Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein

Article abstract of DOI:10.1016/j.bmc.2011.02.039

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with ¹⁸F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of

Full text of DOI:10.1016/j.bmc.2011.02.039

Bioorganic & Medicinal Chemistry 19 (2011) 2190–2198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar
as a positron emission tomography tracer for P-glycoprotein
and breast cancer resistance protein
Bernd Dörner a,b, Claudia Kuntner c, Jens P. Bankstahl d, Thomas Wanek c, Marion Bankstahl d,
Johann Stanek b, Julia Müllauer c, Florian Bauer a, Severin Mairinger a,b,c, Wolfgang Löscher d,
b,c
Donald W. Miller e, Peter Chiba f, Markus Müller b, Thomas Erker a, , Oliver Langer
⇑
a Department of Medicinal Chemistry, University of Vienna, Austria
b Department of Clinical Pharmacology, Medical University of Vienna, Austria
c Molecular Medicine, AIT Austrian Institute of Technology GmbH, Seibersdorf, Austria
d Department of Pharmacology, Toxicology & Pharmacy, University of Veterinary Medicine Hanover, Germany
e Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada
f Institute of Medical Chemistry, Medical University of Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein
(BCRP) inhibitor elacridar (1) with 18F to provide a positron emission tomography (PET) radiotracer to
visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a–
e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to
display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line
Received 11 December 2010
Revised 18 February 2011
Accepted 19 February 2011
Available online 26 February 2011
overexpressing human Pgp (MDCKII-MDR1). 1-[18F]fluoroelacridar ([18F]4b) was synthesized in
a
Keywords:
decay-corrected radiochemical yield of 1.7 0.9% by a 1-step no-carrier added nucleophilic aromatic
18F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [18F]4b was performed in naïve
rats, before and after administration of unlabelled 1 (5 mg/kg, n = 3), as well as in wild-type and
Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice (n = 3). In PET experiments in rats, administration of unlabelled 1 increased
brain activity uptake by a factor of 9.5 (p = 0.0002, 2-tailed Student’s t-test), whereas blood activity levels
remained unchanged. In Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice, the mean brain-to-blood ratio of activity at 60 min
after tracer injection was 7.6 times higher as compared to wild-type animals (p = 0.0002). HPLC analysis
of rat brain tissue extracts collected at 40 min after injection of [18F]4b revealed that 93 7% of total
radioactivity in brain was in the form of unchanged [18F]4b. In conclusion, the in vivo behavior of
PET
1-[18F]Fluoroelacridar
P-glycoprotein
Breast cancer resistance protein
Blood–brain barrier
[
18F]4b was found to be similar to previously described [11C]1 suggesting transport of [18F]4b by Pgp
and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo
defluorination will limit the utility of [18F]4b as a PET tracer.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
endothelium of the blood–brain barrier (BBB) preventing brain up-
take of a wide range of lipophilic molecules by active ATP-driven ef-
The adenosine triphosphate (ATP) binding cassette (ABC) trans-
porters P-glycoprotein (Pgp, ABCB1) and breast cancer resistance
protein (BCRP, ABCG2) act as gatekeepers at the level of the vascular
flux transport.1 Changes in ABC transporter function and expression
are believed to be involved in several neurological disorders, such as
therapy refractory epilepsy, Parkinson’s and Alzheimer’s disease.1
Visualization of ABC transporters with the non-invasive nuclear
imaging method positron emission tomography (PET) might help
to better understand disease related changes in transporter func-
tion/expression.2 Two classes of radiotracers for visualization of
Pgp have been described so far. Radiolabelled Pgp substrates such
⇑
Corresponding author. Tel.: +43 1 4277 550 03; fax: +43 1 4277 9 551.
E-mail addresses: thomas.erker@univie.ac.at (T. Erker), oliver.langer@ait.ac.at
(O. Langer).
Abbreviations: BBB, blood–brain barrier; BCRP, breast cancer resistance protein;
IC50, half-maximum inhibitory concentration; MDCKII-MDR1, Madin-Darby canine
kidney epithelial cells stably transfected with the human MDR1 gene; Mdr1a/b(ꢀ/ꢀ)
Bcrp1(ꢀ/ꢀ) mouse, Pgp/Bcrp1 knockout mouse; PET, positron emission tomography;
Pgp, P-glycoprotein; R123, rhodamine 123; SPE, solid-phase extraction; SUV, stan-
dardized uptake value; TAC, time-activity curve.
as (R)-[11C]verapamil3 or
radiolabelled Pgp inhibitors such as [11C]laniquidar,5
dar,6,7 11C]tariquidar8,9 and [11C]MC1810 (Fig. 1). Radiolabelled
Pgp inhibitors were initially developed to provide PET tracers
[
11C]-N-desmethyl-loperamide4 and
[
11C]elacri-
[
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.039

B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
2191
11CH3
N
CN
O
OCH3
OCH3
11CH3
HO
R
N
N
H3CO
H
OCH3
Cl
11C]-N-desmethyl-loperamide
(R)-[11C]verapamil
O
[
O
O
O
O
N
O
H311CO
O
N
N
H
N
H
H311CO
O
N
H
NH
O
11C]tariquidar
[
11C]elacridar ([11C]1)
[
N
O
O
O11CH3
O
N
N
N
N
O
11CH3
11C]MC18
O
[
11C]laniquidar
N
[
Figure 1. Chemical structures of Pgp substrate and inhibitor PET tracers.
which should bind to Pgp without being transported and therefore
allow for visualization of Pgp expression levels as opposed to sub-
strates which visualize Pgp function. However, the in vivo behavior
of most radiolabelled Pgp inhibitors tested to date in rodents was
found to be ‘substrate-like’ in that brain activity uptake in baseline
scans was equally low or even lower than for radiolabelled sub-
strates and several times increased after administration of unla-
(4b) and 2-fluoroelacridar (4e) were synthesized as shown in
Scheme 1.6,11 As radiolabelling precursors, 1- and 2-chloro substi-
tuted derivatives 4a and 4d as well as 1-nitro derivative 4c were
synthesized according to Scheme 1.6,11 In small-scale experiments
precursor molecules 4a, 4c and 4d were reacted with the
K[18F]F–K222 complex using different solvents (DMF, DMSO), differ-
ent reaction temperatures (120–180 °C) and different reaction
times (5–60 min). Derivatives 4a and 4d failed to provide any
18F-incorporation under all conditions tested, whereas 4c gave
the corresponding 1-[18F]fluoro-derivative [18F]4b in an incorpora-
tion yield of 10–20% based on radio-thin layer chromatography
(radio-TLC) analysis of crude reaction mixture. The synthesis of
belled inhibitor. Our recent experiments with
[
11C]elacridar
([11C]1, Fig. 1) in rats and transporter knockout and wild-type mice
suggested that this radiotracer was efficiently transported by Pgp
and/or BCRP at the rodent BBB.6 Given its favorable metabolic pro-
file with radiometabolites not being taken up into brain tissue,
[
11C]16,7 appeared as an interesting candidate probe to investigate
[
[
18F]4b was automated in a TRACERlab FX synthesis module.
Pgp and BCRP. Due to the short radioactive half-life of 11C
(20.4 min) the use of 11C-labelled PET tracers is restricted to cen-
ters with an onsite cyclotron. In order to facilitate a broader
applicability of this radiotracer we aimed at labelling 1 with the
longer-lived radionuclide 18F (half-life: 109.8 min).
18F]4b, ready for intravenous (iv) injection, was obtained in a
decay-corrected radiochemical yield of 1.7 0.9% (n = 7) based on
18F]Fꢀ in a total synthesis time of 147 min. Radiochemical purity
was greater than 98% and the specific activity at the end of
synthesis was 8 4 GBq/
mol (n = 7). The identity of [18F]4b was
[
l
Here we report on the synthesis of a series of new halogen- and
nitro-substituted derivatives of 1, which we found to possess com-
parable potency to 1 in inhibiting Pgp transport in vitro. Moreover,
we report on the radiosynthesis and small-animal PET evaluation
of 1-[18F]fluoroelacridar ([18F]4b), which we showed to display
similar in vivo behavior as [11C]1.
confirmed by high-performance liquid chromatography (HPLC)
coinjection with unlabelled reference compound 4b.
2.2. Assessment of Pgp inhibitory activity
Pgp inhibition of compounds 4a–e was determined relative to
reference compound 1 using the rhodamine 123 (R123) accumula-
tion assay in a cell line which overexpressed human Pgp (MDCKII-
MDR1) (Table 1). Four compounds displayed comparable potency
to 1 in inhibiting R123 efflux with half-maximum inhibitory
2. Results
2.1. Chemistry and radiolabelling
concentrations (IC50) in the 0.5–2 lM range (4b–e). For instance,
1-fluoro-derivative 4b, which was also tested in vivo in 18F-l
abelled form (see below), had an only about four times higher
IC50 than 1. It is noteworthy that all derivatives were less effective
The para- and the meta-position relative to the carboxylamide
group in the acridone moiety of 1 were selected for no-carrier-added
nucleophilic aromatic substitution with [18F]Fꢀ. 1-Fluoroelacridar

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B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
Table 1
0.9–1.3 min after tracer injection. At 25 min after tracer injection,
brain activity uptake had declined to 0.14 0.02 SUV. In response
to administration of unlabelled 1 at 60 min after injection of
Effects of compound 1 and the new modulators 4a–e on R123 efflux from MDCKII-
MDR1 cell monolayers
Maximal inhibitory effectb (%)
100
a
[
18F]4b there was a steep increase in brain activity concentration,
Compound
IC50
(lM)
log IC50
reaching a SUV of 1.14 0.14 at 140 min after tracer injection
(Fig. 2A), whereas blood activity levels remained unchanged
(Fig. 2B). In group 2, which was scanned at 2 h after administration
of unlabelled 1, brain activity uptake at 25 min after tracer injec-
tion was 9.5 times higher (p = 0.0002) as compared to group 1
(Fig. 2A) with similar blood activity levels as in group 1 (Fig. 2B).
In Figure 3, representative PET summation images are shown for
scans recorded before and after inhibitor administration. There
was appreciable uptake of radioactivity in bone tissue which
pointed to in vivo defluorination of [18F]4b (Fig. 3). In the first
group of rats, activity uptake in humerus was 1.42 0.37 and
4.59 1.00 SUV at 1.8 and 140 min after injection of [18F]4b,
respectively.
1 (elacridar)
0.4
3.8
1.7
1.6
1.4
0.6
ꢀ0.36 0.06
0.58 0.04
0.24 0.04
0.21 0.07
0.14 0.08
ꢀ0.21 0.08
4a
4b
4c
4d
4e
74
81
50
88
69
3
4
4
5
1
a
log IC50 values (fitted parameter standard error) were estimated using
GraphPad Prism software.
b
Maximal inhibitory effects (%) are expressed as inhibition caused by the highest
concentration of the compound tested (4b, 4d, 4e: 10
to the inhibitory effect caused by 3.2 M 1 (100% inhibition).
lM; 4a, 4c: 100 lM) relative
l
than 1 in terms of increasing intracellular R123 concentration in
MDCKII-MDR1 cells with maximal inhibitory effects relative to 1
<100% (Table 1).
Radiometabolites of [18F]4b in plasma were assessed in a sepa-
rate group of rats (n = 3) by a solid-phase extraction (SPE) assay. At
10, 20, 30 and 40 min after injection of [18F]4b into rats, 6 4%,
2.3. Small-animal PET in rats and mice
6
3%, 12 5% and 13 4% of total plasma activity was recovered
in SPE fractions 1 and 2 (corresponding to polar radiometabolites
of [18F]4b) and 94 4%, 94 3%, 88 5% and 87 4% in fraction 3
(corresponding to unchanged [18F]4b and its lipophilic radiome-
tabolites). Fraction 3 from the 40 min sample was further analyzed
by HPLC, which showed that 72 27% of total radioactivity was in
the form of unchanged [18F]4b, whereas the remainder of radioac-
tivity eluted before [18F]4b on reversed-phase HPLC. [18F]4b was
98.0 1.7% (n = 3) protein-bound in rat plasma. Brain extracts col-
lected at 40 min after injection of [18F]4b into rats were also ana-
lyzed by HPLC showing that 93 7% of total radioactivity in brain
was in the form of unchanged [18F]4b.
[
18F]4b was evaluated in two groups of rats. The first group
underwent a 150 min PET scan, during which unlabelled 1 was iv
administered at a dose of 5 mg/kg at 60 min after injection of
[
18F]4b. The second group of rats was scanned at 2 h after admin-
istration of 1. In the first group of rats, brain uptake of activity was
low before unlabelled 1 was administered (Fig. 2A). Peak brain
uptake was 0.46 0.12 (standardized uptake value, SUV) at
In Figure 4, PET summation images and brain time-activity
curves (TACs) of [18F]4b in wild-type and Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ)
mice are shown. Brain TACs were several times higher in Mdr1a/
b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice as compared to wild-type animals (Fig. 4B).
Brain activity uptake at 60 min after tracer injection was normal-
ized to blood activity levels measured after the PET scan. The mean
brain-to-blood ratio of activity at 60 min after tracer injection was
7.6 times higher (p = 0.0002) in Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice than in
wild-type animals (brain-to-blood ratios, wild-type: 1.5 0.2;
Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ): 11.6 1.4).
3. Discussion
The aim of this study was to label the potent dual Pgp/BCRP
inhibitor 1 with the positron emitting radionuclide 18F in order
to develop a longer-lived PET radiotracer, based on 1, than previ-
ously described [11C]16,7 to investigate Pgp and BCRP in vivo. Dur-
ing the preparation of this manuscript a study by Kawamura et al.
has appeared in the literature, which also reported the synthesis of
an 18F-derivative of 1 (i.e., the corresponding 5-[18F]fluoroethoxy
derivative), but this radiotracer was labelled in a different position
than in our work.12 We found that the acridone moiety of 1 gave
straightforward access to the introduction of appropriate leaving
groups (Cl, NO2) in the 1- and 2-positions for 18F-substitution
(Scheme 1). Derivatives 4a–e were prepared as reference com-
pounds and radiolabelling precursors for 18F-fluorination of 1
(Scheme 1). As 4a–e are previously unknown close structural ana-
logues of 1 they were tested for their Pgp inhibitory potencies in
the R123 accumulation assay (Table 1). All compounds except for
1-chloro-derivative 4a were remarkably potent in the Pgp inhibi-
Figure 2. TACs (mean SUV SD) of [18F]4b in whole brain (A) and arterial blood (B)
of rats (group 1: open squares, group 2: filled squares). In group 1, unlabelled 1 was
administered as an iv bolus over 60 s at 60 min after injection of [18F]4b. The time
point of administration of 1 is indicated by an arrow. Group 2 was scanned at 2 h
after administration of 1. For comparison, whole-brain TACs (mean SUV SD, n = 3)
previously obtained with [11C]16 are also shown in A (group 1: open circles, group
2: filled circles).
tion assay with IC50 values in the 0.5–2.0
comparable to 1 (IC50: 0.4 M). As the primary imaging target
was Pgp, BCRP modulation of derivatives 4a–e was not tested.
lM range, which was
l

B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
2193
Figure 3. Exemplary coronal (left), horizontal (center) and sagittal (right) PET summation images in rats, (A) before (group 1: 0–60 min) and (B) after administration of
unlabelled 1 (5 mg/kg). The radiation scale is set from 0.1 to 2.0 SUV. Anatomical structures are indicated by arrows (br, brain; e, eye; h, humerus; sg, submandibular gland;
pg, pituitary gland).
drastic reaction conditions (60 min heating at 175 °C) (Scheme
1). Surprisingly, the specific activity of [18F]4b at end of synthesis
(8 4 GBq/
added nucleophilic aromatic 18F-substitution reaction (>37 GBq/
mol). The most likely explanation for the low specific activity of
18F]4b was that elimination of the NO2 group in precursor mole-
lmol) was lower than expected for a typical no-carrier
l
[
cule 4c had occurred under the employed labelling conditions giv-
ing rise to unlabelled 1, which we failed to separate from 4b on all
tested HPLC systems. The obtained amounts of [18F]4b were suffi-
ciently high to allow for conducting small-animal PET experiments.
The primary aim of the PET examination was to assess if in vivo
[
18F]4b behaved similarly to previously developed [11C]1. There-
fore we chose a similar study set-up as we had previously used
for [11C]1, which was studied in naïve rats by performing paired
PET scans before and after administration of unlabelled 1.6 How-
ever, due to the longer half-life of 18F it was not possible to perform
paired PET scans in the same animals with [18F]4b and therefore
separate groups of rats were examined. As previously observed
for [11C]16 the in vivo behavior of [18F]4b was consistent with that
of a transported substrate (Fig. 2A). Brain activity uptake of [18F]4b
was low in the first group of rats, which was scanned before
administration of unlabelled 1. In response to administration of 1
there was a steep increase in brain activity uptake (Fig. 2A). In
the second group of rats, which was scanned after administration
of 1, brain activity uptake was 9.5 times higher as compared to
group 1. It seems unlikely that the rather low specific activity of
[
18F]4b should have significantly affected the PET results. Kawam-
ura and co-workers have previously studied uptake of [11C]1 into
mouse brain in the presence of increasing amounts of unlabelled
1 (0.1–10 mg/kg, iv) and found that brain uptake of [11C]1 started
to increase at doses greater than 0.1 mg/kg with a half-maximum
effect dose of 1.55 mg/kg.7 As the doses of unlabelled carrier
administered in our in vivo experiments in rats and mice were
<0.05 mg/kg the relatively low specific activity of [18F]4b should
not have influenced its brain uptake. As a second approach to the
rat studies, in which Pgp/BCRP was pharmacologically inhibited
with unlabelled 1, we performed PET experiments with [18F]4b
in wild-type and in transporter knockout mice, which lacked both
Pgp and Bcrp1. Absence of Pgp/Bcrp1 at the BBB was reflected by a
7.6 times higher brain-to-blood ratio of activity as compared to
wild-type mice. Overall, the in vivo behavior of [18F]4b was almost
Figure 4. (A) Sagittal PET summation images (0–60 min) of [18F]4b in wild-type
and Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice. The radiation scale is set from 0.1 to 2.0 SUV.
Anatomical structures are indicated by arrows (br, brain). (B) TACs (mean SUV SD,
n = 3 per mouse type) of [18F]4b in whole brain of wild-type (open squares), and
Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice (filled squares). For comparison TACs (mean SUV SD,
n = 3) previously obtained with [11C]124 are also shown (wild-type mice: open
circles, Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice: filled circles).
Radiolabelling experiments revealed that only the 1-position of
the acridone moiety was moderately activated for 18F-substitution.
1-[18F]fluoroelacridar ([18F]4b) was synthesized in low radiochem-
ical yield of 1.7 0.9% based on starting [18F]Fꢀ employing rather

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B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
R1
R1
O
O
O
R1
OH
a: R1=Cl, R2=H
b: R1=F, R2=H
c: R1=NO2, R2=H
d: R1=H, R2=Cl
e: R1=H, R2=F
R2
R2
R2
A
B
OH
NH2
+
Br
N
H
N
H
O
O
O
O
OH
O
OH
O
OH
2a-2e
3a-3e
18F
O
R1
O
R2
O
O
O
C
D
N
N
N
H
O
N
H
O
O
O
N
H
O
N
H
4a-4e
[
18F]4b
Scheme 1. Synthesis of nitro- and halogen-substituted derivatives of 1 (4a–e) and 18F-labelling of 4c to give [18F]4b. Reagents and conditions: (A) Cu0/K2CO3, EtOH, reflux; (B)
POCl3, CH3CN, reflux; (C) TBTU, Et3N, DMF, rt; (D) K[18F]F-K222, DMSO, 175 °C.
identical to that of [11C]1 as reflected by quite similar TACs of both
radiotracers in rats and mice (Figs. 2A and 4).
of the PET experiments as reflected by the high amount of radio-
activity in bone tissue which would hamper the quantification of
Our in vivo results obtained with [18F]4b are to a certain ex-
tent surprising as 1 has been previously characterized as a non-
transported inhibitor of Pgp. It has been shown that 1 does not
stimulate adenosine triphosphatase (ATPase) activity and has an
efflux ratio that is <2 in cell monolayer transport assays, consis-
tent with a lack of Pgp transport activity.13,14 However, very re-
cent data suggest that the structurally related Pgp inhibitor
tariquidar (Fig. 1), which we have also labelled with 11C and
which was shown to behave similarly to [11C]1 and [18F]4b
in vivo,8 is an avid substrate of BCRP.15 Kannan et al. found that
accumulation of 3H-labelled tariquidar (1 nM) was several fold-
lower in a cell line overexpressing human BCRP relative to the
parental cell line and increased to similar levels as in parental
cells after co-incubation with the BCRP inhibitor fumitremorgin
C, which was consistent with BCRP transport of tariquidar.15 On
the other hand, when a cell line which overexpressed human
Pgp, was incubated with [3H]tariquidar, cellular uptake was high-
er relative to parental cells, which pointed to binding of [3H]tari-
quidar to Pgp. In addition, recent PET results obtained with
[
18F]4b uptake in organs with low uptake which are surrounded
by bone structures.
4. Material and methods
4.1. General
Unless otherwise stated, all chemicals were purchased from
Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), TCI Europe
(Zwijndrecht, Belgium), Merck (Darmstadt, Germany) or Apollo
Scientific Ltd (Bredbury, UK) at analytical grade and used without
further purification. Isoflurane was obtained from Baxter Vertriebs
GmbH (Vienna, Austria). The hydrochloride salt of 1 was obtained
from Glaxo SmithKline (Research Triangle Park, NC, USA). For
administration 1 hydrochloride was dissolved freshly on each
experimental day in a 20% aqueous (aq) EtOH solution and injected
at a volume of 2 mL/kg. Aqueous [18F]Fꢀ was produced in a General
Electrics PETtrace cyclotron (General Electric Healthcare, Uppsala,
Sweden) via the 18O(p,n)18F nuclear reaction by irradiation of a
2 mL water target containing 95.9% enriched [18O]H2O (Hyox18,
Rotem Industries, Beer Sheva, Israel) with 16.5 MeV proton beam.
Typical irradiation times were 60 min with a beam current of
[
11C]tariquidar and [11C]1 in Mdr1a/b(ꢀ/ꢀ), Bcrp1(ꢀ/ꢀ) and Mdr1a/
b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice suggest that the two radiotracers are trans-
ported by Bcrp1 and possibly Pgp at the murine BBB.7,8 Given
the close structural similarity between 1 and tariquidar and the
similar in vivo behavior of [11C]1 and [18F]4b on one hand and
40 l
A, which yielded a [18F]Fꢀ amount of about 110 GBq at end
of bombardment. 1H and 13C NMR spectra were recorded on a
Bruker Advance DPx200 (200 and 50 MHz). Chemical shifts are
reported in d units (ppm) relative to Me4Si line as internal standard
(s, br s, d, m, Cq for singlet, broad singlet, doublet, multiplet and
quaternary carbon, respectively) and J values are reported in Hertz.
Mass spectra (MS) were obtained with a Shimadzu (GC-17A;
MS-QP5050A) spectrometer. Purity of the intermediates 2a–e and
3a–e was established by combustion analysis with a Perkin-Elmer
2400 CHN elemental analyzer and of the key compounds 4a–e by
analytical HPLC confirming a purity >95%.
[
11C]tariquidar on the other hand it seems very likely that the
‘substrate-like’ behavior of [18F]4b was caused by BCRP and pos-
sibly Pgp transport. This is also in good agreement with the
observation that pharmacological doses of 1, which are known
to inhibit BCRP and Pgp transport at the BBB,16 significantly in-
creased brain uptake of [18F]4b in rats. As no radiotracers have
been described to date to measure BCRP transport in vivo it
definitely appears worthwhile to further characterize PET tracers
based on
1 and tariquidar with respect to such a future
application. Moreover, dual Pgp/BCRP substrate radiotracers could
help to better understand the interplay of these two transporters
at the BBB. This class of radiotracers appears particularly
attractive as it has been shown that they lack brain uptake of
radiometabolites7,9 which has been found to be a considerable
limitation of the Pgp substrate probes (R)-[11C]verapamil17 and
4.2. General synthesis procedure for compounds 2a–e
A
suspension of 2-amino-3-methoxybenzoic acid (1.67 g,
10 mmol), 2-bromobenzoic acid derivative (11 mmol, 1.1 equiv),
K2CO3 (2.77 g, 20 mmol, 2 equiv) and copper powder (0.125 g,
2 mmol, 0.2 equiv) was stirred in EtOH (20 mL) and heated to re-
flux for 1.5 h. The suspension was cooled to room temperature
and H2O (20 mL) was added. The mixture was filtered on cellite
to remove the copper. The filter bed was washed with H2O and
the resulting solution was acidified with concentrated HCl to a
[
11C]-N-desmethyl-loperamide.18 In line with previous results
obtained with [11C]16,7 and [11C]tariquidar,8,9 we could show that
>90% of total activity in rat brain was due to unchanged [18F]4b.
However, a considerable drawback of [18F]4b was the significant
degree of in vivo defluorination observed during the time course

B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
2195
pH of 2–3. The resulting suspension was stirred for 1 h at 10 °C, the
solid was filtered and washed with H2O. The product was recrystal-
lized in EtOH/H2O and dried under vacuum.
crystals. Mp: 209–214 °C. MS m/z: 305 (58%, M+), 242 (100%),
214 (95%), 83 (35%), 57 (53%). 1H NMR (DMSO-d6): d 3.74 (s, 3H,
OCH3), 6.30–6.41 (m, 1H), 7.10–7.35 (m, 3H), 7.42–7.59 (m, 2H),
9.93 (br s, 1H), 13.12 (br s, 2H). 13C NMR (DMSO-d6): d 55.5
(OCH3), 113.3 (d, Cq, J = 6.4 Hz), 115.6 (CH), 115.7 (d, CH,
J = 22.8 Hz), 116.5 (d, CH, J = 7.3 Hz), 120.4 (d, CH, J = 22.8 Hz),
122.3 (CH), 124.1 (CH), 125.7 (Cq), 129.5 (Cq), 143.4 (d, Cq),
J = 1.3 Hz), 153.3 (Cq), 153.6 (d, Cq, J = 232.5 Hz), 167.9 (COOH),
168.1 (d, COOH, J = 2.5 Hz).
4.2.1. 2-((2-Carboxy-5-chlorophenyl)amino)-3-methoxybenzoic
acid (2a)
The compound was synthesized according to the general proce-
dure using 2-bromo-4-chlorobenzoic acid (2.59 g, 11 mmol,
1.1 equiv) as 2-bromobenzoic acid derivative. Yield: 2.95 g (92%)
white crystals. Mp: 250–251 °C. MS m/z: 323 (23%, M+, 37Cl), 321
(62%, M+, 35Cl), 258 (100%), 230 (74%), 74 (58%). 1H NMR (DMSO-
d6) d 3.79 (s, 3H, OCH3), 6.27 (s, 1H), 6.77 (d, 1H, J = 8.4 Hz),
7.27–7.44 (m, 2H), 7.45–7.55 (m, 1H), 7.85 (d, 1H, J = 8.4 Hz),
10.08 (s, 1H), 13.17 (br s, 2H). 13C NMR (DMSO-d6) d 55.7 (OCH3),
111.4 (Cq), 113.6 (CH), 115.8 (CH), 116.7 (CH), 122.3 (CH), 125.4
(CH), 127.0 (Cq), 128.0 (Cq), 132.8 (CH), 138.1 (Cq), 148.1 (Cq),
153.6 (Cq), 167.7 (COOH), 168.6 (COOH).
4.3. General synthesis procedure for compounds 3a–e
Compounds 2a–e (5 mmol) were dissolved in CH3CN (15 mL)
and heated to reflux. Phosphorus(V)oxychloride (1 mL, 11 mmol,
2.2 equiv) was added over a period of 1 h. The solution was re-
fluxed for further 2 h and then cooled to 10–15 °C. H2O (10 mL)
was added and the mixture heated to reflux for additional 2.5 h.
The suspension was cooled to 10 °C and filtered. The solid was
washed with H2O and CH3CN and then dried under vacuum.
4.2.2. 2-((2-Carboxy-5-fluorophenyl)amino)-3-methoxybenzoic
acid (2b)
The compound was synthesized according to the general proce-
dure with 2-bromo-4-fluorobenzoic acid (2.41 g, 11 mmol,
1.1 equiv) as 2-bromobenzoic acid derivative. Yield: 3.00 g (98%)
pale white crystals. Mp: 206–209 °C. MS m/z: 305 (49%, M+), 242
(83%), 213 (100%), 184 (35%), 93 (47%). 1H NMR (DMSO-d6) d 3.78
(s, 3H, OCH3), 5.94–6.03 (m, 1H), 6.46–6.60 (m, 1H), 7.27–7.39 (m,
2H), 7.40–7.49 (m, 1H), 7.84–7.96 (m, 1H), 10.11 (s, 1H), 13.04 (br
4.3.1. 1-Chloro-5-methoxyacridone-4-carboxylic acid (3a)
Cyclization of compound 2a (1.61 g, 5 mmol) yielding the title
compound was carried out using the general approach. Yield:
1.12 g (73%) green solid. Mp: 315–320 °C. MS m/z: 305 (25%, M+,
37Cl), 303 (75%, M+, 35Cl), 257 (66%), 151 (88%), 62 (100%). 1H
NMR (DMSO-d6) d 4.03 (s, 3H, OCH3), 7.13–7.36 (m, 3H), 7.67 (d,
1H, J = 8.2 Hz), 8.24 (d, 1H, J = 8.2 Hz), 12.5 (s, 1H). 13C NMR
(DMSO-d6) d 56.5 (OCH3), 112.9 (CH), 113.5 (Cq), 116.9 (CH),
117.3 (Cq), 122.1 (CH), 122.1 (Cq), 123.1 (CH), 129.6 (Cq), 136.0
(CH), 139.6 (Cq), 142.8 (Cq), 147.2 (Cq), 168.8 (COOH), 175.4 (CO).
s, 2H). 13C NMR (DMSO-d6)
d 55.7 (OCH3), 100.1 (d, CH,
J = 25.9 Hz), 104.0 (d, CH, J = 22.2 Hz), 109.2 (Cq), 115.7 (CH), 122.2
(CH), 125.4 (CH), 127.1 (Cq), 127.9 (Cq), 133.8 (d, CH, J = 11.5),
149.6 (d, Cq, J = 12.2), 153.6 (Cq), 165.4 (d, CF, J = 246.1 Hz), 167.7
(COOH), 168.6 (COOH).
4.3.2. 1-Fluoro-5-methoxyacridone-4-carboxylic acid (3b)
Cyclization of compound 2b (1.53 g, 5 mmol) to 3b was carried
out using the general approach. Yield: 1.16 g (81%) green/brown
solid. Mp: 275–278 °C. MS m/z: 287 (100%, M+), 269 (74%), 254
(67%), 241 (71%), 170 (37%). 1H NMR (DMSO-d6) d 4.03 (s, 3H,
OCH3), 6.92–7.07 (m, 1H), 7.15–7.28 (m, 1H), 7.34 (d, 1H,
J = 7.8 Hz), 7.67 (d, 1H, J = 7.8 Hz), 8.31–8.42 (m, 1H), 12.39 (s,
1H). 13C NMR (DMSO-d6) d 56.5 (OCH3), 107.0 (d, CH, J = 21.6 Hz),
110.7 (d, Cq, J = 9.0 Hz), 110.7 (d, Cq, J = 4.2 Hz), 113.0 (CH), 116.6
(CH), 122.1 (Cq), 138.2 (d, CH, J = 13.2 Hz), 142.9 (d, Cq,
J = 5.2 Hz), 147.3 (Cq), 165.0 (d, CF, J = 268.6 Hz), 168.6 (COOH),
174.8 (d, CO, J = 1.7 Hz).
4.2.3. 2-((2-Carboxy-5-nitrophenyl)amino)-3-methoxybenzoic
acid (2c)
The compound was synthesized according to the general
procedure with 2-bromo-4-nitrobenzoic acid (2.71 g, 11 mmol,
1.1 equiv) as 2-bromobenzoic acid derivative. Yield: 3.19 g (96%)
orange crystals. Mp: 279–284 °C. MS m/z: 332 (67%, M+), 269
(67%), 89 (66%), 75 (100%), 51 (75%). 1H NMR (DMSO-d6) d 3.78
(s, 3H, OCH3), 7.02–7.07 (m, 1H), 7.28–7.47 (m, 2H), 7.48–7.57
(m, 2H), 8.08 (d, 1H, J = 8.8 Hz), 10.35 (br s, 1H), 13.43 (br s, 2H).
13C NMR (DMSO-d6) d 55.8 (OCH3), 109.2 (CH), 110.6 (CH), 116.0
(CH), 117.6 (Cq), 122.6 (CH), 125.5 (CH), 126.3 (Cq), 127.8 (Cq),
132.6 (CH), 147.1 (Cq), 150.2 (Cq), 153.2 (Cq), 167.7 (COOH),
168.0 (COOH).
4.3.3. 5-Methoxy-1-nitroacridone-4-carboxylic acid (3c)
Cyclization of compound 2c (1.66 g, 5 mmol) yielding the title
compound was carried out using the general approach. Yield:
1.29 g (82%) brown solid. Mp: 284–290 °C. MS m/z: 314 (31%,
M+), 313 (34%), 121 (47%), 57 (100%), 51 (55%). 1H NMR (DMSO-
4.2.4. 2-((2-Carboxy-4-chlorophenyl)amino)-3-methoxybenzoic
acid (2d)
The compound was synthesized according to the general proce-
dure using 2-bromo-5-chlorobenzoic acid (2.57 g, 11 mmol) as 2-
bromobenzoic acid derivative.19 Yield: 2.80 g (87%) ochre crystals.
Mp: 225–229 °C. MS m/z: 323 (24%, M+, 37Cl), 321 (63%, M+, 35Cl),
260 (35%), 258 (100%), 230 (68%), 44 (85%). 1H NMR (DMSO-d6):
d 3.75 (s, 3H, OCH3), 6.35 (d, 1H, J = 9 Hz), 7.15–7.39 (m, 3H),
7.40–7.52 (m, 1H), 7.75–7.79 (m, 1H), 10.16 (br s, 1H), 12.97 (br
s, 2H). 13C NMR (DMSO-d6): d 55.6 (OCH3), 113.9 (Cq), 115.6
(CH), 116.7 (CH), 120.0 (Cq), 122.2 (CH), 124.7 (CH), 126.4 (Cq),
128.6 (Cq), 129.7 (CH), 132.7 (CH), 145.7 (Cq), 153.4 (Cq), 167.8
(COOH), 168.0 (COOH).
d6) d 4.02 (s, 3H, OCH3), 7.19–7.41 (m, 2H), 7.51 (d, 1H,
J = 8.0 Hz), 7.64 (d, 1H, J = 7.8 Hz), 8.50 (d, 1H, J = 8.0 Hz), 12.49
(s, 1H). 13C NMR (DMSO-d6) d 56.5 (OCH3), 111.2 (Cq), 113.5
(CH), 114.1 (CH), 116.5 (CH), 117.3 (Cq), 121.0 (Cq), 122.7 (CH),
130.1 (Cq), 137.2 (CH), 141.1 (Cq), 147.4 (Cq), 151.6 (Cq), 167.9
(COOH), 173.4 (CO).
4.3.4. 2-Chloro-5-methoxyacridone-4-carboxylic acid (3d)
Cyclization of compound 2d (1.61 g, 5 mmol) to 3d was carried
out using the general approach.19 Yield: 1.21 g (79%) dark green so-
lid. Mp: 309–314 °C. MS m/z: 305 (35%, M+, 37Cl), 303 (100%, M+,
35Cl), 285 (85%), 270 (52%), 257 (76%). 1H NMR (DMSO-d6): d
4.05 (s, 3H, OCH3), 7.19–7.43 (m, 2H), 7.69–7.77 (m, 1H), 8.25–
8.38 (m, 2H), 12.07 (s, NH). 13C NMR (DMSO-d6): d 56.5 (OCH3),
113.2 (CH), 116.8 (CH), 117.3 (Cq), 120.7 (Cq), 122.1 (CH), 122.6
(Cq), 124.5 (Cq), 130.6 (Cq), 130.7 (CH), 135.7 (CH), 138.8 (Cq),
147.4 (Cq), 167.8 (COOH), 175.1 (CO).
4.2.5. 2-((2-Carboxy-4-fluorophenyl)amino)-3-methoxybenzoic
acid (2e)
The compound was synthesized according to the general proce-
dure with 2-bromo-5-fluorobenzoic acid (2.41 g, 11 mmol) as 2-
bromobenzoic acid derivative.19 Yield: 2.84 g (93%) light yellow

2196
B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
4.3.5. 2-Fluoro-5-methoxyacridone-4-carboxylic acid (3e)
Cyclization of compound 2e (1.53 g, 5 mmol) yielding the title
compound was carried out using the general approach.19 Yield:
1.22 g (85%) ochre solid. Mp: >350 °C. MS m/z: 287 (100%, M+),
269 (58%), 254 (63%), 241 (67%), 92 (51%). 1H NMR (DMSO-d6): d
4.05 (s, 3H, OCH3), 7.18–7.41 (m, 2H), 7.68–7.77 (m, 1H), 8.08–
8.23 (m, 2H), 12.04 (s, NH). 13C NMR (DMSO-d6): d 56.5 (OCH3),
112.9 (CH), 116.6 (d, CH, J = 22.5 Hz), 116.7 (CH), 117.3 (d, Cq,
J = 6.5 Hz), 120.0 (Cq), 121.8 (CH), 122.5 (d, Cq, J = 6.0 Hz), 124.1
(d, CH, J = 25.7 Hz), 130.7 (Cq), 137.1 (Cq), 147.3 (Cq), 155.3 (d,
CF, J = 239.0 Hz), 167.8 (d, COOH, J = 2.3 Hz), 175.4 (d, CO,
J = 2.6 Hz).
yellow solid. Mp: 188–195 °C. HRMS (ESI/MS) calcd for
34H32O5N3ClH: 598.2109, found: 598.2105. 1H NMR (CDCl3) d
2.72–3.02 (m, 8H, 4 ꢁ CH2), 3.67 (s, 2H, CH2), 3.85 (s, 6H,
2 ꢁ OCH3), 4.06 (s, 3H, OCH3), 6.56 (s, 1H), 6.61 (s, 1H), 7.08–7.32
(m, 4H), 7.63–7.76 (m, 2H), 7.95–8.13 (m, 1H), 8.31 (d, 1H,
J = 2.2 Hz), 8.60 (d, 1H, J = 2.2 Hz), 9.92 (s, 1H), 12.25 (s, 1H).
C
4.4.5. N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-
yl)ethyl)phenyl)-2-fluoro-5-methoxyacridone-4-carboxamide
(2-fluoroelacridar, 4e)
Starting from 3e (0.287 g, 1 mmol) the title compound was syn-
thesized according to the general procedure. Yield: 0.399 g (69%)
dark-orange solid. Mp: 152–157 °C. HRMS (ESI/MS) calcd for
4.4. General synthesis procedure for key compounds 4a–e
C
34H32O5N3FH: 582.2404, found 582.2398. 1H NMR (DMSO-d6) d
2.56–2.96 (m, 8H, 4 ꢁ CH2), 3.57 (s, 2H, CH2), 3.70 (s, 6H,
2 ꢁ OCH3), 4.03 (s, 3H, OCH3), 6.60–6.69 (m, 2H), 7.16–7.40 (m,
4H), 7.61–7.73 (m, 2H), 7.73–7.80 (m, 1H), 8.10–8.18 (m, 1H),
8.38–8.47 (m, 1H), 10.63 (s, 1H), 12.26 (s, 1H).
To a solution of 6,7-dimethoxy-2-(4-aminophenethyl)-1,2,3,4-
tetrahydroisochinoline (0.312 g, 1 mmol), which has been pre-
pared as described elsewhere,6 and compound 3a–e (1 mmol,
1 equiv) in DMF (2 mL) O-(benzotriazol-1-yl)-N,N,N0,N0-tetrameth-
yluronium hexafluorophosphate (HBTU, 0.379 g, 1.05 mmol,
1.05 equiv) and Et3N (0.29 mL, 2.1 mmol, 2.1 equiv) were added.
The solution was stirred for 2 h at rt, and then a mixture of H2O
and isopropyl alcohol (1/1, v/v, 2 mL) was added. The mixture
was stirred for additional 30 min. The obtained slurry was filtered,
washed with MeOH and H2O, and dried under vacuum.
4.5. General procedure for small-scale [18F]fluorination of
compounds 4a, 4c and 4d
Aqueous [18F]Fꢀ ion from the cyclotron target was collected into
a 3 mL Wheaton V-vial (Wheaton Science Products, Millville, USA)
containing a solution of kryptofix 2.2.2 in CH3CN (120 mg/mL,
100–200
lL; 12.0–24.0 mg, 32.7–65.4
lmol) and a solution of
4.4.1. 1-Chloro-N-(4-(2-(6,7-dimethoxy-1,2,3,4-
K2CO3 in H2O (200 mg/mL, 20–40
l
L; 4.0–8.0 mg, 28.9–57.8 l
mol).
tetrahydroisoquinolin-2-yl)ethyl)phenyl)-5-methoxyacridone-
4-carboxamide (1-chloroelacridar, 4a)
The mixture was exposed to a stream of argon and concentrated to
dryness at 130 °C under repeated addition of CH3CN (3 ꢁ 0.5 mL).
To the dried K[18F]F–K222 complex, the respective precursor (4a,
The compound was synthesized according to the general proce-
dure with 3a (0.304 g, 1 mmol) as reactant. Yield: 0.335 g (56%)
ochre solid. Mp: 183–189 °C. HRMS (ESI/MS) calcd for C34H32
O5N3ClH: 598.2109, found: 598.2102. 1H NMR (DMSO-d6) d 2.69–
2.91 (m, 8H, 4 ꢁ CH2), 3.58 (s, 2H, CH2), 3.70 (s, 6H, 2 ꢁ OCH3),
4.02 (s, 3H, OCH3), 6.61–6.69 (m, 2H), 7.17–7.43 (m, 5H), 7.64 (d,
2H, J = 8.0 Hz), 7.74 (d, 1H, J = 8.0 Hz), 8.31 (d, 1H, J = 8.0 Hz),
10.63 (s, 1H), 12.66 (s, 1H).
4c, 4d: 3–12 lmol) dissolved in dry DMSO or DMF (0.5 mL each)
was added and the resulting solution stirred for 5–60 min at
120–180 °C. An aliquot of the reaction mixture was analyzed by
TLC. For TLC analysis Merck Silica Gel 60 F254 plates were used with
ethyl acetate/MeOH 2:1 (v/v) as mobile phase. TLC plates were
analyzed using a phosphor imager (Cyclone, Packard Instruments,
Meriden, CN, USA). Retardation factors (Rf): [18F]Fꢀ: 0.0; [18F]4b:
0.6–0.7.
4.4.2. N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-
yl)ethyl)phenyl)-1-fluoro-5-methoxyacridone-4-carboxamide
(1-fluoroelacridar, 4b)
4.6. 1-[18F]Fluoroelacridar ([18F]4b)
Starting from 3b (0.287 g, 1 mmol) the title compound was syn-
thesized according to the general approach. Yield: 0.337 g (58%)
brown solid. Mp: 215–220 °C. HRMS (ESI/MS) calcd for
The synthesis of [18F]4b was automated in a TRACERlab FX syn-
thesis module (General Electric Healthcare). After delivery of the
irradiated [18O]H2O to the synthesis module, [18F]Fꢀ was trapped
on a Chromafix 30-PS-HCO3 ion exchange cartridge, which had
been pre-activated with EtOH (2 mL) and H2O (2 mL). [18F]Fꢀ was
eluted to the synthesis reactor by rinsing the cartridge with a mix-
C
34H32O5N3FH: 582.2404, found: 582.2414. 1H NMR (DMSO-d6) d
2.63–3.10 (m, 8H, 4 ꢁ CH2), 3.65 (s, 2H, CH2), 3.69 (s, 6H,
2 ꢁ OCH3), 6.59–6.73 (m, 2H), 7.02–7.22 (m, 2H), 7.24–7.35 (m,
3H), 7.58–7.75 (m, 3H), 8.36–8.52 (m, 1H), 10.58 (s, 1H), 12.65 (s,
1H).
ture of kryptofix 2.2.2. (24 mg, 63.8
lmol) in CH3CN (0.9 mL) and
K2CO3 (3.5 mg, 57.1 mol) in H2O (0.1 mL). The solvent was evap-
l
orated under vacuum for 5 min at 75 °C, then CH3CN (1 mL) was
added and the mixture heated under vacuum for 5 min at 130 °C
to remove the remaining H2O. To the dried K[18F]F–K222 complex,
4.4.3. N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-
yl)ethyl)phenyl)-5-methoxy-1-nitroacridone-4-carboxamide
(1-nitroelacridar, 4c)
radiolabelling precursor 4c (6 mg, 9.9 lmol) dissolved in dry DMSO
According to the general procedure the compound was synthe-
sized using 3c (0.314 g, 1 mmol, 1 equiv) as reactant. Yield: 0.305 g
(50%) brown solid. Mp: 167–172 °C. HRMS (ESI/MS) calcd for
(1 mL) was added and the reaction mixture heated for 60 min at
175 °C. The reaction mixture was then cooled to 40 °C, diluted with
a mixture of CH3CN and 0.1% aq trifluoroacetic acid (TFA) (1:1, v/v,
3 mL) and injected into the built-in HPLC system. A Hamilton-PRP1
C
34H32O7N4H: 609.2349, found: 609.2355. 1H NMR (DMSO-d6) d
2.70–3.04 (m, 8H, 4 ꢁ CH2), 3.67–3.83 (m, 8H, CH2, 2 ꢁ OCH3),
4.05 (s, 3H, OCH3), 6.65–6.75 (m, 2H), 7.25–7.44 (m, 4H), 7.56–
7.84 (m, 4H), 8.52–8.63 (m, 1H), 10.81 (br s, 1H), 12.61 (br s, 1H).
column (500 ꢁ 8 mm, 10
lm) was eluted at a flow rate of 4 mL/min
with a gradient (0–20 min) from CH3CN/0.1% aq TFA 35:65 (v/v) to
45:55. The HPLC eluate was monitored in series for ultraviolet (UV)
absorption at a wavelength of 257 nm and radioactivity. On this
system radiolabelling precursor 4c and product [18F]4b eluted with
retention times of 23 and 19–20 min, respectively. The product
fraction was diluted with H2O (100 mL) and passed over a C18
Sep-Pak Plus cartridge (Waters cooperation, Milford, MA), which
had been pre-activated with EtOH (5 mL) and H2O (10 mL). The
4.4.4. 2-Chloro-N-(4-(2-(6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinolin-2-yl)ethyl)phenyl)-5-methoxyacridone-
4-carboxamide (2-chloroelacridar, 4d)
The title compound was synthesized using the general approach
starting from 3d (0.304 g, 1 mmol) as reactant. Yield: 0.353 g (59%)

B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
2197
cartridge was then washed with H2O (10 mL), followed by elution
of [18F]4b with EtOH (3 mL). The EtOH was removed by heating at
90 °C under a stream of argon and the product formulated in a mix-
ture of 5 mg sodium ascorbate in 0.9% aq NaCl solution/EtOH/poly-
ethylene glycol 300 (50:15:35, v/v/v) for iv injection into animals.
Radiochemical and chemical purity and specific activity of [18F]4b
were determined by analytical radio-HPLC using a Hamilton-PRP1
Animal Welfare Committee and all study procedures were per-
formed in accordance with the Austrian Animal Experiments Act.
4.10. Small-animal PET imaging and PET data analysis
The anesthetized animals were positioned in the imaging cham-
ber of the microPET Focus220 scanner (Siemens, Medical Solutions,
column (290 ꢁ 4 mm, 10
l
m) eluted at a flow rate of 1 mL/min
Knoxville, USA). Animals were injected with
33 3 MBq (mean standard deviation, SD) in a volume of about
0.3 mL corresponding to 9.6 6.5 nmol or 20 12 g/kg body
weight unlabelled 4b; mice: 6 1 MBq in a volume of about
0.1 mL corresponding to 2.0 1.1 nmol or 39 22 g/kg unlabelled
[
18F]4b (rats:
using a gradient (0–25 min) from CH3CN/0.1% aq TFA 35:65 (v/v)
to 45:55. UV detection was performed at
a
wavelength of
l
257 nm. The retention time of [18F]4b was 16–18 min on this HPLC
system.
l
4b) as an iv bolus over approximately 40 s. At the start of radio-
4.7. Cell line and culture condition
tracer injection, dynamic PET imaging was initiated.
Two groups of rats were scanned with [18F]4b (n = 3 per group).
One group underwent a 150 min PET scan with [18F]4b during
which 1 (5 mg/kg) was administered iv over approximately 60 s
at 60 min after injection of [18F]4b. The other group underwent a
60 min PET scan at 2 h after injection of 1. During experiments in
Madin-Darby canine kidney epithelial cells stably transfected
with the human MDR1 gene (MDCKII-MDR1), which were obtained
from the National Cancer Institute (Bethesda, MD, USA), were used
for the fluorescent screening studies. Cells were cultured in Dul-
becco’s Modified Eagles medium (DMEM) supplemented with
80 ng/mL colchicine to maintain high expression of Pgp as previ-
ously described.20 Cells were seeded onto 24-well culture plates
at a density of 65,000 cells/cm2 and used for functional assays
upon reaching confluency (typically 3 days).
rats, 5-lL arterial blood samples were withdrawn manually with
pre-weighted micropipettes from the femoral artery (approxi-
mately every 5 s) during the first 3 min after radiotracer injection,
followed by further 10-lL samples taken at 5, 10, 20, 30, 40, 50, 60,
70, 80 and 90 min (last three time points for group 1 only). Mice
(n = 3 per mouse type) underwent single 60-min PET scans with
4.8. Assessment of Pgp inhibitory activity
[
18F]4b. At the end of the PET scan, venous blood was withdrawn
from mice by retro-orbital puncture into pre-weighted micropi-
pettes. Blood samples from rats and mice were weighted and
counted for activity in a 1-detector Wallac gamma counter (Perkin
Elmer Instruments, Wellesley, USA), which had been cross-cali-
brated with the PET camera. Blood activity data were corrected
for radioactive decay and corrected for injected dose per gram
body weight and expressed as SUV.
Pgp inhibition of derivatives 4a–e was determined as previously
described.21 Briefly, confluent MDCKII-MDR1 monolayers were
pretreated with pH 7.4 Tyrodes balanced salt solution (TBSS):
136 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.36 mM NaH2PO4,
5.56 mM
D-glucose and 5 mM HEPES. Various concentrations of
4a–e (4b, 4d, 4e: 0.1–10
l
M; 4a, 4c: 0.1–100 M, n = 3 per concen-
l
tration) were added to the TBSS and the cells were incubated for
30 min at 37 °C. Following the preincubation period, the TBSS
was removed and replaced with fresh TBSS containing 3.2 lM
R123 to assess drug efflux transporter activity. Cellular accumula-
tion of the fluorescent dye was monitored over a 60 min period,
under control conditions and in the presence of various concentra-
tions of 4a–e (see above). After this time, cells were washed three
times with ice-cold phosphate buffered saline solution and the
cells were solubilized in 1.0% Triton-X100 (0.5 mL). Aliquots
PET images were reconstructed by Fourier rebinning followed
by 2-dimensional filtered back projection with a ramp filter. The
standard data correction protocol (normalization, attenuation, de-
cay correction and injection decay correction) was applied to the
data. Whole brain and humerus (in rats) were manually outlined
on multiple planes of the PET summation images using the image
analysis software Amide and TACs, expressed as SUV, were calcu-
lated. For the mouse data, the SUV values measured with PET in
brain tissue of individual animals during the last time frame (50–
60 min after radiotracer injection) were divided by the SUV value
measured in whole blood at the end of the PET scan to obtain
brain-to-blood ratios of activity. For all outcome parameters, dif-
ferences between groups were tested with a 2-tailed Student’s t-
test. The level of statistical significance was set to p <0.05.
(100 lL) of the solubilized cell solutions were removed for deter-
mination of intracellular R123 accumulation using a Synergy HT
fluorescent plate reader. Protein content was determined using
the Pierce BCA method and the data were expressed as the amount
of fluorescent probe (nmol) per mg cell protein. Concentration re-
sponse curves were fitted using GraphPad Prism 5 software
(GraphPad Software, Inc., San Diego, CA, USA) and compared to
4.11. Metabolism and plasma protein binding of [18F]4b
maximal inhibition achieved using 1 at a concentration of 3.2 lM.
A group of three rats was injected with [18F]4b (74 23 MBq in
4.9. Animals
a volume of about 0.3 mL corresponding to 17.2 3.9 nmol or
37 10 lg/kg body weight unlabelled 4b) without performing
Adult female Sprague–Dawley rats weighing 260–300 g were
obtained from Harlan Netherlands (Horst, Netherlands). Female
FVB (wild-type) and Mdr1a/b(ꢀ/ꢀ)Bcrp1(ꢀ/ꢀ) mice weighing 27–
35 g were purchased from Taconic Inc. (Germantown, USA). Prior
to each experiment, the animals were placed into an induction
box and anesthetized with 2.5% isoflurane. When unconscious,
the animals were taken from the box and kept under anesthesia
with 1.5–2% isoflurane administered via a mask during the whole
experiment. The animals were warmed throughout the whole
experiment at around 38 °C. Rats were implanted with catheters
into the femoral artery (for blood sampling) and vein (for adminis-
tration of [18F]4b and 1). In mice, a lateral tail vein was used for
radiotracer administration. The study was approved by the local
PET examination in order to assess metabolism of [18F]4b. At 10,
20 and 30 min after radiotracer injection 0.5 mL blood samples
were collected into heparinized vials. At 40 min after radiotracer
injection rats were sacrificed, their brains removed and a terminal
blood sample (5 mL) collected. Plasma collected at all time points
was centrifuged (3000g, 5 min, 21 °C) and analyzed for radiome-
tabolites of [18F]4b using a previously described SPE assay.22 In
brief, arterial plasma was spiked with a solution of unlabelled 4b
in DMSO (1 mg/mL, 10 lL) and acidified with 5 M aq. HCl (40 lL)
and loaded on a Sep-Pak vac tC18 cartridge (Waters Corporation,
Milford, USA), which had been pre-activated with MeOH (3 mL)
and H2O (5 mL). The cartridge was first washed with H2O (5 mL)
and then eluted with MeOH (4 mL). Radioactivity in all three

2198
B. Dörner et al. / Bioorg. Med. Chem. 19 (2011) 2190–2198
fractions (plasma, H2O, MeOH) was measured in the 1-detector
Wallac gamma counter. For the 40 min blood sample, the MeOH
fraction was 1:1 diluted with 0.1% aq TFA and analyzed by HPLC
(injected volume: 2 mL). A Chromolith Performance RP 18-e
(100–4.6 mm) column was eluted at flow rate of 5 mL/min using
a gradient (0–20 min) from CH3CN/0.1% aq TFA 20:80 (v/v) to 25/
75. UV detection was performed at a wavelength of 257 nm. The
retention time of [18F]4b was 13–15 min on this HPLC system.
For validation of the SPE assay, [18F]4b dissolved in mouse plasma
(0.5 mL) was subjected to the SPE procedure showing that all
radioactivity was quantitatively recovered in the MeOH fraction.
Rat brain was washed twice with ice-cold H2O and homoge-
nized in 0.9% aq. NaCl solution (0.8 mL) using an IKA T10 basic
Ultra-turrax (IKA Laboratory Equipment, Staufen, Germany). The
brain homogenate was mixed with CH3CN (1.5 mL) and centri-
fuged (3 min, 4 °C, 13,000g). The supernatant was diluted 1:1 with
0.1% aq TFA and injected into the same HPLC system used for anal-
ysis of radiometabolites in plasma (injected volume: 2 mL). Plasma
protein binding of [18F]4b was determined by incubating fresh
plasma samples obtained from naïve rats with [18F]4b during
30 min at 37 °C, followed by ultrafiltration using Amicon Microcon
YM-10 centrifugal filter devices (Millipore Corporation, USA) as de-
scribed previously.23
(Seibersdorf Laboratories GmbH) for their skilful help with labora-
tory animal handling and the staff of the radiochemistry laboratory
(Seibersdorf Laboratories GmbH) for continuous support. 1 hydro-
chloride was kindly provided by Glaxo SmithKline (Research Trian-
gle Park, NC, USA).
References and notes
1. Löscher, W.; Potschka, H. Prog. Neurobiol. 2005, 76, 22.
2. Kannan, P.; John, C.; Zoghbi, S. S.; Halldin, C.; Gottesman, M. M.; Innis, R. B.;
Hall, M. D. Clin. Pharmacol. Ther. 2009, 86, 368.
3. Luurtsema, G.; Molthoff, C. F.; Windhorst, A. D.; Smit, J. W.; Keizer, H.;
Boellaard, R.; Lammertsma, A. A.; Franssen, E. J. Nucl. Med. Biol. 2003, 30, 747.
4. Lazarova, N.; Zoghbi, S. S.; Hong, J.; Seneca, N.; Tuan, E.; Gladding, R. L.; Liow, J.
S.; Taku, A.; Innis, R. B.; Pike, V. W. J. Med. Chem. 2008, 51, 6034.
5. Luurtsema, G.; Schuit, R. C.; Klok, R. P.; Verbeek, J.; Leysen, J. E.; Lammertsma, A.
A.; Windhorst, A. D. Nucl. Med. Biol. 2009, 36, 643.
6. Dörner, B.; Kuntner, C.; Bankstahl, J. P.; Bankstahl, M.; Stanek, J.; Wanek, T.;
Stundner, G.; Mairinger, S.; Löscher, W.; Müller, M.; Langer, O.; Erker, T. J. Med.
Chem. 2009, 52, 6073.
7. Kawamura, K.; Yamasaki, T.; Konno, F.; Yui, J.; Hatori, A.; Yanamoto, K.;
Wakizaka, H.; Takei, M.; Kimura, Y.; Fukumura, T.; Zhang, M. R. Mol. Imaging
Biol. 2011, 13, 152.
8. Bauer, F.; Kuntner, C.; Bankstahl, J. P.; Wanek, T.; Bankstahl, M.; Stanek, J.;
Mairinger, S.; Dörner, B.; Löscher, W.; Müller, M.; Erker, T.; Langer, O. Bioorg.
Med. Chem. 2010, 18, 5489.
9. Kawamura, K.; Konno, F.; Yui, J.; Yamasaki, T.; Hatori, A.; Yanamoto, K.;
Wakizaka, H.; Takei, M.; Nengaki, N.; Fukumura, T.; Zhang, M. R. Ann. Nucl. Med.
2010, 24, 403.
10. van Waarde, A.; Ramakrishnan, N. K.; Rybczynska, A. A.; Elsinga, P. H.; Berardi,
F.; de Jong, J. R.; Kwizera, C.; Perrone, R.; Cantore, M.; Sijbesma, J. W. A.;
Dierckx, R. A.; Colabufo, N. A. J. Med. Chem. 2009, 52, 4524.
5. Conclusions
11. Sharp, M. J.; Mader, C. J.; Strachan, C. PCT Int. Appl. WO 98/52923 A1, 1998.
12. Kawamura, K.; Yamasaki, T.; Konno, F.; Yui, J.; Hatori, A.; Yanamoto, K.;
Wakizaka, H.; Ogawa, M.; Yoshida, Y.; Nengaki, N.; Fukumura, T.; Zhang, M. R.
Bioorg. Med. Chem. 2011, 19, 861.
13. Colabufo, N. A.; Berardi, F.; Cantore, M.; Perrone, M. G.; Contino, M.; Inglese, C.;
Niso, M.; Perrone, R.; Azzariti, A.; Simone, G. M.; Porcelli, L.; Paradiso, A. Bioorg.
Med. Chem. 2008, 16, 362.
14. Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J. B.; Webster, L.
O.; Serabjit-Singh, C. S. J. Pharmacol. Exp. Ther. 2001, 299, 620.
15. Kannan, P.; Telu, S.; Shukla, S.; Ambudkar, S. V.; Pike, V. W.; Halldin, C.;
Gottesman, M. M.; Innis, R. B.; Hall, M. D. ACS Chem. Neurosci. 2011, 2, 82.
16. Agarwal, S.; Sane, R.; Ohlfest, J. R.; Elmquist, W. F. J. Pharmacol. Exp. Ther. 2011,
336, 223.
17. Luurtsema, G.; Molthoff, C. F.; Schuit, R. C.; Windhorst, A. D.; Lammertsma, A.
A.; Franssen, E. J. Nucl. Med. Biol. 2005, 32, 87.
18. Seneca, N.; Zoghbi, S. S.; Shetty, H. U.; Tuan, E.; Kannan, P.; Taku, A.; Innis, R. B.;
Pike, V. W. Nucl. Med. Biol. 2010, 37, 335.
19. Stankiewicz-Drogon, A.; Dörner, B.; Erker, T.; Boguszewska-Chachulska, A. M. J.
Med. Chem. 2010, 53, 3117.
In an attempt to label the potent dual Pgp/BCRP inhibitor 1
with 18F we synthesized a series of new 1- and 2-halogen- and
nitro-substituted derivatives of 1, which were found to display
comparable potency to 1 in inhibiting Pgp transport of R123 in
MDCKII-MDR1 cells. The 1-[18F]fluoro derivative of 1 ([18F]4b)
was synthesized in moderate radiochemical yield and character-
ized in rats and mice with small-animal PET. [18F]4b was found
to display comparable in vivo behavior to previously described
[
11C]1 in that brain activity uptake was low in baseline scans and
several times increased after administration of unlabelled 1,
suggesting that [18F]4b is efficiently transported by BCRP and/or
Pgp and at the rodent BBB. However, the significant degree of
in vivo defluorination observed with [18F]4b together with low
radiochemical yields will in all likelihood limit its future utility
as a PET tracer.
20. Bachmeier, C. J.; Trickler, W. J.; Miller, D. W. Drug Metab. Dispos. 2006, 34, 998.
21. Bachmeier, C. J.; Miller, D. W. Pharm. Res. 2005, 22, 113.
22. Mairinger, S.; Langer, O.; Kuntner, C.; Wanek, T.; Bankstahl, J. P.; Bankstahl, M.;
Stanek, J.; Dörner, B.; Bauer, F.; Baumgartner, C.; Löscher, W.; Erker, T.; Müller,
M. Nucl. Med. Biol. 2010, 37, 637.
Acknowledgments
The research leading to these results has received funding from
the European Community’s Seventh Framework Programme (FP7/
2007-2013) under grant agreement number 201380 (‘Euripides’)
and from the Austrian Science Fund (FWF) project ‘Transmem-
brane Transporters in Health and Disease’ (SFB F35). The authors
thank Michael Sauberer (AIT), Thomas Filip and Maria Zsebedics
23. Kuntner, C.; Bankstahl, J. P.; Bankstahl, M.; Stanek, J.; Wanek, T.; Stundner, G.;
Karch, R.; Brauner, R.; Meier, M.; Ding, X. Q.; Müller, M.; Löscher, W.; Langer, O.
Eur. J. Nucl. Med. Mol. Imaging 2010, 37, 942.
24. Kuntner, C.; Bankstahl, J. P.; Bankstahl, M.; Stanek, J.; Wanek, T.; Dörner, B.;
Bauer, F.; Mairinger, S.; Erker, T.; Müller, M.; Löscher, W.; Langer, O.
Neuroimage 2010, 52, S25. symposium abstract.