Structure-activity relationships of radioiodinated diphenyl derivatives with different conjugated double bonds as ligands for α-synuclein aggregates

Article abstract of DOI:10.1039/c6ra02710e

It is generally recognized that aggregates of α-synuclein (α-syn) in the brain are closely associated with the pathogenesis of Parkinson's disease (PD). Therefore, the development of in vivo imaging probes targeting α-syn aggregates is currently expected.

Full text of DOI:10.1039/c6ra02710e

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Structure–activity relationships of radioiodinated
diphenyl derivatives with different conjugated
Cite this: RSC Adv., 2016, 6, 44305
double bonds as ligands for a-synuclein
aggregates†
a
Masahiro Ono, Yuki Doi,a Hiroyuki Watanabe,a Masafumi Ihara,b Akihiko Ozakic
*
and Hideo Sajia
It is generally recognized that aggregates of a-synuclein (a-syn) in the brain are closely associated with the
pathogenesis of Parkinson's disease (PD). Therefore, the development of in vivo imaging probes targeting a-
syn aggregates is currently expected. In this study, we investigated the structure–activity relationships of
radioiodinated diphenyl (IDP) derivatives with different conjugated double bonds as ligands for a-syn
aggregates. An in vitro binding assay revealed that the binding affinity of the derivatives to a-syn
aggregates increased as the length of the conjugated double bonds in their molecule extended. In
contrast, brain uptake of the derivatives in biodistribution studies decreased in accordance with the
extension of the double bonds. These findings from structure-relationship studies suggested that the
length of the conjugated double bonds in the IDP derivatives plays an important role in the binding
affinity for a-syn aggregates and uptake in the brain. Also, the present study may provide valuable
information on molecular design for the development of new imaging probes targeting a-syn aggregates
in the future.
Received 29th January 2016
Accepted 25th April 2016
DOI: 10.1039/c6ra02710e
www.rsc.org/advances
of the aggregates of these misfolded proteins may offer valuable
information for the early diagnosis and monitoring of disease
progression.5 A number of PET imaging probes targeting Ab
plaques are developed, and three probes have been approved by
the FDA at this time.6–9 Promising PET imaging probes targeting
tau aggregates, including THK-5351, T807, and PBB-3, have
been used in clinical trials.10–14 However, there has been no
report on the development of useful probes targeting a-syn
aggregates that can be utilized clinically.
In 2009, Fodero-Tavoletti et al. reported on the in vitro
characterization of the binding of BF-227, which was developed
as a PET imaging probe for Ab plaques, to a-syn aggregates.15 In
vitro binding studies in this report revealed that [18F]BF-227
Introduction
As the population ages worldwide, the number of patients with
neurodegenerative disorders including Alzheimer's disease
(AD) and Parkinson's disease (PD) has increased rapidly
throughout the world. It is currently accepted that the accu-
mulation of aggregates of misfolded proteins in the brain is
related to the onset of these disorders.1 In the brain of AD
patients, senile plaques composed of b-amyloid (Ab) proteins
and neurobrillary tangles composed of hyperphosphorylated
tau proteins are observed.2 In contrast, another protein
observed in PD patients' brains as the aggregated form is a-
synuclein (a-syn), which is a major component of Lewy bodies
(LBs) and Lewy neurites (LNs).3,4 Since these proteins found in
AD and PD patients' brains play an important role in the
pathogenesis of the diseases, in vivo detection of accumulation
shows high affinity for two binding sites on Ab brils (KD1
¼
1.31 and KD2 ¼ 80 nM, respectively) and to one class binding site
on a-syn brils (KD ¼ 9.63 nM), indicating that [18F]BF-227 is
not a selective imaging probe for a-syn aggregates. Thereaer,
Yu et al. reported the synthesis and evaluation of a new series of
phenothiazine derivatives as a-syn ligands.16 The in vitro
binding assay of that study indicated that phenothiazine 11b
showed high affinity for a-syn brils (Ki ¼ 32 nM) and pheno-
thiazine 11d, 16a, and 16b displayed moderate affinity for a-syn
brils ranging from Ki values of 50 to 100 nM. From the data,
they concluded that further optimization of the chemical
structure of the phenothiazine derivatives may lead to the
development of compounds with high affinity and selectivity for
aDepartment of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical
Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto
606-8501, Japan. E-mail: ono@pharm.kyoto-u.ac.jp; Fax: +81-75-753-4568; Tel: +81-
75-753-4608
bDepartment of Stroke and Cerebrovascular Diseases, National Cerebral and
Cardiovascular Center, 5-7-1 Fujishirodai, Suita-shi, Osaka 565-8565, Japan
cDepartment of Neurology, Osaka Saiseikai Nakatsu Hospital, 2-10-39 Shibata, Kita-
ku, Osaka 530-0012, Japan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra02710e
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a-syn aggregates. A recent paper from the same group reported
a more detailed biological characterization of [125I]SIL23
(named 16b in ref. 16).17 They stated that the affinity of SIL23 for
a-syn aggregates and its selectivity for a-syn against Ab and tau
aggregates is not optimal for the in vivo detection of a-syn
brils, although competitive binding assays using SIL23 can be
applied to screen for more promising ligands. More recently,
Chu et al. reported the design, synthesis, and characterization
of a series of 3-(benzylidene)indolin-2-one derivatives as ligands
for a-syn brils.18 Among the derivatives, the uorine
compound 46a showed the most potent and selective affinity for
a-syn brils (Ki ¼ 2 nM, 70- and 40-fold selectivity versus Ab and
tau aggregates, respectively). However, they stated that [18F]46a
would not be useful for in vivo imaging of a-syn brils in PD
patients' brains due to its high lipophilicity and potential of the
nitro group to be reduced to the primary amino group in vivo.
In this current situation regarding the development of
imaging probes targeting a-syn aggregates, we planned to
search for new scaffolds that can function as a-syn imaging
probes. Our previous study suggested that several avonoids
including avone,19 chalcone,20 and aurone,21 which can inhibit
the formation of Ab aggregates, may be useful candidates as Ab
imaging probes. In addition, it is well-known that these avo-
noids can also inhibit the formation of a-syn aggregates,22–24
indicating that the avonoids can bind to or interact with a-syn
aggregates. From these previous ndings, we rstly decided to
apply the chalcone derivatives with a simple chemical structure
as a-syn imaging probes. We also suggested that probes with
a longer conjugated p system showed higher affinity for Ab
aggregates in our previous paper.25 Another recent paper
regarding the development of tau imaging probes revealed that
the length of molecules may play an important role in the
affinity for tau aggregates, indicating that the molecular length
should also be an important consideration in the molecular
design of a-syn imaging probes.26
Fig. 1 Chemical structures of 125I-labeled IDP derivatives evaluated in
this study.
system (LC-20AD pump with an SPD-20A UV detector, l ¼ 254
nm) using a Cosmosil C18 column (Nacalai Tesque, 5C18-MS-II,
4.6 ꢀ 150 mm) and CH3CN/H2O as the mobile phase at a ow
rate of 1.0 mL minꢁ1. All key compounds were proven by this
method to show 95% purity.
Chemistry
In the present study, we designed and synthesized four 125I-
labeled IDP derivatives with different conjugated double bonds Scheme 1 shows the synthetic route of IDP derivatives.
including radioiodinated 1,3-diphenyl-2-propen-1-one (IDP-1, Compounds 2, 3, and 4 (IDP-1) were synthesized from 1 as
a chalcone derivative),20 1,5-dipenyl-2,4-pentadien-1-one (IDP- a starting material according to our method reported previ-
2), 1,7-diphenyl-2,4,6-heptatrien-1-one (IDP-3), and 1,9- ously.20 Compounds
9 and 13 were synthesized from
diphenyl-2,4,6,8-nonatetraen-1-one (IDP-4) (Fig. 1), and inves- a commercially available compound 5 according to a previously
tigated their structure–activity relationships as ligands for the in reported method.27
vivo imaging of a-syn aggregates.
(2E,4E)-1-(4-Bromophenyl)-5-(4-(dimethylamino)phenyl)penta-
2,4-dien-1-one (6). To a solution of (E)-3-(4-(dimethylamino)
phenyl)acrylaldehyde (5) (88 mg, 0.502 mmol) in a mixed
solvent (5 mL, 4 : 1 ethanol/DMF mixture) was added 4-bro-
Materials and methods
All reagents were commercial products and used without moacetophenone (100 mg, 0.502 mmol). The mixture was stir-
further purication unless indicated otherwise. Smart Flash red for 10 min in an ice bath. Then, 2.0 mL of 10% KOH aq. was
EPCLC W-Prep 2XY (Yamazen Corporation) was used for silica slowly added dropwisely to the reaction mixture. The reaction
gel chromatography. 1H and 13C NMR spectra were recorded solution was stirred at room temperature for 6 h. A precipitate
using a JEOL JNM-ECS400 or JEOL ECA-500 spectrometer, and was collected and washed with ethanol to give 110 mg of 6
1
chemical shis were recorded in d (ppm) relative to TMS as an (61.8%). H NMR (500 MHz, CDCl3) d 7.83 (d, J ¼ 8.3 Hz, 2H),
internal standard. Coupling constants are reported in Hertz. 7.66–7.60 (m, 3H), 7.40 (d, J ¼ 8.9 Hz, 2H), 6.98 (d, J ¼ 15.2 Hz,
Multiplicity was dened as singlet (s), doublet (d), triplet (t), or 1H), 6.93 (d, J ¼ 14.6 Hz, 1H), 6.85 (dd, J ¼ 15.5, 11.2 Hz, 1H),
multiplet (m). Mass spectra were obtained on a SHIMADZU 6.68 (d, J ¼ 8.9 Hz, 2H), 3.02 (s, 6H). 13C NMR (100 MHz, CDCl3)
LCMS-2010 EV. High-resolution mass spectra were obtained on d 189.3, 151.2, 147.0, 143.7, 137.4, 131.7, 129.8, 129.0, 127.2,
a JEOL JMS-SX 102A QQ. HPLC was performed with a Shimadzu 124.0, 122.2, 121.9, 112.0, 40.2. MS (ESI) m/z 356 [MH]+.
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Scheme 1 Synthesis of IDP derivatives. Reagents: (i) 4-bromoacetophenone, 10% KOH, EtOH, DMF, rt; (ii) (Bu3Sn)2, (Ph3P)4Pd, dioxane, Et3N, 95
ꢂC; (iii) 4-iodoacetophenone, 10% KOH, EtOH, DMF, rt; (iv) (1) (1,3-dioxolan-2-yl)methyl-triphenylphosphonium bromide, 18-crown-6, NaH,
THF, (2) 20% HCl, THF.
chromatography (AcOEt/hexane ¼ 1/4) to give 221 mg of 7
(2E,4E)-5-(4-(Dimethylamino)phenyl)-1-(4-(tributylstannyl)
1
(46.4%). H NMR (500 MHz, CDCl3) d 7.88 (d, J ¼ 7.4 Hz, 2H),
phenyl)penta-2,4-dien-1-one (7). A mixture of 6 (300 mg, 0.84
7.64–7.58 (m, 3H), 7.40 (d, J ¼ 8.9 Hz, 2H), 6.97 (m, 2H), 6.85
mmol), (BuSn3)2 (1.46 g, 2.52 mmol), and (Ph3P)4Pd (194 mg,
0.168 mmol) in a mixed solvent (42 mL, 2 : 1 dioxane/Et3N
mixture) was stirred under reux for 2 h. The solvent was
removed, and the residue was puried by silica gel
(dd, J ¼ 15.2, 10.9 Hz, 1H), 6.68 (d, J ¼ 8.9 Hz, 2H), 3.02 (s, 6H),
1.58–1.51 (m, 6H), 1.37–1.29 (m, 6H), 1.10–1.07 (m, 6H), 0.89 (t, J
¼ 7.3 Hz, 9H). 13C NMR (100 MHz, CDCl3) d 190.7, 151.0, 148.8,
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146.1, 142.9, 138.1, 136.4, 128.8, 127.1, 124.1, 122.6, 122.4, 1H), 6.49–6.36 (m, 2H), 3.00 (s, 6H). 13C NMR (100 MHz, CDCl3)
111.9, 40.1, 29.0, 27.3, 13.6, 9.6. MS (ESI) m/z 568 [MH]+.
d 189.4, 152.2, 146.4, 137.8, 131.7, 130.6, 129.9, 129.8, 129.0,
(2E,4E)-5-(4-(Dimethylamino)phenyl)-1-(4-iodophenyl)penta- 128.4, 128.1, 127.1, 122.4, 116.1, 112.2, 112.0, 111.8, 40.1. MS
2,4-dien-1-one (8, IDP-2). To a solution of (E)-3-(4-(dimethyla- (ESI) m/z 408 [MH]+.
mino)phenyl)acrylaldehyde (5) (492 mg, 2.00 mmol) in a mixed
(2E,4E,6E,8E)-9-(4-(Dimethylamino)phenyl)-1-(4-(tributylstannyl)
solvent (20 mL, 4 : 1 ethanol/DMF mixture) was added 4- phenyl)nona-2,4,6,8-tetraen-1-one (15). The same reaction as
iodoacetophenone (350 mg, 2.00 mmol). The mixture was stir- described above to prepare 7 was used, and 11 mg of 15 was
1
red for 10 min in an ice bath. Then, 8 mL of 10% KOH aq. was obtained in a 9.0% yield from 14. H NMR (400 MHz, CDCl3)
slowly added dropwisely to the reaction mixture. The reaction d 7.86 (d, J ¼ 8.1 Hz, 2H), 7.58 (d, J ¼ 7.8 Hz, 2H), 7.52 (dd, J ¼
solution was stirred at room temperature for 6 h. A precipitate 14.6, 11.6 Hz, 1H), 7.33 (d, J ¼ 9.0 Hz, 2H), 6.95 (d, J ¼ 15.1 Hz,
was collected and washed with ethanol to give 100 mg of 8 1H), 6.75 (dd, J ¼ 17.8, 9.9 Hz, 1H), 6.71–6.60 (m, 5H), 6.49–
1
(12.4%). H NMR (500 MHz, CDCl3) d 7.83 (d, J ¼ 8.0 Hz, 2H), 6.40 (m, 2H), 2.99 (s, 6H), 1.58–1.51 (m, 6H), 1.38–1.28 (m, 6H),
7.68 (d, J ¼ 8.0 Hz, 2H), 7.63 (dd, J ¼ 14.6, 11.2 Hz, 1H), 7.40 (d, J 1.11–1.07 (m, 6H), 0.89 (t, J ¼ 7.4 Hz, 9H). 13C NMR (100 MHz,
¼ 8.9 Hz, 2H), 6.99–6.81 (m, 3H), 6.68 (d, J ¼ 8.9 Hz, 2H), 3.02 (s, CDCl3) d 190.7, 150.4, 149.3, 145.0, 143.0, 139.1, 138.0, 136.6,
6H). 13C NMR (100 MHz, CDCl3) d 189.6, 151.2, 147.0, 143.7, 136.1, 130.0, 129.5, 128.0, 127.1, 125.2, 124.4, 112.2, 40.3, 29.1,
138.0, 137.7, 129.7, 129.0, 124.0, 122.2, 121.8, 112.0, 99.9, 40.2. 27.4, 13.7, 9.7. MS (ESI) m/z 620 [MH]+.
HRMS (EI) m/z calcd for C19H18INO (M+) 403.0433, found
403.0435.
(2E,4E,6E,8E)-9-(4-(Dimethylamino)phenyl)-1-(4-iodophenyl)
nona-2,4,6,8-tetraen-1-one (16, IDP-4). The same reaction as
(2E,4E,6E)-1-(4-Bromophenyl)-7-(4-(dimethylamino)phenyl) described above to prepare 8 was used, and 27 mg of 16 was
hepta-2,4,6-trien-1-one (10). The same reaction as described obtained in a 66.5% yield from 13. 1H NMR (400 MHz,
above to prepare 6 was used, and 44 mg of 10 was obtained in CDCl3) d 7.83 (d, J ¼ 8.1 Hz, 2H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.52
1
a 45.5% yield from 9. H NMR (500 MHz, CDCl3) d 7.82 (d, J ¼ (dd, J ¼ 14.9, 11.3 Hz, 1H), 7.33 (d, J ¼ 8.7 Hz, 2H), 6.88 (d, J ¼
8.6 Hz, 2H), 7.61 (d, J ¼ 8.6 Hz, 2H), 7.55 (dd, J ¼ 14.7, 11.7 Hz, 14.8 Hz, 1H), 6.79 (dd, J ¼ 14.6, 11.3 Hz, 1H), 6.68–6.60 (m,
1H), 7.35 (d, J ¼ 8.9 Hz, 2H), 6.90–6.84 (m, 2H), 6.75–6.74 (m, 5H), 6.48–6.36 (m, 2H), 3.00 (s, 6H). 13C NMR (100 MHz,
2H), 6.67 (d, J ¼ 8.9 Hz, 2H), 6.49 (dd, J ¼ 14.3, 11.5 Hz, 1H), 3.01 CDCl3) d 189.5, 150.7, 146.1, 144.6, 138.7, 137.7, 129.8, 128.4,
(s, 6H). 13C NMR (100 MHz, CDCl3) d 189.2, 150.7, 146.1, 144.5, 128.3, 128.1, 124.6, 123.7, 122.6, 112.2, 112.1, 100.0, 40.3.
138.6, 137.3, 131.7, 129.8, 128.4, 128.3, 127.3, 124.6, 123.7, HRMS (EI) m/z calcd for C23H22INO (M+) 455.0743, found
122.6, 112.1, 40.2. MS (ESI) m/z 382 [MH]+.
455.0746.
(2E,4E,6E)-7-(4-(Dimethylamino)phenyl)-1-(4-(tributylstannyl)
phenyl)hepta-2,4,6-trien-1-one (11). The same reaction as Radiolabeling
described above to prepare 7 was used, and 20 mg of 11 was
The radioiodinated forms of compounds [125I]4 ([125I]IDP-1),
[
1
obtained in a 26.0% yield from 10. H NMR (400 MHz, CDCl3)
125I]8 ([125I]IDP-2), [125I]12 ([125I]IDP-3), and [125I]16 ([125I]IDP-
d 7.87 (d, J ¼ 7.6 Hz, 2H), 7.59–7.51 (m, 3H), 7.35 (d, J ¼ 9.0 Hz,
2H), 6.95 (d, J ¼ 14.5 Hz, 1H), 6.84 (dd, J ¼ 15.3, 8.4 Hz, 1H), 6.75–
6.66 (m, 4H), 6.50 (dd, J ¼ 14.1, 11.9 Hz, 1H), 3.00 (s, 6H), 1.58–
1.48 (m, 6H), 1.36–1.30 (m, 6H), 1.11–1.07 (m, 6H), 0.89 (t, J ¼ 7.3
Hz, 9H). 13C NMR (100 MHz, CDCl3) d 190.8, 150.6, 149.1, 145.3,
143.8, 138.1, 138.0, 136.6, 128.6, 128.3, 127.1, 124.8, 123.9, 123.5,
112.1, 40.3, 29.1, 27.4, 13.7, 9.7. MS (ESI) m/z 594 [MH]+.
4) were prepared from the corresponding tributyltin deriva-
tives by iododestannylation (Scheme 2). Briey, to initiate the
reaction, 50 mL of H2O2 (3%) was added to a mixture of a trib-
utyltin derivative (100 mg/100 mL EtOH), [125I]NaI (3.7–14.8 MBq,
specic activity 81.4 TBq mmolꢁ1), and 50 mL of 1 N HCl in
a sealed vial. The reaction was allowed to proceed at room
temperature for 5 min and terminated by the addition of satu-
rated NaHSO3 aq. (100 mL). Aer neutralization with sodium
hydrogen carbonate and extraction with AcOEt, the extract was
dried by passing through an anhydrous Na2SO4 column. The
solution was blown dry with a stream of nitrogen gas. The
radioiodinated ligand was puried by HPLC on a Cosmosil C18
column with an isocratic solvent of CH3CN/H2O ¼ 7/3 (for IDP-1
and IDP-2) or 8/2 (for IDP-3 and IDP-4) at a ow rate of 1.0 mL
(2E,4E,6E)-7-(4-(Dimethylamino)phenyl)-1-(4-iodophenyl)
hepta-2,4,6-trien-1-one (12, IDP-3). The same reaction as
described above to prepare 8 was used, and 29 mg of 12 was
1
obtained in a 45.6% yield from 9. H NMR (500 MHz, CDCl3)
d 7.83 (d, J ¼ 8.7 Hz, 2H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.55 (dd, J ¼
14.7, 11.7 Hz, 1H), 7.36 (d, J ¼ 9.0 Hz, 2H), 6.90–6.83 (m, 2H),
6.75–6.74 (m, 2H), 6.68 (d, J ¼ 9.0 Hz, 2H), 6.49 (dd, J ¼ 14.6,
11.5 Hz, 1H), 3.01 (s, 6H). 13C NMR (100 MHz, CDCl3) d 189.5,
150.7, 146.1, 144.5, 138.6, 137.9, 129.7, 128.4, 128.3, 124.6,
123.7, 122.5, 112.1, 40.2. HRMS (EI) m/z calcd for C21H20INO
(M+) 429.0595, found 429.0590.
minꢁ1
.
(2E,4E,6E,8E)-1-(4-Bromophenyl)-9-(4-(dimethylamino)phenyl)
nona-2,4,6,8-tetraen-1-one (14). The same reaction as described
above to prepare 6 was used, and 81 mg of 14 was obtained in
a 45.3% yield from 13. 1H NMR (400 MHz, CDCl3) d 7.82 (d, J ¼
8.4 Hz, 2H), 7.61 (d, J ¼ 8.7 Hz, 2H), 7.53 (dd, J ¼ 14.8, 11.3 Hz,
1H), 7.33 (d, J ¼ 9.0 Hz, 2H), 6.89 (d, J ¼ 14.8 Hz, 1H), 6.79 (dd, J
¼ 14.4, 11.6 Hz, 1H), 6.73–6.65 (m, 4H), 6.62 (dd, J ¼ 9.3, 7.8 Hz, Scheme 2 125I labeling of IDP derivatives.
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University. A saline solution (100 mL, 20 kBq) of each [125I]IDP
derivative containing EtOH (10 mL) was injected directly into the
tail vein of ddY mice (5 weeks old, male). The mice were sacri-
ced at 2, 10, 30, and 60 min postinjection. The organs of
interest were removed and weighed, and the radioactivity was
measured with a g counter (Wizard 1470, PerkinElmer).
Binding saturation assay using recombinant a-syn and Ab1–42
aggregates
a-syn and Ab were purchased from rPeptide (Bogart, GA, USA)
and the Peptides Institute, Inc. (Osaka, Japan), respectively. a-
syn aggregates were prepared by incubating recombinant a-syn
monomer (1.67 mg mLꢁ1 in 20 mM Tris–HCl, 100 mM NaCl, pH
7.48) at 37 ꢂC for 144 h with shaking at 1000 rpm. Ab aggregates
were prepared by incubating the Ab1–42 peptide (0.25 mg mLꢁ1
in PBS, pH 7.4) at 37 ꢂC for 42 h with gentle and constant
shaking. The reaction mixture contained increasing concen-
trations of [125I]IDP-1–4 (0.39–200 mM, 100 mL, EtOH), a xed
concentration of a-syn aggregates (57 nM, 50 mL, PBS) or Ab
aggregates (55 nM, 50 mL, PBS), and 10% EtOH (850 mL, con-
taining 0.1 w/v% BSA). Nonspecic binding was dened in the
presence of 1 mM nonradioactive IDP-1–4. Aer incubating for 3
h at room temperature, the solution was ltered through GF/B
lters using an M-24 cell harvester. The radioactivity of the
bound [125I]IDP-1–4 was measured with a g counter (Wizard
1470, PerkinElmer). The dissociation constants (Kd) and Bmax
were determined by Scatchard analysis using GraphPad Prism
5.0 (GraphPad Soware, San Diego, CA, USA).
Results and discussion
Chemistry
As shown in Scheme 1, bromo (2, 6, 10, and 14) and iodo (4, 8,
12, and 16) compounds were synthesized by the conventional
aldol condensation reaction between the corresponding alde-
hydes (1, 5, 9, and 13) and 4-bromoacetophene or 4-iodoaceto-
phene. The tributyltin derivatives (3, 7, 11, and 15) were
prepared from the corresponding bromo compounds using the
exchange reaction of bromine to tributyltin catalyzed by Pd(0).
These tributyltin compounds were also used as the starting
materials for radioiodination in the preparation of the corre-
sponding 125I-labeled derivatives. Four 125I-labeled diphenyl
derivatives were obtained by an iododestannylation reaction
using [125I]NaI and hydrogen peroxide as the oxidant under
acidic conditions (Scheme 2). The no-carrier-added derivatives
are expected to be produced as a nal product with a theoretical
specic activity of 81.4 TBq mmolꢁ1, similar to that of 125I. The
radiochemical identity of the radioiodinated ligands was
conrmed by co-injection with non-radioiodinated compounds
from their HPLC proles. Four 125I-labeled diphenyl derivatives
were obtained in a radiochemical yield of 60.0, 54.2, 51.8, and
19.0% for [125I]IDP-1, [125I]IDP-2, [125I]IDP-3, and [125I]IDP-4,
respectively. Aer purication by HPLC, these probes with
a radiochemical purity of >95% were successfully prepared.
Fluorescent staining of PD brain sections
Experiments involving human subjects were performed in
accordance with relevant guidelines and regulations and were
approved by the ethics committee of Osaka Saiseikai Nakatsu
Hospital. Informed consent was obtained from all subjects in
this study. Postmortem brain tissues from autopsy-conrmed
cases of PD (female, 69 years old) were obtained from Osaka
Saiseikai Nakatsu Hospital. Eight-micrometer-thick serial
sections of paraffin-embedded blocks were used for staining.
The sections were subjected to two 15 min incubations in
xylene, two 1 min incubations in 100% EtOH, one 1 min incu-
bation in 90% EtOH, and one 1 min incubation in 70% EtOH to
completely deparaffinize them, followed by two 2.5 min washes
in water. The sections were incubated with 12 (IDP-3) or 16 (IDP-
4) (20 mM, 50% EtOH) for 10 min. Aer two 1 min incubations in
50% EtOH, the sections were observed with a uorescence
microscope (FSX100, Olympus Corp., Tokyo, Japan) equipped
with a U-MWIG3 lter set.
Binding saturation assay using recombinant a-syn and Ab1–42
aggregates
It is generally recognized that a-syn aggregates colocalize with
Ab aggregates in the brain.28 Therefore, it is necessary to develop
in vivo imaging probes that can differentiate between a-syn and
Ab aggregates. In order to evaluate the binding affinity of IDP-1–
4 for a-syn and Ab aggregates quantitatively, we performed
a binding saturation assay using recombinant a-syn and Ab
aggregates. Table 1 shows the Kd values of the IDP derivatives
Immunohistochemical staining of a-syn in human PD brain
sections
Aer uorescent staining, the sections were activated by 90%
formic acid and incubated with anti-phosphorylated a-syn
primary antibody (pSyn#64) at 4 ꢂC for 2 h. Aer three 5 min
incubations in PBS-Tween 20, they were incubated with a uo-
rescent secondary antibody labeled with Alexa Fluor 647 at 4 ꢂC for
1 h. Aer three 3 min incubations in PBS-Tween 20, the sections
were observed with a uorescence microscope (FSX100, Olympus
Corp., Tokyo, Japan) equipped with a U-DM-CY5-3 lter set.
Table 1 Kd values of IDP derivatives for recombinant a-syn and Ab
aggregates
a
Kd (nM)
Compounds
a-syn
Ab
IDP-1
IDP-2
IDP-3
IDP-4
n.d.
12.88 ꢃ 2.02
37.14 ꢃ 10.80
13.35 ꢃ 5.67
16.24 ꢃ 4.78
217.87 ꢃ 73.87
23.28 ꢃ 2.78
5.40 ꢃ 1.50
In vivo biodistribution in normal mice
a
Values are the mean ꢃ standard error of the mean for three
The experiments with animals were conducted in accordance
with our institutional guidelines and approved by Kyoto
independent experiments.
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for a-syn and Ab aggregates. Although [125I]IDP-1 and [125I]IDP-2 corresponded to the results of immunohistochemical stain-
displayed no marked binding affinity for a-syn aggregates, [125I] ing. In contrast, IDP-1 and IDP-2, which showed low binding
IDP-3 and [125I]IDP-4 exhibited high binding affinity for a-syn affinity to a-syn aggregates, did not display marked uorescent
aggregates at Kd values of 23.3 and 5.4 nM, respectively. In accumulation in the PD brain sections (Fig. S1†). The results
contrast, all [125I]IDP derivatives showed high binding affinity suggested that both IDP-3 and IDP-4 can bind to not only
for Ab aggregates ranging from 12.9 to 37.1 nM. The results recombinant a-syn aggregates in vitro but also Lewy bodies
suggest that the binding affinity of IDP derivatives for a-syn composed of a-syn aggregates in brain sections from a PD
aggregates depends on the length of double bonds in the patient.
molecule, while they exhibit a high binding affinity for Ab
In the present study, we evaluated the binding affinity of our
aggregates despite different lengths of their double bond. The probes with recombinant a-syn aggregates, not with homoge-
ratio of the Kd value of the derivatives for a-syn aggregates nates from PD brains, because we could not obtain a sufficient
against Ab aggregates was 5.9, 2.2, and 0.3 for IDP-2, IDP-3, and amount of PD patients' brains. However, the Kd values of the
IDP-4, respectively. Accordingly, only [125I]IDP-4 showed a more probes calculated with recombinant a-syn aggregates were well
selective binding affinity for a-syn aggregates than Ab aggre- correlated with the results of in vitro uorescent staining with
gates, although [125I]IDP-1–3 displayed a higher binding affinity the brain sections from a PD patient. The ndings also suggest
for Ab aggregates than a-syn aggregates. However, the results that the in vitro binding assay system with recombinant a-syn
revealed that the selective binding affinity of IDP-4 was not as aggregates may be useful for screening promising ligands with
high as that of compound 46a reported in a previous study, high binding affinity for a-syn aggregates in PD brains.
although the binding assay system we used was different.18
In vivo biodistribution in normal mice
To evaluate the uptake of [125I]IDP-1–4 into the brain, we carried
Fluorescent staining of PD brain sections
out a biodistribution study in normal mice (Fig. 3 and Table
To conrm the affinity for Lewy bodies in PD patients' brains,
S1†). [125I]IDP-1 showed the highest uptake into the brain at 2
we carried out uorescent staining experiments using IDP-3 and
min aer the injection (brain2 min) (2.43% ID per g), whereas
IDP-4 with high binding affinity for a-syn aggregates in an in
[
125I]IDP-2, [125I]IDP-3, and [125I]IDP-4 displayed a low initial
vitro binding assay. Several uorescent spots were observed in
the section from the midbrain of a PD patient (Fig. 2). When the
same brain section was immunostained with an antibody
against a-syn, and compared with the uorescence of IDP-3 and
IDP-4, the uorescent spots obtained with IDP-3 and IDP-4
uptake (0.81, 0.56, and 0.45% ID per g respectively) (Fig. 3). It is
generally accepted that many factors including the lipophilicity,
molecular weight, molecular charge, and chemical structure
affect the penetration of the blood–brain barrier by the probes.
Therefore, the decrease in the brain uptake of IDP derivatives
may be attributable to their increases of the molecular weight
and size by extension of the conjugated double bonds. There-
aer, the radioactivity derived from the derivatives was cleared
from the brain with time. The radioactivity observed in the
brain at 60 min postinjection was 0.19, 0.38, 0.66, and 0.42% ID
per g for [125I]IDP-1, [125I]IDP-2, [125I]IDP-3, and [125I]IDP-4,
respectively. Some previous reports suggested that imaging
probes with less than 1% ID per g barely detect Ab aggregates in
vivo.29,30 Therefore, it may be difficult to apply them to image a-
syn aggregates even if [125I]IDP-2–4 shows a sufficiently high
binding affinity for a-syn aggregates in an in vitro binding assay.
Fig. 2 Fluorescent staining of IDP-3 (A) and IDP-4 (C) in brain sections
from a PD patient. Immunohistochemical staining with fluorescent
antibody against a-syn of a section adjacent to that used in the staining
of IDP-3 or IDP-4 ((B) and (D) for IDP-3 and IDP-4, respectively). Scale Fig. 3 Comparative brain uptake of radioactivity after the intravenous
bar ¼ 50 mm.
injection of 125I-labeled IDP derivatives.
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Notably, although [125I]IDP-4 displayed the highest and most
selective binding to a-syn aggregates among the four ligands, its
low brain uptake may make it difficult to apply as a promising a-
syn imaging probe.
Together with the results of the in vitro binding assay, the
ndings in the biodistribution study suggest that the length of
the double bonds in the molecule play an important role in the
binding affinity for a-syn aggregates. However, the increases in
the molecular size caused by extension of the conjugated double
bonds also led to a decreased brain uptake of the derivatives.
Therefore, to advance further molecular design for the devel-
opment of more useful imaging probes targeting a-syn aggre-
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Acknowledgements
The study was supported by a grant from the Japan Society for
the Promotion of Science (JSPS) through the “Funding Program
for Next Generation World-Leading Researchers (NEXT
Program)”, initiated by the Council for Science and Technology
Policy (CSTP), Takeda Science Foundation, and the Mochida
Memorial Foundation for Medical Pharmaceutical Research.
We thank Dr Hiroyuki Kimura (Kyoto Pharmaceutical Univer-
sity) for measuring the high resolution mass of IDP derivatives.
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