5746
M. Ono et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5743–5748
Table 1
HPLC retention times of 99mTc/Re compounds and log P of 99mTc compounds
Re compounds
Retention timea (min)
99mTc compounds
Retention timea (min)
Log P of 99mTc compoundsb
9
19
9.5
14.6
10
20
11.1
16.6
2.77 0.04
2.23 0.04
a
Reversed-phase HPLC using a mixture of H2O–acetonitrile (2:3) as a mobile phase.
The measurement was done in triplicate and repeated three times. Each value represents the mean SD for three independent experiments.
b
as described for the synthesis of the flavone derivative 8. After
deprotection of the thiol groups in 8 and 18 in TFA and triethylsi-
lane, the Re complexes (9 and 19) were prepared through a reac-
tion with (PPh3)2ReOCl3. The corresponding 99mTc complexes, 10
([99mTc]BAT-FL) and 20 ([99mTc]BAT-AR), were prepared by a ligand
exchange reaction employing the precursor 99mTc-glucoheptonate
(GH). The resulting mixture was analyzed by reversed-phase HPLC,
showing that a single radioactive complex formed with radiochem-
ical purity higher than 95% after purification by HPLC. The identity
of the complex was established by comparative HPLC using the
corresponding Re complexes as a reference (Table 1). The retention
times for [99mTc]BAT-FL and [99mTc]BAT-AR on HPLC (radioactivity)
were 11.1 and 16.6 min, respectively. The retention times of the
corresponding Re complexes on HPLC (UV detection) were 9.5
and 14.6 min, respectively.
aggregates in binding assays in vitro. The labeling pattern was con-
sistent with that observed with thioflavin S (Fig.3C and D). These
results suggest that [99mTc]BAT-FL and [99mTc]BAT-AR would bind
to b-amyloid plaques in the mouse brain in addition to having
affinity for synthetic Ab(1–42) aggregates. Although [99mTc]BAT-
AR showed greater affinity than [99mTc]BAT-AR in the binding as-
says in vitro, no marked difference in binding between
[
99mTc]BAT-FL and [99mTc]BAT-AR was observed in the fluorescent
staining experiments.
99mTc]BAT-FL and [99mTc]BAT-AR were examined as to their
[
biodistribution in normal mice (Table 2). A biodistribution study
provides important information on brain uptake. The ideal probe
for imaging b-amyloid should penetrate the blood–brain barrier
well enough to deliver a sufficient dose into the brain while clear-
ing rapidly from normal regions so as to achieve a high signal to
noise ratio in the AD brain. Previous studies suggest that the opti-
mal lipophilicity for entry into the brain is obtained with log P val-
ues of between 1 and 3. [99mTc]BAT-FL and [99mTc]BAT-AR had
log P values of 2.77 and 2.23, respectively (Table 1), but showed
less uptake, 0.64 and 0.79%ID/g at 2 min postinjection, than ex-
In vitro binding experiments to evaluate the affinity of
[
99mTc]BAT-FL and [99mTc]BAT-AR for Ab(1–42) aggregates were
carried out in solutions. The percent radioactivity of
[
99mTc]BAT-FL and [99mTc]BAT-AR bound to aggregates increased
dependent on the dose of Ab(1–42), while [99mTc]BAT showed no
marked affinity for the aggregates (Fig. 2). At all concentrations
of Ab aggregates, [99mTc]BAT-AR showed significantly greater
affinity than [99mTc]BAT-FL. In these binding experiments, the
non-specific binding of [99mTc]BAT-FL and [99mTc]BAT-AR was
pected. Thereafter, the radioactivity of
[
[
99mTc]BAT-FL and
99mTc]BAT-AR that accumulated in the brain was rapidly elimi-
nated (0.23 and 0.11%ID/g at 60 min postinjection). Recently, we
have reported that the 99mTc-labeled chalcone derivative showed
high uptake (1.48%ID/g at 2 min postinjection) into and rapid
clearance (0.17%ID/g at 60 min postinjection) from the brain, a
highly desirable property for imaging agents for b-amyloid pla-
ques.29 The pharmacokinetics of the 99mTc-labeled chalcone deriv-
ative in the brain appears superior to that of any 99mTc-labeled
probes reported previously, indicating that this compound should
be investigated further as a potentially useful probe for imaging
b-amyloid. Compared with that of the 99mTc-labeled chalcone,29
the radioactivity of [99mTc]BAT-FL and [99mTc]BAT-AR appears
insufficient for the imaging of b-amyloid plaques in the brain. Since
the affinity of [99mTc]BAT-FL and [99mTc]BAT-AR for Ab aggregates
was as high as that of 99mTc-labeled chalcone derivatives,29
improvement of the uptake of [99mTc]BAT-FL and [99mTc]BAT-AR
is an important prerequisite to developing more useful 99mTc-la-
beled probes. Therefore, additional structural changes in the fla-
vone and aurone scaffold are needed to further improve the
pharmacokinetics of [99mTc]BAT-FL and [99mTc]BAT-AR in vivo.
In conclusion, we successfully designed and synthesized novel
99mTc/Re complexes based on flavone and aurone for the detection
estimated at 1.62–1.85%. The affinity of
[
99mTc]BAT-FL and
[
99mTc]BAT-AR was less than that of 99mTc-labeled chalcone
derivatives reported previously (Fig. 1).29 The order in terms of
strength of binding corresponded with that of radioiodinated
flavonoids,23–25 indicating that the scaffolds of the [99mTc]BAT
complexes did not play an important role in the affinity for Ab
aggregates.
To confirm the affinity for b-amyloid plaques in the mouse
brain, neuropathological fluorescent staining with Re derivatives
(9 and 19) was carried out using Tg2576 mouse brain sections
(Fig. 3). Many b-amyloid plaques were clearly stained with the
derivatives (Fig. 3A and B), as reflected by the high affinity for Ab
of b-amyloid plaques in the brain. Both
[
[
99mTc]BAT-FL and
99mTc]BAT-AR showed high affinity for synthetic Ab(1–42) aggre-
gates. In experiments in vitro using sections of brain from Tg2576
mice, Re complexes intensely stained b-amyloid plaques. In addi-
tion, [99mTc]BAT-FL and [99mTc]BAT-AR displayed good uptake into
and a rapid washout from the brain after their injection in normal
mice. This combination of affinity for b-amyloid plaques, and good
uptake and clearance makes [99mTc]BAT-FL and [99mTc]BAT-AR
promising probes for the detection of b-amyloid plaques in the
brain, although additional modifications are required to enhance
their uptake. The results of the present study should provide infor-
mation useful for the development of 99mTc-labeled probes for the
imaging of b-amyloid plaques in the brain.
Figure 2. Binding assay of [99mTc]BAT-FL, [99mTc]BAT-AR, and [99mTc]BAT with
Ab(1–42) aggregates. Values are the mean standard error of the mean for three
independent experiments.