18F-Labeled BODIPY Dye: A Potential Prosthetic Group for Brain Hybrid PET/Optical Imaging Agents

Article abstract of DOI:10.1021/acschemneuro.8b00480

There are few hybrid positron emission tomography (PET)/fluorescence imaging agents available for brain imaging. For this purpose, BODIPY dye is very attractive because one of its fluorine atoms can be readily exchanged with ¹⁸F, and it can be modified to produce red-shifted fluorescence. In this study, therefore, we synthesized and investigated a ¹⁸F-labeled red-shifted BODIPY dye as a prosthetic group for brain hybrid PET/optical imaging agents and determined the optimal dose of this radioligand for hybrid imaging. The red-shifted BODIPY dye (1) was synthesized, and one of its fluorine atoms was exchanged with ¹⁸F using SnCl₄ in high yield. Partition coefficients of ¹⁸F-labeled BODIPY dye ([¹⁸F]1) and 1 were measured using its radioactivity and fluorescence, respectively, which were shown to be suitable for brain penetration. Optimal dose for hybrid imaging was determined by analysis of PET/CT and optical images of Balb/C nude mice injected with [¹⁸F]1 and 1, respectively. Hybrid PET/optical images of mice injected with optimal dose of [¹⁸F]1 showed strong radioactivity and fluorescence signal in the brain at 2 min after injection, with rapid clearance by 30 min. Tissue distribution data confirmed the in vivo and ex vivo PET/optical imaging data, indicating desirable brain pharmacokinetics of the radioligand. Taken together, the results of this study suggest that [¹⁸F]1 can be widely used as a prosthetic group for brain hybrid PET/optical imaging agents.

Full text of DOI:10.1021/acschemneuro.8b00480

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Article
18
F-Labeled BODIPY Dye: A Potential Prosthetic
Group for Brain Hybrid PET/Optical Imaging Agents
Hyunjung Kim, Kyul Kim, Sang-Hyun Son, Joon Young Choi, Kyung-
Han Lee, Byung-Tae Kim, Youngjoo Byun, and Yearn Seong Choe
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00480 • Publication Date (Web): 28 Dec 2018
Downloaded from http://pubs.acs.org on December 31, 2018
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18F-Labeled BODIPY Dye: A Potential Prosthetic Group for Brain Hybrid PET/Optical
Imaging Agents
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Hyunjung Kim,† Kyul Kim,§ Sang-Hyun Son,§ Joon Young Choi,‡ Kyung-Han Lee,†,‡ Byung-
Tae Kim,‡ Youngjoo Byun,*,§ and Yearn Seong Choe*,†, ‡
†Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul
06351, Korea
‡Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University
School of Medicine, Seoul 06351, Korea
§College of Pharmacy, Korea University, Sejong 30019, Korea
Submitted to: ACS Chemical Neuroscience
*Corresponding authors: Email: ysnm.choe@samsung.com; Tel: +82-2-3410-2623; Fax: +82-
2-3410-2667; Email: yjbyun1@korea.ac.kr
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ABSTRACT
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There are few hybrid positron emission tomography (PET)/fluorescence imaging agents
available for brain imaging. For this purpose, BODIPY dye is very attractive because one of
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its fluorine atoms can be readily exchanged with F, and it can be modified to produce red-
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shifted fluorescence. In this study, therefore, we synthesized and investigated a F-labeled
red-shifted BODIPY dye as a prosthetic group for brain hybrid PET/optical imaging agents
and determined the optimal dose of this radioligand for hybrid imaging. The red-shifted
BODIPY dye (1) was synthesized, and one of its fluorine atoms was exchanged with 18F
using SnCl4 in high yield. Partition coefficients of F-labeled BODIPY dye ([18F]1) and 1
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were measured using its radioactivity and fluorescence, respectively, which were shown to be
suitable for brain penetration. Optimal dose for hybrid imaging was determined by analysis of
PET/CT and optical images of Balb/C nude mice injected with [18F]1 and 1, respectively.
Hybrid PET/optical images of mice injected with optimal dose of [18F]1 showed strong
radioactivity and fluorescence signal in the brain at 2 min after injection, with rapid clearance
by 30 min. Tissue distribution data confirmed the in vivo and ex vivo PET/optical imaging
data, indicating desirable brain pharmacokinetics of the radioligand. Taken together, the
results of this study suggest that [18F]1 can be widely used as a prosthetic group for brain
hybrid PET/optical imaging agents.
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KEYWORDS: BODIPY, 18F, PET/optical, brain, prosthetic group, optimal dose
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INTRODUCTION
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Nucleophilic substitution reaction is commonly used for synthesis of 18F-labeled
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compounds by substituting sulfonate esters, halides, etc with F. Isotopic exchange reaction
is also used, particularly for preparation of radioiodinated compounds such as [123I]MIBG and
[131I]MIBG.1-3 This reaction produces the radiolabeled compounds in high radiochemical
yields but with low molar activity. Recently, several groups reported 18F-labeling methods of
BODIPY dyes, which are BF2 complexes of dipyrrole derivatives. BODIPY dye has been
used as a fluorescent prosthetic group for conjugation to biologically active molecules
because it has unique characteristics, such as neutral nature, photostability, fluorescence in
diverse regions of the spectrum.4-6 Therefore, the 18F/19F exchange on one of the fluorine
atoms of BODIPY dye allows extension of its application in fluorescence imaging to versatile
hybrid imaging fields.
Hybrid PET/optical imaging has been the focus of considerable interest in recent years.7-9
While PET imaging provides sensitive and quantitative information, optical imaging gives
real-time information with high sensitivity. Moreover, PET radioligands can be easily
translated for clinical use, and optical imaging dyes can be used for intraoperative image-
guided surgery. Therefore, hybrid PET/optical imaging can provide complementary
information by compensating the drawbacks of each imaging modality.
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The first 18F-labeling of a BODIPY dye was carried out using a compound where a
difluoroboron moiety was replaced with a hydroxo(phenyl)boron moiety; this precursor was
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labeled with F in aqueous solution, which had been heated with KHF2 (70 °C, 10 min), at
room temperature for 15 min under acidic conditions, and the radiochemical yield was 22 ± 3%
and molar activity was 0.93 ± 0.15 GBq/μmol after HPLC purification.10 Under no-carrier-
added conditions, the precursor (0.85 μmol) was activated using trimethylsilyl
trifluoromethanesulfonate (TMSOTf) and then reacted with nBu4N[18F]F at 60 °C for 5 min
to produce [18F]fluoro(phenyl)boron-containing BODIPY dye in 61% radiochemical yield.10
Another group substituted one of the fluorine atoms of BODIPY with 4-
dimethylaminopyridine and then carried out 18F-labeling under harsh conditions, which
resulted in 67.6 ± 22.9% radiochemical yield.11 They further found the mild conditions for
18F/19F exchange reaction; one of the fluorine atoms of BODIPY dye was substituted with a
triflate group using TMSOTf, which was stable under organic base such as 2,6-lutidine or
N,N-diisopropylethylamine (DIPEA), and then the triflate-activated BODIPY dye was easily
converted into 18F-labeled compound at room temperature within 1 min.11 Using a much
smaller amount of the precursor (0.01 μmol vs. 0.25 μmol), 18F-labeled BODIPY dye was
achieved in higher radiochemical yield (66.7% vs. 37%) and with higher molar activity
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(35.52 GBq/μmol vs. 2.59 GBq/μmol). In order to facilitate F-labeling of BODIPY dyes,
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Liu, et al. tried diverse Lewis acids and found that SnCl4 was the choice of Lewis acid for
18F/19F isotopic exchange (room temperature, 10 min), which resulted in 79% labeling yield
with an estimated (calculated) molar activity of 1.30±0.37 GBq/μmol.12 To extend the
application of 18F-labeled BODIPY dyes for biological use, 18F-labeled ammonium BODIPY
dyes were synthesized for myocardial perfusion imaging due to their lipophilic cationic
properties,13,14 and 18F-labeled BODIPY dyes were conjugated to biomolecules such as
peptides and antibodies as well as small molecules for tumor imaging.11,12,15,16 Technically,
18F-labeled BODIPY dye enables hybrid PET/fluorescence imaging, and moreover, it has
advantages as the hybrid imaging agent, because 18F-labeled BODIPY dye and its non-
radioactive form are the same compound with the difference of one neutron.
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There are few 18F-labeled dyes available for brain hybrid PET/optical imaging. Brain
imaging agents should meet important criteria, such as size, lipophilicity, and brain
permeability. Particularly, red-shifted fluorescence dyes are required for brain imaging due to
the presence of the skull; however, there are few fluorescent dyes that can cross the blood-
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brain barrier (BBB). In this context, F-labeled red-shifted BOIDPY dyes that are small in
size and have appropriate lipophilicity may have potential for brain hybrid PET/optical
imaging. In addition, optimal dose suitable for hybrid PET/optical imaging has never been
determined.
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In the present study, therefore, we synthesized and evaluated 18F-labeled red-shifted
BODIPY dye as a prosthetic group for brain hybrid PET/optical imaging agents, and
determined the optimal dose for hybrid imaging.
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RESULTS AND DISCUSSION
BODIPY dye (1) was synthesized on a large scale from commercially available reagents in
five steps (Scheme 1 and Figures S1-S4 in the Supporting Information). Briefly, compound 4
was synthesized in 3 steps via Horner-Wadsworth-Emmons reaction, catalytic hydrogenation,
and Vilsmeier Haack formylation. Reaction of pyrrole carboxaldehye with
methyl(triphenylphosphoranylidene)acetate in benzene at room temperature provided the
alkene 2 in 63% yield. The E-configuration of 2 was confirmed by large coupling constants
1
(15.9 Hz) of vinylic protons on H NMR spectrum. Compound 2 was converted into
compound 3 under a conventional catalytic hydrogenation condition (Pd/C, H2).17
Formylation of the pyrrole 3 to afford compound 4 was achieved in 92% yield using
Vilsmeier reagent (DMF and POCl3) in 1,2-dichloroethane.18 2,2'-Bipyrrole (5) was prepared
using the reported method.19 Condensation of 4 with 5 under an acid-catalyzed condition and
subsequent complexation with boron trifluoride provided the final BODIPY dye 1 in 36%
yield.
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18F/19F exchange reaction on 1 was carried out as described in the literature with slight
modifications;12 lesser amounts of 1 (0.25 μmol) and higher amounts of SnCl4 (6 μmol) were
used (Scheme 2). Radiochemical yield of [18F]1 increased from 15-20% to 70-80% based on
TLC analysis when the mole ratio of SnCl4 to 1 increased from 12 to 24. The radiochemical
yield after HPLC purification was 35-50%. It is expected that the radiochemical yield would
increase if the radioligand is purified using a sep-pak cartridge rather than HPLC. Molar
activity of [18F]1 was 0.8-1 GBq/μmol when 1 was labeled with 370 MBq of 18F. Radioligand
with high molar activity is required for PET imaging of low density targets in the brain, such
as receptors. For this purpose, molar activity higher than 37 GBq/μmol is generally required
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for the radioligand.20 As reported in the literature, the amounts of starting F and the molar
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activity of F-labeled BODIPY dye are linearly correlated.21 As such, it was reported that
isotopic exchange on BODIPY dye using increased amounts of starting radioactivity (1,850
MBq) and low amounts of precursor (0.04 μmol) elevated estimated (calculated) molar
activity of the product to approximately 42 GBq/μmol.12 Similar results were also reported in
that use of smaller amounts of BODIPY dye (0.01 μmol) provided the 18F-labeled product in
high molar activity (35.52 GBq/μmol).11 Therefore, 18F-labeled BODIPY dye can be
synthesized at this high molar activity by using higher amounts of 18F and reduced amounts of
1. In contrast, imaging of high density targets can be achieved using 18F-labeled BODIPY dye
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with lower molar activity.
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BODIPY dye 1 displayed an excitation peak at 580 nm and an emission at 590 nm in
methanol (Figure 1), which was shown to be suitable for brain optical imaging. In order to
predict brain permeability, partition coefficients of [18F]1 and 1 were measured using
radioactivity and fluorescence, which were within the same range. Log P value of [18F]1 was
1.96 ± 0.01. Partition coefficients of 1 were carefully measured because fluorescence
properties are solvent-dependent. Therefore, we re-extracted the water layer with 1-octanol
after each extraction of 1 with a mixture of 1-octanol and water. Log P value was then
calculated from the fluorescence data of 1 in the same solvents. Log P value of 1 was 1.94 ±
0.07. The log P values obtained using radioactivity and fluorescence were also close to Clog
P value (1.79). Brain imaging agents should be lipophilic enough to penetrate across the BBB
with measured log P values of 0.1-3.5.22 [18F]1 used in this study was shown to be suitable for
penetration through the BBB.
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The BODIPY dye has intrinsic fluorescence, and one of its fluorine atoms can be readily
exchanged with 18F, which enables fluorescence and PET imaging using the same compound.
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As a result, F-labeled BODIPY dye always contains non-radioactive 1, which gives lower
molar activity compared with other radioligands synthesized by nucleophilic radiofluorination.
In this study, therefore, we determined the optimal dose of [18F]1 suitable for brain hybrid
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PET/optical imaging.
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Optical imaging was performed in hairless Balb/C nude mice. Strong fluorescence signal
was detected in mouse brains at 2 min after injection of 1, which demonstrated penetration of
1 across the BBB as well as penetration of fluorescence signal through the intact skull, and
then it faded rapidly within 30 min after injection (Figure 1). Because 1 is a fluorescent dye
without a targeting compound conjugated, it is expected that the dye penetrated across the
BBB and then was rapidly washed out. This result showed the desirable brain
pharmacokinetics of 1. Optimal dose of 1 for optical imaging was also investigated; a dose
higher than 16 μg (46.6 nmol) per mouse was required, and a lower dose than this (12 μg;
35.0 nmol) did not show sufficient fluorescence signal in mouse brains.
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For PET/CT imaging, [18F]1 with relatively high molar activity (0.8-1 GBq/μmol) was
injected into a Balb/C nude mouse. Sagittal and coronal PET/CT images showed high
radioactivity accumulation in the brain at 2 min after injection, with fast wash-out by 30 min
after injection, indicating desirable brain pharmacokinetics of the radioligand (Figures 2A and
2B). PET ROI analysis of the brains revealed 4.6% ID/g at 2 min, 1.2% ID/g at 30 min, and
0.51% ID/g at 60 min. Persistent retention of high radioactivity was detected in the liver,
kidneys, and intestines. The mouse was also subjected to optical imaging, but no fluorescence
signal was detected in the mouse brain (Figure 2C), which is not surprising because [18F]1
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(4.8 MBq) containing 1.8 μg (5.25 nmol) of 1 was injected into the mouse, and an amount
higher than 16 μg was determined as the optimal dose for optical imaging. This result
demonstrated that [18F]1 with relatively high molar activity did not provide hybrid imaging of
mouse brains. These results suggest that [18F]1 with molar activity of approximately 0.1
GBq/μmol is suitable for hybrid PET/optical imaging of mice.
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Hybrid PET/optical imaging of mice was then performed using the optimal dose of [18F]1
(Figure 3). PET images of mice showed high radioactivity accumulation in the brain, liver,
kidney, and intestines at 2 min after injection, and the radioactivity uptake in the brain
disappeared at 30 min (Figure 3A). PET ROI analysis of the brains revealed % ID/g values of
4.7 ± 0.2 at 2 min and 1.1 ± 0.1 at 30 min, which indicates fast clearance in mice. Optical
images of the mice at 2 min demonstrated strong fluorescence signal in the brain, which
decreased rapidly by 30 min (Figure 3B). Due to the limited penetration depth of the
fluorescence imaging, other organs were not clearly detected.
Ex vivo imaging was also performed on major tissues obtained from mice after injection of
[18F]1. PET images showed that strong radioactivity uptake was detected in the brain at 2 min
after injection, and the uptake decreased to the background level at 30 min (Figure 4A). High
radioactivity uptake was also detected in the liver, kidney, and intestines at 2 min, and the
uptake was retained in the liver, but a much stronger signal was detected in the intestines at
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30 min, which suggests excretion of the radioligand through the liver and intestines. Ex vivo
optical images confirmed ex vivo PET images: strong fluorescence signal at 2 min and no
measurable signal at 30 min in the brains (Figure 4B).
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Tissue distribution data demonstrated high radioactivity accumulation of [18F]1 in the
brains of mice at 2 min after injection, with rapid wash-out by 30 min (Table 1); the
respective brain uptakes at 2 and 30 min were 4.51 ± 0.43% ID/g and 0.83 ± 0.20% ID/g with
a high 2 min-to-30 min uptake ratio of 5.43 (***P < 0.001), indicating good penetration of the
radioligand through the BBB and favorable brain pharmacokinetics.22,23 This result meets the
requirements for brain PET imaging agents such as for diagnosis of Alzheimer’s disease: high
initial brain uptake at 2 min and fast wash-out at 30 min in normal mice.23-25 [18F]1 was
excreted through the liver and intestines. Low and constant femur uptake indicated little
metabolic defluorination of the radioligand, which is consistent with the reported results
showing no measurable radioactivity uptake in the bone even at 4 h after injection.10,11 These
data confirmed the in vivo and ex vivo PET/optical imaging data.
There are few brain hybrid PET/optical imaging agents because most fluorescent dyes
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cannot cross the BBB. In this context, F-labeled BODIPY dyes have great advantages over
other fluorescent dyes. The BODIPY dye we used in this study is relatively small compared
to other BODIPY dyes and also red-shifted. In addition, the propionyl side chain is short and
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flexible. There are a few studies on BODIPY dyes developed for brain imaging. One study
showed ex vivo optical images of the brains from a transgenic mouse (Tg2576) and an age-
matched wild-type mouse injected with BODIPY dye.6 In another study, the brain tissue
sections were obtained after injection of BODIPY dye into mice and then immunostained.26
However, these data did not provide quantitative information on brain pharmacokinetics. In
addition, there is a report on the radiolabeled BODIPY dye, which had poor brain
permeability despite its suitable lipophilicity.27 Therefore, the substituents on the BODIPY
skeleton may play a major role in determining brain permeability of the compound.
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To the best of our knowledge, this is the first study on dose optimization of radiolabeled
fluorescent dye for brain hybrid PET/optical imaging. Although [18F]1 enables hybrid
PET/optical imaging of mouse brains, single PET and optical imaging are also available using
[18F]1 and 1, respectively, particularly when high molar activity of the radioligand is required.
CONCLUSION
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In this study, F-labeled BODIPY dye was synthesized in high yield and was shown to
have good brain permeability in vitro and in vivo. Furthermore, hybrid PET/optical images
demonstrated that the radioligand has favorable brain pharmacokinetics in vivo. Because 18F-
labeled and non-radioactive BODIPY dyes have the same chemical structures, with the
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difference of one neutron, the optimal dose of the radioligand for hybrid PET/optical imaging
was determined by analyzing PET/CT and optical images of mouse brains. Taken together,
these results suggest that [18F]1 can be widely used as a prosthetic group for development of
various hybrid PET/optical imaging agents.
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Chemistry. Reagents were purchased from Merck (Darmstadt, Germany), TCI (Tokyo,
Japan), and Alfa Aesar (Ward Hill, MA, USA) and were used without further purification.
Reaction progress was monitored by TLC analysis using a UV lamp and/or KMnO4
staining. 1H NMR spectra were recorded at room temperature on a Bruker BioSpin Avance
300 MHz spectrometer (Rheinstetten, Germany), and chemical shifts () were reported as
ppm downfield of tetramethylsilane as an internal standard. 13C NMR spectrum was obtained
using the same NMR spectrometer and was calibrated with CDCl3 (δ = 77.16 ppm).
Electrospray ionization (ESI) mass spectrum was obtained on an Agilent 6530 Accurate-Mass
Q-TOF LC/MS system (Santa Clara, CA, USA). The purity of compound 1 was measured
using HPLC (Agilent 1260 Infinity) equipped with an analytical column (Phenomenex
Gemini C18, 4.6 × 150 mm, 5 μm).
Methyl
(E)-3-(1H-Pyrrol-2-yl)acrylate
(2).
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methyl(triphenylphosphoranylidene)acetate (14.3 g, 42.8 mmol) in benzene (45 mL) was
added pyrrole-2-carboxaldehyde (2.03 g, 21.3 mmol) at room temperature. After stirring for 3
h, the organic layer was separated, and the aqueous phase was extracted with ethyl acetate.
The combined organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography (50:1 to 5:1 toluene–EtOAc) to afford
compound 2 (2.03 g, 13.4 mmol, 63%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 9.31
(brs, 1H), 7.50 (d, J = 15.9 Hz, 1H), 6.83 (brs, 1H), 6.47 (brs, 1H), 6.18 (brd, J = 2.4 Hz, 1H),
6.00 (d, J = 15.9 Hz, 1H), 3.68 (s, 3H).
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Methyl 3-(1H-Pyrrol-2-yl)propanoate (3). Compound 2 (2 g, 13.2 mmol) was dissolved in
methanol (10 mL) and hydrogenated in a Paar hydrogenation apparatus using Pd/C under H2
(1 bar). The reaction mixture was shaken at room temperature for 3 h. At the end of the
reaction, the mixture was filtered over a bed of celite and washed with methanol. The
combined solution was concentrated in vacuo. The remaining residue was purified by flash
column chromatography (110:1 CH2Cl2–CH3OH) to provide compound 3 (1.86 g, 12.2 mmol,
1
92%) as a colorless oil. H NMR (300 MHz, CDCl3): δ 8.61 (brs, 1H), 6.74–6.70 (m, 1H),
6.18 (dd, J = 2.9 and 5.8 Hz, 1H), 6.00–5.98 (m, 1H), 3.77 (s, 3H), 2.98 (t, J = 6.9 Hz, 2H),
2.71 (t, J = 6.9 Hz, 2H).
Methyl 3-(5-Formyl-1H-pyrrol-2-yl)propanoate (4). To N,N-dimethylformamide (180 μL,
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2.34 mmol) was added slowly phosphorus oxychloride (220 μL, 2.34 mmol) at 0 °C under an
argon atmosphere. The solution was stirred for 15 min at the same temperature and then 1,2-
dichloroethane (2 mL) was added. To this mixture was added slowly a solution of 3 (303.4
mg, 1.98 mmol) in 1,2-dichloroethane (8 mL) at 0 °C under an argon atmosphere. The
reaction mixture was stirred under reflux for 15 min. After cooling to room temperature, a
solution of sodium acetate trihydrate (890 mg, 6.5 mmol) in water (10 mL) was added to the
reaction mixture and was allowed to stir under reflux for an additional 15 min. The reaction
mixture was then cooled, the organic layer was separated, and the aqueous layer was
extracted with chloroform. The combined organic layer was washed with saturated aqueous
NaHCO3 and then brine, dried over MgSO4, and concentrated in vacuo. The resulting residue
was purified by flash column chromatography (50:1 to 5:1 toluene–EtOAc) to afford
compound 4 (331 mg, 92%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 10.46 (brs, 1H),
9.38 (s, 1H), 6.89 (dd, J = 2.6 and 3.7 Hz, 1H), 6.11–6.07 (m, 1H), 3.70 (s, 3H), 3.02 (t, J =
7.20 Hz, 2H), 2.71 (t, J = 7.20 Hz, 2H).
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2,2'-Bipyrrole (5). Compound 5 was synthesized as described in the literature.19 Briefly, to
a stirred solution of 1H-pyrrole (187 μL, 2.7 mmol) in CH2Cl2 (15 mL) at –78 °C,
[bis(trifluoroacetoxy)iodo]benzene (PIFA) (387 mg, 0.9 mmol) and trimethylsilyl bromide
(TMSBr) (233 μL 1.8 mmol) were quickly added. The reaction mixture was stirred for 1 h
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and then stirred for another 10 min at room temperature after addition of saturated aqueous
NaHCO3 solution (~10 mL). The mixture was extracted with dichloromethane, and the
organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography (hexane–EtOAc) to give compound 5 (108 mg,
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69%) as a colorless oil. H NMR (300 MHz, CDCl3): δ 8.23 (brs, 1H), 6.79–6.75 (m, 2H),
6.26–6.20 (m, 4H).
4,4-Difluoro-3-(2-methoxycarbonylethyl)-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene
(1). To a solution of 4 (115 mg, 0.64 mmol) and 5 (84 mg, 0.64 mmol) in dichloromethane
(10 mL) was added phosphorus oxychloride (60 μL, 0.65 mmol) at room temperature. After
the reaction mixture was stirred at room temperature for 5.5 h, sequential additions of N,N-
diisopropylethylamine (445 μL, 2.56 mmol) and boron trifluoride dietherate (325 μL, 2.59
mmol) were made. The reaction mixture was stirred at room temperature for an additional 2 h.
The organic layer was washed with brine, separated, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude product was purified by flash column chromatography (7:1
to 4:1 hexane–EtOAc) to give compound 1 (78 mg, 36%) as a dark-purple solid. An aliquot
of 1 was analyzed by HPLC using a gradient from a 90:10 mixture to a 10:90 mixture of
water and acetonitrile over 20 min, followed by isocratic program with a 10:90 mixture over
10 min at a flow rate of 1 mL/min. The eluent was monitored simultaneously using a UV
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detector (254 nm) and a visible light detector (588 nm). 1H NMR (300 MHz, CDCl3): δ 10.42
(brs, 1H), 7.18 (brs, 1H), 7.05 (d, J = 4.8 Hz, 1H), 6.99 (s, 2H), 6.87 (d, J = 4.5 Hz, 1H), 6.84
(d, J = 3.9 Hz, 1H), 6.42–6.34 (m, 1H), 6.28 (d, J = 3.9 Hz, 1H), 3.72 (s, 3H), 3.34 (t, J = 7.5
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Hz, 2H), 2.80 (t, J = 7.5 Hz, 2H). C NMR (75 MHz, CDCl3): δ 173.1, 154.8, 150.7, 137.4,
133.4, 131.6, 126.1, 125.8, 123.7, 123.4, 120.4, 117.8, 116.1, 111.5, 51.8, 33.3, 24.0. ESI-
HRMS (m/z) calcd for C17H16BF2N3ONa+ [M + Na]+, 366.1195; found, 366.1193; Purity
(HPLC; tR= 16.17 min) > 96% (Figures S1 - S4 in Supporting Information).
Radiochemistry. [18F]Fluoride was produced by the 18O(p,n)18F reaction using a GE
Healthcare PETtrace cyclotron (Uppsala, Sweden). TLC of radioligand was analyzed on a
Bioscan radio-TLC scanner (Washington, DC, USA). Purification and analysis of radioligand
[18F]1 were performed using HPLC (Thermo Scientific, Waltham, MA, USA) equipped with
an analytical column (Phenomenex Gemini C18, 4.6 × 150 mm, 5 μm). Radioactivity was
measured in a dose calibrator (Biodex Medical Systems, Shirley, NY, USA).
Synthesis of F-Labeled BODIPY Dye ([18F]1). F/19F Exchange on BODIPY dye 1 was
carried out using the method reported in the literature with slight modifications.12
[18F]Fluoride (170~450 MBq) was placed in a Vacutainer containing nBu4NHCO3. Three
azeotropic distillations were then performed using 200–300 μL aliquots of CH3CN at 90 °C
(oil bath) under a gentle stream of N2. The resulting n-Bu4N[18F]F (25 μL) was added to 1
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(0.25 μmol), which had been mixed with SnCl4 (6 μmol) dissolved in CH3CN (40 μL). After
the reaction mixture was stirred at room temperature for 10 min, it was purified by HPLC,
and the eluent was monitored simultaneously, using UV (360 nm) and NaI(T1) radioactivity
detectors. The desired product eluted between 18 and 19 min was concentrated under a stream
of N2.
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Molar activity was determined by comparing UV peak area of the desired radioactive peak
and those of different concentrations of non-radioactive standard 1 on HPLC using an
analytical column eluted with a 40:60 mixture of water and acetonitrile at a flow rate of 1
mL/min. Identity of [18F]1 was confirmed by co-elution of the radioligand with non-
radioactive standard 1 when they were co-injected into the HPLC system.
Excitation and Emission Spectra. BODIPY dye 1 (1 μM) was dissolved in methanol, and
its excitation and emission spectra were obtained using a Mithras2 LB 943 monochromator
multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany).
Partition Coefficient Measurement. The partition coefficient of [18F]1 was measured
according to the method described in the literature.24 [18F]1 was dissolved in 1-octanol (1 mL)
and diluted with water (1 mL). The solution was vortexed vigorously for 5 min and then
centrifuged. The 1-octanol and aqueous layers were separated out, and 500 μL aliquot of each
layer was removed and measured using a gamma counter. Samples from the 1-octanol and
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aqueous layers were repartitioned until consistent values were obtained. Similarly, the
partition coefficients of BODIPY dye 1 (0.6-1 μM) were measured as described above. The 1-
octanol and water layers were separated out, and then the water layer was vigorously re-
extracted with 1-octanol. The fluorescence intensities of 1 from the resulting 1-octanol layers
were measured at 590 nm using a Mithras2 LB 943 monochromator multimode microplate
reader. Log P was expressed as the logarithm of the ratio of the count per minute (or
fluorescence) for 1-octanol versus that of water (or 1-octanol extract of water). Clog P of 1
was obtained using ChemDraw Ultra 12.0 software.
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In Vivo Studies. Animal experiments were performed according to the National Institutes
of Health Guide for the Care and Use of Laboratory Animals and approved by the
Institutional Animal Care and Use Committee of Samsung Medical Center. MicroPET and
optical images of Balb/C nude mice were acquired at the Center for Molecular and Cellular
Imaging using an Inveon microPET/CT scanner (Siemens Medical Solutions, Malvern, PA,
USA) and a Xenogen IVIS Spectrum (PerkinElmer), respectively. Tissue radioactivity was
measured in a Wizard2 automatic gamma counter (PerkinElmer, Waltham, MA, USA).
Optical Imaging. BODIPY dye 1 was dissolved in 10% DMSO–saline and injected
intravenously through a tail vein into 6-week-old Balb/C nude mice (male), which had been
fed an alfalfa-free diet for one week prior to this experiment. Optical images were acquired
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for 10 sec at 2, 30, and 60 min after injection (excitation, 570 nm; emission, 640 nm). Data
are expressed as photons/s/cm2/sr (sr: steradian).
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MicroPET/CT Imaging. Radioligand [18F]1 was synthesized using 370 MBq of n-
Bu4N[18F]F, whose molar activity was 0.8~1 GBq/μmol. [18F]1 dissolved in 10% DMSO–
saline was injected intravenously into 6-week-old Balb/C nude mice through a tail vein.
MicroPET/CT imaging of mice was performed at 2, 30, and 60 min after injection (5-min
static PET scan followed by 20-min CT scan). Optical imaging of mice was performed for 10
sec prior to each microPET/CT imaging. PET images were reconstructed using 3-D ordered
subset expectation maximization and then processed using Siemens Inveon Research
Workplace 4.2 (IRW 4.2). ROIs of PET images were drawn over brains, and the average
signal levels in ROIs were measured.
Hybrid PET/Optical Imaging. Radiolabeled BODIPY dye ([18F]1) was injected into
Balb/C nude mice via a tail vein. MicroPET images were acquired for 5 min at 2 and 30 min
after injection. Prior to each PET imaging, mice were subjected to optical imaging for 10 sec
(excitation, 570 nm; emission, 640 nm).
Ex Vivo Imaging. Mice were sacrificed at 2 and 30 min after injection of [18F]1, and tissues
of interest were excised and subjected to PET and optical imaging. Data are expressed as
percent injected dose per gram of tissue (%ID/g) and as photons/s/cm2/sr (sr: steradian),
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Tissue Distribution Studies. [18F]1 was injected into Balb/C nude mice via a tail vein. Mice
were sacrificed at 2 and 30 min after injection, and blood and major tissues were immediately
separated, weighed, and counted.
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Statistical Analysis. The results were analyzed using unpaired, two-tailed Student’s t-tests.
Differences at the 95% confidence level (P < 0.05) were considered statistically significant.
ASSOCIATED CONTENT
Supporting Information
1H and 13C NMR spectra, HRMS spectrum, and HPLC chromatograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Author Contributions
18
H.K. performed F-labeling, in vitro, and in vivo experiments. K.K. and S.H.S. performed
organic synthesis. J.Y.C., K.H.L., and B.T.K. supervised the in vivo studies. Y.S.C. and Y.B.
designed and directed the study.
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Funding
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This research was supported by the National Research Foundation of Korea grant funded by
the Korean government (NRF-2017M2A2A7A01070487) and the Korea Health Technology
R&D Projects through the Korea Health Industry Development Institute (KHIDI), funded by
the Ministry of Health & Welfare, Republic of Korea (HI16C0947 and HI16C1827).
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Notes
The authors declare no competing financial interest.
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Table 1. Tissue distribution of [18F]1 in Balb/C nude mice
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Organ
Blood
2 min
30 min
1.01 ± 0.27
1.87 ± 0.84
1.70 ± 0.52
13.39 ± 1.16
1.70 ± 0.45
4.23 ± 1.16
16.22 ± 0.79
0.96 ± 0.73
0.52 ± 0.16
1.26 ± 0.18
0.83 ± 0.20
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60
2.47 ± 0.21
10.88 ± 0.95
6.16 ± 0.44
10.23 ± 1.10
5.66 ± 1.15
14.63 ± 0.41
5.52 ± 0.48
1.74 ± 0.39
0.64 ± 0.11
1.43 ± 0.18
4.51 ± 0.43
Heart
Lung
Liver
Spleen
Kidney
Small intestine
Large intestine
Muscle
Femur
Brain
Values (% ID/g) are given as the mean ± SD of groups, n = 3 mice.
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