Synthesis and antiproliferative evaluation of ferrocenyl and cymantrenyl triaryl butene on breast cancer cells. Biodistribution study of the corresponding technetium-99m tamoxifen conjugate

Article abstract of DOI:10.1016/j.jorganchem.2012.11.035

1-(p-(ferrocenylcarbonylamino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene,1, and 1-(p-(cymantrenylcarbonylamino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene, 2, were synthesized. Both compounds exhibit a significant antiproliferative effect against hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cells (IC₅₀ = 4.5 and 9.4 μM for 1 and 2 respectively on MDA-MB-231 and around 1 μM for 1 and 2 on MCF-7 cells). Interestingly, 2 is the first cymantrenyl complex in this triaryl butene series to show such a high cytotoxic effect. A metal exchange reaction between 1 and ^99mTcO ₄^- was used for the synthesis of 1-(p- (tricarbonylcyclopentadienyl-[^99mTc]-technetium carboxy-amino-phenyl) -1,2-di(p-hydroxyphenyl)-but-1-ene, 3. ^99mTc-3 was obtained in 70-75% yield. The in vivo biodistribution of purified ^99mTc-3 was undertaken on mature female Wistar rats. The uptake by organs follows the order: liver > lung > kidney > heart > spleen > ovaries > bone > muscle > uterus. The ovaries/muscle ratio was 3.89 while that of uterus/muscle ratio was 0.99. Uptake in ovaries was abolished by co-administration of 17β-estradiol while that of most organs devoided of estrogen receptors remained unchanged.

Full text of DOI:10.1016/j.jorganchem.2012.11.035

Journal of Organometallic Chemistry xxx (2012) 1e9
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and antiproliferative evaluation of ferrocenyl and cymantrenyl triaryl
butene on breast cancer cells. Biodistribution study of the corresponding
technetium-99m tamoxifen conjugate
,
b
,
a
b
b
b
b,
Tesnim Dallagi a
, Mouldi Saidi , Anne Vessières , Michel Huché , Gérard Jaouen , Siden Top
**
*
a Laboratoire des Radiopharmaceutiques, Centre National des Sciences et Technologies Nucléaires, Technopôle de Sidi Thabet, 2020 Sidi Thabet, Tunisie
b Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue P. et M. Curie, 75231 Paris cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
1-(p-(ferrocenylcarbonylamino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene ,1, and 1-(p-(cyman-
trenylcarbonylamino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene, 2, were synthesized. Both compounds
exhibit a significant antiproliferativeeffect against hormone-dependent MCF-7 and hormone-independent
Received 13 October 2012
Received in revised form
23 November 2012
MDA-MB-231 breast cancer cells (IC50 ¼ 4.5 and 9.4
mM for 1 and 2 respectively on MDA-MB-231 and
Accepted 26 November 2012
around 1 M for 1 and 2 on MCF-7 cells). Interestingly, 2 is the first cymantrenyl complex in this triaryl
m
butene series to show such a high cytotoxic effect. A metal exchange reaction between 1 and 99mTcO4ꢀ was
used for the synthesis of 1-(p-(tricarbonylcyclopentadienyl-[99mTc]-technetium carboxy-amino-phenyl)-
1,2-di(p-hydroxyphenyl)-but-1-ene, 3. 99mTc-3 was obtained in 70e75% yield. The in vivo biodistribution of
purified 99mTc-3 was undertaken on mature female Wistar rats. The uptake by organs follows the order:
liver > lung > kidney > heart > spleen > ovaries > bone > muscle > uterus. The ovaries/muscle ratio was
3.89 while that of uterus/muscle ratio was 0.99. Uptake in ovaries was abolished by co-administration of
Keywords:
Radiopharmaceutics
Ferrocene
Cymantrene
Technetium
Breast cancer
17
b
-estradiol while that of most organs devoided of estrogen receptors remained unchanged.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
carcinoma and develops drug resistance [6,7]. Switching from
tamoxifen to aromatase inhibitors after 2e3 years of treatment
significantly improves survival in postmenopausal patients [7,8,9].
In order to overcome drug resistance phenomena and to find
new types of drugs, we have developed organometallic analogs of
tamoxifen based on ferrocene derivatives. The first examples were
hydroxyferrocifen (Fc-OH-TAM) and ferrocenyldiphenol (Fc-diOH)
which exhibit an antiproliferative effect against both hormone
dependent and hormone independent breast cancer cell lines with
Breast cancers are the leading cause of cancer death in women
in Europe [1], while in the USA it is only surpassed by lung cancer
[2]. They are traditionally classified according to their estrogen
receptor status: hormone-dependent cancers (estrogen receptor
positive; ERþ), and hormone-independent cancers (estrogen
receptor negative; ERꢀ). This designation arises because hormone-
dependent cancer cells, whose proliferation is controlled by the
natural hormone 17
a specific receptor: the alpha form of the estrogen receptor (ER
b
-estradiol, induced a nuclear accumulation of
IC50 values for MDA-MB-231 of 0.5
[10,11,12].
mM and 0.64 mM, respectively
a
).
This receptor has been used as a target for endocrine therapies of
hormone dependent breast cancers. For example antiestrogens,
such as tamoxifen, are largely used for the treatment of women
suffering of ERþ breast cancer [3,4,5]. It is known that prolonged
treatment with tamoxifen increases the risk of endometrial
Hydroxy amino and hydroxyl amido derivatives are also very
active with IC50 values of 0.55 M and 0.56 M, respectively [13].
m
m
Pursuing our research on ferrocene derivatives, we were
interested in the amido compound 1 for which the ferrocenyl
moiety is attached to the triaryl butene skeleton via an amide
function. Thus, both 1 and hydroxytamoxifen (OH-TAM) present
the same triaryl butene moiety. Another point of interest in this
type of compound is its suitability for a transformation into 3, its
* Corresponding author. Tél.: þ33 1 44 27 66 99.
technetium analog, via
a known metal exchange reaction
** Corresponding author. Laboratoire des Radiopharmaceutiques, Centre National
des Sciences et Technologies Nucléaires, Technopôle de Sidi Thabet, 2020 Sidi
Thabet, Tunisie.
[14,15,16,17,18]. In molecular radioimaging, 99mTc is the most
widely used radionuclide, owing to its cheapness, easy availability
and suitable physical characteristics (t1/2 ¼ 6.0 h, E ¼ 140.5 keV).
g
E-mail address: siden-top@chimie-paristech.fr (S. Top).
0022-328X/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.11.035
Please cite this article in press as: T. Dallagi, et al., Journal of Organometallic Chemistry (2012), http://dx.doi.org/10.1016/
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2
T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
More than 85% of diagnostic scans are done each year in hospitals
with this radionuclide [19,20]. Some technetium radiopharma-
ceuticals are routinely used in hospitals such as 99mTc-sestamibi
[21], 99mTc-terofosmin [22], 99mTc-methylene diphosphonate [23],
99mTc-MDP [24], 99mTc(V)-DMSA [24]. The search for improved or
more tunable radiopharmaceuticals is still currently undertaken
and recent research has been directed to radiopharmaceuticals
based on compounds containing the CpTc(CO)3 group or Tc(CO)3
core because of the high stability of these moieties
[16,17,18,25,26,27,28]. The common precursor for these
compounds is the aqua ion [99mTc(CO)3(H2O)3]þ, prepared by
Alberto et al. in 2001 [29,30]. Taking into account this important
application, we thought it would be interesting to prepare
compound 3 and examine its potential as a radiopharmaceutical.
Rhenium analogs of technetium compounds are usually used to
identify 99mTc compounds by HPLC [17,31,32]. We have been
working with rhenium and manganese compounds and we found
that both compounds showed similar HPLC retention times
[33,34]. Therefore, either rhenium or manganese compounds can
be used for the identification of 99mTc compounds. Here, we have
chosen to use the manganese complex 2 as the non radioactive
analog of 3 (Chart 1). We now report the synthesis of 1, 2 and 3, the
antiproliferative effect of 1 and 2 against MCF-7 and MDA-MB-231
cells, and the in vivo biodistribution of 3 (Chart 2).
Yvette, France. [99mTcO4]ꢀ was eluted from a Mallinckrodt Med.-
Inc. generator. The analytical purity of compounds was determined
using a reversed-phase HPLC (C18 column, 4.6
m
m ꢁ 250 mm,
eluent gradient (A ¼ 0.1% CF3COOH in H2O, B ¼ MeOH): 0e3 min:
100% A; 3e9 min: 75% A; 9.1 min: 66% A; 9.1e20 min: 66% / 0%
A; 20e25 min: 0% A; 25.1e30 min: 100% A). The effluent from the
column was monitored with a variable-wavelength detector set
at 250 nm and a g-ray detector. The Z and E isomers were iden-
tified by the structure determination of Z-5 by 2D-NMR
experiments.
2.2. 4-t-Butyldimethylsiloxy-40-nitro-benzophenone
4-Hydroxy-40-nitro-benzophenone (2.43 g, 10 mmol) and
imidazole (1.36 g) were dissolved in DMF (15 mL). t-Butyldime-
thylsilylchloride (1.80 g, 12 mmol) was added and the mixture was
stirred for 15 h. The mixture was poured in water (100 mL). The
product was extracted with dichloromethane (3 ꢁ 60 mL) and the
combined organic layer was washed once with water (2 ꢁ 50 mL).
The organic layer was dried over MgSO4 and evaporated under
reduced pressure. The crude product (5.20 g) was purified by flash
chromatography on silica gel column giving 4-t-butyldimethylsi-
loxy-40-nitro-benzophenone (white solid, 2.72 g, 76% yield).
Mp ¼ 72 ꢂC. Rf: 0.44 (diethyl ether:pentane 1:4).1H NMR (300 MHz,
CDCl3):
(d, d, 2H, 2H, J ¼ 8.6 Hz, C6H4); 7.89 and 8.33 (d, d, 2H, 2H, J ¼ 8.8 Hz,
C6H4). 13C NMR (75.5 MHz, CDCl3):
d 0.26 (s, 6H, (CH3)2Si); 1.00 (s, 9H, (CH3)3CSi); 6.92 and 7.75
2. Experimental
d
ꢀ4.3 ((CH3)2Si); 18.2
((CH3)3CSi); 25.5 ((CH3)3CSi); 120.1 (CH, C6H4); 123.4 (CH, C6H4);
129.4 (Cq, C6H4); 130.3 (CH, C6H4); 132.4 (CH, C6H4); 143.7 (Cq,
C6H4); 149.5 (Cq, C6H4); 160.9 (Cq, C6H4); 193.5 (CO). IR (CH2Cl2,
cmꢀ1): 1659.5 (CO). MS (CI): 358.2 [MH]þ.
2.1. General
All air-sensitive reactions were carried out under an argon
atmosphere, using standard Schlenk and vacuum-line techniques.
Anhydrous THF and diethyl ether were obtained by distillation
from sodium/benzophenone. TLC chromatography was performed
on silica gel 60 GF254. Flash chromatography was performed on
silica gel Merck 60 (0.040e0.060 mm). Infrared spectra were ob-
tained with an IR-FT BOMEM Michelson-100 spectrometer
equipped with a DTGS detector. 1H and 13C NMR spectra were
recorded on 300 MHz Bruker spectrometers. Mass spectrometry
was performed with a Nermag R 10-10C spectrometer. Melting
points were measured with a Kofler device. Elemental analy-
ses were performed by the service de microanalyse I.C.S.N., Gif sur
2.3. Z and E-1-(p-aminophenyl)-1,2-di(p-t-
butyldimethylsiloxyphenyl)-but-1-ene (E- and Z-4)
Titanium tetrachloride (3.30 mL, 30 mmol) was added dropwise
to a suspension of zinc powder (3.90 g, 60 mmol) in 80 mL of THF at
0
ꢂC. The mixture obtained was heated at reflux for 2 h and then
allowed to cool to room temperature. A second solution was
prepared by dissolving 4-t-butyldimethylsilyl-propiophenone [35]
(1.596 g, 6 mmol) and 4-amino-40-t-butyldimethylsilyl-benzophe-
none (2.14 g, 6 mmol) in 30 mL of THF. This latter solution was
added dropwise to the first solution and then the resulting mixture
was heated at reflux for 12 h. After cooling to room temperature,
the mixture was poured into water (200 mL). The solution was
neutralized with 20% K2CO3 solution and extracted with diethyl
ether (3 ꢁ 200 mL). The organic layer was washed with water, dried
over magnesium sulphate, filtered and evaporated under reduced
pressure. The crude product was chromatographed on silica gel
column with diethyl ether:petroleum ether 1:2 as eluent. The first
fraction was identified as Z-4, yellow oil, 0.870 g (26% yield). Rf:
R1
R1 = OH ; R2 = O(CH2)3NMe2
: Fc-OH-TAM
: Fc-diOH
R1 = OH ; R2 = OH
R1 = NH2 ; R2 = OH
Fe
R2
R1 = NHCOMe ; R2 = OH
0.75 (diethyl ether:pentane 1:1). 1H NMR (300 MHz, CDCl3):
d 0.14
and 0.22 (s, s, 6H, 6H, 2 (CH3)2Si); 0.91 (t, 3 H, J ¼ 7.4 Hz, CH3); 0.96
and 1.00 (s, s, 6H, 6H, 2 (CH3)3Si); 2.42 (q, 2H, J ¼ 7.4 Hz, CH2); 3.30
(broad s, 2H, NH2); 6.32 and 6.64 (d, d, 2H, 2 H, J ¼ 8.6 Hz, C6H4);
6.63 and 6.96 (d, d, 2H, 2 H, J ¼ 8.6 Hz, C6H4); 6.78 and 7.06 (d, d, 2H,
Chart 1. Ferrocenyl diaryl butenes.
O
2 H, J ¼ 8.6 Hz, C6H4). 13C NMR (75.47 MHz, CDCl3):
d
ꢀ4.4 (CH3Si);
NH
R
13.7 (CH3); 18.2 and 18.3 (t-BuC); 25.7 (t-Bu); 28.9 (CH2); 114.1,
119.4, 119.5, 130.5, 130.6, 130.7, 131.9 (6 CH, 3 C6H4); 134.0, 135.9,
137.3, 137.6, 139.7, 143.9, 153.6, 153.1, (8 Cq, 3 C6H4 þ C]C). IR
(CH2Cl2, cmꢀ1): 3380 (NH2). Mass MS (EI): 559.34 [M]þ, 544.36
[M ꢀ Me]þ. The second fraction corresponds to E-4, yellow oil,
0.396 g (12% yield) Rf: 0.58 (diethyl ether:pentane 1:1) 1H NMR
99m
R =
Fe
Mn(CO)3
Tc(CO)3
1
2
3
HO
OH
(300 MHz, CDCl3):
d 0.10 and 0.16 (s, s, 6H, 6H, 2 (CH3)2Si); 0.95 (t,
Chart 2. Fe, 99mTc and Mn diaryl butenes complexes.
3 H, J ¼ 7.4 Hz, CH3); 0.94 and 0.96 (s, s, 6H, 6H, 2 (CH3)3Si); 2.47 (q,
Please cite this article in press as: T. Dallagi, et al., Journal of Organometallic Chemistry (2012), http://dx.doi.org/10.1016/
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T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
3
2H, J ¼ 7.4 Hz, CH2); 3.80 (broad s, 2H, NH2); 6.46 and 6.70 (d, d, 2H,
2 H, J ¼ 8.6 Hz, C6H4); 6.62 and 6.92 (d, d, 2H, 2 H, J ¼ 8.6 Hz, C6H4);
6.65 and 7.01 (d, d, 2H, 2 H, J ¼ 8.5 Hz, C6H4).13C NMR (75.47 MHz,
C6H4N); 8.87 (s, 1H, NH). 13C NMR (100.62 MHz, CD3COCD3):
ꢀ4.43 ans 4.40 (CH3Si); 13.8 (CH3); 18.6 and 18.7 (t-BuC); 25.8 and
25.9 (t-Bu); 29.2 (CH2); 69.2 (2 CH, C , C5H4); 70.3 (Cp); 71.2 (2 CH,
, C5H4); 77.7 (Cip, C5H4); 119.6 and 132.5 (C6H4O); 120.2, 131.5
(C6H4O); 120.3, 130.3 (C6H4N); 136.2, 137.4, 138.6, 138.9, 139.4,
141.5, 154.3, 154.7, (8 Cq, 3 C6H4 þ C]C), 168.8 (CO). Mass MS (CI):
772.47 [M þ H]þ. Analyse: C45H57FeNO3Si2 cald.: C, 70.01; H, 7.44;
N, 1.81. Found: C, 70.05; H, 7.39; N, 1.72.
d
a
CDCl3):
d
ꢀ4.4 (CH3Si); 13.7 (CH3); 18.1 and 18.2 (t-BuC); 25.7 (t-
Cb
Bu); 28.8 (CH2); 114.7, 118.7, 119.4, 130.5, 130.7, 131.9 (6 CH, 3 C6H4);
134.4, 135.7, 136.9, 137.8, 140.1, 144.7, 153.3, 153.6, (8 Cq, 3
C6H4 þ C]C). IR (CH2Cl2, cmꢀ1): 3384 (NH2). MS (EI): 559.43 [M]þ,
544.59 [M ꢀ Me]þ.
2.4. E-1-(p-ferrocenylcarbonylamino-phenyl)-1,2-di(p-tert-
butyldimethylsiloxyphenyl)-but-1-ene (E-5)
2.6. (Z þ E)-1-(p-ferrocenylcarbonylamino-phenyl)-1,2-di(p-
hydroxyphenyl)-but-1-ene (Z þ Eꢀ1)
Oxalyl chloride (2 mL) was added to ferrocene carboxylic acid
(230 mg, 1 mmol) cooled by ice bath. 5 min after, the cooled bath
was removed and the solution was stirred at room temperature
for 3 h. Excess of oxalyl chloride was removed under vaccuo.
Dichloromethane (4 mL) was added. The mixture obtained was
added into a solution of E-4 (130 mg, 0.3 mmol) and pyridine
(79 mg, 1 mmol) in dichloromethane (4 mL). The mixture was
stirred for 1.5 h and poured in water (40 mL). The compound was
extracted with 2 ꢁ 40 mL of dichloromethane and washed with
40 mL of water. The solution was dried over magnesium sulphate,
filtered and evaporated. The crude product obtained was purified
by flash chromatography with silica gel column using
CH2Cl2:petroleum ether 3:1 as an eluent. E-5 was obtained as an
orange solid (90 mg, 51% yield). Mp ¼ 148 ꢂC (diethyl ether/
hexane). Rf: 0.66 (diethyl ether:pentane 1:1). 1H NMR (300 MHz,
E-5 (90 mg, 0.12 mmol) was dissolved in THF (3 mL). 0.15 mL
(0.15 mmol) of nBu4NF (solution 1 M in THF) were added. After
15 min of stirring, the solvent was evaporated. The crude product
obtained was dissolved in 1 mL of methanol and 2 mL of CH2Cl2 and
was purified by flash chromatography with silica gel column using
CH2Cl2:diethyl ether 3:1 as an eluent. E-1 was obtained as an
orange solid of a mixture of E:Z isomers in a 57:43 proportion
(55 mg, 84% yield). Rf: 0.67 (CH2Cl2/diethyl ether 2/1). 1H NMR
(300 MHz, CD3COCD3) for E-1:
d
0.93 (s, 3H, J ¼ 7.4 Hz, CH3); 2.46 (q,
2H, J ¼ 7.4 Hz, CH2); 4.19 (s, 5H, Cp), 4.37 and 4.90 (t, t, 2H, 2H,
J ¼ 1.9 Hz, C5H4); 6.67 and 6.84 (d, d, 2H, 2 H, J ¼ 8.7 Hz, C6H4); 6.82
and 6.99 (d, d, 2H, 2 H, J ¼ 8.6 Hz, C6H4); 7.06 and 7.40 (d, d, 2H, 2H,
J ¼ 8.6 Hz, C6H4N); 8.70 (s, 1H, NH). 13C NMR (75.47 MHz,
CD3COCD3):
d 15.6 (CH3); 29.5 (CH2); 69.3 (2 CH, C5H4); 70.4 (Cp);
CDCl3):
d
0.16 and 0.22 (s, s, 6H, 6H, 2 (CH3)2Si); 0.93 (t, 3H,
71.3 (2 CH, C5H4); 77.8 (Cip, C5H4); 115.6, 115.8, 119.7, 131.4, 131.6,
131.8 (6 CH, 3 C6H4); 134.3, 136.0, 137.8, 138.5, 139.9, 141.7, 156.6,
J ¼ 7.4 Hz, CH3); 0.95 and 1.00 (s, s, 6H, 6H, 2 (CH3)3Si); 2.46 (q,
2H, J ¼ 7.4 Hz, CH2); 4.23 (s, 5H, Cp), 4.39 and 4.73 (broad s, broad
s, 2H, 2H, C5H4); 6.66 and 6.83 (d, d, 2H, 2H, J ¼ 8.5 Hz, C6H4); 6.81
and 6.97 (d, d, 2H, 2 H, J ¼ 8.5 Hz, C6H4); 7.08 and 7.23 (d, d, 2H,
157.0, (8 Cq, 3 C6H4 þ C]C), 168.8 (CO). 1H NMR for Z-1:
d 0.93 (s,
3H, J ¼ 7.4 Hz, CH3); 2.46 (q, 2H, J ¼ 7.4 Hz, CH2); 4.24 (s, 5H, Cp),
4.42 and 4.96 (t, t, 2H, 2H, J ¼ 1.9 Hz, C5H4); 6.52 and 6.73 (d, d, 2H,
2 H, J ¼ 8.7 Hz, C6H4); 6.65 and 6.98 (d, d, 2H, 2 H, J ¼ 8.7 Hz, C6H4);
2H, J ¼ 8.5 Hz, C6H4N). 13C NMR (75.47 MHz, CDCl3):
d
ꢀ4.4
(CH3Si); 13.6 (CH3); 18.2 and 18.3 (t-BuC); 25.7 (t-Bu); 29.0 (CH2);
68.5 (2 CH, C5H4); 70.2 (Cp); 71.2 (2 CH, C5H4); 118.6, 119.6, 119.7,
130.6, 130.7, 131.6 (6 CH, 3 C6H4); 135.4, 135.35, 136.7, 137.3, 139.4,
141.3, 153.8, 154.3 (8 Cq, 3 C6H4 þ C]C), 168.2 (CO). IR (CH2Cl2,
cmꢀ1): 1671 (CO). MS (EI): 771.24 [M]þ, 714.26 [M-tBu]þ. Analyse:
7.16 and 7.74 (d, d, 2H, 2H, J ¼ 8.6 Hz, C6H4N); 8.88 (s, 1H, NH). 13
C
NMR for Z-1: 13.9 (CH3); 29.5 (CH2); 69.4 (2 CH, C5H4); 70.4 (Cp);
71.4 (2 CH, C5H4); 77.8 (Cip, C5H4); 115.1, 115.6, 120.5, 130.4, 131.6,
132.7 (6 CH, 3 C6H4); 134.3, 135.7, 138.5, 138.8, 139.9, 141.2, 156.6,
157.0, (8 Cq, 3 C6H4 þ C]C), 168.9 (CO). IR (CH2Cl2, cmꢀ1): 1671
(CO). MS (EI): 543.18 [M]þ
C
45H57FeNO3Si2$H2O cald.: C, 68.42; H, 7.53; N, 1.77. Found: C,
68.15; H, 7.53; N, 1.68.
2.5. Z-1-(p-ferrocenylcarbonylamino-phenyl)-1,2-di(p-tert-
2.7. Z-1-(p-ferrocenylcarbonylamino-phenyl)-1,2-di(p-
butyldimethylsiloxyphenyl)-but-1-ene (Z-5)
hydroxyphenyl)-but-1-ene (Z-1)
Oxalyl chloride (3 mL) was added to ferrocene carboxylic acid
(460 mg, 2 mmol) cooled by ice bath. 5 min after, the cooled bath
was removed and the solution was stirred at room temperature for
Z-5 (175 mg, 0.23 mmol) was dissolved in THF (6 mL). 0.5 mL
(0.5 mmol) of nBu4NF (solution 1 M in THF) were added. After
15 min of stirring, 30 mL of methanol and 130 mL of ethyl acetate
were successively added. The mixture was washed with 2 ꢁ 30 mL
of water, dried over MgSO4, filtered and evaporated. After one night
at room temperature, orange crystals were formed from the oily
crude product. Crystals were washed with diethyl ether and
pentane giving 100 mg of Z-1 containing less than 6% of E-1
(100 mg, 80% yield). Mp ¼ 186 ꢂC (CH2Cl2/hexane). Rf: 0.53 (CH2Cl2/
3
h. Excess of oxalyl chloride was removed under vaccuo.
Dichloromethane (8 mL) was added. The mixture was added into
a solution of Z-4 (423 mg, 0.75 mmol) and pyridine (158 mg,
2 mmol) in dichloromethane (8 mL). The mixture was stirred for
1.5 h after which 50 mL of dichloromethane were added. The
mixture was washed with 2 ꢁ 30 mL of water. The solution was
dried over magnesium sulphate, filtered and evaporated. The crude
product obtained was purified flash by chromatography with silica
gel column using CH2Cl2:petroleum ether 2:1 as an eluent. Z-5 was
obtained as an orange solid (372 mg, 64% yield). Mp ¼ 182 ꢂC. Rf:
0.53 (diethyl ether:pentane 1:1). The Z structure of the compound
was identified by NOESY 2D-NMR experiments. IR (CH2Cl2, cmꢀ1):
diethyl ether 2/1). 1H NMR (300 MHz, CD3COCD3):
d 0.92 (s, 3H,
J ¼ 7.4 Hz, CH3); 2.46 (q, 2H, J ¼ 7.4 Hz, CH2); 4.24 (s, 5H, Cp), 4.42
and 4.96 (t, t, 2H, 2H, J ¼ 1.9 Hz, C5H4); 6.52 and 6.73 (d, d, 2H, 2 H,
J ¼ 8.7 Hz, C6H4); 6.65 and 6.98 (d, d, 2H, 2H, J ¼ 8.7 Hz, C6H4); 7.16
and 7.74 (d, d, 2H, 2H, J ¼ 8.6 Hz, C6H4N); 8.88 (s, 1H, NH). 13C NMR
(75.47 MHz, CD3COCD3): d 13.3 (CH3); 29.5 (CH2); 68.7 (2 CH, C5H4);
1671 (CO). 1H NMR (400 MHz, CD3COCD3):
d
0.14 and 0.19 (s, s, 6H,
69.8 (Cp); 70.7 (2 CH, C5H4); 77.1 (Cip, C5H4); 114.4, 114.9, 119.8,
129.7, 130.9, 132.1 (6 CH, 3 C6H4); 133.6, 135.1, 137.8, 138.1, 139.5,
140.6, 155.5, 155.8, (8 Cq, 3 C6H4 þ C]C), 168.5 (CO). IR (CH2Cl2,
6H, 2 (CH3)2Si); 0.94 and 0.98 (s, s, 6H, 6H, 2 (CH3)3Si); 0.96 (overlap
with (CH3)3Si), (CH3); 2.50 (q, 2 H, J ¼ 7.4 Hz, CH2); 4.26 (s, 5H, Cp),
4.43 (d, 2H, J ¼ 2.0 Hz, H
a
, C5H4); 4.98 (d, 2H, J ¼ 2.0 Hz, H
b
, C5H4);
cmꢀ1): 1670 (CO). MS (CI): 544.25 [M
þ
H]þ. Analyse:
6.55 and 6.77 (d, d, 2H, 2 H, J ¼ 8.6 Hz, C6H4O); 6.70 and 7.02 (d, d,
C33H29FeNO3.1/2H2O cald.: C, 71.75; H, 5.47; N, 2.39. Found : C,
2H, 2H, J ¼ 8.6 Hz, C6H4O); 7.20 and 7.77 (d, d, 2H, 2H, J ¼ 8.6 Hz,
72.03; H, 5.51; N, 2.45.
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4
T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
2.8. E-1-(p-tricarbonylcyclopentadienylmanganese carboxy-
amino-phenyl)-1,2-di(p-tert-butyldimethylsiloxyphenyl)-but-1-ene
(E-6)
and MDA-MB-231 cells were from the Human Tumor Cell Bank.
Glutamine, 17 -estradiol and protamine sulphate were from Sigma.
b
2.11.2. Determination of the relative binding affinity (RBA) of 1 and
2 for the alpha form of the estrogen receptor (ER
RBA values were measured on ER purchased from Pan Vera
(Madison, WI, USA). A volume of 10 L of the solution containing
3500 pmoL/mL were added to 16 mL of buffer (10% glycerol, 50 mM
Bis-Tris-Propane pH 9, 400 mM KCl, 2 mM DTT, 1 mM EDTA, 0.1%
Oxalyl chloride (2 mL) was added to tricarbonylcyclopentadie-
nylmanganese carboxylic acid (400 mg, 1.61 mmol). The mixture
was heated at 40 ꢂC for 2 h after which the solution became clear.
Excess of oxalyl chloride was removed under vaccuo. Dichloro-
methane (5 mL) was added. The mixture obtained was added into
a solution of E-4 (190 mg, 0.34 mmol) and pyridine (79 mg,1 mmol)
in dichloromethane (5 mL). The mixture was stirred for 2 h and
poured in water (50 mL). The compound was extracted with
3 ꢁ 40 mL of dichloromethane and washed with 40 mL of water.
The solution was dried over magnesium sulphate, filtered and
evaporated. The crude product obtained was purified by flash
chromatography with silica gel column using diethyl ether:-
petroleum ether 1:1 as an eluent. E-7 was obtained as an yellow oil
(123 mg, 46% yield). IR (CH2Cl2, cmꢀ1): 2030 and 1948 (MnCO),
1680 (NHCO). The compound was directly used to prepare E-2
without further characterization.
a)
a
m
BSA) in a silanized flask. Aliquots (200
mL) of this solution in
polypropylene tubes were incubated for 3 h at 0 ꢂC with [6,7-3H]-
estradiol (2 ꢁ 10ꢀ9 M, specific activity 1.62 TBq/mmol, NEN Life
Science, Boston MA) in the presence of nine concentrations of 2. At
the end of the incubation period, the free and bound fractions of the
tracer were separated by protamine sulphate precipitation. The
percentage reduction in binding of [3H]-estradiol (Y) was calculated
using the logit transformation of Y (logitY: ln[y/1 ꢀ Y]) versus the
log of the mass of the competing steroid. The concentration of
unlabeled steroid required to displace 50% of the bound [3H]-
estradiol was calculated for each steroid tested, and the results
expressed as RBA. The RBA value of estradiol is by definition equal
to 100%.
2.9. E-1-(p-tricarbonylcyclopentadienylmanganese carboxy-
amino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene (E-2)
2.11.3. Culture conditions
Cells were maintained in monolayer culture in DMEM with
phenol red/Glutamax I, supplemented with 9% of decomplemented
fetal calf serum and 0.9% kanamycin, at 37 ꢂC in a 5% CO2 air
humidified incubator. For proliferation assays, cells were plated in
24-well sterile plates with 1.5 ꢁ 104 cells for MDA-MB-231 and of
3 ꢁ 104 cells for MCF-7 in 1 mL of DMEM without phenol red,
supplemented with 9% of fetal calf serum desteroided on dextran
charcoal, 0.9% Glutamax I and 0.9% kanamycin, and were incubated
for 24 h. The following day (D0), 1 mL of the same medium con-
taining the compounds to be tested diluted in DMSO, was added to
the plates (final volumes of DMSO: 0.1%; 4 wells for each condi-
tions). After three days (D3), the incubation medium was removed
and 2 mL of fresh medium containing the compounds was added.
At different days (D3, D4, D5 and D6), the protein content of each
well was quantified by methylene blue staining as follows. Cell
monolayers were fixed and stained for 1 h in methanol with
methylene blue (2 mg/mL), and then washed thoroughly with
water. Two milliliters of HCl (0.1 M) was then added, and the plate
was incubated for 1 h at 37 ꢂC. Then the absorbance (proportional
to the amount of proteins) of each well was measured at 655 nm
with a Biorad microplate reader. The results are expressed as the
percentage of proteins presents in each wells versus the proteins of
the control (untreated cells). IC50 values are obtained by linear
regression of the appropriated data. Experiments were performed
at least in duplicate.
E-6 (110 mg, 0.14 mmol) was dissolved in 5 mL of ethanol
(110 mg, 0.14 mmol). 0.1 mL of HCl (conc) was added. The solution
was stirred at 70 ꢂC for 20 min. The heating bath was removed and
50 mL of diethyl ether was added. The mixture was washed with
2 ꢁ 20 mL of water, dried over MgSO4, filtered and evaporated
under reduced pressure. The crude product obtained was purified
by flash chromatography with silica gel column using diethyl
ether:petroleum ether 4:1 as an eluent. E-2 was obtained as
a yellow solid (80 mg, 62% yield). Mp ¼ 142 ꢂC (diethyl ether/
hexane). Rf: 0.71 (diethyl ether). 1H NMR (400 MHz, CD3COCD3):
d
0.92 (s, 3H, J ¼ 5.5 Hz, CH3); 2.46 (q, 2H, J ¼ 5.5 Hz, CH2); 5.06 (s,
2H, C5H4), 5.74 (s, 2H, C5H4), 6.65 and 6.83 (d, d, 2H, 2 H, J ¼ 8.7 Hz,
C6H4); 6.83 and 6.97 (d, d, 2H, 2 H, J ¼ 8.7 Hz, C6H4); 7.05 and 7.35
(d, d, 2H, 2H, J ¼ 8.6 Hz, C6H4N); 8.18 ans 8.34 (s, s, 1H, 1H, 2 OH);
9.01 (s, 1H, NH). 13C NMR (100 MHz, CD3COCD3):
d 13.8 (CH3); 29.9
(CH2); 84.4 (2 CH, C5H4); 86.4 (2 CH, C5H4); 92.7 (Cip, C5H4); 115.5,
115.7, 119.8, 131.2, 131.5, 131.7 (6 CH, 3 C6H4); 134.1, 135.7, 136.8,
138.2, 140.6, 141.9, 156.5, 156.9, (8 Cq, 3 C6H4 þ C]C), 162.1 (CO). IR
(CH2Cl2, cmꢀ1): 2030 and 1948 (MnCO), 1682 (CO). MS (EI): 561.12
[M]þ, 477.22 [M-3CO]þ. Analyse: C31H24MnNO6.1/2H2O cald.: C,
65.38; H, 4.25; N, 2.46. Found: C, 65.62; H, 4.56; N, 2.39.
2.10. Z-1-(p-tricarbonylcyclopentadienyl-[99mTc]-technetium
carboxy-amino-phenyl)-1,2-di(p-hydroxyphenyl)-but-1-ene (Z-3)
Z-1 (1 mg) and Mn(CO)5Br (1 mg) were dissolved in 150
mL of
2.11.4. Biological pH stability study
dimethyl formamide in a 10-mL glass tube, and 150
m
L [99mTcO4]ꢀ
Purified 3 (100
buffer (900
mL, 50 mCi) was added to potassium phosphate
in saline was added. The vial was sealed and purged with nitrogen
for 10 min and heated in an oil bath at 150 ꢂC for 1 h. The mixture
was purified by chromatography on a silica gel plate (TLC) (diethyl
ether) to give Z-3. Radio-TLC (diethyl ether) gave a peak with an
Rf ¼ 0.69 (yield 70e75%). HPLC retention time ¼ 24.36 min.
m
L, 0.1 M) at pH 7.4. The vial was sealed and incubated at
37.0 ꢂC for the duration of study. The solution was examined by
radio-HPLC at 30 min, 1 h, 2 h, 3 h and 22 h during incubation to
determine the effective stability of the compound.
2.11.5. In vitro stability study
2.11. Biochemical experiments
An in vitro stability study was carried out in isolated heparinized
whole rat blood (mature female Wistar rats). The whole blood
2.11.1. Materials
(5 mL) was incubated with 70 mCi 3 in 100 mL ethanol for 30 min,
Stock solutions (1 ꢁ 10ꢀ3 M) and serial dilutions of the
compounds to be tested were prepared in DMSO just prior to use.
Dulbecco’s modified eagle medium (DMEM), fetal calf serum,
glutamine and kanamycin were obtained from Invitrogen. MCF-7
1 h, 2 h, 3 h, 6 h and 22 h at 37.0 ꢂC. A sample of blood (1 mL) was
taken from the solution each time. The sample was centrifuged at
5000 rpm for 5 min to separate plasma and cellular fractions, and
individual fractions were counted in a well counter. The plasma was
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T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
5
then treated with CH3CN (1 mL) and centrifuged as before and the
under slight ether anesthesia. The dose employed was 80
animal (in 300
with radiotracer, together with a blocking dose (15
m
Ci per
supernatant was separated from the precipitated debris. The frac-
tions were again counted, with the bulk of activity residing in the
plasma supernatant. The radioactive species in the supernatant
were analysed by radio-HPLC.
m
L volume), and one set of animals was coinjected
m
g) of estradiol.
At the specified time points postinjection of the 3, groups of three
animals each were sacrificed by heart puncture; tissues of interest
were removed and weighed; and radioactivity was counted. The
injected dose (ID) was calculated by comparison with dose stan-
dards prepared from the injected solution of appropriate counting
rates, and the data were expressed as percentage of ID per gram of
tissue (% ID/g of tissue). For each animal group, the data were
averaged and standard deviation was calculated. Data were
2.11.6. In vivo biodistribution
The biodistribution studies were carried out according to the
relevant national regulation using mature (5e6 weeks old) female
Wistar rats weighing between 180 and 200 g. Purified 3 was dis-
solved in 15% ethanolesaline buffer and injected via the tail vein
NO2
O
O
tBuMe2SiO
OSiMe2tBu
Zn, TiCl4
THF, reflux
NH2
OSiMe2tBu
tBuMe2SiO
OSiMe2tBu
tBuMe2SiO
NH2
E -4
Z-4
FcCOCl
FcCOCl
CH2Cl2, py
CH2Cl2, py
O
NH
OSiMe2tBu
Fe
O
tBuMe2SiO
OSiMe2tBu
tBuMe2SiO
NH
E -5
Z-5
Fe
nBu4NF
THF, rt
nBu4NF
THF, rt
O
NH
OH
NH
Fe
O
HO
OH
HO
E -1
Z-1
Fe
Scheme 1. Synthesis of ferroceny triaryl butene 1.
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6
T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
O
OSiMe2tBu
OSiMe2tBu
Cl
Mn(CO)3
CH2Cl2, py
O
tBuMe2SiO
NH
tBuMe2SiO
NH2
E-4
-6
E
Mn(CO)3
HClconc
Ethanol
OH
O
HO
NH
-2
E
Mn(CO)3
Scheme 2. Synthesis of manganese triaryl butene E-2.
analysed by multifactorial analysis of variance (ANOVA, SPSS 12.0
for Windows) and significant differences among treatments were
considered using Student’s t test at the P < 0.05 level.
Amine 4 should be prepared from McMurry coupling between
4-t-butyldimethylsilyl-propiophenone and 4-amino-40-t-butyldi-
methylsilylbenzophenone. However, it has been demonstrated that
aminobenzophenone is not convenient for this coupling reaction as
the yield of alkene was very low [13]. To overcome this difficulty,
the nitro compound was used instead of the amine compound.
Thus, the coupling between 4-t-butyldimethylsilyl-propiophenone
and 4-nitro-40-t-butyldimethylsilyl-benzophenone led to the
formation of amine 4. The formation of 4 results from the in situ
reduction of nitro function into amine function. The two isomers Z
and E were separated by column chromatography and obtained
with 26% and 12% yields, respectively. The identification of Z and E
forms of the two isomers was achieved by chemical correlation to
Z-5. Isomer E-4 reacts with ferrocene carboxy chloride in
dichloromethane in the presence of pyridine to produce amide E-5
in 51% yield. In the same conditions, isomer Z-4 gave Z-5 in 64%
yield. The structure of Z-5 was identified by NOESY 2D NMR tech-
nique and this identification allows the structural attribution for all
isomers in this paper. The deprotection of the phenol groups was
done by using nBu4NF reagent. Compound Z-1 was obtained in 80%
yield from Z-5 with less than 6% of E-1 while the deprotection of E-5
gave 1 with a proportion E:Z ¼ 57:43 in 84% yield. It is clear that the
isomerization of the E isomer is a fast process. The isomerization of
Z isomer is slower than that of the E isomer, after one day in
methanol the proportion between Z and E isomers was Z:E ¼ 66:34
and became Z:E ¼ 50:50 after 4 days.
2.11.7. Modelling studies
Molecular modelling studies were carried out using the
programs Spartan and Odyssey [36]. Docking experiments were
performed using the crystal structure of human ERa (hERa) crys-
tallized with hydroxytamoxifen (OHTAM) (pdb 3erd) [37]. Only the
amino acids that constitute the wall of the cavity have been
retained. The hydroxytamoxifen molecule was removed from the
cavity and replaced successively with 1, 3 and 2. Energy minimi-
zation was then carried out using Merck molecular force field
(MMFF). All the heavy atoms of the amino acids of the cavity wall
were then immobilized and the side chain of His524 was left for
free movement. This requires calculation of the energies of
bioligandecavity pair, of the cavity itself, and of the ligand, the
latter two in the conformations they had in the molecular assem-
blies to give the DrHꢂ enthalpy variations for the reactions.
3. Results and discussion
3.1. Synthesis
The synthetic pathway of ferrocene compound 1 is shown in
Scheme 1.
O
O
NH
NH
Fe
-
99mTcO4 , 150°C, 1h
99m
Tc(CO)3
DMF, Mn(CO)5Br
HO
OH
HO
OH
Z -1
Z-3
Scheme 3. Synthesis of 99mTc triaryl butene Z-3.
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T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
7
previous results [34], the cyclopentadiene ReCOe(C5H5), precursor
of ReCOe(C5H4)ꢀ, should be obtained from the photochemical
decomplexation of cymantrene 2. However, due to the easy poly-
merization of ReCOe(C5H5), it was not possible to isolate the
monomer compound from the photochemical reaction. Therefore,
the metal exchange reaction from ferrocene compound appeared to
be a useful alternative method. Thus, Z-1 was heated with 99mTcO4ꢀ
and Mn(CO)5Br in DMF:saline solution 1:1 at 150 ꢂC for 1 h (Scheme
3). Compound Z-3 was obtained in 70e75% after purification by TLC
and identified by comparing its chromatogram to that of the
manganese compound E-2 (Fig. 1). Z-3 and E-2 show single peaks at
24.3 and 23.1 min, respectively. The chromatogram shows a high
purity of the technetium compound.
Another possibility for the synthesis of Z-3 is the reaction of Z-1
with [99mTc(CO)3(H2O)3]þ generated from 99mTcOꢀ4 [29]. We have
previously succeeded in preparing 99mTc compounds by using this
second method [17,18]. Surprisingly, the direct reaction of Z-1 with
[
99mTc(CO)3(H2O)3]þ did not form Z-3 in good yield.
3.2. Biochemical studies
The relative binding affinity (RBA) of 1 and 2 for the estrogen
receptor has been measured and the values found (3.1 and 4%
respectively) show that these compounds are recognized by the ER
even if these values are relatively modest.
The effect of compounds 1 and 2 on the proliferation of cancer
cells has been tested on hormone-dependent MCF-7 breast cancer
cells and hormone-independent MDA-MB-231 breast cancer cells.
MCF-7 cells were incubated with 1
m
M of (Z þ E)-1 or E-2. (Z þ E)-1
from the synthesis of E-1 was used because of the ease of isomer-
ization of 1 in solution. As the isomerization of E-2 also occurs in
solution, its antiproliferative effect should be attributed to
a mixture of Z and E isomers. The results obtained are reported in
Table 1.
Fig. 1. (a) UV-HPLC (254 nm) chromatogram of E-2 and (b) Radio-HPLC chromatogram
of Z-3.
On MCF-7 cells, after 5 days of culture, the proliferation was
reduced from 100% to 59.1 ꢃ 2% for 1 and 52.3 ꢃ 3.3% for 2 at 1
Thus, both compounds show almost the same antiproliferative
activity with IC50 values around 1 M. Therefore, these two
compounds are only slightly less active than ferrocenyldiphenol
M). The cymantrene compound 2 is as active as the
ferrocene compound 1 against MCF-7 cells, this is quite surprising
as the cymantrene diphenol complex was previously described as
non cytotoxic on this cell line [41]. With IC50 values on MDA-MB-
mM.
The synthetic pathway of the manganese compound 2 is shown
m
in Scheme 2.
Isomer E-4 reacts with (h
5-C5H4COCl)Mn(CO)3 in dichloro-
(IC50 ¼ 0.7
m
methane and in the presence of pyridine to give amide E-6 in 46%
yield. The deprotection reaction using nBu4NF led to the formation
of several compounds. By contrast, the reaction with concentrated
HCl is cleaner, heating E-6 with HCl in ethanol at 70 ꢂC for 20 min
gave E-2 with 62% yield. The isomerization of E-2 is slower than
that of ferrocene compound E-1, after 1 day in methanol the
proportion between E and Z isomers was Z:E ¼ 88:12 and became
E:Z ¼ 59:39 after 4 days.
231 of 4.53 ꢃ 0.34
m
M for 1 and 9.4 ꢃ 0.12
mM for 2, both
compounds appear to be less active on this cell line than against
MCF-7 cells with 1 being twice more active than 2.
As 3 and 2 have similar structures, their biological properties
should be similar, but the main interest in 3 lies in its possible use
as a radiopharmaceutical. Z-3 was obtained in pure form in 70e75%
yield. Owing to the ease of isomerization process, 3 must be
considered as a mixture of Z and E isomers. Its in vitro stability was
studied in isolated whole rat blood following the procedure
described in the literature [42]. Around 93% of the radioactivity was
found in the supernatant separated from the pellet over the period
The synthesis of technetium compound Z-3 was carried out
according to the method firstly developed by Wenzel [14,15] and
which has proved to be suitable for synthesis of technetium
cyclopentadienyl tricarbonyl complexes [38,39]. An alternative
method was the reaction of cyclopentadienide ReCOe(C5H4)ꢀ with
99mTcOꢀ4 , method developed by Alberto et al. [40]. According to our
Table 1
Antiproliferative effect of compounds 1 and 2 on MCF-7 and MDA-MB-231 breast cancer cells.
Compound
RBA (%) on ER
a
Effect on the growth of cancer cellsa
MCF-7
MDA-MB-231
1
m
M
10
m
M
1
m
M
10
26.2 ꢃ 3.3% (IC50 ¼ 4.53 ꢃ 0.34
48.9 ꢃ 0.7% (IC50 ¼ 9.4 ꢃ 0.12
mM
(Z þ E)-1
(Z þ E)-2
3.1
4.2
59.1 ꢃ 2%
19.7 ꢃ 1.5%
14.8 ꢃ 1%
81.1 ꢃ 6.2%
95.8 ꢃ 0.4%
m
M)
M)
52.3 ꢃ 3.3%
m
a
Control ¼ cells without added compounds after 5 days of culture, set at 100%.
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8
T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
Table 2
liver > lung > kidney > heart > spleen > ovaries > bone > muscle >
uterus. After 15 min the uptake by liver and lung (respectively, 17.47
and 13.89%) is significantly higher than that of other organs. There is
a significant uptake by ovaries (1.76 ꢃ 0.10%) but this uptake is lower
thanthatofkidney,heart,andspleen.Interestingly,theuptakeof3by
uterusissimilartothatofthetechnetiumchelatepreparedbyHunter
et al. [43] but lower that than of [77Br]-estradiol [44] and [131I]-
tamoxifen [45] (Chart 3). This could be due to the use in our experi-
ment of mature female rat in which estrogen receptors are occupied
byendogenousestradiol[46].Here,theuptakebyuterusislowerthan
that of ovaries and in the same range as for muscle and bone. Inter-
estingly, theovaries/muscleratiois3.89buttheovaries/bloodratiois
1.27, thus opening the possibility of imaging.
Tissue biodistribution of 3 in mature female rats e percent injected dose/g of
tissue ꢃ s.d. (% ID/g of tissue).
% ID/g of tissue ꢃ s.d. (n ¼ 3)
15 min (3
alone)
15 min
1 h
6 h
(3 þ E2)a
Blood
Liver
Bone
Muscle
Lung
Uterus
Ovaries
Kidney
Spleen
Heart
Ovaries\blood
Ovaries \Muscle
Uterus\blood
Uterus\Muscle
1.42 ꢃ0.28
1.28 ꢃ0.02 0.45 ꢃ0.09 0.04 ꢃ0.02
17.47 ꢃ1.80 17.15 ꢃ0.97 4.54 ꢃ0.31 5.32 ꢃ0.44
0.51 ꢃ0.06
0.45 ꢃ0.05
0.34 ꢃ0.09 0.24 ꢃ0.08 0.03 ꢃ0.001
0.24 ꢃ0.07 0.22 ꢃ0.02 0.06 ꢃ0.01
13.89 ꢃ1.20 14.00 ꢃ0.79 2.34 ꢃ0.70 0.23 ꢃ0.04
0.44 ꢃ0.15
1.76 ꢃ0.10
4.32 ꢃ0.15
2.43 ꢃ0.31
2.44 ꢃ0.38
1.27 ꢃ0.32
3.89 ꢃ0.35
0.30 ꢃ0.05
0.99 ꢃ0.40
0.13 ꢃ0.05 0.20 ꢃ0.05 0.04 ꢃ0.01
0.25 ꢃ0.04 0.42 ꢃ0.03 0.12 ꢃ0.02
1.73 ꢃ0.24 1.48 ꢃ0.31 0.47 ꢃ0.03
2.73 ꢃ0.68 0.70 ꢃ0.13 0.17 ꢃ0.04
1.17 ꢃ0.10 0.55 ꢃ0.05 0.22 ꢃ0.03
0.19 ꢃ0.03
To determine whether this uptake was mediated by estrogen
receptors or not, 18
mg/g of 17b-estradiol were co-injected with
80 Ci of 3 in order to prevent its binding to the estrogen receptors.
m
1.11 ꢃ0.47
0.1
ꢃ0.04
The results are shown in Table 2. Clearly, at 15 min the uptake by
organs containing estrogen receptors was dramatically reduced
(from 0.44 to 0.13 in uterus and 1.76 to 0.25 in ovaries) while no
obvious decrease was observed in liver, lung and spleen. This result
0.61 ꢃ0.06
a
Co-administration with 15
m
g 17
b-estradiol (E2).
OH
OH
MeO
MeO
125I
77Br
O
HO
HO
N
H
OCH2CH2NMe2
O
O
N
N
S
O
99mTc
S
Chart 3. Selected radioactive derivatives of estradiol and tamoxifen used for in vivo studies.
of 3 h, indicating little binding of 3 to cells or blood cellular
proteins. According to the radio-HPLC analysis of the supernatant,
Z-3 was the only radioactive species observed during 30 min of
incubation. After this time, the isomerization of Z-3 started to be
visible. The same result was observed when Z-3 was incubated in
phosphate buffer at 37 ꢂC for 22 h.
supports the hypothesis that the observed uptake is mediated by
estrogen receptors and is in accordance with the RBA values found
for the complexes (vide supra).
An additional proof of the existence of an interaction between 2
(and its technetium analog 3) and the estrogen receptor is provided
by molecular docking experiments performed by using the crystal
Purified Z-3 was dissolved in 15% ethanol-saline buffer and
injected into mature female Wistar rats via the tail vein. The dose of
structure of human ERa (hERa) crystallized with OH-TAM as
described before [37,47]. Briefly, OH-TAM was removed from the
cavity and replaced successively with the three complexes (bio-
ligands) 1, 2, 3. Energy minimization was then carried out using
Merck molecular force field (MMFF) and the DrHꢂ enthalpy varia-
tions for the reactions: bioligand þ cavity / molecular assembly
was calculated (Table 3).
For all compounds, binding to the ER is thermodynamically
favored, as evidenced by the negative enthalpy of formation for the
ligandereceptor complex. However, these enthalpy variation
values are much lower than that of Z-OH-TAM (ꢀ151.2 kcal molꢀ1).
The result shows that the E conformation is more favored than the Z
conformation. The enthalpy variation values for all compounds are
in the same range suggesting that 1, 2 and 3 have similar affinities
Z-3 employed was 80
mCi/animal. Animals were sacrificed at
15 min, 1 h, and 6 h post-injection. Due to isomerization of
compound in solution over the time, the uptake result must be
attributed to a mixture of Z and E isomers of 3. Table 2 presents the
uptake of 3 in selected tissues of mature (180e200 g) female Wistar
rats. The uptake values were calculated as the percent of injected
dose per gram of tissue.
Theuptakewasmeasuredfrom15minto6h.At15minpost-injection,
the uptake by organs reaches the maximum and follows the order:
Table 3
Enthalpy variation values (DrHꢂ) of 1, 3, and 2 docked in
for the estrogen receptor ERa.
the alpha form of the human estrogen receptor (hERa).
Compound
DrHꢂ (kcal molꢀ1
)
4. Conclusion
Z-OH-TAM
Z-1
E-1
Z-3
E-3
ꢀ151.2
ꢀ70.4
ꢀ98.5
ꢀ61.9
ꢀ91.8
ꢀ62.2
ꢀ81.5
We have prepared ferrocenyl, and cymantrenyl triaryl butenes.
These compounds show a facile isomerization between Z and E
isomers that makes their separation not essential for a possible use
in biology. Both compounds, 1 and 2, show significant anti-
proliferative activities on MCF-7 and MDA-MB-231 (IC50 ¼ 4.53 and
Z-2
E-2
Please cite this article in press as: T. Dallagi, et al., Journal of Organometallic Chemistry (2012), http://dx.doi.org/10.1016/
j.jorganchem.2012.11.035

T. Dallagi et al. / Journal of Organometallic Chemistry xxx (2012) 1e9
9
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9.4 mM for Z-1 and E-2 and around 1 mM on MCF-7 cells). Complex 2
is the first cymantrenyl derivative in the triaryl butene series found
to have a cytotoxic effect. As the isomerization of these two
compounds occurs in solution, these IC50 values are those for the
mixture of Z and E isomers and not for pure isomers. We succeeded
in the synthesis of [99mTc]-technetiumcyclopentadienyltricarbonyl
compound Z-3 from ferrocenyl compound Z-1 via a double metal
exchange reaction. Tissue distribution of 3 showed a higher uptake
by ovaries when compared to uterus. The fact that this uptake is
significantly reduced when the receptor was blocked by co-injection
of large excess of estradiol, means that this uptake is mediated by
estrogen receptors. With respect to structure similarity, the uptake of
1 and 2 should be similar to that of 3. Further studies are in progress
to increase the selectivity of the uptake between ovary and uterus.
Interestingly a crystal structure of (C5H4eR)Re(CO)3 arylsulfonamide
bound to human carbonic anhydrase IX shows hydrophobic inter-
actions with Phe, Leu, Pro inside the binding cavity [28].
Acknowledgements
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We thank the Ministère de l’Enseignement Supérieur et de la
Recherche,theCentreNationaldelaRechercheScientifiquefor financial
support and M.-N. Rager for identification of structures by 2D-NMR
technique.
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Please cite this article in press as: T. Dallagi, et al., Journal of Organometallic Chemistry (2012), http://dx.doi.org/10.1016/
j.jorganchem.2012.11.035