Development of dual targeting inhibitors against aggregations of amyloid-β and tau protein

Article abstract of DOI:10.1016/j.ejmech.2014.07.095

Aggregations of both amyloid-β (Aβ) and hyper-phosphorylated tau proteins are recognized as key pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of aggregation show promise as drug candidates. Seventeen oligo h

Full text of DOI:10.1016/j.ejmech.2014.07.095

European Journal of Medicinal Chemistry 85 (2014) 228e234
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of dual targeting inhibitors against aggregations
of amyloid- and tau protein
b
,
Shinichiro Fuse a *, Keisuke Matsumura a, Yuki Fujita b, Hachiro Sugimoto b,
Takashi Takahashi c
a Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8552, Japan
b Graduate School of Brain Science, Doshisha University, 4-1-1, Kizugawadai, Kizugawa-shi, Kyoto 619-0225, Japan
c Yokohama College of Pharmacy, 601, Matano-cho, Totsuka-ku, Yokohama-shi 245-0066, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Aggregations of both amyloid-
b (Ab) and hyper-phosphorylated tau proteins are recognized as key
Received 26 April 2014
Received in revised form
25 July 2014
Accepted 25 July 2014
Available online 26 July 2014
pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of ag-
gregation show promise as drug candidates. Seventeen oligo heteroaromatic compounds were rapidly
synthesized via a one-pot, 3- or 4-component coupling procedure. Evaluations showed that compounds
E16 and E18 were the most potent inhibitors of A
b
and tau aggregations (E16: IC50s ¼ 0.38, 0.29
M against A , tau, respectively).
© 2014 Elsevier Masson SAS. All rights reserved.
mM
against A
b
, tau, respectively, E18: IC50s ¼ 0.55, 0.30
m
b
Keywords:
Alzheimer's disease
Amyloid-
b
Tau
Aggregation
SuzukieMiyaura coupling
1. Introduction
evaluation of their inhibitory activity against the aggregations of A
and tau proteins. The dyes E16 and E18 showed high anti-
aggregating activity (IC50s against A and tau < 1 M), and they
b
Thirty-five million people suffer from Alzheimer's disease (AD)
worldwide, and the number of patients is estimated to triple by
2050. Moreover, in 2010 alone AD is estimated to have resulted in
$604 billion in global healthcare expenditures [1,2]. Senile plaques
(SPs) and neurofibrillary tangles (NFTs) are considered the key
b
m
were more potent than methylene blue.
2. Results and discussion
pathological hallmarks of AD. Aggregated amyloid-
hyper-phosphorylated tau proteins are the main components of SPs
and NFTs, respectively. Compelling evidence suggests that both A
and tau aggregations cause AD [1,3]. Several compounds, 1e6,
which targeted both the A and tau aggregations (IC50s < 3
against A and tau) were reported (Fig. 1) [4]. All the compounds
retained planer structures, and can be regarded as functional dyes.
Among these dyes, only methylene blue (3) has reached the level of
a phase III trial [2,5]. It should be noted that several dual targeting
b (Ab) and
Inspired by the fact that functional dyes 1e6 retained potent
inhibitory activity against A and tau protein aggregations, we
briefly tested the inhibitory activities of thiophene-based organic
dye 7 [8e12], which is a conventional donor- -acceptor dye for
dye-sensitized solar cells. The goal was to identify a new template
for Alzheimer's drug discovery. In addition, the structure of 7 could
be easily modified, which was needed in order to tune its properties
for drug development. Surprisingly, dye 7 showed a level of
inhibitory activity that compared favorably to those of methylene
blue (Fig. 2). Thus, we decided to synthesize a small library based
on 7.
b
b
p
b
mM
b
inhibitors against
Ab aggregation and acetyl/butyryl choline
esterase have been reported recently [6,7].
Herein, we wish to report the rapid synthesis of 17 functional
dyes via a one-pot, 3- or 4-component coupling procedure, and the
We have reported library synthesis based on SuzukieMiyaura
coupling [13e15]. Gruttadauria and co-workers have demonstrated
a
one-pot, 3-component coupling (SM coupling/Knoevenagel
condensation) [16]. In our study, a one-pot, 3- or 4-component
coupling (SM coupling/SM coupling or SM coupling/SM coupling/
Knoevenagel condensation) approach was employed for the library
* Corresponding author.
E-mail address: sfuse@apc.titech.ac.jp (S. Fuse).
http://dx.doi.org/10.1016/j.ejmech.2014.07.095
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
229
Fig. 1. (a) Structures of azure A (1), azure B (2), methylene blue (3), myricetin (4), gossypetin (5), and ferric-dehydroporphyrin IX (6), which retained potent inhibitory activities
against A (IC50s: 1; 0.4 M, 2; 0.3 M, 3; 2.3 M, 4; 0.9 M, 5; 1.3 M, 6; 0.2 M) and tau protein (IC50s: 1; 2.6 M, 2; 1.9 M, 3; 1.9 M, 4; 1.2 M, 5; 2.0 M, 6; 1.4 M) aggregations.
b
m
m
m
m
m
m
m
m
m
m
m
m
[20]} were examined. It seemed that the selection of a base was
important in this reaction. The use of K3PO4 afforded better yields
(entries 1, 3, and 5) compared with the use of Na2CO3 (entries 2, 4,
and 6), probably due to the better solubility of K3PO4 against the
toluene/EtOH solvent. The use of an electron-rich and bulky ligand,
[(t-Bu)3PH]BF4 for the first SM coupling resulted in low yields
(entries 5 and 6) due to the overreaction of A1 with the first
coupling product, A1-B1, to generate the undesired A1-B1-A1. The
combination of Pd2(dba)3 and Xantphos as a Pd catalyst and a
phosphine ligand for the first SM coupling afforded the best result
(entry 3). The conditions shown in entry 1 also afforded results
comparable to the best combination (entry 1) (Table 1).
Fig. 2. (a) Structure of the thiophene-based organic dye 7, and its inhibitory activities
against A and tau proteins aggregations.
b
The building blocks were coupled using the developed one-pot
conditions (Table 2). As a result, 8 compounds E3, E5, E7, E10, E11,
E13, E17, and E18 were successfully synthesized from the one-pot,
4-component coupling procedure, and 6 aldehydes, E2, E6, E9, E12,
E16, E19 were synthesized from the one-pot, 3-component
coupling procedure. In regard to the E- and Z-geometries of al-
kenes generated by Knoevenagel condensation between aldehydes
and cyanoacetamide (D2), 1H NMR analysis revealed that the
products retained the E-alkene form [21,22]. Unexpectedly, E4, E8,
E14, and E15 could not be obtained from the one-pot procedure. In
the case of E4, the building block D3 was not soluble against the
toluene/EtOH solvent that was utilized for the one-pot procedure;
synthesis, as shown in Fig. 3. We planned to couple donor block A
with the aromatic scaffold B at first because the CeBr bond of the
first coupling product would be less reactive compared with the
corresponding CeBr bond of the aromatic scaffold B, and, therefore,
an undesired overreaction affording an A-B-A compound would be
suppressed. The following SM coupling and Knoevenagel conden-
sation would afford the desired dyes.
The reaction conditions for the one-pot, 4-component coupling
of A1, B1, C, and D1 were explored (Table 1). Combinations of bases
(K3PO4 and Na2CO3), Pd catalysts {Pd(PPh3)4 [17] and Pd2(dba)3
[18]}, and phosphine ligands {Xantphos [19] and [(t-Bu)3PH]BF4
Table 1
One-pot, 4-component coupling of A1, B1, C, and D1.
Entry
Pd cat.
Ligand
Base
Yieldd
a
a
1
2
3
4
5
6
Pd(PPh3)4
Pd(PPh3)4
Pd2(dba)3
Pd2(dba)3
Pd2(dba)3
Pd2(dba)3
e
K3PO4
Na2CO3
K3PO4
Na2CO3
K3PO4
Na2CO3
38%
9%
40%
12%
6%
e
Xantphosc
Xantphosc
[(t-Bu)3PH]BF4
[(t-Bu)3PH]BF4
b
b
b
b
c
c
6%
a
1 mol%.
0.5 mol%.
2 mol%.
b
c
Fig. 3. Synthesis of structurally diverse organic dyes via a one-pot, 3- or 4-component
coupling (SM coupling/SM coupling or SM coupling/SM coupling/Knoevenagel
condensation) approach.
d
Isolated yield (one-pot, 3-step yield based on B1).

230
S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
thus, Knoevenagel condensation did not proceed. Therefore,
Knoevenagel condensation of the isolated aldehyde E2 (one-pot, 3-
component coupling product) with the building block D3 under
AcOH solvent conditions (D3 was soluble against AcOH) was carried
out to obtain the desired compound E4 (95% yield). In the case of
E8, the desired product was obtained with multiple undesired
compounds, and the purification was difficult due to the insuffi-
cient solubility of E8. Therefore, Knoevenagel condensation of the
isolated aldehyde E6 was carried out to obtain a purer form of E8.
As expected, simple short-pad column chromatography of the
Knoevenagel condensation product afforded E8 in a 53% yield. In
the cases of E14 and E15, the desired products could not be purified
due to their poor solubility.
retained potent inhibitory activity against both Ab and tau aggre-
gations. All the structural components in the dyes E16 and E18
seemed to be important for anti-aggregating activity against both
Ab and tau: structural alterations of the aldehyde and the cyano-
acryl amide to malononitrile (E16 to E17, E18 to E17), and struc-
tural alterations of the 4-methoxy benzene to triphenyl amine (E16
to E9, E18 to E11) decreased the anti-aggregating activities of E16
and E18 against both A
tert-butyl dithienopyrrole in E16 to thiophene (E16 to E12) also
decreased the anti-aggregating activities against both A and tau.
b and tau. In addition, structural alteration of
b
The IC50s of the most active compounds, E16 and E18, were
carefully determined (Table 4). The IC50s of methylene blue (3) and
dye 7 were also determined to be positive controls. To our delight,
The anti-aggregating activity against the A
b
[23, 24] and the tau
both dyes E16 and E18 retained a level of anti-Ab aggregation ac-
[25] of 17 synthesized organic dyes was briefly tested (Table 3). As a
result, 5 dyes, E2, E3, E13, E16, and E18, exerted IC50s of less than
tivity that compared favorably to those of methylene blue (3) and
dye 7. Moreover, both dyes E16 and E18 exerted approximately 10
times more potent anti-tau aggregation activity. As far as we could
ascertain, dyes E16 and E18 are the first compounds to show sub-
10
blocks and showed no potent anti-aggregating activity against A
and tau proteins (IC50s > 10 M). This detrimental effect of the
mM, whereas the 5 dyes, E4eE8 retained D3, D4, or B2 building
b
m
micromolar IC50s against the aggregation of both A
protein.
b and tau
building blocks D3, D4, and B2 on the inhibitory activities might be
attributable to a decrease in molecular planarity, because these
building blocks tended to twist the p-conjugated plane in order to
reduce steric repulsions between adjacent aromatic rings. In terms
of inhibitory activity against tau aggregation, 2 dyes, E16 and E18,
retained A2 and B3 and 1 dye, E19, retained an A3 building block,
and these exerted high inhibitory activity (inhibitory rate of E16:
3. Conclusion
In summary, we demonstrated the rapid synthesis of 17
structurally-diverse, organic dyes via our originally developed one-
pot, 3- or 4-component coupling procedure (SM coupling/SM
coupling or SM coupling/SM coupling/Knoevenagel condensation).
Anti-aggregating activities of the synthesized dyes were evaluated,
and 2 compounds, E16 and E18, showed sub-micromolar IC50s
74% at 1
remaining 14 dyes showed no potent anti-aggregating activity
against tau proteins (IC50s > 10 M). It is interesting that active dyes
E16, E18, and E19 retained pyrrole and bithiophene moieties. Three
dyes E2, E3, and E13 showed inhibitory activity only against A
mM, IC50 of E18 ¼ 1.4
mM, IC50 of E19 ¼ 1.6
mM), whereas the
m
b
against the aggregation of both Ab and tau proteins. As far as we
aggregation. Among the 17 synthesized dyes, only 2, E16 and E18,
could ascertain, these compounds are the first to demonstrate this
quality. Thus, we have successfully identified a totally new template
for Alzheimer's drug discovery.
Although we tried to measure the log Ps of the most active dyes
E16 and E18, they could not be determined due to their very poor
solubility against water. It is important to improve the water sol-
ubility of these dyes by introducing hydrophilic groups to further
develop them for use as an Alzheimer's drug.
Table 2
One-pot, 3- or 4-component coupling for the synthesis of functional dyes.
Entry
Substrate
Product
Yield
A
B
D
E
1
2
3
4
5
6
7
8
A1
A1
A1
A1
A1
A1
A1
A1
A1
A1
A2
A2
A2
A2
A2
A2
A2
A3
B1
B1
B1
B1
B2
B2
B2
B3
B3
B3
B1
B1
B1
B2
B3
B3
B3
B1
e
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
55%a
29%b
(95%)c
23%b
74%a
56%b
(53%)d
28%a
30%b
27%b
48%a
22%b
ee
D2
D3
D4
e
4. Experimental
4.1. Chemistry
D1
D2
e
NMR spectra were recorded on a JEOL Model ECP-400 (400 MHz
for 1H, 100 MHz for 13C) instrument for the indicated solvent.
Chemical shifts were reported in units of parts per million (ppm)
relative to the signal for internal tetramethylsilane (0.00 ppm for
1H) for solutions in CDCl3. The 1H NMR spectral data were reported
as follows: CDCl3 (7.26 ppm), DMSO-d6 (2.49 ppm). 13C NMR
spectral data were reported as follows: CDCl3 (77.0 ppm), DMSO-d6
(39.5 ppm). Multiplicities were reported using the following ab-
breviations: s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
m, multiplet; br. broad; J, coupling constants in Hertz. The IR
spectra were recorded on a Perkin Elmer Spectrum One FT-IR
spectrophotometer. Only the strongest and/or structurally impor-
tant absorption data are reported as the IR data given in cmꢀ1. All
reactions were monitored by thin-layer chromatography carried
out on 0.2 mm E. Merck silica gel plates (60F-254) with UV light,
visualized by p-anisaldehyde solution, ceric sulfate or 10% ethanolic
phosphomolybdic acid. Column chromatography was performed
on Kanto Chemical Co. silica gel 60 (0.063e0.200 mm). ESI-TOF
Mass spectra were measured using a Waters LCT Premier™ XE.
The HRMS (ESI-TOF) was calibrated using leucine enkephalin.
9
D1
D2
e
10
11
12
13
14
15
16
17
18
D1
D2
e
ee
e
34%a
34%b
11%b
42%a
D1
D2
e
a
Isolated yields of one-pot, 3-component SM coupling based on B.
Isolated yields of one-pot, 4-component coupling based on B.
The building block, D3 was poorly soluble against toluene/EtOH solvent,
b
c
therefore, the desired product, E4, was not obtained via one-pot, 4-component
coupling. E4 was synthesized from Knoevenagel condensation of the isolated E2
and the building block D3 (one-pot, 3-component coupling product) under AcOH
solvent conditions. The yield of Knoevenagel condensation is shown in parenthesis.
d
The desired product of one-pot, 4-component coupling, E8, could not be sepa-
rated from the undesired products due to its insufficient solubility. Knoevenagel
condensation (piperidine, CH2Cl2/MeCN ¼ 2/1, 40 ꢁC) of the isolated E6 and the
following short-pad column chromatography afforded the desired E8. The yield of
Knoevenagel condensation is shown in parenthesis.
e
The desired products could not be purified due to their poor solubility.

S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
Table 3 (continued )
231
Table 3
Inhibitory activities of the synthesized 17 compounds against
aggregations.
Ab and tau
Entry Compound
Structure
IC50
[mM] IC50 [mM]
against A against tau
Entry Compound
Structure
IC50
[mM] IC50 [mM]
against A against tau
12
13
14
E12
E13
E16
>10
0% at
>10
18% at
10
m
Ma
10 m
Ma
1
2
E1
E2
>10
>10
43% at
14% at
10
m
Ma
10 m
Ma
<1
>10
28% at
78% at
1
m
Ma
10 m
Ma
7.1
>10
12% at
10
Ma
m
<1
61% at
Ma
<1
74% at
1
m
1 m
Ma
3
4
5
E3
E4
E5
1.5
>10
29% at
10
Ma
m
15
E17
>10
18% at
>10
27% at
>10
0% at
10
Ma
>10
12% at
10
Ma
10
m
Ma
10 m
Ma
m
m
16
17
E18
E19
1.8
1.4
1.6
>10
18% at
10
Ma
>10
7% at
Ma
m
0
m
>10
46% at
10 m
Ma
6
7
8
9
E6
E7
E8
E9
>10
44% at
10
Ma
>10
11% at
m
0 m
Ma
18
7
<1
1.7
2.2
82% at
1
m
Ma
>10
30% at
10
Ma
>10
19
Methylene
blue (3)
0.9
15% at
m
0 m
Ma
a
Inhibitory rates at indicated concentrations of compounds.
4.2. General procedure for one-pot, 3-component coupling (SM
coupling/SM coupling)
>10
46% at
10
Ma
>10
24% at
m
10 m
Ma
A solution of A block (1.10 equiv.), B block (1.00 equiv.),
Pd(PPh3)4 (0.0100 equiv.), and K3PO4 (2.00 equiv.) in a mixture of
toluene (5.00 mL/mmol) and EtOH (5.00 mL/mmol) was degassed
with argon. After being stirred at 70 ꢁC for 8e10 h (monitored by
TLC), the mixture was cooled to room temperature, then 5-
formylthiophenyl-2-boronic acid (C) (1.40 equiv.), Pd2(dba)3
(0.0300 equiv.), [(t-Bu)3PH]BF4 (0.0500 equiv.), and K3PO4
(2.00 equiv.) were added. After being stirred at room temperature
for 2e10 h (monitored by TLC), the mixture was poured into water,
and the aqueous layer was extracted with two portions of chloro-
form. The organic layer was dried over MgSO4, and evaporated in
vacuo. The residue was purified by column chromatography on
silica gel with toluene and further purified by recrystallization with
chloroform:hexane to give the product.
>10
36% at
10
Ma
>10
15% at
10
Ma
m
m
10
11
E10
E11
>10
16% at
10
Ma
>10
18% at
10
Ma
m
m
4.2.1. Compound E2 [8,10,11,26e30]
>10
36% at
10
Ma
>10
33% at
10
Ma
Orange solid; yield 55%; Mp 137e138 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
9.84 (s, 1H), 7.64 (d, 1H, J ¼ 4.4 Hz), 7.44 (d, 2H, J ¼ 8.7 Hz),
m
m
7.30e7.24 (m, 5H), 7.22 (d, 1H, J ¼ 4.4 Hz), 7.15 (d, 1H, J ¼ 4.4 Hz),
7.12 (d, 4H, J ¼ 8.7 Hz), 7.07e7.04 (m, 4H); 13C NMR (100 MHz,

232
S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
Table 4
132.5, 128.2, 128.1, 126.7, 124.4,124.0, 123.3, 119.3, 117.3, 112.1, 101.7;
IC50s of compounds E16 and E18 against Ab and tau proteins aggregation.
FT-IR (neat): 3272, 1633, 1438, 1382, 1317, 1232, 1105, 1066, 1047,
881, 782, 767, 680, 576, 482 cmꢀ1; HRMS (ESI-TOF): calcd. for
Entry
Compound
IC50
[
m
M]a against A
IC50 [m
M]a against tau
C
17H12NOS2 [MþH]þ 310.0360, found 310.0341.
1
2
3
4
E16
E18
7
0.38
0.55
0.41
0.55
0.29
0.30
3.6
4.3. General procedure for one-pot, 4-component coupling (SM
coupling/SM coupling/Knoevenagel condensation)
3
2.4
a
Average values from two independent experiments.
A solution of A block (1.00 equiv.), B block (1.10 equiv.),
Pd(PPh3)4 (0.0100 equiv.), and K3PO4 (2.00 equiv.) in a mixture of
toluene (5.00 mL/mmol) and EtOH (5.00 mL/mmol) was degassed
with argon. After being stirred at 70 ꢁC for 8e10 h (monitored by
TLC), the mixture was cooled to room temperature, then 5-
formylthiophenyl-2-boronic acid (C) (1.40 equiv.), Pd2(dba)3
(0.0300 equiv.), [(t-Bu)3PH]BF4 (0.0500 equiv.), and K3PO4
(2.00 equiv.) were added. After being stirred at room temperature
for 2e10 h (monitored by TLC), D block (malononitrile, cyanoa-
crylamide, or N,N-dimethylbarbituric acid, 3.00 equiv.) was added.
After being stirred at room temperature for 1e2 h (malononitrile or
N,N-dimethylbarbituric acid) or 5e6 h (cyanoacetamide), the
mixture was poured into water, and the aqueous layer was
extracted with two portions of chloroform. The organic layer was
dried over MgSO4, and evaporated in vacuo. The residue was puri-
fied by column chromatography on silica gel with toluene and
further purified by recrystallization with chloroform:hexane to give
the product.
CDCl3):
d 182.3, 148.0, 147.4, 147.2, 146.2, 141.2, 137.4, 134.0, 129.4,
127.2, 127.0, 126.6, 124.8, 123.7, 123.4, 123.2, 123.1; FT-IR (neat)
3065,1736,1661,1592,1522,1490,1455,1327,1282,1229,1050, 882,
830, 791, 753, 696, 528, 478 cmꢀ1; HRMS (ESI-TOF): calcd. for
C
27H20NOS2 [MþH]þ 438.0986 found 438.0986.
4.2.2. Compound E6 [31,32]
Red solid; yield 74%; Mp 169e172 ꢁC; 1H NMR (400 MHz, CDCl3):
d
9.98 (s, 1H), 8.22 (d, 1H, J ¼ 3.9 Hz), 8.05 (d, 1H, J ¼ 7.8 Hz), 7.89 (d,
2H, J ¼ 8.7 Hz), 7.86 (d, 1H, J ¼ 3.9 Hz), 7.75 (d, 1H, J ¼ 7.8 Hz), 7.30
(dd, 4H, J ¼ 8.8, 7.3 Hz), 7.22e7.18 (m, 6H), 7.09 (t, 2H, J ¼ 7.3 Hz); 13
C
NMR (100 MHz, CDCl3):
d 182.9, 153.8, 152.7, 148.9, 148.6, 147.3,
143.3, 136.7, 134.6, 130.0, 129.9, 129.4, 127.8, 127.6, 126.7, 125.1,
124.2, 123.6, 122.4; FT-IR (neat): 3034, 2317, 1664, 1591, 1510, 1487,
1443, 1331, 1283, 1222, 1050, 881, 818, 754, 697, 668, 623, 513 cmꢀ1
;
HRMS (ESI-TOF): calcd. for C29H20N3OS2 [MþH]þ 490.1048, found
490.1021.
4.3.1. Compound E1 [27,29]
4.2.3. Compound E9
Black solid; yield 38%; Mp 182e184 ꢁC; 1H NMR (400 MHz,
Red solid; yield 28%; Mp 207e210 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
7.74 (s, 1H), 7.62 (d, 1H, J ¼ 4.4 Hz), 7.46 (d, 2H, J ¼ 8.8 Hz),
CDCl3):
d
9.83 (s, 1H), 7.65 (d,1H, J ¼ 3.8 Hz), 7.50e7.48 (m, 3H), 7.34
7.40 (d, 1H, J ¼ 3.9 Hz), 7.31e7.26 (m, 5H), 7.21 (d, 1H, J ¼ 3.9 Hz),
(s, 1H), 7.29e7.26 (m, 4H), 7.22 (d, 1H, J ¼ 3.8 Hz), 7.13 (d, 4H,
7.13 (d, 4H, J ¼ 7.7 Hz), 7.10e7.05 (m, 4H); 13C NMR (100 MHz,
J ¼ 7.8 Hz), 7.09e7.03 (m, 4H), 1.84 (s, 9H); 13C NMR (100 MHz,
CDCl3):
d 149.8, 149.7, 148.6, 148.0, 147.3, 139.9, 133.2, 129.5, 128.4,
CDCl3):
d 182.1, 149.2, 147.4, 145.1, 143.0, 142.7, 140.5, 137.6, 131.6,
126.8, 126.7, 125.1, 124.1, 123.7, 123.5, 122.9, 114.3, 113.5, 75.9; FT-IR
(solid): 3022, 2924, 2855, 2215, 1567, 1534, 1485, 1439, 1322, 1230,
1143, 1055, 935, 806, 754, 727, 694, 516 cmꢀ1; HRMS (ESI-TOF):
calcd. for C30H20N3S2 [MþH]þ 486.1099, found 486.1098.
129.3, 129.2, 126.4, 124.6, 123.6, 123.2, 122.9, 118.2, 115.1, 113.5,
109.9, 58.4, 30.7; FT-IR (neat): 2983, 1658, 1591, 1505, 1494, 1420,
1370, 1281, 1229, 1094, 1051, 798, 753, 697, 513 cmꢀ1; HRMS (ESI-
TOF): calcd. for C35H29N2OS3 [MþH]þ 589.1442, found 589.1491.
4.3.2. Compound E3
4.2.4. Compound E12
Red solid; yield 29%; Mp 229e230 ꢁC; 1H NMR (400 MHz,
Yellow solid; yield 48%; Mp 166e169 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
8.34 (s, 1H), 7.82 (d, 1H, J ¼ 3.9 Hz), 7.76 (br-s, 1H), 7.66
CDCl3):
d
9.85 (s, 1H), 7.66 (d, 1H, J ¼ 3.8 Hz), 7.54 (d, 2H, J ¼ 8.7 Hz),
(br-s, 1H), 7.62 (d, 2H, J ¼ 8.8 Hz), 7.57 (d, 1H, J ¼ 3.9 Hz), 7.53 (d, 1H,
J ¼ 3.9 Hz), 7.47 (d, 1H, J ¼ 3.9 Hz), 7.34 (dd, 4H, J ¼ 8.6, 7.3 Hz),
7.11e7.06 (m, 6H), 6.96 (d, 2H, J ¼ 8.8 Hz); 13C NMR (100 MHz,
7.31 (d, 1H, J ¼ 3.9 Hz), 7.23 (d,1H, J ¼ 3.8 Hz), 7.15 (d, 1H, J ¼ 3.9 Hz),
6.93 (d, 2H, J ¼ 8.7 Hz), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3):
d
182.4, 159.8, 147.4, 146.3, 141.3, 137.4, 134.1, 127.2, 126.3, 123.7,
DMSO-d6):
d 162.6, 147.5, 146.6, 145.0, 144.3, 143.2, 139.9, 133.9,
123.1, 114.5, 55.4; FT-IR (neat): 2934, 1660, 1607, 1506, 1444, 1286,
1260, 1179, 1114, 1027, 880, 831, 795, 752, 482 cmꢀ1; HRMS (ESI-
TOF): calcd. for C16H13O2S2 [MþH]þ 301.0357, found 301.0338.
133.2, 129.7, 128.0, 126.6, 126.4, 124.6, 124.2, 123.7, 122.5, 116.9,
100.7; FT-IR (solid): 3347, 3189, 3064, 2207, 1735, 1673, 1577, 1542,
1490, 1445, 1377, 1282, 1052, 794, 754, 696, 517 cmꢀ1; HRMS (ESI-
TOF): calcd. for C30H22N3OS2 [MþH]þ 504.1204, found 504.1184.
4.2.5. Compound E16
Orange solid; yield 34%; Mp 225e227 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
9.84 (s, 1H), 7.66 (d, 1H, J ¼ 4.4 Hz), 7.57 (d, 2H, J ¼ 8.8 Hz),
4.3.3. Compound E5
7.49 (s, 1H), 7.33 (s, 1H), 7.23 (d, 1H, J ¼ 4.4 Hz), 6.94 (d, 2H,
This compound was purified by column chromatography on
silica gel with 90:10 toluene:ethyl acetate and further purified by
recrystallization with toluene:hexane.
J ¼ 8.8 Hz), 3.85 (s, 3H), 1.85 (s, 9H); 13C NMR (100 MHz, CDCl3):
d
182.1, 159.4, 149.2, 145.1, 143.1, 142.6, 140.5, 137.5, 131.5, 128.1,
Dark red solid; Mp 253e254 ꢁC; 1H NMR (400 MHz, CDCl3):
126.9, 122.9, 118.1, 115.0, 114.4, 113.5, 109.9, 58.4, 55.4, 30.7; FT-IR
(neat): 2984, 1659, 1526, 1506, 1469, 1421, 1370, 1293, 1230, 1180,
1056, 1028, 831, 805, 752, 662, 511 cmꢀ1; HRMS (ESI-TOF): calcd. for
d
8.59 (s, 1H), 7.75 (d, 1H, J ¼ 3.9 Hz), 7.49e7.45 (m, 3H), 7.33e7.27
(m, 5H), 7.20 (d,1H, J ¼ 3.9 Hz), 7.13 (d, 4H, J ¼ 7.3 Hz), 7.09e7.05 (m,
4H), 3.43 (s, 3H), 3.40 (s, 3H); 13C NMR (100 MHz, CDCl3):
162.8,
C
24H22NO2S3 [MþH]þ 452.0813, found 452.0825.
d
162.1, 154.0, 151.4, 148.3, 148.1, 147.5, 147.2, 147.1, 135.2, 134.3, 129.4,
128.2, 126.9, 126.6, 124.9, 124.2,123.6, 123.5, 122.9, 108.5, 28.8, 28.1;
FT-IR (solid): 2924, 1724, 1665, 1590, 1563, 1538, 1489, 1421, 1352,
4.2.6. Compound E19
Yellow solid; yield 42%; Mp 190e193 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
11.27 (s,1H), 9.86 (s,1H), 7.96 (d,1H, J ¼ 3.9 Hz), 7.89 (s,
1324, 1286, 1158, 1079, 969, 828, 796, 752, 697, 522, 493 cmꢀ1
;
1H), 7.55 (d, 1H, J ¼ 3.9 Hz), 7.49e7.40 (m, 5H), 6.49 (br-s, 1H); 13
C
HRMS (ESI-TOF): calcd. for C33H26N3O3S2 [MþH]þ 576.1416, found
NMR (100 MHz, DMSO-d6):
d
183.5, 147.8, 146.0, 140.6, 139.2, 136.0,
576.1409.

S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
233
4.3.4. Compound E7
(ESI-TOF): calcd. for
518.1006.
C
27H24N3O2S3 [MþH]þ 518.1031, found
Black solid; yield 56%; Mp 246e250 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
8.27 (d, 1H, J ¼ 4.4 Hz), 8.10 (d, 1H, J ¼ 7.2 Hz), 7.91 (d, 2H,
J ¼ 8.7 Hz), 7.87 (d, 1H, J ¼ 4.4 Hz), 7.85 (s, 1H), 7.76 (d, 1H,
4.4. Synthesis of compound E8
J ¼ 7.2 Hz), 7.31 (dd, 4H, J ¼ 8.7, 7.2 Hz), 7.21e7.19 (m, 6H), 7.10 (t, 2H,
J ¼ 7.2 Hz); 13C NMR (100 MHz, CDCl3):
d
153.8, 152.6, 151.1, 150.4,
A solution of compound E6 (40.0 mg, 0.0820 mmol, 1.00 equiv.),
cyanoacetamide (D2) (13.8 mg, 0.164 mmol, 2.00 equiv.), and
148.8, 147.2, 139.0, 135.4, 135.3, 130.1, 129.6, 129.5, 128.2, 128.1,
126.6, 125.2, 123.8, 123.2, 122.3, 114.2, 113.4; FT-IR (neat): 3036,
2223, 1590, 1569, 1537, 1487, 1434, 1327, 1273, 1196, 1112, 815, 757,
697, 609, 510 cmꢀ1; HRMS (ESI-TOF): calcd. for C32H20N5S2 [MþH]þ
538.1160, found 538.1140.
piperidine (0.0410 mmol, 4.00
mL, 0.500 equiv.) in a mixture of
dichloromethane (1.00 mL) and acetonitrile (0.500 mL) was stirred
at 40 ꢁC for 6.5 h under argon. The mixture was poured into satu-
rated aq. NH4Cl and the aqueous layer was extracted with three
portions of dichloromethane. The organic layer was dried over
MgSO4 and evaporated in vacuo. The residue was purified by col-
umn chromatography on silica gel with chloroform to give the
product E8 (24.0 mg, 0.0432 mmol, 53%).
4.3.5. Compound E10
Black solid; yield 30%; 1H NMR (400 MHz, CDCl3):
d 7.68 (s, 1H),
7.54 (d, 1H, J ¼ 3.9 Hz), 7.52 (s, 1H), 7.49 (d, 2H, J ¼ 8.7 Hz), 7.33 (s,
1H), 7.28 (dd, 4H, J ¼ 8.7, 7.3 Hz), 7.21 (d, 1H, J ¼ 3.9 Hz), 7.13 (d, 4H,
J ¼ 8.7 Hz), 7.09e7.04 (m, 4H), 1.85 (s, 9H); 13C NMR (100 MHz,
Red solid; Mp 273e274 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d 8.41
(s, 1H), 8.31 (d, 1H, J ¼ 7.2 Hz), 8.27 (d, 1H, J ¼ 4.3 Hz), 7.99e7.92 (m,
CDCl3):
d 151.8, 149.5, 147.7, 147.3, 146.2, 144.4, 143.1, 140.7, 132.3,
4H), 7.82 (br-s, 1H), 7.71 (br-s, 1H), 7.36 (dd, 4H, J ¼ 8.3, 7.8 Hz),
130.8, 129.4, 128.9, 126.4, 124.7, 123.4, 123.3, 123.2, 119.8, 115.1,
114.8, 114.3, 114.0, 109.8, 73.8, 58.6, 30.8; FT-IR (neat): 3024, 2218,
1590, 1570, 1517, 1463, 1417, 1372, 1347, 1282, 1232, 1063, 785, 752,
725, 694, 603, 509 cmꢀ1; HRMS (ESI-TOF): calcd. for C38H29N4S3
[MþH]þ 637.1554, found 637.1493.
7.12e7.07 (m, 8H); 13C NMR (100 MHz, DMSO-d6):
d 162.6, 153.0,
151.8, 147.8, 146.7, 146.1, 143.2, 138.2, 136.7, 133.0, 130.2, 129.7, 127.7,
127.6, 127.1, 124.6, 123.7, 123.3,121.9, 116.8, 101.8; FT-IR (neat): 3172,
2209, 1679, 1576, 1512, 1486, 1441, 1381, 1330, 1286, 1225, 902, 813,
754, 698, 511 cmꢀ1; HRMS (ESI-TOF): calcd. for C32H22N5OS2
[MþH]þ 556.1266, found 556.1218.
4.3.6. Compound E11
Black solid; yield 27%; 1H NMR (400 MHz, DMSO-d6):
d
8.32 (s,
4.5. Synthesis of compound E4
1H), 7.85 (s, 1H), 7.80 (d, 1H, J ¼ 3.9 Hz), 7.74e7.59 (m, 6H), 7.31 (dd,
4H, J ¼ 8.2, 7.7 Hz), 7.08e7.03 (m, 6H), 6.98 (d, 2H, J ¼ 8.7 Hz), 1.81
A solution of compound E2 (10.0 mg, 0.0229 mmol, 1.00 equiv.),
barbituric acid (2.9 mg, 0.023 mmol, 1.0 equiv.), and acetic anhy-
(s, 9H); 13C NMR (100 MHz, DMSO-d6):
d
162.7, 146.8, 146.7, 146.5,
145.0,143.0,142.8,141.9,139.8,133.1,131.0,129.5,128.8,126.3,124.1,
123.9, 123.3, 123.1, 117.0, 116.7, 114.1, 114.0, 110.7, 99.8, 58.4, 30.1; FT-
IR (neat): 3183, 2205, 1685, 1574, 1519, 1494, 1419, 1368, 1284, 1094,
941, 797, 754, 697, 611, 509 cmꢀ1; HRMS (ESI-TOF): calcd. for
dride (11.3 mL, 0.115 mmol, 5.00 equiv.) in acetic acid (1.00 mL) was
refluxed for 3.5 h. The mixture was evaporated in vacuo. The residue
was purified by column chromatography on silica gel with chlo-
roform to give compound E4 (11.9 mg, 0.0218 mmol, 95%).
Dark red solid; Mp 291e293 ꢁC; 1H NMR (400 MHz, DMSO-d6):
C
38H31N4OS3 [MþH]þ 655.1660, found 655.1636.
d
11.25 (s, 1H), 11.24 (s, 1H), 8.46 (s, 1H), 8.14 (d, 1H, J ¼ 3.9 Hz), 7.67
4.3.7. Compound E13
(d, 1H, J ¼ 3.9 Hz), 7.65e7.61 (m, 3H), 7.50 (d, 1H, J ¼ 3.9 Hz), 7.34
(dd, 4H, J ¼ 8.2, 7.8 Hz), 7.12e7.07 (m, 6H), 6.96 (d, 2H, J ¼ 8.7 Hz);
Dark red solid; yield 22%; Mp 193e197 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
7.74 (s, 1H), 7.63 (d, 1H, J ¼ 3.9 Hz), 7.55 (d, 2H, J ¼ 8.7 Hz),
13C NMR (100 MHz, DMSO-d6):
d 163.4, 163.3, 151.1, 150.3, 147.6,
7.39 (d, 1H, J ¼ 3.8 Hz), 7.26 (d, 1H, J ¼ 3.9 Hz), 7.20 (d, 1H,
147.4, 146.6, 145.8, 145.2, 134.7, 133.6, 129.7, 128.6, 126.7, 126.3,
124.6, 124.4, 123.7, 122.3, 110.0; FT-IR (solid): 3203, 3068, 2924,
2853, 2382, 1740, 1660, 1568, 1487, 1398, 1264, 1199, 1093, 795, 754,
697, 621, 545, 517 cmꢀ1; HRMS (ESI-TOF): calcd. for C31H22N3O3S2
[MþH]þ 548.1103, found 548.1097.
J ¼ 3.8 Hz), 6.94 (d, 2H, J ¼ 8.7 Hz), 3.86 (s, 3H); 13C NMR (100 MHz,
CDCl3)
d 160.3, 149.9, 149.7, 147.9, 139.9, 133.2, 128.4, 127.3, 126.1,
124.1, 123.5, 114.7, 114.3, 113.5, 75.9, 55.4; FT-IR (neat) 2936, 2218,
1603,1570,1508,1488, 1428,1294,1230,1175,1145, 1059, 1030, 932,
843, 813, 799, 788, 604, 495 cmꢀ1; HRMS (ESI-TOF); calcd. for
C19H13N2OS2 [MþH]þ 349.0469, found 349.0435.
4.6. Preparation of tau protein (3R MBD)
4.3.8. Compound E17
The His-tagged 3R MBD of human brain tau expressed in E. coli
BL21(DE3) was kindly provided by Professor T. Ishida (Osaka Univ.
of Pharmaceutical Sciences) [25]. Bacterial culture was grown with
shaking at 37 ꢁC until the OD600 reached 0.5. The expression of 3R
MBD was induced by adding IPTG. After shaking for 4 h at 37 ꢁC,
bacterial cells were collected by centrifuge. Bacterial pellets were
resuspended in 50 mM TriseHCl, pH 7.6, 50 mM NaCl and soni-
cated. Then the homogenates were centrifuged and the superna-
tants were purified on the Ni-chelating chromatography using Ni
Sepharose 6 Fast Flow (GE Healthcare, Amersham, UK). 3R MBD
was eluted with 50 mM TriseHCl, pH 7.6, 500 mM NaCl, and
100 mM imidazole. The eluted solution containing 3R MBD was
dialyzed against 100 mM ammonium acetate and freeze-dried. The
purified tau protein (3R MBD) was stored at ꢀ30 ꢁC until use.
Black solid; yield 34%; Mp 289e291 ꢁC; 1H NMR (400 MHz,
CDCl3):
7.24 (d, 1H, J ¼ 3.9 Hz), 6.95 (d, 2H, J ¼ 8.7 Hz), 3.86 (s, 3H), 1.87 (s,
9H); 13C NMR (100 MHz, CDCl3):
159.6, 151.9, 149.6, 146.2, 144.4,
d 7.71 (s, 1H), 7.58e7.56 (m, 3H), 7.53 (s, 1H), 7.33 (s, 1H),
d
143.0,140.7, 132.4,130.7, 128.0,127.0, 123.2, 119.8,115.0, 114.8,114.5,
114.4, 114.0, 109.8, 73.9, 58.6, 55.4, 30.8; FT-IR (neat): 2926, 2212,
1568, 1510, 1460, 1413, 1370, 1342, 1295, 1234, 1177, 1094, 1065, 824,
803, 693, 519 cmꢀ1; HRMS (ESI-TOF): calcd. for C27H22N3OS3
[MþH]þ 500.0925, found 500.0891.
4.3.9. Compound E18
Black solid; yield 11%; 1H NMR (400 MHz, DMSO-d6):
d 8.31 (s,
1H), 7.84 (s, 1H), 7.80 (d, 1H, J ¼ 3.8 Hz), 7.69e7.57 (m, 6H), 6.98 (d,
2H, J ¼ 8.7 Hz), 3.79 (s, 3H), 1.82 (s, 9H); 13C NMR (100 MHz, DMSO-
d6):
d
162.7, 158.9,146.6, 145.0, 143.0, 142.6,142.1, 139.8, 133.1, 130.8,
4.7. Inhibition of Ab1e42 aggregation [23,24]
127.4, 126.5, 123.8, 117.0, 116.7, 114.5, 114.1, 113.6, 110.5, 99.8, 58.4,
55.2, 30.2; FT-IR (neat): 3137, 2208, 1683, 1575, 1518, 1466, 1418,
1369, 1288, 1249, 1233, 1176, 1082, 1056, 798, 752, 684 cmꢀ1; HRMS
A
b
1e42 peptide (Peptide Institute, Inc.) was dissolved in 0.1% aq.
NH3 and diluted to 20 M in PBS (solution A). The synthesized
m

234
S. Fuse et al. / European Journal of Medicinal Chemistry 85 (2014) 228e234
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mM in the assay shown in Table 3, and
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(3R-MBD) and 10 mM heparin (SigmaeAldrich Corporation, Japan)
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0.1e10
mM. Following incubation, the fluorescence intensity of the
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mM ThT
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Appendix A. Supplementary data
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095. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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