Synthesis and evaluation of fluoroethyl cyclofenil analogs: Models for potential estrogen receptor imaging agent

Article abstract of DOI:10.1016/j.jfluchem.2012.04.005

Cyclofenil analogs (2a-2f) and their fluorine-containing derivatives (3a-3f) were synthesized and evaluated as candidate ligands for positron emission tomography (PET) imaging of estrogen receptors. Most of them show relatively high binding affinities comparable with estradiol (E₂). (4-Fluoroethoxyphenyl)-(4-hydroxyphenyl) methylenecyclopentane (3a) showed both the highest binding affinity for ERs (88.6 for ERβ, 13.8 for ERα) and highest β/α ratio (β/α for 6.4-fold). The radioactive compound [¹⁸F]3a was prepared via displacement of the corresponding mesylate precursor 4 with [¹⁸F]fluoride (¹⁸F: β⁺; 96.7%, T_1/2 = 109.8 min). The biodistribution studies in immature female SD rats demonstrated that the uptake in the uterus and ovaries were 1.358 ± 0.089% ID/g, 1.439 ± 0.214% ID/g, respectively, both of the ratios of uterus/blood and ovaries/blood was less than 2:1. Micro-PET imaging of immature female SD rats has also been reported.

Full text of DOI:10.1016/j.jfluchem.2012.04.005

Journal of Fluorine Chemistry 139 (2012) 46–52
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis and evaluation of fluoroethyl cyclofenil analogs: Models for
potential estrogen receptor imaging agent
Hua Zhu a,c, Zhi Yang a, Jian-Guo Lin c, Shi-Neng Luo c, Yu-Mei Shen b,
*
a Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute,
Beijing 100142, China
b Shanghai Center for Systems Biomedicine, Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
c Key Laboratory of Nuclear Medicine (Ministry of Health), Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Cyclofenil analogs (2a–2f) and their fluorine-containing derivatives (3a–3f) were synthesized and
evaluated as candidate ligands for positron emission tomography (PET) imaging of estrogen receptors.
Most of them show relatively high binding affinities comparable with estradiol (E2). (4-Fluoroethox-
yphenyl)-(4-hydroxyphenyl) methylenecyclopentane (3a) showed both the highest binding affinity for
Received 26 December 2011
Received in revised form 19 March 2012
Accepted 3 April 2012
Available online 15 April 2012
ERs (88.6 for ER
b, 13.8 for ERa) and highest b/a ratio (b/a for 6.4-fold). The radioactive compound
[
18F]3a was prepared via displacement of the corresponding mesylate precursor 4 with [18F]fluoride (18F:
Keywords:
b+; 96.7%, T1/2 = 109.8 min). The biodistribution studies in immature female SD rats demonstrated that
the uptake in the uterus and ovaries were 1.358 ꢀ 0.089% ID/g, 1.439 ꢀ 0.214% ID/g, respectively, both of
the ratios of uterus/blood and ovaries/blood was less than 2:1. Micro-PET imaging of immature female SD
rats has also been reported.
Cyclofenil
Estrogen receptor
Relative binding affinity
Fluorine-18
ß 2012 Elsevier B.V. All rights reserved.
1. Introduction
haemolytic, anaemia, and ovulation [23–27]. Furthermore, cyclo-
fenil-type ligands labeled with radionuclide may enable non-
Estrogen receptor (ER), which is widely distributed in many
tissues and human breast tumors, is a member of the super family
of ligand-regulated nuclear transcription factors that mediates the
actions of estrogens [1,2]. The development of estrogen derivatives
is an attractive target for the diagnosis and treatment of breast
invasive monitoring ER expression in breast cancer by PET and
single-photon emission computed tomography (SPECT) imaging.
The examples of 18F labeled cyclofenil analogs ([18F]FCF) and 11C
labeled cyclofenil-ester ([11C]CCFE) have been reported for
subtype-specific imaging of ER
a or ERb by PET (Fig. 1). However,
cancer [3]. There are two structurally similar subtypes ER
with different biological properties [4,5]. It is important to find
subtype-specific imaging of ER or ER because the level of ER
declines with breast cancer progression
a
or ER
b
these high affinity ligands for the ER failed to show receptor-
mediated uptake into the uterus [28,29].
a
b
b
Structural studies on the ERs have suggested there is ample
unoccupied space within the ligand binding pocket (Fig. 1) [30,31].
Recently we investigated the 99mTc cyclofenil compounds as
potential breast cancer imaging agents by SPECT [31]. And we also
made great effort to synthesis and labeling of cyclofenil with 18F as
PET probes [32,33]. In the study reported here, we not only
undertook investigation of substituted cyclofenil ligands to
relative to that of ER
[6–10].
a
As one of the most important endogenous ligands for estrogen
receptors, 17-estradiol and its derivatives have been received
much attention. A series of estradiol derivatives labeled with 18F,
77Br, 125I and 11C have been developed as PET imaging agents
[11–18]. While most of these agents are based on steroidal
estrogens, a few investigations have focused on selective ER
modulators [19–22].
establish a structure–activity relationship (SAR) relating ER
a/
ER
b
binding affinities but radio-synthesis, evaluation of 18F labeled
cyclofenil analogs as potential PET image agents.
Cyclofenil and its derivatives, having high binding affinity for
ERs and ER
b selectivity as well as estrogen agonist/antagonist
2. Results and discussion
activity, have been used to treatment of scleroderma, hepatitis,
2.1. Chemical synthesis
The compounds we have synthesized can be divided into three
* Corresponding author. Tel.: +86 21 34204561; fax: +86 21 34204561.
E-mail addresses: yumeishen@sjtu.edu.cn, zhuhuacas@live.cn (Y.-M. Shen).
types (Scheme 1). First series of analogs (type I, compounds 2a–c)
0022-1139/$ – see front matter ß 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2012.04.005

H. Zhu et al. / Journal of Fluorine Chemistry 139 (2012) 46–52
47
Fig. 1. ER ligands and proposed cyclofenil-ER pharmacophore model.
have different cycloalkyl core units, ranging from cyclopentane to
cycloheptane. Second series (type II, compounds 2d–e) have a fixed
cyclohexyl moiety onto which we have introduced methyl
substituent at the C3 or C4 position. Type III (compound 2f) has
an isobutyl core unit. Under the optimized conditions, a series of
reference compounds, cyclofenil-FEts were synthesized in high
yields as molecular probe precursors of ERs.
previous work, the modification of one hydroxyl function and
keep the other was successful, we found cyclofenil-Re(CO)3
compounds bind to ERs with an affinity comparable to that of
estradiol [31]. Preliminary result of synthesis and evaluation of
cyclofenil-FEt derivatives showed potential radioactive PET image
agents [32,33]. However, it was not obvious what effect the ring
size core units with the same substituted hydroxyl group had on
binding affinity.
2.2. Estrogen receptor binding assay
All of the cyclofenil 2b and all of their derivatives (2a, 2c–f)
were selected due to affinities in previous report. In general, the
The [3H]-E2 binding inhibition curves were established for each
cyclofenil-FEt compound shown in Fig. 2. The effective concentra-
tion (EC 50) values of estradiol and cyclofenil derivatives were
calculated by Graphpad Prism software shown in Table 1. The
relative binding affinity (RBA) was expressed as the binding
affinity relative to that of estradiol (100%) [30–32]. RBA values of
the twelve cyclofenil derivatives (2a–2f and 3a–3f) are listed in
Table 2.
RBA values range from 19.5 to 335 for ER
for ER
In short, the RBA values of 3a–f range from less than 0.01 to 88.6
for ER and from 13.8 to 26.3 for ER . In all cases, any substitution
at phenol-hydroxyl group reduced binding affinity to both ERs,
however the degree to which binding was lowered depended on
substituent size and polarity. In general, groups that were small
had the highest binding affinity. Of greatest interest to this study,
the cyclopentyl compound (3a) still had good binding affinity
compared to the cyclofenil parent (2a, approximately 75% of that of
b and from 17.7 to 114.6
a
.
b
a
Structural studies on the ERs have suggested that there is ample
unoccupied space within the ligand binding pocket. By our
n
n
HO
n
F
HO
HO
OH
O
2a, n=1; 2b, n=2; 2c, n =3.
3a, n=1; 3b, n=2; 3c, n =3.
O
R1
O
R1
n=1,2,3.
R2
R2
O
1
FEtOTs
HO
K2CO3,Acetone
R2
TiCl4,Zn/THF
R1
F
HO
OH
OH
HO
O
R1=H, R2=CH3 or
2d, R1=H, R2=CH3;
2e, R1=CH3, R2=H.
R1=CH3, R2=H
3d R1=H, R2=CH3; 3e R1=CH3, R2=H.
H
H
O
H
F
HO
HO
O
2f
3f
Scheme 1. Synthesis of cyclofenil (2a–2f) and cyclofenil-FEt (3a–3f).

48
H. Zhu et al. / Journal of Fluorine Chemistry 139 (2012) 46–52
Fig. 2. Determination of specific binding of cyclofenil-FEt (3a–3f) compounds in purified full-length human ER. (A) 3a–3c for ER
a; (B) 3a–3c for ERb; (C) 3d–3f for ERa; and
(D) 3d–3f for ER . Each data point was from the average of three measurements and the bar represents the standard deviation.
b
lead structure). While others compounds (3b–3e) after the
modification of hydroxyl group with a carbon side chain, the
RBA was moiety reduced (approximately 25% of that of lead
structure, 2b–2e). The isobutyl compound (3f) show the greatly
of phenol-hydroxyl group. It was estimated that the –OMs or –Br
group at 2-methanesulfony bromoethyl would be replaced by
hydroxyl group. However, when we used conditions with the
reaction mixture stirred for 3 h at 52 8C, the pure compound 4 was
obtained as a main product. This interesting result made it possible
that cyclofenil-OMs could directly react with fluorine-18 ions with
high labeling yield and simple purification procedure in certain
conditions.
reduction in ERb, dropped about 2000-fold, due to its Z/E structure.
However, the modification structure 3f might be a good ER
a
selectivity compound.
In all, the principal interest to the study is the fact that we have
identified cyclofenil-FEt compound 3a, that has a binding affinity
Total radiochemical yield of [18F]3a was 30% (start from 18F
anion, total synthetic time of 50 min, decay-corrected end of
synthesis (EOS), measured by radio-HPLC). The aimed product was
identified by radio-HPLC, The retention time of [18F]3a (Fig. 3D)
was about 9.8 min (the same as reference compound 3a,
Rt = 9.75 min, Fig. 3B) and well separated from other chemical
for ER
b nearly comparable to that of estradiol. It is a competitive
candidate for PET imaging of ERs in breast cancer.
2.3. Radio-synthesis of [18F]3a
As shown in Scheme 2. The synthesis strategy of 4 was similar to
that in synthesis of cyclofenil-FEt with a nucleophilic substitution
Table 2
Relative binding affinities (RBA) of cyclofenil and its derivatives at 25 8C.
Table 1
Compounds
RBA (E2 = 100)a
ER
b/
ab
Effective concentration (EC 50) value of cyclofenil derivatives at 25 8C.
a
ERb
Compounds
EC 50 value
ER
2a
2b
2c
2d
2e
2f
17.7 ꢀ 1.8
114.6 ꢀ 15.0
101.3 ꢀ 12.0
109.5 ꢀ 8.0
60.5 ꢀ 8.2
69.5 ꢀ 3.0
13.8 ꢀ 2.2
26.3 ꢀ 2.8
19.3 ꢀ 1.2
22.4 ꢀ 1.8
21.7 ꢀ 2.0
25.7 ꢀ 3.3
121.8 ꢀ 8.0
274.8 ꢀ 15.0
339.0 ꢀ 19.0
220.8 ꢀ 10.0
224.0 ꢀ 18.2
19.5 ꢀ 2.0
88.6 ꢀ 6.8
49.5 ꢀ 3.0
72.2 ꢀ 4.6
41.4 ꢀ 4.4
61.0 ꢀ 3.2
<0.01
6.9
2.4
3.3
2.0
3.7
0.28
6.4
1.9
3.7
1.9
2.8
a
ERb
E2
2a
2b
2c
2d
2e
2f
1.48Eꢁ08
8.36Eꢁ08
1.29Eꢁ08
1.46Eꢁ08
1.35Eꢁ08
2.45Eꢁ08
2.13Eꢁ08
1.07Eꢁ07
5.63Eꢁ08
7.67Eꢁ08
6.61Eꢁ08
6.82Eꢁ08
5.76Eꢁ08
4.07Eꢁ08
3.34Eꢁ08
1.48Eꢁ08
1.20Eꢁ08
1.84Eꢁ08
1.82Eꢁ08
2.09Eꢁ07
4.59Eꢁ08
8.22Eꢁ08
5.64Eꢁ08
9.83Eꢁ08
6.67Eꢁ08
4.07Eꢁ08
3a
3b
3c
3d
3e
3f
3a
3b
3c
3d
3e
3f
<0.004
a
Determined by a competitive radiometric binding assay with [3H] estradiol,
using full-length human ER
a
and ER
b.
b
Under these conditions, the Kd of estradiol is 0.4 nM for ER
a
and 1.0 nM for ERb.

H. Zhu et al. / Journal of Fluorine Chemistry 139 (2012) 46–52
49
110oC, 30 min
18F-/K222
HO
MsCl
NEt3
2a
K2CO3
Br
Br
OH
OMs
O
O
HO
18F
4
18F]3a
OMs
[
Scheme 2. Radiosynthesis of [18F]3a.
Fig. 3. HPLC analyses of cyclofenil and its derivatives. (A) Precursor compound 4; (B) Reference compound 3a; (C) fluorine-18 ions; and (D) [18F]3a.
species (for precursor compound 4a, Rt = 7.5 min, Fig. 3A; 18Fꢁ,
Rt = 2.3 min, Fig. 3C).
37 8C within 180 min, and at least 80% of compound [18F]3a still
keeps the original structures in serum.
Then the reaction products were purified by semi-preparative
HPLC column. The analytical HPLC and radio-thin layer chroma-
tography (TLC) were used for quality control (radiochemical purity
of [18F]3a was >95%, Fig. 3D). The effective specific activity
estimated by comparing UV peak (UV = 254 nm) intensity of
purified [18F]3a and corresponding radioactivity was 380 Ci/mmol.
According to the research by Prossnitz and coworkers, the
appropriately reducing lipophilicity can be favorable to target
tissues uptake in ER-expressing tumors [34]. The value of log P for
compound [18F]3a is 2.19, while the reported value of log P is 3.30
for estradiol [35,36].
2.5. Biodistribution study
2.4. In vitro stability studies and partition coefficients
From the biodistribution studies shown in Table 3, the
compound [18F]3a showed selective uptake in target tissue (uterus
and ovaries), its target/nontarget (blood, muscle and brain) ratio
As shown in Fig. 4, the radio-labeled compound [18F]3a keeps
excellent in vitro stability both in PBS and fetal calf serum (FCS) at
Table 3
Biodistribution of [18F]3a in immature SD female rats (% ID/g).
Tissue
% ID/g
1 h
2 h
Heart
0.657 ꢀ 0.072
1.78 ꢀ 0.122
0.711 ꢀ 0.050
0.905 ꢀ 0.122
0.775 ꢀ 0.101
1.054 ꢀ 0.194
0.619 ꢀ 0.025
0.880 ꢀ 0.137
0.556 ꢀ 0.079
0.628 ꢀ 0.140
0.818 ꢀ 0.104
0.587 ꢀ 0.090
1.439 ꢀ 0.214
1.358 ꢀ 0.089
0.398 ꢀ 0.049
0.851 ꢀ 0.164
0.411 ꢀ 0.033
0.419 ꢀ 0.016
0.338 ꢀ 0.022
0.863 ꢀ 0.162
0.360 ꢀ 0.070
0.558 ꢀ 0.202
0.257 ꢀ 0.036
0.333 ꢀ 0.094
0.391 ꢀ 0.061
0.332 ꢀ 0.020
0.897 ꢀ 0.108
0.720 ꢀ 0.078
Liver
Spleen
Lung
Kidney
Bone
Muscle
Intestine
Brain
Fats
Blood
Thymus
Ovaries
Uterus
Fig. 4. In vitro stability of [18F]3a in PBS and FES.

50
H. Zhu et al. / Journal of Fluorine Chemistry 139 (2012) 46–52
Fig. 5. The uptake in target tissue [uterus (left), ovaries (right)] compared with nontarget tissue [blood, muscle and brain].
Fig. 6. Representative summed images of 65–69 frames in immature female SD rats. Injection of 100
m
Ci [18F]3a coronal (up); sagittal (middle); horizontal (down) in 30 min.
was calculated between 1.7 and 2.6 at 1 h and 2 h after injection,
and the ovaries/brain ratio was up to 3.5 at 2 h as shown in Fig. 5
[28,34]. The radiolabeled compound accumulated in ER-positive
tissues such as kidneys, spleen, ovaries, and uterus. As a small
molecular probe, it showed some accumulation in brain due to its
ability to penetrate the blood–brain barrier.
(down)). Prominent up take of [18F]3a was observed in the liver-
kidneys and urinary bladder, suggesting that this radiotracer might
be mainly excreted through the renal route. These preliminary
results might provide valuable information for exploring ER
mediated PET imaging agents.
We noticed that Seo et al. found some uterine selective uptake
whilenospecificreceptormediateduptakeusingdifferentcyclofenil
analogs [28]. We began with the selective modification of phenolic
hydroxylgroup. Wefocusedonthedesign, synthesisandrevaluation
of 18F labeled cyclofenil. However, atpresentwe havenoexperiment
data whether it is receptor mediated uptake using this kind of
cyclofenil analogs despite it is uterine selective uptake.
3. Conclusion
By design, synthesis, and revaluation of cyclofenil derivatives,
we undertook an investigation of substituted cyclofenil ligands to
establish a structure–activity relationship (SAR) relating ERa/ERb
binding affinities to the substituted group. Compound 3a with high
binding affinity for ERs was labeled by 18F in good radiochemical
purity. The biodistribution studies in immature female SD rats
demonstrated that the uptake in the uterus and ovaries was
1.358 ꢀ 0.089% ID/g, 1.439 ꢀ 0.214% ID/g, respectively, both uterus/
blood and ovaries/blood ratios were less than 2:1.
These preliminary results suggested that the cyclofenil analogs
might be potential PET imaging agents for ERs. Further structure
modification and detailed evaluation is underway and will be
reported in due course.
2.6. Micro-PET imaging of immature female SD rats
The micro-PET imaging was obtained by injection of 100
mCi of
[
18F]3a into immature female SD rats. The representative summed
images of 65–69 frames in immature female SD rats in Fig. 6 also
showed the major uptake of [18F]3a in liver, ovaries, and uterus
from different views (coronal (up); sagittal (middle); horizontal

H. Zhu et al. / Journal of Fluorine Chemistry 139 (2012) 46–52
51
4. Experimental
saturation binding affinity and to 5 nM for competition binding
affinity respectively in which HAP was used to absorb the
receptor–ligand conjugate. The compound (3a–3f or 2a–2f) was
dissolved respectively in DMF, and then diluted with binding
buffer to obtain concentrations ranging from 0.1 nM to 1 mM. Then
All experiments were performed under the specified tempera-
ture conditions. 1H NMR was recorded on a Bruker AC-500
(400 MHz) instrument with Me4Si as an internal standard in the
indicated solution described below, respectively. High resolution
mass spectrums Electron (HRMS) were obtained on Micromass
GCTTM. IR spectra were recorded on an Avataar 370 FT-IR
spectrometer (250–4000 cmꢁ1).
30
synthetic compound was added to the test tube followed by buffer
to a final volume of 300 L. The mixture was incubated at 25 8C for
m
L of the ER solution, [3H]-E2 solution, estradiol and the
m
2 h. HAP slurry (30% solution) was added and the mixture was
vortexes 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 radioactivity was counted the next day in the
scintillation counter (Beckman) with 43% counting efficiency. All
numeric data were expressed as the mean of the values ꢀ the SEM.
Graphpad Prism, Version 4, was used for statistical analysis.
Purified full-length human ER
PanVera/Invitrogen (Carlsbad, CA, USA). [6,7-3H]Estra-1,3,5(10)-
triene-3,17-
-diol ([3H]-E2), 44.8 Ci/mmol, was from Perkin Elmer
a and ERb were purchased from
b
(Boston, MA, USA). Hydroxyapatite (HAP) was from Aladdin
(China). Borosilicate glass tubes were from VWR International
(West Chester, PA, USA). Fluorine-18 was obtained from the 18O (p,
n)18F reaction on an enriched water target (Sumitomo Heavy
Industries, Co. Ltd.). High performance liquid chromatography
(HPLC) analyses of the fluorine-18 analogs were performed using a
Dionex P680 system equipped with a tunable absorption detector
and a PDA-100 photodiode-array detector.
4.5. In vitro stability studies and partition coefficients
Stability of the [18F]3a was evaluated by measuring the
radiochemical purity using radio-HPLC at different time intervals.
[
18F]3a was added to a test tube containing phosphate buffered
4.1. Synthesis of reference compounds 3a–f
saline (PBS) solution or fetal calf serum. The mixture was incubated
by shaking at 37 8C in a Thermo-mixer. The radiochemical purity
was measured at 10 min, 30 min, 45 min, 60 min, 120 min and
180 min by radio-HPLC (Hypersil BDS C-18 reversed-phase column
The synthesis of cyclofenils (2a–f and 3a–f) is shown in Scheme
1. Cyclofenils (2a–f) were prepared from 4,40-dihydroxybenzo-
phenone 1 and ketones by McMurry reaction [30,31].
Compounds 3a–f were synthesized according to the following
general procedure:
(250 mm ꢂ 4.6 mm, 5
m
m), 60% CH3CN/40% H2O, and flow rate
1.0 mL/min).
For partition coefficients, the mixture of 10
m
L aliquot of
The reaction of cyclofenils (2a–f) with 2-fluoroethyl-4-methyl-
benzenesulfonate (FEtOTs, 1.5 equivalents) in the presence of
K2CO3 afforded FEt-cyclofenils (3a–f) in high yields [32,33].
aqueous solution of labeled compound, 1 mL n-octanol and 1 mL
PBS was vigorous shaken for 3 min at room temperature using a
vortex miner, then the solution was incubated for 30 min. This
workup was conducted for five times. The organic layer and the
phosphate buffer solution were collected, and the radioactivity
4.2. Synthesis of precursor compound 4
was measured with
g counter.
A flask containing cyclofenil 2a (1.0 mmol), 2-methanesulfony
bromoethyl-sulfonate (240 mg, 1.2 mmol), K2CO3 (205 mg,
1.5 mmol) was fitted with a reflux condenser, and charged with
nitrogen gas. After acetone (5 mL) was added, the solution was
stirred and heated to 50 8C for 3 h. Purified by column chromatog-
raphy (EtOAc/hexane = 1/5) to afford the product 4. Yield 51.0%,
4.6. Biodistribution studies
Immature female SD rats were used for biodistribution studies.
The radioactivity was injected via tail vein under isoflurane
anesthesia, 200
diluted with 1.8 mL of saline (10% ethanol/saline solution). Two
sets of animals (n = 5) were injected with 20 Ci/200
L of [18F]3a
m mL ethanol and
Ci of [18F]3a was dissolved in 200
colorlessoil. UV(EtOH):
l
228, 275 nm;IR (KBr):
n3210, 2855, 1319,
1169, 1161, 908, 652 cmꢁ1; 1H NMR (CDCl3)
d: 7.10 (2H, d, J = 8.6 Hz,
m
m
aromatic), 7.02 (2H, d, J = 8.5 Hz, aromatic), 6.81 (2H, d, J = 8.6 Hz,
aromatic), 6.75 (2H, d, J = 8.5 Hz aromatic), 4.56 (2H, t, J = 4.4 Hz, –
CH2OMs), 4.22 (2H, t, J = 4.4 Hz, –OCH2–), 3.08 (3H, s, OMs), 2.36–
2.38 (4H, m, C2–H), 1.64–1.70 (4H, m, C3–H); ESI-HRMS calcd. for
and sacrificed at the noted time points (1 h, 2 h). Radioactivity
was counted in a Wallac (Perkin-Elmer) 1470 Wizard Gamma
Counter, and activity in each tissue was calculated as % ID per gram
{organ uptake = [organ radioactivity/(total radioactivity ꢂorgan
weight)] ꢂ 100%}.
C
21H24O5SNa [M+Na]+: 411.1242; found: 411.1240.
4.3. Radio-synthesis of [18F]3a
4.7. Micro-PET imaging
The solution with [18F] fluoride radioactivity was transferred to
an automated synthesis unit (Type PET-MF-2V-IT-I) that contained
Micro-PET scans and image analysis were performed using an
Inveon Dedicated PET (DPET) scanner (Siemens Medical Solutions,
Malvern, PA). The scanner has a computer controlled bed and
12.7 cm trans axial fields of view (FOVs) and 1.4 mm of resolution at
Kryptofix-2.2.2 (18 mg, 48
mmol) and K2CO3 (4.5 mg, 30 mmol).
The reaction product was dissolved in acetonitrile (1.0 mL) and
transferred to a test tube that contained precursor 4 (3.8 mg,
the center of FOV. All rats were injected via the tail vein with100
mCi
of [18F]3a and anesthetized by inhalation of isoflurane/oxygen,
placed head first prone for imaging, and the acquisition time was set
for 30 min. The acquired data were rebinned with a 2D ordered-
subsets expectation maximum (OSEM) algorithm. Representative
images shown are static scans of single mouse (arrows indicate
target tissue), which is representative of 3 mice tested in each group.
10.0
mmol) and a glass bead. The test tube was capped in a reactor
at 110 8C for 30 min. The reaction mixture was purified by RP-HPLC
(Agilent 300SB-C3 Semi-Prep HPLC Column 250 mm ꢂ 9.4 mm,
5
m
m, 60% CH3CN/40% H2O, flow rate 5.0 mL/min) to give 18F
labeled compound [18F]3a.
4.4. Estrogen receptor binding affinity test
Acknowledgements
The ER
a and ERb were diluted to 2 nM in binding buffer
(50 mM Tris–HCl, pH 7.4, 150 mM KCl, 1 mM EDTA). [3H]-E2 was
extracted with ethanol and diluted in Tris–HCl to 0.5–30 nM for
This work was supported by the Natural Science Foundation
of China (No. 81071250, No. 81071198, No. 81172082 and

52
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