Development of 2-(2-(3-(4-([18F]Fluoromethoxy- d2)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione for Positron-Emission-Tomography Imaging of Phosphodiesterase 10A in the Brain

Article abstract of DOI:10.1021/acs.jmedchem.8b01366

Phosphodiesterase 10A (PDE10A) is a newly identified therapeutic target for central-nervous-system disorders. 2-(2-(3-(4-([¹⁸F]Fluoroethoxy)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([¹⁸F]MNI-659, [¹⁸F]5) is a useful positron-emission-tomography (PET) ligand for imaging of PDE10A in the human brain. However, the radiolabeled metabolite of [¹⁸F]5 can accumulate in the brain. In this study, using [¹⁸F]5 as a lead compound, we designed four new ¹⁸F-labeled ligands ([¹⁸F]6-9) to find one more suitable than [¹⁸F]5. Of these, 2-(2-(3-(4-([¹⁸F]fluoromethoxy-d₂)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([¹⁸F]9) exhibited high in vitro binding affinity (K_i = 2.9 nM) to PDE10A and suitable lipophilicity (log D = 2.2). In PET studies, the binding potential (BP_ND) of [¹⁸F]9 (5.8) to PDE10A in the striatum of rat brains was significantly higher than that of [¹⁸F]5 (4.6). Furthermore, metabolite analysis showed much lower levels of contamination with radiolabeled metabolites in the brains of rats given [¹⁸F]9 than in those given [¹⁸F]5. In conclusion, [¹⁸F]9 is a useful PET ligand for PDE10A imaging in brain.

Full text of DOI:10.1021/acs.jmedchem.8b01366

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Article
Development of 2-(2-(3-(4-([18F]Fluoromethoxy-d2)phenyl)-7-
methyl- 4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-
isopropoxyisoindoline-1,3-dione for Positron Emission
Tomography Imaging of Phosphodiesterase 10A (PDE10A) in Brain
Wakana Mori, Tomoteru Yamasaki, Masayuki Fujinaga, Masanao Ogawa,
Yiding Zhang, Akiko Hatori, Lin Xie, Katsushi Kumata, Hidekatsu Wakizaka,
Yusuke Kurihara, Takayuki Ohkubo, Nobuki Nengaki, and Ming-Rong Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01366 • Publication Date (Web): 05 Dec 2018
Downloaded from http://pubs.acs.org on December 6, 2018
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course of their duties.

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Development of 2-(2-(3-(4-([ F]Fluoromethoxy-d )phenyl)-7-methyl-4-
2
oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-
dione for Positron Emission Tomography Imaging of Phosphodiesterase
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0A (PDE10A) in Brain
†
,‖
†,‖
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†,‡
Wakana Mori, Tomoteru Yamasaki, Masayuki Fujinaga, Masanao Ogawa, Yiding
†
†
†
†
§
Zhang, Akiko Hatori, Lin Xie, Katsushi Kumata, Hidekatsu Wakizaka, Yusuke
†
,‡
†,‡
†,‡
†,
Kurihara, Takayuki Ohkubo, Nobuki Nengaki, and Ming-Rong Zhang *
†Department of Radiopharmaceuticals Development, National Institute of Radiological
Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-
9
-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan
‡SHI Accelerator Service, Ltd, 1-17-6 Osaki, Shinagawa-ku, Tokyo, 141-0032, Japan
§Department of Radiation Measurement and Dose Assessment, National Institute of
Radiological Sciences, National Institutes for Quantum and Radiological Science and
Technology, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan
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*
Corresponding author:
Ming-Rong Zhang, PhD
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Department of Radiopharmaceuticals Development, National Institute of Radiological
Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-
9
-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
Tel: +81 43 382 3709; Fax: +81 43 206 3261
E-mail: zhang.ming-rong@qst.go.jp
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ABSTRACT
Phosphodiesterase 10A (PDE10A) is a newly identified therapeutic target for central
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nervous
dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione
[18
F]5) is a useful positron emission tomography (PET) ligand for imaging of PDE10A
in the human brain. However, the radiolabeled metabolite of [ F]5 can accumulate in the
system
disorders.
2-(2-(3-(4-([ F]Fluoroethoxy)phenyl)-4-oxo-3,4-
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([ F]MNI-659,
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brain. In this study, using [ F]5 as a lead compound, we designed four new F-labeled
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ligands ([ F]6–9) to find one more suitable than [ F]5. Of these, 2-(2-(3-(4-
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(
[ F]fluoromethoxy-d )phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-
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isopropoxyisoindoline-1,3-dione ([ F]9) exhibited high in vitro binding affinity (K = 2.9
i
nM) to PDE10A and suitable lipophilicity (LogD = 2.2). In PET studies, the binding
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potential (BP ) of [ F]9 (5.8) to PDE10A in rat brains was significantly higher than that
ND
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of [ F]5 (4.6). Furthermore, metabolite analysis showed much lower levels of
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contamination with radiolabeled metabolites of [ F]9 in the brain than those of [ F]5. In
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conclusion, [ F]9 is a useful PET ligand for PDE10A imaging in brain.
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INTRODUCTION
Cyclic nucleotide signaling regulates a wide variety of cellular functions.
Phosphodiesterases (PDEs) act in every known neuronal population by catalyzing the
breakdown of 3’,5’-cyclic adenosine monophosphate or 3’,5’-cyclic guanosine
monophosphate (cGMP) and are characterized into 12 superfamilies according to
enzymatic characteristics, distribution, and pharmacological profile.1–4 Among these,
PDE10A is newly identified and contains two amino-terminal domains that are similar to
the cGMP-binding domains of PDE2, PDE5, and PDE6 and that are highly expressed in
medium spiny neurons in the striatum.1,5,6 Because of the localization and regulatory
functions of PDE10A, therapeutic studies have been conducted to investigate the effects
of PDE10A inhibitors on multiple central nervous system disorders, such as
schizophrenia, Huntington’s disease, and Parkinson’s disease (PD).7–11 Moreover, the
biological importance of PDE10A expression in PD progression and severity has been
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reported. Despite the behavioral and biological studies that have been conducted, direct
evidence on the interaction between PDE10A functions and multiple neurodegenerative
and neuropsychiatric diseases is scarce.
Positron emission tomography (PET) imaging with radioligands specific to
targeted molecules is frequently used to visualize in vivo brain activity related to the
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targeted molecules and provide detailed in vivo information about their relationship with
brain functions and diseases. Since 2010, some research groups have developed carbon-
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1 ( C)- or fluorine-18 ( F)-labeled radioligands for PDE10A in the brain, some of
which have shown some promising results in preclinical PET imaging studies.13–29 To the
best of our knowledge, the following five PET ligands have been used for visualization
of PDE10A in the human brain to date: 1-[2-fluoro-4-(tetrahydro-2H-pyran-4-yl)phenyl]-
1
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11
5
-[ C]methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one
([ C]T-773,
[11
C]1),30
5,8-dimethyl-2-[2-((1-[ C]methyl)-4-phenyl-1H-imidazol-2-yl)-ethyl]-
11
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31
[
1,2,4]triazolo[1,5-a]pyridine ([ C]Lu-AE92686, [ C]2), ((R)-5-(3-fluoropyrrolidin-
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-yl)-2-(3-methylquinoxalin-2-yl)-N-[ C]methyl-N-(tetrahydro-2H-pyran-4-
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12,32–35
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yl)pyrazolo[1,5-a]pyrimidin-7-amine) ([ C]IMA107, [ C]3),
2-[[4-[1-(2- F-
fluoroethyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]phenoxy]methyl]-3,5-dimethylpyridine
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36,37
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(
[ F]JNJ42259152, [ F]4),
and 2-(2-(3-(4-([ F]fluoroethoxy)phenyl)-4-oxo-3,4-
1
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dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione
([ F]MNI-659,
[18
F]5) (Figure 1).19,38–41
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Figure 1. PET radioligands for clinical PDE10A imaging.
In general, 18F-labeled ligands give higher quality PET images with higher
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spatial resolution than C-ligands. Moreover, F is convenient for multi-scans, storage
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and transportation to other facilities. Among the PDE10A radioligands, [ F]4 and [ F]5
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are more convenient for PET measurement than [ C]1–3. Compared to [ F]4, [ F]5 is
frequently used for PDE10A imaging in human studies because of its promising results
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of first-in-human study. PET with [ F]5 showed high in vivo availability (BP ) in the
ND
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human brain. However, PET quantitative assessments with [ F]5 have shown varying
results. For example, the coefficient of variation (CV) in measurements of the binding
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potential (BP ) of [ F]5 with the Logan graphical method was higher than 30% in non-
ND
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human primate brains. Further, it was reported that some PET ligands containing a 2-
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[18
F]fluoroethoxy group are easily metabolized via the Cytochrome P450
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monooxygenase system in the liver, resulting in [ F]fluoroacetaldehyde.
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Subsequently, [ F]fluoroacetaldehyde is rapidly oxidized to [ F]fluoroacetate, which is
easily accumulated in brain and has even been used as an imaging tool for detection of
cerebral ischemia.45,46 Thus, high variation in quantitative PET measurements of [ F]5
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may be caused by contamination with [ F]fluoroacetate in the brain.
In this study, we aimed to develop a more suitable PET ligand for quantification
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of PDE10A concentrations than [ F]5. Using 5 as a lead compound, we designed four
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new fluoroalkyloxy candidates, 6–9, and subsequently radiolabeled them with F (Figure
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). In place of the fluoroethoxy group in [ F]5, a 3-fluoropropoxy, (3-fluoro-2-
hydroxy)propoxy, fluoromethoxy, or fluoromethoxy-d group was introduced to generate
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the new radioligands [ F]6–9. Introduction of [ F]fluoroalkyloxy groups may reduce
contamination of the brain with radiolabeled metabolites by avoiding the production of
[18
F]fluoroacetate.
Herein, we perform the chemical syntheses of 6–9, radiosyntheses of [ F]6–9,
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in vitro binding assays with PDE10A, and in vivo PET imaging studies of the four
radioligands. To validate their usefulness, we further conducted biodistribution studies
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and metabolite analyses of [ F]8 and [ F]9. Although [ F]5 has been used in clinical
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studies, detailed preclinical data including in vivo metabolite analysis in brain have not
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been reported. We report the results of a preclinical evaluation of [ F]5 in rodents for the
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first time and compare the results with those of [ F]6–9.
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Figure 2. Chemical structures of compounds 6–9 and PET ligands [ F]6–9 in this study.
RESULTS AND DISCUSSION
Chemistry
The novel fluoroalkyloxy analogs 6–9 were synthesized using the same desmethyl phenol
precursor:
2-(2-(3-(4-hydroxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-ꢀ
4
7
isopropoxyisoindoline-1,3-dione (10) (Scheme 1, Supplemental Information Table S1
and Figure S1). The reactions of 10 with fluoropropyl tosylate (11) and fluoromethyl
tosylate (13) in the presence of K CO at 70° C produced 6 and 8 with 52% and 60%
2
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yields, respectively. The cycle-opening reaction of epifluorohydrin 12 with 10 and K CO
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Scheme 1. Chemical synthesis of compounds 6–9a
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a
Reagents and conditions: (a) K CO , DMF, 70° C, 11 h; (b) K CO , DMF, 80° C, 18
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h; (c) K CO , DMF, 70° C, 18 h; (d) silver p-toluenesulfonate, CH CN, reflux, 18 h;
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(
e) TBAF, CH CN, reflux, 12 h; (f) K CO , DMF, 70° C, 13 h.
3 2 3
at 80° C for 18 h produced 7, which contains the (3-fluoro-2-hydroxy)propoxy group. To
prepare the fluoromethoxy-d analog 9, fluoromethyl-d tosylate (16) was synthesized by
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a unique route . Diiodomethane-d (14) was heated with silver tosylate in acetonitrile
2
(
CH CN) to generate bis(tosyloxy)methane-d (15), followed by treatment with
3
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tetrabutylammonium fluoride (TBAF) to yield 16. Similarly to 8, compound 9 was
synthesized by fluoromethylation of 10 with 16 and K CO ; this process had 24% yield.
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In vitro Profiles of PET Ligand Candidates for PDE10A
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The in vitro binding affinities of PET ligand candidates for PDE10A 6–9 were
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measured using a binding assay with [ F]5 in rat brain homogenates. These compounds
exhibited binding affinities (K ) for PDE10A on the single-digit nM order, with a range
i
of 2.6–8.1 nM (Table 1). Changing the fluoroethoxy group to the relatively large
fluoropropoxy group in 6 or 3-fluoro-2-hydroxypropoxy group in 7 slightly weakened
affinities for PDE10A. The K values of 8 and 9 with fluoromethoxy and fluoromethoxy-
i
d2 groups remained 2.6 and 2.9 nM, respectively, which were slightly stronger than that
of 5 (MNI-659: 3.0 nM). These results suggest that only relatively small moieties
substituted at the 4-position of the benzene ring in these analogs can fit into the PDE10A
domain’s binding site.
The values of lipophilicity (LogD) were measured by the shake flask method,49
1
8
18
as shown in Table 1. The LogD values of [ F]5–9 following radiolabeling with F were
2
.2–2.9. These values are in the range normally considered as suitable for PET ligands in
5
0
brain imaging. When the size of the fluoroalkyl group shrank, lipophilicity was also
reduced in this series of compounds.
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1
2
3
4
5
6
7
8
9
Table1. In vitro binding affinity and lipophilicity of compounds 6–9.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Lipophilicity
Ki (nM)a
Compounds
R
ꢀ
(LogD)a
4
.2
2.90
6
7
8
9
CH CH CH F
2
2
2
(
2.3–7.4)
(2.85–2.94)
8.1
2.83
CH CH(OH)CH F
2
2
(
3.3–15.3)
.6
1.5–4.6)
.9
(1.5–5.3)
.0
1.9–5.0)
(2.81–2.85)
2
2.29
CH2F
CD2F
(
(2.28–2.30)
2
2.15
(2.14–2.16)
3
2.48
5
(MNI-659)
CH CH F
2
2
(
ꢀ
(2.43–2.52)
aAssays were duplicated.
Because of these compounds’ promising in vitro profiles, we radiolabeled 6–9
18
with F and performed in vivo evaluation of PET imaging of PDE10A in rat brain.
Radiochemistry
1
8
Radiosyntheses of [ F]6–9 were performed using a homemade automated
5
1
synthesis system (Scheme 2). Our automated system included two steps: 1) preparation
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
agents52:
[18F]fluoropropyl
18
53
of
[ F]fluoroalkylating
bromide
([ F]17),
[18
[18
[18
F]epifluorohydrin ([ F]12),
18
54
[18F]fluoromethyl bromide ([ F]18),
18
48
and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18
55
F]fluoromethyl bromide-d ([ F]19) ; 2) reaction of phenol precursor 10 with the
2
18
F]fluoroalkylating agents. As the first step, the [ F]fluoroalkylating agents were
1
8
18
−
produced by [ F]fluorination of triflate 20, tosylate 21, or bromides 22 or 23 with [ F]F .
1
8
18
18
After the [ F]fluorination, [ F]17 (boiling point [bp]: 61° C) or [ F]12 (bp: 84° C) was
distilled from the reaction mixture and directly trapped into another reaction vessel for
1
8
18
18
the following [ F]fluoroalkylation. As for [ F]18 (bp: 15° C) or [ F]19 (bp: 15° C), the
1
8
crude [ F]fluorinating products were distilled from the reaction mixture, immediately
passed through three short silica Sep-Pak columns to delete volatile unreacted 22 or 23,
1
8
and trapped in another vessel for [ F]fluoroalkylation.
1
8
18
18
18
The reactions of 10 with [ F]17, [ F]12, [ F]18, or [ F]19 were heated at 90–
1
8
1
30° C for 5–20 min. Utilization of these distilled [ F]fluoroalkylating agents resulted in
clear reaction mixtures. Moreover, as compared with the conventional tosylate precursor
1
8
used for direct [ F]fluorination, only 1–2 mg of phenol 10 was sufficient for efficient
[18
F]fluoroalkylation. Under the automated synthesis system, [ F]6–9 were easily
18
separated from the reaction mixtures using semi-preparative reversed phase HPLC
1
8
(
Supplemental Information Figure S2). After HPLC separation and formulation, [ F]6
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6
7
8
9
1
8
(
0.24–0.35 GBq) or [ F]7 (0.10–0.13 GBq) was produced as an injectable solution with
4
% ± 1% (n = 3) and 2% (average, n = 2) radiochemical yields at the end of synthesis
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
−
(
EOS), starting from the cyclotron-produced [ F]F radioactivity of 7.4 GBq. In addition,
[18
F]8 (0.59–0.88 GBq) and [ F]9 (0.66–0.94 GBq) were produced with 7% ± 2% (n =
18
1
8
−
7
) and 7% ± 3% (n = 9) radiochemical yields at EOS, respectively, starting from [ F]F
radioactivity of 12.3–15.6 GBq. Because of the difference in electrophilicity between the
[18
F]fluoromethyl and [ F]fluoropropyl groups, [ F]18 and [ F]19 showed higher
18
18
18
1
8
18
reactivity with phenol 10 than [ F]17 and [ F]12 did. In the finally formulated product
1
8
solutions, the molar activities and radiochemical purities of [ F]6–9 were 200–400
GBq/mol and greater than 99% at EOS (Supplemental Information Figure S3).
Moreover, these products did not show radiolysis at room temperature for 120 min after
formulation, indicating their radiochemical stability within the duration of at least one
1
8
PET scan. All of the analytical results of [ F]6–9 were in compliance with our in-house
quality control/assurance specifications for radiopharmaceuticals produced in our facility
for preclinical and clinical uses.
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
a
Scheme 2. Radiosynthesis of [ F]6–9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aReagents and conditions: (a) o-dichlorobenzene, 130° C, 2 min; (b) NaOH, DMF,
20° C, 10 min; (c) o-dichlorobenzene, 130° C, 2 min; (d) NaOH, DMF, 130° C, 20
1
min; (e) CH CN, 100° C, 5 min; (f) NaOH, DMF, 90° C, 5 min; (g) CH CN, 100° C,
3
3
5
min; (h) NaOH, DMF, 90° C, 5 min.
Small-Animal PET Assessments
Figure 3 shows averaged PET/MRI images (A) and tissue time–activity curves
1
8
(
tTACs) (B) of [ F]5–9 in rat brains. It has been reported that PDE10A mRNA can be
heterogeneously detected in the brain.56,57 The highest expression of PDE10A has been
shown in the striatum. On the other hand, negligible PDE10A levels have been found in
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8
9
the mid-brain and pons.57
As shown in the PET images, [ F]6, [ F]8, and [ F]9 showed a similar
1
8
18
18
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
distribution of radioactivity to the PDE10A-specific [ F]5. Significant radioactivity was
observed in the striatum. No uptake was observed in other brain regions. The images
1
1
reflecting significant striatal uptake were similar to the PET rat brain image using [ C]1
(
Figure 1), a PDE10A-specific radioligand, as reported previously.58 These results
suggested that current PET ligands may have specific binding for striatal PDE10A but
not for other brain region’s PDE10A at low density.
1
8
18
Radioactive uptake in the striatum had the following rank order: [ F]8 ≈ [ F]9
1
8
18
18
>
[ F]5 > [ F]6 > [ F]7 (Figure 3A). The sequence of these radiologands’ striatal uptake
was constituent with that of their in vitro binding affinities to PDE10A. To our surprise,
1
8
the striatal uptake of [ F]7 was negligible (< 0.3 SUV). Although the in vitro binding
affinity of 7 with PDE10A was the weakest among the present radioligands, its affinity
(
K = 8.1 nM) is still considered relatively strong. These results suggest that brain uptake
i
1
8
of [ F]7 may be limited by blood-brain barrier, resulting in a very low initial uptake of
radioactivity.
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
Figure 3. Averaged PET/MRI images (A) and tTACs (B) of [ F]5–9. Horizontal PET
images were reconstructed by summation between 0 and 60 min. The tTACs were
obtained from ROIs located on the striatum (filled circles) and midbrain (open circles).
Radioactivity is expressed as standardized uptake value (SUV). St, striatum; Mb, mid-
brain.
In contrast, there were very low levels of radioactivity in the mid-brain during
the PET scans for each radioligand. Thus, noninvasive PET kinetic analyses for
estimation of ligand availability to PDE10A, represented as BPND in the current PET
studies, were performed using the tTAC of the midbrain as a reference region.
Table 2 shows the striatal kinetic parameters (R1 and BP ) based on the
ND
simplified reference tissue model (SRTM)59 using the tTAC of the mid-brain as a
reference region. Parameter R1 (= K /K ’, the ratio of tracer delivery) was 1.0 for all
1
1
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Page 17 of 52
Journal of Medicinal Chemistry
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5
6
7
8
9
1
8
radioligands except [ F]7, which indicated equal input functions (K ) in both the interest
1
(
striatum) and reference (midbrain) regions. The BPND values of the two
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[18
F]fluoromethoxy radioligands were 5.6 for [ F]8 and 5.8 for [ F]9. The values were
18
18
1
8
18
18
significantly higher (P < 0.01 for [ F]8 and [ F]9) than that of the lead compound [ F]5
(
4.6), although the difference between the three compounds’ in vitro affinities was small.
1
8
18
Because of the very low BPND values of [ F]6 and [ F]7 for visualizing
PDE10A, we ceased evaluation of those two radioligands in the following studies.
1
8
Table 2. In vivo availability (BP ) of [ F]5–9 to PDE10A (Mean ± s.d.)
ND
[18F]6 (n = 1)
18
[18F]8 (n = 3)
[18F]9 (n = 5)
[18F]5 (n = 4)
Parameters
R1
ꢀ [ F]7 (n = 1)
ꢀ
ꢀ
ꢀ
1.0
0.3
1.0
±
±
0.2
1.0
5.8
±
±
0.1
1.0
4.6
±
±
0.1
0.6
BPND
3.8
ꢀ
0.6
ꢀ 5.6
0.5**
ꢀ
0.2***
1
8
*
*P < 0.01, ***P < 0.001 (vs [ F]5)
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Page 18 of 52
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2
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
Figure 4. Averaged PET/MRI images and tTACs in chase-blocking study for [ F]9
1
8
(
A and B) and [ F]5 (C and D). Blocking agent (MNI-659 of 5 mg/kg) was
administrated 20 min after the injection of the radioligand (black arrows). Horizontal
PET images were reconstructed in summation of pre-administration (0-20 min) and
post-administration (30–60 min), respectively. The tTACs were obtained from ROIs
located on the striatum (filled circles) and mid-brain (open circles). Radioactivity is
expressed as SUV. St, striatum; Mb, mid-brain.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
To confirm specific binding to PDE10A, chase-blocking PET studies for [ F]9
1
8
and [ F]5 were performed. Figure 4 shows PET images and tTACs in the brains of rats
1
8
administrated with unlabeled 5 (MNI-659, 5 mg/kg) 20 min after the injection of [ F]9
1
8
(
A and B) and [ F]5 (C and B). In both PET images of the post-blocking, high radioactive
signals in the striatum were significantly washed out compared to the pre-blocking (Figs.
A and C). Radioactivity in the striatum was decreased to 0.1 SUV in both radioligands
4
by administration with the blocking agent. These results suggest that radioactive
accumulation in the striatum indicated specific binding of radioligand for PDE10A.
Biodistribution and Dosimetric Studies
1
8
18
To evaluate the potent availability of [ F]8 and [ F]9 to PDE10A, their
biodistribution throughout the whole body of mice and effective doses of radioactivity
1
8
were compared with those of [ F]5. Tables 3–5 show the radioactive concentrations
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Journal of Medicinal Chemistry
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2
3
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5
6
7
8
9
1
8
18
18
(
%ID/g) of [ F]8, [ F]9, and [ F]5 at various time points (5, 15, 30, 60, and 120 min)
in each tissue. These radioligands showed similar distribution patterns of radioactivity
throughout the whole body of mice. At 5 min after radioligand injection, radioactive
uptake was mainly distributed in the small intestine and liver, which reached roughly
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
0%ID/g. Subsequently, these organs’ radioactivities decreased with time after the initial
accumulation. Their relatively low renal uptake suggests that these radioligands are
eliminated by enterohepatic circulation. At 60 min, the radioactivity levels in all tissues
except the small intestine and stomach had decreased to <1%ID/g.
1
8
60
In general, low bone uptake is a requirement for useful F-labelled ligands.
Low uptake values (0.58–0.77%ID/g) in bone were determined at 5 min after radioligand
1
8
injection. The radioactive bone accumulation of [ F]5 decreased to a very low value
1
8
18
(
0.1%ID/g) until 120 min. Although the radioactive accumulations of [ F]8 and [ F]9 in
bone gradually increased with time, their bone uptakes maintained a low level of 1%ID/g
at 120 min. These results suggest that in vivo defluorination of the two fluoromethoxy
ligands was not significant.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
Table 3. Biodistribution (%ID/g tissue) of [ F]8 in mice (Mean ± s.d., n = 3).
Time after injection
30 min
0.19
Tissue
5
min
±
15 min
0.36
60 min
0.06
120 min
0.03 0.01
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Blood
Heart
0.88
1.01
1.24
11.01
1.12
0.77
2.19
0.71
49.50
1.03
0.40
0.59
0.56
0.62
0.32
0.35
0.43
1.01
0.31
0.14
0.47
0.38
11.14
0.28
0.10
0.27
0.06
0.12
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.04
0.02
0.04
0.28
0.03
0.04
0.17
5.85
9.87
0.07
0.01
0.22
0.04
0.01
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.03
0.03
0.04
0.27
0.01
0.05
0.10
2.11
5.48
0.04
0.04
0.01
0.05
0.25
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
0.02
0.02
0.04
0.02
0.01
0.02
6.16
4.13
0.29
0.11
0.07
0.03
0.21
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.37
0.44
4.83
0.42
0.28
0.87
5.45
38.25
0.60
0.24
0.37
0.32
0.58
0.20
0.22
1.45
0.18
0.17
0.48
4.09
16.71
0.29
0.15
0.21
0.21
0.91
0.07
0.08
0.70
0.08
0.05
0.16
8.53
21.21
0.37
0.14
0.10
0.15
0.87
0.03
0.05
0.54
0.03
0.02
0.08
2.08
7.47
0.05
0.04
0.04
0.09
1.06
0.00
0.02
0.36
0.00
0.00
0.01
0.45
3.12
0.02
0.01
0.01
0.01
0.13
Lung
±
Liver
±
Pancreas
Spleen
Kidney
Stomach
S. Intestine
L. Intestine
Testis
±
±
±
±
±
±
±
Muscle
Brain
±
±
Bone
±
1
8
Table 4. Biodistribution (%ID/g tissue) of [ F]9 in mice (Mean ± s.d., n = 3).
Time after injection
30 min
0.24
Tissue
5 min
1.12
15 min
0.38
60 min
120 min
0.04
Blood
Heart
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.14
0.17
0.31
1.75
0.28
0.19
0.82
2.00
9.69
0.54
0.15
0.15
0.15
0.20
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.05
0.12
0.09
0.90
0.08
0.09
0.25
3.60
6.83
0.08
0.05
0.04
0.06
0.20
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.07
0.07
0.09
0.22
0.06
0.02
0.22
0.71
1.36
0.02
0.01
0.07
0.02
0.06
0.11
0.12
0.13
1.03
0.14
0.09
0.31
2.37
9.21
0.31
0.10
0.10
0.18
0.92
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
0.02
0.02
0.16
0.00
0.01
0.04
1.38
0.28
0.14
0.03
0.02
0.01
0.07
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
0.00
0.01
0.29
0.03
0.01
0.01
1.52
4.63
1.07
0.02
0.01
0.01
0.03
1.24
1.60
13.02
1.50
1.00
3.03
2.06
46.20
1.46
0.60
1.01
0.73
0.77
0.52
0.60
4.53
0.49
0.35
1.23
3.08
37.00
0.46
0.31
0.30
0.37
0.76
0.28
0.33
1.92
0.28
0.20
0.78
1.16
19.63
0.22
0.20
0.31
0.27
0.88
0.04
0.05
0.89
0.06
0.03
0.10
1.98
10.18
0.99
0.04
0.05
0.11
1.06
Lung
Liver
Pancreas
Spleen
Kidney
Stomach
S. Intestine
L. Intestine
Testis
Muscle
Brain
Bone
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1
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Table 5. Biodistribution (%ID/g tissue) of [ F]5 in mice (Mean ± s.d., n = 3).
Time after injection
Tissue
5
min
±
15 min
30 min
60 min
120 min
0.14
Blood
Heart
1.10
1.49
1.55
13.33
1.74
0.97
2.73
2.60
43.83
1.08
0.46
0.95
0.54
0.58
0.18
0.21
0.30
0.14
0.22
0.04
0.42
2.71
31.86
0.25
0.05
0.16
0.05
0.09
0.33
0.48
0.51
5.45
0.55
0.35
1.08
2.69
46.06
0.66
0.30
0.41
0.29
0.19
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.03
0.05
0.04
1.49
0.04
0.01
0.08
2.28
12.57
0.39
0.06
0.15
0.02
0.03
0.21
0.31
0.33
2.20
0.35
0.20
0.62
5.18
18.45
0.28
0.21
0.32
0.26
0.15
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.02
0.04
0.03
0.22
0.02
0.03
0.12
3.77
10.43
0.11
0.08
0.12
0.08
0.02
0.07
0.08
0.08
0.57
0.08
0.06
0.13
1.11
5.65
0.18
0.10
0.08
0.13
0.07
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.01
0.01
0.01
0.06
0.01
0.01
0.02
0.50
2.22
0.06
0.04
0.02
0.02
0.01
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.15
0.01
0.01
0.08
0.02
0.01
0.01
1.19
4.46
0.31
0.03
0.01
0.01
0.00
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
±
0.06
0.06
0.86
0.05
0.04
0.09
3.03
10.60
0.73
0.06
0.04
0.08
0.11
Lung
±
Liver
±
Pancreas
Spleen
Kidney
Stomach
S. Intestine
L. Intestine
Testis
±
±
±
±
±
±
±
Muscle
Brain
±
±
Bone
±
To estimate the effective dose for standard human, we subsequently performed
whole body dosimetry analyses using the biodistribution data from mice. The effective
1
8
18
doses (mSv/MBq) in standard human were 0.013 for [ F]8, 0.013 for [ F]9, and 0.014
1
8
for [ F]5 (Supporting Information Table S2). The values were comparable to that
1
8
61
reported for [ F]FMeNER-d (0.017 mSv/MBq), a clinically used PET ligand for brain
2
1
8
18
imaging. Although some defluorination of [ F]8 and [ F]9 was observed, their bone
uptake was low (≈ 1%ID/g at 120 min after the injection). In fact, there was no difference
1
8
18
18
in the effective dose among [ F]8, [ F]9, and [ F]5.
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9
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2
2
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3
3
3
3
4
4
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Metabolite Analysis in the Plasma and Brain
1
8
18
Figure 5 shows the percentages of the unchanged forms of [ F]8, [ F]9, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[18
F]5 in rat plasma (A) and brain (B). The respective fractions corresponding to the
unchanged forms in plasma decreased from 25%–37% at 20 min to 21%–24% at 60 min
after injection. Two polar radiolabeled metabolites [retention time (t ) = 1.5–2.5 min]
R
were observed for each radioligand on the plasma samples’ HPLC charts (Supporting
1
8
Information Figure S4). At 60 min after injection, the unchanged forms of [ F]8 and
[18
[18
F]9 had retained 96% of total radioactivity in the brain, while the unchanged form of
F]5 had retained 83% of total radioactivity in the brain. A certain level of the
1
8
18
radiolabeled metabolite of [ F]5 in the brain was assumed to be [ F]fluoroacetate, which
1
8
18
might affect the quantitative analysis of PDE10A in brain. In contrast, [ F]8 and [ F]9
showed low levels of contamination by their radioactive metabolites in the brain,
suggesting that their polar metabolites in plasma did not enter the brain easily and could
1
8
not be converted to brain-permeable metabolites, such as [ F]fluoroacetate. The
extraction efficiency (Supporting Information Table S2) of metabolite analyses with
[18
F]8, [ F]9, or [ F]5 was obtained with over 84% in the brain at each time point, which
18
18
strongly supported accuracy for this assessment.
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8
18
The results of metabolite analyses indicate that [ F]8 and [ F]9 showed
1
8
18
improved metabolism over [ F]5 in the brain. Considering that PET with [ F]9 produced
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
the highest BPND values among the newly developed radioligands and [ F]5, we decide
1
8
to proceed with [ F]9 for the further evaluations.
18
18
18
Figure 5. Percentages of unchanged forms of [ F]8, [ F]9, and [ F]5 in the
1
8
plasma (A) and brain (B). **P < 0.01 (vs [ F]5)
CONCLUSION
In the present study, using [ F]5 as a lead compound, we developed four new PET
1
8
1
8
radioligands [ F]6–9 for imaging of PDE10A and evaluated their in vitro and in vivo
1
8
profiles using rodents. Of these PET ligands, two fluoromethoxy analogs ([ F]8 and
[18
F]9) showed potent affinity for PDE10A and adequate lipophilicity. Furthermore, we
found the highest in vivo availability (BP ) to PDE10A in the PET study with the
ND
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
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4
4
4
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5
5
5
5
5
6
[18
F]fluoromethoxy-d ligand [ F]9, which showed low levels of contamination with its
18
2
1
8
radiolabeled metabolites in the brain. Thus, [ F]9 may be a reasonable PET ligand for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
clinical studies of PDE10A imaging in human brains. We are planning to translate this
radioligand for its first human study and then compare in vivo performance between
[18
F]5 and [ F]9 using head-to-head PET studies.
18
EXPERIMENTAL SECTION
All chemical reagents and organic solvents were purchased from Sigma-Aldrich (St.
Louis, MO, USA), FUJIFILM Wako Pure Chem. (Osaka, Japan), or Nacalai Tesque
1
8
(
Kyoto, Japan) and used without further purification. F was produced using a cyclotron
(
CYPRIS HM-18; Sumitomo Heavy Industries, Tokyo, Japan). Melting points (mp) were
measured using a micro melting point apparatus (MP-500P, Yanaco; Tokyo, Japan) and
1
were uncorrected. H NMR (300 MHz) spectra were recorded on a JEOL-AL-300
spectrometer (JEOL, Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard.
All chemical shifts (δ) are reported as ppm downfield relative to the TMS signal. Signals
are quoted as s (singlet), d (doublet), t (triplet), br (broad), or m (multiplet). High-
resolution fast atom bombardment mass spectra (HRMS) were acquired using a JEOL
NMS-SX102 102A spectrometer. Silica gel column chromatography was performed
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5
6
7
8
9
using Wakosil C-200 (FUJIFILM Wako Pure Chem.). HPLC separation and analysis
were performed using a JASCO HPLC system (JASCO, Tokyo, Japan). All semi-
preparative HPLC separations were performed using Capcell Pak C columns (10 mm ×
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
2
50 mm, Shiseido, Tokyo, Japan). All HPLC analyses were performed by using Capcell
Pak C columns (4.6 mm i.d. × 250 mm, Shiseido) to determine the chemical purities
1
8
(>98%) of 6–9. The analytic conditions were as follows: 6: 1.0 mL/min, CH CN/H O
3 2
(
75/25, v/v); 7: 1.0 mL/min, CH CN/H O/Et N (55/45/0.1, v/v/v); 8: 1.0 mL/min,
3
2
3
CH CN/H O (65/35, v/v); 9: 1.0 mL/min, CH CN/H O (65/35, v/v). The radiochemical
3
2
3
2
1
8
purities of [ F]6–9 were analyzed by the same HPLC and detector to monitor
1
8
radioactivity under the following conditions: [ F]6: 1.0 mL/min, CH CN/H O (75/25,
3
2
1
8
18
v/v); [ F]7: 1.0 mL/min, CH CN/H O/Et N (55/45/0.1,v/v/v); [ F]8: 1.0 mL/min,
3
2
3
1
8
CH CN/H O (65/35, v/v); [ F]9: 1.0 mL/min, CH CN/H O (65/35, v/v). The authentic
3
2
3
2
sample of 5 (MNI-659) was synthesized by reaction of 10 with 2-fluoroethyl-4-
1
8
methylbenzenesulfonate. For the in vitro binding assay and preclinical evaluation, [ F]5
1
8
was synthesized by a reaction of 10 with [ F]fluoroethyl bromide in house, as reported
previously.47
Chemical Synthesis
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
-(2-(3-(4-(3-Fluoropropoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-
yl)ethyl)-4-isopropoxyisoindoline-1,3-dione (6). A mixture of 1047 (150 mg, 0.31
mmol), 11 (79 mg, 0.34 mmol), and K CO (64 mg, 0.46 mmol) in DMF (3 mL) was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
heated at 70° C for 11 h. After cooling, the reaction mixture was extracted with CH Cl ,
2
2
and the organic layer was washed with aqueous saturated K CO solution, dried with
2
3
Na SO , and evaporated under reduced pressure. Column chromatographic separation of
2
4
the residue using hexane/ethyl acetate (AcOEt) (4/1, v/v) produced 6 (132 mg, 78%) as a
1
white solid. mp: 161−163° C. H−NMR (CDCl ): δ 1.38 (6H, d, J = 6.3 Hz), 2.13–2.17
3
(
1H, m), 2.21–2.26 (1H, m), 2.46 (3H, s), 2.73 (2H, t, J = 6.9, 7.8 Hz), 4.07 (2H, t, J =
.2, 7.2 Hz), 4.14 (2H, t, J = 6.0, 6.0 Hz), 4.58 (1H, t, J = 5.7, 5.7 Hz), 4.66–4.76 (2H,
m), 7.01 (2H, d, J = 9.3 Hz), 7.14–7.22 (4H, m), 7.33 (1H, s), 7.36 (1H, d, J = 7.5 Hz),
.58 (1H, t, J = 7.2, 8.4 Hz), 8.12 (1H, d, J = 8.1 Hz). HRMS m/z: 544.4276 (calculated
for C H O N F: 544.2248).
7
7
3
1
30
5
3
2
-(2-(3-(4-(3-Fluoro-2-hydroxypropoxy)phenyl)-7-methyl-4-oxo-3,4-dihydro-
quinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione (7). A mixture of 10 (150
mg, 0.31 mmol), 12 (26 mg, 0.34 mmol), and K CO (64 mg, 0.46 mmol) in DMF (3 mL)
2
3
was heated at 80° C for 18 h. After cooling, the reaction mixture was extracted with
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2
3
4
5
6
7
8
9
AcOEt and the organic layer was washed with brine, dried with Na SO , and evaporated
2
4
under reduced pressure. Column chromatographic separation of the residue using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hexane/AcOEt (1/1, v/v) produced 7 (52 mg, 30%) as a white solid. mp: 104–106° C.
1
H−NMR (DMSO-d ): δ 1.26 (6H, d, J = 6.0 Hz), 2.41 (3H, s), 2.66 (2H, t, 2H, J = 7.2,
6
7
4
.2 Hz), 3.85 (2H, t, 2H, J = 7.5, 6.9 Hz), 4.03–4.11 (3H, m), 4.39–4.47 (1H, m), 4.54–
.63 (1H, m), 4.73–4.81 (1H, m), 5.49 (1H, d, J = 4.8 Hz), 7.10 (2H, d, J = 8.4 Hz), 7.24
(1H, s), 7.30–7.44 (4H, m), 7.70 (1H, t, J = 7.8, 7.8 HZ), 7.96 (2H, d, J = 7.5 Hz). HRMS
m/z: 560.2162 (calculated for C H O N F: 560.2197).
3
1
31
6
3
2
-(2-(3-(4-(Fluoromethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-
yl)ethyl)-4-isopropoxyisoindoline-1,3-dione (8). A mixture of 10 (80 mg, 0.17 mmol),
1
3 (37 mg, 0.18 mmol), and K CO (34 mg, 0.25 mmol) in DMF (1.5 mL) was heated at
2 3
7
0° C for 18 h. After cooling, the reaction mixture was extracted with CH Cl , and the
2 2
organic layer was washed with aqueous saturated K CO solution, dried with Na SO ,
2
3
2
4
and evaporated under reduced pressure. Column chromatographic separation of the
residue using hexane/AcOEt (3/2, v/v) produced 8 (23 mg, 27%) as a white solid. mp:
1
1
7
90–192° C. H−NMR (CDCl ): δ 1.38 (6H, d, J = 6.3 Hz), 2.47 (3H, s), 2.73 (2H, t, J =
3
.8, 6.9 Hz), 4.06 (2H, t, J = 7.2, 7.5 Hz), 4.66–4.75 (1H, m), 5.68 (1H, s), 5.86 (1H, s),
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.17–7.37 (8H, m), 7.60 (1H, t, J=8.1, 7.8 Hz), 8.11 (1H, d, J = 8.1 Hz). HRMS m/z:
5
16.1937 (calculated for C H O N F: 516.1935).
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-(2-(3-(4-(Fluoromethoxy-d )phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-
2
yl)ethyl)-4-isopropoxyisoindoline-1,3-dione (9). A mixture of 10 (50 mg, 0.10 mmol),
1
6 (40 mg, 0.19 mmol), and K CO (21 mg, 0.16 mmol) in DMF (2 mL) was heated at
2 3
7
0° C for 13 h. After cooling, the reaction mixture was extracted with CH Cl , and the
2 2
organic layer was washed with aqueous saturated K CO solution, dried with Na SO ,
2
3
2
4
and evaporated under reduced pressure. Column chromatographic separation of the
residue using hexane/AcOEt (3/1, v/v) gave crude 9, which was further purified using the
semi-preparative HPLC column (CH CN/H O = 65/35, 10 mL/min) to produce 9 (13.1
3
2
1
mg, 24%) as a white solid. mp: 193–195° C. H−NMR (CDCl ): δ 1.38 (6H, d, J = 6.0
3
Hz), 2.47 (3H, s), 2.65 (2H, t, J = 7.8, 6.9 Hz), 4.05 (2H, t, J = 7.2, 7.5 Hz), 4.68–4.72
(1H, m), 7.13–7.38 (8H, m), 7.57–7.62 (1H, m), 8.10 (1H, d, J = 7.5 Hz). HRMS m/z:
5
18.2016 (calculated for C H D O N F: 516.1935).
2
9
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2
5
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Methylene-d -bis(4-methylbenzenesulfonate) (15). A mixture of 14 (270 mg, 1 mmol)
2
and silver p-toluenesulfonate (607 mg, 2.2 mmol) in CH CN (2 mL) was heated at reflux
3
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Journal of Medicinal Chemistry
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for 18 h. After cooling, the solvent was removed at reduced pressure. The residue was
dissolved in warmed CH Cl and filtered to remove the unreacted silver p-
2
2
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toluenesulfonate. The organic solvent was removed under reduced pressure, affording a
crude product, which was recrystallized from ethanol to yield 15 (235 mg, 65%) as a
1
white solid. mp: 109–111° C. H−NMR (CDCl ): δ 2.45 (6H, s), 7.24 (4H, d, J = 8.7 Hz),
3
7
.59 (4H, d, J = 8.1 Hz).
Fluoromethyl-d -4-methylbenzenesulfonate (16). A solution of 15 (228 mg, 0.64
2
mmol) in CH CN (3 mL) was added to a solution of TBAF in THF (1 M, 1 mL, 1 mmol).
3
Subsequently, the reaction mixture was heated at reflux for 26 h. The organic solvents
were removed under reduced pressure, and the residue was extracted with AcOEt and
water. The organic layer was washed with brine, dried over MgSO , and removed under
4
reduced pressure. Column chromatographic separation of the residue using
1
hexane/AcOEt (6/1, v/v) produced 12 (42 mg, 31%) as a colorless oil. H−NMR (300
MHz, CDCl ): 2.46 (3H, s), 7.36 (2H, d, J = 8.4 Hz), 7.83 (2H, d, J = 8.4 Hz).
3
Radiochemistry
1
8
2
-(2-(3-(4-(3-[ F]Fluoropropoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-
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