Differential Effects of Structural Modifications on the Competition of Chalcones for the PIB Amyloid Imaging Ligand-Binding Site in Alzheimer's Disease Brain and Synthetic Aβ Fibrils

Article abstract of DOI:10.1021/acschemneuro.5b00266

Alzheimer's disease (AD) is a complex brain disorder that still remains ill defined. In order to understand the significance of binding of different clinical in vivo imaging ligands to the polymorphic pathological features of AD brain, the molecular chara

Full text of DOI:10.1021/acschemneuro.5b00266

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI
Article
Differential effects of structural modifications on the competition
of chalcones for the PIB amyloid imaging ligand-binding
site in Alzheimer’s disease brain and synthetic A# fibrils
Marina Y. Fosso , Katie L. McCarty, Elizabeth Head, Sylvie Garneau-Tsodikova, and Harry Levine, III
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00266 • Publication Date (Web): 18 Dec 2015
Downloaded from http://pubs.acs.org on December 21, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Differential effects of structural modifications on the
competition of chalcones for the PIB amyloid
imaging ligand-binding site in Alzheimer’s disease
brain and synthetic Aβ fibrils
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Marina Y. Fosso,a Katie McCarty,b Elizabeth Head,b,c Sylvie Garneau-Tsodikova,a,* Harry
LeVine, IIIc,d,e,
*
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789
b
South Limestone Street, Lexington, KY, 40536-0596, USA. Department of Pharmacology and
Nutritional Sciences, c Center on Aging, d Molecular and Cellular Biochemistry, and e Center for
Structural Biology, School of Medicine, University of Kentucky, Lexington, KY, 40536-0230,
USA.
KEYWORDS Displacement assay, Neurodegenerative disorder, Pittsburgh Compound B,
Radioactive assay, Structure activity relationships.
ACS Paragon Plus Environment
1

ACS Chemical Neuroscience
Page 2 of 29
1
2
3
4
ABSTRACT
5
6
7
8
9
Alzheimer’s disease (AD) is a complex brain disorder that still remains ill defined. In order to
understand the significance of binding of different clinical in vivo imaging ligands to the
polymorphic pathological features of AD brain, the molecular characteristics of the ligand
interacting with its specific binding site need to be defined. Herein, we observed that tritiated
Pittsburgh Compound B (3H-PIB) can be displaced from synthetic Aβ(1-40) and Aβ(1-42) fibrils
and from the PIB binding complex purified from human AD brain (ADPBC) by molecules
containing a chalcone structural scaffold. We evaluated how substitution on the chalcone
scaffold alters its ability to displace 3H-PIB from the synthetic fibrils and ADPBC. By comparing
unsubstituted core chalcone scaffolds along with the effects of bromine and methyl substitution
at various positions, we found that attaching a hydroxyl group on the ring adjacent to the
carbonyl group (ring I) of the parent member of the chalcone family generally improved the
binding affinity of chalcones toward ADPBC and synthetic fibrils F40 and F42. Furthermore, any
substitution on ring I at the ortho-position of the carbonyl group greatly decreases the binding
affinity of the chalcones, potentially as a result of steric hindrance. Together with the finding
that neither our chalcones nor PIB interact with the Congo Red/X-34 binding site, these
molecules provide new tools to selectively probe the PIB binding site that is found in human AD
brain, but not in brains of AD pathology animal models. Our chalcone derivatives also provide
important information on the effects of fibril polymorphism on ligand binding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
2

Page 3 of 29
ACS Chemical Neuroscience
1
2
3
4
INTRODUCTION
5
6
7
In 2004 the benzothiazole aniline Aβ amyloid fibril ligand Pittsburgh Compound B (PIB) labeled
8
11
with C for positron emission tomography (PET) imaging was shown in a small study of living
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
patients to be retained in brain areas known to accumulate Aβ pathology in Alzheimer’s disease
(AD) brains, but not in normal brains.1 Subsequent studies showed that PIB uptake tracked with
disease progression starting decades before cognitive symptoms were evident.2, 3 As the most
widely clinically used Aβ deposition tracer, PIB is generally considered a benchmark for
amyloid PET imaging agents.
The effort to find agents for early diagnosis of AD by imaging Aβ pathology in living
4
patients has produced a variety of small molecule and peptide ligands (reviewed in ). While
many such molecules have been identified, little is known about which features of Aβ pathology
are being reported and which ligands are recognizing the same feature or binding to the same
site. Lockhart and colleagues defined distinct sites for ligands on synthetic Aβ fibrils based on
their ability to compete for binding.5, 6 Other studies elaborated and confirmed the concept of
multiple distinct sites on Aβ fibrils for ligands to bind.7, 8
The significance of this multiplicity of ligand-binding sites was brought into sharp focus by
the initial observation by Klunk and co-workers that the benzothiazole aniline ligand PIB bound
with high affinity in large amounts to AD brain, but only very little bound to similar amounts of
3
Aβ pathology in a transgenic mouse model of AD.9 High affinity H-PIB binding was also
negligible in other genetic and natural nonhuman primate and canine animal models of AD Aβ
pathology.10 By contrast, ligands such as Congo Red and X-34 (Scheme 1D), which do not
ACS Paragon Plus Environment
3

ACS Chemical Neuroscience
Page 4 of 29
1
2
3
4
5
6
7
8
9
compete for PIB binding and, therefore, bind to a site distinct from where PIB binds to Aβ
pathology, bound normally in these animal models. Since humans are the only animal that
suffers the full progression of AD, these findings suggested that the site to which PIB binds was
unique and that elucidation of how it was formed and its structure might provide insight into the
disease process and potential therapeutic avenues. Our determination that PIB binding Aβ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pathology was an isolatable fraction of the total AD brain Aβ pathology with different physical
properties and protein composition11 indicated that high affinity PIB binding addressed a
reasonably discrete population of peptide conformers present at high levels in human AD brain
and only low levels in animal models.
These observations suggest that imaging ligands that bind to Aβ pathology at sites distinct
from the PIB binding site have the potential to be reporting different processes or to miss events,
both of which would foster confusion potentially leading to misinterpretation in diagnosis and in
defining progression mechanisms. Ni and colleagues showed in AD tissue homogenates that the
two 18F-labeled stilbene derivatives florbetapir (Amyvid™) and florbetaben (Neuraceq™)
(Scheme 1D) now used in the clinic, compete for PIB binding to AD brain tissue, as does
Vizamyl™ (flutemetamol; 2-[3-[18F]fluoro-4-(methylamino) phenyl]-6-benzothiazolol), a PIB
derivative.12 Thus, imaging in AD brain with these ligands should give similar results to those
with 11C-PIB.
PIB competition results have not been reported for the majority of ligands shown in
preclinical studies to bind Aβ pathology. Based on the fact that the chalcone scaffold (1,3-
diphenyl-2-propen-1-one, DPP, Scheme 1D) highly resembles the florbetaben and florbetapir
core, with the exception of the additional carbonyl group in DPP, and also based on the fact that
ACS Paragon Plus Environment
4

Page 5 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
the N,N-dimethylamino/N-monomethylamino moieties of various molecules have been
demonstrated to be critical to Aβ, we hypothesized that chalcone dertivatives containing an N,N-
dimethylamino group could potentially compete for the PIB binding site in Aβ. Here, we
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
demonstrate that molecules based on the chalcone scaffold displace H-PIB from the
Alzheimer’s disease PIB binding complex (ADPBC) purified from AD brain, as well as from
more widely available model fibrils assembled from synthetic Aβ(1-40) and Aβ(1-42) peptides.
We also show that a prototypical chalcone of our series, 9a, stains Aβ plaque pathology
selectively, and like PIB, does not stain tau pathology in tissue sections from hippocampus of
AD brain. Several series of electron-withdrawing, electron-donating, and bulky substituents on
ring I produced molecules with EC50 values displaying consistent structure-activity relationships
3
(SAR). H-X-34 binding was unaffected by these chalcones (EC50 values > 10 μM).
3
Quantification of H-PIB binding to synthetic peptide Aβ(1-40) and Aβ(1-42) fibrils (indicated
from here on as F40 and F42, respectively) indicated a high Aβ:PIB stoichiometry, many Aβs per
high affinity PIB binding site, while the purified ADPBC from AD brain showed nearly a 1:1
stoichiometry.10
We report that the SAR of chalcones for synthetic F40 and F42 fibrils differs from that of the
AD brain site, and that the F40 and F42 fibrils have SARs distinct from one another. This suggests
that the details of the binding site for chalcones that compete for PIB binding differ among the
different fibril populations.
RESULTS AND DISCUSSION
Chemistry
ACS Paragon Plus Environment
5

ACS Chemical Neuroscience
Page 6 of 29
1
2
3
4
5
6
7
8
9
Chalcones have long been known as a prolific class of compounds, displaying a vast array of
biological activities.14 With the aim of investigating their effects on the formation and
dissociation of Aβ oligomers, we recently reported on the synthesis of three series of chalcones,15
differing from each other by the nature of ring I (Scheme 1C). They either bear a pyridine
(compounds 3a-i), a phenol (compounds 6a-h), or an aniline (compounds 7a, b, h) moiety as
ring I. Electron-withdrawing, electron-donating, and bulky substituents, such as a methyl or a
bromo group, were also introduced at various positions on ring I. We further expanded this series
of chalcones by including compounds 8i (Scheme 1A) and 9a (Scheme 1B), which were
obtained in 62 and 86% yields, respectively, by reacting 4-(dimethylamino)benzaldehyde, 2,
with the corresponding ketone (1i and 4a, respectively) in the presence of a base (KOH or
NaOH). Overall, the modifications on ring I were selected with the goals of (i) mimicking the
pyridine ring of florbetapir and (ii) attaching handles that we could later potentially use to further
derivatize our molecules and generate radiolabeled or fluorescently labeled compounds as
imaging agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Investigation of unsubstituted core scaffolds
3
After establishing that our chalcones did not compete for binding with H-X-34 (all EC50 values
against 3H-X-34 > 10 μM), we investigated the binding of the unsubstituted core scaffolds (DPP,
3a, 6a, 7a, and 9a) to ADPBC. The parent member of the chalcone family, DPP (Scheme 1D),
3
was first evaluated and it was found to have better affinity for displacing H-PIB from ADPBC
than synthetic F40 and F42 fibrils (Table 1, entry 23). While not as potent as the best benzothiazole
aniline ligands (PIB: EC50 = 2.6 nM; BTA-1: EC50 = 3 nM),11 it was sub-micromolar despite
ACS Paragon Plus Environment
6

Page 7 of 29
ACS Chemical Neuroscience
1
2
3
4
missing the N,N-dimethylamino/N-monomethylamino moiety on the aromatic ring critical for the
5
6
benzothiazole aniline ligands.16
7
8
9
Selectivity of chalcone scaffold for AD brain Aβ plaque pathology.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sections of hippocampal tissue from AD brain containing both Aβ plaque pathology and tau
neurofibrillary tangle pathology were stained with chalcone 9a to verify its selectivity for the Aβ
pathology (Figure 1). Thioflavin S stains both Aβ plaques (white triangles) and tau pathology
(white arrows) in panel A. By contrast, like PIB and its analogue 6-CN-PIB, the chalcone stains
only the Aβ plaque pathology (white triangles) in an adjacent tissue section shown in panel B.
This selectivity convinced us of the potential ability of chalcones to effectively displace PIB
from its binding site in AD brain and in synthetic peptide model systems, and led us to
investigate the effects of substitution on the structural scaffold of DPP. The ratios of EC50 values
3
shown in Table 1 for displacement of H-PIB from the different Aβ fibril sources also provide
insight into the relative similarity of the ligand-binding site on those fibrils. Large or small ratios
indicate that the compound binds differently to each type of fibrils, whereas ratios close to 1
indicate compounds with comparable affinity to the types of fibrils compared.
Upon attachment of a N,N-dimethylamino moiety on ring II and replacement of the benzene
ring I in DPP by a pyridine (3a, entry 1), phenol (6a, entry 10), or aniline (7a, entry 18) moiety,
we observed that the core scaffold 6a binds better than the core scaffold 3a, which in turn binds
better than DPP to ADPBC, F40, and F42 fibrils. Furthermore, 6a and 7a have comparable binding
affinity toward F40 and F42 fibrils, but the core scaffold 7a does not bind as well as 6a and 3a to
3
ADPBC. It thus appears that having a phenol as ring I provides the strongest competitor to H-
PIB in ADPBC, F40, and F42 fibrils, while the presence of the benzene ring alone imparted the
ACS Paragon Plus Environment
7

ACS Chemical Neuroscience
Page 8 of 29
1
2
3
4
5
6
7
8
9
3
weakest displacement of H-PIB in all cases. Furthermore, when the hydroxyl group on 6a was
moved away from position 1 to 2, as in 9a (Table 1, entry 22), the binding affinity and fibril
selectivity were altered. Compound 6a was better than 9a at binding synthetic F40 fibrils, while
the opposite was observed for synthetic F42 fibrils. Not surprisingly though, the EC50 values for
the two compounds were similar in ADPBC, which may reflect the additional components
(lipids, other proteins) in the ADPBC. As a result, we decided to pursue our structural
modifications on ring I of core scaffolds 3a, 6a, and 7a.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Direct comparison of methylated and brominated core scaffolds
By adding a methyl or bromo group at positions 2-5 of ring I, we were able to compare chalcone
derivatives with the same substituent at the same position in that ring. In the presence of F40
fibrils, the EC50 values of chalcone derivatives 6a-h (Figure 2B) with the phenol ring (entries 10-
17) were the smallest (highest affinity), in comparison to 3a-h (Figure 2A) with a pyridine
moiety (entries 1-8) and 7a, b, and h with an aniline (entries 18-20). In the case of F42 fibrils, any
substitution at position 2 or 5 will still make the chalcone derivatives with the phenolic ring
(entries 11, 14, and 15) better (lower EC50, higher affinity) than their corresponding analogues
with the pyridine (entries 2, 5, and 6). On the other hand, if a methyl group is at position 3 or 4,
the chalcone derivatives from the pyridine (scaffold 3; entries 3-4) and phenol (scaffold 6; entries
12-13) series have comparable affinities; meanwhile a bromo group at position 3 or 4 will make
the chalcone derivatives with a pyridine moiety (entries 7-8) better competitor than the ones with
the phenol (entries 16-17). It thus appears that the presence of a weak electron-withdrawing
group (EWG) at position 3 or 4 will switch which one is better in F42 fibrils. In the case of
ADPBC, substitution did not seem to have a large effect, since the chalcone derivatives with the
phenol moiety had higher affinity than their counterparts with a pyridine or an aniline moiety,
ACS Paragon Plus Environment
8

Page 9 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
with the exception of 6d (entry 13) and 6h (entry 17), which had higher EC50 values (lower
affinity) than their respective pyridine analogues 3d (entry 4) and 3h (entry 8). Overall, 6a-h
with the phenol moiety had higher affinity for ADPBC, F40, and F42 fibrils (Table 1, entries 10-
17) than their counterparts with a pyridine (3a-h, entries 1-8) or an aniline moiety (7a, b, and h,
entries 18-20), which confirms our initial findings that having a phenol moiety as ring I
conferred higher affinity binding to ADPBC, F40, and F42 fibrils. It is important to note that,
although not in all cases, overall, the more lipophilic compounds (higher log P values in Table
S1) displayed higher binding affinity to Aβ.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Comparison of the effect of bromine versus methyl substitution
When comparing the methyl group versus the bromo group as substituents on ring I, it appears
that the chalcone derivatives with the bromo group display comparable or higher affinity than
their methyl counterparts towards ADPBC, F40, and F42 fibrils, with the exception of 6f (entry
15), 6g (entry 16), and 6h (entry 17), which did not bind synthetic F42 fibrils as well as their
methyl counterparts 6b (entry 11), 6c (entry 12), and 6d (entry 13), respectively. This suggests
that a weak EWG such as a bromo group might enhance the binding affinity of the chalcones.
Comparison of the effect of substitution pattern on core scaffolds
Regarding the effect on binding affinity of various positions for substitution on ring I of all three
core scaffolds (3, 6, and 7), we noticed that for synthetic F40 fibrils, position 3 was generally
preferred over position 2, which in turn was preferred to position 4. In the case of ADPBC and
F42 fibrils, position 2 was generally preferred over position 3, which in turn was preferred to
position 4 for the chalcone derivatives with the phenol moiety. Overall, position 5 appears to be
the least favored in most cases, probably due to the steric hindrance that might be created. We
ACS Paragon Plus Environment
9

ACS Chemical Neuroscience
Page 10 of 29
1
2
3
4
5
6
7
8
9
also observed that moving the nitrogen of the pyridine ring away from the carbonyl moiety of the
chalcone, as in compound 8i (entry 21), is disfavored and series 8 was therefore not further
investigated.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16
Previous SAR with the benzothiazole aniline series11,
suggests a relatively sterically
restricted binding pocket with a parallel SAR for ADPBC, F40, and F42 fibrils. The more flexible
chalcone scaffold is relatively tolerant of substituents. Interestingly, the chalcone derivatives
investigated here markedly distinguish among the ADPBC, F40, and F42 fibrils. This behavior
could reflect a different binding mode for chalcones, which probes the structural organization of
the fibrils around while occluding a subsite that binds the benzothiazole aniline moiety.
Chalcones could be especially sensitive to Aβ fibril polymorphism around the PIB binding site,
which is distinct from the pan-amyloid fibril-binding site for Congo Red and X-34.
CONCLUSIONS
In summary, we found that the chalcone scaffold widely reported as Aβ imaging tracers4
3
competes for H-PIB binding, but not for H-X-34 binding to synthetic Aβ(1-40) and Aβ(1-42)
3
fibrils and to the PIB-binding fraction of Aβ isolated from AD brain. As hypothesized,
exploration of a series of ring I substitutions revealed a consistent pattern of differential effects
on competition for 3H-PIB binding that differentiated the two synthetic peptide fibrils from each
other as well as from the AD brain material. Overall, the pattern of the EC50 values of chalcone
displacement of 3H-PIB from PIB binding fraction from AD brain most closely resembled that of
the synthetic Aβ(1-42) fibrils (Table 1). Since the chalcones studied did not interact with the
Congo Red/X-34 binding site, they provide tools to explore details of Aβ fibril structure around
ACS Paragon Plus Environment
10

Page 11 of 29
ACS Chemical Neuroscience
1
2
3
4
the PIB binding pocket and effects of fibril polymorphism on ligand binding, which may explain
5
6
the low binding of PIB to the Aβ pathology of animal models.
7
8
9
METHODS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Materials and instrumentation. Human autopsy tissue for preparation of the ADPBC was
obtained from the University of Kentucky Center on Aging Brain Bank of the Alzheimer’s
Disease Center in accordance with federal and institutional IRB guidelines with informed
consent, and samples were de-identified to ensure the anonymity of subjects. The study conforms
to The Code of Ethics of the World Medical Association. All chemicals were purchased from
Sigma Aldrich (St. Louis, MO) and used without further purification. Chemical reactions were
monitored by thin layer chromatography (TLC) using Merck, Silica gel 60 F254 plates.
Visualization was achieved using UV light and KMnO4 stain (1.5 g KMnO4, 10 g K2CO3, 1.25
1
13
mL 10% NaOH, 200 mL H2O). H and C NMR spectra were recorded at 400 and 100 MHz,
respectively, on a Varian 400 MHz spectrometer, using the indicated solvents. Chemical shift (δ)
is given in parts per million (ppm). Coupling constants (J) are given in hertz (Hz), and
conventional abbreviations used for signal shape are as follows: s = singlet; d = doublet; t =
triplet; m = multiplet; dd = doublet of doublets; dt = doublet of triplets. Liquid chromatography-
mass spectrometry (LCMS) was carried out using an Agilent 1200 series Quaternary LC system
equipped with a diode array detector, and Eclipse XDB-C18 column (250 mm x 4.6 mm, 5 µm),
and an Agilent 6120 Quadrupole MSD mass spectrometer (Agilent Technologies, Santa Clara,
CA). LCMS M + H signals were consistent with the expected molecular weights for all of the
reported compounds.
ACS Paragon Plus Environment
11

ACS Chemical Neuroscience
Page 12 of 29
1
2
3
4
5
6
Synthesis of chalcone ligands. The synthesis and structural characterization of chalcones 3a-i,
6a-h, and 7a, b, h were performed as previously described.15
7
8
9
Synthesis of chalcone 8i. A solution of 3-acetyl-2-bromopyridine (1i) (201 mg, 1.0 mmol) and
4-(dimethylamino)benzaldehyde (2) (150 mg, 1.0 mmol) in EtOH (5 mL) was treated with 3 mL
of a 20% aqueous KOH solution and allowed to stir at rt for 3 h. Upon completion of the
reaction, H2O (5 mL) was added and the solid residues that formed were filtered out, rinsed with
H2O and ice-cold EtOH, and recrystallized from CH2Cl2/hexanes to give compound 8i (Rf 0.17 in
Hexanes:EtOAc/3:1) as dark yellow needles (226 mg, 68% yield): 1H NMR (400 MHz, CDCl3) δ
8.46 (dd, J1 = 4.8 Hz, J2 = 2.0 Hz, 1H, aromatic), 7.70 (dd, J1 = 7.2 Hz, J2 = 2.0 Hz, 1H,
aromatic), 7.46 (dt, J1 = 9.2 Hz, J2 = 2.0 Hz, 2H, aromatic), 7.36 (d, J = 15.6 Hz, 1H, HC=CH-
Ph), 7.37 (dd, J1 = 7.2 Hz, J2 = 4.8 Hz, 1H, aromatic), 6.89 (d, J = 15.6 Hz, 1H, HC=CH-Ph),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
6.69 (dt, J1 = 9.2 Hz, J2 = 2.0 Hz, 2H, aromatic), 3.05 (s, 6H, N(CH3)2); C NMR (100 MHz,
CDCl3) δ 192.7, 152.5, 150.6, 148.8, 138.9, 138.7, 137.4, 130.9 (2 carbons), 122.5, 121.6, 120.4,
111.8 (2 carbons), 40.1 (2 carbons); m/z calcd for C16H15BrN2O 330.0; found 331.0 [M+H]+.
Synthesis of chalcone 9a. The known compound 9a was prepared as previously described.19 A
solution of 3'-hydroxy acetophenone (4a) (68 mg, 0.5 mmol) and 4-
(dimethylamino)benzaldehyde (2) (75 mg, 0.5 mmol) in EtOH (1.5 mL) was treated with NaOH
pellets (400 mg, 10.0 mmol). The reaction was stirred at rt overnight until completion. Most of
the solvent was then removed and 1 N aqueous HCl was added. The precipitate was filtered to
give the known compound MFY-4-8 (Rf 0.26 in Hexanes:EtOAc/3:1) as an orange solid (110
mg, 82% yield): 1H NMR (400 MHz, CDCl3, which matches the lit.19) δ 7.78 (d, J = 15.6 Hz, 1H,
HC=CH-Ph), 7.56-7.50 (m, 4H, aromatic), 7.35 (t, J = 8.0 Hz, 1H, aromatic), 7.29 (d, J = 15.6
ACS Paragon Plus Environment
12

Page 13 of 29
ACS Chemical Neuroscience
1
2
3
4
Hz, 1H, HC=CH-Ph), 7.03 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H, aromatic), 6.69 (d, J = 9.2 Hz, 2H,
5
6
aromatic), 5.30 (s, 1H, OH), 3.04 (s, 6H, N(CH3)2).
7
8
9
Preparation of Aβ fibrils. AD brain ADPBC was purified from autopsy human AD frontal
cortex as previously described11 and was stored in aliquots at -75 °C. For the recombinant Aβ
(rPeptide, Bogart, GA) 1 mg each of Aβ(1-40) (cat. A-1153-2, lot #6050840H) and Aβ(1-42)
(cat. A-1163-2, lot #4230842H) obtained as films dried from HFIP were dissolved in 900 µL of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ice-cold distilled H2O and kept on ice for 30 min with intermittent vortexing. 100 µL of 10x PBS
(final concentration 20 mM sodium phosphate, 145 mM NaCl, pH 7.4) were added with
vortexing followed by 20 µL of 2% w/v NaN3. The solutions were transferred to separate screw
cap polypropylene tubes, sealed, and incubated at 37 °C for one week, vortexing briefly once
each day. Fibril formation was assessed by sedimentable Thioflavin T fluorescence.13, 17 Aliquots
of fibrils (1 mg/mL) were frozen at -20 °C and thawed immediately before use.
Radioligand binding assays:
3
3H-PIB binding. H-PIB binding was assessed in Aβ preparations from AD brain and in fibrils
prepared from Aβ(1-40) and Aβ(1-42) recombinant peptides (F40 and F42, respectively).11 For
binding studies 20 µL of PBS containing purified ADPBC equivalent to 133.3 µg wet weight of
original tissue, 100 ng F40, or 50 ng F42 were added to each of duplicate wells of a 96-well
polypropylene plate (Costar 3365). Under the conditions of the assay these quantities of Aβ gave
3
similar total amounts of H-PIB binding, 10-15% of the input radioactivity. 200 µL of 1.2 nM
3H-PIB (cat. VT 278 specific radioactivity = 70.2 Ci/mmol, Vitrax (Placentia, CA)) containing a
dose response of nonradioactive competitor at a final concentration of DMSO of 1% v/v in PBS
ACS Paragon Plus Environment
13

ACS Chemical Neuroscience
Page 14 of 29
1
2
3
4
5
6
7
8
9
+ 5% v/v EtOH was added to each well. Samples were incubated for 3 h at room temperature
without shaking, transferred to a 96-well Millipore Multiscreen HTS Hi Flow FB (GF/B) filter
plate, and filtered on a multi-well plate vacuum manifold (Millipore Corporation, Bedford, MA).
The filters were rapidly washed three times with 200 µL of PBS + 5% v/v EtOH, dried, removed
from the plate, and placed in scintillation vials. 2 mL of BudgetSolve scintillation fluid were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
added, and the vials capped and shaken before counting for H in a Packard TriCarb 2500 TR
scintillation counter. Specific binding was calculated as (mean CPM of the two filters from wells
containing radioactive PIB + competitors) minus (CPM value from wells containing radioactive
PIB + 1 µM nonradioactive BTA-1 competitor). EC50 values were determined by titrating
3
increasing concentrations of unlabeled test compound against constant (1.3 nM) H-PIB. The
3
EC50 is the concentration at which 50% of the specifically bound H-PIB is displaced. Although
the EC50 values are slightly greater than the IC50 values because more than 10% of the total
radioligand is bound, the EC50 values can be compared with each other since they were
determined under the same conditions.
3
3
3H-X-34 binding. H-X-34 binding18 was performed similarly to PIB binding with 5 nM H-X-
34, 23 Ci/mmol, custom tritiated by Vitrax (Placentia, CA)) with 10 μM X-34 or Congo Red as
nonradioactive X-34 competitor.
Chalcone and thioflavin S histochemistry. Formalin-fixed hippocampal sections from an AD
case (77 year old female, Braak VI, post mortem interval 4.15 h, ApoE 4/4), 8 µm thick were
deparaffinized and rehydrated through decreasing concentrations of alcohol until washing in
PBS. Sections were incubated in a room temperature humidifier with 2 mM chalcone 9a in 20%
v/v DMSO in PBS for 1 h in the dark. After 3x1 min PBS washes, sections were treated with
ACS Paragon Plus Environment
14

Page 15 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
TrueBlack Lipofuscin Autofluorescence Quencher (30s-Biotium, Hayward, CA) and then
coverslipped using Everbrite Hardset Mounting Medium (Biotium, Haywad, CA). An adjacent
section was stained with thioflavin S (0.5% w/v in 50% v/v EtOH) for 5 min, then washed in 50%
EtOH, followed by ddH2O, and then coverslipped using Vectashield with DAPI (Vector
Laboratories, Burlingame, CA). Images were captured using an Olympus BX51 fluorescence
microscope with a wide band pass filter.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information. The supporting information includes a copy of the 1H and/or 13C NMR
spectra for compounds 8i and 9a. It also includes a table containing the log P values for all of our
compounds (Table S1). This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* Harry LeVine, III: E-mail: hlevine@email.uky.edu
or
Sylvie Garneau-Tsodikova: E-mail: sylviegtsodikova@uky.edu
Funding Sources
This work was supported by startup funds from the University of Kentucky (to S.G.-T.) and by
NIH grant AI90048 (to S.G.-T.), and by NIH grant 1R21 NS080576-01A1 (to H.L.), and NIH
ACS Paragon Plus Environment
15

ACS Chemical Neuroscience
Page 16 of 29
1
2
3
4
5
6
7
8
9
Center Core grant P30AG028383 (Alzheimer’s Disease Center). The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National
Institutes of Health. The authors declare no competing financial interest.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACKNOWLEDGMENTS
This work was supported by startup funds from the University of Kentucky (to S.G.-T.) and by
NIH grant AI90048 (to S.G.-T.) and by NIH grant 1R21 NS080576-01A1 (to H.L.), and NIH
Center Core grant P30AG028383 (Alzheimer’s Disease Center). We thank Dr. David S. Watt for
initial compounds implicating chalcones as PIB-displacing Aβ ligands. We acknowledge Linda
Van Eldik, Ph.D. Sanders-Brown Director, Peter Nelson, M.D., Ph.D., Neuropathology Core
Director, and Sonya Anderson, Brain Bank Coordinator for human brain tissue and their
contributions on behalf of the ADC. We are forever in debt to the patients whose brain donations
made this investigation possible.
REFERENCES
1. Klunk, W. E., Engler, H., Nordberg, A., Wang, Y., Blomqvist, G., Holt, D. P., Bergstrom,
M., Savitcheva, I., Huang, G. F., Estrada, S., Ausen, B., Debnath, M. L., Barletta, J., Price, J.
C., Sandell, J., Lopresti, B. J., Wall, A., Koivisto, P., Antoni, G., Mathis, C. A., and
Langstrom, B. (2004) Imaging brain amyloid in Alzheimer's disease with Pittsburgh
Compound-B. Ann. Neurol. 55, 306-319.
2. Jack, C. R., Jr., Knopman, D. S., Jagust, W. J., Petersen, R. C., Weiner, M. W., Aisen, P. S.,
Shaw, L. M., Vemuri, P., Wiste, H. J., Weigand, S. D., Lesnick, T. G., Pankratz, V. S.,
Donohue, M. C., and Trojanowski, J. Q. (2013) Tracking pathophysiological processes in
ACS Paragon Plus Environment
16

Page 17 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
Alzheimer's disease: an updated hypothetical model of dynamic biomarkers. Lancet Neurol.
12, 207-216.
7
8
9
3. Jack, C. R., Jr., Barrio, J. R., and Kepe, V. (2013) Cerebral amyloid PET imaging in
Alzheimer's disease. Acta Neuropathol. 126, 643-657.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4. Eckroat, T. J., Mayhoub, A. S., and Garneau-Tsodikova, S. (2013) Amyloid-beta probes:
Review of structure-activity and brain-kinetics relationships. Beilstein J. Org. Chem. 9, 1012-
1044.
5. Lockhart, A., Ye, L., Judd, D. B., Merritt, A. T., Lowe, P. N., Morgenstern, J. L., Hong, G.,
Gee, A. D., and Brown, J. (2005) Evidence for the presence of three distinct binding sites for
the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide
fibrils. J. Biol. Chem. 280, 7677-7684.
6. Ye, L., Morgenstern, J. L., Gee, A. D., Hong, G., Brown, J., and Lockhart, A. (2005)
Delineation of positron emission tomography imaging agent binding sites on beta-amyloid
peptide fibrils. J. Biol. Chem. 280, 23599-23604.
7. Lockhart, A. (2006) Imaging Alzheimer's disease pathology: one target, many ligands. Drug
Discov. Today 11, 1093-1099.
8. LeVine, H., 3rd. (2005) Multiple ligand binding sites on A beta(1-40) fibrils. Amyloid 12, 5-
14.
9. Klunk, W. E., Lopresti, B. J., Ikonomovic, M. D., Lefterov, I. M., Koldamova, R. P.,
Abrahamson, E. E., Debnath, M. L., Holt, D. P., Huang, G. F., Shao, L., DeKosky, S. T.,
Price, J. C., and Mathis, C. A. (2005) Binding of the positron emission tomography tracer
Pittsburgh compound-B reflects the amount of amyloid-beta in Alzheimer's disease brain but
not in transgenic mouse brain. J. Neurosci. 25, 10598-10606.
ACS Paragon Plus Environment
17

ACS Chemical Neuroscience
Page 18 of 29
1
2
3
4
5
6
7
8
9
10. Rosen, R. F., Walker, L. C., and Levine, H., 3rd. (2011) PIB binding in aged primate brain:
enrichment of high-affinity sites in humans with Alzheimer's disease. Neurobiol. Aging 32,
223-234.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11. Matveev, S. V., Spielmann, H. P., Metts, B. M., Chen, J., Onono, F., Zhu, H., Scheff, S. W.,
Walker, L. C., and LeVine, H., 3rd. (2014) A distinct subfraction of Abeta is responsible for
the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain. J.
Neurochem. 131, 356-368.
12. Ni, R., Gillberg, P. G., Bergfors, A., Marutle, A., and Nordberg, A. (2013) Amyloid tracers
detect multiple binding sites in Alzheimer's disease brain tissue. Brain 136, 2217-2227.
13. LeVine, H., 3rd. (1993) Thioflavin T interaction with synthetic Alzheimer's disease beta-
amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404-410.
14. Batovska, D. I., and Todorova, I. T. (2010) Trends in utilization of the pharmacological
potential of chalcones. Curr. Clin. Pharmacol. 5, 1-29.
15. Fosso, M. Y., LeVine 3rd, H., Green, K. D., Tsodikov, O. V., and Garneau-Tsodikova, S.
(2015) Effects of structural modifications on the metal binding, anti-amyloid activity, and
cholinesterase inhibitory activity of chalcones. Org. Biomol. Chem. 13, 9418-9426.
16. Klunk, W. E., Wang, Y., Huang, G. F., Debnath, M. L., Holt, D. P., Shao, L., Hamilton, R.
L., Ikonomovic, M. D., DeKosky, S. T., and Mathis, C. A. (2003) The binding of 2-(4'-
methylaminophenyl)benzothiazole to postmortem brain homogenates is dominated by the
amyloid component. J. Neurosci. 23, 2086-2092.
17. LeVine, H., 3rd. (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin
T. Methods Enzymol. 309, 274-284.
ACS Paragon Plus Environment
18

Page 19 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
18. Matveev, S. V., Kwiatkowski, S., Sviripa, V. M., Fazio, R. C., Watt, D. S., and LeVine, H.,
3rd. (2014) Tritium-labeled (E,E)-2,5-bis(4'-hydroxy-3'-carboxystyryl)benzene as a probe for
beta-amyloid fibrils. Bioorg. Med. Chem. Lett. 24, 5534-5536.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19. Zhao, P. L., Liu, C. L., Huang, W., Wang, Y. Z., and Yang, G. F. (2007) Synthesis and
fungicidal evaluation of novel chalcone-based strobilurin analogues. J. Agric. Food Chem.
55, 5697-700.
ACS Paragon Plus Environment
19

ACS Chemical Neuroscience
Page 20 of 29
1
2
3
4
SCHEME LEGEND
5
6
7
8
9
Scheme 1. Synthetic scheme for the preparation of A. chalcone 8i and B. chalcone 9a. C.
Structures of chalcones 3a-i, 6a-h, and 7a, b, h. D. Structures of DPP, PIB, BTA-1, X-34,
florbetaben (18F), and florbetabir (18F).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FIGURE LEGENDS
Figure 1. Thioflavin S and chalcone 9a labeling in the hippocampus of an AD case. A.
Thioflavin S binds to plaques (white triangles) and neurofibrillary tangles (white arrows) in area
CA1 of the hippocampus. B. In contrast, chalcone 9a binds to plaques (white triangles), but not
neurofibrillary tangles in an adjacent section from the same case. Scale bar = 40 µm.
3
Figure 2. Displacement of H-PIB from ADPBC by A. chalcones 3a-i and B. chalcones 6a-h.
Percent maximal 3H-PIB binding = (binding in absence of competitor – binding in presence of 1
μM BTA-1). Mean of two assays on separate days ± SDEV.
TABLE LEGENDS
3
Table 1. EC50 values (µM) of chalcone competition for H-PIB binding to Aβ preparations.
ACS Paragon Plus Environment
20

Page 21 of 29
ACS Chemical Neuroscience
1
2
3
4
Scheme 1.
5
6
A
O
7
8
H
O
O
N
9
2
I
II
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aq. KOH, EtOH
rt
68%
N
Br
N
Br
N
1i
8i
B
O
H
O
O
N
HO
HO
2
I
II
NaOH, EtOH
rt
82%
N
N
4a
9a
O
X
O
C
1
1
N
6
6
2
2
3
I
II
I
II
3
5
5
N
4
4
R
R
3a-i
6a-h (X = OH)
7a, b, h (X = NH2)
R: a = H; b = 2-Me; c = 3-Me; d = 4-Me; e = 5-Me; f = 2-Br; g = 3-Br;
h = 4-Br; i = 5-Br
O
N
D
NH
NH
S
HO
I
II
PIB
DPP
N
S
BTA-1
HO
OH
CO2H
HO2C
X-34
NH
NH
18F
O
O
O
O
Florbetaben (18F)
N
18F
O
O
Florbetapir (18F)
ACS Paragon Plus Environment
21

ACS Chemical Neuroscience
Page 22 of 29
1
2
3
4
Figure 1.
5
6
A
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B
ACS Paragon Plus Environment
22

Page 23 of 29
ACS Chemical Neuroscience
1
2
3
4
Figure 2.
5
6
7
8
120
100
80
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
60
3a
3b
3c
3d
3e
3f
3g
3h
3i
40
20
0
0.001
0.01
0.1
1
10
[chalcone], ꢀM
120
100
80
60
40
20
0
6a
6b
6c
6d
6e
6f
6g
6h
-20
0.001
0.01
0.1
1
10
[chalcone], ꢀM
ACS Paragon Plus Environment
23

ACS Chemical Neuroscience
Page 24 of 29
1
2
3
4
5
6
Table 1. EC50 values (µM) of chalcone competition for 3H-PIB binding to Aβ preparationsa.
Ratio of EC50 valuesb
7
8
9
ADPBC
F40/F42
3.3
F40/ADPBC F42/ADPBC
Entry Cpd #
F40
F42
0.037 ± 0.005
> 10
31
c
9.5
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3a
1.150 ± 0.071 0.350 ± 0.057
4.200 ± 0.283 1.850 ± 0.636
2.100 ± 0.707 0.097 ± 0.005
2.3
2
3b
3c
0.465 ± 0.078
0.013 ± 0.001
6.050 ± 1.344
0.795 ± 0.035
0.142 ± 0.069
0.008 ± 0.001
8.000 ± 1.414
0.010 ± 0.003
0.009 ± 0.003
0.056 ± 0.021
0.185 ± 0.035
1.650 ± 0.071
0.014 ± 0.004
0.032 ± 0.001
0.150 ± 0.042
0.059 ± 0.002
0.010 ± 0.002
> 10
22
c
4.5
c
0.2
10
3
4
3d
3e
> 10
0.125 ± 0.007
1.4
2.1
7.3
20
0.8
1.4
5.4
64
0.6
0.7
0.7
3.3
0.5
12
5
5.100 ± 1.556 3.700 ± 0.707
1.150 ± 0.071 0.555 ± 0.021
0.765 ± 0.049 0.105 ± 0.007
0.515 ± 0.120 0.026 ± 0.006
9.000 ± 1.414 4.300 ± 1.414
0.030 ± 0.004 0.120 ± 0.028
0.083 ± 0.014 0.063 ± 0.010
0.058 ± 0.001 0.110 ± 0.014
0.570 ± 0.339 0.170 ± 0.042
2.250 ± 0.212 1.800 ± 0.849
0.150 ± 0.014 0.125 ± 0.007
0.050 ± 0.000 0.410 ± 0.014
0.220 ± 0.014 0.520 ± 0.028
0.035 ± 0.006 0.110 ± 0.014
0.270 ± 0.085 0.082 ± 0.040
1.050 ± 0.071 0.675 ± 0.078
6
3f
7
3g
8
3h
3i
2.1
0.3
1.3
0.5
3.4
1.3
1.2
0.1
0.4
0.3
3.3
1.1
3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6a
9.2
1.0
3.1
1.4
11
7.0
2.0
0.9
1.1
8.9
13
6b
6c
6d
6e
6f
1.6
1.5
0.6
6g
3.5
1.9
6h
7a
27
c
8.2
c
7b
7h
8i
1.6
c
c
c
> 10
> 10
9.000 ± 1.414
0.013 ± 0.001
0.650 ± 0.071
4.6
5.4
13
15
2.8
2.7
9a
0.165 ± 0.064 0.036 ± 0.008
9.500 ± 0.707 1.750 ± 0.495
0.015 ± 0.007 0.040 ± 0.005
0.026 ± 0.007 0.031 ± 0.002
DPP
PIB
BTA-1
0.003 ± 0.001
0.003 ± 0.001
0.4
0.8
4.3
8.8
11.6
10.5
a F40 = Aβ(1-40) fibrils; F42 = Aβ(1-42) fibrils; ADPBC = PIB binding site isolated from AD brain (see Methods).
b
Large or small ratios indicate that the compound binds differently to each fibril type, whereas ratios close to 1
indicate compounds with comparable affinity to the fibril types compared.
3
Due to solubility-micellarization concerns which interfere with H-PIB binding measurements, compounds were
c
not tested at concentrations greater than 10 µM and no ratio can be calculated in this case.
ACS Paragon Plus Environment
24

Page 25 of 29
ACS Chemical Neuroscience
1
2
3
4
Table of Contents Graphic and Synopsis.
5
6
7
Y
X
O
8
9
N
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R = H, Me, or Br
3H-PIB displacement:
(X = C; Y = OH) > (X = C; Y = NH2) > (X = N) > (X = C; Y = H)
Novel molecules compete for PIB binding to Aβ: Our synthesized chalcones are very specific
to the PIB binding site, with the phenolic moiety imparting the greatest affinity.
ACS Paragon Plus Environment
25

ACS Chemical Neuroscience
Page 26 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1 tif
160x317mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 27 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1 tif
77x104mm (300 x 300 DPI)
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 28 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2 tif
80x118mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 29 of 29
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Image for Table of content tif
28x11mm (300 x 300 DPI)
ACS Paragon Plus Environment