 17a
-(40-fluorophenylethynyl)estra-
1,3,5(10)-triene-3,17b-diol (14c) as a light brown solid (66 mg,
50%), mp. 85–88 °C.
column chromatography (ethyl acetate:n-hexane, 1:1) to give 17a-
(40-iodophenylethynyl)estra-1,3,5(10)-triene-3,17b-diol (15c) as a
colourless solid (31 mg, 37%), mp. 219–222 °C.
1H NMR (300 MHz, CDCl3) d: 0.92 (s, 3H, CH3), 1.22–2.40 (m,
12H), 2.88 (m, 3H), 6.56 (d, 1H, 4J = 2.4 Hz), 6.63 (dd, 1H,
3J = 8.4 Hz, 4J = 2.4 Hz), 6.99 (m, 2H), 7.14 (d, 1H, 3J = 8.4 Hz), 7.41
(m, 2H). 13C NMR (75 MHz, CDCl3) d: 13.0, 23.0, 26.6, 27.4, 29.9,
33.0, 39.2, 39.6, 43.7, 47.4, 49.7, 74.4, 80.2, 87.7, 113.0, 115.5,
115.8 (d, JCF = 22 Hz), 126.8, 132.7, 133.8 (d, JCF = 8.2 Hz), 138.5,
153.6, 162.7 (d, JCF = 248.3 Hz). 19F-RMN (282 MHz CDCl3) d:
1H NMR (CDCl3, 300 MHz) d: 0.83 (s, 3H, CH3), 1.24–2.42 (m,
12H), 2.79 (s, 1H), 6.54 (d, 1H, 4J = 2.4 Hz), 6.60 (dd, 1H,
3J = 8.4 Hz, 4J = 2.4 Hz), 7.14 (m, 3H), 7.61 (d, 2H, 3J = 8.4 Hz). 13C
NMR (CDCl3, 75 MHz) d: 12.9, 22.6, 26.4, 27.2, 29.4, 33.1, 39.4,
39.9, 43.6, 47.7,49.8, 79.0, 83.9, 95.0, 112.7, 115.0, 122.5, 126.6,
129.5, 133.2, 137.4, 139.3, 154.9. ESI/MS: calculated for
ꢀ111.41 (quint.). ESI/MS: calculated for
C
26H27O2F: 391.2
C
26H27O2I: 499.1 [M+H]+; found: 498.9 [M+H]+. C26H27O2I: calcd.
C, 62.66; H, 5.46. Found. C, 62.61; H, 5.55.
[M+H]+; found: 391.2 [M+H]+. C26H27O2F: calcd. C, 79.97; H, 6.97.
Found. C, 80.06; H, 6.84.
2.3. Estimation of lipophilicity
2.2.22. 7
triene-3,17b-diol (15a)
solution of
a
-Methoxy-17
a
-(40-iodophenylethynyl)estra-1,3,5(10)-
The lipophilicity, which is referred to as logPo/w (where logPo/
w = octanol/water partition coefficient), of the compounds 14a/b
and 15a/b was determined from the log K0w values (Kw0 = chromato-
graphic capacity factor at 100% aqueous solution). The log K0w val-
ues were determined by reversed-phase HPLC on a C-18 column
according to the method described by Minick [41].
A
7
a
-methoxy-17 -ethynylestra-1,3,5(10)-trien-
a
3,17b-diol (13a) (30 mg, 0.09 mmol), 1,4-diiodobenzene (21.6 mg,
0.09 mmol), CuI (1 mg, 3.6 ꢂ 10ꢀ3 mmol) and Pd[PPh3]4 (2.1 mg,
1.8 ꢂ 10ꢀ3 mmol) was refluxed in a mixture of dry THF (5 mL)
and dry triethylamine (5 mL) for 3 h under nitrogen atmosphere.
After cooling to room temperature, the solvent was evaporated,
and the residue was purified by column chromatography (ethyl
Measurement of the chromatographic capacity factors (k0) for
each compound was carried out at various concentrations in the
range 85–70% methanol (containing 0.25% octanol) and an aqueous
phase consisting of 0.15% n-decylamine in 0.02 M MOPS (3-mor-
pholinopropanesulfonic acid) buffer pH 7.4 (prepared in 1-octa-
nol-saturated water). The compounds were dissolved in
acetate:n-hexane, 1:1) to give 7a-methoxy-17a
-(40-iodophenyle-
thynyl)estra-1,3,5(10)-triene-3,17b-diol (15a) as a colourless solid
(19 mg, 40%), mp. 180–182 °C.
1H RMN (300 MHz, CDCl3) d: 0.83 (s, 3H, CH3), 1.22–2.47 (m,
10H), 2.59–2.65 (m, 1H), 2.76–2.81 (m, 1H), 3.05 (d, 1H,
2J = 17.5 Hz), 3.34 (s, 1H), 3.54 (m, 1H), 4.80 (s, 1H), 6.54 (d, 1H,
4J = 2.4 Hz), 6.61 (dd, 1H, 4J = 2.4 Hz, 3J = 8.4 Hz), 7.15 (m, 3H),
7.62 (d, 2H, 3J = 8.4 Hz). 13C RMN (75 MHz, CDCl3) d: 12.9, 23.0,
26.9, 33.2, 33.3, 36.4, 39.2, 43.4, 47.9, 57.3, 75.1, 80.6, 85.5, 94.4,
113.3, 116.1, 122.7, 127.1, 132.2, 132.2, 135.4, 137.7, 153.7. ESI/
MS: calculated for C27H29O3I: 527.1 [M-H]ꢀ; found: 527.3 [MꢀH]ꢀ.
methanol (1 mg/mL) and about 5 lg were injected onto the col-
umn. The k0 values are defined as (tr ꢀ t0)/t0, where tr and t0 are
the retention times of the compounds and non-retained species
(solvent), respectively. The log k0w values were obtained from linear
extrapolation of log k0 vs
nol) data acquired in the region 0.70 6
u
methanol (
u
u
= volume fractions metha-
methanol 6 0.85. The log-
Po/w values of the compounds were determined from a standard
curve (log Po/w vs logk0w) constructed with data of standard
compounds.
C27H29O3I: calcd. C, 61.37; H, 5.53. Found. C, 61.69; H, 5.23.
2.2.23. 7
triene-3,17bdiol (15b)
solution of
a
- Propoxy-17
a
-(40-iodophenylethynyl)estra-1,3,5(10)-
2.4. Receptor-binding affinity
A
7
a
-propoxy-17 -ethynylestra-1,3,5(10)-trien-
a
3,17b-diol (13b) (30 mg, 0.08 mmol), 1,4-diiodobenzene (17.8 mg,
0.08 mmol), CuI (0.6 mg, 3.2 ꢂ 10ꢀ3 mmol) and Pd[PPh3]4
(1.8 mg, 1.6 ꢂ 10ꢀ3 mmol) was refluxed in a mixture of dry THF
(5 mL) and dry triethylamine (5 mL) for 3 h under nitrogen atmo-
sphere. After cooling to room temperature, the solvent was evapo-
rated, and the residue was purified by column chromatography
The ER
a
competitive binding assay was performed according to
bind-
a described method with minor modifications [42]. The ER
ing buffer used for dilution of the receptor preparations consisted
of 10% glycerol, 2 mM dithiothreitol, 1 mg/mL BSA and 10 mM
Tris–HCl (pH 7.5). The ER
a
a washing buffer contained 40 mM
Tris–HCl and 100 mM KCl (pH 7.4). The hydroxyapatite (HAP) slur-

1130
C. Neto et al. / Steroids 77 (2012) 1123–1132
ry was adjusted to a final concentration of 50% (v/v) by using a
50 mM Tris–HCl solution (pH 7.4). The reaction mixture contained
reactions of 7 with carbon nucleophiles such as with (n-Bu)2-
Cu(CN)Li2 were found to be not completely regioselective, the reac-
tion of 6 with LiAlH4 led exclusively to 7 [56]. Again, the
stereochemistry of 7 at C7 was determined by NOE experiments,
50
binding buffer, 45
and 5 L (0.25 pmol) of ER
by the tritiated estradiol was determined by the addition of a
50 M concentration of the nonradioactive estradiol (E2). The
binding mixture was incubated at 4 °C for 16–18 h. At the end of
the incubation, 200 L of the HAP slurry was added, and tubes
l
L of varying concentrations of the test compound in the ER
L of a solution of tritiated estradiol (23.8 nM)
protein solution. Non-specific binding
a
l
l
a
and the compound was confirmed to be the 7
[57]. Additionally, the coupling constants 3J of H(C7) at d 4.13 (in
CDCl3) with H /Hb(C6) and H(C8) are very small, indicative of
a-hydroxyl derivative
l
a
H(C7) at an equatorial position as one would expect larger coupling
constants between neighbouring axially positioned protons [58].
Etherification of 7 was carried out using NaH as base and alkyl
halides such as methyl iodide and n-propyl iodide as alkylating
agents. In the next step the steroidal esters 8a/8b were debenzylat-
ed at O–C3 by catalytic hydrogenation. Steroidal ethers 9a and 9b
were deprotected at C17 by transacetalisation (acetone, p-TsOH, rt)
to give 10a/10b. The phenolic function at C3 of 10a/10b was pro-
tected once again as its TBDMS ether by treatment with imidazole
in dry DMF, followed by silylation of the phenoxide with tert-
butyldimethylsilyl chloride (TBDMSCl) to give 11a and 11b,
respectively.
The C7-substituted estratetraene derivatives 11a/11b were re-
acted with lithium trimethylsilylacetylide to give 12a and 12b,
after column chromatographic separation on silica gel. Subse-
quently, 12a and 12b were reacted with tetra-n-butyl ammonium
fluoride in THF for 1 h for complete desilylation, giving compounds
13a and 13b in high yield.
l
were incubated on ice and vortexed three times for 15 min. An ali-
quot of 1 mL of washing buffer was added, mixed and centrifuged
at 10,000g for 10 min, and the supernatants were discarded. This
wash-step was repeated twice. The HAP pellets were then re-sus-
pended in 750 lL cold ethanol, vortexed three times for 20 min,
centrifuged and the supernatants were transferred to scintillation
vials for measurement of 3H radioactivity in a liquid scintillation
counter (Packard Tri-CARB 3170 TR/SL). The data obtained from
triplicate measurements were expressed as the percent specific
binding of [3H]E2 vs the log molar concentration of the competing
compound. The IC50 values (calculated using GraphPad Prism soft-
ware) represent the concentration of the test compound required
to reduce the [3H]E2 binding by 50%. Data were then expressed
as a relative binding affinity percentage (RBA) determined in com-
parison with E2.
For the preparation of fluorinated 14a–14c and the iodinated
estradiol derivatives 15a–15c to be used for the in vitro biological
evaluation studies, a Sonogashira coupling reaction of 13 with
1,4-diiodobenzene and with 1,4-fluoroiodobenzene, respectively,
was used [59–61].
3. Results and discussion
3.1. Chemistry
Initially, the synthesis of the 7
a
-alkoxyestradiol derivatives 10
The desired coupling products 14a/14b were obtained in high
chemical purity and good chemical yield (80% and 50%, respec-
tively). Also, the iodinated derivatives, 15a/15b were obtained in
high chemical purity and fair chemical yield (40% and 39%, respec-
tively). These compounds were used for the subsequent biological
followed the general route published earlier, albeit with a different
protective group at C3 [43]. The synthetic route is outlined in
Scheme 1.
The C3 phenol function of estrone was protected as its benzyl
ether, according to a described procedure, to give 3-O-benzyles-
tra-1,3,5(10)-trien-3-ol-17-one, 1 [44]. The acetalization of the
C17 keto group in 1 was carried out with neopentyl glycol (NPG)
under standard conditions to give the fully protected 2 [45].
In order to access the C7-position in estranes of type 2 it was
necessary to activate C6. For this purpose, C6 was oxidized. The di-
rect oxidation of estrane derivatives to 6-ketoestranes has been
previously reported and a variety of reagents has been used
namely, CrO3, CH2Cl2, 2,5-dimethylpyrazole [46], AcOH, CrO3,
H2O [47–49], pyridinium chlorochromate (PCC), benzene [50]
and CrO3, H2SO4 [51]. However, in our hands, the direct oxidation
of fully protected 3 could best be achieved with KMnO4 under PTC
conditions (Adogen 464, benzene, aq. NaHCO3) [45,52]. The out-
come of the reaction is dependent on reaction temperature and
reaction time. Nevertheless, the yield of 3 is similar, when the reac-
tion is run at 80 °C for 2 h or when it is run at 50 °C for 12 h.
Next, 3 was reduced with NaBH4 in a mixture of MeOH/Et2O to
give 4. NOE experiments have shown that the stereochemistry of 4
studies. Also, the non 7
derivatives (14c and 15c) were prepared under the same condi-
tions using 17 -ethynylestradiol as starting material and were ob-
a-substituted fluorinated and iodinated
a
tained in 50 and 37% yield, respectively.
3.2. In vitro studies
3.2.1. Estimation of lipophilicity
The lipophilicity of compounds can affect their tissue perme-
ability, which can impact their localization in target tissues. Lipo-
philicity may also affect binding to low affinity, nonspecific sites
that can compromise target tissue to background tissue ratios. To
anticipate the potential of the synthesized compounds to be stud-
ied further in vivo (cell lines, animal experiments) their octanol/
water partition coefficients were estimated by a reversed-phase
HPLC method described by Minick [41]. As expected on the basis
of their chemical structures, the estimated values (Table 1) indi-
cated that the novel 17a
-(40-halophenylethynyl)estradiol deriva-
at C6 was
a-(hydroxyl). A small amount of b-isomer was also
tives are more lipophilic than estradiol (logPo/w. = 3.82 0.09)
formed. 4 was subjected to dehydration with p-TsOH (benzene, re-
flux) to give 3-O-benzyl-estra-1,3,5(10),6(7)-tetraen-3-ol-17-one
17,17-(20-[50,50-dimethyl-10,30-dioxane]) 5. In the reaction, small
amounts of neopentylglycol were added in order to maintain the
protective group at C17. 5 was then reacted with m-chloroperben-
zoic acid in a phosphate buffer. The reaction is stereoselective and
suggesting their ability to penetrate cell membranes.
3.2.2. Receptor binding affinity studies
The in vitro ERa binding affinity was determined by competitive
radiometric binding assay using [3H]estradiol as tracer. Incuba-
tions were done overnight at 4 °C and hydroxylapatite was used
to separate bound receptor–ligand complex. The relative binding
affinity (RBA) for each compound was calculated against estradiol
(E2) by using the following equation: RBA = IC50 for E2/IC50 for each
compound x 100 and are presented in Table 1.
gives 6a,7a-epoxyestra-1,3,5(10)-trien-3-ol-17-one 6. Again, the
stereochemistry at C6/C7 was determined by NOE experiments.
In order to better attribute the proton signals, a 1H–1H COSY exper-
iment was carried out beforehand.
Benzocycloalkene 1,2-oxides are known to undergo regioselec-
tive reductive ring opening with complex hydrides [53–55]. While
To investigate whether the replacement of the terminal proton
in the acetylene unit by a 40-halophenyl group would lead to com-

C. Neto et al. / Steroids 77 (2012) 1123–1132
1131
Table 1
data indicate that the introduction of a methoxy group into the
C7 position of the ethynylestradiol framework, as in 13a, main-
tained a high ER binding affinity of the molecule (150% vs 154%).
Also, the introduction of a 40-fluorophenylethynyl group on the
estradiol framework, as in 14c, is well tolerated by the receptor
Relative binding affinities (RBA) and log P values for the estradiol derivatives.
R2
OH
(81%). Moreover, the simultaneous addition of a 7a-methoxy and
a 40-fluorophenyl on the ethynylestradiol, as in 14a, did not signif-
icantly diminish the ER binding affinity (79%). In contrast, the pres-
ence of 40-iodophenylethynyl substituents is not tolerated by the
estrogen receptor, excluding the possibility of using their 123I-la-
belled analogues as SPECT imaging agents for estrogen receptor
densities in tumours.
HO
R1
Compound
Substitution
RBA (%)
Log Po/w
Estradiol
Ethynylestradiol
100
3.82 0.09
3.74 0.13
2.93 0.09
3.83 0.13
4.36 0.09
5.20 0.17
5.01 0.15
5.12 0.09
6.15 0.15
5.65 0.11
154.68 0.36
150.08 0.01
15.60 0.01
78.88 0.01
15.82 0.01
80.87 0.02
0.42 0.01
n.d.
13a
13b
14a
14b
14c
15a
15b
15c
R
1 = OCH3; R2 = H
R1 = OC3H7; R2 = H
1 = OCH3; R2 = PhF
1 = OC3H7; R2 = PhF
1 = H; R2 = PhF
1 = OCH3; R2 = PhI
1 = OC3H7; R2 = PhI
1 = H; R2 = PhI
While the 17a
-(40-fluorophenylethynyl)estradiol derivatives
R
R
R
R
R
R
may be explored further as potential PET biomarkers for imaging
of ER expressing tumours, the more important consequence of
the study is that as 7
binding affinity to ER
a
-methoxyestradiols such as 13a show high
4.56 0.01
a, these molecules could potentially be used
as a platform for radiolabeled steroids, where the radiohalogen is
introduced in either the steroidal frame or the alkoxy side-chain.
Hence, studies in this direction are currently underway.
pounds that still retain ER binding affinity, the RBA values were
compared for both the estradiol and the 7 -alkoxy-estradiol series.
The binding affinity enhancing effect of the ethynyl substituent
itself is evident in comparing RBA values of estradiol and 17 -eth-
ynylestradiol (100% vs 154%). Both in the estradiol (14c) and in the
-methoxy series (14a) the introduction of a 40-fluorophenyle-
thynyl moiety is well tolerated by the ER (80% vs 79%). However,
in both series, the introduction of a 40-iodophenylethynyl group
(15c and 15a) drastically diminishes the binding affinity, rendering
these derivatives unlikely candidates for SPECT imaging. This lack
of binding affinity might be explained by the presence of the bulky
iodine atom, not very far away from the steroidal frame.
a
Acknowledgements
a
The authors gratefully acknowledge to DAAD/FCT (0811983/
441.00) for financial support. C. Neto thanks Fundação para a Ciên-
cia e Tecnologia (FCT) for a Ph.D. grant (SFRH/BD/31319/2006). The
authors thank Dr. Joaquim Marçalo and Dr Vânia Sousa for per-
forming mass spectra analysis and elemental analysis, respectively.
The authors are grateful to Dr João Abrantes for b measurement in
the RBA assays. Yosef Al Jasem, UAE University, is thanked for dis-
cussions regarding ligand–receptor interactions. The QITMS instru-
ment was acquired with the support of the Programa Nacional de
Reequipamento Científico (Contract REDE/1503/REM/2005-ITN)
of FCT and is part of Rede Nacional de Espectrometria de Massa
(RNEM).
7a
In this study, we have also compared the effect of the 7
oxy vs 7
-propoxy group on the ethynyl (13a and 13b) and 40-flu-
orophenylethynyl (14a, 14b, 14c) series. Addition of the 7
a-meth-
a
a
-
methoxy group to ethynylestradiol or to 14c, leading to 13a or
14a still maintains the high binding affinity of the compounds. In
contrast, adding a 7a-propoxy group to ethynylestradiol, leading
to 14b, did decrease the ER binding affinity to values of about
15% of that of estradiol, which, however, can still be considered
an adequate binding constant. This data suggests that an elonga-
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