Synthesis and evaluation of indolinyl- and indolylphenylacetylenes as PET imaging agents for β-amyloid plaques

Article abstract of DOI:10.1016/j.bmcl.2008.07.077

Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indoline 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting β-amyloid (Aβ) plaques have been prepared. In vitro binding carr

Full text of DOI:10.1016/j.bmcl.2008.07.077

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4823–4827
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of indolinyl- and indolylphenylacetylenes
as PET imaging agents for b-amyloid plaques
Wenchao Qu a, Seok-Rye Choi c, Catherine Hou a, Zhiping Zhuang c, Shunichi Oya a, Wei Zhang c,
Mei-Ping Kung a, Rajesh Manchandra c, Daniel M. Skovronsky c, Hank F. Kung a,b,
*
a Department of Radiology, University of Pennsylvania, 3700 Market Street, Room 305, Philadelphia, PA 19104, USA
b Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA
c Avid Radiopharmaceuticals, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indo-
line 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting b-amy-
loid (Ab) plaques have been prepared. In vitro binding carried out in tissue homogenates prepared
from postmortem AD brains with [125I]IMPY (6-iodo-2-(40-dimethylamino-)phenyl-imidazo[1,2-a]pyri-
dine) as the radioligand indicated good binding affinities (Ki = 4.0 and 1.5 nM for 8 and 14, respectively).
Brain penetration of the corresponding radiofluorinated ligands, evaluated in the normal mice, showed
good initial brain penetration (4.50 and 2.43% ID/g (injected dose/gram) for [18F]8 and [18F]14 at 2 min
after injection) with moderate to low washout rates from the brain (1.71% ID/g at 2 h and 2.10% ID/g
at 3 h, respectively). Autoradiography and homogenate binding studies demonstrated the high specific
binding of [18F]14 to the Ab plaques; however, [18F]8 showed low specific binding. These preliminary
results identified that indolylphenylacetylene, 14, may be a good lead for further structural modification
to develop a useful Ab plaque imaging agent.
Received 23 June 2008
Revised 21 July 2008
Accepted 22 July 2008
Available online 24 July 2008
Keywords:
Alzheimer’s disease
In vivo metabolism
Brain and PET
Ó 2008 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a neurodegenerative disease which
currently affects millions of elderly people worldwide. The key
pathological features of AD are the formation of senile plaques
aggregated by b-amyloid peptide and neurofibrillary tangles piled
from phosphorylated tau protein in the brain. Based upon the amy-
loid cascade hypothesis, formation of Ab plaques in the brain is the
primary influence driving AD pathogenesis.1–3 Imaging techniques
such as positron emission tomography (PET) and single photon
emission tomography (SPECT) are potentially useful for diagnosis
of AD through imaging Ab plaques in the brain. Various radiola-
beled ligands (including the most well-known agent, [11C]-PIB,
N-methyl-[11C]2-(40-methylaminophenyl)-6-hydroxybenzothiazole)
had been tested clinically and demonstrated potential usage
(Fig. 1).4–10
Most of the reported ligands own a common structural feature,
they contain a terminal p-N-methyl- or p-N,N-dimethylaminophe-
nyl-group, and these groups are critical for successful binding affin-
ity.6,8 In many situations, however, it is speculated that the relatively
low metabolic stability of these radioligands was mainly due to a ra-
pid N-demethylation in vivo. Until now, efforts have been made to
overcome this obstacle by adding an extra neighboring substituent,
such as methyl, bromide, chloride, or fluoride group, ortho- to the N-
methylamino-group on the phenyl ring to reduce the in vivo N-
demethylation while maintaining the desired Ab plaque binding
(Fig. 2 (a)).11,12 N-[11C]-labeled aminophenylbenzothiazoles substi-
tuted with fluorine in different positions have been synthesized
and evaluated. It was suggested that the substitution pattern of
the phenyl ring and the benzothiazole moiety has an influence on
slowing down the in vivo metabolism, which in turn has an effect
on the brain uptake kinetics.11 The data suggest that a strategy of
reducing in vivo N-demethylation may improve the brain kinetics
for agents targeting Ab plaques in the brain.
Recently, the pegylation methodology has been adapted in the
design and synthesis of PET Ab plaque imaging agents in order to
adjust the ligands’ pharmacokinetic properties.13,14 We have since
reported a series of fluoro-pegylated diphenylacetylene and iodin-
ated aza-diphenylacetylene derivatives as potential PET and SPECT
Ab plaque imaging agents (Fig. 2 (b)).15,16 Following these success-
ful results, we decided to further extend our search for PET imaging
agents using this diphenylacetylene core structure. By fusing the
N-methylamino group with the phenyl ring, we hope to prevent
the probability of in vivo N-demethylation. In order to do so, ind-
olinyl and indolyl groups were chosen to replace the p-N-methyl-
or p-N,N-dimethylamino phenyl-group. As such by locking the N-
methyl group into a heterocyclic indole or indoline system, we
* Corresponding author. Tel.: +1 215 662 3096; fax: +1 215 349 5035.
E-mail address: kunghf@sunmac.spect.upenn.edu (H.F. Kung).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.077

4824
W. Qu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4823–4827
NC CN
H311C
H
N
H311C
N
N
S
OH
18F
OH
H
N
11C]6-OH-BTA-1 (PIB)
[
11C]SB-13
[
[
18F]FDDNP
H3C
H
18F
3
N
H3C
N
18F
3
O
O
[
18F]FPEGN3-SB
H
AV-138
AV-1 (BAY-94-9172)
Figure 1. Reported five agents for imaging Ab plaques in the brain.
hope to effectively prevent the in vivo N-demethylation (Fig. 2(c)).
We reported herein the synthesis and initial biological evaluation
of two phenylacetylene derivatives as improved probes for imaging
Ab plaques in the brain.
1.5 0.3 nM (three determinations were made for each Ki value),
respectively.
Encouraged by the binding data observed for these two ligands,
we carried out further biological evaluations with the [18F]-labeled
probes. Standard nucleophilic substitution reactions of [18F]fluo-
ride with the corresponding tosylate precursors 6 and 17, in the
presence of Kryptofix 222 (K222) as phase transfer catalyst (PTC),
were successfully performed.18 For the product [18F]8, however,
one extra microwave heating step had been executed for the
N-Boc deprotection (Scheme 3). The subsequent HPLC purified
radioligands, [18F]8 and [18F]14, showed greater than 97% radio-
chemical purities with 16% and 11% overall yields (decay cor-
rected) and ꢀ670 Ci/mmol and ꢀ1900 Ci/mmol specific activities,
respectively. The partition coefficient (PC, commonly measured
as logP) of two radiofluorinated ligands at pH 7.4 was measured
(logP = 2.95 and 2.56).19 The data illustrate that both ligands
own suitability as a brain imaging agent.
The synthetic procedures for these two target molecules are
illustrated in Schemes 1 and 2. Copper and palladium co-catalyzed
Sonogashira coupling reaction plays a vital role in the assembly of
target molecules 8 and 14. Starting from 5-bromoindoline 1, the
tert-butoxycarbonyl (Boc) group protection, microwave heating
promoted Sonogashira coupling, and following basic deprotection
of trimethylsilyl (TMS) group afforded the intermediate 3. The
Sonogashira reaction was employed a second time to couple inter-
mediates 3 and 4. This was followed by tosylation, fluorination,
and finally N-Boc deprotection. Only four steps were needed to
achieve the desired product 8 from 3. For the synthesis of indolyl-
phenylacetylene 14, a similar synthetic route was adopted with
two exceptions: (1) the synthesis of intermediate 11 only requires
very mild reaction condition (room temperature, 0.7 h) and (2) a
relatively harsh condition (microwave heating to 140 °C) was uti-
lized for the final N-Boc deprotection step.
Two radiofluorinated ligands, [18F]8 and [18F]14, displayed good
initial penetrations of the blood–brain barrier with excellent initial
brain uptakes (4.50% and 2.43% ID/g at 2 min after tracer injection)
in normal mice. However, these two radioligands only displayed
moderate to slow washouts with 1.71% ID/g remaining in the brain
at 2 h after the tracer injection and 2.10% ID/g at three hours,
respectively (Fig. 3). These results show promise; but the rate of
The binding affinities of two non-radioactive ligands 8 and 14
were tested by a competitive binding assay (with [125I]IMPY in
postmortem AD brain homogenates).17 Both new ligands displayed
excellent binding affinities; the Ki values are 4.0 0.8 and
R3'
a
R2
R6
R1
N
R'
N
S
N
N
H
R''
N
R8
R1 = OH, F, H
R' = H, Me, CF3CO; R''= H, Me
R2 = H, Br, Cl, F, CH3
R3= H, Me, Br
R
6 = I, Br, Cl, F, OCH3, NO2, S(CH2)2OH, etc
R8=H, I, CN
b
c
I
F
F
HN
O
O
N
O
3
3
N
HN
O
O
F
F
3
3
H2N
F
3
HN
Figure 2. Examples of potential Ab plaque imaging agents: (a) N-methyl group ortho-substituted benzothiazole and imidazo[1,2-a]pyridine derivatives. (b) Fluoro-pegylated
diphenylacetylene and iodinated aza-diphenylacetylene derivatives. (c) Designed ring-fusing targets based on N-methylamino phenylacetylene: indolinyl- and
indolylphenylacetylenes.

W. Qu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4823–4827
4825
I
Br
Br
c
b
a
OH
3
+
N
O
N
H
N
Boc
3
Boc
4
1
2
d
BocN
e
O
BocN
O
O
O
O
O
6
TsO
O
HO
5
f
g
BocN
HN
O
O
O
O
O
F
F
7
8
Scheme 1. Reagents and conditions: (a) (Boc)2O, THF, rt, 24 h; (b) Pd(PPh3)4, CuI, trimethylsilylacetylene, diethylamine, DMF, 130 °C, microwave heating, 0.5 h; (c) KOH,
MeOH/THF, rt, 0.5 h; (d) Pd(PPh3)4, CuI, Et3N, CH3CN, 0 °C to rt, 1.5 h; (e) TsCl, Et3N, DMAP, DCM, 0 °C to rt, 2 h; (f) TBAF, THF, 75 °C, 1 h; (g) TMSOTf, 2,6-lutidine, DCM, 0 °C to
rt, 1.8 h.
I
I
I
a
b, c
OH
3
+
d
N
O
Boc
N
N
H
Boc
11
4
9
10
e
BocN
O
BocN
O
O
O
O
O
12
13
TsO
HO
f, g
HN
O
O
O
14
F
Scheme 2. Reagents and conditions: (a) (Boc)2O, DMAP, DCM, rt, 1.25 h; (b) Pd(PPh3)4, CuI, trimethylsilylacetylene, diethylamine, DMF, rt, 0.7 h; (c) KOH, MeOH/THF, rt,
2.5 h; (d) Pd(PPh3)4, CuI, Et3N, CH3CN, 0 °C to rt, 1.0 h; (e) TsCl, Et3N, DMAP, DCM, 0 °C to rt, 3 h; (f) TBAF, THF, 75 °C, 1.5 h; (g) microwave heating, 140 °C, 0.5 h.
a, b
BocN
HN
O
O
O
O
O
O
6
TsO
I
*F
[
18F]8
I
c, d
+
OTs
3
N
H
O
N
15
H
16
8
e
f
HN
HN
O
O
O
O
O
O
17
*F
TsO
[
18F]14
Scheme 3. Reagents and conditions: (a) [18F]KF, K222, K2CO3, DMSO, 120 °C, 4.0 min; (b) microwave heating, 150 °C, 2.5 min; (c) Pd(PPh3)4, CuI, trimethylsilylacetylene,
diethylamine, DMF, rt, 0.7 h; (d) KOH, MeOH/THF, rt, 2.5 h; (e) Pd(PPh3)4, CuI, Et3N, CH3CN, 0 °C to rt, 1.0 h; (f) [18F]KF, K222, K2CO3, DMSO, 120 °C, 4.0 min.
brain washout is not optimal for an Ab plaque-targeting imaging
agent since a fast washout rate may help generate the better sig-
nal-to-noise ratios and therefore may be better for Ab plaque
detection.20

4826
W. Qu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4823–4827
5
4.5
4
25
TB
NSB
[18F]-8
20
15
10
5
[18F]-14
3.5
3
2.5
2
1.5
1
0
0.5
0
AD/Grey
Con/Grey
Con/White
0
50
100
150
200
25
20
15
10
5
Time (min)
TB
NSB
Figure 3. Brain uptake and washout of [18F]8 and [18F]14 in normal mice. Data are
presented as % ID/g of three mice standard deviation.
0
AD/Grey
Con/Grey
Con/White
Figure 5. Specific binding of (a) [18F]8 and (b) [18F]14 to AD and control (Con) brain
Figure 4. In vitro autoradiography of frozen human brain sections of AD patients
with (a) [18F]8 and (b) [18F]14. [18F]14 showed excellent binding to the Ab plaques
with very low background labeling.
tissue homogenate. Grey and white matter was dissected from the cortical regions.
[
18F]14 showed high specific binding mainly in the gray matter of AD brain. (NSB,
non-specific binding; TB, total binding; two determinations were made.)
vitro autoradiography of postmortem AD brain sections and brain
tissue homogenate binding assay depicted that radioligand
[
the results suggest that the indolylphenylacetylene ligand, 14, may
be a lead for further structural modification in order to improve the
in vivo stability and in vivo kinetics desirable for a useful Ab plaque
imaging agent.
To confirm the specific binding of radiofluorinated ligands 8 and
14 to Ab plaques, we performed the in vitro film autoradiography.
As shown in Figure 4, [18F]14 distinctively labeled plaques on AD
brain sections with low background labeling. On the contrary, in
addition to plaque labeling, [18F]8 displayed significant white mat-
ter labeling.
18F]14 showed specific Ab plaque labeling signal. Taken together,
Furthermore, the in vitro binding assay using homogenates pre-
pared from AD and control brain tissues was conducted to evaluate
the binding specificity of these two radioligands to Ab plaques (Fig.
Acknowledgments
5).21 18F]14 showed a very high specific binding in homogenate
[
This work was supported by grants from the National Institutes
of Health (AG-022559 to H.F.K.) and Avid Radiopharmaceuticals.
The authors thank Pathology Core Laboratories at The Children’s
Hospital of Philadelphia for assembling the human macro-array
brain sections.
prepared from gray matter of an AD patient with low non-specific
binding. In contrast, [18F]8 displayed similar binding in homoge-
nates prepared from an AD patient and control with high non-spe-
cific binding. These results are consistent with in vitro binding
results derived from autoradiography of AD brain sections. Preli-
minary in vivo and in vitro metabolism data of [18F]14 showed a
complex pattern of metabolism in plasma and in liver (data not
shown). It is likely that the indole or indoline ring may have re-
moved the chances of N-demethylation; however, other metabolic
reaction(s) at different sites of these two molecules may have oc-
curred. Further exploration of the structure-activity relationship
of this series of agents will be needed in the future. Nonetheless,
the novel core structures showing excellent binding affinities to
Ab aggregates in human brain tissue provide potential insight for
developing useful imaging agents.
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