Discovery of an Orally Bioavailable Benzofuran Analogue That Serves as a β-Amyloid Aggregation Inhibitor for the Potential Treatment of Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.7b00844

We developed an orally active and blood-brain-barrier-permeable benzofuran analogue (8, MDR-1339) with potent antiaggregation activity. Compound 8 restored cellular viability from Aβ-induced cytotoxicity but also improved the learning and memory function of AD model mice by reducing the Aβ aggregates in the brains. Given the high bioavailability and brain permeability demonstrated in our pharmacokinetic studies, 8 will provide a novel scaffold for an Aβ-aggregation inhibitor that may offer an alternative treatment for AD.

Full text of DOI:10.1021/acs.jmedchem.7b00844

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Brief Article
Discovery of an Orally Bioavailable Benzofuran Analogue
that Serves as a #-amyloid Aggregation Inhibitor
for the Potential Treatment of Alzheimer’s Disease
Heejin Ha, Dong Wook Kang, Hyuk-Min Kim, Jin-Mi Kang, Jihyae Ann, Hyae
Jung Hyun, Joon Hwan Lee, Sae Hee Kim, Hee Kim, Kwanghyun Choi, Hyun-
Seok Hong, Young Ho Kim, Dong-Gyu Jo, Jiyoun Lee, and Jeewoo Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00844 • Publication Date (Web): 21 Nov 2017
Downloaded from http://pubs.acs.org on November 22, 2017
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Journal of Medicinal Chemistry
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Discovery of an Orally Bioavailable Benzofuran Analogue that Serves
as a β-amyloid Aggregation Inhibitor for the Potential Treatment of
Alzheimer’s Disease
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Hee-Jin Ha, ‡,§ Dong Wook Kang,† Hyuk-Min Kim,† Jin-Mi Kang,† Jihyae Ann,† Hyae Jung Hyun,∥ Joon
Hwan Lee,∥ Sae Hee Kim,∥ Hee Kim,‡ Kwanghyun Choi,‡ Hyun-Seok Hong,‡ YoungHo Kim,‡ Dong-
Gyu Jo,§ Jiyoun Lee,⊥ Jeewoo Lee*,†
† Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul
08826, Republic of Korea
‡ Medifron DBT, Sandanro 349, Danwon-gu Ansan-si, Gyeonggi-do 15426, Republic of Korea
§ School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Ko-
rea
∥ Daewoong Pharmaceutical,72 Dugye-ro, Pogok-eup, Cheoin-gu, Yongin-si, Gyeonggi-do 17028, Republic of Korea
⊥ Department of Global Medical Science, Sungshin University, 76 Dobong-ro, Gangbuk-gu, Seoul 01133, Republic of Korea
ABSTRACT: We developed an orally active and blood-brain barrier-permeable benzofuran analogue (8, MDR-1339) with potent
anti-aggregation activity. Compound 8 restored cellular viability from A-induced cytotoxicity but also improved the learning and
memory function of AD model mice by reducing the A aggregates in the brains. Given the high bioavailability and brain permea-
bility demonstrated in our pharmacokinetic studies, compound 8 will provide a novel scaffold for an A-aggregation inhibitor that
may offer an alternative treatment for AD.
A antibodies might be trapped in the blood stream, allowing
■ INTRODUCTION
only limited quantities to reach the target.5 Despite these clini-
Protein aggregation, once thought to be a rare phenomenon
in living organisms, not only occurs ubiquitously but also con-
tributes to the pathogenesis of more than 50 human diseases.1
Though complete mechanisms of how protein aggregates are
formed and how these aggregates are involved in the etiology
of various diseases have not been fully understood, age-related
impairment of protein quality control has been suspected as a
major factor in these processes, which accounts for the fact that
many disease markers found in senile disorders are protein ag-
gregates. For example, amylin in type II diabetes, -synuclein
in Parkinson’s disease, and amyloid  (A) peptides in Alz-
heimer’s disease (AD) form insoluble fibers or toxic soluble ol-
igomers, which cause the loss of normal cellular function and
accelerate aggregation even further.
cal trial failures, anti-amyloid therapies are still considered to
be a promising alternative to currently available AD treatments
because they have the potential to slow down or prevent neuro-
degeneration.6 Selective brain targeting with improved delivery
methods may also shift the fate of the antibodies that have pre-
viously failed.7,8 Several long-term prevention trials and dose-
adjusted clinical trials of A antibodies are still ongoing, and
early results of these trials have suggested positive outcomes.9-
11
Over the past decade, many protein aggregation inhibitors
have been developed as a part of anti-amyloid strategies. While
several small molecules have advanced to clinical trials,12,13
benzofuran analogues have been extensively studied due to
their high affinity and selectivity toward A fibrils.14-16 More
importantly, as an alternative approach to currently developed
AD therapies, recent studies have focused on multifunctional
ligands and hybrid drugs.17-21 These hybrid drugs combine in-
hibitors of cholinesterase or antioxidants with an anti-aggrega-
tion inhibitor such as SKF-64346 (1).22,23 While 1 is an excel-
lent aggregation inhibitor, its in vivo activity and pharmacoki-
netic parameters have never been reported. We speculated that
novel aggregation inhibitors may streamline the effort to de-
velop highly effective therapeutic agents for AD treatment.
Among these pathogenic aggregates, A peptides are proba-
bly the most extensively studied because they are central to the
amyloid hypothesis, one of the leading theories of AD patho-
genesis.2 Many anti-amyloid therapies, such as - and -secre-
tase inhibitors, anti-A antibodies, and protein aggregation in-
hibitors, have been developed and tested as potential treatment
options for AD. However, recent clinical trial failures cast doubt
on the validity of these therapies. It has been suggested that
secretase inhibitors suppress various other pathways in the brain
and the peripheral tissues, exhibiting severe side effects3,4; anti-
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the amount of A fibrils, we calculated the percentage of the
Therefore, we investigated a diverse series of benzofuran ana-
1
2
3
4
5
6
7
8
logues as A aggregation inhibitors and discovered MDR-1339
(8), 2-(3,4-dimethoxyphenyl)-5-(3-methoxypropyl)benzofuran,
as a clinical candidate (Figure 1). In this work, we aimed to
determine the anti-amyloidogenic activity of compound 8 in
vitro and in vivo and to assess its therapeutic effects in AD
model mice. We evaluated the anti-aggregation and disaggrega-
tion properties of 8 by using preformed aggregates and soluble
A oligomers. In addition, we examined the in vivo activity of
8 by performing cognitive functional assays and quantifying the
amounts of A in the brains of acute and transgenic AD model
mice. The anti-amyloidogenic activity of 8 was further verified
by in-depth studieson pharmacokinetics and brain permeability.
ThT fluorescence of each sample based on the fluorescent sig-
nals of untreated controls. We also performed the assays by us-
ing quercetin (QC), a known aggregation inhibitor, as our posi-
tive control. As shown in Figure 2a, 8 blocked the formation of
A aggregates in a dose-dependent manner, inhibiting the for-
mation of A fibrils up to 50% compared to the control. In ad-
dition, when we incubated 8 with solutions containing pre-
formed A aggregates, 8 appeared to disaggregate A fibrils,
reducing the amountof A aggregates by 40% compared to that
of the control (Figure 2b). On the basis of these observations,
we believe that 8 not only blocked the formation of A fibrils
but also effectively disaggregated preformed A aggregates.
Aggregation of A fibrils and disaggregation by compound 8
during the ThT assays was also confirmed by TEM images
(Supporting Information, Figure S1).
9
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(a)
(b)
1
1
5
0
5
0
0
0
0
1
1
5
0
5
0
0
0
0
Figure 1. -Amyloid Aggregation Inhibitors
*
*
*
*
*
*
*
*
*
■ RESULTS AND DISCUSSION
Chemistry. A highly efficient scale-up synthesis of 8 was ac-
complished in five steps. The Wittig reaction of 3,4-(dimethox-
ybenzyl)triphenylphosphonium chloride 3 with available alde-
hyde 2, the oxidative intramolecular cyclization24 of 2-hy-
droxystilbene 4 to benzofuran 5, the basic hydrolysis of ester 5,
the reduction of acid 6 with NaBH4, and the final methylation
of 7 led to the production of target compound 8 with an overall
yield of 45.4% and a purity >99%. This convenient and eco-
nomical procedure is remarkably applicable for multi kilogram-
scale production.
C
o
n
t
3
.1
1
2
.5
2
5
5 0
C
o
n
t
Q
0
C
3
. 1
1
2
. 5
2
5
5 0
( 1
u M )
8
c o n c . [ u M ]
8
c o n c . [ u M ]
Figure 2. Effect of 8 on (a) the aggregation of monomeric A1-42 and (b)
the disaggregation of A fibrils in vitro. ThT assays were performed by
incubating 25 μM of monomeric A1-42 solutions (Fig 2a) or preformed A
aggregates (Fig 2b) with 8 at the indicated concentrations. The data are pre-
sented as the mean+-SD from three independent experiments. One-way
ANOVA, Dunnett test, [* p<0.001: vs. control]
To investigate whether 8 can protect cells from A-induced
toxicity, we carried out cytotoxicity assays using 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We
treated HT22 cells, a neuronal cell line derived from the mouse
hippocampus, with 8 at different concentrations and subse-
quently incubated these cells with preformed A aggregates.
The cell viability of each sample was assessed by calculating
percent values based on the changes of the signal intensity be-
tween untreated cells and A-treated cells as a control. As
shown in Figure 3a, the treatment of A aggregates alone re-
duced the cell viability to 40% of that observed in normal cells.
Scheme 1. Scale-up synthesis of 8a
(a)
(b)
1
1
5
0
5
0
0
0
0
1
1
5
0
5
0
0
0
0
aReagents and conditions: (a) DBU, CH3CN, r.t, 3 h; (b) I2, K2CO3, THF,
reflux, 4 h; (c) LiOH, MeOH-H2O, 50 °C, 3 h; (d) (i) NaBH4, THF, reflux,
1 h, (ii) I2, THF, reflux, 4 h; (e) MeI, t-BuOK, THF, r.t, 2 h
*
In vitro activity. To determine whether 8 can inhibit the ag-
gregation of Amonomers, we incubated monomeric A1-42 so-
lutionswith 8 at different concentrations, and we quantified the
amount of A aggregates by performing Thioflavin T (ThT) as-
says. Because ThT generates fluorescent signals proportional to
C
o
n
t
0
1
.2
5
2
.5
5
1 0
C
o
n
t
1
.2
5
2
.5
5
1
0
[ u
M
]
+
A  4 2
[ u
M ]
Figure 3. (a) Effect of 8 on the cell viability of the HT22 cells exposed to
A aggregates. (b) Effect of 8 on cell viability without the treatment of A
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Journal of Medicinal Chemistry
aggregates. HT22 cells were treated with preformed A aggregates that
In addition, we performed Y-maze tests to further verify the
in vivo activity of 8. As described in Figure 5b, vehicle-treated
mice showed a significantly suppressed percentage of sponta-
neous alternation compared to that in normal mice, indicating
severe impairments in spatial memory; however, the treatment
of 8 again restored alternation behavior in a dose-dependent
manner, with an ED50 value of 0.18 mg/kg. More importantly, 8
appeared to fully recover the percentage of alternation at a dose
of 1 mg/kg (71.22 ± 3.14) and a dose of 10 mg/kg (73.07 ±
2.83).
were prepared by using 25 M of monomeric A solutions. The data were
presented as the mean+-SD from three independent experiments. One-way
ANOVA, Dunnett test, [* p<0.001: vs. control]
1
2
3
4
5
6
7
8
The treatment of 8 protected cells from this A-induced tox-
icity in a dose-dependent manner, maintaining cell viability to
normal levels at 10 M. We also measured the cytotoxicity of 8
in HT22 cells without A aggregates, confirming that 8 alone
did not affect cell viability at the indicated concentrations (Fig-
ure 3b). These results suggest that 8 suppressed the formation
of toxic A aggregates, protecting cells from A-induced tox-
icity.
9
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(b)
(a)
Next, we wanted to further verify that 8 can suppress the for-
mation of A aggregates. Therefore, we incubated A aggre-
gates with 8 and analyzed the size distribution using SDS-
PAGE followed by silver staining. In addition, we also incu-
bated A aggregates with congo red, a diazo dye with anti-am-
yloidogenic activity,25 as a positive control for comparison.As
shown in Figure 4, when we started the incubation, all samples
contained a mixture of monomeric, dimeric and trimeric A
peptides. After the samples were incubated for 48 h, we found
that these monomeric peptides remained in both congo red- and
8-treated samples. On the other hand, these low molecular
weight bands mostly disappeared in the vehicle-treated sam-
ples, implying that they transformed into high molecular aggre-
gates. These results suggest that 8 prevented oligomer for-
mation ofA peptides and reduced the formation of A fibrils
to a similar extent as congo red.
9 0
8 0
7 0
6 0
5 0
4 0
3
2
1
0
0
0
0
0
0
0
*
* *
* *
#
#
#
#
C
o
n
t
V
e
h
0
. 1
1
1 0
C
o
n
t
V
e h
0 . 1
1
1 0
[ m
g / k g ]
[ m
g
/ k
g ]
Figure 5. Behavioral effects of 8 in an acute AD model mice. (a) Passive
avoidance tests to assess learning and memory impairments; (b) Y maze
tests to assess spatial memory function (## P<0.01 vs. Control, * P<0.05,
**P<0.01 vs. Vehicle)
On the basis of these observations, we decided to test 8 with
a double transgenic AD mouse model, APP/PS1. In APP/PS1
mice, extracellular Aβ deposition can be detected at the age of
2.5 months and apparent dysfunction of learning and memory
can be monitored at the age of 6 to 8 months.27 While we ob-
served the significant improvement in the Y-maze tests at the
dose of 10 mg/kg in our acute AD model mice, we speculated
that the double transgenic animal model might require much
higher doses since they were in the further advanced state of
AD. Therefore, we decided to treat 29-week-old APP/PS1 mice
with 8 at doses of 30 mg/kg and 100 mg/kg for eight weeks by
daily oral administration and performed Y-maze tests. As de-
scribed in Figure 6, vehicle-treated mice demonstrated a severe
reduction in the percentage of spontaneous alternation
(52.64±4.15), which was even lower than the values observed
for the acute AD model mice; however, the treatment with 8
significantly improved spontaneous alternation at both doses
without any apparent toxicity: 63.73±1.06% for 30 mg/kg, and
63.93±2.24% for 100 mg/kg.
Figure 4. Effect of 8 on preformed Aaggregates.
In vivo activity. To verify the anti-amyloidogenic activity of
8, we evaluated its efficacy in animal models of AD. First, we
assessed learning and memory function in an acute AD mouse
model by performing step-through passive avoidance tests. In
this model, A aggregates were directly injected into the brains
of normal mice to induce acute neurotoxicity. After orally ad-
ministering 8 for three consecutive days, the passive avoidance
responses were evaluated by following the standard protocol.26
As shown in Figure 5a, vehicle-treated mice demonstrated sig-
nificantly reduced step-through latency (sec) due to the acute
impairment of cognitive function; however, the treatment of 8
effectively restored the passive avoidance responses in a dose-
dependent manner, with an ED50 value of 0.19 mg/kg. Specifi-
cally, the step-through latency in the sessions with mice treated
with a 10 mg/kg dose (201.2±29.4) was restored close to the
control level.
8 0
* *
*
6 0
4 0
2 0
[ m
g / k g ]
V
e h
3 0
1 0 0
Figure 6. Y-maze tests following 8 treatment in APP/PS1 mice (*P<0.05,
**P<0.01 vs. Vehicle)
To confirm whether the anti-amyloidogenic activity of 8 re-
sulted in these improvements in cognitive function in APP/PS1
mice, we quantified the levels of A aggregates in the brain by
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aAUCinf, total area under the plasma concentration time curve
immunoassay. As demonstrated in Figure 7, when compared to
the vehicle-treated mice, mice treated with 8 appeared to have
1
2
3
4
5
6
7
from time zero to time infinity (hr*ng/mL); AUClast, total area un-
der the plasma concentration time curve from time zero to the last
measured time (hr*ng/mL); Cmax, peak plasma concentration
(ng/mL); T1/2, terminal half-life (hr); Tmax, time to reach Cmax(hr);
BA, bioavailability (%).
dose-dependent reductions in the amounts of A1-40 and A1-42
,
while mice treated with 100 mg/kg had the most significant re-
ductionof A1-42 (70% of vehicle group). We believe that these
results support that 8 effectively reduced the overall amount of
A aggregates, thus improving cognitive function in AD model
mice.
Table 2. Pharmacokinetic parameters of 8 in rat, dog, mon-
key and mouse.a
8
9
I.V.
Cl
33
29
7
Oral
Vss
10
(a)
(b)
Species
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
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T1/2
3.4
1.1
Cmax AUC
421 4203
1607 4186
T1/2
3.8
1.4
F
1 5 0
1 0 0
5 0
1 5 0
1 0 0
5 0
Rat
51
25
Dog
2.7
3.8
* *
Monkey
Mouse
12.9 969
2.75 944
13747 12.9 70
13178 5.87 83.3
10.5 2.5
a AUC, area under curve(ng∙h/ mL);Cmax, maximum concentra-
tion (ng/mL); T1/2, half-life (h); Tmax, time to reach Cmax; CL, time-
averaged total body clearance (mL/min/kg); Vss, apparent volume
of distribution at steady state (L/kg); F, bioavailability(%).
0
0
V
e h
3 0
1 0 0
V
e h
3 0
1 0 0
[ m / k g
[ m
g / k g ]
g
]
Figure 7. (a) A1-40 and (b) A1-42 levels in the brains of APP/PS1 transgenic
mice treated with 8. (**P<0.01 vs. Vehicle; The brain A were quantified
by using the antibodies specific to monomeric A, after brain tissues were
homogenized and amyloid fibrils were solubilized to monomers)
4 0 0
3 5 0
3 0 0
2 5 0
2 0 0
1 5 0
1 0 0
5 0
K
= 1 .5  0 . 4
p
Pharmacokinetics and brain permeability. To further vali-
date the in vivo activity of 8, we determined pharmacokinetic
parameters and summarized these values in Table 1 and Table
2. The bioavailability of 8 increased up to 79.1% (30 mg/kg) in
a dose-dependent manner, indicating that our compound is sys-
tematically well absorbed, and suitable for oral administration
in mice (Table 1). When we administered 8 to various other
species, we also observed high bioavailabilities and a relatively
long half-life among rats and monkeys, which is consistent with
the results from ICR mice (Table 2).
B
P
r a i n
K
=
1 .2

0 . 1
p
l a s m
a
K
= 1 .2  0 . 1
p
K
=
1 .2

0 . 1
K
p
=
1 .4  0 . 5
p
0
[ h r ]
After confirming the high bioavailability of 8, we evaluated
the blood-brain barrier (BBB) permeability of 8 by oral admin-
istration in ICR mice over the course of nine hours. As shown
in Figure 8, 8 demonstrated consistent brain to plasma ratio
(Kp) values that ranged from 1.2 to 1.5, indicating that 8 effi-
ciently crossed the BBB. This high bioavailability and BBB
permeability again support our hypothesis that 8 effectively re-
duced A aggregates in the brains of AD mice and restored their
cognitive function.
0 . 3 3 3
1
3
6
9
Figure 8. Blood-brain barrier permeability of 8. ICR mice (n=3) were orally
administered10 mg/kg of 8. Kp indicates AUCbrain/AUCplasma
Cytochrome P450 (CYP) inhibition. To evaluate the tox-
icity profile of 8, we determined the inhibition of cytochrome
P450 using the human liver microsome. As shown in Table 3,
8 did not significantly inhibit a panel of CYP isozymes, while it
slightly inhibited CYP2C8 (IC50 = 31.4 M). This result sug-
gests that 8 is unlikely to cause drug-drug interactions via me-
tabolism.
Table 1. Pharmacokinetic parameters of 8a
I.V.
Oral
PK
Table 3. Human CYP inhibition of 8
parameters
3 mg/kg
1.4
3 mg/kg 10 mg/kg 30 mg/kg
T1/2
2.0
1.6
3.3
CYP450 1A2 2A6 2C8 2C9 2C19 2D6 3A4
Tmax
Cmax
-
0.7
0.7
3.0
>50 >50
31.4 >50 >50
>50
>50
IC50 (M)
4869.9 (C0)
101.9
660.1
1700.6
AUClast
1303.5
245.6
2238.8
10429.5
AUCinf
BA
1325.1
-
283.1
21.4
2293.8
51.9
10475.7
79.1
■ CONCLUSION
In this work, we developed an orally active and BBB-
permeable A aggregation inhibitor as a potential therapeutic
option for Alzheimer’s disease. Compound 8 demonstrated
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Methyl 3-(2-(3,4-dimethoxyphenyl)benzofuran-5-yl)propano-
anti-amyloidogenic activity against preformed A aggregates
1
and soluble oligomers in vitro. Compound 1 also protected neu-
ronal cells from A-induced cytotoxicity in a dose-dependent
manner. To further validate the anti-amyloidogenic activity, 8
was orally administered to AD model mice, and it restored
learning and memory function to a level close to that of the con-
trol without any apparent toxicity. When we examined the
amounts of brain A aggregates in these AD mice, 8 signifi-
cantly reduced the amount of A1-42 aggregates, suggesting that
the anti-amyloidogenic activity of 8 improved the cognitive
function of AD model mice. In addition, pharmacokinetic pa-
rameters indicated that 8 has high oral bioavailability in mice,
rats, and monkeys and also showed consistently high brain to
plasma ratio (Kp) values over the course of nine hours. Taken
together, these results suggest that 8 is a highly effective A
aggregation inhibitor with excellent bioavailability and brain
permeability, and it is currently under clinical development. We
believe that inongoing clinical phase I study, 8 will provide a
novel scaffold for an A aggregation inhibitor that may offer a
promising alternative treatment for AD in the near future.
ate (5).Iodine (359.7 kg) was added to 4 (323.5 kg) in THF
(1600 L) and K2CO3 (391.7 kg) was added. The mixture was
refluxed. After 4 h, the mixture was concentrated in vacuo and
dissolved in ethylacetate (1600 L). A saturated Na2S2O3 solu-
tion (Na2S2O3 140 kg/water 1000 L) was added to the mixture
and stirred for 10 min. The organic compound was extracted
with ethylacetate and dried over MgSO4 (40 kg) and concen-
trated in vacuo (40-50 °C). The concentrate was dissolved in
dichloromethane (300 L) and crystallized by adding MeOH
(300 L). Stirring was performed at room temperature for 2 h,
and the crystals were filtered, washed with MeOH (32 L), and
dried at 40 °C over 12 h, yielding 5 (125.4 kg, 78% yield, >95%
purity).1H NMR (CDCl3, 500 MHz) δ 7.48-7.43 (m, 4H), 7.13
(dd, 1H, J = 8.40 Hz, 2.10 Hz), 6.97 (d, 1H, J = 8.40 Hz), 6.89
(s, 1H), 4.30 (s, 3H), 3.97 (s, 3H), 3.73 (s, 3H), 3.09 (t, 2H, J =
7.60 Hz), 2.73 (t, 2H, J = 7.70 Hz).
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3-(2-(3,4-Dimethoxyphenyl)benzofuran-5-yl)propanoic acid
(6).LiOH∙H2O (13.6 kg) was dissolved in water (385 L) and it
was added to compound 5 (55 kg) in MeOH (440 L) and re-
fluxed for 3 h. The mixture was allowed to cooled to ambient
temperature, acidified to pH 2.0-3.0 with 6 N HCl. The solution
was stirredroom temperature for 30 min and at 0-5 °C for 1 h.
Crystalized product was filtered, washed with water (55 L), and
dried at 60 °C over 24 h, yielding 6 (52.2 kg, 99% yield, >99%
purity).1H NMR (CDCl3, 500 MHz) δ 12.03 (s, 1H), 7.49-7.40
(m, 4H), 7.15 (dd, 1H, J = 8.30 Hz, 2.00 Hz), 6.98 (d, 1H, 8.30
Hz), 6.89 (s, 1H), 4.30 (s, 3H), 4.00 (s, 3H), 3.10 (t, 2H, J = 7.70
Hz), 2.78 (t, 2H, J = 8.10 Hz).
■ EXPERIMENTAL SECTION
1
General Methods. H and 13C NMR spectra were recorded
on a Bruker Analytik, DE/AVANCE Digital 500 at 500 and 125
MHz. Mass spectra were recorded on a VG Trio-2 GC−MS in-
strument and a 6460 Triple Quad LC−MS instrument. All final
compounds were purified to >95% purity, as determined by
high-performance liquid chromatography (HPLC). HPLC was
performed on an Agilent 1120 Compact LC (G4288A) instru-
ment using an Agilent TC-C18 column (4.6 mm × 250 mm, 5
μm).
3-(2-(3,4-Dimethoxyphenyl)benzofuran-5-yl)propan-1-ol (7).
NaBH4 (12.1 kg) and THF (364 L) were placed in a reactor and
stirred. 6 (52 kg) was slowly added (generation of H2 gas) to the
mixture, THF (26 L) was added, and the resulting mixture was
heated to reflux at 65-75°C for 1 h. The reaction solution was
cooled to 30-40°C, and I2 (28.3 kg) and THF (26 L) were added.
The reaction solution was heated to reflux to 65 -75 °C for 4 h,
and the reaction was terminated. The reaction solution was
cooled to 10-15 °C, and ethylacetate (364 L) was added. Water
(364 L) was added while paying attention to gas generation. The
pH of the solution was adjusted to 3.0-4.0 using 6 N HCl. The
layers were separated; the organic layer was stored, and
ethylacetate (104 L) was added to the water layer. After layer
separation, the water layer was discarded, and the organic layer
was combined with the stored organic layer. The organic layer
was washed with 10% NaHSO3 (NaHSO3 36.4 kg/water 364 L),
dehydrated with MgSO4 (26 kg), and washed with ethylacetate
(52 L). The reaction solution was concentrated under reduced
pressure at 40-50 °C, and isopropylalcohol (260 L) was added
to the concentrate, heated to 70-80 °C and dissolved. Next, the
concentrate was slowly cooled to 20-25 °C, thus producing
crystals. Stirring was performed at 20-25 °C for 2 h, and the
crystals were filtered, washed with 52 L of isopropylalcohol,
and dried at 40 °C for 12 h or longer, yielding 7 (42 kg, 84.3%
yield, >99% purity).1H NMR (CDCl3, 500 MHz) δ7.48-7.40 (m,
4H), 7.10 (dd, 1H, J = 8.40 Hz, 2.10 Hz), 6.96 (d, 1H, J = 8.40
Hz), 6.85 (s, 1H),4.30 (s, 3H), 4.00 (s, 3H), 3.74 (t, 2H, J = 6.40
Hz), 2.84 (t, 2H, J = 7.30 Hz), 1.99 (q, 2H, J = 6.40 Hz), 1.71
(s, 1H).
Methyl 3-(3-(3,4-dimethoxy-styryl)-4-hydroxyphenyl) pro-
panoate (4). 3,4-dimethoxybenzylchloride (155.8 kg), tri-
phenylphosphine (203.8 kg), and sodium iodide (89.6 kg) were
placed in the first reactor, and then, acetonitrile (655 L) was
added. The resulting mixture was heated to reflux at 75-85 °C
for 2 h to terminate the reaction. The reaction solution was
cooled to ambient temperature. Then, methyl 3-(3-formyl-4-hy-
droxyphenyl)propanoate (2) (131 kg) was placed in a second
reactor, and acetonitrile (655 L) was added. The resulting mix-
ture was stirred at room temperature, and then, 1,8-diazabicy-
clo[5.4.0]-7-undecene (DBU, 114.9 kg) was added. Next, the
(3,4-dimethoxybenzyl)triphenylphosphonium chloride (3) pre-
pared in the first reactor was added dropwise into the second
reactor for 30 min. After 3 h, the reaction was terminated. The
reaction solution was cooled to room temperature and concen-
trated under reduced pressure. The concentrate was dissolved in
ethylacetate (1400 L) and stirred with the addition of water
(1270 L). The organic layer was separated, dehydrated with
MgSO4 (40 kg), and concentrated under reduced pressure. The
concentrate was cooled to room temperature, dissolved in
ethylacetate (400 L), and crystallized by adding n-hexane (400
L). Stirring was performed at room temperature for 2 h, and the
crystals were filtered, washed with ethylacetate/n-hexane = 1/3
(65.5 L), and dried in a vacuum at 40 °C, yielding 4 (326.6 kg,
75.8% yield).1H NMR (CDCl3, 500 MHz) δ 7.39 (s, 1H), 7.32-
6.85 (m, 5H), 6.78 (d, 1H, J = 8.20 Hz), 3.98 (s, 3H), 3.94 (s,
3H), 3.72 (s, 3H), 2.95 (t, 2H, J = 7.40 Hz), 2.68 (t, 2H, J = 8.00
Hz).
2-(3,4-Dimethoxyphenyl)-5-(3-methoxypropyl)benzofuran
(8).t-BuOK (76.3 kg) and THF (594 L) were placed in a reactor
and stirred. 7 (84.9 kg) was added, and then, THF (84.9 L) was
added. The resulting solution was cooled to 10-15 °C, and MeI
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Page 6 of 7
(50.8 L) was added at 30 °C or less. The reaction solution was (2) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of
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stirred at 20-25 °C for 2 h, and the reaction was subsequently
terminated. The reaction solution was cooled to 10-15 °C, and
ethylacetate (594 L) was added. Water (594 L) was added while
paying attention to gas generation. The pH of the solution was
adjusted to 3.0-4.0 using 6 N HCl. After layer separation, the
organic layer was stored, and ethylacetate (170 L) was added to
the water layer. After a subsequent layer separation, the water
layer was discarded, and the organic layer was combined with
the stored organic layer. The organic layer was washed with
10% NaHSO3 (NaHSO3 59.4 kg/water 594 L), dehydrated with
MgSO4 (42.5 kg) and washed with ethylacetate (84.9 L). The
reaction solution was concentrated under reduced pressure at
40-50 °C. EtOH (424.5 L) was added to the concentrate. The
concentrate was heated to 70-80 °C and dissolved. Next, the
concentrate was slowly cooled to 20-25 °C, thus producing
crystals. Stirring was performed at 20-25 °C for 2 h, and the
crystals were filtered, washed with EtOH (84.9 L), and dried at
40 °C for 8 h or longer, yielding 8 (81.7 kg, 92.1% yield, >99%
alzheimer's disease: Progress and problems on the road to
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1
purity) as a white solid. mp = 101-105 °C. H NMR (CDCl3,
500 MHz) δ 7.48-7.32 (m, 4H), 7.09 (d, 1H, J = 8.50 Hz), 6.94
(d, 1H, J = 8.00 Hz), 6.85 (s, 1H), 3.99 (s, 3H), 3.93 (s, 3H),
3.41 (t, 2H, J = 5.50 Hz), 3.36 (s, 3H), 2.78 (t, 2H, J = 7.50 Hz),
1.94 (t, 2H, J = 7.00 Hz). 13C NMR (200 MHz, CDCl3) δ 156.1,
153.3, 149.4, 149.1, 136.5, 129.5, 124.5, 123.6, 119.9, 117.8,
111.3, 110.6, 108.0, 99.8, 71.9, 58.5, 55.9, 32.2, 22.6, 14.1.
MS(FAB) m/z 327 [M+H]+. HRMS(FAB) calc. for C20H22O4
[M+H]+ 327.1591, found 327.1594.
(9) Mullard, A. Sting of alzheimer's failures offset by upcoming
prevention trials. Nat. Rev. Drug Discovery 2012, 11, 657-660.
(10) Reardon, S. Antibody drugs for alzheimer's show glimmers
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(11) Sevigny, J.; Chiao, P.; Bussiere, T.; Weinreb, P. H.;
Williams, L.; Maier, M.; Dunstan, R.; Salloway, S.; Chen, T.;
Ling, Y.; O'Gorman, J.; Qian, F.; Arastu, M.; Li, M.; Chollate,
S.; Brennan, M. S.; Quintero-Monzon, O.; Scannevin, R. H.;
Arnold, H. M.; Engber, T.; Rhodes, K.; Ferrero, J.; Hang, Y.;
Mikulskis, A.; Grimm, J.; Hock, C.; Nitsch, R. M.; Sandrock,
A. The antibody aducanumab reduces abeta plaques in
alzheimer's disease. Nature 2016, 537, 50-56.
■ ASSOCIATED CONTENT
Supporting Information. The Supporting Information is avail-
able free of charge on the ACS Publication website at DOI: De-
tailed experimental procedures for in vitro assays and in vivo
animal studies, and molecular formula strings.
(12) Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.;
Huang, K. Inhibiting toxic aggregation of amyloidogenic
proteins: A therapeutic strategy for protein misfolding diseases.
Biochim. Biophys. Acta 2013, 1830, 4860-4871.
(13) Eisele, Y. S.; Monteiro, C.; Fearns, C.; Encalada, S. E.;
Wiseman, R. L.; Powers, E. T.; Kelly, J. W. Targeting protein
aggregation for the treatment of degenerative diseases. Nat.
Rev. Drug Discovery 2015, 14, 759-780.
(14) Howlett, D. R.; Perry, A. E.; Godfrey, F.; Swatton, J. E.;
Jennings, K. H.; Spitzfaden, C.; Wadsworth, H.; Wood, S. J.;
Markwell, R. E. Inhibition of fibril formation in beta-amyloid
peptide by a novel series of benzofurans. Biochem. J. 1999, 340
( Pt 1), 283-289.
■ AUTHOR INFORMATION
Corresponding Author
* Phone, 82-2-880-7846; E-mail, jeewoo@snu.ac.kr.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENT
(15) Ono, M.; Kawashima, H.; Nonaka, A.; Kawai, T.;
Haratake, M.; Mori, H.; Kung, M. P.; Kung, H. F.; Saji, H.;
Nakayama, M. Novel benzofuran derivatives for pet imaging of
beta-amyloid plaques in alzheimer's disease brains. J. Med.
Chem. 2006, 49, 2725-2730.
This study was supported by a grant (HI11C0918) of the Korea
Technology R&D Project, Ministry of Health & Welfare, Re-
public of Korea and partly Cooperative Research Program for
Agriculture Science & Technology Development (PJ00910303)
by RDA.
(16) Hayne, D. J.; Lim, S.; Donnelly, P. S. Metal complexes
designed to bind to amyloid-β for the diagnosis and treatment
of alzheimer's disease. Chem. Soc. Rev. 2014, 43, 6701-6715.
(17) Bajda, M.; Guzior, N.; Ignasik, M.; Malawska, B. Multi-
target-directed ligands in alzheimer's disease treatment. Curr.
Med. Chem. 2011, 18, 4949-4975.
(18) Leon, R.; Garcia, A. G.; Marco‐Contelles, J. Recent
advances in the multitarget‐directed ligands approach for the
treatment of alzheimer's disease. Med. Res. Rev. 2013, 33, 139-
189.
■ ABBREVIATIONS
A-amyloid; AD, Alzheimer’s disease;ThT, thioflavin T;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis; BBB, blood-brain barrier; CYP, cyto-
chrome P450
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