Synthesis and evaluation of stilbenylbenzoxazole and stilbenylbenzothiazole derivatives for detecting β-amyloid fibrils

Article abstract of DOI:10.1016/j.bmcl.2007.10.004

This paper describes a novel series of stilbenylbenzoxazole (SBO) and stilbenylbenzothiazole (SBT) derivatives for β-amyloid specific binding probes. These 24 compounds were synthesized and evaluated by competitive binding assay against β-amyloid 1-42 (Aβ42) aggregates using [¹²⁵I]TZDM. All the derivatives displayed higher binding affinities with K_i value in the subnanomolar range (0.10-0.74 nM) than Pittsburgh Compound-B (PIB) (0.77 nM). Among these derivatives, SBT-2, 5-fluoroethoxy-2-{4-[2-(4-methylaminophenyl)vinyl]phenyl}benzothiazole, showed lowest K_i value (0.10 nM). In conclusion, the preliminary results suggest that these compounds are implying a possibility as a probe for detection of Aβ fibrils in Alzheimer's disease (AD) patients.

Full text of DOI:10.1016/j.bmcl.2007.10.004

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1534–1537
Synthesis and evaluation of stilbenylbenzoxazole and
stilbenylbenzothiazole derivatives for detecting b-amyloid fibrils
Ji Hoon Lee,a,b Seong Rim Byeon,a Soo Jeong Lim,c Seung Jun Oh,c Dae Hyuk Moon,c
Kyung Ho Yoo,a Bong Young Chungb,* and Dong Jin Kima,*
aCenter for Chemoinformatics Research, Korea Institute of Science and Technology,
PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bDepartment of Chemistry, Korea University, Anam-dong, Seongbuk-Gu, Seoul 136-701, Republic of Korea
cAsan Medical Center, Department of Nuclear Medicine, 388-1, Pungnap-2-Dong, Songpa-Gu, Seoul 138-736, Republic of Korea
Received 9 July 2007; revised 1 October 2007; accepted 3 October 2007
Available online 5 October 2007
Abstract—This paper describes a novel series of stilbenylbenzoxazole (SBO) and stilbenylbenzothiazole (SBT) derivatives for
b-amyloid specific binding probes. These 24 compounds were synthesized and evaluated by competitive binding assay against
b-amyloid 1–42 (Ab42) aggregates using [125I]TZDM. All the derivatives displayed higher binding affinities with Ki value in the subn-
anomolar range (0.10–0.74 nM) than Pittsburgh Compound-B (PIB) (0.77 nM). Among these derivatives, SBT-2, 5-fluoroethoxy-2-
{4-[2-(4-methylaminophenyl)vinyl]phenyl}benzothiazole, showed lowest Ki value (0.10 nM). In conclusion, the preliminary results
suggest that these compounds are implying a possibility as a probe for detection of Ab fibrils in Alzheimer’s disease (AD) patients.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a neurodegenerative disease
characterized as progressive memory loss and decrease
of cognitive function. In 1907, the first demented patient
was identified to have senile plaques (SPs) of b-amyloid
protein (Ab) aggregates and neurofibrillary tangles
(NFTs) formed of highly phosphorylated tau proteins
in the post-mortem brain tissue.1–3 Since then, SPs and
NFTs have become the two major pathological hall-
marks characteristic of AD and provided the basic for
the definitive diagnosis of AD. However, yet the diagno-
sis of this disease based on neurological observations is
often difficult and unreliable. Therefore, an increasing
focus on early identification and prevention highlights
a need for simpler diagnostic tools and robust biological
markers. At present, the Ab-aggregate-specific radiola-
beled imaging agents, using single photon emission com-
puted tomography (SPECT) or positron emission
tomography (PET), are needed for early detection or
monitoring of the progression and effectiveness of AD
treatment.4–6 A number of groups have studied to devel-
op Ab-specific binding probes, however those efforts
have been limited by low levels of specific binding in
brain regions and poor blood–brain barrier (BBB)
penetration.
Recently,
hydroxybenzothiazole ([11C]PIB),7,8 E,E-1-iodo-[125I]2,5-
bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene
([125I]-
N-methyl-[11C]2-(40-methylaminophenyl)-6-
IMSB),9,10 and [11C]4-N-methylamino-40-hydroxystilben
([11C]SB-13)11,12 displayed high binding affinities toward
Ab aggregates. PIB, a modified molecule of thioflavin-T
(Th-T), exhibited that neutral benzothiazole–aniline deriv-
atives could bind to amyloid with low nanomolar affinity,
enter brains in sufficient amounts for imaging via PET,
and clear rapidly from normal brain in animal studies
(Fig. 1).
125I
MeO
HO2C
OMe
CO2H
125
[
I]IMSB
11CH3
NH
HO
S
HO
11CH3
NH
N
11
11
Keywords: Alzheimer’s disease; b-Amyloid fibrils; PET imaging.
Corresponding authors. Tel.: +82 2 958 5142; fax: +82 2 958 5189;
e-mail: djk2991@kist.re.kr
[
C]PIB
[
C]SB-13
*
Figure 1. Structures of IMSB, PIB, and SB-13.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.004

J. H. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1534–1537
1535
5
target. The agent should have adequate affinity toward
the target and show rapid clearance of free and nonspe-
cific bound compounds from the brain. Based on the
backbone structures of PIB and SB-13, we have success-
fully developed highly conjugated SBO and SBT deriv-
atives (Fig. 2). Due to the short half-life of 11C, to
broaden the utility of a PET imaging agent, we have
then focused our effort on developing 18F-labeled imag-
ing agent (t1/2 = 110 min). In the beginning, we designed
fluoroethyl and fluoropropyl substituted SBO, SBT
derivatives. All the synthesized compounds were evalu-
ated by competitive binding assays against Ab aggre-
gates using [125I]TZDM.
N
X
R1
R2
6
SBO-1~12, SBT-1~12
SBO-1 : X=O, R1=5-O(CH2)2F, R2=NH2
SBO-2 : X=O, R1=5-O(CH2)2F, R2=NH(CH3)
SBO-3 : X=O, R1=5-O(CH2)2F, R2=N(CH3)2
SBO-4 : X=O, R1=6-O(CH2)2F, R2=NH2
SBO-5 : X=O, R1=6-O(CH2)2F, R2=NH(CH3)
SBO-6 : X=O, R1=6-O(CH2)2F, R2=N(CH3)2
SBO-7 : X=O, R1=5-O(CH2)3F, R2=NH2
SBO-8 : X=O, R1=5-O(CH2)3F, R2=NH(CH3)
SBO-9 : X=O, R1=5-O(CH2)3F, R2=N(CH3)2
SBO-10 : X=O, R1=6-O(CH2)3F, R2=NH2
SBT-1 : X=S, R1=5-O(CH2)2F, R2=NH2
SBT-2 : X=S, R1=5-O(CH2)2F, R2=NH(CH3)
SBT-3 : X=S, R1=5-O(CH2)2F, R2=N(CH3)2
SBT-4 : X=S, R1=6-O(CH2)2F, R2=NH2
SBT-5 : X=S, R1=6-O(CH2)2F, R2=NH(CH3)
SBT-6 : X=S, R1=6-O(CH2)2F, R2=N(CH3)2
SBT-7 : X=S, R1=5-O(CH2)3F, R2=NH2
SBT-8 : X=S, R1=5-O(CH2)3F, R2=NH(CH3)
SBT-9 : X=S, R1=5-O(CH2)3F, R2=N(CH3)2
SBT-10 : X=S, R1=6-O(CH2)3F, R2=NH2
SBO-11 : X=O, R1=6-O(CH2)3F, R2=NH(CH3) SBT-11 : X=S, R1=6-O(CH2)3F, R2=NH(CH3)
SBO-12 : X=O, R1=6-O(CH2)3F, R2=N(CH3)2 SBT-12 : X=S, R1=6-O(CH2)3F, R2=N(CH3)2
The synthesis of SBO and SBT derivatives is outlined in
Scheme 1. The first step of synthetic 4-(diethoxyphos-
phorylmethyl)benzoic acid ethyl ester was achieved with
4-(bromomethyl)benzoic acid and triethylphosphite via
Arbuzov reaction.13 Compound 3 was readily prepared
from compound 2 and 4-nitrobenzaldehyde via Horn-
er–Wadsworth–Emmons reaction and hydrolysis. The
key step for the formation of the benzoxazole and ben-
zothiazole backbones was accomplished via the intra-
molecular cyclization reaction14 between compound 3
and 2-aminophenol or 2-aminothiophenol derivatives.15
The free amino derivatives, compounds 5a–d were pre-
pared from the nitro compounds 4a–d via reduction
Figure 2. Structures of SBO and SBT derivatives.
However labeling of 11C radioisotope is limited to short
half-life (t1/2 = 20 min). SB-13 demonstrated similar
binding properties to those of PIB. IMSB showed lower
initial brain uptake in normal mice (0.14% ID/organ at
5 min after injection) than radioiodinated Th-T deriva-
tives (0.6–3.5% ID/organ at 2 min), but it displays po-
tent binding affinities for Ab aggregates.
Our focus was that a sufficient amount of an imaging
agent should internalize into the brain to bind to the
OEt
(a)
Br
(b)
P
HO2C
OEt
O
NO2
HO2C
(c)
EtO2C
1
2
3
N
X
N
X
(d)
R1
R1
NO2
NH2
4a: X=O, R1=5-OMe
4b: X=O, R1=6-OMe
4c: X=S, R1=5-OMe
4d: X=S, R1=6-OMe
5a~d
(g)
N
(e)
(f)
R1
(g)
N
X
X
NH
R1
R2
6a~d
8a : X=O, R1=5-OH, R2=NH2
8b : X=O, R1=5-OH, R2=NH(CH3)
8c : X=O, R1=5-OH, R2=N(CH3)2
8d : X=O, R1=6-OH, R2=NH2
8e : X=O, R1=6-OH, R2=NH(CH3)
8f : X=O, R1=6-OH, R2=N(CH3)2
8g : X=S, R1=5-OH, R2=NH2
8h : X=S, R1=5-OH, R2=NH(CH3)
8i : X=S, R1=5-OH, R2=N(CH3)2
8j : X=S, R1=6-OH, R2=NH2
N
R1
(g)
X
N
7a~d
N
X
(h)
R1
8k : X=S, R1=6-OH, R2=NH(CH3)
8l : X=S, R1=6-OH, R2=N(CH3)2
R2
SBO-1~12, SBT-1~12
Scheme 1. Synthesis of SBO and SBT derivatives. Reagents and conditions: (a) P(OEt)3, 140 ꢁC, 20 h; (b) i)—4-nitrobenzaldehyde, NaH, THF, rt,
2 h; ii)—NaOH, MeOH/H2O, reflux, 2 h; (c) i)—thionyl chloride, reflux, 1 h; ii)—2-aminophenol or 2-aminothiophenol derivatives, N,N-
dimethylaniline, monochlorobenzene, reflux, 1 h; iii)—p-TsOH, trichlorobenzene, reflux, 8 h; (d) SnCl2, EtOH, reflux, 24 h; (e) p-formaldehyde,
NaOMe, MeOH/THF, NaBH4, reflux, 3 h; (f) p-formaldehyde, NaBH3CN, AcOH, rt, 12 h; (g) BBr3, CH2Cl2, reflux, 12 h; (h) 1-fluoro-2-
tosyloxyethane or 1-fluoro-3-tosyloxypropane, K2CO3, DMF, 90 ꢁC, 2 h.

1536
J. H. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1534–1537
Table 1. Ki values of SBO and SBT derivatives against [125I]TZDM
for binding affinities to Ab42 aggregates
fully synthesized. These SBO and SBT derivatives
displayed excellent binding affinities to Ab aggregates.
In particular, SBT-2 exhibited the best binding affinity
(Ki = 0.10 nM) implying a possibility as a probe for
detection of Ab fibrils in AD brain. Based on the result,
further studies on synthesis and in vivo pharmacokinet-
ics of 18F-labeled compounds are progressing for the
development of AD imaging probe.
Compound
Kia (nM)
SBO-1
SBO-2
SBO-3
SBO-4
SBO-5
SBO-6
SBO-7
SBO-8
SBO-9
SBO-10
SBO-11
SBO-12
SBT-1
SBT-2
SBT-3
SBT-4
SBT-5
SBT-6
SBT-7
SBT-8
SBT-9
SBT-10
SBT-11
SBT-12
PIB
0.32
0.74
0.44
0.47
0.50
0.45
0.45
0.47
0.45
0.59
0.68
0.46
0.41
0.10
0.35
0.59
0.52
0.57
0.42
0.38
0.49
0.55
0.48
0.12
0.77
Acknowledgments
We are grateful to the Ministry of Science and Technol-
ogy (MOST) and Ministry of Commerce, Industry and
Energy (MCIE) of Korea for financial support.
References and notes
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a Ki was calculated by the Cheng–Prusoff equation (Ki = IC50/(1 + [L]/
Kd))20 using Graphpad Prism software.
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with SnCl2. Conversion of compounds 5a–d, to the
monomethylamino derivatives, compounds 6a–d, was
achieved via a method previously reported.16 Com-
pounds 5a–d were also converted to the dimethylamino
derivatives, compounds 7a–d, via an efficient method
with paraformaldehyde, sodium cyanoborohydride,
and acetic acid.17,18 The O-methyl group of compounds
5a–d, 6a–d, and 7a–d was removed by reacting with
BBr3 to give compounds 8a–8l. The desired SBO and
SBT derivatives were prepared from compounds 8a–8l
and 1-fluoro-2-tosyloxyethane or 1-fluoro-3-tosyloxy-
propane by a nucleophilic substitution reaction.19
Specific binding affinities of synthesized compounds to
Ab fibrils were evaluated by an in vitro Ab fibril binding
assay. In vitro competitive binding assay using pre-
formed Ab42 aggregates demonstrated that SBO-1–12,
SBT-1–12 competed against radioligand such as
13. Chaplais, G.; Bideau, J. L.; Leclercq, D.; Vioux, A. Chem.
Mater. 2003, 15, 1950.
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[
125I]TZDM.21–23
The result shown in Table 1 demonstrates that most of
the synthesized compounds displayed lower Ki values
(Ki = 0.10–0.74 nM) than PIB compound. In the struc-
ture–activity relationship, SBO and SBT derivatives
did not show significant difference of binding affinity.
Furthermore, 5-position compounds were slightly better
than 6-position compounds. Among them, 5-fluoroethyl
substituted SBT-224 compound exhibited the highest
binding affinity.
15. Hodson, S. J.; Bishop, M. J.; Speake, J. D.; Navas, F., III;
Garrison, D. T.; Bigham, E. C.; Saussy, D. L., Jr.; Liacos,
J. A.; Irving, P. E.; Gobel, M. J.; Sherman, B. W. J. Med.
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16. Barluenga, J.; Bayon, A. M.; Asensio, G. J. Chem. Soc.
Chem. Commun. 1984, 1334.
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Biol. 2002, 29, 633.
19. 1-Fluoro-2-tosyloxyethane and 1-fluoro-3-tosyloxypro-
pane were purchased from FutureChem Co., Ltd.
20. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
In conclusion, a series of novel fluoroethyl and fluoro-
propyl substituted SBO, SBT compounds were success-

J. H. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1534–1537
1537
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EtOH at room temperature. Filters containing the bound
radioligand were counted in a gamma-counter (Cobra-II).
The result of inhibition assays was subjected to nonlinear
regression analysis using software Graphpad Prism by
which Ki values were calculated.
23. We estimated Kd value (0.13 nM) of [125I]TZDM for Ab42
aggregates. For inhibition studies, the reaction mixture
contained 50 lL of Ab42 aggregates (11.5 nM in the final
concentration), 50 lL of inhibitors (10ꢀ6–10ꢀ12 M in
DMSO), 50 lL of [125I]TZDM (in 40% EtOH, 0.05 nM
in the final concentration), and 10% EtOH in a final
volume of 1 mL. Nonspecific binding was defined by
adding 2 lM Th-T for [125I]TZDM binding. The mixture
was incubated at room temperature for 3 h and the bound
and the free radioactivity were separated by a vacuum
filtration through Whatman GF/B filters using a Brandel
M-24R cell harvester followed by 2· 3 mL washes of 10%
24. Selected data. SBT-2: H NMR (DMSO-d6, 300 MHz) d
1
2.71 (d, J = 4.9 Hz, 3H), 4.36 (dt, J = 4.0, 30.3 Hz, 2H),
4.80 (dt, J = 4.0, 47.9 Hz, 2H), 6.01 (s, NH), 6.56 (d,
J = 8.4 Hz, 2H), 7.00 (d, J = 16.1 Hz, 1H), 7.12 (d,
J = 9.1 Hz, 1H), 7.26 (d, J = 16.5 Hz, 1H), 7.41 (d,
J = 8.3 Hz, 2H), 7.63 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H),
8.01 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H); 13C
NMR (DMSO-d6, 300 MHz) d 29.98, 67.85, 81.51, 106.73,
112.11, 115.84, 122.03, 123.19, 124.56, 126.55, 126.85,
127.82, 128.59, 131.17, 131.72, 141.63, 150.66, 155.45,
158.16, 168.87; HRMS m/z Calcd for C24H22FN2OS (M)+
405.1431. Found: 405.1433.