Structure-activity relationship of imidazo[1,2-a]pyridines as ligands for detecting β-amyloid plaques in the brain

Article abstract of DOI:10.1021/jm020351j

A series of novel β-amyloid (Aβ) aggregate-specific ligands, 2-(4′-dimethylaminophenyl)-6-iodoimidazo[1,2-a]pyridine, 16(IMPY), and its related derivatives were prepared. An in vitro binding study with preformed Aβ aggregates showed that 16(IMPY) and its

Full text of DOI:10.1021/jm020351j

J . Med. Chem. 2003, 46, 237-243
237
Str u ctu r e-Activity Rela tion sh ip of Im id a zo[1,2-a ]p yr id in es a s Liga n d s for
Detectin g â-Am yloid P la qu es in th e Br a in
Zhi-Ping Zhuang,† Mei-Ping Kung,† Alan Wilson,§ Chi-Wan Lee,† Karl Plo¨ssl,† Catherine Hou,†
David M. Holtzman, and Hank F. Kung*,†,‡
Departments of Radiology and Pharmacology, University of Pennsylvania, 3700 Market Street, Room 305,
Philadelphia, Pennsylvania 19104, Department of Psychiatry, University of Toronto, PET Centre, Centre for
Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8, and Departments of Neurology,
Molecular Biology and Pharmacology, Washington University, St. Louis, Missouri 63110
Received August 14, 2002
A series of novel â-amyloid (Aâ) aggregate-specific ligands, 2-(4′-dimethylaminophenyl)-6-
iodoimidazo[1,2-a]pyridine, 16(IMPY), and its related derivatives were prepared. An in vitro
binding study with preformed Aâ aggregates showed that 16(IMPY) and its bromo derivative
competed with binding of 2-(4′-dimethylaminophenyl)-6-iodobenzothiazole, [125I]7(TZDM), a
known ligand for Aâ aggregates, with high binding affinities (Ki ) 15 and 10 nM, respectively).
In vitro autoradiography of brain sections of a transgenic mouse (Tg2576) with [125I]16(IMPY)
displayed high selective binding to amyloid-like structures, comparable to that observed by
staining with thioflavin-S visualized under fluorescence. In vivo biodistribution after an
intravenous injection of [125I]16(IMPY) in normal mice showed a high initial brain uptake and
fast washout, indicating a low background activity associated with this iodinated ligand. Taken
together, the data suggests that [123I]16(IMPY) may be useful for imaging Aâ aggregates in
patients with Alzheimer’s disease.
In tr od u ction
min; 511 keV) and 18F (T1/2 ) 110 min; 511 keV) are
commonly used for positron emission tomography (PET).
Development of Aâ aggregate-specific imaging agents
is often based on highly conjugated dyes, such as Congo
Red (CR) and Chrysamine G (CG) (Chart 1). Thiofla-
vins-S and -T, as well as CR, have been used in
fluorescent staining of plaques and tangles in postmor-
tem AD brain sections. More abbreviated derivatives of
CG, styrylbenzenes (SB), such as 1,4 2,5 3,6 and 47
(Chart 1), have been reported. Because ionic and bulky
molecules are not able to penetrate intact blood-brain
barrier (BBB), attempts to develop labeled CG and CR
derivatives as imaging agents were, in general, unsuc-
cessful8-10 even though these molecules are highly
conjugated, show high binding affinity, and display
some lipophilicity.
Formation of â-amyloid (Aâ) plaques in the brain is
a pivotal event in the pathology of Alzheimer’s disease
(AD). Significant circumstantial evidence suggests that
fibrillary Aâ plaques consisting predominantly of ag-
gregates of Aâ40 and Aâ42 peptides play a major role
in AD pathogenesis.1,2 The accumulation of Aâ plaques
is now considered one of the most significant factors in
AD. Aâ aggregate-specific probes for in vitro and in vivo
studies of Aâ plaques are potentially important for
diagnosis and monitoring of therapeutic effects of drugs
aiming at lowering the Aâ burden in the brain by
noninvasive imaging.1,3 There are several potential
benefits of imaging Aâ aggregates in the brain. The
imaging technique will improve diagnosis by identifying
potential patients with Aâ plaques in the brain, who
are likely to have AD. It may also be useful to monitor
the progression of the disease. When antiplaque drug
treatments become available, imaging Aâ plaques in the
brain will be useful for monitoring treatment. The in
vivo imaging of Aâ plaques may provide information to
understand better AD symptoms vs Aâ burden in the
brain. Such information is very difficult to obtain in the
living human brain by other imaging methods.
To achieve a high brain penetration, it is normally
considered prudent to use neutral, small, and lipophilic
compounds. Interestingly, a neutral and highly lipo-
philic probe, [18F]5(FDDNP) (Chart 1), for binding
tangles and plaques has been reported. Preliminary
imaging study in humans appears to suggest that [18F]-
5(FDDNP) showed a higher retention in regions of brain
suspected of having tangles and plaques.11,12 A neutral
thioflavin derivative, 6(BTA-1) (Chart 1), was recently
reported. In an in vitro binding assay with Aâ40
aggregates, 6 showed an excellent affinity (Ki ) 11
nM).13 When [11C]6 was injected (iv) into normal mice,
it showed excellent brain penetration.13-15 Alternative
iodinated and neutral thioflavins, [125I]7(TZDM) and 8,
were prepared in our laboratory (Chart 1). These neutral
benzothiazole derivatives showed excellent binding af-
finities, with Kd values of <1 nM for aggregates of Aâ40.
Interestingly, under a competitive-binding assaying
condition, different binding sites on Aâ aggregates,
Currently, small-molecule-based diagnostic imaging
agents for Aâ aggregates in the brain can be labeled
with a suitable isotope for PET or SPECT imaging.
99mTc (T1/2 ) 6 h; 140 keV) and 123I (T1/2 ) 13 h; 159
keV) are routinely used for single photon emission
computed tomography (SPECT), whereas 11C (T1/2 ) 20
* To whom correspondence should be addressed. Tel.: (215) 662-
3096. Fax: (215) 349-5035. E-mail: kunghf@sunmac.spect.upenn.edu.
† Department of Radiology, University of Pennsylvania.
‡ Department of Pharmacology, University of Pennsylvania.
§ University of Toronto.
Washington University.
10.1021/jm020351j CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/18/2002

238 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Zhuang et al.
Ch a r t 1
Sch em e 1
Sch em e 2
which are mutually exclusive, were observed for styryl-
benzenes (SB) and thioflavins or benzothiazoles (TZ).7
It is observed that 1-4 bind to the SB sites with high
affinity, while 7(TZDM) and 8 bind to the TZ sites.
Significantly, biodistribution studies in normal mice
after an iv injection showed that [125I]7 and 8 exhibited
excellent brain uptake and retention, which were much
higher than those of [125I]3 and 4.7,16 To further modify
the benzothiazole series of compounds, a group of
stilbenes was prepared.17 Among the stilbenes, 9 (Chart
1) displayed an excellent binding affinity. Binding of
that imidazo[1,2-a]pyridine derivatives containing the
desired N,N-dimethylaminophenyl group, such as 16-
(IMPY), have binding properties similar to those of the
other iodinated benzothiazole ligands. It shares the
same critical structural component, the N,N-dimethy-
laminophenyl group, known to have good binding af-
finity. Reported herein are the synthesis and the
structure-activity relationship of this series of deriva-
tives. Initial in vivo and in vitro characterizations of
[
125I]9 to aggregates of Aâ40 is saturable, and the
dissociation constant (Kd) is 0.19 nM, which is compa-
rable to that observed for 7.7 In normal mice, [125I]9
showed excellent brain penetration (>1% dose/g after
an iv injection in mice).17 On the basis of known
compounds showing good binding affinity to Aâ ag-
gregates, it can be hypothesized that they all share a
common structural feature: all of the active compounds
contain either an N-methylamino- or N,N-dimethylami-
nophenyl group on one end of the molecule. The struc-
tural feature required for binding to Aâ aggregates
appears to be simple and unique.
[
125I]16(IMPY) as an Aâ aggregate-specific imaging
agent are reported.
Resu lts a n d Discu ssion
Ch em istr y. Syntheses of imidazo[1,2-a]pyridine de-
rivatives were achieved by reactions shown in Schemes
1-3. The initial formation of the imidazo[1,2-a]pyridine
ring was readily accomplished by a fusion reaction
between a bromoketone18 and 2-aminopyridines under
In the search for novel ligands for Aâ aggregates
based on the benzothiazole ring system, it was observed

Novel â-Amyloid Aggregate-Specific Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 239
Ta ble 1. Inhibition Constants (Ki, nM)a and Spectroscopic Propertiesb of IMPY and Its Derivatives
Ki
(nM)
λ (nm),
log ꢀ
φ (in EtOH)
compd
12c
R1
R2
H
R3
(λex [nm], λem [nm])b
H
H
NHMe
>1000
355, 4.14
281, 4.06
217, 4.31
336, 4.25
277, 4.38
not measured
not measured
356, 4.26
285, 4.44
234, 4.52
355, 4.21
283, 4.41
229, 4.50
323, 3.97
253, 4.55
210, 4.38
327, 3.90
276, 4.35
340, 4.14
277, 4.38
227, 4.48
358, 4.10
286, 4.35
246, 4.40
366, 4.29
0.24 (345, 446)
13
H
NMe2
>2000
0.25 (337, 450)
14c
15
16(IMPY)
CH3
CH3
I
H
H
H
NHMe
NMe2
NMe2
>2000
242 ( 20
15 ( 5
not measured
not measured
0.039 (339, 446)
17
18
Br
H
H
NMe2
Br
10.3 ( 1.2
638 ( 30
0.071 (350, 460)
0.20 (320, 370)
CH3
19
20
NMe2
H
H
I
Br
339 ( 40
>2000
0.064 (330, 414)
0.0043 (340, 440)
NMe2
22
I
I
NMe2
>2000
too low to be
measured
7(TZDM)
0.9 ( 0.2
0.21 (360, 425)
a
Inhibition constants were measured by in vitro binding assay using preformed Aâ40 aggregates and [125I]7 (TZDM) as the ligand.
b
Referenced to a quinine hydrochloride solution in 0.1 N H2SO4 (φ ) 0.55).
Sch em e 3
15 in good yields. Treatment of compound 13 with iodine
in chloroform led to the formation of the 3-iodo deriva-
tive, 20 (Scheme 2). Apparently, the 3-position is the
most electron-rich position for an iodination reaction.
This sensitivity for electrophilic substitution will play
an important role in the iodination of the series of
compounds.
The starting material for radioiodination, the tri-n-
butyltin derivative 21, was prepared with a palladium-
catalyzed reaction (Scheme 3). Preparation of radioio-
dinated [125I]16(IMPY) was carried out by an iododestan-
nylation reaction catalyzed by hydrogen peroxide (Scheme
3). The desired tracer, [125I]16(IMPY), was readily
purified by HPLC (radiochemical yield 20-60%, radio-
chemical purity >95%). To characterize the tracer fully,
a comparable reaction in a higher chemical amount was
attempted by reacting 21 with “cold” iodine in chloro-
form. Surprisingly, the product from this reaction
showed no binding affinity at all. Upon closer inspection,
it was found that the “cold” iododestannylation reaction
produced a 3,6-diiodo compound, 22, instead of 16(IMPY).
An alternative synthesis by reaction of 10b with 2-amino-
5-iodopyridine, 11e, yielded 16(IMPY) unambiguously.
a mild basic condition to give the desired imidazo[1,2-
a]pyridines in good yields (Scheme 1). The fusion
reaction shown in Scheme 1 is versatile, and it has been
applied in the synthesis of derivatives bearing various
substitution groups on the ring system. The starting
materials, bromoketone and 2-aminopyridine, are gen-
erally available commercially, whereas 2-amino-5-(N,N-
dimethylamino)pyridine was prepared by a known
method reported previously.19 The nitro derivatives, 12a
and 14a , where R3 ) NO2, were readily reduced by
sodium sulfide to amino derivatives, 12b and 14b,20,21
in excellent yields. Subsequently, the amino group of
12b and 14b was converted to an N-methylamino group
by a selective monomethylation reaction using formic
acetic anhydride at ambient temperatures for 30 min,
followed by a reduction reaction in the presence of
LiAlH4. The monomethylated 12c and 14c were again
methylated by paraformaldehyde and sodium borohy-
dride to give the N,N-dimethylamino derivatives 13 and
Under a no-carrier-added condition, the iododestan-
nylation reaction produced only the expected monoiodo
compound, 16(IMPY). It was reported previously that
the imidazo[1,2-a]pyridine ring will readily undergo an
electrophilic substitution reaction at the 3-position.22
The result showed that, even under a very mild reaction
condition, I2 in chloroform, the electrophilic substitution
reaction at the 3-position will take place. The iodination
reaction of the imidazo[1,2-a]pyridine ring produced

240 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Zhuang et al.
Ta ble 2. Biodistribution of Radioactivity in Normal Mice (ICR) after an Intravenous Injection of [125I]16(IMPY) (% Dose/Organ)a
organ
2 min
30 min
1 h
2 h
6 h
24 h
blood
heart
muscle
lung
kidney
spleen
liver
6.41 ( 0.77
0.79 ( 0.14
13.8 ( 3.44
1.56 ( 0.33
4.75 ( 0.49
0.40 ( 0.06
20.9 ( 2.63
5.72 ( 0.90
2.88 ( 0.25
2.44 ( 0.36
0.16 ( 0.02
6.08 ( 0.59
0.31 ( 0.07
1.51 ( 0.27
0.09 ( 0.02
6.32 ( 0.55
4.69 ( 1.06
0.26 ( 0.00
2.50 ( 0.11
0.12 ( 0.02
5.03 ( 1.03
0.34 ( 0.08
1.17 ( 0.29
0.08 ( 0.01
5.88 ( 0.85
4.28 ( 0.25
0.21 ( 0.03
1.82 ( 0.21
0.08 ( 0.01
2.96 ( 0.84
0.20 ( 0.05
0.53 ( 0.05
0.05 ( 0.01
2.90 ( 0.21
3.14 ( 0.51
0.14 ( 0.03
1.40 ( 0.27
0.04 ( 0.01
1.46 ( 0.42
0.12 ( 0.05
0.25 ( 0.05
0.04 ( 0.01
1.54 ( 0.08
2.19 ( 0.63
0.06 ( 0.02
0.18 ( 0.02
0.01 ( 0.00
0.27 ( 0.11
0.05 ( 0.03
0.05 ( 0.01
0.01 ( 0.00
0.61 ( 0.11
0.22 ( 0.06
0.02 ( 0.00
skin
brain
a
Average of 3 or 4 mice per time point ( standard deviation.
very different results from other comparable fused ring
systems, such as benzothiazole, 7(TZDM).
Biologica l Stu d ies. In an in vitro binding assay,
16(IMPY) competed with [125I]7 (TZDM) binding to Aâ40
aggregates, showing an excellent binding affinity (Ki )
15.0 ( 5.0 nM, Table 1). A comparable binding affinity
value was obtained for the corresponding bromo deriva-
tive, 17 (Ki ) 10.3 ( 1.2 nM). Other modifications of
16(IMPY) appeared to decrease the binding affinity to
Aâ aggregates, as measured by in vitro competition with
[
125I]7 (Table 1). Elimination of the halogen groups at
the 6-position (compounds 12 and 13) reduced the
binding affinity dramatically (Ki > 2000 nM); replacing
the 6-halogen with a methyl group also reduced the
binding significantly (Ki for 15 was 242 nM). It is also
worth noting that the 6-methyl derivative, 18, or the
6-(N,N-dimethlyamino)-2-(4′-bromophenyl) derivative,
19, showed low binding affinities (Ki ) 638 and 339 nM,
respectively). The overall molecular size of 16(IMPY)
is similar to that of 7 or 9. Similar to the findings
reported previously,21,23 the imidazo[1,2-a]pyridine com-
pounds showed strong absorbance, with λmax ) 320-
360 nm, with a quantum yield (φ value) of 3.97-4.26
(Table 1). The quantum yields (φ) of fluorescence were
measured using quinine as a standard.24 The φ values
ranged from 0 to 0.25. The compounds containing
halogens, 16(IMPY), 17, 20, and 22, showed the lowest
values, whereas compounds 12c, 13, and 18 showed φ
values of 0.20-0.25, comparable to that of 7 (φ ) 0.21).
The dramatic decrease in intensity of fluorescence upon
introduction of halogens such as iodine or bromine is
well known.25 This series of compounds with halogen
substitution at the 6-position in general exhibited a
higher binding affinity toward the aggregates. It is
important to note that the UV absorption and the
fluorescent properties of this series of compounds are
not coupled to the binding affinity toward the Aâ
aggregates (Table 1). Apparently, the high fluorescent
properties needed for staining the plaques in vitro is
not a prerequisite for binding to the Aâ aggregates.
Brain sections of a 16-month-old Tg2576 mouse were
labeled with [125I]16(IMPY). A distinctive plaque-label-
ing pattern was clearly visualized with a low back-
ground activity (Figure 1). When the same brain section
was stained with the fluorescent dye, thioflavin-S, the
same Aâ plaques showed prominent fluorescent label-
ing, consistent with the results of an autoradiogram.
A biodistribution study in normal mice after an iv
injection showed that [125I]16(IMPY) exhibited excellent
brain uptake (2.9% ID/organ at 2 min) and fast washout
(0.2% ID/organ at 1 h), which is highly desirable for Aâ
plaque imaging agents (Table 2). At 6 and 24 h, there
was essentially no remaining brain uptake. The blood
F igu r e 1. Film autoradiogram of brain sections from a
Tg2576 mouse (right) labeled with [125I]16(IMPY). The plaques
were confirmed with an adjacent section fluorescently stained
with thioflavin-S (left).
levels are relatively low at all time points measured.
The tracer seems to distribute in high blood flow areas,
such as liver, kidney, muscle, and skin (Table 2). The
partition coefficient (PC) of [125I]16 is 100 (1-octanol/
buffer),26 which is comparable to that of [125I]7 (PC )
70).7 A relatively high lipophilicity may be critical for
the initial brain penetration by a simple diffusion
mechanism. The most important feature of this novel
ligand, [125I]16(IMPY), is the faster rate of washout from
the normal brain. This fast rate is due to a lack of
trapping mechanisms in the normal brain (no Aâ
plaques). As expected, the Aâ plaques in the brain of a
Tg2576 mouse can be distinctly visualized and detected
in vivo by using [125I]16(IMPY).26
In summary, this novel imidazo[1,2-a]pyridine de-
rivative, [125I]16(IMPY), is a radioiodinated compound
showing excellent in vitro binding and in vivo biodis-
tribution data. Derivatives of 16(IMPY) generally show
lower binding affinity, suggesting that highly selective
substitution groups are needed on specific locations of
the imidazo[1,2-a]pyridine ring. The in vivo biodistri-
bution of [125I]16(IMPY) in normal mice showed excel-
lent brain uptake and washout. The initial observation
in normal mice is confirmed in the transgenic mice
engineered to develop excess Aâ plaques. It appears that
this ligand will have highly desirable properties: fast
kinetics of high initial brain uptake and rapid washout
in the non-Aâ plaque-containing areas. Tracers with
such properties may provide the highest target-to-
nontarget ratio; therefore, they are most likely to be
successful as imaging agents for Aâ plaques in the brain.
Exp er im en ta l Section
Reagents used in the syntheses were purchased from
Aldrich Chemical Co. or Fluka Chemical Co. and were used
without further purification, unless otherwise indicated. Pre-
parative thin-layer chromatography (PTLC) was performed on

Novel â-Amyloid Aggregate-Specific Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 241
silica gel plates with a fluorescent indicator that was visualized
with light at 254 nm. 1H NMR spectra were obtained on
Bruker spectrometers (Bruker DPX 200 and AMX 500).
Chemical shifts are reported as δ values and referenced to
CDCl3 (7.26 ppm for 1H). Coupling constants are reported in
hertz. Mass spectrometry was performed by the Mass Spec-
trometry Center, University of Pennsylvania. Elemental analy-
sis was performed by Elemental Analysis Center, University
of Pennsylvania. UV absorbance was measured on a Beckman
DU640 spectrophotometer, and the fluorescence was recorded
on a Perkin-Elmer LS55 luminescence spectrometer. Two high-
pressure liquid chromatography (HPLC) systems were used
to confirm the purity of some compounds listed in this section.
In system A, a reversed-phase PRP-1 column (Hamilton, 250
× 4.6 mm) was eluted with CH3CN/dimethyl glutarate buffer,
5 mM, pH 7.0 (9:1) at a flow rate of 1 mL/min. In system B, a
normal-phase Partisil 10 column (Whatman, 250 × 4.6 mm)
was eluted with hexanes/ethyl acetate (1:1) at a flow rate of
2.0 mL/min.
Absorbance (UV/vis) was measured with a spectrophotom-
eter (Beckman DU 640). Fluorescence spectra were obtained
with a luminescence spectrometer (Perkin-Elmer LS 55). Cells
of 1 cm path length were used. Fluorescence quantum yields
were obtained by comparing the area under the emission
spectrum with that of a standard with a known quantum yield
(quinine hydrochloride solution in 0.1 N H2SO4, φ ) 0.55).24
Emission and excitation slits were set to 5 nm and the scan
speed 500 nm/min. The temperature was ambient (24 °C). All
of the compounds were dissolved in absolute ethanol, and
concentrations between 10-5 and 10-6 M were used for absor-
bance measurements.
reduction reaction produced 14b, and subsequent monomethy-
lation yielded 14c with a yield of 52%. The purity was
evaluated on two HPLC systems and found to be greater than
95% (system A, retention time ) 4.0 min; system B, retention
time ) 10.4 min).
1H NMR (500 MHz, DMSO-d6): δ 2.24 (s, 3H), 2.69 (d, J )
4.9 Hz, 3H), 5.75 (q, J ) 4.9 Hz, 1H), 6.55 (d, J ) 9.0 Hz, 2H),
7.00 (d, J ) 9.0 Hz, 1H), 7.37 (d, J ) 9.0 Hz, 1H), 7.65 (d, J )
9.0 Hz, 2H), 8.00 (s, 1H), 8.22 (s, 1H).
2-(4′-Dim eth ylam in oph en yl)im idazo[1,2-a ]pyr idin e (13).
To a mixture of 12c (23 mg, 0.11 mmol) and (CH2O)n (100 mg)
in THF (5 mL) was added NaBH4 (100 mg) in one portion at
room temperature, followed by TFA (2 mL) dropwise. The
resulting mixture was stirred at room temperature overnight
and poured into an ice-cold NaOH solution (25%). The mixture
was extracted with CH2Cl2, dried, filtered, and concentrated
to give a crude product which was purified by PTLC (EtOAc/
Hex ) 1:1 as developing solvent) to give 15 mg of product 13
(58% yield). The purity was evaluated on two HPLC systems
and found to be greater than 95% (system A, retention time
) 4.5 min; system B, retention time ) 6.1 min).
1H NMR (200 MHz, CDCl3): δ 2.99 (s, 6H), 6.70 (t, J ) 6.8
Hz, 1 H), 6.78 (d, J ) 8.8 Hz, 2H), 7.10 (t, J ) 6.8 Hz, 1H),
7.58 (d, J ) 9.5 Hz, 1H), 7.71 (s, 1H), 7.83 (d, J ) 8.7 Hz, 2H),
8.05 (d, J ) 6.7 Hz, 1H).
2-(4′-Dim et h yla m in op h en yl)-6-m et h ylim id a zo[1,2-a ]-
p yr id in e (15). The same procedure for preparation of 13 was
performed to prepare 15, starting from 14c. The purity was
evaluated on two HPLC systems and found to be greater than
95% (system A, retention time ) 5.2 min; system B, retention
time ) 5.4 min).
1H NMR (200 MHz, CDCl3): δ 2.29 (s, 3H), 2.99 (s, 6H),
6.78 (d, J ) 8.8 Hz, 2H), 6.96 (d, J ) 9.2 Hz, 1 H), 7.58 (d, J
) 9.2 Hz, 1H), 7.64 (s, 1H), 7.81 (d, J ) 8.8 Hz, 2H), 7.85 (s,
1H).
2-(4′-Nitr op h en yl)im id a zo[1,2-a ]p yr id in e (12a ). This
compound was prepared by a previously reported method.20
Yield 74%, mp 269-270 °C (lit.20 mp 268-270 °C).
1H NMR (500 MHz, CDCl3): δ 6.86 (dt, J ) 1.1, 7.9 Hz, 1H),
7.22-7.28 (m, 1H), 7.68 (dd, J ) 1.0, 9.2 Hz, 1H), 8.00 (s, 1H),
8.12 (d, J ) 9.0 Hz, 2H), 8.17 (dt, J ) 1.1, 6.7 Hz, 1H), 8.31 (d,
J ) 9.0 Hz, 2H).
2-(4′-Dim eth yla m in op h en yl)-6-iod oim id a zo[1,2-a ]p yr i-
d in e, 16(IMP Y). A mixture of 2-bromo-4′-dimethylaminoac-
etophenone 10b (484 mg, 2 mmol) and 2-amino-5-iodopyridine
11e (440 mg, 2 mmol) in EtOH (25 mL) was stirred under
reflux for 2 h. NaHCO3 (250 mg) was added after the mixture
was cooled. The resulting mixture was stirred under reflux
for 4 h. The mixture was cooled and filtered to give 348 mg of
product, 16(IMPY) (48% yield).
1H NMR (200 MHz, CDCl3): δ 3.00 (s, 6H), 6.77 (d, J ) 8.8
Hz, 2H), 7.27 (dd, J ) 9.4, 1.5 Hz, 1H), 7.38 (d, J ) 9.5 Hz,
1H), 7.66 (s, 1H), 7.79 (d, J ) 8.8 Hz, 2H), 8.32 (d, J ) 0.7 Hz,
1H). Anal. 16, (C15H14IN3).
6-Br om o-2-(4′-d im eth yla m in oph en yl)im ida zo[1,2-a ]p y-
r id in e (17). A mixture of 2-bromo-4′-dimethylaminoacetophe-
none 10b18 (968 mg, 4 mmol) and 2-amino-5-bromopyridine
11d (692 mg, 4 mmol) in EtOH (25 mL) was stirred under
reflux for 2 h. NaHCO3 (500 mg) was added after the mixture
was cooled. The resulting mixture was stirred under reflux
for 4.5 h. The mixture was cooled and filtered to give 655 mg
of product 17 (52% yield).
1H NMR (200 MHz, CDCl3): δ 3.00 (s, 6H), 6.78 (d, J ) 8.7
Hz, 2H), 7.17 (dd, J ) 9.5, 1.7 Hz, 1H), 7.49 (d, J ) 9.5 Hz,
1H), 7.69 (s, 1H), 7.80 (d, J ) 0.7 Hz, 2H), 8.21 (dd, J ) 1.7,
0.8 Hz, 1H). Anal. 17, (C15H14BrN3).
6-Meth yl-2-(4′-n itr oph en yl)im idazo[1,2-a ]pyr idin e (14a).
This compound was prepared by a previously reported method.20
Yield 85%, mp 245-247 °C (lit.20 mp 235 °C).
1H NMR (500 MHz, CDCl3): δ 2.35 (s, 3H), 7.10 (dd, J )
1.7, 9.3 Hz, 1H), 7.57 (d, J ) 9.3 Hz, 1H), 7.91 (s, 1H), 7.94 (d,
J ) 1.7 Hz, 1H), 8.09 (d, J ) 9.0 Hz, 2H), 8.29 (d, J ) 9.0 Hz,
2H).
2-(4′-Am in op h en yl)im id a zo[1,2-a ]p yr id in e (12b). This
compound was prepared by a previously reported method.21
Yield 86%.
1H NMR (500 MHz, CDCl3): δ 6.72-6.78 (m, 1H), 6.75 (d,
J ) 8.5 Hz, 2H), 7.11-7.15 (m, 1H), 7.59 (dd, J ) 0.7, 9.2 Hz,
1H), 7.74 (s, 1H), 7.76 (d, J ) 8.5 Hz, 2H), 8.08-8.10 (m, 1H).
2-(4′-Meth ylam in oph en yl)im idazo[1,2-a ]pyr idin e (12c).
A stirred suspension of 12b (0.5 g, 2.39 mmol) in THF (5 mL)
was treated dropwise with formic acetic anhydride (2 mL) at
ambient temperature under nitrogen. After 30 min, the
mixture was poured into 50 mL of ice-cold saturated aqueous
Na2CO3. The precipitate was collected by vacuum filtration
and dried in vacuo. The formamide was added in small
portions to a stirred solution of LiAlH4 (0.2 g, 5.3 mmol) in
THF (10 mL), and the mixture was heated to reflux for 1 h.
Upon cooling, the solution was cautiously quenched with
aqueous KOH (1 N, 0.5 mL), diatomaceous earth was added,
and the mixture was filtered. Solids were washed with acetone,
and the combined filtrates were evaporated to give a light
brown product (0.33 g, 62%). The purity of 12c was evaluated
on two HPLC systems and found to be greater than 95%
(system A, retention time ) 3.6 min; system B, retention time
) 9.8 min).
2-(4-Br om oph en yl)-6-m eth ylim idazo[1,2-a ]pyr idin e (18).
This compound was prepared by a procedure reported previ-
ously.27
2-(4-Br om op h en yl)-6-(d im eth yla m in o)im id a zo[1,2-a ]-
p yr id in e (19). The same procedure described for the prepara-
tion of 17 was performed to prepare 19, starting from R-bromo-
4-bromoacetophenone 10a (1.5 g) and 2-amino-5-(dimethy-
lamino)pyridine 11a (0.74 g), to give 19 in 38% yield.
1H NMR (200 MHz, CDCl3): δ 2.88 (s, 6H), 7.05 (dd, J )
2.34, 9.81 Hz, 1H), 7.37 (d, J ) 1.89 Hz, 1H), 7.45-7.55 (m,
3H), 7.73-7.81 (m, 3H). 13C NMR (50 MHz, CDCl3): δ 41.53,
107.85, 108.64, 117.03, 119.67, 121.23, 127.18, 131.68, 133.20,
140.13, 142.13, 143.90. Anal. 19, (C15H14BrN3).
1H NMR (500 MHz, DMSO-d6): δ 2.80 (s, 3H), 6.91 (d, J )
5.5 Hz, 2H), 7.47 7.50 (m, 1H), 7.87-7.96 (m, 4H), 8.64 (s, 1H),
8.86 (d, J ) 6.5 Hz, 1H).
6-Meth yl-2-(4′-m eth yla m in op h en yl)im id a zo[1,2-a ]p y-
r id in e (14c). The same procedure described above for the
preparation of 12c was performed. Starting from 14a , a
2-(4′-Dim eth yla m in op h en yl)-3-iod oim id a zo[1,2-a ]p yr i-
d in e (20). To a solution of 13 (20 mg, 0.08 mmol) in CHCl3-

242 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Zhuang et al.
(10 mL) was added I2 solution (0.5 mL, 1 M in CHCl3) dropwise
at room temperature. The mixture was stirred at room
temperature for 1 h. NaHSO3 solution (2 mL, 5%) was added,
followed by KF (2 mL, 1 M in MeOH). The organic phase was
separated, dried, filtered, and concentrated to give a crude
product, which was purified by PTLC (Hex/EtOAc ) 1:1 as
developing solvent) to give 16 mg of product 20 (52%).
1H NMR (200 MHz, CDCl3): δ 3.02 (s, 6H), 6.82 (d, J ) 7.1
Hz, 2H), 6.88 (t, J ) 6.8 Hz, 1H), 7.22 (t, J ) 6.8 Hz, 1H), 7.60
(d, J ) 9.0 Hz, 1H), 7.98 (d, J ) 9.0 Hz, 2H), 8.20 (d, J ) 6.8
Hz, 1H). Anal. 20, (C15H14I2N3).
2-(4′-Dim eth ylam in oph en yl)-6-(tr ibu tylstan n yl)im idazo-
[1,2-a ]p yr id in e (21). To a solution of 6-bromo-2-(4′-dimethy-
laminophenyl)imidazo[1,2-a]pyridinee 17 (80 mg, 0.26 mmol)
in 1,4-dioxane (10 mL) and triethylamine (2 mL) was added
(Bu3Sn)2 (0.2 mL, neat), followed by Pd(Ph3P)4 (20 mg). The
mixture was stirred at 90 °C overnight. Solvent was removed,
and the residue was purified by PTLC (Hex/EtOAc ) 1:1 as
developing solvent) to give 23 mg of product 21 (17%).
1H NMR (200 MHz, CDCl3): δ 0.90 (t, J ) 7.2 Hz, 9H), 1.10
(t, J ) 8.0 Hz, 6H), 1.33 (hex, J ) 7.1 Hz, 6H), 1.54 (pen, J )
7.2 Hz, 6H), 3.00 (s, 6H), 6.78 (d, J ) 8.9 Hz, 2H), 7.11 (d, J
) 8.8 Hz, 1H), 7.57 (d, J ) 8.8 Hz, 1H), 7.71 (s, 1H), 7.84 (d,
J ) 8.8 Hz, 2H), 7.95 (d, J ) 0.8 Hz, 1H). HRMS: m/z calcd
for C27H42N3Sn (M+ + H) 528.2400, found 528.2402. Anal. 21,
(C27H41N3Sn‚2H2O).
incubated at room temperature for 3 h, and the bound and
the free radioactivities were separated by vacuum filtration
through Whatman GF/B filters using a Brandel M-24R cell
harvester, followed by 2 × 3 mL washes with 10% ethanol at
room temperature. Filters containing the bound I-125 ligand
were counted in a γ counter (Packard 5000) with 70% counting
efficiency. The results of inhibition experiments were subjected
to nonlinear regression analysis using EBDA software,29 and
Ki values were calculated.
In Vitr o Au tor a d iogr a p h y a n d F lu or escen t Sta in in g
of Br a in Section s of a Tg 2576 (AP P SW) Mou se. The brain
of a 16-month-old Tg2576 mouse was removed and frozen in
powdered dry ice.30 After equilibration to -20 °C, consecutive
20-µm coronal sections were cut on a cryostat (Hacker Instru-
ments, Fairfield, NJ ), thaw-mounted on Fisher Superfrost Plus
slides, and stored at -70 °C until use. The sections were
thawed, labeled with 0.3 nM [125I]16(IMPY) at room temper-
ature for 1 h, and washed 2 × 3 min with saturated Li2CO3 in
40% EtOH, 2 min in 40% EtOH, and 30 s in H2O. After drying,
the labeled sections were exposed to Cronex MRF film for 72
h, along with a strip of I-125 standard (0.5 cm wide) made of
polymer containing 156, 81, 39.3, 20, 9.85, and 4.98 nCi/mg of
activity (Amersham, Buckinghamshire, England). The films
were developed and digitized using a computer-based image
analysis system (NIH image, version 1.61). The presence of
radiolabeled amyloid deposits in the sections was confirmed
in the same section labeled with thioflavin-S (TF-S). Fluores-
cence staining of the brain tissue sections with TF-S was
achieved by the following steps: staining with TF-S for 3 min
(0.0125% thioflavin-S in 40% EtOH/60% PBS). Sections are
quickly differentiated in 50% EtOH/50% PBS for 3 min, PBS
for 1 min, and H2O for 5 min prior to imaging by fluorescent
microscopy (BioRad 1024-ES and Nikon Eclipse E-600) using
an FITC cube with an exitation filter of 350-390 nm and an
emission filter of 530 ( 15 nm.
2-(4′-Dim eth yla m in op h en yl)-3,6-d iiod oim id a zo[1,2-a ]-
p yr id in e (22). The same procedure described for the prepara-
tion of 20 was performed, starting from 21 to give 22 in 32%
yield.
1H NMR (200 MHz, CDCl3): δ 3.02 (s, 6H), 6.82 (d, J ) 7.1
Hz, 2H), 7.37 (d, J ) 0.92 Hz, 2H), 7.98 (d, J ) 7.0 Hz, 2H),
8.42 (d, J ) 1.1 Hz, 1H). Anal. 22, (C15H13I2N3).
P r ep a r a t ion of R a d ioiod in a t ed Liga n d s:
(IMP Y) a n d [125I]7(TZDM). The radioiodinated compound,
125I]7(TZDM), was prepared according to the method described
[
125I]16-
In Vivo Biod istr ibu tion in Nor m a l Mice. While under
ether anesthesia, male ICR mice (2-3 months old, average
weight 20-30 g) received an injection directly into the tail vein
of 0.15 mL of a 0.1% bovine serum albumin solution containing
[
previously.7 The desired [125I]16(IMPY) was prepared using
iododestannylation reactions with tributyltin precursor 21.28
Hydrogen peroxide (50 µL, 3% w/v) was added to a mixture of
50 µL of the corresponding tributyltin precursor (1 µg/µL
EtOH), 50 µL of 1 N HCl, and [125I]NaI (1-5 mCi, purchased
from NEN) in a sealed vial. The reaction was allowed to
proceed for 10 min at room temperature and terminated by
addition of 100 µL of saturated NaHSO3. The reaction mixture
was extracted with ethyl acetate (3 × 1 mL) after neutraliza-
tion with saturated sodium bicarbonate solution. The combined
extracts were evaporated to dryness. The residues were
dissolved in 100 µL of EtOH and purified by HPLC using a
reversed-phase column (PRP-1, 250 × 4.6 mm) with an
isocratic solvent consisting of 90% acetonitrile/10% 3,3-dim-
ethylglutaric acid (5 mM, pH 7.0) at a flow rate of 1.0 mL/
min. The no-carrier-added products were evaporated to dry-
ness and redissolved in 100% EtOH (1 µCi/µL), The final
[
125I]16 (5-10 µCi). The mice were sacrificed by cardiac
excision at various time points postinjection. The organs of
interest were removed and weighed, and the radioactivity was
counted with an automatic γ counter (Packard 5000). 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 and muscle were calculated
under the assumption that they were 7% and 40% of the total
body weight, respectively.
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health (NS
18509, H.F.K.; AG 13956, D.M.H.) and the Institute for
the Study of Aging (M.-P.K).
[
125I]16(IMPY), with a specific activity of 2200 Ci/mmol and
>95% radiochemical purity, was stored at -20 °C up to 6
weeks for in vitro binding and autoradiography studies.
Bin d in g Assa ys Usin g Aggr ega t ed Aâ40 P ep t id e in
Solu tion . The solid form of peptide Aâ40 was purchased from
Bachem (King of Prussia, PA). Aggregation of peptide was
carried out by gently dissolving the peptide (0.5 mg/mL) in a
buffer solution (pH 7.4) containing 10 mM sodium phosphate
and 1 mM EDTA. The solutions were incubated at 37 °C for
36-42 h with gentle and constant shaking. Binding studies
were carried out in 12- × 75-mm borosilicate glass tubes
according to the procedure described previously.7 For inhibition
studies, 1 mL of the reaction mixture contained 40 µL of
inhibitors (concentration range between 10-5 and 10-10 M,
diluted in 10% EtOH), 50 µL of aggregated fibrils (10-50 nM
in the final assay mixture), and 0.05 nM radiotracer in 40%
EtOH. Ethanol is needed for this assay; without it, some of
the “cold” ligands evaluated were not soluble. Nonspecific
binding was defined in the presence of 2 µM thioflavin-T.
Under the assay conditions, the specifically bound fraction was
less than 15% of the total radioactivity. The mixture was
Refer en ces
(1) Selkoe, D. J . Alzheimer’s disease: genes, proteins, and therapy.
Physiol. Rev. 2001, 81, 741-766.
(2) 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.
(3) Selkoe, D. J . The origins of Alzheimer disease: A is for amyloid.
J AMA-J . Am. Med. Assoc. 2000, 283, 1615-1617.
(4) Styren, S. D.; Hamilton, R. L.; Styren, G. C.; Klunk, W. E. X-34,
a fluorescent derivative of Congo Red: a novel histochemical
stain for Alzheimer’s disease pathology. J . Histochem. Cytochem.
2000, 48, 1223-1232.
(5) Skovronsky, D. M.; Zhang, B.; Kung, M.-P.; Kung, H. F.;
Trojanowski, J . Q.; Lee, V. M.-Y. In vivo detection of amyloid
plaques in a mouse model of Alzheimer’s disease. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 7609-7614.
(6) Lee, C.-W.; Zhuang, Z.-P.; Kung, M.-P.; Plo¨ssl, K.; Skovronsky,
D.; Gur, T. L.; Hou, C.; Trojanowski, J . Q.; Lee, V. M.-Y.; Kung,
H. F. Isomerization of (Z,Z) to (E,E)1-bromo-2,5-bis-(3-hydroxy
carbonyl-4-hydroxy)styrylbenzene in strong base: probes for
amyloid plaques in the brain. J . Med. Chem. 2001, 44, 2270-
2275.

Novel â-Amyloid Aggregate-Specific Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 243
(7) Zhuang, Z.-P.; Kung, M.-P.; Hou, C.; Skovronsky, D.; Gur, T.
L.; Trojanowski, J . Q.; Lee, V. M.-Y.; Kung, H. F. Radioiodinated
Styrylbenzenes and Thioflavins as Probes for Amyloid Ag-
gregates. J . Med. Chem. 2001, 44, 1905-1914.
(8) Dezutter, N. A.; Dom, R. J .; de Groot, T. J .; Bormans, G. M.;
Verbruggen, A. M. 99mTc-MAMA-chrysamine G, a probe for beta-
amyloid protein of Alzheimer’s disease. Eur. J . Nucl. Med. 1999,
26, 1392-1399.
(9) Han, H.; Cho, C.-G.; Lansbury, P. T., J r. Technetium complexes
for the quantitation of brain amyloid. J . Am. Chem. Soc. 1996,
118, 4506-4507.
(18) Diwu, Z.; Beachdel, C.; Klaubert, D. H. A Facile protocol for the
convenient preparation of amino-substituted R-bromo- and R,R-
dibromo arylmethylketones. Tetrahedron Lett. 1998, 39, 4987-
4990.
(19) Fox, G. J .; Hepworth, J . D.; Hallas, G. Bromination of 3-amino-
and 3-(dimethylamino)pyridine. J . Chem. Soc., Perkin Trans. 1
1973, 68-69.
(20) Sundberg, R. J .; Dahlhausen, D. J .; Manikumar, G.; Mavunkel,
B.; Biswas, A.; Srinivasan, V.; King, F., J r.; Waid, P. Preparation
of 2-aryl and 2-aryloxymethylimidazo[1,2-a]pyridines and related
compounds. J . Heterocycl. Chem. 1988, 25, 129-137.
(21) Tomoda, H.; Hirano, T.; Saito, S.; Mutai, T.; Araki, K. Substitu-
ent Effects on Fluorescent Properties of Imidazo[1,2-a]pyridine-
Based Compounds. Bull. Chem. Soc. J pn. 1999, 72, 1327-1334.
(22) Enguehard, C.; Renou, J . L.; Collot, V.; Hervet, M.; Rault, S.;
Gueiffier, A. Reactivity of 3-iodoimidazo[1,2-a]pyridines using
a Suzuki-type cross-coupling reaction. J . Org. Chem. 2000, 65,
6572-6575.
(23) Knoelker, H. J .; Boese, R.; Hitzemann, R. Imidazole derivatives.
III. Regioselective synthesis, structure, and fluorescence proper-
ties of highly substituted imidazo[1,2-a]pyridines and pyrido-
[1,2-a]benzimidazoles. Chem. Ber. 1990, 123, 327-339.
(24) Chen, R. F. Some characteristics of the fluorescence of quinine.
Anal. Biochem. 1967, 19, 374-387.
(25) Lakowicz, J . R. Principles of Fluorescence Spectroscopy; Kluwer
Academic/Plenum Publisher: New York, NY, 1999.
(26) Kung, M. P.; Hou, C.; Zhuang, Z.-P.; Zhang, B.; Skovronsky, D.
M.; Gur, T.; Lee, V. M.-Y.; Trojanowski, J . Q.; Kung, H. F.
IMPY: An improved thioflavin-T derivative for in vivo Labeling
of b-amyloid plaques. Brain Res. 2002, 956, 202-210.
(27) Katsifis, A.; Mattner, F.; Zhang, Z.; Dikic, B.; Papazian, V.
Synthesis of [123I]N′,N′-dimethyl-6-methyl-(4′-iodophenyl)imi-
dazo[1,2-a]pyridin e-3-acetamide for the study of Peripheral
Benzodiazepine Receptors using SPECT. J . Labelled Compd.
Radiopharm. 2000, 43, 385-394.
(28) Kung, M. P.; Hou, C.; Oya, S.; Mu, M.; Acton, P. D.; Kung, H. F.
Characterization of [123I]IDAM as a novel single-photon emission
tomography tracer for serotonin transporters. Eur. J . Nucl. Med.
1999, 26, 844-853.
(29) Munson, P. J .; Rodbard, D. LIGAND: a versatile computerized
approach for characterization of ligand-binding systems. Anal.
Biochem. 1980, 107, 220-239.
(10) Zhen, W.; Han, H.; Anguiano, M.; Lemere, C. A.; Cho, C. G.;
Lansbury, P. T., J r. Synthesis and amyloid binding properties
of rhenium complexes: preliminary progress toward a reagent
for SPECT imaging of Alzheimer’s disease brain. J . Med. Chem.
1999, 42, 2805-2815.
(11) Agdeppa, E. D.; Kepe, V.; Liu, J .; Flores-Torres, S.; Satyamurthy,
N.; Petric, A.; Cole, G. M.; Small, G. W.; Huang, S. C.; Barrio, J .
R. Binding characteristics of radiofluorinated 6-dialkylamino-
2-naphthylethylidene derivatives as positron emission tomog-
raphy imaging probes for â-amyloid plaques in Alzheimer’s
disease. J . Neurosci. 2001, 21, RC189.
(12) Shoghi-J adid, 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-naphthyl-
ethylidene derivatives as positron emission tomography imaging
probes for beta-amyloid plaques in Alzheimer disease. Am. J .
Geriatr. Psychiatry 2002, 10, 24-35.
(13) Mathis, C. A.; Bacskai, B. J .; Kajdasz, S. T. B. M. C.; McLellan,
M. E.; Frosch, M. P.; Hyman, B. T.; Holt, D. P.; Wang, Y.; Huang,
G. F.; Debnath, M. L.; Klunk, W. E. A lipophilic thioflavin-T
derivative for positron emission tomography (PET) imaging of
amyloid in brain. Bioorg. Med. Chem. Lett. 2002, 12, 295-298.
(14) Wang, Y.; Klunk, W. E.; Huang, G.-F.; Debnath, M. L.; Holt, D.
P.; Mathis, C. A. Synthesis and Evaluation of a Radioiodinated
Benzothiazole Derivative as a Radiologand for In Vivo Quanti-
tation of b-Amyloid Deposits in Aging and Alzheimer’s Disease.
J . Labelled Compd. Radiopharm. 2001, 44, S239-S241.
(15) Klunk, W. E.; Wang, Y.; Huang, G.-f.; Debnath, M. L.; Holt, D.
P.; Mathis, C. A. Uncharged thioflavin-T derivatives bind to
amyloid-beta protein with high affinity and readily enter the
brain. Life Sci. 2001, 69, 1471-1484.
(16) Zhuang, Z.-P.; Kung, M.-P.; Hou, C.; Plossel, K.; Skovronsky,
D.; Gur, T. L.; Trojanowski, J . Q.; Lee, V. M.-Y.; Kung, H. F.
IBOX(2-(4′-dimethylaminophenyl)-6-iodobenzoxazole): a ligand
for imaging amyloid plaques in the brain. Nucl. Med. Biol. 2001,
28, 887-894.
(17) Kung, H. F.; Lee, C.-W.; Zhuang, Z. P.; Kung, M. P.; Hou, C.;
Plossl, K. Novel stilbenes as probes for amyloid plaques. J . Am.
Chem. Soc. 2001, 123, 12740-12741.
(30) Hsiao, K.; Chapman, P.; Nilsen, S.; Eckman, C.; Harigaya, Y.;
Younkin, S.; Yang, F.; Cole, G. Correlative memory deficits, Ab
elevation, and amyloid plaques in transgenic mice. Science 1996,
274, 99-102.
J M020351J