Synthesis and evaluation of neuroprotective 4-O-substituted chrysotoxine derivatives as potential multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1039/c5ra21313d

A series of 4-O-substituted chrysotoxine (CTX) derivatives were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease (AD). In vitro assays indicated that four ring substituted compounds (2a, 2b, 3i and 3j)

Full text of DOI:10.1039/c5ra21313d

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Synthesis and evaluation of neuroprotective 4ꢀOꢀsubstituted
chrysotoxine derivatives as potential multifunctional agents for the
treatment of Alzheimer’s disease
Li Guan,a Yanfeng Hao,a Lei Chen,a MengꢀLin Wei,b Qin Jiang,a WenꢀYuan Liu,b YanꢀBo Zhang,c
Jie Zhang,a Feng Feng*ad and Wei Qu. *ad
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
China
b
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical
University), Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, China
c School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10
Sassoon Road, Pokfulam, Hong Kong, China
d Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing
211198, China
* Corresponding authors
Eꢀmail addresses: fengfeng@cpu.edu.cn (F. Feng), weiqcpu@126.com (W. Qu).
Abstract: A series of 4ꢀOꢀsubstituted chrysotoxine (CTX) derivatives were designed, synthesized
and evaluated as multifunctional agents for the treatment of Alzheimer’s disease (AD). In vitro
assays indicated that four ring substituted compounds (2a, 2b, 3i and 3j) exhibited significant
neuroprotective effects against Aβ25ꢀ35ꢀinduced toxicity in PC12 cells. The four compounds also
inhibited selfꢀ and Cu2+ꢀinduced Aβ1ꢀ42 aggregation and acted as biometal chelators. In particular,
compound 2a was a potential lead compound for AD treatment (cell viability up to 100.78% at 50
ꢁM in Aβ25ꢀ35ꢀtreated PC12 cells, 51.88% and 58.03% inhibition at 25 ꢁM for selfꢀ and
Cu2+ꢀinduced Aβ1ꢀ42 aggregation, respectively). Metal chelating experiment manifested that
compound 2a had a moderate interaction with Cu2+ and Al3+. Moreover, western blot analysis
showed that compound 2a attenuated Aβꢀinduced tau protein hyperphosphorylation at Ser199/202

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and Ser396 sites. Furthermore, compound 2a could efficiently cross the bloodꢀbrain barrier (BBB)
by parallel artificial membrane permeability assay (PAMPA). In summary, these results suggested
that compound 2a was a promising multifunctional compound for AD therapy.
1. Introduction
Alzheimer’s disease (AD), characterized by a progressive loss of memory and cognitive
impairments, is one of the most common neurodegenerative diseases mainly occurring in the
elderly.1 The World Alzheimer Report 2015 showed that 46.8 million people have been suffering
from AD and this number is expected to exceed 131.5 million in 2050.2 Current therapeutic drugs
for AD include acetylcholinesterase inhibitors (tacrine, donepezil, rivastigmine and galantamine)3
and NꢀmethylꢀDꢀaspartate (NMDA) receptor antagonists (memantine).4 Although these
singleꢀtarget drugs have a modest and transient improvement in memory and cognitive function,
they cannot prevent progressive neurodegeneration.3 The etiology of AD still remains elusive, but
several factors, such as βꢀamyloid (Aβ) deposits,5 τꢀprotein hyperphosphorylation,6 metal
dyshomeostasis,7 oxidative stress8 and low levels of acetylcholine,9 have been greatly implicated
in the AD pathogenesis.
Among the multiple factors that induce AD, Aβ plays a primary role.10 The Aβ hypothesis
states that the production and accumulation of Aβ peptides in the brain are central events in the
AD pathogenesis.11ꢀ12 Aβ is a peptide chain cleaved from amyloid precursor protein (APP) ranging
in length from 39 to 43 amino acids.13 Aβ1ꢀ40 and Aβ1ꢀ42 are the main isoforms of Aβ peptides.14
A
sustained imbalance between the production and clearance of Aβ40ꢀ42 fragments leads to the
accumulation of Aβ peptide monomers, oligomers, and finally promotes the large aggregated Aβ
plaques, which are thought to initiate the pathogenic cascade, ultimately leading to neuronal loss
and dementia.15ꢀ17 Another hallmark of AD pathology is the intracellular accumulation of
hyperphosphorylated tau protein, which leads to neurofibrillary tangles in the brain.18 Therefore,
an inhibition of Aβ aggregation or tau hyperphosphorylation has been recognized as an effective
therapeutic strategy for AD.
In addition, recent studies have indicated that excessive biometals (such as Cu2+, Fe2+, Zn2+
and Al3+) exist in the brains of AD patients.19 The abnormal accumulation of biometal ions is
closely associated with the formation of Aβ plaques and neurofibrillary tangles, which have been
found to induce neurotoxicity.20ꢀ21 The modulation of brain biometals represents an additional

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rational approach for the treatment of AD.
A major impediment to the development of effective compounds for AD therapy is that
essentially 100% of largeꢀmolecule drugs and >98% of smallꢀmolecule drugs fail to cross the
bloodꢀbrainꢀbarrier (BBB).22 Increasing the lipid solubility of target compounds is regarded as a
promising way to improve their BBB permeability.23
Chrysotoxine (CTX), a natural bibenzyl (4ꢀhydroxyꢀ3,5,3’,4’ꢀtetramethoxybibenzyl), has
been isolated from plants in genus Dendrobium, the source of a valuable and scarce traditional
medicine named “Shihu” in China.24ꢀ26 Recent studies have revealed that CTX can inhibit
SHꢀSY5Y cells apoptosis induced by neurotoxins 6ꢀhydroxydopamine and 1ꢀmethylꢀ4ꢀphenyl
pyridinium (MPP+) in Parkinson’s disease model, which suggested its potential protective effect
against neurodegeneration.27ꢀ28 Similar to resveratrol, CTX contains two aromatic end groups and
a linker region in the middle, which are critical for the efficient bond at the Aβ peptide guided
πꢀstacking.29 However, the scarce source of CTX restrict its further development.
In this paper, we synthesized CTX and its derivatives modified by several series of
hydrophobic moieties as potential neuroprotective agents for AD. Then we chose four compounds
with significant neuroprotective effects to explore their potential multifunctional properties for AD
therapy by the pharmacological evaluations, including selfꢀ and Cu2+ꢀinduced Aβ aggregation, tau
phosphorylation and metal chelation. Meanwhile, theoretical prediction and experimental assay
were performed to explore their BBB permeability.
2. Result and Discussion
2.1. Chemistry
The natural compound CTX was totally synthesized with Wittig reaction as a key step, which was
widely
used
for
the
synthesis
of
bibenzyl
skeleton.30
Syringaldehyde
(4ꢀhydroxyꢀ3,5ꢀdimethoxylbenzaldehyde, 2) was used as the starting material. Based on the
acetylation protection of hydroxyl group, 4ꢀhydroxymethylꢀ2,6ꢀdimethoxyphenyl acetate (4) was
obtained by aldehyde reduction with sodium borohydride. With further hydroxyl chlorination of
compound 4, we got the intermediate 4ꢀchloromethylꢀ2,6ꢀdimethoxyphenyl acetate (5), which was
reacted with triphenylphosphine to prepare the phosphonium salt (6), the key material for the
Wittig reaction. The phosphonium salt was dissolved completely with 3,4ꢀdimethoxybenzaldehyde

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in THF and deprotonated with NaH to yield a mixture of Zꢀ and Eꢀstilbenes, which were reduced
by treatment with catalytic amounts of 10% PdꢀC over H2 (4 atm) to afford bibenzyl skeleton.
After deacetylation with sodium methylate, CTX was finally obtained through a total 7ꢀstep
procedure, and the whole yield of our 7ꢀstep reaction was up to 54.1%, which was summarized in
Scheme 1.
Scheme 1 Synthesis steps of lead compound chrysotoxine (CTX). a) Acetic anhydride, DCM,
DMAP, rt, 30 min; b) NaBH4, EtOH, 0 °C, rt, 30 min; c) SOCl2, Et3N, DCM, 0 °C, rt, 3 h; d) PPh3,
toluene, reflux, 5 h; e) 3,4ꢀdimethoxybenzaldehyde, NaH, THF, Ar, 0 °C, rt, overnight; f) H2, 10%
Pd/C, EtOAc, 4 atm; g) MeONa / MeOH, rt, 30 min.
The chrysotoxineꢀ4ꢀOꢀsubstituted esterified derivatives 1a~1f were catalyzed by EDC·HCl in
just one step, while compounds 2a~2e were obtained through the esterification with the acid
chloride intermediate. These procedures were shown in Scheme 2. The 4ꢀOꢀsubstituted
chrysotoxine ether derivatives 3a~3j were synthesized by the combination of halon compounds
and CTX in DMF, which was described in Scheme 3.
All compounds were purified by recrystallization or chromatography technology. The
analytical and spectroscopic data confirmed their structures, which were shown as details in the
experimental section.

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Scheme 2 Synthetic procedure of 4ꢀOꢀester substituted CTX derivatives.
Scheme 3 Synthetic procedure of 4ꢀOꢀether substituted CTX derivatives.
2.2. Pharmacology
2.2.1. Neuroprotective activity against Aβ25ꢀ35ꢀinduced toxicity in PC12 cells.
To investigate the neuroprotective activity of all designed compounds, we used MTT assay to
explore their effects on Aβ25ꢀ35ꢀinduced toxicity in PC12 cells.31ꢀ33 Cells were pretreated with
varying concentrations (50 ꢁM, 25 ꢁM and 10 ꢁM) of test compounds for 0.5 h, and then Aβ25ꢀ35
(10 ꢁM) was added for an additional 24 h. For model group, only Aβ25ꢀ35 was added. Data were
expressed as percent of control group. As shown in Table 1, Aβ25–35 significantly decreased the
cell viability compared with control group (53.82% of control, P < 0.001). Four ring substituted
compounds (2a, 2b, 3i and 3j) showed significant neuroprotective activity with cell viability

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higher than 70% compared to model group. Among the four compounds, 2a (R=benzoyl)
displayed the most potent neuroprotective activity (cell viability up to 100.78% at 50 ꢁM) and 2b
(R= pꢀchlorobenzoyl) exhibited slightly weaker activity than 2a. Replacement of benzoyl group
(2a, 2b) by pyridinecarbonyl group (2d, 2e) killed the neuroprotective activity, which revealed
that pyridine ring had negligible effect on Aβꢀinduced toxicity. When benzoyl substitution (2a, 2b)
was replaced by alkyl chain substitution (1aꢀ1f and 3aꢀ3h), the neuroprotective activity
dramatically decreased. However, the activity reversed when alkyl chain substitution was replaced
by benzyl (3i) or cyclohexyl (3j), which confirmed that ring substitution was beneficial to the
neuroprotective activity. The most possible reason was that there was an interaction between
tricyclic system of CTX derivatives and Aβ peptides. Therefore, the four ring substituted
compounds (2a, 2b, 3i and 3j) with significant neuroprotection were selected to assess their
potential multifunctional properties for AD treatment.
Table 1 Neuroprotective activity of 23 bibenzyl derivatives against Aβ25ꢀ35ꢀinduced toxicity in
PC12 cells.a
Cell viability (%) b
25ꢁM
Cell viability (%) b
25ꢁM
Com.
Com.
50 ꢁM
10 ꢁM
50 ꢁM
10 ꢁM
CTX
CTXꢀ0
1a
58.89±2.47
48.87±3.51
44.40±6.21
49.37±4.02
45.91±1.91
48.63±3.14
49.57±4.15
60.02±1.89(*)
55.02±2.91
50.01±4.01
44.21±4.30
50.12±2.21
47.33±3.54
41.90±2.91
50.40±2.04
55.21±2.71
52.73±4.18
48.47±3.93
45.31±5.11
54.89±3.56
49.34±2.81
49.73±4.18
49.83±1.01
50.11±1.85
58.18±2.09
56.41±3.04
54.00±1.85
52.97±3.76
2e
3a
3b
3c
3d
3e
3f
46.56±1.42
46.15±3.14
48.81±1.55
53.44±1.80
49.89±2.47
48.75±2.02
49.48±3.72
52.18±1.99
58.88±3.51
50.71±2.85
50.92±3.42
49.11±1.43
52.03±3.44
50.12±1.96
49.73±4.72
50.16±4.70
50.25±3.08
54.78±2.51
53.53±3.51
53.84±2.57
49.21±2.62
51.79±3.09
54.53±1.88
49.02±3.61
50.21±1.51
50.67±1.40
51.03±2.77
54.77±2.79
58.22±2.84
100.05±3.12
1b
1c
1d
1e
1f
3g
3h
3i
2a
100.78±2.51(***) 80.12±2.56(***)
2b
95.62±1.22(***)
49.84±1.09
54.60±3.02
a
69.24±1.52(**)
50.22±2.47
72.64±5.12(**) 61.02±1.05(*)
71.00±2.45(***) 60.89±1.67(*)
2c
3j
2d
50.10±6.07
Model 53.82±2.72(###)
Control
10 ꢁM Aβ25ꢀ35 was added to all the test groups and model group to develop the nerve damage
model for 24 h. Compounds were added 0.5 h prior to Aβ25–35 addition. Cell viability was assessed
by measuring the MTT reduction.
b Data were the mean ± SD expressed as percent of the untreated Aβ25–35 control group. Results are
the mean of 3 independent experiments in triplicate. ###P<0.001 vs. control; ***P<0.001,
**P<0.01, *P<0.05 vs. model (ANOVA followed by NewmanꢀKeuls Test).

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2.2.2. Inhibition of selfꢀinduced Aβ1ꢀ42 aggregation by compounds 2a, 2b, 3i and 3j
34
Thioflavin T (ThT) fluorescence assay
was used to assess the ability of the four selected
compounds (2a, 2b, 3i and 3j) to inhibit the selfꢀinduced Aβ1ꢀ42 aggregation. Curcumin (Cur) and
resveratrol (Res) were used as reference compounds (Fig 1). From the results, four compounds
showed moderateꢀtoꢀgood activities (inhibition ranged from 34.52% to 57.20% at 50 ꢁM) and
exhibited concentrationꢀdependent effects. Benzoyl substituted CTX derivatives 2a and 2b
displayed better activities than 3i and 3j, which was consistent with their neuroprotective activities.
Notably, the inhibition of compound 2a was 51.88% at 25 ꢁM (57.20% at 50 ꢁM), which was
competitive with the two reference compounds (Cur 42.70% and Res 50.20% at 25 ꢁM).
Therefore, compound 2a might serve as a potent Aβ1ꢀ42 selfꢀaggregation inhibitor for AD
treatment.
Fig 1 Inhibition of Aβ1ꢀ42 selfꢀinduced aggregation by compounds 2a, 2b, 3i and 3j. Curcumin
(Cur) and resveratrol (Res) were reference compounds. The thioflavin T fluorescence method was
used and the measurements were carried out in the presence of test compounds in different
concentrations. The mean ± SD values from three independent experiments were shown.
2.2.3. Inhibition of Cu2+ꢀinduced Aβ1ꢀ42 aggregation by compounds 2a, 2b, 3i and 3j
To investigate the inhibition of these selected compounds in Cu2+ꢀinduced Aβ1ꢀ42 aggregation, we
conducted a ThTꢀbinding assay.35 Resveratrol and curcumin were chosen as reference compounds
(Fig 2). The fluorescence of Aβ1ꢀ42 treated with Cu2+ is 148.78% compared to that of Aβ1ꢀ42 alone,
which indicated that Cu2+ could accelerate Aβ1ꢀ42 aggregation. By contrast, the fluorescence of

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Aβ1ꢀ42 treated with Cu2+ and test compounds decreased dramatically (2a, 58.03% inhibition of
Cu2+ꢀinduced Aβ1ꢀ42 aggregation; 2b, 54.13% inhibition; 3i, 45.81% inhibition; 3j, 43.17 %
inhibition; Cur, 61.35% inhibition; Res, 66.13% inhibition).These results suggested that these four
compounds could inhibit Cu2+ꢀinduced Aβ aggregation by chelating Cu2+.
Fig 2 Inhibition of Cu2+ꢀinduced Aβ1ꢀ42 aggregation by compounds 2a, 2b, 3i and 3j. The final
concentration of Aβ1ꢀ42, Cu2+ and test compounds were 25 ꢁM. Values are reported as the mean ±
SD of three independent experiments.*p < 0.05, **p < 0.01 vs. Aβ alone group.
2.2.4. Metal chelating effect of compound 2a
Cu2+ꢀinduced Aβ1ꢀ42 aggregation assays showed that four compounds 2a, 2b, 3i and 3j inhibited
Aβ aggregation by chelating Cu2+. Since compound 2a exhibited the most potency, it was chosen
to study its metal chelating effect. The chelating effect of compounds 2a for metals such as Cu2+
and Al3+ in methanol was studied by UVꢀvis spectrometry with wavelength ranging from 200 to
500 nm.36 In Fig 3a, UVꢀvis spectra of compound 2a with increasing Cu2+ concentrations from 2
to 50 ꢁM was shown as an example. The increase in absorbance, which could be clearly estimated
by an inspection of the differential spectra (Fig 3b), indicated that there was an interaction
between Cu2+ and compound 2a. Similar behavior was also observed when using Al3+. These
observations revealed that compound 2a could effectively chelate Cu2+ and Al3+ and could serve
as a metal chelator in treating AD. The ratio of ligand/metal ion in the complex was investigated

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by mixing the fixed amount of metal ion with increasing ligand. The maximum intensity of
difference spectra was reached at about 1:1 ratio, which was taken as an indication of the
stoichiometry of the complex.
Fig 3 (a) UVꢀvis (200ꢀ500 nm) absorption spectra of compound 2a (25 ꢁM in methanol) alone or
in the presence of Cu2+ (2ꢀ50 ꢁM in methanol); (b) The differential spectra due to 2aꢀCu2+
complex formation obtained by numerical subtraction from the above spectra of those of Cu2+ and
2a at the corresponding concentrations.
2.2.5. Effect of compound 2a on Aβꢀinduced tau protein hyperphosphorylation
Aβꢀinduced tau protein hyperphosphorylation, a cardinal feature of AD, can induce destabilization
of microtubules and eventual death of the neurons.37ꢀ38 Therefore, we tested the effect of
compound 2a on Aβꢀinduced tau protein hyperphosphorylation by western blot analysis. As shown
in Fig 4, the phosphorylation of tau protein at Ser199/202 and Ser396 sites increased after
treatment with 10 ꢁM Aβ25–35 for 6 h. However, phosphorylation of tau protein was prevented by
pretreatment with compound 2a (25 and 50 ꢁM) for 30 min. Meanwhile, the total tau protein
didn’t change much after Aβ25–35 exposure with or without pretreatment with compound 2a,
resulting in an attenuation of pꢀtau/ total tau. Based on the results, we concluded that compound
2a could inhibit tau protein hyperphosphorylation, consequently resulting in a neuroprotective
effect on Aβꢀinduced PC12 cells.

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Fig 4 Compound 2a attenuated Aβ25–35ꢀinduced tau protein hyperphosphorylation in PC12 cells.
PC12 cells were pretreated with compound 2a (10 and 25 ꢁM) for 30 min, and then treated with
Aβ25–35 (10 ꢁM) for 6 h. Western blot analysis of phosphorylated tau protein (at Ser199/202 and
Ser396 sites) and total tau was performed. Values are mean ± S.D. from triplicate independent
experiments. #p < 0.05 vs. control; *p < 0.05, **p < 0.01 vs. Aβ25–35 treatment group.
2.2.6. Docking study of compound 2a with Aβ1ꢀ42
To further study the interaction mode of compound 2a with Aβ1ꢀ42, molecular modeling
calculations and docking studies were performed using Molecular Operating Environment (MOE)
software version 2009.10 (Chemical Computing Group, Montreal, Canada). The Xꢀray crystal
structure of Aβ1ꢀ42 (PDB 1IYT) used in the docking study was obtained from the Protein Data
Bank (www.rcsb.org).13 In Fig 5, (a) showed the interaction conformation between 2a and Aβ1ꢀ42
;
(b) showed that the substituted benzene ring of compound 2a had a πꢀπ interaction with Aβ (Tyr10)
with the distance centroid of 2.27 Å and it could insert into the hydrophobic pocket (colored
yellow, Glu3, His6, Asp7 and Tyr10). This docking simulation theoretically indicated the
formation of Aβ/compound 2a complex by an effective πꢀπ interaction.

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Fig 5 Docking results of compound 2a and Aβ1ꢀ42 (PDB code: 1IYT). (a) Cartoon representations
of compound 2a (colored purple) interacting with Aβ1ꢀ42 (colored yellow); (b) Association of
compound 2a (colored grey) and Aβ1–42 obtained from docking calculations. The interactions
between the ligand and residue Tyr10 are indicated by the green line.
2.2.7. Prediction of BBB penetration based on Lipinski’s rules
A major barrier to the development of effective antiꢀAβ compounds for AD therapy is that
essentially >98% of smallꢀmolecule compounds failed to cross the BBB.39ꢀ40 For drugꢀlike
properties, molecules should satisfy the terms of Lipinski’s rules 41: molecular weight (MW) less
than 500, the calculated logarithm of the octanolꢀwater partition coefficient (Clog P) less than 5,
the number of hydrogen bond donor atoms (HBD) less than 5, the number of hydrogen bond
acceptor atoms (HBA) less than 10 and the small polar surface area less than 90 Å2. Filter for
prediction of BBB penetration involves calculation of logBB by means of the equation shown in
the footnote of Table 2 and compounds with logBB higher than ꢀ1.0 could cross BBB.42 Defined
by the terms of Lipinski’s rules and calculated logBB for potential applications in brains,
compounds 2a, 2b, 3i and 3j fulfill drugꢀlike criteria in general and could possibly penetrate the
BBB better than CTX.
Table 2 Physical properties of compounds 2a, 2b, 3i, 3j and CTX.
Compounds
MW a
318
Clog P a
3.211
5.105
5.818
5.018
5.314
≤5.0
HBA a
HBD a
PSA a
57.15
63.22
63.22
46.15
46.15
≤90
Log BB a
ꢀ0.218
ꢀ0.021
0.079
CTX
6
6
1
0
2a
422
2b
456
6
0
3i
386
5
0
0.210
3j
Rules
a
408
5
0
0.264
≤500
≤10
≤5
≥ꢀ1.0
MW, molecular weight; Clog P, calculated logarithm of the octanolꢀwater partition coefficient;
HBA, hydrogenꢀbond acceptor atoms; HBD, hydrogenꢀbond donor atoms; PSA, polar surface area;
Log BB= ꢀ 0.0148 × PSA + 0.152 × Clog P + 0.139 42
.
2.2.8. In vitro bloodꢀbrain barrier permeability assay
In order to verify the in vitro BBB permeability of the four active compounds, the parallel
artificial membrane permeability assay of BBB (PAMPAꢀBBB) described by Di et al. was
performed.43 This method could give reasonably good predictions on the passive BBB
permeability of commercial centrally active drugs except for compounds which involved active

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uptake/efflux transport or were heavily metabolized in vivo. First, we compared the permeability
of 10 commercial drugs with reported values to validate the assay (shown in Table 3). A plot of
the experimental data versus the reported values produced a good linear correlation: Pe(exp.) =
1.054Pe(bibl.) + 0.5582 (R² = 0.987) (see the Supporting Information, Fig S2). From this equation
and considering the limit established by Di et al. for BBB permeation, we classified compounds as
follows: compounds with Pe (10ꢀ6 cm sꢀ1) > 4.8 for high BBB permeation (CNS+), compounds
with Pe (10ꢀ6 cm sꢀ1) < 2.7 for low BBB permeation (CNSꢀ) and compounds with 2.7< Pe (10ꢀ6 cm
sꢀ1) < 4.8 for uncertain BBB permeation (CNS±). Finally, compounds 2a, 2b, 3i, 3j and CTX were
tested through PAMPAꢀBBB (summarized in Table 3). Compounds 2a, 2b, 3i and 3j showed
higher Pe value than 4.8, which indicated that they were able to cross the BBB and may reach the
therapeutic targets in the CNS.
Table 3 Permeability (Pe, 10ꢀ6 cm sꢀ1) of 10 commercial drugs and active compounds in the
PAMPAꢀBBB assay.
Commercial drugs
Chlorpromazine
Clonidine
Bibliography a
Pe (10ꢀ6 cm sꢀ1) b Compounds Pe (10ꢀ6 cm sꢀ1) b
6.5
7.1±0.9
6.5±1.5
14.8±3.3
0.9±0.4
10.5±1.8
0.3±0.1
1.5±0.5
3.9±1.1
18.1±1.9
16.5±1.7
CTX
4.1±0.5(CNS±)
5.3
2a
2b
3i
3j
ꢀ
6.9±0.7(CNS+)
Desipramine
Lomefloxacin
Progesterone
Dopamine
12.0
1.1
6.2±0.5(CNS+)
7.6±1.1(CNS+)
9.3
8.2±0.9(CNS+)
0.2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Ofloxacin
0.8
ꢀ
Piroxacin
2.5
ꢀ
Testosterone
17.0
ꢀ
Verapamil
16.0
ꢀ
a Data obtained from reference. 43
b Experimental data are the mean±SD of 3 independent experiments, with PBS:EtOH(70:30) as
solvent.
3. Conclusion
In summary, a series of 4ꢀOꢀsubstituted chrysotoxine (CTX) derivatives were designed,
synthesized and evaluated as multifunctional agents for the treatment of Alzheimer’s disease (AD).
Among the four ring substituted compounds (2a, 2b, 3i and 3j) with good neuroprotective
activities, compound 2a exhibited significant inhibition of selfꢀ and Cu2+ꢀinduced Aβ1ꢀ42
aggregation and metalꢀchelating ability. Moreover, compound 2a was also capable of crossing the

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BBB in a parallel artificial membrane permeability assay. Furthermore, the result of western blot
analysis showed that compound 2a was able to reduce Aβꢀinduced tau protein
hyperphosphorylation. Taken together, these results suggest that compound 2a is a promising lead
compound for multifunctional AD treatment. Further investigations of AD therapeutic candidates
based on these results are in progress.
4. Experimental
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Aladdin Shanghai Biochemical
Technology Co., Ltd. or Sinopharm Chemical Reagent Co., Ltd. (China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254
(Qingdao Haiyang Chemical Plant, Qingdao, China) plates and the spots were detected under UV
light (λ=254 nm). IR spectra were recorded on a Nicolet iS10 spectrophotometer using KBr disks.
Melting point was measured on an XTꢀ4 micro melting point apparatus and uncorrected.
1H and 13C NMR spectra were measured on a Bruker 300 MHz spectrometer at 298T and
referenced to TMS. Chemical shifts were reported in ppm using the residual solvent line as
internal standard. Splitting patterns were designed as s (singlet), d (doublet), t (triplet), m
(multiplet).
Mass spectra were obtained on an MS Agilent 1100 Series LC/MSD Trap mass spectrometer
(ESIꢀMS). A Shimadzu LCꢀ2010 series HPLC system (Kyoto, Japan) was used for the purity
detection. Samples were separated on a YMCꢀPack ODSꢀA column (50 mm × 4.6 mm, 3 ꢁm,
◦
YMC Co. Ltd., Kyoto, Japan) with the column temperature set at 30 C. The mobile phase
consisted of acetonitrile–water (90:10, v/v), delivered at a flow rate of 1 mL/min. The injection
volume was 10 ꢁL. Column chromatography was performed on silica gel (200ꢀ300 mesh; Qingdao
Marine Chemical Inc.)
4.1.1. Synthetic intermediates
4.1.1.1. 4ꢀformylꢀ2,6ꢀdimethoxyphenyl acetate (3)
Syringaldazine (8.33 g, 0.046 mol) and catalyst DMAP were dissolved in DCM (10 mL), then
acetic anhydride (4.75 mL, 0.051mol) was slowly added. After the mixture was stirred at room
temperature for 1 h, the solution was washed with diluted water (10 mL × 3). Organic layer was

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dried over anhydrous Na2SO4 and concentrated to get white crystals 10.19 g, 99.8 % yield; mp
118ꢀ119 ºC; IR vmax (KBr, cmꢀ1): 3099, 2940, 1759, 1691, 1603, 1499; 1HꢀNMR (300 MHz, CDCl3)
δ 9.93(1H, s, ꢀCHO), 7.17(2H, s, Hꢀ3, Hꢀ5), 3.92(6H, s, 2×ꢀOCH3), 2.38(3H, s, ꢀCOCH3);
13CꢀNMR (75 MHz, CDCl3) δ 191.1, 168.1, 152.8, 134.3, 115.0, 106.0, 56.3, 20.4; ESIꢀMS m/z:
225.1 [M+H]+ .
4.1.1.2. 4ꢀ(hydroxymethyl)ꢀ2,6ꢀdimethoxyphenyl acetate (4)
To a solution of 3 (8.6 g, 0.038 mol) in EtOH (10 mL), NaBH4 (0.53g, 0.014mol) was slowly
added under ice bath. After stirred at room temperature for 30 min, 5% HCl was added to adjust
pH to 7. Concentrated under vacuum, residue was extracted with DCM (10 mL × 3), then organic
layer was dried over anhydrous Na2SO4 and concentrated to get white solid 8.34 g, 96.5 % yield;
1
mp 98ꢀ99 ºC; IR vmax (KBr, cmꢀ1): 3530, 3126, 2944, 1748, 1605, 1508; HꢀNMR (300 MHz,
CDCl3) δ 6.66 (1H, s, Hꢀ3, Hꢀ5), 4.69 (2H, s, ArꢀCH2ꢀ), 3.84 (6H, s, 2×ꢀOCH3), 2.36 (3H, s,
ꢀCOCH3); 13CꢀNMR (75 MHz, CDCl3) δ 168.8, 152.1, 139.6, 115.3, 103.1, 65.2, 56.1, 20.5;
ESIꢀMS m/z: 227.1 [M+H]+.
4.1.1.3. 4ꢀ(chloromethyl)ꢀ2,6ꢀdimethoxyphenyl acetate (5)
To a solution of 4 (8.3 g, 0.037 mol) in DCM (10 mL), Et3N (6.2 mL, 0.043mol), SOCl2 (3.0 mL,
0.043mol) was slowly dropped under ice salt bath. After stirred at room temperature for 3 h, the
solution was washed with diluted water (10 mL × 3). Organic layer was dried over anhydrous
Na2SO4 and concentrated to get light yellow solid 7.96 g, 88.7 % yield; mp 75ꢀ76 ºC; IR vmax (KBr,
1
cmꢀ1): 3162, 2971, 1759, 1606, 1508; HꢀNMR (300MHz, CDCl3) δ 6.85 (1H, s, Hꢀ3, Hꢀ5), 4.73
(2H, s, ArꢀCH2ꢀ), 3.76 (6H, s, 2×ꢀOCH3), 2.24 (3H, s, ꢀCOCH3); 13CꢀNMR (75 MHz, CDCl3) δ
168.3, 152.0, 139.4, 115.0, 103.2, 60.7, 56.1, 20.4; ESIꢀMS m/z: 227.1 [M+H]+.
4.1.1.4. (4ꢀacetoxyꢀ3,5ꢀdimethoxybenzyl) triphenylphosphonium chloride (6)
5 (7.8 g, 0.032 mol) and PhP3 (8.4g, 0.032 mol) were dissolved in toluene (5 mL) under reflux for
6 h. After cooled to temperature, the mixture was filtrated and washed with toluene under vacuum.
1
Dried under infrared light and 14.63 g white powder was obtained, 90.2 % yield; HꢀNMR (300
MHz, DMSOꢀd6) δ 7.92 (3H, m, ArH), 7.76 (6H, dt, J = 3.6Hz, 8.1Hz, ArH), 7.69 (6H, m, ArH),
6.30 (2H, d, J = 2.4Hz, Hꢀ3, Hꢀ5), 5.09 (2H, J = 15Hz, ArꢀCH2ꢀ), 3.37 (6H, s, 2×ꢀOCH3 ), 2.21
(3H, s, ×ꢀOCH3); 13CꢀNMR (75 MHz, DMSOꢀd6) δ 168.4, 152.0, 151.9, 135.5, 134.8, 134.6,
130.6, 130.4, 126.9, 126.7, 118.8, 117.6, 108.5, 108.4, 56.2, 29.4, 28.8, 20.6; ESIꢀMS m/z: 541.2

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[M+Cl]ꢀ.
4.1.1.5. 4ꢀ[(Z / E)ꢀ3,4ꢀdimethoxystyryl]ꢀ2,6ꢀdimethoxyphenyl acetate (7)
6 (3.0 g, 5.920 mmol) and 3,4ꢀdimethoxybenzaldehyde were dissolved in anhydrous THF (3 mL)
under Ar protection. NaH (0.15 g, 6.25 mmol) was added slowly under ice salt bath. After
overnight reaction in room temperature, the solution was concentrated. Then the mixture of transꢀ
and cisꢀ stilbenes was recrystallized in methanol with 70.4 % yield. Transꢀ and cisꢀ stilbenes could
be separated by fast chromatography, and the ratio of transꢀ and cisꢀ was 3:1.
1
4ꢀ[(Z)ꢀ3,4ꢀdimethoxystyryl]ꢀ2,6ꢀdimethoxyphenyl acetate: HꢀNMR (300 MHz, CDCl3) δ 7.06
(3H, m, ArH), 6.97 (1H, d, J = 8.9 Hz, ꢀCH=CHꢀ), 6.89 (1H, d, J = 8.9 Hz, ꢀCH=CHꢀ), 6.76 (2H, s,
Hꢀ3, 5), 3.97 (3H, s, ꢀOCH3), 3.93 (3H, s, ꢀOCH3), 3.90 (6H, s, 2×ꢀOCH3), 2.37 (3H, s, ꢀCOCH3);
13CꢀNMR (75 MHz, CDCl3) δ 168.7, 151.9, 148.3, 135.8, 130.4, 129.5, 128.5, 122.1, 111.6, 110.7,
106.0, 105.4, 56.0, 55.8, 55.7, 20.5; ESIꢀMS m/z: 359.1 [M+H]+; 4ꢀ[(E)ꢀ3,4ꢀ
1
dimethoxystyryl]ꢀ2,6ꢀdimethoxyphenyl acetate: HꢀNMR (300 MHz, CDCl3) δ 6.87 (1H, dd, J =
1.8, 8.1 Hz, Hꢀ6’), 6.84 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.78 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.57 (2H, s, Hꢀ3,
5), 6.54 (1H, d, J = 12.0 Hz, ꢀCH=CHꢀ), 6.49 (1H, d, J = 12.0 Hz, ꢀCH=CHꢀ), 3.88 (3H, s, ꢀOCH3),
3.69 (6H, s, 2×ꢀOCH3), 3.68 (3H, s, ꢀOCH3), 2.34 (3H, s, ꢀCOCH3); 13CꢀNMR (75 MHz, CDCl3) δ
168.9, 152.2, 149.1, 149.0, 136.0, 130.1, 128.8, 126.6, 122.0, 111.2, 108.7, 102.9, 56.1, 56.0, 55.9,
20.5; ESIꢀMS m/z: 359.1 [M+H]+.
4.1.2. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl acetate (CTXꢀ0)
The mixture of stilbenes (1g, 2.79 mmol) was reduced by treatment with catalytic amounts of 5%
PdꢀC over H2 (4 atm) in ethanol (5 mL) to quantitatively afford 8 with bibenzyl skeleton. White
crystal, mp 124ꢀ125 ºC; IR vmax (KBr, cmꢀ1): 3422, 2924, 1760, 1603, 1517; 1HꢀNMR (300 MHz,
CDCl3) δ 6.82(1H, d, J = 8.1Hz, Hꢀ5’), 6.75(1H, dd, J = 1.8, 8.1Hz, Hꢀ6’), 6.67(1H, d, J = 1.8Hz,
Hꢀ2’), 6.41(2H, s, Hꢀ3, 5), 3.88(3H, s, ꢀOCH3), 3.86(3H, s, ꢀOCH3), 3.80(6H, s, 2×ꢀOCH3),
2.88(4H, s, ꢀCH2CH2ꢀ), 2.86(3H, s, ꢀCOCH3); 13CꢀNMR (75 MHz, CDCl3) δ 168.9, 151.7, 148.6,
147.2, 141.2, 140.1, 134.0, 120.2, 111.8, 111.1, 105.0, 56.0, 55.8, 55.7, 38.6, 37.4, 20.4; ESIꢀMS
m/z: 361.1[M+H]+; HPLC purity: 99%, tR = 2.13 min.
4.1.3. chrysotoxine (CTX)
CTXꢀ0 (1g, 2.78 mmol) was first dissolved in methanol (5 mL), and then catalytic amount of
MeONa was added and stirred for 30 min in the room temperature. 5% HCl was added to adjust

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pH=7. After concentration under vacuum, the residue was extracted with DCM (10 mL × 3), dried
with Na2SO4 and then purified by fast chromatography (PE /aceton 5:1) to get a light yellow solid
0.88g, 99.8% yield. mp 98ꢀ99 ºC; IR vmax (KBr, cmꢀ1): 3462, 3133, 2933, 1609, 1517; 1HꢀNMR
(300 MHz, CDCl3) δ 6.81 (1H, d, J = 8.1Hz, Hꢀ5’), 6.72 (1H, dd, J = 1.8, 8.1Hz, Hꢀ6’), 6.68 (1H,
d, J = 1.8Hz, Hꢀ2’), 6.38 (2H, s, Hꢀ3, 5), 5.39 (1H, s, ꢀOH), 3.88 (6H, s, 2×ꢀOCH3), 3.87 (6H, s,
2×ꢀOCH3), 2.85 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR (75 MHz, CDCl3) δ 146.9, 146.3, 140.4, 133.8,
132.3, 127.8, 119.9, 111.4, 110.7, 104.6, 55.7, 55.4, 55.3, 37.9, 37.3; ESIꢀMS m/z: 319.1 [M+H]+,
336.1 [M+NH4]+; HPLC purity: 100 %, tR = 1.71 min.
4.1.4. General procedure for preparation of 4ꢀOꢀalkylsubstituted CTX ester derivatives
(1a~1f)
To a mixture of CTX (30mg, 0.094 mmol), EDC•HCl and catalytic amount of DMAP in DCM (5
mL), corresponding shortꢀchain carboxylic acid was added. The reaction mixture was stirred for
12ꢀ20 h in room temperature. After complete reaction, the solvent was evaporated under reduced
pressure. The residue was purified on a fast chromatography using the mixture of PE/EtOAc
(20:1ꢀ8:1) as eluent, by which we obtained the corresponding 4ꢀOꢀmodified CTX derivatives.
4.1.4.1. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl propionate (1a). Yield 63.7%; white
solid, mp 101ꢀ102 ºC; IR vmax (KBr, cmꢀ1): 3422, 2922, 1758, 1604, 1515; 1HꢀNMR (300 MHz,
CDCl3) δ 6.82 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, dd, J = 1.8, 8.1 Hz, Hꢀ6’), 6.67 (1H, d, J = 1.8
Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.87 (3H, s, ꢀOCH3), 3.79 (6H, s, 2×ꢀOCH3),
2.88 (4H, s, ꢀCH2CH2ꢀ), 2.66 (2H, q, J = 7.8 Hz, ꢀCOCH2ꢀ), 1.29 (3H, t, J = 7.5 Hz, ꢀCH3);
13CꢀNMR (75MHz, CDCl3) δ 172.0, 151.4, 148.3, 146.8, 139.6, 133.7, 119.8, 111.4, 110.7, 104.7,
55.6, 55.5, 55.3, 38.2, 37.0, 26.7, 8.8; ESIꢀMS m/z: 392.3 [M+NH4]+ , 375.2 [M+H]+ ; HPLC
purity: 100 %, tR = 2.22 min.
4.1.4.2. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl butyrate (1b). Yield 42.1%; yellow
1
powder; mp 60ꢀ61 ºC; IR vmax (KBr, cmꢀ1): 3446, 2953, 1758, 1603, 1508; HꢀNMR (300 MHz,
CDCl3) δ 6.82 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, dd, J = 1.8, 8.1 Hz, Hꢀ6’), 6.67 (1H, d, J =
1.8 Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.79 (6H, s,
2×ꢀOCH3), 2.88 (4H, s, ꢀCH2CH2ꢀ), 2.61 (2H, t, J = 7.2 Hz, ꢀCOCH2ꢀ), 1.82 (2H, m, J = 7.2 Hz,
ꢀCH2ꢀ), 1.07 (3H, t, J = 7.2 Hz, ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 171.1, 151.4, 148.5, 139.6,
133.7, 119.8, 114.5, 111.4, 110.7, 104.7, 55.6, 55.5, 55.3, 38.2, 37.0, 35.3, 18.2, 13.1; ESIꢀMS m/z:

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406.3 [M+NH4]+ , 389.2 [M+H]+; HPLC purity: 98 %, tR = 2.44 min.
4.1.4.3. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl isobutyrate (1c). Yield 49.2%;
yellow powder, mp 80ꢀ81 ºC; IR vmax (KBr, cmꢀ1): 3433, 2932, 1760, 1601, 1509, 1117; 1HꢀNMR
(300 MHz, CDCl3) δ 6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, dd, J = 1.8, 8.1 Hz, Hꢀ6’), 6.67
(1H, d, J = 1.8 Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.87 (3H, s, ꢀOCH3), 3.78 (6H,
s, 2×ꢀOCH3), 2.89 (1H, m, ꢀCOCHꢀ) 2.88 (4H, s, ꢀCH2CH2ꢀ), 1.34 (6H, d, J = 6.9 Hz, 2×ꢀCH3);
13CꢀNMR (75 MHz, CDCl3) δ 174.7, 151.4, 148.5, 146.8, 139.4, 133.7, 119.8, 114.5, 111.4, 110.7,
104.8, 55.6, 55. 5, 55.3, 38.2, 37.0, 33.4, 18.6; ESIꢀMS m/z: 406.3 [M+NH4]+ , 389.2 [M+H]+;
HPLC purity: 100 %, tR = 2.47 min.
4.1.4.4. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl pentanoate (1d). Yield 63.3%; white
1
crystal; mp 77ꢀ78 ºC; IR vmax (KBr, cmꢀ1): 3448, 2956, 1741, 1601, 1509, 1128; HꢀNMR (300
MHz, CDCl3) δ 6.82 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, dd, J = 1.8, 8.1 Hz, Hꢀ6’), 6.67 (1H, d,
J = 1.8 Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.78 (6H, s,
2×ꢀOCH3), 2.88 (4H, s, ꢀCH2CH2ꢀ), 2.62 (2H, t, J = 7.2 Hz, ꢀCOCH2ꢀ), 1.78 (2H, m, J = 7.2 Hz,
ꢀCH2ꢀ), 1.48 (2H, m, ꢀCH2ꢀ), 0.98 (3H, t, J = 7.2 Hz, ꢀCH3); 13CꢀNMR (75MHz, CDCl3) δ 181.1,
151.4, 148.3, 146.8, 139.6, 134.9, 119.8, 111.4, 110.7, 104.7, 55.6, 55.5, 55.3, 38.2, 37.0, 33.1,
26.7, 21.7, 13.3; ESIꢀMS m/z: 420.3 [M+NH4]+ , 403.2 [M+H]+; HPLC purity: 100 %, tR = 2.70
min.
4.1.4.5. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl 3ꢀmethylbutanoate (1e). Yield
60.6%; white crystal; mp 102ꢀ103 ºC; IR vmax (KBr, cmꢀ1): 3422, 2923, 1758, 1607, 1506, 1124;
1HꢀNMR (300 MHz, CDCl3) δ 6.82 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, dd, J = 1.8, 8.1 Hz,
Hꢀ6’), 6.67 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s,
ꢀOCH3), 3.78 (6H, s, 2×ꢀOCH3), 2.88 (4H, s, ꢀCH2CH2ꢀ), 2.49 (2H, d, J = 7.2 Hz, ꢀCOCH2ꢀ), 2.29
(1H, m, ꢀCHꢀ), 1.09 (6H, d, J = 6.6 Hz, 2×ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 170.5, 151.4,
148.2, 146.8, 139.6, 133.65, 119.8, 111.4, 110.7, 104.7, 55.5, 55.4, 55.3, 42.5, 38.2, 37.0, 25.6,
21.8; ESIꢀMS m/z: 420.3 [M+NH4]+ , 403.2[M+H]+; HPLC purity: 100 %, tR = 2.69 min.
4.1.4.6. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl 2ꢀpropylpentanoate (1f). Yield
48.7%; white crystal; mp 63ꢀ64 ºC; IR vmax (KBr, cmꢀ1): 3417, 2914, 1758, 1607, 1513, 1129;
1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, dd, J = 1.8, 8.1Hz,
Hꢀ6’), 6.66 (1H, d, J = 1.8Hz, Hꢀ2’), 6.38 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s,

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ꢀOCH3), 3.76 (6H, s, 2×ꢀOCH3), 2.87 (4H, s, ꢀCH2CH2ꢀ), 2.67 (1H, m, ꢀCOCHꢀ), 1.80 (2H, m,
ꢀCH2), 1.56 (2H, m, ꢀCH2), 1.52 (4H, m, 2×ꢀCH2ꢀ), 0.97 (6H, t, J = 8.1Hz, 2×ꢀCH3); 13CꢀNMR (75
MHz, CDCl3) δ 173.8, 151.4, 148.3, 146.8, 139.5, 133.7, 119.8, 111.4, 110.7, 104.7, 55.5, 55.4,
55.3, 44.8, 38.2, 37.0, 34.4, 19.9, 13.6; ESIꢀMS m/z: 462.3 [M+NH4]+ , 445.2 [M+H]+; HPLC
purity: 96 %, tR = 2.02 min.
4.1.5. General procedure for preparation of 4ꢀOꢀcyclicsubstituted CTX ester derivatives
(2a~2e)
Corresponding cyclicꢀsubstituted acid was reacted in SOCl2 (3 mL) under reflux for 2 h. After
concentration under vacuum, chloride powder was obtained for the next procedure. To a mixture
of CTX (30 mg, 0.094 mmol) and corresponding chloride in aceton (5 mL), Et3N (200 ꢁL) was
dropped slowly under ice bath. The solution refluxed for 4 h and then was evaporated under
reduced pressure. Water was added in the residue, and DCM (10 mL × 3) was used for extract.
Organic layer was dried with Na2SO4 and then was evaporated to get the crude target. Silica gel
chromatography was applied for purification with elution of PE / EtOAc (20:1ꢀ3:1).
4.1.5.1.1 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl benzoate (2a). Yield 90.3%; white
crystal; mp 136ꢀ137 ºC; IR vmax (KBr, cmꢀ1): 3448, 2935, 1741, 1601, 1509, 1130; 1HꢀNMR (300
MHz, CDCl3) δ 8.26, 7.61, 7.52 (5H, m, ArH), 6.83 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.76 (1H, dd, J =
1.8, 8.1Hz, Hꢀ6’), 6.70 (1H, d, J = 1.8Hz, Hꢀ2’), 6.46 (2H, s, Hꢀ3, 5), 3.89 (6H, s, 2×ꢀOCH3), 3.79
(6H, s, 2×ꢀOCH3), 2.91 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR (75 MHz, CDCl3) δ 184.1, 151.6, 148.2,
145.7, 139.8, 133.7, 132.8, 131.2, 129.9, 127.9, 119.8, 111.4, 110.7, 104.8, 55.7, 55.5, 55.4, 38.3,
37.1; ESIꢀMS m/z: 440.3 [M+NH4]+ , 423.2 [M+H]+; HPLC purity: 99 %, tR = 2.53 min.
4.1.5.2. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl 4ꢀchlorobenzoate (2b). Yield 87.2%;
light yellow powder; mp 130ꢀ131 ºC; IR vmax (KBr, cmꢀ1): 3447, 2933, 1743, 1601, 1508, 1127;
1HꢀNMR (300 MHz, CDCl3) δ 8.19 (2H, d, J = 8.4 Hz, ArH), 7.49 (2H, d, J = 8.4Hz, ArH), 6.83
(1H, d, J = 8.1Hz, Hꢀ5’), 6.76 (1H, dd, J = 1.8, 8.1Hz, Hꢀ6’), 6.70 (1H, d, J = 1.8Hz, Hꢀ2’), 6.45
(2H, s, Hꢀ3, 5), 3.91 (6H, s, 2×ꢀOCH3), 3.82 (6H, s, 2×ꢀOCH3), 2.91 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR
(75 MHz, CDCl3) δ 187.3, 151.5, 149.5, 147.0, 140.0, 135.9, 133.7, 131.3, 128.4, 128.3, 119.8,
111.4, 110.7, 104.7, 55.6, 55.5, 55.4, 42.8, 38.3, 37.0, 33.5; ESIꢀMS m/z: 474.2 [M+NH4]+ , 457.1
[M+H]+; HPLC purity: 95 %, tR = 3.06 min.
4.1.5.3. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl 3ꢀphenylpropanoate (2c). Yield

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85.7%; light yellow powder; mp 65ꢀ66 ºC; IR vmax (KBr, cmꢀ1): 3423, 2923, 1759, 1605, 1508,
1127; 1HꢀNMR (300 MHz, CDCl3) δ 7.31 (5H, m, ArH), 6.82 (1H, d, J = 8.1Hz, Hꢀ5’), 6.74 (1H,
dd, J = 1.8, 8.1Hz, Hꢀ6’), 6.67 (1H, d, J = 1.8Hz, Hꢀ2’), 6.40 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3),
3.86 (3H, s, ꢀOCH3), 3.76 (6H, s, 2×ꢀOCH3), 3.12 (2H, t, J = 6.9 Hz, ꢀCH2ꢀ), 2.95 (2H, t, J = 6.9
Hz, ꢀCH2ꢀ), 2.87 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR (75 MHz, CDCl3) δ 170.4, 151.3, 148.5, 146.8,
140.0, 139.7, 133.6, 128.0, 127.9, 125.7, 119.8, 111.4, 110.7, 104.7, 55.6, 55.5, 55.3, 38.3, 37.0,
34.9, 30.5; ESIꢀMS m/z: 468.2 [M+NH4]+, 451.1 [M+H]+; HPLC purity: 99 %, tR = 2.66 min.
4.1.5.4. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl picolinate (2d). Yield 72.7%; yellow
powder; mp 132ꢀ133 ºC; IR vmax (KBr, cmꢀ1): 3131, 2944, 1757, 1601, 1514; 1HꢀNMR (300 MHz,
CDCl3) δ 8.89, 8.32, 7.95, 7.57 (4H, pyridine), 6.83 (1H, d, J = 8.1Hz, Hꢀ5’), 6.75 (1H, dd, J = 1.8,
8.1Hz, Hꢀ6’), 6.69 (1H, d, J = 1.8Hz, Hꢀ2’), 6.44 (2H, s, Hꢀ3, 5), 3.89 (3H, s, ꢀOCH3), 3.88 (3H, s,
ꢀOCH3), 3.78 (6H, s, 2×ꢀOCH3), 2.91 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR (75 MHz, CDCl3) δ 180.1,
151.9, 150.1, 148.9, 146.0, 140.4, 137.0, 134.2, 127.2, 125.9, 120.3, 111.9, 111.2, 105.1, 56.1,
55.9, 55.8, 38.8, 37.5; ESIꢀMS m/z: 424.1 [M+H]+ ; HPLC purity: 99%, tR = 2.00 min.
4.1.5.5. 4ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2,6ꢀdimethoxyphenyl isonicotinate (2e). Yield 87.7%;
white crystal; mp 118ꢀ119 ºC; IR vmax (KBr, cmꢀ1): 3131, 2932, 1735, 1601, 1514; 1HꢀNMR (300
MHz, CDCl3) δ 8.86 (2H, d, J = 5.7 Hz, Pyridine), 8.06 (2H, d, J = 5.7 Hz, Pyridine), 6.83 (1H, d,
J = 8.1Hz, Hꢀ5’), 6.75 (1H, dd, J = 1.8, 8.1Hz, Hꢀ6’), 6.69 (1H, d, J = 1.8Hz, Hꢀ2’), 6.45 (2H, s,
Hꢀ3, 5), 3.89 (3H, s, ꢀOCH3), 3.88 (3H, s, ꢀOCH3), 3.78 (6H, s, 2×ꢀOCH3), 2.91 (4H, s, ꢀCH2CH2ꢀ);
13CꢀNMR (75 MHz, CDCl3) δ 181.5, 155.4, 151.8, 150.6, 141.1, 136.8, 134.1, 123.7, 120.3, 111.9,
111.2, 105.2, 56.1, 56.0, 55.9, 38.8, 37.5; ESIꢀMS m/z: 424.1 [M+H]+ , 406.1 [MꢀOH]+ ; HPLC
purity: 98%, tR = 2.42 min.
4.1.6. General procedure for preparation of 4ꢀOꢀalkyl and 4ꢀOꢀcyclic substituted CTX ether
derivatives (3a~3j)
CTX (30 mg, 0.094 mmol) and corresponding bromoꢀsubstituted hydrocarbons were under reflux
in DMF (3 mL) for 6 h. To the solution EtOAc (30 mL) was added, and water was used to wash
DMF for 5 times. The organic layer was dried with Na2SO4 and the solvent was removed under
reduced pressure. Silica gel chromatography was applied for purification with elution of PE /
EtOAc (20:1ꢀ10:1).
4.1.6.1. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2ꢀethoxyꢀ1,3ꢀdimethoxybenzene (3a). Yield 52.9 %;

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1
yellow crystal, mp 68ꢀ69 ºC; IR vmax (KBr, cmꢀ1): 3132, 2957, 1589, 1515; HꢀNMR (300 MHz,
CDCl3) δ 6.82 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.74 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8
Hz, Hꢀ2’), 6.38 (2H, s, Hꢀ3, 5), 4.04 (2H, q, J = 7.2 Hz, ꢀOCH2ꢀ), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s,
ꢀOCH3), 3.83 (6H, s, 2×ꢀOCH3), 2.87 (4H, s, ꢀCH2CH2ꢀ), 1.37 (3H, t, J = 7.2 Hz, ꢀCH3); 13CꢀNMR
(75 MHz, CDCl3) δ 153.3, 148.7, 146.9, 137.2, 134.3, 120.3, 115.1, 111.9, 111.1, 105.6, 74.2, 56.1,
55.9, 55.8, 38.6, 37.6, 31.1; ESIꢀMS m/z: 347 [M+H]+ , HPLC purity: 99 %, tR = 2.38 min..
4.1.6.2. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxyꢀ2ꢀpropoxybenzene (3b). Yield 61.8 %;
light yellow oil; IR vmax (KBr, cmꢀ1): 3000, 2958, 2935, 1589, 1513; 1HꢀNMR (300 MHz, CDCl3)
δ 6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’),
6.38 (2H, s, Hꢀ3, 5), 3.92 (2H, t, J = 6.9 Hz, ꢀOCH2ꢀ), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3),
3.82 (6H, s, 2×ꢀOCH3), 2.86 (4H, s, ꢀCH2CH2ꢀ), 1.78 (2H, m, ꢀCH2ꢀ), 1.01 (3H, t, J = 7.5 Hz,
ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.3, 148.7, 146.4, 137.2, 134.3, 120.3, 115.1, 111.9, 111.1,
105.6, 75.1, 56.1, 55.9, 55.8, 38.6, 37.6, 23.3, 10.4; ESIꢀMS m/z: 378 [M+NH4]+, 361[M+H]+;
HPLC purity: 97 %, tR = 2.70 min.
4.1.6.3. 2ꢀbutoxyꢀ5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxybenzene (3c). Yield 65.2%;
yellow oil; IR vmax (KBr, cmꢀ1): 3132, 2955, 1581, 1514; 1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H,
d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s,
Hꢀ3, 5), 3.95 (2H, t, J = 6.9 Hz, ꢀOCH2ꢀ), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.82 (6H, s,
2×ꢀOCH3), 2.86 (4H, s, ꢀCH2CH2ꢀ), 1.76 (2H, m, ꢀCH2ꢀ), 1.49 (2H, m, ꢀCH2ꢀ), 0.97 (3H, t, J = 6.5
Hz, ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.3, 148.7, 147.2, 137.2, 134.3, 120.3, 111.9, 111.2,
105.6, 73.2, 56.1, 56.0, 55.8, 38.6, 37.6, 32.2, 19.1, 13.9; ESIꢀMS m/z: 392.2 [M+NH4]+ , 375.2
[M+H]+ ; HPLC purity: 99 %, tR = 3.06 min.
4.1.6.4. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2ꢀisobutoxyꢀ1,3ꢀdimethoxybenzene (3d). Yield 49.8%;
yellow oil; IR vmax (KBr, cmꢀ1): 3132, 2935, 1588, 1514; 1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H,
d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s,
Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.82 (6H, s, 2×ꢀOCH3), 3.71 (2H, d, J = 6.9 Hz,
ꢀOCH2ꢀ), 2.86 (4H, s, ꢀCH2CH2ꢀ), 2.07 (1H, m, ꢀCHꢀ), 1.03 (6H, d, 2×ꢀCH3); 13CꢀNMR (75 MHz,
CDCl3) δ 153.2, 148.8, 147.3, 137.1, 134.3, 120.3, 111.9, 111.1, 105.9, 80.2, 56.2, 56.0, 55.8, 38.5,
37.6, 29.0, 19.2; ESIꢀMS m/z: 392.2 [M+NH4]+, 375.2 [M+H]+; HPLC purity: 100 %, tR = 3.21
min.

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4.1.6.5. 2ꢀ(secꢀbutoxy)ꢀ5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxybenzene (3e). Yield 42.7 %;
yellow oil; IR vmax (KBr, cmꢀ1): 3132, 2955, 1584, 1515; 1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H,
d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.67 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s,
Hꢀ3, 5), 4.11 (1H, m, ꢀCHꢀ), 3.88 (3H, s, ꢀOCH3), 3.85 (3H, s, ꢀOCH3), 3.80 (6H, s, 2×ꢀOCH3),
2.85 (4H, s, ꢀCH2CH2ꢀ), 1.80ꢀ161 (2H, m, ꢀOCH2ꢀ), 1.23 (3H, d, J = 6.3 Hz, ꢀCH3), 0.99 (3H, t, J
= 7.5 Hz, ꢀ CH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.5, 148.7, 142.1, 136.9, 129.3, 120.3, 115.0,
105.6, 80.1, 56.0, 55.9, 55.8, 38.6, 37.6, 29.5, 19.5, 10.0; ESIꢀMS m/z: 392.2 [M+NH4]+ , 375.2
[M+H]+; HPLC purity: 98 %, tR = 3.06 min.
4.1.6.6. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2ꢀ(isopentyloxy)ꢀ1,3ꢀdimethoxybenzene (3f). Yield 44.9%;
yellow oil; IR vmax (KBr, cmꢀ1): 3132, 2924, 1589, 1518; 1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H,
d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s,
Hꢀ3, 5), 3.97 (2H, t, J = 6.9 Hz, ꢀOCH2ꢀ), 3.88 (3H, s, ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.82 (6H, s,
2×ꢀOCH3), 2.86 (4H, s, ꢀCH2CH2ꢀ), 1.89 (1H, m, ꢀCHꢀ), 1.67 (2H, t, J = 6.9 Hz, ꢀCH2ꢀ), 0.96 (6H,
d, J = 6.6 Hz, 2×ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.3, 148.7, 147.2, 137.2, 134.3, 120.3,
111.9, 111.1, 105.6, 71.9, 56.1, 55.9, 55.8, 38.9, 38.6, 37.6, 24.8, 22.6; ESIꢀMS m/z :406.2
[M+NH4]+, 389.2 [M+H]+; HPLC purity: 100 %, tR = 3.47 min.
4.1.6.7. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxyꢀ2ꢀ(pentanꢀ2ꢀyloxy) benzene (3g). Yield
1
37.4 %; yellow oil; IR vmax (KBr, cmꢀ1): 3133, 2935, 1587, 1517; HꢀNMR (300 MHz, CDCl3) δ
6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’),
6.36 (2H, s, Hꢀ3, 5), 4.19 (1H, m, ꢀOCHꢀ), 3.88 (3H, s, ꢀOCH3), 3.85 (3H, s, ꢀOCH3), 3.80 (6H, s,
2×ꢀOCH3), 2.86 (4H, brs, ꢀCH2CH2ꢀ), 1.76, 1.51 (4H, m, ꢀCH2CH2ꢀ), 1.22 (2H, t, J = 6.3 Hz,
ꢀCH2ꢀ), 0.95 (3H, t, J = 6.9 Hz, ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.5, 148.6, 147.2, 136.9,
134.4, 120.3, 111.9, 111.1, 105.5, 78.5, 56.0, 55.9, 55.8, 39.1, 38.6, 37.6, 20.0, 18.8, 14.2; ESIꢀMS
m/z: 406.2 [M+NH4]+, 389.2 [M+H]+; HPLC purity: 99 %, tR = 3.53 min.
4.1.6.8. 5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2ꢀ[(2ꢀethylhexyl) oxy]ꢀ1,3ꢀdimethoxybenzene (3h). Yield
1
39.4%; yellow oil; IR vmax (KBr, cmꢀ1): 3133, 2958, 1637, 1513; HꢀNMR (300 MHz, CDCl3) δ
6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.72 (1H, dd, J = 1.8, 8.1 Hz, Hꢀ6’), 6.68 (1H, d, J = 1.8 Hz, Hꢀ2’),
6.36 (2H, s, Hꢀ3, 5), 3.88 (3H, s, ꢀOCH3), 3.85 (3H, s, ꢀOCH3), 3.82 (2H, d, J = 6.9 Hz, ꢀOCH2ꢀ),
3.80 (6H, s, 2×ꢀOCH3), 2.86 (4H, brs, ꢀCH2CH2ꢀ), 1.40ꢀ1.72(9H, m), 0.95 (6H, t, J = 6.9 Hz,
2×ꢀCH3); 13CꢀNMR (75 MHz, CDCl3) δ 153.5, 149.6, 146.9, 137.2, 134.5, 120.3, 111.9, 111.1,

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105.8, 75.8, 56.1, 55.9, 55.8, 40.2, 38.5, 37.6, 30.2, 29.0, 23.5, 23.2, 14.2, 10.9; ESIꢀMS m/z:
448.2 [M+NH4]+, 431.2 [M+H]+; HPLC purity: 98 %, tR = 6.65 min.
4.1.6.9. 2ꢀ(cyclopentyloxy)ꢀ5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxybenzene (3i). Yield
25.8%; yellow crystal, 51ꢀ52 ºC; IR vmax (KBr, cmꢀ1): 3416, 2361, 1637, 1420, 1231, 1122;
1HꢀNMR (300 MHz, CDCl3) δ 6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.73 (1H, d, J = 1.8, 8.1 Hz, Hꢀ6’),
6.67 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s, Hꢀ3, 5), 4.76 (1H, t, J = 5.4 Hz, ꢀOCHꢀ), 3.88 (3H, s,
ꢀOCH3), 3.85 (3H, s, ꢀOCH3), 3.80 (6H, s, 2×ꢀOCH3), 2.86 (4H, brs, ꢀCH2CH2ꢀ), 1.91, 1.67, 1.58
(8H, m); 13CꢀNMR (75MHz, CDCl3) δ 153.8, 149.7, 148.5, 138.3, 137.2, 134.5, 120.3, 111.9,
111.1, 105.7, 84.3, 56.1, 55.9, 55.8, 38.6, 37.5, 32.7, 23.7; ESIꢀMS m/z: 404.2 [M+NH4]+ , 387.2
[M+H]+ ; HPLC purity: 98 %, tR = 3.22 min.
4.1.6.10. 2ꢀ(benzyloxy)ꢀ5ꢀ(3,4ꢀdimethoxyphenethyl)ꢀ1,3ꢀdimethoxybenzene (3j). Yield 96.8%;
yellow crystal, 77ꢀ78 ºC; IR vmax (KBr, cmꢀ1): 3416, 2937, 1589, 1514, 1128, 1028; 1HꢀNMR (300
MHz, CDCl3) δ 7.52ꢀ7.31 (5H, m, ArH), 6.81 (1H, d, J = 8.1 Hz, Hꢀ5’), 6.71 (1H, d, J = 1.8, 8.1
Hz, Hꢀ6’), 6.67 (1H, d, J = 1.8 Hz, Hꢀ2’), 6.37 (2H, s, Hꢀ3, 5), 5.00 (2H, s, ꢀCH2ꢀ), 3.88 (3H, s,
ꢀOCH3), 3.86 (3H, s, ꢀOCH3), 3.81 (6H, s, 2×ꢀOCH3), 2.87 (4H, s, ꢀCH2CH2ꢀ); 13CꢀNMR (75 MHz,
CDCl3) δ 153.3, 148.8, 147.3, 138.0, 137.6, 134.3, 128.5, 128.1, 127.8, 120.4, 111.9, 111.2, 105.6,
75.1, 56.1, 56.0, 55.8, 38.6, 37.6; ESIꢀMS m/z :426.2 [M+NH4]+, 409.2 [M+H]+; HPLC purity:
100 %, tR = 3.53 min.
4.2. Pharmacology
4.2.1. Cell culture.
Aβ25ꢀ35 (Sigma) was dissolved in deionized distilled water to obtain a stock solution (1 mM). After
aggregation induced by incubating at 37 ºC for 4 days, 44 the stock solution was stored at ꢀ20 °C.
The PC12 cells were purchased from the Institute of Biochemistry and Cell Biology
(Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with penicillin (100 U/ml), streptomycin (100 ꢁg/ml), and 10 % (vol/vol) fetal bovine serum (FBS)
at 37 °C in an atmosphere containing 5% CO2. DMEM and FBS were obtained from Gibco
(GrandIsland, NY). The other reagents were purchased from Beyotime Institute of Biotechnology
(Shanghai, China).
For the experimental studies, the PC12 cells were subcultured in 96 or 6ꢀwell plates at a
density of 1.5×104 or 2×105 cells / well and allowed to adhere and grow. When cells reached the

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required confluence, they were placed in serumꢀfree medium and pretreated with different
concentrations of test compounds for 0.5 h, 45 and then Aβ25ꢀ35 (10ꢁM) was added to the medium
for the corresponding time.
4.2.2 Cell viability assay.
The neuroprotective effects of test compounds on Aβ25ꢀ35ꢀinduced PC12 cells were determined by
the MTT assay.46 After the treatments with test compounds and Aβ25ꢀ35, MTT (0.5 mg/mL, final
concentration) was added and incubated for 2 hours at 37 °C. Formazan crystals were dissolved by
DMSO, and absorbance was measured at a wavelength of 570 nm by a 1500 microplate reader
(Thermo Fisher Scientific Co.). Cell viability was expressed as a percentage of the nonꢀtreated
control. Values were represented as the mean±SD of 3 independent experiments in triplicate. The
significance of differences between groups was assessed by a oneꢀway analysis of variance
(ANOVA).
4.2.3. Inhibition evaluation of selfꢀinduced Aβ1ꢀ42 aggregation.
Thioflavin T (ThT) fluorescene assay34 was used to assess the ability of four chrysotoxine
derivatives to inhibit the selfꢀinduced Aβ1ꢀ42 aggregation with resveratrol and curcumin as
reference compounds. Aβ1ꢀ42 (Anaspec Inc) was dissolved in HFIP (1 mg/mL) and incubated for
24 h at room temperature. Then it was stored at ꢀ80 °C after solvent was evaporated. Solutions of
test compounds were prepared in DMSO at 25 mM for storage and diluted with phosphate buffer
solution (pH 7.4) before use.
For the inhibition of selfꢀinduced aggregation assay, 20 ꢁL Aβ1ꢀ42 (25 ꢁM, final concentration)
was incubated with 20 ꢁL of test compounds (25 ꢁM, final concentration) in 50 mM phosphate
buffer solution (pH 7.4) at 37 °C for 24 h. To minimize evaporation effect the wells were sealed by
a transparent heatꢀresistant plastic film. After incubation, 160 ꢁL of 5 ꢁM thioflavin T (TCI
(Shanghai) Development Co., Ltd.) in 50 mM glycineꢀNaOH buffer (pH 8.5) was added.
Fluorescence was measured on a Varioskan Flash Multimode Reader (Thermo Scientific) with
excitation and emission wavelengths at 446 nm and 490 nm, respectively. The fluorescence
intensities were compared and the percent inhibition due to the presence of the inhibitor was
calculated by the following formula: 100ꢀ (IFi/IFc*100), where IFi and IFc were the fluorescence
intensities obtained for Aβ1ꢀ42 in the presence and in the absence of inhibitor, respectively. Each
assay was run in triplicate.

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4.2.4. Inhibition evaluation of Cu2+ꢀinduced Aβ1ꢀ42 aggregation.
To investigate the ability of the 4 active compounds to inhibit Cu2+ꢀinduced Aβ1ꢀ42 aggregation, a
ThTꢀbinding assay was used similar as before.35 Resveratrol and curcumin were chosen as
reference compounds. Aβ1ꢀ42 was diluted in 20 mM HEPES (pH 6.6) with 150 mM NaCl. The
mixture of the peptide (10 ꢁL, 25 ꢁM, final concentration) with or without copper (10 ꢁL, 25 ꢁM,
final concentration) and the test compound (10 ꢁL, 25 ꢁM, final concentration) was incubated at
37 ºC for 24 h. 30 ꢁL of the sample was diluted to a final volume of 200 ꢁL with 50 mM
glycineꢀNaOH buffer (pH 8.0) containing thioflavin T (5 ꢁM). The detection method was the same
as that of selfꢀinduced Aβ aggregation experiment.
4.2.5. Metal chelating effect.
The study of metal chelation was performed in methanol at 298 K using UVꢀvis
spectrophotometer (SHIMADZU UVꢀ2450PC) with wavelength ranging from 200 to 500 nm. 36, 47
The difference UVꢀvis spectra due to complex formation was obtained by numerical subtraction of
the spectra of the metal alone and the compound alone (at the same concentration used in the
mixture) from the spectra of the mixture. A fixed amount of compound 2a (final concentration, 25
ꢁM) was mixed with growing amounts of copper ion or aluminum ion (final concentration, 2ꢀ50
ꢁM), and the difference UVꢀvis spectra was tested to investigate the ratio of ligand/metal in the
complex
4.2.6. Western blot analysis on Aβꢀinduced tau protein hyperphosphorylation.
After PC12 cells were lysed with RIPA buffer, total protein concentration was estimated by
Bradford assay. Total cell lysates (20 ꢁg) were resolved by 10% SDSꢀPAGE and transferred to
nitrocellulose membrane. Membranes were incubated in a blocking buffer (PBS, 5% nonꢀfat milk)
for 1 h at 20 °C. After overnight incubation (4 °C) with primary antibody(tau 5 at 1:1500; pꢀtau
S396 at 1:500; S199/202 at 1:500), the blots were washed in Tween 20ꢀTBS (TBST) (0.02%
Tween 20,100 mM Tris, pH 7.5, 150 nM NaCl) for 20 min and then incubated with secondary
antibody for 1 h at room temperature. The blots were washed in TBST for 20 min and incubated
with ECL chemiluminescent reagent (Thermo Scientific) for 3 min. Quantification of pixel
intensity was done with ImageꢀPro Plus 6.0 software. βꢀactin was used as a control to normalize
tau protein.
4.2.7. Docking study of compound 2a with Aβ1ꢀ42

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Molecular modeling calculations and docking studies were performed using Molecular
Operating Environment (MOE) software version 2009.10 (Chemical Computing Group, Montreal,
Canada). The Xꢀray crystal structure of Aβ1ꢀ42(PDB 1IYT) used in the docking study was obtained
from the Protein Data Bank (www.rcsb.org). Heteroatoms and water molecules in the PDB file
were removed at the beginning, and all hydrogen atoms were added to the protein. Amber99 force
field was assigned to the enzyme and the partial charges were calculated with the same force field.
Protonated states of the enzyme at pH 7.4 were obtained by following the Protonate 3D protocol
in which all configurations were set as default. Compound 2a was drawn in MOE with all
hydrogen atoms added. During the docking procedure, pose of compound 2a was initially
generated by Triangle Matcher method, and scored with london dG function. 30 Poses of the
compound were dedicated to the next refinement procedure. All poses were fineꢀtuned with the
force field refinement scheme. The best 10 poses of molecules were retained and scored. After
docking, the geometry of resulting complex was studied using the MOE's pose viewer utility.
4.2.8 In vitro bloodꢀbrain barrier permeability assay
The BBB penetration of compounds was evaluated using the parallel artificial membrane
permeability assay (PAMPA) described by Di et al.43 Commercial drugs, dodecane and DMSO
were purchased from Sigma and Aladdin. Porcine brain lipid (PBL) was obtained from Avanti
Polar Lipids. The donor microplate (96ꢀwell filter plate, PVDF membrane, pore size of 0.45 ꢁm)
and acceptor microplate were purchased from Millipore. The 96ꢀwell UV plate (COSTAR®) was
obtained from Corning Incorporated. The acceptor 96ꢀwell microplate was filled with 300 ꢁL of
PBS/EtOH (7:3), and the filter membrane was impregnated with 4 ꢁL of PBL in dodecane (20
mg/mL). Compounds were dissolved in DMSO at 5 mg/ml and diluted 50ꢀfold in PBS/EtOH (7:3)
to a final concentration of 100 ꢁg/mL. Then, 200 ꢁL of the solution was added to the donor wells.
The acceptor filter plate was carefully placed on the donor plate to form a sandwich, and the plates
were incubated undisturbed for 18 h at 25 ºC. After incubation, the donor plate was carefully
removed, and the concentration of the compounds in the acceptor wells was determined using a
UV plate reader (Flexstation® 3). Each sample was analyzed at five wavelengths in four wells and
in at least three independent runs. Pe was calculated by the following formula:
Pe=ꢀVd×[Va(Vd+Va)A×t]×ln(1ꢀdrugacceptor/drugequilibrium), where Vd is the volume of the donor
well, Va is the volume of the acceptor well, A is the filter area, t is the permeation time, drugacceptor

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is the absorbance obtained in the acceptor well, and drugequilibrium is the theoretical equilibrium
absorbance. The results are given as the mean ± SD. In the experiment, 10 quality control
standards with known BBB permeabilities were included to validate the analysis set. A plot of the
experimental data versus the literature values gave a strong linear correlation: Pe(exp.) =
1.054Pe(bibl.) + 0.5582 (R² = 0.987). From this equation and the limit established by Di et al.
(Pe(lit.)=4.0×10ꢀ6 cm sꢀ1) for BBB permeation, we concluded that compounds with a permeability
greater than 4.8×10ꢀ6 cm sꢀ1 can cross the BBB.
Acknowledgements
This research work was financially supported by the National Natural Science Foundation of
China (No. 81373956, No.81274064, No.81502950 and No.81573557), Seed Funding Program for
Basic Research from HKU (201111159043), the Priority Academic Program Development of
Jiangsu Higher Education Institutions, the Fundamental Research Funds for the Central
Universities (2015ZD010), Huahai Graduate Innovation Fund (NO. CX14Bꢀ007HH), College
Students Innovation Project for the R&D of Novel Drugs (J1030830), National Students
Innovation and Entrepreneurship Training Program (G15073).
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Graphical Abstract