Design, synthesis and biological evaluation of estradiol-PEG-linked platinum(II) hybrid molecules: Comparative molecular modeling study of three distinct families of hybrids

Article abstract of DOI:10.1016/j.steroids.2010.09.004

The synthesis of a series of 17β-estradiol-platinum(II) hybrid molecules is reported. The hybrids are made of a PEG linking chain of various length and a 2-(2′-aminoethyl)pyridine ligand. They are prepared from estrone in only 5 chemical steps with an overall yield of 22%. The length of the PEG chain does not influence the solubility of the compounds as it remains relatively constant throughout the series. MTT assays showed that the derivative with the longest PEG chain showed the best activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231). The novel PEG-hybrids are also compared in terms of activities with two other families of 17β-estradiol- platinum(II) hybrids that we reported in previous studies. Molecular modeling study performed on a representative member of each family of hybrids reveals distinct molecular interactions with the estrogen receptor α which further corroborates their notably contrasting cytocidal activities on breast cancer cell lines. This study also shows that lipophilicity and the orientation of the tether chain between the estrogenic portion and the platinum(II) core contribute markedly to the biological activity of the various families of hybrids. The most active hybrids are those possessing an alkyl tether chain at position 16β of the steroid nucleus. For example, derivative 3 (p = 6) is about 16 times more potent on MCF-7 breast cancer cells than the corresponding 16α-PEG-hybrids (2b) made in this study.

Full text of DOI:10.1016/j.steroids.2010.09.004

Steroids 76 (2011) 94–103
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Design, synthesis and biological evaluation of estradiol-PEG-linked platinum(II)
hybrid molecules: Comparative molecular modeling study of three distinct
families of hybrids
Josée Provencher-Mandeville , Chhanda Debnath , Sanat K. Mandalb,c, Valérie Leblanca,
a
a
a
a
a,∗
Sophie Parent , Éric Asselin , Gervais Bérubé
Groupe de Recherche en Oncologie et Endocrinologie Moléculaires, Département de Chimie-Biologie, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, Canada
G9A 5H
Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, Newfoundland, Canada A1B 3V6
Division of Science & Technology, College of the North Atlantic, Clarenville Campus, Clarenville, Newfoundland, Canada A5A 1V9
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 August 2010
Received in revised form
The synthesis of a series of 17␤-estradiol-platinum(II) hybrid molecules is reported. The hybrids are
made of a PEG linking chain of various length and a 2-(2 -aminoethyl)pyridine ligand. They are prepared
ꢀ
from estrone in only 5 chemical steps with an overall yield of 22%. The length of the PEG chain does
not influence the solubility of the compounds as it remains relatively constant throughout the series.
MTT assays showed that the derivative with the longest PEG chain showed the best activity against two
human breast cancer cell lines (MCF-7 and MDA-MB-231). The novel PEG-hybrids are also compared
in terms of activities with two other families of 17␤-estradiol-platinum(II) hybrids that we reported
in previous studies. Molecular modeling study performed on a representative member of each family
of hybrids reveals distinct molecular interactions with the estrogen receptor ␣ which further corrobo-
rates their notably contrasting cytocidal activities on breast cancer cell lines. This study also shows that
lipophilicity and the orientation of the tether chain between the estrogenic portion and the platinum(II)
core contribute markedly to the biological activity of the various families of hybrids. The most active
hybrids are those possessing an alkyl tether chain at position 16␤ of the steroid nucleus. For example,
derivative 3 (p = 6) is about 16 times more potent on MCF-7 breast cancer cells than the corresponding
1
4 September 2010
Accepted 16 September 2010
Available online 30 October 2010
Keywords:
1
Cisplatin
Breast cancer
Estrogen receptor
Biological activity
Molecular modeling
7ˇ-Estradiol-platinum(II) complex
1
6␣-PEG-hybrids (2b) made in this study.
©
2010 Elsevier Inc. All rights reserved.
1
. Introduction
The platinum anticancer drugs have attracted considerable
Even if thousands of platinum analogs have been made to date
and tested for anticancer activity, there are only a few selected
compounds used today in clinics. They are cisplatin, carboplatin,
oxaliplatin, nedaplatin, iobaplatin and heptaplatin [5,9,10]. The
specific uses of these drugs vary from one to another. However,
the most recent platinum drugs have not shown any distinct and
significant advantages over cisplatin and carboplatin [5,9,10].
Recent literature reviews present a broad overview of the actual
knowledge of platinum-based antitumor agents as well as their
action mechanisms [5,9–11]. The antitumor activity of platinum
drugs is a consequence of their interaction with DNA. Cisplatin
binds readily to the N7 position of the guanine bases of DNA
molecules to form mainly 1,2- or 1,3-intrastrand crosslinks, and, to
a lesser extent, interstrand crosslinks. These cisplatin-DNA lesions
cause various cellular responses such as inhibition of replication
and/or transcription, cell cycle arrest, DNA repair and ultimately
induction of apoptosis [3,5,7,10]. Other lesions to the cell are also
occurring and contribute to the global cisplatin activity [4,7].
Targeting platinum anticancer drugs is a subject of intense
research. Many strategies are used to reduce systemic toxicity [12].
interest because of their great therapeutic value in treating a num-
ber of cancers [1,2]. They have been mainly used for the treatment of
solid tumors, particularly small cell lung, ovarian, testicular, head
and neck tumors. Unfortunately, their use has been limited by a
number of severe side effects, particularly nephrotoxicity and neu-
rotoxicity [3,4] and also by the emergence of resistance to the
drug [5,6]. The major mechanisms of resistance to cisplatin (and
analogs) are (1) inactivation by glutathione, metallothionein or
other sulfur-containing molecules, (2) increased DNA repair, (3)
reduced accumulation by alteration of drug uptake/reflux and (4)
increased platinum adducts tolerance and failure of apoptotic path-
ways [7,8].
∗ Corresponding author.
E-mail address: Gervais.Berube@uqtr.ca (G. Bérubé).
0
039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.09.004

J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
95
Several research groups have been investigating the combination
OH
of a platinum complex to an estrogenic moiety in order to target
the estrogen receptor (ER) in hormone-dependent diseases, partic-
ularly breast cancer [13–15]. Obviously, the main goal is to improve
the selectivity and efficacy of this type of drug and, consequently,
to minimize its toxic side effects. Three recent reviews have pre-
sented the many studies on the use of platinum complexes for
hormone-dependent cancers treatment [13–15].
OH
OH
NH
N
O
Pt
Cl Cl
n
HO
1
OH
NH N
Pt
The estrogen receptor is a biological target that has attracted
considerable attention over the years. It is expressed by several
type of cancers; breast (60–70%), uterus (70–73%) as well as ovar-
ian (61%) [16–18]. Together, they represent 36% of all cancers
diagnosed in women and display a 22% mortality rate [19]. The
biological affinity between 17␤-estradiol and its cognate receptor
can theoretically be used to direct a cytotoxic agent to the target
cells.
p
Cl Cl
2
HO
HO
OH
p
NH
H
Cl Pt
Cl
N
3
This manuscript gives the detailed description of the synthesis
p = 2 to 12 carbon atoms
n = 1(a), 2(b), 3(c), 4(d) or 5(e)
of five 17␤-estradiol-PEG-platinum(II) (E -Pt(II)) hybrid molecules
2
1
(Fig. 1) succinctly reported in an earlier communication [20]. The
novel platinum complexes are linked with a polyethylene glycol
PEG) chain at position 16␣ of the steroid nucleus and bear a 16␤-
Fig. 1. Structure of the 17␤-estradiol-platinum(II) complexes studied in our labo-
(
ratory.
hydroxymethyl side chain. They are made from estrone in only
five chemical steps with an overall yield of 22%. The PEG-chain
was used in order to obtain compounds with relatively constant
solubility throughout the series and to study more accurately the
true influence of the length of the chain on the biological activ-
ity. This class of compounds completes our previous work done on
similar derivatives; see compound 2 and 3, which used a carbon
chain tether (Fig. 1) [21–25]. This manuscript also analyses and
compares, using molecular modeling calculations, the three class
of estrogen-platinum(II) hybrids studied in our laboratory.
(
electrospray ionization) using the positive mode (ESI+) (Université
du Québec à Montréal).
Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian 200 MHz NMR apparatus. Samples were dissolved in deute-
rochloroform (CDCl ) or in deuteroacetone (acetone-d ) for data
acquisition using tetramethylsilane as internal standard (TMS, ı
0
cation was clarified by using DEPT (Distortionless Enhancement by
Polarization Transfert) technique. Chemical shifts (ı) are expressed
in parts per million (ppm), the coupling constants (J) are expressed
in hertz (Hz). Multiplicities are described by the following abbrevi-
ations: 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.
3
6
.0 ppm for 1H NMR and C NMR). If necessary, carbon identifi-
13
The objective of the present study was also to determine the
cytotoxic effect of these novel molecules using estrogen depen-
+
dent (estrogen receptor positive; ER ) and independent (estrogen
−
receptor negative; ER ) human breast cancer cells. The biological
activity of these compounds was evaluated in vitro using an MTT
cell proliferation assay [26,27]. The MTT assay was performed over
an incubation period of 72 h. The affinity for the estrogen receptor
alpha (ER␣) was also determined for derivative 1b.
2
2
2
.1.1. Preparation of the diiodo PEG-chains
.1.1.1. Synthesis of ˛,␻-diiodo-PEG (7a). Sodium iodide (3.14 g,
1.0 mmol) was dissolved in a flask with 15 mL of anhydrous ace-
tone (dried by MgSO ) and then 2-chloroethyl ether 5 (0.82 mL,
2
. Experimental
4
7
.0 mmol) was added. The flask was covered with an aluminum
paper in order to protect from daylight. The mixture was heated
to reflux and stirred for 3 days. The solvent was evaporated and
the product was dissolved with diethyl ether. The resulting organic
phase was first washed with water (100 mL) to which was added
2
.1. Chemistry
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 [28]. Anhydrous reac-
tions required an inert atmosphere of dry nitrogen. Estrone was
purchased from Steraloids Inc., Wilton, NH, USA and the diverse
polyethylene glycol chains were purchased from Sigma-Aldrich
Canada Ltd., Oakville, Ontario, Canada. All reactions were moni-
6
1
mL of 5% sodium thiosulfate solution and after with water (4×
00 mL). The organic phase was dried over anhydrous magnesium
sulfate, filtered and evaporated that gave the product with 81%
yield.
tored by UV fluorescence or staining with iodine on Sigma T 6145
commercial TLC plates (polyester silica gel 60 A˚ , 0.25 mm). Purifica-
tions were done using flash column chromatography according to
the method of Still et al. [29] on Silicycle UltraPure Flash Silica Gel,
2.1.1.2. Synthesis of ˛,␻-diiodo-PEG (7b-e). Each of the ethy-
lene glycol 6b-e (tri, tetra-, penta-, hexa-; 6.8 mmol, 1 equiv.)
was dissolved in diethyl ether (50 mL) in presence of triethy-
lamine (2.38 mL, 1.7 mmol) under nitrogen atmosphere. The
resulting solution was cooling down with an ice bath; after-
wards methanesulfonyl chloride (CH SO Cl, 1.1 mL, 13.6 mmol)
4
0–63 ␮m mesh. Hexanes and acetone were distilled before their
use as chromatography eluant.
3
2
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
was added drop-wise to the mixture. The mixture was stirrred at
0 C for 1 h and then at room temperature for 5 h. After 5 h the sol-
vent was evaporated and then 70 mL of dry acetone (dried with
◦
7
070 HS instrument using ionization energy of 70 eV (University
MgSO ) was added and stirred for 5 min. The ammonium chlo-
4
of Sherbrooke). Chemical ionization (NH ) was required for a few
ride salt was filtered (rinsed with 30 mL of acetone). Then, sodium
iodide NaI (4.1 g, 27.0 mmol) was added to the filtrate (8b-e) and the
mixture was heated to reflux with stirring for 20 h under a nitro-
gen atmosphere. Afterwards the mixture was cooled down; sodium
3
compounds. The platinum(II) complexes were analyzed using a MS
model 6210, Agilent technology instrument. The high resolution
mass spectra (HRMS) were obtained by TOF (time of flight) using ESI

9
6
J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
thiosulfate was added and stirred for 5 min, dried with anhydrous
magnesium sulfate. The mixture was filtered on a silica gel (2.5 cm)
and celite (0.5 cm) pad using hexanes (200 mL) mixed with a little
amount of dichloromethane as the eluent. The solvent was evapo-
rated and the yield of diiodo-PEG chain 7b-e was 79%.
1611 and 1505 (C C, aromatic), 1247 and 1043 (C–O–C, aryl alkyl)
1
and 1127 (C–O–C, aliphatic). H NMR (CDCl , ı ppm): 7.19 (1H, d,
3
J = 8.6 Hz, 1-CH), 6.84 (2H, m, 2-CH and 4-CH), 5.39 (1H, t, J = 2.93 Hz,
CH, THP), 3.92 and 3.58 (2H, 2 m, CH O, THP), 2.87 (2H, m, 6-CH ),
2
2
2.55-1.36 (19 H, several m, 3 × CH and 8 × CH ), 0,90 (3H, s, 18-CH ).
2
3
1
3
C NMR (CDCl , ı ppm): 220.9 (C-17), 155.3 (C-3), 137.9 (C-5),
3
2
.1.1.3. Spectral data for 1,5-diiodo-3-oxapentane (7a). IR (NaCl,
133.2 (C-10), 126.4 (C-1), 116.8 (C-4), 114.4 (C-2), 96.6 (CH, THP),
−1
ꢀ
max, cm ):1465(CH , symmetric deformation)and1102(C–O–C,
62.2 (CH O, THP), 50.7, 48.2, 44.3, 38.6, 36.1, 31.8, 30.7, 29.8, 26.8,
2
2
1
+•
aliphatic). H NMR (CDCl , ı ppm): 3.72 (4H, t, J = 6.9 Hz, CH O),
26.1, 25.5, 21.8, 19.0, 14.1 (C-18). MS (m/e), C23H30O : 354 (M ),
271 (M-C5H7O) . Exact mass: calculated for C23H30O = 354.2195;
3
2
3
1
3
+•
3
.21 (4H, t, J = 6.9 Hz, CH I).
C NMR (CDCl , ı ppm): 71.6
2
3
3
+
•
(
2 × CH O), 2.8 (2 × CH I). MS (m/e), C H I O : 326 (M ). Exact
found = 354.2200.
2
2
4
8
2
1
mass: calculated for C H I O = 325.8665; found = 325.8673.
4
8 2
1
2
.1.2.3. Step 2 (activation). In a double necked flask, anhydrous
2
.1.1.4. Spectral data for 1,8-diiodo-3,6-dioxaoctane (7b). IR
tetrahydrofuran (30 mL) was added to potassium hydride (30% in
oil) (KH pure: 1.95 g, 48.6 mmol, with oil: 6.5 g) under nitrogen
atmosphere and the suspension was stirred at room temperature.
Dimethylcarbonate (3.35 mL, 40.5 mmol) was then added to the
potassium hydride suspension. Separately, the protected estrone
−1
(
(
NaCl, ꢀmax, cm ): 1465 (CH , symmetric deformation) and 1102
2
C–O–C, aliphatic). 1H NMR (CDCl , ı ppm): 3.72 (4H, t, J = 6.6 Hz,
3
OCH CH I), 3.61 (8H, m, OCH CH O), 3.21 (4H, t, J = 6.6 Hz, CH I).
2
2
2
2
2
13
C NMR (CDCl , ı ppm): 72.1 (2 × OCH CH I), 70.3 (OCH CH O),
3
2
2
2
2
+
•
3
.5 (2 × CH I). MS (m/e), C H I O : 370 (M ). Exact mass: calcu-
2
6
12
2
2
(
(
from Step 1) (5.74 g, 16.2 mmol) was dissolved in tetrahydrofuran
45 mL) under nitrogen atmosphere. The estrone solution was then
lated for C H I O = 369.8927; found = 369.8930.
6
12 2
2
added to reaction mixture and the overall mixture was refluxed
for 3–5 h. Afterwards, the mixture was cooled down. Tert-butanol
2
.1.1.5. Spectral data for 1,11-diiodo-3,6,9-trioxaundecane (7c).
−1
IR (NaCl, ꢀmax, cm ): 1465 (CH2, symmetric deformation)
and 1112 (C–O–C, aliphatic). H NMR (CDCl , ı ppm): 3.77
(2 mL), methyl alcohol (2 mL) and 20 mL of water were added suc-
1
3
cessively and the resulting solution was stirred for 20 min between
each addition. The mixture was then diluted with ethyl acetate
(
(
(
(
4H, t, J = 6.8 Hz, OCH CH I), 3.67 (8H, m, OCH CH O), 3.21
4H, t, J = 6.9 Hz, CH I).
2
2
1
2
2
3
C
NMR (CDCl3,
ı
ppm): 72.2
2
(
100 mL) and the organic phase was washed with a saturated NH Cl
4
2 × OCH CH I), 70.9 and 70.5 (2 × OCH CH O), 3.3 (2 × CH I). MS
2
2
2
2
2
solution and then with water (5× 100 mL). The organic phase was
dried with anhydrous magnesium sulfate, filtered and evaporated
to give the crude product. The residue was purified by flash chro-
matography with a mixture of hexanes and acetone (90:10) to give
the activated estrone in 90% yield.
+
+
m/e), C H I O : 415 (M+H) , 432 (M+NH ) . Exact mass: calcu-
8
16
2
3
4
lated for C H I O + H = 414.9267; found = 414.9275.
8
16 2
3
2
.1.1.6. Spectral data for 1,14-diiodo-3,6,9,12-tetraoxatet-
−1
radecane (7d). IR (NaCl, ꢀmax, cm ): 1465 (CH , symmetric defor-
mation) and 1107 (C–O–C, aliphatic). H NMR (CDCl , ı ppm): 3.67
2
1
3
2.1.2.4. Spectral data for Step 2 (3-tetrahydropyrannyloxy-16ˇ-
(
4H, t, J = 7.0 Hz, OCH CH I), 3.57 (12H, m, OCH CH O), 3.17 (4H,
2 2 2 2
13
(methoxycarbonyl)-1,3,5(10)-estratrien-17-one) (10). IR (NaCl,
t, J = 7.0 Hz, CH I). C NMR (CDCl , ı ppm): 71.9 (2 × OCH CH I),
2
3
2
2
−
1
ꢀ
max, cm ): 1757 (C O, ester), 1731 (C O, ketone), 1619 and
7
C
0.7, 70.6 and 70.2 (2 × CH -OCH CH O), 3.3 (2 × CH I). MS (m/e),
+ +
2
2
2
2
1496 (C C aromatic), 1224 and 1035 (C–O–C, aryl alkyl) and 1143
H I O : 459 (M+H) , 476 (M+NH ) . Exact mass: calculated
20 2 4 4
10
1
(
C–O–C, aliphatic). H NMR (CDCl , ı ppm): 7.17 (1H, d, J = 8.6 Hz,
3
for C10
H I O + H = 458.9529; found = 458.9525.
20 2 4
1
-CH), 6.81 (2H, m, 2-CH and 4-CH), 5.36 (1H, m, CH, THP), 3.92
and 3.58 (2H, 2 m, CH O, THP), 3.76 (3H, s, COOCH ), 3.20 (1H, t,
2
3
2
.1.1.7. Spectral data for 1,17-diiodo-3,6,9,12,15-pentaoxahept-
−1
J = 9.2 Hz, 16-CH), 2.88 (2H, m, 6-CH2), 2.40–1.41 (17H, several m,
adecane (7e). IR (NaCl, ꢀmax, cm ): 1460 (CH , symmetric defor-
mation) and 1102 (C–O–C, aliphatic). H NMR (CDCl , ı ppm): 3.70
2
1
3
1
3 × CH and 7 × CH2), 0.95 (3H, s, 18-CH3). C NMR (CDCl3, ı ppm):
3
2
12.2 (C-17), 170.1 (COOCH ), 155.3 (C-3), 137.7 (C-5), 132.9 (C-10),
3
(
4H, t, J = 6.8 Hz, OCH CH I), 3.60 (16H, m, OCH CH O), 3.20 (4H,
2 2 2 2
13
126.4 (C-1), 116.8 (C-4), 114.3 (C-2), 96.5 (CH, THP), 62.1 (CH2O,
t, J = 7.0 Hz, CH I). C NMR (CDCl , ı ppm): 72.1 (2 × OCH CH I),
2
3
2
2
THP), 54.3 (COOCH ), 52.7, 49.1, 48.0, 44.2, 38.1, 32.1, 30.6, 29.8,
3
7
0.8 (2C), 70.7 (4C) and 70.4 (2C) (4 × OCH CH O), 3.3 (2 × CH I).
+ +
2
2
2
26.8, 26.6, 26.0, 25.5, 19.0, 13.5 (C-18). MS (m/e), C25H32O5: 413
MS (m/e), C12
H I O : 503 (M+H) , 520 (M+NH ) . Exact mass:
24 2 5 4
+
+
+
(
M+H) , 381 (M–CH O) , 328 (M–C5H7O) . Exact mass: calculated
3
calculated for C12
H I O5 + H = 502.9791; found = 502.9780.
24 2
for C25H32O5 + H = 413.2328; found = 413.2319.
2
2
.1.2. Synthesis of platinum complexes 1a–e
.1.2.1. Synthesis of 3-tetrahydropyrannyloxy-16ˇ-(methoxycarbo-
2.1.2.5. General procedure for the synthesis of 16˛-[␻-iodo-PEG]-
16ˇ-(methoxycarbonyl)-3-(tetrahydropyran-2-yloxy)-1,3,5(10)-est-
ratrien-17-one (11a-e). The activated product 10 (1.2 mmol) and
the diiodo chains 7 (a, b, c, d or e) (4.8 mmol) were dissolved with
20 mL of dry tetrahydrofuran (THF) in a flask. Cesium carbonate
(0.43 g, 1.3 mmol) was then added to the solution and the resulting
mixture was stirred at reflux, under nitrogen atmosphere, the
reaction vessel sheltered from daylight, for 5 h. Afterwards, the
solvent was evaporated. The mixture was diluted with diethyl
ether (100 mL) and washed initially with a saturated ammonium
chloride solution (100 mL) and then with distilled water (5×
100 mL). The organic phase was dried with magnesium sulfate,
filtered and evaporated to give the crude product. The final
product was purified by flash chromatography with a mixture of
hexanes/acetone of 90:10 for the compounds 11a, 11b and 11c
(n = 1–3) and of 85:15 for 11d and 11e (n = 4 and 5). The optimized
output of the pure product was obtained with 63% yield.
nyl)-1,3,5(10)-estratrien-17-one (10): Step 1 (Protection): synthe-
sis of 3-(tetrahydropyran-2-yloxy)-1,3,5(10)-estratrien-17-one. To a
solution of estrone (9) (4.40 g, 16.2 mmol) in dichloromethane
(
45 mL), pyridinium p-toluenesulfonate (100 mg, 0.4 mmol) and
3
,4-dihydro-2H-pyran (3.7 mL, 40.5 mmol), were added. The result-
◦
ing mixture was stirred at room temperature (22 C) for 24 h.
Afterwards, solution was neutralised with a small amount of solid
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 a mixture of
dichloromethane/diethyl ether (50:50) as the eluant. The result-
ing filtrate was evaporated and set to vacuum for an hour to give a
beige solid, 100% yield.
2
1
.1.2.2. Spectral data for Step
,3,5(10)-estratrien-17-one): IR (NaCl, ꢀmax, cm ). 1738 (C O),
1 (3-(tetrahydropyran-2-yloxy)-
−
1

J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
97
2
.1.2.6. Spectral data for 16˛-[5-iodo-3-oxapentyl]-16ˇ-
214.0 (C-17), 171.9 (COOCH ), 155.2 (C-3), 137.6 (C-5), 132.9
3
(
methoxycarbonyl)-3-(tetrahydropyran-2-yloxy)-1,3,5(10)-
(C-10), 126.3 (C-1), 116.7 (C-4), 114.2 (C-2), 96.4 (CH, THP), 72.1,
−
1
estratrien-17-one (11a). IR (NaCl, ꢀmax, cm ): 1757 (C O,
ester), 1726 (C O, ketone), 1609 et 1501 (C C, aromatic), 1240
70.8, 70.7, 70.6, 70.3 and 68.0 (8 × OCH on PEG chain, 70.8 and
2
70.7 represent 2C), 62.0 (CH O, THP), 58.3 (COOCH ), 52.7, 49.5,
2
3
1
(
C–O–C, aryl alkyl) and 1117 (C–O–C, aliphatic). H NMR (CDCl , ı
46.0, 44.2, 38.0, 34.9, 32.3, 31.1, 30.5, 29.7, 26.6, 25.8, 25,4, 18.9,
3
+
ppm): 7.18 (1H, d, J = 8.6 Hz, 1-CH), 6.83 (2H, m, 2-CH and 4-CH),
5
3
14.3 (C-18), 3.2 (CH I). MS (m/e), C35H51I O : 743 (M+H) , 760
2
1
9
+
.39 (1H, t, J = 3.1 Hz, CH, THP), 3.92 (1H, m, CHaH O on THP),
(M+NH ) . Exact mass: calculated for C35H I O + H = 743.2656;
b
4
51 1
9
.72 (3H, s, COOCH ), 3.62 (5H, m, CHaH O on THP and 2 × OCH
found = 743.2642.
3
b
2
on PEG chain), 3.20 (2H, t, J = 6.6 Hz, CH I), 2.88 (2H, m, 6-CH ),
2
2
2
.45–1.26 (19H, several m, 3 × CH et 8 × CH ), 0.95 (3H, s, 18-CH ).
2.1.2.10. Spectral
pentaoxaheptadecanyl]-16ˇ-(methoxycarbonyl)-3-
data
for
16˛-[17-iodo-3,6,9,12,15-
2
3
1
3
C NMR (CDCl , ı ppm): 214.1 (C-17), 171.9 (COOCH ), 155.2
3
3
(
(
C-3), 137.6 (C-5), 132.8 (C-10), 126.3 (C-1), 116.7 (C-4), 114.2
(tetrahydropyran-2-yloxy)-1,3,5(10)-estratrien-17-one
IR (NaCl, ꢀmax, cm ): 1752 (C O, ester), 1721 (C O, ketone),
(11e).
−
1
C-2), 96.4 (CH, THP), 71.5 and 67.5 (2 × OCH on PEG chain), 62.0
2
(
CH O, THP), 58.3 (COOCH ), 52.8, 49.5, 46.0, 44.2, 37.9, 34.9, 32.3,
1614 and 1496 (C C, aromatic), 1240 (C–O–C, aryl alkyl) and 1107
2
3
1
3
(
1.3, 30.5, 29.6, 26.7, 25.8, 25.4, 18.9, 14.3 (C-18), 3.0 (CH I). MS
(C–O–C, aliphatic). H NMR (CDCl , ı ppm): 7.15 (1H, d, J = 8.6 Hz,
2
3
+
•
+•
m/e), C29H39I O : 610 (M ), 526 (M–C5H O ) . Exact mass:
1-CH), 6.78 (2H, m, 2-CH and 4-CH), 5.36 (1H, t, J = 3.1 Hz, CH, THP),
1
6
8
1
calculated for C29
H
39 1
I O = 610.1791; found = 610.1780.
3.92 (1H, m, CHaH O on THP), 3.68 (3H, s, COOCH ), 3.62 (21H, m,
6
b
3
CHaH O on THP and 10 × OCH on PEG chain), 3.22 (2H, t, J = 6.8 Hz,
b
2
2
.1.2.7. Spectral data for 16˛-[8-iodo-3,6-dioxaoctyl]-16ˇ-
CH I), 2.88 (2H, m, 6-CH ), 2.45–1.25 (19H, several m, 3 × CH and
2
2
13
(methoxycarbonyl)-3-(tetrahydropyran-2-yloxy)-1,3,5(10)-
8 × CH ), 0.91 (3H, s, 18-CH ). C NMR (CDCl , ı ppm): 214.2 (C-
2
3
3
−
1
estratrien-17-one (11b). IR (NaCl, ꢀmax, cm ): 1752 (C O,
17), 172.0 (COOCH ), 155.2 (C-3), 137.7 (C-5), 132.9 (C-10), 126.3
3
ester), 1721 (C O, ketone), 1609 and 1496 (C C, aromatic), 1240
(C-1), 116.7 (C-4), 114.3 (C-2), 96.4 (CH, THP), 72.2, 70.8, 70.8, 70.7,
1
(
C–O–C, aryl alkyl) and 1117 (C–O–C, aliphatic). H NMR (CDCl , ı
70.4, 70.3 and 68.0 (10 × OCH on PEG chain, 2 signals of 70.8 rep-
3
2
ppm): 7.12 (1H, d, J = 8.6 Hz, 1-CH), 6.75 (2H, m, 2-CH and 4-CH),
.3 (1H, m, CH, THP), 3.86 (1H, m, CHaH O on THP), 3.67 (3H,
resent 5C), 62.1 (CH O, THP), 58.4 (COOCH ), 52.8, 49.6, 46.1, 44.2,
2
3
5
38.0, 35.0, 32.4, 31.2, 30.6, 29.7, 26.7, 25.9, 25.4, 18.9, 14.3 (C-18),
b
+
s, COOCH ), 3.52 (9H, m, CHaH O on THP and 4 × OCH on PEG
3.2 (CH I). MS (m/e), C37H55I O : 804 (M+NH ) . Exact mass:
3
b
2
2
1
10
4
chain), 3.19 (2H, t, J = 6.8 Hz, CH I), 2.83 (2H, m, 6-CH ), 2.40–1.38
calculated for C37H55I O + NH = 804.3183; found = 804.3190.
2
2
1 10 4
1
3
(
19H, several m, 3 × CH and 8 × CH ), 0.90 (3H, s, 18-CH ).
C
2
3
NMR (CDCl , ı ppm): 213.9 (C-17), 171.8 (COOCH ), 155.0 (C-3),
2.1.2.11. General procedure for the synthesis of 16ˇ-hydroxymethyl-
16˛-[␻-iodo-PEG]-1,3,5(10)-estratrien-3,17ˇ-diol (12a-e).. Step 1
(Reduction): Synthesis of 16␤-16-hydroxymethyl-16␣-[␻-iodo-
PEG]-3-(tetrahydro-pyran-2-yloxy)-1,3,5(10)-estratrien-17␤-ol.
The alkylated product 11 (0.6 mmol) was dissolved in anhydrous
diethyl ether (10 mL), under nitrogen atmosphere. The resulting
solution was cooled down in an ice bath. After a few minutes, the
lithium borohydride LiBH4 (82 mg, 3.8 mmol) was added in two
portions separated by approximately 5 min and the reaction vessel
3
3
1
37.4 (C-5), 132.7 (C-10), 126.1 (C-1), 116.5 (C-4), 114.0 (C-2), 96.2
(
CH, THP), 71.9, 70.1 and 67.8 (4 × OCH , 70.1 represents 2C), 61.8
2
(
CH O, THP), 58.2 (COOCH ), 52.6, 49.4, 45.8, 44.0, 37.8, 34.7, 32.1,
2
3
3
1.0, 30.4, 29.5, 26.5, 25.7, 25.2, 18.8, 14.2 (C-18), 3.1 (CH I). MS
2
+
•
+•
1
(m/e), C31H43I O : 654 (M ), 570 (M–C5H O ) . Exact mass:
1
7
8
calculated for C31
H I O7 = 654.2053; found = 654.2042.
43 1
2
1
1
.1.2.8. Spectral data for 16˛-[11-iodo-3,6,9-trioxaundecanyl]-
◦
6ˇ-(methoxy-carbonyl)-3-(tetrahydropyran-2-yloxy)-
sheltered from daylight. The mixture was kept at 0 C and under
,3,5(10)-estratrien-17-one (11c). IR (NaCl, ꢀmax, cm−1): 1757
O, ester), 1726 (C O, ketone), 1614 and 1511 (C C, aromatic),
nitrogen atmosphere for 1 h and then at room temperature for 11 h.
Afterwards, the solution was neutralized with a small amount of
sodium sulfate decahydrate. The product was extracted with ethyl
(
C
1
1
240 (C–O–C, aryl alkyl) and 1112 (C–O–C, aliphatic). H NMR
(
CDCl , ı ppm): 7.18 (1H, d, J = 8.6 Hz, 1-CH), 6.83 (2H, m, 2-CH
acetate (100 mL) and washed first with a saturated NH Cl solution
3
4
et 4-CH), 5.39 (1H, t, J = 3.1 Hz, CH, THP), 3.92 (1H, m, CHaH O on
(100 mL) and then with water (5× 100 mL). The organic phase was
dried with anhydrous magnesium sulfate, filtered and evaporated
to give the crude product. The compound was then used as such
for the next step; the removal of the protective group at C-3.
b
THP), 3.71 (3H, s, COOCH ), 3.62 (13H, m, CHaH O on THP and
3
b
6
× OCH on PEG chain), 3.21 (2H, t, J = 7.0 Hz, CH I), 2.88 (2H,
2
2
m, 6-CH ), 2.45–1.25 (19H, several m, 3 × CH and 8 × CH ), 0.94
2
2
13
(
(
3H, s, 18-CH ). C NMR (CDCl , ı ppm): 214.2 (C-17), 172.0
3
3
COOCH ), 155.2 (C-3), 137.7 (C-5), 132.9 (C-10), 126.3 (C-1),
2.1.2.12. Step 2 (deprotection): to a solution of crude product
(0.6 mmol) in 10 mL of ethanol, pyridinium p-toluenesulfonate (50 mg,
0.2 mmol) was added. The resulting mixture was stirred at reflux,
the reaction vessel sheltered from daylight, for 4 h. When the reac-
tion was completed, 5 mL of saturated ammonium chloride solution
3
1
16.7 (C-4), 114.3 (C-2), 96.4 (CH, THP), 72.1, 70.7, 70.3 and 68.0
(
6 × OCH on PEG chain, 70.7 and 70.3 represent 2C), 62.1 (CH O,
2
2
THP), 58.4 (COOCH ), 52.8, 49.6, 46.0, 44.2, 38.0, 34.9, 32.3, 31.2,
3
3
0.5, 29.7, 26.7, 25.8, 25.4, 18.9, 14.3 (C-18), 3.1 (CH I). MS (m/e),
2
+
•
+•
1
C33H47I O : 698 (M ), 614 (M–C5H O ) . Exact mass: calculated
(NH Cl sat.) was added and stirred for 5 min. The product was
1
8
8
4
for C33
H
47 1
I O = 698.2315; found = 698.2303.
extracted with ethyl acetate (100 mL) and washed first with a sat-
8
urated NH Cl solution (100 mL) and then with water (5× 100 mL).
4
2
.1.2.9. Spectral
tetraoxatetradecanyl]-16ˇ-(methoxycarbonyl)-3-
tetrahydropyran-2-yloxy)-1,3,5(10)-estratrien-17-one
data
for
16˛-[14-iodo-3,6,9,12-
The organic phase was dried with anhydrous magnesium sulfate,
filtered and evaporated to give the crude product. The compound
was finally purified by flash chromatography while using a mix-
ture of hexanes and acetone 70:30 for the product 12a, 12b, 12c
and 12d and of 60:40 for the product 12e. The optimized output of
the reaction was of 70% yield.
(
(11d).
−1
IR (NaCl, ꢀmax, cm ): 1752 (C O, ester), 1731 (C O, ketone),
1
614 and 1501 (C C, aromatic), 1245 (C–O–C, aryl alkyl) and 1112
1
(
C–O–C, aliphatic). H NMR (CDCl , ı ppm): 7.16 (1H, d, J = 8.6 Hz,
3
1
-CH), 6.80 (2H, m, 2-CH and 4-CH), 5.37 (1H, t, J = 3.1 Hz, CH,
THP), 3.92 (1H, m, CHaH O on THP), 3,69 (3H, s, COOCH ), 3.63
2.1.2.13. Spectral data for 16ˇ-hydroxymethyl-16˛-[5-iodo-3-
oxapentyl]-1,3,5(10)-estratrien-3,17ˇ-diol (12a). IR (NaCl, ꢀmax,
b
3
(
17H, m, CHaH O on THP and 8 × OCH on PEG chain), 3.23 (2H,
b
2
−
1
t, J = 6.8 Hz, CH I), 2.88 (2H, m, 6-CH ), 2.45-1.25 (19H, several m,
cm ): 3365 (O–H), 1609 and 1501 (C C, aromatic) and 1102
2
2
1
3
1
3
× CH et 8 × CH ), 0.92 (3H, s, 18-CH ). C NMR (CDCl , ı ppm):
(C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 7.93 (1H, s,
2
3
3
6

9
8
J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
phenol-OH), 7.09 (1H, d, J = 8.6 Hz, 1-CH), 6.59 (1H, dd, J = 8.6 Hz and
.7 Hz, 2-CH), 6.52 (1H, d, J = 2.7 Hz, 4-CH), 4.25 (1H, d, J = 4.3 Hz,
CHaH OH), 3.80–3.39 (6H, m, CHOH, CHaH OH and 2 × OCH on
6.59 (1H, dd, J = 8.4 Hz and 2.5 Hz, 2-CH), 6.52 (1H, d, J = 2.4 Hz,
2
4-CH), 4.20 (1H, d, J = 4.3 Hz, CHaH OH), 3.76–3.43 (22H, m, CHOH,
b
CHaH OH and 10 × OCH on PEG chain), 3.32 (2H, t, J = 6.4 Hz,
b
b
2
b
2
PEG chain), 3.35 (2H, t, J = 6.4 Hz, CH I), 2.75 (2H, m, 6-CH ),
CH I), 2.75 (2H, m, 6-CH ), 2.23-1.10 (15H, several m, 2 × OH,
2
2
2
2
13
2
.25–1.11 (15H, several m, 2 × OH, 3 × CH and 5 × CH ), 0.88 (3H,
3 × CH and 5 × CH ), 0.87 (3H, s, 18-CH ). C NMR (acetone-d ,
2
2
3
6
13
s, 18-CH ). C NMR (acetone-d , ı ppm): 155.1 (C-3), 138.5 (C-
ı ppm): 156.1 (C-3), 138.5 (C-5), 132.2 (C-10), 127.1 (C-1), 116.1
(C-4), 113.7 (C-2), 90.3 (C-17), 72.7, 71.4, 71.4, 71.2, 71.0, 71.0
69.2 and 67.5 (CH OH and 10 × OCH on PEG chain, two signals
3
6
5
7
4
), 132.2 (C-10), 127.1 (C-1), 116.1 (C-4), 113.7 (C-2), 90.4 (17-C),
2.3, 68.9 and 67.6 (CH OH and 2 × OCH on PEG chain), 48.4, 46.9,
2
2
2
2
5.8, 45.0, 39.9, 39.3, 39.0, 35.4, 30.5, 28.5, 27.3, 12.6 (C-18), 4.8
of 71.4 represents 5C), 48.4, 47.1, 45.9, 45.0, 40.1, 39.3, 39.1, 36.1,
+
•
(
CH I). MS (m/e), C23H33I O : 500 (M ). Exact mass: calculated
30.4, 28.4, 27.3, 12.7 (C-18), 4.6 (CH I). MS (m/e), C31H49I O : 677
2
1
4
2
1
8
+
for C23
H
33 1
I O = 500.1423; found = 500.1419.
(M+H) . Exact mass: calculated for C31H I O + H = 677.2550;
4
49 1 8
found = 677.2540.
2
.1.2.14. Spectral data for 16ˇ-hydroxymethyl-16˛-[8-iodo-3,6-
dioxaoctyl]-1,3,5(10)-estratrien-3,17ˇ-diol (12b). IR (NaCl, ꢀmax,
2.1.2.18. General procedure for the synthesis of 16ˇ-hydroxymethyl-
16˛-[␻-(2-pyridylethylamino)-PEG]-1,3,5(10)-estratrien-3,17ˇ-diol
dichloroplatinate(II) (1a-e). Step 1 (Addition of aminoethylpyridine).
To a solution of derivative 12 in methanol (4 mL) was added
2-aminoethylpyridine (0.64 mL, 5.4 mmol) under nitrogen atmo-
sphere. The resulting mixture was stirred at reflux, the reaction
vessel sheltered from daylight, for 4 h. Then, the methanol was
evaporated and the residue was diluted with ethyl acetate (50 mL)
and washed with distilled water (5× 50 mL). The organic phase
was dried with magnesium sulfate, filtered and evaporated to
yield the crude product 4a-e which was used as such at the next
step.
−
1
cm ): 3369 (O–H), 1616 and 1506 (C C, aromatic) and 1099
1
(
C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 7.92 (1H, s,
6
phenol-OH), 7.09 (1H, d, J = 8.6 Hz, 1-CH), 6.59 (1H, dd, J = 8.6 Hz et
.5 Hz, 2-CH), 6.52 (1H, d, J = 2.3 Hz, 4-CH), 4.20 (1H, d, J = 4.3 Hz,
CHaH OH), 3.78–3.39 (10H, m, CHOH, CHaH OH and 4 × OCH
2
b
b
2
on PEG chain), 3.34 (2H, t, J = 6.4 Hz, CH I), 2.75 (2H, m, 6-CH ),
2
2
2
.31–1.11 (15H, several m, 2 × OH, 3 × CH and 5 × CH ), 0.88 (3H,
2
13
s, 18-CH ). C NMR (acetone-d , ı ppm): 155.9 (C-3), 138.4 (C-5),
3
6
1
7
4
4
32.1 (C-10), 127.0 (C-1), 115.9 (C-4), 113.6 (C-2), 90.2 (C-17), 72.5,
0.9, 70.6, 69.1 and 67.4 (CH OH and 4 × OCH on PEG chain), 48.2,
2
2
6.9, 45.7, 44.8, 40.0, 39.1, 38.9, 35.8, 30.2, 28.3, 27.2, 12.5 (C-18),
+
•
+•
.1 (CH I). MS (m/e), C25H37I O : 544 (M ), 526 (M–H O) . Exact
2
1
5
2
mass: calculated for C25
H I O5 = 544.1686; found = 544.1675.
37 1
2.1.2.19. Step 2 (complexation). A solution of crude aminopyridine
(0.37 mmol) in DMF (6 mL) was treated with a solution of potas-
2
3
.1.2.15. Spectral data for 16ˇ-hydroxymethyl-16˛-[11-iodo-
,6,9-trioxaundecanyl]-1,3,5(10)-estratrien-3,17ˇ-diol (12c). IR
sium tetrachloroplatinate(II) (0.187 g, 0.5 mmol) in water (2 mL)
and the mixture was stirred at room temperature (22 C), the reac-
tion vessel kept in the dark. The pH was measured and it usually
varied between pH 8–9. After 3 days of stirring in the darkness,
pH was reached to about 5. Then, 4–5 drops of DMSO were added
◦
−1
(NaCl, ꢀmax, cm ): 3380 (O–H), 1619 and 1506 (C C, aromatic)
1
and 1107 (C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 7.99
6
(
1H, s, phenol-OH), 7.08 (1H, d, J = 8.6 Hz, 1-CH), 6.59 (1H, dd,
J = 8.2 Hz and 2.7 Hz, 2-CH), 6.52 (1H, d, J = 2.4 Hz, 4-CH), 4.26 (1H,
d, J = 3.9 Hz, CHaH OH), 3.76–3.42 (14H, m, CHOH, CHaH OH and
to the resulting mixture to destroy the excess of K PtCl4 and the
2
solution was further stirred for 3 h. Next, a saturated KCl solution
(4–5 mL) was added to the mixture and stirred for 1 h. A small
amount of solid KCl was then added and the mixture was strongly
stirred for 18 h in order to pulverize the lumps of the precipitated
Pt(II) complex. The solid suspension was then filtered; the product
was washed with water and then with diethyl ether to accelerate
the drying process. Of note, only a small amount of diethyl ether
should be used as the platinum complex is slightly soluble. The
solid is dried in desiccators for 1 day. The residue was finally dis-
solved in a mixture of acetone and methanol and adsorbed on silica
gel. Then, it was purified by flash chromatography with hexanes
and acetone (45:55) for 1a, 40:60 for 1b, 1c and 1d and 35:65 for
1e. The optimized output of the pure product 1 was obtained 55%
yield.
b
b
6
× OCH on PEG chain), 3.32 (2H, t, J = 6.6 Hz, CH I), 2.74 (2H, m, 6-
2
2
CH ), 2.30–1.10 (15H, several m, 2 × OH, 3 × CH and 5 × CH ), 0.87
2
2
13
(
(
7
3H, s, 18-CH ). C NMR (acetone-d , ı ppm): 155.9 (C-3), 138.3
3 6
C-5), 132.0 (C-10), 127.0 (C-1), 116.0 (C-4), 113.6 (C-2), 90.2 (C-17),
2.5, 71.2, 71.1, 70.9, 70.8, 69.1 and 67.4 (CH OH and 6 × OCH on
2
2
PEG chain), 48.2, 46.9, 45.8, 44.8, 40.0, 39.1, 38.9, 35.9, 30.3, 28.3,
+
•
2
7.2, 12.6(C-18), 4.5(CH I). MS(m/e), C27H41I O : 588(M ). Exact
2
1
6
mass: calculated for C27
H I O = 588.1948; found = 588.1940.
41 1 6
2
3
.1.2.16. Spectral data for 16ˇ-hydroxymethyl-16˛-[14-iodo-
,6,9,12-tetraoxatetradecanyl]-1,3,5(10)-estratrien-3,17ˇ-diol
−1
(12d). IR (NaCl, ꢀmax, cm ): 3370 (O–H), 1614 and 1506 (C C,
1
aromatic) and 1102 (C–O–C, aliphatic). H NMR (acetone-d6,
ı ppm): 7.95 (1H, s, phenol–OH), 7.08 (1H, d, J = 8.6 Hz, 1-CH),
6
4
.59 (1H, dd, J = 8.2 Hz and 2.7 Hz, 2-CH), 6.52 (1H, d, J = 2.4 Hz,
2.1.2.20. Spectral data for 16ˇ-hydroxymethyl-16˛-[5-(2-
pyridin-2-yl-ethylamino)-3-oxapentyl]-1,3,5(10)-estratrien-
3,17ˇ-diol dichloroplatinum(II) (1a). IR (KBr, ꢀmax, cm−1):
-CH), 4.24 (1H, d, J = 4.3 Hz, CHaH OH), 3.76–3.41 (18H, m, CHOH,
b
CHaH OH and 8 × OCH on PEG chain), 3.32 (2H, t, J = 6.6 Hz, CH I),
b
2
2
2
.75 (2H, m, 6-CH ), 2.23–1.10 (15H, several m, 2 × OH, 3 × CH
3430–3170 (O–H and N–H), 1611 and 1499 (C C, aromatic) and
2
1
3
1
and 5 × CH ), 0.87 (3H, s, 18-CH ). C NMR (acetone-d , ı ppm):
1109 (C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 9.16
2
3
6
6
1
1
6
4
56.0 (C-3), 138.4 (C-5), 132.1 (C-10), 127.0 (C-1), 116.0 (C-4),
13.7 (C-2), 90.2 (C-17), 72.6, 71.3, 71.2, 71.1, 70.9, 70.8, 69.1 and
7.5 (CH OH and 8 × OCH on PEG chain, 71.2 represents 2 C),
(1H, d, J = 5.9 Hz, a-CH), 8.04 (1H, t, J = 7.2 Hz, c-CH), 7.93 (1H, s,
phenol-OH), 7.54 (1H, d, J = 7.4 Hz, d-CH), 7.43 (1H, t, J = 6.6 Hz,
b-CH), 7.08 (1H, d, J = 8.2 Hz, 1-C), 6.59 (1H, dd, J = 8.1 Hz and
2.5 Hz, 2-CH), 6.53 (1H, d, J = 2.3 Hz, 4-CH), 6.12 (1H, s, NH) 4.21
2
2
8.3, 47.0, 45.8, 44.9, 40.0, 39.2, 39.0, 36.0, 30.3, 28.4, 27.2, 12.6
+
(
C-18), 4.6 (CH I). MS (m/e), C29H45I O : 633 (M+H) . Exact mass:
(1H, m, CHaH OH), 4.12–2.90 (12H, several m, CHOH, CHaH OH,
2
1
7
b
b
calculated for C29
H
I O7 + H = 633.2288; found = 633.2278.
2 × OCH on PEG chain and OCH CH NHCH CH -pyridine),
45 1
2
2
2
2
2
2
.75 (2H, m, 6-CH ), 2.69–1.16 (15H, several m, 2 × OH, 3 × CH
2
1
3
2
3
.1.2.17. Spectral data for 16ˇ-hydroxymethyl-16˛-[17-iodo-
,6,9,12,15-pentaoxaheptadecanyl]-1,3,5(10)-estratrien-3,17ˇ-
and 5 × CH ), 0.86 (3H, s, 18-CH ).
C NMR (acetone-d , ı
6
2
3
ppm): 159.9 (C-e), 155.3 (C-3), 153.7 (C-a), 139.4 (C-c), 137.8
−1
diol (12e). IR (NaCl, ꢀmax, cm ): 3385 (O–H), 1619 and 1505
(C-5), 131.5 (10-C), 126.4 (C-1), 124.8 (C-d), 123.9 (C-b), 115.3
1
(
C
C, aromatic) and 1102 (C–O–C, aliphatic). H NMR (acetone-d ,
(C-4), 113.0 (C-2), 89.3 (17-C), 69.0, 68.4 et 66.8 (CH OH and
6
2
ı ppm): 7.93 (1H, s, phenol–OH), 7.09 (1H, d, J = 8.6 Hz, 1-CH),
2 × OCH2 on PEG chain), 55.1, 47.6, 46.5, 46.0, 45.1, 44.2, 39.8,

J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
99
3
(
8.9, 38.5, 38.2, 34.7, 29.6, 27.7, 26.5, 11.9 (C-18). ESI + HRMS:
29.6, 27.7, 26.5, 11.9 (C-18). ESI + HRMS: (M+H)+ and (M+Na)+ cal-
M+H)+ and (M+Na)+ calculated for C30H43Cl N O Pt and
culated for C36H55Cl N O7Pt and C36H54Cl N NaO7Pt: 893.3021
2
2
4
2
2
2
2
C30H42Cl N NaO Pt: 761.2233 and 783.2052; found = 761.2220
and 915.2841; found = 893.3018 and 915.2837, respectively.
2
2
4
and 783.2047, respectively.
2.1.2.24. Spectral data for 16ˇ-hydroxymethyl-16˛-[17-(2-
2
.1.2.21. Spectral data for 16ˇ-hydroxymethyl-16˛-[8-(2-
pyridin-2-yl-ethylamino)-3,6,9,12,15-pentaoxaheptadecanyl]-
pyridin-2-yl-ethylamino)-3,6-dioxaoctyl]-1,3,5(10)-estratrien-
1,3,5(10)-estratrien-3,17ˇ-diol dichloroplatinum(II) (1e). IR
,17ˇ-diol dichloroplatinum(II) (1b). IR (KBr, ꢀmax, cm−1):
(KBr, ꢀmax, cm ): 3430–3170 (O–H and N–H), 1611 and 1498
−1
3
3
1
1
430–3170 (O–H and N–H), 1611 and 1502 (C C, aromatic) and
(C C, aromatic) and 1100 (C–O–C, aliphatic). H NMR (acetone-d ,
6
1
096 (C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 9.12 (1H,
ı ppm): 9.13 (1H, d, J = 6.1 Hz, a-CH), 8.04 (1H, t, J = 6.6 Hz, c-CH),
7.96 (1H, s, phenol–OH), 7.55 (1H, d, J = 7.8 Hz, d-CH), 7.42 (1H, t,
J = 6.6 Hz, b-CH), 7.09 (1H, d, J = 8.6 Hz, 1-C), 6.59 (1H, dd, J = 8.4 Hz
and 2.5 Hz, 2-CH), 6.53 (1H, d, J = 2.7 Hz, 4-CH), 5.95 (1H, s, NH),
6
d, J = 5.9 Hz, a-CH), 8.04 (1H, t, J = 7.6 Hz, c-CH), 7.96 (1H, s, phenol-
OH), 7.55 (1H, d, J = 7.8 Hz, d-CH), 7.42 (1H, t, J = 6.6 Hz, b-CH), 7.08
(
(
1H, d, J = 8.6 Hz, 1-C), 6.60 (1H, dd, J = 8.4 Hz and 2,5 Hz, 2-CH), 6.53
1H, d, J = 2.7 Hz, 4-CH), 6.12 (1H, s, NH), 4.24 (1H, m, CHaH OH),
4.20 (1H, d, J = 3.9 Hz, CHaH OH), 4.10–2.86 (28H, several m, CHOH,
b
b
3
.10–2.81 (16H, several m, CHOH, CHaH OH, 4 × OCH on PEG
CHaH OH, 10 × OCH on PEG chain and OCH CH NHCH CH -
b
2
b
2
2
2
2
2
chain and OCH CH NHCH CH -pyridine), 2.75 (2H, m, 6-CH ),
2
s, 18-CH ). C NMR (acetone-d , ı ppm): 159.9 (C-e), 155.3 (C-3),
pyridine), 2.75 (2H, m, 6-CH ), 2.70–1.06 (15H, several m, 2 × OH,
2
2
2
2
2
2
1
3
.57–1.21 (15H, several m, 2 × OH, 3 × CH and 5 × CH ), 0.89 (3H,
3 × CH and 5 × CH ), 0.87 (3H, s, 18-CH ). C NMR (acetone-d , ı
2
2
3
6
13
ppm): 160.0 (C-e), 155.3 (C-3), 153.7 (C-a), 139.4 (C-c), 137.8 (C-5),
3
6
1
53.7 (C-a), 139.4 (C-c), 137.8 (C-5), 131.5 (C-10), 126.4 (C-1), 124.8
131.5 (C-10), 126.4 (C-1), 124.8 (C-d), 123.8 (C-b), 115.3 (C-4),
(
C-d), 123.8 (C-b), 115.3 (C-4), 113.0 (C-2), 89.6 (C-17), 70.0, 68.8,
112.9 (C-2), 89.5 (C-17), 70.5, 70.3, 70.2, 69.2, 68.5 and 66.8 (CH OH
2
6
2
2
8.7 and 66.7 (CH OH and 4 × OCH on PEG chain, 70.0 represents
and 10 × OCH2 on PEG chain, 70.5, 70.3 and 70.2 represents 8C),
54.8, 47.6, 46.4, 46.3, 45.1, 44.2, 39.6, 39.3, 38.5, 38.3, 35.3, 29.6,
27.7, 26.5, 11.9 (C-18). ESI + HRMS: (M+H)+ and (M+Na)+ calcu-
lated for C38H59Cl N O Pt and C38H58Cl N NaO Pt: 937.3284 and
2
2
C), 55.7, 47.6, 46.6, 46.4, 45.1, 44.2, 39.7, 39.4, 38.5, 38.4, 35.6,
9.6, 27.7, 26.6, 12.1 (C-18). ESI + HRMS: (M+H)+ and (M+Na)+ cal-
culated for C32H47Cl N O5Pt and C32H46Cl N NaO5Pt:804.25043
2
2
2
2
2
2
8
2
2
8
and 826.23237; found = 804.25045 and 826.23244, respectively.
959.3104; found = 937.3281 and 959.3099, respectively.
2
.1.2.22. Spectral data for 16ˇ-hydroxymethyl-16˛-[11-(2-
2.2. Biology
pyridin-2-yl-ethylamino)-3,6,9-trioxaundecanyl]-1,3,5(10)-
estratrien-3,17ˇ-diol dichloroplatinum(II) (1c). IR (KBr, ꢀmax,
2.2.1. In vitro cytotoxic activity
−
1
cm ): 3430–3170 (O–H and N–H), 1614 and 1499 (C C, aromatic)
The cytotoxicity of the 17␤-estradiol-PEG-platinum(II) com-
1
+
and 1100 (C–O–C, aliphatic). H NMR (acetone-d , ı ppm): 9.13
plexes (1a–e) was evaluated on MCF-7 (ER ) and MDA-MD-231
6
−
(
1H, d, J = 5.5 Hz, a-CH), 8.03 (1H, t, J = 7.6 Hz, c-CH), 7.92 (1H, s,
(ER ) breast cancer cell lines. MTT (3-(4,5-dimethylthiazol-2-yl)-
phenol-OH), 7.54 (1H, d, J = 7.0 Hz, d-CH), 7.41 (1H, t, J = 6.8 Hz,
b-CH), 7.09 (1H, d, J = 8.2 Hz, 1-C), 6.58 (1H, dd, J = 8.2 Hz and
phenyl-tetrazolium bromide) assay, a standard colorimetric test,
was used for measuring cellular proliferation [27]. Tumor cell lines
2
.7 Hz, 2-CH), 6.53 (1H, d, J = 2.7 Hz, 4-CH), 6.05 (1H, s, NH), 4.17
were added into 96-well tissue culture plates in culture medium
◦
(
1H, m, CHaH OH), 4.08–2.86 (20H, several m, CHOH, CHaH OH,
and incubated at 37 C in a 5% CO atmosphere. Dilutions were done
b
b
2
6
× OCH on PEG chain and OCH CH NHCH CH -pyridine), 2.75
using chromophore:ethanol (1:1) solution. Cells were incubated
with or without drugs for 72 h. Culture plates were processed using
MTT for 3.5 h. Afterwards SDS solubilisation solution (HCl 0.010 M,
sodium dodecyl solution 10%) was added. The absorbance was read
using a scanning multiwell spectrophotometer (FLUOStar OPTIMA)
at 565 nm. All measurements were carried out in triplicate. The
results were compared with those of a control reference plate fixed
on the treatment day, and the growth inhibition percentage was
calculated for each drug contact period.
2
2
2
2
2
(
2H, m, 6-CH ), 2.66–1.11 (15H, several m, 2 × OH, 3 × CH and
2
1
3
5
1
1
1
× CH ), 0.87 (3H, s, 18-CH ). C NMR (acetone-d , ı ppm):
2
3
6
60.0 (C-e), 155.3 (C-3), 153.7 (C-a), 139.4 (C-c), 137.8 (C-5),
31.5 (C-10), 126.4 (C-1), 124.8 (C-d), 123.8 (C-b), 115.3 (C-4),
12.9 (C -2), 89.5 (C-17), 70.4, 70.3, 70.2, 69.0, 68.5 and 66.7
(
4
2
CH OH and 6 × OCH on PEG chain, 70.2 represents 2C), 55.4,
2
2
7.6, 46.4, 46.3, 45.1, 44.2, 39.7, 39.4, 38.5, 38.3, 35.3, 29.6, 27.7,
6.5, 12.0 (C-18). ESI + HRMS: (M+H) and (M+Na)+ calculated
+
for C34H51Cl N O Pt and C34H50Cl N NaO Pt: 849.2758 and
2
2
6
2
2
6
8
71.2578; found = 849.2747 and 871.2574, respectively.
2.2.2. Estrogen receptor binding affinity
The estrogen receptor alpha (ER˛) affinity assay was per-
2
.1.2.23. Spectral data for 16ˇ-hydroxymethyl-16˛-[14-(2-
formed using recombinant hER˛ (Calbiochem/EMD BioSciences,
TM
pyridin-2-yl-ethylamino)-3,6,9,12-tetraoxatetradecanyl]-
,3,5(10)-estratrien-3,17ˇ-diol dichloroplatinum(II) (1d). IR
Darmstadt, Germany) and the HitHunter
Enzyme Fragment
1
Complementary (EFC) Estrogen Receptor Assay kit (Discoverex Cor-
−1
(KBr, ꢀmax, cm ): 3430–3170 (O–H and N–H), 1615 and 1502
poration, Fremont, CA) according to manufacturer’s protocol [30].
1
TM
(
C
C, aromatic) and 1106 (C–O–C, aliphatic). H NMR (acetone-d ,
HitHunter
EFC technology is based on a genetically engineered
6
ı ppm): 9.13 (1H, d, J = 5.8 Hz, a-CH), 8.03 (1H, t, J = 7.8 Hz, c-CH),
.92 (1H, s, phenol-OH), 7.54 (1H, d, J = 6.6 Hz, d-CH), 7.42 (1H, t,
J = 6.8 Hz, b-CH), 7.09 (1H, d, J = 8.6 Hz, 1-C), 6.59 (1H, dd, J = 8.6 Hz
and 2.7 Hz, 2-CH), 6.52 (1H, d, J = 2.7 Hz, 4-CH), 5.95 (1H, s, NH),
ˇ-galactosidase enzyme that consists of two fragments termed
enzyme acceptor (EA) and enzyme donor (ED). Briefly, different
concentrations of estrogen analog (1b) were added to wells con-
taining ES (Estrogen Steroid) Receptor + ED in a 96-well black plate.
Incubation provided competition for the estrogen receptor binding
against labelled Enzyme Donor-Estrogen Steroid hormone conju-
gate (ED-ES conjugate), a small peptide fragment of ˇ-galactosidase
(ˇ-gal). Then, EA, an inactive ˇ-gal protein fragment, and a Fluo-
rescent 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 (FLU-
OStar OPTIMA). The excitation wave is 530 nm and luminosity is
detected at 620 nm. The amount of free ED conjugate in the assay
7
4
.18 (1H, d, J = 4.3 Hz, CHaH OH), 4.15–2.86 (24H, several m, CHOH,
b
CHaH OH, 8 × OCH on PEG chain and OCH CH NHCH CH -
b
2
2
2
2
2
pyridine), 2.75 (2H, m, 6-CH ), 2.70-1.07 (15H, several m, 2 × OH,
2
1
3
3
× CH and 5 × CH ), 0.87 (3H, s, 18-CH ). C NMR (acetone-d ,
2
3
6
ı ppm): 160.0 (C-e), 155.3 (C-3), 153.7 (C-a), 139.4 (C-c), 137.8
(
(
C-5), 131.5 (C-10), 126.4 (C-1), 124.8 (C-d), 123.8 (C-b), 115.3
C-4), 112.9 (C-2), 89.5 (C-17), 70.5, 70.4, 70.3, 70.2, 69.1, 68.5 and
6
2
6.8 (CH OH and 8 × OCH on PEG chain, 70.4 and 70.2 represents
2
2
C), 55.3, 47.6, 46.4, 46.3, 45.1, 44.2, 39.7, 39.4, 38.5, 38.3, 35.3,

1
00
J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
Table 1
Calculated Log P of the aminopyridine ligands 4a–e, of the final E2-Pt(II) hybrids
Table 2
Inhibitory concentration of cisplatin and 1a–e on both ER+ and ER breast cancer
a
−
1a–e and of 17␤-estradiol.
cell lines.
Compoundsa
c Log Pb
Compoundsa
c Log Pb
+
Compounds
MCF-7 (ER )
IC50, ␮M
MDA-MB-231
(ER ) IC50, ␮M
Chain length n
a
−
a
4
4
4
4
4
1
a
b
c
d
3.98
3.81
3.64
3.48
3.32
4.00c
1a
1b
1c
1d
1e
4.40
4.24
4.07
3.91
3.74
Cisplatin
18.97 ± 0.43
68.50 ± 3.01
33.44 ± 1.79
38.04 ± 2.02
35.64 ± 0.84
20.61 ± 0.94
17.33 ± 2.28
38.67 ± 4.15
17.86 ± 1.33
27.29 ± 4.61
17.52 ± 1.47
13.90 ± 1.87
–
1
2
3
4
5
1a
1b
1c
1d
e
7␤-Estradiol
1
e
a
b
c
Chain length: a (n = 1), b (n = 2), c (n = 3), d (n = 4) and e (n = 5).
Calculated Log P as obtained with CaChe work system pro, 2006.
Reported literature value for 17␤-estradiol is 3.90 [33]
OH
a
Inhibitory concentration (IC50, ␮M) as obtained by the MTT assay. Experiments
were performed in duplicates and the results represent the mean ± SEM of three
independent experiments. The cells were incubated for a period of 72 h.
OH
NH
N
O
n
chloroethyl ether (5) and from PEG of various lengths (6b–e) (Fig. 2).
Thus, treatment of 2-chloroethyl ether with sodium iodide in
refluxing acetone for 3 days gave 2-iodoethyl ether (7a) in 81% yield.
Derivatives 7b–e were obtained in a two-step reaction sequence.
The PEG chains were treated with mesyl chloride and triethylamine
HO
4
.
◦
is proportional to the concentration of estrogen analogs bound to
the estrogen receptor [30]. A standard curve of 17ˇ-estradiol was
run in parallel. All assays were done in triplicate.
in diethyl ether at 0 C to give the bis-mesylate intermediates 8b–e.
The bis-mesylates 8b–e were subsequently treated, without purifi-
cation, with excess sodium iodide in refluxing acetone to give the
diiodo-PEG chains 7b–e in 79% average yield.
As shown in Fig. 3, estrone (9) was initially protected as a
tetrahydropyranyl ether (R = THP) under standard reaction con-
ditions. Accordingly, estrone was treated with dihydropyran in
dichloromethane in the presence of pyridinium p-toluenesulfonate
2
.3. Molecular modeling
All calculations (molecular mechanics, MM2) and modeling
were performed on Scigress Explorer Ultra Version 7.7.0.47, Fujitsu,
USA [31]. The active site of the estrogen receptor (PDB 1ERE) was
available on the Protein Data Bank web site [32].
[
34]. The yield of the protection reaction is 99%. The intermediate
was further transformed into the ␤-keto-ester 10 upon treatment
with dimethyl carbonate in the presence of a mixture of KH in dry
tetrahydrofuran [35,36]. Derivative 10 was obtained with 90% yield.
Treatment of derivative 10 with a suitable diiodo-PEG chains 7a–e
and with cesium carbonate in tetrahydrofuran gave compounds
3
. Results and discussion
3.1. Synthesis of 17ˇ-estradiol-platinum(II) complexes (7a–e)
11a–e in 63% yield. The iodo-PEG chain were added to the less hin-
dered ␣ face of the molecule as shown by the presence of a single
As shown in Table 1, the calculated Log P (c Log P) of the
aminoethylpyridine ligands 4a–e is relatively constant throughout
the series varying from 3.98 for 4a to 3.32 for 4e (Table 1). Estradiol
peak for the 18-CH at ı 0.90 in the 1H NMR spectrum and at ı
3
1
4.2 ppm in the 13C NMR spectrum. Reduction of the ␤-ketoester
◦
moiety with lithium borohydride in dry ether at 0 C followed by
itself has a c Log P of 4.00. The final E -Pt(II) hybrids 1a–e should
2
the cleavage of the tetrahydropyranyl ether gave the triol 12a–e
also possess relatively constant solubility. The cLogP of the final
platinum(II) complexes vary from 4.40 for 1a to 3.74 for 1e. Thus,
a slight increase of solubility is predicted for derivatives 1 as the
chain is lengthening. Consequently, we speculated that the differ-
ence in cytotoxicity, if any, should only reflect the influence of the
length of the PEG tether chain on the final hybrid molecules.
The synthesis involves only five chemical steps starting from
estrone as the steroid template. The 17␤-estradiol-Pt(II) com-
plexes 1a–e were obtained efficiently in high yield (22% overall)
using an efficient reaction sequence. First, the preparation of the
PEG chains was accomplished from commercially available 2-
[
21,34]. It was obtained in 70% overall yield as a single 17␤-hydroxy
isomer as shown by a sole signal for the 18-CH at ı 0.88 in the
3
1
13
H NMR spectrum and at ı 12.5 ppm in the C NMR spectrum.
The stereochemistry of the 17␤-hydroxy function was confirmed
by comparison with 13C NMR spectral data of known 17␤- and
1
7␣-estradiol derivatives [37].
The final 17␤-estradiol-linked Pt(II) complexes 1a-e were
obtained in a two-step chemical sequence [21]. Initially, the triol
ꢀ
1
2a–e was treated with excess 2-(2 -aminoethyl)pyridine to give
derivatives 4a–e for a yield of 80–100%. It is noteworthy that a short
reaction period is necessary for this reaction in order to avoid the
decomposition of the PEG tether chain. Thus, this reaction is per-
formed in only 4 h instead of about 20 h when ␣,␻-dibromoalkane
chain are used [21,23,25]. Then, the triol-aminopyridine interme-
diates 4a–e were treated with potassium tetrachloroplatinate in a
mixture of dimethylformamide and water to give the correspond-
ing 17␤-estradiol-PEG-linked Pt(II) complexes 1a–e with n = 1, 2, 3,
4 and 5. The yield of this two-step sequence was 55%. As a result,
the new cytotoxic molecules possess a PEG tether chain varying
from 5 to 17 atoms long. All new compounds synthesized were
characterized by IR, NMR spectroscopy and mass spectrometry.
NaI, acetone
reflux, (81%)
Cl
I
O
5
O
Cl
I
7a
OH MsCl, Et N
3
OMs
O
O
HO
n
o
n
Et2O, 0 C, N2
MsO
6
b-e
8
n
b-e
NaI, acetone
reflux, (79%)
I
O
I
3.2. In vitro cytotoxic activity
7
b-e
As shown by the MTT assays, the new Pt(II) complexes present
no specific toxicity towards ER breast cancer cells (Table 2). How-
+
Fig. 2. Preparation of bis-diodo-PEG chains 7a–e.

J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
101
O
O
O
OCH3
a
H
THPO
HO
9
10
b
O
O
OH
OCH3
OH
O
c
O
n
I
n
I
THPO
HO
1
1a-e
12a-e
d
OH
OH
H
N
n = 1(a), 2(b), 3(c), 4(d) or 5(e)
O
n
Cl Pt
Cl
HO
N
1
a-e
◦
Fig. 3. Synthesis of 17␤-estradiol-linked platinum(II) complexes. Reagents. (a) (1) DHP, PPTs, CH2Cl2, 22 C, 24 h; (2) KH, dimethyl carbonate, THF, reflux, 3 h, 90%; (b) 7a–e,
◦
◦
◦
ꢀ
Cs2CO3, THF, reflux, 4 h, 63%; (c) (1) LiBH4, Et2O, 0 C, 1 h and 22 C, 11 h; (2) PPTs, EtOH, 22 C, 4 h, 70%; (d) (1) 2-(2 -aminoethyl) pyridine, CH3OH, reflux, 4 h; (2) K2PtCl4,
DMF: H2O (2:1), 22 C, 2 days, 55%.
◦
ever, it is important to indicate that the desired selectivity towards
ER cancer cells might be expressed more clearly (and possibly
3.3. Estrogen receptor binding affinity
+
The estrogen receptor binding studies showed good affinity for
derivative 1b to the estrogen receptor alpha. The reference deriva-
tive, i.e. cisplatin presents, as expected, no affinity for the ER␣ (see
Table 3). The estrogen-PEG-Pt(II) hybrid molecule 1b has an EC50
of 4.25 nM compared to 0.66 nM for 17␤-estradiol, the natural lig-
and. This type of molecule presents lower affinity than the previous
classes of derivatives 2 and 3 reported [21–24]. As indicated in
Table 3, derivatives 2 (p = 6) and 3 (p = 6) have an EC50 of 1.79 and
only) in vivo as it was previously demonstrated for similar types
of derivatives [38,39].
ꢀ
The estrogen-PEG-Pt(II) hybrid molecules carry
a
2-(2 -
aminoethyl)pyridine ligand which was found to be the best
ligand for biological activity for hybrids of formulae 2 and 3.
However, the hybrids 1a–e were generally less cytotoxic than
cisplatin itself. The length of the side chain seems to be opti-
mal at n = 5 where we observed an IC50 of 20.6 and 13.9 ␮M,
respectively for the MCF-7 and MDA-MB-231 cancer cells. The
derivative with shorter side chains (n = 1) is the least active
estradiol-PEG-hybrid of the series. Moreover, it is observed that
the cytocidal activity is generally more important on the hormone-
independent breast cancer cells. If selectivity can be achieved
in animal models, derivative 1e might become an alternative of
choice for site-specific treatment of hormone-dependent breast
cancer.
1
.52 respectively.
3.4. Comparative molecular modeling studies
As mentioned earlier, the new estrogen-PEG-Pt(II) hybrid
molecules 1a–e are relatively less potent than the previous related
platinum complexes linked with an alkyl chain (see structures
2 and 3). Of note, the best 17␤-estradiol-PEG-Pt(II) hybrid (1e)
Table 3
Comparison of different families of 17␤-estradiol-Pt(II) complexes. Inhibitory concentration of cisplatin 1b, 2 (p = 6) and 3 (p = 6) on both ER+ and ER breast cancer cell lines
a
−
and binding affinity on ER␣.
+
Compounds
Cisplatin
Value of
n or p
Total number
of atoms
MCF-7 (ER )
IC50, ␮M
Ratio
CisPt/drug
MDA-MB-231
(ER ) IC50, ␮M
Ratio
CisPt/drug
ER␣ affinity
EC50, nM
a
−
a
–
–
8
8
8
18.97 ± 0.43
33.44 ± 1.79
5.80 ± 3.00
2.18 ± 0.11
–
17.33 ± 2.28
17.86 ± 1.33
8.60 ± 0.1
–
–
1
2
3
b This study
[21,22]
[23,24]
n = 2
p = 6
p = 6
0.59
3.27
8.70
0.97
2.01
8.02
4.25
1.79
1.52
2.16 ± 0.16
a
Inhibitory concentration (IC50, ␮M) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent the mean ± SEM of three
independent experiments. The cells were incubated for a period of 72 h.

1
02
J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
Fig. 4. Superimposition of the energy minimized structures of compounds 1b (n = 2)
blue), compound 2 (p = 6) (pink), and compound 3 (p = 6) (brown), platinum and
(
chlorine atoms are shown as white and green colors, respectively. (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of the article.)
Fig. 5. Orientation of compounds 1b (n = 2), 2 (p = 6), and 3 (p = 6) in the ER alpha
binding pocket: showing the positional differences in the reactive site (PtCl2) portion
and similarity of steroid portion.
was already analyzed by molecular modeling in a previous com-
munication [20]. Thus, to understand the differences in potency
between these three series of platinum complexes, we performed
molecular modeling and docking experiments using the opti-
mized structures of these classes of compounds (compounds 1b,
2
and 3) and the crystal structure of estrogen receptor alpha
(
(
ER␣).
For this purpose, three specific derivatives were selected (1b
n = 2), 2 (p = 6) and 3 (p = 6)) among the families and their struc-
tures are shown in Fig. 1. It is noteworthy that the overall length
of the linking arm between the estrogenic portion and the plat-
inum(II) moiety is, in each case, 8 atoms (Table 3). Their respective
cytocidal activities on hormone-dependent (MCF-7) and hormone-
independent (MDA-MB-231) breast cancer cells are presented in
Table 3. Table 3 also gives the CisPt/drug ratios as well as the
binding affinity of the compounds. It is quite clear that there is
a substantial difference of activities among the three estrogen-
platinum(II) derivatives. In fact, it becomes obvious that in both
types of cells activity increases with lipophilicity of the deriva-
tives, and the cytocidal activity follows this order; 1b < 2 < 3. The
estradiol-platinum(II) derivative 3 (p = 6) is about 8 times more
cytotoxic than cisplatin. Derivative 2 (p = 6) is about 2 to 3.27 times
more active than cisplatin and derivative 1b (n = 2) is slightly less
active than cisplatin. On MCF-7 cells, derivative 3 (p = 6) is about
Fig. 6. Docking view of compounds 1b (n = 2), 2 (p = 6), and 3 (p = 6) to the active site
of ER alpha: ER alpha binding pocket is shown with lines only (blue lines) and the
folding of rest of the ER alpha molecule is shown as solid ribbon. (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of the article.)
compounds to the ER alpha binding pocket. In this diagram, it is
clear that the binding of the steroid portion is similar while the
orientation and the position of the reactive sites were significantly
different. Fig. 6 shows the positions of the reactive sites of the three
compounds. In compound 3 (p = 6), the Cl groups of the reactive
site is exposed outside of the ER alpha where water molecules are
readily available for hydrolysis and, the Pt moiety would be freely
available for binding to the target molecule. These reasons could
explain the greater potency of compound 3 compared to other two
derivatives. Also, this diagram shows the folding of ER alpha upon
binding of these molecules.
1
6 times more potent than the corresponding estrogen-PEG-Pt(II)
hybrid 1b (n = 2). The potency of the various estradiol-platinum(II)
hybrids is also in relation with the binding affinity with compound
3
(p = 6) having the best affinity for the ER␣ (Table 3).
Fig. 4, the superimposition view of compounds 1b (n = 2), 2
(
p = 6), and 3 (p = 6), shows that the orientation of compound 3,
bearing a beta linking arm, is significantly different from the other
two derivatives (compounds 1b and 2) while compounds 1b and
2
are rather similar with their alpha linking arm. However, the
positions of the portion of the reactive site (PtCl ) in all three com-
2
pounds are different while the conformation of the steroid portion
of all three molecules remains the same. These structural differ-
ences could be one of the reasons for the variations in potency
among these three compounds.
Due to the structural disparities among these three compounds,
their respective binding affinities to the ER alpha are naturally dis-
tinct and, to understand this binding difference, we performed the
docking experiments. Fig. 5 shows the docking view of these three
4. Conclusion
This manuscript presents a new series of cytotoxic 17␤-
estradiol-PEG linked Pt(II) hybrid molecules (1a-e). They are readily
available from estrone in only 5 chemical steps with excellent
yields. The biological activity is lower than that of the hybrids of
first and second family, see derivatives 2 and 3. The most promis-

J. Provencher-Mandeville et al. / Steroids 76 (2011) 94–103
103
ing compound of the series is derivative 1e which is equipotent to
cisplatin itself. Despite a relatively low cytocidal activity, the novel
hybrids could have interesting in vivo biological potential due to
their enhanced solubility as compared to the first two prototype
hybrids (2 and 3).
Three 17␤-estradiol-platinum(II) complexes were selected for
comparative analysis in terms of cytocidal activities and molecular
modeling. Molecular modeling studies show that the orientation of
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Acknowledgments
The authors wish to thank the Fonds de Recherche sur la Nature
et les Technologies du Québec (FQRNT) and the Canadian Institutes
for Health Research (CIHR) for useful financial support. The NSERC
student fellowship for Josée Provencher-Mandeville is gratefully
acknowledged.
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