Synthesis and characterization of novel radiofluorinated probes for positron emission tomography imaging of monoamine oxidase B

Article abstract of DOI:10.1002/jlcr.3779

Monoamine oxidase B (MAO-B), predominantly expressed in glial cells, plays an important role in neurotransmitter regulation, and MAO-B activity relates to several neuronal diseases. Here, we aimed to develop a radiofluorinated MAO-B imaging probe based on the structure of a selective MAO-B inhibitor, MD-230254. We synthesized and evaluated a series of compounds in vitro and in vivo. A series of fluorinated analogs of MD-230254 were synthesized and evaluated for inhibitory potency and selectivity toward MAO-B. 5-[4-(2-[¹⁸F]Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (2-[¹⁸F]FBPO) was synthesized from a corresponding tributylstannyl precursor and [¹⁸F]CH₃COOF. Biodistribution after intravenous injection of 2-[¹⁸F]FBPO was evaluated in male ddY mice with or without pretreatment by inhibitors. Among the compounds synthesized and evaluated, 2-FBPO showed high inhibitory potency and selectivity toward MAO-B comparable with MD-230254. 2-[¹⁸F]FBPO was successfully synthesized by an electrophilic reaction with a high radiochemical purity of more than 99%. 2-[¹⁸F]FBPO was efficiently taken up by the brain and showed rapid blood clearance, which provided a brain/blood radioactivity ratio of 3.7 at 90 minutes postinjection. The brain radioactivity was significantly decreased by pretreatment with an MAO-B selective inhibitor. The great potential of 2-[¹⁸F]FBPO as an MAO-B imaging probe, applicable to a variety of diseases, is indicated.

Full text of DOI:10.1002/jlcr.3779

Temma Takashi (Orcid ID: 0000-0002-7345-6004)
Synthesis and characterization of novel radiofluorinated probes
for Positron Emission Tomography imaging of monoamine oxidase B
Mitsuyoshi Yoshimoto, PhD1,2)*; Masahiko Hirata, PhD1)*; Shinya Kagawa, PhD1,3)
Yasuhiro Magata, PhD4,5); Yoshiro Ohmomo, PhD1)†; Takashi Temma, PhD1)†
;
1) Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-
1094, Japan
2) Division of Functional Imaging, Exploratory Oncology Research & Clinical Trial Center,
National Cancer Center, Kashiwa, Chiba, Japan
3) Division of PET Imaging, Shiga Medical Center Research Institute, 5-4-30 Moriyama,
Moriyama, Shiga 524-8524, Japan
4) Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine,
Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
5) Department of Molecular Imaging, Institute for Medical Photonics Research,
Preeminent
Medial Photonics Education and Research Center, Hamamatsu University School of
Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
* M.Y. and M.H. contributed equally to this manuscript.
†Corresponding author
Address correspondence to:
Takashi Temma, PhD
Professor of Department of Biofunctional Analysis, Osaka University of Pharmaceutical
Sciences; 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Tel.: +81-72-690-1040, Fax: +81-72-690-1041
E-mail: ttemma@gly.oups.ac.jp
Yoshiro Ohmomo, PhD
Professor of Education and Research Center for Pharmaceutical Sciences, Osaka University
of Pharmaceutical Sciences; 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Tel.: +81-72-690-1274, Fax: +81-72-690-1027
E-mail: ohmomo@gly.oups.ac.jp
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Short title: 70 characters
Fluorinated probe for MAO-B PET
Type of article:
Research article
Financial support:
This work was partly supported by a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (08770752). There are no conflicts of interest.
Abstract
Objective
Monoamine oxidase B (MAO-B), predominantly expressed in glial cells, plays an important
role in neurotransmitter regulation, and MAO-B activity relates to several neuronal diseases.
Here, we aimed to develop a radiofluorinated MAO-B imaging probe based on the structure of
a selective MAO-B inhibitor, MD-230254. We synthesized and evaluated a series of
compounds in vitro and in vivo.
Methods
A series of fluorinated analogues of MD-230254 were synthesized and evaluated for
inhibitory potency and selectivity toward MAO-B. 5-[4-(2-[18F]Fluorobenzyloxy)phenyl]-3-
(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (2-[18F]FBPO) was synthesized from
a
corresponding tributylstannyl precursor and [18F]CH3COOF. Biodistribution after intravenous
injection of 2-[18F]FBPO was evaluated in male ddY mice with or without pretreatment by
inhibitors.
Results
Among the compounds synthesized and evaluated, 2-FBPO showed high inhibitory potency
and selectivity toward MAO-B comparable to MD-230254. 2-[18F]FBPO was successfully
synthesized by an electrophilic reaction with a high radiochemical purity of more than 99%. 2-
[18F]FBPO was efficiently taken up by the brain and showed rapid blood clearance, which
provided a brain/blood radioactivity ratio of 3.7 at 90 min post injection. The brain radioactivity
was significantly decreased by pretreatment with an MAO-B selective inhibitor.
Conclusions
The great potential of 2-[18F]FBPO as an MAO-B imaging probe, applicable to a variety of
diseases, is indicated.
Key words: 10 words
Monoamine oxidase; MAO; MAO-B; Positron Emission Tomography; PET; SPECT; MD-
230254
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Introduction
Monoamine oxidase (MAO), a flavin-containing enzyme predominantly expressed in glial
cells (1), catalyzes an oxidative deamination reaction of endogenous and exogenous amines (2-
4). MAO plays an important role in neurotransmitter regulation, which leads to the association
of MAO with several neuronal diseases such as Parkinson’s disease, depression, and certain
psychiatric disorders (5-9). MAO has two subtypes, MAO-A and MAO-B. The activity of
MAO-B, in particular, increases with normal aging and the above-mentioned diseases (10, 11),
which suggests that MAO-B could be a potential biomarker of disease.
In a previous study, we developed a radioiodinated ligand, 2-[125I]IBPO, as a potential
MAO-B probe for Single Photon Emission Computed Tomography (SPECT) imaging, that was
based on the structure of a selective MAO-B inhibitor, MD-230254 (12) (Figure 1). 2-IBPO
showed high inhibitory potency and selectivity for MAO-B and radioiodination was achieved
through an iododestannylation reaction from a corresponding tin precursor. 2-[125I]IBPO was
rapidly delivered into the mouse brain with the highest brain to blood radioactivity ratio
obtained at 15-30 min post iv injection, which demonstrated the high potential of 2-[125I]IBPO
as a SPECT probe for MAO-B imaging. In addition, 2-[125I]IBPO was eligible for
derivatization toward a radiofluorinated probe for Positron Emission Tomography (PET)
imaging of MAO-B because of the rapid brain uptake profile. Although [11C]L-deprenyl-D2
has been widely used as a PET probe for MAO-B imaging in clinical settings (13-16), the
development of an 18F-labeled probe with a longer half-life would be beneficial to increase the
availability of MAO-B imaging for patients. Recently, several 18F-labeled probes based on the
structure of L-deprenyl and rasagiline were imaged successfully in vivo in cynomolgus monkey
brains (17, 18). Thus, in this study we aimed to develop a novel radiofluorinated probe based
on the findings of 2-[125I]IBPO with the higher MAO-B selectivity of 2-IBPO and MD-230254
in comparison to L-deprenyl (12) for PET imaging of such MAO-B associated brain diseases.
We synthesized a series of fluorinated compounds and evaluated the performance in vitro and
in vivo.
Experimental
Reagents and instruments
The melting points were determined on a Yanagimoto micro-melting point apparatus and
were uncorrected. The infrared (IR) spectra were taken on a JASCO IR-700 spectrometer. The
1H-NMR spectra were recorded on a Varian Gemini-200 (200 MHz) spectrometer, and the
chemical shifts were recorded in ppm downfield from an internal tetramethylsilane standard.
The high-resolution mass spectra (HRMS) were obtained on a Hitachi M-80 instrument. The
high performance liquid chromatography (HPLC) system included a Waters M-600 pump, a
Lambada-Max 481 ultraviolet detector, a Beckman 170 NaI radioactivity detector, and a
Cosmosil 5C18-AR column (10 x 250 mm, Nacalai Tesque). The radioactivity was measured
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using an Aloka ARC-300 NaI (Tl) gamma counter. All chemicals used were of reagent grade
and were used without purification.
Male ddY mice and male Wistar rats were obtained from Japan SLC (Shizuoka, Japan) and
were housed for at least one week with free access to food and water before the experiments.
The animal experiments in this study were conducted in accordance with the animal experiment
guidelines of the Osaka University of Pharmaceutical Sciences.
Synthesis (Scheme 1)
Substituted ethyl 4-(benzyloxy)benzoate (2a-c)
A solution of ethyl 4-hydroxybenzoate (0.10 mol) in dry N,N-dimethylformamide (DMF)
(40 ml) was added dropwise to a stirred and cooled suspension of sodium hydride (2.4 g, 0.10
mol) in DMF (20 ml). The stirred mixture was allowed to stand for 30 min at room temperature.
Then, a solution of benzyl bromide derivatives in dry DMF (10 ml) was added dropwise. The
reaction mixture was stirred for 5 h. The solvent was evaporated and the crude ester was washed
with 100 ml ice water and then filtered, dried, and recrystallized from EtOH.
Ethyl 4-(2-fluorobenzyloxy)benzoate (2a): Yield 33.0%, mp 48-50oC, IR (KBr): 1697, 1508,
1
1109, 875, 633 cm-1. H-NMR (DMSO) δ: 1.38(3H, t, CH2CH3), 4.35(2H, q, CH2CH3),
5.18(2H, s, CH2O), 7.00, 7.23, 7.75, and 8.01(each 2H, d. q. t. d., aromatics). Anal. Calcd for
C16H15FO3: C, 70.06; H, 5.51. Found: C, 70.06; H, 5.49. HRMS calcd for m/z: 274.1005.
Found: 274.1008.
Ethyl 4-(3-fluorobenzyloxy)benzoate (2b): Yield 50.4%, mp 69-70oC, IR (KBr): 1679, 1513,
1118, 871, 634 cm-1. 1H-NMR (DMSO) δ: 1.31 (3H, t, CH2CH3), 4.30 (2H, q, CH2CH3), 5.23
(2H, s, CH2O), 7.09, 7.16, 7.53, and 7.86 (each 2H, d. t. d. dd.，aromatics). Anal. Calcd for
C16H15FO3: C, 70.06; H, 5.51. Found: C, 70.08; H, 5.48. HRMS calcd for m/z: 274.1005.
Found: 274.1001.
Ethyl 4-(4-fluorobenzyloxy)benzoate (2c): Yield 33.0%, mp 60-62oC, IR (KBr): 1700, 1510,
1121, 864, 638 cm-1. 1H-NMR (DMSO) δ: 1.36 (3H, t, CH2CH3), 4.27(2H, q, CH2CH3), 5.16
(2H, s, CH2O), 7.12, 7.23, 7.55, and 7.91 (each 2H，d. t. t. d., aromatics). Anal. Calcd for
C16H15FO3: C, 70.06; H, 5.51. Found: C, 70.05; H, 5.50. HRMS calcd for m/z: 274.1005.
Found: 274.1006.
Substituted [4-(benzyloxy)benzoyl]hydrazine (3a-c)
A mixture of 2a-c (5 mmol) and hydrazine monohydrate (2.4 ml) in 1-propanol (15 ml) was
stirred to reflux for 24 h. After cooling, the solvent was evaporated and the crude compound
was resolved in CHCl3. The solution was washed with 100 ml ice water, then the organic layer
was dried and evaporated. The obtained solid was recrystallized from MeOH.
[4-(2-Fluorobenzyloxy)benzoyl]hydrazine (3a): Yield 46.0%, mp 114-115oC, IR (KBr):
1
3312, 3192, 1620, 1258, 837 cm-1. H-NMR (DMSO) δ: 4.44 (2H, s, NHNH2), 5.20 (2H, s,
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CH2O), 7.08, 7.22, 7.61, and 7.82 (each 2H, d. q. t. d., aromatics), 9.64 (NHNH2). Anal. Calcd
for C14H13FN2O2: C, 64.61; H, 5.03; N, 10.76. Found: C, 64.60; H, 5.05; N, 10.76. HRMS
calcd for m/z: 260.0961. Found: 260.0963.
[4-(3-Fluorobenzyloxy)benzoyl]hydrazine (3b): Yield 79.5%, mp 139-143oC, IR (KBr):
l
3313, 3195, 1615, 1255, 834 cm-1. H-NMR (DMSO) δ: 4.41 (2H, s, NHNH2), 5.17 (2H, s,
CH2O), 7.06, 7.21, 7.53, and 7.84 (each 2H, d. t. d. dd., aromatics), 9.67 (NHNH2). Anal. Calcd
for C14H13FN2O2: C, 64.61; H, 5.03; N, 10.76. Found: C, 64.63; H, 5.03; N, 10.74. HRMS
calcd for m/z: 260.0961. Found: 260.0960.
[4-(4-Fluorobenzyloxy)benzoyl]hydrazine (3c): Yield 49.8%, mp 152-154oC, IR (KBr):
l
3312, 3192, 1613, 1251, 835 cm-1. H-NMR (DMSO) δ: 4.47 (2H, s, NHNH2), 5.23 (2H, s,
CH2O), 7.01, 7.11, 7.48, and 7.79 (each 2H, d. t. t. d., aromatics), 9.59 (NHNH2). Anal. Calcd
for C14H13FN2O2: C, 64.61; H, 5.03; N, 10.76. Found: C, 64.62; H, 5.05; N, 10.77. HRMS
calcd for m/z: 260.0961. Found: 260.0964.
Substituted 1-[4-(benzyloxy)benzoyl]-2-(2-cyanoethyl)hydrazine (4a-c)
A mixture of 3a-c (1.01 g) and acrylonitrile (1.83 ml, 27.5 mmol) in EtOH was refluxed for
24 h. The solvent was evaporated. The obtained solid was recrystallized from MeOH.
1-[4-(2-Fluorobenzyloxy)benzoyl]-2-(2-cyanoethyl)hydrazine (4a): Yield 59.5%, mp 113-
1
116oC, IR (KBr): 3260, 3209, 2251, 1601, 1366 cm-1. H-NMR (DMSO) δ: 2.63 (2H, t,
CH2CN), 3.01 (2H, q, CH2CH2), 5.20 (2H, s, CH2O), 5.46 (1H, q, NHCH2), 7.10, 7.23, 7.61,
and 7.83 (each 2H, d. q. t. d., aromatics), 9.99 (NHNH). Anal. Calcd for C17H16FN3O2: C,
65.17; H, 5.15; N, 13.41. Found: C, 65.20; H, 5.14; N, 13.40. HRMS calcd for m/z: 313.1226.
Found: 313.1222.
1-[4-(3-Fluorobenzyloxy)benzoyl]-2-(2-cyanoethyl)hydrazine (4b): Yield 43.2%, mp 75-
83oC, IR (KBr): 3263, 3211, 2248, 1605, 1362 cm-1. 1H-NMR (DMSO) δ: 2.59 (2H, t, CH2CN),
3.05 (2H, q, CH2CH2), 5.21 (2H, s, CH2O), 5.41 (1H, q, NHCH2), 7.09, 7.15, 7.58, and 7.83
(each 2H, d. t. d. dd., aromatics), 9.97 (NHNH). Anal. Calcd for C17H16FN3O2: C, 65.17; H,
5.15; N, 13.41. Found: C, 65.16; H, 5.15; N, 13.39. HRMS calcd for m/z: 313.1226. Found:
313.1229.
1-[4-(4-Fluorobenzyloxy)benzoyl]-2-(2-cyanoethyl)hydrazine (4c): Yield 51.8%, mp 132-
1
135oC, IR (KBr): 3260, 3207, 2252, 1611, 1370 cm-1. H-NMR (DMSO) δ: 2.61 (2H, t,
CH2CN), 3.00 (2H, q, CH2CH2), 5.15 (2H, s, CH2O), 5.44 (1H, q, NHCH2), 7.06, 7.18, 7.54,
and 7.81 (each 2H, d. t. t. d., aromatics), 10.00 (NHNH). Anal. Calcd for C17H16FN3O2: C,
65.17; H, 5.15; N, 13.41. Found: C, 65.17; H, 5.17; N, 13.40. HRMS calcd for m/z: 313.1226.
Found: 313.1223.
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Substituted 5-[4-(benzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (5a-c)
Compounds 4a-c (0.29 g) were dissolved in dry THF, then triphosgene (0.075 g, 0.25 mmol)
in dry THF was added dropwise. The reaction mixture was stirred for 3 h at room temperature.
The solvent was evaporated. The obtained solid was recrystallized from MeOH.
5-[4-(2-Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (5a): Yield
82.1%, mp 167-168oC, IR (KBr): 2250, 1776, 1614, 1244, 984 cm-1. 1H-NMR (DMSO) δ: 3.07
(2H, t, CH2CH2), 4.08 (2H, t, CH2CN), 5.29 (2H, s, CH2O), 7.28, 7.46, 7.63, and 7.83 (each
2H, d. q. t. d., aromatics). Anal. Calcd for C18H14FN3O3: C, 63.70; H, 4.16; N, 12.39. Found:
C, 63.74; H, 4.15; N, 12.40. HRMS calcd for m/z: 339.1019. Found: 339.1024.
5-[4-(3-Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (5b): Yield
65.7%, mp 152-159oC, IR (KBr): 2251, 1775, 1617, 1248, 989 cm-1. 1H-NMR (DMSO) δ: 3.04
(2H, t, CH2CH2), 4.01 (2H, t, CH2CN), 5.19 (2H, s, CH2O), 7.11, 7.21, 7.64, and 7.89 (each
2H, d. t. d. dd., aromatics). Anal. Calcd for C18H14FN3O3: C, 63.70; H, 4.16; N, 12.39. Found:
C, 63.73; H, 4.16; N, 12.38. HRMS calcd for m/z: 339.1019. Found: 339.1020.
5-[4-(4-Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (5c): Yield
65.1%, mp 164-166oC, IR (KBr): 2255, 1772, 1609, 1250, 979 cm-1. 1H-NMR (DMSO) δ: 3.01
(2H, t, CH2CH2), 4.02 (2H, t, CH2CN), 5.18 (2H, s, CH2O), 7.18, 7.23, 7.56, and 7.77 (each
2H, d. t. t. d., aromatics). Anal. Calcd for C18H14FN3O3: C, 63.70; H, 4.16; N, 12.39. Found: C,
63.68; H, 4.14; N, 12.38. HRMS calcd for m/z: 339.1019. Found: 339.1022.
Radiosynthesis (Scheme 2)
5-[4-(2-[18F]Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one
([18F]5a, 2-[18F]FBPO)
A tin precursor compound 6a was synthesized as indicated in a previous report (12).
20
Fluorine-18 was produced by a Ne(d, α)18F reaction with an in-house HM-12 (Sumitomo
Heavy Industries, Ltd., Tokyo, Japan) 12 MeV cyclotron, followed by [18F]CH3COOF
synthesis in a multipurpose synthetic system (19) according to a method previously reported
(20). [18F]CH3COOF from a multipurpose synthesizer was added to dry ice-cooled
trichlorofluoromethane containing 6a (10 mg/mL), and reacted for 5 min with stirring. After
the temperature was gently returned to room temperature, EtOH (1 ml) was added, followed
by evaporation and HPLC purification. The HPLC solvent was MeOH : H2O = 8 : 2 with 3
mL/min flow rate.
Estimation of MAO inhibitory potency
Rat livers were homogenized in 0.25 M sucrose phosphate buffer (pH 7.4) under cooling.
The homogenates were centrifuged at 1,000 g (10 min, 4oC) to remove debris. The protein
content in the supernatant was measured by the biuret method (21). The MAO activity was
measured following a method reported by Krajl et al (22) using kynuramine as an assay
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substrate with the rat liver homogenate. In the assay, L-deprenyl, a potent and selective MAO-
B inhibitor, was used as a positive control.
In vivo biodistribution study
2-[18F]FBPO (100 μL, 185 kBq in saline) was injected into the male ddY mice via the tail
vein. The mice were sacrificed at various times. Samples of blood and the organs of interest
were excised and weighed. The radioactivity was measured using a well-type NaI (Tl) gamma
counter. The radioactivity accumulation in each tissue was expressed as a percentage of the
total dose per gram of tissue (%dose/g tissue).
In order to evaluate the specificity of 2-[18F]FBPO accumulation in tissue, an in vivo
pretreatment study was performed. First, L-deprenyl (a selective irreversible inhibitor of MAO-
B, 10 mg/kg), Ro 16-6491 (a selective reversible inhibitor of MAO-B, 0.2 mg/kg), or clorgyline
(a selective irreversible inhibitor of MAO-A, 10 mg/kg) was intravenously injected into the
mice, followed by an injection of 2-[18F]FBPO 1 hour later in a similar manner as described
above. At 90 min after 2-[18F]FBPO administration, the blood and tissues of interest were
excised, weighed, and the radioactivity measured to obtain %ID/g tissue. The data were
presented in comparison to that of the non-pretreated group.
Statistics
Data are shown as a mean ± standard deviation (sd). Statistical analysis was performed using
the Mann-Whitney U test. Atwo-tailed value of p < 0.05 was considered statistically significant.
Results & Discussion
Synthesis (Scheme 1)
Fluorinated derivatives of MD-230254 were synthesized according to Scheme 1 constructed
from previous reports (12, 23). Ethyl 4-hydroxybenzoate was converted into 2a-c by treatment
with sodium hydride in dry DMF followed by reaction with appropriate benzyl bromide
derivatives. Compounds 2a-c were treated with hydrazine hydrate to produce hydrazides 3a-c.
A Michael reaction between hydrazides 3a-c and acrylonitrile gave 4a-c. The fluorinated
derivatives of MD-230254 5a-c were synthesized by a cyclization reaction of hydrazines 4a-c
with bis(trichloromethyl)carbonate (triphosgene) (24) in a high yield.
In vitro study
The inhibitory potencies of fluorinated compounds 5a-c and the lead compound MD-230254
against MAO-A and MAO-B activities were measured using a rat liver mitochondria fraction
prepared from normal Wistar rats. The data, expressed in IC50 values for the corresponding
inhibitory curves of the compounds 5a-c, are summarized in Table 1 together with the
previously reported data from MD-230254 and L-deprenyl (12). The compounds 5a-c showed
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high and selective MAO-B inhibitory potencies in comparison with L-deprenyl used as a
positive control, and in particular, 5a showed comparably potent inhibitory activity to the lead
compound MD-230254. While our previous study revealed that the introduction of an iodine
atom at the para position diminished the inhibitory potency for MAO-B (12), 5c had similar
values to those of 5a-b in this study. This was presumably caused by the size difference between
iodine and fluorine, which brought significant steric hindrance between the iodinated
compound and the protein. Based on these results, the radiosynthesis of [18F]5a (2-[18F]FBPO)
was performed and was followed by the in vivo experiments.
Radiosynthesis (Scheme 2)
2-[18F]FBPO was synthesized by a metal substitution reaction of a tributylstannyl precursor
with [18F]CH3COOF prepared by the multipurpose synthesizer. The molar activity of
[18F]CH3COOF was 150-250 MBq/μmol. The synthesis of 2-[18F]FBPO was confirmed by
radio HPLC analysis showing a peak at 16 min of retention time in comparison with the UV
chromatogram of nonradioactive 2-FBPO which showed a peak at a similar retention time. The
total time needed for the 2-[18F]FBPO synthesis including HPLC purification and the
evaporation steps was less than 60 min from the end of the [18F]CH3COOF synthesis. The
radiochemical yield obtained in the three trials was 5.3-7.1% without decay correction. Both
the radiochemical and chemical purities at the end of the synthesis time (EOS) were estimated
to be more than 99% by radio and UV HPLC analyses. The molar activity of 2-[18F]FBPO was
42-60 MBq/μmol.
In vivo study
In vivo radioactivity biodistribution was evaluated in male ddY mice at 5-90 min post
injection of 2-[18F]FBPO (Table 2). The administered radioactivity was rapidly delivered
throughout the body and peaked at 5-15 min in most tissues that were evaluated including the
brain, which is the main target of the probe, with the exception of the liver and the bone. The
radioactivity accumulation in the brain was highest with 2.20%ID/g at 15 min and gradually
decreased to 1.30%ID/g at 90 min. The rapid radioactivity clearance in the blood was clearly
indicated by the values of 2.58 and 0.35%ID/g at 5 and 90 min, respectively. The heart showed
a similar accumulation profile as the brain, peaking at 15 min (5.40%ID/g) and decreasing
thereafter (2.57%ID/g at 90 min), and the liver showed rapid uptake (9.19%ID/g at 5 min) and
a gradual increase (14.20%ID/g at 90 min). This pattern was probably due to two different
accumulation mechanisms, which are target specific delivery and excretion related
accumulation. The brain/blood radioactivity ratio, which is recognized as an index for in vivo
PET imaging, thus gradually increased during the study period to reach 3.73 at 90 min post
injection of 2-[18F]FBPO. This steady increase indicates the high potential of 2-[18F]FBPO for
in vivo PET imaging of MAO-B expression in disease. It should also be noted that a gradual
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increase of radioactivity accumulation in the bone is a drawback of the probe, because it
suggests that defluorination might occur to a certain extent in vivo.
An in vivo blocking experiment using MAO-B selective (L-deprenyl, Ro 16-6491) or MAO-
A selective (clorgyline) inhibitor treatment prior to 2-[18F]FBPO administration was then
performed (Figure 2). The data are expressed as percent of the control %ID/g value.
Radioactivity accumulation in the brain, heart and liver with a certain amount of MAO
expression reported, was significantly decreased by pretreatment of L-deprenyl and Ro 16-6491
but not by clorgyline, which clearly indicates the MAO-B selectivity of 2-[18F]FBPO in vivo.
Conclusions
The newly designed fluorinated derivatives of MD-230254 showed high and selective MAO-
B inhibitory activities comparable to MD-230254 and an iodinated compound (2-IBPO) we
previously reported (12). The radiofluorination of 2-FBPO was successfully achieved using
[18F]CH3COOF with a corresponding tributylstannyl precursor. 2-[18F]FBPO was actively
delivered to targeted tissues such as the brain owing to a MAO-B dependent mechanism with
rapid blood clearance, which provided the high brain/blood ratio for in vivo PET imaging. In
conclusion, the results indicate the great potential of 2-[18F]FBPO as a MAO-B imaging probe
applicable to a variety of diseases.
Acknowledgements
This work was partly supported by a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (08770752). There are no conflicts of interest.
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Table 1 Inhibitory potencies of compounds toward MAO activities.
IC50 (nM)
Compounds
MAO-B
MAO-A/MAO-B
MAO-A
> 100000
> 100000
> 100000
> 100000
920
1.9
5.0
3.3
1.8
8.8
> 53000
> 20000
> 30000
> 56000
104
5a
5b
5c
MD-230254*
L-Deprenyl*
*Data from the previous report (12).
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Table 2 Radioactivity biodistribution after intravenous administration of 2-[18F]FBPO in normal ddY mice.
Time after administration (min)
5
15
30
60
90
Blood
Pancreas
Spleen
Liver
2.58 ± 0.12
3.02 ± 0.22
1.50 ± 0.15
9.19 ± 1.09
8.17 ± 1.79
4.45 ± 0.82
9.16 ± 1.96
4.73 ± 1.02
2.02 ± 0.31
0.78 ± 0.10
1.21 ± 0.51
2.16 ± 0.53
0.94 ± 0.44
12.61 ± 1.67
5.69 ± 4.38
5.40 ± 1.51
7.27 ± 1.32
8.30 ± 1.24
2.20 ± 0.60
1.88 ± 0.24
0.52 ± 0.15
1.43 ± 0.47
0.42 ± 0.18
8.81 ± 5.49
2.44 ± 1.02
3.20 ± 0.58
4.88 ± 1.46
8.35 ± 1.77
1.47 ± 0.18
2.92 ± 0.40
0.33 ± 0.02
1.17 ± 0.43
0.30 ± 0.02
10.82 ± 2.17
1.12 ± 0.35
2.12 ± 0.52
2.71 ± 0.69
8.82 ± 0.68
1.15 ± 0.20
3.50 ± 0.85
0.35 ± 0.05
1.22 ± 0.21
0.31 ± 0.07
14.20 ± 0.57
1.63 ± 0.79
2.57 ± 0.22
2.84 ± 0.46
9.12 ± 1.35
1.30 ± 0.14
3.73 ± 0.74
Kidney
Heart
Lung
Bone
Brain
Brain/Blood†
Data are presented as % injected dose per gram. Each value represents the mean ± sd for 4 animals at each interval.
†Brain to blood radioactivity ratio.
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Scheme 1. Synthesis of compounds 5a-c.
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Scheme 2. Radiosynthesis of [18F]5a (2-[18F]FBPO).
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Figure 1. Chemical structures of MD-230254 and 2-IBPO previously reported (12).
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Figure 2. Effects of pretreatment of inhibitors (L-deprenyl, Ro 16-6491, and clorgyline) on
the uptake of 2-[18F]FBPO in living mice. Each value represents mean ± sd of four mice as
percent of the control value. *p < 0.05.
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