Synthesis of 17β-estradiol-linked platinum(II) complexes and their cytocidal activity on estrogen-dependent and -independent breast tumor cells

Article abstract of DOI:10.1016/j.bioorg.2004.06.009

The synthesis of two new highly potent 17β-estradiol-linked platinum(II) complexes is described. The new molecules are linked at position 16 of the steroid nucleus with an alkyl chain. They are made from estrone in nine chemical steps with an overall yiel

Full text of DOI:10.1016/j.bioorg.2004.06.009

Bioorganic Chemistry 33 (2005) 1–15
www.elsevier.com/locate/bioorg
Synthesis of 17b-estradiol-linked platinum(II)
complexes and their cytocidal activity
on estrogen-dependent and -independent
breast tumor cells
a
Valerie Perron , Daniel Rabouin , Eric Asselin ,
a
a
´
´
Sophie Parent , Rene C.-Gaudreault , Gervais Berube
a
b
a,
*
´
´
´
a
De´partement de Chimie-Biologie, Universite´ du Que´bec a` Trois-Rivie`res, C.P. 500,
`
Trois-Rivieres, Que., Canada, G9A 5H7
b
´ ´
Unite des Biotechnologies et de Bioingenierie, Centre de Recherche, C.H.U.Q.,
´
ˆ
Hopital Saint-Franc¸ois d ÕAssise, 10 Rue de l’Espinay, Quebec, Que., Canada G1L 3L5
Received 5 May 2004
Available online 6 August 2004
Abstract
The synthesis of two new highly potent 17b-estradiol-linked platinum(II) complexes is
described. The new molecules are linked at position 16 of the steroid nucleus with an alkyl
chain. They are made from estrone in nine chemical steps with an overall yield exceeding
10%. The biological activity of these compounds was evaluated in vitro on estrogen dependent
and independent (ER+ and ERꢀ) human breast tumor cell lines: MCF-7 and MDA-MB-231.
The novel compounds prove to be highly cytotoxic against breast cancer cell lines. The most
cytotoxic derivative shows high affinity for the estrogen receptor alpha.
ꢀ 2004 Elsevier Inc. All rights reserved.
Keywords: Antitumor agents; Breast cancer; Estradiol; Platinum(II) complexes; Hybrid estradiol-Pt(II)
complexes
*
Corresponding author. Fax: +1-819-376-5084.
E-mail address: Gervais_Berube@uqtr.ca (G. Berube).
´
´
0045-2068/$ - see front matter ꢀ 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2004.06.009

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V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
1. Introduction
Breast cancer is a major health problem among women in the world [1]. The suc-
cessful treatment of this disease is limited by the fact that essentially all breast can-
cers become resistant to chemotherapy and endocrine therapy [2,3]. Moreover, it is
also known that 40% of patients with estrogen receptor positive (ER+) tumors do not
respond to endocrine manipulation [4]. Recent evidence suggests that the progression
to hormone resistance in some breast tumors is due to a variety of cellular changes
mediated via the estrogen receptor-signalling pathway [5]. There is a need to develop
new chemotherapeutic agents with increased efficacy and decreased systemic toxicity.
Our goal is to design, based on our knowledge of triphenylethylene platinum(II)
complexes (a non-steroidal cytotoxic estrogen) and on literature precedents, new
highly potent 17b-estradiol-linked platinum(II) complexes. The estrogenic portion
of the molecule would be used to direct the cytotoxic Pt(II) moiety toward the target
cells thereby increasing specificity and reducing systemic toxicity.
The mechanism of growth regulation of breast cancer is a very complex process
[6]. Nevertheless, it is well established that, in hormone-dependent breast cancer cell,
there is an interaction of the native ligand 17b-estradiol (E2) with the estrogen recep-
tor (ER). This event leads to an array of cellular transformations, which culminate to
cell growth. Agents that could interfere with this process could eventually give addi-
tional tools to treat breast cancer. More precisely, the synthesis of new antiestrogens
bearing a cytotoxic moiety could be of prime interest because of their potential un-
ique interaction with the ER and with DNA. This manuscript describes the synthesis
of a new family of highly potent 17b-estradiol-linked Pt(II) complexes (see general
structure 1). It also reports the in vitro cytotoxic activity of these compounds on
two neoplastic human breast cancer cell lines: MCF-7 and MDA-MB-231 (ER+
and ERꢀ). For further evaluation, the novel Pt(II) complexes were tested on skin
(B16-F10) and on intestine (HT-29) cancers.
cis-Diamminedichloroplatinum(II) (2, cisplatin) is one of the most successful che-
motherapeutic agents used in the treatment of several human cancers. It is generally
accepted that the biological effects of platinum(II) complexes are due to the forma-
tion of DNA adducts capable of blocking replication and/or transcription [7]. Unfor-
tunately, these drugs present deleterious side effects, particularly nephrotoxicity [7].
Several new antiestrogen-linked Pt(II) complexes have been described recently in the
literature [8]. Interestingly, depending on the nature of the antiestrogenic moiety the
in vitro and in vivo biological activity do not necessarily coincide when tested on

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
3
ER+ cancers. Much work is needed to better understand the biological behaviour of
this type of cytotoxic agent. We have been investigating for some time the synthesis
of new triphenylethylene Pt(II) complexes designed for the treatment of breast can-
cer. We have shown that the presence of two or three hydroxyls groups on the tri-
phenylethylene moiety of the molecule (see compound 3) is essential for binding
affinity to the ER [9]. Also, it was observed that the length of the side chain bearing
the cytotoxic Pt(II) portion should be 11 or 12 carbon atoms long for optimal bio-
logical activity. More recently, we have incorporated several diamine ligands on a
common triphenylethylene backbone in order to improve the cytotoxicity of this
type of molecules. We have discovered that the Pt(II) complex 3 derived from 2-
(20-aminoethyl)pyridine possesses excellent in vitro cytotoxic activity [10]. Com-
pound 3 (IC50 = 5lM) is as active as cisplatin on MCF-7 breast cancer cells. Based
on these results, we decided to incorporate the same pyridine derivatives on the 17b-
estradiol nucleus (see compound 1).
It is known that in 17b-estradiol, the 3-hydroxy group is more important for bind-
ing than the 17b-OH. While 17b-estradiol possesses an ER relative binding affinity
(RBA) of 100%, 17-deoxyestradiol has an RBA of 14%, whereas 3-deoxyestradiol
has a RBA of 1.7% [11]. The relative importance of the 3- and 17-hydroxy functions
can also be illustrated via a close examination of the RBA values of two known
estradiol-linked Pt(II) complexes 4 and 5 (see structures below) [12]. The Pt(II) deriv-
ative 4 with a free phenol function possesses a RBA value of 6% while the Pt(II)
derivative 5 with the free 17b-OH function possesses a RBA value of less than
1%. From these results, it is obvious that the presence of both hydroxyl functions
on the estradiol nucleus is essential for high affinity to the ER. Therefore, the pro-
posed Pt(II) complexes linked at C-16 would necessarily be better ligands for the ER.

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2. Materials and methods
Anhydrous reactions were performed under an inert atmosphere, the setup assem-
bled and cooled under dry nitrogen. Unless otherwise noted, starting material, reactant
and solvents were obtained commercially and were used as such or purified, and dried
by standard means [13]. Organic solutions were dried over magnesium sulfate
(MgSO4), evaporated on a rotatory evaporator and under reduced pressure. All reac-
tions were monitored by UV fluorescence or staining with iodine. Commercial TLC
˚
plates were Sigma T 6145 (polyester silica gel 60A, 0.25mm). Preparative TLC was per-
˚
formed on 1mm silica gel 60A, 20 · 20 plates (Whatman, 4861 840). Flash column
chromatography was performed according to the method of Still et al. [14] on Merck
grade 60 silica gel, 230–400 mesh. All solvents used in chromatography had been
distilled.
The infrared spectra were taken on a Nicolet Impact 420 FT-IR. Mass spectral
assays were obtained using a VG Micromass 7070 HS instrument using ionization
energy of 70eV (University of Sherbrooke).
Nuclear magnetic resonance (NMR) spectra were recorded either on a Bruker
AMX-II-500 equipped with a reversed or QNP probe (Pharmacor) or (when
indicated) on a Varian 200MHz NMR apparatus. Samples were dissolved in deu-
terochloroform (CDCl3), deuteroacetone (acetone-d6) or deuterodimethylsulfoxide
(DMSO-d6) for data acquisition using tetramethylsilane or chloroform as internal
1
standard (TMS, d 0.0ppm for H NMR and CDCl3 d 77.0ppm for 13C NMR).
Chemical shifts (d) 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 quar-
tet, m for multiplet, #m for several multiplets, and br s for broad singlet.

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
5
3. Synthesis of 17b-estradiol-linked Pt(II) complexes
3.1. Synthesis of 3-benzyloxy-1,3,5(10)-estratrien-17-one (7)
To a solution of estrone 6 (1.00g, 3.70mmol) in dichloromethane (10mL), was
added benzylbromide (0.53mL, 4.44mmol), tetrabutylammonium hydrogen sulfate
(100mg), and a solution of sodium hydroxide (10% w/v, 5mL). The reaction mixture
was stirred vigorously at reflux for 24h. Then, the mixture was diluted with diethyl
ether (30mL) and water (30mL) and washed with water (4·75mL). The organic
phase was dried with magnesium sulfate, filtered, and evaporated to yield a solid
compound. The residue was triturated with hexanes to give a white solid in 99% yield
which was homogeneous by TLC. It was used without further purification at the next
step. M.P.: 128–129ꢁC. IR (KBr, mmax, cmꢀ1): 1731 (C@O), 1614 (C@C), 1230, and
1
1008 (C@O). H NMR (CDCl3, dppm): 7.41 (2H, d, J = 7.6Hz, a-CH), 7.36 (2H, t,
J = 7.5Hz, b-CH), 7.29 (1H, t, J = 7.2Hz, c-CH), 7.18 (1H, d, J = 8.6Hz, 1-CH),
6.78 (1H, dd, J = 2.5Hz and J = 8.5 Hz, 2-CH), 6.71 (1H, d, J = 2.1Hz, 4-CH),
5.01 (2H, s, CH2Ph), 2.88 (2H, m, 6-CH2), 2.50–1.39 (13H, #m, 3· CH and 5·
CH2), 0.89 (3H, s, 18-CH3).13C NMR (CDCl3, d ppm): 220.5 (17-C), 156.8 (3-C),
137.7 (CCH2O), 137.2 (5-C), 132.2 (10-C), 128.4 (b-C), 127.7 (c-C), 127.3 (a-C),
126.2 (1-C), 114.8 (4-C), 112.3 (2-C), 69.8 (CH2Ph), 50.3, 47.9, 43.9, 38.3, 35.8,
31.5, 29.6, 26.5, 25.8, 21.5, 13.8 (C-18). MS (m/e): 360 (M+), 269 (M+AC7H7). Exact
mass. Calculated for C25H28O2 = 360.2089; found = 360.2095.
NB: a-CH, b-CH, and c-CH are ortho, meta, and para protons on the benzyl pro-
tecting group. Similarly, a-C, b-C, and c-C are the corresponding carbons on the
benzyl group.
3.2. Synthesis of 3-benzyloxy-16a,b-(methoxycarbonyl)-1,3,5(10)-
estratrien-17-one (8)
A solution of 3-benzyloxy-1,3,5(10)-estratrien-17-one (7) (4.00g, 11.1mmol) in
dry THF (5mL) was added over a period of 30min to a solution of dimethylcarbon-
ate (2.34mL, 27.8mmol) and potassium hydride (1.42g, 34.7mmol) in dry THF
(40mL). Then, the mixture was heated to reflux for a period of 3h. Most of the sol-
vent was then evaporated and the residue was diluted with ethyl acetate (100mL) and
treated with a saturated ammonium chloride solution (50mL). The organic phase
was washed with water (6 · 40mL), dried and evaporated to give a yellowish solid.
Trituration of the residue with a mixture of acetone: hexanes (1:1) yielded the title
compound in 90% yield as a white solid which was homogeneous by TLC. It was
used without further purification at the next step. M.P.: 152–154ꢁC. IR (NaCl, mmax
,
cmꢀ1): 1747 (C@O, ester), 1721 (C@O, ketone), 1609 (C@C), 1226 (CAO). 1H NMR
(CDCl3, d ppm): 7.46 (2H, d, J = 7.4Hz, a-CH), 7.41 (2H, t, J = 7.5Hz, b-CH), 7.35
(1H, t, J = 7.2Hz, c-CH), 7.22 (1H, d, J = 8.7Hz, 1-CH), 6.83 (1H, dd, J=2.5Hz and
J=8.5Hz, 2-CH), 6.77 (1H, s, 4-CH), 5.06 (2H, s, CH2Ph), 3.79 (3H, s, COOCH3),
3.24 (1H, t, J = 9.2Hz, 16a-CH), 2.92 (2H, m, 6-CH2), 1.31–2.46 (11H, #m, 3· CH,
4· CH2), 1.02, and 0.99 (3H, 2s, 18-CH3, 16a,b (1:4)). 13C NMR (CDCl3, d ppm),

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V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
major isomer 16b-CO2CH3: 211.9 (17-C), 169.8 (COOCH3), 156.9 (3-C), 137.6
(CCH2O), 137.1 (5-C), 131.9 (10-C), 128.4 (b-C), 127.8 (c-C), 127.3 (a-C), 126.2
(1-C), 114.8 (4-C), 112.4 (2-C), 69.8 (CH2Ph), 54.0 (COOCH3), 52.4, 48.8, 47.8,
43.9, 37.8, 31.8, 29.5, 26.4, 26.3, 25.7, 13.2 (18-C). MS (m/e): 418 (M+), 386
(M+ACH3O). Exact mass: Calculated for C27H30O4 = 418.2144; found = 418.2136.
3.3. Synthesis of 3-benzyloxy-16a,b-(methoxycarbonyl)-
16a,b-(110-tetrahydropyranyloxyundecanyl)-1,3,5(10)-estratrien-17-one (9)
A stirred solution of derivative 8 (0.98g, 2.33mmol), 1-tetrahydropyranyloxy-11-
bromoundecane (3.12g, 9.32mmol), benzyltriethylammonium chloride (150mg) and
sodium hydroxide 10% w/v (8mL) in dichloromethane (12mL) was heated to reflux
for 20h. Then, the mixture was diluted with diethyl ether (40mL) and extracted with
a saturated ammonium chloride solution (2 · 20mL) and with water (4 · 50mL).
The organic phase was dried, filtered, and concentrated to an oil. Purification by
flash chromatography with a mixture of hexanes and acetone (9:1) gave 1.12g
(70%) of a viscous oil. IR (NaCl, mmax, cmꢀ1): 1747 (C@O, ester), 1722 (C@O, ke-
1
tone), 1604 (C@C), 1231, and 1031 (CAO). H NMR (CDCl3, d ppm): 7.43 (2H,
d, J = 7.6Hz, a-CH), 7.38 (2H, t, J = 7.4Hz, b-CH), 7.32 (1H, t, J = 7.3Hz, c-
CH), 7.19 (1H, d, J = 8.5Hz, 1-CH), 6.79 (1H, dd, J = 2.0Hz and J=8.8Hz, 2-
CH), 6.74 (1H, s, 4-CH), 5.04 (2H, s, CH2Ph), 4.58 (1H, t, J = 3.6Hz, OCHO),
3.90–3.36 (4H, 4m, CH2OCHOCH2) 3.73 (3H, s, COOCH3), 2.91 (2H, m, 6-CH2),
2.41–1.20 (37H, #m, 3· CH, 17· CH2), 0.93 and 0.91 (3H, 2s, 18-CH3, 16a,b-
CO2CH3 (1:6)). 13C NMR (CDCl3, d ppm), major isomer 16b-CO2CH3: 214.0 (17-
C), 171.8 (COOCH3), 156.9 (3-C), 137.7 (CCH2O), 137.2 (5-C), 132.1 (10-C),
128.5 (b-C), 127.8 (c-C), 127.4 (a-C), 126.2 (1-C), 114.8 (4-C), 112.4 (2-C), 98.8
(OCHO), 69.9 (CH2Ph), 67.6 (CH2OCH on THP ring), 62.3 (CH2OCH on aliphatic
chain), 60.1, 52.5 (COOCH3), 50.4, 49.4, 45.9, 44.0, 37.9, 35.5, 32.0, 31.5, 30.7, 30.5,
29.74, 29.70, 29.5, 29.4, 29.3, 26.5, 26.2, 25.7, 25.5, 25.4, 19.7, 14.0 (18-C). MS (m/e):
672 (M+), 587 (M+AC5H9O), 497 (M+@C5H8O and C7H7). Exact mass: Calculated
for C43H60O6 = 672.4390; found = 672.4398.
3.4. Synthesis of 3-benzyloxy-16a,b-(110-hydroxyundecanyl)-1,3,5(10)-
estratrien-17-one (10)
A solution of 3-benzyloxy-16a,b-(methoxycarbonyl)-16a,b-(110-tetrahydropyrany-
loxyundecanyl)-1,3,5(10)-estratrien-17-one (9) (0.41g, 0.61mmol), lithium chloride
(0.57g, 13.37mmol), and water (0.24mL, 13.37mmol) in N,N-dimethylformamide
(8mL) was stirred and heated to reflux for 20h. Afterwards, the solvent was partly
evaporated and the residue transferred into an extraction funnel using ethyl acetate
(40mL) and water (30mL). The organic phase was washedtwice with hydrochloric acid
(20mL, 10% v/v) and with water 4 · 50mL. The organic phase was dried, filtered, and
concentrated to an oil. Purification by flash chromatography with a mixture of hexanes
and acetone (9:1) gave 80% of the final product as an oil. IR (NaCl, mmax, cmꢀ1): 3200–
3600 (OAH), 1731 (C@O), 1604 (C@C), 1231, and 1021 (CAO). 1H NMR (CDCl3, d

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
7
ppm): 7.45 (2H, d, J = 7.3Hz, a-CH), 7.39 (2H, t, J = 7.4Hz, b-CH), 7.33 (1H, t,
J = 7.3Hz, c-CH), 7.21 (1H, d, J = 8.6Hz, 1-CH), 6.80 (1H, dd, J = 2.0Hz and
J = 8.8Hz, 2-CH), 6.75 (1H, s, 4-CH), 5.05 (2H, s, CH2Ph), 3.65 (2H, t, J = 6.6Hz,
CH2OH), 2,91 (2H, m, 6-CH2), 2.47–1.25 (33H, #m, 4· CH, 14· CH2, OH), 0.96
and 0.88 (3H, 2s, 18-CH3, 16a, b-110-hydroxyundecanyl (1:1.7)). MS (m/e): 530
(M+ + H+), 439 (M+ ꢀ C7H6). Exact mass: Calculated for C36H50O3 (M + H+) =
530.3768; found = 530.3760.
3.5. Synthesis of 3-benzyloxy-16a,b-(110-hydroxyundecanyl)-1,3,5(10)-
estratrien-17b-ol (intermediate precursor of 11)
To a solution of derivative 10 (0.23g, 0.43mmol) in dry THF (8 mL) at ꢀ78ꢁC
under an inert nitrogen atmosphere, was slowly added a solution of lithium alumi-
num hydride 1M/THF (4.3mL, 4.3mmol). The resulting mixture was stirred for
1h. Then, ethyl acetate (1mL) was added to destroy the excess LiAlH4. The reaction
mixture was diluted with ethyl acetate (40mL), extracted with a solution of hydro-
chloric acid (10% v/v, 3 · 20mL) and with water (5 · 50mL). The organic phase
was dried, filtered, and evaporated to an oil. Purification by flash chromatography
with a mixture of hexanes and acetone (4:1) gave a total of 0.18g (78%) of a viscous
oil. In this specific experiment, the two isomers were isolated 60% (0.14g) of the 16a
isomer and 18% (0.04g) of the 16b isomer. However, on larger scale, the mixture of
isomers was used for the synthesis of the final platinum(II) complexe. IR (NaCl,
1
m
max, cmꢀ1): 3650–3100 (OAH), 1609 (C@C), 1221, and 1022 (CAO). H NMR
(CDCl3, d ppm): 7.44 (2H, d, J = 7.5Hz, a-CH), 7.38 (2H, t, J = 7.4Hz, b-CH),
7.32 (1H, t, J = 7.2Hz, c-CH), 7.19 (1H, d, J = 8.6Hz, 1-CH), 6.78 (1H, dd,
J = 1.9Hz, and 8.1Hz, 2-CH), 6.72 (1H, d, J = 0.8Hz, 4-CH), 5.03 (2H, s, CH2Ph),
3.79 (1H, d, J = 11.2Hz, CHOH), 3.62 (2H, t, J = 6.7Hz, CH2OH), 3,50 (1H, d,
J = 10.9Hz, CHOH, 16a), 3,45 (1H, s, CH2OH), 2.82–2,80 (2H, m, 6-CH2), 2.30–
1.07 (32H, #m, 4· CH, 14· CH2), 0.88 (3H, s, 18-CH3, 16a). 13C NMR (CDCl3,
d ppm), 16a pure isomer: 156.7 (3-C), 137.9 (CCH2O), 137.2 (5-C), 132.9 (10-C),
128.5 (b-C), 127.8 (c-C), 127.4 (a-C), 126.2 (1-C), 114.8 (4-C), 112.2 (2-C), 90.5
(CHOH), 69.9 (CH2Ph), 62.9 (CH2OH), 47.6, 46.7, 44.8, 43.8, 39.3, 37.92, 37.89,
33.1, 32.7, 30.5, 29.7, 29.59, 29.57, 29.5, 29.3, 27.4, 26.2, 25.7, 24.7, 11.9 (18-C).
MS (m/e): 532 (M+), 423 (M+-H2O and C7H7). Exact mass: Calculated for
C36H52O3 = 532.3905; found = 532.3916.
3.6. Synthesis of 3-benzyloxy-16a,b-(110-bromoundecanyl)-1,3,5(10)-
estratrien-17b-ol (11)
The diol intermediate (0.16g, 0.30mmol) was solubilized in dichloromethane
(10mL), then carbon tetrabromide (0.40g, 1.20mmol) and triphenylphosphine
(0.31g, 1.20mmol) are added to the diol. The reaction mixture under an inert atmo-
sphere of nitrogen was stirred at room temperature for 1–2h. The organic phase was
diluted with diethyl ether (50mL) and washed with a saturated ammonium chloride
solution (25mL) and with water (5 · 60 mL). The ethereal phase was dried, filtered,

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V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
and evaporated to an oil. The residue was purified by flash chromatography with a
mixture of hexanes and acetone (9:1) to give 0.11g (64%) of the title derivative as an
oil. IR (NaCl, mmax, cmꢀ1) : 3650–3150 (OAH), 1604 (C@C), 1232, and 1022 (CAO).
1H NMR (CDCl3, d ppm): 7.47 (2H, d, J = 7.4Hz, a-CH), 7.42 (2H, t, J = 7.5Hz, b-
CH), 7.35 (1H, t, J = 7.1Hz, c-CH), 7.24 (1H, d, J = 8.5Hz, 1-CH), 6.83 (1H, dd,
J = 1.7 and 8.1Hz, 2-CH), 6.76 (1H, s, 4-CH), 5.07 (2H, s, CH2Ph), 3.77 (0,7H, d,
J = 10.1Hz, CHOH, 16b), 3.44 (2H, t, J = 6.9Hz, CH2Br), 3.30 (0,3H, d,
J = 7.6Hz, CHOH, 16a), 2.89 (2H, m, 6-CH2), 2.35–1.02 (33H, #m, 4· CH, 14·
CH2, -OH), 0.80, and 0.78 (3H, 2s, 18-CH3, 16a,b (1:2.4)). MS (m/e) : 594
(M+), 504 (M+AC7H6). Exact mass: Calculated for C36H51O2Br = 594.3045;
found = 594.3072.
3.7. Synthesis of 16a,b-(110-bromoundecanyl)-1,3,5(10)-estratrien-3,17b-diol
(precursor of derivatives 12a and 12b)
A stirred suspension of 3-benzyloxy-16a, b-(110-bromoundecanyl)-1,3,5(10)-estra-
trien-17b-ol (11, 300mg, 0,50mmol) and 10% Pd/C (150mg) in dry THF (4mL) was
stirred under hydrogen atmospheric pressure for 3–6h. The reaction was followed by
TLC until completion. The insoluble material was filtered off with diethyl ether
(70mL) and the filtrate was concentrated to give the crude product. It was purified
by flash chromatography (hexanes: acetone (4:1)) to give 225mg (90%) of a white so-
lid. M.P.: 128–130ꢁC. IR (NaCl, mmax, cmꢀ1): 3600–3100 (OAH), 1604 (C@C), 1247,
1
and 1068 (CAO). H NMR (CDCl3, d ppm): 7.15 (1H, d, J = 8.5Hz, 1-CH), 6.62
(1H, d, J=8.7Hz, 2-CH), 6.56 (1H, s, 4-CH), 4.75–4.35 (1H, br s, 3-OH), 3.73
(1H, d, J = 10.1Hz, CHOH, 16b), 3.41 (2H, t, J = 6.8Hz, CH2Br), 3.27 (1H, d,
J = 7.6Hz, CHOH, 16a), 2.82 (2H, m, 6-CH2), 2.30–0.97 (33H, #m, 4· CH, 14·
CH2, 17-OH), 0.80 and 0.77 (3H, 2s, 18-CH3, 16a,b (1:3.9)).13C NMR (CDCl3, d
ppm), major 16b isomer: 153.5 (3-C), 138.1 (5-C), 132.5 (10-C), 126.4 (1-C), 115.2
(4-C), 112.7 (2-C), 82.4 (CHOH, 16b), 48.5, 44.1, 44.0, 39.9, 38.3, 37.6, 34.0, 32.8,
32.3, 31.4, 29.8, 29.61, 29.56, 29.5, 29.4, 28.7, 28.6, 28.1, 27.4, 26.2, 12.3 (18-C).
MS (m/e): 504 (M+), 426 (M+A78Br). Exact mass: Calculated for C29H45O2Br =
504.2592; found = 504.2603.
3.8. Synthesis of 16a,b-[110-(200-picolylamino)undecanyl]-1,3,5(10)-
estratrien-3,17b-diol (12a)
The title compound was made as described for the synthesis of the amine 12b be-
low. In this case the following quantities were used: 16a, b-(110-bromoundecanyl)-
1,3,5(10)-estratrien-3,17b-diol (70mg, 0.14 mmol), 2-(aminomethyl)pyridine (80lL,
0.84 mmol), methanol (2 mL). The reaction mixture was heated to reflux for 21h un-
der an inert atmosphere of nitrogen. The extraction was done using a mixture of
diethyl ether and dichloromethane (4:1, 30mL). The crude amine was obtained
quantitatively as a yellowish oil. The title amine was used without further purifica-
tion in the next step. IR (NaCl, mmax, cmꢀ1): 3650–3100 (OAH and NAH), 1588
(C@C), 1246, and 1072 (CAO). 1H NMR (CDCl3, d ppm): 8.56 (1H, d,

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
9
J = 4.9Hz, a0-CH), 7.64 (1H, t, J = 8.2Hz, c0-CH), 7.30 (1H, d, J = 7.8Hz, d0-CH),
7.20–7.14 (2H, m, b0-CH and 1-CH), 6.61 (1H, dd, J = 2.2 and J = 8.7Hz, 2-CH),
6.56 (1H, s, 4-CH), 3.93 (2H, s, CH2pyridyl), 3.73 (1H, d, J = 10.0Hz, CHOH,
16b), 3.26 (1H, d, J = 7.3Hz, CHOH, 16a), 2.82 (2H, m, 6-CH2), 2.67 (1H, t,
J=5.5Hz, NH), 2.60–0.86 (36H, #m, 4· CH, 15· CH2, 2· OH), 0.80 and 0.77
(3H, 2s, 18-CH3, 16a,b (1:4)). MS (m/e): 532 (M+), 426 (M+AC6H6N2). Exact mass:
Calculated for C35H52O2N2 = 532.4022; found = 532.4029.
3.9. Synthesis of 16a,b-[110-(200-pyridylethylamino)undecanyl]-1,3,5(10)-
estratrien-3,17b-diol (12b)
A stirred solution of 16a, b-(110-bromoundecanyl)-1,3,5(10)-estratrien-3,17b-diol
(0.15g, 0.30mmol) and 2-(2-aminoethyl)pyridine (0.36mL, 3mmol), in methanol
(5mL) was heated to reflux for 3 days under an inert atmosphere of nitrogen. Then,
the solvent was evaporated and the residue dissolved in diethyl ether (30mL) was
washed with water (5 · 50mL). The aqueous phases are extracted with diethyl ether
(2 · 15mL). The combined organic phases were dried, filtered, and evaporated to an
oil. The crude amine was obtained in 80% yield and was used without further puri-
fication at the next step. IR (NaCl, mmax, cmꢀ1): 3550–3050 (OAH and NAH), 1588
(C@C), 1241, and 1072 (CAO). 1H NMR (CDCl3, d ppm): 8.48 (1H, d, J=4.2Hz, a0-
CH), 7.63 (1H, t, J = 7.7Hz, c0-CH), 7.18 (2H, m, d0-CH and b0-CH), 7.12 (1H, d,
J = 8.2Hz, 1-CH), 6.63 (1H, d, J = 8.3Hz, 2-CH), 6.57 (1H, s, 4-CH), 3.73 (1H, d,
J = 10.0Hz, CHOH, isomer 16b), 3.26 (1H, d, J = 7.2Hz, CHOH, isomer 16a),
3.16 (4H, m, NHCH2CH2pyridyl), 2.79 (4H, m, (CH2)10CH2NH and 6-CH2),
2.29–1.00 (35H, m, 4· CH, 14· CH2, 2· OH and NH), 0.80 and 0.76 (3H, 2s, 18-
CH3, 16a,b (1:4)). 13C NMR (CDCl3, d ppm), major isomer 16b: 159.4 (pyridyl-
C), 154.0 (3-C), 148.8 (a0-C), 138.1 (5-C), 137.0 (c0-C), 132.2 (10-C), 126.4 (1-C),
123.6 (d0-C), 121.9 (b0-C), 115.4 (4-C), 112.9 (2-C), 82.5 (CHOH, 16b), 48.8, 48.6,
48.2, 44.1, 44.0, 40.0, 38.4, 37.7, 32.4, 31.4, 29.8, 29.7, 29.6, 29.5, 29.2, 28.6, 28.3,
27.5, 27.0, 26.3, 12.4 (18-C). MS (m/e): 546 (M+), 454 (M+AC6H4N). Exact mass:
Calculated for C36H54O2N2 = 546.4198; found = 546.4185.
3.10. Synthesis of 16a,b-[110-(200-picolylamino)undecanyl]-1,3,5(10)-
estratrien-3, 17b-diol dichloroplatinate (II) (1a)
This platinum(II) complex was made as described for the synthesis of the complex 1b
below. In this case the following quantities were used : amine 12a (63mg, 0.12mmol),
potassium tetrachloroplatinate (II) (60mg, 0.14mmol), DMF: H2O (2:1, 3mL). Puri-
fication by flash chromatography with hexanes: acetone (1:1) gave the title compound
in 61% yield in the form of yellow crystals. M.P.: > 124ꢁC decomposed. IR (NaCl,
1
m
max, cmꢀ1): 3600–3050 (OAH and NAH), 1609 (C@C), 1241 and 1062 (CAO). H
NMR (CDCl3 + Acetone-d6 (9 :1), d ppm): 9.20 (1H, d, J = 5.6Hz, a0-CH), 8.13
(1H, t, J = 7.8Hz, c0-CH), 7.84 (1H, s, 3-OH), 7.69 (1H, d, J = 7.8Hz, d0-CH), 7.45
(1H, t, J = 6.7Hz, b0-CH), 7.15 (1H, d, J = 8.4Hz, 1-CH), 6.67 (1H, d, J = 8.2Hz, 2-
CH), 6.62 (1H, s, 4-CH), 6.27 (1H, br s, NH), 4.95 (1H, dd, J = 6.2Hz, and

10
V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
J = 16.5Hz, NHCHxHypyridyl), 4.28 (1H, d, J = 16.3Hz, NHCHxHypyridyl) 3.80
(1H, d, J = 9.8Hz, CHOH, isomer 16b), 3.32 (1H, d, J = 7.4Hz, CHOH, isomer
16a), 3.16 and 2.83 (2H, 2m, (CH2)10CH2NH), 2.88 (2H, m, 6-CH2), 2.34–1.05 (32H,
#m, 3· CH, 14· CH2, 17-OH), 0.88, and 0.86 (3H, 2s, 18-CH3, 16a, b (1:3.7)). 13C
NMR (CDCl3: Acetone-d6 (9:1), d ppm), major isomer 16b: 161.6 (pyridyl-C), 154.0
(3-C), 147.4 (a0-C), 137.8 (5-C), 136.9 (c0-C), 130.7 (10-C), 125.4 (1-C), 123.4 (d0-C),
121.2 (b0-C), 114.4 (4-C), 112.0 (2-C), 81.1 (CHOH, 16b), 59.9, 54.8, 47.9, 43.3, 42.7,
39.6, 37.8, 37.1, 35.3, 31.7, 30.9, 30.1, 28.0, 26.8, 26.7, 25.8, 25.6, 11.7 (18-C).
3.11. Synthesis of 16a,b-[110-(200-pyridylethylamino)undecanyl]-1,3,5(10)-
estratrien-3,17b-diol dichloroplatinate (II) (1b)
To a solution of 16a, b-[110-(200-pyridylethylamino)undecanyl]-1,3,5(10)-estratrien-
3, 17b-diol (12b, 0.13g, 0.24mmol) in DMF (1mL) at 23ꢁC was added potassium tet-
rachloroplatinate (II) (0.11g, 0.26mmol) dissolved in a mixture of DMF/H2O (2:
1.6ml). The resulting mixture (pH 8–9) was stirred in the dark for 2–3 days until the
pH value reached 4–5. Then, a drop of dimethylsulfoxide was added to destroy the ex-
cess K2PtCl4 and the stirring was continued for 2–3h. The solvent was evaporated and
the residue was stirred vigorously in a saturated aqueous potassium chloride solution
(5mL) for 15min. A vigorous stirring was essential in order to pulverize the lumps of
precipitated platinum(II) complex. The resulting suspension was filtered, washed with
water (100mL), and dried in a desiccator for a 1 day. The product was further purified
by flash column chromatography (hexanes: acetone, 3:2) to give the title compound in
the form of yellow crystals (52% yield). M.P.: >148ꢁC decomposed. IR (NaCl, mmax
,
cmꢀ1): 3600–3100 (OAH and NAH), 1610 (C@C), 1211, and 1063 (CAO). 1H NMR
(Acetone-d6, 310K, d ppm): 9.13 (1H, d, J = 5.9Hz, a0-CH), 8.02 (1H, t, J = 7,8Hz,
c0-CH), 7,53 (1H, d, J = 7,8Hz, d0-CH), 7,41 (1H, t, J = 6,7Hz, b0-CH), 7.08 (1H, d,
J=8.5Hz, 1-CH), 6.59 (1H, dd, J = 1.9Hz and J=8.5Hz, 2-CH), 6.53 (1H, s, 4-
CH), 5.87 (1H, br s, NH), 3.72 (1H, d, J = 9.8Hz, CHOH, isomer 16b), 3.66–3.61,
3.23–3.17, 3.00–2.90, and 2.90–2.80 (7H, 4m, RCH2NHCH2CH2pyridyl and CHOH,
isomer 16a), 2.80–2.72 (2H, m, 6-CH2), 2.40–1.00 (34H, #m, 4· CH, 14· CH2, 2· OH),
0.81 and 0.78 (3H, 2s, 18-CH3, 16a,b (1:3.8)). 13C NMR (Acetone-d 6, d ppm), major
isomer 16b: 160.5 (pyridyl-C), 156.0 (a0-C), 154.4 (3-C), 140.0 (c0-C), 138.5 (5-C),
132.3 (10-C), 127.1 (1-C), 125.5 (d0-C), 124.6 (b0-C), 116.0 (4-C), 113.7 (2-C), 82.4
(CHOH, 16b), 57.3, 49.7, 46.8, 45.1, 41.5, 40.4, 39.6, 38.8, 33.5, 32.8, 30.8, 30.7,
28.7, 28.5, 27.3, 13.2 (18-C).
4. Results and discussion
4.1. Synthesis of 17b-estradiol-linked Pt(II) complexes
As shown in Scheme 1, the synthesis involves nine chemical steps starting from
estrone (6) as the steroid template. The 17b-estradiol Pt(II) complexes 1a and 1b
were obtained using a straightforward reaction sequence.

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
11
Scheme 1. Reagents: (a) BnBr,Bu4N+HSO4ꢀ, NaOH 10% p/v, CH2Cl2, reflux, 24h, 99%; (b) KH,
dimethyl carbonate, THF, reflux, 3h, 90%; (c) Br(CH2)11OTHP, Et3N+BnClꢀ, NaOH 10% p/v, CH2Cl2,
reflux, 20h, 70%; (d) LiCl, DMF: H2O, reflux, 20h, 80%; (e) (1) LiAlH4 1M/THF, ꢀ78ꢁC, 1h, 78%; (2)
CBr4, PPh3, CH2Cl2, 22ꢁC, 1–2h, 64%; (f) (1) H2, Pd/C, 10% p/p, THF, 22ꢁC, 3–6h, 90%; (2) 2-
aminomethyl pyridine (n=1) or 2-(20-aminoethyl)pyridine (n = 2), CH3OH, reflux, 3 days, 90%; (g)
K2PtCl4, DMF, H2O (2:1), 22ꢁC, 2–3 days, 57%.
Initially, estrone (6) was protected as a benzyl ether using phase transfer catalysis
(PTC) methodology. Accordingly, estrone was treated with benzyl bromide and tet-
rabutylammonium hydrogensulfate in dichloromethane in the presence of a 10%

12
V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
aqueous sodium hydroxide solution [15]. The yield of the protection reaction is 99%.
The derivative 7 was transformed into the b-ketoester 8 upon treatment with di-
methyl carbonate in the presence of a mixture of NaH/KH in dry tetrahydrofuran
[16,17]. Derivative 8 was obtained with 90% yield. Treatment of derivative 8 and
1-tetrahydropyranyloxy-11-bromoundecane under PTC reaction conditions gave
compound 9 in 70% yield. The bromoalkane side chain was added mainly to the less
hindered a face of the molecule (16a,b; 6:1) as shown by the presence of two singlets
1
for the 18-CH3 at d 0.93 and 0.91 in the H NMR spectrum. Decarboalkoxylation
with simultaneous deprotection of the tetrahydropyranyl ether of derivative 9 was
achieved upon treatment with lithium chloride in a mixture of N,N-dimethylforma-
mide and water at reflux for 20h. The cleavage of the tetrahydropyranyl ether under
these reaction conditions is a known process [18]. The alcohol 10 was obtained in
80% yield as a mixture of a and b isomers (16a,b; 1:1.7). Reduction of derivative
10 with lithium aluminum hydride in dry THF gave the intermediate diol. The ste-
reochemistry of the 17b-hydroxy function as well as the 16-a or 16-b side chain posi-
tion was confirmed by comparison with 13C NMR spectral data of known 16-a and
16-b substituted 17b-estradiol derivatives [19]. Selective bromination of the primary
alcohol was performed with a mixture of carbon tetrabromide, triphenylphosphine
in dichloromethane. The hydroxy-halide 11 was obtained in 64% overall yield.
The final 17b-estradiol-linked Pt(II) complexes 1a and 1b were obtained in three
additional chemical reactions. Initially, hydrogenolysis of the benzyl ether found in
compound 11 with 10% Pd/C in tetrahydrofuran gave the phenol intermediate, which
was treated with an excess 2-aminoalkylpyridine to give derivative 12 in 80% yield. The
diol-aminopyridine intermediates were treated with potassium tetrachloroplatinate in
a mixture of dimethylformamide and water to give the corresponding 17b-estradiol-
linked Pt(II) complexes 1, n = 1(a), 2(b). All new compounds synthesized were charac-
terized by their respective IR, 1H NMR and 13C NMR spectra.
4.2. In vitro cytotoxic activity
The toxicity of the 17b-estradiol-linked Pt(II) complexes was evaluated on two
human breast tumor cell lines using the Sulforhodamine B colorimetric assay
[20,21]. The cytotoxicity of the compounds was tested along with controls (cisplatin,
estradiol and tamoxifen) on both estrogen-receptor positive (ER+, MCF-7) and
estrogen-receptor negative (ERꢀ, MDA-MB-231) human mammary carcinomas
[22]. They were also tested on skin (B16-F10) and intestine (HT-29) cancers for addi-
tional comparison of activities.
As shown by the SRB assays on the two human breast cancer cell lines, the new
Pt(II) complexes are very toxic toward breast cancer cells (Table 1). The Pt(II) com-
plex 1a is twice as toxic as tamoxifen on MCF-7 cells and five times more toxic on
MDA-MB-231 cells, showing an IC50 of 5.9 and 4.1lM, respectively. Moreover, it
presents an activity three times greater than cisplatin itself on both types of cells.
The Pt(II) complex 1b presents a greater activity than tamoxifen; it is 22 times more
toxic on MCF-7 cells and 37 times more toxic on MDA-MB-231 cells. Furthermore,
it is 32 times more toxic on MCF-7 and 25 times more toxic on MDA-MB-231 than

V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
13
Table 1
Inhibitory concentration of drug on breast (ER+ and ERꢀ), skin, and intestine cancer cell lines
Compounds
IC50 (lM)a
MCF-7 (ER+)
MDA-MB-231 (ERꢀ)
B16-F10
HT-29
Cisplatin
Estradiol
Tamoxifen
16.1
31.5
11.1
5.9
12.8
>100
18.9
4.1
4.5
28.9
7.0
10.3
85
11.0
13.5
2.1
1a
1b
a
2.7
0.5
0.5
0.5
Inhibitory concentration as obtained by the SRB assay. Experiments were performed in octuplicate.
cisplatin. Platinum complex 1b shows an IC50 of 0.5lM on all the cells at the excep-
tion of the HT-29 intestine cancer cells with an IC50 of 2.1lM. These data confirm
that the Pt(II) complex bearing the 2-(20-aminoethyl)pyridine ligand presents higher
activity than those bearing the 2-aminomethylpyridine ligand. This was previously
observed with the triphenylethylene Pt(II) complexes [10]. The cytotoxic activity of
1b is remarkable as it is significantly more toxic than cisplatin. Both Pt(II) complexes
1a and 1b are more toxic toward the skin cancer cells (B16-F10) as compared to the
intestine cancer cells (HT-29). It is also observed that estradiol is essentially inactive
on all the cell lines when compared to the estradiol-linked Pt(II) complexes.
The estrogen receptor alpha (ERa) affinity assay was performed using the
HitHunter EFC Estrogen Fluorescence assay kit (Discoverx, Fremont, CA) accord-
ing to manufacturerÕs instructions [23]. Only the most promising estrogen-Pt(II)
complex 1b was tested in this experiment.
The estrogen receptor binding studies showed a strong affinity for 1b, the most
cytotoxic molecule, to the estrogen receptor a (see Fig. 1). The reference derivative
Fig. 1. Estrogen receptor binding affinity (ERa). ED-Estradiol, Enzyme Donor-Estradiol; E2, 17b-
estradiol. Cisplatin presents no affinity for the ER.

14
V. Perron et al. / Bioorganic Chemistry 33 (2005) 1–15
(i.e., cisplatin) presents, as expected, no affinity for the ERa. The estrogen-Pt (II) hy-
brid molecule 1b shows an IC50 of 0.35nM compared to 4.79nM for 17b-estradiol,
the natural ligand. It has a remarkably high affinity which is likely to lead the cyto-
toxic Pt(II) moiety to the ERa-expressing target cells in an in vivo study that will be
performed in the near future.
5. Conclusion
In summary, this manuscript presents a facile synthesis of highly toxic 17b-estra-
diol Pt(II) complexes. They are made form estrone in nine chemical steps with an
overall yield exceeding 10%. Using this strategy a large variety of Pt(II) complexes
could be easily synthesized either with an alkyl side chain or a polyethylene glycol
side chain. Furthermore, other diamine ligands could, without difficulty, be coupled
to the bromide intermediate 11. This kind of estrogen-linked Pt(II) complexes could
present several advantages over the known cisplatine analogs. Theoretically, the
estrogenic portion of the molecule could direct the cytotoxic Pt(II) moiety toward
the target cells via the ERa, increasing specificity and reducing systemic toxicity.
The most promising estrogen-Pt(II) complexe 1b displays high affinity for the
ERa. This preliminary investigation suggests that these 17b-estradiol Pt(II) com-
plexes are quite cytotoxic towards breast cancer cell lines as compared to cisplatin.
Further research will be necessary to assess the full biological potential of these
new cytotoxic agents. Particularly, the synthesis of optically pure 16a and 16b iso-
mers is now warranted considering the biological results obtained with the mixture
of epimers at position 16.
Acknowledgments
´
This work was supported by the Universite du Quebec a Trois-Rivieres. We are
grateful to Mr. Jacques Lacroix for biological evaluation studies. We also thank
´
`
`
1
Dr. Gilles Sauve and Mr. Nicolas LeBerre, Pharmacor Inc. for the H and 13C
´
NMR spectra and Mr. Gaston Boulay, Universite de Sherbrooke, for the mass
spectra.
´
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