68Ga-Bivalent Polypegylated Styrylpyridine Conjugates for Imaging Aβ Plaques in Cerebral Amyloid Angiopathy

Article abstract of DOI:10.1021/acs.bioconjchem.6b00127

Aβ plaques deposited on blood vessels are associated with cerebral amyloid angiopathy (CAA). In an effort to selectively map these Aβ plaques, we are reporting a new series of ⁶⁸Ga labeled styrylpyridine derivatives with high molecular weights. In vitro binding to Aβ plaques in post-mortem Alzheimer's disease (AD) brain tissue showed that these ⁶⁸Ga labeled bivalent styrylpyridines displayed good affinities and specificity (K_i < 30 nM). In vitro autoradiography using post-mortem AD brain sections showed specific binding of these ⁶⁸Ga complexes to Aβ plaques. Biodistribution studies in normal mice showed very low initial brain uptakes (<0.3% dose/g) indicating a low blood-brain barrier (BBB) penetration. The preliminary results suggest that ⁶⁸Ga labeled bivalent styrylpyridines may be promising candidates as PET imaging radiotracers for detecting CAA.

Full text of DOI:10.1021/acs.bioconjchem.6b00127

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Article
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Ga-Bivalent Polypegylated Styrylpyridine Conjugates for
Imaging A# Plaques in Cerebral Amyloid Angiopathy (CAA)
Zhihao Zha, Jin Song, Seok Rye Choi, Zehui Wu, Karl Ploessl, Megan Smith, and Hank F. Kung
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00127 • Publication Date (Web): 04 Apr 2016
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68GaꢀBivalent Polypegylated Styrylpyridine Conjugates for Imaging Aβ Plaques in
Cerebral Amyloid Angiopathy (CAA)
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Zhihao Zhaa,c, Jin Songa,c, Seok Rye Choib,c, Zehui Wua,c, Karl Ploesslb,c, Megan Smithb,
Hank Kunga,b,c*
aBeijing Institute for Brain Disorders, Capital Medical University, Beijing, 100069,
b
c
China; Five Eleven Pharma Inc., Philadelphia, PA 19104; Department of Radiology,
University of Pennsylvania, Philadelphia, PA 19104, USA
*Corresponding author contact information:
Hank F. Kung, Ph.D. Department of Radiology, University of Pennsylvania, 3700
Market Street, Room 305, Philadelphia, PA 19104, USA Tel: +1 215 662 3989; Fax: +1
215 349 5035; Eꢀmail: kunghf@sunmac.spect.upenn.edu (H.F. Kung)
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Abstract:
Aβ plaques deposited on blood vessels are associated with cerebral amyloid
angiopathy (CAA). In an effort to selectively map these Aβ plaques, we are reporting a
new series of 68Ga labeled styrylpyridine derivatives with high molecular weights. In
vitro binding to Aβ plaques in postmortem Alzheimer’s disease (AD) brain tissue showed
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that these Ga labeled bivalent styrylpyridines displayed good affinities and specificity
(Ki < 30 nM). In vitro autoradiography using postmortem AD brain sections showed
specific binding of these 68Ga complexes to Aβ plaques. Biodistribution studies in normal
mice showed very low initial brain uptakes (< 0.3% dose/g) indicating a low bloodꢀbrain
barrier (BBB) penetration. The preliminary results suggest that 68Ga labeled bivalent
styrylpyridines may be promising candidates as PET imaging radiotracers for detecting
CAA.
Key Words: cerebral amyloid angiopathy, Aβ plaques, Alzheimer’s disease, positron
emission tomography, molecular imaging, autoradiography and biodistribution.
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Introduction:
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Cerebral amyloid angiopathy (CAA) is recognized as an important cause of lobar
intracerebral hemorrhage (ICH) and microbleeds (MBs) in older people (1-4). It is a
disease in which Aβ plaques deposited on the blood vessel walls in the brain may lead to
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(5)
stroke . Amyloidꢀstaining deposits in blood vessels are commonly composed of 39ꢀ43
amino acid peptides similar to that of Alzheimerꢀrelated senile plaques (6). There is a
close relationship between CAA and Alzheimer’s disease (AD) and they share the same
genetic risk factors (7, 8). CAA has a prevalence of 78ꢀ98% in individuals with AD, but
only 25% of patients with AD showed moderate to severe CAA (9, 10). CAA may occur in
the absence of AD. A recent report on the autopsy of brains of individuals with AD or
ADꢀrelated pathology (ADRP) showed that the presence of CAA might contribute to
neurodegeneration in AD (11). Additionally, it is likely that Aβ plaques may accumulate
earlier in blood vessel walls as well as within the parenchyma. The Aβ plaques may exits
indside and outside the endothetial. Therefore, it is important to develop a nonꢀinvasive
method for mapping Aβ plaques accumulating outside the endothelial of cerebral blood
vessels. Imaging Αβ plaques in CAA will allow an improved understanding of Αβ
aggregation and deposition in the cerebral blood vessels, supporting development of both
diagnostic and therapeutic agents.
In the past decades, several Positron Emission Tomography (PET)
radiopharmaceuticals targeting amyloid plaques in patients have been reported, including
florbetapir (Amyvid; [18F]AVꢀ45, Ki = 2.87 nM) (12-16), florbetaben (Neuraceq;
[18F]BAY94ꢀ9172, Ki = 2.22 nM) (17-20), and flutemetamol (Vizamyl; [18F]GEꢀ067, Ki =
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(21-24)
0.74 nM)
(Figure 1). Recent reports showed that amyloid imaging agents, such as
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11C labeled Pittsburgh Compound B (PiB), could image Aβ plaques in the blood vessels
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(25-31)
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of CAA patients
(Figure 1). However, CꢀPIB binds to the Aβ aggregates located
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in both blood vessels and parenchymal brain tissues, therefore further improvements for
selective βꢀamyloid imaging tracers associated with CAA are necessary for accurately
mapping Αβ aggregates in blood vessels. New 99mTc(CO)3 labeled benzothiazole
derivatives were reported to show selective binding to Aβ deposits located in cerebral
arterioles(32)
.
Figure 1. Chemical structures of various PET ligands targeting Αβ plaques in the brain.
Recently, many advances have been reported with respect to the binding affinity
enhancements that were achieved through multivalent binding (33-35). Multivalent binding
relies on multivalent ligand receptor interactions, which allow multiple and simultaneous
binding to several receptors (36, 37). The selfꢀassembled βꢀsheet strain of peptides displays
multiple and identically spaced binding sites toward the surface of the amyloid fibrils.
Thus, multivalency may be useful for enhancing the binding affinity of the molecular
imaging ligand for Αβ aggregates. Iikuni et al. recently reported 99mTcꢀhydroxamamide
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99mTcꢀHam) complexes with a bivalent ligand, such as [99mTc]5. Data suggests the
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(
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enhancement of the binding affinity occurs by application of bivalency (38) (Figure 2).
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Figure 2. Chemical structures of bivalent ligands and IMPYꢀtargeting Aβ plaques in the
brain. All of the compounds have been previously reported. (38-40)
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Our group has previously reported Fꢀlabeled bivalent ligands (6aꢀc) containing
mutiple AVꢀ45 (styrylpyridine) binding cores, which provided selective detection of Αβ
aggregates in blood vessel walls (39) (Figure 2). The binding affinity was measured using
postmortem AD brain homogenates against [125I]6ꢀiodoꢀ2ꢀ(4′ꢀdimethylaminoꢀ)phenylꢀ
imidazo[1,2ꢀa]pyridine ([125I]IMPY) (40). It is assumed that the styrylpyridine moieties are
responsible for inserting into the binding sites within the Aβ plaques. This bivalency,
using two styrylpyridines, will likely improve binding, increase the molecular weight,
and reduce the ability to penetrate intact bloodꢀbrain barrier (BBB) while targeting the
binding to Aβ plaques only on the blood vessel walls.
68Ga (t1/2 = 68 min, β+ = 89%) is an attractive isotope for PET imaging due to its
promising physical characteristics and distinctive advantage over 18F, the most commonly
used radionuclide for PET imaging. The availability and commercialization of 68Ga from
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a Ge/68Ga generator allows the simple and routine preparation of Ga imaging agents
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without the need of a nearby cyclotron for production. N,N'ꢀbis[2ꢀhydroxyꢀ5ꢀ
(carboxyethyl)benzyl]ethylenediamineꢀN,N'ꢀdiacetic acid (HBEDꢀCC) is a highly
effective complexing agent for Fe(III) and Ga(III), which forms complexes with log Kd =
39.7 and 39.6, respectively (41). 68Ga HBEDꢀCCꢀAhxꢀLysꢀureaꢀGlu complexes have been
widely used in humans for imaging prostateꢀspecific membrane antigen (42, 43). The
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resulting Ga HBEDꢀCC derivatives are sufficiently bulky, therefore they can not cross
the BBB.
We envision that 68Ga labeled agents targeting Aβ plagues may provide several
advantages: a) the selected HBEDꢀCC core derivatized with styrylpyridinyl groups,
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allows bivalent binding of Aβ plaques; b) the Ga complex will not penetrate BBB and
stay outside the brain parenchymal for binding of Aβ aggregates on the blood vessel
walls; c) 68Ga complex can be readily prepared in minutes using simplified kit
formulation; d) 68Ga is a generatorꢀbased isotope: it can be made widely available
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without the need of a nearby cyclotron. Therefore, successful Ga CAA imaging agents
may lead a widespread clinical application.
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On the basis of using bivalent styrylpyridines and Ga HBEDꢀCC complexes, we
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herein report the development and evaluation of several Gaꢀlabeled βꢀamyloidꢀtargeted
PET imaging agents, [68Ga]7aꢀe, for CAA (Figure 3).
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Figure 3. Structure of bivalent ligands [68Ga]7aꢀe, based on styrylpyridine cores. The
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novel compounds are designed to bind to Aβ aggregates via multiple binding sites.
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Results
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Chemistry. The synthesis of natGa styrylpyridine complexes is summarized in
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Schemes 1ꢀ3. Compound 9 was prepared by Oꢀmethylation (esterification) of carboxylic
acids in good yield (84%). The methyl ester was treated with MgCl2 and
paraformaldehyde to give salicylaldehyde, 10, in excellent yield (90%). Condensation of
salicylaldehyde with ethylenediamine produced Schiff base without need for further
purification. The corresponding secondary amine, 11, was obtained from the Schiff base
after a reduction reaction with sodium borohydride in 69% yield. The secondary amines
were condensed with an excess amount of tertꢀbutyl bromoacetate to afford 12 in 87%
yield. The methyl ester group of compound 12 was selectively removed by NaOH
hydrolysis to give acid, 13, in 96% yield.
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The intermediates 21aꢀe and 22 were synthesized by a coupling reaction of 13
with the corresponding stryrylpyridine, 20aꢀe, in 29ꢀ59% yield. The tertꢀbutyl and Boc
protection groups were then removed by trifluoroacetic acid (TFA) to give 7aꢀe and 8 in
28ꢀ44% yield. The precursors, 7aꢀe and 8, were used for radiolabeling and for the
synthesis of “cold compounds” [natGa]7aꢀe and [natGa]8.
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Radiosynthesis of [68Ga]7aꢀe and [68Ga]8. 68Ga conjugates were readily prepared
by incubating an excess of corresponding starting materials, 7aꢀe or 8 (~20 µM), with
ꢀ
[68Ga]GaCl4 (2.5ꢀ3.0 mCi in 0.05 N HCl, obtained from a 68Ge/68Ga generator) and
NaOAc buffer, resulting in specific activities ranging from 236 to 283 Ci/mmol. The
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labeling efficiency of Ga conjugates was greater than 93%, as confirmed by TLC and
HPLC analysis. TLC with citric acid showed Rf = 0 for 68Ga conjugates and 68Ga colloid
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and Rf = 0.9ꢀ1.0 for free of [68Ga]GaCl4 . In the second solvent system, Ga conjugates
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migrated to the solvent front, whereas the expected [68Ga]GaCl4 and 68Ga colloid stayed
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at the origin. The retention times obtained for each compound from HPLC were identical
to authentic “cold” standards. The [68Ga]7aꢀe and [68Ga]8 preparations were found to be
stable for more than 3 h at room temperature (rt) in phosphateꢀbuffered saline (PBS).
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Furthermore, no significant degradation of any Ga conjugates was observed up to 3 h
postꢀpreparation, when incubated with human plasma at 37 °C, thus suggesting high in
vitro stability.
In vitro binding studies using Aβꢀaggregates in the AD brain tissue
homogenates. An in vitro competitive binding assay was conducted to measure the
inhibition of [125I]IMPY, a known Aβꢀaggregates ligand (44), binding to Aβꢀaggregates in
the AD brain tissue homogenates. Inhibition constants (Ki, nM) of various ligands (free
ligands and natGa conjugates) against the binding of [125I]IMPY to Aβꢀaggregates are
shown in Table 1. The HBEDꢀbased bivalent ligands (7aꢀe) displayed high binding
affinities (Ki = 1.80, 3.53, 4.52, 1.52 and 1.71 nM, respectively), whereas the associated
natGa conjugates showed lower binding affinities (Ki = 30.6, 13.4, 18.1, 6.70 and 10.7
nM, respectively) (Table 1). The length of the pegylation chain showed no remarkable
effect on the binding affinities. However, the monovalent ligands showed dramatically
lower binding affinities (8 and [natGa]8, Ki = 239 and 185 nM, respectively). These
monovalent Ga conjugates were not studied any further.
Table 1. Inhibition constant (Ki, nM) of Aβ plaques targeting styrylpyridine derivatives
against binding of [125I]IMPY, a known Aβꢀaggregates ligand (44)
Compound Name
Ki (nM)*
Compound Name Ki (nM)*
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[natGa]7a (n = 3)
[natGa]7b (n = 4)
[natGa]7c (n = 5)
[natGa]7d (n = 6)
[natGa]7e (n = 7)
[natGa]8a
1.80 ± 0.24
30.6 ± 6.97
13.4 ± 3.71
18.1 ± 4.32
6.70 ± 1.70
10.7 ± 4.45
185 ± 80
7a (n = 3)
7b (n = 4)
7c (n = 5)
7d (n = 6)
7e (n = 7)
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3.53 ± 1.21
4.52 ± 2.42
1.52 ± 0.20
1.71 ± 0.25
239 ± 38
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florbetapir (AVꢀ45), 1 2.87 ± 0.17
*Measured against [125I]IMPY binding to Aβ plaques in postmortem AD brain
homogenates, n= 3.
In vitro autoradiography of AD brain sections. The postmortem AD brain
sections containing Aβ plaques were labeled intensely by all of the bivalent ligands,
[68Ga]7aꢀe. The adjacent sections were labeled by [125I]IMPY and showed different
images to those of [68Ga]7aꢀe (Figure 4). Furthermore, the autoradiographic signals were
almost completely blocked after incubating the sections in the presence of excess of
IMPY (28 ꢁM), demonstrating [68Ga]7aꢀe were competing for the same Aβ plaque
binding sites. As expected, the monovalent ligand, [68Ga]8, did not show any Aβ plaques
labeling, which was consistent with the low binding affinity measured by an in vitro
binding assay using AD brain homogenates.
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Figure 4. In vitro autoradiography of brain sections from AD patients labeled with
[125I]IMPY, [68Ga]7aꢀe, and [68Ga]8. [125I]IMPY and [68Ga]7a binding in AD brain
sections blocked in the presence of 28 µM IMPY.
In vivo biodistribution study in CDꢀ1 mice. After an iv injection in normal mice,
[68Ga]7aꢀe showed low brain uptake (0.1–0.3% ID/g at 2 min post injection) (table 2).
Compared to the brain uptake of [18F]1, the bivalent ligands, [68Ga]7aꢀe, clearly
displayed a very low brain penetration. This may allow a selective labeling of Aβ plaques
deposited on the walls of cerebral blood vessels, not in the Aβ plaques in the
parenchymal brain tissues.
Table 2. Biodistribution in normal CDꢀ1 mice after an iv injection of [68Ga]7aꢀe and
[68Ga]8 in saline (% dose/g, n = 3)
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[68Ga]7a
[68Ga]7b
organ
blood
muscle
kidney
liver
2 min
60 min
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3.10 ± 0.27
0.26 ± 0.02
3.71 ± 0.33
25.7 ± 3.25
0.12 ± 0.05
0.48 ± 0.04
0.88 ± 0.15
0.15 ± 0.11
1.87 ± 0.52
22.5 ± 2.63
0.08 ± 0.09
0.40 ± 0.08
4.57 ± 0.07
0.47 ± 0.10
4.94 ± 0.44
28.5 ± 3.24
0.17 ± 0.05
0.74 ± 0.12
0.59 ± 0.82
0.15 ± 0.04
1.97 ± 0.13
15.4 ± 1.07
0.04 ± 0.00
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brain
bone
0.44 ± 0.11
[68Ga]7c
[68Ga]7d
organ
blood
muscle
kidney
liver
2 min
60 min
2 min
60 min
9.48 ± 3.63
0.68 ± 0.18
10.7 ± 2.09
43.3 ± 17.8
0.31 ± 0.09
1.02 ± 0.34
7.24 ± 4.21
0.35 ± 0.03
3.32 ± 0.23
26.7 ± 2.43
0.11 ± 0.03
0.82 ± 0.13
7.85 ± 1.95
0.42 ± 0.07
4.01 ± 0.11
15.9 ± 0.61
0.21 ± 0.05
0.98 ± 0.03
2.35 ± 0.54
0.34 ± 0.05
2.03 ± 0.10
12.8 ± 1.80
0.11 ± 0.01
0.74 ± 0.08
brain
bone
[68Ga]7e
[68Ga]8
organ
blood
muscle
kidney
liver
2 min
60 min
2 min
60 min
6.87 ± 0.86
0.41 ± 0.05
4.75 ± 0.24
12.6 ± 0.86
0.22 ± 0.03
0.75 ± 0.08
2.88 ± 0.64
0.35 ± 0.05
2.40 ± 0.41
8.12 ± 0.31
0.07 ± 0.01
0.46 ± 0.02
3.22 ± 0.44
0.40 ± 0.03
6.33 ± 0.65
23.3 ± 2.06
0.11 ± 0.01
0.57 ± 0.11
1.01 ± 0.10
0.14 ± 0.01
1.63 ± 0.12
2.17 ± 0.24
0.03 ± 0.00
0.26 ± 0.03
brain
bone
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Discussion
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In the present study, we reported a series of novel 68GaꢀHBED complexes
containing bivalent polypegylated styrylpyridine as PET imaging agents for mapping Aβ
plaques in the blood vessels of CAA patients. They are useful as alternatives to 18F
labeled imaging agents. Compared to 18F, 68Ga is more conveniently produced by
68Ge/68Ga generators without an onꢀsite cyclotron. In addition, 68Ga provides a simple,
rapid radiolabeling method, which is suitable for using a convenient lyophilized kit
formulation (45). Many potential chelating agents have been reported for complexation of
68Ga to targeted carrier molecules (46). The predominantly used bifunctional 68Ga chelator
is 1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid (DOTA), and it is considered
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the “gold standard” for chelating radioactive metals (47, 48). However, Ga complexation
with DOTA requires heating at elevated temperatures or longer reaction times, which
might have detrimental effects on heatꢀlabile ligands such as proteins or other
biomolecules. Therefore other chelators such as 1,4,7ꢀtriazacyclononaneꢀtriacetic acid
(NOTA) and (2,2ꢀ(7ꢀ(1ꢀcarboxyꢀ4ꢀ(2ꢀmercaptoethylamino)ꢀ4ꢀoxobutyl)ꢀ1,4,7ꢀtriazonaneꢀ
1,4ꢀdiyl)diacetic acid) (NODAGA) have been evaluated to circumvent these problems (49)
(50, 51). Among them, HBED was described recently as a highly efficient chelator allowing
fast and easyꢀtoꢀperform labeling at room temperature (52). Previously reported synthesis
of HBED complexes employed a Fe complex of HBEDꢀCC as the intermediate (53). The
reaction scheme was relatively inefficient, and a new scheme involving a more efficient,
simpler means to carry out, without the use of Fe(III) HBEDꢀCC complex was described
in Scheme 1. As described here, the 68Ga complexation of all ligands, [68Ga]7aꢀe and
[68Ga]8, resulted in high radiochemical yields of 93ꢀ98% after 5 min reaction time at
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room temperate, which led to less than 20 min of preparation time for radiolabeling and
quality control. As a consequence, radiotracers were subsequently used for in vitro and in
vivo experiments without further purification. All 68Ga conjugates used in this study
proved to be stable in PBS and human plasma for the studyꢀrelevant time period of 3 h.
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One of the major preꢀrequisites for the function of the chelator in
radiopharmaceuticals is that the structure and physical properties of the metal complex do
not have a large impact on receptor binding (46). The change of overall charge and
lipophilicity of the radiometal complexes often influence pharmacokinetics of targeting
moiety, especially for small vectors. The HBEDꢀCC styrylpyridines showed the desired
(13, 14)
high binding affinity comparable to that of [18F]AVꢀ45 ([18F]1)
and previously
reported 18Fꢀlabeled CAA imaging agents ([18F]6aꢀc). We also developed other 68Ga
complexes with different chelating groups, such as DOTA and 6ꢀaminoꢀ6ꢀ
methylperhydroꢀ1,4ꢀdiazepinetetraacetic acid (AAZTA) (54, 55), to compare with [68Ga]7aꢀ
c in terms of βꢀamyloid binding characteristics. Surprisingly, compared to the HBED
complexes [68Ga]7aꢀc, the DOTA and AAZTA complexes showed significantly lower
binding affinities (data not shown). This decrease may have been caused by the change of
hydroxybenzyl groups of the ligands. Further investigations with other chelating groups
such as NOTA, NODAGA, and 2ꢀ[[6ꢀ(carboxy)ꢀpyridinꢀ2ꢀyl]ꢀmethylamino]ethane
(H2dedpa) will be required to confirm the validity of this assumption.
Various multivalent ligands have been reported for enhancing peptide affinity (33)
.
An interesting study comparing binding affinity with amyloid aggregates between
bivalent and monovalent ligands showed that the bivalent scaffold correlates with
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improved binding (38). For this report, we have prepared and examined the Αβ aggregate
binding affinity of HBEDꢀCC styrylpyridines, 7aꢀe, 8, and corresponding natGa
conjugates. Similarly, the high binding affinities observed for bivalent ligands (7aꢀe
(dimer), Ki = 1.7ꢀ4.5 nM vs. 8 (monovalent), Ki = 239 nM), suggest that there are
bivalent interactions in binding to neighboring binding sites, and a bivalent ligand shows
better binding affinity than the comparable monomer. The comparison of the inhibition
constants of 7aꢀe suggested the length of the PEG scaffold did not interfere with the
binding. However, the binding affinity of Ga conjugates, [natGa]7aꢀe, showed a slight
reduction (Ki = 6ꢀ30 nM), compared to the precursors. This indicates that the change of
geometry after natGa complex formation might change the interaction with aggregated
amyloid peptides and reduce the binding affinity.
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In vitro labeling of Aβ plaques in AD brain sections showed that [68Ga]7aꢀe were
capable of labeling Aβ aggregates in brain sections, indicating the feasibility of using
them as PET imaging agents for detecting Aβ plaques in CAA patients. Of note, whereas
[125I]IMPY exhibited a discrete binding pattern, [68Ga]7aꢀe displayed a more blurry
image. Presumably, this discrepancy is due to lower auger electrons from 125I and the
relatively longer traveling distance of positrons emitted by 68Ga. As expected, [68Ga]8
showed no marked labeling to Aβ plaques. The data of in vitro autoradiography of AD
brain sections are consistent with those of in vitro binding studies.
Conclusion
A new series of 68GaꢀHBEDꢀCC conjugates with bivalent styrylpyridine targeting
Aβ aggregates on the walls of blood vessels was successfully prepared and tested. The
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bivalent ligands, [68Ga]7aꢀe, showed highly promising Αβ aggregateꢀbinding affinity.
The enhancement of binding affinity was confirmed by in vitro autoradiography of
postmortem AD brain sections. Furthermore, [68Ga]7aꢀe displayed low BBB penetration,
showing low uptake in the brains of normal mice. These results support that similar to
[18F]6aꢀc (39), [68Ga]7aꢀe are equally effective and are considered as promising candidates
for PET imaging agents for Αβ plaques on the walls of blood vessels in CAA.
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Experimental sections
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General. All chemicals were purchased from Aldrich Chemical Co. and used
without further purification unless otherwise indicated. Solvents were dried through a
molecular sieve system (Pure Solve Solvent Purification System; Innovative Technology,
Inc.). Normal CDꢀ1 mice (20ꢀ26 g) were used for the biodistribution studies. The
protocol requiring the use of mice was reviewed and approved by the Institutional
Animal Care and Use Committee (University of Pennsylvania). Postmortem human
samples were obtained from the National Disease Research Interchange (NDRI,
Philadelphia, PA).
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The synthesis of bivalent and monovalent styrylpyridine derivatives, 7aꢀe and 8,
was outlined in Schemes 1ꢀ3. The details of the procedure for synthesis of 14ꢀ20 are
included in the supplemental information.
Chemistry. Methyl 3ꢀ(4ꢀhydroxyphenyl)propanoate (9). To a solution of 3ꢀ(4ꢀ
hydroxyphenyl)propanoic acid (3.0 g, 18.1 mmol) in 50 mL MeOH was added BF3•Et2O
(0.3 mL). After stirring at rt for 6 h, the solvent was removed, and the residue was
purified by flash chromatography (FC) (ethyl acetate (EtOAc)/hexane = 2/8) to give 9 as
1
a white solid (yield: 2.72 g, 83.5%). HNMR (400 MHz, CDCl3) δ: 7.07(d, 2H, J = 8.4
Hz), 6.76 (d, 2H, J = 8.4 Hz), 4.72 (s, 1H), 3.68 (s, 3H), 2.89 (t, 2H, J = 7.6 Hz), 2.60 (t,
2H, J = 7.6 Hz); HRMS calcd. for C10H13O3 (M + H)+: 181.0865, found 181.0815.
Methyl 3ꢀ(3ꢀformylꢀ4ꢀhydroxyphenyl)propanoate (10). To a solution of 9 (2.72
g, 15.1 mmol) in 70 mL acetonitrile (ACN), MgCl2 (2.87 g, 30.2 mmol),
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paraformaldehyde (3.66 g, 120.8 mmol), and triethylamine (Et3N, 6.1 g, 60.4 mmol) were
added at rt. The mixture was heated under reflux for 8 h, diluted with water (25 mL),
followed by acidification using HCl (5%, 100 mL), and extraction with ether (50 mL ×
3). The organic layer was then dried over MgSO4 and filtered. The filtrate was
concentrated, and the residue was purified by FC (EtOAc/hexane = 2/8) to give 10 as a
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white solid (yield: 2.85 g, 90.4%). HNMR (400 MHz, CDCl3) δ: 10.89 (s, 1H), 9.88 (s,
1H), 7.37ꢀ7.40 (m, 2H), 6.94 (d, 1H, J = 9.6 Hz), 3.68 (s, 3H), 2.95 (t, 2H, J = 7.4 Hz),
2.64 (t, 2H, J = 7.4 Hz); HRMS calcd. for C11H13O4 (M + H)+: 209.0814, found
209.0825.
Dimethyl 3,3'ꢀ(((ethaneꢀ1,2ꢀdiylbis(azanediyl))bis(methylene))bis(4ꢀhydroxyꢀ
3,1ꢀphenylene))dipropanoate (11). Ethylenediamine (0.371 g, 6.18 mmol) was added to
a solution of 10 (2.84 g, 13.6 mmol) in 60 mL MeOH at rt. After stirring at 50 °C
overnight, the mixture was cooled in an iceꢀbath. NaBH4 (1.05 g, 27.81 mmol) was added
in portions to the reaction mixture, and the resulting solution was allowed to warm to rt.
After being stirred at rt for 24 h, the reaction was quenched with 100 mL H2O and
extracted with ethyl acetate (150 × 3 mL). The organic layer was dried over MgSO4 and
filtered. The filtrate was then concentrated, and the residue was purified by FC
(dichloromethane (DCM)/MeOH/NH4OH = 90/9/1) to give 11 as a colorless oil (yield:
2.08 g, 68.8%). 1HNMR (400 MHz, CDCl3) δ: 7.00 (dd, 2H, J = 2.0 Hz, J = 8.4 Hz), 6.82
(d, 2H, J = 2.0 Hz), 6.76 (d, 2H, J = 8.4 Hz), 3.97 (s, 4H), 2.67 (s, 6H), 2.83ꢀ2.87 (m,
8H), 2.58 (t, 4H, J = 7.8 Hz); HRMS calcd. for C24H33N2O6 (M + H)+: 445.2339, found
445.2139.
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Dimethyl
3,3'ꢀ(((2,2,13,13ꢀtetramethylꢀ4,11ꢀdioxoꢀ3,12ꢀdioxaꢀ6,9ꢀ
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diazatetradecaneꢀ6,9ꢀdiyl)bis(methylene))bis(4ꢀhydroxyꢀ3,1ꢀ
7
8
9
phenylene))dipropanoate (12). To a solution of 11 (1.8 g, 4.05 mmol) in 50 mL ACN,
tertꢀButyl bromoacetate (1.66 g, 8.51 mmol) and Na2CO3 (1.71 g, 16.2 mmol) were
added. The mixture was then heated at 60 °C overnight before being cooled to rt and
filtered. The filtrate was concentrated, and the residue was purified by FC (EtOAc/hexane
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= 1/1) to give 12 as a colorless oil (yield: 2.36 g, 86.6%). HNMR (400 MHz, CDCl3) δ:
9.55 (s, 2H), 7.00 (dd, 2H, J = 2.0 Hz, J = 8.4 Hz), 6.77 (d, 2H, J = 8.4 Hz), 6.74 (d, 2H,
J = 2.0 Hz), 3.70 (s, 4H), 3.67 (s, 6H), 3.17 (s, 4H), 2.83 (t, 4H, J = 7.8Hz), 2.69 (s, 4H),
2.57 (t, 4H, J = 7.8 Hz), 1.46 (s, 18H); HRMS calcd. for C36H53N2O10 (M + H)+:
673.3700, found 673.3662.
3,3'ꢀ(((2,2,13,13ꢀTetramethylꢀ4,11ꢀdioxoꢀ3,12ꢀdioxaꢀ6,9ꢀdiazatetradecaneꢀ6,9ꢀ
diyl)bis(methylene))bis(4ꢀhydroxyꢀ3,1ꢀphenylene))dipropanoic acid (13). A solution
of 12 (1.6 g, 2.38 mmol) in 10 mL MeOH/NaOH (1 N) (1/1) was stirred at rt for 2 h. HCl
(1 N) was then added to the reaction mixture to pH = 4ꢀ5. The resulting mixture was
extracted with EtOAc (50 mL × 3). The organic layer was then dried over MgSO4 and
filtered. The filtrate was concentrated, and the residue was purified by FC
(DCM/MeOH/NH4OH = 90/9/1) to give 13 as a white solid (yield: 1.2 g, 78.2%).
1HNMR (400 MHz, CDCl3) δ: 7.03 (dd, 2H, J = 2.0 Hz, J = 8.4 Hz), 6.80 (d, 2H, J = 8.4
Hz), 6.71 (d, 2H, J = 2.0 Hz), 3.56 (s, 4H), 3.26 (s, 4H), 2.84 (t, 4H, J = 7.0 Hz), 2.62 (t,
4H, J = 7.0 Hz), 2.56 (s, 4H), 1.48(s, 18H); HRMS calcd. for C34H49N2O10 (M + H)+:
645.3387, found 645.3417.
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Diꢀtertꢀbutyl
2,2'ꢀ((E)ꢀethaneꢀ1,2ꢀdiylbis((5ꢀ(3ꢀ((2ꢀ(2ꢀ(2ꢀ((5ꢀ((E)ꢀ4ꢀ((tertꢀ
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butoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ethoxy)ethoxy)ethyl)amino)ꢀ
3ꢀoxopropyl)ꢀ2ꢀhydroxybenzyl)azanediyl))diacetate (21a). To a solution of 13 (118.3
mg, 0.183 mmol) in 5 mL DMF, N,Nꢀdiisopropylethylamine (DIPEA, 189.6 mg, 1.47
mmol), 1ꢀhydroxybenzotriazole hydrate (HOBt, 0.55 mmol, 92.8 mg), Nꢀ(3ꢀ
dimethylaminopropyl)ꢀNꢀethylcarbodiimide hydrochloride (EDC, 105 mg, 0.55 mmol)
and 20a (168 mg, 0.367 mmol) were added at 0 °C. The mixture was stirred at rt for
overnight before 30 mL EtOAc was added to the reaction mixture. It was then washed
with H2O (10 mL × 2) and brine (10 mL), dried over MgSO4, and filtered. The filtrate
was concentrated, and the residue was purified by FC (DCM/MeOH/NH4OH = 95/5/0.5)
to give 21a as a colorless oil (yield: 140 mg, 50.2%): 1HNMR (400 MHz, CDCl3) δ: 9.50
(br s, 2H), 8.17 (d, 2H, J = 2.4 Hz), 7.78 (dd, 2H, J = 2.4 Hz, J = 8.8 Hz), 7.45 (d, 4H, J =
8.8Hz), 7.24 (d, 4H, J = 8.4 Hz), 6.97ꢀ7.02 (m, 6H), 6.73ꢀ6.77 (m, 6H), 6.23 (br s, 2H),
4.50 (t, 4H, J = 4.8 Hz), 3.85 (t, 4H, J = 4.8 Hz), 3.67ꢀ3.73 (m, 12H), 3.53 (t, 4H, J = 5.0
Hz), 3.43 (t, 4H, J = 5.2 Hz), 3.28 (s, 6H), 3.18 (s, 4H), 2.84 (t, 4H, J = 7.8 Hz), 2.67 (s,
4H), 2.42 (t, 4H, J = 7.8Hz), 1.48 (s, 18H), 1.46 (s, 18H); HRMS calcd. for C84H115N8O18
(M + H)+: 1523.8329, found 1523.8373.
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Diꢀtertꢀbutyl
2,2'ꢀ((E)ꢀethaneꢀ1,2ꢀdiylbis((5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ((tertꢀ
butoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ13ꢀoxoꢀ3,6,9ꢀtrioxaꢀ12ꢀ
azapentadecanꢀ15ꢀyl)ꢀ2ꢀhydroxybenzyl)azanediyl))diacetate (21b). Compound 21b
(yield: 43 mg, 49.5%) was prepared from 13 (43 mg, 0.0667 mmol), DIPEA (68.9 mg,
0.534 mmol), HOBt (33.8 mg, 0.2 mmol), EDC (38.2 mg, 0.2 mmol), and 20b (67 mg,
1
0.1334 mmol) following the same procedure described for compound 21a. HNMR (400
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MHz, CDCl3) δ: 9.50 (br s, 2H), 8.17 (d, 2H, J = 2.4 Hz), 7.77 (dd, 2H, J = 2.4 Hz, J =
8.8 Hz), 7.44 (d, 4H, J = 8.8Hz), 7.23 (d, 4H, J = 8.4 Hz), 6.96ꢀ7.00 (m, 6H), 6.73ꢀ6.77
(m, 6H), 6.30 (br s, 2H), 4.48 (t, 4H, J = 4.8 Hz), 3.84 (t, 4H, J = 4.8 Hz), 3.56ꢀ3.72 (m,
20H), 3.51 (t, 4H, J = 5.0 Hz), 3.40 (t, 4H, J = 5.2 Hz), 3.27 (s, 6H), 3.17 (s, 4H), 2.82 (t,
4H, J = 7.8 Hz), 2.66 (s, 4H), 2.41 (t, 4H, J = 7.8Hz), 1.46 (s, 18H), 1.45 (s, 18H);
HRMS calcd. for C88H123N8O20 (M + H)+: 1611.8854, found 1611.8998.
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Diꢀtertꢀbutyl
2,2'ꢀ((E)ꢀethaneꢀ1,2ꢀdiylbis((5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ((tertꢀ
butoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ16ꢀoxoꢀ3,6,9,12ꢀtetraoxaꢀ
15ꢀazaoctadecanꢀ18ꢀyl)ꢀ2ꢀhydroxybenzyl)azanediyl))diacetate (21c). Compound 21c
(yield: 40 mg, 51.2%) was prepared from 13 (29.7 mg, 0.046 mmol), DIPEA (47.4 mg,
0.368 mmol), HOBt (23.3 mg, 0.138 mmol), EDC (26.3 mg, 0.138 mmol), and 20c (50
1
mg, 0.092 mmol) following the same procedure described for compound 21a. HNMR
(400 MHz, CDCl3) δ: 9.50 (br s, 2H), 8.17 (d, 2H, J = 2.0 Hz), 7.77 (dd, 2H, J = 2.4 Hz, J
= 8.8 Hz), 7.44 (d, 4H, J = 8.8Hz), 7.22 (d, 4H, J = 8.4 Hz), 6.96ꢀ7.01 (m, 6H), 6.73ꢀ6.78
(m, 6H), 6.29 (br s, 2H), 4.47 (t, 4H, J = 4.8 Hz), 3.83 (t, 4H, J = 4.8 Hz), 3.56ꢀ3.71 (m,
28H), 3.50 (t, 4H, J = 5.0 Hz), 3.42 (t, 4H, J = 5.2 Hz), 3.27 (s, 6H), 3.16 (s, 4H), 2.83 (t,
4H, J = 7.8 Hz), 2.66 (s, 4H), 2.41 (t, 4H, J = 7.8Hz), 1.46 (s, 18H), 1.45 (s, 18H);
HRMS calcd. for C92H131N8O22 (M + H)+: 1699.9378, found 1699.9348.
Diꢀtertꢀbutyl
2,2'ꢀ((E)ꢀethaneꢀ1,2ꢀdiylbis((5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ((tertꢀ
butoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ19ꢀoxoꢀ3,6,9,12,15ꢀ
pentaoxaꢀ18ꢀazahenicosanꢀ21ꢀyl)ꢀ2ꢀhydroxybenzyl)azanediyl))diacetate
(21d).
Compound 21d (yield: 60 mg, 58.9%) was prepared from 13 (36.8 mg, 0.057 mmol),
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21a. HNMR (400 MHz, CDCl3) δ: 9.50 (br s, 2H), 8.17 (d, 2H, J = 2.0 Hz), 7.78 (dd,
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2H, J = 2.4 Hz, J = 8.8 Hz), 7.44 (d, 4H, J = 8.4Hz), 7.22 (d, 4H, J = 8.8 Hz), 6.96ꢀ7.01
(m, 6H), 6.73ꢀ6.79 (m, 6H), 6.22 (br s, 2H), 4.48 (t, 4H, J = 4.8 Hz), 3.85 (t, 4H, J = 4.8
Hz), 3.57ꢀ3.71 (m, 36H), 3.50 (t, 4H, J = 5.0 Hz), 3.42 (t, 4H, J = 5.2 Hz), 3.27 (s, 6H),
3.17 (s, 4H), 2.83 (t, 4H, J = 7.8 Hz), 2.66 (s, 4H), 2.41 (t, 4H, J = 7.8Hz), 1.46 (s, 18H),
1.45 (s, 18H); HRMS calcd. for C96H139N8O24 (M + H)+: 1787.9902, found 1787.9882.
Diꢀtertꢀbutyl
2,2'ꢀ((E)ꢀethaneꢀ1,2ꢀdiylbis((5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ((tertꢀ
butoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ22ꢀoxoꢀ3,6,9,12,15,18ꢀ
hexaoxaꢀ21ꢀazatetracosanꢀ24ꢀyl)ꢀ2ꢀhydroxybenzyl)azanediyl))diacetate
(21e).
Compound 21e (yield: 83 mg, 55.3%) was prepared from 13 (51.6 mg, 0.08 mmol),
DIPEA (82.6 mg, 0.64 mmol), HOBt (40.5 mg, 0.24 mmol), EDC (45.8 mg, 0.24 mmol)
and 20e (100 mg, 0.16 mmol), following the same procedure described for compound
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21a. HNMR (400 MHz, CDCl3) δ: 9.50 (br s, 2H), 8.18 (d, 2H, J = 2.0 Hz), 7.79 (dd,
2H, J = 2.4 Hz, J = 8.8 Hz), 7.44 (d, 4H, J = 8.8Hz), 7.22 (d, 4H, J = 8.4 Hz), 6.97ꢀ7.01
(m, 6H), 6.73ꢀ6.80 (m, 6H), 6.29 (br s, 2H), 4.49 (t, 4H, J = 4.8 Hz), 3.85 (t, 4H, J = 4.8
Hz), 3.56ꢀ3.72 (m, 44H), 3.51 (t, 4H, J = 5.0 Hz), 3.42 (t, 4H, J = 5.2 Hz), 3.27 (s, 6H),
3.17 (s, 4H), 2.84 (t, 4H, J = 7.8 Hz), 2.67 (s, 4H), 2.42 (t, 4H, J = 7.8Hz), 1.47 (s, 36H);
HRMS calcd. for C100H147N8O26 (M + H)+: 1876.0427, found 1876.0447.
(E)ꢀ3ꢀ(3ꢀ(((2ꢀ(tertꢀButoxy)ꢀ2ꢀoxoethyl)(2ꢀ((2ꢀ(tertꢀbutoxy)ꢀ2ꢀoxoethyl)(5ꢀ(1ꢀ
((5ꢀ(4ꢀ((tertꢀbutoxycarbonyl)(methyl)amino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ13ꢀoxoꢀ3,6,9ꢀ
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trioxaꢀ12ꢀazapentadecanꢀ15ꢀyl)ꢀ2ꢀhydroxybenzyl)amino)ethyl)amino)methyl)ꢀ4ꢀ
hydroxyphenyl)propanoic acid (22). Compound 22 (yield: 33 mg, 28.9%): was
prepared from 13 (90.4 mg, 0.14 mmol), DIPEA (72.2 mg, 0.56 mmol), HOBt (35.4 mg,
0.21 mmol), EDC (40.1 mg, 0.21 mmol), and 20b (70 mg, 0.14 mmol), following the
same procedure described for compound 21a. 1HNMR (400 MHz, CDCl3) δ: 8.17 (d, 1H,
J = 2.0 Hz), 7.77 (dd, 1H, J = 2.4 Hz, J = 8.8 Hz), 7.44 (d, 2H, J = 8.8Hz), 7.22 (d, 2H, J
= 8.4 Hz), 6.96ꢀ7.03 (m, 4H), 6.71ꢀ6.78 (m, 5H), 6.55 (br s, 1H), 4.47 (t, 2H, J = 4.8 Hz),
3.84 (t, 2H, J = 4.8 Hz), 3.50ꢀ3.72 (m, 16H), 3.40ꢀ3.42 (m, 2H), 3.27 (s, 3H), 3.24 (s,
4H), 2.82 (t, 4H, J = 7.4 Hz), 2.70 (s, 4H), 2.59 (t, 2H, J = 7.4 Hz), 2.44 (t, 2H, J = 7.8
Hz), 1.46 (s, 27H); HRMS calcd. for C61H86N5O15 (M + H)+: 1128.6120, found
1128.6105.
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2,2'ꢀ((E)ꢀEthaneꢀ1,2ꢀdiylbis((2ꢀhydroxyꢀ5ꢀ(3ꢀ((2ꢀ(2ꢀ(2ꢀ((5ꢀ((E)ꢀ4ꢀ
(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ethoxy)ethoxy)ethyl)amino)ꢀ3ꢀ
oxopropyl)benzyl)azanediyl))diacetic acid (7a). A solution of 21a (30 mg, 0.020
mmol) in 1 mL TFA was stirred at rt for 4 h. The reaction mixture was evaporated in
vacuo, and the residue was purified by semiꢀpreparative HPLC to give 8.6 mg of a
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yellowꢀcolored solid, 7a (yield: 37.6%). HNMR (400 MHz, MeOD) δ: 8.24 (d, 2H, J =
2.0Hz), 8.02 (dd, 2H, J = 2.4 Hz, J = 8.8 Hz), 7.70 (d, 4H, J = 8.4 Hz), 7.40 (4H, J = 8.4
Hz), 7.07ꢀ7.18 (m, 8H), 6.87 (d, 2H, J = 8.8Hz), 6.80 (d, 2H, J = 8.8Hz), 4.46 (t, 4H, J =
4.8Hz), 4.10 (s, 4H), 3.85 (t, 4H, J = 4.6 Hz), 3.57ꢀ3.72 (m, 12H), 3.47 (t, 4H, J = 5.4Hz),
3.30ꢀ3.32 (m, 8H), 3.06 (s, 6H), 2.80 (t, 4H, J = 7.4Hz), 2.43 (t, 4H, J = 7.4Hz); HRMS
calcd. for C66H83N8O14 (M + H)+: 1211.6029, found 1211.6028.
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2,2'ꢀ((E)ꢀEthaneꢀ1,2ꢀdiylbis((2ꢀhydroxyꢀ5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ
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(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ13ꢀoxoꢀ3,6,9ꢀtrioxaꢀ12ꢀazapentadecanꢀ15ꢀ
yl)benzyl)azanediyl))diacetic acid (7b). Compound 7b (yield: 8.4 mg, 34.0%): was
prepared from 21b (31 mg, 0.019 mmol) and 1 mL TFA, following the same procedure
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described for compound 7a. HNMR(400 MHz, D2O) δ: 8.43(d, 2H, J = 9.2 Hz), 8.24(s,
2H), 7.71(d, 4H, J = 8.4Hz), 7.51(d, 4H, J = 8.4Hz), 7.35(d, 2H, J = 8.8Hz), 7.05ꢀ7.21(m,
8H), 6.81(d, 2H, J = 8.0Hz), 4.55(t, 4H, J = 4.0Hz), 4.21(s, 4H), 3.97(t, 4H, J = 4.0Hz),
3.71ꢀ3.80(m, 12H), 3.56ꢀ3.66(m, 8H), 3.50(s, 4H), 3.46(t, 4H, J = 5.4Hz), 3.26(t, 4H, J =
5.2Hz), 3.13(s, 6H), 2.77(t, 4H, J = 7.4Hz), 2.44(t, 4H, J = 7.4Hz); HRMS calcd for
C70H91N8O16 (M + H)+: 1299.6553, found 1299.6506.
2,2'ꢀ((E)ꢀEthaneꢀ1,2ꢀdiylbis((2ꢀhydroxyꢀ5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ
(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ16ꢀoxoꢀ3,6,9,12ꢀtetraoxaꢀ15ꢀazaoctadecanꢀ
18ꢀyl)benzyl)azanediyl))diacetic acid (7c). Compound 7c (yield: 9.0 mg, 28.2%): was
prepared from 21c (40 mg, 0.023 mmol) and 1 mL TFA following the same procedure
described for compound 7a. 1HNMR (400 MHz, MeOD) δ: 8.26 (d, 2H, J = 2.0Hz), 8.07
(dd, 2H, J = 2.4Hz, J = 8.8Hz), 7.71 (d, 4H, J = 8.4Hz), 7.43 (d, 4H, J = 8.4Hz), 7.08ꢀ
7.20 (m, 8H), 6.92 (d, 2H, J = 8.8 Hz), 6.81 (d, 2H, J = 8.8 Hz), 4.45 (t, 4H, J = 4.8 Hz),
4.13 (s, 4H), 3.84 (t, 4H, J = 4.8 Hz), 3.53ꢀ3.70 (m, 28H), 3.45 (t, 4H, J = 5.4 Hz), 3.29ꢀ
3.35 (m, 8H), 3.07 (s, 6H), 2.81 (t, 4H, J = 7.4 Hz), 2.44 (t, 4H, J = 7.4 Hz); HRMS
calcd. for C74H99N8O18 (M + H)+: 1387.7077, found 1387.7093.
2,2'ꢀ((E)ꢀEthaneꢀ1,2ꢀdiylbis((2ꢀhydroxyꢀ5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ
(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ19ꢀoxoꢀ3,6,9,12,15ꢀpentaoxaꢀ18ꢀ
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azahenicosanꢀ21ꢀyl)benzyl)azanediyl))diacetic acid (7d). Compound 7d (yield: 22 mg,
43.8%) was prepared from 21d (60 mg, 0.034 mmol) and 1 mL TFA, following the same
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procedure described for compound 7a. HNMR (400 MHz, MeOD) δ: 7.96 (d, 2H, J =
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2.0Hz), 7.82 (dd, 2H, J = 2.4Hz, J = 8.8Hz), 7.39 (d, 4H, J = 8.4Hz), 7.14 (d, 4H, J =
8.4Hz), 6.77ꢀ6.88 (m, 8H), 6.67 (d, 2H, J = 8.8 Hz), 6.50 (d, 2H, J = 8.8 Hz), 4.15 (t, 4H,
J = 4.6 Hz), 3.87 (s, 4H), 3.53 (t, 4H, J = 4.4 Hz), 3.44 (s, 4H), 3.20ꢀ3.36 (m, 32H), 3.09ꢀ
3.14 (m, 8H), 2.98 (t, 4H, J = 5.4 Hz), 2.76 (s, 6H), 2.49 (t, 4H, J = 7.4 Hz), 2.13 (t, 4H, J
= 7.4 Hz); HRMS calcd. for C78H104N8O20 (M + H)+: 1475.7602, found 1475.7187.
2,2'ꢀ((E)ꢀEthaneꢀ1,2ꢀdiylbis((2ꢀhydroxyꢀ5ꢀ(1ꢀ((5ꢀ((E)ꢀ4ꢀ
(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ22ꢀoxoꢀ3,6,9,12,15,18ꢀhexaoxaꢀ21ꢀ
azatetracosanꢀ24ꢀyl)benzyl)azanediyl))diacetic acid (7e). Compound 7e (yield: 18.9
mg, 37.8%) was prepared from 21e (60 mg, 0.032 mmol) and 1 mL TFA following the
same procedure described for compound 7a. 1HNMR (400 MHz, MeOD) δ: 8.28 (d, 2H,
J = 2.0Hz), 8.11 (dd, 2H, J = 2.4Hz, J = 8.8Hz), 7.72 (d, 4H, J = 8.4Hz), 7.46 (d, 4H, J =
8.4Hz), 7.11ꢀ7.25 (m, 8H), 6.96 (d, 2H, J = 8.8 Hz), 6.82 (d, 2H, J = 8.8 Hz), 4.47 (t, 4H,
J = 4.8 Hz), 4.16 (s, 4H), 3.86 (t, 4H, J = 4.8 Hz), 3.55ꢀ3.73 (m, 44H), 3.45 (t, 4H, J = 5.4
Hz), 3.31ꢀ3.37 (m, 8H), 3.08 (s, 6H), 2.82 (t, 4H, J = 7.4 Hz), 2.45 (t, 4H, J = 7.4 Hz);
HRMS calcd. for C82H115N8O22 (M + H)+: 1563.8126, found 1563.8157.
(E)ꢀ3ꢀ(3ꢀ(((Carboxymethyl)(2ꢀ((carboxymethyl)(2ꢀhydroxyꢀ5ꢀ(1ꢀ((5ꢀ(4ꢀ
(methylamino)styryl)pyridinꢀ2ꢀyl)oxy)ꢀ13ꢀoxoꢀ3,6,9ꢀtrioxaꢀ12ꢀazapentadecanꢀ15ꢀ
yl)benzyl)amino)ethyl)amino)methyl)ꢀ4ꢀhydroxyphenyl)propanoic
acid
(8).
Compound 8 (yield: 9.5 mg, 37.1%) was prepared from 22 (31 mg, 0.028 mmol) and 1
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mL TFA, following the same procedure described for compound 7a. 1HNMR (400 MHz,
MeOD) δ: 8.26 (d, 1H, J = 2.0Hz), 8.11 (dd, 1H, J = 2.4Hz, J = 8.8Hz), 7.72 (d, 2H, J =
8.4 Hz), 7.40 (d, 2H, J = 8.4Hz), 7.10ꢀ7.21 (m, 6H), 6.80ꢀ6.91 (m, 3H), 4.47 (t, 2H, J =
4.4 Hz), 4.16 (s, 2H), 4.11 (s, 2H), 3.86 (t, 2H, J = 4.4Hz), 3.56ꢀ3.71 (m, 14H), 3.48 (t,
2H, J = 5.0Hz), 3.37 (s, 4H), 3.08 (s, 3H), 2.83 (t, 4H, J = 7.4Hz), 2.56 (t, 2H, J = 7.6Hz),
2.46 (t, 2H, J = 7.4Hz); HRMS calcd. for C48H62N5O13 (M + H)+: 916.4344, found
916.4436.
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[natGa]7a. A solution of GaCl3 (1.13 M) in 8 ꢁL 0.1 N HCl was added to the
compound 7a (10 mg, 7.7 ꢁmol) in 1 mL H2O. The mixture was stirred at rt for 1 h. The
final complex was determined using LCꢀMS. HRMS calcd. for C66H80GaN8O14 (M + H)+:
1277.5050, found 1277.5081. The resulting aqueous solution of the natGa complex was
further diluted and used in the in vitro binding affinity study without further processing.
[natGa]7b. Compound [natGa]7b was prepared from GaCl3 (1.13 M) in 9 ꢁL 0.1 N
HCl and 7b (11 mg, 8.1 ꢁmol) following the same procedure described for compound
[natGa]7a. HRMS calcd. for C70H88GaN8O16 (M + H)+: 1365.5574, found 1365.5779.
[natGa]7c. Compound [natGa]7c was prepared from GaCl3 (1.13 M) in 8 ꢁL 0.1 N
HCl and 7c (10 mg, 7.2 ꢁmol) following the same procedure described for compound
[natGa]7c. HRMS. calcd for C74H96GaN8O18 (M + H)+: 1453.6098, found 1453.6178.
[natGa]7d. Compound [natGa]7b was prepared from GaCl3 (1.13 M) in 7 ꢁL 0.1 N
HCl and 7d (10 mg, 6.8 ꢁmol) following the same procedure described for compound
[natGa]7a. HRMS calcd. for C78H104GaN8O20 (M + H)+: 1541.6623, found 1541.6689.
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[natGa]7e. Compound [natGa]7e was prepared from GaCl3 (1.13 M) in 7 ꢁL 0.1 N
HCl and 7e (10 mg, 6.4 ꢁmol) following the same procedure described for compound
[natGa]7a. HRMS calcd. for C82H112GaN8O22 (M + H)+: 1629.7147, found 1629.7166.
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[natGa]8. Compound [natGa]8 was prepared from GaCl3 (1.13 M) in 8 ꢁL 0.1 N
HCl and 8 (7 mg, 7.6 ꢁmol) following the same procedure described for compound
[natGa]7a. HRMS calcd. for C48H59GaN5O13 (M + H)+: 982.3365, found 982.3417.
Radiosynthesis of [68Ga] 7aꢀe and [68Ga]8, and stability test. Galliumꢀ68 was
obtained from a 68Ge/68Ga generator (iTG, Germany). A stock solution of ligand (7aꢀe
and 8, 1 mg in 1 mL 0.1 N NaOAc) was prepared and used for the radiolabelling studies.
68Ga labeling was performed in aq. NaOAc buffer (15 µL, 2.0 N) by combining the
ligand solution (13 µL) and 68Ga solution (500 µL in 0.05 N HCl, 2.5ꢀ3.1 mCi). The final
pH of the mixture was 4.10. Radiolabeling yields were 93ꢀ98% after maintaining at rt for
5 min. Radiochemical yields were determined by two TLC systems: A: ITLCꢀSG
(Agilent) developed with 0.1 N citric acid; B: ITLCꢀSG plates developed with a solvent
mixture (H2O/EtOH/Pyridine = 4/2/1). The HPLC system was developed using an
Agilent ECꢀC18 column (A: 0.1% TFA in H2O, B: ACN; 0ꢀ6 min 100ꢀ0% A), 2 mL/min.
The stability of [68Ga]7aꢀe and [68Ga]8 was determined by incubating the
preparation in PBS (pH = 7.4, 3 h at rt) and human plasma (3 h at 37 °C). 68Ga conjugates
and free 68Ga levels were determined using radio TLC and HPLC as described above.
In vitro binding studies using Aβꢀaggregates in the AD brain tissue
homogenates. [125I]IMPY with 2200 Ci/mmol specific activity and more than 98%
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