Syntheses and evaluation of novel isoliquiritigenin derivatives as potential dual inhibitors for amyloid-beta aggregation and 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2013.05.015

A series of new isoliquiritigenin (ISL) derivatives were synthesized and evaluated as dual inhibitors for amyloid-beta (Aβ) aggregation and 5-lipoxygenase (5-LO). It was found that all these synthetic compounds inhibited Aβ (1-42) aggregation effectively with their IC₅₀ values ranged from 2.2 ± 1.5 μM to 23.8 ± 2.0 μM. These derivatives also showed inhibitory activity to 5-LO with their IC₅₀ values ranged from 6.1 ± 0.1 μM to 35.9 ± 0.3 mM. Their structure-activity relationships (SAR) and mechanisms of inhibitions were studied. This study provided potentially important information for further development of ISL derivatives as multifunctional agents for Alzheimer's disease (AD) treatment.

Full text of DOI:10.1016/j.ejmech.2013.05.015

Accepted Manuscript
Syntheses and Evaluation of Novel Isoliquiritigenin Derivatives as Potential Dual
Inhibitors for Amyloid-beta Aggregation and 5-Lipoxygenase
Yi-Ping Chen, Zi-Ying Zhang, Yan-Ping Li, Ding Li, Shi-Liang Huang, Lian-Quan Gu,
Jun Xu, Zhi-Shu Huang
PII:
S0223-5234(13)00315-2
10.1016/j.ejmech.2013.05.015
EJMECH 6193
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2013
Revised Date: 12 May 2013
Accepted Date: 13 May 2013
Please cite this article as: Y.-P. Chen, Z.-Y. Zhang, Y.-P. Li, D. Li, S.-L. Huang, L.-Q. Gu, J. Xu, Z.-S.
Huang, Syntheses and Evaluation of Novel Isoliquiritigenin Derivatives as Potential Dual Inhibitors for
Amyloid-beta Aggregation and 5-Lipoxygenase, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.05.015.
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ACCEPTED MANUSCRIPT
Syntheses and Evaluation of Novel Isoliquiritigenin Derivatives as Potential Dual
Inhibitors for Amyloid-beta Aggregation and 5-Lipoxygenase
Yi-Ping Chena,b, Zi-Ying Zhanga, Yan-Ping Lia, Ding Lia, Shi-Liang Huanga,*, Lian-Quan Gua,
Jun Xua, Zhi-Shu Huanga,*
IC50 = 3.2±1.2 ꢀM for Aβ (1-42) aggregation
IC50 = 6.1±0.1 ꢀM for 5-LO
A series of new isoliquiritigenin derivaives were synthesized and evaluated as a dual inhibitor of Aβ
self-induced aggregation and 5-lipoxygenase (5-LO). Compound 4d exhibited strong inhibitory
potency against both targets.

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Reseach highlights
► A series of novel Isoliquiritigenin (ISL) derivatives were synthesized.
► Most of synthesized compounds had better inhibition on Aβ aggregation and 5-LO.
►The amide derivatives(4b-4d) exhibit strong inhibitory potency against both targets .

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Title page
Syntheses and Evaluation of Novel Isoliquiritigenin Derivatives as Potential Dual Inhibitors
for Amyloid-beta Aggregation and 5-Lipoxygenase
Yi-Ping Chena,b, Zi-Ying Zhanga, Yan-Ping Lia, Ding Lia, Shi-Liang Huanga,*, Lian-Quan Gua, Jun Xua, Zhi-Shu
Huanga,*
aSchool of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China
bSchool of Pharmaceutical Sciences, Guangxi University of Chinese Medicine, Nanning 530001, People’s Republic of
China
* To whom correspondence should be addressed. Tel.: 8620-39949052, Fax: 8620 39943056, E-mail addresses:
lsshsl@mail.sysu.edu.cn (S.-L. Huang), ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
Abstract
A series of new isoliquiritigenin (ISL) derivatives were synthesized and evaluated as dual inhibitors for
amyloid-beta (Aβ) aggregation and 5-lipoxygenase (5-LO). It was found that all these synthetic compounds
inhibited Aβ (1-42) aggregation effectively with their IC50 values ranged from 2.2 ± 1.5 µM to 23.8 ± 2.0 µM.
These derivatives also showed inhibitory activity to 5-LO with their IC50 values ranged from 6.1 ± 0.1 µM to
35.9 ± 0.3 µM. Their structure-activity relationships (SAR) and mechanisms of inhibitions were studied. This
study provided potentially important information for further development of ISL derivatives as
multifunctional agents for Alzheimer’s disease (AD) treatment.
Keywords:
Isoliquiritigenin derivatives; Anti-Alzheimer agent; Amyloid-beta aggregation; 5-lipoxygenase; Inhibitors
Abbreviations:
AD: Alzheimer’s disease
Aβ: amyloid beta protein
APP: amyloid precursor protein
ADAM10: a disintegrin and metalloproteinase domain-containing protein 10
BACE1: beta-site APP cleaving enzyme 1
CD: circular dichroism
CNS: Central Nervous System
CREB: cAMP-response element binding protein
EDC: 1-ethyl-3-(3-dimethylaminoprpyl) carbodiide
EM: electron microscopy
FLAP: 5-lipoxygenase activating protein
HOBt: N-hydroxybenzotriazole
ISL: isoliquiritigenin
5-LO: 5-lipoxygenase
MTT: methyl thiazolyl tetrazolium
NDGA: nordihydro-guaiaretic acid
Resv: resveratrol
SAR: structure-activity relationships
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1. Introduction
Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a progressive
neurodegenerative brain disorder. It is characterized by dementia, cognitive impairment, and
memory loss [1-3]. The pathology of AD includes Tau protein hyperphosphorylation, aggregated
amyloid beta protein (Aβ) deposits, cholinergic system dysfunction and so on [4]. Although many
influencing factors have been found to be implicated in AD, its etiology and pathogenesis remain
unclear. Amyloid plaques, widely accepted as the key pathological feature of AD, are mainly
constituted by aggregation of the Aβ peptide which derived from the amyloid precursor protein
(APP) and consists of 39–43 amino acid residues [5]. Based on the amyloid cascade hypothesis, the
aggregation of Aβ has been considered to be a crucial step in the etiology of AD. The current
therapy strategies have focused on modifying the amyloid plaques formation, and clearing the
accumulation of this potentially neuron-toxic peptide. Therefore, the inhibition of amyloid plaques
formation and clearance of potential neurotoxic peptide by small molecular compounds are
promising strategies for the prevention and treatment of AD. Various compounds including anti-Aβ
aggregation agents, Aβ peptide production blockage agents (β- and γ-secretase inhibitors), Aβ
clearance agents (Aβ immunization and drugs modulating micro RNAs) [6], and anti-inflammatory
drugs [7-9] have been reported to show these properties and prevent Aβ neurotoxicity.
The 5-lipoxygenase enzyme (5-LO) is widely distributed within the central nervous system
(CNS) and its activity is regulated by 5-lipoxygenase activating protein (FLAP) [10]. A peculiar
aspect of the 5-LO/FLAP pathway is the fact that its expression levels are significantly increased in
CNS with aging, which is also region-specific in the hippocampus [11]. It has been confirmed that
5-LO activity was up-regulated in AD and the 5-LO protein levels in AD brains was higher than
healthy ones [12, 13]. Firuzi et al. studies showed that 5-LO targeted gene disruption or its in vivo
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selective pharmacological inhibition might lead to a significant reduction of Aβ deposition in the
brains [14]. Furthermore, their study also revealed that 5-LO and its biologically active metabolites
could affect CNS and AD via a possible mechanism involving the modulation of the γ-secretase
activity. In addition, Chu et al. studies [15-17] showed that 5-LO regulated the formation of Aβ by
activating the cAMP-response element binding protein (CREB) and preventing CREB activation by
pharmacologic inhibition or dominant negative mutants, which blocks the 5-LO-dependent
elevation of Aβ formation, γ-secretase mRNAs and protein levels. Chu et al. also provided
evidences that FLAP was involved in the same AD-like amyloidotic phenotype in vivo, and they
found that treatment with MK-591, a FLAP inhibitor, or Zileuton, a specific 5-LO inhibitor, both
had similar effects in the 5-LO enzymatic pathway of the pathogenesis of AD-like amyloidosis and
could result in significant reduction of the γ-secretase complex, but did not induce any change in the
steady state levels of APP, beta-site APP cleaving enzyme 1 (BACE1) or a disintegrin and
metalloproteinase domain-containing protein 10 (ADAM10) [16-17]. Therefore, 5-LO/FLAP
pharmacological inhibition may be beneficial for the treatment and prevention of AD.
Because of the importance and connection in multiple mechanism of 5-LO/FLAP and Aβ in AD
pathogenesis. In this study, we present the idea of designing novel dual-target agents which target
both 5-LO and Aβ aggregation.
It has been reported that many flavonoids and polyphenols including quercetin, curcumin, and
nordihydroguaiaretic acid (NDGA), inhibit not only Aβ aggregation [18-20], but 5-LO activity [21]
as well. These compounds have also shown beneficial effects in animal models of neurodisease.
Their structures are shown in Fig.1. Thereinto, chalcone is a member of flavonoids family,
containing a structurally similar moiety to curcumin (Fig.1). Recently, it has been reported that
radiolabeled chalcone derivatives can be used as amyloid imaging probes with high binding affinity
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to Aβ aggregates, high uptake into the brain, and rapid clearance from the brain [22]. Some recent
studies have indicated that some electron-donating groups such as amino, methylamino,
dimethylamino, methoxyl or hydroxyl groups play critical roles for the binding of chalcone to Aβ
aggregates [23-25].
Isoliquiritigenin (4, 2′, 4’-trihydroxychalcone, ISL, Fig.1), with the simple chalcone structure,
one of the components of Licorice (Radix Glycyrrhizae), has exhibited a variety of pharmacological
properties including antioxidative [26], anti-inflammatory [27], estrogenic [28], chemopreventive
[29] and antitumor activities [30]. Licorice is widely used in medical formulations including
anti-AD with a long application history in traditional Chinese medicine.
In the present study, a series of new ISL derivatives were designed and its dual inhibitors for
amyloid-beta aggregation and 5-LO (Scheme 1) were both investigated. Their in vitro biological
evaluations including inhibition of Aβ aggregation, cytotoxicity and anti-oxidant activity were
carried out. Their structure-activity relationships were studied. The mechanisms for inhibiting
β-sheet aggregation of Aβ (1–42) were further investigated by using circular dichroism (CD) and
electron microscopy (EM) experiments. Molecular docking study with MOE was also carried out to
understand the binding mode. Our studies provide potentially important information for further
development of ISL derivatives as novel multifunctional agents for AD treatment.
2. Chemistry
A series of ISL derivatives were prepared by using Claisen–Schmidt condensation. As shown in
Scheme 1, compounds 4 were prepared through condensation, hydrolysis, and amidation reactions
[31]. The key step was the conversion of compound 3 to 4 through reacting with various
corresponding amines in the presence of N-hydroxybenzotriazole (HOBt) and 1-ethyl-3-
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(3-dimethylaminoprpyl)carbodiide (EDC) producing various amides 4 with yields ranged from 30%
to 79%.
Compounds 5 were prepared through reactions of compound 1 with various aryl aldehydes 7 (as
shown in Scheme 1). Most of these reactions gave ordinary condensation products, but some
reactions only produced their further Williamson etherification products. For example, compounds
5a, 5c, 5f were generated from corresponding aldehydes 7a1, 7b, 7c2, respectively. Some aryl
aldehydes 7 (7a1, 7a2, 7b, 7c1, 7c2 and 7d) were prepared through the reaction of the starting
material aldehydes 6 with various alkyl bromides and K2CO3/DMF. The structures of all these target
1
13
compounds were determined by using H NMR, C NMR, HRMS, and their purity was measured
by using HPLC.
3. Results and Discussion
3.1. Aβ(1-42) self-induced aggregation inhibitory activity
The inhibitory activity of all compounds to Aβ(1-42) were tested following the method of Thio
flavinT fluorescence (ThT) assay in vitro [32] using resveratrol (Resv, Fig.1) as a reference. As
shown in Table 1, most of these compounds presented better inhibitory activity (with a range of IC50
value of 2.2 ± 1.5~ 23.8 ± 2.0 µM) than the original compound ISL (IC50 19.7± 0.8 µM) except for
2, 4f and 4j, and part of derivatives (4a-4d, 4k-4n, 5d, 5e, and 5g-5i) showed better inhibitory
activity compareing with Resv (IC50 15.9 ± 0.50 µM). Compounds 4b and 4d, which respectively
had 4-substituted side chain of N-methylpiperazin-1-yl and morpholine group in A ring, displayed
the most potent Aβ(1-42) aggregation inhibitory effects with IC50 values of 2.2 ± 1.5 µM and 3.2 ±
0.5 µM, respectively.
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Analyzing the structure-activity relationships (SAR), it was found that the introduction of
4-substituted side chain in A ring could increase inhibitory activity of compounds, and the structure
of side chain had an significant effects on their activity. It demonstrated the activity trend
following: compounds 4b-4d (R = six-member cyclic amines) > 4k-4n (R = aryl amines ) 䏌 5a-5i
(R′ = ethers) > 4e-4i (R = alkyl amines) 䏌 3 (R = OH) > 2 (R = OMe).
3.2. In vitro 5-LO inhibition
Inhibitory activity of all targeted compounds for 5-LO were determined by using potato 5-LO
(Cayman Chemical, USA) and an enzyme immuno assay (EIA) kit (Cayman Chemical, USA)
according to manufacturer’s instructions with NDGA (Fig. 1) as a reference. As shown in Table 1,
most of these compounds presented similar or better inhibitory activity to 5-LO (with a range of
IC50 value of 6.1 ± 0.1~ 35.8 ± 0.3 µM) than the original compound ISL (IC50 18.6 ± 0.2 µM)
except for 2, 3, 4e, 4j and 4l. And part of derivatives (4a-4d, 5d, and 5f) showed better and others
showed similar or weak inhibitory activity comparing with positive reference compound NDGA
(IC50 12.4 ± 1.3 µM). Among these compounds, 4b-4d, with a 4-substituted side chain of
N-methylpiperazin-1-yl, and piperidine and morpholine group in A ring, displayed the best
inhibitory activity to 5-LO with IC50 values ranged from 6.1 ± 0.1 to 7.9 ± 0.1 µM.
The SAR demonstrated that their activity follow the trend of compounds 4a-4d䟉R = cyclic
amines䟍IC50 = 6.1~10.5 ꢀM䟊> 5a-5i䟉R′ = ethers, IC50 = 9.7䠟16.8ꢀM䟊> 4j-4n䟉R = aryl amines䟍
IC50 =11.4䠟24.9 ꢀM䟊≈ 4e-4i䟉R = alkyl amines䟍IC50 =12.9䠟22.1 ꢀM䟊> 3 (R = OH ) > 2 (R =
OMe).
3.3. Molecular docking studies
To further study the interaction mode and analysis the SAR profile of these derivatives for
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Aβ(1-42) and 5-LO, molecular docking study was performed using software package MOE2010.
The X-ray crystal structure of the human 5-LO complex (PDB code 3O8Y) [33] and protein
Aβ(1-42) structure (PDB code 1IYT) [34] used in the docking study were obtained from the Protein
Data Bank (www.rcsb.org). Our docking studies indicated that most of these compounds exhibited
mainly σ-π interaction and hydrogen bonding with 5-LO or Aβ(1-42).
A binding mode study of compound 4d with 5-LO (Fig. 2) showed that there were two
intermolecular hydrogen bond interactions. One is hydrogen bond between His600 residue and the
carbonyl oxygen in the side-chain of A ring of compound 4d (N-O distance: 2.82 Å), and the other
one is between His367 residue and the carbonyl oxygen in the chalcone structure of 4d (N-O
distance: 2.87 Å). This indicates that hydrogen bond interactions play an important role in their
binding. Two reasons are concluded to address the strong inhibitory activity of 4d to 5-LO. Firstly,
the length and flexibility of substituents affect the formation and stability of hydrogen bonding and
thus influence the activities. Hydrogen bondings between 4d and His600, His367 residues are
contributed to the binding affinity of this compound to 5-LO. Impairment or destruction of the
hydrogen bonds could fundamentally lead to a descent inhibition. Secondly, the side chains of
compounds wedge in the deep cavity of the pocket, the size fitness of which directly influences
their activities. 4d with a 6-member ring fits perfectly to the pocket, which may lead to its high
activity.
The interactions were also observed in the complex of compound 4d /Aβ(1–42) (PDB code:
1IYT). B ring of compound 4d was combined with the central hydrophobic region Leu17-Ala21
(LVFFA), while the side-chain morpholine ring resided in the ring area Asp23-Lys28 (DVGSNK). A
σ-π interaction was found between B ring of 4d and Leu17 residue (Fig. 3), and a hydrogen bond
was found between His13 residue and the oxygen of hydroxyl group in the B ring (N-O distance:
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3.14Å). The result indicated that van der waals force together with hydrogen bond play important
roles in the stability of the 4d /Aβ(1–42) complex.
3.4. Effect of compound 4d on Aβ β-sheet formation
It has been reported that Aβ(1–42) adopted a conformational mixture of σ-helix, β-sheet, and
random coil in the aqueous solution and formed a intramolecular β-sheet structure in the fibrillation
through a conformational change [35]. In order to investigate the effect of compound 4d on the
structural transition of Aβ(1–42), Circular dichroism (CD) spectroscopy was employed to monitor
the changes of the secondary structure of Aβ(1–42). It has been showed that the contents of
β-sheet structure (occurrence of a peak around 195 nm) and σ-helix structure (occurrence of a broad
minimum around 217 nm) changed after the 48 h incubation (Fig. 4).
As shown in Fig. 4A, there was no aggregation of Aβ(1–42) before incubation. During 2 days
incubation (Fig. 4B and 4C), the CD spectrum of Aβ(1–42) with (red line) and without 4d (black
line) were both found to have negative bands around 217 nm and positive bands around 195 nm.
However, the presence of 4d in Aβ(1–42) sample resulted in a significant decrease of β-sheet
structure at all tested time, with slight change to the intensity of the band around 217 nm. These
results revealed that compound 4d could reduce or retard the β-sheet structure formation, without
significant effect on the content of σ-helix structure of peptide.
3.5. Effect of compound 4d on abundance of Aβ fibrils
In order to complement the ThT binding assay and CD assay, electron microscopy (EM) assay
was employed to monitor and clarify the effect of compound 4d on Aβ aggregation. As shown in
Fig. 5, After 4 days incubation, a large number of fibrils were observed in the control sample of
Aβ(1-42) alone (Fig. 5A). In contrast, only a few fibrils were found in the Aβ(1-42) sample
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incubated with compound 4d (Fig. 5B). The EM results were in agreement with the results of ThT
and CD studies, which were strongly further proving that compound 4d can inhibit and slow down
the Aβ(1–42) fibrils formation.
3.6. Cytotoxicity and antioxidation assay
The cytotoxicity of the ISL derivatives was examined in human neuro-blastoma SH-SY5Y cells.
In methyl thiazolyl tetrazolium (MTT) assays, the cells were treated with different concentrations of
compounds (0-100 µM), and our data indicated that all the compounds have their IC50 values above
100 µM. It is implied that all the target compounds have lower cytotoxicity. Their in vitro
anti-oxidative activity was also examined, but all exhibited lower anti-oxidative activity than Trolox
(data no shown).
Finally, it should be pointed out that all the data we received were obtained in vitro, and the
necessary IC50 values may be extremely difficult to meet with in vivo testing. We will continue to
explore this series of compounds in order to optimize the structural features and to discover more
potent derivatives with high efficacy.
4. Conclusion
In the present study, a series of new ISL derivatives have been synthesized and their biological
activities were evaluated. The SAR indicated that most of compounds had more effective and
similar inhibitory potencies against self-induced Aβ(1-42) aggregation and 5-LO than Resv and the
parental compound ISL except for compounds 2 and 3. Additionally, it was interesting that 4b-4d
present strong inhibitory potency against both targets mentioned above, they are amides with
4-substituted side chain of cyclic amine group in A ring . Further investigations of compound 4d by
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using CD and EM experiments confirmed that compound 4d could inhibit β-sheet aggregation and
fibril formation. In summary, our study provided potentially important information for further
development of ISL derivatives as new multifunctional agents for AD treatment.
5. Experimental Section
5.1. Chemistry
General procedures:
Commercially available solvents and reagents (chemicals) were used without further
purification, unless otherwise stated. Analytical thin-layer chromatography (TLC) was done with
HSGF 254. All target products were characterized by using NMR and High resolution mass spectra
(HRMS). NMR spectra were recorded using TMS as the internal standard in CDCl3 with a Bruker
Avance III 400 spectrometer. The chemical shifts are reported in parts permillion (ppm), the
coupling constants (J) are expressed in hertz (Hz) and signals are described as singlet (s), doublet
(d), triplet (t), quartet (q), quint, broad (br), as well as multiplet (m).
The purities of synthesized compounds were confirmed to be higher than 95% by using
analytical HPLC performed with a LC-20A system equipped with a Ultimate QB-C18 column
(4.6×250 mm, 5 ꢀm particle size) and eluted with acetonitrile/water (65:35-80:20) containing 0.1%
TFA at a flow rate of 0.5 mL/min, with calculation of the relative purity of each compound based on
its absorption at 254 nm.
5.1.1. Synthesis of ISL derivatives 2 and 3
5.1.1.1. (E)-methyl-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)benzoate (2)
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1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) (16.6 g, 100 mmol) and methyl 4-formylbenzoate
(16.4 g, 100 mmol) were dissolved in MeOH (500 mL), and 8% KOH in H2O (100 mL) was added.
The reaction mixture was stirred at room temperature for 48 h, and large amount of yellow solid
was precipitated. The yellow precipitate was filtered and washed with appropriate amount of water,
1
then recrystallized with MeOH to afford pure product 2 (24.5 g, 78%) as a yellow solid. H NMR
(400 MHz, CDCl3) δ 13.33 (s, 1H), 8.10 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.85 (d, J =
8.7 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 15.5 Hz, 1H), 6.52 (dd, J = 8.4, 2 Hz, 1H), 6.50
(d, J = 2 Hz, 1H), 3.96 (s, 3H), 3.89 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 191.4, 166.8, 166.5,
166.43, 142.7, 139.0, 131.6, 131.3, 130.2 (2C), 128.3 (2C), 122.6, 114.1, 107.9, 101.1, 55.6, 52.3.
ESI-HRMS m/z: calcd for C18H16O5 [M+Na]+ 335.0886, found 335.0890. Purity: 95.5% by HPLC.
5.1.1.2. (E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-enyl) benzoic acid (3)
Compound 2 (2.84 g, 10 mmol) was mixed with 10% KOH in H2O (20 mL), and heated on a
water bath at 50-60 °C until compound 2 completely dissolved. Then 10% HCl solution was added
to neutralize the solution to generate yellow precipitate. The yellow precipitate was filtered and
washed with appropriate amount of water, and recrystallized with MeOH to afford pure product 3
(2.6 g, 87%) as a yellow solid. 1H NMR (400 MHz, d6-DMSO) δ 13.34 (s, 1H), 13.13 (s, 1H), 8.30
(d, J = 9.1 Hz, 1H), 8.12 (d, J = 15.5 Hz, 1H), 8.03 (d, J = 8.9 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H),
7.85 (d, J = 15.5 Hz, 1H), 6.59 (dd, J = 9.0, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 3.87 (s, 3H). 13C
NMR (101 MHz, d6-DMSO) δ 191.5, 166.7, 166.1, 165.7, 142.4, 138.5, 132.7, 132.1, 129.6 (2C),
128.9 (2C), 123.3, 113.8, 107.4, 100.8, 55.7. ESI-HRMS m/z: calcd for C17H14O5 [M-H]- 297.0754,
found 297.0763. Purity: 96.5% by HPLC.
5.1.2. General procedure for synthesis of amide compounds 4a-4n
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A mixture of compound 3 (298 mg, 1.0 mmol), HOBt (270.2 mg, 2.0 mmol), and EDCI (383.4
mg, 2.0 mmol) was dissolved with CH2Cl2, and stirred for 10 min. The mixture was added with
amine (2.0 mmol), and stirred at the room temperature for 2h. The reaction mixture was
concentrated in vacuo to give the crude product. The crude product was purified by chromatography
with EtOAc/PE, and crystallized with EtOAc to afford pure products.
5.1.2.1.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(pyrrolidine-1-carbonyl)phenyl)-prop-2-en-1-one
(4a)
Compound 3 was reacted with pyrrolidine following the general procedure to give the desired
product 4a (159 mg, 52%) as a yellow solid, 1H NMR (400 MHz, CDCl3) δ 13.38 (s, 1H), 7.88 (d, J
= 15.5 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 15.0 Hz, 1H), 7.59
(d, J = 8.0 Hz, 2H), 6.51 (dd, J = 8.0, 2.5 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 3.88 (s, 3H), 3.67 (t, J =
6.9 Hz, 2H), 3.46 (t, J = 6.6 Hz, 2H), 2.04 – 1.95 (m, 2H), 1.95 – 1.86 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 190.5, 167.8, 165.7, 165.3, 142.2, 137.9, 135.1, 130.3, 127.3 (2C), 126.8 (2C), 120.5,
113.0, 106.8, 100.1, 54.6, 48.5, 45.3, 25.4, 23.4. ESI-HRMS m/z: calcd for C21H21NO4 [M-H]-
350.1389, found 350.1392. Purity: 96.7% by HPLC.
5.1.2.2.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(4-methylpiperazine-1-carbonyl)phenyl)prop-2-en-
1-one (4b)
Compound 3 was reacted with 1-methylpiperazine following the general procedure to give the
1
desired product 4b (228 mg, 60%) as a yellow solid. H NMR (400 MHz, d6-DMSO ) δ 13.37 (s,
1H), 7.88 (d, J = 15.5 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 15.5
Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 6.51 (dd, J = 8.0, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 3.88 (s,
3H), 3.75-3.82 (br, 2H), 3.54–3.40 (br, 2H), 2.55–2.36 (br, 4H), 2.34 (s, 3H). 13C NMR (101 MHz,
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d6-DMSO) δ 191.7, 168.3, 166.1, 165.7, 143.0, 137.8, 135.5, 132.7, 129.03 (2C), 127.4 (2C), 122.3,
113.9, 107.4, 100.9, 55.7, 54.6, 54.3, 46.9, 45.5, 41.4. ESI-HRMS m/z: calcd for C22H24N2O4
[M-H]- 379.1652, found 379.1658. Purity: 95.8% by HPLC.
5.1.2.3.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(piperidine-1-carbonyl)phenyl)prop-2-en-1-one
(4c)
Compound 3 was reacted with piperidine following the general procedure to give the desired
product 4c (129 mg, 35%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.37 (s, 1H), 7.88 (d, J
= 15.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 15.5 Hz, 1H), 7.46 (d,
J = 8.2 Hz, 2H), 6.51 (dd, J = 8.0, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 3.88 (s, 3H), 3.73 (br, 2H),
3.37 (br, 2H), 1.71 (br, 4H), 1.55 (br, 2H). 13C NMR (101 MHz, CDCl3) δ 190.5, 168.4, 165.7,
165.4, 142.2, 137.3, 134.8, 130.3, 127.5 (2C), 126.5 (2C), 120.3, 113.0, 106.8, 100.1, 54.6, 47.7,
42.2, 25.5, 24.6, 23.5. ESI-HRMS m/z: calcd for C22H23NO4 [M-H]- 364.1544, found 364.1549.
Purity: 99.7% by HPLC.
5.1.2.4.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(morpholine-4-carbonyl)phenyl)prop-2-en-1-one
(4d)
Compound 3 was reacted with morpholine following the general procedure to give the desired
product 4d (238 mg, 65%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.35 (s, 1H), 7.87 (d, J
= 15.5 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 15.5 Hz, 1H), 7.48 (d,
J = 8.2 Hz, 2H), 6.50 (dd, J = 8.0, 2.5 Hz, 1H), 6.47 (d, J = 2.5 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 4H),
3.66 (s, 2H), 3.47 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 191.40, 169.49, 166.68, 166.34, 142.87,
137.02, 136.20, 131.31, 128.56 (2C), 127.79 (2C), 121.62, 113.96, 107.77, 101.06, 66.77 (2C),
13

ACCEPTED MANUSCRIPT
55.58, 48.05, 42.67. ESI-HRMS m/z: calcd for C21H21NO5 [M-H]- 366.1345, found 366.1341.
Purity: 95.2% by HPLC.
5.1.2.5.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-isopropylbenzamide (4e)
Compound 3 was reacted with propan-2-amine following the general procedure to give the
1
desired product 4e (122 mg, 36%) as a yellow solid. H NMR (400 MHz, CDCl3 ) δ 13.36 (s,1H),
7.88 (d, J = 15.4 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.3 Hz,
2H), 7.63 (d, J = 15.5 Hz, 1H), 6.51 (dd, J = 8.0, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 5.98 (d, J =
8.5 Hz, 1H), 4.30 (m, 1H), 3.88 (s, 3H), 1.30 (d, J = 6.5 Hz, 6H).13C NMR (101 MHz, CDCl3 ) δ
191.5, 166.7, 166.4, 165.89, 142.9, 137.4, 136.5, 131.3, 128.5 (2C), 127.5 (2C), 121.9, 114.0, 107.9,
101.1, 55.6, 42.1, 22.8. ESI- HRMS m/z: calcd for C20H21NO4. [M-H]- 338.1396, Found 338.1392.
Purity: 95.9% by HPLC.
5.1.2.6.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-propylbenzamide (4f)
Compound 3 was reacted with propan-1-amine following the general procedure to give the
1
desired product 4f (102 mg, 30%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 13.33 (s, 1H),
7.87 (d, J = 15.7 Hz, 1H), 7.83 (d, J = 7.4 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.8 Hz,
2H), 7.62 (d, J = 15.5 Hz, 1H), 6.51 (dd, J = 8.0, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 6.20 (br,
1H), 3.87 (s, 3H), 3.44 (q, J = 8.0 Hz, 2H), 2.27–1.51 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 191.5, 166.8, 166.7, 166.4, 142.8, 137.5, 136.4, 131.3, 128.5 (2C), 127.5 (2C),
121.9, 114.0, 107.9, 101.1, 55.6, 41.9, 22.9, 11.4. ESI-HRMS m/z: calcd for C20H21NO4. [M-H]-
338.1394, Found 338.1392. Purity: 99.2% by HPLC.
5.1.2.7.(E)-N-dodecyl-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)benzamide (4g)
14

ACCEPTED MANUSCRIPT
Compound 3 was reacted with dodecan-1-amine following the general procedure A to give the
1
desired product 4g (272 mg, 58%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 13.35 (s, 1H),
7.88 (d, J = 15.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.3 Hz,
2H), 7.62 (d, J = 15.5 Hz, 1H), 6.51 (dd, J = 8.8, 2.5 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 3.87 (s, 3H),
3.47 (q, J = 7.1 Hz, 2H), 1.49–1.21 (m, 21H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3)
δ 191.5, 166.8, 166.7, 166.5, 142.9, 137.5, 136.4, 131.3, 128.5 (2C), 127.5 (2C), 121.9, 114.0, 107.9,
101.1, 55.6, 40.3, 31.9, 29.6 (2C), 29.6 (2C), 29.6, 29.5, 29.3, 27.0, 22.7, 14.1. ESI-HRMS m/z:
calcd for C29H39NO4 [M-H]- 464.2805, found 464.2801. Purity: 97.5% by HPLC.
5.1.2.8.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-phenethylbenzamide (4h)
Compound 3 was reacted with 2-phenylethanamine following the general procedure to give the
1
desired product 4h (320 mg, 79%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 13.34 (s, 1H),
7.87 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz,
2H), 7.63 (d, J = 15.5 Hz, 1H), 7.36 (t, J = 7.3 Hz, 2H), 7.32–7.22 (m, 3H), 6.52 (dd, J = 8.5, 2.5 Hz,
1H), 6.48 (d, J = 2.5 Hz, 1H), 6.18 (t, J = 4.9 Hz, 1H), 3.88 (s, 3H), 3.76 (q, J = 6.7 Hz, 2H), 2.97 (t,
13
J = 6.8 Hz, 2H). C NMR (101 MHz, CDCl3) δ 191.5, 166.8, 166.7, 166.5, 142.8, 138.8, 137.6,
136.1, 131.3, 128.8 (2C), 128.7 (2C), 128.6 (2C), 127.5 (2C), 126.7, 122.0, 114.0, 107.9, 101.1,
55.6, 41.2, 35.6. ESI-HRMS m/z: calcd for C25H23NO4 [M-H]- 400.1582, Found 400.1554. Purity:
96.3% by HPLC.
5.1.2.9.(E)-N-benzyl-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)benzamide (4i)
Compound 3 was reacted with phenylmethanamine following the general procedure to give the
1
desired product 4i (265 mg, 68%) as a yellow solid. H NMR (400 MHz, CDCl3 ) δ 13.33 (s, 1H),
15

ACCEPTED MANUSCRIPT
7.88 (d, J = 13.4 Hz, 1H), 7.85 (d, J = 6.1 Hz, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.3 Hz,
2H), 7.63 (d, J = 15.5 Hz, 1H), 7.38 (m, 5H), 6.51 (dd, J = 8.8, 2.8 Hz, 1H), 6.48 (d, J = 2.8 Hz, 1H),
6.44 (t, J = 5.1 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ
191.7, 166.1, 165.7, 165.5, 142.8, 139.5, 137.1, 135.7, 132.8, 128.9, 128.25 (2C), 127.7, 127.2 (4C),
126.7, 122.7, 113.9, 107.5, 100.9, 55.7, 42.7. ESI-HRMS m/z: calcd for C24H21NO4 [M-H]-
386.1386, Found 386.1392. Purity: 98.7% by HPLC.
5.1.2.10.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-methyl-N-phenylbenzamide
(4j)
Compound 3 was reacted with N-methylaniline following the general procedure to give the
1
desired product 4j (183 mg, 47%) as a yellow solid. H NMR (400 MHz, CDCl3). δ 13.33 (s, 1H),
7.77 (d, J = 6.2 Hz, 1H), 7.74 (d, J = 11.8 Hz, 1H), 7.50 (d, J = 15.5 Hz, 1H), 7.44 (d, J = 8.3 Hz,
2H), 7.35 (d, J = 8.3 Hz, 2H), 7.28–7.22 (m, 2H), 7.17 (tt, J = 7.2, 2 Hz, 1H), 7.05 (d, J = 8.4 Hz,
13
2H), 6.47 (dd, J = 7.0, 2.5 Hz, 1H), 6.45 (d, J = 2.5 Hz, 1H), 3.85 (s, 3H), 3.52 (s, 3H). C NMR
(101 MHz, CDCl3) δ 191.5, 169.7, 166.7, 166.3, 144.6, 143.1, 137.8, 135.8, 131.3, 129.4 (2C),
129.3 (2C), 127.8 (2C), 126.9 (2C), 126.7, 121.4, 113.9, 107.7, 101.1, 55.6, 38.4. ESI-HRMS m/z:
calcd for C24H21NO4 [M-H]- 386.1385, Found 386.1392. Purity: 97.9% by HPLC.
5.1.2.11.(E)-N-(2,4-dimethylphenyl)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)benza
mide (4k)
Compound 3 was reacted with 2,4-dimethylaniline (2.0 mmol) following the general procedure
to afford 4k (253 mg, 63%) as a yellow solid. 1H NMR (400 MHz, CDCl3). δ 13.33 (s, 1H), 7.93 (d,
J = 8.3 Hz, 2H), 7.90 (d, J = 16.4 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.76 (d, J = 7.8 Hz, 3H), 7.65
16

ACCEPTED MANUSCRIPT
(d, J = 15.5 Hz, 2H), 7.07 (m, 2H), 6.51 (m, 2H), 3.87 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H). 13C NMR
(101 MHz, CDCl3 ) δ 191.5, 166.9, 166.5, 164.8, 142.7, 138.0, 136.5, 135.5, 132.9, 131.3, 131.3,
129.7, 128.7 (2C), 127.8 (2C), 127.5, 123.5, 122.3, 114.1, 107.9, 101.2, 55.7, 20.9, 17.8.
ESI-HRMS m/z: calcd for C25H23NO4 [M-H]- 400.1543, Found 400.1549. Purity: 95.7% by HPLC.
5.1.2.12.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-(2-methoxyphenyl)benzami
de (4l)
Compound 3 was reacted with 2-methoxyaniline following the general procedure to give the
desired product 4l (285 mg, 71%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.36 (s, 1H),
8.59 (s, 1H), 8.53 (dd, J = 8.0, 1.5 Hz, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 15.5 Hz, 1H), 7.83
(d, J = 8.9 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 15.5 Hz, 1H), 7.11 (td, J = 7.6, 2 Hz, 1H),
7.04 (td, J = 7.6, 2 Hz, 1H), 6.94 (dd, J = 8.1, 1.2 Hz, 1H), 6.51 (dd, J = 8.9, 2.5 Hz, 1H), 6.48 (d, J
= 2.4 Hz, 1H), 3.95 (s, 3H), 3.86 (s, 3H). 13C NMR (101 MHz, ) δ 190.3, 165.7, 165.4, 163.2, 147.1,
141.6, 136.8, 135.6, 130.3, 127.6 (2C), 126.6 (2C), 126.5, 123.1, 121.1, 120.1, 118.8, 112.9, 108.9,
106.8, 100.1, 54.8, 54.6. ESI-HRMS m/z: calcd for C24H21NO5 [M-H]- 402.1335, Found 402.1341.
Purity: 98.5% by HPLC.
5.1.2.13.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-(2-hydroxyphenyl)benzami
de (4m)
Compound 3 was reacted with 2-aminophenol following the general procedure to give the
1
desired product 4m (183 mg, 47%) as a yellow solid. H NMR (400 MHz, d6-DMSO) δ 13.34 (s,
1H), 9.70 (br, 1H), 9.63 (s, 1H), 8.32 (d, J = 9.1 Hz, 1H), 8.13 (d, J = 15.5 Hz, 1H), 8.09–8.02 (m,
4H), 7.87 (d, J = 15.5 Hz, 1H), 7.64 (dd, J = 7.9, 1.5 Hz, 1H), 7.05 (td, J = 8.0, 1.4Hz, 1H), 6.93 (dd,
17

ACCEPTED MANUSCRIPT
J = 8.1, 1.4 Hz, 1H), 6.83 (td, J = 7.8, 1.4 Hz, 1H), 6.59 (dd, J = 9.0, 2.5 Hz, 1H), 6.54 (d, J = 2.5
Hz, 1H), 3.85 (d, J = 6.2 Hz, 3H). 13C NMR (101 MHz, d6-DMSO) δ 191.6, 166.8, 166.2, 165.7,
142.5, 138.6, 132.8, 132.2, 129.7 (2C), 129.0 (2C), 127.8, 127.3, 124.4, 123.5 (2C), 119.1, 113.9,
109.5, 107.5, 100.9, 55.8. ESI-HRMS m/z: calcd for C23H19NO5 [M-H]- 388.1179, Found 388.1185.
Purity: 99.1% by HPLC.
5.1.2.14.(E)-4-(3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-N-(2-hydroxy-5-nitrophenyl)b
enzamide (4n)
Compound 3 was reacted with 2-amino-4-nitrophenol following the general procedure to give
the desired product 4n (210 mg, 48%) as a yellow solid. 1H NMR (400 MHz, d6-DMSO) δ 13.30 (s,
1H), 8.33 (d, J = 9.0 Hz, 1H), 8.24 – 8.17 (m, 3H), 8.13 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 2.4 Hz, 1H),
7.96 (dd, J = 9.2, 2.4 Hz, 1H), 7.90 (d, J = 15.5 Hz, 1H), 6.85 (d, J = 9.1 Hz, 1H), 6.78 (s, 2H), 6.60
(dd, J = 9.0, 2.5 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H), 3.86 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ
191.6, 166.2, 165.7, 163.8, 148.3, 142.3, 139.5, 135.1, 134.5, 132.9, 130.6 (2C), 130.2, 128.9 (2C),
124.0, 123.7, 119.7, 113.9 (2C), 107.6, 100.9, 55.8. ESI-HRMS m/z: calcd for C23H19N2O7 [M-H]-
433.1038, Found 433.1036. Purity: 97.6% by HPLC.
5.1.3. Syntheses of various aryl aldehydes 7a1–7a2, 7b, 7c1-7c2 and 7d
General procedure for the syntheses of various aryl aldehydes 7a1–7a2, 7b, 7c1-7c2 and 7d.
To an aryl aldehyde 6 (10 mmol) and powdered K2CO3 (1.6 g) in DMF (15 mL), an appropriate
amount of dibromoalkane derivative (40 mmol) or mono bromoalkane derivative (15 mmol) was
added. After stirring at 80 °C for 4 h, white solid was precipitated. The precipitate was filtered and
washed with appropriate amount of water to afford products 7a1–7a2, 7b, 7c1-7c2 and 7d, and the
products were pure enough for the next step of the reaction without further purification.
18

ACCEPTED MANUSCRIPT
5.1.4. Synthesis of ISL derivatives 5a-5i
General procedures for the preparation of compounds 5a-5i.
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) (166 mg, 1 mmol) and various aryl aldehyde 7 or
commercially available aldehyde (1 mmol) were dissolved in EtOH (50 mL), in which was added
10% NaOH solution(10 mL). The reaction mixture was stirred at room temperature for 24 h, and
yellow solid was precipitated. The yellow precipitate was filtered and washed with appropriate
amount of water, the crude products recrystallized with MeOH to afford pure products.
5.1.4.1.(E)-3-(4-(3-ethoxypropoxy)-3-methoxyphenyl)-1-(2-hydroxy-4-Methoxyphenyl)prop-2-en-1-
one (5a)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7a1 following the general
1
procedure to give the desired product 5a as a yellow solid (280 mg, 65%). H NMR (400 MHz,
d6-DMSO). δ 13.53 (s, 1H), 7.85 (d, J = 5.7 Hz, 1H), 7.82 (s, 1H), 7.43 (d, J = 15.4 Hz, 1H), 7.23 (d,
J = 8.3 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.51–6.47 (m, 2H), 4.19 (t, J =
6.4 Hz, 2H), 3.94 (s, 3H), 3.86 (s, 3H), 3.61 (t, J = 6.1 Hz, 2H), 3.50 (q, J = 7.0 Hz, 2H), 2.13 (quint,
J = 6.3 Hz, 2H), 1.20 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 191.8, 166.6, 166.1, 151.3,
149.6, 144.6, 131.1, 127.7, 123.3, 117.9, 114.2, 112.7, 111.0, 107.6, 101.1, 66.9, 66.3, 66.2, 56.2,
55.5, 29.6, 15.2. ESI-HRMS m/z: calcd for C22H26O6 [M-H]- 385.1654, found 385.1657. Purity:
98.6% by HPLC.
5.1.4.2.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(3-methoxy-4-(3-phenoxypropoxy)phenyl)prop-2-en-1
-one (5b)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7a2 following the general
1
procedure to give the desired product 5b as a yellow solid (290 mg, 67%). HNMR (400 MHz,
19

ACCEPTED MANUSCRIPT
CDCl3) δ 13.53 (s, 1H), 7.85 (d, J = 4.4 Hz, 1H), 7.82 (d, J = 14.8 Hz, 1H), 7.43 (d, J = 15.4 Hz,
1H), 7.30 (d, J = 7.9 Hz, 2H), 7.23 (d, J = 8.7 Hz, 1H), 7.16 (s, 1H), 6.94 (m, 4H), 6.49 (m, 2H),
4.28 (t, J = 6.3 Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H), 3.93 (s, 3H), 3.86 (s, 3H), 2.34 (quint, J = 6.0 Hz,
13
2H). C NMR (101 MHz, CDCl3) δ 191.8, 166.7, 166.1, 158.8, 151.0, 149.6, 144.6, 131.1, 129.5
(2C), 127.9, 123.2, 120.8, 118.1, 114.5 (2C), 114.2, 112.7, 110.9, 107.6, 101.1, 65.7, 64.2, 56.09,
55.57, 29.20. ESI-HRMS m/z: calcd for C26H26O6 [M-H]- 433.1648, found 433.1651. Purity: 95.9%
by HPLC.
5.1.4.3.(E)-3-(3-(2-ethoxyethoxy)phenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en -1-one (5c)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7b following the general
1
procedure to give the desired product 5c as a yellow solid (185 mg, 54%). H NMR (400 MHz,
CDCl3) δ 13.41 (s, 1H), 7.85 (d, J = 15.5 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 15.5 Hz,
1H), 7.34 (t, J = 7.9 Hz, 1H), 7.25 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 7.01 (dd, J = 8, 2.4
Hz, 1H), 6.51 (dd, J = 9.0, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 4.19 (t, J = 5.2 Hz, 2H), 3.88 (s,
3H), 3.83 (t, J = 5.2 Hz, 2H), 3.64 (q, J = 7.0 Hz, 2H), 1.27 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 191.8, 166.7, 166.3, 159.3, 144.3, 136.2, 131.3, 129.9, 121.5, 120.6, 116.9, 114.4, 114.1,
107.7, 101.1, 68.9, 67.7, 66.9, 55.6, 15.2. ESI-HRMS m/z: calcd for C20H22O5 [M-H]- 341.1388,
found 341.1389. Purity: 95.4% by HPLC.
5.1.4.4.(E)-3-(4-(benzyloxy)phenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (5d)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7c1 following the general
procedure to give the desired product 5d as a yellow solid (180mg, 50%).1H NMR (400 MHz) δ
13.54 (s, 1H), 7.86 (d, J = 15.4 Hz, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H),
7.49–7.34 (m, 6H), 7.02 (d, J = 8.7 Hz, 2H), 6.53–6.45 (m, 2H), 5.12 (s, 2H), 3.86 (s, 3H). 13C
20

ACCEPTED MANUSCRIPT
NMR (101 MHz, CDCl3) δ 191.9, 166.6, 166.1, 160.9, 144.2, 136.4, 131.1, 130.4 (2C), 128.7 (2C),
128.2, 127.8, 127.5 (2C), 118.0, 115.4 (2C), 114.2, 107.6, 101.2, 70.2, 55.6. ESI-HRMS m/z: calcd
for C23H20O4 [M-H]- 359.1275, found 359.1283. Purity: 97.6% by HPLC.
5.1.4.5.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5e)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 4-methoxybenzaldehyde
following the general procedure to give the desired product 5e as a yellow solid (174 mg, 32%). 1H
NMR (400 MHz, CDCl3) δ 13.55 (s, 1H), 7.84 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.59
(d, J = 8.4 Hz, 2H), 7.43 (d, J = 15.4 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.55–6.37 (m, 2H), 3.84 (s,
3H), 3.83 (s, 3H).13C NMR (101 MHz, CDCl3) δ 191.9, 166.6, 166.0, 161.8, 144.2, 131.1, 130.4
(2C), 127.5, 117.8, 114.5 (2C), 114.2, 107.6, 101.1, 55.5, 55.4. ESI-HRMS m/z: calcd for C17H16O4
[M-H]- 283.0966, found 283.0970. Purity: 97.3% by HPLC.
5.1.4.6.(E)-3-(4-(2-ethoxylethoxy)phenyl)-1-(2-hydroxyl-4-Methoxyphenyl)prop-2-en-1-one (5f)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7c2 following the general
1
procedure to give the desired product 5f as a yellow solid (170 mg, 50%). H NMR (400 MHz,
CDCl3) δ 13.54 (s, 1H), 7.86 (d, J = 15.4 Hz, 1H), 7.83 (d, J = 9.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H),
7.46 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.55–6.41 (m, 2H), 4.18 (t, J = 5.2 Hz, 2H), 3.86
13
(s, 3H), 3.82 (t, J = 5.2 Hz, 2H), 3.61 (q, J = 7.0 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H). C NMR (101
MHz, CDCl3) δ 191.9, 166.6, 166.0, 161.1, 144.2, 131.1, 130.3 (2C), 127.6, 117.9, 115.1 (2C),
114.2, 107.6, 101.1, 68.8, 67.6, 66.9, 55.6, 15.1. ESI-HRMS m/z: calcd for C20H22O5 [M-H]-
341.1381, found 341.1389. Purity: 96.2% by HPLC.
5.1.4.7.(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (5g)
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1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 2,4,5- trimethoxybenzaldehyde
following the general procedure C to give the desired product 5g as a yellow solid (179 mg, 52%).
1H NMR (400 MHz, CDCl3) δ 13.69 (s, 1H), 8.16 (d, J = 15.5 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H),
7.52 (d, J = 15.5 Hz, 1H), 7.11 (s, 1H), 6.52 (s, 1H), 6.49–6.45 (m, 2H), 3.94 (s, 3H), 3.92 (s, 3H),
3.90 (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 192.3, 166.6, 165.8, 154.9, 152.7, 143.3,
139.8, 131.1, 118.1, 115.4, 114.3, 111.8, 107.4, 101.0, 96.8, 56.62, 56.3, 56.1, 55.5. ESI-HRMS
m/z: calcd for C19H20O6 [M-H]- 343, found 343.1182. Purity: 97.3% by HPLC.
5.1.4.8. (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5h)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 3,4,5-trimethoxybenzaldehyde
following the general procedure to give the desired product 5h as a yellow solid (190 mg, 55.4%).
1H NMR (400 MHz, CDCl3). 13.46 (s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 15.5 Hz, 1H), 7.45
(d, J = 15.4 Hz, 1H), 6.87 (s, 2H), 6.58–6.41 (m, 2H), 3.93 (s, 6H), 3.91 (s, 3H), 3.87 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ 191.6, 166.7, 166.2, 153.5 (2C), 144.5, 140.6, 131.2, 130.3, 119.5,
114.1, 107.7, 105.8 (2C), 101.0, 61.0, 56.2 (2C), 55.6. ESI-HRMS m/z: calcd for C19H20O6 [M-H]-
343.1181, found 343.1182. Purity: 97.5% by HPLC.
5.1.4.9.(E)-3-(4-(benzyloxy)-3-bromo-5-methoxyphenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-
1-one (5i)
1-(2-Hydroxy-4-methoxyphenyl)ethanone (1) was reacted with 7d following the general
1
procedure to give the desired product 5i as a yellow solid (150 mg, 32%). H NMR (400 MHz,
CDCl3) δ 13.39 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 15.4 Hz, 1H), 7.55 (d, J = 6.7 Hz, 2H),
7.49 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 15.4 Hz, 1H), 7.42–7.33 (m, 3H), 7.08 (d, J = 1.8 Hz, 1H),
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6.50 (dd, J = 8.8, 2.5 Hz, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.12 (s, 2H), 3.94 (s, 3H), 3.87 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 190.3, 165.7, 165.3, 152.9, 146.2, 141.6, 135.8, 130.9, 130.2, 127.5
(2C), 127.3 (2C), 127.2, 123.9, 119.6, 117.7, 112.9, 110.8, 106.8, 100.1, 73.9, 55.2, 54.6.
ESI-HRMS m/z: calcd for C24H21O5Br [M-H]- 467.0501, found 467.0494. Purity: 98.6% by HPLC.
5.2. Biological assays
5.2.1. Inhibition of Aβ (1-42) self-induced aggregation
The thioflavin-T fluorescence method was used as described by LeVine [32] with minor
modification. Experiments were performed by incubating the peptides in 50 mM phosphate buffer
(pH 7.4) at 37 °C for 5 days (120 h, Aβ(1-42) 40 ꢀM) with the tested compounds at different
concentrations (0, 2.5, 5, 10, 20, 40, 80 µM). After incubation, the samples were diluted to a final
volume of 180 µL with 50 µM glycine-NaOH buffer (pH 8.5) containing 5 µM Thioflavin-T.
Fluorescence signal was measured (excitation wavelength 450 nm, emission wavelength 485 nm
and slit widths set to 5 nm) on a monochromators based multimode microplate reader (INFINITE
M1000), adapted for 96-well microtiter plates. Each inhibitor was examined in triplicate. The
fluorescence intensities were recorded, and the percentage of inhibition on aggregation was
calculated by using the following equation: (1 – IFi / IFc) * 100% in which IFi and IFc were the
fluorescence intensities obtained for absorbance in the presence and absence of inhibitors,
respectively, after subtracting the background fluorescence of the 5 ꢀM Thioflavin-T solution.
5.2.2. In vitro 5-lipoxygenase inhibition assay
The IC50 values of all compounds were determined using potato 5-LO (Catalog No. 60401,
Cayman Chemical, Ann Arbor, MI, USA) and an enzyme immuno assay (EIA) kit (Catalog No.
760700, Cayman Chemical, Ann Arbor, MI, USA) according to manufacturer’s instructions and Li
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et al. [36] with minor modification.
The lipoxygenase inhibitor screening assay measured the hydroperoxides produced in the
lipoxygenation reaction. Pre-assay preparation was carefully performed according to the
instructions. To a 90 µL solution of 5-LO enzyme in 0.1 M Tris-HCl buffer, pH 7.4, 10 µL of
various concentrations of testing drug solutions (0, 2.5, 5, 10, 25, 40, 80 µM in a final volume of
210 µL) was added, respectively. The 5-LO reaction was initiated by the addition of 10 µL substrate
of linoleic acid (the final concentrations of 100 µM in the well). After putting the 96-well plate on a
shaker for 5 min, 100 µL of chromogen was added, and the plate was put on the shaker for another
5 min. The 5-LO activity was determined by measuring absorbance at a wavelength of 490 nm. The
percentage inhibition was calculated through the comparison of enzymatic activity with or without
inhibitor. The concentration required for the tested compound to cause 50% inhibition (IC50, µM)
was calculated from the concentration–inhibition response curve. Each assay was repeated at least
twice, and the average of measured data was reported.
5.2.3. Molecular modeling
All calculations and analysis were carried out with Molecular Operating Environment (MOE)
program (Chemical Computing Group, Montreal, Canada). The X-ray crystal structure of Aβ (1-42)
(PDB code 1IYT) and the human 5-LO complex (PDB code 3O8Y) used in the docking study were
obtained from the Protein Data Bank (www.rcsb.org).
Heteroatoms and water molecules in the PDB files were removed at the beginning, and all
hydrogen atoms were subsequently added to the proteins. Amber99 force field was assigned to the
enzymes and the partial charges were calculated with the same force field. Protonation states of
both enzymes at pH 7 were obtained by following the Protonate 3D protocol in which all
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configurations were set as default. Compounds were drawn in MOE with all hydrogen atoms added.
During the docking procedure, poses of compounds were initially generated by Triangle Matcher
method, and scored with Affinity dG and london dG function for 5-LO and Aβ(1-42), respectively.
30 Poses of each compound were dedicated to the next refinement procedure. All poses were fine
tuned with the Forcefield Refinement scheme with MMFF94x force field, and rescored with the
same function. A maximum of 5 poses for each compound were preserved eventually.
Finally, some poses were separately delivered to a further restrained minimization processing in
that potential hydrogen bondings might be overlooked in the rigid docking procedure.
5.2.4. CD assay
The CD assay was used as described by Chen et al. [37] and Yan et al. [38] with minor
modification, and 20 µM Aβ(1-42) (Anaspec Inc) was mixed with and without 10 ꢀM 4d in 50 mM
sodium phosphate buffer (pH 7.4). All solutions were incubated at 37 °C for 2 days. CD spectra
were obtained using a Jasco-810-150S spectropolarimeter (Jasco, Japan). A quartz cell with 1 mm
optical path was used, and spectra were recorded at 25 °C between 190 and 260 nm with a
bandwidth of 0.5 nm, a 3 s response time, and scan speed of 10 nm/min. Background spectra and
when applicable, spectra of compound 4d were subtracted.
5.2.5. EM assay
Aβ (1-42) peptide was dissolved in 50 mM phosphate buffer (pH 7.4), which was incubated in
the presence and absence of 4d at 37 °C. The final concentrations of Aβ(1-42) and 4d were 40 ꢀM
and 20 ꢀM, respectively. After 4 days incubation, aliquots of 10 ꢀL samples were placed on
carbon-coated copper/rhodium grid. After 1 min, the grid was washed with water and negatively
stained with 2% uranyl acetate solution for 1 min. After draining off the excess of staining solution
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by means of a filter paper, the specimen was transferred for examination in a transmission electron
microscope (JEOL JEM-1400).
5.2.6. Cell culture and MTT assay
SH-SY5Y cells were purchased from American Type Culture Collection (ATCC TIB71,
Manassas, VA) and cultured as described by Li et al. [39] with minor modification. Cells were
cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented
with 10% fetal bovine serum (GIBCO), 1 mM glutamine, 100 U /mL penicillin, and 100 µg/mL
streptomycin under 5% CO2 at 37 °C in humidified atmosphere. To measure cell viability, MTT
assay was performed as described previously. SH-SY5Y cells were sub-cultured in 96-well plates at
a seeding density of 10,000 cells per well. After 24 h, they were treated with different
concentrations of compounds (0-100 µM). After 48 h, the survival of cells was determined with
MTT assay. Briefly, 20 ꢀl of MTT (5 mg/mL) was added to each well and incubated for 4 h. The
MTT medium in each well was carefully removed and 100 µL DMSO was added into each well,
followed by incubation at 37 °C for 10 min with horizontal shaking. The absorbance of each well
was measured with a micro culture plate reader at the wavelength of 570 nm.
Acknowledgments
We thank the Natural Science Foundation of China (81273433) and Specialized Research Fund
for the Doctoral Program of Higher Education of China (20110171110051) and for financial
support.
Reference
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