Small molecules that protect against β-amyloid-induced cytotoxicity by inhibiting aggregation of β-amyloid

Article abstract of DOI:10.1016/j.bmc.2012.06.045

Aggregated β-amyloid (Aβ) plays crucial roles in Alzheimer's disease (AD) pathogenesis, therefore blockade of Aβ aggregation is considered as a potential therapeutic target. We designed and synthesized small molecules to reduce Aβ-induced cytotoxicity by inhibiting Aβ aggregation. The small molecules were screened via ThT, MTT, and cell-based cytotoxicity assay (Aβ burden assay). Selected compounds 1c, 1d, 1e, and 1f were then investigated by evaluating their effects on cognitive impairment of acute AD mice model. Learning and memory dysfunction by injection of Aβ(1-42) was recovered by administration of these molecules. Especially, 1d showed the best recovery activity in Y-maze task, object recognition task, and passive avoidance task with dose dependent manner. These results suggest that 1d has high potential as a therapeutic agent for AD.

Full text of DOI:10.1016/j.bmc.2012.06.045

Bioorganic & Medicinal Chemistry 20 (2012) 4921–4935
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecules that protect against b-amyloid-induced cytotoxicity
by inhibiting aggregation of b-amyloid
Yun Suk Lee a,b, Hye Yun Kim a, YoungSoo Kim a, Jae Hong Seo c, Eun Joo Roh a, Hogyu Han b,
Kye Jung Shin c,
⇑
a Korea Institute of Science and Technology, 39-1 Hawolgog-dong, Sungbuk-gu, Seoul 136-791, Republic of Korea
b Department of Chemistry, Korea University, 1 Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea
c Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si,
Gyeonggi-do 420-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Aggregated b-amyloid (Ab) plays crucial roles in Alzheimer’s disease (AD) pathogenesis, therefore block-
ade of Ab aggregation is considered as a potential therapeutic target. We designed and synthesized small
molecules to reduce Ab-induced cytotoxicity by inhibiting Ab aggregation. The small molecules were
screened via ThT, MTT, and cell-based cytotoxicity assay (Ab burden assay). Selected compounds 1c,
1d, 1e, and 1f were then investigated by evaluating their effects on cognitive impairment of acute AD
mice model. Learning and memory dysfunction by injection of Ab(1–42) was recovered by administration
of these molecules. Especially, 1d showed the best recovery activity in Y-maze task, object recognition
task, and passive avoidance task with dose dependent manner. These results suggest that 1d has high
potential as a therapeutic agent for AD.
Received 2 May 2012
Revised 25 June 2012
Accepted 26 June 2012
Available online 3 July 2012
Keywords:
Alzheimer disease
b-Amyloid
Aggregation inhibitor
Bis-styryl aromatic compound
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ing b, c-secretases, increasing Ab clearance, or blocking Ab aggre-
gation with peptides, antibodies, and small molecules.4
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder and its clinical symptoms are characterized by dysfunc-
tion in cognition and memory. One of the major pathologic hall-
marks of AD is accumulation of extracellular b-amyloid (Ab)
peptide. A number of studies have provided strong evidences that
aggregation of Ab induces neurodegeneration, neurotoxicity, and
oxidative stress in AD brains.1
There are numbers of small molecules reported to have anti-
neurodegenerative activities. Styryl benzene (SB) derivatives have
excellent binding affinities to b-sheet rich Ab fibrils (Fig. 1). Also,
SB type molecules are known to bind to oligomeric Ab which is
relatively smaller and more soluble fibrils, leading to prohibit the
formation of larger and insoluble fibrils.5 Ferulic acid (FA), a
well-known anti-oxidant, inhibited fibril formation of Ab(1–40)
and Ab(1–42) and neutralized Ab(1–42)-induced toxicity.6 It was
also demonstrated that long-term administration of FA exhibited
anti-dementia activity in vivo.7 Resveratrol, a polyphenol com-
pound, exhibited a wide range of biological and pharmacological
activities including anti-oxidant, anti-inflammatory, anti-muta-
genic, anti-carcinogenic, and anti-angiogenic effects.8 Resveratrol
inhibited Ab fibril formation and exerted neuroprotective effect
against cell death induced by Ab.9 Curcumin has potent anti-oxi-
dant and anti-inflammatory activities and can suppress oxidative
damage, inflammation, and amyloid accumulation. It directly binds
to small Ab species, blocking aggregation and fibril formation
in vitro and in vivo.10
Ab is derived from the sequential proteolytic processing of amy-
loid precursor protein (APP) by b-secretase and
cleavage of APP at different positions by -secretase produces
c
-secretase.2 The
c
two predominant Ab peptide residues, Ab(1–40) and Ab(1–42).
The most abundant variant in vascular Ab is Ab(1–40), but Ab(1–
42), the longer form, is more fibrillogenic and neurotoxic than
Ab(1–40). Although production of Ab is a normal process, the
over-production of Ab or an increased proportion of Ab(1–42) ap-
pears to cause early onset of AD.3
Since the progressive accumulation of Ab aggregates is widely
believed to be fundamental to the initial development of neurode-
generative pathology, many therapeutic approaches in AD are fo-
cused on reducing neurotoxicity by decreasing the concentration
of cerebral Ab. Various approaches to decrease Ab include inhibit-
The recent studies suggest that fibrils are formed through olig-
omeric Ab assembly intermediates, which are more toxic than fi-
brils.11 In the light of these findings, a need for inhibitors of
pathogenic oligomeric assembly formation has been recognized.
We previously introduced a small molecule, KMS4005, with decent
⇑
Corresponding author. Tel.: +82 2 2164 4060; fax: +82 2 2164 4059.
E-mail address: kyejung@catholic.ac.kr (K.J. Shin).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.045

4922
Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
OH
COOH
HO
HO
HO
OH
H3CO
HOOC
COOH
FA
SB
Resveratrol
OH
H
O
O
O
O
HO
OH
OCH3
HO
OH
OCH3
HO
OH
OCH3
OCH3
OCH3
OCH3
keto form
enol form
KMS4005
Curcumin
R
X
X
R'
R'
R''
R''
New molecule
Figure 1. Structures of styryl benzene (SB), ferulic acid (FA), resveratrol, KMS4005, curcumin, and new molecule.
binding affinity and specificity to the non-fibrous and monomer-
like Ab.12 By extension of these results, we here designed and
synthesized new molecules which are derived from previously
reported KMS4005. A central benzene ring of KMS4005 into pyrim-
idine ring was modified and substituents on central ring were
added. According to our earlier study, hydroxyl groups on outer
rings may play an important role in its anti-amyloidogenic activity
and is considered as necessary pharmacophore to have protective
effect on Ab-induced cytotoxicity. Therefore, we added naturally
occurring substituents such as hydroxy and methoxy, and lipo-
philic substituents such as dimethylamino group on outer ring.
Several clinical studies proved that curcumin has a chemother-
apeutic activity in AD and is non-toxic even at very high doses.13 In
spite of its pharmacological efficacy on AD and safety, curcumin
has not yet been approved as a therapeutic agent due to its poor
bioavailability, high rate of metabolism, and instability under neu-
tral-basic conditions.14 Newly designed molecules have central
pyrimidine or benzene ring which is equivalent to the hexagonal
shape of enol form of curcumin.
In this study, we synthesized new small molecules as described
above (Fig. 1). Their activities to prevent fibril formation and alle-
viate Ab-induced cytotoxicity were investigated using fluorescence
assay in vitro and cell-based assay. We utilized acute AD mouse
model to assess the recovery abilities of titled compounds against
Ab-induced behavioral abnormality.
Figure 2. Effect of compounds on Ab oligomerization. Monomerized Ab(1–42)
(25 M) was incubated for 2 h at 37 °C in the presence of curcumin, 1c, 1d, 1e, and
1f (2 M). After cross-linking of peptide using PICUP technique, the mixture was
l
l
applied on SDS–PAGE gel.
2. Results and discussion
2.1. Chemistry
R
R
a
N
N
HN
NH2
Compounds 1a–5f are shown in Fig. 2 and were prepared
according to Schemes 1–5. Cyclization of O-methylisourea 6a and
S-methylisothiourea 6b with acetylacetone in aqueous K2CO3 solu-
tion afforded 2-methoxypyrimidines 7a and 2-thiomethylpyrimi-
dine 7b, respectively (Scheme 1). Halogenation of benzyl alcohol
8 followed by Arbuzov reaction gave diethyl phosphonates 10
6a. R = OMe
6b. R = SMe
7a. R = OMe
7b. R = SMe
7c. R = H
Scheme 1. Reagents and conditions: (a) acetylacetone, aqueous K2CO3, reflux.

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4923
dimethylamino group were prepared from nitrobenzoic acid 14a
and 14b, respectively (Scheme 4). Condensation of 7 with 12,
13e, and 13f in aqueous NaOH solution, and Horner-Emmons reac-
tion of 10 with 13 in the presence of t-BuOK as a base afforded pro-
tected molecules 1a0–5f0 (Scheme 5). Removal of PMB and MOM
groups afforded the target compounds 1a–5f.
R
R
b
EtO
EtO
OEt
OEt
P
P
X
X
O
O
8. R = OH, X = OH
9a. R = OH, X = I
9b. R = H, X = Br
10a. R = OH
10b. R = H
a
2.2. Anti-amyloidogenic activity and cytotoxicity
To determine anti-amyloidogenic activity and cytotoxicity of
the compounds, a fluorescence intensity assay using thioflavin T
(ThT) and MTT assay were performed.15 Most of compounds
showed inhibitory effects on Ab fibril formation (Table 1). It ap-
peared that the central pyrimidine and benzene rings were respon-
sible for the inhibition of Ab fibril formation, but R substituents of
the central ring did not have a meaningful activity. Generally, com-
pounds 1 and 5 except compounds b and g showed a higher inhib-
itory activity than others. The inhibitory activity depended rather
strongly on R0 and R00 substituents of outer rings. Non-substituted
compounds 2g–5g at outer ring inhibited only 20% of fibril forma-
tion (1g: 47.4%, 2g: 18.8%, 3g: 11.6%, 4g: 19.8%, 5g: 19.8%). Intro-
duction of e-donating groups at R0 and R00 had a tendency to
lower fibril formation. In proportion to the strength of e-donating
group at outer ring substituents (H < OMe < NMe2), inhibitory
activity on Ab fibril formation was increased (a < c < e, b < d < f).
Although e-donating group on both R0 and R00 was critical for inhi-
bition of Ab fibril formation, e-donating group on R0 played a more
important role than that on R00 (a > b, c ꢀ d, e < f). Summing up
in vitro data, compounds f had the best inhibitory activity com-
pared to others, due to relatively high binding affinity to Ab with
N,N-dialkylamino group.16 According to the cell cytotoxicity data
obtained in treating compounds to HT22 cell line, 1a–2f except
2b and 2c had no cytotoxicity. Among them, 1a–1f except 1b
and 2f were non-toxic, and also effectively blocked fibrillogenesis
in vitro.
Scheme 2. Reagents and conditions: (a) BF3ꢁEt2O, KI, 1,4-dioxane, rt; (b) P(OEt)3,
toluene, reflux.
O
O
a or b
R'
R'
R''
R''
11a. R' = OH, R'' = H
11b. R' = H, R'' = OH
12a. R' = OPMB, R'' = H
12b. R' = H, R'' = OPMB
11c. R' = OH, R'' = OMe 12c. R' = OPMB, R'' = OMe
11d. R' = OMe, R'' = OH 12d. R' = OMe, R'' = OPMB
13a. R' = OMOM, R'' = H
13b. R' = H, R'' = OMOM
13c. R' = OMOM, R'' = OMe
13d. R' = OMe, R'' = OMOM
Scheme 3. Reagents and conditions: (a) PMB-Cl, K2CO3, DMF, 60 °C; (b) MOM-Cl,
Et3N, THF, 0 °C.
O
O
OH
OMe
a
R'
R'
R''
R''
14a. R' = OH, R'' = NO2
14b. R' = NO2, R'' = OH
15a. R' = OH, R'' = NO2
15b. R' = NO2, R'' = OH
IC50 values of compounds with over 80% inhibition activity at
10
of 0.8–3.7
most effectively (IC50 = 0.8 and 1.1
l
M were shown in Table 2, and they had IC50 values in the range
M. Compounds 2f and 3f blocked fibril formation of Ab
M, respectively).
l
b
l
O
O
2.3. Protective effect against Ab-induced cytotoxicity
OMe
OMe
c
For the further investigation of activity, we selected compounds
with % inhibition over 80% and cytotoxicity under 10%, and exam-
ined a protective effect of these compounds on Ab-induced cyto-
toxicity with HT22 cell line. Ab(25–35) was used to induce Ab
cytotoxicity. The truncated fragment of Ab is often used as a com-
ponent for amyloid-induced neurotoxicity assays for drug screen-
ing. Ab(25–35) is known to generate toxicity similar to Ab(1–42),
and causes neuronal cell death and oxidative stress in cell cul-
tures.17 As shown in Table 3, Ab(25–35)-induced cytotoxicity de-
creased the cell viability by 44%. Compounds 1c–1f and 2f had a
protective effect on Ab-induced cytotoxicity (67–103%). In particu-
lar, Compound 1c, which was structurally similar to the enol form
of curcumin, had no cytotoxicity, and alleviated Ab-induced cyto-
toxicity (103%). Compound 2f that blocked fibril formation of Ab
in ThT assay most effectively (IC50 = 0.8) showed a little protective
effect (67%) against Ab compared with compounds 1c–1f.
R'
R'
R''
R''
17a. R' = OMOM, R'' = NMe2
17b. R' = NMe2, R'' = OMOM
16a. R' = OMOM, R'' = NO2
16b. R' = NO2, R'' = OMOM
d
OH
O
e
R'
R'
R''
R''
18a. R' = OMOM, R'' = NMe2 13e. R' = OMOM, R'' = NMe2
18b. R' = NMe2, R'' = OMOM 13f. R' = NMe2, R'' = OMOM
Scheme 4. Reagents and conditions: (a) concd H2SO4, MeOH, reflux; (b) MOM-Cl,
Et3N, THF, 0 °C; (c) H2, 10% Pd/C, formaldehyde, MeOH; (d) LiAlH4, THF, 0 °C; (e)
MnO2, CH2Cl2.
2.4. Inhibitory activity on formation of oligomeric species
We previously reported that KMS4005 (4c) exhibited specific
interaction with non-fibrous and monomeric Ab and contributed
to the inhibitory effect on the pathogenic oligomer or fibril forma-
tion.12 Non-effect of central ring on Ab aggregation inhibition and
structural analogousness with 4c implies that specific interaction
(Scheme 2). The protection of commercial hydroxybenzaldehydes
11 with p-methoxybenzyl (PMB) or methoxymethyl (MOM) groups
afforded O-protected benzaldehydes 12a–12d and 13a–13d,
respectively (Scheme 3). Benzaldehydes 13e and 13f possessing

4924
Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
O
R
N
N
+
R
R
a
R'
X
X
X
X
R''
c or d
7
12, 13e, 13f
R
O
b
R'
R'
R'
R'
R''
R''
R''
R''
+
EtO
EtO
OEt
OEt
R'
P
P
O
1a' - 5f'
1a - 5f
O
R''
13
10
Scheme 5. Reagents and conditions: (a) Bu4NHSO4, 5 N NaOH, reflux; (b) t-BuOK, THF, 0 °C; (c) EtOH, 1 N HCl, reflux; (d) TFA, CH2Cl2, rt.
Table 1
Inhibitory activity on Ab fibril formation and cytotoxicity of compounds: thioflavin T (ThT)a and MTT assayb
R
X
X
R'
R'
R''
R''
Compd
X
R
R0
R00
% Inhibition of fibril
Viability (%)
Compd
X
R
R0
R00
% Inhibition of fibril
Viability (%)
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
SMe
SMe
SMe
OH
H
H
OH
OMe
OH
NMe2
OH
H
OH
OMe
OH
NMe2
OH
64.3 7.0
14.1 19.7
79.3 5.4
80.9 0.6
83.5 2.6
93.7 1.8
67.4 7.3
ꢂ28.7 11.6
68.0 11.6
55.0 9.2
74.1 7.3
90.4 3.7
67.4 7.4
ꢂ53.3 16.3
67.6 12.5
104.2 7.1
142.5 5.6
215.6 3.5
135.6 6.1
146.2 26.6
132.8 41.1
101.2 31.8
45.9 4.2
75.2 12.5
98.4 2.7
96.6 13.7
101.4 2.5
65.3 6.0
3d
3e
3f
4a
4b
4cc
4d
4e
4f
5a
5b
5c
5d
5e
5f
N
N
N
SMe
SMe
SMe
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OMe
OH
NMe2
OH
H
OH
OMe
OH
NMe2
OH
H
OH
OH
NMe2
OH
H
OH
OMe
OH
NMe2
OH
H
OH
75.2 11.1
78.6 10.3
88.8 4.2
58.4 6.2
ꢂ28.7 8.2
71.5 1.1
67.9 1.1
63.9 10.1
56.0 8.3
80.7 1.1
7.1 12.4
79.0 2.4
84.5 3.8
74.2 5.8
83.3 0.7
78.8 9.3
77.3 15.1
85.3 8.1
102.1 3.9
47.0 6.7
74.8 1.2
69.5 4.2
95.7 8.3
100.8 7.6
37.4 4.7
59.2 15.4
80.9 13.1
73.5 14.1
66.0 12.1
64.4 4.2
OH
OMe
OH
NMe2
OH
H
OH
OMe
OH
NMe2
OH
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
OMe
OH
NMe2
OH
3a
3b
3c
H
OH
OMe
OMe
OH
NMe2
43.7 6.6
79.0 7.9
OH
a
b
c
Ab (25
l
M) and compounds (10
l
M) were incubated at rt for 1 h, before adding ThT solution.
Cytotoxicity of compounds (10
KMS4005.
lM) in HT22 cell.
Table 2
IC50 of compounds with over 80% inhibition at 10
Table 3
Protective effect of compounds on Ab-induced cytotoxicity: Ab burden assaya
lM concentration in ThT assay
Entry
Compd
IC50 (lM)
Entry
Compd
Protective effect (%)
1
2
3
4
5
6
7
8
9
1c
1d
1e
1f
2f
3f
5a
5d
5f
3.2
2.1
3.7
3.4
0.8
1.1
1.6
1.6
3.3
1
2
3
4
5
6
1c
1d
1e
1f
2e
2f
103.0 13.0
91.8 8.8
85.3 0.1
76.6 12.1
59.0 5.7
67.4 5.1
56.0 0.6
Ab(25–35)
a
Ab(25–35) (25
l
M) and compounds (10 lM) were incubated for 18 h.
of our new compounds with monomeric Ab might induce an equi-
librium shift in favor of monomers, consequently inhibiting forma-
tion of pathogenic oligomers and fibrils.
To confirm the inhibitory activity of compounds 1c, 1d, 1e, and
1f against oligomers, we further characterized the aggregation
state in the presence and absence of compounds using SDS–PAGE

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4925
(sodium dodecyl sulfate polyacrylamide gel electrophoresis) and
PICUP (photo-induced cross-linking of unmodified proteins) tech-
nique (Fig. 2). For this study, compounds were prepared at final
erence index = 0.64). However, they recovered by 7–13% (prefer-
ence index = 0.59–0.61, but no statistical significance) when
administered with the compounds, 1c, 1d, and 1f (Fig. 4b). Com-
pound 1e showed no effect in the object recognition task. Treating
group with 1c showed similar activity to control group (preference
index = 0.61) for the new object task.
concentration of 2 lM, based on their IC50 values (2.1–3.7 lM).
In the absence of compounds, Ab was spontaneously aggregated
into high molecular weight oligomers (HMW oligomers, bands
ranging from 150 to 225 kDa and discrete bands above 52 kDa)
and the low molecular weight oligomers (LMW oligomers, bands
below 17 kDa corresponding to dimmers, trimers, and tetramers).
In the presence of compounds, inhibitory effect on the formation
of HMW oligomers was observed. Compounds 1d–1f prevented
formation of the HMW oligomers strongly. However, compound
1c showed weak inhibitory effect on the formation of oligomers
than compounds 1d–1f. It shows that there is a good relationship
in ThT assay and SDS–PAGE results of compounds 1d–1f.
In passive avoidance task,21 long-term memory was assessed by
measuring a natural tendency (step-through latency) of mice to
enter darkened space. Ab(1–42) injected mice showed reduced
step-through latency by 53% (90 s) compared to control (190 s),
but they recovered latency (118–144 s, but no statistical signifi-
cance) when administered with the compounds (Fig. 4c). A group
of treatment with 1d also showed the highest effect (144 s) for
step-through latency.
Results of the watermaze task designed to test spatial learning
ability are depicted in Fig. 4d.22 As seen in Fig. 4d, the escape la-
tency of mice injected with Ab(1–42) was longer than that of con-
trol group. When administered with compounds 1c, 1d, 1e, and 1f,
the latencies of compound-treated groups were shortened than
those of mice injected with Ab(1–42) in the fourth and fifth train-
ing sessions. In the watermaze study, impairment of spatial learn-
ing abilities caused by Ab(1–42) was restored by administration of
compounds.
Taken together, the results obtained in acute AD mouse model
study demonstrated that the four compounds 1c–f enhanced
learning and memory ability impaired by injection of Ab(1–42)
(see Fig. 4a–d). Especially, acute AD mice administrated with 1d
showed the highest improvement compared to others (1d, 72% vs
the rest, 67–71% in Fig. 4a; 1d, 93% vs the rest, 83–96% in Fig. 4b;
1d, 76% vs the rest, 62–73% in Fig. 4c). From results of ThT assay,
SDS–PAGE, and Ab burden assay, compound 1d inhibited Ab aggre-
gation (% inhibition of fibril formation: 1d, 19% in Table 1) and alle-
viated Ab-induced cytotoxicity (protective effect: 1d, 91% in
Table 3). It suggests that the improvement effect of 1d on learning
and memory in vivo is due to alleviation of cytotoxicity by inhibit-
ing Ab aggregation. As shown in Fig. 5, 1d also showed learning and
memory improvement effects in a dose-dependent manner over
50–200 mg/kg po, suggesting that these improvement effects came
from administration of 1d. Administration of 1d could prevent cog-
nitive deficits at the lower dose than curcumin (500 mg/kg curcu-
min chow) and ferulic acid (daily 14–19 mg/kg for 2–4 weeks).7,23
2.5. Efficacy in acute AD animal model
Several behavioral tests were developed for learning and mem-
ory in mice model. To examine the efficacy of compounds for the
animal model, behavioral tests were performed to test whether
they could recover the Ab-induced learning and memory impair-
ment in ICR mice. Intracerebroventricular (ICV) injection of Ab is
well known to cause memory deficits.7,18 Therefore, we used this
method to evaluate efficacy of compounds for in vivo mouse
model.
The experimental schedules are summarized in Fig. 3. All sam-
ples were administered via oral routes (100 mg/kg) or food pellets.
After the ICV injection of Ab(1–42), the compounds were adminis-
tered for 2 days (for Y-maze and the object recognition), 3 days (for
passive avoidance), or 7 days (for watermaze) to the mice, then
they were subjected to the Y-maze on day 2, the object cognition
task on days 2–3, the passive avoidance task on days 3–4, and
the watermaze task on days 3–7.
Short-term memory was assessed by spontaneous alternation
behavior in the Y-maze task.19 Spontaneous alternation behavior
was considered to reflect spatial working memory. As shown in
Fig. 5a, mice injected with Ab(1–42) had impaired memory (62%),
and recovered memory with statistical significance in the com-
pound-treated groups (67–72%). Treating group with 1d displayed
the highest effect (72%) with a decrease of alternation behavior.
Non-spatial and long-term memory recovery activity was eval-
uated by time that spends to explore the object in the object recog-
nition task.20 Memory was assessed by measuring mice’s ability to
discriminate the familiarity of objects previously encountered.
Acute AD mice showed reduced preference for the new object by
about 17% (preference index = 0.53) compared to the control (pref-
3. Conclusion
Ab aggregates play crucial roles in AD pathogenesis and block-
ade of Ab aggregation is a promising target. It is reported that sol-
uble oligomeric intermediates are more toxic than fully formed
mature amyloid fibrils and are recognized as a key pathogen in
AD.3,24 To reduce Ab aggregation and alleviate toxicity, a number
of small molecules and peptides that specifically focused on solu-
ble and toxic oligomers were reported.4e,25,26 We synthesized
new compounds and evaluated their inhibitory ability and protec-
tive effect on Ab aggregates. We showed that compounds, espe-
cially 1c, 1d, 1e, and 1f, can inhibit Ab aggregation, reduce Ab-
induced cytotoxicity in vitro, and attenuate the Ab-induced learn-
ing and memory impairment in acute AD mouse model. Among
them, 1d is most effective, and a promising drug candidate to inhi-
bit fibril formation of Ab for treatment and prevention of AD.
4. Experimental section
4.1. General methods
All chemicals were obtained from commercial supplies and
used without further purification. Solvents were distilled (THF
Figure 3. Experimental schedules of acute AD model.

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Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
Figure 4. Recovery effect of 1c, 1d, 1e, and 1f on the Ab(1–42)-induced impairment in learning and memory ability of acute AD mice. After the injection of aggregated Ab(1–
42) (10 nM), mice (n = 10) was administered with the compounds of 1c, 1d, 1e, and 1f (100 mg/kg po). The data are expressed as mean SEM. (a) Y-maze task. (b) Object
recognition task. (c) Passive avoidance task. (d) Watermaze task. ⁄t-test, P <0.05, #t-test, P <0.05.
Figure 5. Dose dependence of 1d. After the injection of aggregated Ab(1–42) (10 nM), mice (n = 10) was administered with the various doses of 1d. The data are expressed as
mean SEM. (a) Y-maze task. (b) Object recognition task. (c) Passive avoidance task. ⁄t-test, P <0.05, #t-test, P <0.05.
from Na/Benzophenone; CH2Cl2 from CaH2). Flash column chroma-
tography was performed with Silica Gel 60 (40–63 m, 230–
0.29 mmol) was refluxed. After cooling, the precipitates were fil-
tered off. The products were purified by recrystallization from
EtOAc. Otherwise, the mixture was extracted with CH2Cl2. Then,
the organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash column chromatography to give
the products.
l
400 mesh, Merck). Thin layer chromatography (TLC) was per-
formed Silica Gel 60 F254 precoated plates (0.25 mm thickness,
Merck, Darmstadt). All 1H and 13C NMR spectra were recorded on
a Brucker Avance 400 spectrometer. Chemical shifts were reported
in part per million relative to internal tetramethylsilane (TMS). All
13C NMR spectra were recorded with complete proton decoupling.
4.2.2. General procedure B
Preparation of 4a0–5f0. To a stirred solution of 1,3-bis(diet-
hylphosphonomethyl)benzene or 3,5-bis(diethylphosphonometh-
yl)phenol (2.00 mmol) in dry THF (50 mL) was added
benzaldehydes (4.00 mmol). Then, potassium tert-butoxide was
added at 0 °C and the mixture was stirred for 2 h at the same tem-
perature. The excess H2O was added into the mixture. If the prod-
ucts precipitated, it was collected by filtration. Otherwise, the
4.2. Synthesis procedures and spectral data
4.2.1. General procedure A
Preparation of 1a0–3f0 and 1g–5g. A mixture of pyrimidines
(2.00 mmol) and benzaldehydes (4.00 mmol) in 5 N NaOH solution
(20 mL) containing tetrabutylammonium hydrogen sulfate (0.10 g,

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4927
mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The res-
idue was purified by flash column chromatography to give the
products.
4.2.9. 2-Methoxy-(E,E)-4,6-bis[40-(400-methoxybenzyloxy)styryl]
pyrimidine (2a0)
Compound 2a0 was prepared from 7a and 12a according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 2a0 (0.57 g, 49%). 1H NMR (400 MHz, CDCl3) d
7.83 (d, J = 15.9 Hz, 2H), 7.65 (d, J = 8.7 Hz, 4H), 7.38 (d, J = 8.6 Hz,
4H), 7.23 (s, 1H), 7.07 (d, J = 15.9 Hz, 2H), 7.05 (d, J = 8.7 Hz, 4H),
6.94 (d, J = 8.6 Hz, 4H), 5.06 (s, 4H), 3.97 (s, 3H), 3.75 (s, 6H).
4.2.3. (E,E)-4,6-Bis[40-(400-methoxybenzyloxy)styryl]pyrimidine
(1a0)
Compound 1a0 was prepared from 4,6-dimethylpyrimidine and
12a according to general procedure A. The crude product was puri-
fied by crystallization from EtOAc to give 1a0 (0.93 g, 84%). 1H NMR
(400 MHz, CDCl3) d 9.06 (s, 1H), 7.85 (d, J = 15.9 Hz, 2H), 7.56 (d,
J = 8.7 Hz, 4H), 7.37 (d, J = 8.7 Hz, 4H), 7.22 (d, J = 0.9 Hz, 1H),
7.00 (d, J = 8.7 Hz, 4H), 6.94 (d, J = 15.9 Hz, 2H), 6.93 (d, J = 8.7 Hz,
4H), 5.04 (s, 4H), 3.83 (s, 6H).
4.2.10. 2-Methoxy-(E,E)-4,6-bis[30-(400-methoxybenzyloxy)styryl]
pyrimidine (2b0)
Compound 2b0 was prepared from 7a and 12b according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 2b0 (0.53 g, 45%). 1H NMR (400 MHz, DMSO-d6)
d 7.89 (d, J = 15.9 Hz, 2H), 7.38 (d, J = 8.6 Hz, 4H), 7.31 (dd, J = 8.0,
8.0 Hz, 2H), 7.21–7.20 (m, 4H), 7.02–6.91 (m, 9H), 5.03 (s, 4H),
4.11 (s, 3H), 3.82 (s, 6H).
4.2.4. (E,E)-4,6-Bis[30-(400-methoxybenzyloxy)styryl]pyrimidine
(1b0)
4.2.11. 2-Methoxy-(E,E)-4,6-bis[30-methoxy-40-(400-methoxyben-
zyloxy)styryl]pyrimidine (2c0)
Compound 1b0 was prepared from 4,6-dimethylpyrimidine and
12b according to general procedure A. The crude product was puri-
fied by crystallization from EtOAc to give 1b0 (0.84 g, 76%). 1H NMR
(400 MHz, DMSO-d6) d 9.05 (d, J = 0.7 Hz, 1H), 7.89 (d, J = 16.0 Hz,
2H), 7.66 (d, J = 0.8 Hz, 1H), 7.41–7.27 (m, 12H), 7.01 (dd, J = 1.3,
7.9 Hz, 2H), 6.94 (d, J = 8.6 Hz, 4H), 5.07 (s, 4H), 3.74 (s, 6H).
Compound 2c0 was prepared from 7a and 12c according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 2c0 (0.73 g. 57%). 1H NMR (400 MHz, DMSO-d6)
d 7.82 (d, J = 15.9 Hz, 2H), 7.37 (d, J = 8.6 Hz, 4H), 7.35 (d, J = 1.8 Hz,
2H), 7.21 (dd, J = 8.4, 1.8 Hz, 2H), 7.12 (d, J = 15.9 Hz, 2H), 7.09 (d,
J = 8.4 Hz, 2H), 6.95 (d, J = 8.6 Hz, 4H), 5.04 (s, 4H), 3.98 (s, 3H),
3.83 (s, 6H), 3.75 (s, 6H).
4.2.5. (E,E)-4,6-Bis[30-methoxy-40-(400-methoxybenzyloxy)styryl]
pyrimidine (1c0)
Compound 1c0 was prepared from 4,6-dimethylpyrimidine and
12c according to general procedure A. The crude product was puri-
fied by crystallization from EtOAc to give 1c0 (1.0 g, 81%). 1H NMR
(400 MHz, CDCl3) d 9.04 (d, J = 0.8 Hz, 1H), 7.81 (d, J = 15.9 Hz, 2H),
7.37 (d, J = 8.6 Hz, 5H), 7.16 (d, J = 1.7 Hz, 2H), 7.11 (dd, J = 8.3,
1.7 Hz, 2H), 6.93 (d, J = 15.9 Hz, 2H), 6.91 (d, J = 8.3 Hz, 2H), 6.91
(d, J = 8.6 Hz, 4H), 5.12 (s, 4H), 3.93 (s, 6H), 3.80 (s, 6H).
4.2.12. 2-Methoxy-(E,E)-4,6-bis[40-methoxy-30-(400-
methoxybenzyloxy)styryl]pyrimidine (2d0)
Compound 2d0 was prepared from 7a and 12d according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 2d0 (0.71 g, 55%). 1H NMR (400 MHz, DMSO-d6)
d 7.81 (d, J = 15.9 Hz, 2H), 7.46 (d, J = 1.6 Hz, 2H), 7.41 (d, J = 8.6 Hz,
4H), 7.25 (s, 1H), 7.24 (dd, J = 8.4, 1.6 Hz, 2H), 7.12 (d, J = 15.9 Hz,
2H), 7.02 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.6 Hz, 4H), 5.08 (s, 4H),
3.98 (s, 3H), 3.79 (s, 6H), 3.75 (s, 6H).
4.2.6. (E,E)-4,6-Bis[40-methoxy-30-(400-methoxybenzyloxy)styryl]
pyrimidine (1d0)
Compound 1d0 was prepared from 4,6-dimethylpyrimidine and
12d according to general procedure A. The crude product was puri-
fied by crystallization from EtOAc to give 1d0 (1.0 g, 81%). 1H NMR
(400 MHz, DMSO-d6) d 9.03 (s, 1H), 7.78 (d, J = 15.9 Hz, 2H), 7.40 (d,
J = 8.5 Hz, 4H), 7.22 (s, 1H), 7.19 (s, 2H), 7.18 (d, J = 8.6 Hz, 2H), 6.92
(d, J = 8.5 Hz, 4H), 6.91 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 15.9 Hz, 2H),
5.12 (s, 4H), 3.91 (s, 6H), 3.81 (s, 6H).
4.2.13. 2-Methoxy-(E,E)-4,6-bis(40-methoxymethoxy-30-N,N-
dimethylaminostyryl)pyrimidine (2e0)
Compound 2e0 was prepared from 7a and 13e according to gen-
eral procedure A. The crude product was purified by flash column
chromatography (MeOH/CH2Cl2 = 1:50) to give 2e0 (0.58 g, 55%). 1H
NMR (400 MHz, CDCl3) d 7.86 (d, J = 15.8 Hz, 2H), 7.20–7.13 (m,
6H), 6.93 (s, 1H), 6.91 (d, J = 15.9 Hz, 2H), 5.30 (s, 4H), 4.12 (s,
3H), 3.54 (s, 6H), 2.86 (s, 12H).
4.2.7. (E,E)-4,6-Bis(40-methoxymethoxy-30-N,N-dimethylamino-
styryl)pyrimidine (1e0)
4.2.14. 2-Methoxy-(E,E)-4,6-bis(30-methoxymethoxy-40-N,N-
dimethylaminostyryl)pyrimidine (2f0)
Compound 1e0 was prepared from 4,6-dimethylpyrimidine and
13e according to general procedure A. The crude product was puri-
fied by flash column chromatography (EtOAc/n-hexane = 2:1) to
give 1e0 (0.51 g, 52%). 1H NMR (400 MHz, DMSO-d6) d 8.97 (d,
J = 1.0 Hz, 1H), 7.87 (d, J = 15.9 Hz, 2H), 7.67 (d, J= 1.0 Hz, 1H),
7.24 (dd, J = 8.9, 1.9 Hz, 2H), 7.23 (d, J = 1.9 Hz, 2H), 7.11 (d,
J = 15.9 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H), 5.24 (s, 4H), 3.42 (s, 6H),
2.77 (s, 12H).
Compound 2f0 was prepared from 7a and 13f according to gen-
eral procedure A. The crude product was purified by flash column
chromatography (MeOH/CH2Cl2 = 1:50) to give 2f0 (0.53 g, 51%).
1H NMR (400 MHz, CDCl3) d 7.84 (d, J = 15.8 Hz, 2H), 7.39 (s,
2H), 7.21 (d, J = 8.1 Hz, 2H), 6.92 (d, J = 8.1 Hz, 2H), 6.91 (s, 1H),
6.89 (d, J = 15.8 Hz, 2H), 5.29 (s, 4H), 4.10 (s, 3H), 3.56 (s, 6H),
2.86 (s, 12H).
4.2.8. (E,E)-4,6-Bis(30-methoxymethoxy-40-N,N-dimethylamino-
styryl)pyrimidine (1f0)
4.2.15. 2-Methylthio-(E,E)-4,6-bis[40-(400-methoxybenzyloxy)
styryl]pyrimidine (3a0)
Compound 1f0 was prepared from 4,6-dimethylpyrimidine and
13f according to general procedure A. The crude product was puri-
fied by flash column chromatography (EtOAc/n-hexane = 1.5:1) to
give 1f0 (0.42 g, 43%). 1H NMR (400 MHz, DMSO-d6) d 8.94 (s,
1H), 7.82 (d, J = 15.9 Hz, 2H), 7.66 (s, 1H), 7.35 (d, J = 1.8 Hz, 2H),
7.26 (dd, J = 8.3, 1.8 Hz, 2H), 7.03 (d, J = 15.9 Hz, 2H), 6.90 (d,
J = 8.3 Hz, 2H), 5.26 (s, 4H), 3.45 (s, 6H), 2.80 (s, 12H).
Compound 3a0 was prepared from 7b and 12a according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 3a0 (0.84 g, 70%). 1H NMR (400 MHz, DMSO-d6)
d 7.83 (d, J = 15.9 Hz, 2H), 7.65 (d, J = 8.7 Hz, 4H), 7.37 (d, J = 8.6 Hz,
4H), 7.27 (s, 1H), 7.05 (d, J = 8.7 Hz, 4H), 7.05 (d, J = 15.9 Hz, 2H),
6.94 (d, J = 8.6 Hz, 4H), 5.06 (s, 4H), 3.74 (s, 6H), 2.58 (s, 3H).

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Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4.2.16. 2-Methylthio-(E,E)-4,6-bis[30-(400-methoxybenzyloxy)
styryl]pyrimidine (3b0)
DMSO-d6) d 7.80 (s, 1H), 7.44–7.33 (m, 3H), 7.28 (s, 2H), 7.26 (d,
J = 16.4 Hz, 2H), 7.17 (d, J = 16.4 Hz, 2H), 7.09 (dd, J = 8.3, 1.5 Hz,
2H), 7.05 (d, J = 8.3 Hz, 2H), 5.15 (s, 4H), 3.84 (s, 6H), 3.38 (s, 6H).
Compound 3b0 was prepared from 7b and 12b according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 3b0 (0.79 g, 66%). 1H NMR (400 MHz, CDCl3) d
7.85 (d, J = 15.9 Hz, 2H), 7.38 (d, J = 8.6 Hz, 4H), 7.31 (dd, J = 8.1,
8.1 Hz, 2H), 7.21–7.19 (m, 4H), 6.99 (d, J = 15.9 Hz, 2H), 6.99–6.95
(m, 3H), 6.93 (d, J = 8.6 Hz, 4H), 5.04 (s, 4H), 3.83 (s, 6H), 2.68 (s,
3H).
4.2.24. (E,E)-3,5-Bis(40-methoxy-30-methoxmethoxystyryl)
benzene (4d0)
Compound 4d0 was prepared from 10a and 13d according to the
general procedure B to give4d0 (0.69 g, 75%). 1H NMR (400 MHz,
DMSO-d6) d 7.79 (s, 1H), 7.34–7.32 (m, 5H), 7.23 (d, J = 16.4 Hz,
2H), 7.21 (dd, J = 8.5, 1.9 Hz, 2H), 7.07 (d, J = 16.4 Hz, 2H), 7.01 (d,
J= 8.5 Hz, 2H), 5.20 (s, 4H), 3.78 (s, 6H), 3.41 (s, 6H).
4.2.17. 2-Methylthio-(E,E)-4,6-bis[30-methoxy-40-(400-methoxy-
benzyloxy)styryl]pyrimidine (3c0)
Compound 3c0 was prepared from 7b and 12c according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 3c0 (1.20 g, 91%). 1H NMR (400 MHz, CDCl3) d
7.79 (d, J = 15.9 Hz, 2H),7.37 (d, J = 7.4 Hz, 5H), 7.14 (s, 2H), 7.09
(d, J = 8.2 Hz, 2H), 6.92–6.84 (m, 8H), 5.12 (s, 4H), 3.93 (s, 6H),
3.81 (s, 6H), 2.67 (s, 3H).
4.2.25. (E,E)-3,5-Bis(40-methoxymethoxy-30-N,N-dimethylamino-
styryl)benzene (4e0)
Compound 4e0 was prepared from 10a and 13e according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:1) to give 4e0 (0.68 g,
70%). 1H NMR (400 MHz, DMSO-d6) d 7.80 (s, 1H), 7.43–7.32 (m,
3H), 7.24 (d, J = 16.4 Hz, 2H), 7.14 (d, J = 1.8 Hz, 2H), 7.11 (dd,
J = 8.2, 1.8 Hz, 2H), 7.10 (d, J = 16.4 Hz, 2H), 7.00 (d, J = 8.2 Hz,
2H), 5.19 (s, 4H), 3.41 (s, 6H), 2.75 (s, 12H).
4.2.18. 2-Methylthio-(E,E)-4,6-bis[40-methoxy-30-(400-methoxy-
benzyloxy)styryl]pyrimidine (3d0)
Compound 3d0 was prepared from 7b and 12d according to gen-
eral procedure A. The crude product was purified by crystallization
from EtOAc to give 3d0 (1.24 g, 94%). 1H NMR (400 MHz, DMSO-d6)
d 7.81 (d, J = 15. 9 Hz, 2H), 7.47 (d, J = 1.6 Hz, 2H), 7.40 (d, J = 8.6 Hz,
4H), 7.29 (s, 1H), 7.25 (dd, J = 8.3, 1.6 Hz, 2H), 7.10 (d, J = 15.9 Hz,
2H), 7.01 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.6 Hz, 4H), 5.07 (s, 4H),
3.78 (s, 6H), 3.74 (s, 6H), 2.60 (s, 3H).
4.2.26. (E,E)-3,5-Bis(30-methoxymethoxy-40-N,N-dimethylamino-
styryl)benzene (4f0)
Compound 4f0 was prepared from 10a and 13f according to gen-
eral procedure B (0.88 g, 90%). 1H NMR (400 MHz, DMSO-d6) d 7.78
(s, 1H), 7.41–7.29 (m, 3H), 7.26 (d, J = 1.7 Hz, 2H), 7.22 (d,
J = 16.3 Hz, 2H), 7.16 (dd, J = 8.2, 1.7 Hz, 2H), 7.04 (d, J = 16.3 Hz,
2H), 6.88 (d, J = 8.2 Hz, 2H), 5.24 (s, 4H), 3.44 (s, 6H), 2.74 (s, 12H).
4.2.19. 2-Methythio-(E,E)-4,6-bis(40-methoxymethoxy-30-N,N-
dimethylaminostyryl)pyrimidine (3e0)
4.2.27. (E,E)-3,5-Bis(40-methoxymethoxystyryl)phenol (5a0)
Compound 5a0 was prepared from 10b and 13a according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:2) to give 5a0 (0.47 g,
57%). 1H NMR (400 MHz, DMSO-d6) d 9.45 (s, 1H), 7.53 (d,
J = 8.7 Hz, 4H), 7.23 (s, 1H), 7.15 (d, J = 16.3 Hz, 2H), 7.02 (d,
J = 8.7 Hz, 4H), 7.02 (d, J = 16.3 Hz, 2H), 6.83 (d, J = 1.1 Hz, 2H),
5.19 (s, 4H).
Compound 3e0 was prepared from 7b and 13e according to gen-
eral procedure A. The crude product was purified by flash column
chromatography (THF/n-hexane = 1:1) to give 3e0 (0.47 g, 44%). 1H
NMR (400 MHz, CDCl3) d 7.81 (d, J = 15.9 Hz, 2H), 7.20–7.13 (m,
6H), 6.97 (s, 1H), 6.89 (d, J = 15.9 Hz, 2H), 3.54 (s, 6H), 2.86 (s,
12H), 2.69 (s, 3H).
4.2.20. 2-Methylthio-(E,E)-4,6-bis(30-methoxymethoxy-40-N,N-
dimethylaminostyryl)pyrimidine (3f0)
4.2.28. (E,E)-3,5-Bis(30-methoxymethoxystyryl)phenol (5b0)
Compound 5b0 was prepared from 10b and 13b according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:2) to give 5b0 (0.45 g,
54%). 1H NMR (400 MHz, DMSO-d6) d 9.61 (br s, 1H), 7.32–7.22
(m, 7H), 7.17 (d, J = 1.5 Hz, 4H), 6.93–6.91 (m, 2H), 6.88 (d,
J = 1.1 Hz, 2H), 5.22 (s, 4H), 3.38 (s, 6H).
Compound 3f0 was prepared from 7b and 13f according to gen-
eral procedure A. The crude product was purified by flash column
chromatography (EtOAc/n-hexane = 1:2) to give 3f0 (0.54 g, 51%).
1H NMR (400 MHz, CDCl3) d 7.79 (d, J = 15.9 Hz, 2H), 7.39 (d,
J = 1.6 Hz, 2H), 7.21 (dd, J = 8.3, 1.6 Hz, 2H), 6.95–6.86 (m, 4H),
5.30 (s, 4H), 3.56 (s, 6H), 2.89 (s, 12H), 2.68 (s, 3H).
4.2.21. (E,E)-3,5-Bis(40-methoxymethoxystyryl)benzene (4a0)
Compound 4a0 was prepared from 10a and 13a according to
general procedure B. The crude product was purified by crystalliza-
tion from CH2Cl2/n-hexane (1:8) solution to give 4a0 (0.50 g, 63%).
1H NMR (400 MHz, DMSO-d6) d 7.77 (s, 1H), 7.54 (d, J = 8.6 Hz, 4H),
7.43–7.31 (m, 3H), 7.26 (d, J = 16.4 Hz, 2H), 7.11 (d, J = 16.4 Hz, 2H),
7.03 (d, J = 8.6 Hz, 4H), 5.20 (s, 4H), 3.37 (s, 6H).
4.2.29. (E,E)-3,5-Bis(30-methoxy-4-methoxymethoxystyryl)
phenol (5c0)
Compound 5c0 was prepared from 10b and 13c according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:1) to give 5c0 (0.61 g,
64%). 1H NMR (400 MHz, DMSO-d6) d 7.26 (d, J = 1.4 Hz, 3H), 7.14
(d, J = 16.3 Hz, 2H), 7.08 (dd, J = 8.4, 1.7 Hz, 2H), 7.07 (d,
J = 16.3 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 1.1 Hz, 2H),
5.14 (s, 4H), 3.83 (s, 6H), 3.38 (s, 6H).
4.2.22. (E,E)-3,5-Bis(30-methoxymethoxystyryl)benzene (4b0)
Compound 4b0 was prepared from 10a and 13b according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:5) to give 4b0 (0.70 g,
88%). 1H NMR (400 MHz, DMSO-d6) d 7.86 (s, 1H), 7.50–7.23 (m,
13H), 6.93 (dd, J = 7.7, 1.3 Hz, 2H), 5.23 (s, 4H), 3.39 (s, 6H).
4.2.30. (E,E)-3,5-Bis(40-methoxy-30-methoxymethoxystyryl)
phenol (5d0)
Compound 5d0 was prepared from 10b and 13d according to
general procedure B. The crude product was purified by crystalliza-
tion from EtOAc/n-hexane (1:3) to give 5d0 (0.49 g, 52%). 1H NMR
(400 MHz, DMSO-d6) d 9.43 (s, 1H), 7.32 (d, J = 1.8 Hz, 2H), 7.25
(s, 1H), 7.19 (dd, J = 8.4, 1.8 Hz, 2H), 7.12 (d, J = 16.3 Hz, 2H), 7.00
(d, J = 8.4 Hz, 2H), 6.98 (d, J = 16.3 Hz, 2H), 6.81 (d, J = 0.8 Hz, 2H),
5.19 (s, 4H), 3.78 (s, 6H), 3.41 (s, 6H).
4.2.23. (E,E)-3,5-Bis(30-methoxy-40-methoxmethoxystyryl)
benzene (4c0)
Compound 4c0 was prepared from 10a and 13caccording to the
general procedure B to give 4c0 (0.77 g, 84%). 1H NMR (400 MHz,

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4929
4.2.31. (E,E)-3,5-Bis(40-methoxymethoxy-30-N,N-dimethylamino-
styryl)phenol (5e0)
uct was purified by recrystallization from EtOH/H2O to give 1c
(0.46 g, 71%). 1H NMR (400 MHz, DMSO-d6) d 9.48 (br s, 2H), 8.95
(s, 1H), 7.83 (d, J = 16.0 Hz, 2H), 7.57 (s, 1H), 7.32 (d, J = 1.6 Hz,
2H), 7.13 (dd, J = 8.1, 1.6 Hz, 2H), 7.09 (d, J = 16.0 Hz, 2H), 6.82 (d,
J = 8.1 Hz, 2H), 3.84 (s, 6H); 13C NMR (100 MHz, DMSO-d6) d
162.8, 157.9, 149.1, 148.4, 138.0, 127.4, 122.8, 122.7, 116.1,
115.6, 111.3, 56.1. HRMS: calcd for C22H20N2O4 + H+, 377.1501;
found (ESI, [M+H]+), 377.1496. Mp 242 °C.
Compound 5e0 was prepared from 10b and 13e according to
general procedure B. The crude product was purified by flash col-
umn chromatography (EtOAc/n-hexane = 1:2) to give 5e0 (0.44 g,
44%). 1H NMR (400 MHz, DMSO-d6) d 9.44 (s, 1H), 7.28 (s, 1H),
7.16–7.00 (m, 10H), 6.83 (d, J = 0.9 Hz, 2H), 5.20 (s, 4H), 3.42 (s,
6H), 2.76 (s, 12H).
4.2.32. (E,E)-3,5-Bis(30-methoxymethoxy-40-N,N-dimethylamino-
styryl)phenol (5f0)
4.2.38. (E,E)-4,6-Bis-(30-hydroxy-40-methoxystyryl)pyrimidine
(1d)
Compound 5f0 was prepared from 10b and 13f according to gen-
eral procedure B. The crude product was purified by flash column
chromatography (EtOAc/n-hexane = 1:2) to give 5f0 (0.69 g, 69%).
1H NMR (400 MHz, DMSO-d6) d 9.42 (s, 1H), 7.24 (s, 1H), 7.24 (d,
J = 1.8 Hz, 2H), 7.14 (dd, J = 8.3, 1.8 Hz, 2H), 7.12 (d, J = 16.3 Hz,
2H), 6.94 (d, J = 16.3 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H), 6.79 (d,
J = 1.0 Hz, 2H), 5.24 (s, 4H), 3.43 (s, 6H), 2.74 (s, 12H).
Compound 1d0 (1.06 g, 1.70 mmol) was deprotected in EtOH/
1 N HCl (48 mL) according to the general procedure C. The crude
product was purified by recrystallization from EtOH/H2O to give
1d (0.60 g, 93%). 1H NMR (400 MHz, DMSO-d6) d 9.17 (s, 2H),
8.95 (d, J = 1.0 Hz, 1H), 7.78 (d, J = 15.9 Hz, 2H), 7.62 (d, J = 1.0 Hz,
1H), 7.13 (d, J = 1.9 Hz, 2H), 7.11 (dd, J = 8.3, 1.9 Hz, 2H), 6.98 (d,
J = 15.9 Hz, 2H), 6.97 (d, J = 8.3 Hz, 2H), 3.80 (s, 6H); 13C NMR
(100 MHz, DMSO-d6) d 162.8, 158.6, 149.7, 147.2, 137.0, 128.9,
123.9, 120.8, 116.1, 114.1, 112.5, 56.0. HRMS: calcd for
4.2.33. General procedure C
Removal of p-methoxybenzyl and methoxymethyl protecting
groups in 1a0–5f0 for the preparation of 1a–5f. A solution of 1a0–
5f0 in EtOH/1 N HCl (3:1) was refluxed. When the reaction was
completed, the reaction mixture was allowed to cool to room tem-
perature and excess H2O was added. (Compounds containing
dimethylamino group were neutralized with NaHCO3 before add-
ing H2O) If the product precipitated, it was collected by filtration.
Otherwise, the reaction mixture was extracted with EtOAc. The or-
ganic layer was washed with brine and concentrated in vacuo. The
residue was purified by recrystallization or flash column chroma-
tography to give the products.
C
22H20N2O4 + H+, 377.1501; found (ESI, [M+H]+), 377.1503. Mp
202 °C.
4.2.39. (E,E)-4,6-Bis-(40-hydroxy-30-N,N-dimethylaminostyryl)
pyrimidine (1e)
Compound 1e0 (0.51 g, 1.00 mmol) was deprotected in EtOH/1 N
HCl (48 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 1e
(0.31 g, 77%). 1H NMR (400 MHz, DMSO-d6) d 9.62 (br s, 2H), 8.93
(s, 1H), 7.81 (d, J = 15.9 Hz, 2H), 7.59 (s, 1H), 7.19 (d, J = 1.8 Hz,
2H), 7.16 (dd, J = 8.1, 1.8 Hz, 2H), 7.01 (d, J = 15.9 Hz, 2H), 6.81 (d,
J = 8.1 Hz, 2H), 2.73 (s, 12H); 13C NMR (100 MHz, DMSO-d6) d
163.0, 158.6, 152.1, 141.7, 137.4, 127.4, 123.1, 123.0, 117.7,
116.3, 115.6, 43.0. HRMS: calcd for C24H26N4O2 + H+, 403.2134;
found (ESI, [M+H]+), 403.2133. Mp 129 °C.
4.2.34. General procedure D
Removal of p-methoxybenzyl and methoxymethyl protecting
groups in 1a0–5f0 for preparation of 1a–5f. To a stirred solution of
1a0–5f0 in CH2Cl2 was added trifluoroacetic acid (TFA) and the mix-
ture was stirred at room temperature for 1–2 h. When the reaction
was complete, CH2Cl2 and trifluoroacetic acid was evaporated. The
residue was purified by recrystallization or flash column chroma-
tography to give the products.
4.2.40. (E,E)-4,6-Bis-(30-hydroxy-40-N,N-dimethylaminostyryl)
pyrimidine (1f)
Compound 1f0 (0.43 g, 0.87 mmol) was deprotected in EtOH/1 N
HCl (48 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 1f
(0.28 g, 80%). 1H NMR (400 MHz, DMSO-d6) d 9.48 (br s, 2H), 8.93
(s, 1H), 7.76 (d, J = 15.9 Hz, 2H), 7.63 (s, 1H), 7.07 (s, 2H), 7.06 (d,
J = 8.0 Hz, 2H), 6.91 (d, J = 15.9 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H),
2.75 (s, 12H); 13C NMR (100 MHz, DMSO-d6) d 162.8, 158.6,
149.9, 143.0, 137.0, 129.1, 123.3, 120.7, 118.1, 115.8, 114.2, 42.6.
HRMS: calcd for C24H26N4O2 + H+, 403.2134; found (ESI, [M+H]+),
403.2132. Mp 189 °C.
4.2.35. (E,E)-4,6-Bis-(40-hydroxystyryl)pyrimidine (1a)
Compound 1a0 (1.00 g, 1.80 mmol) was deprotected in EtOH/1 N
HCl (80 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 1a
(0.55 g, 96%). 1H NMR (400 MHz, DMSO-d6) d 9.96 (br s, 2H), 8.99
(s, 1H), 7.90 (d, J = 16.0 Hz, 2H), 7.67 (s, 1H), 7.58 (d, J = 8.6 Hz,
4H), 7.06 (d, J = 16.0 Hz, 2H), 6.84 (d, J = 8.6 Hz, 4H); 13C NMR
(100 MHz, DMSO-d6) d 162.1, 160.6, 154.2, 141.3, 130.7, 126.4,
119.9, 116.5, 115.3. HRMS: calcd for C20H16N2O2 + H+, 317.1290;
found (ESI, [M+H]+), 317.1289. Mp 292 °C.
4.2.41. (E,E)-4,6-Bis-styrylpyrimidine (1g)
4.2.36. (E,E)-4,6-Bis-(30-hydroxystyryl)pyrimidine (1b)
Compound 1g was prepared from 4,6-dimethylpyrimidine and
benzaldehyde according to general procedure A. The crude product
was purified by recrystallization from EtOAc to give 1g (0.43 g,
Compound 1b0 (0.90 g, 1.60 mmol) was deprotected in EtOH/1 N
HCl (48 mL) according to the general procedure C. The crude product
was purified by recrystallization from EtOH/H2O to give 1b (0.39 g,
78%). 1H NMR (400 MHz, DMSO-d6) d 9.57 (br s, 2H), 9.04 (d,
J = 0.7 Hz, 1H), 7.84 (d, J = 16.0 Hz, 2H), 7.74 (d, J = 0.8 Hz, 1H),
7.25–7.13 (m, 6H), 7.07 (d, J = 1.7 Hz, 2H), 6.79 (dd, J = 7.9, 1.7 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) d 162.7, 158.3, 158.2, 137.7,
137.1, 130.4, 125.9, 119.2, 117.2, 116.8, 114.5. HRMS: calcd for
76%). 1H NMR (400 MHz, CDCl3)
d 9.11 (s, 1H), 7.91 (d,
J = 15.9 Hz, 2H), 7.63–7.61 (m, 4H), 7.43–7.34 (m, 6H), 7.30 (s,
1H), 7.08 (d, J = 15.9 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 162.7,
158.7, 137.0, 135.7, 129.4, 128.9, 127.6, 125.8, 116.4.
4.2.42. 2-Methoxy-(E,E)-4,6-bis-(40-hydroxystyryl)pyrimidine
(2a)
C
20H16N2O2 + H+, 317.1290; found (ESI, [M+H]+), 317.1289. Mp
Compound 2a0 (0.57 g, 0.97 mmol) was deprotected with TFA
(6 mL) in CH2Cl2 (60 mL) according to the general procedure D.
The crude product was purified by recrystallization from EtOAc
to give 2a (0.17 g, 51%). 1H NMR (400 MHz, DMSO-d6) d 9.87 (br
s, 2H), 7.79 (d, J = 15.9 Hz, 2H), 7.54 (d, J = 8.5 Hz, 4H), 7.20 (s,
1H), 6.98 (d, J = 15.9 Hz, 2H), 6.81 (d, J = 8.5 Hz, 4H), 3.96 (s, 3H);
218 °C.
4.2.37. (E,E)-4,6-Bis-(40-hydroxy-30-methoxystyryl)pyrimidine
(1c)
Compound 1c0 (1.06 g, 1.70 mmol) was deprotected in EtOH/1 N
HCl (48 mL) according to the general procedure C. The crude prod-

4930
Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
13C NMR (100 MHz, DMSO-d6) d 165.3, 165.0, 159.5, 137.4, 130.0,
126.9, 122.6, 116.3, 110.5, 54.6. HRMS: calcd for C21H18N2O3 + H+,
347.1396; found (ESI, [M+H]+), 347.1393. Mp 162 °C.
3.95 (s, 3H), 2.74 (s, 12H); 13C NMR (100 MHz, DMSO-d6) d 165.6,
165.2, 149.8, 143.0, 137.1, 129.0, 123.3, 120.8, 118.1, 114.2,
110.6, 54.5, 42.6. HRMS: calcd for C25H28N4O3 + H+, 433.2240;
found (ESI, [M+H]+), 433.2234. Mp 102 °C.
4.2.43. 2-Methoxy-(E,E)-4,6-bis-(30-hydroxystyryl)pyrimidine
(2b)
4.2.48. 2-Methoxy-(E,E)-4,6-bis-styrylpyrimidine (2g)
Compound 2b0 (0.10 g, 0.17 mmol) was deprotected in EtOH/
1 N HCl (8 mL) according to the general procedure C. The crude
product was purified by flash column chromatography (EtOAc/n-
hexane = 1:2) to give 2b (0.02 g, 34%). 1H NMR (400 MHz, DMSO-
d6) d 9.58 (br s, 2H), 7.79 (d, J = 15.9 Hz, 2H), 7.37 (s, 1H), 7.22
(dd, J = 7.8, 7.8 Hz, 2H), 7.12 (d, J = 6.0 Hz, 2H), 7.11 (d,
J = 15.9 Hz, 2H), 7.05 (s, 2H), 7.78 (dd, J = 6.0, 1.8 Hz, 2H), 3.98 (s,
3H); 13C NMR (100 MHz, DMSO-d6) d 165.6, 165.2, 158.2, 137.1,
137.0, 130.4, 126.3, 119.2, 117.1, 114.4, 111.5, 54.6. HRMS: calcd
for C21H18N2O3 + H+, 347.1396; found (ESI, [M+H]+), 347.1394.
Mp 229 °C.
Compound 2g was prepared from 7a and benzaldehyde accord-
ing to general procedure A. The crude product was purified by flash
column chromatography (EtOAc/n-hexane = 1:10) to give 2g
(0.50 g, 80%). 1H NMR (400 MHz, CDCl3) d 7.94 (d, J = 15.9 Hz,
2H), 7.61–7.60 (m, 4H), 7.42–7.33 (m, 6H), 7.03 (d. J = 15.9 Hz,
2H), 6.94 (s, 1H), 4.12 (s. 3H); 13C NMR (100 MHz, CDCl3) d 165.7,
165.0, 136.9, 135.8, 129.3, 128.8, 127.6, 125.7, 111.3, 54.6.
4.2.49. 2-Methylthio-(E,E)-4,6-bis-(40-hydroxystyryl)pyrimidine
(3a)
Compound 3a0 (0.52 g, 0.86 mmol) was deprotected with TFA
(4 mL) in CH2Cl2 (40 mL) according to the general procedure D.
The crude product was purified by recrystallization from diethyl
ether to give 3a (0.29 g, 93%). 1H NMR (400 MHz, DMSO-d6) d
9.90 (br s, 2H), 7.79 (d, J = 15.9 Hz, 2H), 7.55 (d, J = 8.6 Hz, 4H),
7.24 (s, 1H), 6.96 (d, J = 15.9 Hz, 2H), 6.81 (d, J = 8.6 Hz, 4H), 2.58
(s, 3H); 13C NMR (100 MHz, DMSO-d6) d 170.9, 163.2, 159.4,
137.2, 130.0, 126.9, 122.7, 116.2, 111.9, 13.9. HRMS: calcd for
4.2.44. 2-Methoxy-(E,E)-4,6-bis-(40-hydroxy-30-methoxystyryl)
pyrimidine (2c)
Compound 2c0 (0.63 g, 0.97 mmol) was deprotected with TFA
(7 mL) in CH2Cl2 (70 mL) according to the general procedure D.
The crude product was purified by recrystallization from EtOAc
to give 2c (0.28 g, 71%). 1H NMR (400 MHz, DMSO-d6) d 9.53 (br
s, 2H), 7.79 (d, J = 15.9 Hz, 2H), 7.30 (d, J = 1.7 Hz, 2H), 7.22 (s,
1H), 7.12 (dd, J = 8.1, 1.7 Hz, 2H), 7.04 (d, J = 15.9 Hz, 2H), 6.81 (d,
J = 8.1 Hz, 2H), 3.97 (s, 3H), 3.84 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) d 165.2, 164.9, 149.0, 148.4, 138.0, 127.4, 122.8, 122.6,
116.1, 111.3, 110.3, 56.0, 54.7. HRMS: calcd for C23H22N2O5 + H+,
407.1607; found (ESI, [M+H]+), 407.1606. Mp 121 °C.
C
21H18N2O2S + H+, 363.1167; found (ESI, [M+H]+), 363.1159. Mp
219 °C.
4.2.50. 2-Methylthio-(E,E)-4,6-bis-(30-hydroxystyryl)pyrimidine
(3b)
Compound 3b0 (0.79 g, 1.30 mmol) was deprotected in EtOH/
1 N HCl (100 mL) according to the general procedure C. The crude
product was purified by flash column chromatography (EtOAc/n-
hexane = 1.5:1) to give 3b (0.24 g, 51%). 1H NMR (400 MHz,
DMSO-d6) d 9.63 (br s, 2H), 7.89 (d, J = 15.9 Hz, 2H), 7.81 (s, 1H),
7.27–7.09 (m, 8H), 6.81 (d, J = 8.0 Hz, 2H), 2.94 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 176.0, 167.8, 163.0, 142.3, 141.8, 135.1,
130.7, 124.0, 121.9, 119.2, 117.4, 18.7. HRMS: calcd for
4.2.45. 2-Methoxy-(E,E)-4,6-bis-(30-hydroxy-40-methoxystyryl)
pyrimidine (2d)
Compound 2d0 (0.22 g, 0.03 mmol) was deprotected with TFA
(1.5 mL) in CH2Cl2 (15 mL) according to the general procedure D.
The crude product was purified by column chromatography
(MeOH/CH2Cl2 = 1:50) to give 2d (0.11 g, 79%). 1H NMR
(400 MHz, DMSO-d6) d 9.18 (br s, 2H), 7.75 (d, J = 15.9 Hz, 2H),
7.28 (s, 1H), 7.13–7.09 (m, 4H), 6.98–6.92 (m, 4H), 3.96 (s, 3H),
3.80 (s, 6H); 13C NMR (100 MHz, DMSO-d6) d 165.5, 165.3, 149.7,
147.2, 137.1, 128.8, 123.8, 120.8, 114.1, 112.5, 110.8, 56.0, 54.5.
HRMS: calcd for C23H22N2O5 + H+, 407.1607; found (ESI, [M+H]+),
407.1607. Mp 140 °C.
C
25H18N4O2S + H+, 363.1167; found (ESI, [M+H]+), 363.1170. Mp
205 °C.
4.2.51. 2-Methylthio-(E,E)-4,6-bis-(40-hydroxy-30-methoxy-
styryl)pyrimidine (3c)
Compound 3c0 (0.75 g, 1.10 mmol) was deprotected with TFA
(5 mL) in CH2Cl2 (50 mL) according to the general procedure D.
The crude product was purified by recrystallization from Et2O to
give 3c (0.21 g, 45%). 1H NMR (400 MHz, DMSO-d6) d 9.47 (s, 2H),
7.78 (d, J = 15. 9 Hz, 2H), 7.30 (d, J = 1.6 Hz, 2H), 7.25 (s, 1H), 7.12
(dd, J = 8.1, 1.6 Hz, 2H), 7.02 (d, J = 15.9 Hz, 2H), 6.80 (d,
J = 8.1 Hz, 2H), 3.83 (s, 6H), 2.58 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d 171.0, 163.3, 148.9, 148.4, 137.6, 127.5, 123.1, 122.5,
116.1, 111.7, 111.3, 56.0, 13.9. HRMS: calcd for C23H22N2O4S + H+,
423.1379; found (ESI, [M+H]+), 423.1372. Mp 145 °C.
4.2.46. 2-Methoxy-(E,E)-4,6-bis-(40-hydroxy-30-N,N-
dimethylaminostyryl)pyrimidine (2e)
Compound 2e0 (0.58 g, 1.11 mmol) was deprotected in EtOH/1 N
HCl (40 mL) according to the general procedure C. The crude prod-
uct was purified by column chromatography (MeOH/
CH2Cl2 = 1:40) to give 2e (0.41 g, 87%). 1H NMR (400 MHz,
DMSO-d6) d 9.58 (br s, 2H), 7.76 (d, J = 15.9 Hz, 2H), 7.24 (s, 1H),
7.17 (d, J = 1.8 Hz, 2H), 7.13 (dd, J = 8.0, 1.8 Hz, 2H), 6.94 (d,
J = 15.9 Hz, 2H), 6.79 (d, J = 8.0 Hz, 2H), 3.95 (s, 3H), 2.71 (s,
12H); 13C NMR (100 MHz, DMSO-d6) d 165.6, 165.5, 152.1, 141.6,
137.5, 127.4, 123.0, 122.9, 117.8, 116.3, 110.3, 54.5, 43.0. HRMS:
4.2.52. 2-Methylthio-(E,E)-4,6-bis-(30-hydroxy-40-methoxy-
styryl)pyrimidine (3d)
calcd for
C
25H28N4O3 + H+, 433.2240; found (ESI, [M+H]+),
Compound 3d0 (1.25 g, 1.90 mmol) was deprotected in EtOH/
1 N HCl (100 mL) according to the general procedure C. The crude
product was purified by flash column chromatography (EtOAc/n-
hexane = 1.5:1) to give 3d (0.24 g, 30%). 1H NMR (400 MHz,
DMSO-d6) d 9.17 (br s, 2H), 7.74 (d, J = 15.9 Hz, 2H), 7.31 (s, 1H),
7.13 (d, J = 1.9 Hz, 2H), 7.10 (dd, J = 8.3, 1.9 Hz, 2H), 6.96 (d,
J = 8.3 Hz, 2H), 6.92 (d, J = 15.9 Hz, 2H), 3.80 (s, 6H), 2.57 (s, 3H);
13C NMR (100 MHz, DMSO-d6) d 171.0, 163.2, 149.7, 147.1, 137.2,
128.8, 123.7, 120.9, 114.1, 112.5, 112.1, 56.0, 13.9. HRMS: calcd
for C23H22N2O4S + H+, 423.1379; found (ESI, [M+H]+), 423.1376.
Mp 216 °C.
433.2237. Mp 115 °C.
4.2.47. 2-Methoxy-(E,E)-4,6-bis-(30-hydroxy-40-N,N-dimethyla-
minostyryl)pyrimidine (2f)
Compound 2f0 (0.53 g, 1.01 mmol) was deprotected in EtOH/1 N
HCl (40 mL) according to the general procedure C. The crude prod-
uct was purified by column chromatography (MeOH/
CH2Cl2 = 1:40) to give 2f (0.32 g, 74%). 1H NMR (400 MHz, DMSO-
d6) d 9.36 (br s, 2H), 7.71 (d, J = 15.8 Hz, 2H), 7.29 (s, 1H), 7.06–
7.05 (m, 4H), 6.85 (d, J = 15.8 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H),

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4931
4.2.53. 2-Methylthio-(E,E)-4,6-bis-(40-hydroxy-30-N,N-
dimethylaminostyryl)pyrimidine (3e)
13C NMR (100 MHz, DMSO-d6) d 148.3, 147.2, 138.3, 129.4, 129.3,
129.1, 125.7, 125.3, 123.9, 120.6, 116.0, 110.2, 56.0. HRMS: calcd
for C24H22O4 + H+, 375.1596; found (ESI, [M+H]+), 375.1588. Mp
181 °C.
Compound 3e0 (0.50 g, 0.90 mmol) was deprotected in EtOH/1 N
HCl (60 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 3e
(0.17 g, 42%). 1H NMR (400 MHz, DMSO-d6) d 9.60 (br s, 2H), 7.76
(d, J = 15.9 Hz, 2H), 7.29 (s, 1H), 7.17 (d, J = 1.7 Hz, 2H), 7.15 (dd,
J = 8.1, 1.7 Hz, 2H), 6.94 (d, J = 15.9 Hz, 2H), 6.79 (d, J = 8.1 Hz,
2H), 2.71 (s, 12H), 2.57 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
171.0, 163.3, 152.2, 141.6, 137.7, 127.3, 123.1, 122.7, 117.8,
4.2.59. (E,E)-3,5-Bis-(30-hydroxy-40-methoxystyryl)benzene (4d)
Compound 4d0 (0.69 g, 1.80 mmol) was deprotected in EtOH/
1 N HCl (80 mL) according to the general procedure C. The crude
product was purified by recrystallization from EtOH/H2O to give
4d (0.52 g, 77%). 1H NMR (400 MHz, DMSO-d6) d 9.03 (s, 2H),
7.74 (s, 1H), 7.41–7.29 (m, 3H), 7.16 (d, J = 16.3 Hz, 2H), 7.04 (d,
J = 1.9 Hz, 2H), 6.98 (dd, J = 8.3, 1.9 Hz, 2H), 6.98 (d, J = 16.3 Hz,
2H), 6.91 (d, J = 8.3 Hz, 2H), 3.77 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) d 148.2, 147.1, 138.1, 130.5, 129.4, 129.1, 126.3, 125.5,
124.3, 118.9, 113.4, 112.6, 56.0. HRMS: calcd for C24H22O4 + H+,
375.1596; found (ESI, [M+H]+), 375.1593. Mp 255 °C.
116.3, 111.6, 43.0, 13.9. HRMS: calcd for
C
25H28N4O2S + H+,
449.2011; found (ESI, [M+H]+), 449.2009. Mp 168 °C.
4.2.54. 2-Methylthio-(E,E)-4,6-bis-(30-hydroxy-40-N,N-
dimethylaminostyryl)pyrimidine (3f)
Compound 3f0 (0.54 g, 1.00 mmol) was deprotected in EtOH/1 N
HCl (60 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 3f
(0.26 g, 61%). 1H NMR (400 MHz, DMSO-d6) d 9.41 (br s, 2H), 7.73
(d, 2H), 7.35 (s, 1H), 7.08–7.06 (m, 4H), 6.88–6.84 (m, 4H), 2.75
(s, 12H), 2.65 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 171.0,
163.1, 149.8, 142.7, 137.3, 129.2, 123.2, 120.8, 118.2, 114.3,
111.8, 42.7, 13.9. HRMS: calcd for C25H28N4O2S + H+, 449.2011;
found (ESI, [M+H]+), 449.2007. Mp 123 °C.
4.2.60. (E,E)-3,5-Bis-(40-hydroxy-30-N,N-dimethylaminostyryl)
benzene (4e)
Compound 4e0 (0.35 g, 0.70 mmol) was deprotected in EtOH/1 N
HCl (40 mL) according to the general procedure C. The crude prod-
uct was purified by flash column chromatography (EtOAc/n-hex-
ane = 1:1) to 4e (0.18 g, 64%). 1H NMR (400 MHz, DMSO-d6) d 9.25
(br s, 2H), 7.74 (s, 1H), 7.37–7.27 (m, 3H), 7.18 (d, J = 15.6 Hz, 2H),
7.10 (s, 2H), 7.03 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 15.6 Hz, 2H), 6.75
(d, J = 8.1 Hz, 2H), 2.71 (s, 12H); 13C NMR (100 MHz, DMSO-d6) d
150.5, 141.5, 138.3, 129.6, 129.3, 125.3, 125.2, 123.8, 121.3, 116.7,
116.2, 43.1. HRMS: calcd for C26H28N2O2 + H+, 401.2229; found
(ESI, [M+H]+), 401.2230. Mp 94 °C.
4.2.55. 2-Methylthio-(E,E)-4,6-bis-styrylpyrimidine (3g)
Compound 3g was prepared from 7b and benzaldehyde accord-
ing to general procedure A. The crude product was purified by crys-
tallization from EtOAc to give 3g (0.37 g, 56%). 1H NMR (400 MHz,
CDCl3) d 7.88 (d, J = 15.9 Hz, 2H), 7.61–7.59 (m, 4H), 7.42–7.35 (m,
6H), 7.01 (d, J = 15.9 Hz, 2H), 6.96 (s, 1H), 2.68 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 172.0, 162.7, 137.0, 135.8, 129.3, 128.8,
127.7, 125.8, 112.0, 14.2.
4.2.61. (E,E)-3,5-Bis-(30-hydroxy-40-N,N-dimethylaminostyryl)
benzene (4f)
Compound 4f0 (0.90 g, 1.80 mmol) was deprotected in EtOH/1 N
HCl (80 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 4f
(0.54 g, 75%). 1H NMR (400 MHz, DMSO-d6) d 9.11 (s, 2H), 7.73 (s,
1H), 7.39–7.28 (m, 3H), 7.15 (d, J = 16.3 Hz, 2H), 6.99 (d,
J = 1.7 Hz, 2H), 6.96 (dd, J = 8.1, 1.7 Hz, 2H), 6.94 (d, J = 16.3 Hz,
2H), 6.82 (d, J = 8.1 Hz, 2H), 2.69 (s, 12H); 13C NMR (100 MHz,
DMSO-d6) d 150.1, 141.2, 138.1, 131.3, 129.4, 129.2, 126.0, 125.4,
124.2, 118.9, 118.4, 113.5, 43.0. calcd for C26H28N2O2 + H+,
401.2229; found (ESI, [M+H]+), 401.2225. Mp 167 °C.
4.2.56. (E,E)-3,5-Bis-(40-hydroxystyryl)benzene (4a)
Compound 4a0 (0.50 g, 1.20 mmol) was deprotected in EtOH/1 N
HCl (60 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 4a
(0.37 g, 100%). 1H NMR (400 MHz, DMSO-d6) d 9.58 (br s, 2H),
7.70 (s, 1H), 7.42 (d, J = 8.5 Hz, 4H), 7.36–7.28 (m, 3H), 7.18 (d,
J = 16.4 Hz, 2H), 7.00 (d, J = 16.4 Hz, 2H), 6.76 (d, J = 8.5 Hz, 4H);
13C NMR (100 MHz, DMSO-d6) d 157.8, 138.3, 129.4, 129.1, 128.5,
128.3, 125.4, 125.2, 124.0, 116.0. HRMS: calcd for C22H19O2 + H+,
315.1385; found (ESI, [M+H]+), 315.1371. Mp 243 °C.
4.2.62. (E,E)-3,5-Bis-styrylbenzene (4g)
Compound 4g was prepared from 10a and benzaldehyde
according to the general procedure B to give 4g (0.33 g, 59%). 1H
NMR (400 MHz, CDCl3) d 7.65 (s, 1H), 7.55–7.53 (m, 4H), 7.44–
7.33 (m, 7H), 7.29–7.25 (m, 2H), 7.19–7.10 (m, 4H); 13C NMR
4.2.57. (E,E)-3,5-Bis-(30-hydroxystyryl)benzene (4b)
Compound 4b0 (0.71 g, 1.70 mmol) was deprotected in EtOH/
1 N HCl (80 mL) according to the general procedure C. The crude
product was purified by recrystallization from EtOH/H2O to give
4b (0.31 g, 58%). 1H NMR (400 MHz, DMSO-d6) d 9.44 (s, 2H),
7.82 (s, 1H), 7.48–7.33 (m, 3H), 7.23 (d, J = 16.4 Hz, 2H), 7.17 (dd,
J = 7.7, 7.7 Hz, 2H), 7.15 (d, J = 16.4 Hz, 2H), 7.01 (d, J = 7.7 Hz,
2H), 6.98 (s, 2H), 6.68 (dd, J = 7.7, 2.1 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 158.1, 138.7, 137.8, 130.1, 129.5, 129.4,
128.4, 126.3, 124.8, 117.9, 115.4, 113.5. HRMS: calcd for
(100 MHz, CDCl3)
d 137.7, 137.2, 129.0 (overlapped), 128.7,
128.5, 127.7, 126.5, 125.7, 124.7.
4.2.63. (E,E)-3,5-Bis-(40-hydroxystyryl)phenol (5a)
Compound 5a0 (0.48 g, 1.10 mmol) was deprotected in EtOH/1 N
HCl (60 mL) according to the general procedure C. The crude prod-
uct was purified by flash column chromatography (EtOAc/n-hex-
ane = 1:1) to give 5a (0.34 g, 94%). 1H NMR (400 MHz, DMSO-d6)
d 9.56 (br s, 2H), 9.38 (br s, 1H), 7.41 (d, J = 8.5 Hz, 4H), 7.16 (s,
1H), 7.07 (d, J = 16.3 Hz, 2H), 6.91 (d, J = 16.3 Hz, 2H), 6.78 (s,
2H), 6.75 (d, J = 8.5 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) d
158.2, 157.7, 139.4, 128.8, 128.5, 128.3, 125.7, 116.0, 115.8,
C
22H18O2 + H+, 315.1385; found (ESI, [M+H]+), 315.1389. Mp
189 °C.
4.2.58. (E,E)-3,5-Bis-(40-hydroxy-30-methoxystyryl)benzene (4c)
Compound 4c0 (3.13 g, 6.70 mmol) was deprotected in EtOH/1 N
HCl (320 mL) according to the general procedure C. The crude
product was purified by recrystallization from EtOH/H2O to give
4c (2.14 g, 85%) without purification. 1H NMR (400 MHz, DMSO-
d6) d 9.16 (s, 2H), 7.73 (s, 1H), 7.39–7.29 (m, 3H), 7.20 (d,
J = 1.8 Hz, 2H), 7.19 (d, J = 16.3 Hz, 2H), 7.06 (d, J = 16.3 Hz, 2H),
6.99 (dd, J = 8.1, 1.8 Hz, 2H), 6.76 (d, J = 8.1 Hz, 2H), 3.82 (s, 6H);
112.1. HRMS: calcd for
C
22H19O3 + H+, 331.1334; found (ESI,
[M+H]+), 331.1335. Mp 204 °C.
4.2.64. (E,E)-3,5-Bis-(30-hydroxystyryl)phenol (5b)
Compound 5b0 (0.38 g, 1.00 mmol) was deprotected in EtOH/
1 N HCl (40 mL) according to the general procedure C. The crude

4932
Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
product was purified by recrystallization from EtOH/H2O to give 5b
(0.25 g, 86%). 1H NMR (400 MHz, DMSO-d6) d 9.48 (s, 1H), 9.42 (s,
2H), 7.28 (s, 1H), 7.15 (dd, J = 7.8, 7.8 Hz, 2H), 7.12 (d, J = 16.2 Hz,
2H), 7.05 (d, J = 16.2 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H), 6.95 (s, 2H),
6.86 (s, 2H), 6.68–6.65 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d
158.3, 158.0, 138.9, 138.7, 130.1, 129.1, 128.7, 118.0, 116.5,
115.3, 113.5, 113.1. HRMS: calcd for C22H19O3 + H+, 331.1334;
found (ESI, [M+H]+), 331.1334. Mp 190 °C.
6.90 (s, 2H), 4.81 (br s, 1H); 13C NMR (100 MHz, CDCl3) d 156.0,
139.3, 137.1, 129.5, 128.7, 128.1, 127.8, 126.6, 118.3, 112.3.
4.2.70. 2-Methoxy-4,6-dimethylpyrimidine (7a)
Acetylacetone (50.0 g, 0.5 mol) and K2CO3 (138.0 g, 1.0 mol)
were dissolved in H2O (250 mL). O-Methylisourea sulfate (61.5 g,
0.25 mol) was added and the reaction mixture was refluxed with
stirring. After cooling, more K2CO3 (70 g) was added and the reac-
tion mixture was left at 25 °C for 48 h. The formed oil was sepa-
rated, combined with the ether, extracted with the aqueous
phase, and dried over MgSO4. The organic layer was concentrated
in vacuo. Vacuum distillation gave 7a (48.3 g, 70%) as a yellow
oil. 1H NMR (400 MHz, CDCl3) d 6.65 (s, 1H), 3.97 (s, 3H), 2.40 (s,
6H).
4.2.65. (E,E)-3,5-Bis-(40-hydroxy-30-methoxystyryl)phenol (5c)
Compound 5c0 (0.61 g, 1.20 mmol) was deprotected in EtOH/1 N
HCl (60 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 5c
(0.42 g, 89%). 1H NMR (400 MHz, DMSO-d6) d 9.39 (s, 1H), 9.13
(br s, 2H), 7.19 (s, 1H), 7.18 (d, J = 1.5 Hz, 2H), 7.08 (d, J = 16.3 Hz,
2H), 6.97 (dd, J = 8.1, 1.5 Hz, 2H), 6.97 (d, J = 16.3 Hz, 2H), 6.78 (s,
2H), 6.75 (d, J = 8.1 Hz, 2H), 3.81 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) d 158.2, 148.2, 147.1, 139.4, 129.1, 129.1, 125.9, 120.6,
116.0, 115.6, 112.2, 110.2, 56.0. HRMS: calcd for C24H22O5 + H+,
391.1545; found (ESI, [M+H]+), 391.1540. Mp 119 °C.
4.2.71. 4,6-Dimethyl-2-methyl-mercapyrimidine (7b)
Compound 7b was prepared from methylisothiourea sulfate
(69.6 g, 0.25 mol) and acetylacetone (50.0 g, 0.5 mol) as described
for 7a (57.8 g, 75%). 1H NMR (400 MHz, CDCl3) d 6.68 (s, 1H),
2.56 (s, 3H), 2.40 (s, 6H).
4.2.66. (E,E)-3,5-Bis-(30-hydroxy-40-methoxystyryl)phenol (5d)
Compound 5d0 (0.50 g, 1.00 mmol) was deprotected in EtOH/
1 N HCl (40 mL) according to the general procedure C. The crude
product was purified by recrystallization from EtOH/H2O to give
5d (0.37 g, 94%). 1H NMR (400 MHz, DMSO-d6) d 9.40 (br s, 1H),
9.00 (br s, 2H), 7.20 (s, 1H), 7.05 (d, J = 16.4 Hz, 2H), 7.02 (d,
J = 1.9 Hz, 2H), 6.97 (dd, J = 8.3, 1.9 Hz, 2H), 6.90 (d, J = 8.3 Hz,
2H), 6.99 (d, J = 16.4 Hz, 2H), 6.79 (d, J = 1.0 Hz, 2H), 3.77 (s, 6H);
13C NMR (100 MHz, DMSO-d6) d 157.6, 147.6, 146.5, 138.6, 129.9,
128.3, 125.9, 118.3, 115.4, 112.8, 112.0, 111.9, 55.5. HRMS: calcd
for C24H22O5 + H+, 391.1545; found (ESI, [M+H]+), 391.1544. Mp
163 °C.
4.2.72. 3,5-Di(iodomethyl)phenol (9a)
To
a solution of 3,5-di(hydroxymethyl)phenol 8 (38.5 g,
0.25 mol) in dry 1,4-dioxane (300 mL) were added BF3ꢁEt2O
(61.7 mL, 0.50 mol) and KI (83.0 g, 0.5 mol) were added and the
resulting mixture was stirred at room temperature. After 25 h,
the reaction mixture was poured into cold water and extracted
with diethyl ether. The combined organic layer was washed with
water and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography
(EtOAc/n-hexane = 1:10) to give 9b (81.3 g, 87%). 1H NMR
(400 MHz, CDCl3) d 6.90 (s, 1H), 6.67 (d, J = 1.0 Hz, 2H), 5.24 (s,
1H), 4.29 (s, 4H).
4.2.67. (E,E)-3,5-Bis-(40-hydroxy-30-N,N-dimethylaminostyryl)
phenol (5e)
4.2.73. 1,3-Bis(diethylphosphonomethyl)benzene (10a)
To a solution of
a,
a0-dibromo-m-xylene 9a (59.4 g, 0.225 mol)
Compound 5e0 (0.43 g, 0.85 mmol) was deprotected in EtOH/1 N
HCl (40 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 5e
(0.29 g, 67%). 1H NMR (400 MHz, DMSO-d6) d 9.36 (br s, 1H), 9.24
(br s, 2H), 7.20 (s, 1H), 7.08 (d, J = 1.7 Hz, 2H), 7.06 (d, J = 16.3 Hz,
2H), 7.01 (dd, J = 8.0, 1.7 Hz, 2H), 6.90 (d, J = 16.3 Hz, 2H), 6.76 (s,
2H), 6.74 (d, J = 8.0 Hz, 2H), 2.69 (s, 12H); 13C NMR (100 MHz,
DMSO-d6) d 158.2, 150.4, 141.4, 139.4, 129.3, 128.9, 125.5, 121.3,
116.7, 116.1, 115.6, 112.1, 43.1. HRMS: calcd for C26H28N2O3 + H+,
417.2178; found (ESI, [M+H]+), 417.2170. Mp 127 °C.
in toluene (500 mL) was added triethylphosphite (77.1 mL,
0.4 mol) and the reaction mixture was refluxed overnight. The ex-
cess toluene was removed by evaporation to give 10a (72.3 g, 85%)
as a yellow oil. 1H NMR (400 MHz, CDCl3) d 7.27–7.17 (m, 4H),
4.04–3.96 (m, 8H), 3.12 (d, J = 21.9 Hz, 4H), 1.23 (t, J = 7.0 Hz, 12H).
4.2.74. 3,5-Bis(diethylphosphonomethyl)phenol (10b)
Compound 10b was prepared from 9b (81.3 g, 0.217 mol) and
triethylphosphite (74.4 mL, 0.434 mol) as described for 10a
(76.1 g, 89%). 1H NMR (400 MHz, CDCl3) d 6.82 (d, J = 1.5 Hz, 2H),
6.63 (s, 1H), 4.04–3.96 (m, 8H), 3.07 (d, J = 21.6 Hz, 4H), 1.24 (t,
J = 7.0 Hz, 12H).
4.2.68. (E,E)-3,5-Bis-(30-hydroxy-40-N,N-dimethylaminostyryl)
phenol (5f)
Compound 5f’ (0.69 g, 1.30 mmol) was deprotected in EtOH/1 N
HCl (80 mL) according to the general procedure C. The crude prod-
uct was purified by recrystallization from EtOH/H2O to give 5f
(0.35 g, 65%). 1H NMR (400 MHz, DMSO-d6) d 9.43 (br s, 1H), 9.34
(s, 2H), 7.23 (s, 1H), 7.07 (d, J = 16.3 Hz, 2H), 7.02–7.00 (m, 6H),
6.90 (d, J = 16.3 Hz, 2H), 6.82 (s, 2H), 2.77 (s, 12H); 13C NMR
(100 MHz, DMSO-d6) d 158.3, 150.2, 139.1, 138.9, 132.9, 128.7,
127.1, 119.1, 118.8, 116.1, 113.8, 112.6, 43.4. HRMS: calcd for
4.2.75. General procedure E
4.2.75.1. Protection of hydroxyl group with p-methoxybenzyl
group.
A mixture of hydroxybenzaldehyde (1.0 equiv) and p-
methoxybenzyl chloride(1.1 equiv) in DMF containing K2CO3
(1.2 equiv) was heated at 60 °C overnight. The reaction mixture
was poured into cold H2O and the precipitate was collected by fil-
teration to give the product.
C
26H28N2O3 + H+, 417.2178; found (ESI, [M+H]+), 417.2173. Mp
4.2.76. General procedure F
148 °C.
4.2.76.1. Protection of hydroxyl group with methoxymethyl
group.
To a stirred solution of phenol (1.0 equiv) and triethyl-
4.2.69. (E,E)-3,5-Bis-styrylphenol (5g)
amine (1.2 equiv) in dry THF was added chloromethyl methyl ether
(1.1 equiv) dropwise at 0 °C under N2. When the reaction was com-
pleted, the reaction mixture was extracted with H2O and EtOAc.
The organic layer was washed with aqueous 1 N HCl, 1 N NaOH,
and brine, dried over MgSO4, and concentrated in vacuo to give
the product.
Compound 5g was prepared from 10b and benzaldehyde
according to the general procedure B. The crude product was puri-
fied by flash column chromatography (EtOAc/n-hexane = 1:10) to
give 5g (0.41 g, 69%). 1H NMR (400 MHz, CDCl3) d 7.52–7.50 (m,
4H), 7.38–7.34 (m, 4H), 7.29–7.22 (m, 3H), 7.14–7.03 (m, 4H),

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4933
4.2.77. 4-(4-Methoxybenzyloxy)benzaldehyde (12a)
4.2.88. 3-(Methoxymethyl)-4-nitrobenzoic acid methyl ester
(16b)
Yield: 95%. 1H NMR (400 MHz, CDCl3) d 9.89 (s, 1H), 7.84 (d,
J = 8.7 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H),
6.93 (d, J = 8.7 Hz, 2H), 5.08 (s, 2H), 3.83 (s, 3H).
Compound 16b, obtained as a yellow solid, was prepared from
15a (27.0 g, 0.13 mol) as described in general procedure F (92%
yield). 1H NMR (400 MHz, CDCl3) d 7.96 (d, J = 1.5 Hz, 1H), 7.80
(d, J = 8.3 Hz, 1H), 7.75 (dd, J = 8.3, 1.5 Hz, 1H), 5.34 (s, 2H), 3.95
(s, 3H), 3.53 (s, 3H).
4.2.78. 3-(4-Methoxybenzyloxy)benzaldehyde (12b)
Yield: 89%. 1H NMR (400 MHz, CDCl3) d 9.98 (s, 1H), 7.48–7.43
(m, 3H), 7.38 (d, J = 8.5 Hz, 2H), 7.25–6.95 (m, 1H), 6.94 (d,
J = 8.5 Hz, 2H), 5.06 (s, 2H), 3.83 (s, 3H).
4.2.89. N,N-3-(Dimethylamino)-4-(methoxymethyl)benzoic acid
methyl ester (17a)
To a solution of 16a (5.55 g, 23.0 mmol) in MeOH (150 mL) was
added 10% Pd/C (1.0 g) and the mixture was stirred at room tem-
perature under H2. When reduction of nitro group was complete,
formaldehyde (37% solution in H2O, 30.0 mL, 0.37 mol) was added.
The mixture was stirred under H2 overnight. The reaction mixture
was filtered throughout Celite 545 and the filtrate was concen-
trated in vacuo to give the product (5.28 g, 96%). 1H NMR
(400 MHz, CDCl3) d 7.67–7.63 (m, 2H), 7.13 (d, J = 2.2 Hz, 1H),
5.30 (s, 2H), 3.88 (s, 3H), 3.52 (s, 3H), 2.83 (s, 6H).
4.2.79. 3-Methoxy-4-(4-methoxybenzyloxy)benzaldehyde (12c)
Yield: 92%. 1H NMR (400 MHz, CDCl3) d 9.78 (s, 1H), 7.36 (d,
J = 1.7 Hz, 1H), 7.34 (dd, J = 8.1, 1.7 Hz, 1H), 7.31 (d, J = 8.6 Hz,
2H), 6.95 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 5.11 (s, 2H),
3.88 (s, 3H), 3.75 (s, 3H).
4.2.80. 4-Methoxy-3-(4-methoxybenzyloxy)benzaldehyde (12d)
Yield: 91%. 1H NMR (CDCl3, 400 MHz) d 9.83 (s, 1H), 7.47 (d,
J = 1.8 Hz, 1H), 7.46 (dd, J = 8.7, 1.8 Hz, 1H), 7.38 (d, J = 8.7 Hz,
2H), 6.98 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.7 Hz, 2H), 5.12 (s, 2H),
3.95 (s, 3H), 3.81 (s, 3H).
4.2.90. N,N-4-(Dimethylamino)-3-(methoxymethyl)benzoic acid
methyl ester (17b)
Compound 17b was prepared from 16b (5.33 g, 22.1 mmol) as
described for 17a (4.54 g, 86%). 1H NMR (400 MHz, CDCl3) d 7.71
(d, J = 1.9 Hz, 1H), 7.66 (dd, J = 8.3, 1.9 Hz, 1H), 6.90 (d, J = 7.2 Hz,
1H), 5.25 (s, 2H), 3.86 (s, 3H), 3.52 (s, 3H), 2.90 (s, 6H).
4.2.81. 4-(Methoxymethoxy)benzaldehyde (13a)
Yield: 65%. 1H NMR (400 MHz, CDCl3) d 9.90 (s, 1H), 7.83 (d,
J = 8.7 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 5.25 (s, 2H), 3.49 (s, 3H).
4.2.91. N,N-3-(Dimethylamino)-4-(methoxymethyl)benzyl
alcohol (18a)
4.2.82. 3-(Methoxymethoxy)benzaldehyde (13b)
To a suspension of LiAlH4 (1.1 g, 27.6 mmol) in dry THF (50 mL)
was added dropwise 17a (5.0 g, 20.9 mmol) in dry THF (50 mL) at
0 °C. The reaction mixture was stirred for 1 h, and quenched with
H2O (2 mL). Then 15% NaOH solution (2 mL) and H2O (6 mL) were
added and the reaction mixture was stirred at ambient tempera-
ture for 1 h. The precipitate was removed by filter and the filtrate
was evaporated to give 18a (4.23 g, 96%) as a oil. 1H NMR
(400 MHz, CDCl3) d 7.11 (d, J = 8.1 Hz, 1H), 6.98 (s, 1H), 6.92 (dd,
J = 8.1, 1.6 Hz, 1H), 5.25 (s, 2H), 4.61 (s, 2H), 3.52 (s, 3H), 2.81 (s,
6H).
Yield: 54%. 1H NMR (400 MHz, CDCl3) d 9.98 (s, 1H), 7.55–7.43
(m, 3H), 7.30 (ddd, J = 8.0, 2.5, 1.2 Hz, 1H), 5.23 (s, 2H), 3.49 (s, 3H).
4.2.83. 3-Methoxy-4-(methoxymethoxy)benzaldehyde (13c)
Yield: 86%. 1H NMR (400 MHz DMSO-d6) d 9.84 (d, J = 1.0 Hz,
1H), 7.50 (dd, J = 8.2, 1.7 Hz, 1H), 7.43 (d, J = 1.4 Hz, 1H), 7.24 (d,
J = 8.2 Hz, 1H), 5.28 (s, 2H), 3.83 (s, 3H), 3.38 (s, 3H).
4.2.84. 4-Methoxy-3-(methoxymethoxy)benzaldehyde (13d)
Yield: 75%. 1H NMR (400 MHz, DMSO-d6) d 9.81 (s, 1H), 7.60
(dd, J = 8.3, 1.8 Hz, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.20 (d, J = 8.3 Hz,
1H), 5.22 (s, 2H), 3.87 (s, 3H), 3.38 (s, 3H).
4.2.92. N,N-4-(Dimethylamino)-3-(methoxymethyl)benzyl
alcohol (18b)
Compound 18b was prepared from 17b (5.0 g, 20.9 mmol) as
described for 18a (4.28 g, 97%). 1H NMR (400 MHz, CDCl3) d
7.18–6.96 (m, 3H), 5.27 (s, 3H), 4.61 (s, 2H), 3.53 (s, 3H), 2.83 (s,
6H).
4.2.85. 4-Hydroxy-3-nitrobenzoic acid methyl ester (15a)
To
a solution of 4-hydroxy-3-nitrobenzoic acid (25.0 g,
0.13 mol) in MeOH (150 mL) was added concd H2SO4 (5 mL) and
the mixture was refluxed overnight. The reaction mixture was al-
lowed to cool to room temperature and concentrated in vacuo.
The residue was poured into cold H2O and the precipitate was col-
lected by filteration to give 15a (23.2 g, 88%) as a yellow solid. 1H
NMR (400 MHz, CDCl3) d 10.89 (s, 1H), 8.82 (d, J = 2.1 Hz, 1H),
8.24 (dd, J = 8.7, 2.1 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 3.94 (s, 3H).
4.2.93. N,N-3-(Dimethylamino)-4-
(methoxymethyl)benzaldehyde (13e)
To a solution of 18a (18.0 g, 0.085 mol) in CH2Cl2 (500 mL)
was added MnO2 (66 g, 0.759 mol) and the mixture was refluxed
overnight. MnO2 was removed by filtration throughout Celite
545 and the filtrate was concentrated in vacuo. The residue
was purified by flash column chromatography (EtOAc/n-hex-
ane = 1:5) to give 13e (9.96 g, 56%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) d 9.86 (s, 1H), 7.47 (dd, J = 8.2, 1.9 Hz, 1H),
7.44 (d, J = 1.9 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 5.33 (s, 2H),
3.53 (s, 3H), 2.84 (s, 6H).
4.2.86. 3-Hydroxy-4-nitrobenzoic acid methyl ester (15b)
Compound 15b was prepared from 3-hydroxy-4-nitrobenzoic
acid (25 g, 0.13 mol) as described for 15a (25.0 g, 97%). 1H NMR
(400 MHz, CDCl3) d 10.48 (br s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.81
(d, J = 1.8 Hz, 1H), 7.60 (dd, J = 8.8, 1.8 Hz, 1H), 3.95 (s, 3H).
4.2.87. 4-(Methoxymethyl)-3-nitrobenzoic acid methyl ester
(16a)
4.2.94. N,N-4-(Dimethylamino)-3-
(methoxymethyl)benzaldehyde (13f)
Compound 16a, obtained as a yellow solid, was prepared from
15a (23.2 g, 0.11 mol) as described in general procedure F (96%
yield). 1H NMR (400 MHz, CDCl3) d 8.47 (d, J = 2.1 Hz, 1H), 8.17
(dd, J = 8.8, 2.1 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 5.36 (s, 2H), 3.94
(s, 3H), 3.54 (s, 3H).
Compound 13f was prepared from 18b (20.2 g, 0.095 mol) as
described for 13e (18.6 g, 94%). 1H NMR (400 MHz, CDCl3) d 9.80
(s, 1H), 7.57 (d, J = 1.6 Hz, 1H), 7.47 (dd, J = 8.2, 1.7 Hz, 1H), 6.93
(d, J = 8.0 Hz, 1H), 5.26 (s, 2H), 3.53 (s, 3H), 2.96 (s, 6H).

4934
Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4.3. Preparation of Ab protein for fibrillization studies
(1 mM, Sigma–Aldrich) in sodium phosphate buffer (10 mM, pH
7.4) were added into assay solution. The mixtures were irradiated
3 times (1 s each) using a 200 W incandescent lamp and quenched
with a reducing PAGE sample buffer. The mixtures were boiled for
5 min, then loaded onto a 1.0 mm-thick 10–20% tris–tricine gradi-
ent gel (Criterion™ Precast Gel, Biorad), followed by the electro-
phoresis at 130 V in tris/tricine/SDS running buffer. Separated Ab
bands were stained via silver staining kit (PlusOne silver staining
kit, Amersham Biosciences) to visualize.
Purified Ab(1–42) was purchased from Bachem (United states).
Ab(1–42) stock solutions (250 lM) were prepared by completely
dissolving the peptide in DMSO. Ab(25–35) purchased from US
peptide was prepared as 10 mM stock solution in DMSO and di-
luted 10-fold with phosphate-buffered saline (PBS) to induce
aggregation for 1 h at room temperature.
4.4. ThT fluorescence assay for Ab fibril formation
4.8. Experimental design for in vivo tests
Aggregation of Ab(1–42) peptide was measured by the ThT as-
say, in which the fluorescence intensity reflects the degree of Ab fi-
bril formation. To each well of a 96-well microplate (black) were
added 250 lM Ab(1–42) (5 lL) and PBS buffer (45 lL). The mixed
solution was added to compounds and incubated for 1 h at room
Male ICR mice weighing 25 g purchased from OrientBio Co.
(Korea) were bred as five mice per cage and the cage was kept with
following condition maintaining the temperature of 22 2 °C and
the relative humidity of 50 5% under the regularly controlled
light/dark condition with an interval of 12 h.
temperature. ThT was diluted with 50 mM glycine buffer solution
to be
(150
5
l
M. To each well was added diluted ThT solution
Ab(1–42) (Bachem) was dissolved in DMSO in order to be
250 lM solution, diluted with PBS to 10 nM, and aggregated at
l
L). After shaking together for 10 s, the ThT fluorescence
was measured by using TECAN spectrofluorometer (Safire, TECAN)
with excitation at 450 nm and emission at 480 nm.
37 °C for 4 days (Passive avoidance task) or 6 days (Y-maze task).
Aggregated Ab(1–42) was administrated into the mice according
to the procedure disclosed in the literature.27 50
lL of pre-aggre-
4.5. Cell culture
gated Ab(1–42) was injected into the 2.4 mm depth of bregma
region with Hamilton micro-syringe equipped with 26-gauge
needle.
HT22, a murine cell line of hippocampal origin, was cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum and 1% penicillin/streptomycin (Sigma) at 37 °C and
5% CO2. At the outset, 90% confluent cells were dissociated and pla-
ted at a density of 5 ꢃ 103 cells/well in a 96-well plate. When cells
were attached to the plate, the medium was replaced with plain
DMEM.
After the injection of Ab(1–42), Y-maze, object recognition, pas-
sive avoidance, and watermaze task were performed. Y-maze task
and object recognition task were performed 2 days after the
administration of Ab and passive avoidance and watermaze task
were performed 3 days after the administration of Ab. The com-
pounds were administered orally at 100 mg/kg into the mice once
a day. In case of watermaze task, the feed was administered by pel-
let feed for 7 days and one pellet was designed to contain 3 mg of
the compound.
4.6. Cytotoxicity assay and Ab burden assay
The innate cytotoxicity of compounds was measured by using
MTT assay. Before compound treatment, HT22 cells were incu-
bated in DMEM without serum for 1 h. Following treatment, cells
were incubated for 18 h. 5 mg/mL of MTT solution was added to
the culture medium and the culture further incubated for 4 h at
37 °C. Cells were then solubilized in 50% dimethylformamide and
10% sodium dodecyl sulfide (pH 4.7) for 18 h. The degree of cell
survival was determined by checking conversion of MTT to forma-
zan. The absorbance was measured at 570/630 nm using a plate
reader (Sunrise, TECAN) and the percentage cytotoxicity was calcu-
lated. For Ab burden assay, 1 h after compounds treatment, aggre-
4.9. Y-maze task
The Y-maze apparatus was made of black acrylic and was com-
posed three arms (L: 40 cm, W: 5 cm, H: 10 cm) having identical
angle each other. Mice were positioned at the center of the maze
and let to move freely within the maze for 8 min. Thereafter, the
entering order of the mice into the pathway was observed and
the entering was determined when 4 limbs of the mice were en-
tered within the pathway. To determine the spatial memory, the
determined spontaneous alternation behavior was calculated by
following empirical formula and the actual alternation was as-
signed to one time at the time that mice was entered three path-
ways continuously.
gated Ab(25–35) was added to the concentration of 25 lM and
cells were incubated for 18 h to induce cell necrosis. Cell viability
was checked in the same manner as described above for MTT assay.
4.7. SDS–PAGE with PICUP technique
4.10. Object recognition task
Ab(1–42) peptide was treated in pre-chilled 1,1,1,3,3,3,-hexa-
fluoro-2-propanol (HFIP) to a final concentration of 0.5 mM for
monomerization of Ab(1–42) peptides. After the sonication for
5 min at room temperature, it was vortexed gently and then incu-
bated for 30 min at room temperature. To remove HFIP, the tubes
were left opened in a clean hood overnight. The monomerized
Ab(1–42) peptides were dissolved in anhydrous DMSO to prepare
Ab stock solution (0.5 mM). The Ab aggregation reaction was initi-
ated by adding Ab stock solution into phosphate buffered saline
The object recognition task was performed by using an open-
field box consisting of black acrylic (L: 50 cm, W: 50 cm, H:
30 cm). Two objects were placed in a symmetrical position about
5 cm away from the wall. During the acquisition trial (first trial),
the mouse was exposed to two identical objects for 3 min. The
mouse was then returned to its home cage. After a delay of 24 h,
the mouse was exposed to one of the familiar objects and a novel
object for 3 min. All combinations and locations of objects were
used in a balanced manner to reduce potential biases due to pref-
erences for particular locations or objects. Care was taken to avoid
olfactory stimuli by cleaning the objects carefully. Exploration
behavior was calculated by following formula and exploration
was defined as follows: directing the nose to the object at a dis-
tance of no more than 2 cm and/or touching the object with the
nose.
(PBS, pH 7.3) in the absence or presence of compounds (25
lM fi-
nal Ab(1–42) concentration and 2 M final compound concentra-
l
tion). The assay solutions were incubated for 2 h at 37 °C,
followed by photo-induced cross-linking of unmodified proteins
(PICUP) technique. 1.2
Sigma–Aldrich) and
l
M
of ammonium persulfate (20 mM,
tris(2,2’-bipyridyl)dichlororuthenium(II)

Y. S. Lee et al. / Bioorg. Med. Chem. 20 (2012) 4921–4935
4935
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Acknowledgments
23. Frautschy, S. A.; Hu, W.; Kim, P.; Miller, S. A.; Chu, T.; Harris-White, M. E.; Cole,
G. M. Neurobiol. Aging 2001, 22(6), 993.
We thank the Digital biotech scientists, Dr. Y.H. Kim and Ms.
Hee Kim, for their support of biological assay data. This work
was financially supported by Research Fund 2011 of The Catholic
University of Korea.
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