A novel probe for imaging amyloid-β: Synthesis of F-18 labelled BF-108, an acridine orange analog

Article abstract of DOI:10.1002/jlcr.716

The synthesis of 3-(2-[¹⁸F]fluoroethyl)ethylamino-6-diethylaminoacridine ([¹⁸F]BF-108), a potential positron-labelled probe for imaging amyloid-β is described. The precursor tosylate derivative was fluorinated with [¹⁸F]KF

Full text of DOI:10.1002/jlcr.716

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 765–772.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.716
Research Article
A novel probe for imaging amyloid-b: Synthesis of
F-18 labelled BF-108, an Acridine Orange analog
Hiroshi Shimadzu*, Takahiro Suemoto, Masako Suzuki, Tsuyoshi
Shiomitsu, Nobuyuki Okamura, Yukitsuka Kudo and Tohru Sawada
BF Research Institute, Inc. c/o National Cardiovascular Center 7-1, 5-Chome,
Fujishirodai, Suita, Osaka 565-0873, Japan
Summary
The synthesis of 3-(2-[18F]fluoroethyl)ethylamino-6-diethylaminoacridine
([18F]BF-108), a potential positron-labelled probe for imaging amyloid-b is
described. The precursor tosylate derivative was fluorinated with [18F]KF/
Kryptofix 222 in acetonitrile, and the crude product was purified by semi-
preparative HPLC to give the radiolabelled BF-108. The radiochemical purity
was >95% and the maximum specific activity was 33.9 TBq/mmol at the end
of the synthesis (EOS). The synthesis time was 130 min from the end of
bombardment (EOB). Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: 18F; 3-(2-fluoroethyl)ethylamino-6-diethylaminoacridine; BF-108;
amyloid-b; PET
Introduction
Assessing the deposition of amyloid-b in the living brain is important
for the diagnosis of Alzheimer’s disease (AD) at an early stage.
Radioactive probes which have a high binding affinity to amyloid-b are
required for non-invasive imaging techniques, such as positron emission
*Correspondence to: H. Shimadzu, BF Research Institute, Inc. c/o National Cardiovascular Center
7-1, 5-Chome, Fujishirodai, Suita, Osaka 565-0873, Japan. Email: shimadzu@po.iijnet.or.jp.
Contract/grant sponsor: Program for Promotion of Fundamental Studies in Health Sciences of the
Organization for Pharmaceutical Safety and Research of Japan.
Copyright # 2003 John Wiley & Sons, Ltd.
Received 13 January 2003
Revised 26 February 2003
Accepted 20 March 2003

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H. SHIMADZU ET AL.
tomography (PET) or single photon emission computed tomography
(SPECT) which are expected to facilitate the evaluation of AD. Many
amyloid-b imaging probes, such as FDDNP,1 BSB,2 TZDM3 and 6-
OH-BTA-1,4 have recently been reported. We have also searched
randomly for compounds which specifically bind to amyloid-b(1-40)
with a b-sheet structure using the thioflavin T fluorescence method
developed by LeVine5 and Wood,6 and found that 3,6-bis(diethylami-
no)acridine (BF-009) inhibits amyloid-b(1-40)-thioflavin T binding
more than Congo Red, a positive control. Moreover, BF-009 has a
partition coefficient (octanol-water) much larger than that of Congo
Red. This finding suggests that BF-009 has an increased permeability
across the blood brain barrier. To ascertain the validity of the design for
the radiolabelling, 3-(2-fluoroethyl)ethylamino-6-diethylaminoacridine
(BF-108), a fluorinated derivative of BF-009, was synthesized and it was
confirmed that this compound has a binding activity and a partition
coefficient as large as BF-009. Thus, we chose [18F]BF-108 as a
candidate for the radioactive probe for imaging the amyloid-b peptide.
Results and discussion
The synthetic route to the authentic BF-108 (6) is shown in Figure 1.
The precursor tosylate (7) was prepared by reacting a 3-(2-hydro-
xyethyl)ethylamino derivative (5) with an excess amount of p-
toluenesulfonic anhydride in pyridine.7 As the tosylate (7) was found
to be hygroscopic and unstable, it had to be stored in a freezer. It was
converted to an unknown compound when left standing at room
temperature. Furthermore, it is sensitive to basic reagents and reacts
readily, for example, with potassium carbonate to give compound 5.
This property may cause a problem in the synthesis of radioactive BF-
108, because, in the radiofluorination, an excess amount of potassium
carbonate, which is used for detachment of the radioactive fluoride
anion from the ion-exchange resin, is always present in the reaction
mixture. So use of a minimum amount of potassium carbonate and an
addition of KH2PO4 to neutralize the reaction solution were thought to
be advantageous for obtaining the desired product. The non-radioactive
fluorination was successfully carried out by reacting 7 with powdered
potassium fluoride in the presence of Kryptofix 222 in acetonitrile at
808C for 40 min. The reaction mixture was concentrated to dryness and
the residue was purified by column chromatography on silica-gel to give
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 765–772

A NOVEL PROBE FOR IMAGING
767
60°C 1.5h
NaBH4
AcOH
NaBH4
ClCH2COOH
y=63%
EtHN
N
NHEt
H2N
EtHN
Et2N
NH2
N
y=62%
2
1
110°C 12h
1)AcONa-AcOH
DMS
60°C 1.5h
NaBH4
Cl
Cl
AcOH
Et2N
N
N
N
N
2)50°C 30min
8N HCl
ClCH2CH2Cl
y=62%
Et
Et
y=45%
3
4
-50°C→rt 4h
OH
F
N
N
Et2N
N
N
DAST/DME
y=53%
Et
Et
5
(COOH)2
6(BF-108 · Free base)
80°C 1h
Ts2
80°C 40min
8(BF-108 · Oxalate)
O
KF
OTs
Et2N
N
N
Pyridine
y=75%
Kryptofix 222
CH3CN
Et
7
y=34%
Figure 1. Synthetic routes to BF-108
6 in 34% yield. Compound 6 thus obtained was compared to an authentic
sample of 6 prepared from 5 and (diethylamino)sulfur trifluoride
(DAST). Their HPLC retention times and NMR spectra were identical.
The radiosynthesis was performed in a manner similar to that of the
non-radiosynthesis described above except that KH2PO4 was further
added (Figure 2). The desired product was isolated by semi-preparative
reversed-phase HPLC (Figure 3). The radioactive fraction containing
[18F]6 was collected, concentrated under reduced pressure and
transferred to a product vial. The synthesis time was 130 min from
EOB. The analytical HPLC showed that the radiochemical purity was
>95% and the maximum specific activity was 33.9 TBq/mmol.
Biodistribution of [18F]6 was determined in Slc:ICR mice (male,
28–32 g, SLC). The mice were decapitated at specific times (2, 10, 30, 60
and 180 min) after intravenous injection of 0.3–0.4 MBq of [18F]6 in
saline. The organs of interest were removed and weighed, and the
amount of radioactivity was counted with an automatic g-counter
(Wizard 1480, Turku, Finland). The percentage injection dose per gram
(%ID/g) was calculated by comparison of the tissue counts to the
weights of tissues and standards. Each %ID/g value is an average
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 765–772

768
H. SHIMADZU ET AL.
80°C 20min
[
18F]KF / K.222
OTs
18F
KH2PO4 / CH3CN
Et2N
Et2N
N
N
N
N
Et
Et
7
[18F]6
Figure 2. Synthesis of [18F]BF-108
Figure 3. Semi-preparative HPLC separation of [18F]BF-108
ꢀ standard deviation (SD) of three or four separate experiments. A
high brain uptake (1.53% ID/g) was obtained at 30 min post injection as
shown in Figure 4.8 This result suggests that [18F]6 has the potential to
be used as a radiotracer for the early diagnosis of AD.
Experimental
General method
Compounds: 3-(2-hydroxyethyl)ethylamino-6-diethylaminoacridine (5)
and 3-(2-fluoroethyl)ethylamino-6-diethylaminoacridine oxalate (8,
Copyright # 2003 John Wiley & Sons, Ltd.
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A NOVEL PROBE FOR IMAGING
769
organ
brain
2min
10min
30min
60min
120min
180min
0.42 0.09
2.89 0.56
7.82 1.51
1.13 0.25
2.65 0.68
6.28 1.39
1.53 0.36
2.57 0.51
8.08 7.74
1.39 0.13
2.32 0.27
2.99 0.33
6.42 0.58
2.17 0.31
5.36 0.80
2.02 0.16
9.51 0.76
5.41 1.77
1.48 0.16
1.83 0.20
1.34 0.31
1.91 0.32
2.45 0.43
2.86 0.20
1.89 0.33
3.06 0.63
1.50 0.27
9.75 1.60
8.00 2.28
1.27 0.27
1.57 0.28
1.04 0.25
1.56 0.43
1.75 0.32
3.81 3.35
1.37 0.24
2.61 0.58
1.10 0.15
4.38 0.51
8.96 0.82
0.91 0.18
1.12 0.17
blood
liver
kidney
heart
14.26 2.84 14.54 0.56 10.79 3.01
8.87 1.98
3.47 0.28
2.60 0.38
7.52 0.76
2.78 0.31
7.97 2.16
4.31 1.06
1.82 0.27
2.15 0.39
lung
20.06 1.93 13.89 2.77
spleen
intestine
bone
3.07 0.62
2.60 0.84
1.84 0.32
2.65 0.51
1.05 0.15
3.91 0.50
5.32 1.11
2.95 0.58
2.50 0.38
2.11 0.35
muscle
skin
All values showing the %ID/g; mean of three or four mice standard deviation.
Figure 4. Biodistribution in mice after i.v. injection of [18F]BF-108
BF-108 ꢁ oxalate) were prepared from 3,6-diaminoacridine as shown
Figure 1 and also supplied by Tanabe R&D Service Co., Ltd., Osaka City.
All other chemicals used in this study were purchased from
commercial suppliers. NMR spectra were recorded on a Varian
GEMINI-300 spectrometer. A JASCO PU-987 pump and a JACSO
UV-970 UV/Vis detector were used for the semi-preparative HPLC, and
a Shimadzu C-R4AX Chromatopac, a Shimadzu SPD-6A UV detector,
a JASCO PU-980 pump and an Aloka RLC-700 radioanalyzer were
used for the analytical HPLC.
3-(2-p-Toluenesulfonyloxyethyl)ethylamino-6-diethylaminoacridine (7)
Compound 5 (50 mg, 0.12 mmol) was dissolved in pyridine (25 ml). To
this solution, p-toluenesulfonic anhydride (500 mg, 1.56 mmol) was
added and the mixture was stirred at 808C for 1 h. The reaction mixture
was evaporated to dryness, and the residue was purified by silica gel
column chromatography (eluent: chloroform/methanol 10:1(v/v)) to
give 7 (55 mg,0.04 mmol) as a brown hygroscopic solid in 39% yield. 1H-
NMR (CDCl3) d: 1.26 (3 H, t, J ¼ 7:2 Hz) 1.31 (6 H, t, J ¼ 7:2 Hz) 2.35
(3 H, s) 3.57 (2 H, t, J ¼ 7:2 Hz) 3.58 (4 H, q, J ¼ 7:2 Hz) 3.83 (2 H, t,
J ¼ 5.1 Hz) 4.33 (2 H, t, J ¼ 4:8 Hz) 6.95–7.05 (2 H, m) 7.24–7.28
(3 H, m) 7.35 (1 H, br.s) 7.64–7.71 (2 H, m) 7.73 (2 H, d, J ¼ 6:6 Hz) 8.34
(1 H, s); Anal. Calculated for C28H33N3O3S ꢁ H2O: C, 65.99; H, 6.92; N,
8.24. Found: C, 66.05; H, 6.78; N, 8.03.
Copyright # 2003 John Wiley & Sons, Ltd.
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H. SHIMADZU ET AL.
3-(2-Fluoroethyl)ethylamino-6-diethylaminoacridine (6, BF-108)
Method A.A solution of DAST (0.83 ml, 6.28 mmol) in 1,2-dimethox-
yethane (20 ml) was added to a solution of 5 (1.06 g, 3.14 mmol) in 1,2-
dimethoxyethane (40 ml) at –508C under stirring. After 10 min, the
cooling bath was removed and the reaction mixture was allowed to
warm up to room temperature. After 4 h, to the resulting reaction
mixture, chloroform and an aqueous potassium carbonate solution were
added and the separated organic phase was evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography (eluent: chloroform/methanol/NH4OH 100:10:1) to
give 550 mg (53%) of 6 as a brown solid. The oxalate (8) was prepared
by treating 6 with a solution of oxalic acid in a mixture of methanol and
diethyl ether.
1
Free base (6); H-NMR (CDCl3) d:1.32 (9 H, t, J ¼ 7:2 Hz) 3.58
(4 H, q, J ¼ 7:2 Hz) 3.68 (2 H, q, J ¼ 7:2 Hz) 3.87 (2 H, td, J ¼ 4:5,
25.8 Hz) 4.77 (2 H, td, J ¼ 4:8, 47.4 Hz) 7.01 (1 H, dd, J ¼ 2:4, 9.6 Hz)
7.04 (1 H, dd, J ¼ 2:4, 8.1 Hz) 7.33 (1 H, br.s) 7.49 (1 H, br.s) 7.67 (1 H,
d, J ¼ 9:6 Hz) 7.68 (1 H, d, J ¼ 9:6 Hz) 8.34 (1 H, s).
1
Oxalate (8); H-NMR (DMSO-d6) d:1.23 (9 H, t, J ¼ 6:6 Hz) 3.5–3.7
(6 H, m) 3.91 (2 H, td, J ¼ 4:5, 25.8 Hz) 4.77 (2 H,td, J ¼ 4:5, 47.1 Hz)
6.79 (1 H, s) 6.87 (1 H, s) 7.26 (1 H, d, J ¼ 9:3 Hz) 7.30 (1 H, d,
J ¼ 9:3 Hz) 7.94 (2 H, d, J ¼ 9:3 Hz) 8.83 (1 H, s) Elemental anal.
Calculated for C21H26N3F ꢁ C2H2O4 ꢁ 0.2H2O: C, 63.79; H, 6.61; N, 9.70;
F 4.39. Found: C, 63.68; H, 6.33: N, 9.66; F 4.27; mp 203–2058C.
Method B.To a solution of 7 (6.0 mg) in acetonitrile (2 ml), powdered
potassium carbonate (6 mg) and Kryptofix 222 (15 mg) were added, and
the mixture was stirred at 808C for 40 min. The reaction mixture was
evaporated to dryness under reduced pressure, and the residue was
purified by silica gel column chromatography (eluent: chloroform/
methanol 10:1) to give 1.4 mg (34%) of 6 as a brown solid. The
compounds obtained by Methods A and B were identified by means of
NMR, HPLC and TLC.
3-(2-[18F]Fluoroethyl)ethylamino-6-diethylaminoacridine ([18F]BF-108 ꢁ free
base, [18F]6)
[18F]fluoride prepared by the 18O(p,n)18F nuclear reaction (15 mA,
20 min) in a CYPRIS HM-18 cyclotron (Sumitomo Heavy Industries
Ltd., Tokyo) on an enriched water (ca.95%18O) target was adsorbed
onto an anion exchange resin (AG1-X8, BIO-RAD) and was eluted with
Copyright # 2003 John Wiley & Sons, Ltd.
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A NOVEL PROBE FOR IMAGING
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7.5 mM potassium carbonate (0.3 ml ꢂ 2) and a solution of 20 mg of
Kryptofix 222 in 3.0 ml of acetonitrile was added. The resulting solution
was transferred to a glassy carbon vessel and 0.5 mg of potassium
dihydrogen phosphate in 0.05 ml water was added. Then, water and
acetonitrile were removed in a stream of helium at 1158C under reduced
pressure. To the residue, a solution of the precursor 7 (ca.1 mg) in 1.0 ml
of acetonitrile was added, and the mixture was stirred at 808C for
20 min. After cooling, 1.0 ml of 0.01 M phosphate buffer was added and
the mixture was injected onto a semi-preparative HPLC column (YMC,
ODS-AQ-323,10 ꢂ 250 mm
+ SHISEIDO CAPCEL PAK C18
10 ꢂ 150; eluent: CH3CN/0.01 M sodium phosphate buffer (pH6.5)
60:40; flow rate: 4.0 ml/min) The effluent was monitored with both UV
(270 nm) and radioactivity detectors and the fraction containing the
desired product at 25–30 min was collected and concentrated under
reduced pressure to less than half of the initial volume, and transferred
to a vial. The product activity was 200–850 MBq at EOS. The synthesis
time was 130 min from EOB. HPLC analysis (Wakosil-II5C18 HG
4.6 ꢂ 150 mm; CH3CN/0.01 M sodium phosphate buffer (pH6.5) 60:40;
1.0 ml/min: UV detector at 270 nm; radioactivity detector) showed that
the radiochemical purity was >95% and the maximum specific activity
was 33.9 TBq/mmol at EOS.
Acknowledgements
This study was supported by the Program for Promotion of
Fundamental Studies in Health Sciences of the Organization for
Pharmaceutical Safety and Research of Japan. The authors thank
Dr Yoshinori Miyake and Ms Mikako Ogawa of the National
Cardiovascular Center Japan for their valuable advice on radiosynthesis
and Mr Norimasa Ejima of the Institute for Biofunctional Reseach Ltd.
for the cyclotron operation and for his assistance in dealing with the
automated synthesis apparatus.
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