Synthesis and In Vivo Evaluation of a Kit-Type 99mTc-labeled N-(2-Aminoethyl)-3-(4-(2-hydroxy-3-(isopropylaminopropoxy)phenyl)propanamide as a Selective β1-Adrenoceptor-binding SPECT Radiotracer

Article abstract of DOI:10.1002/bkcs.11027

To develop a novel β₁-adrenoceptor selective radiotracer, ^99mTc-labeld N-(2-aminoethyl)-3-(4-(2-hydroxy-3-(isopropylaminopropoxy)phenyl)propanamide ([^99mTc]1) was prepared by incorporating [^99mTcO₄]^? into the precursor 3. Radiotracer [^99mTc]1 was optimized in several reaction conditions for kit-type preparation of ^99mTc-radiopharmaceuticals. The radiochemical yield was around 95% (non-decay corrected) within 30 s labeling time and radiochemical purity showed over 98%. In image studies, [^99mTc]1 provided a good heart accumulation, whereas liver and lung uptakes showed relatively low at initial time. Furthermore, [^99mTc]1 images at 25 min post-injection provided a clearly visible cardiac image with high contrast. To confirm the β₁-adrenoceptor-binding specificity of [^99mTc]1, a blocking experiment was performed. In vivo blocking with excess esmolol (18 mg/kg) or atenolol (2 mg/kg) resulted in significantly reduced radioactivity uptake in the heart around 92 and 76%, respectively. The results suggested that [^99mTc]1 might be of worth applying to the assessment of β₁-adrenoceptor expression in the heart diseases.

Full text of DOI:10.1002/bkcs.11027

Article
DOI: 10.1002/bkcs.11027
BULLETIN OF THE
Y. Lu et al.
KOREAN CHEMICAL SOCIETY
Synthesis and In Vivo Evaluation of a Kit-Type 99mTc-labeled
N-(2-Aminoethyl)-3-(4-(2-hydroxy-3-(isopropylaminopropoxy)phenyl)
propanamide as a Selective β1-Adrenoceptor-binding SPECT
Radiotracer
Yingqing Lu,† Jae Ho Jung,‡ Hong Jin Lee,‡ Byung Seok Moon,‡ Byung Chul Lee,‡,§,
*
and Sang Eun Kim†,‡,§,
*
†Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology,
Seoul National University, Seoul 08826, Korea. *E-mail: kse@snu.ac.kr
‡Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul National
University Bundang Hospital, Seongnam 13620, Korea. *E-mail: leebc@snu.ac.kr; kse@snu.ac.kr
§Center for Nanomolecular Imaging and Innovative Drug Development, Advanced Institutes of
Convergence Technology, Suwon 16229, Korea
Received October 7, 2016, Accepted November 2, 2016, Published online December 2, 2016
To develop a novel β1-adrenoceptor selective radiotracer, 99mTc-labeld N-(2-aminoethyl)-3-(4-(2-hydroxy-
3-(isopropylaminopropoxy)phenyl)propanamide ([99mTc]1) was prepared by incorporating [99mTcO4]− into
the precursor 3. Radiotracer [99mTc]1 was optimized in several reaction conditions for kit-type preparation
of 99mTc-radiopharmaceuticals. The radiochemical yield was around 95% (non-decay corrected) within
30 s labeling time and radiochemical purity showed over 98%. In image studies, [99mTc]1 provided a
good heart accumulation, whereas liver and lung uptakes showed relatively low at initial time. Further-
more, [99mTc]1 images at 25 min post-injection provided a clearly visible cardiac image with high con-
trast. To confirm the β1-adrenoceptor-binding specificity of [99mTc]1, a blocking experiment was
performed. In vivo blocking with excess esmolol (18 mg/kg) or atenolol (2 mg/kg) resulted in signifi-
cantly reduced radioactivity uptake in the heart around 92 and 76%, respectively. The results suggested
that [99mTc]1 might be of worth applying to the assessment of β1-adrenoceptor expression in the heart
diseases.
Keywords: β1-Adrenoceptor, Technetium-99m, Single photon emission computed tomography, Cardiac
imaging
Introduction
Even though β2-adrenoceptor is prominent, the density of
β1-adrenoceptor is decreased in patients with congestive
heart failure.6 Recently, most studies confirmed that the
failing human hearts often perform a selective reduction in
β1-adrenoceptor without change in β2-adrenoceptor den-
sity.7 On account of its crucial effect to cardiac disease,
β1-adrenoceptor is an ideal target for specific cardiac ima-
ging and also for the development of fixed point therapy.
As shown Figure 1, (S)-[11C]CGP 12177 (4-(3-tert-buty-
lamino-2-hydroxypropoxy)benzimidazol-2-one), the first
reported radiotracer in 1991 by Delforege et al., was devel-
oped for assessment of β-adrenoceptor density by positron
emission tomography (PET), but the benzimidazol-2-one
analog has not enough binding affinity for imaging of
β1-adrenoceptor as the radiotracer.8–11 As another the
benzimidazol-2-one analog, (S)-[11C]CGP 12388, has been
presented more easily accessible for the β1-adrenoceptor
but it also showed non-selective receptor targeting
in vivo.12 For measuring of β1-adrenoceptor density usually
abnormally changes in cardiac disease, a selective β1-
It is generally accepted that β-adrenoceptors play a decisive
role in various cardiac diseases such as heart failure.1
β-Adrenoceptors belong to the G protein-coupled receptor
(GPCR) super-family usually related to various cardiovas-
cular functions which can be subdivided into three different
subtypes including β1-, β2- and the atypical β3-adrenocep-
tor.2,3 In β-adrenoceptor family, β1-adrenoceptor are found
on cardiac muscle cells whereas β2-adrenoceptor are found
on arterial and bronchial smooth muscle at a ratio of about
7:3.4 β1-Adrenoceptor agonists stimulate the heart rate and
cardiac contractility representing roughly 70% of the total
β-adrenoceptor density,1c while selective β2-adrenoceptor-
binding agents mainly monitor bronchodilation and vaso-
depression. The atypical β3-adrenoceptor participate in
lipolysis and the assumed subtype β4-adrenoceptor
mediated by the β1-adrenoceptor were also identified in the
cardiac tissue.5 β1-Adrenoceptor stimulation is one of the
most effective measures for maintaining cardiac output.
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includes a much more convenient and instantaneous radiola-
beling procedure, a kit-type of radiolabeling procedure.
Kit-type preparation for radiopharmaceuticals can date back
to 1980s when Dougan et al. managed to label [123I]mIBG
by kit form, and gradually play a significant role in opti-
mized labeling process with high reliability, especially for
radiopharmaceuticals.20 Not only dramatically shortens the
reaction time, kit-type labeling also largely improves
the safety for reaction as well as stability on storage. Herein,
the preliminary biological evaluation of [99mTc]1 for heart
imaging based on β1-adrenoceptor expression was carried by
serial SPECT imaging experiments.
Experimental
Figure 1. Chemical structure of current radiotracers and blockersa
for β1-adrenoreceptor.
All chemicals, reagents, and solvents for the synthesis used
were analytical grade and purchased from Sigma-Aldrich
(Darmstadt, Germany). Esmolol provided from Dongwoo
Syntech Co., Ltd. (Seongnam, Korea). Flash column chro-
matography was performed with silica gel (230–400 mesh,
ASTM) from Merck (Darmstadt, Germany). All reactions
were controlled by thin-layer chromatography (TLC),
which was performed on Merck silica gel glass plates
(60F254). Proton nuclear magnetic resonance (1H NMR)
spectra were analyzed by an INOVA-400 (400 MHz) at
room temperature using CDCl3 or D2O as solvent. Chemi-
cal shifts were recorded in parts per million (ppm, δ units).
Electrospray mass spectrometry (ESI-MS) was performed
adrenoceptor-binding radiotracer is imminently needed on
cardiac image for cardiopathic diagnosis and therapy
effective evaluation as well as prognosis judgment. How-
ever, specific and selective β1-adrenoceptor radiopharma-
ceuticals for clinical use are still very rare so far.
Subsequently, only few radiotracers, for example,
(Æ)-[11C]HX-CH 44,13 (S)-[11C]bisoprolol,14 [11C]CGP
20712A,15 and ICI 8940616 were developed. Nevertheless,
their applications are mostly restricted due to high non-
specific binding, instability in vivo or a tissue uptake that
does not reflect binding to β1-adrenoceptor.14,15,17 Further-
more, these radiotracers usually need to be prepared in
harsh conditions, complicated procedures, and multiple
subsequent purification processes which lead to low radio-
chemical yields.18 Among all possible commercialized
drugs reacting with β1-adrenoceptor, we chose esmolol, an
early example of a well-designed cardioselective and an
ultra-short acting β1-adrenergic blocking agent, which has
been widely used in the sympathetic nervous system.19
Esmolol can be easily available to modify its structure with
no touching the functional group reacting with β1-adreno-
ceptor. In addition, we focused on the radionuclide, single
photon emitting 99mTc to develop a selective β1-adrenocep-
tor-binding radiotracer because of its ideal physical proper-
ties (141 keV, t1/2 = 6 h) and convenient preparation for
routine clinical use. As a chelating functional group for
99mTc, ethylenediamine was embedded to esmolol instead
of methoxy group, which can be easily labeling with
99mTc. It is a strategic 99mTc chelating moiety, which has
been effectively applied in targeting receptors for minimiz-
ing its interruption to receptor-binding site. Therefore, the
present study describes a new β1-adrenoceptor-binding single
photon emission computed tomography (SPECT) radiotra-
cer, 99mTc-labeled N-(2-aminoethyl)-3-(4-(2-hydroxy-3-(iso-
propyl amino-propoxy)phenyl)propanamide, 99mTc-labeled
esmolol analog, for diagnostic purpose in cardiac dysfunc-
tion related β1-adrenoceptor expression. Also, this study
on
a
LC/MS spectrometer (Agilent 6130 Series).
Na99mTcO4 was eluted on a daily basis from 99Mo/99mTc
generators (Samyoung Unitech, Seoul, Korea). HPLC was
carried out on a Thermo Separation Products System
(Fremont, CA, USA) equipped with an analytical column
(YMC triart C18, 4.6 mm × 250 mm) and equipped with a
UV detector (wavelength set at 280 nm) and a gamma-ray
detector (Bioscan). HPLC-grade Solvents (J. T. Baker,
Pennsylvania, USA) are used for HPLC purification
through a 0.22 μm filtering (Whatman, Maidstone, UK).
The column was eluted with a mixture of 15% acetonitrile–
0.1 M phosphate buffered saline (pH = 6.0) at a flow rate
of 0.5 mL/min. Radio-TLC was carried out by a Bioscan
radio-TLC scanner (Washington, DC, USA). All radioacti-
vitive counting were analyzed by a VDC-505 activity cali-
brator from Veenstra Instruments (Joure, The Netherlands).
All of the animal experiments using Sprague-Dawley
(SD) rats were approved by the Institutional Animal Care
and Use Committee of the Seoul National University Bun-
dang Hospital (SNUBH).
N-(2-Aminoethyl)-3-(4-(2-hydroxy-3-(isopropylamino)-
propoxy)phenyl)propanamide dihydrochloride (3). To
compound 2 (600 mg, 2.03 mmol) in 10 mL of methanol,
ethylenediamine (1.22 g, 20.3 mmol) was added and the
solution was heated at reflux for 30 h. Upon completion,
the reaction mixture was filtered and washed with metha-
nol. Filtrate was concentrated under reduced pressure at
55ꢀC. The residue was dissolved in toluene and
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concentrated under reduced pressure at 65ꢀC. Ethyl acetate
(10 mL) was added to the residue and the mixture was stir-
red at room temperature for 12 h. The precipitate was fil-
tered, washed with ethyl acetate, and dried under reduced
pressure at 55ꢀC, to give N-(2-aminoethyl)-3-(4-(2-
hydroxy-3-(isopropyl-amino)propoxy)phenyl)propanamide
(513 mg, 78.2%) as a white solid: MS (ESI) m/z 324.02 [M
+H]+. Subsequently, the obtained compound (500 mg,
1.55 mmol) in 10 mL of methanol, isopropyl alcohol
(0.888 mL) were added. The solution was filtered, washed
with 10 mL of methanol, and cooled to 0ꢀC. The hydrogen
chloride solution (230 mg of hydrogen chloride in 713 mg
of isopropyl alcohol) was added slowly to cooled solution
and then stirred for 1 h at room temperature. Isopropyl
alcohol (10 mL) was added additionally and the solution
was stirred for 3 h at room temperature. The resulting pre-
cipitate was filtered, washed with isopropyl alcohol (5 mL),
and dried under reduced pressure at 55ꢀC to give crude
mixture (301 mg). The crude mixture in 5 mL of isopropyl
alcohol was heated at 75ꢀC for 30 min. The solution was
filtered and washed with isopropyl alcohol. Filtrate was
cooled slowly to 20ꢀC for 3 h. The precipitate was filtered
and washed with isopropyl alcohol again. The product was
dried under reduced pressure at 55ꢀC to give white crystal-
line solid product 3 (271 mg, 48.4%): m.p. 124.5–127.3ꢀC;
1H NMR (D2O, 400 MHz) δ 7.24 (d, J = 11.6 Hz, 2H),
7.01 (d, J = 11.6 Hz, 2H), 4.33–4.29 (m, 1H), 4.16–4.07
(m, 2H), 3.55–3.49 (m, 1H), 3.41 (t, J = 6.2 Hz, 2H),
3.36–3.22 (m, 2H), 3.04 (t, J = 6.2 Hz, 2H), 2.89
(t, J = 7.4 Hz, 2H), 2.58 (t, J = 7.4 Hz, 2H), 1.38–1.36
(m, 6H); 13C NMR (D2O, 100 MHz) δ 176.8, 156.4, 133.5,
129.6, 114.8, 69.6, 65.6, 51.0, 46.6, 38.9, 37.3, 36.5, 30.2,
18.2, 17.8. MS (ESI) m/z 324.24 [M+H−2HCl]+.
Reagents solution B includes SnF2 (25 mg) in 25 mL of
0.1 N HCl. In aseptic room, the mixed solution (solutions
A and B) was filtered by PVDF (polyvinylidene difluoride)
membrane filter (0.22 μm) and divided by micropipette for
1.25 mL per vial. In result, every kit-vials contained 1 mg
of compound 3, 1 mg of ascorbic acid, 5 mg of mannitol,
0.25 mg of SnF2, and 0.02 N HCl and dried over vacuum
freeze-drying at −50ꢀC. Pure N2 (99.9999%) was used to
protect ingredients from O2 and all vials were sealed com-
pletely with rubber cap. In preparation of kit-vials for NaCl
(5 mg) or cyclodextrin (5 mg) as other stabilizers, all pro-
cesses were performed as same described above. For syn-
thesis of [99mTc]1, Na99mTcO4 (approximately 740 MBq,
1 mL of saline) and 0.1 N HCl (63 μL) were added in an
empty vial. After voltexing for few seconds, the mixture
was injected into kit-vial and voltexed at room temperature
for 30 s. The solution was filtered by PVDF membrane fil-
ter (0.22 μm, 13 mm). The radiochemical yield of [99mTc]1
was more than 95% and there was no byproduct peak
except for negligible free 99mTcO4 which had checked by
−
radio-TLC and HPLC. The identity was confirmed by coin-
jection with authentic [Re]1 in the analytical HPLC system
(YMC triart C18, 4.6 mm × 250 mm, 15% acetonitrile–
0.1 M phosphate buffered saline (pH 6.0), flow rate = 0.5
mL/min). For biological evaluation by SPECT in rat,
[
99mTc]1 was injected as below one-tenth of total volume
(v/v) per rat.
SPECT/CT Imaging. Animal SPECT imaging study was
performed using low-energy and high-resolution pyramid
collimator (4 × 9, 1.4 mm pinhole) in SPECT/CT (Single
photon emission computed tomography/ Computed tomog-
raphy) scanner system (NanoSPECT/CT, Bioscan Inc.,
Washington, DC, USA). [99mTc]1 (approximately 74 MBq,
n = 3) was administrated to normal SD rat (8–9 weeks)
through the intravenous injection under anesthesia with 2%
isoflurane. Whole body images were obtained in 24 projec-
tions over 10 min periods. For measurement of heart, lung,
and liver uptake of [99mTc]1, HiSPECT (version 1.0, Bios-
can Inc., Washington, DC, USA) and InVivoScope (version
1.43, Bioscan Inc., Washington, DC, USA) were used for
image reconstruction and quantification, respectively. The
reconstructed SPECT was described with an image size of
176 × 176 × 136 and a voxel size of 0.2 mm × 0.2 mm ×
0.2 mm (x, y, z). The accumulated radioactivity of each
organs was determined by drawing regions of interest
(ROI) using the reference source (99mTc-pertechnate,
37 MBq). The percentage of the injected dose per gram of
weight (% ID/g) was calculated from the radioactivity con-
tained in the ROI divided by the dose administered to
the rat.
Re-coordinated
N-(2-Aminoethyl)-3-(4-(2-hydroxy-3-
(isopropylamino)propoxy)phenyl)propanamide ([Re]1).
To ReOCl3[P(Ph)3]2 (trichlorooxobis(triphenyl phosphine)
rhenium(V), 333 mg, 0.4 mmol) in 4.0 mL of methanol,
aqueous solution of sodium hydroxide (48 mg, 1.2 mmol)
and compound 3 (158 mg, 0.4 mmol) in 2.0 mL of water
were added (water:methanol:0.3 N NaOH = (v/v/v) 1:2:2).
The mixture solution was heated at 90ꢀC for 30 min. Upon
completion, reaction mixture was cooled to 0ꢀC, then fil-
tered, and washed with methanol (2.0 mL × 2). The solid
was dissolved in 10 mL of H2O and filtered again. Filtrate
was frozen and dried to give white solid (178 mg, 85.2%).
1H NMR (D2O, 400 MHz) δ 7.14 (d, J = 10.4 Hz, 4H),
6.89 (d, J = 10.4 Hz, 4H), 4.02-3.99 (m, 4H), 3.93–3.89
(m, 2H), 3.02 (t, J = 7.8 Hz, 4H), 2.87–2.71 (m, 8H),
2.64–2.58 (m, 2H), 2.47–2.37 (m, 8H), 0.98–0.96
(m, 12H). MS (MALDI) m/z 866.05, 868.06 [M+H]+.
Three Different Stabilizers Contained Kit Formulation
and General Method for 99mTc-labeled N-(2-Ami-
noethyl)-3-(4-(2-hydroxy-3-(isopropylamino)propoxy)
phenyl)propanamide ([99mTc]1). Reagents solution A
includes compound 3 (100 mg), ascorbic acid (100 mg),
and mannitol (500 mg) in 100 mL of distilled water.
Blocking Study. Normal SD rats (8–9 weeks) were admi-
nistered with esmolol (18 mg/kg) or atenolol (2 mg/kg)
into the tail vein before 10 min the injection of [99mTc]1.
After that, the rats were anesthetized with 2% isoflurane in
a 7:3 mixture of N2/O2 and the intravenous injection of
[
99mTc]1 (approximately 74 MBq, n = 3) was performed.
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Whole body SPECT images were acquired at a mid-scan
time of 25 min for scan duration of 10 min, followed by a
CT scan of body for attenuation correction.
1.95 Æ 0.3% ID/g and the lowest uptake in the liver
(1.10 Æ 0.2% ID/g) at 5 min post-injection compared to
those of other stabilizers as shown Figure 2.
Conversely, lung uptake of [99mTc]1 was 0.38 Æ 0.1%
ID/g and slowly decreased with elapsed time by urinary
excretion. In case of NaCl, the uptake of [99mTc]1 in the
heart was decreased (40% reduction vs. the mannitol trea-
ted group) and the uptake in the liver reached up to 4.7%
ID/g which was fourfold higher than that of mannitol.
Even though the cyclodextrin treated group showed the
similar heart uptake, the liver uptake showed 3.9%
ID/g. Consequently, we established that mannitol was the
best stabilizer for preparing a kit-type [99mTc]1 superior to
NaCl and cyclodextrin by measuring of heart-to-lung and
heart-to-liver ratios.
Results and Discussion
The precursor 3 was simply prepared by the amide conden-
sation from esmolol (2) and diethylamine as shown in
Scheme 1. The obtained N-(2-aminoethyl)-3-(4-(2-hydroxy-
3-(isopropylamino)propoxy)phenyl)propanamide was trea-
ted with hydrogen chloride, resulted in the chlorine salt
form 3. The “cold” rhenium coordination reaction with the
precursor was performed in a water–methanol–sodium
hydroxide mixture at 90ꢀC using ReOCl3(PPh3)2. The rhe-
1
nium complex [Re]1 was characterized by H NMR and
In terms of purity, [99mTc]1 could be prepared in high
radiochemical yield over 95% with more than 98% of radi-
ochemical purity only through filtration of PVDF filter
mass spectrum. The coordination of the 99mTc radioisotope
to the precursor 3 was performed in aqueous media from
99mTc-pertechnetate in the presence of SnF2, ascorbic acid,
and stabilizers.
(0.22 μm) after incorporation of 99mTcO4 and the precur-
−
sor (3) in the presence of SnF2, ascorbic acid, and mannitol
at room temperature for 30 s. Radio-TLC was used to iden-
tify [99mTc]1. The Rf value of [99mTc]1 and unreacted
99mTc was 0.0 with 99.1% and 1.0 with 0.9%, respectively,
using an absolute acetone as developing solvent. In HPLC
analysis, [99mTc]1 was eluted at 4.9 min while the retention
As the ingredient in a kit formulation, SnF2 was served
as an alternative reducing agent to SnCl2 to avoid 99mTc-
colloidal phenomenon. Though SnCl2 is a very common
used reducing agent in various 99mTc-labeled procedures,
the use of SnCl2 induced a mass of 99mTc-colloidal resulted
in high liver uptake of [99mTc]1 in SPECT/CT images. An
anti-oxidant ascorbic acid was also employed to enhance
the in vivo stability of [99mTc]1 for this reaction. In addi-
tion, various stabilizers were investigated to improve target
to non-target ratio, i.e., heart-to-liver and heart-to-lung.
Figure 2 summarize the in vivo imaging results of the
of 99mTcO4 was 10.9 min. Without additional purification
−
process such as HPLC or the C18 Sep-Pak cartridge,
[
99mTc]1 was readily prepare to use directly for biological
experiment or in vivo images by simple filtering (Figure 3).
After determining the optimal kit formulation, we obtained
high-resolution scans (12 frames) of each rat from 0 to
120 min after the intravenous injection of [99mTc]1 (approxi-
mately 74 MBq). In SPECT images, [99mTc]1 showed high
accumulation in the heart and kidneys, while the liver
and lung uptakes were relatively low as shown Figure 4.
Timed organ distributions (% ID/g) was addressed in the
Table 1. The desirable scan time, 25 min post-injection
was derived from serial SPECT imaging study by consid-
ering of the ratios of heart-to-lung and heart-to-liver.
To confirm the β1-adrenoceptor-binding specificity of
[
99mTc]1 without or with different stabilizer. Among four
images, [99mTc]1 exhibited significantly the high liver accu-
mulation at non-use of stabilizer. Comparison of the serial
SPECT/CT images of [99mTc]1 with stabilizer-dependent
was performed in normal rats (n = 3), respectively.
Through comparative experiments, an optimal stabilizer
was chosen from the most excellent performance focusing
on cardiac SPECT images. Among three SPECT/CT
images by different stabilizers contained kit-vials, mannitol
induced the highest uptake of [99mTc]1 in the heart about
[
99mTc]1, the blocking experiment was performed with
esmolol and atenolol, respectively, which was well known
as β1-adrenoceptor blockers.19,21 Using excess esmolol
(18 mg/kg) or atenolol (2 mg/kg),22 both images resulted in
significantly reduced radioactivity uptake in the heart
(92 and 76% reduction in heart uptake vs. control, respec-
tively). Our results indicated that the heart images of
[
99mTc]1 was mediated selectively by β1-adrenoceptor as
shown in Figure 5.
In summary, To better prepare a kit-type 99mTc-labeled
esmolol derivative ([99mTc]1), several attempts were inves-
tigated with different reagents and stabilizers. [99mTc]1 can
be prepared extremely simple and instantaneous by reacting
99mTcO4− with the precursor in the presence of SnF2, ascor-
bic acid and mannitol at room temperature. Total prepara-
tion time was around 5 min including radiolabeling and
Scheme 1 (a) ethylenediamine, MeOH, reflux, 30 h; (b) 0.6 N
HCl, iso-propyl alcohol, MeOH, rt, 4 h; (c) ReOCl3(PPh3)2,
NaOH, 90ꢀC, 30 min for [Re]1; Na99mTcO4, ascorbic acid, stabi-
lizer, SnF2, 0.02 N HCl, rt, 30 s for [99mTc]1.
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Figure 2. Time-activity curves (a–d), ratios of heart-to-lung and heart-to-liver (e), and representative SPECT/CT images (f ) of [99mTc]1
without or with different stabilizers.
filtration. Compared to the reported β1-adrenoceptor-
binding radiotracers, 99mTc-labeled esmolol derivative is
much more attractive in terms of its longer half-life (t1/
separation or any other further purification processes. The
kit-type radiolabeling method takes great potentials for
commercialization and development of clinical radiophar-
maceuticals. It can be also readily automated using a com-
mercial radiosynthetic module fleetly and largely without
burdensome operation or sophisticated diversiform instru-
ments. In SPECT imaging studies, [99mTc]1 showed rela-
tively high radiotracer uptake in the heart in comparison
= 6 h), convenient to acquire from generator and simple
2
radiolabeling procedure. In this work, 99mTc-labeled esmo-
lol derivative was prepared by a kit-type radiotracer, which
was able to perform as the final in vivo injection through
filtration of PVDF filter (0.22 μm) without HPLC further
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Figure 3. Identification of [99mTc]1 using radio-TLC scanner and HPLC.
Simultaneously, in vivo blocking experiments with excess
esmolol displayed remarkable reduction of radioactivity
uptake in the heart. From the other side, it demonstrated
that [99mTc]1 inherits a series of advantages from esmolol.
It illustrates that ethylenediamide modification does not
impact the original cardioselective β1-adrenoceptor blocker
function of esmolol, i.e., short duration of action and no
significant intrinsic sympathomimetic performance.23 The
short duration of action of esmolol is based on rapid
hydrolysis by plasma esterase on the ester-methyl chain.
This metabolism produces a free acid and methanol. In a
similar, [99mTc]1 exhibits relatively little slower but still
rapid clearance rate as the peptide bond would be broken
by enzymes first, then releases ethylenediamide moiety and
99mTc, radioactive marker, immediately. These results indi-
cate that [99mTc]1 is a highly sensitive radiotracer for β1-
adrenoceptor imaging in the heart. Based on the success
modification using ethylenediamide, we will focus on the
optimized formula linked with esmolol and radioactive
markers, e.g., Ga-68, Cu-64 and In-111 to further enhance
the quality of cardiac images. Furthermore, animal models
for cardiovascular disease will be selected to evaluate the
application of [99mTc]1.
Figure 4. Serial SPECT/CT images of [99mTc]1 in the presence of
mannitol.
Table 1. Uptake (% ID/g) of [99mTc]1 in major organs of normal
rats.a
% ID/g
Organs
5 min
25 min
55 min
115 min
Lung
Liver
Heart
Hr/Lub
Hr/Lic
a
0.38 Æ 0.1
1.10 Æ 0.2
1.88 Æ 0.2
4.94 Æ 0.7
1.73 Æ 0.2
0.21 Æ 0.1
0.85 Æ 0.2
1.02 Æ 0.2
4.98 Æ 0.9
1.22 Æ 0.2
0.13 Æ 0.1
0.75 Æ 0.2
0.73 Æ 0.1
5.98 Æ 1.3
0.99 Æ 0.2
0.07 Æ 0.1
0.65 Æ 0.2
0.50 Æ 0.2
7.61 Æ 2.2
0.77 Æ 0.1
Conclusion
In conclusion, 99mTc-labeld esmolol derivative, [99mTc]1,
was successfully synthesized in excellent yield by “kit”-
type formulation. Also, the presented preclinical biological
study suggests that [99mTc]1 may have potential as a useful
SPECT radiotracer for assessment of β1-adrenoceptor in
heart diseases.
The data expressed the percentage of injected dose per gram
(% ID/g, n = 3).
b
c
The ratio of heart to liver.
The ratio of heart to lung.
Acknowledgments. We would like to gratefully and sin-
cerely thank Professor David J. Yang in university of Texas
MD Anderson cancer center for his assistance and under-
standing 99mTc-ethylendiamine moiety radiolabeling. Also,
we would like to give a special thanks to DongWoo Syn-
tech Co., Ltd., which has provided the necessary esmolol
derivatives. This study was funded by the Ministry of
Health & Welfare and the Korean Research Foundation
grants (HI14C-1072-010014, HI12C-0035-030015 and
NRF-2014R1A2A2A01007980).
with liver and lung. Compared to the initial scan time, the
20–30 min summed (25 min post-injection) image was
shown a clearly visible image of the heart with high con-
trast. The radioactivity of heart at 55 min post-injection
was decreased to half (0.92 Æ 0.15%ID/g) of that uptake in
5 min post-injection and then rapidly washout via mainly
urinary excretion. Appropriate clearance rate greatly eases
the risk of the negative influence due to a long time of
radiotracer left in the heart and other vital organs.
Bull. Korean Chem. Soc. 2016, Vol. 37, 2029–2035
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 2034

BULLETIN OF THE
Article
SPECT Radiotracer for β1-Adrenoceptor
KOREAN CHEMICAL SOCIETY
Figure 5. Axial heart SPECT/CT images of [99mTc]1 without or with esmolol or atenolol.
2003, 140, 1; (d) J. R. Arch, Br. J. Pharmacol. 2004, 143,
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Bull. Korean Chem. Soc. 2016, Vol. 37, 2029–2035
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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