Synthesis and Evaluation of Two 18F-Labeled 6-Iodo-2-(4′ -N,N-dimethylamino)phenylimidazo[1,2-a]pyridine Derivatives as Prospective Radioligands for β-Amyloid in Alzheimer's Disease

Article abstract of DOI:10.1021/jm030477w

This study evaluated ¹⁸F-labeled IMPY [6-iodo-2-(4′ -N,N-dimethylamino)phenylimidazo[1,2-a]pyridine] derivatives as agents for imaging β-amyloid plaque with positron emission tomography (PET). The precursor for radiolabeling and reference compounds was synthesized in up to five steps from commercially accessible starting materials. One of the two N-methyl groups of IMPY was substituted with either a 3-fluoropropyl (FPM-IMPY) or a 2-fluoroethyl (FEM-IMPY) group. FPM-IMPY and FEM-IMPY were found to have moderate affinity for Aβ-aggregates with K_i = 27 ± 8 and 40 ± 5 nM, respectively. A "one-pot" method for ¹⁸F-2-fluoroethylation and ¹⁸F-3-fluoropropylation of the precursor was developed. The overall decay-corrected radiochemical yields were 26-51%. In PET experiments with normal mouse, high uptake of activity was obtained in the brain after iv injection of each probe: 6.4% ID/g for [ ¹⁸F]FEM-IMPY at 1.2 min, and 5.7% ID/g for [¹⁸F]FPM-IMPY at 0.8 min. These values were similar to those of [¹²³I/ ¹²⁵I]IMPY (7.2% ID/g at 2 min). Polar and nonpolar radioactive metabolites were observed in both plasma and brain homogenates after injection of [¹⁸F]FEM or [¹⁸F]FPM-IMPY. In contrast to the single-exponential washout of [¹²³I/¹²⁵I]IMPY, the washouts of brain activity for the two fluorinated analogues were biphasic, with an initial rapid phase over 20 min and a subsequent much slower phase. Residual brain activity at 2 h, which may represent polar metabolites trapped in the brain, was 4.5% ID/g for [¹⁸F]FEM-IMPY and 2.1% ID/g for [¹⁸F]FPM-IMPY. Substantial skull uptake of [¹⁸F]fluoride was also clearly observed. With a view to slow the metabolism of [ ¹⁸F]FEM-IMPY, an analogue was prepared with deuteriums substituted for the four ethyl hydrogens. However, D₄-[¹⁸F]FEM-IMPY showed the same brain uptake and clearance as the protio analogue. Metabolism of the [¹⁸F]FEM-IMPY was appreciably slower in rhesus monkey than in mouse. Autoradiography of postmortem brain sections of human Alzheimer's disease patients with [¹⁸F]FEM-IMPY showed high displaceable uptake in gray matter and low nonspecific binding in the white matter. This study demonstrates that the IMPY derivatives have favorable in vivo brain pharmacokinetics and a moderate affinity for imaging β-amyloid plaques; however, further improvements are needed to reduce radioactive metabolites, increase binding affinity, and reduce lipophilicity.

Full text of DOI:10.1021/jm030477w

2208
J . Med. Chem. 2004, 47, 2208-2218
Syn th esis a n d Eva lu a tion of Tw o 18F -La beled
6-Iod o-2-(4′-N,N-d im eth yla m in o)p h en ylim id a zo[1,2-a ]p yr id in e Der iva tives a s
P r osp ective Ra d ioliga n d s for â-Am yloid in Alzh eim er ’s Disea se
Lisheng Cai,*,† Frederick T. Chin,† Victor W. Pike,† Hiroshi Toyama,† J eih-San Liow,† Sami S. Zoghbi,†
Kendra Modell,† Emmanuelle Briard,† H. Umesha Shetty,† Kathryn Sinclair,† Sean Donohue,† Dnyanesh Tipre,†
Mei-Ping Kung,‡ Claudio Dagostin,§ David A. Widdowson,§ Michael Green,# Weiyi Gao,| Mary M. Herman,|
Masanori Ichise,† and Robert B. Innis†
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, 10 Center Drive,
Bethesda, Maryland 20892, Department of Radiology, University of Pennsylvania, Pennsylvania 19104, Department of
Chemistry, Imperial College, London, U.K., Nuclear Medicine Department, NIH Clinical Center, National Institutes of Health,
Bethesda, Maryland 20892, and Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of
Health, Bethesda, Maryland 20892
Received September 23, 2003
This study evaluated 18F-labeled IMPY [6-iodo-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-
a]pyridine] derivatives as agents for imaging â-amyloid plaque with positron emission
tomography (PET). The precursor for radiolabeling and reference compounds was synthesized
in up to five steps from commercially accessible starting materials. One of the two N-methyl
groups of IMPY was substituted with either a 3-fluoropropyl (FPM-IMPY) or a 2-fluoroethyl
(FEM-IMPY) group. FPM-IMPY and FEM-IMPY were found to have moderate affinity for Aâ-
aggregates with Ki ) 27 ( 8 and 40 ( 5 nM, respectively. A “one-pot” method for
18F-2-fluoroethylation and 18F-3-fluoropropylation of the precursor was developed. The overall
decay-corrected radiochemical yields were 26-51%. In PET experiments with normal mouse,
high uptake of activity was obtained in the brain after iv injection of each probe: 6.4% ID/g for
[18F]FEM-IMPY at 1.2 min, and 5.7% ID/g for [18F]FPM-IMPY at 0.8 min. These values were
similar to those of [123I/125I]IMPY (7.2% ID/g at 2 min). Polar and nonpolar radioactive
metabolites were observed in both plasma and brain homogenates after injection of [18F]FEM
or [18F]FPM-IMPY. In contrast to the single-exponential washout of [123I/125I]IMPY, the washouts
of brain activity for the two fluorinated analogues were biphasic, with an initial rapid phase
over 20 min and a subsequent much slower phase. Residual brain activity at 2 h, which may
represent polar metabolites trapped in the brain, was 4.5% ID/g for [18F]FEM-IMPY and 2.1%
ID/g for [18F]FPM-IMPY. Substantial skull uptake of [18F]fluoride was also clearly observed.
With a view to slow the metabolism of [18F]FEM-IMPY, an analogue was prepared with
deuteriums substituted for the four ethyl hydrogens. However, D4-[18F]FEM-IMPY showed the
same brain uptake and clearance as the protio analogue. Metabolism of the [18F]FEM-IMPY
was appreciably slower in rhesus monkey than in mouse. Autoradiography of postmortem brain
sections of human Alzheimer’s disease patients with [18F]FEM-IMPY showed high displaceable
uptake in gray matter and low nonspecific binding in the white matter. This study demonstrates
that the IMPY derivatives have favorable in vivo brain pharmacokinetics and a moderate
affinity for imaging â-amyloid plaques; however, further improvements are needed to reduce
radioactive metabolites, increase binding affinity, and reduce lipophilicity.
1. In tr od u ction
pathological features for postmortem AD are the abun-
dance of neuritic plaques composed of Aâ-amyloid and
neurofibrillary tangles made of PHF-tau aggregates.1
Recent efforts to identify positron emission tomography
(PET) Aâ-amyloid probes have generated a few leads,
including [11C]-2-(4-(methylamino)phenyl)benzo[d]thia-
zol-6-ol ([11C]6-OH-BTA-1) 2 and [18F]-2-(1-(2-(N-(2-fluo-
roethyl)-N-methylamino)naphthalen-6-yl)ethylidene)-
malononitrile ([18F]FDDNP, Figure 1).3 Some derivatives
of 6-iodo-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-
a]pyridine (IMPY, Figure 1) have also been identified
as single-photon emission computed tomography (SPECT)
There are a number of reasons for the development
of noninvasive biomarkers for Alzheimer’s disease (AD).
Diagnosis and staging of the disease are of immediate
interest for patients. In addition, quantitation of â-amy-
loid in brain would presumably allow presymptomatic
identification of patients and monitoring of putative
neuroprotective effects of novel treatments that are
currently being investigated. Two of the characteristic
* To whom correspondence should be addressed. Phone: (301) 451-
3905. Fax: (301) 480-5112. E-mail: cail@intra.nimh.nih.gov.
† Molecular Imaging Branch, National Institute of Mental Health.
‡ University of Pennsylvania.
probes for Aâ-amyloid.4
[
123I/125I]IMPY has a high
§ Imperial College.
uptake in the normal mouse brain (peak: 7.2% dose/g
at 2-5 min) and fast clearance (to <0.35% dose/g after
2 h of injection). Brain radioactivity in the Tg2576 AD
#
Nuclear Medicine Department, NIH Clinical Center.
| Clinical Brain Disorders Branch, National Institute of Mental
Health.
10.1021/jm030477w CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/20/2004

IMPY Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2209
FT-IR spectrometer, and UV-vis spectra were recorded using
a Lambda 40 UV-vis spectrometer. Mass spectra were ac-
quired using either Thermo Finnigan LCQDECA LC-MS (MS-
HPLC column, Luna C18, 5 µm, 2.0 mm × 150 mm, Phenom-
enex; flow rate, 150 µL/min; eluent, MeOH and H2O mixture)
or Thermo Finnigan PolarisQ GC-MS (GC column, capillary
RTX-5ms 30 m × 0.25 mm; flow rate, 1 mL/min; carrier gas,
He). High-resolution mass spectra were acquired under elec-
tron ionization (EI) conditions using a double-focusing high-
resolution mass spectrometer (AUTOSPEC, Micromass Inc.)
with samples introduced through a direct insertion probe.
Elemental analyses of selected compounds were carried out
by Midwest Microlab (Indianapolis, IN) or Galbraith Labora-
tories, Inc. (Knoxville, TN).
F igu r e 1. Structures of leading tracers for â-amyloid.
transgenic mouse model is ∼3.3 times higher than that
in the age-matched control at 4 h after injection. This
desirable pharmacokinetics makes [125I]IMPY an at-
tractive agent for mapping brain Aâ plaques. Here, we
report the synthesis and evaluation of two 18F-labeled
analogues of IMPY, in which one of its N-methyl groups
is replaced with [18F]3-fluoropropyl ([18F]FPM-IMPY) or
[18F]2-fluoroethyl ([18F]FEM-IMPY), as prospective PET
radioligands for â-amyloid.
4-Meth yla m in oa cetop h en on e (2). 4-Aminoacetophenone
(15.0 g, 111 mmol), sodium carbonate (35.3 g, 333 mmol), and
iodomethane (13.8 mL, 31.5 g, 222 mmol) in water (400 mL)
were stirred at gentle reflux for 5 h. [CAUTION! The io-
domethane has a low boiling point (41-43 °C), so it refluxes
energetically and tends to evaporate off.] The mixture was then
cooled, and an NaOH solution (20% w/v, 80 mL) was added to
quench the reaction. The mixture was stirred for another 15
min. Water was added to dissolve the inorganic salts. This
mixture, containing the white organic solids insoluble in water,
was extracted three times with CH2Cl2, and the collected
organic phase was dried over Na2SO4. The solvent of the
filtrate was removed by rotary evaporation to afford a crude
solid (15 g), which was purified by flash chromatography (FCC)
(silica gel, hexane-AcOEt ) 3:1 v/v) to give the title compound
as a slightly pale-yellow solid (4.1 g, 27 mmol, 25%) and
4-(N,N-dimethylamino)acetophenone (5.0 g, 30 mmol, 27%).
2. Ma ter ia ls a n d Meth od s
2.1. P r ep a r a tion of Com p ou n d s. Ch em ica ls, Rea gen ts,
a n d Solu t ion s. [125I]-4-(6-iodobenzo[d]thiazol-2-yl)-N,N-di-
methylbenzenamine ([125I]TZDM) was prepared as described
previously.5 A solid form of the peptide Aâ40 monomer in the
form of a trifluoroacetate salt was purchased from Biosource
International Inc. (Camarillo, CA). Reagents used in the
syntheses were purchased from Aldrich Chemical Co. or Fluka
Chemical Company (Milwaukee, WI) and were used without
further purification unless otherwise indicated. Water was
purified through a Millipore water purification system, com-
prising a combination of two filters, one Rio, one reservoir,
and one Milli-Q synthesis system (Bedford, MA). Acetonitrile
(HPLC grade) was obtained from Fisher Scientific (Pittsburgh,
PA). Phosphate buffer (pH 7.4) was made through mixing
solutions of NaH2PO4 and Na2HPO4 (81:19 v/v). Brain tissue
from deceased Alzheimer’s disease (AD) patients was obtained
from Brain Bank of the Clinical Brain Disorders Branch,
National Institute of Mental Health, National Institutes of
Health. Both entorhinal cortex and hippocampal regions were
used.
3
1H NMR (400 MHz, CDCl3): δ 7.82 (2H, d, J HH ) 9.0 Hz),
3
6.54 (2H, d, J HH ) 9.0 Hz), 4.26 (1H, bs, NH), 2.88 (3H, d,
3J HH ) 4.5 Hz, N-Me), 2.49 (3H, s). 13C{1H} NMR (400 MHz,
CDCl3): δ 196.4 (-CO-), 153.0 (Ph-1), 130.7 (Ph-2,6), 126.5
(Ph-4), 111.0 (Ph-3,5), 30.1 (CH3-N), 26.0 (CH3-CO). MS, m/z
(CI): 167 [M + NH4]+, 150 [M + H]+. Anal. Calcd for C9H11
-
NO: C, 72.50; H, 7.40; N, 9.40. Found: C, 72.39; H, 7.27; N,
9.26.
N -(4-Ace t ylp h e n yl)-2,2,2-t r iflu or o-N -m e t h yla ce t a -
m id e (3). Compound 2 (1.25 g, 8.28 mmol) and triethylamine
(TEA) (4.17 g, 41.4 mmol) were dissolved with stirring in
anhydrous CH2Cl2 (45 mL) under dinitrogen. The mixture was
cooled to -10 °C with an acetone-CO2 bath. A cooled (-10
°C) solution of trifluoroacetic anhydride (6.95 g, 33.1 mmol)
in anhydrous CH2Cl2 (15 mL) was added via
a syringe
dropwise over 15 min. The mixture was allowed to warm to
room temperature (RT), and the stirring was continued for 4
h. The reaction was quenched with NH4Cl solution (1 M, 30
mL). The mixture was stirred vigorously for 15 min. The
organic phase was separated off, and the aqueous phase was
extracted with CH2Cl2 (2 × 20 mL). The combined organic
phase was washed with water and brine and dried over Na2-
SO4. The solvent of the filtrate was removed by rotary
evaporation. The crude solid (3.76 g) was purified by FCC
[silica gel, hexane-CH2Cl2 (1:1 v/v) + 3% AcOEt] to yield the
title compound as a transparent oil, which crystallized over
In str u m en t a n d Gen er a l Con d ition s. Analytical HPLC
except for radiosynthesis was performed using a reverse-phase
column (Luna C18, 5 µm, 10.0 mm × 250 mm; Phenomenex)
eluted with concentrated ammonia (0.25%) in acetonitrile-
water at 6.2 mL/min. The chromatography system was fitted
with a continuous wavelength UV-vis detector (Beckman
System Gold 168 detector) and an autosampler (Beckman
System Gold 508 autosampler). For semipreparative Beckman
HPLC, a reverse-phase column (Luna C18, 5 µm, 21.2 mm ×
250 mm; Phenomenex) was eluted at 20.0 mL/min. The HPLC
system was fitted with a manual injector (5 mL injection loop).
The purity of compounds was determined with HPLC moni-
tored for UV absorbance at 280 nm (for IMPY derivatives) or
254 nm (for other aromatic compounds) and expressed as area
percentage of all peaks. The 1H and 13C NMR spectra of all
compounds were acquired on Bruker 400 MHz spectrometers
using the chemical shifts of residual deuterated solvent as the
internal standard; chemical shift (δ) data for the proton and
carbon resonance were reported in parts per million (ppm)
relative to the internal standard. Thin-layer chromatography
(TLC) was performed using silica gel 60 F254 plates from EM
Science, and compounds were visualized under UV light. Flash
chromatography was carried out using a Biotage Horizon
HPFC system (Charlottesville, VA, column sizes: 12 mm ×
150 mm, 25 mm × 150 mm, 40 mm × 150 mm) with hexanes
and ethyl acetate (EtOAc) as eluents with chromatographic
solvent proportions expressed on a volume:volume basis. IR
spectra were recorded using a Perkin-Elmer Spectrum One
1
24 h to a pale-yellow solid (1.92 g, 7.84 mmol, 95%). HNMR
3
(400 MHz, CDCl3): δ 8.01 (2H, d, J HH ) 8.5 Hz), 7.34 (2H, d,
3J HH ) 8.5 Hz), 3.37 (3H, s, N-CH3), 2.62 (3H, s). 13C{1H} NMR
3
(400 MHz, CDCl3): δ 209.5 (-CO-), 156.6 (q, J CF ) 36.4 Hz,
-C(O)CF3), 149.1 (Ph-1), 144.7 (Ph-4), 129.7 (Ph-2,6), 127.6
2
(Ph-3,5), 116.2 (q, J CF ) 288 Hz, CF3), 39.4 (N-CH3), 26.6
(CH3-CO). MS, m/z (CI): 263 [M + NH4]+, 246 [M + H]+. Anal.
Calcd for C11H10F3NO2: C, 53.88; H, 4.11; N, 5.71. Found: C,
53.71; H, 4.20; N, 5.72.
N-[4-(2-Br om oa cetyl)p h en yl]-2,2,2-tr iflu or o-N-m eth yl-
a ceta m id e (4). Compound 3 (1.9 g, 7.7 mmol) and tetra-n-
butylammonium tribromide (4.45 g, 9.2 mmol) were dissolved
in methanol (150 mL). The reaction was stirred at RT
overnight. The solvent was then removed under reduced
pressure, and the residue was dissolved in AcOEt (60 mL).
The mixture was washed twice with water (2 × 30 mL) and
once with brine. The organic solution was then dried over Na2-

2210 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Cai et al.
SO4, and the solvent from the filtrate was removed. The crude
product was purified using FCC (hexane-CH2Cl2 ) 1: 1 v/v/
+ 2% AcOEt), yielding the title compound as a transparent
oil (1.81 g, 5.58 mmol, 72%). 1H NMR (400 MHz, CDCl3): δ
min. The retention time of the compound was 13.0 min. The
solvent of the collected portion was removed under reduced
pressure to generate a white powder (32.2 mg, 33%). 1H NMR
(400 MHz, CDCl3): δ 8.60 (s, 1H), 8.05 (d, 3J HH ) 8.9 Hz, 2H),
3
3
3
3
8.06 (2H, d, J HH ) 8.6 Hz), 7.38 (2H, d, J HH ) 8.2 Hz), 4.43
7.92 (s, 1H), 7.62 (d, J HH ) 9.4 Hz, 1H), 7.52 (d, J HH ) 9.4
3 2
(2H, s, CH2Br), 3.40 (3H, s, CH3N). 13C{1H} NMR (400 MHz,
Hz, 1H), 7.0 (d, J HH ) 8.9 Hz, 2H), 4.86 (dt, J FH ) 48 Hz,
3
3
3J HH ) 5.6 Hz, 2H, CH2F), 3.93 (dt, J FH ) 24 Hz, J HH ) 5.6
3
CDCl3): δ 190.1 (-CO-), 156.6 (q, J CF ) 36.4, -C(O)CF3),
Hz, 2H, CH2N), 3.30 (s, 3H, NCH3). 19F NMR (400 MHz,
145.2 (Ph-1), 134.0 (Ph-4), 130.4 (Ph-2,6), 127.9 (Ph-3,5), 116.1
2
2
CDCl3): δ (relative to CFCl3), -222.5 (hept, J HF ) 45.2 Hz,
(q, J CF ) 288 Hz, CF3), 39.4 (N-CH3), 30.4 (CH2-Br). MS,
3J HF ) 22.6 Hz). UV-vis (EtOH, nm): 355 (ꢀ ) 2.65 × 104),
285 (ꢀ ) 3.63 × 104), 234 (ꢀ ) 4.49 × 104). LC-MS, m/z: 396.2
[M + H]+. HRMS Calcd, m/z (FAB+): C16H16IFN3 ) 396.0373.
Found: 396.0378.
m/z (EI): 323-325 [M]+, 230 [M - CH2Br]+. Anal. Calcd for
C
11H9BrF3NO2: C, 40.77; H, 2.80; N, 4.32. Found: C, 40.87;
H, 2.75; N, 4.18.
2,2,2-Tr iflu or o-N-[4-(6-iod oim id a zo[1,2-a ]p yr id in -2-yl)-
p h en yl]-N-m eth yla ceta m id e (5). Compound 4 (1.8 g, 5.5
mmol) and 5-iodo-2-aminopyridine (1.26 g, 5.73 mmol) were
dissolved in absolute ethanol (65 mL) and stirred under reflux
for 2 h. The mixture was then cooled, and NaHCO3 (0.836 g,
9.94 mmol) was added. The reaction mixture was refluxed for
another 4 h. The solvent was then removed under reduced
pressure, and the residue was collected with AcOEt (70 mL)
and washed with water (40 mL). The organic phase was then
dried with Na2SO4 and the solvent was removed by rotary
evaporation, affording a crude product (2.8 g). This was
purified using FCC (silica gel, hexane-CH2Cl2-AcOEt ) 2:
2: 1 by volume), yielding the title compound as a pale-yellow
solid (1.51 g, 3.4 mmol, 62% yield). The product was recrystal-
lized from Et2O-EtOH. 1H NMR (400 MHz, CDCl3): δ 8.40
6-Iod o-2-[4′-N -(2-flu or op r op yl)m e t h yla m in o]p h e n -
ylim id a zo[1,2-a ]p yr id in e (8, F P M-IMP Y). The same syn-
thetic procedure as that used for FEM-IMPY was used except
that 3-fluoropropyl triflate replaced 2-fluoroethyl triflate. HM-
IMPY (57.4 mg, 0.164 mmol) and 3-fluoropropyl triflate (129.1
mg, 0.614 mmol) were used to generate a yellow solution. The
HPLC retention time of the compound was 13.8 min. A pale-
yellow product was obtained (26.1 mg, 39%). 1H NMR (400
MHz, CDCl3): δ 8.15 (s, 1H), 7.61 (d, 3J HH ) 8.9 Hz, 2H), 7.48
3
3
(s, 1H), 7.21 (d, J HH ) 9.4 Hz, 1H), 7.11 (dd, J HH ) 9.4 Hz,
2
3
4J HH ) 1.6 Hz, 1H), 4.34 (dt, J FH ) 47.2 Hz, J HH ) 5.6 Hz,
3
3
2H), 3.36 (t, J HH ) 7.0 Hz, 2H, CH2-N), 1.81 (dpent, J FH
)
33.4 Hz, 3J HH ) 6.7 Hz, 2H, CH2), 2.83 (s, 3H, NCH3). 19F NMR
(400 MHz, CDCl3): δ (relative to CFCl3), -221.4 (2J HF ) 48.9
3
3
Hz, J HF ) 30.1 Hz). UV-vis, in ethanol (nm): 355 (ꢀ ) 2.08
(1H, s, H-5), 8.02 (2H, d, J HH ) 8.4 Hz, H-2′,6′), 7.82 (1H, s,
× 104), 285 (ꢀ ) 3.30 × 104), 234 (ꢀ ) 3.99 × 104). LC-MS,
3
3
H-3), 7.42 (1H, d, J HH ) 9.4 Hz, H-8), 7.35 (1H, dd, J HH
)
410.2 [M + H]+. HRMS Calcd, m/z (FAB+): C17H18IFN3
)
4
3
9.5, J HH ) 1.6 Hz, H-7), 7.29 (2H, d, J HH ) 8.2 Hz, H-3′,5′),
3.37 (3H, s, CH3). 13C{1H} NMR (400 MHz, CDCl3): δ 156.9
(q, 3J CF ) 35.2 Hz, -C(O)CF3), 144.8, 144.3, 140.4, 134.1, 133.1,
130.6, 127.8 (C-H, Ph), 127.2 (C-H, Ph), 118.7, 116.4 (q, 2J CF
) 286 Hz, CF3), 108.2, 75.5 (C-I), 39.6 (N-CH3). MS, m/z
(CI): 446 [M + H]+, 320 [M - I + H]+. Anal. Calcd for C16H11F3-
IN3O: C, 43.17; H, 2.49; N, 9.44. Found: C, 43.25; H, 2.40; N,
9.32.
410.0529. Found: 410.0534.
D4-Eth ylen e Glycol Bis-tosyla te (9).6 To a mixture of
anhydrous ethylene-d4 glycol-d2 (1.0 g, 15 mmol) and dry
pyridine (4.0 mL) was added p-toluenesulfonyl chloride (5.9
g, 31 mmol) portionwise, keeping the temperature at 0 °C. The
mixture was stored in a refrigerator overnight. The mixture
was then poured into ice-water (60 mL), and the resulting
white precipitate was collected by filtration and washed with
cold water (2 × 25 mL). The solid was recrystallized from
EtOH to give 4.2 g (76%) of the desired product as white
[4-(6-Iodoim idazo[1,2-a ]pyr idin -2-yl)ph en yl]-N-m eth yl-
a m in e (6, HM-IMP Y). Compound 5 (0.9 g, 2.0 mmol) and K2-
CO3 (1.12 g, 8.1 mmol) were added to a solution of MeOH-
H2O (20:1 v/v, 70 mL). This solution was refluxed under
stirring for 1 h. The MeOH was then removed by rotary
evaporation, and the organic residue with some water was
extracted with Et2O (4 × 50 mL). The combined organic phase
was washed with water and dried over Na2SO4. The volume
of the filtrate was reduced to about 30 mL. The precipitate
formed in the concentrated solution was decanted off and
washed twice with Et2O (2 × 10 mL). The ethereal mother
liquor was further concentrated until another batch of the
precipitate appeared. The procedure above was repeated to
afford collectively a pale-yellow solid (0.615 g, 1.7 mmol, 88%).
1
crystals. Mp: 125 °C. H NMR (400 MHz, CDCl3): δ 2.46 (s,
3
3
6H), 7.34 (dm, 4H, J HH ) 8.0 Hz), 7.74 (dm, 4H, J HH ) 8.0
Hz). 13C NMR (400 MHz, CDCl3): δ 21.67, 127.96, 129.95,
132.42, 145.25. GC-MS: 373.86. IR (KBr): 553, 564, 576, 661,
693, 709, 819, 860, 908, 972, 1020, 1067, 1082, 1101, 1179,
1194, 1299, 1311, 1371, 1598 cm-1. HRMS Calcd, m/z (FAB+):
C
16H15D4O6S2 ) 375.0875. Found: 375.0865.
P r od u ction of [18F ]flu or id e. No carrier added (NCA) [18F]-
fluoride in 18O-enriched water was provided by the PET
Center, National Institutes of Health. Briefly, it was obtained
from the 18O(p,n)18F reaction by the proton irradiation of 18O-
enriched water (>95 atom %). This is the most effective method
of generating NCA [18F]fluoride.7
1H NMR (400 MHz, CDCl3): δ 8.30 (1H, s, H-5), 7.74 (2H, d,
3
3J HH ) 8.6 Hz, H-2′,6′), 7.63 (1H, s, H-3), 7.35 (1H, d, J HH
)
3
4
2.2. P r ep a r a tion of Ra d ioliga n d s. NCA [18F]fluoride
(100-250 mCi in 0.25 mL of H218O) was added with Kryptofix
222 (5 mg, 13.3 µmol) and K2CO3 (0.5 mg, 3.6 µmol) in MeCN-
water (95:5 v/v, 0.1 mL) and dried azeotropically by addition-
evaporation cycles (4 × 2 mL of MeCN). Ethylene glycol bis-
tosylate or 1,3-propandiol bis-tosylate (2.0 mg) in MeCN (250
µL) was then added, and the closed glass reaction vessel was
heated at 110 °C for 10 min to generate [18F]2-fluoroethyl
9.4 Hz, H-8), 7.26 (1H, dd, J HH ) 9.5, J HH ) 1.5 Hz, H-7),
3
6.64 (2H, d, J HH ) 8.6 Hz, H-3′,5′), 3.85 (1H, s, N-H), 2.87
(3H, s, CH3). 13C{1H} NMR (400 MHz, CDCl3): δ 149.6, 147.1,
144.0, 132.0, 130.2, 127.3 (C-H, Ph), 122.2, 118.1, 112.5 (C-
H, Ph), 106.3, 74.5 (C-I), 30.7 (N-CH3). MS, m/z (CI): 350
[M + H]+, 224 [M - I + H]+. HRMS Calcd, m/z (CI): C14H13
IN3 ) 350.015 425. Found: 350.016039. Anal. Calcd for C14H12
-
-
IN3: C, 48.16; H, 3.46; N, 12.03. Found: C, 48.06; H, 3.26; N,
11.89.
tosylate ([18F]F(CH2)2OTs) or
[
18F]3-fluoropropyl tosylate
([18F]F(CH2)3OTs), respectively.8 The above solution was trans-
ferred to HM-IMPY (2-5 mg) in a V-vial, and the reaction
mixture was concentrated to ∼50 µL. This residue was heated
at 135 °C for 45 min and then diluted with MeCN-water (3:
1 v/v, 1.5 mL) for direct injection onto an HPLC column (Luna
C-18, 10 mm × 250 mm, Phenomenex) eluted with MeCN +
0.25% triethylamine (TEA) and H2O + 0.25% TEA (isocratic
50:50, v/v) at 6.0 mL/min, with eluate monitored for absorbance
at 365 nm and radioactivity. For detection of radioactive
compounds, a γ-ray detector (Bioscan Flow Count fitted with
a NaI(Tl) detector) was used in series with the UV absorbance
detector. The radioactive fraction with the same retention time
6-Iod o-2-[4′-N -(2-flu or oe t h yl)m e t h yla m in o]p h e n yl-
im id a zo[1,2-a ]p yr id in e (7, F EM-IMP Y). Compound 6 (HM-
IMPY) (87.0 mg, 0.249 mmol) and 2-fluoroethyl triflate (143.3
mg, 0.731 mmol) were dissolved in 2.5 mL of dry CH3CN in a
microwave tube (10 mL volume). The mixture was subjected
to CEM microwave irradiation (200 W, 100 °C, 10 min). A
yellowish-orange solution was generated. The mixture was
injected onto an HPLC column (21 mm × 250 mm; Phenom-
enex), eluted with an aqueous ammonia solution (0.25% v/v
of concentrated ammonia and water)-MeCN ammonia solu-
tion (0.25% v/v of concentrated ammonia and MeCN) gradient
(20-100% MeCN ammonia solution over 15 min) at 20 mL/

IMPY Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2211
as the respective reference ligand (tR: FEM-IMPY, 18 min;
FPM-IMPY, 21 min) was collected. H2O (∼100 mL) was added
to the collected portion of the product. The solution was passed
through a C-18 Sep-Pak cartridge (Waters, Milford, MA),
which was washed at least twice with H2O. The radioactive
product was washed out of the cartridge by absolute ethanol
(1.0 mL) into a V-vial with Tween 80 (∼25 µL). The solvent
was evaporated under a stream of nitrogen at RT. Formulation
of [18F]FEM-IMPY or [18F]FPM-IMPY in saline (0.9% w/v, 0.5
mL) provided a concentrated solution (2-18.4 mCi/mL) for
mouse, rat, and monkey PET experiments.
The final product was analyzed on a Luna column (4.6 mm
× 250 mm; Phenomenex) eluted with MeCN + 0.25% TEA and
H2O + 0.25% TEA (isocratic at 60: 40 v/v) at 2.0 mL/min (tR:
FEM-IMPY, 6 min; FPM-IMPY, 8 min). Radioactivity and
absorbance (365 nm) were monitored to confirm the radio-
chemical and chemical purity of the [18F]FEM-IMPY or [18F]-
FPM-IMPY and to measure its specific radioactivity.
2.3. In Vitr o Bin d in g Assa y a n d Non sp ecific Bin d in g
in Hu m a n AD Br a in Hom ogen a te. The Aâ40 monomer was
aggregated by dissolving this peptide (0.5 mg/mL) in a buffer
[sodium phosphate (pH 7.4, 10 mM) containing EDTA (1 mM)].
The solution was incubated at 37 °C for 46 h with gentle and
constant shaking at 30 strokes per minute. After formation of
the Aâ40 aggregates, the solution was dispensed into separate
vials and stored at -80 °C for future use. Binding was assayed
in 12 mm × 75 mm borosilicate glass tubes as described in ref
4. For inhibition studies, the reaction mixture contained Aâ40
aggregates (10-20 nM in the final mixture, 50 µL) or human
AD brain homogenate (100 µg in the final mixture, 100 µL),
inhibitors (10-5-10-10 M in 10% ethanol, 50 µL), [125I]TZDM
(in 40% EtOH, 0.05 nM in the final mixture, 50 µL, 100 000
dpm/tube), and aqueous EtOH (10% v/v) in a final volume of
1.0 mL. Nonspecific binding was defined by adding thioflavin-T
(10 µM) for [125I]TZDM binding in the same assay tubes. The
mixture was incubated at RT for 3 h for 125I-labeling or 40
min for 18F-labeling, and the bound and the free radioactivity
was separated by a vacuum filtration through Whatman GF/B
filters using a Brandel M-24R cell harvester followed by
washes with aqueous ethanol (10% v/v, 2 × 3 mL) at RT.
Filters containing the bound 125I-labeled ligand were counted
in a γ-counter (Packard 5000) with 70% counting efficiency.
Under the assay conditions, the nonspecifically bound fraction
was less than 15% of the total radioactivity. The results of
inhibition experiments of synthetic amyloid aggregates were
subjected to nonlinear regression analysis using software
EBDA9 by which Ki values were calculated.
F igu r e 2. Determination of specific vs nonspecific binding of
[18F]FEM-IMPY in postmortem AD brain tissue homogenate.
Each data point came from the average of four measurements,
and the bar represents the standard deviation (SD).
FEM-IMPY, 1.0 mg, was dissolved in 1.265 mL of ethanol,
resulting in a stock solution of 2.00 × 10-3 M, which was then
further diluted to 1 × 10-3 M with 10% ethanol. One of the
Alzheimer’s disease tissue slides (Figure 3b) was pretreated
with FEM-IMPY stock solution for 60 min at room tempera-
ture and washed with water for 30 s. All slides were then
placed flat at the bottom of a glass dish and were incubated
for 25 min at RT in [18F]-FEM-IMPY solution (made up with
0.1 mCi in about 0.1 mL of ethanol added to 10 mL of 0.9%
saline). They were then dipped in distilled water for 30 s, then
immersed in ethanol for 15 min and lastly in water for 30 s.
The slides were dried on a hot plate with a stream of cold air
and placed in a cassette with the exposed sides facing up. The
phosphor imaging plate was held with the blue side down. The
entire cassette was placed in the dark at RT overnight for 18F
labeling. Digital autoradiography was acquired using a FUJ I
BAS 5000 phosphorimager (FUJ I, Tokyo, J apan) with
a
resolution of 25 µm.
2.5. P a r tition Coefficien t Deter m in a tion . Ligand (3-5
mg) was dissolved in sodium phosphate buffer (0.1 M, pH 7.4,
6.0 mL) and n-octanol (1.0 mL). The septum-sealed mixture
was vortexed for 1 min, shaken vigorously for another min,
filtered, stood at RT for 10 min, and centrifuged at 5000g for
10 min. An aliquot of organic or aqueous layer was taken
through the septum by syringe. An aliquot of octanol layer (5.0
µL) was added to MeCN (5.0 mL) to prepare a stock solution.
Both aqueous and organic phases (1.0 mL each) were analyzed
by HPLC using MeCN and water with 0.25 vol % of concen-
trated ammonia solution in each as a gradient mixture (5-
95% of the MeCN solution in 15 min) at 2.0 mL/min. Partition
coefficients (PC) were determined by the concentration ratio
of the the organic phase to the aqueous phase.
Binding was also assayed in the same way with [18F]FEM-
IMPY as radioligand and incubation for 40 min. The inhibition
curve of [18F]FEM-IMPY by FEM-IMPY in AD brain tissue
homogenates was fitted [KaleidaGraph 3.5] to the following
equation, which is derived from the modified Hill equation:10
M(Ki)H
[L]H + (Ki)H
F(x) )
+ N
where F(x) is the radioactivity bound relative to control, M is
the maximal percentage of [18F]FEM-IMPY bound specifically,
H is the Hill coefficient (close to 1), [L] is the concentration of
inhibitor, and N is the percentage of nonspecific binding.
2.4. Au tor a d iogr a p h y. Coronal sections of cerebrum from
a confirmed case of Alzheimer’s disease (female, 59-years old,
postmortem interval (PMI) of 47.5 h) and a normal control
(female, 64-years old, PMI of 28.5 h) were frozen rapidly in a
50:50 mixture of dry ice and isopentane, sealed in a plastic
bag, and stored at -76 °C before sectioning. Frozen blocks of
the medial temporal lobe (hippocampal region) were sectioned
at a thickness of 14 µm, mounted on gelatin-coated glass slides,
dried, and stored under desiccant at -76 °C. Slide-mounted
tissue sections were removed from the freezer, thawed at room
temperature for 20 min, and air-dried. They were then
2.6. In Vivo Biod istr ibu tion in Nor m a l Mice. For
radioligand injection, a 30 gauge needle was attached to a
polyethylene catheter (PE 10) and inserted into the tail vein
of male ICR mice (2-3 months old, average of 20-30 g). The
needle and catheter were secured with tissue adhesive. For
arterial blood sampling, femoral artery catheter (PE 10)
insertion with 1% isoflurane anesthesia was performed. Sub-
cutaneous tunneling and tethering at the nape secured the
arterial catheter. When fully anesthetized in the induction
chamber, the mice were placed on a heating pad. For dynamic
scanning, heads and bodies of mice and rats were fixed with
tape and kept with 1% isoflurane anesthesia via a nose cone.
Body temperature was monitored with a rectal probe and
maintained at 36-37 °C. Then NCA radioligand (400-450 µCi)
in saline (0.9% w/v, 0.03-0.15 mL) with Tween solution (0.1%)
was injected and flushed with saline (0.070 mL).
immersed in
a 10% neutral phosphate-buffered formalin
solution at room temperature for 1 h, delipidized in xylene for
40 min, and rehydrated through ethanols to water.

2212 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Cai et al.
We used a small animal PET camera [Advanced Technology
Laboratory Animal Scanner (ATLAS)] with an 11.8 cm diam-
eter aperture, 2 cm axial field-of-view ring type research
scanner surrounding the animal with 15 mm deep, 18 LGSO
(7 mm)/GSO (8 mm) phoswich detector modules.11 This
phoswich design achieved good resolution [1.1-1.8 mm full
width at half-maximum (fwhm)] at the center with 3D OSEM
(ordered subset expectation maximization) and 2D OSEM/
single slice rebinning (SSRB) reconstruction methods, while
preserving a sensitivity of 2.7% (100-650 keV).
For dynamic scanning, sequential brain scanning was
performed in the same bed position immediately after iv
injection of the radioligand; the scan lasted 1-2 h. Sequential
time-activity curve (TAC) data were acquired immediately
after radioligand injection to evaluate the brain uptake and
washout in each mouse. The %ID/brain, %ID/g, and %ID-kg/g
were calculated by both TAC of the PET scan and in vitro
tissue counts (determined under section 2.7).
2.7. Meta bolism of Ra d ioliga n d s in Nor m a l Mice.
Consecutive blood samples (each 10-35 µL for mouse) that
were taken during PET scanning and dissected homogenized
brain taken after scanning were analyzed to evaluate the
metabolites and parent radioligand in the blood and brain. (As
a general rule, 10% of the total blood volume can be collected
in one experiment every 2-4 weeks. Total blood volume can
be calculated as approximately 7.5% of body weight.)
The metabolites were determined with reverse-phase HPLC
analysis on a column (Nova-Pak C18 Radial-Pak Cartridge, 4
µm, 8 mm × 10 mm; Waters Corp., Milford, MA) and with
radial compression module RCM-100 and eluted with
a
mixture of MeOH, H2O, and TEA (70:30:0.1) at 1.0 mL/min.
The HPLC was equipped with a Beckman System Gold 126
solvent module and a flow-through NaI(Tl) scintillation detec-
tor (model FC-4000) and rate meter (Bioscan, Washington,
DC).
2.8. In Vivo Biod istr ibu tion in Rh esu s Mon k ey. A male
rhesus monkey (7.12 kg) was initially immobilized with
ketamine (15 mg/kg) and subsequently anesthetized with
isoflurane (1.5%) for the duration of the PET scan. The monkey
was placed prone in the PET camera (GE Advance tomograph;
GE Medical Systems, Waukesha, WI). A urinary catheter was
inserted and clamped so that the activity overlaying the
bladder represented the total urinary excretion during the
scanning interval. ECG (Electrocardiogram), body tempera-
ture, and heart and respiration rates were measured through-
out the experiment. Before injection of the radioligand, each
animal received a brain transmission scan (32 min) with two
rotating rods containing 68Ge for subsequent attenuation
correction of the emission image. Brain emission scans were
performed following the intravenous administration of [18F]-
FEM-IMPY (2.9 mCi). Serial dynamic transaxial images were
acquired for a total of 120 min for the head in 3D mode (33
frames with scan duration ranging from 30 s to 5 min). The
tomographic images were analyzed with PMOD 2.4 (pixelwise
modeling computer software; PMOD Group, Zurich, Switzer-
land).12 All frames of the original reconstructed PET data were
summed, and this summed image was coregistered to a T1-
weighted magnetic resonance (MR) image acquired separately
on a GE 1.5 T Signa MR scanner (GE Medical Systems,
Waukesha, WI), using image analysis software MEDx (Sensor
Systems Inc., Sterling, VA). The summed PET image was then
fused with the coregistered MR image using an image fusion
tool in PMOD. Several regions of interest (ROIs) for the source
organs were then manually defined on this fused image with
anatomical structures identified on the MR image.
3. Resu lts
F igu r e 3. Autoradiography of [18F]FEM-IMPY and immuno-
fluorescent (IF) detection of â-amyloid plaques in postmortem
brain tissue sections of medial temporal lobe from an AD
patient and an age-matched control: (a) AD tissue + [18F]FEM-
IMPY; (b) AD tissue + IF protocol; (c) AD tissue pretreated
with FEM-IMPY + [18F]FEM-IMPY; (d) control tissue + [18F]-
FEM-IMPY.
3.1. Ch em ica l Syn th esis. The nucleus of imidazo-
[1,2-a]pyridines is generally made by condensation
between a 2-aminopyridine and an R-halogenoketone or
aldehyde in the presence of a mild base such as sodium
bicarbonate according to Scheme 1. The pyridine nitro-
gen attacks the R-carbon of the ketone, displacing the

IMPY Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2213
Sch em e 1
monoiodination of 4-methylaminoacetophenone with I2
and Selectfluor17 in MeOH gave a mixture of compounds
with many ring-iodinated byproducts.
An alternative synthesis of HM-IMPY is the mono-
demethylation of the imidazopyridine, IMPY.15 This
reaction was attempted in different ways according to
methods reported in the literature for the demethylation
of N,N-dimethylanilines (Scheme 5). In the first ap-
18
proach TiCl4/CH2Cl2 gave only polymerized products.
In the second, tetra-n-butylammonium periodate, a
potent oxygen donor, was used to hydroxylate one of the
methyl groups in the presence of a metalloporphyrin
catalyst.19 The R-amino alcohol was then expected to
decompose easily to give the secondary amine and
formaldehyde. However, this method had to be aban-
doned because the reaction was low-yielding with many
side reactions. The third approach20 was more effective
and allowed us to obtain the desired imidazopyridine
in 30% yield. The proposed mechanism consists of a
double single-electron-transfer oxidation of the nitrogen
center by the iodosobenzene, giving iodobenzene and an
iminium ion. The iminium ion should be attacked by
halide and forming a pyridinium salt. The salt is
deprotonated by the weak base. The imine group gener-
ated attacks the carbonyl group intramolecularly to give
an amino alcohol intermediate, which dehydrates spon-
taneously to form the imidazole.
The synthesis of HM-IMPY is shown in Scheme 2. The
secondary amine 2 was protected using trifluoroacetic
anhydride.13 The trifluoroacetyl group gives multiple
advantages. The bromination can be done in one step
and under mild conditions, using tetra-n-butylammo-
nium tribromide in MeOH according to a modified
procedure.14 This bromination is regioselective, occur-
ring only on the methyl group, thus avoiding byproducts
from bromination on the ring. This occurs because the
trifluoroacetyl group depresses the ER (electron reso-
nance) effect of the N-methylamino group. The reaction
solvent appears to be critical for this reaction. The
trifluoroacetyl group as a protecting group also provides
for easy removal at the end of synthesis by weak bases
under mild conditions. The condensation reaction be-
tween the aminopyridine derivative and bromoketone
was conducted under mild basic conditions.15 2-Amino-
5-iodopyridine was available either commercially or
through electrophilic iodination at the 5-position of
2-aminopyridine using I2 and periodic acid in acid media
(Scheme 3).16 Ideally, HM-IMPY might be derived from
the condensation reaction between 5-iodo-2-aminopyri-
dine and 2-bromo-4′-methylaminoacetophenone, but the
attempt to synthesize the latter by the usual process
(Scheme 4) was frustrated by the poor yield and complex
mixture of products at the dibromination step. The
-
the N3 ion, forming an aminomethylene azide deriva-
tive. Quenching the reaction mixture with an aqueous
solution of NaHCO3 allows the azide to be displaced by
H2O to form the R-amino alcohol, which dissociates
quickly to form formaldehyde and the secondary amine.
The problem with this procedure is that the reaction is
not reproducible enough on scale-up. Moreover, the
product is quite labile under the oxidative reaction
conditions.
3.2. Ra d iola belin g. A “one-pot” radiosynthesis of
[18F]FEM-IMPY or [18F]FPM-IMPY was developed ex-
perimentally and gave radiochemical yields ranging
from 26% to 51% (mean, 35%), corrected to the start of
radiosynthesis, and in >93% radiochemical purity
(Scheme 6). Specific radioactivity was determined to be
2.3-17.9 Ci/µmol at EOS (end of synthesis).
3.3. Bin d in g to Aâ40 Aggr ega tes in Solu tion a n d
in AD Br a in Hom ogen a tes. Both FEM-IMPY and
FPM-IMPY compete well with [125I]TZDM for binding
to Aâ-40 aggregates, a previously established selective
probe.5 This is one of three binding sites reported in the
literature for thioflavin-type ligand series. The other two
are for a Congo red series5 and an FDDNP series.21 The
Sch em e 2

2214 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Cai et al.
Sch em e 3
Sch em e 4
F igu r e 4. Time-activity curve in brain and skull after iv
injection of [18F]FEM-IMPY or D4-[18F]FEM-IMPY in normal
mice. Each point represents the average of four mice. The
standard error of each point for the brain is within 3-12%
and that for the skull is 5-25% after 1.2 min. For clarity, the
error bar is not shown. D4-skull represents data from D4-
[18F]FEM-IMPY.
Sch em e 5
this and subsequent data expressed as the mean ( SD).
This binding constant compares well with that deter-
mined from the synthetic amyloid aggregates (Ki ) 27
( 8 nM).
3.4. La belin g AD Br a in Section s. The pattern of
radioactivity in the adjacent coronal AD brain specimen
without competitor showed specific binding of [18F]FEM-
IMPY to cortical areas containing neuritic plaques (NP)
(Figure 3a). Specific binding of [18F]FEM-IMPY to
regions of cortical gray matter with NPs was eliminated
in the AD specimen pretreated with nonradioactive
FEM-IMPY, compared to autoradiography without com-
petitor (Figure 3c). No specific binding was observed for
a control brain specimen (Figure 3d).
Ta ble 1. The log D and Ki Values of IMPY Derivatives against
[
125I]TZDM for Binding to Aâ(1-40) Aggregates
ligand
log D
Ki [nM]
IMPY
FEM-IMPY
FPM-IMPY
3.58 ( 0.44
4.41 ( 0.10
4.60 ( 0.19
15.0 ( 5.0
27 ( 8
40 ( 5
3.5. Stu d ies in Nor m a l Mice. In the averaged TAC
for four individual mice administered with [18F]FEM-
IMPY (Figure 4), the brain uptake of the activity (%ID-
kg/g) peaks between 0.5 and 2 min after injection. The
elimination of the activity follows an exponential decay
but is near zero after about 20 min. The individual TAC
data were well fitted to the double-exponential formula
Ki values decrease and the experimentally determined
log D values (log P at pH 7.4) increase with increasing
length of the side chain on amino group (Table 1).
With increasing concentrations of FEM-IMPY, [18F]-
FEM-IMPY binding to Aâ-amyloid plaques of the hu-
man AD brain homogenates should decrease and level
off after all specific binding sites have been displaced.
We observed that about half of all binding was displace-
able (or specific, Figure 2). The curve was fitted to a
modified Hill equation to accommodate the nonspecific
binding of the ligand, yielding Ki ) 83 ( 69 nM (with
y ) a - b exp(-ct) + d exp(-et)
where y is the activity, a is the residual radioactivity
trapped inside the brain, b and d are constants, c is the
Sch em e 6

IMPY Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2215
Ta ble 2. Curve-Fitting Constants of TAC Obtained after Intravenous Administration of Radiolabeled IMPY or Derivatives to Mice
residual activity
administered
radioligand
max activity
[(ID%-kg)/g]
time at max
activity [min]
washout
at 120 min,
rate of bone uptake,
rate [min-1
]
102 × [(ID%-kg)/g]
103 × [min-1
]
[
[
[
125I]IMPY4
0.216 ( 0.010
0.161 ( 0.019
0.157 ( 0.018
2
0.50
1.83
0.122 ( 0.024
0.334 ( 0.021
0.127 ( 0.006
1.08 ( 0.22
11.1 ( 0.05
4.79 ( 0.11
NA
1.60 ( 0.294
7.28 ( 2.85
18F]FEM-IMPY
18F]FPM-IMPY
F igu r e 5. Time-activity curve in brain and skull after iv
injection of [18F]FPM-IMPY in normal mice. Each point
represents the average of four mice. The standard error of each
point for the brain is within 2-12% and that for the skull is
7-38% after 1.2 min. For clarity, the error bar is not shown.
F igu r e 6. Time-activity curves of different regions of brain
after iv injection of [18F]FEM-IMPY in a rhesus monkey.
Ta ble 3. Uptake and Elimination Rate Constants of
Radioactivity in Various Brain Regions of a Rhesus Monkey
after Intravenous Administration of [18F]FEM-IMPY
component
elimination [min-1
]
uptake [min-1
]
rate of uptake, e is the rate of elimination, and t is the
time after injection. The first exponential portion in the
equation represents the uptake of the activity in brain,
and the second exponential portion represents the
elimination of the activity.
frontal cortex
striatum
brainstem
cerebellum
jaw bone
0.0606 ( 0.0024
0.0859 ( 0.0037
0.0488 ( 0.0020
0.0854 ( 0.0040
NA
0.859 ( 0.030
0.972 ( 0.040
1.00 ( 0.040
1.09 ( 0.050
0.373 ( 0.058
The average TAC from four individual mice injected
with [18F]FPM-IMPY is shown in Figure 5. Assuming
an average brain weight of 0.40 and 25 g body weight,
analogous fitting was performed for [125I]IMPY data
from Kung et al.4 The constants from the individual
curve fittings are summarized in Table 2.
The residual activity in brain at 2 h was much greater
for [18F]FEM-IMPY than for [125I]IMPY.4 Much less
residual radioactivity was left for [125I]IMPY itself
during the time course of the measurement, while a
substantial amount of radioactivity was trapped inside
the brain for [18F]FEM-IMPY. Values for curve-fitting
constants for [18F]FPM-IMPY fall between [18F]IMPY
and [18F]FEM-IMPY with respect to residual brain
activity and clearance.
Substantial bone uptake of radioactivity was observed
in the skull after the administration of [18F]FEM-IMPY
or [18F]FPM-IMPY to normal mice. The averaged bone
uptake TACs for each radioligand in four individual
mice are shown in Figures 4 and 5. The bone uptake
TAC of [18F]FEM-IMPY can be fitted to a linear function
except for a few initial points (less than 5 min), while
that of [18F]FPM-IMPY is fitted more accurately with
an exponential curve. The average rate for bone uptake
of radioactivity is faster for [18F]FPM-IMPY than for
[18F]FEM-IMPY (Table 2). A significant reduction in
skull bone uptake of radioactivity was observed for [18F]-
D4-FEM-IMPY (Figure 4 and Table 2). The radioactivity
in the skull bone was reduced by ∼34% over the span
of the experiment upon substitution of the 4-ethylene
protons by deuteriums. However, there was no effect of
deuterium substitution on brain uptake or clearance of
radioactivity.
3.6. Stu d ies in Mon k ey. The TACs of [18F]FEM-
IMPY in a rhesus monkey are shown in Figure 6 and
Table 3. Brain activity increased quickly and reached
substantial levels, with peak values of 0.26-0.35 %ID-
kg/g at 3-6 min after injection. Washout of brain
activity was similar in all brain regions with a mono-
exponetial washout rate of 0.05-0.09 min-1 (corre-
sponding to a half-life T1/2 ) 8-14 min). Activity in bone
increased in an exponential fashion and reached levels
of ∼0.1 %ID-kg/g, similar to residual activity in brain
(Figure 6).
3.7. Meta bolite An a lysis. 3.7.1. Meta bolism in
Blood in Vivo. The distribution of radioactivity among
chemical species following injection of the radioligand
was monitored by HPLC. The TACs for several compo-
nents of activity in whole blood are shown in Figure 7.
Curves for parent, lipophilic metabolite (L-metabolite),
and total radioactivity were fitted to a single-exponen-
tial decay, and the curve for polar metabolite (P-
metabolite) was fitted to a double-exponential function.
The half-lives for all radioactive components were less
than 2 min. This is much shorter than those within the

2216 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Cai et al.
F igu r e 7. Time-activity curves of different radioactive
components after iv injection of [18F]FEM-IMPY in the in vivo
whole blood of normal mice.
F igu r e 8. Time-activity curves of different radioactive
components after incubation of [18F]FEM-IMPY in the in vitro
brain homogenate of normal mice.
Ta ble 4. Elimination Rate Constants for [18F]FEM-IMPY and
Radioactive Derivatives in Vivo in Whole Blood
Sch em e 7
component
parent
P-metabolite
L-metabolite
uptake [min-1
]
elimination [min-1
]
NA
1.14 ( 0.16
0.576 ( 0.980
NA
NA
0.575 ( 2.21
0.722 ( 0.347
0.801 ( 0.158
total radioactivity
brain of the same animal. The rate constants are
summarized in Table 4. No significant difference was
found for [18F]D4-FEM-IMPY (data not shown), com-
pared with [18F]FEM-IMPY. Only a single polar radio-
active metabolite was seen in plasma at <30 min after
injection of [18F]FPM-IMPY.
and parent [18F]FEM-IMPY. The retention time of
P-metabolite was very close to that of added [18F]fluoride
under the same conditions. Each fraction was collected,
allowed to decay, and analyzed by LC-MS (electro-
spray). The potential metabolites of FEM-IMPY were
examined in each fraction according to the proposed loss
or addition of fragment(s) from the FEM-IMPY mol-
ecule, namely, (1) hydroxylation by either the addition
of an oxygen atom at one of the nitrogen atoms or the
insertion of an oxygen atom into one of the C-H bonds
(m/z 412), (2) N-demethylation at the tertiary amino
group (m/z 382), (3) N-defluoroethylation at the tertiary
amino group (m/z 350), and (4) simultaneous N-de-
methylation and N-defluoroethylation at the tertiary
amino group (m/z 336). The chromatographic separation
followed by electrospray analysis generated m/z 382 and
m/z 350 ions specific for metabolites of N-demethylated
and N-defluoroethylated FEM-IMPY, respectively, but
not for hydroxylated metabolites in the HPLC fractions.
No significant amount of primary amine, resulting from
complete N-dealkylation of FEM-IMPY, was observed.
The metabolic pathways consistent with the MS analy-
sis are shown in Scheme 7.
3.7.2. Meta bolism in Br a in in Vivo. HPLC analysis
of brain homogenates obtained 2 h after injection of [18F]-
FEM-IMPY showed virtually no parent compound. The
only radioactive species observable were polar (P-
metabolite), presumably fluoride ion or a related small-
molecule ionic species such as [18F]FCH2CO2-. Analo-
gous metabolites were observed for [18F]FPM-IMPY.
3.7.3. Meta bolism in Br a in Tissu e in Vitr o. Incu-
bation of mouse brain homogenates with each radio-
ligand at 37 °C for various lengths of times resulted in
radioactive metabolites. HPLC analysis showed the
same pattern of metabolism as described for whole blood
in vivo. The TACs for [18F]FEM-IMPY are displayed in
Figure 8. For unknown reasons, degradation of the
radiolabeled ligand did not go to completion but only to
about 10% conversion. The curves were fitted to mono-
and biexponential functions (to account for the ac-
cumulation and consumption). No significant difference
was observed for the disappearance of [18F]FEM-IMPY
(t1/2 ) 15 min) compared to that of [18F]D4-FEM-IMPY
(t1/2 ) 14 min). However, [18F]FPM-IMPY (t1/2 ) 11 min)
degraded more quickly than [18F]FEM-IMPY.
4. Discu ssion
An efficient chemical synthesis for the common pre-
cursor, HM-IMPY, has been developed among several
available synthetic routes to the compound. This allows
the syntheses of FEM-IMPY and FPM-IMPY, either as
reference compounds or as 18F-labeled ligands. HM-
IMPY is soluble in acetone, CH2Cl2, and CHCl3 but is
insoluble in MeCN and water. It is stable in solid form
but decomposes gradually in solution when exposed to
3.7.4. LC-MS An a lysis of In ter m ed ia tes. Incuba-
tion of a [18F]FEM-IMPY and FEM-IMPY (carrier, 15
µg) mixture with mouse brain homogenate provided
sufficient material for the isolation of the radioactive
metabolites by HPLC in three fractions: polar metabo-
lite (P-metabolite), lipophilic metabolite (L-metabolite),

IMPY Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2217
air. The most reactive sites of the molecule toward
electrophiles such as alkyl halides and triflates are the
aromatic amino group and the aromatic imino group,
with the reactivity of the latter rarely observed. It also
reacts with acid, but the exact location is unclear at this
time; it is probably the imino site. Both FEM-IMPY and
FPM-IMPY are more lipophilic than HM-IMPY or
IMPY. The order of the retention times on reverse-phase
HPLC is HM-IMPY < IMPY < FEM < FPM.
HM-IMPY reacts slowly with ethylene glycol bis-
tosylate at 130-150 °C. For example, the reaction is less
than 5% complete after 10 min under microwave condi-
tions (300 W and 150 °C). However, addition of [K-
(Kryptofix 222)]+[F]- accelerates the alkylation to gen-
erate FEM- or FPM-IMPY in a 70-80% yield under
anhydrous conditions with the same microwave condi-
tions. Alkylation also occurs if 2-fluoroethyl triflate or
3-fluoropropyl triflate or the corresponding tosylate is
used. This may explain the success of the “one-pot”
synthesis of [18F]FEM-IMPY and [18F]FPM-IMPY, where
excess ethylene glycol bis-tosylate or 1,3-propanediol bis-
tosylate does not interfere with the radiosynthesis.
Indeed, premixing of HM-IMPY, ethylene glycol bis-
tosylate or 1,3-propanediol bis-tosylate, and [K-(Krypto-
fix 222)]+[18F]- generated the same [18F]FEM- and
[18F]FPM-IMPY in a truly “one-pot” reaction. This
suggests that FCH2CH2OTs, not the N-tosylate of IMPY,
is the reactive intermediate in the radiosynthesis.
exponential washout of [123I/125I]IMPY, the washout of
radioactivity from each probe examined here was bi-
phasic, being rapid over the first 20 min and much
slower thereafter. Residual brain radioactivity was 4.5%
ID/g for [18F]FEM-IMPY and 2.1% ID/g for [18F]FPM-
IMPY at 2 h after injection.
Homogenates of ex vivo mouse brain after injection
of the radioligand were analyzed by HPLC. The parent
compounds of both ligands disappeared rapidly. The
residual activity represented polar metabolite(s), one of
which is probably the [18F]fluoride ion. This means that
the residual radioactivity observed for both [18F]FEM-
IMPY and [18F]FPM-IMPY came from the polar me-
tabolite and not from nonspecific binding. Lipophilicity
of the ligands does not play any role in this case.
Detailed analysis of the in vivo whole blood of mice
showed that the metabolism in blood is even faster than
that in the brain. The contribution of the lipophilic
intermediate (L-intermediate) is unclear at this time.
Given its low concentration observed both in vivo and
in vitro, it may not contribute significantly to the TACs.
The in vivo instability of the ligands within mouse
brain is also observed in vitro. Identical metabolites for
[18F]FEM-IMPY and [18F]FPM-IMPY were observed
with both in vivo and in vitro experiments. However,
the in vitro metabolism ended after about a 10%
consumption of the radioligand, which may have been
caused by instability of the enzymes or depletion of
cofactors responsible for the metabolism of the radioli-
gand. In the brain, the enzyme may be monoamine
oxidase, which has been shown to be involved in the
metabolism of dopamine and other amine compounds.22
In the periphery, the enzyme responsible could be MAO
or a P-450 subtype.
The in vitro metabolites were isolated with the help
of added carrier. Mass spectrum analysis of the me-
tabolites demonstrated the presence of a dealkylated
aromatic amino group, consistent with the assumption
of the instability of tertiary amino groups in an oxidative
environment. Indeed, an analogous oxidative reaction
was used in the synthesis of HM-IMPY (Scheme 5).
The binding affinities of IMPY, FEM-IMPY, and
FPM-IMPY to synthetic Aâ aggregates are comparable
but decrease gradually in the same order. This suggests
that there is increasingly unfavorable steric interaction
between IMPY derivatives and the protein pocket with
an increase in alkyl chain length. However, removing
one of the alkyl groups on the amino group dramatically
reduces the binding affinity. The Ki values for HM-
IMPY and FEH-IMPY (6-iodo-2-[4′-N-(2-fluoroethyl)-
amino]phenylimidazo[1,2-a]pyridine) are 140 ( 20 and
161 ( 30 nM (our present work), a 5-fold lower affinity
than those of FEM-IMPY and FPM-IMPY. This suggests
that the binding pocket around the amino group is
highly hydrophobic. If the amino group is replaced by a
hydroxy group, the Ki is greater than 1000 nM (this
work). The Ki is 451 ( 59 nM if the amino group is
substituted by a methoxy group (this work). Both the
number of alkyl groups and their electron-donating
ability contribute to the binding affinity of IMPY
derivatives to Aâ40 aggregates. In conclusion, the
binding pocket around the amino group is a small
hydrophobic pocket.
Substantial uptake of [18F]fluoride in the skull was
also clearly observed for both [18F]FEM-IMPY and [18F]-
FPM-IMPY. A deuterium isotope effect is common for
both chemical and biological reactions and has been
observed for the pharmacokinetics of certain PET ra-
diotracers.23 [18F]D4-FEM-IMPY was synthesized and
evaluated for its effect on metabolism in both brain and
periphery. No deuterium effect was observed for the
brain uptake of the radioligand, and no clear reduction
of residual brain radioactivity was found. However, a
significant reduction of the bone uptake of radioactivity
in the skull was clearly identified. This suggests that
the metabolism for the IMPY derivative in the periphery
but not in the brain is sensitive to small structural
changes in the molecule.
All of the IMPY derivatives reported here have quite
high lipophilicity, with log D values between 3.6 and 4.6
at pH 7.4. This high lipophilicity may explain the
relatively high nonspecific binding of [18F]FEM-IMPY
to human AD brain homogenates, which is approxi-
mately 50% of the total binding.
The results of in vivo biodistribution studies in normal
mice showed that both FEM-IMPY and FPM-IMPY
easily penetrate the blood-brain barrier after intra-
venous injection. In PET experiments, high uptake of
activity was obtained for both probes (6.4% ID/g for [18F]-
FEM-IMPY and 5.7% ID/g for [18F]FPM-IMPY at 0.5-1
min). These values compare well with [123I/125I]IMPY
(7.2% ID/g in 2 min).4 However, in contrast to the single-
In vivo distribution studies of the radioligands in
rhesus monkeys showed species differences in the rate
of metabolism of these radioligands. Both residual brain
activity and bone uptake were substantially less in
rhesus monkey compared to mice. No significant differ-
ence in activity uptake among regions of rhesus brain
was observed, consistent with the absence of amyloid
plaques in these normal monkeys.

2218 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
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In a preliminary manner, we double-labeled indi-
vidual tissue sections with [18F]FEM-IMPY and with
fluorescent-labeled antibody (DAKO) to amyloid (Figure
3b). The sections showed a general correspondence of
colabeling. However, the phosphorimager plate demon-
strated clumps of activity that were enriched in the gray
matter, with individual spots about 100 µm in size and
roughly spherical in shape. Thus, the resolution of the
plate was degraded relative to the primary distribution
of radioligand perhaps because of the distance traveled
by the energetic â+ positron before annihilation by an
electron. In any case, the general pattern of the auto-
radiographic labeling (each area of activity measuring
about 100 µm in diameter) showed a strong visual
correlation with higher densities of fluorescently labeled
amyloid plaques (each area about 10 µm in diameter).
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FEM-IMPY. The nonspecific binding of [18F]FEM-IMPY
in AD brain sections is much less compared with that
determined from in vitro binding assays (50%). This
may come from the xylene defatting step used for the
AD brain sections pretreatment.
Con clu sion s
The two 18F-labeled IMPY derivatives enter the brain
of normal mice readily and quickly. However, the
washout shows a biphasic behavior. The first phase is
similar to the behavior of [125I]IMPY. The brain reten-
tion of activity may be due to trapping of polar metabo-
lite(s). The probes are quickly metabolized in plasma,
with substantial defluorination and bone uptake. Rhesus
monkey showed much slower metabolism for these
derivatives than mouse. These results suggest that
IMPY derivatives are promising candidates as PET
imaging agents for Alzheimer’s disease, based on their
favorable pharmacokinetics and their high specific
labeling of â-amyloid plaques in vitro. However, more
work is required to fine-tune these structures in order
to reduce metabolism, increase affinity, and decrease
lipophilicity. Radiolabeling beyond the N-alkyl chains
in these structures is likely to be preferred.
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Ack n ow led gm en t. We gratefully thank Dr. Shuiyu
Lu (MIB, NIMH) for his help in the microwave reac-
tions, Mr. J insoo Hong (MIB, NIMH) for assistance in
determining the specific radioactivity of the radioli-
gands, Mr. Kun Park (MIB, NIMH) for determination
of log D, Dr. Senda Beltaifa (CDBD, NIMH) for the
initial brain tissue processing, and Dr. J oel E. Kleinman
(CDBD, NIMH) for access and discussion of postmortem
brain tissues.
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