Novel estradiol based metal complexes of Tc-99m

Article abstract of DOI:10.1016/j.jinorgbio.2012.03.001

Aiming to contribute to the design of technetium imaging agents for estrogen receptor (ER) positive breast tumors, we have synthesized and evaluated the novel organometallic estradiol complexes (fac-[M(CO)₃(κ ³-10)]⁺ and f

Full text of DOI:10.1016/j.jinorgbio.2012.03.001

Journal of Inorganic Biochemistry 111 (2012) 1–9
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel estradiol based metal complexes of Tc-99m
a
a
Carina Neto a, Maria Cristina Oliveira a, , Lurdes Gano , Fernanda Marques ,
⁎
Thies Thiemann b, Isabel Santos a
a
Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10,
2686-953 Sacavém, Portugal
b
Faculty of Science, United Arab Emirates University, United Arab Emirates
a r t i c l e i n f o
a b s t r a c t
Article history:
Aiming to contribute to the design of technetium imaging agents for estrogen receptor (ER) positive breast
tumors, we have synthesized and evaluated the novel organometallic estradiol complexes (fac-[M(CO)3
(κ3-10)]+ and fac-[M(CO)3(κ3-12) M=Re/99mTc) using two different bifunctional tridentate ligands (4 and
8). The rhenium complexes (13 and 14) were fully characterized by IR, 1H NMR, 13C NMR, mass spectrometry
and elemental analyses. The 99mTc complexes (15 and 16) were obtained with high radiochemical purity and
exhibited high in vitro radiochemical stability. To get a first insight into the relevance of these complexes for
targeting ER positive tumors, ER binding affinity assays and cellular internalization studies in an ER expressing
cell line, MCF-7, have also been performed suggesting a non ER mediated uptake.
Received 22 December 2011
Received in revised form 5 March 2012
Accepted 5 March 2012
Available online 13 March 2012
Keywords:
Estrogen receptor
Breast cancer
Tricarbonyl complexes
Technetium
© 2012 Elsevier Inc. All rights reserved.
Rhenium
SPECT imaging
1. Introduction
In spite of the better resolution of PET imaging, SPECT still remains
the more practical approach for routine diagnostics in nuclear medi-
Breast cancer is the most malignant form of diagnosed cancer
among women and still remains a major cause of death in the western
world. Due to its propensity to metastasize often, even before its
detection by mammography, an early detection of breast cancer is
determinant for the patients' survival rate. The well documented over-
expression of estrogen receptors (ER) in the majority of breast cancers
makes them relevant biomarkers in diagnosis, prognosis and prediction
of the therapeutic response in ER positive [ER(+)] breast tumors [1,2].
Given the high sensitivity of the nuclear techniques, namely single
photon emission computerized tomography (SPECT) and positron
emission tomography (PET), efforts have been undertaken to design
radioactive probes directed to estrogen-receptors for breast tumor
imaging [3,4].
cine due to longer-lived radionuclides such as iodine-123 and
techentium-99m. During the last decades several efforts have been
made to synthesize estradiol derivatives that could be labeled with
such γ-emitters. Several radioiodinated estradiol derivatives have
been studied [3,4,14]. Among them both isomers of 11β-methoxy-
(17α,20E/Z)-[123I]iodovinylestradiol have been clinically assessed,
with the 20Z isomer giving the better images of ER-positive human
breast tumors. While both primary and metastatic tumors were
detected with good sensitivity and selectivity, extensive correlations
between imaging and clinical outcome have not been provided so
far [15–19]. Efforts have also been directed to the design of steroidal
estrogen derivatives labeled with 99mTc, due to its favorable nuclear
decay properties, low cost and availability [20–30].
Concerning PET-imaging, fluorine-18, a cyclotron-produced radio-
nuclide, has been the most explored radiohalogen for in vivo imaging
studies of estrogen receptors [5–10]. 16α-[18F]-estradiol (18F-FES) is
the most promising candidate for in vivo imaging of estrogen
receptor-positive tumors and is currently in phase II of a study to
predict response to first line hormone therapy in women with ER(+)
metastatic breast cancer [11–13].
Due to the known tolerance of the estrogen receptors for large sub-
stituents at the 7α- and 17α-positions of the estradiol framework,
several radioligands were prepared bearing diverse inorganic and
organometallic rhenium/technetium units tethered to these positions.
Many of these compounds retained high affinity for the cognate
receptor, but the corresponding 99mTc-complexes did not show a
receptor-mediated mechanism in vivo, apparently due to their high
lipophilicity, bulkiness and fast metabolism [21–30]. So far, the most
promising agent is a Re(I)/99mTc(I)-17α-estradiol-pyridin-2-yl hydra-
zine complex described and evaluated by Nayak et al. This complex
showed a receptor-mediated uptake in normal target organ as well as
in human breast adenocarcinoma MCF-7 tumors [31,32]. However, in
⁎
Corresponding author.
E-mail address: cmelo@itn.pt (M.C. Oliveira).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.03.001

2
C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
spite of the encouraging results, further structural modifications are still
needed to improve the imaging.
chromatography on silica gel (ethyl acetate) to give 2 as a yellow oil
(3.3 g, 31%). 1H NMR (CDCl3, 300 MHz) δ: 2.29 (s, 3H, CH3), 2.32 (s,
3H, CH3), 4.26 (t, 2H, 3J=6.8 Hz), 4.53 (t, 2H, 3J=6.8 Hz), and 5.93
(s, 1H).
Aiming to contribute for the design of technetium based radiotracers
as molecular probes for the estrogen receptor, we have prepared
and evaluated two novel Re(I)/99mTc(I)-complexes containing 17α-
substituted estradiol derivatives. These complexes were prepared by
reacting the organometallic fragment fac-[M(CO)3]+ (M=Re, 99mTc)
with 17β-estradiol bearing bifunctional chelators with different donor
atom sets. Herein, we will describe the synthesis and characterization
of the novel estradiol conjugates, the corresponding Re(I)/99mTc(I)
tricarbonyl complexes and their in vitro biological behavior.
2.2.2. 2-[(O)-tert-Butylcarboxamidoethylaminoethyl]-3,5-dimethylpyrazole
(3)
To a solution of 2 (3.29 g, 0.016 mol) in dry acetonitrile (18 mL),
tert-butyl-2-aminoethylcarbamate (3.87 g, 0.02 mol), K2CO3 (3.34 g,
0.02 mol) and KI (0.13 g, 0.8 mmol) were added. The reaction
mixture was heated overnight under reflux. Afterwards, it was cooled
to room temperature, and the solvent was removed in vacuum. The
residue was purified by chromatography on silica gel (CHCl3/MeOH
95/5) to give 3 as an oil (3.9 g, 85%). 1H NMR (CDCl3, 300 MHz) δ:
1.38 (s, 9H, But), 2.15 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.67 (t, 2H,
J=6 Hz), 2.93 (t, 2H, J=6 Hz), 3.14 (m, 2H, J=6 Hz), 3.99 (t, 2H),
5.10 (s, 1H), and 5.73 (s, 1H).
2. Experimental
2.1. General
Unless stated otherwise, all chemicals were of reagent grade and
were used without further purification. 1H NMR and 13C NMR spectra
were recorded at room temperature on a Varian Unity 300 MHz spec-
trometer. 1H NMR and 13C NMR chemical shifts are reported relatively
to residual solvent signals or tetramethylsilane (TMS) as reference.
Electrospray ionization/quadropole ion trap mass spectrometry (ESI/
QITMS) was acquired from a Bruker HCT Mass Spectrometer. Elemental
analyses were performed on an EA 110 CE Instrumental automatic
analyzer. IR spectra were recorded on a Bruker Tensor 27 spectrometer,
using KBr pellets. Chemical reactions were monitored by thin-layer
chromatography (TLC) on Merck plates pre-coated with silica gel
60F254. Column chromatography was performed on silica gel 60
(Merck). Sodium pertechnetate, Na[99mTcO4], was eluted from a
commercial 99Mo/99mTc generator (MDS Nordion S.A.) using 0,9% saline
solution.
2.2.3. N-[2-(4-Bromophenyl)ethyl]-N-{2-[(O)-tert-butylcarboxamido]
ethyl}-N-[2-(3,5-dimethylpyrazol-2-yl)ethyl]amine (4)
1-Bromo-4-(2-bromoethyl)benzene (300 μL, 2 mmol), tetrabuty-
lammonium bromide (TBAB, 2 mg, 0.01 mmol) and a solution of 40%
NaOH (0.2 mL) were added to compound 3 (200 mg, 0.708 mmol).
The reaction mixture was heated under reflux for 3 h. Then, the mixture
was poured into water and extracted with CHCl3 (10 mL), dried over
MgSO4, and the solvent was removed in vacuum. The residue was puri-
fied by chromatography on silica gel [gradient of CHCl3–MeOH (100:0
to 98:2)] to give 4 as a brown oil (173 mg, 52%). 1H NMR (CDCl3,
300 MHz) δ: 1.41 (s, 9H, But), 2.14 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.61
(m, 6H), 2.85 (m, 2H), 3.01 (m, 2H), 3.01 (m, 2H), 4.84 (s, 1H), 5.75
(s, 1H), 6.90 (d, 2H, J=8.1 Hz), and 7.32 (d, 2H, J=8.1 Hz). 13C NMR
(CDCl3, 75 MHz) δ: 11.3, 13.8, 28.7, 33.3, 38.8, 47.0, 53.6, 56.3, 63.5,
79.2, 105.3, 120.0, 130.8, 131.6, 133.4, 139.4, 144.9, and 156.3. ESI/MS
HPLC analysis and purification of the ligands and of the Re/99mTc
complexes were performed on a Perkin Elmer system, equipped with
a biocompatible quaternary pump (LC 200), an UV/Vis detector (LC
290, Perkin Elmer) and a radioactivity detector (LB 509, Berthold). The
ligands and the Re complexes were detected by UV (λ=254 nm) and
99mTc complexes were identified by gamma detection. HPLC analyses
of the unlabelled and labeled compounds were achieved on a reverse-
phase (RP) Nucleosil Column (250×4 mm, 5 μm) eluted with a binary
gradient system with a flow rate of 1 mL/min using the same binary
system — eluents: A-methanol, B-aqueous 0.1% TFA, method 1:
0–5 min 25% A, 5–20 min 25–100% A, 20–30 min 100% A. Method 2:
0–5 min 30% A, 5–5.1 min 30–50% A, 5.1–8.1 50% A, 8.1–13.1 min 50–
70% A, 13.1–16.1 min 70% MeOH, 16.1–21.1 min 70–100% A, and
21.1–30 min 100% A. HPLC purifications were performed on a semi-
preparative reverse-phase VP-Nucleosil C18 column (250×8 mm,
7 μm) with a flow rate of 2 mL/min using the same binary system —
eluents: A-methanol, B-aqueous 0.1% TFA solution, method 3:
0–6 min 70% A; 6–8 min 70–85% A; and 8–30 min 85%A.
m/z: 467.0 (calcd. 467.2) [M+H]+
.
2.2.4. 5-(Bromopyrid-2-yl)-methanol (6)
To solution of 5-(bromopyrid-2-yl)-carbaldehyde (700 mg,
a
3.76 mmol) in dry methanol (8 mL) was added, slowly, NaBH4
(286 mg, 7.53 mmol), and the resulting reaction mixture was stirred
overnight at room temperature. Afterwards, the solvent was removed
in vacuum, the residue was dissolved in ethyl acetate (20 mL), and the
resulting solution was washed with a solution of Na2CO3 (3×20 mL).
The organic layer was separated, dried over MgSO4 and the solvent
was removed to give 6 as a white solid (657 mg, 93%).1H NMR
(CD3OD, 300 MHz) δ: 4.64 (s, 2H), 7.48 (d, 1H, J=8.4 Hz), 7.99 (dd,
1H, J=2.1 Hz, J=8.4 Hz), and 8.55 (d, 1H, J=2.1 Hz). 13C NMR
(CD3OD, 75 MHz) δ: 63.9, 118.8, 122.4, 134.0, 149.4, and 160.2. ESI/
MS m/z: 188.1 (calcd. 188.0) [M+H]+
.
The rhenium starting complex [Re(CO)3(H2O)3]Br was prepared
as reported elsewhere [33]. The radioactive precursor fac-[99mTc(CO)3
(H2O)2]+ was prepared using an Isolink® kit (Mallinckrodt), according
to the manufacturers. Radioactivity measurements were carried out on
an ionization chamber (Aloka Curiemeter, IGC-3, Japan) or a gamma-
counter (Berthold, LB 2111, Germany).
2.2.5. 2-Bromomethyl-5-bromopyridine (7)
A solution of triphenylphosphine (PPh3, 2.01 g, 7.68 mmol) in dry
THF (8 mL) was added, slowly, to a mixture of 6 (657 mg, 3.49 mmol)
and CBr4 (2.3 g, 6.98 mmol). The reaction mixture was stirred over-
night at room temperature. Then, the mixture was filtered, and the
solvent was removed in vacuum. The residue was purified by chroma-
tography on silica gel (CH2Cl2/MeOH 98:2) to give 7 as a brown oil
(507 mg, 58%). 1H NMR (CD3OD, 300 MHz) δ: 4.54 (s, 2H), 7.45 (d,
1H, J=8.8 Hz), 7.96 (dd, 1H, J=8.8 Hz, J=2.4 Hz), and 8.57 (d, 1H,
J=2.4 Hz). 13C NMR (CD3OD, 75 MHz) δ: 31.8, 120.1, 125.4, 140.5,
2.2. Chemistry
2.2.1. N-(2-bromoethyl)-3,5-dimethyl-1H-pyrazole (2)
To a solution of 3,5-dimethylpyrazole (5.0 g, 0.05 mol) in 1,2-
dibromoethane (45 mL, 0.52 mol), 40% aq. NaOH solution (15.6 mL)
and tetrabutylammonium bromide (0.432 g, 1.0 mmol) were added.
The reaction mixture was heated for 2 h under reflux. Thereafter,
the reaction mixture was cooled to room temperature, the phases
were separated, the organic phase was dried over MgSO4, and the
solvent was removed in vacuum. The residue was purified by
150.2, and 156.0. ESI/MS m/z: 274.0 (calcd. 273.9) [M+Na]+
.
2.2.6. Ethyl 2-[(5-bromopyrid-2-yl)methylamino]acetate (8)
To a solution of 7 (55 mg, 0.22 mmol) in dry acetonitrile (5 mL), gly-
cine ethyl ester hydrochloride (31 mg, 0.22 mmol), K2CO3 (60.7 mg,
0.44 mmol) and KI (2 mg, 0.011 mol) were added. The reaction mixture
was heated overnight under reflux. Thereafter, the reaction mixture

C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
3
was cooled to room temperature, and the solvent was removed in vac-
uum. The residue was purified by chromatography on silica gel (CHCl3/
MeOH 95:5) to give 8 as a brown oil (28 mg, 47%). 1H NMR (CD3OD,
300 MHz) δ: 1.25 (t, 3H, J=7.2 Hz), 3.42 (s, 2H), 3.87 (s, 2H), 4.17 (q,
2H, J=7.2 Hz), 7.38 (d, 1H, J=8.4 Hz), 7.94 (dd, 1H, J=2.4 Hz,
J=8.4 Hz), and 8.58 (d, 1H, J=2.4 Hz). 13C NMR (CD3OD, 75 MHz) δ:
13.4, 49.4, 52.9, 60.7, 119.0, 124.2, 139.8, 149.8, 157.8, and 172.0. ESI/
80.4, 83.4, 96.1, 112.8, 115.3, 121.7, 126.5, 132.2, 138.2, 139.3, 151.7,
153.7, 159.1, and 170.2. ESI/MS m/z: 489.3 (calcd. 489.3) [M+H]+
.
2.2.10. 17α-[2-(Hydroxycarbonylmethylaminomethyl)pyrid-5-yl-ethynyl]
estra-1,3,5(10)-triene-3,17β-diol (12)
A solution of 11 (45 mg, 0.10 mmol) in THF (10 mL) was added to
a solution of NaOH (92 mg) in water (4 mL), and the resulting reac-
tion mixture was heated overnight under reflux. Then, the solution
was neutralized, and the solvent was removed. The residue was redis-
solved in THF (10 mL), filtered, and the solvent was removed, to give
12 as a brown oil (40 mg, 94%). 1H NMR (CD3OD, 300 MHz) δ: 0.83 (s,
3H, CH3), 1.15–2.40 (m, 13H), 2.77 (m, 2H), 3.58 (s, 2H), 4.36 (s, 2H),
6.47 (d, 1H, 4J=2.7 Hz), 6.53 (dd, 1H, 4J=2.7 Hz, 3J=8.4 Hz), 7.09
(d, 1H, 3J=8.4 Hz), 7.41 (d, 1H, 3J=8.4 Hz), 7.85 (dd, 1H, 4J=
2.1 Hz, 3J=8.4 Hz), and 8.65 (d, 1H, 4J=2.1 Hz). 13C NMR (CD3OD,
75 MHz) δ: 12.2, 22.7, 26.5, 27.3, 29.5, 34.2, 38.7, 39.8, 43.7, 47.0,
47.3, 48.7, 50.2, 79.9, 83.5, 98.0, 112.6, 114.9, 122.5, 125.0, 126.2,
128.4, 131.2, 137.6, 139.8, 151.4, 154.7, and 167.6. ESI/MS m/z:
MS m/z: 297.0 (calcd. 297.0) [M+Na]+
.
2.2.7. 17α-[(2-tert-butoxycarbamido-ethyl)-(2-(3,5-dimethyl-1H-
pyrazol-1-yl)-ethyl)amino)-4-ethylphenyl]-ethynyl-estra-1,3,5(10)-
triene-3,17β-diol (9)
A solution of Pd(PPh3)2Cl2 (15.1 mg, 0.02 mmol) in diisopropyla-
mine (5 mL) was stirred for 10 min under a nitrogen atmosphere.
CuI (4.1 mg, 0.02 mmol) and 4 (100 mg, 0.215 mmol) were added.
After the mixture was stirred for 5 min, 17α-ethynylestradiol
(63.7 mg, 0.215 mmol) was added and the solution was stirred at
55 °C for 4 h. The volatiles were removed in vacuum, and the residue
was purified by column chromatography on silica gel (eluant: CH2Cl2:
MeOH 100:0 to 95:5), to give 9 (80 mg, 55%) as a brown oil.1H NMR
(CDCl3, 300 MHz) δ: 0.83 (s, 3H, CH3), 1.20–2.37 (m, 11H), 1.34 (s,
9H, But), 2.21 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.52–2.63 (m, 6H),
2.77 (m, 3H), 2.86 (m, 2H), 3.03 (m, 2H), 3.93 (t, 2H), 5.77 (s, 1H),
6.55 (d, 1H, 4J=2.4 Hz), 6.61 (dd, 1H, 4J=2.4 Hz, 3J=8.4 Hz), 7.01
(d, 2H, J=8.1 Hz), 7.13 (d, 1H, 3J=8.4 Hz), and 7.31 (d, 2H,
J=8.1 Hz). 13C NMR: (CDCl3, 75 MHz) δ: 10.9, 12.8, 13.2, 22.9, 26.4,
27.2, 28.4, 29.6, 33.0, 33.2, 38.9, 39.4, 43.3, 46.6, 47.5, 47.8, 49.6,
53.0, 53.4, 55.6, 80.4, 85.8, 92.4, 105.1, 112.8, 115.3, 120.5, 126.3,
128.6, 131.6, 137.8, 139.3, 140.4, 147.5, and 154.2. ESI/MS m/z:
461.3 (calcd. 461.2) [M+H]+
.
IR υmax (KBr)/cm−1
:
1584
(υ(C_O)). Elemental analysis (%) for C28H32N2O4: Found: C, 73.08;
N, 6.00; and H, 6.95. Calculated: C, 73.02; N, 6.08; and H, 7.00.
2.2.11. Synthesis of Re(I) complexes
2.2.11.1. General procedure. [Re(H2O)3(CO)3]Br was reacted with equi-
molar amounts of 10 or 12 (0.10 mol) in refluxing methanol (5 mL)
for 18 h. Then, the solvent was removed in vacuum and the desired
complexes were purified by chromatography.
681.2 (calcd. 681.4) [M+H]+
.
2.2.11.2. fac-[Re(CO)3(κ3−10)]+ (13). The complex was purified by
HPLC-RP (method 3) to give 13 (33%). 1H NMR (CD3OD, 300 MHz)
δ: 0.91 (s, 3H, CH3), 1.28–2.51 (m, 12H), 2.37 (s, 3H, CH3), 2.45 (s,
3H, CH3), 2.51 (m, 1H), 2.82 (m, 5H), 2.92 (m, 2H), 3.55 (m, 4H),
3.76 (m, 1H), 4.10 (m, 1H), 4.27 (m, 1H), 4.55 (m, 1H), 5.50 (m,
1H), 6.22 (s, 1H), 6.47 (d, 1H, 4J=2.4 Hz), 6.53 (dd, 1H, 4J=2.4 Hz,
3J=8.4 Hz), 7.10 (d, 1H, 3J=8.4 Hz), 7.31 (d, 2H, J=8.1 Hz), and
7.42 (d, 2H, J=8.1 Hz). 13C NMR (CD3OD, 75 MHz) δ: 10.4, 12.3,
14.9, 22.7, 26.6, 27.5, 29.6, 31.2, 33.3, 38.8, 40.0, 42.5, 44.1, 50.0,
53.5, 62.2, 79.7, 84.8, 93.4, 108.1, 112.6, 114.9, 122.2, 126.1, 129.0,
131.3, 131.8, 137.7, 138.0, 144.2, 153.9, 154.8, 192.4, 193.7, and
194.1. ESI/MS m/z: 851.1 (calcd. 851.3) [M]+. Elemental analysis (%)
for C40H48N4O5Re.CF3COOH. Found: C, 49.01; N, 4.64; and H, 5.27. Cal-
culated: C, 49.02; N, 4.58; and H, 5.20. IR νmax (KBr)/cm−1 1912;
2030 (C`O).
2.2.8. 17α-[(N-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-ethyl)ethane-1,2-
diamino)-4-ethylphenyl]-ethynyl-estra-1,3,5(10)-triene-3,17β-diol (10)
To a solution of 9 (111 mg, 0.16 mmol) in methanol (5 mL), was
added, slowly, a solution of HCl (1 mL, 12 M), and the reaction mix-
ture was stirred for 24 h at room temperature. Then, the solvent
was removed in vacuum. The residue obtained was purified by
HPLC-RP (method 1), to give 10 (32 mg, 34%). 1H NMR (CD3OD,
300 MHz) δ: 0.86 (s, 3H, CH3), 1.15–2.32 (m, 11H), 2.16 (s, 3H,
CH3), 2.31 (s, 3H, CH3), 2.76–2.87 (m, 7H), 3.07 (m, 2H), 3.17 (m,
2H), 3.74 (m, 2H), 4.27 (m, 2H), 5.96 (s, 1H), 6.46 (d, 1H,
4J=2.4 Hz), 6.52 (dd, 1H, 4J=2.4 Hz, 3J=8.4 Hz), 7.07 (d, 2H,
J=8.1 Hz), 7.16 (d, 1H, 3J=8.4 Hz), and 7.35 (d, 2H, J=8.1 Hz).13
C
NMR: (CDCl3, 75 MHz) δ: 11.2, 13.3, 13.5, 23.7, 27.5, 28.4, 30.6, 34.3,
35.2, 39.8, 39.9, 40.9, 41.1, 43.5, 44.7, 45.1, 51.1, 51.9, 56.1, 70.7,
80.8, 84.6, 94.7, 109.5, 113.7, 116.0, 123.6, 127.2, 130.3, 132.4, 132.9,
137.2, 138.6, 138.7, 148.1, and 155.8. ESI/MS m/z: 581.8 (calcd.
581.4) [M+H]+. Elemental analysis (%) for C37H48N4O2: Found: C,
61.09; N, 6.95; and H, 6.30. Calculated: C, 60.88; N, 6.93; and H, 6.23.
2.2.11.3. fac-[Re(CO)3(κ3−12)] (14). The complex was purified by col-
umn chromatography on silica gel (CHCl3/MeOH/NH4OH 80:20:2) to
give 14 (57%). 1H NMR (CD3OD, 300 MHz) δ: 0.83 (s, 3H, CH3), 1.28–
2.38 (m, 13H), 2.77 (m, 2H), 3.72 (m, 2H), 4.29 (s, 1H), 4.56 (s, 2H),
6.47 (d, 1H, 4J=2.4 Hz), 6.53 (dd, 1H, 4J=2.4 Hz, 3J=8.4 Hz), 7.08
(d, 1H, 3J=8.4 Hz), 7.68 (d, 1H, 3J=8.4 Hz), 8.05 (dd, 1H, 4J=
2.1 Hz, 3J=8.0 Hz), and 8.82 (d, 1H, 4J=2.1 Hz). 13C RMN (CD3OD,
75 MHz) δ: 12.3, 22.7, 26.6, 27.4, 29.6, 33.4, 38.6, 40.0, 43.9, 47.0,
50.2, 53.8, 62.2, 69.9, 79.7, 82.0, 112.6, 114.9, 122.3, 123.5, 126.1,
131.2, 137.6, 141.9, 154.0, 154.8, 158.9, 182.9, 195.2, 196.0, and
196.8. ESI/MS m/z: 731.3 (calcd. 732.3) [M+H]+. Elemental analyses
(%) for C31H32N2O7Re. Found: C, 51.00; N, 3.75; and H, 4.50. Calculat-
ed: C, 50.95; N, 3.83; and H, 4.41. IR νmax (KBr)/cm−1: 1889, 2028
(C`O), 1612 (C_O).
2.2.9. 17α-[2-(Ethoxycarbonylmethylaminomethyl)pyrid-5-yl-ethynyl]
estra-1,3,5(10)-triene-3,17β-diol (11)
A solution of Pd(OAc)2 (4.2 mg, 0.02 mmol) and PPh3 (9.7 mg,
0.04 mmol) in diethylamine (5 mL) was stirred for 10 min under a
nitrogen atmosphere. Then, CuI (7 mg, 0.37 mmol) and 8 (100 mg,
0.37 mmol) were added, and the mixture was stirred for 5 min.
17α-ethynylestradiol (110 mg, 0.37 mmol) was added and the solu-
tion was stirred at 55 °C for 4 h. The volatiles were removed in vacuum,
and the residue was purified by column chromatography on silica gel
(CH2Cl2/MeOH 98:2) to give 11 (112 mg, 68%). 1H NMR (CDCl3,
300 MHz) δ: 0.84 (s, 3H, CH3), 1.23 (t, 3H, J=6.9 Hz), 1.20–2.40 (m,
13H), 2.77 (m, 2H), 3.43 (s, 2H), 3.93 (s, 2H), 4.17 (q, 2H), 6.52 (d, 1H,
4J=2.4 Hz), 6.59 (dd, 1H, 4J=2.4 Hz, 3J=8.4 Hz), 7.12 (d, 1H,3J=
8.4 Hz), 7.29 (d, 1H, 3J=8.4 Hz), 7.68 (d, 1H, 4J=2.4 Hz, 3J=8.4 Hz),
and 8.60 (d, 1H, 4J=2.4 Hz). 13C NMR (CDCl3, 75 MHz) δ: 12.9, 14.2,
23.0, 26.5, 27.2, 29.6, 33.2, 38.9, 39.5, 43.6, 47.7, 49.9, 50.8, 54.1, 60.9,
2.3. Synthesis of 99mTc(I) complexes (15, 16)
2.3.1. General procedure
In a nitrogen-purged glass vial, 100 μL of 10−3 or 10−4 M ethanolic
solution of 10 or 12 was added to 900 µL of a solution of
fac-[99mTc(OH2)3(CO)3]+ (1–2 mCi) in PBS at pH=7.4. The reaction

4
C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
mixture was incubated for 30 min at 100 °C and analyzed by HPLC (γ
detection). tr=23.37 min (method 1) for fac-[99mTc(CO)3(κ3-10)]+
(15). Yield >96%, tr=23.93 min (method 2) for fac-[99mTc(CO)3(κ3-
12)] (16).
slurry was added and tubes were incubated on ice and vortexed
three times for 15 min. An aliquot of 1 mL of washing buffer was
added, mixed and centrifuged at 10,000×g for 10 min, and the super-
natants were discarded. This wash step was repeated twice. The HAP
pellets were then resuspended in 750 μL cold ethanol, vortexed three
times in 20 min, centrifuged and the supernatants were transferred to
scintillation vials for measurement of 3H radioactivity in a liquid scin-
tillation counter (Packard Tri-CARB 3170 TR/SL). The data obtained
from triplicate measurements were expressed as the percent specific
binding of [3H]E2 vs. the log molar concentration of the competing
compound. The IC50 values (calculated using GraphPad Prism soft-
ware) represent the concentration of the test compound required to
reduce the [3H]E2 binding by 50%.
2.4. In vitro assays
2.4.1. Partition coefficient measurements
The log Po/w values of complexes 15 and 16 were determined by
the “shake flask” method under physiological conditions [n-octanol/
0.1 M phosphate-buffered saline (PBS), pH 7.4] [34]. The HPLC-
purified compounds (100 μL, approximately 3.7 MBq, 100 μCi) were
added to a test tube containing 1 mL of n-octanol and 1 mL of a PBS
solution. The tube was vortexed for 1 min and centrifuged for 5 min
at 3500 rpm. After centrifugation, 500 μL of the n-octanol was trans-
ferred to another tube and further extracted with 500 μL of aqueous
phase, as described for the first extraction. After separation of the
phases, 50 μL aliquots of each phase were taken for radioactivity
measurements (in duplicate) using a gamma-counter. The partition
coefficient (Po/w) was calculated on the basis of the ratio (activity in
the n-octanol layer)/(activity in the aqueous layer) and is expressed
3. Results and discussion
Several 99mTc-complexes bearing estradiol derivatives have been
previously studied and, for most of them, the conjugation of the
metal fragment to the estradiol was performed through the 7α or
17α position [21–23,26,27,31]. Despite the promising biological data
obtained for the 17α substituted estradiol derivatives none of the
reported analogs was ideal for imaging, due to their low binding affin-
ity, high lipophilicity or low specific activity of the 99mTc-complexes
[32]. Several linkers between the 17α-position and the metal frag-
ment were studied and the ethynyl group was found to be the best,
as it provides an effective separation of the steroid and the metal,
without introducing excessive conformational flexibility [24]. The
introduction of this linker also reduced the affinity of the estrogen
derivatives for α-fetoprotein and sex-hormone steroid-binding
protein, resulting in more favorable in vivo pharmacokinetics [36].
Taking into account these results, we have designed and evaluated
novel estradiol derivatives bearing tridentate chelators, namely a
pyrazolyl-diamine [37,38] and a pyridine-aminocarboxylate [39],
which were linked to the 17α-position through an ethynyl group.
as log Po/w
.
2.4.2. In vitro cellular and nuclear internalization studies
An ER (+) human breast cancer cell line, MCF-7 (ATCC), was used
in this assay. Cells were grown in Dulbecco's Modified Eagle Medium
(DMEM, Gibco), supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin under a humidified 5% CO2 atmosphere. For
the internalization studies, cells were plated at a density of approxi-
mately 2×105 cells per well in 24-well tissue culture plates and
allowed to attach overnight. The cells were incubated in humidified
5% CO2 at 37 °C for a period spanning from 15 min to 4 h with approx-
imately 200,000 cpm of radiocomplex in 0.5 mL of assay medium
(Minimum Essential Medium Eagle (MEM) without phenol red and
0.2% bovine serum albumin). Incubation was terminated by washing
the cells twice with ice-cold PBS. Cell surface bound radioactive com-
plex was removed by two steps of acid wash (50 mM glycine·HCl/
100 mM NaCl, pH 2.8) at room temperature for 3 min. pH was neu-
tralized with cold PBS with 0.2% bovine serum albumin (BSA) and,
subsequently, the cells were lysed in 500 μL of lysis buffer (Tris
10 mM, MgCl2 3 mM, NaCl 10 mM, Nonidet P-40 0.5%, pH 7.5–8.0).
After 30 min of incubation, the cell suspension was removed and cen-
trifuged at 1300 g at 4 °C for 5 min. At different time points over the
4 h incubation period, the radioactivity associated to cell surface
membrane, supernatant (activity outside the nucleus, cytoplasm)
and of the precipitate (activity in the nucleus) were measured (at
least 3 replicates) in a gamma-counter. Radioactivity associated to
each fraction at each time point was expressed as the percentage of
the total activity added to the cells and presented as an average
plus the standard deviation.
3.1. Chemistry
3.1.1. Synthesis of bifunctional ligands (4 and 8)
The bifunctional ligand tert-butyl-2-(4-bromoethylphenyl)-(2-
(3,5-dimethyl-1H-pyrazol-1-ylethyl)amine)ethylcarbamate (4) was
prepared in a three step synthesis as depicted in Scheme 1. 2-
Bromoethyl-3,5-dimethyl pyrazole (2), obtained by N-alkylation of
pyrazole (1) with excess 1,2-dibromothane, underwent a nucleophil-
ic substitution with tert-butyl-2-aminoethylcarbamate, in the pres-
ence of potassium carbonate and potassium iodide, to give tert-
butyl-2[2(3,5-dimethyl-1H-pyrazol-1-yl)ethylamine]ethylcarbamate
(3) [40]. Before coupling the ligand to 17α-ethynylestradiol, the cen-
tral amino group of 3 was functionalized by N-alkylation with 1-
bromo-4-(2-bromoethyl)benzene. The bifunctional ligand
4 was
obtained in 52% overall yield, after purification by column chromatog-
raphy on silica gel (chloroform/methanol). Compound 4 was charac-
terized by NMR spectroscopic techniques (1H/13C NMR) and mass
spectrometry.
2.4.3. Receptor-binding affinity
The ERα competitive binding assay was performed according to a
described method with minor modifications [35]. The ERα binding
buffer used for dilution of the receptor preparations consisted of
10% glycerol, 2 mM dithiothreitol, 1 mg/mL BSA and 10 mM Tris–
HCl (pH 7.5). The ERα washing buffer contained 40 mM Tris–HCl
and 100 mM KCl (pH 7.4). The hydoxyapatite (HAP) slurry was
adjusted to a final concentration of 50% (v/v) by using a 50 mM
Tris–HCl solution (pH 7.4). The reaction mixture contained 50 μL of
varying concentrations of the test compound in the ERα binding buff-
er, 45 μL of a solution of tritiated estradiol (23.8 nM) and 5 μL
(0.25 pmol) of ERα proteins solution. Non specific binding by the tri-
tiated estradiol was determined by the addition of a 50 μM concentra-
tion of the nonradioactive E2. The binding mixture was incubated at
4 °C for 16–18 h. At the end of the incubation, 200 μL of the HAP
The bifunctional ligand 8 was synthesized from precursor 5-
bromopyridine-2-carbaldehyde (5) that was reduced with sodium
borohydride to give 5-bromopyridine-2-methanol (6) as depicted in
Scheme 2. Bromination of the hydroxyl group of 6 under Appel condi-
tions with triphenylphosphine and tetrabromomethane gave 5-
bromo-2-bromomethylpyridine (7), which was subsequently reacted
with ethyl glycate hydrochloride, by direct N-alkylation, in the pres-
ence of potassium carbonate and potassium iodide. The bifunctional
ligand 8 was obtained in 47% yield, after chromatographic purification
on silica gel (eluant: chloroform/methanol) and was charac-
terized by NMR spectroscopic techniques (1H/13C NMR) and mass
spectrometry.

C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
5
Scheme 1. Synthesis of bifunctional ligand tert-butyl-2-(4-bromoethylphenyl)-(2-(3,5-dimethyl-1H-pyrazol-1-ylethyl)amine)ethylcarbamate (4).
Scheme 2. Synthesis of bifunctional ligand ethyl 2-[(5-bromopyrid-2-yl)methylamino]acetate (8).
spectroscopic techniques (1H/13C NMR), mass spectrometry and
elemental analyses.
3.1.2. Synthesis of 17α-substituted estradiol conjugates (10 and 12)
The coupling of the bifunctional ligands (4 and 8) to 17α-
ethynylestradiol (EE) was achieved by Sonogashira reaction with
minor modifications, as depicted in Scheme 3 [41]. The first attempt
to conjugate 4 to EE was based on a previously published procedure,
which has used a mixture of tetrakis(triphenylphosphine)palladium
(0) and copper iodide as catalyst and triethylamine as base [42].
However, under these conditions the yield of the reaction was very
low (b5%). To improve the yield, the use of different palladium cata-
lysts and bases was further investigated. The best results were
obtained when bis(triphenylphosphine)palladium chloride and diiso-
propylamine were used. The resulting compound 9 was obtained in
55%, after purification by column chromatography using silica gel
and a mixture of dichloromethane and methanol as eluent. Initially,
a mixture of trifluoroacetic acid and dichloromethane was used to re-
move the BOC protective group, however, a very complex mixture
was obtained. The subsequent use of a solution of hydrochloric acid
in methanol removed the BOC group efficiently yielding 17α-[(N-(2-
(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)ethane-1,2-diamine)-4-ethyl-
phenyl]-ethynyl-estra-1,3,5(10)-triene-3,17β-diol (10) in 34% yield,
after HPLC purification. The conjugate 10 was characterized by NMR
The estradiol derivative 12 was synthesized as depicted in
Scheme 4. The coupling of 8 to 17α-ethynylestradiol was performed
using bis(triphenylphosphine)palladium diacetate, generated in situ,
and copper iodide as catalyst in presence of diethylamine. The conju-
gate 11 was obtained in 68% yield after purification by column chro-
matography on silica gel (eluant: dichloromethane/methanol). The
ester group was removed by base catalyzed hydrolysis with aq.
NaOH, and the final conjugate 17α-[2-(hydroxycarbonylmethylami-
nomethyl)pyrid-5-yl-ethynyl]-estra-1,3,5(10)-triene-3,17β-diol (12)
was characterized by spectroscopic techniques (IR and 1H/13
NMR), mass spectrometry and elemental analyses.
C
3.1.3. Synthesis of tricarbonyl M(I) complexes (M=Re: 13 and 14;
M= 99mTc: 15 and 16)
The synthesis of the rhenium complexes fac-[Re(CO)3(κ3-10)]+
(13) and fac-[Re(CO)3(κ3-12)] (14) was carried out by reacting fac-
[Re(H2O)3(CO)3]Br with 10 and 12 in refluxing methanol
(Scheme 5). Complexes 13 and 14 were analyzed by spectroscopic
techniques (IR and 1H/13C NMR), mass spectrometry and elemental
Scheme 3. Synthesis of conjugate 17α-[(N-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)ethane-1,2-diamine)-4-ethylphenyl]-ethynyl-estra-1,3,5(10)-triene-3,17β-diol (10).

6
C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
Scheme 4. Synthesis of conjugate 17α-[2-(hydroxycarbonylmethylaminomethyl)pyrid-5-yl-ethynyl]-estra-1,3,5(10)-triene-3,17β-diol (12).
analyses, which allowed for the unambiguous identification of their
chemical structures. The IR spectra of 13 and 14 showed intense ab-
sorption bands between 1889 and 2030 cm−1, easily assigned to
the ν(C`O) stretching modes of fac-[Re(CO)3]+ unit. The 1H NMR
data obtained corroborated a facial coordination through the pyrazo-
lyl ring, the central and the terminal amino groups for 13 and through
the pyridine ring, the central amino group and the terminal carboxyl-
ate for 14, since all the spectra show a set of multiplets for the methy-
lenic protons of the framework of the pyrazolyl-diamine and the
pyridine-aminocarboxylate ligand, which is consistent with the dia-
stereotopic character of the protons.
12) at 100 °C for 30 min at neutral pH (pH 7.4) (Scheme 5). The reac-
tions were almost quantitative (radiochemical yield>95%) and the
99mTc complexes were obtained with high radiochemical purity
(>95%). The chemical identity of 15 and 16 was ascertained by
comparison of their HPLC profiles with those of the corresponding
rhenium complexes 13 and 14, respectively (Fig. 1).
3.2. In vitro assays
The in vitro evaluation of 99mTc complexes 15 and 16 included the
determination of their lipophilicity, their radiochemical stability in
physiological solutions and in human serum as well as the assessment
of their internalization in MCF-7 cells. Binding affinity studies of Re(I)
complexes towards the estrogen receptor are also described.
The 99mTc complexes fac-[99mTc(CO)3(κ3-10)]+ (15) and fac-
[
99mTc(CO)3(κ3-12)] (16) were obtained in aqueous solution by reac-
tion of fac-[99mTc(H2O)3(CO)3]+ with the appropriate ligand (10 or
Scheme 5. Synthesis of tricarbonyl M(I) complexes (M=Re: 13 and 14; M=99mTc: 15 and 16).

C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
7
Fig. 1. Radiochromatograms of the co-elution of 99mTc complexes 15 and 16 with their Re analogs 13 and 14.
3.2.1. Lipophilicity
confirmed that both complexes were stable in these conditions
during the incubation period. The internalization kinetics of both
complexes into the whole cell (radioactivity retained in the cyto-
plasm and nucleus) with and without receptor blockade is presented
in Fig. 3.
The lipophilicity of the complexes 15 and 16 was assessed by mea-
surement of the respective octanol/water partition coefficient using
the “shake-flask” methodology [34]. The octanol/water partition coef-
ficient of estradiol was reported by Pomper et al. as log P=3.26 [5]. The
values of log PO/W of the two 99mTc(I), complexes were found to be up to
almost 100 times lower than that of estradiol (log PO/W(15)=1.08
0.01, log PO/W(16)=1.25 0.07). Nevertheless this reduced lipophilici-
ty can be favorable to improve target tissue selectivity in vivo [32]. Com-
paring these values with the reported value for 17α-ethynylestradiol
(log PO/W=3.42) one can observe a pronounced decrease in lipophilic-
ity, probably due the hydrophilic character of the ligands [5,43,44].
The total cellular internalization of both complexes in MCF-7 cells
was time-dependent with a more evident increase for complex 15,
but the overall rate of internalization is relatively low, even after 4 h
of incubation (2.0 0.3 and 3.2 0.2% for complexes 16 and 15, re-
spectively). Despite the similar lipophilic character of both 99mTc
complexes, the higher rate of internalization of complex 15 possibly
results from its overall positive charge as it is well-established that li-
pophilic cationic complexes tend to localize into tumor cells [45].
Moreover there was no evidence of a relevant decrease in the cellular
uptake of any of the radioactive complexes when cells were previ-
ously treated with estradiol.
3.2.2. Radiochemical stability
The radiochemical stability of the 99mTc complexes (15 and 16)
was evaluated by HPLC analysis of samples at certain time periods
after incubation in physiological saline at 37 °C. Both complexes
were shown to be radiochemically stable up to 4 h in physiological sa-
line. The 99mTc complexes were also incubated with samples of
human serum at 37 °C, after which the samples were treated with
ethanol to precipitate the proteins. HPLC analyses of the ethanolic
extracts have demonstrated high radiochemical stability of the com-
plexes for up to 4 h (Fig. 2).
The nuclear internalization of the 99mTc-complexes in comparison
with their total internalization inside the cells is presented in Fig. 4.
3.2.3. Biological assays
3.2.3.1. Cellular and nuclear internalization. To find out whether the
radiolabelled complexes are effectively taken up in vitro by tumor
cells, cellular and nuclear internalization studies were performed in
an ER positive human breast cancer cell line, MCF-7. Since estrogen
receptors have been identified as nuclear transcription factors, the
degree of cellular/nuclear internalization of the complexes is an
important parameter to predict their ability to be retained into the
tumor cells by a receptor mediated process. In order, to assess specific
cellular uptake similar experiments were performed in parallel by
incubation of the labeled complexes in the presence of 10 μM unla-
belled estradiol to assure receptors saturation. Prior to these cellular
studies the stability of radioactive complexes was evaluated by incu-
bation in the cell growth medium at 37 °C up to 4 h. HPLC analysis
Fig. 2. Radiochemical stability of 99mTc complexes 15 and 16 in human serum.

8
C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
Fig. 5. Estrogen receptor binding affinity of complexes 13 and 14 in comparison with
estradiol.
Fig. 3. Internalization of 15 and 16 with and without simultaneous incubation with
unlabelled estradiol in whole MCF-7 cells (% of total activity inside cytoplasm and
nucleus).
The binding affinities were determined by in vitro competitive
radiometric binding assays using [3H]-estradiol as tracer. Thus, affin-
ities correspond to the inhibition of the binding of [3H]-estradiol to
the recombinant human ERα and were expressed as IC50 and relative
binding affinity (RBA) where estradiol is set to 100. The IC50 values for
each complex were calculated according to the sigmoid inhibition
curves represented in Fig. 5. The binding results are presented in
Table 1.
As shown in the figure, the nuclear internalization of complex 15
increases over time in parallel with the corresponding total internal-
ization in whole cell. In addition the percentage of activity inside the
nucleus was always higher than the activity in the remaining cyto-
plasm suggesting that after crossing the cellular membrane the com-
plex is able to reach the nucleus and to accumulate inside. On the
other hand, the nuclear internalization of complex 16 is much lower
than the internalization in cytoplasm and remains almost constant
over time indicating that the complex is not able to be transported
or to be retained into the nucleus.
The whole set of results indicate that both complexes exhibited
a low binding affinity for ERα (RBA (13)=0.4 0.1%, RBA (14)=
0.7 0.2%) explaining the non-ER-mediated process observed in cel-
lular studies. Animal studies were not undertaken due the low rate of
cellular/nuclear internalization of the complexes and low ER binding
observed.
Data from these cellular studies indicate that the conjugation of the
biomolecule to the bifunctional chelators 4 and 8 led to final complexes
with a decreased ability to penetrate the cell membranes probably due
to the evident reduction on lipophilicity when compared with the estra-
diol itself and other 99mTc-tricarbonyl complexes bearing a 17α-
estradiol derivative [32]. Furthermore, the evidence that the internali-
zation rate for both complexes was not decreased in cells treated with
estradiol, suggests that the uptake mechanism is not an ER-mediated
process.
4. Conclusions
We have described the synthesis of two bifunctional chelators with
different donor atom sets and charge (4 and 8). The conjugation of 4
and 8 to 17α-ethynylestradiol was performed successfully using the
Sonogashira methodology. Using these novel estradiol-ligand conju-
gates, the Re(I)/99mTc(I)-tricarbonyl complexes (13/15 and 14/16)
were synthesized. The 99mTc complexes 15 and 16 were obtained in
high radiochemical yield and characterized by comparing their HPLC
profiles with the ones obtained for the corresponding Re analogs (13
and 14), which were fully characterized. The 99mTc complexes show a
high stability in physiological saline, human serum and cell medium.
Cellular studies showed that 15 has a higher ability to accumulate in
the nucleus when compared with 16 most probably due to its cationic
overall charge. However, estradiol saturation studies indicate a non
ER-mediated uptake process for both complexes. These results were
corroborated with the low RBA determined for the Re analogs.
3.2.3.2. Receptor binding affinity of Re(I) complexes. Despite the disap-
pointing cellular internalization data, we decided to determine the
relative binding affinity of the Re complexes (13 and 14) for the iso-
lated recombinant human ERα trying to understand those results as
these two assays measure different properties and give complemen-
tary information. While the receptor binding assay with the free ER
just assesses the receptor binding, the cellular binding assays evaluate
not only the receptor binding but also the ability of the complexes to
enter into the cell and nucleus or any binding to other biomolecule in
the cell that would interfere in the ER binding [29].
Acknowledgments
C. Neto thanks Fundação para a Ciência e Tecnologia (FCT) for a
Ph.D. grant (SFRH/BD/31319/2006). The authors thank Dr Joaquim
Marçalo and Dr Vânia Sousa for performing mass spectra analysis
and elemental analysis, respectively. The authors are grateful to Dr
João Abrantes for β measurement in the RBA assays. The QITMS
instrument was acquired with the support of the Programa Nacional
de Reequipamento Científico (Contract REDE/1503/REM/2005-ITN)
Table 1
ER binding data for rhenium complexes.
Complexes
13
11
14
IC50 SD (μM)
1
7
1
RBA SD (%)
0.4 0.1
0.7 0.2
Fig. 4. Activity of 15 (black) and 16 (gray) internalized inside the nucleus versus total
internalized activity.
SD: Standard deviation (n=3).

C. Neto et al. / Journal of Inorganic Biochemistry 111 (2012) 1–9
9
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