Synthesis and evaluation of [18F]Fluorobutyl ethacrynic amide: A potential PET tracer for studying glutathione transferase

Article abstract of DOI:10.1016/j.bmcl.2012.04.091

[¹⁸F]Flurobutyl ethacrynic amide ([¹⁸F]FBuEA) was prepared from the precursor tosylate N-Boc-N-[4-(toluenesulfonyloxy)butyl] ethacrynic amide with a radiochemical yield of 3%, a specific activity of 48 GBq/μmol and radiochemical purity of 98%. Chemical conjugation of [ ¹⁸F]FBuEA with glutathione (GSH) via a self-coupling reaction and enzymatic conjugation under catalysis of glutathiontransferase alpha (GST-α) and π provided about 41% yields of radiochemical conjugated product [¹⁸F]FBuEA-GSH, 85% and 5-16%, respectively. The catalytic selectivity of this tracer toward GST-alpha was addressed. Positron emission tomography (PET) imaging of [¹⁸F]FBuEA in normal rats showed that a homogeneous pattern of radioactivity was distributed in the liver, suggesting a catalytic role of GST. By contrast, PET images of [¹⁸F]FBuEA in rats with thioacetamide-induced cholangiocarcinoma displayed a heterogeneous pattern of radioactive accumulation with cold spots in tumor lesions. PET imaging with [¹⁸F]FBuEA could be used for early diagnosis of hepatic tumor with a low GST activity as well as liver function.

Full text of DOI:10.1016/j.bmcl.2012.04.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3998–4003
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of [18F]Fluorobutyl ethacrynic amide: A potential PET
tracer for studying glutathione transferase
Ho-Lien Huang a, , Chun-Nan Yeh b, , Kang-Wei Chang c, Jenn-Tzong Chen c, Kun-Ju Lin d, Li-Wu Chiang a,
Kee-Ching Jeng e, Wei-Ting Wang f, Ken-Hong Lim f,g, Caleb Gonshen Chen f,g, Kun-I Lin a,h
,
Ying-Cheng Huang i, Wuu-Jyh Lin c, Tzu-Chen Yen d, Chung-Shan Yu a,j,
⇑
a Department of Biomedical Engineering and Environmental Sciences, National Tsing-Hua University, Hsinchu 300, Taiwan
b Department of Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taiwan
c Institute of Nuclear Energy Research, Taoyuan 32546, Taiwan
d Department of Nuclear Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taiwan
e Taichung Veterans General Hospital, Taichung 40705, Taiwan
f Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan
g Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, and Department of Medicine, Mackay Medical College, Taipei 104, Taiwan
h Departments of Obstetrics & Gynecology, Chang Bing Show Chwan Memorial Hospital, Lukang Zhen, Changhua County, Taiwan
i Department of Neurosurgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taiwan
j Institute of Nuclear Engineering and Science, National Tsing-Hua University, Hsinchu 300, Taiwan
a r t i c l e i n f o
a b s t r a c t
18F]Flurobutyl ethacrynic amide ([18F]FBuEA) was prepared from the precursor tosylate N-Boc-N-[4-(tol-
uenesulfonyloxy)butyl]ethacrynic amide with a radiochemical yield of 3%, a specific activity of 48 GBq/
mol and radiochemical purity of 98%. Chemical conjugation of [18F]FBuEA with glutathione (GSH) via
a self-coupling reaction and enzymatic conjugation under catalysis of glutathiontransferase alpha
(GST- ) and
provided about 41% yields of radiochemical conjugated product [18F]FBuEA–GSH, 85%
Article history:
Received 22 February 2012
Revised 3 April 2012
Accepted 19 April 2012
Available online 30 April 2012
[
l
a
p
and 5–16%, respectively. The catalytic selectivity of this tracer toward GST-alpha was addressed. Positron
emission tomography (PET) imaging of [18F]FBuEA in normal rats showed that a homogeneous pattern of
radioactivity was distributed in the liver, suggesting a catalytic role of GST. By contrast, PET images of
Keywords:
Imaging
GST-
a
[
18F]FBuEA in rats with thioacetamide-induced cholangiocarcinoma displayed a heterogeneous pattern
Cholangiocarcinoma
Tumor
Cold spot
of radioactive accumulation with cold spots in tumor lesions. PET imaging with [18F]FBuEA could be used
for early diagnosis of hepatic tumor with a low GST activity as well as liver function.
Ó 2012 Elsevier Ltd. All rights reserved.
Glutathion transferase (GST) represents a major group of detox-
ification enzymes.1 All eukaryotic species possess multiple
cytosolic and membrane bound GST isoenzymes, each of which
displays distinct catalytic as well as noncatalytic binding proper-
ties. The cytosolic enzymes are encoded by seven distantly related
gene families (designated class alpha, pi, mu, theta, omega, zeta,
and sigma). The most abundant mammalian GSTs are the class
alpha, mu, and pi enzymes. The biological control of these enzymes
is complicated as they exhibited sex-, age-, tissue-, species-, and
tumor-specific patterns of expression. GSTs are regulated by struc-
turally diverse xenobiotics. Many inducers can further affect tran-
scriptional activation of GST genes, for example, antioxidant
responsive element (ARE) and the xenobiotic-responsive element
(XRE). In addition, compounds that induce GST are themselves sub-
strates for these enzymes, or are metabolized to compounds that
can serve as GST substrates. The majority of human tumors and
human tumor cell lines expressed significant amounts of pi GST.
The human alpha class GSTs consist of 5 genes, hGSTA1–hGSTA5.
The homodimer hGSTA1-1 (and to a lesser extent hGSTA2-2)2 cat-
alyzes the GSH-dependent detoxification of carcinogenic metabo-
lites of environmental pollutants, tobacco smoke, and several
alkylating chemotherapeutic agents. hGSTA1-1 and hGSTA2-2 are
expressed at high levels in liver, intestine, kidney, adrenal gland,
and testis. Alpha class GSTs have been suggested to be involved
in susceptibility to diseases with environmental causes (such as
cancer, asthma, and cardiovascular disease) and in response to
chemotherapy. Although hGSTM1, hGSTT1, and hGSTP1 have been
associated with such diseases, alpha class GSTs has not been well
studied in this respect and its molecular basis for the variation is
less clear.
Abbreviations: CCA, cholangiocarcinoma; TAA, thioacetamide; NERI, Nuclear
Energy Research Institute; CGMH, Chang-Gung Memorial Hospital.
⇑
Corresponding author.
E-mail address: csyu@mx.nthu.edu.tw (C.-S. Yu).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.091

H.-L. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3998–4003
3999
Cholangiocarcinoma (CCA) is a cancer derived from bile duct
epithelium (i.e. cholangiocytes). It is characterized by a great diver-
sity of symptoms commonly occurring in the late course of the
disease, and therefore making treatment difficult.3 CCA is related
to a wide range of risk factors, such as infestation with liver flukes,
primary sclerosing cholangitis, and hepatolithiasis, liver cirrhosis
and causes various incidence rates of CCA in different areas of
the world.4 However, data have shown that the incidence and mor-
tality rates of CCA have been rising worldwide over past decades,
particularly the intrahepatic CCA.5–7 An oral thioacetamide
(TAA)-induced model of rat CCA has been used to study the thera-
peutics responses with tumor measurement and molecular imag-
ing using 2-deoxy-2-[18F]fluoroglucose (FDG) with positron
emission tomography (PET).8 The average PET signal of tumor to
liver ratio was 1.60. Given the fact that all the human, rat and mice
for example, receptors without encountering toxicity concerns.
Introduction of 18F can be mediated through a direct substitution
reaction or an indirect reaction via a bifunctional group.17,18 The
former includes a nucleophilc or electrophilic pathway. Taken
together, we attempt to prepare 18F-labeled FBuEA analog 3 as a
substrate to assess its potential use as an in vivo imaging agent
for studying GST isoenzymes in CCA rats. The structure of FBuEA
3 showed that fluorine at the end position of the butyl group
should not alter the enone functionality. In addition, the precursor
for radiofluorination is a primary alcohol-derived tosylate, which
has been widely used for radiofluorination in radiochemistry.
Preparation of precursor (8). The preparation of the desired alco-
hol 12 was initially started from a methyl ester of EA, which was
prepared by using CH2N2 and EA (Scheme 1 of Supplementary
data). Whereas the ester could be obtained in satisfactory yield
(70%), subsequent coupling with the unprotected 4-aminobutyl
alcohol provided the desired amide coupling product in only 20%
yield due to the lack of regioselectivity and the less reactive ester
(Scheme 1 of Supplementary data). Hence, by adopting the usual
HBTU-mediated amide coupling protocol in association with the
source carboxylic acid 2 and the well-protected O-TBDMS butyl
amine 4,19 a satisfactory yield of 70% of amide 5 was obtained
(Scheme 1). Recently, the preparation of 3-hydroxypropyl ethacry-
nic amide has been prepared from EA acyl chloride without
protecting the hydroxy group.20
Prior to the next step, the synthetic strategy was examined.
When the tosyl group is substitutable by [18F]fluoride ion from tet-
rabutyl ammonium fluoride, the imido proton of the tautomer
could be removed by the basic fluoride and resulted in a good
nucleophilic state readily for intramolecular cyclization. The anti-
cipitated ring closure that forms a 7-membered ring might reduce
the radiochemical yield, because we observed in a similar case
with 5-membered ring formation when performing a fluorination
for 5-(2-tolylsulfonylethyl)-20-deoxyuridine analog.21 Therefore,
an adequate protection at the amide group is needed.
An attempt to protect the amide group with acetyl group using
isoproprenyl acetate failed to provide the N-acetyl product 5 and
produced only the undesired O-acetyl byproduct, probably due to
the instability of the silyl group (Scheme 2 ofSupplementary data).
Hence, through an alternative treatment with (Boc)2O, the desired
Boc-protected product 6 could be obtained in 76% yield. By remov-
ing the silyl group with the combination of tetrabutyl ammonium
fluoride (TBAF) and AcOH, the desired product 7 could be obtained
in quantitative yield. However, in a parallel experiment under the
presence of 4 Å MS, the cyclized hemiacetal byproduct 14 was
solely obtained in 30% yield (Scheme 3 of Supplementary data).
With the alcohol 7 prepared, either the subsequent fluorination
with DAST to provide the nonradioactive compound 9 or the prep-
aration of tosylate 8 using TsCl can be performed. The cold
compound 9 and cold FBuEA 3 were both used as authentic
contain 5 subclasses of GSTa and one subclass of GSTp (two GSTp
isoforms in mice), an animal model of TAA-induced CCA would be
the choice for developing potential compounds for studying the li-
ver function.
There are two criteria of an ideal probe for studying the poten-
tial marker GSTp 9,10
:
firstly it should be a good substrate to be
conjugated with glutathione (GSH) under catalysis of GST . Sec-
p
ondly, the molecule should be tagged with appropriate moieties
and thus it can be traceable by detecting the emitting photons
which are energetically enough to penetrate the animal tissues. A
number of compounds showed marked differential affinities
toward GST isoenzymes-catalyzed conjugation with GSH; for
5
example, 4 androstene-3,17-dione, selective for rGSTA1 and/or
A2 subunits; 4-hydroxynonenal, selective for rGSTA4; 1,2-dichol-
oro-4-nitrobenzene (DCNB), selective for rGSTM1.1 1-Choloro-
2,4-dinitrobezene (CDNB) and ethacrynic acid (EA) are classified
as the relatively nonselective substrates. CDNB shows broad affin-
ities toward almost all GST isoenzymes. By contrast, EA shows a
moderate substrate affinity toward rGSTP1-1. Hence, the recent re-
port of a butyl ethacrynic amide,11–13 a butyl analog of EA, which
showed a modified cytotoxicity against tumor cells, attracts our
attention (Fig. 1).14
Regarding the second criterion, the substrate should be tagged
with a photon emitter with a size small enough to not to hamper
its original biological activity. Positron emitters, for example, 18F
emitting two energetic photons (511 keV) in an angle of 180° has
been coupled with PET for a wide use in diagnostic medicine.15,16
The characteristics of 18F, such as low radiation doses, short tissue
range, feasibility of multistep synthesis and extendable scanning
protocols are attributed to a relatively low energy of 0.64 MeV
and a relatively long half-life (t1/2 = 109.7 min). The adequate
atomic size due to being a member of the second periodic atoms
makes 18F a suitable atom for mimicking oxygen or hydrogen.
The high sensitivity of 18F allows the use of a very low concentra-
tion (10ꢀ12 M) of radiolabeled tracer for imaging cellular markers,
H
N
H
N
OH
O
O
C4H9
F
2
O
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
O
O
O
1
2
3
Ethacrynic acid
BuEA
FBuEA
IC50 = 8 μM14
IC50 = 84 μM
IC50 = 13 μM
Figure 1. Cytotoxicities of ethacrynic acid analogs against A549 cells.

4000
H.-L. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3998–4003
R
N
OH
H2N
O
OTBDMS
O
2
OTBDMS
2
Cl
Cl
O
Cl
Cl
O
TBAF/THF
4
quantitative
HBTU, 67%
O
O
5
6
R = H
Boc2O
76%
2
R = Boc
R
N
R1
N
O
X
2
O
OR2
2
Cl
O
Cl
Cl
O
9
DAST, 35% for
Cl
[
18F]TBAF, 46% for [18F]9
O
O
9 X = F, R = Boc; from 7
18F]9 X = 18F, R = Boc; from 8
1 = Boc, R2 = H
1 = Boc, R2 = Ts
[
7
8
R
R
TFA/CH2Cl2
70%
TsCl
76%
TFA/CH2Cl2
3
FBuEA R = H
18
3
F]FBuEA R = H
[
3% (based on
[
18F]F-)
Scheme 1. Synthesis of nonradioactive compound 9 and deprotected FBuEA 3 as authentic samples for radiochemical synthesis of [18F]9 and [18F]FBuEA 3.
samples throughout the radiochemical synthesis for optimizing the
radiochemical yield.
ical purity of 98% and specific activity of greater than 48 GBq/
[Fig. 2(B)].
lmol
In order to obtain a satisfactory radiochemical yield from radio-
fluorination, it is critical to have sufficiently pure tosylate 8. There-
fore, samples were collected from centered fractionations of
column chromatography with a number of tosylate 8 preparations
and the purity was met with criteria by elemental analysis.
Radiosynthesis of [18F]FBuEA 3. The preparation was carried out
by using the tosylate 8 (10 mg) and [18F]FꢀN+Bu4ꢁ [18F]9 was ob-
tained in an average radiochemical yield of 46%. The subsequent
removal of the Boc group using trifluoro acetic acid (TFA) at ambi-
ent temperature was accomplished in 7% yield, whereas a more
rigorous condition of using 50 °C in association with reduced
equivalents of TFA did not produce the desired [18F]FBuEA 3. The
HPLC chromatogram of the product mixture using a normal phase
column showed an UV active peak at tR = 8.5 min, suggesting the
released leaving group [Fig. 2(A)]. Whereas this UV active sub-
stance and the nonpolar radioactive unknown substance
(tR = 5.0 min) may not disturb the PET imaging outcomes of
Interestingly, hydrolyzed byproduct or the byproduct from
elimination was not observed in the chromatogram either before
or after HPLC purification, which might be attributed to the previ-
ous manipulation of the cartridge settings. The present protocol for
preparing [18F]FBuEA 3 (ready for tail vein injection), which in-
volved the two-step radiochemical synthesis including deprotec-
tion, collection of the fractions isolated from HPLC and
concentration under reduced pressure was accomplished with a
radiochemical yield of 2.3% (decay corrected) within 1.5 h (end of
bombardment, EOB).
Radiosynthesis of [18F]FBuEA–GSH (10). The radiochemical yield
(41%) of [18F]10 obtained from selfconjugation of [18F]FBuEA 3 with
GSH under pH 8.022 is less than the yield that reported previ-
ously.23,24 In contrast to the radio TLC estimation reported by these
literatures, the current yield calculation was based on the isolated
product from HPLC purification. On the other hand, the nonradio-
active authentic sample FBuEA–GSH 10 was prepared under the
same reaction condition (see Supplementary data) and the yield
was slightly greater than 41%. The two diastereomers were
[
18F]FBuEA 3, additional purification with semi-preparative HPLC
was used, and the isolated fractions obtained reached a radiochem-
Figure 2. Chromatogram for [18F]FBuEA before (A) and after (B) HPLC purification. A normal phase semipreparative column (Si-100) was employed. AU: Arbitrary Unit.
Eluting condition: [(A), see Supplementary data], CPS: counts per second.

H.-L. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3998–4003
4001
observed from 1H NMR in a ratio of 3:2. A further separation using
chiral column will be carried out in future study.
distribution was reached as early as the first 5 min in several or-
gans including liver, kidney and bladder (data not shown).
Although [18F]FBuEA 3 was initially assumed to be capable of intra-
cellular accumulation in tumors, a vast abundance of GSH and the
Conjugation of [18F]FBuEA (3) with GST under catalysis of GST-
p
and GST-a
. The method reported by Lo et al.25 was adopted to per-
form this conjugation (Scheme 2). In their work, a prolonged reac-
tion time (2 h) could assure that the reaction achieved completion.
The conjugating experiment using [18F]FBuEA 3 and GSH under
presence of GST especially GST-a in liver could overwhelmingly
dominate the formation of [18F]FBuEA–GSH 10 complexes. The
accumulation of [18F]FBuEA 3 was homogeneously distributed over
the liver of the normal rat [Fig. 5(A)]. By contrast, a heterogeneous
distribution of [18F]FBuEA 3 in the liver of the CCA rat was noted
[Fig. 5(B)]. The cold spots with no radioactivity accumulation sug-
gested a low level of GST. The two rats were then sacrificed and
their photographs [Fig. 5(C and D)] showed significantly patholog-
ical differences. The initial assessment of [18F]FBuEA showed a
negative correlation between GST isoenzymes and the tumor
progression of the infiltrative liver of CCA rat.
catalysis by GST-
another assay). By contrast, a high conversion yield of 85% was ob-
served in the case of GST-
(Fig. 3B). The selectivity of [18F]FBuEA 3
toward GST- may allow the study of the functions of liver since it
constitutes the major portion of GST enzyme in liver. The present
p
gave [18F]FBuEA–GSH 10 in 16% yield (5% from
a
a
findings are different from our initial aim for targeting GST-p and
now it is clear that the application of [18F]FBuEA 3 should be con-
fined to the study of liver function. An appropriate liver disease
model is needed therefore. The recently reported CCA rat with liver
tumors induced by TAA emerges as the suitable animal model for
the preliminary assessment of the probe.
Being a promising imaging probe, [18F]FBuEA 3 should be
capable of detecting a disease at its early stage. Since an oversatu-
ration of the imaging signal of normal rat was observed with a time-
frame averaging from 0 to 120 min [Fig. 5(A)], a shorter imaging
Metabolite analysis. Before carrying out the animal imaging
experiment, the stability of [18F]FBuEA 3 in vivo was assessed by
HPLC measurement of the radioactivity remaining in the blood.26,27
As shown in Figure 4, the in vivo half-life of [18F]FBuEA 3 was
determined to be 46 min (t1/2). Compared to the plasma half-life
of 0.5–1 h of the parent ethacrynic acid,28 the hydrophobic butyl
moieties of [18F]FBuEA 3 did not significantly alter the duration
time.
Small Animal PET imaging of [18F]FBuEA (3). Figure 5 shows small
animal PET images averaged from 0 to 120 min timeframes postin-
jection of [18F]FBuEA 3. After intravenous application of [18F]FBuEA
3, the radiotracer was rapidly distributed. The liver is the main site
of accumulation of [18F]FBuEA 3, which could be explained by the
formation of the [18F]FBuEA–GSH 10 complex as well as its subse-
quent transformation by membrane transporters. Due to the size
limitation of the MicroPET scanner of NERI, only local imaging
could be studied on the rat. However, when mouse was investi-
gated, a whole-body image was obtained and a rapid radioactivity
Figure 4. Metabolite analysis of [18F]FBuEA 3. Time-activity relationship obtained
by integrating the counts of the peak corresponding to [18F]FBuEA 3 from the
chromatogram taken at each time point. Plasma T1/2 = 46 min. CPS: counts per
second.
Cl
O
Cl
O
O
Cl
Cl
O
O
O
18F
*
N
H
O
18F
N
H
HOOC
S
85%
18
3
[
F]FBuEA
+
NH
O
GST-α
COOH
N
HOOC
HS
H
NH
O
O
NH2
10 (two diastereomers)
COOH
NH2
N
H
GSH
Scheme 2. Formation of the [18F]FBuEA–GSH 10 complex under the catalysis of GST-
a.
Figure 3. RP-HPLC chromatogram of the mixtures of [18F]FBuEA–GSH (tR = 17.5 min) and the residual [18F]FBuEA (tR = 23.7 min) obtained from selfconjugation under pH 8
[Fig. 3(A)] and under catalysis of GST- [Fig. 3(B)]. The peak at tR = 5.8 min was suggested to be GST-
a
a.

4002
H.-L. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3998–4003
Figure 5. (A) PET images of the normal rat were normalized from 0 to 120 min post injection. Injection dose: 717 lCi/1 mL. The frames are composed of four subframes which
were denoted individually by upper left subframe: transverse cross section, upper right subframe: sagittal cross section, lower left subframe: coronal cross section, and lower
right subframe: coronal cross section with contrast. (B) PET images of the rat of CCA (feeding with TAA for 24 weeks) were normalized from 0 to 120 min post injection.
Injection dose: 569
liver, respectively.
lCi/0.6 mL. The two rats were then sacrificed and photographs were taken for both (C) and the CCA rat (D). Labels: M and L denote middle liver and left
Figure 6. (A) PET images of the normal rat using [18F]FBuEA were average of dynamic images of time frame from 0 to 30 min. Injection dose: 265 uCi/0.25 mL. (B) PET images
of the CCA rat (feeding with TAA for 18 weeks) using [18F]FBuEA were average of dynamic images of time frame from 0 to 30 min post injection. Injection dose: 486
lCi/
0.23 mL. Arrow indicates the lesion of tumor. (C) PET images of the same CCA rat as that of (B) were taken by using [18F]FDG post 90 min. Arrow indicates the lesion of tumor.
This PET image was carried out in Chang-Gung Memorial Hospital. (D) PET images of the normal rat were average of dynamic images of time frame from 5 to 10 min post
injection. Injection dose: 764
lCi/0.8 mL. (E) PET images of the same rat as that described in (B) with a prolonged feeding to 23 weeks of TAA were average of dynamic images
of time frame from 5 to 10 min post injection. Injection dose: 801
lCi/0.5 mL.
time frame was adopted. As shown in Figure 6(C), PET images of
CCA-rat receiving TAA for 18 weeks using [18F]fluorodeoxyglucose
([18F]FDG) indicated a significant hot spot implying a tumor lesion.
Then the CCA rat along with the normal rat were subsequently im-
aged with [18F]FBuEA 3 5 days later. As shown in Figure 6(B), the
same region of the CCA rat that highlighted by [18F]FDG showed a
cold spot instead and with a more diffused signal pattern that sug-
gested either a deficiency in GST-a expression or an extraordinary
function of GST. The same CCA rat after feeding for 23 weeks of
TAA was imaged again with [18F]FBuEA 3 but the imaging time

H.-L. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3998–4003
4003
was shortened to 5–10 min for optimization test [Fig. 6(D and E)].
Supplementary data
Whereas the tumor lesion was still dark and the cold spot was sim-
ilar to that of 18 week rat. From the above comparisons, the opti-
mized imaging sampling times appears to be 0–30 min. These
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
04.091.
results suggest that regulation of GST-a synthesis was disturbed.
A work reported by Ling et al. has addressed a similar finding of de-
creased synthesis of GST-a during the process of hepatocarcinogen-
References and notes
esis.29 Although the cytotoxicity of the nonradioactive FBuEA 3
against A549 might imply its preferential accumulation in tumors
through GST catalysis and the corresponding radioactive
1. Hayes, J. D.; Pulfor, D. J. Crit. Rev. Biochem. Mol. Biol. 1995, 30(6), 445.
2. Sies, H.; Packer, L. Human alpha class glutathione S-transferase genetic
polymorphism, expression, and susceptibility to disease In Methods in
Enzymology; Coles, B. F., Kadlubar, F. F., Eds.; Elsevier Academic Press: San
Diego, USA, 2005; Vol. 41, p 9.
3. Liver Cancer Study Group of Japan. Classification of primary liver cancer. 1st
English ed. Tokyo: Kanehara Shuppan, 1997.
4. Shaib, Y.; El-Serag, H. B. Semin. Liver Dis. 2004, 24, 115.
[
18F]FBuEA 3 displaced a hot spot, another cytotoxic mechanism
via Wnt signaling pathway could also be involved.20 The level of
expression of GST, specifically GST- , might be negatively corre-
lated to that of Wnt signaling proteins.
a
5. Shaib, Y. H.; Davila, J. A.; McGlynn, K.; El-Serag, H. B. J. Hepatol. 2004, 40, 472.
6. Patel, T. BMC Cancer 2002, 2, 10.
7. Chang, K. Y.; Chang, J. Y.; Yen, Y. J. Natl. Compr. Canc. Netw. 2009, 7, 423.
8. Yeh, C. N.; Lin, K. J.; Hsiao, I. T.; Yen, T. C.; Chen, T. W.; Jan, Y. Y.; Chung, Y. H.;
Lin, C. F.; Chen, M. F. Mol. Imaging Biol. 2008, 10, 209.
Whereas the current cold spot prevents its use from tracing the
metastasis to other organs, [18F]FBuEA 3 might be capable of
assessing the liver function based on GST activity. For example,
the clinical efficacy of current chemotherapeutic drugs, for exam-
ple, mephalan and temozolomide by exerting their cytotoxicities
via alkylation mechanism is inevitably retarded by GST of the liver.
Liver cancer or other types of cancers with low GST activity might
be suitable for treatment with this type of drugs.
9. Townsend, D. M.; Tew, K. D. Oncogene 2003, 22, 7369.
10. McIlwain, C. C.; Townsend, D. M.; Tew, K. D. Oncogene 2006, 25, 1639.
11. Chiang, L.-W.; Pei, K.; Chen, S.-W.; Huang, H.-L.; Lin, K.-J.; Yen, T.-C.; Yu, C.-S.
Chem. Pharm. Bull. 2009, 57, 714.
12. Wang, K.; Li, C.; Song, D.; Zhao, G.; Zhao, L.; Jing, Y. Cancer Res. 2007, 67, 7856.
13. Su, Y. H.; Chiang, L. W.; Jeng, K.-C.; Huang, H.-L.; Chen, J.-T.; Lin, W.-J.; Huang,
C.-W.; Yu, C.-S. Bioorg. Med. Chem. Lett. 2011, 21, 1320.
In brief, the radiolabeled compound [18F]FBuEA 3 was prepared
based on an initial cellular-based screening of a library of ethacry-
nic acid-derived amide formation products using solution phase
14. Unpublished results. IC50 of FBuEA against MCF7 and A549 were 3 and 8
l
M,
parallel assay: IC50 of FBuEA against A549 and IMR-90
control) were 21 and 18 M, respectively. IC50 of BuEA
M, respectively.
respectively.
A
(fibroblast as
a
l
parallel synthesis.30 18F]FBuEA 3 was prepared via the nucleo-
[
philic radiofluorination of the precursor tosylate 8, from ethacrynic
acid via a 4-step synthesis. A sufficient radiochemical yield of 3%
and specific activity of 48 GBq/lmol was obtained from these pro-
cedures. The liver is the major organ for the tracer uptake, and glu-
tathione and GST enzymes play a role in the metabolism of this
tracer. An in vivo half-life of 46 min for [18F]FBuEA 3 obtained from
a preliminary in vivo stability test for [18F]FBuEA 3 is shorter than
the half-life of 18F. The adequate clearance rate is capable of pro-
viding an acceptable contrasting image for the TAA-treated CCA
rat. [18F]FBuEA 3 was found to be accumulated homogeneously
in the liver of normal rat using PET. However, an extraordinary
change in the liver image was observed in the CCA rat at the early
stage of tumor development and suggested its diagnostic potential.
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Acknowledgments
We are grateful to the National Science Council of Taiwan,
CGMH_NTHU Joint Research and Chang-Gung Medical Research
Project for providing financial support of the Grant numbers of
NSC-98-2113-M-007-012, NSC-97-2314-B-182A-020-MY3, CGTH9
6N2342E1, CMRPG390931, CMRPG391512 and CMRPG3B0361.
Technical assistance by Mr. Yean-Hung Tu and Ms. Li-Yuan Huang
are acknowledged. We acknowledge Dr. Shui-Tein Chen from
Institute of Biological Chemistry at Academia Sinica for his review
of this manuscript.
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