Tritium-labeled (E,E)-2,5-bis(4′-hydroxy-3′-carboxystyryl)benzene as a probe for β-amyloid fibrils

Article abstract of DOI:10.1016/j.bmcl.2014.09.075

Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[³H]-2,5-bis(4′-hydroxy-3′-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils.

Full text of DOI:10.1016/j.bmcl.2014.09.075

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5534–5536
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tritium-labeled (E,E)-2,5-bis(40-hydroxy-30-carboxystyryl)benzene as
a probe for b-amyloid fibrils
Sergey V. Matveev a,b, , Stefan Kwiatkowski a,c, , Vitaliy M. Sviripa a,c, Robert C. Fazio e, David S. Watt a,c,d,
,
⇑
Harry LeVine III a,b,
⇑
a Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40536-0509, United States
b Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536-0230, United States
c Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0596, United States
d Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0093, United States
e ViTrax Radiochemicals, 660 S. Jefferson Street, Unit E, Placentia, CA 92870, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2014
Revised 11 September 2014
Accepted 24 September 2014
Available online 6 October 2014
Accumulation of Ab in the brains of Alzheimer disease (AD) patients reflects an imbalance between Ab
production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by
processing proteases generates soluble APP fragments including the neurotoxic amyloid Ab40 and
Ab42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended
time-frame, and the early detection and modulation of plaque formation are areas of active research.
Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important
for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurode-
generative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[3H]-2,5-bis(40-hydroxy-
30-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously
reported radioiodinated analog for radiometric quantification of Ab plaque and tau pathology in brain tis-
sue and in vitro studies with synthetic Ab and tau fibrils.
Keywords:
Tritium-labeled probe
Alzheimer’s disease
b-Amyloid
Detection of amyloid fibrils
Ó 2014 Elsevier Ltd. All rights reserved.
Accumulation of extracellular Ab senile plaques in brain tissue
and tangles of hyperphosphorylated tau protein inside brain neu-
rons are the classical histopathological signs of Alzheimer’s disease
(AD).1,2 According to the AD b-amyloid hypothesis,3,4 the imbalance
between the production and clearance of Ab from brains of AD
patients results from the alternative processing of the amyloid pre-
prevention13–16 of Ab deposition are of considerable importance.
The development of probes for amyloid plaques and tangles17,18
provides one avenue for the noninvasive diagnosis and evaluation
of AD progression in living subjects and its differentiation from
age-related cognitive decline in AD patients.19,20 We report the
synthesis of a radiolabeled Congo Red analog, (E,E)-1-[3H]-2,5-
bis(40-hydroxy-30-carboxystyryl)benzene (1a), which has not been
reported previously, and its nonradiolabeled counterpart21 1b,
which is commonly known as X-34 (Fig. 1). Both ligands bind to
fibrillar forms of both Ab and tau, and we demonstrate the utility
of 1a in studies of synthetic Ab40 and Ab42 fibrils. We also report a
cautionary note regarding the oxidative sensitivity of an intermedi-
ate in this route, namely 2,5-bis(40-hydroxy-30-carbomethoxysty-
ryl)-1-iodobenzene (2) (Fig. 1) and by analogy, a radioiodinated
version of 2, which has also been reported22 as a probe for Ab
deposition.
cursor protein5,6 (APP) by b- and
c-secretases resulting in the gen-
eration of soluble APP fragments:7 amyloid Ab38, Ab40, and Ab42
,
which assemble into plaques.8 Tau protein hyperphosphorylation
is induced by Ab leading to neurofibrillary tangle formation.9,10
The soluble oligomers of Ab42 that result from the assembly of
monomer produced by the sequential proteolytic cleavage of APP
by b- and c
-secretase are neurotoxic,11 cause neuroinflammation
and neuronal death, and ultimately result in cognitive impairment,
irreversible memory loss, and disorientation in AD.
Because Ab plaque formation occurs over a lengthy time period,
methods for the early and conclusive detection12, monitoring, and
Plans for the synthesis of 1a or 1b focused on the regioselective,
catalytic hydrogenolysis of a suitable iodinated 4-styrylstilbene,
namely (E,E)-2,5-bis(40-hydroxy-30-carbomethoxystyryl)-1-iodo-
benzene (2) (Fig. 2), using tritium gas and a palladium catalyst or
sodium borohydride and a palladium catalyst, respectively. Initial
efforts, as a consequence, focused on the synthesis of 2. Following
⇑
Corresponding authors. Tel.: +1 859 218 1026; fax: +1 859 257 6030 (D.S.W.);
tel.: +1 859 218 3329; fax: +1 859 323 2866 (H.L.).
E-mail addresses: dwatt@uky.edu (D.S. Watt), harry.levine@uky.edu (H. LeVine).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.09.075
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. V. Matveev et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5534–5536
5535
bromobenzene (12). Oxidative cyclization23–25 of other 1,4-bis
(styryl)benzenes to 3-styrylphenanthrenes suggested at least one
avenue for the oxidation of 2, and indeed, the treatment of non-
chromatographed, well characterized 2 with hydrogen peroxide
alone led to a plethora of products. In summary, we developed
an unambiguous route to 2,5-bis(40-hydroxy-30-carbomethoxysty-
ryl)-2-iodobenzene (2) that did not involve an oxidative process,
and we utilized 2, without further purification, in the synthesis
of the desired 1a and 1b, as discussed below. In our opinion, inves-
tigators opting to use the radioiodinated [125I]-version22 of 2 as a
probe for Ab deposition should be aware of this chemical instabil-
ity, avoid chromatographic purification on silica gel, and consider
the tritiated ligand 1a (Fig. 1) as a preferable alternative. It should
also be noted that the ‘cold’ ligand 1b is sufficiently fluorescent to
find application in its own right as a probe for Ab deposition.2
Catalytic hydrogenolysis26 of iodinated 4-styrylstilbene 2 using
sodium borohydride and a catalytic amount of tetrakis(triphenyl-
phosphine)palladium(0) furnished 2,5-bis(40-hydroxy-30-carbo-
methoxystyryl)benzene (1b) or X-34,2,27 as it is commonly known
in the literature (Fig. 3). The ‘cold’ 4-styrylstilbene (1b) was also
synthesized independently using the Horner–Emmons condensa-
tion of 5-formylsalicylic acid with p-xylenediphosphonic acid
tetraethyl ester.27 In order to achieve high specific activity, hydrog-
enolysis of 2 was performed using tritium gas and a palladium
catalyst to provide radiolabeled (E,E)-1-[3H]-2,5-bis(40-hydroxy-
30-carbomethoxystyryl)benzene (1a) at 23 Ci/mmole. Tritiated
sodium borohydride and the palladium catalyst could be used to
secure 1a, albeit at lower specific activities than those reported
here.
X
HO
HO2C
OH
CO2H
1a X = 3H
1b X = H
2
X =
I
Figure 1. Iodinated and tritiated (E,E)-bis-1,4-styrylbenzenes required as imaging
agents.
the procedure of Zhuang,22 the benzylic bromination of 2-bromo-
para-xylene (3) and Arbusov reaction with triethyl phosphite
provided tetraethyl (2-bromo-1,4-phenylene)bis(methylene)-
diphosphonate. Wadsworth–Emmons condensation of this
phosphonate with 3-carbomethoxy-4-methoxybenzaldehyde and
demethylation of the intermediate bisanisole 4 secured the bisphe-
nol 5 (Fig. 2). Efforts, however, to effect the tri-n-butylstannylation
of 5 were unsuccessful, contrary to a report22 claiming a 25% yield.
Suspecting that this failure was a consequence of the phenolic
hydroxyl groups in 5, we converted 5 to the bisacetate 6 and were
successful in converting 6 to the arylstannane 7, albeit only in
18–23% yields. Iodination of 7 using sodium iodide and hydrogen
peroxide, and saponification of 8 furnished the iodinated 4-styryl-
stilbene 2 in very poor yield. The low yield in the iodination
reaction was unexpected until subsequent work, as discussed
below, brought to our attention the oxidative sensitivity of 2.
The poor yields in the stannylation and iodination reactions led
us to explore an alternate route to the iodinated 4-styrylstilbene 2.
Elaboration of 2-iodo-p-xylene (9) to the iodinated bisphenol 11
followed a similar sequence to that described earlier (Fig. 2). The
acetylation of 11 afforded the bisacetate 8 that was identical to
material prepared from the route originating with 2-bromo-p-
xylene (3). The saponification of 8 and subsequent acidification
gave a precipitate that was unambiguously identified as the iodin-
ated 4-styrylstilbene 2 according to NMR and mass spectral data.
Efforts, however, to purify 2 by recrystallization or chromatogra-
phy were complicated by the apparent instability of 2 on exposure
to air. For example, the 1H NMR of a chromatographed sample of
the iodinated 4-styrylstilbene 2 did not display the sharp signals
seen in either the nonchromatographed sample of 2 or in the
brominated analog, 2,5-bis(40-hydroxy-30-carbomethoxystyryl)-1-
The binding of tritiated 1a was selective for the Congo Red-bind-
ing site on Ab40 and Ab42 fibrils and was displaced only by those
unlabeled ligands, including the ‘cold’ 1b that mimicked Congo
Red (Table 1). Nonspecific retention of 1a on the GF/B filter material
was very low, and as expected, ligands with structures similar to
Congo Red (i.e., possess extended
p systems) vied for binding
against 1a with efficacy values (EC50, Table 1) for either Ab40 or
Ab42 fibrils (i.e., displayed varied EC50 values) that varied with
substitution patterns. In contrast, benzothiazole ligands, such as
Thioflavine T, Pittsburgh Compound B (PIB), or 2-(40-methylamin-
ophenyl)benzothiazole (BTA-1), were ineffective in displacing 1a
as were several other molecules reported to bind to Ab fibrils but
with structures unlike the Congo Red structure. From the Scatchard
analysis, the binding stoichiometry of tritiated 1a approached one
ligand per two peptides. The similarity of the Scatchard Kd to the
EC50 for ‘cold’ 1b competition against tritiated 1a suggested
X
X
that the EC’50s were equivalent to Kd values, namely for Ab40
Kd = 0.60 M and the ratio of molecules of 1b/molecules of Ab40
(as monomers) = 0.43 and for Ab42, Kd = 0.25 M and the ratio of
,
a-c
l
H3C
CH3
RO
OR
l
4
5
X = Br, R = CH3
X = Br, R = H
d
d
3 X = Br
9 X = I
H3CO2C
CO2CH3
molecules of 1b/molecules of Ab42 (as monomers) = 0.63.
10 X = I, R = CH3
11 X = I, R = H
X
X
HO
HO2C
CHO
e
a
AcO
H3CO2C
OAc
CO2CH3
6
7
X = Br
HO
HO2C
OH
CO2H
f
g
X = Sn(n-Bu)3
1a X = 3
1b X = H
H
10 X = I
b or c
X
I
h
HO
OH
HO
HO2C
OH
CO2H
2
X = I
2
12 X = Br
HO2C
CO2H
Figure 2. Synthesis of (E,E)-2,5-bis(40-hydroxy-30-carboxystyryl)-1-iodobenzene
(2). (a) NBS (recrystallized), AIBN (cat), CCl4; (b) P(OCH2CH3)3; (c) NaOCH3, 3-
carbomethoxy-4-methoxybenzaldehyde, CH3OH; (d) BBr3, CH2Cl2, ꢀ78 °C; (e) Ac2O,
Py; (f) Pd(PPh3)4, (n-Bu)3SnSn(n-Bu)3; (g) NaI, H2O2; (h) NaOH, aq CH3OH.
Figure 3. Synthesis of (E,E)-1-[3H]-2,5-bis(40-methoxy-30-carboxystyryl)benzene
(1a) and its ‘cold’ analog (1b or X-34). (a) NaOCH3, p-xylenediphosphonic acid
tetraethyl ester; (b) tritium gas, proprietary palladium catalyst (ViTrax, Placentia,
CA) or [3H]-NaBH4, Pd(PPh3)4 to give 1a; (c) NaBH4, Pd(PPh3)4 to give 1b.

5536
S. V. Matveev et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5534–5536
Table 1
The contents are solely the responsibility of the authors and do
not necessarily represent the official views of the NIH, the NINDS,
or the NIGMS.
Displacement of 5 nM of (E,E)-1-[3H]-2,5-bis(40-hydroxy-30-carbomethoxystyryl)ben-
zene (1a) from synthetic Ab fibrils by unlabeled compounds. [3H]-X-34 is another
name for (E,E)-1-[3H]-2,5-bis(40-hydroxy-30-carbomethoxystyryl)benzene (1a); X-34
is another name for (E,E)-2,5-bis(40-hydroxy-30-carbomethoxystyryl)benzene (1b);
BSB, (E,E)-1-bromo-2,5-bis(40-hydroxy-30-carbomethoxystyryl)benzene; ISB, (E,E)-1-
iodo-2,5-bis(40-hydroxy-30-carbomethoxystyryl)benzene; K114, (E,E)-1-bromo-2,5-
bis(40-hydroxystyryl)benzene; BMB, (E,E)-1-methoxy-2,5-bis(40-aminostyryl)benzene
or as it also known 1,4-bis(4-aminophenylethenyl)-2-methoxybenzene; PIB,
Pittsburgh compound B; BTA-1, 2-(40-methylaminophenyl)benzothiazole; IMPY,
2-(40-dimethylaminophenyl)-6-iodo-imidazo[1,2-a]pyridine; ThT, Thioflavine T; ThS,
Thioflavin S
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.075.
References and notes
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Ab(1–40) EC50
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l
M
Ab(1–42) EC50, lM
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possesses an extended
p system like the pan-fibrillar amyloid,
Congo Red ligand, tritiated 1a will be useful for standardizing the
quantitative assessment of relationships between chemical struc-
tures of existing and future amyloid probes with the ligand binding
site types present on amyloid deposits.30,31 Ligands, like the one
reported here, will be important for understanding the molecular
mechanisms of deposition linked to AD and other neurodegenera-
tive diseases with misfolded protein pathology.
Acknowledgments
H.L. was supported by the Coins for Alzheimer’s Research Trust
(C.A.R.T.) of the Rotary Clubs of North Carolina, South Carolina, and
Georgia; the National Institute of Neurological Disorders and
Stroke (R21 NS080576); an unrestricted ‘Grants4Targets’ Grant
from Bayer Healthcare; and a University of Kentucky Research Sup-
port Grant. D.S.W. was supported by the Office of the Dean of the
College of Medicine and by NIH Grant Number P20 RR020171 from
the National Institute of General Medical Sciences to L. Hersh, P.I.
28. 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.; Mathis, C. A. J. Neurosci. 2005, 25, 10598.
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