New diphenylacetylenes as probes for positron emission tomographic imaging of amyloid plaques

Article abstract of DOI:10.1021/jm070090j

A series of ¹⁸F fluoropegylated diphenylacetylenes as probes for binding to Aβ plaques were successfully prepared. These relatively rigid acetylenes, 12a, 12b, 14a, and 14b, displayed high binding affinities in postmortem AD brain homogenates (

Full text of DOI:10.1021/jm070090j

J. Med. Chem. 2007, 50, 2415-2423
2415
New Diphenylacetylenes as Probes for Positron Emission Tomographic Imaging of Amyloid
Plaques
Rajesh Chandra,† Shunichi Oya,† Mei-Ping Kung,† Catherine Hou,† Lee-Way Jin,‡ and Hank F. Kung*,†,§
Departments of Radiology and Pharmacology, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104, and Department of Pathology,
UniVersity of California Health System, Sacramento, California 95817
ReceiVed January 23, 2007
A series of 18F fluoropegylated diphenylacetylenes as probes for binding to Aâ plaques were successfully
prepared. These relatively rigid acetylenes, 12a, 12b, 14a, and 14b, displayed high binding affinities in
postmortem AD brain homogenates (Ki ranging from 1.2 to 2.9 nM). In vivo biodistribution in normal mice
exhibited excellent initial brain penetrations (4.42, 4.55, 5.41, and 6.78% dose/g at 2 min for [18F]12a, 12b,
14a, and 14b, respectively). [18F]12b and [18F]14b, with a longer fluoropegylated unit, that is, n ) 3, showed
faster brain washout at 30 min postinjection (0.42 and 1.57% dose/g) as compared to the shorter
fluoropegylated chain ligands, that is, [18F]12a and [18F]14a (1.03 and 3.69% dose/g). Autoradiography and
homogenate binding confirmed the high binding signal due to Aâ plaques. These preliminary results suggest
that the novel diphenylacetylenes may be potentially useful for imaging of Aâ plaques in the brain of patients
with Alzheimer’s disease.
Introduction
are some mild cognitive impairment cases that convert to AD,
while those with lower PIB uptake in the cortex appear to have
less propensity to convert to AD.20-22 Additional tracers labeled
with 18F (T1/2 ) 110 min, â+, commonly produced by a
cyclotron) may be even more useful as PET imaging agents
for detection and quantification of Aâ aggregates since the half-
life of 18F (110 min) is 5.5 times longer than 11C (T1/2 ) 20
min, â+). Using 18F tracers, the manufacturing and distribution
can be centralized, which will significantly simplify the clinical
application.
A highly lipophilic tracer, [18F]FDDNP, 3 (Figure 1), for
binding to both tangles and plaques has been reported.23
Preliminary studies in humans suggested that [18F]FDDNP
showed a higher retention in regions of the brain suspected of
having tangles and plaques,17,24-26 therefore, it is not selective
for measuring Aâ burden in the AD brain. Fluorinated PIB and
related neutral thioflavin derivatives, such as BTA-1, have also
been reported.11,27 However, clinical studies of the 18F-labeled
PIB have not yet been published in a full paper.
Alzheimer’s disease (ADa) is a neurodegenerative disease
affecting millions of elder people. Major neuropathology
observations of postmortem AD brains depict the presence of
senile plaques (containing â-amyloid (Aâ) aggregates) and
neurofibrillary tangles (highly phosphorylated tau proteins). The
exact mechanisms leading to the development of AD are not
fully understood, however, the formation of Aâ plaques,
consisting of â-sheets of Aâ protein aggregates, in the brain is
a pivotal event in the pathology of Alzheimer’s disease.1-7
Developing specific probes for in vivo imaging studies of Aâ
plaques may be important for the diagnosis and monitoring of
AD patients.1,8-11 The imaging technique could improve
diagnosis by identifying potential patients with Aâ plaques in
the brain that are likely to develop AD. When antiplaque drug
treatments become available, the imaging of Aâ plaques in the
brain may serve as an essential tool for monitoring the
progression and the treatment of the disease.
Development of Aâ plaque-specific imaging agents has been
reported previously (for review see refs 10-14). Different PET
(positron emission tomography) and SPECT (single photon
emission tomography) tracers, such as [11C]PIB, 1,15 [11C]SB-
13, 2,16 [18F]FDDNP, 3,17,18 and [123I]IMPY, 4,19 have been
tested clinically and demonstrated the potential utility of in vivo
imaging of Aâ plaque deposition in the brain. Using PIB/PET
to study the relationship between Aâ plaque burden and AD
neurological measurements, the results seem to suggest that there
We have recently prepared potential PET imaging probes
based on stilbene, 5, and styrylpyridines,28-30 6, (see Figure
1). The fluoropegylated group or simply an ethylene glycol
group was retained to reduce the lipophilicity and maintain
neutrality. As part of the continuing effort in developing probes
for imaging Aâ plaques in the brain, we have prepared a series
of fluoropegylated diphenylacetylenes, which mimic the struc-
ture of fluoropegylated stilbenes. The diphenylacetylene deriva-
tives employed in the present study are a simplified version of
SB-13, 2; the double bond in the SB-13, 2, is replaced by a
triple bond. Two reasons prompted us to investigate this class
of compounds. They can be synthesized by Sonogashira
reaction, which is tolerant to a wide variety of functional groups.
The second and more important reason is the nonexistence of
geometrical isomers, which is an often-encountered problem
with their double-bonded counterparts.31 The diphenylacetylenes
are relatively rigid; therefore, the degree of freedom around the
triple bond is limited, leading to a tight fit to the binding pocket
at the â-sheets. An additional impetus came from the fact that
none of the Aâ plaque imaging agents so far reported have been
based on a triple-bonded (diphenylacetylene) structure.
* To whom correspondence should be addressed. Hank F. Kung, Ph.D.,
Department of Radiology, University of Pennsylvania, 3700 Market Street,
Room 305, Philadelphia, PA 19104. Tel.: (215) 662-3096. Fax: (215) 349-
5035. E-mail: kunghf@sunmac.spect.upenn.edu.
† Department of Radiology, University of Pennsylvania.
‡ Department of Pathology, University of California Health System.
§ Department of Pharmacology, University of Pennsylvania.
a Abbreviations: SPECT, single photon emission computed tomography;
PET, positron emission tomography; AD, Alzheimer’s disease; MCI, mild
cognitive impairment; Aâ, â-amyloid; PIB, 2-(4′-(methylaminophenyl)-6-
hydroxybenzothiazole; SB-13, 4-N-methylamino-4′-hydroxystilbene; FDD-
NP,
2-(1-{6-[(2-fluoroethyl)methyl-amino]-2-naphthyl}ethylidend)-
malononitrile; IMPY, 6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2-
a]pyridine.
10.1021/jm070090j CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/21/2007

2416 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Chandra et al.
Figure 1. Chemical structures of various probes previously reported for imaging Aâ aggregates.
eluted at flow rates of 1.0 mL/min. The chromatographic systems
were fitted with UV detectors set at 280 nm.
The desired diagnostic imaging agents of AD based on Aâ
plaque-specific binding can be defined as ligands with the
following properties: (a) high binding affinity to Aâ aggregates;
Ki < 10 nM; (b) high binding selectivity; Ki for other sites >
100-fold; (c) readily labeled with nuclide for imaging; (d) small
(mol wt < 600), lipophilic (P.C. ) 100-1000) and neutral
molecules; (e) desirable pharmacokinetics; good initial brain
uptake (>4% dose/g at 2 min post iv injection in normal mice)
and fast washout (less than 30% of initial uptake remaining at
30 min in the brain of normal mice); (f) in vivo stability as
evaluated by in vitro metabolism studies (metabolite in the brain
is less than 5% of parent ligand).
Initially, we have prepared limited p-N,N-dimethylamino-
diphenylacetylene derivatives, which have not been reported
previously, to see if they showed reasonably good binding
affinities toward Aâ plaques. Once it became apparent that
suitably substituted diphenylacetylene structures indeed showed
excellent binding to Aâ plaques in vitro, we proceeded to
synthesize fluorine-containing derivatives. These molecules have
two distinct parts, planar or easily planarized diphenylacetylene,
bearing a nucleophilic group (NH2, NHMe, NMe2, OH, or OMe)
for binding and the second part containing a fluoropolyethyl-
eneglycol (FPEG) for carrying the radio tracer, [18F]fluorine,
in the present study. It is also worth mentioning that the
lipophilicity of these molecules could be fine-tuned by varying
the length of the PEG chain until a molecular weight ceiling of
600 Da is reached. We herein report a series of 18F-labeled
diphenylacetylenes as PET imaging probes for detecting Aâ
plaques in the brain.
General Procedure for Sonogashira Reaction: Method A:
This procedure is followed when the acetylene used in the reaction
is 4-ethynylaniline (7a). To a mixture of PdCl2(PPh3)2 (1 mol %
with respect to iodoarene), CuI (2 mol % wrt iodoarene), and
iodoarene (0.5 mmol) in 3 mL of anhyd THF (degassed with argon)
was added, at room temperature under argon, 4-ethynylaniline 7a
(0.6 mmol). Ammonia solution (0.5 M aq; 2 mL, 1 mmol) was
then added dropwise, and the mixture was stirred at room
temperature for 4 h. Diethyl ether (20 mL) and water (20 mL) were
then added to the reaction. The organic layer was separated and
the aqueous layer was further extracted with ether (3 × 10 mL).
The combined organic layer was dried (MgSO4) and concentrated,
and the residue was purified by silica gel column chromatography
(appropriate mixture of hexane-ethyl acetate).
Method B: This procedure is followed when the acetylene used
in the reaction is 4-ethynyl-N,N-dimethylaniline (13). A mixture
of PdCl2(PPh3)2 (2 mol % with respect to iodoarene), CuI (2 mol
% wrt iodoarene), and iodoarene (0.5 mmol) were taken in a round-
bottomed flask equipped with a reflux condenser, and the whole
set up was degassed and back-filled with a gaseous mixture of
approximate 1:1 H2 and Ar. THF followed by TEA were then
syringed in and the mixture was heated to 60 °C. A solution of
4-ethynyl-N,N-dimethylaniline (0.5 mmol) in 2.5 mL of THF was
then added under the reducing atmosphere of 1:1 H2 and Ar. After
refluxing the mixture for 16 h, the solvents were evaporated and
the residue was purified by silica gel column chromatography
(appropriate mixture of hexane-ethyl acetate).
4-(4-Amino-phenylethynyl)-phenol (9a). Compound 9a was
prepared according to method A: yield 54%. 1H NMR (200 MHz,
CD3OD, δ ppm): 6.64 (d, J ) 8.7 Hz, 2H), 6.74 (d, J ) 8.8 Hz,
2H), 7.18 (d, J ) 8.7 Hz, 2H), 7.27 (d, J ) 8.8 Hz, 2H). HRMS
calcd for C14H11NO (M+), 209.0841; found, 209.0851.
Experimental Section
1
General Methods. H NMR spectra were recorded on Bruker
4-(4-Methylamino-phenylethynyl)-phenol (9b). Compound 9b
1
DPX 200 MHz spectrometer in CDCl3 or CD3OD, chemical shifts
were reported as δ values with respect to residual solvent protons
unless otherwise mentioned. The coupling constants (J) were
reported in Hz. The multiplicity is defined by s (singlet), d (doublet),
t (triplet), br (broad), and m (multiplet). High-resolution mass
spectrometry (HRMS) was performed at the McMaster Regional
Centre for Mass Spectrometry, McMaster University, using a
micromass/Waters GCT instrument (GC-EI/CI time-of-flight mass
spectrometer). The microwave reactions were carried out in biotage
initiator microwave synthesis system. Commercially available
reagents were used as received without further purification. Crude
products were purified by either flash chromatography using 230-
400 mesh silica gel (Aldrich grade 9385, 60 Å) or preparative TLC
(Analtech, 20 × 20 cm, 2000 microns). The reported chemical
yields were not optimized. The purities of compounds 9(a,b),
11(a,b), 12(a,b), 14(a,b), 17, 19b, 20(a,b), and 23(a,b), which were
used for biological evaluations, were determined using both reverse
phase and normal phase HPLC (Agilent 1100 series LC), and all
of them were determined to be >95% pure. Analytical HPLC was
performed using a Hamilton PRP-1 reverse phase column (4.1 ×
250 mm, 10 µm) eluted with an acetonitrile/aq buffer (1 mM
ammoniumformate, pH 7) mobile phase mixture or a Phenomenex
Silica normal phase column (4.6 × 250 mm, 5 µm) eluted with an
ethylacetate/hexane mobile phase mixture. Both columns were
was prepared using similar procedure as for 12a: yield 88%. H
NMR (200 MHz, CDCl3, δ ppm): 2.74 (s, 3H), 6.53 (d, J ) 8.7
Hz, 2H), 6.74 (d, J ) 8.7 Hz, 2H), 7.18-7.30 (m, 4H). HRMS
calcd for C15H13NO (M+), 223.0997; found, 223.0996.
4-(4-Methoxy-phenylethynyl)-phenol (9c). Compound 9c was
prepared according to method A: yield 52%. 1H NMR (200 MHz,
CDCl3, δ ppm): 3.82 (s, 3H), 6.75-6.90 (m, 4H), 7.36-7.47 (m.
4H). HRMS calcd for C15H12O2 (M+), 224.0837; found, 224.0841.
4-(4-Amino-phenylethynyl)-phenylamine (9d). Compound 9d
1
was prepared according to method A: yield 68%. H NMR (200
MHz, CDCl3, δ ppm): 6.58-6.64 (m, 4H), 7.26-7.33 (m, 4H).
HRMS calcd for C14H12N2 (M+), 208.1000; found, 208.1011.
4-(4-Methoxy-phenylethynyl)-phenylamine (9e). Compound 9e
1
was prepared according to method A: yield 50%. H NMR (200
MHz, CD3OD, δ ppm): 3.80 (s, 3H), 6.62 (d, J ) 8.7 Hz, 2H),
6.73 (d, J ) 8.8 Hz, 2H), 7.18 (d, J ) 8.7 Hz, 2H), 7.24 (d, J )
8.8 Hz, 2H). HRMS calcd for C15H13NO (M+), 223.0997; found,
223.0991.
4-(4-(2-(2-Fluoro-ethoxy)-ethoxy)-phenylethynyl)-phenyl-
amine (11a). Compound 11a was prepared according to method
1
A: yield 68%. H NMR (200 MHz, CDCl3, δ ppm): 3.72-3.91
(m, 4H), 4.15 (t, J ) 4.4 Hz, 2H), 4.59 (dt, J ) 47.6, 4.2 Hz, 2H),
6.62 (d, J ) 8.6 Hz, 2H), 6.87 (d, J ) 8.8 Hz, 2H), 7.31 (d, J )

Diphenylacetylenes as Probes for Amyloid Plaques
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2417
Scheme 1a
1-Ethynyl-4-(2-(2-(2-fluoro-ethoxy)-ethoxy)-ethoxy)-ben-
zene (16). To a solution of 15 (162 mg, 0.5 mmol) in methanol (8
mL) was added KOH (66 mg, 1 mmol) in methanol (4 mL). The
mixture was stirred at room temperature for 10 h. The residue, after
the evaporation of the volatiles, was purified using silica gel column
chromatography (20% ethyl acetate in hexanes) to afford 16 in 82%
yield. H NMR (200 MHz, CDCl3, δ ppm): 2.98 (s, 1H), 3.64-
3.88 (m, 8H), 4.05-4.13 (m, 2H), 4.55 (dt, J ) 47.6, 4.2 Hz, 2H),
6.85 (d, J ) 8.8 Hz, 2H), 7.41(d, J ) 8.8 Hz, 2H).
1
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, 0.5 M NH4OH, THF,
rt, 4 h; (ii) (a) NaOMe, (CH2O)n, MeOH, reflux, 2 h; (b) NaBH4, reflux, 1
h.
4-(4-(2-(2-(2-Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethynyl)-
phenol (17). Compound 17 was prepared according to method A:
yield 58%. H NMR (200 MHz, CDCl3, δ ppm): 3.66-3.88 (m,
1
8H), 4.11 (t, J ) 4.4 Hz, 2H), 4.56 (dt, J ) 47.6, 4.1 Hz, 2H), 6.78
(d, J ) 8.6 Hz, 2H), 6.83 (d, J ) 8.8 Hz, 2H), 7.36 (d, J ) 8.6 Hz,
2H), 7.42 (d, J ) 8.8 Hz, 2H). HRMS calcd for C20H21FO4 (M+),
344.1424; found, 344.1426.
8.6 Hz, 2H), 7.41 (d, 8.8 Hz, 2H). HRMS calcd for C18H18FNO2
(M+), 299.1322; found, 299.1313.
4-(4-(2-(2-(2-Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethynyl)-
phenylamine (11b). Compound 11b was prepared according to
method A: yield 72%. 1H NMR (200 MHz, CDCl3, δ ppm): 3.65-
3.86 (m, 8H), 4.09-4.16 (m, 2H), 4.56 (dt, J ) 49.7, 3.5 Hz, 2H),
6.62 (d, J ) 8.6 Hz, 2H), 6.86 (d, J ) 8.8 Hz, 2H), 7.30 (d, J )
8.6 Hz, 2H), 7.41 (d, J ) 8.8.Hz, 2H). HRMS calcd for C20H22-
FNO3 (M+), 343.1584; found, 343.1572.
2-(2-(4-(4-Amino-phenylethynyl)-phenoxy)-ethoxy)-ethanol
(19a). Compound 19a was prepared according to method A: yield
1
52%. H NMR (200 MHz, CDCl3, δ ppm): 3.64-3.88 (m, 6H),
4.09-4.16 (m, 2H), 6.62 (d, J ) 8.4 Hz, 2H), 6.86 (d, J ) 8.7 Hz,
2H), 7.30 (d, J ) 8.4 Hz, 2H), 7.41 (d, J ) 8.7 Hz, 2H).
2-(2-(2-(4-(4-Amino-phenylethynyl)-phenoxy)-ethoxy)-ethoxy)-
ethanol (19b). Compound 19b was prepared according to method
(4-(4-(2-(2-Fluoro-ethoxy)-ethoxy)-phenylethynyl)-phenyl)-
methylamine (12a). Under argon atmosphere, sodium methoxide
(33 mg, 0.60 mmol) was added to a solution of compound 11a (40
mg, 0.12 mmol) in methanol (8 mL), followed by paraformaldehyde
(18 mg, 0.60 mmol). The solution was heated to reflux for 2 h.
After cooling the mixture to 0 °C, sodium borohydride (23 mg,
0.60 mmol) was added in portions, and the mixture was refluxed
further for 1 h. The mixture was then poured into crushed ice and
extracted with ethyl acetate (3 × 10 mL). The combined ethyl
acetate layers were dried over MgSO4, concentrated, and purified
using preparative thin layer chromatography to afford 12a in 88%
1
A: yield 64%. H NMR (200 MHz, CDCl3, δ ppm): 3.58-3.75
(m, 8H), 3.83-3.88 (m, 2H), 4.09-4.15 (m, 2H), 6.62 (d, J ) 8.6
Hz, 2H), 6.86 (d, J ) 8.8 Hz, 2H), 7.30 (d, J ) 8.6 Hz, 2H), 7.41
(d, J ) 8.8 Hz, 2H). HRMS calcd for C20H23NO4 (M+), 341.1627;
found, 341.1621.
4-(4-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
phenylethynyl)-phenylamine (19c). Compound 19c was prepared
1
according to method A: yield 62%. H NMR (200 MHz, CDCl3,
δ ppm): 0.07 (s, 6H), 0.89 (s, 9H), 3.59-3.63 (m, 2H), 3.76-
3.88 (m, 4H), 4.09-4.14 (m, 2H), 6.62 (d, J ) 8.6 Hz, 2H), 6.86
(d, J ) 8.8 Hz, 2H), 7.31 (d, J ) 8.6 Hz, 2H), 7.41 (d, J ) 8.8 Hz,
2H).
4-(4-(2-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
ethoxy)-phenylethynyl)-phenylamine (19d). Compound 19d was
prepared according to method A: yield 66%. 1H NMR (200 MHz,
CDCl3, δ ppm): 1H NMR (200 MHz, CDCl3, δ) 0.61 (s, 6Η), 0.89
(s, 9Η), 3.53-3.88 (m, 10H), 4.10-4.15 (m, 2H), 6.61 (d, J ) 8.6
Hz, 2H), 6.86 (d, J ) 8.8 Hz, 2H), 7.31 (d, J ) 8.6 Hz, 2H), 7.40
(d, J ) 8.8 Hz, 2H).
1
yield. H NMR (200 MHz, CDCl3, δ ppm): 2.85 (s, 3H), 3.72-
3.76 (m, 1H), 3.87-3.91 (m, 3H), 4.14 (t, J ) 4.4 Hz, 2H), 4.59
(dt, J ) 47.6, 4.2 Hz, 2H), 6.54 (d, J ) 8.7 Hz, 2H), 6.87 (d, J )
8.9 Hz, 2H), 7.34 (d, J ) 8.7 Hz, 2H), 7.42 (d, J ) 8.9 Hz, 2H).
HRMS calcd for C19H20FNO2 (M+), 313.1478; found, 313.1467.
(4-(4-(2-(2-(2-Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethynyl)-
phenyl)-methylamine (12b). Compound 12b was prepared in 90%
yield from 11b following the same procedure as described for 12a.
1H NMR (200 MHz, CDCl3, δ ppm): 2.84 (s, 3H), 3.66-3.88 (m,
8H), 4.11 (t, J ) 4.4 Hz, 2H), 4.56 (dt, J ) 47.6, 4.2 Hz, 2H), 6.54
(d, J ) 8.7 Hz, 2H), 6.86 (d, J ) 8.8 Hz, 2H), 7.34 (d, J ) 8.7 Hz,
2H), 7.41 (d, J ) 8.8 Hz, 2H). HRMS calcd for C21H24FNO3 (M+),
357.1740; found, 357.1724.
2-(2-(4-(4-Methylamino-phenylethynyl)-phenoxy)-ethoxy)-
ethanol (20a). Compound 20a was prepared in 85% yield from
1
19a following the same procedure as described for 12a. H NMR
(200 MHz, CDCl3, δ ppm):2.84 (s, 3H), 3.64-3.88 (m, 6H), 4.11-
4.16 (m, 2H), 6.54 (d, J ) 8.6 Hz, 2H), 6.87 (d, J ) 8.7 Hz, 2H),
7.34 (d, J ) 8.6 Hz, 2H), 7.42 (d, J ) 8.7 Hz, 2H). HRMS calcd
for C19H21NO3 (M+), 311.1521; found, 311.1521.
(4-(4-(2-(2-Fluoro-ethoxy)-ethoxy)-phenylethynyl)-phenyl)-
dimethylamine (14a). Compound 14a was prepared according to
method B: yield 80%. 1H NMR (200 MHz, CDCl3, δ ppm): 2.97
(s, 6H), 3.74 (t, J ) 4.2 Hz, 1H), 3.87-3.91 (m, 3H), 4.13-4.17
(m, 2H), 4.59 (dt, J ) 47.6, 4.2 Hz, 2H), 6.65 (d, J ) 8.8 Hz, 2H),
6.87 (d, J ) 8.7 Hz, 2H), 7.35-7.44 (m, 4H). HRMS calcd for
C20H22FNO2 (M+), 327.1635; found, 327.1635.
2-(2-(2-(4-(4-Methylamino-phenylethynyl)-phenoxy)-ethoxy)-
ethoxy)-ethanol (20b). Compound 20b was prepared in 92% yield
1
from 19b following the same procedure as described for 12a. H
NMR (200 MHz, CDCl3, δ ppm): 2.84 (s, 3H), 3.59-3.75 (m,
8H), 3.83-3.88 (m, 2H), 4.09-4.16 (m, 2H), 6.55 (d, J ) 8.6 Hz,
2H), 6.86 (d, J ) 8.8 Hz, 2H), 7.33 (d, J ) 8.6 Hz, 2H), 7.42 (d,
J ) 8.8 Hz, 2H). HRMS calcd for C21H25NO4 (M+), 355.1784;
found, 355.1789.
(4-(4-(2-(2-(2-Fluoro-ethoxy)-ethoxy]-ethoxy)-phenylethynyl)-
phenyl]-dimethylamine (14b). Compound 14b was prepared
1
according to method B: yield 84%. H NMR (200 MHz, CDCl3,
δ ppm): 2.97 (s, 6Η), 3.65-3.68 (m, 8Η), 4.14 (t, J ) 4.8 Hz,
2H) 4.56 (dt, J ) 48.5, 3.2 Hz, 2H), 6.65 (d, J ) 8.8 Hz, 2H), 6.86
(d, J ) 8.8 Hz, 2H), 7.35-7.43 (m, 4H). HRMS calcd for C22H26-
FNO3 (M+), 371.1897; found, 371.1882.
(4-(4-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
phenylethynyl)-phenyl)-methylamine (20c). Compound 20c was
prepared in 90% yield from 19c following the same procedure as
(4-(2-(2-(2-Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethynyl)-tri-
methylsilane (15). To a solution of 10b (354 mg, 1 mmol) and
TMSA (0.207 mL, 1.5 mmol) in triethylamine (10 mL) was added
PdCl2(PPh3)2 (5 mol %) and CuI (3 mol %) at 0 °C under an argon
atmosphere. The mixture was stirred at 0 °C for 2 h and then at
room temperature overnight. After evaporation of the solvent, the
crude residue was purified using silica gel column (20% ethyl
acetate in hexanes) to afford 15 in 78% yield. 1H NMR (200 MHz,
CDCl3, δ ppm): 0.22 (s, 9H), 3.66-3.86 (m, 8H), 4.12 (t, J ) 4.4
Hz, 2H), 4.54 (dt, J ) 47.6, 4.2 Hz, 2H), 6.82 (d, J ) 8.8 Hz, 2H),
7.38 (d, J ) 8.8 Hz, 2H).
1
described for 12a. H NMR (200 MHz, CDCl3, δ ppm): 0.07 (s,
6H), 0.89 (s, 9H), 2.85 (s, 3H), 3.60-3.63 (m, 2H), 3.76-3.88
(m, 4H), 4.09-4.14 (m, 2H), 6.54 (d, J ) 8.7 Hz, 2H), 6.86 (d, J
) 8.9 Hz, 2H), 7.34 (d, J ) 8.7 Hz, 2H), 7.41 (d, J ) 8.9 Hz, 2H).
(4-(4-(2-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
ethoxy)-phenylethynyl)-phenyl)-methylamine (20d). Compound
20d was prepared in 94% yield from 19d following the same
1
procedure as described for 12a. H NMR (200 MHz, CDCl3, δ
ppm): 0.66 (s, 6H), 0.89 (s, 9H), 2.83 (s, 3H), 3.56 (t, 5.3 Hz,
2H), 3.69-3.87 (m, 8H), 4.10-4.14 (m, 2H), 6.53 (d, J ) 8.6 Hz,

2418 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Chandra et al.
Scheme 2a
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, 0.5 M NH4OH, THF, rt, 4 h; (ii) (a) NaOMe, (CH2O)n, MeOH, reflux, 2 h; (b) NaBH4, reflux, 1 h.
Scheme 3a
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, TEA, THF, Ar + H2, 60 °C, 16 h.
Scheme 4a
4.16 (m, 2H), 4.37-4.42 (m, 2H), 6.86 (d, J ) 8.7 Hz, 2H), 7.21
(d, J ) 8.5 Hz, 2H), 7.42-7.47 (m, 4H).
Methanesulfonic Acid 2-(2-(2-(4-(4-(tert-Butoxycarbonyl-
methyl-amino)-phenylethynyl)-phenoxy)-ethoxy)-ethoxy)-eth-
yl Ester (22d). Compound 22d was prepared in 94% yield from
1
21d following the same procedure as described for 22c. H NMR
(200 MHz, CDCl3, δ ppm): 1.45 (s, 9H), 3.04 (s, 3H), 3.26 (s,
3H), 3.68-3.87 (m, 8H), 4.11-4.16 (m, 2H), 4.35-4.39 (m, 2H),
6.87 (d, J ) 8.6 Hz, 2H), 7.20 (d, J ) 8.4 Hz, 2H), 7.42-7.47 (m,
4H).
2-(2-(4-(4-Dimethylamino-phenylethynyl)-phenoxy)-ethoxy)-
ethanol (23a). Compound 23a was prepared according to method
B: yield 80%. 1H NMR (200 MHz, CDCl3, δ ppm): 2.97 (s, 6H),
3.65-3.89 (m, 6H), 4.10-4.16 (m, 2H), 6.65 (d, J ) 8.8 Hz, 2H),
6.86 (d, J ) 8.8 Hz, 2H), 7.34-7.44 (m, 4H). HRMS calcd for
C20H23NO3 (M+), 325.1678; found, 325.1675.
2-(2-(2-(4-(4-Dimethylamino-phenylethynyl)-phenoxy)-ethoxy)-
ethoxy)-ethanol (23b). Compound 23b was prepared according to
method B: yield 82%. 1H NMR (200 MHz, CDCl3, δ ppm): 2.97
(s, 6H), 3.47-3.65 (m, 8H), 3.79-3.84 (m, 2H), 4.10-4.17 (m,
2H), 6.70 (d, J ) 8.9 Hz, 2H), 6.94 (d, J ) 8.8 Hz, 2H), 7.32 (d,
J ) 8.9 Hz, 2H), 7.39 (d, J ) 8.8 Hz, 2H). HRMS calcd for C22H27-
NO4 (M+), 369.1940; found, 369.1934.
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, TEA, 0 °C-rt, 16 h;
(ii) KOH, MeOH, rt, 16 h; (iii) p-iodophenol, PdCl2(PPh3)2/CuI, 0.5 M
NH4OH, THF, rt, 4 h.
2H), 6.85 (d, J ) 8.6 Hz, 2H), 7.33 (d, J ) 8.6 Hz, 2H), 7.41 (d,
J ) 8.6 Hz, 2H).
(4-(4-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
phenylethynyl)-phenyl)-methyl-carbamic Acid tert-Butyl Ester
(21c). A solution of 20c (106 mg, 0.25 mmol), di-tert-butyl
dicarbonate (110 mg, 0.50 mmol), and catalytic DMAP (10 mg)
was refluxed in anhydrous THF (4 mL) for 16 h. Another portion
of di-tert-butyl dicarbonate (55 mg, 0.25 mmol) was then added,
and the mixture was refluxed again for 10 h. The volatiles were
then removed under reduced pressure, and the resulting residue was
purified by silica gel column chromatography (20% EtOAc in
hexanes) to afford 12c in 52% yield. 1H NMR (200 MHz, CDCl3,
δ ppm): 0.73 (s, 6H), 0.89 (s, 9H), 1.45 (s, 9H), 3.26 (s, 3H),
3.60-3.65 (m, 2H), 3.77-3.89 (m, 4H), 4.10-4.15 (m, 2H), 6.88
(d, J ) 8.7 Hz, 2H), 7.20 (d, J ) 8.5 Hz, 2H), 7.42-7.47 (m, 4H).
(4-(4-(2-(2-(2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy)-ethoxy)-
ethoxy)-phenylethynyl)-phenyl)-methyl-carbamic Acid tert-Butyl
Ester (21d). Compound 21d was prepared in 58% yield from 20d
following the same procedure as described for 21c. 1H NMR (200
MHz, CDCl3, δ ppm): 0.63 (s, 6H), 0.89 (s, 9H), 1.45 (s, 9H),
3.26 (s, 3H), 3.56 (t, 5.4 Hz, 2H), 3.64-3.88 (m, 8H), 4.11-4.16
(m, 2H), 6.88 (d, J ) 8.7 Hz, 2H), 7.20 (d, J ) 8.5 Hz, 2H), 7.41-
7.46 (m, 4H).
Methanesulfonic Acid 2-(2-(4-(4-Dimethylamino-phenylethy-
nyl)-phenoxy)-ethoxy)-ethyl Ester (24a). A solution of 23a (20
mg, 0.06 mmol) in dry DCM was cooled to 0 °C, and TEA (22
µL, 0.15 mmol) was added followed by MsCl (6 µL, 0.07 mmol).
The reaction mixture was stirred initially at 0 °C and then at room
temperature for 2 h. After quenching the reaction with cold water,
the organic layer was separated. The aqueous layer was further
extracted with DCM (2 × 5 mL). The combined organic extracts
were dried (MgSO4) and concentrated, and the residue was purified
by PTLC (50% EtOAc in hexanes) to afford 24a in 78% yield. 1H
NMR (200 MHz, CDCl3, δ ppm): 2.97 (s, 6H), 3.08 (s, 3H), 3.65-
3.75 (m, 4H), 3.82-3.89 (m, 2H), 4.07-4.16 (m, 2H), 6.65 (d, J
) 8.9 Hz, 2H), 6.85 (d, J ) 8.8 Hz, 2H), 7.34-7.44 (m, 4H).
Methanesulfonic Acid 2-(2-(2-(4-(4-Dimethylamino-phenyl-
ethynyl)-phenoxy)-ethoxy)-ethoxy)-ethyl Ester (24b). Compound
24b was prepared in 84% yield from 23b following the same
Methanesulfonic Acid 2-(2-(4-(4-(tert-Butoxycarbonyl-methyl-
amino)-phenylethynyl)-phenoxy)-ethoxy)-ethyl Ester (22c). Com-
pound 21c (36 mg, 0.07 mmol) was dissolved in 1 mL dry THF,
and the solution was cooled to 0 °C. Tetrabutylammonium bromide
(0.17 mL, 1 M solution in THF) was then added, and the mixture
was stirred at room temperature for 2 h and after which was
quenched with ice. The mixture was then extracted with EtOAc (3
× 8 mL), and the combined organic layer was dried (MgSO4) and
concentrated. The crude residue was dissolved in dry dichlo-
romethane (4 mL) and cooled to 0 °C. Triethyl amine (40 µL, 0.28
mmol) and MsCl (17 µL, 0.21 mmol) were added to the reaction
mixture and stirred at room temperature for 3 h. After quenching
with cold water, the reaction mixture was extracted with DCM (3
× 5 mL). The combined organic layer was dried, concentrated,
and purified by PTLC (60% EtOAc in hexanes) to afford 22c in
1
procedure as described for 24a. H NMR (200 MHz, CDCl3, δ
ppm): 2.97 (s, 6H), 3.09 (s, 3H), 3.62-3.84 (m, 8H), 4.12-4.18
(m, 2H), 4.32-4.37 (m, 2H), 6.71 (d, J ) 8.9 Hz, 2H), 6.95 (d, J
) 8.8 Hz, 2H), 7.32 (d, J ) 8.9 Hz, 2H), 7.40 (d, J ) 8.8 Hz, 2H).
(4-(4-(2-(2-[18F]Fluoro-ethoxy)-ethoxy)-phenylethynyl)-phen-
yl)-methylamine, [18F]12a. [18F]Fluoride was produced by the JSW
typeBC3015 cyclotron using 18O(p,n)18F reaction and passed
through a Sep-Pak Light QMA cartridge (waters) as an aqueous
solution in [18O]-enriched water. The cartridge was dried by airflow,
and the 18F activity was eluted with 2 mL of Kryptofix 222 (K222)/
K2CO3 solution (13.2 mg of K222 and 3.0 mg of K2CO3 in CH3-
CN/H2O 1.12/0.18). The solvent was removed at 120 °C under an
argon stream. The residue was azeotropically dried with 1 mL of
anhydrous CH3CN twice at 120 °C under a nitrogen stream. A
1
80% yield over two steps. H NMR (200 MHz, CDCl3, δ ppm):
1.45 (s, 9H), 3.04 (s, 3H), 3.26 (s, 3H), 3.81-3.90 (m, 4H), 4.12-

Diphenylacetylenes as Probes for Amyloid Plaques
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2419
Scheme 5a
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, 0.5 M NH4OH, THF, rt, 4 h; (ii) (a) NaOMe, (CH2O)n, MeOH, reflux, 2 h; (b) NaBH4, reflux, 1 h; (iii)
(Boc)2O, DMAP, THF, relux, 24 h; (iv) (a) TBAF (1 M in THF), 0 °C-rt, 2 h; (b) MsCl, TEA, DCM, 0 °C-rt, 3 h.
Scheme 6a
a Reagents and conditions: (i) PdCl2(PPh3)2/CuI, TEA, THF, Ar + H2, 60 °C, 16 h; (ii) MsCl, TEA, DCM, 0 °C-rt, 2 h.
Scheme 7a
a Reagents and conditions: (i) (1)18F-/K222, DMSO; (2) microwave, 100 W, DMSO, (ii) 18F-/K222, DMSO.
solution of mesylate precursor 22c (1 mg) in DMSO (0.5 mL) was
added to the reaction vessel containing the dried 18F activities. The
mixture was heated at 120 °C for 4 min. To remove the Boc
protecting group, the mixture was further irradiated with microwave
at 100 W (Resonance Instruments Model 521) at 150 °C for 3 min.
Water (5 mL) was added, and the mixture was passed through a
preconditioned OASIS HLB cartridge (3 cm3; Waters). The
cartridge was washed with 10 mL of water, and labeled compound
was eluted with 2 mL of CH3CN. Eluted compound was purified
by HPLC. [Phenonemex Gemini C18 semi-prep column (10 × 250
mm, 5 µm), CH3CN/water 7/3, flow rate 3 mL/min, rt ) 9.6 min.]
The entire preparation procedure took approximately 90 min. The
radiochemical yield was 20% (decay corrected). The radiochemical
purity and specific activity were determined by analytical HPLC.
[Phenomenex Gemini C18 analytical column (4.6 × 250 mm, 5
µm), CH3CN/ammonium formate buffer (1 mM) 8/2; Flow rate 1
mL/min; rt ) 5.3 min.] The radiochemical purity was >99%.
Specific activity was estimated by comparing UV peak intensity
of purified [18F]-labeled compound with reference nonradioactive
compound of known concentration. The specific activity was in
the range of 600-1300 mCi/µmol after the preparation.
(4-(4-(2-(2-[18F]Fluoro-ethoxy)-ethoxy)-phenylethynyl)-phen-
yl)-dimethylamine (14a), [18F]14a. The procedure for [18F]12a was
followed for precursor 2a up to the initial reaction of 2a with 18K/
K222 by heating. After the initial heating was complete, water (5
mL) was added and the mixture was passed through a precondi-
tioned OASIS HLB cartridge (3 cm3; Waters). The cartridge was
washed with 10 mL of water, and the labeled compound was eluted
with 2 mL of CH3CN. The eluted compound was purified and
analyzed by HPLC (same condition). Rt (semi-prep) ) 13,8 min
(4 mL/min), (analytical) ) 7.6 min. The preparation took ap-
proximately 60 min. The radiochemical yield was 30% (decay
corrected). The radiochemical purity was >99%. Specific activity
was in the range of 1100-4600 mCi/µmol after the preparation.
(4-(4-(2-(2-(2-[18F]Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethy-
nyl)-phenyl)-dimethylamine (14b), [18F]14b. Same procedure for
[18F]14a was applied for precursor 12b. The final compound was
purified and analyzed by HPLC. Rt (semi-prep column) ) 14.0
min (4 mL/min), (analytical) ) 7.3 min. The preparation took
approximately 60 min. The radiochemical yield was 30% (decay
corrected), and the radiochemical purity was >97%. Specific
activity was approximately 980-4000 mCi/µmol after the prepara-
tion.
(4-(4-(2-(2-(2-[18F]Fluoro-ethoxy)-ethoxy)-ethoxy)-phenylethy-
nyl)-phenyl)-methylamine (12b), [18F]12b. The above-described
procedure for [18F]12a was applied for precursor 22d. The
final compound was purified and analyzed by HPLC (same
conditions). Rt (semi-prep) ) 9.3 min, (analytical) ) 5.2 min. The
preparation took approximately 90 min. The radiochemical yield
was 20% (decay corrected). The radiochemical purity was >99%.
Specific activity was approximately 600 mCi/µmol after the
preparation.
Preparation of Brain Tissue Homogenates. AD postmortem
brain tissues were obtained from University of Washington Alzhe-
imer’s Disease Research Center, and neuropathological diagnosis
was confirmed by current criteria (NIA-Reagan Institute Consensus
Group, 1997). Homogenates were then prepared from dissected gray
matters from four pooled AD patients in phosphate buffered saline
(PBS, pH 7.4) at the concentration of approximately 100 mg wet
tissue/mL (motor-driven glass homogenizer with setting of 6 for

2420 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Chandra et al.
the assumption that they were 7% of the total body weight. The %
dose/g of samples was calculated by comparing the sample counts
with the count of the diluted initial dose.
Partition Coefficient. Partition coefficients were measured by
mixing the [18F]tracer with 3 g each of 1-octanol and buffer (0.1
M phosphate, pH 7.4) in a test tube. The test tube was vortexed
for 3 min at room temperature, followed by centrifugation for 5
min. Two weighed samples (0.5 g each) from the 1-octanol and
buffer layers were counted in a well counter. The partition
coefficient was determined by calculating the ratio of cpm/g of
1-octanol to that of buffer. Samples from the 1-octanol layer were
repartitioned until consistent partitions of coefficient values were
obtained. The measurement was done in triplicate and repeated three
times.
Figure 2. HPLC profiles of [18F]14b (top) and 14b (bottom). HPLC
condition: Agilent 1100 series; Gemini C-18 column CH3CN/am-
monium formate (10 mM) 8/2 1 mL/min, 254 nm, tR ) (UV) 7.04
min, (_) 7.29 min. The slight difference in retention time between the
radioactive peak and the UV peak is due to the configuration of detector
system).
Results and Discussion
Chemistry and Radiochemistry. The synthesis of the
diphenylacetylene core structures was easily achieved as given
in Scheme 1. The key step in the synthesis of these compounds
was the Pd(0)/Cu(I)-catalyzed (Sonogashira) coupling of the
suitably substituted iodobenzenes 8(a-c) with appropriately
functionalized phenyl acetylenes 7(a,b). Dimerization of acety-
lene was a side reaction under these conditions, and dimers are
typically formed in 10-20% yield.
The coupling of amino-substituted phenylacetylene 7a with
para-F-PEG iodobenzenes 10(a,b) in THF using 0.5 M NH4-
OH (Method A,33 see Experimental Section) as base afforded
compounds 11(a,b). Subsequent monomethylation of the NH2
group in 11(a,b) under standard conditions afforded monom-
ethylated derivatives 12(a,b; Scheme 2).
However, the coupling of N,N-dimethyl-substituted acetylene
13 with 10(a,b) under similar conditions yielded appreciable
amounts of dimer, the desired products were formed only in
negligible amounts. To circumvent this, Sonogashira reaction
was carried out under a reducing atmosphere.34 Thus, under a
gaseous mixture of hydrogen (10-50%) and argon, the reaction
proceeded to afford the desired products 14(a,b) in good yields
(Scheme 3).
The synthesis of hydroxy-substituted derivative 17 started
with the coupling of TMS acetylene with 10b, which afforded
the substituted phenylacetylene 16. The subsequent removal of
the TMS group in 15 followed by the coupling with para-
iodophenol afforded the hydroxy derivative 17 (Scheme 4).
The synthesis of the precursors to the corresponding [18F]-
labeled compounds of N-monomethyl derivatives is depicted
in Scheme 5. Compounds 19(a-d), obtained by the coupling
of acetylene 7a with iodocompounds 18(a-d), were monom-
ethylated under the standard conditions to afford 20(a-d). The
amino groups in the TBS-protected compounds 20(c,d) were
then protected with Boc group. The subsequent removal of the
TBS group followed by the mesylation of the resulting free
hydroxy groups yielded precursors 22(c,d). Compounds 22(c,d)
serve as the immediate precursors for the [18F]-labeled 12(a,b)
(Scheme 5).
30 s). The homogenates were aliquoted into 1 mL portions and
stored at -70 °C up to 2 years without loss of binding signal.
Binding Studies. [125I]IMPY, 4, with 2200 Ci/mmol specific
activity and greater than 95% radiochemical purity was prepared
using the standard iododestannylation reaction and purified by a
simplified C-4 mini column as described previously.32 Competition
binding assays were carried out in 12 × 75 mm borosilicate glass
tubes. The reaction mixture contained 50 µL of pooled AD brain
homogenates (20-50 µg), 50 µL of [125I]IMPY, 4, (0.04-0.06 nM
diluted in PBS), and 50 µL of inhibitors (10-5-10-10 M diluted
serially in PBS containing 0.1% bovine serum albumin) in a final
volume of 1 mL. Nonspecific binding was defined in the presence
of 600 nM IMPY, 4, in the same assay tubes. The mixture was
incubated at 37 °C for 2 h, and the bound and the free radioactivity
were separated by vacuum filtration through Whatman GF/B filters
using a Brandel M-24R cell harvester followed by 2 × 3 mL washes
of PBS at room temperature. Filters containing the bound I-125
ligand were counted in a gamma counter (Packard 5000) with 70%
counting efficiency. Under the assay conditions, the non-specific-
ally bound fraction was less than 15% of the total radioactivity.
Inhibition experiments were repeated three times, and the results
were subjected to nonlinear regression analysis using equilibrium
binding data analysis, which Ki values were calculated.
Similarly, specific binding of [18F]12b (0.001-0.4 nM) to
homogenates, prepared from gray matters of four pooled AD
patients, were carried out as described above. Nonspecific binding
was determined in the presence of 800 nM of nonradioactive 12b.
Film Autoradiography. To compare different probes using
similar sections of human brain tissues, a human brain macro-array
from six confirmed AD cases and one control subject was
assembled. The presence and localization of plaques on the sections
were confirmed with immunohistochemistry stained with mono-
clonal Aâ antibody 4G8 (Signet Lab. Inc. Dedham, MA). The
sections were incubated with [18F]tracers (300 000-600 000 cpm/
200 µL) for 1 h at room temperature. The sections were then dipped
in saturated Li2CO3 in 40% EtOH (2 min wash × 2) and washed
with 40% EtOH (2 min wash × 1), followed by rinsing with water
for 30 s. After drying, the 18F-labeled sections were exposed to
Kodak Biomax MR film overnight.
The precursors for [18F]14(a,b) were obtained by the coupling
of the phenylacetylene 13 with iodobenzene derivatives 18(a,b)
under reducing conditions (Scheme 6). The mesylation of the
hydroxyl groups present in 23(a,b) afforded the precursors
24(a,b) (Scheme 7).
Organ Distribution in Normal Mice. While under isoflurane
anesthesia, 0.15 mL of a 0.1% bovine serum albumin solution
containing [18F]tracers (5-10 µCi) was injected directly into the
tail vein of ICR mice (22-25 g, male). The mice (n ) 3 for each
time point) were sacrificed by cervical dislocation at designated
time points postinjection. The organs of interest were removed and
weighed, and the radioactivity was counted with an automatic
gamma counter. The percentage dose per organ was calculated by
a comparison of the tissue counts to suitably diluted aliquots of
the injected material. Total activities of blood were calculated under
To obtain [18F]12a and [18F]12b, precursors (22c and 22d,
respectively) were reacted with [18F]fluoride in the presence of
Kryptofix 222 and potassium carbonate in DMSO at 100 °C
for 4 min. The resulting [18F]-labeled intermediates were
irradiated with microwaves to deprotect the Boc group to
provide the desired labeled compounds (Scheme 7). The crude
product was purified by HPLC. The procedure took 90 min for

Diphenylacetylenes as Probes for Amyloid Plaques
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2421
Table 1. Inhibition Constants (Ki, nM) of Compounds on [125I]IMPY, 4, Binding to Amyloid Plaques in ad Brain Homogenatesa
a Values are mean ( SEM of three independent experiments, each in duplicate.
Table 2. Biodistributions in ICR Mice after iv Injections of
profile consistent with the “cold” carrier (Figure 2). To obtain
[18F]Tracersa
[18F]14a and [18F]14b, the desired products were obtained in a
one step reaction from 22a and 22b, respectively. The crude
product was purified by HPLC. The procedure took 60 min for
both compounds, and the specific activity at the end of synthesis
was 1100-4600 mCi/µmol for [18F]14a and 980-4000 mCi/
µmol for [18F]14b (radiochemical purity > 97%, radiochemical
yield ∼ 30%, decay corrected).
[18F]12a (Log P ) 3.48)
organ
2 min
30 min
1 h
2 h
blood
heart
muscle
lung
kidney
spleen
liver
skin
brain
bone
7.89 ( 1.12
4.73 ( 1.06
0.79 ( 0.22
6.57 ( 0.51
5.55 ( 0.75
3.74 ( 0.38
29.6 ( 1.36
0.94 ( 0.11
4.42 ( 0.88
1.54 ( 0.28
7.90 ( 0.93
2.84 ( 0.10
1.26 ( 0.38
4.77 ( 0.45
4.78 ( 0.66
3.60 ( 0.61
18.3 ( 2.21
2.11 ( 0.36
1.03 ( 0.15
1.46 ( 0.21
10.3 ( 0.11
3.54 ( 0.20
1.38 ( 0.25
6.01 ( 0.41
6.17 ( 0.38
3.78 ( 0.33
15.6 ( 2.43
2.55 ( 0.22
0.99 ( 0.09
1.92 ( 0.16
7.84 ( 0.33
2.60 ( 0.06
1.26 ( 0.06
4.67 ( 0.31
5.55 ( 0.68
3.30 ( 0.04
11.7 ( 0.91
2.04 ( 0.12
0.82 ( 0.03
2.07 ( 0.14
Biological Evaluation. The binding affinities (Ki, nM) of the
diphenylacetylene compounds are evaluated via competition
with [125I]4 binding for Aâ plaques, and the results are presented
in Table 1. Most of these derivatives showed excellent binding
affinities toward Aâ plaques. The presence of a nucleophilic
group such as amino or hydroxy, attached directly to one of
the phenyl rings, is necessary but not sufficient for these
structures to show desirable binding characteristics. By compar-
ing the Ki values for the core structures (left-hand side of the
table), it is clear that diphenylacetylene (9) without any
substituents showed a moderate affinity (175 ( 35 nM).
Simultaneously introducing NH2 and OH groups (9a) at
the 4-position on both phenyl rings increased the binding affinity
(Ki ) 106 ( 6 nM). However, adding an N-methyl group to
the amine moiety (9b) increased the binding affinity (Ki ) 1.5
( 0.1 nM) about 100-fold. A comparable increase in binding
affinity is also observed when the OH group in 9a is replaced
by an OMe group (9e; Ki ) 2.9 ( 0.4 nM). The intro-
duction of a methyl group to the NH2 or OH group plays a
critical role in improving the binding affinities of PEGylated
derivatives as well (Table 1). Compounds 11a and 11b, having
an unsubstituted amino group, showed Ki values of 12 and 21.5
nM, respectively. In comparison, their N-methylamino analogs,
12a and 12b, have Ki values of 1.2 and 1.9 nM, respectively.
This significant improvement in binding affinities may be
attributed to the increase in lipophilicity of the molecule, with
the introduction of the methyl group. However, adding one
more methyl group to the amine moiety, that is, the N,N-
dimethylamino derivatives, did not bring about any appreciable
change in the binding properties compared to the monomethyl
derivatives. The FPEGylated compound 17, where the nucleo-
philic group is OH, however, showed a comparatively lower
binding affinity (Ki ) 16.5 ( 3.5, see Table 1). Consistent
with the stilbene series of ligands,28,31 the hydroxyl substitution
of the fluoro group at one end of phenyl ring by a pegylated
chain did not affect the binding affinity toward Aâ plaques
(20a, 23a vs 12a and 14a; 20b and 23b vs 12b and 14b). It
is important to note that these seemingly extremely simple
diphenylacetylenes appeared to bind â-sheets formed by Aâ
peptide aggregates. The binding affinity of such simple diphen-
ylacetylenes to peptide aggregates has not been reported before.
[18F]14a (log P ) 3.12)
organ
2 min
30 min
1 h
2 h
blood
heart
muscle
lung
kidney
spleen
liver
skin
brain
bone
7.33 ( 0.43
10.5 ( 1.13
0.91 ( 0.26
11.1 ( 0.99
12.9 ( 1.51
5.12 ( 0.95
19.3 ( 1.14
0.85 ( 0.15
5.41 ( 0.84
1.78 ( 0.15
6.13 ( 0.35
2.82 ( 1.13
1.38 ( 0.15
4.66 ( 0.37
4.75 ( 0.13
2.94 ( 0.41
11.2 ( 0.49
2.23 ( 0.36
3.69 ( 0.25
1.41 ( 0.08
5.68 ( 0.75
2.48 ( 0.36
1.21 ( 0.17
3.82 ( 0.55
4.79 ( 0.82
2.38 ( 0.43
9.99 ( 1.76
1.71 ( 0.70
2.06 ( 0.33
1.36 ( 0.29
5.17 ( 0.39
2.11 ( 0.13
1.03 ( 0.09
3.38 ( 0.43
4.16 ( 0.69
2.31 ( 0.27
8.85 ( 0.72
1.67 ( 0.44
1.34 ( 0.12
1.65 ( 0.27
[18F]12b (log P ) 3.07)
organ
2 min
30 min
1 h
2 h
blood
heart
muscle
lung
kidney
spleen
liver
skin
brain
bone
3.28 ( 0.63
4.21 ( 0.79
1.97 ( 1.15
4.64 ( 0.76
4.74 ( 1.22
2.69 ( 0.60
25.8 ( 2.80
1.24 ( 0.29
4.55 ( 0.82
1.50 ( 0.16
3.28 ( 0.57
1.40 ( 0.17
0.61 ( 0.10
2.10 ( 0.30
2.90 ( 0.99
1.22 ( 0.04
14.8 ( 4.12
1.40 ( 0.23
0.42 ( 0.13
0.66 ( 0.12
3.22 ( 0.19
1.24 ( 0.19
0.62 ( 0.07
2.04 ( 0.28
2.40( 0.22
0.95 ( 0.04
10.2 ( 4.00
0.95 ( 0.11
0.37 ( 0.02
0.65 ( 0.09
3.17 ( 0.62
0.96 ( 0.19
0.39 ( 0.14
1.72 ( 0.27
1.80 ( 0.62
1.02 ( 0.15
9.14 ( 3.24
0.60 ( 0.17
0.38 ( 0.07
0.82 ( 0.15
[18F]14b (log P ) 3.18)
organ
2 min
30 min
1 h
2 h
blood
heart
muscle
lung
kidney
spleen
liver
skin
brain
bone
5.72 ( 0.51
9.97 ( 1.51
0.79 ( 0.24
8.38 ( 1.28
12.4 ( 2.37
4.61 ( 0.76
23.2 ( 3.53
0.68 ( 0.17
6.78 ( 1.16
1.51 ( 0.34
2.97 ( 0.10
1.49 ( 0.14
0.67 ( 0.10
2.26 ( 0.25
2.68 ( 0.08
1.39 ( 0.14
13.8 ( 0.92
1.41 ( 0.24
1.57 ( 0.13
0.64 ( 0.04
2.51 ( 0.35
1.04 ( 0.07
0.51 ( 0.08
1.26 ( 0.90
2.53 ( 0.21
1.13 ( 0.07
8.13 ( 1.69
1.28 ( 0.06
0.77 ( 0.07
0.56 ( 0.10
2.35 ( 0.39
0.84 ( 0.10
0.42 ( 0.05
1.47 ( 0.19
2.02 ( 0.50
1.17 ( 0.17
6.58 ( 0.43
0.68 ( 0.18
0.42 ( 0.03
0.70 ( 0.11
a Percent dose/g, avg of three mice ( SD.
both compounds, and the specific activity at the end of synthesis
was 600-1300 mCi/µmol for [18F]12a and ∼600 mCi/µmol
for [18F]12b (radiochemical purity > 99%, radiochemical yield
∼ 20%, decay corrected). The purified product showed an HPLC
Based on the encouraging binding data obtained with 12a,
12b, 14a, and 14b, we chose to further carry out the biological

2422 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Chandra et al.
considered as potential imaging agents targeting Aâ plaques in
vivo.
We carefully constructed human macro-array sections from
six confirmed AD cases plus one control subject. After
sectioning, adjacent sections would reflect comparable patho-
physiology. In vitro film autoradiography was carried out with
18F-labeled diphenylacetylene probes and reported using these
brain sections. Consistent with 4G8 (antibody for Aâ) immu-
nohistochemical labeling, four diphenylacetylene probes, [18F]-
12a, 12b, 14a, and 14b, all exhibited distinctive and matching
plaque labeling (Figure 3) with minimal background labeling.
The control case (marked by the arrowhead, Figure 3) was
clearly void of any notable Aâ labeling, suggesting the
specificity of plaque labeling with these novel 18F-labeled
diphenylacetylene probes. Furthermore, [125I]IMPY, 4, a well
characterized SPECT imaging agent for amyloid plaques,35
displayed a similar pattern of Aâ plaque labeling on these array
sections. These results confirmed the specific binding of these
diphenylacetylene probes for Aâ plaques. To further characterize
the specific nature of plaque binding, we chose [18F]14b to carry
out a direct in vitro binding assay using AD brain homogenates.
As expected, [18F]14b displayed a specific and a saturable high
binding (data not shown).
In conclusion, a new series of novel 18F-labeled dipheny-
lacetylene derivatives, containing an end-capped fluoropoly-
ethylene glycol (n ) 2 and n ) 3), were successfully prepared
as potential PET imaging agents for AD. These fluorinated
diphenylacetylenes displayed excellent binding affinities to Aâ
plaques (Ki in the nM range). The radiofluorinated probes
showed desirable in vivo kinetic properties in mouse brain. A
specific plaque-labeling signal was clearly indicated by these
probes in postmortem AD brain sections as well as in brain
tissue homogenates. Taken together, the results suggest that
novel diphenylacetylene series ligands could be potentially
useful for in vivo PET imaging of Aâ plaques in living human
brain.
Figure 3. In vitro autoradiography of macroarray brain sections
constructed from six postmortem AD cases plus one control (marked
by arrowhead). An autoradiogram of a series standards is included for
comparison. Section labeling was carried out with four 18F-labeled
diphenylacetylenes. Immunohistochemistry stained with 4G8 confirmed
the plaque presence in the sections. [125I]IMPY, 4, a well characterized
SPECT ligand35 targeting Aâ plaques, was included for comparison.
Clearly, the Aâ plaques can be visualized for all four 18F dipheny-
lacetylenes with low background labeling.
evaluations using 18F-labeled diphenylacetylenes as probes for
Aâ aggregates. All of the diphenylacetylenes measured under
the experimental conditions showed relatively high partition
coefficients (P.C. ) 3.07-3.48), a reflection for the lipophilic
property of the 18F diphenylacetylenes. Nonetheless, biodistri-
bution studies done in the normal mice (Table 2) clearly
indicated that all four diphenylacetylenes, [18F]12a, 12b, 14a,
and 14b, readily penetrated intact blood-brain barrier showing
excellent initial brain uptakes (4.42, 4.55, 5.41, and 6.78%
dose/g for [18F]12a, 12b, 14a, and 14b, respectively, at 2 min
after a tracer injection). The high brain uptakes were subse-
quently followed by rapid washouts (except [18F]14a) with less
than 1% dose/g remaining in the brain at 2 h after injection
(Table 2). The difference in brain kinetics between chain length
of PEG was also noted: n ) 3 ligands [18F]12b and [18F]14b
exhibited faster brain washouts as compared to n ) 2 ligands,
[18F]12a and [18F]14a. The brain washout for N,N-dimethy-
lamino ligands, that is, [18F]14a and [18F]14b, was also slower
as compared to N-methylamino ligands, that is, [18F]12a and
[18F]12b (Table 2). The in vivo defluorination, as reflected by
the bone uptake, for all four diphenylacetylene probes is low;
particularly for probes with a longer PEG unit, that is, [18F]-
12b and [18F]14b, the bone uptake was low (<1% dose/g at 2
h after injection). Consistently, blood levels for [18F]12a and
[18F]14a ligands appeared to be higher than [18F]12b and [18F]-
14b, and the blood activity was sustained throughout the
experimental period (up to 2 h). Comparing N,N-dimethylamino
with N-methylamino ligands, we also noted that the latter
showed better and faster kinetics. A good initial brain uptake
combined with a rapid washout in normal mouse brain (presum-
ably no Aâ plaques for extra binding of these probes) is a highly
desirable property for Aâ plaque targeting imaging agents
similar to that reported for stilbene28 or styrylpyridine analogs.30
The diphenylacetylene probes reported here, especially [18F]-
12b and [18F]14b, met the criteria and could be favorably
Acknowledgment. This work was supported by grants from
the National Institutes of Health (AG022559 to H.F.K). The
authors thank Pathology Core laboratories at Children’s Hospital
of Philadelphia to assemble the human macro-array brain
sections. The authors also thank Drs. Daniel Skovronsky and
Rajesh Manchanda for their helpful discussion.
Supporting Information Available: Schemes and procedures
for the synthesizing intermediates 10(a,b) and 18(a,d) mentioned
in the paper; the purity and HPLC data (using normal as well as
reverse phase columns) of compounds 9(a,b), 11(a,b), 12(a,b),
14(a,b), 17, 19b, 20(a,b), and 23(a,b), which were used for the
biological evaluations. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s
disease: Progress and problems on the road to therapeutics. Science
2002, 297, 353-356.
(2) Hardy, J. Has the amyloid cascade hypothesis for Alzheimer’s disease
been proved? Curr. Alzheimer Res. 2006, 3, 71-73.
(3) Armstrong, R. A. Plaques and tangles and the pathogenesis of
Alzheimer’s disease. Folia Neuropathol. 2006, 44, 1-11.
(4) Golde, T. E. The Abeta hypothesis: Leading us to rationally-designed
therapeutic strategies for the treatment or prevention of Alzheimer
disease. Brain Pathol. 2005, 15, 84-87.
(5) Goedert, M.; Spillantini, M. G. A century of Alzheimer’s disease.
Science 2006, 314, 777-781.
(6) Roberson, E. D.; Mucke, L. 100 years and counting: Prospects for
defeating Alzheimer’s disease. Science 2006, 314, 781-784.

Diphenylacetylenes as Probes for Amyloid Plaques
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2423
(7) Braak, H.; Alafuzoff, I.; Arzberger, T.; Kretzschmar, H.; Del Tredici,
K. Staging of Alzheimer disease-associated neurofibrillary pathology
using paraffin sections and immunocytochemistry. Acta Neuropathol.
2006, 112, 389-404.
(22) Edison, P.; Archer, H. A.; Hinz, R.; Hammers, A.; Pavese, N.;
Tai, Y. F.; Hotton, G.; Cutler, D.; Fox, N.; Kennedy, A.; Rossor,
M.; Brooks, D. J. Amyloid, hypometabolism, and cognition in
Alzheimer disease. An [11C]PIB and [18F]FDG PET study. Neurol-
ogy 2006.
(23) Barrio, J. R.; Huang, S.-C.; Cole, G.; Satyamurthy, N.; Petric, A.;
Phelps, M. E.; Small, G. PET imaging of tangles and plaques in
Alzheimer’s disease with a highly hydrophobic probe. J. Labelled.
Compd. Radiopharm. 1999, 42 (Suppl. 1), S194.
(24) Shoghi-Jadid, K.; Barrio, J. R.; Kepe, V.; Huang, S. C. Exploring a
mathematical model for the kinetics of beta-amyloid molecular
imaging probes through a critical analysis of plaque pathology. Mol.
Imaging Biol. 2006, 8, 151-162.
(25) Kepe, V.; Barrio, J. R.; Huang, S. C.; Ercoli, L.; Siddarth, P.; Shoghi-
Jadid, K.; Cole, G. M.; Satyamurthy, N.; Cummings, J. L.; Small,
G. W.; Phelps, M. E. Serotonin 1A receptors in the living brain of
Alzheimer’s disease patients. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
702-707.
(26) Small, G. W.; Kepe, V.; Ercoli, L. M.; Siddarth, P.; Bookheimer, S.
Y.; Miller, K. J.; Lavretsky, H.; Burggren, A. C.; Cole, G. M.; Vinters,
H. V.; Thompson, P. M.; Huang, S. C.; Satyamurthy, N.; Phelps, M.
E.; Barrio, J. R. PET of brain amyloid and tau in mild cognitive
impairment. N. Engl. J. Med. 2006, 355, 2652-2663.
(27) Mathis, C. A.; Holt, D. P.; Wang, Y.; Huang, G. F.; Debnath, M. L.;
Klunk, W. E. 18F-Labeled thioflavin-T analogs for amyloid assess-
ment. J. Nucl. Med. 2002, 43, 166P.
(28) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung, H.
F. F-18 PEG stilbenes as PET imaging agents targeting Aâ aggregates
in the brain. Nucl. Med. Biol. 2005, 32, 799-809.
(29) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung, H.
F. F-18 stilbenes as PET imaging agents for detecting beta-amyloid
plaques in the brain. J. Med. Chem. 2005, 48, 5980-5988.
(30) Zhang, W.; Kung, M. P.; Oya, S.; Hou, C.; Kung, H. F. (18)F-Labeled
styrylpyridines as PET agents for amyloid plaque imaging. Nucl. Med.
Biol. 2007, 34, 89-97.
(31) Stephenson, K. A.; Chandra, R.; Zhuang, Z. P.; Hou, C.; Oya, S.;
Kung, M. P.; Kung, H. F. Fluoro-pegylated (FPEG) imaging agents
targeting Aâ aggregates. Bioconjug. Chem. 2007, 18, 238-246.
(32) Kung, M.-P.; Hou, C.; Zhuang, Z.-P.; Cross, A. J.; Maier, D. L.;
Kung, H. F. Characterization of IMPY as a potential imaging agent
for â-amyloid plaques in double transgenic PSAPP mice. Eur. J. Nucl.
Med. Mol. Imaging 2004, 31, 1136-1145.
(33) Mohamed, Ahmed, M. S.; Mori, A. Sonogashira coupling with
aqueous ammonia directed to the synthesis of azotolane derivatives.
Tetrahedron 2004, 60, 9977-9982.
(34) Elangovan, A.; Wang, Y.-H.; Ho, T.-I. Sonogashira coupling reaction
with diminished homocoupling. Org. Lett. 2003, 5, 1841-1844.
(35) Kung, M.-P.; Hou, C.; Zhuang, Z.-P.; Skovronsky, D.; Kung, H. F.
Binding of two potential imaging agents targeting amyloid plaques
in postmortem brain tissues of patients with Alzheimer’s disease.
Brain Res. 2004, 1025, 89-105.
(8) Selkoe, D. J. The origins of Alzheimer disease: A is for amyloid.
JAMA, J. Am. Med. Assoc. 2000, 283, 1615-1617.
(9) Selkoe, D. J. Alzheimer’s disease: Genes, proteins, and therapy.
Physiol. ReV. 2001, 81, 741-766.
(10) Blennow, K.; Zetterberg, H. Pinpointing plaques with PIB. Nat. Med.
2006, 12, 753-754.
(11) Mathis, C. A.; Wang, Y.; Klunk, W. E. Imaging â-amyloid plaques
and neurofibrillary tangles in the aging human brain. Curr. Pharm.
Des. 2004, 10, 1469-1492.
(12) Nichols, L.; Pike, V. W.; Cai, L.; Innis, R. B. Imaging and in vivo
quantitation of beta-amyloid: An exemplary biomarker for Alzhe-
imer’s disease? Biol. Psychiatry 2006, 59, 940-947.
(13) Schmidt, B.; Braun, H. A.; Narlawar, R. Drug development and PET-
diagnostics for Alzheimer’s disease. Curr. Med. Chem. 2005, 12,
1677-1695.
(14) Huddleston, D. E.; Small, S. A. Technology Insight: Imaging amyloid
plaques in the living brain with positron emission tomography and
MRI. Nat. Clin. Pract. Neurol. 2005, 1, 96-105.
(15) Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.;
Holt, D. P.; Bergstrom, M.; Savitcheva, I.; Huang, G.-f.; Estrada,
S.; Ausen, B.; Debnath, M. L.; Barletta, J.; Price, J. C.; Sandell, J.;
Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni, G.; Mathis, C. A.;
Langstrom, B. Imaging brain amyloid in Alzheimer’s disease with
Pittsburgh compound-B. Ann. Neurol. 2004, 55, 306-319.
(16) Verhoeff, N. P.; Wilson, A. A.; Takeshita, S.; Trop, L.; Hussey, D.;
Singh, K.; Kung, H. F.; Kung, M.-P.; Houle, S. In vivo imaging of
Alzheimer disease beta-amyloid with [11C]SB-13 PET. Am. J.
Geriatr. Psychiatry 2004, 12, 584-595.
(17) Shoghi-Jadid, K.; Small, G. W.; Agdeppa, E. D.; Kepe, V.; Ercoli,
L. M.; Siddarth, P.; Read, S.; Satyamurthy, N.; Petric, A.; Huang, S.
C.; Barrio, J. R.; Liu, J.; Flores-Torres, S.; Cole, G. M. Localization
of neurofibrillary tangles and beta-amyloid plaques in the brains of
living patients with Alzheimer disease: Binding characteristics of
radiofluorinated 6-dialkylamino-2-naphthylethylidene derivatives as
positron emission tomography imaging probes for beta-amyloid
plaques in Alzheimer disease. Am. J. Geriatr. Psychiatry 2002, 10,
24-35.
(18) Small, G. W. Diagnostic issues in dementia: neuroimaging as a
surrogate marker of disease. J. Geriatr. Psychiatry Neurol. 2006,
19, 180-185.
(19) Newberg, A. B.; Wintering, N. A.; Plossl, K.; Hochold, J.; Stabin,
M. G.; Watson, M.; Skovronsky, D.; Clark, C. M.; Kung, M. P.;
Kung, H. F. Safety, biodistribution, and dosimetry of 123I-IMPY:
A novel amyloid plaque-imaging agent for the diagnosis of Alzhe-
imer’s disease. J. Nucl. Med. 2006, 47, 748-754.
(20) Rentz, D. M.; Becker, J. A.; Moran, E.; Sperling, R. A.; Manning,
L.; Bonab, A.; Mathis, C. A.; Klunk, W.; Fischman, A. J.; Johnson,
K. A. Amyloid imaging in AD, MCI, and highly intelligent older
adults with Pittsburgh compound-B (PIB). J. Nucl. Med. 2006, 289p
(abstract).
(21) Villemagne, V. L.; Ng, S.; Gong, S. J.; Ackermann, U.; Pike, K.;
Savage, G.; Klunk, W.; Mathis, C.; Masters, C. L.; Rowe, C. C. 11C-
PIB PET imaging in the differential diagnosis of dementia. J. Nucl.
Med. 2006, 74p (abstract).
JM070090J