Metal-chelating benzothiazole multifunctional compounds for the modulation and 64Cu PET imaging of Aβ aggregation

Article abstract of DOI:10.1039/d0sc02641g

While Alzheimer's Disease (AD) is the most common neurodegenerative disease, there is still a dearth of efficient therapeutic and diagnostic agents for this disorder. Reported herein are a series of new multifunctional compounds (MFCs) with appreciable affinity for amyloid aggregates that can be potentially used for both the modulation of Aβ aggregation and its toxicity, as well as positron emission tomography (PET) imaging of Aβ aggregates. Firstly, among the six compounds tested HYR-16 is shown to be capable to reroute the toxic Cu-mediated Aβ oligomerization into the formation of less toxic amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS) caused by Cu2+ ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to PET imaging agents by pre-chelation with the 64Cu radioisotope, and the Cu complexes of HYR-4 and HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo. Importantly, the 64Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ± 0.04 percentID per g. Overall, by evaluating the various properties of these MFCs valuable structure-activity relationships were obtained that should aid the design of improved therapeutic and diagnostic agents for AD. This journal is

Full text of DOI:10.1039/d0sc02641g

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Metal-chelating benzothiazole multifunctional
6
4
compounds for the modulation and Cu PET
Cite this: DOI: 10.1039/d0sc02641g
imaging of Ab aggregation†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
b
a
b
Yiran Huang,
Hong-Jun Cho, Nilantha Bandara,
Liang Sun,
Diana Tran,
b
ac
Buck E. Rogers* and Liviu M. Mirica
*
While Alzheimer's Disease (AD) is the most common neurodegenerative disease, there is still a dearth of
efficient therapeutic and diagnostic agents for this disorder. Reported herein are a series of new
multifunctional compounds (MFCs) with appreciable affinity for amyloid aggregates that can be
potentially used for both the modulation of Ab aggregation and its toxicity, as well as positron emission
tomography (PET) imaging of Ab aggregates. Firstly, among the six compounds tested HYR-16 is shown
to be capable to reroute the toxic Cu-mediated Ab oligomerization into the formation of less toxic
amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS)
2+
caused by Cu ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to
64
PET imaging agents by pre-chelation with the Cu radioisotope, and the Cu complexes of HYR-4 and
HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo.
64
Received 12th May 2020
Accepted 7th July 2020
Importantly, the Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ꢀ 0.04 %
ID per g. Overall, by evaluating the various properties of these MFCs valuable structure–activity
DOI: 10.1039/d0sc02641g
relationships were obtained that should aid the design of improved therapeutic and diagnostic agents for
AD.
rsc.li/chemical-science
1
8,20
21
synaptic receptors
affect neurotransmission,
In addition, the amyloid deposits contain uncommonly high
that inuence intracellular systems and
Introduction
22,23
leading to neurodegeneration.
Alzheimer's disease (AD) is the most common neurodegenera-
2
+
2+
2+ 24,25
tive disease, associated with loss of memory and cognitive concentrations of metal ions such as Fe , Cu and Zn ,
decline. An estimated 5.8 million Americans of all ages are and it has been found that these metal ions promote the
living with Alzheimer's dementia currently. The presence of formation of neurotoxic Ab aggregates.
1
2
26–29
Cu and Fe ions can
amyloid plaques and neurobrillary tangles in the brain is the also cause the formation of reactive oxygen species (ROS), which
3
25,30–32
hallmark of AD. Amyloid b (Ab) peptides, the main component exacerbates Ab toxicity.
Previously, we have reported that
2
+
4–7
of amyloid plaques, are formed from the cleavage of amyloid Cu ions can slow down Ab brillization and stabilize Ab
33,34
precursor protein (APP) by b- and g-secretases. The main allo- oligomers,
and thus small molecules that can inhibit the
forms of Ab are Ab40 and Ab , containing 40 and 42 amino interaction between metal ions and Ab peptides, can be used as
42
8
35–38
acids, respectively. Even though Ab40 is present in the deposits potential therapeutic compounds for AD.
in larger amounts, Ab exhibits higher neurotoxicity and
aggregates more easily.
Moreover, the development of novel diagnostic agents is
In the past two decades, soluble Ab essential for the prevention and treatment of AD. Recently,
4
–12
2
9
oligomers have been found to be the most toxic form among all several positron emission tomography (PET) compounds have
13–19
Ab species
through their interactions with membrane and been approved by FDA and can be used to visualize amyloid
plaques in AD patients. However, these radiolabeled agents are
1
1
18
employing short-lived radionuclides, such as C and F (t1/2
¼
39–45
20.4 min and 109.8 min,
respectively), thus limiting their
a
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. widespread use. Thus, the development of longer-lived radio-
Mathews Avenue, Urbana, Illinois 61801, USA. E-mail: mirica@illinois.edu
labeled compounds is essential for further expanding the use of
PET imaging in healthcare, and diagnostic agents employing
b
Department of Radiation Oncology, Washington University School of Medicine, St.
Louis, Missouri 63108, USA
64
+
longer-lived radionuclides such as Cu (t1/2 ¼ 12.7 h, b ¼ 17%,
c
Hope Center for Neurological Disorders, Washington University School of Medicine,
ꢁ
b
¼ 39%, EC ¼ 43%, E
¼ 0.656 MeV) are viewed as optimal
max
6,47
St. Louis, MO 63110, USA
4
PET imaging agents.
†
Electronic supplementary information (ESI) available: Experimental section and
Fig. S1–S8. See DOI: 10.1039/d0sc02641g
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We have previously reported rst-generation multifunctional
compounds (MFCs) that contain Ab binding and metal
Results and discussion
Design, synthesis and characterization of MFCs
33,48
chelating fragments
and these MFCs were found to have the
ability to modulate the aggregation of Ab42 species, although Following up on our previous results, we set out to develop
some led to the formation of neurotoxic soluble Ab42 oligo- second-generation MFCs aimed to better alleviate the metal-
3
3,48
mers.
Moreover, some MFCs could be converted into PET induced Ab toxicity, while also obtaining valuable structure–
6
4
imaging agents through pre-chelation with Cu and have with activity relationships (SAR) that should give us insight into the
MFCs, PET imaging agents could be obtained for the diagnosis design of improved therapeutic and imaging agents for AD.
of Alzheimer's disease.
49,50
Herein, we report a series of second-
Previously, 2-aryl-benzothiazole derivatives have been found
generation multifunctional compounds (MFCs) containing to have appreciable Ab binding affinity and uorescence prop-
33,48
metal-binding and Ab-interacting fragments that also exhibit erties,
and thus we continued to use such molecular
additional key properties (Fig. 1). In contrast to the rst- frameworks. Inspired by the structure of Pittsburgh compound
generation MFCs, which were shown to promote the forma- B (PiB, Fig. 1), a widely-used amyloid-binding compound with
33,48
39
tion of neurotoxic oligomeric Ab species,
these MFCs are high Ab binding affinity, we introduced a hydroxyl group in
capable of re-routing the neurotoxic metal-stabilized Ab oligo- the 6-position of the benzothiazole aromatic ring (Fig. 1). In
mers into less toxic aggregates, while also decreasing the addition, dimethylamino and monomethylamino groups were
0
formation of ROS. Moreover, these MFCs can be easily con- introduced at the 4 position of the phenyl ring, since such
6
4
verted into PET imaging agents by chelation with the Cu functional groups are present in many Ab binding compounds
radionuclide, and ex vivo labeling studies using AD mouse brain such as Thioavin T (ThT) and Florbetapir.
43,51
Then, we have
sections reveal that the MFCs and their Cu complexes can introduced a 1,4-dimethyl-1,4,7-triazacyclononane (tacn) metal-
clearly label the amyloid plaques. In addition, biodistribution chelating group attached to the benzothiazole aromatic ring in
6
4
studies show that the Cu-radiolabeled compounds cross the order to generate a MFC that can modulate the metal–Ab
blood–brain barrier (BBB) efficiently and thus should be able to interactions. Importantly, previous studies have shown that the
act as potential therapeutic or imaging agents in vivo. Finally, tacn-phenolate metal-chelating fragment can bind more tightly
52–54
structure–activity relationship (SAR) studies suggest that the to Cu versus other metal ions such as Zn and Fe.
Accord-
presence of a monomethylamine group leads to increased ingly, HYR-1 and HYR-4 were developed, and we have also
0
specicity for binding to the Ab aggregates, while the intro- designed HYR-14 and HYR-16 as 3 -pyridyl analogues in order to
duction of a pyridyl group is essential for modulating the probe the effect of a hydrogen-bond acceptor pyridyl group vs.
43,51
neurotoxicity of metal–Ab species. Most importantly, reposi- a phenyl group.
Moreover, it was previously found that a PiB
tioning of the hydroxyl group and the metal-chelating azama- derivative with a hydroxyl group in the 4-position of benzo-
crocycle on the benzothiazole ring in HYR-17 has a dramatic thiazole framework performed similarly as PiB in Ab binding
6
4
55
effect on improving the brain uptake of the corresponding Cu and bio-distribution studies, and thus we have designed HYR-
complex, which could be a useful design approach for the 17, which contains a 4-hydroxyl substituent and has the tacn
development of improved PET imaging agents.
azamacrocycle connected to the 5 position of the benzothiazole
ring (Fig. 1). Finally, the MFC HYR-18 that contains two 2-aryl-
benzothiazole fragments attached to one tacn azamacrocycle
was also synthesized, in order to probe whether additional Ab
binding fragments will improve the affinity for Ab aggregates.
The synthesis of the MFCs HYR-1, -4, -14, -16, -17 and -18
follows a stepwise sequence of steps that typically includes an
oxidative
cyclization
step
between
a
2-amino-
methoxybenzenethiol derivative and a benzaldehyde or ben-
zoic acid derivative to generate the 2-aryl-benzothiazole frag-
ments (Scheme 1). In cases where the 2-amino-methoxy-
benzothiazole precursor was readily available, hydrolysis
under
basic
conditions
afforded
the
2-amino-
methoxybenzenethiol starting materials, while for the pyridyl
derivatives 2-amino-5-(triuoromethyl)pyridine was employed
in the oxidative cyclization reaction. Reduction of the nitro
group to the aniline derivative using tin(II) chloride (if needed)
and subsequent N-monomethylation using paraformaldehyde
and sodium borohydride or N-dimethylation using para-
formaldehyde and sodium cyanoborohydride generated the 2-
Fig. 1 Structures of Pittsburgh compound B (PiB) and the developed
multifunctional compounds (MFCs) HYR-1, -4, -14, -16, -17 and -18.
The metal-binding and Ab-interacting fragments are shown in blue
0
(4 -aniline-aryl)-benzothiazole derivatives. Deprotection of the
methoxy group using boron tribromide afforded the hydroxyl-
and red, respectively. The numbering of the different positions on the benzothiazole derivatives, while the last synthetic step for all
aromatic rings is shown only for the first structure.
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Scheme 1 Synthesis of the investigated MFCs HYR-1, -4, -14, -16, -17 and -18. The metal-binding and Ab-interacting fragments are shown in
ꢂ
blue and red, respectively. Reagents and conditions: (1a) KOH, H
2
O, ethylene glycol, reflux, 48 h; (1b) DMSO, 170 C, 30 min; (1c) BBr
3
, DCM, rt,
O)
TACN,
, DCM, rt, 24 h; (3d)
, DCM, rt, 24 h; (3g)
, DCM, rt, 24 h; (4d)
ꢂ
2
4 h; (1d) Me
2
HTACN, (CH
2
O)
n
, MeCN, reflux, 24 h; (2a) DMSO, 125 C, overnight; (2b) SnCl
2
, EtOH, conc. HCl, reflux, 3 h; (2c) (i) (CH
O) , MeCN, reflux, 24 h; (2f) MeH
CN, acetic acid, rt, overnight; (3c) BBr
2
n
,
ꢂ
NaOMe, MeOH, reflux, 2 h; (ii) NaBH
CH O)
HTACN, (CH
4
, 0 C to rt, 1 h; (2d) BBr
3
, DCM, rt, 24 h; (2e) Me
O) , NaBH
, NaOMe, MeOH, reflux, 2 h; (ii) NaBH
2
HTACN, (CH
2
n
2
ꢂ
(
2
n
, MeCN, reflux, 24 h; (3a) NaOH (1 M), 90 C, 3 h; (3b) (CH
2
n
3
3
Me
Me
Me
2
2
2
2
O)
O)
O)
n
, MeCN, reflux, 24 h; (3e) (i) (CH
, MeCN, reflux, 24 h; (4a) KOH, H
, MeCN, reflux, 24 h.
O)
2 n
4
, reflux, 1 h; (3f) BBr
O, ethylene glycol, reflux, 48 h; (4b) DMSO, 125 C, 30 min; (4c) BBr
3
ꢂ
HTACN, (CH
HTACN, (CH
2
n
2
3
2
n
compounds is the Mannich reaction with paraformaldehyde the other MFCs it was difficult to obtain reproducible K
i
values,
and 2,4-dimethyl-1,4,7-triazacyclononane under reux to likely due to the larger structural differences between these
generate the targeted MFCs (Scheme 1).
MFCs and ThT and thus the inability to compete with ThT for
ThT uorescence competition assays
In order to measure the binding affinity of MFCs toward
amyloid brils, ThT uorescence competition assays were per-
formed. The Ab40 peptide was used in these experiments, since
56,57
it is known that Ab40 forms well-dened amyloid brils.
Excitingly, HYR-1 and HYR-4 exhibit nanomolar affinities for
the Ab40 brils with K values of 11 ꢀ 7 nM and 85 ꢀ 9 nM,
i
respectively, indicating that the MFCs can replace ThT effi-
Fig. 2 ThT fluorescence competition assays for MFCs HYR-1 and
ciently and bind tightly to the Ab40 brils (Fig. 2). However, for HYR-4 with ThT-bound Ab40 fibrils ([Ab] ¼ 5 mM, [ThT] ¼ 2 mM).
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the same binding site(s) on the amyloid brils and that not lead amyloid precursor protein (APP) and presenilin 1, develop
to an appreciable decrease in ThT uorescence. In addition, we amyloid plaque deposits at an younger age, and show progres-
consider that probing the affinity of the MFCs toward native sive cognitive impairment similar to that found in AD in
58
amyloid plaques by performing ex vivo binding studies with humans.
transgenic AD mice brain sections should provide more physi-
Interestingly, incubation of the 5ꢃFAD mouse brain sections
ologically relevant results and also rule out any non-specic with the different MFCs for 1 hour reveals signicant uores-
binding (see below).
cent staining of the amyloid plaques, as conrmed by co-
staining with Congo Red (CR), a well-known amyloid-binding

uorescent dye (Fig. 3). Among the MFCs, HYR-1, -4, -17, and
Fluorescence imaging of amyloid plaques in 5ꢃFAD mice
-18 show signicant amyloid staining at low concentration (25
mM), suggesting an appreciable Ab binding affinity. The most
specic amyloid-binding MFCs are HYR-4 and HYR-18 (and
HYR-17 to a slightly lesser extent), which bind to the dense core
of the amyloid plaques and thus show the best colocalization
with CR. By contrast, HYR-1 seems to label other endogenous
proteins, both in AD and WT brain sections (Fig. 3 and S5,†
respectively). Also, HYR-1 can label other regions in wild type
mice brain section (Fig. S5†), which means its specicity is
extremely low. By comparison, the pyridyl-containing MFCs
HYR-14 and HYR-16 exhibit appreciable amyloid plaque stain-
ing only at high concentrations (250–500 mM), suggesting that
their Ab binding affinity is somewhat reduced and thus these
two compounds were not employed in radiolabeling studies
brain sections
Brain sections collected from 8 month-old 5ꢃFAD transgenic
mice were employed in these ex vivo Ab binding studies. The
transgenic 5ꢃFAD mice overexpress mutant forms of the
(
see below).
2
+
We have also probed the ability of the Cu complexes of
HYR-4, -17 and -18 to label the amyloid plaques in 5ꢃFAD mice
brain sections (Fig. 4). In this case, higher ligand to Congo Red
2
+
ratios were used since the Cu ions lead to some uorescence
2
+
quenching for our MFCs (Fig. S5 and S6†). However, the Cu
complexes of HYR-4, -17 and -18 can still label the amyloid
plaques efficiently (Fig. 3), with the complexes of HYR-4 and -18
Fig.
sections incubated with multifunctional compounds HYR-1, -4, -14,
16, -17 and -18 (left panels), Congo Red (CR, middle panels), and Fig.
merged images (right panels, with the Pearson's coefficient R shown). sections incubated with Cu complexes of HYR-4, -17 and -18 (left
Scale bar: 100 mm. The concentration of MFC and MFC : Congo Red panels), Congo Red (middle panels), and merged images (right panels).
3
Fluorescence microscopy images of 5ꢃFAD mice brain
-
4
Fluorescence microscopy images of 5ꢃFAD mice brain
2+
ratio used are listed at the top.
Scale bar: 100 mm.
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analysed by native gel electrophoresis/western blot analysis and
transmission electron microscopy (TEM, Fig. 6). The former
analysis method reveals the presence of smaller, soluble Ab
aggregates and their MW distribution, while the latter method
Fig.
5
Fluorescence microscopy images of 5ꢃFAD mice brain
sections incubated with MFCs HYR-4, -17 and -18 or their Cu(II)
complexes (left panels), CF594-6E10 antibody (middle panels), and
merged images (right panels). Scale bar: 100 mm.
showing higher specicity compared with the Cu-HYR-17
complex. Finally, the uorescently labeled CF594-6E10 antibody
–
which binds to a wide range of Ab species, was employed to
2
+
conrm that our MFCs and their Cu complexes are able to
specically label the Ab42 species (Fig. 5). It is important to note
that we have previously determined the stability constants for
the Cu complex of another 1,4,7-triazacyclononane-phenolate
ligand, which was shown to bind Cu very tightly.
2
+
2
+
49,50
Since the
structural changes for the different MFCs described herein are
remote from the Cu-binding site, we envision that the stability
2
+
constants for the corresponding Cu complexes will not vary
signicantly.
2+
2+
Fig. 6 Effects of the compounds on Cu -free and Cu -induced
Ab42 aggregation. (a) Scheme of the inhibition experiment: freshly
Modulation of metal-free and metal-induced Ab aggregation
2+
prepared Ab42 (25 mM) in the presence or absence of Cu (25 mM) and
ꢂ
The ability of these MFCs to modulate the aggregation of Ab42 with or without MFCs (25 mM) was incubated at 37 C for 24 h with
2
+
constant agitation. (b) Native gel/western blot analysis of the resulting
Ab42 species using the 6E10 anti-Ab antibody. (c) Representative TEM
images of the Ab42 aggregates upon incubation with or without MFCs
was then explored, both in the absence or presence of Cu ions.
The Ab42 peptide was used since it was shown to form neuro-
1
2,13,18
toxic soluble Ab42 oligomers.
Freshly prepared monomeric
(
scale bar, 200 nm). (d) Representative TEM images of the Ab42
2
+
2+
Ab42 solutions were treated with MFCs, Cu , or both, and
aggregates upon incubation with Cu and with or without MFCs
ꢂ
incubated for 24 hours at 37 C, and the resulting samples were (scale bar, 200 nm).
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provides the characterization of the larger, insoluble Ab aggre- concentration, and thus these three MFCs were employed in
3
3,34
gates that cannot penetrate the gel, and thus allowing us to Ab -induced cytotoxicity studies. As we have shown before,
4
2
2
+
visualize all Ab42 aggregates of various types and sizes.
the presence of both Cu ions and Ab42 leads to pronounced
While the aggregation of Ab42 in the absence of Cu ions cell toxicity (ꢄ50% cell viability), likely due to the formation of
leads to well-dened Ab42 brils – as conrmed by TEM neurotoxic Ab42 oligomers (Fig. 8). Interestingly, HYR-16 can
2
+
2
+
(
Fig. 6c), the Ab42 aggregation in the presence of Cu ions signicantly alleviate the toxicity of Cu-stabilized Ab42 oligo-
generates a limited amount of Ab brils (Fig. 6d), and native gel/ mers and increase the cell viability up to 80% vs. a 1% DMSO
2
+
western blotting shows that the aggregation of Ab42 with Cu
ions yields mostly soluble Ab42 oligomers of various sizes accelerate the aggregation of toxic Ab42 oligomers into nontoxic
Fig. 6b). These results are consistent with our previous studies Ab42 aggregates. By comparison, HYR-4 and HYR-14 do not
control, which is consistent with in vitro results that HYR-16 can
(
2
+
which suggest that Cu can stabilize the soluble Ab oligomers seem to signicantly reduce the neurotoxicity of the Cu-Ab42
4
2
33,59
and slow down the Ab42 aggregation.
species (Fig. 8), suggesting that the 2-monomethylamino-
All MFCs HYR-1, -4, -14, -16 and -17 did not seem to signif- pyridyl fragment found in HYR-16 is needed for the efficient
2
+
icantly inhibit the Ab42 aggregation in the absence of Cu , even alleviation of Ab42 oligomer neurotoxicity.
though some morphological changes were observed for the Ab42

brils (Fig. 6b and c). Interestingly, the presence of HYR-16 had
Antioxidant properties
2
+
a dramatic effect on the Cu -mediated oligomerization of Ab42
and promoted the formation of larger Ab42 aggregates, as
observed by TEM (Fig. 6d). Moreover, native gel/western blot-
ting analysis reveals the presence of large, insoluble Ab42
aggregates at the top of the gel, which were not observed for the
Ab42 aggregation in presence of only Cu ions. Thus, HYR-16 is
expected to control the neurotoxicity of Ab42 species by accel-
erating the aggregation of toxic Ab42 oligomers into nontoxic
Ab42 aggregates. By comparison, the other MFCs do not show
2
+
Several previous reports have shown that the Cu ions can
interact with various Ab species and lead to formation of reac-
tive oxygen species (ROS) such as H O and hydroxyl radicals
2
2
31,60
(OHc).
In this regard, the antioxidant capacity of the pyridine
derivatives HYR-14 and HYR-16 – the least toxic MFCs investi-
gated herein, was rst evaluated via the Trolox equivalent
antioxidant capacity (TEAC) assay. Both HYR-14 and HYR-16
2
+
61
showed better performance on scavenging the free radical
+
0
ABTS c (ABTS ¼ 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid)) than glutathione – a well-known antioxidant, at all the
selected time points (1–15 min, Fig. 9a), with HYR-16 showing
an antioxidant capacity similar to Trolox.
2
+
a dramatic effect on Cu -mediated Ab aggregation (Fig. 6b,
le panels).
4
2
In addition, the ability of HYR-14 and HYR-16 to quench the
Cu-induced hydroxyl radical (OHc) generation was measured by
Cytotoxicity of MFCs and modulation of Ab42 neurotoxicity
The neurotoxicity of the MFCs and their ability to alleviate the
Cu-induced Ab42 toxicity was performed using mouse neuro-
blastoma (N2a) cells. First, we examined the toxicity of all MFCs
at various concentrations ranging from 2 to 20 mM (Fig. 7).
Among the different MFCs, HYR-4, -14 and -16 exhibit no
appreciable cell toxicity (>80% cell viability) up to 10 mM
concentration, with HYR-16 showing no cell toxicity up to 20 mM
62
using coumarin-3-carboxylic acid (CCA) antioxidant assay. In
2+
+
this assay, the Cu ions are reduced by ascorbic acid to Cu ions
that then react with O to produce OHc. The non-uorescent
2
2+
Fig. 8 The effect of MFCs on Cu -induced Ab42 cytotoxicity, re-
ported vs. a 1% DMSO control. N2a cells were treated with Ab42
(
20 mM), CuCl
2
(20 mM), and MFCs (10 mM) and incubated for 40 h at
ꢂ
Fig. 7 Toxicity of MFCs at various concentrations in N2a cells, re- 37 C. The error bars indicate the standard deviations based on at least
ported vs. a 1% DMSO control. The error bars indicate the standard three samples per group, and the statistical analysis was evaluated
deviations based on at least five samples per group.
according to one-way ANOVA (****p < 0.0001).
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CCA reacts with OHc to produce the uorescent 7- Table 1 Molecular weights for MFCs and log Doct values for the cor-
64
responding Cu-labeled complexes
hydroxycoumarin-3-carboxylic acid (CCA-OH), and CCA-OH
formation can be monitored to evaluate the efficacy of ligand
binding to Cu and inhibition of OHc generation. At 80 mM, both
ꢁ1
Ligand
MW (g mol
)
log Doct
2
+
of HYR-14 and HYR-16 show a strong Cu binding affinity HYR-1
439.6
425.6
440.6
426.6
439.6
679.9
1.08 ꢀ 0.11
1.29 ꢀ 0.19
0.58 ꢀ 0.05
0.56 ꢀ 0.17
1.30 ꢀ 0.15
1.09 ꢀ 0.11
(
corresponding to a 2 : 1 MFC : Cu ratio) and limit the genera- HYR-4
HYR-14
tion of OHc (Fig. 9b and c), while HYR-16 is able to limit the
HYR-16
HYR-17
HYR-18
generation of OHc even at 40 mM (corresponding to a 1 : 1
MFC : Cu ratio). Overall, these studies reveal that HYR-16 is the
most potent anti-oxidant among the investigated MFCs, and
along with its ability to efficiently destabilize the neurotoxic
soluble Ab42 oligomers strongly suggest that HYR-16 may
potentially exhibit therapeutic properties in animal studies.
Radiolabeling and log Doct value determination
One crucial factor for developing imaging agents for neurode-
generative diseases is that they should be able to effectively
cross the blood–brain barrier (BBB). To determine the hydro-
phobicity of the radiolabeled compounds, the octanol/PBS
partition coefficient values log Doct were determined for the
6
4
Cu complexes of HYR-1, -4, -14, -16, -17 and -18. The obtained
6
4
log Doct values for the Cu-radiolabeled complexes HYR-1, -4,
17 and -18 are in the range of 1.08–1.30 (Table 1), which
supports their potential ability to cross the BBB, since log D
-
63
values between 0.9 and 2.5 are considered optimal. In
6
4
contrast, the Cu complexes of HYR-14 and -16 exhibit log Doct
values of ꢄ0.6, suggesting that 2-pyridyl-benzothiazole deriva-
tives may be too hydrophilic to cross the BBB. Thus, only the
MFCs HYR-1, -4, -17 and -18 were employed in the subsequent
radiochemistry studies.
Ex vivo autoradiography studies
Ex vivo autoradiography studies using brain sections of trans-
genic 5ꢃFAD mice were also performed to determine the
Fig. 9 (a) The antioxidant activity of Trolox, glutathione, HYR-14, and
HYR-16 (25–100 mM) at 1, 3, 6, and 15 minutes assessed by TEAC assay.
The TEAC values of glutathione, HYR-14, and HYR-16 were normalized
to the Trolox activity. The inhibitory activity of (b) HYR-14 and (c) HYR-
1
6 toward Cu-mediated hydroxyl radical generation, evaluated via the
] ¼ 40 mM;
CCA antioxidant assay. Conditions: [CCA] ¼ 100 mM; [CuSO
4
[
3
ascorbic acid] ¼ 400 mM; [HYR-14/HYR-16] ¼ 0–160 mM; lex
¼
95 nm; lex ¼ 450 nm. All experiments were performed in triplicate
and the error bars indicate the standard deviation for each Fig. 10 Autoradiography images of 5ꢃFAD mice brain sections with
measurement.
1
the absence and presence with a known Ab blocking agent (B ).
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64
Fig. 12 Various organs uptake (%ID per g) of Cu-labeled HYR-4 and
HYR-17 from the biodistribution studies in CD-1 mice, at 2, 60, and
240 min post injection.
Fig. 11 Brain uptake (% injected dose per gram, %ID per g) results from
the biodistribution studies in CD-1 mice, at 2, 60, and 240 min post
injection.
Biodistribution studies
Encouraged by the promising in vitro radiolabeling and
amyloid-binding studies, in vivo biodistribution experiments
6
4
6
4
were performed to probe the uptake of Cu-labelled HYR-4
and -17 complexes using normal CD-1 mice. The retention
specic binding to the amyloid plaques of the Cu-labeled
HYR-1, -4, -17 and -18. The brain sections were stained,
washed, and radioimaged as described in the experimental
section. When compared with WT brain sections that show
6
4
and accumulation of the Cu-labeled complexes in selected
organs was evaluated at 2, 60, and 240 minutes aer tracer
6
4
6
4
administration. Excitingly, Cu-HYR-17 shows an appre-
ciable brain uptake of 0.99 ꢀ 0.04 % injected dose per gram
a limited background intensity (Fig. 10, rst row), the Cu-
labeled complexes of HYR-4, -17 and -18 show an increased
autoradiography intensity for the 5ꢃFAD mouse brain sections
(
0
%ID per g) at 2 min post injection, which drops to 0.20 ꢀ
6
4
.01 %ID per g at 60 min (Fig. 11 and Table 2). By compar-
(
Fig. 10, second row). The Cu-labeled HYR-1 also exhibits non-
6
4
ison, Cu-HYR-4 shows a relatively low brain uptake of 0.16
specic binding as appreciable autoradiography intensity is
observed for WT brain sections, which is consistent with the
brain section uorescent imaging studies described above.
Finally, the specic binding to amyloid plaques of the Cu-
labeled MFCs was conrmed by blocking with the non-
radioactive blocking agent B (Fig. S6†), which led to a mark-
edly decreased autoradiography intensity (Fig. 10, third row).
Overall, these autoradiography results strongly suggest that the
6
4
ꢀ
0.02 %ID per g at 2 min post injection, although Cu-HYR-
6
4
4
shows a similar blood uptake to that of Cu-HYR-17
6
4
(
Fig. 12), and suggesting that the structure of HYR-17 con-
taining the 4-hydroxyl substituent and tacn azamacrocycle
connected to the 5 position of the benzothiazole ring should
lead to improved brain uptake properties for the corre-
1
6
4
sponding
Cu-labeled complexes. Overall, these bio-
6
4
6
4
distribution studies strongly suggest that the Cu-HYR-17
complex can efficiently cross the BBB and thus could serve as
a PET imaging agent for the detection of Ab aggregates in
vivo. Importantly, the rapid clearance from the brain of WT
mice suggest that these radiolabeled MFCs do not release
Cu-labeled MFCs HYR-4, -17 and -18 have the necessary
amyloid-binding specicity to be used as imaging agents in vivo.
The MFC HYR-18, the bis-(2-phenylbenzothiazole) analogue of
HYR-4, exhibits a slightly lower log Doct value than HYR-4, and it
was also perceived to have a too large MW for an efficient brain
uptake. Since HYR-4 already exhibits a promising log Doct value
and specic binding to amyloid plaques, we decided to not
include HYR-18 in the in vivo biodistribution studies.
6
4
Cu in the brain to an appreciable extent, and thus should
not lead to a signicant background PET signal in WT or
healthy controls.
64
Table 2 Overall biodistribution results of Cu-Labeled HYR-4 and HYR-17 for the three time points evaluated (2, 60, and 240 min; % injected
dose/gram, mean ꢀ SEM)
HYR-4
HYR-17
Organ
2 min
60 min
240 min
2 min
60 min
240 min
Blood
Lung
Liver
Kidney
Muscle
Brain
Bone
Tail
2.85 ꢀ 0.52
3.77 ꢀ 0.88
32.48 ꢀ 3.94
30.27 ꢀ 0.44
0.37 ꢀ 0.08
0.16 ꢀ 0.02
0.54 ꢀ 0.11
15.91 ꢀ 2.84
1.02 ꢀ 0.12
3.36 ꢀ 0.25
8.84 ꢀ 0.98
9.39 ꢀ 2.02
0.30 ꢀ 0.02
0.10 ꢀ 0.01
0.45 ꢀ 0.01
12.8 ꢀ 5.38
0.86 ꢀ 0.10
4.47 ꢀ 0.49
9.57 ꢀ 1.19
8.24 ꢀ 1.95
0.30 ꢀ 0.04
0.15 ꢀ 0.03
0.59 ꢀ 0.07
3.69 ꢀ 1.92
26.82 ꢀ 1.59
11.02 ꢀ 0.52
11.50 ꢀ 0.69
15.96 ꢀ 0.81
0.89 ꢀ 0.04
0.99 ꢀ 0.04
1.59 ꢀ 0.07
2.53 ꢀ 0.16
3.70 ꢀ 0.06
3.65 ꢀ 0.16
14.79 ꢀ 1.71
34.17 ꢀ 2.87
0.62 ꢀ 0.02
0.20 ꢀ 0.00
0.59 ꢀ 0.09
3.20 ꢀ 0.29
1.24 ꢀ 0.08
4.47 ꢀ 0.49
12.16 ꢀ 1.28
21.40 ꢀ 2.27
0.45 ꢀ 0.01
0.15 ꢀ 0.02
0.64 ꢀ 0.07
1.05 ꢀ 0.05
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University in St. Louis. The animal protocol #20190073
including all the animal studies performed herein was
Conclusions
Herein we report several metal-chelating benzothiazole reviewed and approved by the Institutional Animal Care and
multifunctional compounds (MFCs) and investigate their Use Committee of Washington University in St. Louis.
various biochemical, in vitro, and in vivo properties to bind to
various Ab species, modulate Ab aggregation and its neuro-
6
4
toxicity, and potentially act as Cu PET imaging agents for in Conflicts of interest
vivo detection of Ab aggregates. During these studies, we
have obtained important structure–activity relationships
There are no conicts to declare.
(
SAR) that will guide us to develop improved MFCs as
potential therapeutic or diagnostic agents for AD. Firstly,
when comparing HYR-4 vs. HYR-1 and HYR-16 vs. HYR-14, we
can conclude that the compounds containing a mono-
Acknowledgements
L. M. M. acknowledges research funding from NIH
methylamino vs.
a dimethylamino group exhibit an
(R01GM114588), the Alzheimer’s Association (NIRG 12-259199),
increased specicity for the Ab aggregates as well as reduced
cytotoxicity. Secondly, the introduction of a pyridyl group in
MFCs such as HYR-14 and HYR-16 dramatically reduces their
cytotoxicity and improves their antioxidant properties.
Taken together, the MFC HYR-16 containing the 2-
monomethylamino-pyridyl fragment is most effective at
alleviating the Ab42 oligomer neurotoxicity and modulating
Ab aggregation. Thirdly, all investigated MFCs can be effi-
ciently radiolabeled with the Cu radioisotope, and the Cu
complexes of HYR-4, HYR-17, and HYR-18 exhibit acceptable
lipophilicity and specic radiolabeling of amyloid plaques ex
vivo.
and the Washington University Knight Alzheimer’s Disease
Research Center (NIH P50AG05681). The authors would like to
thank the small animal imaging facility at Washington
University School of Medicine for excellent technical assistance,
Cedric Mpoy for valuable assistance with animal studies, and
the Isotope Production Group at Washington University for on
64
time weekly production of Cu.
6
4
64
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