Design, synthesis, and evaluation of cyclofenil derivatives for potential SPECT imaging agents

Article abstract of DOI:10.1007/s00775-010-0627-0

To develop technetium- and rhenium-labeled nonsteroidal estrogen imaging agents for estrogen receptor (ER) positive breast tumors, two groups of rhenium and technetium cyclofenil derivatives were synthesized and characterized. The binding affinities of the rhenium complexes for ERs were determined. The tricarbonyl rhenium complex showed the highest binding affinity for ERs (81.2 for ERβ, 16.5 for ERα). Tricarbonyl technetium cyclofenil complexes were obtained in high radiochemical purity and radiochemical yields. The results of studies of their octanol/water partition and in vitro stability are presented. These results demonstrate that these radiolabeled cyclofenil derivatives may be considered as potential breast cancer imaging agents. SBIC 2010.

Full text of DOI:10.1007/s00775-010-0627-0

J Biol Inorg Chem (2010) 15:591–599
DOI 10.1007/s00775-010-0627-0
ORIGINAL PAPER
Design, synthesis, and evaluation of cyclofenil derivatives
for potential SPECT imaging agents
•
Hua Zhu Liliang Huang Yuanqing Zhang
•
•
•
Xiaoping Xu Yanhong Sun Yu-Mei Shen
•
Received: 17 November 2009 / Accepted: 26 January 2010 / Published online: 1 March 2010
Ó SBIC 2010
Abstract To develop technetium- and rhenium-labeled
nonsteroidal estrogen imaging agents for estrogen receptor
(ER) positive breast tumors, two groups of rhenium and
technetium cyclofenil derivatives were synthesized and
characterized. The binding affinities of the rhenium com-
plexes for ERs were determined. The tricarbonyl rhenium
complex showed the highest binding affinity for ERs (81.2
for ERb, 16.5 for ERa). Tricarbonyl technetium cyclofenil
complexes were obtained in high radiochemical purity and
radiochemical yields. The results of studies of their octa-
nol/water partition and in vitro stability are presented.
These results demonstrate that these radiolabeled cyclofe-
nil derivatives may be considered as potential breast cancer
imaging agents.
Introduction
Estrogen receptor (ER) is an attractive target in the treat-
ment of breast cancer, in which it has two structurally
similar subtypes ERa and ERb with different biological
properties [1, 2]. It is notable that the level of ERb relative
to that of ERa declines with breast cancer progression
[3–6]. A number of estradiol derivatives labeled with
bromine, iodine, fluorine, rhenium, and technetium have
been developed as tumor imaging agents [7–16]. Although
most of these agents are based on steroidal estrogens, a few
investigations have focused on nonsteroidal estrogens or
antiestrogens, including selective ER modulators [17–22].
Cyclofenil and its derivatives have high binding affinity
for ER, often comparable to or greater than that of estra-
diol, and bis(4-hydroxyphenyl)methylidenecyclopentane
showed the highest ERb to ERa ratio [23]. Notably, it has
mixed agonist–antagonist activity typical for a nonsteroidal
selective ER modulator such as tamoxifen or raloxifene
[22, 24]. More recently, 18F-labeled fluorocyclofenil ana-
logues have been synthesized and investigated for subtype-
specific imaging of ERa or ERb by using positron emission
tomography (PET). Unfortunately, these 18F-labeled high-
affinity ligands for ER failed to show receptor-mediated
uptake into the uterus [25]. The corresponding 11C-labeled
cyclofenil ester has also been studied. These chemistry
results encouraged the further in vivo biological evaluation
of 11C-labeled cyclofenil derivatives as new potential PET
imaging agents [26].
Keywords Cyclofenil ꢀ Tricarbonyl rhenium ꢀ
Radiolabeling ꢀ Nonsteroidal ꢀ Estrogen receptor
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-010-0627-0) contains supplementary
material, which is available to authorized users.
H. Zhu ꢀ L. Huang ꢀ Y. Zhang ꢀ X. Xu ꢀ Y. Sun ꢀ Y.-M. Shen
Shanghai Institute of Applied Physics,
Chinese Academy of Sciences, Shanghai 201800, China
H. Zhu ꢀ L. Huang ꢀ Y. Zhang ꢀ X. Xu
Graduate School of the Chinese Academy of Sciences,
Beijing 100049, China
The PET radionuclides must typically be generated by
an on-site cyclotron and used as soon as possible owing to
their short half-lives. Single photon emission computed
tomography (SPECT) imaging with 99mTc alleviates some
of the problems encountered with PET owing to its con-
venient 6-h half-life and its wide availability. 99mTc has
Y.-M. Shen (&)
Shanghai Center for Systems Biomedicine,
Shanghai Jiao Tong University,
800 Dongchuan Road,
Shanghai 200240, China
e-mail: shenyumei@sinap.ac.cn; ymshencsu@yahoo.com
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J Biol Inorg Chem (2010) 15:591–599
obtained with a Thermo-MAT95XP mass spectrometer
under electron impact ionization conditions. NMR spectra
were recorded using a Bruker Avance 400 or 500 MHz
spectrometer. IR spectra were recorded with an Avataar
370 Fourier transform IR spectrometer (250–4,000 cm-1).
The elemental analyses were conducted using an Elementar
Analysensysteme (Germany) vario EL III.
N
M
N
CO
CO
CO
O
m
N
Purified full-length human ERa and ERb were purchased
from PanVera/Invitrogen (Carlsbad, CA, USA). [6,7-3H]
Estra-1,3,5,(10)-triene-3,17-b-diol ([3H]-E2), 44.8 Ci/mmol,
was from PerkinElmer (Boston, MA, USA). Hydroxyapatite
(HAP) was from Aladdin (China). Borosilicate glass tubes
were from VWR International (West Chester, PA, USA).
The organometallic precursor [Et4N]2[Re(CO)3Br3] and the
radioactive precursor [99mTc(H2O)3(CO)3]? were prepared
as reported before [35, 36]. Na[99mTcO4] was eluted from a
99Mo/99mTc generator (Shanghai Yuanpu Isotope Technology)
using 0.9% saline. High performance liquid chromatography
(HPLC) analyses of the rhenium and technetium-99m
complexes were performed using a Dionex P680 system
equipped with a tunable absorption detector and a PDA-
100 photodiode-array detector using a Hypersil BDS C-18
reversed-phase column (5 lm, 250 mm 9 4.6 mm). The
HPLC solvents were methanol (solvent A) and aqueous
triethylammonium phosphate buffer, pH 2.76 (solvent B).
The HPLC eluting conditions were as follows: 0–3 min,
15% solvent A; 3–6 min, 15–25% solvent A; 6–9 min,
25–35% solvent A; 9–22 min, 35–98% solvent A;
22–25 min, 98–25% solvent A; 25–30 min, 25–15% sol-
vent A. The flow rate was 1 mL/min.
HO
ER pocket
n
M= 99mTc or Re
m = 1, 3 or 5
n = 1, 2 or 3
Chelating
system
Warhead
Receptor
Linker
Ligand
Fig. 1 Structure and proposed mechanism of binding of cyclofenil
derivatives with estrogen receptor (ER)
emerged as a preeminent radionuclide, and it is used in
over 80% of all routine diagnostic nuclear medicine pro-
cedures [27].
Structural studies on the ERs have suggested that there
is ample unoccupied space within the ligand binding
pocket [28]. An important requirement for high affinity for
the ERs is that at least one hydroxyl function should remain
on the estrogenic nucleus [29–31]. As shown in Fig. 1, a
reasonable pharmacophore model has been advanced to
guide the design of cyclofenil for a potential SPECT
imaging agent [32–34].
Starting from 4, 40-dihydroxybenzophenone, we pre-
pared a series of cyclofenil derivatives based on a tridentate
ligand chelating system by x-bromoalkylation and sub-
sequent nucleophilic substitution. The corresponding
cyclofenil rhenium complexes were synthesized and char-
acterized. These tricarbonyl rhenium complexes showed
excellent binding affinities for the ERs when compared
with the lead structure. Tricarbonyl technetium cyclofenil
complexes were synthesized and preliminarily revaluated
with effective specific activities sufficient for in vivo bio-
distribution studies. All these studies were designed to
develop labeled cyclofenil derivatives as new potential
SPECT nonsteroidal estrogen radioligands for imaging ER
in breast cancer.
Chemical synthesis
Syntheses of compounds 2a, 2b, and 2c
The syntheses of compounds 2a, 2b, and 2c (Scheme 1)
were performed according to the following procedure.
Cyclofenil and its analogues were prepared via McMurry
coupling. The low-valence titanium was formed by
reaction of TiCl4 (3.28 g, 17.5 mmol) with zinc power
(2.4 g, 36 mmol) in absolute tetrahydrofuran (25 mL) at
100 °C. A solution of 1 (1.00 g, 4.67 mmol) and each
ketone (5.0 mmol) dissolved in absolute tetrahydrofuran
(15 ml) was injected by syringe, and the reaction mixture
was refluxed for 1.5 h. The cooled reaction mixture was
slowly poured into a NaHCO3 solution (200 mL) with
vigorous stirring. The heterogeneous solution was filtered
through Celite. The aqueous layer was extracted with
EtOAc (100 mL) three times and finally purified by
column chromatography. Under these optimized condi-
tions, all of the reactions gave reasonable yields of
78–85%, which are higher than the yields reported in the
literature [23].
Experimental
Materials and methods
All experiments were performed under the specified tem-
perature conditions. Solvents were distilled from the
appropriate drying agents and degassed before use. Melting
points were determined using a WRS-IA apparatus and
were uncorrected. High-resolution mass spectra were
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593
n
n
O
1, ω- Dibromide Comp.
2,2'-Dipicolylamine
CH3COCH3
Br
ketone
CH3COCH3
TiCl4,Zn/THF
O
OH
OH
HO
HO
m
3a n = 1 m = 3
3a' n = 1 m = 5
3a'' n = 1 m = 7
3b n = 2 m = 3
3c n = 3 m = 3
HO
2a n = 1
2b n = 2
2c n = 3
1
n
n
γ
δ
β
g
e
i
σ
CO
Br-
CO
N
f
d
α
α
[Et4N]2[Re(CO)3Br3]
j
N
N
h
N
CH3OH
Re
m
c
N
O
N
a
m
HO
k
O
m
CO
HO
b
l
4a n = 1 m = 3
4a' n = 1 m = 5
4a'' n = 1 m = 7
4b n = 2 m = 3
4c n = 3 m = 3
σ
5a n = 1 m = 3
5a' n = 1 m = 5
5a'' n = 1 m = 7
5b n = 2 m = 3
5c n = 3 m = 3
β
δ
γ
Scheme 1 Synthetic strategy for cyclofenil–Re(CO)3 conjugates. THF tetrahydrofuran
Syntheses of compounds 4a, 4a0, 4a00, 4b, and 4c
Analytical data for compound 2a (a white solid): Yield
81%, white solid, m.p. 193–194 °C; IR (KBr) m: 3,280,
2,949, 1,613, 1,592, 1,429, 1,332, 840, 562 cm-1; 1H NMR
(dimethyl-d6 sulfoxide) d: 9.25 (s, 2H, –OH), 6.91(d, 4H,
aromatic, J = 8.6 Hz), 6.66 (d, 4H, aromatic, J = 8.6 Hz),
2.28 (t, 4H, C2–H, J = 6.8 Hz), 1.61 (dt, 4H, C3–H,
Compounds 3a, 3a0, 3a00, 3b, and 3c (1 mmol), K2CO3
(280 mg, 2 mmol), and KI (36 mg, 0.2 mmol) were dis-
solved in 15 mL acetone, then 2,20-dipicolylamine
(300 mg, 1.5 mmol) in acetone (5 mL) was added slowly.
After the reaction mixture had been stirred at 44 °C for
18 h, the reaction was stopped, and the reaction mixture
was filtered, evaporated under reduced pressure, and puri-
fied by column chromatography.
3
2JH–H = 6.6 Hz, JH–H = 2.7 Hz). Analytical data for 2b
and 2c are given in the electronic supplementary material.
Syntheses of compounds 3a, 3a0, 3a00, 3b, and 3c
Analytical data for compound 4a (colorless oil): Yield:
63%; 1H NMR (CDCl3, 500 MHz) d: 8.51 (d, J = 8.6 Hz,
2H, r-CH, Py), 7.63 (t, J = 6.0 Hz, 2H, c-CH, Py), 7.54
(d, J = 7.6 Hz, 2H, b-CH, Py), 7.14 (t, J = 6.2 Hz, 2H,
d-CH, Py), 7.06 (d, J = 8.4 Hz, 2H, ArH), 7.04
(d, J = 8.0 Hz, 2H, ArH), 6.77 (d, J = 8.4 Hz, 2H, ArH),
6.74 (d, J = 8.4 Hz, 2H, ArH), 3.86 (s, 4H, –N(CH2)2),
3.81 (t, J = 5.7 Hz, 2H, 10-CH2), 2.60 (t, J = 6.4 Hz, 2H,
40-CH2), 2.38–2.40 (m, 4H, 2-CH2 and 5-CH2), 1.60–1.80
(m, 8H); IR (KBr) m: 2,432, 1,593, 1,471, 1,364, 1,164,
1,046, 833, 756, 580 cm-1; ESI-HRMS: calcd. for
C34H38N3O2 [M ? H]? 520.2964, found 520.2966. Ana-
lytical data for 4a0, 4a00, 4b, and 4c are given in the elec-
tronic supplementary material.
Compound 2a, 2b or 2c (2 mmol), and K2CO3 were dis-
solved in 15 mL acetone, then 1, x-dibromide compound
(2.4 mmol) dissolved in another 10 mL acetone was slowly
dropped into the mixture. After the reaction mixture had
been stirred for 8 h at 46 °C, the reaction was stopped, and
the reaction mixture was filtered, evaporated under reduced
pressure, and purified by column chromatography.
Analytical data for compound 3a (straw yellow oil):
Yield: 62%; 1H NMR (CDCl3, 400 MHz) d: 7.09
(d, J = 8.1 Hz, 2H, ArH), 7.05 (d, J = 8.0 Hz, 2H, ArH),
6.81 (d, J = 8.4 Hz, 2H, ArH), 6.75 (d, J = 8.1 Hz, 2H,
ArH), 4.83 (s, 1H, –OH), 3.99 (t, J = 6.6 Hz, 2H, 10-CH2),
3.50 (t, J = 6.4 Hz, 2H, 40-CH2), 2.39 (s, 4H, 2-CH2 and
5-CH2), 2.06–2.10 (m, 2H, 20-CH2), 1.94–1.96 (m, 2H,
30-CH2), 1.66–1.69 (m, 4H, 3-CH2 and 4-CH2); IR (KBr)
m: 2,949, 2,876, 1,601, 1,507, 1,168, 927, 837, 605,
579 cm-1; electrospray ionization (ESI) high-resolution
mass spectrometry (HRMS): calcd. for C22H25O2BrK
[M ? K]? 439.0675, found 439.0684. Analytical data for
3a0, 3a00, 3b, and 3c are given in the electronic supple-
mentary material.
Syntheses of complexes 5a, 5a0, 5a00, 5b, and 5c
Complexes 5a, 5a0, 5a00, 5b, and 5c (0.2 mmol) were
synthesized according to the following general procedure.
(Et4N)2[Re(CO)3Br3] (180 mg, 0.24 mmol) and ligands 4a,
4a0, 4a00, 4b, and 4c were dissolved in ethanol and
the mixture was stirred at room temperature for 2 h. The
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reaction mixture was evaporated and the residue was
purified by flash chromatography. Compounds for bioassay
were recrystallized from CH2Cl2 and hexane.
the mixture was vortexed and centrifuged. The supernatant
was removed and discarded and the pellet was washed
three times with Tris–HCl (0.05 M, pH 7.4). The radio-
activity was counted the next day with a scintillation
counter (Beckman) with 43% counting efficiency. All
numeric data were expressed as the mean of the
values the standard error of the mean. Graphpad Prism,
version 4, was used for statistical analysis.
Analytical data for compound 5a (pale-yellow solid):
1
Yield: 85%, melting point more than 200 °C; H NMR
(CDCl3, 500 MHz) d: 8.63 (d, J = 5.4 Hz, 2H, r-CH, Py),
7.88 (d, J = 7.8 Hz, 2H, c-CH, Py), 7.74 (t, J = 7.7 Hz,
2H, b-CH, Py), 7.17 (t, J = 6.58 Hz, 2H, d-CH, Py), 7.04
(d, J = 8.6 Hz, 2H, ArH), 6.97 (d, J = 7.2 Hz, 2H, ArH),
6.75–6.83 (m, 4H, ArH), 6.03 (s, 1H, –N(CH2)2), 5.99 (s,
1H, –N(CH2)2), 4.35 (s, 1H, –N(CH2)2), 4.31 (s, 1H,
–N(CH2)2), 4.00 (t, J = 5.8 Hz, 2H, 10-CH2), 3.84 (t,
J = 8.4 Hz, 2H, 40-CH2), 2.35 (d, J = 7.2 Hz, 4H, 2-CH2
and 5-CH2), 2.23 (t, J = 5.9 Hz, 2H, 30-CH2), 1.87
(t, J = 6.23 Hz, 2H, 20-CH2), 1.60–1.70 (m, 4H, 3-CH2
and 4-CH2); 13C NMR (CD3OD,125 MHz) d: 197.7 (3C,
fac-Re(CO)3), 162.6 (2C, Py-aC), 158.9 (1C, Ar-kC), 157.0
(1C, Ar-cC), 153.6 (2C, Py-rC), 142.1 (2C, Py-cC), 138.4
(2C, Ar-iC and Ar-mC), 137.0 (2C, Ar-eC and Ar-aC),
134.4 (2C, Ar-jC and Ar-lC), 131.8 (2C, Ar-dC and
Ar-bC), 127.3 (2C, Py-bC), 127.3 (2C, Py-dC), 125.1 (1C,
C1), 116.5 (2C, Ar-hC and Ar-fC), 115.7 (1C, gC), 72.3
(1C, C10), 69.1 (2C, PyCH2), 69.0 (1C, C40), 34.4 (2C, C2
and C5), 28.4 (2C, C3 and C4), 28.0 (1C, C20), 23.9 (1C,
C30); IR (KBr) m: 2,921, 2,848, 2,026, 1,916, 1,605, 1,503,
1,242, 1,029, 829, 764 cm-1; ESI mass spectrometry (MS):
calcd. for C37H37O5N3Re [M-Br]? 789.91, found 790.20;
anal. calcd. for [C37H37N3O5Re]Brꢀ1/2CH2Cl2: C 49.37, H
4.20, N 4.61; found C 49.46, H 3.85, N 4.62. Analytical
data for 5a0, 5a00, 5b, and 5c are given in the electronic
supplementary material.
Evaluation of radioactive cyclofenil–99mTc(CO)3
conjugates
Preparation of radioactive cyclofenil–99mTc(CO)3
conjugates
99mTc(CO)3(H2O)?3 was prepared as reported before [40–
42]. In our experiment the radiochemical purity of the
[
99mTc(CO)3(H2O)3]? intermediate exceeded 95% as
determined by radio-HPLC. To a 5-mL serum vial, 0.2 mL
of aqueous [99mTc(CO)3(H2O)3]? solution was added,
followed by 100 lL of 4a, 4b, or 4c (10-4 M). The reac-
tion mixture was heated at 42 °C for 60 min (Scheme 2).
After the mixture had been cooled to room temperature, the
radiotracer was purified by HPLC using conditions iden-
tical to those for [99mTc(CO)3(H2O)3]? analysis.
Octanol/water partition coefficient
of cyclofenil–99mTc(CO)3
The final partition coefficient was expressed as log P. Log P
of cyclofenil–99mTc(CO)3 complexes was determined by
measuring the distribution of radioactivity in 1-octanol and
phosphate-buffered saline (PBS). A 10-lL sample of
cyclofenil–99mTc(CO)3 in PBS was added to a vial which
contained 1 mL of 1-octanol and 1 mL of PBS. After the
mixture had been vortexed for 8 min, the vial was centri-
fuged for 5 min to ensure complete separation of layers.
Then, 10 lL of each layer was pipetted into the other test
tubes, and log P values were calculated using the formula
log P = log(counts in octanol/counts in water) [43].
Binding affinity assay
The equilibrium and competition binding affinity assays
were performed as described by Katzenellenbogen et al.
[37, 38] with some modifications [39]. ERa and ERb were
diluted to 2 nM in binding buffer [50 mM tris(hydroxy-
methyl)aminomethane (Tris)–HCl, pH 7.4, 150 mM KCl,
1 mM EDTA, 0.1% bovine serum albumin]. [3H]-E2 was
extracted with ethanol and diluted in Tris–HCl to 0.5–
30 nM for the saturation binding affinity assay and to 5 nM
for the competition binding affinity assay. HAP was used to
absorb the receptor–ligand complexes and free ligand was
washed away. Each compound (2a, 2b, 2c, 5a, 5a0, 5a00, 5b,
and 5c) was dissolved in dimethylformamide and then
diluted with binding buffer to obtain concentrations rang-
ing from 0.1 nM to 1 mM. Thirty microliters of the ER
solution, [3H]-E2 solution, estradiol, and each test com-
pound was added to a test tube, followed by binding buffer
to a final volume of 300 lL. The mixture was incubated at
25 °C for 2 h. HAP slurry (30% solution) was added and
In vitro stability of cyclofenil–99mTc(CO)3
The stability of cyclofenil–99mTc(CO)3 complexes was
studied by measuring the radiochemical purity using radio-
HPLC at different time intervals after preparation. The
complex was added to a test tube with PBS. The mixture
was incubated by shaking the test tube at 37 °C in an
incubation apparatus. The radiochemical purity was mea-
sured at 30 min and at 1, 2, 4, and 6 h by radio-HPLC. The
same procedure was applied in an experiment using histi-
dine and cysteine [44].
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595
CO
99mTc CO
N
N
N
N
N
HO
O
HO
HO
HO
O
O
O
4 a
6 a
N
CO
CO
99mTc CO
N
[
99mTc(H2O)3(CO)3]+
1h, 10-4M, 42 0C
N
HO
O
N
CO
4 b
6 b
CO
N
99mTc CO
N
HO
O
N
CO
4c
6 c
Scheme 2 Radiolabeling of cyclofenil–99mTc(CO)3 (6a, 6b, 6c)
Results and discussion
and elemental analysis. The protons of the methylene
groups adjacent to the pyridines are equivalent by virtue of
their symmetry for ligands 4a, 4a0, 4a00, 4b, and 4c. After
ligands 4a, 4a0, 4a00, 4b, and 4c had coordinated to rhe-
nium, the splitting pattern of these methylene protons
became more complicated, resulting in multiplets in the
Design and synthesis of cyclofenil derivatives
Clearly, the hydroxylation pattern is important for receptor
binding and in vitro activity [45]. The presence and the
nature of the basic amine substituent is critical in deter-
mining estrogen antagonist activity [46]. We began with
the selective modification of phenolic hydroxyl group.
Then dipicolyamin chelators system were introduced for
further coordination to fac-[M(CO)3]? (Scheme 1).
1
4.0–6.0-ppm range. The H NMR spectra of Py–CH2– of
ligands 4a, 4a0, 4a00, 4b, and 4c show a single peak at
3.9 ppm. Rhenium complexes 5a, 5a0, 5a00, 5b, and 5c
show four single peaks at 6.06, 6.00, 4.35, and 4.00 ppm.
The 13C NMR spectra of the five rhenium complexes show
chemical shifts of three carbonyl peaks in the range 195.9–
197.9 ppm. All the conditions show that the rhenium core
lacking electrons makes the entire molecular electric field
move towards the rhenium core. So many proton signals
show a downfield shift. The IR spectra of complexes 5a,
5a0, 5a00, 5b, and 5c exhibit a sharp, strong band in the
2,026–2,030-cm-1 range and two broad, intense absorp-
tions at 1,910 and 2,036 cm-1, attributed to m(C–O) of the
fac-Re(CO)3 unit [47, 48]. The absorptions are significantly
blueshifted compared with the absorptions of the starting
material [Re(CO)3Br3]2- (1,998, 1,871 cm-1). The ESI–
MS of 5a, 5a0, 5a00, 5b, and 5c showed cleavage of bromide
anion, whereas the corresponding rhenium core is rela-
tively stable. These features indicate the tridentate coor-
dination mode of ligands 4a, 4a0, 4a00, 4b, and 4c via the
tertiary amine and the two pyridine nitrogens. Elemental
analyses of the compounds gave definite ultimate compo-
sitions of 5a, 5a0, 5a00, 5b, and 5c. Owing to the purity of
Cyclofenil (2a, 2b, 2c) contains two phenols, which
react with 1,x-dibromide to form 3a, 3a0, 3a00, 3b, and 3c.
In the reaction we chose K2CO3 as the base and acetone as
the solvent. Firstly, all of the starting materials were mixed
together at room temperature, then the mixture was stirred
at 46 °C for 12 h, the reaction was stopped, and the product
(3a, 3a0, 3a00, 3b, and 3c) was obtained with 50–62% iso-
lated yield. With the same procedure a series of cyclofenil
derivatives (different lengths of the alkyl chains as well as
variation of the ring size in the lead structure) could be
easily prepared for further reaction. Their structures were
1
characterized by IR spectroscopy, H NMR spectroscopy,
and ESI-HRMS.
The tridentate ligands 4a, 4a0, 4a00, 4b, and 4c were
1
characterized by IR spectroscopy, H NMR spectroscopy,
and HRMS. The rhenium complexes 5a, 5a0, 5a00, 5b, and
5c were unambiguously characterized by IR spectroscopy,
1H NMR spectroscopy, 13C NMR spectroscopy, ESI–MS,
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what effect the substituted hydroxyl group had on binding
affinity. Since cyclofenil (2a, 2b, and 2c) shows better
binding affinity than estradiol (Table 1), we prepared the
first group of cyclofenil–Re(CO)3 conjugates (5a, 5a0, 5a00,
5b, and 5c) to evaluate the RBAs of cyclofenil compounds
having different ring size core units with the same substi-
tuted hydroxyl group (four carbon alkyl chains). In short,
the RBAs range from 13.3 to 44.8 for ERa and from 33.8 to
81.2 for ERb (Table 1).
As the ring size increased from cyclopentyl (5a) to
cycloheptyl (5c), the binding affinities for ERb increased,
and cycloheptyl (5c) showed the highest ERb affinity.
However, the RBAs for ERa did not show this tendency,
and 5b show the highest ERa affinity. All compounds
(5a, 5a0, 5a00, 5b, and 5c) show slight selectivity for ERb
from the calculated ERb to ERa ratio. On this scale,
cycloheptyl compound (5c) was 4.9-fold ERb-selective.
Examples of the [3H]-E2 inhibition curves used for the RBA
estimations are presented in Figs. 2, S1, and S2. These data
suggest that after the modification of hydroxyl group with a
carbon side chain, the RBA was slightly reduced (approx-
imately 30% of that of lead structure). However, affinities
comparable to the affinity of estradiol are maintained.
To investigate the relation between the lengths of the
alkyl chains and ER affinity, further modification of 2a
were present. 2a has the smallest ring size and it might be
more suitable to ‘‘enter’’ the ER pocket (Fig. 1). Rhenium–
cyclofenil complexes with a chain of four to eight carbons
(5a, 5a0, and 5a00) were synthesized. In general, the RBAs
ranged from 7.7 to 13.3 for ERa and from 33.8 to 62.6 for
ERb (Table 1).
Table 1 Relative binding affinities (RBA) of cyclofenil and its
derivatives at 25 °C
Complexes
RBAa
ERb/ERab
ERa
ERb
2a
2b
2c
17.7 1.8
114.6 5.0
101.3 4.0
13.3 1.2
9.43 0.8
7.75 0.6
44.8 1.8
16.5 1.2
121.8 4.0
274.8 5.0
339.0 9.0
33.8 1.8
62.6 2.0
57.4 2.2
66.7 2.6
81.2 2.4
6.9
2.4
3.3
2.5
6.7
7.4
1.5
4.9
5a
5a0
5a00
5b
5c
The RBA is expressed as the binding affinity relative to that of
estradiol (100%)
a
Determined by a competitive radiometric binding assay with
[3H]estradiol, using full-length human estrogen receptor a (ERa) and
estrogen receptor b (ERb). The values are reported as the mean
range (n = 2)
b
Under these conditions, Kd of estradiol is 0.4 nM for ERa and
1.0 nM for ERb
the raw products by recrystallization, different numbers of
CH2Cl2 groups were left on the surface of the crystal.
Binding affinity assay of cyclofenil–Re(CO)3
conjugates
The [3H]-E2 binding inhibition curves were established for
each compound. The relative binding affinity (RBA) was
expressed as the binding affinity relative to that of estradiol
(100%) [36, 49]. The results are shown in Table 1.
As the number of carbons in the chain increased from
four (5a) to eight (5a00), the binding affinity for ERa
gradually decreased, whereas for 5a0 and 5a00 show similar
affinity for ERb, double that of 5a, greatly improving the
selectivity for ERb. Let us take 5a00 as an example. It is
For an ER pharmacophore, the most important require-
ment for high affinity for the ER is to maintain the integrity
of the hydroxyl functions. However, it was not obvious
Fig. 2 Determination of specific binding of estradiol (E2) and rhenium–cyclofenil (5a, 5b, and 5c) complexes in purified full-length human ER.
Left for ERa; right for ERb. Each data point was from the average of three measurements and the bar represents the standard deviation
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J Biol Inorg Chem (2010) 15:591–599
597
Fig. 3 Determination of specific binding of 5a and rhenium
complexes with different carbon chain lengths (5a0 and 5a00) in
purified full-length human ER. Left for ERa; right for ERb. Each data
point came from the average of three measurements and the bar
represents the standard deviation
7.4-fold ERb-selective and has highest binding affinity of
all the compounds, comparable to that of the original
compound (2a, ERb to ERa ratio of 7.7 [23]). Examples of
the [3H]-E2 inhibition curves used for the RBA estimations
are presented in Fig. 3. As the carbon chain length
increased, the binding affinity of ERb also increased;
however, the situation is opposite that of ERa.
HPLC. Radio-HPLC also readily distinguished cyclofe-
nil–99mTc(CO)3 (6a, 6b, and 6c) from 99mTc(CO)3(H2O)?3
and 99mTcO4-, whose retention times were 5.51 and
10.30 min, respectively. The retention times for the cy-
clofenil–Re(CO)3 analogues (5a, 5b, and 5c) were 23.12,
24.37, and 24.12 min, respectively (Fig. 4).
In term of binding affinity, our modification was
successful, because we found the cyclofenil–Re(CO)3
compounds 5a0, 5b, 5c, and 5a0 bind to ERs with an affinity
comparable to that of estradiol.
Octanol/water partition coefficient
of cyclofenil–99mTc(CO)3
To evaluate the aqueous solubility of the complex, the
octanol/water partition coefficient was determined. The
values of log Po/w for cyclofenil–99mTc(CO)3 are shown in
Table 2. The octanol/water partition of estradiol was
reported as log P = 3.26 [13] or log P = 3.30 [15]. It is
well known that the lipophilicity can affect the tissue
permeability properties of a ligand, thus affecting its ability
to enter target tissues. By altering the lipophilicity of the
cyclofenil derivatives through synthetic modification, one
can change the tissue permeability by allowing more or less
of the ligand to enter cells in ER-rich target tissues or
nontarget tissues. The oil/water partition coefficients of the
cyclofenil–99mTc(CO)3 complexes were up to almost 100
times lower than that of estradiol. According to research by
Nayak et al. [50], appropriately reducing radiopharma-
ceutical lipophilicity can be favorable to target tissue
uptake of ER-expressing tumors.
Evaluation of cyclofenil–99mTc(CO)3 conjugates
Preparation of cyclofenil–99mTc(CO)3
To further study their water solubility and stability, we
selected radioactive 99mTc(CO)3(H2O)?3 instead of the
stable rhenium complex. In the periodical table of ele-
ments, technetium and rhenium are in the same group, and
they have extremely similar properties, so 6a, 6b, and 6c
show almost the same chemical character as 5a, 5a0 5a00,
5b, and 5c, respectively.
The radiolabeling of cyclofenil derivatives is shown in
Scheme 2. The radiochemical purity of the 99mTc(CO)3
(H2O)?3 intermediate exceeded 95% as determined by
radio-HPLC. The intermediate was used directly without
further purification. The radiochemical purity of the crude
cyclofenil–99mTc(CO)3 product exceeded 90%.
In vitro stability study
The retention times were 22.75 min for 4a, 23.75 min
for 4b, and 23.80 min for 4c and 23.15 min for 6a,
24.55 min for 6b, and 24.24 min for 6c. The retention
times are long enough for the technetium complexes to be
separated from unreacted chelate by semipreparative
The in vitro stability in PBS, histidine, and cysteine was
tested, and the results are shown in Fig. S3. The radioactive
conjugates for cyclofenil–99mTc(CO)3 maintain excellent in
vitro stability in PBS at 37 °C within 6 h. Decomposition or
123

598
J Biol Inorg Chem (2010) 15:591–599
Fig. 4 High performance liquid
chromatography analyses of
cyclofenil–Re(CO)3 (5a, 5b,
and 5c) and c-trace of the
radioactive
cyclofenil–99mTc(CO)3
conjugates (6a, 6b, and 6c)
Table 2 Octanol8water partition coefficient of cyclofenil–99mTc(CO)3
conjugates
cyclofenil–99mTc(CO)3 conjugate has excellent in vitro
stability for potential nuclear medical applications.
Complexes
P
Log P
6a
9.30 0.94
10.4 0.95
11.0 1.07
9.38 0.95
9.44 0.95
0.968 0.02
1.02 0.02
1.04 0.03
0.972 0.02
0.975 0.02
Conclusion
6a0
6a00
6b
6c
The strategy for the preparation of a monophenolhydroxyl-
substituted 1,1-diarylethylene unit has been developed. A
tridentate chelating system was introduced for labeling
with fac-[M(CO)3]? (M is 99mTc, Re). Rhenium–cyclo-
fenil complexes show binding affinity comparable to that
of estradiol in binding to ERs. THe octanol/water partition
coefficient and the in vitro stability of 99mTc–cyclofenil
complexes were evaluated. We did not obtain a compound
having outstanding affinity preference for either ERa or
ERb. Further work on the optimization of the structures of
dissociation of the complexes to either [99mTc(CO)3]? or
other side products was not observed for all of the com-
plexes under the conditions used. In both histidine and
cysteine, more than 90% of the conjugate still maintains the
original structure within 6 h. These results indicate that the
123

J Biol Inorg Chem (2010) 15:591–599
599
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Acknowledgments This work was supported by the Hundred Talent
Program of the Chinese Academy of Sciences (no. 26200601), the
Natural Science Foundation of Shanghai (no. 3109ZR1438400), and
the Natural Science Foundation of China (no. 10905087).
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