Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta

Article abstract of DOI:10.1016/j.steroids.2008.04.009

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER⁺ and ER^-) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17β-estradiol (E₂) on both estrogen receptors (ERs), α and β. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E₂-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17β-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.

Full text of DOI:10.1016/j.steroids.2008.04.009

steroids 7 3 ( 2 0 0 8 ) 1077–1089
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Improved synthesis of unique estradiol-linked platinum(II)
complexes showing potent cytocidal activity and affinity
for the estrogen receptor alpha and beta
Caroline Desc oˆ teaux, Val e´ rie Leblanc, Geoffroy B e´ langer, Sophie Parent,
∗
´
Eric Asselin, Gervais B e´ rub e´
Groupe de Recherche en Biopathologies Cellulaires et Mol e´ culaires, D e´ partement de Chimie-Biologie, Universit e´ du Qu e´ bec a`
Trois-Rivi e` res, C.P. 500, Trois-Rivi e` res, Qu e´ bec, G9A 5H7 Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the
female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an
eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in
nine chemical steps with 10% overall yield. This new compound has been tested in vitro on
Received 5 December 2007
Received in revised form
26 March 2008
+
−
Accepted 13 April 2008
estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER and ER ) cell lines. Inter-
estingly, the biological activity was quite significant, more potent than that of cisplatin, the
compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA)
of this compound was very similar to that of 17␤-estradiol (E2) on both estrogen receptors
(ERs), ␣ and ␤. In order to further study this type of molecule, we have decided to synthesize
several analogs with the same estrogenic scaffold but with various chain lengths separat-
ing the estradiol from the toxic part of the molecule. This was planned in order to study
the effect of the length of the linking chain on the biological activity of the hybrids. Four
E2-Pt(II) hybrid molecules having 6–14 carbon atoms linking chain have been synthesized
using a new synthetic methodology. They are synthesized in only eight chemical steps with
Published on line 25 April 2008
Keywords:
17␤-Estradiol-linked platinum(II)
complex
Breast cancer
Estrogen receptor
Cisplatin
Biological activity
2
1% overall yield. The 17␤-estradiol-linked platinum(II) complexes have been tested for their
receptor binding affinity as well as for their cytocidal activity on several breast cancer cell
lines. The synthesis and biological results are reported herein.
©
2008 Elsevier Inc. All rights reserved.
1
.
Introduction
it spreads throughout the whole body and affects healthy
organs, causing numerous side effects such as nausea and
vomiting, kidney toxicity, blood test anomalies, anaemia and
increased risk of infection [4].
Bearing in mind the severe side effects following
chemotherapy with platinum-based drugs and other
chemotherapeutic agents, the search for site specific treat-
ment is still, to this day, underway. The specific delivery of
anticancer drugs to the sites needing treatment would, not
Cisplatin (cis-diamminedichloroplatinum(II)) (1) is a small
anticancer agent useful in the treatment of several type of
cancers, including lung, head and neck, ovarian, bladder and
testicular tumors [1]. Cisplatin (1) binds to the DNA of fast
growing cells, such as cancerous cells, and stops cell pro-
liferation leading to apoptosis [2,3]. This compound is very
active but presents no specificity. Thus, when administered,
∗
Corresponding author.
E-mail address: Gervais.Berube@uqtr.ca (G. B e´ rub e´ ).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.04.009

1
078
steroids 7 3 ( 2 0 0 8 ) 1077–1089
only improve efficacy, but also minimize toxic side effects.
The strategy of drug targeting and delivery could be applied
to tumours that exhibit biochemical differences (biomarkers)
from normal tissues [5].
responsible for binding to the receptors [5]. An estradiol ligand
modified in the 16 position of the steroid backbone, the 17␤-
estradiol-platinum(II) complexe (7, n = 9, Fig. 1), was described
in an early report [15].
Most of breast cancer diseases are classified as hormone-
dependent (60–70%) due to an overexpression of estrogen
receptors (ERs) at mammalian cells level [6]. The number of
estrogen receptor (ER) in hormone-dependent (ER ) breast
cancer cells is in the range of 10 000–30 000 (20 000 on average)
The distance separating the natural ligand from the
cytotoxic moiety seems to play an important role in the bio-
logical activity. It was observed that, on several non-steroidal
estrogen-Pt(II) complexes, the length of the side chain bearing
the cytotoxic Pt(II) portion should be 11 or 12 carbon atoms
long for optimal biological activity [16]. The 17␤-estradiol-
platinum(II) (7, n = 9) reported earlier has an 11 carbon atoms
chain at position 16␤ of the steroid nucleus [15].
The new 17␤-estradiol-platinum(II) derivative (7, n = 9)
showed high affinity for the estrogen receptor ␣ even bet-
ter than 17␤-estradiol itself [15]. Hybrid 7 (n = 9) showed an
IC50 of 0.35 nM compared to 4.79 nM for the natural ligand 2.
This strong affinity confirms that the modified structure could
theoretically target the ER␣-expressing cancer cells in an in
vivo experiment and, consequently, carry the cytotoxic moi-
ety at the sites needing treatment. Also, the in vitro biological
activity studies revealed a very active compound. The new
estradiol-platinum(II) complex is 32 times more active than
cisplatin against MCF-7 ER-dependent cells (1, IC50 = 16.1 ␮M;
7c, IC50 = 0.5 ␮M) and 26 times more active with MDA-MB-231
ER-independent cells (1, IC50 = 12.8 ␮M; 7c, IC50 = 0.5 ␮M) [15].
These results were obtained using the Sulforhodamine B col-
orimetric assay.
+
[
7]. The ER␣ is well characterized and is present in mammalian
gland, as well as in ovarian and in uterine tissues [8]. The
ER␣ is present at different levels of expression in a variety of
normal and cancer tissues. For examples, ER+ positive breast
cancer is an example of a high expression level tissue while the
expression level in normal mammary gland is relatively low
[
9]. Considering those facts, the ER␣ has already been used as a
biological target in cancer therapies [10]. Thus, using estradiol
E2) (2) as a carrier molecule, an anticancer drug could, theo-
(
retically, be directed accurately to hormone-dependent breast,
uterus and ovarian cancer cells.
Several estrogenic compounds have already been used to
link a platinum(II)-based toxic moiety at different position of
the steroid nucleus [10]. Only a few of the molecules reported
in the review paper are described herein in order to exemplify
the general concept of estrogen-linked platinum(II) strategy
for the treatment of hormone-dependent cancers. For exam-
ple, more than a decade ago, compounds (3, 4) have been
synthesized using the alcohol function (either at position 3
or 17) as an anchor point for adding a cytotoxic moiety [11,12].
Relative binding affinities (RBA) for those complexes were rel-
atively low, with RBA values of 0.53 for compound 3 and of
Taking into consideration the structure of the 17␤-
estradiol-platinum(II) complex (7c), its great cytotoxic activity
and its potential for the selective treatment of ER+ cancers,
this hybrid molecule was synthesised with a shorter and more
efficient methodology, resulting in an increase overall yield.
Three other 17␤-estradiol-platinum(II) complexes bearing 6, 8
and 14 carbon atoms in the alkyl chain (compounds 7a, 7b and
7d) were also synthesized in order to study the influence of the
length of the alkyl chain upon the biological activity.
6
.0 (compound 4). Taking into account that the hydroxyl func-
tions were playing an important role in receptor recognition,
another compound (5) was then synthesized in order to main-
tain these functions free to bind to the estrogen receptor [13].
In this case, the cisplatin component was added to the 17␣
position. Unfortunately, the RBA values were less than 1%,
yet again probably due to steric hindrance at the 17␤-hydroxy
function. Cisplatin dimers (6) have also been designed in
order to target estrogen receptor-positive cells [14]. Hence, sev-
eral estrogen-tethered orally active platinum(IV) complexes
were designed to release the toxic moiety by intracellular
esterases. The reducing environment of the cell converts the
platinum(IV) to platinum(II), in this case cisplatin itself. The
This manuscript gives the detailed description of the syn-
thesis of four 17␤-estradiol-platinum(II) complexes, bearing
an hexyl, octyl, undecyl and tetradecyl tether chain. The cyto-
cidal activity on four neoplastic human breast cancer cell
+
lines, MCF-7 (ER ), MDA-MB-231, MDA-MB-468 and MDA-MB-
−
436 (ER ) is reported herein. The estrogen receptor binding
affinities (ERBA) (ER␣ and ER␤) are also reported for three of
the four hybrids.
+
design rationale was inspired by the observation that ER cells
exposed to the hormone are sensitized to cisplatin (1). This
type of molecule (dimer 6) seems to show substantial activity
in vitro. These molecules are a few examples of estrogen-linked
platinum(II) complexes, additional examples are provided in
the review manuscript [10].
2
.
Experimental
2.1.
Chemistry
As one can noticed, an important requirement for high
affinity for the ER is to maintain the integrity of the hydroxyl
functions on the estrogenic nucleus. In fact, experiments
have revealed that the replacement of the hydroxyl groups at
positions 3 and 17␤ of the steroid backbone is generally detri-
mental to the receptor interaction as they play a crucial role in
the receptor recognition. Thus, it is important to reduce steric
hindrance at both the 3- and 17-hydroxyl binding pocket of the
receptor. Furthermore, it was reported that a rigid spacer kept
the platinum core well away from the two hydroxyl groups
All reactions were performed with ACS Fisher solvents.
In some cases, solvent, as well as starting materials and
reactants, were first purified and dried by standard means
[17]. Anhydrous reactions required an inert atmosphere of
dry nitrogen. Estrone was purchased from Steraloids Inc.,
Wilton, NH, USA and the n-alkyl chains were purchased from
Sigma–Aldrich Canada Ltd., Oakville, Ontario, Canada. All
reactions were monitored by UV fluorescence or staining with
iodine on Sigma T 6145 commercial TLC plates (polyester silica
gel 60 A˚ , 0.25 mm). Purification were done using flash column

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1079
Fig. 1 – Structure of cisplatin (1), 17␤-estradiol (2) and estrogen-linked platinum(II) compounds (3–7).
chromatography according to the method of Still et al. [18] on
Silicycle UltraPure Flash Silica Gel, 40–63 ␮m mesh. Hexanes
and acetone were distilled before their use as chromatography
eluant.
The infrared spectra were taken on a Nicolet Impact 420
FT-IR. Sodium chloride or potassium bromide pellets were
used for analysis. Mass spectral assays were obtained using
a VG Micromass 7070 HS instrument using ionization energy
of 70 eV (University of Sherbrooke). Chemical ionization (NH3)
was required for a few compounds.
Nuclear magnetic resonance (NMR) spectra were recorded
on a Varian 200 MHz NMR apparatus. Samples were dissolved
in deuterochloroform (CDCl3) or in deuteroacetone (acetone-
d6) for data acquisition using tetramethylsilane as internal

1
080
steroids 7 3 ( 2 0 0 8 ) 1077–1089
standard (TMS, ı 0.0 ppm for 1H NMR and 13C NMR). Carbon
identification was sometimes clarified by using DEPT (Distor-
tionless Enhancement by Polarization Transfert) technique.
Chemical shifts (ı) are expressed in parts per million (ppm),
the coupling constants (J) are expressed in hertz (Hz). Mul-
tiplicities are described by the following abbreviations: s for
singlet, d for doublet, dd for doublet of doublets, t for triplet, q
for quartet, m for multiplet, #m for several multiplets and br
s for broad singlet.
+
+
(M −C4H9O), 275 (M −C5H9O2). Exact mass: calculated for
C19H37BrO2 − H = 375.1898; found = 375.1901.
2.1.1.5. General procedure for the preparation of the iodide THP
chains 12a–d. Anhydrous acetone (70 mL) and sodium iodide
(6.41 g, 42.8 mmol) were added to the protected alkyl chain
(10 or 11). The resulting mixture was stirred at reflux, under
nitrogen atmosphere, sheltered from daylight, for 3 days. Ace-
tone was then evaporated. The mixture was diluted with
diethyl ether (30 mL) and washed first with a 5% sodium
thiosulfate solution (30 mL) and after with water (4 × 75 mL).
The organic phase was dried with magnesium sulfate, fil-
tered and evaporated to yield the desired compound with 98%
yield.
2
.1.1. General procedure for the preparation of the THP
chains 10a,b and 11a,b
To solution of an appropriate n-alkyl chain (8a, 8b,
a or 9b) (6.66 mmol) in dichloromethane, 3,4-dihydro-2H-
pyran (2.81 mL, 30.8 mmol) and pyridinium p-toluenesulfonate
a
9
(
100 mg, 0.40 mmol), were added. The resulting mixture was
2.1.1.6. Spectral
data
for
1-tetrahydropyrannyloxy-6-
◦
−1
stirred at room temperature (21 C) for 18 h. Afterwards, solu-
tion was neutralised with a small amount of solid sodium
bicarbonate. Magnesium sulfate was added for drying and the
mixture was vigorously stirred. The mixture was filtered on
a silica gel (3 cm) and celite (1 cm) pad using hexanes as the
eluant. The resulting filtrate was evaporated to an oil. The
THP-alkyl chains (10a, 10b, 11a or 11b) were obtained with
iodohexane (12a). IR (NaCl, ꢀmax, cm ): 1132 and 1035
1
(C–O–C). H NMR (Acetone-d6, ı ppm): 4.56 (1H, t, J = 2.7 Hz,
OCHO), 3.85–3.35 (4H, #m, 2× CH2O), 3.29 (2H, t, J = 6.8 Hz,
1
3
CH2I). C NMR (Acetone-d6, ı ppm): 98.4 (OCHO), 67.0 (CH2O,
THP), 61.5 (CH2O, chain), 7.3 (CH2I). MS (m/e), C11H21IO2:
+
+
311 (M−H) , 211 (M −C5H9O2). Exact mass: calculated for
C11H21IO2 − H = 311.0508; found = 311.0510.
1
00% yield.
2
.1.1.7. Spectral data for 1-tetrahydropyrannyloxy-8-iodooctane
−
1
1
2
.1.1.1. Spectral
chlorohexane (10a). IR (NaCl, ꢀmax, cm ): 1137 and 1020
C–O–C). 1H NMR (Acetone-d6, ı ppm): 4.54 (1H, t, J = 2.3 Hz,
OCHO), 3.78–3.27 (4H, 4m, 2× CH2O), 3.58 (2H, t, J = 3.3 Hz,
data
for
1-tetrahydropyrannyloxy-6-
(12b). IR (NaCl, ꢀmax, cm ): 1143 and 1025 (C–O–C).
NMR (CDCl3, ppm): 4.46 (1H, apparent, J = 3.9 Hz,
OCHO), 3.76–3.21 (4H, 4m, 2× CH2O), 3.08 (2H, t, J = 7.0 Hz,
H
−1
ı
t
(
1
3
CH2I).
C NMR (CDCl3, ı ppm): 98.9 (OCHO), 67.6 (CH2O,
1
3
CH2Cl), 1.78 (2H, m, CH2CH2Cl).
C NMR (Acetone-d6, ı
THP), 62.4 (CH2O, chain), 7.3 (CH2I). MS (m/e), C13H25IO2:
+
+
ppm): 98.8 (OCHO), 67.4 (CH2O, THP), 61.9 (CH2O, chain), 45.5
339 (M−H) , 239 (M −C5H9O2). Exact mass: calculated for
C13H25IO2 − H = 339.0821; found = 339.0816.
+
+
(
CH2Cl). MS (m/e), C11H21ClO2: 219 (M−H) , 119 (M −C5H9O2).
Exact mass: calculated for C11H21ClO2 − H = 219.1152;
found = 219.1148.
2.1.1.8. Spectral
iodoundecane (12c). IR (NaCl, ꢀmax, cm ): 1127 and 1040
(C–O–C). H NMR (CDCl3, ı ppm): 4.47 (1H, t, J = 3.3 Hz, OCHO),
data
for
1-tetrahydropyrannyloxy-11-
−
1
1
2
.1.1.2. Spectral
data
for
1-tetrahydropyrannyloxy-8-
−1
chlorooctane (10b). IR (NaCl, ꢀmax, cm ): 1143 and 1025
3.75–3.20 (4H, 4m, 2× CH2O), 3.07 (2H, t, J = 7.0 Hz, CH2I).
1
13
(
C–O–C). H NMR (CDCl3, ı ppm): 4.46 (1H, t, J = 3.5 Hz, OCHO),
C NMR (CDCl3, ı ppm): 98.8 (OCHO), 67.7 (CH2O, THP),
+
3
.79–3.21 (4H, 4m, 2× CH2O), 3.41 (2H, t, J = 6.6 Hz, CH2Cl).
C NMR (CDCl3, ı ppm): 98.9 (OCHO), 67.6 (CH2O, THP),
62.3 (CH2O, chain), 7.3 (CH2I). MS (m/e), C16H31IO2: 382 (M ),
1
3
+
+
381 (M−H) , 183 (M −C12H23O2). Exact mass: calculated for
C16H31IO2 − H = 381.1290; found = 381.1296.
6
2
2.3 (CH2O, chain), 45.1 (CH2Cl). MS (m/e), C13H25ClO2 :
47 (M−H) , 147 (M −C5H9O2). Exact mass: calculated for
+
+
C13H25ClO2 − H = 247.1465; found = 247.1470.
2.1.1.9. Spectral
iodotetradecane (12d). IR (NaCl, ꢀmax, cm ): 1137 and
1030 (C–O–C). H NMR (CDCl3, ı ppm): 4.52 (1H, t, J = 3.5 Hz,
data
for
1-tetrahydropyrannyloxy-14-
−
1
1
2
.1.1.3. Spectral data for 1-tetrahydropyrannyloxy-11-
−1
bromoundecane (11a). IR (NaCl, ꢀmax, cm ): 1143 and 1040
OCHO), 3.69–3.29 (4H, 4m, 2× CH2O), 3.12 (2H, t, J = 7.0 Hz,
1
13
(
C–O–C). H NMR (CDCl3, ı ppm): 4.52 (1H, t, J = 3.3 Hz, OCHO),
CH2I).
C NMR (CDCl3, ı ppm): 98.9 (OCHO), 67.8 (CH2O,
3
.81–3.26 (4H, 4m, 2× CH2O), 3.34 (2H, t, J = 6.8 Hz, CH2Br).
THP), 62.4 (CH2O, chain), 7.4 (CH2I). MS (m/e), C19H37IO2: 424
1
3
+
+
+
C NMR (CDCl3, ı ppm): 99.0 (OCHO), 67.8 (CH2O, THP), 62.4
(M ), 423 (M−H) , 296 (M−HI) . Exact mass: calculated for
C19H37IO2 − H = 423.1760; found = 423.1757.
+
(CH2O, chain), 35.7 (CH2Br). MS (m/e), C16H31BrO2: 335 (M+H) ,
+
+
3
33 (M−H) , 233 (M −C5H9O2). Exact mass: calculated for
C16H31BrO2 − H = 333.1429; found = 333.1432.
2.1.2. Synthesis of 17ˇ-estradiol-platinum(II) complexes
7
a–d
2.1.2.1. Synthesis
estratrien-17-one (14). To
2
.1.1.4. Spectral data for 1-tetrahydropyrannyloxy-14-
of
3-tetrahydropyrannyloxy-1,3,5(10)-
solution of estrone (13)
−
1
bromotetradecane (11b). IR (NaCl, ꢀmax, cm ): 1148
and 1040 (C–O–C). H NMR (CDCl3, ı ppm): 4.53 (1H, t,
a
1
(10.12 g, 37.43 mmol) in dichloromethane, pyridinium p-
toluenesulfonate (1.0 g, 4.0 mmol) and 3,4-dihydro-2H-pyran
(8.54 mL, 93.6 mmol), were added. The resulting mixture
J = 3.3 Hz, OCHO), 3.82–3.27 (4H, 4m, 2× CH2O), 3.35 (2H,
1
3
t, J = 6.8 Hz, CH2Br). C NMR (CDCl3, ı ppm): 99.0 (OCHO),
◦
6
7.8 (CH2O, THP), 62.4 (CH2O, chain), 34.1 (CH2Br). MS (m/e),
was stirred at room temperature (21 C) for 20 h. Afterwards,
+
+
+
C19H37BrO2: 377 (M+H) , 375 (M−H) , 305 (M −C4H8O) and 303
solution was neutralised with a small amount of solid

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1081
sodium bicarbonate. Magnesium sulfate was added for drying
and the mixture was stirred vigorously. The mixture was
filtered on a silica gel (3 cm) and celite (1 cm) pad using
hexanes as the eluant. The resulting filtrate was evaporated
and set to vacuum for an hour to give a beige solid, 100%
yield.
2.1.2.5. Synthesis
of
3-THP-16ˇ-(methoxycarbonyl)-16˛-
(THP-alkylchain)-estrone (16a–d). Activated estrone (15)
(0.500 g, 1.22 mmol), dichloromethane (8 mL), triethylben-
zylammonium chloride (100 mg), 1-tetrahydropyrannyloxy-
n-iodohexane (4.89 mmol) and a 10% (w/v) NaOH solution
(5 mL) were put together in a flask. The mixture was heated
to reflux and strongly stirred for 24 h. The resulting solution
was diluted with diethyl ether (60 mL). The organic phase
was washed with saturated NH4Cl solution (2× 70 mL) as well
as with water (4× 70 mL). The organic phase was dried with
anhydrous magnesium sulfate, filtered and evaporated. Flash
chromatography with a mixture of hexanes and acetone (92:8)
gave yellow oil with 92% optimized yield.
2
.1.2.2. Spectral data for 3-tetrahydropyrannyloxy-1,3,5(10)-
−
1
estratrien-17-one (14). IR (NaCl, ꢀmax, cm ): 1744 (C O), 1613
1
(
(
C
C), 1247 and 1043 (C–O). H NMR (CDCl3, ı ppm): 7.19
1H, d, J = 8.6 Hz, 1-CH), 6.85 (1H, dd, J = 2.3 Hz and J = 8.6 Hz, 2-
CH), 6.81 (1H, d, J = 2.7 Hz, 4-CH), 5.39 (1H, t, J = 2.9 Hz, OCHO),
3
#
.98–3.55 (2H, 2m, CH2O), 2.90 (2H, m, 6-CH2), 2.57–1.37 (19H,
m, 3× CH and 8× CH2), 0.90 (3H, s, 18-CH3). 13C NMR
2.1.2.6. Spectral data for 3-tetrahydropyrannyloxy-16ˇ-
(
(
(
CDCl3, ı ppm): 220.9 (17-C), 155.3 (3-C), 137.8 (5-C), 133.2
10-C), 126.4 (1-C), 116.8 (4-C), 114.4 (2-C), 96.6 (OCHO), 62.2
CH2O), 50.7, 48.2, 44.3, 38.6, 36.1, 31.8, 30.7, 29.8, 26.8, 26.1,
ꢀ
(
1
methoxycarbonyl)-16˛-(6 -tetrahydropyrannyloxyhexyl)-
,3,5(10)-estratrien-17-one (16a). IR (NaCl, ꢀmax, cm 1): 1757
−
+
(C O, ester), 1731 (C O, ketone), 1609 and 1573 (C C), 1050
2
5.5, 21.8, 19.0, 14.1 (C-18). MS (m/e), C23H30O3: 354 (M ), 271
1
+
(C–O). H NMR (Acetone-d6, ı ppm): 7.16 (1H, d, J = 8.6 Hz,
(
M −C5H7O). Exact mass: calculated for C23H30O3 = 354.2195;
1-CH), 6.80 (1H, d, J = 8.6 Hz, 2-CH), 6.75 (1H, m, 4-CH), 5.37
found = 354.2200.
(
(
2
1
1H, m, OCHO, steroid), 4.55 (1H, m, OCHO, chain), 3.84–3.27
6H, 6m, 3× CH2O), 3.68 (3H, s, OCH3), 2.82 (2H, m, 6-CH2),
2
.1.2.3. Synthesis
of
3-tetrahydropyrannyloxy-16˛,ˇ-
(15). In
.38–1.12 (33H, #m, 3× CH, 15× CH2), 0.98 and 0.93 (3H, 2s,
(
methoxycarbonyl)-1,3,5(10)-estratrien-17-one
a
13
8-CH3, 16␣,␤ (12: 1)). C NMR (Acetone-d6, ı ppm): 213.0
double necked flask, tetrahydrofuran (30 mL) was added to
potassium hydride (30% in oil, 15.06 g, 112.7 mmol) under
nitrogen conditions. The solution was stirred at room tem-
perature. Dimethylcarbonate (7.91 mL, 93.9 mmol) was then
added to the potassium hydride suspension. Separately,
protected estrone (14) (13.3 g, 37.6 mmol) was dissolved in
tetrahydrofuran (70 mL) under nitrogen atmosphere. The
estrone solution was then added to reaction mixture and the
overall was heated to reflux for 2.5 h. Afterwards, the mix-
ture was cooled down. Tert-butanol (10 mL), methyl alcohol
(
1
(
17-C), 171.7 (COOCH3), 155.4 (3-C), 137.6 (5-C), 133.0 (10-C),
26.3 (1-C), 116.8 (4-C), 114.2 (2-C), 98.4 (OCHO, chain), 96.2
OCHO, steroid), 67.1 (CH2O, THP, side chain), 61.6 (CH2O, hexyl
chain), 61.5 (CH2O, THP, steroid), 60.2, 52.0 (OCH3), 49.4, 46.1,
4
2
5
4.2, 38.1, 35.3, 32.4, 30.88, 30.73, 30.56, 29.83, 26.6, 26.2, 25.9,
+
5.8, 25.4 19.6, 15.6, 13.8 (18-C). MS (m/e), C36H52O7: 596 (M ),
+
+
29 (M −C4H3O), 513 (M −C5H7O). Exact mass: calculated for
C36H52O7 = 596.3713; found = 596.3704.
2
(
1
1
.1.2.7. Spectral data for 3-tetrahydropyrannyloxy-16ˇ-
(
20 mL) and water were added and the resulting solution was
ꢀ
methoxycarbonyl)-16˛-(8 -tetrahydropyrannyloxyoctyl)-
stirred for 15 min between each adding. The mixture was
then diluted with ethyl acetate (80 mL) and the organic phase
was washed with saturated NH4Cl solution (2× 90 mL) as well
as with water (4× 90 mL). The organic phase was dried with
anhydrous magnesium sulfate, filtered and evaporated to
give the crude product. The residue was first triturated with
hexanes and purified by flash chromatography with a mixture
of hexanes and acetone (92:8) to give the activated estrone in
,3,5(10)-estratrien-17-one (16b). IR (NaCl, ꢀmax, cm 1):
−
762 (C O, ester), 1726 (C O, ketone), 1614 and 1573 (C C),
1
1
250 and 1040 (C–O). H NMR (CDCl3, ı ppm): 7.18 (1H, d,
J = 8.6 Hz, 1-CH), 6.85 (1H, dd, J = 2.3 Hz and J = 8.6 Hz, 2-CH),
.81 (1H, d, J = 2.3 Hz, 4-CH), 5.39 (1H, t, J = 3.1 Hz, OCHO,
steroid), 4.57 (1H, t, J = 3.1 Hz, OCHO, chain), 3.92–3.35 (6H, 6m,
6
3
× CH2O), 3.72 (3H, s, OCH3), 2.89 (2H, m, 6-CH2), 2.38–1.13
(37H, #m, 3× CH, 17× CH2), 0.92 and 0.88 (3H, 2s, 18-CH3, 16␣,␤
(
9
0% yield.
1
3
7: 1)). C NMR (CDCl3, ı ppm): 214.3 (17-C), 172.1 (COOCH3),
55.3 (3-C), 137.8 (5-C), 133.0 (10-C), 126.4 (1-C), 116.8 (4-C),
1
2
.1.2.4. Spectral data for 3-tetrahydropyrannyloxy-16˛,ˇ-
114.3 (2-C), 99.1 (OCHO, chain), 96.5 (OCHO, steroid), 67.9
(CH2O, THP, side chain), 62.5 (CH2O, octyl chain), 62.2 (CH2O,
THP, steroid), 60.4, 52.8 (OCH3), 49.7, 46.2, 44.3, 38.1, 35.8,
32.3, 30.99, 30.72, 30.62, 29.93, 29.77, 29.61, 29.51, 26.8, 26.4,
25.9, 25.7, 25.6, 25.5, 19.6, 18.9, 15.5, 14.3 (18-C). MS (m/e),
(
methoxycarbonyl)-1,3,5(10)-estratrien-17-one (15). IR (NaCl,
−1
ꢀ
max, cm ): 1763 (C O, ester), 1740 (C O, ketone), 1605 (C C),
1
1232 (C–O), 1155 and 1035 (C–O–C). H NMR (CDCl3, ı ppm):
7.18 (1H, d, J = 8.6 Hz, 1-CH), 6.85 (1H, dd, J = 2.7 Hz and J = 8.6 Hz,
2-CH), 6.81 (1H, d, J = 2.3 Hz, 4-CH), 5.39 (1H, t, J = 2.9 Hz, OCHO),
3.96–3.54 (2H, 2m, CH2O), 3.76 (3H, s, OCH3), 3.20 (1H, t,
+
+
+
C38H56O7: 624 (M ), 594 (M −CH2O), 542 (M −C5H6O). Exact
mass: calculated for C38H56O7 = 624.4026; found = 624.4014.
J = 9.2 Hz, 16-CH), 2.88 (2H, m, 6-CH2), 2.42–1.39 (17H, #m, 3×
CH and 7× CH2), 0.98 and 0.95 (3H, 2s, 18-CH, 16␣,␤ (2.5: 1)).
2.1.2.8. Spectral data for 3-tetrahydropyrannyloxy-16ˇ-
1
3
ꢀ
C NMR (CDCl3, ı ppm): 212.4 (17-C), 170.1 (COOCH3), 155.3
(methoxycarbonyl)-16˛-(11 -tetrahydropyrannyloxyundecyl)-
(
(
3-C), 137.8 (5-C), 132.9 (10-C), 126.5 (1-C), 116.7 (4-C), 114.3
2-C), 96.5 (OCHO), 62.2 (CH2O), 54.3, 52.8 (OCH3), 49.2, 48.1,
1,3,5(10)-estratrien-17-one (16c). IR (NaCl, ꢀmax, cm 1): 1759
−
(C O, ester), 1721 (C O, ketone), 1617 (C C), 1231 (C–O). 1
H
4
4.3, 38.1, 32.2, 30.6, 29.8, 26.6, 25.5, 19.0, 14.6 (18-C). MS (m/e),
NMR (CDCl3, ı ppm): 7.16 (1H, d, J = 8.6 Hz, 1-CH), 6.84 (1H,
dd, J = 2.3 Hz and J = 10.9 Hz, 2-CH), 6.79 (1H, s, 4-CH), 5.37
(1H, t, J = 3.1 Hz, OCHO, steroid), 4.56 (1H, t, J = 3.1 Hz, OCHO,
+
C23H32O5: 413 (M+H) , 381 (M−CH3O), 328 (M−C5H9O). Exact
mass: calculated for C25H32O5 + H = 413.2328; found = 413.2319.

1
082
steroids 7 3 ( 2 0 0 8 ) 1077–1089
+
+
chain), 3.90–3.34 (6H, 6m, 3× CH2O) 3.71 (3H, s, OCH3), 2.87
353 (M −OH), 270 (M −H2O and C6H10). Exact mass: calculated
(
2H, m, 6-CH2), 2.37–1.25 (43H, #m, 3× CH, 20× CH2), 0.90
for C24H34O3 = 370.2508; found = 370.2512.
1
3
and 0.87 (3H, 2s, 18-CH3, 16␣,␤ (3.5: 1)). C NMR (CDCl3, ı
ppm): 214.3 (17-C), 172.1 (COOCH3), 155.3 (3-C), 137.8 (5-C),
2.1.2.12. Spectral data for compound 17b. IR (NaCl, ꢀmax,
cm ): 3200–3600 (O–H), 1711 (C O), 1629 and 1583 (C C),
1255 and 1071 (C–O). H NMR (Acetone-d6, ı ppm): 7.10 (1H,
−
1
1
33.0 (10-C), 126.4 (1-C), 116.8 (4-C), 114.3 (2-C), 99.0 (OCHO,
1
chain), 96.5 (OCHO, steroid), 67.9 (CH2O, THP, side chain),
6
5
3
2
2.5 (CH2O, undecyl chain), 62.1 (CH2O, THP, steroid), 60.4,
2.8 (OCH3), 49.7, 46.2, 44.3, 38.1, 35.8, 32.3, 31.8, 31.0, 30.72,
0.62, 29.97(2× C), 29.75 (3× C), 29.57, 26.75, 26.46, 25.91,
5.72, 25.65, 25.49, 22.9, 19.9, 19.0, 14.34 (18-C). MS (m/e),
d, J = 8.2 Hz, 1-CH), 6.60 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH),
6.55 (1H, s, 4-CH), 3.51 (2H, t, J = 5.9 Hz, CH2OH, 16␤), 2.82 (2H,
m, 6-CH2), 2.40-1.33 (28H, #m, 4× CH, 11× CH2, 2× OH), 0.94
1
3
and 0.85 (3H, 2s, 18-CH3, 16␣,␤ (1: 4)). C NMR (Acetone-d6, ı
ppm): 220.9 (17-C), 155.4 (3-C), 137.7 (5-C), 131.0 (10-C), 126.4
(1-C), 115.3 (4-C), 113.0 (2-C), 61.8 (CH2OH), 49.17, 49.01, 48.24,
44.38, 38.33, 33.13, 32.49, 32.28, 29.5, 26.9, 26.0, 13.7 (18-C).
+
+
+
C41H62O7: 666 (M ), 635 (M −CH3O), 582 (M −C5H8O), 498
+
(
M −2C5H8O). Exact mass: calculated for C41H62O7 = 666.4496;
found = 666.4488.
+
+
+
MS (m/e), C26H38O3: 398 (M ), 383 (M −CH3), 270 (M −H2O
and C8H14). Exact mass: calculated for C26H38O3 = 398.2821;
found = 398.2824.
2
.1.2.9. Spectral data for 3-tetrahydropyrannyloxy-16ˇ-
ꢀ
(
methoxycarbonyl)-16˛-(14 -tetrahydropyrannyloxytetradecyl)-
−
1
1
,3,5(10)-estratrien-17-one (16d). IR (NaCl, ꢀmax, cm ): 1757
2.1.2.13. Spectral data for compound 17c. IR (NaCl, ꢀmax, cm 1):
−
(
(
C
O, ester), 1726 (C O, ketone), 1619 (C C), 1224 and 1030
1
C–O). H NMR (CDCl3, ı ppm): 7.18 (1H, d, J = 8.6 Hz, 1-CH),
3200–3600 (O–H), 1732 (C O), 1670 (C C), 1255 and 1093 (C–O).
1
6
5
.84 (1H, dd, J = 2.7 Hz and J = 8.6 Hz, 2-CH), 6.81 (1H, s, 4-CH),
.39 (1H, t, J = 3.1 Hz, OCHO, steroid), 4.57 (1H, t, J = 3.1 Hz,
H NMR (Acetone-d6, ı ppm): 7.10 (1H, d, J = 8.2 Hz, 1-CH), 6.60
(1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.55 (1H, s, 4-CH), 3.51
(2H, t, J = 5.9 Hz, CH2OH, 16␤), 2.82 (2H, m, 6-CH2), 2.40–1.33
(28H, #m, 4× CH, 11× CH2, 2× OH), 0.94 and 0.85 (3H, 2s, 18-
OCHO, chain), 3.91–3.31 (6H, 6m, 3× CH2O), 3.72 (3H, s,
OCH3), 2.88 (2H, m, 6-CH2), 2.38–1.15 (49H, #m, 3× CH, 23×
1
3
13
CH2), 0.91 and 0.88 (3H, 2s, 18-CH3, 16␣,␤ (4: 1)). C NMR
CH3, 16␣,␤ (1: 4)). C NMR (Acetone-d6, ı ppm): 220.9 (17-C),
(
1
9
CDCl3, ı ppm): 214.7 (17-C), 172.2 (COOCH3), 155.3 (3-C),
37.8 (5-C), 133.0 (10-C), 126.4 (1-C), 116.7 (4-C), 114.3 (2-C),
9.1 (OCHO, chain), 96.5 (OCHO, steroid), 67.9 (CH2O, THP,
155.4 (3-C), 137.7 (5-C), 131.0 (10-C), 126.4 (1-C), 115.3 (4-C),
113.0 (2-C), 61.8 (CH2OH), 49.17, 49.01, 48.24, 44.38, 38.33, 33.13,
32.49, 32.28, 29.5, 26.9, 26.0, 13.7 (18-C). MS (m/e), C29H44O3: 440
+
+
+
side chain), 62.6 (CH2O, tetradecyl chain), 62.2 (CH2O, THP,
steroid), 60.4, 52.8 (OCH3), 49.7, 46.2, 44.3, 38.1, 35.8, 34.9,
(M ), 422 (M −H2O), 270 (M −H2O and C11H20). Exact mass:
calculated for C29H44O3 = 440.3290; found = 440.3297.
3
2
2.3, 31.8, 31.0, 30.6, 29.98, 29.84, 29.73, 29.61, 26.8, 26.5,
5.92, 25.73, 25.67, 25.49, 22.9, 19.9, 19.0, 14.35, 14.28 (18-
2.1.2.14. Spectral data for compound 17d. IR (NaCl, ꢀmax,
C). MS (m/e), C44H68O7: 726 (MNH4)+, 624 (M−C5H8O) .
Exact mass: calculated for C44H68O7 + NH4 = 726.5308;
found = 726.5326.
+
−1
cm ): 3200–3600 (O–H), 1737 (C O), 1511 (C C), 1214 (C–O).
1
H NMR (Acetone-d6, ı ppm): 7.12 (1H, d, J = 8.6 Hz, 1-CH), 6.60
(1H, dd, J = 2.7 Hz and J = 8.6 Hz, 2-CH), 6.56 (1H, s, 4-CH), 3.51
(
2H, t, J = 5.9 Hz, CH2OH, 16␤), 2.81 (2H, m, 6-CH2), 2.38–1.13
2
1
.1.2.10. General procedure for the synthesis of 3-hydroxy-
6˛,ˇ-(hydroxyalkyl)-1,3,5(10)-estrone (17a–d). Alkylated
(40H, #m, 4× CH, 17× CH2, 2× OH), 0.95 and 0.86 (3H, 2s, 18-
13
CH3, 16␣,␤ (1: 1.2)). C NMR (Acetone-d6, ı ppm): 220.4 (17-C),
155.4 (3-C), 137.8 (5-C), 131.3 (10-C), 126.4 (1-C), 115.3 (4-C),
112.9 (2-C), 62.0 (CH2OH), 49.20, 49.01, 48.24, 44.38, 38.33, 33.13,
33.00, 32.49, 32.28, 31.45, 31.14, 29.5, 26.9, 26.0, 13.7 (18-C). MS
estrone 16a–d (2.62 mmol), lithium chloride (2.43 g, 57.6 mmol)
and water (1.04 mL, 57.6 mmol) were made soluble in DMF
(
15 mL). The solution was stirred to reflux for 20 h. After-
+
+
+
wards, ethyl acetate was added (90 mL) and the organic
phase was washed with 10% (v/v) HCl solution (2× 70 mL)
as well as with water (4× 70 mL). The organic phase was
then dried with anhydrous magnesium sulfate, filtered and
evaporated. Purification was done by flash chromatography
with a mixture of hexanes and acetone (70:30). We obtained a
decarboalkoxylated and bis-deprotected compound with 71%
optimized yield.
(m/e), C32H50O3: 482 (M ), 464 (M −H2O) and 270 (M −H2O
and C14H26). Exact mass: calculated for C32H50O3 = 482.3760;
found = 482.3755.
2.1.2.15. General procedure for the synthesis of 16˛,ˇ-
(hydroxyalkyl)-17ˇ-estradiol
(18a–d). Compound
17a–d
(1.3 mmol) was dissolved in diethyl ether (10 mL),
dichloromethane (3 mL) and anhydrous THF (5 mL) under
nitrogen atmosphere. The resulting solution was cooling
down with an ice and water bath, afterwards lithium boro-
hydride (0.17 g, 7.9 mmol) was added. The mixture was kept
2
.1.2.11. Spectral data for compound 17a. IR (NaCl, ꢀmax,
−
1
cm ): 3200–3600 (O–H), 1711 (C O), 1624 and 1578 (C C),
255 and 1076 (C–O). 1H NMR (Acetone-d6, ı ppm): 7.10 (1H,
d, J = 8.2 Hz, 1-CH), 6.60 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH),
.55 (1H, s, 4-CH), 3.52 (2H, t, J = 5.9 Hz, CH2OH 16␤), 2.83 (2H,
◦
1
at 0 C for 3 h and then, at room temperature for 3 days.
When reaction completed, sodium sulfate decahydrate (0.5 g)
was added. Work-up was done by diluting with diethyl
ether (40 mL) and washing the organic phase with saturated
NH4Cl solution (2× 20 mL) and with water (4× 50 mL). The
organic phase was dried with anhydrous magnesium sulfate,
filtered and evaporated. A white solid was obtained quanti-
tatively and was used without further purification at the next
step.
6
m, 6-CH2), 2.26–1.32 (24H, #m, 4× CH, 9× CH2, 2× OH), 0.94
1
3
and 0.85 (3H, 2s, 18-CH3, 16␣,␤ (1: 1.9)). C NMR (Acetone-
d6, ı ppm): 221.0 (17-C), 155.4 (3-C), 137.7 (5-C), 131.0 (10-C),
1
4
2
26.4 (1-C), 115.3 (4-C), 113.0 (2-C), 61.8 (CH2OH), 49.18, 49.01,
8.25, 44.37, 38.69, 38.33, 33.07, 32.46, 32.28, 29.5, 28.6, 28.2,
6.9, 26.04, 25.92, 13.7 (18-CH3). MS (m/e), C24H34O3: 370 (M ),
+

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1083
2
.1.2.16. Spectral data for compound 18a. IR (NaCl, ꢀmax,
complete dissolution, carbon tetrabromide (1.81 g, 5.44 mmol)
and triphenylphosphine (1.46 g, 5.44 mmol) were added.
The mixture was stirred at room temperature (21 C), under
nitrogen atmosphere, for 5 h. The organic phase was then
extracted with diethyl ether (50 mL) and washed with a
saturated NH4Cl solution (2× 60 mL) as well as with water (2×
60 mL). The ethereal phase was dried, filtered and evaporated.
Flash chromatography with a mixture of hexanes and acetone
(80:20) gave the desired product in 62% optimised yield.
−
1
cm ): 3200–3600 (O–H), 1621 (C C), 1255 and 1072 (C–O).
1H NMR (Acetone-d6, ı ppm): 7.90 (1H, s, 3-OH), 7.09 (1H, d,
J = 8.2 Hz, 1-CH), 6.60 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH),
◦
6
.53 (1H, s, 4-CH), 3.62 (1H, m, 17-OH), 3.52 (2H, t, J = 6.1 Hz,
CH2OH), 3.40 (1H, m, 17-CH), 2.86 (1H, s, CH2OH), 2.75 (2H, m,
6
2
-CH2), 2.30–1.29 (22H, #m, 4× CH, 9× CH2), 0.81 and 0.78 (3H,
s, 18-CH3, 16␣,␤ (1: 2)). 13C NMR (Acetone-d6, ı ppm): 155.3 (3-
C), 137.8 (5-C), 131.5 (10-C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C),
8
3
1.7 (17-C), 61.9 (CH2OH), 49.0, 44.34, 44.30, 40.7, 38.9, 38.1, 33.2,
2.7, 32.0, 30.2, 29.7, 29.0, 27.7, 26.6, 26.1, 12.5 (18-C). MS (m/e),
2.1.2.21. Spectral data for compound 19a. IR (NaCl, ꢀmax,
+
+
+
−1
C24H36O3: 372 (M ), 354 (M −H2O), 285 (M −C5H11O). Exact
cm ): 3650–3150 (O–H), 1624 and 1501 (C C), 1230 and 1071
1
mass: calculated for C24H36O3 = 372.2664; found = 372.2662.
(C–O). H NMR (Acetone-d6, ı ppm): 7.90 (1H, s, 3-OH), 7.09 (1H,
d, J = 8.6 Hz, 1-CH), 6.58 (1H, dd, J = 2.3 Hz and J = 8.2 Hz, 2-CH),
6.53 (1H, s, 4-CH), 3.74 (1H, m, 17-CH), 3.49 (2H, t, J = 6.6 Hz,
CH2Br), 2.90 (1H, s, 17-OH), 2.76 (2H, m, 6-CH2), 2.25–1.03 (22H,
#m, 4× CH, 9× CH2), 0.81 and 0.78 (3H, 2s, 18-CH3, 16␣,␤ (1: 23)).
2
.1.2.17. Spectral data for compound 18b. IR (NaCl, ꢀmax,
−
1
cm ): 3200–3600 (O–H), 1619 and 1506 (C C), 1245 and 1066
1
(
C–O). H NMR (Acetone-d6, ı ppm): 7.95 (1H, s, 3-OH), 7.09 (1H,
1
3
d, J = 8.6 Hz, 1-CH), 6.56 (1H, dd, J = 2.7 Hz and J = 8.6 Hz, 2-CH),
.53 (1H, s, 4-CH), 3.68-3.47 (2H, 2m, 17-OH and 17-CH), 3.52 (2H,
t, J = 5.5 Hz, CH2OH), 2.96 (1H, s, CH2OH), 2.75 (2H, m, 6-CH2),
.23–1.07 (26H, #m, 4× CH, 11× CH2), 0.81 and 0.78 (3H, 2s, 18-
C NMR (Acetone-d6, ı ppm): 155.3 (3-C), 137.8 (5-C), 131.5 (10-
6
C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C), 81.7 (17-C, 16␤), 49.0,
44.33, 44.31, 40.7, 38.9, 38.1, 34.2, 33.0, 32.7, 32.0, 29.73, 29.45,
29.06, 28.26, 27.73, 26.6, 12.5 (18-C). MS (m/e), C24H35BrO2:
2
1
3
+
CH3, 16␣,␤ (1: 2.7)). C NMR (Acetone-d6, ı ppm): 155.3 (3-C),
37.8 (5-C), 131.5 (10-C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C), 81.7
17-C), 61.9 (CH2OH), 48.9, 44.34, 44.30, 40.7, 38.9, 33.1, 32.75,
434 (M ). Exact mass: calculated for C24H35BrO2 = 434.1820;
1
(
found = 434.1815.
3
1
2.09, 30.4, 30.12, 30.07, 29.90, 29.85, 29.74, 29.69, 29.03, 26.07,
2.1.2.22. Spectral data for compound 19b. IR (NaCl, ꢀmax,
cm ): 3650–3150 (O–H), 1614 and 1501 (C C), 1255 and 1066
(C–O). 1H NMR (Acetone-d6, ı ppm): 7.94 (1H, s, 3-OH), 7.09
(1H, d, J = 8.6 Hz, 1-CH), 6.58 (1H, dd, J = 2.7 Hz and J = 8.2 Hz,
+
+
−1
2.5 (18-C). MS (m/e), C26H40O3: 400 (M ), 382 (M −H2O), 354
+
(
M −C2H6O). Exact mass: calculated for C26H40O3 = 400.2977;
found = 400.2964.
2
-CH), 6.53 (1H, s, 4-CH), 3.69 (1H, m, 17-CH), 3.49 (2H, t,
−
1
2
3
.1.2.18. Spectral data for compound 18c. IR (NaCl, ꢀmax, cm 1):
J = 6.6 Hz, CH2Br), 2.88 (1H, s, 17-OH), 2.75 (2H, m, 6-CH2),
2.23–1.02 (26H, #m, 4× CH, 11× CH2), 0.81 and 0.78 (3H, 2s,
200–3600 (O–H), 1600 and 1506 (C C), 1245 and 1066 (C–O). H
NMR (CDCl3, ı ppm): 7.27 (1H, s, 3-OH), 7.14 (1H, d, J = 7.8 Hz,
-CH), 6.61 (1H, dd, J = 2.7 Hz and J = 8.6 Hz, 2-CH), 6.56 (1H,
s, 4-CH), 3.85–3.45 (1H, m, 17-CH), 3.64 (2H, m, CH2OH), 2.80
2H, m, 6-CH2), 2.70–0.88 (34H, #m, 4× CH, 14× CH2, 2× OH),
.79 and 0.76 (3H, 2s, 18-CH3, 16␣,␤ (1: 1.2)). 13C NMR (CDCl3,
ı ppm): 153.6 (3-C), 138.5 (5-C), 132.9 (10-C), 126.7 (1-C), 115.5
4-C), 112.9 (2-C), 82.8 (17-C), 63.4 (CH2OH), 44.35, 44.20, 40.2,
1
3
18-CH3, 16␣,␤ (1: 16)). C NMR (Acetone-d6, ı ppm): 155.3 (3-
C), 137.8 (5-C), 131.5 (10-C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C),
81.7 (17-C, 16␤), 48.9, 44.34, 44.30, 40.7, 38.9, 38.1, 34.2, 33.0,
32.7, 32.1, 29.82, 29.72, 29.43, 28.94, 28.16, 27.72, 26.6, 12.5 (18-
1
(
0
+
C). MS (m/e), C26H39BrO2: 462 (M ). Exact mass: calculated for
C26H39BrO2 = 462.2133; found = 462.2129.
(
3
1
2.1.2.23. Spectral data for compound 19c. IR (NaCl, ꢀmax, cm 1):
3650–3150 (O–H), 1614 and 1516 (C C), 1255 and 1071 (C–O). H
NMR (CDCl3, ı ppm): 7.26 (1H, s, 3-OH), 7.15 (1H, d, J = 8.2 Hz,
−
1
8.6, 33.0, 32.6, 30.54, 30.12, 29.90, 29.85, 29.74, 29.63, 28.9, 25.9,
+
+
2.6 (18-C). MS (m/e), C29H46O3: 442 (M ), 424 (M −H2O). Exact
mass: calculated for C29H46O3 = 442.3447; found = 442.3449.
1-CH), 6.62 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.56 (1H,
2
.1.2.19. Spectral data for compound 18d. IR (NaCl, ꢀmax,
s, 4-CH), 3.74 (1H, d, J = 9.8 Hz, 17-CH), 3.41 (2H, t, J = 7.0 Hz,
CH2Br), 2.80 (2H, m, 6-CH2), 2.60–1.02 (33H, #m, 4× CH, 14×
CH2, 1× OH), 0.80 and 0.76 (3H, 2s, 18-CH3, 16␣,␤ (1: 3.5)).
−
1
cm ): 3650–3100 (O–H), 1609 (C C), 1230 and 1061 (C–O).
1H NMR (Acetone-d6, ı ppm): 7.94 (1H, s, 3-OH), 7.09 (1H, d,
J = 8.2 Hz, 1-CH), 6.58 (1H, dd, J = 2.7 Hz and J = 8.6 Hz, 2-CH), 6.53
1
3
C NMR (CDCl3, ı ppm): 153.6 (3-C), 138.5 (5-C), 133.0 (10-C),
(
1H, s, 4-CH), 3.69–3.42 (2H, 2m, 17-OH and 17-CH), 3.52 (2H,
126.7 (1-C), 115.5 (4-C), 112.9 (2-C), 82.8 (17-C, 16␤), 48.8, 44.35,
44.21, 40.2, 38.6, 37.9, 34.3 (2× C), 33.1 (2× C), 32.6, 31.7, 30.1,
28.99 (2× C), 28.87, 28.41 (2× C), 27.63, 27.42, 26.52, 12.6 (18-
m, CH2OH), 2.94 (1H, s, CH2OH), 2.76 (2H, m, 6-CH2), 2.23–1.03
(
(
40H, #m, 4× CH, 18× CH2), 0.81 and 0.79 (3H, 2s, 18-CH3, 16␣,␤
13
+
1: 4.5)). C NMR (Acetone-d6, ı ppm): 155.2 (3-C), 137.8 (5-
C). MS (m/e), C29H45BrO2: 504 (M ). Exact mass: calculated for
C), 131.5 (10-C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C), 81.7 (17-C),
C29H45BrO2 = 504.2603; found = 504.2592.
6
1.9 (CH2OH), 49.0, 44.34, 44.31, 40.7, 38.9, 38.1, 33.1, 32.8, 32.1,
3
0.15, 29.86, 29.79, 29.66, 29.45, 29.03, 27.7, 26.6, 26.1, 25.5, 12.5
2.1.2.24. Spectral data for compound 19d. IR (NaCl, ꢀmax,
+
+
−1
(
(
18-C). MS (m/e), C32H52O3: 484 (M ), 466 (M −H2O) and 397
cm ): 3650–3150 (O–H), 1619 and 1506 (C C), 1260 and 1081
+
1
M −C5H11O). Exact mass: calculated for C32H52O3 = 484.3916;
(C–O). H NMR (Acetone-d6, ı ppm): 7.92 (1H, s, 3-OH), 7.09 (1H,
found = 484.3922.
d, J = 8.5 Hz, 1-CH), 6.58 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH),
6.53 (1H, s, 4-CH), 3.71 (1H, m, 17-CH), 3.48 (2H, t, J = 6.6 Hz,
2
(
(
.1.2.20. General procedure for the synthesis of 16˛,ˇ-
bromoalkyl)-17ˇ-estradiol (19a–d). Compound 18a–d
1.4 mmol) was dissolved in dichloromethane (15 mL). After
CH2Br), 2.98, (1H, s, 17-OH), 2.76 (2H, m, 6-CH2), 2.23–1.03 (38H,
#m, 4× CH, 17× CH2), 0.81 and 0.79 (3H, 2s, 18-CH3, 16␣,␤ (1:
1
3
11.5)). C NMR (Acetone-d6, ı ppm): 155.3 (3-C), 137.7 (5-C),

1
084
steroids 7 3 ( 2 0 0 8 ) 1077–1089
1
1
3
31.5 (10-C), 126.4 (1-C), 115.3 (4-C), 112.9 (2-C), 81.7 (17-C,
6␤), 49.0, 44.35, 44.31, 40.7, 38.9, 38.1, 34.2, 33.0, 32.8, 32.1,
2.1.2.28. Spectral data for compound 20c. The spectral data for
this particular compound were reported elsewhere and are in
agreement with the literature [15]. The proportion of the 16␣,␤
epimers for derivative 20c is 1:4.
0.19, 29.91, 29.83, 29.77, 29.70, 29.07, 28.2, 27.8, 26.6, 12.5 (18-
+
C). MS (m/e), C32H51BrO2: 546 (M ). Exact mass: calculated for
C32H51BrO2 = 546.3072; found = 546.3079.
2
.1.2.29. Spectral data for compound 20d. IR (NaCl, ꢀmax,
ꢀ
2
.1.2.25. General procedure for the synthesis of 16˛,ˇ-[n -
−1
1
cm ): 3550–3050 (O–H and N–H), 1598 (C C), 1224 (C–O).
H
ꢀ
ꢀ
(
2 pyridylethylamino)alkyl]-1,3,5(10)-estratrien-3,17ˇ-diol
NMR (Acetone-d6, ı ppm): 8.48 (1H, dd, J = 2.0 Hz and J = 4.7 Hz,
compound (20a–d). The bromide 19a–d (0.46 mmol)
ꢀ
ꢀ
a -CH), 7.65 (1H, tt, J = 2.0 Hz and J = 7.4 Hz, c -CH), 7.25 (1H,
ꢀ
was dissolved in anhydrous methanol (7 mL). 2-(2 -
Aminoethyl)pyridine (0.56 mL, 4.6 mmol) was added to
the bromide solution. The mixture was stirred and heated
to reflux under nitrogen atmosphere for 4 days. The solvent
was then evaporated and the residue was diluted in diethyl
ether (30 mL). The organic phase was washed with water (6×
ꢀ
dd, J = 1.2 Hz and J = 7.8 Hz, d -CH), 7.16 (1H, tt, J = 1.6 Hz and
J = 4.7 Hz, b -CH), 7.09 (1H, d, J = 8.2 Hz, 1-CH), 6.96 (1H, s, 3-OH),
ꢀ
6
4
.58 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.52 (1H, d, J = 2.7 Hz,
-CH), 3.72 (1H, d, J = 9.4 Hz, 17-CH, 16␤), 3.61–2.82 (6H, #m,
NHCH2CH2pyridyl, 16-CH, 17-CH 16␣), 2.76 (2H, m, 6-CH2), 2.59
(
2H, t, J = 6.6 Hz, (CH2)13CH2NH), 2.24–1.06 (39H, #m, 3× CH, 17×
5
0 mL). Resulting aqueous layers were washed again with
13
CH2, 17-OH, NH), 0.81 and 0.79 (3H, 2s, 18-CH3, 16␣,␤ (1:9)).
C
diethyl ether. Organic phases were combined, dried, filtered
and evaporated to a yellow viscous oil. The crude amino
intermediates were obtained quantitatively with excellent
purity (>95%). No further purification was needed.
NMR (Acetone-d6, ı ppm): 161.1 (pyridyl-C), 155.3 (1-C), 149.3
ꢀ
ꢀ
(
(
a -C), 137.7 (5-C), 136.3 (c -C), 131.4 (10-C), 126.4 (1-C), 123.3
d -C), 121.3 (b -C), 115.3 (4-C), 112.9 (2-C), 81.5 (17-C, 16␤), 65.5,
ꢀ
ꢀ
5
3
2
1.2, 49.7, 49.6, 49.0, 44.35, 44.28, 40.7, 39.6, 38.9, 38.4, 38.1, 34.3,
2.7, 32.1, 30.18, 30.06, 29.72, 29.44, 29.06, 28.98, 27.74, 27.44,
6.6, 15.0, 12.5 (18-C). MS (m/e), C39H60N2O2: 588 (M ). Exact
2
.1.2.26. Spectral data for compound 20a. IR (NaCl, ꢀmax,
+
−
1
cm ): 3550–3050 (O–H and N–H), 1598 (C C), 1255 and 1086
mass: calculated for C39H60N2O2 = 588.4655; found = 588.4658.
(
C–O). 1H NMR (Acetone-d6, ı ppm): 8.48 (1H, dm, J = 5.1 Hz,
ꢀ
a -CH), 8.02 (1H, s, 3-OH), 7.66 (1H, td, J = 1.6 Hz and J = 7.5 Hz,
c -CH), 7.25 (1H, dd, J = 1.2 Hz and J = 7.8 Hz, d -CH), 7.16 (1H,
2.1.2.30. General
procedure
16˛,ˇ-[n -(2 pyridylethylamino)alkyl]-1,3,5(10)-estratien-
3,17ˇ-diol dichloroplatinate(II) (7a–d). To solution of
for
the
synthesis
of
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
td, J = 1.2 Hz and J = 5.1 Hz, b -CH), 7.08 (1H, d, J = 8.6 Hz, 1-
a
CH), 6.58 (1H, dd, J = 2.3 Hz and J = 8.2 Hz, 2-CH), 6.52 (1H, d,
J = 2.3 Hz, 4-CH), 3.72 (1H, d, J = 9.8 Hz, 17-CH, 16␤), 3.26 (1H,
d, J = 4.3 Hz, 17-CH, 16␣), 2.94 (4H, m, NHCH2CH2pyridyl), 2.75
2-pyridylethylamino-17␤-estradiol 20a–d (0.37 mmol) in
DMF (3 mL) at room temperature (21 C), was added potas-
sium tetrachloroplatinate(II) (0.16 g, 0.39 mmol) dissolved in
◦
(
(
0
2H, m, 6-CH2), 2.60 (2H, t, J = 6.6 Hz, (CH2)5CH2NH), 2.24–1.90
3H, m, 16-CH, 17-OH, NH), 1.46–1.06 (21H, m, 3× CH, 9× CH2),
mixture of DMF/H O (3.0 mL:1.5 mL). The starting pH value
was 9. After 5 days stirring in darkness, pH reached 5. Then, 5
2
.78 (3H, s, 18-CH3, 16␤). 13C NMR (Acetone-d6, ı ppm): 161.0
drops of DMSO were added to the resulting mixture to destroy
the excess of K PtCl and the solution was stirred for 3 h more.
ꢀ
ꢀ
(
pyridyl-C), 155.4 (3-C), 149.3 (a -C), 137.7 (5-C), 136.4 (c -C),
2
4
ꢀ
ꢀ
1
31.4 (10-C), 126.4 (1-C), 123.3 (d -C), 121.3 (b -C), 115.4 (4-
Next, a saturated KCl solution (4 mL) was added followed by
solid KCl (1 g). The mixture was strongly stirred overnight in
order to pulverize the lumps of precipitated Pt(II) complex. The
solid suspension was then filtered, washed with water and
dried in a desiccator for 1 day. The residue was adsorbed on sil-
ica gel and purified by flash chromatography with hexanes and
acetone (1:1) as the eluant to give 17␤-estradiol-platinum(II) 7
(a–d) as yellow crystals with 58% optimized yield.
C), 113.0 (2-C), 81.6 (17-C, 16␤), 49.8, 49.6, 49.0, 44.35, 44.30,
4
2
3
0.7, 38.9, 38.3, 38.1, 32.8, 32.1, 30.14, 30.08, 29.84, 29.75,
+
9.45, 27.51, 26.6, 12.5 (18-C). MS (m/e), C31H44N2O2: 476 (M ),
+
+
98 (M −C5H4N), 384 (M −C6H5N). Exact mass: calculated for
C31H44N2O2 = 476.3403; found = 476.3408.
2
.1.2.27. Spectral data for compound 20b. IR (NaCl, ꢀmax,
−
1
cm ): 3550–3050 (O–H and N–H), 1609 (C C), 1219 and 1061
C–O). 1H NMR (Acetone-d6, ı ppm): 8.48 (1H, dm, J = 4.7 Hz,
−1
1
(
2.1.2.31. Spectral data for compound 7a. IR (NaCl, ꢀmax, cm ):
ꢀ
ꢀ
a -CH), 7.65 (1H, tt, J = 2.0 Hz and J = 7.4 Hz, c -CH), 7.25 (1H,
dd, J = 1.2 Hz and J = 7.8 Hz, d -CH), 7.16 (1H, dd, J = 1.2 Hz and
J = 4.7 Hz, b -CH), 7.09 (1H, d, J = 8.6 Hz, 1-CH), 6.58 (1H, dd,
J = 2.7 Hz and J = 8.6 Hz, 2-CH), 6.52 (1H, d, J = 2.3 Hz, 4-CH),
3600–3100 (O–H and N–H), 1614 (C C), 1240 and 1076 (C–O). H
ꢀ
NMR (Acetone-d6, ı ppm): 9.15 (1H, dd, J = 1.2 Hz and J = 6.3 Hz,
ꢀ
ꢀ
ꢀ
a -CH), 8.04 (1H, td, J = 1.6 Hz and J = 7.8 Hz, c -CH), 7.90 (1H,
ꢀ
s, 3-OH), 7.54 (1H, d, J = 7.8 Hz, d -CH), 7.43 (1H, td, J = 1.6 Hz
and J = 7.4 Hz, b -CH), 7.08 (1H, d, J = 8.6 Hz, 1-CH), 6.58 (1H,
ꢀ
3
.72 (1H, d, J = 10.0 Hz, 17-CH, 16␤), 3.26 (1H, d, J = 4.3 Hz, 17-
CH, 16␣), 3.60–2.81 (4H, m, NHCH2CH2pyridyl), 2.75 (2H, m,
dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.53 (1H, d, J = 2.7 Hz, 4-CH),
6.05 (1H, br s, NH), 3.78–3.68 (1H, m, 17-CH, 16␤), 3.50–2.81
(7H, #m, CH2NHCH2CH2 pyridyl, and 17-CH, 16␣), 2.78–2.74
(2H, m, 6-CH2), 2.40–0.84 (23H, #m, 4× CH, 9× CH2 and 17-
6
1
-CH2), 2.59 (2H, t, J = 6.6 Hz, (CH2)7CH2NH), 2.36–1.87 (3H, m,
6-CH, 17-OH, NH), 1.46–1.02 (25H, m, 3× CH, 11× CH2), 0.81
1
3
and 0.78 (3H, 2s, 18-CH3, 16␣,␤ (1:15)). C NMR (Acetone-d6,
ı ppm): 161.0 (pyridyl-C), 155.4 (3-C), 149.3 (a -C), 137.7 (5-C),
ꢀ
13
OH), 0.80 and 0.77 (3H, 2s, 18-CH3, 16␣,␤ (1: 22)). C NMR
ꢀ
ꢀ
ꢀ
ꢀ
1
36.4 (c -C), 131.4 (10-C), 126.4 (1-C), 123.3 (d -C), 121.3 (b -
(Acetone-d6, ı ppm): 160.0 (pyridyl-C), 155.2 (a -C), 153.7 (3-
ꢀ
C), 115.4 (4-C), 113.0 (2-C), 81.6 (17-C, 16␤), 49.8, 49.6, 49.0,
C), 139.4 (c -C), 137.8 (5-C), 131.5 (10-C), 127.8 (1-C), 126.4
ꢀ
ꢀ
4
2
5
4.35, 44.30, 40.7, 38.9, 38.3, 38.1, 32.8, 32.1, 30.14, 30.08, 29.84,
(d -C), 124.9 (b -C), 123.9, 115.3 (4-C), 112.9 (2-C), 81.7 (17-
C, 16␤), 56.6, 49.0, 45.9, 44.33, 44.30, 40.6, 39.9, 38.9, 38.1,
32.7, 31.9, 29.7, 28.7, 27.93, 27.75, 26.68, 26.61, 12.5 (18-C). MS
9.75, 29.45, 27.51, 26.6, 12.5 (18-C). MS (m/e), C33H48N2O2:
+
04 (M ). Exact mass: calculated for C33H48N2O2 = 504.3716;
+
found = 504.3726.
(m/e), C31H44Cl2N2O5Pt: 742.2 (M+H) . Exact mass: calculated

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1085
for C31H44Cl2N2O5Pt = 742.25003 (M+H)+; found = 742.24692
and calculated for C31H44Cl2N2O5Pt = 764.23198 (M+Na)+;
found = 764.23100.
MDA-MB-468 (ER ) and MDA-MB-436 (ER ) breast cancer cell
lines. MTT (3-(4,5-dimethylthiazol-2-yl)-phenyl-tetrazolium
bromide) assay, a standard colorimetric test, was used for
measuring cellular proliferation [19]. Tumor cell lines were
added into 96-well tissue culture plates in culture medium
−
−
−
1
2
.1.2.32. Spectral data for compound 7b. IR (NaCl, ꢀmax, cm 1):
3
600–3100 (O–H and N–H), 1624 (C C), 1250 and 1076 (C–O). H
◦
and incubated at 37 C in a 5% CO2 atmosphere. Dilution
NMR (Acetone-d6, ı ppm): 9.15 (1H, dd, J = 1.2 Hz and J = 5.9 Hz,
were done using cremophore:ethanol (1:1) solution. Cells were
incubated with or without drugs for 24 h, 48 h and 72 h. Cul-
ture plates were processed using MTT for 3.5 h afterwards
SDS solubilisation solution (HCl 0.010 M, sodium dodecyl solu-
tion 10%) was added. The absorbance was read using a
scanning multiwell spectrophotometer (FLUOStar OPTIMA)
at 565 nm. All measurements were carried in triplicates.
The results were compared with those of a control ref-
erence plate fixed on the treatment day, and the growth
inhibition percentage was calculated for each drug contact
period.
ꢀ
ꢀ
a -CH), 8.02 (1H, td, J = 1.6 Hz and J = 7.8 Hz, c -CH), 7.90 (1H, br
ꢀ
s, 3-OH), 7.53 (1H, d, J = 7.8 Hz, d -CH), 7.43 (1H, td, J = 1.6 Hz
ꢀ
and J = 7.4 Hz, b -CH), 7.08 (1H, d, J = 8.6 Hz, 1-CH), 6.58 (1H,
dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.53 (1H, d, J = 2.7 Hz, 4-
CH), 6.11–6.06 (1H, br s, NH), 3.83–3.70 (1H, m, 17-CH, 16␤),
3
.45–2.81 (7H, #m, CH2NHCH2CH2 pyridyl and 17-CH, 16␣), 2.75
(
2H, m, 6-CH2), 2.40–0.97 (27H, #m, 4× CH, 11× CH2 and 17-
13
OH), 0.80 and 0.77 (3H, 2s, 18-CH3, 16␣,␤ (1: 14)). C NMR
(
1
ꢀ
Acetone-d6, ı ppm): 160.0 (pyridyl-C), 155.2 (a -C), 153.7 (3-C),
ꢀ
ꢀ
39.5 (c -C), 137.8 (5-C), 131.5 (10-C), 126.4 (1-C), 124.9 (d -
ꢀ
C), 123.9 (b -C), 115.3 (4-C), 112.9 (2-C), 81.7 (17-C, 16␤), 56.6,
5
2
4.4, 48.9, 45.9, 44.3, 39.9, 38.9, 38.1, 32.8, 32.1, 29.90, 29.76,
2
.2.2. Estrogen receptor binding affinity
9.51, 29.37, 29.00, 27.97, 27.77, 26.71, 26.63, 12.6 (18-C). MS
The estrogen receptor alpha and beta (ER␣ and ER␤) affin-
ity assay was performed using recombinant hER␣ and hER␤
+
(
m/e), C33H48Cl2N2O5Pt: 770.3 (M+H) . Exact mass: calculated
for C33H48Cl2N2O5Pt = 770.28133 (M+H)+; found = 770.28107
and calculated for C33H48Cl2N2O5Pt = 792.26328 (M+Na)+;
found = 792.26258.
(
Calbiochem/EMD BioSciences, Darmstadt, Germany) and the
TM
HitHunter
Enzyme Fragment Complementary (EFC) Estro-
gen Receptor Assay kit (Discoverex Corporation, Fremont,
CA) according to manufacturer’s protocol [20]. HitHunterTM
EFC technology is based on a genetically engineered ␤-
galactosidase enzyme that consists of two fragments termed
Enzyme Acceptor (EA) and Enzyme Donor (ED). Briefly, dif-
ferent concentration of estrogen analogs (7a–c) were added
to wells containing ES (Estrogen Steroid) Receptor + ED in
2
.1.2.33. Spectral data for compound 7c. The spectral data
for this particular compound were reported elsewhere and
are in agreement with the literature [15]. The propor-
tion of the 16␣,␤ epimers for derivative 7c is 1:9. MS
+
(
m/e), C36H54Cl2N2O5Pt: 812.3 (M+H) . Exact mass: calculated
for C36H54Cl2N2O5Pt = 812.32828 (M+H)+; found = 812.33215
and calculated for C36H54Cl2N2O5Pt = 834.31023 (M+Na)+;
found = 834.30901.
a
96-well black plate. Incubation provided competition
for the estrogen receptor binding against labelled Enzyme
Donor–Estrogen Steroid hormone conjugate (ED–ES conju-
gate), a small peptide fragment of ␤-galactosidase (␤-gal).
Then, EA, an inactive ␤-gal protein fragment, and a fluorescent
substrate were added to each well. Unbound ED–ES bind to EA
to form an active enzyme, which subsequently hydrolyse the
fluorescent substrate for EFC detection by a microplate reader
2
3
.1.2.34. Spectral data for compound 7d. IR (NaCl, ꢀmax, cm−1):
600–3100 (O–H and N–H), 1600 (C C), 1212 and 1076 (C–O).
1
H NMR (Acetone-d6, ı ppm): 9.14 (1H, dd, J = 0.8 Hz and
ꢀ
ꢀ
J = 5.9 Hz, a -CH), 8.03 (1H, td, J = 1.6 Hz and J = 7.8 Hz, c -CH),
7
td, J = 1.6 Hz and J = 5.9 Hz, b -CH), 7.09 (1H, d, J = 8.6 Hz, 1-
CH), 6.59 (1H, dd, J = 2.7 Hz and J = 8.2 Hz, 2-CH), 6.54 (1H,
d, J = 2.7 Hz, 4-CH), 6.04 (1H, br s, NH), 3.84–2.80 (8H, #m,
CH2NHCH2CH2pyridyl and 17-CH, 16␣ and 16␤), 2.76 (2H, m,
ꢀ
.91 (1H, s, 3-OH), 7.54 (1H, d, J = 7.8 Hz, d -CH), 7.42 (1H,
(
FLUOStar OPTIMA). The excitation wave is 530 nm and lumi-
ꢀ
nosity is detected at 620 nm. The amount of free ED conjugate
in the assay is proportional to the concentration of estrogen
analogs bound to the estrogen receptor [20]. A standard curve
of 17␤-estradiol was run in parallel. All assays were done in
triplicates.
6
-CH2), 2.31–1.08 (39H, #m, 4× CH, 17× CH2 and 17-OH), 0.80
13
and 0.77 (3H, 2s, 18-CH3, 16␣,␤ (1: 15)). C NMR (Acetone-
d6, ı ppm): 159.9 (pyridyl-C), 155.3 (a -C), 153.7 (3-C), 139.4
ꢀ
ꢀ
ꢀ
(c -C), 137.8 (5-C), 131.5 (10-C), 126.4 (1-C), 124.9 (d -C), 123.8
3.
Results and discussion
ꢀ
(b -C), 115.3 (4-C), 112.9 (2-C), 81.7 (17-C, 16␤), 56.6, 49.0, 45.9,
4
4.34, 44.30, 40.7, 39.8, 38.9, 38.1, 32.8, 32.1, 30.1, 29.85, 29.76
3.1.
Synthesis of 17ˇ-estradiol-platinum(II) complexes
(
(
4× C), 29.34, 29.02, 27.96, 27.75, 26.67, 26.62, 12.5 (18-C). MS
(7a–d)
+
m/e), C39H60Cl2N2O5Pt: 854.4 (M+H) . Exact mass: calculated
for C39H60Cl2N2O5Pt = 854.37524 (M+H)+; found = 854.37162
and calculated for C39H60Cl2N2O5Pt = 876.35718 (M+Na)+;
found = 876.35655.
As shown in Scheme 1, the ␻-chloro-alcohols (8a,b) and the
␻-bromo-alcohols (9a,b) were first transformed into the cor-
responded THP protected derivatives 10 and 11 (a–b) under
standard reaction conditions. These derivatives were con-
verted to their respective iodo analogs 12a–d. Thus, the
alkyl chains were treated with 3,4-dihydro-2H-pyran and
pyridinium p-toluenesulfonate in dichloromethane. After an
appropriate work-up, the crude THP-products were treated
with sodium iodide in acetone at reflux for 3 days to give the
2
.2.
Biology
2
.2.1. In vitro cytotoxic activity
The cytotoxicity of the 17␤-estradiol-platinum(II) complexes
+
−
(7a–d) was evaluated on MCF-7 (ER ), MDA-MD-231 (ER ),

1
086
steroids 7 3 ( 2 0 0 8 ) 1077–1089
Scheme 1 – Synthesis of protected iodo alkyl side chains.
desired protected ␻-iodo-THP chains 12a–d with an average
overall yield of 98%.
dride in dichloromethane and tetrahydrofuran to provide
quantitatively the appropriate triols 18a–d as a single. It is
worth mentioning that the hydride reduction of either pure
16␣-substituted or 16␤-substituted estrone derivatives gave
stereospecifically the corresponding 17␤-estradiol derivatives,
without any trace of the formation of the 17␣-estradiol deriva-
tives [23]. This is also what was observed in our experiments as
only the 17␤-estradiol derivatives (18a–d) were obtained. The
intermediates were subjected to selective bromation of the
primary alcohol. Compounds 18a–d were treated with carbon
tetrabromide and triphenylphosphine in dichloromethane to
give the hydroxy-bromides 19a–d with 62% optimised yield.
SN2 nucleophilic substitution of the primary bromide upon
treatment with an excess 2-aminoethylpyridine in methanol
gave the desired amino-pyridine products 20a–d in 97%
yield. The amino-pyridines were then treated with potas-
sium tetrachloroplatinate in a mixture of DMF and water to
give the corresponding 17␤-estradiol-linked Pt(II) complexes
7a–d with 58%. The estradiol-platinum(II) complexes were
obtained in eight chemical steps with an optimized overall
yield of 21%. This new synthetic methodology represents a
great improvement of the initial synthesis of 7c done in nine
chemical steps and 11% overall yield. All the new compounds
As shown in Scheme 2, four 17␤-estradiol-platinum(II)
complexes (7a–d) bearing a tether chain of different length
were obtained using a straightforward reaction sequence. Ini-
tially, estrone (13) was protected as a tetrahydropyrannyl (THP)
ether. Thus, estrone was treated with 3,4-dihydro-2H-pyran
and pyridinium p-toluenesulfonate in dichloromethane. The
yield of the protection reaction is 100%. The THP is the
right choice of protecting group as it was also used for the
protection of the ␻-iodo-THP chains. It was rationalized that,
at a later step, it will be possible to deprotect them both at the
same time. Derivative 14 was further transformed into the
␤-ketoester 15 upon treatment with dimethylcarbonate in
the presence of KH in dry tetrahydrofuran. Compound 15 was
obtained with 90% yield. The pure activated estrone was then
alkylated using phase transfer catalysis (PTC) methodology
[
21]. Accordingly, derivative 15 was treated with protected
iodo-chain (12a–d) and triethylbenzylammonium chloride in
dichloromethane in the presence of a 10% aqueous sodium
hydroxide solution, to give the desired product 16a–d in 92%
optimised yield. The next chemical step led to the concurrent
transformations of three functional groups on the molecule.
Hence, decarboalkoxylation and two THP deprotections were
achieved upon treatment with lithium chloride in DMF and
water at reflux for 20 h. We were aware that the cleavage of
the tetrahydropyrannyl ether under these reaction conditions
is a known process [22]. The diol derivatives 17a–d were
obtained with an overall yield of 71%. The pure compounds
were then reduced in the presence of lithium borohy-
were fully characterized by their respective IR, 1H NMR, 13
NMR and mass spectra.
C
The final products and the intermediates are a mixture
of only two stereoisomers: 16␣ and 16␤. However, the final
E2-Pt(II) hybrid molecules were greatly enrich by flash chro-
matography to the major 16␤ stereoisomer, the main orienta-
tion of the tether chain. It was established that the proportion

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1087
Scheme 2 – Synthesis of estradiol-platinum(II) complexes 7a–d.
of the 16␣,␤-side chain varied from 1:9 to 1:22. This is a level
of purity adequate to perform the biological assays and to
get meaningful results on these experimental anticancer
agents. It is possible to easily establish the 16-C side chain
orientation (␣ or ␤) using the 17-C and 18-C chemical shifts
reported in the literature [23]. Substituted 17␤-estradiol at the
as the final product since the protonation of the enolate inter-
mediate occurs at the ␣ side of the steroid. For all molecules,
the stereochemistry of the 16-␣ or 16-␤ side chain orientation
was confirmed by comparison with 13C NMR spectral data
of known 16-␣ and 16-␤ substituted 17␤-estradiol derivatives
[23].
1
1
6␣ position gives a 17-C and 18-C signals at ı 87.11 ppm and
2.20 ppm, respectively, whereas those same carbons appear
3
.2.
In vitro cytotoxic activity
at ı 81.91 ppm and 12.93 ppm when it is 16␤ substituted.
Moreover, in 1H NMR the 18-CH3 singlet appear at ı 0.80 ppm
for the 16␣ isomer and ı 0.77 ppm for the 16␤ isomer. It was
then possible to establish the ratio of the two stereoisomers
with two of the most distinctive NMR features: 16␣, 13C NMR
The cytotoxicity of the complexes was evaluated on several
tumor cell lines using the MTT colorimetric assay [19]. The
cytotoxicity of the compounds was tested along with cisplatin
+
as the control on both estrogen-receptor positive (ER , MCF-7)
1
for 17-C; ı 87.11 ppm, H NMR for 18-CH3; ı 0.80 ppm and 16␤,
−
and estrogen-receptor negative (ER , MDA-MB-231, MDA-MB-
1
3
1
C NMR for 17-C; ı 81.91 ppm, H NMR for 18-CH3; ı 0.77 ppm.
468 and MDA-MB-436) human mammary carcinomas [24].
According to the spectral data, it was establish that the
alkylation step (15→16) was done with the side chain added
mainly to the less hindered ␣ face of the molecule (16␣,␤;
As shown by the MTT assays, the 17␤-estradiol-platinum(II)
hybrid molecules 7a–d were more cytotoxic than cisplatin
4–9 times), on MCF-7 and MDA-MB-231 breast cancer cell
lines (Table 1). However, for the MDA-MB-468 and MDA-MB-
36 breast cancer cell lines the hybrid molecules 7a–d are
mostly less cytotoxic than cisplatin itself. At best, hybrid 7c
(
4
:1–12:1). This was shown by the presence of two singlets for
the 18-CH3 at ı 0.92 ppm and 0.88 ppm. The subsequent decar-
boalkoxylation reaction gave mainly the 16-␤ stereoisomers
4

1
088
steroids 7 3 ( 2 0 0 8 ) 1077–1089
Table 1 – Inhibitory concentrations of cisplatin and the hybrids (7a–d) on both ER+ and ER breast cancer cell lines
−
+
−
−
Compounds
MCF-7 (ER )
MDA-MB-231
(ER ) IC50 (␮M)
MDA-MB-468 (ER )
MDA-MB-436 (ER )
Chain length (n + 2)
IC50 (␮M)a
−
IC50 (␮M)
IC50 (␮M)
Cisplatin (1)
18.97 ± 0.43
4.22 ± 0.18
2.18 ± 0.11
2.29 ± 0.13
3.81 ± 0.05
17.33 ± 2.28
3.59 ± 0.17
2.16 ± 0.16
2.06 ± 0.06
1.88 ± 0.04
0.99 ± 0.06
1.81 ± 0.05
1.48 ± 0.08
1.08 ± 0.01
1.02 ± 0.06
3.28 ± 0.38
3.66 ± 0.18
1.85 ± 0.16
1.68 ± 0.03
2.21 ± 0.13
–
6
8
11
14
7
7
7
7
a
b
c
d
a
Inhibitory concentration (IC50, ␮M) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent the
mean ± S.E.M. of three independent experiments. The cells were incubated for a period of 72 h.
is about twice as cytotoxic as cisplatin on the MDA-MB-436
cells. Generally, the hybrid 7c presents a useful cytotoxic activ-
ity throughout the different cancer cells tested with potential
for in vivo evaluation. This is in agreement with our previ-
ous results [15]. Overall, the addition of a toxic moiety at the
1
6␤ position of the steroid nucleus does not interfere with the
cytocidal activity of the molecule.
This study was undertaken to verify the influence of the
length of the alkyl chain on the biological activity. For these
particular E2-Pt(II) hybrid molecules, the length of the alkyl
chain does not influence very much the activity. However,
we can observed that the Pt(II) complexes bearing the short-
+
ness chain 7a is the least effective towards ER as well as
−
ER breast cancer cell lines. This observation can be ratio-
nalized by the hypothesis that a short linkage can cause
additional steric hindrance around the 17-hydroxyl group of
the steroid, which is necessary for binding to the estrogen
receptor [6]. Unfortunately, the in vitro assays show no clear
specific action on estrogen-dependent cells as compared to
estrogen-independent cells. Interestingly, the in vivo studies
show that this class of E2-Pt(II) hybrid molecules are specific
towards homone-dependent breast and ovarian cancers. All
the in vivo results will be soon reported in a subsequent paper.
3
.3.
Estrogen receptor binding affinity
Fig. 2 – Estrogen receptor binding affinity (ER␣ and ER␤).
ED-estradiol: enzyme donor-estradiol; E2: 17␤-estradiol.
Estradiol and 17␤-estradiol-platinum(II) complexes (7a–7c)
present similar affinity for ERs. Cisplatin presents no
affinity for the ERs.
The estrogen receptor alpha and beta (ER␣ and ER␤) affin-
ity assay was performed using the HitHunterTM EFC Estrogen
Fluorescence assay kit (Discoverx, Fremont, CA) according to
manufacturer’s instructions [20]. The results are presented
in Fig. 2. The binding affinities were measured for only for
hybrids 7a, 7b and 7c. As expected, the estrogen receptor
binding studies showed strong affinities for 17␤-estradiol with
EC50 of 0.40 nM and 0.93 nM, respectively, for the ER␣ and the
ER␤. Interestingly, those three E2-Pt(II) hybrids demonstrated
similar binding affinity for the ER␣ and ER␤, close to that of
4
.
Conclusion
In summary, this manuscript presents a new and improved
methodology for the synthesis of E2-Pt(II) hybrid molecules.
The novel compounds are made at position 16 of the steroid
nucleus. There are 6, 8, 11 or 14 carbon atoms separating the
cytotoxic Pt(II) moiety from the steroid nucleus. The E2-Pt(II)
hybrids were made from estrone in only 8 chemical steps with
an overall yield of 21%. This represents a huge improvement
from an earlier synthesis of hybrid 7c which was made in 9
steps with only 10% overall yield. The THP protective group
at the beginning of the synthesis proved to be a judicious
choice as it allowed in a single synthetic step three chemi-
cal transformations (16a–d to 17a–d); decarboalkoxylation and
bis-deprotection. The previous synthesis used a benzyl protec-
tive group which required an additional synthetic step [15].
1
7␤-estradiol. They have EC50 varying from 1.52 nM to 2.75 nM
for ER␣ and from 1.49 nM to 1.83 nM for ER␤. Hence, those
results demonstrate that the length of the tether chain does
not interfere that much with the estrogen receptor binding
affinity. It is noteworthy that cisplatin has no affinity for the
ER [15]. Consequently, this type of E2-Pt(II) hybrids have the
potential to target the ER in an in vivo model and show selec-
tive anticancer activity as well as reduce systemic toxicities.
The strong cytocidal activity of E2-Pt(II) hybrids 7a, 7b and 7c
may be, at least partly, attributed to their interactions with the
ER␣ or ER␤.

steroids 7 3 ( 2 0 0 8 ) 1077–1089
1089
Moreover, the new synthetic methodology allowed the
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1
enrichment of the final hybrids to the 16␤ stereoisomer. Never-
theless, work is in progress in our laboratory to isolate and test
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[
[
+
E2-Pt(II) hybrids showed potent cytocidal activity on ER (MCF-
[
[
−
7
) and ER (MDA-MB-231) breast cancer cell lines. They are
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The length of the alkyl chain separating the estradiol from the
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for site specific treatment of hormone-dependent breast can-
cers. Further investigation will be necessary to assess the full
biological potential of these new E2-Pt(II) hybrid molecules.
Several in vivo assays have been carried out with hybrid 7c
on nude mouse xenografted model. Hybrid 7c show strong
and selective anticancer activity on both, hormone-dependent
breast and ovarian cancers and the results will be reported
elsewhere.
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The authors wish to thank the Fonds de Recherche sur la
Nature et les Technologies du Qu e´ bec (FQRNT), the Natural
Sciences and Engineering Research Council of Canada (NSERC)
and the Canadian Institutes for Health Research (CIHR) for
generous financial support. The FRSQ student fellowship for
Caroline Desc oˆ teaux is gratefully acknowledged. The authors
also wish to thank Daphne Efford for her help in preparing this
manuscript.
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[