Synthesis and in vivo evaluation of 18F-fluoroethyl GF120918 and XR9576 as positron emission tomography probes for assessing the function of drug efflux transporters

Article abstract of DOI:10.1016/j.bmc.2010.12.004

The purpose of this study was to synthesize two new positron emission tomography (PET) probes, N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2- isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[¹⁸F]fluoroethoxy-9-oxo-4- acridine carboxamide ([¹⁸

Full text of DOI:10.1016/j.bmc.2010.12.004

Bioorganic & Medicinal Chemistry 19 (2011) 861–870
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vivo evaluation of 18F-fluoroethyl GF120918 and XR9576
as positron emission tomography probes for assessing the function of drug
efflux transporters
Kazunori Kawamura a, , Tomoteru Yamasaki a, Fujiko Konno a, Joji Yui a, Akiko Hatori a,
⇑
Kazuhiko Yanamoto a, Hidekatsu Wakizaka b, Masanao Ogawa a,c, Yuichiro Yoshida a,c
,
Nobuki Nengaki a,c, Toshimitsu Fukumura a, Ming-Rong Zhang a
a Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
c SHI Accelerator Service Ltd, 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The purpose of this study was to synthesize two new positron emission tomography (PET) probes,
N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[18F]fluoroethoxy-
9-oxo-4-acridine carboxamide ([18F]3) and quinoline-3-carboxylic acid [2-(4-{2-[7-(2-[18F]fluoroethoxy)-
6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxyphenyl]amide ([18F]4),
andto evaluate the potentialofthese PET probesfor assessingthe functionoftwomajor drugefflux transporters,
P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). [18F]3 and [18F]4 were synthesized by
18F-alkylation of each O-desmethyl precursor with [18F]2-fluoroethyl bromide for injection as PET probes. In
vitro accumulation assayshowed that treatment with P-gp/BCRP inhibitors (1and2) enhanced the intracellular
accumulation capacity of P-gp- and BCRP-overexpressing MES-SA/Dx5 cells. In PET studies, the uptake
(AUCbrain [0–60 min]) of [18F]3 and [18F]4 in wild-type mice co-injected with 1 were approximately sevenfold
higher than that in wild-type mice, and the uptake of [18F]3 and [18F]4 in P-gp/Bcrp knockout mice were eight-
to ninefold higher than that in wild-type mice. The increased uptake of [18F]3 and [18F]4 was similar to that of
parent compounds ([11C]1and [11C]2) previously described, indicating that radioactivity levels in the brain after
injection of [18F]3 and [18F]4 are related to the function of drug efflux transporters. Also, these results suggest
that the structural difference between parent compounds ([11C]1 and [11C]2) and fluoroethyl analogs ([18F]3
and [18F]4) do not obviously affect the potency against drug efflux transporters. In metabolite analysis of mice,
the unchanged form in the brain and plasma at 60 min after co-injection of [18F]4 plus 1 were higher (95% for
brain; 81% for plasma) than that after co-injection of [18F]3 plus 1. [18F]4 is a promising PET probe to assess
the function of drug efflux transporters.
Received 6 October 2010
Revised 1 December 2010
Accepted 2 December 2010
Available online 5 December 2010
Keywords:
Positron emission tomography (PET)
Fluorine-18
P-Glycoprotein (P-gp)
Breast cancer resistance protein (BCRP)
Drug efflux transporter
GF120918 (elacridar)
XR9576 (tariquidar)
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The acridone carboxamide derivative GF120918 (1, elacridar,
Scheme 1) and anthranilic acid derivative XR9576 (2, tariquidar,
Drug efflux transporters have a major impact on the pharmaco-
logical behavior of drugs, critically affecting its absorption, disposi-
tion, and elimination from organs and tissues.1–4 These transport
ers are known to limit absorption across many biological barriers
and restrict entry into important pharmacological sites.3 The most
extensively characterized drug efflux transporters are ATP-binding
cassette transporters, which include P-glycoprotein (P-gp), multi-
drug resistance-associated proteins (MRPs), and breast cancer resis-
tance protein (BCRP).2,5 Furthermore, multidrug resistance (MDR) is
primarily caused by cellular extrusion of drugs by drug efflux
transporters.
Scheme 1) were developed as third-generation MDR inhibitors.6,7
Compound 1 is active against P-gp, which is about 100-fold as
potent as cyclosporin A and verapamil.6 It can also reverse BCRP-
mediated drug resistance in human and murine cell lines.8,9
Compound 2 inhibits the basal ATPase activity associated with
P-gp, suggesting that the modulating effect of 2 is derived from
the inhibition of substrate binding, ATP hydrolysis, or both.10 Inhib-
itory activity of 2 against P-gp function is higher than that of typical
P-gp modulators, although 2 slightly inhibits BCRP function.11
[
11C]1 and [11C]2 (Scheme 1) have been developed as positron
emission tomography (PET) probes for evaluating drug efflux
transporters.12–15 In PET studies using [11C]1 and [11C]2, the radio-
activity level for 60 min after injection in P-gp/Bcrp knockout mice
were nine- and 11-fold higher than that in wild-type mice,
⇑
Corresponding author. Tel.: +81 43 206 3192; fax: +81 43 206 3241.
E-mail address: kawamur@nirs.go.jp (K. Kawamura).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.004

862
K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
O
CH3
O R
N
O
O
O
O
O
O
CH3
CH3
H3C
H3C
N
H
N
N
H
NH
O
R
O
N
H
O
N
1: R = CH3
3: R = CH2CH2F
[11C]1: R = 11CH3
[18F]3: R = CH2CH218F
2: R = CH3
4: R = CH2CH2F
[11C]2: R = 11CH3
[18F]4: R = CH2CH218F
Scheme 1. Chemical structures of GF120918 (1), XR9576 (2), and fluoroethyl analogs 3 and 4.
respectively.13,14 We demonstrated that increased radioactivity
levels in P-gp/Bcrp knockout and wild-type mice for 60 min after
injection of [11C]1 and [11C]2 is correlated with the function of drug
efflux transporters. We suggest that [11C]1 and [11C]2 are useful
PET probes and accumulation of these probes helps assess the
function of drug efflux transporters.13,14
2.3. Radiosynthesis
All synthetic sequences of [18F]3 and [18F]4 were carried out
using an in-house automated synthesis system.16 18F]2-Fluoroeth-
[
yl bromide was synthesized as the alkylating intermediate by the
reaction of cyclotron-produced [18F]fluoride ion (Fꢀ) with 2-trif-
Here we developed
[
18F]fluoroethyl analog of 1, N-(4-(2-
luoromethanesulfonyloxyethylbromide.17 18F]3 and [18F]4 were
[
(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-
9,10-dihydro-5-[18F]fluoroethoxy-9-oxo-4-acridine carboxamide
([18F]3, Scheme 1) and [18F]fluoroethyl analog of 2, quinoline-3-
carboxylic acid [2-(4-{2-[7-(2-[18F]fluoroethoxy)-6-methoxy-3,
4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dime-
thoxyphenyl]amide ([18F]4, Scheme 1) to enhance the effectiveness
of probes for evaluating the function of drug efflux transporters.
synthesized by heating each O-desmethyl precursors (5 or 9) with
[
18F]2-fluoroethyl bromide in anhydrous DMF containing tetrabu-
tylammonium hydroxide as a base at 90–120 °C for 10 min. Radio-
synthesis times of [18F]3 and [18F]4 were 60–70 min from the end
of irradiation. Decay-corrected radiochemical yields from [18F]Fꢀ at
the end of synthesis (EOS) were 4.7 1.9% (n = 6) and 1.5 0.9%
(n = 6), respectively. The specific activity of [18F]3 and [18F]4 was
140–490 TBq/mmol at EOS. Radiochemical yield and specific radio-
activity were at levels suitable for injection as PET probes. [18F]3
and [18F]4 were identified on analytical high performance liquid
chromatography (HPLC) by co-injection with unlabeled 3 and 4.
The radiochemical purity of [18F]3 and [18F]4 was >99% and
remained equal to 95% after maintaining the formulated product
for at least 3 h at room temperature.
2. Results
2.1. Chemistry
Compounds 3 and 4 were synthesized according to the reaction
sequences delineated in Schemes 2 and 3. Alkylation of 513 with
1-iodo-2-fluoroethane in N,N-dimethylformamide (DMF) contain-
ing potassium carbonate at 80 °C for 2 h gave 3 with a chemical
yield of 62%. Compound 4 was synthesized in three-steps as
mentioned below. Alkylation of 614 with 1-iodo-2-fluoroethane
containing potassium carbonate as a base in DMF at 80 °C for 2 h
gave 7, and reductive amination of 7 gave 8. Reaction of 8
with quinoline-3-carbonyl chloride in dichloromethane at room
temperature for 7 h gave 4 with a chemical yield of 24% from 6.
2.4. In vitro accumulation assay
Figure 1 shows increased intercellular accumulationof [18F]3 and
[
18F]4 in P-gp- and BCRP-overexpressing MES-SA/Dx5 cells18–20
treated for 120 min at 37 °C with one of 1, 2, 3 and 4 at a concentra-
tion of 10 M compared to untreated cells (control cells). Treatment
l
with all investigated compounds enhanced the tracer accumulation
capacity to MES-SA/Dx5 cells. The percentage of increased accumu-
lation of [18F]3 and [18F]4 in cells treated with 1 was higher than that
treated with other investigated compounds (2–4). The increased
accumulation of [18F]3 and [18F]4 in cells treated with 2 was similar
to that treated with 3 and 4.
2.2. Computation of c Log D
c Log D (at pH 7.4) values for 3 and 4 were 5.62 and 5.12,
respectively.
O
O
O
O
a or b
CH3
CH3
CH3
N
N
CH3
N
H
O
N
H
O
OH
O
R
O
N
H
O
N
H
5
3: R = CH2CH2F
[18F]3: R = CH2CH218F
Scheme 2. Chemical synthesis of 3 and radiosynthesis of [18F]3. Reagents and conditions: (a) 1-iodo-2-fluoroethane, potassium carbonate, N,N-dimethylformamide (DMF),
80 °C, 2 h, yield 62%; (b) [18F]2-fluoroethyl bromide, tetrabutylammonium hydroxide, DMF, 80 °C, 10 min, 4.7% radiochemical yield (RCY) from [18F]Fꢀ.

K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
863
A
O
O
O
CH3
CH3
N
F
N
a
O
O
OH
O
O
O
O
N
H
2
H3C
H3C
N
H
2
H3C
H3C
NO
NO
7
6
O
O
CH3
O
O
N
F
CH3
O
N
F
O
O
b
c
O
H3C
H3C
N
H
O
O
N
H
NH2
H3C
H3C
NH
O
N
8
4
B
O
O
CH3
OH
CH3 18
N
N
F
O
O
O
O
O
d
O
H3C
H3C
N
H
H3C
H3C
N
H
NH
O
NH
O
O
N
N
9
[18F]4
Scheme 3. Chemical synthesis of 4 (A)and radiosynthesis of [18F]4 (B). Reagents and conditions:(a) 1-iodo-2-fluoroethane, potassium carbonate, DMF, 80 °C, 2 h;(b) ammonium
formate, 5% palladium carbon, methanol, reflux, 6 h; (c) quinoline-3-carbonyl chloride, dichloromethane, rt, 7 h, yield 24%; (d) [18F]2-fluoroethyl bromide, tetrabutylammonium
hydroxide, DMF, 80 °C, 10 min, 1.5% RCY from [18F]Fꢀ.
Control
A
Control
B
Treatment with 1
Treatment with 1
Treatment with 2
Treatment with 2
Treatment with 3
Treatment with 4
50
25
0
50
25
0
*
*
*
*
Figure 1. Increased intercellular accumulation of [18F]3 (A) or [18F]4 (B) in MES-SA/Dx5 cells treated with 1, 2, 3 and 4 (10
lM) for 120 min at 37 °C compared to untreated
cells (control cells). Data was triplicate. ⁄Significant difference (P <0.05) compared with the control (one-way ANOVA and Dunnett’s post hoc tests). Abbreviation: N.D., not
determined.
2.5. Biodistribution in mice
and small intestine gradually increased, and levels in the blood,
heart, lung, kidney, and muscle gradually decreased, and levels in
the bone and brain were maintained at a constant level after initial
uptake. In contrast, the radioactivity level in the liver gradually in-
creased until 15 min after injection of [18F]4 and was then main-
tained at a constant level.
To evaluate whether drug efflux transporters mediated penetra-
tion of radioactivity in the brain and testis after injection of [18F]3
or [18F]4, we investigated the effects of co-injection with 1 in the
brain, testis, and blood after injection of [18F]3 and [18F]4 in mice
(Fig. 2). Co-injection with 1 (5 mg/kg) induced approximately a
Tables 1 and 2 summarize tissue distribution of radioactivity
after injection of [18F]3 and [18F]4 in mice. As for [18F]3, the radio-
activity level in the brain was the lowest of all tissues investigated.
While radioactivity levels in the pancreas, liver and small intestine
gradually increased, levels in the blood, heart, lung, and muscle
gradually decreased, and levels in the spleen, kidney, bone, and
brain were maintained at a constant level after initial uptake. As
for [18F]4, the radioactivity level in the brain was also the lowest
of all tissues investigated. While levels in the pancreas, spleen,

864
K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
Table 1
Tissue distribution of radioactivity after injection of [18F]3
Radioactivity levela (%ID/g)
5 min
15 min
30 min
60 min
90 min
Blood
Heart
Lung
Liver
Pancreas
Spleen
Kidney
Small intestine
Muscle
Bone
1.39 0.12
6.65 0.55
19.60 0.96
4.88 0.36
6.12 1.87
4.76 0.51
20.65 1.58
4.51 0.39
1.75 0.14
1.99 0.27
0.18 0.00
0.72 0.09
2.43 0.15
13.32 1.05
5.99 0.73
9.68 0.71
5.34 0.50
24.17 2.08
7.09 0.57
1.18 0.18
1.66 0.21
0.16 0.01
0.65 0.37
1.38 0.09
7.21 0.50
5.35 0.38
11.15 2.52
5.82 0.34
19.45 1.05
7.60 1.29
0.62 0.03
1.63 0.22
0.14 0.01
0.40 0.04
1.19 0.06
6.16 1.27
11.60 1.26
15.14 1.58
5.74 0.55
23.44 2.44
12.48 1.07
0.53 0.04
1.43 0.38
0.17 0.02
0.36 0.04
0.85 0.02
4.09 0.45
14.82 1.04
15.21 1.44
4.71 0.24
19.98 1.32
17.15 2.25
0.38 0.05
1.30 0.22
0.13 0.02
Brain
a
Mean SD (n = 4); percentage of injected dose/gram tissue (%ID/g).
Table 2
Tissue distribution of radioactivity after injection of [18F]4
Radioactivity levela (%ID/g)
30 min
5 min
15 min
60 min
90 min
Blood
Heart
Lung
Liver
Pancreas
Spleen
Kidney
Small intestine
Muscle
Bone
1.39 0.09
7.44 0.85
22.50 2.58
29.33 2.30
6.05 0.60
5.12 0.67
18.00 1.79
5.67 0.67
2.23 0.24
2.17 0.20
0.13 0.02
0.88 0.05
3.49 0.31
20.55 1.94
31.06 0.63
8.27 0.84
6.73 0.10
16.09 0.98
9.42 1.42
1.61 0.18
1.85 0.23
0.11 0.01
0.63 0.04
2.86 0.24
17.89 1.29
23.23 3.31
8.56 0.35
6.34 0.40
11.34 1.17
14.09 1.61
1.04 0.12
1.45 0.32
0.11 0.00
0.61 0.02
2.09 0.13
16.06 1.53
25.40 2.82
12.45 0.68
7.18 0.27
11.29 0.52
22.42 1.57
0.92 0.13
1.62 0.20
0.10 0.00
0.57 0.02
1.74 0.17
15.76 2.05
22.18 2.26
13.56 1.09
7.42 0.94
10.17 0.73
24.45 2.38
0.68 0.06
1.45 0.12
0.10 0.01
Brain
a
Mean SD (n = 4); percentage of injected dose/gram tissue (%ID/g).
10- and seven-fold increase in the brain-to-blood and testis-to-
blood ratios of radioactivity after injection of [18F]3, respectively.
Similarly, co-injection with 1 induced a 10- and fivefold increase
in the brain-to-blood and testis-to-blood ratios of radioactivity
after injection of [18F]4, respectively.
(Table 3). The percentage of unchanged form in the brain and plas-
ma at 30 min after co-injection of [18F]3 plus 1 was higher than
that at 60 min after co-injection. In contrast, a significant differ-
ence was not observed in the percentage form in the brain and
plasma at 30 and 60 min after co-injection of [18F]4 plus 1. In the
plasma, the percentage of unchanged form after co-injection of
2.6. Metabolite analysis in the brain and plasma of mice
co-injected with 1
[
18F]4 plus 1 was higher than that after co-injection of [18F]3 plus
1 at both 30 and 60 min. In the brain, the percentage of unchanged
form at 60 min after co-injection of [18F]4 plus 1 was higher than
that after co-injection of [18F]3 plus 1.
Radiolabeled metabolites with high polarity were observed on
each HPLC chart. The eluting time from the column of metabolite
for [18F]3 and [18F]4 were 1.8 and 1.9 min, respectively. After
injection of [18F]3, recovery of radioactivity from brain tissue and
To evaluate the role played by radiolabeled metabolites in in-
creased accumulation after co-injection of [18F]3 or [18F]4 by inhib-
iting drug efflux transporters, we investigated the percentages of
unchanged forms in the brain and plasma at 30 and 60 min after
co-injection of [18F]3 plus 1 (5 mg/kg) or [18F]4 plus 1 (5 mg/kg)
A
B
Control
Control
Co-injection with 1
*
3.0
Co-injection with 1
3.0
2.0
1.0
0.0
*
*
2.0
*
1.0
0.0
[18F]4
[18F]3
[18F]4
[18F]3
Figure 2. Effects of co-injection with 1 on the brain-to-blood (A) and testis-to-blood (B) ratios at 30 min after injection of [18F]3 and [18F]4 into mice (n = 4 per group).
Injected dose of 18F -labeled ligands was 1.0–4.8 MBq. Co-injected dose of 1 was 5 mg/kg. ⁄Significant difference (P <0.05) compared with the control (one-way ANOVA and
Dunnett’s post hoc tests).

K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
865
plasma into acetonitrile for deproteinized treatment were
91.4 5.3% (n = 7) and 94.1 1.2% (n = 7), respectively. After injec-
tion of [18F]4, recovery of radioactivity from brain tissue and plas-
ma into acetonitrile for deproteinized treatment were 92.5 1.6%
(n = 7) and 95.3 1.3% (n = 7), respectively. Recovery of radioactiv-
ity from HPLC analysis was essentially quantitative.
P-gp/Bcrp knockout mice was 8.3-fold higher than that in wild-type
mice (32.9 2.5 for P-gp/Bcrp knockout mice; 4.0 0.2 for wild-type
mice; n = 4 each group), and that in P-gp knockout mice after injec-
tion of [18F]4 was 2.3-fold higher than that in wild-type mice
(9.1 1.8 for P-gp knockout mice, n = 4). However, AUCbrain [0–60 min]
in Bcrp knockout mice after injection of [18F]4 was almost identical
to that of wild-type mice (6.5 0.8 for Bcrp knockout mice, n = 4).
2.7. PET studies in wild-type and P-gp/Bcrp knockout mice
Also, AUCbrain
[
in wild-type mice after co-injection of
[0–60 min]
18F]4 plus 1 was 7.1-fold higher than that in wild-type mice
To evaluate whether brain penetration of [18F]3 and [18F]4 is
related to the function of drug efflux transporters, we further
performed PET studies using [18F]3 and [18F]4 in wild-type mice
co-injected with or without 1 and in P-gp and/or Bcrp knockout
mice (Figs. 3 and 4). In wild-type mice, the radioactivity level in
the brain after injection of [18F]3 or [18F]4 was low (Fig. 3). Co-
injection with 1 induced an increase in the radioactivity level of
the brain after injection of [18F]3 or [18F]4 (Fig. 3). In P-gp/Bcrp
knockout mice, the radioactivity level in the brain after the injec-
tion of [18F]3 and [18F]4 was distributed throughout the brain
(Fig. 3). In P-gp/Bcrp knockout mice, the radioactivity level in the
brain after injection of [18F]3 was maintained at a constant level
after initial uptake for 60 min after injection (Fig. 4A). After injec-
tion of [18F]4, radioactivity levels in the brain increased gradually
after initial uptake (Fig. 4B). In P-gp knockout mice, the radioactiv-
ity level in the brain after injection of [18F]3 and [18F]4 was gradu-
ally decreased after initial uptake, and that was maintained at
higher level than that in wild-type mice for 60 min after injection
(Fig. 4). In Bcrp knockout mice, the radioactivity level in the brain
after injection of [18F]3 and [18F]4 was gradually decreased after
initial uptake, and that was almost identical to that of wild-type
mice (Fig. 4). In wild-type mice co-injected with 1, the radioactivity
level in the brain after injection of [18F]3 decreased gradually after
initial uptake for 60 min after injection (Fig. 4A). After injection of
(28.1 3.3 for wild-type mice co-injected with 1, n = 4). No signifi-
cant differences were observed in the AUC of ROI in the blood from
0 to 60 min (AUCblood [0–60 min]) after injection of [18F]3 among
wild-type (25.6 5.3), P-gp/Bcrp knockout (25.0 3.6), P-gp knock-
out (23.5 4.6) and Bcrp knockout mice (25.3 6.9). Also, there are
no significant differences in AUCblood
after injection
[0–60 min]
of
[
18F]4 among wild-type (33.2 5.7), P-gp/Bcrp knockout
(33.9 5.0), P-gp knockout (45.0 8.1) and Bcrp knockout mice
(42.2 11.6).
3. Discussion
Compound 3 was synthesized by alkylation of its O-desmethyl
precursor 5 with 1-iodo-2-fluoroethane and commercially avail-
able materials. We attempted direct alkylation of 9 with 1-iodoflu-
oroethane or 1-bromofluoroethane in the presence of a base, but
the reaction did not proceed efficiently because of hydrolysis of
the amide between quinoline and dimethoxybenzene. We deter-
mined another three-step route to accomplish the synthesis of 4.
Fluoroethylation of 6 followed by reduction of the nitro group gave
8, which was acylated with quiniline-3-carbonyl chloride to pro-
duce 4.
[
18F]3 and [18F]4 were readily synthesized by alkylation of each
O-desmethyl precursor (5 or 9) with [18F]2-fluoroethyl bromide,
which was synthesized from the cyclotron-produced [18F]Fꢀ and
2-trifluoromethanesulfonyloxyethylbromide. Purification by HPLC
gave [18F]3 and [18F]4 with radiochemical yields, radiochemical
purity, and specific radioactivity suitable for injection as PET
probes. Formulated PET probes were radiochemically stable.
Previously, de Vries et al. and we suggested that P-gp and Bcrp act
in concert to limit brain penetration of drugs,21 such as [11C]1 and
[
18F]4, radioactivity level in the brain increased gradually after ini-
tial uptake and maintained constant level for over 30 min after
injection (Fig. 4B). In the blood, the time–activity curve (TAC) in
wild-type mice after injection of [18F]3 and [18F]4 were similar pat-
tern to that in P-gp and/or Bcrp knockout mice (Fig. 5).
The area under the time-activity curve (AUC) of the region of
interest (ROI) in the brain from 0 to 60 min (AUCbrain [0–60 min]) after
injection of [18F]3 in P-gp/Bcrp knockout mice was 9.0-fold higher
than that in wild-type mice (39.8 1.8 for P-gp/Bcrp knockout mice;
4.4 0.5 for wild-type mice; n = 4 each group), and that in P-gp
knockout mice after injection of [18F]3 was 2.4-fold higher than that
in wild-type mice (10.6 0.6 for P-gp knockout mice, n = 4). How-
[
11C]2.13,14 In addition, Kannan et al. suggested that some inhibitors
of ABC transporters may lose selectivity for one ABC transporter at
high concentrations.22 For example, low concentrations (6100 nM)
of tariquidar appear to be selective for P-gp, but higher concentra-
tions (>1 lM) show cross reactivity for BCRP. Therefore, we consid-
ered that it is important to assess affinity for both P-gp and BCRP to
evaluate the function of drug efflux transporters.22 Parent com-
pound 1 is a dual P-gp and BCRP inhibitor, and parent compound 2
is a P-gp inhibitor. However, recent in vivo studies suggest that tra-
cer amounts (injected dose, >0.5 nmol) of [11C]1, [11C]2, [11C]N-
desmethylloperamide and [11C]laniquidar (MDR inhibitor) act as a
substrate for drug efflux transporters.13,14,22,23 Tracer amounts of
ever, AUCbrain
[
in Bcrp knockout mice after injection of
[0–60 min]
18F]3 was almost identical to that of wild-type mice (5.0 0.7 for
Bcrp knockout mice, n = 4). Also, AUCbrain [0–60 min] in wild-type mice
after co-injection of [18F]3 plus 1 was 6.9-fold higher than that in
wild-type mice (30.2 2.0 for wild-type mice co-injected with 1,
n = 4). In addition, AUCbrain
after injection of [18F]4 in
[0–60 min]
[
18F]3 and [18F]4 are also predicted to act as a substrate for drug ef-
Table 3
fluxtransporters. Furthermore, compounds 3 and4maydisplaysim-
ilar in vitro and in vivo property to compounds 1 and 2, because of
the molecular similarity and bioisosteric property of O-CH3CH2F
with O-CH3 group. Therefore, we evaluated the usefulness of [18F]3
and [18F]4 as PET probes to assess the function of drug efflux trans-
porters by in vivo studies.
In the in vitro accumulation assay, treatment of P-gp- and
BCRP-overexpressing MES-SA/Dx5 cells18–20 with 1 resulted in a
remarkable increase in the accumulation of [18F]3 and [18F]4
(Fig. 1), indicating active transport and efflux of [18F]3 and [18F]4
by MES-SA/Dx5 cells. On the other hand, the increased accumula-
tion of [18F]3 in cells treated with 3 (26%) was lower than that
of [18F]3 treated with 1 (41%) ( Fig. 1). Also, the increased
Metabolite analysis in the brain and plasma of mice at 30 and 60 min after co-
injection of [18F]3 plus 1 (5 mg/kg) and [18F]4 plus 1 (5 mg/kg)
Unchanged forma (%)
Brain
Plasma
30 min
60 min
30 min
69.6 1.5
60 min
[
[
18F]3 plus 1 (5 mg/kg) 95.9 0.8 88.2 2.7*
48.8 4.5*
18F]4 plus 1 (5 mg/kg) 95.7 0.3 95.4 2.9** 89.3 1.2** 80.8 5.8**
a
Mean SD (n = 3–4).
*
Significant difference (P <0.05) in the percentages of unchanged form in the brain
and plasma at 30 and 60 min after injection.
**
Significant difference (P <0.05) between co-injections of [18F]3 plus 1 and [18F]4
plus 1.

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K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
Figure 3. Typical coronal PET images showing [18F]3 (A) and [18F]4 (B) in the brain of wild-type mouse, wild-type mouse co-injected with 1, and a P-gp/Bcrp knockout mouse.
The injected dose of [18F]3 and [18F]4 was 3.3–4.9 MBq. Co-injected dose of 1 was 5.0 mg/kg. PET images were obtained from 0 to 60 min after injection. Mice were
anesthetized with isoflurane and fixed in a prone position on the bed of the scanner. The radioactivity level was expressed as the standardized uptake value (SUV).
A
B
Wild-type
Co-injection with 1 (wild-type)
P-gp/Bcrp knockout
P-gp knockout
Bcrp knockout
Wild-type
Co-injection with 1 (wild-type)
P-gp/Bcrp knockout
P-gp knockout
Bcrp knockout
1.0
0.5
0.0
1.0
0.5
0.0
0
30
60
0
30
60
Time after injection (min)
Time after injection (min)
Figure 4. Time–activity curves of the brain after injection of [18F]3 (A) and [18F]4 (B) in wild-type mice, wild-type mice co-injected with 1, P-gp/Bcrp knockout mice, P-gp
knockout mice and Bcrp knockout mice (n = 4–6 per group). The injected dose of [18F]3 and [18F]4 was 2.8–5.6 MBq. The radioactivity level was expressed as SUV.
accumulation of [18F]4 in cells treated with 4 (23%) was lower than
that of [18F]4 treated with 1 (32%) (Fig. 1). These results suggest
that the inhibitory effect on the function of drug efflux transporter
by treatment with 3 or 4 is weaker than that by the parent com-
pound 1. Based on these in vitro results, it is suggested that
mice co-injected with 1 were approximately sevenfold higher than
that in wild-type mice, and AUCbrain [0–60 min] in P-gp/Bcrp knockout
mice were eight- to ninefold higher than that in wild-type mice
(Fig. 4). Previously, we evaluated [11C]1 and [11C]2 as PET probes
to assess the function of drug efflux transporters using small-
[
18F]3 and [18F]4 are modulated by the function of drug efflux
animal PET measurements.13,14 AUC
in P-gp/Bcrp
brain [0–60 min]
transporters as substrates.
knockout mice after injection of [11C]1 and [11C]2 were approxi-
mately nine- and 11-fold higher than that in wild-type mice,
respectively.13,14 The increased uptake after injection of [18F]3
and [18F]4 was similar to that of [11C]1 and [11C]2. As previously
described for [11C]1 and [11C]2, these results indicate that radioac-
tivity levels in the brain after injection of [18F]3 and [18F]4 are
related to the function of drug efflux transporters. Also, these
results suggest that the structural difference between parent
In in vivo tissue distribution, the radioactivity level in the brain
at 30 min after injection of [18F]3 and [18F]4 was the lowest of all
tissues investigated ( Tables 1 and 2), due to relatively high
lipophilicity (c Log D, 5.6 for 3; 5.1 for 4). Co-injection with 1
(5 mg/kg) induced a significant increase in the brain uptake at
30 min after injection of [18F]3 and [18F]4 (Fig. 3). Furthermore,
AUCbrain [0–60 min] after injection of [18F]3 and [18F]4 in wild-type

K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
867
In PET studies, the radioactivity level in the brain of P-gp/Bcrp
knockout mice after injection of [18F]3 was maintained at a con-
stant level after initial uptake for 60 min after injection, and that
in the brain of wild-type mice co-injected with 1 decreased gradu-
ally after initial uptake for 60 min after injection (Fig. 3). On the
other hand, the radioactivity level in the brain after injection of
A
B
Wild-type
Wild-type
P-gp/Bcrp knockout
P-gp knockout
Bcrp knockout
P-gp/Bcrp knockout
P-gp knockout
Bcrp knockout
10
5
10
5
[
18F]4 had a tendency to accumulate after initial uptake (Fig. 4).
This variation in kinetics of the brain between [18F]3 and [18F]4
can be explained by radiolabeled metabolites in the brain and plas-
ma. In the metabolite study, the percentage of unchanged form in
the brain at 60 min after co-injection of [18F]4 plus 1 (95%) was
higher than that of [18F]3 plus 1 (88%) (Table 3). In addition, the
percentage of unchanged form in the plasma at 30 and 60 min after
co-injection of [18F]4 plus 1 (89% for 30 min, 81% for 60 min) was
higher than that of [18F]4 plus 1 (70% for 30 min, 49% for 60 min)
(Table 3). [18F]4 exhibits a higher metabolic stability than [18F]3.
Therefore, [18F]4 may be more suitable than [18F]3 to evaluate
in vivo the function of drug efflux transporters.
0
0
0
30
60
0
30
60
Time after injection (min)
Time after injection (min)
Figure 5. Time–activity curves of the blood after injection of [18F]3 (A) and [18F]4
(B) in wild-type mice, P-gp/Bcrp knockout mice, P-gp knockout mice and Bcrp
knockout mice (n = 4–6 per group). The injected dose of [18F]3 and [18F]4 was 2.8–
5.6 MBq. The radioactivity level was expressed as SUV.
4. Conclusions
compounds ([11C]1 and [11C]2) and fluoroethyl analogs ([18F]3 and
[
We synthesized and evaluated new PET probes, [18F]3 and
18F]4) do not obviously affect the potency against drug efflux
[
18F]4, to assess the function of drug efflux transporters. [18F]3
and [18F]4 were synthesized by 18F-alkylation of each O-desmethyl
precursors (5 and 9) with [18F]2-fluoroethyl bromide for injection
as PET probes. Radioactivity levels after injection of [18F]3 and
transporters. In the metabolite study using mice co-injected with
1, [18F]3 and [18F]4 showed a high metabolic stability at 30 and
60 min after injection in the brain (Table 3), indicating that the
radioactive metabolites scarcely affect the uptake of [18F]3 and
[
18F]4 in mice brain increased by inhibiting the function of drug ef-
18F]4 in the brain. These results suggest that excretion of [18F]3
flux transporters. The increased uptake after injection of [18F]3 and
[
or [18F]4 itself from the brain is related to drug efflux transporters.
Therefore, radio-metabolites after injection of [18F]3 or [18F]4 in
the plasma are considered to have an little effect on the radioactiv-
ity level in the brain, although the percentage of unchanged [18F]3
(70%) and [18F]4 (89%) in plasma were lower than that of [11C]1
(96%)13 or [11C]2 (93%)14 at 30 min after injection. In general, the
short half-life of the 11C-labeled probe often limits its usefulness
if a dynamic PET experiment has a turnover time longer than
[
18F]4 was similar to that of [11C]1 and [11C]2. In metabolite anal-
ysis of mice brain and plasma, [18F]4 was more metabolically sta-
ble than [18F]3. [18F]4 may be more suitable than [18F]3 to
evaluate in vivo the function of drug efflux transporters. Conse-
quently, [18F]4 is a promising PET probe to assess the function of
drug efflux transporters.
5. Materials and methods
100 min. Since 18F has the advantages of
a longer half-life
(110 min vs 20 min) and lower positron energy (650 keV vs
960 keV) compared to 11C, the 18F-labeled probe produce high
quality images with a high spatial resolution in PET measurements.
Moreover, long-distance transportation of 18F to other facilities can
be conveniently performed. Therefore, [18F]3 and [18F]4 are more
useful PET probes than [11C]1 and [11C]2 to assess the function of
drug efflux transporters.
5.1. General
Melting points were uncorrected. Proton nuclear magnetic res-
onance (1H NMR) and carbon-13 nuclear magnetic resonance (13
C
NMR) spectra were recorded on the JNM-AL300 spectrometer (Jeol,
Tokyo, Japan) using tetramethylsilane as an internal standard. All
chemical shifts (d) were reported in parts per million (ppm) down-
field from the standard. High resolution fast atom bombardment-
mass spectrometry (FAB-MS) was obtained on a JEOL NMS-SX102
spectrometer (Jeol). Column chromatography was performed using
Kieselgel Gel 60 F254 (70–230 mesh; Merck, Darmstadt, Germany)
or Wakogel C-200 (100–200 mesh; Wako Pure Chemical Industries,
Osaka, Japan). HPLC was performed using a Jasco HPLC system
(Jasco, Tokyo, Japan). Effluent radioactivity was monitored using
a NaI (Tl) scintillation detector system. Compounds 1 and 2 were
synthesized in our laboratory as described previously with some
modifications.27,28,7 Reagents and organic solvents were commer-
cially available and used without further purification. If not
otherwise stated, radioactivity was determined using an IGC-3R
Curiemeter (Aloka, Tokyo, Japan).
In PET studies, the AUCbrain [0–60 min] after injection of [18F]3 and
[
18F]4 in P-gp knockout mice were approximately 3.6- to 3.8-fold
lower than that in P-gp/Bcrp knockout mice, although the AUC-
value in P-gp knockout mice was approximately
brain [0–60 min]
2.3- to 2.4- fold higher than that in wild-type mice (Fig. 4). As pre-
viously described for [11C]1 and [11C]2, the expression of Bcrp in
P-gp knockout mouse might be sufficient to limit brain penetration
of [18F]3 and [18F]4, because the mRNA levels of Bcrp in P-gp
knockout mice were three times higher than that in wild-type
mice.24 Additionally, it has been suggested that P-gp and Bcrp
functions in the brain concertedly limit brain penetration of shared
substrates.21,25 Therefore, it is considered that P-gp and Bcrp act in
concert to limit brain penetration of [18F]3 and [18F]4.
Defluorination is a major issue for some 18F-labeled probes. The
[
18F]Fꢀ formed by defluorination of 18F-labeled probes binds avidly
to the bone, including the skull.26 Considering biodistribution in
mice, radioactivity levels in the bone at 60 min after injection of
5.2. Chemistry
18F]3 and [18F]4 were relatively low ([18F]3, 0.40 standardized
5.2.1. N-(4-(2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-
isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-fluoroethoxy-9-
oxo-4-acridine carboxamide (3)
A mixture of N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-iso-
quinolinyl)ethyl)phenyl)-9,10-dihydro-5-hydroxy-9-oxo-4-acri-
[
uptake value (SUV); [18F]4, 0.42 SUV) (Table 1 and 2). These radioac-
tivity levels in the bone were same as that at 60 min after injection of
[
11C]1 and [11C]2 (0.55 SUV and 0.45 SUV).13,14 These results suggest
that defluorination of [18F]3 and [18F]4 does not occur in mice.

868
K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
dine carboxamide13 (5, 52.0 mg, 0.095 mmol), 1-iodo-2-fluoroe-
thane (24.7 mg, 0.142 mmol), potassium carbonate (19.6 mg,
0.142 mmol) in DMF (3 mL) was heated at 80 °C for 2 h. The reac-
tion was quenched with water (5 mL) and extracted with ethyl
acetate (5 mL, three times). The organic layer was washed with
water and brine, dried over magnesium sulfate, and evaporated
in vacuo. The residue was recrystallized from the mixture of
chloroform and n-hexane, yielding N-(4-(2-(1,2,3,4-tetrahydro-
6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-flu-
oroethoxy-9-oxo-4-acridine carboxamide (3, 35.0 mg, 62%) as a
yellow solid. Melting point: 211–212 °C. 1H NMR (300 MHz,
CDCl3): d (ppm) 2.77–2.87 (m, 6H), 2.92–2.97 (m, 2H), 3.67 (s,
2H), 3.85 (s, 6H), 4.44 (t, J = 4.0 Hz, 1H), 4.53 (t, J = 4.0 Hz, 1H),
4.87 (t, J = 4.0 Hz), 5.02 (t, J = 4.0 Hz, 1H), 6.56 (s, 1H), 6.61 (s,
1H), 7.16-7.24 (m, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz,
2H), 8.01 (dd, J = 1.5, 7.7 Hz, 1H), 8.06 (dd, J = 2.2, 7.3 Hz, 1H),
8.11 (brs, 1H), 8.65 (dd, J = 1.0, 7.7 Hz,1H), 12.24 (s, 1H). 13C NMR
(75 MHz, CDCl3): d (ppm) 28.67, 33.49, 51.03, 55.72, 56.00, 56.05,
59.97, 68.61 (d, JC–F = 21.7 Hz), 81.84 (d, JC–F = 170.8 Hz), 109.87,
111.74, 113.90, 118.93, 119.21, 119.74, 121.02, 121.26, 122.07,
122.66, 126.31, 126.64, 129.37, 131.51, 131.85, 132.14, 135.70,
137.35, 140.22, 146.88, 147.47, 147.79, 166.44, 177.80. High-reso-
lution MS(FAB), m/z: 596.2522 (calcd for C35H35FN3O5: 596.2561).
Chemical purity: 98.2% (see Supplementary data).
J = 4.4 Hz, 1H), 4.82 (t, J = 4.4 Hz, 1H), 6.60 (s, 1H), 6.62 (s, 1H),
7.11 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.60–7.65 (m, 1H), 7.61 (d,
J = 8.4 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 8.16
(d, J = 7.7 Hz, 1H), 8.45 (br s, 1H), 8.52 (s, 1H), 8.73 (d, J = 2.0 Hz,
1H), 9.52 (d, J = 2.0 Hz, 1H), 12.44 (br s, 1H). 13C NMR (75 MHz,
CDCl3): d (ppm) 28.73, 33.42, 50.92, 55.55, 55.95, 56.03, 56.23,
60.06, 68.74 (d, JC–F = 21.1 Hz), 81.97 (d, JC–F = 170.2 Hz), 104.86,
109.81, 112.13, 112.27, 112.99, 121.17, 122.74, 126.63, 126.85,
127.14, 127.43, 127.66, 129.16, 129.28, 129.32, 131.42, 135.51,
135.73, 137.24, 144.50, 145.96, 148.36, 148.73, 149.30, 152.61,
163.84, 167.47. High-resolution MS(FAB), m/z: 679.2888 (calcd
for C39H40FN4O6: 679.2932). Chemical purity: 95.7% (see Supple-
mentary data).
5.3. Computation of c Log D
c Log D (at pH 7.4) for 3 and 4 were computed using the pro-
gram Pallas 3.4 (CompuDrug, Sedona, AZ, USA).
5.4. Radiosynthesis of N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimeth
oxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[18F]fluoro
ethoxy-9-oxo-4-acridine carboxamide ([18F]3) and quinoline-3-
carboxylic acid [2-(4-{2-[7-(2-[18F]fluoroethoxy)-6-methoxy-
3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-
dimethoxyphenyl]amide ([18F]4)
5.2.2. Quinoline-3-carboxylic acid [2-(4-{2-[7-(2-fluoroethoxy)-
6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylc
arbamoyl)-4,5-dimethoxyphenyl]amide (4)
The [18F]Fꢀ was produced from the cyclotron (CYPRIS HM-18;
Sumitomo Heavy Industries, Tokyo, Japan) by the 18O (p, n) 18F
reaction on 95 atom % H218O using 18 MeV protons (14.2 MeV on
target) and was separated from [18O]H2O using a QMA short col-
umn (Waters, Milford, MA, USA). [18F]Fꢀ was eluted from the resin
with aqueous K2CO3 (3.3 mg/0.3 mL) into a vial containing a solu-
tion of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexaco-
sane (Kryptofix 222, 25 mg) in CH3CN (1.5 mL) and transferred
into another reaction vessel in the hot cell. An aqueous [18F]Fꢀ
solution was dried at 120 °C for 15 min to remove H2O and CH3CN.
Subsequently, trifluoromethanesulfonic acid 2-bromoethyl ester17
A
mixture of N-{4-[2-(7-hydroxy-6-methoxy-3,4-dihydro-
1H-isoquinolin-2-yl)-ethyl]-phenyl}-4,5-dimethoxy-2-nitrobenza-
mide14 (6, 82 mg, 0.16 mmol), 1-iodo-2-fluoroethane (62 mg,
0.36 mmol), and potassium carbonate (33 mg, 0.24 mmol) in
DMF (5 mL) was heated at 80 °C for 2 h. The reaction mixture
was quenched with water (5 mL), and extracted with ethyl acetate
(5 mL, two times). The organic layer was washed with water (3 mL)
and brine (3 mL), dried over magnesium sulfate, and evaporated in
vacuo. The residue was purified by column chromatography on
silica gel using a mixture of chloroform and methanol (100:1, v/v)
as the mobile phase, yielding N-(4-{2-[7-(2-fluoroethoxy)-6-meth-
oxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenyl)-4,5-dimethoxy-
2-nitrobenzamide (7, 42 mg) as a yellow solid.
Ammonium formate (100 mg) and 5% palladium carbon (20 mg)
were added to a solution of 7 (100 mg, 0.18 mmol) in methanol
(5 mL). The mixture was refluxed for 6 h. The reaction mixture
was filtered through Celite, and evaporated in vacuo. The residue
was diluted with chloroform (5 mL), washed with 5% aqueous
ammonia (2 mL) and water, and dried over magnesium sulfate.
The extract was evaporated in vacuo, yielding 2-amino-N-(4-
{2-[7-(2-fluoroethoxy)-6-methoxy-3,4-dihydro-1H-isoquinolin-2-
yl]ethyl}-phenyl)-4,5-dimethoxybenzamide (8, 80 mg) as a pale
yellow solid.
(8 lL) in o-dichlorobenzene (0.4 mL) was added to the radioactive
mixture. [18F]2-Fluoroethyl bromide in this vessel was distilled un-
der a helium flow (90–100 mL/min) at 130 °C for 5 min and bub-
bled into another vessel containing the O-desmethyl precursor (5
or 9; 1.0 mg) and 0.33 M tetrabutylammonium hydroxide in meth-
anol (7
lL) in anhydrous DMF (0.35 mL) at ꢀ15 °C, and the reaction
mixture was heated and maintained at 120 °C for 10 min. Prepara-
tive HPLC purification was performed on a Capcell Pak C18 UG 80
column (10 mm internal diameter ꢁ 250 mm length; Shiseido,
Tokyo, Japan) using a mobile phase of acetonitrile/water/triethyl-
amine (60:30:0.1, v/v/v, for [18F]3; 65:35:0.1, v/v/v, for [18F]4) at
a flow rate of 3.5 mL/min for [18F]3 and 5.0 mL/min for [18F]4.
Retention times of [18F]3 and [18F]4 were 10 and 9 min, respec-
tively. HPLC fractions of [18F]3 or [18F]4 were collected into a flask
Quinoline-3-carbonyl chloride (82.1 mg, 0.43 mmol) in dichlo-
romethane (1 mL) was added to a slurry of 8 (160 mg, 0.31 mmol)
in dichloromethane (10 mL) and cooled with an ice-water bath.
The resulting solution was cooled to room temperature, and stirred
for 7 h. The reaction mixture was diluted with dichloromethane,
washed with 1 M sodium carbonate and water, dried over magne-
sium sulfate, and evaporated in vacuo. The residue was purified by
column chromatography on silica gel using a mixture of chloro-
form and methanol (50:1, v/v), yielding quinoline-3-carboxylic
acid [2-(4-{2-[7-(2-fluoroethoxy)-6-methoxy-3,4-dihydro-1H-iso-
quinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxy-
to which Tween 80 (75 lL) in ethanol (0.3 mL) had been added be-
fore radiosynthesis, and these fractions were then evaporated to
dryness. The residue was dissolved in physiological saline.
The products were analyzed by HPLC using a Capcell Pak C18
UG 80 column (4.6 mm internal diameter ꢁ 250 mm length; Shise-
ido). Elution was performed using the same mobile phase as that
used for preparative HPLC at a flow rate of 2.0 mL/min. Retention
times of [18F]3 and [18F]4 were 4.3 and 4.5 min, respectively.
5.5. In vitro accumulation assay
phenyl]amide (4, 124 mg, 23.7% from 6) as a pale yellow solid.
Melting point: 128–129 °C. 1H NMR (300 MHz, CDCl3): d (ppm)
2.74–2.94 (m, 8H), 3.63 (s, 2H), 3.73 (s, 3H), 3.82 (s, 3H), 3.90 (s,
3H), 4.17 (t, J = 4.4 Hz, 1H), 4.26 (t, J = 4.4 Hz, 1H), 4.66 (t,
The doxorubicin-resistant human uterus sarcoma cell line
(MES-SA/Dx5)18 was obtained from the American Type Tissue Cul-
ture Collection (ATCC, Rockville, MD, USA). Cell lines were grown
as a monolayer in McCoy’s 5A medium (ATCC) supplemented with

K. Kawamura et al. / Bioorg. Med. Chem. 19 (2011) 861–870
869
10% fetal calf serum (ATCC) in a humidified atmosphere of 5% CO2
in air at 37 °C.
ultraviolet detector at 254 nm on a Novapak C18 column (8 mm
internal diameter ꢁ 100 mm length; Waters) contained within a
radial compression module (RCM-100, Waters). Elution was per-
formed using a mixture of acetonitrile and 0.1 M sodium acetate
buffer (pH 4.7) (45:55, v/v, for [18F]3; 50:50, v/v, for [18F]4) at a
flow rate of 2.0 mL/min. Retention times of [18F]3 and [18F]4 were
5.6 and 5.7 min, respectively. Radioactivity in the supernatants,
residual precipitates after centrifugation, and waste solution from
HPLC were measured using the automatic gamma counter. Per-
centages of unchanged form were then determined.
MES-SA/Dx5 cells were plated on 24-well plates (105 cells/well)
and incubated at 37 °C with complete growth medium in a humid-
ified atmosphere of 5% CO2. After a monolayer was formed, cells
were incubated for 120 min in medium containing 7.4 MBq/ml tra-
cer ([18F]3 or [18F]4) with or without 10
lM one of 1, 2, 3 and 4.
After removing the medium, cells were washed five times with
pre-chilled phosphate buffered saline and dissolved with 0.2 M
NaOH. Radioactivities of cell lysates were measured using the
automatic gamma counter (Wizard 300 1480; Perkin Elmer, Wal-
tham, MA, USA). After measuring radioactivity, protein concentra-
tions of cell lysates were measured using a DC protein assay kit
(Bio-Rad). Accumulation of tracer in MES-SA/Dx5 cells treated with
MDR inhibitor was expressed as the percentage of increased accu-
mulation of tracer compared to untreated cells (control cells) using
the following formula: percentage of increase = 100 ꢁ [percent
incubation dose per mg protein in cells (%ICD/mg protein) treated
with MDR inhibitor ꢀ %ICD/mg protein in the control cells]/%ICD/
mg protein in cells treated with MDR inhibitor.29,30
5.9. PET study in mice
PET scans were obtained using an Inveon Dedicated PET Scan-
ner (Siemens Medical Solutions, Knoxville, TN, USA). P-gp/Bcrp
knockout mice (age, 16–26 weeks; weight, 29–36 g), P-gp knock-
out mice (age, 18–39 weeks; weight, 27–37 g), Bcrp knockout mice
(age, 18–39 weeks; weight, 26–36 g) and wild-type mice (age, 7–
22 weeks; weight, 23–35 g) were used in the PET study.
Mice were anesthetized with isoflurane (1.0–1.5%, v/v) and
placed in a prone position on the bed. [18F]3 (3.7–5.4 MBq/26–
39 pmol) or [18F]4 (2.8–5.6 MBq/20–40 pmol) was intravenously
co-injected with or without 1 (5 mg/kg) in mice (n = 4–6 per group).
A time-sequential scan was performed for 60 min in the three-
dimensional (3D) list mode with an energy window of 350–
650 keV. List-mode data were sorted into 3D sinograms (6 ꢁ 10 s,
4 ꢁ 15 s, 5 ꢁ 1 min, 4 ꢁ 2 min, 3 ꢁ 5 min, and 3 ꢁ 10 min), which
were then Fourier rebinned into two-dimensional sinograms. Dy-
namic images were reconstructed with filtered back projection
using a ramp filter. Decay-corrected radioactivity was expressed
as SUV, (tissue radioactivity/milliliter of tissue)/(injected radioac-
tivity/gram of body weight)]. ROIs were masked on the brain using
the ASIPro VM software (Siemens Medical Solutions). ROIs in the
blood were masked on the ventricular cavity using the frame of
first 5 seconds after the administration. AUC of ROIs in the brain
(AUCbrain, SUV min) was calculated starting from 0 to 60 min.
5.6. Animals
Animals were maintained and handled in accordance with the
recommendations by the US National Institutes of Health and in-
house guidelines (National Institute of Radiological Sciences, Chiba,
Japan). The animal experiments were approved by the Animal
Ethics Committee of the National Institute of Radiological Sciences.
Male FVB mice were purchased from CLEA Japan (Tokyo, Japan).
Male P-gp knockout [Mdr1a/1b(ꢀ/ꢀ)],31 Bcrp knockout [Abcg2
(ꢀ/ꢀ)]32 and P-gp/Bcrp knockout [Mdr1a/1b(ꢀ/ꢀ)-Abcg2(ꢀ/ꢀ)]
mice33 were purchased from Taconic Farm (Hudson, NY, USA).
5.7. Biodistribution in mice
Tissue distribution of radioactivity after injection of [18F]3 and
[
18F]4 were investigated. 18F]3 or 18F]4 (1.0–4.8 MBq/7.1–
[ [
34 pmol) was intravenously injected in normal FVB mice (age,
8–10 weeks; weight, 25–29 g). Mice were sacrificed by cervical dis-
location at 5, 15, 30, 60, or 90 min after injection (n = 4 per group).
Inhibitory effects of the function of drug efflux transporters on
tissue distribution of [18F]3 and [18F]4 were investigated. [18F]3
or [18F]4 (1.0–4.8 MBq/7.1–34 pmol) was intravenously co-injected
with or without 1 (5 mg/kg) in normal FVB mice (age, 8–10 weeks;
weight, 25–29 g). Mice were sacrificed by cervical dislocation at
30 min after injection (n = 4 per group).
Blood samples were collected by heart puncture, and the tissues
were dissected and weighed. Radioactivity in samples was mea-
sured using the automatic gamma counter. Radioactivity level
was decay corrected to injection time and was expressed as the
percentage of injected radioactivity per gram of tissue (%ID/g).
5.10. Statistical analysis
Quantitative dataareexpressedasthemean standarddeviation
(SD). In the in vitro accumulation assay and in vivo distribution
study, differences between control and treatment groups were
determined by one-way analysis of variance (ANOVA) and Dunnett’s
post hoc tests. In metabolite analysis and PET study, differences be-
tween all pairs of groups were determined by one-way ANOVA and
Bonferroni’s multiple comparison tests. Data was analyzed using the
GraphPad Prism 5 software (GraphPad Software, San Diego, CA,
USA). Differences were considered significant if P <0.05.
Acknowledgments
We are grateful to the staff of Cyclotron Operation Section NIRS
for their technical assistance in radioisotope production, and the
staff of the Departmentof MolecularProbesNIRS for theirassistance.
5.8. Metabolite analysis in the brain and plasma of mice
[
18F]3 (17–29 MBq/0.12–0.21 nmol) or 18F]4 (9–36 MBq/
[
0.064–0.26 nmol) was intravenously co-injected with 1 (5 mg/kg)
into normal FVB mice (age, 9–13 weeks; weight, 24–31 g; n = 3–4
per group). Mice were sacrificed by cervical dislocation at 30 and
60 min after injection. Blood samples were collected into a hepa-
rinized syringe by heart puncture, and the whole brain was dis-
sected out. Deproteinization was performed using previously
described methods mentioned below.34 Plasma and homogenized
brain tissues were deproteinized with the same volume of ice-cold
acetonitrile. The mixture was then vortexed and centrifuged at
20,000g for 2 min, and the supernatant was collected. Supernatants
were analyzed by HPLC using a radioactivity detector35 and an
Supplementary data
Supplementary data (the purity of 3 and 4 was determined by
HPLC) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2010.12.004.
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