Bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors for Aβ fibril formation

Article abstract of DOI:10.1016/j.bmcl.2006.10.090

New bis-styrylpyridine and bis-styrylbenzene derivatives were designed and synthesized. These 34 compounds were evaluated by Aβ fibril formation inhibitory assay using thioflavin T as a dye (named ThT assay). Most of them showed excellent inhibitory activ

Full text of DOI:10.1016/j.bmcl.2006.10.090

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1466–1470
Bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors
for Ab fibril formation
Seong Rim Byeon,a,b Ji Hoon Lee,a Ji-Hoon Sohn,c Dong Chan Kim,a Kye Jung Shin,a
Kyung Ho Yoo,a Inhee Mook-Jung,c Won Koo Leeb and Dong Jin Kima,*
aMedicinal Chemistry Research Center, Korea Institute of Science and Technology, P.O. Box 131,
Cheongryang, Seoul 130-650, Republic of Korea
bDepartment of Chemistry and Interdisciplinary Program of Integrated Biotechnology, Sogang University,
Seoul 121-742, Republic of Korea
cDepartment of Biochemistry and Cancer Research Institute, Seoul National University College of Medicine,
Seoul 110-799, Republic of Korea
Received 18 September 2006; revised 25 October 2006; accepted 28 October 2006
Available online 2 November 2006
Abstract—New bis-styrylpyridine and bis-styrylbenzene derivatives were designed and synthesized. These 34 compounds were eval-
uated by Ab fibril formation inhibitory assay using thioflavin T as a dye (named ThT assay). Most of them showed excellent inhib-
itory activities for Ab fibril formation at IC50 of 0.1–2.7 lM which is comparable to curcumin (IC50 of 0.8 lM). Among them, nine
compounds were screened for their cytotoxicities on HT-22 cell by MTT assay at 1, 10, and 50 lM. In particular, I-7 and II-2 exhib-
ited the best combination of inhibitory activity and compound cytotoxicity.
Ó 2006 Elsevier Ltd. All rights reserved.
Alzheimer disease (AD) is a sort of dementia and Alz-
heimer patients progressively undergo brain disorder
that destroys cognitive functions and daily activities.
fibrils, and plaques. Ab 42 aggregates such as oligomers
and fibrils deposit in the brain and cause strong neuro-
nal toxicity. On the other hand, it was recently reported
that the precursors of fibrils, oligomers might be more
toxic than fibrils.7
Since cholinergic deficit in the central nervous system
has been observed in AD patients, inhibitors for acetyl-
choline esterase (AchE) such as tacrin, donepezil, and
rivastigmine are used for the clinical treatment of AD
symptoms in current therapeutic approaches. Although
these are the only FDA-approved AD therapies on the
market, they cannot solve fundamental problems but
can be only palliative solutions.1
Consequently, we attempted to design and synthesize
small molecules that prevent the formation of insoluble
Ab fibrils by specific binding with Ab 42 monomers or
relatively smaller oligomers. Based on the previous re-
sults,8 we synthesized (E,E)-1,3-bis-30-methoxy-40-
hydroxystyrylbenzene which was suggested to shift the
equilibrium of multimeric Ab, inhibiting the pathogenic
oligomer or fibril formation. In this research, we de-
signed and synthesized various new bis-styrylpyridine
and bis-styrylbenzene derivatives following the below
Scheme (Fig. 1).
Pathomorphologically important hallmarks in AD are
senile plaques (SP) composed of amyloid b (Ab)2–4
and neurofibrillary tangles (NFTs). Therefore, reducing
deposition of Ab plaques in the brain is believed to be
pivotal to treat the AD patients.5 Based on Ab hypoth-
esis,6 Ab 42 monomers divided by b-, c-secretases from
APP are gradually aggregated to oligomers, protofibrils,
All the synthesized compounds were evaluated by Ab fi-
bril formation inhibitory assay using thioflavin T as a
dye (named ThT assay).9 IC50 value was estimated after
% inhibition screening at 20 lM, then 2 lM of the com-
pounds. Among them, nine compounds were screened
for their cytotoxicities on HT-22 cell by MTT assay at
1 and 10 lM (Fig. 4).10
Keywords: Ab fibril formation inhibitor; Alzheimer disease; Bis-styryl-
pyridine; Bis-styrylbenzene; Amyloid b.
*
Corresponding author. Tel.: +82 2 958 5142; fax: +82 2 958 5189;
e-mail: djk2991@kist.re.kr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.090

S. R. Byeon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1466–1470
1467
Figure 1. Structures of bis-styrylpyridine and bis-styrylbenzene derivatives.
R1
X
R1
X
R1
R1
(a)
(b)
(c-i)
X
X
Br
Br
(EtO)2OP
PO(OEt)2
R3
IV
III
R3 R3
R3
R2
R2
R2
R2
Aldehyde V-1: I-13'~18', II-15'~16' (to route c-ii)
V-2: I-6', I-12', II-1', II-10' (to route c-i)
V-3: I-9, II-3
I-1, 6, 12
II-1, 5, 10
V-4: I-11, II-9
V-5: I-1', II-5' (to route c-i)
V-6: II-12~14
(c-ii)
(d)
(e)
V-7: II-11
R1
R1
X
R1
X
X
I-13~18
II-15, 16
I-2, 7
II-2, 6
I-4
II-7
R3
R3 R3
R3
R3
R3
R2
R2
R2
R2
R2
R2
(f)
I-13' : R1=H, R2=OMe, R3=OPMB
I-14' : R1=Br, R2=OMe, R3=OPMB
I-15' : R1=Cl, R2=OMe, R3=OPMB
I-6' : R1=H, R2=OMe, R3=NO2
I-12' : R1=Br, R2=OMe, R3=NO2
II-1' : R1=H, R2=OMe, R3=NO2
R1
I-16' : R1=OMe, R2=OMe, R3=OPMB II-10' :R1=Br, R2=OMe, R3=NO2
I-17' : R1=Me, R2=OMe, R3=OPMB
I-1' : R1=H, R2=NO2, R3=OMe
X
I-18' : R1=NMe2, R2=OMe, R3=OPMB II-5' : R1=H, R2=NO2, R3=OMe
II-15' : R1=Br, R2=OMe, R3=OPMB
II-16' : R1=I, R2=OMe, R3=OPMB
R3
R3
R2
R2
I-3, 5, 8, 10
II-4, 8
Scheme 1. Synthesis of bis-styrylpyridine and bis-stylrylbenzene derivatives. Reagents and conditions: (a) triethylphosphite, 135 °C, 2.5 h; (b) 1 M
tKOBu in THF, aldehyde (V-1–7), THF, rt, 0.5 h; (c—i) SnCl2, EtOH, reflux, 3 h; (c—ii) 1 N HCl, EtOH, reflux, 3 h; (d) NaOMe, (CH2O)n, MeOH,
NaBH4, DMSO, reflux, 2 h; (e) AcOH, (CH2O)n, NaCNBH3; (f) BBr3, CH2Cl2, rt, 8 h.

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S. R. Byeon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1466–1470
Regarding the chemicals, new bis-styrylpyridine and bis-
styrylbenzene derivatives were synthesized as shown in
Scheme 1. Various synthetic 1,3-bis(bromomethyl)pyri-
dines (or benzenes) (III) were reacted with triethyl phos-
phite by Arbuzov reaction to give the corresponding
1,3-bis(diethylphosphono-methyl)pyridines (or benz-
enes) (IV). The Horner–Emmons reaction of phosponates
(IV) with synthetic aldehydes (V) (Fig. 2) is the key step to
obtain only trans–trans (E,E) isomer in the presence of
potassium tert-butoxide as a base, respectively.11,12 To
obtain a series of styryl compounds having hydroxyl
group at C-4 and methoxy at C-3 in the terminal aromatic
ring (Fig. 1), PMB-protected intermediates were consecu-
tively deprotected (by c-ii in Scheme 1). 8 To obtain C-3
(or C-4) mono or dimethylaminostyrylpyridine (or ben-
zene) derivatives, the intermediates bearing nitro group
at C-3 (or C-4) position were reduced (by c-i in Scheme
1), then subsequently were served in reductive amination
(by d or e in Scheme 1).13 Finally, styryl compounds with
both alkylamine and hydroxyl groups were prepared by
demethylation using BBr3 (by f in Scheme 1).11,12
In the design of the target compounds, the features of
IMSB (Fig. 3)11,12 were considered. Although IMSB
has the excellent binding affinity for Ab fibrils, its inhib-
itory activity for Ab fibril formation was relatively low
(IC50 of 8 lM) and brain uptake was hampered by the
polar carboxylic acid group. Therefore, we replaced
the carboxylic acid with a hydroxyl or alkylamine group
to increase lipophilicity for penetration of blood–brain
barrier (BBB) and enhance in vivo pharmacokinetic sta-
bility with high solubility. We then changed p-position
of the styryl conjugated system to m-position as in cur-
cumin.15 It was reported16 that low dose of curcumin
effectively disaggregates Ab as well as prevents fibril
and oligomer formation in vitro and in vivo (aged Tg
2576 mice). Especially it is widely used as a food additive
and has undergone extensive toxicological screening and
preclinical investigation in vivo. Therefore, structural
similarity of the synthetic compounds with curcumin
possesses good opportunity to be a potent AD drug
candidate.
O
V- 1 : R2=OMe, R3=OPMB
V- 2 : R2=OMe, R3=NO2
V- 3 : R2=OMe, R3=NMe2
V- 4 : R2=OMe, R3=N(CH2CH2)2
V- 5 : R2=NO2, R3=OMe
V- 6 : R2=H, R3=NMe2
H
R3
R2
V- 7 : R2=OMe, R3=OMe
V
Figure 2. Synthetic aldehydes (V-1–7).
I
HO
OH
CO2H
HO2C
IMSB
O
O
Shown in Table 1, 27 compounds have an excellent
inhibitory activity with IC50 of 0.1–2.7 lM and the cur-
cumin, a reference compound, was assessed with IC50 of
0.8 lM.
HO
OH
OCH3
OCH3
Curcumin
Figure 3. Structures of IMSB and curcumin.
Table 1. Biological activity of bis-styrylpyridines (I-1–18) and bis-styrylbenzenes (II-1–16) for inhibition of Ab 42 fibril formation
Compound
Inhibition of fibril formation IC50 (lM)a
Compound
Inhibition of fibril formation IC50 (lM)a
I-1
I-2
0.6
1.1
0.5
0.9
2.3
0.7
0.5
0.9
0.8
0.9
-
II-1
0.6
0.6
1.0
0.7
0.3
1.3
1.1
2.7
>20
1.8
’20
>20
>20
>20
0.1
—
II-217
II-3
I-3
I-4
II-4
II-5
I-5
I-6
II-6
II-7
I-717
I-8
II-8
II-9
I-9
I-10
I-11b
I-12
I-13
I-14
I-15
I-16
I-17
I-18
II-10
II-11b
II-12
1.1
0.8
1.1
1.1
1.7
1.4
1.9
II-13
II-14
II-15
II-16b
Curcumin
Acridine orange14
IMSB
0.8
0.6
8
IC50 was calculated from nonlinear regression by Graphpad Prism software.
a ThT assay.
b These were not suitable to ThT assay because of their own high-fluorescence interference effect.

S. R. Byeon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1466–1470
1469
Figure 4. Compound (finally 10 lM) cytotoxicities by MTT assay on HT-22 cell. The cells were treated with the compounds in media with 10% fetal
bovine serum and 1% penicillin/streptomycin (totally 0.5% DMSO). In control, the cells were only treated with the same media (0.5% DMSO).
In the structure–activity relationship, the central pyridine
or benzene did not affect the IC50 and R1 substituents in
the central ring also did not show a meaningful effect (I-
13–18 in Table 1). The value of IC50 was increased slightly
in order of halogen < Me < OMe < NMe2. The com-
pounds (II-12, 13, and 14) with only one substituent (dim-
ethylamine) at C-4 position in the terminal aromatic ring
showed lower activity than compound (II-3) with two
substituents (dimethylamine and methoxy) both at C-3
and C-4.
Additional assays including in vitro BBB penetration,
protection effect of toxicity induced by Ab 42 fibrils,
in vivo pharmacokinetics, and behavior tests on APP/
PSI mice are currently in progress.
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.
Changing –OMe at C-4 (II-11) to –OH (II-15) or –NH2
(II-10) significantly enhanced the inhibitory activity.
These results suggest that the presence of two electron
donating groups both at C-3 and C-4 positions probably
plays a critical role to display higher activity, and at
least one of them at C-3 or C-4 is necessary to be –
NH2, –NHMe, –NMe2 or OH.12 When R3 substituent
was replaced with NH2, NMe2 or N(CH2 CH2)2, inhib-
itory activity was decreased from 0.6, 1.0 to >20 lM (in
II-1, 3, and 9).
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lengths were 450 and 482 nm with 10 nm slit (LS-55
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Selected nine compounds were initially screened at low
concentration of 1 lM for the compound cytotoxicity
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In conclusion, most of the synthesized compounds in
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high concentration. In particular, I-7 and II-2 exhibited
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nyltetrazolium bromide) was added into the HT-22 cell.

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S. R. Byeon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1466–1470
Cell viability was estimated by measuring the absorbance
17. Selected data. Compound I-7: 1H NMR (CD3OD,
300 MHz) d 2.86 (s, 6H), 3.93 (s, 6H), 6.59 (d, J = 7.83,
2H), 7.03 (d, J = 16.15, 2H), 7.12 (d, J = 8.35, 2H), 7.07 (s,
2H), 7.37 (d, J = 7.81, 2H), 7.52 (d, J = 16.14, 2H), 7.67 (t,
J = 7.51, 1H); 13C NMR (CDCl3, 75 MHz) d 30.16, 55.46,
106.78, 108.65, 118.45, 122.29, 124.05, 125.33, 133.35,
136.58, 139.98, 146.87, 156.18; HRMS m/z Calcd for
C25H27N3O2 (M)+ 401.2103. Found: 401.2182. Anal.
Calcd for C25H27N3O2: C, 74.79; H, 6.78; N, 10.47.
Found: C, 74.51; H, 6.77; N, 10.12. II-2: 1H NMR
(CDCl3, 300 MHz) d 2.91 (s, 6H), 3.93 (s, 6H), 6.58 (d,
J = 8.09, 2H), 6.94 (d, J = 16.23, 2H), 7.01 (d, J = 1.64,
2H), 7.07 (dd, J = 8.20, 1.71, 2H), 7.11 (d, J = 16.25, 2H),
7.33 (t, J = 4.63, 1H), 7.34 (d, J = 1.18, 2H), 7.61 (s, 1H);
13C NMR (CDCl3, 75 MHz) d 30.44, 55.49, 106.66,
109.30, 121.11, 123.10, 124.40, 124.52, 127.36, 128.84,
129.39, 138.63, 139.11, 147.12; HRMS m/z Calcd for
C26H28N2O2 (M)+ 400.2151, Found 400.2151; Anal. Calcd
for C26H28N2O2: C, 77.97; H, 7.05; N, 6.99. Found: C,
78.41; H, 7.18; N, 6.57.
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